STUDIES ON CAROI'ENOIDS AND THEIR PRECURSORS

A thesis presented by

JOHN BERNARD DAVIS

in part fulfilment of the requirements for

admittance to the

DIPLOMA OF MEMBERSHIP

OF

IMPERIAL COLLEGE

1962 2

ABSTRACT

Section (I) commences with a review of the previous work on the structure of the alleged natural precursors of the . The structures of , , and neurosporene are unambiguously elucidated with the aid of nuclear magnetic resonance spectroscopy. The number of possible formulae that can now be proposed for 4r-(zeta-) is reduced to two.

Most schemes of carotogenesis involve these four compounds; a reappraisal of these schemes is given in the light of this new knowledge.

Section (II). The occurrence, alleged biological activity, and biogenesis of are discussed. The first total synthesis of the closely related polyene astacene, which might also occur in Naturev is described.

The use of reversed-phase chromatoplates is advocated in work involving highly oxygenated carotenoids of this type.

Section (III) commences with a review of the occurrence of the epoxides (and their furanoid oxide derivatives) in Nature; the characteristic properties of these compounds are mentioned. An improved synthesis of A-carotene diepoxide is described. The nuclear magnetic resonance spectra of some of these compounds are measured and discussed: the presence of an epoxide (or furanoid oxide) end-group in a carotenoid gives rise to a characteristic band pattern in the spectrum, and this should be of use in recognising these types of carotenoids in future.

Section (IV). Improved syntheses of two recently discovered carotenoids, 3 isorenieratene and renierapurpurin, are described.

Appendix (I). The various techniques which have been used in the past to isolate from seaweed are discussed; the yields obtained were often poor or inconsistent. An isolation procedure which combines some of the better features of these methods is developed; this procedure gives consistent results, and the pigment so isolated is pure.

Appendix (II) describes an attempt to complete the first total synthesis of azafrin; a pigment is obtained which is very similar to, but not identical with, azafrin. 4

ACKNOWLEDGMENTS

I would first like to thank Professor Weedon for his constant guidance and encouragement during the course of this work.

The first two years of this course were spent at Imperial

College and I am very grateful to Professor Barton for laboratory facilities there. Thanks are also due to Dr. Jackman and his colleagues

(especially Drs. Lown, Pratt, and Thompson, and Messrs. Foster and Serkis) for the measurement of the nuclear magnetic resonance spectra. I would also like to thank Mr. Watson and the staff of Room 80 for excellent technical assistance, Dr. Erskine and Mrs. Boston for some of the infrared and ultraviolet spectra and Miss Cuckney and her staff for some of the microanalyses. This work was completed at Queen Mary College and thanks are due to Mr. Cook for the measurement of infrared spectra there.

I am indebted to Hoffmann-La Roohe A.G. (Basel) and to Roche

Products Ltd. (Welwyn Garden City) for generous gifts of chemicals and for financial support. Thanks are also due to Dr. Allen for the Chlorella vulgaris, the Nutritional Research Associates, Inc. for the carrot oil, and Drs. Rabourn, Nakayama, and Arigoni for some of the other materials used in Section I; Dr. Eugster kindly provided the sample of taraxanthin mentioned in Section III.

Thanks are also due to colleagues in both colleges for some helpful discussions, and in particular to Mr. (now Dr.) Mike Barber for 5 his suggestions during the early stages of this work.

Finally, I would like to express my gratitude to my parents for their invaluable help and encouragement.

Chemistry Department, 19a414 ("Top laboratory"), PcemeR 1741

Queen Mary College,

London, E.1. 6

NOTES

Whenever possible, experiments involving polyenes were carried out in an atmosphere of pure, dry nitrogen, and all solvents were evaporated either under reduced pressure or under the influence of a stream of nitrogen. Solutions of polyenes were not exposed unduly to bright light.

Unless otherwise stated, light petroleum refers to the fraction of b.p.

60-80°, 'alumina (II)' to commercial "activated alumina" (Spence, type H - 1‘ which was found to be of approximately grade II activity ), and 'alumina (iv) ' 2 1 to acid-washed, grade IV alumina. The special precautions which were taken when working with some of the substances described in this thesis are described at the beginning of the experimental parts of the relevant Sections.

Melting points suffixed f(a were determined on a Kofler block, and are corrected. All other melting points were determined in capillary tubes, and for polyene materials these capillaries were sealed under vacuum; whether these melting points are corrected or not is indicated in the text.

Visible- and ultraviolet-light absorption spectra were determined on a Unicam S.P. 500, a Perkin-Elmer Spectracord, a Unicam S.P. 700, a second Unlearn S.P. 700, or a Unicam S.P. 600. Nearly all the quantitative measurements were obtained using one of the first three of these instrments;the spectrum obtained from a test solution of a carotenoid (isorenieratene) on each of these three instruments was identical (within experimental error) with respect to both wavelength and extinction coefficient. The Unicam S.P. 600 and the Spectracord have previously been shown to give spectra which were "normally identical".3 Solvents used for these spectral measurements are indicated either in the text or in the introduction to the

Experimental sections. The most intense band in a given spectrum is underlined, and inflections are -indicated by enclosing the relevant wavelength value in parentheses [e.6.(420)).The underlining principle has not

been used when reporting the many bands present in a complex spectrum (as

obtained on a multicomponent mixture).

Infrared spectra were determined on a Grubb-Parsons. double beam

S-4 spectrometer with sodium chloride optics (at Imperial College) and on a

Grubb-Parsons double beam grating spectrometer, type GS-2A (at Queen Mary

College). Most of the extinction coefficients were obtained using the former instrument; in those cases where the extinction coefficients of the peaks in a spectrum obtained with one instrument are compared with those from the other, some indication is given in the text of the extent to which a direct comparison is justified.

Nuclear magnetic resonance spectra were determined with a

Varian 4300 spectrometer with a 40 or 56.4 megacycle oscillator. The spectra were measured in dilute solutions (2-5%) using carbon tetrachloride, chloroform, or deuterochloroform as solvents. The line positions in each solvent have been shown to be identical within experimental error.4 The spectra were calibrated, using the usual side-band technique,5 against tetramethylsilane'as internal reference. The results, expressed as 5 6 T-values' are the averages of at least three separate determinations. a

Diagrams have been drawn with the field increasing from left to right, as

is the modern convention.5

Chloroform for all spectral determinations was freed from ethanol

(and any traces of acid) immediately before use by passing the commercial solvent through alumina (II).

Arabic numerals after titles in the experimental sections refer to

page numbers in laboratory notebooks (the pagination in these was consecutive).

In the references quoted at the end of each section, those

references citing Chemical Abstracts as the source were not checked against

the original paper as the latter could not be obtained (this applies

especially to papers which have appeared in bulletins issued from foreign

universities and research institutes).

Carbon atoms in carotenoid formulae have been numbered using 8 Winterstein's modification? of Karrer's scheme, as illustrated for y-carotene below (cf. Goodwin9).

y -carotene

References

1. Brockmann and Schodder, Ber., 1941, a, 73.

2. Cheesman et al., J., 1949, 3120. 9

3. Barber, Ph.D. Thesis, London, 1960, p. 5.

4. Barber, Davis, Jackman, and Weedon, J., 1960, 2870.

5. cf. Jackman, "Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry", Pergamon, London, 1959.

6. Tiers, J.Phys.Chem., 1958, 62, 1151.

7. Winterstein et al., Angew.Chem., 1960, 902. 8. Karrer, Bull.Soc.Chim.biol., 1948, 12, 150.

9. Goodwin, "The Comparative Biochemistry of the Carotenoids", Chapman and Hall, London, 1952, p.2. 10 OTT-1 TS

SECTION I (Carotenoid precursors) Page

Introduction, review, and discussion .. 12

Experimental 121

References 146

SECTION II (Astacene)

Introduction, review, and discussion 156

Experimental ;)21

References 237

SECTION III (Carotenoid epoxides)

Introduction, review, and discussion 246

Experimental 296

References 301

SECTION IV (Isorenieratene and renierapurpurin)

Introduction, review, and discussion 306

Experimental 336

References 353

APPENDIX I (Fucoxanthin)

Introduction, review, and discussion 359 Experimental 364

References 371

APPENDIX II (Azafrin)

Introduction, review, and discussion 373 Experimental 379 References 386 11

SECTION I

THE NATURAL PRECURSORS OF THE CAROTENOIDS 12

The Natural Precursors of the Carotenoids

The first comprehensive scheme of carotenoid biogenesis was

propounded in 1950. Four of the five C hydrocarbons which were 40 postulated, in this scheme, to be involved in carotcgenesis had been

discovered in Nature in the period 1930 - 1950. The first part of this

review will outline the discovery of these substances, and indicate some of

the natural sources from which they have since been isolated. Other

reviews concerned with various aspects of this subject are occasionally cited here.

In 1939, Strain5 isolated, from carrots, a carotenoid whose

longest wavelength absorption band (in light petroleum) was 425 9.1 - an

unusually low value as compared with other carotenoids known at that time.

The substance appeared as a lemon-yellow, diffuse zone, above 0-carotene,

but below 6-carotene, on a magnesia column. It behaved as a hydrocarbon

both on a chromatogram, and on partition between light petroleum and 90%

methanol.7a It gave a blue colour with sulphuric acid, and also with the

Carr-Price reagent ,7b These observations suggested that the substance was

apolyene hydrocarbon. Strain succeeded in crystallising the material

(from light petroleum-methanol); the freshly prepared crystals were orange rbut contained traces of a white insoluble impurity; he did not record

their melting point. On exposure to air, they rapidly became colourless.

However, the freshly prepared crystals, on dissolution in light petroleum,

gave an absorption spectrum (with maxima at 425 and 400 mi ) which showed good fine structure suggesting that the crystalline solid, was a genuine

polyene and not an oxidation product; Strain did not record extinction 13 coefficients, however, so that the purity of the crystals he obtained cannot be assessed.

6 Strain has since repeated some of this work and named this substance '-(zeta-) carotene. (It is interesting to note that, until recently, most of the workers in this field have been unable to crystallise

r -carotene; attempts to do so have usually resulted in the formation of 8 19 a white, gummy oxidation product; ' see later discussion). It should be noted here that a substance with an absorption spectrum very similar to that of Strain's C'-carotene had been isolated, also from carrots, in 1932.9

17,21 C -carotene has since been isolated, not only from carrots, but also from a wide variety of other natural sources; for example, palm 10 11 12,13,16 14,15 oil, Valencia oranges, fungi, and bacteria. In addition, 18,19,22 Porter and his co-workers have succeeded in breeding new strains of tomato plants which bear fruit containing relatively large amounts of r -carotene (and other "more saturated" polyenes; see below), so providing a usefule source of this compound. (More recently, carrot oil has been 8 20 found to provide a convenient source of large quantities of C-carotene; ' 8 the r -carotene content of this oil has been reported to be as high as ca. 6g./kg.).

The early work on the structure of C-carotene was carried out by Nash et al.'24 on material isolated from hybrid tomatoes. Thus they showed that r-carotene had no vitamin-A activity,23 and analyses suggested that it was a C40 hydrocarbon with a methyl-branched (or "polyisoprenic") 24 structure. Microhydrogenation suggested the presence of nine or ten 14

19 double bonds; its visible spectrum showed that only seven of these were conjugated.24 On the basis of these observations, Nash, Quackenbush, and 24 Porter suggested, in 1948, that te -carotene was an octahydrolycopene.

They were unable to establish the position of the heptaene chromophore but, by analogy with , they suggested it was located centrally and, accordingly, proposed the following structure (I):-

(1)

This structure has since been disproved (see later discussion ).

In his paper of 1939, mentioned previously, Strain5 found not only C-carotene in his carrot extract but also some colourless, fluorescent substances. These were present in relatively large amounts, and appeared below the a-carotene zone on a magnesia chromatogram.

Evaporation of the solvent from the fluorescent fractions gave oils which

Strain was unable to crystallise, and which oxidised very rapidly on exposure to air. He did not record the absorption spectra of these 25 substances, but it seems likely that the strongly fluorescent substance 26 isolated by Zechmeister and Polgar in 1944, and subsequently named 25 ftphytofluene", was at least one component of Strain 's mixture of fluorescent substances. Indeed, it is possible that these were a mixture 27 26 of phtyofluene stereoisomers. However, Zechmeister and Polgar are generally credited with the discovery of phytofluene since they not only isolated it in a relatively pure state, but also described some of its 15

properties, and conducted a limited survey of its occurrence in Nature.

Thus, they demonstatrated the presence of phytofluene in the flowers of several different plants, and in oranges, carrots,and tomatoes. They concluded that phytofluene was a polyene hydrocarbon from its behaviour on a chromatogram, in partition tests, and in tests with the usual acidic 27 28 reagents. '

Since then, phytofluene has been found in tomatoes29-32 (in both 18\ the commercial varieties and in the fruit of specially bred plants ); in 11,33 oranges, and in some other fruits;33 in palm oil;10 in four types of fungus;12'34-37 and in the flowers, fruits and other organs of plants from

19 different families.25'39 In addition, and rather surprisingly on the 25 basis of earlier work, phytofluene has also been found in the chlorophyll- 38, rich tissues of some plants; 44,44a (earlier it had appeared to be notably absent in chlorophyllous tissue being present only in those plant organs which produce large amounts of carotenoid pigments in the absence of chlorophy1125).

It is noteworthy that Strain,55 in 1936, isolated two intensely fluorescent substances from a wide variety of leaf extracts. They also appeared Just below a-carotene on a magnesia chromatogram. However, although both substances absorbed light in the ultraviolet, neither gave a spectrum with maxima and minima, sugesting that neither was homogeneous.

There has been some speculation as to whether either of these was 25 phytofluene; Zechmeister and Sandoval thought not, though Porter and 30 56 Zscheile, and more recently Zechmeister himself, have included Strain's 1_6 materials in their references to the occurrence of phytofluene in Nature.

The fact that Strain obtained both his substances as solids does not entirely preclude the idea that initially he had isolated phytofluene and then this had been converted into a solid oxidation product during its manipulation.

In 1946, Zechmeister and Sandoval32 showed that phytofluene was a C isoprenic compound with, apparently, seven double bonds of which 40 five were conjugated; they postulated no definite structure for phytefluene at this stage.

Meanwhile, in 1946, Porter and Zscheile" had isolated two 22 colourless polyenes from an extract of their specially bred tomatoes; one of these polyenes was intensely fluroescent and was, almost certainly, phytofluene. The other polyene, however, was almost non-fluorescent

(a highly concentrated solution displayed a pale blue fluorescence). This new polyene appeared below phytofluene on a magnesia column; it gave a characteristic ultraviolet-light absorption spectrum whose maxima were at even shorter wavelengths than those of phytofluene. Its other properties were similar to those of phytofluene: the substance was an oil which was rapidly oxidised on exposr.re to air, and it behaved like a polyene 18 hydrocarbon. It was named "phytoene" by Porter and Lincoln who isolated it from the fruit of hybrid tomatoes. Since then, phytoene has been found in carrot oil 20,40 in a wide variety of fruits4 1'42 and vegetables;41 in tomatoes (in both the commercial varieties,43 and in hybrids18); in palm 10 41 oil; in chlorophyllous tissue ( alpha alpha, and Hevea brasiliensis 17 leayss44);44a and in a variety of fungi.45-49

In 1949, Haxo35 isolated a new, crystalline pigment from the fungus Neurospora crassa (wild type). The pigment, which he named

"neurosporene", had the properties expected of a polyene hydrocarbon. Its absorption spectrum indicated the presence of nine double bonds in conjugation. Analyses suggested that neurosporene was a C compound, and 40 the formular C H + 2H was tentatively assigned to it. Haxo showed that 40 58 — neurosporene was probably identical with an unidentified, minor component

(called "pigment A") of a carotenoid mixture obtained three years previously

(by Zechmeister et a1.36) from a mutant of another fungus (the red yeast,

Rhodotorula rubra); the two pigments showed no separation on a mixed chromatogram (cf. p. 39, however), and they had qualitatively identical visible absorption spectr05

As yet, neurosporene has only been isolated from a few other sources - mainly from the two fungi Neurospora crassa (see above), 50'51 13 37 52 10 and Phycouyces blakesleeanus; ' ' other sources include palm oil, a green mutant of the bacterium Rhodopseudomonas spheroides,53 ripe 61-65 Pyracantha berries,54 and some X-ray mutants of the alga Chlorella vulgaris.

(In several of these papers the pigment was reported as "tetrahydrolycopene" since until recently it was assumed that neurosporene was a tetrahydrolycopene). The "Unidentified Carotene I" isolated by Porter 30 54a and Zscheile from tomatoes was probably proneurosporene. 18

Theories of carotogenesis

As early as 1934, Zechmeister57 had suggested that the final

chemical step in the formation of polyene pigments in Nature might involve

the dehydrogenation of a colourless precursor. Support for this idea came

from the knowledge that the formation of carotenoid pigments in tomatoes and other fruit requires the presence of oxygen,57 - an observation which

has been frequently confirmed since that time.1'51

25 The discovery of phytofluene encouraged Zechmeister to expand

this idea - especially when it was found that phytofluene had a C 40 32 isoprenic type of structure similar to that of the carotenoids. In 1946, 36 Zechmeister et al. suggested that the following scheme might operate

during the biogenesis of the carotenoids found in the red yeast Rhodotorula

rubra and some of its mutants:-

Ma- >torulene I •Unknown precursor(s) --> phytofluene trb >p- and y-carotene, etc.

Step I was apparently blocked in the colourless mutants, whilst step IIa

was largely blocked in two of the other (coloured) mutants since both of these contained only traces of but normal amounts of the other carotenoids.

18 In 1950, Porter and Lincoln developed this idea for, by that time, four hydrocarbons (phytoene, phytofluene, C-carotene, and neurosporene) were recognised and, although none of their structures was known with certainty, it was apparent that three bf these, at least, were C compounds 40 19

(from molecular weight determinations). Also, it was known from an

examination of their ultraviolet and visible absorption spectra that these

substances contained chromophores of three, five, seven, and nine

conjugated double bonds, respectively. In addition, all four had been

found in Nature in association with true carotenoids so that it appeared

likely that they might be concerned in carotogenesis.

Porter and Lincoln carried out some genetical studies on tomatoes.

Extensive cross-breeding of various types of natural tomato produced plants

which yielded fruit containing unusually large amounts of phytoene,

phytofluene, and T--carotene. An examination of the relative quantities

of these three substances and of lycopene present in the fruit of the

various strains of hybrid tomatoes, suggested that lycopene was being

formed from the three more saturated substances; in particular, it seemed

Unlikely that the lycopene was being converted into the more saturated

substances by hydrogenation.

At that time (1950), r-carotene was considered to be an 24 octahydrolyoopene (C H ) and the results obtained by Zechmeister and 40 64 32 Sanoval and by Porter et al.58 suggested that phytofluene was a

dodecahydrolycopene (C401168). Porter and Lincoln 18 considered

neurosporene to be identical with what they (erroneously) called

tetrahydrolyeopene (040H60). It was assumed (presumably on the basis

of ultraviolet absorption spectra, and by analogy with the rest of the

series) that phytoene had the molecular formula C401%. A new substance

that they had found in the first fraction from the chromatograms of

20 tomato extracts contained, apparently, only isolated double bonds.

Porter and Lincoln suggested that this was a tetrahydrophytoene and assigned it a tentative structure (II).

They postulated that it is formed in Nature from two C20 units, and that it then undergoes the following series of reactions - four hydrogens being

lost at each step:-

72?)__7:11L.> Tetrahydrophytoene (II, C40 phytoene (C4011 -411 phytofluene (C H 7) -411 >r carotene 401164?) 40 68. (C

-411 neurosporene (C H lycopene (C4056). 40 60:7)

In addition, they suggested that a-, 3-, and y- were formed from lycopene by the following series of isomerisations:-

Lycopene—>y-carotene—>p carotene---->a carotene

(It is noteworthy that Turian,59 also in 1950, made similar though

less detailed suggestions regarding carotogenesis in the bacterium Mycobacterium phlei).

Porter and Lincoln pointed out that, in 1950, there had been

no reports of the presence of phytoene, phytofluene, fp -carotene, or

lycopene in green leaves suggesting that a different biosynthetic mechanism

might be operating there. Since then, however, both phytoene and

phytofluene have been found in green leaves,38,44 and Eny44 suggested, 21. therefore, that there was no need to restrict the Porter-Lincoln 60a hypothesis to non-chlorophyllous tissue. Mackinney, however, considered it most unlikely that carotogenesis in all organisms is governed by one main scheme.

During the past ten years, many experiments have been carried out by Goodwin, Mackinney, Stanier, and others to determine to what extent, if any, carotogenesis involves sequential desaturation of the kind la proposed by Porter and Lincoln. Much of this work was done with organisms and plants which had been interfered with in some way so as to cause the organism to produce unnaturally large quantities of the members of the phytoene series (phytoene, phytofluene, C-carotene, and neurosporene). This was done using one of two main methods:-

(a)by causing genetic mutations. This was done by irradiation with 36 61-65 ultraviolet light or X-rays; the most promising (ie. least coloured) areas of the growth were isolated and then allowed to grow. Extensive 18 cross-breeding as used by Porter and Lincoln has also been used; this

method has the advantage that some degree of control can be exerted on the process, whereas irradiation methods tend to produce rather genetically haphazard results; occasionally, spontaneous albino mutants have been 66 obtained from "normal" tomatoes by repetitive breeding;

(b)by growing the organism in the presence of a trace of diphenylamine

(DPA). Turian,59 in 1950, developed the earlier observations of Kharasch 67 et a1. and showed that the addition of a trace (ca. 1/40,000) of DPA to the culture medium supporting a growth of MYcobacterhumphlei inhibited the

normal biosynthesis of carotenoids in that organism. It has since been 22

14 68 shown ' that this inhibtion in the synthesis of normal carotenoids is accompanied by a marked increase in the concentration of the phytoene series 13 (usually present in only trace amounts14 ) in the organism. Goodwin found that the same effect occurred in the fungus Phycomyces blakesleeanus

(which normally contains only very small amounts of the phytoene series); the effect of DPA on carotogenesis in a variety of other organisms has 69 since been investigated.

1,70b,72,73 Both Mackinne y6° and Goodwin have been critical of the Porter-Lincoln theory of carotogenesis. Goodwin,13 in a study on the effect of DPA on carotogenesis in Phycomyces blakesleeanus, showed that

DPA inhibits the production of the most unsaturated polyenes (a-, p-, and y-carotenes, and lycopene) and stimulates the synthesis of phytofluene, r -carotene, and (to a slight extent) neurosporene; (the presence of DPA, and also of naturally derived impurities, in the phytoene fraction from the chromatograms of DPA-cultured fungi precluded the possibility of obtaining any reliable data on phytoene biosynthesis). Goodwin pointed out that these results could be interpreted in two different ways: either as a confirmation of the Porter-Lincoln theory, or as evidence that a different "parallel" type of biosynthesis of the following kind was occurring:-

phytoene A , phytofluene (Route A, unaffected by DPA) CP -carotene neurosporene Common Precursor a-carotene B ;) p-carotene (Route B, blocked by DPA) y-carotene lycopene 23

Thus, blocking the synthesis (Route B) of the fully unsaturated polyenes by DPA treatment wOuld make more of the common (unknown) precursor available for the synthesis of the more saturated polyenes (Route A) which would then accumulate. On this argument, the phytoene series would not be intermediates in the synthesis of the carotenes or of lycopene. Further 74 work by Goodwin et al. on P. blakesleeanue provided strong support for the parallel biosynthesis scheme postulated above. In particular, the results obtained indicated that it was most unlikely that n-carotene arose from a biosynthetic pathway which included phytofluene,. -carotene, and neurosporene; in other words, the pathways of synthesis of the phytofluene series and of n-carotene appeared to be entirely separate. This conclusion was strongly supported by a study of the polyene content of ripening tomatoes. Samples of excised tomatoes were ripened at various temperatures and the polyene content of each sample determined at intervals during the ripening process. It was shown that if the tomatoes were ripened attemperatures above 300, the synthesis of both the phytofluene series (phytofluene, °-carotene, and neurosporene contents were determined) and also of lycopene, • was inhibited iwhilst the synthesis of 75 ar and p-carotenes was barely affected.

66 Mackinney et al. also carried out some work on the polyene content of ripening tomatoes. They studied a wide variety of different types of fruit including a naturally-occurring albino mutant which contained as much phytoene as normal red fruit contained lycopene. The quantities of phytoene, 13-carotene, and lycopene present in the tomatoeo were measured at various stages during the ripening of the fruit. The 24 results were reported to be "difficult to reconcile" with a biochemical pathway involving dehydrogenation of phytoene to lycopene. Shneour and 76 Zabin came to a similar conclusion after investigating the incorporation of [2-14C]-mevalonic acid into ripening tomatoes; they added that their results were better explained by a "parallel pathways" scheme.

In all work discussed here, previous to the report of Shneour and Zabin's, the quantities of polyenes present in the eluates from chromatograms had been estimated spectrally; however, Shneour and Zabin based their estimation of polyene content, in part, on the specific activities of the various radioactive polyene zones. Recently, Anderson,

Norgard, and Porter77 have shown that after chromatography of crude tomato extracts on magnesia, many of the polyene zones were grossly contaminated with radioactive, non-carotene substances which could only be removed by chromatography on a stronger adsorbent such as alumina. Hence any 76 conclusions based on results obtained by workers (such as Shneour and Zabin; and Purcell et al.78/\ who used magnesia as adsorbent and then estimated the polyene content of the various zones by measuring their radioactivity, 78 should be treated with reserve. For example, Purcell et al reported that the label of [1-140-acetate and, to a greater extent, of [2-14C]-

mevalonatewasincorporated into all the polyenes in ripening tomato fruit.

The activity of the phytoene zone, however, was considerably less than that of any other zones from the chromatogram of the tomato extract. This was particularly noticeable when mevalonate was used: the activity of the

phytoene zone was negligible when compared with that of the phytofluene and

r-carotene zones. This led the authors to suggest that phytofluene and 25 r-carotene, but not phytoene, were precursors of the carotenes in tomatoes.

The more recent studies of Anderson et al.77 (see below) suggest that the reason for the very high activity of the phytofluene and r-carotene zones noted by Purcell et al.78 was that these two zones were both grossly contaminated with labelled, non-carotene substances. The chromatographic adsorbents used by Purcell et al.78 had failed to remove these impurities, except in the case of phytoene which was subjected to chromatography on active alumina. Thus; the apparently low value obtained for phytoene represented a proper measure of the activity incorporated into that substance; the higher values obtained for the phytofluene and r-carotene zones were grossly exaggerated by the impurities in them.

The results obtained by Anderson et al.,77 following a careful study of the adsorbents needed to purify their tomato extracts, are, therefore, far more reliable. They showed that [2-14C]-mevalonic acid was incorporated into phytoene, phytofluene,tr-carotene, neurosporene, lycopene, and f3- and y-carotenes when-administered to ripening tomatoes.

However, they reported that their results neither confirmed nor disproved the Porter-Lincoln theory, though they added that they considered the circumstantial evidence in favour of it was very strong.

Braithwaite and Goodwin79 have recently been very cautious in interpreting the results they have obtained in a study of the fate of the

(previously labelled) phytoene series of compounds in the fungus Phycomzces blakesleeanus. Their diffidence was again caused by the problem of knowing how much of the apparent activity of an uncrystallisable Oil Wab caused by traces of highly active contaminants. Similar experiments by 26

these workers had earlier been used as evidence for the probable

incorrectness of the Porter-Lincoln theory.72

Meanwhile, some evidence had been obtained which supportedthe 80 Porter-Lincoln theory. Thus Schlegel's results suggested that phytoene

was being converted into phytofluene in Mycobacterium phlei. In addition,

Schlegel showed that labelled phytoene was converted in small amount into

(labelled) myxoxanthophyll - a highly unsaturated, oxygenated carotenoid;

the pigment was shown to be pure by reprecipitating it to constant activity.

81 Jensen, Cohen-Bazire, Nakayama, and Stanier have concluded,

quite definitely, that the Porter-Lincoln series is involved in the

biosynthesis of carotenoids in a bacterium they have studied. These

authors have carried out a thorough investigation of carotogenesis in the

photosynthetic bacterium Rhodospirillum rubrum. This bacterium was shown

to contain many polyenes, both true carotenoids and the more saturated

compounds, although under normal culture conditions the latter were present in only very small amount. It was found that most of the hydrocarbon

polyenes were accompanied by mono- and sometimes di-hydroxy derivatives.

Jensen et al. used the term "group" to indicate the combination of a

hydrocarbon and its hydroxy derivatives. The carotenoids present in normal, rapidly growing cells were the lycopene group (lycopene, "hydroxy- lycopene", and "dihydroxy-lycopene"), the P481 group [P481 (a pigment of unknown structure) and its hydroxy derivative], and the spirilloxanthin group ("hydroxy-spirilloxanthin", and spirilloxanthin). Experiments were carried out which had been designed to reveal the interconversiong that these carotenoids and also the more saturated compounds underwent in the 27 cells of the bacteria.

Normal, rapidly growing cells were harvested, and then suspended in a medium which did not support growth (i.e. a medium unable to provide an exogeneous source of carbon), and illuminated anaerobically.

The quantities of each of the carotenoids present were measured (by spectral estimation following chromatographic separation) at intervals. It was found that the concentration of the spirilloxanthin group increased steadily throughout the period. Initially, the concentration of the P481 group also rose whilst the concentration of the lycopene group (already low) dropped, quite quickly, to practically zero. As soon as that had happened, the concentration of the P481 group also started to fall. For the remainder of the time the spirilloxanthin group increased at the same rate as the P481 group decreased (with the lycopene group steady at a near-zero concentration). Overall, there was a stoichiometric balance between the increase in the spirilloxanthin group and the decrease in the lycopene and

P481 groups. It was therefore concluded that spirilloxanthin was being synthesised as follows:-

lycopene----4 P481------irspirilloxanthin

The effect of diphenylamine (DPA) was then investigated (cf. p.21).

If normal, rapidly growing cells were treated with DPA the synthesis of the normal carotenoids was completely arrested, except in that any members of the lycopene or P481 groups were converted into spirilloxanthin just as above when the exogeneous carbon source was removed (this shows, incidentally, that DPA at the low concentration used here has no effect on the biosynthetic 28 steps involved in the conversion of lycopenefift spirilloxanthin outlined above, although it blocks completely the neurosporene—>lycopene step: see later). Simultaneously, there was a rapid build up of the more saturated polyenes [the rate of this build up was in the order phytoene

(fastest), •C-carotene, phytofluene, neurosporene: the figures for

phytoene are a little doubtful, however, due to the presence of DPA which is not easy to remove from the phytoene fraction without involving repeated chromatography of the phytoene zone with invevitable small losses].

The more saturated polyenes were accompanied by variable quantities of, what appeared to be, their hydroxy-derivatives (compounds which were spectrally indistinguishable from the parent hydrocarbons but behaved as hydroxylated compounds on chromatograms and in partition tests).

The cells were grown in the presence of DPA until they had accumulated large stocks of the more saturated polyenes. At this stage the cells were harvested, washed free of DPA, and suspended in a medium unable to provide exogenous carbon, and illuminated anaerobically (as in the first experiment). Photosynthesis started as soon as the cells were exposed to light, and during the first few hours gross changes occurred in the pigment composition of the cells (this is probably why Goodwin and 81a Osman missed many of the more important polyene interconversions gloing on in the bacteria; their first measurements were taken 24 hours after the cells were first illuminated).

During the first twenty minutes of anaerobic photosynthesig only a small amount of true carotenoid (lycopene) was synthesised, However, the various concentrations of the more saturated polyenes changed markedly. 29

There was a notable increase in the neurosporene group and a concomitant decrease in the g)-carotene and phytofluene groups, the increase in the neurosporene group being almost the same as the total decrease in the other two. During the following 24 hours, there was a rapid rise in the concentration of the true carotenoids and a simultaneous decrease in the concentration of the more saturated polyenes (as before, the phytoene figures were not included due to the difficulty of removing all the DPA).

The total synthesis of the former was stoichiometrically equivalent to the total drop in the concentration of the latter.

Jensen et al.geoncluded (a) that lvcopene was formed from phytofluene (and possibly this, in turn, from phytoene)through a series of 18 dehydrogenation steps as suggested by Porter and Lincoln; (b) that the observed accumulation of more saturated polyenes on treatment of the cells with DPA, is caused by the DPA blocking the step involving the conversion of neurosporene into lycopene [rather than the alternative interpretation which is that the blockage of carotenoid synthesis causes a diversion of the biosynthetic intermediates,(which normally lead to the carotenoidg)to side-products (the more saturated polyenes) which are not normally formed at all by the cell and (c) that the accumulation of the more saturated polyenes in other organisms when normal carotenoid synthesis is blocked by

DPA, indicates that the same general pathway of carotenoid synthesis normally exists in those organisms as well; since some of the organisms where this effect has been observed biosynthesise alicyclic carotenoids (eg. p-carotene 74 in Phycomyces blakesleeanus ), Jensen et al. concluded that one general pathway existed for the biosynthesis of a wide variety of carotenoids, both acyclic and alicyclic (they did not, however, suggest at which stage in this 36 biosynthetic series ring closure occurred to give the alicyclic carotenoids).

As mentioned before, the exact measurement of the concentration of the phytoene was rendered difficult in the DPA-grown cells by the DPA itself. However, since the authors were able to account for the synthesis of the true carotenoids on the basis of the consumption of the phytofluene,

47-carotene, and neurosporene, it appeared that phytoene might not be involved in carotene biosynthesis at all. However, Jensen et al. were not dogmatic about this and have since modified their opinions (see below and p.117).

Since the work described above was carried out (in 1958), the structures of many of the polyenes involved have been revised. This has prompted the authors to propose a comprehensive scheme of carotogenesis in the purple bacteria based on the correct chemical structures of the 82 compounds involved. Some of these structures were finally confirmed during work in which the present author was involved;92 this work has yet to be discussed. For this reason, these very recent proposals of Jensen 82 et al. will be considered later in this discussion along with other biosynthetic schemes that incorporate the correct structures of the participating polyenes (see p.114). 51 Recently, Grob has used a modified form of the Porter-Lincoln theory to explain the results he obtained in a study of carotogenesis in

Neurospora crassa. In his scheme, Grob postulated the conversion of 18 lycopene to ?-carotene as Porter and Lincoln did; however, he omitted to 31 make any reference to phytofluene which appears from other authors' work to be directly involved in carotogenesis.

61-65 During the last few years, Claes has published a series of papers dealing with the polyene content of various X-ray-induced mutants of the alga Chlorella vulgaris. Most of the mutants accumulated large quantities of one or more of the four compounds phytoene, phytofluene, g'-carotene, and proneurosporene. For example, one mutant (5/871) accumulated only phytoene whereas another (5/515) accumulated phytoene, 61 phytofluene, and -carotene. Many of the mutants synthesised no true carotenolds at all. One that did (9a) however, synthesised and also phytoene, phytofluene, and g'-carotene, but no (or very little) 61 a- and p-carotenes. This suggested that the xanthophylls had been synthesised by dehydrogenation of a more saturated, oxygenated precursor a- or p- (rather than by oxygen introduction at theAcarotene stage). Part of this 86 87 work has been reviewed by Mase and Goodwin; some of the results have 86 been used to devise a comprehensive scheme of carotogenesis (cf. pJ05).

The present author has used an X-ray mutant of C. vulgaris as a source of the more saturated polyenes (see the Experimental section).

Finally, Zechmeister and Koe83'84 have claimed that they have succeeded in duplicating in vitro the four steps in the original

Porter-Lincoln series involving the conversion of phytoene into lycopene. 85 Dale had earlier shown that treatment of with N-bromosuccinimide produced a mixture of polyenes containing up to eleven double bonds in conjugation. Similar treatment of phytoene83,84 resulted in the rapid formation of a complex mixture of products one of which resembled natural 32 phytofluene. Zechmeister and Koe83'84 then studied the effect of

N-bromosuccinimide on the other "more saturated polyenes". They claimed that this reagent converted phytofluene, r-carotene, and neurosporene into r-carotene, neurosporene, and lycopene, respectively (the yields were reasonably good in all but the last mentioned case). Evidence for the nature of the reaction products was provided by a comparison of their visible and ultraviolet absorption spectra with those of natural specimens, by elemental analysis (except for lycopene), and by comparison with natural specimens in mixed chromatograms.84 However, this last mentioned test is by no means infallible and has led to wrong conclusions before

(cf. p.39 ). The other tests mentioned above do not give any indication of the position of the chromophores in the reaction products so that it is advisable to be cautious in interpreting the results obtained. However, the results of these experiments have proved to be a valuable guide to further work to he done in this field.

It seemedlikely, therefore, that carotenoid biosynthesis might involve at least part of the original Porter-Lincoln series. The major point of dispute with the original sequence was that the conversion of lycopene into y- and p-carotenesby ring closure seemed most unlikely to 71a occur. This view had resulted from a consideration of both theoretical

(e.g., lycopene is thermodynamically more stable than p-carotene; cf. p.105) and experimental evidence. The experimental evidence has been reviewed recently by Goodwin;71a,87 a paper by Mackinney in which he 66 came to the same conclusion should also be mentioned. An example of this work is as follows. Goodwin and Jamikorn75 have reported that if

33 fruit of the normal tomato plant were ripened at 300 or above, the synthesis of both the phytofluene series of compounds (phytofluene,

f-carotene, and neurosporene) and of lycopene was inhibited, whilst that of a- and p-carotenes was barely affected.*

This disadvantage of the Porter-Lincoln theory (i.e., that regarding the conversion of lycopene into p-carotene) has been avoided in 71a,72 Goodwin's "parallel pathways" scheme. Goodwin has recently developed his earlier scheme and has made one important amendment, namely, that he placed lycopene in the same "branch" of the scheme as the other (c$ p.22) acyclic polyenes (phytoene, etc.); earlier/the had included it with the 13 cyclic compounds. His revised scheme is as follows:-

(a) (a) (a) >0 cyclic—>unknown --->p-carotene 40 precursor intermediates

MVA -4C precursor— 40 (perhaps lycopersene) ( b) (b) (b) ('OVA mevalonate) phytoene---->phytofluene--->r-carotene

(b) (a) (a) spirilloxanthin4--lycopene.(----neurosporene

[Steps (a) - inhibited by DPA; steps (b) stimulated by DA].

X However, it has been claimed recently that lycopene can undergo

conversion into p-carotene in tk4a : please see p.111 . 34

If it is assumed that steps of type (a) are inhibited by diphenylamine and those of type (b) are stimulated, then this scheme will explain several experimental results.71a'87 For example, when Phycomyces blakesleeanus was grown in the presence of DPA, the synthesis of p-carotene was arrested whilst the more saturated polyenes accumulated; if the culture was washed free of DPA and suspended in buffer solution,the culture synthesised 3-carotene (whether the buffer contained a carbon source or not) but no simultaneous loss of the more saturated polyenes was detected.74

A similar scheme to that above was put forward by Mackinney et al.37 to explain the results they obtained in a study of the stimulation of polyene synthesis produced by adding p- to P. blakesleeanus (the action is stimulatory - no p-iononsis incorporated as such 88,89)

More recent work on the elucidation of structures

During the ten years that elapsed between the original suggestion of Porter and Lincoln regarding carotogenesis and Goodwin's scheme noted above, several workers had been investigating the structures of the four members of the phytoene series (phytoene, phytofluene,

r-carotene, and neurosporene). In 1950, only -carotene had been given a structure, and this has since been shown to be incorrect. Since that time a variety of different structures have been proposed for each of the four compounds as new pieces of experimental evidence became available.

This, of course, was very confusing to those studying the biogenesis of these compounds, particularly when a new molecular formula was proposed for 35 one of the compounds which changed its hydrogenation level relative to the other members of the series. For example, at one stage, the structures assigned to phytoene, phytofluene, and g7-carotene were all isomeric

(C H )7C)c so that if the Porter-Lincoln series did operate in Nature, 40 64 two of the steps were apparently isomerisations and did not involve dehydrogenation as had been postulated. A similar situation has existed 87 until very recently. It was therefore of considerable importance to determine the correct structures of all four compounds so that the hydrogenation level of each was known.

The elucidation of the structures of these compounds was particularly hampered by their unusual sensitivity to atmospheric oxidation; this sensitivity is particularly marked in the cases of phytoene, phytofluene, and '-carotene. This will be discussed briefly later; suffice to say here that the reason for the low values obtained by some of the earlier workers for extinction coefficients and for hydrogen uptake may well be due to the sample having been contaminated by oxidation product by exposure to air(during weighing operations, etc.) It is also likely that some of the earlier chromatographic procedures were not sufficiently rigorous to remove all traces of impurity (cf. ref. 77).

The first definite evidence that phytoene had a C isoprenic 40 skeleton similar to those of phytofluene, '-carotene, and neurosporene was provided by Zechmeister's observation that it could be converted into phytofluene by treatment with N-bromosuocinimide.84 In 1954, Rabourn, 20 Quackenbush, and Porter obtained phytoene in considerable quantities from 36 carrot oil and tomato paste; they reported on some of the properties of phytoene and also showed that samples of phytoene from the two sources 40 were identical. Two years later, Rabourn and Quackenbush elucidated the structure of phytoene (III) by the extensive use of the classical analytical and degradative prodedures, coupled with spectral evidence.

The infrared spectrum of phytoene showed a strong band near

12.9 ju which is characteristic of the grouping C.= ; compounds 90 containing methyl-substituted cis double bonds do not give a band at 12.9)4.. 20 In addition, phytoene failed to give a thiourea adduct, whereas "all-trans"- squalene (but not any of its cis isomers) readily forms an adduct.93 This suggested that natural phytoene has a "central-cis" configuration, although it was notable that no evidence of stereoisomerisation could be obtained on treating the natural material with a wide variety of the usual 20 stereoisomerising reagents. Recent work by Siddons and Weedon91 suggests that the triene chromophore of natural phytoene does contain a cis double bond; these workers synthesised the "all-trans" isomer of phytoene and the extinction coefficient of tneir synthetic product had a markedly higher value than that of natural phytoene.

The only dubious point in the structure assigned to phytoene by

Rabourn and Quackenbush40 was the positioning of the triene chromophore. 3'7

Although ozonolysis yielded glyoxal but not methylglyoxal (as is to be expected if the triene is placed centrally), the same authors have, more recently, been unable to detect any methylglyoxal on ozonolysis of c-carotene and phytofluene although these compounds should yield two and 6 96 one moles of methylglyoxal, respectively. ' The yield of levulinaldehyde, also obtained from ozonolysis of phytoene, was not unreasonable for a compound with structure (III). A consideration of all the available evidence (including the infrared determination) shows that there is considerable support for the suggested structure of phytoene, although it cannot be said that the evidence is conclusively in favour of it.

The correct structure of phytofluene was first suggested by 56 Zechmeister in 1958; (he simultaneously withdrew his earlier suggestion

(IV)94 which was apparently based on the early analytical work on phytofluene.32,95). Brief mention has already been made of the (probable) formation of phytofluene following the treatment of phytoene with

N-bromosuccinimide, and the analagous reactions with phytofluene,r-carotene, and neurosporene.84 Each component of the complex reaction products obtained from these experiments contained an odd number of conjugated double bonds; it appeared, therefore, that each step increased the length of the chromophore by two double bonds, and never one. To explain why this should 38

56 be so, the suggestion was made that at each step one of the previously isolated double bonds was brought into conjugation with the main chromophore. Hence, as soon as phytoene had been shown to have the 56 structure (III) above, Zechmeister suggested that phytofluene was probably 11, 12-dehydrophytoene (V):

(V), phytofluene

Support for this suggestion was soon provided by Rabourn and 96 Quackenbush. They carried out a study of phytofluene, involving 2,3 degradative and analytical work, and their results were in accord with the formulation suggested above. However, since no methylglyoxal, and only a poor yield of glyoxal, was isolated from ozonolytic degradation, the evidence for the position of the chromophore was weak. Natural phytofluene is a cis isomer which is fairly atable to heat, but which is rapidly atereoisomerised into a mixture rich in the "all-trans" form on exposing the compound to light;27 tentatively, it has been assigned the 13',14'; 15 ,15'- 96 di-cis- configuration by Rabourn and Quackenbush; Zechmeister,136 however, has suggested that the natural isomer might have the 15,15'-mono- cis-configuration.

The available evidence suggested that it was neurosporene that had been formed by the in vitro dehydrogenation of 4.-carotene, thus suggesting a close structural relationship between these two compounds. 39

At this time it was considered that r-carotene was an octahydrolycopene

[(I); see previous discussion]; it was apparent (from spectral

measurements) that neurosporene had nine double bonds in conjugation as opposed to the seven in c-carotene, so it was suggested that neurosporene had the following (tetrahydrolycopene) structure (VI):97

(vi)

Karrer and his co-workers97 synthesised this compound (VI) and reported that it showed no separation in a mixed chromatogram with natural neurosporene. However, the mixed chromatogram test is by no

means infallible and has led to wrong conclusions before (and it is now 99,121 known that it did so in this case).3,98, Thus, if two compounds do separate in a mixed chromatogram then it is certain that they are different in some respect (perhaps, even, only stereochemically different);

but failure to show separation does not necessarily indicate that the two compounds are identical. Structure (VI) has two asymmetric carbon atoms

(at positions 5 and 5'). Karrer et al?7pointed out that their compound

might be expected to be a racemic or meso form, whereas the natural compound [if it did have this structure (VI)] would be an optically active form. It was suggested that it was because of this difference that the synthetic material had a rather lower meltin,s. point than the natural. 53 Since then, Nakayama has reported that natural neuroeporene shows no optical rotation; and we now know that Karrer's "neurosporene" had two 40 more hydrogen atoms in it than has the natural material.

8 Recently, Rabourn and Quackenbush have completed a more thorough investigation into the structure of t'-carotene than that previously reported [and which gave rise to an incorrect structure (I) being assigned to C1 -carotene]. From the results they obtained, Rabourn and Quackenbush suggested that c-carotene is a tetrahydrolycopene, and assigned it a symmetrical structure (VII). They apparently did not consider the asymmetrical analogue (VIII) although their results would fit this structure equally well.

(VIII)

As has already been mentioned, these workers were unable to detect any methylglyoxal (2 moles would be expected on the basis of either of the above structures) on ozonolysis of °°-carotene, although they did find levulinaldehyde and glyoxal successfully; the reason for this discrepancy remains unknown.

This revision in the structure assigned to r-carotene necessitated a revision in the structure (VI) previously assigned to neurosporene, since the structure of the latter was based on that of the 100 former. Rabourn and Quackenbush have, therefore, assigned a new, dihydrolycopene structure (IX) to neurosporene although they have put forward no chemical evidence for it. It should be noted that whichever of the two structures ((VII) and (VIII)] for c -carotene is correct, the structure of neurosporene is almost certain to be as follows:

(IX), neurosporene

It is noteworthy that the analytical figures obtained in 1954 for a sample of neurosporene prepared by N-bromosuccinimide dehydrogenation of r -carotene, agreed much better with a dihydrolycopene (C40H58) formulation than with the tetrahydrolycopene (C40 H60) formulation that it was then assumed to have.84

As can be seen, none of the four structures proposed for phytoene(III), phytofluene(V), -carotene(VII), and neurosporene(IX), respectively, has been established with certainty. In particular, the positions of the chromophores in the first three of these compounds were based on ozonolysis experiments. In most cases the yields of the fragments obtained on ozonolysis were low, and with phytofluene and

C-carotene (which should give, on the basis of the structures assigned to these substances, one and two moles of methylglyoxal, respectively) no methylglyoxal at all was detected. Thus the various alternative structures that could be considered for any one of these substances should not be dismissed solely on the grounds that ozonolysis would then have been 42 expected to yield fragments which were not, in fact, detected in practice.

The ozonolysis results obtained by Rabourn and Quackenbush show that the positions assigned to the chromophores in these molecules are no less likely than any other postions. However, to establish unequivocally the

positions of the chromophores, it would be necessary to isolate each of the expected fragments from ozonolysis in high yield. Later in this discussion, a description of the contribution made by the present author to this problem will be given. First, however, mention should be made of the first member of the original Porter-Lincoln series ("tetrahydrophytoene",

(II); see earlier discussion).

It is now apparent that if there is a basic C40 precursor in the series "before" phytoene, then it is unlikely to have the structure that la was assigned (by Porter and Lincoln ) to tetrahydrophytoene. Until recently, little had been done in the way of investigating its chemical nature. This apparent lack of interest was probably partly caused by the inability of some spectrophotometers to record, satisfactorily, absorption maxima in the region (200-210 my) where a compound with isolated double bonds absorbs. Interest in this problem has recently been stimulated by the discovery that radioactive acetate and mevalonate are incorporated into carotenoids in a variety of plants to give the same type of labelling 104 pattern as that found in squalene biosynthesised from the same substrates.

It seemed reasonable to suppose, therefore, that the C skeleton of the 40 carotenoids might be biosynthesised in the same kind of way as the C30 squalene skeleton.

The mechanisms of most of the steps involved in the biosynthesis

43 of squalene have recently been elucidated with a fair degree of :71e,105-1 certainty 07

++ cii\• Mn CHiN 3 C—CH---CH OH _-4,C- CH--- CH--- ° P P HOOC.CH/ 2 2 \ 2 2 ' ' 2 I II— O—C—ii CH/ 2 OH r If > (OP ATP ADP 0 H+ Mevalonic acid (3 steps) ao. CO 2 (from acetylcoenzyme A71c) H PO - 2 4 CH3\ V C CH —CH-0.P.P. — 2 2 + 2 H i lSoPEArrFivYL (xi) r YROPHOSPHRTE O.P.P. CH N CH 3N (xi)-4 ,c=CH—C C—CH—CH -0.P.P. /' H2 II 2 CH CH H 3 2 (XIa) DIPIETHYLALLYL PYROPHOSPHATE

-)1.11 P 2 27 V CH3 CH3 I C:=CH.CH2.CH2.C===CH.CH2.0.P.P. Geranyl pyrophosphate, (XII) CH 3 ( x )

H P C V 2 2 7 CH, I H3 1 H3 C===CH.CH .CH .C===CH.CH .CH .C==CH.CH .0.P.P. CH'" 2 2 2 2 2 3 Farnesyl pyrophosphate,(XIII) 44 0- ?- ( - O.P.P. represents the pyrophosphate residue,--01-0-T-OH )

0 0

105 According to Popjak, Cornforth, and their co-workers, the

last step in the biosynthesis of squalene involves the tail-to-tail

condensation of one molecule each of farnesyl pyrophosphate (XIII) and

nerolidyl pyrophosphate (itself formed by an allylic-type rearrangement of

farnesyl pyrophosphate). Popjgk et al.10 -5 also showed that the joining of

the two fragments was accompanied by a simultaneous reduction, in the form

of the donation of a hydride ion from reduced triphosphopyridine nucleotide 1b (TPNH, or NADPH2 as it has since been called ); various mechanisms for 105 this step were suggested. By analogy with the above, it might be

expected that the two corresponding C20 compounds [geranylgeranyl

pyrophosphate (XIV) and its isomerisation product (XV)] could condense (in

the presence of TPNH) to give the C analogue (XVI) of squalene.87 This 40 108 compound (XVI) was named "lycopersene" by Karrer and Kramer in 1944 when they reported its first synthesis (this was before there was any

suggestion that it might have biological significance; the synthetic

material was probably a mixture of stereoisomers, whereas natural

lycopersene, if it exists, would be expected to possess an "all-trans" 130 configuration: cf. squalene93' ).

CH CH 3\ 1 3 —CH —C CH 0.P.P. ,C=CH—[CH2 2 2 CH 3 45 CH CH, I 3 3 C=CH--ECH —CH —C=CH--]--C-C CH —CH H—CH /'' 2 2 2 2 2 — 2 CH3 (XV) O.P.P.

(The existence of neither (XIV) nor (XV) has been demonstrated unequivocally as yet, but evidence for 109 the existence of (XIV) has been obtained by Grob et al; see below).

It can be seen from its structure that lycopersene would fit

into the Porter-Lincoln theory very well [assuming, that is, that the

structures assigned to phytoene, etc. by Rabourn and Quackenbush (see above)

are correct]. The conversion of lycopersene (XVI) to neurosporene (IX)

would then involve a series of dehydrogenation steps [(XVI)-->(IE0-400-4

(VII)--3(IK)1 with two hydrogen atoms (rather than four, as originally

proposed18\ ) being lost at each step.

109 Recently, Grob, Kirschner, and Lynen have sought to

demonstrate the existence of the biosynthetic system that, it was suggested

above, might lead to the formation of lycopersene. They obtained

evidence that geranylgeranyl pyrophosphate could be prepared enzymically, and that it could be converted, in vivo, into a hydrocarbon with the

properties expected for lycopersene.

A yeast preparation containing isopenten^Y1 pyrophosphate

isomerase and farnesyl pyrophosphate synthetase (and probably other 46

106'110 enzymes) was first treated with iodoacetamide to deactivate selectively the isomerase.m The yeast preparation was then incubated

with [1-14C]-isopentenyl pyrophosphate and synthetic (unlabelled) farnesyl

pyrophosphate. This produced a new, radioactive, water-soluble compound. which was acid labile. This compound was probably geranylgeranyl

pyrophosphate since, on treating it with the enzyme phosphatase, it was

cleaved, and yielded inorganic phosphate and geranylgeraniol (identified as a crystalline derivative).

The labelled geranylgeranyl pyrophosphate was incubated with an extract of Neurospora crassa in the presence of TPNH. After one hour, the reaction was quenched with methanolic alkali, and the petrol-soluble' fraction was filtered through alumina to remove the geranylgeraniol formed as a by-product (from reaction of the pyrophosphate with the

phosphatase in the F. crassa). The eluate contained a small amount of labelled squalene as impurity (cf. the footnote on this page). Synthetic

(unlabelled) "all-trans" samples of squalene and lycopersene were added to the eluate as carriers and the mixture was separated into two components using reversed-phase paper chromatography. The two zones were cut out

3f This treatment was designed to prevent the isomerase catalysing the isomerisation of the [1 -14C]-isopentenyl pyrophosphate (cf. XI) to [1-14C]- 106 dimethylally1 pyrophosphate (cf. XIa) which would then have triggered off the series of reactions leading to squalene. As can be seen, however, this treatment was not completely effective and a small quantity of squalene was formed [this is not unexpected: cf. ref. (110)]. 47 and assayed for radioactivity. The lycopersene zone was much more active than the squalene zone. The only evidence that Vie compound formed was, in fact, lycopersene was provided by the observation that the major labelled product from the enzymic reaction showed no separation from the added (unlabelled) lycopersene on the paper chromatogram. No indication was given as to the effectiveness of this system in separating \ 109 lycopersene from similar compounds (other than squalene).

Evidence of a lycopersene-like compound has also been provided 77 by Anderson et al. These authors were carrying out a study of the 14 incorporation of [2- C)-mevalonic acid into ripening tomatoes.

Chromatography of the crude extracts resolved the mixtures into many components one of which was rapidly eluted and preceded phytoene on the columns. This substance (which was labelled) was unsaturated, and on ozonolysie gave labelled levulinic acid which contained 71% of the activity of the parent substance (expected for lycopersene, 75%;; and for squalene, 67%). A similar substance was extracted from tomatoes by 20 Rabourn et al.

Further work is obviously required before it can be said whether or not either of the substances described above was lycopersene.

In this respect, n.m.r. spectroscopy should prove very useful, as will be shown later. The present author has isolated a lycopersene-like substance from carrot oil; the n.m.r. spectrum of the substance showed, however, that it is most unlikely to be lycopersene (see p.99).

A further report of, what appears to be, a carotene precursor 78 should be mentioned. Purcell et al. have investigated the incorporation 48 of [1-14C]-acetate and [2-14C]-mevalonate also into ripening tomatoes (cf. Anderson et al.77 above). Repeated chromatography of the extract separated the complex mixture into several crude fractions. These were the crude phytoene zone, crude phytofluene, crude '-carotene, and the true carotenes. [As mentioned earlier (p.24), this work was really concerned with a study of caroto;7enesis in tomatoes but, because of incomplete purification of the polyenes, the results were of dubious significance]. The crude phytoene zone was rechromatographed on active alumina (as recommended by Anderson et al.77) and yielded, in addition to phytoene, four zones wMch were eluted faster than phytoene. These were called fractions Ia, Ib, IIa, and IIb. Practiors Ia and Ib were found to incorporate acetate very readily, but mevalonate hardly at all. On the other hand, fractions IIa and IIb (especially IIa) incorporated mevalonate very readily, but incorporated very little acetate. Both fractions IIa and 78 IIb appeareto be dienes (A 208 and 231 91). max. 111 More recently, the same group have studied these substances in more detail. Fractions Ia and Ib appeared to be the same substance,

Wlich was called fraction I. Fraction I was not the lycopersene-like substance observed by Anderson et al.77 (see previous discussion) since it was inert to ozone. (The fact that it failed to incorporate mevalonate significantly suggested to the authors that it was a saturated hydrocarbon derived from a fatty acid whose biosynthesis does not require mevalonic acid71d).

111 Purcell et al. also showed that fractions IIa and IIb were probably isomeric since on repeated chromatography one was, apparently, 49 gradually converted into the other (cf. the behaviour of cis and trans isomers of polyenes); the authors reported that two other dienes

(farnesene and phytadiene) that they had worked withlbehaved similarly.

In general, a mixture of fractions IIa and IIb was used for chemical tests

(and was called fraction II).

Molecular weight and spectral determination showed that fraction

II was a C diene. The presence of a methylene group in II (inferred 20 from its infrared spectrum) was confirmed by a direct determination.

Ozonolysis gave three products all of which were labelled; these were acetone (ca. 0.1 mole), levulinic acid (0.8 mole), and malonic acid (0.55 111 mole). From these results, Purcell et al. concluded that the most probable structure for fraction II was:-

[fraction II(?)]

[atoms marked •ie are those which one would expect to be derived from the

(labelled) C of mevalonate]. 2

This structure is consistent with the results obtained except in that no pyruvic acid was found on ozonolysis. Each of the ozonolysis products which was isolated was labelled, as expected; the yields were low but this is not unusual. The above structure obviously bears a close relationship to geranylgeranyl pyrophosphate (XIV).

111 Purcell et al. then carried out a series of tests to determine how the quantity of fraction II present in tomatoes varied during the 50 ripening process. The tomatoes were gathered at various stages of

maturity, inoculated with labelled substrate, incubated for 24 hours, and then extracted.

Immature fruit contained substantial amounts of fraction II but much of it disappeared before any significant ripening had occurred. No concomitant synthesis of the polyenes (phytoene, phytofluene, lycopene„ and n-carotene) occured during this time. (N.B. The purity of the phytofluene zone was questionable; cf. ref. 77). The quantity of fraction I (the fully saturated substance) present followed a similar trend to fraction II but was present throughout in far larger quantities.

Thus it appeared that fraction II might be a carotenoid precursor, but, at the same time, it was apparently being converted not into phytoene but into another substance (perhaps an intermediate between fraction II and phytoene) which was not detected. This intermediate

might contain a pyrophosphate residue which, during the processing of the tomato extract, would be hydrolysed; the resulting alcohol would not 1,0 eluted in the first fractions from the column. Apparently the tomato synthesises a large stock of frE'ztion II early on in growth; it is then consumed during the ripening process. Unless it is being simultaneously synthesised by the plant, fraction II is unlikely to be very important in carotene synthesis since the amount of carotenoids formed is much greater than the amount of fraction II apparently consumed.

It is interesting to note that Anderson et al.7,7 as mentioned above, obtained a single (lycopersene-like) substance from the eluate 51

78,111 preceding phytoene whereas Purcell et al. above obtained two substances entirely different from that obtained by Anderson. Anderson et al. harvested their tomatoes at the full-ripe stage. At this stage,

Purcell's tomatoes contained very little fraction II; however, they apparently did not contain any of Anderson's lycopersene-like substance either, and they did contain a saturated hydrocarbon (fraction I) in quite considerable quantities (although not so much as when the tomatoes were green). The reason for this difference is unknown although it should be noted that the tomatoes used were probably not of exactly the same variety, and the fruit processing procedures were slightly different (and, therefore, some chemical changes might have occurred in the constituents of the tomatoes in one case and not the other).

Substances with absorption maxima similar to those of fradtion II, and which were eluted faster than phytoene, have been observed by Rabourn 20 112 and Quackenbush, by Suzue, and by the present author [in extracts of both Chiorella vulgaris and of carrot oil (fraction 1,2); see Experimental section, Pages 127 and 139]. 112a In 1959, Suzue reported on the effect of culture conditions 112b). on carotogenesis in a micro-organism (Staphylococcus auereus The organism synthesised polyenes (tentatively identified as phytoene, C-carotene,

6-carotene, and ) in various amounts depending on the conditions in particular, on the amount of air allowed into the system. However, whatever conditions were used, a substance 91) which absorbed (') Imax.232'5 light in the diene region was invariably found. Suzue suggested that it 52 was a carotene precursor, but provided little evidence in support of this idea.

Thus it appears that, as yet, there is no definite proof of the existence of lycopersene. It will be interesting to see if Grob et al.

(see above) 109can substantiate their claim. This apparent absence of lycopersene in Nature does not preclude its existence. To start with, the search for this substance has not been very intensive; also, it is possible that, once formed, it is dehydrogenated so rapidly and efficiently to phytoene that lycopersene never accumulates in an amount sufficient to be detected. 131 Finally, it should be noted that Wright et al. have isolated a diene (which they called neophytadiene) from naturally-aged tobacco leaves. This source also contained carotenes, including phytoene and phytofluene, in association with the diene; but it was shown that the latter is most unlikely to be a carotenoid precursor in that, apart from the 132 diene chromophore, it is saturated:

CH CH CH 3 2 CH3 3 CH.CH2.CH2.CH2ARCH2.CH2.CH2.CH.CH2.CH2.CH2.C--CH=TCH2 CH 3

Neophytadiene

132 Rowland suggested that it was formed from phytol (by the loss of one molecule of water). It could, alternatively, have arisen by partial saturation of Purcell's diene ("fraction II",see above) though this is unlikely since it is difficult to see why the diene chromophore in Purcell's 53 diene should survive while the remaining (isolated) double bonds became saturated.

