I

ASPECTS OF ERGOT ALKALOID BIOSYNTHESIS

A thesis presented by

FRANCOISE QUIGLEY

In part fulfilment of the requirements for the degree of Doctor of Philosophy, of the University of London

Imperial College November 1973 Biochemistry Department ABSTRACT

Methods of isolation and purification of ergot alkaloids were investigated with reference to the following chromatographic techniques: thin layer chromatography (T.L.C.), gas liquid chromatography (G.L.C.) and the use of neutral and ion-exchange chromatography. Sephadex SP C-25 ion-exchange resin, in appropriate ionic form, selectively absorbed ergot alkaloids from culture extracts and gave not only a separation of the basic and amphoteric fractions but also separated the peptide and clavine ergot alkaloids. G.L.C. analysis of Claviceps fusiformis extracts was achieved by trifluoroacetylation and silylation.

The three major ergot alkaloids present in the amphoteric fraction of Claviceps fusiformis extracts were identified as 4-dimethylallyl- tryptophan, clavicipitic acid, and N-methyl-4-dimethylallyltryptophan. The latter, a new isolate, was observed to accumulate under anaerobic conditions. The inter-relationship of the clavine alkaloids was investigated in 14 Claviceps fusiformis by a kinetic feeding experiment using (2- C) tryptophan as the precursor. Results agreed with the generally accepted scheme of ergot alkaloid biosynthesis in Claviceps spp. tryptophan dimethylallyl- mevalonic ---4 tryptophan ___4 chanoclavine-I ---> agroclavine acid elymoclavine

The presence of other biosynthetic intermediates, hydroxylated clavines, _ 14 was also suggested. Feeding of (3'-2H3, 2- C) mevalonate confirmed the general scheme above and provided further evidence for the loss from ) C-3 of mevalonate,of 1 and 2 protons in the enzymatic formation of chanoclavine I and agroclavine respectively.

The major ergot alkaloids present in Sphacelia sorghi culture extracts were identified as chanoclavine;T, festuclavine,dihydroelymoclavine, dihydrolysergic acid and dihydroergosine. There was evidence for minor dihydroalkaloids of the clavine type and the remainder of the extract consisted mostly of peptides, which appeared to be unrelated to any known ergot alkaloids. The biosynthetic pathway leading to ergot alkaloids in 14 Sphacelia sozr2....ii was investigated. Feeding of (2- C) mevalonate, (2-14C) tryptophan and combined feeding and trapping experiments with

festuclavine, dihydroelymoclavine and dihydrolysergic acid suggested the pathway:

tryptophan mevalonic chanoclavine -I ) festuclavine --dihydroelymoclavine acid

dihydrolysergic acid dihydroergosine minor dihydro-peptide alkaloids.

This pathway parallels that of unsaturated ergot alkaloids biosyitheis in Claviceps spp. and seems to be specific to aia.2211a sorghi. The negative incorporation of agroclavine into the peptides alkaloids suggested that no unsaturated clavine alkaloids were involved in the pathway. However, agroclavine and elymoclavine were found to have an inhibitory effect on the biosynthesis of the dihydro-peptides in this fungus. - IV -

CONTENTS

Page ABSTRACT II CONTENTS IV ACKNOWLEDGEMENTS V PART I : REVIEW I.Introduction 1 II.Chemistry 2 III.Biosynthesis 7 PART II : MATERIALS AND METHODS I.Physical measurements 29 II.Strains and culture 29 III.Extractions methods 32 IV.Chromatographic methods 33 V.Identification and quantitative estimation of alkaloids 38 VI.Feeding of precursors 38 VII.Radioactivity measurements 39 VIII.Compounds and derivatives 39 IX.Experimental 42 PART III : INVESTIGATION OF METHODS I.Introduction 50 II.Thin Layer Chromatography 50 • III.Resin Chromatography 56 IV.Gas Liquid Chromatography 69 PART IV : CLAVICEPS FUSIFORMIS I.Introduction 82 II.Study of the clavine alkaloid fraction 83 III.Study of the amphoteric alkaloid fraction 89 IV.[2- 14C1 tryptophan time sequence feeding 95 ' 2 14 , V. L33L '-2 HH3,2-,2 d mevalonic acid feeding 101 PART V : SPHACELIA SORGHI I.Introduction 109 II.Sphacelia sorghi alkaloids 110 III.Sphacelia sorghi biosynthesis 111 REFERENCES 149 ACKNOWLEDGEMENTS

I am grateful to Professor E. B. Chain for being able to work in his department. I am much endebted to my supervisor , Dr. K. Barrow , for his guidance throughout this project. I must thank Dr. P. Mantle for helpful. advices , and also Dr. G. Mellows.

I like to thank C. H. Freshwater and R. Freer for their occasional technical assistance.

I gratefully acknowledge the S. R. C. for its financial assistance. - VI -

To Paul VII -

PART I 1. INTRODUCTION The main sources of ergot alkaloids are the different species of the fungus Claviceps which grows parasitically on rye and other (1, 2) grasses . Ergot alkaloids occur as well in other related fungi: (3,4) Aspergillus, Rhizopus Penicillium (5) and in higher plants belonging to the Convolvulaceae mainly in the genera Ipomea, Rivea and Argyreia (2'6).

The name ergot refers to the sclerotia of Claviceps purpurea (Fr.) Tul. which appears as a dark-coloured extension on the ears of rye. This specie is responsible for the long history of ergot alkaloids (7,8) . During the Middle Ages it was responsible for widespread poisoning epidemics known as "St. Anthony's Fire" or "Ergotism", but at the same time their medicinal importance was recognized and has been mentioned as early as 1582. Their use has been increasing ever since, especially in the 19th century when the first crystalline .preparations of ergot alkaloids were isolated from sclerotia. Today the ergot alkaloids and their derivatives have found many applications in pharmacological fields. Apart from their classical use in obsterics, (2,9,10) they are being utilized in neurology and psychiatry 9,10) and their potential as an anti-fertility agent is being investigated (11) This development has been made possible by a greater availability of ergot alkaloids due to the introduction of saprophytic culture. Until recently ergot alkaloids were isolated from sclerotia coming from spontaneously or artificially infected rye fields. The first fungi grown saprophytically with success were those producing alkaloids of the clavine type (12) and they could be grown either by surface or sub- merged cultures depending on the strain (13' 14' 15). The most impor- tant step was the growth in submerged cultures of the lysergic acid derivatives producing fungus: Claviceps paspali (16), thus giving access to the large scale production of the pharmaceutically important ergot alkaloids (e.g. 17).

Another aspect of the culture of ergot alkaloid producing fungi has been the recent development of cell-free systems that synthesize or (19) transform alkaloids from Claviceps spp. and these vary from crude preparations to very specific ones (20, 21, 22) There are several reviews covering the mycology, pharmacology (2 15, 11, 18) and chemistry of the ergot alkaloids ' 8, emphasising one or more of these aspects. The biosynthesis of these alkaloids has been reviewed recently by Agurell (23) Voigt X24) Ramstad(25) (26) Grtiger Thomas and Bassett (27)

2. CHEMISTRY The ergot alkaloids belongs to the large group of indole alkaloids. They are distinguished in that group by being 3, If substituted indoles instead of 2, 3. The majority have the tetracyclic structure known as ergoline (28)

Indole Ergoline

They can be conveniently divided into 2 groups on the basis of their structure; the lysergic acid derivatives and the clavines.

Lysergic acid derivatives Clavine derivatives - 3 -

Table I :Simple amide peptide alkaloids.

0 R --c

R

(+)-lysergic acid -OH ergine (+)-lysergic acid -hydroxyethyl amide 4'41112 tt 3 (lysergylmethyl carbinolamide) 011 ergonovine (ergobasines ergometrine) -NH-CH-CH3 CHOH lysergic acid L-valine methyl ester -NH-CH-CH(CH3)2 COOCH3

ergosecaline • Table 2 • Tripeptide cyclic alkaloids.

R R R"

ergotamine;) H H -CH2-C6H5 ergosine H H -CH2-CH(CH3)2 ergocristine CH3 CH3 -CH-C2 6h 5 a-ergokryptine CH CH -CH -CH(cli ) 3 3 2 3 2 p-ergokryptine CH3 CH3 ;CH(CH3)0H2CH3 ergocornine CH ) CH3 3 -CH(CH3 2 ergostine H CH 3 -CH2-C6H5 ergovaline H -CH(CH3)2

(Plus the epimers at C-8). 5

Table 3 Examples of clavine alkaloids.

17 0-1L—R

N-C H3 H 1-1

R Rt An

A8 =9-ergolenes agroclavine H H elymoclavine H OH

A9'1° -ergolenes 1O lysergene(& 8'17 andt.xA 9' ) - H lysergine H H H lysergol OH H H setoclavine H OH H penniciavine OH OH H

ergolines (D-ring saturated ) festuclavine h. H H H pyroclavine H H H dihydroelymoclavine H .H OH (dihydrolysergol)

( epimeric at 0-8) The first group is composed of the derivatives of lysergic acid and isolysergic acid (the stereoisomer at C8). It can be further divided into the sample amide derivatives and the cyclic peptide derivatives. (Table 1 and 2)

The clavine derivatives can be divided in 3 groupsA8-9 ergolenes A 9-10 ergolenes and those with a saturated D ring (table 3). A few (29) clavines have an open D ring the chanoclavines and rugulovasineP°).

OH OH

N 1-ICH I-1

1111111

Isochanoclavine-I

Chano clavine-I OH 0

(-) chanoclavine II (-1- enantiomer) rugulovasinesA and B

The structure of ergot alkaloids has been elucidated by both physical and chemical methods. The total synthesis of lysergic acid (31) and ergotamine (32) has been achieved. The absolute configuration of lysergic acid has been determined by rotatory dispersion(33) and by (34) chemical degradation to an amino acid of known configuration , Structure and stereochemistry of ergot alkaloids could be derived by relating chemically one compound to another: clavines via derivatives (18). of lysergic acid or dihydrolysergic acid Ergot alkaloids were found to be related toD lysergic acid with a 5R, 8R configuration and ring C and D trans to each other.

3. BIOSYNTHESIS

Early precursors It has now been well established that the ergoline skeleton precurs ors are tryptophan, mevalonic acid and methionine (fig. 1).

CO 0 k 0 - methionine p

H

H mevalonic acid agroclavine

L-tryptophan

Fig. 1

The role of tryptophan as a precursor has been demonstrated by high incorporation rates of labelled tryptophan into clavine alkaloids (10.6, 39.4%) 36' 37) and peptide alkaloids (36% (38) 59% (39)), but the incorporation efficiency varies with the fungus strain and a low efficiency, in some cases, might be due to the degradation of tryptophan by other pathways (23) The whole of the tryptophan molecule, except the carboxyl group is found to be incorporated into the ergoline nucleus (35' 36). Double labelling experiment showed that all the rest of the carbon, hydrogen and nitrogen atoms of the side chain ofL,tryptophan incorporated into elymoclavine and that D tryptophan although incorporated must first be (40) converted to the L isomer This has recently been confirmed by using a cell-free system and testing for the efficiency of both L and D tryptophan as precursors of the- ergot alkaloids (22) Feeding experiments with compounds related to tryptophan showed that precursors such as anthranilic acid, indole and indolyl-3-pyruvic acid were reauily incorporated (37) but not the various derivatives (41), (42) tried which included 4 hydroxytryptophan 5 hydroxytryptophan and 4 hydroxytryptamine (23). In agreement with this last result the retention of label at 6 and 5 position and its loss at the 4 position was observed in clavine alkaloids after feeding the corresponding (37). deutero tryptophan precursor Therefore, the activation of the C4 position of tryptophan does not involve hydroxylation. The non- (43) (43, 44) incorporation of tryptamine, N methyltryptamine and (44) N methyltryptophan axeevidences for either methylation or decarboxylation occuring at a later stage after the condensation with the mevalonic moiety. (45) Double labelling feeding experiments by Baxter showed that N methylation is done via methionine by the usual transmethylation reaction.

The identity of the third precursor of the ergoline nucleus as 46, 47) mevalonic acid was established simultaneously by 3 groups with the incorporation of labelled mevalonic acid in clavine as well as (48) peptide alkaloids. As expected, the carboxyl group is lost and (37, 19) (23, 49) isopentenylpyrophosphate and dimethylpyrophosphate were also found to be precursors, the last one being the most efficient 22) and consequently the active unit in the alkylation of tryptophan ( (fig. 2).

•

0

mevalonic acid dimethylallyl isopentenyl pyrophosphate pyrophosphate

Fig. 2 Lower rates of incorporation of labelled mevalonic acid into alkaloids (50,51,52). were found than for tryptophan and ranged from 0.3 to 21/0 It may reflect the fact that mevalonic acid is in greater demand in the fungus than tryptophan and must be diverted through other pathways. It is also known that only one of the mevalonic acid isomer is incorporated(38) in fact, the 3R isomer (52,53)

Incorporation studies using mevalonic acid labelled specifically in different positions have yielded many particularly interesting results and have been very useful in determining the mechanistic details.

Degradation studies following incorporation of [2-/4C] mevalonic acid into ergot alkaloids have shown the label to be distributed between the C-17 (90%) and C-7 (10%) positions in 38) (52), tetracyclic alkaloids elymoclavine (51, and ergosine but for the clavines With an open D ring most of the label (90%) is in the C methyl which is the c,is methyl for Chanoclavine I and II and the (55 tran$methyl for Isochanoclavine ' (fig.38) 3).

chanoclavine -I Isochanoclavine Chanoclavine-II enantiomer

R = OH Elymoclavine

R = H Agroclavine

Fig. 3 Incorporation of (2-14C) mevalonic acid into clavine alkaloids. - 10 -

The fate of the hydrogen atoms of mevalonic acid have been followed by incorporating a mixture of [3 RC S -.2 14c]. mevalonate and mevalonate tritiated in selected positions. Both C-2 hydrogens showed complete retention in festuclavine and agroclavine(48) while there was only a 50% retention for the C-4 and C-5 hydrogens in (48)as (23). these alkaloids well as chanoclavine I The stereo specificity of the lost or gained protons has been determined by using mevalonates stereospecifically labelled. (fig. 4). The feeding of 5R and 5S tritiated mevalonates resulted in the incor- poration of the 5S label and the loss of the 5 R in the chamoclavines, agroclavino and elymoclavine(53'54)Ithe label should be situated at 0-10 in the case of elymoclavine(53) • In the same way it was found that the 4R proton of mevalonate which 54 56) was retained while the 4S was lost(38' ' ,but it was retained 100% in chanoclavine there was only 70% of the label. left inelymoclavine.

All these results have to be taken into account in the discussion of the formation of the ergoline nucleus, but at that stage the retention of the 4 R hydrogen in ergot alkaloid is particularly significant. Isomer- isaticn of mevalonic acid to dimethylallylbyrophosphate has been shown to occur with retention of the 4 R hydrogen and the C-2of mevalonate becoming the trans methyl group of dimethylallylpyrophosphate(5) (fig.2). As in chanoclavine I, the label is found in the cis position, there must be a trans isomerisation between the condensation and chanoclavine stages.

Very little is know of the condensation mechanism itself from the evidence reviewed above. It must occur between dimethylallylpyro- phosphate and L tryptophan unexpectedly activated at the C-4 position. It seems that the isoprenylation of tryptophan might proceed with (58). inversion of configuration at the allylic carbon atom It must aldo take place at an early stage before decarboxylation of the side chain or methylation of the nitrogen. The product of such a condensation is dimethylallyltryptophan. (fig. 5).

This compound has only been shown recently to be a natural metabolite. It has been isolated from cultures where it had accumulated in one case because of anaerobic conditions, .and in the other, due to inhibition of N - methylation by methionine (59)

OH OH

I

OH OH

)

IL IC

Fig. 4 Incorporation into chanoclavine I (III) and elymoclavine (IV) of mevalonic acid stereospecifically labelled with tritium at the C-4 position (I) and the C-5 position (II) - 12 -

dimethylallyl pyrophosphate

4-dimethylallyl- tryptophan L-tryptophan

Fig. 5 Condensation of dimethylallyl pyrosphate and L-tryptophan.

Earlier feeding experiments using synthetic labelled dimethylallyl- (60 61 49) tryptophan as well as more recent ones with labelled (62, 22) )4methylallyltryptophan isolated from cultures showed an incorporation rate of about 15% in clavine alkaloids agroclavine and _elymoclavinet thus being a more efficient precursor then tryptophan.

The isolation of a cell-free system from a Claviceps spp. capable (22) of catalysing the formation of 4 dimethylallyl tryptophan has confirmed the scheme shown in fig. 5 dimethylallyl pyrophosphate and L tryptophan were the best precursor, - and as expected the dimethylallyl tryptophan synthetised had the L configuration. Subh a system will certainly lead to a greater knowledge of the condensation mechanism itself.

The next stages in the biosynthetic pathway must include decarboxy- lation and closure of ring C. The decarboxylation product of dimethylallyltryptophan is dimethyallyltryptamineand such a synthesised compound labelled in the allylic side-chain was found to be incorpo- (4 rated very poorly in clavine alkaloids 9), although when the -- experiment was repeated with the label in the alanine side chain, (63) this compound was incorporated more efficiently.63) OH

11H2.

A

1

4-dimethylally1 tryptophan

R = H agroclavine

R = OH elymoclavine

dimethylallyltryptamine

Fig. 6 Proposed pathway by Plieninger et.al.(63). 14

The closure of ring C involving either 4 dimethylallyl tryptophan or 4 dimethylallyltryptaminewould give the compound deoxychanoclavine or nordeoxychanoclavine if methylation of the nitrogen had occured.

R = H nor deoxychanoclavine-I

R = CH3 deoxychanoclavine-I

Both compounds have been synthesized but (6o, 64) they are not incorporated in alkaloids (64, 65). This means that hydroxylation of the allylic side chain must occur before cyclisation.

Plieninger et. al. have synthesised labelled hydroxy derivatives dimeFhylallyl iTypta m ine of dimethylallyl tryptophan A andAp and shown their incorporation into agroclavine and elymoclavine,B being much more efficient than A which made them put forward the scheme in fig. 6.

However, B is an isomer of the expected intermediate: 1i ) OH

- 15 -

OH

OH OX OH ° NHCH3

11101 Si Pi\

cha nocl a v ne I

•OH als R=H or COOH; —CH,- N o ,X=HPO H ' 3 2 CHLOP

Fig. 7 : Hypothetical scheme for ring C formation during ergoline biosynthesis 54. - 16 -

It might be mentioned here that none of these proposed intermediates have yet been found in inhibition studies discussed earlier which resulted in the accumulation of dimethylallyltryptophan, only one other metabolite was isolated after inhibition with ethionine. This is clavicipitic acid (66) whose structure is now under debate. However, this metabolite is not incorporated into ergot alkaloids (66) A model of ring C closure which takes into account the cis-trans isomerisation of the allylic chain and the retention of the 4R and 5S protons of the mevalonic acid at C-10 and C-8 respectively has been put forward (54)(fig. 7).

It involves an activation of the C-10 position by hydroxylation, but this model could only explain the formation of chanoclavine I and II.

The exact stage of methylation is unknown, but it must occur just before or during ring C closure.

