The Microbiological Transformation of Diterpenoids

J. R . Hanson School of Molecular Sciences, University of Sussex, Brighton, Sussex BN 7 9QJ

Reviewing the literature between 1973 and June 1991

1 Introduction 2 Xenobiotic Transformations

2.1 Bicyclic Diterpenoids 2.2 Tetracyclic Diterpenoids

3 Biosynthetically-directed Microbiological

3.1 Transformations with Gibberella fujikuroi Transformations

3.1.1 Biosynthetic Pathways in Gibberella fujikuroi 3.1.2 Transformations of Steviol 3.1.3 Transformations of ent-Kaurene C-19 Esters 3.1.4 Transformations of ent-Kaurenes Modified on Ring A 3.1.5 Transformations of ent-Kaurenes Modified on Ring B 3.1.6 Transformations of ent-Kaurenes Modified on Ring c 3.1.7 Transformations of ent-Kaurenes Modified on Ring D 3.1.8 Transformations of Other Diterpenoid Skeleta 3.1.9 Transformations of Gibberellins

3.2 Transformations with Trichothecium roseum 3.3 Transformations with Cephalosporium aphidicola

4 Conclusion 5 References

1 Introduction Microbiological transformations fall into two major types. On the one hand there are xenobiotic transformations in which the substrate is completely alien to the micro-organism. These transformations utilize enzyme systems of relatively low substrate-specificity but with a definite regio-specificity that is often a characteristic of the individual micro-organism. The second type of transformation are those that are bio- synthetically directed and are sometimes known as analogue biosyntheses. In these the substrate is an analogue of a biosynthetic intermediate and the transformation relies on the flexibility of the existing biosynthetic pathways. Steroid microbiological hydroxylationsl are well-known examples of xenobiotic transformations. Typically these transformations involve only one or two enzymic steps. In contrast analogue biosyntheses may well involve a series of steps and consequently the overall efficiency of the process may well be less than that of the xenobiotic transformation. Analogue biosynthesis has recently been used to good effect in the study of the mechanism of penicillin biosynthesis.2

The diterpenoids are a large family of natural products3 possessing a variety of skeletal types. They display a wide range of biological activities. Amongst the diterpenoids are tumour inhibitory substances, antibiotics, hypertensive agents, psycho- tropic agents, sweeteners, bitter principles, perfumery inter- mediates, insecticides, anti-feedants, phytotoxic compounds, and plant growth hormones. Some diterpenoids are readily available whilst others are rare. The branched-chain nature of their isoprenoid backbone leads to quaternary centres in their structure which provide a block to many conventional chemical manipulations and hence the remote functionalization available through bio-transformation is of considerable value in the partial synthesis of bio-active compounds. Furthermore, the regio-specificity of such processes may contrast with that available from photochemical transannular reactions.

(1) R’ = H2, R2= H (2) R’ = a - H , P-OH, R2 = H (3) R’ = 0, R2 = H (4) R’ = H2, R2 =OH (5) R’ = HE, R2 = H, 7a,8a epoxide

Systematic studies of steroid microbiological hydroxylations have led to their rationalization in terms of geometrical relationships between a binding site and the site of hydr~xylation.~ Whilst some diterpenoids possess a formal similarity to the steroids, their stereochemical differences have shed further light on the geometrical requirements for hydroxylation. The variety of diterpenoid skeleta have also been used to shed light on the stereochemical constraints of biosynthetic processes through analogue biosynthesis.

In this review we will illustrate these various facets of microbiological transformations from recent fungal work on the diterpenoids. The work prior to 1973 has been reviewed5 in ‘ Microbial Transformations of Non-Steroid Cyclic Com- pounds ’ by Kieslich.

2. Xe n o b i ot i c Transformations 2.1 Bicyclic Diterpenoids Plants of the genus Grindelia (Compositae) produce a resin containing the diterpenoid grindelic acid (1). Derivatives of grindelic acid possess insect anti-feedant properties.6 In studies directed at enhancing this activity, grindelic acid has been converted’ in good yield by Aspergillus niger and Penicillium brevi-compactum into the 3P-hydroxy derivative (2). The 3- ketone (3), 18-alcohol (4), and 7a,8a-epoxide (5) were minor products obtainede from large-scale fermentations with A . niger. The bio-transformation has been extendedg to methyl 7a,8a-epoxygrindelate and 6,8( 17)-dehydrogrindelic acid.

A similar functionalization of ring A of the perfumery intermediate sclareol (6) to form the 3P-alcohol (7), the 3- ketone (8), and the 2a-alcohol (9) has been achievedlO with Septomyxa aflnis. A Cunninghamella species will hydroxylate sclareol to form 3P-hydroxy-(7) and 18-hydroxysclareol (10) in 36 and 50% yield respectively. A similar transformation of sclareol and manool is effected1’. l2 by Mucor plumbeus. Other transformation^'^, by bacteria (Nocardia restricta), lead to degradation of the side chain, elimination of the 8a-hydroxyl group, and oxidation at C-18. The more extensive degradation observed with bacteria, such as the cleavage of the side chain, is typical of the difference between fungal and bacterial biotransformations observed in the steroids.

139

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140 NATURAL PRODUCT REPORTS, 1992

OH

(6) R' =R3=H, R2=H2 (7) R' = R3 = H, R2 = a-H, P-OH (8 ) R' = R3 = H, R2 = O (9) R' =OH, R2 = He, R3 = H

(10) R' = H, R2= H2, R3 =OH

(1 1) R' = R2 = OH, R3 = AC (12) R' = R2 = H, R3 = AC (13) R' = R2 = R3 = H (14) R' = R2=OH, R3 = H

Forskolin (l l) , obtained', from the roots of the Indian medicinal herb Coleus forskohlii, is a potent activator of adenylate cyclase in various tissues. It exhibits anti-hyper- tensive, positive inotropic, bronchospasmolytic, and anti- thrombotic activity with potential therapeutic application in the treatment of glaucoma, cardiac disease, and asthma.15 Consequently, compounds of this type have been the target of considerable synthetic activity.16 In the plant, forskolin (1 1) is accompanied by significant amounts of the inactive 1,9-dideoxy derivative (12). Despite the apparent unlikelihood of finding an organism that would carry out the interconversion a Scopulari- opsis species was f o ~ n d , l ~ ~ ~ ~ in a screening programme of 263 fungi, which converted 7-deacetyl- 1,9-deoxyforskolin (1 3) into 7-deacetylforskolin (14) albeit in low yield. The functionaliz- ation of ring A of 1,9-deoxyforskolin has also been reported using Mortierella i~abe l l ina~~ and Neurospora crassa.zo Although forskolin is rare, manoyl oxide derivatives are quite common. Since forskolin possesses functionality in all three rings, microbiological transformation provides a useful ap- proach to structure:activity studies. A series of bio- transformations of ent-13-epimanoyl oxides have been de- scribed in this context. Thus incubation of ent-3P-hydroxy- 13- epi-manoyl oxide (ribenol) (1 5) with Curvularia lunata gavez1 its ent-6P-hydroxy (16) and ent- la,6P-dihydroxy- (17) derivatives. Other hydroxylations of ent- 18-acetoxy- 16-hydroxy- 13- epimanoyl oxide (1 8) and its ent-6a-acetoxy derivative using Rhizopus nigricans ledz2. z3 to functionalization at C-3 and C-20.

