XXXI. A SURVEY OF . III. NOTES ON THE DISTRIBUTION OF LEUCO-ANTHOCYANINS. BY GERTRUDE MAUD ROBINSON AND ROBERT ROBINSON. From the Dyson Perrins Laboratory, Oxford. (Received December 31st, 1932.) IN the course of an investigation of the pigment of young vine leaves, Rosen- heim [1920] noted the presence in the leaves of a water-soluble colourless sub- stance which yielded a colouring matter regarded as on treatment with hot 20 % hydrochloric acid. This important discovery threw light on the cause of some unexplained observations of Willstiitter and Nolan [1915] who found that a methyl alcoholic hydrogen chloride extract of Rosa gallica showed a gradual increase of tinctorial intensity to about twice its original value on keeping; this change occurred in the absence of oxygen. Rosenheim called the new class of colourless generators of "leuco-anthocyanins " and advanced the hypothesis that they are glucosides of the pseudo-bases of the anthocyanidins. He found that leuco- occurs in the skins of white grapes and that the formation of anthocyanidin on hydrolysis is just as rapid in a stream of carbon dioxide as in air. The present authors, having meanwhile found that colourless progenitors of anthocyanidin occur in almost every kind of plant material, enquired of Dr Rosenheim as to whether a further publication was contemplated. The reply was, respecting the immediate future, in the negative; but Dr Rosenheim kindly communicated that "he has followed up the relative distribution of leuco- anthocyanins, anthocyanins and flavones in a vine during a whole year from spring to autumn. He examined the young and mature (pigmented) leaves, the ripe and unripe fruit, the seeds, bark, wood and roots; he has also isolated a white powder containing leuco-anthocyanin from the skins of muscat grapes and noted the occurrence of leuco-anthocyanin in various parts of rose trees." Although we propose to retain the name leuco-anthocyanin for the colourless substances which we have found in a variety of woods, seeds and other parts of plants, it is unfortunate in one respect in that the prefix usually connotes re- duction of a dyestuff and in this case it is probable that the leuco-anthocyanin and the anthocyanidin are in the same state of oxidation. The leuco-antho- cyanins are normally stable in the presence of 10-15 % aqueous hydrochloric acid in the cold, although alcoholic hydrogen chloride of similar concentration, or even weaker, brings about gradual formation of anthocyanidin. This degree of stability to the action of a mineral acid excludes, in our opinion, the hypo- thesis of Rosenheim that the leuco-anthocyanins are saccharides of the antho- (or substituted anthocyanidin) pseudo-bases. Substitution of the hydrogen of the carbinol hydroxyl by alkyl groups does not protect the pseudo- bases derived from flavylium salts against the action of even quite weak acids. LEUCO-ANTHOCYANINS 207 Thus dilution of an alcoholic solution of O-pentamethylcyanidin chloride by absolute alcohol causes slow decoloration and such a solution must contain the ethyl ether of the pseudo-base (I or II). Reconstitution of the anthocyanidin 0 GEt OMe 0 ~~OMe MeO Me oMe MeO OMe |COMe CH(OEt) MeO MeO (I) (II) colour-salt is rapidly brought about by the addition of 5 % aqueous hydro- chloric acid. Other anthocyanidins and also anthocyanins behave similarly, but the naturally occurring members of the group are not easily changed to pseudo- bases in pure alcohol; they tend to give rather stable colour-bases. In order to avoid this difficulty we suggest that the leuco-anthocyanins con- tain the group -CH(OH) . CH(OH)- at position (3: 4) in the pyran ring. Thus the immediate precursor of cyanidin chloride is assumed to be (III), ring-chain tautomeric with (IV); conversion to the anthocyanidin then demands dehydra- tion (loss of hydroxyl at 4 and hydrogen at 3) and as the hydroxyl at 4 is in the O OH OH OH OH HO / \OH HO co- OH 6H(OH) I|CH(OH) YH(OH) HEH/(OH) (III) (IV) f-position to a carbonyl group this process will certainly proceed in