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Tall fescue (Festuca arundinacea Schreb.) infested with the endophytic fungus, Acremonium coenophialum Morgan-Jones and Gams, is responsible for fescue toxicoses in cattle, sheep and horses. A number of ergopeptine and related alkaloids are produced by the fungus which may, individually or in combination, contribute to these toxicoses. The fact that ergovaline, the most predominate ergopeptine alkaloid, is present at biologically active concentrations in endophyte-infested (E+) tall fescue, has led to renewed scientific interest in these alkaloids as they relate to animal health and economic consequences. Biosynthesis of the ergopeptine alkaloids begins with the formation of chanoclavine from tryptophan, source of the indole ring structure, and dimethylallylpyrophosphate (.)

Covering: 2000 to 2010 Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. They have been identified in two orders of fungi and three families of higher plants. The most important producers are fungi of the genera Claviceps, Penicillium and Aspergillus (all belonging to the Ascomycota). Chemically, ergot alkaloids are characterised by the presence of a tetracyclic ergoline ring, and can be divided into three classes according to their structural features, i.e.amide- or peptide-like amide derivatives of D-lysergic acid and the clavine alkaloids. Significant progress has been achieved on the molecular biological and biochemical investigations of ergot alkaloid biosynthesis in the last decade. By gene cloning and genome mining, gene clusters for ergot alkaloid biosynthesis have been identified in at least 8 different ascomycete species. Functions of most structure genes have been assigned to reaction steps in the biosynthesis of ergot alkaloids by gene inactivation experiments or biochemical characterisation of the overproduced proteins.

The ergot alkaloids are a diverse class of fungal-derived indole alkaloid natural products with potent pharmacological activities. The biosynthetic intermediate chanoclavine-I aldehyde 1 represents a branch point in ergot biosynthesis. Ergot alkaloids festuclavine 2 and agroclavine 3 derive from alternate enzymatic pathways originating from the common biosynthetic precursor chanoclavine-I aldehyde 1. Here we show that while the Old Yellow Enzyme homologue EasA from the ergot biosynthetic gene cluster of Aspergillus fumigatus acts on chanoclavine-I aldehyde 1 to yield festuclavine 2, EasA from Neotyphodium lolii, in contrast, produces agroclavine 3. Mutational analysis suggests a mechanistic rationale for the switch in activity that controls this critical branch point of ergot alkaloid biosynthesis.

β-Galactosides of elymoclavine, chanoclavine, lysergol, 9,10-di-hydrolysergol, and ergometrine were prepared using β-galactosidase from Aspergillus oryzae .

The total enantioselective synthesis of (−)-chanoclavine I 2 in twelve steps from indole-4-carboxaldehyde 4 is described The key-step 3 → 6 which involves the formation of the C ring by creation of the C5-C10 bond is catalyzed by a chiral palladium (0) complexes. The chiral ergoline synthon 6 is produced with an excellent diastereo-and enantioselectivity (up to 95% ee).

Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaFS has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaFS comprises 290 amino acids with a molecular mass of about 32.1 kDa. The coding region of fgaFS consisting of three exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE70 and overexpressed in E. coli. The soluble monomeric His(6)-FgaFS was purified by affinity chromatography and used for enzyme assays. It has been shown that FgaFS is responsible for the conversion of chanoclavine-I aldehyde to festuclavine in the presence of the old yellow enzyme FgaOx3. The structure of festuclavine including the stereochemistry was unequivocally elucidated by NMR and MS analyses. Festuclavine formation was only observed when chanoclavine-I aldehyde was incubated with FgaOx3 and FgaFS simultaneously or as a tandem-reaction with a sequence of FgaOx3 before FgaFS. In the absence of FgaFS, two shunt products were formed and did not serve as substrates for FgaFS reaction.

Ergot fungi in the genus Claviceps and several related fungal groups in the family Clavicipitaceae produce toxic ergot alkaloids. These fungi produce a variety of ergot alkaloids, including clavines as well as lysergic acid derivatives. Ergot alkaloids are also produced by the distantly related, opportunistic human pathogen Aspergillus fumigatus . However, this fungus produces festuclavine and fumigaclavines A, B, and C, which collectively differ from clavines of clavicipitaceous fungi in saturation of the last assembled of four rings in the ergoline ring structure. The two lineages are hypothesized to share early steps of the ergot alkaloid pathway before diverging at some point after the synthesis of the tricyclic intermediate chanoclavine-I. Disruption of easA , a gene predicted to encode a flavin-dependent oxidoreductase of the old yellow enzyme class, in A. fumigatus led to accumulation of chanoclavine-I and chanoclavine-I-aldehyde. Complementation of the A. fumigatus easA mutant with a wild-type allele from the same fungus restored the wild-type profile of ergot alkaloids. These data demonstrate that the product of A. fumigatus easA is required for incorporation of chanoclavine-I-aldehyde into more-complex ergot alkaloids, presumably by reducing the double bond conjugated to the aldehyde group, thus facilitating ring closure. Augmentation of the A. fumigatus easA mutant with a homologue of easA from Claviceps purpurea resulted in accumulation of ergot alkaloids typical of clavicipitaceous fungi (agroclavine, setoclavine, and its diastereoisomer isosetoclavine). These data indicate that functional differences in the easA -encoded old yellow enzymes of A. fumigatus and C. purpurea result in divergence of their respective ergot alkaloid pathways.