In short, just because an unsaturated C hydrocarbon is found in 20 association with carotenoids in a plantI one should not assume it is concerned with carotojenesis; it might well be the precursor, or a degradation product, of non-carotenoidal natural products. 82,113 The apparent absence of lycopersene in Nature prompted Stanier, 86 and others, to suggest that the basic C precursor is phytoene and not 40 lycopersene. A possible mechanism by which lycopersene (if it exists) might be formed has already been discussed (see p.414). It involved the tail-to-tail condensation of geranylgeranyl pyrophosphate (XIV) with its isomerisation product (XV) in the presence of T?NH as reducing agent. If this reaction proceeded without TPNII participating, the product would be phytoene. The nucleus of the conjugated double bond system would then be present in the first member of the Porter-Lincoln series and dehydrogenation would give phytofluene. If lycopersene were the first member of the series it is difficult to see why the first dehydrogenation should give phytoene

(with the triene system in the centre of the molecule) exclusively as it apparently does. There seems little reason why a mixture of isomeric trienes should not be formed; only one isomer, phytoene, has ever been reported.

The application of n.m.r. spectroscopy

The purpose of the present work was to isolate pure samples of 54 phytoene, phytofluene, t' -carotene, and neurosporene from one or more natural sources, and to determine the n.m.r. spectrum of each of these compounds. From the results obtained,it was hoped that it would be possible to show whether or not the structures assigned to these compounds

[(III), (V),( II), and (IX), respectively] by Rabourn and Quackenbush

(see previous discussion) were correct. In addition, it was hoped to screen the first fractions from chromatogram in an attempt to detect, and if possible isolate, any ]ycopersene-like substances.

The main distinguishing feature in the various structures proposed for phytoene, phytofluene, r -carotene, and neurosporene, is the presence in some of these structures [cf. the earlier proposals:

"hexadecahydrolycopene", (IV), (I), and(VI), respectively] of a methyl group attached to a saturated carbon atom (XVII). This feature is absent 10 from the structures suggested by Rabourn and Quackenbush for these compounds [(III), (V), (VII), and (IX), respectively] in which all methyl groups are attached to unsaturated carbon atoms [as in (XVIII)]. In two

C 40 molecules, otherwise identical, the presence of a group of type (XVII)

1H3 H3

C, N. ,r

Dwun rather than of type (XVIII) would make little difference to the molecular formula of the molecule. This slight difference (i.e. two hydrogen atoms in a C molecule of molecular weight 540) would be difficult to detect 40 55 with certainty by elemental analysis, although it should be possible to detect by microhydrogenation. However, the difficulties encountered in the purification and manipulation of these unstable substances make it difficult to obtain accurate and reproducible analytical data of any kind.

Even the results obtained by the ozonolytic degradation of phytoene,40 96 8 phytofluene, and C'-carotene are not completely incompatible with these molecules containing one group of type (XVII). However, groups of this type (XVII) can now be readily detected by n.m.r. spectroscopy.

A recent survey114 of the n.m.r. spectra of a wide variety of polyenes enabled the authors of that paper to show that a useful indication could be obtained of the molecular environment of a methyl group in a polyene from the position of the band it gave in an n.m.r. spectrum.

The results obtained which will be of particular value in the present context are as follows.

The band caused by a methyl group attached to an olefinic carbon atom invariably appeared in the region 7.8 to 8.45 p.p.m., whereas it is known that the band given by a methyl group attached to saturated carbon [as in (XVII)] occurs near p.p.m.11 -5 In addition, it was shown that the various types of methyl group attached to an olefinic carbon could be differentiated as follows.

"In-chain" methyl groups (XIX) cave an absorption band near

8.05 p.p.m. whereas "end-of-chain" methyl groups (XX) absorbed near 8.2 p.p.m. "In-chain" methyls (and probably also "end-of-chain" methyls, though this was not tested) could be resolved from "isoprenoid" methyl

56

groups (XXI) which gave a band near 8.1 p.p.m!14

CH3 C H3 H3

(xx) (xxr)

In addition, it was shown that a methyl group attached to a

carbon atom carrying an isolated double bond absorbed near 8.4 p.p.m., and

this type could be classified still further. Thus it was shown that it was

possible to distinguish between methyl groups which were cis and those which

were trans to the alkyl substituent (R) at C in unconjugated trisubstituted

double bonds. These two types of methyl group were called cis-olefinic

(XXII) and trans-olefinic (XXIII) methyl groups, respectively.92 (Hence,

by the definition of these terms, the methyl group on a conventional

methyl-substituted trans double bond is a cis-olefinic methyl group). Bates 116 and Gale examined the n.m.r. spectra of a variety of isoprenairl compounds

Me ///R Mb H N 'N. / /Ca C c C i3 13 \ / a CH H --CH \11 -- 2 2

cia-olefinic methyl (XXIII), trans-olefinic methyl

of known stereochemistry. Invariably, the cis-olefinic methyl groups in

these compounds gave a band at slightly higher fields (by ca. 0•07 p.p.m.) 57 than the trans-olefinic methyls." For example, it is known that all four non-terminal double bonds in natural squalene (XXIV) possess a trans 03 configuration.' '130 Each isopropylidene group contains one cis- and one 116 trans-olefinic methyl group. The n.m.r. spectrum of squalene in the

0.4 p.p.m. region showed two peaks, one at 8.405 p,p.m. and the other at

(oov)

8.335 p.p.m., of relative intensities 3:1. These peaks were, therefore, assigned to the cis-olefinic methyls (six) &-nd the trans-olefinic methyls 116 (two), respectively. This finding was confirmed independently in the course of the present work ( Tvalues found: 8.42 and 8'35 p.p.m.; see p.80 ; the infrared spectrum of the sample11 7 showed that it was free of the group 0 which is sometimes present in squalene samples and is C / \ CH formed by the isomerisation of the 1 3 groups present in the natural 116a /A•' material ). Similarly, the 8.4 p.p.m. region in the n.m.r. spectrum of lycopene (XXV) shows two peaks of equal intensity (at 8.38 and 8.31 114 p.p.m.; see p.80); lycopene contains only two isolated double bonds both of which are present as isopropylidene groups; each of these contains x This differentiation in band positions is not apparently shown in compounds where "R" represents a polyene chain, so that this technique cannot be used to ascertain the stereochemistry of the double bonds in the polyene 91 chromophore. 58 one cis- and one trans-olefinic methyl.

The n.m.r. spectrum of lycopersene [(XVI), cf. p.++) would be expected to be similar to that of squalene except in that the relative intensities of the peaks at 8.42 and 8.35 p.p.m. should be in the ratio 8;2 (for squalene, the ratio is 6:2).

(xvi)

It now remained to isolate samples of the four polyenes (phytoene, phytcfluene, r-carotene, and neurosporene) in a pure state, and it should then have been possible using the knowledge outlined above to assign each of the bands in the 9.5 to 8 p.p.m. region of their spectra unequivocally to one of the above types of methyl absorption.(Olefinic proton absorption is less well understood and will be discussed later when the n.m.r. spectra of these four polyenes are discussed). In the event, there was no need to isolate any neurosporene as Dr. Nakayama kindly supplied a sample which he had isolated from a green mutant of the bacterium Hhodopseudomonas 53 53 spheroides; Dr. Nakayama had already shown that his sample was 53 (probably) identical with samples of neurosporene both he and Haxo35 had isolated from the fungus Neurospora crassa [no separation was observed in mixed chromatograms, but see p.39 regarding the reliability of this test; the melting point of Dr. Nakayama's sample was similar to (but slightly lower than) the melting point recorded by Haxo35].

The other three polyenes (phytoene, phytofluene, and r" -carotene) 59

were isolated by the present author from natural sources using the methods

shortly to be discussed. First, however, some of the properties of these

polyenes will be described.

Neurosporene is a red crystalline solid,35 phytoene is a

colourless viscous oil which is said to fluoresce pale blue in very 30 concentrated solutions (this has been disputed;136 cf. ref. 20), and

phytofluene is a yellow viscous oil which exhibits an intense green 26 96 136 fluorescence. ' ' Strain5 claimed to have crystallised C4-carotene;

all subsequent workers up until recently reported that c-carotene was an

orange viscous oil which was converted into a white oxidation product 8,19 whenever attempts were made to crystallise it. Recently, both 129 150 Petzold, and Siddons and Weedon, have succeeding in crystallising

Z.-carotene.

The four polyenes behave like normal (i.e. more unsaturated)

polyenes to the extent that they are unstable to acid but stable to base, and their cis-isomers are stereoisomerised in solution by the influence of 136 light, especially in the presence of iodine as catalyst.27 ' However, the characteristic property of the "more saturated" polyenes is their extreme lability towards oxygen - a property which is amply demonstrated

by the following figures. On exposing dry samples of phytoene, phytofluene, and r-carotene to air at room temperature, the samples showed gains in weight (presumably due to oxygen absor—ption) corresponding to an oxygen uptake (per mole of polyene) of 5 atoms of oxygen in 24 hours (phytoene),2°

5 to 8 atoms (for the "all-trans" and neo-B forms,respectively) of oxygen 60

96 . in 24 hours (phytofluene), and 12 atoms of oxygen in 6 hours (r-carotene).

The samples were viscous oils and therefore the rate of oxygen uptake depends to some extert on how thin a layer of substance is tested. It also depends on how much illumination the sample receives. Thus, Zechmeister 32 and Sandoval have shown that phytofluene is oxidised more rapidly in the light than in the dark.

The rate of oxygen uptake apparently falls off exponentially 20 with time, so that appreciable oxidation occurs within seconds of exposure to oxygen (or air). The oxidation products were reported to be sticky, 20,96'8 hard substances which were insoluble in hexane but soluble in acetone.

The absorption spectra of these oxidation products have been found to be completely different from those of the original polyenes; in both examples studied by the present author, the absorption maxima shifted towards shorter wavelengths by a considerable amount showing that oxidation had attacked the chromophore quite irestically. Thus phytofluene (A . 367, x, and 332 mix) gave, after several days in the air, a product (A max 225.5 71) with a . single broad absorption band. la -Carotene (maxima at 400, and 378 D54) gave, after 12 hours, a substance whose absorption maximum had already shifted to 334 T.137

Phytoene, phytofluene, and r-carotene could, however, be stored satisfactorily if kept in solution (preferably in a non-polar solvent); solutions that had to be stored for some time were maintained at low 96 temperature in the dark and under nitrogen. Rabourn and Quackenbush reported that they had stored a solution of crude phytofluene in hexane at si

o -20 over a layer of hydroquinone for five years without apparent loss.

The present author kept a solution of phytoene in hexane at room temperature in a stoppered flack in the dark for six months without there being any significant change in the quantitative absorption spectrum. Even so, some crude phytofluene concentrates stored at 20° started to deposit a colourless solid on the walls of the flasks after a few days - a sure sign that the polyene ws being converted into colourless, hexane-insoluble, decomposition products. The general impression gained was that phytoene was the least unstable of these three polyenes.

Less experience was gained with neurosporene; however, this polyene appeared to be rather more stable than phytoene, phytofluene, and

° -carotene, but rather less stable than a normal carotenoid. Por example, gummy, colourless oxidation products were soon formed if a neurosporene long without solution was kept the normal precautions being taken (cf. above). On the other hand, the substance did melt sharply on a Kofler block.

Presumably, these polyenes are well protected from oxidation 131 in biological systems. Indeed, Wright et al. have reported finding phytoene and phytofluene in naturally aged tobacco leaves which had been harvested, apparently, at least two years previously. N.,

The reason for the extreme lability towards oxygen of these

"more saturated" polyenes appears to be unknown; indeed, the problem has 138 attracted little attention. Zechmeister and Sandoval have observed that some of the artificial partial-reduction products of the carotenoids are likewise prone to oxidation. 62

The major problem likely to be encountered in this work could therefore be predicted. This was how to develop a technique whereby a small quantity (ca.5-10 mg.) of a purified, extremely oxygen-sensitive polyene dissolved in a large volume of eluate from a chromatogram could be transferred to a few drops of another solvent in a n.m.r. sample tube. The difficulty was aggravated by the inexplicable fact that all these polyenes

(with the possible exception of phytoene) gave unusually diffuse zones on a chromatogram (this was true even of the stereochemically pure compounds: cf. p.134); this caused the volume of eluate to be even larger than might usually be expected. This problem will be discussed more fully after the first (unsuccessful) attempt at isolating pure c-carotene has been described.

The choice of the natural material to be used as the source of the phytoene, phytofluene,and C7-carotene, was governed by several factors. It was desirable that all three polyenes should be obtained from one source, and in sufficient quantity to obtain enough (10 mg., if possible) of each polyene to obtain a satisfactory n.m.r. spectrum of each. (It has since been shown that, under favourable conditions, as little as 4 mg. a would giveA satisfactory spectrum in the methyl proton region; more sample would be required, however, for the olefinic proton region). Two sources which might be expected to fulfil these requirements, and which were also comparatively accessible, were a mutant of the alga Chlorella vulgaris, and carrot oil (both of these sources have been used by other workers in this 61-65; 8,40,96). field. Eventually, it was found to be necessary to use both sources. 63

All manipulations with the polyenes isolated from these

sources were performed in very dim light and, as far as possible, in the

cold, in the hope that the naturally-occurring stereoisomers might be isolated.

The precaution taken to protect the polyenes from atmospheric oxidation will

be discussed later.

The various stereoisomers of r-carotene and phytofluene which

were isolated, were identified, wherever possible, by comparing their

adsorption properties and visible absorption spectra with those reported

for the various stereoisomers isolated by Rabourn and Quackenbush.8'96

Thus, the r-carotene isolated from carrot oil by both Rabourn and 8 Quackenbush and by the present author was a mixture of four different

stereoisomers, each of which had distinctive adsorption and absorption

properties. Each isomer was named by comparing its properties with the

data published by Rabourn and Quackenbush; this procedure is justified in

this case since the same source was used by both Rabourn and Quackenbush

and by the present author. This procedure has, for convenience, been

extended to theC-carotene stereoisomers isolated from the algae, although

it is not claimed that the isomer from this source with properties similar

to, say, neo-C-carotene-C from carrot oil is necessarily identical with it.

The four stereoisomers of r-carotene isolated from carrot oil

by Rabourn and Quackenbush were designated by them: "all-trans" (425),

neo-A(419), nco-B(423), and neo-C(413), in the order that they appeared on

an alumina column from top to bottom. The figures represent the reported

positions (in fix) of the longest wavelength band in the spectrum of each

(in hexane solution). Stereomutation of any of these isomers by exposure 64 to light (with iodine catalysis) was reported to give a product containing the only two stereoisomers. These were reported to berall-trans" and 8 neo-B forms by Rabourn and Quackenbush. Lately, it has been claimed that the stereoisomer which accompanies the "all-trans" form is not the neo-B isomer but the neo-A.91 This confusion has little bearing on the present work except in that any isomer which is isolated from Nature other than the two isomers obtained by stereomutation, might be expected to have been formed as such in Nature (and not to be an artefact formed during the isolation procedure). The present author found comparatively large amounts of the neo-C isomer in the ° -carotene isolated from both Chlorella vulgaris and from carrot oil, and this, therefore, might be the (or one of the) naturally formed stereoisomers. (If this is the case, then the neo-C isomer must be thermally stable since the carrot oil is heated during processing: see p.74).

The first material to be studied was an X-ray mutant (no. G77) of the alga Chlorella vuliris, which had been grown and harvested by

Dr. Allen in America. The polyenos were solvent-extracted in the colds Was \ 118 and the chlorophyllAremoyed by saponification (also in the cold).

Repeated chromatography of the pentane extract separated the polyene mixture into the following components (in order of elution): a substance

77 Anderson et al. have recently suggested that a more drastic method

than that usually used for plant materials (as above) is required for

the efficient extraction of carotenes from the cells of Chlorella. 65

(A max.230 DT) whose absorption spectrum was suggestive of. a diene (cf. p.48), followed by phytoene (the major component), phytofluene (in small

amount only), and a yellow zone which had an unusually complex absorption

spectrum (maxima at 421, 2210 and 377 IT with, in addition, a relatively weak shoulder near 450 9.1). The yellow zone was rechromatographed and

the eluate monitored spectrally. This revealed that the complex spectrum

was not caused by one pigment but by a mixture, since the intensity of the

shoulder near 450 mp (relative to the major peaks in the spectrum) varied

as the zone was eluted. The major component of this mixture was apparently

C-carotene (as a mixture of stereoisomers of which the neo-C form

constituted, probably, as much as one third of the total). The other

pigment present in the mixture gave a pinkish cast to the lower part of the yellow zone and, so, was called the"pink contaminant: In addition to

these coloured constituents, the zone also contained relatively large

quantities of colourless materials (perhaps lipids). Both the phytoene and

the crude '-carotene zones were treated with lithium aluminium hydride

(cf. ref. 20) to convert any lipids present into compounds strongly adsorbed by alumina. The two zones were separately chromatographed. The

phytoene was isolated as a viscous, colourless oil; its n.m.r. spectrum

was determined and the result is discussed later (cf. p.79). This

treatment removed the colourless impurities from the r-carotene but the latter still contained the pink contaminant.

By this stage in the isolation procedure, only a small amount

(ca. 2 mg.) of the C-carotene initially present (ca. 9 mg. in the

non-saponifiable fraction of the crude chlorella extract) remained. At 66 least some of this loss can be attributed to the decomposition of the polyene during chromatographic purifications. (Mackinney et al.37 have suggested that chromatography of the "more saturated" polyenes causes their decomposition to a small extent; support for this suggestion has since come from work by the present author on the r-carotene isolated from carrot oil: cf. later discussion). It was decided, therefore. not to attempt to remove the pink contaminant from the C-carotene, since this was expected to involve prolonged chromatography on active alumina ( cf. 16 Goodwin and Osman ). Instead, it was decided to determine the n.m.r. spectrum of the mixture. If this showed no bands attributable to methyl protons attached to saturated carbon, then one of the objects of this work would have been satisfied. In fact, the spectrum consisted of a strong peak near 8.75 p.p.m. and a series of smaller peaks on the low-field side of this. The significance of the 8.75 p.p.m. peak is discussed below; it is in the region where the hydrogen atoms on methylene groups in saturated, 115 long-chain hydrocarbons absorb. In addition, the sample showed strong carbonyl absorption in the infrared. It was concluded that the ° -carotene contained large quantities of an impurity (other than the pink contaminant).

However, it is worth noting that the band pattern in the region 8.0 to 8.5 p.p.m. was almost identical with that in a spectrum since obtained on a sample of pure C-carotene isolated from carrot oil (see later discussion).

At this stage, it was decided to abandon the attempt at isolating a sample of pure C-carotene from the chlorella. Thus, the chlorella had provided only one (phytoene) of the three polyenes required in sufficient quantity and purity for a n.m.r. determination. The 67 phytofluene was present in only trace amounts. Attention was therefore directed to the other source mentioned (carrot oil). First, however, mention should be made of the nature of the pink contaminant observed above and of the significance of the strong band at 8.7 p.p.m. in the n.m.r. spectrum.

As mentioned above, the r-carotene zone had an unusually complex visible absorption spectrum; it was recognised that this was probably caused by a contaminant with a band near 450 9p (and, presumably, a further band near 425 9p masked by the long wavelength band of the r-carotene). "Carotenes" with absorption spectra of this type have been 13 found before in other natural materials. For example, Goodwin found such a "carotene" in the fungus Phycomyces blakesleeanus. He reported that repeated chromatography had no effect on the relative intensities of the peaks in the spectrum and concluded, therefore, that the peak near

450 my was not due to an impurity but was genuine. Soon afterwards, 16 however, Goodwin and Osman succeeded in resolving the "carotene" into c-carotene and another pigment (perhaps identical with the "pink contaminant" above) with absorption maxima at 452, 421, and 400 911; this separation was achieved by chromatography on active alumina from ether-light petroleum (1:3). It seems likely that the "carotene" isolated from corn, 122 119 120 and variously called "K-carotene", "Unnamed Carotene 1", "Pigment C3a, 123 and "Unidentified I", was also, in fact, a mixture of C -carotene and another pigment absorbing near 450 my (possibly the same one as above).

Recently, Quackenbush and his co-workers124 have re-investigated 68 the carotene content of corn. They found that chromatography of the extract on magnesia failed to separate the c-carotene from the pigment absorbing near 450 ET. However, by using a more active adsorbent (cf. 16 Goodwin and Osman's method, above), they succeeded in resolving the mixture into three main constituents. These were, in order of elution,

13-carotene; a pigment with absorption maxima near 450, 425, and 400 ay; and, finally, C'-carotene (the order of the last two named is the same as 16 reported by Goodwin and Osman, when they resolved the crude C7-carotene from P. blakesleeanus into two constituents: this suggests that the "pink contaminant" in both cases is the same). Repeated chromatography of the middle zone on lime separated it into two major constituents which were called a-zeacarotene (lower zone on the column) and p-zeacarotene (upper zone); several minor constituents were also observed. Treatment of the 2 zeacarotenes with N-brorfuccinimide84 converted them into 6-carotene and y-carotene; respectively.124 Hence p-zeacarotene would appear to have structure (XXVII). Kargl and Quackenbush125 have recently reinvestigated the constitution of 6-carotene and they concluded that it has the structure 126 (XXVIII) originally assigned to it by winterstein. (rather than the , alternative suggestion of Porter and Murphy127 ). Assuming this conclusion 124 to be correct, Quackenbush et al. concluded that a-zeacarotene has structure (XXIX): -

(XXVII), p-zeacarotene (?) 69

(XXVIII),6-carotene

(XXIX),a-zeacarotene (?)

p-zeacarotene, but not a-zeacarotene, was found to show vitamin-A activity (p-zeacarotene had ca. 25% of the activity of 124 p-carotene). This would be expected if the structures suggested above for these carotenoids are correct. It is noteworthy that "K-carotene" 019 (see above) was reported to have vitamin-A activity, whereas,' -carotene 23 itself has not.

A total synthesis of p-zeacarotene has recently been claimed by 128 Isler et al. The synthetic product was obtained crystalline, whereas Quackenbush et al.124 were unable to crystallise the natural material. The synthetic sample has not yet been compared directly with natural p-zeacarotene; however, it was reported to behave in the same way as 128 p-zeacarotene on treatment with N-bromosuccinimide (cf. above). Pigments with absorption spectra similar to those of the zeacarotenes have 61 been isolated by Claes from an X-ray mutant (9a) of Chlorella vulgaris, and by various authors from DPA—cultures of Mycobacterium phlei.14'66'70c 70

It will be remembered that the n.m.r. spectrum of the crude

'-carotene isolated from the chlorella showed a strong band near 8.75 p.p.m. in the infrared - The same sample showed strong carbonyl absorptioq(at 1718 cm.1, suggestive of a saturated ketone); however, no hydroxyl absorption was observed.

Since that time, many n.m.r. spectra have been run on samples of .-carotene and the other "more saturated" polyenes and in many of these spectra a small peak was observed near 8.75 p.p.m. In each of these spectra, however, the peak near 8.75 p.p.m. was much smaller than the Other peaks in the spectrum. It was usually more noticeable when very small samples were used; with larger samples (say, 10 mg.) it was often so weak as to be undetectable unless the spectrometer was operated at high "gain" when most of the other peaks in the spectrum would be off-scale. Since the peak was of variable intensity depending on the sample it seems certain that it was spurious. Fewer infrared spectra have been determined but, especially in the liquid film spectra, a very weak carbonyl absorption band was usually observed.

It is now thought that these spurious peaks were caused by the presence of small amounts of autoxidation products in the samples since it is known (cf. p. 59) that all these polyenes are very susceptible to atmospheric oxidation; indeed, n.m.r. spectroscopy probably provides the most sensitive test for the presence of autoxidation products. Other explanations put forward during the course of this work were that the peaks were caused by lipids, traces of solvents (hexane or benzene), or grease present in the samples. These suggestions can probably be discounted. The lipids should have been removed by the treatment with lithium aluminium hydride; also, similar spurious peaks have been observed in entirely synthetic samples of these pOlyenes.141 The samples were (apart from neurosporene) oils but there is little likelihood of them containing any solvents since they were always dried (as fairly thin films) under high vacuum for at least three hours before spectral determinations were done.

Also, contamination by hydrocarbon solvents would not account for the carbonyl absorption in the infrared. Every care was taken to exclude all traces of grease as it was realised that it might well be difficult to remove it from a sample by chromatography; a sample of "Apiezon L" showed no carbonyl absorption in the infrared.

It was apparent that the technique used tc manipulate the ncarotene isolated from the chlorella was not sufficiently refined to exclude all traces of oxygen from the sample. This, of course, was a test case since there was so little polyene in the sample. The phytoene sample, isolated using the same technique, gave an n.m.r. spectrum almost entirely free of a peak near 8.75 p.p.m. The phytoene sample was several-fold larger in weight than the L'-carotene sample and so exposure to a trace of oxygen would not be so apparent; also, phytoene appeared, throughout this work,to be rather less sensitive to atmospheric oxidation than g'-carotene (cf. p.59). The technique first used was briefly as follows.

The eluate containing the r-carotene from the column was evaporated to a small volume (in the cold and under nitrogen in the usual way), finally in a small flask. The vacuum was released with nitrogen and WAS the concentrateAtransferred (in hexane) to a small test tube. This was then suspended in a flask which was cautiously evacuated with a water pump until most of the solvent had evaporated (this usually took about 30 minutes). The remaining traces of solvent were removed under high vacuum. This method of evaporating the solvent had three disadvantages; the solution tended to 72

bump; the polyene was left as a smear round the inside of the tube (it was desirable to have the sample more compact so that only the minimum of solvent would be required to transfer it to an n.m.r. tube later); and the sample was exposed, as the solvent evaporated, to air at a pressure of about 15 mm. which might allow some autoxidation to occur during the 30

minutes that the evaporation required. This method did, at least, ensure that the sample tube was well separated from any grease on the ground glass

joints.

In later work, the following modifications were introduced. The concentrate was filtered in hexane through a small pad of active alumina resting on a cotton wool plug in the nozzle of a "dropper" made from glass tubing. The alumina removed any oxidation product (as a pale yellow ring of material held fast to the top of the alumina) which had been formed during previous operations. The filtrate was collected in a small tube, and most of the solvent was blown off by directing a stream of dry nitrogen on to the surface of the solution. As soon as the solvent had evaporated, the material deposited on the inside wall of the tube was rinsed down with the minimum of fresh solvent. This was evaporated and then the tube was wrapped in aluminium foil leaving only a small hole for the nitrogen inlet.

The nitrogen inlet was removed, the small hole covered with foil, and the tube immediately transferred to a flask which had been filled previously with nitrogen. Since there was only a trace of solvent left, the flask could straightaway be put under high vacuum. The aluminium foil served to slow down the diffusion of air into the tube during the brief period that the tube was being transferred to the flask.

Towards the end of this work the following procedure was used. 73

The sample was filtered through the alumina after the hexane had been removed using the same solvent (carbon tetrachloride) as was to be usedfcrthe spectral determination. The filtrate was sometimes collected in the n.m.r. tube itself; otherwise, it was concentrated (with a nitrogen stream, as above) to a small volume and then transferred to the n.m.r. tube. This would appear to be the best method since any autoxidation product was removed after all the other manipulations had been completed; however, this method was not tried out sufficiently with the polyenes to recommend it unconditionally.

The spurious peak near 8.75 p.p.m. was insignificantly small in all the spectra determined with the exception of that of the r-carotene isolated from the chlorella.

Samples of phytoene, phytofluene, and c-carotene were isolated from carrot oil;' at the same time, at least three other substances

(probably all hydrocarbons) were obtained which, as far as can be ascertained, have not been described in detail before. Spectral observations suggested that these substances were a tetraene, a triene, and a substance containing isolated double bonds. The latter may not have been pure, but it is unlikely that it contained any "lycopersene". The"triene", however, appeared to be heavily contaminated with another substance (the mixture has been called the "triene fraction") which may, conceivably, have been

"lycopersene".

Carrot oil is a viscous, dark red oil. It is a commercial product and the sample used in the present work was obtained from the same 74

142 8,20,40,96 source as that used by Rabourn and Quackenbush. Carrot oil is 143 prepared commercially as follows.

Carrots are harvested, washed, and then "steam cooked" (the temperature reached in this process is not specified, but, presumably, approaches 1000: sufficiently hot to stereoisomerise any thermolabile stereoisomers present). The mixture is ground to a pulp and most of the

moisture is expressed as a colourless juice in a hydraulic press. The

residue is ground, air-dried, and the dry powder is extracted with light

petroleum. The solvent is allowed to evaporate, finally under vacuum. This

leaves a viscous dark red oil; one ton of carrots yields one half to one

kilogram of oil. The oil is allowed to stand for several weeks during which

time much of the p-carotene (along with plant waxes) separates out and is

removed by filtration. The residual oil, "carrot oil", contains large

quantities of phytoene, phytofluene, and '-carotene. The amounts present

probably vary from batch to batch but Rabourn and Quackenbush estimated the C-carotene content to be ca. 6.2 g./kg. of oil. Spectral examination of the crude oil used in the present work suggested that it contained (per kilogram of oil): phytoene (20 g.), phytofluene (10 g.), CD-carotene (7 g.), and an unidentified pigment (probably a , . 470, 440 irp)

(7 g.). These values were probably grossly exaggerated by the presence of

light-absorbing substances other than these (cf. the "tetraene" and the

"triene" isolated by the present author).

The procedure used to extract the polyenes was a modification of 20 96 that used by Rabourn and Quackenbush. ' At the time the present work

was done it was not realised that the carrot oil used had been heated ?5 during processing, and an attempt was made to isolate the natural stereoisomers of these compounds. Rabourn and Quackenbush's instructions were modified accordingly. Thus, the hexane solution of the oil was saponified by prolonged shaking with methanolic alkali at relatively low temperature, rather than by boiling the mixture for a shorter period as suggested. This divergence from the normal procedure might have contributed to the thick, very stable emulsions encountered in the subsequent stages of the processing(higher temperatures might ensure a more complete break down of cell tissue, etc.) Thus, washing the hexane solution free of alkali and methanol caused a series of emulsions. As much as possible of the polyene in the emulsions was washed out with hexane

(now that it is realised that warming the mixture would have had little further effect on the polyene contents, it is apparent that continuous extraction with pentane could have been used here, perhaps with advantage).

The partly emulsified concentrate was clarified by filtering it through a desiccant - a procedure which proved rather wasteful of polyene. Rabourn 20 and Quackenbush chromatographed their 'Crude extracts at this stage and subsequently treated each of the required zones with lithium aluminium hydride. Since all the zones were wanted for study in the present work, it was decided to treat the whole of the hexane extract with lithium aluminium hydride, without chromatographing the extract first. A larger quantity of hydride was used than suggested. This resulted in the formation of a very bulky precipitate (from the decomposition of the excess hydride with acetone) which occluded coloured substances very tenaciously. (It appeared, therefore, that it would have been better to have removed some of the 76 impurities by chromatographing the mixture before the treatment with hydride as Rabourn and Quackenbush originally suggested).

The concentrate was chromatographed (on weak alumina); each zone had to be rechromatographed (on active alumina) at least once to remove impurities from the major constituents; the compounds then obtained (as they would appear on a column, from top to bottom) were ae follows [spectra are quoted for hexane solutions, except for (n)]: (a) "all-trans"-ncarotene (ca. 10 mg.)

(b) a substance, eN max. 470 and 441 my (trace), which overlapped

with (a). (c) neo-r-carotene-A (ca. 9 mg.) (d) neo-'-carotene-B (trace, only; possibly an artefact since it was found only in a fraction that had been exposed to the light).

(e) neo-'-carotene-C (ca. 4 mg.).

(f) a substance of unknown nature, 418, (398) my (ca.0.2mig.) (g) phytofluene (probably "all-trans")(ca. 10 mg.). (h) a cis-phytofluene (ca. 20 mg.). (i) phytoene (ca. 250 mg.). a substance of unknown nature, rall (trace). (j) A max. 356, 338 327, and 300 yj. (k) a "tetraene" (ca. 8 mg.), 3k max. al, (1) a fraction containing a "triene" (not phytoene)(ca. 50 mg.), . 295, 282, and 268 T. max (m) a series of substances with absorption maxima at 210, 220; 210, 230; and near 210, 260 T.

(n) a substance (or substances) containing only isolated double bonds, . (in ethanol) 206 IT, which will be called "P 206". 77

The figures given are only qualitative estimates because some solutions had to be stored longer than others before they were rechromatographed; in some cases, the solutions deposited some colourless solid (presumably autoxidation or polymerisation products).

N.m.r. spectra were determined as follows:

(i) a mixture of the "all-trans" and neo-A isomers of r-carotene

(ca. 1:1) containing a trace (ca.2-3%) of the substance with

absorption maxima at 470 and 441 my;

(ii)stereochemically pure neo-r-carotene-A, free of the substance

. 470, 441 mp) mentioned above; 96‘ (iii)the cis-phytofluene (probably the neo-B form );

(iv)phytoene;

(v) the "triene";

(vi)the substance (or substances) called "P 206".

Spectrum (i) was quite satisfactory in that the peak (near 8.75 p.p.m.) caused by autoxidation product in the sample was barely detectable and the spectrum was of the type expected (based on the spectrum obtained on the phytoene previously isolated from Chlorella vulgaris). However, it was considered desirable to determine the n.m.r. spectrum of a sample of r-carotene which had been freed of the trace of impurity ( A max. 470 and 441 m)1). Chromatography of a mixture of r-carotene stereoisomers and the impurity on active alumina served to separate the three cis isomers (which were eluted from the column) from the "all-trans" isomer which remained, mixed with the impurity, on the column. Prolonged development of the column led to a partial separation of the pink impurity from the C-carotene, 78 the pink zone emerging just below the yellow C-carotene zone. However, the two zones were only partly separated and, although the upper part of the zone was free of impurity, the recovery of pure '-carotene was low. The n.m.r. spectrum of the purified "all-trans"-'-carotene was determined but was too weak to be of much use.

The attempt to purify the c-carotene by prolonged chromatography had the disadvantage that it was apparent that I7-carotene slowly decomposed on the column (cf. ref. 37). This was once demonstrated by allowing the chromatogram to stand overnight and then continuing the development of the column on the following day. Although most of the C-carotene zone was eluted slowly down the column as before, there remained on the column a yellow zone, which was not eluted, where the r-carotene zone had been.

It was found to be easier to remove the pink impurity from the neo-A isomer than it was from the "all-trans" isomer. Thus, on chromatography of crude r-carotene, the pure neo-A isomer was eluted before the pink impurity and was collected.' The n.m.r. spectrum of the neo-C=carotene-A was determined and was found to be essentially identical with the spectrum (i) determined on the mixture of "all-trans" and neo-A isomers (cf. the discussion below).

The n.m.r. spectrum of the phytoene obtained from the carrot oil

(above) was identical with that given by a sample of phytoene (also from 20 carrot oil) kindly supplied by Drs. Rabourn and Quackenbush, which, in turn, was identical with that given by the sample of phytoene isolated by the present author from Chlorella vulgaris. This constitutes the first 79 real evidence for the identicality of the phytoene present in one of the chlorella mutants with the phytoene which occurs in carrot oil (and, therefore, in carrots). Previously, identification has been on the basis of a comparison of ultraviolet spectra; this only showed that each of the two compounds contained a triene chromophore (one obvious alternative structure that might be found in Nature is the cyclic analogue of phytoene; this would be readily distinguishable from phytoene itself by a comparison of n.m.r. spectra).

The n.m.r. spectrum of the cis-phytofluene (which was probably the neo-B isomer%) was identical with that of a cis-phytofluene (also from carrot oil) kindly supplied by Drs. Rabourn and Quackenbush.

The n.m.r. spectra of phytoene, phytofluene, andr-carotene will be discussed together, along with the spectrum given by the neurosporene kindly supplied by Dr. Nakayama (see pages 58 and144). The four spectra are reproduced on page 80. First, it will be seen that none of the spectra shows any bands near 9.1 p.p.m., where methyl groups attached to saturated carbon atoms would absorb, if present (cf. p. 55). Therefore, all suggested structures for these compounds which include such a feature

(cf. p.54) can be immediately eliminated.

Each of the four spectra shows bands in the same regions of the n.m.r. spectrum as the others. 80 Lri "'t m m o Op e3 Co

CAROTENE

eis et d 0 am' co Co Co Co

PHYTOENE NEUROSPORENE

••••• co (T) O to 111".. 1." Csi m oD 0.0 00 to 00 do

PHyTOFLUCIVE ZYCOPENE

THE N.M.R. SPECTRA or THE COMPOUNDS MENTIONED IN THE rasLE ON ffice 85 ON THE 7.5- To 8.5 r-P.m. RECION,0NLY), AND ALSO of 5GUALENE (SEE PACE 5 7).

[WHERE THE SPECTRUM or THAN ONE SAMPLE of THE SAME COMPOUND WAS 1)RTERIIINEP THE 7-VALUES QUOTED fog THE COMPOun ABOVE ARE THE MEANS OF THE. VALUES os-rAINED ON EACH OF THE sevas (alp, 85 )1. 8i

These are, progressing from the low field up to the high field end of the spectrum,146 as follows:- (i) T near 3.8 p.p.m. In each of the four spectra, two or more peaks of variable intensity appeared in this region; they are all ascribed to protons attached to non-terminal, acyclic, conjugated double bonds (cf. XXX) .115 Phytoene gave two peaks of similar intensity (at 3.78 and 3.95 p.p.m.); phytofluene gave one main peak (3.75 p.p.m.) with three very weak neighbouring peaks; in the spectra ofr-carotene and neurosporene, groups of five peaks occurred, the central peak (at 3*80 and 3.66 p.p.m., respectively) being the strongest in each case (the two outlying peaks in

C.-carotene were too weak and broad for their lrvalues to be measured accurately and so these values are not given in the Experimental section). (ii) p.p.m. A single, rather broad band occurred consistently in this region in all four spectra; it is due to absorption by protons attached to non-terminal, acyclic, non-conjugated double bonds

(cf. XXXI).115 Me Me Me

H

(XXX) (xXXI) (iii) T7.75 to 8.1 p.p.m. This is the region where the methylene protons in squalene absorb (see p.80). The methylene protons in the 115 system CH3-CH2-CIPgC:: would be expected to absorb near 7.83 p.p.m. In squalene, there are two different types of methylene group. These are: (a) those(of type -CaH2-, below) which do have a neighbour carrying an olefinic 82 proton (with which the methylene protons can undergo spin-spin interaction), and (b) those (of type -CA-) which do not have a neighbour of this type.

The protons of either of these two types of methylene group can undergo

CO2's,„ -T0-12

(xxxu) extensive spin-spin coupling with the protons of the neighbouring methylene an group and, if the group is of the type -CA- , withAolefinic proton as well. The result is a broad, methylene-absorption band extending from

7.70 to 8.1 p.p.m. in squalene, with a prominent peak at 8.03 p.p.m.

A similar broad band occurs in the spectra of phytoene, phytofluene, (-carotene, and neurosporene; in all but neurosporene, a strong band stands out from the methylene absorption band near 8.03 p.p.m. rather as in the spectrum given by squalene. In phytofluene and c'-carotene, this band near 8.03 p.p.m. is just resolved from a band (due to "in-chain" methyls, see below) at slightly higher field; this second band is not observed in phytoene and this suggests there are no "in-chain" methyls in this compound (see below). In neurosporene, only the strong "in-chain" the methyl band is observed; this has probably maskey.03 p.p.m. band which, in any case, might be expected to be weaker in this compound because it contains fewer methylene groups.

The polyene spectra often showed other peaks (in addition to the

8.03 p.p.m. peak mentioned above) standing out from the methylene absorption band. However, they were always of weak intensity and their 83

appearance was inconsistent, depending to a certain extent on how well the spectrometer was functioning. When it is reported in the present work that two spectra "were identical", there may have been slight differences in the methylene absorption band but these differences have been disregarded. Thus, the two cis-phytofluenes which were isolated (by 96 Rabourn and Quackenbush, and by the present author) from carrot oil, showed quite marked differences in their n.m.r. spectra in this region, although there is every reason to believe that they are identical compounds, 91 % except possibly in stereochemistry (others have had the same experience ).

Indeed, it might be that differences in the stereochemistry of the end-of- chain double bond of a chromophore affect the extent to which the protons of the methylene group allylic to this double bond are deshielded and so cause these small differences. Until the spectra of several pure samples of stereoisomers of known configurations are determined and compared with one another, it is unwise to make too many of these speculations. The methylene region contains no bands of significance required for the assignments of structures in the present investigation.

(iv) T 8.1 to 1:8.5 p.p.m. It is in this region that the protons of methyl groups attached to various types of unsaturated carbon atom absorb.114

Four bands appeared in this region in each spectrum (with the exception of phytoene which only gives three). The exact positions varied from one spectrum to another but the mean values were 8.09, 8.21, 8.35, and 8.41 p.p.m. These can be assigned to "in-chain" methyls, "end-of-chain" methyls,

"trans-olefinic" methyls, and "cis-olefinie" methyls,respectively (these assignments have been discussed previously; see p.55). The peak at 84

8.35 p.p.m. sometimes appeared as a shoulder on the side of the more intense 8.41 p.p.m. peak; usually, however, it was resolved as a just perceptible individual peak close to the side of the 8.41 peak. This depended to a large extent on how well the spectrometer was operating. It was noticeable that the 8.35 peak in phytofluene never appeared as more than a rather indistinct shoulder; why this should be so is unknown; since spectra were run on this compound on three different occasions it is unlikely to be entirely the fault of the spectrometer. In general, one would have expected that the more nearly equal in intensity the 8.35 and

8.41 peaks were, the more likely that two distinct peaks would be observed.

This was borne out to the extent that the two peaks were very well resolved in the neurosporene spectrum.

It was from the measurements of the areas enclosed by each of these peaks in the spectra given by the four polyenes that the structures of these compounds were deduced. These peak-area values are given in the following table. The table also gives the approximate positions at which these methyl bands occurred, as calculated by averaging the values obtained foi each of the four polyenes. The actual values obtained for each compound are summarised below the table, and are given in full in the

Experimental Section.

It was noticeable that the l'-values obtained for each of the methyl bands varied slightly from one compound to another: this was especially true of "end-of-chain" and "in-chain" methyl absorption bands which appeared at slightly higher fields when attached to short systems of conjugated double bonds than when attached to longer systems. It seems that when these methyls are attached to short polyene chains they are not quite so deshielded by the conjugated double bonds as when attached to more

8 5 extended systems. Nuclear magnetic resonance spectra (methyl bands only).

Assignments! Type of Nre group In-chain End-of-chain trans-Olefini cis -Olefinic Ch6 CH3 CH` 3 //,R 0•. Partial structure C==C am C==C // // —CH2 —CH2

Approx. band position+ 8.09 8.21 8'35 8.41 Relative number of Me groups found: Phytoene 0 2 2 6 Phytofluene 1 2 2 5 C-Carotene 2 2 2 4 Neurosporene 3 2 2 3 114 Lycopene 4 2 2 2 +Actual band positions were as follows:- Band positions found

Compound Source 17:-valuesN Phytoene Chlorella vul ris(a) ONO 8.25 8.33 8'41 a Carrot oil 8.26 8.36 8.42 (b) Carrot oil 8.25 8.35 8'41 11-qtufluene Carrot oil(a) 8.13 8.22 8.36 8.42 Carrot oil(b) 8.10 8.22 8'35 8.41 (a) r-carotene Carrot oil 8.09 8.20 8.36 8.42 (c) Neurosporene R. spheroides 8.05 8.19 8.32 8.40 0) Lycopene114 Synthetic 8.03 8.18 8.31 8.38

Notes: (a) Extracted by the present author: see Experimental Section. (b)Kindly supplied by Drs. Rabourn and Quackenbush. (c)From a sample isolated from Rhodopseudomonas spheroides by Dr. Nakayama. (d)See ref. 145; spectrum determined by Barber, Davis, Jackman, and Weedon.114 (cf. p.90) *The spectrwere determined for carbon tetrachloride or deuterochloroform solutions at 56-4 Mc./sec. and calibrated against tetramethylsilane as an internal standard. Band positions are given as T-values, as defined by Tiers (J. Phys. Chem., 1958, 62, 1151). The band assignments have already been discussed (on p.55). The conclusions that can be drawn from the above results in the light of these assignments are as follows.

As has already been pointed out, there can be no methyl- substituted saturated carbon atoms in any of these four polyenes. The

peaks at 8.41 and 8.35 p.p.m. account, between them, for all the methyl

groups other than those attached to the polyene chain. A comparison of

the relative intensities of these two peaks in each spectrum shows that the the remainder 8.35 p.p.m. peak can account for no more than two of these methyl groups,/

being at 8.41 p.p.m. Since each of these polyenes has two isopropylidene

end groups, each of which must contain one trana-olefinic methyl group

(near 8.35 p.p.m.), it is apparent that all the other methyls attached to

isolated double bonds are of the cis-olefinic type. Thus, the methyl substituents on all non-terminal, unconjugated double bonds are of this

type and, so, the bonds themselves all have the trans-configuration (as in

natural squalene: see p.5'7).

At first sight, it might appear that these isolated double bonds

could occupy either of two positions, relative to the methyl substituent,

and still satisfy the requirement that the methyl must be attached to an

unsaturated carbon. Thus, the double bond associated with the C carbon 5 could be disposed in either of the following two ways [(XXXIII) and (XXXIV)]:-

87

(Xc.xru) (xxxrv) However, the following correlations are well established:115 I I I CH —cg -CH=C( , 1- near 7.8; =C-CH -C. lr.- near 7-1;=C-CH -CH -CH - 3 2 H 2 ' 2 2 2 -C=' l' .. H

near 8.7 p.p.m.

No bands were observed at either 7.1 or 8.7 p.p.m. in the spectra

of the four polyenes being discussed (however, cf. p.70 regarding the peak

given by the autoxidatiqproduct:this peak was virtually non-existent in the

spectra used for these structure determinations). However, strong absorption always occurred near 7.8 p.p.m. Thus, all the methylene groups

in these polyenes must be of the type: -C=C-CH2-CH2-2= as in (XXXIII).

Hence, if it is assumed that the 0111-dimethyl end group is of the

isopropylidene type rather than the isopropenyl type (and the evidence in favour of this assumption is overwhelming), the isolated double bonds must

be disposed as in partial formula(XXXIII) throughout the molecule.

The peak assigned to the trans-olefinic methyls (now known to be two in number) is of the same magnitude, in each of the spectral as that due to the "end-of-chain" methyls (T, near 8.21) which, therefore, must be two in number. This immediately rules out the possibility of any of the polyenes possessing a structure with a chromophoric system which does not include the centre section of the molecule (i.e. carbons C to C inclusive) fully 13 13 and symmetrically unsaturated (as in (xxxv), below) 8 8

c 13 C V I 13r

This is a geometrical requirement caused by the "head-to-tail" arrangement

of isoprene residues in the centre of the molecule. The only structures

that can now be written for a triene and a pentaene, embodying the above

requirements, are those which have been assigned to phytoene (III) and

phytofluene (V), respectively, by Rabourn and Quackenbush.

Similarly, only two different structures can be written for

neurosporene [(IX) and (XXXVI)]. Of these, the latter can immediately be

eliminated since one of the isopropylidene groups is now in conjugation with

the chromophore and therefore this group alone would give rise to two

"end-of-chain" methyl bands (just as the end groups of the hydrocarbon

(XXXVII) and its C30 analogue do 114). Neurosporene must therefore have

(x xxvii)

structure (IX). This constitutes the first direct evidence that neurosporene

has this type of structure, and simultaneously defines the structure as that 89 represented by formula (IX). Of the four polyenes being discussed, only r--carotene remains to be considered. Here, two possible structures can be written down [(VII) and (VIII), cf. p.40) both of which are compatible with both the n.m.r. work described here and the analytical and degradative 8 work described by Rabourn and Quackenbush.

No mention has been made of the "in-chain" methyl bands in the above discussion, since the existence of the two "end-of-chain" methyls in each of the spectra coupled with a knowledge of the length of the chromophores present in each, establishes the structures of these compounds as near as it is possible to do. This is perhaps fortunate since there is no need to consider a somewhat more complicated region of the spectra

(8.0 to 8.1 p.p.m.) where not only "in-chain" methyls appear but also (had they been present) "isoprenoid" methyls (cf. p.55); in addition, the various methylene absorption bands build up to a climax near 8.03 p.p.m. in phytoene,phytofluene, and '-carotene and give a quite well-marked peak near this -r-value in these three compounds [this does not occur in neurosporene and lycopene probably because, in these compounds, there are fewer methylene groups to give bands]. However, it should be added that a peak (equivalent to one methyl group) appears in the spectrum of phytofluene, in the region expected for "in-chain" methyl bands, which is notably absent from the spectrum of phytoene. This is assigned to the "in-chain" methyl which must be present in phytofluene. This "in-chain" methyl band increases in size on progressing from phytofluene along the series to lycopene in exactly the way expected for the proposed formulae. 90

It has, therefore, been shown unequivocally that the structures of phytoene, phytofluene, and neurosporene are as follows:-

Phytoene, (III)

Phytofluene, (V)

/A( Neurosporene, (IX)

These structures have been assigned on the basis of the measurement of the n.m.r. spectrum, the molecular weight (to give the number of carbon atoms), and the visible absorption spectrum (to give the number of conjugated double bonds) of each of the polyenes. These measurements would require, in all, only 10 mg. (or perhaps a little less) of each compound.

Further confirmation of the formulae of phytoene and phytofluene came from performing area counts. The relevant sections of the curves

[recorded on broad (12.5 cm.) chart) were cut out and weighed; at least three determinations were made on the same sample and the mean values taken.

Three areas were determined. These were the group of peaks near 3.8 p.p.m.

(protons attached to non-terminal, acyclic, conjugated double bonds), the single peak near 4.9 p.p.m. (protons attached to non-terminal, acyclic, 91

non-conjugated double bonds) and the group of peaks in the region 7.5 to

8.5 p.p.m. (protons attached to saturated carbon). No attempt was made to

subdivide each group up into its individual constituent bands since these were

rarely sufficiently well separated. The values obtained for phytoene and

phytofluene were in good agreement with the values expected [found and

expected values for these two compounds were, in the order given above:

4, 6.5, and 93.9; 4,6, and 54 (for phytoene; found and expected values, respectively); and: 6.1, 5.2, and 50.7; 7,5, and 50 (for phytofluene, as

above)).

As already mentioned, the structure of g7-carotene remained in

doubt after these n.m.r. determinations. The results obtained were

compatible with either structure (VII) or structure (VIII), since both of

these structures have identical numbers of each of the various types of

methyl group found to be present in r-carotene.

(m)

(viii)

150 Siddons has now synthesised each of these compounds. The

"all-trans" stereoisomer of the synthetic compound (VII) had a qualitatively 92

with that identical visible-light absorption curveiof the "all-trans" natural

C-carotene isolated by the present author, and the two samples showed no

separation on a chromatoplate. However, this evidence is by no means

conclusive and Siddons has since carried out a direct comparison of the

properties of each of the two synthetic compounds [(VII) and (VIII)] with

those of natural c-carotene. He has come to the conclusion that

C-carotene is, in fact, the symmetrical compound (VII). His evidence for

this conclusion was largely based on a study of.the spectral properties of

the products obtained by iodine-catalysed stereomutation of the "all-trans"

forms of (VII), (VIII), and natural C-carotene, [infrared, visible- and

ultraviolet-light absorption spectra were all measured; it was confirmed

that the n.m.r. spectra of (VII) and (VIII) were essentially identical].

Siddons also succeeded in crystallising "all-trans"-r-carotene 190 (cf. Petzold129). A full report of this work will appear elsewhere; 92 meanwhile a preliminary communication has been published.

190 Siddons has also completed unambiguous total syntheses of

"all-trans"-phytofluene, "all-trans"-neurosporene, and phytoene (as a.

mixture of stereoisomers). The natural (central-cis) stereoisomer of

phytoene has yet to be prepared but the n.m.r. spectrum of the synthetic

mixture of isomers was reported to be identical with that of natural

phytoene.92 The "all-trans" samples of phytofluene and neurosporene were

identified with samples of phytofluene and neurosporene isolated from

natural sources by a comparison of spectra (infrared, ultraviolet- and

visible-light absorption and n.m.r. spectra) and of chromatographic

properties (and mixed melting point in the case of neurosporene). 92,150 93

The present author isolated several substances from carrot oil other than those already mentioned. The chromatographic properties of these substances suggested that they were hydrocarbons (the possibility that the more strongly adsorbed members of this group might contain epoxide groups can be excluded since the mixture had been treated with lithium aluminium hydride during processing using conditions which would reduce an epoxide to the alcohol156). In that these substances were found in the same natural environment as phytoene, phytofluene, and c'—carotene, and are also hydrocarbons, they are of some interest. Some of the properties of these compounds will now be discussed. The positions they occupy on a chromatogram, relative to the polyenes already discussed, have been indicated previously (see p.76).

The pigment labelled fraction (f)(on p. 76, X max. 445, 418, and (398) mdµ) is notable for the unusual shape (for a carotenoid) of its absorption spectrum, the longest wavelength band being the most intense.

The zone containing this pigment was monitored spectrally as it was eluted 22 30 and was found to be homogeneous. Porter and Zscheile ' found a carotene

(which they called "Unidentified Carotene II") with a similar absorption curve in some varieties of tomato.

The "tetraene[fraction (k) on p.76) was isolated as a colourless, viscous oil which in dilute solution (1 mg./100 ml., in hexane) fluoresced pale green on exposure to ultraviolet light. It appeared to be reasonably stable to atmospheric oxidation in that no increase in the weight' of a sample was observed during weighing. However, old solutions of the 94 substance showed a tendency to deposit a colourless solid (admixed with the oil) if the solvent was evaporated; the colourless solid was readily removed by chromatography of the mixture.

The substance was tentatively identified as a tetraene by comparing its absorption spectrum (maxima, in hexane, at 327, 2j1, and 300 91) with those of the triene, phytoene, and the pentaene, phytofluene.

The spectrum of this "tetraene" showed rather more fine structure than

phytoene, and wasvery similar in shape to the spectra of two synthetic tetraenes, isoaxerophthene[(XXXVIII), maxima, in ethanol,31 at 324, 221, and 296 9.0151 and isodesmethylaxerophthene [(XXXIX), maxima, in ethanol,' at W2 320, 226, and 292 91]. The "tetraene" absorbed at rather longer wavelengths

than either of these compounds but hardly at long enough wavelengths to have a chromophore like that of axerophthene(XL).153

(XL)

If the "tetraene" has the same extinction coefficients as isodesmethylaxerophthene,then the molecular weight of the "tetraene" must be approximately 550, suggesting that it is a C compound. However, it is 40 quite possible that it is contaminated with non-polyene impurities in the same way as the "triene"1 discussed belowl seems to be. 8 The corresponding values in light petroleum will be 0-1 mix less. 95

A tetraene (maxima, in iso-octane, at 318, 303.5, and 291 71) 131 has been found in naturally-aged tobacco leaves; this substance was

non-fluorescent and absorbed at shorter wavelengths (by ca. 9 9.1) than the

tetraene isolated by the present author. A substance which behaved like

a hydroxy-tetraene was found by Eny44 in the leaveS of Hevea brasiliensis.

The "triene" fraction (fraction (1) on p.76; maxima, in hexane,

at 295, 282, and 268 mp) was isolated, in comparatively large amounts, as

a colourless, viscous, non-fluorescent oil. The n.m.r. spectrum of the (see p.97) substance/was similar to that of squalene not only in the positions of the

bands but also in their relative intensities. The major difference was

that the methylene absorption band in the "triene" only extended down to

ca. 7.85 p.p.m. (it extends down to 7.7 p.p.m. with squalene); the triene

also showed a weak band at 8.75 p.p.m. and a group of three weak bands at

9-9.2 p.p.m., which are not present in the squalene spectrum. These bands

are probably due to methylene groups in a saturated environment (8.75 p.p.m.),

and to methyl groups attached to saturated carbon (at 9-9.2 p.p.m.). The

bands at 8.41 and 8.35 p.p.m. (relative areas, 3:1) in the triene spectrum

were well resolved. The sample had to be stored several weeks in solution

(in the cold) before the following tests could be carried out (this is

mentioned because similar treatment had a marked, and as yet unexplained,

effect on the fraction called "P 206"; see below).

An attempt to determine the molecular weight of the "triene"

fraction was unsuccessful - the sample apparently decomposed under the

conditions used (dissolved in boiling benzene). Therefore, the extinction

coefficients of the substance could not be determined from its ultraviolet 96

spectrum. However, the positions of the bands (maxima, in hexane, at

295, 282, and 268 riya) indicated the presence of a triene (phytoene absorbs at similar, but slightly longer, wavelengths). However, it was apparent that the "triene" (E1%cm. at 282 IT was only 143) must either have a very high molecular weight or, more likely, have been contaminated with an impurity if it were to have an extinction coefficient (E) in the range normally expected of a triene. Indeed, it seems likely that the major component of the "triene" fraction was a substance with isolated double

bonds and that this fraction contained relatively little of the "triene".

This conclusion is supported by the infrared spectrum of the "triene" fraction. The only peak detected in the 6)A. region was at a position - \ (1668 cm.1) expected for an isolated carbon-carbon double bond rather than for a conjugated (triene) system of such bonds [whicn would show absorption at rather lower frequency:154 cf. the two bands given by phytoene, at

1665 cm:1 (isolated double bonds) and 1631 cm:1 (conjugated double bonds)155].

It is noteworthy that the n.m.r. spectrum was, as mentioned, similar to that of squalene and, therefore, was not unlike that predicted for

"lycopersene" (cf. p.44). However, the material would have to be obtained free of the triene component (and also, perhaps, subjected to ozonolytic

degradation) before one could say whether it were "lycopersene" or not. m

3t According to Anderson et al.77, squalene (and presumably, therefore,

"lycopersene") cannot be eluted from alumina with spectroscopically pure

hexane. This would appear to exclude the possibility of "lycopersene"

occurring in this fraction; however, Anderson's alumina might have

been more active than that used by the present author. 97

"p206 , see p.140 for details, (also p.98).

A SERIES of WEAK, 1C.1.-DcfrNED priors wERE OSSEAvED 1N THE 47 toy-3 Y.P.M. REG ION.

"ThE SpEcTRUti Viol 8r "P.2 06 " fifTER 1115 5035 7-nticE ilflp SEEN STORED fir 0°Itv CC14 foR 6 WEE ICS : SEE PP. 100 AND 14-1,

O Do

"••••••••••••••••••••• 00

774E "TRIENE"FRACTION, SEE PP. 95, 141.

0 ao co cr, tr) co

co

8

-rHE N. M. R. SPECTR A OF "P-206 d AND 77/E "7—RIENE n FRACTION ISOLATED FROM CARROT OIL.. 98

(See p.97) The n.m.r. spectrumAof the "triene" fraction contained weak

bands at 3.98 p.p.m. and near 8.2 p.p.m. which might have been caused by an olefinic proton and an "end-of-chain" methyl, respectively, attached to the chromophore of the triene component of this fraction.

The triene component was not phytoene since that compound was eluted after, and was well separated from,the "triene" fraction; and it is unlikely to have been a cis isomer of phytoene since the latter already has 13 a central-cis configuration. Goodwin isolated a substance (or mixture of substances), which he called the "phytoene-like fraction", from the fungus

Phycomyces blakesleeanus. Goodwin's material had a similar absorption spectrum (maxima, in light petroleum, at 296, 285, and 272 91) to that of the "triene" fraction here, and the composition of it, too, remained unknown.

Fraction (m) (see p.76) consisted of a mixture of substances which were not separable under the conditions used. One component (maxima at 230 and 210 mr) was perhaps a diene; the occurrence of dienes in Nature

has already been discussed (see p.48).

The most readily eluted fraction collected from the carrot oil chromatogram, "P 206" (p.776), was isolated as a colourless oil. It

contained only isolated double bonds, and was the second most abundant

component found in the carrot oil. It gained weight slowly on exposure to air, and, therefore, was purified by passing it through alumina immediately

before any tests were carried out on it (as for the polyenes).

It will be remembered that a peak occurred near 8.75 p.p.m. in

the n.m.r. spectrum of those polyene samples which had been exposed to the 99

(site p.97) air. A peak occurred at this position in the spectrum of "P 206'put it

was shown to be genuine since the spectrum was unaffected by alumina

treatment (cf. above). An area count on the spectrum, taken in conjunction

with a molecular weight determination, suggested that "P 206" contained two

olefinic protons and 30 protons attached to saturated carbon. The spectrum

contained a group of bands near 9.1 p.p.m. indicative of methyl groups

(probably two) attached to saturated carbon. A feature of the spectrum was

a pair of weak bands (equivalent to one proton) near 7.75 p.p.m. ?hese R can probably be assigned to proton H in the group d/ CH= , the band R due to this proton (ii) having been split by spin-spin coupling with the 115 neighbouring olefinic proton. The two major bands in the spectrum (at

8.40 and 8.34 p.p.m.) can be assigned to cis- and trans-olefinic methyl

groups (probably three in all).

In the olefinic proton region there was a group of bands

(equivalent to two protons in all) in the region 4.75 to 5.3 p.p.m. It is

unlikely that any of these bands are due to the presence of =CH2 protons 5.35 p.p.m.115. ( 1-• value, ) since the infrared spectrum of "P 206" showed no -1. N. absorption in the region (near 900 cm. ) where d.C=CH2 groups give rise to strong bands.154 Little other information could be gleaned from the -1 infrared spectrum; the band at 974 cm. could probably be assigned to the

trans -CHi.CH- (or trans --CMe..CH-) group.