the direction leading to flavylium salt formation rather than in that leading to a flavone. In the actual leuco-anthocyanins any of the hydroxyl groups may bear carbo- hydrate residues or be acylated. Although the greater number of the leuco-anthocyanins are readily water- soluble, a certain number of them are obtained in the first instance in colloidal solution and after flocculation cannot be redissolved. We suggest that in these cases the potential anthocyanidin molecule is attached to a polysaccharide (see Eucalyptu8 mnrgincata). The actual details will be found in the experimental section and the plan adopted has been the identification of the anthocyanidins obtained from the leuco-anthocyanins, the methods being those of Part I [1931], and Part II [1932] of this series. In order to compare the properties of a new anthocyanidin from the wood of Peltogyne spp. and from the wood of Copaifera pubiflora with authentic speci- mens, new syntheses of rhamnetinidin chloride (V) and 5-0-methylcyanidin chloride (VI) have been effected. C1 C' O OH 0 _OH- MeO OH HO OH OH OVH HO Me (V) (VI) 208 G. M. ROBINSON AND R. ROBINSON The condensation of 4-methoxy-2: 6-dihydroxybenzaldehyde and W-3: 4- trihydroxyacetophenone with the help of hydrogen chloride gave a product (V) having exactly the same properties as the rhamnetinidin chloride which Robert- son and Robinson [1927] prepared by the reduction of rhamnetin. Hence the statement of Malkin and Nierenstein [1930] that rhamnetinidin chloride is a bisflavylium salt cannot be accepted. We have long been of the opinion that the major (sparingly soluble or insoluble) products of the reduction of flavonols in acid solution are produced from pinacols but these substances are accompanied by true crystallisable anthocyanidins in smaller relative amount. Thus the pre- paration of cyanidin chloride from by reduction under the conditions prescribed by Willstatter and Mallison [1914] is reproducible and, as in the case of rhamnetinidin, we have identified cyanidin chloride made in this manner with cyanidin chloride synthesised by way of benzoylcyanidin chloride [Robertson and Robinson, 1928]. The remarkably wide distribution of leuco-anthocyanins opens up many interesting questions of biogenesis and possible physiological function, but it is too early to speculate even on the nature of the relation between the leuco- anthocyanins and the anthocyanin pigments of flowers. This is certainly not so simple and direct as it appears at first sight.

EXPERIMENTAL. Extraction of leuco-anthocyanins. In many cases the leuco-anthocyanin can be extracted by means of distilled water, various woods yielding acid solutions. Boiling 1 % hydrochloric acid has, however, usually been employed because this also extracts, preserves and indicates free anthocyanin or anthocyanidin. Should free anthocyanin be present it is possible to effect the complete hydrolysis of the glucosidal pigment before attacking the leuco-anthocyanin. The anthocyanidin is then removed by means of amyl alcohol and the separated aqueous solution, on concentration by boiling, affords further anthocyanidin, the result of decom- position of the leuco-anthocyanin. Extraction by means of alcohol or alcoholic hydrogen chloride was always more effective than the use of water. In many cases the anthocyanidins required drastic purification, and removal of impurities by extraction with ethyl acetate was frequently practised. When ethyl alcohol or ethyl acetate was employed especial care was taken to remove every trace of these solvents before carrying out distribution-ratio experiments with the anthocyanidin solutions. Summary of results. Aesculus hippocastanum. The crushed endocarp was extracted with boiling 1 % hydrochloric acid, the ffitered solution made up to 10 % hydrochloric acid, boiled for 2 minutes and the antho- cyanidin purified. This is the standard process unless otherwise stated and in this case the antho- cyanidin proved to be cyanidin. Arachis hypogaea. The testa is rich in leuco-anthocyanin affording