摘要:

Natural Product Reports Editorial Board Professor T. J. Simpson (Chairman) Dr J. R. Hanson Dr R. 6. Herbert Professor J. Mann Professor D. J. Robins Dr C. J. Schofield Dr D. A. Whiting Editorial Staff Dr Sheila R. Buxton Managing Editor Dr Roxane M. Owen Deputy Editor Miss Nicola P. Coward Production Editor Dr Anthony f? Breen Mr Michael J. Francis Tech n ica I Editors Mrs Mandy Scripps Miss Karen White Editorial Secretaries University of Bristol U n iversi ty of Sussex University of Leeds University of Reading University of Glasgow University of Oxford University of Nottingham Editorial Office The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cam bridge UK CB4 4WF Telephone +44 (0)1223 420066 Facsimile +44 (0) 1223 420247 E-mail (Internet) rscl@rsc.org RSC Server http://c h em ist ry.rsc.0 rg/rsc/ Natural Product Reports is a bimonthly journal of critical reviews.It aims to foster progress in the study of bioorganic chemistry by providing regular and comprehensive reviews of the relevant literature published during well-defined periods. Topics include the isolation structure biosynthesis biological activity and chemistry of the major groups of natural products -alkaloids terpenoids and steroids aliphatic aromatic and 0-heterocyclic compounds. This is augmented by frequent reviews of the wider context of bioorganic chemistry including developments in enzymology nucleic acids genetics chemical ecology primary and secondary metabolism and isolation and analytical techniques which will be of general interest to all workers in the area.Articles in Natural Product Reports are commissioned by members of the Editorial Board or accepted by the Chairman for consideration at meetings of the Board. Natural Product Reports (ISSN 0265-0568) is published bimonthly by The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge UK CB4 4WF. 1996 Annual Subscription Price EEA €325.00 USA $615.00 Rest of World f333.00.Change of address and orders with payment in advance to The Royal Society of Chemistry The Distribution Centre Blackhorse Road Letchworth Herts. UK SG6 IHN. Air Freight and mailing in the USA by Publications Expediting Service Inc.200 Meacham Avenue Elmont NY 11003. US 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 UK are by Bulk Airmail within Europe and Accelerated Surface Post outside Europe. Printed in the UK. Members of the Royal Society of Chemistry should order the journal from The Membership Manager The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge UK CB4 4WF. 0 The Royal Society of Chemistry 1996 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. Printed in Great Britain by the University Press Cambridge Subscription rates for 1996 EEA f325.00 USA $615.00 Rest of World f333.00

ISSN:0265-0568    

DOI:10.1039/NP99613FX001

出版商:RSC    

年代:1996

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP99613BX003

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Natural Product Reports Editorial Board Professor T. J. Simpson (Chairman) Dr J. R. Hanson Dr R. B. Herbert Professor J. Mann Professor D. J. Robins Dr C. J. Schofield Dr D. A. Whiting Editorial Staff Dr Sheila R. Buxton Managing Editor Dr Roxane M. Owen Deputy Ed itor Miss Nicola P. Coward Production Editor Dr Carmel M. McNamara Technical Editor Ms Dawn J. Webb Miss Karen L. White Editorial Secretaries Editorial Office University of Bristol University of Sussex University of Leeds University of Reading University of Glasgow University of Oxford University of Nottingham The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge UK CB4 4WF Telephone +44 (0)1223 420066 Facsimile +44 (0) 1223 420247 E-mail perkin@ rsc.org RSC Server http://c hem istry. rsc.org/rsc/ Natural Product Reports is a bimonthly journal of critical reviews. It aims to foster progress in the study of bioorganic chemistry by providing regular and comprehensive reviews of the relevant literature published during well-defined periods. Topics include the isolation structure biosynthesis biological activity and chemistry of the major groups of natural products -alkaloids terpenoids and steroids aliphatic aromatic and 0-heterocyclic compounds. This is augmented by frequent reviews of the wider context of bioorganic chemistry incl ud ing developments in enzymology n ucleic acids genetics c hem ica I ecology primary and secondary metabolism and isolation and analytical techniques which will be of general interest to all workers in the area.Articles in Natural Product Reports are commissioned by members of the Editorial Board or accepted by the Chairman for consideration at meetings of the Board. Natural Product Reports (ISSN 0265-0568) is published bimonthly by The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge UK CB4 4WF. 1997 Annual subscription rate f355.00; US$640.00. Customers in Canada will be charged the sterling price plus a surcharge to cover GST. Change of address and orders with payment in advance to The Royal Society of Chemistry The Distribution Centre Blackhorse Road Letchworth Herts. UK SG6 IHN. Air Freight and mailing in the USA by Publications Expediting Service Inc.200 Meacham Avenue Elmont NY 11003. US 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 UK are by Bulk Airmail within Europe and Accelerated Surface Post outside Europe. Printed in the UK. Members of the Royal Society of Chemistry should order the journal from The Membership Manager The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge UK CB4 4WF. 0 The Royal Society of Chemistry 1997 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. Printed in Great Britain by the University Press Cambridge

