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