cyanidin. Baphia nitida. Barwood contains a leuco-anthocyanin which gives rise to an anthocyanidin not conforming to the usual tests and requiring further examination. Bertholletea excelsa. The endocarp (shell of brazil nut) yields a little leuco-anthocyanin to boiling 1 % hydrochloric acid but treatment with hot 15 % alcoholic hydrogen chloride results in the formation of a relatively large amount of cyanidin chloride. Cae8alpinia granadillo. Results similar to those with Baphia nitida from the heartwood. Since this is a well-known redwood containing brazilin it is possible that the anomalous results are due to contamination with isobrazilein salts. LEUCO-ANTHOCYANINS 209 Castanea sativa. Endocarp. Leuco-A. -->cyanidin. Cocos nucifera. The fibres of the coconut contain much leuco-A. -+ cyanidin. Copaifera pubiflora. The 1 % hydrochloric acid extract of the ground heartwood contains already some anthocyanidin of undetermined nature and this is removed by the minimum of amyl alcohol. In this case the leuco-anthocyanin can be almost completely extracted from the aqueous layer by means of ethyl acetate. It may therefore be non-glucosidic or at most monoglucosidic. The substance may be rendered to 1 % hydrochloric acid after addition of light petroleum to the ethyl acetate and, on boiling with 10 % hydrochloric acid, it affords an anthocyanidin tallying in every detail with peltogynidin (see Peltogyne below). Diospyros sp. Ebony of W. African origin extracted by means of alcoholic hydrogen chloride gave anthocyanidin having the same reactions as that from Caesalpinia granadillo. Entandrophragema macrophyllum. The leuco-A. of mahogany affords mainly with indications of admixed methylated derivative or cyanidin. Erica gracili8. The flowers, stem-wood and roots contain (apparently) the same leuco-A. yield- ing a complex (i.e. acylated) anthocyanidin which is Fe +. It is unusually difficult to hydrolyse by means of alkalis but, on prolonged treatment, cyanidin is produced. Eucalyptu8 fa8cicula. The wood was extracted by means of cold 18 % alcoholic hydrogen chloride and the anthocyanidin thus produced was cyanidin. It was independently proved that the pigment arises from leuco-A. E. marginata. Powdered jarrah gives an acid extract with cold distilled water, and on the addition of i vol. of concentrated hydrochloric acid a colloid, which passes through an ordinary filter-paper, is precipitated. On shaking with ethyl acetate this is obtained in a more solid form, and after collection it cannot be redissolved in water. This solid material and also the residual aqueous solution afford delphinidin on treatment with hot 10 % hydrochloric acid. The bark, sapwood and heartwood of this eucalypt contain leuco-A. -+ delphinidin; the cortex is the richest and the sapwood the poorest in potential anthocyanidin content. E. tereticornis. The wood of this species also contains leuco-A. -- delphinidin, but admixture with cyanidin or methylated delphinidin was indicated. Fagus sylvatica. Beech-nuts contain a leuco-A. affording an unusual anthocyanidin which tallies with, butof course cannot be yet definitely identified with, 6-hydroxycyanidin chloride. This substance has recently been synthesised in collaboration with Dr E. H. Charlesworth (unpublished work). The Fe + reaction is violet and the distributions and behaviour in the oxidation test are intermediate between cyanidin and delphinidin. The alkali colour reactions are much redder than with cyanidin or delphinidin (violet instead of blue; red-violet instead of blue-violet). Helichrysum bracteatum. The red flowers contain a leuco-A. -- complex anthocyanidin (Fe +). Hydrangea hortensis. Faded flowers of a red variety were found to contain much leuco-A. complex anthocyanidin. On treatment of the aqueous solution of the leuco-A. with sodium hy- droxide, boiling half a minute, and then decomposing with hot 10 % hydrochloric acid, cyanidin chloride resulted. In this case it is clear that the leuco-anthocyanin yields an anthocyanidin different from that present in the