β-Galactosides of elymoclavine, chanoclavine, lysergol, 9,10-di-hydrolysergol, and ergometrine were prepared using β-galactosidase from Aspergillus oryzae .

A method for transfructosylation of ergot alkaloids elymoclavine, chanoclavine, lysergol, 9, 10-dihydrolysergol using commercial yeast β-fructofuranosidase and sucrose is described

A new synthetic route involving the fragmentation reaction of 3-amino alcohols for the total synthesis of 6,7-secoergoline alkaloids was developed and then successfully applied to the total syntheses of (±)-chanoclavine-I and (±)-isochanoclavine-I.

Clavine alkaloid production in submerged cultures of various Claviceps strains was inhibited by 0.25 mM 5-fluorotryptophan (5-Ftrp). The inhibition was removed by washing the cultures, or by addition of 50 mM tryptophan. Lower doses of tryptophan (0.25–25.0 mM) were ineffective. Addition of cycloheximide and/or 8-hydroxyquinoline (8-HOQ) to be inhibited cultures 24 and 48 h prior washing increased the rate of alkaloid synthesis in washed cultures (superinduction) and the rate of clavine synthesis was proportional to the length of 8-HOQ pretreatment. The addition of 0.25 mM tryptophan in washed cultures also stimulated alkaloid synthesis, but only for 2–3 days, then production in the cultures without tryptophan was higher. Cultures of Claviceps strains 59 and EK-10 inhibited with 5-Ftrp were capable of converting chanoclavine into further clavine pathway products. 5-Fluorotryptophan probably acts as an inhibitor of the first enzyme of the clavine pathway. However, the superinduction suggests the presence of a labile regulatory protein controlling clavine biosynthesis.

A new strain, Claviceps purpurea 88-EP-47, with high invertase activity was selected. Free and Calginate immobilized cultures of this strain were used for fructosylation of ergot alkaloids. By bioconversion from their aglycones, elymoclavine-O-β-d-fructofuranosyl(2→1)-O-β-d-fructofuranoside, and elymoclavine-O-β-d-fructoside, the respective fructosides of chanoclavine, lysergol and dihydro-lysergol monofructosides were obtained. These substances are formed by β-d-fructofuranosidase present in Claviceps cells. The bioconversion activity of the enzyme system (fructose transfer) is strongly dependent on pH, substrate (sucrose) concentration and the developmental profile of invertase activity. The pH optimum for elymoclavine fructosylation is 6.5, for chanoclavine 5.7, and the optimal sucrose concentration is 75 g/l. Fifteen-day-old production cultures had the best glycosylation activities. Fructosylation of alkaloids can be stimulated in production cultures of C. purpurea or C. fusiformis forming elymoclavine or chanoclavine by a pH shift to 6.5 at the end of the production phase. Glycosylating Claviceps strains producing elymoclavine eliminate the free alkaloid into glycosides. The feedback inhibition of alkaloid synthesis by elymoclavine is then strongly reduced, helping to further improve elymoclavine yields. Elymoclavine can be liberated simply by invertase activity of baker's yeast.

Summary Lipids influence ergot alkaloid biosynthesis in Claviceps purpurea mainly as structural elements of membranes. Chlorophenoxy acids (CPA) were applied to C. purpurea cultures to modify cellular lipid composition. Mutant strain C. purpurea 59 accumulating tricyclic clavines and two parental strains 35 and 129 were used. All the CPA - clofibrate, 2-(4-chlorophenoxy)-acetate and 2-(4-chlorophenoxy)-propionate (CPP) lowered lipid content. Fatty acid (FA) unsaturation index increased indicating a stimulation of FA-desaturation and also FA-elongation. Sterol biosynthesis was impaired mainly in the C(4)- and C(14)-demethylation resulting in a decrease of “membrane functional” (quasiplanar) sterols. Relative content of phosphatidylethanolamine decreased 1 Of old. Changes in lipids correlated with the decrease of microviscosity of cell membranes (measured as a steady state fluorescence anisotropy of the protoplast membranes with DPH and TMA-DPH as probes). CPP in subtoxic doses stimulated alkaloid production and changed alkaloid composition in the C. purpurea 59 strain impaired in chanoclavine cyclase function.