A comparison of the n.m.r. spectra of "P 206" (as found) and of

"lycopersene" (as predicted, on p. 58) shows that "P 206" is not

"lycopersene". It is possible, however, that "P 206" was a mixture 100 containing "lycopersene" as one of the components; if this is so, then it could only have been a minor component.

Analytical facilities were not available at the time the n.m.r. spectrum was determined, and so the sample had to be stored, in solution, for several weeks before the following determinations were carried out. and contained only The sample, after purification, then analysed for C18H30 one double bond, implying the presence of three rings in the substance.

The n.m.r. spectrum of the hydrogenation product, however, suggested that it contained no five- or six-membered alicyclic rings115 (only weak absorption was observed in the 8.5-8.6 p.p.m. region - probably due to mothine protons); the two major bands are attributed to methyl and methylene groups (ratio, ca. 1:3) in saturated environments.

Meanwhile, the n.m.r. spectrum of the substance was determined in the absence of the tetramethylsilane usually added as an internal standard, so that protons attached to cyclopropane rings could be detected if present 115 (these give bands near 9.8 p.p.m. and might be obscured in the presence of tetramethylsilane); none was detected. However, this spectrum (see p.97) was markedly different from that originally obtained on a sample of "P 206".

In particular, the olefinic-methyl bands were of much reduced intensity whereas the 8.75 p.p.m. peak had increased in magnitude and was now the most intense band by far. The analytical figures suggested that the substance being analysed was not so unsaturated as "P 206" which itself must have contained at least two double bonds to accommodate the three olefinic methyl groups and two olefinic protons found to be present in it. 101

The reason for this change is unknown. Two of the

possibilities are (a) that "P 206" was a mixture, one component of which

decomposed during storage; (b) that "P 206" underwent cyclisation,

catalysed by a trace of acid in the solvent or in the alumina used to

purify the substance. Whatever the reason, there seemed little point in

attempting to elucidate the structure of the substance.

Thus, the structuresof "P 206", the "triene" fraction, and the

"tetraene" remain unknown. Of course, some or all of these compounds

might be artefacts formed during the processing of the carrot oil; in

particular, they might be products of the lithium aluminium hydride

reduction of the polyenes. In any future work on these compounds, therefore,

it would be advisable to investigate the possibility of separating these

readily eluted substances from the lipids also present just by using

chromatography, without invoking the hydride-reduction treatment.

Meanwhile, it is interesting to note a report regarding the

behaviour of some unsaturated on active, acid washed alumina. 157 Stedman et al. have chromatographed freshly distilled squalene (XXIV)

on acid-washed alumina which had been activated by heating it (at 160° for

15 hours). Fractions of the eluate from the column were collected; each

fraction had the same infrared spectrum but this was different from the

spectrum given by a sample of the squalene that was put on to the column. -1 Thus, the broad band in the squalene spectrum near 830 cm. (due to the

group -C(CH3).CH- 154) had disappeared and a new band had appeared at -1 889 cm. (assigned to the group -C(=CH2)-CH2- 154). Similarly, the band

at 1664 cm:1 (-C(CH3 )=CH-) in the spectrum of squalene had been replaced 10 2 by a band at 1647 cm:1 (-C(=CH2)-CH2-); in addition, a slight shoulder appeared at 234 ny. in the ultraviolet spectrum. Similar (but not wholly identical) spectral differences have been observed between squalene and

"regenerated squalene" (which is, itself, the product obtained by pyridine \.158 treatment of the solid hexahydrochloride derivative of squalene)

Evidently, the "regenerated squalene" was not identical with the squalene itself, which had, in fact, undergone isomerisation during the course of what was designed to be a purification procedure.

157 Stedman et al. also observed that neophytadiene (see p.52] underwent isomerisation on chromatography in the same way as squalene.

It appears, therefore, that the isolation from Nature of hydrocarbons with only a few double bonds might be unexpectedly complicated.

On the one hand, it would seem desirable to avoid the use of lithium aluminium hydride to remove the lipids which should, instead, be removed by chromatography on an active adsorbent (this might be necessary, in any case, for the separation of one hydrocarbon from another). On the other hand, the use of an active adsorbent could give rise to the hydrocarbons undergoing isomerisation so that the hydrocarbon isolated might be an isomer, or mixture of isomers, of the compound actually present in Nature. Indeed, it is conceivable that even more fundamental changes in the structure of such compounds might occur during chromatographic purification on active alumina.

In the present work, the "active" alumina,(cf. p. 6) used had not been freshly heated nor had it been acid-washed. As it was, only phytoflUene of the four alleged carotenoid precursors was chromatographed on 103

"active" alumina before measuring its n.m.r. spectrum. The spectrum given by phytofluene was identical with that given bya sample of phytofluene isolated independently (see previous discussion); and the spectrum of r—carotene was unaffected by chromatography on this adsorbent.

It might be fitting to make a few remarks here regarding the relative merits of the chlorella and of the carrot oil as sources of phytoene, phytofluene, and C.-carotene.

Carrot oil is a commercial product and is prepared in large quantities as a matter of course. On the other hand, the growing of chlorella mutants is not at all easy and much labour goes into the production of a relatively small 4uantity of algae. The dried algae contain much less of the required compounds than carrot oil. Part of this deficiency in the case of the chlorella might be attributable to incomplete extraction (cf. the footnote on p.640; this difficulty does not arise in the extraction of the carrot oil, of course, since the oil is merely diluted with solvent. Of the more saturated polyenes present in the chlorella„ phytoene was the most abundant and was followed by r—carotene, and, finally, phytofluene. Several other polyenes were present, one of which could have been neurosporene; this compound has been reported in some other mutants of 61 chlorella vulgaris. The r-carotene from the chlorella was heavily contaminated with an impurity (the "pink contaminant") with similar adsorption properties to those of r-carotene; the r-carotene from carrot oil contained a different impurity which was present in much smaller amount. The main advantage in using chlorella would be that, according contain to the limited experience gained in the present work, it does not seem to/ 104 the large number of other compounds that carrot oil does. Admittedly, since there was less phytoene, phytofluene, and ''-carotene in chlorella, it might be there was less of these other materials and they went by undetected.

However, it was very noticeable that the phytoene from the chlorella could be obtained, quite easily, in a perfectly colourless state whereas the phytoene from carrot oil was invariably tinged yellow by a trace of one of these unidentified substances which was not removed b_✓ chromatography. The sample of phytoene (also isolated from carrot oil) kindly supplied by

Dr. Rabourn was also very pale yellow.

(PYWRAPO FRoll RIOS)

(XLI)

(MAO (XLIV) 105

Carotogenesis - a reappraisal

In 1957, Rabourn and Quackenbush proposed a scheme of carotogenesis which included most of the carotenoid hydrocarbons known in

Nature at that time. Phytoene, phytofluene, and "-carotene were represented by the structures they had proposed on the basis of their degradative and analytical work (described earlier); neurosporene was represented by a structure which had, apparently, been derived from a consideration of its relationship to C-carotene.1°I° Now that these structures have been shown to be correct, the original scheme will be discussed. The scheme itself is outlined overleaf. It should be noted that the structures of ?j-carotene (isolated from the berries of Lonicera japonica by Goodwin39) and C-carotene (isolated from a marine diatom,

Navicula torquatum ) are by no means proven.1'3 It has been shown, however, that 6-carotene, whose structure has been in doubt for some years, is as 124 shown.

All the steps involved in Rabourn and Quackenbush's scheme fall into one of three types - dehydrogenation, ring closure, and double bond migration (the isomerisation of an a-ionone to a p-ionone end-group). All the ring closure steps in their scheme involved cyclisation of the "more saturated" type of end group (XLI) [to give (nu)], rather than of the lycopene type (XLIII)[which would give (XLIV)1 The latter reaction would not be favoured thermodynamically3'86 since it involves a reduction in the length of the conjugated double bond system [cf. the criticism of the suggestion that lycopene cyclises to s-carotene during biosynthesis (p. 32

However, a biosynthetic schemeinvolving this type of cyclisation has been proposed by Winterstein (and is discussed on p.111).159

alb ••• ••• MO. •110 • Figures (x1.1)-Nuiv) are on p.104. 106

PHYTOENE

PHYTOFLUENE

C.-CAROTENE

l/RoSPORENE N.‘ LYCOPENE

tX- CAROTENE

/3-CAROTENE

T-CRROTENE

"A Unified Concept of Carotene Photosynthesis" (Rabourn and 'xiackenbush, 1957 1000, ). 107

It is obvious that several more carotenes could be fitted into

Rabourn and Quackenbush's scheme since, for each chromophore, one can postulate a series of isomers differing only in the type of end group. With three types of end group available (acyclic, a-ionone, and p-ionone) and assuming that the two end groups on a molecule could be either the same or different, it is apparent that up to six isomers could occur. These would all have identical visible absorption spectra, except in those cases where isomerisation of the end-group from the a-ionone to the p-ionone type would bring the main chromophore into conjugation; in these cases the spectrum would be displaced towards the red end of the spectrum (by ca. 8 or) with a simultaneous loss of fine structure.

A multitude of uncharacterised carotenes has been reported over the years. Some of these are almost certainly identical with an already known compound (or one of its stereoisomers); also many of the unidentified carotenes are probably the same substance occurring in different environments. However, there are many "vacancies" in the biosynthetic pathways which might be filled by some of these, as yet uncharacterised, carotenes. The elucidation of the structures of these substances is often hampered by their occurring in only trace amounts. However, n.m.r. spectroscopy would be of value in this respect since relatively little sample is required (ca. 5 mg. should be sufficient for a simple hydrocarbon).

The fact that these substances occur in very small quantities might be because they are by-products of the pathways leading to the major carotenoids or because they are rapidly and efficiently converted into the next member 108 of the series, and therefore do not accumulate. Some of these unoharacterised carotenes are as follows. (The zeacarotenes and the "pink contaminant" which both absorb near 450 , 425, and 400 mp. (corresponding to eight double bonds in conjugation) are discussed elsewhere: see pp.65, 67].

Spectra, when quoted, are for hexane or light petroleum solutions.

Two pigments (93" and "C") with absorption maxima at positions 54a similar to those of y-carotene were found in tomatoes by Trombly and Porter.

Pigments with absorption maxima near 470, 440, and 415 haVe been found in tomatoes ("Pigment A"54a), and carrot oi1!33 A pigment

("P 450"), whose spectrum suggests the presence of a nonaene chromophore in conjugation with a ring double bond, has been found in DPA-grown cells of 81 66- Rhodospirillum rubrum. Mackinney's "Pigment I" might be neurosporene.

Two groups of workers have found an unidentified pigment (called 81 "P 412", maxima at 438, 412, and 388 ny) in DPA-grown cells of 81 81a Rhodospirillum rubrum. '

Mackinney's "Pigment II" had absorption maxima at positions near 66 149 to those of C-carotene, but was not that compound. Turian and Hex° have found what they claim to be a new carotene (" 0-carotene") in

Neurospora crassa; it had properties similar to, but not identical witht those of 7--carotene. Nakayama148 has since detected a pigment with similar properties in some mutants of the bacterium Rhodopseudomonas spheroides.

Nakayama134 found two fluorescent substances in two species of red yeast (Rhodotorula peneaus and R. Flutinie) with maxima at 408, 385, 109 and 365 ar (suggestive of hexamechromophores conjugated with ring double bonds).

A polyene which probably has a hexaene chromophore has been 135 found in Valencia oranges.

A substance with an absorption spectrum almost identical with that of phytofluene but which was too strongly adsorbed to be trans- phytofluene, was found in tomatoes by Trombly and Porter.54a Minor fluorescent zones with absorption spectra identical to the spectrum of phytofluene were noted by Petraceck and Zechmeister27 in tomato pastes.

Goodwin39 suggested that the fluorescent substance which he found in the berries of Lonicera japonica might be the cyclic analogue of phytofluene; this claim has yet to be substantiated.

Two recently characterised carotenes which could easily be 124 fitted into the Rabourn and Quackenbush scheme are the a- and p-zeacarotenes. The structures of these two compounds have already been discussed (cf.p.68).

a-Zeacarotene (XXVIII) could be formed from C-carotene as shown overleaf. The conversion of a- into p-zeacarotene is a straightforward isomerisation; a-zeacarotene might also give 6-carotene (a reaction that can be performed in vitro by treatment with N-bromosuccinimide). 124

(3-Zeacarotene (XXVI) might then be transformed either to v-carotene (by dehydrogenation - a reaction that has also been performed in vitro124) or to a-carotene and then (3-carotene. If this route to (3-carotene (with a-, y-, and 6-carotenes, as by-products or intermediates) does operate in Nature, it provides an alternative to the route, suggested by Rabourn and Quackenbush, 110

C-carotene (VII)

"Unknown B"

71 -carotene a-zeacarotene(F)- (cfp.106) (XXIX)

8-carotene (Jcnna) -zeacarotene (?) (XXVI I)

y -carotene @fp%) "Unknown C"

a-carotene

p-carotene

A hypothetical scheme of carotogenesis involving the zeacarotenes. 111 which involves Y1- and E-carotenes as intermediates. This might be part 1 of the reason why p-carotene is so widespread in Nature whilst the alleged intermediates appear comparatively rarely: p-carotene could be the end product of several biosynthetic pathways of the types discussed above originating from one of the more saturated polyenes (in the two routes discussed here, '—carotene), so that there is a high probability that at least one of these routes is operating in any given plant.

Wintersteini" has suggested a rather different scheme to explain the biosynthesis of the zeacarotenes. His scheme starts from neurosporene and includes the thermodynamically unfavourable type of cyclisation discussed above. When his paper was published (1960) the biosynthetic schemes contained therein could, perhaps, have been criticised on this count. 160 However, since then Decker and Uehleke have claimed to have demonstrated the conversion of lycopene into n-carotene in isolated chioroplasts. They reported that radioactive carbon from biosynthetically labelled lycopene was incorporated into p-carotene; they claimed that this did not involve 0 the re-synthesis of p-carotene from primary breakdown products of lycopene.

(They also reported, incidentally, that u-carotene was converted into lycopene in tomato parenchymatous tissue). This does not mean that the main route to n-carotene in all natural materials goes necessarily through lycopene. However, it does appear that the main objection to the original

Porter-Lincoln scheme (i.e. that p-carotene is most unlikely to be formed from lycopene: see p.32) has been removed.*

However, the recent results of Davies175 (obtained during a study of carotogenesis in a variety of the fungus Rhizophlyctis rosea) suggest that 112

159 161 Winterstein and Isler et al. have postulated a

comprehensive scheme of carotenoid biosynthesis based on the Porter-Lincoln

idea. They have extended the original scheme to its logical conclusion by

including the following two steps after the lycopene stage:

lycopene----monodehydrolycopene---bisdehydrolycopene

The conversion of lycopene into bisdehydrolyoopene in vitro by

treatment with N-bromosuccinimide was reported several years ago by Karrer 162 and Rutschmann; monodehydrolycopene (XLV) has recently been prepared in 163 a similar way but using milder conditions.

Both the mono- and the bis-dehydro-compounds have recently been 163 isolated from natural sources for the first time by Winterstein et al.

The bisdehydro-compound was (probably) isolated (in trace amounts) from

Valencia blood-oranges; the authors stated that it was identical with a

sample prepared synthetically (cf. above). Monodehydrolycopene was

isolated from an (unspecified) micro-organism, and is reportedto have agreed

in adsorption and absorption properties with a sample prepared synthetically

from lycopene (as above). The same organism also yielded two other

carotenoids with fully unsaturated end groups; these were torulene (XLVI)

and a C polyene aldehyde (XLVII). The aldehyde was identified by 40 comparison with a synthetic sample. Torulene was first isolated nearly

thirty years ago by Lederer164 from a yeast Rhodotorula rubra, and ever

[footnote, continued from previous page] y-carotene is no& formed from lycopene by ring closure, at least not in

this organism; it seems likely that these two oigments (which constitute

Woof the total carotenoids in mature cultures of this organism) are

biosynthesised through separate pathways. 113

(XLV), monodehydrolycopene

(XLVI), torulene

(XLVII). 3.,4''clehydro-17'-oxo-y-carotene

1 since has been assumed to be a methoxylated carotenoid. However, Isler 128 et al. have recently synthesised the hydrocarbon represented by structure

(XLVI) above and found it to be identical in all respects with natural torulene. The acidic pigment torularhodin, which accompanies torulene

(and other carotenes) in the yeast R. rubra, is now known to be the acid 165 corresponding to the aldehyde (XLVII).

Thus, in all, five carotenoids containing fully unsaturated end- groups have now been discovered, and so compounds containing this feature can no longer be considered a rarity in Nature. Torulene might arise by sequential desaturation of p-zeacarotene (XXVII)or, now that there is no 114 longer the strong objection to invoking the cyclisation of lycopene type end groups (XLIII), it might arise by ring closure of monodehydrolycopene

(XLV); in that torulene accompanies the latter pigment in Winterstein's 163 "micro-organism", this latter possibility is, perhaps, the more likely of the two. The polyene aldehyde (XLVII) and the corresponding acid, torularhodin, presumably arise from enzymic oxidation of one of the isopropylidene methyl groups in torulene.

82 Jensen, Cohen-Bazire, and Stanier have recently reconsidered the results which they (see p.26) and other workers had obtained during studies of the carotenoid content of the purple bacteria, in the light of recent work on the chemical structures of many of the participating polyenes.

Thus the structures of phytoene, phytofluene, '—carotene, and neurosporene are now known with certainty following the work of Jackman, Siddons, Weedon, N 92 and the present author (cf. the Experimental section). The structure of 166167 spirilloxanthin has been revised and is now known to be as follows:

Meo

Spirilloxanthin 82 The "hydroxy-lycopene" of Jensen et al. (cf. p.26 ) has been shown to be identical with rhodopin [probably, (XLVIII)] and not lycoxanthin (the N 99,168 169 3-hydroxy analogue of rhodopin). The structures of P 481, its hydroxy-derivative,170 and "hydroxy-spirilloxanthin"167 have been largely established and are, probably, represented by (XLIX), (L), and (LI), 115

82 82 respectively. The doubt associated with the position of the

chromophore in P 481 and its hydroxy derivative could be resolved by a

study of their n.m.r. spectra.

Ho

MeO

OH

Me0

So far, only those bacteria (for example, Rhodoepirillum rubrum,

discussed on p. 26) which synthesise the lycopene, P 481, and spirilloxanthin

groups of pigments have been discussed; all these pigments taken together 82 have been called the Group A pigments. Other species of purple bacteria

(for example, Rhodopseudomonas spheroidee) are known, however, which do not

produce these pigments but instead synthesise two pigments ("Y" and "R"; 139,140 the latter is now known as spheroidenone) and their hydroxy-derivatives; 116

82 these four carotenoids are known as the Group B pigments and the

relative amounts of each which are formed depend on the culture conditions

used; in particular the presence of oxygen promotes the formation of

pigment R and its hydroxy-derivative at the expense of pigment Y and its

hydroxy derivative.82,139,140

It has been shown that both RhodosQirillum rubrum (which

synthesises group A pigments) and Rhodopseudomonas epheroides (which

synthesises group B) contain, in addition to the true carotenoids, traces of 81 171 the more saturatedpolyenes. ' In addition, a green mutant of the

latter organism, which is totally blocked in the synthesis of pigments Y

and R, accumulates neurosporene and a hydroxy-neurosporene known as

chloroxanthin.53 The structures of pigment Y (LII), pigment R

(spheroidenone) (LIII), and of chloroxanthin (LIV) have recently been

established.140,172 This has enabled Jensen, Cohen-Bazire, and Stanier82 to put forward a comprehensive scheme of darotogenesis which covers most of

the polyenes (15 in number) present in the two types of purple bacteria.

They have suggested that in both types, phytoene is converted into

Me 0

(L11) 117 \./NN ' HO

neurosporene by sequential desaturation as in the scheme of Rabourn and 18 quackenbush mentioned above (cf. Porter and Lincoln ) and that hereafter the pathways diverge. On the one hand, dehydrogenation could lead to lycopene and the other Group A pigments; on the other, hydration of one of the isopropylidene groups could lead to chloroxanthin and the Group B pigments. It is interesting to note that, earlier, Jensen et al.81 were somewhat sceptical of the supposition that phytoene is one of the carotene 82 precursors. In this more recent paper, however, they considered it to be the basic C precursor; they made no reference to "lycopersene". The 40 transformations involved in their scheme fall into five categories (the full scheme is given in ref. 82) :-

1. Dehydrogenation, 18 (cf. Porter and Lincoln )

OH 2. Dehydrogenation,

3. Hydration

OH 4. Methylation >a/t/

OR OR 5. Introduction of (RAH orMe) a keto-group 0 118

81 As mentioned before (see p.114), Jensen et al. have found, in

DPA-grown cells of Rhodospirillum rubrum, several substances which appeared

to be hydroxy-derivatives of the more saturated polyenes. These were

"hydroxy-phytofluene", "mono-" and "dihydroxy-r-carotenes", and "hydroxy-

neurosporene".

Substances which resembled "hydroxy-phytofluene" and "monohydroxy-

C-carotene" had previously been observed in DPA-grown cells of Chromatium98 81a and R. rubrum, respectively. A substance resembling "hydroxy-phytofluene"

was found (in very small quantities) in tomato paste by Zechmeister and

Pinckard;173 it was called "phytofluenol" and was probably the first example

to be discovered of a hydroxy derivative of a more saturated polyene. The

"hydroxy-neurosporene" might have been identical with the "hydroxy-

neurosporene" (chloroxanthin) which was postulated in Jensen's scheme (above)

as an intermediate in the biosynthesis of the Group B pigments. The light yellow, hypophanic substance which Grob51 observed after exposing a culture

of Neurospora crassa"wild Au to light for a short period might also be a

hydroxyneurosporene. E ny44 detected what appeared 'to be a hydroxy-tetraene

in the leaves of Heves. brasiliensis.

Indeed, these hydroxy-polyenes might be rather more widespread in

Nature than is thought, since, by using the chromatographic conditions

normally employed to separate the hydrocarbons, the hydroxy-derivatives would

remain tightly adsorbed to the top of the column along with polyene

decomposition products. However, it should be noted that Zechmeister and 173 Pinckard were unable to detect "phytofluenol" in a variety of natural iia materials other than tomato paste.

81' Jensen et al. 82 were unable to define the role of the hydroxy-derivatives in carotogenesis. In general, changes in the concentration of the hydroxy-derivatives ran roughly parallel with those in the parent hydrocarbons.

The problem of elucidating the mechanism by which oxygenated carotenoids are formed in Nature has received less attention than carotene biosynthesis, and in this review has been purposely omitted (with the exception of the work of Jensen et al., above) as it is not within the scope of the work performed. However, it is of interest to note a recent report that hydroxy-carotenoids appear to be synthesised direct from 160 carotenes in tomatoes.

It is fitting to conclude this review with some recent remarks made by Quackenbush, [who, with Rabourn, put forward the carotogenetic scheme discussed earlier (p.105)]. 174He and Krzeminski have studied the incorporation of a variety of labelled substrates into the carotenoids of

Neurospora crassa. Mevalonic acid and six other substrates were incorporated, to varying degrees, into all the C polyenes present. From 40 the results they obtained, Krzeminski and Quackenbush174 concluded that the substrates were converted into intermediates of relatively high

molecular weight, and that each of the polyenes was synthesised from the intermediates (or, the intermediate) by a pathway "largely independent of the others".

So it would appear that theories of carotogenesis are still 120 very much in a state of flux. It is interesting to note that there have now been one or more reports of the occurrence in natural, carotenoid- containing materials of hydrocarbons with two, four, six, and eight conjugated double bonds (cf. the previous discussion). It is tempting to suggest that these compounds form a series of carotene precursors analogous to the (alleged) Porter-Lincoln series in some types of environment. 121_

EXPERIMENTAL

In the work desoribed in this section special precautions were

taken to free solvents from traces of acid and non-volatile impurities, using methods similar to those of other workers in this field.20 '40.96

"Light petroleum" refers to the fraction of b.p. 60-800, "pentane" to "AnalaR" grade petroleum spirit of b.p. 30-400, and "hexane" to commercial n-hexane. "Pure hexane" implies n-hexane freed from aromatic

materials by allowing the solvent to percolate down through a column of 176 freshly roasted (at 200°) silica gel. The eluate from the column was

monitored spectrophotometrically at 210 mr_ and only that an absorbance wit111 of lees than 0.30 (using 1 cm. cells) against distilled water in the reference beam was collected. The silica gel could be reactivated and used repeatedly; 1 kilo of either fresh or recovered silica gel was sufficient to treat 2 - 2-i- litres of n-hexane. The use of this specially purified solvent was essential in the search for compounds containing isolated double bonds and for conjugated dienes and was desirable in work involving trienes.

The four solvents mentioned above, and also benzene (which was sulphur free), were all distilled from potassium hydroxide pellets through ia short column containing a glass wool plugl before use. The ether used in the lithium aluminium hydride reductions was boiled under reflux with lithium aluminium hydride or calcium hydridel and then distilled; the acetone was i 2 2

boiled with zinc wool, or a mixture of sodium sulphite and sodium

carbonate, and then distilled. Methanol was freshly distilled. '

Apparatus was rinsed out with aqueous sodium hydrogen carbonate,

water, and, finally, redistilled solvent before use. The work was

performed in a very dim light (and, whenever possiblet in an atmosphere of

pure, dry nitrogen) in the hope that the naturally occurring stereoisomer of zeta(Z)-carotene might be isolated. Solvents were evaporated from a water

bath at 30-40° (ca. 20° for cis compounds) under reduced pressure, normally

using a nitrogen bleed.

Visible and ultraviolet light absorption spectra are quoted for 96 hexane solutions. Literature values for phytoene,0 phytofluene, and 8 J -carotene are("all -trans"isomers): max. 295, 286, 278; 367, .3.AL 332; and 1355 400, 378 my., respectively. The various cis-isomers of .7 -carotene 8 absorb at up-to 12 my. shorter wavelengths than the all-trans form.

Extinction coefficients used to calculate the polyene content of various 49. 96 000 for phytoene; t: 348=89,000 for phytofluene; solutions were: i%286"46' and E =138,000 for (Y-carotene.8 400 The Extraction of Chlorella vulgaris, mutant G.77.

The algae were grown and freeze-dried by Dr. Allen in America o and were stored at -10 for 10 weeks before extraction.

The green, fibrous material (50 g.) was crushed to a powder and 1.23

steeped in a mixture of pentane (120 ml.) and methanol (120 ml.) at 25° for 24 hr. The mixture was then filtered, the solid on the filter being repeatedly washed with pentane. The solid was re-extracted for 24 hr. with pentane-methanol and then twice more using pentane only (120 ml. portions for 3 hr. periods). By then, the solid no longer fluoresced on exposure to ultraviolet light and extraction was assumed to be complete. The combined filtrates were diluted with an equal volume of water and the aqueous methanol layer removed. The pentane solution was dried (Na SO ) 2 4 ' filtered, and concentrated (to 150 ml.). The residual dark green solution was added to 20% methanolic potassium hydroxide solution (200 ml.) and left at 25° for 15 hr. during which time all the green colour (chlorophyll) was transferred to the methanol layer leaving the pentane solution a pale yellow-brown. The mixture was diluted with water (300 ml.) and the two were layers/separated. The pentane solution was washed with water until free of alkali, and then dried (Na2SO4), and filtered. Spectral analysis of this solution (N, . 446, 423, 399, 377,(360),(355),(294), 284,273,(232) mlx ) indicated the presence of -carotene (ca. 6.5 mg.), phytoene

(ca. 36 mg.), traces ofplvtofluene, and an unwanted pigment with an absorption band near 446 my. (and, presumably, a further band near 425 my_ masked by the t -carotene) with an intensity of ca. 36% of the 423 m)A. band.

The emulsions which formed at the interface during the processing described above were extracted with pure hexane and the extracts were combined 124

with the main solution. The aqueous methanolic wash liquors separated, on standing, into two layers the upper one of which contained further quantities of c! -carotene(2.5 mg.) and phytoene (11 mg.). The upper layer was separated off, and the liquors extracted with pure hexane (2 x 80 ml.); the combined extracts were carefully washed with water, dried (Na2SO4),. filtered, and combined with the main solution. This solution was concentrated (to,20 ml.) whereupon a white, waxy solid separated out. The mixture was cooled to 00 and the solid (38 mg., m.p. (K) 128-1400) was filtered off.

On subjecting the filtrate to chromatography on alumina (IV,

20 x 2 cm.) from pure hexane, the phytoene, phytofluenel and -carotene were quickly eluted in that order, though separation of the zones was not complete. The chromatogram was followed by spectral monitoring of the eluate and by the characteristic intense green fluorescence exhibited by the phytofluene on exposure to ultraviolet light. Five coloured zones

remained on the column and were (from top to bottom of the column) red,

pale yellow, orange-red, pale pink, and pink. They were eluted with 5,%

acetone in petrol but were not studied further. The JP-carotene zone

consisted of a mixture of cis and trans isomers and also ca. 30% of the

near 448 mtL). Concentration of the unwanted pink pigment (X max. 41 -carotene zone and rechromatography on alumina (IV, 20 x 2 cm.) from y hexane removed traces of phytoene and phitofluens but failed to remove the 1.25

pink contaminant. Absorption spectra taken on the eluate (i) at the beginning, (ii) about half way through, (iii) towards the end, and (iv) at the end of the elution of the Y-carotene zone had the following maxima: (i) 416, 221, 374 My-(similar to Rabourn's "neon-carotene C"8); (ii)4 17, 221, 374 myk(as above); (iii)421, 396.5, 376 my.; (iv)414, 399, 378'5 1191(presumablynall-transqloarotens).

An additional peak (due to the pink contaminant) near 446 my. was present in each of the four spectra and was especially noticeable in the first part (i) of the zone. Since the object of this work was, essentially, to demonstrate the absence of any methyl groups attached to saturated carbon in j-carotene, it was decided to run the n.m.r. spectrum on the crude ,J -carotene without attempting to remove the pink contaminant. The whole of the j-carotene band from the chromatogram was concentrated to a small volume, and then, transferred (in hexane) to a small tube suspended in a flask. The solvent was removed by evacuating, first to 10 mm. for 30 min., and then to 103 mm. for 10 hr. The bulk of the residual orange-red oil was far greater than would be expected for the 3 mg. or so of polyene estimated (spectrally) to be present. The n.m.r. spectrum (which showed an anomalous strong peak at 8.75 p.p.m.) confirmed the presence of colourless impurities (probably lipids)in the sample. The sample was 126

recovered and treated with lithium aluminium hydride (3 g.) in refluxing ether (80 ml.) for 1 hr. to convert any lipids present to compounds 20 strongly adsorbed by alumina. The excess hydride was decomposed with ethyl acetate, and the solvents were evaporated. The yellow residue was repeatedly extracted with hexane until the extract was colourless. The hexane solution was washed with water (6 x 100 ml.), dried (Na2SO4), filtered, concentrated to small volume and chromatographed on alumina

(IV, 20 x 2 cm.) from hexane. This gave one broad, yellow band which was collected (X. . 420, 396, 375 Rik, indicating it to be mainly cis isomer(s) of Jr -carotene). Spectral estimation (assuming 6=110,000 for 8 the 400 my. band ) indicated the presence of Jr-carotene (1.9 mg.) and pink contaminant (ca. 0.6 mg.). The solvent was evaporated, the sample war dried as before, and the n.m.r. spectrum run (in chloroform). In the

8 - 8.5 p.p.m. region, the spectrum was almost identical with the spectrum of a sample of pure -carotene since obtained from carrot oil. However, an anomalous strong peak (near 8.7) was also present. The sample was

1622 (w), recovered and its infrared spectrum run (in chloroform): V.max. 1590 (w), 965 (s) cm.-1; there was also a strong band at 1718 cm.-1.

The attempt to obtain a pure specimen of Jr-carotene from this

Source was abandoned at this stage. Instead,attention was turned to the isolation of the phytoene present in the orignal extract.

All solutions from the various chromatograms already performed' 127

containing any phytoene or phytofluene were combined and evaporated to dryness. The residual oil was treated with lithium aluminium hydride

(0.5 g.) in ether (250 ml.). The mixture was boiled under reflux for 1 hr., allowed to cool, and the excess hydride was decomposed with ethyl acetate. (3)(80ml.), The solvent was evaporated, the grey residue was extracted with pure hexaneA

4) and the hexane solution was washed with water (6x 80 ml.), dried (Na2SO , and filtered. The filtrate was concentrated (to 10 ml.), and chromatographed on alumina (IV, 16x 2 cm.) from pure hexane. The mixture separated into the following components (detected, separated, and assayed, by spectral monitoring of the eluate) eluted in the following order:-

substance with A 230 lay, in only small amount and (a) max. contaminated with other substances showing weak absorption at

260 my and in the 280-340 my region;

(b) a band showing a faint blue fluorescence on exposure to

ultraviolet light, with 297, 286, 276 51; phytoene (20 mg.); max.

(c) a band showing an intense green fluorescence in ultraviolet light,

.24, 368 DT; phytofluene (ca. 0.7 mg.). max. 332,

The phytoene solution [zone (b)1 was evaporated to dryness and the residual colourless oil was dried at 103 mm. for

12 hr. The sample was dissolved in carbon tetrachloride and its n.m.r. spectrum determined: T 8.41, 8.33, 8.25, 8.03 (relative 128

intensities 3:1:1:1.5) with a broad (methylene absorption) band from

8 to 7.78 p.p.m. A trace of oxidation product was just detectable but only when operating the instrument at high sensitivity; it gave a small "blip" on the base line near 8.7 p.p.m. The sample was recovered and its ultraviolet absorption spectrum shown to,be unchanged. Infrared absorption spectrum: V max. (in carbon tetrachloride solution) 1662 (w., VI isolated C=C), 1631 (m., conjugated C="1C) cm.-1. Soon aftelards a sample of phytoene extracted from carrot oil was kindly sent to us by Drs. Rabourn and Quackenbush. The n.m.r. spectrum of their sample (see p.137) was identical with that obtained on the phytoene from Chlorella.

The Extraction of Carrot 011(1129) 142 The carrot oil was obtained from the same source as that of 8 Rabourn and Quackenbush and the extraction procedure based on their work '2° Again an attempt was made to isolate the natural isomer(s) of Ir-carotene 143 but it has since been discovered that during processing the carrots are heated to 80-100° so that any native, thermolabile isomer will stereomutate under these conditions to the equilibrium mixture of stereoisomers.

Carrot oil (50 g.) was dissolved in pure hexane (100 ml.). 129

Spectral analysis of the solution ( 1 . 276, 285'5, 296; 332.5, 349,

368.5; 400.5, 423; 442, 471 my.) showed the presence of phytoene (ca. 1.1 g.), phytofluene (ca. 500 mg.), Y-carotene (ca. 350 mg.), and a substance

absorbing near 440, 470 my (ca. 350 mg.; probably mainly xanthophylls since only a little survived the partitioning procedures described below). The

solution was shaken with a solution of potassium hydroxide (40 g.) in

methanol (150 ml.) and water (40 ml.) at 40° for 10 hr.s then at 27° for 60 hr.

Aqueous potassium hydroxide (35%; 60 ml.) and pure hexane (400 ml.) were

added and the mixture was shaken well. This gave an emulsion which showed

no signs of separating even after standing for 24 hr. Water (100 ml.) was

added and the mixture was left 2 days during which time it partially was separated into 2 layers. The aqueous phase was tapped off, the hexane liver/ *V, keptl and the voluminous emulsion between these two layers was repeatedly

extracted wit}, pure hexane until the extract was colourless. The combined

hexane solutions were washed with 90% aqueous methanol (1 x 200 ml.) and

then water (0-51.) was added. The water precipitated out a white solid from

the hexane layer; the solid was dissolved by adding methanol (150 ml.).

The mixture was then diluted with water (2.5 1.) and left 3 days, during

which time the emulsion cleared.

The hexane layer was separated, and washed to neutrality with

water (2 x 2 1.). The hexane solution was partially emulsified and so was

filtered through batches of sodium sulphate to remove the water causing the 130

emulsion. Much desiccant was required and, despite repeated washing of it after use(with pure hexane)t it retained a yellow colour and a relatively large amount of polyene was lost in the process. The hexane solution was concentrated (to ca. 40 ml.), and washed with 90% aqueous methanol (3 x 20m1.) and then water (3 x 200 ml.). The water-washing caused emulsions to form at the interface. They were combined and repeatedly extracted with pure hexane as before, the hexane extracts being then combined, washed cautiously with water, dried (Na SO ), and combined with the main solution. 2 4 The hexane solution was evaporated to dryness, and the residue

Was treated with lithium aluminium hydride (1 g.) in refluxing ether

(200 ml.) for 1 hr.; fresh reagent (ca. 4 g.) in ether (50 ml.) was added and the mixture refluxed 1 hr., allowed to cool, and the excess hydride was decomposed with acetone (40 ml.). The solvents were evaporated leaving a grey sludge to which pure hexane (200 ml.) was added. The use of a large excess of hydride to ensure the complete removal of lipids had the disadvantage of causing a very bulky precipitate on decomposition with acetone. The filtration of this mixture took several hours and although the residue was repeatedly washed with pure hexane it seems likely that further quantities of material were lost at this stage. (Decantation, rather than filtration, would have been a better technique to use at this stage).

The filtrate was concentrated to 50 ml.l and the solution was subjected to chromatography on an alumina column (IV, 25 x 3.5 cm.) which i 3 1

had first been washed free of traces of materials absorbing near 220 mr, with pure hexane. Seven fractions were collected. Spectral monitoring of the eluate showed these to contain:-

(I) substance(s) showing strong absorption near 205 myLfollowed by a substance absorbing near 268 mj and finally a mixture of substances:

max. near 285, 2265 312, 327 miL;

(II) mainly phytoene:A 275, 287, 297 IT; Max. (III)phytoene, followed, and overlapped by, phytofluene: . 332, 348, 367 mil); {Amax (IV)some phytofluene, but mainly cis forms ofc-carotene of

A max. 417-8, 394-5,373 intt ; (V) a mixture of r-carotene isomers:A from 419, 394'5, 373 to MaX . 422.5, 291i., 377.5 (cis peaks at 296, 285; E42211296==7.4)71/4; (VI)a mixture of 1-carotene isomers (10 mg.) : A . 422•5, 398, 377.5 to 414, Afli, 378'5 mr;

(VII)all-trans-f-caroteneA . 424'59 400, 379 T.

The last four fractions all contained1 in addition traces of a near 440 and 470 but this was present in contaminant.( max. my.) relatively small amount, especially in (VI) where it amounted to 2-3% of the i-carotene. Only faint yellow and pink zones remained on the column. The various fractions were treated as follows. 132

Fraction (VI). . 423, 392., 378, 296, and 285 T. 8 A consideration of the data given by Rabourn and Quackenbush for the positions and relative intensities of the peaks near 425 and 400 my and of the cis peaks at 296 my in the 4 stereoisomers of r-carotene they isolated suggests that (VI)Ivasa mixture of the "all-trans" and neo —A forms (ca. 1:1).

The solvent was evaporated, the residual dark yellow oil was dried at 105mm. for 6 hr., the sample (10 mg.) was dissolved in carbon tetrachloride, and the n.m.r. spectrum was run: 7 8.42, 8.36, 8.20, 8.095, 8.06 p.p.m.; relative intensities, 4:2:2:2:ca.1; in addition, there was broad (methylene) absorption in the 8.0 to 7.75 region. Peaks due to olefinic protons were observed at 774.95 (broad, protons attached to non-conjugated acyclic C==C bonds115) and at 4'06, 3.81, and 3.62 (all three due to protons attached to conjugated C==C bonds115). Relative areas were 3.8:10.2 (protons attached to non-conjugated, and to conjugated, C==C bonds, respectively). The proposed formula for r -carotene requires 4:10. The sample was recovered, weighed, and its infrared spectrum was determined (in chlorofrom): ))max. 1629 (w., conj. C==C), 1586 (w), and 968 cm:1 (6495; trans-CH=CH-); no absorption due to oxygenated functions was discernible. The spectrum was similar to that of lycopene (with 11 conjugated double bonds): .9 .1626 (w), 1595 (w), 1553 (w), and 967 cm:1(060). If Samples of all-trans-S-carotene and neoZ-carotene-A were isolated stereochemically pure and free from the pink contaminant in the following way.

It was first shown that a mixture ofr-carotene stereoisomers (from fractions (V) and (VII)] could be separated on alumina (II) using benzene-light petroleum mixtures as solvent.

Fractione(VI) and (VII) were combined and rechromatographed on alumina (II, 20 x 2.5 cm.). Development with light petroleum eluted 133

a small amount (ca. 0.2 mg.) of a spectrally homogeneous yellow zone,

max. A45, 418, (398) 9µ (relative E values: 1.0, 0.64, 0.25); this was not studied further. The eluant was changed to benzene-light petroleum (1:9). This eluted three yellow zones with the following absorption maxima:-

zone(VI+VII,1), X max. 413, 390, 368, my, neo-r-carotene-0 (lit., , mp), in very small amount; max. 41 3, 219., 370 zone(VI+VII 2), which overlapped zone(VI+VII,1), max. 424, 398-400, 379 my, neo-5-carotene-B (ca. 0.2 mg.) (lit.,8

MAX.423, 398, 378 my); Z011e(VI+VII,3), just separated from zone(VI+VII,2), MAX. 419, 396, 375, 358 (shoulder) my with cis peaks at 296, 265 T (E /E 296 419 =0.28); ne4-carotene-A (ca. 0.7 mg.) (lit.,6 A max. 419, 395, 374, (357), 296, 286 ny. E =0.31). 296419

Prolonged development of the column with the same solvent mixture finally eluted the "pink contaminant" and the"all-transtg-carotene; three fractions were collected:-

zone(VI+VII,4), pink contaminant, Xmax. 470, 441 pp (trace); zone(VI+VII,5), a mixture of the above and"all-transtr-carotene; ff zone(VI+VII,6), pure all-transtr-carotene. 134

Fraction (7I+VII,6) was evaporated and rechromatographed on alumina (II) using benzene-light petroleum (1:9) (to wash out any traces of stereomutation products formed during the collection and evaporation of the zone). Thefiall-transtrcarotene was eluted with benzene-light petroleum (1:3), and had PA . 424.5, 400.5, 379.5, 361 (shoulder) my (no cis peak near 296 mr.). A sample of syntheticuall-transcarotene (prepared by Mr. P. T. Siddons) had a qualitatively identical absorption spectrum. A mixed chromatogram of the two samples on alumina (II, 25 x 1 cm.) in benzene-light petroleum (1:9) showed no separation. However, the zones on this medium were broad so a mixed chromatoplate was also run. A calcium hydroxide-kieselgel plate was used with light petroleum as developer. No separation was apparent and on this medium the spots were reasonably small:

RF (mean of 4 values each), 0.46 (natural), 0.45 (synthetic), 0.46 (mixture).

Fractions (IV) and (V). These were combined (r-carotene content, 20 mg.) and chromatographed on alumina (II, 16 x 2.5 cm.) from benzene-light petroleum (1:9). This eluted:- zone (IV+V,1) phytofluene (5 mg.); zone (IV+V,2) crude neo-C-carotene-C (ca. 3.5 mg.), Amax. 416.5, 373 91, followed, and overlapped,by: zone (IV+V,3) neo-r-carotene-A (no neo-r-carotene-B was found in this chromatogram). The early part of this fraction was contaminated with fraction (IV+V,2); the eluate was

considered to be pure neo -A isomer (A max. 419, 6, 375 9µ) once the ratio E 396 409 (reported to be 2.04 for the 135

8 pure stereoisomer) attained a constant value of 2.02-

2.03. The latter part of the zone was contaminated with another pigment, .X max, near 475 and 445 ny, zone (IV+V,4) mall-trana-carotene (trace).

Zone (IV+V,3) (3 mg.) was combined with zone (VI+VII,3) from the previous chromatogram, and the solvent was evaporated. On chromatography on alumina (II, 6.5 x 2 cm.) from benzene-light petroleum (1:9)/ traoes of the neo-C isomer (eluted first) and also the pigment absorbing near 475 and 445 Imp (which remained on the column) were removed. The pure neoLcarotene- A solution was concentrated, filtered through a small pad of alumina (IV) to remove oxidation product (which was retained as a light, yellow zone on the top of the alumina), and the filtrateA reduced to small volume by blowing off the solvent with nitrogen. The residual oil was dried(3.5 hr. at

10-4 mm.), the sample (ca.3mg.) was dissolved in carbon tetrachloride/ and the n.m.r. spectrum of the solution determined; 17.. 8.41, 8.34 (shoulder), 8.19, 8.08, 8.05 (intensities, 4:2:2:2:ca.1 ), with broad (methylene) absorption in the 8 to 7.76 p.p.m. region. The spectrum was virtually identical with that obtained from the mixture ofuall-transuand neo-A isomers; the major difference being that, due to the small amount of sample (and consequent loss of sensitivity), resolution of the 8.34 peak (which was just attained before) was no longer obtained; instead it appeared as a shoulder on the side of the 8.41 peak; similarly,the 8.05, 8.08 peaks were barely resolved.

The sample was recovered and had absorption maxima at 418.5,

325, 374, 357 (shoulder), 296, and 285 5 (E395/E409, 2.03; E296/E4105,0.34) 136

thus showing it to be still pure neo-r-oarotene-A.

Fraction (III). This contained (by spectral assay) phytoene (ca. 90 mg.), phytofluene (ca. 100 mg.), and a substance (trace) absorbing near 310 91. On chromatography on alumina (II, 15 x 3.5 cm.), the constituents separated into:-

zone(III,1) phytoene (70 mg.), eluted first, in light petroleum; zone(III,2) a zone eluted with benzene-light petroleum (1:10), showing an intense green fluorescence on exposure to ultraviolet light;

zone(III,3) eluted with benzene-light petroleum (1:6), also fluorescent, max. 368, 14§, 332 14 phytofluene (probably the "all-trans" isomer).

Zone (III,1) was evaporated to small volume, the concentrate was filtered through a pad of alumina (II, 2.0 x 0.5 cm.) in hexane, and the filtrate was blown to dryness with a stream of nitrogen. The sample was dried (10-3mm.) to give phytoene (70 mg.) as an oil [slightly yellow due to a trace (ca. 2) of an impurity, 'Ximax. 330 117:1. The sample had the following spectral properties: .A 297, 286, and 276 91 (10-3 max. 1E 27.8, 41.2, and 32.4, respectively) [lit. (figures estimated from a diagram in -3 ref. 20),A 298, 286, and 275 IT (10 max. E ca.33, 46.3, and ca. 38, respectivelyth max. (in carbon tetrachloride solution) 1665 (E 25, isolated C:=0, 1631 (E 56, conjugated C:=C) cm.-1. [The infrared spectrum of a sample of phytoene isolated from fraction (II) was run as a liquid film:

Vmax. 1667 (w.), 1638 (m.), and 766 (s., cis)aH=TCHO cm.-1]; T(in carbon 137

tetrachloride solution) 8.42, 8.36 (shoulder), 8.26 p.p.m. with a broad

(methylene absorption) band from 8.1 to 7.78 from which peaks at 8.03 (strong), 7.92 (weak), and 7.86 (weak) p.p.m., stood out.

The n.m.r. spectrum of a sample of phytoene (also from carrot oil) kindly supplied by Drs. Rabourn and Quackenbush was also determined: 8.41, ca. 8.35 (shoulder), 8.25 (relative intensities, 3 : ca.l:1), with a broad (methylene absorption) band from 8.1 to 7.76 from which peaks at 8.03 (strong), 7.92 (weak), and 7.87 (weak), stood out; peaks due to olefinic protons appeared at 4.95 (broad, protons attached to non-conjugated n5 C=0 bonds), and at 3.95 and 3.78 (protons attached to conjugated C:=C bonds 175 )plim.Relative areas of the bands in the 8.5 - 7.5 p.p.m. region, the 4.95 p.p.m. band, and the two bands near ir3.9, were 53.5 : 6.5 : 4 required for the proposed formula for phytoene, 54 6 : 4.

Zone (I1I,2) was considered from its position on the chromatogram [below zone (III,3)1 to be a cis-phytofluene; this was confirmed by its ultraviolet absorption spectrum, which also suggested that it was the neo-B isomer rather than the neo-A : Amax. 367, 347.5, 331.5, (318), (304), 96, 257 ("211-Peek"), and 249 ny.(E347 F367 , 1.18) [lit. (for neophytofluene-B), i 257 mjp; 347 E367 1.16 (for neo-A, 3Nmax. 367, 347-8, 331, (318), (303), E 1.11)1. The solution was evaporated to dryness, and dried at 13 347/E366 = 10-4 mm., to leave the phytofluene as a yellow oil. The oil was dissolved in carbon tetrachloride, the solution was filtered through a pad of alumina

(II), and its n.m.r. spectrum was determined: q78.42, ca. 8.36 (shoulder),

8.22, 8.13 p.p.m. (relative intensities, 5:ca.2:2:1), with a broad (methylene absorption) band from 8.1 to 7.75 from which peaks at

138

8-06 (strong); 7.90 (weak), and 7.84 (weak), stood out; peaks due to olefinic protons appeared at 4.93 (broad), and 3.75 (showing multiplicity)154m11. Relative areas of the bands in the 8.5 - 7.5 p.p.m. region (due to protons attached to saturated carbon), the olefinic protons band at 4'93 p.p.m. (protons attached to non-conjugated C=C bonds115), and the olefinic protons 115 band at 3.75 p.p.m. (protons attached to conjugated C=C bonds ) were 50.7

: 5.2: 6.1; required for the proposed formula for phytofluene, 50:5:7.

The n.m.r. spectrum of a sample of phytofluene (also from

carrot oil) kindly supplied by Drs. Rabourn and Quackenbush was also

determined (in deutrochloroform) :1;8.41, ca. 8.35 (shoulder), 8.22, 8.10

p.p.m. (relative intensities. 5 : ca.2 : 2 : 1), with a broad (methylene absorption) band from 8.06 to 7.73 p.p.m. from which a strong peak at 8.01

stood out; peaks due to olefinic protons were present at 4.91 (broad), and

3.71 (showing multiplicity) p.p.m. Their sample was shown to be

predominantly a cis form (or forms): A max. 366, 347, 331 , (317), 257 ("cis- peak"), 248 51. (E3.4E . (in carbon disulphide solution) 41 366' 1.13); 1) 1678 (w., isolated C.C), 1621 (m., conjugated C.C), 960 (s., trans-CH.CH-), - 1 (E773/E960 2). and 773 (m., cis -C11.20H-) cm. , 0.3 Fraction (II). This was mainly phytoene (210 mg.), which was

purified by chromatography on alumina (II, 25 x 3.5 cm.) from hexane. This

gave, in order of elution:- zone(II,I), a mixture of two substances with absorption maxima at 298,

282, and 268 my; and at 326, 313, and 300 mp: evaporation

to dryness left an oil (ca. 15 mg.). Chromatography of 139

this oil from hexane on alumina (II, 4 x 20 cm.) failed

to separate the components which were perhaps cis forms

of the "triene" and "tetraene" (respectively) found in

fraction (I) (see below);

a substance ( A near 338, 356 71) in small amount, zone(II,2), max. followed, and overlapped, by -

zone(II,3), phytoene. Throughout the collection of this zone the phytoene contained traces of the substance absorbing near

338 and 356 9R. The proportion of this contaminating

impurity fell from an initial value of ca.1O% to a steady

2%. Only that phytoene containing less than 3% of this

impurity was kept. This solution, on evaporation, gave

a viscous oil (120 mg.; pale yellow due to the trace of

impurity) which was used for the infrared (liquid film)

determination mentioned previously.

Fraction (I). Chromatography of this fraction on alumina (II, 4 x 15 cm.) from nure hexane separated the mixture into 3 main components; the following fractions were collected, in order of elution:- (ca. 100 mg.); zone(I,1), a substance rapidly eluted, X max.206 my zone(I,2), a series of materials not separated from each other with absorption maxima: near 210 and 260 mp, near 210 and 230

and near 210 and 220 Iv; these were not investigated

further;

zone(I,3), a "triene" ( .295, 282, 268 my; 40 mg.) which was

not phytoene; 140

zone(I,4), a mixture of the triene and a "tetraene"; zone(I,5), the tetraene ( Xuax.326, al, 300 9;1; 3.4 mg.).

Zone(I,1) (= "P 206": cf. p.98) was evaporated to dryness. The residual colourless oil [ca. 100 mg., mol.wt. (in boiling benzene) 225], in carbon tetrachloride, was filtered through a pad of alumina (II, 2 x 1 cm.), and the filtrate was blown to dryness using a stream of nitrogen. The residual oil was dried at 104mm., and then had the following spectral properties: A . (in ethanol) 206 9µ; max max. (in carbon tetrachloride) 2857 (v.s.), 2806 (s.), 1528 (m.), 1451 (m.), 1371 (m.), 1241 (w.), 1208 (w.), 998 (w.), ana 974 (w.) cm:1; T (in carbon tetrachloride) 9.17, 9.08, 9.01, 8.74, 8.40, 8.34, 7.99 (shoulder), 7.95, 7.80, 7.71, and 7.32 (broad) p.p.m.; olefinic protons at T 5.30, 5.23, 5.12, 4.94, and 4.75 p.p.m. Relative areas (protons attached to saturated carbon: olefinic protons, respectively) were 15.2:1. (This spectrum is reproduced on page 97).

Similar determinations (using the same solvents as above) were 117 made on a sample of pure squalene: 208 IT (10 31 )1 max. 5 27); /)max.2874 (v.s.), 2827 (s.), 1657 (w., E40, c=c), 1439 (m.), 1374 (m.), 1146 (v.w.), 3 )(,,H...154) cm:1 1104 (w.), and 976 (v.w., possibly trans -C(CH (There was -1 containing the)C=0H2 group: no absorption near 890 cm., suggesting that the sample was free of isomers/ 154,157 see p. 57); 't 8-42, 8.35 p.p.m., (relative intensities, 3:1); there was, also, a broad (methylene absorption) band from 8.1 to 7.70 p.p.m. from [lit., T8 which a peak at 8.03 p.p.m. stood out. 116 .405, 8.335 p.p.m. (relative intensities, 3:1)].

All solutions containing zone(I,1) were combined and filtered 141

through a pad of alumina. The filtrate was evaporated to dryness, finally at 10 4 mm. The residual oil was dissolved in carbon tetrachloride, and stored for 8 weeks under nitrogen at 00. The solution was then concentrated (to 0.5 ml.), and filtered through a pad of alumina. The

filtrate was evaporated to dryness (finally at 10-3 mm. for 12 hr.) to leave a colourless oil which was analysed. Found: C, 87.6; H, 12.0,10; mol.wt. (in boiling benzene), 251; hydrogen no., 248. (Calc. for C18 H30: C,87.7; (1 .2D1 _ 12-3(A mol.wt., 246). • The sample 1.588) had the following spectral properties: 9 (in carbon tetrachloride) 2905 (v.s.), 2850 (s.), max. 1678 (v.w.), 1637 (v.w.), 1450 (m.), and 1377 (m.), cm:1 (further bands -1 appeared in the 1300-900 cm. region but all were very weak); T (in carbon tetrachloride) 9.18, 9.12, 8.76, 8.42,8-37,and 8.04 p.p.m. (see p•97)• The

hydrogenation product (from the analysis) was recovered, dried (10-3mm., 3.5 hr.), and dissolved in carbon tetrachloride: T9.15 (showing signs of multiplicity), 8.76 p.p.m. (relative areas, 1:1.9, respectively).

Zone(I,3), the "triene" fraction, was evaporated to dryness

(finally at 10-3mm. for 10 hr.). The residual, colourless oil was dissolved in carbon tetrachloride and- the solution was filtered through a pad of alumina

(II, 1 x 0.5 cm.). The filtrate was concentrated, by blowing off most of was the solvent with a nitrogen stream, and its n.m.r. spectrum4determined:1:9.19, 9.12, 9.01, 8.75, 8.41, 8.35, 8.04, and 7.98 (shoulder); olefinic protons

at 4.92 and 3.98 p.p.m. Relative areas 30:3:1 [for protons attached to saturated carbon: protons attached to non-conjugated C.0 (15, 4.92): protons

attached to conjugated C.0 (2-, 3.98), respectivelyj (This spectrum is

reproduced on page 97). The sample used for the n.m.r. determination was 142 diluted with carbon tetrachloride, and stored at 00 under nitrogen for 6 weeks. The solution was then filtered through a pad of alumina, in the usual way, to remove any oxidation products. The filtrate was evaporated to dryness (finally at 104mm. for 14 hr.) to give a colourless oil: V

(in carbon tetrachloride) 2917 (v.s.), 2852 (s.), 1668 (v.w., Elcm.0.20),

1449 (m.), 1379 (m.), 1108 (w.), 985 (w.), and 970 (w.) em7;the last two of these bands can perhaps be ascribed to trans -CH CH-154; 295, 282, Nmax. and 268 mR (Eicm.133, 143, and 117, respectively). The solution used for the latter determination (6 mg./100 ml., in pure hexane) didnnt fluoresce when exposed to ultraviolet light.

An attempt to determine the mol. wt. of the substance (in boiling benzene) was unsuccessful; the substance apparently decomposed under the conditions used.

o Zone(I,5), containing the "tetraene", was stored at 0 under nitrogen for 8 weeks. The solution was then evaporated to dryness. The residual yellow oil was dissolved in hexane and the solution was filtered through a pad of alumina in the usual way. Evaporation of the solvent

(finally at 103 mm. for 8 hr.) gave a colourless, viscous oil (3.0 mg.), X .327, 313, 300, and 286 (shoulder) mji (Eicm.726, 792, 633, and 530, respectively); the spectrum also contained a broad, weak band at 231 9R

(possibly caused by a diene present as an impurity). The solution

(concentration lmg./100 ml. pure hexane) exhibited a pale green fluorescence on exposure to ultraviolet light. Irradiation of the solution (40 w. lamp,

1 foot distant, for 5 min.) in the presence of a trace of iodine caused no change in the positions of the absorption maxima; however, some of the fine 143

values decreased (to 580, structure of the spectrum was lost, and the E1cm. 626, 536, and 484, respectively). The tetraene showed no tendency to gain in weight (by oxygen absorption) during weighing.

An attempt to separate a further quantity of this tetraene from zone(I,4) by repeated chromatography was unsuccessful. 144

Neurosporene

The material used in this work had been isolated from a green

mutant of Rhodopseudomonas spheroides by Dr. Nakayama in America; he had

already shown53 that it was probably identical with neurosporene from

Neurospora crassa35 (cf. p.58).

The solid (ca.15 mg.) was purified by chromatography on alumina

(IV, 20 x 2 cm.) from light petroleum. The major zone was bright yellow and very diffuse but it was well separated from a series of minor yellow, red,

and brown zones near the top of the column. No separation into stereoisomers

was apparent. It was eluted with benzene-light petroleum (1:9), and the

solvent was evaporated. The residual solid was rapidly chromatographed on

a short column of alumina(N, sx 1 cm.) from light petroleum. The major zone

was collected, the solvent was evaporated, and the residual red

microcrystalline (needles) solid was dried (0.05 mm. for 15 min.); m.p.

116-118° (K.) [lit.,53'35 m.p. 117°(corr.)(from R. spheroides);53 m.p. 124°

(corrN(from N. crassa)351: A .(lightpetroleum) 466'9, 437.9, 414, and

(395) m? [lit., .(light petroleum) 4.2, 437.5, and 413 "1;93 A . (light petroleum) 470, and 441.5 my35]; A .(benzene) 479, 449.5, 424, and (405) T.

The bulk of the neurosporene (after being dried as above) was was immediately dissolved in deuterochloroform and its n.m.r. spectrum/determined:

T8.40, 8.32, 8.19, and 8.05 p.p.m. (relative intensities, 3:2:2:3); two

further peaks (due to methylene absorption) were present, at 7.90 and 7.87

p.p.m. (barely resolved, relative intensity ca. 1.5). Further peaks, all 145

due to olefinic protons, occurred at 4.92 (broad; protons attached to non-conjugated C=C bonds) and at 4'17, 3'96, 3.66, 3.43, and ca. 3.28 p.p.m. (the last named was rather indistinct), of which that at 1C 3.66 was the most intense; these last five peaks were in a group and are due to protons attached-to conjugated C=C bonds. i 4 6

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The squalene, which was of natural origin and had been purified by repeated distillation. was kindly sulalied by Dr. Arigoni of Eidg. Technische Hochschule,Ziirich, Switzerland.

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125. Kargl and Quackenbush, Arch.Biochem.Biophys.. 1960, 88, 59; Kargl, Diss.Abs., 1960, 20, 2533. 126. Winterstein, Z.physiol.Chem., 1933, Eat 249. 152

127. Porter and Murphy, Arch.Biochem.Biophys., 1951, 12, 21.

128. Ruegg, Schwieter, Ryser, Schudel, and Isler, Helv.Chim.Acta, 1961, AA„ 994. 129. Petzold, Diss.Abs., 1959, 20, 62. 130. Nicholaides and Laves, J.Amer.Chem.Soc., 1954, 76, 2596.

131. Wright, Burton, and Berry, Arch.Biochem.Biophys., 1959, 82, 107. 132. Rowland, J.Amer.Chem.Soc., 1957, 12, 5007; see also refs. cited under ref. 131. 133. See Experimental Section,p. 153. 134, Nakayama: a personal communication to Mackinney, referred to in ref. 60b. 135. Nataradan and Mackinney, quoted in ref. 700. 136. Zechmeister, Fortschr.Chem.org.Naturstoffe, 1960, 18, 223. 137. Davis, unpublished observations.

