Tropic acid ester biosynthesis in Datura stramonium and related species David O’Hagana and Richard J. Robinsb a Department of Chemistry University of Durham Science Laboratories South Road Durham UK DH1 31E b etabolismes CNRS UPRES-A 6006 Universit Laboratoire d’Analyse Isotopique et Electrochimique de M� e de � Nantes D�epartement de Chimie 2 rue de la Houssiniere 44072 Nantes cedex 03 France The origin of the tropic acid ester moiety found in some of the tropane alkaloids but particularly in hyoscyamine and scopolamine has been a subject of discussion and investigation in biosynthesis for many years. Recently it has been shown in Datura stramonium root cultures that hyoscyamine arises by isomerisation of the tropane alkaloid littorine. The mechanism of this isomerisation process is not obvious and in this review we present our recent results and current thinking on this process.1 Introduction In the last few years new experimental evidence has forced a reevaluation of the biosynthesis of the tropate ester moiety of the tropane alkaloids hyoscyamine and scopolamine. Robert Me N Me N O O O O O 1' 3' 2' OH OH scopolamine hyoscyamine David O’Hagan was born in Glasgow in 1961. He was an undergraduate at the University of Glasgow (1982) and carried out his doctoral research (1985) on antibiotic biosynthesis at the University of Southampton with Professor John A Robinson. After a postdoctoral year with Professor Heinz G Floss (Ohio State University) he took up a position at the University of Durham where he is now a Senior Lecturer.His main research interests are in the biosynthesis of secondary metabolites and in bio-organic fluorine chemistry. Richard J. Robins David O’Hagan CD3 N O O um OH hyoscyamine Robinson recognised in 19281 that the tropate ester is an isomer of the phenylpropionoid skeleton a structural motif common in plant alkaloids and other plant metabolites such as the flavanoids. The phenylpropionoid moiety derives from (S)-phenylalanine and Robinson later (1955) proposed2 that tropic acid may originate by a rearrangement of the phenylpropanoid skeleton. In a definitive experiment twenty years later (Scheme 1) Leete demonstrated3 in Datura innoxia plants that (R,S)- phenyl[1,3-13C2]alanine was indeed incorporated intact into the tropate moiety of hyoscyamine.Importantly this experiment demonstrated an intramolecular rearrangement where the two isotopes became contiguous in the resultant tropate. Thus the origin of the tropate ester from phenylalanine was established. A number of intriguing questions remained however concerning this conversion. Although phenylalanine is a good precursor the true substrate for the rearrangement and the stereochemistry remained ill-defined until recently. The rearrangement belongs to a small class of enzyme-mediated carbon skeletal isomerisations and the mechanism of these enzymes has attracted wide interest. The rearrangement has a RO O O D. innoxia O– plants OH NH3 + ( RS)-phenyl[1,3-13C2] alanine Scheme 1 Richard J.Robins read Biochemistry at the University of Oxford (1971–75) and carried out doctoral work on the digestive processes of carnivorous plants with Dr B. E. Juniper University of Oxford. After a post-doctoral study of the mechanism of amino acid absorption with Professor D. D. Davies (University of East Anglia) he joined (1981) the AFRC Institute of Food Research to study the biosynthesis and control of secondary product formation in plant tissue cultures. In 1995 he moved to Nantes to become Director of the CNRS Unit of Isotopic and Electrochemical Studies of Metabolism. His current research interests include the biosynthesis of secondary metabolites and the use of natural-abundance isotopic techniques to study metabolism.207 Chemical Society Reviews 1998 volume 27 superficial similarity to those mediated by some co-enzyme B12-dependant mutases however the process does not appear to be B12-dependent. 