BIOSYNTHESIS OF INDOLE-DITERPENOIDS

AND OTHER ISOPRENOIDS BY THE

RYEGRASS ENDOPHYTE, Acremonium loliae

A thesis presented in fulfilment of the requirements for

the degree of Ph.D. of the University of London

and the Diploma of Imperial College.

CHRISTOPHER MICHAEL VEEDON

1987

Department of Biochemistry

Imperial College of Science and -Technology

London SV7 2AY 2

ABSTRACT

Acremanium loliae, the ryegrass endophyte, is implicated in causing ryegrass staggers (RGS) - a neurological syndrome of ruminants grazing ryegrass-dominant pastures, particularly in New Zealand.

Tremorgenic substituted indole-diterpenoids, lolitrems A-D, isolated from A. loliae-infected perennial ryegrass iLolium perenne), are incriminated as the toxins responsible for RGS. However, the precise biosynthetic contribution to lolitrems made by the fungus and the grass is unknown, and the principal objective of this study has been to explore this, with the long-term aim of divising a strategy to avoid RGS.

Although lalitrems were never detected in culture, A. loliae was shown to biasynthesise the closely related indole-diterpenoid tremorgen, paxilline. Paxilline was produced in certain culture conditions, long after growth was complete, and was detectable in culture only for a few hours on one day. Paxilline was also found, for the first time, in endophyte-infected ryegrass seed containing lolitrem B (the most abundant lolitrem). Direct transformation of

1^C-paxilline to lolitrem B could not be demonstrated, either in A. loliae culture or in endophyte-infected ryegrass seedlings.

Nevertheless, paxilline remains a potential biosynthetic intermediate of lolitrems, implying a major role for A. loliae in the biosynthesis of lolitrems.

A. loliae> as well as the main paxilline-producing fungus,

Penicillium paxillir was shown to biosynthesise several indole- diterpenoids, closely related in structure to paxilline. These include one, that could be a biosynthetic precursor of paxilline, and another that is a paxilline isomer. 14Qf-hydroxypaxilline was also isolated and 3 was shown to arise directly from 1 'C-paxilline given to A. loliae cultures. It is postulated that lolitrem A, which has not formerly been characterized further than oxo-lolitrero B, may be 1 AO£r hydroxylolitrera B. 14G*-hydroxylatian of paxilline gave much reduced tremorgenic activity and is the first recorded 19G*-hydroxy indole- diterpenoid that is not a \ patent tremorgen. Furthermore, the new compounds may allow some revision of the indole-diterpenoid biosynthetic pathway as applied to paspalicine and the paspalinines of

Claviceps paspali.

Mutagenic treatment of A. loliae led to the isolation of a variant strain, that reproducibly yielded paxilline, whether or not in the special culture conditions found necessary for production by the native endophyte. Detailed characterization of the mutant remains to be carried out. Nevertheless, the feature of consistent paxilline biosynthesis might greatly facilitate the screening for a strain incapable of paxilline biosynthesis, following mutagenic treatment.

When re-introduced into ryegrass, such a non-tremorgenic endophyte would theoretically be incapable of causing RGS but might still confer the benefits of native A . loliae.

The principal sterol of A. loliae was 5ctf-ergosta-7,22E-dien-

3/3-ol, which is sufficiently uncommon that it can,not only provide a biochemical means of differentiating from the closely related tall fescue endophyte A . coenophialum} but may also enable detection of A. loliae in ryegrass.

The indolic metabolite tryptophol was found to be a major constituent of A. loliae culture filtrate and, as a growth promoter of certain plants, may account for the enhanced vigour conferred d h ryegrass by A, loliae. 4

CONTENTS

PAGE

ABSTRACT ...... 2

CONTESTS ...... 4

INDEX OF FIGURES...... 6

INDEX OF T A B L E S ...... 10

ACKNOWLEDGEMENTS . . . '...... 12

1. INTRODUCTION...... 13

2. MATERIALS AND METHODS

2.1. Fungal Culture

2.1.1. Source of Fungi ...... 29

2.1.2. Media ...... 31

2.1.3. Inoculation and Incubation ...... 33

2.1.4. Microscopy ...... 34

2.1.5. Culture Growth Rate Assessment ...... 35

2.2. Sample Component Analysis

2.2.1. Sample Preparation ...... 35

2.2.2. Chromatography ...... 36

2.2.3. Spectroscopy ...... 40

2.3. Use of Metabolic Precursors

2.3.1. Addition and Incubation of 1AC-Radiolabelled

Mevalonic Acid and Amino A c i d s ...... 40

2.3.2. Addition and Incubation of Paxilline ...... 41

2.3.3. Autoradiography ...... 43

2.4. Mutagenic Treatment of A. l o l i a e ...... 43 3 . RESULTS

3.1. Confirmation of Identity of Isolated Ryegrass Endophyte

as Acremonium l o l i a e ...... 44

3.2. A . loliae Culture in Optimum Growth Conditions

3.2.1. Determination of Optimum Growth Conditions .... 43

3.2.2. Products of A,loliae in Optimum Growth Conditions . 51

3.3. Preparation of 1^C-Radiolabelled Paxilline

3.3.1. Penicillium paxilli Culture ...... 82

3.3.2. Additional Compounds Isolated from P. paxilli

Culture...... 91

3.4. Pectin-Supplemented A. loliae Culture ...... 115

3.5. Incubation of Paxilline with A. loliae

3.5.1. Incubation of Paxilline with A. loliae in Optimum'

Growth-Rate Conditions ...... 125

3.5.2. Incubation of 1^C-Paxilline with A. loliae

in Paxilline Production Medium ...... 131

3.6. Tremorgen Investigation in Lolium perenne

3.6.1. Examination of Ryegrass Seeds ...... 139

3.6.2. Introduction of 1^C-Radiolabelled Paxilline

into A. loliae-Infected Seedlings ...... 139

3.7. Administration of Indole-Diterpenoids to M i c e ...... 144

3.8. Mutagenic Treatment of A. lo l i a e ...... 144

4. DISCUSSIOM...... 147

5. REFERE5CES...... 151 6

IflDEX OF FIGURES

PAGE

Figure 1. The Indole-Diterpenoids ...... 17

Figure 2. Peramine...... 19

Figure 3. Phaseolin...... 19

Figure 4. Verruculogen, 15-Acetoxyverruculogen and

Fumitremorgin A ...... 24

Figure 5. Proposed Mechanism of Cyclization of Geranyl

Geranyl Pyrophosphate in Indole-Diterpenoid

Biosynthesis...... 26

Figure 6. Proposed Mechanism of Attachment of an Isoprenoid

Moiety to the Indolic Benzene Ring of Indole-

Diterpenoids ...... 27

Figure 7. Proposed Mechanism of Lolitrem B Biosynthesis via

Paxilline...... 28

Figure 8. Emergence of Mui Endophyte from Ryegrass Seedling . . . 30

Figure 9. Cultures of isolated Nui endophyte and a typical

strain of A. l o l i a e ...... 44

Figure 10. Effect of Incubation Temperature on A, loliae Growth . 49

Figure 11. The Major Acetone-Soluble Products of A. loliae .... 54

Figure 12. Thin Layer Chromatography of Acetone-Soluble

Products of A. l o l i a e ...... 56

Figure 13. High Performance Liquid Chromatography of the

Major Acetone-Soluble Products of A . loliae ...... 58

Figure 14. Electron Impact Mass Spectroscopy of the Major

Acetone-Soluble Products of A. loliae ...... 62 7

Figure 15. Autoradiographs and Thin Layer Chromatographs of

Acetone Extracts of A. loliae Cultures after

Incubation with 1AC-Radiolabelled Primary

Metabolites...... 71

Figure 16. P. paxilli C u l t u r e...... 85

Figure 17. Paxilline Standard Curve ...... 86

Figure 18. Time-Course of Paxilline and Biomass Yield in P.

paxilli Culture ...... 88

Figure 19. Autoradiographs and Thin Layer Chromatographs of

Acetone Extracts of P, paxilli Cultures After

Incubation with 1AC-Mevalonate and 1^C-Benzene

Ring Tryptophan...... 89

Figure 20. The Indole-Diterpenoids of A. loliae and P,

p a x i l l i...... 91

Figure 21. Thin Layer Chromatography of Acetone Extracts of

A. loliae and P. paxilli Cultures...... 92

Figure 22. High Performance Liquid Chromatography of the

Indole-Diterpenoids of A. loliae and P. paxilli .... 93

Figure 23. El Mass Spectrum of Paxilline (70eV)...... 95

Figure 24. 1HMMR Spectrum of Paxilline (DMSO, 500MHz> ...... 97

Figure 25. Proton-Proton Couplings and Full Stereochemistry

of Paxilline...... 99

Figure 26. El Mass Spectrum of Prepaxilline-16/3-ol (70eV) .... 100

Figure 27. 1HNMR Spectrum of Prepaxilline-16/3-ol CDMSO,

500MHz)...... 102

Figure 28. Proton-Proton Couplings and Full Stereochemistry

of Prepaxilline-16j^-ol...... 101

Figure 29. El Mass Spectrum of 140Phydroxyprepaxilline

(70eV)...... 104 8

Figure 30. 1 HNMR Spectrum of 14Qf-hydroxyprepaxi 11 ine (DMSO,

500MHz)...... 106

Figure 31. Proton-Proton Couplings and Full Stereochemistry

of 14QJ-hydroxypre paxilline...... 108

Figure 32. El Mass Spectrum of 14C£-hydroxypaxilline (70eV).... 109

Figure 33. 1HNKR Spectrum of 14Q£-hydroxypaxilline (CDCl--.,

250MHz)...... Ill

Figure 34. Proton-Proton Couplings and Full Stereochemistry

of 14Qf-hydroxypaxilline...... 113

Figure 35. Effect of Agitation and Inoculum on A. loliae

Morphology in G P Y E ...... 119

Figure 36. HPLC Paxilline Analysis of a Single A . loliae

Culture on Consecutive Days...... -. 122

Figure 37. Autoradiograph and Thin Layer Chromatograph of

Acetone Extracts of A. loliae Mycelium and

Culture Filtrate, After Incubation with 1^C-

Paxilline in a GPYE Culture...... 126

Figure 38. Thin Layer Chromatograph of Acetone Extracts of

A. loliae Mycelium and Culture Filtrate, After

Incubation with Paxilline in GPYE Cultures for

Periods of 24 Hours on Consecutive Days

(Including Time-Course of Main A. loliae Acetone

Soluble Products) ...... 128

Figure 39. Time-Course of Mycelium and Culture Filtrate Dry

Weights of A. loliae After Incubation with

Paxilline in GPYE Cultures for Periods of 24

Hours on Consecutive Days...... 127 9

Figure 40. Autoradiograph and Thin Layer Chromatograph of

Acetone Extracts of A. loliae Mycelium and

Culture Filtrate After Incubation with 1 **C-

Mevalonate and Non-Radiolabelled Paxilline in

GPYE Culture...... 130

Figure 41. Autoradiographs and Thin Layer Chromatographs of

Acetone Extracts of A. loliae Mycelium and

Culture Filtrate After Incubation with 1AC-

Paxilline in Paxilline Production Medium ...... 134

Figure 42. HPLC of 2:1 CHCls/methanol Extract of Ware

Ryegrass Seeds ...... 140

Figure 43. Autoradiographs and Thin Layer Chromatographs of

Extracts of L. perenne Seedlings After Incubation

with 1 AC-Paxilline...... 142

Figure 44. Thin Layer Chromatograph of Acetone Extracts of

A. loliae UV Survivor and Native A . loliae

Mycelium and Culture Filtrate After Incubation in

G P Y E ...... 145

Figure 45. Proposed Pathway of Hydroxylation in the

Formation of Indole-Diterpenoids of A. loliae . . . 157 10

INDEX OF TABLES

PAGE

Table 1. Comparison of Isolated Ryegrass Endophyte with

Authentic A. l o l i a e ...... 46

Table 2. Effect of Carbon and Nitrogen Source, and Pre-

Sterilization Medium pH on A. loliae Biomass

Accumulation ...... 47

Table 3. *4. loliae Optimum Growth Conditions...... 48

Table 4. Penicillium paxilli Culture Characteristics and

Paxilline Production in Different Culture Media .... 83

Table 5. El Mass Spectral Data of Paxilline...... 96

Table 6. 1HNMR Data of Paxilline in D M S O ...... 98

Table 7. El Mass Spectral Data of Prepaxilline-16j0-ol...... 101

Table 8. 1 HNMR Data of Prepaxilline-16/3-ol in D M S O ...... 103

Table 9. El Mass Spectral Data of 14Q£-hydroxyprepaxilline .... 105

Table 10. 'HNMR Data of 140f-hydroxyprepaxilline in D M S O ...... 107

Table 11. El Mass Spectral Data of 140f-hydroxypaxilline ...... 110

Table 12. 'HNMR Data of 14Qf-hydroxypaxilline in C D C I 3 ...... 112

Table 13. Effect of Pectin : GPYE Ratio on A. loliae

Longevity, Morphology and Metabolite Production .... 114

Table 14. The Effect of Pectin : Glucose Ratio on Paxilline

Production by A. l o l i a e ...... 116

Table 15. Effect of Pectin Sterilization Method and Glucose

Inclusion on Paxilline Production ...... 117

Table 16. Effect of Amount of Fully-Grown Inoculum on

Morphology and Viability of A . loliae in GPYE .... 118 11

Table 17. Cultural Conditions Employed to Follow Paxilline

Production Time-course but in which Paxilline was

Never Detected...... 121

Table 18. Paxilline and 14C£-Hydroxypaxilline Production

Time-courses ...... 120

Table 19. Time-course of Paxilline Transformation by A.

loliae in Optimum Growth Conditions ...... 124

Table 20. Transformation of Paxilline after Incubation in A.

loliae GPYE Cultures of different Ages...... 129

Table 21. Concentrations of Paxilline and 14QP-

hydroxypaxilline in Endophyte Culture to which had

been added 1^C-Radiolabelled Paxilline at

Different Stages...... *. 132

Table 22. Diagrammatic Summary of Figure 3 2 ...... 138

Table 23. Occurrence of Paxilline in Cultures of A. loliae

UV Survivors...... 146 12

ACKNOWLEDGEMENTS

This period of research has been particularly enjoyable to me,

owing largely to the good nature of many people in the Life Sciences

Division and Chemistry Department of Imperial College; to all of them

I offer my thanks.

My special thanks are to Dr P.G. Mantle, for his consistent

enthusiasm and outstanding supervision; Mrs S.E. Yeulet-North, for

endless, constructive discussion and technical assistance, and for her

infectious brightness and thoughtfulness (and for the use of a word-

processor!); Mr J.N. Bilton, whose relentless work on mass

spectrometry and structure elucidation has run far beyond the call of

duty; Mr R.N. Sheppard for NMR spectroscopy of the highest standard;

Mr G. Millhouse for photographing and printing all the autoradiographs; and Ms D.E. Frederickson for her invaluable help with

micro-photography.

My grateful acknowledgement also goes to the SERC for funding

this research.

This thesis is dedicated to Barry, my father, whose discussions

with me, over the odd pint of natural products perhaps, have

contributed immeasurably to the quality of my work; and to Penny

Holmes, my fiancee, who has offered me the priceless gift of stability

two ’seers of Quality' who seem in continual, respectful,

conflicting agreement with each other.

No thought, no reflection, no analysis, No cultivation, no intention, Let it settle itself.

(The six precepts of Tilopa.)

There is always an alternative approach. 13

1. IHTRODUCTIOH

Ryegrass staggers (RGS> Is a neurological syndrome of ruminants grazing certain perennial ryegrass (Lolium perenne L. ) pastures, notably in New Zealand. Typical acute symptoms include pronounced incoordination on rapid movement, culminating in collapse (Cunningham and Hartley, 1959); head or whole-body tremor is also evident. The disorder regresses within several days of moving to different pasture.