138. See ref. 32 and refs. there cited.

139. Goodwin, Land and Sissins, Biochem.J., 1956, €4., 486.

140. Barber, Ph.D. Thesis, London, 1960. 141. Mr. P.T. Siddons, personal communication; cf. refs. 2 and 92.

142. Nutritional Research Associates, Inc., South Whitley, Indiana, U.S.A. 143. Details obtained from pamphlets issued by Nutritional Research Associates, Inc. (see ref. 142); and also a personal communication from Dr. Germann, President of N.R.A. 1 43 144. The common, field-grown carrot, Daucus carota, is used. 145. Isler, Gutmann, Lindlar, Montavon, Ruegg, Ryser, and Zeller. Helv.Chim.Acta, 1956, 121 463. 146. It is conventional to present n.m.r. spectra with the field increasing from left to right (see ref. 115, Preface).

147. Raxo, Fort.Chem.org.Naturstoffe, 1955, 12, 169 (p. 175). i 5 3

148. Nakayama, Arch.Biochem.Biophys., 1958, 12, 356. 149. Turian and Haxo, quoted by Haxo in ref. 147. 150. Mr. P.T. Siddons, private communication; Siddons and Weedon, forthcoming publication; see ref. 92 for a preliminary report on this work.

151. Karrer, Karanth, and Benz, Helv.Chim.Acta, 1949, 436. 152. Karrer, Karanth, and Renz„ Helv.Chim.Acta, 1949, 31, lo36. 153. Karrer and Benz, Helv.Chim.Acta, 1949, 32, 232; idem ibid., 1948, a. 1048. 154. Bellamy, "The Infrared Spectra of Complex Molecules", Methuen, London, 1958.

155. See the Experimental Section. 156. Gaylord, "Reduction with Complex Metal Hydrides", Interscience, New York, 1956. 157. Stedman, Swain, and Rusaniwskyj, J.Chromatog., 1960, Ao 252. 158. Dauben, Bradlow, Freeman, Kritchevsky, and Kirk, J.Amer.Chem.Soc., 1952, 1A0 4321. 159. Winterstein, Angew.Chem., 1960, ya, 902. 160. Decker and Uehleke, Z.physiol.Chem., 1961, 21, 61.

161. Isler, Ruegg, and Schudel, Chimia(Switz.), 1961, 1.2, 208.

162. Karrer and Rutschuann, Helv.Chim.Acta, 1945, EL 793. 163. Winterstein, Studer, and Ruegg, Ber., 1960, 231., 2951. 164. Lederer, quoted in ref. 1, p. 105. 165. Isler, Guex, Ruegg, Ryser, Saucy, Schwieter, Walter, and Winterstein, Helv.Chim.Aota, 1959, Aa, 864. 166. Barber, Jackman, and Weedon, Proc.Chem.Soc., 1959, 96; Jensen, Acta Chem.Scand., 1959, 11 381.

167. Jensen, Acta Chem.Scand., 1960, 953.

168. Jensen, Acta Chem.Scand., 1959, 12, 842. 154

169. Jensen, Acta Chem.Scand., 1961, EL, xxxx. 170. Jensen, Acta Chem.Scand., 1959, 2143.

171. Cohen-Bazire, unpublished observations, quoted in ref. 82. 172. Barber, Jackman, Weedon, and Yokoyama: quoted in ref. 92. 173. Zechmeister and Pinckard, Experientia, 1948, 4, 474. 174. Krzeminski and Quackenbush, Arch.Biochem.Biophys., 1960, 88, 287. 175. Davies, Biochem.J., 1961, 80, 48P. 176. Graff, O'Connor, and Skau, Ind.Eng.Chem.Anal., 1944, 1.6., 556. 155

SECTION II

THE SYNTHESIS OF ASTACENE 156

The Synthesis of Astacene

Astacene is a hirial- oxygenated polyene. 040 Until recently,'it \ -1-3 was assigned the tetraketone structure (I) although it was recognised

that under certain condition's two of the carbonyl groups (at c and C ) 3 31 1 might undergo enolisation. Recently, infrared spectral evidence has suggested that it exists essentially in the cnolised form (11).4'5 Although 0

(I) 0--

(II) astacene itself might not normally occur in Nature, its close relative 2 6 astaxanthin (III) is a widespread natural carotenoid.' Kuhn et al. reported that if a solution of astaxanthin was made alkaline and exposed to

014

HO 0 (III), astaxanthin oxygen the astaxanthin was rapidly and irreversibly converted into astacene

(cf. below.) Those investigators who reported isolating astacene from

Nature invariably incorporated a saponification step in their isolation 157 procedure. Unless special precautions were taken to excludenir from the saponified mixture until after it was acidified, any astaxanthin rrosent in the mixture would have been oxidised to sstacene and this is the pigment 2 which would be isolated. This led Goodwin to conclude that the astacene reported as being isolated from a wide variety of natural sources always originated from astaxanthin (or its esters). Perhaps one should not be too dogmatic on this point unless it can be shown that all reported sources of astacene did not contain any of this pigment itself. However, in the following brief review of the reported isolation of these pigments, the term

"astaxanthin" will be used with the understanding that in some reports astacene was the pigment actually isolated but that it may have been formed from astaxanthin during the isolation process. Samples of astaxanthin and astacene (especially if impure) would be difficult to differentiate since 2 their absorption spectra and melting points are rather similar.

Astaxanthin is the characteristic carotenoid of the marine animals. It was first isolated as a crystalline (although perhaps impure) solid in 1876 by Pouchet7 from the lobster. Subsequently, the red pigments of several other crustaceans were also investigated.8 The pigments were given various names (cf. Goodwin2 ) but were probably all essentially 2 astaxanthin. However, it was not until the period 1933-1938 that any real progress was made on the elucidation of the structure of astaxanthin.

In 1933, Kuhn and Lederer9 investigated the pigments of the eggs, shell, and skin of the lobster Astacus Bammarus (= Homarus pmmarus9). They found that the addition of acetone to the eggs decomposed the greenpiginent 158

6 (a chromoprotein, subsequently named "ovo'verdin") in them with the

simultaneous liberation of a red pigment which dissolved in the acetone.

The red pigment was obtained crystalline, and was found to be hypophasic

(towards 904 methanol"). It was called "ovoester" since treatment with

alkali apparently saponified it to give a different pigment, "astacene".

The (red) skin, or hypodermis, also yielded a red pigment when extracted 11 with acetone, but this pigment was epiphasic. Saponification of this

pigment also gave astacene. It appeared, therefore, that the two red

pigments were two different esters of astacene, the nature of the acid

fragment determining the solubility of the ester formed. Astacene was also

obtained from the brown chromoprotein of the lobsters' shells after treating

these with acid (to remove the protein) And then alkali (the saponification 1 \ step often incorporated in carotenoid extraction procedures ). Astacene

was obtained crystalline from all three sources; preliminary analytical

work suggested the formula C2 132039 (this was later proved to be incorrect:

see below).

32 "ovoester" was also obtained by Kuhn et al. in 1933 from the

eggs of another crustacean, Pfaja squinado. The properties of "ovoverdin",

end suggestions regarding the structure of this and similar chromoproteins, 112 6 have been reviewed by others. ' 6 In 1938, Kuhn and Sorensen showed that "ovoester" was not an

ester of astacene but was a new pigment, which they named "astaxanthin".

They showed that if a pyridine solution of astaxanthin was treated with lartanolic potassium hydroxide solution in the absence of air, then a deep 159

blue colour appeared in the solution. On exposure to oxygen, the solution became red with the simultaneous rapid absorption of two moles of oxygen

(per mole of astaxanthin); the product wan astacene. Prolonged shaking of the red solution in oxygen gave rise to only very slow oxygen absorption (of. p. 169).

The structure of astacene had meanwhile been elucidated by Karrer 12 13 and his co-workers, ' who were the first to recognise that astacene was 12a a C comoound (a low carbon analysis had led Kuhn9 to assign it the 40 12 formula C27113203: see above). Karrer et al. applied the classical l2b degradative and analytical techniques to astacene and its bisphenazine derivative." On the basis of this work they assigned the tetraketo- 13 6 structure (I) to astacene. Kuhn and Sorensen therefore concluded that astaxanthin must have the corresponding di-a-ketol structure (III). The alternative structure, 4,4'-dihydroxy-3,3'-diketo-P-carotene, was excluded on the grounds that the carbonyl group would be expected to be in conjugation with the polvene chain (in addition, a compound wit" this alternative structure would absorb at rather shorter wavelengths than astaxenthin is 2\ observed to do ).

The epiphasic red pigment isolated by Kuhn and Lederer9 from 13 lobster (see above) was, however, a true ester. Karrer et a1. investigated this compound (in 1935 before the relationship between "ovoester" and astacene had been discovered); they suggested that it was astacene dipalmitate. It is more likely, however, to have been the dipalmitate of astaxanthin. 160

4 Astaxanthin has since been found widely in marine animals, 1 2 16 1 16 especially in crustaceans, and echinoderms '2' (the starfishes, in 17-20). particular, have received considerable attention in recent years It occurred as a chromoprotein complex (similar to "ovoverdin"), esterified, or as the free pigment depending on the source: often it occurred in different forms in the different parts of the body of the same animal. The occurrence 1 2 16 16 of astaxanthin in fish has been reviewed by several authors; '' Fox concluded that the pigment was derived from the astaxanthin-containing crustaceans which constitute part of the diet of the fish.

Astaxanthin is not limited in occurrence to the marine kingdom. 21 22 The "euglenarhodone" isolated by Tischer ' from two species of algae has 15 23 has been shown ' to be identical with astaxanthin. Astaxanthin has also 24a'25 1,24b been found in some insects and in the feathers of birds; the striking pink colour of the ferthers of the flamingo has been shown to be 26 27 caused by astaxanthin. ' The occurrence of this pigment in bird feathers 28 has recently (1960) been reviewed by Volker.

The mechanism by which the highly oxygenated end-group of 24c astaxanthin is formed in Nature is, as yet, unknown. Goodwin concluded that it arose in animals by enzymic oxidation of ingested carotenoids; he pointed out that it is unlikely that animals synthesisecarotenoids from smaller molecular species although they must be able to convert one carotenoid to another (and also oxidatively degrade carotenoids).

One pigment system which comes to mind in connection with the biological oxidation of carotenoid end-groups is that encountered in, 161

29 for example, Rhodopseudomonas spheroides. Anaerobically cultured bacteria of this type are yellowish in colour and the major pigment present has been called "pigment Y". On admitting oxygen to the cultures, they rapidly become reddish-purple nnd this has been shown to be caused by the conversion of "pigment Y" into "pigment R" (spheroidenone). The change that occurs can be represented:-

(for R' , see p.116).

"Pigment Y" "Pigment R"

If astaxanthin was formed similarly, its immediate precursor would be zeexanthin (or its dimethyl ether). However, up until 1952 at least, had only been found in P very fer of the large number of marine invertebrates examined, whereas many of them had been 24d shown to contain astaxanthin. It should be added that many of these animals did contain , the closely related isomer of zeaxanthin.

30 Goodwin has reviewed his work on the carotenoids of some varieties of locust. He has shown that astaxanthin exists only in the skin of these insects whereas the other major carotencid present (P-carotene) was present in most parts of the body of the locust. Goodwin suggested that n-carotene was converted into astaxanthin in the skin. A study of the carotenoid content of the locusts' eggs during the incubation period strongly suggested that fl-carotene was being converted into astaxanthin there as well. Similarly, de Nicola31 suggested that p-carotene was being 162

converted into astaxanthin in the asteroid echinoderm Ophidiaster ophidianus; she postulated the following biosynthetic scheme:-

[c22. H26] 22 E1261

(IV), P-carotene (V), cryptoxanthin

[C*22 H G22 H263

HO OH (vi) (VII)

astaxanthin

De Nicola reported ihat the echinoderm eIntnired astaxanthin,

P-carotene, cryptoxanthin, and, in addition, two pigments (both, apparently, newly described) which had the properties which would be expected for compounds with structures (VI) and (VII), above. It should be emphasised that the structures assigned to these pigments were based only on adsorption and visible-absorption properties.

20 De Nicola has since studied another asteroid, Asterina panceri.

The shell of this animal was found to contain astaxanthin (as the major carotenoid), P-carotene,two pigments of unknown structure, and ; no cryptoxanthin was detected. It was suggested that the two pigments of unknown structure were the same as those [tentatively assigned structures

(VI) and (VII)] present in the asteroid (O. ophidianus) previously studied.

The chromatographic properties-of one of these pigments resemble those 25 reported for astaxanthin, as expected for a compound with structure (VII).

163

De Nicola suggested that astaxanthin was also formed from P.-carotene in 20 A. panceri; the route suggested was:

3-carotene --->echinenone--->(vI)--->(VII)-->astaxanthin

This is the same scheme as that postulated for the biosynthesis

of astaxanthin in 0. ophidinnus (see allove) excepting that the first

oxygenated carotenoid in the series was apparently echinenone (4-keto-P-

carotene; cf. above) in A. panceri rather than cryp"toxanthin (3-hydroxy-

.P-carotene) as in 0. ophidinnus. A carotenoid which may have had structure 20a (VI) was also found in the eggs of the echinoderm Peracentrotis lividus.

1a Goodwin and Jamikorn-3 have investigated the carotenoids of the

alga Haematococcus pluvialis. This alga produces cysts at its "resting

stage" and at the same tine its colour changes from green to orange or red.

This change in colour was shorn to be accompanied by the formation of large

amounts of astaxanthin. However, this pigment was apparently not formed

at the expense of the 3-carotene (or other carotenoids) already present in the

alga,nor is it likely to have been formed from phytoene or phytofluene (cf.

Section I) since neither of these compounds was present in detectable

quantities.

1 32 33 Its occurrence in the eggs of some crustaceans,' 9 ' insects 35 (cf. p.161 30), and fishes,34 led to the suggestion that astaxanthin 2 might be concerned with the reproductive cycle of these creatures. However,

it would be unwise to be more specific than this until more definite 1 2 evidence is available. Both Karrer And Goodwin have reviewed, briefly, 164

this aspect of the possible bioloical function of astaxanthin. More 36 recently, Massonet has reported that administration of astaxanthin to rats which had been starved of vitamin A, had a marked effect on their reproductive abilities. However, since the Pigment was administered dissolved in an oil containing c-tocopherol (as an antioxidant), it is difficult to ascertain how much of the observed effect was caused by the astaxanthin.

During the lost few veers, the suggestion has been made repeatedly that astaxanthin might act as a precursor for vitamin A in some creatures.

This suggestion was first put forward following investigations which showed that although the livers of some species of plankton-eating fish contained large quantities of vitamin A,1,37,38 the plankton themselves

(mainly small crustaceans) contained much too little p-carotene to account for the amount of vitamin present in the fishes.38 However, since the

plankton did contain relatively large amounts of astaxanthin, it was this suggested thatAcarotenoid was also being converted into vitamin A by the fishes.39-41 This hypothesis, which was strongly criticised by Goodwin,38 may no longer be necessary as it has since been shown that although some of

these crustaceans contain only small amounts of A-carotene, others (for 43, 44 example, the euphausiids42 end some of the other crustaceans, including

the lobster45) contain relatively large amounts of vitamin A itself. 42 Interestingly enough, the euphausiids were reported to contain no A-carotene and so it appeared that they did not acquire their vitamin A from that source 165

A2 However, the euphausiids did contain astaxanthin so it was suggested that the postulated conversion'of astaxanthin into vitamin A occurred not so much in the fish as in some of the crustacea (the euphausiids) that constitute part of the food of the fish.46

Despite this discovery, Grangaud and his colleagues have continued to carry out their experiments designed to demonstrate the conversion of astaxanthin into vitamin A in Gambusia hoibrooki (a mall fresh-water fish). 47 Grangaud's earlier work was reviewed in 1951. An example of some of the more recent work of Grangaud et el. is as follows. A large number of

G. holbrooki were fed on a diet which was free of vitamin A until the intestines of these fish could be shown to be entirely free of this vitamin.

The remainder were then given a food containing a large amount of astexanthin; samples of the fish were sacrificed et intervals, the intestines were collected, and a search was made for carotenoids therein. • It was reported that three hours after feeding the fish, their intestines contained 11-carotene and vitamin A1, and that subsequently they contained 48-50 vitamin P1 and, possibly, traces of vitamin A2 more recent work by 57 Grangaud et al. had shown that if any vitamin A2 was formed, it did not arise from vitamin Al). However, no mention was made of the purity of the astaxanthin at the time (although it has recently been claimed that it was 36 N entirely free from vitamin A and A-carotene ), and the a-carotene SFAS only identified by its visible-light absorption spectrum and a mixed chromatogram (the latter test is by no means infallible: cf. p.39)

However, it must be admitted that had the astaxanthin merely suffered 166

oxidative degradation to a compound with a similar absorption spectrum to that of p-carotene, this degradation product would have been expected to be much more strongly adsorbed than f!-carotene.

4 Grangaud, T:assonet, and their colleboretors 8-51,53 also investigated the effect of a mixture of astaxanthin end its esters [as 52 isolated from the shrimp Aristeomorphn folincea (- Penaeus foliaceus38 )1 on the condition of rats w'ich had been deprived of their normal supplies of vitamin A. They reported that the crude astaxanthin allayed some or all of the deficiency symptoms in the rats (vitamin-A deficiency in rats manifests itself in several ways, including the cessation of normal growth and the appearance of an eye disorder - xerophthelmia). However, Fisher et al.42 reported that astaxanthin (as a crude extract from the small crustacean

Thysanoessa raschii) was completely inactive as a precursor of vitamin A in the rat.

The following reports further illustrate the confue-ion that pervades some of this work on the alleged activity of astaxanthin as a 54a precursor for vitamin A. In 195C, 1assonet reported that a mixture of astaxanthin esters (isolated from the prawn Aristeus nntennatus) had insignificant growth-promoting prnperties but had marked anti-xerophthalmic activity in rats deprived of vitemin A. Yowever, in 1956 the same author reported that a mixture of astaxanthin esters derived from the same prawn promoted nearly normal growth in vitamin-A deficient rats (but failed to relieve some other symptoms of deficiency).54b

That astaxanthin might be a precursor for vitamin A in air 167

38 organism was considered unlikely by Goodwin since, as he pointed out, a

prerequisite for biological activity of this type in a carotenoid is generally 2 considered to be the presence of an unsubstituted P-ionone ring therein.*

This definition included those cerotenoids which contain a p-ionone type of end- group carrying a 5,6-epoxide ring, since there was evidence to suggest that the oxygen was sufficiently labile for these epoxides to be reduced in vivo

to the corresponding deoxygenated compounds with the necessary unsubstituted

end-group.55 It is much more difficult to conceive that the highly

oxygenated end-group of astaxanthin could be reduced to a P-ionone end-group

(although this is what the results of Grangaud et al.48-50 suggested). Since

the conversion of astaxanthin into vitamin A is so unlikely an occurrence, it

is not likely to be accepted as a fact unless it can be demonstrated that the

astaxanthin administered to the various animals was pure and that the substance detected was definitely vitamin A. It seems, in particular, that

the purity of the astaxanthin was somewhat dubious in some of the experiments mentioned.

Astacene is a deep purple crystalline solid which, until the

present report, had only been prepared by the alkali-catalysed autoxidetion of astaxanthin. It has been found to be only sparingly soluble in most of

the common organic solvents except pyridine, chloroform, and dioxen.9 The

number of suitable crystallising solvents is therefore limited and it has

In recent years. it has been shown that some aromatic analogues of 56 vitamin A show (mainly slight) growth-promoting properties: this does not detract from the point made above. 168 usually been crystallised from pyridine-water (as tiny, curved needles: for a photograph, cf. ref. 9);1'9 the present author has found a chloroform-ethanol mixture to be a more convenient solvent.

Astacene has been reported to be very stable towards atmospheric 1 oxygen - a property that has also been noted in the present work. However,

it should be added that the present author has observed that exposing

astacene to bright light causes the colour of the pigment to fade rather

rapidly under certain conditions (cf. p.234). It is noteworthy that Lrumm

and his co-workersr19 reported that the red pigment (described as astacene,

but probabl.i astaxanthin) of the prawn Palaemonetes vulgaris was rapidly

destroyed on exposing the animal, immersed in water, to bright light. ?he susceptibility of astacene (or astaxanthin) to atmospheric oxidation in the

lobster caused some difficulties to those concerned with the canninr, of this an.tal.zga l. ".pus,Thus, if freshly cooked lobsters were canned, and then Ietored for prolonged periods at low temperature, the highly pigmented tips of the

lobsters' claws gradually turned yellow. This discoloration subsequentle spread over much of the pi6mented surface of the animals. This effect

could be minimised by excluding air, and by the incorporation of antioxidants in the canned lobster. The yellow pigment was similar to that produced 66 by the action of hydrogen peroxide on astacene. It was apparent that the t he fats were oxidised first; x resulting peroxidesmay have been responsible for 66 the oxidative degradation of the astacene. Similar effects were found on storing shrimps67 and fish.98'68

The visible-light absorption spectrum of astacene has been shown 1 2 61 to consist of one broad band showing no fine structure whatsoever. ' '

169

This lack of fine structure is most unusual in the absorption spectrum of 1 3\ a carotenoid (which usually shows two or three distinct peaks ); the 61 probable reason for it has recently been outlined by Zechmeister. It

should he added that the spectrum of astaxanthin was originally reported

to show three peaks in pyridine solution, although the two subsidary peaks 6 were ill-defined. Goodwin and :risukh29'33 have since reported that they

were unable to detect any fine structure in the spectrum.

Astaxanthin can be distinguished from astacene most readily by

the way in which it reacts with alkali. This reaction has been used by 63 u 6r; several authors as a test for the presence of astaxanthin. Thus, Volker

showed that dissolution of astaxanthin in butanolic potassium hydroxide

solution in the absence of air gave rise to a deep blue solution which

i7Imediately turned red if oxygen was allowed access to it. As already

ontimed, Kuhn et al.6 have shown that this reaction involves the

Insorption of oxygen (two moles per mole of astaxanthin) and gives rise to

azt7.?ene. It has been suggested that the blue colour is caused by the

presence of a potassium salt of astaxanthin.1 '6 It should be added that the

present author has discovered that astacene itself reacts with oxygen in the

presence of a strong base; however, this is a very slow reaction as

compared with that mentioned for astaxanthin, above.

The acidic properties of astacene were first demonstrated by

Kuhn et al.9'32 who showed that on partitioning astacene between light

petroleum and methanol, the Pigment was more soluble in alkaline than in

neutral methanol (cf. ref. 1); in addition, the diacetate (which no longer 170 contains an enolic hydrogen) was shown not to have this preference for alkaline media.32 Partition tests have been used by some authors as the 59 basis of a characteristic test for astacene. Thus, Drumm and his co-workers tested for astacene in a solution of a mixture of pigments in methanolic alkali by taking a portion of the solution, covering it with light petroleum, and acidifying it with acetic acid. Under these conditions, any astacene present in the original solution (as its potassium salt) was precipitated at.the interface. Goodwin and Jamikorn3la used a modification cf this procedure. They diluted the alkaline solution with ether and then added water slowly until two layers formed. All the carotenoids present went into the ether layer except the astacene which was deposited as its potassium the salt at,(ether-aqueous ethanol interface; the deposit was a fluffy, brownish- red material. The present author used a similar test in the early stages of the present work to test for the presence of astacene in crude reaction products (see later).

Chromatographic tests have also shown that astacene can behave as an acid. Thus, it apparently forms a salt with alumina since it has been reported that the pigment cannot be eluted from this adsorbent even with one 1 29 of the strongest eluting solvents, pyridine. Goodwin and Srisukh, ' and 20 de Nicola have compared the chromatographic properties of astaxanthin with 25 those of astacene. Thus, Goodwin and Srisukh reported that astaxanthin could be separated from astacene by adsorbing a mixture of the two pigments on "weakened" (methanol-washed) alumina and developing the column first with ethanolic acetic acid (which eluted the astaxanthin) and then with ethanolic 20 potassium hydroxide (which eluted the astacene). De Nicola reported that the two pigments could be separated on pure calcium carbonate (cf. below); 171

she reported that astaxanthin was eluted with an ether-light petroleum (1:1) mixture whereas astacene required ethanol to elute it.

69 It has been pointed out that the calcium carbonate used as adsorbent for chromatographing astacene should be of "AnalaR" grade since the "reagent grade" material may contain small amounts of calcium oxide (or hydroxide) with which astacene would be expectrd to form a salt. The effect of chromatographing astacene on the two types of calcium carbonate has been briefly investigated by the present author. Adsorption of astacene on a column of reagent-grade calcium carbonate gave rise to a dark-red zone at the top of the column. Development of the column with benzene eluted some of the pigment down the column but instead of it being eluted as a well- defined zone, the pigment was merely spread out down the column as a broad, uniform, pink-red zone. Although the column was washed with benzene for a long time, none of the pigment could be eluted. Apparently, as the ascacene advanced down the column, it was being continually consumed(and converted irt/s a salt)by the traces of calcium oxide present in the calcium carbonate until no free pionent remained; the front of the advancing pigment zone would then stop moving down the column. However, astacene could be chromatographed successfully on a column of "AnalaR" calcium carbonate from a mixture (1:1) of benzene and light petroleum. The pigment could be eluted in the normal way although it should be added that it did give a rather broad zone. In general, a better system for the chromatography of astacene was found to be powdered sucrose using a benzene-light petroleum (1:4) mixture as solvent.

71 Karrer andJucker have listed several carotenoids of unknown structure which were reported to have properties reminiscent of astaxanthin 172

or astacene but which were apparently not identical with either of these

pigments. Strain72 detected two keto-carotenoids which were similar to,

but not identical with, astaxanthin in the alga Protosiphon botrYoides.

One of the new keto-carotenoids [(VII) on p.1621 described by de Nicola2031 probably had one end-group of the astaxanthin type. 74 Krinsky and Goldsmith73 have recently undertaken a reinvestigation of the carotenoids of the alga uglena gracilis. Three carotenoids [which were probably (zeaxanthin monoepoxide),\ 75p-carotene, and neoxanthin75) accounted for 99/, of the carotenoid present in the alga.

However; traces of six other carotenoids were also detected, including echinenone and two apparently-new carotenoids which were named euglenanone and hydroxy-echinenone. The visible-light absorption spectrum of euglenanone and of hydroxy-echinenone consisted of a single, broad absorption 61 oand,73 similar to that of astacene. The shape of these absorption

curves would make it difficult to determine the exact position of the absorption maximum in them. The values reported are made even more

uncertain by the fact that neither pigment was isolated in sufficient

quantity to obtain it in a pure, crystalline state. It is unwise, therefore, to attach too much significance to those suggestions regarding the structures of these compounds which were based on comparisons of the positions of their absorption maxima with the values reported by other workers for compounds with similar spectra. Thus, the fact that the absorption maximum for euglenanone was reported to fall at slightly shorter wavelengths than that of may or may not be of any structural significance. 173

Reduction of either euglenanone or hydroxy-echinenone with sodium

borohydride resulted in the formation of a product with an absorption spectrum like that of p-carotene, suggesting that in both pigments the

polyene chain was conjugated at one end, at least, with a 4-(or keto

group.73

Euglenanone was nearly equally distributed between hexane and

99% methanol. Hydroxy-echinenone was found to be hypophasic; also it was

markedly more soluble in alkaline aqueous methanol than in acidic aqueous

methanol, so demonstrating the acidic properties of the pigment (cf. the

behaviour of astacene, mentioned earlier). The acidic nature of hydroxy-

echinenone was further demonstrated by its chromatographic properties. Thus,

it could be eluted easily from calcium carbonate, but glacial acetic acid

was required to elute it from alumina, indicating that hydroxy-echinenone

was capable of forming a salt with alumina. Euglenanone, on the other

hand, was eluted fairly readily from alumina. A comparison of the

properties of these two pigments with those of other keto-carotenoids led

Krinsky and Goldsmith73 to suggest that euglenanone and hydroxy-echinenone

light have the following structures:-

(VIII),euglenanone (?)

HO (IX),hydroxy-echinenone (?) 174

As has already been mentioned, it seems likely that end-groups of the astacene type exist primarily in the enol form [cf. (II)], and compounds containing such an end-group would be expected to form a salt with basic adsorbents. Thus, it is immediately apparent that euglenanone

(which was reported to be eluted from alumina comparatively easily) cannot be represented by the structure (VIII) assigned to it by Krinsky and

Goldsmith.73 The possibility that euglenanone might be identical with the well-known carotenoid canthaxanthin (4,4'-diketo-p-carotene) was dismissed by Krinsky and Goldsmith mainly on the grounds that the proportion of euglenanone observed in the epiphase when this pigment was partitioned between hexane and 95; methanol was different from the value reported by

Zechmeister76 for canthaxanthin under similar conditions. However, it should be noted that the difference between the two values was insignificantly larger than the discrepancy between the value reported by

Ilrinsky nnd Goldsmith73 for a sample of cryptoxanthin (isolated from E. 76 aa-,• acilis) and the value reported for this pigment by Zechmeister.

It should be added that the chromatographic properties of

hydroxy-echinenone were not incompatible with the structure (IX) assigned to this pigment by Krinsky and Goldsmith: those authors reported that glacial acetic acid was required to elute hydroxy-echinenone from their "weakened" alumina; Goodwin reported that astaxanthin could be eluted from his variety of "weakened" alumina by a mixture of ethanol and acetic acid. The

possibility that hydroxy-echinenone might have the structure (VIII) assigned by Krinsky and Goldsmith to euglenanone has been discounted recently by Cooper and deedon.77 These workers have synthesised 1.75

3',4'-diketo-p-carotene (VIII), using a reaction first used in the carotenoid field by the present author in his synthesis of astacene (cf. below). Preliminary reports77 have suggested that neither euglenanone nor hydroxy-echinenone is identical with Cooper and 4eedon's synthetic polyene (VIII).

It would obviously be of considerable interest to compare euglenanone and hydroxy-echinenone with de Nicola's keto-carotenoids {cf. 20 compounds (VI) and (VII) mentioned earlier). Thus de Nicola's compound

"A (VII, or its autoxidation products)?] would appear to bear a close 6" [. resemblance to Krinsky and. Goldsmith's hydroxy-echinenone. It is noteworthy that Krinsky and Goldsmith found no astaxanthin or astacene associated with their hydroxy-echinenone in glena gracilis; however, they did report finding cryptoxanthin and, in small amount, echinenone (cf. de Nicola's • work mentioned earlier).

Until quite recen- ly, the isolation of a carotenoid with a snectrunshowing a single, broad absorption band with a maximum, in pyridine, near 495 mix was taken to be characteristic of astacene (or astaxanthin).

It would be interesting to know if any of the earlier workers in this field who reported detecting astacene in Nature had, instead, found one of the other natural carotenoids (mentioned above) now known with a spectrum similar to that of astacene.

Echinenone (X), canthaxanthin (XI), and astaxanthin (III) have all been positively identified as natural products. These carotenoids can be considered to be three members of a group of five polyenes which can be 176

imagined as being built up from the end-groups of '--carotene,

canthaxanthin, and astsxanthin. The remaining two members of this group

would be represented by structures (XII) and (Lx) (the latter is the

structure proposed for hydroxy-echinenone by Krinsky and Goldsmith).

Neither of these pigments has yet been positively identified in I:ature, nor

echinenone

O

O (X1), canthaxanthin

HO (m) 177 have they been synthesised in the laboratory.

20 31 The structures [(VI) and (VII)] assigned by de Nicola ' to her two keto-carotenoids are the 3-hydroxy derivatives of echinenone (X) and compound (IX), above.

It is noteworthy that the chromatographic nroperties of a pigment with structure (IX) might be expected to he similar to those of astaxanthin. It would differ from astaxanthin, however, in possessirg an unsubstituted p-ionone ring, and would, therefore be active as a orecursor 2 of vitamin A. It is tempting to suggest that the vitamin-A activity attributed to astaxanthin by r',1.7.,.ud r,t, el. (sos ePrlier ''.isousFinr„) due to the presence of the esters of this, as yet unidentified, pigment (I: in Crangaud's "mixture of astaxanthin esters".

The first routes envisaged for the unthesis of astacere utilised

building-up principle. The series of reactions involved the C1004C2O+C1 78 would have been similar to those used by Warren and "eedon in their synthesis of canthaxanthin (KI).

Warren and rifeedon first syntlecized the keto-acid (XIII). The

1:eto-.roue was then protccted by converting the compound into its etIy -1,:meLL;xy-derivati-.-e(X.IV); this was to prevent the keto group reacting with the methyl-lithium in the next stage, and also to nrevent it "activating" the methylene groups at :35 and C, [cf. (XV)] in the stage subsequent to that.

Treatment of the ethylenedioxy-derivative (XIV) with methyl-lithium gave the methyl ketone (XV) which condensed with dial (XVI) in the presence of ethanolic alkali to give the nonaenedione (XVII). Removal of the

178

(x Iv) (xv)

(XVI), crocetin dial 0 0

i'roteciing ethylenedioxy groups gave the corresponding tetraketone. The

methylene groups at C2 and C4 [cf. (XVII)] then had an adjacent keto group and under the influence of a base, therefore, either of these could give

rise to a carbanion.

On treatment of the tetraketone with methanolic alkali, it was, as would be expected, the methylene at C2 which condensed with the keto group at C [cf. XVII)] to give the corresponding cyclohexenone, 7 canthaxanthin (XI). [A similar series of reactions starting with the condensation of the ketone (XV) with 3-apo-8'-carotenal (XVIII) gave echinenone (X)].

(XVIII), p-apo-W-carotenal 179

The C 10 compound required as the end-group in a synthesis of astacene would be a derivative of the triketone (XIX) in which the 6-, and 1 4 ° (X IX)

0 perhaps also the 9-, keto groups were protected [it was thought that there may be no need to protect the 9-keto group: if this group was left unprotected the carbanion which would be formed at C in the base-catalysed 4 reaction with crocetin dial (XVI) would, it was expected, be prevented by steric hindrance effects from competing very effectively with the carbanion at C for the aldehyde groups of the crocetin dial). Base-catalysed 1 condensation of the derivative of the triketone (XIX) with crocetin dial

(XVI), followed by removal of the ketone protecting groups would give a hexaketone (XX); this, it was hoped, might furnish astacene (I,II) on 0

0 treatment with methanolic alkali.

The key intermediate in the synthesis of the derivative of the triketone (XIX) was the half-ester acid chloride (XXI). This was synthesissiusing the following series of reactions (cf. Warren and Weedon;79 the yields quoted are those obtained by the author; they are similar to those reported by Warren and Weedon):- 180

(i)KCN in aq.EtO[I (ii) (cH3) 2c=o + CH2CN-0O2Et ;) (CH ) C.C(CW-00 Et 3 2 2 (7M (76%)

Ye0H/H+, KOH/Me0H CO H 0, Me CO,jle 2 (66%) • (553/4)

CO H CO e C 0C1 2 2 M C 02H (VI)

The product (XXI) was stored in benzene solution; it was not distilled in case the heating caused it to disproportionate.

Meanwhile, the diethyl ester of monoethylmalonic acid was 80 prepared and it was converted (75';,) into the half-ester acid (XXIIa) using 81 the method of audinger and Bereza. The half-ester was added to a 82\ solution of freshly distilled dihydropyran (cf. L'ohniepp and Geller ) in 83 benzene containing a trace of phosphorous oxychloride or sulphuric acid.84

Tjn,:'er these conditions the tetrahydropyranyl ester (XXIIb) should have been 54 formed. A vigorous, exothermic reaction did occur as expected, but, because of the ease with which these esters pyrolyse,84 the product could not be distilled and characterised. The crude ester was obtained as a pale Ci o2R

COzEt (XXII) al R=H CO2Et C01E t CO2 Et CO2Et 6, R= xxiii) (xxrv) (xxv) (xxvi) 181 yellow, viscous oil. This was dissolved in benzene and the solution was 85 divided into two parts. Each solution was added to sodium powder and, as soon as the metal had dissolved, the two solutions were treated with redistilled isobutyryl chloride (as a model reaction) and the half-ester acid chloride (XXI), respectively. The products [which should have been

(XXIII) and (XXIV), respectively] were treated with acid and should have given p-ketoesters [(XXV) and (XXVI), respectively] (cf. ref. 84). However, neither of the crude products gave a colour with ferric chloride. The -1 model reaction gave one main product (b.p. 44 0/0.4mm..11): . 1724 cm. ).

This gave a 2,4-dinitrophenylhydrazone which showed no carbonyl absorption in the infrared, so ruling out the possibility that the expected p-ketoester

(XXV)had been formed initially but had condensed to a dehydroacetic acid derivative (XXVII) during distillation due to the presence of ti trace of alkali (which catalyses that type of reaction86). Analyses suggested that the product woo a diketane, C101-11c,04; the two remaining oxyger atom acid chloride appeared to 1,:e present as ether linkages. The tithe; 77.7) ,,ave a siJailer produr2t along with the half-ester of dim,,Q1y1succinic acid (formed by hydrolysis of the acid chloride (XXI) during the working-up Ilrocedure).

The reactions it was prcposed to carry oat with the 0-ketester

(XXVI)were fLliows. The ester was to 're converted into the 37 nhenylhydrazone (XXVIII) using the Japp-Klinoemann reaction. Hydrolysis of the ester and then troaimetit with methyl-lithium should then have given the methyl ketone (XXIX) which would have been condensed with crocet:in dial 182

(XVI). The phenylhydrazone residue in the product (XXX) was to be

of the 68 removed by oneptardard methods and the resulting hexaketone (XX)

CO. Me

N.NHPh N.NHPh

(xxviii)

treated with alkali to give, it was hoped, astacene (1,11). 183

An alternative approach which was considered was to convert

Warren and Weedon's half-ester acid chloride (XXI) into the ketoester

(XXXI),79 and then to oxidise this to the diketone (XXXII) with selenium 89 dioxide. The a-diketone system could have been protected by converting it into the quinoxaline derivative (XXXIII); this would then have been treated in the same way as the phenylhydrazone (XXVIII), above. However, the quinoxaline ring "protecting group" would have had to be removed at one of the final stages and a method of doing this would have had to be devised.

2 3 CO2Me > C 02 Me K CO2 Me

C;iN11/"- 0 (XXX II) (XXXIII)

It will be noted that both routes presuoposed that a satisfactory method of preventing the keto groups at C4 and C, from causing the respective

I-methylene groups [in (XXVIII) and (XXXIII), respectively] to condense with the crocetin dial (during the base-catalysed condensation of the dial with the end-group) was to convert the keto groups into derivatives containing the carbon-nitrogen double bond, in place of the carbon-oxygen bond. The present author carried out experiments designed to determine whether the dial would condense with methyl groups in a position neighbouring the carbon atom of a carbon-nitrogen double bond (cf. (XXXIV)]. Two compounds containing this feature were tested. These were 2,3-dimethylquinoxaline and 2-methylquinoline. The condensation of crocetin dial (XVI) with a 184 •

methyl ketone has usually been effected by treating a mixture of the dial

and a. large excess of the methyl ketone with 5% ethanolic or methanolic

potassium hydroxide solution, usually at room temperature.78,79,90,91

Similar conditions were applied to the two nitrogen—ous bases mentioned

above, the visible-light absorption spectrum of each mixture being determined

at intervals: the formation of a condensation product between the dial and

the methyl group of the base [cf. (XXXIV)] would be revealed in the

production of a shoulder on the long wavelength side of the crocetin dial

-N= C-CH3

(XXXIV) (xxxv)

absorption spectrum. No such additional absorption could be detected even

after keeping a mixture of the dial and either of the bases at 20° with

excess 5;,% methanolic potassium hydroxide for 7 days. However, a mixture

of 2,3-dimethylquinoxaline and the dial did react on maintaining the reaction

mixture near 70° for several hours. During this period, a small shoulder

(near 920 mil) appeared in the absorption spectrum of the reaction mixture;

the intensity of this shoulder increased as time went on, until after 6 hours

it was nearly half as intense as the main crocetin dial peak. At this stage

the reaction was "quenched" and the crude product was chromatographed.

Apart from recovered 2,3-dimethylquinoxaline and crocetin dial, there was

also obtained a trace of a rather tightly adsorbed material [maxima, in

benzene, at c49, 916, and(487) mp] and a rather larger amount of a substance

which had the absorption !maxima, in benzene, at 522, 488, and (464) IT] and 185

79 adsorption properties expected of a nonaene dione. It is suggested that this is the product (XXXV, above) of the condensation of diacetyl (slowly liberated from the quinoxaline) with the crocetin dial. An attempt to prove that this was so was made by trying to effect the condensation of diacetyl itself with crocetin dial. However, treatment of this mixture polymerisation with methanolic alkali merely caused the rapid/of the diacetyl; none of the required product was detected. On treating a mixture of diacetyl and the dial in ethanol with either pineridine92 or piperidine acetater no reaction occurred - although under the same conditions cinnamaldehyde was found to condense readily with diacetyl (cf. ref. 92).

In connection with these remarks concerning the condensation of methyl ketones with crocetin dial, it is noteworthy that commercial

"activated alumina" has been found to effect this condensation under certain conditions. Thus, it was found that if crocetin dial was adsorbed from benzene on to a column of alumina (II), and then the column was washed with acetone-benzene (1:100, then 1:90), then the material eluted was not crocetin dial but a compound with an absorption spectrum (maxima near 917 and 487 3k1 91 in benzene) almost identical to that of dial. It seems likely that the product was the nonaene dione (XXXVI). A condensation product obtained in tLis way should be reasonably pure since,as it is eluted from the top of the column (where the condensation occurs), it would be subjected to chromatographic purification. 186

90 Meanwhile, Akhtar and Weedon announced the first convenient

high-yield synthesis of canthaxanthin, and it seemed possible that astacene

might best be synthesised in good yield from readily available starting

materials by the oxidation of the c;:clohexenone end-groups of canthaxanthin.

The first unambiguous total synthesis of the naturally occurring

keto-carotenoid9°4 canthaxanthin was reported by 'Marren and Weedon7B in

1958, using the Ci...0+C20/*C10 building-up principle. The ketone (XXXVII ,

synthesised from a,a-dimethylglutaric acid) was condensed with crocotin

dial (XVI), the protecting ethylenedioxy groups were removed, and the

product (XXXVIII) on treatment with base underwent ring closure to give

canthaxanthin (XI). Soon afterwards, Isler et al.94 synthesised

(xxxviii)

canthaxanthin by an alternative route starting from p-ionone and based on

the C19+C2+C19 building-up principle. The oxygen was introduced at the

C4 positions by the allylic rearrangement of the dehydro-compound (XXXIX)

with acetic acid; this gave the diacetate (XL) which was saponified to the diol; this was then oxidised to dehydrocanthaxanthin.

OH (ma) 187

OAc

OAc (XL)

96 In 1956, Petracek and Zechmeister95 ' isolated canthaxanthin

from the complex mixture of oxy- and dehydro-p-carotenes they obtained on 97 treating n-carotene with N-bromosuccinimide in ethanol-containing chloroform

(this work first gave rise to the suggestion that canthaxanthin was 4,4'-

diketo-p-carotene). The yield of canthaxanthin obtained (l'A) has since been

slightly improved (to 3')) by Entschel and Karrer.98 It should be added

that on treatment of 13-carotene with N-bromosuccinimide in the absence of ethanol-free ethanol (for example, in/ chloroform or in carbon tetrachloride99) only

dehydro-p-carotenes were obtained.97 '99 The introduction of oxygen

apparently requires the presence of ethanol; the probable reason for this 100 has been discussed by ikhtar; some other alcohols can replace the

'ethanol.97

None of the above routes to canthaxanthin can be said to constitute

a convenient, high-yield synthesis of this carotenoid. The contribution of 90 Akhtar and Weedon was therefore, particularly valuable. These authors

prepared the C diketone (XLI) by the base-catalysed condensation of crocetin 30 dial (XVI) with isopropyl methyl ketone in 70(;_, yield. .The cyclohexenone 101 rings were formed by the use of Robinson's Mannich base method. Thus,

treatment of the C30 diketone (XLI) with the methiodide [(XLII), prepared

188

from propionyl chloride in three steps901 in ethanolic potassium ethoxide

gave (40)0 15,15'-dehydrocanthaxanthin [cf. (XI)] directly.

E t2Mets14...,

(XLIT) (XL I)

Work on the closely similar synthesis of 19,15'-dehydroechinenone

[cf. (x)]90,100 showed that ethyl vinyl ketone could be used in place of

the methiodide (XLII) in this last step with only a small decrease in the

yield; this suggests that astaxanthin (III) might be conveniently

synthesised using this route from the acetoxy-derivative (XLIII) of ethyl

vinyl ketone. A closely related compound (XLIVa) has been synthesised as 102,103 follows [the methoxy-analogue (XLIVb) was also prepared];- (1) Br2102 ' (ii) KOH ".1 KOAc/Et0H 103> Ac elyN Br Ac0 O 0 (XL3 I) (XLIVa)

The bromo-derivative (XLIVc) of methyl vinyl ketone has been

reported to be produced in good yield by brominating the ketone in cold

water and then steam distilling the intermediate. 104

tleO O (XLIVb)

Akhtar and Weedon's synthesis90 of canthaxanthin has made this 189 compound one, of the most readily available synthetic carotenoids. The efforts of the present author to effect the conversion of canthaxanthin into astacene will now be described.

Dehydrocanthaxanthin was first treated with a slight excess of

N-bromosuccinimide in commercial (ethanol-containing) chloroform at -200

Using the conditions described by Zechmeister95,97 in his synthesis of keto-p-carotenes by allylic oxidation of a-carotene (cf. above). The mixture was treated with N-ethylmorpholine,98 and was chromatographed on alumina (grade IV). Benzene eluted a series of yellow and orange zones; this left the uppermost layer of the column (where any dehydroastacene, if present, would have been) only a very pale yellow.

A sample of dehydrocanthaxanthin was then treated with selenium dioxide (one equivalent) in refluxing tetrahydrofuran for seven hours.

Chromatography of the product did this time give rise to a red zone held tightly to the top of the column. 'Phis was present in only small amount, however, and was not investigated further since a better method for the 109 oxidation of (some) ketones to a-diketones was simultaneously reported.

This reaction was discovered during work on the elucidation of 106 the structure of the bitter principle, limonin. Barton and Templeton showed that on shaking a t-butanol solution of limonin in oxygen in the presence of potassium t-butoxide, the cyclohexanone ring (XLV) in limonin was autoxidised to give the corresponding diosphenol (XLVI). Some closely related ketones reacted similary.106a In each case, 1 mol. of oxygen was absorbed; yields were usually in the range 40 to 60%. It appeared to be

190 advantageous to have the base present as a concentrated solution (ca. 1N) 106-100 and in a large excess.

It seems certain that this reaction proceeds through a hydroperoxide since in certain cases this was the product isolated. Thus 110 Bailey, Elks, and Barton have treated several 20-ketosteroids (XLVII) lacking substituents at positions 17 and 21 with oxygen under the above conditions and the products isolated were 17a-hydroperoxides (XLVIII). 21 20 0

17 HO O O (m) (XLVI) (XLVII) (XLVIII) 111 The mechanism of the reaction is probably as follows:- N / CH CH CH BASE _22_4 R' (But() But t H '`0=0 111 ,o HO +2 toe 0 oe O (XLIX) (L) a, R = H; b, R = alkyl a, R = H; b, R - alkyl

In compounds containing two hydrogens at C2 [cf. (XLIXa)1, the

hydroperoxide (La) decomposes (perhaps during the working-up process) to

the diosphenol (LI), possibly as follows:- ti N/ / CH CH

HO HOB O

(La) (LI)

Li! 0119 or Oil 191

In ketones containing only one hydrogen at C, [cf. (XLIXb)1, as in the 20-ketosteroids mentioned above, the final product is the hydroperoxide, as would be expected if the above mechanism is correct.

Optimal conditions for the production of a diosphenol from its ketone have yet to be reported in detail. In particular, it might be that bases other than potassium t-butoxide in t-butanol (which is one of the strongest bases known) could be used as the anionising medium. As yet, only preliminary investigations along these lines have been made; sodium t-butoxice and t-pentyloxide have both been used successfully in place of potassium 110 t-butoxide in one of the preparations mentioned above.

It should be added that previous to the above reports there had been several references in the literature to the susceptibility of ketones 111,112 to base-catalysed autoxidation. Usually, however, the hydroperoxide first formed underwent cleavage of one of the carbon-carbonyl bonds, and so a diosphenol was not produced and the reaction was of no practical value.

However, Howe and Mcquillin113 did show that two a,p-unsaturated ketones of the type (Lila) could be made to undergo base-catalysed autoxidation without the molecule undergoing simultaneous decomposition. With most basic catalysts tried, the product was the 6-hydroxy compound (LIIb), but, when sodium t-butoxide was used, the major product was an acidic compound which, it was intimated, was probably the vinylogous a-diketone (LIII). 9 0 .0 CHMe2

R fl"! (LII) (LIV),R= -CH2.CH2.CHM62 a, R = H; b, R OH a, R'. H; b, R' = 0.0H. 19.2

, 114 Stevens and ':Iright have reported that hexahydrocolupulone

(LIVa) undergoes autoxidation in neutral solution to give the hydroperoxide

(LIVO. A reaction apparently related to those described above is the recently reported base-catalysed autoxidation of a 1,4-diketone (LV) to the 115 corresponding conjugated enedione (LVI).

O O (1,v) (Iv') (Lvii)

At first, it seemed unlikely that the base-catalysed autoxidation reaction of Barton and Templeton could be of much value in the 1 carotenoid field since, as is well known, the polyene chains in these compounds are susceptible to oxidative degradation. Thus, it was thought that even if the cyclohexenone ring system (LVII) present in canthaxanthin

(XI) was amenable to base-catalysed autoxidation [to give (LVIII)], the expected simultaneous oxidative degradation of the polyene chain would compete too effectively with the formation of the diosphenol [(LVIII), astacene (II) in this case] for it to be isolated in reasonable yield. However, since the formation of astacene in on1:: a comparatively small yield would represent an improvement over the yield expected from other methods, it was decided to carry out a series of small scale oxygenation experiments on canthaxanthin.

It seemed unlikely that it would be poSsible to follow the oxygenation of canthaxanthin in the way that the oxygenation of other ketones

(cf. above) has been followed - that is by measuring the uptake of oxygen. 193

ileasurement of the absorption spectrum of the mixture too was expected to be of little value in following the reaction since the bathochromic shift (of ca. 12 my) expected if astacene was formed would probably be counteracted to a large extent by a hypsochremic shift due to the formation of degradation products absorbing at n:i•orter wavelengths than canthaxanthin. It was decided, therefore, to follow the reaction by periodically taking samples of the reaction mixture and subjecting them to partition between hexane and

900; methanol; the effect of further diluting the methanol layer would then be noted. It was apparent from reports in the literature (see previous discussion) that the behaviour of astacene in these tests would differ markedly from that of canthaxanthin.

A sample of canthaxanthin was mixed with a large (100-fold) excess of potassium t-butoxide in t-butanol solution, a little benzene was added (to dissolve some of the canthaxanthin), and the mixture was stirred in oxygen. Oxygen was absorbed; samples were per5odically withdrawn and were subjected to the partition test described above. It was shown that the starting material in the reaction (canthaxanthin) was essentially epiphasic to K., methanol although the methanol layer did contain some of the pigment as well; this, however, was quickly transferred to the hexane

(epiphase) on further dilution of the methanol with water. As the absorption of oxygen in the above experiment proceeded, it became apparent that the canthaxanthin was being replaced by a pigment with solubility properties similar to those of astacene. Thus, after an amount of oxygen considerably in excess of the two cols. theoretically required for the 194

formation of astacene had been absorbed, the reaction mixture was found to be entirely hypophasic to 9(DI methanol. In addition, on further diluting the methanol with water, none of the pigment was transferred to the hexane; instead, most of it was deposited at the interface as a brownish-red film of'material. The pale yellow colour which remained in the aqueous methanol layer was probably due to the presence of degradation products (for example, polyene acids would be soluble in the aqueous methanol since this would be weakly alkaline from the sample of strongly alkaline reaction mixture added to it). The brownish-red deposit mentioned above was isolated, washed free of traces of alkali, and dissolved in pyridine. It had the 1,2 same absorption spectrum as has been reported for naturally-derived astacene.

In addition, it was more strongly adsorbed on calcium carbonate than canthaxanthin, it could not be eluted from alumina with pure methanol, and it reacted with o-phenlenediamine to give a product which could be satisfactorily chromatogrephed on alumina. The above experiment was repeated and the product was obtained as an amorphous black solid by carefully diluting a concentrated pyridine solution of the crude reaction product with water (cf. Kuhn and Lederer9). The infrared spectrum was not 016 incompatible with the presence of a diosphendl end-group (LVIII) although at this stage the spectrum given by natural astacene was unknown,so a direct comparison was not possible.

The results obtained on following the reaction using partition tests had suggested that a considerable excess of oxygen had to be absorbed before all the canthaxanthin was consumed - the excess being used, presumably, in the 195 oxidative degradation of the pigments present. Had the exact excess of oxygen been known, the optimal yield of astacene could have been obtained; absorption of oxygen by the mixture after all the canthaxanthin had been consumed could only be expected to lower the yield of the astacene-like pigment. Unfortunately, it was found to be difficult to correlate partition behaviour with oxygen uptake on a small scale; each time a sample was withdrawn from the reaction mixture for partition tests the reaction flask subsequently had to be flushed nut with oxygen and on restarting the stirrer there was always a rapid, instantaneous uptake of oxygen. [At this stage it was thought that this might be because the rate-determining step was the conversion of the solid canthaxanthin into the soluble enolate ion; it has since been shown that this effect occurs even in the absence of canthaxanthin (see later discussion)].

Therefore, when the above small scale experiments were repeated on a large scale, the amount of oxygen that was allowed to be absorbed was the rather arbifary quantity of a fivefold excess over that theoretically required for the conversion of canthaxanthin into astacene. On this larger scale the reaction mixture was shaken since, even on a small scale, magnetic stirring of the mixture was not very efficient because of its viscosity.

This time the product was finally crystallised from chloroform-ethanol.

This time, it was shown without doubt that the product was identical with astacene. This identification was considerably strengthened 117a by the acquisition of a small sample of naturally-derived astacene with which the synthetic product could be compared directly. 196

The natural and synthetic products showed no depression of melting point on admixture, and they did not separate on a mixed chromatogram on sucrose (sucrose was found to be more satisfactory than "AnalaR" calcium carbonate for this purpose). In addition, their visible-light absorption spectra were qualitatively identical, and their n.m.r. spectra were virtually the sane (cf. below). Their infrared speCtra were quantitatively superimposable, and it was established (cf. below) that astacene exists ess, mtially in the diosphenol form (II) rather than the tetraketo-form (I).

Samples of the synthetic product reacted with o-phenylenediamine in hot acetic acid and with acetic anhydride in pyridine to give, respectively, a phenazine derivative and an acetate in the same way that natural astacene has been reported to do.15'32 On treatment of canthaxanthin with each of the two reagents mentioned under the same conditions, the canthaxanthin was recovered quantitatively. The n.m.r. spectra of the two derivatives showed the features which would be expected of tne bisphenazine ant diacetate derivatives of astacene. The phenazine derivative could be chromatographed satisfactorily on alUmina but the acetate could not; instead, the acetate behaved in the same way as astacene, being tightly adsorbed to the top of the column with even methanol failing to elute it. Presumably, the acetate which,being an enul acetate,wouid be expected to be very sensitive to acid, decomposed (to astacene) on the alumina, despite the fact that this adsorbent apparently gave an alkaline reaction to ethanolic phenolphthalein (cf. the 119b) similar behaviour of carotenoid epoxides The bisphenazine and diacetate derivatives of the naturally-derived astacene were prepared and strong evidence for the identity of their synthetic counterparts with them 197

was obtained.

The synthetic astacene was entirely hypophasic on distribution between hexane and a saturated solution of potassium hydroxide in 910 methanol. Even if the lower layer was diluted with water until only 5Z methanol, the pigment remained hypophasic. Further dilution of the methanol precipitated the pigment at the hexane-aqueous methanol interface as a brownish-red film of material (cf. earlier discussion).

Apparently-pure samples of synthetic astacene were submitted for

"direct oxygen" determinations on three occasions. All three results were within 0.6 of the value required for astacene. However, although several carbon and hydrogen analyses were made (including determinations on the same samples as used for the oxygen determinations), the carbon values were 62 always 1 to 4% low. Warren also found that oxygenated carotenoids frequently gave satisfactory oxygen analyses but gave low carbon values.

In the present case, it might be caused by the chelation of a trace of a foreign. material (for example, heavy metal ion) by the diosphenol grouping

[dehydroastacene (see later) gave the correct analytical figures].

The visible absorption spectra of synthetic astacene, its diacetate, and its bisphenazine derivative all consisted of a single broad absorption band with maximal absorption near 500 TT (in pyridine); as would at be expected, the bisphenazine derivative absorbed/slightly longer wavelengths and had a rather higher molar extinction coefficient (6) than either astacene or its diacetate.

The n.m.r. spectra of astacene, astacene diacetate, and the 198 bisphenazine derivative of astacene will now be discussed; each of the spectra was obtained with an entirely synthetic sample of the pigment. It should be added that the spectrum obtained on a sample of natural astacene was virtually identical with the spectrum given by a pure sample of synthetic astacene, the only difference being that the peak near 8.0 p.p.m. in the natural-astacene spectrum was slightly more incense relative to the other two peaks than in the synthetic-astacene spectrum. This small difference can be ascribed to the fact that the sample of naturally-derived astacene used had been recovered from other tests and there was too little to crystallise it more than once, and it was not entirely pure (it melted seven degrees lower than pure astacene). Since each of the three compounds whose spectrum is to be discussed is symmetrical about its central 1q,1 1 -carbon- carbon bond, one half of each molecule will be discussed in the following section.

Firstly, it should be noted that previous work on the n.m.r. spectra of carotenoids has shown that the introduction of a keto group at position 4 into a p-end group [as in n-carotene (cf. LIX)] has a marked 118 effect on the n,:n.r. spectrum of this end-group. This is illustrated in the following diagrams which show one half of p-carotene (LIX) and of canthaxanthin (LX), and the positions (in p.p.m.) at which the methyl protons in these molecules absorb. The values r iven are actually for carbon tetrachloride solutions of these carotenoids but in general the values obtained in carbon tetrachloride, chloroform, or deuterochloroform solution were identical within experimental error (± 0'025 p.p.m.), 118 199

8.98 8-98 $-05 8.05 8.81 8-81 9-04- 8.04

N NNN

8.31 8.21

(Lx)

The reasoning which has led to the assignments shown for

[3-carotene has been outlinedin other Sections of this thetis11 and 118 elsewhere. It has been shown that protons lying in conical regiont

extending above and below the plane of a carbonyl bnnd experience a positive

shielding effect. Elsewhere in the neighbourhood of the bond, a proton

experiences a de-shielding effect and this is most marked in the plane of

the carbonyl bond . A very similar effect occurs in the neighbourhood of 120 a carbon-carbon double bond (cf. below). The protons of the 5-methyl

group in canthaxanthin [cf. (LX)], which lie roughly in the plane of the

4-keto carbonyl bond, will, therefore, be de-shielded. A similar effect

has been observed in some types of acyclic polyenes. Thus, the "end-of-chain" 119c methyls in polyene esters and aldehydes of the following types [(LXI) 118 • and (LXII)] usually absorb at 0.1 to 0.29 p.p.m. lower fields than the 118 121 "end-of-chain" methyls in hydrocarbon polyenes (LXIII), '

T Me

C H, C H5 CH5 (LXI) (LXII) (LXIII)

The observed de-shielding of the gem-methyls in canthaxanthin

[cf. (LX)] can be ascribed to the long-range de-shielding effect12° of the 200

4-keto group.

The end-group of astacene [cf. (LXIV)] contains a second carbon- carbon double bond and this would be expected (cf. above) to cause further de-shielding of the three end-group methyls (none of these methyls lies in the region above the plane of any of the double bonds where protons experience positive shielding: cf. above). The n.m.r. spectrum of astacene contained peaks at 7.90, 7.98, and 8.70 p.p.m. with relative intensities of 1:2:2.

For the reason outlined above, the 8.70 p.p.m. peak is ascribed to the gem- 119c methyls. The "in-chain" methyl groups attached to a polyene chain have 110 previously been found to absorb near 0.0 p.p.m. If the peak at 7.98 p.p.m. is ascribed to the "in-chain" methyls in astacene, then the remaining peak in the spectrum (that at 7.90 p.p.m.) can only be due, to the 5-methyl group. The assignments for astacene would then be as in (LXIV). However, it is conceivable (though less likely) that the "in-chain" methyl at C 9 120 could experience a long-range de-shielding effect from one (or more) of the double bonds in the end-group and so give rise to the 7.90 p.p.m. peak

(cf. the spectrum of isorenieratone discussed on p.355); the 1.98 p.p.m. peak would then be ascribefi to the 9-methyl. Whichever is the case, it

(which is far removed from seems certain that the "in-chain" methyl at C13 any de-shielding effects exerted by the end-group) absorbs at 7.98 p.p.m., and also that the de-shielding received by the 5-methyl in the astacene end-group (LXIV) is markedly greater than that received in the canthaxanthin end-group (LX). A peak at 3.94 p.p.m. of intensity one sixth of the 8.70 201

p.p.m. peak has tentatively been ascribed to the olefinic proton at C2. A series of ill-defined peaks at 3.4 to 3-6 p.p.m. are ascribed. to the remaining olefinic protons: that is, those attached to the conjugated double bonds of the polyene chain (cf. ref. 120).

Me spectrum of astacene diacetate [cf. (LXV)] contained peaks at

7.71 ; 7.955, with a shoulder at 7.98; and 6-6.'", p.p.m., with relative intensities of 1 :2:ca.1 (shoulder):2. The 6.69 p.p.m. can only be ascribed to the gem-methyls which are apparently rather more de-shielded here than in astacene (possibly by the carbonyl group of the acetyl function). The 7.71 p.p.m. peak is ascribed to the acetate methyl group. This type of methyl 120 usually absorbs at somewhat higher fields (near 8.0 p.p.m. ); however, the methyl group under - discussion might be expected to be subjected to the de-shielding influence of one or more of the three double bonds in the end-group to which the acetyl function is attached. It appears that the

methyls attached to carbons 5,9, and 13 are, all de-shielded, fortuitously, to approximately the same extent.

