摘要:

Natural Product Reports Editorial Board Professor G.Pattenden (Chairman) University of Nottingham Dr. D. V. Banthorpe University College London Professor M. F. Grundon University of Ulster at Coleraine Professor F. D. Gunstone University of St. Andrews Dr. J. R. Hanson University of Sussex Dr. R. B. Herbert University of Leeds Dr.T. J. Simpson University of Edinburgh Natural Product Reports is a journal of critical reviews published bimonthly which is intended to foster progress in the study of natural products by providing reviews of the literature that has been published during well-defined periods. For any individual topic successive reviews will deal with consecutive periods and while the coverage that was provided by the Specialist Periodical Reports on 'Aliphatic and Related Natural Product Chemistry' 'The Alkaloids' 'Biosynthesis' and 'Terpenoids and Steroids' will be continued this will be supplemented by occasional reviews of areas of both general and specific interest to workers in these and in other fields.Bimonthly publication allows greater flexibility than the annual or biennial publication of volumes of each of the series of Specialist Periodical Reports mentioned above in that individual reviews can be published as they become available. All articles in Natural Product Reports are commissioned by members of the Editorial Board. Natural Product Reports (ISSN 0265-0568) is published bimonthly by The Royal Society of Chemistry Burlington House London W1V OBN England. 1985 Annual Subscription Price U.K.f 125.00 Rest of World f 131.OO,USA. $242.00.Change of address and orders with payment in advance to The Royal Society of Chemistry The Distribution Centre Blackhorse Road Letchworth Herts. SG6 lHN England. Air Freight and mailing in the U.S. by Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11003. U.S. Postmaster send address changes to Natural Product Reports Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11003. Second-Class postage paid at Jamaica NY 11431-9998. All other despatches outside the U.K. are by Bulk Airmail within Europe and Accelerated Surface Post outside Europe. Printed in the U.K. 0The Royal Society of Chemistry 1985 All Rights Reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photographic recording or otherwise without the prior permission of the publishers. Computer typeset by SB Datagraphics. Printed in Great Britain by Spottiswoode Ballantyne Printers Ltd Subscription rates for 1985 U.K. f 125.00 Overseas f 131.OO U.S.A. USS242.00 Subscription rates for back issues (1984) are U.K. f 120.00 Overseas f 126.00 U.S.A. USS240.00 Members of the Royal Society of Chemistry should order the journal from The Membership Officer The Royal Society of Chemistry 30 Russell Square LONDON WC1B 5DT England

ISSN:0265-0568    

DOI:10.1039/NP98502FX001

出版商:RSC    

年代:1985

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Printed 0 contains udto 60 highly topical and important references per monthly issue drawn from current scientific and technical literature worldwide. references each include a document title biblio-graphic citation and FULL ABSTRACT making it easy to identify documents of interest. issues are produced by a combination of manual and computerised techniques to ensure compre- hensive cove rage. issues each contain a Subject and a Chemical Index enhanced and cumutated annually to facilitate your search for items of interest. saves you the time effort and expense of per-forming your own literature searches. FREE SAMPLE ISSUES and further details from The Royal Society of Chemistry The University Nottingham NG7 2RD England. Online LHB online -is the computer-readable equivalent of printed LHB and contains about 3,400 references to date searchable by Chemical Name CAS Registry Number Subject Area Use LHB online for both current awareness and retrospective searching- contact your chosen host today for further details ! LHB is available online via Pergamon lnfoline Ltd. 12 Vandy Street London EC2A ZDE England. ESA Information Retrieval Service ESR IN Via Gallileo Gallilei 00044 Frascati Italy. Data-Star Plaza Suite 114 Jermyn Street London SW1Y 6HJ England.

