摘要:

ISSN 0265-0568 NPRRDF 14(4) 309-432 (1997) Natural Product Reports A journal of current developments in bioorganic chemistry Volume 14 Number 4 CONTENTS ... 111 Hot off the press Robert A. Hill and Andrew R. Pitt Reviewing the recent literature on natural products and bioorganic chemistry 309 Chemistry and biosynthesis of clavulanic acid and other clavams Keith H. Baggaley Allan Brown and Christopher J. Schofield 335 Biosynthesis of fatty acids and related metabolites Bernard J. Rawlings Reviewing the literature published up to the end of 1994 359 Biosynthesis of plant alkaloids and nitrogenous microbial metabolites Richard B. Herbert Reviewing the literature published in 1995 373 Steroids reactions and partial synthesis James R. Hanson Reviewing the literature published in 1995 387 Phenethylamine and isoquinoline alkaloids Kenneth W.Bentley Reviewing the literature published between July 1995 and June 1996 413 Recent progress in the chemistry of non-monoterpenoid indole alkaloids Masataka Ihara and Keiichiro Fukumoto Reviewing the literature published between July 1995 and June 1996 431 Book review Analysis ofsteroids by L. John Gould and Toshihiro Akihisa (reviewed by James R. Hanson) 432 Corrigendum Cumulative Contents of Volume 14 Number 1 1 Brassinosteroids Shozo Fujioka and Akira Sakurai 11 Quinoline quinazoline and acridone alkaloids (July 1994 to June 1995) Joseph P. Michael 21 Indolizidine and quinolizidine alkaloids (July 1994 to June 1995) Joseph P.Michael 43 Lignans neolignans and related compounds (January 1994 to December 1995) Robert S. Ward 75 Cyclopeptide alkaloids (January 1985 to December 1995) Dimitris C. Gournelis Gregory G. Laskaris and Robert Verpoorte Number 2 83 Recent advances in chemical ecology (July 1992 to December 1995) Jeffrey B. Harborne 99 The role of carbohydrates in biologically active natural products Alexander C. Weymouth-Wilson 11 1 The biosynthesis of C,-C2 terpenoid compounds (1993 to 1995) Paul M. Dewick 145 Natural sesquiterpenoids (1 995) Braulio M. Fraga 163 Fatty acids fatty acid analogues and their derivatives (1988 to 1995) Marcel S. F. Lie Ken Jie Mohammed Khysar Pasha and M. S. K. Syed-Rahmatullah 191 Diterpenoid and steroidal alkaloids (mid-1994to the beginning of 1996) Atta-ur-Rahman and M.Iqbal Choudhary Number 3 205 Synthesis of amino acids incorporating stable isotopes (1990 to mid 1996) Nicholas M. Kelly Andrew Sutherland and Christine Willis 221 The biosynthesis of the gibberellin plant hormones (up to September 1996) Jake MacMillan 245 Diterpenoids (1995)James R. Hanson 259 Marine natural products (1995)D. John Faulkner 303 Amaryllidacae alkaloids (1995)John R. Lewis Number 4 309 Chemistry and biosynthesis of clavulanic acid and other clavams Keith H. Baggaley Allan Brown and Christopher J. Schofield 335 Biosynthesis of fatty acids and related metabolites (up to end 1994) Bernard J. Rawlings 359 Biosynthesis of plant alkaloids and nitrogenous microbial metabolites (1995)Richard B. Herbert 373 Steroids reactions and partial synthesis (1995)James R.Hanson 387 Phenethylamine and isoquinoline alkaloids (July 1995 to June 1996) Kenneth W. Bentley 413 Recent progress in the chemistry of non-monoterpenoid indole alkaloids (July 1995 to June 1996) Masataka Ihara and Keiichiro Fukumoto 431 Book review Analysis of’steroids by L. John Gould and Toshihiro Akihisa (reviewed by James R. Hanson) 432 Corrigendum Articles that will appear in forthcoming issues include Monoterpenoids (part 1993 all 1994 part 1995) David H. Grayson Natural products derived from unusual variants of the shikimate pathway Heinz G. Floss Biosynthesis of polyketides (mid 1993 to end 1994) Bernard J. Rawlings Coumarins (January 1995 to December 1996) Ana EstCvez-Braun and Antonio G. Gonzalez Secondary metabolites from marine microorganisms bacteria protozoa algae and fungi Francesco Pietra Triterpenoids (July 1995 to June 1996) Joe Connolly and Robert Hill Isopentenyl diphosphate isomerase a core enzyme in isoprenoid biosynthesis. a review of its biochemistry and function Robert van der Heijden Ana C. Ramos-Valdivia and Robert Verpoorte Recent progress in the chemistry of the monoterpenoid indole alkaloids (1996) J. Edwin Saxton Pyrolizidine alkaloids (I 995) J. Richard Liddell

ISSN:0265-0568    

DOI:10.1039/NP99714FP007

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Natural Product Reports Edit0 ria I 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 Uniiversity 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 Nicola P. Coward Cambridge Production Editor UK CB4 4WF Dr Carmel M. McNamara Technical Editor Telephone +44 (0) 1223 420066 Mrs Dawn J. Webb Facsimile +44 (0) 1223 420247 Miss Karen L. White E-mail perkin@ rsc.org Editorial Secretaries RSC Server h tt p://c hem ist ry.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 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. 1997 Annual subscription rate €355.00; 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 1HN. Air freight and mailing in the USA by Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11 003.US Postmaster send address changes to Natural Product Reports Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11 003. Periodicals 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 Henry Ling Ltd at the Dorset Press Dorchester Dorset.

ISSN:0265-0568    

DOI:10.1039/NP99714FX017

出版商:RSC    

年代:1997

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP99714BX019

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Chemistry and biosynthesis of clavulanic acid and other clavams ~ Keith H. Baggaley," Allan G. Brownb and Christopher J. Schofield' '3 Blackstone Hill Redhill Surrey UK RHI 6BE 'Petersgate Avenue Road Cranleigh Surrey UK GU6 7LL "The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences University of Oxford South Parks Road Oxford UK OX1 3QY 1 Introduction 2 Discovery and development of clavulanic acid 2.1 P-Lactamases 2.2 Detection of P-lactamase inhibitors 2.3 Structure determination 2.4 Biochemical and microbiological properties 3 Other naturally occurring clavams 4 Other metabolites from S. cluvuligerus 5 Chemistry 5.1 Derivatives 5.2 Synthesis 5.3 Structure-ac tivi ty relations hips 6 Synthetic analogues 7 Mode of action 8 Riosynthesis 8.1 Clavulanic acid biosynthesis 8.2 Biosynthesis of other clavams 9 References 1 Introduction Almost since the first clinical application of the early members of the p-lactam family of antibiotics their continued utility has been haunted by the spectre of resistance.Today the threat of bacterial resistance to the therapeutic efficacy of p-lactams and other antibiotics looms larger than ever before. The discovery and application of clavulanic acid 3 demonstrates one approach to combating antibiotic resistance that has been successfully applied in the clinic. H OMe NH2 0 CH2R C02H 1 Cephamycins 1 p-2 0 43 2 Clavam ring system 3 Clavulanic acid 7-oxo-4-oxa-1 -azabicyclo[3.2.0] heptane Subsequent to its isolation and identification' as a producer of 7a-methoxycephalosporin derivatives (cephamycins) 1 the actinomycete Streptomyces cluvuligerus has served as a source of a series of new p-lactam antibiotics based on the 7-oxo-4- oxa-l-azabicyclo[3.2.O]heptaneor clavam 2 ring system.P2 One of these clavulanic acid 3 is a potent antibiotic inhibitor of 'serine' (or Classes A C and D) p-lactamases.Clavulanic acid 3 is now used as its potassium salt in conjunction ?The numbering indicated on structures 2 and 3 is based on the clavam ring system and does not relate to the IUPAC system. with amoxycillin and prescribed clinically as co-amoxiclav (Augmentin@) and with ticarcillin as Timentin@.3p'0 The aims of this review are to (i) highlight the discovery development and chemistry of clavulanic acid 3 and its deriva- tives with brief comment on their biological properties; (ii) describe the biosynthetic pathway to clavulanic acid 3; and (iii) outline the biochemistry of the known enzymes involved in the biosynthetic pathway leading to 3 and the relation of these enzymes to those implicated in the biosynthesis of other second- ary metabolites including the penicillins and cephalosporins.2 Discovery and development of clavulanic acid 2.1 p-Lactamases The discoveries of the 'classical' p-lactam antibiotics namely the penicillins and cephalosporins are prime examples of the importance of serendipity in the identification of novel chemi- cal entities with useful biological properties as well as being instances of chance favouring the prepared mind.' ' The iso-lation of clavulanic acid 3 (and concurrently the olivanic acidkhienamycin or carbapenem family of p-lactam anti-biotics) serves as a paradigm for how the careful application of a pioneering approach can lead to the discovery of novel structures with powerful medicinal properties.' Early in the studies relating to the development of an efficient process for the isolation and purification of penicillin and well before its widespread therapeutic use Abraham and Chain recognised that bacteria were capable of producing an enzyme which could inactivate penicillin. '' The enzyme activity was defined as a penicillinase and is now more usually called a P-lactamase.Following exposure to most clinically important semi-synthetic penicillins and cephalosporins the ability of a wide range of pathogenic bacteria to evolve highly efficient P-lactamases with different substrate specificities provides the major mechanism by which resistance develops to p-lactam antibiotics. P-Lactamases inactivate penicillins and cepha-losporins by hydrolysing their p-lactam rings to give penicilloic acid derivatives and analogous degradation products in the case of cephalosporins. In the late 1940s and early 1950s extensive use of penicillin G 4 led to a dramatic decrease in the susceptibility of Staphylococci to this antibiotic due to P-lactamase production. In order to overcome this problem of P-lactamase mediated resistance a number of semi-synthetic penicillins were developed.These included methicillin 5 and the isoxazolyl penicillins cloxacillin 6 and flucloxacillin 7 which were less susceptible to hydrolysis by the Stuphylococcus p-lactarna~e.'~-'~ Similarly the first generation cephalosporins (e.g. cephaloridine 8 and cephalexin 9) were developed follow- ing the discovery that cephalosporin C 10 exhibited some resistance to P-Iactamase inactivation. '6*'7 Since P-lactamases play a major role in the development of resistance to penicillins and cephalosporins in both Gram- positive and Gram-negative bacteria their production and mechanism of action have been the subject of widespread inves- tigation~.""~ They have been classified according to sequence similarities (whether encoded for on plasmids or chromosomes) their substrate selectivities and other biochemical parameters by Baggaley Brown and Schojield Chemistry and biosynthesis of clavulanic acid and other clavams ,.*. PLactamases of Gram-negative Bacteria RCoNHwSU @iq' 0 C02H 4 Benzyl penicillin R = CH2Ph or Penicillin G OMe CH-(;~~~ I 35 Amoxycillin R = I-IOfi~HP 36 Ticarcillin R = 0 C02H ,-~AHTvH~ 9 Cephalexin R -8 Cephaloridine R1 = CrCH2-R2= -ND Chromosomally-mediated Plasmid-mediated Cephalosporinase Penicillinase Broad TEM Spectrum Inducible Constitutive OXA PSE Class I Class II Class IV Class Ill Class V Bacterial P-Lactamases (b) I Class A Class B Class C Class D 'penicillinases' 'metallo-enzymes' 'cephalosporinases' 'oxacillinases' contains contains active contains contains nucleophilic site metal e.g.zinc; nucleophilic nucleophilic active site e.g. from active site active site serine; Bacillus cereus serine; serine; e.g. from S. aureus and Aeromonas hydrophilia e.g. from E. coli and e.g. from E.coli and E. coli (TEM); Pseudomonas Pseudomonas Pseudomonas aeruginosa aeruginosa a eruginosa Fig. 1 Classifications of p-lactamases according to (a) Sykes el UZ.,'"~~,~' (b) Ambler et al.25,28*29 Classification according to substrate specificity partially reflects historical considerations. Adapted from ref. 19. R2 = H 10 Cephalosporin C Ri = D-6-(a-aminoadipoyl); R2 = OCOCH3 Staphylococci but also by pathogenic Gram-negative organ- Richmond and Sykes,20 Sykes and Matthews,21 B~sh~~-~~ Many semi-synthetic p-lactams were (and still are) and ism~.~~ Ambler.25 The Ambler clas~ification~~ separates the metallo- enzymes (Class B) from those that possess a nucleophilic serinyl residue at their active sites.Currently the serine p-lactamases are more clinically important although resistance mediated via the metallo-enzymes of which there are few reported selective inhibitors is becoming more prevalent. The reaction mechanism of all the serine p-lactamases mini- mally proceeds through a three-step mechanism involving for- mation and hydrolysis of an acyl-enzyme complex covalently linked to the enzyme via the active site serine.However unlike the trypsin or subtilisin families of serine proteases,26 the serine p-lactamases do not contain a catalytic triad (Asp/Ser/His) of active site residues. It is possible that the serine P-lactamases more closely resemble the recently proposed family of Ser/Lys catalytic dyad pro tease^.