Submerged culture of Claviceps fusiformis supplemented with chanoclavine I produced chanoclavine I O-β-D-fructofuranoside and chanoclavine I O-β-D-fructofuranosyl-(2→1)-O-β-D-fructofuranoside. The structures of the two new glycosides were deduced from mass and nmr spectral data

EA have been a major benefit, and a major detriment, to humans since early in recorded history. Their medicinal properties have been used, and continue to be used, to aid in childbirth, with new uses being found in the treatment of neurological and cardiovascular disorders. The surprisingly broad range of pharmaceutical uses for EA stems from their affinities for multiple receptors for three distinct neurotransmitters (serotonin, dopamine, and adrenaline), from the great structural diversity of natural EA, and from the application of chemical techniques that further expand that structural diversity. The dangers posed by EA to humans and their livestock stem from the ubiquity of ergot fungi (Claviceps species) as parasites of cereals, and of related grass endophytes (Epichloe, Neotyphodium, and Balansia species) that may inhabit pasture grasses and produce toxic levels of EA. Further concerns stem from saprophytic EA producers in the genera Aspergillus and Penicillium, especially A. fumigatus, an opportunistic pathogen of humans. Numerous fungal species produce EA with a wide variety of structures and properties. These alkaloids are associated with plants in the families Poaceae, Cyperaceae, and Convolvulaceae, apparently because these plants can have symbiotic fungi that produce EA. Pharmacological activities of EA relate to their specific structures. Known as potent vasoconstrictors, the ergopeptines include a lysergic acid substituent with an amide linkage to a complex cyclol-lactam ring structure generated from three amino acids. Simpler lysergyl amides and clavines are more apt to have oxytonic or psychotropic activities. One of the lysergyl amides is LSD (5), the most potent hallucinogen known. The EA biosynthetic pathway in Claviceps species has been studied extensively for many decades, and recent studies have also employed epichloes and A. fumigatus. The early pathway, shared among these fungi, begins with the action of an aromatic prenyl transferase, DMATrp synthase, which links a dimethylallyl chain to L-tryptophan. When the dmaW gene encoding DMATrp synthase was cloned and sequenced, the predicted product bore no identifiable resemblance to other known prenyl transferases. The dma W genes of Claviceps species are present in clusters of genes, several of which also have demonstrated roles in EA biosynthesis. In many other fungi, dma W homologues are identifiable in otherwise very different gene clusters. The roles of DMA Trp synthase homologues in these other fungi are probably quite variable. One of them is thought to prenylate the phenolic oxygen of L-tyrosine, and another catalyzes the unusual reverse prenylation reaction in the biosynthesis of fumigaclavine C(10), an EA characteristic of A. fumigatus. The second step of the EA pathway is N-methylation of DMATrp (12) to form 13, which is then subjected to a series of oxidation/oxygenation and reduction reactions to generate, in order, chanoclavine-I (16), agroclavine (19), and elymoclavine (6). Shunt reactions generate a wide variety of other clavines. Two epimerizations occur in this pathway: one from 12 to 16, the other from 16 to 19. Further oxidation of 6, catalyzed by the cytochrome-P450 CloA, generates lysergic acid (1). An unusual NRPS complex, lysergyl peptide synthetase (LPS), is responsible for linking 1 to three hydrophobic L-amino acids to generate the ergopeptide lactams. The LPS complex includes two polypeptides, one (LPS 2) possessing a single module for activation of 1, and the other (LPS 1) possessing three modules, each specifying one of the L-amino acids. Variations in LPS 1 sequences are associated with variations in the incorporated amino acids, leading to differences between strain chemotypes, and even multiple ergopeptines within strains. For example, C. purpurea P1 produces two distinct ergopeptines (ergotamine (4) and ergocryptine (Table I)), each of which is believed to be generated by multiple LPS 1 subunits encoded by separate, but related, genes (lpsA1 and lpsA2). The main ecological roles of EA in nature are probably to protect the fungi from consumption by vertebrate and invertebrate animals. The EA produced by plant-symbiotic fungi (such as epichloe endophytes) may protect the fungus by protecting the health and productivity of the host, which may otherwise suffer excessive grazing by animals. The EA, at levels typical of plants bearing these symbionts, can negatively affect the health of large mammals as well herbivorous insects. Some clavines have substantial anti-bacterial properties, which might protect the fungus and, in some cases, their host plants from infection. However, the fact that a large number of epichloe, and even several Claviceps species, produce no detectable EA indicates that the selection for their production is not universal. An unfortunate fact for many livestock producers is that some of the most popular forage grasses tend to possess EA-producing epichloe endophytes. Such endophytes are easily eliminated, but confer such fitness enhancements to their hosts that their presence is often preferred, despite the toxic EA. The future looks promising for continued interest in EA. Research continues into their pharmacological properties, medicinal uses, and structure-function relationships. New clavines and lysergic acid derivatives are identified regularly from new sources, such as marine animals. Also, programs are well underway to modify or replace epichloe endophytes of forage grasses in order to produce new grass cultivars that lack these toxins.