INTERMEDIATES Because of their open D ring, the chanoclavines were obvious intermediates in ergot alkaloid biosynthesis. The situation was confused (52, 23) until the four chanoclavines were properly characterised by Stauffacher and Tscherter (29) and this structure confirmed (67) Isochanoclavine I although the most likely precursor because of its configuration,6 (55, 5) was in fact found not to be incorporated in (38). elymoclavine 65) nor was chanoclavine II Chanoclavine I is the most efficient precursor- (55, 68, 69) with a high incorporation level of up to 40%. In feeding experiments chanoclavine I is also characterized by a high specific activity and it has been found to be incorporated as efficiently as agroclavine into elymoclavine. It has been mentioned earlier that the synthesis of elymoclavine and agroclavine from chanoclavine I occur with a cis-trans isomerisation at the allylic 1 double bond as the labelL2-C2- Cj mevalonic acid is found at the C-7 of chanoclavine I and C-17 of elymoclavine and other tetracyclic ergot alkaloids (fig. 3). .Refeeding of chanoclavine I labelled at C-7 also gave elymoclavine with 90% labelled at C-17 (38). These results cancel out a possibility of ring D closure by reaction between the methyl group and nitrogen but rather suggest that it is the hydroxy methyl group which is involved. - 17

It has been advanced that the hydroxyl group is first oxidised to the aldehyde. The observed loss of a methylene proton at C-17 (56) r14 has been verified by using chanoclavine C, 17-3H] giving 50% • (70) retention of the tritium label in elymoclavine . Indeed the synthesized aldehyde chanoclavine I aldehyde labelled with tritium at C-17 was better incorporated in elymoclavine than chanoclavine I and without loss of the tritium label which was found as expected in the C-7 positon. If chanoclavine I aldehyde is a true intermediate the cis-trans isomerisation must occur at that stage. In this process we must take into account the 70 retention of the tritium label at the C-9 position of elymoclavine during its formation from chanoclavine I. (56) It has been suggested that this isomerisation is catalyzed by an enzyme Enz-X-H which adds and removesprotons to the C-9 position, thus transferring labelled protons from 1 substract molecule to another, some exchange with solvent protons would explain the relative loss in label (fig. 89. In support of this scheme are the results of double labelling studies with 4-2H, 2-13C]mevaJonate and detection of incorporations by mass spectrometry. Chanoclavine I showed incorporation of either 13C or 2 H but not both into the same molecule. However,elymoclavine isolated from the same feeding experiment contained single molecules possessing both the 2H and 13C labels (70) The cis-trans isomerisation can be simultaneous. with ring formation (path a) or preceeds it (path b). Ring closure via. chanoclavine I aldehyde makes of agroclavine an obligatory intermediate, but direct conversion of chanoclavine I into elymoclavine has also been observed in a cell-free system from a Claviceps spp. (20) Although, it proceeded with a 20% efficiency no agroclavine was detected or able to act as substrate. The need for free oxygen in the system suggested an epoxy intermediate (fig. 819. This scheme takes into account the loss of a C-9 proton but not the C- 7 one, But in a cell-free system from another strain of Claviceps which is able to catalyse the con- version of tryptophan into chanoclavines I and II, agroclavine and elymoclavine, agroclavine was incorporated into elymoclavine with 15% efficiency (21) 1.8

H

chanoclavine I Enz - X-H

Enz-X-T

'X-Ena • 7-

NCH

H

- Enz-X-T

agroclavine

Fig.8a: Closure of ring D (scheme 1)56.. 19

chanoclavine I

OH

elymoclavine

Rt Closure of ring B (scheme 2 20 - 20 -

INTER-RELATIONSHIP BETWEEN CLAVINE AND PEPTIDE ALKALOIDS

The main features of the relationship between ergot alkaloids have been worked out (23) and agroclavine has been established as the primary ergoline, precursor of both clavine and peptide alkaloids (fig. 9).

This involved the sequence:

Chanoclavi ne I->agroclavine-elymoclavine4peptide alkaloids. It can be noted that although chanoclavine I gives elymoclavine as seen (68) before it is not a reversily process . The position of elymo- clavine has been debated. There seems to be a direct pathway for its synthesis from chanoclavine I in a strain of Clavicem as agroclavine was neither product or substrate in the cell-free system of this (20)1 strain but most evidence.makes agroclavine a precursor of elymoclavine. Specific activity studies also agree with that (23, 68) scheme . The oxidation of agroclavine has been observed to be (23). a fast and efficient process A cell-free system catalysing that (21). reaction has been obtained It is a non-reversible process. The mechanism itself is not understood, C-17 oxidation is mostly (72) specific to Claviceps spp. and does not seem to involve a peroxi- (73)w dase as the level of this enzyme in culture is low hile the concentration of elymoclavine is quite high. An oxygenase is more likely, these enzymes seem to be implicated in ergot alkaloid (74). biosynthesis in vivo The enzymic cell-free system catalysing that reaction needed NADPH and areobic conditions.- The hydroxyl group (75) oxygen has been shown to come from molecular oxygen . Recently, oxidation of agroclavine to elymoclavine has been achieved using a non,- Claviceps spp. cell-free extract, but arnammalian microsomal system (76) involving cytochrome P450

Other clavine alkaloids are derived from agroclavine by hydroxy- lation of the C-8 position. This hydroxylation in contrast with the one seen previously is not very specific and can be achieved by several fungi as well as plant homogenates containing the enzyme peroxidase(77) In vitro experiments using a purified peroxidase: hydrogen peroxidase showed that this enzyme can catalyse the conversion of agroclavine (78,79) (80) and elymoclavine to their respective 8-hydroxy derivatives . The 10-hydroxy derivatives which are also products of the reaction re-arrange easily to give the 8-hydroxy derivatives, creating an asymmetric centre at the C-8 position. This accounts for the stereoisomer derivatives.of agroClavine: setoclavine isosetoclavine and those of

— 21 —

lysergine dihydroely moclavine lysergol isolysergine (dihydrolysergo1-1) isolysergol

OH OH

chanoclavine-1 agroclavine elymoclavine lysergene

z OH \/

festuclavine setoclavine penniclavine pyroclavine isosetoclavine isopenniclavine

norsetoclavine

Fig. 9 : Interrelationships of the clavine alkaloids. - 22 -

10 hydoxy derivative

8 hydroxy derivative

8-9 9-10 epoxy derivatives

R=OH , elymoclavine ,

R=H, agroclavine ,

Fig. 10 : Peroxidase conversions of A8,9 clavines. 23

elymoclavine:penniclavine and isopenniclavine (fig. 10). In ergot cultures the level of peroxidase is low and that of catalase high (73), and explains the low concentraction of these derivatives in cultures. In aerobic conditions the peroxidase catalyses as well the formation of the different epoxy derivatives which occurs naturally in small amounts. Norsetoclavine, another minor metabolite (81) is explained by the demethylation of setoclavine by the peroxidase (79) Hydrogenation of theL18-9 double bond in culture yields the two (82). stereoisomers festuclavine and oyroclavine The origin of dihydrolysergol and costaclavine is uncertain (23)

The last family of clavine alkaloids is produced by the isomerisation of the double bond from L1 8-9 to theA 9 -10 position. Lysergol has been shown to derive from elymoclavine in ergot (82) cultures but for its stereoisomer isolysergol and the equivalent derivatives of agroclavine: lysergine-isolyserginel not much is known (23). concerning their origin Lysergene has been isolated from nature but its origin is not certain and might be a precursor of lysergol and 8 hydroxy elymoclavine (82' 83) Clavine alkaloids have been demonstrated many times to be the precursors of simple amide and more complex peptide alkaloids e.g.: Chanoclavine I into lysergamide, ergotamine and ergotoxine (68) elymoclavine into lysergic acid (83) lysergamide (8k) and ergometrine (85) land agroclavine into ergometrine and ergotamine (85)

The incorporation of these labelled precursors occurs quite efficiently with the label being found in the ergoline moiete of the lysergic acid derivative. More specifically the activity of C -17 labelled precursors is found at the same position in the (84, 86) derivatives : the carboxyl group of lysergic acid. These feeding experiments gave evidence for the accepted biosynthetic sequence shown in fig. 11 and was confirmed as well by Agurell in competitive feeding experiments with the different intermediates from the sequence from tryptophan to lysergyl methyl carbinolamide (ox hydroxy ethylamide) (87, 88, 89, 61) - 24

OH

Elyrnoclairine Agroclavine

Ni-l-R

NCH3 R = peptide units

peptide alkaloids Lysergic acid

Fig. 11

A major step is the oxidation of elymoclavine to lysergic acid. It involves 2 stages, the oxidation of the C-17 hydroxyl group and the shift of theA8-9 double bond to thea-10 position. This last one is not the first step as lysergene, lysergol, isolysergol and penniclavine do not seem to be precurs ors (85, 86). The alternative would involve.6-methyl-8-ergolen-8-carboxylic acid.

OH

6 methyl-8 -ergolen-8-carboxylic acid 25

This acid which does accumulate in certain strains(23) can be incor- porated in lysergic amide derivatives but not as efficiently as lysergic acid(89), and no known natural derivatives of this compound exist. An aldehyde intermediate has been proposed by Floss (86) with the double bond isomerisation taking place at that stage. The synthetic compound and acetate of lysergaldehyde with a tritiated indole moiete was found to be incorporated in ergotoxine but only as (9o)( efficiently as elymoclavine fig. 12).

OH OAc. 1\1H R 0 NCH3 H NCH3

5yr)Lne.,12 Claviceps purpurea.

elymoclavine ergotoxine

R = peptide units

Fig. 12

The evidence discussed previously make lysergic acid a likely precursor of the ergoline moiete of the amide and peptide alkaloids. 14 3 This is supported by direct feeding of labelled ( C or H ) lysergic acid which was incorporated in ergometrine and lysergyl (89, 91) methyl carbinolamide . It was found also to be incorporated in ergotamine and ergokryptine with a good efficiency (15.8%) (92)

The conversion of simple amide derivatives into the more complex peptide alkaloids as well as their inter-relationship have been studied, but the results are still sparse. They are summarised in fig. 13. 2 compounds which have been put forward as likely precursors of the peptide alkaloids: lysergyl alanine and ergometrine (23, 93) (92, 94) .seem, in fact, not to be incorporated into these alkaloids 26

H3 ly sergamide

0 cHs OH tiH-CH-0O2,H

lysergyl methyl cerbirsola.micle

H-CH -CH,OH lysergylalanine lysergic acid 0 N CH3

94 e rgometrine.

92. '93 91- CHs :v 10 NH

0 NCH 0 / riir 11 61- IL Ph

ergota.mine

Fig. 13: Inter-relationship of the simple amide pep;„ide alkaToids. - 27 -

Lysergamide, the simplest derivative, seems to be a degradation product of lysergyl methyl carbinolamide. Its accumulation in some strains of Claviceps paspali might be due to the lack of an enzyme which hydrolyses amides of lysergic acid but not lysergamide or the cyclic peptide alkaloids (95). This enzyme was isolated from Claviceps purpurea and in this strain some peptide alkaloids could be recycled through that pathway (25)

The origin of the side chain has been examined by feeding likely amino-acid precursors. In some cases it gave the expected results: Phenyl alanine was shown to be incorporated in the side chain of (96) ergotamine and proline in the side chain of ergotoxine (97) (ergocornine and ergokryptine) but the origin of the (X hydroxyamino acid is less clear. Both the amino acid and its hydroxy- derivative have been tested as precursors and one or the other seem to be favoured in different cases, valine was precurssor of ergocornine and ergokryptine(7)while alanol was precursor of than alanine in ergokryptine (92), for ergometrine alanine seems the best precursor (92, 94) -28-

'ART T.R - 29 -

1. PHYSICAL MEASUREMENTS Melting point were measured on a Koffler block and were corrected. Ultra violet/absorption (U.V) was measured with an Unicam SP 200 U.V. spectrophotometer and infra-red (I.R.) absorption with a Unicam SP 200 G IR spectrophotometer - (K Br discs).

Mass spectra were obtained with a M.S.902 mass spectrometer.

2. STRAINS AND CULTURE

(i)STRAINS

Clavice s fusiformis strain 1a/2LIG Claviceps fusiformis strain 139/2

Claviceps fusiformis strain 139/2/ IG produces enzymes (3 glucanase and pglucosidase which autolyse theB 1-3 glucan produced by that fungus.

Claviceps fusiformis strain 139/2 is the original strain without this enzyme system (98, 99)

Unless specified, Claviceps fusiformis strain 139/2/ IG will be used in experiments.

Sphacelia sorghi The strains of Sphacelia sorghi used were selected for their alkaloid producing properties from an original strain obtained from sclerotia of Sphacelia sorghi (McRae) parasitic of Sorghum vulg. (Pers.) (100). in Nigeria 4 main strains were used.

(ii) CULTURE Claviceps fusiformis culture

Thel strain was preserved in pyrex ampoules under liquid nitrogen at - 196°. These were obtained from 5-6 days old submerged culture with 10% glycerol added(1 ml inoculum /ampoule).

Slope cultures were grown at 24° and subcultured every 2 weeks.

Batch culture Flask fermentation :

First stage seed cultures.

A spore suspension (containing 2-6 x 107 spores /ml) was prepared from 14 day old slope cultures. The usual procedure was by homogenising 2 slopes mycelia in distilled water (10 ml) with a Vortex mixer and filtering through cheese cloth all under sterile conditions. The suspension was divided in 2 (2-3 ml) portions and used to inoculate 2 flasks. This could be done on a bigger scale using same ratio of water: slopes.

The submerged cultures were incubated on a rotary o shaker at 200 rpm and 9 cm eccentric throw at 27 for 5 days.

Second stage culture.

10 ml of the seed culture was transferred to new flasks of medium. These were grown under the same conditions.

Pilot plant

1st stage laboratory cultures in 500 ml Erlenmeyer flasks (as above)

15 days 2nd stage laboratory culture 100 ml of 1st stage culture 1 litre medium / 3 litre flasks 14 days

5 litre New Brunswick fermenters 300 ml 2nd stage cultures 3.1 litre medium

14 days

60 litre fermenter

2 x 5 litre fermenter to 54 litre medium

14 days

400 litre fermenter

1+0 litre culture from 60 1 fermenter to 360 litre medium - 31 -

These fermentations were performed at 27° with aeration and agitation. An anti-foam (P2000, Dow Chemical Company, London W.1) was added to the medium 0.01% (V/V) before sterelization.

Sphacelia sorghi culture

Slope cultures are grown at 24° and subcultured every 2 weeks.

Surface culture: The mycelium from 1 slope culture was used to inoculate 1 flask. A minimum of - slope culture per flask could be used. The mycelium was divided finely manually and the flasks were left standing at 27°.

(iii) Medium

The same medium was used for both strains. For liquid culture 100 ml of medium were used per 500 ml flask. The solid medium for culture slopes contained 2% agar. The medium was then sterilised.

Medium : (modified from Stoll et.al. 101)

Sucrose 100 g L apparagine 10 g Ca (NO 4H 0 3)2 2 1 g Mg SO4 7a20 .25 g K H PO .25 g 2 4 K Cl .125 g FeS0 4 7H20 1% 3.3 ml ZnSO, 1% q 7H20 2.7 ml cysteine hydrochloride 0.01 g yeast extract 0.1 g

made to PH = 5.2 with 10% sodium hydroxide and made to 1 litre with distilled water.

Agar: 15 g of Oxoid Agar No. 3 for every litre of medium. --32

3. EXTRACTION METHODS All homogenisations were performed with a Vortex'mixer. (centrifugations were done with a PULSE HS 18 centrifuge (8000 rpm, 20 minutes, 50). If the extract volume was above 1200 ml a PR II centrifuge was used instead (5000 rpm, 1 hour 10°) The following main methods of extraction were used: Aqueous extract of broth and mycelium Culture mycelium and medium were homogenised after being made basic with ammonium hydroxide with an equal amount of methanol / distilled water 50:50 (v/v) 3 times. The debris was centrifuged each time. The extracts were collected and the methanol as well as some of the ammonia were evaporated on a rotary evaporator.

Basic alkaloids If the mycelium was not extracted, it was first centrifuged off. Otherwise alkaline (by addition of ammonium hydroxide) mycelium and medium were homogenised with an equal volume of chloroform / methanol 80:20 (v/v) (3 times). The 2 phases were centrifuged between each extraction. The organic phase was filtered through phase filter paper(Whatman SPI) and evaporated down. Basic alkaloids were also extracted as above using chloroform / isopropanol 3:1 v/v) as an alter- native solvent.

Amphoteric alkaloids The aqueous extract from the chloroform extraction was made acid to PH2 by adding concentrated hydrochloric acid. It was extracted 3 times with an equal amount of butanol. Extracts were collected and evaporated down.

Extraction of pilot plant fermentation The Broth (400 1 ) was extracted at PH 10 with isobutyl methyl ketone (i-volume)giving the basic alkaloid fraction. The aqueous phase was then made to PH 4.5 and extracted with butanol. Both extracts were concentrated down. - 33 -

4. CHROMATOGRAPHIC METHODS

(0 Thin layer chromatography (TLC)

TLC was performed using glass plates (5 x 10 cm) coated with silica gel GF 254 or PF 254 (Merck) (0.5 mm thickness). For preparative TLC 20 x 20 or 40 x 20 cm plates were used coated with PF 254 or sometimes HF 254 (1 mm thick). The plates were activated at 110° for 1 hour before use. Pre-coated plates (Polygram Sil q/uv 254 Mackerey-Nagel and Co Wren, Germany were used most of the time for all chromatography apart from preparative TLC.

Solvent systems used

Chloroform/methanol/ammonia (0.88) 95:5:4 (v/v) Chloroform/methanol/ammonia (0.88) 90:10:4 Chloroform/methanol/ammonia (0.88) 80:20:6 Ammoniacal chloroform/isopropahol 80:20 Ammoniacal"chloroform/t. butanol 3:1 Acetone/ethylacetate/N dimethyl formamide 5:5'1 Ethyl acetate/isopropanol/ammonia (0.88) 45:35:15 Chloroform/methanol/glacial acetic acid 70:20:10

The plates were developed in a tank lined with Whatman paper No. 1. The eluent solvent was made up fresh before use and kept one day maximum. It was shaken thoroughly to ensure the mixing of the different phases.

In preparative TLC alkaloids were eluted from the silica with ammoniacal methanol. - 34 -

(ii) Column Chromato llala The material used for column chromatography included silica gel MFC (100-200 mesh), aluminium oxide (100-200 mesh), Dowex 50W-X8 (reform). 4mberlite XAD-2, Sephadex SPC-25 and Sephadex LH2O.

Details of the methods not described below can be found in the experimental section. Silica gel MFC and aluminium oxide were packed in the starting eluent solvent.

Dowex 50W-X8

Two small columns were prepared following the method described (102). in ref. One was packed with the Dowex 50W-X8 in hydrogen + (H ) form. The resin was rinsed with distilled water. The other column was packed with the resin and rinsed with 2N ammonium chloride (10 column volumes) and washed with distilled water (10 to 20 column volumes) until the effluent was PH 8-9. The resin was then in ammonium (NH4} form). The capacity of the resin was for a column (7 x 1.5 cm) size 1000 jig amino nitrogen or 20 mg alkaloid. The alkaloid mixture was dissolved in ethanol., 2N ammonium hydroxide and made to PH 7.1 with conc. hydrochloric acid. The mixture was loaded on the[NH4lcolumn which was connected to the[e]column. The resins were washed with (4x 20 ml) volume of distilled water. Both columns were eluted separately. TheNE4+1column was eluted successively with : 2N ammonium hydrox4de (160 ml), distilled water (80 ml), 0.5N hydrochloric acid (500 ml), and 6N hydrochloric acid (100 ml). The[e]column was eluted with 2N ammonium hydroxide until just basic (15 ml) followed by distilled water (80 ml) it was alternatively eluted with 2N ammonium hydroxide (5 ml) followed by 4N ammonium hydroxide (10 ml). The fractions eluted from the I\THIllcolumn were made basic with ammonium hydroxide and extracted 3 times with chloroform and checked for alkaloid content by T.L.C. Amberlite XAD-2 neutral The resin was prepared by soaking in ethanol (Methanol and acetone have also been used) and washing with distilled water (1 litre for 5 g of resin). - 35 -

The capacity of the resin was 1.5 to 2.5 mg alkaloid/g of resin Two absorption procedures were compared:

Batch procedure. After the resin had been filtered, the sample was added,dissolved in distilled water, and let to absorb overnight. The resin was filtered or centrifuged and put on a column. It was then washed with distilled water (1 litre for 20 g of resin).

Column procedure. The sample was loaded on the column with the prepared resin and put through several times. The resin was washed as before.

Two main different solvent systems were used for elution distilled water /ethanol 50:50 (v/v) with some ammonium hydroxide until basic. (Total volume eluent 600 ml) and dimethyl formamide / ammonia 50:4 (70 ml). The effluent was checked for alkaloid content by reaction with Van Urk reagent and by evaporation followed by TLC.

Sephadex SPC 25 Theammonium (NH.4 ) form of the resin was obtained by the following procedure. The resin (25 g) was allowed to swell in distilled water overnight, packed in the column, rinsed with 2N ammonium hydroxide until basic then with distilled water until the effluent was at PH 8-9. The resin was regenerated by washing with 2N ammonium hydroxide (2 litres) followed by distilled water until the effluent PH was 8-9. The hydrogen (He ) form of the resin was obtained by washing the packed swollen resin (5g dry weight) with 0.01N hydrochloric acid (PH 2) until the effluent was acid and then with distilled water until ' neutral.

The[111 form was regenerated by rinsing with 2N ammonium hydroxide (1 litre) followed by 0.01N hydrochloric acid and distilled water as before. The apparatus consisted of 2 columns, Sephadex K15 and K26.[NT141 form resin column size (2.6 x 40 cm) NH4 column ,[B 1j form resin column size (1.5 x 30 cm) "H." column. The effluent from the columns could be passed through to a L.K.B. Uvicord (type 4701A) (254 nm) which was connected to a chart recorder (Bryans). (speed 5 min/cm). The effluent was collected either in flasks or by the means of a fraction collector (L.K.B. 7000A).

The material to be chromatographed was pumped on to the columns, + the first column (NH4 column) being connected to the second (H column) with a peristaltic pump(L.K.B. Perpex pump). Both columns were washed the same way with distilled water (1-2 litres) then disconnected and eluted separately with 2N ammonium hydroxide.

The alkaloid material (20 to 30 mg) was applied to the (NH4 ) column in aqueous phase at neutral PH. Cultures broth were centrifuged first, then adjusted to PH7 with conc. hydrochloric acid and ammonium hydroxide. Extracts were made totally free of solvent by evaporation under vacuum, dissolved into dilute hydrochloric acid and made to PH7 by adding ammonium hydroxide.