^^ The serine P-lactamases may be further divided into Classes A C and D.28329 The Class A enzymes (molecular weights ca. 29 kDa) examples of which are the p-lactamases of Staphylococcus aureus and the Escherichia coli TEM plasmid-mediated enzymes are normally selective for penicillins. Class C p-lactamases (molecular weights ca. 39 kDa) normally hydrolyse cephalosporins more readily than penicillins and display no significant sequence homology with the Class A or Class D enzymes.The Class C p-lactamases include chromosomally encoded p-lactamases from organisms such as Escherichia coli Pseudomonas aerugi- nosa and Enterobucter spp. The Class D enzymes preferentially hydrolyse oxacillins.28 A simplified division of p-lactamases from Gram-negative organisms into various types or classes is summarised in Fig. 1. For more detailed discussion on the biochemistry and epidemiology of P-lactamases the reader is referred to specialist publication^.^' -32 2.2 Detection of p-lactamase inhibitors During the 1960s chemists microbiologists and biochemists worked together in the hope of discovering new p-lactams with a degree of stability to the P-lactamases produced not only by 3 10 Natural Product Reports 1997 prepared by acylation of 6-aminopenicillanic acid 11 (6-APA) and 7-aminocephalosporanic acid 12 (7-ACA) and consider- able efforts were applied to the chemical manipulation of these intermediates in order to design novel semi-synthetic p-lactam based structures.Simultaneously there was a general interest in looking for microbial metabolites with activities against resistant organisms. H2NFJ Re> 0 C02H 0 CH20Ac C02H 11 6-APA R = NH2 12 7-ACA 17 Penicillanic acid R = H In the mid-l960s as a result of detailed studies on the production of p-lactamases by bacteria such as Klebsiella pneumoniae (aerogenes) Escherichia coli Enterobacter cloacae strains of Pseudomonas aeruginosa Proteus spp. and Serratia marcescens and the microbiological evaluation of many semi- synthetic penicillins and cephalosporins individually and in combination Dr George Rolinson (Beecham Pharmaceuticals) considered screening microorganisms for naturally occurring p-lactamase inhibitors.A new microbiological screen based on the traditional agar hole-in-plate assay was applied to a series of culture filtrates derived from fungi and actinomycetes isolated mainly from soil samples obtained from a variety of worldwide locations.2 The assay utilised a p-lactamase producing strain of Klebsiella aeruginosa and a fixed amount of penicillin G 4. On incu- bation if no p-lactamase inhibitor was present benzyl penicil- lin 4 was destroyed and a lawn of Klebsiella was obtained. If however a solution of an inhibitor was placed in a well cut in 2.3 Structure determination the agar plate diffusion occurred and now on incubation a clear zone of inhibition resulted around the well due to p-lactamase inhibition.Cloxacillin 6 was used as the control inhibitor in the early use of the assay. Application of this screen to cu. 1000 Actinomycetes and fungi gave a series of isolates which exhibited potent p-lactamase inhibitory activity. All the isolates were found to be strains of Streptornyces olivaceus though they were derived from different soil samples collected from localities in Africa Israel New Zealand and Spain. Studies initiated in the late 1960s on the isolation and characterisation of the inhibitory substances from these S.olivaceus strains were hampered by the instability of prep- arations with loss of activity occurring especially during concentration of active solutions. At the time these investigations were in progress pharma- ceutical companies other than Beechams were also attempting to develop new semi-synthetic penicillins and cephalosporins with better stability to P-lactamases and increased potency to the P-lactamase producing strains emerging in the clinic; reports from Lilly and Merck laboratories described the cephamycin 1 or 7a-methoxycephalosporin family of p-lactam antibiotics e.g. cephamycin A 13 cephamycin B 14 cepha-mycin C 15 and 7a-methoxycephalosporin C 16.'.33-35 These C02H 13 Cephamycin A R = 0 COC=COOS03H -OMe 14 Cephamycin B R = OCOC=CHeOH OMe 15 Cephamycin C R = OCONH2 16 7a-Methoxycephalosporin C R = OCOMe compounds had an increased stability to serine p-lactamases and showed some interesting activity against Gram-negative organisms.The screens used to detect these new p-lactams were based on the use of highly sensitive strains of Salmonella and p-lactamase sensitivity patterns. In order to compare the p-lactamase inhibitory activity with the crude inhibitors from S. olivuceus the cephamycin producing cultures were purchased by Beecham workers from the American Type Culture Collection; the species examined were isolates of S. clavuligerus S. lipmanii S. lactamdurans and S. griseus. When culture filtrates from these species were screened those from S.clavuligerus were found to produce a metabolite with pronounced p-lactamase inhibitory activity. Chromato- graphic comparisons indicated that this metabolite was not one of the cephamycins derived from the Lilly or Merck cultures using cephamycin markers supplied by these companies. It was also clear from these chromatographic comparisons that the metabolite was not one of the products being produced by S. olivaceus. The p-lactamase inhibitory compound from S. clavuligerus was initially known as MM (or BRL) 14151 and was subsequently named clavulanic acid 3 by analogy with penicillanic acid 17.2-36,37 The inhibitory substances produced by the S. olivaceus strains were also shown to be neither cephamycins 1 The early to mid 1970s was a remarkable period in the discovery of new naturally occurring p-lactam ring sys-tem~.~',~~ Initially there were the reports of the cephamycin family 13-16 of cephalosporins whose structures feature a 7a-methoxy group.1,33 Then followed clavulanic acid 3 which can be considered as the first naturally occurring example of a truly non-traditional bicyclic p-lactam ring ~ystern.~,~~ The structure of clavulanic acid 3 was deduced following its isolation as its sodium potassium or lithium salts and prep- aration of its methyl 18 benzyl 19 p-nitrobenzyl 20 and h-N4 0 C02R p-bromobenzyl 21 esters.37 Scrutiny of 'H and I3C NMR infrared spectroscopic and mass spectrometric data allowed a novel bicyclic p-lactam structure to be proposed for 3. This was confirmed and defined in absolute terms by X-ray analysis of the p-nitrobenzyl 20 and p-bromobenzyl 21 esters.These experiments established the structure of clavulanic acid as Z-(2R,5R)-3-(2-hydroxyethylidene)-7-oxo-4-oxa- 1-azabicyclo [3.2.0]heptane-2-carboxylic acid 3. The X-ray structure studies reveals that the bicyclic lactam of the clavams is even more strained than that of the penicillin and cephalosporin ring systems. This presumably reflects (a) the substitution of an oxygen atom for sulfur (b)the lack of an acylamino substituent at C-6 and (c) the presence of an exo-p-hydroxyethylidene function at C-2. The discoveries of the cephamycins 13-16 and clavulanic acid 3 were succeeded by the isolation from Nocardia uniformis subsp. tsuyamonensissOof the nocardicins e.g.22 and 23 with U RcoNH71Fro" 22 Nocardicin A R = H02CCH(NH2)(CH2)20 23 Nocardicin G a substituted monocyclic p-lactam as their core structure and the olivanic acids 24-26 30-3339-42 and thienamycin group 27-2943,46 of closely related carbapenems from Streptornyces spp. In addition Woodward reported on the synthesis of the penem nucleus 34." New naturally occurring p-lactams which followed in the early 1980s were the monoba~tams~~'~~ and the 7a-formamidocephalosporins.54-56 2.4 Biochemical and microbiological properties In Section 2.1 a brief outline was given of the classification or clavulanic acid 3. They were subsequently shown to be closely related members of the olivanic a~id,~,~~~~ and of P-lactamases produced by bacteria of major clinical im- thienamy~in,~~or carbapenem family of novel naturally portance.Table 1 summarises the p-lactamase inhibitory 46 occurring p-lactam antibiotics most of which possess potent properties of clavulanic acid 3 against examples of serine p-P-lact amase inhibitory properties. 6347 lactamases from Gram-negative organisms and Staphylococcus Baggaley Brown and SchoJield Chemistry and biosynthesis of clavulanic acid and other clavams 311 Me from the Gram-positive organisms Staphylococcus aureus and H-.l H r! Bacillus cereus I P-lactamase are also significantly readily M;* R inhibited. Clavulanic acid 3 does not inhibit the Class I type (or Ambler type B) of chromosomal metallo-p-lactamases H03s0mR 0 C02H generated by certain pathogens such as Enterobacter spp., 0 C02H Citrobacter spp.various Proteus spp. Pseudomonas aerugi- 0-nosa and Serratia marcescens. 24 R= S+ / 27 R= S-N H2 A number of penicillins and some cephalosporins which ONHCOMe MM 4550 Thienamycin 25 R= '+NHCOMe 28 R= S-NHCOMe MM 13902; Epithienamycin E N-Acety lthienamycin 26 R= '-NHCOMe 29 R= '~NHCOM~ MM 17880; Epithienamycin F N-Acetyldehydrothienamycin H;* R M;* R n v 0 C02H C02H 30 R= Se 32 R= S,-,,-, NHCOMe NHCOMe MM 22380; Epithienamycin A MM 22381 ; Epithienamycin C 31 R = S d 33 R =S* NHCOMe NHCOMe MM 22382; Epithienamycin B MM 22383; Epithienamycin D ROCNH C02H 34 Table 1 (3-Lactamase inhibition spectrum for clavulanic acid 3; benzylpenicillin 4 substrate (1 mg ml -I)'' ICso value"/ Source of (3-lactamase pg ml-' ~ ~ Escherichia coli JT4' 0.07 Escherichia coli JT20' 0.3 Klehsiella arogenes Ba95' 0.07 K.uerogenes E70 0.015 Proteus mirabilis 03x9 0.03 Serratia marcescens U39' 0.4 Pseudomonas aeruginosa 4482' 0.35 Ps. aeruginosa Dalglieish 0.007 Huemophilus injtuenzae 4482' 0.07 Staphylococcus uureus Russell 0.06 Bacillus cereus (mixture of types 1 and 11) 17 "Adapted from ref. 57. %hibitor incubated for 15min with enzyme at pH 7.3 before addition of substrate. "Contains resistance plasmid of TEM type. aureus as described by IC, measurement5 i.e. a measure of the ability of 3 to inhibit the rate of hydrolysis of a stan- dard penicillin or cephalosporin substrate by so%,either with or without preinc~bation.~~,~~ Clavulanic acid 3 is a potent progressive inhibitor of certain p-lactamases and especially (i) the clinically important TEM group of plasmid-mediated P-lactamases produced by Enterobacteriaceae Haemo-philus inzuenzae and Neisseria gonorrhoeae and (ii) the chromosomally-mediated p-lactamases from Klebsiella pneumoniae (aerogenes) Pro teus mirabilis Pro teus vulgaris Branhamella catarrhalis and Bacteroides fragilis.P-Lactamases 3I2 Natural Product Reports 1997 exhibit degrees of instability to p-lactamases can be protected from inactivation by clavulanic a~id.,~,~' Amoxycillin 35 in particular shows striking in vitro (i.e. using various anti- bacterial assay systems) synergistic antibacterial activity in conjunction with clavulanic acid 3 against bacteria producing clavulanic acid-sensitive p-lactarna~es.~~~'~~ Table 2 illustrates the effect of clavulanic acid 3 on the activity of amoxycillin 35 against examples of such bacteria.Similar dramatic protection and synergy is found for ticarcillin 36 in combination with clavulanic acid 3.7359Amoxycillin 35 and ticarcillin 36 sensitive strains do not show an increased sensitivity to these com- pounds in the presence of clavulanic acid 3. P-Lactamase stable penicillins and cephalosporins do not show a synergistic anti- bacterial effect in the presence of clavulanic acid 3. It should also be noted that methicillin-resistant strains of Staphylo-coccus auras (MRSA) do not exhibit a significantly enhanced sensitivity to amoxycillin 35 plus clavulanic acid 3 as the resistance of these strains is the result of non-p-lactamase mediated mechanisms such as an altered penicillin binding protein (PBP).Clavulanic acid 3 itself has only weak though broad- spectrum antibacterial activity. Its antibacterial mode of action is via inhibition of penicillin binding protein 2 (PBP 2).6' Clavulanic acid 3 is well absorbed and distributed in a number of animal species and in man. It also possesses satisfactory metabolic toxicological and clinical pharmaco- logical properties to enable it to be developed as a chemo- therapeutic agent in combination with certain p-lactamase susceptible p-lactam antibacterial^.^^,^^.