ISSN:0265-0568    

DOI:10.1039/NP99613FX025

出版商:RSC    

年代:1996

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP99613BX027

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

The DAP Pathway to Lysine as a Target for Antimicrobial Agents Russell J. Cox School of Chemistry University of Bristol Cantock's Close Bristol BS8 1TS UK Reviewing the literature published up to September 1995 1 Introduction The Diaminopimelate Pathway to L-Lysine 1.1 Enzymes of the Diaminopimelate Pathway 2.0 L-Dihydrodipicolinate Synthase EC 4.2.1.52 dapA 2.1 L-Dihydrodipicolinate Reductase EC 1.3.1.26 dapB 2.2 Succinyl CoA L-Tetrahydrodipicolinate N-Succinyl- transferase dapD 2.3 N-Succinyl-LL-DAP Aminotransferase EC 2.6.1.17 dapC 2.4 N-Succinyl-LL-DAP Desuccinylase EC 3.5.1.18 dapE 2.5 meso-DAP Epimerase EC 5.1.1.7 dapF 2.6 meso-DAP Dehydrogenase EC 1.4.1.16 ddh 2.7 meso-DAP Decarboxylase EC 4.1.1.20 lysA 3 Antibiotic Properties of DAP Pathway Inhibitors 4 Overview 5 References 1 Introduction The Diaminopimelate Pathway to L-Lysine The enzymes which catalyse the synthesis of L-lysine in plants and bacteria have attracted'interest from two directions; firstly from those interested in inhibiting lysine biosynthesis as a (1a) R' strategy for the development of novel antibiotic or herbicidal n compounds (vide infra);' and secondly from those wishing to enhance lysine yields in over-producing organisms.2-4 Mammals are deficient in lysine biosynthesis and thus require a dietary source of this amino acid for protein ~ynthesis.~ In plants and bacteria lysine is synthesized de novo and is utilized for protein biosynthesis.Crucially however most bacteria also require either lysine or its biosynthetic precursor diaminopimelate (DAP),6 as a component of the peptidoglycan (la) layer of the cell wall. This polymeric compound consists of chains of alternating N-acetylglucosamine and N-acetylmuramic acid cross linked by short pep tide^.^ For almost all bacteria the cross links are formed by diamines; either lysine or DAP.8-10 The cross links give the cell wall the strength to resist lysis caused by high intracellular osmotic pressures.ll Inhibitors of lysine or DAP biosynthesis could therefore be very effective antibiotics like other successful drugs targeted towards cell wall bio- synthesis such as p-lactams and g1ycopeptides.l. l2 The absence of the DAP pathway in mammals offers the potential for the development of novel antibiotics with low mammalian toxici- ties.Peptidoglycan monomers such as the potent cytotoxin (1 b) from Bordetella pertussis and Neisseria g~norrhoeae,'~ and related DAP-containing peptides possess a range of biological effects including cytoto~icity,~~ antitumour activities15 and angio tensin converting enzyme (ACE) inhibition. l6 The investigations into lysine biosynthesis have classically focused on the enzymes themselves their properties mech- anisms and modes of reg~1ation.l~ More recently much more information has come from genetic studies. This has allowed (7a,b) comparisons of protein sequences from diverse organisms and has ultimately resulted in cloning of the biosynthetic genes and their over-expression.The availability of lysine biosynthetic proteins in substantial quantities means that the enzymes are becoming amenable to more widespread study. It is the intention of this review to highlight opportunities and stimulate AcHN O4 HNY O*NH &02H A' = D-ala-mDAP-oglu-L-ala-(MurNAc-GlcNAc),; R2= D-ala; = polymer (1b) R' = H; R2 = OH; n = monomer efforts for the further study of enzyme mechanism and inhibition in this area. The later steps of peptidoglycan biosynthesis have received attention e1sewhere.l' l8 1.1 Enzymes of the Diaminopimelate Pathway The early steps of the pathway (reduction of L-aspartate to L-aspartate semialdehyde) are common with methionine and threonine biosynthesis and will not be dealt with here." The first step unique to lysine biosynthesis (Scheme 1) is the condensation of pyruvate (2) with L-aspartate semialdehyde (L- ASA) (3) to form ~-2,3-dihydrodipicolinate(L-DHDP) (4) catalysed by L-DHDP synthase.20 The heterocyclic product is reduced by L-DHDP reductase to form ~-2,3,4,5-tetra-hydrodipicolinate (L-THDP) (5).At this point the pathway splits and two main routes have been identified in different bacterial species.21 The longer of the two routes proceeds via acylation of L-THDP (3,yielding acyl-blocked cc-amino-e- ketopimelate (6a,b). In many bacterial species including Escherichia coli the blocking group is succinate while in many species of Bacillus acetate is utilized.8 The e-keto group thus freed is subject to transamination by a pyridoxal phosphate (PLP) dependent aminotransferase utilizing glutamate as the amino-donor.2z Removal23 of the blocking acyl-group from then affords LL-DAP (8a) which is subsequently epimerized to meso-DAP (8b).24 In the shorter pathway rneso-DAP (8b) is produced directly from L-THDP (5) by meso-DAP dehydrogenase; this enzyme is less widespread but is found in Bacillus sphaericus for example.Dual pathways are present in some bacterial strains ;for example both the succinyl-blocked and dehydrogenase pathways are presentz5 and operative26 in 29 NATURAL PRODUCT REPORTS 1996 mco2H-H02C'0 ;3CO2H NH2 NH2 H2NOICO*H H02CQCO2" (2)pyruvate Ii (3) L-ASA (9) L-Lysine viii (3) L-ASA (4) L-DHDP 02pH 7.41 t H02C 0C02H H02CQC02H HOzCY-+7C02H (104 NH2 NH2 (4) L-DHDP (8b) meso.DAP NAD(P)H I J,ii vii vi n H02C' (5) L-THDP (8a) LL-DAP RSCoA t ROH V CoASH 1iii 1 L-glU a-kg JLL.H02GWCO2H Ho2C~Cu2H iv 0 NHR NH2 NHR (6a) R = COCH2CH2C02H (7a) R = COCH2CH2C02H (6b) R=COMe (7b)R=COMe The DAP pathway to L-lysine. Enzymes and corresponding genetic loci i Dihydrodipicolinate Synthase EC 4.2.1.52 dupA ;ii Dihydro- dipicolinate Reductase EC 1.3.1.26 dupB; iii Acyl CoA Tetra-hydrodipicolinate N-Acyltransferase dapD ; iv N-Ac~I-LL-DAP Aminotransferase EC 2.6.1.17 dupC; v N-ACYLLL-DAP Deacylase EC 3.5.1.18 dupE; vi meso-DAP Epimerase EC 5.1.1.7 dupF; vii meso-DAP Dehydrogenase EC 1.4.1.16 ddh; viii meso-DAP De- carboxylase EC 4.1.1.20 lysA Scheme 1 the industrially important lysine producer Corynebacterium glutamicum.In the last step of the pathway L-lysine (9) is synthesized directly from meso-DAP (8b) by decarboxylation catalysed by the PLP dependent meso-DAP decarb~xylase.~' The situation in plants is less clear. Both L-DHDP synthase and L-DHDP reductase have been obtained from plant extracts as have meso-DAP epimerase and meso-DAP decarboxylase. The presence of enzymes from the middle section of the pathway such as meso-DAP dehydrogenase,28 L-THDP acyl- transferase or LL-N-acyl-DAP deacylase is more contro-~ersial.~~ This has led to the suggestion that direct trans-amination of L-THDP (5) may be occurring to afford LL-DAP @a) though definitive proof is lacking.30 Evidence suggests that lysine biosynthesis in plants may occur largely or entirely within ~hloroplasts.~~ This is in accord with current evidence supporting a bacterial origin for these organelles.32 2 L-Dihydrodipicolinate Synthase EC 4.2.1.52 dapA L-Dihydrodipicolinate (L-DHDP) synthases have been isolated and purified to homogeneity from many source^,^^-^^ and the dapA locus has been cloned from both plants36 and bacteria.".38 The enzyme is a homotetramer in the majority of cases with a subunit M of between 28000 and 35000. The pea (Pisurn sativum) enzyme is reported to be a homotrimer of subunit M Scheme 2 43OO0.39Sequence analysis has revealed 30 YOidentity between the wheat (Triticum aestivum) and E. coli enzymes,*O and 33 O/O identity between E.coli and Brevibacterium lactofermenturn enzyme^.^' Significant homology exists with another pyruvate binding enzyme the B. lactofermenturn enzyme shares 29 Yo identity with E. coli N-acetylneuraminate lyase.*l Three assays have been reported for L-DHDP ~ynthase.~~ Observation of the characteristic absorbance of an adduct between the reaction product and o-aminobenzaldehyde at 540 nm has been used as has a coupled assay utilizing the NAD(P)H dependent L-DHDP reductase (Section 2.1). The most widely used assay relies on rapid spontaneous oxidation at pH 7.4 of the product L-DHUP (4) to dipicolinate (IOa) which can be detected spectrophotometrically at 270 nm (Scheme 2). This procedure is suitable for detailed kinetic analysis because the synthesis of highly pure substrate L-ASA (3) has been achieved,*3 and the product of the reaction unambiguously shown to be dipicolinate (10a).44 Michaelis constants for many of the enzymes have been determined; they range between 0.4 and 3.1 mM for L-ASA (3) and between 0.5 and 11.8 mM for pyruvate (2).Kinetic analysis of the reactions catalysed by the E. ~oli,'~ wheat4s and maize (Zea mays)*6 enzymes suggests that pyruvate (2) binds to the enzyme first with the loss of water followed by binding and reaction of L-ASA (3). The active site of the enzyme has been probed in several ways. Early studies showed that pyruvate (2) forms a reducible imine (NaBH is inhibitory) with the €-amino group of a lysine residue and that for the E.coZi tetramer one equivalent of pyruvate (2) binds per subunit.20 Imine formation with pyruvate (2) has recently been observed directly by electrospray mass spectrometry.14 The enzyme catalyses the reversible exchange of tritium between p-3H pyruvate and water indicative of the formation of an enamine at the active site. Lysine-161 of the E. coli enzyme has been identified as the active site residue through sequencing of tryptic digests of reduced imino protein and in fact this is the only conserved lysine in all known L-DHDP synthase sequence^.^' On the basis of these observations the reaction mechanism has been formulated as in Scheme 3. The E. coli pzyme has been crystallized and a crystal structure at 2.5 A resolution obtained.48 The actiye site lysine is accessible to solvent lying at the bottom of a 10 A deep by 30 A long crevice.Substrates could approach lys-161 through an entrance formed by adjacent aspartate residues Asp-1 87 and Asp-188. These two residues are also conserved between the known L-DHDP synthases ; this may indicate a mechanistic role for these residues possibly in general acid/base catalysis. Numerous studies of substrate analogues and inhibitors have been carried out. The enzyme displays high specificity for both substrates. For the E. coli enzyme analogues of pyruvate (2) such as phosphoenolpyruvate (PEP) phenylpyruvate a-ketobutyrate a-ketoglutarate oxaloacetate and fluoropyru- vate are not recognized. L-ASA (3) analogues such as L-glutamate semialdehyde N-acetyl-L-ASA and succinate semi- aldehyde are also not substrates.D-ASA is not a substrate for NATURAL PRODUCT REPORTS 1996R. J. COX 5 H02C'0 NH;! H02C'NH 5 (2) Enz Enz lr-L-ASA H02C 1 Enz - Enz OH H20 H02C 0C02H (4) L-DHDP Enz Scheme 3 the wheat enzyme. None of these compounds shows any inhibitory activity although fluoropyruvate weakly inhibits the wheat enzyme only (ICso > 10 mM).49 Bromopyruvate alkyl- ates the enzymes and causes inhibition (Ki= 1.6 mM for E. coli 1.8 mM for wheat).49 Alkylation may be at or near the active site for the wheat enzyme since the presence of excess pyruvate (2) affords protecti~n.~~ Alkylation is not at the active site of the E. coli enzyme; studies of inhibition by bromo- pyruvate using electrospray mass spectrometry have shown that each protein subunit is monoalkylated after incubation with four equivalents of this compound.Peaks corresponding to imine formation between lys- 16 1 and bromopyruvate were not detected. Furthermore monoalkylated enzyme subunits retained 72% of their activity; significant loss of activity occurred only on p~lyalkylation.~~ lnvestigations with the E. coli enzyme have revealed that below pH 8 acetopyruvate (1 1) is an effective slow-binding inhibitor (K = 5pM).50 A drop in potency of inhibition at higher pH values however indicates that the protonated form of (1 1) is the active species. Clearly acetopyruvate (1 l) when bound at the enzyme active site could mimic one of the reaction intermediates since it contains structural elements of each substrate.For the plant and some bacterial enzymes the final product of the DAP pathway lysine (9) is regulatory. For the E. coli and wheat enzymes lysine (9) is a non-competitive inhibitor with respect to pyruvate (2) but it inhibits competitively with respect to L,-ASA (3) suggesting that the lysine and L-ASA (3) binding sites overlap. In E. coli and Bacillus sphaericus5' inhibition by lysine (9) is weak (ICso E. coli = 1 mM; K B. sphaericus = 0.6 M) and has not been observed in other bacterial enzymes.s2 The plant enzymes however are charac- terized by potent allosteric inhibition by lysine (ICs, values wheat germ 11 pM ;53 tobacco (Nicotiana sylvestris) 15 pM;54 spinach (Spinacea oleracea) 20 ,lcM;j5 maize 23 pM;46 wheat 5 1 pM4').Lysine analogues show similar patterns of inhibition but are somewhat less effective; for wheat threo-P-hydroxy-L- lysine (THL) (12) is inhibitory (ICso = 141 pM) as is (2-aminoethy1)-L-cysteine (AEC) (13) (ICso = 288 PM).~~ Other plant L-DHDP synthases show similar patterns of behaviour e.g. AEC inhibits the tobacco synthase (ICso = 120PM),~~ and the spinach homologue (ICso= 400 PM).'~ The pea enzyme is inhibited by ~-a-(2-aminoethoxyvinyl)glycine (AVG) (14) (ICso = 155pM). Interestingly activators that are analogues of DAP (though not DAP itself) have also been discovered for the pea enzyme. The phosphono-DAP analogue (15) gave 13 YO (1 1) (12) THL (13) AEC (14) AVG (15) rate enhancement at 1.2 mM while P-hydroxy-DAP isomer (57b) gave 38 % rate enhancement at 1.2 mM.39 Heterologous expression of the unregulated L-DHDP synthases from Coryne-bacterium glutamicum in canola and soybean5' and from E.coli in tobacco chloroplast^^^ leads to increased levels of L-lysine synthesis in these plants clearly indicating how feedback control of the native plant enzymes limits overall lysine production. The observation that the E. coli enzyme is inhibited by dipicolinate (10a) (ICso = 1.2 mM)47 has encouraged a sys-tematic investigation of numerous heterocycles as potential inhibitor^.^^ Substituted pyridines and piperidines (Figure 1) were found to be moderate inhibitors (ICsovalues < 1 mM). The best inhibitors are compounds such as the ditetrazole (16) diimidate (17) the dinitrile (18) and the N-oxide (19).Compounds (1 8) and (19) inhibit noncompetitively against either substrate. Esters inhibit more strongly than their acid congeners and a planar disposition of the substituents is preferred. These relationships are clearly shown for compounds (10a,b) and (20a-d). L-DHDP synthases isolated from Bacillus spp. show some- what different properties towards inhibitors. Dipicolinate (10a) is a constituent of spores from these species and is synthesized from L-DHDP (4) during sp~rulation.'~ Dipicolinate (10a) is not an inhibitor of these enzymes although a-,€-diketopimelate (2 1) is a good competitive inhibitor [Ki = 220 pM versus L-ASA (3) and Ki = 156 pM against pyruvate (2)].35 N MeOAOMe I 'N N.N NH NH (16) ICm= 0.25 mM (17) IC = 0.2 mM NCOCN 0-(18) (19) ICW= 0.3 mM ICgj = 0.2 mM K = 1.25 mM vs L-ASA(3) & = 0.29 mM vs L-ASA(3) K,= 0.35 mM vs pyruvate (2) 4 = 0.06 mM yspyruvate (2) RO2C 0C02R (loa) R = H; ICw = 1.2 mM (lob) R = Me; ICs0= 0.7 mM RO,C*"+ 0C02R R02C QC02R H H (20a) R = H; no inhibition (2Oc) R = H; no inhibition (20b) R = Me; ICW= 5.2 mM pod) R = Me; ICs0 = 0.7 mM Figure 1 Heterocyclic inhibitors of L-DHDP synthase H* L-DHDP reductase Scheme 4 2.1 L-Dihydrodipicolinate Reductase EC 1.3.1.26 dapB L-DHDP reductases have been purified from E.~oli,~~ maize60 and Bacillus ~pp.,~' and activity detected in Chlamydomonas corn soybean and tobacco.29 All of the enzymes studied so far catalyse the irreversible reduction of L-DHDP (4) utilizing either NADPH or NADH as an electron source.Two assays have been described utilizing synthetic L-DHDP (4) (this material may be generated chemically or enzymatically and is reported to be stable if stored at pH > 10).59.60For crude samples and as an aid to screening restoration of DAP biosynthesis to a lysate of an E. coli dapB mutant (deficient in L-DHDP reductase) has been observed. For quantitative activity measurements oxidation of NADPH may be mon- itored at 340 nm (Scheme 4). The E. coli enzyme is a homotetramer of approximate M 110000 to 120000. The E. coli dapB gene has been sequenced by two groups the predicted subunit being a polypeptide of 273 amino acids.62 Nucleotide sequencing of the chromosomal gene predicted a M of 28 798.62 The gene has been cloned directly by PCR and the product overexpressed in E.coV3 The PCR product sequence predicts M 28 757 which matched the M of the cloned and overexpressed gene product of 28 758 & 8 measured by electrospray mass spe~trometry.~~ The maize enzyme which resembles the E. coli enzyme in its behaviour towards substrates and inhibitors is smaller having a M of approximately 80 OO0.60 The availability of overexpressed protein in E. coli has facilitated mechanistic and crystallographic sudie~.~~ The cofactor donates its 4-p0-R hydride to the substrate at its y-position. Michaelis constants have been determined for the substrate L-DHDP (4) (K = 50 f12 pM) and the cofactor NADPH (K = 8.0f2.5 pM).Kinetic analyses suggest that the enzyme binds the cofactor and the substrate sequentially before reaction and release of product and oxidized cofactor. Dipicolinate (10a) is a linear competitive inhibitor with respect to the substrate (Ki = 26 & 6 pM) but inhibits uncompetitively against the cofactor (Ki = 330 & 50 pM). Binding by (10a) at the L-DHDP binding site indicates that the substrate (4) is probably bound in its cyclic state. Product inhibition by NADP' was also observed being competitive with respect to NADPH (Ki = 190& 35 pM) and noncompetitive versus sub- strate (Ki = 700f200 pM). Much weaker inhibition has been observed for other substrate analogues such as iso-phthalic acid (22) (ICs0-2 mM) and compounds with only one carboxylate such as pipecolic (23) and picolinic (24) acids are very poor inhibitors (IC5,,> 20 mM).Piperidine dicarboxylic acids (20a,c) are not inhibitory. The E. coli enzyme is unusual in that either NADPH or NADH are accepted as cofactor. In early work it was noted that with NADH as cofactor the Vm, was approximately half of the value observed when NADPH was Values for V/K7however show that NADH is the better substrate by a factor of two because of its four-fold lower Km.63The conclusions from the kinetic studiesoare supported by the X-ray crystal structure obtained at 2.2 A resolution. The subunit consists of a cofactor binding domain and a substrate binding domain.NADPH is bound in the crystal and the proposed binding site of the substrate would juxtapose the substrate and cofactor in the correct orientation for reaction.64 The maize enzyme is similar to the E. coli enzyme in its NATURAL PRODUCT REPORTS 1996 0 Ho2c-Co2H 0 H02C C02H H 0C02H C02H -N N-response to inhibitors and its kinetic characteristics. This situation contrasts with enzymes isolated from Bacillus spp. where dipicolinate (IOa) is an important constituent of spores and is produced by oxidation of L-DHDP (4). The Bacillus reductases are large (approximately 150000 Da) and differ in their response to dipicolinate (10a).61 This compound is a good inhibitor (Ki = 85 pM for B. cereus) but it inhibits non-competitively versus L-DHDP (4) suggesting a regulatory role.Kinetic analyses and the observation of product inhibition at high NAD(P)+ concentrations indicate a sequential ordered mechanism analogous to that determined for the E. coli enzyme. A third class of L-DHDP reductase has been isolated from sporulating B. s~bti1i.s.~~ This enzyme is a homotetramer of subunit M -18500 which again may use either NADPH or NADH. It differs markedly from the other L-DHDP reductases in containing flavin mononucleotide (FMN). Dipicolinate (1Oa) is again inhibitory (IC50 -0.4mM); it inhibits non/ uncompetitively with respect to L-DHDP (4) but competitively against NAD(P)H.66 Mechanistic studies have revealed that a ternary complex is not formed; rather NAD(P)H binds first and reduces FMN before the loss of NAD(P)+ followed by the binding and reduction of L-DHDP (4).Inhibition by di-picolinate (10a) and o-phenanthroline (25) (1C5, -70 pM) is positively cooperative. The enzyme reduces various synthetic dyes and therefore may not be a dedicated L-DHDP reductase. 2.2 Succinyl CoA :L-TetrahydrodipicolinateN-Succinyl-transferase dapD The succinyltransferase has been purified to homogeneity from E. ~oli.~~ While chemical syntheses of the substrate L-THDP (5) have recently been initial investigations of this enzyme had to await the development of a reliable enzymatic synthesis utilizing meso-DAP dehydrogenase (Section 2.6). Existence of the acetyl transferase variant in Bacillus spp. has been inferred from the observation of N-acetyl-LL-DAP (7b) accumulation in a B.megaterium mutant lacking DAP-deacylase activity.69 The standard assay utilizes radio-labelled L-THDP (5) and an in situ generation of succinyl CoA. Aliquots of the reaction mixture are acidified to pH 2 and the radioactivity determined of fractions eluted from a cation exchange column. The eluted fraction consists of the product N-succinyl-a-amino-e-ketopimelate (6a); protonated L-THDP (5) remains bound to the resin. The reaction may be more conveniently followed by observation of the release of free thiol from succinyl CoA utilizing Ellman's reagent 5,5'-dithiobis (2-nitrobenzoic acid) monitored at 21 5 nm.70 The E. coli enzyme is a homodimer of subunit M 31 000 determined by gel electrophoresis; this is in agreement with the value of 30040 calculated from the dapD nucleotide sequence.71 The apparent K, for L-THDP (5) is 22 pM for succinyl CoA 15 pM with k,, 43.3 s-l.The reaction is reversible under standard conditions the initial forward reaction proceeds some 380 fold faster than the reverse. The acyclic compound L-a-aminopimelate (L-cz-AP) (26) has been used as an alternative substrate for the enzyme [K = 1 mM k,,,/K = 1.3YOthat of L-THDP (5)].72 The enantiomer D-a-aminopimelate (27) is a competitive inhibitor [Ki = 0.76 mM against L-THDP (5) 0.31 mM against L-a-AP (26)] NATURAL PRODUCT REPORTS 1996R.J. COX (5) L-THDP (35) succinylCoA Ho2CmC02H AB XY (28a) B = OH; X = NH,; A = Y = H (28b) A=OH; X=NH2; B=Y=H (28~) B=OH; Y=NHz; A=X=H (28d) A=OH; Y=NH2; B=X=H A (29) (30) DHT which appears to bind to the enzyme more tightly.These observations led to an investigation of binding by a number of acyclic substrate analogues. It was found that the enzyme was specific for diacids having a carbon chain length of seven. The presence of an a-hydroxy moiety was preferred slightly over a-amino for binding. Thus LL-a-amino-e-hydroxypimelate(28a) was succinylated at 43% of the rate of L-~-AP (26) but the statistical mixture of isomers (28a-d) was succinylated 21 YO faster than L-a-AP. Since the D-a-amino configuration (28c,d) is not succinylated and the LL isomer (28a) is succinylated slowly these results suggest that the a-L-amino-e-D-hydroxy- pimelate (28b) isomer is a very good substrate but further measurements were not made.The conformationally restricted (ring-like) compound (2E,SE)-y-ketohepta-2,5-dienedioic acid (29) is an inhibitor [y= 0.53 mM against L-~-AP (26)]. Cyclic L-THDP analogues have also been tested against the succinyltransferase. One substrate was found 3,4-dihydro-2H-1,4-thiazine-3,5-dicarboxylicacid (DHT) (30) [K",pp = 2 mM k,,,/K",PP = 0.5 % that of L-THDP (5)]. Other cyclic compounds are generally poor inhibitors (Figure 2). It is noteworthy however that unsaturated compounds bearing trans carboxyl groups are better inhibitors than those with carboxylates disposed cis. One compound m-a-hydroxytetrahydropyran-a,€-dicarboxylate (HTHP) (34) is a very potent competitive inhibitor [Ki = 58 nM against L-u-AP (26)].These observations have been used to propose a stereo-chemical model for the mechanism of the succinyltransferase (Scheme 5).72 The model proposes that the enzyme binds L-THDP (5) in its cyclic form then catalyses the addition of water to the re face of the imine. The trans-piperidine dicarboxylate intermediate (35) then reacts with succinyl CoA H02C 0k02H HO2C (20a) X = NH; K = 2.0 mM (20b) X = NH; K,= 63 mM (31a) X = 0; K = 0.68 mM (31 b) X = 0; 6= 3.9 mM (32a) X=CH2; K,=144mM (32b) X=CH2; &=265mM (33a) X=S; K,= 1.1 mM (33b) X = S; K,= 4.4 mM H02C 0C02H (loa) X=N; $=12.8mM (22) X= CH; K,=8.5mM Figure 2 Inhibitors of succinyl CoA :L-tetrahydrodipicolinatesuccinyl-transferase H02CI {I (27) O-a-AP II H20 (34) DL-HTHP (28b) Stereochemical model for the mechanism of succinyl CoA L-tetrahydrodipicolinate succinyltransferase Scheme 5 and the ring is opened.A feature of this model is that the acyclic substrates and inhibitors must bind in a ring-like manner in which the carboxyl groups are disposed in the same trans conformation as L-THDP (5). This conformation forces the disposition of the a and e substituents as shown in Scheme 5 and accounts for both the apparently good substrate activities of (28b) and the inhbition by D-~-AP (27) and DL-HTHP (34) since these compounds can mimic the conformation of the intermediate (35). 