2.2 Tetracyclic Diterpenoids The gibberellin plant growth hormones have attracted con- siderable attention. Their biosynthesis, which has been studied in detail in Gibberella fujikuroi (see below) involves ent- kaurenoid intermediates some of which are more readily available from ent-kaurenes obtained from higher plants. The microbiological hydroxylation of these ent-kaurenes to in- troduce appropriate functionality particularly on ring B has been the subject of a number of ~ tud ie s .~~-~O The results of these are summarized in Table 1.

The hydroxylation of steroid substrates by Rhizopus nigricans has been rationalized4 in terms of an enzyme system in which there are three participating sites each with a binding or hydroxylating capability. These are located in a triangular arrangement corresponding to C-3, C-11, and C-16 (sites A, B, and C respectively) of the steroid nucleus, (22). In an extensionz5 of this scheme, it was supposed that site A was below the plane of the steroid, site B was below or co-planar, and that site C was above. Utilizing this model it was possible to account for the sites of hydroxylation of 17-norkauran- 16-one and ent- 17- norkauran- 16-one by the fungus.

In the course of these investigations the bio-transformations provided the opportunity for resolving a structural problem. The kaurene and phyllocladene skeleta are closely related and

possess similar spectroscopic properties. However, the ent- kaurene skeleton is, by far, the most widespread. Calliter- penone, from the Indian medicinal plant Callicarpa macro- phylla, had consequently been assigned31 the structure (23). However, the microbiological transformation of 17-norphyllo- cladan-16-one (21) led to the 3,16-dione which was identical to the product obtained from calliterpenone. The structure of the latter was therefore revised to (24).26*cf. 3 z This reveals another facet of xeno bio tic fungal micro biological trans for ma tions, namely that the carbon skeleton is only rarely modified and thus it can provide a means of correlating structures when the carbon skeleton may be in doubt.

The key gibberellin biosynthetic intermediate gibberellin A,, 7-aldehyde (26) is conveniently prepared by the chemical ring- contraction of derivatives of 7~-hydroxykaurenolide (25). Whereas in some higher plants the hydroxylation of gibberellins on ring c, particularly at C-13, takes place at this C,, level, hydroxylation of C-13 is the last stage in gibberellic acid biosynthesis in Gibberella fujikuroi, i.e. 13-hydroxylated ent- kaurenes and kaurenolides are not major natural metabolites of G. fujikuroi. In order to study gibberellin biosynthesis in higher plants, a 13-hydroxylated derivative of gibberellin A,, 7- aldehyde was required. Hydroxylation of both 7a- and 7/3- hydroxykaurenolides (25) and (27), by Rhizopus a r r h i z ~ s , ~ ~ , 34

and 3/3,7P-dihydroxykaurenolide (28) by R. n i g r i ~ a n s ~ ~ gave the 13-hydroxy derivatives (20)-(31), together with some 1 la- hydroxy derivatives (e.g. 32). These hydroxylations also fit the model proposed for the steroid hydroxylations with the 7- hydroxyl group binding at either site B to give 13-hydroxylation from site A, or site C to give 11-hydroxylation from site B. These bio-transformations were to prepare labelled material.

The microbiological hydroxylation of the more readily accessible gibberellins to produce their rarer analogues has hitherto not been particularly successful, possibly because the compounds are too polar. Whereas incubation of gibberellin A, with Rhizopus nigricans merely led to hydration of the double-bond, hydroxylation of the methyl ester (33) afforded36 the rarer gibberellins GA,, (34), GA,, (39, and GA,, (36) as their methyl esters.

The shape of the ent-kaurenoid diterpenoids lends itself to mapping the active-site of the microbial hydroxylases. A number of ent-kaurenones have been e ~ a r n i n e d ~ ~ - ~ ~ in this context. Rhizopus nigricans, Curvularia lunata, and Aspergillus niger converted the 16-enes to the 16,17-glycols. R. nigricans and C. lunata reduced the ketones at C-3 and C-7 to give the P- alcohols. With ent-kaur- 15-enes R. nigricans also introduced hydroxyl groups at C-13 and at C-3 and, together with C. lunata, at C-17.

Stevioside is an intensely sweet glycoside which is used in some parts of the world as a non-nutritive sweetener. The microbiological transformation of the methyl ester of its aglycone, steviol(37), by Rhizopus stolonifer (syn. R. nigricans)

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NATURAL PRODUCT REPORTS, 1992--5. R. HANSON 141

Table 1

Position of hydroxylation

Substrate ent-17-norkauran-16-one (19)

17-norkauran- 16-one (20)

17-norphyllocladan- 16-one (21) ent- 17-norkauran-3-one

ent-3P-hydroxy- 17-nor-kauran- 16-one

ent- 17-norkauran-3,16-dione ent- 19-hydroxy- 17-nor-kauran- 16-one

ent- 17-nor- 16-oxokauran- 19-oic acid

ent-kaur- 16-en- 19-oic acid

ent- 17-hydroxykauran- 19-oic acid

C. decora 1 a, 6P3’ la , 7p

-

-

7a3’ 13 7a, 11p 7a3’

7a3’ 1 2 9

7a 129 7a 7P 7aZ9 15a 7a, 1501 7aZ8 7P

R . nigricans A . niger/A. ochraceus 3aZ5 3aZ6 la, 301 3a, 7a 3a, 7p 3p25 3P5 78 3P, 701 3P, 9a - 3P26

3~24, 26

- -

1 a30 6 P 0 7a 7a

1 2 9 1 6P-OHZ9 7a 1 a 2 9 13” 7a 13,16P-OH 7P

- -

7p29 1601, 17”

R’

Qq-) .../ -

C02H CHo

(33) R’ = R2 = R3 = H (34) R’ = R2 = H, R3 = OH (35) R’ =OH, R2= R3 = H (36) R’ = R3 = H, R2 = OH

(25) R’ = R3 = R4 = H, R2 = a-OH, P-H

(28) R’ = OH, R3 = R4 = H, R2 = a-H, P-OH (29) R’ = R4 = H, R3 = OH, R2 = a-OH, P-H (30) R’ = R4 = H, R3 = OH, R2 = a-H, P-OH (31) R’ = R3 = OH, R4 = H, R2 = a-H, P-OH (32) R’ = R3 = H, R4 = OH, R2 = a-OH, P-H

(27) R’ = R3 = R4 = H, R2 = a-H, p-OH

12 OH

l o

i a . - - C02Me

19

(37)

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142 NATURAL PRODUCT REPORTS, 1992

OH

17

1

(39)

&- I

602H

has been examined40 not only in the context of identifying its mammalian metabolites but also with the object of introducing substituents onto rings A-c for structural correlations within this series of diterpenoids. In this case the C-7p and C-9p hydroxylation products were obtained.