2 Plants and transformed roots of Datura stramonium Much of the recent work on the biosynthetic origin of tropic acid has exploited transformed root cultures as experimental material. These cultures are generated by infecting the lightlydamaged surface of sterile leaf or stem from tropic acidproducing plants such as D. stramonium with a suspension of the pathogenic bacterium Agrobacterium rhizogenes. The bacterium inserts into the plant cells a short section of DNA (the Ri-DNA) which stimulates cell division to cause root formation.The emergent roots can be removed treated with antibiotic to kill the remaining bacteria and cultured in perpetuity in a sterile liquid medium. A small part of the culture is transferred to fresh medium every two or three weeks (see ref 4 for a review of the properties of these cultures). Figure 1 This system offers many advantages over the use of whole plants; * large amounts of genetically identical material can be generated. * the roots grow at a constant rate. * the experiment is conducted under asceptic conditions. * precursors can readily be added and their absorption by the tissue monitored directly and multiple additions can be made. * experiments can be conducted on small amounts of material allowing considerable savings on the quantity of isotopically labelled precursor required.* very high specific incorporations can be obtained rendering analysis by GCMS and NMR relatively straightforward. Chemical Society Reviews 1998 volume 27 208 Figure 2 Root cultures are not however suitable for all studies. In D. stramonium plants scopolamine is a major product in the leaves yet only traces of this alkaloid are recovered from root cultures. In other species however such as Hyoscyamus muticus or a Brugmansia hybrid scopolamine accumulates in the root cultures as a major alkaloid. A key requirement for using root cultures is that this tissue must be the site of biosynthesis in the plant. For example terpenes in Mentha species are made in leaf glands and shoot cultures5 are required in this case.3 The role of (R)-phenyllactate (S)-Phenylalanine is efficiently incorporated into the tropate ester moiety of hyoscyamine.3 Other experiments6 with 14C-labelled phenylpyruvate and phenyllactate indicated that all of these compounds become similarly incorporated into the tropate ester moiety of the alklaloids. This observation is readily rationalised if these compounds interconvert in vivo. The importance of phenyllactic acid as an intermediate was confirmed by feeding (R,S)-phenyl[1,3-13C2]lactate to D. stramonium transformed root cultures7 or plants.8 High incorporation was obtained and the observed spin-spin coupling of the two 13C nuclei in the extracted hyoscyamine7 and scopolamine8 confirmed both the intramolecular rearrangement shown by Leete3 and the putative role of phenyllactic acid in the formation of hyoscyamine.It remained therefore to identify the most direct precursor of the three. In an effort to resolve this issue we studied the incorporation of deuterium from (R,S)-[2-13C 2H]phenyllactate. If phenyllactate is oxidised to phenylpyruvate then the deuterium atom will be lost prior to incorporation into tropic acid. If however phenyllactate is utilised directly then the deuterium atom will be retained. An initial feeding experiment supplementing D. stramonium root cultures with (R,S)phenyl[2-13C,2H]alanine resulted9a in a substantial retention of deuterium isotope attached to carbon-13 (17%) at C-3A of the tropate ester moiety of hyoscyamine.In a further experiment9b resolved (R)- and (S)-phenyl[2-13C 2H]- alanines were added to D. stramonium cultures. Feeding the (R)-isomer resulted in hyoscyamine showing retention of the dual 13C-2H isotopes (28.9%) indicating that the bond had remained intact during the biosynthesis. For the (S)-isomer there was a significant 13C-enrichment but all of the deuterium was lost indicating that this bond had been broken during hyoscyamine formation. These results demonstrate that (R)- and not (S)-phenyllais the stereoisomer used during the biosynthesis. (S)-Phenyllactate must be converted to the 13 (R)-isomer presumably via phenylpyruvate prior to incorporation into tropic acid. The experiment also established that the C-2 hydrogen atom of (R)-phenyllactate is retained during the rearrangement.Experiments using (R)- and (S)-phenyl[1,3- 2]lactates fed to whole plants10 have also indicated on the C basis of differential incorporation levels that only the (R)- isomer is the precursor of hyoscyamine and scopolamine. 