flower-pigment since the latter is a delphinidin derivative (see Part II). Leuco-anthocyanin was also found in fresh green leaves. Hymenaea Courbaril. The sawdust contains leuco-A. -+ cyanidin. Juglans regia. The walnut is interesting in that the endocarp contains leuco-A. -> 3-acylated cyanidin simulating delphinidin and the testa affords leuco-A. -- acylated cyanidin -- cyanidin. Juniperus virginiana. The sawdust gave leuco-A. -* cyanidin. In the case of J. procera the final anthocyanidin was cyanidin with some admixture, possibly of a methylated cyanidin. Lathyrus odoratus. There seems to be a relation between the leuco-anthocyanin of the seeds of sweet-peas and the colour of the flowers, but an insufficient number of cases has been investigated to establish this. "2 LO" (scarlet flowers with bioside) has a seed-coat containing leuco-A. pelargonidin or and more probably the former. "Model" (white flowers) gave leuco-A. -- cyanidin. " Olympia" (purple flowers) gave leuco-A. -* or cyanidin-delphinidin mixture and probably the former. A remarkable feature is the high content of leuco-anthocyanin in all these seed-coats. Biochem. 1933 xxviI 14 210 G. M. ROBINSON AND R. ROBINSON

Mimusops Heckelii. The sawdust gave leuco-A. -- cyanidin. Payena utilis. Very little can be extracted from the sawdust by hot aqueous hydrochloric acid, but alcoholic hydrochloric acid yields leuco-A. -- cyanidin. Peat and miscellaneous. Alcoholic hydrochloric acid acting on peat produces cyanidin, doubt- less by decomposition of leuco-A. Even a fibrous lignite gave indications of the presence of leuco- anthocyanin. Boxwood, alderwood, elm wood and teak were found to contain a little leuco-A. Richer are the wood of Rhamnusfrangula, the seeds and stem of Phoenix dactylifera (date), seeds of Lupinus polyphyllus, of Citrus aurantium, of C. nobilis Tangerana and of C. medica Limonum. Leuco-anthocyanin also occurs in the grain of barley and oats. Peltogyne pubescens and P. porphyrocardia, also a third unnamed "purpleheart," gave exactly the same results. By extraction with cold 1 % hydrochloric acid some free anthocyanidin was obtained, and this was increased on boiling. Normally this was not separated and the leuco-A. was decomposed by boiling 10 % hydrochloric acid. The resulting anthocyanidin exhibits characteristic properties distinguishing it from all known substances of a similar type; it is therefore proposed to name it peltogynidin. Solutions of peltogynidin salts have a particularly bright cyanidin-like bluish-red colour and the Fe + reaction is bright blue. The solution in concentrated sulphuric acid fluoresces green (resemblance to fisetinidin), but it has a pink colour (resemblance to cyanidin; distinction from fisetinidin). Stability in the oxidation test resembles that of cyanidin. Sodium acetate added to the amyl alcoholic solution gives a much redder shade than cyanidin, but in aqueous sodium carbonate the colour is pure blue. Its distribution to the delphinidin reagent is very high (100) and to the cyanidin reagent it is low (< 5). Ethyl acetate extracts it from a solution containing salt. In all these properties peltogynidin resembles very closely 5-0-methylcyanidin (VI). There is, however, one difference and that is the curious point that peltogynidin is not completely ex- tracted by amyl alcohol from 1 % aqueous hydrochloric acid. Conversely continued washing of an amyl alcoholic solution with the aqueous acid gradually removes the colouring matter. This is such a constant property that we cannot attribute it to the effects of foreign substances. It is not due to insufficient acid hydrolysis, and if it is the result of acylation the group in question is not removed by boiling the solution after the addition of sodium hydroxide and subsequent addition of hydrochloric acid. This process is best accomplished by the use of the leuco-A. extracted by means of methyl alcoholic hydrogen chloride. It is almost certain that peltogynidin is either a 5- or a 7-0-substituted-cyanidin; there may be one or more hydroxyl