RGS is closely associated with potent neurotoxins (lolitrems) in ryegrass that is also infected with an endophytic fungus, ryegrass endophyte (Mortimer et al., 1982). Good correlation between occurrence of lolitrems and the presence of endophyte has been established but the extent of involvement of the fungus and the grass in lolitrem biosynthesis is unknown. The principal objective of the present study has been to explore this question, approached mainly from a hypothesis that the endophyte plays a major role in the biosynthesis of lolitrems.

RGS affects mainly sheep but also cattle, horses and occasionally wapiti deer (Gilruth, 1906; Cunningham and Hartley, 1959;

Mackintosh et al.t 1982). Although the syndrome shows highest incidence in New Zealand, it also occurs sporadically in Australia, the USA and UK (Gilruth, 1906; Munday and Mason, 1967; Shaw and Muth,

1949; Clegg and Watson, 1960). It appears only during late summer and autumn.

Although the predominance of perennial ryegrass is essential, the nature of pastures on which RGS has been recorded varies considerably. For example, the pasture may be seeding, lush and leafy 14 or very short and consisting mainly of dead material (Gilruth, 1906;

Cunningham and Hartley, 1959; Mortimer et al. , 1984; Armstrong, 1956).

A particularly careful study (Keogh, 1973) demonstrated conclusively that sheep crazing the lower 2.5cm of the grass were very much more

likely to develop symptoms than were animals eating the upper parts.

The syndrome arises owing to perturbation of the CNS, probably involving biochemical changes in nerve impulse transmission (Mortimer

et al., 1982), disturbing the gross motor coordination of the animal.

Ho consistent histological changes have been observed in naturally- affected animals (Cunningham and Hartley, 1959; Clegg and Watson,

1960; Munday and Mason, 1967) and symptoms are reversible; the mode of action of the causative agent is certainly subtle.

In sheep, mildly affected animals exhibit only slight incoordination when farced to run 50 to 100 metres (Cunningham and

Hartley, 1959). In the most severe instances animals may be recumbent with pronounced tetanic, muscular spasm, neck extended and legs rigid.

Therefore, although RGS is not directly fatal, it does lead to deaths from accidents such as drowning, or being trampled by the mob or in transit. RGS is thus of some economic significance on account of mortalities and is especially troublesome in making stock management difficult - several thousand animals may habitually be managed by ' a man and dog' in hill-country terrain.

The cause of RGS has been elusive since the syndrome was first reported at the beginning of this century (Gilruth, 1906). However, in

1981 a strong correlation between incidence of the disease and the presence of an intercellular fungus in perennial ryegrass was noticed

(Fletcher and Harvey, 1981). This finding was subsequently thoroughly corroborated (Mortimer et al. < 1982; Fletcher, 1983; Mortimer et al. ,

1984). The fungus itself was probably first observed by McLennan

(1920). Subsequent studies revealed that its natural existence is 15 confined to perennial ryegrass. It is found between, or occasionally inside cells (Philipson and Christey, 1986), and is generally systemic throughout the plant, although predominantly in the region 2cm above ground level (Musgrave, 1984), living as an obligate biotroph (Siegel et al. , 1987).

Although it has been suggested that dissemination of the endophyte is potentially "horizontal" - through vegetative propagation of the grass in which the fungus is systemically distributed (Sampson,

1935) - it is probable that the only (Latch, 1983) means of natural spread is "vertical" - via the grass seed. The position of the endophyte in vegetative tillers is mainly in the region 0-5cm above ground. Contrastingly, in reproductive tillers the endophyte is found predominantly in the flowering stem (Musgrave and Fletcher, 1984)'.

This is possibly a reflection of the fact that the endophyte maintains a position in or near the meristematic apical growing points of the plant, which ensures its presence between, and at times inside, the cells of each ovary as it develops (Philipson and Christey, 1986).

Consequently, seed-borne dissemination is made possible. Fertilization thus leads to formation of seeds containing the fungus - up to 20% w/w of the seed may be due to the endophyte (Musgrave, 1984). The bulk of the endophyte exists in a region between the aleurone layer and the pericarp/testa (McLennan, 1920; Philipson and Christey, 1986) but the most important location, with respect to effective dissemination, is in the embryo. As the embryo grows and differentiates the endophyte enters the shoot apex. Therefore, seed distribution and germination in the usual manner result in progeny containing endophyte. The fungus is thus able to persist from one generation to the next without ever leaving the plant or producing spores.

/ 16

The rare incidence of reproductive structures in axenic culture hindered classification of the endophyte until, in 1984, Latch et ai. described a sparing culture and named the ryegrass endophyte

Acremonium loliae Latch, Christensen and Samuels. The fungus is distinct from all other Acremonium species, except A. coenophialum

Morgan-Jones and Gams, by having no septa at the base of its conidiogenous ceils, periclinal thickening absent at the tip of such cells (which are either closed or bear a single developing conidium) and also by there being only one conidium, or rarely two, transversely positioned, at the tip of the conidiogenous cell.

At about the same time that the correlation between A. loliae and RGS was observed, a group of potent neurotoxic substances was identified in perennial ryegrass (Gallagher et al., 1981). Like the fungus, the toxins correlated closely with the occurrence of RGS

(Mortimer et al., 1982). They were named lolitrem A, B, C and D, and the structures of B and C have been fully elucidated (Gallagher et al>> 1984) - see Figure 1. The most abundant is lolitrem B, which was subsequently shown to be both qualitatively and quantitatively linked with A. loliae in ryegrass (Mortimer et al., 1982).

One year after the finding of lolitrems in ryegrass, the additional discovery was made that A. loliae was responsible for the natural resistance of L. perenne to certain insect pests (Prestidge et al., 1982; Funk et al., 1983; Barker et al., 1983; Gaynor et al,,

1983). Subsequently, an insect-antifeedant, peramine (Figure 2), was isolated from A. loliae-iniected ryegrass and shown to be produced by the endophyte in axenic culture (Rowan et al., 1986). However, prior to the commencement of the present study, A. loliae had not been shown to produce any biologically active compounds, including indole- diterpenoids (such as the lolitrems).

k 1 7

Figure 1. The Indole-Diterpenoids (T = tremorgen)

Ri Re R:

Paspalicine H H H Paspali nine1' OH H H Paspalinines* Paspalitrem AT OH -CHe-CH=C(CH3);2 H Paspalitrem BT OH -CH=CH-COH(CH3>e H Af latrem1" OH H •C(CH:,-

Aspergillus flaws (Wilson and Wilson, 1964). Claviceps paspali (Fehr and Acklin, 1966; Cole et al., 1977a).

Paxil line1"

<•= position of 1 AC when radiolabelled.) Penicillium paxilli (Cole et al, , 1974). Emericella striata, E. desertorum (Seya et al.f 1986). 18

Paspaline

Claviceps paspali (Fehr and Acklin, 1966; Gysi et al., 1973; Leutwiler, 1973).

,0H

Janthitrem ET H OH Janthitrem FT -CO-CHa OH Janthitrem Gr -CO-CHa H

Penicillium janthinellum (de Jesus et al., 1984).

Penitrem AT Cl OH (17a, 180f-epoxide) Penitrem BT H H (17a, 180-epoxide) Penitrem CT Cl H Penitrem DT H H Penitrem ET H OH (17a, 18a-epaxide) Penitrem FT Cl H (17a, 18Qf-epoxide)

Penicillium cyclopium CWilson et al.t 1968). P. aurantiogriseum> P. granulatum> P. viridicatum (Ciegler and Pitt, 1970). P. janczewskii (Mantle et al., 1978). P. crustosum (Pitt, 1979; de Jesus et al. , 1983a-c; Mantle et al., 1983; ). P. duclauxii (Patterson et al. , 1979). P. puberulum (Vagener et al., 1980). 1 9

Figure 2. Peramine

Acremonium loliae (Rowan et al., 1986).

Figure 3. Phaseolin

Phaseolus vulgare (Mann, 1978). 2 0

Nevertheless, the finding' that lolitrem B can be isolated from ryegrass only if the grass is infected with A, loliae, implied involvement of the fungus in the biosynthesis of these tremargens. Yet detailed knowledge of the relationship between A. loliae and lolitrem biosynthesis remained obscure. There are three possibilities for the biosynthesis of lalitrems: solely by the hast plant in response to presence of the endophyte, solely by the endophyte, or jointly by the host plant and the endophyte (both contributing to the molecule).

The first possibility is the only one that does not include contribution of a biosynthetic precursor by the endophyte, and would be analogous to the production of phytoalexins by higher plants in response to invasion by parasitic fungi. The biological activity of phytoalexins - such as phaseolin (Figure 3) from the bean — is as antifungals; and, like lolitrems, they are meroterpenoids and polycyclic.

There is no evidence, however, that lolitrems are antibiotic.

Furthermore, far from being harmful, A. loliae seems to be beneficial to the host plant. Apart from insect feeding-deterrency, A. loliae confers the additional advantages of substantially increasing ryegrass growth and persistence. Leaf, pseudostem and root yields, leaf area, and tiller number are all considerably greater in A, loliae-infected grass compared to its clonal, uninfected counterpart (Latch et al. ,

1985). Similarly, recovery from summer climatic pressures is more rapid, and resistance from invading weeds more effective, in infected plants (Funk et al., 1985). These additional benefits may be caused by an activity of A. loliae to behave in a manner reminiscent of mycorrhizae, providing the host with otherwise inaccessible nutrients.

However, at least in the case of increased growth vigour, the question has been raised concerning the possibility of production, by A. loliae, of a plant growth-promoting Mhormoneu (Latch et al., 1985). 2 1

From the important differences between the lolitrems and. phytoalexins, and A. loliae and phytoalexin-inducing fungi, it seems that the possibility of production of lolitrems being solely by the plant can be ruled out.

The other two possibilities concerning the origin of lolitrems propose a direct biosynthetic involvement of A. loliae in producing either all or part of the lolitrem molecule. To resolve which is correct, a thorough understanding of the biosynthesis, structure and biological properties of the lolitrems is essential. To this end it is relevant to consider a related neurological syndrome of ruminant livestock - paspalum staggers - which also results indirectly from a parasitic fungus/grass interaction.

Paspalum staggers CPS) occurs mainly in cattle, but also in sheep and horses, grazing flower-heads of paspalum grass CPaspalum dilatatum Pair, and/or P. distichum L.), heavily infected with scleratia (ergots) of Claviceps paspali Stevens and Hall (Cole et al. ,

1977a; Mantle et al., 1978). The syndrome occurs in countries where

Paspalum species form a major pasture constituent, including New

Zealand, Australia, South Africa, Italy, Portugal, the USA, and sub­ tropical countries of South America. Symptoms are essentially indistinguishable from RGS, often occurring annually during about the same season.

The strong implication of ergots in the etiology of PS led to the isolation from the ergots of the causal agents (Cole et al,,

1977a; Mantle et al., 1978) and their identification as paspalinines

(see Figure 1). This established that a fungus can be the sole producer of potent toxins causing a natural staggers syndrome.

The striking similarity between the symptoms and occurrence of

RGS and PS compels suspicion of analogous causation. That is, since a fungus causes PS by producing paspalinines, perhaps RGS is also caused 2 2

by a fungus, producing toxins similar in structure and activity to paspalinines. If so, then A. loliae may well be the fungus and

lolitrems the toxins. Clearly, such an argument is strongly in favour

of a fiTngal role in lolitrem biosynthesis, whether the fungus is in

culture or is in association with ryegrass.

The question of the biological origin of lolitrems may also be

addressed by considering the origins of all of the compounds closely-

related in structure. The paspalinines, for example, are markedly

similar to the lolitrems. They are all derived from just two

biomolecular precursor types: an indole moiety and several penta-

carbon units. The 5-C units derive from isopentenyl pyrophosphate

(IPP) and/or dimethylallyl pyrophosphate (DMAPP), both of which are

structurally similar to isoprene (2-methylbuta-l,3-diene). End to end

linkage of two such isoprenoid units forms a "monoterpene" moiety from

which diterpenoids are farmed by chain extension with IPP.

Biosynthetic linkage with an indole nucleus then forms "indole-

diterpenoids" .

Indole-diterpenoids include (in order of increasing structural

complexity): paspaline, paxilline, paspalicine, paspalinines

including paspalitrems), aflatrem, janthitrems, penitrems and

lolitrems (see Figure 1 for structures). These differ in the types and

means of attachment of the functional groups bound to the indole and

diterpenoid moieties. The majority of indole-diterpenoids possess a

tertiary hydroxyl group at the 19 position, thought to be necessary

for their tremorgenicity (Moss, 1979); these are the indole—

diterpenoid tremorgens. It is of considerable significance that all indole- diterpenoids, of which the biosynthetic source is proven, are produced only by fungi, and only by the genera Claviceps, PeniciIlium and

Aspergillus. Since lolitrems contain the same indole-diterpenoid carbon skeleton, they too would appear to be of fungal origin.

Furthermore, the functional similarity between the lolitrems and other indole-diterpenoid tremorgens is also evidence in support of a fungal origin for lolitrems. An essential factor in the

incrimination of paspalinines in PS causation was that parenteral administration of the pure tremorgens into mammals (Cole et ai.,

1977a), or ingestion of sclerotia by sheep and cattle (Mantle et al,>

1978), led to symptoms identical to those of the natural syndrome.

Similarly, ingestion by mammals of ryegrass seed material containing

lolitrem B (Gallagher et al., 1982; Munday et al.} 1985) caused symptoms indistinguishable from RGS. Moreover, intraperitoneal administration of pure lolitrem B to mice caused a similar response and, remarkably, greatly prolonged effects compared with other

tremorgens (Gallagher and Hawkes, 1986). The fact that paspalinines

and lolitrems experimentally cause very similar symptoms reinforces

the hypothesis that lalitrems are of fungal origin.

Thus a strong prima facie case is made for A. loliae, rather

than the perennial ryegrass host, being the main, if not the only,

biosynthetic source of lolitrems and, therefore, for focusing

investigations chiefly on the endophyte.

The structural analogies between lolitrem and other indole-

diterpenoid molecules may well imply similarity of biosynthetic

mechanisms. Thus by considering the manner of formation of other

indole-diterpenoids it may be possible to postulate a mechanism for

lolitrem biosynthesis. In so doing, information might be gained

concerning the structures of passible biosynthetic intermediates. 2 4

i

Figure 4. Verruculogen, 15-Acetoxyverruculogen and Fumitremorgin A

Ri R:

Verruculogen H H 15-Acetoxyverruculogen -QCO-CH3 H Fumitremorgin A H -che-ch=<

Penicillium verruculosum; P. paraherquei\ P. piscarium, P. janthinellum; Aspergillus fumigatus; A. caespitosus (Cole et al.t 1972; Yashizaga et al., 1976; Cole et al., 1977b; Cole et al., 1977c; Gallagher and Latch, 1977; Yamazaki et al., 1979; Schroeder et al., 1975; Yamazaki et al., 1971). 25

Studies employing ' ':C- and ' -'C-precursor administration in two different fungi producing indole-diterpenoids have confirmed the origin of the indole nucleus from tryptophan (de Jesus et al., 1983a) and each C atom of the diterpenoid moiety from geranyl geraniol (Cole et al.% 1977a and references cited therein). Thus it has been passible to devise mechanistic schemes for the biosynthesis of paspaline, paspalicine, paspalinine (Acklin et al. , 1977) and paxilline

(Yamasaki, 1980). These proposals venture no suggestions as to intermediates in either the transformation of paspaline to paspalinine or the equivalent transformation to paxilline. Figure 5 shows a possible scheme of paspaline and paxilline biosynthesis, based on these proposals and classical mechanisms of organic synthesis.

Isoprenoid attachment to the indolic benzene ring also exists in several compounds related to lolitrems, including the derivatives of paspalinines - paspalitrems A and B (see Figure 1). A proposed mechanism of such attachment is shown in Figure 6 . Similar reactions are likely in the analogous attachments of other indole-diterpenoids such as aflatrem and penitrems. The presence of an isoprenoid unit bound to the diterpenoid moiety is unique to lolitrems, although some non-indole-diterpenoid fungal tremorgens, ie the verruculogens and fumitremorgin A (Figure 4), contain a similar isoprenoid unit bound to two hetero-atoms, as is the case in lolitrems. Many plant compounds, such as phaseolin (Figure 3), have an isoprenoid attached to a single

0 atom.