4 Stereochemistry of the rearrangement During the rearrangement of phenyllactate to tropate two bonds are broken and two are formed. In a given reaction bonds are normally broken/formed with either retention or inversion of configuration. A recent stereochemical analysis11 on tropate biosynthesis has revealed that both of the bonds are broken/ formed with inversion of configuration. O O– NH3 + ( S)-phenylalanine O O– O –D phenylpyruvate –D O O– D OH ( R)-phenyl[2-13C]lactate ( R)-phenyl[2-13C 2H]lactate RO O D OH 13C - 2H-labelled tropate Scheme 2 Firstly radiolabelled phenyl[2-3H]lactate was incubated with D.stramonium root cultures.11a The resultant hyoscyamine retained tritium at the 3-pro-S position of the tropate ester. This conclusion was drawn after diluting the isolated hyoscyamine with ‘cold’ unlabelled alkaloid and converting the carbon atom carrying the tritium into a chiral methyl group by introduction of deuterium in a stereospecific manner. The strategy is shown in Scheme 3. Oxidation of 2-phenylpropanol carrying the chiral methyl group generated a sample of chiral acetic acid. Although O O– OH D ( S)-phenyl[2-13C 2H]lactate O O– H OH RO O H OH 13C - labelled tropate cold carrier was added such that sufficient material could be manipulated through the derivatisation protocol it is important to note that all of the molecules carrying tritium had come through the biosynthetic experiment and that only these molecules give rise to chiral acetic acid.Enzymatic assay of the resultant chiral acetic acid indicated that the methyl group had an R-configuration (98% ee). Thus by deduction the tritium must have occupied the 3-pro-S site in the hyoscyamine isolated after the biosynthetic experiment. In view of the fact that (R)- and not (S)-phenyllactate is utilised then it was concluded that there is an overall inversion of configuration at this centre during the rearrangement.Me N O D. stramonium O O– O OH T T OH 50 mgs isolated of 1 500 mgs cold carrier Ba(OH) CH2N2 2 Mesyl Cl MeO MeO O O DMAP T T O-SO2Me OH 78% LiAlD4 stereochemical inversion D HO D O H KIO4 H HO T T KMnO4 D D 82% 16%, (98%ee) ( R)-acetic acid 6.0 mCi mmol Scheme 3 The stereochemistry at the other migration terminus was established after feeding experiments with (2R,3R)- and (2R,3S)-phenyl[3-2H]lactates. In the event only deuterium from 3-pro-S (2R)-phenyllactate was retained. However this deuterium –carbon bond had become configurationally inverted in the resultant hyoscyamine. In the complementary experiment the 3-pro-R hydrogen was lost during the rearrangement.With this information we concluded that the bond breaking/forming at this carbon atom proceeds with an inversion of configuration. The overall stereochemical course of the rearrangement is summarised in Scheme 4 inversion of configuration occurring at both migration termini. This study has corrected a previous stereochemical analysis12 of this system in plants and supported an even earlier study by Haslam13 who also showed an inversion of configuration at C-2 of the tropate ester. 209 Chemical Society Reviews 1998 volume 27 inversion RO O O HS HS HR H* OR OH H* HO inversion HR Scheme 4 5 Substrate for the rearrangement process The above experiments proved a clearly defined role for (R)- phenyllactate in the biosynthesis of the tropate ester moiety.However the true substrate for the putative isomerase is not free (R)-phenyllactate. It is poignant that the co-produced alkaloid littorine the tropine ester of (R)-phenyllactate is found widely in tropane-alkaloid forming species. So clearly (R)-phenyllactate can couple to tropine in D. stramonium raising the possibility that littorine is the substrate for the enzyme. Indirect evidence to support this conclusion was obtained from experiments7 in which added unlabelled tropic acid failed to diminish the incorporation of label from (R,S)- phenyl[1,3-13C2]lactate. Thus free tropic acid was found unlikely to be an intermediate of hyoscyamine formation. Similarly 14C-tropic acid was found14 to be a very poor precursor for hyoscyamine compared with 14C-phenylalanine in root cultures of Duboisia leichhardtii.The role of littorine as a direct precursor of hyoscyamine was established in an experiment14 using littorine isotopically labelled in both the tropane ring and the tropate ester moiety. This study demonstrated unequivocally that littorine can rearrange in vivo to hyoscyamine. The precursor littorine was labelled by incorporating three 2H nuclei in the N-methyl of the tropine moiety and two 13C