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus , a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea . We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW , the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus , and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea , as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3′-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similiarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxido-reductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway.

金沢大学大学院自然科学研究科生理活性物質科学, 金沢大学薬学部

The first pathway-specific step of ergot alkaloid biosynthesis in the fungus, Claviceps purpurea , is catalyzed by the prenyltransferase, 4-(γ,γ-dimethylallyl)tryptophan synthase. Partial sequence information was obtained for the purified enzyme and a degenerate oligonucleotide mixture was used to identify and amplify segments of the gene, dmaW . The complete gene and near-full-length cDNA were cloned and sequenced. The cDNA was cloned in a yeast expression vector in sense and antisense orientations relative to the inducible GAL1 promoter. Extracts of yeast transformants with the sense constructs, but not antisense constructs or cloning vector, catalyzed production of 4-(γ,γ-dimethylallyl)tryptophan. The sequence of dmaW and its cDNA indicated that it encoded a 455 amino acid polypeptide with a predicted molecular mass of 51,824 Da and a putative prenyl diphosphate binding motif.

Ergot alkaloids are mycotoxins produced mainly by the genera Claviceps, Aspergillus and Penicillium. Natural and semisynthetic ergot alkaloids are used as drugs in modern medicine. Chanoclavine-I aldehyde was found to be the branch point in the biosynthesis of different ergot alkaloid types. In Claviceps purpurea, it was converted to agroclavine by agroclavine synthase EasG in the presence of reduced glutathione, while festuclavine synthase FgaFS and the old yellow enzyme FgaOx3 from Aspergillus fumigatus catalysed the formation of festuclavine by two reduction steps. In this study, pyroclavine was detected as a minor product in the incubation mixture with enzymes from A. fumigatus under optimised HPLC conditions. Furthermore, we demonstrated that the homologues of FgaFS and FgaOx3 from Penicillium commune, FgaFSpc and FgaOx3pc, also catalysed the formation of pyroclavine and festuclavine, but with a significantly higher yield for pyroclavine. In addition, EasG was also shown to convert chanoclavine-I aldehyde to pyroclavine and festuclavine in the presence of the old yellow enzyme FgaOx3 or FgaOx3pc. More importantly, the ratio of pyroclavine to festuclavine was controlled by the reduction of an imine intermediate catalysed by FgaFS, FgaFSpc or EasG, which was proven by using different combinations of these enzymes in the presence of FgaOx3 or FgaOx3pc. The structure of pyroclavine was unequivocally elucidated by NMR and MS analyses.

Stipa robusta (= Stipa vaseyi) is a perennial grass found in certain areas of the southwestern United States. It is commonly known as sleepygrass, as horses that ingest this grass may become profoundly somnolent or stuporous for periods of time lasting up to several days. In an attempt to determine the active principle(s), fractionation of a methanolic extract of sleepygrass infected with an Acremonium endophyte has yielded lysergic acid amide (20 micrograms/g dry wt), isolysergic amide (8), 8-hydroxylsergic acid amide (0.3), ergonovine (7), chanoclavine-I (15), and N-formylloline (18). Related alkaloids have been found in many endophyte-infected grasses. The dominant alkaloid constituent in sleepygrass, lysergic acid amide, has not previously been identified in a grass in such high concentration. Lysergic acid amide is likely to be the basis for the extreme sedative effects on animals, given past pharmacological work on the compound from the ergot fungus Claviceps paspali.

Novel three synthetic routes to (±)-6,7-secoagroclavine were developed from either methyl 3-(3-formylindol-4-yl)acrylate, indole-4-carbaldehyde, or 4-iodoindole-3-carbaldehyde. The total syntheses of (±)-chanoclavine-I, (±)-chanoclavine-II, and (±)-agroclavine-I were accomplished as well from the synthetic intermediates involved in the synthesis of (±)-6,7-secoagroclavine, culminating in establishing an efficient and common synthetic method for ergot alkaloids.