ISSN:0265-0568    

DOI:10.1039/NP98502BX003

出版商:RSC    

年代:1985

数据来源: RSC

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Enzymology in Biosynthesis Mechanistic and Stereochemical Studies of p-Lactam Biosynthesis and the Shikimate Pathway J. A. Robinson and D. Gani Chemistry Department The University Souihampton SO9 5NH Reviewing the literature published to December 1984 1 The Biosynthesis of p-Lactam Antibiotics 1.1 Penicillins and Cephalosporins 1.2 Nocardicins 2 The Shikimate Pathway 3 References This review is not a formal extension of previous coverage of the literature but provides an up-to-date (to the end of 1984) account of recent published experiments focusing on mechan- istic and/or stereochemical aspects of enzyme-catalysed pro- cesses in the two areas of the biosynthesis of p-lactam antibiotics and the shikimate pathway up to phenylalanine and tyrosine.Similar reviews in different areas will appear in due course. 1 The Biosynthesis of P-Lactam Antibiotics 1.1 Penicillins and Cephalosporins Stereochemical and mechanistic studies over the past few years have dramatically improved understanding of the biosynthetic processes leading to the penicillin and cephalosporin groups of p-lactam antibiotics. The intermediates on this pathway that have been discovered to date together with their inter- relationships are shown in Scheme 1. The outstanding progress in this area reviewed here was facilitated initially by the development of cell-free systems derived from a species of the genus Cephalosporiurn and from related micro-organisms that are capable of sustaining the biosynthesis of antibiotics and most recently as a result of the purification and characteriza- tion of the key enzymes that are involved in the formation of the p-lactams.Several excellent reviews describing the experiments that led to the definition of the pathway that is shown in Scheme 1 are already available. -5 In the early stages of the biosynthesis the natural L-isomers of valine cysteine and u-aminoadipic acid are utilized to generate the tripeptide (l) possessing the L,L,D absolute configuration. The precise mechanistic path of these coupling processes is not known although it has been shown6 that one of the carboxyl oxygen atoms in an l8O-labelled L-valine precursor is lost during incorporation into penicillin-V (8) by intact mycelia of Penicilliurn chrysogenum (Scheme 2) whereas the conversion of LLD-ACV (1) into isopenicillin-N (2) by a cell- free extract of Cephalosporiurn acrernoniurn occurred without incorporation of oxygen label from an isotopically enriched Given that the biosynthetic pathways in the two organisms are the same it follows that the oxygen atom is lost during the incorporation of L-valine into the tripeptide (l) possibly by a process involving the activation of the carboxyl of valine as a thioester derivative.As pointed out by Thomas,6 it is conceivable that the mechanisms of activation and coupling resemble those that are known to occur during the formation of the peptide ionophore grami~idin-s.~ It is of interest that the C- 2 centre in valine is inverted during the formation of (l) which parallels the known incorporation of L-phenylalanine into the corresponding D-residue in gramicidin-S.The incorporation of L-valine that is isotopically labelled at C-2 into the tripeptide (l) as might be expected then occurs with complete loss of this label. On the other hand no further stereochemical changes occur in the amino acid building blocks during the formation of LLD-ACV (1). It is important to note for discussion later that the C-3 centre in L-valine is not disturbed at this stage of the biosynthesis. Thus (2s)-[ 1-I4C 3-3H]valine was incorporated into the LLD-ACV dimer with full retention of tritium label at C-3 in the D-valine residue' and (2S,3S)-[4-' 3C]- valine when added to a P-lactam-negative mutant of C.acrernoniurn afforded a tripeptide which contained a (2R,3S)-4- 13C-enriched valine residue' (see Scheme 3). During the past decade the precise role of LLD-ACV in the formation of penicillins has been clearly defined. Of particular note were the first reports that only the tritiated L,L,D- stereoisomer (and not the L,L,L or D,L,D forms) was efficiently incorporated into penicillin-like materials in cell-free extracts that had been prepared by lysis of protoplasts of C. acrernoniurn.* Moreover the L,L,D-tripeptide was isolated by extraction from the mycelia of Penicillium chrysogenurn and C. acrernoniurn.l3 The use of cell-free extracts circumvents the permeability barrier to exogenous labelled tripeptide that is posed by the intact cell membrane.Alternative solutions to this problem involving the use of cells of C. acrernoniurn that had been made permeable by treatment with diethyl ether have also been reported.I4 Later it was demonstrated that LLD-ACV (1) is transformed by cell-free extracts of C. acrernoniurn into isopenicillin-N (2) as the first-formed p-lactam antibiotic,' 5-1 and complementary results have been demonstrated in cell-free extracts of Penicilliurn chrysogenum' * and Streptornyces clavuli- gerus.I9 Also of great interest is the direct observation by *HZ0and by I3Cz1 n.m.r. spectroscopy of the conversion of tripeptides into isopenicillin-N (2) in cell-free extracts of C. acremoniurn. In these experiments no evidence was seen for the existence of enzyme-free intermediates at least within the limitations that are imposed by the sensitivity of the method of observation.Finally h.p.1.c. has proved to be a useful tool for the detection and quantification of turnover of the tripeptide not only into isopenicillin-NZ2 but also the further transforma- tion into penicillin-N (3)23 and into the cephalosporins (4)-(6).24 An unstable epimerase has been dete~ted*~,~~ in protoplast lysates of C. acrernoniurn that converts isopenicillin- N into penicillin-N prior to its conversion into cephalosporins. The conversion of penicillin-N (3) into deacetoxycephalo- sporin-C (4)in a cell-free system from C. acrernoniurn was first reported by Demain and c~-workers,~~-~~ and this important result was later reinforced by the specific incorporation of 3H- and IT-labelled penicillin-N into deacetoxycephalosporin-C in protoplast lysates of C.a~rernonium,~~ by the conversion of penicillin-N into deacetoxycephalosporin-C by permeabilized and sonicated cells of a high-producing mutant of C. ~crernoniurn,~and importantly by the cell-free conversion of isopenicillin-N into deacetoxycephalosporin-CZ6 The ob-served conversion3 of penicillin-N (3) into cephalosporins in cell-free extracts of S. clavuligerus supports the postulate that the identical biosynthetic pathway (Scheme 1) operates in this Gram-positive bacterium also. These experiments clearly demonstrate the linear nature of the biosynthetic pathway and the role of penicillin-N (3) as the immediate precursor of deacetoxycephalosporin-C (4).293 NATURAL PRODUCT REPORTS 1985 SH -02'n'i .&Me -Me + NH3 L-a-Aminoadipic acid L -Cysteine L -Valine -+ + -+ 0 I I C02 H g-(-L-a-aminoadipyl 1-L-Cysteinyl-D-valine (1) (LLD -ACV) I -0 cYyg$ -02cTEZ&: NH3 0 I 'NH3 0 I I C02H C02H Penicillin -N (3) Isopenicillin -N (2 1 C02H CO2H Deacetoxycephalosporin-C (4) Deacetylcephalosporin -C (5) C02 H C02 H 7 a-Methoxycephalosporin -C (7) Cephalosporin -C (6) Scheme 1 H and by Demain and Wolfe and their co-worker~.~~.~~ PhOCH2 N>> In each case the protein that was isolated (M 37 000-40 000) showed one major band by polyacrylamide gel electrophoresis H3i+ i (PAGE) and required Fe?+ O? and ascorbate for maximum activity.No enzyme-free intermediates in the turnover of the 0 I I L,L,D-tripeptide(1) into isopenicillin-N (2) have been detected coj COOH although a stoicheiometric requirement for molecular oxygen has been e~tablished,~~ which is consistent with the loss of four ( 0 ='80 1 Penicillin-V (8) hydrogen atoms from the tripeptide during oxidative bis- Micro-organism i Penicillium chrysogenum cyclization. Isopenicillin-N synthetases have also been partial- Scheme 2 ly purified from or detected in P. chrysogenurn and S. cla~uligerus.~~ On the other hand the partial purification of the Before reviewing mechanistic and stereochemical aspects of ring-expansion enzyme from C. acrernoniurn which catalyses the formation of isopenicillin-N and of cephalosporin-C it is the conversion of penicillin-N (3) into deacetoxycephalosporin- The ring-expansion process appropriate to consider the most recent advances in the C (4)has been des~ribed.~~,~~,~~ enzymology of these (and the remaining) conversions on this [(3)-+ (4)]and the subsequent hydroxylation of (4)to produce (5) pathway.Thus the purification of isopenicillin-N synthetase each show the same absolute requirement for a-ketoglutarate from C. acrernoniurn has been described by the groups at Fe2+,02,and a reducing and the Ciba group have NATURAL PRODUCT REPORTS 1985-5. A. ROBINSON and D. GANI 295 suggested38 that both reactions may be catalysed by the same enzyme no conversion into a p-lactam antibiotic was observed. (or closely similar) proteins since during purification the Indeed it is of interest to note that the N-hydroxy-peptide synthetase and hydroxylase activities could not be separated acts as an inhibitor of the formation of isopenicillin-N from but migrated as a single band (M,ca 33 000) in SDS-PAGE.0-(l) as does 6-(~-a-aminoadipyl)-~-cysteinyl-3,4-didehydro-~-Acetylation of (9,utilizing a~etyl-CoA,~l then leads to ~aline.~~ v4* cephalosporin-C (6). The further transformation of cephalo- sporin-C into 7cc-methoxycephalosporin-C (7) has been ob- x served in Streptomyes clauuligerus and the methoxyl oxygen is s2 * again derived from molecular oxygen43 in an a-ketoglutarate- H H linked dioxygenase process whereas the methyl group origin- RNy ates from methi~nine.~~ The conversion of (6) into (7) in extracts of S.clauuligerus in the presence of S-adenosylmeth-0 Me I ionine a-ketoglutarate Fe2+ and a reducing agent has been dem~nstrated,~~ as has the intermediacy of 7a-hydroxycepha- co; CO H losporin-C.46 From the bio-organic viewpoint two aspects of these H biosynthetic processes have attracted special interest; these are the isopenicillin-N synthetase reaction and the transformation of penicillin-N into deacetoxycephalosporin-C and deacetylce- phalosporin-C. In each case the corresponding conversions I have not been achievable in uitro although several relevant co; model systems have been des~ribed.~~-~O CO H Labelling experiments have established the fate of the (2S,3S 1 carbon hydrogen and oxygen atoms in the L,L,D-tripeptide (1) during its conversion into isopenicillin-N (2) and the subse- (‘Me=13CH3) quent conversion into deacetoxycephalosporin-C (4).The formation of the p-lactam ring occurs in a stereospecific fashion with loss of 3-H but without loss of the hydrogen at C- 2 in the cysteine residue (Scheme 4).Given the R configuration at position 5 in penicillin-N it follows that the p-lactam ring is closed stereospecifically with retention of configurati~n.~ The Reagents i Cephalosporium acremonium (p-lactam-negative mutant); carbon skeleton of the L-a-aminoadipyl residue of the ACV ii oxidation tripeptide is incorporated intact and 70/1 *O-labelling of the carbonyl oxygen of the amide group has been empl~yed~.~ Scheme 3 to show that no loss of this oxygen atom occurs during the SH * formation of isopenicillin-N (Scheme 5).This result renders H U H untenable any mechanism for P-lactam ring-closure that RN requires a dehydration-rehydration step (such as those de- picted in Scheme 5) and which proceeds via a thiazoline or Me Me thiazoline sulphone. Further evidence against the involve- I ment of an N-hydroxy-tripeptide such as (9) as a transient I enzyme-bound intermediate was provided by Scott and co- worker~,~~ who synthesized (10) by the route that is shown in Scheme 6. When (10) was incubated with the partially purified Scheme 4 SH I C02H I- co, I L Scheme 5 NATURAL PRODUCT REPORTS 1985 Me H mbocN 02; HO' '&MeI I C02 bz / '\ CO2H HO It Ill H bocN nbz02C ~ iv I \ C02 H I HO COZ bt (10) R = H (9 1 R = S-Enzyme boc NH mbocN nbzOZC OCO;!Et H$bz C02 H (11) (12) ( boc = benzyloxycarbonyl mboc = 4-methoxybenzyloxycarbonyl bz = benzyl,nbz = 4-nitrobenzyl) Reagents i dicyclohexylcarbodi-imide,DMF (1 1); ii HCl MeNO,; iii (12) N-methylmorpholine CH,CI,; iv Na liq.NH3 Scheme 6 SH HOzC H02 C Scheme 7 SH HA HI H RN 0)x+0AC , C02 H COZH CO zH Cephalosporin -C Scheme 8 It is the valine residue in the L,L,D-tripeptide (1) that showed that upon incorporation label in 3-MeR of valine undergoes the most profound changes during formation of the appeared in 2-Mes of isopenicillin-N,lol whereas label in 3-penicillins and cephalosporins.However not only is the carbon Me of valine appeared in 2-MeR in i~openicillin-N~~* loo (see skeleton of this residue incorporated intact but also the seven Scheme 8). Since it is also knownl1V1* (see above) that no hydrogen atoms at C-2 C-4 and C-4' are not disturbed during change occurs at C-3 in L-valine during its incorporation into the conversion into isopenicillin-N (2). This result although the L,L,D-tripeptide it follows that the thiazolidine ring of first rep~rted~~-~~ a decade ago has been confirmed recentlys7 isopenicillin-N is formed with stereochemical retention. On the (by *H n.m.r. spectroscopy) with tripeptide that had been other hand during the formation of cephalosporin-C from deuteriated at C-2 and in the methyl groups of the valine (3R)-[4-13C]valine* the 13C label appears in the thiazine residue using an efficient cell-free conversion prepared from ring,96197.lol whereas from (3S)-[4-' 3C]valine the label is extracts of C. acremonium (Scheme 7). The C-H bond at C-3 in incorporated into the acetoxymethyl position (Scheme 8). Since valine however is replaced by a C-S bond during the the linear nature of the biosynthetic pathway to penicillins and biosynthesis and this replacement could occur with stereoche- cephalosporins is now well established these results demon-mica1 retention or inversion. To resolve this point several group~~~-l O0 synthesized L-valine that contained an isotopic * Note that replacement of the 3(pro-3R)-methyl group (3-MeR) in valine by a label in only one of the diasteteotopic methyl groups and methyl group that contains I3C affords (3R)-[4-13C]valine.NATURAL PRODUCT REPORTS 1985-5. A. ROBINSON and D. CAN1 297 strate that the 2cr-methyl group (i.e. 2-MeR) in isopenicillin-N virtue of isotopic labelling in only one methyl group. The undergoes hydroxylation whereas the 2P-methyl group (it. 2-addition does not occur to equal extents on each diastereotopic Me,) participates in the ring-expansion process and this aspect face and in fact a clear preference for 3re,4si addition on the 2s component and for 3si,4re attack on the 2R component was of the biosynthesis occurs in a completely stereospecific manner. A more profound stereochemical ambiguity concerns the course of the substitutions at these methyl groups as in each case a hydrogen atom is formally replaced by a heteroatom during the conversion of penicillin-N (3) into cephalosporin-C (6).To reveal the steric course of such processes requires the stereospecific synthesis of a precursor containing methyl groups that are chiral by virtue of isotopic substitution with both deuterium and tritium together with an appropriate analytical method to reveal the location of these labels at the new stereoheterotopic positions in the product.58 The synthesis of valine that contains chiral methyl groups has been described by Crout and co-w~rkers,~~~~~ as well as by Townsend and co- workers,61 and their routes are shown in Schemes 9 and 10 respectively. The former method proceeds via a 3,4-didehydro- valine derivative that is stereospecifically deuteriated at the double-bond terminus.The reduction of this double-bond by catalytic hydrogenation can occur through syn-addition of hydrogen to either face of the double-bond. When tritium gas is used the syn nature of the addition inexorably links the absolute configuration of the newly generated chiral methyl group to that of the centre C-3 which is itself now chiral by CO2H xi -xiii \ 3H 3H H 2H*H + MeJyNH3 co co; co; (2 RSI3S,4R) ( 2 RS 3RJ4S) 3H 3H H 2H t b02 H co; (2RS,3SJ4S) ( 2RSI3R,4R) Reagents i NaH; ii Na amalgam 2H,0; iii LiAlH,; iv TosC1 Et3N; v KCN; vi MeOH HCl; vii NaOCl; viii NaOMe MeOH; ix dilute HCI; x Ac,O K2C03; xi [3H]H2 [(PPh3)3RhCI]; xii AcOH Ac,O; xiii H+ H,O noted.Nevertheless the biologically active 2s isomer will contain a mixture of molecules having only the (3S,4R) and (3R,4S) absolute configurations and this fulfils the first stereochemical requirement given that the fate of each 1i,ii CD2 OH CD,-OCO COMe Me-+H Me-+-H CH2 Ph CH2 Ph I ivv H vi,vii Me H CHzPh viii-x VIII-X H+T D Me T~~ :#+ H kH3 hH3 co co (3R,4S 1 (3R,4R) Reagents i CHIN2; ii LiAID,; iii ClCOCOMe pyridine; iv hv PhH; v horse liver alcohol dehydrogenase NADH; vi N2(CO2Et), PPh3 PhC02H; vii NaOMe MeOH; viii MeSO2CI pyridine; ix LiBH, HTO; x 03,at -78 "C silica gel Scheme 9 Scheme 10 NATURAL PRODUCT REPORTS 1985 H H 'H \/ U H 3H HO L ..HO; Micro-organism i Cephalosporium acremonium Scheme 11 H Reagents i 03,H20; ii NaOH; iii glycolate oxidase catalase Scheme 12 diastereotopic methyl group in L-valine is known during the biosynthesis (see above). The incorporation of these chiral methyl valines into cephalosporin-C was reported by Abraham and Crout and their co-w~rkers,~~~~~ and the position of tritium in the product was established directly by 3H n.m.r. spectros- copy. In the ideal case a stereospecific substitution into the methyl group proceeding with a normal intramolecular kinetic isotope effect will lead to an unequal distribution of tritium label between the two positions at the new methylene group an excess being associated with a =CDT group which arises preferentially by loss of protium.Also the stereoheterotopic position of the excess 3H label will depend crucially on the configuration of the starting material and on the steric course of the reaction. With respect to the exocyclic acetoxymethyl group these expectations were fully realized since the 3H n.m.r. spectrum revealed that in cephalosporin-C that had been biosynthesized from each sample of chiral methyl valine there was an 80:20 distribution of tritium between the two positions in the new methylene group (see Scheme 11 which shows the result of turnover of doubly labelled molecules in the idealized case where an infinite kinetic isotope effect is operative). Clearly therefore the hydroxylation [i.e.(4) -+ (5)] proceeds in a highly stereospecific fashion and with a substantial intramolecular kinetic isotope effect (kH/kD3 5).Unfortunately the overall steric course (i.e. retention versus inversion) could not be established by this method since the tritium (and proton) n.m.r. signals for 3'-H and 3'-Hs have not yet been assigned. In the case of the endocyclic -CH,-S- group the relevant 3H n.m.r. signals in samples of cephalo-sporin-C that had been biosynthesized from the chiral methyl valines were almost identical in the two experiments the distribution of 3H between the two positions at the new methylene group being not essentially different63 from 50 :50. This is not the result that would be expected of a stereospecific reaction but rather indicates that a loss of stereocontrol has occurred during the reaction which in some turnovers has proceeded with retention and in others with inversion of configuration (i.e.overall racemization at the labelled centre). These results have been confirmed and extended in an independent investigation by Townsend and c~-workers.~* In particular valines that contained chiral labelled methyl groups were synthesized (Scheme 10) and incorporated into cephalo- sporin-C. These materials were then degraded (by ozonolysis and saponification) to afford samples of glycolic acid that was derived from the important centres C-3 and C-3' (see Scheme 12). The location of the excess tritium label at C-2 in glycolate could then be established by its conversion into glyoxylic acid catalysed by glycolate oxidase.It is known65 that this enzymic oxidation involves the stereospecific loss of only ~-HR. The oxidation of the glycolate that had been derived from the feeding experiment using the (3S,4S)-valine resulted in loss of 82% of tritium label to the medium whereas only ca 25% of the (13) COZH H 0hNeoH COzH Scheme 13 tritium was lost in the complementary experiment using the (3S,4R)-valine. Again therefore a substantial isotope effect is evident during the hydroxylase-catalysed process and this has now been shown to proceed stereospecifically with retention. Enzyme-catalysed reactions that proceed with a loss of stereochemical control are of special interest. Amongst the substitution processes at a methyl group that are known to occur with apparent loss of stereocontrol are those utilizing coenzyme B, in several of the so-called vicinal interchange rearrangements.Since some of the reasons for the loss of stereospecificity in these cases are understood it may be helpful to consider these processes when discussing the mechanism of the ring-expansion that is catalysed by deacet- oxycephalosporin-C synthase. In particular the established intermediacy of radical species that are generated at unactiv- ated positions in the B ,-dependent reactions suggests but certainly does not prove that similar entities may be present transiently at the active site of the synthase during the formation of the cephalosporin. The contrasting stereochemical fates of the two methyl groups in penicillin-N during the formation of cephalosporin-C are particularly clear and rather intriguing given that both are transformed on closely similar proteins which in each case are Fe2+- and a-ketoglutarate-linked dioxygenases.However the ring-expansion enzyme is not a typical dioxygenase in that an oxygen atom is not incorporated into the product. The mechanisms of the two processes may nevertheless have features in common. In the case of the hydroxylation reaction if removal of a hydrogen atom from the methyl group proceeds by a radical process the stereospecific nature of the transfor- mation argues against the transient generation of a torsiosym- metric methylene radical (1 3) shown in Scheme 13. Either such rotation is restricted by the protein or more probably the lifetime of the free species is too short.Delocalization may be important or (in the extreme) a non-radical process may be NATURAL PRODUCT REPORTS 1985-5. A. ROBINSON and D. GANI envisaged involving the insertion of oxygen into the C-H bond perhaps by an oxenoid-type species similar to that which is thought to be involved in other enzymic hydroxylations and stabilized here by interaction with iron. During the ring-expansion process on the other hand the existence of a radical such as (14) (see Scheme 14) at the unactivated position may now have a lifetime that is long enough for torsion to occur and this would account for the observed loss of stereocontrol. Such a radical may interact with molecular oxygen to generate a hydroxymethyl group at the active site (Path a) that is suitably placed to participate in ring-expansion by rearrangement.The direct migration of sulphur to afford the new tertiary radical (Path b) is unlikely in view of the unfavourable nature of 1,2-radical rearrangements al-though a two-step process involving an elimination-addition path is possible. Alternatively further oxidation to afford a carbo-cation may occur (Path c) prior to capture by the sulphur atom. After rearrangement a new tertiary carbo-cation would H H CO2H (14) C02H 'OH I (Path c) (Path bi/ [-e'l 1 H /H COz H t-eY \ H R0Np& CH3 COzH (4 1 C02H COzH (15) (16 1 (" 1 = -O+NH3z C m be generated that may collapse by direct loss of a proton or be trapped by water.It is of interest to note that the 3p-hyroxycepham (15) has been isolatedz4 from the filtered broth of C. acremonium although it is apparently not converted into cephalosporin-C when added to active cell-free extracts; this indicates either that (15) is a shunt metabolite (and is not on the direct path for bioconversion) or that it cannot enter the catalytic cycle at this point. The corresponding 3a-hydroxyce-pham has not been discovered or tested in this way. However the 3-exomethylenecephalosporin-C (16) has been synthe-sized,66 and although its conversion into deacetoxycephalo-sporin-C (4) was not obser~ed,~~.*~ converted into it was deacetylcephalosporin-C (5) in a cell-free extract of C.acrernonium that contained the synthase and hydroxylase a~tivities.~' The significance of this observation is at present unclear. Finally the cx-and 0-sulphoxides of penicillin-N have also been prepared but no bioconversion into cephalosporins was observed when they were incubated with deacetoxycepha-losporin-C ~ynthase.~~.~~ Returning to the formation of isopenicillin-N (2) from the tripeptide precursor several new results that bear crucially on the mechanism and timing of the bis-cyclization have been reported recently by the groups at Oxford. The synthesis of compounds that may be enzyme-bound intermediates in the conversion of (1) into (2) has received attention. For example an earlier proposal68that the conver-sion may proceed through the thiazepine-containing peptide (18) and the 3-hydroxyvaline-containing peptide (I 7) was tested through the synthesis69 of (17) (18) and (19) which when tested in an active cell-free extract were not converted into isopenicillin-N (see Scheme 15).The extremely unstable SH H I RN H RN ___d 0XJMe I I I COzH COZH I I I j. SH H RN COzH H RN 0x--.Me I Me I COZH H C02H (19) Scheme 14 Scheme 15 NATURAL PRODUCT REPORTS 1985 COP PMB CO2 PMB C02 PMB ( PMB = 4 -methoxybenzyl) Ng> iv -02c -0zc -0 ! CO;! H CO; H (20) Reagents i HgC12,HOCH2CMe2CH20H acid cr-(4-methoxybenzyl)ester ethyl 2-ethoxy-l,2-;ii NEt, N-(4-methoxybenzoyl)-~-cr-aminoadipic dihydroquinoline-1-carboxylate;iii PhH PhOMe CF,C02H; iv H2S Scheme 16 NHBoc I A RCOE 5% - ... I. II ... Ill ____) -Ozc= 0 iv (20) "ti3 0 I I I C02 Bzh CO;! BZh (21) (Bzh = benzhydryl Boc = t -butoxycarbonyl ) Reagents i 1M-HCl MeOH; ii I, NaHCO, aq. THF; iii CF,CO,H PhOMe; iv dithiothreitol or Zn DCI D20 Scheme 17 monocyclic p-lactam tripeptide (20) has also been synthesized by Baldwin and co-w~rkers,~~ following the route that is shown in Scheme 16 and independently by Scott and CO-workers,' as shown in Scheme 17. The unstable nature of (20) whose half-life at pH 6.95 is <3 minutes renders the significance of an earlier report1* of its occurrence in extracts of Penicillium chrysogenun? questionable. Also when the stable disulphide I C02H (21) and dithiothreitol were added to cell-free extracts of either C02H r3H lNaBH4 C.acremoniurnor P.chrysogenum no conversion into isopenicil-8-(L-a -amino adipy1)-\iO' lin-N was detected showing that the enzyme-free monocyclic J p-lactam cannot enter the normal catalytic cycle of isopenicil-L-cysteinyl glycine lin-N synthetase.Nevertheless important evidence that the 0-(ACG) (22) lactam ring is the first to be formed during the biosynthesis of L isopenicillin-N has been obtained. Thus the substrate ana-Inact ivat ion logue 6-(L-cr-aminoadipyl)-L-cysteinylglycine (ACG) (22) upon incubation with purified isopenicillin-N synthetase leads to inactivation7* of the enzyme by irreversible inhibition (see Scheme 18). Also tritium is released to the medium from [~ysteinyl-3-~H,]ACG at virtually the same rate as the inactivation process.Finally the incubation of [cysteinyl-U-C02H 14C]ACGand [3H]NaBH (together) with the enzyme leads to the specific incorporation of tritium into ACG (22) at C-3 in the [cysteiny/-3 3~ IACG cysteine residue. This result clearly shows that processing of C-3 of the cysteine residue can occur first in the catalytic cycle. Scheme 18 NATURAL PRODUCT REPORTS 1985-5. A. ROBINSON and D. GANI 30 1 SH SH Moreover it is reasonable to assume that the analogue causes inhibition of the enzyme because the structural unit that is necessary for the normal formation of a thiazolidine ring is absent allowing one of the two oxidizing equivalents from molecular oxygen to act destructively (see Scheme 18).In complementary experiment^'^ which strengthen the first I conclusion incubation of a 1 :1 mixture of unlabelled LLD-ACV I C02H C02H (1) and [~ysteinyl-3-~H,]ACV (23) with the purified synthetase resulted in preferential conversion of the protiated substrate Icy~teinyl-3-~H~] ACV (23) [va/ine-3-*HI ACV (24) over the dideuteriated substrate into isopenicillin-N. In a similar vein a 1 :1 mixture of unlabelled LLD-ACV (1) and H RN S-S-Enz [~aline-3-~H]ACV (24) gave isopenicillin-N without isotopic discrimination between the two available substrates. Since in competitive experiments of this type the effect of Vmax/Kmis H monitored only those changes in the nature of the carbon- hydrogen bond that take place between the starting material I [i.e.tripeptide (l)] and the transition state of the rate-limiting C02H step can be exposed.74 The appearance of an isotope effect with the cysteinyl-labelled tripeptide (but not with the ualine-labelled (25) tripeptide) therefore indicates that it is cleavage of the HS C(3)-H bond of the cysteine residue that represents the first ..H rate-limiting transition state and this precedes cleavage of the RN C(3tH bond of the valine residue. Although the mechanism of these early stages in the I formation of isopenicillin-N is as yet unclear the results that 0)xSieJ have been described to date support the formation of an C02H enzyme-bound p-lactam such as (25) at the active site (Scheme 19).However as noted by Baldwin and co-worker~,~~ an (2) alternative (if less likely) scenario might involve the formation Scheme 19 of an enzyme-bound thioaldehyde equivalent such as (26). On SH I COzH (27)R1= Me R2= Et (28) R1= Et Rz= Me (29)R1= H Rz= Me H H H H RN RN RN RN Et I ! COZH do2H COz H COzH (30) (34) (31) (32) + H H RN RN 0&xMe + I Me I C02H ratio (30) (34):(35) was ca 10 1:l ratio (32) (33) was ca 3:l Scheme 20 NATURAL PRODUCT REPORTS 1985 SH -n r H C02H C02H (36)R1= D R2= H (38) (37)R'= H R2= D Scheme 21 the other hand convincing evidence for the formation of the thiazolidine ring via a homolytic pathway has been found. The ability to trick the enzyme by supplying modified substrate has in particular revealed hitherto undisclosed aspects of this catalytic cycle.One of the first reports describing the transformation of structural analogues of LLD-ACV (1) in a cell-free extract from C. acremonium focused on the turnover of (27) (28) and (29) (Scheme 20).75 The isoleucine-containing tripeptides (27) and (28) were converted into the corresponding penicillins (30) and (31) with stereochemical retention*O at C-3 of the isoleucine residue. This stereochemical result parallels that which is seen with the natural substrate (1). The analogues however are not good substrates although the isomer (27) is transformed more efficiently than is (28) and both act as inhibitors of the conversion of ACV tripeptide (1) into isopenicillin-N.The peptide (29) containing D-a-aminobutyrate is of special interest. In a cell-free extract from C.acremonium this analogue was transformed largely into the 2-demethylisopenicillin-N (32) with only small amounts of the 2-epimer being detected. The turnover of these analogues by a highly purified preparation of the isopenicillin-N synthetase however al- lowed a more detailed analysis of the product to be made. Firstly,76 the analogue (29) (containing a D-a-aminobutyrate residue) when incubated with the enzyme in the presence of cofactors was transformed into two detectable p-lactams one being the previously noted penam (32) and the other surprisingly being the cepham (33) the ratio of (32) :(33) being ca 3 :1 (Scheme 20).Apparently the enzyme is able to catalyse the oxidative cyclization of the substrate analogue (29) to afford p-lactams that contain either a five- or a six-membered ring. Presumably the mechanistic path that is used for removal of a hydrogen atom from C-3 of the valine residue within (1) now operates to allow abstraction of hydrogen from either C-3 or C-4 of the aminobutyrate residue within (29). The steric course of the C-S bond-closure in the formation of isopeniciliin is known and it was of great interest to investigate also the steric course of the formation of the C-S bond that leads to the penam (32). Two diastereomeric monodeuteriated tripeptides (36) and (37) (Scheme 21) were therefore prepared by Baldwin and co- worker~,~~ and were incubated with the purified synthetase and various cofactors.The penam product was isolated from each experiment and these samples were shown to be identical and to have the structure (38). The hydrogen isotopomer (32) was not detected indicating that a substantial kinetic isotope effect was operating and leading in each case to the removal of protium from C-3 of the valine residue. It follows therefore that formation of the C-S bond occurs with retention at this C-3 during turnover of (37) but with inversion when starting from (36) i.e. the steric course is determined by the position of deuterium in the substrate. These unusual observations bear crucially on the mechanism of formation of the thiazolidine ring. An attractive rationale that is consistent with all of the data that have been described so far involves the removal of a hydrogen atom from C-3 of the valine residue of the substrate (1) in a process that must occur after the irreversible formation of a p-lactam ring.The tertiary radical that is generated (see Scheme 22) should not then undergo torsion around the C(2)-C(3) bond presumably due to the steric influence of the protein or iron atom and coupling to SH I CO 2H C02H 1 I H RN Me 0bN\ 7- ~e '0' I C02H CO2H (39) Scheme 22 the sulphur atom will afford the five-membered ring with overall retention at C-3. In the case of the deuteriated analogues (36) and (37) the substrate again experiences the formation of a P-lactam ring but now the butyryl side-chain can in principle exist in two conformations in which the methyl group occupies either of the sites that are normally filled by the two methyl groups of the side-chain of the valine residue in (1) (see Scheme 23).If at this stage the conformation of the butyryl side-chain is such that deuterium is presented to the radical site a kinetic isotope effect will disfavour cleavage of a C-D bond and allow a re-organization of the substrate such that protium is suitably placed for abstraction. The simplest (but not the only) interpretation is that rotation around the C(2)-C(3) bond is now possible at this stage of the catalytic cycle and the intramolecular isotope effect leads to selective loss of protium. In addition however a conformational change is also required at the stage that follows the abstraction of protium to account for the formation of the (2S)-2-demethyl- isopenicillin-N (38) from the (3s) labelled substrate (36).As indicated in Scheme 23 the secondary radicals that are generated by loss of protium from (36) and (37) can be formulated as (41) and (40),respectively. Interconversion of these (favouring the location of the larger methyl group in the 3P-position) followed by active-site-directed coupling to sul- phur in the preferred conformer (40) would lead to the observed (2S)-2-demethylisopenicillin-N(32). This explanation also finds support (and precedent) among related observations in reactions that are catalysed by coenzyme-B ,-dependent enzymes.65 The formation of the cepham (33) also from the D-a-aminobutyrate-containing tripeptide (29) fits less easily into this stereochemical picture but again presumably arises from the reduced conformational control in the substrate analogue (29) during the stages leading to the formation of the C-S bond with generation of a primary NATURAL PRODUCT REPORTS.1985-5. A. ROBINSON and D. GANI H RN S -Enz XJHB 0 I Me COzH 0AAaMe I I COzH (40) COz H (32) Scheme 23 radical now competing with the H SH H RN S-Enz I I C02H I COzH C02H J/ (29) I H H H RN RN RNvs-Ec I Me 0hNflHA COzH (411 normal formation of a Reagents i isopenicillin-N synthetase Scheme 24 H I CO?H H 0AJ I I C02H (42) + trace Reagents i isopenicillin-N synthetase Scheme 25 The formation of the hydroxymethylcepham (50) and of the hydroxyhomocepham (51) is less easy to reconcile with the mechanistic scheme that has been developed so far and they may be formed in an unrelated manner by the enzyme.It is not yet clear whether the hydroxyl groups in (50) and (51) are derived from the co-substrate dioxygen or from the medium although the former seems more likely. If this is the case the enzyme appears to be functioning with this analogue both as an oxidase in the formation of penams and cephams and as a mono-oxygenase in the formation of (50) and (51)! The secondary radical (Scheme 24). The incorporation of one deuteriated isotopomer of the D-a-aminobutyrate-containing tripeptide (29) into the cepham (33) has been reported,76 although no stereochemical details of the formation of the cepham are yet available.Returning now to the substrate analogue (27) containing D-isoleucine both this and two other tripeptide analogues (42) and (49 one containing D-norvaline and the other D-allylglycine have been incubated with the highly purified isopenicillin-N synthetase with remarkable results. The D-iSOleUCine-COntaininganalogue (27) of LLD-ACVwas tran~formed’~ and into the penam (30) as reported ea~lier,’~ also into the novel cephams (34) and (35) (Scheme 20). On the other hand the D-norvaline-containing peptide (42) was converted by the enzyme into the novel cephams (43) and (44) as shown in Scheme 25 [the ratio (43) :(44) was CQ 7 :11 and the D-allylglycine-containing analogue (45) gave rise to the six distinct P-lactam-containing that are shown in Scheme 26.The structures of all metabolites although each was generated in sub-milligram amounts in solution were rigor-ously established by a combination of spectroscopic and degradative methods. Assuming in all cases that the formation of the p-lactam ring is initiated and that it follows the normal mechanistic course then in each of the analogues (27) (29) (42) and (45) the enzyme is presented with a tertiary a secondary or an allylic carbon for ring-closure. The transformation products that have been derived from (27) (29) and (42) presumably reflect a balance between the formation of five-and six-membered rings uia the corresponding primary secondary or tertiary radicals that are generated at the active site.The formation of the two 2-vinylpenicillins (46) and (47) from (45) is consistent with the intermediacy of a secondary allylic radical (shown in Scheme 27) which could also account for the formation of the homocephams (48) and (49). The formation of the last two compounds may well be stereospecific with respect to C-2 of the C-terminal residue even though a mixture of epimers is observed since (48) and (49) were seen to interconvert spontaneously under the conditions of the enzymic reaction. NATURAL PRODUCT REPORTS 1985 H H RN RN bp + ' 0bN+ 0 I COI H I C02H (46) * -02c mEsHJ ( 50)* 'NH 3 0 I w ; + H RN H RN + 0H>-OH CO;! H / I I C02 H (47) (51) ratio(46):(47):(48):(49):(50):(51) wasca 4 1 :lO:l:2:5 (*for these metabolites,an a-configuration for the carboxyl group was assumed but not proven 1 Reagents i isopenicillin-N synthetase Scheme 26 SH H RN S-Em ti H RN H RN S-Enz I 0HJ -RNliJy? 0)iJ 0 1 I I I I CO;!H C02H CO;! H C02H (45) JI1 (50) + (51) (46)+ (47) Reagents i isopenicillin-N synthetase Scheme 27 multifarious factors influencing the courses of these reactions 1.2 Nocardicins are complex and not yet fully understood although the The nocardicins are a small group of monocyclic p-lactam importance of bond-dissociation energies and of steric and antibiotics that are produced by species of the genus other radical-stability effects has been highlighted and Nocardid5 The seven known nocardicins (54)-(60) may be discussed.78.79 viewed as N-acyl derivatives of (-)-3-aminonocardicinic acid Finally tripeptide analogues have been in (53).Apart from the p-lactam ring the most abundant which the &(L-a-aminoadipyl) moiety has been altered. These metabolite [nocardicin-A (54)] contains an ether-linked include the twelve tripeptides (52a)-(521). Only the analogues D-homoserine residue an oxime and the rare (4-hydroxy-(52b) (52c) and (52d) upon incubation with the purified pheny1)glycine unit. The biosynthesis of nocardicin-A has been isopenicillin-N synthetase gave products having antibacterial studied by Townsend and co-workers,86-88and the peptide activity against Staphylococcus aureus.From this it was origin of the antibiotic has been established by feeding concludedg0 that the minimum structural requirement for N-experimentsg6to whole cultures of Nocardia uniformis subsp. acyl-L-cysteinyl-D-valine tripeptides to be converted into tsuyarnanensis. In particular the efficient incorporation of L-penicillin products by the synthetase is that the N-acyl group methionine L-serine L-tyrosine and (4-hydroxypheny1)glycine has a six-carbon or equivalent chain terminating in a carboxyl has led to the suggestion that the tripeptide (61) is an group hence implying the location of a substrate-binding site intermediate in the biosynthesis of nocardicin-A (54) although that is separate from the catalytically active site of the enzyme.this remains to be proven. The apparent similarity to the The transformation of 6-(D-a-aminoadipy1)-L-cysteinyl-D-biosynthesis of penicillins is made more striking by the valine into p-lactam antibiotics by a partially purified extract of apparent inversion of configuration that occurs during the C. acremonium has also been described.lo2The major product incorporation of ~-(4-hydroxyphenyl)glycine(62) into the N-was characterized as deacetoxycephalosporin-C. The cell-free terminal D-residue in (54) as shown in Scheme 28. Indeed the extract was shown to contain isopenicillin-N synthetase and a-hydrogen of both L-and ~-(4-hydroxyphenyl)glycineis lostg6 the ring-expanding enzyme but no epimerase.during incorporation into both sites in (54). No reports of cell- 305 NATURAL PRODUCT REPORTS 1985-5. A. ROBINSON and D. CAN1 SH R-NdJMe H 0 Me I I C02 H +NH3 0 =H02cY-n-% k; N HAc 0 1; R =H I COz H (53) R = H (54) R= (58) R = 0 HO\N (59) R = HO-0 free systems that are capable of sustaining the formation of corporated into nocardicin-A (54) without significant loss of nocardicins have yet been disclosed. tritium label indicating that the oxidation state of C-3 of serine Of special interest is the mechanism of formation of the remains unaltered. Also double-labelling experiments with p-lactam ring since the experiments that have been de-serine that was tritiated at C-2 showed the retention of ca 20% s~ribed~~,~~ clearly show that this is different to the mechanism of tritium label upon incorporation which was reasonably of formation of the 0-lactam ring in the biosynthesis of interpreted as disfavouring the intermediacy of a 2,3-penicillins.In particular (2S,3RS)-[3-14C,3-3H]serine is in- didehydroalanine residue. The steric course at C-3 in L-serine NATURAL PRODUCT REPORTS. 1985 .. OH Hou \ co; I -OZCvSMe "-NHz H3iJH I I1 A +AH3 (621 CO H coz-F+NH~ o ~ '0 $ ~\ O (61) I C02 H 1 c $ COzH Scheme 28 during the formation of the p-lactam ring has been investi-gated ;87,88 for this stereospecifically deuteriated (2S,3R)-[2,3-2H2]serineand (2S,3S)-[3-ZH,]serinewere prepared.The H n.m.r. signals corresponding to 3-H 4-H, and 4-H in nocardicin-A have been assigned from an analysis of the observed coupling constants in the AMX system and this assignment has received independent supports9 from the synthesis of stereospecifically deuteriated model compounds. The incorporation of deuteriated serines into (54) could then be followed by 2H n.m.r. spectroscopy and this revealed that the p-lactam ring is closed with inversion of configuration (see Scheme 29). This result stands in contrast to that which was obtained from studies on the biosynthesis of penicillins where a complex oxidative transformation occurs proceeding with overall retention at C-3 in cysteine (see above). It appears therefore that a likely mechanism of closure of the p-lactam ring in this case would involve direct displacement of the hydroxyl group of the serine residue (perhaps activated for example by prior phosphorylation) by the amide nitrogen.This process has a sound chemical basis which has been demonstratedg0in successful model reactions in vim involving the cyclization of the peptide (63) under the conditions that have been described by Mitsunobu91 (Scheme 30). Finally the fate of doubly labelled [2-l3C 5N]-~-(4-hydroxy-pheny1)glycine upon incorporation into nocardicin-A has been followed by I3C n.m.r. spectros~opy.