nuclei in the phenyllactoyl moiety (Scheme 5). A specific incorporation into hyoscyamine of between 4.5 and 6.5% of the quintuply-labelled molecule was measured by GCMS analysis. Some hydrolysis of littorine occurred resulting in labelling of both tropine (at the M + 3 ion) and phenlylactate methyl ester (at the M + 2 ion).From the percent isotopic excess in these products it could be estimated however that a route involving hydrolysis of the ester followed by reincorporation could only account for about 0.2% isotopic excess in the isolated hyoscyamine. Furthermore neither added cold tropine nor phenyllactate diluted the percent isotopic incorporation. Confirmation that the rearrangement is intramolecular was shown by NMR the isolated hyoscyamine showing a high level of 13C spin-spin coupling due to the adjacent enriched nuclei at the C-1A and C-2A positions. Thus after many years of speculation the substrate for the isomerisation is now established as the tropane alkaloid littorine. CD3 CD3 N N O O O D.stramonium O OH OH hyoscyamine littorine Scheme 5 6 Some ideas on the mechanism of rearrangement There is a superficial similarity between co-enzyme B12 processes and the rearrangement of littorine to hyoscyamine. However in the related co-enzyme B12 processes a vicinal Chemical Society Reviews 1998 volume 27 210 interchange process is apparent. This is illustrated typically for (R)-methylmalonyl-CoA mutase15 in Scheme 6 where the thioester carboxylate migrates to the vicinal carbon and the hydrogen that is removed from this carbon is relocated at the O SCoA SCoA methylmalony-CoA mutase co-enzyme B12 O H O– H O– 1,2-vicinal interchange O O succinyl-CoA R-methylmalonyl-CoA Scheme 6 original carboxy site.It had been reported12 that the hydrogen at C-3 (of phenyllactate) which is removed during the process was relocated at the C-3A position of hyoscyamine. This conclusion was drawn after a 3H/14C labelling study and the observation of an apparent vicinal interchange process clearly implied a role for co-enzyme B12 in the rearrangement process. However our stable isotope study11b did not reveal any evidence for a vicinal interchange process i.e. the 3-pro-R hydrogen of littorine is not relocated at the 3-pro-R site of hyoscyamine. This observation and the apparent lack of coenzyme B12 in plants lays to rest the putative involvement of this co-factor. In many plant systems iron-oxo species operate to generate radicals.For example Sankawa has shown16 that the isoflavone synthase of Pueraria lobata cell cultures is a cytochrome P450-mediated reaction as illustrated in Scheme 7. An important OH Fe(V)=O OH Fe(IV)-OH HO O O HO O O OH HO O O HO Fe(IV)-OH Fe(III) • oxygen rebound O O OH OH 2,7,4'-trihydroxyflavanone –H2O dehydratase O HO O OH daidzein Scheme 7 • rearrangement observation in that system is the ‘oxygen rebound’ process where it is proposed that the rearranged radical is quenched by an hydroxyl radical from Fe(iv)-OH to generate 2,7,4A-trihydroxyisoflavone. In cell free extracts this intermediate was isolable and the new hydroxy group was labelled from 18O2. A dehydratase then acts to generate the isoflavanone daidzein.We have recently demonstrated that the P-450 inhibitor chlotrimazole appears to inhibit the conversion of littorine to O O– HO D D. stramonium Me N Me N D. stramonium O O O 25-29% loss D O oxygen-18 HO D OH littorine hyoscyamine 71-75% retention Scheme 8 hyoscyamine17 in roots of D. stramonium. So it became relevant to explore the possibility of an oxygen rebound process operating in the rearrangement of littorine to hyoscyamine. Our most recent results18 utilising (R,S)-phenyl[2-2H,18O]lactate have shed some light on this issue but do not provide convincing evidence for an oxygen rebound process operating during the rearrangement of littorine to hyoscyamine. After supplementing D. stramonium cultures with (R,S)-phenyl[2- 2H,18O]lactate as shown in Scheme 8 GCMS analysis demonstrated that both littorine and hyoscyamine had enriched M + 3 ions showing that the oxygen-18 and deuterium atoms were both incorporated but to different extents in each of the metabolites.The relative ratio of M + 1 (2H only) to M + 3 (18O + 2H) in the molecular ions of hyoscyamine and littorine demonstrated that ~ 71–75% of the oxygen-18 was retained and recriprocally that ~ 