^^ Its combinations with amoxycillin 35 and ticarcillin 36 are effective in mice against experimental infections caused by P-lactamase pro- ducing Oral and injectable formulations of potassium clavulanate with amoxycillin 35 (approved name co-amoxiclav) and ticarcillin 36 are marketed as Augmentin@ and Timentin@ respectively.Augmentin and Timentin are used against urinary tract skin and soft tissue and respiratory infections as well as bone and joint infections. In general the p-lactamase inhibitory effect of potassium clavulanate extends the antibacterial spectrum of amoxycillin 35 and ticarcillin 36 to include many p-lactamase producing bacteria normally resistant to amoxycillin and ticarcillin and other p-lactam antibiotics. 3 Other naturally occurring clavams The publication of several patent applications in the mid 1970s revealed that a number of groups had detected metabolites based on the clavam ring system 2.In particular it was obvious that Beecham and Glaxo research workers had detected isolated and characterised clavulanic acid 3 simultaneously with the initial Beecham patent application preceding that of Glaxo by a matter of a few months. Clavulanic acid 3 has also been isolated from S.jumonjinen-sis,66 S. katsuraharnanensi~~~ and an unnamed Streptomyces SP.,~~ and the P-hydroxypropionyl derivative 37 of clavulanic acid was obtained (though only isolated as its benzyl ester) from S. cl~vuligerus.~~ Clavaminic acid 38 was isolated by Elson et al. and shown to be a biosynthetic precursor of clavulanic acid 3 and to have the opposite i.e.3S,5S stereo-chemistry (see Section 8.1).70 A mutant of S. clavuligerus blocked in the production of clavulanic acid was found to accumulate three N-acyl derivatives 39 40 and 41 of clavaminic acid 38.7'The clavam derivatives 42 43 4472 and Ro22-5417 4573have also been obtained from S. clavuligerus. Table 2 Antibacterial spectrum of amoxycillin 35 in the presence of clavulanic acid 3 compared with amoxycillin and clavulanic acid alone against p-lactamase) producing organismsn MIC values/pg ml ' ' ~ Amoxycillin Amoxycillin + Clavulanic acid clavulanic acid Escherichiu coli NCTC 1 1560 >512 8 16 Escherichia coli Atcc 352 18' >512 4 16 Klehsiella pneumoniue 12 64 2.0 32 Proteus mirabilis 889 >512 4 32 Proteus vulgaris 75 10 Yersinru enterocoliticu 10723 512 32 2.0 8 32 32 Aeromonus hydrophila U53 256 32 16 Buc trroides fragih B3 32 0.5 32 Bucteroides vulgutus 940Buiw-oides meluninogenicus 109 16 16 0.1 0.1 32 32 Bacteroides thetuiotaomicron 4873 32 0.5 16 Huemophilus inyuenzae NEMC 1' 128 0.5 32 Neisxeriu gonorrhoeae AX 1729' 16 1.o 4 Moruxellu cutarrhulis 200 1E' 16 0.25 8 Stuphylococcts aureus ATCC 292 13 8 0.25 16 Stuphylococcus uureus NCTC 11561' 256 1.o 16 Stuphylococcus aureus V532'," 156 16 512 Stuphylococcus epidermidis 810 128 1.o 8 Stuphylococsus epidermidis 254'~~ 256 4 4 "Adapted from ref.6. "Expressed as a concentration of amoxycillin 35; MIC minimum inhibitory concentration. 'Strains producing a plasmid-mediated p-lactamase.dp-Lactamase-producing methicillin-resistant. 38 Clavaminic acid R = H 39 R=COCH3 40 R = COCH2NHCOCH3 41 R = COCH2NH2 H 0' 42 R=C02H 43 R = CH20H 44 R=CH20CHO 45 R = CH~CH(NH~)COZH 46 R = CH2CH20H 47 R = CH2CHCH(OH)C02H I NHCOCH(NH2)CHMe2 48 R = CH2CH(OH)CHCO2H I NHCOCH(NH2)CHMez Two clavams the hydroxyethyl clavam 4674 and valclavam have also been isolated from S. untibioticus. Valclavam was initially assigned the structure 4775but this has been reassigned to the regioisomer 48.76The clavamycin family (A to E) 49-54 is produced by Streptomyces hygroscopicus strain^.'^ Clavamy-cin A has also been isolated from S. platensis FERM 8313,78 clavamycins D and E from S. lavendulae FERM 831479 and clavamycin E from S.hrunneogriseus ssp. bannaensis FERM 706.80 Notably the clavams and clavamycins all lack the C-3 carboxy function of clavulanic acid 3 and clavaminic acid 38 and all possess the common 2R stereochemistry. It would appear that other than for clavulanic acid 3 (its biosynthetic intermediate 173) and possibly 37 all the reported naturally occurring clavams i.e. 3W6 and 48-54 possess the 49 ClavamycinA 50 Clavamycin B 51 R = COCH(NH2)CH(OH)CH(OH)CH2NH2 Clavamycin C 52 R = L-Valyl; Clavamycin D 53 R = L-Alanyl; Clavamycin E 54 R = ~-N~-Acetylornithyl; Clavamycin F opposite (i.e. 5S instead of 5R) ring junction configuration at C-5 to clavulanic acid 3.8*,82No other naturally occurring p-lactams with the S absolute stereochemistry at the bridge- head position have so far been reported.Whilst some of the clavams with the 5S-stereochemistry are antibiotics (e.g. the clavamycins 49-54 and valclavam 48) their antibacterial mode of action may not involve direct interruption of bacterial cell wall synthesis nor are they p-lactamase inhibitors. Some of the Baggaley Broaiw and SchoJi'eld Chemistry and biosynthesis of clavulanic acid and other clavams 5s-clavams (e.g. 48 and 46) also possess weak antifungal activity. 4 Other metabolites from S. clavuligevus As mentioned earlier S. cluvuligerus was originally noted for its production of cephamycin C 15. It was also found to produce penicillin N 55 deacetoxycephalosporin C 56 de-acetylcephalosporin C 57 and the 3-carbamoyloxymethyl HH HO&CH(NH2)(CH2)3CONH (D) 0’ C02H 55 Penicillin N H02CCH(NH2)(CH2)&ONH j3.J (D) 0 CH2R s a 0 C02H H 56 R=H 59 Holomycin 57 R=OH 58 R=OCONH2 \ OH OH 60 Tunicamycin n=8,9,10,or11 analogue 58 of cephalosporin C.1,83,s4 Non-P-lactam metabo- lites derived from S.cluvuligerus include holomycin 59 and an antibiotic complex MM 19290 related to tunicamycin 60.85 Recently Jensen and co-workers by examining discrepan- cies in estimates of clavulanic acid 3 in culture filtrates using bioassays and spectrophotometric p-lactamase inhibition assays have described a P-lactamase-inhibitory protein (BLIP) from S. clavuligerus.86 5 Chemistry 5.1 Derivatives The availability of quantities of clavulanic acid 3 prepared via large scale fermentation and isolation as an alkali metal salt or as its methyl 18 benzyl 19 or p-nitrobenzyl esters 20 allowed the modification of its functional groups to be examined in detai1.9,37,87-89 The aim of such studies was to explore structure-activity relationships of clavulanic acid 3 derivatives in order to determine if any had improved properties over the parent P-lactamase inhibitor 3.Esterification of the carboxylic acid function of 3 can be readily carried out using a diazoalkane by alkylation of an alkali metal salt or reaction with an alcohol or thiol and a carbodiimide in a suitable solvent. Benzyl 19 and p-nitrobenzyl clavulanate 20 were the most frequently used intermediates for derivatisation studies since these esters can be readily cleaved via catalytic hydrogenolysis in the presence of base leading to a salt of the derivative.Thus free clavulanic acid 3 can be generated by hydrogenolysis of benzyl clavulanate 19 in 314 Natural Product Reports 1997 ethanol at room temperature and pressure while sodium clavu- lanate was prepared under similar conditions in aqueous etha- nol containing an equivalent of sodium hydrogen carbonate. Amides 61 were obtained from the free acid 3 and an amine using a carbodiimide coupling agent. The free carboxylic acid 3 H H 61 R’ = CONHR3; R2 = H 66 62 R1=CH20H; R2=H H 63 R1= OMe; R2= H -Po\ 64 R’=OAc; R2=OAc t!d pCH2OH 65 R’ = OAC; R2 = H 67 R’ = R2 = H H ’R 68 R=H 70 R=C02H can be reduced to a primary alcohol 62 by reduction using lithium borohydride.Oxidative decarboxylation of clavulanic acid 3 by electrolysis in methanol gave clavam 63 while use of lead tetraacetate allowed the preparation of the clavam deriva- tives 64 65 and 66 though catalytic decarboxylation of 3 with mercuric acetate formed the two isomeric clavams 67 and 68.89-91 Controlled catalytic hydrogenation of methyl 18 or benzyll9 clavulanate produced the corresponding dihydroclavulanate esters 69 as a mixture of C-2 epimers. Hydrogenolysis of benzyl H 69 R = Me or CH2Ph 71 -n 0p YCH20H v d’ H’ C02CH2COPh 72 73 clavulanate 19 under modified conditions resulted in the formation of isoclavulanic acid 70 the E-isomer of 3 and 9-deoxyclavulanic acid 71.Isoclavulanate analogues were also prepared by photolysis of the allylic double bond of clavu- lanate derivatives. The tetracyclic p-lactam 72 was derived by photolysis of the phenacyl ester 73.87788 Exploitation of the chemistry of the allylic hydroxy function showed that it could be transformed into esters 74 ethers 75 74 X = OCOR2 81 X=NH2 75 X=OR2 82 X=NHCOR2 76 X = SR2; SOR2; S02R2 \ /N,, NN 77 X = OCONHR; OCSNHR 83X= \-I I 78 X=S03H 84 X = NHCONHR2 79 X=CIorBr 85 X=NHS02R2 80 X=N3 sulfides sulfoxides and sulfones 76 carbamates and thio- carbamates 77 and sulfonic acids 78.89,92,93 Conversion of the allylic alcohol into the 2-chloro or -bromo derivatives 79 gave access by replacement with amino carbon and sulfur nucleo- philes and via azide 80 reduction to amines 81 acylamines 82 triazoles 83 ureas 84 and sulfonamides 85.89,92,93 The inter- mediacy of the diene 86 in the allylic nucleophilic displacement 3y 0Lr>CHO 0 C02R' H C02R1 86 87 88 89 90 reactions was examined by Glaxo researchers who concluded that an overall 1,4-addition to diene 86 occurs regio- and stereo-specifically to regenerate the original R-chirality at C-3 and the 2-exocyclic alkene.92,93 Oxidation of the allylic alcohol function using pyridinium chlorochromate gave the aldehyde 87 whereas under Pfitzner-Moffat conditions elimi- nation occurred with formation of the diene 86.94Oxidation with rn-chloroperbenzoic acid led to the epoxide 88 while ozonolysis led to cleavage of the double bond with resultant formation of lactone 89.The p-lactam carbonyl of clavulanic acid derivatives also possess sufficient ketonic character pre- sumably due to its strained bicyclic structure to react with stabilised Wittig reagents to yield olefins e.g. 90 from the appropriate ester.95 Clavulanic acid 3 was also transformed into a number of non-p-lactam produ~ts.'~,~~,~~ The pyrroles 91 and 92 were OH OH / 91 92 generated vicr treatment of the ester 19 with triethylamine and acetic acid. The amino ketone 93 together with the liberation of carbon dioxide was a product of the hydrolysis of clavulanic acid 3 in alkaline neutral or acidic solutions. On long exposure to alkaline and neutral conditions further products including the pyrazines 94 and 95 were generated (Scheme 1).Amino-ketone 93 and the reduced pyrrole 96 are considered to be major metabolites of clavulanic acid 3 as they have been isolated from the urine of rats and mice following its admin- istration by various routes.98 Ring expansion of clavulanic acid led to the formation of the products 97 and 98.99,'0* More radical modification of p-nitrobenzyl deoxyclavu- lanate 99 led to interesting chemistry which gave a direct route into clavem 100 penem 101 and ethylidenepenam 102 deriva-tives. This chemistry has been reviewed by Newall; the princi- pal reactions are summarised as follow^.^^,^^ Treatment of 99 with triethylamine gave the crystalline racemic betaine 103 in equilibrium with racemic 2-ethylclavem 104.Optically pure 104 could be prepared by careful hydrogenation of the diene ester 105. Variations of this betaine chemistry depending upon the starting clavulanate derivative 106 were developed to provide a new route to penem antibiotics. Thus 'one-pot' conversion of Ho2cxoH 93 H 96 i HO&/froH 94 CH3CHO I HO HOA;ToHz OH 95 Scheme 1 97 98 the betaine intermediates 107 to the methanesulfonates 108 followed by reaction with hydrogen sulfide and triethylamine to form the thiobetaines 109 and then heating gave the racemic 2-ethylpenem or 2-( 1-ethoxyethy1)penem 110 (Scheme 2). Such chemistry enabled ready access to a variety of penem derivatives. These were found to have antibacterial activity but were poor p-lactamase inhibitors.5.2 Synthesis Given its medicinal and commercial importance the novelty of its ring structure with only two chiral centres and its low molecular weight (179 Da C,H,NO,) it is remarkable that the synthesis of clavulanic acid 3 has not received more attention. This a perhaps due to the ready availability of 3 by fermen- tation giving little rationale for its synthesis. The complemen- tary synthetic approach to novel clavulanic acid 3 analogues and derivatives was pursued within Beecham and Glaxo research laboratories in the mid- 1970s. The first formal total synthesis (Scheme 3) of racemic clavulanic acid 3 was achieved starting from the azetidinone 111."' Alkylation of 111 with the 2-bromo derivative of Bagguley Bro,zw and Schofield Chemistry and biosynthesis of clavulanic acid and other clavams 315 CH2C02Me 0$>OH C02Me C02Me NB 113 99 103 111 112 / ti / H r! C02Me I C02Me 114 115 V 0flyR JL+R H H 0 C02H C02H 101 1 02 0py C02pNB 105 106 107 0&3HzR-0 &?;re C02pNB C02pN B 109 108 I + a>cH2R e.g.R = H or OCH(Et)Me etc. 110 Scheme 2 dimethyl 3-oxoglutarate gave the diester 112 which exists predominantly in the enolic form. Reaction of 112 with chlorine substituted the thiomethyl to give chloride 113 which was cyclised on treatment with potassium carbonate to form a clavam derivative as a single isolated double bond isomer 114.This on irradiation formed a 3:2 mixture of 114 and 115 which on reduction with diisobutyl-aluminium hydride gener- ated 116 and 117 in low yield. Chromatographic resolution of this mixture provided ( f) methyl clavulanate 117 and ( f) methyl isoclavulanate 116. Since methyl clavulanate 18 had been previously hydrolysed to clavulanic acid 3 the synthesis of ( f) methyl clavulanate 117 confirmed the structure of 3. An alternative synthesis also by Bentley et al. started by reaction of the azetidinone 118 with vinylacetyl chloride to give the ester enol 119 which on chlorinolysis and cyclisation formed the diene 120.lo2 Ozonolysis of 120 and hydrogenolysis of the intermediate ozonide produced ( f) methyl clavulanate 117 and the aldehyde 121.316 Natural Product Reports 1997 116 117 Scheme 3 Reagents i NaH DMF MeO,CHBrCOCH,Me; ii Cl, CCI,; iii K,CO,; iv hv C,H,; v Bu’,ALH MeC,H, -70 “C. C02Me 118 119 pyH p yv H 0 0 0 C02Me C02Me 120 121 The chemistry developed in the above routes to 7-oxo-4-oxa- l-azabicyclo[3.2.0]heptanes allowed analogues of clavulanic acid to be prepared and access to a range of synthetic clavam and clavem derivatives which were not available by ready modification of the natural product. In particular compounds lacking the C-3 carboxylate function and with a number of substituents at C-2 were prepared and evaluated as inhibitors of p-lactamases. Io3 5.3 Structure-activity relationships The p-lactamase activity profile of clavulanic acid derivatives (Table 3) has been studied in depth and their chemotherapeutic potential in combination with various P-lactamase susceptible penicillins and cephalosporins e~aluated.