2.3 N-Succinyl-LL-DAPAminotransferase EC 2.6.1.17 dapC The aminotransferase has been purified to homogeneity from E.coli and its activity detected in both Gram-negative and Gram-positive bacteria.73 The E. coli enzyme is a homodimer of subunit M 39900.74 The enzyme is PLP dependent and requires L-glutamate (36) as the amine donor. Early studies utilized substrate (6a) isolated from an E. coli mutant blocked in deacylase a~tivity.'~ The assay consisted of measuring LL-DAP (8a) production by a reaction mixture containing an excess of DAP desuccinylase (Section 2.4). A more convenient continuous spectrometric assay has been described in which the forward reaction is coupled to glutamate dehydrogenase (EC 1.4.1.4) in the presence of NADPH and ammonium ions (Scheme 6).74 Under physiological conditions the reaction is freely reversible and the reverse reaction may be monitored by observing the disappearance of N-succinyl-LL-DAP (7a).The Michaelis constants for the natural substrates have been measured for L-glutamate (36) K = 1.21 mM; for L-N-succinyl-a-amino-e-ketopimelate(6a) K = 0.18 mM k,, = 86 s-l. Kinetic analyses suggest a sequential reaction mechanism in which L-glutamate (36) reacts with the pyridoxal phosphate form of the enzyme donating its amino group via aldimine quinonoid and ketimine intermediates (Scheme 7). The sub- strate (6a) then reacts in the reverse direction regenerating the PLP form of the enzyme with transfer of the amino group onto NATURAL PRODUCT REPORTS 1996 Ho2Cmco2H Ho2CYco2H 0 0 HNPCo2H NH2 0 7-7- (74 HOpCwC02H I NH2 0 Ho2cY-co2H (36) + H20 u NADP’CI-ii NADPH + NH&I Assay for N-succinyl-LL-DAP aminotransferase.Enzymes i N-succinyl-LL-DAP aminotransferase ; ii glutamate dehydrogenase EC 1.4.1.4 Scheme 6 H02CVR NH2 H02CvR Enz-NH2 0$4H f H Michaelis complex external aldimine Enz-NH3+ &H H02C I( H02C 0 H Enz-NH2 (NH2 Enz-NH2 (YH quinonoid intermediate m*-+0 H H Michaelis complex ketimine Scheme 7 the product (7a). These properties are typical of PLP-dependent CBZ (38) (kcat/Pzp= 23.5 YO) and dihydrocinnamoyl (39) aminotransferases and are consistent with those of the ‘model’ (kCa,/K”,PP = 7.7 %) are also acceptable. Interestingly the sub- system of aspartate amino transferase (EC 2.6.1.1).76 stitution of succinyl by acetyl (40) the presumed natural Early studies showed the E.coli enzyme to be specific for the substrate for the Bacillus spp. aminotransferase is less favoured natural substrates L-glutamate (36) and L-N-succinyl-a-amino- (k,,,/Pzp = 4.5 YO).The amide funtionality may even be s-ketopimelate (6a). The enzyme tolerates modifications of the deleted :the racemic substrate analogue (41) showed detectable acyl side chain with the substitution of succinyl by cinnamoyl activity (k,,,/K”,pp = 0.7 YO).Other modifications are not toler- (37) being particularly favoured (kca,/KzP= 70 YOof the value ated removal of a carboxyl results in compounds such as for the natural substrate). Other aromatic substituents such as L-e-N-CBZ lysine (42) and N-succinyl-s-amino-a-ketohexanoic HO2CW.-H02C y-yCO;HB H02c-3 0 HN HNK 0 HN 0 0 0 H02c~C02 HNK Ho2CY-YC0,H H Ho2cp oa 0 HNY n 0 NATURAL PRODUCT REPORTS 1996-R.J. COX 35 ""'Y-l Ho2cYC02H H02CY-Yco2H 0 HNl-Co2H HNPCo2H HNK-Co2H OH 0 0 0 (43) (44) (45) Ho2CmCo2H HNPco2H HNR HNNH 0 0 (Ma) R' = succinyl; R2 = H (47) (48) (46b) R' = H; R2 = succinyl c."c02H NH2 NH2 H02C wCo2H (84 HNPco2H CO H Enz-NH2 0$H NH2 0 H02C-Enz-N9 f (74 H02C I H H Michaelis complex stable hydrazone Scheme 8 PEP ~COSCOA H02C Assay for N-succinyl-LL-DAP desuccinylase. Enzymes i N-succinyl- acid (43) which are neither substrates nor inhibitors. The c-LL-DAP desuccinylase EC 3.5.1.18; ii porcine succinate thiokinase keto/amino substituent is also of key importance compounds EC 6.2.1.4; iii pyruvate kinase EC 2.7.1.40 ;iv lactate dehydrogenase lacking it such as DL-N-succinyl-a-aminopimelate (44) and L-EC 1.1.1.27 N-succinyl-a-amino-ehydroxypimelate(49 showed no de-Scheme 9 tectable inhibition.Both a-carbons must possess L-configu- ration since a mixture of N-succinyl-meso-DAP isomers (46a,b) was inactive with respect to the reverse reaction. The enzyme like other PLP-dependent aminotransferases is in good agreement with a calculated subunit M of 41129 inhibited by hydroxylamine hydrazine and their substituted determined from the nucleotide sequence of the E. coli dapE analogues. These compounds react with pyridoxal phosphate locus.s4 The dapE locus from Corynebacterium glutamicum has or hydra~one'~ also been ~equenced;~~ inferred amino acid sequence at the active site forming a stable nitr~ne~~ the which cannot react further and which is also resistant to indicates a subunit M of 39942.hydrolysis (Scheme 8). This fact was exploited in the design of The E. coli enzyme requires a metal ion ideally cobalt(I1) (K specific potent inhibitors of the enzyme. Substituted hydrazino = 4.0 pM) but zinc (K = 1.2 pM) is also effective though 2.2 substrate analogues have been synthesized with the motifs fold less so. Early studies showed that iron(m) nickel@) and required for good recognition specifically a DAP skeleton with manganese@) ions can substitute with similar efficacies to zinc. a succinyl(47) or CBZ (48) substituted amine. These compounds The enzyme is functionally similar to other carboxypeptidases,s6 are potent tight slow-binding inhibitors of the enzyme [q and shows 38.7 % sequence similarity with the cobalt(I1)- values:'' 22 nM for (47); 54 nM for (48)].74 Substrate binding dependent acetylornithine deacetylase EC 3.5.1.16 from E.coli by the model PLP enzyme aspartate aminotransferase is and 3 1.5% sequence similarity with Pseudomonas sp. G2- accompanied by a conformational closure of the active site carb~xypeptidase.~~ The deduced amino acid sequence of the which promotes fast reaction and product release.s0 In the case C. glutamicum desuccinylase is 23% homologous with the E. of inhibition of N-succinyl-LL-DAP aminotransferase by these coli enzyme.85 substituted hydrazines a similar but prolonged closure of the Assays have been developed to detect either succinate or LL-active site may be occurring.This fact is reflected in the long DAP (8a) liberation. A spectrometric method has been off-rate half times79 for these two inhibitors 78 and 45 min described for the quantification of succinate production respectively. (Scheme 9). This coupled system relies on the reaction of released succinate with coenzyme A catalysed by porcine succinate thiokinase (EC 6.2.1.4). Inosine 5'-triphosphate (ITP) 2.4 N-Succinyl-LL-DAP Desuccinylase EC 3.5.1.18 dapE is concomitantly converted into inosine 5'-diphosphate (IDP) The product of the aminotransferase N-succinyl-LL-DAP (7a) which in turn reacts with phosphoenoylpyruvate (PEP) to form in the case of E.coli N-acetyl-LL-DAP (7b) in the case of many pyruvate (2) catalysed by pyruvate kinase (EC 2.7.1.40). Bacillus sp~.,~ is subject to deacylatiom81 The E. coli de-Pyruvate (2) is then reduced by lactate dehydrogenase (EC succinylase has been purified to homogeneity,s2 and DAP 1.1.1.27) and the consequent decrease in NADPH concentration deacylase activity detected in numerous bacterial species.83 The is observed at 340 nm. E. coli enzyme is dimeric at low concentrations (< 1 mg ml-l) Michaelis constants for the natural substrate (7a) have been but forms a tetramer at higher concentrations; the subunit M determined (K = 410 pM k,, = 266 s-l) and systematic has been determined to be approximately 40000. This figure is studies of potential substrate analogues carried out.The E. coli NATURAL PRODUCT REPORTS 1996 mco2H Ho2c-Y-Yo2H Ho2cm HNPCo2H HNPCo2H0 NH2 0 NH2 NH2 HNPC02H 0 (49) (50) (51) Ho2cY-Yco2H YNHHNT-Co2H' 0 0 (9) desuccinylase hydrolysed a statistical mixture of N-succinyl- DD-DAP (8c) is not a substrate. The stereochemistry of the non- DAP stereoisomers (49) rapidly releasing 25 % of the succinate reacting a-carbon therefore controls the substrate specificity of and LL-DAP (8a). A further 25 YOof the succinate was released the enzyme since catalysis at the reacting a-carbon is fully slowly concomitantly with meso-DAP (8b) but no further reversible (Scheme 10). The Michaelis constants for LL-DAP hydrolysis was observed even with prolonged incubation. (8a) (K = 160 pM k,, = 84s-l k,,,/K,, = 525000 M-ls-l) These observations were rationalized by assuming initial rapid and meso-DAP (8b) (K = 360pM k,, = 67 s-l k,,,/K,, = hydrolysis of the LL-N-succinyl-DAP isomer (7a) followed by 186000 M-ls-l) have been determined and the equilibrium slow hydrolysis of the N-succinyl-meso-DAP isomer with the L-constant was shown by HPLC analysis of products to be 2.0 amide (approximately 30% of the rate of the LL isomer at (reflecting the statistical distribution of LL and meso isomers).10 mM). The 50% of isomers bearing D-configured a-amido Early investigations showed that the enzyme does not require carbons were presumably not substrates for the reaction. Both PLP for activity and that it is not inhibited by hydrazine or carboxyls are required for substrate activity L-e-N-succinyl-hydroxylamine.Furthermore a reducible imine is not an lysine (50) was not a substrate while L-a-N-succinyllysine (5 1) intermediate since sodium borohydride is not inhibitory as it is was a very poor substrate (0.016 % of the rate of the natural with L-DHDP synthase (Section 2.0). Nicotinamide and flavin substrate at 10 mM). A mixture of isomers of N-succinyl-a- cofactors are unnecessary and metals appear to play no part in aminopimelate (44)was also a very poor substrate (0.036 YOthe the reaction. Tritium at the substrate a-position is exchanged rate of the natural substrate at 10 mM) with a K of 16 mM. rapidly with solvent. Dithiothreitol must be present for the The L-N-acetyl substrate analogue (7b) (the substrate for the enzyme to remain active and time dependent inhibition by Bacillus spp.deacylases) showed no substrate activity; neither iodoacetamide was observed. One equivalent of iodoacetamide did L-N-succinyl-a-amino-e-ketopimelate (6a) or N-acety1-N'- is bound per enzyme and activity is protected by the presence succinyl-DAP (52). of substrate. These observations suggested that meso-DAP epimerase operates via base catalysed a-proton abstraction. A thorough investigation of the exchange of tritium between 2.5 meso-DAP Epimerase EC 5.1.1.7 dapF the substrate a-carbon and the solvent has been carried The epimerase has been detected in both Gram-positive and Each turnover is accompanied by the loss of an a-proton to 89 Gram-negative bacteriaa8 as well as in plant species.29+ solvent and a solvent derived proton is preferentially delivered Purification of the enzyme has proven problematical in part to the substrate.It was found that the kinetic isotope effects for because of the presence of a free thiol residue necessary for tritium exchange differed for LL and meso substrates. The activity. The Bacillus megaterium enzyme has been partially results are consistent with a mechanism in which two bases purified (20 fold),g0 but the E. coli enzyme has been purified to must be at work (Scheme 11); a single base mechanism The involving deprotonation from one face followed by repro- > 85 Ohpurity and has thus attracted the most attenti~n.~' E. coli dapF locus has been cloned,92 sequencedg3 and tonation from the opposite face is not supported exper- The epimerase is a monomeric polypeptide of imentally.meso-DAP epimerase resembles the well studied overexpre~sed.~~ M 34000 as determined by gel filtration; this figure has been enzyme proline racemase in this respect.95 However in the case refined to 30265 from the dapF nucleotide sequence. The of proline racemase where both bases are thiols the kinetic enzyme has been assayed by monitoring the release of 3H from isotope effects for each substrate enantiomer are nearly a-tritiated DAP or via the NADP' dependent oxidation of identical. This suggests that the bases involved in meso-DAP meso-DAP (8b) to L-THDP (5) by meso-DAP dehydrogenase epimerase are likely to be different. It has been argued on (section 2.6). purely kinetic grounds that one of the meso-DAP epimerase The enzyme interconverts LL-DAP (8a) and meso-DAP (8b); bases is in fact a thiol.This is consistent with the observation H02C w C 0 2 H I I H02c-To2H =Ho2Cmco2H =I-NHP NH2 NH2 NH2 NH2 NH2 DD-DAP (8a) LL-DAP (8b) meso-DAP (8~) Scheme 10 CO2H * C02H H-B+ -=B-H ,I~H -B H< B d NH2 d NH2 Scheme 11 NATURAL PRODUCT REPORTS 1996-R. J. COX H02C& C02H Ho2cnH:2H HN (53) azi-DAP NH2 AB NH2 (56a)B=NH2; Y=F; A=X=H (56b) A=NH2; Y=F B=X=H (56c)B=NH2; X=F A=Y=H (56d)A=NH2; X=F; B=Y=H Scheme 12 Ho2cY-Yco2H NH2 NH2 NH2 -H02C QCO, (5) L-THDP (54) (55) Scheme 13 that the irreversible inhibitor azi-DAP (53) (Ki2 25 pM) specifically covalently labels Cys-73 of meso-DAP epimerase (Scheme 12).96 Other epimerases such as mandelate racemase (EC 5.1 .XQg7 operating by 'two-base ' mechanisms have been intensively studied because of the apparent paradox of relatively poor active-site bases being able to abstract very weakly acidic protons from the substrate a-carbon.Recently the concept of short strong hydrogen bondsgs between an active site residue and the substrate carboxylate in the transition state (or similar intermediate) has been validated e~perimentally.~~ These 'low barrier' hydrogen bonds can stabilize the transition state significantly thus influencing the pK of the a-proton and it may be that a similar process allows meso-DAP epimerase to exchange substrate a-protons as observed. The proposed 'two-base' mechanism could proceed in a stepwise or concerted fashion but in either case negative charge should be concentrated at the a-carbon during reaction.This feature has directed the design of possible meso-DAP epimerase inhibitors towards compounds unstable to elimination (i.e. /3 or N-substituted) or towards compounds that are planar at the a-carbon which could mimic the putative planar transition state (Scheme 11). Thus a mixture of /3-chloro-DAP (54) stereo- isomers potently inhibits the epimerase (Ki = 200 nM).loo Inhibition is reversible and competitive with substrate. At low inhibitor concentrations however a time dependent decrease of inhibition was observed indicative of inhibitor turnover. The product of this reaction was reduced by meso-DAP dehydrogenase in the presence of NADPH; it was thus identified as L-THDP (5).It is likely therefore that the epimerase catalyses elimination of HCl from P-chloro-DAP forming the intermediate (55) (Scheme 13) which is planar at the reacting a-carbon and which is therefore a mimic of the postulated transition state (Scheme 11). This enamine then slowly isomerizes to L-THDP(5),which is not inhibitory. Since meso- DAP dehydrogenase is specific for L-THDP (5) (Section 2.6) the substrate for the epimerase must have possessed L-configuration at the distal (non reacting) end. This is consistent with the known substrate specificity of the epimerase. The synthesis in stereochemically pure form of the four stereoisomers of P-fluoro-DAP (56a-d) possessing e-L con-F F H H H H Stereochemical rationalization of reactions of P-fluoro-DAP stereo-isomers (56a-d) catalysed by rneso-DAP epimerase.Scheme 14 figuration has been achieved.lol All of these compounds are good inhibitors of the epimerase [IC, values (56a) 10pM; (56b) 25pM; (56c) 4pM; (56d) 8pM].As expected the elimination of HF from each isomer is catalysed by the epimerase and the eventual product is L-THDP (5). The characteristics of these elimination reactions vary however. The rates of HF elimination and epimerization at the reacting a-carbon have been independently determined.l0' For one pair of isomers (56c,d) the elimination is slow but epimerization is fast and the two isomers are in rapid equilibrium. For the other pair (56a,b) only fast elimination is observed.These results suggest the stereochemical course shown in Scheme 14 in which the position of the charged groups and the bulky substituent R are fixed in the enzyme active site [the same conclusions may be drawn from a scheme in which the a+ bond of (56a-d) has been rotated by 180" yielding the intermediate (E)-(55)]. Fast elimination is expected when H and F are fixed either syn or NATURAL PRODUCT REPORTS 1996 OH H02C~0-~C02HH02CwC02H H02C*C02H N-0 NH2 N-0 NH NH2 NH2 (574 N-0 N-0 Ho2CY-YCo2H NH2 HN. NH2 (64) (65) (59) Ho2cmH:H NH2 NH Ho2CmP(oH)2 (67) (684 B=X=NH2; A=Y=H AB X H02cmco2H Y (60) (61a) B =X= NH2; A=Y=H (61b) B=Y=NH,; A=X=H (61~)A =X = NH2; B=Y=H (61d) A = Y = NH2; B=X=H anti-coplanar in the enzyme active site as in stereoisomers (56a,b).lo2 When H and F are fixed gauche as in stereoisomers (56c,d) epimerization occurs rapidly and HF elimination is slow.P-Hydroxy DAP (57a,b) isomers have also been synthe- Sized.lOO,101,103 These compounds are poor inhibitors of the epimerase (IC50= 2.5 and 4 mM respectively) since H,O is not eliminated; (57b) is not epimerized at all and (57a) is epimerized at the a-centre distal from the hydroxy group. A mixture of stereoisomers of N-hydroxy-DAP (58) reversibly and com-petitively inhibited the epimerase (K,= 56 pM).lo4 N-Amino- DAP (59) was a much poorer inhibitor (K,= 2.9 mM). It has been suggested that elimination of water from (58) would lead to the planar imine (60) which could either bind to the epimerase tightly per se or reversibly react with one of the active site bases.Another strategy for the design of possible inhibitors of DAP-epimerase relies on the perturbation of the a-proton pK by the substitution of carboxylate by phosphonate. The doubly charged phosphonate could also mimic a possible aci-carbanion transition state as is observed in the potent slow-binding inhibition of alanine racemase (EC 5.1.1.I) by phosphono lo5 It analogues of ala~~ine.~~' was thought that a similar approach could yield useful meso-DAP epimerase inhibitors.lo6 Four stereochemically pure phosphono-DAP isomers (6 1 a-d) have been synthesized.lo7 These compounds are relatively poor competitive inhibitors of meso-DAP epimerase (K,= 3.9-7.2 mM) however and none of them are substrates.Heterocyclic compounds (62-66) in which a planar configu- ration about the a-carbon is rigidly held have also been synthesized as potential inhibitors of the epimerase.los Similar compounds can inhibit other 'two-base' epimerases such as proline racema~e.'