Aphidicolin (38) is a specific inhibitor of DNA polymerase a and it has attracted interest because of its anti-tumour and anti- viral activity. A number of micro-organisms were screened for their ability to transform aphidicolin. Whilst some organisms selectively acetylated or oxidized the existing hydroxyl groups, Streptomyces punipalus gave a 54 % yield of 6P-hydroxy- aphid i~ol in .~~. 42 With similar objectives in mind, the hydroxyl- ation of the relative stemodin (39) to the 7a,7P- and 1401- hydroxy derivatives by Cunninghamella echinulata and to the 19-hydroxy and 17,19-dihydroxystemodins by Polyangium cellulosum has been

3 B iosy nt het ica I I y - d i rected M icro b io log ica I Transformations Diterpenoids are produced by a number of fungi which have been used for biosynthetically-directed transformations in the light of biosynthetic studies.

602H C02H

(42) J J (43)

J &flm -.-*I

=-.*.+- =-.*.*- HO ; _. I - OH 0

C02H CHo C02H Co2H : OH b 2 H

(44)

I __F

OH

co-0

(48)

I fl -.-*I

OH HOCH2 I

CO-0

(46)

t . CHO

HO

co-0

(47)

t O H

(51)

O H

(54) Scheme 1

(55)

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NATURAL PRODUCT REPORTS, 1992--5. R. HANSON 143

OR2 OH

(56) R1 = R2 = H (57) R’ = H, R2 = AC (58) R1 = Me, R2 = H

C02H

C02Me

(63) R‘ =OH, R2 = H (64) R’ = H, R2 =OH

3.1 Transformations with Gibberella fujikuroi The fungus Gibberella fujikuroi produces the diterpenoid gibberellin plant hormones such as gibberellic acid ( 5 5 ) together with a number of other tetracyclic diterpenoids related to ent- kaurene (40) including the kaurenolide lactones (48) and (52).

3. I .I Biosynthetic Pathways in Gibberella fujikuroi The biosynthesis of these metabolites in G. fujikuroi has been studied in a number of laboratories and this has been r e v i e ~ e d . ~ ~ . ~ ~ There are a sequence of events involving a series of divergent pathways (see Scheme 1). Cyclization of geranyl- geranyl pyrophosphate in two stages affords ent-kaurene (40) which is then converted to the gibber ell in^.,,-,^ Hydroxylation and oxidation of C- 19 gives ent-kaur- 16-en- 19-oic acid (41)49* 50

which may undergo either hydroxylaton at C-751 or 6,7- dehydrogenation. 52 The former pathway leads, via ent-7a- hydroxykaur- 16-en- 19-oic acid (42) and an oxidative ring- contraction involving the abstraction of the 6P-hydrogen5, to the gibberellins whilst the latter leads via (44) to the kaurenolide 19 -+ 6 lactones (48). Hydroxylation and oxidation of ent-7a- hydroxykaur-16-en-19-oic acid (42) leads via the diol (45) to fujenal(49). The product of ring-contraction, gibberellin A,, 7- aldehyde (43), is5, either oxidized to gibberellin A,, (47) and then converted to the 3-desoxygibberellins (e.g. 51) or it is hydroxylated to form gibberellin A,, 7-aldehyde (46). The latter is then converted to the 3-hydroxylated gibberellins. There is little crossover between these Although C-20 is lost as C02,58359 the substrate for the conversion of the C,, to the C,, gibberellins is probably the C-20 aldehyde gibberellin A,, (50).,O Gibberellins such as gibberellin A,, (53) appear to be terminal metabolites. At the C,, level, gibberellin

t CIC H2*C H2*& Me)3

CI -

A, (54) is first dehydrogenated to form gibberellin A, and then the 13-hydroxyl group is inserted to form gibberellic acid (55).55756 There are some other minor hydroxylations that the fungus also carries out. In some higher plants the C-13 hydroxyl group is inserted much earlier in the biosynthesis and there are also some differences in the formation of ring A. About 80 gibberellins are now known, mostly occurring in very small amounts. Hence there is a need to prepare them from more readily available substances in order to assess their biological properties and their role in the biosynthesis of other gibberellins in higher plants.

3.1.2 Transformations of Steviol The structure of steviol (56) embodies two features that are involved in gibberellin biosynthesis. The 19-carboxyl group represents an early stage and the 13-hydroxyl group is the last stage in the fungal pathway. Before the sequence of these biosynthetic events was known steviol (56) was considered“ as a precursor of gibberellic acid. However, initial experiments showed that Gibberella fujikuroi did not convert steviol into gibberellic acid but metabolized it into a gibberellin-like compound of unknown structure. Further experiments led6’ to the isolation of 7p, 13-dihydroxykaurenolide (59) from the neutral metabolites.

These early experiments illustrated a major problem of analogue biosynthesis, that of separating the metabolites of exogenous substrates from the endogenous natural metabolites. Two methods have been used to overcome this difficulty. The first utilizes a mutant of G. fujikuroi (B41-la) which is blocked for normal gibberellin biosynthesis at the stage of oxidation of 19-aldehyde to the 1 9 - ~ a r b o x y l . ~ ~ , ~ ~ In this instance the artificial substrates need to possess a 19-carboxyl group for successful analogue biosynthesis. The second method rests on the observation that a number of plant growth regulators block en t- kaurene syn t he tase. 65-68 These compounds do not significantly affect the post-kaurene stages of diterpenoid metabolism in the fungus. Hence the second method is to incubate the substrate with G. jujikuroi in the presence of an inhibitor such as AMO-1618 (60) or CCC (61) which suppresses the formation of the natural metabolites and facilitates the isolation of the metabolites of the exogenous substrate.

The metabolism of steviol (56),,, its 13-acetate (57),,O and isosteviol (62) was subsequently thoroughly investigated using the mutant system and GC-MS for the identification of the metabolites. A number of 13-hydroxygibberellins, which are characteristic of higher plants rather than the fungal system, were identified. The 13-acetate suppressed the 3-hydroxylation and ring A desoxygibberellins (e.g. GA,, (34)) were produced. This bio-transformation was ~sed’O-~~ to prepare labelled giberellins from the more easily labelled steviol. The metabolism of steviol in the presence of plant growth retardants,, and of some steviol derivatives has been examined further. 74

3.1.3 Transformation of ent-Kaurene C-19 Esters Methylation of the 19-carboxylic acid of steviol prevented ring contraction and bio-transformation of the methyl ester (58) led to 7- , 1 1-, and 15-oxygenated metabolites such as (63) and (64).,,* 75 This effect of esterification at C-19 and the requirement

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144 NATURAL PRODUCT REPORTS, 1992

C H20CC H2CH2C02H I I 0

(65) R = a-H, P-OH (66) R = a-OH, P-H (67) R = H2

,.--I fl CH,OH

(74) (75) R’ = OH, R2 = R3 = H (76) R’ = R2 = OH, R3 = H (77) R’ = R2 = R3 = OH (78) R’ = R 2 = H, R3=OH

(79) R’ = CHzOH, R2 = CX-OH, P-H, R3 = H (80) R’ = CH20H, R2 = a-OH, P-H, R3 = OH (81) R’ =CHO, R2= 0, R3 = H (82) R’ = c o 2 ~ , fq2=0, R~ = H (83) R’ = C02Me, R2 = a-OH, P-H, R3 = OH

of a free carboxyl group for ring-contraction had been noted previously. 76 Thus ent-3P-hydroxykaur- 16-en- 19-yl succinate (65) was only hydroxylated at C-76, whilst the ent-3a-epimer (66) was hydroxylated at either the C-7p or C-6p positions. The 19-succinyloxy function was a less-efficient block on its own and thus the succinate (67) was converted not only .to the ent- 7a-hydroxy- and ent-ba,7a-dihydroxy derivatives, but also as a result of hydrolysis to gibberellic acid. The P-hydroxypropionic acid ester of ent-kaur-16-en-19-oic acid was also used to prepare the ent-7a-monohydroxy- and ent-6a,7a-dihydroxy derivatives.