On the basis of Figures 5 and 6 , and the analogous processes projected for the formation of other tremorgens, the mechanism of biosynthesis of lolitrem B may be postulated as that shown in Figure

7. Significantly, one - and only one - indole-diterpenoid tremorgen, paxilline, may thus be regarded as a possible intermediate in lolitrem formation. 26

Figure 5. Proposed Mechanism of Cyclization of Geranyl Geranyl Pyrophosphate in Indole-Diterpenoid Biosynthesis

'O' *= after Tanabe (see Yamazaki, 1980); Paxilline possibly occurs via paspaline 2 7

Figure 6 . Proposed Mechanism of Attachment of an Isoprenoid Moiety to the Indolic Benzene Ring of Indole-Diterpenoids

3-methyl-2-butenylpaspalinine (Paspalitrem A)

3-hydroxy-3-methyl-1-butenylpaspalinine (Paspalitrem B) 2 8

Figure 7. Proposed Mechanism of Lolitrem B Biosynthesis via Pani11ine 2 9

2. MATERIALS AID METHODS

2.1. Fungal Culture

2.1.1. Source-.of Fungi

a. Isolation of Endophyte from Seed.

Perennial ryegrass seed was used as a convenient source of ryegrass endophyte. Seed was obtained from sites on which RGS had been observed recently, or from ryegrass plants taken from affected pastures and allowed to set seed in the following year, ie from New

Zealand (cv 'Nui'), the USA (cv 'Repell'), Cooling Marshes in North

Kent, and Stamford in Lincolnshire. The endophyte was enabled to grow

out of the germinating seedling, using the method of Neill (1940).

Adherent flower parts were removed from seed with fine forceps.

Seed was surface-sterilized in 0.1% HgCls for 5 minutes, washed five

times in sterile distilled water, and placed on PDA (about 5 seeds per

Petri dish) at 27°, to allow germination and assess surface-sterility.

Uncontaminated, germinated seeds (shoot length l-6mm, root

length 0-5mm) were transferred to test-tubes, containing 5cm depth of

GPYE medium at 23°. Fungus was subcultured to fresh growth medium when

sufficiently prominent on seedlings.

Surface-sterilization of seeds was found to be completely

effective. The extent of seedling germination ranged from 0-100%

(average 56%, assessed from 97 groups of 5 or more seeds). The rate of

seed germination varied widely; most seedlings were transferred to

GPYE after 3-10 days on PDA but others after up to 26 days. 30

Figure 8. Emergence of Nui Endophyte from Ryegrass Seedling

8.1. Emergence from the coleoptile tip (20 days) 31

Emergence of endophyte (Figure 8) usually occurred 10-20 days after seedling transfer to GPYE, although on several occasions the first signs were seen as early as day 5 or as late as day 30. Initial emergence was usually either from the coleoptile tip or, slightly less commonly, the root tip, although sometimes it occurred from other parts of the root or shoot. The protruding endophyte appeared as a cream-coloured, feathery, roughly spherical mass of hyphae, radiating from the plant. No correlation was evident between rate of, or percentage, germination and endophyte presence in the seed. Incubation of the newly emerged endophyte for a further 1-2 weeks, allowed sufficient growth to permit subculturing.

Endophyte isolated from Nui ryegrass seed was used in all investigations unless otherwise stated.

b. Culture Collections.

Two Acremonium loliae cultures recorded as ’typical* of tie species (Latch et al., 1984) were obtained from the CMI (Commonwealth

Mycological Institute), Kew (isolate designations and CMI accession numbers; R100, 296973 and R103, 296974). These had been isolated in

New Zealand from Lolium perenne leaf tissue in 1982.

Penicillium paxilli was obtained from the CSIRQ (Commonwealth

Scientific and Industrial Research Organization) culture collection in

Australia (isolate number FRR 1900, ATCC 26601). Original isolation was from pecan nuts (Cole et al.y 1974).

2.1.2. Media

All media components were sterilized by autoclaving (120°, 20 minutes) when combined, unless stated otherwise. Components are recorded in % (ie g/100ml>. 32

PDA potatoes (infusion from): 20. 0

(Bacto) dextrose: 2.0

agar: 1.5

(tetracycline (optional): 0.05)

CMA commercial maize meal: 3. 0

nutrient agar (Bacto): 2.0

(dextrose (optional); CMDA: 0.2 or 2 .0)

(s)PYE sugar (glucose, G; sucrose, S;

fructose, F; or mannitol, M): 4. 0

peptone: 1.0

yeast extract: 0.5

CD YE Czapek Dox broth.

yeast extract: 0.5

(CaCli>. 2Hi;0 (optional); weighed

into flasks before autoclaving

medium: 2. 0)

Paxilline- pectin (Sigma - citrus): 1. 0

Productioii G: 2.0

Medium P: 0.5

YE: 0.25

(pectin autoclaved separately from

"GPYE" and subsequently combined)

Medium T sucrose-asparagine medium (see

Stoll et al., 1954) 33

G: 4. 0

JHUOH: to pH 6.2

succinic acid: 2. 0

KH^POn: 0.025

MgSCU: 0.025

tap water

G: 4. 0

glycine: 2. 0

NaOH/HsSCU: to pH 6.2

KH^POn: 0. 025

MgSCU: 0. 025

tap water

2.1.3. Inoculation and Incubation

a. Agar Media.

25ml universal battles or Petri dishes contained 10ml or 20ml medium, respectively. Slopes were inoculated with endophyte mycelium or spores of P. paxilli. Cultures were incubated at 27° and subsequently stored at 3°; A. loliae was transferred only after 3-4 weeks. Homogeneously sporing P. paxilli was obtained by growing for 24 hours at 27°, the culture shaken to spread spores, and incubated for a further 2 days before cold-storage. Cultures stored in this way remained viable for at least 6 months. 34 b. Liquid Media.

Standard conditions for A. loliae and P. paxilli fermentations involved inoculation of 100ml medium in 500ml erlenmeyer flasks by a

17, v/v transfer of fully grown culture. The endophyte was routineLy incubated at 23° and F. paxilli at 27°, on a rotary shaker (200rpm,

10cm eccentric throw).

F. paxilli seed-stage culture used baffled flasks (CDYE), which were inoculated with the spores from an agar slope and grown for 48 hours, ensuring submerged culture of all mycelia.

Inoculation of endophyte cultures for investigation of optimum growth conditions commenced with aseptic centrifugation of a shake culture. The pellet was resuspended in 100ml sterile distilled water and homogenized (400ml Sarvall Omni-mixer) to form inoculum for 1% transfer to appropriate media.

Inocula for investigation of optimum agitation and inoculum for desirable morphology were formed from 8-day-old Nui endophyte in GPYE submerged culture, either undiluted, or diluted 11 or 121 times. 1ml of each dilution was used to inoculate two 500ml conical flasks containing 100ml GPYE, incubated at either fast (200rpm, 10cm eccentric throw) or slow (lOOrpm, 3cm eccentric throw) rotation speed.

2.1.4. Microscopy

Liquid culture samples were usually mounted in culture medium.

Agar-cultured material was mounted in distilled water. Some material for microscopy (Leitz Ortholux) was mounted in aniline blue <0. 05%, in phosphate buffer, pH 8.4), boiled momentarily to accelerate staining of cytoplasm. 35

2.1.5. QaI jaxt^ J I ^ qvjX h^Roie—AsssjaSiseiLt.

The diameter of colonies on PDA was measured through the

underside of Petri dishes.

Liquid media culture growth was followed by dry weight

measurement or optical density (EEL colourimeter, filter 626,

distilled water blank) of cultures, incubated in 500ml conical flasks

with a suitable side-arm attachment.

2.2. Sample Component Analysis

2.2.1. Sasapla.Preparation

a. Routine Preparation of Fungal Culture Extracts.

Mycelium was separated from broth by pouring whole cultures under vacuum through a 9cm diameter Sinta-3 glass filter, together with a Whatman Mo. 1 filter paper. Mycelia and culture filtrates were transferred to separate, weighed round-bottomed flasks and freeze- dried (0.01-0.OOlmBar).

Weighed, dry samples were extracted under acetone overnight, filtered and the mycelium or solutes re-extracted in acetone for a further 15-30 minutes. Combined acetone extracts were evaporated to dryness, the residues treated with CHCla and transferred to individual

4ml stoppered glass vials. CHCla extracts were evaporated to dryness pending analysis.

A 3001 fermenter (Snewin, 1984) yielded about 5kg dry A. loliae mycelium and 2601 culture filtrate, thoroughly separated from each other using a Manton-Gaulin press. Known masses of dry mycelium were extracted in appropriate solvents as required. The culture filtrate was extracted by solvent partition using IBMK, which was evaporated 36 under vacuum, leaving 200ml residue. This was extracted with hexane

(to remove P2000 antifoam) and the hexane-insoluble material extracted with acetone. Evaporation under vacuum thus provided 2.3g culture filtrate acetone extract.

b. Preparation of Ryegrass Seed Extracts.

Extraction of tremorgens from seed was carried out either with or without a clean-up step in the case of lolitrem B or paxilline, respectively.

Seeds, including adherent flower parts, were oven-dried at 45° overnight. They were then milled by 5 passes through a Glen Creston sample mill, lg of this homogenate was gently shaken (Ludham bench shaker-table, mark 3) in 15ml CHCls/methanol (2:1) in a stoppered

vessel for 1 hour. The mixture was filtered and the extract evaporated

to dryness prior to clean-up and/or chromatography.

Sample clean-up (Gallagher et al., 1985) involved extract

equivalent to l/15g of seed, processed through a silica Sep-Pak

cartridge (Waters Associates).

2.2.2. Chromatography

a. 'Flash' Column Chromatography (FCC). / 400ml mobile phase (CHCla/acetone mixtures chosen to resolve

relevant substances between Rf 0.2-0.7 on silica TLC) was stirred with

75g column silica (Kieselgel 60, 0.040-0.063mm, 230-400 mesh ASTM

Merck) to form a translucent slurry. This was carefully poured into a

5cm diameter glass column (slanted, so as to prevent void formation), 3 7 fitted with a Sinta-glass base and PTFE stop-flow tap, to control mobile phase flaw. The column was then held vertically and mobile phase run through under gravity to a depth of 3mm above the silica bed.

The acetone extract of, for example, Nui endophyte mycelium after culture in 4 x 11 GPYE, was dissolved in CHCI3 (approx. 50ml) , shaken with column silica (approx. lOg) and rotary evaporated to a crumbly powder texture.

The silica-bound sample was applied evenly to the top of the column using a nickel spatula. Mobile phase was then added down the column side, using a Pasteur pipette, to a depth of 2cm above the silica. This was reduced to 3mm using pressure applied from rubber

"bellows" fitted by quick-fit attachment to the top of the column.

This rebuilding and lowering of the mobile phase layer was repeated until the 3mm solvent above the silica surface was completely colourless. The column was filled with mobile phase and 50ml fractions collected, controlling the flow of solvent with the bellows, and stopping flow between fractions using the tap. Fractions were monitored by TLC; appropriate fractions were combined and evaporated to dryness.

b. Thin Layer Chromatography (TLC).

Analytical TLC used Polygram SIL G/UV^s* (0.25mm layer silica gel containing fluorescent indicator UV^s*) coated on plastic sheets

(Camlab). Mobile phases are specified with the corresponding chromatograms. Chromatograms were observed for: quenching (254nm - dashed outline) of the fluorescent indicator, fluorescence excited at

360nm (solid outline), and reaction with spray reagents. 38

TLC Spray Reagents:

Ehrlich's Reagent 2% p-dimethylaminobenzaldehyde (DMAB) in HC1; (5

short bursts from an aerosol spray at 1 second

intervals from 30-40cm). Many N- and O-containing

compounds: shades of green (including paxilline

and close structural relatives), blue, purple,

red, grey.

Phenol Stain 1% aqueous potassium hexacyanoferrate KaFeCCN)© /

2% aqueous ferric chloride FeCls (mixed 1:1 just

prior to spraying). Phenolic compounds: deep blue

on contact.

Preparative TLC was carried out over silica, either pre-coated

or hand-poured on glass (Whatman, PK6F silica gel, 1.0mm layer or

Merck Kieselgel 60 GFSS4, approximately 1.0mm layer, respectively) or

pre-coated on aluminium (Whatman, AL SIL G/UV, 1.0mm layer).

Appropriate bands were selected using standards, UV observation and

spraying the edge of a chromatogram. Selected bands were scraped off

the backing substrate and submerged in a suitable solvent (usually

10:1 CHCl3/propan-2-ol) for 1/2-2 hours. Elution was completed by

filtration under vacuum through a Pyrex G2 Sinta-glass filter, and

followed by evaporation of filtrates to dryness. 39 c. High Performance Liquid Chromatography (HPLC).

All HPLC was isocratic. Unless otherwise specified next to particular chromatographs, mobile phase flow rate was 4ml/min and chart-recorder speed was 5mm/min. Other details, including column type, detection wavelength, mobile phase constitution and sample solvent are presented with the corresponding chromatographs.

Normal phase HPLC employed a Waters Z-module radial compression separation system with a Radial-PAK /iBondapak NH^ cartridge or a

Radial-PAK Resolve silica cartridge (both 10cm x 0.8cm, 10/im particle size). A Gilson pump (model 302), manometric module (model 802) and UV detector (Holochrome, ll/ll flow-cell volume) were linked in series with the column, and a Gallenkamp Euroscribe dual-pen chart-recorder connected to the detector. A Rheodyne injection valve, with either a

20/11 (analytical) or 200/11 (preparative) injection loop, was fitted between the raanometric module and a pre-column filter (2/lm pore size) directly upstream of the column.

Reversed phase HPLC involved an Altex Ultrasphere 25 x 1cm QDS

(5/im particle size) column, or a Radial-PAK Resolve Cis cartridge

(10cm x 0.8cm, 5/lm particle size). An Altex pump (110A) and injection valve (210) fitted with a 20/11 (analytical) or 250/11 (preparative) loop, were situated in series just upstream of a pre-column filter

(2/im pore size). Detection was made with a Pye-Unicam UV detector

(PU4020, 8/ll flow-cell) connected to an MSE Fisons Vitatron dual-pen chart-recorder.

Fluorimetric detection used a Perkin-Elmer fluorimeter (1000M,

8/ll flow-cell) placed in series immediately upstream of the UV detector in normal phase HPLC conditions. Narrow band-pass interference filters were used to provide wavelengths of excitation 40 and emission of 268nm (Glen Creston) and 450nm (Perkin-Elmer), respectively. The fluorimeter was connected to the chart-recorder via the same voltage as the UV detector.

a. Mass Spectrometry (MS).

Electron Impact (El) MS and mass measurement used a VG 7070E

double focusing mass spectrometer. Samples were loaded in methanol on

to a glass probe.

b. Proton Nuclear Magnetic Resonance ^HNMR).

1HNMR used a fourier-transform Bruker 250MHz or 500Mhz

spectrometer. Samples were dissolved in appropriate deuterated

solvents (TMS absent) ie benzene, CHCla, methanol or DMSO.

2.3. Use of Metabolic Precursors

2.3.1. Addition and Incubation of 1^C-Radiolabelled Mevalonic Acid and

Amino Acids

After sterile addition of radiolabelled compounds, (using a

Gilson Pipettman), cultures were incubated under routine conditions

(see 2. 1,3. b).