~~ None of these compounds showed sig- nificant inhibition perhaps because of the steric bulk of the ring or possibly because of the inability of the ring substituents to take up the correct conformation to mimic the postulated transition state geometry. Several other miscellaneous compounds have been tested. The epimerase has been suggested to be the target of the antibacterial compound y-methylene DAP (67),"O although this material is only a moderate inhibitor (K,=0.95 mM).lo4 The sulfur containing DAP analogue meso-lanthionine (68b) is also a moderate mixed competitive inhibitor (K,=0.18 mM).Its LL isomer (68a) inhibits competitively (K,=0.42 mM) while the DD analogue (68c) shows no inhibition. Oxidation of the sulfur to a sulfoxide (69) or sulfone (70) led to progressively poorer inhibitors (Ki = 11 and 21 mM respectively for meso isomers).lo4 (68b) A=X=NH2; B=Y=H (6&) A=Y=NH2; B=X=H 2.6 meso-DAP Dehydrogenase EC 1.4.1.16 ddh The biosynthesis of meso-DAP (8b) via acylated intermediates and meso-DAP epimerase as described above is bypassed in some organisms. In this variation of the pathway the direct reductive amination of L-THDP (5) to meso-DAP (8b) is catalysed by the NADPH dependent meso-DAP dehydro- genase.This enzyme occurs in Bacillus sphaericuslll (which lacks meso-DAP epimerase and enzymes of the acylated pathway)l12 and some other Gram-negative and Gram-positive bacteria.'l37 114 An early report suggested the presence of meso-DAP dehydrogenase in plants,28 although more recent work seems to have refuted this finding.29 The enzyme has been purified to homogeneity from Corynebacterium gl~tarnicum'~~ and Brevibacterium spp.'15 but the most highly studied enzyme is that from B. sphaericus,l16 which has a high dehydrogenase activity.l13 The reaction is reversible maximal velocity in the forward direction of reductive amination is displayed at pH values of 7.5-8.5.Maximal reverse velocity occurs at pH values between 9.0 and 10.5,"' although under forcing conditions (i.e. using NADP' with meso-DAP in the absence of NH,) the reverse reaction may be studied at pH 7.8. The reaction is conveniently assayed by observation of cofactor consumption in either direction or via a sensitive coupled reaction with formazan in the reverse direction.lls The bacterial enzymes are homodimers of subunit M approximately 39000 and two active sites are present per dimer. The ddh locus from C. glutamicum has been cloned and sequenced,ll9 and the inferred peptide has a M of 35 099.lZ0 Michaelis parameters have been determined for the natural substrates and cofactors for reactions catalysed by the B. sphearicus C.glutamicum and Brevibacterium spp. enzymes K values for L-THDP (5) -0.2 mM; NADPH -0.2 mM; NH = 12-62 mM; meso-DAP (8b) =2.5-6 mM; NADP' = 0.83 pM for B. sphaericus -0.1 mM for the others. Detailed kinetic analyses of reactions catalysed by the B. sphaericus enzyme have revealed that the reaction is sequentially ~rdered,"~ like the classical glutamate dehydrogenase mech- anism. In the forward reaction NADPH binds first followed by L-THDP (5) and then ammonia. After reaction meso-DAP is released followed by NADP'. The reverse sequence is observed for the oxidative deamination of meso-DAP. The reaction is unusual in that oxidation takes place at a D centre; meso-DAP is transformed into L-THDP (5) in the reverse reaction. Since neither DD-(8c) nor LL-DAP (8a) are substrates for this reaction it is evident that L stereochemistry is required at the non reacting a-carbon as in the case of meso- DAP epimerase (Section 2.5).The forward reaction is likely to NATURAL PRODUCT REPORTS 1996-R. J. COX n NH3 IFNADPH H02C-CO2H Scheme 15 proceed via ring opening of L-THDP (5) by ammonia forming a planar imine intermediate (60) (Scheme 15). Imine reduction then forms the wcentre of meso-DAP. The 4-pro-S hydrogen of NADPH is transferred stereospecifically in the rate-determining step. Like meso-DAP epimerase substrate specificity is tight ; DAP analogues lacking carboxylate or amine groups or shortened by a methylene group are not substrates. meso-Lanthionine (68b) is a poor substrate ( VmaX/Km = 0.2O/O of the value for meso-DAP) and oxidation of the central sulfur lowered substrate activity further.lo4 This result was surprising in view of the fact that lanthionine (68) is only 5 O/O longer than DAP and that at least one of the stereoisomers of the bulkier DAP analogue y-methylene-DAP (67) is a somewhat better substrate (y,ldx/Km = 0.4% of the value for meso-DAP).The observed results may be due to the decreased pK,s of the protonated carboxylate and imine functionalities of the lanthionine imino intermediate. Oxidation of sulfur could reduce these pK,s further explaining the extremely poor activities of the sulfone and sulfoxide.lo4 Two compounds N- hydroxy-DAP (58) and N-amino DAP (59) were substrates; mixtures of stereoisomers turned over at 22 and 4% of the rate for meso-DAP respectively.Fluoro-DAP stereoisomer (56b) an analogue of the natural substrate meso-DAP (Sb) showed neither substrate nor inhibitor activity against the B. sphaericus dehydrogenase.lO' Phosphono-DAP analogues (6 la4) also show no substrate activities although binding evidently occurs since these compounds are weakly inhibitory. It has been suggested that inhibition may occur because of the reduced propensity of the amine adjacent to the phosphono group to undergo oxidation or to cyclize onto the a-imino carbon at the reacting end. The two nzeso isomers bind more tightly than either LL (61a) (K = 12 mM) or DD (61d) (K,= 26 mM) analogues. The best of them (61c) (K,= 4.3 mM) would place the L-phosphono group at the non reacting end if specificity is assumed to be similar to DAP analogues.The other meso analogue (61b) (K,= 7.4 mM) would place the phosphono group at the reacting end. DAP isomers themselves are competitive inhibitors of the forward reaction with K values of 3.1 4.0 and 4.2 mM for LL (8a) DD (8c) and meso-DAP (8b) respectively."' The isoxazoline (62) shows potent inhibitory activity against meso-DAP dehydrogenase from B. sphaericus. At pH 7.8 this compound is a potent inhibitor of both the forward [K,= 4.2 pM versus L-THDP (5)] and reverse reactions [K,= 23 pM with respect to nzeso-DAP (8b)] although inhibition falls off with rising pH (ICs0= 4.1 mM at pH 10.5). Initially it was thought that this compound might be a good mimic of the postulated imine intermediate (60) with the L-amine filling the non-reacting site and the planar oxime bound at the reacting end.However kinetic analysis showed that for the reverse reaction (62) inhibited noncompetitively versus meso-DAP and uncompetitively against NADP' (K = 9.2 pM). In the forward direction (62) is a competitive inhibitor against L-THDP (5). Hence the isoxazoline inhibitor competes only for the L-THDP (5) binding site and does not occupy the meso-DAP binding site. These results imply separate binding sites for the two substrates. The L-THDP (5) binding site may contain ionizable residues since both inhibition by (62) and substrate activity of L-THDP (5) fall off dramatically at high pH.Inhibition by (62) was not merely due to to the isoxazoline moiety. Similar compounds with alternative side chains were very poor inhibitors. In particular the isoxazoline (63) differing only in ring junction stereochemistry showed a poor 13 YO inhibition at 1 mM. The distal amino acid moiety is clearly required because removal of the side chain (64) or replacement by carboxyl (65) led to even poorer inhibitors of the dehydrogenase (7.5 and .c 2 % inhibition respectively at 7.75 mM). The imidazole analogue (66) was also a very poor inhibitor (< 7% inhibition at 1 mM). The B. sphaericus enzyme contains one sulfhydryl group per subunit and is susceptible to inhibition by charged or bulky sulfhydryl reagents such as Hg2+ or Ellman's reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).Enzyme inhibited by formation of a disulfide with DTNB could be reactivated by treatment with cyanide forming an enzyme-cyanide adduct. It was concluded therefore that the sulfhydryl residue was not required for enzymic activity.12' 2.7 meso-DAP Decarboxylase EC 4.1.1.20 ZysA Decarboxylation of meso-DAP (8b) is catalysed in a PLP- dependent reaction to produce L-lysine (9) as the ultimate product of the pathway. meso-DAP decarboxylase is extremely widespread in both plants and bacteria; it has been purified from a number of sources including E. coli,122, 123 Bacillus sphaericus,lZ4 duckweed (Lemna perp~silla)~~~ and wheat (Triti- cunz vulgaris).126 The B. sphaericus enzyme is a homodimer of approx M 80000,whereas the E.colienzyme is a homotetramer with a subunit M of 46099 determined by genetic analysis.12' The lysA loci from a range of other bacterial species including 129 B. methan~licus,~~~ B. subtilis,128. Mycobacterium tubercu- Ios~s,~~~ and Pseudomonas aeru- Corynebacterium gl~tamicuml~~ gino~al~~ have also been cloned and sequenced. The genes encode similarly sized proteins (M = 4500W9000) with significant sequence ~imi1arities.l~~ The reaction has been assayed in several ways. Early studies relied on manometric measurement of CO evolution or colorimetric determination of lysine (9) by its reaction with ninhydrin. Neither method is reliable enough for kinetic measurements however and more recent studies have measured the release of 14C02from radio-labelled DAP,lS5 or determined lysine (9) formation using the enzyme L-lysine-a-ketoglutarate e-aminotransferase (EC 2.1.6.36) in non-continuous Assays have also been developed for the simultaneous determination of epimerase and decarboxylase activity.13' The decarboxylase is specific for meso-DAP (K -1.7 mM for most bacterial enzymes 0.16-0.35 mM for plant homologues).A mixture of lanthionine isomers (68a-c) was turned over by the B. sphaericus enzyme at approximately 5% of the rate for meso-DAP but although many other amino acids and their analogues have been examined no other substrates have been found. Since the enzymes studied to date accept neither LL (8a) nor DD-DAP (8c) as substrates or inhibitors it may again be concluded that L configuration is required at the non reacting a-carbon.The enzymes are unique among PLP-dependent amino acid decarboxylases in catalysing reaction at a D centre. This fact is reflected by protein sequence studies which suggest that DAP- decarboxylases are closely related as a group but seem to be unrelated to most other PLP-dependent enzymes.1"". 1'34,13*. 139 The stereochemical course of the reaction catalysed by the B. ~phaericus'~~ and wheat enzymes has been studied in detail. In both cases decarboxylation occurs with inversion of stereo- chemistry again contrary to the state of affairs observed for other PLP-dependent decarboxylases where reaction is ac- NATURAL PRODUCT REPORTS 1996 external aldimine Reactions catalysed by PLP-dependent L-amino acid decarboxylases (path Scheme companied by retention of stereochemistry.This apparent dichotomy in the stereochemical preference and course of reaction has been accounted for in two ways. A 'swinging door' mechanism has been proposed in which a rotation of the substrate-PLP complex occurring after loss of CO would allow protonation from the same direction as CO loss. This model would involve a drastic conformational change of the enzyme bound substrate however. An alternative hypothesis would link the mechanism of meso-DAP decarboxylase with other PLP-dependent decarboxylases if a common quinonoid intermediate (71) was formed in the enzyme active site (Scheme 16). Protonation of this intermediate from the re face at the a-carbon in each case would result in the observed stereochemical outcome (i.e.inversion at D centres by DAP decarboxylases and retention at L-centres in other PLP-dependent decarboxyl- ases). The design of effective inhibitors for DAP-decarboxylase has been hampered by the apparently very tight substrate specificity. Neither LL nor DD-DAP inhibit the reaction,24 but L-lysine (9) and its analogues; such as AEC (13) are generally weak competitive inhibitors of many of the enzymes with IC, values > 20 mM.141* 142 meso-Lanthionine (68b) is a somewhat better inhibitor of the enzymes from B. sphaericus and wheat germ with IC,,s of 10 mM and 14 mM respectively. The lanthionine sulfoxides (69) are better still with IC, values of -1 mM.Further oxidation of the sulfur does not increase potency the sulfone analogues (70) showed poorer inhibition (IC50 -10 mM).143 As expected the enzyme is inhibited by 'carbonyl' reagents such as hydroxylamine and isonicotinic acid hydrazide (72) (isonia~id),~~. 144 but detailed investigations have not been carried out. It would be expected that DAP analogues of these compounds could be potent and specific inhibitors of meso-DAP decarboxylase in analogy to the inhibition by similar compounds of the PLP-dependent N-swccinyl-LL-DAP amino- transferase in time-dependent reactions (Section 2.3). Mixtures of isomers of N-amino-DAP (59) and N-hydroxy-DAP (58) however were found merely to be effective competitive inhibitors of the enzymes from B.sphaericus (Ki = 100 and 84 pM respectively) and wheat germ (Ki = 910 and 710 pM respectively). a-Difluoromethyl amino acids are also known inhibitors of PLP-dependent enzymes. These compounds can undergo a series of elimination reactions catalysed in the active site resulting in irreversible enzyme ina~tivati0n.l~~ The a-difluoro- methyl DAP analogue (73) was however merely a weak competitive inhibitor (1C5 -10 mM) of the decarboxylases from wheat germ and B. sphaericus. a-Methyl-DAP isomers H H quinonoid intermediate (71) NH2 L) and meso-DAP decarboxylases (path D) 16 H "yN *NH2 (72) (73) Ho2CmCo2H Ho2cY-YCo2H Me NH2 NH2 NH2 NH2 (74) (75) (76) (77) (74) were also poor inhibitors."O These findings underline the tight substrate specificity of meso-DAP decarboxylase since many other PLP-dependent decarboxylases can accept suitable a-methyl or a-difluoromethyl substrate analogues into their active Unsaturated substrate analogues could also be expected to be good inhibitors and a number of them have been tested against the decarboxylase from E.coli. The best of these compounds the mixture of stereoisomers (75) was a moderate competitive inhibitor (Ki = 180 pM) but structural modification for example y-methylation (76) led to much poorer inhibitors. The individual isomers of y-methylene-DAP (67) are also uniformly poor inhibitors (-30 % inhibition at 10 mM).l1° 3 Antibiotic Properties of DAP Pathway Inhibitors Naturally occurring DAP analogues with biological activities would appear to be very rare possibly reflecting the critical importance of the products of the DAP pathway to bacterial growth and development.An alanyl dipeptide (77) of a-hydroxymethyl-DAP which shows antibiotic activity against E. coli has been isolated from Micromonospora chalcea how-ever.147 The activity against E. coli on minimal media is synergistically enhanced by a number of peptidoglycan bio- synthesis inhibitors such as penicillin-G and chloro-D-alanine NATURAL PRODUCT REPORTS 1996-R. J. COX H02C0C02H HO2C4$CO2H OH OH 0 suggesting that inhibition of DAP biosynthesis may be occurring. These effects are not evident on nutrient agar which may contain DAP or lysine. Efficient transport of potential DAP biosynthesis inhibitors through the cell membrane is clearly desirable.DAP itself would appear to be transported via the cystine uptake mechani~m,'~~ at least in E. coli 14g and Salmonella typhi- murium.150 DAP analogues may also be transported as di- or tri-peptides which are taken up by efficient cellular transport systems. This strategy has been demonstrated to be effective for other peptidoglycan biosynthesis inhibitors such as the peptidic antibiotic alaphosphin which is cleaved in vivo to release the alanine racemase (EC 5.1.1.1) inhibitor pho~phonoa1anine.l~~ In cell-free protein extracts of E. coli the succinyltransferase inhibitor L-OI-AP (26) blocks DAP biosynthesis and causes a build-up of the precursor L-THDP (5).L-OI-AP(26) itself showed no antibacterial activity however perhaps because of poor transport into the cell. When L-OI-AP was included in alanyl dipeptides good antibacterial activity was observed. The peptide (78) was the most potent with MICs of 1-16 pg ml-l against a range of Gram-negative bacteria. High internal cellular concentrations of up to 30mM L-OI-AP (26) were measured when bacterial cells had been treated with these alanyl dipeptides ; this is a concentration significantly higher than the K, of L-OI-AP (1 mM). The dipeptides caused depression of DAP biosynthesis in 'resting' E. coli cells at 2.4 mM and caused lysis of growing Enterobacter cloacae at 0.2 mM. These effects were reversed in the presence of LL-DAP @a) showing that DAP biosynthesis was indeed the likely target of action.These encouraging results suggest that even modest enzyme inhibitors (in fact L-OI-AP is a poor substrate of the succinyl- transferase) can be effective antibiotics if properly delivered. It would be expected that alanyl peptides of more potent enzyme inhibitors could be extremely effective antibiotics. This line of reasoning has recently encouraged the synthesis of a depsi-peptide analogue (79) of enantiomerically pure L-HTHP (80) [Ki of racemate 58 nM against L-OI-AP (26)] along with other potential transition state inhibitors (81) and (82) of the s~ccinyltransferase.~~~ The strategy has been adopted for other potential DAP biosynthesis inhibitors such as phosphono- DAP analogues. These generally show little or no antibacterial activity although the meso-compound (61b) inhibits the growth of Salmonella tryphimurium at 1 pg ml-l.This organism has a low intracellular pool of DAP,150 and growth inhibition is reversed by LL-DAP @a) meso-DAP (8b) and L-cysteine suggesting phosphono-DAP uptake is via the usual cystine mechanism.lo7 The tripeptide (83) is a more effective growth inhibitor and is active against a wider range of bacteria (e.g. MICs of 4-32 pg ml-' versus E. coli and Citrobacter freundii). Other compounds targeted against DAP biosynthesis have been tested. N-Amino (59) and N-hydroxy-DAP (58) good inhibitors of meso-DAP epimerase and poor substrates of meso-DAP dehydrogenase inhibit the growth of Bacillus megaterium at 20 pg m1-l' while y-methylene-DAP (67) (not a particularly good inhibitor of any of the later enzymes) inhibits the growth of E.coli and Pseudomonas aeruginosa at 4 pg ml-l. The heterocyclic dehydrogenase inhibitor (62) does not inhibit growth of Proteus vulgaris or Corynebacterium glutamicum which do not require the dehydrogenase for DAP biosynthesis but it inhibits growth of Bacillus sphaericus which does. Inhibitors of meso-DAP epimerase such as P-chloro-DAP (54) and P-fluoro-DAP (56) stereoisomers are inactive or weak growth inhibitors of E. coli although where activity is seen it is reversed in the presence of DAP.lol Other similar compounds can actually substitute for DAP in mutants auxotrophic for DAP. Compounds such as /I-hydroxy-DAP (57) a very weak inhibitor of the epimerase can be incorporated into the peptidoglycan of E.coli in the absence of DAP. It is thought that the promiscuity of the DAP-condensing enzyme acting during the synthesis of muramyl peptides is responsible. Lanthionine (68) y-methyl-DAP (84) and cystathionine (85) may also be incorp~rated.'~~~~~~ LL-DAP (8a) can also be incorporated into the peptidoglycan of E. coli instead of meso- DAP (8b) in dapF mutants lacking meso-DAP-epimerase.15* Indeed the engineered increase in biosynthesis of alternative substrates for the DAP-condensing enzyme can overcome the absence of DAP in bacterial strains in which dapA (coding for L-DHDP synthase) has been deleted although L-lysine must still be supplied to allow protein ~ynthesis.'~~ 4 Overview Investigations into a wide range of bacteria have revealed two main routes between L-THDP (5) and meso-DAP (8b) directly via the dehydrogenase or indirectly via acylated intermediates.The succinylated and acetylated pathways may be quite distinct since acetylated intermediates are very poor substrates for at least two of the enzymes from the succinyl-blocked pathway. Further work is required on the enzymes from the acetylated pathway chiefly occurring in Bacillus spp. Some enzymes from plants are still 'missing' in particular those linking L-THDP (5) with LL-DAP (8b). The plant enzymes studied to date are remarkably similar in catalytic mechanism to their bacterial homologues although in most cases plant enzymes tend to be more highly regu1ated.l' Understanding of the underlying regulation processes has led to expression of genes for unregulated lysine biosynthetic enzymes in transgenic organ- isms.This has allowed the production of plant and bacterial strains with enhanced lysine production. The enzymes of the DAP pathway to L-lysine have provided many challenges to chemists and biochemists. At the same time novel features of enzyme mechanism and structure have been revealed. For example the stereochemical course of meso- DAP dehydrogenase is unique amongst dehydrogenases. Other enzymes are also very unusual meso-DAP decarboxylases are not related to most other PLP dependent decarboxylases from a structural or stereochemical viewpoint. meso-DAP epimerase a member of the small class of 'two-base' amino acid racemases is another fascinating enzyme and many mechanistic questions have yet to be answered.These three functionally unrelated enzymes are of particular interest for the way the stereo- 42 chemistry of the non-reacting substrate a-carbon controls the substrate specificity and for the apparent very tight dis-crimination for the DAP skeleton. Until recently attention has focused mainly on those enzymes of the pathway where substrates or proteins have been available. The lack of readily available substrates has stimulated efforts towards synthesis of the three optically pure isomers of DAP (8a-c).15s-159 Synthetic effort in this area has been considerable and has resulted in the generation of a library of DAP analogues.Development of specific inhibitors for lysine biosynthetic enzymes has also relied heavily on novel syntheses. In many cases the elucidation of mechanism and the de- velopment of substrates and inhibitors has occurred syner- gistically. Molecular biological techniques are beginning to provide DAP pathway enzymes for studies requiring consistent sources of protein such as crystallography. These techniques will also allow site-directed mutagenesis to facilitate further mechanistic studies. Coupled with the bank of synthetic substrates and inhibitors these methods will lead inevitably to a better understanding of the structures and functions of these enzymes. This will further aid the development of potential inhibitors especially for those enzymes towards the end of the pathway where substrate specificity appears to be very tight.Better candidates for inhibition may ultimately be enzymes from the middle of the pathway such as the succinyltransferase and aminotransferase. Substrate specificity of these enzymes is less rigorous and very potent (nM range) inhibitors have been developed. Additionally since some compounds with DAP skeletons can be incorporated into peptidoglycan it would be beneficial to block DAP biosynthesis at an early stage. In conclusion while potent broad spectrum antimicrobial compounds have not yet emerged from the study of DAP pathway inhibition the scene is set for rapid development. 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White Meth. Enzymol. 1971 17B 140. 124 Y. Asada K. Tanizawa Y. Kawabata H. Misono and K. Soda Agric. Biol. Chem. 1981 45 1513. 125 Y. Shimura and H. J. Vogel Biochim. Biophys. Actu 1966 118 396. 126 M. Mazelis and R. K. Creveling J. Food Biochem. 1978 2 29. 127 P. Stragier 0.Danos and J.-C. Patte J.Mol. Biol. 1983 168,321. 128 J. Yamamoto M. Shimizu and K. Yamane Agric. Biol. Chem. 1991 55 1615. 129 J. Yamamoto M. Shimizu and K. Yamane Nucleic Acids Res.1989 17 10105. 130 D. A. Mills and M. C. Flickinger App. Environ. Microbiol. 1993 59 2927. 131 A. B. Andersen and E. B. Hansen Gene 1993 124 105. 132 P. Yeh A. M. Sicard and A. J. Sinskey Mol. Gen. Genet. 1988 212 112. 133 C. Martin B. Cami P. Yeh P. Stragier C. Parsot and J.-C. Patte Mol. Biol. Evol. 1988 5 549. 134 E. Sandmeier T. I. Hale and P. Christen Eur. J. Biochem. 1994 221 997. 135 J. G. Kelland M. M. Palcic M. A. Pickard and J. C. Vederas Biochemistry 1985 24 3263. 136 T. Hammer and R. Bode J. Basic Microbiol. 1992 32 21. 137 A. N. C. Weir C. Bucke G. Holt M. D. Lilly and A. T. Bull Anal. Biochem. 1989 180 298. 138 C. Momany R. Ghosh and M. L. Hackert Prof. Sci. 1995 4 849. 139 N. V. Grishin M.A. Phillips and E. J. Goldsmith Prot. Sci. 1995 4 1291. 140 Y. Asada K. Tanizawa S. Sawada T. Suzuki H. Misono and K. Soda Biochemistry 198 1 20 688 1. 141 D. P. Grandgenett and D. P. Stahly J. Bacteriol. 1971 105 1211. 142 A. Rosner J. Bacteriol. 1975 121 20. 143 J. G. Kelland L. D. Arnold M. M. Palcic M. A. Pickard and J. C. Vederas J. Biol. Chem. 1986 261 13216. 144 H. P. Willett Am. Rev. Resp. Dis. 1959 81 653. 145 D. Schirlin J. B. Ducep S. Baltzer P. Bey F. Piriou J. Wagner J. M. Hornsperger J. G. Heydt M. J. Jung C. Danzin R. Weiss J. Fischer A. Mitschler and A. De Cian J. Chem. Soc. Perkin Trans. 1 1992 1053. 146 R. Poulin L. Lu B. Ackermann P. Bey and A. E. Pegg J. Biol. Chem. 1992 267 150. 147 J. Shoji H. Hinoo T.Kato K. Nakauchi S Matsuura M. Mayama Y. Yasuda and Y. Kawamura J. Antibiotics 1981 34 374. 148 L. Leive and B. D. Davis J. Biol. Chem. 1965 240 4362. 149 L. Leive and B. D. Davis J. Biol. Chem. 1965 240 4370. 150 S. Cooper and N. Metzger FEMS Microbiol. Lett. 1987,36 191. 151 J. G. Allen F. R. Atherton M. J. Hall C. H. Hassall S. W. Holmes R. W. Lambert L. J. Nisbet and P. S. Ringrose Nature (London) 1978 272 56. 152 J. L. Roberts J. Borgese C. Chan D. D. Keith and C.-C. Wei Heterocycles 1993 35 115. 153 D. Mengin-Lecreulx D. Blanot and J. van Heijenoort J. Bacteriol. 1994 176 4321. 154 D. Mengin-Lecreulx C. Michaud C. Richaud D. Blanot and J. van Heijenoort J. Bacteriol. 1988 170 2031. 155 C. Richaud D. Mengin-Lecreulx S. Pochet E. J. Johnson G. N. Cohen and P. Marliere J. Biol. Chem. 1993 268 26827. 156 R. C. Holcomb S. Schow S. Ayral-Kaloustian and D. Powell Tetrahedron Lett. 1994 35 7005. 157 G. Bold T. Allmendinger P. Herold L. Moesch H-P. Schar and R. 0. Duthaler Helv. Chim. Acta 1992 75 865. 158 R. M. Williams and C. Yuan J. Org. Chem. 1992 57 6519. 159 K. Agouridas J. M. Girodeau and R. Pineau Tetrahedron Lett. 1985 26 3115.