3.1.4 Transformations of ent-Kaurenes Modified on Ring A

The transformation of ent-kaur-2,16-dien- 19-01 (68) afforded7’ a number of gibberellin metabolites including 2,3-dehydro- gibberellin A,, (69), 2,3-dehydrogibberellin A, (70), their 2p,3/3- epoxides, together with the lactone (71). The formation of the latter is reminiscent of the formation of the kaurenolide lactones and presumably arises through opening of the epoxide. Some allylic oxidation at C-1 was also detected7a in the kaurenoid metabolites (e.g. 72).

The transformation of ent-2a-, ent-2P-, and ent-3 a-hydroxy- kaur- 16-en- 19-01s and 2a,3a-dihydroxykaur- 16-en- 19-oic acid by G. fujikuroi has been studied,, using GC-MS. ent-3a- hydroxykaur- 16-en- 19-oic acid (73) gave gibberellins A,, A,, and AI3, 3P,7P-dihydroxykaurenolide and a 3-hydroxylated fujenal derivative. Interestingly, these studies showed7, that an alternative pathway to gibberellic acid could be induced involving ent-2P, 19-dihydroxykaur- 16-ene (74). However,

ent-3p-hydroxykaur- 16-ene (75) was not metabolizedao whilst ent-3,8,18-dihydroxykaur- 16-ene (76) gave ent-3P,7a, 18-tri- hydroxykaur-16-ene (foliol) (77). The 16-ene of the latter was subsequently hydrated. Thus a 3a-hydroxyl group, which is on the same face of the molecule as C-19, appears to exert an inhibitory effect on transformations involving oxidation at C- 19.

3.1.5 Transformations of ent-Kaurenes Mod$ed on Ring B

The oxidation of ring B plays a central role in the divergent gibberellin and kaurenolide pathways. Incubation of ent-7a- hydroxykaur- 16-ene (78) with G. fujikuroi gavea1 gibberellic acid, gibberellins A, and A,, and fujenal, but no kaurenolides, i.e. the order of events, hydroxylation at C-7 and oxidation at C-19, may be inverted. Detectable quantities of 19-desoxy- metabolites were not formed from (78) indicating that oxidation of (2-19 to the level of a carboxylic acid is an important prerequisite of further metabolism of ring B. The absence of kaurenolides may be understood in terms of the divergence of the kaurenolide pathway through the dehydrogenation of ent- kaur- 16-en- 19-oic acid.

The normal biosynthesis of the seco-ring B metabolite fujenal (49) lies through the diol (45)82*83 although some fujenal IS

formed via the kaurenolides. Incubation of a series of 6- oxygenated ent-kaurenes (79)-(82) gavea4 fujenal (49) but no products of ring contraction. Interestingly, the methyl ester (83) was not metabolized.

The kaurenolide lactones (e.g. 48) are biosynthesized from ent-kaur-6,16-dien- 19-oic acid (44).85 It was suggested that a

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NATURAL PRODUCT REPORTS, 1992--5. R. HANSON 145

(84) R’ = R2 = H (85) R’ = H, R2=OH (86) R’ =OH, R 2 = H (87) R’ = R2 = OH

(88) R = H (89) R = O H

R’ R2

fl I

CO-0

6,7-epoxide was formed first and that this underwent a trans- diaxial opening with participation of the 19-carboxyl group to generate the lactone ring. However, it was not possible to detect the formation of the epoxide, presumably due to its rapid hydrolysis. Incubation of ent-kaur-6,16-diene (84) gaves6 7- hydroxykaurenolide (48) and ent- 18-hydroxykaur-6,16-diene (85) gave 7,18-dihydroxykaurenolide (52) and thus the 6-ene was directing microbiological transformation to the kaureno- lide pathway. Use was then made of the fact that a 3a-hydroxyl group inhibited microbiological oxidation at C- 19, by incubat- ing ent-3P-hydroxy- (86)87 and ent-3P, 18-dihydroxykaur-6,16- dienes (87)s6 with G. fujikuroi. These gave the corresponding 6/3,7P-epoxides, ( 8 8 ) and (89), in accordance with the putative role of the epoxide in kaurenolide formation.

Some further aspects of kaurenolide metabolism were revealeds8 through biotransformation studies. 7/3, 18-Dihydroxy- kaurenolide (52) is the major kaurenolide metabolite of G. fujikuroi. It is accompanied by smaller amounts of 7-hydroxy- kaurenolide (48) whilst other kaurenolides such as 3p,7P- dihydroxy-(90), 7P, 1 la-dihydroxy-(91), 7P, 13-dihydroxy-(92) and 4/?,7/3-dihydroxy- 18-norkaurenolide have been detected. Kaurenolide (93) itself has, understandably, not been recorded as a natural metabolite of the fungus. 7P-Hydroxykaurenolide (48) is converted to fujenal (49) and 7,8,18-dihydroxykaureno- lide (52). However, incubation of 7P, 18-dihydroxykaureno- lide (52) gave products arising from oxidation at C- 18 whilst no compounds of the 18-hydroxyfujenal type were detected. 18- H ydroxykaurenolide (94), lacking the 7-hydroxyl group, gave products arising from oxidation at C- 18 and hydroxylation at C- 1 1. There was no insertion of a hydroxyl group at C-7 and no cleavage of ring B. The lack of hydroxylation at C-7 may be understood in terms of the different geometry of the kaureno- lides compared to the kaurenes.

Epicandicandiol (95) is similar in structure to 7P,18- dihydroxykaurenolide (52) but lacks the 19-6a-lactone ring. Incubation of (95) with G . fujikuroi gaveas ent-7a,18,19- trihydroxykaur-16-ene (96) and the 19-acid (97). On the other

(95) R’ =Me, R2=OH (96) R’ = CHZOH, R2 =OH (97) R’ = C02H, R2= OH (98) R’ = Me, R2 = H

R C02H

(101) R = M e (I 02) R = C02H

(103) R = a-OH, P-H (104) R = O

hand ent-18-hydroxykaur- 16-ene (98), lacking the 7-hydroxyl group, gaves1 both the acid (97) and 7P,18-dihydroxy- kaurenolide (52). This difference may be rationalized in terms of the role of the 6-ene in kaurenolide formation. However, equally significant was the absence of gibberellin and fujenal metabolites and it appeared that an 18-hydroxyl group inhibited the oxidative removal of a hydrogen atom from the C-6 position and thus ring-contraction or hydroxylation and ring cleavage. A similar pattern of results was obtaineds1 with the A15-double bond isomer of epicandicandiol (sideridiol). This gave the corresponding ent-7a, 18,19-triol and 19-acid.