~~ A clear retention of the * 3C-15Nbond at both C-5 and C-2 was evident (see Scheme 31) from the observed scalar couplings. The required epimerization at C-2 in the (4-hydroxypheny1)glycine during incorporation into (54) does not therefore involve loss of the amino-group (paralleling observations that were made of the biosynthesis of peni~illin-G~~), and the oxime function at C-2' must be generated by direct oxidation of the intact amino function rather than via an a-keto-amide intermediate that is generated for example by transamination.602 H Micro-organism i Nocardia unijormis subsp. tsuyamanensis Scheme 29 Ft>NaOCH2Ph 0 H OH I C02Me Ft H (63) 0 (Ft = phthalimido ) COZ Me Reagents i (Et02C)2N, PPh, THF Scheme 30 15 ' +NH3 Ii J-OH (0J3C) Micro-organism i Nocardia uniformis subsp. tsuyamanensis Scheme 31 2 The Shikimate Pathway It is well known that the shikimate pathway in plants and micro-organisms leads to the formation of the aromatic a-amino acids tyrosine phenylalanine and tryptophan.Al-though the steps that are shown in Scheme 32 are well established and all of the enzymes that are involved have now been partially or fully purified two aspects in particular have attracted considerable attention recently. These include on the one hand the mechanistic and stereochemical problems that are associated with key reactions and on the other the organizational differences at the enzymic and genetic levels that exist between prokaryotic and eukaryotic organisms. Thus five of the enzyme activities in certain fungi appear as a multifunctional O6 encoded by one complex gene cluster (the arum gene cluster) whereas in Escherichia coli the same five activities are on separate proteins encoded by five separate genes.07-1O9 The potential value of compounds that selectively inhibit this pathway thereby dramatically influ-encing the growth of the plant or microbe has also been recognized and exploited. NATURAL PRODUCT REPORTS 1985-5. A ROBINSON and D. GANI CO;!HI I OH I bH HO' 3 -Deoxy -D -arabino -heptulosonic acid 7 -phosphate t)H (DAHP 1 f--HO" I OH I I I I AH OH OH Shikimic acid Dehydroshikimic acid Deh yd roq uin ic acid (DHQ 1 iii _____) @o'O &OAC02H bH I OH 5 -Enolpyruvylshi kimic acid 3 -phosphate CO H ToH I d L -Phenylalanine Chorismic acid L -Tyrosine )t I I 1 OH ! Prephenic acid J.3 * L-Tryptophan OH Anthranilic acid Reagents i DAHP synthase; ii DHQ synthase; iii EPSP synthase phosphoenolpyruvate; iv chorismate mutase; v prephenate dehydratase; vi prephenate deh yd rogenase Scheme 32 The condensation of phosphoenolpyruvate and D-erythrose 4-phosphate is catalysed by 3-deoxy-~-arabino-heptulosonate-7-phosphate synthase (DAHP synthase). This enzyme is subject to allosteric control by products of the shikimate pathway in particular tyrosine and recently two isozymes of DAHP synthase (a major one of M 137 000 and a minor component of M 175000) have been isolated from Pseudornonasaeruginosa and characterized. O An important observation concerning the mechanism was made by DeLeo and Sprinson,lll and independently by Nagano and Zalkin,' * who showed (by *O-labelling) that it is the C-0 bond in phosphoenolpyruvate and not the P-0 bond that is broken during the new carbon-carbon bond-forming process.Moreover when the enzymic reaction is conducted in [3H]Hz0 tritium label is incorporated into the product at C-3. The steric course of the reaction was studied by Floss and co-workers,' l3 who showed that (22)-[3-3Hl]phosphoenolpyruvate, upon incubation with a cell-free extract from Aerobacter aerogenes was converted into [6-3H,]shikimic acid with predominantly the (6R) absolute configuration. The si face of phosphoenolpyruvate (PEP) thus adds to the re face of the carbonyl group in erythrose 4- phosphate but more importantly the hydrogens at C-3 in PEP largely retain their stereochemical integrity during the synthase reaction (see Scheme 33).This observation is clearly inconsis- tent with a mechanism (shown in Scheme 34) in which PEP is loaded onto the enzyme by an addition-elimination sequence that proceeds through an intermediate (64) which possesses a freely rotating methyl group. The next stage in the pathway involves the conversion of 3-deoxy-D-arabino-heptulosonicacid 7-phosphate into dehydro- quinic acid (DHQ) catalysed by 3-dehydroquinate synthase. This multi-step transformation shows' l4 an absolute require- ment for NAD+ and Coz+,and the enzyme (M = 57 000) has been purified from cells of E. coli B. Only low levels of enzyme are normally present in cell-free extracts and this fact has undoubtedly hindered mechanistic studies on this reaction.Most recently however the purification of DHQ synthase from E. coli K- 12 has been reported' together with details of the construction of two plasmid-bearing strains' 26 that over- produce the enzyme by factors of 20 and 1000. The latter strain was generated by subcloning the aroB gene encoding the synthase behind a tac promoter in a plasmid that carried the p-lactamase (ampicillin-resistance) gene. The host cells were E. coli strain RB791; these carry a mutation that requires the production of DHQ synthase to be induced by the addition of the lactose analogue isopropyl 1-thio-P-D-galactopyranoside (IPTG) to the medium. The IPTG-induced cells then produce 1000 times as much enzyme as the wild strain and this enzyme constitutes ca 5% of the water-soluble protein.The homogen- eous enzyme is thus available in large amounts for mechanistic studies. The isolated enzyme is smaller (M ca 40-44 000) than that which was reported in an earlier isolation but identical to the enzyme that has been isolated from the wild strain of E. coli. Prior to this work important experiments that focused on the steric course and mechanism of the enzymic reaction were reported by Sprinson and co-workers.' l4 The incubation of (7RS)-[7-3H,]DAHP with the partially purified enzyme re- sulted in the formation of DHQ without loss of label; when the reaction was conducted in [3H]Hz0 the conversion of un- labelled DAHP afforded unlabelled DHQ.Similarly (7R)- and (7S)-[7-3Hl]DAHP were each converted into DHQ without loss of label but upon further conversion into dehydroshiki- mate the material that had been derived from the (7R)-isomer completely lost the tritium label whereas tritium was retained in the dehydroshikimate from the (7S)-isomer (see Scheme 35). Since it was known' l6 that the 3-dehydroshikimic acid is formed from DHQ by syn-elimination involving 2-HR it follows that the overall steric course of the synthase reaction at C-7 in DAHP is inversion. Moreover it is clear also that any intermediates that contain a free methyl group at C-7 are ruled out. The turnover of DAHP that is labelled specifically at C-4 C- 5 and C-6 has revealed further important clues to the reaction NATURAL PRODUCT REPORTS 1985 COz H I -H HO' HO' OH t)H Enz Enz 1 -I Scheme 33 Enz Enz + + product Scheme 34 mechanism.* In particular a kinetic isotope effect (kH/kT) '9 of 1.8 on the turnover rate was observed for substrate that was labelled with tritium at C-5 or at C-6 indicating that a redox change at C-5 (presumably mediated by bound NAD+) participates in the catalytic cycle. In support of this quenching the reaction of pure enzyme and DAHP with [3H]NaBH4 affords' the 3-deoxyheptonic acid 7-phosphate (66) (see Scheme 35) which is labelled with tritium at C-5 presumably through interception of the enzyme-bound 5-dehydro-inter- mediate. The formation of quinic acid (67) was also observed but this contained no tritium at C-4 (which is derived from C-5 of DAHP) thereby suggesting either that the 3,4-bisdehydro- quinate (68) is not an intermediate or that its available concentration on the enzyme is very low (see Scheme 36).A mechanistic scheme that is consistent with the data that are available to date is shown in Scheme 36. Evidence for the correctness of this scheme must now await further detailed studies. A new synthetic route to 3-deoxy-~-arabino-heptulosonic acid 7-phosphate has been described by Frost and Knowles.lzO The route which is shown in Scheme 37 starts with 2-deoxy-~- glucose and proceeds in 6% overall yield. A related procedure has been reported by Aldersberg and Sprinson.' Finally the synthesis of phosphonate and homophosphonate analogues of 3-deoxy-~-arabino-hep tulosonic acid 7-p hosp hate and their NATURAL PRODUCT REPORTS 1985-5.A. ROBINSON and D. GANI 309 CO;! H CO;! H I H B A H [-HA] H& t-H;!01 OH HO' , I I bH AH AH 0 T CO;! H CO;!H I I NAD+ NADH HO' -+OH HOfiO H HOG O H t)H 0 \i,ii I CO;! H HO C02H HO C02H + ' H A o H HO" OH HO I Oh I O H NAD+ NADH OH I OH bH 0 (66) (67) Dehydroquinic acid (68) Scheme 36 i ii $. ['HI HCOZH Reagents i DHQ synthase; ii [3H]NaBH,; iii NaIO Scheme 35 interaction with the DHQ synthase from E. coli have been iii-v reported. The conversion of dehydroquinic acid into dehydroshikimic 1 acid was the first demonstrated case1 of an enzyme-catalysed syn-elimination and proceeds' 23 with loss of 2-HR.Rather indirect evidence for a mechanism involving an imine and then formation of an enamine at the active site as shown in Scheme 38 has been formulated by Butler et al.,I 24 who showed that the addition of NaBH to the enzyme together with substrate leads to irreversible deactivation of the enzyme. The aroD gene that encodes the dehydroquinase has been cloned in E. coli.lz5 Shikimic acid is produced from dehydroshikimate by a reduction that is mediated by the NADPH-dependent enzyme vi vii shikimate dehydrogenase. This activity has been purified from i several sources most recently from tomatoes,' 27 and the subcloning of the shikimate dehydrogenase gene (aroE) of E.coli from the transducing bacteriophage hspcl has also been described. * viii-x Shikimate 3-phosphate is converted into 5-enolpyruvylshiki- -.OH* P(OPh)2 mic acid 3-phosphate (EPSP) in an unusual reaction which HHOO q c o 2 CO2 Me involves formally the condensation of shikimic acid 3-phosphate with phosphoenolpyruvate. This reversible process OH 0Me catalysed by EPSP synthase has attracted considerable attention. Important clues to the mechanism of the reaction DAHP came from *O-labelling studies,' 29. 30 which showed that it is Reagents i HCI HS[CH,]$H EtOH; ii Me,CO H,SO,; iii BuLi; the C-0 bond in PEP that is cleaved. Thus PEP that was iv CIC0,Me; v N-bromosuccinimide Me,CO; vi HBr MeOH; labelled with l80 in the bridge position produced [180]phos- vii (PhO),POCl pyridine; viii Pt,O Hz; ix KOH; x Dowex 50 phate; when the reaction was carried out in [3H]H20or 2H20 resin (H+ form) isotopic hydrogen was incorporated into the pyruvyl moiety of the product and into re-isolated PEP.129 On this basis the Scheme 37 310 mechanism that is shown in Scheme 39 was proposed which involves the intermediate (69) possessing a methyl group as C- 3.Consistent with this view when [3-2H2]PEP was used as the substrate in H,O protium was incorporated into the methyl- ene group of EPSP equally at both the 9(pro-E)- and 9(pro-2)- positions indicating that the methyl group is torsiosymmetric and able to rotate freely at the active site. A detailed analysis of the isotope effects that are operating in this reaction has been reported by Knowles and co-workers,' * who demonstrated an apparent isotope effect of ca 2 favouring loss of protium over deuterium from the methyl group in (69) that had been derived from [3-'H2]PEP.Moreover the appearance of small portions (-8.8%) of unlabelled EPSP from [3-2H2]PEP after short incubation times indicated that loss of isotope to the solvent could occur by wash-out from the intermediate (69) possibly by the routes indicated in Scheme 39. On the other hand the deuterium content of PEP that was re-isolated from such experiments was almost unchanged indicating that debinding of PEP is relatively slow and that the intermediate partitions preferentially towards the product. Similarly the transforma- tion of unlabelled substrates in [3H]H20 generated after 50% reaction a product with a specific activity of 17% of that of the solvent whereas the re-isolated PEP had a specific activity of only 1.8% of that of the solvent.Since the isotope effect will favour retention of tritium in the elimination step that leads to the product the lower than expected wash-in of tritium indicates that a significant isotope effect also operates during the addition step that leads to (69). The transformation of other doubly labelled precursors in HzO and 2H20 also showed retention of tritium ; this observation further supports the existence of these isotope effects. The mechanism of the EPSP synthase reaction has also been studied by Abeles and co-workers.'32 They pointed out the similarity of this reaction to that which is catalysed by UDP- GlcNAc-pyruvyl transferase during which PEP is attached to OV I OH I OH OH Enz-NH2 I i HO C0,H CO H I YAl O H I Enz 6H Enz OH Scheme 38 CO;!H CO;!H I I A It NATURAL PRODUCT REPORTS 1985 UDP-GlcNAc during the biosynthesis of the bacterial cell wall.In this process however evidence for the formation of an intermediate 'enzyme-PEP' adduct has been obtained. EPSP synthase will normally catalyse the exchange of the hydrogens at position 3 in PEP with protons of the solvent only in the presence of both substrates. However when EPSP is replaced by the substrate analogue 4,5-dideoxyshikimic acid 3-phos- phate which lacks the key hydroxyl group to which PEP should be attached an enzyme-catalysed exchange at C-3 in PEP is again observed albeit at a much reduced rate (225 times slower).This implies that addition of the substrate hydroxyl to PEP is also not required for the normal exchange process to occur. An alternative mechanism analogous to that which has been proposed for UDP-GlcN Ac-pyruvyl transferase has therefore been suggested (as shown in Scheme 40),involving here also a PEP-derived enzyme-bound intermediate. The steric course of the EPSP synthase reaction at C-3 of phosphoenolpyruvate has been elucidated independently by the groups of Knowles' 33 and Floss. 34 During the reaction a methyl group is generated transiently at this position; for this to become chiral both deuterium and tritium must be introduced stereospecifically into the substrate.If it is assumed that both the addition and the elimination step each proceed in a completely stereospecific manner (i.e. syn or anti fashion) and that a substantial isotope effect operates to favour loss of protium during the elimination step then the position of deuterium and tritium in the product i.e. at C-9 will be informative for the overall steric course (see Scheme 41). If the addition and elimination steps have opposite steric courses the configuration at the end will be the same as at C-3 in PEP whereas the opposite configuration will result if the addition and elimination steps have the same steric courses. To complete the analysis a catalytic homogeneous hydrogenation was introduced to reduce the enoyl group in the product stereospecifically (by syn addition of hydrogen).This generates new chiral methyl groups in those molecules which still retain both deuterium and tritium label and their absolute configura- tions will be linked to those of the adjacent chiral centres. It is of interest to note that a kinetic isotope effect in the elimination step of the synthase reaction is not essential to the successful outcome of the experiment since under such circumstances the product of the enzymic reaction will contain an equal mixture of the protio-tritio-and deuterio-tritio-isotopomers (non-tritiated material can be safely ignored) (see Scheme 41). Only the latter isotopomer will generate a new chiral methyl group upon catalytic hydrogenation although the formation also of achiral tritiated methyl groups at this stage will severely reduce the clarity of the Luthy-Cornforth configuration assay.58 In practice stereospecifically labelled (E)-and (Z)-[3-2H, 3- 3HI]PEP were each converted into chorismate in a two-step process using a partially purified preparation of EPSP synthase and chorismate synthase.' 33 The labelled chorismates were then transformed as shown in Scheme 42 into doubly labelled chiral acetates and the absolute configuration of each was established by the methods of Luthy et ~1.'~' and Cornforth C02H I H1 H H-B+ It exchange exchange Scheme 39 NATURAL PRODUCT REPORTS 1985-5.A. ROBINSON and D. CAN1 31 1 H RoIco2H HH RO H Scheme 40 HOZC-OR HOzC-OR @o D"T:" " H (anti) or @oxco2H D T -Note ; the anti-syn pathway is indistinguishable from the syn-anti pathway,as is the anti-anti path from the syn-syn path.Scheme 41 CO;! H C02 H I I OH 1Z) ii,iii H - H 3. H T+D T+D T+D iv. v I + I co;,H Reagents i DMSO at r.t. for 12 hours; ii H2 Wilkinson's catalyst; iii Birch reduction; iv L-lactate dehydrogenase; v Kuhn-Roth oxidation Scheme 42 et a1.'63 In this way the (2)-isomer afforded (2S)-[2-2H1 2- 3Hl]acetate (F value = 0.39) whereas the (E)-isomer gave (2R)-[2-2H, 2-3H,]acetate (F value = 0.62). Elsewhere (1R,2R)-[ l-2H, l-3Hl]glycerol was prepared by the route that is shown in Scheme 43 and was administered (together with excess shikimate) to the chorismate-producing mutant of Klebsiellapneumoniae.34 This approach relies on the formation of (2E)-[3-2H, 3-3H,]PEP in tlivo and makes use of the known steric course of the enolase reaction.65 Again the doubly labelled chorismate was degraded by the same route that is shown in Scheme 43 to produce samples of chiral acetic acid the one that was derived from (2s)-lactate being predominantly (2s) (F value = 45). The different experimental approaches thus produce com- plementary and reinforcing results showing that (2E)-[3-2H 3-3H,]PEP is incorporated into chorismate that has the 9E configuration. It is logical to conclude from this (see Scheme 41) that in the EPSP synthase reaction the addition step has an opposite steric course to that for the elimination step.The absolute stereochemical course of each step cannot yet be defined but either the addition is anti and the elimination is syn or vice versa. On the other hand it is of interest to consider the significance of this result in the light of the mechanism that is shown in Scheme 40 and which was discussed earlier. The implication of an enzyme-bound PEP that can exchange the phosphoryl group by substrate hydroxyl with overall retention prior to the elimination step would lead to a different conclusion. The inclusion of these steps combined with the stereochemical results that are discussed above would then require either that both the addition and the elimination stage proceed in an overall anti fashion or that both should proceed in an overall syn fashion.From a chemical viewpoint this may be regarded as the preferred steric course for each process although this preference may be overruled by the enzyme. New advances in this area will now no doubt follow from studies with purified EPSP synthase. This enzyme has been isolated and characterized from several sources including NATURAL PRODUCT REPORTS 1985 OH /iv CO;!H I v,vi H H H H OMe xi,xii xiii xi1 I .1 H v CO2H Reagents i LiAID,; ii liver alcohol dehydrogenase [l-3H]ethanol; iii H30+; iv Klebsiella pneumoniae 62-1 ; v pyridinium dichromate DMF; vi CH,N,; vii [(PPh,),RhCl] H,; viii NaOH MeOH; ix Na liq. NH,; x H,O+; xi L-lactate dehydrogenase NAD+; xii HzOz; xiii D-lactate dehydrogenase Scheme 43 (most recently) Escherichia coli 35 Klebsiella pneumoniae,' 36 and seedlings of Pisum sativum.37 Moreover the subcloning of the E. coli gene aroA which codes for EPSP synthase,' 26 using a multicopy plasmid (PAT1 53) has now been achieved and the transformed cells of E. coli over-produce the synthase by 100- fold.138 The cloned enzyme has been isolated in substantial amounts and appears to be identical (M 40 000) to that which has been isolated from the wild strain. The complete amino- acid sequence of the enzyme of E. coZi [M,(calculated) for 427 amino-acid residues = 46 1 121 has been reported 39 although the important regions of the active site have yet to be identified.The EPSP synthase has also attracted attention because the enzyme from several sources (including K. pneumoniae 40 E. coli,' 38 Neurospora crassa,' 37 seedlings of Pisum sativum,'37 and cultured cells of higher plants' 42) is selectively and powerfully inhibited by N-(phosphonomethy1)glycine (glyphosate). Gly- phosate (70) is now marketed as a powerful broad-spectrum non-selective post-emergence herbicide. The inhibition is reversible and competitive with respect to PEP but not against EPSP or shikimate 3-phosphate and the K is typically 1 pmol dm-3 at pH 6.8.140,141 The nature of the inhibition is of considerable interest and has been extensively studied. 40 The inhibitor is capable of binding to the enzyme in the absence of shikimate 3-phosphate and the magnitude of the inhibition increases sharply with a rise in pH.It has been suggested that glyphosate acts as a transition-state-analogue inhibitor and forms a strong salt bridge with the conjugate base in the active site that mediates the addition-elimination sequence (see Schemes 39 and 40 and Figure 1). Enolpyruvylshikimic acid 3-phosphate is converted into chorismic acid in a 1,4-elimination of phosphoric acid that proceeds in an overall anti fashion142-144 with the loss of 6-HR. c02-I N+/CH2 'CH2 I O\P-*H Figure 1 A possible structure for the complex between glyphosate (70) and the active site of 5-enolpyruvylshikimate-3-phosphatesynthase C02H c0,-I + I OH Chorismic acid + I ' NH3' c0,-(73) I + o-""'+ CH3COC02-Anthranilic arid Reagent i anthranilate synthase Scheme 44 CHz-Cl I o=c c0,-c02-(71) (72 1 In certain micro-organisms chorismic acid also functions as a precursor of anthranilic acid which itself is a key intermediate in the biosynthesis of tryptophan.The conversion of chorismic acid into anthranilate (see Scheme 44) is catalysed by anthranilate synthase. Monofunctional anthranilate syn- thase from prokaryotic organisms contains dissimilar subunits designated AS1 and ASII.145 The AS1 protein catalyses a reaction between chorismate and ammonia affording anthran- ilic acid whereas AS11 binds glutamine and catalyses the transfer of the amide nitrogen to AS1 as ammonia where it can be utilized directly for the formation of anthranilate.A covalent y-glutamyl-AS11 thioester intermediate is formed during the catalytic cycle. 46 Anthranilate synthase is inacti- vated14' by (71) and by (72) (a metabolite of Streptomyces sviceus) which bind reversibly to the glutamine-binding site on AS11 and then alkylate the cysteine residue in the active site that is essential for the utilization of glutamine. The amino-acid sequences of both the AS1 and AS11 subunits from several bacterial sources have been deduced. 48-1 Chemical modifications145.151 of the active site of AS1 have indicated that arginine histidine lysine and cysteine residues are essential for the binding and turnover of substrate. The more complex anthranilate synthase from eukaryotic organ- isms has also been isolated and studied.*52-156 NATURAL PRODUCT REPORTS 1985-5.A. ROBINSON and D. GANI Although the roles of essential amino acids at the active site of AS1 have not yet been determined the course of the reaction initially leads to the intermediate (73) (Scheme 44). The involvement of (73) in the formation of anthranilate has long been surmised and the amino-alcohol (74) is produced by a strain of Streptomyces aureofaciens. 57 However firm evidence for the role of (73) as an enzyme-bound intermediate in the anthranilate synthase reaction has been provided only recently through its synthesis and the observation of its turnover into anthranilate by the AS1 protein from Serratia marcescens. The synthetic routes that have been developed by Ganem' 58 and by Berchtold and WalshlS9 are shown in Schemes 45 and 46 respectively.The observed values of K (-0.1 mmol dm-3) and V,, (600 nmol min-' mg-') convincingly establish (73) as a kinetically viable intermediate although previous attempts to isolate and to characterize (73) from the enzymic reaction have been unsuccessful. A possible explanation of this may be that (73) is unstable in solution and rearranges rapidly to (75) which is an effective inhibitor of the enzyme. s8 No cofactors appear to be involved in the 1,5-substitution process. Given the trans configuration of (73) the second step of the transformation must then involve an overall syn-elimination of pyruvic acid to afford anthranilate. It has been established that the hydrogen atom at C-6 in (73) is not incorporated into the pyruvate that is formed,160.161 thereby negating a concerted pericyclic syn-elimination.The steric course how- ever as it affects C-3 of the pyruvyl moiety has been elucidated by Floss and co-workers,' 34 and this work formed part of their studies on the stereochemistry of the action of EPSP synthase as it has been described above. Chorismic acid was biosynthe- sized from (1R,2R)-[ l-2Hl l-3Hl]glycerol in K. pneumoniae (Scheme 47). The configuration at C-9 of (76) that is doubly labelled with 'M and 3H follows from the known steric course of the reaction of EPSP synthase (see above) and this with C02Me i ,ii v ELNH2 OH (74) ... I I I I-v C02 H I &zcoz"-vi,vii I C02H COzH I I @""".(73) H02CA 0 anthranilate synthase and excess L-lactate dehydrogenase and NADH,afforded (S)-lactate (see Scheme 47). This lactate now containing a chiral methyl group was degraded to acetate and analysed by the methods of Luthy et a1.16* and Cornforth et The F value164 was ca 44.0 indicating that the sample contained an excess of (2S)-[2-ZH1,2-3H1]acetate. A proton is therefore delivered to the re face of the enolpyruvyl side-chain. Apart from the well-known pathways leading to the aromatic a-amino acids intermediates on the shikimate pathway have also been implicated in other areas of secondary metabolism. For example the biosynthesis of the phenazines (77) and (78) is thought to proceed through the intermediate (73),16s*166 whose nitrogen atom again derives from glutamine (Scheme 48).Also the important ansamycin and mitomycin groups of antibiotics such as rifamycin-S and porfiromycin contain a C7N unit whose origin has now been shown to be 3-amino-5-hydroxyben- zoic (see Scheme 49).A possible pathway to this intermediate from dehydroquinate has been discussed. 69 C02Me ,COz Me I I/NHBoc I NHBoc t enant iomer ii,iii ~ iv &NHBoc OH (?I-( 73 1 (BOC = Bu'OCO) Reagents i heat; ii m-chloroperoxybenzoic acid CH:CI,; iii I ,8-diazabicyclo[5.4.0]undec-7-ene, THF; iv steps iii -viii of Scheme 45 Scheme 46 OH CO;! H COz H I 6oxT I I C02H C02H I bH OH iii (76) C02H I H H (BOC = Bu'OCO) (75) Reagents i Bu'OCON=C(Ph)CN; ii CH2N2; iii (Me0,C)2C=N2, + Rh,(OAc), PhH at 80°C; iv H,C=NMe I-; v MeI CH,CI,; vi NaOH aq.MeOH; vii aq. NaOH THF; viii Reagents i Klehsiellapneumoniae 62-1 ;ii anthranilate synthase; iii L-CFAC02H lactate dehydrogenase NADH; iv Cr?072-,H+ Scheme 45 Scheme 47 Chorismic acid stands at a major branch point in the shikimate pathway and of special interest is its transformation into prephenic acid catalysed by chorismate mutase (Scheme 50). This reaction has received much attention and formally represents the only known example of a [3,3] sigmatropic rearrangement in intermediary metabolism. The rearrange- ment also proceeds thermally and calculations show that the uncatalysed process is accelerated about 106-fold by enzymes from Streptomyces aureofaciens and Aerobacter aero-genes.70,1 Although the concerted nature of the enzymic process remains unproven it has been suggested that the observed enhancement of the rate may derive from a combination of factors. These include the stabilization in the active site of the unfavourable conformation [depicted in (79)] that has an axial enolpyruvyl group (calculated to be 7 kcal mol-l less stable than the corresponding equatorial conformer' 70) the 'freezing-out' of other degrees of rotational L (73) J I I OH 0-(77) Micro-organisms i Pseudomonas aureojaciens; ii Brevibacterium iodinum Scheme 48 C02 H mit o mycins OH ansamycins OH Scheme 49 CO ZH I I OH HO (791 NATURAL PRODUCT REPORTS 1985 freedom and possibly also acid-base catalysis by protein- bound residues.However the maximum velocity of the mutase reaction is independent of both ionic strength and pH although these variables do affect the value of K for chorismate. * 72 The concertedness of both the enzymic and non-enzymic rearrangements has been probed by Knowles and co-workers' 73 by measuring secondary tritium isotope effects in competitive turnover experiments using [7-I4C 5-3H]- and [7-'4c 9-3H]-chorismates. In the uncatalysed process kH/kTis 1.149 for the bond-breaking site and 0.992 for the bond-making site. Since substitution with tritium at C-9 should accelerate the rearrangement if the new bond is formed at the transition state whereas substitution with tritium at C-5 should retard the process if the C(5)-0 bond is partially broken at the transition state the observed isotope effects indicate that the transition state is 'asymmetric' with the C(5)-0 bond substantially broken and little (if any) formation of the new carbon-carbon that is eventually formed.Unfortunately the values of kH/kT for the enzymic process are unity at both positions. As it is unlikely that the transition state is so early that virtually no bond-making or bond-breaking has occurred the rate-limiting transition state probably precedes the isotopically sensitive steps. A likely explanation was suggested' 73 in which chorismate binds to the enzyme in its stable equatorial conformation and then in a slow step undergoes a conforma- tional change which places the enolpyruvyl group in an axial position for rearrangement to occur.Alternatively it is conceivable if less likely that the kinetically significant transition state is derived by binding the small equilibrium proportion of the free axial conformer of chorismate. Although both the chair and boat transition states of the rearrangement are allowed processes the geometry of the enzyme-catalysed transition state is of considerable interest and has been investigated in several ways. Chair transition states are generally favoured in Cope rearrangements although Huckel and MIND0/3 calculations have indicated that this could be by less than 2 kcal mol-' in the present case.174,1 75 Structural analogues of putative chair-like and boat-like transition states have been synthesized and investigated as inhibitors of the mutase.74 76,177 In particular the 6-exo-hydroxybicyclo[3.3.l]nonane-l,3-exo-dicarboxylic acid (80) is a good inhibitor (Ki= 3.0 x mol dm-3) of the chorismate mutase-prephenate dehydrogenase from E. coli and A. aero- genes whereas the epimer (81) is not. However amongst other compounds that have been tested' 76 as inhibitors of the mutase is the phosphonic acid (82) which gives 50% inhibition at 70 pmol dm-3. The tight binding of (80)suggests that the active site of the mutase stabilizes the chair form preferentially. Confirmation of this has come recently from elegant stereo- chemical investigations. The geometry of this transition state can be elucidated in principle by following the steric course of the reaction at C-9 in chorismate as illustrated in Scheme 51.The essential (albeit HO HO (80) (81) I I OH Scheme 50 NATURAL PRODUCT REPORTS 1985-5. A. ROBINSON and D GAN COzMe COzMe I I -2 2H Boat ~ vi-/ /xv vii,xii bH HO C02 Me HB -02c-/t-"A 02c*HA Chair ix-xiv IX ,x ,xvi ,xi\-xiv w-Pcoy I I I HO HO COZH C02H Scheme 51 demanding) requirements of the analysis include the prepara- tion of chorismate that is stereospecifically labelled at this position with isotopes of hydrogen together with an analytical technique to show the final stereoheterotopic position of these labels in prephenate.A stereospecific synthesis of [9-2Hl]chorismate and [9-3Hl]chorismate has been reported recently by Hoare and Berchtold,'78 and their synthetic route is shown in Scheme 52. However the labelled material can also be derived biosyntheti- cally from [3-2H1 3-3H1]phosphoenolpyruvate. As described earlier the EPSP synthase reaction proceeds stereospecifically by an addition-elimination mechanism and the intramolecular kinetic isotope effect that operates in the elimination step leads preferentially to chorismate that contains an excess of 3H label (75:25) in the 9(pro-E)-position when starting from (2E)-[3- 3H, 3-2Hl]PEP and in the 9(pro-Z)-position when starting from (2Z)-[3-2H, 3-3Hr]PEP (see Scheme 41). Fortunately an appropriate stereochemical assay of the product was also available through the work of Retey and co- workers 79,1 8o who showed that phenylpyruvate tautomerase stereospecifically catalyses the enolization of phenylpyruvate by removal of the proton 3-HR (see Scheme 53).The rate of spontaneous enolization is rather low at pH 6.2 but is increased several hundred-fold by the enzyme. Accordingly both the synthetic and biosynthetically derived [9-3H]chorismates could be incubated with chorismate mutase and phenylpyruvate tautomerase at pH < 6.181*182 The labelled prephenate that is generated in each incubation undergoes spontaneous decarboxylative elimination at this pH to afford phenylpyruvate and the phenylpyruvate tautomerase then acts to remove 3-HR stereospecifically; this atom appears in the solvent (see Scheme 54).If the tritium label is located at 3-HR,a burst of tritium should appear in the medium whereas from 3-Hs the loss of tritium label occurs more slowly by spontaneous tautomerization. The analysis works convincingly in practice using both the synthetically and biosynthetically derived chorismates. Taking into account the spontaneous slow loss of tritium in the products the sizes of the bursts when extrapolated to zero time were 20% 51% and 67% for the synthesized (Z)-[9-3H ,]chorismate randomly labelled (E/Z)-[9- 3H l]chorismate and enzymically synthesized (E)-[9-3H1]chor- ismate respectively. Since the (E)-[9-3H,]chorismate produces OH OH Reagents i N-bromosuccinimide CCI,; ii NaI Me,CO; iii m-c hloroperbenzoic acid C H C1 ; iv 1,8-d iazabicyclo[ 5.4.01undec- 7-ene CH2CI,; v (Me0,C)2C=N2 Rh,(OAc),; vi Me,kH I- Et,N; vii MeI; viii DMSO at 80 "C; ix Brz CH,Cl,; x DBN CH,Cl,; xi Zn/Ag couple tetrahydrofuran DzO ([3H]H,0); xii aq.NaOH tetrahydrofuran; xiii PhSe- MeOH; xiv H,Oz 3,5-dimethoxyaniline; xv Me,NH DCDO (TCHO); xvi Zn/Ag couple tetrahydrofuran H,O Scheme 52 0 OH Reagent i phenylpyruvate tautomerase Scheme 53 H02C I 'C02 H I HO I OH C02 H I mainly (R)-[3-3H I Iphenylpyruvate whereas (2)-[9-3H,]choris- mate affords mainly (S)-[3-3H1]phenylpyruvate it follows that the enzymic rearrangement proceeds via a transition state with the chair-like geometry. 0' The conversion of chorismic acid into phenylalanine or tyrosine is mediated by at least two different activities.In Escherichia coli Alcaligenes eutrophus Aerobacter aerogenes Reagent i phenylpyruvate tautomerase and Salmonella typhimurium a single bifunctional enzyme Scheme 54 catalyses the conversion of chorismate into prephenate and of prephenate into phenylpyruvate although in several other organisms the two enzymes are distinct proteins. The choris- mate mutase-prephenate dehydratase of E. coli is coded by the pheA gene and kinetic'72*183 and mutant studies,Ig4 as well as amino-acid modifications to the protein indicate that each of the activities occurs at distinct active sites. Studies using labelled chorismate also show that prephenate dissociates from the mutase active site and equilibrates with the medium before combining at the dehydratase site.83 On the other hand tyrosine is also produced in Escherichia coli by a single bifunctional enzyme chorismate mutase-prephenate dehydrogenase. This enzyme requires NAD+ for activity and has been isolated recently from a wild strainIgs and from E. coli that carries a multicopy tyrA-containing plas- mid.Ig6 The level of enzyme in the recombinant strain was 5000 times higher than that from the wild strain and under optimal conditions for growth it constitutes 4% of the dry weight of the cells. The enzyme has M 88 000 consists of two identical subunits and conforms to Michaelis-Menten kinetics. Also steady-state kinetic data indicate' 88 that the substrates 877 bind to the enzyme in a random manner unlike most NAD+-dependent dehydrogenases (whose NAD+ binds to the enzyme before the second substrate is bound).Tyrosine is an end- product inhibitor of this enzyme and it is as yet unclear whether catalysis occurs at separate closely interacting sites or at one common site.189 However recent studiesIg0 on the inhibition of the enzyme by substrate analogues suggest that the chorismate- and prephenate-binding sites share common features in the protein and can be considered to overlap. This contrasts with the distinct and separate sites on the chorismate mutase-prephenate dehydratase. The bifunctional chorismate mutase-prephenate dehydro-genase from a regulatory mutant of E. coli K12 has been isolatedlg1(M = 78 000).This enzyme is also a homodimer and contains three reactive cysteine residues per subunit one of which is particularly susceptible towards alkylation and which is essential for both catalytic activities.The mechanism of the reaction of prephenate dehydrogenase has been probed in an elegant series of experiments by Cleland and co-workers. 92 Two equally feasible mechanisms can be formulated one being a stepwise process in which transfer of a hydride ion to afford a vinylogous P-keto-acid precedes decarboxylation as shown in Scheme 55 (compare this with the known stepwise mechanisms of malic enzyme 93 6-phospho-gluconate dehydrogenase,' 94and isocitrate dehydrogenaselg5) and the other a concerted process whose transition state may be lowered by the ensuing process of aromatization (Scheme 56).These possibilities were distinguished by determining the 3C isotope effects on the value of V/K with both a deuteriated and an unlabelled substrate and the deuterium isotope effect on V/K.From these data it is possible to ascertain whether or not the 3C-sensitive and 2H-sensitive steps are the same.Ig3 Thus the 13C isotope effect that is of interest is that originating from cleavage of the C-C bond during decarboxylation. In a concerted process this effect would be increased after deuteriation of the substrate at C-4 since both the I 3C and 2H isotope effects will operate on the same step; deuteriation effectively makes the 3C-sensitive step more rate-limiting. However in a stepwise reaction the I3C isotope effect will be less with the deuteriated substrate since deuteriation slows down a step other than the I3C-sensitive one.For convenience deoxoprephenate (83) which is also a substrate for the enzyme (V is 5% and V/Kis 0.7% that of the natural substrate) was specifically deuteriated at C-4 and used in this study. Since the I3C isotope effect was less with unlabelled than with deuteriated deoxoprephenate it follows that both the 3C and the ZHisotope effects operate on the same step and the reaction therefore occurs in a concerted manner. Moreover a detailed analysis of kinetic data indicated that in the transition state the hydride is roughly symmetrically placed between C-4 of deoxoprephenate and the si-face at C-4 of NAD,Ig2whereas NATURAL PRODUCT REPORTS.1985 NA CIf NADH II H OH 0 p... OH Scheme 55 0 0 0 C02H OH + NADH + CO2 Scheme 56 C02H OH H I -T H OH -O*6OZH 0*602 0 Reagent i prephenate dehydrogenase Scheme 57 the bond between C-1 and the carboxyl group is only slightly stretched indicating that the transition state for cleavage of the C-C bond occurs early. Interestingly deoxydihydroprephenate (84) is also a substrate for the enzyme but it undergoes only an oxidation to afford 1-carboxy-4-oxocyclohex-2-ene-l -propan-oate (85); no decarboxylation is observed. Thus although the NATURAL PRODUCT REPORTS 1985-J. A. ROBINSON and D enzyme provides the correct environment for oxidative decarboxylation to proceed with prephenate and deoxopre- phenate it is a change in the reactivity of the substrate that with the dihydro-derivative leads to a different mechanistic course (see Scheme 57).Further experiments revealed also that the fully saturated analogue of deoxoprephenate is a very slow substrate (V = 0.07%; V/K = that of prephenate) and pH profiles show both a group (pK = 8.3) that must be protonated if substrate is to be bound and a catalytic group (pK = 6.5)that is a cationic acid (probably histidine) which accepts a proton from the hydroxyl group at C-4 of prephenate concomitant with the oxidation. In contrast the acid-catalysed (non-enzymic) decarboxyla- tive dehydration of prephenate and deoxoprephenate (tt z 3.7 min at low pH) is a stepwise reaction with a carbonium ion intermediate since *Ois incorporated into both the substrate and its 4-epi-isomer during reaction in H2I80.Analogues of prephenate with one double-bond or no double-bonds in the ring are stable towards acids as are the ketones to which they are oxidized by prephenate dehydrogenase. These and other observations that have been reported by Hermes et a1.192thus significantly extend the understanding of the mechanism of the reaction of prephenate dehydrogenase. 3 References 1 D. J. Aberhart Tetrahedron 1977 33 1545. 2 E. P. Abraham J. Antibiot. 1977 30,Suppl. S1. 3 S. W. Queener and N. Neuss in 'The Chemistry and Biology of p-Lactam Antibiotics' ed. R. B. Morin and M. Gorman Academic Press New York 1982 Vol.3 p. 1. 4 J. O'Sullivan and E. P. Abraham in 'Antibiotics' ed. J. Corcoran Springer-Verlag Berlin 198 1 Vol. 4 p. 101. 5 A. L. Demain in 'Handbook of Experimental Pharmacology' ed. A. L. Demain and N. A. Solomon Springer-Verlag Berlin 1983 Vol. 67 Part I p. 189. 6 J. S. Delderfield E. Mtetwa R. Thomas and T. E. Tyobeka J. Chem. Soc. Chem. Commun. 1981 650. 7 R. M. Adlington R. T. Aplin J. E. Baldwin L. D. Field E.-M. M. John E. P. Abraham and R. L. White J. Chem. Soc. Chem. Commun. 1982 137. 8 R. M. Adlington R. T. Aplin J. E. Baldwin B. Chakravarti L. P. Field E.-M. M. John E. P. Abraham and R. L. White Tetrahedron 1983 39 I06 1. 9 For a review see K. Kurahashi in 'Antibiotics' ed. J. Corcoran Springer-Verlag Berlin 1981 Vol.4 p. 325. 10 B. W. Bycroft C. M. Wels K. Corbett A. P. Maloney and D. A. Lowe J. Chem. Soc. Chem. Commun. 1975 923. 11 J. E. Baldwin and T. S. Wan Tetrahedron 1981 37 1589. 12 R. L. Baxter A. I. Scott and M. Fukumura J. Chem. Soc. Chem. Commun. 1982 66. 13 P. A. Fawcett J. J. Usher J. A. Huddleston R. C. Bleaney J. J. Nisbet and E. P. Abraham Biochem. J. 1976 157 651. 14 H. R. Felix J. Nuesch and W. Wehrli FEMS Microbiol. Lett. 1980 8 55. 15 J. O'Sullivan R. C. Bleaney J. A. Huddleston and E. P. Abraham Biochem. J. 1979 184 421. 16 T. Konomi S. Herchen J. E. Baldwin M. Yoshida N. A. Hunt and A. L. Demain Biochem. J. 1979 184 427. 17 Y. Sawada J. E. Baldwin P. D. Singh N. A. Solomon and A. L. Demain. Actimicrob. Agents Chemother.1980 18 465. 18 B. Meeschaert P. Adriaens and H. Eyssen J. Antibiot. 1980,33 722. 19 S. E. Jensen D. W. S. Westlake and S. Wolfe J.Antibiot. 1982 35 483. 20 G. Bahadur J. E. Baldwin L. D. Field E.-M. M. Lehtonen J. J. Usher C. A. Vallejo E. P. Abraham and R. L. White J. Chem. Soc. Chem. Commun. 1981 917. 21 J. E. Baldwin B. L. Johnson J. J. Usher E. P. Abraham J. A. Huddleston and R. L. White J. Chem. Soc. Chem. Commun. 1980 1271. 22 N. Neuss D. M. Berry J. Kupka A. L. Demain S. W. Queener D. C. Duckworth and L. L. Huckstep J.Antibiot. 1982,35 580. 23 S. E. Jensen D. W. S. Westlake and S. Wolfe J. Antibiot. 1982 35 1026. GANI 317 24 R. D. Miller L. L. Huckstep J. P. McDermott S. W. Queener S. Kukolja D. 0.Spry T. K. Elzay S. M. Lawrence and N.Neuss J. Antibiot. 1981 34 984. 25 G. S. Jayatilake J. A. Huddleston and E. P. Abraham Biochem. J. 1981 194 645. 26 J. E. Baldwin J. W. Keeping P. D. Singh and C. A. Vallejo Biochem. J. 1981 194 649. 27 M. Kohsaka and A. L. Demain Biochem. Biophys. Res. Commun. 1976 70 465. 28 Y. Sawada N. A. Hunt and A. L. Demain J.Antibiot. 1979,32 1303. 29 M. Yoshida T. Konomi M. Kohsaka J. E. Baldwin S. Herchen P. Singh N. A. Hunt and A. L. Demain Proc. Natl. Acad. Sci. USA 1978 75 6253. 30 J. E. Baldwin P. D. Singh M. Yoshida Y. Sawada and A. L. Demain Biochem. J. 1980 186 889. 31 H. R. Felix H. H. Peter and H. J. Treichler J.Antibiot. 1981,34 567. 32 S. E. Jensen D. W. S. Westlake R. J. Bowers and S. Wolfe J. Antibiot. 1982 35 1351.33 C.-P. Pang B. Chakravarti R. M. Adlington H.-H. Ting R. L. White G. S. Jayatilake J. E. Baldwin and E. P. Abraham Biochem. J. 1984 222 189. 34 J. Kupka Y.-Q. Shen S. Wolfe and A. L. Demain Can. J. Microbiol. 1983 29 488. 35 I. J. Hollander Y.-Q. Shen J. Heim A. L. Demain and S. Wolfe Science 1984 224 610. 36 R. L. White E.-M. M. John J. E. Baldwin and E. P. Abraham Biochem. J. 1982 203 791. 37 J. Kupka Y.-Q. Shen S. Wolfe and A. L. Demain FEMS Microbiol. Lett. 1983 16 1. 38 A. Scheidegger M. T. Kuenzi and J. Nuesch J. Antibiot. 1984 37 522. 39 D. J. Hook L. T. Chang R. P. Elander and R. B. Morin Biochem. Biophys. Res. Commun. 1979 87 258. 40 M. K. Turner J. E. Farthing and S. J. Brewer Biochem. J.,1978 173 839. 41 H. R.Felix J. Nuesch and W. Wehrli FEMS Microbiol. Lett. 1980 8 55. 42 Y. Fujisawa and T. Kanzaki Agric. Biol. Chem. 1975 39 2043. 43 J. O'Sullivan R. T. Aplin C. M. Stevens and E. P. Abraham Biochem. J. 1979 179 47. 44 J. G. Whitney D. R. Brannon J. A. Mabe and K. J. Wicker Antimicrob. Agents Chemother. 1972 I 247. 45 J. O'Sullivan and E. P. Abraham Biochem. J. 1980 186 613. 46 J. D. Hood A. Elson M. L. Gilpin and A. G. Brown J. Chem. Soc. Chem. Commun. 1983 1 187. 47 J. E. Baldwin and T. S. Wan J.Chem. Soc. Chem. Commun. 1979 249. 48 J. E. Baldwin and A. P. Davis J. Chem. Soc. Chem. Commun. 1981 1219. 49 C. J. Easton J. Chem. Soc. Chem. Commun. 1983 1349. 50 C. J. Easton and N. J. Bowman J. Chem. Soc. Chem. Commun. 1983 1193. 51 D. J.Morecombe and D. W. Young J. Chem. Soc. Cherii. Commun. 1975 198; D. W. Young D. J. Morecombe and P. K. Sen Eur. J.Biochem. 1977,75 133; D. J. Aberhart L. J. Lin and J. Y.-R. Chu J. Chem. Soc. Perkin Trans. I 1975 2517. 52 A. I. Scott S. E. Yoo S. K. Chung and J. A. Lacadie Tetrahedron Lett. 1976 1137. 53 R. L. Baxter G. A. Thomson and A. I. Scott J.Chem.Soc.,Chem. Commun. 1984 32. 54 D. J. Aberhart J. Y.-R. Chu N. Neuss C. H. Nash J. Occolowitz L. L. Huckstep and N. De La Higuera J. Chem.Soc. Chem. Commun. 1974 564. 55 H. Kluender F.-C. Huang A. Fritzberg H. Schnoes C. J. Sih P. Fawcett and E. P. Abraham J. 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Robinson ‘Stereospecificity in Organic Chemistry and Enzymology’ Verlag Chemie Weinheim 1982. 66 J. E. Baldwin B. Chakravarti M. Jung N. J. Patel P. D. Singh J. J. Usher and C. Vallejo J. Chem. Soc. Chem. Commun. 1981 934. 67 R. M. Adlington J. E. Baldwin B. Chakravarti M. Jung S. E. Moroney J. A. Murphy P. D. Singh J. J. Usher and C. Vallejo J. Chem. Soc. Chem. Commun. 1983 153. 68 S. Wolfe R. J. Bowers S. K. Hasan and P. M. Kazmaier Can.J. Chem. 1981 59 406. 69 G. Bahadur J. E. Baldwin T. Wan M. Jung E. P. Abraham J. A. Huddleston and R. L. White J.Chem. Soc.,Chem. Commun. 1981 1146. 70 E. P.Abraham R. M. Adlington J. E. Baldwin M. J. Crimmin L. D. Field G. S. Jayatilake and R. L. White J. Chem. Soc. Chem. Commun. 1982 1130. 71 S. K. Chung R. Shankaranarayan and A. I. Scott Tetrahedron Lett. 1983 24 2941. 72 J. E. Baldwin E. P. Abraham C. G. Lovel and H.-H. Ting J. Chem. Soc. Chem. Commun. 1984 902. 73 J. E. Baldwin R. M. Adlington S. E. Moroney L. D. Field and H.-H. Ting J. Chem. 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ISSN:0265-0568    