ISSN:0265-0568    

DOI:10.1039/NP9961300029

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

ISSN 0265-0568 NPRRDF 5 1-1-1-74 (1996) Natural Product Reports A journal of current developments in bioorganic chemistry Volume 13 Indexes CONTENTS ... 111 Preliminary pages for Volume 13 1-1 Index of authors cited 1-37 Subject index ISSN 0265-0568 Coden NPRRDF Natural Product Reports A journal of current developments in bioorganic chemistry Volume 13 1996 The Royal Society of Chemistry Cambridge ~~ ~~~~~~~ Natural Product Reports (ISSN 0265-0568) @ The Royal Society of Chemistry 1997 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.Printed in Great Britain by the University Press Cambridge ISSN 0265-0568 NPRRDF 13 1-468; 1-1-1-74 (1996) Natural Product Reports A journal of current developments in bioorganic chemistry Volume 13 CONTENTS 1 Modern Bioassays using Metal Chelates as Luminescent Probes Peter G. Sammes and Gokhan Yahioglu 29 The DAP Pathway to Lysine as a Target for Antimicrobial Agents Russell J. Cox Revietving the literature published up to September 1995 45 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites Richard B. Herbert Reviewing the literature published in 1994 59 Diterpenoids James R. Hanson Reviewing the literature published in 1994 73 Book Reviews Anticancer Drugs from Animals Plants and Microorganisms by George R.Pettit Fiona H. Pierson and Cherry L. Herald (reviewed by John Mann); Oxidative Stress and Antioxidant Defenses in Biology ed. S. Ahmad (reviewed by David J. Robins); Advances in Nitrogen Heterocycles (Volume I) ed. C. J. Moody (reviewed by Joseph P. Michael) 75 Marine Natural Products D. John Faulkner Revierzing the literature published in 1994 127 P-Phenylethylamines and the Isoquinoline Alkaloids Kenneth W. Bentley Reviewing the literature published between July 1994 and June 1995 151 Triterpenoids Joseph D. Connolly and Robert A. Hill Reviewing the literature published in 1994 171 Amaryllidaceae and Sceletium Alkaloids John R. Lewis Revierving the literature published in I994 177 The Biosynthesis and Degradation of Thiamin (Vitamin B,) Tadhg P.Begley Reviewing the literature published between January 1986 and January 1996 187 Pyrrolizidine Alkaloids J. Richard Liddell Reviewing the literature published between July 1994 and June 1995 195 Monoterpenoids David H. Grayson Reviewing the literature published in 1991 1992 and part of 1993 227 Steroids Reactions and Partial Synthesis James R. Hanson Revieit'ing the literature published in 1994 24 1 Recent Progress in the Chemistry of Non-monoterpenoid Indole Alkaloids Masataka Ihara and Keiichiro Fukumoto Reviewing the literature published between July 1994 and June 1995 263 Book Review Enzyme Catalysis in Organic Synthesis A Comprehensive Handbook by K. Drauz and H. Waldmann (reviewed by David R. Kelly) 265 Dietary Antioxidants in Disease Prevention Michael H.Gordon 275 Recent Advances in Annonaceous Acetogenins Lu Zeng Qing Ye Nicholas H. Oberlies Guoen Shi Zhe-Ming Gu Kan He and Jerry L. McLaughlin Reviewing the literature published up to January 1996 307 Natural Sesquiterpenoids Braulio M. Fraga Reviewing the literature published in 1994 327 Recent Progress in the Chemistry of the Monoterpenoid Indole Alkaloids J. Edwin Saxton Reviewing the literature published between July 1994 and December 1995 365 Decanolides 1O-membered Lactones of Natural Origin Gerald Drager Andreas Kirschning Ralf Thiericke and Marion Zerlin Reviewing the literature published between 1975 and 1995 377 Natural Guanidine Derivatives Roberto G. S. Berlinck Reviewing the literature published in 1994 and 1995 41 1 Oligomeric Proanthocyanidins Naturally Occurring O-Heterocycles Daneel Ferreira and Riaan Bekker Reviewing the literature published between January 1992 and December 1995 435 Muscarine Imidazole Oxazole Thiazole and Peptide Alkaloids and Other Miscellaneous Alkaloids John R.Lewis Reviewing the literature published between July 1993 and June 1994 469 The Glycopeptide Story -How to Kill the Deadly ‘Superbugs’ Dudley H. Williams 479 Catalytic Antibodies -Reaching Adolescence? Neil R. Thomas Reviewing the literature published up to the end of February 1996 513 Pigments of Fungi (Macromycetes) Melvyn Gill Reviewing the literature published between September 1992 and February I996 529 The Sesterterpenoids James R.Hanson Reviewing the literature published between November 1991 and March 1996 Natural Product Reports EditoriaJ Board Professor T. J. Simpson (Chairman) University of Bristol Dr J. R. Hanson University of Sussex Dr R. B. Herbert University of Leeds Professor J. Mann University of Reading Professor D. J. Robins University of Glasgow Dr C. J. Schofield University of Oxford Dr D. A. Whiting University of Nottingham Editorial Staff Editorial Office Dr Sheila R. Buxton The Royal Society of Chemistry Managing Editor Thomas Graham House Dr Roxane M. Owen Science Park Deputy Editor Milton Road Miss Nicoila P. Coward Cambridge Produeti0n Editor UK CB4 4WF Dr Carmel M. McNamara Telephone +44 (0) 1223 420066 Technical Editor Facsimile +44 (0) 1223 420247 Ms Dawn J.Webb E-mail perkin@rsc.org Miss Karen L. White RSC Server http://c hem ist ry.rsc.or g /rsc/ Editorial Secretaries Natural Product Reports is a bimonthly journal of critical reviews. It aims to foster progress in the study of bioorganic chemistry by providing regular and comprehensive reviews of the relevant literature published during well-defined periods. Topics include the isolation structure biosynthesis biological activity and chemistry of the major groups of natural products -alkaloids terpenoids and steroids aliphatic aromatic and 0-heterocyclic compounds. This is augmented by frequent reviews of the wider context of bioorganic chemistry including developments in enzymology nucleic acids genetics chemical ecology primary and secondary metabolism and isolation and analytical techniques which will be of general interest to all workers in the area.Articles in Natural Product Reports are commissioned by members of the Editorial Board or accepted by the Chairman for consideration at meetings of the Board. Contributors to Volume 13 Begley. Tadhg P. 177 Gordon Michael H. 265 Oberlies Nicholas H. 275 Bekker Riaan 41 1 Bentley Kenneth W. 127 Berlinck Roberto G. S. 377 Grayson David H. 195 Gu Zhe-Ming 275 Hanson James R. 59 227 529 Sammes Peter G. 1 Saxton J. Edwin 327 Shi Guoen 275 Connolly Joseph D. 151 Cox Russell J. 29 Drager Gerald 365 Faulkner D. John 75 Ferreira Daneel. 41 1 Fraga Braulio M. 307 Fukumoto Keiichiro 241 Gill Melvyn 513 He Kan 275 Herbert Richard B.45 Hill Robert A. 151 Ihara Masataka 241 Kirschning Andreas 365 Lewis John R. 171 435 Liddell J. Richard 187 McLaughlin Jerry L. 275 Thiericke Ralf 365 Thomas Neil R. 479 Williams Dudley H. 469 Yahioglu Gokhan 1 Ye Qing 275 Zeng Lu 275 Zerlin Marion 365 Nomenclature It is the policy of The Royal Society of Chemistry to en- courage the use of IUPAC" and IUBMB* Recommendations on nomenclature. Many of the appropriate nomenclature documents are included in the following compilations. Compilations 1 Nomenclature of Organic Chemistry Sections A B C D E F and H 1979 edition a 550-page hardcover volume published in 1979 available from Pergamon Press Oxford. Section F of this volume covers general principles for the naming of natural products.2 A guide to IUPAC Nomenclature of Organic Compounds (Recommendations 1993 a 182-page softcover volume published in 1993 available from Blackwell Scientific Publications Oxford to be used in conjunction with item 1. 3 Biochemical Nomenclature and Related Documents A Compendium 2nd edition 1992 a 348-page softcover manual published in 1992 by Portland Press Ltd for TUBMB and available from the publisher (59 Portland Place London W1N 3AJ UK). 4 Compendium of Chemical Terminology IUPAC Recornmendations a 456-page volume published in 1987 available in hardcover and softcover from Blackwell Scientific Publications Oxford. Specific Recommendations A selection of specific recent recommendations [many of which are included in Biochemical Nomenclature and Related Documents see above] that will be of particular interest to those who investigate bioorganic chemistry or the chemistry occurrence or biosynthesis of natural products includes Extension of Rules A-1.1 and A-2.5 concerning numerical terms used in organic nomenclature (Recommendations 1986) Pure Appl.Chem. 1986 58 1693-1696. [The original versions of these Rules are in Nomenclature of Organic Chemistry I979 edition see above] Nomenclature of steroids (Recommendations 1989) Eur. J. Biochem. 1989 186 429-458. Nomenclature of tetrapyrroles (Recommendations 1986) Pure Appl. Chem. 1987 59 779-832. Nomenclature and symbols for folic acid and related compounds (Recommendations 1986) Pure Appl. Chem. 1987 59 833-836; Eur..I. Biochem. 1987 168 251-253. Nomenclature of prenols (Recommendations 1986) Pure Appl. Chem.. 1987 59 683-689; Eur. J. Biochem.. 1987 167 181-184. Nomenclature of retinoids (Recommendations 198l) Pure Appl. Chem. 1983 55 721-726; Eur. J. Biochem. 1982 129 1-5. Nomenclature of vitamin D (Recommendations 1981) Pure Appl. Chem. 1982 54 1511-1516; Eur. J. Biochem. 1982 124 223-227. Nomenclature of tocopherols and related compounds (Recommendations 1981) Pure Appl. Chem. 1982 54 1507-1510; Eur. J. Biochem. 1982 123 473-475. Recommendations for the presentation of thermodynamic and related data in biology (1985) Eur. J. Biochenz. 1985 153 429-434. Enzyme Nomenclature (Recommendations 1984) Supplement 1 Corrections and additions Eur.J. Biochem. 1986 157 1-26. Enzyme Nomenclature 1992 (Recommendations of the Nomenclature Committee of the International Union of Biochemistry on the nomenclature and classification of enzyme- catalysed reactions) Academic Press Orlando Florida 1992. Nomenclature for multienzymes (Recommendations 1989) Eur. J. Biochem. 1989 185 485486. Symbolism and terminology in enzyme kinetics (Recommendations 1981) Eur. J. Biochem. 1982 128 281-291. Symbols for specifying the conformation of polysaccharide chains (Recommendations 1981) Pure Appl. Chem. 1983 55 1269-1272; Eur. J. Biochenz.. 1983 131 5-7. Polysaccharide nomenclature (Recommendations 1980) Pure Appl. Chem. 1982 54 1523-1526; Eur. J. Biochem. 1982 126 439-441. Abbreviated terminology of oligosaccharide chains (Recommendations 1980) Pure Appl.Chem. 1982 54 1517-1522; Eur. J. Biochem. 1982 126 433437. Nomenclature of glycoproteins glycopeptides and peptidoglycans (Recommendations 1985) Eur. J. Biochenz. 1986 159 1-6. Nomenclature and symbolism for amino acids and peptides (Recommendations I983) Pure Appl. Chenz. 1984 56 595-624; Eur. J. Biochem. 1984. 138 9-37 (see also Eur. J. Biochem. 1985 152 1 and the Newsletter 1985 of NC-IUB and JCBN ibid. 1985 146 pp. 238 and 239 and the Newsletter 1986 ibid. 1986 154 pp. 485 and 486). Nomenclature for incompletely specified bases in nucleic acid sequences (Recommendations 1984) Eur. J. Biochem. 1985 150 1-5 (see also Eur. J. Biochem. 1986 157 1). Abbreviations and symbols for the description of conformations of polynucleotide chains (Recommendations 1982) Pure Appl.Chem. 1983 55 1273-1280; Eur. J. Biochenz. 1983 131 9-15 (see also the Newsletter 1984 of NC-IUB and JCBN Eur. J. Biochrm. 1984 138 p. 7). A list of restriction endonucleases and their isoschizomers (updated annually) is given in R. J. Roberts and D. Macelis Nucleic Acids Res. 1991 19 2077-2109. Glossary for chemists of terms used in biotechnology Pure Appl. Chem. 1992 64 143. Selection of terms symbols and units related to microbial processes Pure Appl. Chem. 1992 64,1047. Organism Nomenclature Recent codes of nomenclature for organisms include International Code oj Nomenclature of Bacteria and Statutes oj the Internatiorial Committee on Systematic Bacteriology (1989 Revision) ed.P. H. A. Sneath V. B. D. Skerman and V. McGowan American Society for Microbiology Washington DC USA 1976. [Appendix 2 of this publication (Approved Lists of Bacterial Names) appeared in Int. J. Syst. Bacteriol. 1989 39.1 International Code of Botanical Nomenclature (I 988) ed. W. Greuter H. M. Burdett W. G. Chaloner V. Demoulin R. Grolle D. L. Hawksworth D. H. Nicholson P. C. Silva F. A. Stafleu E. G. Voss and J. McNeill Koeltz Scientific Books Konigstein Germany 1988. International Code of Zoological Nomenclature 3rd edn ed. W. D. L. Ride C. W. Sabrosky G. Bernardi R. V. Melville J. 0. Corliss J. Forest K. H. L. Key and C. W. Wright International Trust for Zoological Nomenclature in association with the British Museum (Natural History) London UK and the California Press Berkeley and Los Angeles USA 1985.* IUPAC International Union of Pure and Applied Chemistry. IUBMB International Union of Biochemistry and Molecular Biology.