D, L-[ 2-1 AC] mevalonic acid (40/lCi)* was generated as the free

acid from the lactone by adding slightly in excess of a molar

equivalent of NaOH, after evaporating the benzene used as a storage

solvent (under a stream of Qs-free Ns gas, blown over the surface of

the solution). C COOH- 1 'lC3 anthranilic acid (50/lCi)1, L-[ CH3- 1 UC] methionine

(5/MCi>*, L-C U-1 nC] proline (20jLtCi),:, L-C U- wlC] phenylalanine (10/£Ci ) k,

L-[ U-1 rtC] tyrosine (10/lCi)* and D, L-C CH--1 •iC] tryptophan (20/lCi)1' were added in 2% aqueous ethanol, except anthranilate and methionine, which were added in ethanol and water, respectively.

(*: added, in two equal portions (days 8 and 10), to A. loliae cultures, subsequently harvested on day 12. )

2.3.2. Mdl.tiQn.and Incubation of Paxilline

a. Addition to A. lollae cultures.

1AC-radiolabelled paxilline (see Figure 1 for positions of '*0 was dissolved in redistilled ethanol (lmg/50/ll). The tip of a' 23cm

Pasteur pipette was drawn to a very fine capillary (0. lmm diameter) and inserted below the surface of stirred sterile distilled water

(20ml in a 100ml conical flask on a Gallenkamp magnetic stirrer, speed level 6). Ethanolic solution of paxilline was introduced into tie pipette using a Gilson Pipettman and expressed slowly into tie swirling water with a rubber teat. A further 50fll ethanol was used to wash out the pipette similarly. The very fine suspension of paxilline in water was poured directly into an appropriate culture, aseptically.

Incubation proceeded as detailed in 3.5.

b. Administration to Ryegrass Seedlings.

60 L, perenne L. seedlings (developed from seeds shown to te

100% infected with live Rui endophyte), grown in garden soil for 10 weeks in spring, were washed in water and their roots cut to about

0.75cm. Seedlings were approximately 30cm from cut root to leaf blade tip. They were placed upright in a small glass vessel with roots in water (25ml), to which 14.56mg 1AC-radiolabelled paxilline (specific 42 activity 2.14 x 10'l/lCi mol"'1) had been added (in 250/U ethanol 96, placed dropwise into the water, stirred at 40°). The paxilline became partially colloidal as the solution cooled.

After incubation in daylight at room temperature for 1.5 days with occasional swirling of the suspension, the level of the suspension dropped substantially. So a further 20ml water was added and incubation continued for 1.5 days. The seedlings were then repotted (having washed the roots into the incubation vessel using distilled water) and maintained in moist John Innes potting compost for 4 days.

The plants were washed with distilled water and each was transversely separated at the junction between the first leaf and its leaf sheath. Thus one combined portion consisted mainly of short'roots and lower leaf sheaths, and the other of leaf blades (post incubation weights 7.3g and 10.4g, respectively).

Each portion was finely ground with a pestle and mortar, before being freeze-dried, extracted in CHCla/methanol (2:1) overnight, filtered (as described in 2.2.1.a) and re-extracted for 1 hour. The combined extracts of each portion were dried under reduced pressure, transferred to 4ml vials using 4 x 1ml extraction solvent, and taken to dryness, prior to chromatography and autoradiography.

c. Administration to Mice.

The tremorgenicity of paxilline and 140f-hydroxypaxilline was compared in mice. Duplicate intraperitoneal injections of lOOjhg of each compound (in lOOfll 50% aqueous DMSO, warmed in the hand to enhance dissolution) were made into 25g, female mice. A control experiment employed carrier solvent only. Subsequent observation was carried out over several hours noting, in particular, tremors and incoordination of movement, especially when encouraged to be active, 43

2 . 3. 3. hsii&radiogLs^ihyL

Appropriate thin layer chromatograms were taped to a clean 20 x

20cm glass plate. Under red light, an 18 x 13cm sheet of NIF Fuji RX

x-ray film was similarly attached to cover the chromatogram. A second

glass plate completed the sandwich, which was enclosed in black

plastic and placed in the dark at -170°. Exposed film was developed in a Kodak industrial X-OMAT processor, model 3.

2.4. Xutagenic Treatment of A. loliae

8-day-old 'Nui' endophyte shake culture in GPYE <50ml) was homogenized (Sorvall Omnimixer, 90ml vessel) for 15 seconds at

QOOOrpm, repeated eight times at 30 second intervals. 1ml homogenate was diluted in 100ml GPYE (rather than water, to minimize osmotic shock) and 25ml suspension transferred aseptically to a Petri dish.

With lid removed and frequent agitation, the suspension was irradiated

(254nm) 48cm below a Hanovia Chromatolite UV lamp. 0.1ml aliquots of suspension were removed at intervals and spread over PDA in Petri dishes, which were incubated at 23° for 2 weeks. 44

3. RESULTS

3.1. Confirmation of Identity of Isolated Ryegrass Endophyte as

Acremonium loliae

Isolated ryegrass endophyte was confirmed as Acremonium loliae\ no significant differences were evident (see Figures 9 and 12, and

Table 1) between the isolated fungus and a typical strain of A. loliae

(Latch et al.t 1984).

Figure 9. Cultures of Isolated Nui Endophyte (left) and a Typical Strain of A. loliae

9.1. GPYE (6 days, x 160 magnification) 45

9.3. GPYE (7 days)

) 46

CRITERION USED TO COMPARE ISOLATED RG ENDOPHYTE WITH A, loliae FUNGAL CHARACTERISTICS

Microscopic Hyphal Diameter: 1.8-2.2/fm.

Appearance Hyphae septate, but not at base of side-branches. 1

Staining with aniline blue shows vacuoles, particularly

in older hyphae.

Spores very rare and sparse (CMA, 15°, 7 weeks; one

culture only): 2.6 x 6.5^lm, elliptical or slightly

reniform, no abscission scar.

Culture PD-Agar: small, hemispherical or highly convoluted

Appearance colonies; cream-white, gelatinous; glossy or

matt surface, with or without aerial hyphae.

CM(D)-Agar: bell-shaped or flat colonies; white,

gelatinous, glossy.

GPYE Shaken Broth: cream-white, suspension of hyphal

fragments and small mycelial pellets.3

Rate of Colony

Diameter (PDA)

Increase 0.9mm/week (27°); no growth <5°, >32°.n

Metabolites Mainly sterols and triglycerides, giving characteristic

pattern on TLC.3

Table 1. Comparison of Isolated Ryegrass Endophyte with Authentic A. loliae, (1: see Figure 9.1; 2: see Figure 9.2; 3: see Figure 9.3; 4: see Figure 10. Discrepancies between these rates and those published

(Latch et al., 1934) for A, loliae are probably due to much smaller inoculum colony size used in the present work - ie 3mm; 5: see Figure 12.) 47

NUTRIENT INVESTIGATED GROWTH MYCELIUM OR PSM pH MEDIUM'1 DRY WEIGHT

Sucrose SPYE 0. 17

Glucose GPYE 0.92

Fructose FPYE 0.75

Mannitol MPYE 0. 17

STH*- GNS 0. 00

Glycine GGly 0. 00

NO3" CD 0. 00

Asparagine Medium T 0. 13

4. 0 GPYE 0. 00

5. 0 II 0.67

6. 0 II 0. 80

7.0 II 0.57

7.5 M 0. 63

Table 2. Effect of Carbon and Nitrogen Source, and Pre-Sterilization

Medium (PSM) pH on A. laliae Biomass Accumulation. (1: see 2.1.2 for medium composition; 2: reciprocated cultures, 27°, harvested on day

8, 2 replicates per culture condition.) ! 3.2. A. loliae Culture in Optimum Growth Conditions

3.2.1.

From the results of the replicated investigations (Table 2, and

Figure 10) the conditions shown in Table 3 were found to be optimal for A. loliae growth. Incubation of the endophyte for 3 days at 36° was lethal, whereas at 31°, although growth was not detected, the fungus remained viable, as shown when transferred to 27° (Figure

10.2).

Although not exhaustive, this investigation provided adequate conditions for commencing studies on the biosynthetic potential of

.4. loliae.

CULTURE INCUBATION OPTIMUM CONDITION PARAMETER FOR A. loliae GROWTH

Medium (reciprocated) GPYE

Pre-Sterilization Medium pH 6 . 0

Incubation Temperature 23-27'

Table 3. A. loliae Optimum Growth Conditions iue 0 Efc o Icbto eprtr on Temperature Incubation of Effect 10. Figure 49 RATE OF COLONY Culture PDA on Effect 10.1. DIAMETER INCREASE (mm/week) . loliae A. Growth

ABSORBANCE OF A. loliae CULTURE 10.2. Effect on GPYE Culture ( * = cultures incubated at 31° and 36'’ and 31° at incubated cultures = ( * Culture GPYE on Effect 10.2. rnfre t 7; nuain eprtrs ▼= 3; s 27°;$ = ▼ 23°;= 36°) ▲ = 31°;= temperatures: incubation 27°; to transferred

50

51

3.2.2

a. Predominant Acetone-Soluble Products.

Figure 11 shows the predominant acetone-soluble products of A. loliae after full growth in GPYE under submerged conditions (see

Figures 12-14 for chromatographic and spectroscopic results).

Triglyceride

EIMS of the material, which appeared as a clear orange oil, showed m/z 676 as the probable molecular ion. Several 14, 26 and 28 mass unit differences between peaks suggested consistent losses of

CH;i, CH=CH and CHs-CHs, respectively. Many 2 mass unit differences were also evident suggesting different degrees of unsaturation within the molecule. Unsaturation was also indicated by the sample staining with iodine vapour after TLC. The El mass spectrum bore close

resemblance to those of other triglycerides, such as the lauro

derivative.

1HNMR (spectrum not presented) showed no aromatic region.

Signals indicative of glycerol were clear, as were those of

unsaturation and terminal methyl groups near a carbon double bond.

Dihydroergosterols

Samples appeared as needle-shaped crystals or as a white

amorphous solid. EIMS gave a spectrum identical to 5QJ-ergosta-7,22E-

dien-3/3-ol (National Bureau of Standards EIMS library reference number

2465-11-4) except for a peak at m/z 314, which was most likely to have

arisen from contaminating 50f-ergosta-7,23-dien-3j8-ol. 52

Ergosterol Peroxides

EIMS indicated a mixture of at least two compounds (sterols are notoriously difficult to resolve by simple chromatography), the material appearing as a white amorphous solid. Peaks corresponded exactly to several ergosterol peroxides, that were not distinguishable by ElMS (Gunatilaka, 1981):

M'f' 426: 5(2, 8(2-epidioxy-24 (S) -methylcholesta-6, 9(11), 22-trien-3/3-ol ;

M ,_ 428: 5(2, 80f-epidioxy-24 (i?)-methylcholesta-6, 22-dien-3jS~ol;

5(2, 80f-epidioxy-24 (S)-methylcholesta-6,22-dien-3/3-ol.

Glycerol Monoglucoside

The native compound crystallized readily to needles or plates, and proved unsuitable for EIMS. EIMS of the peracetylated compound gave no molecular ion (predicted as m/z 506) but showed prominent signals at 361 (361.1133, calculated for CisHr/Oio - error: -0.5pjm) and 289 (289.0918, calculated for Ci^Hiv'Oe - error: -1.9ppm). The spectrum was identical to authentic glycerol monoglucoside (Smale,

1968).

Tryptophol

The compound, a white amorphous solid, was chromatographically indistinguishable from authentic material (obtained from Sigma

Chemical Co. Ltd.), and its El mass spectrum was identical to tlat published (Rayle and Purves, 1967). 53

Tyrosol

Crystals of this compound were needle-shaped and had a melting

point of 92° (authentic tyrosol has a melting point of 92-93':’ - Cross

et al. , 1963), reacting to the phenol stain on TLC. EIMS showed m/z

138 (133.0695, calculated for C^HioO.:: - 133.0681) for the molecular

ion, and m/z 107 and the corresponding m/z 31 due to loss of CH--0H.

Bis-tyrosol

The material was a white amorphous solid, that gave a positive

phenol test, and EIMS (spectrum not presented) indicated fragmentation

as shown in Figure 14.7 indicating the given structure for the novel compound bis-tyrosol.

/3-Hydroxyphenylalanyl prolyl diketopiperazine

EIMS of this white amorphous solid gave peaks corresponding to

the fragments shown in Figure 14.8. The position of the hydroxyl group required for the McLafferty rearrangement necessary for this

fragmentation was supported by the compound being unreactive to the phenol spray on TLC, precluding the possibility that the compound was a tyrosine derivative and confirming its identification as the novel metabolite j(3-hydroxyphenylalanyl prolyl diketopiperazine. 5 4

Figure 11. The Major Acetone-Soluble Products of A. loliae

11.1. Mycelium Products o c h 2-o -c -(ch2)„-c h =c h -c h 3 c h -o -c o -(c h 2)-c h =c h -c h 3 CH2-0-C -(^H 2)n-CH=CH-CH3 6 Triglyceride (structures not fully elucidated)

R ~ 20 22 23 24 25 50f-ergosta-7,22E-dien-3jS-ol -CH (CH3 ) -CH=CH-CH (CH3 ) -CH (CH3 ) „ 20 22 23 24 25 5QJ-ergasta-7,23-dien-3/>-ol -CH (CH3 ) -CHs-CH=C (CH3 > -CH (CH3 )

50; 8Gf-epidioxy-24 (S)-methylcholesta-6,9(11), 22-trien-3j8-ol 50, 80f-epidioxy-24 (j?)-methylcholesta-6, 22-dien-3jS-ol 5 a 8C£-epidiaxy-24 (S)-methylcholesta-6, 22-dien-3jS-ol

c h 2-oh c h 2-oh CH-OH c h 2-o - HO. OH OH Glycerol Monoglucoside 55

11.2. Culture Filtrate Products

.OH

Tryptophol

Agrobacterium tumefaciens (Kaper and Veldstra, 1958); Acetobacter xvlinum (Larsen et al. , 1962); Diplodia natalensis (Bailey and Gentile, 1962); Bacillus cereus (Perley and Stowe, 1966a); Taphrina deformans (Perley and Stowe, 1966b); Cucumis sativus (Rayle and Purves, 1967).

Tyrosol

Ceratocystis fimbriata (Stoessl, 1969); Cochliobolus lunata (Nukina et al., 1978); Gibberella fujikuroi (Cross et al., 1963); Pyricularla oryzae (Devys et al., 1976).

HO OH

Bis-tyrosol

Novel

OH j0-hydroxyphenylalanyl prolyl diketopiperazine

Novel 56

Figure 12. Thin Layer Chromatography of Acetone-Soluble Products of A. loliae

12.1. Mycelium Products (19:1 CHCla/acetone; after Ehrlich's Spray)

1 = triglyceride 2 = dihydroergosterols 3 = ergosterol peroxides 4 = tryptophol 5 = sterols (unidentified) 6 = glycerol monoglucaside

r} |*YC \ \\ c : ,, rcCtS ("',L

i 57 12.2. Culture Filtrate Products (8:1 CHCls/acetone)

1 = tryptophol 2 - tyrosol and bis-tyrosol 3 - p-hydroxyphenylalanyl prolyl diketopiperazine a. After Ehrlich's Spray b. After Phenol Spray 58

Figure 13. High. Performance Liquid Chromatography of the Major Acetone-Soluble Products of A. loliae

13.1. Mycelium Products column = NHs jXBondapak ‘Z-Module' Cartridge mobile phase = CHj-Cl^ solvent = CHsCl^

1 = dihydroergosterols 2 = ergosteroi peroxides 59

A 246

1 2 3 mins. 60

13.2. Culture Filtrate Products column = Altex ODS Ultrasphere 250 x 10mm mobile phase 2:1 methanol/water solvent = methanol

1 = tyrosol (TY) and tryptophol (TR) 2 = bis-tyrosol (B-TY) 3 = j$-hydroxyphenylalanyl prolyl dihetopiperazine SUjlU 8Z.9St'£Zl 11 01 6 8 £ 9 S P 2 Z l 9 S t £ Z l i ■ ■ ■ ■ -1 i i i , I I I ------L. I l l ------1------<------*- j__i___i___I— i-----1---- I

zzz v

t

19 62

Figure 14. Electron Impact Mass Spectroscopy of the Major Acetone- Soluble Products of A. laliae

14.1. Triglyceride 14.2. Dihydroergosterols

m/z 450 14.3. Ergosterol peroxides 100 14.4. Glycerol Monoglucoside (peracetylated)

m/z

100-

90-

80-

70 -

60- RA so- % 40-

30- 289

20- 361

10- 272 0-1*- 260 280 300 320 340 3& ) m / z ' 380 400 ' 4^0 440 460

u> U1 66

14.5. Tryptophol 67

14.6. Tyrosol 6 8

14.7. Bis-Tyrosol

+•

-CH.OH - z ----> m * 215-5

m/z 274

m *208-3 " H 2 °

+

-h2o < m * 190-4 6 9

14.3. hydroxyphenylalanyl prolyl diketopiperazine

+ • OH

400 m/z 7 0 b. Incubation of 1 '‘C-Radiolabelled Primary Metabolites with A. loliae

Cultured in GPYE.