DOI:10.1039/NP9850200293

出版商:RSC    

年代:1985

数据来源: RSC

摘要:

The Biosynthesis of Polyketides T. J. Simpson Department of Chemistry University of Edinburgh West Mains Road Edinburgh EH9 3JJ Reviewing the literature published between July 1983 and June 1984 (Continuing the coverage of the literature in Natural Product Reports 1984 Vol. 1 p. 281) 1 Introduction 2 Fatty Acids 3 Triketides 4 Tetraketides 5 Pentaketides 6 Hexaketides 7 Heptaketides 8 Octaketides 9 Nonaketides 10 Decaketides 11 Macrolides 12 Polyether Ionophores I3 Miscellaneous Metabolites 14 References 1 Introduction This report follows the format of previous reports appearing in the ‘Biosynthesis’ series of Specialist Periodical Reports’ and in Natural Product Reports‘ and it covers the literature appearing in the period between July 1983 and June 1984.During the review period the application of oxygen-18 and deuterium labelling using isotope-induced shifts in 3Cn.m.r. spectra to detect the isotope label has continued to develop and provides increasingly useful and often very subtle information on the intermediate processes in the biosynthesis of polyketides. In this context the recent symposium-in-print3 on ‘N.m.r. Spectroscopic Techniques for Studying Metabolic Processes,’ as well as containing several articles that are discussed below and which are of immediate relevance to the biosynthesis of polyketides also makes essential reading for anyone involved in applying n.m.r. techniques to biosynthetic studies. In general polyketide biosynthesis continues to be an active and healthy area.Several of the studies that are discussed below are worthy of special mention. These include Schwab’s elegant stereochemical studies on the P-hydroxydecanoyl-thioester-dehydrase-catalysed allylic rearrangement of (E)-dec- HS HR HA 2-enoate to (Z)-dec-3-enoate the detection of a hexanoate ‘starter’ effect in the biosynthesis of averufin by Townsend and Moreau’s demonstration that the tetrahydrofuranoid myco-toxin botryodiplodin is in fact a further product of the metabolism of orsellinic acid. Finally Parry’s results which indicate a triketide origin for the unusual a-amino acid furanomycin and the unusual labelling patterns in myxovires- cin A that have been reported by Trowitzsch and Hofle show that there are still many surprising and intriguing results to be revealed in the biosynthesis of polyketides.The long-awaited second edition of Turner’s ‘Fungal Metabolites’ has appeared.4 Polyketide-derived compounds are particularly characteristic of micro-organisms and a major part of this book is devoted to them. Along with the first editi~n,~ it provides an incompar- able source of structural and biosynthetic information on polyketides and metabolites of other biogenetic origins. 2 Fatty Acids 0-Hydroxydecanoyl thioester dehydrase which is the pivotal enzyme in the biosynthesis of unsaturated fatty acids in anaerobic micro-organisms mediates the interconversion of acyl-carrier-protein thioesters of (R)-3-hydroxydecanoic acid (I) (E)-dec-2-enoic acid (2) and (Z)-dec-3-enoic acid (3) as indicated in Scheme 1.The key allylic rearrangement which interconverts (2) and (3) has been studied using the dehydrase that is produced by Escherichia coli DM51 A (a cloned over- producing mutant strain). Scheme 2 shows the route by which (4R)- and (4S)-(E)-[4,5,5-2H,]dec-2-enoic acids were synthe- sized. These as their N-acetylcysteinamine thioesters were converted by the dehydrase into the corresponding (2)-dec-3- enoyl thioesters which were isolated after reduction with sodium borohydride and conversion of the resulting alcohols into the correspondingp-phenylbenzoates(4). Analysis of these by ’H n.m.r. showed that the 4(pro-4S)-hydrogen of (2) was retained and that the 4(pro-QR)-hydrogen was removed in the course of the rearrangement to (3).6 The stereochemical course at C-2 was then studied by preparation of the corresponding thioester of (E)-[2-’H]dec-2-enoic acid and studying its conver- R / (4)R =H or D fl SR 1 Ph H Scheme I NATURAL PRODUCT REPORTS 1985 c5H11xco2Me ..... Ill c5H’1xcD0 c5H”Xc02Me c5H”XCD20H HH i_ DD DD DD cfiII+OH R2 R’ c5H11&c0zH x,xi / DD DO (4R)-[4,5,5 -‘H31 ;R’ = H R2 = D (4s)-[ 4,5 5-’H,] ;R’ = D R2= H Reagents i NaOMe MeOD; ii LiAID4 EtzO; iii CrO, pyridine; iv 9-borabicycio[3.3.1]nonane(9-BBN) (+)-a-pinene (for R’ v 9-BBN (-)-a-pinene (for R’ = D R‘ = H); vi TsCI pyridine; vii LiC-CCHzOThp MeOH; ix LiAIH, NaOMe THF; x MnO, pentane; xi NaCIOz Bu‘OH Scheme 2 sion into the ester of (Z)-dec-3-enoic acid by the dehydrase.’ h The configuration of the deuterium label at C-2 of this ester was determined by ’H n.m.r.using the method of Parker.* The labelled thioester was converted into the corresponding satu- rated alcohol which was then re-oxidized to the acid and esterified to the methyl ester (5) of (S)-2-[(3Z)-dec-3-enoyl1-2-phenylacetic acid in which the diastereotopic hydrogen atoms at C-2 are now resolved. A complementary experiment was carried out in which (E)-dec-2-enoic acid was used with de- hydrase in ?HzO. These experiments clearly show that the enzyme-catalysed protonation occurs at the si face at C-2 so that the hydrogen at C-2 in (2) occupies the 2(pro-2R)-position in (3) and the proton from the medium the 2(pro-2S)-position.Thus the enzyme-catalysed allylic rearrangement is a supra- juciul process and the involvement of a single base at the active site of the enzyme in an intra-molecular protein transfer seems likely. These results differ from those previously reported’ for the dehydrase from Brevibacteriurn arnrnoniugenes and suggest that a re-examination of this system may be required. Carbon- 13 n.m.r. and c.d. analysis of ’ 3C,’H-enriched fatty acids from [2-I3C,zH3]acetate-supplementedcultures of the yeast Sac-charornyces cerevisiae the blue-green alga Anacystis nidulans the green alga Chlordla pyrenoidosu and the diatom Phaeodac- of tylum tricornuturn have established the regiochemical distribu- tion of 13C-?H bonds the efficiency of incorporation of ’H at labelled sites and the chiral purity of -13C1HzH- groups.Conclusions on the specificity of the enoyl reductase and desaturase enzymes and on the stereospecificity of hydrogen- exchange processes during the biosynthesis of fatty acids can be drawn from these results.q Analysis of the c.d. of the palmitic acid (6) that was isolated from the cultures has established that the ’H of the monodeuteriomethylene group C-2 (and by inference at the other even-numbered carbons) in the palmitic acid from the three algae is in the pro-S position and that from the yeast is in the pro-R position. Thus in the enoyl reductase ciu an step the hydrogen from the medium is inserted in the pro-R position by re attack (path u of Scheme 3) in the algae and in the pro-S position by si attack (path b of Scheme 3) in the yeast.This then allowed the stereospecificity of the desaturase in each of these organisms to be deduced. The complete absence of ’H label at the olefinic carbon in the palmitoleic acid (7) from the yeast and the retention of this label at similar sites in the unsaturated acids from the three algae means that the desaturase in ull of these organisms stereospecifically removes the pro-R-hydrogen from the even-numbered positions. From these studies it was also concluded that the loss of ’H label varies in amount along the chain in a way that is Characteristic for each enzyme but that the loss which occurs through exchange processes is stereospecific and must occur after transfer of malonate to the acyl-carrier protein.The hydrogen iv (v) vi ix c5H11& R2 p’ c5H11$0Ts t \\ OH vii ,viii f-- D D D D = H Rz= D); (Thp = tetrahydropyran-2-yl) HMPT; viii TsOH CO2H (61 0 R4SE”Z 4-‘\ DH 0 F+Enz HD Scheme 3 C02H (7) that is lost by exchange corresponds to the 2(pro-2S)-hydrogen malonate. The only enzyme that is involved in the biosynthesis of fatty acids which cleaves a C-H bond is the 0-hydroxyacyl thioester dehydrase which is known to remove the 2(pro-2S)-hydrogen of the P-hydroxyacyl thioester (8) stereo-specifically during the dehydration step [Scheme 4(a)]. There- fore it is proposed that this exchange occurs by the action of the p-hydroxyacyl thioester dehydrase on the enzyme-bound malonate (91 as shown in Scheme 4(b).Feeding experiments using doubly labelled [ 1-IJC 9,10-acid (10) show that it is incorporated intact into the 3HH,]oleic long-chain hydrocarbons (1 1) of the green unicellular alga Borryococcus brauni. Their biosynthesis appears to take place elongation-decarboxylation mechanism (Scheme S) analogous to that which operates in higher plants.’” The stereochemistry of hybridalactone (1 8) (an icosanoid that has been isolated from the marine alga Luurencia hybridu) has been established by X-ray crystallography.’ Prior to this the stereochemistry was deduced by a combined analysis of molecular-mechanics calculations and H n.m.r. data and from biogenetic considerations. Based on the absolute stereo- chemistry that was deduced a complete synthesis was initiated.The biogenetic pathway that is outlined in Scheme 6 was proposed. The first step is lipoxygenase-mediated oxidation of the icosapentaenoic acid (1 2) to give the (I 2S)-12-hydroperox-ide (1 3) which is then converted into the epoxy-allylic carbo- cation (14). After rotations to relieve internal non-bonded repulsions this can rearrange [via the cyclopropyl and cyclobutyl carbo-cations (1 5) and ( 16)] to (17); internal NATURAL PRODUCT REPORTS 1985 -T. J. SIMPSON r\ A-H+ A H20 1 [exchange of pro -2s -hydrogen with the medium] A?H A-H+ - SEnz SEnz SEnz H(R1 HS(m) HR Scheme 4 * * 0 C8Hi,CH=CHICH2I6CH2CH2[CH2InCH=CH2 Scheme 5 (11) n = 7 9 or 11 * (12) (13)I- q:;c02H t t t H HA +H H H' (16) (17) H-? (18) Scheme 6 NATURAL PRODUCT REPORTS 1985 nucleophilic attack by the carboxylate group generates *.hybridalactone (18) with the correct absolute configuration. Me-cozNa} *COzH Am 3 Triketides MeCHHC02Na Triketides are very un~ommon,~,~ the only established repre- sentatives being triacetic acid lactone (19)12 and colletodiol (21 1 (20) and related metabolites. Several pyrones (see radicinin citreopyrone and alternaric acid below) possibly contain J a triketide moiety though this has not yet been satisfac- y2 torily demonstrated. However incorporation studies with [ 1-l 3C]propionate and with singly and doubly 13C-labelled acetates indicate that the antibiotic furanomycin (22) which is a metabolite of Streptomyces threomyceticus is derived (Scheme m* 7) from the triketide (21).14 This contrasts with the structurally (22) similar agaric toxin muscarine (23) which is biosynthesized Scheme 7 + N Me3 I -P" OH 0 (19) (20) (23) (25) [ -coz1 H-0 Scheme 8 MeO H~~ (25) R=OH (26) R = H 7 H0 [4,51 OH H I (27) (29) OMe HOkh (30) Scheme 9 NATURAL PRODUCT REPORTS 1985 -T.J. SIMPSON from pyruvate and glutamate.' [U-I4C]Lactate was not in- corporated into (22) so that the necessary hydroxylation at C-2 of the propionate moiety occurs after rather than before assembly of the carbon chain. [l-14C1 ;o,2-3H0,1]Propionate was incorporated with 45% retention of 3H so the possible formation of a keto-function at C-2 of the propionate-derived carbon chain during biosynthesis can also be ruled out.4 Tetraketides Two interesting studies involving novel tetraketides have been reported. Botryodiplodin (24) first isolated from Botryodiplodia theo- bromae has recently been isolated from a toxigenic strain of Penicillium roquefbrti. Incorporation studies with 3C-labelled acetates gave a labelling pattern in (24) that is consistent with its formation uiu ring-cleavage of orsellinic acid (25) as indicated in Scheme 8. This was confirmed by synthesizing [2- 3C,carboxy-I 3C]- and [3,4-' 3C,$orsellinic acids and studying their incorporation. As required by Scheme 8 these labelled botryodiplodin respectively at both C-4 and C-6 and at C-7 only.I8 The biosynthesis of verrucarin E (27)19 can also be explained via ring-cleavage of orsellinic acid.*O Botryodiplodin (and possibly verrucarin E) therefore joins the increasing group of compounds [which includes penicillic acid (28),5 patulin (29),5 and the recently isolated astepyrone (30)2] which are all formed by oxidative ring-cleavage of orsellinic acid (25) or 6-methylsalicylic acid (26) as indicated in Scheme 9. It is interesting to note that a strain of P. roqueforti is reported to produce either penicillic acid or patulin depending on the culture conditions.z1 Asticolorins A (31) B (32) and C (33) are novel mycotoxins isolated from toxic extracts of cultures of Aspergillus multicolor on whole maize." Incorporation studiesz3 on asticolorin C (33) using a variety of 3C-and 'H-labelled precursors indicate a biosynthetic pathway involving one molecule of mevalonate and four molecules of orcinol (34).The labelling patterns from [1,2-l 3Cz]- and [I-*3C,2H3]-acetates are summar- ized in Scheme 10. These are consistent with a biosynthesis of asticolorin C as shown in Scheme 11. Oxidative coupling of orcinol which is formed by decarboxylation of orsellinic acid HO& '0 / O! 0 t II Ho I HO OH II Ho yields pannarol (39,'" which is then prenylated to (36) and hydroxylated to (37). Further oxidative coupling between (36) and (37) followed by formation of the C-15-C-31 bond as indicated allows formation of the D and E rings of the asticolorins as shown.However the observed stereochemistry and labelling patterns would also be consistent with an intermolecular cycloaddition between the diene (38) and the orthoquinone (39) formed from (36) and (37) respectively. 5 Pentaketides The biosynthesis of diplosporin (40) which is a mycotoxin that is elaborated by the maize contaminant Diplodia macrospora has previously been shown to involve the incorporation of methionine-derived carbon atoms into both the carbocyclic and the heterocyclic ring as shown in Scheme 12. In an effort to obtain information on the mechanisms of formation of the rings the origins and fates of the oxygen and hydrogen atoms in diplosporin have been investigated using 2H- and 80-labelled precursor^.'^ In the proton-noise-decoupled (p.n.d.) 3C n.m.r.spectrum of diplosporin that had been derived from [l-13C *H3]acetate the resonance due to C-9 shows P-2H isotope- induced shifts indicating that 1 to 3 *H atoms are incorporated at C-10 and so confirming its derivation from the acetate 'starter' unit of the polyketide chain. Interestingly no acetate- derived 'H was incorporated elsewhere in the molecule. On incorporation of [1-' 3C,1 801]acetate only the C-3 resonance showed an l80 isotope-induced shift in the 13C n.m.r. (31) R = H;X = H -OH (32) R = H;X = 0 (33) R = OH ;X = 0 OH I &-Lo" \ Me-C02Na D.c@ C02Na D DDD Scheme 10 NATURAL PRODUCT REPORTS 1985 HO OH HO OH OH I (38) CD3 C02Na .I .A Me C02Na * i [CD31 methionine A (39) J * I I Scheme 11 so that the involvement of intermediates with the same oxidation level at these centres is ruled out. Citreothiolactone (41) is an unusual sulphur-containing metabolite which has been isolated from the mycelium of Penicillium citreo-uiride BiourgeZ6 along with citreopyrone (42) and pyrenocine B (43) (a phytotoxin that had previously been isolated from Pyrenochaeta terrestri~~'). The labelling pattern resulting from incorporation of [1,2-' 3C2]acetate into these metabolites has been determined by obtaining the I3C n.m.r. spectrum by using an INADEQUATE pulse sequence.28 On the basis of these results a two-chain pathway (shown in Scheme 14) has been proposed.Condensation of a triketide moiety with a diketide as shown in path (a) would give citreothiolactone whereas an alternative condensation between these two moieties [path (b)] would give citreopyrone and pyrenocine B. It is suggested that the sulphur atom originates from the enzyme-bound thiocrotonate. This Scheme embodies a number of features each of which is unusual (i) there is a lack of firmly established precedent for involvement of diketide or triketide moieties in biosynthesis; (ii) multi-chain pathways are rare; and (iii) the existence of alternative modes of condensation of two pre-formed chains in one organism is unprecedented. An alternative pathway which would account for the formation of these compounds from a common intermediate is shown in Scheme 15.This involves alternative ring-cleavages of a pentaketide-derived coumarin (44). The 0-methyl ether of (44) is a known fungal metab~lite.'~ Further studies to test which of these pathways operates would be very worthwhile. Previously reported incorporation experiments' with 3C-labelled acetates and methionine gave a labelling pattern that is consistent with the intermediacy of the symmetrical dialdehyde (47) in the biosynthesis of austdiol(49) which is the main toxic metabolite of a strain of Aspergil/us ustus. By analogy with previous results for citrinin and ascochitin,' a reasonable pathway to (47) would involve reduction of an enzyme-bound thioester (45) to give (46) followed by oxidation of the methyl group that had been derived from methionine (Scheme 16).This has been tested by synthesizing a sample of the keto- aldehyde (46) that was specifically labelled with 'H at the (40) Scheme 12 spectrum. The lack of l8O at C-1 is surprising. On incorpora- tion of [methy/-2H3]methionine 'H label was observed (by ZH n.m.r) at C-1 1 and C-12 showing that oxidation of these two carbons does not go beyond the aldehyde level during the biosynthesis. On the basis of these results it is suggested that C- 1 1 arises by C-methylation at C-2 rather than by 0-methylation at C-5. No overall pathway is proposed but a reasonable mechanism for the biosynthesis consistent with the above observations is shown in Scheme 13. It is noteworthy that no randomization of labelling between C-1 and C-1 1 is observed NATURAL PRODUCT REPORTS 1985-T.J. SIMPSON 0 0 HocHzx% pJ OH Scheme 13 OMe 0 OMe I HO 0 OMe 00 Scheme 14 OMe =-'Uu.b,.-+ (42) _.) (43) 0a02H Scheme 15 methyl group C-13. This was fed to A. ustus and 2H n.m.r. on C-7 and C-8that are derived from the same molecule of showed that label from (46) was specifically incorporated into 802.Surprisingly no I8O isotope-induced shift on the the methyl group C-13 of austdiol with ca 10% efficiency. resonance for the aldehyde group C-12 was observed in the 3C Further confirmation that (46) was an obligatory intermediate n.m.r. spectrum. The authors explain this as being due to facile was provided by an 'isotopic trap' experiment using [Me-14C]-exchange of the oxygen in carbonyl groups as has been noted in methionine and unlabelled (46).30 In a further the incorporation of 802 resulted in an isotope-induced shift being observed in the I3Cn.m.r.for C-7 only. This suggests an oxidation mechanism involving a mono-oxygenase-mediated addition of an oxygen atom to C-7 of either the dialdehyde (47) or the quinone methide (48) as shown in Scheme 17(a) for the quinone methide. The involvement of an intermediate [e.g. (SO)] that is derived through the action of a dioxygenase [Scheme 17(b)] would result in austdiol that contains l8Oatoms previous biosynthetic experiments involving 80-labelled precursor^.^' If this is so then I8O label might equally well be lost from the 0x0 function at C-8 in the intermediate (5 1) on the dioxygenase pathway! Full details have appeared of the incorporation of 2H l8O and I7O from [1-13C,1802]- [2-13C,2H3]- and [1-13C,170]- acetates into citrinin (52) by cultures of Penicillium citrinum.jz Deuterium and oxygen-18 were detected by observation of 3C-2Hcouplings and isotope-induced shifts in the 3C n.m.r.NATURAL PRODUCT REPORTS 1985 i' Me ow Cl/' 0 0 OH COSR (45) CHO1 Me CHO 0 CHO OH 13 OH (471 (46) THO CHO I Me 0 ow 12 OH (48) (49) Scheme 16 CHO 1 (49) c t CHO (50) Scheme 17 spectra of the enriched metabolite. The 3C-17Ocoupling was too small to be detected by I3C n.m.r. spectroscopy but the incorporation of I7Owas measured directly by 170n.m.r.spectroscopy. The results are consistent with the previously established' pathway that is shown in Scheme 18 and confirm that the oxygen atoms that are attached to C-3 C-6 and C-8 originate from acetate. CD3 C02Na * MeC02 Na U 1 i' I' / Me COSR C1 00 OH 1 Ho* CHO C HO Hov OH Me Me 0 HO2C %WCD3 Me *OH (52 1 Scheme 18 OH (531 (54) R HO (55) R = H (57) (56) R = OH Aspyrone (53) and asperlactone (54) are closely related metabolites of Aspergillus melleus. Their biosynthesis has been extensively studied. A full account of previously reported results and details of further studies have ap~eared.~~-~~ Three possible pathways were postulated as a basis for a series of detailed studies using 3C- I4C- *H- and 3H-labelled simple precursors and potential advanced intermediates.These pathways are summarized in Scheme 19. Pathways (a) and (b) are based on the observation that mellein (59 4-hydroxymel-lein (56) and penicillic acid (57) are all co-metabolites of aspyrone and asperlactone. Incorporation of 4C-labelled acetate and malonate into aspyrone followed by comprehen- NATURAL PRODUCT REPORTS 1985 -T. J. SIMPSON 329 sive degradations established that C-9 and C-10 were derived from an acetate ‘starter’ unit. On incorporation of [3H]acetate extensive retention of 3H was observed [particularly at C-7 of Me-C02Na (53)] which immediately ruled out pathway (a)in Scheme 19. Compounds (58)-(61) were each synthesized with a I4C label in the methyl group but none was significantly incorporated into (53) making pathway (b) unlikely.This was subsequently confirmed by incorporating [2-l 3C,2H3]acetate into aspyrone followed by analysis of the 3C n.m.r. spectrum of the product which showed that two acetate-derived hydrogen atoms were incorporated at C-7; therefore intermediates in which C-7 is part of an aromatic ring are excluded. This experiment also showed that up to three acetate-derived hydrogen atoms are incorporated at C-10 confirming its origin from the ‘starter’ acetate of the ~olyketide.~~ Thus pathway (c) involving the rearrangement of a linear pentaketide remained the only possibility (if the possibility that there are two chain mechanisms is excluded).Stereochemical studies on aspyrone and asperlactone established the absolute stereochemistries shown in (53) and (54) and indicated that the 6-and y-lactone rings could be formed by alternative ring-openings of an epoxide intermediate (62) by carboxylate as shown in Scheme RO 20.34On incorporation of [ 1,2-l 3C2]acetate into asperlactone a two-bond 3C-1 3C coupling between C-2 and C-8 was detected indicating their derivation from a rearrangement involving an HOfi HO&i originally intact acetate unit.35 This confirms previous obser- vations for a~pyrone.~~ Finally *H n.m.r. analysis of [2H3]ace- tate-enriched asperlactone confirmed the retention of acetate- (58) R = OH (60) R = OH derived hydrogens on C-5 C-7 C-8 and C-10.A (59) R = H biosynthetic pathway (Scheme 20) has been proposed to (61) R = H accommodate all of the above results and to account for the formation of all of the pentaketide-derived metabolites of A. melleus. The steps that are proposed in this pathway bear comparison with those in the postulated biosynthesis of monensin and other polyether antibiotics where ring-closure onto epoxide intermediates is suggested for the formation of five- and six-membered oxygen-containing rings. Clearly, q2 I*O-labelling studies would be of value in providing further 1 evidence for this pathway. Rice blast disease which is caused by the fungus Pyricularia Hov oryzae is the most serious disease of rice. Several anti-blast COSR -chemicals e.g. tricyclazole (63) are very effective for control of blast disease in vim but are not toxic to P.~ryzue.~~ They appear to act by inhibition of the biosynthetic pathway to melanin OH melanization of the cell wall being essential for pathogeni- Studies with P. oryzue have resulted in the biosynthetic I pathway shown in Scheme 21 being proposed for the formation of melanin. In this 1,3,6,8-tetrahydroxynaphthaIene(64) (derived from a pentaketide precursor) is reduced to scytalone (65); elimination of water to give 1,3,8-trihydroxynaphthalene (66) and a second sequence of reduction and elimination converts (66) ria vermelone (67) into 1,8-dihydroxynaphtha- lene (68) which is then polymerized to melanin. Treatment of (62) cultures with tricyclazole inhibits the reduction step resulting \ in the accumulation of shunt products inter uliu flaviolin (69) isosclerone (70) and 2-hydroxyjuglone (7 1).Recent work has shown that this pathway is common to a large number of ascomycetes and fungi imperfe~ti.~~ The regio- and stereo- specificity of incorporation of label from [’H,]acetate into scytalone (65) in Phiulophora lugerbergii has been studied. yx Deuterium label was incorporated at C-4 and C-5 only with no detectable label at C-2 or C-7 (Scheme 22). The 2H was OH incorporated into both axial and equatorial positions at C-4 so reduction of the established intermediate (64) to the dihydro- (53) (54) naphthalene level is not stereospecific. [2-’ 3C]Malonate was Scheme 20 incorporated with equal efficiency into all of the positions that are indicated in Scheme 22.No ‘starter’ effect was observed. On the basis of this result and the non-incorporation of 2H at C-2 or C-7 it is suggested that scytalone may not be a pentaketide-derived metabolite but may be formed via deacet-ylation of a hexaketide-derived naphthol (72) as indicated in Scheme 2L40 The co-occurrence of the naphthol (73) and (63 1 330 NATURAL PRODUCT REPORTS 1985 HO 0 0 OH HO OH 4Melanin HO OH HO f-+& & 0 OH 0 (68) (69) (70) (711 T t t HO OH HO 0 HO OH HO OH HO OH HO HO (641 Scytalone (65) (66 1 (67) Scheme 21 Scyt alone t CD3 C02Na .C H,(CO Et l2 SR HO OH (721 Scheme 22 * MeC02Na HO 0 (731 (74) *( * OH ii COSR 0 HO *'%TOH HO 0 (75) (76) 5,6 -dihydro 1 nepodin (74) in Rumex alpinus provides some precedent for this (75) proposal.41 Scheme 23 Alterperylenol (75) and dihydroalterperylenol (76) are metabolites of an unidentified species of the genus Alternaria 6 Hexaketides which show activity against the plant-pathogenic fungus Valsa 3-epi-Deoxyradicinol (77) has been isolated along with Incorporation of [2-l 3C]acetate indicates a deoxyradicinin (78); these are new phytotoxic metabolites of ~eratosperrna.~~ pentaketide origin for (75) and (76) via dimerization of 1,3,8- Alternaria helianthi which is the causative agent of seedling trihydroxynaphthalene as shown in Scheme 23.blight and leaf spot (these are major diseases of ~unflower).~~ NATURAL PRODUCT REPORTS 1985 -T.J. SIMPSON Radicinol (80) has also been isolated along with the known compound radicinin (79) from the closely related Alternaria chry~unthemi.~~ Radicinin is of interest as it was the first compound to have its biosynthesis studied by direct 3C n.m.r. methods. The incorporation of [ 1-l 3C]- and of [2-' 3C]-acetate into deoxyradicinin has been reported.45 A two-chain pathway has been generally accepted but a ring-cleavage pathway as shown in Scheme 24 could account for the formation of these metabolites. Studies on the inter-relationships among this group of metabolites would be of interest as would 2H- and I80-labelling studies to ascertain the oxidation levels of the enzyme-bound intermediates.LL-D253a which is a chromanone that was first isolated from Phoma pigmentivora and subsequently from several other organism^,^ was originally assigned the structure (81). This has been revised to (82) on the basis of a complete analysis of the fully proton-coupled 3C n.m.r. spectrum of LL-D253a diacetate (83) and confirmed by synthesis.46 The biosynthesis of the chromanone has been studied by incorporation of I3C- ?H- and sO-labelled acetates; the resulting labelling patterns are summarized in Scheme 25."' A particularly interesting feature was the partial randomization of label from singly 3C-labelled acetates between C-10 and C-11 in the hydroxyethyl side-chain. On incorporation of [I-' 3C,2H2]acetate two 2H atoms were incorporated at both C-10 and C-1 1 and only one at C-3.Taken with the I8Olabelling (Scheme 25) this indicated that the chromanone ring was formed by conjugate addition of a phenolic hydroxyl group to the corresponding ap-unsatu- rated ketone (Scheme 26). Because LL-D253a is optically active the ring-closure is stereospecific with respect to C-2 but analysis of the 'H n.m.r. spectrum showed that the two hydrogen atoms at C-3 were labelled to an equal extent so that protonation of the intermediate enolate must occur with equal facility from both faces as indicated in Scheme 26. This contrasts with the corresponding ring-closure by which a chalcone is converted into a flavanone which is known to be stereospecific with respect to both positions. LL-D253a must be biosynthesized via two pre-formed polyketide chains.One possibility is shown in Scheme 27. The observed randomization 0 OH 00 (77) (78) R = H 1 (77) etc. Scheme 24 33 1 of labelling in 80% of the molecules is accounted for by the formation of a symmetrical cyclopropyl intermediate (84) as shown. This intermediate can undergo hydrolytic ring-opening at either the a-or the P-carbon. According to this Scheme the 20% of the molecules that are not undergoing randomization should have the 11-hydroxyl group derived from the atmos- phere; in accord with this fermentation in an atmosphere of 1802resulted in an Is0 isotope shift being observed on the resonance due to C-1 1 in the I3C n.m.r. spectrum the intensity of the shifted peak being ca 20% that of the unshifted peak.It is not clear whether the randomization is a process that occurs in vivo or in vitro. ""* M eO OR (811 (82) R = H (83) R = AC Me0 0 "O} ~60.40 .:0 - H* D CD3'3C02Na - Me'3C'802Na H0%?D3D OH Scheme 25 1 Scheme 26 NATURAL PRODUCT REPORTS 1985 7 Heptaketides 0 HO 1 20 *I* 1 HO& v:*OH OACO H+ Scheme 27 HO The incorporation of [2-' 3C]- and [ 1,2-]3C2]-acetates into marticin (85) which is a phytotoxic metabolite of Fusarium martii gave the labelling pattern that is shown in Scheme 28. This is consistent with the addition of a C3unit (derived from an intermediate in the Krebs cycle) to a heptaketide which is probably related to the intermediate leading to fusarubin (86) and related corn pound^.^^ No mechanism is proposed but a possible sequence is shown in Scheme 28.It should be possible by appropriate labelling studies to establish which C4 dicarboxylic acid is involved ; oxaloacetate or malate would appear to be the most likely compounds. Incorporation of I 3C- 2H- and *O-labelled acetates into monocerin (87) by cultures of Drechslera ravenelii and analysis by I3C and *H n.m.r. spectroscopy gave the labelling patterns that are shown in Scheme 29.49 A particularly interesting feature is the retention of two acetate-derived hydrogen atoms at C-10 which suggests that reduction of the P-ketoacyl intermediate to the corresponding P-hydroxyacyl intermediate takes place during assembly of the carbon chain. Only one of the diasterotopic hydrogen atoms at C-12 is labelled but the absolute stereochemistry remains to be established.The trihydroxylated moiety (88) is proposed as the likely enzyme- bound precursor (Scheme 30). The 80-labelling pattern (see Scheme 29) means that the benzopyrone ring must be formed by nucleophilic attack at the terminal carboxyl moiety by the hydroxyl group at C-9. It is likely that the cyclization takes place on the thioester (89) to give (90) as the first enzyme-free intermediate. The retention of the acetate carbon-oxygen bond at C-11 indicates that the tetrahydrofuran ring is formed by nucleophilic attack of a hydroxyl function at C-11 on C-8. A mechanism for this would be nucleophilic addition onto a quinone methide intermediate (92) that is formed by oxidation of (91) which is the hydroxylated derivative of (90).This is supported by the co-occurrence of monocerin and the fusaren- tin methyl ether (93) in Fusarium larvarumso Brefeldin A (94) is a metabolite of fenicillium brefeldianum. Its biosynthesis has been much studied as a model for that of the macrolide antibiotics also due to its structural similarity to the prostaglandins and to determine the relationships (if any) between the biosynthesis of fatty acids and of polyketides. In 0 OH (86 1 - Me-CO2Nait Oy?l&Y COSR COSR 0 OH OH0 OH Scheme 28 NATURAL PRODUCT REPORTS 1985 -T. J. SIMPSON D this regard the stereochemistry of labelling from [2-13C,*H3]-acetate in the fatty acids of P. brefeldianum has been determined and compared with the labelling of the correspond- ing positions in brefeldin A.This studys1 was based on the hypothesis that labelling in different positions in the brefeldin CD3-C02Na A molecule might correspond to the labelling in the different MeO intermediates of the biosynthesis of fatty acids. Thus as shown in Scheme 31,positions 2 3 and 11 of (94) may correspond to the enoylthioester intermediate (97); positions 4,6,8 and 12 to the a-carbon of the saturated thioester intermediate (98) ; positions 5 7 9 and 13 to the P-carbon of (98); position 14 to 0- I Me the a-carbon of the P-ketothioester intermediate (95); and Me13C -"02Na position 15 to the P-hydroxy-thioester intermediate (96). The labelling patterns in stearic and oleic acids of P. brefeldianum were determined. The labelling for palmitic acid (99) was Me0 Scheme 29 OH 0 assumed to be the same and is compared with that of brefeldin A in Scheme 31.The stereochemistry at C-6 and C-8 in brefeldin A differs from that of the fatty acid suggesting that the configuration at these centres is determined by some g M =14 1 Yo W (87) HO 89 -H \ HO HO 0 (92) (91) R = H Scheme 30 (93)R = Me v * e4-C-C-SEnr H I - NADPH. V!jEnz SEnz H H' *A H' (95) (96) L * H/" 'COz H A 1[-HOH') Ho 0 NADPH A f-- V S E n z .H20 q S E n z H H H* (98) (97) Scheme 31 process that is unique to the formation of the cyclopentane ring. The epoxy-lactones (100) and (101) have been proposed as the immediate biosynthetic precursors of brefeldin A. Both of these and their alkene precursors (102) and (103) have been synthesized,s2 presumably as a preliminary to testing them as biosynthetic precursors.These results will be awaited with great interest. 8 Octaketides Betaenones A (104) B (109 and C (106) are phytotoxins that have recently been isolateds3 from Phoma betae which is the fungus that is responsible for leaf spot disease of beetroot. They are closely related to the phytotoxins stemphyloxin I (107)s4 and diplodiatoxin (108).5s Although the highly branched structures are suggestive of a propionate origin the incorpora- tion of 3C-labelled acetates and methionine into betaenone B indicates an origin via an acetate-derived polyketide with five methyl groups being derived from the C1pool as indicated in Scheme 32.56It would appear that betaenone A is formed via an (100) NATURAL PRODUCT REPORTS 1985 intramolecular aldol condensation between C-1 and C-17 of betaenone C (106) which would also be the precursor (by reduction at C-18 and oxidation at the methyl group C-15 respectively) of betaenone B (105) and stemphyloxin I (107).A large number of polyketides containing a highly deoxygenated decalin ring system have been isolated recently. These include mevin~lin,~~ c~mpactin,~~ nargenicin A ,s9 chlorothricin,60 kijanimicin,6 the tetrocarcins,62 and ilicicolin H.63A variety of mechanisms such as intramolecular aldol condensation intramolecular cycloadditions and electrophilic cyclization of polyolefins have been proposed for the formation of the carbocyclic systems.Studies to elucidate these mechanisms are in progress (see below) and should produce interesting results. Previous studies on the biosynthesis of streptolydigin (log) which is an acyltetramic acid antibiotic that is produced by Streptomyes lydicus had shown that the methyl groups C-15 C- 16 and C-17 as well as C-18 were derived from propionate. However establishing which parts of the structure were derived from acetate proved difficult because the incorporation of singly labelled [13C]acetate was too low to demonstrate R’ H02C 18 HO (105) R’ = CH20H R2 = Me 15 (108) (106) R’ = CHO R2= Me (104) 18 -*a Me-C02 Na A * [Me] methionine A * A OH OH 00 0 Scheme 32 18 - McCH2C02Na - Me-COz Na ** ___) OH (109) Scheme 33 NATURAL PRODUCT REPORTS 1985 -T.J. SIMPSON enrichment of individual carbon atoms and because there is no degradation scheme that would be suitable for I4C studies. However by feeding [ 1,2-l 3C,]acetate and detecting low-level 13C-13C coupling satellites in the I3C n.m.r. spectrum the origin of carbons 2‘ and 3‘ 1 and 2,9 and 10 and 13 and 14 from intact acetate units was established. The results to date are summarized in Scheme 33. The remainder of the tetramic acid moiety is probably derived from P-methylaspartic acid as glutamate which is known to be a precursor to P-methylaspar- tate and seems to be incorporated exclusively into this part of the molecule.64 Germichrysone (1 10) (an octaketide-derived hydroanthra- cene) is produced in high yields by callus cultures of Cassia forosa.It is also the characteristic pigment of the seedlings whereas the main pigment in the seeds is an anthraquinone glycoside. However the main pigment in six-week-old callus cultures was the xanthone pinselin (1 1 1) and the germichrysone content was markedly decreased suggesting that pinselin is derived via germichry~one.~~ Ilicicolin H (1 14) is a metabolite of Cylindrocladiurn ilicicola containing a 5-(4-hydroxyphenyl)-a-pyridonechromophore. Feeding experiments with 3C-labelled acetates and with 5N-and T-labelled phenylalanine indicate a biosynthetic path- way (Scheme 34) in which the decalin system is derived by cyclization of a bis-C-methylated octaketide precursor which then condenses with phenylalanine to give an acyltetramic acid intermediate (1 12).Rearrangement of the tetramic acid uia an intermediate quinone methide (1 13) would then generate the a-pyrid~ne.~~ The incorporation of ’H from [’HJacetate into the alkyl side-chains of 2-n-hexyl-5-n-propy1resorcino1,which is a polyketide metabolite of Pseudornonas sp. B-9004 has been 0 OH OH Me02C 0 OH HO studied.66 Different levels of incorporation were found as indicated in (1 15). The ’H content was established by ‘H n.m.r. studies on the deuteriated n-hexyl(pheny1)carbinol (1 16) and n- propyl(pheny1)carbinol (1 17) which were obtained from the enriched metabolite as shown in Scheme 35. The (1s) enantiomers were resolved by preparative g.1.c.of their (-)-menthoxycarbonyl esters and recovered by cleavage of the esters with LiAIH4. Deuteron n.m.r. studies in the presence of E~(fod)~ revealed that deuterium was incorporated specifically into the 2(pro-2S)-position of (1 17) and the 3(pro-3S)-position of (1 16) using a method that is described in an accompanying paper6’ and which is generally applicable to determining enantiomeric composition and absolute configuration of 2-and/or 3-deuteriated 3-alkyl-propan-1-01s. 9 Nonaketides The incorporation of [ 1,2-l 3C,]acetate into alternaric acid (1 18) by cultures of Alternaria solani gave the labelling pattern that is shown in Scheme 36 68 Alternaric acid is derived from nine intact acetate units with the remaining carbon atoms being derived from the C pool.The levels of incorporation into C-13 and C-14 and into C-3’ and C-4’ were slightly higher than into the remaining carbon atoms indicating that there are small acetate ‘starter’ effects consistent with a two-chain pathway for the biosynthesis of alternaric acid. To try to distinguish between the two possible pathways (a) and (6) in Scheme 36 [1,2-13C2]acetate was fed to the fungus along with unlabelled triacetic acid lactone (1 19) its dihydro-derivative (120) sodium acetoacetate and sodium 3-hydroxybutyrate (as possible specific precursors for C or C4side-chains). However in each case the enrichment was essentially the same as for acetate alone. Similarly feedings of ‘H-labelled (I 19) or 3-hydroxybutyrate gave alternaric acid which was unlabelled (according to ‘H n.m.r.).It seems that recourse to cell-free enzyme systems will be required to resolve this problem which is common to the biosynthesis of a number of polyketides for which a two-chain pathway has been invoked. The antibiotics nodusmicin and its pyrrolecarboxylate ester nargenicin A (122) represent a novel group of macrolide [Me] methionine + I* HO (1 12) AMe ___) (113) (114) Scheme 34 NATURAL PRODUCT REPORTS 1985 2.7 *I* i ii O3CTOH 00 03c??7c OD (1 16) iii iv -+ H (1 17) Reagents i RuO, NaIO,; ii LiAlH,; iii pyridinium chlorochromate NaOAc CH,Cl,; iv PhMgBr Scheme 35 Me=CO2Na 000000 tt tt t c1 c1 C1 c1 c1 a b I I (118) Scheme 36 OH OH shown in Scheme 37.The observed labelling of the pyrrolecar- boxylate moiety is consistent with the conversion of propionate into succinate and thence into dehydroproline via a-ketoglutar-ate. Incorporation of [I-' 3C,1 *O,]-acetate and -propionate indicated that the oxygen atoms that are attached to C-1 and 0AM=,h Me C-11 were derived from acetate and those attached to C-9 and (119) (120) antibiotics that has recently been isolated from Saccharopofy-spora hirsuta and Nocardia argentinensis respectively. The derivation of the carbon skeletons of these antibiotics from acetate and propionate has been established by two separate st~dies,~~,~~ using 14C- and * 3C-labelled precursors to be as C-17 were derived from pr~pionate.~~ The remaining oxygen atoms are presumably derived from molecular oxygen.The observed derivation of the ether and carbonyl oxygens of the lactone from separate acetate and propionate units is consistent with previous observations for macrolides. The fact that neither the oxygen at C-9 nor that at C-11 is derived from molecular oxygen rules out various ring-forming mechanisms NATURAL PRODUCT REPORTS 1985 -T. J. SIMPSON based on cyclizations of epoxy-olefins and epoxy-alcohols and I the absence of propionate-derived oxygen on C-13 suggests that the C-4-C-13 bond has not been formed by an aldol reaction as has been suggested for the formation of the cyclohexane ring in the biosynthesis of avermectins.Therefore an intramolecular MeC02Na Diels-Alder reaction of an intermediate (1 21) is proposed. This would account for the observed labelling stereochemistry and functionality. Cultures of Fusarium roseum were treated with [‘“C]- zearalenone (1 23) [ SC]-ar-zearalenol (1 24) and [ “C]-p-zeara-lenol (1 25) to determine whether precursor-product relation- ships existed amongst them. Compounds (1 24) and (125) were converted into zearalenone (123) within 7 and 14 days respectively but (123) was not converted into other compounds. Recent 3C n.m.r. studies’ have established the biosynthetic origins of the carbon skeleton of oxytetracycline (128) in Streptomyces rimosus. Further results on the incorporation of ’H from [1-l 3C,2H3]acetate have been rep~rted.~’ The obser- vation of P-’H isotope-induced shifts for C-6a and C-8 indicates the acetate origin of the hydrogen atoms that are attached to C-7 and to C-9.The lack of ’H at C-4 is consistent with the H introduction of the amine functionality onto an aromatic precursor such as 6-methylpretetramid (1 26). The biosynthesis of oxytetracycline is generally thought to deviate from that of tetracycline (129) after dehydrotetracycline (127) has been (122) Scheme 37 OH 0 Me ,H formed (see Scheme 38). The conversion of (127) into (128) involves an oxidative step leading to the introduction of a 5a-hydroxyl group. Stereospecific hydroxylation with either retention or inversion of configuration would necessitate the elimination of either the a-or the P-hydrogen atom at this prochiral centre in (127) only one of which is likely to be derived from the 5-H of 6-methylpretetramid (126) and hence HO (123) X (124) X (125) X = 0 = H Q -OH = H 8 -OH X acetate.Assuming that 2H from acetate is incorporated at C-5 of (I 26) with comparable efficiency to the incorporation that is observed at C-7 and C-9 of (1 28) it follows that only one of the diastereotopic hydrogen atoms at C-5 of (1 27) is derived from acetate and that this is stereospecifically eliminated when (127) is hydroxylated to form oxytetracycline (1 28). Thus the D MeD D 0 0 0 0 0 O,C-CO t 0 Na I D \ D HO yH NMe2 (127) NH2 H (129) (128) Scheme 38 338 NATURAL PRODUCT REPORTS 1985 determination of the stereospecificity of incorporation of 2H origin from a decaketide precursor with acetyl-CoA as the from acetate at C-5 of tetracycline (1 29) or 7-chlorotetracycline ‘starter’unit as shown in Scheme 39.73The ‘starter’ unit for the would allow the stereochemistry of the hydroxylation step in polyketide chain in the tetracyclines is malonamyl-CoA which the biosynthesis of oxytetracycline to be elucidated.suggests that acetoacetyl-CoA (rather than acetyl-CoA) may in fact be the actual chain-initiating unit in cetocycline though this might be fairly difficult to establish. No incorporation of 10 Decaketides label from [ l-I3C]propionate was observed so the methyl Cetocycline (130) is a broad-spectrum antibiotic that is closely groups that are attached to C-6 and C-9 are probably derived related to the tetracyclines.The incorporation of [1-’ 3C]acetate from the C pool. into cetocycline in cultures of Nucardia sulphurens indicated its Aklanonic acid (131) has been isolated from cultures of a species of the genus Streptomyces. Its status as a possible intermediate in the biosynthesis of anthracyclines has been tested by studying its transformation by daunorubicin-negative mutants of daunorubicin-producing strains of Streptumyces 0 MeCOzNa gri~eus.’~ Strain 0,P7 converted aklanonic acid and its methyl ester into E-rhodomycinone (132) 7-deoxy-~-rhodomycinone (133) and glycosides of daunomycinone (134) as shown in 00000 Scheme 40. A second mutant strain 1P5 converted aklanonic acid into the fully aromatic substance 1P/II (135). Oxygen-18-and deuterium-labelling studies might provide evidence for or against the intermediacy of fully aromatic tetracyclic com-pounds in the biosynthesis of anthracyclines.Carbon-13-labelled acetates and propionate were incorpor-ated into the antitumour antibiotic ravidomycin (136) by a Streptomyces species.7s The pairs of coupled carbon atoms in the I 3C n.m.r. spectrum of [ 1 ,2-‘-TC,]acetate-enriched ravido-mycin were identified by a two-dimensional INADEQUATE pulse sequence. [1-I 3C]Propionate enriched C-4 indicating that the Me Me vinyl group is derived from a propionate ‘starter’ unit. On the basis of the observed labelling pattern the biosynthetic pathway that is shown in Scheme 41 has been proposed. Condensation of a decaketide chain with loss of oxygen from (130) C-7 and introduction of oxygen at C-1 gives the tetracyclic Scheme 39 intermediate (1 39).Oxidative cleavage of (139) followed by 0 CO H I1 I HO 0 OH OH 0 0 OH C02Me 0hH\ / + \ I I OH 0 OH OH OH 0 OH OH HO 0 OH OH (135) (132) R =OH (133) R = H (134) Scheme 40 OH OMe Me2 HO AcO (137) R = CH=CH;I (136) (138) R = Me NATURAL PRODUCT REPORTS 1985 -T. J. SIMPSON 3 39 decarboxylation gives (140); bond rotation and lactonization (1 38) from [2-' 3C]propionate was observed. [2-' 3C]Acetate of this gives (141) which is converted into ravidomycin by 0-enriched both carbon atoms of the vinyl group but this can be methylation dehydrogenation of the ethyl group and C-accounted for by the recycling of acetate through the Krebs glycosylation.Similar results have been reported for the closely cycle and production of propionate from succinate via related compounds gilvocarcin V (137) and gilvocarcin M methylmalonate as shown in Scheme 42. To explain the (138) which are antitumour substances that have been isolated labelling a pathway that is shown in Scheme 43 was proposed. from Streptomyces gilvotan~reus.~~ Carbon-1 3-labelled acetates In this a common tetracyclic intermediate is alkylated by and propionates were incorporated into (1 37) and (1 38) which either acetate or propionate to generate either gilvocarcin M or were not separated but were analysed as a ca 7 :1 mixture. It is gilvocarcin V respectively. However the results for ravidomy- clear that the 8-methyl group in (138) and the 8-vinyl cin (see above) rule out this pathway.The fault in this study is substituent in (1 37) are derived from acetate and propionate that the 3C n.m.r. analysis was carried out on a mixture. This respectively. It was found that [2-l3C]- and [3-13C]-propionate has obscured the derivation of C-8 in (138) from C-l of specifically enriched the a-and P-carbons respectively of the propionate rather than acetate as would be required by the vinyl group in (137). A small enrichment of the 8-methyl in pathway that is shown in Scheme 43. OH OH ;OH COSR CO2 H (139) OH OH HO OH (136) f-\* */ * HO * 0 (141) Scheme 41 .CH,CO,H -0CHZCOZ H 0 .I 0. MeC02Na _j + Me-CH _I) MeCHzCOSCoA ieCOSCoA -I -.I .CH~COSCOA CHzC02H I COSCoA Scheme 42 000 000 MeCo2Na *o } -oq+ oq AA MCHzCO2Na 0 0 0 RCH~COSCOA (R = H or Me) HO OMe OH 0 OH 0 0 OH 0 OH (137) X = A+ * (138) X = Me Scheme 43 NATURAL PRODUCT REPORTS 1985 As usual there have been several papers on the biosynthetic to standing cultures after 48 72 and 96 hours followed by pathway leading to the most important mycotoxin aflatoxin B work-up after a further 24 This is a most significant (153).By far the most significant of these is the reported result as it provides the first firm evidence for the previously observation of a hexanoate ‘starter’ effect in the biosynthesis of postulated involvement of linear ‘starter’ units of C or longer averufin (145). Feeding of [ 1-I 3C]hexanoate to Aspergillus carbon chains in the biosynthesis of polyketides.Previous parasiticus (ATCC 24 55 1) resulted in high specific incorpora- attempts to incorporate these compounds using 4C-labelled tion of label at C-1’ of averufin. Some incorporation but at a precursors have mostly resulted in rapid catabolism of the much lower level was also observed at positions that would be precursors by P-oxidation resulting in the secondary incorpora- derived from C-1 of acetate if this were produced as the result of tion of label via acetate. some breakdown of hexanoate to acetate. Two different In addition the observation rationalizes some hitherto feeding protocols were used (a)addition of labelled hexanoate curious observations on the biosynthesis of aflatoxins such as to 48-hour-old mycelial pellets that had been resuspended in a the first isolable intermediate on the pathway being norsolor- replacement medium that contained a low level of sugar and inic acid (143) followed by averantin (144) and then averufin which were shaken for a further 48 hours and (b)pulse addition (145) as shown in Scheme 44.Thus norsolorinic acid can be H-OH HO 0 0 Me 6’ 0 (146) Averufin (145) J -HO (1 47) (1 48) 1 OH 0 OH H* ij (1 51 1 H*$&D H*$&I3 6 OH 0 OMe (152) Aflatoxin B1 (153) Scheme 44 NATURAL PRODUCT REPORTS 1985 -T. J. SIMPSON formally regarded as being formed by the addition of seven malonate units to a separately formed hexanoate ‘starter’ to give an octaketide intermediate (142).However an alternative explanation which cannot be totally excluded at present is that a single polyketide synthetase is involved; this produces the initial C segment which is able to exchange with free hexanoyl-CoA. Interestingly no specific incorporation could be obtained when the experiment was repeated with [1-3C]butyrate [1-13C]-5-oxohexanoate or [1-I 3C]-3-oxo-octan-34I (154) prior degradation to [ 1-13CIa~etate.~~ oate; these all resulted in low uniform enrichments due to their These results also help to explain the long-standing observation that the carbon atoms in HO ;; the bis-furanoid moiety of sterigmatocystin (1 52) are labelled to a lower extent (ca 10%)than those of the xanthone nucleus (ca 1 1 %) if [1-I 4C]acetate is fed to Aspergillus uer~icolor.’~ The incorporation of [4’-’ 3C]- and of [l’-I 3C l’-2H]-averufin into versicolorin A (1 51) by Aspergillus parasiticus (ATCC 36 537) has also been The former enriched C-4 of versicolorin A; in the p.n.d.I3C n.m.r. spectrum of versicolorin A that had been enriched from the latter the resonance for C-1’ showed a triplet due to 13C-ZH coupling. This confirms that ’H label at C-1’ of averufin is retained during the rearrange- ment steps in which the linear C side-chain of averufin is converted into the branched C side-chain of versicolorin A and of aflatoxin B itself. It is proposed that the next intermediate after averufin (145) is nidurufin (146). The relative stereochemistry of the 2’-hydroxyl group of nidurufin has been revised from endo to exo by synthesis of both epimers.This enables the sequence that is shown in Scheme 44 to be proposed for the key rearrangement step. In the former structure for nidurufin the bond between C-2’ and oxygen was orthogonal to the migrating bond between C-2 and C-1’ whereas in the revised structure these bonds are essentially antiperiplanar and so ideally disposed stereoelectronically for rearrangement to yield the oxonium ion (147) which would be hydrolysed to the aldehyde (148)and would cyclize to the hemiacetal (149). Baeyer-Villiger oxidation of (149) would generate versiconal acetate (1 SO) which on hydrolysis and oxidation would yield versicolorin A (1 5 1). Further evidence for the mode of incorporation of averufin into aflatoxin B has been provided by the synthesis of [5,6- 3C,]- and [8,11 -I 3C,]-averufin and by their incorporation into aflatoxin B by cultures of A.parasiticus (ATCC 15 517). As expected analysis of the 3C n.m.r. spectra shows that the C-8- C-11 bond of averufin (145) becomes the C-2-C-3 bond of aflatoxin B (1 53) whereas C-6 of averufin is transformed into C-5 of aflatoxin B with loss of C-5 of averufin in the process.80 A large number of metabolites that are related to the aflatoxin pathway have been isolated from Bipolaris sorokin- iana. Averufanin (1 54) versicolorin C (1 59 and bipolarin (I 56) were isolated along with sterigmatocystin (1 52) from B. sorokiniana that had been cultured on maize. When cultured on a liquid medium several other metabolites were also produced.These are averufin ( 145) versiconol (1 57) versiconol acetate (1 58) versiconal acetate (1 50),and a novel xanthone (1 59).81 No bipolarin was isolated and a re-examination of the data suggests that the previously isolated compound that was formulated as bipolarin (156) was in fact versiconol (157). Versiconal acetate and versiconol acetate were previously only obtained by treatment of aflatoxin-producing cultures of Aspergillusjavus and A. parasiticus with the enzyme inhibitor dichlorvos. This report of their production under natural conditions strongly supports their intermediacy in the biosyn- thesis of aflatoxins and of sterigmatocystin. The austocystins e.g. austocystin D (160) are toxic metabolites of Aspergillus US~US.Interestingly these xanthones show a linear fusion of the xanthone and bis-dihydrofuran moieties in contrast to the angular fusion in sterigmatocystin (1 52).The incorporation of [ 1 ,2-’ 3C,]acetate into austocystin D results in the labelling pattern that is shown in Scheme 45 consistent with a biosynthesis via ring-cleavage of an anthra- quinone e.g. versicolorin A (I 51). In support of this averufin 0 (1 55) Q (156) 0 (157) R = H (158) R = AC HOCHZ CHzOH Me+Me 9 H Me-COZNa + I I1 I OH OH 0 OH (160) Scheme 45 versicolorin C and 8-deoxy-6-0-methyiversicolorin A are co- metabolites of austocystin D in A. ustus.8’ 11 Macrolides Tylosin (1 61) is a sixteen-membered-ring macrolide antibiotic whose carbon skeleton is derived from acetate propionate and butyrate.The origin of the oxygen atoms in tylactone (162) which is a biosynthetic precursor of tylosin has been determined by feeding [1-13C,1802]-acetate,-propionate and -butyrate to a blocked mutant of Streptomyces Jradiae. The labelling patterns that are shown in Scheme 46 were ob-tained.83[1-I3C,I801]Butyrate,as well as labelling the oxygen that is attached to C-5 also enriched (to a lower extent) the propionate-derived oxygen atoms that are attached to C-3 and to C-15. The results are consistent with macrolide ring-closure by direct displacement of thiol from a C-1 thioester by a hydroxyl group at C-15. Full details of studies that were designed to elucidate the origins of the carbon skeleton and the oxygen atoms of the erythromycins have been reported.8s Feeding of [1-13C]-and [2-' 3C]-propionates to cultures of Streptomyces erythreus gave erythromycin A (163) and erythromycin B (164) with the labelling pattern that is shown in Scheme 47.A slightly greater enhancement of the signals due to C-13 in the 13C n.m.r. spectrum of each sample indicated a propionate 'starter' effect. In agreement with these observations Kuhn-Roth oxidation of a labelled erythromycin that was obtained from feeding [l-'Tlpropionate to the bacterium gave propionic acid whose p-phenylphenacyl ester had 21.3% of the specific activity of the intact macrolide. A number of assignments in the 13C n.m.r. spectra were ambiguous.These were resolved by feeding experiments with [2,3-13C,]succinate. This acts in ciuo as a precursor of [2,2'-' 3C,]methylmalonyl-CoA which is generat-ed by the action of methylmalonyl-CoA mutase and it can therefore be considered as an equivalent in vim of [2,3-0 NATURAL PRODUCT REPORTS 1985 3C,]propionate. The resultant 3C-13C couplings in the I 3C n.m.r. spectrum enabled most of the dubious assignments to be resolved. Incorporation of [ 1-I 3C,1802]propionateresulted in the incorporation of '*O into the oxygen atoms that are attached to C-1 C-3 C-5 C-9 C-11 and C-13. Variable (but small) amounts of oxygen exchange were observed to occur at most sites resulting in a partial loss of I80 label relative to 13C. The ketone carbonyl oxygen at C-9 showed more exchange and this was attributed to the basic conditions (pH 9) that are required during isolation.From these results it is clear that each of the six oxygen atoms that are present in the initially formed aglycon (165) is derived from propionate. The ring-closure must occur by addition of a hydroxyl group at C-13 onto a carboxyl function (C-1). The four secondary alcohol or ether functions on the macrolide ring were all labelled irrespective of their configuration i.e. D (C-13) or L (C-3 C-5 and C-11). These results suggest that the oxidation level that is observed in the aglycon (I 65) is established during elongation of the carbon chain thereby excluding alternative oxidation or dehydration-re-hydration mechanisms which have been previously proposed.3-Amino-5-hydroxybenzoic acid (166) has been identified as the key amino acid 'starter' for the biosynthesis of a number of ansamycin antibiotics. Yields of actamycin (1 71) were in-creased 4.6-fold when actamycin-producing cultures of Strepto-myes sp. E/784 were supplemented with (166). However addition of the 4-chloro- 6-chloro- N-methyl and 0-methyl OH 0 0 0 0 MeCOzNa * MeCH2COzNa A MeCH2CHZCOzNa 2 Scheme 46 A 0 0 - * OA Me-CH2 CO Na t C02Et EtOzC- (164) R = OH Scheme 47 NATURAL PRODUCT REPORTS 1985 -T. J. SIMPSON analogues (167)-( 170) reduced the yield of actamycin but did not cause any structurally modified actamycins to be pro- duced.8s These results suggest that the corresponding chlorine N-methyl and 0-methyl substituents that are present in the nuclei of various ansamycins are introduced at biosynthetic stages beyond the level of the amino acid (166).The biosynthetic origin of the milbemycins which are important insecticidal and anthelmintic substances that are produced by Streptomyces hygroscopicus has been studied.86 HO "Oz 'VNH OH (166) OH OH % * Me6 (172) 0 0 % *' MeO (173) MeC02Na -MeCH2C02Na * [Me] methionine Scheme 48 A OMe Twenty milbemycins have been isolated but strain Au-3 which was chosen for the biosynthetic study produces milbemycin a2 (172) milbemycin cis (173) and milbemycin D (174) as its major metabolites.[1-I 3C]Isobutyrate DL-[2-*3C]valine [ 1-3C]acetate [ 1-' 3C]- and [3-'3C]-propionate and L-[methyl-13C]methionine were all fed. This revealed that except for C-25 the carbon skeletons of milbemycins a2 a, and D are derived from seven acetate units and five propionates as indicated in Scheme 48. The methyl ethyl and isopropyl 0 OH (171) OH OMe 0 % OH (174) . .. (175) MeCH2C02Na A [Me] methionine A HO Me Me Scheme 49 344 groups at C-25 are derived from acetate propionate and isobutyrate (itself derived from valine) respectively and the methoxyl group at C-5 of milbemycins az and a4 is derived from methionine. 12 Polyether lonophores Cationomycin (175) is a polyether antibiotic that is produced by a rare actinomycete Actinomadura a~urea.~’ ally unique in having an aromatic acyl substituent.Carbon-13- labelled acetate propionate and methionine were incorpor- ated to give the labelling pattern that is shown in Scheme 49. Label from [2-l 3C]acetate was extensively randomized giving rise to multiply labelled propionate so that extensive coupling was observed among the propionate-derived carbon atoms. This may be rationalized by multiple passages of acetate through the Krebs cycle followed by methylmalonate via succinate as shown Somewhat surprisingly methoxy-6-methylsalicylate was incorporated. The macrotetrolide ionophore antibiotics (176)-( 180) are macrocyclic tetraesters that are built up from both enantiomers of nonactic acid (1 81) and homononactic acid (1 82) which in turn are derived from acetate propionate and succinate as I Q R4 (176) R’ = R2 = R3 = R4 = Me (177) R’ = Et R2= R3 = R4= Me (178) R1 = R2 = Et ,R3 = R4 = Me (179) R1=R2=#=Et,R4= Me (180) R’ = R2= R3= R4 = Et 0 MeCO2Na MeCH2C02Na R 6 + H02CCH2CH2C02Na R*OH (+)-(181) R = Me (+)-(182) R = Et It is structur- 3C-1 the formation of in Scheme 42.neither labelled orsellinate nor 4-NATURAL PRODUCT REPORTS 1985 indicated in Scheme 50. [1-13C,1802]-Acetateand -propionate have been incorporated into nonactin (1 76) and monactin (1 77) by cultures of Streptomyces griseus ETHA 7796.88 The incorporation of was determined by the reduction of (176) and (177) to the diols (183) and (184) respectively which were converted in to their (-)-a-me t hox y-a-trifluorome t h ylp henyl- acetyl esters (185)-( 188) for separation by h.p.1.c.and subsequent 3Cn.m.r. analysis. Incorporation of [I-’ 3C,1802]-acetate led only to enrichment of the oxygen that is attached to C-8 in (185) and (186) whereas [1-13C,1802]pr~pionate en-riched the oxygen atoms that are attached to C-1 C-6 and C-8 in (1 87) and (1 88) and to C-1 and C-6 in (1 85) and (1 86). These results are consistent with the conversion of propionate into succinate (see Scheme 42) and with the formation of the thioester (191) by reduction of an intermediate such as (189) which is converted [via reduction to the diol (190) and intramolecular Michael reaction] into (191) as shown in Scheme 51.Since the observed retention of I8O relative to I3C at C-1 in samples of (1 87) and (1 88) that had been derived from [1-13C,1802]pr~pionate is >50% it follows that homononactic acid (182) [and therefore nonactic acid (181)] is not an obligatory intermediate on the biosynthetic pathway to the macrotetrolides but that a thioester intermediate such as (191) can react to generate an ester bond by direct displacement of the thiol activating group by the hydroxyl group that is attached to C-8 of a second building block. Thus the biosynthesis probably proceeds from acetate propionate and succinate without the intervention of free unactivated intermediates despite the fact that they can be isolated from cultures and incorporated into the macrotetrolides.13 Miscellaneous Metabolites Full details of an extensive study using stable isotopes to R’%odo investigate the biosynthesis of the antibiotic virginiamycin M (192) in Streptomyces virginiae have appeared.89 The skeleton is derived from valine seven acetate units glycine serine and proline as summarized in Scheme 52. The methyl group at C- 32 is derived from methionine but the methyl at C-33 is derived by a novel pathway involving decarboxylation of an acetate unit as shown in Scheme 53.That the methyl group is derived from an acetate unit that is added to a pre-formed polyketide SRO +R &OH (R = Me or Et) Scheme 50 R+OH (183) R = Me (185) R = Me from (+) -(181) (184) R = Et (186) R = Me from (-1 -(181) (187) R = Et from (+) -(182) (188) R = Et from (-1 -(182) Scheme 51 NATURAL PRODUCT REPORTS 1985 -T.J. SIMPSON chain rather than by one of the alternative mechanisms that have been proposed was demonstrated by a rather subtle experiment in which [3-' 3C]serine was used as a delayed source of [2-' 3C]acetate. [2-' 3C]Acetate enriched carbons 4,6 1 1 13 15 17 and 33 to the same extent. However while [3-I 3C]serine enriched carbons 4,6 11 13 15 and 17 to the extent of 0.8% the enrichment at C-33 was 1.9%. This experiment depends on the conversion of serine into acetyl-CoA via dehydration and deamination to pyruvate. It would have been interesting to see 0 Me mC02Na A [ Me 1 methioni ne if feeding [2-* 3C]malonate also gave differential labelling.The oxazole ring is formed from serine presumably by a pathway such as shown in Scheme 54. A similar derivation of a methyl group from C-2 of an acetate unit which is added to a pre-formed polyketide has been demonstrated during the biosynthesis of two metabolites that have been isolated from gliding bacteria. The first of these is myxopyronin A (193) which is an antibiotic that has been isolated from Myxococcus fu/v~s.~* Incorporation studies with 0 11 13 HNHZ COZH 32 '-COZH HOZC bH Scheme 52 r -COZI HOzC Scheme 53 0 0 H02CYNH2 L OH -0H Scheme 54 * Me=CO2Na HOzCvNHz A [Me] methionine &A WSRL O SRO N"Z or 0 SR 0 NH2 * T NH-C02 Me A Ge-G 17 12 *Me A (193) Scheme 55 NATURAL PRODUCT REPORTS 1985 A I Me *.Me-COZNa A [Me] methionine _i) 8 HzNCH2C02 H OH * he OH (194) Scheme 56 I 3C-labelled acetate glycine and methionine indicated that the carbon skeleton is derived from two polyketide chains (Scheme 55). On incorporation of [13C2,1 SN]glycine C-12 showed both 3C-1 3C and * 3C-15Ncouplings so that glycine is incorporated intact as a ‘starter’ unit in one of the chains. The methyl groups C-8 and C-17 are derived from methionine whereas C-21 is enriched from [2-I3C]acetate. A similar resultg1 was observed for C-33 in the antibiotic myxovirescin A (194) which is a metabolite of Myxococcus uirescens.Carbon- 13-labelled acetates and methionine are incorporated as shown in Scheme 56 C-33 being derived from C-2 of a cleaved acetate unit. Even more curiously both carbon atoms of the ethyl group that is attached to C-13 are derived from C-2 of acetate. Although no pathway is suggested for this derivation uia an intermediate in the Krebs cycle appears to be likely. Glycine functions as the ‘starter’ unit of a polyketide chain which is then alkylated at acetate-derived methylenes (C-2 and C-4) and carbonyls (C-13 and C-17). The remaining moiety appears to be a C5 hydroxy-acid that has been derived from acetate and methionine but at what stage it is incorporated [and which bond (ester or amide) is formed first] is not yet known. Further results on this intriguing compound will be awaited with eager anticipation.14 References 1 T. J. Simpson in ‘Biosynthesis’ ed. R. B. Herbert and T. J. Simpson (Specialist Periodical Reports) The Royal Society of Chemistry London 1983 Vol. 7 p. 1. 2 T. J. Simpson Nat. Prod. Rep. 1984 1 281. 3 ‘Tetrahedron Symposia-in-Print’ ed. A. I. Scott Tetrahedron 1983 39 pp. 3441-3591. 4 D. C. Aldridge and W. B. Turner ‘Fungal Metabolites II’ Academic Press London 1983. 5 W. B. Turner ‘Fungal Metabolites’ Academic Press London 1971. 6 J. M.Schwab and J. B. Klassen J. Chem. SOC. Chem. Commun. 1984 296. 7 J. M. Schwab and J. B. Klassen J. Chem. SOC. Chem. Commun. 1984 298. 8 D. Parker J. Chem. SOC. Perkin Trans. I 1983 83. 9 A. G. McInnes J. A. Walter and J.L. C. Wright Tetrahedron 1983 39 3515. 10 J. Templier C. Largeau and E. Casadevall Phytochemistry 1984 23 1017. 11 E. J. Corey B. De J. W. Ponder and J. M. Berg Tetrahedron Lett. 1984 25 1015. 12 R. Bentley and P. M. Zwitkowitz J.Am. Chem. SOC. 1967,89,676. 13 J. MacMillan and T. J. Simpson J. Chem. Soc. Perkin Trans. I 1973 1487; R. C. Ronald and S. Gurusiddiah Tetrahedron Lett. 1980 21 681. 14 R. J. Parry and H. P. Buu J. Am. Chem. SOC. 1983 105 7446. 15 K. Nitta R. J. Studelmann and C. H. Eugster Helv. Chim. Acta 1977 60 1747. 16 G. P. Arsenault J. R. Althaus and P. V. Direkar Chem. Commun. 1969 1414. 17 S. Moreau A. Lablache-Combier J. Biguet C. Foulon and M. Delfosse J. Org. Chem. 1982 47 2358. 18 F. Renauld S. Moreau and A.Lablache-Combier Tetrahedron 1984 40,1823. 19 K. K. Chexal C. Snipes and C. Tamm Helzi. Chim. Acra 1980,63 761. 20 J. S. E. Holker personal communication to T. J. Simpson. 21 F. J. Olivigni and L. B. Bullerman Appl. Environ. Microbiol. 1978 35 435. 22 C. J. Rabie T. J. Simpson P. S. Steyn P. H. van Rooyen and R. Vleggaar J. Chem. SOC. Chem. Commun. 1984 764. 23 P. S. Steyn R. Vleggaar and T. J. Simpson J. Chem. SOC. Chem. Commun. 1984 765. 24 B. Akermark H. Erdtman and C. A. Wachtmeister Acta Chem. Scand. 1959 13 1855. 25 C. P. Gorst-Allman P. S. Steyn and R. Vleggaar J. Chem. Soc. Perkin Trans. 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Staunton J. Chem. SOC. Perkin Trans. I 1984 1027. 36 J. S. E. Holker and T. J. Simpson J. Chem. SOC. Perkin Trans. I 1981 1397; M. Tanabe M. Uramoto T. Hamasaki and L. Cary Heterocycles 1976 5 355. 37 C. P. Woloshuk P. M. Wolkow and H. D. Sisler Pestic. Sci. 1981 12 86. 38 C. P. Woloshuk H. D. Sisler M. C. Tokousbalides and S. R. Dutky Pestic. Biochem. Physiol. 1980 14 256. 39 M. H. Wheeler Trans. Br. Mycol. Soc. 1983 81 29. 40 E. Bardshiri and T. J. Simpson Tetrahedron 1983 39 3539. 41 H.-J. Bauch R. P. Labadie and E. Leistner J. Chem. SOC. Perkin Trans. I 1975 689. 42 T. Okuno I. Natsume K. Sawai K. Sawamura A. Furusaki and T.Matsumoto Tetrahedron Lett. 1983 24 5653. 43 D. J. Robeson and G. A. Strobel Phytochemistry 1982 21 1821. 44 D. J. Robeson G. R. Gray and G. A. Strobel Phytochemistry 1982 21 2359. 45 D. J. Robeson and G. A. Strobel Phytochemistry 1984 23 767. 46 C. R. McIntyre and T. J. Simpson J. Chem. SOC. Chem. Commun. 1984 704. 47 C. R. McIntyre T. J. Simpson L. A. Trimble and J. C. Vederas J. Chem. SOC. Chem. Commun. 1984 706. 48 J. E. Holenstein H. Kern A. Stoessl and J. B. Stothers Tetrahedron Lett. 1983 24 4059. 49 F. E. Scott T. J. Simpson L. A. Trimble and J. C. Vederas J. Chem. SOC. Chem. Commun. 1984 756. NATURAL PRODUCT REPORTS 1985 -T. J. SIMPSON 50 J. F. Grove and M. Pople J. Chem. Soc. Perkin Trans. I 1979 2048. 51 C. R.Hutchinson S.-W. Li A. G. McInnes and J. A. Walter Tetrahedron 1983 39 3507. 52 T. Ohta M. Sunagawa K. Nishimaki and S. Nozoe Heterocycles 1983 20 1567. 53 A. Ichihara H. Oikawa K. Hayashi S. Sakamura A. Furusaki and T. Matsumoto J.Am. Chem. Soc. 1983,105,2907; A. Ichihara H. Oikawa M. Hashimoto S. Sakamura T. Haraguchi and H. Nagano Agric. Biol. Chem. 1983 47 2965. 54 I. Barash S. Manulis Y. Kashman J. P. Springer M. H. M. Chen J. Clardy and G. A. Strobel Science 1983 220 1065. 55 P. S. Steyn P. L. Wessels C. W. Holzapfel D. J. J. Potgieter and W. K. A. Louw Tetrahedron 1972 28 4775. 56 H. Oikawa A. Ichihara and S. 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ISSN:0265-0568    