~’ To retain potent p-lactamase inhibitory properties in a derivative certain empirical structure-activity relationships were deduced the principal ones being; (i) only replacement of the hydroxy function at C-9 by small hydrophilic functions provided com- pounds with activity approaching or bettering that of 3; (ii) any modification of the double bond by reduction addition or isomerisation resulted in loss of activity; (iii) loss of the C-3 carboxylate function or substitution at C-6 resulted in consid- erably reduced P-lactamase inhibitory activity; and (iv) com- pounds with the opposite stereochemistry to clavulanic acid at C-5 were poor inhibitors (i.e.the 5R stereochemistry is required for activity). lo In addition to demonstrate potential synergistic properties in combination with p-lactamase suscep- tible penicillins and cephalosporins derivatives had also to be able to penetrate the cell wall of Gram-negative organisms. Table 3 p-Lactamase-inhibitory properties of derivatives of clavulanic acid 3" ~~ Staphylococcus Escherichiu Klebsiellu Proteus Citrobucter uureus Russell coli JT4 pneumoniue E70 mirubilis C889 ,freundii Manti p-lactamaseb TEM (3-lactamase" p-lactamaseh p-Iactamaseh p-lactamase' ~~ Clavulanic acid 3 0.06 0.07 0.03 0.03 10 -5 Deoxyclavulanic acid 71 0.12 0.09 0.05 -5 Isoclavulanic acid 70 0.6 1.o 0.45 Acetate 74 (X=OCOCH, R'=H) 0.04 ->0.4 -0.4 Carbamate 77 (X =OCONHCH, R' =H) 1.5 2.5 2.5 -0.45 Methyl ether 75 (X=OMe R'=H) 0.05 0.18 0.07 0.01 8.5 Benzyl ether 75 (X=OCH,Ph) 0.005 0.1 0.04 0.02 4.4 Thioether 76 (X=SMe R1=H) 0.1 1 0.04 0.13 0.01 > 10" Amine 81 (X= NH, R' =H) 0.002 0.04 0.08 0.01 0.62 "Adapted from ref.47. "'Penicillinase'. "Cephalosporinase'. dp-Lactamase from Enrerobacter cloacae P99. Needless to say the retention of the p-lactam ring system Mode of action studies indicated that while it was an irrevers- was essential for activity. Of the derivatives studied in depth ible inhibitor of the TEM P-lactamase it was also a substrate the two with some promising activity to rival clavulanic for the enzyme.Oral absorption in humans of 124 is poor but acid 3 were sodium 9-deoxyclavulanic acid 122 and useful blood levels are obtained when it is given parenterally 9-aminodeoxyclavulanic acid 123.Io4 Of these the amino being well tolerated with good urinary recovery. Mutual pro-drugs 125 (sultamicillin) and 126 linking 124 with ampicillin 127 and mecillinam have also been pre-pyv H 3 pl+YH2NHz pared.108,109 Sultamicillin 125 (Unasyn@) is well absorbed H 0 0 C02Na C02H 122 123 compound 123 was found to be more effective than 122 and clavulanic acid 3 in vitro and in vivo as a synergist in combi- nation with amoxycillin. This derivative however was found in toxicological studies to cause liver and kidney damage in animals following oral dosage and was not progressed as an alternative to clavulanic acid 3.uu CGH&H(NH~)CONH wsk' n4-N-( ... 6 Synthetic analogues W C02H The discovery and development of clavulanic acid 3 as a 127 Ampicillin P-lactamase inhibitor of clinical significance prompted con- siderable efforts towards the discovery of alternative structures with similar or improved P-lactamase inhibitory pharmaco- logical and toxicological properties. Most of those prepared from the gastrointestinal tract and then hydrolysed into 124 were derivatives of penicillanic acid 17. and ampicillin 127. Sulbactam 124 is also used in non-covalent Penicillanic acid sulfone 124 (CP-45,899; sulbactam) was combination with ampicillin 116 and cefoperazone 128. identified by the Pfizer group as the first synthetic p-lactam H 09 HO-0 C02H 124 Sulbactam analogue with inhibitory characteristics to rival clavulanic acid \ Et 3.lo5 It was prepared by oxidation of penicillanic acid or by the 128 Cefoperazone hydrogenolysis of 6,6-dibromo- or 6a-bromo-penicillanic acid sulfone.The properties of this penicillanic acid derivative as an inhibitor of a range of p-lactamase types and as a synergist in combination with a variety of penicillins and cephalosporins Examples of other penicillanic acid derivatives with inhi- have been the subject of many papers.1067107 In general terms it bitory activity include 6P-bromo- 129 (bromobactam) and 6p-is a less effective inhibitor than clavulanic acid 3 of the plasmid iodo-penicillanic acids 130."03' I Other active sulfones are mediated P-lactamases produced by E.coli and K. aerogenes. the 6a-chloro-compound 131,'l2 and the P-methyl modified Baggaley Brown and Schofield Chemistry and biosynthesis of clavulanic acid and other clavams 317 penicillanates 132 133' 133' l4 and 134 with the latter tazo-bactam (YTR 830),'15 being the most interesting as a possible alternative to 3. From detailed p-lactamase inhibition studies against the most clinically relevant plasmid mediated p-lactamases it was shown that tazobactam 134 and clavulanic acid 3 were gener- ally equipotent inhibitors and more effective than sulbactam 124.lI6This ranking of activity was also reflected in assays to determine the effectiveness of p-lactam-p-lactamase inhibitor combinations against ampicillin-resistant clinical isolates.' ' C02H " C02H 129 R=Br 131 130 R=I 132 R=CI 133 134 R= 'N ,N+ N Ll Tazobactam CNk0 Et 135 Pipericillin The kinetics of tazobactam 134 inhibition have been examined in detail."' It has undergone clinical studies in combination with the broad-spectrum penicillin piperacillin 135. C6-Substituted methylene penams such as 136 and 137 have shown striking inhibitory activity.' 19,120 SmithKline Beecham workers have developed the 6-triazolylmethylene R IH )I-Lr% 0 C02H CO2H 136 R=OCH3 138 BRL 42715 137 R = COCH3 penem 138 (BRL 42715) as a very potent broad spectrum inhibitor of a wide range of p-lactamases including Ambler Class B p-lactamases. 121,122 The two recently reported sulfones 139 and 140 also have promising inhibitory properties.123,124 With the more recent observation of an increasing number of examples of bacterial resistance developing via the plasmid mediated transfer of J3-lactamases of metallo-enzyme type inhibitors of these metal requiring 0-lactamases (Ambler Class B) are being sought. '25,126 Two inhibitors of this type of 141 142 p-lactamase typified by p-lactamase I1 of B. cereus are N-(carboxymethyl)-6-aminopenicillanic acid 141 and the bicyclic &lactam 142. The mechanism of action of these compounds has still to be determined. '27 Trifluoromethyl-ketones and alcohols have also recently been shown to be inhibitors of the metallo p-lactamases. '28 7 Mode of action Extensive studies on the kinetics of and mechanism of interac- tion and inactivation of various p-lactamases by clavulanic acid 3 have been carried out by Reading and co-~orkers'~~-'~' and Kn~wles'~~ Most experiments have used TEM P-lactamase from E.coli though the kinetics of the inhibition of P-lactamases from Staphylococcus aureus Klebsiella pneu- moniae Branhamella catarrhalis and Bacillus cereus I have also been investigated. The E. coli TEM 1 TEM 2 and pBR 322 S. aureus ,and Bacillus cereus I Class A J3-lactamases show a high degree of active site homology including a conserved nucleo- philic serine residue.'33 For each molecule of inactivated TEM I1 P-lactamase produced by clavulanate 3 inhibition ca. 115 molecules of 3 are required; for p-lactamase inhibition by sulbactam 124 ca.7000 molecules are required. 132,134 Kinetic studies using UV-VIS spectroscopy on the clavulanic acid mediated inhibition of the S. aureus Class A p-lactamase led to a proposed mechanism (Scheme 4).' '2,128.129,135,136Reading and Hepburn also considered decarboxylation of the P-keto acid intermediate(s) 143 and 144 to give 145 and speculated on coo-3 41 0, /N-PCH3 I 143 144 Scheme 4 318 Natural Product Reports 1997 0 rHNhOH A 00 *W 145 146 A 00 & 148 the possibility of clavulanate 3 induced cross linking of the p-lactamase between Ser-70 and a lysine residue to give 146. 129 Detailed studies on the E. coli TEM enzyme by Knowles and co-workers described the production of three irreversibly inacti- vated enzyme complexes and a transiently inhibited form.137 Labelling experiments indicated that for the four inactivated species the carbons of both rings of clavulanic acid 3 were retained in the transiently inhibited species and in two of the three irreversibly inhibited species. 13* The other irreversibly in- hibited species was assigned as the cross linked enamine 147.'39 A crystallographic study on a complex formed from S. aureus PCI 0-lactamase and clavulanic acid 3 was consistent with the production of a Ser-70 (using the Ambler numbering system' 33) linked cis-enamine 143 and the decarboxylated E-enamine 145. I4O However cross-linking of the p-lactamase was not apparent.Modelling studies,I4' based on the 28 resolution crystal structure of the Bacillus licheniformis 3 143 andlor 145 TEM-2 with -C3H403 dehydroalanyl f 130 152 00 I t&ml TEM-2 with +H20 H.C=~ 148 dehydroalanyl -C3H403 130 ?O & 153 p-lactamase led to the proposal that Ser-130 was the only likely residue to be involved in cross-linking of the p-lactamase to form a vinyl ether 148. Direct evidence for production of the Ser-70 to Ser-130 cross linking vinyl ether 148 has come from use of electrospray ionisation mass spectrometric (ESIMS) analysis of TEM 2 P-lactamase after treatment with clavu- lanate 3. ESIMS studies demonstrated that minimally four different modifications are made to the P-lactamase upon incu- bation with 3.The shortest lived of these whose mass corre- sponded to decarboxylated clavulanate inhibited enzyme was assigned structure 149. Mass increments corresponding to al- dehyde 150 hydrated aldehyde 151 and a cross-linked species such as 147 or 148 were also observed. The putative cross- linked species was the most stable of the complexes observed. ESIMS analysis of peptides obtained after protease mediated fragmentation of clavulanate inhibited P-lactamase demon- strated modifications to both Ser-70 and Ser- 130 consistent with the production of the proposed cross-linked vinyl ether 148. Surprisingly under the acidic purification conditions evi- dence also accrued to indicate that the major decomposition pathway open to the vinyl ether 148 is fragmentation to give a dehydroalanyl residue at Ser- 130.The relative stability of the vinyl ether 148 suggests that its formation will effectively cause irreversible inhibition under physiological conditions. A mech-anism for clavulanate 3 inhibition of the TEM 2 p-lactamase based on the spectroscopic' 12-129,135-139 and ESIMS142 studies is summarised in Scheme 5.'42 Initial acylation of Ser-70 by clavulanate is followed by rapid oxazolidine ring opening and decarboxylation to give 149. This intermediate may undergo hydrolysis to form aldehyde 150/hydrated aldehyde 151 which may be further hydrolysed to generate catalytically active en- zyme. Alternatively 149 may react to form the cross-linked vinyl ether 148 which fragments (under acidic conditions) to give an aldehyde 152/hydrated aldehyde 153 with a dehydro- alanyl residue 130.These slowly hydrolyse to give Ser-130 dehydrated p-lactamase. * coo-h\;7d\\Y andlor 15* *q 150 Scheme 5 Baggaley Brown and Schojield Chemistry and biosynthesis ofclavulanic acid and other clavams 8 Biosynthesis 8.1 Clavulanic acid biosynthesis Studies on the origins of clavulanic acid 3 over the last couple of decades have revealed a remarkable and unprecedented biosynthetic pathway. Several reports on early studies have been p~blished.'