ISSN:0265-0568    

DOI:10.1039/NP99613FP033

出版商:RSC    

年代:1996

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP99613BP041

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites Richard B. Herbert School of Chemistry University of Leeds Leeds LS2 9JT UK Reviewing the literature published in 1994 (Continuing the coverage of literature in Natural Product Reports 1995 Vol. 12 p. 445) 1 Pyrrolidine and Piperidine Alkaloids 1.1 Tropane Alkaloids 1.2 Cyclizidine and Swainsonine 1.3 Pyrrolizidine and Quinolizidine Alkaloids 2 Isoquinoline Alkaloids 2.1 Morp hinan A1 kaloids 2.2 Protoberberine and Benzophenanthridine Alkaloids 2.3 Colchicine and Ipecac Alkaloids 3 Metabolites derived from Tryptophan 3.1 Terpenoid Indole Alkaloids 3.2 Eudistomins Pyrrolnitrin Xenorhabdus Metabolites and Indolic Phytoalexins 4 Other Metabolites of the Shikimate Pathway 4.1 Pyoverdines and Gliovirin 4.2 Ephedra Alkaloids and TaxoP (Paclitaxel) 4.3 Pentabromopseudilin Ascomycin and Naphthomycin 4.4 DIMBOA 4.5 Phenazines and Actinomycins 5 P-Lactams 5.1 Clavulanic Acid and Valclavam 5.2 Penicillins 6 Miscellaneous Metabolites 6.1 And rimid Lac t acys tin and Eus ta tins 6.2 3-Nitropropionic Acid and Valanimycin 6.3 Tetraponerine-8 and Lysolipins 6.4 Nucleoside Antibiotics 6.5 Coronatine 7 References Year by year some groups of secondary metabolites remain like hardy perennials the subject of consistent and interesting research whilst some groups burst fresh upon the biosynthetic scene and flower briefly; both types can reveal exciting new aspects of secondary metabolism.Increasingly there is a shift to more biologically based investigations. One way or another it is a delight to read and report on the ever evolving biosynthetic studies of this group of nitrogen-containing metabolites. The time-honoured pattern of previous reports is continued in this year's account with appropriate specific reference to earlier reports in the series and to other reviews for background material.l-s Access to the literature was obtained in substantial measure through the excellent IS1 Data Service at Bath. The stereochemistry of enzymic reactions which take place in association with a-amino acids has been very nicely re~iewed.~ The biomolecular and genetic aspects of nicotine and iso- quinoline terpenoid indole and tropane alkaloids -for which there is now extensive such information -has been surveyed.'O 1 Pyrrolidine and Piperidine Alkaloids 1.1 Tropane Alkaloids The biosynthesis of the tropic acid (2) moiety in tropane alkaloids such as hyoscyamine (3) begins with phenylalanine (1).In the course of biosynthesis the skeleton of (1) rearranges such that the carboxyl group undergoes an intramolecular 1,2- (3) R = 'HYoH 0 ?H (4) R = pph 0 (5) R = 0 shift with retention of configuration at C-3 and that the %pro- S hydrogen of (1) migrates in the reverse direction.s Recently it has been shown that phenyllactic acid (6) is a key late intermediate; it is a more immediate precursor than phenyl- pyruvic acid (ref.5 p. 446). This latter conclusion has been confirmed by the results of an experiment with (RS)-[2-13C,-2-2H]-3-phenyllactic acid [as (6)].11 Incubation of this material with transformed root cultures of Datura stramonium gave labelled littorine (4) and hyoscyamine (3). Detailed examination (NMR MS) of the latter showed that the 13C-2H bond of the precursor was incorporated intact into the hydroxymethyl group of the tropate moiety in (3). Significant loss of deuterium was also observed for both (3) and (4)which may be attributed to interconversion of (6) and phenylpyruvic acid. Furthermore [1,2-13C2]phenyllactic acid [as (6)] was incorporated in D.stramonium into the tropic acid moieties of hyoscyamine (3) and scopolamine (7) in a manner such that the two labels become contiguous (labelling of C-1' and C-2'),12 i.e.intra-molecular 1,2-shift of the carboxyl group as observed for phenylalanine [see above cf. (I) (2) and (3)]. In transformed Datura root cultures tropic acid was found to inhibit alkaloid production whilst phenyllactic acid was less inhibitory; the former had little influence on the specific incorporation of phenyllactic acid which was an efficient alkaloid precur~or.~~ This indicates strongly that free tropic acid is not an alkaloid precursor and this is supported by additional definitive results. NMe (RS)-Phenyl[1,3-*3C2]lactoyl[rnethyl-2H3] tropine [as(4)] on incubation with root cultures gave quintuply labelled hyos- cyamine (3) and apoatropine (9 i.e.intact incorporation ; 45 some of the label in the alkaloids formed and of the littorine recovered arose from hydrolysis and recombination ;neither of the hydrolysis products phenyllactic acid and tropine when added to the cultures led to dilution of the quintuply labelled (3) and (5) which was produced; the rearrangement of the labelled phenyllactoyl moiety was observed in the expected way.14 Clearly littorine (4) can serve as a direct precursor in vivo for hyoscyamine (3) and is thus the substrate on which rearrangement can occur. (An additional path through the CoA ester of phenyllactate cannot yet be excluded. The early stages of tropane alkaloid biosynthesis can involve putrescine (8) as an intermediate (cf.ref. 3 p. 575) which becomes N-methylated to give N-methylputrescine as the next biosynthetic intermediate. Further work on the enzyme responsible putrescine N-methyltransferase has been reported. The enzyme was purified 700-fold and characterized. The latter included testing the activity of analogues of putrescine there was a requirement for two amino groups in a trans conformation separated by four carbon atoms; the C diamine cadaverine was a very poor substrate.l5 (For the ensuing stages of tropane biosynthesis see ref. 5 p. 445 and earlier reports.) Two tropinone reductases have been identified in vivo; one reversibly converts tropinone (9) into tropine (10) and the other yields Y-tropine [the C-3 epimer of (lo)] irreversibly (ref.4 p. 55). Both of these compounds have been found to accumulate in transformed root cultures of Atropa belladonna. Results of feeding experiments indicate that tropine and Y-tropine do not isomerize and only the former is incorporated into hyoscyamine (3).16 The two reductases have been purified from the transformed root cultures and characterized.l63 Basic simi- larities were seen with tropinone reductases from other sources mainly regarding kinetic properties and analogue acceptance ; there were also clear differences." NMe NMe 5l 0 (9) The gene for hyoscyamine 6P-hydroxylase [see (3)] from Hyoscyamus niger has been expressed in hairy routes of A. belladonna (ref. 5 p. 446). This gene fused to a reporter gene has been expressed in A.belladonna H. niger and Nicotiana tabacum.18 Expression of the hydroxylation gene was examined and it was concluded from the results that it is controlled by some genetic regulation specific to scopolamine-producing plants [6/?-hydroxyhyoscyamine is a precursor for scopolamine (711. Tropane alkaloid patterns in plants and hairy roots of Hyoscyamus albus have been examined in detail.Ig The incorporation of 14C-labelled phenylalanine ornithine and arginine into tropane alkaloids in suspension-cultured cells and aseptic roots of intact A. belladonna has been studied." The following have also been examined scopolamine production in root cultures of Duboisia myoporoides obtained by repeated selectionz1 and by a new two-stage culture method;22 the effects of oxygen on the production of nicotine and tropane alkaloids in cultured roots of D.my~poroides.~~ Anatoxin-a (1 1) is produced by a number of cyanobacterial species including Anabaena jlos-aquae. It has been found that like other pyrrolidine alkaloids,8 it is derived (in part) from arginine ornithine putrescine (13) and A'-pyrroline (12) (incorporation of 'T-labelled materials and identification of appropriate enzyme activities in A.fl~s-aquae).~~ NATURAL PRODUCT REPORTS 1996 N-H 1.2 Cyclizidine and Swainsonine Cyclizidine (14) is an unusual metabolite containing a cyclo- propane ring produced by a Streptomyces species. It is biosynthesized from three propionate units (this includes the provenance of the cyclopropane ring) and four acetates [see ( 14)].8-25 New evidence26 demonstrates (a) that the oxygen at C-2 is carried over from a molecule of acetate (presumably the other two oxygen atoms originate in aerial oxygen); (b) C-16 as well as C-9 and C-17 originate in C-3 of propionate; (c) in providing C-15 of (14) C-2 of propionate is utilized with retention of only one proton that is the 2-pro-S and in addition inversion of configuration occurs (determined with deuterium labelling).A mechanism consistent with these data is shown in Scheme 1; the initial steps account for observation (c). COSCoA COSCoA COSCoA )-H* )V-HR ->;H~* Me Hs* X S Scheme 1 Swainsona galegifolia produces the indolizine alkaloid swain- sonine.It has been found that the addition of copper sulfate reduction of medium pH and feeding with swainsonine precursors enhanced swainsonine production in transformed root cultures of S. galegifolia; stimulation of the release of swainsonine into the culture medium was also 1.3 Pyrrolizidine and Quinolizidine Alkaloids A notable contribution has been made to our understanding of the biosynthesis of pyrrolizidine alkaloids through the identi- fication in Senecio vulgaris of homospermidine synthase (HSS) which catalyses the combination of two molecules of putrescine (1 5) to give homospermidine (1 7) an intermediate in pyrrolizidine alkaloid biosynthesis. The reaction is NAD' dependent (ref. 5 p. 447). Welcome further progress has now been reported.28 Both plant and bacterial HSS have been examined.First it was shown that label from both (S)-and (R) -[1-*H]putrescine could be completely retained in the homospermidine produced that is to say hydrogen is transferred to and from C-1 of putrescine in what is essentially an intramolecular process (by way of NADSNADH). Some conflict with previous results using whole plants was noted. Second in the presence of putrescine (1 5) spermidine (1 6) could substitute for one of the molecules of (1 5) required to make (1 7). It is suggested that in NATURAL PRODUCT REPORTS 1996R. B. HERBERT the biosynthesis of pyrrolizine alkaloids in vivo half of (1 7) has its provenance in (15) the other in (16). The way it is deduced that the enzyme works is shown in Scheme 2.Protein N I I I I H2Nd NH3 + or H2N-NH2 Transaldimination I 1 I 1 NH2 I+ I I NH2 Scheme 2 Details were recently reported of a study on the biosynthesis of Lythraceae alkaloidsz9 (ref. 5 p. 447) and work has again been published in this area. Incorporation of alkaloid pre- cursors as [3H]lysine/[14C]phenylalanine and [3H]lysine/[14C]- p-coumaric acid has been used to establish relationships between the three alkaloid types found in Heimia salicifolia plants.30 Whereas within an alkaloid type both the cis- and trans-fused variations originate from a common metabolic pool the three types of alkaloid are formed from different precursor pools. Where they overlap the earlier resultszg are consistent with the findings reported here.30 Quinolizidine alkaloids are constituted from multiples of cadaverine (18) units two units for instance being used to construct lupinine (19).The fate of the cadaverine protons on /OH & 4 3 incorporation into a range of quinolizidine alkaloids has been examined in detail. Results for the incorporation of [l-2H]- and 4-zH,]Cadaverine [as (1 S)] gave lupinine (22) labelled as shown; (-)-sparteine was also isolated in this experiment and it had a labelling pattern similar to that found previously for the enantiomeric compound. The chirally labelled compounds (20) and (21) gave lupinine with the labelling patterns (23) and (24) respectively. It is notable that the carbon which becomes C-1 of (19) is associated with stereospecific retention of a 2-pro-R proton from cadaverine and loss of a 2-pro-S proton.Retention of deuterium at C-1 and C-3 means that imine/enamine equilibration does not occur during biosynthesis as it does during the biosynthesis of other quinolizidine alkaloids such as sparteine with consequent deuterium loss. R' d:: R2 D~; D (20)R' = D;R2 = H (21) R1=H; R2=D Work on actyltransferases associated with the biosynthesis of quinolizidine alkaloids (ref. 5 p. 447) has been extended with the isolation purification (to homogeneity) and charac- terization of a novel 0-trigoyltransferase from Lupinus termis seedlings.33 Two isoforms of the enzyme were identified both of which were specific in catalysing transfer of tiglic acid from tigloyl CoA to (-)-13a-hydroxymultiflorine (25) thus yielding (26) and to (+)-13a-hydro~ylupanine.~~ Both of these compounds are tetracyclic with an axial hydroxy group at C- 13 and are of 7S,9S configuration.No enzyme activity was detected towards the 7R,9R alkaloid (-)-baptfoline or towards the bicyclic quinolizidine alkaloids (+)-epilupinine and (-)-lupinhe. (-)-3P-Hydroxy- 13a-tigloyloxylupanine has been isolated as a new alkaloid from seedlings of Cytisus scoparius and tigloyltransferase activity associated with the formation of the alkaloid was detected in cell-free extracts of this plant.34 Clarification has been given to the interspecies distribution of acyltransferases in relation to alkaloid pattern in the various plants.33 2 lsoquinoline Alkaloids 2.1 Morphinan Alkaloids In a remarkable body of work nigh on all the enzymes involved in the biosynthesis of morphine have been described (ref.5 p. 446; and earlier reports). One of the remaining enzymes has been isolated this past year (from poppy cell cultures) and has been purified to homogeneity and characterized. It catalyses the closure of the oxide bridge in the conversion of salutaridinol (27) into thebaine (29)3s and there is a surprise associated with this. Enzyme activity was found to be associated with intact [3-zH]cadaverine into lupinine have been reported previo~sly.~~ mitochondria. Since intact mitochondria are known to generate In new work the incorporation of [2-2H]cadaverine [as( 18)] ATP this suggested that phosphate might serve as a leaving into lupinine in Lupinus luteus has been [2,2,4 group in the ring closure.But when an enzyme preparation NATURAL PRODUCT REPORTS 1996 was incubated with ATP/Mg2+ or any other nucleoside triphosphate conversion was not enhanced. On the other hand use of a cofactor mix which included coenzyme A resulted in a 100°/~ enhancement. Quite surprisingly it turns out that the enzyme (acetyl coenzyme A salutaridinol-7-0-acetyltrans-ferase) is acetyl CoA dependent and that salutaridinol 7-0-acetate (28) is formed which then undergoes spontaneous ring closure in vivo at slightly alkaline pH to give (29) with acetic acid as the leaving group [an enzyme catalysing this step could not be found; the C-7 epimer of (27) was inert with the acetylating enzyme].It may be noted that one other step in morphine biosynthesis namely the conversion of neopinone into codeinone (ref. 5 p. 447) is also spontaneous. Me0 HO& Mz$,NMe - NMe Me0 ,' Me0 Hd H Acd H 1 Me0 ' Me0 S\ NMe 2.2 Protoberberine and Benzophenanthridine Alkaloids The biosynthesis of sanguinarine (3 1) involves many steps the last of which takes place with the oxidation of dihydro- sanguinarine (30).36 Five further enzyme-catalysed steps beginning with (30) lead to macarpine (36) (Scheme 3). The first L6 (30) Lo (32) R=H 1 E(33) R = Me I (34) R=H C(35)R=Me J OMe Scheme 3 two steps involve hydroxylation to (32) then 0-methylation to (33).The enzymes for these steps have been characterized; they are highly substrate specifi~.~'Two novel enzymes dihydrochelirubine- 12-hydroxylase and SAM 12-hydroxy-dihydrochelirubine- 12-@methyltransferase have been found in cell-free extracts of yeast-elicited ThaZictrum bulgaricum (and also Eschscholtzia calif~rnica).~~ They are responsible for the next two steps (33) +(34) +(35) in macarpine biosynthesis. The hydroxylase is a microsomal-associated cytochrome P-450-dependent mono-oxygenase specific for C-12 of (33). The 0-methyltransferase was purified; it appears to be highly specific for (34) as substrate. The last step (35) +(36) is catalysed by non-specific dihydrobenzophenanthridine oxi-da~e.~~ In the biosynthesis of macarpine (36) highly substrate- specific microsome-bound cytochrome P-450 enzymes play a major role in catalysing a number of steps.All of these enzymes are activated (three to more than ten-fold) by yeast elicitation. On the other hand the cytosolic methyltransferases are not activated by the elicitation process. It is that there is interplay between cytosolic and membrane-associated enzymes which may have regulatory roles. The bio-synthesis of macarpine has been authoritatively reviewed.40 Evidence has been obtained that an external source of calcium ions is required for benzophenanthridine alkaloid accumulation which has been induced by a fungal elicitor in cell-suspension cultures of Sanguinaria canadensi~.~~ was It suggested therefore that calcium and possibly calmodulin and/or protein kinase C may participate in a signal transduction system which leads to benzophenanthridine alkaloid pro-duction.Early in the course of the biosynthesis of benzyl- isoquinoline alkaloids e.g. berberine (39) norcoclaurine (37) undergoes 0-methylation to give (38) (ref. 2 p. 512; see Scheme 8). The effect of cytochinins on the activities of early biosynthetic enzymes in ThaZictrum minus cell cultures has been examined and it has been found that of these enzymes norcoclaurine-6-0-methyltransferasewas markedly activated by cytochinins and especially by 6-ben~ylaminopurine.~~ The results suggest that the induction of berberine production in these cell cultures by cytochinins is primarily attributable to the increase in this 0-methyltransferase activity.Other work has been reported on the stimulation of berberine production in T. minus cell "H <% HO 0Y ''H 0 OMe ' HO '*' OMe (37) R = H (39) Berberine (38) R = Me S-Adenosyl-L-methionine:tetrahydroberberine-cis-N-methyl-transferase catalyses the specific cis-N-methylation of e.g. canadine (40) to give the N-methyl derivative (41). This 0 OMe OMe uOMe uOMe enzyme has been purified to homogeneity from S. canadensis cell cultures and has been ~haracterized.~~ Of several substrates tested the enzyme was only fully active with (40) that is to say it is highly substrate specific; surprisingly stylopine (42) was not a substrate (cf. ref. 40). Previously an N-methyltransferase NATURAL PRODUCT REPORTS 1996-R.B. HERBERT catalysing the same reaction had been partially purified from E. ~alifornica.~~ Stylopine(42)and canadine (40)were found to be equally effective and the best substrates for this enzyme. The relationship of the enzymes from the two sources remains to be explored further. It has been that the results with the E. californica enzyme arise from the use of partially purified protein. Stylopine (42) is biosynthesized in two steps from scoulerine (43) each step involving the formation of a methylenedioxy group by oxidation of an O-methoxyphenol. The two enzymes from E. californica,which are involved have been characterized they are P-450 enzymes (ref. 3 p. 578; ref. 2 p. 511) and thus ring closure probably involves (formally) the radical (45) rather than the cation (46).A P-450enzyme has been detected in microsomal preparations from different Ranunculaceae and Berberidaceae cell cultures and partly characterized in prep- arations from a Thalictrum tuberosum cell line.46 This enzyme catalyses the conversion of (S)-tetrahydrocolumbamine (44) into (Qcanadine (40) and is hghly specific for this substrate. This conversion represents the penultimate step in the bio- synthesis of berberine (39) and this enzyme is the last to be characterized in the biosynthetic pathway. The substrate specificity of the enzyme confirms the deduced role for canadine (40) as an intermediate in berberine biosynthesis. The cDNA from E. californica that encodes berberine-bridge enzyme (see e.g.ref 1 p. 188) has been heterologously expressed in a cell culture of the fall army worm Spodoptera fr~giperda.~’ The expression resulted in overproduction of the plant enzyme in a catalytically active form. Jasmonic acid (47) is deduced to be an integral part of a general signal transduction system which regulates inducible defence genes in plants (ref. 5 p. 449). Induction of berberine- bridge enzyme was part of this study. It has likewise been part of a study in which coronatine (48) was found to mimic octadecanoid (jasmonic acid) signalling in higher plants ;(48) does not elicit the accumulation of endogenous jasmonic acid. Most interestingly modelling reveals that coronatine is a structural analogue of the cyclopentane octadecanoid pre- cursors [as (49) cis isomer] of jasmonic 0 (49) Clear evidence has been obtained that tetrahydroproto- berberine alkaloids with the unusual 14-R configuration [as (50)] are derived by reduction of alkaloids which are at the oxidation level of berberine (39) (ref.3 p. 578). This has now been confirmed in precursor feeding experiments in Corydalis cava for corydaline (51) tetrahydrocorysamine (52) and thalictricavine (53).49 A partially enriched protein fraction from C. cava was found to catalyse reduction and methylation of berberine (39) to give (53) (other protoberberines showed varying substrate acceptability). The conversion is dependent on SAM and NADPH (B-type reductase). 7,8-Dihydro- berberine (54) was an excellent enzyme substrate.Thus the sequence to e.g. thalictricavine (53) is very reasonably (39) + (54) -+ (53). (51) R1 = R2= R3 = R4 = Me (52) R1R2= R3R4= CH2 (53) R1R2= CH2; R3 = R4 = Me The X-ray crystal structure of a salt of (-)-corycavinium (56) has been carried This alkaloid and its desoxy derivative (55) were shown to be efficiently transformed into corynoline (57) and its C-14 epimer in tissue cultures of C. cava;(55) was also transformed into (56). (For earlier related work see ref. 5 p. 450; ref. 2 p. 514; ref. 1 p. 189.) (55) R = H (SS)R=OH The aberrant bioconversion of unnatural 2'-aminoreticuline (58) into unnatural alkaloids such as 12-aminoberberine [as (39)] has been reported; incorporation as measured by radioactivity was less than 0.7 %.51 MeomNMe HO OMe 2.3 Colchicine and the Ipecac Alkaloids The bioconversion of colchicine and thiocolchicine into their 3-demethyl derivatives has been studied in plant cell and bacterial cultures.52 The effect on cephaeline and emetine production of adding known precursors of ipecac alkaloids to tissue cultures of Cephaelis ipecacuanha has been 3 Metabolites derived from Tryptophan 3.1 Terpenoid Indole Alkaloids The cDNA encoding strictosidine synthase from RauwolJia serpentina has been heterologously expressed in a cell culture of the fall army worm.47 It has been shown that the indole-diterpenoid paxilline (59) is an efficient precursor for the penitrems of Penicillium janczewskii e.g.penitrem A (60) (ref. 1 p. 192). Recent results confirm this finding; (59) was a similarly efficient precursor for the janthitrems of Penicillium janthinellum e.g. janthitrem B (61).54 Excellent incorporations of 1OP-hydroxypaxilline (62) into both series of metabolites were also observed; the 10a- epimer of (62) was poorly incorporated which indicates that (62) is normally involved in biosynthesis with enzymic attention being paid to the stereochemistry at C-10. 10P-0-Acetyl- paxilline was incorporated at a modest level presumably after in vivo hydrolysis to (62). 7a-Hydroxy- 13-desoxypaxilline [as (59)] and 10P-hydroxy- 13-desoxypaxilline [as (62)] have been identified as novel metabolites of P. pa~illi.~~ 7a-Hydroxy-paxilline [as (59)] was isolated from P.paxilli and was shown to derive from paxilline. Acremonium lolii was found to produce metabolites similar to P. paxilli. Possible biosynthetic relationships between the different indole-diterpenoids have been 3.2 Eudistomins Pyrrolnitrin Xenorhabdus Metabolites and Indolic Phytoalexins Results of feeding experiments with radioactive precursors demonstrate that eudistomin I (63) is biosynthesized in the Floridian tunicate Eudistoma olivaceum from L-tryptophan via tryptamine and from L-proline ; L-ornithine and L-arginine were not used significantly for bio~ynthesis.~~ Aromatic nitro groups are found rarely in secondary metabolites examples being chloramphenicol,8 obafluorin (ref. 4 p. 60) and pyrrolnitrin (66).s These nitro groups arise by oxidation of amino functions and a valuable preliminary contribution to our understanding of what is going on has been NATURAL PRODUCT REPORTS 1996 H (59) H H OH made.A chloroperoxidase has been isolated from cultures of the pyrrolnitrin producer Pseudumonas pyrrocinia which chlori- nates (64) to give (65).57 The pure enzyme (cloned and overexpressed) has now been found to oxidize (65) into pyrrolinitrin (66) in the presence of hydrogen peroxide.58 Further developments are awaited with interest. (For recent work on the formation of 3-nitropropionate see Section 6.2). Tryptophan (68) is incorporated with retention of C-3 and loss of C-1 into the indole metabolites (67) of the entomo- pathogenic bacterium Xenorhabdus nematophil~s.