8.65 845 ca..7 96 co— 7- 96 CH 3 CH3T CHH3 CH3 9

7.71 co.. 7- 96 CHJ.00.0 CH 3 (L XV)

202

vt. The spectrum of t'-,e 1:ispherne derivative of astacene [cf. (LXVI)] showed peaks at 6-08 40 7.61, 7.94, 7.9e, end 8.79 p.p.m., with relative intensities A 2:3;3:3:6. The 7.94, 7.98, and 8.79 p.p.m. peaks can only be assigned as

shown in the figure (LXVI) below; the "in-chain" methyl at 09 certainly

experiences a small (but distinct) de-shielding effect from the end-group

in this compound (cf. the suggestion regarding similar de-shielding in astacene,

above), although this might be caused by the aromatic rings in this, rather

special, case.

To determine the effect of the nitrogen ring system on the

resonant frequency ( /7-value) of the nearby protons in the phenazine end-

group [cf. (LXVI)], the n.m.r. spectrum of 2,3-dimethylquinoxaline.(LXVII)

was measured. The two methyls in this compound gave rise to a single, sharp

band at 7.37 P.P.m- Methyls attached to a carbon-carbon double bond p.p.m. 118,119a,121 (LXVIII) have previously been shown to absorb close to 8.4

The methylene protons in system (MIX) would be expected. to absorb near to 7.8 p.p.m.122 These values are illustrated below. It becomes apparent,

therefore, that the methylene protons attached to C2 in the bisphenazine

derivative [cf. (LXVI)] of astacene would be expected to absorb near 6.8

p.p.m. [cf.(LX.X)]. The assignment of the 6-88 p.p.m. band in the n.m.r.

_Me rhr7-4) , , / , CH ( CH ("t"----.8-4) CH (-r It- 7.8) cH (T^-- 6.8 p) -2 2 I 3 I 3 C CH /CH \ / ,,C\ / / C \ / N C —c c --C N C II 1 i 1 II N N

(Lxvii) (Lxviii) (mix) Lxx)

.•••• •••• * See footnote on p.22 7. 203

spectrum of the phenazine derivative :o the methylene protons at C2 [cf.

(LXVI)] is therefore justified on the grounds of both the observed intensity

and position of this band. A single sharp band is observed because there

are no protons on the neighbouring carbons with which the C2 methylene

protons can undergo spin-spin coupling (end-group methylene protons in

carotenoids usually give a broad absorption band which is essentially an

"envelope" of the many small closely-spaced bands caused by complex spin-spin 118\ interactions ).

The only band remaining unassigned in the 6 to 9 p.p.m. region of

the spectrum of the phenazine derivative is that at 7.61 p.p.m. which must

therefore be caused by the 5-methyl group. This group is formally of the

"in-chain" type now, but it is de-shielded even more than a normal "in-chain" 110\ methyl (T p.p.m. ) presumably because it is in the plane of the 123 nearby aromatic system.

( 4 See footnote on p. 227.) 204

-1 The 1700 to 1500 cm. region of the infrared spectrum of astacene showed bands (in chloroform solution) at 1686 (vw), 1612 (vs), and 1551 (m) cm:1 The 1686 cm:1 band was also observed in spectra determined in a potassium bromide disc, and was then of the same intensity relative to the other bands as in the solution spectrum. Both Sorensen4'31 and the present author have observed this band in the spectrum given by naturally-derived astacene. A similar band was observed in the spectra of dehydroastacene and astacene diacetate, but it was absent from the spectrum of the bisphenazine derivative of astacene. This suggests that it might be associated with the stretching of the C2- C3 double bond [cf. (LXXII), (UXIII)). However, this bond is part of a vinyl ether (or 124 117 acetate) system and an a&-unsaturated ketone system and would, -1 therefore, be expected to absorb in the 1610-1630 cm. region (and to be -1' • -1 obscured by the 1612 cm. band) rather than at 1686 cm.

If The spectrum reproduced in Sorensen's paper (1450-1800 cm. only) also showed a compani-on peak of similar intensity at slightly higher frequencies; this was not observed by the present author using pure astacene. 205

-1 The 1612 cm. band is ascribed to the conjugated carbonyl groups.

It was found to be markedly more intense (E 1200) than the corresponding

band in canthaxanthin (61,"":900)125 and occurred at 40 cm:1 lower frequency7.8

The reason for the frequency shift is probably composite. Thus, the

corresponding band in astacene diacetate [cf. (Lain)] occurred at only

12 cm. lower frequency than carthaxanthin (cf. (LXXI)) and this can 117 probably be ascribed to the normal effect of cross-conjugation (with the double bond) on the infrared frequency of the 4-keto group. A C2-C3 similar effect would be expected to operate in astacene itself but this -1 \ would account for only part of the observed shift (40 cm. ). The additional low-frequency shift in the astacene spectrum is probably due to

hydrogen bonding (LXXII) between the 4-keto group and the enolic hydroxyl 127 group. The occurrence of a relatively sharp hydroxyl band near 3410

CH3.00.0 } 0 (LXXIII) 1 cm. in the astacene spectrum also suggests the presence of an intramolecular

hydrogen bond [cf. (LXXII)], although it should be added that this band 128 would be expected to occur at rather higher frequencies than this.

Barton and Templeton's diosphenols [cf. (LXXIV)]106 showed hydroxyl 1 absorption near 3480 cm.-1 ; the diosphenol keto-group absorbed near 1690 cm.

- that is, at rather lower frequencies than would be expected for a normal 1 cyclohexenone. The 1551 cm. band in the astacene spectrum can be assigned 206

78 90 91 to the conjugated carbon-carbon double bonds. ' '

' 'C

11 o c) HO 0 (Lxxv) (LXXVI) a, R H; b, R = Nie

4 Sorensen has compared the diosphenol end-group in astacene 129 (LXXII) with the tropolone ring system (LXXVa); (the characteristic- 130,131 frequency range of the tropolone carbonyl group has been given as -1 \ 1605-1624 cm. ). He pointed out that the carbonyl frequency in both systems is unusually low and suggested that this might be characteristic of the cross-conjugated diosphenol keto-group (LXXVI). However, this analogy is 131 not a good one since Scott and Tarbell have reported that the carbonyl 1 stretching frequency in some tropolones falls near 1620 cm. whether or not there is any possibility of hydrogen bonding between the ketone and a • hydroxyl group [that is, in compounds of both type (LXXVa) and of type

(LXXV1)].

-1 The bands at 1063, 998, and 969 cm. in the astacene spectrum were also present in the spectra of dehydroastacene, astacene diacetate, and the bisphenazine derivative of astacene (although in the latter case the band 1 near 1063 cm. was rather weaker than in the other spectra). Peaks near

969 and 998 cm:1 are common in polyene spectra78'90 but a strong band near -1 1060 cm. is an unusual feature.

The spectrum of dehydroastacene was similar to that of astacene 207

except in that it contained, in addition, a very weak band at 2146 Oulti 90 1 (acetylene), minor bands near 1370 cm. , and an ill-defined peak on the

low frequency side of its 970 cm.` band. This last-mentioned feature is

characteristic of compounds which contain a poiyene chain incorporating an 90 -1 acetylene bond. Both spectra showed a strong band near 1249 cm. [which

has been tentatively assigned to the stretching vibration of the enolic

carbon-oxygen bond (cf. Scott and Tarbe11131)] and a medium-intensity band 1 117 near 785 cm. caused by the gem-methyls.

Once it was known that astacene was indeed being produced by the

autoxidation of canthaxanthin, efforts were made to determine the conditions

necessary to give the best yield of astacene. As was mentioned earlier,

oxygen was nhsorbed by the oxidative degradation reactions as well as by

the autoxidation process so that it was not possible to follow the usual

practice of just allowing the theoretical quantity of oxygen to be

absorbed. Instead it was necessary to determine at what stage all the

canthaxanthin had been consumed; it was thought that at this stage the yield of astacene would be optimal and that further oxidation would merely

destroy some of the astacene (cf. later discussion,however). To do this,

samples of the reaction mixture would have to be taken periodically and

analysed for canthaxanthin and astacene content. It was apparent that a

newly announced chromatographic technique might be applied to this problem

with advantage.

132 In 1998, Stahl reported that a mixture could be conveniently

separated into its components (on a micro-scale) on what was essentially 208

a very small-scale column chromatogram. Instead of the adsorbent being

confined in a glass tube, it was smeared (uniformly) on to a flat strip or sheet of glass, which was then dried to give what has been termed a

"chromatoplate". The technique used to resolve a mixture into its

components was to "spot on" the mixture(dissolved in a volatile solvent)

near one edge of the plate. This edge was then dipped into the developing

solvent so that the solvent level did not quite reach the "spotting on"

position. As the solvent rose up the adsorbent on the plate (by capillary

action, rather as in ascending paper chromatography), the mixture was

carried with it and subjected to a chromatographic separation. Stahl133 134 has since reviewed the potential applications of the method and Randerath

has used a similar technique to resolve mixtures of nucleic acids into their

components, using an ion-exchange resin as the adsorbent.

1 39 In 1999, Demole - described the resolution of mixtures of

carotenoids on chromatoplates using silicic acid as adsorbent. He also

gave details of the experimental techniques used in chromatoplate work and

mentioned some of the advantages of the method [two advantages being that

it is a rapid method of analysis and that only 5-10 yg,of carotenoid are

required (or less, see below)]. The use of chromatoplates in the 136 carotenoid field has been illustrated by Isler et a1. Winterstein and 137 Hegedus found that as little as 0.03 mg. of retinene or p-apo-8 -

carotenal could be detected on a chromatoplate, after development, by

spraying the plate with rhodanine and a base.

138 In 1960, Winterstein et al. showed that highly oxygenated 209

carotenoids could be chromatographed or "reversed-phase" chromatoplates.

This was of particular interest to the preSent author since it was expected

that astacene would require a very strong developing solvent on the

"normal" plates described previously. The "reversed-phase" plates were

made by soaking a "Kieselgel"-coated chromatoplate in a light petroleum

solution of "liquid paraffin". The adsorbent was then essentially a

hydrocarbon (the liquid paraffin left behind as the light petroleum

evaporated) so that on chromatographing a mixture -of carotenes and

oxy-carotenoids on the plate, the hydrocarbons remained on the base line

(R 0.0) whereas the oxy-carotenoids tended to move with the solvent used F as developer. This is the reverse rf the behaviour on "normal" plates.

The present author therefore undertook a study of the conditions

whereby canthaxanthin and astacene could be separated on a chromatoplate

from each other and from any degradation products. It was hoped, in

addition, that it would be possible to separate the trioxy-compound (LXXVII)

which it is assumed must be an intermediate in the oxidation of canthaxanthin

(XI) into astacene (II). It was essential for the results to be 0 • • N • • • • • •

HO (LXXVII) 0 meaningful, of course, that the system chosen should, on developing the

plate, allow all the components of the mixture to move off the base line

but for none of them to be eluted on the solvent front. (On some occasions 210

this could not be achieved on one plate, so two plates were run using different developers).

A series of trial runs were made on a mixture of canthaxanthin and 138 synthetic astacene using "reversed-phase" plates and various concentrations of aqueous acetone as developing solvents. At first the results were very disappointing. Occasionally, a good separation would be achieved but all attempts to reproduce the result using apparently identical conditions would then fail - the spots either not moving at all (RF 0.0) or

moving on the solvent frontthroughout (Rp / .0). It was eventually discovered that many different factors could affect the result obtained, and that it was essential to keep all these constant. The preparation of a typical "reversed-phase" plate is outlined in the Experimental section.

The following general points are, in addition, worthy of note. These remarks refer to the technique used in the more recent stages of the work with chromatoplates when the results obtained were essentially reproducible.

The plates were always stored in a dry atmosphere. After the

"normal" plates were converted into "reversed-phase" plates, the latter were stored before use for several hours in dry air to allow all traces of solvent (redistilled light petroleum) to evaporate. The acetone used

(after suitable dilution with water) as the developer, was obtained consistently from the same source. The use of "recovered" acetone was found to give rise to erratic results presumably because this "acetone" also contained other solvents and a variable (and unknown) quantity of water. To saturate the aqueous acetone with liquid paraffin, the aqueous 2,11

acetone was twice shaken with a 5% light petroleum solution of liquid

paraffin. Because the acetone was soluble in both phases, the relative

volumes of the two phases was kept consistent so that the same quantity of

acetone was washed out of the acetone layer by the light petroleum each

time this procedure was carried out.

Each of the developing solvents used for these plates contained,

therefore, acetone, water, light petroleum, and liquid paraffin in varying

proportions (depending on the strength of the original aqueous acetone

solution). Since acetone is more volatile than water, if one of the

developing solvents was left uncovered its power as an eluting agent slowly

diminished due to the evaporation of the acetone. The development of the

plates was carried out in a covered tank. Even if the top of the tank

was only briefly removed to allow one elate to be removed and a second plate

to be inserted, sufficient acetone a-nnarently evaporated for there to be a

marked drop in the eluting power of the solvent in the tank (for example,

using freshly prepared 75 aqueous acetone the R value of astacene fell

from 0.60 on the first plate to 0.92 on the second). Thus, it was

impracticable to use the same solvent for more than two or three plates since

after this the solvent became too weak. The same effect was observed on

prolonged storing of the solvent in the tank since although the tank had a

. cover it was not air-tight. Because of these uncertainties in the solvent

concentration, it was important to calibrate each plate by including a

known substance along with those to be studied.

With the above precautions, the RF values obtained for a given 212

compound were found to be reasonably reproducible. Thus, on one occasion using freshly-prepared 77 aqueous acetone as developer, astacene and canthaxanthin had R values of 0.97 and 0.24, respectively. A repetition F of the experiment using a freshly prepared plate and freshly-prepared solvent gave R values of 0.90 and 0.22: This technique was used F successfully'during the work on fucoxanthin (Appendix I), azafrin (Appendix

II), astacene, and a further oxidation product of canthaxanthin (the

"pentaketone", see below); suitable solvent strengths were found to be

725 4 715, 77,x, and 75,L aqueous acetone, respectively. Indeed, during the work on azafrin two pigments were obtained which failed to separate on an alumina (II) column but showed distinct signs of separating on a reversed- phase plate (although the spots were not completely resolved). Also, dehydro compounds showed obvious signs of separation from their polyene analogues and here the difference in the colour of the two compounds demonstrated that partial separation had occurred.

Finally, it should be added that the technique evolved by the present author was eE-sentially an arbitrary one which happened to give, eventually, satisfactory and reproducible results. Further work may well show that better separations can be achieved and, in particular, more

"stable" solvent systems can be devised than those mentioned here.

In connection with the work on the autoxidation of canthaxanthin, reversed-phase chromatoplates were of considerable assistance. In particular, they afforded a far more rapid and convenient method of analysis than chromatography on sucrose columns (in addition, much less sample was 213 required for the chromatoplates).

Firstly, a mixture of canthaxanthin (1 mol.), a small quantity of benzene, and a large excess of potassium t-butoxide (100 mols.) in t-butanol was shaken in oxygen to obtain the oxygen-absorption curve; samples of the mixture were not taken on this occasion. The curve obtained is reproduced on p. 214. Although a time-scale is shown this only applied to the experiment in question: the rate at which oxygen absorption proceeded varied quite markedly freel experiment to experiment. Eome idea of this variation is given in the Table on page 229 in the Experimental section. The reason for this variation was not investigated; it might have been-caused by variations in the concentration and the purity of the base. It will be noted that there was a rapid initial uptake of oxygen which was complete within 90 seconds of starting the shaker. This was a common feature of all the oxygenation reactions carried out. The same effect occurred in the absence of canthaxanthin (p.233), and is ascribed to the absorption of oxygen by the solvent mixture. An allowance has always been made for this whenever oxygen absorption figures are given.

p /-*•• The above effect was the cause of come conl us len in the early stages ef this work (or. p. 195 ) •

A similar experiment was then performed but this time samples of the reaction mixture were withdrawn periodically. The composition of each sample was determined using reversed-phase chromatoplates. The canthaxanthin and astacene spots were identified by running mixed chromatoplates with the starting material and with the synthetic astacene

114s. of oxtvoN ABSORBED koRREcrED To A17:R)

1‘..) 0, \ '40 0 0 0 0 0 0 0 0

I

()I 0

0

0

P O

ID A+

tA to

eT to to

tO

O ti

t.o4 t.x4

0o 6 0 1:4

ev

CO

171 215

already prepared. The canthaxanthin gave a small orange or orange-red

spot whereas both the astacene and the "pentaketone" (see below) gave

slightly elongated purple-pink spots(cEref.151).

The results obtained are summarised in the diagram on page 216.

The repeated interruption of the reaction and the continual withdrawal of

samples made it impracticable to obtain accurate oxygen-absorption values.

Instead the results of the sample analyses have been plotted against time.

For the purposes of the present work, it was assumed that every component of each sample was colouredl so that the total of coloured

pigments in any one sample was equal to "100 (this was, probably, essentially true for much of the duration of the experiment; it was almost certainly not true towards the end of the reaction when relatively large qucntities of faintly coloured substances were probably nroduced: see below).

The relative amounts of each component separated on the plate from a given sample was estimated by eye. The results obtained are summarised in the diagram (see p. 216). The rapid decrease in the concentratien of the canthaxarthin was accompanied, an expected, by the concomitant formation of astacene. Tlowever quite early on in the reaction, traces of a new substance wore fcrmed which, from its beh4.viour on the chromatoplates, appearecitobeeve nraore hipmly avgenatedtywastacene (vpieala_ vslues x A consideration of the composition of the crude reaction products obtained from other(Similar)reactions has enabled a rough estimate of these values to be made; they are appended to the diagram in question.

MOLS. OXIKEN ABSORBED PER Mot. of CANTHAXANTHIN: APPROXIMATE VALUES ONLY :SEE Foorwore oN PAcE 215.

t00% 100%

MATURE OF YELLOW DEGRADATION PRODUCTS 5370 07. o

100% 100%

"PENTA KETONE" 5oZ

0%

100% 100%

ESTACENE .0 z o% 0%

100% -t00%

CANT11/007 HIN 5-07.

40 0

0 0 4o 80 120 160 200 240 280 32 •"r/ME HouRs : 8ur SEE P.21.3) --->

or THE REACTION MIXTURE. CANTHAXANTHIN 02 /PorRssium i-BoroxIDE : THE COMPOSITION 217

using 75./. aqueous acetone were 0.15, 0.45, and 0.75 for canthaxanthin,

astacene, and the "new substance", respectively). This new substance was

therefore called the "pentaketone" so as to suggest its highly oxygenated

state. It should be emphasised that although attempts have been made to

elucidate the structure of this compeund, there is no definite evidence that it contains five keto-groups (or 3 ,etc-groups and two enolic hydroxy

groups). ?his "pentaketone" was apparently formed at the expense of the

astacene, according to the scheme:-

Canthaxanthin------>astacene-----y'pentaketone" (---4polyene acids ?)

Oxidative degradation products were also formed during the

reaction. On one occasion these were briefly investigated (cf. below).

It appeared. that relatively large quantities of only faintly coloured degradation products accumulated towards the end of the reaction

period investigated, Thus the final analysis of the reaction described above suggested that the reaction mixture at that stag consisted of approximately 60/, of the "pentaketone", 20/0 of degradation products, and a trace of astacene, However, on the twe occasions when the reaction was allowed to proceed to nearly this stage and then worked-up,only relatively small quantities of the "pentaketone" were isolated. In addition, the weight of the acid fraction isolated simultaneously was almost as much as the yield of "pentaketone", and the major constituent of this acid

fraction absorbed near 345 rp (in benzene) and so would not have been detected on the chromatoplate. the The structure ofA"pentaketone" remains unknown. The chromophore 21.8 must be similar to that of astacene; in particular, it cannot have been shortened significantly by oxidative degradation. Its chromatographic and partition properties suggest that one group is of the astacene type

[for example, the "pentaketone" forms a salt with alumina, but is not soluble in alkali (cf. p.233 )j. In addition, its infrared spectrum bears a marked resemblance in certain regions to that of astacene. Thus the

"pentaketone", like astacene, gave a rather broad hydroxyl peak near 3410 -1 cm. , a conjugated-carbonyl absorption peak near 1610, and. a peak near 1 1062 cm. ; all these peaks had 6 values of approximately one half of those found in the astacene spectrum, suggesting that one end-group of the

"pentaketone" is the same as that in astacene. The other end-group (which can only contain three oxygen atoms if the other end-group is of the astacene type) would then be the one which gave rise to bands in the -1 "pentaketone" but not in astacene: that is the sharp band at 3590 cm. whose position and intensity (€83) suggest the presence of two free hydroxyl groups and the 1704 cm:1 band with an evalue (655) suggestive of two 91 saturated carbonyl functions, or possibly one conjugated ester. The spectrum of the phenazine derivative of the "pentaketone" shows a carbonyl -1 1 absorption band at 1704 cm. of the same intensity relative to the 970 cm. band as occurs in the "pentaketone" itself, showing that treatment with o-phenylenediamine has no effect on the carbonyl group(s) in the

"pentaketone" responsible for the 1704 cm:1 hand.

The n.m.r. spectrum of the "pentaketone" is also compatible with the presence in that molecule of one end-group identical with the astacene end-group. Thus the spectrum includes those peaks present in the astacene 219 spectrum and additional peaks at 4'59, 4.84, 8'775, and near 7.98 p.p.m.

Unfortunately, the proximity of the bands in each group of methyl-proton

bands made accurate area measurements difficult. lit is noteworthy that

the two protons attached to a furanoid oxide ring (cf. SectionIII) give a

band near 4'84 p.p.m.l. It would be unwise to speculate on the structure of the "pentaketone" at this stage. However, it is unlikely that the

"pentaketone" is the rearrangement product (LXXVIII), since it was found to

be insoluble in alkali. The chromatographic properties of the mixture

NO (L" xxyin) CO2H of methyl esters obtained by methyl-esterification of the acid fraction

strongly suggested that the majority of the constituents of the "acid

fraction" each contained one end-group of the astacene type. The formation

of large quantities of these acids along with, and following, the formation

of the "pentaketone" suggested that the final step in the reaction sequence

might be the break-down of one of the end-groups in the "pentaketone" (one

end-group of wLich is probably of the astacene type: see above). This

would give a polyene acid and, subsequently, (by the progressive removal of

small groups of carbon atoms), a series of polyene acids with progressively

Shorter chromophores. The fact that the mixture of esters showed an -1 absorption band at 3590 cm. (which did not occur in the astacene spectrum

but which did occur in the "pentaketone" spectrum) suggests that the "new"

end-group (that is, that not of the astacene type) in the "pentaketone"

survives in the oxidative degradation of this substance and that it is the

astacene end-group which undergoes degradation. 220

It will be noted in the Experimental section that the working-up procedure is more simple than that described in the early investigations.

Experience showed that the partition procedures used originally (and incorporated so as to remove unchanged canthaxanthin and also degradation products) could be dispensed with providing the reaction was allowed to proceed until most of the canthaxanthin had been consumed. Thus, in the procedure used more recently, the degradation products were either washed out of the chloroform during working-up or were sufficiently soluble in ethanol to be easily removed from the crude pigment by crystallisation from a chloroform-ethanol mixture (this solvent was found to be more convenient than that previously used for astacene, pyridine-water9). Finally, it 106 should be mentioned that Barton and Templeton's isolation procedure relied on the ability of aqueous alkali to extract their diosphenols from a chloroform solution. Attempts to apply this procedure to astacene were unsuccessful: on shaking a chloroform solution of astacene with aqueous alkali the pigment was removed from the organic layer but it was not transferred to the alkali; instead, it was precipitated at the interface

(on acidification of the mixture, the pigment returned to the chloroform P. layer)(cq1 9 7). 221 EXPhRIMENTAL

V.m.r. spectra were ,letermined at 56.4 Mc. Infrared spectra

measured on the Imperial College spectrometer (see the notes at the

beginning of this Thesis) are marked "(IC)", and those on the Queen Mary

College machine, "(QiC)"; Evalues obtained on the 2 instruments are not

strictly comparable.

Reversed-:phase chromatc'Thlates were prepared as follows.

The clean, glass plates were covered with an even layer of a

freshly prepared cream of "Kieselgel" (Merck)(30 g.) and distilled water

(60 ml.), and were then dried (105 , f hour). The plates were allowed

to cool and were then carefully immersed in a 5% solution (v/v) of liquid

paraffin in light petroleum. The plates were stored in dry air for at

least 4 hr. (to allow solvent to evaporate). The developing solvent was

prepared by shaking aq. acetone (380 ml.) of the required concentration

(cf. discussion) with 2 successive portions (of 100 ml. each) of 5;.:, liquid

paraffin in light petroleum. The mixtures to be analysed were spotted on as concentrated solutions in chloroform 20 mm. distant from the lower

edge of the plate which was then dipped into the developing solvent in a

closed tank. The solvent front was allowed to rise (during ca. 20 min.)

40 mm. above the spotting-on line, and then the plate was examined.

Exppriments with natural astacene (995, 1401).

117a Crude naturally-derived astacene [30 mg., m.p. 198-203o(K.)] was recrystallised (with filtration) from chloroform-ethanol to give a deep purple, glittering, crystalline solid (the crystalshaving the same characteristic shape as those of synthetic astacene; see below), m.p. 222

1 231-233° (evac.cap., uncorr.)(for natural Psi-scene, most authors give

(in carbon disulphide) m.p. 228°); Max. (in pyridine) 498 TT , 2\ Max (IC) ( N152 514 my; V ,in chloroform) 3420 (rather broad, 6 10,D), 1688 (vw), mnx. 1614 (vs, 1170), 1552(s), 1330(s), 1063(s), 998(m), and 969(vs, & 070)cm:/

The hydroxyl region of the infrared spectrum was determined in dilute

(6mg./25m1.) carbon tetrachloride solution using a 10 cm. path-length cell;

sharper than in The sample the OH peak was chloroform: V (IC)max. 3400 cm:1 was recovered, once crystallised [m.p. 215-219°(<.)], and its n.m.r. (inC1-1C13)'#3 spectrum was determinedtir 7.91, 7.99, 8.70 p.p.m. (relative areas ca.

1:2:2, but see p.198).

The residues from the above work were combined, the solvents 145a were evaporated, and the residual crude astacene (ca. 25 mg.) was acetylated in the same way as for synthetic estacene, below. The crude nroduct was crystallised once from cloroform-efhanol to the dincete 4 e (4 mr• )•

(evac. A(pyri6ine) 498 mp, m.p. 223-2250 cap., uncorr.),143 MPX. ' max. (in chloroform)143'144 1759 (mf 1675 (vw), 1642 (vs, E 1 070), 1055 (s), 991 (w), and 971 (s) cmtl.

117a , o A further quantity (18 mg. of crude as lm.p. 203-207

(K.)1 was treated 1451) with o-phenylenediamine in acetic ocid in the same way as for the synthetic astecend, below. The crude solid (ca.4 rig.) obtained from the chromatogram had -A (in carbon disulphide) 514 91; the infrared spectrum [in chloroform (IC)], although weak, was essentially. similar to that of the phenazine derivative of synthetic astacene (see below); nc carbonyl or hydroxyl absorption was discernible; the n.m.r. spectrum was 223

too weak to be meaningful. The sample was used for the mixed chromatogram

test (below).

Preparation of astacene (1649).

The following procedure was used to clean potassium which had

been stored under "liquid Paraffin". Potassium (ca. 8.5 g.) was melted by

heating it in benzene to 80° in a tared flask. mixture was allowed to

cool, and the solvent was decanted threegh a loosely-inserted glass stopper.

This procedure was rep-eated, and then tl,e flask was immediately evacuated

and, as soon as the last traces of benzene had evaporated, the vacuum was

released with dry nitrogen. The potassium was weighed (7.68 r., 1c)6 m/mole)

in the flask under nitrogen, and t-butenol (commercial, 140 ml,) was added.

The mixture was heated on the steam 'oet in a dry atmosphere (usually

nitrogen) until all the metal had dissolved; the resulting solution (1.4011)

was used within 1-2 days of preparation.

To canthr”xanthin [m.p. 215-2160 (evac.cap.,uncorr.): cf. ref.78J

(1.C3 g., 1.82 m/moles) in dry benzene (15 ml.) was added the potassium

t-butoxide solution (1.40N, 130 ml., 50-fold excess). The flask containing

the mixture was attached to a "hydrogenating apparatus", flushed out with

oxygen, and re-evacuated. The temperature of the mixture (which had fallen

during the mixing of the benzene and the t-butoxide solution, and during

the subsequent evacuation of the flask) was equilibrated with room

temperature by shaking the evacuated flask for a few min. Oxygen was then admitted, end the mixture was shaken at room temperature under oxygen et a 139 pressure just in excess of 1 atmosphere. After 30 hr., 221 ml. of 224

oxygen [reduced to Y.T.P., equiv. to 5.4 mols. of oxygen (cf. the notes on

p. 228) per mol. of canthxanthin] had been absorbed, and the reaction was

stopped. Water (200 ml.) was added, and the mixture was transferred to a

separating funnel with 0.5N hydrochloric acid (800 ml.)150 and chloroform

(35C ml.). The mixture was shaken, and the organic layer was separated,

washed with saturated aoueous sodium hydrogen r,:arbona.te (3 x 300 nl.), water

(300 ml.), and, finally, distilled rater (300 ml.). The chloroform solution

(cf. the notes on p. 227) was diluted wit. benzene (ca. 50 ml.), and the

solvents v.ere evaporated ieavina. r ,Itrk red residue. Crystallisation from

chloroform-ethanol (1:5) rave (cf. the notes below) astacene (540 mg., 50 , m.p. 216-219° (evac.crp.,uncorr.) - raised by recrystallisation to 252-233°

(evea.cap.,uncorr.), 227-231° (K.); mixed m.p. with naturally-derived

astacene (m.p. 231-233°), 252-235° (evac.cap.,uncorr.)(Found: 0, 10.82, 10.84,

and 10.74. C 011 0 reqrres 0,10.8M). The product was a deep purple, 4 48 4 glittering, crystalline solid; under the microscope, the crystals were

seen to be of the shape 1 Ko separation was observed in a mixed chromatogram with the naturally-derived sample on sucrose (finely powdered)

from benzene-light petroleum (1:4) (under these conditions, canthaxanthin

was separated readily from astacene, and was eluted first). F.pectral

(in pyridine) 498 8 100,000; in C:)' in hexane, in properties: •max. my, 2' ethanol, and in ethanolic KOH, the absorption maximum occurred at 513, 477,

483, and 470 IT, respectively; t (in euterochloroform) 7.90, 7.9P,

and 8.70 p.p.m. (relative peak areas 1:2:2), r-Ith.nlefinic proton bands at

ca. 3.45, 3.55, 3.60 (shoulder) 142and at 3.94 P.D.M.; the spectrum was

similar to that given by the nalural -derived astacene (see above end 225

152 p• 198 ); (IC) / in chloroform) 3410 (rather broad, 6 1684 (vvr, X

& 100), 1610 (Vs, g 120*), 1550 (RI), 1327 (s), 1062 (S), 998 (m), and 969 \ -1 (s, 895) cm. ; the spectrum was -.,.•:Perimposahle (within experimental error) on that obtained on a sample of naturally-derived astacene (see above). In a KC1 disc spectrum, further bands were revealed at 1244 (vs) end 787 (m) cm:1 : (IC) [in carbon tetrachloride, 2.7 mi./14.2 ml., 5 cm. path-length may. cells (cf. under "natural astacene")] 3410 cm-.1 (sharp,Elkll 25).

sample of crude astacene [m.p. 211-218° (evac.cap.,uncorr.)] • was convertedi4la into its diacetPte by a modification of the method of

Kuhn et a1.32 A solution of the crude astacene (68 mg.) in pyridine (dry,

2.0 ml.) and acetic anhydride (0.043 ml.) was kept at 25° for 24 hr. The excess anhydride vas then decomposed by adding methanol (0.020 ml.); water

(1.0 ml.) was added dropwise, nd the mixture was left at.0°. The crude product (46 mg.) was collected, washed with 50`', aqueous pyridine, and crystallised from chloroform-ethanol (1:5) to give the diacetate (27 me:.), 0 m.p. 232-233° (decomp.)(evac.cap.,uncorr.)[Kuhn et al.32 Five m.p. 235

(decomp.)(evac.cep.,uncorr.) for :,4 -r:e derivative of naturally-derived astacene]; X (in pyridine) 497 u, E 92,000; 15(in chloroform) 7.71, MDX. 7.955, 7.98 (shoulder), and 8.65 p.p.m. [relative peak areas, 1:2:ca.1

(in chloroform)140 1756 (m., E 530, enol acetate), (shoulder):2]; 9 (IC)max. 1678 (vv), 1639 (vs, S 1150, conj. c=0), 1555 (m), 1055 (s), 992 (w), and 972 (s, trans )cm:1 The infrared spectrum was virtually identical144 with that of the diacetate prepared similarly from naturally- derived astacene (cf. above). 226

On treatment of carthexanthin with. acetic anhydride and pyridine under the same conditions Ps above, ihe canthaxenthin [identified by a mixed chromatogram (conditions as below, under phenazine derivative)] was recovered quantitatively.

141b Crude astacene [m.p. 211-22e(K.)1 was converted into its bisphenazine derivative using a modification of the method of Kuhn et al.15 85 A solution of crude astacene (125 mg.) and freshly purified o-phenylene- diamine (157 mg.) in "glacial" acetic acid (10 ml.) was heated (in the dark) at 1000 for 90 min. The mixture was allowed to cool, and was then transferred to a separating funnel using water (200 ml.) and chloroform

(40 ml.). The mixture was shaken, the organic layer vies separated, and was then cached with 1N hydrochloric acid (2 x 50 ml.), saturated aqueous sodium hydrogen carbonate (100 ml.), water (150 ml.), and distilled voter (150 ml.).

The chloroform solution was diluted with benzene (50 ml.), end the solvents were evaporated. The residual solid was then chromatographed on alumina

(IV, 16 x 3.5 cm.) from benzene-light petroleum (40:60). The main purple- red band was collected, and ''he solvent was evaporated, leaving a dark purple solid (39 mg.), m.p. 218-225°(K.). This was crystallised from carbon disulphide-ethanol (1:5) and then chloroform-ethanol (1:1) and gave the bisphenazine derivative (21 mg.) as a purple microcrystalline (tiny, 15 curved needles) solii;, m.p. 229-230°(K.), 231°(evac.cap.ouncorr.) [Kuhn et al. give m.p. 224-2250 (probably cvac.cap.,uncorr.) for tile derivative of "natural astacene"]. (Found: N,7.6. Cale. for C52H56114: Np7.6%). No separation was observed in a mixed chromatogram [on alumina(IV) from benzene-light 227

petroleum (40:60)] with Ule derivative of the naturally-derived astacene

(see above). Spectral properties: (in pyridine) 503 140,000; Al max. mp, 6 kmax.(in carbon disulphide; 517 ni; lr (in deuterochloroform) 6.88,1' 7.61,

7.94, 7.98, 8.79 (relative peak areas rere 2:3:3:3:6); (in chloroform) 11250w), 1550 (m.,conj.C.C), 1391 (s), 1364 (m), 1344 (ff),i(1066. (m), 1000 (w), and 969(s, E 800)em71 ; no carbonyl or hydroxyl absorption was discernible; the

infrared spectrum was similar to that obtained on the phenazine derivative

of naturally-derived astacene (see above).

On treatment of canthaxanthin with o-phenylenediamine and acetic acid under the same conditions as above, the canthanxanthi.n [identified by a mixed chromatogram on alumina(W) from benzene-light petroleum (1:1) - conditions which quickly separated canthaxenthin from the bisehenazine (or diacetate) derivative of estacenel was recovered, quantitptively.

The crude chloroform extract (marked : see p. 224) was shorn

(reversed-phase chromatoplate/74;laq.acetone) to contain approximately: canthaxanthin (2%), astacene (72%), pentaketone (20`;), and yellow

(decomposition) product (6q. Thus the major impurity in the astacene was the pentaketone. The above procedure could have been improved by allowing

t Results obtained from another spectrum (determined on a slightly impure sample of the compound) suggest that the true position of this band might be at slightly higher fields (near "1" 6.91 p.p.m.). 228

less oxygen to be absorbed (e.g. 4.0 mols. instead of 5.4 mo1s.); canthaxanthin would then have been the major impurity and this would have been easier to remove by crystallisatiou than the pentaketone (whose solubility croperties are similar to those of astacene). It is suggested 3 and after that chromatoplate tests should be made afterh4 mols. of oxygen have been absorbed, and the results obtained used to stop the reaction at the stage where the reaction mixture still contains 20-25 canthaxanthin.

Although only a small part of the canthaxanthin dissolved in the benzene, it was found to be essential to add the benzene (or similar inert solvent) otherwise only traces of canthaxarthinwent into solution and oxygen uptakewas slow and erratic. Astacene was found .to be adsorbed by Na2SO4 and similar desiccants: solutions were, therefore, dried azeotropically. To obtain a good recovery of either astacene or the "pentaketone" (see below) on crystallisation, it was, apparently, necessary to leave the solution for several days at 00: crystallisation occurred gradually over a period of days.

X That is, 4.8 mots. in this particular reaction - not necessarily in a repeat experiment since it ap7earcithat the composition of the product obtained from a given uptake of oxygenwas not necessarily the same an apparently identical experiment. 229

Table showing the variation in the rate of the autoxidation of canthaxanthin (cf. p.213 )

0 absorption ' Astacene Page Canthaxanthin K t-butoxide 2 31 Weight m/mole Conon. Mole. Mols. Hours Yield No.

362 mg. 0•64 1.4 144 10.0 in 76 hr. ca. 30 mg. 941

403 mg. 0.71 1.57 104 8.6 in 90 hr. ' 130 mg.(34cA) 981 1.02 g. 1.81 1.42 150 8.7 in 40 hr. ! 250 mg.(23ei) 1067

1.03 g. 1.83 1.31 100 5'4 in 30 hr. . 540 mg.(5CP 1649

Page no. in laboratory notebooks (which have consecutive pagination).

The preparation of the "pentaketone" (1747)

The experimental details are similar to those used in the preparation of tstacene except that here the reaction was allowed to proceed for longer so that more oxygen was absorbed.

To canthaxanthin (1-00 g., 1.77 m/mole) in dry benzene (15 ml.) was added potassium t-butoxide in t-butanol (1.37N, 130 ml., 50-fold excess), 139 and the mixture was shaken in oxygen until, after 85 hr., 524 ml. of oxygen (reduced to N.T.P., equiv. to 13.2 mols. per mol. of canthaxanthin) had been absorbed. The reaction was stopped, and the mixture was diluted 146 with water, acidified, and extracted with chloroform. The chloroform solution** was washed with saturated aqueous sodium hydrogen carbonate

(3 x 300 ml., the first of these extracts being kept - the "acid fraction": see below), and then distilled water (2 x 300 ml.). The solution was diluted with benzene (ca. 50 ml.), the solvents were evaporated, and the residual solid was crystallised (cf. the notes on p.228 ) from chloroform- 230

ethanol (1:6) to give the "pentaketone" (164 mg.) as a very dark purple

microcrystalline powder m.p. 200 - 2080 (evac.cap., corr.). Evaporation of the mother liquors to small volume, and dilution with ethanol gave a further quantity (30 mg.) of product, with the same m.p. Total yield

194 mg. (18'A, based on the formula C40H5005: see below). The solid was recrystallised from chloroform-ethanol to m.p. 207 - 208.5° (evac.cap., corr.) (Found: C, 78.5; H, 8.1; 0, 13-0. Calc. for C H 0 : C, 78.65; H, 8.25; 40 50 5 0, 13,1; and for C40H4805: C, 79.0; H, 7.95; 0, 13.1'iiL Spectral properties ( E values based on the formula C ): .(in pyridine) 40 H50 05 max 497 7, £105,000; 17(in chloroform) 7.89, 7.97, 7'99, 8.69, 8.775 p.p.m. (approx.relative areas, 1:2:2:2:1) with olefinic protons at 3'45, 3'57, 3.95, 4.59, 4.84 P.P.m. [approx. relative peak areas of the last 3 peaks, 1:3:2 (on this scale, the remaining band, with maxima at 3.45 and 3.57 p.p.m. and small

associated peaks, had an intensity of ca. 12)]; V max. (in chloroform) 3593 (sharp, 83; free OH), 3413 [rather broad, g 56; H-bonded OH ( -0H----0-C)], •1786 ''w), 1704 (vs,E 655; probably satd. carbonyl), 1696 (vw), 1616 (vs, 6700, conj. k'tone) 1608 (vs, shoulder,690), 1555 (s, conj. C-C), 1525

(shoulder,m), 1095 (w), 1064 (m), 1 043 (w), 999 (m), and 970 (vs,g 805) cm.-1 (the labt two bands are assigned to trans-CH=CH- ). In a KBr disc spectrum, -1 an additional band was revealed at 1248(vs) cm. .

Notes regarding the preparation of the "pentaketone".

The crude chloroform extract (marke?W) contained (according to

chromatoplates146) astacene (20), "pentaketond" (755), and yellow degradation 231

products A cleaner product could have been obtained by allowing

the reaction to proceed rather longer [until ca.16 mols. of oxygen had been

absorbed (but cf. footnote on p. 2.28n, although the yield of"pentaketone"

might have been less (even though crystallisation losses incurred in removing

astacene would have been less) since the oxidative-degradation reaction

appeared to compete very effectively with the formation of the "pentaketone"

(from the astacene) at this stage.

The "acid fraction" from the working-up of the "pentaketone" (1756)

The NaHCO extract containing the "acid fraction" was washed with 3 chloroform; and was then just acidified (3N hydrochloric acid). The weakly

acid solution was extracted with chloroform, and then the chloroform extract

was washed with water, diluted with benzene, and the solvents were evaporated,

leaving a dark red solid (13U mg.) which was shown to be a mixture [reversed

-phase plate/7qL aq.acetone: 3 spots, RF 0.84, 0.92 (major product), 0.961.

The solid, in tetrahydrofuran (100 ml.),was treated with excess ethereal

diazomethane (6 m/mole) at 25° for 3 hr. The solution was evaporated to

dryness [ .(in benzene) near with inflexions near 410 and 450 max 345 91,

91]' A sample of the residue was adsorbed on alumina (IV). Only minor zones could be eluted; most of the pigment was held tenaciously to the top

of the column and could not be eluted, even with pyridine (cf. the behaviour

of astacene), whereas it was rapidly eluted through a starch or sucrose t As mentioned earlier, this is a measure of the coloured pigments present

- the large quantities of faintly coloured or colourless degradation products would not be detected. 232

column with benzene. Attempts to resolve the mixture by crystallisation were also unsuccessful. The crude pigment had )) .(in chloroform) 3590 max -1 (sharp,. w), 3425 (rather broad, vw), 1720 (vs), 1628 (m), and 971 (m) cm.

Preparation of the phenazine derivative of the "pentaketone" (1773)

A mixture of the crude"pentaketonen[m.p. 200-2080 (evac.cap.,corr.); 57 mg.] and o-phenylenediamine (155 mg.) was dissolved in "glacial" acetic acid (10 ml.) and heated (in the dark) at 1000 for 90 min. The mixture was allowed to cool, diluted with chloroform (60 ml.) and 0.2 N hydrochloric acid (100 ml.), and, after shaking, the chloroform layer was separated, and was washed with 1N hydrochloric acid (50 ml.), saturated aqueous sodium hydrogen carbonate (100 ml.), and, finally, water (150 ml.). The chloroform solution was diluted with benzene (20 ml.), the solvents were evaporated, and the residue was chromatographed on alumina (IV, 20 x 3.5 cm.) from benzene. A series of minor zones was eluted with benzene, and then the major product was eluted (rather slowly) with ethanol-benzene (1.5:98-5). was The solvent/ evaporated, and the residual solid (20 mg.) was rechromatographed.

The central part of the main red band was collected, the solvent was evaporated, and the residueN (16.3 mg.) was dissolved in chloroform:))(QMC) max.\ (in chloroform) 3581 (m, sharp), 1704 (s), 1070 (m), 1043 (m), 1000 (w), and 970 (s) cm:1 ; A (in pyridine) 496 nyi. max.

Some properties of the "pentaketone" and itspllenazine derivative

On a reversed-phase plate with 75% aq.acetone as developer, the

0 This was inadvertently exposed to air at 50 for a short time. 233

following fl values were observed: astacene, 0'40; "pentaketone", 0.30;

phenazine derivative of the "pentaketone", 0.39. The line nta. ke t one" not (like astacene) couldre eluted from alumina (IV) with ethanol. On

shaking a chloroform solution of the ntaketonewith aqueous alkali, the

"pentaketond"(like astacene) was deposited at the interface(cf.p.220).

Oxygen absorption curve, and a study of the varying composition of the reaction mixtureL (1571, 1630

(i) The oxygen absorption curve (see p. 2p4.) was that obtained during

the preparation of the "pentaketone", above. On another occasion, a solution

of potassium (8 g.) in t-butanol (150 ml.) containing dry benzene (18 ml.)

was shaken in oxygen. The mixture absorbed oxygen very rapidly during the

first 2 min. Oxygen absorption (reduced to N.T.P.) at 0, 1, 2, and 11 min.

was: 0, 27.5, 29.0, and 29.0 ml., respectively. This accounts for the

observed, very rapid, initial absorption of oxygen in oxygenation experiments

(cf. diagram on 11.214-).

(ii) A mixture of canthaxanthin (203 mg., 0.36 m,/moles), dry benzene

(2.4 ml.), and potassium t-butoxide in t-butanol solution (0.98N, 55

79 molar excess) was shaken in oxygen for a period of 320 hr. At intervals

during this period, samples (1 ml. each) of the reaction mixture were taken

against a counter-current of oxygen. In all, 18 samples were taken of

which 11 were analysed on reversed-phase chromatoplates (see earlier) using

77% or 75% aq. acetone (for astacene-rich and "pentaketone"-rich mixtures,

respectively). The canthaxanthin and astacene spots on each plate were

identified by running mixed chromatoplateo. The "pentaketone" spots all

had the same R value relative to the astacene spot, and had the same R„ F 234

crystallised.(p.230) value as theAupentaketone" run wider identical conditions. The proportion of each of the coloured pigments (canthaxanthin, orange-red; astacene and 151 "pentaketone", purple-pink and slightly more elongated than is usual for carotenoids) on the plates was estimated by eye.

I, t Treatment of dehydrocanthaxanthin with oxygen/h0Eu : oxygen absorption curve, etc.0861T— a To dehydrocanthxanthin (428 mg., 0.76 m/moles) in dry benzene

(6.5 ml.) was added potasSium t-butoxide in t-butanol (1.36N, 56 ml., 90-fold 146 excess), and the mixture was shaken in oxygen. In this case the polyene ketone (dehydrocanthaxanthin) dissolved almost completely [the analogous polyene ketone, canthaxanthin, was only partly soluble (see p.228), the rest remaining in suspension]. An oxygen-absorption curve was plotted and, in addition, samples of the reaction mixture were taken and analysed using 146 chromatoplates. After 1.69 mols.139 of oxygen (90-90 min.) had been absorbed (on 2 runs the values were 1.63 and 1.68 moms., respectively), the reaction mixture started to deposit a solid. At this stage, only ca. 19 ax of the dehydrocanthxanthin remained: the major product had the Tilp value expected149 of dehydroastacene. After 1.9 mols.of oxygen had been absorbed, only this new substance (now known to have been dehydroastacene) could be detected (and the rate of gas absorption did appear to drop slightly). The reaction was allowed to proceed for a further 425 hr., by which time 24 mols. of oxygen had been absorbed and all the dehydroastacene had been destroyed.

However, despite a careful scrutiny of the reaction mixture at intervals during this time using chromatoplates, no "pentaketone" - like substance was detected, Each plate showed one major spot (dehydroastacene) near Rie 0.4 235

(see below) and a faint yellow spot (Re 1.0) caused by degradation products.

It was apparent that to obtain the optimum yield of dehydroastacene, the

reaction should be stopped as soon as all the dehydrocanthaxanthin had been

consumed. subsequent to this stage, the yield of dehydroastacene would be

expected to drop owing to its being gradually converted into oxidative

degradationproducts.Re values (reversed-phase plate/75 aq. acetone):

canthaxanthin (orange-red), 0.20; dehydrocanthaxanthin (yellow), 0.26;

astacene (purple-pink), 0.52; dehydroastacene (pinky-orange), ca. 0'6. With

72 aq. acetone, the R values were: ca. 0.09; 0.09; 0.32; 0.37

(respectively).

Preparation of dehydroastacene (1981)

A mixture of dehydrocanthaxanthin (in pyridine) [Aun. 455 T.; powdered, 1-20 g., 2.13 m/mole], benzene (dry, sulphur-free, 18 ml.), and

potassium t-butoxide in t-butanol (1.33N, 160 ml., 50-fold excess) was shaken 146 in oxygen. The reaction was stopped soon after a chroinatoplate test had shown that no dehydrocanthaxanthin remained. Total oxygen absorption,

87.0 m1.139 (reduced to Y.T.P., equivalent to 1.83 cools. of oxygen per cool.

of dehydrocanthaxanthin) in 140 min. Water (300 ml.) was added, and the 150 mixture was transferred to a separating funnel with 0.5N hydrochloric acid

(950 ml.) and chloroform (300 ml.). The chloroform extract was wasned (as under astacene), and was then diluted with benzene (sulphur-free, 50 ml.), and the solvents were evaporated to leave 15, 15'-dehydroastacene as a red crystalline residue (1.18 g., 93), m.p. 199-201° (evac.cap.,corr.), /‘ max.

(in pyridine) 467 Ey (showing a shift of 12 mkt from the starting material,

236

On ao expected), which gave only a single spotlachromatoplate (reversed phase/

72.5! aq. acetone, RF 0.35 : cf. above). The solid was crystallisoci from

benzene-ethanol to m.p. 197-198° (evac.cap.,corr.) (Found: C, 01.5; H, 7%9;

0, 11.0.40 H ,0 requires C, 81.3; H, 7.85; 0, 10-8%); )‘. (in pyridine) 4r 4 max (wC) 466 T., 6 9o,00co) (in chloroform) 3423 (8 110, OH), 2146 (vw, 1688 (vw), 1616 (vs, E1180, conj. C-0), 1565 (m, con j. c.c), 1332 (s), 1064

(vs), 1000 (m), 970 (s), and 957 (m) cm:1 (the last three bands are assigned

to trans -CH.CH-90). In a KC1 disc, further bands were revealed at 1247 1 (vs, =C 0- ?) and 783 (m, t,- cm. The spectrum was very similar to

that of astacene (see discussion section).

2,3-Dimethyrquinoxaline (1).

147 One of the acre recent literature methods was modified in the following way.

To a solution of o-pheny]enediamine (21.6 g.) in. hot (70°) water

(320 ml.) was added a zolutia of diacetyl (17.2 g.) and sodium metabisulphite

(38.0 g.) in hot water (240 ml.). The mixture was kept at 70° for 15 min.,

and then, cooled. to 20°. "odium carbonate (68.9 g.) was added, and the

mixture was extracted with ether. The ethereal solution was dried (Na2004)

immediately, and the ether 'Pr1:.;- evaporated to give a brown solid. This was

treated with water, and the mixture was steam distilled. The product (22 g., 0 70) separated from the distillate as white needles, m.p. 106 (uncorr.) (IC) flit 148 106°]; chl,Droform) 3310 (broad, water of crystallisation, max. of. ref. 148), 169 (w), 1487 (m), 1396 (m), 1349 (w), 1331 (m), 1319 (m),

1159 (m), 1151 (m), 992 (m), 96r) (w) cm:1 ; lr(in chloroform) 7.37 p.p.n. 237 REFERENCES

1. Karrer and Jucker, "Carotenoids", (transl. Braude), Elsevier, 1950.

2. Goodwin, "The Comparative Biochemistry of the Carotenoids", Chapman and Hall, London, 1952.

3. Zechmeister, Fortschr.Chem.org.Naturstoffe, 1958, 15, 31 (p. 68).

4. Liaaen and 4rensen, paper presented at the 2nd. International Symposium on Seaweeds, 1956.

5. See Experimental Section.

6. Kuhn and Sorensen, Ber., 1938, 71, 1879.

7. Pouchet, quoted in ref. 9.

8. For reviews of this early work, see refs. 9 and 10.

9. Kuhn and Lederer, Ber., 1933, 66, 488; cf. ref. 32. 10. Lederer, "Les Carotenoides des Animaux", Hermann, Paris, 1935 (quoted in ref. 2).

11. Ref. 1, p. 21.

12. Reviewed by Karrer and Jucker, ref. 1, pp. 234 - 235.

12a.Karrer and Loewe, 1934, 17, 745.

12b.Karrer and Benz, Helv.Chim.Acta, 1934, 1/, 412.

13. Karrer, Loewe, and NUbner, Helv.Chim.Acta, 1935, 18, 96.

14. For a review of all sources of astaxanthin known in 1 939, see refs. 15 and 9.

15. Kuhn, Stene, and Sorensen, Ber., 1939, /a, 1688.

16. Fox, Fortschr.Chem.or.Naturstoffe, 1948, 5, 20.

17. De Nicola and Goodwin, Chem.Abs., 1954, I, 11661b.

18. Millott and Vevere, J.Marine Biol.Assoc., 1955, a, 279.

19. Nishibori, Chem.Abs., 195, 42, 5698f.

20. De Nicola, Experimental Cell Research, 191;6, 10, 441.

20a. De Nicola and Goodwin, Experimental Cell Research, 1954, 7,, 23. 238

21. Tischer, Z.physiol.Chem., 1936, 257.

22. Tischer, Z.physiol.Chem., 1937, 250, 147; 1938, 252, 225. 23. Tischer, Z.physiol.Chem., 1941, La, 281 ; cf. ref. 31a. 24. Reviewed by Goodwin, ref. 2; (a) p. 223 (b) pp. 261-3 (c) p. 173 (d) p. 181.

25. Goodwin and Srisukh, Biochem.J., 1949, 45, 263. 26. Fox, Nature, 1955, 175, 942. 27. Fox, Chem.Abs., 1957, 22, 5939a. 28. Volker, Fortschr.Chem.org.Naturstoffe, 1960, 18, 177 (pp. 182-186, 192-3). 29. See p.116 of this thesis; cf. Barber, Ph.D. Thesis, London, 1960.

30. Goodwin, Biochem.J., 1949, 45, 472; cf. ref. 2, pp. 219-223; ref. 25. 31. De Nicola, Biochem.J., 1954, 56, 555. 31a. Goodwin and Jamikorn, Biochem.J., 1954, 51, 376.

32. Kuhn, Lederer, and Deutsch, Z.physiol.Chem., 1933, 220, 229.

33. Goodwin and Srisukh, Biochem.J., 1949, A5, 268.

34. Hartmann, Medem, Kuhn, anti. Bielig, Z.Naturforsch., 1947, 2b, 330. 35. Glover, Morton, and Rosen, Biochem.J., 1952, 50, 425. 36. Massonet, Arch.Sci.physicl., 1957, 11, 223.

37. Ref. 16, pp. 33-34. 38. Ref. 2, p. 173.

39. Morton, Chem.and Ind., 1940, 5, 301. 40. Lovern, Chem.and Ind., 1942, 61, 222. 41. cf. Inhoffen and Pommer in "The Vitamins", ed. by Sebrell and Harris, Academic Press, New York, 1954, Vol. 1, p. 100.

42. Fisher, Kon, and Thompson, J.! nine Biol.Assoc., 1952, 229. 239

43. Nielands, Arch.Biochem., 1947, 415.

44. Kon and Thompson, Biochem.J., 1949, 12, xxxi.

45. Kon and Thompson, Arch.Biochem., 1949, 24, 233.

46. Fisher, Kon, and Thompson, J.Marine Biol.Assoc., 1954, a, 589.

47. Grangaud ("Astaxanthin: a new vitamin A factor"), Chem.Abs., 1996, CO 5992b; 1903, AL 2302e.

48. Grangaud and Massonet, Compt.rend., 1999, al, 1087.

49. Grangaud, Vignais, Massonet, and Moatti, Compt.rend., 1956, EAJ„ 1170.

50. Grangaud, Vignais, Massonet, and Moatti, Bull.Soc.Chim.biol., 1957, 22, 1271.

51. Grangaud and Massonet, Compt.rend., 1950, 230, 1319.

52. Grangaud, Chechan, and ::assonet, Compt.rend.Soc.Diol., 1950, 144, 1022.

53. lAassonet, Compt.rend.oc.Biol., 1949, 143, 1178.

54. Massonet, Compt.rend-Soc.Idol., (a) 1950, ail, 1020; (b) 1956, 190, 529.

55. See p.257 of this thesis and also ref. (2), p. 271.

96. Lowe, Torto, and Weedon, J., 1958, 1855, and refs. there cited.

57. Grangaud, Vignais, and Moatti, Arch.Sci.nhysiol., 1957,'11, 231.

58. Tarr and Dean, J.Fisheries Res.Board of Canada, 1948, ", 221.

59. Drumm, O'Connor, and Renouf, Biochem.J., 1945, Z1, 208; cf. ref. 2, p. 172.

60. See the Experimental Section for details.

61. Zechmeister, Fortschr.Chem.org.Naturstoffe, 1998, 19, 31, (pp. 68-70); cf. ref. 62.

62. Warren, Ph.D. Thesis, London, 1957.

63. See, for example, refs. 25,64, and 65.

64. Abolins, Acta Zoolo&ica (Stockholm), 1957, 22, 223. 240

6. Volker, Naturwiss., 1994, Alo 405.

66. Bligh, Dyer, and Horne, J.Fisheries Res.'3oard of Canada, 1957, 21, 637. 67. Faulkner and Watts, Food Technol., 1955, 2, 632. 68. See ref. 66 for a brief review.

69. Lederer, Biochim.Biophys.Acta, 1952, 2, 92 (cf. ref. 70). 70. Hodgkins, Liston, Goodwin, and Jamikorn, J.Cen.fficrobiol.,1954, 11, 438. 71. Ref. 1, pp. 295-340; see also ref. 2. 31a 72. Strain (1951) quoted by Goodwin and Jamikorn.-

73. Krinsky and Goldsmith, Arch.Dicchem.Biophys., 1960, 21., 271.

74. For earlier work with this alga, cf. refs. 2 and. 73.

75. cf. Section IT1 of this thesis. 76. Petracek and ZechmOster, Analyt.Chem., 1956, 1484.

77. Cooper an We don, unpu::lishe3 results.

78. Warren and. Weedon, J., 1953, 3966.

79. Warren and Weedon, J., 1959, 3972.

80. cf. Weiner, Org.S.ynth., 1943, Coil. vol. Ii 279.

61. Staudinger and Bereza, Ber., 1909, AL 4913.

82. Schniepp and Geller, J.Amer.Chem.Soc., 1946, 68 1646.

83. cf. Henbest, Jones, and Walls, 1., 1950, 3646; Woods and Kramer, J.Amer.Chem.Soc., 1947, 2246.

84. Bowman and Fordham, J., 1952, 3945.

85. Vogel, "Practical Organic Chemistry", Longmans, London, 1954.

86. Chem.Abs., 1956, 50, P2568a; 1955, Ala, P10739g, 11723h; 1 954, 48, 7644a.

87. Japp and Klingemann, Annalen, 1888, 247, 190 (p. 218); cf. Reynolds and Van Allan, Or.6.Synth., 1952, 21, 84. 241

88. Strain, J.Amer.Chem.Soc., 1935, a, 758 (p. 760); Demaecker and liArtin, Nature, 1954, 173, 266; De Puy and Ponder,. J.Amer.Chem.Soc., 1959, 81, 4629.

89. cf. Evans, Ridgion, and Simonsen, J., 1 934, 1 37; Organic Reactions, 2, 331.

90. Akhtar and Weedon, J., 1999, 4058.

90a. For detailed reviews concerned with the structure elucidation and synthesis of the keto-carotenoids, cf. refs. 1,2,62,78,97,100.

91. Barber, Ph.D. Thesis, London, 1960.

92. cf. Karrer and Cochand, Helv.Chim.Acta, 1945, 28 1181.

93. Bohlmann, Ber., 1951, !IA, 860; Kuhn and Grundmann, Ber., 1937, 70, 1318; Ann.Reports, 1941, Z1, '170.

94. Isler et al., Helv.Chim.Acta, 1959, Az, 841. 95. Petracek and Zechmeister, J.Amer.Chem.Soc., 1956, 1427.

96. Petracek and Zechmeister, Arch.Biochem.HpThys., 1956, 61, 137.

97. cf. Zechmeister, Fortschr.Chem.org.Naturstoffe, 1958, 15, 31.

98. Entschel and Karrer, Helv.Chim.Acta, 1958, 41, 402.

99. Karmakar and Zechmeister, J.Amer.Chem.Soc., 1955, f, 55.

100. Akhtar, Ph.D. Thesis, London, 1959.

101.- cf. Robinson et al., J., 1935, 1285; 1937, 53.

102. Claisen, Annalen, 1876, 180, 11.

103. Pauly and Lieck, Ber., 1900, EL, 500.

104. German Patent 708, 371.

105. The author is greatly indebted to Professor D.H.R. Barton, F.R.S., for informing him of these results before their 'publication; cf. refs. 106-109.

106. Barton and Templeton (J.F.), unpublished results, cf. refs. 107-109.

106a. See refs. 107, 108, 109a, 109b. 242

107. Arigoni, Barton, Corey, Jeger, and their co-workers, Experientia, 1960, 26, 41.

108. Barton, Pradhan, bternhell, and ToMpleton (J.F.), J., 1961, 255.

109. Barton, Pure and Applied Chemistry, 1961, 2; (a) 558; (b) 562.

110. Bailey, Elks, and Barton, Proc.Chem.Soc., 1960, 214. 111. cf. ref. 110.

112. For reviews, see Doering et al., J.Amer.Chem.Soc., 1946, 68, 586; 1954, E.5 482. 113. Howe and Mcquillin, J., 1958, 1513.