groups in the substituent. The sapwood of Peltogyne spp. gave much the same results as the heartwood, and in both cases, as with Eucalyptus marginata, the leuco-A. was obtained in part in a solid form, capable of yielding peltogynidin on further decomposition. After extraction with aqueous acid is completed, further amounts of pigment can be obtained by the use of alcoholic hydrogen chloride. Phaeolus multilrus. The seeds of the scarlet runner bean "Champion" contain leuco-A. cyanidin. The substance is concentrated in the pigmented seed-coat. Pinus sp. A soft pine wood contained leuco-A. -- cyanidin. Polystichum aculeatum. The sporangia of the prickly shield fern gave leuco-A. -->cyanidin. The roots and the epidermal layer of the young fern gave similar results. Prunus Amygdalus. The endocarp contains leuco-A. cyanidin. P. communis, cortex and heartwood, contain leuco-A. cyanidin and a much larger proportion of anthocyanidin tallying with 6-hydroxycyanidin. The cyanidin was removed by extraction with cyclohexanol-toluene and separately examined. P. spinosa (blackthorn), heartwood, gave leuco-A. -- supposed 6-hydroxycyanidin. Again we do not claim to have identified the new anthocyanidin, but it is curious that the properties are in such close agreement with those of the proposed model. The sapwood gave leuco-A. cyanidin. Pterocarpus tinctorius, Pt. soyceuxii and Pt. Osun. These woods are closely related to the dye- wood "Sanderswood" (Pt. santalinus) and doubtless owe their red colour to a pigment of the santalin type. We have not observed the presence of leuco-anthocyanin of the usual kind, but noted that the red aqueous acid extracts yield a yellow, green fluorescent, solution on shaking with ethyl acetate. On the addition of light petroleum the yellow pigment passes back into the aqueous LEUCO-ANTHOCYANINS 211 layer and with a red colour. This behaviour is reminiscent of some substituted 4-phenylflavylium salts which we happen to have had in our hands, and with the help of a carboxyl group and due regard to the work of A. G. Perkin [1899] and Cain and Simonsen [1912; 1914] on santalin, it is not difficult to construct a plausible constitutional formula for the pigment of the species of Pterocarpus mentioned above. Pyrus malus. The heartwood gave leuco-A. -+ eyanidin. The seed-coats (Jonathan apple) are rich in leuco-A. --. eyanidin; extraction with alcoholie hydrogen chloride was necessary. Quercus pendunculata. The pericarps (acorns) contain leuco-A. -- eyanidin. Q. suber. Quite a large amount of leuco-A. -+ eyanidin can be obtained from ordinary corks, especially on treatment with alcoholic hydrogen chloride; hence the familiar discoloration. Rosa. The outer red parts of the haws of roses do not yield leuco-A. to boiling aqueous hydro- chloric acid, but the substance is present and can be extracted by alcoholic hydrochloric acid; the final anthocyanidin is eyanidin. The colourless inner portions afford more readily leuco-A. - cyanidin. Scolopendrium vulgare. The sporangia gave leuco-A. -- delphinidin. Taxus baccata. The bark and heartwood of the yew were best worked up by means of alcohol. The leuco-A. gives an anthocyanidin closely resembling 6-hydroxycyanidin in its properties. Trachylobium Hornemanianum. This wood gave the same results as Peltogyne spp. and Co- paifera pubiflora. Typha latifolia. The seeds on the spike ofjthe reed-mace and also the stem gave leuco-A. cyanidin. Viburnum tinus laurustinus. The wood gave leuco-A. -e cyanidin. Possibly some methyl ethers are also present because the distribution to the delphinidin reagent was somewhat too high for pure eyanidin. Vitisvinifera. The green leaves of the Corinth vine gave leuco-A. -- cyanidin and the colourless flesh of Alicante grapes gave the same results. The colourless flesh and skins of Almeria grapes were best worked up by means of alcoholic hydrogen chloride and ultimately afforded eyanidin. The coating of the seeds was found to be rich in its content of leuco-A. -e cyanidin. The description given by Rosenheim [1920] of his colouring matter from young vine leaves does not support the suggestion that the pigment was a pure anthocyanidin, and it was more probably a mixture with quercetin or quercitrin [Neubauer, 1873], such mixtures being extremely difficult to resolve by crystalli- sation from aqueous hydrochloric acid. The view that free anthocyanidin exists in the leaves is also not rigidly proved because it rested mainly on the high dis- tribution to amyl alcohol. Owing to the courtesy of Dr Rosenheim we have been able to examine a small specimen of the pigment crystallised along with much flavonol derivative. It is completely extracted from aqueous solution by amyl alcohol like an anthocyanidin, but it does not pass from 1 % hydrochloric acid into ethereal picric acid. After successive hydrolysis with sodium hydroxide and hydrochloric acid a true anthocyanidin is recoverable, and this is completely extracted from watery solutions by means of ethereal picric acid. It appears therefore that the pigment of young vine leaves is a complex anthocyanin, but the quantity was insufficient for complete identification of the underlying anthocyanidin. Synthesis of rhamnetinidin chloride (V). co-3: 4-Trihydroxyacetophenone (0-12 g.) was dissolved in the minimum of boiling ethyl acetate, the solution cooled and, after the introduction of 4-methoxy-2: 6-dihydroxybenzaldehyde (0-10 g.), saturated with hydrogen chloride during 1-5 hours. A part of the salt crystallised and the remainder was obtained as picrate by washing the solution with water and adding saturated picric acid to the aqueous layer. The picrate was collected, added to the crystallised chloride and the whole dissolved in boil- ing alcohol containing a little hydrogen chloride. The salt was precipitated by means of ether, collected and crystallised by slow evaporation of a solution in 14-2 212 G. M. ROBINSON AND R. ROBINSON aqueous alcoholic hydrochloric acid as described by Robertson and Robinson [1927]. The chloride had all the properties mentioned by these authors as cha- racteristic of rhamnetinidin chloride, and a direct comparison of colour reactions and distribution ratios showed that the salts were indeed identical. In addition to the properties already described rhamnetinidin is very largely extracted by the delphinidin reagent, wholly from dilute solutions; and it is more readily ex- tracted by the cyanidin reagent than is cyanidin, but not so readily as peonidin. In sulphuric acid rhamnetinidin gives a pink solution with a faint green fluor- escence. Synthesis of 5-0-methylcyanidin chloride (VI). This salt was prepared like the foregoing, but using 2-methoxy-4: 6-dihydroxybenzaldehyde in place of the isomeride; the whole salt (which did not crystallise), was converted into picrate and this derivative was crystallised several times from half-saturated aqueous picric acid containing a few drops of alcohol. The orange-brown, slender clustered needles (found in material dried at 1000: C, 50-2; H, 2-9; N, 7-9. C22H15O13N3 re- quires C, 49-9; H, 2-8; N, 7*9 %) dissolve in sulphuric acid to a pink solution exhibiting an intense green fluorescence. The chloride prepared in the usual manner is very similar to rhamnetinidin chloride, the only distinction being that the colour in aqueous sodium carbonate is greenish blue (rhamnetinidin-blue without green tinge) and the green fluorescence in sulphuric acid solution is much the more intense in the case of the 5-methyl ether. Undoubtedly peltogynidin resembles cyanidin 5-methyl ether more closely than it does the 7-methyl ether; it should be noted that attachment of a sub- stituent to positions 3, 3', or 4' is excluded. Our grateful thanks are due to Miss M. M. Chattaway and Dr L. Chalk of the Department of Forestry, University of Oxford, and also to Mr B. J. Rendle and Mr W. Campbell of the Forest Products Research Laboratory, Princes Ris- borough, for invaluable assistance on the botanical side and for the generous provision of authentic material.

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