25–29% of the oxygen-18 was lost during the conversion from littorine to hyoscyamine. This can be accounted for by several mechanistic possibilities as illustrated in Schemes 9 10 and 12. A process initiated by iron-oxo abstraction of hydrogen will generate a substrate radical. Rearrangement to a product radical followed by oxygen rebound in the classical manner [Scheme 9 and process (a) Scheme 10] would then generate an aldehyde hydrate as an intermediate.If this is the case then the collapse of this hydrate to an aldehyde prior to reduction by a dehydrogenase may be partially stereospecific as only ~ 25–29% of the original C-2A oxygen of littorine is lost or fully stereospecific forwarding retention of the labelled oxygen with loss of isotope occurring by exchange of the aldehyde oxygen with the aqueous medium prior to reduction. A non-ster- 2 1 Fe(V)=O CO2R • CO2R isomerisation HO Fe(IV)-OH Fe(IV)-OH Scheme 9 • OH O eospecific process would lead to 50% loss and this is not observed. An alternative explanation [Scheme 9 and process (b) Scheme 10] invokes disproportionation of the putative Fe(iv)- OH intermediate and the product radical to generate an aldehyde directly.Such a conclusion has been discussed in a P-450 mediated oxidation operating during oestrogen biosynthesis19,20 where a similar level (80%) of oxygen-18 retention was observed in the oxidation of a primary alcohol to an aldehyde as shown in Scheme 11. Again the high retention of oxygen-18 here either requires a collapse of a diol hydrate (generated after oxygen rebound) or disproportionation. In the latter case the ~ 20% loss of oxygen- 18 can be accounted for by some exchange of the aldehyde carbonyl with the aqueous medium. In the light of the high level of retention of oxygen-18 in going from littorine to hyoscyamine and also in the case in Scheme 11 the oxygen rebound process perhaps appears less likely than disproportionation as it requires both systems to display the same stereoselectivity and to favour retention of the original C–O bond.Alternatively a two electron oxidation of littorine to generate a carbocation as illustrated in Scheme 12 offers an appealing mechanism. The generation of carbocations in iron-oxo systems is not judged so common but such intermediates are implicated for example during the biosynthesis of prostacylin and thromboxane, 21 in two closely related heme-thiolate enzymes. Also carbocations have been recently implicated22 in the generation of minor side products in reactions of mechanistic probes in P-450 enzyme hydroxylations.Scheme 12 illustrates a two electron oxidation of littorine 1 to a substrate carbocation. Rearrangement and the collapse of the product carbocation to an aldehyde would not require oxygen loss and is consistent with the experimental observation with labelled oxygen if accompanied by some exchange at the aldehyde level. An attractive feature here is that the substrate oxygen • OH Fe(IV)-OH a HO OH Fe(III) rebound H H + disproport- • OH O Fe(IV)-OH b Fe(III) H2O ionation H H Scheme 10 HO O P-450 80% retention oxygen-18 O O Scheme 11 CO2R dehydrogenase Fe(III) + H2O O disproportionation CO2R OH Fe(III) OH oxygen rebound 211 Chemical Society Reviews 1998 volume 27 CO2R dehydrogenase O 1 H2O Fe(V)=O (two electron oxidation) CO2R + + HO OH O Fe(III) + –OH Fe(III) + –OH Scheme 12 + cation ring opening + OMe OMe • • radical ring opening OMe OMe Scheme 13 2 CO2R isomerisation benzylic carbocation is predicted to rearrange to the more stable product carbocation,22,23 an oxonium ion.This is most clearly illustrated by the methylcyclopropane ring opening reactions of Newcomb23 shown in Scheme 13 which show a common cyclopropane ring being opened under radical and carbocation conditions. The stabilising substituents are aryl and oxygen and the system closely models the putative intermediates in the littorine to hyoscyamine rearrangement. It was demonstrated that the methylcyclopropane carbocation opens towards oxygen in the same direction as the rearrangement of littorine to hyoscyamine whereas the methylcyclopropane radical opens towards the aryl ring the opposite direction to the rearrangement.So such models suggest a carbocation process. In conclusion our working hypothesis proposes the involvement of two enzymes (mutase + dehydrogenase). The experimental evidence