Incorporation into sterols was prominent for all the radiolabelled precursors administered (see Figure 15 for autoradiographs and corresponding thin layer chromatograms).

Incorporation of radiolabelled amino acids into the identified sterols

(presumably after catabolism to acetate) was at least as pronounced as incorporation into passible amino acid derived metabolites. However, several compounds seemed to incorporate non-catabolised radiolabel, particularly anthranilate and proline, but were present in amounts too low to allow their identification. It is likely that those incorporating 1^C-mevalonate were sterols. The metabolites isolated from the culture filtrate (see Figure 11) were not detectably labelled. Specific incorporation into amino acid derivatives might have been achieved by administering the precursors later in the culture incubation. Figure 15. Autoradiographs and Thin Layer Chromatographs of Acetone Extracts of A . loliae Cultures after Incubation with 1AC- Radiolabelled Primary Metabolites

1 = triglyceride and sterols (unidentified) 2 = sterol (unidentified) 3 = dihydroergosterols 4 = ergosterol peroxides 5 = passible derivative of uncatabolised 1AC-precursor (unidentified) u = unmetabolised 1AC-precursar i = impurity of 1^C-precursar 72

15.1. Incubation with '‘‘C-Mcvaionate

15.1.1. eluent: 25:25:2 CHC1 ,/hexane/acetcme

1

2

2

I wte.. e x fos*re o favitc n \ £ V i ^ u . 6 )•• l •• 0-09 H a l CtouP) t / d / lm lC f ) Mttoug 15,1.2. left: eluent: 19:1 CHC1. ^/acetone

right: eluent: CHCla 74 75

15.2. Incubation with 1AC-Anthranilate - A. loliaes left; P. aurantiogriseum, right (Yeuiet, 1986) a.= auranthine left: eluent: 2:1 CHC1 ace tone right: eluent: 93:7 CHCls/acetone 76 >

~ a

4 77

15.3. Incubation with 1 "'C-Proline eluent: 19:1 CHCla/acetone 73

15.4. Incubation with 1^C-Methionine (MET), 1^C-Phenylalanine CPHE), 1'xC-Tyrasine (TYR) and 1AC-Tryptophan (TRP) 15.4.1. eluent: 1:1 CHC13 /hexane 80

15.4.2. eluent: 19:1 CHCLs/acetone

; y ' ■ t \ C -V *. i.'A' V 81

15.4.3. eluent: 2:1 CHCla/acetone 82 I 3.3. Preparation of 1^C-Radiolabelled Paxilline

3.3. 1. FejijLG±Llmm_paxUJ i. Cu 1t u eg.

Table 4 describes features of Fenici11ium paxilli cultured in a

variety of incubation conditions (see also Figure 16). Shaken CDYE

provided a relatively nigh yield of paxiliine (see Figure 20 for

structure) and a mycelium morphology ideal for dispersal of added

'-'C-radiolabelled precursors. The time-courses of biomass and

paxiliine yield (Figure 18) indicated the ideal time of addition of

precursors (ie B, L-C 2- 1 ‘'Cl mevalonic acid: 5/fCi added to one 100ml

culture on day 3 and 5, harvested day 6; D,L-[benzene ring-U-

1 ■1C] tryptophan: 0.5/XCi, 1.5/XC-i and 0.5jU,Ci added (Kyriakis, 1987) to

one 100ml culture on day 1, and early and late on day 2, respectively,

harvested day 5) to ensure maximum incorporation into paxilline (see

Figure 19 for autoradiographs). Thus 19.16mg pure 1^C-radiolabelled

paxilline (specific activity 2.62 x 104''/fCi mol '1) was obtained (clear,

colourless flat or needle-shaped crystals, or amorphous, white

crystalline powder, tinged yellow in larger quantity). 83

Table 4. PaniciIlium paxilli Culture Characteristics and Paxilline

Production in Different Culture Media. (1; incubation time = 16 days,

except shaken CDYE and shaken CDYE + CaCl^ - 14 and 19 days,

respectively. [Paxilline] assessed from standard curve (Figure 17); 2:

separation of well grown mycelium from broth by filtration caused pink

colouration of the filter paper after removal of the mycelium. This

colour was bleached or turned blue in the fumes of HC1 or ammonia,

respectively.) 84

[PAXILLINEJ O CULTURE INCUBATION CULTURE CELLS (mg/g dry MEDIUM TIME (days) APPEARANCE mycelium)'

PDA 4 Spores grey-green, covering agar;

no colour added to medium.

Static CDYE 7 Thick,convoluted mycelial mat;

spores pale brown, covering mat;

no colour added to medium.

16 As day 7, but medium dark brown. 17.23

Static GPYE 7 As static CDYE, except spores

pale grey-green. 3.83

Shaken CDYE2 2-6 Cream-white, homogeneous

(standard suspension of small, white

fermentation) mycelial pellets.

7-8 Mycelium green-brown (no spores).

9-19 Mycelium yellow-brown, becoming

dark brown red. 21. 12

Shaken CDYE + 1 Mycelium as day 2 shaken CDYE,

CaCl* except pale green tinge.

2-19 Mycelium distinct pale green,

becoming slightly darker (green

colour due to spores);

no colour added to medium. 0. 04 85

Figure 16. P. paxilli Culture

16.1. PDA <5 days) 16.2. CDYE (2 days) 86

Figure 17. Paxilline Standard Curve (from average of 3 HPLC peaks) column = NH-- //tEondapak ' Z-Moaule* Cartridge detection wavelength = 281nm range = 0. 05 injection volume = 20/11 mobile phase = 100: 1 CH:;: C1 / pr a pa n- 2 - o 1 solvent = C H :;;'j Cl flow rate = 4ml/min. chart speed = 5mm/min. AVERAGE PEAK HEIGHT (cm)

MASS OF PAXILLINE (JW.9) 19 Figure 18. Time-Course of Paxilline and Biomass Yield in P. paxilli Culture ( H = [paxiliinel; • = mycelium dry weight) 100ml culture) 100ml [PAXILLINE] (mg/ [PAXILLINE]

INCUBATION TIME (days) 89 iue 9 AtrdorpsadTi Lyr hoaorps f Acetone of Chromatographs Layer Thin and Autoradiographs 19. Figure 14Qf-hydroxypaxilline =4 CO (unidentified) to indole-diterpenoid probable = 1 paxilline = = prepaxilline-i prepaxilline-i 6/3= 14Gthydroxyprepaxiiline and -ol eaoae lf) n 1 = (eluent and (left) "lC-BenzeneTryptophan Mevalonate Ring xrcs of Extracts 19: CHCl.^/acetane)1 . paxilli P. Clue Atr nuainwt 1^C- with Incubation After Cultures

90

v

1 1

3 4 4 f r i«. *«.<**/ 9 1

3 . 3 . 2 .

In addition to paxilline, three structurally related, less abundant metabolites were isolated from P. paxilli mycelium, and identified as prepaxilline-16/J-ol, 14G£-hydroxyprepaxilline and 140f- hydroxypaxilline (see Figure 20, 21 and 22 for structures, TLC and

HPLC, respectively). These are strong candidates for being intermediates and/or co-products of the paxilline biosynthetic pathway.

Figure 20. The Indole-Diterpenoids of A. loliae and P, paxilli

H

Prepaxilline-16/3-ol (novel)

R t R*

Prepaxilline' (hypothetical) H H Paxilline OH H 14QFhydroxyprepaxi11ine (novel) H OH 14Q£- hydr oxypaxi 11 i ne (novel) OH OH 92

Figure 21. Thin Layer Chromatography of Acetone Extracts of A. loliae (left) and P. paxilli Cultures

21.1. eluent = 19:1 CHCI3:acetone

21.2. eluent = 12:1 CHC1 :acetone

1 = probable indole-diterpenoid (unidentified) 2 = probable indole-diterpenoid (unidentified) 3 = paxilline 4 = prepaxilline-16p-oi and 14Qf-hydroxyprepaxilline 5 = 14a- hydroxypaxi11 i ne 93

Figure 22. High. Performance Liquid Chromatography of the Indole- Diterpenoids of A. loliae and P. paxilli

1 = paxilline 2 = prepaxilline-16|$-al 3 = 14Qf-hydraxyprep’axilline 4 = 14Gf-hydroxypaxilline

1 : column - HH;,: /-tEondapak 'Z-Module' Cartridge detection wavelength 2Slnra mobile phase = 100; 1 CH:;.::Cl,:;:/prOpan-2-Ol solvent = CH^Ci.;:;

2, 3, 4: column = Resolve ODS 'Z-Module' Cartridge ,detection wavelength 230nm mobile phase = 4; 1 methanol;water solvent = methanol 94 95

Figure 23. El Mass Spectrum of Eaxiiline (70eV)

10O-,

RA %

50' 43

58

182

p i— —II..---- |4U.-- --- rjlL ■44 Hf. -V 20 200 260 Paxillinc

m/z FRAGMENT

435 M'

420 M‘ - CH.-:-,

417 M' - H,;:0

402 M" - CH,:: - H.:,0

377 M" - CH ,-CQ-CH.:.:

362 M"“ - CH,-C0-CH,: - CH:-:

359 M'- - CH..3-C0-CH3 - H,.:;:0

344 JT - CH:,-C0-CH..: - CH-: H:.,0

182 cleavage across Cl 1-12 and C20-21

130 indole

58 CH.::■-CD-CHa

43 CH-3-CO

Table 5. El Mass Spectral Data of Paxilline - see Figure 23.

In assigning the various protons of 1HIIMR spectra (chemical

shifts given relative to IMS) the different resonance patterns

produced by axial and equatorial protons on 'chair' conformation six-

membered rings was of particular importance in a number of cases.

Axial protons tend to give broader patterns (overall) than equatorial protons because they are able to couple more strongly to the protons of adjacent C atoms since these protons are trans-coplanar, unlike tie arrangement of equatorial protons. Figure 24. 1HNMK Spectrum of Paxilline (DMSO, 500MHz)

r~1 1 1 8 0 4.0 5 .0 0.0 1 .0 PPM 9 8

CHEMICAL SIGNAL NO. OF PROTON SHIFT (ppm) MULTIPLICITY PROTONS' ASSIGNMENT

10. 70 s 1 1 NH

7. 22 m 2 4, 7

6. 90 m 2 5, 6

5.75 d 1 17

4.95 s 1 OH

4.82 t 1 140

4.30 s 1 OH

3. 66 d 1 150

2. 73 m 1 22/5

2. 60 dd 1 23/5

2.57 m 1 120

2.31 dd 1 230

2.23 m 1 130

1.60 - 2.00 m 6 200, 2C/5, 210, 21/5,

1.24 s 3 280 CHa

1.20 s 3 25 CHa2

1. 15 s 3 26 CHa2

0.90 s 3 27/5 CHa

Table 6. 1HNMR Data of Paxiliine in DMSQ - see Figure 24. <1: from

integration of NMR signal; 2: may be interchanged.) 99

Proton-prat: on decoupling experiments were conducted an paxilline in CDC1 .. Irradiation at 5 3.66 (150! H) sharpened the triplet at 5 4.32 (140! H). Irradiation of the broad triplet at 5 4.32

(140! H) collapsed the doublet at 5 5.75 <17 vinyl H) to a singlet, the multipiet at 5 2.23 (130! H) to a broad double double doublet, and the mult inlet at 5 1.35 (13 (3 H) to a double double doublet. Irradiating at 5 5.75 (17 H) sharpened the triplet at 5 4.82 (140! H). Figure 25 shows these couplings.

Figure 25. Proton-Proton Couplings and Full Stereochemistry of

Paxilline (coupling constants in Hz) 100

Figure 26. El Mass Spectrum of Prepaxilline-16j$-ol (70eV) L 01

Prepaxilline-16/3-ol

m/z FRAGMENT

421 M' i re 406 3S o

388 M" - CH„ - H.,0

182 cleavage acros:: Cll-12 and C20-21

130 indole

Table 7. El Mass Spectral Data of Prepaid 1 line-16/3-a 1 - see Figure 26.

COSY was carried out on prepaid lline-16/3-ol, revealing the

following proton-proton couplings (see Figure 28): 17 vinyl to 160!;

16/3 OH to 160!; 24 OH to 150; 160 to 150; 140 to 130 and 13/3; 22/3 to

2.30, 23/3 and 210; 23/3 to 230; 1 9 0 to 2 0 0 and 2q(3; 130 to 13/3 and 12/3; and 12/3 to 120. Notice that unlike paxilline this metabolite exhibited

no coupling between the 140 and 150 protons.

Figure 28. Proton-Proton Couplings and Full Stereochemistry of

Prepaid lline-i (^(3-ol (coupling constants in Hz) Figure 27. 1HNMR Spectrum of Prepaxi11ine-16/3~ol (DMSO, 500MHz)

J l ______>

, ,-r-m -T • ■ — , ------1 r ■ 1 1 I ' ■ '"r-p ™ I' r,T ™ ■ , ■ ■ ■ '-| ' ' 10.0 9.0 8.0 7,0 6.0 9.0 PPM LOS

CHEMICAL SIGNAL NO. OF PROTON SHIFT (ppm) MULTIPLICITY PROTONS' ASSIGNMENT

10.70 3 - 1 NH

7.25 dd 2 4. 7

6.90 m 2 5, 6

5.44 d 17

4.80 d ]_ 16/3 OH

4. 48 s 1 24 OH

4. 05. dt 1 16a

3.91 m 1 14Q!

2. 98 a 1 15a

2.70 m 1 22/3

2.59 dq 1 23/3

2.30 dd 1 23a

2. 19 d (broad) 1 19a

2. 04 m 1 1 3 a

1.98 m 1 12/3

1.82 dq 1 1 2 a

1.75 m 1 13/3

1.70 m 1 21/3

1.60 dq 1 2 ia

1.52 m 1 20 a

1.44 dq \ 20/3

1.21 3 3 25 C H 3

1.16 S 3 26 CH.3

0.97 S 3 28a CH:

0.88 S 3 27/3 CH;

Table 8. 1HNMR Data of Prepaxiliine-16/3~ol in DMSO - see Figure 27.

(1: from integration of NMR signal; 2: may be interchanged.) 104

Figure 29. El Mass opectruin of 140Hiydraxyprepaxi11ine (70eV) 140-hydroxyprepax i. 11 i no

m/ ?RAGUES!

43- M'

420 M' - CH..:

417 M" ~ H 0

402 H' - CH.". - H 0

377 M' - CH ,-CO-CK":

362 M' - CH.-.-CO-CH-, - CH J;

359 M- - CH ,-CG-CH .• - H.;,0

344 M" - CH.--CO-CH::::: - CH.:: " H:;;>0

182 Cl'eavage across Cl1-12 and C20-21

130 indole i 1 0 o 58 CK: o

43 CH:2-CO

Table 9. El Mass Spectral Data of 140-hydroxyprepaxiiline - see Figure

29.