DOI:10.1039/NP9850200321

出版商:RSC    

年代:1985

数据来源: RSC

摘要:

Carotenoids and Polyterpenoids G. Britton Department of Biochemistry University of Liverpool P.O. Box 147 Liverpool L69 3BX Reviewing the literature published between September 1982 and August 1984 (Continuing the coverage of literature in Natural Product Reports 1984 Vol. 1 p. 67) 1 Carotenoids 1.1 Introduction 1.2 New Structures and Stereochemistry 1.2.1 Carotenoids 1.2.2 New Natural Products Related to Carotenoids 1.3 Synthesis and Reactions 1.3.1 Carotenoids 1.3.2 Retinoids 1.3.3 Carotenoid-like Compounds 1.4 Carotenoid-Protein Complexes 1.5 Physical Methods 1.5.1 Separation and Assay 1S.2 N.M.R. Spectroscopy 1.5.3 Circular Dichroism 1.5.4 Raman and Infrared Spectroscopy 1S.5 Electronic Absorption Spectroscopy 1.5.6 Photoacoustic Spectroscopy 1S.7 Mass Spectrometry 1.5.8 X-Ray Methods 1.5.9 Miscellaneous Physical Chemistry 1.6 Photoreceptor Pigments 1.7 Biosynthesis and Metabolism 2 Polyterpenoids and Quinones 2.1 Polyterpenoids 2.2 Isoprenylated Quinones 3 References ~~ ~~~~ ~~ 1 Carotenoids 1.1 Introduction The major publication in the carotenoid field in 1984 was the second volume of Goodwin’s book ‘The Biochemistry of the Carotenoids’ which deals with the structures distribution metabolism and functions of carotenoids in animals.’ A chapter in another book’ on the biochemistry of natural pigments describes the general features of carotenoid struc- tures properties biosynthesis and functions.A general review article3 outlines the chemistry of p-carotene and vitamin A and an assessment of trends in research on cis-carotenoids4 reflects the increasing interest in the chemistry and biochemistry of these geometrical isomers. Several other review articles have been published dealing with the structures biosynthesis and functions of carotenoids in animals and plant^,^ fungi,6 and molluscs7 and with carotenoids as food colorants.8 The published proceedings of the 6th International Photosynthesis Congress held in Brussels in 1983 contain many articles on the biosynthesis distribution and localization of carotenoids in pigment-protein complexes of the photosynthetic apparatus of plants algae and ba~teria.~ Extensive details of the biosynthe- sis of terpenoids including carotenoids are given in a two- volume work.’O The wide current interest in the chemistry and applications of vitamin A and related compounds is reflected in a two- volume monograph on ‘The Retinoids’.’ ’ The first volume contains chapters on the chemistry and physical properties of retinoids,’ la the synthesis of retinoids that are labelled with radioisotopes Ib and methods for the extraction separation and chemical analysis of retinoidsllc whilst chapters on the biosynthesis absorption and hepatic metabolism of retinol’ Id and the metabolism of natural and synthetic retinoids’ le are included in the second volume. Vitamin A and its derivatives are also the subject of other review articles.I2+I3 The IUPAC- IUB recommendations (1981) for the nomenclature of retinoids have been published in several journals.The carotenoid-like substance abscisic acid is the subject of two and of chapters in a book on plant growth substances.’’ Steady rather than spectacular progress continues in all areas of carotenoid chemistry and biochemistry. High-performance liquid chromatography (h.p.1.c.) is now used routinely as the method of choice for the analysis and purification of carotenoids. A major result of using this technique has been the discovery of the widespread Occurrence of cis-isomers in natural extracts. The large number of papers on resonance Raman spectroscopy of carotenoids pure or in uiuo indicates that the enormous potential of this technique in the carotenoid field is beginning to be exploited.The general importance of studying the properties of carotenoids in uiuo or in association with other molecules rather than simply as pure compounds in solution is also beginning to be realised. In the field of synthesis some new or modified procedures for construction or extension of the polyene chain are still being developed but most synthetic effort is being devoted to the preparation of chiral end-group synthons for carotenoids and to the production of a large number of analogues of retinal (for studies of visual pigments and bacteriorhodopsin) and of vitamin A and other retinoids (to satisfy the demands of pharmaceutical research).1.2 New Structures and Stereochemistry I .2.I Curotenoids Some novel end-groups have been identified in carotenoids that were isolated from algae and characterized by detailed ‘H n.m.r. study. The 3,4,7,8-tetradehydro-P-end-group was pre- sent in the two compounds (3R)-3’,4’-anhydrodiatoxanthin [3’,4’,7’,8’-tetradehydro-(,~-caroten-3-01] (1) and eutreptiellan- one [3,6-epoxy-3’,4’,7’,8’-tetradehydro-5,6-di hydro-P,P-caro-ten-4-onel (2) from Eutreptiellu gymnusticu,’ the latter compound being the first example of a naturally occurring carotenoid that contains a 3,6-epoxy-P ring. Prasinoxanthin (= ‘xanthophyll K’) which is the characteristic carotenoid of the Prasinophyceae has been shown to have a novel substituted y end-group and was identified as 3,6,3’-trihydroxy-7,8-dihydro-y,e-caroten-8-one (3) the (3RY3’R,6’R) chirality being assumed on biogenetic grounds.Carotenoids with unusual structural modifications continue to be discovered in invertebrate animals. Sidnyaxanthin from the tunicate Sidnyum argus is related to prasinoxanthin and has been assigned the structure 3,6’-dihydroxy-7,8-didehydro-7’,8’-di hydro-P,e-carotene-3’,8’-dione (4).’O Spectroscopic methods have been used to show that the very polar carotenoid mactraxanthin from the edible surf clam (Muctru chinensis) has the symmetrical structure 5,6,5’,6’ -tetrahydro-p,p -carotene -3,5,6,3‘,5‘,6’ -hexaol (5).?l Detailed u.v.-visible i.r. n.m.r. (IH and I3C) and c.d. spectroscopic studies and chemical correlations have allowed the stereochemistry of bastaxanthin c (3,19,17’-trihydroxy-7,8-didehydro-P,~-carotene-3’,6’-dione 3-sulphate] which is the NATURAL PRODUCT REPORTS 1985 R2 0 b e f HO&I? i i (1)R’=a,R2=b (2) R’ = c R2= b (3)R’= d,R2= e (4)R’ = f R2= g major carotenoid sulphate from the sponge Ianthella basta,22 to be assigned as (3R,l’R,SR) (9).23 This structure corrects that which was given in the previous Report.24 The minor components of this sponge which are bastaxanthins b b2 c2 d e and f have been identified as (3R,l’R,5’R)-3,19-dihydroxy-3’,6’-dioxo-7,8-didehydro-~,~-caroten-3-sul-17’-a1 phate (10) (3R,3’R,5’R)-3,19-dihydroxy-7,8-didehydro-P,~-carotene-3’,6’-dione 3-sulphate (1 l) (3R,3’R,SR)-3,19,3’-trihydroxy-7,8-didehydro-P,~-caroten-6’-one 3-sulphate (12) (3R 1’R,3’R,5’R)- 3,19,3’ 17’- tetrahydroxy -7,8 -didehydro-P,~ -caroten-6’-one 3-sulphate (1 3) (3R l’R,SR)-3,19-dihydroxy- 3’,6’-dioxo-7,8-didehydro-~,~-caroten-17’-oic 3-sulphate acid (1 4) and (3R l’R,3’R,5’R)-3,19,3’-trihydroxy-7,8-didehydro-6’-oxo-~,~-caroten-17’-oic acid 3-sulphate (1 5) respecti~ely.~~ Bastaxanthol b (= bastaxanthin b lacking the sulphate group) was also isolated.The main pigment of Brevibacterium linens which is characterized by a colour change from orange to pink on treatment with alkali was foundt6 to be identical to the phenolic carotenoid 3,3’-dihydroxyisorenieratene[+,+-taro-tene-3,3’-diol] (6) that had previously been encountered only in Streptomyces medi~lani.~’Reassessment of their chemical properties and ‘H n.m.r.spectra has led to a revision of the proposed structures2s for the C, carotenoids c.p. 450 and c.p. 473 of Corynebacterium poinsettiae to 2,2‘-bis-(4-hydroxy-3-me thy1 bu t-2-enyl)-P,p-carotene (7) and 2-(4-hydroxy-3-methyl- but-2-enyl)-2’-(3-methylbut-2-enyl)-3’,4’-didehydro-~,$-caro- ten-1-01 (8),respe~tively.~~ The absolute configurations of capsanthin [3,3’-dihydroxy- P,~-caroten-6’-one] (1 6) and capsorubin [3,3’-dihydroxy-~,~- carotene-6,6’-dione] (1 7) have been confirmed as (3R,3’S,5’R) and (3S,5R,3’S,5’R) respectively by their stereospecific synthesis from ( +)-camphor.30 The chiralities of two isomers of tunaxanthin have been established by h.p.1.c. and c.d. OC d h (5) R’ = R2 = h (6) R’ = R2= i (7) R’ = R2= j (8)R’ = j ,R2 = k the (3R,6S,3’R,6’S)-isomer (19) [ = ‘oxyxanthin The absolute configuration of the major carotenoid of members of the Chlorophyceae i.e.loroxanthin [(3R,3’R,6’R)-P,~-carotene-3,19,3’-triol] (20) has been confirmed as identical to that of lutein [(3R,3’R,6‘R)-P,~-carotene-3,3’-diol] (21) by an extensive ‘H n.m.r. study to establish the 3’,6‘-trans configuration and by c.d. correlation with synthetic (3R,6‘R)-P,~-carotene-3,19-diol (22).33 Flexixanthin [3,1 ’-dihydroxy-3’,4’-didehydro-1’,2’-dihydro-P,$-caroten-4-one] (23) and its 2’-hydroxy-derivative (24) have been re-isolated from a species of the genus Flexibacter and their absolute configurations confirmed as (3s) and (3S,2’S) respectively on the basis of IH n.m.r.and c.d. comparison with rubixanthin [P,$-caroten-3-01] (25) and other related compounds.34 The partly racemic nature of the insect pigments P,P-caroten-2-01 (26) 2’-hydroxy-P,P-caroten-2-one (27) and P,P-carotene-2,2’-diol (28) as indicated by c.d. measurements has been confirmed by H n.m.r. investigation of their methoxy(pheny1)trifluoromethylacetate esters in the presence of shift reagents.35 Circular dichroism and H n.m.r. spectroscopic studies have confirmed that the carotenoids of red algae (Rhodophyceae) have the same chiralities as in higher plants.36 In this work a-cryptoxanthin [(3’R,6’R)-P,~-caroten- 3’-011 (29) was fully characterized for the first time. Considerable effort has been devoted to investigating the chirality of epoxy-carotenoids both natural and synthetic or semi-synthetic.Thus c.d. comparison with the synthetic compounds has proved the (2s) and (2’s) configurations for 1,2-epoxy-1,2-dihydro-$,$-carotene(30) and 1’,2’-epoxy-1’,2’- dihydro-P,$-carotene (3 l) respectively which are the natural 1,2-epoxide and 1’,2’-epoxide of lycopene and y-carotene and which were isolated from to ma toe^.^^^^^ High-performance liquid chromatography methods have been devised for separat- ing diastereoisomeric and epimeric carotenoid furanoid oxides and c.d. and n.m.r. analysis of the purified products has allowed correlations. Thus tunaxanthin A is (~S,~S,~’S,~’S)-E,E-C~~O-determination of the absolute configurations of tene-3,3’-diol (18) (= ‘oxyxanthin 45’13’ and tunaxanthin C is antheraxanthin [ 5,6-epoxy-5,6-dihydro-P,P-carotene-3,3’-diol] 351 NATURAL PRODUCT REPORTS 1985-G BR ITTON CH20H I HO a b C 0 HO” w HO HO‘ d e f HOp0 HO 9 (9) R’ = a R2 = b(X = CH20H;Z = 0) (10)R’ =u,R*=~(X=CHO;Z=O) (11)R’ =a,R2=b(X=Me;Z=0) (12) R’ = u R2 = b (X = Me; Z = H,OH) (13) R’ = u,R2 = b (X = CH20H;f= H,OH) (14) R’ = 0 R2 = b (X = C02H;f = 0) (15) R’ = a R2 = b (X = C02H;Z = H,OH) (16) R’ = c (Y =Me),R2 = b(X = Me;Z =H,B (32) and the d i astereoisomeric mu tatoxanthins [5,8-epoxy-5,8-dihydro-P,P-carotene-3,3’-diols] (3 3)39 and of the neoc hromes (= trollic hromes) [5’,8’-epoxy-6,7-dide hydro-5,6,5’ 8’-te tra- hydro-p,p-carotene-3,5,3’-triols](34) and dinochromes [= neoc hrome 3-acet ate^].^^ Some novel carotenes from sponges have been identified as the cis-isomers (7Z)-+,+-carotene (42) and (7Z)-+,~-carotene (43) (from Tethya amamensi~)~’ and (72,7’Z)-+,+-carotene (44) (from Suberites seri~eus)~~ and given the unfortunate trivial names ‘isorenieracistene’ ‘renieracistene’ and ‘isorenieradi- cistene’.Details of the assignment of the stereochemistry of the biosynthetic intermediates leading to prolycopene [(72,92,7’2,9’2)-$,$-carotene](45)in the tangerine-fleshed strain of tomato ‘Ailsa Craig’ and in other strains have been published and aspects of the biosynthetic mechanism for formation of the cis double-bonds disc~ssed.~~,~~ Amongst the many carotenoids that have been isolated from the hips of Rosa pomijera were cis-isomers of phytoene [7,8,11,12,7’,8‘ 1l’ 12’-octahydro-$,$-carotene] (46) phytofluene [7,8,11,12,7’,8’-hexahydro-$,$-carotene] (47) <-carotene [7,8,7’,8’-tetrahydro- $,$-carotene] (48),neurosporene [7,8-dihydro-$,$-carotene] (49) lycopene [$,$-carotene] (39 &,$-carotene (36) p,$-caro- tene (37) P-cryptoxanthin [p,p-caroten-3-01] (38) zeazanthin [P,P-carotene-3,3’-diol] (39) and rubixanthin (25).45 The most interesting of these were the (52)-isomers or (5’2)-isomers.Their possible role as precursors of cyclic carotenoids was discussed. The (5’2)- and (S’E)-isomers of rubixanthin were easily separated by h.p.1.c. and differentiated by low-tempera- ture c.d.The 15-cis [=(152)]configuration of a natural isomer of v iolaxan t hi n [5,6;5’,6’-diepoxy-5,6,5’ 6-tetrahydro-P,P-caro- tene-3,3’-diol] (40) has been confirmed by 3C n.m.r. spectros- h i (17)~’=R~=~(X=M~;Z=H.~-OH) (l8)R’ = R2= d (19)~’= R*= e (20) R’ = c(Y =CHzOH) ,R2 = f (21) R1= c(Y = Me),R2 = f (22) R’ = c(Y = CH20H),R2 = g (23) R’ = h,R2 = i (X = H) OH) (24) R’ = h R2 = i (X =OH) copy,46 and cis-isomers (mainly 9-cis identified by h.p.1.c.) have been found to constitute a major part of the canthaxanthin [P,P-carotene-4,4-dione](41) in eggs and other tissues of a brine shrimp (Artemia SP.).~’ A survey of the C30 diapocarotenoids of Staphylococcus aureus strain 209P has been published.48 The main xanthophyll was identified as a 4-~-~-glucopyranosyloxy-4,4’-diaponeuro-sporen-4-oic acid ester (50) which differs from the main diaponeurosporenoic acid derivative ‘staphyloxanthin’ (5 1) that had previously been reported49 in strain S41 of S.aureus. Two related compounds diglucosyl 4,4’-diapo-$,$-carotene-4,4’-dioate (52) and glucosyl 4,4-diapo-$,$-caroten-4-oate-4-oic acid (53) both acylated were isolated from a red strain of a species of the genus Rhizobi~m.~~ The stigma of Crocus neapolitanus has afforded two new glycosyl esters of the C2, compound crocetin (54) ;after detailed two-dimensional H n.m.r. work these have been characterized as crocetin p-gentiobiosyl 0-neapolitanosyl ester and crocetin di-(P-neapoli- tanosyl) ester,5 and contain the previously unknown trisac- charide neapolitanose (O-P-~-glucopyranosyl-(1+2)-0-[P-D-glucopyranosyl-( 1+6)]-~-glucose).A novel carotenoid disaccharide that is present in some species of the Dinophyceae has a complex structure and has only been partly characterized by mass spe~trometry.~~ 1.2.2 New Natural Products Related to Carotenoids Several new natural products and metabolites containing ring structures that are similar to those of carotenoids have been reported. (Note in this article the carotenoid numbering scheme will be used whenever such compounds are described.) NATURAL PRODUCT REPORTS 1985 HOJp++ Q HO" &+ b C d e f (25) R' (26) R' (27) R' (28) R' (29) R' (30) R' (31) R' (32) R' (33) R' = o,R2= b = C(X = H,OH) R~ = c(x = H,H) = c(X = O),R2 =C (X = H,H) = R2 = c(X = H,OH) = c(X = H,H),R2 = d = e,R2 = b = c(X = H,H),R2 = e = f,R2 = Q = g,R2= a (34) R' = h,R2= g (35) R' = R2 = b (36) R'= i,R2 = b (37) R' = c (X = H,H),R2 = b (38) R' = o,R2 = c(X = H,H) (40)R' = R2 = f (39) R' = R~ = a (41) R' = ~2 = j a b C d e f 9 (42) R' = Q,R' = b (43) R' = Q,R' = C (44)R' = RZ = Q (45) R1 = R2 = d (46) R' = R2 = c (47) R' = c,R2 = f (48) R1 = R2= f (49) R' = f RZ = g NATURAL PRODUCT REPORTS 1985-6.BRITTON CH2OH CH20X CH2OH ’*% HO HO H,OCH24 Ho%HO “HO S HHO Oi+ a O\Ck II b 0 C (50) R’ (51) R’ (52) R’ (53) R’ = COzR R2 = a = 6(X=COCl4H2,1,R2= Me = R2 = c ; 07’ = c ,R2= CO,H ;A7’ (56) A7 (57 1 (65) X = OH Y = H (66) X=H,Y=OH It has previously been considered that ionones and irones that had been obtained from various plant sources could have been derived from intact carotenoid molecules but it has now been shown that the or-irone (56) and the y-irone (58) and their dihydro-derivatives [(57) and (59)] from Iris germanica I.pallida and I. Jlorentina arise by oxidative degradation of met h yl-su bsti tu ted trite rpenes. These precursors which are a-and y-irigermanal[(60) and (63)] iripallidal(61) desoxyiripalli- dal (62) and iriflorental (64) have been isolated and characteri~ed~~.~~ and the stereochemistry of each has been determined,ss along with that of the irones and dihydro-irones and the ‘acyclic’ intermediates iridogermanal(65) and isoirido- germanal (66).Different enantiomeric forms of the irones were found in Iris oils of different origin. The stereostructure of (+)-(2S,6S)-trans-a-irone (67) has been confirmed by its synthesis from (.-)-a-pinene and the preferred conformations of (67) and of (-)-(2S,6R)-cis-a-irone (56) and the or-ionone (68) in solution have been deduced from H and 13Cn.m.r. studies.s6 The novel compound 3- h yd roxy-7,8-d ide hyd ro-p-ionone (69) has been isolated from leaves of Lycium chinen~e.~’ Tobacco continues to yield new ionone-related compounds e.g. 4-hydroxy-P-damascone (70)58 and (3S,5R,6R,9R)-3,6-epoxy-5-hydroxyionol (72) the structure and stereochemistry of which were determined by X-ray crystallography of its 9-p-nitroben- ~oate.~~ Leaves of various species of the genus Nicotiana have yielded glycosides of 3-0x0-a-ion01 (73) the 5,6-epoxy-3-hydroxy-5,6-di hydro-&ion01 (75),3-hydroxy-P-damascone (71) blumenol A (74) blumenol C (76) and 6,7-didehydroblumenol C (77).60 Several new relatives and metabolites of the plant growth substance abscisic acid (78) have been identified as the hydroxymethyl structure (79)61*6’ (from cell suspension cul- tures of Nigefla darnascena) 3-dihydroabscisic acid (80) [from immature seeds of the broad bean (Vici~fbba)],~~ and the 3-043- D-glucoside of dihydrophaseic acid (81) [from the avocado (the fruit of Persea arneri~ana)].~~ 354 NATURAL PRODUCT REPORTS.1985 OCOzH 0 COz H 0 HO 1.3 Synthesis and Reactions 1.3.1 Carotenoids A review of industrial applications of the Wittig reaction includes information that is relevant to the synthesis of carotenoids and retinoids.h5 Routes for the synthesis of the cyclopentane carotenoids capsorubin (1 7) and capsanthin (16) in both optically inactive and optically active forms have been described.30.6h In the synthesis of the optically inactive compounds benzylic acid rearrangement of the diosphenol compound (82) in dilute alkali gave the cyclopentanedicarboxy- lic acid intermediate (83) and thence the ketone (84; optically inactive).This ketone was obtained in optically active form from (+)-camphor in many steps.-30 In an alternative synthe- sis,hh the chiral ketone (84) was constructed by hydroboration [with ( +)-di-isopinocamphenylborane] of the unsaturated acetal (85) which was also obtained in many steps from (+)-camphor.In each case aldol condensation of the ketone with the C20compound crocetindial(55) was used to give racemic or (3S,5R,3’S,5’R)-capsorubin. The ketone (84) was also used to prepare capsanthin (1 6) and cryptocapsin [(3’S,5’R)-3’-hy- droxy-P,~-caroten-6’-one](86) by aldol condensation with the appropriate aldehydes. Oppenauer oxidation then gave the corresponding 3-ketones capsorubone [~,~-carotene-3,6,3’,6’-tetrone] (87) capsanthone [3-hydroxy-P,~-carotene-3’,6’-dione] (88) and cryptocapsone [P,~-carotene-3’,6’-dione] (89).66 3,4,3’,4’-Tetradehydro-P,P-carotene-2,2’-dione (90) has been X &&OH I HO (67)X = Me (69) (70)X = H,Y =OH (72) (68)X = H (71 ) X = OH ,Y = H 0*OH 0P O H (73)X = H (75) (76) (74)X = OH X P C O zH Glc 0 0yo.(77) (78) R = Me; X = 0 (79) R = CHzOH ;X = 0 (80)R = Me;X = H,OH %ZH COMe >4 5 OH prepared by standard condensation of the Wittig salt (92) [which was obtained in several steps from p-ionone (93)] with the C, dial (95).h7 A chiral and enantioselective synthesis of (4R,4’R)-isozeaxanthin [(4R,4’R)-P,P-carotene-4,4-diol] (91) used the reverse procedure i.e. condensation of the C, Wittig salt (96) with the protected CISend-group aldehyde (97),68 which was obtained from (-)-(4R)-4-hydroxy-P-ionone (94) cia silylation and condensation with triethyl phosphonoacetate. Several papers report work on the construction of 1- and 2- substituted and of modified acyclic end-groups and the synthesis of acyclic and monocyclic carotenoids from them.Thus the chiral end-group synthons (98) and (99)-( 103) were prepared from D-mannitol and L-serine re~pectively.~~ Com-pound (103) was then converted into the C,,,Wittig salt (104) which on condensation with the C,, 8’-apo-P-caroten-8’-al (109 gave (2’s)-plectaniaxanthin [(2’S)-3‘,4’-didehydro-l’,2’-dihydro-P,$-carotene-l’,2’-diol](106).?O Another chiral end- group synthon (107) was prepared from the chiral diol (108) which was in turn obtained by chiral reduction of the ketone (109) with baker’s yeast or after resolution (by h.p.1.c.) of the diastereoisomeric esters of the diol with camphanic acid. Compound (107) was used to prepare the lycopene and y-carotene derivatives (2S)-1,2-epoxy-l,2-dihydro-$,ll/-carotene (30) and (2’S)-1’,2’-epoxy-1’,2’-dihydro-~,$-carotene (31) iden- tical to the natural products from tomatoes.38 The methylene- carotenoid aleuriaxanthin [ l’ 16’-didehydro-1’,2’-dihydro-P,$-caroten-2’-01] (1 15) was synthesized in an enantiomeric form NATURAL PRODUCT REPORTS 1985-G.BRITTON X&Po*@ I I a C OH b d (66) R' = u(X = H),R2 = 6(Z = H,p-OH) (87) R' = R2 = b(Z= 0) (88) R' = u(X = OH),R2 = b(Z = 0) (89) R' = u (x = H),R~= b(z = 0) (90)R' = R2 = c (91) R' = R2 = d (93) X = H (95) R = CHO OSiMe3 (94) X = OH (96)R = CH26Ph3 Br-(97) i ?H Hok &OH0 $0 OH (98) (99) (100) (101) (102) R = CH2I (104) (103) R = CHO R (105) R = CHO OH Br-(107) (108) X = H.OH (109) x = 0 R -0Ac 0 OAc (1 10) OH NATURAL PRODUCT REPORTS 1985 [A+pL.“eo)o 4 56 \\\\ \o\ 4 5 -2 30 “‘Ok0 I a b C d e f (127) R’ =R2= (130) R’ =a,R2 =c (133) R’ =f,R2 =g (128) R’= R2= b (131) R’ =b,R2 =d (134) R’ =c,R2 =g (129) R’= a,R2=b (132) R’ =R2 =e (135) R’ =d,R2 =g from the synthon (114).The latter was obtained in several steps from (1 10) uiu asymmetric epoxidation with the Me,COOH-optically active dimethyl tartrate reagent [to give (1 1 l)] tosylation iodination and reduction of this by a hydride [to form (112)] cleavage of the epoxide ring [to give the diol (1 13)] protection of the secondary hydroxy-group by acetyla- tion and deh~dration.~’ The octenediol derivatives (1 16)- (1 18) have been prepared from the C,-substituted heptene-dione derivative (1 19) as acyclic C end-groups for the synthesis of chiral C4 and C, car~tenoids.~~ The sesqui-a- ionone (1 20) and sesqui-P-ionone (1 21) which are potentially useful end-groups for the synthesis of the CS0 analogues (122) and (1 23) of &,&-carotene and P,P-carotene have been prepared by a route involving acid-catalysed cyclization of (124).73 Cyclization in the presence of H2S0 gave the bromo-ketone (1 25) and thence sesqui-P-ionone whereas with H3PO4 the bromo-ether (1 26) was obtained which could be converted into the sesqui-wionone.Continuing their extensive work on cyclic carotenoid epoxides Eugster and his co-workers have synthesized the dias tereoi someric (5R,8R,5’R,8’R)- (5R,8S,S’R,8’s)- and (5R78R,5’R,8’S)-aurochromes [(127)-( 129) respectively] and the rneso-forms (5R 8R 5’S,8’s)-and (5R78S S’S,8’R)-auro- chrome [(130) and (131)] by condensation of the C10 Wittig salt (96) with the appropriate isomer of the C,s aldehyde (136).74 These aldehydes were obtained uiu isomerization of the 5,6-epoxide (137) in the presence of BF,.Et,O and their structures were confirmed by X-ray crystallography .7 Aurochrome isomers were also obtained via isomerization of (1 38) in strong base or by isomerization of the 5,6;5’,6’-diepoxy-5,6,5’,6’-tetrahydro-P,P-carotene (1 32).74 The latter compound as its (5R,6S75‘R,6S)-isomer was prepared by Wittig condensation of the Clo salt (96) with the epoxy-aldehyde (139) which was obtained via chain-extension of 5,6-epoxy-P-ionone (140) with triethyl phosph~noacetate.’~ Acid-catalysed isomerization of (5S,6R)-5,6-epoxy-5,6-di hydro-P,P-carotene (1 33) similarly NATURAL PRODUCT REPORTS 1985-G.BRITTON (137)X = CHCH20C(O)Ph (138)X = CHCH2P(O)(OEt)2 (139) X = CHCHO (140)X = 0 (145) gave the 8-epimeric mutatochromes (1 34) and (1 35) [(SS,SS)-and (5S,8R)-5,8-epoxy-5,8-dihydro-P,P-carotene], which were fully characterized by spectroscopic methods.77 The synthesis of several carotenoids that bear a hydroxyl group at C-19 has been achieved by a route involving the I ,2-addition of reactive vinyl anions [which were prepared by the Shapiro reaction of carotenX-al (105) underwent hydroxylation at C-4 whereas retinal (156) and P-ionone (93) both of which have shorter chains did not.Lycopene was totally oxidized to products that were not identified.82 When the reaction was carried out with P,P-carotene in the presence of H2 80,the hydroxyl group that was introduced contained 80,and a 3,4-dehydrogenation- (phenylsulphony1)hydrazones of polyenones] to p~lyenals.~~ hydration mechanism was Thus the phenylsulphonylhydrazone(141) with BuLi gave the lithium derivative (142); with the aldehyde (143) this formed the carotenoid intermediate (1 44) and after hydrogenation and acid-catalysed rearrangement P,~-carotene-3,19-diol (1 45). The synthesis of (1 52)-rubixanthin [(152)-(25)] by standard methods has been described.4s The series of diterpenes (146)- (149) which could be biosynthetic precursors of the C, carotenoid crocetin (54) has been prepared by standard Wittig condensation procedures.79 For continuing studies of the transfer of energy between carotenoids and porphyrins in relation to the functioning of carotenoids in photosynthesis two further model complexes i.e.a caroteno-pyropheophor-bide a (1 50)80and a carotenoid-porphyrin-quinone (15 have been synthesized. The ability of the supposedly inert chromatographic adsor- bent microcel C to catalyse the introduction of hydroxyl groups at C-4 of O-ring carotenoids has been investigated further. The reaction took place most efficiently with light petroleum as solvent and hydroxylation occurred only at positions that were allylic to a long main polyene system.Thus a-carotene [P,E-carotene] (1 52) was hydroxylated only in the P ring and gave 4- hydroxy-a-carotene (and not the 3’-hydroxy- 4,4’-dihydroxy- or 4,3’-dihydroxy-products) and the long-chain 8‘-apo-P-indicated.83 The epoxidation of canthaxanthin with perbenzoic acid produced a mixture of the 9,lO- 11,12- and 13,14-epoxides (157) (159) and (161).* Reduction of these with NaBH gave the corresponding isozeaxanthin epoxides (1 58) (160) and (1 62) whereas reduc- tion of the 9,lO-epoxide (157) with LiAIH4 gave 9,lO-dihydro- P,P-carotene-4,9,4’-triol(1 63). The structures of ‘deoxyluteins I 11 and III’ which were obtained by dehydration of lutein have been characterized spectroscopically (ir.I H and I 3Cn.m.r. c.d. and m.s.) as the isomeric 3’,4’-didehydro-P,g-caroten-3-01(1 53) 3’,4’-didehy- dro-P,y-caroten-3-01(154) and 2’,3’-didehydro-P,&-caroten-3-01 (155).85Thermal degradation of P,P-carotene gave the series of shorter chromophore analogues (1 64)-( 167).86 Thermal iso- merization of (all-E)-P,P-carotene produced four mono-2- five di-Z- and one tri-2-isomers together with at least six further uncharacterized isomers.87 Amongst the products were the first reported hindered (72)-isomers to be characterized. The thermocyclized product (1 68) and its (9’2)-isomer were also obtained. The products of the iodine-catalysed isomerization of natural and semi-synthetic antheraxanthin (32) have been identified by I3C n.m.r.spectroscopy as 13’4s (neoanthera- xanthin A’) 13-cis (neoantheraxanthin A”) 9’4s (neoanthera- xanthin B’) and 94s (neoantheraxanthin B”).88 NATURAL PRODUCT REPORTS 1985 Me a b C (152) R' = u,R' = b (153) R' = c,R2 = d CHO X (1 56) A study has been reported8" of methods for determining the enantiomeric composition of partly racemized carotenols. The camphanate ester methodg0 was suitable only for a-ketols. For simple carotenols no h.p.1.c. separation was achieved of the camphanate esters nor of the diastereoisomeric esters with methoxy(pheny1)trifluoromethylacetic acid but H n.m.r. spectroscopy of the latter products in the presence of the shift R* d c f (154) R' = c,R2 = e (155) R' = c,R2 = f (157) X =O (158) X = H,OH reagent Eu(fod) allowed quantitative determination of the enantiomeric compositions of carotenols with 2-hydroxy-0 and 3-hydroxy-P end-groups.A sensitive procedure has been devised for qualitative and quantitative determination of enantiomers of zeaxanthin (39) and lutein by h.p.1.c. separation of the diastereoisomeric dicarbamates that were obtained by reaction with (+)-(S)-a-(1-naphthyl)ethyl i~ocyanate.~' NATURAL PRODUCT REPORTS 1985-G. BRITTON X x (159) X = 0 (160) X = H,OH X X (161) X = 0 (162) X = H,OH OH I (164) x = CH=CH (165) X = CMe=CH OH R (169) R =CH20H (170) R = C02Et (171) R = CH~OAC (168) (172) R = C02Me (173) R = CHO LC0,Et R\ + + (178) R = CHO + (174) R = CH2P (Ph)z polystyryl Br-(176) R = CHzP(Ph)z polystyryl Br-(179) R = CH2PPh3 Br-(175) R = CH2Br (177) R = CHzOH I .3.2 Retinoilis A chapter in a monograph on the retinoids surveys the chemistry (including syntheses) and properties of these compounds.la An extensive and detailed article9’ reviews the photochemistry and synthesis of stereoisomers of vitamin A (retinol) (1 69). Another review of chemical synthesis with vinylallenes includes work on re ti no id^.^^ More methods have (180) R = CHZCI (181) R = CH(0Et)Z been developed for constructing the polyene chain of vitamin A and its derivatives. An interesting development is the use of polymer-supported Wittig reagents to synthesize ethyl retin- oate (1 70).9JThe insoluble polystyryl Wit.tig reagents (I 74) and (1 76) were prepared by the reaction of (1 75) or (1 77) with the diphenyl(polystyry1)phosphine or its hydrobromide.Genera- tion of the polymer-bound phosphoranes with NaOEt-EtOH in the presence of the aldehydes (178) and (182) respectively gave stereoisomeric mixtures of ethyl retinoate. Another new development is the use of electro-organic procedures for Wittig synthesis of retinyl acetate (171) from the C Wittig salt (179) and the aldehyde (1 83).9s Electrogenerated base was produced from the pro-base ethyl 2-cyano-2-(fluoren-9-ylidene)acetate with Li+ as the cation of the electrolyte. Yields of up to 40% were achieved of retinyl acetate in a cis :trans ratio of 3 :1 to 4 :1.More conventional sulphone procedures have been used to prepare methyl retinoate (172) from the C, sulphone (184) by alkylating it with (186)96 or by the addition of the aldehyde (187),97 which was obtained by the Sharpless oxidation of methyl geranoate. Several procedures have been described for the synthesis of various Z-isomers of retinal (173). Wittig condensation of the C, phosphonium salt (185) with (2E,62)- 2,6-dimethyl-8-triphenylsilyloxyocta-2,6-dien-4-yn-(188) 1-a1 produced the (1 32)-didehydroretinol (189). On reduction with Cr2+ this gave predominantly (1 3Z)-retinol whereas reduction with Lindlar catalyst afforded 93% of the (1 12,13Z)-isomer NATURAL PRODUCT REPORTS 1985 condensation with acetaldehyde and extension of the side-chain with ethyl lithio(trimethylsily1)acetate.O (72,9Z,1 1E,13E)-1 2-Fluororetinal(l96) was also obtained by a route involving condensation of (1 95) with (EtO)?P(O)CFH- C0,Et. A method for extension of a polyene chain by condensation of No-unsaturated aldehydes with 1-trimethylsilyl-oxy-1,3-dienes [e.g.(197)] in the presence of a ZnC1 catalyst has been used to form the vitamin A synthetic intermediate (198; all-E or Z/E mixture) from the C,,-aldehyde acetal (1 8 1). O2 The P-cyclocitral acetal (199) similarly gave the C product (200). Many papers describe detailed procedures for the synthesis of a wide range of analogues of retinal for use in studies of the binding of the retinal chromophore to opsin in the visual pigment rhodopsin and to the corresponding protein in bacteriorhodopsin.The potential therapeutic importance of analogues and derivatives of retinol and of retinoic acid has also stimulated the synthesis of a wide variety of related oxidation with MnO then giving the corresponding retinal~.~~ compounds. Relatively simple structural variations for which Coupling of the propargyl benzoate (190) with the mixed syntheses are described include compounds with bulky sub- cuprate [ButMe,SiOCH,CH2CMe=CHCuC=C-C(OMe)-stituent groups at C-4 e.g. 4-butylretinal (2Ol),lo3 the Me,]- Li+ gave the vinylallene (191) which on heating gave three isomers of retinol these being the (1 12)-and the highly hindered (1 1Z 132)- and (92,11Z,13Z)-i~omers.~~ The (92,11Z)-isomer could not be isolated and underwent cycliza- tion to give the tricyclic compound (192).The alternative allene 'isoretinol' (1 93) was prepared by methylation of the chloro-derivative (194) which had been obtained by the reaction of the C chloride (180) with a (chlorodihydrofury1)-copper reagent. This allene also gave a stereoisomeric mixture of retinals by oxidation with dimethyl sulphoxide-dicyclohex- ylcarbodi-imide.loo The (72,92,112)- and (72,92,112,132)- isomers of retinal have been prepared from the CI5 aldehyde (195) by a series of reactions including chloromethylenation (1 82) u LCHzOAc (184) R = CHZSOzPh OHC (185) R = CH26Ph3 Br-(188) (189) F acetylenic 7,8-didehydroretinal (207) and its 5-demethyl- and 1,1,5-tridemethyl-analogues[(208) and (2O9)],lo4 and the 1,1,5- tridemethyl- (202),'05 5-demethyl- 9-demethyl- 13-demethyl- and 9,13-didemethyl-retinals (203)-(206).O6 A new method for addition of deprotonated ketimines (210) to aldehydes and ketones has been used to prepare the methyl-substituted intermediates (21 1) and (212) from p-ionone and P-ionylidene- acetaldehyde. O7 Standard Wittig procedures for preparing 9- demethyl- 13-demethyl- and 9,13-didemethyl-retinals were modified by replacement of the acetoxy-aldehyde (21 3) by 5-(acetoxymethyl)furfural (214) to give 13-demethyl-l l,14-epoxyretinal (2 15). O8 Similar procedures were used to make a series of 1 l-14-bridged retinals (21 6)-(220) and their (9Z)- (187) I CH20H (1 90) (191) (194) (195) NATURAL PRODUCT REPORTS 1985-G.BRITTON 36 1 Metor H) /cC H20Ac OHCoCH20Ac 0 OHC \ ' 'CHO vR /CH(OEt)2 fi (215) R = H ,X = 0 s/ pc*cyc+c CHO (216) R = H ,X = NH 0 (222) RUCHO (227) R = CH=CHCH=CMe2 (228) R = CH=CHCH2CHMe2 (229) R = CH=CHMe (225) R' = Et ,R2 = Me (230) R = CH=CH2 (226) R' = R2 = Me (231) R = Me isomers. Io9 In a synthesis of the 10-14-sulphur-bridged analogue (221) of retinal the key step was the cyclization- sulphidation of (222) with thiourea in dimethylformamide at 230 "C affording the thiopyranone (223).' lo The various acyclic analogues (224)-(226) of retinal have been synthe- sized,' I ' as have the shorter analogues (227)-(231) lacking the six-membered ring and differing in the length of the conjugated polyene chain.I Many analogues of retinal with aromatic ring systems have been prepared including the bromobenzyl compound (232) and the bicyclic compound (235),' I3 the naphthylretinal (236),' l5 and the collection of substances (233) (234) and (237)-(240) and their 9-demethyl derivatives as both (all-E)- and (9Z)-isomers. I Much effort has been devoted to the synthesis of analogues of retinal and of retinol in which the configuration of the polyene chain is fixed by being in a five- six- or seven-membered ring. Thus thermally induced [ 1,5]-sigmatropic rearrangement of the fused vinylallenols (241) gave the fixed 12-s-cis products NATURAL PRODUCT REPORTS 1985 C HO (237) R = H (238) R = Me (241) n = 1 or 2 (242) n = (243) n = (244) n = (245) n = (246) /I = (240) 1 ,R' = H R~= CH,OH (247) 1 ,R' = CHzOH R2 = H 2 ,R'= H RZ= CH2OH 2 R' = CH20H R2= H 2 R' = H R2 = CHO CHO (248) (249) &%A& y' U RZ c 104-(242)-(245) and their corresponding aldehydes.The cyclo- heptane aldehyde (246) underwent thermal isomerization and photoisomerization to form (247),I the (9s 13E)- (9Z 13E)- (9E 132)- and (92,13Z)-isomers of which were used in spectroscopic studies. * Synthesis of the five-membered-ring compounds (248) and (249) provided retinals with fixed 13- trans and 13-cis configurations. Other interesting analogues [(250) and (251)l have been prepared in which a benzene ring replaces part of the polyene system.' 20.1 21 The preparation of the spiro-retinal(252) and the corresponding methyl retinoate in which the methyl substituents at C-1 are replaced by a cyclopropane ring has been described.I Many Schiff-base and protonated Schiff-base derivatives of retinal and its analogues have been made for spectroscopic studies related to the functioning of visual pigments and bacteriorhodopsin. Thus the reaction of retinal and its CI5 homologue (178) with a range of amino acids H2N[CH21,- CO2H (n = 1,3 or 5) or with proline gave imines (Schiff bases) (250) C HO c10,-which were shown by n.m.r. studies to be in equilibrium with the zwitterionic form (253).' 23 The iminium salts (254)-(265) of various analogues of retinal with cyclic bases have been prepared for an extensive study of the effects of acidic (C02-) or additional basic (NMe,) groups in different parts of the molecule on the electronic absorption spectroscopic proper- ties.121 127 Similar studies involved the synthesis of n-butylamino-substituted derivatives of the protonated n-butyl- amine Schiff bases [(266) and (267)] of retinalIzx and of benzenoid analogues (268).?') Several derivatives of retinal and related compounds have been prepared. The treatment of all-?runs-retinal with N-bromosuccinimide in acetic acid gave 4-acetoxyretinal (269) which was converted into the 4-hydroxy-derivative (270) and the 4-0x0-derivative (27 I). 30 The 4,4-difluoro-derivatives (272) of retinol and retinoic acid and esters of the latter have been obtained by treatment of 4-oxoretinoic acid with diethylaminosulphur trifluoride.I 31 With the same reagent NATURAL PRODUCT REPORTS 1985-G. BRITTON 9"; 7' C02H a (258) R' = H ,R2 = 0 (259) R' = C02H .RZ= Me (260) R' = C02H R2 = u (261) R' = H ,R2 = Me cLO, eo-Me2N (263) (265) n =l or 2 NHBu I-1 (267) (268) n = 1 or 2 R = H or Me (266) X = Cl or CF3CO2 (269) R = CHO ; X = H,OAc (274) X = CHrNOR (270) R = CHO; X = H,OH (275) X = CONHR (271) R = CHO; X = 0 (276) X = COCL (272) R = CH20H or C02H; X = F,F (281) R' = CHzOH R2 = C02H (282) R' = CO2H ,R2 = CH20H (278) X = H2,Y =O (283) R' = C02Me R2 = COzH (279) X = 0.Y = H2 (284) R' = COzH RZ = C02Me (285) (280)X = Y =O (286) R' = R2 = CHzOH (287) R' = Rz = COzMe retinoic acid gave retinoyl fluoride (273).' 32 5,8-Epoxyretinal retinoyl chloride (276) with the appropriate iimine.' 35 The (277) has been prepared,' JJ and the 5,8-ci.s and 5,8-trrrtz.s (I 3Z)-retinamides were similarly obtained from (1 3Z)-retinoic diastereoisomers were separated by h.p.1.c.The (1 12 5,8-acid rirr either its acid chloride or its imidazolide. fruns) (I 32 5,8-fruns),and (I 32 5,8-cis) products were ob- The 6-lactones [(278) and (279)] of (I 32)-12-(hydroxy-tained by irradiation. Condensation of (all-i?)-or (I 3Z)-retinal methyl)-retinoic acid (28 1) and (I 32)- 12-carboxyretinol (282) with the appropriate substituted hydroxylamine (HONHR) [and the &lactones of their (1 1E 13Z)-isomers] have been gave the retinylnitrones (274; R = Me Bur Pr' cyclohexyl or prepared from the half-esters (132)- 12-carbomethoxyretinoic Ct,F5CH2).'