This incubation of substances that interfere with the oxidative metabolism of ring B has been exploreds0 further in the context of the development of plant growth regulators. A number of compounds that are powerful inhibitors of gibberellin plant hormone biosynthesis have been found. The ring B-nor- and ring B seco-kaurenes (e.g. 99 and 100) which inhibit these steps in gibberellin biosynthesis are slowly metabolized by G. fujikuro, by hydration of the 16-ene.

3.1.6 Transformations of ent-Kaurenes ModiJied on Ring c A number of rare plant gibberellins possess oxygen functions on ring c. Since this ring is relatively inaccessible for chemical correlations the microbiological transformation of substituted ent-kaurenes has been used in the preparation of these compounds. In one study ent-12P-hydroxykaur-16-ene (101) was convertedsl~ s2 to 12a-hydroxy-GA,,, 1 2a-hydroxy-GA2,, and 12a-hydroxy-GA,, together with ent-7a, 12P-dihydroxy- and ent-6a,7a- 12P-trihydroxykaurenoic acids. A more extensive studyg3 of the microbiological conversion of 12-oxygenated derivatives of ent-kaur- 16-en-19-oic acid (e.g. 102) led to the identification of 1 2a-hydroxy-GA4 (GA,,) and 7P, 12a- hydroxykaurenolide. This transformation, for example, facili- tated the identification of the gibberellins of Cucurbita maxima. Unlike the 12-oxygenated kaurenoic acids, ent-1 1P- (103) and 1 1-0x0 (104) acids were only metabolized to C,, gibberellins,

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NATURAL PRODUCT REPORTS, 1992 146

OH

fl OH

R

(109) R = C02H (1 10) R = CH20H

OH

(112) R‘=OH, R2=R3=R4=H (1 13) R’ = R4 = OH, R2 = R3 = H (114) R1=R3=R4=H, R2=OH (115) R’=R3=H, R2=R4=OH (116) R ’=R2=R4=H, R3=OH (117) R1=R2=H, R3=R4=OH

(118) R ’=R3=H, R2=Me (1 19) R’ = H, R2 = Me, R3 = OH (120) R’ = OH, R2 = Me, R3 = H (121) R’ = R3 =OH, R2 = C02H

OH

CO-0

e.g. 1 la-hydroxy-GA,, (105) and 1 1-0x0-GA,, (106). No C,, gibberellins were suggesting that an 1 1-oxygen function blocked the loss of C-20.

The C-14 position is also relatively inaccessible in the gibberellin series. 14P-Hydroxy-GA, (108) has been obtainedg4 through the biotransformation of ent- 14a,19-dihydroxykaurene (107) with G. fujikuroi.

3.1.7 Transformations of ent-Kaurenes Modified on Ring D

A number of gibberellins possess an oxygen function at C-15. The structure of one of these, GA,, (1 11) from Pyrus communis, was establishedg5 through its preparation by the bio-trans- formation of ent- 1 5a-hydroxykaur- 16-en- 19-oic acid (109) using G. fujikuroi. Other 1 5P-hydroxygibberellins have also been detectedg2. 93 in this biotransformation and in that of the corresponding ent- 15a, 19-dihydroxykaur- 16-ene (1 10).

There is an interesting effect from the l5a-epimer in that it appears to inhibit oxidation at C-19 by G. fujikuroi. Thus incubation of ent- 15P, 18-dihydroxykaur- 16-ene (candidiol) (1 12) with G. fujikuroi affordedg6 ent-1 la,15,8,18-tri- hydroxykaur- 16-ene (1 13). Further studiesg7 with ent- 15/3,18- dihydroxykaur-6,16-diene gave products of hydroxylation at C-1 lp and epoxidation of the 6,7-ene but no oxidation at C-19.

R’

(1 24) R’ = R3 = H, R2 = C02H (125) R’ = R3 = H, R2 = Me (126) R’ =H, R2= Me, R3=OH (1 27) R’ = R3 = H, R2 = CH20H (128) R’ = R3 = OH, R2 = Me (129) R’ = R3 = OH, R2 = CH20H

O H

Even incubation of ent-l5P,19-dihydroxykaur-l6-ene (1 14) gave ent- 1 la, 15P, 19-trihydroxykaur- 16-ene (1 15) and sur- prisingly ent-7P, 1 1 a, 1 5p, 19-tetrahydroxykaur- 16-ene but no products of further oxidation at C-19. Incubation of ent- 7a, 1 5P-dihydroxykaur- 16-ene (1 16) gaveg8 products arising from attack at C-11 (e.g. 117) and only very limited hydroxylation at C-19. These observations are of interest since ent-kauran- 16P, 17-epoxide isg9 an inhibitor of gibberellin biosynthesis at the kaurene stage. A common by-product from bio-transformations with G. fujikuroi is hydration of the A16- double bond. The 16a-alcohols do not appear to be further transformed and this appears to be a ‘dumping mechanism’ on this pathway.

Whilst ent-kaur- 16-ene is a key precursor of the gibberellin plant hormones, the dwarf d, mutant of maize which is blocked for gibberellin biosynthesis, produces ent-kaur- 15-ene. It has been SuggestedlOO that the gibberellin biosynthetic system in the dwarf maize is unable to handle the double-bond isomer ent- kaur- 1 Sene. However, incubationlo’ of ent-kaur- 15-ene (1 18) and ent-7a-hydroxykaur-15-ene (1 19) gave a range of A15- isomers of the natural metabolites of G. fujikuroi together with ent-7a-hydroxy- 15P, 16P-epoxykaurane (1 22). ent- 18- Hydroxykaur- 15-ene (1 20) gave ent-7a, 18-dihydroxykaur- 15- en- 19-oic acid (1 2 1) and 7P, 18-dihydroxykaur- 15-enolide (1 23).

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NATURAL PRODUCT REPORTS, 1992--5. R. HANSON 147

R' co-0

(131) R = H (132) R=OH

CH20H

(1 39)

R

C02H

(133) R'=OH, R2=H (134) R' = R2 = H (135) R' = H, R2 = OH

(140) R' = R2 = H (141) R' = H, R2=OH (142) R' =OH, R2= H

co-0 (148) R = H (149) R=OH

C02H

(150) R = O (151) R = a-OH, P-H (152) R=H2

Thus the fungal system can accept 15-enes. The formation of the 15,16-epoxide may be a modification of the hydration at C- 16.