COSY determined the following proton-proton couplings within

140-hydroxyprepaxiliine (see Figure 31): 17 vinyl to 190; 24 OH to

15QJ; 22(3 to 210, 230 and 23/3; 190 to 200 and 20/3: 23/8 to 230; irresolvable 'incestuous' coupling between 120 12/8, 130 and 13/8; and

21/3 to 210.

i40-hydroxyrrepaxilline was subjected to noe (nuclear overhauser effect) thus confirming the stereochemistry of many of tie substituents - in particular the 14 OH group - as that shown in Figure

31. Irradiation of 27/8 CH.--, caused a response at the 22(3 double doublet, the 12 H and 13 H region (probably due to noe to the 12/8 H), and a weak response to the 1IH. Irradiation of 280 CH~.- caused a signal Figure 30. 'HUME Spectrum of 14Q£-hydroxyprepaxilline (DMSO, 500MHz) 106 107

CHEMICAL SIGNAL NO. OF PROTON SHIFT (ppm) MULTIPLICITY PROTONS' ASSIGNMENT

10.70 3 1 1 NH

7.25 dd 2 4, 7

6.90 m o 5, 6

6.53 3 1 140! OH

5.62 d 1 17

4.21 3 1 24 OH

4.08 S 1 150!

2.70 dd + m o 19Q!, 22/3 2.62 dd 1 23/3 2.31 dd 1 23a

2.13-1.93 m (complex) 4 120; 12/3,

1.73 m 1 21/3

1.65 dq 1 20/3

1.56 m 1 200!

1.49 dq 1 210!

1. 16 2s 6 25, 26 CH .3

1.00 s 3 280! CHa

0.87 s 3 27/3 CH-3

T ab le 10. 1 HNMR Data of 14Qf-hydroxyprepaxilline in DMSG - see Figure

30. (1: from integration of NMR signal.) LOS at the 210! double double t, revea led m e LOG! K as a GQU ble doublet at 5

2.70, and caused a response in t a nd 13 H region (probab1y due to noe to the 120! H) . Irratiiation of th-a 17 vi nyi H produced a weak response at the 200! H Irrad iation of the 150! K enhanced the

r e h o n a no e 5 o r c n e 25 and 26 CH. : grou ps , as we : L ,3. — those of the rv/o

hydroxyls, suggestiivy m e stereochemistry of one 14 QK to be a.

Irradiation of the 14 OK gave a strong response at the 150! H, confirming the OL sie reochemistry of the 14 OH - and a negative response at the 24 OK, probably due to exchange effects. Irradiation at the 24 OH caused a weak negative response at the 14Q! OK, and a strong response at 150!.

Figure 31. Proton-Protan Couplings and Full Stereochemistry of 14Gb hydroxyprepaxiliine (coupling constants in Hz)

H H 109

Figure 32. El Ka; D0( i40Haydrcxypaxi illne (7 0eV>

130 L _ illll l AlL L Jllh.-j.Jill .iL .tk L -i.-i tl.J . ikJuj_ l l L U U, Ml . I .1 140*-hydroxy paxi L line

m/ z FRA■ jMENI

45 1 }['

436 M' - CH

433 M' - K.,0

413 M"‘ - CH 7: - H,0

415 M‘ - 2H.,Q

400 M" - 2H...G - CH.,

393 M ’ - CH. ,-CQ-CH.-,

375 M' - CH.,-CG-CH, - H:,G

360 M- - CHr.-CQ-CK,, - CK., - H,::Q

357 M !' - CH ,-CC-CH,; - 2H..,0

342 M" - CH.:,-CG-CH„ - CK,, - 2H.,Q

i.1 U'X.OO CJX0- l -avage across Cl1-12 and C20-

130 indole

58 CK.„-CG-CH.;,

43 CH,,-CO

Table 11. El Mass Spectral Data of 140Hiydraxypaxilline - see Figure

32.

Noe of 14Qf-hydro:-iypaxilline revealed the stereochemistry of many of the substituents as that shown in Figure 34, but left the stereochemistry of the 14 uH inconclusive, this being assigned as Oi on the assumption that it is the same as 14Qf-hydro::yprepaxilline. Noe (in

DMSCO at 14 OH and the 15CK H should resolve this matter. Irradiation at the 230' K produced a response at the geminal 23/5 H, and at the c is

230' C H . Irradiation at the triplet of doublets of the 13/5 H caused 111

Figure 33. 1HNMR Spectrum of 14Q£"hydroxypaxilline (CDCls, 250HHz) CHEMICAL SIGNAL MO. OF PROTOM SHIFT (ppm) MULTIPLICITY PROTOMS1 ASSIGNMENT

7.68 s 1 1 NH

7.33 m 1 Ar

7.2 m 1 Ar

7. 01 m 2 Ar

5.32 s (sharp) I 17

4.30 s (sharp) 1 1 5 a

3.92 s (broad) 1 OH

3.30 s (broad) 1 OH

3.62 s (broad) 1 OH

2.78 m 1 220

2. 68 dt + dd 2 120, 230

2.39 dq 1 230

2.25 dt 1 130

2. 07 dd 1 130

2. 02 m 1 210

1.93-1.67 m (complex) 3 200, 200, 210

1.43 m 1 12 0

1. 10 s 3 280 CH3

1.32 s 3 25 CHs2

1. 19 s 3 26 CHs2

1.02 s 3 270 CH3

Table 12. 1 HNMR Data of 14Qf-hydroxypaxilline in CDCls - see Figure

(1: from integration of NMR signal; 2: may be interchanged. ) a 2:1 doub le doublet,response at the yeminai i2 C L H, revealing it as3 a 2:1 double doublet,doublet,response and also at the 27(3 CH.:. Irradiation oi the 27/3 CK .• confirmed the reciprocal response at 12(3 and also caused a signal at the 22(3 multiplet that is otherwise partially obscured by the 23(3 dd.

sep aratel y produc ed a singleng the 23 and 26 CH . groups teseparately produced a singleng

at the 130i H in both cases. I

the 1201 H as a double triolet,

the 23(3 n in the normal spectrum. at the 23Oi H, and a weak response at the 21Q£ H.

Figure 34. Proton-Proton Couplings and Full Stereochemistry of 140f-

hydroxypaxiiiine (coupling constants in Hs) VOLUME (ml j p jv p p p t MYCEL i VM CULTURE FILTRATE <£ .'c? P i i VIABLE DIAMETER DRY DRY ADDED TO AT MOSTLY WEIGHT WEIGHT 100ml GPYS HARVEST >/< 1mm (g) STEROL PAX (g) STEROL PAX Coo. day a 4 ) 100* yes ' > 0 1 ^ - 6. 09 ++

100 yes > 0.62 + 2.49 ++ +++++

50 no < 0.26 ■f *f"t — 2. 19 T +

25 no < 0. 48 ttrrr 4- 1.65 -f-r

15 no < 0.66 + + + + 1.60 + +

10 no 0. 48 -r + -b + + 1.73 + + —

0 no < 0. 43 1.43 -j- X _

Table 13. Effect of Pectin : GFYE Ratio on A. lo li as Longevity,

Morphology and Metabolite Production, ( PAX = paxilline; #■: added to glucose-free medium PYE. ) 1

3.4. Pectin-SupplGmentcd A. lolias Culture

The inclusion of pec tin in 33 YE s u o t a n t l a l 1 y i noreased .4.

loliae longevity and mycelial oe11e t sice, and effected the unprecedented producti on of a tremor yenic indoie-diter pens id - caxiiline

Pectin was much less readily utilised as a carbon source than glucose, reflected by the mycelial and culture filtrate dry weights of cultures grown in ?YE and GPYE. As might be expected, the main yield of sterol occurred in the mycelium in most cases, although sterols were also present in the culture filtrate, probably owing to cell autoiysis. However, one culture, which had a relatively high mycelium dry weight, contained only a low level of sterol in its mycelium, there being more sterol in its culture filtrate at harvest. This was one of the two cultures incubated in media containing the largest amount of pectin , both of which differed from all ethers by being viable even after 34 days, when harvested. These cultures also differed by consisting predominantly of large mycelial pellets, that were probably internally anaerobic. Significantly, it was these cultures too that produced considerable yields of paxilline, the culture containing glucose being by far the more productive

2.5mg/100ml. Several, less prominent, possible structural relatives of paxilline (ie yellow or green with Ehrlich’s reagent) were also

bserved □n c hr o ma t o g rams of the acetone extract of the culture

iitrate of this culture. Three of these were indistinguishabie from the F. paxiHi products, prepaxi 1 line- 16j8_o l , 140f-hydroxyprepaxiliine and 14Gf-hydraxvDaxi1line (see Figure 21>, therefore exposing remarkably similar biosynthetic facility in A. ialias and F. paxilli. 116

. ;w u x c u 4 *£_. [GLUCOSE] MO PECTIN “ 1% PECTIN :N MEDIUM <%; IN MEDIUM IN MEDIUM

0. 0 0. 0 0. 0

0 .2 II

0 .6

1. 0

i c=; i . . J

2. 0

2. 5

3. 0

3. 5 0. 0

4. 0

Table 14. The Effect of Pectin : Glucose Ratio on Paxilli ne Production by A. laliae. (1: peptone = 0.5%, yeast extract = 0.25%, medium volume

= 100ml. At harvest (day 35) only those cultures cont aining pectin

were viable; 2: conditions previously found suitable for paxilline

production - see Table 13.) Ill certain circumstances significant paxilline production

occurred even in the absence of pectin (Table 14). Further, the

inclusion of glucose and the sterilisation of pectin separately from

GPYE were found to be beneficial to production of the tremorgen,

which, however, rapidly disappeared from cultures after biosynthesis

(Table 15).

[ PAXILL INE3 (mg/100ml culture) PECTIN PECTIN PECTIN AUTOCLAVED AUTOCLAVED AUTOCLAVED INCUBATION SEPARATELY SEPARATELY' TOGETHER1 TIME (days) FROM PYE FROM GPYE WITH GPYE

1 0.00 0. 00 0. 00

5 M If If

8 It II II

12 II II II

19 If II 11

27 0. 04 0. 44 II

34 TRACE TRACE 0. 02

40 0.00 v It TRACE

47 " nv 0. 00 0.00

57 fl II II

65 NOT TESTED II II

92 If II M

103 0.00 " V " V

Table 15. Effect of Pectin Sterilisation Method and Glucose Inclusion

on Paxil! ine Production. (i: media components were at levels optimal

for paxil line production (ie Table 13, line 2); v: culture viable; nv;

culture non-viable.) PELLET DIAMETER MOSTLY >/< 1mm INOCULUM AGITATION VIABILITY LOST INCUBATION TIME (davs) ■ DILUTION'1 SPEED (rpm) BETWEEN DAYS 6 23 30 41 60 i. UNDILUTED 200z 10-23 no no t 2. /II II 10-23 no no

3. /121 M 2 30-41 yes no no no

4. UNDILUTED 1003 23-30 no no no

5. /II M 3 30-41 yes yes yes no

6. /121 .. 3 41-60 yes yes yes yes no

Table 16. Effect of Amount of Fully-Grown Inoculum on Morphology and

Viability of A. loliae in GPYE. <1: 1ml of 0PQ ’r' dilution used for inoculation of 100ml GPYE; 2: 10cm eacentric throw; 3: 3cm eccentric throw.) 119

Reduced agitation and amo nt O inocul um caused increased

longevity, and allowed the format! n of 1arge mycel ia i pell ets (Table

16 and Figure 35), similar to th se f ound associated with paxi11ine production in Table 13. However, pa:-£1111ne wras no + detec ted in any culture, although it might have been revealed with more frequent sampiing.

Figure 35. Effect of Agitation and Inoculum on A. loliae Morphology in

GPYE (numbers as in Tab: e 16> - day 6 INCUBATION l PAXILLINE] 14QB HYDROXY P AXIL LINE j 11 MS Mays.* 1 (mg-/10 Oml c u lt u r e ) (mg/100ml culture)

19 0. 00 0. 00

22 II M

23 II II

24 0.28 0. 04

25 0. 00 0. 00

26 M 11

27 11 11

29 II II

30 II II

Table 18. Paxilline ana 140f-Hydraxypaxi11ine Production Time-courses.

(1: samples were taken from a single 200ml culture, which remained viable throughout.) 1 9 1

The coni'irmation chat she occurrence in .4. loliae culture of both paxilline and the novel indole-diterpenoid - identified as loa­ hydroxypaxi11ine (see Figure 20) - is transient dable 13 and Figure

36), implies some profound catabolic fate, or an anabolic role for these compounds as biosynthetic intermediates, Clearly, paxilline biosynthesis may easily be missed - which might have been the case in she investigations recorded in Tables 13-17 unless cultures are analysed at least daily.

[ MEDIUM COMPONENT! (%) VIABILITY LOST PECTIN GLUCOSE PEPTONE Y. E BETWEEN DAYS 3AKPL ING DAYS’ p 3.5 0.5 0.25 67+ 2, 7, 11, 14, 17,

3.0 II II 32-36 ► 21, 24, 23, 32, 36,

2.5 II II dr\ UC o ✓■p 39, 43, 51, 67 j 1.0 2.0 If II 88+ 0,5, 10,15, 20,26,56, 75,83

Table 17. Cultural Conditions Employed to Follow Paxilline Production

Time-course but in which Paxil line was Never Detected. (1: samples were examined from single 200ml cultures.) column = column detection wavelength detection Figure 36. Figure 122 oiepae = phasemobile let = olvent njection volume = volumeI--1-njection 10 PCPxlie AnalysiPaxillineHPLC osctv as (pax DaysConsecutive

Qfll 2Q 2Slnm M CK;,:C1:,: 100: 1 2 : fid (ie 1ml wholeculture)extractthe of of CH-Ci:,,;7prQpan-2-ol naa '2-Module'ondapak Cartridge

f Single a of paxilline)

. loliae A.

Culture on Culture

A 281

mins. 124

CULTURE PAXILLINE' __ INDQLE-DITERF5N0ID LEVEL AGE AT INCUBATION ___MYCELIUM _____ CULTURE FILTRATE HARVEST(d) TIME(HOURS) PAXILLINE 14QP0HPAX PAXILLINE 14Qf-QHPAX

1 26. 0 4 4 4 4 - - — 44 -

2 23. 0 ~b*f T 4~ - 4 t 4 -

J, o 29. 5 + + T - -

£ 26.5 rVT - -

0 33,5 4444 - •f“T -

n 22. 0 t Tt + - 44 -

r? / 26.5 444 4 - tt -

a 26. 0 4444 - + + -

9 29. 0 4444 - 44 -

10 24. 0 4444 + 4 -

13 66.0 *f* + + 44 --

14 23. 5 4444 + T-

15 23. 5 4444 4“ 4 -

16 23. 5 4“++ + 4 --

4 r? 23.5 4444 "t* --

IS 25. 0 ■ 4444 4. --

19 39.5 4444 4 --

21 24. 0 4444 4 --

22 25. 0 4444 -f --

Table 19. Time-course of Paxil line Transformation by .4. loliae in

Optimum Growth Conditions. (1: ie G.25mg paxilline incubated in

separate cultures for about 24 hours, on each day of a 22-day culture

period; 2; - = not detected, + = evident,... 4444 = prominent.) 125

3.5. Incubation of Paxilline with A. laliae

The main paxilline transformation product in GPYE was identified as 14Gf-hydroxypaxilline (see Figures 20-22 and 32-34 far structure, and chromatographic and spectroscopic data). Thin layer chromatography and autoradiography (Figure 37) demonstrated that transformation was almost solely to this product. On this assumption, the calculated transformation was 24%. Transformation was cell- associated; several radiolabelled components were evident in the culture filtrate but only in trace amounts, precluding their identification.