~~-'~~ This section reviews the presently available evidence and is presented in approximately chrono- logical order of discovery. Following its discovery and application biosynthetic studies on clavulanic acid 3 were initiated and pioneered in the Beecham Pharmaceutical Lab- oratories (now SmithKline Beecham) at Brockham Park and Worthing.Since crude clavulanic acid 3 is unstable in labelling studies it is usually isolated as its benzyl 19 or p-bromobenzyl 21 esters. In the first experiments carried out by the Beecham group [1-',C] ace ta te [2- ' Clace t a te [I,2-C,] ace t a te [1-' C] propionate and [I3C]bicarbonate were fed to fermentations of S. cluvuligerus ATCC 20764 in the production phase of 3.'46 Labelling patterns determined by ',C-NMR spectroscopy indicated that the acetates had been metabolised through the tricarboxylic acid cycle (TCA) and that carbon atoms 2 3 8,9 and 10 i.e. the C unit of 3 were probably derived from 2-oxoglutarate (a-KG) whereas the three carbon skeleton of the p-lactam ring i.e.the C unit had originated from the TCA cycle via gluconeogenesis. Feeding [1,3-' 3C2]glycerol 154 * CH20H 2 F O H HO+H * * CH20H 0 'C02H 154 3 155 = '3C gave good incorporation levels into C-5 and C-7 of 3 with long range ',C-' spin-spin couplings suggesting that the p-lactam ring may be derived via incorporation of intact glycerol. Hence it was deduced that the carbon atoms of the p-lactam ring were derived from gluconeogenesis products,'43 possibly pyruvate. The deduction that the C unit originated from a-KG suggested initially the intermediacy of glutamate. The proposal was reinforced by feeding racemic [3,4-'3C,]glutamate 155 which labelled C-2 and C-8 of 3.'47I3C-I3C Spin-spin coupling experiments confirmed that the C-3 and C-4 bond of the glutamate remained intact during incor- poration into C-2 and C-8 of 3 which ruled out the possibility that C-8 and C-9 of 3 were incorporated from a separate two carbon unit.These early experiments thus demonstrated that 3 was of mixed biosynthetic origin. If glutamate is a precursor of clavulanic acid 3 then at some time its y-carboxy group must be reduced to an hydroxymethyl group. Hence 2-amino-5-hydroxyvalerate 156 was considered as a potential intermediate. The incorporation of both 13C and fHR H2N 'CO2H H2N 'CO2H 156 157 R = H Ornithine 158 R = C(NH)NH2 Arginine I4C labelled versions of this amino acid was investigated and in each case was found to be very low although specific to the C unit of 3.145,148 In order to examine further the origin of the C unit Townsend and Ho studied the incorporation of the radio- labelled C amino acids ~-[U-'~C]glutamate ~-5-hydroxy-[U-''C]norvaline (2-amino-5-hydroxyvalerate) ~-[U-'~C]proline 14* L-[U-''C]ornithine and ~-[U-'~C]arginine.They concluded that the urea amino acids ornithine 157 and arginine 158 and particularly ornithine 157 were better utilised than the other amino acids with minimum randomisation into the C unit of 3. Shortly after this report Romero et ul. described studies on the incorporation of [l-'4C]ornithine [5-'4C]ornithine L-[U- 14Clglutamic acid and ~-[U-'~C]arginine into 3.'49 It was found that these amino acids were well incorporated and that glutamate was utilised 30 to 40 fold less efficiently than arginine or ornithine.Both arginine and ornithine stimulated the production of 3. It was also reported that the enzyme arginase which hydrolyses arginine to ornithine was present in the cells during production of 3. This group concluded that arginine is converted into ornithine and that this C amino acid instead of glutamate is the direct precursor of 3 support-ing Townsend and Ho's finding~.'~' Townsend and Ho investigated the stereochemistry of orni- thine oxidation during its incorporation into 3 by feeding (2RS,5RS)-[5-,H U-'4C]ornithine.'48 It was found that half of the tritium label was lost on incorporation into 3 suggesting intermediate transamination or amine oxidation to an aldehyde 159 which on eventual reduction would lead to the hydroxy- methyl group of 3.The stereoselectivity of this transformation was investigated by the synthesis of the two epimeric ornithines H2N. C02H 160 HA=H; HB=~H 161 HA= 3H; Hg= H I 162 (2RS,SR)-[5-'H]ornithine 160 and (2RS,5S)-5-3H]ornithine 161 which were fed to S. clavuligerus fermentation^.'^' The label of the 5-pro-R (HB) ornithine was largely retained whilst it was lost in the 5-pro-R (HA) case. Degradation of the labelled clavulanate ester 21 to glycolic acid derived from the C-8 and C-9 atoms of 162 followed by assay with glycolate oxidase demonstrated that the retained tritium label occupied the (9-pro-S) position in 162 and hence that the 5-pro-R hydrogen of ornithine was retained in biosynthesis with overall inversion of stereochemistry.The hydrogen atom H in 162 was presumed to have been delivered to the re-face of a hypothetical aldehyde intermediate such as 159 as opposed to a sequence involving elimination or displacement. In a related study the Beecham group found that the labelled carbon atom of D,L-[~-~H,,~- I3C]ornithine 163 was well incorporated into 3 but no detectable levels of deuterium were found in 3.15' Following on from Elson's con~lusions'~~~' 46 that the C unit was derived from a glycolytic intermediate possibly pyru- vate Townsend and Ho investigated the incorporation of radiolabelled glycerol pyruvate serine and glycerate.They concluded that of the possible C glycolytic intermediates 320 Natural Product Reports 1997 OH 164 D-glycerate 164 was most directly utilised to become the P-lactam carbons of 3 confirming the mixed biosynthetic origin of 3. In order to determine the fate of the diasterotopic protons HA and H of glycerol 165,the incorporation of (1R,2R)- and 165 3 (1S,2R)-[1-3H,1,3-14C]glycerol was studied.153 The glycerol 1-pro-R tritium was retained in 3 whilst the 1-pro-S label was lost indicating that the diasterotopic tritium labels in 165 do not become homotopic in the biosynthetic process. It was concluded that the absolute sense of chirality at the glyc- erol C-1 centre is retained during biosynthesis of 3 from the labelled glycerol and occurs with overall retention of the stereochemistry at C-5 of 3.Since the oxidation levels of the carbon atoms in P-hydroxypropionate are at the same level as those of the corresponding atoms in 3 and P-hydroxypropionate is a known product of glycerol metaboli~m,'~~.~~~ Gutman and co-workers investigated the incorporation of radiolabelled P-hydroxypropionate into 3.1S6The high retention of label in the C unit of 3 led to the postulate that the first step in the building of the skeleton of 3 involves the coupling of P-hydroxypropionate with an appropriate amino acid such as glutamate or ornithine to form the 'dipeptide' intermediates 166 and 167. It was further proposed that a concerted OkNH+ C02H 166 R=C02H 167 R=CH2NH2 oxidation and ring closure was involved in the later stages and that 166 and 167resembled the Arnstein tripeptide the precur- sor of the penicillins and cephalosporins which cooccur with 3 in S.cluvuligerus. 57 Subsequently however Townsend et reported that they obtained only low levels of nonspecific incorporation of P-hydroxypropionate into 3. Almost all knowledge of the biosynthetic pathway up to this point had been deduced from feeding labelled molecules which had to undergo an unknown number of enzymatic steps to yield 3. In order to obtain information regarding the biosynthetic intermediates between the proposed primary metabolic precur- sors ornithine 157 and glycerate 164 and 3 other possible approaches were considered. Since the feeding of whole cells with labelled speculative precursors would involve lengthy syn- thesis of target substrates that may or may not be incorporated other approaches involving cell free technique^,'^^ were adopted by the Beecham A chemical programme syn- thesising unlabelled speculative biosynthetic intermediates was also initiated with the objective of testing the compounds in cell-free systems and assaying for clavam production.IS9 Cell-free studies have been very successfully harnessed in the elucidation of the penicillin and cephalosporin biosynthetic pathways144,157.160 which also occur in S. clavuligerus. Several of the enzymes involved in the biosynthesis of the penicillin and cephalosporin family of antibiotics including isopenicillin N synthase and deacetoxycephalosporin C synthase belong to the family of non-haem ferrous dependent oxygenases.157 These enzymes all have a requirement for ferrous iron as a cofactor and dioxygen as a cosubstrate. Most also require 2-oxoglutarate (a-KG)as a cosubstrate and dithiothreitol or another reducing agent for optimal in vitro activity. The Beecham group specu- lated that related oxygenases may be involved in the biosynthe- sis of clavulanic acid 3 and attempted to demonstrate the production of 3 in ultrasonically disrupted S. clavuligerus cells in the hope of identifying endogenous precursor corn pound^.^^ Combinations of ferrous iron ascorbate dithiothreitol and 2-oxoglutarate were added to the aerated cell free extracts. The production of 3and other clavams was monitored for by assaying for P-lactamase inhibitory activity and by use of HPLC analysis after treatment with imidazole.Imidazole reacts with 3,but not penicillins and cephalosporins to give the adduct 168,which exhibits a characteristic UV absorption at 313 nm.l6I Other clavams also react with imidazole to produce derivatives analogous to 168. 0 1613 Only in the presence of a combination of a-KG Fe2+ ion and 0 was the production of a clavam observed. No produc-tion of clavulanic acid 3 was detected however HPLC analysis detected a peak with a shorter retention time than that due to 3 (i.e. 168) the intensity of which increased with time. The substance responsible for this peak was identified as a new natural product 38,named clavaminic acid.Clavaminic acid 38 does not have P-lactamase inhibitory activity and has the opposite relative stereochemistry at C-3 and C-5 to 3 i.e. the same as other reported clavams 42-46 and 48. Since the amount of 38 increased with incubation time it was reasoned that it was produced enzymatically in the cell-free reaction mixture from an endogenous natural precursor. Large scale fractionation of cell-free extracts by column chromatography led to the discovery of a compound which could be converted into 38 by treatment of column eluates with crude enzyme preparations and appropriate cofactors and cosubstrates. The precursor to 38 was named proclavaminic acid and assigned the structure 169 on the basis of spectroscopic analyses and by 169 Proclavaminic acid comparison with compounds prepared in the speculative chemical programme.All four stereoisomers of 169 were syn- thesised'62 and it was found that only a single unassigned enantiomer was transformed to 38 by the cell-free system. Baggalej? Brown and Schojield Chemistry and biosynthesis of clavulanic acid and other clavams The enzyme which cyclises 169 to 38 was named initially clavaminic acid synthetasef and characterised using conven- tional procedure^.^' a-KG Fe2' ion and 0 were found to be -aYoH essential for the enzymatic conversion of 169 to 38 and stoichi- 0 pN?NH2 '. 0 metric conversion could only be obtained using two equiv. of *'C02H I~-KG.~' In this reaction the a-KG is a cosubstrate being *C02H 170 U Clavarninate synthase (CAS) * Fez+,2 02 2 equiv.2-oxoglutarate 169 38 oxidised to succinate and carbon dioxide. Four hydrogen atoms must be removed from proclavaminic acid 169 to form 38 presumably in two stages. Possible outline mechanisms for the reaction will be discussed below. The isolation of proclavaminic acid 169 clavaminic acid 38 and characterisation of the enzyme clavaminate synthase (CS) which carries out the oxidative cyclisation of one to the other did not in itself prove that these compounds were intermediates in clavulanic acid 3 biosynthesis. The conversion of 169 into 38 was also observed in by cell-free extracts of the clavulanic acid 3 deficient mutant S. clavuligerus dclC 8 which had been shown to produce the acylated derivatives of 38 and 3941 with the (3S,5S) stere~chemistry.