Effluents were collected from the moment the eluent solvent reached the top of the (NH4+) column. 30 to 50 fractions (17 ml) were usually collected from the (NH41") column (total volume 510- 850 ml), and 10 to 20 from the (le) column (total volume: 170-340 ml). Elution was monitored by the Uvicord and proceeded at a flow rate of 2.8 to 4.3 ml/Min.

Individual fractions were checked for their alkaloid content after evaporation either by reaction with van Urk reagent or by T.L.C. - 37 -

(iii) Gas liquid chromatography (G.L.C.) G.L.C. was performed on a Pye 104 with a FID detector, using glass columns (5 feet long). The stationary phase was chromowsorbW (80-100 mesh). The different liquid phase used were 10% SE 30, 1% SE 30, 3% SE 52, 10% OV 17 conditioned with helium. The carrier gas was nitrogen (flow rate 50 ml/min). Temperature conditions are given in table 4.

DERIVATIVES

NTFA COLUMN or NTFA methyl TMS I TMS II ester ■•••••■••••

10% SE 30 200 - 202°

o 10% SE 30 2100 210 5 min 215° 6°min 10 min 200° 2°/min to 261

1% SE 30 179 - 180°

3% SE 52 4 min 198° 8°/min to 227°

10% OV 17 200°, 220°

TABLE 4 ChromatOgraphic conditions of G,L.C. analysis of N trifluoroacetyl -(TFA) and trimethylsilyl (TMS) derivatives of tryptophan and ergot alkaloids. - 38 -

5. IDENTIFICATION AND QUANTITATIVE ESTIMATION OF ALKALOIDS Reagents (103) Van Urk's reagent: 0.125 g.p. dimethylaminobenzaldehyde in 100 ml 65% sulphuric acid and 0.1 ml 5% (w/v)ferric chloride. Erlich's spray reagent: p. dimethylaminobenzaldehyde in ethanol concentrated sulphuric acid.

Ninhydrin spray reagent: Ninhydrin in acetone.

After thin layer chromatography, alkaloids were visualized under U.V.irradiation (254 nm) and by spraying with Erlich's reagent. hi-nhydrin spray was used in some cases for localizing amino acids or amphoteric alkaloids. After preparative T.L.C. alkaloidal bands were localized by spraying the edge of the plate.

Quantitative estimation of the alkaloid yield in aqueous broth was performed by reaction after suitable dilution with Van Urk' reagent in the ratio 2:4 (aqueous/reagent v/v) for 1 hour and colorimetric measurement at 565 nm (by reference with agroclavine). After extraction, alkaloid concentrations were estimated by the U.V. absorption of the material in methanol after suitable dilution with reference to agroclavine absorbance standard curve.

6. FEEDING OF PRECURSORS Precursors were fed usually-on the 4-5th day of the second stage culture for Claviceps fusiformis and on the 14th, 15th day of growth for Sphacelia sorghi.

Labelled precursors such as tryptophan, mevalonate and methionine were fed directly in aqueous-solution. Those obtained biosynthetically were dissolved into a small amount of distilled water / ethanol. Larger amounts of crystallined compounds (10-30 mg) were added as such. For Sphacelia sorghi precursors and compounds were introduced underneath the mycelium mat. - 39 -

RADIOACTIVITY MEASUREMENTS Samples (0.1 ml)or (1 mg) were counted by a Packard tri-carb liquid scintillator after adding 3 ml ethoxyethanol and 10 ml liquid scintillator. Liquid Scintillator. 50 g naphthaline and 6 g Butyl, PBD scintillator (obtained from CIBA) made up to 1 litre with toluene. Autoradiograms were obtained by placing T.L.C. plates in contact with X-ray paper (Kodak X-Ray film.) (Kodirex) for 1 to 2 weeks. Pre-coated T.L.C. plates (Polygram sheets) were used for the direct determination of the radioactivity and concentration of compounds only present in small amounts. After chromatography of a known amount of extract, the bands corresponding:to the different compounds were cut out and the solvents added,scintillation liquid or methanol. After the silica gel had settled radioactivity or concentration were measured as usual.

HYDROLYSIS OF PEPTIDE ALKALOIDS. The peptide alkaloid (.1 mg) was refluxed with 6 N hydrochloric acid (.5 ml) for 24 hours at 120°. The amino acid content was analysed on a micro amino acid analyser.

8. COMPOUNDS AND DERIVATIVES 14 (2- C) mevalonate, (2- C)tryptophanand 14C methionine were obtained from the 'Radiochemical Centre'(Amersham).Elymoclavine was brought from Kock-Light Co. Ltd. Dihydrolysergic acid was given by .Sandoz (Basel) . All other compounds unless stated otherwise were obtained either synthetically or biosynthetically.

Agroclavine Agroclavine was obtained from pilot plant submerged fementation of Claviceps fusiformis. The crude agroclavine was purified on an alumina column by elution with Chloroform -while protecting from the light. It was recrystallised from ethylacetate. Mp 204-205° (lt. 205-2060)2 Festuclavine Agroclavine (20'mg) in acetic acid/ethanol 1:1 (20 ml) was shaken under a constant flow of hydrogen for 4 hours with Pt02 as catalyst (5 mg). Festuclavine was recrystallised from acetone. Pyroclavine was isolated by preparative T.L.C. in ammoniacal Isopro- panol/ch1oroform 20:80. (In that system pyroclavine has an intermediate Rf between agroclavine and festuclavine). (dihydrolysergol - I) Dihydroelymoclavine was obtained by reduction of dihydro- lysergic acid with lithium aluminium hydride as described by Stoll et.a1.(104)

Dihydrolyser2ic acid Dihydroergosine (46.2 mg) (2.1 x 107 dpm/mmole) in methanol (15 ml) was refluxed with 40;gKOH (15 ml) for 2 hours under (105) nitrogen The reaction mixture was then extracted 3 times with butanol after addition of concentrated hydrochloric acid to PH 5. After evaporation the extract was re-dissolved in hot methanol and the precipitated sodium chloride filtered. Pure dihydrolysergic acid was obtained from the filtrate by preparative T.L.C. in chloroform/ methanol/ammonia(0.88) 80:20:6 by identification with reference dihydrolysergic acid (yield: 1.8 mg, 158000dpm , 2.4 x 107 dpm/m mole)

a [2_14-lU.1 agroclavine Obtained biosyntheticaily after feeding [2-14C) tryptophan to Claviceps fusiformis.

1311:festuclavine and I3H. agroclavine Tritiated trifluoro acetic acid (3H ) TFA. Tritiated water (0.2 ml) was added to TFA anhydride cooled with dry ice.

t3Hifestuclavine and agroclavine 31i.ltrifluoro acetic acid (.5 ml) was added to each compound (100 mg) dissolved in chloroform (.5 ml) and left to react for 5 minutes. The reaction mixture was added to Sodium hydrogen carbonate solution (10 ml) and the tritiated alkaloids extracted with chloroform. (3H) Agroclavine was re-crystallised from ethyl acetate, and (3H) festuclavine from acetone. N trifluoroacetyl methylesters of tryptophan and amphoteric alkaloids (NTFA methyl esters) HC1 saturated methanol was obtained by adding acetyl chloride to methanol cooled with dry ice (106). The compound (.5 mg) was reacted with HC1 saturated methanol in a small tube. After stoppering the tube, the reaction mixture was left standing overnight. It was then evaporated under nitrogen and reacted with N trifluoroacetylanhydride after adding or not dichloromethane as a solvent. The reaction mixture was injected directly or after evaporating under nitrogen the derivative was re-dissolved in dichloromethane. An alternative method (107) for methylation was used which consisted of reacting the compound in dichloromethane with N trifluoroacetyl anhydride firstl as described-above. The solvents were evaporated under nitrogen and the methylester was reacted with diazomethane overnight. After evaporation under nitrogen the product was re-dissolved in chloroform. N trifluoroacetate of clavine alkaloids (TFA I and TFA II) Trifluoroacetatyl anhydride was reacted with the compound as above and injected as such or after redissolving in dichloromethane.

Tri-methyl S yl,y7_ ethers (TMSlo han.2...... am clavine alkaloids

2 methods were used. Reactions were carried out in Pierce Reactivials(0.5 ml) with a Teflon lined screw cap. First method (108) TMS I The compound (.5 mg) in dry pyridine was reacted with N, 0 bistrimethylsilyl acetamide (1 ml). The mixture was heated at 70-80° overnight and injected directly. Second method (109) TMS II The compound (1 mg) was reacted with the following mixture of reagents: (.2 ml) bis trimethylsilyltrifluoroacetamide/ N-trimethylsilyldiethylamine /trimethylchlorosilane/ pyridine 99:30:1:100. The reaction mixture was heated for 10 minutes at 100°, left standing overnight and injected as such - 42 -

9. EXPERIMENTAL

(i) Investigation of methods

Efficiency of alkaloid concentration and radioactivity determination ilfilly.E1:92aILLT:LEL122122.

The efficiency of radioactive measurements was assessed in the following way. A sample of Claviceps fusiformis broth from the time sequence feeding experiment was submitted to the same extraction procedure as in that experiment (see below). After chromatography of the basic alkaloids, the corresponding bands were cut out and the radioactivity was measured before and after the addition of a standard 14 ( C) hexodecane (2.26 x 103 dpm/mg). The efficiency of concentration measurements was determined by chromatographying known amounts of agroclavine under the same conditions as in the time sequence feeding experiment. The concentration of the chromatographed agroclavine was determined and the recovery yield estimated.

Sephadex SPC-25 ion exhange chromatography Correlation of alkaloid elution and. U.V. recordi

Sphacelia sorghi broth elution was followed by both U.V. recording and colorimetric measurements of alkaloid concentration in each eluted fraction. In another experiment the elution of tritriated alkaloids from a biosynthetic feeding of Sphacelia sorghi (3H) agroclavine and (3H) festuclavine was followed by measuring the radioactivity of each fraction and correlating to the U.V. chart recording.

Efficiency of recovery from the (NH/++) column

To a cell-free extract of Claviceps fusiformis (150 ml) (1500 pg/mi alkaloids) (3H) agroclavine was added (1.8 mg, 68000 dpm/mg). After stirring for 2 hours the extract was divided in 3 parts (50 ml). One part was extracted with chloroform/methanol 80:20 (v/v) and concentration and radioactivity determined. A second 50 ml fraction was stirred with Sephadex resin in (NH4) form (15g dry weight) after adding distilled water (50 ml) for 45 minutes. It was rinsed twice with distilled water, the resin being separated by centrifugation. Both washings were further absorbed on more (NH4) Sephadex (8 g dry weight). The resin was extracted by stirring with 2N ammonium hydroxide 4 times (80 ml fractions). The first time for 60 minutes and thereafter for 5 minutes. Each fraction was evaporated down and measured. The last 50 ml fraction was extracted with the[NHIlcolumn and concentration and radioactivity of the effluent measured.

Gas-liouid chromatography

Sublimation of N trifluoroaC2ILLagroclavine

The product of the N trifluoroacetylation of agroclavine was heated for 5 hours at 270 - 280° under high vacuum.

Relation between N trifluoroacetylation of Claviceps fusiformis extracts and alkaloid yield.

After measuring the alkaloid yield, Claviceps fusiformis cultures were extracted with chloroform on the 2, 5, 7 and 9th day of growth. A known volume of extract was reacted with N trifluoroacetic anhydride and the product made up to a suitable dilution with dichioromethane before injection. The column used was 10% SE 30 (210°). The chromatographic conditions were the same for all samples.

(ii) Claviceps fusiformis Clavicizitic acid and 4-dimethylallyltrutophan

The butanol extract of a large scale fermentation of Claviceps fusiformis to which ethionine had been added (50 g/400 litres) was concentrated. -

Samples of the crude butanol extract (1-2 g) were applied on columns of silica gel M.F.C. (100 g). Elution with ethyl acetate/ isopropanol/ammonia (0.88) 45:35:15 (v/v) (10 ml fractions) was followed by purification of the crude amphoteric fractions by preparative T.L.C. in the same solvent or by further chromatography on silica gel with ammoniacal methanol as eluent solvent. An alternative eluent solvent chloroform/methanol/benzene 2:1:1 (v/v) was used for the chromatography of another sample (10 g) of crude butanol extract on silica gel M.F.C. (300 g). 200 ml fractions were collected and the amphoteric fraction (5 to 13) was further chromatographed on a column with ethyl acetate/isopropanol/ammonia (0.88) 45:35:15 (v/v) as before. 2 pure amphoteric fractions were obtained which crystallised from methanol.

N-methyldimethylallyltryptophan

The butanol extract of large scale fermentation of Claviceps fusiformis under anaerobic conditions was, after concentration, chromatographed on a column of M.F.C. silica gel (14 Kg) packed in the starting eluent solvent chloroform/methanol/ammonia (.88) 80:20:1 (v/v).

The methanol content of the eluent solvent was gradually increased until a ratio 100:1 methanol/ammonia (0.88) was reached. 1 litre fractions were collected. N methyldimethylallyltryptophan crystallised from the fractions 23-27. It was re-crystallised from methanol. - 45 -

Tirneseat 14 enent. 5 submerged cultures of Claviceps fusiformis were fed on the 14 6 9th day of growth with (2- C) tryptophan (5.5 x 10 dpm each). The flasks were collected at 1, 6, 24, 48, 96 hours after feeding the radioactive label. The mycelium was centrifuged off and extracted with methanol/distilled water/ 2N ammonium hydroxide 30:40: 30 (40 ml). Supernatant and mycelium extract were collected together and the volume recorded. A fraction (30 ml for the first 2 flasks 15 ml onwards) was applied on the Sepahadex columns as described.

The basic and amphoteric alkaloid fractions were collected and a concentrated known proportion chromatographed on pre-coated thin layer silicagel plates using the solvent systems: 1) chloroform- methanol-ammonia (0.88) 95:5:4; 2) chloroform-methanol- ammonia (0.88) 80:20:6 for basic alkaloids and for amphoteric alkaloids respectively. Alkaloid bands were cut out and concentration and radioactivity determined, 1 plate was reserved for spraying with Erlich's reagent. 14 ( C) methionine feeding. (14C) methionine (5 ye) was fed to a 5 day old (secondary stage Claviceps fusiformis culture. The culture was extracted on the 5th day with ammoniacal methanol/distilled water and submitted to Sephadex ion-exchangecolumns. Separation of a small sample of amphoteric and basic alkaloids was achieved by thin layer chromatography on Polygram sheets in the usual way. 14 Anaerobic feeding of (2- C) tryptophan, (214C) mevolonate and (11 +eimethionine . Claviceps fusiformis cultures (2 per precursor) were fed on the 5th day of growth with the following precursors: 6 (14C) methionine (11 x 10 dpm), (2-14C) mevalonate, (5.5 x 106 dpm). (21 tryptophan (5.5 x 106 dpm). The flasks were immediately flushed with nitrogen after adding the precursors and, grown for 5 days without shaking under anaerobic conditions. The cultures were extracted with chloroform-isopropanol (3:1) and butanol. Autoradiograms of the different extracts were obtained and radioactivity of some radioactive bands determined. A known amount r 14 of the butanol extract from theL Coethionine feeding was developed on preparative T.L.C. in chloroform methanol/ammonia (0.88) 80:20:6 v/v and the band corresponding to N-methyl dimethylallyl- tryptophan eluted.

14 (3'- 2 - C) M.V.A. feeding

To each of 8 flasks containing Claviceps fusiformis cultures (100 ml) was added the following precursors: (3' - 2H ) mevalonic 3 acid (25 mg) in ethanol/distilled water (50:50) (2 ml) and 14 6 (2 - 0) mevalonic acid (2.2 x 10 dpm). Deuterated mevalonate was obtained from Professor Ntizon, the synthesis of which is described in ref.(1101 The deuterated mevalonate was 99% D with no measurable D or D1. 3 2 8 control flasks were grown under similar conditions. On the 5th day of growth both sets of flasks were extracted with a) chloroform to remove most of the agroclavine b) chloroform/ isopropanol (3:1). Agroclavine which precipitated out from the chloroform extract, was recrystallised from acetone. The remaining basic alkaloid extract was purified by P.L.C. in the solvent systems: chloroform/methanol/ammonia (0.88) 95:5: , ammoniacal chloroform/ t-butanol 3:1, and ethyl acetate/acetone/dimethylformamide 5:5:1. The T.L.C. pure alkaloids isolated were submitted for mass spectra. All mass spectra were recorded under identical conditions. (iii) Sphacelia Sorghi Isolation of com ounds from Sphacelia sorghi.

1st Isolation 3 cultures of Sphacelia sorghi (2nd strain) were collected after 30 days growth. The alkaloids (150 mg approximatively) were separated into main fractions by SPC-25 Sephadex intl■THilform (60 g dry weight). Further purification was achieved by preparative T.L.C. with the solvent system chloroform-methanol-ammonia (0.88) 95:5:4. - 47 -

2nd Isolation

140 cultures of Sphaceliasorghi were extracted with ammoniacal chloroform-methanol (90:10) (mycelium) and ammoniacal chloroform-isopropanol (3:1) (medium).

The extracts were combined (5 mg alkaloid approximatively) and absorbed on a column of Sephadex resin LH 20 (500 g) swollen in chloroform. Fractions (80 ml) were collected auto- matically. The column was first eluted with chloroform- methanol 95:5 (45 fractions collected), then the ratio of methanol was augmented to 50:50 until fraction 53. The column was then rinsed with methanol (500 ml) followed by methanol plus 5% ammonia (0.88) (400 ml). Alkaloids appeared at fraction 8. Fractions(tubes) similar in alkaloidal content were grouped together into 10 main fractions.

Fraction I 8-9 146 mg VI 15-17 338 mg II 1C .-52 mg VII 18-20 100 mg III 11 188 mg VIII 21 5 mg IV 12-13 935 mg IX 22-23 181 mg V 14 343 mg X 24-42 170 mg XI Washings 218 mg

Further purification of the alkaloid was achieved by preparative T.L.C. in systems chloroform /methanol/ammonia (0.88) 95:5:4 and 90:10:4. For fraction II the system chloroform isopropanol 80:20 ammoniacal was also used.

Feeding experiments Feeding conditions and details of extraction processes are -described in general methods. For most feedings 1 culture flask was used per experiment.Cultures were extracted on the 5th day after the introduction of the label compound. In most experiments alkaloids were obtained by aqueous extraction of medium and mycelium followed by elution on SPC25 Sephadex column in (NH4•) form. Individual alkaloids were separated by preparative T.L.C. in the system chloroform/methanol/ammonia (0.88) 90:10:4. -48 -

To obtain separation of dihydroelymoclavine and chanoclavine-I the system used was ammoniacal chloroform/t-butanol 3:1 and agroclavine and festuclavine were separated byre-chromatography with chloroform/ isopropanol 80:20 made alkaline with 1 ml 0.88 ammonia. Unless stated otherwise, the different alkaloids were estimated quantitati- vely after 1 preparative T.L.C. purification. Only the alkaloid present in the greatest amount were isolated. Extract contents and purity of the isolated fraction were checked by T.L.C. in the same solvent systems. Alternative methods of extraction were used in the different experiments. [3H]festuclavine andr3Hlagroclavine Alkaloids were extracted with chloroform-methanol 80:20 made basic with ay. ammonium hydroxide. 14 (2- C) Mevalonic acid Alkaloids were first extracted with basic chloroform-methanol 80:20 then separated on Sephadex SPC25 in (NH4) form as usual. (2 - 14 C) tryptophanI/lCir5t feed.Lng). 2 flasks were fed with the labelled precursor and extracted on the 4th day with chloroform-methanol 80:20, after being made basic with ammonia (0.88). The extract was separated by preparative T.L.C. in the system chloroform-methanol-ammonia (0.88) 95:5:4. After elution from the silica each fraction was refed to fresh cultures of Sphacelia sorghi which were extracted in the same way.

Dihydrolysergic acid Basic alkaloids were extracted with chloroform isopropanol 3:1 ,after addition of ammonia (0.88) (1 ml). The aqueous phase was then made to PH 5 with concentrated hydrochloric acid and the amphoteric alkaloids extracted with Butanol. The TLC soluent system used for that fraction was chloroform-methanol-ammonia (.88) 80:20:6 - 49 -

PART . III — 50—

I Introduction.

Alkaloid isolation is commonly achieved by extraction with a solvent such as chloroform at basic PH. Further purification frequently involves the use of the chromatographic techniques: silica or alumina column chromatography and thin layer chromatography (TLC). But these methods are not always satisfactory because of lack of selectivity , relatively poor yields and difficulties in separating closely related compounds. Improve- illEmts to these ,methods in particular TLC have been devised and new tech- niques such as resin chromatography or gas liquid chromatography (GLC) have been tentatively applied to the study of alkaloids_Ceg.: ill ). Some of these techniques : TLC, resin chromatography and GLC have been investigated in relation to ergot alkaloids.