114. Stevens and Wright, Proc.Chem.Soc., 1960, 417.

115. Dauben, Boswell, and Templeton (w.), J.Org.Chem., 1960, E5, 1853.

116. cf. later discussion and ref: 117.

117. Bellamy, "The Infra-red Spectra of Complex Taqecules", Methuen, London, 1958.

117a. Grateful thanks are extended to Hoffman:171,a Roche A.G. (Basel) for the donation of this sample.

118. Barber, Davis, Jackman, and Weedon, J., 1960, 2870. 119. See, this Thesis: (a) Section I; (b) Section III; (c) P. 55.

120. Jackman, "Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry", Pergamon, London, 1959.

121. Davis, Jackman, Siddons, and Weedon, Proc.Chem.Soc., 1961, 261.

122. Ref. 120, p. 110.

123. cf. ref. 120, p. 125.

124. cf. Davison and Bates, J., 1953, 2607.

125. Published figures (refs. 78,91,100) vary, but are all near to E= 900.

126. Cross, "Practical Infra-red Spectroscopy", Butterworths, London, 1960. 243

127. For reviews, see ref. 117, P. 144 and ref. 126, p. 38.

128. Ref. 117, pages 100, 102.

129. cf. Dewar, Nature, 1945, 199, 141, 479.

130. Aulin-Erdtman and Theorell, Acta Chem. Scand., 1950, 4, 1490.

131. Scott and Tarbell, J.Amer.Chem.Soc., 1950, E, 240.

132. Stahl, Chem.-Ztg., 1958, 82, 323.

133. Stahl, Angew.Chem., 1961, 22, 646.

134; Randerath, Angew.Chem., 1961, 73, 674.

135. Demole, Chromatographic Reviews, 1959, 1, 1.

136. Isler, Ruegg, and Schudel, Chimia (Switz.), 1961, 15, 200.

137. Winterstein and egedus, Chimia (Switz.), 1960, 14, 18.

138. Winterstein, Studer, and Ruegg, Ber., 1960, 23, 2951.

130a. cf. the alcohols prepared in section IV and also refs.91,100.

139. In excess of the 25-40 ml. of oxygen wrlich was rapidly absorbed (by the solvent: see discussion) as soon as shaking was started.

140. Determined on a crude sample (it melted 10° lower than the pure material); soon after running the spectrum on the pure diacetate, it was shown that the spectrum was unreliable because of a fault in the instrument.

141. See pages (a) 957 (b) 919, 1003 in lab. notebooks.

142. Only one determination was made in this region, so the exact positions of these peaks are uncertain.

143. For lit, values, band as:;ignments, etc., see under the preparation of the corresponding derivatives of synthetic astacene.

144. A close examination of the spectrum of the "natural acetate" suggested that the quantity of solvent in the 2 beams was not exactly balanced; this would have obscured any bands (e.g. near 1550 cm.-1 ) where chloroform shows even moderately strong absorption (cf. ref. 126, p. 28) and this probably explains the apparent absence of the 1550 cm.-1 band in the "natural acetate" spectrum. 244

145. See pages (a) 1415 (b) 997, 1035 in lab. notebooks.

146. Experimental details as under the preparation of astacen,,.

147. Jones and McLaughlin, Org. Synth., 1950, 22, 86: cf. ref. 148.

148. Gabriel and Sonn, Ber., 1907, 40, 4850; cf. Heilbron and Bunbury, "Dictionary of Organic Compounds", 1953, 2, 343.

149. Based on the R values for the polyene analogues, and for dehydrocanthaxanthin.F

150. The crude product is more readily soluble if the reaction mixture is acidified.

151. The colour of the astacene spotsfaded quite quickly on exposing the spots to light; cf. p. 168 .

152. FoR $AND R5SliNtIENTS, SEE 245

SECTION III

THE F.M.R. SPECTRA OF CAROTENOID EPDXIDES

AND THEIR FURANOID OXIDE DERIVATIVES 246

The N.m.r. Spectra of Carotenoid Epoxides and their Puranoid Ox,ide Derivatives*

One of the most recent techniques to come to the aid of those concerned with the elucidation of the structures of organic compounds has been 1 n.m.r. spectroscopy. It soon became apparent that this technique might prove useful in the carotenoid field. Barber, Jackman, Weedon and the present 2 author undertook, therefore, the measurement of the spectra of a large number of polyenes of known structure in the hope that the results obtained might be of use in elucidating the structures of less well characterised polyenes. The object of this work has already been realised to the extent that the information gleaned from this survey of spectra has enabled its authors and their collaborators to elucidate (or confirm) the structures of spirilloxanthin3 and three other bacterial pigments,4 the paprika ketones 9 6 (capsanthin and capsorubin), ' and four of the (alleged) natural precursors of the carotenoids.7,8 (N.m.r. spectroscopy has also been used to deduce the stereochemistry of methyl natural bixin9).

It has been shown, during the last few years, that many of Nature's carotenoids are epoxides, or their furanoid oxide derivatives (see below).110-13

Only 5,6-(mono)-epoxides and 5,6:5',6'-diepoxides (and their corresponding furanoid oxide derivatives) will be discussed in this section (these are the only thoroughly characterised carotenoid oxides known as yet). The terms

"monoepoxide" (or "epoxide" when it is unambiguous) and "diepoxide" will, therefore, be used without adding the numbers which define the positions of attachment of the epoxide rings in the molecule. 247

Three compounds of this type were prepared by the present author and, with the three other compounds of this type already available for spectral 14 determinations, a reasonable representation was obtained of this important class of carotenoids.

It was not until 1945 that it was realised that some of the

carotenoids produced in Nature were epoxides and furanoid oxides. In that year, Karrer and Jucker15 reported that treatment of lutein (I)m (as its acetate) with ethereal monoperphthalic acid gave a crystalline monoepoxide.

It was evident from the shift in the visible absorption spectrum which 4' OH

3 45 (I), lutein H accompanied this reaction (equivalent to the removal of one ring double bond

out of conjugation with the polyene chain) that the product was the 18 ,6-monoepoxide. Shortly afterwards, Karrer et al. showed that this

compound was probably identical with a pigment isolated in 1943 from 8a 18b chrysanthemum flowers. (Karrer et al. have also shown that Hey's 18c "eloxanthin" (isolated in 1937 from the leaves of a pond weed ) was 19 probably lutein epoxide]. Since then, Karrer et al. have reported lutein

N This compound (I) was first isolated from Nature in a pure state by Karrer 16 et al., and named "xanthophyll"; this term has since been used as a

generic term for hydroxy-carotenoids and the name "lutein" assigned to the

compound (I) above;17 in his publications cited above, Karrer retains the

name "xanthophyll" for (I). 248 epoxide to be widely distributed in the vegetable kingdom. However, it should be noted that in some of these papers (see, for example, refs. 18 and

56) the main evidence presented for the suggestion that the pigment was lutein epoxide and not taraxanthin - which has very similar properties - was that the pigment in question reacted with hydrochloric acid to give

(depending on the conditions) either a blue colour or a rearrangement product 66 (see below). Until 1957, it was assumed (on the basis of Kuhn's work ) that taraxanthin was essentially inert to hydrochloric acid; in 1957,

however, it was shown that this acid reacts with both lutein epoxide and taraxanthin in a very similar manner,38 so nullifying its use as a reagent

to distinguish the two.

Karrer and Jucker15 also discovered the characteristic reaction of the epoxides. This is, that treatment of an epoxide (IIa) in solution with a trace of acid causes the epoxide to undergo a rapid and irreversible reaction with the formation of the corresponding furanoid oxide (IIb). [In compound addition, they reported that a small quantity of the parent/(IIc) was formed by the loss of oxygen from the epoxide; since this only became reasonably

(IIa) (IIb) - well authenticated during the work on the p-carotene epoxides, it will be 15 discussed along with those compounds (p.252)]. Karrer and Jucker showed that this rearrangement could be effected merely by dissolving an epoxide in chloroform which had been standing in the light and air for some time (under these conditions traces of hydrochloric acid are produced in chloroform). 249

This reaction has since become a well-known feature of carotenoid chemistry. It has been shown that it is invariably accompanied by a 11,18 hypsochromic shift of approximately 20 my for each epoxide group rearranged, 11 and this has made a useful diagnostic test for epoxides. It should he noted that one or both of two epimers can be formed when an epoxide rearranges to itsfuranoid derivative in this way. An example of this will be given below. The mechanism of this rearrangement, and the effect of concentrated hydrochloric acid on epoxides, will be discussed later (p.261 11 15 25 and p.266, repectively). It was shown by Karrer ' and others that the hydrocarbon, its epoxide, and the furanoid derivative could be separated by chromatography. In general, this is the order in which these three compounds were eluted, using a non-polar eluting solvent (this was not necessarily so 13, if polar solvents were used ); the separation between the hydrocarbon and the epoxide just above it was often rather poor whereas the epoxide was well separated from the furanoid oxide.25'29

15 When Karrer and Jucker treated their synthetic lutein epoxide with acidic chloroform they obtained two products which were identified as 20 21 • and chrysanthemaxanthin. Both these compounds had been 19 isolated previously from Nature, but were of unknown structure. These two compounds were reported to have very similar properties,1 -5 and it was suggested that both pigments could only be represented by formula (III). 15 Karrer and Jucker suggested that in one of these compounds the 3-hydroxyl substituent was disposed cis to the oxygen of the furanoid ring (at C5) whereas in the other pigment it was disposed in the trans manner (cf. the

formation of the two epimers, above).

HO (III) Treatment of zeaxanthin (IV, as its diacetate) with perphthalic acid gave a mixture of two products - a monoepoxide and a diepoxide. These were separated chromatographically, and were shewn to be identical with two OH

NS

HO (IV), zeaxanthin carotenoids that had already been found in Nature. 9 These were antheraxanthin (first isolated in 193522) and (first isolated 23b. crystalline in 1931 from the pansy Viola tricolor 4 respectively.

Violaxanthin was detected spectroscopically (but not crystallised) in Viola tricolor in 1903 by Schunck23a (Schunck's "Y.xanthophyll"). The furanoid oxides corresponding to these two epoxides were mutatoxanthin (zeaxanthin monofuranoid oxide) and auroxanthin (the difuranoid oxide), respectively.

Auroxanthin had been isolated from the pansy Viola tricolor in 1942.24

Mutatoxanthin was not described as a natural product until 1954 when Curl 25 and Bailey (probably) found it in the juice from Valencia oranges.

Mutatoxanthin had been obtained as one of the products from the action of -5a hydrochloric acid in methanol on natural violaxanthin in 19442 (before the epoxidic nature of violaxanthin had been recognised). It was reported at that time that violaxanthin yielded mutatoxanthin, auroxanthin, and zeaxanthin under these conditions (it will be observed that the formation of 251

the mutatoxanthin and zeaxanthin involved the deoxygenation of an epoxide end-group; as already briefly noted this probably does occur to a certain extent in this reaction of epoxides with a trace of acid: cf. the n-carotene epoxides).

Strain57 has since denied that any zeaxanthin and mutatoxanthin are formed by the rearrangement of violaxanthin with acid. However, most of

:.is work was done with ethanolic acetic acid as the catalyst for the rearrangement, and although he mentions that mineral acids effected the rearrangement with much greater rapidity, he does not make it clear whether he also failed to isolate zeaxanthin and mutatoxanthin from the mixture obtained from the latter reaction. It is possible that the strength of the acid affects the composition of the reaction product (just as the composition of the product obtained from acid treatment of a solution of an epoxide differs from that obtained from leaving the solution over alumina: see later • discussion). Strain57 succeeded in isolating two other pigments (probably with the flavoxanthin chromophore: cf. above) from the (slow) rearrangement of violaxanthin as caused by the aPition of ethanolic acetic acid. It would appear likely that they may have been epimers of the half rearrangement product of violaxanthin (i.e., zeaxanthin epoxide —furanoid oxide). The fact that the pigment (which resembled auroxanthin: see above) produced by prolonged action of acetic acid on violaxanthin sometimes tended to separate into two zones on chromatography,suggested that it, too, was a mixture of epimers (as would be expected if the intermediate epoxide-furanoid oxide were). This final product could contain three epimers and it is unlikely 25 that all three would separate. Curl and Bailey - also observed the two 252 intermediate flavoxanthin-like pigments and named them luteoxanthins lay and le.

26 Karrer et a1. also reported that treatment of a-carotene with

per-acids gave a monoepoxide and both this epoxide and its furanoid derivative, flavochrome, were shown to be present in Nature18'26 (mainl y in 19‘ the petals of various flowers ). Also, citroxanthin (a carotenoid first 27\ 28 isolated from orange peel ) was shown to be identical with a furanoid 29 oxide, mutatochrome (V), synthesised from n-carotene (see below).

(V) It may have been noted from the reactions described above that the isolated double bond in the a-ionone end-group of a carotenoid containing both a- and p-ionone end-groups was not affected by per-acid. In general, it has been found that it is the terminal double bonds of the polyene chain 11 which are preferentially attacked in both cyclic10 ' and acyclic3° polyenes.

29 Karrer and Jucker15 also reported that treatment of an epoxide with acid produced not only the corresponding furanoid oxide (in a yield of ca. 4020 but also a small amount (usually about 1) of the hydrocarbon from which the epoxide was first derived. Thus treatment of n-carotene with ethereal perphthalic acid (1.5 atoms of "active" oxygen per mole of polyene) gave a mixture of n-carotene mono- and di-epoxides [and also some of the diepoxide's partial rearrangement product, luteochrome (VI), produced presumably by the action of the weak organic acids (perphthalic and phthalic 29 acids) present in the reaction mixture]. Acid treatment of the 253 monoepoxide gave mutatochrome (V) and p-carotene;

(VI),luteochrome similarly, the diepoxide (VII) gave aurochrome (VIII), an equal quantity of

(VII),p-carotene diepoxide

(VIII),aurochrome mutatochrome (V) (which involves deoxygenation of one of the epoxide rings), and a rather smaller quantity of 13-carotene (which involves deoxygenation 29 of both epoxide rings). The readiness with which the epoxides lose oxygen under these in vitro conditions has important biosynthetic implications (which will be discussed later).

30a The "n-carotene oxide" obtained by Karrer et al. in 1932 (by treating n-carotene with one mole of perbenzoic acid) was probably 11 mutatochrome.

11 Karrer and Jucker have listed ten partially synthetic carotenoids all of which, they have claimed, gave some of the parent hydrocarbon tie well as the furanoid oxide on treatment with acid. In some cases, the only 254 evidence for the identity of the hydrocarbon was a comparison of adsorption and visible absorption properties with previously published values for the hydrocarbons. However, in other cases the formation of the parent hydrocarbon was proved beyond all reasonable doubt (for example, the pigment produced along with flavochrome by acid-catalysed rearrangement of a-carotene monoopoxide was shown to be a-carotene,by crystallising it and performing a 26, mixed melting point determination ). In addition, the isolation of compounds [such AS mutatochrome (V)] where one end-group of a diepoxide had lost oxygen and given the parent cyclohexene ring end-group,provided strong support for the suggestion that deoxygenation of the epoxide can occur.

It is now known that many natural carotenoids are epoxides, or their furanoid oxide derivatives. A carotenoid epoxide is readily recognised by the characteristic effect (mentioned previously) that the addition of a trace of acid has on the absorption spectrum of such a compound (due to its rearrangement to the furanoid form). Because of this lability of carotenoid epoxides towards acid, it is not known how many of the furanoid oxides reported to exist in Nature do exist as such; it seems likely that, at least in some cases, they have been produced by rearrangement of the epoxide during the isolation procedure. It seems probable that those that do exist in Nature have been formed in vivo by 10 rearrangement of epoxides, probably by plant acids. However, whether the furanoid oxides are formed in vivo or during processing does not alter the fact that carotenoid epoxides must now be recognised as being widespread in the vegetable kingdom. (It should be added that there have also been a 255

few reports of carotenoid epoxides occurring in the marine world: cf. the 76 alleged detection of in a skin-borne bacterium of the Arctic Cod, and of a- and p-carotene epoxides in Sardine pilchardus77).

Thus, a broad survey of the carotenoid content of leaves has 31-36 shown that two of the three major carotenoids usually present are epoxides.

'these are violaxanthin (zeaxanthin diepoxide, mentioned previously) and

neoxanthin. Violaxanthin and other epoxides and furanoid oxides [especially taraxanthin, flavoxanthin and chrysanthemaxanthin (III; cf. above), lutein

epoxide, and a-carotene epoxide] also occur widely in flower petals.37a

Violaxanthin, taraxanthin, mutat'ochrome (V), and aurochrome (VII), and other 37b; 34-36 carotenoid oxides have been reported to be present in various fruits. 5,22 Antheraxanthin (zeaxanthin monoepoxide)0 • has been isolated from the anthers of Lilium tifFinum; the antheraxanthin was reported to be accompanied

by capsanthinl 5'22 — this is of considerable biosynthetic interest (see 37c below). Epoxides have been found in the anthers and pollen of other plants.

Of the various epoxides mentioned above, neoxanthin and taraxanthir are of unknown structure. Neoxanthin is a highly oxygenated carotenoid, and, from its chromatographic behaviour, would appear to contain at least two (non-tertiary) hydroxyl groups. As yet, its structure remains unknown although the following proposal (IX) of Goldsmith and Krinsky32 accounts for most of its properties: OH

HO (D) 256

32 It has been suggested that the epoxide foliaxanthin, found by

Cholnoicy et al.34-36 in the leaves and unripe fruits of some varieties of

the yellow and red paprikas, is identical with neoxanthin.

Taraxanthin is also of unknown structure. Chemical evidence

suggests that it is a monoepoxide and contains three hydroxyl groups.38 The

present author has determined the n.m.r. spectrum of a sample39 of

taraxanthin and this suggested that it might have the following structure

(X), although this is by no means certain: OH

In particular, the group of bands near 9 p.p.m. suggested the 2 presence of an end-group of the azafrin (XI) type. Unfortunately, only a

small sample was available and an attempt to effect the acid-induced

rearrangement of taraxanthin to the furanoid form, tarachrome,38 failed to

give sufficient product for a determination of its n.m.r. spectrum.

OZ H

OH (XI), azafrin

The widespread occurrence of epoxides in the vegetable kingdom

has led to the suggestion that they are involved in oxygen transport.34-36

This suggestion receives ready support from the observation (already

discussed)26'29 that treatment of a carotenoid epoxide with acid produces

not only the furanoid derivative but also a small amount of the hydrocarbon 257 with simultaneous loss of oxygen. It is apparent that the conversion of an epoxide into the corresponding hydrocarbon (with loss of oxygen) can also occur in vivo since it has been observed that carotenoids containing an epoxy-3-ionone end group [e.g. p-carotene diepoxide (VII) and luteochrome 40 (VI) show considerable activity as vitamin A precursors. It is generally accepted that a prerequisite of biological activity of this type in a C40 1111,12 12 carotenoid is the presencep of an unsubstituted p-ionone ring therein. however, the footnote on p.167).

34-36 Cholnoky et a1. have studied the carotenoid content of the leaves and fruit of the yellow and red peppers. They found that, during ripening of the fruit, not only did the total carotenoid content increase 37d ) markedly (as is usual for the fruit of plants but also the actual carotenoids present changed. They were able to account for most of their observations on the basis of the existence of two schemes of oxygen transport in the plants.34'35 Both schemes involved carotenoid epoxides; these were assumed to be formed in the plant by oxidation of the carotenes present with oxygen derived from the atmosphere (however, cf. p.264). These schemes were:-

(i) zeaxanthin--->zeaxanthin monoepoxide (antheraxanthin)--41utein

(ii)p-carotene >p carotene monoepoxide--- carotene

In the most recent of the papers under discussion, Cholnoky et 36 al. invoked a third scheme:-

(iii)3-cryptoxanthin (3-hydroxy-p-carotene)-->.p-cryptoxanthin-5,6-

moncepoxide---4a-cryptoxanthin (3-hydroxy-a-carotene)

see footnote on next page. 258

It will be observed that the deoxygenation of the epoxides (which accompanied their donation of oxygen to the plants' photosynthetic apparatus) in these schemes was assumed to give rise to hydrocarbons which were not those from which the epoxides were derived; instead, they were the isomerisation products with a-ionone end-groups. This suggestion is not incompatible with the observed biological activity of the epoxides if it is assumed that the a-end group hydrocarbons were accompanied by the p-ionone analogues which could then have been "recycled" through the schemes.

Alternatively, the deoxygenation mechanism in the plant and in the animal might be sufficiently different as to give different isomers.

Cholnoky et al.34-36 omitted to suggest biosynthetic roles for what they claimed to be a new epoxide (foliaxanthin) and its furanoid derivative (foliachrome) which they isolated from the leaves and unripe fruit (but not from the ripe fruit) of the peppers. These two pigments are of considerable interest since they disappear during the ripening of the 32 fruit. Krinsky has suggested that foliaxanthin is identical with neoxanthin (and, presumably therefore, that foliachrome is identical with

Two monoepoxides can, in theory, be formed from cryptoxanthin; it appears likely that the 5,6-(rather than the 5',6'-)monoepoxide is formed 11 preferentially when cryptoxanthin is treated with per-acid; however, there is no reason to suppose that the mechanism of the epoxidation of carotenoids in Nature (cf. p.264) parallels that of the per-acid epoxidation of these pigments. 259 the furanoid derivative of neoxanthin).

It was assumed that scheme (i) often operated by itself with the other schemes[(ii) and (ill)] only being active when scheme (i) was unable to satisfy the oxygen demands of the plant. Thus in green leaves, all three schemes were assumed to be operating to satisfy the high demand for oxygen, whereas in unripe (chlorophyll-containing) fruit the demand was considered to be less so that only scheme (i) would be required; this would explain, for example, the presence in leaves, but not in unripe fruit, of a-carotene.

As soon as the chlorophyll had disappeared during the ripening process, oxygen would no longer be required by the plant. Scheme (ii) would cease to operate and schemes (i) and (iii) would only operate to the extent that oxygen would be taken up by the zeaxanthin and p-cryptoxanthin and give rise to the two zeaxanthin epoxides (antheraxanthin and violaxanthin) and to p-cryptoxanthin epoxide, respectively. At this stage of ripeness, these epoxides would not give up their (unwanted) oxygen. Instead, the epoxides would either accumulate (in the yellow peppers) or would be converted into thethree polyene ketones:capsorubin, capsanthin, and cryptocapsin (in the red peppers). Apparently, the red peppers possess the ability to effect this conversion, whereas the yellow peppers do not; indeed, this is the reason for the difference in the colour of the ripe fruit of these two kinds of peppers.

Cryptocapsin was described for the first time during the course of this work by Cholnoky et al.34 Both capsanthin and capsorubin, however;

260

had been known for many years, although at the time their structures were

unknown. Many different suggestions had been put forward for the structure

of one or both of these pigments.41 Several of these proposals have since

been excluded by synthetical studies.42 Recently, the structures of both

have been elucidated and shown to be as follows:9'6,43,44 OH

OH OH (XII),capsanthin

(XIII),capsorubin OH This strengthens the suggestions of CholnoXy et al.34-36

regarding the biosynthesis of these compounds. Thus capsanthin might well 5, 6, 43a be formed in Nature from zeaxanthin monoepoxide, perhano 's follows:-

.4,11 —En %....71 • Ho HO HO (XIV) (See below regarding the nature (xv) of RLEn)

Alternatively, the epoxide ring [in (X N)] might be opened first,

and then the resulting glycol could give rise to the cyclopentane ring [in 261

(XV)] through a pinacolic rearrangement.43'45

Similarly, the biosynthesis of capsorubin can be visualised as 36 proceeding through zeaxanthin diepoxide. Cholnoky et al. suggested that crytpocapsin, a minorpolyene pigment of paprika,34-36 might arise from

p-cryptoxanthin monoepoxide. If this hypothesis is correct, then cryptocapsin would differ from capsanthin only in the absence of the hydroxy-substituent in the cyclopentane ring (this compound could be 11 synthesised readily by the base-catalysed condensation5'46 of P-citraurin with 1-acetyl-1,2,2-trimethylcyclopentane5'6). However, this is as yet, speculative, as Cholnoky and his co-workers were unable to prove that the epoxide which they isolated was in fact p-cryptoxanthin monoepoxide, although since it gave, amongst other products, cryptoxanthin on acid treatment, evidence for it being so was strong (cf. however, the footnote on p.258, and also refs. 5 and 44).

+ In the me=chanism suggested above, the symbol g— En represents an enzymic fragment. It seems unlikely that the reaction is catalysed by an acid as such since, as has already been pointed out, carotenoid epoxides are converted by traces of acid into their corresponding furanoid derivatives, probably as follows: 262

It should be noted that Cholnoky's results did suggest that the

formation of capsanthin during the final stage in the ripening of the fruit

of the red peppers was accompanied by the formation of a trace of the

corresponding furanoid oxide (mutatoxanthin, zeaxanthin monofuranoid oxide:

cf. p.25.0). Since, however, the amount of mutatoxanthin formed was only a

fraction (ca. 1%) of the amount of capsanthin formed, it seems unlikely that

the latter was formed as a by-product during the production of the

mutatoxanthin by acid treatment of the corresponding epoxide (antheraxanthin).

Also, nc auroxanthin (zeaxanthin difuranoid oxide) was found to accompany

the capsorubin.

Other biosynthetic processes in which epoxides have been invoked

as intermediates will now be mentioned.

It is well known that the ripening of fruit requires the presence

of oxygen.47 This suggested that the pigments were formed from more 48 saturated precursors by a dehydrogenation process which could only proceed

in the presence of oxygen. Zechmeister49 suggested that this oxygen might

be supplied to the areas of pigment formation through the agency of epoxides.

It has also been suggested that the formation of its epoxide 51 might be the first stage in the natural degradation of a carotenoid. Thus, 50 Glover and Redfearn studied the effect of environment on the carotene

content of excised tomato leaves. The epoxidesof n-carotene, which are 51 only minor conmonents of fresh green leaves, accumulated rapidly in the

leaves if they were left in the dark in the absence of carbon dioxide; there was a simultaneous reduction in the amount of ,9-carotene present.

263

However, as soon as carbon dioxide and light were admitted (so allowing the leaves to resume photosynthesis) the epoxides in the leaves quickly dropped to their normal (low) level but the R-carotene content did not start to increase until after a time lag of about 24 hours. These observations suggested that epoxide formation was the first step in the oxidative degradation of the R-carotene and that the epoxides were not intermediates in the biosynthetic route to 6-carotene:-90-52 (and yet

R-carotene epoxides are apparently converted into 6-carotene fairly efficiently in the animal body judging by their activity as vitamin-A precursors : see p.257).

The observed sudden formation of lutein epoxide in leaves during autumn necrosis has been considered to represent the first stage in the oxidative degradation of lutein - the major leaf carotenoid.33

acid azafrin [(XI), p.256], It has been suggested that the C27 which may be considered to be a degradation product of a C carotenoid, 40 arises by cleavage, in vivo, of a furanoid oxide of the following type

(XVI):_53

0

The mechanism by which epoxides are formed in the plant remains unknown. Karrer et 81.54 suggested that they might be formed by straightforward epoxidation of the cyclohexene ring double bond by a hydrogen peroxide — peroxidase system. However, a recent study of the incorporation 264

of labelled (1802) oxygen and labelled (E/2180) water into two of the

carotenoids (violaxanthin and lutein) of the alga Chlorella vulgaris

suggested that the epoxidic oxygen (in the violaxanthin) was derived, rather

surprisingly, from the water and the 3-hydroxvlic oxygen (in the lutein)

.from the atmospheric oxygen.55 Zechmeister's suggestion (above) regarding

the role of epoxides in the ripening of fruit is not consistent with these

observation: (cf. also p. 257).

Despite all this interest in the biosynthetic role of carotenoid 12 epoxides, the structures of many of these compounds remain unknown. Three 25161,62 epoxides isolated in recent years by Cur1 might be mentioned in this 61 respect. Curl used a counter-current distribution technique in conjunction

with chromatography to separate the complex mixture of carotenoids often

present in fruit into its constituent members. This method was used by 25 Curl and Bailey to isolate several known epoxides es well as two previously epoxides undescribed/from the juice of Valencia oranges. The two new epoxides were

notable for the shortness of their ehromophores. They were named

valenciaxanthin and sinensiaxanthin and probalqy contained five and six

double bonds respectively, in conjugation with the epoxide groups. Both

were shown to be monoepoxides by measuring the shift in their absorption

spectra on treatment with acid. Their furanoid oxides were called

valenciachrome and sinensiachrome, respectively.

62 Cur1, using the technique mentioned above, detected over thirty

polyenes in a variety of the peach. Several epoxides were found including

one (which was named persicaxanthin) with an absorption spectrum identical 2G5 with that of valenciaxanthin but 1-1-lich war not that compound. It seemed likely that valenciaxanthin, sinenFliaxnnthin, and persicrixant.hin were not

C carotenoids. It is tempting to suggest that these epoxides were 40 derivatives of polyenes such as P-apo-14'-carotenal. Winterstein and his 14' 15s• `s. N. • '•. '15' 11-apo-14'-carotenal co-workers63 have found similar polyene aldehydes (but with rather longer polyene chains) in some citrus fruits. c-Carotene (see formula VII, p.91 ) 61 was also found in the juice of Valencia oranges, and sinensiaxanthin might, alternatively, be the 9,10-monoepoxide of this compound.

The structures of neoxanthin,31-33,67 violeoxanthin (from 12 pansies57'64), tareoxanthin (from the dandelion57), and petaloxenthin11' are also unknown; all are apparently epoxides. (Trollixanthin has recently been assigned a structure38).

In particular, it would be most useful to know the structure of neoxanthin - an epoxide already mentioned es being present in most leaves and possibly identical with Cholnolcy's folinxanthin (p.258).

It was apparent that compounds with epoxide and furanoid oxide end-groups should be included in the survey it was proposed to make of the n.m.r. spectra of carotenoids (and related compounds) if this survey were to be at all comprehensive. It was decided, therefore, to prepare the oxides derived from P-carotene. This work also resulted in n-carotene diepoxide being prepared from p-carotene in reasonably good yield for the first time.

The two furanoid oxides [mutntochrome (10 and aurochrome (VIII)] were 266 prepared by acid treatment of the diepoxide (as described by Karrer and 29 \ Jucker ). At the same time, Akhtar53 synthesised two carotenoid epoxides and one furanoid oxide during his attempt to achieve a total synthesis of azafrin (XI). Thus, in all, three epoxides and three furanoid oxides became available for spectraimeasurements. This provided an adequate representation of these two important classes of compound so that the chraracteriatic band pattern due to the two types of end-group can now be recognised in an n.m.r. spectrum.

Before a description is given of the methods available for the preparation of the carotenoid epoxides, a test for these compounds should be mentioned.

10 15 Karrer ' showed that if a solui-ion of an epoxide (or its furanoid derivative), usually in ether, was shaken with concentrated hydrochloric acidl the acid layer became blue. The depth of the colour, the rapidity with which it was formed, and its stability once formed, seemed to depend on the oxide being tested. It should be added that some of these compounds might only give a blue colour if the test is carried out under carefully controlled conditions. For example, lilugster and Karrer38 showed

More recent work has suggested that diepoxides give a more intense colour 11 than monoepoxides, although with the structures of so many epoxides being uncertain this can hardly be said to be a proven rule. The intensity of 25 the colour has been used, however, by Curl and Bailey to distinguish between mono- and di-epoxides using carefully standardised conditions. 267

that taraxanthin readily gave a blue colour providing the acid was

sufficiently concentrated, and the mixture was prevented from becoming hot

during the mixing of the ether solution and the acid by external cooling. 66 Kuhn and Lederer had earlier reported that taraxanthin did not give a blue 76 colour. (cf. Goodwin et s1. who failed to observe the blue colour with

what they supposed to be neoxanthin).

It should be added that this test is not specific for carotenoid

oxides: some polyene aldehydes and some polyhydroxycarotenoids [for example,

azafrin (XI)" and fucoxanthin78a] have also been reported to give a blue

colour with hydrochloric acid. However, the test is useful to the extent

that if negative it shows tat the substance being tested is, almost

certainly, not an epoxide (or furanoid oxide). It should be added that the

blue coloursgiven by mutatochrome and n-carotene diepoxide can be

distinguished readily from that given by fucoxanthin by their visible-light

absorption spectra.78 The present author has shoWn that although all three

spectra consist of a single broad absorption band, the position of maximal

absorption in each of the first two spectra mentioned (572 and 593 IT, respectively78b) differs significantly from that in the spectrum of the blue

solution give by fucoxanthin ( A near 6S0 mp788 ). max. 23a It is interesting to note that as early as 1903,Schunck had

observed that his "Y xenthopiy11" (which was almost certainly violaxanthin)

gave a blue colour with hydrochloric acid. (Schunck also mentioned the way in which the colour of an epoxide solution fades markedly as soon as the acid is added: cf. the Ekperimentni seet4.on). PVPT1 earlier, Sorhy," in 268

1873, had reported obtaining a deep blue colour on treatment of the (yellow)

extract of yellow chrysanthemum flowers (cf. the chrysanthemaxanthin isolated 21 by Karrer et al. ) with hydrochloric acid, and had compared it with the

blue colour given by fucoxanthin under the same treatment.

The present author carried out the following test on the blue

colour given by P-carotene diepoxide. The hydrochloric acid solution of

the blue colour was washed free of any unchanged pigment with ether. The

acid was then treated with dilute alkali whereupon the blue colour was

discharged. A yellow colour sim,ltnneol:sly appeared in the ether (which

had been liberated from the acid solution during basificaion). The yellow

solution showed a rather ill-defined two-banded spectrum with maxima (at

41 5 and 396 up,in ether) at rather shorter wavelengths (by about 5 np) than .5-carotene difuranoid oxide (aurochrome).

At the time that this work was started, there existed two

recognised methods of preparing 5-carotene diepoxide. These were:

(a) by treatment of 5-carotene with ethereal perphthnlic acid according 29 to the directions of Karrer and Jucker. These authors separated the

complex reaction product by repetitive chromatography and eventually they

obtained pure samples of the monoepoxide, the diepoxide, and luteochrome (VI),

all in poor yield (5 to 101, however. This method has recently been used

by Tsukida and Zechmeister" (and equally poor yields were obtained).

(b) by direct aerial oxidation of P-carotene. Thus, Hunter and 65 Krakenberger studied the effect of passing oxygen through a hot solution of

5-carotene. If this treatment was prolonged, the fi-carotene was completely 269 bleached. However, if the reaction was interrupted before this stage a complex mixture of P-carotene epoxides and their rearrangement and degradation products could be isolated. The oxidation occurred much more quickly in benzene than in arachis oil (which contains anti-oxidants). The yields obtained were not reported.

The reported occurrence of furnnoid oxides in the reaction product can probably be ascribed to thn rearrangement of the epoxides, both during the reaction period itself and during the subsequent chromatographic purification. Thus, Tsukida and Zechmeister13 have reported that their samples of the f-carotene epoxides underwent rearrangement to the furanoid forms both in hot solvents and whilst in contact with alumina (see p.p.276 ,

277).

Hunter and Krakenberger's product also contained P-carotenone

(XVIIa) and semi-r-carotenone (XVIIb).65

R.(4), (3

)‹.

R = ( s), (0/II)

It was apparent that of the t:•ro methods [(a) end (b); descried above, the former method would give a rather less complex reaction product.

Accordingly, P-carotene was treated with dry ethereal perphthalic acid 29 [method (a)] according to the directions of Karrer and Jucker. Karrer and

Jucker chromatographed their crude product on lime. Apparently, the various 270

carotenoid oxides present did not separate from each other at all well using lime as adsorbent and so the present suthor used an alumina column instead in the hope that this adsorbent would effect a cleaner separation. Although the alumina was of the "acid-washed" type, it gave an alkaline reaction on shaking with ethanolic phenolphthalein. It was hoped, therefore, that the epoxides would not undergo their characteristic acid-catalysed rearrangement reaction; unfortunately, however, they did.

The major product isolated from the chromatogram was identified

(by spectral comparisons end a mixed melting point determination) as 1.-carotene.

Many other zones were observed higher on the column. The visible absorption spectrum of one of these -suggested that it might have been luteochrome (VI); this zone gave a blue colour with hydrochloric acid (of. above, regarding the- test for epoxides). An accompanying (and minor) zone which appeared just below this,luteochrome-likd zone had absorption maxima desplaced a little towards the ultraviolet, suggesting that it might have been a cis-luteochrome.

Neither of the n-carotene epoxides was detected.

53 Meanwhile, Akhtsr was finding that his attempts to purify another polyene epoxide (XVIII) through chromatography on alumina (of the sane type as used by the present author) were eaually unsuccessful. However, Akhtar went on to show that the epoxide (XVIII) could be prepared in a pure state

CO2Me

without using column chromatography. He treated the corresponding C27-ester 271 with a two-fold excess of perphthelic acid (rather than just a small excess P1 as advocated by Kerrer et a1.- ). The organic acids were then removed in the usual way, and the epoxide (XVIII) was isolated in good yield (60r":) by direct crystallisation.

The present author decided to apply this method to F.-carotene. With this compound, of course, there was the complication that two epoxides could be formed. However, despite this, it was found that the diepoxide could be prepared in fair yield (45%) by treating n-carotene with an excess of perphthalic acid (4 atoms of "active" oxygen per mole of P-carotene, for

24 hours). The product was isolated (in the same ray as above) as a pink-red crystalline solid. This was shown to be free of n-carotene by chromatographing a sample on alumina (IV) -rith lip-ht petroleum as eluant

(under these conditions any deliberately added A-carotene was easily detected).

However, the product did contain a trace of A-carotene monoepoxide. This was demonstrated 1,y chromatoprephing P sample nn lime; it gave only n, single zone on the column but the first few drops of eluate from this zone had absorption maxima at slightly longer wavelength than the rest of the zone.

In nOdition, the n.n.r. spectrum (which will be discussed later) did show two small spurious peaks near 8.28 end 8.97 p.p.m. These could be 2 most readily ascribed to an end-group of the F-carotene type, as does occur in 13-carotene monoepoxide. The intensity of these two peaks suggested that if the impurity causing them was the monoepoxide, then it was present to the extent of 15-240 of the diepoxide (that is, the diepoxide sample was 80-85;:; pure).

An attempt was made to obtain the diepoxide free of the 2 7 monoepoxide by treating P-carotene with per-acid under more vigorous conditions than those used above. However, when s-carotene was treated with a larger excess of et cereal. perphthalic acid (6 atoms of "active" oxygen per mole of n-carotene) for a longer reaction period (60 hours) at room temperature, the product consisted of a mixture of the diepoxide and its rearrangement products (mainly aurochrome). If the diepoxide had been required analytically pure itvrould have probably been better to prepare the

crystalline diepoxide in the usual way (cf. above) and then to have treated

this with a further portion of per-acid.

It would be more difficult to obtain the monoepoxide by the method outlined above since the reaction product would probably be a mixture of

three compounds - r-carotene, the monoepoxide, end the diepoxide. However, in the present work there was no need to prepare the monoepoxide since both mutatochrome (V) end aurochrome (VIII) could be obtained by acid treatment 29 \ of the diepoxide (as described by Karrer and Jnoker ). This was done and the product (which was mainly a mixture of`.?-carotene, mutatochrome, and aurochrome) was readily separated into its constituents through chromatography on alumina. The fun-mold oxides were apparently unaffected by this 29 procedure (cf. the epoxides, above); Karrer and Jucker used lime columns.

The yields obtained by Karrer and Jucker and by the present author were approximately the same (about 25`' for each of the two furnnoid oxides and about 55 for the -carotene).

The fl-carotene diepoxide, mutatochrome, and aurochrome all crystallised well. During the crystallisation of the diepoxide, every 273 effort was made to ensure that the length of time that the solution was hot was restricted to a minimum since it was suspected that prolonged boiling might cause some rearrangement. This suspicion has been confirmed by 13 Zechmeister et al. who have shown that after boiling a solution of the diepoxide for one hour it was partly (to the extent of 11cM converted into a mixture of the three furanoid oxides - luteochrome, aurochrome, and mutatochrome.

Throughout the work with the epoxides it was essential to exclude any traces of acid from the apparatus. In general, glassware was rinsed out with aqueous sodium hydrogen carbonate, then distilled water, acetone, and finally light petroleum which had been distilled from alkali.

These precautions were necessary because of the extreme lability of epoxides towards acid. It is perhaps significant that on the one occasion when these precautions were not taken, the diepoxide sample which was being recrystallised underwent rearrangement during this process; this was heralded by a change in colour of the solution from dark red to a distinctly greenish red.

The pure diepoxide was quite stable (in the absence of acid) both as a solid and in solution. 'Thus the melting point of a sample of the pure diepoxide dropped by only about one degree during a period of three weeks in the air in the dark at 00. A solution of the diepoxide in benzene was once kept at room temperature for three weeks (mainly in the dark); at the end of this period the absorption spectrum of the solution was quantitatively identical with that of the freshly prepared solution. 274

In some ways the two furanoid oxides handled seemed to be rather

less stable than the diepoxide. Thus their solutions tended to assume a

dark greenish-brown colour during crystallisation (a phenomenon also observed 60 by Akhtar when working with another furanoid oxide). On one occasion, a somwhat impure sample of aurochrome which had been collected from a chromatogrem was left in solution (mainly at 0°) for one week. At the end of this period no aurcchrome could be detected (by chromatography) in the sample: it had apparently completely decomposed under these conditions.

Even pure, crystalline Evirochrom was rather unstable; exposure of a sample to air and dim light overnight caused its melting noint to drop by eight degrees. However, as is often the case with carotenoids, it appeared that these compounds were even less stable When impure. Thus a pure sample of mutatochrome was left exposed to the air and light for 24 hours on one occasion without there being any visibly obvious deterioration of the sample; a slightly impure sample (its melting point was four degrees lower than that of the pure material) deve-loped a greenish-brown colour under the same conditions.

Once the diepoxide was obtained in a reasonably pure state some chromatographic tests were carried out on it. It soon became obvious why the earlier attempt to purify the compound by chromatography on alumina had failed. The alumina (which was grade IV79) used in that instance had been previously washed with dilute hydrochloric acid, then water, and finally methano1;79 it had given an alkaline reaction with ethonolic phenolphthalein.

A sample of the crystalline diepoxide was chromatogrephed on a 275 small column of this alumina using a rapid flow rate and a strong eluting agent (strong for the diepoxide: a 50/50 mixture of benzene and light petroleum). Under these conditions, all the pigment put on to the column was eluted within 30 minutes. Two zones formed. The majority of the pigment was in the lower zone which was shown to be unchanged diepoxide (by comparison of absorption spectra; and also see below). The absorption spectrum of the upper zone indicated that it was l'iteochrome [the half- rearrangement product, (ITI)]. It had previously been shown that the sample of diepoxide put on to the column was free -'f luteochromn by determining its n.m.r. spectrum (by which the presence of quite a small quantity of a furanoid oxide impurity in the diepoxide could easily have been detected: see later). On Another occasion when the diepoxide yes chromatographed on alumina, the chromatogram was developed rather more slowly so that the diepoxide zone was on the column for nearly 90 minutes. Despite this, a not inconsiderable amount of the diepoxide was recovered (quantitative experiments were not carried out). On this occasion, the identity of the diepoxide recovered from the column was proved not only by a comparison of absorption spectra but also by crystallising the substance and showing that it gave no melting point depression on admixture with an authentic sample of the diepoxide.

However, if the diepoxide was allowed to remain on the column long, more extensive rearrangement of the diepoxide occurred. Thus, on one occasion a sample of the diepoxide was adsorbed nn alumina(of the same type as above) and left for 18 hours. mhe pigment was then elu,ted with benzene. 276

The . pigment eluted from the column absorbed at almost 50 my shorter wavelengths than the diepoxide, and its absorption spectrum was almost superimposable on the spectrum given by aurochrome (IrIII)I showing that both epoxide rings had rearranged. The spectrum also showed that the pigment eluted contained a negligible amount of mutatochrome. This is rather surprising in view of the fact that acid-catalysed rearrangement of the diepoxide has been shown to give equal quantities of eurochrome and mutatochrome (cf. previous discussion). Obviously, however, the rearrangement described here was not caused by residual acid in the alumina • snce chromatography of the diepoxide on alumina w;tich had been prepared 0y washing commercialnactive"alumina just with methanol (and not with acid as well, as before) also caused the epoxide to undergo some rearrangement. It is most unlikely that this alumina could have contained any traces of acid.

13 Meanwhile, Tsukida and Zechmeister published some work on the stereoisomerisation of the 3-carotene epoxides; their paper included some observations on the stability of these compounds when in contact with active,

"strongly basic" alumina. Their results supported the qualitative observations of the present author in that the epoxides underwent rearrangement to furanoid oxides even on an adsorbent which could not have contained any acid. In oneexperiment, they left a solution of p-carotene diepoxide in hexane over the alumina for 24 hours. The Pigment was then eluted and separated into its constituents (by chromatography on lime). This gave aurochrome (6-5;74) and three of its cis isomers (total, 5%), mutatochrome

(0.3;4), luteoChrome (51/, and F-carotene monoepoxide (2.21; the remainder 277 of the pigment (35;) underwent decomposition. The isolation of a little of the monoepoxide is especially interesting since this provides a the straightforward example of the deoxygenation of an epoxide end-group to giveh corresponding hydrocarbon end-group. These findinns also confirm the suggestion implicit in the present author's observations. This is that under acid catalysis P.-carotene diepoxide rearranges according to the scheme:-

(-carotene diepoxide-->mutatochrome (a deoxygenation product) + aurochrome

(in equal amounts) + a small amount of the hydrocarbon (P-carotene),

whilst on being adsorbed on an active, basic surface the diepoxide rearranges, more slowly than with acid, es follors:-

P-carotene diepoxide-->luteochrome--.)eurochrome, with on17 small

amounts of deoxygenation products (the monoepoxide and mutatochrome).

67 It is noteworthy that Krinsky and Goldsmith reported the apparently successful isolation of the epoxide neoxenthin by chromatography on alumina (the acldve commerical material was used after it had been

"weakened"by treating it with water). It may be that neoxanthin is more stable than the n-carotene epoxides to the influence of active surfaces as catalysts for promoting the rearrangement of epoxidesto furanoid oxides. 13'15'29 25 Lime or magnesia columns would seem preferable for the resolution of mixtures containing carotenoid epoxides.

13 Tsukida and Zechmeister showed that the n-carotene epoxides also underwent some rearrangement to furanoid forms on melting the crystalline solids in vacuo, on heating to 700 it collation, or on illuminating the solutions either in the presence or absence of catalytic 'amounts of 278 iodine. The extent of rearrangement ranged from 20-30°/on melting the solids to 1-5 on illuminating the solutions under various conditions. In the latter case, iodine had a distinct, but small, catalytic effect on the rearrangement reaction. In all cases, some of the pigment sirultaneously underwent decomposition. It is interesting to note that the rearrangement of another epoxide (trollixanthin) to its furanoid derivative (trollichrome) is very susceptible to iodine catalysis. Thus, the addition of a trace of iodine to a solution of trollixanthin resulted in the spectral changes caused by the rearrangement of the epoxide to overshadow those caused by its stereoisomerisation.38'59

The n.m.r. spectra of the (?-carotene dienoxide, mutotocnrome, and aurochrome samples were all determined initially at 40 Mc./sec. However, some time after these measurements had been made the spectrometer vas modified so as to operate at 56.4 ./sec. It was shown2 that better separation of bands could be obtained (and also that less sample was required) at 56.4 Mc., and so the measurements were repeated at this higher frequency.

Any differences observed in the spectra measured at the two different frequencies are mentioned in the text, below. Deuterochloroform was used as the solvent for eaci, cf the determirations. The samples were recovered after each n.m.r. determination; in each case, their visible-light absorption spectra were shown to be unchanged. To do this, the deuterochloroform 'as evaporated and the residue was then disolved in benzene. This check was used to show that the diepoxide had not partly undergone rearrangement during, or before, the measurement of the apectrum(as already mentioned, polyene 279 epoxides are characterised by the readiness with which they undergo rearrangement to the furanoid form on being exposed to traces of acid).

Puranoid oxides have also been shown to be very sensitive to acid, and it was hoped that this check would show whether they had survived the n.m.r. determination unchanged. However, it has since been shown that a furanoid oxide solution which has become distinctly greenish can give a perfectly

"satisfactory" absorption spectrum after being recovered in the way outlined above.

Before the n.m.r. spectra of 1-carotene diepoxide, mutatochrome, and aurochrome are discussed, the bands occurring in the spectrum of the parent hydrocarbon, F-carotene, will be considered.

246 P-Carotere (XL6 haC earlier been shown to absorb (in deuterochloroform) at 8.03, E3.2E3, and 8.97 p.p.m. (with relative peak areas of 2:1:2). A consideration of the n.m.r. spectra of a large number of

8.97 8.97 8.03 8.03

(XIX), ft-carotene polyenes of known structure had led to the conclusion that an "in-chain" methyl group (XX) gives rise to an absorption band near 8*0 p.p.m. (this assignment has already been discussed in Section I of this thesis). 2

Methyl groups attached to saturated carbon have been shown to give 1 rise to bands near9'1 p.p.m. The only methyl groups which fall into this 280 category in the n-carotene molecule are the E2E-methyl groups. The fact that they gave rise to a band at rather lower fields than 9.1 p.p.m. led to

the suggestion that these groups must be de-shielded to a small extent by the neighbouring double bonds (in particular, the ring double bond). The cyclohexene ring would be expected to undergo rapid interchange between two equivalent conformations and this is reflected in the fact that the two gem-methyl groups have been shown to give rise to a single sharp absorption band indicating that the two groups experience identical environments.

The broad survey of the n.m.r. spectra of polyenes (mentioned above) also showed that the "end-of-chain" methyl [cf. (XXI)1 at C5 in an end-group of the P-ionone type (8 "P-end group") normally absorbs near 8.28 2 p.p.m.; the band at 8.28 p.p.m. in the n.m.r. spectrum of f-carotene has, Me

(xxi) therefore, been assigned to the two 5-methyls in this compound.

It is noteworthy that "end-of-chain" methyls in cyclohexene rings have always been found to give rise to a band (near 8.28 p.p.m.) at rather higher fields than those attached to the end of en acyclic polyene chain

(which absorb near 8.20 p.p.m.2'7). The probable reason for this difference, and for the occurrence of the "end-of-chain" methyl band at similarly high fields in furannid oxides(cf. p. 28S), will be briefly elaborated.2

It has been suggested (in order to explain some otherwise anomalous observations) that the n-electrons of a carbon-carbon double bond 281

68 can exert a long-range shielding effect. It was shown that if this effect really did operate, then a proton in the plane of the double bond would be de-shielded (and this would explain the obnervations alluded to above). A comparison with the long-range shielding effects known to be 1 exerted by the double bond of the carbonyl group suggested that a proton in certain regions of the space above (or below) the plane of a carbon- carbon double bond would be positively shielded by the n-electrons; the limited experimental evidence available provided some support for this 168 suggestion. A proton affected in this way would abso:b at higher fields than world otherwise have been expected. Models have indicated that the protons of the "end-of-chain" methyl in a r-end grou'p are located above the plane of the 7,8-double bond of the polyene chain in such a way as to experience this positive shielding effect. This would not be true of

"end-of-chain" methyls in acyclic ,-olyenes; such aroups are not expected to be restricted to taking up only certain conformafions rolaiye to the' rest of the molecule.

The n.m.r. spectrum of the diepoxide as determined at 40 Mc. was identical, within experimental error,K with that determined et 56.4 Mc. The.

T values ouoted in the text below are actually those which were obtained at

56.4 Mc./sec. Both spectra showed small spurious peaks near 8.28 and 8'97 p.p.m. which were probably caused by the presence of traces of monoepoxide in the diepoxide samples (cf. p.271). The sample used for each of the

K [A difference of f 0'025 p.p.m. between two spectra has usually been 1 considered tolerable, although in the present work better agreement than this has usually been obtained]. 282 determinations had been freshly prepared (using the same method); each of the samples had the same physical properties.

The diepoxide (VII) absorbed et 8.05, 8.85, 8.90, and 9.06 p.p.m. with relative peak areas of 2:1:1:1. The peak at 8.05 p.p.m. can only, reasonably, be assigned to the "in-chain" methyl groups (which, in '-carotene, 2 have been shown to absorb at 8.03 p.p.m. ). This peak showed signs of multiplicity end appeared to consist of two equally intense peaks with

17 values almost but not cuite, identical. The corresponding band in

P-carotene is sharp, so it would appear that the observed broadening of this peak in the diepoxide is caused by the epoxidic oxygen atoms influencing, slightly, the protons in the methyl groups attached to carbons 9 and 9' in the polyene chain.

5

n-carotene diepoxide

The end-group methyls in the diepoxide gave rise to a completely different pattern from those in fl-carotene itself. This difference can only be ascribed to the presence of the epoxide ring.

The 5-methyl group in P-carotene diepoXide is attached to an oxygen-bearing carbon atom. It has previously been shown that methyl groups in this type of environment experience a de-shielding effect from the 1 2 electrons of the oxygen atom. ' Thus, the gem-methyl groups in the 69 following two compounds {( XXII) and (XXIII)] also absorb near 8.85 p.p.m. 283

The presence of the epoxide ring in the end group would be

expected to place a restriction on the six-membered ring undergoing rapid

interchange between two eanivalent conformations (as can end does occur in

a normal rend group). Presumably, the proxy-end gr'up takes up that

conformation in which the steric interference between the various groups

present is a minimum. The two Em-methyls in this end group are exposed

to two potential de-shielding influences - the double bonds of the polyPne

chain and the apoxidic oxygen (under certain conditions the polyene chain

might positively shield the gem-methyls: cf. the possibility, already noted,

of a positive shielding effect being exerted by the 7,8-double bond on the

5-methyl group in P-carotene). It might be expected that in the preferred

conformation of the end group, the overall de-shielding effect experienced

by the two ma-methyls might be different, in which case they would give rise to two separate hands. This is what is observed in the spectrum of

fl-carotene diepoxide,where the two gem-methyls give rise to bands at 8.90 and 9.06 n.p.m., indicating that both methyls are de-shielded to a certain

extent but that one is rather more de-shielded (probably by being in the

plane of the double bonds of the polyene chain) than the other.

The band pattern caused by the end-group mei.hyls in another epoxide [(XVIII), see p.2719153 had previously been examined. The spectrum of that compound had been determined at 40 Mc. but evenlso the absorption 284 caused by the end-group methyls was resolved into three distinct peaks

[7- values found, for (XVIII), were 8'86, 8.90, and 9'06 for these three 2 methyl groups J. The band pattern was virtually identical with that caused by the end-group methyls of P-carotene diepoxide. As already mentioned, the absorption caused by the latter methyls _ was also resolved into three bands on measuring the spectrum at the lower frequency (40 Mc.).

It can be concluded, therefore, that the epoxy-P-ionone type of end-group gives rise to three bands of equal intensity near 17values of

9.06, 8.90, and 8.86 p.p.m. These have been assigned, tentatively, es follows (the corresponding values for the unsubstituted P-ionone end-group are included for comparison purposes):

9-06, 8-90 (8.97) (8.97) CH3 CH3 CH3 CH3

CH3 CH3 (8.80 (8.28) It should be added that the actual assignments for the epoxide end-group are tentative; all that is I-ease-m.0)1y certain is that the 9.06 p.p.m. band must be assigned to one of the gem-methyls. It is conceivable

(though less likely) that the 5-methyl group might have caused the 8.90 p.p.m. band (and one of theme-methyls the 8.85 p.p.m. hand). with regard to this last point, it should be added that in an epoxy-aldehyde (XXIV)7° 2 which had earlier been examined, the bands expected near 8.85 and 8.90 p.p.m. were not resolved at 40 Ile.; instead a ningla hand was :7,bncirvnd at 8'96 285 p.p.m. Unfortunately, the spectrum was not measured at 56.4 Mc., so it is not known whether this band might have been resolved into two bands at this higher frequency.

The n.m.r. spectrum of aurochrome (VIII) was, as to be expected, markedly different from that of the diepoxide. The spectrum as obtained at

40 Mc. will be discussed first: the effect of repeating the determination using the enhanced resolving rower obtainable at 56.4 Ir.c. will be discussed afterwards.

The spectrum showed four bands in the methyl proton region; these occurred at 8.07, 8.27, 8.58, and 8.90 p.p.m., with relative peak areas of 1:1:1:2. The band at 8'07 p.p.m. can only be ascribed to the 2 "in-chain" methyls. The band at 8.27 p.p.m. is at rather higher fields than is usual for a methyl group attached to the end of an acyclic, polyene chain (a methyl of this type has usually been founc to absorb near 8.20 p.p.m.2'7). This shift to higher fields is apparently a genuine effect since the "end-of-chain" methyls in two other furanoid oxides [mutatochromeTish

(see below) and an ester (XXV)] also occurred near 8.27 p.p.m. dia-magnetic shift can probably be ascribed to positive shielding of the

x6 2

3 NV5 . o 4- (VIII), aurochrome 286

CCaM e

(KtV)

"end-of-chain" methyls in furanoid oxides by the 6,7-double bond of the furanoid oxide ring (cf. the earlier suggestion regarding the positive shielding of the 5-methyl in a (3-end group by the 7,8-double bond: p.281).

The band at P•90 n.p.m. must be caused by methyl groups attached to saturated carbon. The intensity of the band confirms that it should be assigned to the two gem-methyls which are present in each of the aurochrome end groups. This band showed signs of multiplicity at 40 and was resolved into two major and one minor (cf. below) bands at 56.4 1c. It might be expected that each of the two gem-methyls of the furanoid oxide end group would receive lifferent overall de-shielding effects from the nearby groups (as in the diepoxide), and, therefore, that these groups would give rise to two separate bands. This will be further discussed below.

The P.58 p.p.m. band can, then, only be ascribed to the 5-methyl group. This methyl will be de-shielded by the nearby oxygen atom as it is in the epoxy-end group. However, in the furanoid oxide, the shift in the band position away from that expected for a methyl attached to saturated carbon is twice that observed in the diepoxide. This additional de-shielding

of the r-methyl group in the furanoid form is probably caused by the double

bond in the furanoid oxide ring (cf.above). 287

As has already been mentioned, the formation of a furanoid oxide ring from the parent epoxide can give rise to two different epimers. The extent to which the double bond and the oxygen atom in the furanoid oxide ring de-shield the methyl groups at positions 1,5, and 9 will depend on the spatial disposition of these various groups relative to ono another. This might explain why the bands caused by the methyl groups at each of these positions showed signs of multrolicity, even at 40 Mn. This tendency was even more pronounced at 56.4 Mc.

At 56.4 Mc., the 8.90 p.n.m. band was resolved into three bands, which occurred at 8.89, 8.84, and 8.74 p.p.m. (with relative intensities of ca. 6:5:1);the8.58 p.p.m. band was just resolved into two :-;harp peaks (at

8.56 and 8.54 p.p.m. at 56.4 1'c.) of similar intensity; and the 8.27 p.p.m. band was just resolved into two peaks (at 8.27 and 8.20 p.p.m.), that at

8.20 p.p.m. being markedly weaker than its companion. It should be added that in the region of the 8.27 p.p.m. band there were other weak, ill-defined absorption bands. This feature is not uncommon in carotenoid spectra, and has been ascribed to absorption by methylene protons which, because of complex spin-spin coupling interactions, do not normally give rise to

well-defined absorption bands.2'71 The superposition of the 8.27 p.p.m. on theseweak methylene absorption bands might have given rise to the weak band (at 8.20 p.p.m.) which accompanied the R•27 p.p.m. band. It is unlikely that interference from methylene absorption could explain the splitting of the 8.58 p.p.m. band since here each of the two peaks was relatively strong.

Also, the observed splitting of the 0-9 p.p.m. peak is almost certainly 288

genuine since methylene absorption rarely occurs at fields as high as this

in carotenoid spectra. It is notable that the corresponding band in the

spectrum of mutatochrome, although not resolved into two peaks, did show

distinct signs of multiplicity (see below).

Since aurochrome contains two furanoid oxide end-groups, there is

obviously the possibility that the sample cauld contain several different

epimers. It is, therefore, not surprising that the bands in the 8'9 p.p.m.

region were not so well resolved as those in the diepoxide. At the moment,

it is difficult to say whether the presence of two major hands(at 8.89 and

8.84 p.p.m.) in this region indicates that the two gem-methyls are grossly

non-equivalent (and, as in the diepoxide, give rise to a separate band each),

or whether, by chance, these are equally de-shielded and their splitting is

caused by there being a mixture of epimers present in the sample. The

observed multiplicity of the band near 8.58-p.p.m. and the appearance of a

weak band at 8.74 p.p.m. suggests that, in any case, the sample was a mixture

of epimers.

Finally, it should be added that although the sample used for the

40 Mc. determination was recrystallised in the usual way, that used for the

56.4 Mc. determination was freshly chromatographed but not crystallised.

,7hese different treatments might have had some effect nn the proportion of

various epimers present in the n.m.r. sample.

Mutatochrome (V) was expected to give a particularly interesting

spectrum since this compound contains two different end-groups.

At 56.4 Mc., bands were found at 8.05, 8.28, 8.57, 8.90, and 289

8•97 p.p.m. with relative areas 5:2:1:2:2. Of these, the peak at 8.57 p.p. m. showed distinct signs of multiplicity with a pronounced shoulder at 8.54 p.p.m. (as in the spectrum of aurochrome); the 8.90 p.p.m. peak had a distinct shoulder at 8.84 p.p.m. and, therefore, bore the expected resemblance to the aurochrome spectrum. It is noteworthy that the band at 8.28 p,p.m. in the mutatochrome ppectrum was quite sharp. This band must have been caused by the two "end-of-chain" methyl groups - that in the rend group (the 5-methyl) and that attached to the carbon at position 9 .