-3JThe (all-E)-retinamides .(275; R = alkyl acid (283) and methyl (I 32)- 12-carboxyretinoate (284) which hydroxyalkyl etc.) were obtained by amidation of (al1-E)-were obtained by methanolic saponification of (I 323-1 2- carboxyretinoic anhydride (280) or by partial methylation of (1 1E,132)-12-~arboxyretinoic acid (285).' 36 Reduction of the (1 3Z)-6-lactones (278) and (279) gave (1 32)-12-(hydroxymeth- yl)retinol(286) which could not be made by reduction of (132)-12-carboxyretinoic acid dimethyl ester (287) with LiA1H4 only the (1 lZ,l3E)- and (1 lE,13Z)-isomers being obtained.I3' Many exotic retinoid structures have been synthesized mostly containing aromatic (benzene naphthalene or heterocyclic) rings.These include a series of conformationally restricted benzenoid derivatives (288)-(292),' 38 some containing a 7,8- dihydro- or 7,8-methano-group. Other compounds which have been made include those containing a fluorine substituent or a furan thiophene or pyridine ring [(293)-(298)],] 39 the naphthalenecarboxylic acid derivatives (299) and (300),140 and the 'arotinoids' (301)I4l and related sulphur-containing deriva- tives (302).142 Other papers report syntheses of the bicyclo- retinoates (303)143 and of the cyclopropyl compounds (304h (307)144 from the appropriate C1 precursors. The synthesis of C02 Et NATURAL PRODUCT REPORTS 1985 the retinoidal ylidenebutenolides (308) and (309) and their homologues from P-cyclocitral (3 10) and 3-methoxy-p-cyclo- citral (31 1) has been described.145 The urinary metabolites (3 12) and (3 13) of retinoic acid have been synthesized by a route involving a Grignard reaction.146 Several papers describe the preparation of retinal that is labelled with stable or radioactive isotopes.Thus [ 10-13C]- [ 1 1 3C]- [ 19-13C]-,and [20-' 3C]-all-trans-retinals were pre- pared from p-ionone the 13Cbeing introduced from MeI3CN 3CH3CN or 13CH31 during chain-extension reactions.147 Further work158 gave [14-I3C]- [15-13C]- [14,15-13Cz]- and [ 14-*H]-retinals into which additional *H labelling was then introduced at position 15. In a preparation of [20,20,20-2H3]- and [ 14,20,20,20-2H4]-retinyl acetate the C2H3 group was introduced by aldol condensation of the C P-ionylideneacetal-dehyde (178) with [*H,Jacetone whilst the *H at C-14 was introduced by a Horner reaction between the CIS ketone (314) and (Et0)2P(0)CLH2C02Et.149 The same aldol condensation C02H (296) X = 0 (297) X = S (301) R' = H Me Et ,Pr",Pri or Me0 ; R2 = R3 = H or C02H (303) R = H or Me (302) R'=R3=H; R2=S(0),X n=0-3;X=Na,Et,orNHEt RfiHo (304) R' = COzEt R2 = H ,R3 = Me R (305) R' = H ,R2= COzEt R3 = Me (306) R' = COZEt ,R2 = R3 = H (308) R = H or OMe (130 (310) R = H (307) R' = R3= H R2 = C02Et (309) R = H or OMe (131) (311) R = Me0 NATURAL PRODUCT REPORTS 1985-4. BRITTON CHO &a- 0 R' (312) R = Me (314) (315) R' = OCOCHN2 ,R2 = Me (313) R = CHzOH (316 ) R' = OCOCHN2 R2 = Me ; 9 -cis $CHzOH && 2Et (317) R' = H .R2 = CH2OCOCHNz; 9 -cis CHO 0 R' \ 0\cHo (321) (322) R1= C02Me R2= H (323) R' = H ,R2= C02Me (324) CH2OAC (328) R = CHO (329) R = CH (CNINMe2 (325) (326) (327) (330) R = CH2CHO (331 1 R = CH2CH2COMe was used in the preparation' 50 of [ 10,l 1,12,14,15,20,20,20-'HJretinal the other 2H substituents being introduced by the reaction of p-ionone or the C18 ketone (314) with the anion of Me3SiC2H2C2H=NCMe3.A chapter in the book on retinoidsllb reviews methods for preparing retinoids that are labelled with radioisotopes. Several synthetic routes to [ 11,l 2-3H2]retinoic acid with high specific activity have been described.' 51 Fluorescence irradia- tion of all-trans-[1 l-3H]retinal in MeCN gave a mixture of isomers from which 11-cis-[1 l-3H]retinal was purified by h.p.1.c.52 A transesterification procedure (using MeOH and NaOMe) was used to prepare all-trans-[1 l-3H]retinol from its acetate. 53 3-Diazoacetoxy-all-trans-retinal(315),l54 3-diazo-acetoxy- and 19-diazoacetoxy-9-cis-retinals [(3 16) and (31 7)],' 55 and 4-azidoretinol(3 18)' 56 have been prepared for photoaffin- ity-labelling studies of bacteriorhodopsin visual pigments and retinol-binding protein respectively. Irradiation (at 514.5 nm) of (9Z)- (1 12)- (1 32)- and (all-E)- retinal each adsorbed onto wet silica gel that was suspended in cyclohexane gave reasonably efficient photoisomerization of each isomer.' 57 The photostationary state contained a mixture of the four isomers the (1 1Z) being the major one.The regio- and stereo-selective photoisomerization of retinal methyl retinoate and the analogues (314) and (319)-(325) has been explained in terms of the directed decay of the planar excited species to the respective perpendicular intermediates.'58 (332) R =CH2CH20H (333) R = CH~CH~OAC (334) R = CH2CH2CI Contrary to previously reported results amines (e.g. Et3N) slowed the isomerization of (1 12)- and (13Z)-retinal and of their Schiff bases with n-butylamine. 59 Solvent-dependent photoisomerization of retinonitrile (326) has been studied. * 6o The product ratios were similar to those that were obtained with retinal. I.3.3 Carotenoid-like Compounds Work on the preparation and reactions of carotenoid-like compounds may be relevant to the synthesis and chemistry of carotenoids.Reviews have been presented of the preparation purification etc. of isophorone (327)I6l and of the synthesis and structure-function relationships of abscisic acid (78). 62 A practical synthesis' 63 of y-cyclocitral (328) involves the reaction of the bromide (335) with Me2NCH2CN to give the nitrile (329) which is then hydrolysed in the presence of AgNO,. A new preparation of y-cyclohomogeranial (330) and of dihydro-y-ionone (331) and its derivatives (332)-(334) all of which are volatile components of ambergris utilizes Claisen rearrangement of the vinyl ether (336). 164 (E)-7-Methyl+-ionone (337) has been prepared by methylation of p-ionone with lithium dimethylcuprate.65 The intermediate enolate was trapped with benzeneselenenyl bromide and oxidized with H20z. 7-Methyl-a-ionone (338) was prepared similarly from a-ionone. Condensation of acetone with the stereoisomeric NATURAL PRODUCT REPORTS 1985 (335) R = CH,Br (339) (336) R = CHzOCH=CHz (340) (3411 WR2 X R (353) (354) R = Me (355) R = H 3 0e C 0 2 H 4 (360) R = CH20H (3591 (361) R = OSiMc3 (362) R = OSiMe3; A3 citrals which had been prepared from telomerization products of isoprene gave the (52)-and (5E)-isomers of the enone (339) from which a-,p- and y-ionones were obtained by acid- catalysed cyclization.'66 A 1 :9 mixture of cis-and trans-y-irone [(340) and (341)] and pure trans-y-irone have been synthesized via the intramolecular Diels-Alder reaction starting from a hexa-2,4-dienylaniline derivative (342) and p,p-dimethylacryloyl chloride (343).' 67 A simple preparation of 9-oxodihydro-a-damascone (344) has been achieved' 68 by con- densation of a-cyclogeranoyl chloride (345) with acetone in diethyl ether that contains NaNH,.A new synthesis of a-damascone (346) and of the boll-weevil pheromone (347) used 2-(hydroxymethyl)-4-(phenylthio)but-1 -ene as a building block. 69 In the trisporic acid series an efficient synthesis of deoxytrisporone (348) has been achieved from isobutene and methyl 2,4-dioxohexanoate by a route involving photocycload- dition and chain elongation with the lithium dianionic species (351) (which was generated from 3-methylbut-2-enoic acid and lithium di-isopropylamide),'70 methyl (7E,9Z)-trisporate B (344) R = COCH2COMe (345) R = COCL (347) (346) R = COCH=CHMe 0 (&OH R (356) (357) R = H (358) R = Me (363) A'(7) (366) X = 0 (364) A3 ,A'") (367) X = OH Me (368) (365) A2 (349) has been obtained by a procedure which uses 2-(hydroxymethy1)cyclopropyl phenyl sulphide (as its lithium dianion),' and methyl 4-dihydrotrisporate B (350) has been prepared ria Wittig condensation of the lactone (352) and the triphenylphosphonium bromide (353).The (Z)-a-ionylideneacetic acid (354) which is an intermedi- ate in the synthesis of abscisic acid was prepared in four steps from a-ionone ria ethoxycarbonylation of the acetylene (356) with CIC(OEt), an addition reaction with Me,CuLi and hydrolysis.73 The (Z)-9-demethyl analogue (355) of a-ionyl-ideneacetic acid has also been synthesized,' 74 along with the methyl esters of the 9-demethyl analogues [(357) and (359)] of 5,6-epoxy-P-ionylideneacetic acid and abscisic acid. The (+)-and ( -)-enantiomers of (9Z)-5,6-epoxy-P-ionylideneacetic acid (358) were prepared from the corresponding (+)-and (-)-5,6-epoxy-P-ionones (140) which had been obtained via Katsuki-Sharpless asymmetric epoxidation of P-cyclogeraniol (360). 75 A short synthesis of (+)-3,4-dihydroactinidiolide (363) from the ketone (366) employed Grignard methylation with MeMgI followed by oxidative cyclization of the intermediate (367) with NATURAL PRODUCT REPORTS 1985-G.BRITTON the quinquevalent chromium reagent (bipyH2)CrOCl5 and dehydrogenation.176 3,4-Dihydroactinidiolide actinidiolide (364) and aeginetolide (368) were also prepared uiu the common trimethylsilyloxy precursor (36 I) the key steps involving sequential oxidation with rn-chloroperbenzoic acid and acetylation of the product and its derived siloxy-diene (362).17' The related compound (+)-loliolide (369) has been synthesized uia the intermediates (365) and (371) which were obtained by stereospecific cyclization of homogeranoic acid. I 78 Loliolide dehydrololiolide (370) and 3-oxoactinidiol (372) have also been made by regioselective ozonolysis and epoxidation of the megastigmatrienone (373).' 79 The related bromide (374) was obtained by cyclization (using SnCl in MeCN) of the bromohydrin of homogeranoic acid (375).l8O Other carotenoid-like compounds that have been synthesized include the drimatrienes (376; R = Bun But or Ph) from (377) uia the allenylidene (378),I8l trixagol (379)18' and diumycinol (380)' 83 from the y-cyclo-compounds (381) and (382) respec-tively and the pallescensins (383) and (384).Is4 The intermedi- ate compounds (385) and (386) have been prepared and used for the synthesis of a range of bicyclic terpenoids.Is5 4-0x0-0- (369)X = H,OH (3711 (370)X = 0 (379) cyclogeranoic acid (387) and its methyl ester have been made in two ways (either from cx-and 0-cyclocitrals by N-bromosucci- mide-catalysed photoreaction,Is6 or in five steps from a-iononelS7) for use as intermediates in the synthesis of strigol.Products such as (388) and (389) have been obtained by the CF,C02H-catalysed cyclization of phenylselenium derivatives of terpenes. I 88 Vilsmeier reaction of the methyl-a-ionol (390) (which was obtained by a Grignard reaction between a-ionone and MeMgI) at 100 "C gave or-ionylideneacetaldehyde (392),' 89 whereas the methyl-P-ionol (391) gave the retro-aldehyde (394) at 100 "C but mainly 0-ionylideneacetaldehyde (393) at 28 "C. The substituted 0-ionone (395) and its iminium derivative (396) bearing a carboxyl group that is isolated from the TC-conjugated system have been synthesized for spectroscopic studies.Ig0 The reaction of 0-ionone with the Wittig reagent + Ph3P[CH2],NMe2 gave the amino-alkene (397) as a mixture of (E)-and (Z)-i~omers.'~' A method has been described for oxidation of p-ionone with a peroxydisulphate salt in the presence of the corresponding copper carboxylate to give 4-(380) (381)n = 1 (382)n= 2 X &YR2 Y (383)X=H,Y =OH (385)R' = HIR2 = H ,SiMe ,Br ,or OAc 0 (384)XY = bond (386)R'= SiMe3 or OMe R2= H (387) (388) I kHO (390)A4 (392) A4 (3891 (391) A'(') (393) A'(') (394) acetoxy-P-ionone (398) and 4-propionyloxy-P-ionone (399) and thence the 4-hydroxy-derivative (400).92 The dehydration of or-ionol(401) and of 0-ionol(402) with toluene-p-sulphonic acid has been studied. 93 0-Ionol reacted rapidly at 20 "C to give the retro-hydrocarbon (403) in high yield whereas or-ionol was very slowly converted into the hydrocarbon (404) at this tempera- ture.At 80°C both ionols gave a complex mixture of dehydration products. Refluxing p-ionone with toluene-p-sulphonic acid in benzene or with iodine in CCl, gave 80-90% yield of the trimethyltetralin (405) presumably via isomeriza-tion to the alcohol (406). 194Highly regioselective reduction of orb-unsaturated carbonyl compounds (including P-ionone) to either ap-saturated derivatives or allylic alcohols has been effected by [C1Rh(PPh3),]-catalysed hydrosilylation followed by methanolysis of the resulting adducts. I 95 The regioselecti- vity depended on whether mono- or di-hydrosilanes were used. A procedure has been describedlg6 for the reduction of terpenoids (including a-and 0-ionones) with NaBH, NaI and HOAc or Al(OPri) whilst they are adsorbed on t.1.c.plates. Several papers' 97-1 99 report detailed studies of the selectivity of hydrogenation of 0-ionone on a wide range of catalysts. A method for regioselective epoxidation by a peroxysulphur reagent that was generated from o-or p-nitrobenzenesulphonyl chloride and KOz has been applied to a-ionone and to 0-ionone.200 Catalytic hydroamination of mixtures of a-ionone and P-ionone by aliphatic nitriles and amines gave stereoiso- meric mixtures of the saturated N-substituted products (407; R = Et Pr Bun Bus or cyclohexyl).201 A convenient method for a'-chlorination of ap-unsaturated and conjugated ketones by (397) (395) x = 0 (408) R = CH2C1 X = H (412) X (409) R = Me X = Br (413) X (415) NATURAL PRODUCT REPORTS 1985 slow addition of the methyl ketone to a slight excess of PrI2NLi at O"C and quenching of the resulting a'-enolates by N-chlorosuccinimide at -70 "C has been used to prepare the chloro-0-ionone (408) from P-ionone.20z The reaction of 4- bromo-&ionone (409) with NaOEt gave (410) by a Favorskii- type rearrangement and double-bond migrationto3 whereas its reaction with PhONa or EtzNH gave primarily substitution products.The reaction of P-cyclogeranoyl chloride (41 1) with the organometallic compounds RMnI RzZn or RzCd (in diethyl ether at -15 to -20 "C) gave mainly the normal alkylated product (412) together with some of the a-is~mer.~~~ On heating 0-cyclofarnesoyl chloride (413) at 110 "C in a sealed tube the dimeric product (414) was obtained,205 whereas heating at 140 "C in a flow-type microreactor gave the spiro- compound (41 5).The iron carbonyl complexes (41 6)-(418) were obtained by the reaction of 3,4-didehydro-P-ionone (419) with Fez(C0)9 or Fe(CO)S. The structure of the most stable product which was the unusual iron dicarbonyl complex (41 8) was determined by X-ray crystallography.206 The dehydro-0-ionone was easily liberated from the complexes e.g. from (416) by treatment with HzOz in MeOH that contained NaOH at 0-20 "C for 15 minutes.207 Ene-reactions of trixagol (379) and its acetate with lozhave been studied.208 Many papers have been published reporting detailed and extensive studigs of the photochemistry of substituted ionones and related compounds and large numbers of the products that were formed have been characterized.Thus (E)-7-methyl$- ionone (337) on irradiation (h > 347 nm) in pentane gave a mixture of (a-and (2)-isomers and a tricyclic product whereas = COR = CH2CH2CMe=CHCOCI (414) NATURAL PRODUCT REPORTS 1985-G. BRITTON the main product was the benzopyran (420) if acid was present.?09 Compound (421) which is the 5,6-epoxy-derivative of (337) also underwent geometrical isomerization if h > 347 nm but underwent ring-cleavage if h = 254 nm.210 The initial photoreactions of the pivaloyl analogues (423) and (424) were identical to those of (E)-ol-and 0-ionones but differences were observed in the later stages.21 The n-butyl analogue (425) of a-ionone also behaved like a-ionone,212 but the corresponding 0-ionone derivative (426) at 254 nm gave only the pyran derivative (427).On triplet-sensitized irradiation (E)-ol-and (E)-P-damascones [(346) and (428)] both gave exclusively (2)-isomers whereas direct irradiation in alcoholic solvents gave a range of products.213 Triplet excitation of the (0-epoxide (422) also resulted in geometrical isomerization but singlet excitation at 254 nm afforded products which included the cyclobutene derivative (429). 214 Under basic conditions irradiation of the me thox ycarbon ylQ-ionones (430 ; R = H or C02Me) gave oxatricyclodecane derivative^.^' On irradiation vomifoliol acetate (43 1) underwent a di-n-methane rearrangement to produce (433) ;deoxyvomifoliol acetate (432) similarly gave (434).216 The photochemistry of a wide range of (420) (421)X = 0 (422)X = CH2 I (428) (429) (4301 0L OAc 5,6-epoxy-P-ionone derivatives and analogues has been studied extensively.The compounds that have been investigated include the 2-hydroxy-derivative (439,” the diepoxides (436)-(438),218 the 3,4;5,6-diepoxides (439) and (440),219,220 the methano-compounds (442) and (443),221 and the 4-hydroxy- compound (444).222 In all cases many products were character- ized. Laser flash photolysis (265 nm) of the epoxides (140) (439) (441) and (445)-(448) gave short-lived transients which were identified spectroscopically as carbonyl ylides. 223 1.4 Carotenoid-Protein Complexes Interest in complexes between carotenoids and proteins especially the blue carotenoproteins of invertebrate animals continues to increase and the topic has been Several new natural blue carotenoproteins have been de- scribed e.g.the complexes between astaxanthin [3,3’-dihy- droxy-f3,P-carotene-4,4’-dione](449) and proteins from Ane-monia sul~ata,~~~ from four copepods from the Great Barrier Reef,226 and from the carapace of the crab Carcinus maenast2’ and complexes between proteins and canthaxanthin [p,p-carotene-4,4’-dione] (450) from the anostracan crustacean @Bu 4 (423)R = Bu‘ ; A4 (427) (424)R = Bu‘ ; A’‘‘) (425)R = Bu”; A4 (426)R = Bu”; As(‘) OAc (431)R =OH (4331 (432)R = H (439)x = Y EO (434) (435) (436)X=O (438) (440)X = CH2,V=0 (437)X=CH2 (441)X = 0,Y = CH2 R (442) (443) (444) (445)R =Me (447)x = 0 (446)R = OMc (448)X =CH2 0 370 Branchinecta packardiZz8 and from the freshwater mollusc Unio pictorurn.229 Carotenoproteins that contain astaxanthin canth- axanthin zeaxanthin (39) and isozeaxanthin (9 1) have been obtained from four echinoderm^.'^^ These complexes lend themselves well to detailed spectro- scopic studies on the interactions between the carotenoid chromophore and its protein environment and on the mechanism of the spectral shift. Thus a c.d. study of the pigments ovoverdin a-, p- and y-crustacyanin and the ‘yellow protein’ of the lobster Hamarus americanus has shown that the asymmetry that is induced in the blue chromophore of ovoverdin by binding to the protein differs from that in the crustacyanins and exciton interactions were observed for the chromophore of the yellow pr~tein.’~ No evidence of exciton interactions between carotenoids was obtained from c.d.studies of the astaxanthin-protein complex (h,, 650 nm) from the copepod Anornal~cerapatersonior that from a siphonophore of the genus Porpir~.’~‘The resonance Raman spectra were also reported. Similar c.d. and Raman spectroscopic investiga- tion indicated that in the canthaxanthin-lipovitellin complex of Branchipus sfagnalis although there is only a single carotenoid-binding site the carotenoid molecules are bound asymmetrically in three different configurational states ;233 in the complex from a brine shrimp of the genus Arternia the absorption bands with A,,,, 368 460 and 600 nm may result from twisting of the chromophore which (at least in the species for which A,,,, is 460nm) may be in a twisted cis conforma-tion.234 Carotenoid-protein interactions have been detected by resonance Raman spectroscopy of carotenoids in situ in the pigmented calcareous skeletons of some corals.235 As part of an investigation of the proteins (rather than the chromophores) of carotenoprotein complexes the apoprotein subunits of a-crustacyanin from the lobster Hornarus gammarus and the V,, complex of Velella celella have been compared by peptide mapping.236 A red form of P,P-carotene (h,,, ca. 530 nm) has been obtained from a strain of Phjmrn-pces ~lake.deeanu.~.~~~ Inter-action with protein appears to be important in maintaining the carotene chromophore in this state.There may be similarities between this complex and a red 0,P-carotene species that has been isolated from leaves.238 The red colour of this latter example has been attributed to microcrystalline aggregation of the carotene molecules rather than to a direct effect of protein on the chromophore. 1.5 Physical Methods Many of the papers that have been described above contain details of the chromatographic purification (especially by h.p.1.c.) of carotenoids and related compounds and present extensive u.v.-visible i.r. n.m.r. m.s. and c.d. data for the compounds that are discussed. In general this work will not be surveyed in this section which will consider only those papers that are devoted largely or entirely to the development of assay procedures or to detailed spectroscopic studies or which introduce novel or unusual applications of the techniques.I .5.I Separation and Assay An extensive review on the chromatography of carotenoids and retinoids has been published.239 A chapter in ‘Methods in Enzymology’ presents methods for the extraction separation identification and determination of carotenoids in biological samples by t.1.c. and reversed-phase h.p.l.c.240 A chapter in a comprehensive book on the retinoids surveys the methods that are used for the separation purification and assay of these compounds. IC Two papers describe the hydroxylation of P-ring carotenoids on microcel C emphasizing the need for caution when using this supposedly inert chromatographic adsorbent.2.83 Methods for determining the enantiomeric composition of partly racemized carotenols have been e~aluated.~~ Only a-NATURAL PRODUCT REPORTS 1985 ketols [e.g.astaxanthin (449)] could be separated by h.p.1.c. of the diastereoisomeric camphanate esters.90 No separation was obtained of the diastereoisomeric esters with the chiral methoxy(pheny1)trifluoromethylacetic acid but the enantio- meric compositions of carotenols with 2-hydroxy-p and 3- hydroxy-p end-groups could be determined by ‘H n.m.r. spectroscopy of these esters in the presence of the shift reagent E~(fod)~. A method has been developed for the separation and quantitative determination of the optical isomers of zeaxanthin (39) and lutein (21) by reaction with (+)-(S)-a-(1-naphthy1)- ethyl isocyanate and separation of the diastereoisomeric dicarbamates by h.p.1.c.on a Spherisorb S5-W column 50 cm long.9 1 The separation of carotenoids by reversed-phase h.p.1.c. on Zorbax-ODS with an isocratic non-aqueous solvent system has been studied in detail and procedures have been developed for analysis of the carotenoids from several natural sources with this system.’“’ A similar approach has been used for the purification and assay of retinol.242 A procedure for the analysis of spinach leaf carotenoids also uses Zorbax-ODS columns.’-13 An interface system has been described that can collect spectral data from a wavelength-scanning microproces- sor-controlled h.p.1.c.detector and pass them to a microcom- puter and it has been applied in the analysis of the carotenoids of tobacco.’44 Other h.p.1.c. procedures have been described for the separation of the carotenes of tomatoes,24s the carotenoids of citrus fruits,2S6. 247 and microbial carotenoids,248 and a computer has been used to optimize the solvent composition for separating the carotenoids of Corynehacteriurn poinsettiae. 249 A paper on the identification of the (Z)-isomers of carotenoids from hips of Rosa pomifhrass describes several h.p.1.c. procedures which give impressive separations of groups of closely related and isomeric carotenoids. Especially noteworthy is the use of reversed-phase nitrile columns. The separation of 1,2-epoxides of lycopene P,$-carotene and E,$-carotene by reversed-phase h.p.1.c.and of their isomers on buffered silica columns has been described.2so Reversed-phase h.p.1.c. has also been used to separate lutein esters with fatty acids of different chain lengths.” A procedure for the deconvolution of multicomponent u.v.- visible spectra has been developed as a means of analysing complex mixtures of carotenoids.2s2 A programmed gradient procedure has been described for the separation of retinoids by h.p.l.c.253 A systematic study has been undertaken to determine the optimum solvent composi- tions for separating (92)- (1 1Z)- (1 32)- and (all-E)-retinol by h.p.1.c. on silica.‘54 These isomers have also been separated on Partisil-ODS and Zorbax-CN columns in series.25s The separation of (9Z)- (1 32)- and (all-E)-retinyl palmitates by h.p.1.c.has been described with a warning about isomerization that occurs in chlorinated solvents if the solutions are exposed to light. 25h Other papers describe procedures for the separation of (Z)-/(E)-isomers of 5,6-epoxyretinal (45 1yS7 and of new aromatic retinoids by h.p.l.c.’58 A method for the analysis of deuteriated species of retinol by g.1.c.-m.s. (electron-impact or chemical ionization) has been reported.‘ 5‘) High-performance liquid chromatography g.l.c. and t.1.c. methods for determination of abscisic acid have been evaluated and compared. 2h0 A reversed-phase h.p.1.c. procedure has been described for separating abscisic acid and its metabolites.261 The methyl ester of the (RS)-[10-i4C]abscisate derivative (80) has been resolved on an optically active Pirkle column and the two isomers were converted into (R)-and (S)-abscisic acid (78).262A stereoselective antibody technique has also been (451) NATURAL PRODUCT REPORTS 1985-G.BRITTON 37 1 used to prepare (R)-and (S)-enantiomers of abscisic acid in highly pure Very sensitive monoclonal antibody264 and radioimmun~assay~~~ methods have been developed for abscisic acid. The analysis of conjugated metabolites of abscisic acid by chemical-ionization mass spectrometry has been described. 266 1S.2 N.M.R. Spectroscopy In a detailed study with synthetic model compounds 3C n.m.r. shift data for vinyl methyl and vinyl methylene carbon atoms of polyene (Z)-/(,!?)-isomers have been compiled.The I3Cdata are more useful than the corresponding ‘H n.m.r. data in providing a general method for unambiguous assignment of geometry in polyene isoprenoids especially car~tenoids.’~~ 3C n.m.r. A analysis has been performed to determine the configurations of the double-bonds in several cis-isomers of antheraxanthin.88 A computer-assisted procedure has been used for conformational analysis of carotenoporphyrins and carotenoporphyrin-quin- ones from ‘H n.m.r. data.81.’h8 A detailed report of a two-dimensional ‘H and 13C n.m.r. study of (all-,!?)-retinal has appeared.269 Several papers report extensive n.m.r. analyses of geometrical isomers of retinol and related compounds.Ring-chain conformations of three (72)- isomers of retinol and several related hindered trienes have been examined by ‘H n.m.r. methods including nuclear Overhauser experiments and dynamic n.m.r. Dihedral angles and C-6-C-7 rotational barriers were calculated.270 Proton n.m.r. data have been tabulated for geometrical isomers of 5,6-epo~y-5,6-dihydroretinal.‘~~ N.m.r. data (‘H and I3C) as well as u.v. i.r. and m.s. have been reported for some all-trans- and 13-cis-retinarnide~;~’~ in a H and I3C n.m.r. study of methoxy- retinoids (452) configurations of double-bonds in the polyene chain have been derived.’73 The I3C and temperature-dependent ’ H n.m.r. properties of trans- and cis-3,4-didehydro- 0-ionone (41 9) have been determined and discussed in relation to conformational proper tie^."^ Solid-state magic-angle sample-spinning methods have been used in a I3C n.m.r.study of lyophilized purple membrane (the bacteriorhodopsin-containing membrane of Halobacterium halohiurn) after incorporation of [lo-’ 3C]- [ 1 1 -I 3C]-,or [ 12-13C]-retinal.2’s The results showed the coexistence of the all- trans- and 13-cis-isomers of retinal. When [ 19-I3C]- and [20- 3C]-retinals were used the substantial upfield shift was consistent with the protonated Schiff-base structure. In a similar study of bacteriorhodopsin that had been prepared from [14-’3CC]retinal the large upfield shift was attributed to the 13-cis configuration and a mixture of 13-cis-1 5-sl.n- and all- trans- 15-unti-isomers was identified.’76 The protonated Schiff- base structure has also been studied by solid-state IsN n.m.r.In analysis of E-[ ‘N]lysylbacteriorh~dopsin.’~~ a survey of solid-state ’C and I ’N n.m.r. studies of samples of rhodopsin and of bacteriorhodopsin that had been prepared with 3C-enriched retinals and ’N-labelled lysine it was concluded that the chemical shifts of C-6 and C-I3 were not consistent with the proposed external point-charge interactions of the chromo- phore.2-8 A 1 ’C and ’N n.m.r. study has been undertaken of the effects of protonation and hydrogen-bonding in the n-butylretinylidene Schiff-base as a model for the visual pig men t s. ’‘’ (452) R’ = H Me or OMe R2 = H or OMe R3 = H ,Me or OMe a a R4= CHO C02Me or C0,Et 1S.3 Circular Dichroism As indicated earlier (Section 1.2) c.d.correlations have been used to establish the absolute configurations of a number of natural optically active carotenoids. In the carotenoid field the geometry of the chromophore is fundamentally important for determining c.d. and some cis-trans isomers can be distin- guished. Thus rubixanthin (25) and its (5’Z)-isomer (gazania- xanthin) could be differentiated by c.d. at low temperat~re.~~ The different characteristics of c.d. spectra of the (all-E)- and (7Z)-isomers of 1,2-epoxylycopene (30) and (all-E)-and (7’2)- 1’,2’-epoxy-l’,2’-dihydro-~,~-carotene (3 I) have been discussed.38 The c.d. properties of a carotenoid are greatly influenced by its molecular environment. Thus the c.d.spectra of lutein (21) and zeaxanthin (39) in aqueous ethanol showed that these carotenoids can exist in two different physical states depend- ing on the concentration of water.28o The visible-region circular dichroism of aggregates of lutein in a cationic surfactant solution (dodecyltrimethylammonium bromide) differs from that which is induced by sodium dodecyl sulphate below the critical micelle concentration.’x’ The induced c.d. of 0,P-carotene in human blood lipoproteins has been used to study their thermal behaviour.282 The c.d. spectra of several carotenoprotein complexes have been recorded and tentative conclusions drawn about the geometry and exciton interactions of the carotenoid chromophores.231 ’j4 The c.d. and U.V. spectra of Schiff bases that had been prepared from 0-cyclocitral (310) and the optically active amines EtMeCHNH and Me(CH,0H)CHNH2 show remark- able dependence on temperature and on the solvent; this dependence has been attributed to (s-cis)-(s-trans) isomeriza- tion about the C-6-C-7 single bond and chiral discrimination of the twisted conformations of the conjugated system.2x-’ I S.4 Raman and Infrared Spectroscopy Although most reports are concerned with applications of the resonance Raman technique to detect and investigate the carotenoid (or retinoid) chromophore in z+w or in complexes some fundamental work on the detailed analysis and assign- ment of the resonance Raman spectra of the free compounds has been done.The Raman spectra of (all-E)-and (lSZ)-P,P- carotene in the solid state (at 77 K) and in solution in cyclohexane with exciting light of various wavelengths have been compared.The solid sample gave a large number of sharp (though weak) bands in addition to the well-known bands that were also seen for the sample in solution. All bands at 5800-1800 cm-I were accounted for as overtones or combination tones of strong fundamentals. Information on the mechanism of resonance enhancement was obtained from approximate excitation profiles and degrees of depolarization and differ- ences between the (all-E)- and (1 52)-isomers were analysed.284 Normal-co-ordinate calculations were performed for the (all-E)- (72)- (9Z)- (1 32)- (1 5Z)- (92,132)- (92,13’2)- (9Z 152)- and (1 32,15Z)-isomers of P,P-carotene.Vibrational modes that were assigned to the Raman bands that are characteristic of the various (Z)-isomers were analysed in detail.’*’ Time-resolved pulsed laser Raman spectroscopy has been used to study the position of electronic states of P,p-carotene,‘xh and time-resolved resonance Raman and photo- isomerization studies of (all-E)- and (1 SZ)-P,P-carotene in the electronic ground and excited states have been reported.287 Photoisomerization of (1 52) to (1 5E) was favoured over (1 5E) to (1 52) in CS solution whereas photoisomerization of the (1 52)-isomer did not occur in hexane. Space- and time-resolved resonance Raman spectra have been obtained from a short- lived (several femtoseconds) singlet excited state (S,)of P,P-carotene.288 Effects of temperature and of pressure on resonance Raman excitation profiles of 0,P-carotene in isopentane solution have been investigated.’*” The sensitized photoisomerization of carotenoids and the application of time- resolved resonance Raman spectroscopy to study the lowest NATURAL PRODUCT REPORTS 1985 OH (453) excited triplet states that are involved in the isomerization processes have been reviewed.290 The time-resolved resonance Raman spectra have revealed evidence that identical triplet states are obtained from the (1 5E)-and (1 SZ)-isomers of p,p-carotene. 29 The resonance Raman spectrum of P,P-carotene in a bilayer lipid black film with oleylamine has been deter- mined,292 and resonance Raman methods have been used to study the orientational ordering of P,P-carotene in lipid bilayers and the dependence of this ordering on the structures and compositions of the lipids.293,294 A review has been published of the application of resonance Raman spectroscopy in the study of carotenoid-containing biomolecules and micro-organisms.29 The resonance Raman spectra of several blue canthaxanthin- and astaxanthin-protein complexes have been obtained as part of a detailed investigation of the interactions between the protein and the carotenoid chr~mophore.*~~-~~~ Several papers report reso-nance Raman studies of the geometry of carotenoids in the reaction centres of photosynthetic bacteria. Thus comparison of their Raman lines with those of a range of cis-isomers of p,P-carotene led to the conclusion that the carotenoids in the istics of each isomer discussed.309 Transient Raman spectra have been determined for the lowest excited triplet states of these isomers of retinal.310 Except for the (132)-isomer all isomers gave identical relaxed triplet species and decayed to the (all-E) ground state.310*311 The i.r.stretching frequency for the protonated Schiff-base analogues (254)-(257) of retinal has been correlated with the position of the external positive charge.’ 24 Calculations of the vibrational frequencies of polyene Schiff bases have been related to the resonance Raman spectra of bacteriorhodop- sin.312 Factors influencing C=C and C=N stretching fre- quencies have been discussed in a correlation of vibrational frequencies with light-absorption maxima in polyenes ana- logues of retinal protonated Schiff bases rhodopsin and bacteriorh~dopsin.~ The resonance Raman spectra of con-densation products of retinal with diphenylamine indole and 3-methylindole have been compared with the spectra of l4 bacteriorh~dopsin.~ Resonance Raman and u.v.-visible absorption spectroscopic studies of phototransformations of protonated retinal-n-butylamine Schiff base in hexafluoroiso- propyl alcohol as a result of irradiation with light from various reaction centre have the (1 52)configurati~n.~~~-~~~ From laser lines have been reported.another study of these complexes however it was concluded that the cis-isomeric forms that had been detected by resonance Raman spectroscopy were artefacts which arose from the trans-isomers during the procedures that were used to solubilize the pigment-protein complexes.298 Resonance Raman spectra of the reaction-centre carotenoids in the triplet state were consistent with a cis conformati~n.~~~,~~~ The transfer of energy between carotenoids and other photosynthetic pigments has been studied by resonance Raman spectroscopy on a sub- nanosecond time-~cale.~~~ A technique for resonance Raman microspectrometry ofcells has been used to examine complexes between peridinin [5’,6’-epoxy-3,5,3’-trihydroxy-6,7-dide-hydro-5,6,5’,6’-tetrahydro-lO,ll,20-trinor-~,~-caroten-l9’, 1 1’- olide 3-acetate] (453) chlorophyll and protein in Gonyaulaxpol~+ edru.302 P,P-Carotene in human blood can be detected and determined by Raman lines at 1010 1165 and 1523 cm-’.Molecular aggregation has been shown to affect these line intensities ~ubstantially.~~~ Similar carotenoid-derived vibra- tions at 1530 and 1160 cm-I were detected in the Raman spectra of the lipids of blood platelets.304 The resonance Raman spectra of native and modified blood low-density lipoprotein suggested the presence of several different struc- tural populations of P,P-carotene.30s The spectra were changed by variations in pH and temperature. The Raman spectra of the sciatic nerve of the frog Rana temporaria revealed the presence of carotenoids which were not found in nerves from the rat or the crab Curcinus rnaena~.~O~ The intensities of the carotenoid bands were affected by the functional state of the nerve.Carotenoid-protein interactions have been detected by resonance Raman spectroscopy of carotenoids in situ in the pigmented calcareous skeletons of some corals.235 An extensive survey of the pre-resonance Raman and i.r. spectra of a wide range of 2H- and 13C-labelled species of (1 32)-retinal has allowed all of the major spectral lines and 2H shifts to be assigned rigorously.307 The F.T. i.r. spectra of fifteen purified retinoids have been recorded and compared. 308 Most i.r. and Raman bands of (all-E)- (92)- (1 12)- and (1 32)-retinal in the solid state have been assigned on the basis of normal-co-ordinate calculations and the vibrational character- Two reviews discuss the use of resonance Raman spectros- copy to investigate the structures and primary photochemistry of rhodopsin and bacteriorh~dopsin,~ 6 and many papers report details of such studies.Thus resonance Raman studies of bacteriorhodopsin that had been prepared from 2H-and 13C- labelled retinals have investigated the geometry of the 0640 and K610 intermediates in the photocy~le,~~~~~~~ and led to the conclusion that the primary step in the photocycle involves solely isomerization about the C-13-(2-14 bond.320 Other related work on the photocycle of bacteriorhodopsin is reported in a volume of symposium proceeding^,^^'-^^^ and in a num- ber of paper^.