~~ 0 (67)R' = H or COMe R2= H or Me NATURAL PRODUCT REPORTS 1996-R.B. HERBERT SMe H L-Cysteine Other indolic phytoalexins ii (68)L-Tryptophan H (72) H (73) SMe Scheme 4 The biosynthesis of the cruciferous phytoalexins brassinin (71) cyclobrassinin (73) and spirobrassinin (72) has been examined in detail in UV-irradiated turnip slices. Preliminary results (ref. 3 p. 580) are now incorporated in a full paper.6o The combined data allow a clear description of the biosynthetic pathway to be that shown in Scheme 4. A key putative intermediate is (70) and there is new persuasive evidence that it is a normal turnip metabolite. Thus a turnip homogenate when incubated with sodium methanethiolate gave (71) and also (74); neither metabolite was produced in the absence of methanethiolate.Also the moderately reactive benzyl isot- hiocyanate gave (75) on incubation with the homogenate. L-[methyl-3H,35S]Methionine afforded doubly labelled (71) and (72) without change of isotope ratio i.e. the methylthio group in these metabolites arises intact from methionine an unusual observation. Products obtained from the metabolism of (76) support a role for the epoxide (77) (or equivalent) in the steps beyond (71). Finally a close relationshp in the biosynthesis of glucosinolates [as (69)] and that of the phytoalexins has been deduced. The completed pathway whch involves a key rearrangement of the tryptophan (68) skeleton (see * atoms) is shown as Scheme 4.H PhT%N IfSMe S (74)n= 2 (75)n= 1 ~-@-~~C]Tryptophan and ~-['~CH,]rnethionine have been found to be incorporated into the phytoalexins cyclobrassinone and 1-methoxyspirobrassin in UV-irradiated slices of tubers of kohlrabi6' This is of course consistent with the foregoing. On the other hand in Arabidopsis thaliana it has been found62 that the phytoalexin camalexin (78) is formed directly from 9 H H2N-NmoH N 'OH H oHN \ AH NH2 (79) anthranilic acid rather than its later metabolite tryptophan. It was concluded that the pathway to (78) diverts from an intermediate in primary metabolism that lies between an-thranilic acid and indole. H H (77) (78) 4 Other Metabolites of the Shikimate Pathway The biosynthesis of glucosinolates and cyanogenic glycosides is the subject of detailed discussion in a companion 4.1 Pyoverdins and Gliovirin The biosynthesis of the pyoverdins (ref.4 p. 61) has been reviewed.64 Pseudobactin (79) is a member of this group of metabolites. The aromatic moiety of (79) has been shown to originate in Pseudomonas fiuorescens from DL-tyrosine [as (80)].65 Dopa is not a precursor so further aromatic hy- droxylation occurs after joining of the tyrosine residue to at least part of the rest of the molecule. Diketopiperazine metabolitesg constitute a diverse and interesting group of metabolites. One such metabolite is gliotoxin (82) which has been extensively investigated (ref. 3 p. 580); it originates in part in phenylalanine.Gliotoxin (82) and gliovirin (83) are produced by different strains of the fungus Gliocladium virens. It has now been shown66 that (83) is formed from two molecules of L-phenylalanine (8 1) (labelling of C-1 and C-3 of (83) by the [l-13C]-labelled amino acid). Rm:H (80)R= OH (81) R = H OMe NATURAL PRODUCT REPORTS 1996 4.2 Ephedra Alkaloids and TaxoP (Paclitaxel) A full paper has appeared6’ on the biosynthesis of Ephedra alkaloids whch proceeds via a novel route from phenylalanine by way of benzoic acid (as its CoA ester?) (ref. 5 p. 456). The origin of the N-benzoylphenylisoserine side chain in TaxoP (84) has been the subject of recent scrutiny (ref. 5 p. 455). It has now been demonstrated by making elegant use of multiply deuteriated precursors that the sequence of side- chain attachment in Taxol biosynthesis is baccatin-I11 (86) (intact incorporation of 10-acetyl-2H, 13-2H,-labelled material) HN4’ +(87) (intact incorporation of material deuteriated thrice in the acetyl group and five-fold in the phenyl ring) -P Taxol (84).6s N-Benzoylated phenylisoserine when used as a pre-cursor was hydrolysed prior to incorporation.It was established H H that cephalomannine (85) is formed by N-acylation of (87) (90) (Scheme 5). 0 PhKNH 0 (84) R= MeoYYMe PhU Taxow AH 0 0 (85) R = I OH (86) R =H *p* Me *‘OH (91) t NH2 0 0 PhKSCoA OH Hd (87) I Scheme 5 4.3 Pentabromopseudilin Ascomycin and Naphthomycin Pentabromopseudilin (89) is a potent marine antibiotic which has been isolated together with violacein (90) from Chromo-Hca; bacteria and Alteromonas luteoviolaceus.[For the bio-synthesis of (90) see ref. 5 p. 454.1 Exploratory feeding Hd . OH experiments with differently labelled glucose samples in cultures OH of A. luteoviolaceus showed that the benzene ring of (89) has its provenance in carbohydrate metab~lism.~~ The labelling pat- terns observed for (89) and (90) were unexpected but they could reasonably be attributed to a lack of triosephosphate isomerase in the organism. The results did show however that the shikimate pathway was involved [presumably due to per- meability problems shikimic acid itself was incorporated into neither (89) nor (90)].The labelling patterns for (89) indicated that a symmetrical intermediate was implicated in its bio- synthesis. This was identified as p-hydroxybenzoic acid (88) and it was firmly identified as a biosynthetic precursor in feeding J experiments with 2H-and lT-labelled (88). Curiously the pyrrole ring was not labelled by labelled samples of acetate tryptophan benzoic acid glycerol or glucose.69 -(91) Following work on related metabolites the biosynthesis of the dioxygenated cyclohexane ring in ascomycin (91) has been studied.’* [2-13C]Shikimic acid [as (92)] specifically labelled OH C-34 of (91). Further experiments were carried out with Scheme 6 NATURAL PRODUCT REPORTS 1996R. B. HERBERT deuteriated shikimic acid samples from the results of which the sequence and stereochemistry of the reactions could be deduced; the two acids (93) and (94) were also found to act as precursors and can be located as biosynthetic intermediates.It is interesting to note that there are significant stereochemical differences between this case (Scheme 6) and that of cyclohexanecarboxylic acid which also originates from shikimic acid (ref. 5 p. 456). Results of an extensive study with 13C-labelled precursors demonstrate that naphthomycin A (95) is assembled in Streptornyces collinus via a polyketide pathway from 3-amino- 5-hydroxybenzoic acid (96) as a starter unit plus seven propionate and six acetate chain extension units as A m-C,N unit [as (96)] is common to a number of antibiotics8 and this unit may or may not derive via the shikimate pathway (ref.4 p. 59; ref. 3 p. 584; ref. 2 p. 522; ref. 1 p. 195). Results of a feeding experiment with [13C3]glycerol establish that the rn-C,N unit in (95) is elaborated via the shikimate pathway (see ref. 4 p. 59 for details of the likely pathway). Finally it was shown that [7-13C]-3-amino-5-hydroxybenzoic acid [as (96)] was efficiently and specifically incorporated into (95) (labelling of C-27).'l H0$ 4.4 DIMBOA DIMBOA (99) is a major defence compound in maize. It has long been known to derive from anthranilic acid (97) and ribose. Recently it was shown that tetradeuterio-anthranilic acid (98) was incorporated into (99) with retention of three deuterium atoms consistent with entry of a hydroxyl group at C-1 by a mechanism of hydroxylative decarb~xylation.~~ The effect of growing cultures of maize in D,O has been explored.73 Glucosyltransferase and N-hydroxylase activity associated with DIMBOA biosynthesis has been identified in maize and ~haracterized.,~2H- 1,4-Benzoxzin-3(4H)-one (101) has been valine and good evidence, has been obtained that the necessary inversion of configuration in the course of biosynthesis occurs in the enzyme-bound form of the amino acid.This is of likely relevance to the biosynthesis of other peptidic antibiotics which contain D-amino acids e.g. the penicillins (Section 5.2) and virginiamycins. 5 B-Lactams 5.1 Clavulanic Acid and Valclavam Beautiful work has been reported on the biosynthesis of clavulanic acid (1 06) in Streptomyces clavuligerus most recently the stages leading up to the intermediate claviminic acid (102) (ref.5 p. 458). Two things need to happen in the conversion of (102) into (106); that is to say change of an amino group to a hydroxyl [the 9-oxygen in (106) derives from molecular oxygen indicating the transformation is oxidative] and a curious inversion of stereochemistry at C-3 and C-5. These points are reasonably accommodated by proposing that the aldehyde (103)/(105) is an intermediate between (102) and (106); the change in stereochemistry could occur simply via (104). This aldehyde has now been identified as present in S. clavuligerus cultures and it has the (3R,5R) stereochemistry (105).78 Further an enzyme clavulanic acid dehydrogenase has been isolated from S.clavuligerus which converts (105) into clavulanic acid (106) in the presence of NADPH. The enzyme has been purified and the N-terminal sequence has been determined (it is PSALQGKVALITGASSGIGE -amended seq~ence'~). Evi- dence was obtained that the benzyl ester of (105) undergoes slow spontaneous racemization in solution presumably via the ester of (104). All this evidence points strongly at the aldehyde (105) being an intermediate in clavulanic acid biosynthesis. The aldehyde (105) undergoes ready decarboxylation thus providing a reasoned entry to non-carboxylated clavam~.,~ H H m 0-C02H c02x firmly identified as an intermediate in DIMBOA bio~ynthesis.~~ It was an efficient precursor for (99) and could be isolated in a radioactivity trapping experiment.Further it was converted into (100) in an NADPH- and oxygen-dependent reaction with maize microsomes. R A H (97) R = H (99) R=OMe (101) (98) R = 0 (100) R=H 4.5 Phenazines and Actinomycins The genetic cloning of a phenazine biosynthetic locus from Pseudomonas aureofaciens and analysis of its expression has been reported.s6 Multifunctional actinomycin synthase I1 assembles the intermediate containing the first three residues of the acti- nomycin molecule namely 4-methyl-3-hydroxyanthranilic acid L-threonine and D-valine. The enzyme activates L- but not D- Claviminic acid synthase (CAS) is responsible for the two- step conversion of proclaviminic acid (109) into claviminic acid (102) which occurs via (1 10).CAS also catalyses the earlier step whereby (107) is converted into (108). Following upon earlier work (ref. 5 p. 458; ref. 80) two isozymes of CAS have been purified from S. clavuligerus.81One of the isozymes has been cloned and expressed in Escherichia coli. The recombinant enzyme was able to catalyse the above three steps thus confirming a trifunctional role for the enzyme. U 0JPNKNH2 NH C02H (107) R = H (108) R=OH NATURAL PRODUCT REPORTS 1996 During the purification of ACV synthetase (penicillin biosynthesis) from S. clavuligerus a small protein corre-sponding to an amidinohydrolase was identified which is essential for clavulanic acid production.82 The corresponding gene was located near the penicillin cephamycin and clav- ulanic acid biosynthetic genes.It seems that this protein may be the same as PAH which catalyses the conversion of (108) into (109) (ref. 5 p. 458). Valclavam (1 11) is produced by Streptomyces antibioticus ssp. antibioticus Tii718. Primary precursors83 for (1 11) are L- valine the methyl group of L-methionine a C pool metabolite and significantly arginine rather than ornithine (as for clavulanic acid ref. 5 p. 458). Work by others shows that proclaviminic acid (109) and valine are specific precursor^.^^ A link between the biosynthesis of clavulanic acid and that of (1 11) is apparent. This is strengthened by the identification in S. antibioticus of clavulinate biosynthesis enzymes namely CAS and PAH.85 5.2 Penicillins A review on the reactions of non-haem iron with dioxygen in biology and chemistry includes a discussion of the mechanism of action of isopenicillin N synthase.86 Another review which is on 2-oxoglutarate dependent dioxygenase and related enzymes includes a discussion of penicillin and cephalosporin biosynthesi~.~~ A stimulating essay has been publisheds8 on genetic engineering in the synthesis of natural products.It includes penicillins and cephalosporins. Biomimetic conversion of ACV tripeptide analogues into p-lactams has been rep~rted.~’ ACV synthetase couples together L-a-aminoadipate L-cysteine and L-valine to give L-&(a-aminoadipo1y)-L-cysteinyl-D-valine (ACV). In the search for analogues acceptable as substrates for the synthetase it has been founds0 that (S)-carboxymethylcysteine is an effective substitute for a-aminoadipate and both allylglycine and vinylglycine could substitute for cysteine indicating that the thiol group of the latter is not essential for peptide formation.L-Allo-isoleucine but not L-isoleucine could effectively substitute for valine. In common with other non-ribosomal peptide synthetases ACV synthetase appears to have a relatively broad substrate specificity. Amino acid sequence alignment of the Cephalosporium acremonium isopenicillin N synthase (IPNS) to similar non- haem Fe2+ containing enzymes from 28 different sources reveals a homologous region of high sequence conservation with an invariant histidine residue at position 272 in IPNS.’l Site-directed mutagenesis for IPNS confirms the importance of this amino acid residue it is essential for catalytic activity.For related spectroscopic work see ref. 3 p. 585. Isopenicillin N is the progenitor of other penicillins with different side chains the conversion of the former into the latter involving replacement of the L-a-aminoadipoyl chain in isopenicillin N and this is catalysed by an acyltransferase. The effect of site-directed mutagenesis on proenzyme cleavage and the catalytic activity of the acyl coenzyme A isopenicillin N acyltransferase from Penicillium chrysogenum has been stud- ied.s2 A separation of proenzyme cleavage and catalytic activity was noted. A broad-range disulfide reductase has been isolated from P.chrysogenum which when coupled with IPNS results in the conversion of the disulfide form of ACV into isopenicillin N.93 It is suggested that the reductase might have a normal role in penicillin biosynthesis. Expression of penicillin biosynthetic genes in P. chrysogenum has been found to be regulated by nitrogen repression glucose repression and growth stage a-Aminoadipic acid is a key building block in the biosynthesis of penicillins. It is known to be synthesized in P. chrysogenum from a-ketoglutaric acid and acetyl CoA. It has now been found that catabolism of lysine which may occur by two different routes also affords a-aminoadipic acid in this organism.s5 6 Miscellaneous Metabolites The levels of caffeine and threobromine and the metabolism of [8-14C]adenine in developing leaves of C0Jg-a arabica have been examined.s6 Soluble glyucosyltransferase(s) have been identified in Solunum melongena leaves which catalyse the glucosylation and galactosylation of solasodine and diosgenin and related 6.1 Andrimid Lactacystin and Eustatins Results of experiments with precursors labelled with stable isotopes clearly define the origins of the unusual acylsuccinimide moiety of andrimid (112) in cultures of a marine isolate of Pseudomonas $uorescens ; excellent incorporations were ob- tained.ss [1-l3C]Valine labelled C-1’ of (1 12) [13C2]acetate labelled C-2 and C-3 and curiously C-6 (also C-1”’ through C-8”’,but not C-4 and C-5) whilst glycine provides C-4 C-5 and the nitrogen atom as an intact unit.A pathway consistent with the results is shown in Scheme 7.98 Scheme 7 NATURAL PRODUCT REPORTS 1996R. B. HERBERT Interestingly [1,2-13C,]glycine in addition to giving doubly labelled (1 12) also gave some material whch bore a single label at C-5 (corresponds to C-2 of glycine). This is consistent with operation of the tartronic semialdehyde and glyoxylate path- ways recently observed in terrestrial P. fluorescen~.~~ Streptomyces sp. commonly elaborate metabolites with unique structures. Such a metabolite is lactacystin (1 13). Study of its biosynthesis with precursors labelled with stable isotopes shows that C-4 C-5 C-9 C-10 C-11 and C-12 have their genesis in L-leucine the cysteine unit derives from L-cysteine and C-6 C-7 C-8 and C-13 are provided by isobutyrate ([l-13C]isobutyricacid was found to label C-8 C-1 C-4 and C-14; the latter three labelling sites are accounted for by unexceptional isobutyrate metabolism).loO It is suggested that isobutyrate condenses with leucine via methylmalonic semialdehyde.eH HOpC. ,N. ' $OH lsobutyrate A 12 A Leucine L-Leucine and L-ornithine are precursors for eurystatins A (1 14) and B (1 15) in cultures of Streptomyces eurythermus."' Valine and isoleucine could be substituted for leucine thus affording new eurystatins. H (114) R= Me (115) R= Et 6.2 3-Nitropropionic Acid and Valanimycin Penicilliurn utrovenetum biosynthesizes 3-nitropropionic acid (119) from L-aspartic acid (116) (ref.2 p. 206; ref. 4 p. 64). The incorporation of the diethyl ester (117) into (119) after anticipated hydrolysis in the culture indicates (118) is a biosynthetic intermediate. It has now been shown that hydrolysis of racemic (1 17) by pig liver esterase (chosen for mild hydrolysis) affords (1 19).lo2 Since no decarboxylase activity could be associated with the esterase decarboxylation of (1 18) formed in the hydrolysis must be spontaneous. This raises the question as to whether decarboxylation of (118) is normally spontaneous in P. atrovenetum or is enzyme catalysed. Attempts to resolve this question failed. (For other work on the formation of nitro groups see Section 3.2.) fC02R (117) R= Et (118) R= H The provenance of valanimycin (124) in Streptomyces viri- difaciens is in isobutylamine (1 20) via isobutylhydroxylamine (121) and serine (122) (ref.4 p. 64). The role of the hydroxylamine (1 2 1) in biosynthesis has been strongly sup- ported by the identificationlo3 ofenzyme activity in s.viridifuciens which catalyses the conversion of (120) into (121) in the presence of FAD plus NADH. Of a range of amines tested (120) was the best substrate. Specific incorporation of [4-13C]( 123) and incorporation of [15N,]( 123) without fracture of the N-N bond establishes (123) formed reasonably from the reaction of (121) and (122) as an intermediate in valanimycin biosynthesis.lo3 (121) R =OH (123) 6.3 Tetraponerine-8 and Lysolipins The tetraponerines are a group of tricyclic defence alkaloids produced by Tetraponera ants.Feeding experiments (using about a thousand ants per batch) with 14C-labelled compounds indicate that the origins of tetraponerine-8 (125) are as follows.1o4 The pyrrolidine ring arises from glutamate via L-ornithine and putrescine whilst the remaining 12-carbon chain derives from a combination of six acetate units. HH The lysolipins e.g. lysolipin X (126) are members of a small family of xanthone metabolites. ResultsLo5 of a study of the biosynthesis on the lisolipins revealed two particularly in- teresting features. First the oxygen incorporation pattern [see (126)] was unexpected and will repay further investigation. Second the skeleton is constructed entirely from malonate. It serves as starter unit (C-21 C-23 C-24) for what turns out to be a single polyketide chain.The use of malonate here is in the opposite sense to that in cycloheximide and the tetracyclines; that is to say the activated carbon of malonyl CoA is attached to the ring nitrogen of (126) whereas the C0,-derived carbon atom is the one which initiates the polykedite chain. Further results are awaited with interest. *o-o* 6Me Me 6.4 Nucleoside Antibiotics A-Factor is a small y-lactone which induces streptomycin production in Streptomyces griseus. Based on recent work a model has been proposedlo6 for an A-factor regulation cascade which ultimately switches on the streptomycin biosynthetic genes.lo6 Quite unusually the uracil moiety in sparsomycin (131) origmates in tryptophan (1 27).A plausible route to (1 3 1) would be by way of the kynurenine pathway of tryptophan degra- dation whch involves scission of ring B ; in the case of (13 1) scission of ring A would follow. However two intermediates on this pathway namely N-methylanthranilic acid (ref. 4 p. 58) and N'-formylkynurenine (1 28),lo7 were not intact precursors for (1 3 1) in Streptomyces sparsogenes. This argues against the involvement of the kynurenine pathway in sparsomycin biosynthesis and an alternative is favoured where ring A of (127) is cleaved before ring B.lo7 The pyrimidine (1 29) is a specific precursor for sparsomycin (1 3 1) (ref. 4 p. 58) and its potential normal intermediacy in the biosynthesis of (1 3 1) is considerably strengthened by isolating an enzyme from S.sparsogenes that catalyses the conversion of (129) into (1 30). The enzyme requires NAD' and it is suggested that its mechanism of action is similar to that of IMP dehydrogenase which catalyses the conversion of inosine 5'- monophosphate (IMP) into xanthosine 5'-rnonopho~phate.'~~ 0 0 It has been shown recently that neplanicin A (132) is an intermediate in the biosynthetic pathway to aristeromycin (133) in Streptomyces citricozor (ref. 5 p. 460). In strong support of this enzyme activity has been identified in and partially purified from S. citricolor that catalyses the NADPH-dependent reduction of (132) to (133).lo8 It was shown further that reduction proceeds with anti geometry and involves the 4- pro-R hydrogen atom of NADPH (Scheme 8).In the search for intermediates between glucose and neplanicin A/aristeromycin it has been found that the tetraol (134) is not an intact precursor for aristeromycin (133); the incorporation was very 10w .lo9 *HR Hs NATURAL PRODUCT REPORTS 1996 sine D-glucose L-a-arginine and the methyl group of meth- ionine. Carefully chosen enzyme inhibitors have been used to distort biosynthesis in S. griseochromogenes."O Three known previously minor metabolites [including (1 36) and (1 37)] and two new ones accumulated in usefully substantial quantities. This aided elucidation of the biosynthetic intermediates en route to (135) (137) is the first nucleoside intermediatelll and (136) is the last intermediate;l12 the mechanism and stereo- chemistry of C-3' deoxygenation has been e~tab1ished.l~~ (135) R=Me (136)R = H 6.5 Coronatine Coronatine (138) which has a quite unique structure is a phytotoxin produced by many pathovars of Pseudomonas syringae.The coronafacic acid (139) moiety is a polyketide derived from three acetate units one butyrate unit and one pyruvate unit; the two oxygen atoms are acetate oxygens. The coronamic acid (141) portion is formed from L-isoleucine by way of L-alloisoleucine (140); the nitrogen atom of the latter is retained into (141). Cyclopropane formation occurs with loss of the C-2 proton and one proton from C-6; a C-6 hydroxy derivative is not involved. A mechanism for ring closure involving iron 0x0 species may reasonably be drawn.Finally coronamic acid (141) is a highly efficient precursor for coronatine (1 38). Some of these results which had appeared in preliminary communications (ref. 3 p. 588; ref. 114) are now available in a full paper.l15 The intermediacy of coronamic acid (141) in the biosynthesis of (138) has been confirmed by the work of others.116 Normally present in cultures in minute amounts (141) accumulated in a mutant blocked in cor- onatine biosynthesis. It was also labelled by administered ~-[U-~~C]isoleucine and coronamic acid was an efficient coronatine precursor in a blocked mutant. It is clear that cyclopropane formation precedes linkage to the coronofacic acid moiety of (138). "QH H i (138)R= HNq H02C '-(139)R=OH CONH2 OH 6 R H2N H Me bAd Hob..m I ... a . Hd 'OH HO OH HO' OH (132) (133) (1 34) Ad = adenine Scheme 8 Blasticidin S (135) is an antifungal antibiotic elaborated by Streptomyces griseochromogenes. It is constructed from cyto- Work on the cloning and expression of genes responsible for coronamic acid biosynthesis has been reported.'17 Interestingly the production of (141) can occur in P. syringae which lacks the gene cluster for the synthesis of (138) i.e. production of (139) and (141) can occur independently. NATURAL PRODUCT REPORTS 1996R. B. HERBERT 7 References 1 R. B. Herbert Nat. Prod. Rep. 1991 8 185. 2 R. B. Herbert Nat. Prod. Rep. 1992 9 507. 3 R. B. Herbert Nat. Prod. Rep. 1993 10 575.4 R. B. Herbert Nat. Prod. Rep. 1995 12 55. R. B. Herbert Nat. Prod. Rep. 1995 12 445 6 R. B. Herbert in Rodds Chemistry of Carbon Compoundrs ed. S. Coffey Elsevier Amsterdam 1980 2nd edn. Vol. IV Part L p. 291. 7 R. B. Herbert in Rodd’s Chemistry of Carbon Compounds ed. M. F. Ansell Elsevier Amsterdam 1988 2nd edn. Vol. IV Part L supplement p. 155. 8 R. B. Herbert The Biosynthesis of Secondary Metabolites 2nd edn. Chapman and Hall London 1989. 9 D. W. 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ISSN:0265-0568    