Das 13C-angereicherte C-Atom der 4'-(E)-Methylgruppe im 4-(3-Methyl-2-butenyl)tryptophan (3) erscheint bei der Biosynthese in der C-Methylgruppe des Chanoclavins (4) und im C-17 von Agroclavin (5), Elymoclavin (6) und Lysergsaure (7). Incorporation of [4'-(E)-13C]4-(3-Methyl-2-butenyl)tryptophan into Clavine Alkaloids and Lysergic Acid The 13C-enriched C-atom of the 4'(E)-methyl groups of 4-(3-methyl-2-butenyl)tryptophan (3) appears during the biosynthesis at the C-methyl group of chanoclavine (4) and at C-17 of agroclavine (5), elymoclavien (6) and lysergic acid (7).

L-Tryptophan did not exert any influence on peptide alkaloid formation in an ergotamine and in an ergosine-accumulating C. purpurea strain. A different picture was observed in a series of related C. purpurea strains. Tryptophan showed a slight stimulatory effect on the ergotoxine producer Pepty 695/S. A blocked mutant of it, designated as Pepty 695/ch which was able to accumulate secoclavines gave similar results. In a high-yielding elymoclavine strain Pepty 695/e, the progeny of the former one, tryptophan up to a concentration of 25 mM stimulated remarkably clavine biosynthesis. Furthermore, tryptophan could overcome the block of synthesis by inorganic phosphate. Increased specific activities of chanoclavine cyclase but not DMAT synthetase were observed in cultures of strain Pepty 695/e supplemented with tryptophan. 5-Methyltryptophan and bioisosteres of tryptophan were ineffective in alkaloid stimulation. These results are compared with those obtained with the grass ergot strain SD 58 and discussed with the relation to other induction phenomena.

Panicum repens and P. antidotale were found to be infected with Claviceps sp. This is the first report of ergot on P. repens. The pyrenomycete produced abundant sclerotia on the host plants. The sclerotia contained 0.71 and 0.68% alkaloids, respectively, which predominantly consisted of chanoclavine, festuclavine and agroclavine. The infected grasses were possibly mycotoxic. Submerged cultures of Claviceps strain isolated from Panicum spp. produced significant amount of chanoclavine, festuclavine and agroclavine. No pharmaceutically important alkaloid was found in sclerotia or in submerged culture.

The racemic ergot alkaloids chanoclavine I ( 1 ) and 6,7-secoagroclavine ( 4 ) have been synthesized stereoselectively from indole-4-carboxaldehyde ( 7 ) in overall yields of 14 and 13%, respectively. Further syntheses of isochanoclavine I ( 2 ), paliclavine ( 5 ) and costaclavine ( 6 ), via the same isoxazolidine 18 are described. The key step 16 → 18 (Scheme 4) involves a transient nitrone 17 which undergoes a kinetically controlled, regio- and stereoselective intramolecular cycloaddition to a 1,2-disubstituted olefinic bond.

(E)-3-(1-Acetyl-1,5-dihydrospiro[benz[cd]indol-4(3H),2′-[1-3]dioxolan]-5-yl)-2-methylacrylsaureathylester (9) und ahnliche Benz[cd]indol-Derivate werden mit der Zielsetzung einer Chanoclavin-Synthese dargestellt. Synthesis of Benz[cd]indole Derivatives, III. Preliminary Experiments for a Synthesis of Chanoclavine Ethyl (E)-3-(1-acetyl-1,5-dihydrospiro[benz[cd]indol-4(3H),2′-[1-3]dioxolan]-5-yl)-2-methylacrylate and related benz[cd]indole derivatives are synthesized with the aim of a chanoclavine synthesis.

Ergot alkaloides were extracted from seed of native, wild-type morning glories (i.e. Texas red morning glory (Ipomoea hederifoliaL.), cypress vine morning glory (I. quamoclit L.), red morning glory (I. coccinea L.) and palmleaf morning glory (I. wrightii Gray) and quantified by spectrophotometry as ergonovine maleate equivalents. A horticultural variety (I. tricolor Cav. cv. Heavenly Blue) was utilized for comparison and contained 0.052% total alkaloids in the seed. Total ergot alkaloids in these four native, wild-type morning glories ranged from 0.003 to 0.005% (i.e. 5.97 to 9.77% of the alkaloids found in Heavenly Blue seed) and were not present in sufficient quantity to induce any psychotomimetic hazard. Major alkaloids found in these wild-type morning glory species were separated and identified by two-dimensional thin-layer chromatography and co-chromatography with authenticated standards. Alkaloids identified included chanoclavine, elymoclavine, penniclavine, ergonovinine, agroclavine, ergonovine, ergosine and ergosinine. Alkaloids varied between morning glory species.

The racemic alkaloids chanoclavine I (1) and isochanoclavine I (2) have been synthesized stereoselectively from indole-4-carbaldehyde (3) by a sequence of 11 operations in overall yields of 14% and 2.4%, respectively. The key step 6 8(Scheme 2) involves a transient nitrone 7 which undergoes a regio- and stereoselective intramolecular cycloaddition to a 1,2-disubstituted olefinic bond.