3.1.8 Transformations of Other Diterpenoid Skeleta ent-Kaurene is one of a number of biosynthetically closely related diterpenoid hydrocarbons which also include the ent- atiserene, ent-beyerene, and ent-trachylobane series. The flexibility of the gibberellin biosynthetic pathway in Gibberella fujikuroi has been explored with substrates possessing these different carbon skeleta. Incubation with the pentacyclic trachylobanic acid (1 24) gavelo2 12,16-cyclo-(trachyloba)- gibberellins A, (1 30) and A,, whilst a separate study reportedlo3 the conversion of ent-trachylobane (1 25), ent-7a-hydroxy- trachylobane (126), and ent-19-hydroxytrachylobane (127) into trachylobagibberellins A,, A,, AI3, A2,, and A,,. Whilst 7P-hydroxy and 7P, 18-dihydroxytrachylobanolides (1 3 1) and (1 32) were obtained from ent-trachylobane and ent- trachyloban- 19-01, the presence of a 7P-hydroxyl group directed the metabolism exclusively into the gibberellin pathway. An 18- hydroxyl group as in ent-7a, 18-dihydroxytrachylobane (128) inhibited oxidation at C-6 affording ent-7a, 18,19-trihydroxy- trachylobane (129) as the major metabolite.

Incubation of ent-7a-hydroxyatisenoic acid (1 33) gave only atisa-GA,, (1 36) and atisa-GA,, ( 137).lo4 The bio-transform- ationlo5 of the A15-isomer (1 38) gave A15-isoatisa-GA,2 and A15-isoatisa-GA,, whilst ent- 19-hydroxyatisa-6,15-diene (1 39)

C02H

(136) R = H (137) R=OH

R

COZH H

C02H Co2H

(143) R = H (144) R=OH (145) R = OH, A'*2

(146) R =OH (147) R = H

R3

R'

(1 53) (154) R' = R2= R3 = H (155) R' =OH, R2 = R3 = H (156) R' = R3 = OH, R2 = H (157) R' = R2 = R3 = OH

gave 7P-hydroxy- and 7P, 18-dihydroxyisoatisenolides. Simi- larly no products of oxidation at C-20 were observed on transformationlo1 of ent-atis- 16-en- 19-oic acid (1 34) or ent- 13(S)-hydroxyatis- 16-en- 19-oic acid (1 35). Thus the different c/D-geometry would seem to impede the loss of C-20 and the formation of the C- 19 gibberellins.

The microbiological transformation of ent-beyer- 15-ene (140) into the beyergibberellins A, (143) and A,, (146); and the beyerenolides (148) and (149) and of beyer- 15-en- 19-01 (14 1) into beyergibberellins A, (144), A, (145), A, (143), A,, (146) and A,, (147), and the beyerenolides has been reported.lo6, lo7

Again the inhibitory effect of an 18-hydroxyl group on oxidative transformations at C-6P was noted in the products of the incubation of ent-beyer- 15-en- 18-01 (142). Whereas the ent- beyer- 15-enes gave products of 3-hydroxylation, incubation of isosteviol (1 50) gave70 only 3-desoxy compounds (e.g. 153). This effect has also been observedlo8 with the 16-alcohol (1 5 1) and isostevic acid (152) and may be attributed to the sp3 centre at C-15 impeding the binding to the 3P-hydroxylase. Seco-ring B metabolites related to fujenal were also absent from these incubations.

In an attempt to prepare the enantiomer of gibberellic acid, ( +)-17-14C-kaurene was incubatedlog with G . fujikuroi but no biotransformation was observed.

The cyclization of geranylgeranyl pyrophosphate to ent- kaurene takes place via an ent-labdadienol pyrophosphate. ent- 13-Epimanoyl oxide (olearyl oxide) (154) is produced as a metabolite by G . fujikuroi from this stage. Incubation of ent-19-

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148 NATURAL PRODUCT REPORTS, 1992

(1%) R' = R ~ = H (159) R' = H, R2 = OH (160) R' = OH, R2 = H (161) R' = R2 = OH

C02H

P 2 WR3 -=.*.-- - HO

CO2H

(166) R' = Me, R2 = R3= H (167) R' = Me, R2 = H, R3 = OH (168) R' = R 3 = H, R2 = Me (169) R '=H, R2=Me, R3=OH

hydroxy- 13-epimanoyl oxide (1 55) with G. fujikuroi affordedllO ent- 12a,19-dihydroxy- 13-epimanoyl oxide (1 56) and ent- 7p, 1 2 4 19-trihydroxy- 13-epimanoyl oxide whilst ent-3P- hydroxy- 13-epimanoyl oxide (ribenol) (1 58) gave the derivatives (159)--(161). There were no detectable products of oxidation at C-19.

3.1.9 Transformations of Gibberellins The introduction of fluoro- and alkyl-substituents onto the steroids modifies their biological properties. Modification of the properties of the gibberellins has also been affected by the introduction of these substituents. Methods for introducing these substituents are not always compatible with the sensitive functionality found in the biologically active gibberellins. Hence the microbiological transformation of suitably sub- stituted precursors has formed a method for the partial synthesis of these gibberellin analogues.

18-Fluorogibberellin A,, 7-aldehyde (1 62), prepared from the toluene-p-sulfonate of 7,18-dihydroxykaurenolide, was incubatedll' with G. fujikuroi to give 18-fluoro-GA9 (163) and 18-fluorogibberellic acid although these were not separable from the natural substrates. ent- 1 5P-Fluorokaur- 16-en- 19-oic acid ( I 64) was converted112 into 15a-fluoro-GA4 and 1501- fluoro-GA,, in the presence of an inhibitor. ent-16,16-Difluoro- 17-norkauran- 19-oic acid (165) gave", the 16-gem-difluoro derivatives of 1 7-nor-GA9 and 17-nor-GA7, the latter isolated as its 3-ketone.

The hydroxylation of gibberellin A, at C-13 is the last stage in the biosynthesis of gibberellic acid by G. fujikuroi. This has been used in some biotransformations. Whilst incubation of la-methyl-GA, (166) gave114 la-methyl-GA, (167) as the sole metabolite, incubation of lp-methyl-GA, (1 68) gave 1 -methyl- GA, (170), 1-methyl-GA, (171), and lp-methyl-GA, (169) in accordance with the loss of a la-hydrogen in the normal biosynthesis of GA, from GA,. The bridgehead hydroxylation was also found in the biotransformation of 2,2-dimethyl-GA4 (1 72) into115 2,2-dimethyl-GA1 (1 73). However, incubation of GA, or GA, analogues does not always lead to their hydroxylation at C- 13. Thus the ap-unsaturated ketone derived

(170) R = O H (171) R = H

(172) R = H (173) R = O H

(1 74) R' = Me, R2 = CH20H (175) R' = Me, R2 = C02H

(177) R' = CH20H, R2 = H

(179) R = O (180) R=H2

(176) R' = Me, R2= H

(178) R' = C02Me, R2 = H

from GA, is hydrated at C-1 whilst 16,17-dihydro-GA4 was hydroxylated to give the 1 a-hydroxy derivative. 116

Gibberellin A,, 7-aldehyde (43) and a number of relatives such as the 7,19-diol(174) and the 7-alcohol ( I 75) are efficiently transformed by G. fujikuroi. However, no bio-transformation of a series of 19-nor analogues (176)-(178) could be detected',, except in the case of the 7,20-diol (177) in which a minor amount of the 16-hydrate was formed. This failure to observe biotransformation again indicates the importance of the 19- carboxyl group in the middle stages of gibberellin biosynthesis. Indeed the 19-nor compounds acted as inhibitors of gibberellic acid biosynthesis.