^*^-^^^ In the hR578 form of the related pigment halorhodopsin the (all-E) configuration of the retinal chromophore has been established by resonance Raman spectroscopy.330 A resonance Raman study of octopus hypsorhodopsin has been published.33 Fourier-Transform i.r.difference spectra have been used to investigate transitions of intermediates in the rhodopsin ~y~le~~~*~~~ and the primary photochemistry of bacteriorh~dopsin.~~~ I S.5 Electronic Absorption Spectroscopy Absorbance coefficients have been determined for the triplet states of some car~tenoids.~~ Neurosporene (49) spheroidene [1 -methoxy-3,4-didehydro- 1,2,7’,8’-tetrahydro-$,$-carotene] (454) spheroidenone [ l-methoxy-3,4-didehydro-l,2,7’,8’-tetra-hydro-$,rC/-caroten-2-one] (455) and spirilloxanthin [ 1,1’-di-methoxy-3,4,3’,4-tetradehydro- 1,2,1’,2’-tetrahydro-$,$-caro- tene] (456) gave values of 27.4 30.9 6.06,and 9.20 (x lo4 dm3 mol-I cm-I) respectively at their absorption maxima of 489 510,550 and 550 nm.A triplet absorbance coefficient of 7.30 x lo4 dm3 mol-’ cm-I was obtained for crocetin (54) sensitized by p~oralen.~~~ Triplet-triplet spectra and quantum yields were reported for this aqueous solution. A new absorption band that has been identified in the spectrum of p,p-carotene that was incorporated into biological and liposomal membranes was considered to be due to interactions between the oriented polyene chromophore and neighbouring lipid and calculated values of characteristic wave- NATURAL PRODUCT REPORTS 1985-4. BRITTON MeoApL.A X 0 b retinal Schiff-bases showed that torsion about single or double bonds shifts the value of A,, to shorter or longer wavelengths re~pectively.3~~ The absorption spectra of the 12-s-cis confor- mationally locked analogues of retinal (9E 1 lE 13E)-(247) (92,l lE,13E)-(247) (9E,1 lE,132)-(247) and (92,l 1E713Z)- (247) in polar solvents all exhibited a red shift of the a-band that was of greater magnitude than that which had been observed for the corresponding isomers of retinal.The highly hindered compounds (1 12,13Z)-retinal and (92,11Z,13Z)-retinal exhibited extraordinary absorption spectra which were blue-shifted compared with those of the corresponding alcohols.99 A series of papers describes the absorption spectra of iminium derivatives of analogues of retinal that contain additional charged substituent groups.Thus the values of h,, of the n-butylamine derivative (267) which bears an additional butylamino-group on C- 19 were more strongly blue-shifted than the h,, of (266) in which the butylaminomethyl substituent is at C-12.' 28 Large shifts to longer wavelength were observed' 29 in the absorption spectra of nine protonated HoM Schiff-bases of retinal and its benzenoid analogues (recorded C CHO (4591 lengths and oscillator strengths were in good agreement with the experimental data. Theories about the spectral shift of p,p-carotene from 450 to 1000 nm that occurs when it is complexed with iodine have been discussed and the effects of chlorinated solvents on this absorption band determined.338 Large blue shifts of the absorption bands of carotenoids in chromato- phores and cell membranes of photosynthetic bacteria were obtgined by addition of the hydrophobic anion tetra-phenylborate.39 Polarized U.V. absorption spectra have been obtained at room temperature and at 4.2 K for all-trans-retinal and both crystalline forms of 1 1-cis-1 2-s-cis-retinal in extremely thin microcrystal platelet~.~~O-~~~ Absorption and fluorescence spectra of retinyl acetate methyl retinoate and retinoic acid have been determined343 in solution and in poly(vinylbutyra1) films from 293 to 4.2 K. Absorbance coefficients for the triplet state singlet-triplet intersystem-crossing yields and kinetic data for the triplet state have been determined for the (all-E)- (72)- (9Z)- and (72,9Z)-isomers of the C,5 aldehyde (178) and the c18 ketone (314) in he~ane.~~~ In a similar study absorption-emission spectra fluorescence quantum yields triplet absorption spectra intersystem-crossing yields and photoisomerization of the Cz2 homologue (459) of 11 -&retinal have been investigated and compared with those of 1l-cis-retinal.345 Electric-field-induced changes in absorption spectra have been used to derive values for dipole moments and polarizabilities of excited states of retinal and diphenylpoly- ene~.~~~ Calculations on the absorption spectra of protonated with the base held in a polyethylene matrix) when the counter- ion was changed from C1- to CF,C02-.For a series of iminium analogues [(258)-(262)] of retinal which contained additional C02- groups the value of h,, was dependent on the distance between the iminium nitrogen and the C02- counter-ion and on the polarity of the solvent.125 The effects of a non-conjugated positive charge that was located in various positions in the molecule on the absorption spectra of model iminium derivatives of retinal and the analogues (254)-(265) have been investigated.24-26 27 The U.V. spectroscopic properties of the p-ionone derivatives (395) and (396) have also been deter- mined and the effects of the non-conjugated carboxyl group in shifting A,, of the iminium salt (396) to longer wavelength under alkaline conditions were discussed. 90 Absorption maxima and lifetimes have been recorded for the transient carbonyl ylides that were obtained by laser flash photolysis of the epoxyionone compounds (140) (439) (441) and (445)- (448).223 A bsorption-spectroscopic studies have been reported for rhodopsin and bacteriorhodopsin photoproducts on a pico- second or nanosecond time-~cale.~~*-353 Anew 540 nm side- band in the absorption spectrum of bovine hypsorhodopsin (A,, 435 nm) has been described and discussed.354 The effect of non-ionic detergents on the spectroscopic characteristics of bacteriorhodopsin has been investigated.3s5 Theoretical studies on the absorption spectra of rhodopsin and retinal protonated Schiff-bases have been rep~rted.~~~,~~~ 1S.6 Photoacoustic Spectroscopy The relatively new technique of photoacoustic spectroscopy has been used to study depth profiles of P,P-carotene in mammalian kin,^^^,^^^ carotenoids of the antenna in photo- synthetic bacteria,36o and the photocycle of bacteriorhodopsin and intermediates in the cy~le.~~l,~~* I S.7 Mass Spectrometry A review of mass spectrometry of tobacco isoprenoids includes a consideration of substances that are structurally related to carotenoids.363 1.5.8 X-Raj7 Methods The structures of the diastereoisomeric furanoid oxides (1 36) have been confirmed by X-ray ~rystallography.~ The structure of the tobacco constituent (72) was established by X-ray crystallography of its p-nitrobenz~ate.~~ This technique was also used to determine the structure of the unusual dehydro-P- ionone-iron dicarbonyl complex (41 8).206 X-Ray structural analysis of (all-E)-4-oxoretinal (271) revealed a half-boat NATURAL PRODUCT REPORTS 1985 R2 0 HOJ& xo 0 b C d Q R2= b (X = Ac) b(X =H),R2= c c,R2= d conformation for the ring s-cis geometry for the C-6-C-7 efficient in photosystem-I complexes but not in lipo~omes.~~~ A bond and typical curvature of the polyene chain.36s photoelectrochemical study of this energy transfer in a lipid X-Ray photoelectron-spectroscopic studies have been re-monolayer has been reported.38s One-electron oxidation of ported of all-trans- and 13-cis-fl~orophenylretinals~~~ and of carotene and electron transfer involving carotene cations and analogues of bacteriorhodopsin that had been prepared from chlorophyll in micelles has been The carotene them.366 radical cation has been shown to react with bacteriochlorophyll only in an aqueous phase suggesting that the electron transfer occurs in a micelle that contains one carotene and one bacteriochlorophyll molecule.386 Photoelectric effects of chlo- 1S.9 Miscellaneous Physical Chemistry rophyll and various carotenoids in membranes have been The properties of P,P-carotene as an unusual type of antioxi- in~estigated.~~’ A theoretical examination of the interactions dant which is very effective only at low partial pressures of between P,P-carotene and chlorophyll has been oxygen have been discussed.367 Several papers report studies Triplet- and singlet-state energy transfer involving a carotenoid on the quenching of singlet oxygen (‘0,)by carotenoids or chromophore that is covalently linked to a porphyrin system retinoids.Reactions between P,P-carotene and lo2have been have been in~estigated.~~,~~~ studied by a time-resolved thermal lensing technique.368 Rate The kinetics of fluorescence and the triplet yields of the C2, constants for physical and chemical quenching of lo2by P,P-aldehyde (459) and quenching by aromatic molecules and carotene zeaxanthin or fucoxanthin [5,6-epoxy-3,3’,5’-trihy-alcohols have been st~died.~~O*~~l An investigation has been droxy-6’,7’-didehydro-5,6,7,8,5’,6’- hexahydro-P,P-caroten-8-presented of the kinetics of quenching by P,P-carotene of one 3’-acetate] (460)in CCl suggest that a charge-transfer short-lived triplet states of several conjugated dienes and complex between ‘02and carotenoid contributes highly to the aryl-substituted 01efins.~~’ A rate constant of 4 x lo9 quenching so that a low-lying triplet level is not dm3 mol-I s-l has been determined for triplet energy The rate constants for the reaction of (all-E)-retinol or retinal transfer from biradical intermediates (generated from O-alkyl- with lo2increased with increasing dielectric constant of the substituted aromatic compounds) to P,P-carotene.393 Lasel solvent again suggesting a charge-transfer mechanism.370 The flash photolysis has been used to study the triplet quenching of role of P,P-carotene as a quencher of the photo-oxidation of (all-E)-retinal and related polyenes by the stable free radical di- poly(butadiene) by lo2has been evaluated.371 The reaction of t-butylnitro~yl.~~~ Picosecond fluorescence kinetics of (al1-E)- triplet-state retinol with oxygen gave lo2in low yield.-j7’ and its n-butylamine Schiff base396 have been Sulphur radicals that appeared during the retinal-photosensi- determined.Ultraviolet irradiation of these compounds in tized oxidation of glutathione were recorded by spin trapping. acetonitrile led to isomerization and different species were There was no reaction in the absence of oxygen indicating that obtained by laser irradiation in CF3CH(OH)CF3.397 The lo takes part in the reaction.373 A mechanism has been formation of Schiff bases between (92)- (1 1Z)- and (132)- proposed for the destruction of P,P-carotene and the evolution retinals and n-butylamine aniline or piperidine and the of ‘0 in a system containing lactoperoxidase H,O, and protonation of these Schiff bases strongly enhanced the halide ions.37s measured rates of thermal i~omerization.~~~ Two papers report The properties of carotenoids in lipid bilayers or interfaces calculations on the electronic configuration of excited states of are being studied increasingly.The permeability of P,P-protonated retinal Schiff bases.399.J00 The electrochromism of carotene-containing lecithin multibilayers to oxygen has been (all-E)-retinal in viscous systems (polypropylene matrices) has examined.375 The surface properties (surface pressure rersus been studied and model calculations of the properties of its area data) for monolayers of (all-E)-P-cryptoxanthin zea-excited state have been reported.jo’ Adiabatic potentials have xanthin and lutein and their mono- and di-esters either alone been calculated for (2)-(E) isomerization of the C-11-C-12 or in combination with egg lecithin at the air-water interface bond of the protonated retinal Schiff-base chromophore of Fluorescence-excitation profiles of rhodopsin in the first excited state in the presence of an have been disc~ssed.~~~-~~~ (all-E)-P,P-carotene in solution and in phospholipid mixtures external point negative charge.J0’ These calculations have have been obtained.379 The formation and the stability of two since been criticized.J03 The barrier for thermal isomerization kinds of excitonic complexes of lutein and zeaxanthin in of ( I 32)-retinal protonated Schiff-base as in bacteriorhodop- aqueous ethanol have been examined.380 The absorption sin is reduced by charge localization relative to that of retinal spectra of liposomes that contain carotenoids (P,P-carotene or its Schiff base.4o4 Kinetic studies have been reported on the zeaxanthin astaxanthin or apocarotenals) and chlorophyll iodine-catalysed isomerization of the (all-E)- (92)- (1 1Z)- and change with temperature cooling leading to bathochromic (I 3Z)-isomers of retinal,joS on the fluorescence of retinyl shifts.38 The reinforcement of phospholipid bilayers by acetate in non-polar solvents,,06 and on the oxidati~n~~~.~~~ carotenediols acting as rigid rivets has been discussed.38’ and autoxidationso9 of retinyl acetate and of methyl retinoate Energy transfer from P,P-carotene to chlorophyll a was in the solid state.510 NATURAL PRODUCT REPORTS 1985-G.BRITTON 1.6 Photoreceptor Pigments The visual pigments of animals e.g. rhodopsin and the related photoreceptor pigments of Halobacteria especially bacterio- rhodopsin have retinal as their light-absorbing chromophore. They and their photocyles have been investigated very extensively. Many papers which report spectroscopic and other physical studies on the protonated retinal Schiff-base chromo- phores of these pigments have already been cited (n.m.r.,275- 179 resonance Raman and i.r. ~pectroscopy,~~~~~~ 2-335 elec-tronic absorption spectroscopy,’ 8.1 photoacous-297357-357 tic ~pectroscopy,~~ and physical chemistry396-400~402-404 ) as have several reports on the synthesis of model or labelled analogues of retinal and their incorporation into the photo- receptors.103 106.109.1 1 1 I 15.1 19-1 2 I. 1 23.155-1 56,41 1 Several reviews deal with various aspects of the chemistry and properties of these pigments.512 518 Other papers report various aspects of the photochemistry of the chromophore of these photoreceptorsJ I -525 and the preparation properties and photochemistry of model pigments that were prepared from isomers and analogues of retinal.525-541The photo- chemistry of two rhodopsin-like pigments in two bacterio- rhodopsin-free mutants of Halobacterium halobium has been described.js‘ Neutron-diffraction investigations have shown that fully deuteriated retinal that has been incorporated into bacteriorhodopsin is located in the centre of the 1.7 Biosynthesis and Metabolism Several general reviews on the biosynthesis of carotenoids have been p~blished.~~~-~~~ Some of the books and review articles that are cited in the Introduction (Section 1.1) include sections on this topic.l.‘.s*q*lo Other reviews discuss the enzymology of the biosynthesis of carotenoids in chromoplasts of Capsicum annuumJS8and the effects of bioregulators on the biosynthesis of carotenoids.sJ‘) Aspects of the role of cis-isomers of intermediates in the biosynthesis of carotenoids have been discussed.From assign- ments of the stereochemistry of isomers of phytoene (46) phytofluene (47) <-carotene (48) neurosporene (49) and lycopene (35) that had been isolated from a tangerine-fleshed strain of tomato a scheme was proposed for the biosynthesis of the (7Z,9Z,7’Z,9’Z)-isomer prolycopene (45),43.55 suggesting that in the conversion of phytoene into phytofluene the isomerization to (9’23 is simultaneous with the introduction of the (1 1’E)-double-bond and the C-15-C-15’ and C-9-C-10 double-bonds then undergo simultaneous isomerization (to E and Z respectively) as <-carotene is formed from phytofluene.The C-7-C-8 and C-7’-C-8’ double-bonds are then introduced with cis geometry. The discovery of the (5Z)-isomer of neurosporene has prompted further discussion of the possible role of such (5Z)-isomers as precursors of cyclic carotenoids.55 When the purple photosynthetic bacteria Rhodopseudomonas sphueroides Rho~lopse udomonus gelutinosa and R hodom ic ro- biuni runnielii were grown in the presence of nicotine so that biosynthetic intermediates accumulated and were then incu- bated in ’H20 after removal of the inhibitor the normal main carotenoids that were produced were shown (by mass spec- trometry) to have incorporated ‘H specifically into the 2- position.This gave direct proof of the biosynthetic conversion of lycopene into P,P-carotene and rhodopin [1,2-dihydro-$,$-caroten- 1-01] (457) in Rm. cannielii of neurosporene into spheroidene (454) in Rps. sphaeroides and of spheroidene into hydroxyspheroidene [l’-methoxy-3’,4’-didehydro-1,2,7,8,1’,2’-hexahydro-$,$-caroten- 1-01] (458) in Rps. gelatinosa and confirmed that the hydroxyl group at C-l in these compounds is introduced by hydration of the C- 1-C-2 double-b~nd.~~~ The mechanism of formation of the C37 carotenoid peridinin (453) has been st~died.~~~-~~~ [ ITIZeaxanthin (39) was incorporated efficiently into neoxanthin [5’,6’-epoxy-6,7-dide- hydro-5,6,5’,6’-tetrahydro-P,P-carotene-3,5,3’-triol] (461) di-adinoxanthin [5,6-epoxy-7’,8’-didehydro-5,6-dihydro-P,P-caro-tene-3,3’-diol] (462) and peridinin by a cell-free system from Amphidinium carterae and an efficient conversion of neo-xanthin into peridinin and diadinoxanthin was also demon- strated.It was concluded that peridinin is made by extrusion of a C3 fragment from the C, carotenoid skeleton and that the acetylenic end-group of diadinoxanthin is formed from the allenic end-group of neoxanthin and not the reverse. Incorpor- ation studies with (5R)-[5-3H,]- and (5S)-[5-3H,]-mevalonate in Calendula oficinalis have shown that the hydroxyl groups are introduced into both the p ring and the E ring of lutein (21) by direct stereospecific replacement of hydrogen at the 3-positions of each ringJs3 Work continues on the biosynthesis of carotenoids in cell- free systems but progress is slow.Phytoene synthetase and phytoene desaturase activities have been reported in spinach chloroplast envelope preparations and were not detected in thylakoid~.~~~ Other workers however have concluded that the biosynthesis of carotenoids takes place entirely in the thylakoids of radish chloroplasts.555 Enzyme activity for the synthesis of phytoene from prephytoene diphosphate has been located exclusively within the plastids of leaves of Triticum sativum and the fruits of Capsicum ann~um.‘~~ Inhibitor studies showed that the phytoene synthetase is not synthesized on the 70s ribosome.Cell extracts of wild-type and mutant strains of Phycomyces blakesleeanus incorporated mevalonate into P,P-~arotene.~~~ Oxygen was necessary for desaturation and the synthesis of P,P-carotene was stimulated by retinol. The biosynthesis of carotenoids from isopentenyl diphosphate has been demonstrated with isolated-membranes of Micrococcus I~teus.~~~ Many papers report work on the effects of herbicides and other inhibitory compounds on the desaturation of phytoene in plants algae fungi and cell-free systems,Js9-J68 or inhibitory effects of nicotine CPTA or Flurenol on the cyclization of carotenoids.56” It is noteworthy that in most of this work a stimulatory effect was seen on the biosynthesis of carotenoids overall in addition to the inhibition of the particular enzyme reactions.An unusual effect was seen with methyl jasmonate which in the tomato inhibited the synthesis of lycopene but stimulated that of P,P-car~tene.~~~ In the cyanobacterium Cyanophora parado.ua the protein-synthesis inhibitors cyclo- heximide and chloramphenicol had a differential effect on the biosynthesis of carotenes and xanthophyll~.~~~ Several investigations on the regulation of the biosynthesis of carotenoids in fungi have been reported. The regulatory interactions of trisporic acid and inhibitory and stimulatory proteins in Blakeslea trispora have been studiedJT5 and the stimulation of the formation of carotenoids by phosphate has been rep~rted.~’~.~’~ Stimulatory effects of cyclic AMP on the biosynthesis of carotenoids in Neurospora crussu have been de~cribed~’~,~~~ and new findings on the genetic regulation and on the regulation by light of carotenogenesis in Phjcomjces blakesleeanus have been reported.580,58 Changes in the biosynthesis of carotenoids in Rhodopseudomonas aciriophila that were caused by variations in growth conditions have been exarnined.l8 The metabolism of carotenoids in invertebrate animals and fish is reported in several publications.Pathways have been suggested for the conversion of P,P-carotene into astaxanthin (449) in the crustaceans Diaptomus castor and Eudiaptomus ambljod~n~*~ and the lobster Homarus americanus.18J A comparative metabolic study of carotenoids with 2-0x0 and 2-hydroxy end-groups in different insect species has been reported.J85 Feeding studies have revealed that zeaxanthin is metabolized to rhodoxanthin [4’,5’-didehydro-4,5’-retro-fi,fi-carotene-3,3’-dione] (463) in the fish Tilupiu niloticu.J8h The metabolism of carotenoids to vitamin A in fish has also been investigated.Thus the conversion of lutein (21) uia anhydro-lutein [3’,4’-didehydro-P,P-caroten-3-01] (1 53) into vitamin A2 [3,4-didehydroretinol] (464) and 3-hydroxyretinol(465) in some freshwater fish has been demonstrated.487 These compounds can also be obtained from P-cryptoxanthin (38) in fish that use (463) (4651 (471) vitamin A2 whereas those that use vitamin Al (retinol) can derive this from P-~ryptoxanthin.~~~ The metabolism of vitamin A and other retinoids is the subject of a and of chapters in a book.Id le In an investigation of the biliary metabolites of (all-E)-retinoic acid in the rat the glucuronide and retinotaurine were identified by h.p.l.~.~~O (All-E)-retinoic acid glucuronide and other trans-compounds were the major metabolites of (13Z)-retinoic acid in the rat.491 High-performance liquid chromatography was used to identify metabolites of (all-E)-4,4-difluororetinyl acetate (466) as the corresponding palmitate and other esters together with 4-oxoretinol (467) and 4-oxoretinoic acid (468).492 It now seems clear that the a-irone (56) and the y-irone (58) are formed by oxidative degradation of the C3] precursors iripallidol(61) and iriflorental(64) in rhizomes of species of the genus Iris.53-55 Cyclization of the intermediate iridogermanals (65) and (66) to give the irone ring structure is initiated by the incoming methyl group at C-2. The origin of p-irone has not been determined. A book on plant growth substances includes a chapter on the biosynthesis of abscisic acid (78).493 More details of the biosynthesis of abscisic acid in the fungus Cercospora rosicola NATURAL PRODUCT REPORTS 1985 CHzOH (464 (469) X = 0 (470) X = H OH I ?H I (472) phaseic acid (472).500 After separation of the (R)-and (S)-isomers of abscisic acid by h.p.l.c.262 or by a stereoselective antibody technique,263 it has been shown that the two isomers are metabolized by plants at different rates.2 Polyterpenoids and Quinones 2.1 Polyterpenoids Two reviews have been published on the structures and biosynthesis of polyisopren~ids.~~~ qSo2 The long-chain poly- prenols from several plant sources have been described. Those from Ginkgo biloba were of the betulaprenol type (473) having an average of fourteen to twenty isoprenoid residues with the seventeen- eighteen- and nineteen-residue homologues pre- dominating. The stereochemistry of the isoprene residues was determined by IH and I3C n.m.r. spectroscopy as o-trans-trans-(cis),-~is-OH.5~~ Similar compounds (474) with an average of fifteen to eighteen isoprene residues were obtained from six species within the Pina~eae.~O~ Soya beans (Glycine max) yielded a series of polyprenols with fifteen to 22 isoprene residues.505 These were of the dolichol type with the a-residue saturated and had the overall structure o-trans-trans-(cis),- a(saturated)-OH (476).The CS5 polyprenol from leaves of have been elucidated and the subject has been revie~ed.~~~,~~~ Quercus ilex that was suffering from infection by Microsphaera The immediate precursor 6-deoxyabscisic acid (469) and abscisic acid itself were produced in high yield from (7E,9Z)-cr- ionylideneethanol (47 1) and (7E,9Z)-a-ionylideneaceticacid (354). The (7E,9E)-isomers of these precursors were converted into 6-deoxyabscisic acid but not into abscisic acid.The corresponding P-ionylidene derivatives were not converted.496 The enantioselective oxidation of (354) to the (3,6-trans)-3- hydroxy-derivative (470) and the 0x0-derivative (469) has been dem~nstrated.~~’ Examination of ,C-’ 3C coupling in the n.m.r. spectrum of abscisic acid that had been biosynthesized from [1,2-l 3C2]acetate in Cercosporu rosicola has shown that it is the pro-R methyl substituent at C-1 that arises via C-3’ of meval~nate.~~~ This result is compatible with the same chair- folding stereochemistry of cyclization as has been established for the C40 carotenoids. The biosynthesis of abscisic acid in Cercosporu rosicola is inhibited apparently specifically by ~ytokinins.~~~ In leaves of Xanthium strumarium it has been shown that one I8O atom from I8O2is incorporated into the carboxyl group of abscisic acid and into the hydroxyl group of alphitoides was shown to be identical to ficaprenol-11 [o-(trans),-(cis),-&OH] (475; m = 7).Related C45 C50 and c60 polyprenols with the usual unsaturation of the isoprenoid residues were also pre~ent.~~~.~~~ The mushroom Gyrnnopilus spectabilis has been investigated extensively and a series of new polyhydroxylated polyprenols has been characterized. The structures of gymnoprenols A and B were elucidated as (477) and (478) respectively,508 and the (2S,3R) stereochemistry of the 1,2,3-triol moiety was deduced by correlation of the y-lactone (479) which was obtained as a degradation product of the natural compounds with the (2R,3S)-y-lactone that was obtained by stereocontrolled synthesis.509 An even greater degree of hydroxylation was established for gymnoprenols D and E [(480) and (481)].510 Another compound gymnopilin from the same source was identified as the P-hydroxy-P- methylglutaryl ester of gymnoprenol A.5 Two homologues of this have now been found i.e.gymnopilins A and A, [(482) and (483)] together with gymnopilin B, (484) which is an NATURAL PRODUCT REPORTS 1985-G. BRITTON (473) /I = 3 m = 11 -17 (474)n = 3 m = 12 -15 (475) n = 4 m= 3 -7 (485) ester of gymnoprenol B.512 A new tetraterpene alcohol from Elodea canadensis was shown to have the unsymmetrical C25- C1 structure (485).51 In addition to the CZ0 CZ0diether 2,3-di- 0-phytanyl-sn-glycerol (486) that is considered to be a characteristic membrane lipid of haloalkaliphilic archaebac- teria the C2s C20diether 2-0-sesterterpanyl-3-0-phytanyl-sn-glycerol (487) and the C25 C25 diether 2,3-di-O-sesterterpanyl-l4l5I5 sn-glycerol (488) have been rep~rted.~ In the archaebacterium Sulfolobussolfataricus (syn.Caldariella acido- phila) the major lipids are known to be 72-membered macrocyclic ethers e.g. (489) which are derived by ‘head-to- head’ linkage of the phytanyl chains of two molecules of 2,3-di- 0-phytanyl-sn-glycerol and derivatives of these ethers e.g. (490) in which the C40 chains are folded into cyclopentane rings. Twenty individual molecular species of these lipids have now been separated by h.p.1.c.In addition to the known compounds with two four six and eight cyclopentane rings four new examples (491)-(494) with one three five and seven rings were identified together with their calditol ethers and also the presumed biosynthetic precursor (499 in which only one pair of the C20 chains is linked.516 The properties of these compounds in membranes especially the relationship between the number of rings and the transition temperatures have been investigated.s I (476) n = 3 m= 11 -10 A review has been published of syntheses of polyprenyl compounds with (E) or (2) regio~electivity.~~~ A general method has been described for the stereospecific synthesis of the betulaprenols (473; m = 3-6) in which the all-cis chloro- compound C1[CH2CMe=CHCH2],0CH2Ph was used as the source of the cisoid part of the molecule that was added to the terminal (E)-farnesyl The acetates of the polyprenols (473) from Ginkgo biloba reacted with the (R)or (S)Grignard reagent BrMgCH2CHMeCH2CH20Thp (Thp = tetrahydro-pyran-2-yl) to give the (R)-and (S)-dolichols (476; m = 12-18).520 The [~-(trans)~-(ci~),]-heptaprenol (475; rn = 3) has been synthesized stereospecifically in seven steps from the C20phenyl sulphide (496).After elimination of PhS hydroly- sis amination and further hydrolysis chain-extension was effected by addition of the aldehyde (497; n = 2) to give (498) and thence the heptaprenol.s2* This heptaprenol and a second with different stereochemis try [o-( trans) -(cis)* -trans-OH] (499) have been prepared as synthons for higher polyprenols in many steps from geranyl-linalool (500) or farnesyl bromide (501) via directed aldol condensation of a-lithiated aldimines [e.g.(502)] with protected aldehydes including (497).522 A series of acyclic a,o-bifunctional (2)-and (E)-isoprenoid aldehydes (503) was prepared via the diols (504) which were obtained following ozonolysis of polyis~prene.~~~ (22,62)-8- NATURAL PRODUCT REPORTS 1985 CH20H I CH~OH C H20H I HCO H2CO CHzOH (495) (499) (500) (Benzy1oxy)-1-chloro-2,6-dimethylocta-2,6-diene (505) which is a key C, compound for the construction of cisoid polyprenols was synthesized stereoselectively from nerol (506).524 Procedures for coupling (505) with prenyl p-tolyl sulphone or neryl p-tolyl sulphone to give the (2)-C and (2)-C, prenols (507) and (508) are described to illustrate the synthesis of polyprenols via this reagent.The synthesis of terpenoid compounds by way of Michael addition to conjugat-ed dienyl sulphones has been des~ribed,”~ and illustrated by routes to short-chain prenols. Thus Michael addition of EtOH MeCOCH2C0,But and PhSCH,COMe to the sulphone (509) gave the diallylic sulphones (510) and thence the conjugated terpenoid compounds e.g.(511) and (512). As a model for the synthesis of [o,o-(C H,),]polyisoprenols the la belled geraniol (51 3) and farnesol (514) have been prepared from the terminal aldehydes (515) and the ’H-labelled Wittig reagent.s26 The phosphates (5 I7)-(5 19) of farnesol (5 16) moraprenol (520) and solanesol (521) have been prepareds’7.s28 by the reaction of the terpenols with C1,CCN (to give the trichloroacetami- dates) followed by phosphorylation with H,PO and Et,N.The 3Cn.m.r. spectra of polyisoprenes have been analysed by correlation with those of acyclic terpenes and polyprenols allowing the stereochemistry of the polyene chain to be established as [o-(trans),-(~is),,].~~~ An assignment correlation has been described for the I3C n.m.r. shifts of the methylene carbons that flank transoid (E) double-bonds and epoxide NATURAL PRODUCT REPORTS 1985-G. BRITTON L J0-5 (503) R = CHO; X =o (506) n = 1 (509) n = 1 or 2 (504) R = CHZOH; X =H,OH (507) n = 2 (508) n = 3 0 U O E t 0Et L n (511) (510) n = 1 or 2 R (513) n = 1 (515) n = 1 or 2 (514) n = 2 (518) n = 4,m = 7 R = OPO,(NH,) (519) n = 9 rn = 0,R = OP03(NH,)2 (520)n = 4,m = 7 R = OH (521) n = 9 rn =0 R = OH (522) n = 11 rn = 0 R = diphosphate The functions in seventeen isoprenoid~.~~~spectrometric determination of dolichol and phosphorylated derivatives via the Chugaev colour reaction has been described.531 The role of hydroxymethylglutaryl-CoA reductases in regulating the biosynthesis of dolichols and ubiquinones has been re~iewed.'~' Several papers report experiments which re-examine the stereochemistry of formation of cis-isoprenoid residues in the biosynthesis of long-chain polyprenols.In systems that had previously been studied the formation of an (E)-isoprene unit involved loss of the 4(pro-4S)-hydrogen atom of mevalonate whereas the 4(pro-4R)-hydrogen atom of mevalonate was lost during the formation of a (2)-isoprene unit.In contrast to this it has now been shown that in the biosynthesis of the malloprenols [e.g. (475; m = 5-7)] and related polyprenols the 4(pro-4S)-hydrogen atom of mevalon- (523) the corresponding free or phosphorylated polyprenol in rat liver has been dem~nstrated.~~~.~~~ The kinase from membrane fractions of Dictyostelium discoideum which will phosphorylate a dolichol to dolichyl phosphate has been studied,s4' as has the phosphatase which liberates dolichols from their phosphates in soya beans.50s A method has been described for the efficient enzymic hydrolysis of polyprenyl diphosphates (eg.octa-prenyl) by potato acid phosphatase in aqueous 60% metha- nol.542 Complete hydrolysis was achieved in six hours. 2.2 Isoprenylated Quinones The chemistry and metabolism of vitamins K are surveyed in a general review on these vitamins.5s3 A new menaquinone (524) from Actinomadura madurae has been shown by m.s. and 'H ate is lost in the formation of (2)-isoprene resid~es.~~~-~~~ n.m.r. spectroscopy to be a derivative of menaquinone-9 in Experiments with stereospecifically labelled isopentenyl di- phosphate have shown that the enzymic formation of undeca- prenyl diphosphate (522) by Bacillus subtilis occurs by cis-condensation of isopentenyl diphosphate and with elimination of the 2(pro-2S)-hydrogen atom [ = 4(pro-4S)-hydrogen of meval~nate].~~~ The solubilization and characterization of the polyprenyltransferase that is involved in the biosynthesis of the dolichyl phosphates (523; n = 12-15) have been described.537 Heptaprenyl diphosphate synthase has been separated into two components.s38 The formation of a dolichol its phosphate and its diphosphate by saturation of the a-terminal isoprene unit of which there are three saturated isoprene residues.s54 The closely related octahydro-analogue (525) was identified in Streptomyces ~lbus.~~~ A derivative (526) of menaquinone-6 which has an additional methyl group at either C-5 or C-8 of the benzenoid ring has been isolated from Campylobucter jejuni (and C.fi?tus)546and from Wolinellu succinogenes ('thermoplas-maquinone-6')547 and characterized by m.s.and 'H n.m.r. spectroscopy. The identification of two new closely related isoprenylated quinols compound (527) from the brown alga Cystoseira cae~pitosu~~~ and kombic acid (528) from the seed fat of Pycnanthus k~mbo,~~~ has been reported. The absolute 380 NATURAL PRODUCT REPORTS. 1985 0 (526) R'=Me,R2=H or R'=H,R2=Me OH OH (527) (528) 0 0 OH I OH Meo@Me0\ 1 OMe (529) (530) (531) 0 0 1 (5361 (537) 0 HO H (538) configuration of phylloquinone epoxide (529) has been assigned as (2S,3R) by c.d. correlation with the optically enriched enantiomers that had been obtained by stereoselective synthesis.550 The geometrical configuration (2)or (8,of the phytyl side-chain did not affect the circular dichroism.The preparation and properties of a large number of analogues of the ubiquinones (530) have been described.551 The syntheses involved Claisen rearrangement and reaction with diketene or ethyl orthoacetate and gave a range of compounds in which there were terminal acid amide alcohol and methyl ketone groups in the side-chain. The I-methyl ethers of the ubiquinols (531 ;R = phytyl or geranyl) have been prepared5** by methylating the quinol (532) with Me,SO to give mainly (533) which was then condensed with the isoprenoid side-chain. The preparation of the azido-ubiquin- one derivatives (534) and (535) has been described.553 Studies of the electrochemistry of ubiquinones-1 -3 and -10 [(530; n = l) (530; n = 3) and (530 n = lo)] menaquinones-2 -7 L '8 (539) (540) and -10 [(536; n = 2) (536; n = 7) and (536; n = lo)], and plastoquinones-1 and -9 [(537; n = 1) and (537; n = 9)] and some model compounds in aprotic solvents have been described.554 Of the many procedures that have been described for the separation purification and assay of the main classes of isoprenylated quinones some are particularly noteworthy.A reversed-phase procedure has been described which allows separation of menaquinones with different chain-lengths and degrees of saturation of the side-~hain.~~~ Similar separations have also been achieved by a combination of a reversed-phase column (to separate different chain-lengths) and an Ag+-loaded ion-exchange column (to separate on the basis of the degree of un~aturation).~~" Several procedures for the reversed-phase h.p.1.c.separation of ubiquinones-6 to -10 have been evalu- atedSs7 and a method for separating homologues up to ubiquinone-14 has been reported.558 A reversed-phase h.p.1.c. method for the simultaneous determination of ubiquinones and NATURAL PRODUCT REPORTS 1985-G. BRITTON ubiquinols has been described.559 New electrochemical-fluorometric methods for the detection of vitamins K in h.p.1.c. are substantially more sensitive than the conventional method of detection (by monitoring U.V. absorpti~n).~~~-~~* Electron spin resonance spectra of chromanoxyl and chromenoxyl radicals that were obtained from phylloquinone (538)563 and ubiquinone-1563by oxidizing the phenol precursors with Pb02 in toluene have been recorded and proton hyperfine splittings determined.The thermotropic properties of ubiquinone-10 and its lower homologues have been investigated565 and fluores- cence-probe studies of the distribution of ubiquinones-3 to -10 in bilayers of dipalmitoylphosphatidylcholine have been reported.566 Reviews have been published of the biosynthesis of phylloquinone plastoquinone-9 and other isoprenoids in plants,567-569of menaquinones in bacteria,570 and of ubiquin-ones.57 The incorporation of isopentenyl diphosphate and of ring precursors into ubiquinones in plant mitochondria has been demonstrated. Mevalonic acid 5-diphosphate was not incorporated.572 Isopentenyl diphosphate has also been in- corporated into menaquinones by isolated membranes of Micrococcus luteu~.~~~ The incorporation of the ring precursor (4-hydroxypheny1)pyruvic acid into prenylated quinones by spinach chloroplast stroma has been Two compon- ents of the membrane-bound enzyme system of Escherichiu coli for hydroxylation of the ubiquinone-8 precursor 2-octaprenyl- phenol (539) have been identified.These are a membranous component (cytochrome o) and a cytoplasmic component (a NADPH-cytoc hrome-c reduc tase). The 0-me thy lation of 5-hexaprenyl-3,4-dihydroxybenzoicacid (540) has been identi- fied as a regulated step in the biosynthesis of ubiquinone-6 in Succharomyces ~erevisiae.~~ The enzyme that prenylates menadione to menaquinone-4 has been isolated from the microsomal fraction of rat and chicken livers.576 In studies of the stimulation of the enzyme (from rat liver) which prenylates 4-hydroxybenzoate evidence has been obtained for a cytosolic protein that transports polyprenyl dipho~phate.~~~ 3 References 1 T.W. Goodwin ‘Biochemistry of the Carotenoids Volume 2 Animals’ Chapman and Hall London and New York 1984. 2 G. Britton ‘The Biochemistry of Natural Pigments’ Cambridge University Press 1983. 3 0. Isler and F. Kienzle Kirk-Othmer Encycl. Chem. Technol. 3rd Edn. 1983 24 140. 4 K. Tsukida Bitamin 1984 58 185. 5 J. Garrido-Fernandez and M. I. Minguez Mosquera Grasas Aceites (Seville) 1983 34 339. 6 M. Ruddat and E.D. Garber Mycol. Ser. 1983 5 95. 7 D. L. Fox in ‘Mollusca’ ed. P. W. 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ISSN:0265-0568    