Triadimefon (TDF) (5 mg · liter−1) stimulated the functions of chanoclavine cyclase in submerged cultures of high producing mutant Claviceps purpurea 59 with partially blocked oxidation and cyclization of chanoclavine. The culture growth decreased and specific clavine production was raised. The effect of TDF on growth and production was reversed by plant oil (7.2 g · liter−1), but not by ergosterol (10 mg · liter−1). The oil stimulated growth and lowered both specific alkaloid production and chanoclavin cyclase function. Ergosterol showed the opposite effects. TDF reduced the total mycelial lipids to 66%, sterols to 40%, and phospholipids to 72%. In the cultures supplemented with ergosterol, the sterol content increased 16-fold and phospholipids were lowered to 27%; however, the total lipid content remained unchanged. Oil increased all lipid components. The sterol content was indirectly proportional to the PC PE ratio in all cultures. The membrane fluidity of protoplasts from TDF-treated cultures was higher, in ergosterol-treated cultures lower, and unchanged in the presence of oil. The content of methylated sterols in the TDF-treated cultures was only 2% higher than in controls. The demethylation of sterols in Claviceps occurs first in position C-14, then in C-4. The chanoclavine cyclase function probably does not depend on membrane fluidity, but a correlation between higher activity and low phospholipid content was found.

Clomiphene depressed the growth and enhanced clavine production of Claviceps purpurea strains 129,35 and 59. Mycelial content of 18:2 and 16:0 fatty acids decreased, whereas that of 18:1 and 18:0 acids increased. In the mutant strain 59 clomiphene, triadimefon and ergosterol stimulated the impaired function of chanoclavine cyclase. Their effect was counteracted by plant oil. Clomiphene decreased the content of total lipids (44%), triglycerides (32%), sterols (22%) and sterol/phospholipid molar ratio. The PC/PE ratio was 9X increased. Clomiphene and triadimefon enhanced membrane fluidity of protoplasts, ergosterol and oil reverted their effect.

Two endophytic clavicipitaceous fungi, Balansia epichloë and B. henningsiana, were isolated from several grasses, cultured on laboratory media, and assessed for their ability to produce the major ergot alkaloids agroclavine, chanoclavine (I), ergonovine, and ergonovinine. Data indicated that the ability of these two species of fungi to biosynthesize ergot alkaloids was host related. All isolates of B. epichloë from smut-grass (Sporobolus poiretii) and 59% of the isolates of B. henningsiana from broom-sedge (Andropogon virginicus) produced the major alkaloids in culture. All isolates of B. epichloë from Eragrostis secundiflora and E. hirsuta, and all isolates of B. henningsiana from Panicum tenerum failed to produce alkaloids. The total yield and chemical species of individual alkaloids varied among isolates from a sampling site.

Submerged cultures ofClaviceps purpurea 59 produce predominantly tricyclic chanoclavine and chanoclavine-I-aldehyde; formation of the tetracyclic agroclavine and elymoclavine is limited to 20%. The production cultures were characterized by the oxidation index oi (100% chanoclavine/% chanoclavine) and cyclization index ci (% agroclavine +% elymoclavine/% chanoclavine-I-aldehyde), expressing two functions of chanoclavine cyclase. Both indices were influenced by cultivation temperature and membrane agents. At 20°–24°C, clomiphene increased oi and ci; at 28°C and more it increased oi only. At 24°C nystatin increased oi and decreased ci. During cultivation of vegetative inocula and production cultures at various temperatures (24°, 28°, and 33°C), the lower cultivation temperature of the inoculum increased ci of the production culture with the higher temperature. The higher cultivation temperature of the inoculum decreased oi and particularly ci of the production culture with the lower temperature. In production cultures cultivated from the inoculum of an identical temperature, oi decreased to 50% with increasing temperature above 24°C, whereas ci decreased continuously. The role of the chanoclavine cyclase conformation as a membrane enzyme is discussed.

Four species of Balansia (clavicipitaceous systemic grass pathogens) isolated from pastures where cattle showed signs of ergot toxicity were grown in culture. Balansia epichloe, one isolate of B. claviceps, B. henningsiana and two isolates of B. strangulans produced conidia in submerged culture during the first stage of a two-stage fermentation procedure. When transferred to a glucose/sorbitol/inorganic salts medium during the second stage, these four species produced ergot alkaloids in stationary cultures. The transfer of fungi cultured in the first medium to the second medium was necessary for alkaloid biosynthesis. One isolate of B. claviceps did not produce alkaloids. Balansia epichloe produced chanoclavine (I), agroclavine, penniclavine, elymoclavine, ergonovine and ergonovinine. Balansia claviceps produced chanoclavine (I), ergonovine and ergonovinine. This is the first report of isolating ergonovine and ergonovinine, two lysergic acid derivatives, from fungi outside the genus Claviceps. Only chanoclavine (I) was identified from extracts of B. strangulans and B. henningsiana. Chanoclavine (I) and ergonovine were identified from smut grass (Sporobolus poiretii) parasitized by B. epichloe, indicating that this endophyte produces alkaloids both in vivo and in vitro.