It will be remembered that "end-of-chain" methyls in acyclic systems normally absorb near 8.20 p.p.m., and the positive shielding effect of a nearby 8-90,8-84 CH CHT (4.85)

.71/ CH3 (8 28)

5' N•„// 0 CH3 (8.05) c 145 (&05) CH5 c H3 (4.85) (8-57)cH3 H (8-97) (8.97) (V), mutatochrome

tr.\ O

00

Then.m.r. spectrum of mutatochrome at 56.4 Mc./sec. 290

double bond was invoked to explain the observed diamagnetic shift in each

case (the n-electrons of the 7,8- and the 6',7'-double bonds, respectively,

being responsible). The sharpness of the 8.28 p.p.m. band shows that the

extent of positive shielding in each case is, fortuitously, identical.

The gem-methyls in the 11-end group give rise to the 8.97 p.p.m.

band, and the "in-chain" methyls give rise to the 8.05 p.p.m. band. The

peak near 8.57 p.p.m.. can only be ascribed to the 5'-methyl in the furanoid

oxide end group. The fact that this band shows signs of multiplicity nay

again be ascribed to the presence of different epimers in the sample, as in

the case of aurochrome.

At 40 Mc., alsop the 8.57 p.p.m. band showed signs of multiplicity

with a shoulder on the low-field side of the band; however, the 8.90 p.p.m.

bond was no longer well resolved from the 8.97 p.p.m. band but instead was

present as a pronounced shoulder on the side of the 8.97 p.p.m. band. The samples used for the 40 Mc. determination and for the 56.4 Mc. determination

had identical physical properties.

The spectrum of the furanoid oxide (XXV) that had been determined

previously showed the some general featuresas the two furanoid oxides

discussed ehove.

A conspicuous feature of each of the three furanoid oxide spectra

was a single sharp band in the olefinic proton region near 4.85 p.p.m. The

intensity-of this band was found to be consistently equivalent to two protons

for each furanoid oxide ring present in the molecule. This band was well 291 separated from the other olefinic Protons (which cave a rather broader band near 3.6 p.p.m.) in each of the spectre. It has been assigned to the two protons attached to the furanoid oxide ring; the resonances of these protons must be accidently degene"rate, so that no spin-spin coupling is observed.

The characteristic features of the n.m.r. spectrum of a compound containing a furanoid oxide ring in its end group can be summarised as follows:- (8.89, 8.84) CH CH 3 3 H(4.85) C H (8.27)

0 H(4'8 CH3 5) (857 -9

Notes: K Only resolved at 56.4 ; shows signs of multiplicity (this might on occasion also apply to the 8.27 p.p.m. band).

The infrared spectra of these compounds were also determined. ?2i The spectrum of each compound was compared with that given by P-carotene so that the bands caused by the epoxide and furanoid oxide rings could be recognised. It was expected that these bands would be relatively weak owing to the low molar concentration of these two rings in the C carotenoids 40

The spectrum of the diepoxide differed from that of ?-carotene in -1 the presence of six additional weak bands in the region 1250 - 1040 crn. 292 and bands at 1628, 1381 (discussed later), and 891 cm.1 Other workers have reported that the positions at which bands caused by the epoxide group occur are not at all consistent.73 Little is known regarding the effect on the positions of these bands of placing the epoxide group at the end of a chain of conjugated double -bonds. One report has suggested that this would result in a shift of the absorption bands to rather lower frequencies.73 If this effect operates in n-carotene diepoxide, the bands which occur at 1249,

1234, 1116, 1044, and 891 cm.1 in the spectrum of this compound might all be said to occur near to values assigned to the epoxide group by other workers.

The spectral effect of introducing a furanoid oxide ring into

--carotene was much more dramatic. A iare number of bands, some of quite high intensity, occurred in the furanoid oxide spectra. In all, four of these compounds have been examined. These were aurochrome and mutetochrome prepared by the present author, and two polyene furanoid oxides prepared by

Akhtar. 53Both of Akhter's compounds were polyene esters and the presence of the ester group complicated their spectra; however, by comparing the spectra with those of the parent polyene esters, it was possible to pick out those bands caused by the presence of the furanoid oxide rings. and The spectra of mutatochrome/aurochrome were virtually identical

in that, with few exceptions, the peaks in the spectrum of aurochrome had twice the intensity (6) of those in the srectrum of mutatochrome. The 1 exceptions were the bands near 2920 and near 1450 cm. (methyl and methylene 293

1 bands: also present in P-carotene) and at 965 cm. (trans -CH=CH-). All the remaining significant bands in the spectra were apparently associated with the presence of the furanoid oxide ring. These bends have been listed in the Table below. nost of these bands also occurred in the spectraof the 2 polyene-ester furanoid oxides examined [the few that did not appear were those which occurred in the same positions es the ester absorption bands

(which would obscure the generally weaker furanoid oxide bands)]. The

Table also gives the overall range in which each band occurred (including all four compounds where possible). An indication of the relative intensity of each band is given end refers to the spectrum of surochrome fon the heeds that the 965 cm:1 band in this spectrum was "very strong" (vs)]. The bands in the mutatochrome spectrum were half es intense as the corresponding bands in the aurochrome spectrum. The intensities of the bands in the spectra of the two polyene-ester furanoid oxides were known less accurately, but they were of about the same intensity as the corresponding bands in the mutatochrome spectrum.

Table of infrared_f!mfncies apnnrently characteristic of furanoid oxides.

-1 Overall Rnnze (in cm. )

[1300 - 1301 (w)]41 Notes: The nverailRange includes the

[1217 - 1218 (w) ]x band position in all four compounds

[near 1160 (w)]K except where the bands are marked K

[1154 - 1135 (w)] in these cases, only the values of

1120 - 1123 (m) mutatochrome and nurochrome have been

1082 - 1085 (w) included, since in the esters the 294

1065 - 1066 (s) ester bands interfered with the furanoid

1045 (w)'' oxide bands.

1031 - 1032 (w) The values enclosed in square

1017 Wm brackets indicate that the bands concerned

996 (s) were found to be too weak or ill-defined to

[889-890 (w)] be of diagnostic value. Where only one value

879 - 880 (m) is given as the "Overall Range" for a band, it

should be understood that the band concerned

occurred consistently at this value in all

the compounds.

It is suggested that the characteristic band pattern in the region 1085 - 996 cm:1 might be of use in identifying the presence of a furanoid ring in a polyerie of unknown structure.

It is noteworthy that each of the furanoid oxide spectra contained -1 a weak, but sharp, band near 1645 cm. This band was absent from the spectra of n-carotene and its diepoxide, and it was twice as intense in aurochrome as in mutatochrome. This suggested that it was caused by the isolated double bond in the furanoid ring.

The spectra of p-carotene diepoxide, mutatochrome, and aurochrome -1 13 all contained a band near'1382 cm. Tsukida and Zechmeister also noted a band at this position in the diepoxide and showed that it was also present in n-carotene monoepoxide (they made no reference to the infrared spectra of the furanoid oxides). Before this discovery was made, it had always been assumed that the presence of a band at this position in a carotenoid 295 tie indicated the presence of a "methylated cis double bond" ( ) therein.74 It seems most unlikely that the specimens used by Tsukida and 13 Zechmeister and by the present author were anything but "all-trans" compounds. Due caution should therefore be used when assigning a cis configuration to a carotenoid on the strength of the presence of a band near -1 1380 cm., unless it can be shown that the compound is not an epoxide or furanoid oxide. 296

L..Arl_dti1,ENTAL-

Special precautions were taken to ensure that the apparatus used was free from all traces of acid. Glassware was rinsed out with aqueous sodium hydrogen carbonate, then distilled water, acetone and finally light petroleum which had been distilled from alkali.

Ethereal monoperphthalic acid (289. 617).

This was prepared from hydrogen peroxide and phthalic anhydrideP o yield, 662% The solution was stored at 0 (over MgSO4). Evenso, the normality of the solution dropped steadily (and linearly) during a period of

20 weeks (from 0.63 N to 0-41N). The solution was freshly titrated each time it was used; the use of relatively old perphthalic acid in the reaction below apparently had little affect on the yield obtained. p-Q.Ar.dene diepoxide (391).

29 The method of Karrer and Jucker was modified in the following way.

Ethereal monoperphthalic acid (0.50N, 6.0 ml., 2.1 equiv., 4.2 atom-equiv. of oxygen) was added. to a solution of p-carotene (187 mg.) in ether (400 ml.). The mixture ( A and 451 n5.) was kept at 200 until max.479 there was no further shift in the position of maximal absorption (24 hr.).

The solution ( and w max.468 439)lay aswashed with saturated sodium hydrogen carbonate (4 x 40 ml.), then water (2 x 40 ml.), and was dried (Na2 SO ). Evaporation of the solvent, and crystallisation of the residue from 4 benzene-methanol gave the diepoxide (89 mg.) as pink-red leaflets, m.p. 180-

182° (K.), raised by recrystallif:atir to m.p. 194-165° (Y.), 198-1890 (-'?vac. cap.,uncorr.), 194 - 1950 (evr2,c.cap.,corr.), 2 9 7

(in benzene) 481, 451, and 426 my_ (10 36127, 136, and 90, respectively) A max. ,29 (for infrared spectrum, see below). [lit., m.p. 184°(evac. cap., uncorr.),' \ 13 189 - 190°(evac.cap.corr.); X 29 max.(in benzene) 485 and 456 111.L I.

1C (in deuterochloroform) e.o6, 3.,07, 0.91, and 9.06 p.p.m. at 40

LIc., 304- 3.06,. 2.93 and ).06 p.p.m. .8* 56.4 Mc.; relative peak areas 2:1:1:1. The specimens used to determine the n.m.r. spectra were recovered; their visible-light absorption properties were unchanged.

The diepoxide prepared in this way contained 15-20 of the monoepoxide (see the discussion). Vihen a three-fold excess of per-acid was used (a two-fold excess was used above) and the mixture was left for 60 hr., the epoxide was partly rearranged (mainly into aurochrome).

The diepoxide (1.2 mg.) in ether (8 ml.) was shaken with 25% aqueous hydrochloric acid (17 ml.). A blue colour slowly developed, but even after keeping the solution, with occasional shaking, 3 days, not all of the pigment had dissolved in the acid layer. The latter was then washed free of unreacted pigment (using ether) and then gave a single broad absorption band, X max. (in hydrochloric acid) 593 IT. Basification of the acid solution and extraction with ether gave a yellow solution, X max (in ether) 415 and 396 It.

Mutatochrome and aurochrome29 (447),(467).

Two drops of chloroform saturated with hydrogen chloride were added to a solution of crude p-carotene diepoxide [173 mg., m.p. 175 - 176°

(K.)] in chloroform (150 ml.). The colour of the solution, initially red, 298

80 quickly faded to yellow (and, on some occasions, then became greenish- 82. yellow ). 4 min. after adding the acid to the solution, the solution was poured into saturated sodium hydrogen carbonate (100 ml.), and shaken, whereupon the solution became orange-yellow. The solution was washed with and the solvent was evaporated. water (100 ml.), and then dried (Na2SO4), Chromatography of the residue on alumina (IV, 26 x 2.5 cm.) from benzene- light petroleum (3:7) gave 3 main bands. In order of elution these gave:

(i) p-carotene (ca. 5 mg.);

(ii)mutatochrome (45 mg.) which crystallised from benzene-methanol in orange leaflets, m.p. 159 - 160°(K.), 161 - 162° (evac. cap.. uncorr.),

166 - 167° (evac. cap., corr.), )1 .(in benzene) 463, 437_, and (416) 9„.t.

(10-3 C 101, 113, and 77, respectively)[lit., m.p. 163 - 164° (evac. cap. 13 (in benzene) 470 and uncorr.),29 165 - 166° (evac. cap., corr.); max. 29 440 ri34 1; (iii)aurochrome (45 mg.) which crystallised from benzene-methanol in glistening, golden7yellow leaflets, m.p. 187 - 1890(K.), 195 - 197°(evac. cap., uncorr.),max. 434, 409, and 387 m .(10d E 115, 116, and 73.5, 0 respectively) [Karrer and Jucker29 give m.p. 185 (evac. cap., uncorr.),

max.(in benzene) 440 13,11-1. 29 Karrer and Jucker gave the position of only the long wavelength absorption band in their paper (possibly because of instrumental 13 limitations). Tsukida and Zechmeister have published values for hexane solutions of these compounds; the estimated values for benzene solutions are:

483, 452, and 426; 466, 437, and 414; and 436, 410, and 389 T. for the diepoxide, mutatochrome, and aurochrome, respectively. 299

1.7 values for mutatochrome in deuterochloroform were 4.85, 8.04.

8.28, 8.57, 8.88 (shoulder), and 8.96 p.p.m. at 40 Mc.;-1r 8.05, 8.28, 8.54

(shoulder), 8.57, 8.84 (shoulder), 8.90, 8.97 p.p.m. at 56.4 Mc. (cf. p.289 for peak areas). The sample from the latter determination was recovered, rechromatographed, and the determination repeated. The spectrum was unchanged.

T values for aurochrome in deuterochloroform were 4.84,87,2,.27,

8.58, and 8.90 p.p.m. at 40 Mc.; relative peak areas 2:3:3:3:6; 8.06,

8.20 (bat see p.287), 8.26, 8.54, 8.56, 8.74, 8.84, and 8.89 p.p.m. at

An ethereal solution of mutatochrome was shaken with hydrochloric acid (conditions as for the dienoxide, above). The acid layer slowly developed a blue colour, and after 20 hr. the absorption spectrum of the solution was measured; it consisted of a single, broad absorption band, max.(in hydrochloric acid) 572 T.

Infrared spectra (in carbon tetrachloride solutions).

(Inflections have been indicated by enclosing the relevant value in square brackets). p-Carotene diepoxide: . 3026 (m), 2916 (vs),

2861 (m), 1628 (vw), 1474 (m), [1459 (D)], 1449 (m), 1398 (m), 1380 (m),

1364 (m), 1249 (vw), .1234 (w), 1171 (vw), 1147 (vw), 1116 (m), 1063 (vw), 1043 (w). 1003 (w), 965 (vs, 6 470), 906 (w), and 891 (m) cm:1

3009 (m), 29o8(vm), 2845 (s), 1642 (vw), 1453 (m), Mutatochrome: 11 max. [1445 (m)1, 1395 (w), 1382 (w), 1366 (m), 1311 (vw), 1300 (w), 1217 (vw), 300

1171 (w), 1134 (w), 1121(w), 1082 (w), 1065 (m), 1045 (w), 1032 (w), 1017 (w), 996 (m), 965 (vs, 6 565), 889 (vw), 879 (w), 865 (vw), and 857 (vw)cm:1 Aurochrome: . 9 max 3008 (m), 2912 (vs), 2841 (s), 1646 (w), 1498 (m), [1445 (m)], 1396 (vw), 1 383 (w), 1 367 (s), [1314 (w)], 1301 (m), 1271 (vw), 1218 (w), 1171 (w), 1160 (w), 1135 (w), 1122 (m), 1082 (w), 1065 (s), 1045

(w), 1032 (w), 1017 (m), 996 (s), 965 (vs,E 565), 889 (w), 880 (m), 867 (w), and 857 (w)cm:1 301

REF70,R7,NCES

1. Jackman, "Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry", Pergamon, London, 1959.

2. Barber, Davis, Jackman, end Weedon, J., 1960, 2870.

3. Barber, Jackman, and Weedon, Proc. Chem. Soc., 1959, 96.

4. Barber, Jackman, weedon, and Yokoyema; ouoted in ref. 7; cf. Barber, Ph.D. Thesis, London, 1960.

5. Barber, Jackman, Warren, and Teedon, Proc.Chem.Soc., 1960, 19.

6. Barber, Jackman, ''arren, and ITeedon, J., 1961, 4019.

7. Davis, Jackman, Siddons, and.Teedon, Proc.ehem.:;oc., 1961, 261.

8. See section I of this thesis.

9. Barber, Hardisson, Jackman, and Veedon, J., 1961, 1625; Barber, Jackman, and Weedon, Proc.Chem.Soc., 1960, 23

10. Karrer, Fortschr.Chem.org.Naturstoffe, 1948, a, 1.

11. Karrer and Jucker, "Carotenoids", trans. Braude, Elsevier, New York, 1950.

12. Goodwin, "The Comparative Biochemistry of the Carotenoids", Chapman end Hall, London, 1952.

13. Tsukida and Zechmeister, Arch.Biochem.Bionhm., 1958, 1A, 408.

14. Prepared by M.Akhtar; cf. Akhtar, Ph.D. Thesis, London, 1959. 15. Karrer and Jucker, Eelv.Chim.Acta, 1945, 28, 300.

16. Karrer, Salomon, and Tehrli, Helv.Chim.Acta, 1929, 12, 790; Karrer and Salomon, ibid., 1930, 11, 1063.

17. Kuhn, ':finterstein, and Lederer, Zophvniol.Chem., 1931, 197, 141; cf. ref. 12, p. 4.

18. Karrer, Jucker, Rutschmenn, and Steinlin, Helv.Chim.Actf,19459 28, 1146.

18a. Karrer and Jucker, Helv.Chim.Actal 1943, 26, 626. , 18b. Karrer and Rutschmann, Holv.Chim.Acta, 1945, 28 1526. 18c. Hey, Biochem.J., 1937, a, 532.

302 19. Yor lists r)f natural sources of these carotenoids, see refs. 10 - 12.

20. Kuhn and Brockrnann, Z. physiol.Chern., 1932, 213, 192.

Karrer and Jucker, :elv.Chim.cta,, 1945, 26, 626. Karrer and Osrald, 1935, 18, 1303.

23. (a) Sollunck, Proc.Poy.Soc., 1903, 72, 165; (b) Kuhn and Winterstein, Ber., 1931, 61, 326.

24. Karrer and Rutschmann, He1v.Chiu.Acta, 1542, 25, 1624.

25. Curl and Bailey, J.Agric.Food Chem., 1954, 2, 665.

25a. Karrer and Putschmann, Helv.Chim.Acta, 1944, 27, 1684.

26. Karrer and Jucker, Helv.Chim.1;cta, 1945, 28, 471.

27. Karrer and Jucker, lielv.Chim.Acta, 1944, 27, 1695.

28. Karrer and Krause-Voith, Helv.Chim.Actai 1948, j, 302' 29. Karrer and Jucker, Kelv.Chim.iicta, 1945, 28, 427. 30. Zechmeister, Fortschr.orc,.Chem.Naturstoffe, 1958, 15, 31 (p. 58); Bush and Zechmeister, J.Amer.Chem.Soc., 1958, 00, 2991.

30a. von Euler, I:arrer, and Walker, Helv.Chim.Acta, 1932, lc, 1527.

31. Strain, and others, quoted in ref. 12, p. 14; see also ref. 57.

32. Goldsmith and Krinsky, Nature, 1960, me, 491.

33. Goodwin, Biochem.J., 1958, 68, 503.

34. CholnoV, Gyorgyfy, Nagy, and Pfincz61, Acta Chim.Acad.'Sci.Rung., 1995, 6, 143.

35. Cholnoky, Gyorgyfy, Nagy, and Ancz41, Nature, 1956, 178, 410. 36. Cholnoky, CyorgTfy, Nagy, and lIncz41, Acta Chim.Acad.Sci.7Jung., 1958, 16, 227.

- 47 • For ccmprchensive reviews of the earlier (pre-1952) work, see Goodwin, ref. 12: (a) p. 51; (b) p. 36; (c) p. 53; (d) p. 39.

38. Eugster and Karrer, Helv.Chim.Acta, 1957, 4, 69.

39. Kindly supplied by Dr. Eugster: cf. ref. 38.

40. von Euler and Karrer, Helv.Chim.Acta, 1950, 33, 1401. 303

41. For reviews, see refs. 5,6,11,12,42, and 46.

42. Warren and Weedon, J., 1958, 3972. 43. Frthschel and Karrer, Helv.Chim.Acta, 1960, 42, 09. 43a. Winterstein, Angew.Chem., 1960, 72, 902. AI cf. Cholnoky and Szabolcs, Experiontia, 1960, 16, 433. 45. Faigle and Karrer, Helv.Chim.Acta, 1961, 44, 1297. 46. Barber, Ph.D. Thesis, London, 1960. 47. For a review, see Goodwin, ref. 12. p. 39. 48. Section I of this thesis, p.18.

49. Zechmeister, Fortschr.org.Chem.Naturstoffe, 1958, 15, 31 (p. 40). 50. Glover ana Redfearn, Biochem.J., 1993, 54, viii. 51. Goodwin, Preprint Paper no. 55, read at the 5th International Congress of Biochemistry, Moscow, August 1961.

52. Goodwin, Ann.Rev.Biochem., 1955, 24, 497 (p. 507).

53. Akhtar, Ph.D. Thesis, London, 1999.

54. Karrer et al., quoted in ref. 12, p. 73. 55. Dr. H. Yokoyama, personal communication. 56. Karrer, Krause-Voith, and Steinlin, Helv.Chim.Acta, 1948, 21.7 113. 57. Strain, Arch.Biochem.Biophys., 1954, .49.4, 458. 58. Sorby, Proc.Roy.Soc., 1873, 21, 442 (pp. 459, 462).

59. Zechmeister, Fortschr.org.Chem.Naturstoffe, 1960, 18, 223 (1). 272). 60. Akhtar, personal communication (cf. ref. 53.) 61. Curl, J.Agric.Food Chem., 1953, 1, 456.

62. Curl, Food Res., 1959, 413; and rektbere cited. 63. Winterstein, Studer, and Ruegg, Ber., 1960, Z1, 2951. 64. Strain, Manning, and Hard'in, quoted in ref. 57 (Biol.Bull., 1944, 86, 169). 304

65. Hunter and Krakenberger, J., 1947, 1; cf. Savinov and Svishchuk, Chem.Abs., 1952, 46, 10230c (Ukrain.khim.Zhur., 1950, 16, 57).

66. Kuhn and Lederer, Z.physiol.Chem., 1931, 200, 108.

67. Krinsky and Goldsmith, Arch.Biochem.Bi2phys., 1960, 20 271.

68. Ref. 1, p. 129.

69. Warren and Weedon, J., 1958, 3972.

70. Akhtar and Weedon, unpublished results: cf. ref. 53.

71. cf. Section I of this thesis.

72. Warren, Ph.D. Thesis, London, 1957.

73. cf. Bellamy, "The Infra-red Spectra of Complex Molecules", Methuen, London, 1958.

74. Lunde and Zechmeister, J.Amer.Chem.Soc., 1 955, 77, 1647. 75. Vogel, "Practical Organic Chemistry", Longmans, London, 1954.-

76. Hodgkiss, Liston, Goodwin, and Jamikorn, J.Gen.Microbiol., 1954, 11, 438.

77. De Gouveia and Gouveia, Chem.Ahs., 1954, 48, 2281i. 78. (a) of. Appendix I, p.368 ; (b) cf. the Experimental Section.

79. of. the "Notes" at the beginning of this thesis. 23a 80. A similar effect was noted many years ago by Schunck, and others before him.

81. cf. for example, Karrer and Jucker, Helv.Chim.Acta, 1945, 28, 1143; Karrer, Jucker, and Steinlin, Helv.Chim.Acta, 1947, 12, 531.

82. This tendency for the mixture to develop a green colour was also observed by Akhtar53 working with another epoxide. 305

SECT ION IV

NOVEL SYNTHESES OF IS ORENIERATENE AND RE NIERA PURPUR IN 30 6

Earil.§m1.1222fL211a2re nieratene and Renierapurpurin.

Isorenieratene and renierapurpurin are two isomeric hydrocarbon carotenoids which have recently been found in a Japanese sea sponge. They occur along with a third isomeric carotene, renieratene, and the three compounds are characterised by the fact that they have aromatic end groups. They constitute the first (and, as yet, only) examples of natural carotenoids having this feature.

The carotenoids occurring in the various species of sea sponge 16. have been the subject of many investigations over the past 80 yeare.

However, much of the early work was performed without the aid of column chromatography (a technique virtually unknown in this field before

613‘ 1931 ), so that it is doubtful whether many of these pigments were homogeneous. Also, in many cases, the pigments were only isolated in small amount, so that elemental analyses were rarely performed and tentative identifications were made mainly on the basis of their visible 3,6a. absorption spectra. The inadvisability of this practice is demonstrated in the following description of Yamaguchi's recent, and more precise, investigations in Japan on the carotenoid content of Reniera 307

japonica - an orange coloured sea sponge found widely in the tidal zone of the Japanese sea shore.3 Yamaguchi revealed the presence, at least in that sponge, of several new pigments, three of which had absorption spectra very similar to those of n-carotene, rscarotene,and torulene,respectively, but which he showed to be entirely different from these.3

In all, 15 pigments were isolated by Yamaguchi using the normal chromatographic procedures (although,since the sponge is inevitably contaminated witfl algae, some of the minor components might conceivably have arisen from that source). Of these, six were obtained in only trace amounts; the remaining nine are listed below in the order that they appear on an alumina chromatogram from bottom to top of the column:-3

7... a b First Remarks No. max. m.p. Yield (in my) (in mg.) isolated

1. 507 185° 2 19573 Identified as a-carotene.3 2 2 3 2. 520 183° 20 1954 Identified as 13-carotene. ' 2 3. 520 199° 45 1954 Hydrocarbon, later named4 isorenieratene. 3 4. 532 185° 93 19542 Hydrocarbon, named renieratene. 308

No. X a m.pb Yieldc First Remarks max. (in mo) Ijm ma.) isolated 2 9. 536 2050 1.1 1954 2 .3 6. 544 230° 4.4 1954 Hydrocarbon, named renierapurpurin.

7. 558 161° 2.6 1957 3 Oxygenated carotenoid, named3 renieraxanthin. 3 8. 518 153-4° 0.4 1957 Similar to, but not identical with,3 cryptoxanthin. 9. 530 173-4° 1.0 1957 3 Similar to rubixanthin and gazaniaxanthin but its identity with either is unlikely.3

Notes :

a Position of longest wavelength absorption band in carbon disulphide solution. b In evacuated capillary, uncorrected.

c From 5.7 kg. of wet Reniera japonica.

Of particular interest were the three hydrocarbons (3,4, and 6) characterised by an unusually high carbon to hydrogen ratio; the structures of two of these (isorenieratene4 and renieratene5) were soon elucidated and both contain substituted benzene rings as end groups. This is the first time that such an end group has been found in a naturally occurring carotenoid (although the unsubstituted analogue was synthesised 309 .

prior to this by Karrerl0 ). Neither pigment shows any pro-vitamin A

activity.4,13

The major pigment had the same visible absorption spectrum as y-carotene but was found to be not that but a previously uncharacterised hydrocarbon, "renieratene". Much of the remaining pigmentation was

accounted for by two hydrocarbons with the same absorption maxima as

0-carotene. One was p-carotene but the other was the new hydrocarbon

"isorenieratene". The latter resembled leprotene12 (the characteristic carotenoid of the bacterial family Mycobacteriaceae, but of unknown

31 structure ) in melting point and visible absorption spectrum but differed significantly in molecular formula and hydrogen number. It is interesting

11 to note that if the recent suggestion that Reniera japonica is identical with Halichondria panicea is correct, then the "carotene" isolated by

ILInnberg17 from the latter sponge in 1931 may well have been isorenieratene or a mixture of this with p-carotene. The third member of the new group of hydrocarbons, "renierapurpurin", was found3 in much smaller amount than the other two and this scarcity of material prevented its structure being deduced by the usual degradative methods that were applied to the others. 310

The pigment with an absorption spectrum similar to that of torulene (a cerotenoid hydrocarbon: cf. p.115) was named "renieraxanthin" and. analysed for C40115002. It failed to give the colour test for epoxides with' ydrochloric acid and, according to Yamaguohi,3 probably contains at least one ketone group conjugated with the polyena chain. In that it iS oxygenated and, as yet, of unknown structure, it is of no further interest in the present. context.

The structures of renicratene and isorenieratene followed by the application of the classical techniques.

7 Henleratene analysed for C40H48 or 401150, tne ,.ormer being the more likely. ) It absorbed 15 moles ydroger13'hut its ;risible absorption spectrum fA (in carbon disulphide) 532, max . 4, 463 T, similar to that of double y--carotene) implied that it contained not more than 12/bonds in conjuL:ati.:n.

The infrared spectrum showed only bands characteristic of conjuglted

-.1 trans double bonds (near 1000 cm. and 97c cm. ) , but also a strong band near 800 cm: indicative of a benzene ring. Ozonolysis produced a negligible amount of acetone so ruling out'4 the possibility of the presence

14 of an isopropylidene group - anC also, probably, that of the isopropyl, 311

1.5 a-ionone and p-ionone -groups, all of which give small but readily measurable quantities of acetone, under these conditions. Mild chromic

13 acid oxidation gave, after chromatography of the crude product,

3 crystallisable oxidation products which were (from bottom to top of the chromatogram) : CS2 (±) an aldehyde, "isorenierai", C30H360 , max.526, ; on

catalytic hydrogenation it absorbed 12 moles of hydrogen;

(ii) an aldehyde, "renieral", obtained in somewhat smaller amount,

of which also analysed for C30H360 and absorbed 12 moles hydrogen; CS2 max. 540, 501 my.;

(iii)a compound with properties reminiscent of those of crocetin dial.

More vigorous oxidation of renieratene gave (iii) as the major product (C20112402)and the properties of (iii) itself and its

16 2,4-dinitrophenylhydrazone were identical with those reported for the synthetic C20 dial (I).Both renieral and isorenieral gave (iii) on further oxidation but neither of these two aldehydes could be converted one to the other by oxidation. This degradative work can be summarised: 312

Renieratene

Renieral Isorenieral

Crocetin dial (I) e wherein both R and R are residues each containing 3 double bonds C9H11- and one ring, the latter being a benzene ring in at least one case.

Yamaguchi4 then repeated the above work on the other-hydrocarbon

3 -isorenieratene. This also analysed for more probably C40-48-50'

C 4 it showed absorption maxima (in carbon disulphide) at 520, 484, 40 H48 ;

and 452 m}.1.. It also absorbed_ 15 moles of hydrogen, showed benzenoid absorption in the infrared, and gave no acetone on ozonolysis.

Comparatively vigorous chromic acid oxidation again furnished crocetin 313

dial. Milder oxidation gave only one polyene aldehyde which was shown to

be identical with the isorenieral described above; on this occasion

Yamaguchi was also able to isolate 2,3,6-trimethylbenzaldehyde from the

crude oxidation product. Permanganate oxidation of the pigment also furnished this aldehyde and, in addition, the corresponding acid. From these results it is apparent that isorenieratene is symmetrical and its,

i end group (which is the R of above) is . The only problem remaining was to elucidate the structure of end-group "R" in renieratene.

5 Mild permanganate oxidation of this pigment gave a mixture containing

2,3,4- and 2,3,6-trimethylbenzaldehydes (which were isolated and separated as derivatives), Hence "R" in renieratene is and the two pigments have the following structures:-

Isorenieratene (II)

Renieratene (III)

All experimental observations with one exception are in accord with these structures - the exception is that both pigments4,13 314

gave volatile acid corresponding to 7 moles of acetic acid on "Side Chain

Methyl" determination whereas only 4 would be expected; (the reason for this anomalous result is still unknown).

The differences observed between the absorption spectra of the

10 , two natural pigments (II and III) and of the synthetic analogue (IV) were explained5 on the grounds of steric hindrance. Thus, in polyenes with a

2,3,6-trimethylphenyl end group, steric hindrance between the o-methyls and the polyene chain would be expected to distort the molecule from the planar form which the unsubstituted analogue (IV) can adopt. This would cause a decrease in the degree of conjugation and a consequent shift in the absorption spectrum to shorter wavelengths. This shift would be particularly marked in isorenieratene (II) where both end-groups are of the di-o-substituted type. The difference between the absorption maxima of the two polyene aldehydes renieral and isorenieral is consistent with this argument. (Steric hindrance effects are discussed further on page33l). 315

The structure proposed for isorenieratene was soon confirmed

20 by a total synthesis of the pigment firstly by Yamaguchi and later by

Karrer.21 Both syntheses were based on the method used by Karrer in his

syntheses of the polyenes (111)1° and (V).18 The aryl aldehyde (VI) was

(V)

condensed with acetone in an alkaline medium to give ketone (VII) which

with propargyl magnesium bromide was converted into the acetylenic alcohol

(VIII). The Grignard derivative (IX) of (VIII) was treated with the

C diketone (X)19 in the presence of a cuprous chloride catalyst to give 8

(XI). Semihydrogenation of the acetylenic bonds in the tetraol,

followed by dehydration with 2-toluene sulphonic acid in boiling toluene,

furnished the fully conjugated polyene hydrocarbon (XII).

316

Ar CHO > Ar0 OH (viii)

C M313r [A r OH (ix)

OH ow (xi)

X X XXX X X X X

10 Thus benzaldehyde furnished t})e diphenylpolyene(IV), p-naphthaldehyde gave

/ 18 the analogue kV), and 2,3,6-trimethylbenzaldehyde gave isorenieratene

20,21 (III). That the product obtained synthetically was identical with

20 naturally occurring isorenieratene was proved conclusively by Yamaguchi by direct comparison of natural and synthetic samples (infrared and visible absorption spectra identical, nodepressionof m.p. on admixture 317

•of the two samples, and no separation on a mixed chromatogram). Karrer's

product had the same physical constants but he was unable to perform a

21 direct comparison.

As mentioned earlier,the structure of renierapurpurin could

not be elucidated further using the classical methods applied to

renieratene and isorenieratene since it is present in Nature in

comparatively small amount. All that was known of it was that it

analysed for C4048(or C401150), had m.p.230° and showed absorption

maxima (in carbon disulphide) at 544, 504, and 475 94. On this evidence 11 alone, Yamaguchi recently suggested that renierapurpurin has the same

type of structure as renieratene and isorenieratene, and that neither

of its end-groups is likely to be of the di-o-substituted type since its

absorption maxima occur at even longer wavelengths than those of

renieratene. If both end-groups were 2,3,4-trimethylphenyl-, the series

of hydrocarbons that could be visualised as arising from the two types of

end-group so far encountered (2,3,6- and 2,3,4-trimethylphenyl-) would

be complete.

11 To test this hypothesis, Yamaguchi performed a further 318

synthesis using the same reaction scheme as before but condensing the

C diketone with an equimolar mixture of the two acetylenic alcohols 8

(XIII and XIV) derived from 2,3,6-trimethylbenzaldehyde and

2,3,4-trimethylbenzaldehyde, respectively. This gave a mixture of

C tetraols (XV, XVI, and XVII) which was then converted to the 40 corresponding mixture of three

(xv) 319

(xv

renierapurpurin) hydrocarbons (isorenieratene (II), renieratene(III), and the structure suggested for renierapurpurin(XVIII)). The mixture was separated chromatographically and the hydrocarbons were found in this order from bottom to top of the column. Pigment (III) was found to be identical in all respects with renieratene, so confirming the structure deduced from the degradative work already described. In addition, (XVIII) was indeed shown to be renierapurpurin by direct comparison of physical properties in the usual way. Renierapurpurin was then synthesised in better yield from the alcohol (CIV). This was first converted to the tetraol (XVII) which, in turn, furnished renierapurpurin in the same way as before.

There is one major criticism that can be made of the reaction 320

scheme utilised in the syntheses just described; this is that the yield of pigment from starting material is extremely poor throughout. The first two stages linvolving the conversion of the benzaldehyde (VI) into the

yamaguchi11,20 acetylenic alcohol (VIII)l both proceed in reasonable yield obtained an overall yield of 24% (with both the 2,3,6- and 2,3,4- trimethylphenyl- series of compounds). whilst Karrer21 improved this to

48%. In his synthesis of isorenieratene, Yama guohi2° obtained a yield of 50% for the conversion of the acetylenic alcohol (VIII, XIII) into the tetraol (XI,XV) but the yield in the last step (involving the conversion of the tetraol to the final product (XII,II)) was only 1-7%. Similarly, although Karrer omitted to quote a yield for his chromatographed product, it is obvious that the yield he obtained for this last step was little, if any, better. The yields Karrer obtained in his synthesis of the diphenyl analogue (IV) and the dinaphthyl analogue (V) were similarly 11 10'18 In his synthesis of renierapurpurin, however, Yamaguchi poor. • succeeded in increasing the yield for the last stage to 5%. Even then, the overall yield of carotenoid from readily available starting materials was very low. Even the synthesis of the C8 diketone (X), used as the

321

central unit in the synthesis of the C skeleton, proceeded in poor 40 yield.

19 Karrer synthesised this diketone by condensing glyoxal and

acetoacetic acid (under basic conditions) to give octa-3,5-diene-2,7-dione; 19 the yield, originally32 e, was later improved, but to an unspecified degree. Reduction of the octadione with zinc dust in pyridine and acetic

acid furnished (70-75%) the C8 diketone (X). The same diketone has since

been synthesised (in 12% overall yield) by a new, and better, route

starting from acetylene.33

The stage which proceeded in particulary low yield in the

preparation of these aryl carotenoids was that involved in the formation

of the polyene chain from the tetraol (XI). This difficulty has been

avoided in the new synthetic route utilised by the present author, by

C 10-C40. The central C20 unit was using the building principle Cr.+u 20+C provided by crocetin dial - a compound already containing a heptaene

chromophore. The synthesis of this compound in high overall yield from

the C dial (XIX) was announced by Isler and his co-workers16 in 1956. 10 The C dial (XIX) itself had already been made by the same group34 (and, 10 independently, by Mildner and Weedon58) by condensinga-methylacraldehyde

with acetylene using the Grignard reaction, rearranging the product (XX)

with acid, and oxidising the resulting diol (XXI) with manganese dioxide

to give the C10 dial (XIX), in 50;%:' overall yield.

CH H3 I 3 I 3 I 3 2CH=C.C.QH0 + BrMgCMCItgBr--> CH2C.CROH.CEEC.CHOH.C=CH2

322

C C H I H3 I 3 .CH OH > HOCH2•C=CH-CEEC-CH=C 2

dial (XIX) with ethyl orthoformate gave Treatment of the C10 the corresponding diacetal (XXII) which with ethyl vinyl ether in the presence of zinc chloride underwent chain extension to give (XXIII).

This, on acid hydrolysis, furnished the C dial (XXIV). The C dial 14 14 was then subjected to a similar series of reactions using propenyl vinyl ether and gave dehydrocrocetin dial; semihydrocenation using Lindlar's catalyst,35 and stereomutation of the central-cis product, gave crocetin dial (XXV), in an overall yield of 23% from the C10 dial (XIX), or 11% from acetylene.

Eto 0Eb OEt EtE

OEt OEt Eto OEt Et 0 OEt 3 2 3

(xxv)

This C dial has since been used for several syntheses of 20 carotenoids and related polyenes. Two main methods have been used to

attach the C end groups to the central C unit. These are the 10 20 aldol condensation (using which canthaxanthin 36 and desoxycapsorubin37

have, for example, been made"), and the Wittig reaction.23 The latter

reaction, discovered in 1954, involves the conversion of an alkyl bromide

(XXVI) to the corresponding phosphonium bromide (or "Wittig salt", XXVII)

by treatment with triphenylphosphine. Treatment of the Wittig salt with

phenyl- (or butyl-) lithium gives the phosphorane (XXVIII) and this reacts

with crocetin dial to give the hydrocarbon (XXIX):-

C + - 20dia1 11-CH Br --->R•CH PPh PBr --H)1.R.CH=PPh 2 2 3 3

(xxvi) (xxvii) (xxviii)

R.C H C 324

Using this reaction sequence, Isler and his co-workers have 16 16 formed new synthetic routes to a-carotene, lycopene, andliall-trans"-_____ methyl bixin.39 The present author has now used this reaction to prepare two of the aryl carotenoids described previously - isorenieratene and renierapurpurin - and also the unsubetituted analogue (IV). Both the natural compounds were synthesised from the same aromatic aldehydes as were used by Yamaguchi and Karrer.

2,3,6-Trimethylbenzaldehyde (xXXI) is a well characterised compound and was prepared from 3-bromopseudocumene (XXX), using the 24 Grignard reaction as described by Lowe, Torto, and Weedon. The aldehyde was reduced (in 76% yield) to the corresponding alcohol (XXXII) with lithium aluminium hydride in the usual way. An attempt to prepare the alcohol direct from the Grignard derivative of 3-bromopseudocumene by treatment with formaldehyde was unsuccessful. Two anomalous products, of unknown structure, resulted from the reaction. (It is noteworthy that the reaction of formaldehyde with the Grignard derivative of substituted benzyl 29 halides often proceeds in an unexpected manner ).Treatment of the alcohol with phosphorus tribromide (at -30°)t and conversion of the crude bromide (XXXIII)ints the Wittig salt (XXXIV) using triphenylphosphine, followed in 98% yield. The Wittig salt, without purification, was treated with ethereal butyl - lithium to give the red phosphorane(XXXV). Addition of crocetin dial caused the rapid and efficient formation of isorenieratene

325

(II) which separated out from U-c reaction mixture in a high state of

purity in 89% yield (based on the dial: a slight excess of end-group was

used). Its identity with the natural material was confirmed by comparing

it directly with a sample of natural isorenieratene isolated from

Reniera japonica and kindly donated by Dr. Yamaguchi.

H2Pfh3 8r

(xxxiii)

CH=Pfh C dial (XXV) 3 20 Isorenieratene(II)

The possibility that steric hindrance from the two o-substituted methyl groups in the benzene ring of (XXXV) might prevent the condensation with the C dial (XXV) was not realised. 20 26 Smith and Agre have reported that the bromide (xxx III) can also be prepared in moderate yield (41P direct from prehnitene 326

(1,2,3,4,tetrainethylbenzene) bytreating this hydrocarifiCn with bromine in bright' sunlight (at 140°). Although Smith and Agre claimed to have proved that the structure of the product was, in fact, (XXXIII), the evidence they presented is hardly conclusive enough to warrant the use of this route in an unambiguous synthesis of the kind being attempted by the present author. However, if their claim could be substantiated this reaction would afford a very convenient synthesis of the required Wittig salt.

2,3,4-Trimethylbenzaldehyde was prepared in the following way.

A sample of commercial hemimellitene (XXXVI) was first shown to be free of the other two isomeric trimethylbenzenes (pseudocumene and

mesitylene) by gas-liquid chromatography. The hydrocarbon was then

brominated (using Martin' s41 modification of the procedure of Smith and

Moyle40) and this gave 4-bromohemimellitene (XXXVII) in good yield. The

bromo-compound was then converted, through its Grignard derivative, into

2,3,4-trimethylbenzaldehyde. The aldehyde was converted into the corresponding Wittig salt (XXXVIII) using the same series of reactions as were employed in the synthesis of isorenieratene. The Wittig salt was obtained in 31% overall yield from hemimellitene. On treatment with

propyl-lithium and crocetin dial (XXV), it furnished renierapurpurin (XVIII) 327

+ C HiPPh3 Br

(XXXVII) (xxxviii) E,C20die.1(XXV),M,D

Renierapurpurin, (XVIII).

in 9Y yield. As with the preparation of isorenieratene, the Wittig

reaction occurred rapidly and the product separated in a high state of

purity from the reaction mixture. The pigment had the same physical 3 11 properties as those reported by Yamaguchi ' for renierapurpurin but, as yet, no direct comparison with the natural material has been made.

Luch of Yamaguchi's work on the determination of the

substitution patterns present in the three aryl carotenoids depends on the

pattern in the two trimethyl-substituted benzaldehydes. Not only did

Yamaguchi identify the aromatic aldehydes produced on oxidative

degradation of the natural carotenoids by comparison with these two synthetic aldehydes, but, in addition, he used the synthetic benzaldehydes

as starting materials for his synthetical proofs of the structures of the

natural pigments. Of these two aldehydes, the 2,3,6 isomer is well 24- characterised. The present author (and, apparently, Yamaguchi) obtained

the other aldehyde from the bromo. compound resulting from bromination

of hemimellitene. Of the two positions in hemimellitene available for

attack by an electrophile, the 4 position is the one favoured on 328

mechanistic grounds. That bromination does actually occur at the

4 position was demonstrated by Smith and Moyle40 as follows.

Nitration of the bromohemimellitene under vigorous conditions gave a solid dinitro-compound which was reduced with stannous chloride in acid to give the corresponding diamine. The latter gave a phenazine derivative on treatment with phenanthraquinone. The phenazine contained no halogen (presumably the reduction step had also removed the

as expected. The original bromine) and analysed for C23H18N2 bromo-compound must, therefore, have contained two adjacent positions available for nitration to allow the formation of an o-diamine on reduction of the dinitro-compound. Unfortunately, no yields were quoted by Smith and Moyle so it is possible that the bromo-oompound might have been a mixture of the 4- and 5-bromo-isomers. This is unlikely, however, since the bromo-compound has also been obtained in good yield by the present author (using conditions similar to those employed by Smith and

Moyle) and it gave only one peak on a gas-liquid chromatogram. Further confirmation of the substitution pattern present has been provided by the present work. The infrared spectrum of the benzyl alcohol prepared from the bromohemimellitene gave a strong band (at 815 cm.-1) in the region expected for a compound containing two adjacent hydrogen atoms in a benzene ring; it showed no absorption in the region (860-900 cm.-1) 28 where compounds containing an isolated ring-hydrogen atom absorb. The same 329

is true for the 2,3,6-trimethylbenzyl alcohol which gave a single strong -1 absorption band at 804 cm. in this part of the spectrum.

A direct synthesis of 2,3,4-trimethylbenzaldehyde from hemimellitene (by treating the latter with zinc cyanide and hydrogen 42,43 chloride in the presence of aluminium chloride) has also been described.

However, although the product gave the correct analytical figures, no attempt was made to prove at which position in the benzene ring substitution had occurred. The product obtained by Smith and Stanfield43 did give, .on permanganate oxidation, an acid whose melting point agreed 44-46 closely with that reported for 2,3,4-trimethylbenzoic acid but sufficient doubt still exists regarding the nature of the product to make it unsuitable for its use in the present work which is aimed at achieving unambiguous syntheses.

The third member of the group of aryl carotenoids discovered by Yamaguchit renieratene, has yet to be synthesised by the new route using the Wittig reaction.- One obvious method of preparing it would be to carry out the normal Wittig reaction on An equimotar mixture of the two Wittig salts (XXXIV) and (XXXVIII). This would give a mixture of the three hydrocarbons (isorenieratene, renieratene, and renieraputpurin) which could be separated chromatographically. In fact, renieratene has, as yet, only been prepared as one of 3 products from the mixed reaction carried out by

330

11 Yamaguchi (see previous discussion). One advantage of the Wittig reaction is that it should allow a more specific synthesis of renieratene. Thus addition of the phosphorane corresponding to one end group to a large excess of crocetin dial should give the corresponding polyene aldehyde. Thus the

Wittig salt from 2,3,4-trimethylbenzyl bromide should give aldehyde (XXXIX),

Yamaguchi's "renieral"23This could be isolated from the excess crocetin dial chromatographically, characterisedrand treated with the phosphorane derived from 2,3,6-trimethylbenzyl bromide. This should furnish renieratene.

A "half-condensation" Wittig reaction of this kind has been successfully carried out by Akhtar48 in his synthesis of the aldehyde (XLI) from the phosphorane (XL) and the dehydro-C10-dial (XIX).

F'Ph3 (xu) As a model reaction for the syntheses of isorenieratene and renierapurpurin, the preparation of the unsubstituted analogue (Iv) was also carried out. Benzyl bromide was converted into the corresponding Wittig salt (XLII) and this, on treatment with phenyl-lithium and crocetin dial(, 331

furnished (82X) the hydrocarbon (IV). The meltirgpoint of the 10 hydrocarbon was rather higher than that reported for it by Karrer but

it exhibited the expected spectral and chromatographic properties. On bne

occasion the product from this reaction was isolated chromatographically

and, in addition to the required hydrocarbon (IV), there was also obtained

a small quantity of another substance. This by-product was eluted from

a column much more slowly than the hydrocarbon and had a visible absorption

spectrum distinctly lacking in fine structure as compared with that of the

hydrocarbon (IV). The shape of the spectrum was very similar to that 13 reported by Yamaguchi for his polyene aldehydes renieral and isorenieral. -1 Its infrared spectrum showed the band pattern in the 1(00 to 1500 cm.

region characteristic of a polyene aldehyde47 and it is probable that this

substance was the half-condensation product (XLIII) C Ha PPh 3 Br XLII)

The series of 5 polyene hydrocarbons discussed above

[renierapurpurin (XVIII), renieratene (III), isorenieratene (II), the

diphenyl analogue (IV), and the dinaphthyl compound (V)1, provides an interesting example of the effect of steric hindrance on the properties of a molecule. The longest wavelength absorption maxima of these 332

hydrocarbons (in carbon disulphide) are 544, 532, 520, 536, and 550 mp,

respectively.101118 ' ' In hydrocarbon (IV) there are no substituents on the

benzene ring, and so the whole molecule can adopt a planar configuration

without difficulty. (The same is true for the dinaphthyl compound (V)

though here the extra benzene rings cause a bathochromic displacement as

might be expected).

Each of the three natural pigments has three methyl substituents

on each aryl end-group. This alkyl substitution might be expected to

cause these compounds to absorb at longer wavelengths than the unsubstituted

analogue (IV), just as the absorption maxima of pseudocumene

(1,2,3-trimethylbenzene) are at 15 mp longer wavelengths than those of 46 benzene. However, all three compounds have end-groups in which one or

both of the positions ortho to the polyene chain is occupied by a methyl

eroup. The resulting steric interference (between the o-methyl(s) and the

polyene chain) would be expected to restrict the ability of the molecule

to adopt a planar form.59 This "ortho-effect" is enhanced by the

"buttressing effect"24 of the 3-methyl group present in both 2,3,4 and 2,3,6

Some doubt exists concerning the exact position of the long wavelength 11 absorption band of renierapurpurin. Yamaguchi3' gives the value 544 11.

for the natural material but quotes no figures for his synthetic specimen; 11 instead he gives a diagram in which this band is placed at 538 mp. The

specimen synthesised by the present author gave a value of 536 my.. There

is general agreement, however, on the position of the major band (504 mp)

333

end-groups. Thus, in renierapurpurin (where only one o-position is

occupied), the buttressing effect of the two vicinal methyl groups (at

positions 3 and 4 in the ring) on the o-methyl (at position 2) enhances

the "ortho-effect" to the extent that the expected bathochromic effect of

the trimethyl substitution is almost nullified by the hypsochromic shift

in the absorption spectrum caused by the steric effects; [renierapurpurin absorbs at only slightly longer wavelengths than the unsubstituted analogue (IV)].

Two distinct forms can be drawn for each end of the molecules of

a polyene containing methyl substituted aryl end-groups; in the case of renierapurpurin for example, these two forms are represented by partial formulae (XLIV) and (XLV). The renierapurpurin molecule would be expected

to assume, as near as steric effects will allow, that conformation (XLIV) in which steric interference between the 0-methyl and the polyene

C H 3 CH3

C H3 C H3

(xLv) CH3 H chain is at a minimum. In the 2,3,6-trimethylphenyl end-group, however, the presence of methyl substituents in both o-positions must lead to considerable steric hindrance in both conformationai(XIVI) and (XLVII)]. 3,,'"-7 4

C H3

(x Lvt)

(In addition, one of the o-methyls (that at position 2 in the ring) experiences the buttressing effect of a neighbouring methyl group (that at position 3)1. Compounds containing a 2,3,6 end-group exhibit, therefore, an even larger hypochromic shift due to steric effects than those containing A the 2,3,4 end-group. Thus, renieratene, containing one 2,3,4 end group and one 2,3,6 end-group, absorbs at ca. 10 nyt shorter wavelength than renierapurpurin (in which both end-groups are of the 2,3,4 type). Similarly, isorenieratene (with both end-groups of the 2,3,6 type) absorbs at ca. 10 ay shorter wavelength than renieratene.

Similar effects have been observed in compounds prepared during 24 60 60 the synthesis of the pseudocumyl, mesityl, and o-tolyl analogues of vitamin A.

Solubilities of these hydrocarbons follow the same general pattern: the more distorted from planarity (by steric hindrance) the molecule is, the more soluble is that compound. Thus the most distorted molecule of the group, isorenieratene, is quite soluble, for example, in hot chloroform: sufficiently so, in fact, for a n.m.r. spectrum to be determined quite satisfactorily on the warm saturated solution. 335

Renierapurpurin, however, is markedly less soluble and only a very weak

n.m.r. spectrum could be obtained, much of the pigment having crystallised

out before the spectrum could be determined. The unsubstituted analogue

(IV) is even less soluble and only dissolves slowly on boiling with a large 18 volume of chloroform. Finally, Karrer has reported that the dinaphthyl

compound (V) was only very sparingly soluble in all organic solvents. He

was unable, even, to obtain a satisfactory quantitative visible absorption

spectrum of the compound due to its poor solubility even in boiling carbon

disulphide (less than 3 mg./l. dissolved).

As mentioned previously, the n.m.r. spectrum of only one of these

compounds [isorenieratene (II3 has, as yet, been determined satisfactorily.

The spectrum showed only 3 peaks in the methyl-proton region,at 8.01, 7.91,

and 7.74 p.p.m. (with relative intensities of 1:1:3). The 8.01 band is

typical of "in-chain" methyl groups49,50 and is associated, presumably, with

the two in-chain methyl groups in the centre of the molecule well away from

the influence of the aryl end-groups. The protons in the other two

in-chain methyl groups have been deshielded by the nearby aromatic rings

and so these show a separate peak at 7.91 p.p.m. The three methyl

substituents on the benzene ring end-groups are, apparently, equivalent

since they give a single band (although there are indications of a weak

shoulder on the high field side of this band). This band occurs at a 49 slightly higher field than that produced by the methyl group of toluene

(-r 7.66) indicating that these methyl groups all experience slight positive

shielding by the polyene chain(cf p. 199 and ref. so). 336

EXPERIMENTAL

Benzyltriphenylphosphonium bromide (411),(763).

Solutions of triphenylphosphine (4.98g.) in benzene (35ml.), and benzyl bromide (freshly distilled, b.p.198°/760 mm.; 3.25g.) in benzene (15 ml.), were mixed and left at 20° for 48 hr. The solid which gradually separated during this period was then collected and washed with cold benzene. The crude salt [7.82g., 95%,, m.p. 278-280°(unoorr.)] crystallised from a small volume of ethanol as colourless plates, m.p.

282-283°(Lncorr.)[lit., m.p.280-282°(uncorr.)54; 280.5°(uncorr.)55].

Infra'red absorption (KBr disc):1) max. 1434 (s,,P-Ph28), 1109(v.s.), 28\ -1 and 993(m., possibly P-Ph )cm. [triphenylphosphine had 11 max. (KBr disc) 1474(s.), 1433(e. ,p_ph28), 1089 (m.), and 1026(m.)cm.-1].

1,18-Dipheny1-5,7,12,167tetramethyloctadeca-1,3„5,7,9,11,13,15,17- nonaene (II/ 815, 767) .

(i) An ethereal solution of phenyl-lithium51 (1.0N, 1.96m1., 1.96m/mole) was added (during 2 min.) to a stirred suspension of benzyltriphenylphosphonium bromide (886mg., 2.04m/mole) in dry ether (20 ml.) under nitrogen. The mixture, which soon became yellow, was stirred for 1 hr. and then a solution of crocetin dial (186mg.,

0.63m/mole) in dry methylene chloride (4m1.) was added. A red solid soon started to separate out and, after 30 min., a sample of the 337

reaction mixture was found to have absorption maxima (in benzene) at 500

(shoulder), 473, and (454) rap- (no "cis-peak" near 372 mp. ). The mixture was boiled under reflux for 24 hr. The warm mixture was diluted with methanol (50m1.) and then briefly heated to boiling. The mixture was cooled, left at 00 overnight, and filtered. The red solid was washed with methanolland dried. Yield, 213mg., m.p,225-228° (K).

The filtrate ( A. .(in benzene) (495), (446). 358y. ) was placed in bright daylight in the presence of a trace of iodine for 1 hr. The

(in benzene) 511,8,452 M4 ) was resulting solution (X MAX. evaporated to dryness and the residue was crystallised from benzene- methanol to give a further quantity (20mg., m.p.228-232°(K)) of product. Total yield, 233mg.(82cM. Crystallisation, first from benzene, and then from chloroform-ethanol, gave microscopic copper-red plates, m.p.

230-231° (evac.cap.,uncorr.), 234-235°(K). (Found:C, 91.3; H, 7.75. Calc. for C34 H36'• C, 91.8 ; H, 8-40; X roax (in benzene) 517, 482, and 455 mr. (10 31i 138,157, and 107, respectively: logio lE 5.14, 5.195, and 5.03, respectively); after iodine isomerisation (trace iodine, bright daylight for 5 min.),A .(in benzene) 513, 480, 455, and

372 IA (10-3 e 101, 123, 87.6, and 28.4, respectively); .1) ..(KBr disc) 964 (v.s., trans CH=CH), 747(s.) and 690(s.). (The last two bands can both be assigned to benzene rings containing 5 adjacent 28‘ 10 o hydrogen atoms ). [lit., m.p., 209-210 ( evao.cap.,uncorr.);; max. 338

(in benzene) 517, 462.5, and 455.5 IT, (log10 6. 5.12, 5.18, and 5•01,

respect ively) 1 .

The pigment dissolved in concentrated snlphuric acid to

give an indigo-blue solutiondL uax. 547,806,887 mr, (Karrer and gs 10 gi Eu ter ve max 561 mtk ). .

In a previous experiment (using the same quantities as above)

an attempt was made to isolate the product chromatographically. The

crude reaction mixture was evaporated to dryness and the red residual

solid extracted repeatedly with hot benzene and methanol. The extracts

were combined and then diluted with water. The benzene layer was

separated, and was then washed with 9O methanol (2x50m1.), water

(1x50m1.), and dried (Na2SO4). The solution was concentrated (to ca.

.30m1.), diluted with light petroleum (90 ml.), and chromatographed

on alumina (IV, 24x3-5cm.) from benzene-light petroleum (1:3). At

various stages during these manipulations much of the pigment separated

out from the solution as small copper-red plates (m.p. near 233°(K);

visible and infra-red absorption spectra were identical with those

obtained on the product from experiment (i) described previously). The

chromatogram yielded - (a) a broad red zone which was rapidly eluted

and was shown to be a mixture of cis and all-trans forms of the desired

product; and - (b) a red zone which was eluted with benzene-light

petroleum (3:1); evaporation of the solvent and crystallisation of 339

the residue from benzene-methanol gave a dark red crystalline solid

(7 mg.), m.p. 172-175°(K), \ . (in benzene) 508, max 479, (453) ty (no "cis-peak"); the peaks in this spectrum were much less well defined than those in the spectrum of the required hydrocarbon; iodine

isomerisation gave a spectrum with absorption maxima at 503, 477, and 356 T. Infrared absorption: )) . (in chloroform) 1666 (v.s., E 1cm. 19.3 (equivalent toS 715 for a substance of mol. weight 369)], 1609 (s.), 1566 (m.), 1515 (w.), 1003 (m.), and 970 (s.) cm.-1; the first 3 of these bands have the pattern characteristic of a polyene

aldehyde containing the group)CH=CMe.CHO (rather than>H=CH.CH0)47; -1 the bands at 1003 and 970 cm. are attributed to trans CH:=CH. (It seems likely that this is the half-condensation product : c.f. page331).

2,3,6 -Trimethylbenzaldehyde SXXXI), (1515).

A sample of 3-bromopseudocumene prepared by G. Lowe24

was redistilled and had b.p. 114-118°/21 mm. (lit.,25 b.p. 85.5-86.5° 24 /5 mm.). 3-Bromopseudocumene (23.9 g.) was then converted, using the Grignard reaction, into 2,3,6-trimethylbenzaldehyde. The aldehyde (8.2 g., 4610 was obtained as a colourless oil(900X, pure according to n 2 g.l.c.); b.p. 73-77 . D1 1.5450 (lit.,24 b.p., 74°/1 mm.; °/1 4 tam.- — 26 n D 1'5430); V MaJC . (in carbon tetrachloride) 2868 and 2751 (aldehyde C-11), 1694 (conj. C=0)cm.-1. It gave a chromatographically homogeneous

2,4-dinitrophenylhydrazone (75% yield) which crystallised from ethyl acetate as small orange needles, m.p. 225-226°(corr.) flit.,24 m.p.

222-223°(corr.)1; infrared absorption:9 . (Nujol) 3270 cm.-1 (N-H).

A large amount of pseudocumene (identified by g.l.c.) was 340

also obtained from the above reaction; it had probably been formed by alcoholysis of the intermediate Grignard derivative by the ethanol later shown to be present in the ethyl orthoformate: the simple distillation of this reagent had obviously not completely eliminated the ethanol from it.

2,3,6-Trimethylbenzyl alcohol, (XXXII), (1603).

2,3,6-Trimethylbenzaldehyde (5.74 g.) in ether (15 ml.) was added, during 15 min., to a cold (0°) stirred suspension of lithium aluminium hydride (448 mg.) in ether (20 ml.). The mixture was stirred for 7 hr. at room temperature, the excess hydride was decomposed with

moist ether, and then ammonium chloride (650 mg.) in water (1.6 ml.) was added. The solution was filtered, washed with water, and, dried, and

the ether was distilled off, leaving a white solid (5.94 g.); this, on

crystallisation from light petroleum (b.p. 40-60°) gave the alcohol

(4.40 g., 76%), m.p. 83-84°. Recrystallisation raised the m.p. to 84.5-85° (corr.), [lit.,26m.p. 83'5-85°, but the compound was not

thoroughly characterised]. (Found: C, 79.7; H, 9-3.Calc.for.C10H140 :

C,79-95; H, 9'e0). It gave a single peak on a gas-liquid chromatogram

(Apiezon T, 150°); infrared absorption: V max . (in carbon tetrachloride) 3614 [sharp, 6 42'5 (cf. benzyl alcoho1,28 )) . 3614 cm.-1)1, 804 (v.s., 28, 1 2 adjacent ring hydrogens )cm. . The alcohol formed a 3,5-dinitro-

benzoate (70 yield) which crystallised from aqueous ethanol as needles,

. H 06N2 requires m.p. 147.5-140'5° (corr.) (Found: 0,59'6; H, 4.9.C17-16 - - C, 59'3; H, 4'7%).