~' Also labelled ornithine 163 was in- corporated into 40 with approximately 70% of the molecules bearing I3C at C-9 and a deuterium at C-8.I5l The level of production of 40 in the mutant strain dclC 8 was similar to the titre of 3 in the clavulanic acid producing strain and it was deduced that the biosynthetic pathway in dclC 8 was blocked between 38 and 3 resulting in an intracellular accumulation of 38 which was then acetylated and excreted from the cells.Other microorganisms were examined for the presence of procla-vaminic acid 169 clavaminic acid 38 and CS activity. Evidence for them was found in the clavulanic acid producing strains of S.jumonjinensis ATCC 2986466 and S. katsurahamensis T-27267 and also in the cephamycin producer S. lipmanii NRRL 3584'; S. lipmanii NRRL 3584 does not produce clavulanic acid 3 and has not been reported to produce other clavams. The Beecham group considered it likely that there are at least three biosynthetic steps between 38 and 3 possibly amine-oxo exchange inversion of the C-3 and C-5 stereochemistry and aldehyde reduction although not necessarily in that order. Cell-free experiments in the presence of the cofactors pyri- doxal phosphate pyruvate and NADPH converted 38 into 3 in low (0.3%) yield.'63 In order to obtain stronger evidence that 38 and 169 were on the same biosynthetic pathway both compounds were prepared in doubly labelled I 3C-labelled form for feeding experiments.' One of the racemic diastero- isomers of [1,2-13C2]-169 labelled as shown in 170 was found to be well incorporated into 3. This diasteroisomer contained the natural enantiomer of proclavaminic acid which had been converted into 38 in the cell-free system containing CS and the appropriate cofactors. The other diasteroisomer of 170 was not incorporated and was not converted into 38 in the cell-free system with CS. [3,10-'3C2]-38 i.e. 171 was prepared by con- verting the [1,2-"C2]-170 into 171 in vitro with partially puri- fied CS and feeding the resultant 171 to S. clavuligerus. In the I3C NMR spectra of the isolated benzyl ester 19 of 3 produced by feeding either the doubly labelled diasteroisomer of 170 or 171 I3C-l3C spin-spin couplings were observed between the labelled centres C-3 and C-10 in 3 demonstrating that both $In subsequent publications from the UK groups the name clavaminic acid synthase (CAS) was used.Townsend and co-~orkers'~~ have used the name clavaminate synthase (CS) which is used in this review. H 0 172 171 * = 13c compounds were incorporated intact into 3. The result was confirmed using a triple "C-labelled sample of 38 [3,7,10- "C3]-172 the third label being located at the carbonyl carbon of the p-lactam ring. The I3C NMR spectrum of the isolated clavulanate exhibited l3C-I3C spin-spin coupling between the three labels located at C-3 C-7 and C-10 again indicating that 38 was incorporated intact with no bond breakage occur-ring.163 These results clearly demonstrated that both 38 and 169 were intermediates in the biosynthesis of 3 and that CS was a functional enzyme the first to be identified in the central part of the clavulanic acid biosynthetic pathway.Studies directed towards identifying and cloning the biosyn- thetic genes for clavulanic acid biosynthesis were initiated in Beecham laboratories in the early 1980s. In 1984 Bailey et reported the cloning of the dclC genetic locus which was shown to be involved in clavulanic acid 3 biosynthesis by complementation of a lowhon clavulanic acid 3 producing (dcl) mutant. DNA sequences from the dclC locus were used as probes to identify cosmid clones which encoded clavulanic acid 3 biosynthesis genes.'65 N-Terminal ~equencing'~~,'~~ of purified CS enabled the corresponding gene to be identified within a putative cluster of clavulanic acid 3 biosynthesis genes.165,167,168 The absolute stereochemistry of the bioactive enantiomer of proclavaminic acid 169 was deduced to be (2S,3R)-169 by 693 synthesis from 3- h ydroxyorni thine.I I7O This result led to the conclusion that the CS catalysed oxidative cyclisation of pro-clavaminic acid 169 to clavaminic acid 38 proceeds with reten- tion of stereochemistry at the carbon atom bearing the carboxy group. Independent synthesis of natural proclavaminic acid 169 from L-glutamic acid confirmed this assignment.I7' By feeding ~,~-[3-"c]ornithine to growing cultures of S. clavuligerus under an atmosphere containing I8O2,it was found that efficient and approximately equal incorporation of the "0 label occurred into the oxygen of the oxazolidine ring and that of the hydroxy group of 3.17 This result suggested that the oxazolidine oxygen of 3 was introduced via a hydroxylation of an intermediate prior to 169 in the pathway and that the conversion of 169 to 3 involved an oxidative deamination of the C-9 amino function of 38 to give the aldehyde 173 (Scheme 6).17* This last result reinforced earlier indications that an aldehyde intermediate was involved in the biosynthesis of 3 and led to a mechanistic proposal (Scheme 6) based on precedents from clavam chemi~try,~~,~~ for the remarkable isomerisation of the 3S,5S ring of 38 to the 3R,5R of 3. Firm evidence for the involvement of the aldehyde 173 was later published'73 (see below).Gutman and co-workers followed up their earlier specu- lation~'~~ that 'dipeptides' of the type 166 and 167 may be biosynthetic precursors of 3 by synthesising 166 and 167 for biosynthetic studies. 174 Similarly Negro et al.'75 prepared 167 and 174 followed later by a report'76 on the synthesis of rac-threo-174 and -175. In vitro mechanistic of the cyclisation of pro- clavaminic acid 169 by CS with deuteriated proclavaminic 322 Natural Product Reports 1997 system'80 indicating the possible presence of 181. The exper- H* 38 0 L C02H0 I iment was repeated with the deuteriated substrate 182 in the hope that an isotope effect would slow the formation of the exocyclic double bond and enable the putative saturated intermediate to be isolated.In the event the saturated bicyclic clavam 183 was isolated from this experiment and character- ised. When incubated with CS purified 183 was converted -g++bH0 pNJ,,,,lrNH2R ,7NH2 C02H 0 C02H0 C02H 181 R=H 1 82 173 0 p++ 183 R =2H 1 3 * = 180 Scheme 6 C02H into clavaminic acid 38 demonstrating the intermediacy of 181 in the cyclisation of 169 to 38. Subsequently using recombi- nant CS the intermediate 181 was isolated from in vitro CS incubations. The isolation of the saturated intermediate 181 demonstrates that CS catalyses sequential two electron oxidations of sub- strates in the biosynthesis of 3. Each of these oxidations is coupled to the consumption of one molecule of 2-oxoglutarate and (presumably) of dioxygen.In its cosubstratelcofactor HOJNH2 0 ?eNH2 requirements CS is a member of the ferrous dependent non- HOCH2CH2CONH /*\C02H C02H 174 175 C02H 176 "7NH2 H'"' * = 18c C02H 178 0 = '3C acids 176 177 and doubly labelled material 178 demonstrated that the cyclisation of 169 to 38 did not involve exchange of the protons at C-2 or C-3 (of the side chain) and the oxygen of the oxazolidine ring of 38 was derived from the hydroxy group of 169. When specifically labelled proclavaminates 179 and 180 were incubated with CS the clavaminate resulting from 179 retained (94%) deuterium. 158 Stereoisomer 180 reacted signifi- cantly more slowly and the resulting clavaminic acid 38 contained only ca.12% deuterium at C-5 of the clavaminate 38. From these data it was concluded that the 4'shydrogen HA was selectively (>go%) lost and the 4'R hydrogen H, retained. Thus CS forms the oxazolidine ring of 38 by a desaturative ring closure involving removal of the 4'shydro-gen and the hydroxy hydrogen of proclavaminate 169. Evidence that the oxazolidine ring of 38 is formed prior to the introduction of its double bond was obtained by NMR spectroscopic analysis of the product composition of an incu- bation of proclavaminic acid 169 with partially purified cs.178,179 In addition to signals corresponding to clavaminic acid 38 a minor resonance at ca. 6 5.4 was observed. This was reminiscent of the C-5 proton of the 2,8-dihydroclavulanate haem iron oxygenase and oxidase family.Most of these enzymes have a requirement for a 2-oxoacid cosubstrate normally 2-oxoglutarate. Known exceptions to this require- ment are isopenicillin N synthase (IPNS)' 57 and 1-amino- 1- cyclopropanecarboxylic acid (acc) oxidase. The desaturative cyclisation of 169 to 181 by CS is clearly reminiscent of the conversion of the tripeptide L-6-(a-aminoadipoyl)-L-cysteinyl-D-valine 184 into isopenicillin 185 as catalysed by IPNS (Scheme 7). However in contrast to the IPNS reaction where 184 IPNS-Fe(li) K::20 C02H H H2NT-$Nn-f C02H 0' 't02H 185 Scheme 7 the tripeptide undergoes a four electron oxidation in the CS catalysed reaction a two electron oxidation of 169 is coupled to the oxidation of 2-oxoglutarate to succinate and CO,.A possible outline mechanism for the CS catalysed conversion of 169 to 38 is indicated in Scheme 8. Thus initial generation of a ferryl 0x0 [Fe(~v)=Ol species by reaction of CS-Fe(I1) with 2-oxoglutarate and dioxygen as proposed for other 2-oxoglutarate dependent dioxygenases '82 is followed by binding of 169. It is possible that the hydroxy group of 169 ligates to the active site iron of CS as in 186. The reactive ferryl species then oxidatively inserts into the 4'sC-H bond of 169 to generate an intermediate 187. Ring closure of 187 gives 181 which is released into solution and regenerates CS-Fe(1r). The desaturation of 181 to give 38 is similarly mediated by the Baggaley Brown and SchoJield Chemistry and biosynthesis of clavulanic acid and other clavams cs H O=Fe/ -H20 A II 186 187 0 cs O.+e'i'V I OH As in A 1 coy Ii t 181 38 Scheme 8 intermediacy of a CS bound ferryl 0x0 [Fe(w)=O] species.Note the proposed 'flip' in the orientation of the ferryl intermediate to account for the CS mediated oxidation of the 0-lactam 169 C-4 position and the C-2 hydrogen of 181. Thus CS is the second enzyme (after IPNS) to be discovered which catalyses a reaction forming the nucleus of a bicyclic p-lactam. S. clavuligerus produces penicillins and cephalosporins in addition to clavams but there is no known direct overlap in the enzymatic machinery used in the two pathways. It is unclear to what extent it is coincidence that nature uses non-haem oxygenase-oxidase enzymes which are clearly mechanistically related to effect the strained bicyclic ring structures of both the penicillins and the clavams.A possible explanation for the used of non-haem rather than haem-based enzymes is that only in the former is ligation of the substrate to the metal centre possible. Identification and cloning of the gene encoding for CS led to the over production of CS in E. coli enabling it to be produced on a relatively large scale for biochemical studies. 66 Restric-tion mapping data'65 suggested chromosomal linkage of the cephamycin (i.e. penicillin and cephalosporin) and clavulanic acid 3 biosynthetic gene Probing gene libraries from S. clavuligerus S. jumonjinensis and S. katsurahamensis led to the conclusion that in each of the three Streptomyces spp.examined the penicillin/cephalosporin and clavulanic acid 3 genes are closely linked on the chromosome. It was specu- lated that the clustering of the two pathways may result from a requirement for a common control mechanism or that coproduction of an antibacterial 0-lactam (e.g. penicillin or cephalosporin) together with a 0-lactamase inhibitor may confer a selective advantage in direct analogy to the chemo- therapeutic use of co-amoxiclav (Augmentin@). 65 The Townsend group published a purification protocol and partial characterisation of CS lg3 which was a modification of the Beecham method.70 Significantly the American group found that two isozymes of CS exist in S.clavuligerus. On the basis of kinetic ~tudies''~ they proposed that two stepwise oxidations of 169 occur a proposal consistent with the Oxford-SmithKline Beecham result discussed above. '787179In 324 Natural Product Reports 1997 contrast to the UK group's findings the Townsend group reported that there is not a 'substantial release of an intermedi- ate' from CS. The group cloned and sequenced both isozymes of clavaminate synthase designated CSl and CS2 with the corresponding genes designated csl and cs2.1g5The CS char- acterised by the Beecham group corresponded to the gene product of c~2.I~~ Initial characterisation of the iron binding sites in CSl and CS2 was carried out on material prepared by overexpression.lg6 Both isozymes were found to be very sus-ceptible to self inactivation with both diethyl pyrocarbonate and N-ethylmaleimide inhibiting catalytic activity but not inhibiting inactivation.Co2+ was found to be a potent inhibi- tor of both isozymes and also inhibited inactivation. These results were consistent with the presence of histidine and cysteine residues at or near to the active site and it was proposed that these residues are probably involved in the binding of iron. '86 The presence of metal ligating histidine residues has been previously inferred by chemical modification experiments at the active sites of a number of ferrous dependent oxygenases and oxidases including prolyl-4-hydroxylase proline-4-hydroxylase,'88 and ACC oxidase."' The crystal structure of IPNS complexed to manganese reveals that the active metal is ligated to two histidinyl an aspartyl and a glutaminyl residue.'89 Sequencing of the csl and cs2 genes revealed a high degree of homology (>85% identity).'85 Gene duplication is common in secondary metabolism and has been proposed as the branch point in divergent evolution. Surprisingly however the csl and cs2 genes were reported to be separated by more than 20 kbp in the gen~rne.''