II Thin Layer Chromatography (TLC).

Although every experimental work with ergot alkaloids involves thin layer chromatography at some stage , only a few reports have been publi- shed which investigate the TLC of large number of these alkaloids. Most of the study has been done on small groups of peptide alkaloids specially those important as pharmaceutical or illicit drugs(112,113,114,115) But the study by Agurell covers a large number of clavine and peptide (116) alkaloids A simple solvent system was needed which was capable of giving a good separation of the alkaloids present in cultures extracts of Claviceps fusiformis and Sphacelia sorghi, thus including basic and amphoteric alkaloids. Because of the polarity difference between these two families of alkaloids , two groups of solvent systems were devised. Different solvent systems were tested in each case. The basic alkaloids , including peptide alkaloids , occuring in the two fungi studied , were found to be the best separated by the system : chloroform/ methanol / ammonia (.88 ) C.95:5:4 or 9D:10:4 (Table 5 1. This solvent system seems to be generally (117) the 'most suitable for ergot alkaloids Other solvents systems were found to be more suitable for the separation of related alkaloids. Ammoniacal chloroform / isopropanol (80:20 ) gave a good resolution of agroclavine pyroclavine and festuclavine. Ammoniacal chloroform I t butanol C.3:11_ was found to be suitable for chanoclavine-I dihydroelymoclavine and elymoclavine. — 51 —

Solvent systems **

Compounds CMA I CBA

Agroclavine* .72 .67

Agroclavine 100 100

Elymoclavine 44 75

Dihydroelymoclavine 37 68

Chanoclavine I 37 64

Table 5 : Chromatographic data for'identified basic ergot alkaloids.

CMA I : chloroform / methanol / ammonia ( .88 ) 95:5:4 . CBA : ammoniacal chloroform / t butanol 3:1 .

* Rf value for agroclavine ** Reference agroclavine -52 —

A good resolution of the amphoteric alkaloids was more difficult to obtain . Several basic solvent systems were tested. Chloroform /methanol/ ammonia ( .88 ), (80:20:6)was found to be the most suitable . Varying the ratio of the different components did not bring any improvement . Ano- ther suitable system giving similar results to the above was ethyl acetate/ isopropanol / ammonia (.88 ), (45:35:15 ). Although amphoteric alkaloids in both these systems have low and similar Rft s , they are well defined. ( Table 6 ).

* Solvent systems

Compounds CMA II CMAA EIA

Clavicipitic acid 12.1 51.5 19.0 N-Methyl-dimethyl- allyltryptophan 88.5 45.4 16.6 Dimethylallyl- tryptophan 5.8 27.2 13.0 Tryptophan 2.6 17.0 9.4

Table 6 : Rf values for amphoteric alkaloids isolated from Claviceps fusiformis cultures.

distance origin-alkaloid x100 distance origin-solvent front

Solvent systems: CMA II : chloroform / methanol / ammonia (.88 ) 80:20:6 CMAA : chloroform / methanol /glacial acetic acid 70:20:10 EIA : ethyl acetate / isopropanol / ammonia C. 88 ) 45:35:15. -53—

Acid solvent systems such as the ones used for amino acids separation were not found to be as useful .Although Rfi s values are increased and a better separation is obtained between the different amphoteric alka- loids , spots are broader sometimes with much trailing. The most satis- factory system was found to be chloroform / methanol / acetic acid (70:30:10 ) However in those acid solvent systems some basic alkaloids have similar Rf's to that of amphoteric alkaloids making the interpretation of chromatograms of non-pure fractions difficult. In ammoniacal systems , am- photeric and basic fractions are well defined. Reproducibility of results cannot be readely achieved with solvent systems involving aqueous solvents. It depends- also on the thickness of the plates used .However this matter could be improved by standardisation of the methods: ie: using fresh solvent systems , by using reference compounds during chromatography and by calculating Rf's values from a suitable standart such as agroclavine. Precoated chromatogram sheets : Polygram sil G / UV 254 were found to give particulary good reproducible results and their sensitiveness made them very useful for the detection of small quantities of compounds. Because of both these factors , these plates were used in quantitati- ve studies in determining both concentration and radioactivity of ergot alkaloids . It was therefore neccessary to test the accuracy of these measurements . Both concentration (. fig. 14 ) and radioactivity (Table 7) were found to be measured with accuracy and not to be apparen- tly affected by the presence of silica . Recovery of compound on the scale used was found to be particulary good with a q4 90 efficiency . -54--

Amount applied ( lig )

Fig.14: Recovery of agroclavine from Polygram TLC sheets. -55—

Radioactivity ( dpm ) Compound Alkaloid Alkaloid + Standart % Error

Measured Theory

Agroclavine 874 1491 1515 1.6 154 325 314 3.0 A3 137 306 297 3.Q A4 Elymoclavine 116 288 276 3.0 Chanoclavine 245 582 565 2.6 A7 -I- 72 256 232 6.5

Table 7 : Error on radioactivity determination using Polygram sheets.

Theory - measured % error = x 100 Measured -56—

III Resin chromatography.

Neutral and ion-exchange resins have been used for the chromatography of alkaloids . Ion-exchange resins can bind compounds selectively and could be used for the separation of basic and amphoteric alkaloids . These resins Dowex 50 W-X8 and Amberlite IRA-400 have found most applications in the isolation or purification of amphoteric alkaloids vi4ich are particulary difficult to isolate by solvent extraction . Neutral resins of the same type , eg. : Amberlite XAD-2 can also be used for selective non-ionic binding of alkaloids . Finally,a dextran type resin , Sephadex , can be used in its neutral form for partition chromatography giving a separation based mainly on molecular weight ; but also in its ionic form as an alternative to Dowex 50 W-X8. These different resins were investigated as a mean of isolation and purification of ergot alkaloids from cultures or extracts , with the amphoteric fraction particulary in view .

Dowex 50 W-X8 in ammonium (- NH + 4 ) form and acid ( H ) form has been used for the separation of basic and amphoteric amino acids . The el form has been used also for amphoteric ergot alkaloids extraction n (62,66,22) and purificatio . This resin might have been suitable for the separation of basic and amphoteric alkaloids .

The separation method used for amino acids was therefore applied to an extract of Claviceps fusiformis containing both amphoteric and basic alkaloids . It was found than no Van Urk positive material could be + eluted from the[ NH4 1 resin , only fluorescent material . As-no alka- loids could be detected in the washings , the basic alkaloids must have been irreversiblk bound 'on the resin or decomposed .Amphoteric alkaloids were eluted from the [e] column but not satisfactorily as after a first elution quite an important pourcentage of alkaloids could still be eluted fom the resin which had been left standing . A increase in the basicity of the eluent solvent did not improve that matter .

Consequently , this resin could be of no value for separating basic from amphoteric alkaloids and not very suitable for the purification of amphoteric alkaloids.

These difficulties were likely to be due to the strong ionic groups of the Dowex resin and a similar but neutral resin Amberlite XAD-2 , was tested. This resin,an expended cross linked divinyl benzene styrene co-• -57—

copolymer binds compounds such as alkaloids by Van Der Waals forces and might be used to separate alkaloids fom other compounds such as aqueous solutes . The resin was tested as before with crude extracts of Clavi- ceps fusiformis containing both amphoteric and basic alkaloids.Absorption of the alkaloids on the resin was found to be selective , more efficient for the amphoteric fraction and more polar basic alkaloids . Tryptophan and the less polar clavines took longer to become absorbed .

The PH of the alkaloid extract,if aqueous, was found to influence the speed of elution from the resin.A basic PH resulted in the most rapid elution , but no amphoterics were eluted I probably because not being absorbed on the resin at that PH. Either with batch or column procedure, total elution of the absorbed material was difficult to obtain with both eluent solvent systems tried. Dimethyl formamide / ammonia (50:4) (v/v), resulted in a smaller elution volume . Most alkaloids were eluted in the first 15 ml of that eluent and with distilled water / ethanol (50:50) (v/v) within 300. ml. Acetone was tried as an eluent , but it was not efficient either. In these experiments , agroclavine was found to he the alkaloid remaining bound to the resin. The elution with dimethyl forma- mide /ammonia of radioactive agroclavine was followed by measuring the radioactivity of the fractions eluted . Itwas found as before that most of the label was recovered in the first 10 ml fraction , but radioactir ye material was still slowly eluted afterwards. The low-specific activir ty of the radioactive agroclavine did not allow a calculation of the pourcentage recovery.

The eluent solvents were those recommended for the elution of non- polar amino acids from another similar neutral polystyren resin "Poro- "(118). pak Q Amberlite XAD-2 has been used for the isolation of alkaloids from biological fluids. Solvent systems , at basic PH , such as chloro- 119 form /isopropanol 3:1 (v/v) and 1,2 dichloro ethane / ethyl acetate (120) 4:6 (v/y) , have been recommended. These alkaloids belong to the narcotic family (morphine and related compounds ). Pourcentage recovery was found to vary with the alkaloid from 40% to 90% and with the eluent system used. The alkaloids were lost mostly by non-absorption C1191

Therefore , Amberlite XAD-2 resin would not be suitable for quantkta- tive work. However , good results were obtained in the ' cleaning - up' of amphoteric fractions and this resin might be useful for purification and isolation of alkaloids when qualitative results only are needed. -58—

An ionic form of this resin : Amberlite IRA 400 ( OH ), was used in the separation of basic peyote alkaloids from amphoteric ( phenolic ) ones. Selective elution was obtained by varying the PH of the alcoholic (121,122 ) eluent . However, this method would not be suitable for ergot alkaloids because of their sensitivity to acids.

Another type of resin , Sephadex , was investigated. The matrix of this resin , unlike the ones seen previously , is a dextran polymer , thus reducing interaction between matrix and compound . This resin has been used for the chromatography of labilled substances such as proteins. It partitions compounds on a molecular weight basis. Ion-exchange Sephadex has been used mainly in connection with proteins)nucleic acids and car- bohydrates. Tryptophan and tryptamine derivatives have been separated on (123). an anionic form of Sbphadex : QAE A25 For ergot alkaloids an cationic form was chosen : Sephadex SP C-25 . This resin with both combined properties of ionic binding and molecular weight partitioning might be expected to separate alkaloids from aqueous culture broth (which consists mainly of polysaaharides ) and to resolve basic from amphoteric alkaloids.

Preliminary investigations showed that the resin in (Hlform did ab- sorb the alkaloids from a culture broth of Claviceps fusiformis and that

the{ NH41 form was selective for the basic alkaloids , both amphoterics and carbohydrates being washed off. The general procedure described in 'methods' was therefore adopted.

1 The selective absorption of basic alkaloids by the t NH4 column and of amphoteric alkaloids by the [ el column was successfull for extracts of the three strains tested :Claviceps fusiformis Claviceps purpurea and Sphacelia sorghi . Alkaloids could be applied either as culture broth extracts or as free alkaloids with equal success. The washing fractions were found to contain non alkaloidal material (non van Urk positive ) and in the case of culture broth , mostly carbohydrates which eluted for the greater part within 500 ml of eluent . Examination by TLC of the collected effluent fractions froM each column showed that the two families of ergot alkaloids: amphoteric and basic were effectively separated by this method (Plate I and II ) , in contrast to solvent extraction. Chloroform / iso- propanol 3:1 (v/v) which an improved system extracts a fair proportion of amphoteric alkaloids while the butanol extract still contains some basic alkaloids. The [ H1 column effluent was as well much cleaner than 59-

F

DH E

DHL + CH A DHLY ••• T

16 9 A B

Plat.2 I: C-Iromatograms of basic (A) and amphoteric (B) alkaloid fractions from Sphacelia sorghi cultures + eluted from Sephadex SP C-25 in (E-H4 ) and(H) forms revrectively (see fig. 16).

DHE = dihydroergosine F = festuclavine DHL = dihydroelymoclavine CHA chanoclavine DHLY = dihydrolysergic acid tryptophan

* solvent: chloroform/methanol/ammonia (0.88) 95:5:4 (basic alkaloids, 80:20:6 (amphoterics). - 0 -

E - CHA —

CA — N DMKT DMAT —T

A

Plate II; ,Chromatograms* of clavine (A) and amphoteric (13) fractions from aallcas fusiformis cultures eluted from Sephadex SP C-25 in (NH4+) and (11+) forms respectively. A = agroclavine E = elymoclavine CHA = chanoclavine I CA = clavicipitic acid N-DMAT = N-methyl dimethylallyltryptophan DMAT = dime thylallytryptophan T = typtophan * solvent: chloroform/methanol/ammonia (0.88) 95:5:4 (basic alkaloids), 80:20:6 (amphoterics). -61—

the corresponding butanol extract athough it still included some non Van Urk positive material of similar polarity to the amphoteric alkaloids.

+ When individual fractions eluted from the[ NH4 ] column were examined by TLC, some separation of the basic alkaloids on the [NH41 column was observed. A good resolution between peptide and clavine alkaloids was thus obtained as well as a limited separation of the clavine alkaloids. (figs.16 and 17 ),( plate I ). The order of elution was dihydroergosine. dihydroelymoclavine. and chanoclavine-I, followed by festuclavine and agroclavinei(giving only the alkaloids which, have been identified both in Claviceps fusiformis and Sphacelia sorghi ). The order shows as expected that separation depends mainly on molecular weight. This was confirmed by the order of elution of a mixture of pure alkaloids which included peptide and clavine alkaloids ( fig. 15 ). No such separation was ob- served on the [el column although trytophan and dihydrolysergic acid were eluted at slightly different times( fig. 15 ). Complete separation might be achieved by the use of suitable conditions.

UV absorption chart recording proved to be a suitable system to follow alkaloid elution. Both concentration and radioactivity measurements of alkaloids fom Sphacelia sorghi fitted closely the UV absorption curve (fig. 16 and 17 ) . An UV absorbent material was always present at the beginning of Claviceps fusiformis basic alkaloid elution but it was proved to be a non ergot alkaloid material and so could be discarded. The UV recording was not as suitable towards the end of the alkaloid elution from the NH4+] column. Although agroclavine or festuclavine were still eluted none or little UV absorption was recorded. This is correlated to the fact that these last clavine alkaloids tend to be released slowly from the Sephadex resin and the last peak can show some trailing (fig. is ). The peak shape and therefore separation of the different alkaloids was found to depend on experimental conditions such as flow rate and the way the alkaloidal extract was applied as well as on the quantity or puri- ty of the alkaloidal extract. Best results)as expected, were found for small or pure amounts of compounds ,but still adequate results were obtai- ned with a maximum loading of the resin , because of the efficient separa- tion of peptide and clavine alkaloids .

The available capacity of the[ NH41 form of Sephadex was found to be 1 mg of alkaloid / gram of dry resin ( 2.3 + 0.3 meq/g theorical capacity), 30-60 mg/ grecommended load ). The actual total capacity for ergot alkaloids A

dihydrolysergic + trptophan acid

ergotoXine co

0 co ag rod avine

1 1 1 0 2:0 310 1 0 F R ACTIONS

Fig. 15 ; Separation by Sephadex SI) C-25 in I NO CO and (B) forms of a mixture of basic ergot alkaloids ; ergotwane (11.5 mg), agroclavine (12 mg), and amphoterics : dihydrolysergic acid C9 mg), tryptophan C12 mg). — 63 —

peptides A

clavines

__ __ ---,......

••••• a...... 1'o 2- 0 3 F r act—tons

amphoter ics B

5

I b Fpo,chon 5 I

FIG. 16 : Elution pattern of Sphacelia sorghi alkaloids from [NH411 (A) and [el (B) Sephadex SP C-25. Absorbance ( arbitrary units ), --- Alkaloid concentration ( mg / fraction ). — 6L —

[3H] AGROCLAVINE

x ••••■• o E

o. FESTUCLAVINE

200

fC

4:2

ce n 100 bp r bso A

LO 30 Fr a c t i on s Fig.17 : Elution pattern from Sephadex SP C-25[Nqlcolumn for,tritiated alkaloids following the feeding of [3H ]agro_ clavine and 1 31]] festuclavine to Sphacelia sorghi. absorbance, ----radioactivity) . — 65 —

was found to vary between 60 and 40 mg for the [NH4 +] column ( 25 g dry weight of Sephadex ). Best results were obtained with 20 - 30 mg alkaloid. for culture broth. The reason for such a low capacity must be due , first of all , to the high ionic concentration of the aqueous extracts applied to the column and possibly to the chemical nature of the ergot alkaloids.

The extraction of culture broth of Claviceps fusiformis with solvent and with Sephadex ( batch and column ) was compared by measuring concen- trations and radioactivities ( table 8 ). The column extraction showed the lowest efficiency (35 % recovery ) , ( fig. 18 ), the batch process was better (44% recovery ) , but more akward to used ( fig. 19 ). The low efficiency of extraction with Sephadex was not due to overloading as no significant radioactivity could be recovered from the washings. It did not seem either to be due to retention on the column.It was genera- lly found that no alkaloids were further eluted after the column had been left standing. The slower elution of the clavines agroclavine and festu- clavine could not account either for the poor yield. It is possible that some alkaloids are decomposed by ion-exchange Sephadex . The cationic form of this resin has sulphopropyl groups -C3H6S03 , which are strongly acidic. Ergot alkaloids , in particular unsaturated ones such as agroclavine, are very sensitive to acids. In the work involving dihydro ergot alkaloids diming the study of Sphacelia sorghi biosynthesis, it was noticed on several occasions that solvent and Sephadex extraction gave comparable results.

Therefore the use of ion-exchange Sephadex in extraction and separation of ergot alkaloids present some disadvantages. Because of the small capa- city of the resin forthese alkaloids, it can be used only practically for extraction on a small scale .It was found to be suitable for one single culture extraction of Sphacelia sorghi . The poor yield of that method is compensated , in some cases , by the effectiveness of the separation between the amphoteric and basic fractions and between clavine and pep- tide alkaloids which could not be achieved by any other available method. Sephadex is also quite effective as an extraction method for obtaining easely the alkaloidal fraction free of polysaccharides , thus avoiding the otherwise neccessary centrifugations. This is particulary true for the amphoteric fraction which cannot be obtained free of polysaccharides by straight butanol extraction. ** Extraction method Alkaloids extacted % yield

Amount ( mg ) Radioactivity ( dpm ) Concentration Radioactivity

Solvent 56.3 28000 72.5 68

Batch,Sephadex 37.0 18000 50 44

Column Sephadex 28.0 14000 41.4 34.5

. .

Table 8 Extraction methods . Efficiency of 'recovery .

For details see experimental part .

* * Concentration before extraction : 75 mg/fraction - Radioactivity before extraction t 40800 dpmara.ction. -10

-5

\41

x 0)•••*"‘

tO 2.01 so Fr act ions 40 0 153 33. 1193 Volume (Ml) 663

Fig. 18 : Sephadex SP C-25, Column procedure. Elution of Claviceps fusiformis basic alkaloids with added [31-1] agroclavine from [N1141-] Sephadex SP C-25. UV absorption, radioactivity (dpm x 103 -2 concentration (mg / ml x 10 ), x-----% specific activity (dpm / mg x 10). Data. expressed. for the foriowir.c fraci ions collected toetkeri 2.-10 Y1-16, R- 30, 31- 40 — 68 —

01$

I 100 200 sou 400

Volume (ml)

Fig.19 : S',phadex SP C 25 ; Batch procedure. Elution of Claviceps fusiformis basic alkaloids with f• added PH] agroclavine from the NH resin. IL 4

Concentration (mg x 2 tfra.ctioa) ■ Radioactivity (dpm x103 ) Specific activity (dpm/:ng x102)

Data nive&sartd, f or each elutect, fraction. an_ct ex press e the - v& cd- value of' the volu..w-e of th-e co rresponcicvl -69—

III . Gas liquid chromatography ( GLC ).

The use of gas liquid chromatography is becoming important for the analysis of biological compounds.This technique presents several advan- tages. Only very small amounts of compound are needed and it can be used on a qualitative basis for the separation of complexe mixtures of related compounds as well as quantitatively. GLC can also be used in connection with other techniques such as radioactive counting and mass spectrometry. More recently, GLC and mass spectrometry have been linked with computers for an automatic identification of compounds.

GLC techniques have been used only for a few families of alkaloids, in particular those connected with drug abuse where a technique for identifying quickly small amounts of compound is needed. Opium alkaloids ha- (124),. ve been repetitivelyseparated and detected by GLC Peyote alkaloids from extracts of that cactus have been identified by GLC (125,126 ) GLC-mass spectrum combined have been used for both these families (127,128) (129) (130) Other alkaloids belonging to tryptamine and indole families have been identified in plant extract by that method .