An attempt was also made to prepare the alcohol directly 341 from.the Grignard derivative of 3-bromopseudocumene by treating the 27 latter with formaldehyde. o 22 3-Bromopseudocumene (24.4g., b.p. 100-103 /10mm., n

1.5580, (lit.,25 n2CD 1.5575)), Was convertedinto its Grignard derivative as before.2 4 Dry formaldehyde gas was generated by passing dry nitrogen

over heated (180°) paraformaldehyde (20g.) which had previously been stored

4 weeks in vacuo over periodically renewed batches of phosphorous pentoxide.

A stream of the gas was introduced (during 2 hr. ) over the surface of the

Grignard solution at a rate sufficient to maintain gentle refluxing of the

solution which soon deposited a white solid. The mixture was boiled under

reflux for 3 hr., left overnight at room temperature,and then decomposed

at 0° with 2N-hydrochloric acid (120m1.). The product, isolated in the 27 usual way, was a lachrymatory, pale yellow oil which, on distillation,

gave 3 main fractions:

(a) b.p. 53-54°/9 mm., 2.6g.; (b) b.p. 125-128°/9 mm., n212)

1.5554, 6.3g; (c) b.p. 238-242°/9mm. , n2D 1.5490, 5.4g.

Fraction (a) was shown to be pseudocumene by comparison of its ultraviolet absorption spectrum with that of an authentic specimen

rok max.(in ethanol) 276,(270),267, and (263) 7).1, (E 509,446,492, and 362), and by gas-liquid chromatography.

Fraction (b) distilled as a mobile, colourless liquid which

rapidly darkened on standing. Its infrai.ed spectrum (in carbon

tetrachloride solution, 49mg./ml) showed no absorption in the hydroxyl -1 region: a band at 1727 cm. was far too weak for carbonyl absorption and 342

28 is suggestive of a 1,2,3,5-tetrasubstituted benzene ring. Its

ultraviolet absorption spectrum max.(in ethanol) 242 my.. (E1l%m. [A c 334, showed none of the fine structure expected of either a hydrocarbon or of

a bromobenzene but is very similar to that expected for a methyl

substituted benzyl bromide (or chloride). Such an inference is supported

by the lachrymatory nature of the compound and the rapid formation of a

white precipitate on treating it with aqueous methanolic silver nitrate.

Fraction (c) solidified on keeping; it gave a white granular

solid from ether-light petroleum (b.p. 40-60°), m.p. 46-50°. It contained

no halogen. Its infra-red spectrum (in carbon tetrachloride solution,

61 mg./ml.) showed no hydroxyl or carbonyl absorption. Its ultraviolet

absorption spectrum was similar in shape to that of pseudocumene but

shifted (by ca. 5 mj.) to longer wavelength, )1/4 max.(in ethanol) 281,276, 271,(268) m1A. (E1 cm. 43, 41 , 45, 37 ).

2.1 3,6-Trimethylbenzyltrip_henylphosphonium bromide, (XXXIV) (1613).

Owing to the low solubility of the alcohol (XXV) in light 16 petroleum, the method of Isler was modified in the following way.

To a suspension of 2,3,6-trimethylbenzyl alcohol (1.22g.)

in a mixture of light petroleum (b.p. 40-600; 25 ml.), carbon tetrachloride

(40 ml.), and dry pyridine (0-08 ml.) at -30°, was added (during 30 min.) a solution of phosphorous tribromide (0.31 ml.) in light petroleum 343

(b.p. 40-600; 2 ml.). After stirring a further 30 min. at-25°, all the solid had gone into solution,and the temperature of the mixture was o allowed to rise to 20° during the next hr. It was then cooled to 0 and

poured on to ice water (20 ml.),and light petroleum (100 ml.) was added.

The upper layer was separated, washed free of acid, and dried (Mgs04). The solvent was evaporated off under reduced pressure to leave the bromide as a viscous, lachrymatory oil (1.75g.). This oil (1.73g.) in benzene (2m1.) was added to a solution of triphenylphosphine (2-13g.) in benzene (8 ml.).

The mixture was briefly warmed and then allowed to cool to room temperature.

A white solid was deposited immediately. After standing for 7 days the

phosphonium bromide was sucked off, washed with cold benzene, and dried in

vacuo. Yield, 3'72g. (97% overall yield from the alcohol), m.p.242-5-2450 (corr.). This material was used without purification for the preparation

of isorenieratene. Repeated recrystallisation of a sample from isopropanol

-light petroleum gave a microcrystalline solid, m.p. 243-245°(corr.).

0 H BrP requires C, 70.7; H, 5.9; P,6-57.). (Found: C,70.3; H,509; 13,6-25. 28 28 Infrared light absorption: 1) max. (KBr) 1437(s:, p-Ph28), 1109(v.s.), 997

(m., possibly p-ph28)cm.-1

Isorenieratene (II),(1727). triphepyl A suspension of finely powdered 2,3,67trimethylbenzythosphonium

bromide (1.43g., 3.0 m,/mole) in dry ether (25 mi.) was stirred under

nitrogen. Ethereal butyl-lithium (0.60N; 5.5 ml., 3.3 p/mole) was added, 344

during 1 min.,,against a counter-current of nitrogen. The mixture, which soon became red in colour, was stirred for 90 min. and then dry methylene chloride (0.5 ml.) was added to destroy the small excess of butyl-lithium.

After 5 min., a solution of crocetin dial (294 mF., 1.0 m/mole) in methylene chloride (12 ml.) was added during 5 min. The mixture was stirred in the dark under reflux for a total of 4 hr. During the first hr. a red solid gradually separated out and at the end of this period a (in benzene) 491, A62 my.; no sample of the reaction mixture had A max. "cis-peak" near 350 mu. 4 was discernible. A further sample was taken 94; no "cis-peak"]; it gave only after 3 hr. FA. max. (benzene) 492, 412, one (red) spot (R p 0.0) on a chromatoplate (reversed phase, 75% aqueous acetone) showing that all the crocetin dial (which gave a yellow spot,

R 0.85, under identical conditions) had been consumed at this stage and, also, that the reaction was complete. The mixture was cooled (to 00), and methanol (30 ml.) and methylene chloride (10 ml.)Aere added. The mixture was left at 0° overnight and then filtered. The red solid was washed thoroughly with methanol and dried in vacuo. Yield, 470 mg. (89%).

It consisted of microscopic red needles of m.p. 205-206°(evac.cap.,corr.);

max. (in benzene) 494, 465'9, (442) mrThem.p. was unchanged after storing the solid for 6 wke. at 20 mm. at room temperature. Several crystallisations from chloroform-ethanol gave pure isorenieratene as small, purple-red needles, m.p. 207 - 208° (evac.cap.,corr.). (Found: C, 90.8; 3 4 r:

H, 9.1. Calc. for 040H48: C, 90.85; H, 9-15%). The properties of the synthetic material were compared with those of a sample of natural 30 isorenieratene [small purple-red needles v m.p. 207-2080(evac.cap., corr.)]. Pinely powdered samples of the synthetic isorenieratene, the natural isorenieratene, and a mixture of the two, all had m.p. 204-2050 (evac.cap.,corr.). No separation was observed on a mixed chromatoplate

(Rp 0.71; on calcium hydroxide-kieselgel (4:1) developed with benzene-light petroleum (1:4)). The infrared Spectra of the natural and synthetic samples were identical:V TtlaX . (Nujol) 964 (v.s., trans CH=CH), 815 (w.) and 810 (m.) (2 adjacent hydrogens in an aromatic ring 28) cm:1

The synthetic sample had I% . (in benzene) 493, 465, and

(443)9A (10-3E106, 123, .and 94.7, respectively). [Yamaguchi20 gives

.hmax. (in benzene) 492 and 463 mp. (103E104 and 123, respectively)]. After iodine isomerisation (60 w. lamp, 2 feet, 40 min., trace iodine), the solution had )1 . 488.5, 461, (439), and 348 94,(101790.5, 109, 81.7, and 18.8, respectively). T(in chloroform) 6.01, 7.91, 7.74 (weak shoulder at 7.76) p.p.m; relative intensities 1:1:3.

4-Bromohemimellitene CXXXVII1.(1979).

A sample of commercial hemimellitene was first shown to be essentially free of isomeric trimethylbenzenes by gas-liquid chromatography using conditions (Apiezon T, 80°) under which mesitylene, 346

pseudocumene, and hemimellitene (1,3,5-, 1,2,4-, and 1,2,3- trimethylbenzenes)separated readily (retention times 10.8,12.6, and 16.0 min., respectively). The hemimellitene by itself gave one major peak, a trace of solvent, and a trace (less than 1%) of a compound with the same retention time as pseudocumene.

Hemimellitene (45g.) in chloroform (100m1.) was treated with bromine (65g.) in chloroform (150m1.) at 0° as described by Martin.41 The solvent and excess bromine were removed by distillation and the residue was distilled at 23mm. A small fore-run was rejected.No main fractions were o,n2 collected: (i) b.p.127-130 1.5618(54.8g.), and (ii) b.p. 130-134°, n2;), n215) 1.5627(12.8g.)(lit., b.p. 128°/25mm.41; 1:561840). Both fractions gave only one peak on a gas-liquid chromatogram (squalane, 1000) except when the column was grossly overloaded when traces (totalling less than

1% of the major band) of other compounds were detectable. The column was left running for 60 hr. but only a trace (certainly less than 1%) of a peak which might have been due to the dibromo-derivative was observed.

Total yield, 72.6g. (89g).

2,3,4-Trimethylbenzaldehyde (1891)(cf. ref.24).

• Ethyl orthoformate was dried and twice fractionated through a

Dufton column (310x2 cm.). The distillate (b.p. 144-145°) still contained. some ethanol (ca. N40, by g.l.c.; since the residue from this distillation 347

also showed ethanol present, it might be that the material must be

distilled in dry nitrogen to prevent hydrolysis).

4-Bromohemimellitene (39.0&) in ether (60m1.) was added to a

stirred suspension of magnesium turnings (10.4g.) in ether (25m1.) containing a trace of iodine. Ethyl bromide (21.0g.) in ether (120m1.) was then

added during 90 min. and the mixture, which became dark in colour, was

boiled under reflux for 8 hr. Some magnesium was left undissolved.

Ethyl orthoformate (64g.) in ether (40m1.) was added during 30 min. and

the mixture was boiled under reflux for 10 hr. The ether was distilled

off in a nitrogen stream and the residue treated with a mixture of ice-

water (50m1.) and 5N hydrochloric acid (160m1.) The mixture was refluxed

under nitrogen for 30 min. and then steam distilled under nitrogen. The

distillate was extracted with ether; the ethereal solution was washed with

saturated aqueous sodium hydrogen carbonate, then water, and dried (Mgs04).

The ether was distilled off through a Dufton column (30x2cm4p and the

residual oil was distilled. Two main fractions were collected : (i)

a forerun, b.p. 20-40°/0.8mm., n22 1.5110 (mainly hemimellitene formed by

hydrolysis of the Grignard reagent by the ethanol in the ethyl orthoformate);

(ii)the major fraction (15.0g., 52%), b.p. 120-125°(mainly 120-122°)/0-8mm.; 22 11 o n p 1'5495. (Yamaguchi gives b.p. 133-135 /30mm.). Gas-liquid •

chromatography (Apiezon T, 100°) showed fraction (ii) to contain ca. 90%

of one component. It was characterised as follows. Light absorption: 348

5 56\ Tnax . (in ethanol) 265 and 214.5 my (10" 611-5 and 18.6, respectivelY ) ;

V alax . (in carbon tetrachloride) 2858 and 2720 (aldehydic C-H), 1698 (conj. C=0)cm.-1. A 2,4-dinitrophenylhydrazone was prepared and obtained, after chromatographic purification, in 845, yield. It crystallised from o chloroform-ethanol in red needles, m.p. 230-231 (uncorr.) [Yamaguchi5giv.p.

229° (uncorr.)]; (in chloroform) 387.5 my. (10- 6 28.8)56; 10 A max. max. -1 (Nujol) 3280 cm. . The semicarbazone was also prepared (yield 77'),and crystallised from ethanol: m.p. 240-241° (decomp.)(uncorr.) [Yamaguchi5 gives m.p. 240-242° (uncorr.)].

2,3,4:-Trimethylbenzyl alcohol (1911).

Lithium aluminium hydride (940 mg.) in ether (100 ml.) was added

(during 30 min.) at 0° to a stirred solution of 2,3,4-trimethyloenzaldehyde (8.15 g.) in ether (30 ml.). The temperature was then allowed to rise to 20° the rdxtrevras stirred for 4 hr. at this temperature. The mixture was cooled (to 0°) and the excess hydride was decomposed with moist ether.

Ammonium chloride (1.36 g.) in water (3.4 ml.) was added, the mixture was filtered, and the filtrate was washed and dried in the usual way. Removal of the ether first by distillation through a Dufton column (30 x 2 cm.), and finally by evacuating to 15 mm., left a viscous oil which on cooling gave a solid (8.29 g.). Crystallisation from a small volume of light petroleum (b.p. 40-60°) gave the alcohol (5.2 g., 63%) as needles, m.p. 41-49°(K). 349

0 Recrystallisation from the same solvent raised the m.p. to 48.5-49.5 (uncorr.)[lit.,45 m.p. 49-50°(uncorr.)1 (Found: C, 79.9; H, 9.2. Cale. for C H 0 : C, 79.95; H, 9.0). The compound gave only one peak on a 10 14 gas-liquid chromatogram (dinonyl phthalate, 1500). Infre'red absorption:

1) max.(in carbon tetrachloride) 3614 (sharp, E 39),3468(broad), 1010 (v.a., 28 -1 broad), and 815 (v.s., 2 adjacent ring hydrogens )cm. . The alcohol was further characterised by subjecting a sample (225mg.) to mild alkaline permanaganate oxidation (as described by Reichstein et a1.45) to give 2,3,4-trimethylbenzoic acid (150mg., 61%) which was crystallised from light petroleum (b.p. 100-120°), with filtration, to give the pure acid as prisms, m.p. 16 5-60(uncorr.).[lit., m.p. 166-1680 (uncorr.)45; 167.5° (uncorr.)441; Vmax.(in carbon tetrachloride) 1687 cm.-1.

2.3,4 -Trimethylbenzyltriphenylphoaphonium bromide (XXXVIII),(1929) •

2,3,4-Trimethylbenzyl alcohol (1.22g.) was converted into 2,3,4-trimethylbenzyl bromide in the same way as described for the 2,3,6-isomer previously. The bromide (1.75g.) was obtained as a clear, 24 1.5760. The crude bromide (1.74g.) in benzene (3m1.) colourless oil, n D was added to a solution of triphenylphosphine (2.13g.)inbenzene(8m1.). The mixture was left at 20° for 36 hr. During this period the product, which initially started to separate as small droplets of oil, separated as a white solid. This, the phosphor.ium bromide, was collected, washed with cold benzene, and dried. Yield 3.93g.(100/0 overall yield from the benzyl alcohol). 350

The crude product [m.p. 223 - 2260 (corr.)] was used directly for the next stage. A sample was recrystallised several times from isopropanol-light petroleum to give a microcrystalline solid, m.p.

requires 222 - 223° (corr.). (Found: C, 70.3; H, 6.0; P, 6.6 .0-282 8-r P C, 70.7; H, 5.9; P, 6.5c/). Infrared abGorptionN (KBr disc) mxa. 28\ 28\ 1437(s., P-Ph ), 1110 (v.s.), 996 (m., possibly P.pn )cm, .

Renierainurpurin 2{VIII) ( 19/9 1937).

Ethereal propyl-lithium (1.0N, 2.5 ml., 2.5 m/mole) was added triphenyl (during 2 min.) to a stirred suspension of 2,3,4-trimethylbenzyyphosphonium bromide (finely powdered, 800 mg., 1.68 m/ mole) in ether (20 ml.) under nitrogen. The mixture, which soon became red, was stirred for 90 min. and then methylene chloride (1 ml.) was added. The mixture was stirred for

5 min. and then crocetin dial (206 mg., 0.70 my/mole) in methylene chloride

(6 ml.) was added (during 5 min.). A dark red precipitate started to separate immediately and 10 min. after adding the dial a sample of the mixture showed absorption maxima (in carbon disulphide) at (529) and r;00

The mixture was boiled under reflux for 5 hr. Samples taken during this time had the same absorption spectra as tile one taken 10 min. after adding the dial (and all showed a certain lack of fine structure as compared with 11 \ the spectrum gives by authentic renierapurpurin ). A further quantity of phosphorane (from 160 mg. of Wittig salt) was prepared and added (in ether) to the reaction mixture. The mixture was boiled under reflux for 5 hr., 351

left stirring at 20° overnight, and then diluted with methanol (30 ml.), and left at -20°overnight. The red, microcrystalline solid was filtered

off, washed with methanol, and dried. Yield 335 mg. (90o, based on the crocetin dial) m.p. 232-234° (evac. cap., corr.). Crystallisation from chloroform-ethanol gave renierapurpurin as glistening purple plates, m.p. 237-238° (evac.cap.,corr., on the powdered solid). [Yamaguchi" gives m.p. 230° (evac.cap.uncorr.)1. (Found: C, 90.8; H, 9.1. Cale. for C40H48

C, 90.85; H, 9.15%). Visible light absorption: Nmax. (in carbon disulphide)

536, 504, and (477) my. (10-3 £88.1, 109, and 82.9 respectively): Amex.

(in benzene) 519, 12L, and (464) my. ;:k max. (in light petroleum) 501, An, and (451) mjt [Yamaguchill gives Amax.(in carbon disulphide) Ca.538, 201

and 475 Mi.& (10 3E 97, 119, and 88, respectively): figures estimated from diagram in ref. 111. Infra-red absorption: L) max,(Nujol) 1007 (w.), 973

(v.s.), 966(m.)(trans-CH=CH), 883 (w.), and 836 (m.) and 813 (m.) cm.-1 (the last 2 bands are in the region (800-860 cm.-1) expected for compounds 28 x with 2 adjacent hydrogens in an aromatic ring ); the spectrum showed the

same characteristics as that (published as a diagram in ref. 11) obtained 11 by Yamaguchi.

On a chromatoplate (calcium hydroxide-kieselgel (4:1))% synthetic

isorenieratene separated easily from synthetic renierapurpurin though, due to the low solubility of the latter, the renierapurpurin gave long, "streaky" spots. RF Values: (isorenieratene and renierapurpurin, respectively) 0.79 352

and near 0-3, with benzene-light petroleum (3:7) as developer; and, 0.89 and near 0.6, respectively, using benzene only.

A DDEND1JM

51 52 52 Phenyl-lithium, butyl-lithium, and propyl-lithium,

During this work53the 3 above reagents were used indiscriminately without any gross differences in their efficiency at liberating the

phosphorane being apparent. Of the three, the by-products (which arise by coupling during preparation of the reagents) from the last two are less objectionable (volatile liquids) than those from the phenyl-lithium

(diphenyl, a solid).

In the preparation of the model compound (IV) a slight deficit of phenyl-lithium was used to ensure that all of it was consumed in its reaction with the Wittig salt and none remained to react with the crocetin dial when that was added. Also the solution was titrated as total alkali s whereas all 3 reagents are very moisture sensitive and inevitably contain some lithium hydroxide. In the synthesis of the natural carotenoids, therefore, asmall excens of alkyl-lithium was used and this excess was destroyed with methylene chloride just prior to adding the crocetin dial.

To get a more exact estimate of the alkyl-lithium content of the reagent to

be used, 2 aliquots of the reagent solution were taken. One was added to water and was titrated with standard acid in the usual way, whilst methylene 353

chloride was cautiously added to the other (to destroy all the alkyl-lithium and remove it as a neutral product)pand then this solution was also titrated with acid, This procedure is a modification of that given by

Gilman and Haubein.57

REFERENCES

1. Goodwin, "The Comparative Biochemistry of the Carotenoids", Chapman and Hall, London, 1952, p. 155.

2. Tsumaki, Yamaguchi, and Tsumaki, J.Chem.Soc.Japan,Pure Chem.Sect., 1954, 12, 297. 3. Yamaguchi, Bull.Chem.Soc.Japan, 1957, 12, 111.

4. Yamaguchi, Bull.Chem.Soc.Japan, 1958, j1, 51.

5. Yamaguchi, Bull.Chem.Soc.Japan, 1958, 11., 739.

6. Karrer and Jucker, (tr. Braude), "Carotenoids", Elsevier, 1950; (a) p. 88 (b) p. 23.

7. Lederer, Bull.Soc.chim.biol., 1938, 20, 567.

8. Karrer and Solmssen, Helv.Chim.Acta, 1935, 18, 915.

9. Drumm, O'Connor, and Renouf, Biochem.J., 1945, 12.1, 208.

10. Garbers, Bugster, and Karrer, Helv.Chim.Acta, 1952, 22, 1179, 1850; c.f. Karrer and Eugster, ibid., 1950, 23., 1172. 354

11. Yamaguchi, Bull,Chem.Soc. Japan, 1960, 1560.

12. Grundmann and Takeda, Naturwiss., 1937, 5, 27; Takeda and Ohta, Z.Physiol.Chem., 1939, .2.58, 6; 1940, 265, 233. 13. Yamaguchi, Bull.Chem.Soc.Japan, 1957, 579.

14. Kuhn and Roth, Ber., 1932, 61, 1285.

15. Zechmeister and Schroeder, Arch.Biochem., 1942, 1, 231.

16. Isler, Gutmann, Lindlar, Montavon, Ruegg, Ryser, and Zeller, Helv.Chim.Acta, 1956, 2., 463.

17. Lonnberg, Arkiv for Zoolot. 1931, 22A, no. 14, 1.

18. Linner, Eugster, and Karrer, Helv.Chim.Acta, 1955, 28, 1869.

19. Karrer and Eugster, Helv.Chim.Acta, 1949, 11, 1934.

20. Yamaguchi, Bull.Chem.Soc.Japan, 1959, E, 1171.

21. Khosla and Karrer, Helv.Chim.Acta, 1960, 4, 453.

22. Kindly supplied by Hofmann-La Roche Ltd.

23. Wittig and Schollkopf, Per., 1954, 87, 1318.

24. Lowe, Torto, and Weedon, J., 1958, 1855. 355

25. Smith and Kiess, J.Amer.Chem.Soc., 1939, 61, 284.

26. Smith and Agre, J.Amer.Chem.Soc., 1938, 60, 652.

27. Nigam, Ph.D. Thesis, London, 1955, p. 129.

28. Bellamy, "The Infra-red Spectra of Complex Molecules", 2nd ed., Methuen, London, 1958.

29. Kantor and Hauser, J.Amer.Chem.Soc., 1951, 73, 4122.

30. Kindly supplied by Dr. M. Yamaguchi of Kyushu University, Fukuoka, Japan.

31. Goodwin and Jamikorn, Biochem.J. 1956, 62, 269; Turean, Helv.Chim.Acta, 1953, 36, 937.

32. Karrer, Eugster, and Perl, Helv.Chim.Acta, 1949, E, 1013.

33. Ahmed, Sondheimer, Weedon, and Woods, J., 1952, 4089.

34. Inhoffen, Isler, von der Bey, Raspe, Zeller, and Ahrens, Annalen, 1953, 580, 7.

35. Lindlar, Helv.Chim.Acta, 1952, EL, 446.

36. Warren and Weedon, J.,1958, 3986.

37. Barber, Jackman, Warren, and Weedon, J., 1 961 ,4019.

38. cf. also Warren and Weedon, J., 1958, 3972; Akhtar and Weedon, J., 1959, 4058.

39. Isler, Gutmann, Montavon, Ruegg, Ryser, and Zieller, Helv.Chim.Acta, 1957, Az 1242. 356

40. Smith and Moyle, J.Amer.Chem.Soc., 1936, 5g, 1.

41. Martin, J., 1943, 239.

42. Smith and Agre, J.Amer.Chem.Soc., 1938, 60, 648.

43. Smith and Stanfield, J.Amer.Chem.Soc., 1949, 71, 81.

44. Jacobsen, Ber., 1886, 12, 1214.

45. Reichstein, Cohen, Ruth. and Meldahl, Helv.Chim.Acta, 1936, 22, 412.

46. See the Experimental section.

47. Barber, Ph.D. Thesis, London, 1960.

48. Akhtar, Ph.D. Thesis, London, 1959.

49. of. Jackman, "Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry", Pergamon, London, 1959.

50. Barber, Davis, Jackman, and Weedon, J., 1960, 2870.

51. "Newer Preparative Methods of Organic Chemistry", Interscience, New York, 1948, P. 576.

52. Gilman, Beel, Brannen, Bullock, Dunn, and Miller, J.Amer.Chem.Soc., 1949, /1. 1499. 53. Thanks are particulary due to Mr. P. T. Siddons for helpful discussions about the Wittig reaction.

54. Wittig and Haag, Ber., 1955, 88, 1654. 357

T1 55. Krohnke, Ber., 1950, 91, 291.

56. cf. ref. 24 for data on analogous compounds.

57. cf. oilman and Haubein, J.Amer.Chem.Soc., 1944, 66, 1515.

58. Mildner and Weedon, J., 1953, 3294.

59. Braude, Sondheimer, and Forbes, Nature, 1954, 211, 117.

60. Bharucha and Weedon, J., 1953, 1571. 358

APPENDIX I

THE ISOLATION OF FUC OXANTHIN FROM THE SEAWEED FUCUS VES IC ULOSUS 359

The Isolation of Pucoxanthin from the Seaweed FUCUB vesiculosus.

Fucoxanthin is the principal carotenoid of the brown algae 12 (Phaeophyceae). All types of brown algae so far investigated have contained the pigment - usually in relatively large amounts (0.05 to

0.1% on a dry weight basis).1 Considering the widespread distribution 1 of algae, it is not surprising to note a claim that fucoxanthin is the most abundant carotenoid in Nature. Despite this, its structure remains unknown and, as part of a project concerned with some degradative wf111: on the pigment, a reliable method of extraction was required.

The extraction of the pigment from natural sources in good yield is complicated by its being unusually sensitive to both acid and base; also, it has been reported 3,10 that the pigment in the algae prior to extraction is subject to quite rapid atmospheric oxidation. Two main 2,4 extraction procedures have been used in the past. Both involved mincing the air-dried algae, exhaustively extracting with 90% methanol, and washing the extract free of chlorophyll with light petroleum. The extract werthen either:-

(a) diluted with water and covered with light petroleum whereupon the crude pigment separated at the interface; the pigment was 2 collected, aTVI-mAs then crystallised from aqueous methanol; 360

or (b) the aqueous methanol solution was diluted with ether and water was added to transfer the pigment to the ether layer. The ether was evaporated and replaced by benzene,and the pigment was isolated by chromatography followed by crystallisation from ether-light petroleum.4

With either method the use of hydroxylic crystallising solvents has the disadvantage of producing a pigment containing solvent of crystallisation. Chromatography of the crude pigment is desirable since straightforward crystallisation might be ineffective in removing any other xanthophylls which may accompany the fucoxanthin. Much of the early work might have been on impure pigment preparations and this 2 would explain the wide variation in reported melting points.

Other workers in this fie)d have recently sent us a crude brown solid which they obtained using method (a). However, chromatographic purification of this solid and spectral assay of the eluate, showed that it contained a negligible amount of fucoxanthin.

This failure might have been due to a seasonal variation in the pigment content of the source used (the seaweed Fucus vesiculosus) since, on a previous occasion, the same workers had obtained a solid containing

(by infrared spectral assay) ca.2O fucoxanthin.

The purpose of this section is to describe the successful isolation of fucoxanthin from the same source using a modification of 361

method (b) and to describe the methods used to follow and control the extraction procedure. Straightforward spectral analysis of the extract at various stages is unsatisfactory for assaying the fucoxanthin, since the n-carotene and chlorophyll, also present, interfere. This difficulty was overcome by determining absorption spectra of suitably diluted aliquots, first in neutral (methanolic) solution and then after adding a drop of concentrated hydrochloric acid to the solution in the cell and shaking it. The effect of the acid on the absorption spectra of the 3 principal pigments is:-

(a)n-carotene - no effect;5

(b)fucoxanthin - the spectrum in the 400-520 mt4region vanishes and is

replaced by a single broad band with maximal absorption near 680 y.4

(e) chlorophyll - bands in the 430-450 mµ region shift to shorter wavelengths whilst those near 660 Tshift to slightly longer 6 wavelengths.

Hence the difference in optical density at 480 mr (where fucoxanthin, but not chlorophyll, shows strong absorption) indicated the amount of fucoxanthin destroyed by the acid and, hence, the amount originally present. Thus it was shown that 3 extractions (with methanol) of the dried, powdered seaweed were sufficient to extract most of the fucoxanthin (relative amounts extracted 1.0 0.67 : 0624, respectively).

This method was used to estimate any losses of pigment during processing 362

and the relevant figures are given in the Experimental section. 1-3 It has been claimed that the fucoxanthin in Fucus vesiculosus is sometimes accompanied by smaller quantities of zeaxanthin and violaxanthin (zeaxanthin diepoxide). Heilbron and Phipers3 found that if the seaweed was air-dried for a long period before processing it, only zeaxanthin could be isolated. This led them to suggest that zeaxanthin is a decomposition product of fucoxanthin. 1 Recently, however, Liaaen and S'OXensen °, in Norway, have shown that although their freshly harvested algae contained no zeaxanthin this pigment did accumulate quite rapidly if the algaemere stored under a variety of conditions (all essentially anaerobic) after harvesting. They showed, however, that the zeaxanthin was formed not at the expense of the fucoxanthin (which remained fairly constant) but by the decomposition of the violaxanthin initially present in their samples of algae. The extraction procedure in the present instance was followed on reversed phase chromatoplates (on which fucoxanthin and zeaxanthin show good separation). Only one yellow spot (fucoxanthin) was observed at all stages of the processing.

The three batches of seaweed processed by us were collected 8% (near Inveresk, Scotland ) early in January,)atein February, and late in

March, 1961, respectively. All three batches gave approximately the same yield of fucoxanthin (ca. 0.2g./kg. dry weight) so that over this 3 month 363

period, at least, there was no marked seasonal variation. The yield was less than that obtained by the Norwegian workers (who obtained

0-7g./kg., using an unspecified technique) who suggested that the freshly harvested seaweed should be dried "rapidly at low temperature"; the fucoxanthin content of the algae dried by them in this way showed little change on keeping for several days at room temperature whereas, as has already been mentioned,3 prolonged air-drying (involving the very gradual loss of moisture) apparently effected the complete destruction of the fucoxanthin. In the present instance the seaweed was air-dried for

3-4 days at 20-30°, a process which may well have destroyed a large part of the fucoxanthin originally present.

In the Experimental section, only the processing of the first of the three seaweed samples eventually processed by us will be described 7 in detail. The second and third extractions were performed on seaweed samples of approximately ten times the weight and, accordingly, larger quantities of solvent were used. Any relevant differences between the procedure used on the small scale and that on the larger scale extractions are noted in the text. 3 6 4

EXPERIMENTAL (1671)

Light petroleum was distilled from potassium hydroxide pellets

before use; ether and methanol were of "AnalaR" grade. 8 The seaweed (Pucus vesiculosus, 4.5 kg.) was collected early

in January at Inveresk, Scotland. It was air-dried (24 hr. at 20° followed by 60 hr. at 25-300 ) in Scotland and arrived at the Tropical

Products Institute (where much of the processing was done)9 as a dark green, rather brittle, material (dry weight 1•45kg.). This was mixed with powdered Cardice (to prevent any local heating of the material, during grinding) and was fed through an electric mincer. The resulting

powder was delivered directly into a tank of methanol (6 1.). The

methanol immediately became dark green. The mixture was left 24 hr. in the dark with occasional stirring. The supernatant solution was decanted and fresh methanol (6 1.) was added to the residual sludge. The

mixture was left for 24 hours with occasional stirring as before and then the extract was decanted; the remaining sludge was then finally extracted with a third portion of methanol (6 1.) Each of the three methanolic extracts was kept at 00 for 1-2 days. During this time a little sediment settled out. The extracts were decanted free of this sediment and were then filtered (cotton wool plug).

The methanolic solution (16 1.) was concentrated (to 5 1.) 365

under reduced pressure by feeding it (in 1.5 1. batches) through a

cyclone evaporator. The heat required to cause the evaporation of the

solvent was supplied to the solution by 4 vertical glass tubes immersed

in the circulating solution. Although the uppermost 15cm. of each of

these tubes was steam heated, the solution was only momentarily in contact

with them so that little pyrolysis of the pigment occurred. The

temperature of the bulk of the circulating solution never exceeded 25'.

About 24W/0 of the pigment was apparently lost (spectral assay) at this stage. (A more moderate method of heating the solution has been used

recently; hot water was circulated through the whole length of the tubes

in place of heating just a short length with steam). Any attempt to

effect further concentration of the solution at this stage resulted in

the formation of a dark green precipitate (chlorophyll and its decomposition products) which complicated the next stage in the processing.

The chlorophyll (and hydrocarbon carotenoids) were removed from the methanolic concentrate by continuous extraction with light

petroleum. The petroleum was distilled up a column, condensed in the usual way, and the cold distillate was passed down into a liquid-liquid extractor surmounting the flask containing the methanol solution. The light pettoleum was allowed to return to the distilling flask and was recycled. Initially the extract was deep green but after 70-80 hr. it was only faintly coloured and the process was stopped. About 200 of 366

the pigment was lost at this stage (partly in the interfacial emulsion

which formed and was discarded).

The dark yellow methanol solution (5 1.) was siphoned off,

diluted with ether (2.5 1.), and water (5 1.) was added. Most of the

pigment (ca. 6FA;) was transferred to the ether layer which was siphoned

off. Ether (2.5 1.) was added to the aqueous layer, the mixture was stirred, and the ether layer was separated off as before; this left only a

little pigment in the aqueous layer. The combined ether solutions were

evaporated under reduced pressure (to 500 ml.) in nitrogen from a warm water bath. Benzene (500 ml.) was added and the evaporation was continued

(to ca. 100 ml.). A further portion of benzene (500 ml.) was added and the evaporation was repeated. This treatment sufficed to dry the solution

(azeotropically). Any attempt to reduce the volume of the solution to less than ca.,60 ml. resulted in its becoming viscous and unmanageable.

The dark brown concentrate (80 ml.) was poured on to a column of alumina

( 25 x 3.5 cm.) in benzene. The dark red fucoxanthin zone was held also fast to the the top of the column which carried a diffusel dark green zone.

The column was developed with:-

(a) benzene (700 ml.), to elute a broad, pale greenish zone;

(b) benzene-methanol (99.3 : 0.7, v/v)(4 1.); this slowly eluted the

bright red fucoxanthin zone down "through" the dark green zone - which

remained held fast to the upper part of the column. 3 57

A second red zone, rather ill-defined and much smaller in amount than the main zone, followed the fucoxanthin zone and was collected separately. The appearance of uncharacterised "fucoxanthin 1 2 isomers" has frequently been reported in the literature. '

The main zone was collected and evaporated to dryness in the usual way. The residual dark red oily solid was crystallised from ether-light petroleum (7:1). The fucoxanthin separated out as a brick-red granular solid, m.p. 156-8° (evac.cap.,corr.). Recrystallisation from the same solvent raised the m.p. to 165-7° (corr.). [lit., map.

159-160°(corr.)4; 166-167° (uncorr.)31. The pigment was characterised spectrally:- (a)visible absorption spectrum (in carbon disulphide) :X max.504, 476 my(E il 'in.1735, 2130) flit.,4X .(in carbon disulphide), 508,

478 my. (E 1:;114 1625, 2025)). (b)infrared absorption spectrum (in chloroform) : 3597 (& 91, sharp; non-bonded OH; probably 3 such grobps), V max. 3448 (broad, bonded OH), 1931 (E:91'; allene), 1729 (C 430, sat.ketone), 1654 (C 565,conj.cyclohexenone), 1608 (a.) and 1576(m.)[conj. C=C of

the type R.CO.CR'==Cerather than of the type R.CO.CH=C() ), 970 (v.s.), and 956 (m.) (conj. trans)CH:=CH<)cm. 1. There was no acetylenic absorption near 2180 cm.-1(cf. ref. 4). The spectrum was superimposable on a spectrum obtained (on an authentic specimen of 36

11 fucoxanthin) by M.S.Barber. The figures agree well with those given

by Torto and Weedon.4 In a potassium bromide disc, additional bands -1 appeared at 1259 and 1248cm. (both v.s., possibly 0-0).

(c) treatment of a solution of the pigment in ether (1m1.) with aqueous

hydrochloric acid (25%, 2011.) gave an intense blue colour due to a

single broad absorption band (X max.682 mtL) x. mp.).

The colour faded only slowly; 5% (E682m,t.cfell by ca. during the hour

following the preparation of the solution).

Several recrystallisations of the fucoxanthin (from ether—light petroleum) gave a much deeper coloured solid which was crystalline and had m.p.178-9°(evac.cap.,corr.). The infra-red spectrum

(KBr disc) of this material was essentially identical with that obtained on the lower melting non-crystalline sample.

It was found that a mixture of fucoxanthin and zeaxanthin separated well on a reversed phase chromatoplate (cf.section II) using

72% aqueous acetone as developer Or values 0.47 and 0.30, respectively).

Hydrochloric acid fumes turned the 2 spots blue and green respectively.

Samples were taken during the processing, as follows:- 36'9

(i) the crude methanolic concentrate;

(ii) the methanolic concentrate following extraction with

petroleum;

(iii) the ether concentrate;

(iv) the benzene concentrate;

(v) the crystallised pigment.

No zeaxanthin was detected in any of these samples. All

showed a single orange-yellow spot (fucoxanthin) and samples (i) - (iv)

gave, in addition, a diffuse,faint olive-green spot ('which was rather

"streaky", Rp ca. 0.6). Sample (i) had a dark green spot -Rp 0.0 due to

chlorophyll which, therefore, behaved as a hydrocarbon under these

conditions (cf. 0-carotene which also had R 0.0). • F

The yields of once crystallised pigment were approximately

the same for each of the three batches of seaweed processed. Thus, on

the large scale, 45 kg. of freshly harvested Fucus vesiculosus gave a dry

weight of ca. 16 kg. from which 3 to 3.5 g. of fucoxanthin was obtained.

On the large scale,? the dried seaweed was first extracted with

three portions (each of 20 1.) of methanol. The extract was evaporated

(to 20 1.) and continuously extracted with liJ7ht petroleum in two

(10 1.) portions. Each portion was then, in turn, diluted with ether 3 7 0

(7 1.) and water (40 1.) was added. After separating the layers the aqueous phase was re-extracted with ether (7 1.). The combined ether solutions were concentrated (to 1 1.) and the solvent was replaced by benzene as before. Azeotropic drying of the solution on this scale took some time and an alternative method (which also removed some of the sediment which settled out of the solution) of removing much of the water was used on one occasion. The solution was filtered (in portions of ca. 200 ml.) through a pad of alumina (IV, 10 x 5 cm.) under suction.

The pigment remained held fast to the top layer of alumina whilst much of the water passed through into the filtrate. The top of the pad was then removed and the pigment was eluted with methanol-benzene (5:95).

Final drying of the filtrate was achieved by azeotropic distillation with benzene as before. The concentrate was chromatographed in portions on alumina; an attempt to chromatograph all the concentrate on one large column was unsuccessful; the column became blocked by the traces of pectinoid material in the solution and failed to flow properly. 37 1

REFERENCES

1. Goodwin, "The Comparative Biochemistry of the Carotenoids", London, 1952, pp. 132-138, and refs. cited therein.

2. Karrer and „Tucker (tr.Braude), "Carotenoids", Elsevier, 1950, p. 309. 3. Heilbron and Phipers, Biochem.J.,1935, g51, 1369.

4. 'Torto, unpublished work; cf. Torto and Weedon, Chem. and Ind., 1955, 1219.

5. Ref. 2. page 135.

6. Zscheile. and Comar, Bot.Gaz., 1941, 102, 463.

7. With Dr. H. Yokoyama, in part.

8. Thanks are due to Dr. Booth and the staff of the Seaweed Research Institute, Inveresk, Scotland, for this.

9. We are most grateful for the help and advice afforded us by the staff of the Tropical Products Institute, London.

10. Liaaen and Sifrensen, paper presented at the 2nd. International Symposium on Seaweeds, 1956.

11. Barber, Ph.D. Thesis, London,1960. 3 7 ?

APPENDIX II

THE ATTEMPTED SYNTHESIS OF AZA FRIN 37

The Attempted Synthesis of Azafrin

Azafrin is a yellow C -polyene acid which shows entirely 27 hypophasic properties.' It has only been found,' so far, in two South

American plants (Escobedia scabrifolia and E.linearis). Extensive 2,3 degradative work on the pigment by Kuhn and his co-workers during the period 1931-1933 showed, almost conclusively, that this carotenoid has the structure (I, R=H); this is the only example known of a carotenoid containing the 1,2-diol grouping. Part of the evidence for this formulation was that mild oxidation of methylazafrin gave a diketone which

Kuhn formulated as (III).

Recently,Akhtar and Weedon4 have achieved a total synthesis of the ester (II) and they have shown that both this ester and also natural methylazafrin are converted, through mild chromic acid oxidation, to the same product, the diketone (III). This confirms the suggested structure for methylazafrin as (I, R=14e). These workers have also

CO2R

OH (I)

CO2 Me

(n)

374

CO2 me

0

obtained the first direct evidence for the presence of hydroxyl groups in methylazafrin by determining its infrared absorption spectrum. Comparison of the extinction coefficient of the hydroxyl peak with that of zeaxanthin showed that methylazafrin contains two hydroxyl groups. The sharpness of the peak led them to suggest that these groups are in the diaxial trans conformation (IV), since this is the only conformation which precludes hydrogen bonding.

HO iikMe q14111101‘11k. %s CO2,1-4

OH (IV) 4 Akhtar and Weedon then attempted to convert their synthetic

-ester or its dehydro derivative(V)]into azafrin itself; this would C27 have constituted the first synthesis of azafrin. These authors converted the dehydro derivative (V) of the C27-ester (II) to the corresponding epoxide (VI) by treatment with perphthalic acid. Base hydrolysis of the epoxide would be expected to give the corresponding trans diol [in this case, dehydroazafrin methyl ester (VII)] just as lycopene monoepoxide (VIII) 6 yields the diol (IX). Akhtar and Weedon found, however, that the dehydro-C -ester epoxide (VI) was unexpectedly resistant to hydrolysis 27 3 75

C 0211e

C 02 Me

CO2 Me

H (vii)

0H 376 and even after prolonged treatment with boiling alcoholic potassium

ethoxide (and hydroxide) they were unable to detect any of the required

product. Treatment with acids, with boron trifluoride etherate, or with

potassium acetate in glacial acetic acid all gave the corresponding furanoid oxide (cf. p. 248).

An alternative approach was, therefore, tried in the present work. This was to attempt direct trans-hydroxylation of the 5,6- carbon-carbon double bond of the C -ester using the "Pr;vost reaction". 27 7 Prevost? has shown that iodine reacts rapidly with a suspension of silver

benzoate in benzene with the formation of a pale yellow complex, "silver iodobenzoatc" (X). This reagent converts an olefin to the corresponding dibenzoate (XI) which on alkaline hydrolysis yields the dio1.7'8 It has 13a been suggested 9, that the reaction proceeds as follows:-

12 C=C > 2PhCO2 ---\Ag[(PhCO ) Agr Ag+ [(PhCO ) 2 2 2 2Agr 1+ (X) I O.CO.Ph OH \\ I,/ \ / \ // C --C PhCO2 0--0 Bases. C--C /I \ --- O.CO.Ph O.CO.Ph OH

Silver acetate may be used instead of the benzoate but the 10 iodoacetate is less reactive. Trans-hydroxylation of the double bond 10 11 predominates providing the reaction is performed under dry conditions. '

In the presence of water (e.g., silver iodoacetate in wet acetic acid), 11 12 13\ the cis-diol is formed ' (Woodward's modification ).

The dehydro ester (V) was treated with a suspension of silver 377

iodobenzoate (1 equivalent) in dry, refluxing benzene for 28 hours.

Most of the starting material was recovered unchanged. A small amount

(ca. 1%) of a pigment with chromatographic and spectral properties similar to those expected for dehydroazafrin methyl ester was isolated.

An attempt to increase the yield of this pigment by using more vigorous conditions was unsuccessful. More than half the starting material was still recovered unchanged and, in addition, extensive decomposition of the polyene occurred.

After these two small scale exploratory experiments with the dehydro ester, a larger scale experiment with the polyene ester (II) was performed. Any products from this reaction could then be compared (after esterification) directly with natural methylazafrin. Again vigorous conditions were employed and, this time, fresh portions of reagent were added occasionally during the reaction in case the reagent was being slowly decomposed under the conditions used, or being consumed by polyene decomposition products. The product was a complex mixture of substances which was separated chromatographically. The major constituent of this mixture was, again, unchanged starting material. However, two of the other substances (zone IX formed in 1.5% yield, and zone X formed in 6% yield) showed hydroxyl absorption in the infrared. In addition, one of them (zone X) bore a marked resemblance to natural methylazafrin (infrared spectra almost identical, no separation in a mixed chromatogram, and similar visible spectra). However, it was shown to be different from methylazafrin by comparing their n.m.r. spectra and by running a mixed chromatoplate. 378

An examination of its spectral data suggested that zone X

might be an intermediate hydroxybenzoate (such as, for example, XII) formed by incomplete hydrolysis of the dibenzoate (cf. XI) supposedly

CO2Ple

0.CO.Ph (xii) formed as the initial reaction product. The infrared spectrum of zone X was therefore examined (in carbon aisulphide solution) in tne -1 -1 800-600 cm. region. It showed a stong band at 758 cm. and a weak 21 band at 697 cm.`. (Some cis-compounds show absorption bands near

770 e but the presence of a cis-double bond in thie substance was unlikely). Eeither naturl methylazafrin nor the C -polyene ester 27 showed absorption bands near these positions. (Ethyl and methyl benzoates 22 -1 both show a very strong band near 708 cm. and medium strength bands -1 near 685 and 672 cm. ). Zone X was, therefore, subjected to alkaline hydrolysis using conditions considerably more vigorous than those already used during the usual working up of the initial reaction product. This gave three products none of which (according to chromatoplate work) was identical with either methylazafrin or with zone X. The major product -1 no longer showed infrared absorption at 758 and 697 cm. but no longer shored hydroxyl absorption either. In addition, it behaved as a non-hydroxylated substance on a chromatoplate, and was not studied further.

Since on the main chromatogram of the reaction product all other zones separated from zone X whilst under similar conditions zone X failed to separate from natural methylazafrin, none of these other zones could have been methylazafrin. This conclusion was confirmed (for the zones close to zone X on the column) by running chromatoplates. 379

EXPERIMENTAL

Treatment of methy1-419,13-trimethy1715-(2,6,6-trimethylcyclohex-1-eny1)- decapenta-2,4,800,12,14-hexaene-6-ynoate ("dehydro-C27-ester" )(V) with

silver iodobenzoate (735, 7.0

The ester (prepared by M. Akhtar4) was dried (12 hr. at 0.5 mm.) before use; it was chromatographically homogeneous. The reaction was performed under dry conditions using a slight excess (20%) of silver benzoate to ensure that no free iodine remained to catalyse the stereomutation of the polyene.

A solution of iodine (5.1 mg.) in benzene (2 ml.) was added to a stirred suspension of silver benzoate (12 mg.) in benzene (2 ml.).

The colour of the iodine vanished instantly, and the suspended solid

became pale yellow. The C -ester (V, 7.8 mg.) in benzene (3 ml.) 27 was added and the mixture was refluxed, with stirring, for 28 hr. No change in the absorption spectrum of the reaction mixture was detected during this time. The mixture was filtered, the filtrate was washed with sodium hydroxide solution (5%, 2 x 2 ml.), water (2 x 5 ml.), and was dried (mg804). Evaporation of the solvent left a solid residue which was dissolved in methanol (5 ml.) containing potassium hydroxide

(1.5 mg.). The solution was heated to boiling, and then left at 20° for 36 hr. The mixture was diluted with water, acidified (dilute

hydrochloric acid), and the polyene was extracted with chloroform. solu ti.on The chloroform was washed with water, benzene was added, and the solvents A were evaporated. The residue, in ether, was treated with excess diazomethane to esterify any acid formed by hydrolysis of the methyl 380 ester during the treatment with base.5 The solution was evaporated to dryness, and the residue was chromatographed on alumina (IV, 20 x 2 cm.) from benzene-light petroleum (1:1), and, finally (for zone (iii)), from benzene. This gave 3 main zones in the following order of elution. (absorption spectra were determined on solutions in light petroleum):-

(i) A max. 407 9p, shown to be unchanged starting material (by a mixed chromatogram on alumina) (7 mg., or 90% recovery);

(ii) A max . (424), AlmL, and (388) 9p (0.6%);

(iii) A (422), AM, and (390) 9p (0-9%).

The quantity of pigment present in each zone was estimated spectrally assuming that the molar extinction coefficient of each was equal to that of the C -ester (E , 70,0004). 27 max.

On repeating the experiment using more vigorous conditions

(2.4 equivalents of reagent, 132 hr. in refluxing benzene), the yields of the 3 zones obtained were: 60, 7%, and 1%, respectively. This leaves 32% unaccounted for - presumably decomposition products held fast to the top of the column.

Preparation of methy1-49,13-trimethy1-15-(2.6.6-trimethyleyclohex-1- eny1)-decapenta-2,4,6,8,10,12,14-heptaeneoate (1227-polyene ester") (U),1707

The dehydro C27-ester14 (V, 2.00 g.) in ethyl acetate 15 (90 ml.) was shaken in hydrogen with Lindlar catalyst (1.5 g.) and quinoline (0.55 ml.) until hydrogen absorption ceased (1.2 mol., 18 min.).

The mixture was filtered, and the filtrate was evaporated to dryness.

A solution of the residue in benzene (1.8 1.) containing a trace of iodine was left in bright daylight (4 hr.), and then the solvent was 381

evaporated. Chromatography of the residue on alumina (IV, 28 x 3.5 cm.)

from light petroleum, collection of the main band, and crystallisation t% from a small volume of light petroleum gave the all-trans-C27-polyene ° ester (650 mg.), m.p. 135-7 (corr.); X max. (in light petroleum) 425 9.1 (no "cis-peak" near 310 mil); V max. (in chloroform) 1694 (6 510, conjugated ester), 980, 962 (conj- trans CR=C14) cm.-1 [Lit., m.p.

131-133° (Kofler block, corr.4); A max. (in light petroleum) 426 my51. A further quantity (410 mg.) of thenall-traneester was obtained by

combining the mother liquors from the previous crystallisation with the

cle-C27-ester from the chromatogram, irradiating the mixture, and working

up as before. Total yield, 1.06 g. (5e).

Treatment of the C27:polyene ester (II) with silver iodobenzoate under dry conditions (1717)

To a suspension of silver benzoate (229 mg.) in benzene (1.5 ml.)

was added, with stirring, iodine (127 mg.) in benzene (2.5 ml.). The

silver iodobenzoate so formed (0.5 mol.) was forced under nitrogen into a

mg.) in refluxing benzene stirred solution of the C27-polyene ester (406 (1 ml.). The mixture was stirred at 90° for a total of 108 hr., further

portions of freshly prepared reagent being added at intervals as follows:

after 12 hr., 0.5 mol.; after 28 hr., 0.5 mol.; after 40 hr., 0.5 mol.;

after 68 hr., 2.0 mol. The mixture was filtered, the filtrate was washed

with aqueous sodium hydroxide (5%, 1 x 20 ml.), and water (1 x 100 ml.),

and the solvent was evaporated. The residue was dissolved in benzene

(2 ml.) and treated with potassium hydroxide (180 mg.) in methanol (20 ml.).

The mixture was refluxed for 2 hr., left at 20° for 4 days, and then pdared into water (1 1.) and chloroform (50 ml.), and acidified with 2N 382 hydrochloric acid (5 ml.). The chloroform layer was separated, the aqueous phase was extracted with chloroform (4 x 150 ml.), the combined solutions were diluted with benzene (100 ml.), and were then evaporated to dryness. The residue was dissolved in tetrahydrofuran, treated with excess ethereal diazomethane, and left at 200 for 3 hr. The solution was evaporated to dryness, and the residue was chroma.tographed on alumina

(30 x 3.5 cm.) initially from benzene-light petroleum (1:1) and, finally, frcm benzene. This gave a complex chromatogram consisting of the following zones in order of elution (absorption spectra are quoted for benzene solutions; spectral estimates of yields are based on the assumption that 13/4 E 1 cm.values at the position of maximal absorption vary from 1,750 for ca 1i with maximal absorption near 400 rap to 1,400 for compounds withAmax.near7,60*

I, >I 442 nip, shown to be unchanged starting material (132 mg.) ma. by a mixed chromatogram on alumina (IV) from light petroleum;

TI, X (450), 597, .5L my (3 mg.); max.

III, A Max . 459, 43:), 413 my (17 mg.); 431.5, 410, (391) up (10 mis.);

378.5. L2, 341 .5 up (1 mg.);

max. (439),,42, 389 mp (5 mg.); 442, 414, my (1 mg.);

435, ill 91 (2 mg.);

(427), 400, 112 mp (7 weighed directly);

)max. (462), 441 mp (28 mg., weighed directly). X' Natural methylazafrin absorbs (in benzene) at 456 and 433 T.

On a reversed phase chromatoplate with 72% aqueous acetone as developer it gave a single spot, RE 0.79; the C27-polyene ester, under the same 383

conditions, had an RF value of 0.05 (c f. hydrocarbons which have RF 0.0).

Zone III, whose visible light absorption spectrum was similar to that of

methylazafrin, was shown to be a non-hydroxylated compound (RF, 0.10).

Zone IX gave a single spot (RF, 0.6) on a reversed phase plate

(developer, 72r, aqueous acetone) Which was well separated from that given

by methylazafrin (RF, 0.79). Zone IX was rechromatographed on alumina (IV, 6 x 2.5 cm.) from benzene. One broad yellow zone was obtained which

was not, however, spectrally homogeneous. The centre part of the'band was

collected, the solvent was evaporated, and the residual yellow oil was

dried at -3 mm. The sample[5.8 mg., (in hexane) (417), 388, 10 max. .LZ, 350 mp] was dissolved in chloroform and its infrared spectrum

determined: V 0.67), 1704 (E if 6.5), 1614, 980, and max. 3590 (E lcm. lcm. 968 cm.-1(E =0.10). 3590/E1704 Zone X showed no separation in a mixed chromatogram with

77ethylazafrin[on alumina (II, 12 x 1 cm.) from benzene]. On a mixed

chromatoplate (conditions as above), however, zone X gave a single spot

(RF, 0.70) which showed partial separation from that of methylazafrin

(RF, 0.79). Zone X was evaporated to dryness, finally at 0.01 mm. for

15 hr., to leave a red oil. Infrared, visible, and n.m.r. spectra were

determined and compared directly with those of methylazafrin:-

(in chloroform): 9 max • zone X at 3600 (E tn. 0.63), 1694 (E ta. 7.7), 1615, 1582, 1537,

980, 967 cm.-1 (', 3600/E1694' 0.082); 16 1.57; E 69; non-bonded methylazafrin at 3612 (E lcm. OH), 1694 E 17), (E lam. 11.7; 515; conj. ester), 1615, 1586, 1539 (conj. 0=0 980, 965 (trans 01:1=7,0H) cm.-1 (E361A694 0.14). 384

18 ?k max. I. pure hexane ): 1% zone X at 443, 421.5, (401.5), and 281 mp (Elcm. 904, 1130, 887, and 525, repectively);

methylazafrin16 at 444.5, 419, 398.5, and 243 nap (10-3 E89.9, 104, 75.0, and 11.1, respectively). 1: values:

zone X (in chloroform) 9.10 (not sharp), 8.96, (8.79), 8.74, 8.15, 8.08, 8.02, and 6.25 p.p.m.; relative intensities: ca.1:2:(1):1:1:1: ca.2:1; methylazafrin (in deuterochloroform19) 9.16, 8.87, 8.81, (8.76), 8.44 (not sharp), 8.07, 8.01, and 6.25; relative intensities (excluding the 8.76 and 8.44 bands) 1:2:1:1:2:1.

The samplehof zone X used for the spectral determinations were recovered, the solvents were evaporated, and the residual oil was chromatographed on alumina (IV, 8 x 2.5 cm.) from benzene. The major zone (ca. 80% of the total pigment on the column) was collected and the solvent was removed under reduced pressure (finally at 0.01 mm. for 10 hr.). The (in light petroleum) 445 (shoulder), residual red oil [8.8 mg.,Xmax. 4,(402) 9p] was dissolved in carbon disulphide (1.0 ml.) and its infrared absorption spectrum determined: 3590, 1715 (E ))max. lcm. 8.9), 979, 962, 758 (E 171' 5.3), 697 (E 1.2)cm.-1 The infrared spectra. lcm. lcm. • 14.8), 978, and of both natural methylazafrin [Vmax. 3600, 1712 (E lcm. 11 -1 9.3), 977, and 962 cm. ] and the C27-polyene ester [Vmax. 1712 (E lcm. 963 cm. 1] were also determined in carbon disulphide. Neither showed any bands near 758 and 697 cm.-1. The sample of zone X used for the vidS infrared determination was recovered and the solventA evaporated. Part of 385

the sample (7.5 mg.) was dissolved in benzene (2 ml.) and added to a

solution of potassium hydroxide (2 g.) in methanol (12 ml.). The

solution was boiled under ref lux for 18 hr. and then allowed to cool.

The mixture was acidified (dilute hydrochloric acid), and the polyene was

then extracted with chloroform. The chloroform solution was washed with water, diluted with benzene, and evaporated to dryness. The residual oil

was dissolved in tetrahydrofUran (10 ml.) and treated with a solution of diazomethane(ca. 200 mg.) in ether (30 ml.). The solution was left at o 20 for 3 hr. and then evaporated to dryness. The residual oil was

chromatographed on alumina (IV, 10 x 2 cm.) to give 3 zones:-

zone (i), eluted with benzene-light petroleum (1:9), ? max. (in benzene) 438, (460) my (3.1 mg.);

zone (ii), eluted with benzone-light petroleum (1:3), A max. (in benzene) A22, (466) 9.1 (ca. 0.8 mg.); evaporation

of the solvent gave a solid, m.p. 75-85° (K);

zone (iii), slowly eluted with benzene, (in benzene) max. All, (456) mp (ca. 0.5 mg.).

Zone (i) was evaporated to dryness (finally at 0.01 mm.) : `,1 (in carbon disulphide) 1712 (E lcm. 8.8), 978, and 963 cm.-1. The V max. -1 spectrum showed no bands near 758 and 697 cm. (c zone X). In addition, the sharp band at 3590 cm.-1 (due to hydroxyl absorption) in the spectrum of zone X had also disappeared whilst a new (strong) band had -1 appeared at 1082 cm. .

Zones (i), (ii), and (iii) were 'then compared with natural methylazafrin and with zone X on chromatoplates (reversed phase, using 386

71% aqueous acetone as developer). RF values were as follows: zone (i),

0.04; zone (ii), 0.04; zone (iii), 0.44; methylazafrin, 0.61; zone X, 0.51. Mixed plates of zone (iii) with methylazafrin and with zone X showed partial, but not complete, separation. Thus zones (i) and -polyene (ii) both behaved as non-hydroxylated substances (c f. the C27 0.05 under these conditions). ester has RF

REFERENCES

1. Karrer and Jucker (tr. Braude), "Carotenoids", Elsevier, 1950. 2. Kuhn and Deutsch, Ber., 1933, 66, 883. 3. Kuhn, Winterstein, and Roth, Ber., 1931, 333.

4. Akhtar and Weedon, unpublished work; cf. Akhtar, Ph.D. Thesis, London, 1959; see also ref. 5.

5. RUegg, Montavon, Ryser, Schwieter,and Isler, Hely. Chim. Acta, 1959, Ai, 864. 6. Zechmeister, Fortschr. Chem. org. Naturstoffe, 1958, 15, 31 (p. 58).

7. Pr6vost, Compt. Rend., 1933, 196, 1129. 8. Jeffries and Milligan, J., 1956, 2363.

9. Prevost, Comyt.Rend., 1935, 200, 942. 10. Pr6vost, Compt. Rend., 1933, 197, 1661. 11. Gundstone and Morris, J., 1957, 487. 12. Ann. Reports., 1954, 51, 178 and refs. there cited.

13. See, for example: (a) Ginsburg, J. Amer. Chem. Soc., 1953, 75, 5746; (b) Barkley et al., J. Amer. Chem. Soc.,

1954, 76, 5014. 387

14. Supplied by Hofmann-La Roche Ltd., and synthesised using Isrer's route (see ref. 5). 15. Lindlar, Hely. Chim. Acta, 1952, 446. 16. Supplied by Hofmann-La Roche Ltd., [m.p. 191-193.5° (corr.); (lit.,2 m.p. 193-4° (corr.)]. 17. cf. M. S. Barber, Ph.D. Thesis, London, 1960. 18. Aromatics - free, cf. section I of this thesis. 19. Spectrum determined on a sample kindly supplied by Professor Kuhn; see ref. 20.

20. Barber, Davis, Jackman,and Weedon, J., 1960, 2870. 21. Lunde and Zechmeister, J. kmer. Chem. Soc., 1955, //, 3647. 22. From a collection of standard I.R. data published by Imperial Chemical Industries Ltd., (Bellingham Division).