~ Such a large separation in duplicated and coexpressed genes is unprecedented but may turn out to be more widespread. Since proclavaminic acid 169 appears to accumulate in S. clavuligerus cells it was suggested that the reason for the expression of two CS isozymes was to increase the rate of its conversion to clavaminic acid 38."' However in the light of the discovery that both CS isozymes catalyse an earlier reaction in the pathway such an explanation would seem to be unlikely (see below).The csl and cs2 sequences did not display significant similarity with IPNS the other enzymes of penicillin and cephalosporin biosynthesis nor any other ferrous dependent oxygenases-oxidases leading to the sugges- tion that they evolved to a related site structure-function via a convergent evolutionary process.' 85 Paradkur and Jensen"" have reported results of gene dis- ruption studies on cs2. A cs2 mutant was constructed which did not produce 3 in a starch-asparagine medium but pro- duced low levels of 3 in a soy medium suggesting both isozymes can contribute to the production of 3 consistent with the protein purification studies 166,183which demonstrated the expression of two CS isozymes.However in media in which csl expression is blocked cs2 expression is essential for the biosynthesis of 3. Thus there are differences both in the regulation and transcriptional organisation of the csl and cs2 genes. The cs2 gene is located within the clavulanic acid 3 operon and is transcribed as monocistronic and multicistronic transcripts whereas csl is transcribed as a monocistronic transcript only under certain growth conditions. It was sug- gested that a reason for the two cs genes may be related to the production of more than one clavam (or related metabolite requiring oxidation of an arginine related metabolite) product by S.~lavuligerus.'~" Chemical or ultraviolet light mediated mutation of S. clavuligerus led to mutants which were deficient in the produc- tion of 3.16' The mutants were assigned into seven different groups indicative of the involvement of at least seven different enzymes in clavulanic acid 3 biosynthesis. Complementation studies were used to identify the appropriate location of each of the genes corresponding to each of the mutant classes. A further gene corresponding to pah encoding for proclavaminic acid amidino hydrolase (PAH) (see below) was subsequently identified thus indicating that there are minimally eight differ- ent biosynthetic genes involved in the biosynthesis of 3. Three complete sequences and two partial sequences of five biosyn- thetic open reading frames (ORFs 1-5) were reported.'68 Since mutations on ORFl led to the accumulation of the arginine derivatives 188 and 189 it was proposed to encode for an H CNKNH2 R+ NH 188 R=H 189 R =OH enzyme which catalyses a biosynthetic step prior to the for- mation of 190 (see below).The predicted amino acid sequence was reported to show no significant similarity with any known sequence. ORF2 (pah),for which no mutations were identified lies downstream and adjacent to ORFl and encodes for PAH. ORF3 initiates downstream from ORF2 and corresponds to the cs2 gene encoding for CS. ORF4 which is downstream from ORF3 encodes for a 393 amino acid protein which shows 38% significant identity with orni thine ace tyl transferase.19' It is presently uncertain whether ORF4 encodes for a genuine ornithine acetyltransferase or whether it catalyses a related reaction in the biosynthetic pathway cJ PAH and arginase. Sequence analysis of ORF4 suggested that it may be subjected to regulation by phosphate and arginine. Immediately down- stream of ORF4 lies ORF5 the product of which was reported to display 24% amino acid identity and 48% similarity with peptide transport proteins including the E. coli oligopeptide transport protein.Ig2 The role of the gene product of ORF5 and of the remaining ORFs in clavulanic acid 3 biosynthesis remains open to speculation. To complete the stereochemical details of the conversion of 169 into 38 the stereospecificity of the desaturation of 181 to 38 by CS was determined to be predominately syn by using the stereospecifically labelled proclavaminates 191 and 192.193 Incubation of 191 with CS2 yielded clavaminate retaining deuterium 193 whereas clavaminate 194 from 192 contained no label.This stereochemical result is the same as that observed for the desaturation of stearic acid to oleic acid in bacteria194 and algae.'95 191 (45)HA= H; Hg= 2H 193 R=2H 192 (4R) HA= 2H; Hg= H 194 R=H The first evidence for the pathway prior to proclavaminic acid 169 resulted from ~peculations'~~ that the hydroxy group of 169 was introduced by an a-KG dependent dioxygenase such as CS or a closely related enzyme. It will be recalled that "0 feeding experiment^'^^ had demonstrated that the ring oxygen of clavulanic acid 3 was derived (at least in part) from dioxygen and that the hydroxy oxygen of 169 was not exchanged on conversion into 38.'77 Incubation of 195 with either native CS (a mixture of CS1 and CS2) or recombinant CS2 with the appropriate cofactors gave similar results.A low conversion (c10%) to 169 was observed 181 and 38 forming 190 R = C(NH)NH2 196 195 R=H NH C02H 197 subsequently. The major product was the unsaturated com- pound 196. These results indicated that hydroxylation of 195 by CS2 was unlikely to be a major pathway for the biosyn- thetic production of 169. Since evidence'97 was emerging that the guanyl group of arginine was retained in the early stages of the pathway 190 was incubated with clavaminate synthase and efficient conversion into the alcohol 197 having the same stereochemistry as proclavaminic acid 169 was observed.There was no evidence for unsaturated products in the exper- iment indicating a complete bias of CS2 activity towards hydroxylation. Incubation of 190 with CS2 under an atmos- phere of labelled dioxygen showed a ca. 77% incorporation of a single labelled oxygen atom into the hydroxy group of 197. Incubation in oxygen labelled water led to incorporation of labelled oxygen into 197. Thus it seems that the ferry1 0x0 or other intermediate (Scheme 8) is capable of undergoing oxygen exchange with water. The observation that 190 is an efficient substrate for CS suggests a sequence of events leading to 38 via 190 197 169 and 181 beginning with arginine and not ornithine whereby the CS2 executes three enzymatic steps.They were also suggestive of an enzyme activity capable of converting 197 into 169. Further evidence that arginine and not ornithine was the amino acid taken into the clavulanic acid biosynthetic pathway Baggaley Brown and SchoJeld Chemistry and biosynthesis of clavulanic acid and other clavams was obtained by the SmithKline Beecham group by blocking clavuligerus ATCC 27064 15' indicates that the incorporation enzymes in the urea cycle of S. clavuligerus.'98 Arginine of ornithine into 3 was blocked in the arginine auxotrophs. auxotrophs of S. clavuligerus were prepared by UV irradiation. The proposed explanation of these data was that arginine is Two auxotrophs argB-2 and argC-2 were identified each taken into the clavulanic acid 3 biosynthetic pathway and respectively lacking one of the critical enzymes (arginosucci- ornithine is not a direct precursor but proceeds to be incorpor- nate synthase and ornithine carbamoyl transferase) for the ated into 3 via the urea cycle and arginine.The results conversion of ornithine into arginine (Scheme 9). Both auxo- suggested the early precursor(s) in the pathway would be trophs produced clavulanic acid 3 when grown in complete guanylated. Consequently an amidinohydrolase might operate in the pathway after arginine and before 169. Data from the sequencing of the DNA of the clavulanic acid gene cluster,'68 Ornithine H which had identified an open reading frame which showed 155 -NH2 carbamoyl strong homology to arginase and agmatine ureohydrolase transferase strengthened this argument.Therefore in order to identify ArgC-2 possible intermediates between arginine and 169 mutants of S. clavuligerus blocked in clavulanic acid 3 production were H2N *C02H H2N /\C02H screened for the production of compounds bearing guanidine 157 Ornithine Citrulline groups.'97 From the culture broth of the mutant dclH 65 the two arginine derivatives 188 and 189 were isolated. These com- t Argininosuccinate I pounds are members of the opine class of natural synthase ArgB-2 I The occurrence of 188 and 189 in S. clavuligerus dclH 65 Arginase does not in itself prove their involvement in clavulanic acid 3 biosynthesis since they may arise by hydrolysis of 190 or 197 respectively.Their structures however made them obvious candidates as precursors of 169 suggesting a number of poss- ible biosynthetic sequences to 169 as indicated in Scheme Commencing with 188 at least three biochemical steps are required to generate 169 namely the introduction of a hydroxy group (step A) formation of the p-lactam ring (step B) and H2N *CO2H H2N AC02H hydrolysis of the guanidino group to an amino group (step C). 158 Arginine Argininosuccinate To determine which if any of 188 189 190 197 and 198 Scheme 9 actually lay on the pathway these compounds were synthe- sised in triply I3C-labelled form as indicated in Scheme The five racemic I3C-labelled compounds were fed to S.clavuligerus in the standard manner and the resultant 3 isolated media. If the biosynthetic pathway to 169 and hence 3 leaves as the benzyl ester 19 for I3C NMR analysis. Incorporation of the urea cycle at ornithine both argB-2 and argC-2 should be I3C-label was observed only from feeding 188 190 and 197. capable of incorporating labelled ornithine into 3. If pro- The enrichment was specific to carbons 3 7 and 10 of 3 with clavaminic acid 169 is biosynthesised from arginine the two I3C-l 3C spin-spin coupling being observed between all three auxotrophs would not be able to incorporate labelled ornithine centres indicating that no bond breakage had occurred during into 3. When [U-I4C]ornithine was fed to argB-2 and agrC-2 incorporation. These results indicated that 188 190 and 197 the resulting 3 incorporated only a trace of radiolabel.Com- (or derivatives of them) may act as biosynthetic precursors of parison with the 10.6% incorporation of ornithine into 3 by S. 169 and hence clavulanic acid 3 and logically they occurred in H H JNXNH2 JNXNH2-A 158 188 lB IB NH NH II * = '3C p2H p2H 190 197 169 Proclavaminic acid CS 02. Fez+ 2-oxoglutarate CS 02,Fez+ dJI ...,,,rNH2 2-oxogiutarate 0 =.,10C02H 0 C02H 0 C02H 3 Clavulanic acid 38 Clavaminic acid 181 Dihydroclavaminic acid Scheme 10 326 Natural Product Reports 1997 the pathway in that order. The evidence indicated that 189 did not lie on the biosynthetic pathway and that the production of this compound in the blocked mutant dclH 65 probably results from the hydroxylation of 188 or an ester thereof as a side reaction.The transformation of 197 to 169 was found to be mediated by a new enzyme proclavaminic acid amidino hydrolase (PAI~).~’’ Cell-free preparations of S. clavuligerus were able to convert one of the enantiomers of 197 into 169 and urea. Since the absolute configuration at the 2-position of 169 is S it was concluded that the 2s enantiomer of 197 is the natural substrate. The activity of PAH was enhanced in the presence of Mn2+ as reported for other arginases including those from Bacillus anthracis”‘ and Staphylococcal sources.2o2 (29-190 was also hydrolysed under these conditions but much less rapidly than 197 whereas no hydrolysis was detected for 188 and 189.Clearly PAH is able to discriminate efficiently between the guanidino groups of these early biosynthetic precursors. PAH was purified by conventional techniques (Mr 33 000 from SDS-PAGE electrophoresis) and the pure enzyme did not hydrolyse arginine to ornithine hence it is different to the arginase previously reported from S. clavuligerus.