The GLC of ergot alkaloids has been reported (131), but it was as successful as for the alkaloid families mentioned above. Ergot alkaloids are quite labile and have a relatively high molecular weight . This means long retention times , therefore high column temperatures. The GLC of lysergic acid diethylamide has been attempted on several occa- (132). sions , but only with mixed results These difficulties can be over- come by the use of suitable derivatives which may have also the advantage of being more volatile. Such an approach was used for the morphin alkaloids (124,134). which derived as their trimethylsilylethers (TMS) The alter- native derivative is a trifluoroacetate (N-TFA ) . Both derivation tech- niques have been developped for the GLC analysis of compounds such as tryptophan, tryptamines and related amines (eg.:108,109,134)

Therefore in an attempt to find a suitable method for the GLC analysis of ergot alkaloids, different derivatives and methods of derivation were investigated for both isolated compounds and culture extracts, together with chromatographic conditions such as column materials and temperatures.

Claviceps fusiformis extracts contain as a major product the clavine alkaloid agroclavine. The remaining alkaloids , present in small amounts, can be divided into two fractions : clavines and amphoterics. The ampho- _ 70 —

teric alkaloids are similar in properties to tryptophan and to a certain extent , so are the clavine alkaloids. Tryptophan is isolated with the amphoteric fraction . Much chromatographic data is available for trypto- phan ,as for the other amino acids, both as its trifluoroacetyl methyl (134,106,108) (108,109) ester and trimethylsilyl ether . Tryptophan yeld indeed thesetwo derivatives on trifluoroacetylation and silylation. A good response was obtained on the GLC analysis of these two derivatives (Table 9 ) .

Column and temperature

10% SE 30 . 10% SE 30 10% OV 17

* Derivative 200° 202° 200° 206° 210° 200°

TFA I Me 4.6 6.2 11.8 8.6 ester

TFA II Me 10.8 ester

TMS I 19.2 38

Table 9 : Retention times ( minutes ) of tryptophan derivatives. TFA I Me ester : N trifluoroacetyl methyl ester TFA II Me ester : N trifluoroacetyl methyl ester TMS I silyl ether ( method I ) TMS II silyl ether ( method II ) TFA I : N trifluoroacetate TFA II : N trifluoroacetate (For details see experimental section). -71—

However, it was noticed that the N TFA methyl ester was not very sta- ble and decomposed on TLC due to the presence of methanol as well as on standing. The peak due to N TFA tryptophan methyl ester on the GLC recor- ding disapeared while another peak of higher retention time appeared when the derivative had been left standing a few days. However , when 5 methyl tryptophan was used , no satisfactory N TFA derivative could be obtained only a multiple peak response was obtained on GLC.By contrast, the say' ether derivative of 5 methyl tryptophan ( method 2 ) gave a single peak of retention 12.2 minutes on GLC recording ( column 10% SE 30 , hold 5 minutes at 2150, temperature gradient : 60/ min. to 2610 ).

More complex results were obtained with purified amphoteric alkaloids, as it can be expected from the increasing complexity of the molecule ; (table 10).All compounds gave several peaks on derivation (fig. 20 ). although consistency between number of peaks ,ratios and retentions times could be found for pure alkaloids. Variations in the derivation methods (different silylation agents ) and in the chromatographic conditions did not improved that matter. Identification of a simple mixture of amphotr- teric might possible after N trifluoroacetylation because of the difference in retention times of the three main compounds, but more complex mixtures such as crude butanol extracts of the amphoteric fractions could not be analysed that way. Indeed, derivation of such extracts followed by GLC resulted in verycomplex chromatograms.

Agroclavine, the main basic alkaloid product of Claviceps fusiformis was thoroughly tested for GLC analysis. Several peaks (2 or 3 ) were obtained on derivation with N trifluoroacetic anhydride while one peak was given when silylated (table 11 ). Agroclavine itself , chromatographed readely under the conditions used and it was found that one peak of the chromatogram obtained after N trifluoroacetylation of agroclavine did correspond to agroclavine. One of the N TFA derivative of agroclavine was obtained in a pure form after sublimation of the mixture resulting from trifluoroacetylation (table 12 ). As in the case of tryptophan , the,N trifluoroacetyl derivatives were found to be unstable, decomposing with time and in the presence of some solvents , back to agroclavine (table 13 ). Some solvents like pyridine had a drastic effect. Agrocla- vine could not be derived with pyridine as a solvent, and the addition of that solvent after derivation decomposed the derivative back to agro- clavine. Although the reaction mixture could be injected directly, the Dimethylallyltryptophan Clavicipitic acid Dimethylallyltryptophan (crude) Column Column

temperature TFA I Me TFA II Me TMS I 'TMS II TFA I Me TMS I

200 16 17.6 28.6

10 % 202° 18 9.7 8.4 SE 30 20 11.4 10.2 32 12 11.0 10 min. 200° 2°/min. 32.4 32.8 34.4 35.6 36.8 36.6 5min. 215° o/min. to 6 16.4 261° 17.4 18.6

3% 4omin. 200° 10.2 SE 52 8 /min. to 11.6 227° 13.0

Table 10 ; Retention times ( minutes ) of amphoteric alkaloids derivatives.

—73—

w U)

0

U) w CC

(0 20 30 O 441. MIN.

Fig. 20 : GLC chromatogram of N trifluoroacetyl dimethylallyltryptophan methyl ester ( 10% SE 30, 202° ).

w cr)

0 a_

w a:

10 20 30

Fig. 21 : GLC onromatogram of the trimethyls51y1 ether derivative of Claviceps fusiformis chiniofcrm C 10% SE 30,10 •min. 200,2/mir.).° ° -74—

• Derivative

Column Temperature A ,groclavine TFA I TFA II TMS I ,TMS II (in CH2C12)

202° 58.3 25,47 63.0

o 10% 210 24.8 20,24.4,28.6 SE 30 24.8 38.2

215°(5 min.) e/min.too 16.0 261

10% OV 17 220° 77.5

Table 11 : retention times (minutes) of agroclavine and agroclavine derivatives.

Peak A Peak B

N TFA agroclavine 23.2 45.2

Sublimed N TFA 24 agroclavine

Table 12 : Retention times (minutes) of N trifluoroacetyl derivatives of agroclavine before and after sublimation (column 10% SE 30, temperature 202° ). -75—

derivative was found to be more stable in methylene chloride , probably because of the presence of trifluoroacetic acid in the reaction mixture.

Derivative Conditions Number of peaks Retention times Peak ratios ** (minutes)

N TFA I immediatly 2 25 47.2 2.9

after standing 2 24.6 47.6 1.5

N TFA II derivation solvent : pyridine 0 CH C1 3 20 24.4 2 2 28.6 injection solvent : CH C1 3 20 24.4 2 2 28.6 reaction mixture : 3 20.6 26.1 29.4 pyridine 2 25 28.2

Table 13 : Stability of N trifluoroacetyl derivatives of agroclavine. *

For chromatographic conditions see table 4 ** peak A Peak ratio - (see fig. 22 peak B

The data indicated as well that one of the derivatives was more stable than the other . The mono derivative of the indole moiete would be expec- ted to be more stable than a di-derivative, wether the secondary deri- vation occurs at the indole nitrogen or the methylated nitrogen of the ergoline nucleus. -76—

Methods for improving the yield of one single derivative were inves- tigated by varying both quantity of the reagent and time of reaction (from 22 hours to 48 hours ), but no change in the relative amount of products as well as yield could 1Jeobserved.Itis.natknown why in one case tri- fluoroacetylation gave one derivative , while it gave two derivatives later on when conditions were kept the same , or apparently so.

Trifluoroacetylation or silylation of other clavine alkaloids as well as simple mixtures of these alkaloids gave unsatisfactory results. However, agroclavine, despites its giving multiple peaks on trifluoro- acetylation, was relatively easely derived, and derivation of whole Clavi- ceps fusiformis extracts, either by that method or silylation , could be expected to give satisfactory results , because of its high concentration in such extracts. Indeed, chromatograms of chloroform extracs fom Clavi- ceps fusiformis showed peaks fig. 21 and 22 ) , which could be related to the derivatives of agroclavine ( table 14 ).

Derivative Chromatographic Agroclavine Chloroform extact conditions

N TFA II 10% SE 30 (202°) 25 , 47 - 32 ,45 (210°) 18.6 , 26,6

TMS I 10% SE 30 (210°) 38.2 41 10% SE 30 (10 Thin. :200°, 20/min.) 31.5 3% SE 52 0 (4 min. :198°, 8°/min. to 227°) 10.8

Table 14 : Retention times C minutes ) of derivatives of agroclavine and Claviceps fusiformis chloroform extracts.

The relation between alkaloid concentration in Claviceps fusiformis extracts and the GLC response was studied for trifluoroacetylation. It —77—

U) 0 aU) rz

20 40 min. GLC chromatogram of tri-fluoroacetylated extract. (column 10 % SE 30, temperature 2100 ).

Co 4-1 0

;-1

A 5- ro

0

ro

1000 2000 Alkaloid concentration pg/m1 ) Relation between Peak A area and alkaloid concentration.. (Peak A / Peak B = 2.28 = cste). i'ig.22: N-trifluoroacetylation of Claviceps fusiformis chloroform extracts. - 78 -

was found that derivative peak area and alkaloid concentration were di- rectly proportional ( fig. 22 ) . Derivative and unreacted agroclavine peaks ratios staid constant . Therefore, alkaloid concentration could be determined from GLC analysis after derivation.

Ergot alkaloids , like other alkaloids , are not easely derived because the nitrogens available for derivation are most of the time secondary or tertiary. The presence of two nitrogens in the ergoline nucleus means also the possibility of multiple derivation.

If methods are quite satisfactory for tryptophan and tryptamines, difficulties are encountered with more complex molecules. Trifluoroace- tylation of different ergot alkaloids always yielded more than a single product: for amphoteric as well as clavine alkaloids, but results were quite reproducible. However, dimethylallyltrytophan has been reported to give one single peak after trifluoroacetylation and analysis by GLC(59) It seems that some experimental conditions such as solvents are very impor- tant in these matters. Similar difficulties of mutiple peaks have also (108). been noted for tryptamines Trifluoroacetylation offers the advan- tage of giving more volatile compounds and consequently allow the use of lower column temperatures (200 to 21Q° ).

Silylation of agroclavine gave quite good results. This method has also (123, 133) been used for morphine alkaloids . However , with the amphoteric alkaloid, clavicipitic acid , multiple derivatives were obtained as on (135,136), trifluoroacetylation. Silylation of basic amino acids and (137) trytamine , has also been found to yield several products by succes- sive substitution at both nitrogen atoms ( indole and side chain for tryptamines ). The structure of the derivative was determined by GLC- mass-spectroscopy analysis. The number of products on derivation has been related to experimental conditions, such as reagent (137), solvent(136) (135). and time of reaction However, for clavicipitic acid, varying si- lylation reagent did not improve the derivation process. Silylation has the disadvantage of .giving products with long retention times, of the order of,30 minutes for ergot alkaloids.This was improved to a certain extent by temperature programming, but high column tempera- tures were still neccessary (200,2600). No satisfactory results were obtained with clavine alkaloids Contai-, ning hydroxyl groups . These compounds were found also to chromatograph (125) very badly • -79-

Best chromatographic results for silyl and trifluoroacetyl derivatives as well as agroclavine were obtained with neutral silicon liquid phase SE 30 in a 10% concentration. This material has been proved to the most usefull for the GLC analysis of alkaloids where it is mostly used in a 3 to 5 % concentration of the stationary phase. This low pourcentage has the advantage of reducing very much retention times . However, when 1% SE 30 was used for ergot alkaloids derivatives, peaks showed too much trailing. 3% SE 52 liquid phase gave also good results by decreasing re- tention times and allowing the use of lower temperatures (200 to 230° ). (109,136)as 3 to 5 % OV 17 has been used for amino acids well as trypta- (109,129) mines but it was found to give very long retention times (70 minutes for the silyl ether of agroclavine , 10% OV 17 ), although peak shapes were satisfactory.

From these results and the litterature data ,it seems that derivation methods for compounds such as ergot alkaloids , previous to GLC analysis , are not yet fully reliable. However, it was found that whole culture ex- tracts of Claviceps fusiformis were derived satisfactorally because the main alkaloid present , agroclavine, derived easely and consequently GLC analysis could be used quantitatively foralkaloid concentration deter- mination. However, much more improvement of these methods must be achie- ved before GLC techniques can be used in separation and identification of the various ergot alkaloids present in culture extracts. - 81 -

PART IV 82

I. INTRODUCTION Most of the major features of clavine ergot alkaloid biosynthesis have been elucidated. Although in appearance simple, this pathway is more intricate than it appeared to be. As mentioned in the review, some points remain obscure, in particular ring C and ring D closures, these are in fact the most important stages of the ergoline nucleus biosynthesis.

Several approaches have been used to study these questions using the different methods available for the study of biosynthetic pathways. The approach for studying the ergot alkaloid biosynthesis in Claviceps fusiformis was determined in part by the strain itself. Clavicas fusiformis was found to produce high yield of alkaloids in submerged cultures, a relatively important fraction of which were amphoteric alkaloids The amphoteric fraction was therefore studied in order to characterise early intermediates in ergot alkaloid biosynthesis. The relationship between amphoteric and clavine alkaloids was investigated.

At the same time, the relationship between the clavines themselves was examined. Some aspects of this inter-relationship was examined further by means of specific labelling of these compounds. This investiga- tion meant a need for the characterisation, of different clavine alkaloids. Claviceps fusiformis is a clavine alkaloid producing strain. Very

high yields of these alkaloids (2000-3000 pg/m1) are obtained within ' 8-9 days of growth where alkaloid production reaches a maximum (99) The major alkaloid product is the clavine alkaloid agroclavine which accounts for about 80% of total alkaloid.

Agroclavine - 83 -

Claviceps related strains produces different clavine alkaloids in varying proprortion. The main ones being agroclavine, elymoclavine and chanoclavine I as well as several minor clavine alkaloids.

OH OH

Elymoclavine Chanoclavine -I

Claviceps fusiformis extracts were found to contain about 7 main alkaloids together with a few minor ones and it was necessary to identify and connect some of these alkaloids with known ones in order to be able to study their inter-relationship and possibly some aspects of their biosynthesis.

II. STUDY OF THE CLAVINE ALKALOID FRACTION In conjunction with the feeding of deuteriated mevalonate to Claviceps fusiformis, the mass spectra of the main clavine alkaloids present in extract of that fungus were obtained and used an attempt to identify these compounds (table 15) 814- CMA* CBA** Agroclavine .72 .67 Al 100 100 A2 69 89 A3 61 84 A4 51 8o A5 44 75 A6 37 66 A7 25 62 A8 14 18.4 A9 12 13.6 Table 15 Chromatographic data for the basic alkaloids of Claviceps fusiformis.

Solvent systems chloroform-methanol-ammonia (95:5:4) CMA, ammoniacol chloroform-t-butanol 3:1 CI3A.

* Rf value for agroclavine ** Reference agroclavine, (RA x 100)

3 other compounds can usually be observed on chromatograms of Claviceps fusiformis clavine alkaloids which give a green colour on spraying with Erlich's reagent instead of blue violet as for most other clavine alkaloids. These alkaloids have a Rf in the region of agroclavine, 2 lower and 1 higher. They are likely to be setoclavine, penniclavine, isosetoclavine or related compounds. These alkaloids were not isolated for identification by mass spectrum in that experiment. Of the isolated compounds Al is present in the greatest amount in Claviceps extracts, followed by A3, A4 and A6. The lowest Rf compounds are found in small quantities so is A2 and A5. This can be appreciated qualitatively by chromatography of extracts in the solvent system chloroform( methanol /ammonia which give the best separation of the different components.

The isolated basic alkaloids, pure on T.L.C. gave the following mass spectra:

Al (Agroclavine) molecular ion le m/e 238 (70% M-1),M-1 237. Fragments (inferior to 10%), 154, 167, 223.

A2 Molecular ion m/e 270 fragments: 154, 184, 185, 209, 223, 224, 237, 256. main fragments (more abundant than molecular ion) 154, 223, 224.

A3 molecular ion m/e 256 fragments: 1441 154, 155, 170, 185, 242

main fragments (more abundant than molecular ion) 155, 185 - 85 -

A4 molecular ion m/e 256 fragments: 155 (75%), 183 (85%), 237 (65%) less important fragments: 168, 196

A5 (elymoclavine) molecular ion M m/e 254 (70% M-1) M-1:253 fragments (less than 10%): 154, 167, 223

A6 (chanoclavine I) molecular ion m/e 256 fragments: 171 .(38%), 185 (52%), 213 (32%), 241 (30%)

A7 molecular ion m/e 256 fragments: 144, 155, 170, 185, 228, 237 main fragments (more abundant than molecular ion) 155, 185

A8 molecular ion m/e 256 fragments: 129 (90%), 137, 155, 171, 185 (50%), 213, 228, 237

A9 molecular ion m/e 256 fragments: 139 (85%), 149 (40%), 185, 223, 238

Comparative studies of the mass spectra suggest 2 groups of compounds. A strong molecular ion M and stronger M-1 are together with few fragments of m/e (154, 167) in low abundance characteristics of ergot alkaloids having a closed D ring and an 138 hydrogen at the C-10 position .

Compound Al and Compound A5 present these features and can be assumed respectively to be agroclavine and elymoclavine. They also have the same chromatographic properties than reference agroclavine and elymoclavine.

R = H agroclavine R = OH elymoclavine - 86 -

The other mass spectra present more fragments of higher relative abundance, sometimes more abundant than the respective molecular ions. These features together with the fragment numbers(154,167, 160)are connected with open D ring, unsaturatedA9-10 or C-10 substituted ergot alkaloids(138). There is. also evidence for the presence of hydroxy groups from the molecular ion values and fragments at (M-H 0 ). 2 Fragments of m/e 170 and 185 have been connected with the presence of -0- or -OH grouph8°).The molecular ion of these compounds is predominantly m/e 256 and is likely that some or all chanoclavine isomers are present. Compound A6 is likely to be chanoclavine-I. Its fragmentation pattern is similar to that of chanoclavine -I isolated from Sphacelia sorghi and which was characterised as well by I.R spectra and melting point values. Both compounds show the same Rf . indifferent T.L.C. systems:-

The mass spectra of 2 other compounds: A4 and A8 are also very similar to that of chanoclavine--I and may be respectively isochano- clavine and chanoclavine -II. Chanoclavine -II has been found to have a lower Rf and isochanoclavine a higher one than chanoclavine -I in ( TLC solvent systems similar to the one used in this study 38)

014

Isochanoclavind -I (+) chanoclavine-II - 87 -

An alkaloid clavine 4 with a Rf and mass spectrum similar to A4 was also isolated from Slpluell.a ..s.orahi (see later). Compounds A3 and A7 give both a characteristic bright blue colour with Erlich's reagent. Their macs spectra present some common features, the molecular ion m/e 256 seems particularly unstable.

A9, another compound with a molecular ion m/e 256 shows a characteristic mass spectrum, but could not be identified. The presence of a fragment at 238 suggests an hydroxyl group possibly at the C-10 position.

A molecular weight of 256 corresponds to both chanoclavines and hydroxy-dihydro derivatives. The chanoclavine isomers have been well characterised, but little data is available on hydroxy-dihydro- ergoline derivatives. Only such compound: fumiclavineB has been (139) found in nature

Fumiclavine-B

Compound A2 has a higher molecular ion (m/e 270) and is likely to have a closed D ring,as the fragment 223, 224 indicate a loss of (1 ) side chain in an ergot alkaloid molecule 40 - 88 -

2 structures can be proposed for that compound.:

OH OH

penniclavine

10 -hydroxyelymoclavine isopenniclavine

But 10 hydroxyelymoclavine mass spectra is different in some aspects of that compound A2 and both compound have also a different RfP°12 is possibly penniclavine, Rf values do agree, but A2 was not fluorescent under U.V. light and did not give a green colour with Erlich's reagent as penniclavine or its isomer do.

The clavine alkaloids found in.Claviceps fusiformis extracts could not be fully characterised apart from agroclavine, elymoclavine and chanoclavine I, Mass spectrum and chromatographic data were not enough for the identification of the investigated compounds but suggest that these compounds are hydroxylated, either derivatives of the saturated clavine or belonging to the chanoclavine family. 89 -

III. STUDY OF THE AMPHOTERIC ALKALOID FRACTION

Amphoteric ergot alkaloids occur in small amounts in culture extracts ofallImaap, The relative amount of these alkaloids has been found to be increased by addition of ethionine(59,66)to cultures or growing the fungus under anaerobic conditions(62). Two of the amphoteric alkaloids have been obtained by these methods in sufficient quantity for characterisation. One has been identified as dimethyl- allyltryptophan(59, 62) .

The presence of this compound has been confirmed further by isolating from a.Claviceps mu. an enzyme system capable of synthetising that metabolite(22).

4-dimethylallyltryptophan

The other amphoteric alkaloid was named Clavicipitic acid but its structure is not certain (66) •

Claviceps fusiformis produces a relatively important amphoteric fraction composed of 3 main alkaloids. One of these compounds, corresponding to clavicipitic acid has been isolated from large scale fermentation of this fungus.