Transformation of paxilline to 14Qf-hydroxypaxilline was enhanced by prolonged incubation with A. loliae. It occurred only in cultures that were at least 10 days old at the time of 1AC-paxilline addition (see Table 19 and Figure 38) and therefore had passed the period of maximum biomass proliferation (Figure 39). When paxilline was added during the early stages of culture (days 1-8) transformation never occurred, even after prolonged incubation (Table 20). ! Ofy

Figure 37. Autoradiograph, and Thin Layer Chromatograph of Acetone Extracts of .4. loliae Mycelium (left) and Culture Filtrate, After Incubation with !AC-Paxilline in a GPYE Culture

1 = paxiiline 2 = 14QFhvdroxypaxiIiine

kvu g o i m W c Q>hc + ‘Vc-M* f Ax

rt-.i C-.A MYCELIUM DRY WEIGHT (fl) Figure Time-Courseof39,Mycelium and WeightsDryCultureFiltrate of dry weight;dry o eid f2 or o CneuieDy (Days mycelium Consecutive •= on forofPeriods24Hours loliae A. Atr nuainwt Pxlie in PaxillineCulturesGPYE Incubation Afterwith a filtrate-drycultureweight)

BROTH DRY WEIGHT (g) 128

Figure 38. Thin Layer Chromatograph of Acetone Extracts of .4. loliae Mycelium (top) and Culture Filtrate, After Incubation with

Paxilline in GPYE Cultures for Periods of 24 Hours on Consecutive Days - Including Time-Course of Main A. loliae Acetone Soluble Products

Base-line numbers correspond to number of days of incubation

1 = paxilline 2 = 140Phydroxypaxilline

V J

■ " §

• • .miiM • M imn «I t -tuH

1

•• • • • • • • # . f • 1 ■# • % * # • i 7 (0 t*. •{ t ; s *■ r « -5 (k 17 1* 1*1 Z\ 2 Z OW - PA *

‘A ;» f.» vj.'iv .. *»y Piioft< P^c.-ft - t > p v s O P Nd C. L' ft fV-rs Cl* / r 129

CULTURE AGE DURING WHICH ______INDQLE-DITERPENQID LEVEL___ PAXILLINE WAS NYCELIUM CULTURE FILTRATE INCUBATED (days) PAXILLINE 14QP0HPAX PAXILLINE 14QJ-0HPAX

3-8 ++++'1 ++ -

8-13 +++ + - -

13-17 ++ + - -

Table 20. Transformation of Paxilline after Incubation in A. loliae

GPYE Cultures of different Ages. (1: - = not detected, + = evident,...

++++ = prominent.)

A control experiment (no fungus) demonstrated that any

insoluble paxilline can be removed by the routine method of filtration

(see 2.2.1.a), after incubation in the culture medium. Hence, presence

of the tremorgen with the mycelium may be at least partially accounted

for by its being particulate, but this would only apply in the worst

situation where the fungus was unable to solubilize all added

paxilline.

Co-incubation of D,L-[2-1AC]mevalonic acid (2.5/jCi) and non-

radiolabelled paxilline (1.25mg added days 7 and 9, harvested day 13)

with A. loliae, yielded no radiolabelled products other than those

obtained on incubation of 1^C-mevalonate alone (compare Figures 15.1.2

and 40). 130

Figure 40. Autoradiograph and Thin Layer Chromatograph of Acetone Extracts of A. loliae Mycelium and Culture Filtrate After Incubation with 1^C-Mevalonate and Non-Radiolabelled Paxilline in GPYE Culture (eluent = 19:1 CHCla/acetone)

1 = paxilline 2 = 14Qf-hydroxypaxilline

• 1 • K'H ('VC. P0 ZcrVU +- ? s. +lu-cM/ ■ !+&'/ 131

Livcihba.ti.aa_.of__1 ''C-?axi 11 ine wuii. .A.*. Lol.Li.e_Ln__

Er.adu_c_tiDn_IfeciLu;ii

After addition of paxiliine to A. loliae in paxilline- product ion medium during growth, and before, during and after biosynthesis of paxilline, daily samples were examined by HPLC and autoradiography. The relative intensities of radioactive areas revealed in the autoradiographs (Figure 4i), matched closely the relative abundance of the corresponding compounds on the TLC plates.

Incubation during the main growth phase (Figure 41.1) resulted in rapid disappearance of radiolabel. In contrast, little or no decrease in 1''C-radiolabelled paxilline was observed following its incubation before, during and after paxilline production (Tables 21 and 22, and Figures 41.2-41.4), perhaps owing to inaccessibility of the added metabolite to the necessary enzymes. Consequently, no firm conclusions could be drawn concerning the fate of paxilline biosynthesised by A. loliae. Extraction of freeze-dried mycelium and broth in methanol/water 4;1 yielded no further information. Table 21. Concentrations of Paxiliine in Endophyte Culture to which had been added 1AC-Radiolabelled Paxilline at Different Stages. <1: -

= not tested; 2: = culture harvested; 3: Cpaxiliine] 1 minute before/after addition of 1'xC--radiolabelled paxilline to the culture;

4: small level.) 133

INCUBATION ADDED TO CULTURE STAGE. AT WHICH 1,1C~PAX WAS ADDED TIME (days) CULTURE (fig/ 100ml) GROWTH PRE-PROD CO-PROD POST-PROD

8 GROWTH.- 449.0 — 1 - - -

9 13.3 - - -

10 0. 8 - - -

11 0. 0 - - -

12 II -- -

13 II -- -

14 if - --

15 - --

16 -- -

17 - - -

18 - - -

19 -- -

20 - 0. 0 0. 0

21 0. 0 11 II

22 II II It

23 II II II

24 PRE-PROD: 571.0 ::: 0/28 II II

25 25.6 II +-1

26 POST-PROD: 571.0 19.6- II 0/71

27 17. 9 II 68. 0

28 II 38.8

29 II 14. 7

30 CO-PROD: 714.0 100/428 11. 1*

31 378. 0

32 364. 7

33 349.5- 1 3 4

Figure 41. Autoradiographs and Thin Layer Chromatographs of Acetone Extracts of A. laliae Mycelium and Culture Filtrate After Incubation with 1^C-Paxilline in Paxilline Production Medium

41.1. Incubation During Growth (1^C-paxilline added: day 9; culture harvested day 13; eluent = 19:1 CHCls/acetone)

*•* <\

ft •< 'lL ff ( f i d ) 135

41.2. Incubation Before Paxilline Production (1^C-paxiiline added: day 24; culture sampled day 25, 26, 27, 28; left: CHCl.a extracts; right: methanol extracts; eluent = 14:1 CHCls/acetone)

i'T ft n i8 Vaa*

t * A i fV^c-AAyCcoUoLE,) (Vveow . IV'.( c-.X 136

41.3. Incubation During (left; '^C-paxilline added; day 30; culture sampled; day 31, 32, 33) and After ('AC-paxilline added: day 26; culture sampled: day 27, 28, 29, 30) Paxilline Production (eluent = 19:1 CHCla/acetone; M = mycelium extract, B = broth extract)

*»Aa 3! Jl 32. .‘i m •'» ■»» 8 K c ►v - '* ** 'n 14* &« ^g i8i

sJl 137

41.4. Incubation During (sampled day 31, left) and After (sampled day 27) Paxilline Production (eluent = 16:1 CHC1 .,/acetone)

1 paxilline 2 14Qf-hydroxypaxi 11 ine 138 >

COMPOUND RELATIVE ABUNDANCE CULTURE INCUBATION TIME (days) 1nCPAX g r o w t h . PRE-PRODUCTION: COMPOUND 9 13 25 26 27 23 31 32 33 27 28 29 30

++:- ++ ++ +

PAX3 +++++ ++ + ++++ -f+++ ++++ ++ ++++ ++++ ++++ ++++ +++ +++ + +

OHPAX'1- +++ + ++ ++ ++ ++ ++ ++ ++ +++ +++ +++■ ++■+•

++ ++ ++ ++ + ++ ++ ++ + + +

++ + + + + ++ ++ ++ + + +

ORIGIN + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ -H-

Table 22. Diagrammatic Summary of Figure 41. <1: ie of paxilline by A.

loliae; 2; + = evident,... +++++ = prominent;PAX =paxi1line;OHPAX =

14Q*-hydroxypaxilline. ) 139

3.6. Tremorgen Investigation in Lolium parenno

3.6. 1. E x amlnaiLq n of Ryegrass Seeds

T rylitrem 3 and, for the ii.r s t t, 1me. p a x i 11 i ne v/e r e de t ec t ed

(3.25 and l.BOppm, respect i ve 1 y 30P Pigure 42) in ryegrass seed material shown to be 100% infected with .4. loliae, the seeds havi ng been obta ined from English pasture -Ware) on which RGS had recently occurred.

3.6.2. Introduction of :'JC-Eadiola belled Paxil line. into 4. loll <3

Infected Seedii

Figure 43 shows clear evidence of substantial uptake of noa- transformed paxiiline by ryegrass seedlings; about 14,36mg left the original solution. The presence of the radiolabeiled tremorgen was particularly clear in the blade extract. Possible transformation products were also evident, being more apparent in the root/lower sheath extract. Differential transportation and/or transformation of metabolites (possibly including impurities in the administered paxilline) may account for these discrepancies. Lolitrem 3 was not detected. 140

Figure 42 /methane Hxtl- I” 3j 'traCT':: Sb Seed

>wer fiucrimetri detection; excitation wavelength = 268nm emission wavelength = 450nm scale expansion = x 200 column = silica Resolve ' Z-Module* Cartridge mobile phase 15; 1 CH ;::C 1 / CH.^Cl'I solvent = CHCla flow rate ml/min. column = M - jLlBondapak ‘ Z-Module' Cartridge mobile phase CKsCia solvent = CHC1 flow rate = 2ml/min. 14 L > 268 142

Figure 43. Autoradiographs and Thin Layer Chromatographs of Extracts of L, perenne Seedlings After Incubation with 1^C-Paxilline

43.1. eluent = 10:10:1 CHCla/hexane/acetone

1 = residual 1'1C-paxilline in incubation vessel 2 = root/sheath extract 3 = blade extract 4 = 1AC-paxilline standard

1 2 3 4 43.2. eluent = 8:1 CHCla/acetone

1 = 1AC-paxilline standard 2 = root/sheath extract 3 = residual 1^C-paxilline in incubation vessel 4 = 1AC-paxilline after prolonged exposure to air

• t

i i ,.v»P r-l C:X

.» */ ♦ * «t' % fc { «/< 'V** “t fAx 144

3.7. Administration of Indole-Diterpenoids to Mice

Parenteral administration of paxilline into mice resulted in mild tremorgenic symptoms after 8 minutes, becoming prominent 12 minutes later and continuing for over an hour. Mice were unaffected by administration of an identical amount of 140f-hydroxypaxilline. Clearly

14Gf-hydroxylation, had a profound effect on biological activity.

3.8. Mutagenic Treatment of A.loliae

All survivors of UV irradiation were morphologically indistinguishable from native endophyte. Thin layer chromatograms of culture extracts were also identical, except for those of survivor 6 which consistently showed paxilline whether the strain had been cultured in GPYE or paxilline production medium (Table 23 and Figure

44). Moreover, this paxilline persisted in GPYE cultures of the survivor, even after death. 145

Figure 44. Thin Layer Chromatograph of Acetone Extracts of A. loliae UV Survivor (left) and Native A. loliae Mycelium (M) and Culture Filtrate (B) After Incubation in GPYE (eluent = 38:1 CHCla/acetone) p = paxilline 146

PAXILLINE LEVEL INCUBATION 4. loliae UV- IN CULIURE__ TIME IN SURVIVOR IRRADIATIQN PAX-PROD PAX-PROD NUMBER TIKE (secs) GPYE MEDIUM MEDIUM (days)

1 0 - 36

3 12 - - 35

4 II -- 41

5 II - - 43

6 II ++++ ++++ II

7 16 - + T + II

8 II - + + + II

9 II - - II

10 25 - - II

11 II - - 47

12 II - - II

14 37 - - II

15 It - - II

16 II - II

17 II - - II

18 II - - II

19 II - - II

20 41 - - II

21 61 - - II

22 70 - - II

23 II - - II

24 82 - + + + II

Table 23. Occurrence of Paxilline in Cultures of A . loliae UV

Survivors. (1: cultures harvested on day 35; 2: - = not detected, +t++

= prominent. ) 147

4. DISCUSSION

Significance of A. loliae Sterols

A . loliae, the ryegrass endophyte, and A. coenophialum, an endophyte of tall fescue grass (Festuca arundinacea Schreber), together form a sub-set of Acremonium based on profound morphological differences from all other members of the genus (see Introduction).

These two species are morphologically distinguishable from each other by subtle differences of the spores and spore-bearing cells (Morgan-

Jones and Gams, 1982; Latch et al.} 1984). However, sporulation in A. loliae is rare, making morphological distinction between the two species impractical. In view of this, the differences between'their sterols may ease identification of these fungi. Unfortunately, both species produce ergosterol peroxide (see Results and Davis et al, ,

1986). However, although the dihydraergosterols produced by A. loliae are both very difficult to discern chromatographically from ergosterol, produced by A. coenophialum, these prominent sterols are readily distinguishable by mass spectrometry. Furthermore, the absence from A. loliae culture of the prominent .4. coenophialum sterol, ergosta-4,6,8(14),22-tetraen-3-one (which fluoresces bright green under 365nm light), potentially allows a valuable and very simple means of differentiation between the species by TLC analysis of organic solvent extracts of their mycelia. The prominence of the sterols of A. loliae also potentially allows assessment of infection of ryegrass with the endophyte. 1 4 8

Enhancement of Agronomic Vigour by A. loliae

Tryptophol, unlike the other prominent compounds isolated in substantial quantity from A. loliae culture filtrate, has a range of biological activities (Lingappa at al., 1969; Seed et al., 1978;

Sugawara and Strabel, 1987). Mast significant to the present study is its action as a growth-promoting hormone in several plants (Rayle and

Purves, 1967). This is probably accounted for by the structural similarity of tryptophol to the important plant hormone indole acetic acid. It may be, therefore, that the ability of A. loliae to enhance the agronomic vigour of L. perenne (Latch et ai., 1985) is, at least partly, due to production of tryptophol by the endophyte in vivo.

The Role of A. loliae and Paxilline in Lolitrem Biosynthesis

The determination of the biological origin of lolitrems has been considerably advanced by the finding that A. loliae biosynthesises paxilline. Strong evidence has thus been provided that the endophyte makes a major contribution to both the biosynthesis of the lolitrems and the etiology of RGS, but the biological origin and characterization of the transformation of paxilline to lolitrems still remain to be elucidated.

Paxilline production in A. loliae culture was not coincident with growth and occurred only in well-grown cultures, that remained viable for many days after achieving maximum growth, and in which viability persisted long after paxilline production. The presence of pectin in culture media seemed beneficial to, but not always essential for, paxilline production. It is possible that, by altering the rheological properties of the medium, pectin provided suitable physical conditions for production, perhaps leading to the large mycelial pellet size that also seemed important, possibly by imposing 1 4 9 intra-pellet constraints on aeration. If so, the morphological change found to occur by using a low level of inoculum and reduced agitation might allow paxilline biosynthesis in the absence of pectin.

Paxilline may act as a natural biosynthetic precursor of lolitrems. If so, transformation of paxilline would involve prenylation of both the indole benzene ring and the diterpenoid moiety, as well as the appropriate bonding of 0 atoms, including epoxidation of the 17-18 carbon double bond. The mechanism of this transformation is postulated in Figure 7, and gains much greater importance in the light of the circumstantial evidence suggesting paxilline to be a genuine intermediate in lolitrem biosynthesis.

There are several examples of attachment, by fungi, of isoprenoid units to the benzene ring of indale-diterpenoids’ (see

Figures 1 and 6). The oxygen atom attached to carbons 31 and 32 of lolitrem has analogy with that of the hydroxylated isoprene substituent at the same position on the aromatic ring in paspalitrem

B. Epoxidation of the C17-18 double bond is also evident in indole- diterpenoids (penitrems A, B, E and F). That A. loliae, rather than the ryegrass host, performs the corresponding transformations of paxilline in nature, is clearly suggested by all these precedents.

Attachment of a single isoprenoid substituent to two 0 atoms of the diterpenoid moiety is a unique feature of the lolitrems.