Summary In the Claviceps strain SD58 the anthranilate synthetase was partially purified by fractionation with protamine sulfate and ammonium sulfate. The main activity (showing a 10–15 fold purification) was precipitated in the 30–50% (NH 4 ) 2 SO 4 fraction. The apparent Km values for the substrates chorismic acid, glutamine, NH 4 Cl and the cofactor Mg ++ are ~ 0,04, ~ 4, ~ 8–9 and ~ 0,4 mM respectively. The p H-optima for the glutamine-dependent reaction are p H 7,8 and p H 8,6 respectively. A stimulatory effect of univalent cations on the ASSase was observed with increasing order by K + > Na + , NH 4 +. The influence of bivalent ions was tested. Only Mn ++ - and Co ++ -ions can partially substitute the Mg ++ -ions. The enzyme was inhibited in the presence of tryptophane, chanoclavine, elymoclavine, indoleacrylic acid, prephenic acid and slightly activated in the presence of histidine. Histidine can not compensate the inhibition by tryptophan. The kinetic parameters of these inhibition- and activation reactions are described. In comparison to the strain SD58 the anthranilate synthetase from Claviceps purpurea , strain Pepty 695 (saprophytically alkaloids producing) and strain M5/95 F (saprophytically non-producing) was also studied. The properties of those enzymes are corresponding to the ASSase of the strain SD58.

The conversions of clavine-type alkaloids have been studied by the use of non-labeled alkaloids and the acetone-treated mycelia of the strains of the Claviceps as well as those of the Aspergillus, Penicillium and others which were productive of clavine-type ergot alkaloids. It was found that agroclavine could be converted to elymoclavine not only by ergot fungi but also by the fungus other than ergot fungi. This finding indicates that the theory presented by authors that elymoclavine is the primary product of the ergot alkaloids has become untenable to some extent. While, it was found that, besides the previous cyclic route, elymoclavine→lysergolI→lysergene→agroclavine→elymoclavine, another biosynthetic route from agroclavine to elymoclavine through lysergene, lysergol and an unestablished alkaloid PS might exist in various fungi on the one hand. This finding indicates that agroclavine rather than elymoclavine will be the primary product of the alkaloids, and that chanoclavine (secaclavine) will be a biosynthetic precursor of agroclavine, and therefore, of the remaining alkaloids. Judging from the data of Stauffacher et al., it was presumed that the reversible reaction, chanoclavine_??_agroclavine, which was found in all of the tested strains would be effected through a certain intermediary compound, possibly iso-chanoclavine-(I), and indeed this intermediary compound would be an obligatory precursor of agroclavine. The present finding indicates that the conversion reaction, elymoclavine→peptide-type alkaloids, cannot be effected through lysergol, but possibly through 6-methyl-Δ8, 9-ergolene-8-carboxylic acid which was discovered by Kobel et al. The outline of this study was given at the 3 rd International Symposium on “Biochemistry and Physiology of Alkaloids” held in Halle (Saale), June 24_??_27, 1965.

SUMMARY The emergence of a new stable strain of Claviceps purpurea is described. The strain formed the sphacelial stage very weakly on rye, and was unlikely therefore to be able to initiate an epidemic under field conditions, but the sclerotia contained chanoclavine as the major alkaloid in contrast to the ergotamine component of the parent strain. Isolates of C. purpurea derived from ryegrass ergots were also shown to parasitize rye.

The biogenetic interconversions of clavine alkaloids have been studied by the use of clavine-C14 and clavine-H3 alkaloids labeled biosynthetically in saprophytic culture, and four different ergot strains in saprophytic cultures. Agroclavine was shown to be oxidized irreversibly to elymoclavine. The attack appears to be directly on the methyl group. Agroclavine, but not elymoclavine, was shown to be converted irreversibly to the following clavine alkaloids: festuclavine, pyroclavine, and setoclavine. Elymoclavine was shown to be the precursor of lysergol, isolysergol, penniclavine, and isopenniclavine. These conversion reactions are irreversible. Only three clavine alkaloids were found to be metabolized in ergot strain 47 A, viz., agroclavine, elymoclavine, and lysergene, all of which have a double bond in position 8. Chanoclavine was shown not to be a precursor of the clavine alkaloids. Evidences indicate that it was not a breakdown product of agroclavine or of any other clavine alkaloid derived from agroclavine. Lysergene was shown to be converted to lysergol, isolysergol, penniclavine, and isopenniclavine. No evidence was found suggesting that lysergene is a normal intermediate in the biosynthesis of these compounds from elymoclavine. Each stereoisomer was found to be biosynthesized as such and not to be the result of a chemical or biochemical isomerization process.