DOI:10.1039/NP9961300045

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Diterpenoids James R. Hanson School of Molecular Sciences University of Sussex Brighton Sussex BN I 9QJ,UK Reviewing the literature published in 1994 (Continuing the coverage of literature in Natural Product Reports 1995 Vol. 12 p. 207) 1 Introduction 2 Acyclic and Related Diterpenoids 3 Bicyclic Diterpenoids 3.1 Labdanes 3.2 Clerodanes 4 Tricyclic Diterpenoids 4.1 Abietanes 4.2 Pimaranes 4.3 Vouacapanes 4.4 The Chemistry of the Tricyclic Diterpenoids 5 Te tracyclic Di terpenoids 5.1 Kauranes 5.2 Trachylobanes 5.3 Aphidicolanes 5.4 Gibberellins 6 Macrocyclic Diterpenoids and their Cyclization Products 6.1 Cembranes 6.2 Taxanes 7 Miscellaneous Diterpenoids 8 References 1 Introduction This report follows the pattern of its predecessors and covers the literature published in 1994.l The period has again been dominated by studies on the tumour-inhlbitory diterpenoid TaxoP (also known as paclitaxel) and related compounds.Although outside the scope of this review it is important to record the total synthesis of Taxo1.@2.3 An increasing number of diterpenoids are being detected possessing structures derived by rearrangement and cleavage of the normal diterpenoid carbon skeleta. This has led to the revision of some previously accepted structures. Some recent aspects of the chemistry of the diterpenoids have been re~iewed.~ 2 Acyclic and Related Diterpenoids Phytoene- 1,2-diol(l) has been obtained5 from Artemisia annua (Compositae) the source of the antimalarial sesquiterpenoid artemisinin.Some linear diterpenoids containing a furan ring e.g. 12-hydroxyambliofuran (2) have been isolated6 from a Western Australian sponge. 3 Bicyclic Diterpenoids 3.1 Labdanes Further investigations of the conifer Cryptomeria japonica (Taxodiaceae) (sugi) have yielded' a number of labdanes related to imbricatolic acid (3). ent-Sclareol 13-o-P-D-XylO- pyranoside has been obtained* from Conyza trihecatactis whilst examination of the resin 'Ladano' obtained from Cistus creticus (Cistaceae) afforded9 some labdan- 15-yl esters of malonic acid. (3) The seeds of Aframomum species (Zingiberaceae) are used as a West African food spice.Aframodial (4) (from A. danielli),l0 aulacocarpin A (5) and aulacocarpinolide (6) from A. aulac~carpos)~~ have been isolated from these plants. The synthesis of two epimeric 6,7,8-trihydroxylabdadieneshas confirmed12 the structure (7) of crotomachlin a trio1 obtained from Croton macrostachys. (4) (5) Examination of the Mexican plant Gymnosperma glutinosum (Compositae) gavel3 the ent-labdanetriol (8). Continuing investigations of Halimium viscasum (Cistaceae) have yielded 59 NATURAL PRODUCT REPORTS 1996 some nor-labdanes including (9)14 and some rearranged labdanes (10).15 The roots of the Tibetan plant Phlomis medicinalis (Labiatae) which have been used in folk medicine have afforded16 a series of labdane glycosyl esters the phlomisosides e.g.(1 1).Labdane butenolides have also been obtained1' from the rhizomes of Hedychium coronarium (Zingiberaceae) e.g. (12) whilst the amoenolides e.g. (13) were isolated 18,l9 from Amphiachyris amoena (Compositae). Further studies20 of the medicinal plant Andrographis paniculata have yielded 14-epiandrographolide isoandrographolide (1 4) and some dimeric derivatives which were potent inducers of phagocytosis. Examination of the leaves of Leonotis ocymifolia var. raineriana (Labiatae) gave2' the known labdane leonitin and a relative (15). OPoH 0 A number of labdanes have been obtained from marine organisms. Thus the furan blanesin (16) was isolatedz2 from the Mediterranean sponge Raspaciona aculeata and chloro- lissoclimide (1 7) was from the tunicate Lissoclinum voeltzkowi.The ptychantins e.g. (18) are relatives of forskolin which were isolated24 from the liverwort Ptychanthus striatus. Apiculol lp-hydroxy- 13-epimanoyl oxide was from the roots of the mangrove Rhizophora apiculata whilst some known 13-epimanoyl oxide derivatives were detected2s in a further study of' Ladano' resin (Cistus creticus). Stimulated by the biological activity of the forskolin series the microbiological hydroxylation of ribenone (19) at C-1 C-6 and C-1 1 by Curvularia lunata has been rep~rted.~' The 'Cup-plant' Silphium perfoliatum (Compositae) has been used medicinally in North America. Extraction of the leaves afforded2s chloro- silphanol (20) and silphanepoxol (21). The X-ray crystallo- graphic studies of (20) suggested a need to re-examine the structures of the carterochaetols.Some highly modified labdanes the clutyenes e.g. (22) and (23) have been obtainedz9. 30 from the medicinal plant Cluytia richardiana (Euphorbiaceae). These compounds may be visualized as 6,7 :9,1 O-biseco-6,ll 1,9-bicyclo- and 6,7-seco- 6,ll -cyclo-labdanes. Pallavicinin (24) is a 7,8-secolabdane isolated3' from the liverwort Pallavicinia subciliata. ?H 0 A number of aspects of the chemistry of the labdanes have been examined particularly in the light of the perfumery properties of diterpenoid ethers in this series. These studies include the oxidation of the side chain of sclareol to give ambraketal (25) using ruthenium tetr~xide~~ and an exam- ination of the products of the ring opening of this acetal with boron trifl~oride.~~ The synthesis of some biologically active drimanes including polygodial (26) from sclareol has been NATURAL PRODUCT REPORTS 1996J.R. HANSON reported.34 These drimanes have also been obtained from other OAc labdane~.~~ The preparation of methyl isocopalate from sclareol and of norambreinolide from abienol have been described.36. 37 Some studies on the partial synthesis of prehispanolone from hispanolone have been reported."* 3.2 Clerodanes Many clerodanes have been isolated from the Labiatae and this work has been revie~ed.~~~*~ An account of work on the furanoclerodanes from Teucrium species has been pre~ented.~~ lariaceae). The cytotoxic clerodane (36) has been isolated51 Croton sonderianus (Euphorbiaceae) is a widespread Brazilian from the stem bark of Polyalthia barnesii (Annonaceae).medicinal shrub which is used as a remedy for gastric Copaiba oil has been to contain 7a-acetoxyhardwickiic disturbances. 6a-Hydroxyannonene (27) and the corresponding acid (37). 6a,7/?-diol have been isolatedg2 from a population growing in c6" North East Brazil. Heteroscyphic acid A (28) the 6a-acetoxy derivative and the 3a,4a-epoxide have been obtainedg3 from cultured cells of the liverwort Heteroscyphus planus (Junger-manniales). Roseostachenol (29) is a neoclerodane which was isolated4* from Stachys rosea (Labiatae). Extraction of the Mexican plant Gymnosperma glutinosum (Compositae) afforded not only labdanes but also clerodanes including dihydro- tucumanoic acid (30).45 Further examination of Cistus populi- fulius (Cistaceae) has yielded46 some clerodanes functionalized on ring A including (3 1).Pulicaria salviifolia (Compositae) (fleabane) contains4' the clerodane salvicinin (32). The clero- dane (33) was among the constituents of the dried seed pods of Sindora sumatrana (Leguminosae).*8 p I' OH (29) (30) Vi' HQC OH H0& (33) Although triterpenoids of the limonin variety are common in the Meliaceae diterpenoids are rare. However the clerodane 'OAc HOzC 137) Investigations on Salvia and Teucrium (Labiatae) species have continued to yield novel clerodanes. NMR studies have been on a number of compounds derived from Mexican sources.New clerodanes include infuscatin (38) from S. inf~scata,~* salviandulin D (39) from S. lavand~loides,~~ salvimadrensin (40) from S. madren~is,~~ 3-deacetylteu-micropodine (4 1) from Teucrium polium ssp. au~asianum,~' and teutrifidin (42) from T. triJid~m.~* The stereochemistry of 0Aoj p HO-ys" _..-0 OH (34) has now been found49 in Cipadessa fruticosa. The bH norclerodane (35) along with some other clerodanes has been ~H~OAC CH&AC obtained50 from Linaria saxatilis var. glutinosa (Scrophu- opening of the epoxide ring of some 4a,18-epoxyclerodanes from Teucrium species has been examined.59 The structure of montanin E (43) was confirmed by X-ray crystallography. Some further reactions of eriocephalin have also been investigated.60 These compounds continue to attract interest as insect anifeedants.The biological activity of a range of clerodanes from Baccharis Teucrium and Salvia species has been examined in this context.61 Scutalpin E (44) has been obtained@ from Scutellaria alpina. CH20H CH20Ac (43) (44)R = CCMe=CHMe 0 A number of cis-clerodanes have been isolated. Thus the unsaturated ketone (45) has been from Vellozia bicolor (Velloziaceae). Tinospora species (Menispermaceae) have yielded a number of these compounds. Thus menisperma- cide (46) was obtained64 from T. malabarica and tinotufolin C (47) from T. tuberculata.65 The cordifolisides e.g. (48) are glycosidic constituents of T. cordifolia.66 0 GluO'* C02Me C02Me (47) (48) The clerodane ring system undergoes various rearrangements and examples of structures arising by these reactions have been isolated from natural sources.These include teubrevin A (49) from Teucrium b~evifolium,~' peronemin A from Peronema canexens (Verbenaceae),68 salvileucantholide (50) and salvian- NATURAL PRODUCT REPORTS 1996 dulin E (51) from S. le~cantha.~'Further studies of different populations of S. rhyacophila have afforded7* the 5,6-secoclerodane 7-epirhyacophiline (52). 4 Tricyclic Diterpenoids 4.1 Abietanes The crystal structure of abietic acid has been rep~rted.~' Some dehydroabietic acid derivatives including (53) have been is~lated'~ from Nepeta teydea (Labiatae). Examination of the leaves of Juniperus chinensis (Cupressaceae) aff~rded'~ (54) whilst 3~-hydroxy-l-oxototarol (55) was obtained'* from the roots.13P-Hydroxyabiet-8( 14)-en- 19-a1 has been isolated75 from the berries of J. foetidissima. More highly oxidized abietanes including (55) were the constituents of the leaves of Cryptomeria japonica (Taxodiaceae). Examination of Lepechinia caulescens (Labiatae) which is used in Mexican folk medicine for the treatment of stomach ailments gave" the epoxy acid (56). (53) (54) C02H The catechols (57) montbretol and salvinolone have been shown to be identi~al.'~ A number of abietanes have been isolated from Salvia (Labiatae) species. Thus columbaridione (58) and 1 1,12-di-0-methylrosmanol (59) have been 0 ,' 0 '*.* H @.&$ (57) (58) OMe I NATURAL PRODUCT REPORTS 1996-5.R. HANSON obtained5" from S. columbariae whilst pomiferin F (60) and G (61) have been obtainedg1 from S. pomferu and nemorosin (62) was obtained" from S. nemorosa. Some rearrangement products include coulterone (63) from S. co~lteri,~~ and tilifolidione (64) from S.tiliaef~lia.~~ Model studies on the biogenetic relationships between these oxidized abietanes have been reported.s5 A number of these compounds form the antimicrobial constituents of the roots of the Labiatae. Thus the quinone (65) has been founds6 in the roots of Plectranthus heteroensis. Further studies on Sulvia sclureu have revealed8' the presence of the highly oxidized abietane 2,3-dehydro- salvipisone (66) whilst safficinolide (67) and sageone (68) were obtaineds* from S.oficinalis. Paramiltioic acid (69) in which ring C has been cleaved was isolatedsg from S.paramiltiorrhiza. HO> Ho..& C02H (62) 0 OH 0 (63) (64) "OH OH I The villosins A-C e.g. (70) were obtainedgo from Teucrium divaricutum. Their formation involves a rearrangement of the isopropyl side chain of the abietanes. The Chinese anticancer drug Tripterygium wilfordii (Celastraceae) has continued to be the source of abietanes including the compounds (71)g1and (72).92 The microbiological hydroxylation of abietanes such as (73) by Aspergillus fumigutus has been exploredg3 as a route to these compounds. CH20H 0& 0 (73) 4.2 Pimaranes The structure of the A9'11)-i~~pimaradiene (74)94 and 14a-hydroxysandaracopimara-7,15-dien- 19-oic acid (75)95 ( = callyphyllin from Callicarpa macrophyllag6) have been con- firmed by X-ray crystallography.The phytoalexin oryzalexin F (76) has been producedg7 in rice leaves by UV irradiation. Fermentation of an unidentified coelomycete producedgs a diol maxikdiol (77) which is a powerful agonist of the maxi-K channels. As such it could have therapeutic benefit in the treatment of diseases such as asthma. The rhizomes of Kuempferia pulchra (Zingiberaceae) have been showng9 to contain 2a-acetoxysandaracopimaradien-1a-01 (78). The rearranged isopimarane (79) has been isolatedlo0 from Satureja gilliesii (Labiatae). C02H (75) =*\ & 'I H 4.3 Vouacapanes 6a-Acetoxyvouacapane (80) has been isolatedlOl from the seeds of Dipteryx lacunifera (Leguminoseae).The oil from the fruit of the Brazilian plant Pterodon polygalaeforus is reputed to inhibit the penetration of the skin by the cercaria of Schistosoma mansoni. Examination of this oil has revealedlo2 14,15- epoxygeranylgeraniol as an active component together with 6a-7P-dihydroxyvouacapan- 17p-oic acid (8 1) and its esters. Caesaldekarin A (82) has been isolatedlo3 from the roots of Caesalpinia major (Fabaceae) which are used as an anthelmintic. The erythroxylon diterpenoid 7/3-hydroxyfagonene (83) has been isolatedlo4 from Fagonia bruguieri (Zygophyllaceae). Some dolabrane diterpenoids with a cis A/B ring fusion including NATURAL PRODUCT REPORTS 1996 5 Tet racycl ic Di terpenoids 5.1 Kauranes Euphoranginol B (88) and C (89) are two ent-kauranes that have been obtained114 from Euphorbia wangii (Euphorbiaceae).ent-Kaur- 16-ene-3/3 1SP-diol(88) was also obtained115 from the roots of Gelonium multijlorum (Euphorbiaceae). ent-Kaur- 16-en-19-oic acid has been reported116 to be a trypanocidal component of Mikania obtusa with activity against Chagas disease. The X-ray crystal structure of (16R)-ent-kaurane-2,12- dione (90) obtained from the rhizomes of Alisma orientale has been described.lI7 Methods for the separation and quanti- fication of the sweet diterpenoids stevioside and rebaudioside in plant extracts using HPLC and TLC methods have been described.11* 0 (84) have been isolatedlo5 from the wood of Endospermum diadenum (Euphorbiaceae).$pcH*oH I OH 0@...rcH20H 0 Examination of the constituents of Werneria ciliolata has \ \ OH 4.5 The Chemistry of the Tricyclic Diterpenoids The oxidative decarboxylation of dehydroabietic acid by hydrogen peroxide and a mercury(I1) salt has been described.lo6 The modification of podocarpic acid via the intermediate (85) has been reported.lo7 The copper(1) mediated oxygenation of the aromatic ring of podocarpic acid and totarol has givenlo* o-catechols. Various methods for the benzannulation and cyclopenta-annulation of podocarpic acid derivatives have been reported involving orthometallation and aryne ~hemistrylO~-~~~ and leading to compounds such as (86) and (87) reminiscent of steroids.OMe OH revealedlls the presence of some dimeric diterpenoids based on a combination of ent-manoyl oxide and kauren-17-al. Liverworts are a rich source of diterpenoids. The 6,7- secokaurene (9 1) has been obtained120 from Jungermannia exserifolia ssp. cordifolia. Some dimeric diterpenoids were also found in this species. AcO.. The genus Rabdosia (Isodon) (family Labiatae) comprises about 150 species a number of which figure in Chinese traditional medicine. Examination of these has continued to yield novel diterpenoids. 121Recent isolates include megathyrin A (92) from I. megathyrsus,122 rabdoternin D (93) from R. ternif~lia,~~~ nervosanin A (94) from I. nervo~us,~~~ Me02C NATURAL PRODUCT REPORTS 1994-J.R. HANSON longirabdolide A (95) and C (96) from R.longit~ba,~~~*~~~ and e.g. (104) which underwent further rearrangement in acid to rabdoshikoccin A (97) from R.shikoki~na.'~~ Examination of R. eriocalyx revealed the presence of monomeric compounds such as maoecrystal L (98)128 and dimeric The antibacterial activity of R.trichocarpa particularly against oral micro-organisms has been associated130 with the presence of these di terpenoids. n (97) The toxic principle of Iphiona aucheri (Asteraceae) has been to be atractyloside. 16a 17-Isopropylidene-3- oxophyllocladene has been as a constituent of the Indian medicinal plant Callicarpa macrophylla (Verbenaceae). Bengalensol (99) isolated from Coflea bengalensis has been to be a 16-epicafestol derivative.(99) Rearrangements of the tetracyclic diterpenoids have continued to attract interest. The Favorski rearrangement of the chloroenol-lactone (100) has been to give the isomer (101) of gibberellin A12. The aromatic compound (102) was formed135 in the rearrangement of methyl 9P-hydroxy-11- 0x0-ent-kauran- 19-oate. Acetolysis of ent-7a 18-diacetoxy-14P- mesyloxybeyer-l5-one (103) gave136 12( 13-14)abeo compounds afford (105). The microbiological transformation of ent- 16P 17-epoxy-7- hydroxykaurane by Gibberella fujikuroi gavel3' ent-7a,11a,16p 17-tetrahydroxykaurane rather than gibberellins. The presence of a 16,17-diol appeared to inhibit oxidation at C-19 -a key step in gibberellin biosynthesis.The biotrans- formation of some ent-beyerenones by Rhizopus nigricans and Curvularia lunata has been examined.138 5.2 Trachylobanes The trachylobane diterpenes have been reviewed.13 15-Oxotrachylobanic acid has been isolated140 from a West African population of the tree Xylopia aethiopica (Annonaceae). 5.3 Aphidicolanes Further total syntheses of the tumour inhibitory diterpenoid aphidicolin have been recorded.141* 16p 18-Dihydroxy- aphidicolan- 17-oic acid (106) has been isolated143 from the fungus Cephalosporium aphidicola. The scope of biosynthetically-directed transformations with this fungus has been examined in the context of the biotransformation of sclare01,~~~ ent- 19-hydroxykaur- 16-en- 15-0ne,l,~ some stemo- dane diterpen~ids,,~~ and some aphidicolanes with different substituents on ring A.147 5.4 Gibberellins The quantitative analysis of the gibberellin plant hormones by isotope dilution mass spectrometry has been described.148 Systems for the reverse phase HPLC of permethylated free and glycosylated gibberellins have been reported.,, Details have appeared of the identification of gibberellins A, A and A, in Phaeosphaeria sp. L487,150.151 of GA, and GA, in the growing shoots of the yam Dioscorea b~lbifera,'~~ a range of of gibberellins in Dalbergia d~lichopetala,~~~ and of GA, in clover bro~rnrape.'~~ The possibility was considered that this parasite obtained its GAS from the host plant. GA, (107) and GA, (108) have detected in developing wheat grain and their partial synthesis has been rep0~ted.l~~ 16a 17-Dihydroxy- 16,17- dihydroGA,- 17-O-~-~-ghcopyranoside (109) has been isolated from rice The surprisingly high gibberellin-like biological activity of some tetracyclic diterpenoids of Elaeo-selinum species has been re~0rded.l~~ 6 Macrocyclic Diterpenoids and their Cycl iza tion Products 6.1 Cembranes Soft corals have continued to be a source of cembrane diterpenoids.Some relatives e.g. (1 lo) of sarcophytol A have been from Sarcophyton trocheliophorum (Alcyonaceae) whilst sarcotol(ll1) with a novel 13-membered ring has been isolated159 from another Sarcophyton species. The occurrence of the cembranolide (1 12) has been reportedlG0 in a Lobophyturn species.Some cembranes have been isolated161 from the resin of Western Australian plants that are adapted to desert conditions including Eremophila gilesii and E. viscida. The koumbalones A (1 13) and B are casbane diterpenes that have been found162 in Maprounea africana. oO:* OH 6.2 Taxanes There has been an enormous upsurge of work that has been reported on taxanes in the light of their anticancer activity. Reviews have appeared on the chemistry of tax01,'~~ the phytochemistry of the yew,164 and on structure-activity relationships in this series.165 The determination of the structures of taxanes by tandem mass spectrometryf66. 167 and laser desorption/ionization time of flight mass spectrometry168 has been reported. Rearranged taxanes of the 11(15-+1)- and 2(3+20)-abeo types (114) and (115) respectively have been isolated16g from Taxus baccata.AcO. Ad-*'OH 20 NATURAL PRODUCT REPORTS 1996 Desacetyltaxine A has also been isolated170 from this source whilst cell culture lines of T. baccata have yielded some new biologically-active ta~0ids.l~' The taxane (I 16)172 and the taxuspines e.g. (117) and (1 18),173 have been obtained from T. cuspidata. The revision of the structures of some taxanes from T. brevifolia has been The 11(15-tl)-abeo-taxane structure (1 19) has been e~tablished"~ for taxuchin A obtained from T. chinensis whilst the taxchinins E-K have been assigned similar structures. The rearranged taxane ( and some further taxol derivatives have been isolated from Taxus xmedia c~1tivars.l~~ The structure of wallifoliol(121) from T.wallichiana represents another variant of the underlying taxane skeleton. Yunantaxusin A (122) and some relatives have been from T. yunnanensis whilst yunnanexane has been isolatedlB0 from cell cultures of T. chinensis. AcO Y Et-y-C-Me 0 HO.. Ad OAc AcO.. AcO. PhOCO OAc NMR and molecular modelling studies of taxol analogues in aqueous and non-aqueous solution have been reported.181 A great deal of novel chemistry of taxol and its relatives has been reported in studies on structureactivity relationship^'^^-^^^ including studies on the facile hydrolysis of the C-2 benzoatels5 and the synthesis of C-2 taxol analogues.ls6 Modifications have been reported at C_4,187,188 C-9,189,190 C-10 191-194 C-14195,196 and C-19.1g7 A number of rearrangements of the taxane skeleton have been rep~rted'~~-~~~ and some photochemical NATURAL PRODUCT REPORTS 1996-5.R. HANSON reactions have been 203 The electrochemical modification of taxanes has been described.204* 205 Various improvements to the methods for introducing the side chain and modifications of its structure have been reported.20s-z12 The synthesis of photoaffinity analogues of taxol bearing photo- reactive substituents and their use in labelling tubulin has been described.213. 214 The partial synthesis of the major human metabolites of docetaxel has been 7 Miscellaneous Diterpenoids A wide diversity of diterpenoid structures continue to be isolated from marine organisms.Several spongiane e.g. (123) and isocopalane e.g. (124) diterpenoids have been obtained216 from the Mediterranean sponge Spongia zimocca. A number of dolabellane diterpenoids have been isolated217 from the brown alga Dilophus mediterraneus whilst the cyclocembrane variant (125) has been obtained21s from the coral Sarcophyton trocheliphorum. The isolation of the glucoside (126)219 from Chrozophora obliqua (Euphorbiaceae) appears to be the first report of a dolabellane diterpenoid from a higher plant. Other dolabellanes and dolestanes have been isolated220 221 from marine alga such as Dictyota pardalis. " "O OH P A number of eunicellane (cladiellane) diterpenoids have been described including palmonine F (127) from the gorgonian Eunicella verrucosa,222 the compound (128) from the coral Cladiella australi~,~~~ and litophynol A (129) from a Litophyton species.224 Gorgonian octocorals of the genus Briareum have afforded various briarane and asbestinane diterpenoids in- cluding (1 30) (1 3 1) and (1 32) from B.asbestin~m.~~~, 226 Novel ..OH #OH * OAc (127) R = Me OAc (128) R=CH2 HO'. xenicanes include helioxenicin A (133) from the blue coral and Heliopora ~oerulea~~~acalycigorgin A (1 34) from an Acalycigorgia species.228 Ad@ 0 ACO H (134) Amongst the prenylated sesquiterpenes that have been isolated are lemnabourside (1 35) from the coral Lemnalia bournei (Alcyona~eae),~~~ and the kalihinol isonitriles (1 36) and (1 37),230 231 from the sponge Acanthella cavernosa which was collected from the Seychelles.Palmatol (138) is a prenyl-bicyclogemacrane which was isolated232 from the Mediterranean octocoral Alcyonium palmatum. $ CN (1 37) The unusual structure (139) has been assigned to floridicin which was from the coral Xeniaflorida. It represents an oxidative cyclization product of xeniafaraunol A (140) from Xenia faraunensis. Examination of the soft coral Sinularia dissecta gave235 the unusual mandapamate OHC (141) the structure of which may arise through the internal Diels-Alder cyclization of a furanocembranoid compound of the pukalide series. Fungi have yielded some unusual diterpenoid structures including the 2,3-~econeodolastane trichoaurantin (142) from Tricholoma auranti~m,~~~ and the phomactins e.g.(143) which OH 0 OH (142) are platelet activating factors from a marine Phoma species.237 Some further indole diterpenoids e.g. (144) have been reported23s from Emericella purpurea. Pl Liverworts have proved to be a source of fusicoccane diterpenoids. Recent isolates include fusicogigantepoxide (145) from BryopteriaJili~ina,~~~ fusicorrugatol(l46) from Plagiochila corr~gata,~~~ fusicoauritone from Anastrophyflum and a~ritum.~~~ The unusual structure (147) has been established242 for jamesoniellide C which was isolated from Jamesoniella autumnalis (Hepaticeae). NATURAL PRODUCT REPORTS 1996 A further phorbol derivative has been from Euphorbia laterifolia whilst picrodendrin U (148) is a picro- toxane lactone which was isolated244 from Picrodendron baccatum (Euphorbiaceae) a plant which is used in folk medicine as an insecticide..o HO 8 References 1 J. R. Hanson Nat. Prod. Rep. 1995 12 207. 2 R. A. Holton C. Somoza H. B. Kim F. Liang R. J. Biediger P. Boatman S. Shido C. S. Smith S. Kim H. Nadizadeh Y. Suzuki C. Tao P. Vu S. Tang P. Zhang K. K. Murthi L. N. Gentil and J. H. Liu J. Am. Chem. SOC. 1994 116 1597 et seq. 3 K. C. Nicolaou Z. Yang J. J. Liu H. Ueno P. G. Nantermet R. K. Guy C. F. Claiborne J. Renaud E. A. Couladoures K. 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ISSN:0265-0568    

DOI:10.1039/NP9961300059

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Book Reviews Anticancer Drugs from Animals Plants and Microorganisms By George R. Pettit Fiona H. Pierson and Cherry L. Herald Wiley Interscience New York 1994 ix+670 pp. Price f78 ISBN 0471036579 Between 1960 and 1982 the National Cancer Institute in the USA screened around 114000 plant extracts and a similar number of microbial extracts for anticancer activity. Since this initial programme ended a new programme has evaluated many extracts from plants microorganisms and even animals for not only anticancer activity but also anti-HIV activity. Yet it is estimated that only 15-20 YOof terrestrial plants have been properly studied and since the Earth’s total biodiversity may run to 500 million species there is clearly much yet to be done. Recent successes including the growing use of TaxoP for the treatment of ovarian cancer and refractory breast cancer and the ongoing phase I1 clinical trials with the marine natural product bryostatin I reemphasize that many of the best anticancer drugs are of natural origin.George Pettit has of course made enormous contributions in this area not least with the structure elucidation and synthetic studies of the bryostatins and dolastatins and he is well-placed to produce the present immensely informative book. His earlier books Biosynthetic Products for Cancer Chemotherapy were invaluable and this book continues the literature coverage from January 1986 to January 1989. The long gestation period is a little puzzling though there were a large number of structures and other data to check.~_________ ~_____ ~ ~ ~~ The book commences with a very brief account of the cost of cancer to the economy and then reminds the reader of the growing threats to health posed by HIV and a number of other viruses like ebola virus and by tumour promoters/carcinogens both natural and man-made. But the main service provided by the book comprises the long lists of antineoplastic agents and anti-proliferative agents that have been identified during the period under review. There are three large sections products from terrestrial plants fungi and animals together with those from marine algae and invertebrates; products from marine animals; and products from marine plants. Each entry includes a chemical structure (with full stereochemistry where known) common name organism and source some physical data and leading references to spectral and other data.The book concludes with various indices subject molecular weights and the references. The book is not cheap but is the essential source book of information about recently discovered anticancer agents. It is also an invaluable guide to those synthetic targets which might just attract research funding. John Mann University of Reading UK Advances in Nitrogen Heterocycles ed. C. J. Moody JAI Press Inc. Greenwich Connecticut Volume 1 1995. xi +257 pp. Price f62.50 ISBN 0-89232-864-9 And so another new series is unleashed on the confraternity of organic chemists already groaning under the pressure of the information explosion.The volume under review has as its focus the synthesis and transformations of pyrroles and some of their benzo-fused derivatives an indisputably important group of nitrogen heterocycles in natural products and medicinal chemistry. Coverage ranges from pyrroles and indoles to porphyrins carbazoles and carbolines. The seven chapters deal with a selection of versatile synthetic methods for constructing and modifying the ring systems of interest. In general not only are basic methodological studies described but ample illustrations from the total synthesis of natural products are also provided. For example the short opening chapter on intermolecular and intramolecular reactions of metal-stabilized carbenoids with pyrroles (Davies) is bolstered with examples showing asym- metric entries to indolizidine and tropane alkaloids.Following this is a substantial contribution by Lash on the synthesis of porphyrins with exocyclic rings often present as geological marker pigments in organic-rich sedimentary deposits. This wide-ranging review includes both the synthesis of [b]-and [c]- cycloalkenopyrrole building blocks and a microcosmic survey of methods for coupling pyrrole systems. The next four chapters interrelated by their subject matter deal with palladium-catalysed coupling reactions of indoles (Martin and Zheng) cycloaddition reactions of indole derivatives (Pindur) transition metal-mediated synthesis of carbazole derivatives (Knoelker) and synthesis of [b]-annellated indoles by thermal electrocyclic reactions (Hibino and Sugino).Pharmacologically active natural products whose syntheses are illustrated wholly or in part include ergot and iboga alkaloids protein kinase C inhibitors (e.g.rebeccamycin staurosporine) the marine alkaloid hyellazole and quinonoid fungal metabolites such as the murrayaquinones and kinamycins. Overlap of material in these chapters is minimal no doubt a reflection of perceptive editorial work by Professor Moody. I noticed however a threefold appearance of the electrocyclic reactions of 2,3-divinylindoles (pp. 156 184 207). The final chapter contains a personal and pithy account of Boger’s imaginative total synthesis of the exceptionally potent antitumour antibiotic (+)-duocarmycin SA and its (-)-enantiomer.All the contributions to this book are interesting and all have been well written. Nevertheless I cannot help questioning whether this latest addition to the expanding JAI series in chemistry is really justified. Granted this volume like all those in the JAI series is intended to give leading exponents an opportunity to survey a topical area while at the same time providing them with a showcase for highlighting their own contributions. Granted specialists will appreciate the con-venience of having reviews by leading workers (not to mention the handy collection of references contained therein) in a single volume. However I believe that much of the material in this volume would have been equally at home in say Advances in Heterocyclic Chemistry or in review journals.Furthermore the personal perspective in the articles is not so obtrusive as to make them unpublishable elsewhere for example in Synlett which encourages reviews enlivened by personal viewpoints. Most seriously I believe that the essential features of some of these reviews have in fact been published elsewhere already. 73 Perhaps I am being both churlish and conservative in challenging the decision to launch a new series devoted entirely to nitrogen heterocycles no matter how consequential that broad class of compounds may be. As for the first volume is it timely? Yes. Is it useful? Indubitably. But is it necessary..,? The typesetting of the book is good and virtually error-free and the diagrams are neat clear and accurate.The index is adequate -about as detailed as one expects in a work of this NATURAL PRODUCT REPORTS 1996 type and about as capricious. Nonetheless I do not believe that the volume represents good value for money. If nothing else the rather coarse paper with its ominous yellow tinge fails to match the expectations engendered by the volume’s iniquitous price. Joseph P. Michael University of the Witwatersrand South Africa Oxidative Stress and Antioxidant Defenses in Biology ed S. Ahmad Chapman & Hall New York 1995,xxi+457 pp. Price f69,ISBN 0-412-03971-0 Molecular oxygen is essential to many metabolic processes of all aerobic life forms. This dependence on oxygen paradoxically has resulted in a universal toxicity of oxygen to all aerobic life processes.One electron reduction of oxygen generates the superoxide anion free radical which can undergo further reduction to the hydroxyl radical. The production of these and related species and their harmful reactions with biological macromolecules are discussed in two chapters by Cadenas and by Chen et al. These reactive oxygen species can react with unsaturated groups in membrane lipids to form unstable hydroperoxides which can break down and produce further free radicals. This oxidative stress on low density lipoprotein which can lead to atheroschlerosis is reviewed by Kalyanaraman whereas Stohs concentrates on the oxidative stress caused by environmental contaminants including drugs and pesticides. Berenbaum summarizes what little is known about the metabolic detoxification of prooxidants.The oc- currence of a number of natural products such as vitamins C and E and /?-carotene which act as antioxidants is addressed by Larson. The antioxidant defences displayed by enzymes and proteins are broadly discussed by Ahmad then Cunningham and Ahern detail how these defences operate in prokaryotes Dalton extends this treatment to plants and fungi and Felton completes this set of chapters by considering vertebrates and invertebrates. Finally Ahern and Cunningham outline work carried out on the genetic regulation of antioxidant defences based on studies with bacteria. There is much of interest in these 11 chapters and the book is well produced with a subject index although there is some unevenness in the style and quality of the chemical structural drawings. A number of the authors appear to be uncomfortable with chemical structures and this is evident in a number of errors on pages 101 (impossible resonance stabilized picture) 194 (five-bonded carbon) 197 199 and 380 [mysterious -C(OH)=O-group (three times)]. Nevertheless this is an up to date account of an important area of ongoing research which requires further input from many different disciplines. David J. Robins University of Glasgow UK

ISSN:0265-0568    

DOI:10.1039/NP9961300073

出版商:RSC    

年代:1996

数据来源: RSC