The relative amount of amphoterics was found, as expected, to be increased by addition of ethionine to culture of Claviceps fusiformis. 90 -

Consequently, large scale fermentation of that fungus were performed and inhibited by addition of ethionine or by anaerobic growth in order to determine further the nature of these amphoteric alkaloids.

Following inhibition of alkaloid biosynthesis by ethionine,2 compounds were isolated from the culture extracts by column chromato- graphy and preparative TLC of the crude amphoteric fraction.

AMPH 1 (Clavicipitic acid) (Rf = .12) crystallised from methanol; gives a blue violet colour with Erlich's reagent.

T.V. spectrum: absorption at 225 nm ( E = 24000 ), 275 nm ( E = 4900), 287 nm ( g = 6600), 293 nm ( E = 6600) I.R. spectrum: absorption at: 3410 (broad), 3310, 1660 (s) 1400, 1360, 1140, 690 (broad). Mass spectrum (fig. 23): molecular ion m/e 270 (909) fragments: 44 (100%), 169 (65%), 182 (60%), 269 (67%). (66) This data agrees with the reference • AMPH 2 (4-dimethylallyltryptophan) (Rf = .06) crystallised from methanol; gives a blue green colour with Erlich's reagent. U.V. spectrum: absorption at: 225 nm (. E = 38400), 275 nm (. E = 7900), 280 nm (. E = 7650) and 293 nm ( E = 6200) I.R. spectrum: absorption at : 3340(broad) 2920, 1590 (s), 1500 (s), 1410 (s), 750 cm -I.

The mass spectrum agrees with the reference (59) and AMPH2 chromatographs as authentic dimethylallytryptophan. Both compounds have a non-substitued nitrogen in contrast to all known ergot alkaloids in which this nitrogen is methylated. Ethionine is known to interfere with the methylation step and both . compounds accumulate under these conditions.

* Solvent system for amphoteric alkaloids: chloroform/ methanol/ammonia (0.88) 80:20:6 (v/v). Abundance 100 60 40 2.0 100 80 40 O 0 40

Fig.23: Massspectraofamphoteric alkaloidsfrom 40

44 N Methyl-4-dimethylalltjltryptophan. Compound Amph Compound Clavici piti.c 80 80 91 A 120 120 mp

Claviceps fusiformis. Odd h m/e 156 m 2. 160 154 160 /e

169 az 2.00 2.00 198 215 240 2.40

2.60 270 286 260 - 92 -

Clavicipitic acid is the main component of the amphoteric fraction from Claviceps fusiformis extracts. Apart from dimethyl- allyltryptophan, there is still another amphoteric alkaloid present in about the same amount than dimethylallyltryptophan.

This compound which was not found in. significant amount in culture extracts after additon of ethionine was isolated from large scale culture of Claviceps fusiformis where growth was inhibited by anaerobic conditions. This alkaloid (Rf .09) was obtained in crystalline form and was identified by its mass spectrum (fig. 23 ) (lem/e 286(18%) ) as N methyl dimethylallyltryptophan. This mass spectrum was very similar to that of dimethylallyltryptophan with m/e 198 (100%). It also gave a green colour with Erlich's reagent and wasnLnhydrin positive.

N-methyl 4 dimethylallyltryptophan

No clavicipitic acid was found in significant amounts in the amphoteric fraction indicating that oxygen must be needed for its biosynthesis. Anaerobic conditions stops clavine biosynthesis, the (75) biosynthesis of chanoclavine I has been shown to require free oxygen. Consequently, any non-oxygenated precursor should accumulate, which this case proves to be N-methyldimethylallyltryptophan. Dimethylallyltryptophan has also been found in Claviceps cultures grown anaerobically (62) In order to confirm the nature of the amphoteric alkaloids labelled precursors were fed under anaerobic conditions. Autoradio- grams of the resulting amphoteric and clavine extracts showed that the biosynthesis of the clavine alkaloids was very much reduced or 14 14 stopped while the precursors: (2- 0) tryptophan and ( C) - 93 - methionine were still incorporated into some amphoteric alkaloids.

The 3rd precursor fed, (2 -I C) mevalonic acid, was found not to be incorporated in any ergot alkaloids. Aerobic conditions have been found before to be necessary for providing the energy for con- version to isopentenyl pyrophosphate (62).(14C} methionine was found to be incorporated in mostly one single amphoteric compound which after isolation and chromatography with reference compounds was found to correspond to N-methyl dimethylallyltryptophan. The level of incorporation was high (4 to 8) and comparable to that of ( 14C) methionine into agroclavin.e under aerobic. conditions (see table 16).

Under aerobic conditions amphoteric alkaloids were found to be weakly labelled ( 0.04%).

N methyldimethylallyltryptophan must be utilized for synthesis very quickly. As the other amphoteric alkaloids should not contain any methyl group, the low level of radioactivity exhibited is then normal. 14 (2 - C) tryptophan feeding under _anaerobic conditions resulted in the accumulation of 2 labelled amphoterics which were identified as dimethylallyltryptophan and N methyldimethylallyltryptophan after chromatography with reference compounds ) the latter being more actively labelled. In both feedings the level of clavicipitic acid was found to be much reduced in comparison with normal conditions or the aerobic feeding of methionine. No radioactivity corresponding to that compound could be detected.

These experiments confirm the structure of the new isolated amphoteric alkaloid N-methyldimethylallyltryptophan. The identity of the 3 amphoteric alkaloids found in Claviceps fusiformis extracts can then be put forward as clavicipitic acid, N-methyl 4-dimethyl- allyltryptophan and 4-dimethylallyltryptophan.

These experiments give also a few indications as to the relation- ship of these alkaloids. No other methylated compound was detected under anaerobic condition and it might be expected that N methyl- dimethylallytryptophan is the last intermediate before oxidation. Dimethylallyltryptophan did accumulate as well under these conditions and as this compound also accumulates when N-methylation is inhibited by addition of ethionine, dimethylallyltryptophan must be an immediate precursor of the N-methylated derivative. Precursor fed % incorporation in fractions or compounds amphoteric basic other Amph 1 chanoclavine agroclavine alkaloid amphoteric I

[140) methionine aerobic 2.3 18.7 0.04 0.04 0.17 10 6 (11 x 10 dpm)

[14Cimethionine anaerobic 24.6 17.2 8 (11 x 106 dpm)

L2-14CI\tryptophan (5.5 x 106 dpm) 65 2.5 .4

Table 16: 'Anaerobic feeding of labelled precursors to Claviceps fusiformis - 95 -

Clavicipitic acid biosynthesis is stopped by anaerobic conditions but it accumulated when N-methylation is prevented so it must be derived directly from dimethylallytryptophan, its synthesis being encouraged when the methylation of dimethyalJ.yltryptophan is prevented These facts can be summarised in the following scheme: 0 tryptophan )dimethylallyltryptophan 2 >Clavicipitic acid

methionine

N-methyliii.rn.ahyi all 71 try pto ph an.

02

Clavines

d.imethylallyltryptophan is therefore a very likely percursor of the clavine alkaloids, the next step in biosynthesis involving an oxidation. The next recognised intermediate in ergot alkaloid biosynthesis is chanoclavine I. It is known that molecular oxygen is needed for its formation (75). But it is likely that more intermediates must exist between chanoclavine I and its amphoteric precursors. In theory all these compounds should be N-methylated and would exclude tryptamine derivatives which have been put forward as likely intermediates(49,63) but further evidence in regard of that scheme would be provided by feeding labelled N-methyldimethylallyltryptophan.

IV. (2 -14C) TRYPTOPHAN TIME-SEQUENCE aHIING Some of the relationships between amphoteric and clavine alkaloids has been demonstrated. Synthetic as well as biologically produced dimethylallyltryptophan has been found to be incorporated into clavine alkaloids.(49, 61, 62). Clavicipitic acid, in contrast, did not act as (66) a precursor when tested . - 96 -

The relationship amphoteric, clavine alkaloids was tentatively investigated with a kinetic experiment invloving feeding of a labelled precursor and collecting cultures at different times of growth. In such an experiment the label should be found first of all into the early intermediates of the hypothetical pathway, then successively into the other intermediates depending on their order in the pathway. In theory this method should yield interesting informations concerning the pathway, but difficulties can arise which are due to differences in pool size and rates of synthesis of the various metabolites. Kinetic experiments are particularly suitable when pulse labelling can be used, which means introducing the label for definite periods of time and removing it. The use of labelled carbon dioxide in that respect has been successful in the study of alkaloid biosynthesis in plants, (eg 141). The study ofaulasps-fusiformis biosynthesis with a time sequence experiment was made possible by the efficient separation of amphoteric and clavine alkaloids by Sephadex ion-exchange resin. Of the 2 14 precursors fed, (2 -14C) rievalonic acid and (2 - C)tryptophan the mevalonate was found not to be suitable because of its low rate of 14 incorporation into clavine alkaloids. (2- C) tryptophan is usually a much better precursor and the isolated alkaloids in this feeding experiment were found to be sufficiently labelled for the results to be significant, but as tryptophan is eluted from the Sephadex ion exchange resin in (H) form with the amphoteric fraction, no reliable quan- titative results could be obtained for the different amphoteric alkaloids. The 3 amphoterics and tryptophan have a very similar RF in.the solvent system used and their chance of being contaminated by the very highly radioactive tryptophan is fairly high. However, results for Clavicipitic acid and N-methyldimethylallyltryptophan are tabulated (table 17), these compounds having the highest Rf's. They show a high level of incorporation even after 1 hour which remains relatively constant over the period of time the experiment was performed ,Clavicipitic acid might show a slight tendency to accumula- tion.. Isolated tryptophan level stayed constant throughout the experiment. - 97 -

Total radioactivity(dpm)

Alkaloid 1 6 24 48 96

A9 430 1800 2000 1500 2730

`A8 390 1500 1500 1060 2930

A7 880 3800 3730 2920 4600

Chanoclavine -I 525 2660 5200 7900 11800

Elymoclavine 260 945 1330 2400 7900

A4 350 1720 2730 3870 7800

A3 350 2450 4050 4650 8100

Agroclavine 2420 39500 154000 24000 79000

AMPH I 19000 21300 18400 14000

AMPH II 8500 14600 22500 14000

Tablel7: Time sequence feeding experiment, Total radioactivity of alkaloid fractions after feeding [2-14C]tryptophan (5.5006dpm)-for the periods of time indicated (1 to 96 hours). - 98 -

In relation to the clavine alkaloids, only agroclavine after 6 hours becomes most highly labelled, but no definite conclusion in regard to that relationship can be reached from that experiment. More interesting results are obtained concerning the inter-relationship of the clavine alkaloids for which both total radioactivity and specific activity could be determined, (Table 17, fig. 24 and 25), but this is still subject to some errors due to the small scale on which alkaloids were isolated.

At the time the labelled tryptophan was added to the cultures on the 8th day of growth) alkaloid production had reached or nearly reached its maximum and important variations in amount of the different alkaloids would not be expected. Consequently, changes in specific activities of the compounds should follow closely the radioactivity counts , the latest being less subject to errors. The concentration of compounds present in only small amounts could not be determined very reliably. In fact, only small variations in concentration of the different alkaloids were observed. Some were within the range of error of the method used in particular for compounds such as A8 and A9, which are only present in small amounts.

Both graphs (fig. 25 and 24) (specific activity and total counts) show that a few compounds accumulate and are synthesised steadily, specific activity and total count increasing from the time of the addition of the labelled tryptophan. One of these compounds. A4 was tentatively identified as isochanoclavine I and the other A as a 3 dihydro, hydroxy derivative of agroclavine, which is compatible with their being end-products. Isochanoclavine, in articular, has been (55) shown not to be incorporated in elymoclavine . Agroclavine, in fact, although reaching the highest specific activity very quickly (within 6 hours) seem to attain a maximum level early compared with the other alkaloids (24 hours). The relatively small amount observed at 48 hours is probably due to culture variation rather than to such an actual decrease in synthesis of agroclavine. The level of incorporation of label for that feeding was slightly low for all isolated alkaloids. The levelling of agroclavine biosynthesis coincides in particular with a sharp increase in elymoclavine production and might agree with evidence from other sources for agroclavine being a precursor of elymoclavine, but the compound which had been identified as chanoclavine I earlier on shows also a levelling of label incorporation and specific activity around that time (48 hours), - 99 -

15-

to -

Li II o

0 0

Cr

4 48 96 Time (hours)

Fig. 24: [2-14C)tryptophan_ time sequence feeding. Variation of alkaloid total radioactivities with time. A9, + A8, A AT, n Chanoclavine I, o Elymoclaviner • A4, o A3 (dpm/flask x103), x Agroclavine (dpm/flask x 104). 100 -

24 48 96 Time (hours) Fig.25: {214dtryptophan time sequence feeding. Variation of alkaloid specific activities with time. A A9, A8, a AT, ■ Chanoclavine I, 0 Elymoclavine, • A4, ❑A3,(dpm/mmole x 105), x Agroclavine(dpm/mmole x 2 x105). - 101 -

and its high specific activity makes it a possible precursor. Chanoclavine I has been found to be incorporated in clavine alkaloids, (67,68,69) agroclavine and elymoclavine .In this experiment chanoclavine I was found to have a relatively low specific activity in the early stages (1-6 hours) which might be due to its being always present in relatively large quantities in extracts of Clayiceps fusiformis. This low specific activity might not prevent it from acting as a precursor of agroclavine, as agroclavine accumulates. Both decreases in synthesis of agroclavine and slowing down of that of chanoclavine I might be related. 3 compounds A7, A8, A9 show a relatively high specific activity as well as total count early after the introduction of labelled tryptophan (1, 6 hours). This level remains more or less stable in latter stages. These compounds could be incorporated further. The nature of these alkaloids is not known, one of them, A8, might be chanoclavine II which is supposed to be an end product(38it is possible also that more of these compounds accumulate slowly.or may act as precursor of alkaloids other than agroclavine. This time sequence experiment gave evidence for at least 3 clavine alkaloids being still actively synthesised when agroclavine has reached its maximum level. Two of these compounds, A3, A4, seem to be synthesised independently or at least in a parallel way to agroclavine while elymoclavine, chanoclavine I and agroclavine synthesis seem to be related - both chanoclavine I and agroclavine could be precursors of elymoclavine from the data available. The 3 clavines of low Rf's A7, A8, A9 appear to be early precursors, A7 in particular

2 V. (3L 21., 2 - C) MEVALONIC ACID FEEDING Another aspect of ergot alkaloids biosynthesis in Claviceps fusiformis was investigated by studying the fate of the C-3' hydrogen atoms of the mevalonate precursor. Evidence towards the closure mechanism of ring C and D can be expected from such studies. The other of hydrogen atomsAmevalonic acid (C-4 and C-5) have been studied in much details(48 53,54)• C-3'hydrogens have only been studies in connection with C-4 hydrogens with respect to both chanoclavine I and elymo- clavine (56) - 102 -

For that purpose mevalonic acid fully deuterated at the C-3' position was fed to Claviceps fusiformis. After isolation of the deuterated alkaloids and non-labelled ones from controle cultures, the mass spectra obtained from these compounds were analysed in the way described below. An average value for the abundance of m/e fragments in the molecualr ion region was determined for chanoclavine and agroclavine (fig. 26) and the relative amounts of deuterated species calculated as indicated.

Mt

156

M+I M+2

25? 2.58

Agroclavine chanoclavine -I

Fig. 26 Molecular ion region

- 103 -

Agroclavine

M-1 - M 11+2 237 23 239 Unlabelled Standard 1.000 .588 .097 .025

Abundance (arbitrary units) Labelled 1.760 1.110 .200 .0424 (i) material

The peak at 237 is due to unlabelled species only, therefore, the contribution of unlabelled sample to other peaks is:

1.76 (1.76 x .588) (1.76 x 0.97) (1.76 x 025) = 1.76 = 1.0343 = 0.1708 = .0434 (ii)

substracting (ii) from (i) gives:

0 0.0757 0.0292 .001 (iii) The peak height in le (0.0757) is now due solely to the M-1 peak of the monodeuterated sample, the contributions of the single labelled species to the other peaks are: 0 (0.0757 x 1) (0.0757 x .588) (0.0757 x 0.097) = 0.0757 v-T, .0444 = .0073 (iv) substracting (iv) from (iii) gives: 0 0 - .0152. .0083

There are no D or higher species. 2 io4 -

% of monodeurated species: 0.07,57 (1.76 + 0.0757) x 100 = 4.1%

% of non-deuterated species

= 1.76 x 100 %= 96 (1.76 + 0.0757)

Chanoclavine I

14+1 11+2 256 257 258

Abundance Unlabelled standard 3.82 (arbitrary .724 .08 (1) units) Deuterated 3.59 .671 1.06 xi.o6 material = 3.82 .713 1.13 (ii)

(ii) - (i) 0 - .011 .05

There are no monodeuterated species.

% D species: 2

= .05 x 100 = 1.37% (3.59 + .05)

% of Do species: = 3.82 3.59 .o5 x 100 = 98.6%

Chanoclavine I Agroclavine D 0 2 1.37 D 1 0 4.1 D0 98.6 96

Table 18. Distribution of deuterated species (%) - 105 -

The results of this mass spectrum analysis summarised in Table 18 show that the only deuterated species found in chanoclavine I are di-deuterated while in agroclavine only monodeuterated species are present. Consequently, in the biosynthetic pathway 1 hydrogen must be lost between dimethylallylpyrophosphate and chanoclavine I and still another one between chanoclavine I and agroclavine and the clavine alkaloids (fig. 27). (56) Similar results were obtained by Floss who fed mevalonic acid deuterated in 2 positions:[3)-4 D51 mevalonic acid:.

in order to study the C-8 position of the ergoline nucleus as well, but because of this extra label, the evidence was more difficult to interpret. The loss of 1 hydrogen from the methyl group in the conversion of dimethylallylpyrophosphate to chanoclavine I may occur during the oxida- tion of one methyl group from dimethylallyltryptophan to its hydroxy-- derivative (fig. 27). These results agree with the proposed scheme of ring C closure - probably, by addition of an enzyme on the double bond and not involving the hydroxylated methyl group (fig. 7).

The loss of an hydrogen from the C-17 of chanoclavine I has been 14 3 verified by the use of chanoclavine-I ( CI 17- His This supposes an aldehyde as intermediate (fig. 28), and in fact the aldehyde derivative of chanoclavine I acted as a good precursor of elymoclavine (70) (40% incorporation) -The different evidences agree with the assumption that chanoclavine-I aldehyde is probably a natural inter- mediate.

OH a D NHz o o COOH

OH D N (-Hs

<---- <7---

C hanoc la vine -I

Fig. 27: Incorporation of (3'-2H 2-14C) mevalonate into cliacloclavine -I 3 and agroclavine.

- 107 -

ON

>

chanoclavine-I chanoclavine-I aldehyde

agroclavine

Fig. 28 - 108 -

PART V . - 109 -

I. INTRODUCTION

Sphacelia sorghi (McRae) is a fungus parasitic of Sorghum vulgare. Although, it has not been properly characterised, it has been shown to produce ergot alkaloids and consequently must be related to the Claviceps spp, and other fungi synthetising ergot alkaloids (100)

Sphacelia Aorghi extracts were found to contain peptide as well as clavine alkaloids, but unlike other Claviceps spp. which produce lysergic acid class of ergot alkaloid, the main peptide product was found to be a dihydro peptide alkaloid and has been identified as dihydroergosine, the dihydroderivative of the peptide alkaloid ergosine (142)

dihydroergosine Dihydroergosine had not previously been found in nature. Extracts of Sphacelia sorghi were found to contain dihydroderivatives of clavine alkaloids in smaller amounts. These were suggested to be festuclavine, pyroclavine, dihydroelymoclavine and chanoclavine (100)

Because of this unusual alkaloid pattern the biosynthesis of ergot alkaloids in Sphacelia sorghi might be expected to differ to some extent from that of the other Claviceps spp. - 110 -

R = H festuclavine, pyroclavine Chanoclavine-I

R = OH dihydroelymoclavine

The elucidation of the ergot alkaloid biosynthesis pathway in Sphacelia sorghi was therefore attempted. Also a study of the different alkaloids present in extracts of that fungi was undertaken in order to confirm or determine the structure of these compounds and investigate their possible involvement in the biosynthetic pathway.

II. SPHACELIA SORGHI ALKALOIDS

Chromatographic evidence (plates I and III) suggests the presence of many alkaloids in Sphacelia sorghi extracts, 7 or 8 compounds occuring in larger amounts. Two attempts were made to isolate the different alkaloids.

Isolation of Sphacelia sorghi alkaloids on a small scale by means of the Sephadex columns in (NH4) form and purification by preparative T.L.C. yield the main compounds listed in Table 19. These compounds could be separated in 2 groups: peptides and clavines following their elution from the columns. Mass spectrum examination of the different compounds pure by T.L.C. standards and consideration of the chromato- graphic data led to the identification of several compounds.