The seeds of a number of commercially available varieties of Morning Glory ( Ipomoea and Convolvulus sp.) have been found to contain clavine and lysergic acid alkaloids. Using thin layer and paper chromatographic methods, ergine, isoergine, ergometrine, ergometrinine, elymoclavine, penniclavine, and chanoclavine have been tentatively identified. Not all varieties of seed tested contained these alkaloids: one which contained a substantial amount also contained alkaloid in the leaves and stem of the mature plant.

An isolate of Claviceps purpurea (Fr.) Tul. from sclerotia containing a large amount of polypeptide ergot alkaloids failed to produce alkaloid in vitro. After this isolate had been established in vivo on a rye host, sclerotial subisolates produced a new thick pigmented growth form which produced alkaloid in vitro, having a much greater dry weight than the parent and resembling the tissue of natural sclerotia. This growth form was readily lost during successive transfers in vitro, but was repeatedly recovered following re-establishment of the parasitic phase. Alkaloid production in submerged culture was also associated with a ‘sclerotial’ type of morphology. The constituent alkaloids produced in vitro (mainly Δ8–9 and Δ9–10 lysergic acids and chanoclavine) were consistently different from those produced in vivo (mainly ergo toxine and ergotamine).

From a rye ergot alkaloid fraction rich in chanoclavine we isolated three new crystalline alkaloids, each a stereoisomer of the known chanoclavine. Their configurations could be elucidated by NMR.-spectra. According to the nomenclature of the dihydro-d-lysergic acid series the isolated alkaloids are named: chanoclavine-(I) (formerly named chanoclavine), chanoclavine-(II), rac. chanoclavine-(II) and isochanoclavine-(I).

The reaction sequence tryptophan →chanoclavine-I → agroclavine → elymoclavine →lysergic acid derivatives was demonstrated in Claviceps. Incorporation of chanoclavine-I into ergoline derivatives ranged up to 40 per cent. No conversion of elymoclavine or agroclavine into chanoclavine-I could be demonstrated.

SUMMARY The constituent ergoline alkaloids produced in vitro by a Nigerian strain of Sphacelia sorghi have been identified as dihydroergosine, festuclavine, pyroclavine, dihydroelymoclavine, and chanoclavine. The same alkaloids were present in both the sphacelial stage and the sclerotia when S. sorghi parasitized florets of Sorghum vulgare. The Nigerian fungus appears to be quite different from certain oriental collections bearing the same name, and although forming stromatal initials, failed to develop the sexual stage. Mice successfully raised litters and showed no adverse response when fed on diets containing up to 50% of the ergot sclerotia. The ergot had also no effect on early pregnancy. Some of the alkaloid was excreted in the faeces, but, as injected alkaloid was also shown to be excreted in the faeces, this could have represented alkaloid which had been absorbed from the ingested sclerotia and re-excreted via the bile.

Summary For these investigations the following strains were used a) saprophytically alkaloid producing strains: Li 342 (C. paspali); SD 58 (C. fusiformis) and Pepty 695 (C. purpurea), b) saprophytically non-producing strains: M5/95F and Jammu (C. purpurea). During the course of fermentation the activity of some enzymes of the aromatic pathway was investigated, especially DAHP synthetase, chorismate mutase, prephenate dehydrogenase, prephenate dehydratase, anthranilate synthetase and tryptophan synthetase. The DAHP synthetase levels were considerably higher in producing strains compared with those not able to produce alkaloids. During the transition from growth to alkaloid formation in appropriate strains there was a sharp increase in the levels of anthranilate synthetase and tryptophan synthetase. After the maximum was reached the activity dropped markedly. The influence of various effectors on DAHP synthetase and chorismate mutase activity using purified extracts was studied. DAHP synthetase was inhibited to a different extent by L-tryptophan, L-phenylalanine and L-Tyrosine. Inhibition was also caused in strain SD 58 by chanoclavine and elymoclavine. A slight activation was observed after adding dimethylallyltryptophan, L-tryptophan activated chorismate mutase. The most pronounced effect was seen in strain SD 58. Only a slight inhibition was caused by L-phenylalanine and L-tyrosine depended on the strain used. The same effect showed prephenic acid.