is consistent with an iron-oxo mutase perhaps a heme-thiolate in view of the inhibition by the P-450 inhibitor chlotrimazole17 and an analogy with carbocation generating heme-thiolate enzymes however the mechanism of the rearrangement remains elusive and must await further evaluation and in particular enzyme isolation. More generally little is known of the enzymes implicated on this biosynthetic pathway.Earlier claims made in the literature for enzyme activities that convert phenylalanine to phenylpyruvate and that esterify tropine with free tropic acid (see ref 24 for a review) have proved dubious. A transaminase for phenylalanine reported25 from Hyoscyamus albus transformed roots shows only weak activity and poor kinetic properties. Despite the efforts of several laboratories the conversion of phenylpyruvate to phenyllactate in vitro has not been demonstrated. Similarly the putative CoA-thioligase for phenyllactate and phenyllactoyl-CoA tropine acyltransferase activities have both proved ellusive. Yet these studies have used tissues from Chemical Society Reviews 1998 volume 27 212 7 Acknowledgements We are grateful to all of our co-authors but particularly to Dr Nicola C.J. E. Chesters and to Professor Heinz G. Floss Dr Jack G. Woolley and Dr Nicholas J. Walton for their contributions to the project. We also thank the CIBA foundation for an ACE award. which other enzymes,24 notably hyoscyamine 6b-hydroxylase,26 have been readily extracted and purified. A clear definition in vitro of these activities is required to confirm that the pathway of tropic acid biosynthesis proposed on the evidence of chemical labelling is indeed that which functions in planta. Received 9th January 1998 Accepted 16th January 1998 8 References 1 R. Robinson Proceedings of the University of Durham Philosophical Society 1927–1932 8 14.2 R. Robinson Structural Relations of Natural Products Clarendon Press Oxford 1955. 3 E. Leete N. Kowanko and R. A. Newmark J. Am. Chem. Soc. 1975 97 6826. 4 M. J. C. Rhodes R. J. Robins J. D. Hamill A. J. Parr M. G. Hilton and N. J. Walton in Secondary Products from Plant Tissue Culture eds. B. V. Charlwood and M. J. C. Rhodes Oxford University Press Oxford 5 A. Spencer J. D. Hamill and M. J. C. Rhodes Phytochemistry 1993 32 6 M. Ansarin and J. G. Woolley (a) Phytochemistry 1993 32 1183; (b) 7 R. J. Robins J. G. Woolley M. Ansarin J. Eagles and B. J. Goodfellow 1990 Proc. Phytochem. Soc. Europe 30 201. 911. J. Nat. Prod. 1993 56 1211. Planta 1994 194 86. 8 M. Ansarin and J. G. Woolley Phytochemistry 1994 35 935. 9 (a) N.C. J. E. Chesters D. O’Hagan and R. J. Robins J. Chem. Soc. Perkin Trans. 1 1994 1159; (b) N. C. J. E. Chesters D. O’Hagan and R. J. Robins J. Chem. Soc. Chem. Commun. 1995 127. 10 M. Ansarin and J. G. Woolley J. Chem. Soc. Perkin Trans. 1 1995 487. 11 (a) N. C. J. E. Chesters D. O’Hagan R. J. Robins A. Kastelle and H. G. Floss J. Chem. Soc. Chem. Commun. 1995 129; (b) N. C. J. E. Chesters K. Walker D. O’Hagan and H. G. Floss J. Am. Chem. Soc. 1996 118 925. 12 (a) E. Leete Can. J. Chem. 1987 65 226; (b) E. Leete J. Am. Chem. Soc. 1984 106 7271. 13 V. R. Platt C. T. Opie and E. Haslam Phytochemistry 1984 23 2211. 14 R. J. Robins P. Bachmann and J. G. Woolley J. Chem. Soc. Perkin Trans. 1 1994 615. 15 J. Retey in B12 Biochemistry and Medicine ed. D. Dolphin Wiley 1982 2 357. 16 (a) T. Hakamatsuka M. F. Hashim Y. Ebizuka and U. Sankawa Tetrahedron 1991 47 5969; (b) M. F. Hashim T. Hakamatsuka Y. Ebizuka and U. Sankawa FEBS Lett. 1990 271 219. 17 I. Zabetakis R. Edwards J. T. G. Hamilton and D. O’Hagan Plant Cell Rep. 1998 in press. 18 C. W Wong J. T. G. Hamilton D. O’Hagan and R. J. Robins Chem. Commun. 1998 in the press. 19 M. Akhtar M. R. Calder D. L. Corina and J. N. Wright Biochem. J 1982 201 569. 20 M. Akhtar and J. N. Wright Nat. Prod. Rep. 1991 8 527. 21 V. Ullrich and R. Brugger Angew. Chem. Int. Ed. Eng. 1994 33 1911. 22 M. Newcomb M. H. Le Tadic-Biadatti D. L. Chestney E. S. Roberts and P. F. Hollenberg J. Am. Chem. Soc. 1995 117 12085. 23 M. Newcomb and D. L. Chetney J. Am. Chem. Soc. 1994 116 9753. 24 R. J. Robins and N. J. Walton in The Alkaloids ed. G. A. Cordell Academic Press Orlando 1993 44 115. 25 K. Doerk Disertation zur Doktorgrades Universit�at D�usseldorf 1993 pp. 187. 26 T. Hashimoto and Y. Yamada Eur. J. Biochem. 1987 164