Clavine 2 (M,+ m/e 240) and peptide 1 were identified respectively as festuclavine and dihydroergosine, clavine 5 appeared to be a mixture of chanoclavine-I and dihydroelymoclavine. Further evidence for these compounds was obtained later when they were isolated in greater quantities (see over).. 2 other clavines were characterised:

clavine 3: (M m/0 256) fragments at: 119, 149, 169, 185, 213, 228, is probably dihydrosetoclavine or dihydroisosetoclavine.

Clavine 4 (em/e 256) fragments at: 129, 144, 155, 167, 168, 183, 197, 213, 223, 237 is probably a chanoclavine, isochanoclavine from.the chromatographic data.

TABLE 19

Chromatographic data for reference and isolated alkaloids (solvent system: chloroform-methanol-ammonia (0.88 ) (95:5:4)

Compound Rf value* ** Festuclavine .57 Clavine 2 100 Peptide 1 56 Clavine 3 56 Clavine 4 39 Peptide 2 25 Clavine 5 20 Clavine 6 15 Peptide 3 11 Clavine 7 8

* Reference festuclavine (RF x 100) ** Rf value for festuclavine. - 112

TABLE 20

Compound Rf value * Elution fraction Number

P 1 115 II P 2 70 X P 3 56 III, IV, V, VI P 4 45 VII P 5 4o Ix . P 6 3o x P 7 25 VIII P 8 17 IX P 9 13 xl P 10 13 XI P 11 11 IX P 12 11 VIII P 13 11 VII P 14 4 Ix

Peptide alkaloids eluted from Sephadex LH 20 * Reference festuclavine (RF x 100) in solvent system : chloroform - methanol - ammonia (0.88 ) 95:5:4.

Further identification of Sphacelia sorghi alkaloids was achieved by isolating these compounds on a larger scale using LH 20 Sephadex resin. 2 compounds crystallised directly from the elution fractions III, IV, V, VI and XI respectively. Other compounds were obtained by further purification of the different elution fractions by preparative T.L.C. The compounds were divided into clavine and peptide alkaloids by consideration of their molecular weight (determined by mass spectroscopy) and classified by their chromatographic behaviour as well as their elution from the LH2O resin. - 113 -

It was found that the resin was able to separate compounds having a same Rf value in the chromatographic solvent system used. This (90). has been found for other ergoline compounds These compounds are listed in Table 20 and Table 21.

TABLE 21

Compound Reference Elution Number Number fraction

C 1 100 II C 2 20 IX C3 20 X c 4 8 xi c 5 8 xi

Clavine alkaloids eluted from Sephades LH 20 reference festuclavine (RF x 100).

Clavines : C 1 (festuclavine) Crystalline, fine colourless needles mp 206 - 207 °(lit 242-245)* synthetic : 223 - 228°. IR spectrum (identical to literature(2)and synthetic festuclavine) showed adsorption bands at : 3100, 2940, 2920, 2860, 2780, 1605, 1440, 1340, 1220, 1120, 1040, 800 and 750 cm -I. The mass spectrum (fig. 29) gave a molecular ion m/e 240 and main fragments at 144 (35%), 154 (39%), 197 (22%). It corresponded to literature (138)

* Melt. points done under our conditions (Koffler block) melting point values were often lower than the literature references. The same differenCe for festuclavine was observed (100): 208 - 210o (Koffler block) and 240° (sealed tube).

- 114 -

100 Compound CI 240 Festuclavine %

80.

dance 60_ bun 40.

A 105 144 154 ive t

la 20- Re 0 I I 80 12.0 160 ZOO 290 280 mie

Compound C 2. 100 256 Di hydroelymoclavine %

80..

dance 60. n bu A

40- 144 ive

t 154

la 20 Re o 1 1 I 80 120 160 200 240 280 m/e

Compound C3 100 256 Chanoclavine-1 183 %

80 . 154 nce

da 60- n 168 236

Abu 40 127 e iv t

la 2.0.

Re 1 I 1 I 0 tl 30 120 160 2.00 240 1 2.80 ie

Fig.29: Mass spectra of clavine alkaloids from

Sphacelia sorghi. _ — - 115 -

C 2 (dihydroe3 22)

Small colourless crystals from methanol mp 230 - 240° decomp. (lit. 280 - 283°). The I.R. spectrum was identical to literature 143.

Absorption bands : 3400, 3100 (broad), 2880, 1605, 1440, 1340, 1320, 1220, 1060 and 750 cm -1. Mass spectrum (fig.29) molecular ion m/e 256 main fragments: 144 (33%), 154 (22%) U.V. spectrum:Emax at 875, 282, 295 nm identical to synthetic dihydroelymoclavine or literature (143)

C 3 (chanoclavine I) o (68, 69) ). Crystallised from methanol: mp 215° (lit. 212 - 215 The I.R. spectrum was identical to literature (29) and showed absorption at 3240, 2770, 2700, 1600, 1480, 1440, 1370, 1340, 1070 and 750 cm -1. Mass spectrum (fig. 29): molecular ion m/e 256 main fragments: 154 (69%), 183 (86%).

These 2 last compounds, dihydroelymoclavine and chanoclavine I are not separated by T.L.C. in solvent system chloroform-methanol. ammonia(0.88) 95:5:4 and corresponded to clavine 5 of the previous separation. The next 2 compounds crystallised directly from fraction XI. C4 mp 180°-185° colourless sheets. Infra red spectrum: absorption at 3150 (broad), 3640, 2810, 1740, 1410 (s), 1090, 1030, 940 cm -1.

Mass spectrum: fragments at: 103, 133, 144, 158, 200, 228, 243, 288, 319 molecular ion m/e 232. C mp: 173 ; colourless needles - 5 0-1760 Mass spectrum: molecular ion m/e 232. fragments at: 103, 133, 118, 124, 144, 149, 158, 200, 228, 243, 256, 284. These 2 last compounds are very similar and must be closely related. - 116 -

Peptides P mass spectrum : 240, 289, 372, 432, 786, 804 1 contains traces of festuclavine (240) and was isolated from the same fraction as festuclavine and has similar RF's value. P gave a blue spot with yellow centre on spraying with Erlich's 2 reagent. IR: absorption at 3330 cm-1 (broad), 2930, 2860, 1690 (s) 1460, 1420, 1340, 1290, 1250, 1230, 1150, 1020, 760. cm -1. Mass spectrum: 256, 264, 284, 310, 328, 342, 368, 386, 396, 414, 428, Acid hydrolysis: no yield of amino acids.

P (dihydroergosine) 3 Crystallised directly from the elution fractions as massive crystals ( mp 180-183°, lit. 2120(2)) yield 1.3 g. IR spectrum: absorption at 3320 (broad), 2960, 2880, 2800, 1730, 1650, 1560, 1450, 1220, 1140, 1040, 930, 860, 820, 750 cm "41.

Mass spectrum: 144, 154, 167, 223, 251, 269, 339, 353, 365, 392, 410, 429, 462, 468 - no molecular ion.

DihydroergOsine reference (Sandoz) 144, 154, 167, 223, 224, 225, 269, 282, 296, 314, 339, 396, 410, 414, 424, 514, 531, 549 molecular ion We 549.

The differences observed between these 2 mass spectra might be due to the conditions under which the mass spectra were performed and to the purity of the dihydroergosine isolated.

P4 Mass spectrum: , 256, 269, 326, 351, 368, 382, 411, 424, 428, 442. - 117 -

P Gave a bright blue colour with Erlich's reagent. 5 IR spectrum: absorption at: 3400, 3220, 2960, 2930, 2870, 1730, 1650, 1460,1410, 1390(s) 1050, 760.

Mass spectrum: 154, 185, 256, 269, 288, 354, 368, 424., 452, 508. shows some clavine impurity and yields little amino acid on acid hydrolysis (leucine and proline).

P IR spectrum shows main absorption: 3400 (broad), 3300, 6 2930, 2860, 1660, 1480, 1390, 1160, 1120, 1000, 753,751 cm -1 Mass spectrum: 256, 268, 284, 288, 308, 333, 383, 446; shows some clavine impurity. yield no amino acid or amino acid analysis.

P (peptide 2) IR spectrum: absorption at: 3350 (broad), 2960, 7 2930, 2880, 1730, 1650, 1560, 1460, 1390, 1270, 1210, 1140, 1040, 930, 760 cm -1 . Mass spectrum: 256, 269, 284, 361,376, 394, 428, 446.

P IR spectrum: absorption at: 8 3330 (broad), 3200, 2960, 2930, 2860, 1680 (s),1610, 1460, 1420, 1380, 1040, 840 cm -1.

Spectrum most similar to that of ergometrine,isolysergic acid in region 1680 in the range of peptide alkaloids spectra available.

Mass spectrum: 154, 223, 269, 361, 369, 394, 428, 444. gave leucine and proline in ration 1:1 on acid hydrolysis. - 118 -

P 9

Mass spectrum: 154, 223, 242, 256, 264, 272, 284, 288, 302, 412, 428, 430

P10

Mass spectrum: 208, 242, 256, 284, 311, 354, 367, 382, 394, 398, 425 • gave no amino acid on hydrolysis.

P Crystallised from methanol mp. 138-140o o 11 then 250 , in fact it was a mixture from crystals. IR spectrum: 3320 (broad), 2960, 2930, 2880, 1730, 1650, 1560, 1450, 1210, 1140, 1040, 930, 750 cm -1 This spectrum is very similar to that of dihydroergosine. Mass spectrum: fragments at: 231, 322, 445, 533, 645, 663.

Acid hydrolysis gave a good yield of the amino acid proline and leucine in the ratio 1:1.

P12 Crystallised from methanol mp 165-175 colourless crystals IR spectrum: absorption at: 3330 (broad), 2960, 2920, 2860, 1730, 1650, 1560, 1480, 1080, 760 cm -1 Mass. spectrum: 154, 167, 196, 209, 210, 222, 224, 256, 269, 354, 367, 398, 414, 428, 444, 452.

None or little amino acid yield on acid hydrolysis.

P IR spectrum: absorption at: 3300 (broad), 2960, 2930, 2880, 1750, 13 1650, 1560, 1450, 1220, 1140, 1040, 930, 760 cm -1.

Mass spectrum: 154, 167, 255, 269, 284, 339, 378, 396, 414, 428, 547. - 119 -

P IR spectrum : absorption at: 3380(broad), 2960, 2930, 2870, 14 1730, 1650, 1560, 1450, 1390, 1060, 760 cm -I.

Mass spectrum: 154, 269, 281, 284, 333, 376, 394, 410, 424, 466, 496, 512.

Acid hydrolysis gave leucine and proline in good yield (1:1).

All compounds,unless noted otherwise, gave a characteristic blue colour with Erlich's reagent. As far as it could be observed, the U.V. spectra of all Sphacelia sorghi compounds were very similar with max at 275, 282, and 295 nm, which agree with the other evidence suggesting that the peptide alkaloids produced by this fungus are saturated.

For all the ergot alkaloids of high molecular weight, no molecular ion could be detected with certitude in the mass spectra of those compounds. For such non-volatile compounds the relative abundance of the molecular ion is very small, so the last fragment observed in the mass spectrum might not be the molecular ion and many of the peptide alkaloids might have a higher molecular weight. For dihydroergosine, for example, the last fragment observed was at 468, but the fragment pattern is the same than for reference dihydroergosine and contribute to the identification of this compound, together with its other properties.

A comparison of the mass spectra of the other minor peptide alkaloids seems to point out to 2 main groups of compounds. Most of the mass spectra are very similar to that of dihydroergosine with fragments of m/e: 269, 310, 328 and these compounds are likely to be related to dihydroergosine and derivative of dihydrolysergic acid. (Unsaturated peptides should have the corresponding fragment m/e 267(138)). By contrast, the mass spectra of a few peptides: P1, P6, P10 and P11 do not follow this pattern.

Most of these compounds were obtained on a very small scale and structural evidence from other sources is not always available or reliable.

Proline and leucine were the only amino acids observed on acid hydrolysis, always in the ratio 1:1 as for dihydroergosine, but because of the small scale of the hydrolysis only a good yield of amino- acids made the results reliable. - 120 -

Infra - red spectroscopy provides more evidence for some of the compounds to be related to dihydroergosine, all but a few show a strong absorption at 1730 cm -1. This band is present for the more usual tripeptide alkaloids (2). The compounds which differ in that point are P2 and P8. Their IR spectra are more like those of the simple amide derivatives, such as ergometrine and isolysergic acid amide (2) P P P show the same typical absorption than dihydroergosine 13 11 17 in the region 2800 - 3000 cm -I while all other peptides give a constant but slightly different spectrum in that region. Four compounds Po? P6 P14 and P5 show an absorption at 1390 cm -1 Compounds related to dihydroergosine could either be isomers or degradation products of that compound. It is found that isomers usually have a very similar Rf in T.L.C. chromatography (116) would be a likely candidate, but most of the compounds similar to dihydroergosine have a much lower Rf e.g. P13/ Pik and might be degradation products. Another tripeptide giving leucine and proline on hydrolysis is ergokryptine ,but is not found in association with the other tripeptide ergosine.

CIA CH 3\ / 1 CH OH H 0 =

0 NCH3

ergokryptine

- 121 -

The dihydroderivative of ergokrytine would also have a higher Rf in the chromatographic solvent system used than dihydroergosine, as dihydroderivatives have a lower Rf than the corresponding unsaturated (116) compound and ergosine has the lowest Rf of all known tripeptide (112). ergot alkaloids However, no molecular ion corresponding to ergokryptine or a related compound (553, 577) was observed. The compounds with a high molecular ion e.g. Pi P 11 could not be explained in terms of known tripeptide alkaloids which molecular weight range from 545 -610. The other known peptides alkaloids are the simple amide derivative with much smaller molecular weights (270 to 400). In the intermediate range only one dipeptide ergot alkaloid is known: ergosecaline (436). • C H3 H N 1/° "`,..<:-. 0

0 "N/- ci-ic-H H \c1-4I

ergosecaline

The dihydroderivative of that compound with leucine substituted for valine would agree with.the molecular ion m/e 512 of 1 of the compounds isolated (P ), although no other evidence supporting this 12 hypothesis(acidpeptide hydrolysis) is available.

- 122 -

1-4 N 0-1 1 0 i‘N C.1-t c 1-4 ***•• CH a

leucine - dihydro derivative of ergosecaline

Therefore, the identity of the many compounds which mass spectra show a last fragment of m/e at 430 - 44o is very difficult to elucidate even if this is not the molecular ion. In the clavine group there was evidence for two closely related compounds C5, cle Their mass spectra• show a molecular ion m/e 332. These compounds are chromatographed with the clavine group by Sephadex ion exchange resin. They chromatographed as one single spot on T.L.C. in the usual system and might be isomers of the same compound. The mass spectrum does not show any fragment of m/e at 269, unlike dihydroergosine and related compounds. C4 and C5 could not be related to any known ergot alkaloid of the simple amide and clavine types. Some of the isolated clavine alkaloids isolated were well characteri- sed: festuclavine, dihydroelymoclavine, chanoclavine I. There was evidence for at least two more clavinessuggested as dihydrosetoclavine and isochanoclavine.Pyroclavine which was thought to occur in Sphacelia sorghi was not detected. - 123 -

Consequently, four of the alkaloids, dihydroergosine, chanoclavine Ildihydroelymoclavine and festuclavine could be identified satisfactorily, but chromatographic evidence as well as mass spectrum measurements suggest the presence of more alkaloids, some present only in very small amounts in Sphacelia sorghi extracts.

The characterised alkaloids apart from chanoclavine I are not usual. Festuclavine is the most common, although it usually occurs in small quantities in several ergot alkaloid producing fungi (2) Dihydroelymoclavine (d-Dihydro lysergol-I) has only been isolated from Claviceps gigantea, a maize ergot (143); Dihydropeptide alkaloids although synthesized (2) have not been found in nature. Dihydroergosine was confirmed as the major ergot alkaloid of Sphacelia sorghi and there is chromatographic evidence for the presence of dihydrolysergic acid (plate 1). Most of'the minor peptide alkaloids are likely to be related to dihydrolysergic acid, like dihydroergosine. The unusuality and the small amount available of these compounds make their identi- fication difficult.

The alkaloidal composition of Sphacelia sorghi differs from that of other ergot alkaloid producing fungi from higher plants. No other such pattern has been characterised yet. In the Mexican corn ergot from which dihydroelymoclavine was isolated other dihydro derivatives were found (Table 22), but this fungus does not produce any peptide alkaloids (144, 143) •

Festuclavine 65% D-dihydrolysergol-I 15% Chanoclavine 7% Pyroclavine 3%

Table 22;Alkaloid composition in Claviceps gigantea (144)

The clavine pattern is nevertheless similar to that of Sphacelia sorghi, although no pyroclavine was found in that fungus. It might however, be present in very small amounts. In Sphacelia sorghi, dihydroergosine is obviously the main product and seem to represent about 70% of the total extracted alkaloid even when variationsof the total alkaloid yield or content occur (Table 23). - 124

TABLE 23 Alkaloid composition of Sphacelia Sorghi . (% of total extracted alkaloid)

• Strain 2 Strain 3 Dihydroergosine 66.5 71.5 Peptide 2 2.5 Festuclavine 15 8.2 Dihydroelymoclavine ) Chanoclavine-I 3.5 13.2 Other alkaloids 12.5 7.0

The other alkaloids are present in smaller amount each accounting for 1% or less than of the total content. The contribution of amphoteric alkaloids would be as well of that order. Chanoclavine is found to be in greater amount than daydroelymoclavine when these two compounds are separated. The level of festuclavine was found to vary very much with different strains ( Table 23). In one strain used in feeding experiments festuclavine could not be detected by T.L.C. (see plate v), or on autoradiogram after feeding of labelled precursor as it usually does. Total yield was quite variable between strains and sometimes within as well, although culture and inoculation conditions were the same. It was found to vary between 600 pg/ml (good producing strain) and 100 pg/ ml (poor producing strain) in cultures. The yield could be related directly to the level of dihydroergosine which varied between 3 and 25 mg per culture flask. The variation in the other peptide alkaloids was more difficult to notice as they were only present in small quantities. ' These variations are relevant to biosynthesis studies, but despite differences in total yields, and in the proportion of the alkaloids, it did not influence apreciatively the biosynthetic results - 125 -

III. BIOSYNTHESIS The major ergot alkaloids of1102=21alma have been characterised as dihydroergosine, festuclavine, dihydroelymoclavine and chanoclavine-I. Although this represents an unusual pattern for ergot alkaloids producing fungi, it can be expected that the biosynthesis of these alkaloids and the other non-identified minor ones is similar to that of the more common unsaturated clavine and peptide alkaloids.

Therefore, mevalonic acid, methionine and tryptophan can be considered as primary precursors and the clavine alkaloids possible intermediates in the synthesis of the peptide alkaloids, dihydro- ergosine being the main end product.

The incorporation of labelled tryptophan and mevalonic acid as well as likely alkaloid labelled precursors was studied. 14 14 Incorporation of (2 C) mevalonate and (2- C) tryptophan gave the results shown in Tables 24 and 25. Both incorporation patterns of labelled tryptophan and mevalonic acid into alkaloids are comparable, but mevalonic acid is not as efficient (see Table 24) which has also been found for the other ergot alkaloid producing strains.