3.2 Transformations with Trichothecium roseurn Diterpenoid biosynthetically-directed microbiological trans- formations are not restricted to Gibberella fujikuroi. A series of tricyclic diterpenes exemplified by rosenonolactone ( 179) are produced by Trichothecium roseum. The final stages in the biosynthesis of rosenonolactone involve the hydroxylation of desoxyrosenonolactone (1 80) at C-7 and oxidation to the

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NATURAL PRODUCT REPORTS, 1992-J. R. HANSON 149

OH

ketone.lls Incubation of 7/3,1 O D , 19-trihydroxyros- 15-ene (1 8 1) and its 15,16-dihydro derivative with T. roseum led'19 to selective oxidation at C-7 and the formation of the cor- responding 7-ketones whilst 10/3,19-dihydroxyrosa-7,15-diene (1 82) afforded the 7/3,8P-epoxide in a selective stereospecific epoxidation from the more hindered face of the molecule.

3.3 Transformations with Cep ha Iosporium aphidicola The biosynthesis of the tumour-inhibitor aphidicolin (183) in Cephalosporium aphidicola involves a stepwise series of hydroxylations of aphidicolan-16/3-01 at C-18, C-3a, and C- 1 7.120 The topological relationship between the substrate and the active site of the enzymes responsible for the methyl group hydroxylations has been examined12' by incubating analogues of the natural substrates. The chirality of hydroxylation at C- 17 using an ethyl homologue of 3a, 16P, 18-trihydroxy- aphidicolane was used to establish the stereochemical re- lationship between the substrate and the hydroxylase for the reaction at C-17 whilst the stereochemistry of hydroxylation of a 3a,18-ether provided some evidence for the stereochemistry of hydroxylation of the C-18 methyl group.122 The fungus will also transform derivatives of aphidicolin modified on rings A and D . ' ~ ~

4 Conclusion The xenobiotic transformations of diterpenoids that have been described in this review reveal the potential value of the method not only in preparing compounds for structure : activity studies but also in structural correlations. Although comparison with the steroids has allowed some further mapping of the active sites of hydroxylases, what is still lacking are a reliable series of

1 1

micro-organisms of known regiospecificity for diterpenoid hydroxylations.

The biosynthetically-patterned biotransformations have re- vealed a number of constraints on pathways which would not have been deduced from conventional biosynthetic studies. Although the yields of metabolites are relatively poor, the transformations do have some preparative value in the context of rare or labelled compounds. It has become clear that certain functional groups play a key role in coding for transformation along particular pathways. Other groups, for example a 3a-, 15a, or 18-hydroxyl group in the ent-kaurenes, prevent particular steps and possess an apparent regulatory role. Yet other groups, such as the 16-hydroxyl group, represent a 'dumping' mechanism. Indeed with these studies it is possible to begin to speculate on the role of various hydroxyl groups within the plethora of hydroxylation patterns found in the diterpenoids.

5 References 1 2

3 4 5

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7

8

9

10

I1

12

13

14

15

16

17

18

19

20

21

22

23

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24 A. B. Anderson, R. McCrindle, and J. K. Turnbull, J . Chem. Soc., Chern. Commun., 1973, 143.

25 R. McCrindle, J. K. Turnbull, and A. B. Anderson, J. Chern. Soc., Perkin Trans. I , 1975, 1202.

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27 J. P. Beilby, E. L. Ghisalberti, P. R. Jefferies, M. A. Sefton, and D. N. Sheppard, Tetrahedron Lett., 1973, 2589.

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30 E. L. Ghisalberti, P. R. Jefferies, and M. A. Sefton, J . Chern. Res. (S), 1982, 8.

31 A. Chatterjee, S. K. Desmukh, and S. Chandrasekharan, Tetra- hedron, 1972, 28, 4319.

32 S. A. Ahmad and A. Zaman, Tetrahedron Lett., 1973, 2179. 33 J. R. Hanson, G. Savona, and M. Siverns, J . Chem. Soc., Perkin

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Harvey, J. MacMillan, and B. 0. Phinney, J. Chern. Soc., Perkin Trans. 1, 1976, 178.

37 A. Garcia-Granados, A. Martinez, M. E. Onorato, and J. M. Arias, J . Nut. Prod., 1986, 49, 126.

38 A. Garcia-Granados, A. Martinez, M. E. Onorato, M. L. Ruiz, J. M. Sanchez, and J. M. Arias, Phytochemistry, 1990, 29, 121.

39 A. Garcia-Granados, A. Martinez, A. Ortiz, M. E. Onorato, and J. M. Arias, J . Nut. Prod., 1990, 53, 441.

40 J. R. Hanson and B. H. de Oliveira, Phytochemistry, 1990, 29, 3805.

41 J. Ipsen, J. Fuska, A. Foskova, and J. P. Rosazza, J. Org. Chern., 1982, 47, 3278.

42 J. Ipsen and J. P. Rosazza, J. Nut. Prod., 1984, 47, 497. 43 F. A. Badria and C. D. Hufford, Phytochemistry, 1991, 30, 2265. 44 J. R. Hanson, Biochem. SOC. Trans., 1983, 11, 522. 45 J. R. Bearder, ‘ Biochemistry and Physiology of Gibberellins’, vol.

46 B. E. Cross, R. H. B. Galt, and J. R. Hanson, J . Chern. Soc., 1964,

47 J. R. Hanson and A. F. White, J . Chem. SOC. (C), 1969, 981. 48 R. Evans and J. R. Hanson, J. Chem. Sac., Perkin Trans. I , 1972,

49 T. A. Geissman, A. J. Verbiscar, B. 0. Phinney, and G. Cragg,

50 S. J. Castellaro, S. C. Dolan, P. Hedden, P. Gaskin, and J.

51 J. R. Hanson, J. Hawker, and A. F. White, J. Chem. Soc., Perkin

52 P. Hedden and J. E. Graebe, Phytochemistry, 1981, 20, 101 1. 53 R. Evans, J. R. Hanson, and A. F. White, J. Chem. Soc. (C) , 1970,

54 B. E. Cross, K. Norton, and J. C. Stewart, J . Chem. Soc. (C) ,

55 R. Evans and J. R. Hanson, J . Chem. Soc., Perkin Trans. 1, 1975,

56 J. R. Bearder, J. MacMillan, and B. 0. Phinney, J . Chem. Soc.,

57 P. Hedden, J. MacMillan, and B. 0. Phinney, J. Chern. Soc.,

58 B. Dockerill and J. R. Hanson, Phytochernistry, 1978, 17, 701. 59 P. Lewer and J. MacMillan, Phytochemistry, 1984, 23, 2803. 60 Y. Kamiya and J. E. Graebe, Phytochemistry, 1983, 22, 681. 61 M. Ruddat, E. Heftmann, and A. Lang, Arch. Biochem. Biophys.,

62 J. R. Hanson and A. F. White, Tetrahedron, 1968, 24, 6291. 63 J. R. Bearder, P. Hedden, J. MacMillan, C. M. Wels, and B. 0.