DOI:10.1039/NP9850200349

出版商:RSC    

年代:1985

数据来源: RSC

摘要:

Book Review Metabolites and Metabolism -A Commentary on Secondary Metabolism E. Haslam; 1985; Clarendon Press Oxford; x + 161 pp; f15-00; ISBN 0-19-855377-3 Biosynthetic relationships (both proven and hypothetical) are recognized as keys to the comprehension of the types and varieties of natural products. Indeed biosynthesis has become central to the teaching of natural product chemistry as is reflected in the number of excellent books published in recent years. At first glance ‘Metabolites and Metabolism’ might be taken for another work in the same vein. However Professor Haslam’s aim proves to be more original than this; he seeks to review knowledge of secondary metabolism to discuss its relationship with primary metabolism and its role in the overall biology and biochemistry of the organism.It is acknowledged that such a discussion cannot be conclusive at present. However a review of the pertinent questions and a critique of the answers that have been offered is appropriate and stimulating. Two major strands can be discerned in this work. The first is an account of essential biosynthetic knowledge which does not set out to be detailed or comprehensive. Rather historical development is noted the major routes of the pathway are mapped and signs leading to its byways are set up. A few representative cases and some unresolved questions are presented. The second strand is a discussion of the general characteristics of secondary metabolism -its relationship with growth and development regulation enzymology etc.and of its importance in the overall functioning of an organism. The book -fairly slim (161 pp.) reflecting the generally concentrated and minimal treatment and the author’s resolve not to fill it out with biosynthetic detail -is organized into seven chapters. The introduction (16 pp.) opens with an exposition of the difficulties of the definition of secondary metabolism. More space might have been devoted to this problem clouded as it is by historical patterns of discovery as well as by ignorance of the biological functions of secondary metabolites. A view of the archetypal natural product as one of restricted occurrence in Nature i.e. the specialized response of a few species or genera in metabolic evolution is easier to defend than one based on importance of function.Perhaps ‘special’ metabolism might be a better term than ‘secondary’ metabolism (and ‘general’ rather than ‘primary’ metabolism)? The rise and significance of biogenetic theory is described and attention is called to the fragmentary state of knowledge of the enzymology of secondary metabolism. The methodology of biosynthetic investigation is outlined with comments on some pitfalls of interpretation. It may be observed that the text gives precedence to the wider issues of the subject and in doing so excludes some details valuable to e.g. undergraduates. Thus those inescapable terms ‘incorporation’ and ‘dilution’ are not defined; the range of techniques mentioned is not wide and the inexperienced reader is not given a clear idea when a particular method is appropriate.Polyketides are the first chemical category to be described (21 pp.). This group illustrates perfectly the remarkable number of variations when b-polyketomethylene chains are transformed through Claisen and aldol reactions with modifi- cation through alkylation and redox processes. Little mechanis- tic rationalization is offered. Some specific examples are dealt with and some unanswered problems brought out. The chapter concludes with brief mention of biomimetic synthesis; several examples are given but surprisingly not those which might have a bearing on chemical modes of control of polyketide cyclization. The chief justification of the inclusion of biomime- tic synthesis in a book such as this would seem to be the light which may thus be shed on the nature of regio- and stereo- chemical control of biological reactions i.e.‘chemical’ versus ‘enzymic’ regulation. This theme does not emerge strongly. The longest section (36 pp.) is devoted to alkaloids perhaps in recognition of the role that speculation on their origins played in the development of biogenetic ideas. The narrow range is delineated of reactions -amino-acid transformations the Mannich reaction and acetoacetate Schiff’s base and redox chemistry -which are needed to explain alkaloid biosynthesis. Each major class is mentioned and the theme of “Nature’s impressive economy” in primary metabolism in contrast to “Nature’s unrestrained prodigality” in secondary metabolism is well brought out in discussion of the metabo- lites.A short outline of the secondary metabolism of peptides is included. Only a short chapter (12 pp.) is devoted to plant phenolics; the major groups are pointed out and the importance is emphasized of phenylalanine and tyrosine ammonia-lyases in the regulation of access to cinnamate-based compounds. Here as elsewhere some acquaintance with elementary biochemistry is assumed with ‘ATP’ ‘NADPH’ ‘CoASH’ etc. appearing unexplained. The account of natural products is concluded with a section (31 pp.) on terpenoids and steroids. The difficulties of classifying metabolites as primary or secondary becomes particularly acute with some terpenes e.g. those involved in regulation communication and inter-species interactions.Structural diversity is illustrated here by reference to trypto- phan-derived mycotoxins a slightly curious choice since other tryptophan-terpene condensation products are included as alkaloids. A broad historical perspective is well conveyed -the book overall gives a valuable feeling for the development of ideas in biogenetic theory -and the well-known essentials of the mevalonic acid pathway are summarized. The importance (at least theoretically) of carbo-cation chemistry in structural elaborations (“Nature’s embroidery”) is given due weight. Each terpenoid class is briefly surveyed and some experimental work discussed for a few cases e.g. Overton’s work on y-bisabolene and Arigoni’s investigation of avocettin.With a general survey of specific groups completed the author then turns to the most valuable and original section a discussion (31 pp.) of the general characteristics of secondary metabolism surveyed in the context of its relationship with primary metabolism. Thus the formation of secondary metabo- lites at differing growth phases of an organism is discussed especially for micro-organisms and for tissue culture as well as the manipulations of nutrition and environment that may induce the formation of natural products. The major hypoth- eses which have been advanced to explain the role of secondary metabolism are then reviewed the ‘overflow’ theory which proposes that secondary metabolites arise during a phase of unbalanced growth the process being more important than the compounds; the ‘detoxification’ idea i.e.that natural products arise from the removal of surplus intermediates; the ‘waste- product’ theory; the ‘back-up’ theory suggesting that secon- dary metabolism supports primary when normal substrates are not available; and the ‘survival value’ notion i.e. that secondary metabolites play a part in inter-species relationships e.g. the insect-host balance and thus help individual species to survive in their own environmental niche. In this last context the physiological activity of many natural products is striking as is the parallel between the specific and individual nature of the natural products formed by a species and the specific and individual nature of its environmental niche.The ecological view has been popular but some reasons for caution in ready acceptance of it are adduced here. Questions of storage and toxicity of secondary metabolites are dealt with and the crucial question of the enzymology of secondary metabolism is discussed at some length; the very limited knowledge of enzymes in this area and the nature of their specificity and regulation is brought out. The final chapter (6 pp.) gives a short defence of the ‘overflow’ hypothesis arguing that it is a plausible alternative to the ecological idea in certain cases. It would not be impossible that a given metabolite or group of metabolites within a pathway has both ‘overflow’ and ‘survival value’ roles. A few errors are apparent in the book but they are minor.NATURAL PRODUCT REPORTS 1985 Since the discussions are deliberately brief and not detailed a good reading list seems appropriate and I would have liked to have seen more (and more up-to-date) references given and more guidance to good sources of detailed exposition for the less experienced. Overall Professor Haslam has produced a valuable and original discussion of secondary metabolism and its function. All those involved in biosynthetic studies will find it profitable and this book would be ideal reading for chemists biochemists and biologists who seek a stimulating overview of the area. So much work has been done on structure and on biosynthetic pathways that the need can now be seen for attention to the regulation of the metabolic links to primary processes and to biological function. This book will surely help to provoke interest and work in these neglected areas. D. A. Whiting

ISSN:0265-0568    

DOI:10.1039/NP9850200389

出版商:RSC    

年代:1985

数据来源: RSC

摘要:

Erratum In issue 3 of this volume references 17 to 25 of the article by D. J. Robins were omitted from page 219. These should have read 17 M. F. Mackay M. Sadek and C. C. J. Culvenor Acta Crystallogr. Sect. C 1984 40,470. 18 K. Narasaka T. Sakakura T. Uchimaru an2 D. Guedin-Vuong J. Am. Chem. Soc. 1984 106 2954. 19 S. Mohanraj P. S. Subramanian and W. Herz Phytochemistry 1982 21 1775. 20 H. J. Huizing E. C. Pfauth T. M. Malingre and J. H. Sietsma Plant Cell Tissue Organ Culture 1983 2 227. 21 J. N. Roitman Aust. J. Chem. 1983 36 1203. 22 H. Riieger and M. H. Benn Can. J. Chem. 1983 61 2526. 23 Y. Asada and T. Furuya Chem. Pharm. Bull. 1984 32 475. 24 E. Roder H. Wiedenfeld and A. Hoenig Planra Med. 1983,49,57. 25 A. M. Rizk F. M. Hammouda S. I. Ismail H. A. Ghaleb M. K. Madkour A. E. Pohland and G. Wood Fitoterapia 1983,54 115.

ISSN:0265-0568    

DOI:10.1039/NP9850200391

出版商:RSC    

年代:1985

数据来源: RSC