Autoradiograms of culture extracts following the feeding of these labelled precursors showed 3 main zones of activity correspon- ding to the major alkaloids: festuclavine, dihydroergosine and dihydroelymoclavine and chanoclavine-I. (See Plate III). A few other minor alkaloids were also labelled. Following the first feeding of (2-14C) tryptophan to Sphacelia sorghi, the whole of the basic alkaloid fraction was examined for its alkaloidal content and corresponding activity. The preparative T.L.C. of the chloroform extract showed nine (9) main bands under U.V. irradiation at 254 nm. These were numbered 1-9 with decreasing Rf values. (Table 25). The bands showing the strongest absorption (2, 5, 8) were found to be, by comparison with standards: festuclavine dihydroergosine and dihydroelymoclavine - chanoclavine-I, 2 other bands corresponded to almost pure clavine 1 and peptide 2. The other bands were a mixture of the major alkaloids and minor ones. The lowest Rf bands consisted mainly of 3 alkaloids. Radioactivity Alkaloid fed or recovered recovered % incorporation

dpm mg f 14 , 1/42- C) MVA 920000 Total basic alkaloids 72150 13.2. 7.84 Peptide alkaloids 16100 7.8 1.93 Clavines 5950 1.6 .65 14 6 (2- C) Tryptophan I 22 x 10 (2 flasks) Total basic alkaloids 6500000 56 30 6 Peptide 2.96 x 10 27.6 13.5 6 Clavines . 2.36 x 10 10.22 . 10.5 14 (2- 0) Tryptophan II Fed 4.4 x 106 Total alkaloids ** 2300000 38.4 52 Peptide + amphoterics 6 alkaloids 1.53 x 10 31.0 35.0 Clavine alkaloids 745000 7.4 16.9

TABLE 24 Incorporation of labelled precursors in Sphacelia sorghi Clavine and Peptide fractions. * CHC13 extraction

** Sephadex extraction 6 I (2-14C) tryptophan fed: 22 x 10 dpm Alkaloid recovered 0 FRACTION COMPOUND Radioactivity Amount Specific Activity incorporation (dpm) (mg) (dpm/mmole) radioactivitz 1 ' Clavine 1 150800 .7 2 Festuclavine 1584100 5.65 7.0 x 107 7.2 3 75800 .3 4 17000 .1 5 dihydroergosine 2727900 25.0 6.0 x 107 12.4 ,6 159800 .7 7 peptide 2 77250 .95 .3 8 dihydroelymoclavine* 503000 10.4 x 107 2.28 + chanoclavine-I 1.3 9 470200 2.1 6 II (2-14C)tryptophan fed: 4.4 x 10 dpm Dihydroergosine 497000 15.7 1.74 x 107 11.3 Festuclavine 144900 1.8 1.93 x 107 3.3 Dihydroelymoclavine + chanoclavine-I 327600 2.9 2.87 x 107 7.5 C4 18500 .4 TABLE 25 Incorporation of (2-14C) tryptophan into Sphaceliaporghi alkaloids * The T.L.C..system used for most separations: chloroform/methanol/ammonia 95:5:4 does not resolve these 2 compounds which were isolated as one in most experiments. - 128 -

Plate III: Basic alkaloid fractions from (2-14C) tryptophan (I) and (2-14C) mevalonate (II, III, IV) feedings to Sphacelia sorghi. whole chloroform extract; II, III, IV peptides (p) and clavines (c) fractions eluted from (NH4) Sephadex SP C-25. I, II, IV : autoradiograms; III chromatogram. (T.L.C. solvent: chloroform/methanol/ammonia 0.88, 95:5:4) DHE = dihydroergosine; F = festuclavine; DHL = dihydro- elymoclavine; CHA = chanoclavine-I. —F

DH E

D H L — + C H A oursomilo

P p

I II 12 - 130

Accordingly, most of the label was incorporated into the major alkaloids. The second experiment with feeding of (2-1 4'C) tryptophan showed essentially the same results (Table 25) (2 different strains were used). The higher specific activities of dihydroelymoclavine - chanoclavine-I and festuclavine might suggest in particular that these 2 alkaloids could possibly be intermediates in the biosynthesis of dihydroergosine. Refeeding of all the alkaloidal fractions isolated 14 in the first feeding of (2- C) tryptophan in Sphacelia sorghi agreed with this hypothesis. Autoradiograms corresponding to the different feedings were obtained (Plate IV)and dihydroergosine isolated(Table 26). The peptide alkaloids were confirmed as end product5)neither dihydroergosine nor the unknown peptideswere metabolised any further. In contrast, festuclavine, dihydroelymoclavine - chanoclavine-I and' unknown clavine-I were incorporated very efficiently (18.7%, 51%, 8.7% respectively) into dihydroergosine. Incorporation of the mixed fractions did not yield any positive results. The last fraction (9) was not found to be incorporated significantly in dihydroergosine or any other compound. The precursors used in that experiment were only crude fractions and it was necessary to feed again the same pure labelled alkaloids. Labelled festuclavine and dihydroelymoclavine were then fed and paralell trapping experiments were done with these compounds.

The results are presented in Table 27 and 28 for festuclavinelin Table 29 for dihydroelymoclavine. Festuclavine was incorporated very efficiently into dihydro- ergosine (24-31%). The high specific activity of the resulting dihydroergosine almost certainly implies that festuclavine is an immediate precursor. At least, one of the compounds, and probably both, in the mixture dihydroelymocalvine - chanoclavine-I must be an intermediate between festuclavine and dihydroergosine for the con.. sideration of these same 2 factors. Dihydroelymoclavine was a slightly more efficient precursor (33% incorporation) of dihydroergosine. 131 -

Plate IV: Autoradiograms of the chloroform extracts form Sphacelia sorghi cultures fed with basic alkaloid fractions (1,2,5,7, 8) biosynthetically labelled with (2_14c) tryptophan (see text). T.L.C. solvent system: chloroform/methanol/ammonia 0.88 (95:5:4).

DHE = dihydroergosine - 1 3 --

2_

DNE-

5 7

COMPOUND FED DIRTDROERGOSINE RECOVERED

Fraction COMPOUND Amount Radioactivity Specific Amount % Incorporation Specific Number (mg) dpm Activity (mg) radioactivity activity d•m/m mole (dpm/mmole)

1 Clavine I 1.14 x105.- 12.7 8.7 4.1 x 105

2 Festuclavine 4.2 1.2 x 106 6.8 x 107 14.8' 18.7 8.4 x lo6 6 5 Dihydroergosine 17.8 1.95 x lo 6.o x 107 23.6 47.5 2.14 x 107

8 Dihydroelymoclavine 1.04 4.0 x 105 9.85 x 107 10.2 51 1.1 x 107 Chanoclavine -I

TABLE 26 Refeeding of labelled alkaloid fractions from 12r -141 quryptophan feeding. Fed (3H) festuclavine(1 mg12.6 x 0 dpm, Fed (3H) festuclavine(.95 mg)2.5 x 106 dpm 6.2 x 108 dpm/mmole)- 6.3x 108 dpm/mmole).

COMPOUNDS Amount % incorporation specific activities Amount % incorporation specific activity RECOVERED (mg) radioactivity ,(44/Mmole) (mg) radioactivity (dpm/mole)

Peptides 63 10.1 42 Clavines 8 Dihydroergosine 8.5 24.0 9.8 x 107 4.05 31.0 1.07 x 10 Peptide 2 4.3 6 Festuclavine .58 2.4 2.6 x 107 .35 0.1 1.7 x 10 Dihydroelymoclavine) Ohanoclavine-I ) 1.3. 5.38 2.7 x 107

TABLE 27 Feeding of (3-H) festuclavine 2 - 14C tryptophan 4.4 x 106 dpm C tryptophan 4.4 x 106 dpm Festuclavine 31.5 mg COMPOUND Amount % incorporation specific activity Amount °o incorporation specific activity RECOVERED (mg) radioactivity_ (d m/m'mole) (mg) radioactivity (dpm mole) Peptides 4- amphoterics 35 23.5 29.5 35.0 Clavines 10.9 12.1 7.1 16.8 Dihydroergosine 26 16.8 1.56 x 10 15.7 11.3 1.74 x 10 Festuclavine 2.5 2.1 .9 x 107 1.8 3.3 1.9 x 107 Dihydroelymoclavine 1.7 2.7 1.87 x 107 2.9 7.5 2.87 x 107 4-- Chanoclavine-I

TABLE 28 Festuclavine trapping 14 Fed (2-14C) tryptophan 4.4 x 105 dpm Fed C dihydroelymoclavine dihydroelymoclavine 30.1 mg .21 mg 48000 dpm 5.85 x 107 dpm/

COMPOUNDS Amount % incorporation specific activity Amount % incorporation specific activity RECOVERED (mg) radioactivity dpm/m mole (mg) radioactivity dpm/m mole

Peptides 29 33 5.2 57.5 Clavines 8.5 11.3 1.5 41

6 6 Dihydroergosine 23.5 21 , 2.2 x 10 3.85 33 2.3 x 10 Festuclavine .4 .6 1.5 x 106 Dihydroelymoclavine 4.5 1.5 3.7 x 106 .4 4.5 1.3 x 106 6 Chanoclavine-I 1.5 4.1 3.1 x 10 - 4.5 .8 x 106 *

TABLE 29 Dihydroelymoclavine trapping and feeding

* Specific activity after one preparative T.L.C. separation (likely contamination by dihydroelymoclavine).

s - 137 -

The very low activity of chanoclavine in that experiment agreed with this compound being an early intermediate in the biosynthesis and that a route involving dihydroelymoclavine - dihydroergosine must exist. This was suggested in the results of the festuclavine feeding.

Unfortunately, the strain used in the last experiment did not produce any festuclavine (or very little) and it was not detected on the separation of the basic alkaloids by preparative T.L.C. and so no incorporation of dihydroelymoclavine into festuclavine could be determined. However, it is considered very unlikely that dihydroelymoclavine could act as a precursor of festuclavine. 14 A small part of the activity of (2- C) tryptophan was found to be trapped in added dihydroelymoclavine or festuclavine in respective trapping experiments.

Until now, we have evidence of the role of festuclavine, dihydroelymoclavine and chanoclavine-I in the biosynthesis of dihydro- ergosine, these compounds being the main alkaloids found in basic extracts of Sphacelia sorghi cultures. Amphoteric alkaloids represent only a very small proportion of the total alkaloids and evidence suggested dihydrolysergic acid as the main one. This compound might be expected to be a precursor of dihydroergosinegLabelled dihydro- lysergic acid was found to be incorporated almost directly into dihydroergosine. Autoradiograms of the butanol and chloroform extracts showed only 2 main radioactive bands corresponding to dihydrolysergic acid and dihydroergosine. There were 2 minor bands corresponding to amphoteric fractions of Rf's slightly greater than dihydro- lysergic acid, but at the dilution used no other band showed. This also confirms the fact that the other peptides alkaloids present in Sphacelia sorghi are minor ones. Quantitative results (see Table 30) agree with these findings. Most of the label is found in dihydroergosine (33%) but the specific activity of this compound although high is lower than the recovered precursor dihydrolysergic acid in contrast with preceeding fending experiments of the other . precursors. This might be caused by 2 factors: Fed (2-14C) tryptophan 8.4 x 106 dpm Fed (14C) dihydrolysergic acid dihydrolysergic acid 30.25 mg 1.5 mg 132000 dpm 3.28 x 107 dpm/m mole COMPOUNDS Amount % incorporation specific activity Amount % incorporation specific activity RECOVERED (mg) radioactivity dpm/m mole (mg) radioactivity dpm/m mole

Basic alkaloids 29.5 22.6 8.9 . 41.0 Amphoteric alkaloids 29.8 14.6 8.1 51.0

4

Dihydroergosine 11.8 8.8 3.45 x 107 2.74 32.6 8 x 106 Dihydrolysergic acid 8.9 .8 2.1 x 106 .4 13.8 1.2 x 107 -4

TABLE 30 Dihydrolysergic acid feeding and trappin& - 139 -

This is a later stage in the pathway,or dihydrolysergic does not penetrate the cells as readily as the precedent precursors discussed before. Dihydrolysergic acid is indeed not very soluble and difficulties due to that cause arised during extraction of that compound. 14 TABLE 31 Incorporation of (2- C) tryptophan into dihydroelymoclavine and chanoclavine-I

Com.ound Amount Radioactivity Specific activity (211) __...22)( d i daL_._..1_--Ira mole)

Dihydroelymoclavine .21 39880 4.85 x 107

Chanoclavine-I .7 64250 2.3 x 107

Addition of excess dihydroelysergic acid resulted in the trapping of 14 some activity of the (2- C) tryptophan in the parallel trapping experiment. This time again the overall dihydroergosine synthesis was not visibly affected.

This may be explained by the non-solubility or permeability of this compound, and this might be true as well for dihydroelymoclavine and to a lesser extent for festuclavine in the similar experiments, but for dihydroelymoclavine as well as for dihydrolysergic acid the actual amount of dihydroergosine produced is much greater than in the feeding of the single labelled precursor while the specific activity is high as expected. The added compound seems to promote or stimulatethe, synthesis of the end product.

These experiments, therefore, show definitevely that festuclavine, dihydroelymoclavine and dihydrolysergic acid can act as precursors of dihydroergosine and probably the minor peptides alkaloids of Sphacelia sorghi. Chanoclavine-I,although not fed directly was incorporated into dihydroergosine when fed as a mixture of dihydroelymoclavine and chanoclavine-I. The ratio of these 2 compounds and activity from[2-14 1Cjtryptophan was determined by further purification after feeding this precursor and is shown in Table 31.

Chanoclavine-I, although of lower specific activity would have been detected if not incorporated in dihydroergosine. Dihydro- elymoclavine has always been found to be present in very much smaller amounts than chanoclavine.

- 1140 -

The data discussed above gives very little evidence for establishing a detailed sequence in the biosynthesis of dihydro- ergosine. It can be expressed at: tryptophan chanoclavine -I ---> x -+Iihydrolysergic acid mevalonic acid dihydroergosine

The exact situation Of festuclavine and dihydroelymoclavine indicated by x is not known. There was evidence for 1 more clavine intermediate, but others may exist as well. The pathway of unsaturated ergot peptide alkaloids is thought to occur in the clavines alkaloids in the way represented in Fig. 30.

This suggests an equivalent pathway in Sphacelia sorghi, but with the dihydroderivative equivalents with festuclavine precursor of dihydroelymoclavine. Festuclavine and dihydroelymoclavine have been isolated from culture extracts of other fungi where they appear in fact, to be end products.- Festuclavine is more common, although occuring in small quantity and is believed to be derived from (82). agroclavine This compound and dihydroelymoclavine have also been found in greater quantity, but elymoclavine was not incorporated in extracts of Clavice s gigantea significantly into dihydroelymocla- vine in that fungus 3 . Dihydroelymoclavine could then well be derived from festuclavine as probably occured in this work. Agroclavine then should be the unsaturated link between chanoclavine-I and festuclavine. Consequently, although agroclavine had not been detected in Sphacelia sorghi extracts both feeding of labelled agroclavine and 14 the trapping of this compound after feeding (2 - C) tryptophan were attempted. The results are shown in Table 32 and Fig. 17. These experiments were performed in parallel with similar ones involving festuclavine which have been discussed before. In contrast to the strong incorporation of (3H) festuclavine in dihydroergosine, (3H) agroclavine incorporation was minimal and the low specific activity of the resulting dihydroergosine suggest degradation of the initial labelled material before incorporation. Most of the label was recovered with the festuclavine-agroclavine fraction, none was found in dihydroelymoclavine-chanoclavine-I fraction.; COOH OH OH OH

H 0

mevalonic •acid H Tryptophan Chanoclavine -I Agroclavine Elymoclavine

01-I 0

K R •• peptide

Peptide alkaloid Lysergic acid

Figure 30 . Biosynthesis of ergot peptide alkaloids Fed (3H) agroclavine(1.7 mg i Fed (3H) agroclavine0.95 mg, Fed (201 C) tryptophan 4.4 x 10' dpmi 1 x 105 dpm )1.4 x 107 dpm/m mole). 117000 dpm )1.4 x 107 dpm/m mole). agroclavine 30.9 mg).

COMPOUNDS Amount % specific Amountt % specific Amount /0a specific RECOVERED (mg) incorporation activity (mg) incorporation activity (mg) incorporation activity radioactivity (dpm/mmole) radioactivity (dpm/m mole) radioactivity (dpm/ m mole)

Peptides 5.95 1.7 35 2.3 35 Clavines 16.5 21.0

° Dihydroergosine 1.9 1.8 5.2 x 105 .5 .8 9.9 x 105 4.3 .5 3.o x 106 Peptide 2 1.5 Compound X1 6 .62 2.6 2.7 x 10 •

Agroclavine 6 6 4.4 .02 5.9 x 104 1.28 21.9 4.1 x 10 .46 5.6 x 10 Festuclavine 9.3 1.1 5.2 4.4 x lo7

Compound X2 .63 .2 3.2 x. 106

Dihydroelymo - -_- _ clavine .9 .6 1.7 x 105 Chanoclavine 1 .54 202 4.5 x 10

TABLE 32 Agroclavine feeding and trapping 143

14 In the attempt to trap the activity of added (2- C) tryptophan in agroclavine separation of agroclavine and festuclavine was achieved by preparative T.L.C. The purified agroclavine was inactive although in the parallel experiment the festu-Clavine was highly active.

Examination of the results show that not only agroclavine did not function as a precursor but it inhibited the alkaloid synthesis. Even small quantities of precursors such as the one used in feeding experiments have the effect of lowering thetotal alkaloid yield, in particular that of dihydroergosine. Increasing amounts of agroclavine increased that effect. Results are difficult to compare because of the difference in yields in different cultures and the increasing amount of agroclavine added;but 20 mg of agroclavine were sufficient to stop completely or nearly so the synthesis of dihydro- ergosine while the same amounts of festuclavine and dihydroelymocla- vine seemed in contrast to inhance this synthesis.

Alkaloidal extracts from inhibited cultures have a composition different.from the one usually observed. Only dihydroergosine and chanoclavine-I are still present in significant amounts which are likely the amount present at the time of addition of agroclavine, while some new compounds (compoundsXl and X2)appeared . These last compounds are mainly most likely degradation products of agroclavine (plate V)I old agroclavine solutions give a similar pattern of compounds on T.L.C. One or 2 compolindsonly present in small amounts, of Rf's slightly above chanoclavine-I, have also been detected which could not be related to the usual alkaloids of Sphacelia sbrghi.

From the data obtained in agroclavine feeding (Table 32) there is not much evidence for an accumulation of intermediates: festuclavine or chanoclavine-I, these compounds have nevertheless a much stronger specific activity than normal. As no dihydroelymoclavine could be detected the incorporation of the label from tryptophan must be able to proceed up to festuclavine, agroclavine acting as an inhibitor at that stage.

-144 -

A

DHE

DHL +CHP■

Plate V: Inhibition of alkaloid production is Sphacelia by agroclavine.

Chlomatogram of basic alkaloids from normal culture (II) and from culture after adding 20 mg agroclavine (I) .Reference agroclavine (III).

A = agroclavine; DHE 6 dihydroergosine; DHL = dihydroelymo- clavine; CHA = chanoclavine I.

(T.L.C. solvent system: chloroform/methanol/ammonia (0.88) 95:5:4) - 14-5 L.,

Mycelium from flasks where agroclavine has been added always a grey-.brown colour shows, instead of the usual light grey, which could be due to the degradation of agroclavine. The same phenomena has been observed on the addition of tryptophan.

In Sphacelia sorghi alkaloid biosynthesis seem to follow an unusual pathway which does not involve agroclavine. Although elymoclavine is thought to be derived from agroclavine a direct path from chanoclavine-I to elymoclavine has been found in cell free extracts(20) and elymoclavine could be a possible intermediate in Sphacelia sorghi alkaloid biosynthesis.

In a trapping experiment done with that compound the excess elymoclavine reduced the incorporation of labelled tryptophan into the peptide alkaloids (table 33), but the specific activity of dihydroergosine is high and comparable to control values. The synthesis of this compound appear to have been slawed. down.

With respect to festuclavine, dihydroelymoclavine and chanoclavine-I, elymoclavine has the same effect as agroclavine. Very little activity is trapped in elymoclavine itself.-Elymoclavine was not found to be a precursor of dihydroelymoclavine in Claviceps (143) giAantea and is probably not a precursor of dihydroergosine either. The major clavines alkaloids present in Sphacelia sorghi extracts: festuclavine, dihydroelymoclavine and chanoclavine-I have been shown to be efficient precursors of the peptide alkaloids, dihydroergosine being the main end-product.

The biosynthesis of ergot alkaloids in Sphacelia sorghi is different from that of other peptide alkaloid producing strains. It involves dihydroclavine alkaloids instead of the unsaturated equivalents. Agroclavine does not seem to be part of the pathway and even small quantities of this compound dramatically repress dihydroergosine synthesis. This does not happen with other precursors. Elymoclavine does not seem to be involved either.

In the amphoteric fraction, dihydrolysergic acid has been shown to be converted into dihydroergosine. Extracts from Spthalia sorghi show the presence of more minor clavine alkaloids which have not been identified. One of them: clavine-I has been found to act as a precursor of dihydroergosine. Precursor: (2-14C) tryptophan 4.4 x 105 dpm Elymoclavine 20 mg

Amount % incorporation Specific activity COMPOUNDS (mg) radioactivity (dpm/mmole)

Peptides 16.0 9.2 Clavines 9.4 15.4

Dihydroergosine 4.3 4.7 2.68 x 106 Festuclavine ..8 6.9 9.5 x 106 Elymoclavine 3.5 .4 1.2 x 105 Dihydroelymoclavine - - - 6 Chanoclavine-I 1.1 4.7 4.9 x 10

TABLE 33 Elymoclavine trapping 147

The others, present only in trace amounts could not be identified and used in feeding experiments. Addition of excess intermediate in trapping experiments did not seem to affect much the total biosynthesis. Probably, one a small part of the material may have permeated into the cells. If that is the case, this small amount seems sufficient for agroclavine or elymoclavine to inhibit or reduce the total synthesis, while under similar conditions festuclavine promots alkaloid biosynthesis.

Consideration of specific activities results from the different feeding experiments suggest a possible pathway for the biosynthesis of ergot alkaloids in Sphacelia sorghi, although the presence of other intermediates and the reversibility of some of the steps cannot be excluded (fig. 31). off OH

NCH3

mevalonic acid

H H tryptophan chanoclavine-I festuclavine dihydroelymoclavine NH riCH3 0 cH2.CH(CH3)z.

dihydroergosine dihydrolysergic acid

Fig. 31 Hypothetical pathway for ergot alkaloid biosynthesis in phacelia sorghi. - 11+9 -

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