Isoprenoid attachment to one 0 atom is achieved by plants, in phaseolin, for example. However, the presence of an isoprenoid substituent bound to both an oxygen and a nitrogen atom, in tie verruculogens and fumitremorgin A, favours the fungus, rather than the plant, as the biological source for the corresponding biosynthetic step in lolitrems. 150

The presence of paxilline in culture for only one day admits the possibility of an anabolic fate for the tremorgen, perhaps after substrate-induction of an appropriate enzyme. However, no experimental evidence could be found to support the possibility that paxilline is converted by ,4. loliae to a molecule further along a pathway leading to lolitrems - despite incubation of radiolabelled paxilline with the endophyte during the period of active biosynthesis of paxilline. The present study showed that, at least in optimum growth conditions, the endophyte was unable to add radiolabelled mevalonate to unlabelled paxilline, when the two metabolites were concurrently incubated in culture. This is inconsistent with prenylation of paxilline being performed by -4. loliae. However, it is possible that the exogenous paxilline was unable to reach the enzymic site of transformation of biosynthesised paxilline. Thus clarification of the metabolic fate of paxiliine may be better achieved by feeding 1^C-mevalonate during biosynthesis of the tremorgen. It is clear that there are efficient uptake mechanisms for mevalonate, incorporating it into sterols.

However, there would be little competition from primary metabolism, particularly sterol biosynthesis, at the necessarily advanced culture stage, and the fate of biosynthesised paxilline incorporating radiolabel might be followed.

The view that paxilline may be a natural biosynthetic precursor of lolitrems was further supported by its presence with lolitrem B in

A. lolia&-infected ryegrass seed, obtained from English pasture on which RGS had recently occurred. Furthermore, the finding, in this situation, of an indole-diterpenoid {paxilline) known to be biosynthesised by .4. loliae, is strong evidence in favour of a major role for the endophyte in lolitrem biosynthesis. A need was indicated 151 for further investigation in order to establish consistent correlation between A. loliae and the two tremorgens in perennial ryegrass, and between paxilline and lolitrem incidence.

Paxilline is as acutely potent a tremorgenic neurotoxin in the mouse as is lolitrem B. The tremorgenicity induced by paxilline in the present study was similar in severity to that caused by lolitrem B

(Gallagher and Hawkes, 1986). Clearly, the presence of paxilline in ryegrass seed implies involvement of paxilline in causing RGS. Bearing in mind the fact that the presence of the paspalinines in pasture, in conjunction with their experimentally demonstrable tremorgenicity, was regarded as sufficient evidence to confirm the paspalinines as the cause of PS (Cole et al.t 1977a), it is even possible that, had the production of paxilline by A. loliae been demonstrated before lolitrems had been found in ryegrass, the lolitrems might never have been discovered. Nonetheless, the lolitrems are believed to be the major, if not the only, direct natural cause of RGS by virtue of their tremorgenic effects being greatly prolonged (Gallagher and Hawkes,

1986) compared with all other tremorgens, including paxilline. This property is particularly pertinent in view of the protracted symptomatology of RGS.

After incubation of radiolabelled paxilline with endophyte- infected ryegrass seedlings, no lolitrem B, radiolabelled or otherwise, was detected. This observation seems inconsistent with a role for paxilline in lolitrem biosynthesis, although very substantial transformation of absorbed 1AC-paxilline was evident. New radiolabelled components were observed, possibly resulting from this transformation of paxilline. These were present in amounts too law to allow their characterization, preventing assessment of their involvement in the transformation of paxilline to lolitrems. It is possible that the added paxilline did not become available to the 152 enzymes involved in transformation of paxiiline that may have been biosynthesised in regions of intimate association of endophyte and host cells. Furthermore, there is reason to believe that ryegrass seedlings contain only low levels of endophyte; if A. loliae is

involved in lolitrem formation, infected seedlings are unlikely to be the best experimental subjects for testing such biosynthesis.

Conducting the experiment with much more mature plants, in which the extensive incidence of endophyte mycelium had been demonstrated, might provide more information. Additionally, incubation of 1/JC-paxilline in homogenates or cell-free extracts of infected and uninfected ryegrass may prove informative. However, the present experiment itself deserves repeating on a large scale with investigation of the fate of paxilline in greater detail.

Demonstration of direct transformation of paxiiline to lolitrems seemed impassible to achieve in the situations tested.

Perhaps the in vitro conditions used were totally unsuitable to the expression of steps necessary to convert an indole-diterpenoid such as paxiiline towards the structure of lolitrems. Further investigation is necessary in order to characterize this transformation and to determine its biological origin. In spite of this, the biosynthesis of paxiiline by the endophyte, in conjunction with the earlier arguments, strongly supports the hypothesis that a major contribution is made by

A, loliae in the biosynthesis of lolitrems. 153

Mutagenesis of A. loliae as a Means of Preventing RGS

The isolation, after mutagenic treatment, of an A. loliae variant, capable of producing paxilline even when cultured in GPTE medium, actually enhances the possibility of eventually selecting a non-toxic strain of the endophyte. This paradox will be explained later. Such a strain could be reintroduced into ryegrass with the aim of avoiding RGS.

Assuming the new isolate to be a genuine mutant, two contrasting alternatives exist regarding the nature of the mutation.

The variant may be able to produce paxilline constitutively; it thus yields paxilline without specially formulated nutrients or physical conditions. Alternatively, the strain may be defective in its paxilline metabolism, and hence unable to anabolise or catabolise the paxilline it produces, causing the tremorgen to remain as an "end- product" . Determination of which is correct is essential for designing an effective approach to further selection for a non-toxic strain.

At first sight, it might seem more likely that the mutant isolate produces paxilline constitutively, since the tremorgen has never been detected in GPYE cultures of the native endophyte. However, consider the possibility that the detection of paxilline (which is present only transiently) in cultures of native endophyte (such as pectin-supplemented cultures) is achievable only because of the greatly extended viability of such cultures. If the life-span of batch cultures is shortened, as when using GPYE, paxilline may still be produced, but be present for such a short period that its detection is rendered extremely unlikely, if not impossible. That is, paxilline biosynthesis might occur in all GPYE cultures but the tremorgen might always be rapidly further metabolised. This contrasts with prominent accumulation of paxilline in pectin-supplemented culture. The observed 154 presence of paxilline in GPYE cultures of the mutant variant may, therefore, be due to blockage of the normal paxilline metabolic pathway and not to constitutive production.

The mutant is a prime candidate far further selection of a strain of A. loliae incapable of producing tremorgens. Production of paxilline by the mutant in GPYE is reproducible, making screening for a non-producing strain much easier than when using native endophyte.

Growth in GPYE of cultures derived from isolates that had survived further mutagenic treatment, would enable assessment (using chromatography) of their ability to produce paxilline. However, the value of such an assessment depends upon resolution of the nature of the mutation.

If the original variant - variant 1 - produces paxi'lline constitutively, it is obviously not a non-toxic strain. However, a non-producing mutant - variant 2 - selected following mutagenic treatment of variant 1, would probably be unable to biosynthesise paxilline in any growth medium. In this case inability to detect paxilline production would be a true reflection of the biosynthetic limitations of the strain.

Importantly, variant 2 would be likely to be stable in the parasitic association with ryegrass. The site of operation of any selective advantage has to be the meristematic stem apex, where a revertant would have to grow faster to become dominant in the plant thereafter. Indole-diterpenoid biosynthesis is unlikely to affect growth rate and, therefore, is probably not of any selective advantage to the endophyte in the natural situation. Variant 2 would thus remain non-toxic after introduction to ryegrass - an essential attribute of a strain potentially incapable of causing RGS. Further, more detailed study is essential to more fully characterize the association of A. 155 loliae with the ryegrass meristem, and to determine the importance of the exact position of mycelium in the stem apex with regard to dissemination of the endophyte.

If, on the other hand, the metabolism of paxilline by variant 1 is defective, then variant 1 may be a suitable candidate for potentially preventing RGS without further mutagenesis. This is explained by the possibility, discussed previously, that the formation of lolitrems may necessitate further metabolism of paxilline by A. loliae itself. Variant 1 might be defective in such metabolism and hence lolitrems would never be biosynthesised, even if the variant was in association with ryegrass. Inability to cause RGS, in this case, would depend on the toxic contribution from paxilline in the grass being negligible. Clearly, if variant 1 has a blockage in paxilline metabolism, further mutagenic treatment might simply unblock the defect, and a strain found unable to produce paxilline in GPYE might be a revertant to the native endophyte, which would be undesirable.

Characterization of the mutation of variant 1 is clearly essential if an effective strategy for obtaining a non-toxic endophyte is to be devised. Evidence perhaps favouring the view that paxilline production is constitutive was provided by comparison of the acetone- soluble products of GPYE cultures of native endophyte and variant 1.

Ho substantial differences were observed, except paxilline in variant

1

Despite the absence of thorough characterization of the variant, it remains an extremely interesting strain with respect to preventing RGS. Like native A. loliae, the mutant biosynthesises 156

tryptophoi. An ideal strain of .4. lolije would be incapable of causing

RGS, but still confer all the advantages to the grass of the native endophyte, such as its insect anti-feedant properties and its growth- promoting influence. The ideal strain would be introduced into ryegrass bv artificial infection (Latch and Christensen, 1685’. The

"new cultivar" would then be multiplied, initially by clonal propagation, and later by allowing to run to seed. Seed multiplication would provide a commercial cultivar for generating new ryegrass pastures on which RGS could not occur but which maintained their high grass yield, improved drought tolerance, and insect-feeding deterrency. Vith the discovery of the ability of A. loliae to biosynthesise paxiiline, tryptophoi (both in the present work) and peramine (Rowan et al., 1986), the possibility exists, for the first time, for the screening and selection of the ideal endophyte strain.

Significance of A. loliae Products to Indole-Diterpenoid Biosynthesis

The production, by both A. loliae and P, paxilli, of paxilline and a group of close structural relatives of paxiiline, not only demonstrates very similar biosynthetic capabilities in the two organisms, but also provides new information concerning the mechanism of biosynthesis of indoie-diterpenoias.

Examination of the structures of the four metabolites suggests that prepaxilline-16j8-ol may be a biosynthetic precursor of the other three. Significantly, therefore, prepaxilline-16/3-ol seems to be an intermediate between the hypothetical molecule prepaspaiine (see

Figure 5) and paxilline. Further transformation of prepaxiiiine-16jS-ol may occur via the hypothetical and unisolated molecule prepaxilline.

140f-hydroxypaxilline might be considered to be derived by a single hydroxylation of either paxilline or 14Gf-hydroxyprepaxilline, and Figure 45. Proposed Pathway of Hydroxyiation in the Formation of Indole-Diterpenoids of A. loliae

E = enzyme (ie a hydroxylase) S = substrate

PAXILLINE

PREPASPALINE (s >(£)-($) rds > ( e s ) ------> 14a-HYDROXYPREPAXILLINE

14a- HYDROXY PAXILLINE 1 5 8 hence would seem to come last in the sequence. However, the order of biosynthesis of paxilline and I4Gbhydroxyprepaxilline is less easy to deduce on the basis of structural complexity alone.

Recent thinking relating to the cephalosporins (Baldwin, 1987) revised the approach to assigning the biosynthetic order of "families" of secondary metabolites. It has been proposed that the enzymes forming such families act via indiscriminate functions of enzyme- substrate complexes. Once the complex is formed and activated, slight structural changes may occur at the active site of the enzyme, leading not to one product but to many. The relative abundance of each product is determined by the frequency of the corresponding structural change in the enzyme.

Application of these ideas in the present context leads to the postulated pathway shown in Figure 45. Prepaxilline, or possibly the known molecule prepaxilline-16/3-ol, is first bound to a single hydroxylase. The resulting complex becomes activated. Subsequent conformational changes at the enzyme's active site allow direct formation of the three isolated products from the same enzyme- substrate complex. It may be incorrect, therefore, to place an order of biosynthesis on the three products.

If the essence of these ideas is true, it is clear that the biosynthesis of paxilline would always occurs with that of 140f- hydroxyprepaxilline and 14Qf-hydroxypaxilline, which would consequently appear as co-prcducts, as in the present study.

Incubation of paxilline with A . laliae in GPYE culture caused substantial, but not complete, transformation of the tremorgen to

14Qthydroxypaxilline alone. From the preceding discussion, it is clear that the final stages of biosynthesis of both paxilline and 14Qf- hydroxypaxilline may occur using the same enzyme. This enzyme must thus be assumed to have been constitutive in .4. laliae GPYE culture, 159 despite the apparent absence of biosynthesised paxilline. The transformation of added paxilline to 14Gf-hydroxypaxilline occurred only in the later stage of culture, when autolysis was evident. It seems that only when the hydroxylase was released from the cells was the hydroxylation of exogenous paxilline possible; entry of added paxilline to the fungal cells must have been very difficult. This supports the results of addition of 1nC-radiolabelled paxilline to paxilline-producing A. loliae culture, discussed earlier. In addition, such evidence favours the biosynthesis of paxilline by A. loliae, even in GPYE culture, although the tremorgen may exist only for a very short period. Correspondingly, this implies the endophyte mutant

(variant 1), previously discussed, to be defective in its paxilline metabolism rather than being a constitutive producer.

More information concerning the order of biosynthesis of paxilline-related compounds could be obtained by adding 14Q£- hydroxyprepaxilline to A. loliae GPYE culture. In this case, as in the case of added paxilline, only 14Qf-hydroxypaxilline should result, with much of the added precursor remaining. Addition of prepaxilline-16/3-01 in a similar manner would be predicted to lead to its complete transformation to all three hydroxylated products, probably in th.e relative proportions observed in paxilline-biosynthesising cultures.

The finding that 14C£-hydraxypaxilline is non-tremor gent c apparently conflicts with the assumption that the existence of a hydroxyl group in the 19 position confers tremorgenicity. Furthermore, additional hydroxylation at C14 rendered a tremorgenic 19-hydroxy compound (paxilline) relatively non-toxic.

The 14C£-hydroxy indole-diterpenoids have structures favourable to a condensation reaction resulting in formation of a seventh ring, as in paspalinine and aflatrem, for example. In Claviceps paspali,

14Q{-hydroxyprepaxilline and 140f-hydroxypaxilline may, therefore, be 160 natural biosynthetic intermediates of paspalicine and paspalinine, respectively. It is of note that this conflicts with the proposal

(Gallagher et ai., 1980) that paspalicine is derived by direct transformation of paspalinine.

If paxilline is a genuine intermediate in lolitrem biosynthesis, the fact that A . loliae seems not to allow further cyclization of the 140f-hydroxy indole-diterpenoids may be of paramount importance to the formation of lolitrems. It has been proposed, here, that the 140£-hydroxy compounds are unavoidably biosynthesised at the same time as paxilline. By not allowing formation of a cyclic anhydride, A. loliae ensures that the hydroxyl group of the terminal isoprenoid unit of these 14Qf-hydroxyl at ed products is free, as is that of paxilline, to bind the appropriate isoprenoid unit necessarily added in lolitrem biosynthesis. It follows that a co-product of lolitrem B (and C) would be the corresponding 14Qbhydroxylated lolitrem. Vith this in mind, it is pertinent to consider lolitrem A, the structure of which is as yet unreported. EIMS indicated lolitrei A to bear one 0 atom more than lolitrem B, and showed an intense fragmentation ion at m/e 348 (Gallagher et al.y 1981) due to cleavage of the C10-11 and C20-21 bonds (as is the case for lolitrem E>, indicating the extra 0 atom to be to the "right" of this cleavage point. On the basis of this information and the preceding discussion, it may be predicted that lolitrem A is in fact 14Ctf-hydroxylolitrem B.

Elucidation of the structure of lolitrem A could thus provide other fascinating information concerning indole-diterpenoid biosynthesis, and possibly give mechanistic evidence in support of the hypothesis that paxilline, biosynthesised by A. loliae> is a natural biosynthetic precursor of lolitrems. 161

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