64 J. R. Bearder, J. MacMillan, C. M. Wels, M. B. Chaffey, and

65 D. T. Dennis, C. D. Upper, and C. A. West, Plant Physiol., 1965,

66 M. Ruddat, E. Heftmann, and A. Lang, Naturwiss., 1965,50,267. 67 M. F. Barnes, E. N. Light, and A. Lang, Planta, 1969, 88, 172. 68 B. E. Cross and P. L. Myers, Phytochemistry, 1969, 8, 79. 69 J. R. Bearder, J. MacMillan, C. M. Wels, and B. 0. Phinney,

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2601.

1968, 1054.

663.

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1965, 111, 187.

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B. 0. Phinney, Phytochemistry, 1974, 13, 91 1.

40, 948.

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70 J. R. Bearder, V. M. Frydman, P. Gaskin, J. MacMillan, C. M. Wels, and B. 0. Phinney, J . Chem. Soc., Perkin Trans., I , 1976, 173.

71 T. Gianfagna, J. A. D. Zeevart, and W. J. Lusk, Phytochemistry, 1983, 22, 427.

72 N. Murofushi, S. Nagura, and N. Takahashi, Agric. Biol. Chem., 1979, 43, 1159.

73 N. Murofushi, Y. Shigematsu, S. Nagura, and N. Takahashi, Agric. Biol. Chem., 1982, 46, 2305.

74 J. R. Hanson and B. H. de Oliveira, Phytochemistry, 1990, 29, 3805.

75 Y . Shigematsu, N. Murofushi, and N. Takahashi, Agric. Biol. Chem., 1982, 46, 2313.

76 P. R. Jefferies, J. R. Knox, and T. Ratajczak, Phytochernistry, 1974, 13, 1423.

77 H. J. Bakker, I. F. Cook, P. R. Jefferies, and J. R. Knox, Tetrahedron, 1974, 30, 363 1.

78 I. F. Cook, P. R. Jefferies, and J. R. Knox, Tetrahedron, 1975,31, 251.

79 M. W. Lunnon, J. MacMillan, and B. 0. Phinney, J. Chem. Soc., Perkin Trans. 1, 1977, 2308.

80 B. M. Fraga, A. G. Gonzalez, J. R. Hanson, and M. G. Hernandez, Phytochemistry, 198 1, 20, 57.

81 B. M. Fraga, J. R. Hanson, M. G. Hernandez, and F. Y. Sarah, Phytochemistry, 1980, 19, 1087.

82 B. E. Cross, J. C. Stewart, and B. L. Stoddart, Phytochemistry, 1970, 9, 1065.

83 J. R. Hanson and J. Hawker, Tetrahedron, 1972, 28, 2521. 84 M. Alam, J. R. Hanson, and F. Y. Sarah, Phytochemistry, 1991,

30, 807. 85 M. H. Beale, J. R. Bearder, G. H. Down, M. Hutchison, J.

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87 C. E. Diaz, B. M. Fraga, and M. G. Hernandez, Phytochemistry, 1989, 28, 1053.

88 J. R. Hanson and F. Y. Sarah, J . Chern. Soc., Perkin Trans., I , 1979, 3151.

89 B. M. Fraga, J. R. Hanson, and M. G. Hernandez, Phytochemistry, 1977, 17, 812.

90 J. R. Hanson, K. P. Parry and C. L. Willis, Phytochernistry, 1982, 21, 1575; M. K. Baynham, J. M. Dickinson, and J. R. Hanson, Phytochemistry, 1988, 27, 761.

91 K. Wada and H. Yamashita, Agric. Biol. Chem., 1980, 44, 2249. 92 K. Wada, T. Imai, and H. Yamashita, Agric. Biol. Chem., 1981,

45, 1833. 93 P. Gaskin, M. Hutchison, N. Lewis, J. MacMillan, and B. 0.

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19, 2323. 100 See, for example, R. C. Coolbaugh in ‘Biochemistry and Physi-

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101 B. M. Fraga, M. G. Hernandez, M. D. Rodriguez, C. E. Diaz, P. Gonzalez, and J. R. Hanson, Phytochemistry, 1987, 26, 1931.

102 M. H. Beale, J. R. Bearder, J. MacMillan, A. Matsuo, and B. 0. Phinney, Phytochemistry, 1983, 22, 875.

103 B. M. Fraga, A. G. Gonzalez, M. G. Hernandez, J. R. Hanson, and P. B. Hitchcock, J . Chem. SOC., Chem. Cornmun., 1982, 594; C. E. Diaz, B. M. Fraga, A. G. Gonzalez, P. Gonzalez, J. R. Hanson, and M. G. Hernandez, Phytochernistry, 1984, 23, 2813.

104 J. R. Hanson, F. Y. Sarah, B. M. Fraga, and M. G. Hernandez, Phytochemistry, 1979, 18, 1875.

105 B. M. Fraga and R. Guillermo, Phytochemistry, 1987, 26, 2521. 106 B. M. Fraga, A. G. Gonzalez, M. G. Hernandez, A. San Martin,

2. J. Duri, and J. R. Hanson, Tetrahedron Lett., 1983, 24, 1945. 107 C. E. Diaz, B. M. Fraga, A. G. Gonzalez, J. R. Hanson, M. G.

Hernandez, and A. San Martin, Phytochemistry, 1985, 24, 1489.

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108 M. S. Ali, J. R. Hanson, and B. H. de Oliveira, Phytochemistry, in the press.

109 B. E. Cross, K. Norton, and J. C. Stewart, Phytochemistry, 1968, 7, 83.

110 B. M. Fraga, P. Gonzalez, R. Guillermo, M. G. Hernandez, and J. Rovirosa, Phytochemistry, 1989, 28, 185 1.

111 J . H. Bateson and B. E. Cross, J . Chem. SOC., Perkin Trans. I , 1974, 1131.

112 B. E. Cross and A. Erasmuson, J. Chem. SOC., Perkin Trans. I , 1981, 1918.

1 13 B. E. Cross and P. Filippone, J. Chem. Res. (S), 1981, 166; K. Boulton and B. E. Cross, J . Chem. SOC., Perkin Trans. 1, 1981, 427.

114 J. MacMillan and C. L. Willis, J . Chem. SOC., Perkin Trans. I , 1985, 2177.

115 M. H. Beale, J. MacMillan, C. R. Spray, D. A. Taylor, and B. 0. Phinney, J. Chem. SOC., Perkin Trans., 1, 1984, 541.

116 K. M. Abouamer and J. R. Hanson, unpublished work. 117 M. S . Ali, M. K. Baynham, and J. R. Hanson, Phytochemistry,

118 B. Achilladelis and J. R. Hanson, J . Chem. SOC. (C), 1969, 2010;

119 M. Alam and J. R. Hanson, Phytochemistry, 1990, 29, 3801. 120 M. J. Ackland, J. F. Gordon, J. R. Hanson, B. L. Yeoh, and

A. H. Ratcliffe, J. Chem. SOC., Chem. Commun., 1987, 1492; J . Chem. SOC., Perkin Trans. I , 1988, 1477.

121 J. F. Gordon, J. R. Hanson, and A. H. Ratcliffe, J . Chem. SOC., Chem. Commun., 1988, 6.

122 A. J. Jarvis and J. R. Hanson, unpublished work.

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