‘49 The N-terminal amino acid sequence of PAH was deter- mined and correlated with the open reading frame showing homology to arginaseI6’ that had been located adjacent to the initial SmithKline Beecham cs gene (Townsend’s cs2 gene’”) in the clavulanic acid gene cluster. The lower incorporation of 13C-labelled188 (ca. 1%) compared with 190 (ca. 12%) and 197 (ca. 5%) indicated that either the compound is less efficiently transported into the cells or that the true intermediate is possibly a derivative of 188 such as a coenzyme A thioester which might favour ring closure.The incorporation of 188 suggests that the generation of the I)-lactam ring of 190 is by a biochemically unprecedented process i.e. by amide bond formation whereas in the case of the penicillin^,'^^ the nor- cardicins203 and probably the monobactam~,~’~’~’~ the amide bond is formed by activation prior to ring closure. In these compounds ring closure is believed to occur by displacement of an activated serinyl hydroxy function with inversion. It is perhaps ironic that clavulanic acid 3 a clinically useful p-lactamase inhibitor appears to be biosynthesised by a mech- anism that is formally the reverse of that of p-lactamases! As described above earlier labelling studies had shown that the p-lactam carbons of 3 were derived from the glycolytic pathway with pyr~vate’~~ being specifically and ~-1actate’~~ incorporated into the p-lactam ring.Hence the C moieties of 188 190 and 197 would be derived from the same pathway. It is known that pyruvate and lactate can be biochemically converted to acrylate”’ and malonic semialdehyde.206 Enzy- matically catalysed Michael addition of arginine to acrylate or Schiff s base addition with malonic semialdehyde followed by reduction would yield 188 in either case.2oo The possible derivation of 188 from reaction of arginine with malonic semialdehyde is of particular interest as it bears a strong resemblance to the biosynthesis of the opine metabolites where Schiff’s base formation and subsequent reduction occur.2o7 The labelling experiments with radiolabelled P-indicated that no change occurs in the oxidation state of the pro-R hydroxymethylene in the biosynthetic steps between glycerol (or glycerate) and proclavaminic acid 169.The eluci- dation of the biosynthetic steps between glycerol (or glycerate) and the C-3 substrate for the production of 188 will be of considerable interest. The hydroxylation of 190 by recombinant CS2 was shown to be highly stereospecific using the deuterium labelled samples of 190 199 and 200.209 Incubation of 199 with recombinant CS2 NH NH 201 R’ = R2 =2H 202 R’ =R2=H under the standard reaction conditions gave 201 in >85% conversion.Similarly incubation of 200 under the same con- ditions yielded 202 in >80% conversion indicating that the CS2 mediated hydroxylation of 190 proceeds with a high degree of retention of configuration at C-3 with the removal of the pro-R hydrogen. Other analogous highly stereoselective hydroxylations catalysed by ferrous-dependent oxygenases have been The formation of the oxazolidine ring of 38 from 169 by the insertion of the oxygen at C-4’ is also a similar reaction. 158 In order to explore the binding of substrates to and the mechanism of clavaminate acid synthase (CS) various analogues of the natural substrates have been investigated. The y-lactam analogue 203 of proclavaminic acid 169 on incu- bation with either a mixture of CSl and CS2 or recombinant CS2 yielded the y-lactams 204 and 205 indicating that CS may have a relaxed specificity towards substrates.211 It is notable that despite some efforts the chemical synthesis of y-lactam analogues of 3 has remained elusive.Studies were also carried out wherein the p-lactam ring was replaced by an acetamido group.212 Incubation of N’-acetylornithine with CS yielded a mixture of 206 and 207 (3:l) indicat- ing that replacement of the p-lactam with acetamido biases the CS activity from predominantly desaturation (>10:1) in the case of 195 to mostly hydroxylation in the case of N2-acetylornithine. N2-Acetyl-L-arginine was effectively hydroxylated by CS to yield 208 whereas when N2-acetyl-D- arginine was used as the substrate very little hydroxylation occurred.These results were consistent with previous results hydroxypropionate described by Gutman and co-worker~’~~ 203 204 and discussed earlier deserve further comment. These workers CI found that the labelled P-hydroxypropionate was specifically incorporated into the P-lactam ring of 3 and they proposed that the first step in the biosynthesis involved the coupling of the P-hydroxypropionate with an appropriate amino acid to form a hypothetical ‘dipeptide’ intermediate. An alternative explanation consistent with the results of the SmithKline Beecham group. is that the fermentation conditions employed C02H 205 206 208 I C02H R=H R = C(NH)NH2 by Gutman and co-worker~’~~ induced cells to transform labelled substrates into malonic semialdehyde or acrylate which was then incorporated into the pathway via Schiff’s base formation or Michael addition with arginine as dis- cussed above.Results from the Townsend group,2o8 however 207 Baggaley Brown and Schofield Chemistry and biosynthesis oj clavulanic acid and other clavams which indicated CS converted substrates with 2s stereochem-H istry. It is apparent from these results that CS is able to oxidise simple derivatives of arginine and ornithine at unactivated P 0 G 209 O positions. Indeed N2-acetyl-L-arginineis a convenient replace-ment for proclavaminic acid as a substrate for routine CS assays.2' The oxidation especially hydroxylation of arginine and amino acid residues by 2-0x0 acid dependent dioxygenases is Xa to give active PAH extended at the C-terminus by four a common feature in the biosynthesis of secondary metabo-amino acids.Arginine and N-acetylarginine were not found to lites. The hydroxylation of arginine residues at the P-position be substrates for the recombinant enzyme in support of the has been implicated in the biosynthesis of streptothricin F,213 SmithKline Beecham work.200 and at the y-position in the biosynthesis of the heptapeptide The biosynthetic sequence in which 190 is converted into 38 antibiotics K-582A and B.214In the last example the L-threo-is remarkable in its exploitation of the versatile substrate isomer is exclusively produced. selectivity of CS. CS catalyses three separate oxidative reac-A series of proclavaminic acid analogues was investigated to tions a hydroxylation 190 to 197 an oxidative cyclisation 169 determine whether they could be cyclised to clavams by CS to 181 and a desaturation 181 to 38 (Scheme 11).Whilst the but without success.215Attempts to design mechanism based catalysis of sequential oxidations by ferrous-dependent oxy-inhibitors of CS using chemical moieties that had proved genases is well pre~edented,~'~ the reactions catalysed by CS useful in the probing of isopenicillin N ~ynthase,'~~ also are unusual in the different types of oxidative chemistry carried proved ~nrewarding.~'~ out. What makes the CS catalysed sequence unprecedented is Following the report on the involvement of arginine as the intermediacy of a step catalysed by another enzyme initiating the biosynthetic pathway19' and the role of PAH in proclavaminate amidinohydrolase (PAH).The product of the converting 197 to 169200and prior to the public disclosure of first CS catalysed reaction alcohol 197 is not a substrate for the location of the puh gene by the SmithKIine Beecham CS. PAH then intervenes to convert the guanidino side chain group,'68 two other groups published findings regarding this of 197 to the amino group of proclavaminic acid 169 which is enzyme. Aidoo et uL2l7 found that in the course of purifying a substrate for CS. Thus PAH 'mutates' the product of the 6-(L-a-aminoadipyl)-L-cysteinyl-D-vahe 184 synthetase the first CS catalysed reaction 197 to produce another substrate tripeptide precursor of the penicillin nucleus from S.cluvuli-169. Clearly this indicates a subtle relationship between the gerus a contaminating protein of the same mass initially basic side chain binding interactions at the CS active site and named 6-(L-a-aminoadipyl)cysteinyl-D-valine-relatedprotein the type of oxidative chemistry catalysed. Moreover it would (ACVSR) was copurified. When cloned the gene encoding seem likely that this type of 'substrate mutation' is unlikely to ACVSR ucvsr was found to lie adjacent to the cs2 gene. be confined to the clavulanic acid 3 biosynthesis pathway and Insertional inactivation of this gene greatly reduced the will probably be utilised in the biosynthesis of other secondary production of 3 and it was surmised that ucvsr corresponded to metabolites.the PAH gene. Similarly the Townsend group,218having Experimental evidence to support the that the identified csl and cs2 and wishing to determine whether other aldehyde 173 (Scheme 7) was the ultimate biosynthetic precur-adjacent genes had been duplicated sequenced up and down-sor of clavulanic acid 3 was obtained by the SmithKline stream from each gene. It was found that the open reading Beecham group.173 Fermentation broths of S. cluvuligerus frame adjacent to cs2 showed greater than 40% identity to ATCC 27064 and S. cluvuligerus dclI 111 a clavulanic acid 3 agmantine ureohydrolase and several arginases. The function deficient mutant were shown to contain 173. The aldehyde 173 of this gene was unclear until the report by Elson et al. defining is highly unstable with a half-life of about one hour yielding the role of PAH in converting 197 to 169.200PAH was over the decarboxylated aldehyde 209; it possesses the 5R stereo-produced in E coli in the form of a functional fbsion with chemistry.A synthetic sample of the benzyl ester of 173 was maltose binding protein which could be cleaved using Factor found to racemise suggesting that a biological ester such as an H NH lNXNHZ ? * NH L-Arginine 158 \ ? n Clavaminate synthase 2 K / t NHz 2-Oxoglutarate 02 Fez+ f' ACOpH C3pool metabolite . .. okN<! NH2 C02H COpH COPH 188 190 197 Proclavaminic acid amidino hydrolase (PW Clavarninate synthase 2 Clavaminate synthase 2 dJ..,,,,rNHp4 2-Oxoglutarate 02,Fez+ I 2-Oxoglutarate 02 Fez+ 0 0 COpH COpH COpH 38 181 169 02+? I ~YOH dehydrogenase $*o Clavulanic acid (CAD) NADPH 0 'COpH 0 'COpH 173 3 Scheme 11 328 Natural Product Reports 1997 oxygen or coenzyme A thioester of 173 might mediate a similar inversion in the biosynthetic transformation of the 3S,5S stereochemistry of clavaminic acid 38 to the 3R,5R stereochemistry of 173.It was found that the reduction of 173 to 3 could be effected by the addition of NADPH to broken cells of S. clavuligerus ATCC 27064. The enzyme responsible for this reduction clavulanic acid dehydrogenase (CAD) was purified and the N-terminal amino acid sequence enabled the location of the corresponding DNA sequence (cad) to be identified in the clavulanic acid gene cluster.168 This sequence showed homol- ogy to known NADPH dependent dehydrogenases from both prokaryotes and eukaryotes.220 The results reviewed above are summarised in Scheme 11 and represent the knowledge currently available on the biosyn- thetic pathway to clavulanic acid 3. Although there are several biosynthetic steps remaining to be delineated the questions to be resolved are highly intriguing. Which C precursor is utilised to generate the condensation product with arginine to give the opine 188 if indeed it is a true intermediate? Although further labelling studies of the proposed precursors acrylate or malonic aldehyde may yield some evidence the problem probably requires in vitro studies using pure enzymes.The mechanism of formation of the p-lactam ring of 190 is likely to be unprecedented in biosynthetic studies. The 3'-carboxy group of 38 may require activation via a CoA thioester for the ring closing process to occur. Again a solution may lie in a cell-free approach. Finally and certainly not least is the novel oxidative conversion of clavaminic acid 38 having the 3S,5S stereochemistry to the aldehyde 173 of enantiomeric configu- ration. No evidence has been reported to negate or confirm the involvement of the acylated derivatives 39-41. A major role in unravelling these final questions will undoubtedly be played by the remaining sequence information in the clavulanic acid 3 gene cluster which is there to be harvested! 8.2 Biosynthesis of other clavams As mentioned earlier S.clavuligrrus produces the clavams 42 43 44 and 45 as well as clavulanic acid 3.72The hydroxyethyl compound 46 valclavam 48 and Tu 1718B have been isolated from S. antibioticus ssp. antibioticus Tu 171874 which does not produce clavulanic acid 3. Tu 1718B is a degradation product of valclavam. The structures 47 and 210 initially assigned to valclavam and Tu 1718B have been revised to 48 and 211.76 Valclavam 48 is related to the clavamycins 49-54 produced by S. IzyKrosc~cjpicu.~.~~ HQ Iwata-Reuyl and Townsend22' hypothesised that the origins of 42 43. 44 and 46 could be rationalised starting from clavalanine 45. The structure of 45 suggested the utilisation of L-ornithine in the opposite regiochemical sense to that from ornithine in the clavulanic acid pathway indicating a related but separate pathway to that for 3.Consequently L-[U-''C]ornithine was fed to S. clavuligerus NCIB 11260 which produced both 3 and clavam-2-carboxylate 42 resulting in similar levels of incorporation into both compounds. Feeding of (2S,4S)-[4-'H,5-"C]ornithine however led to no incorpor- ation of label into 42 but showed the expected enrichment at C-9 in 3 suggesting that the ornithine had been incorporated in the same regiochemical sense in both compounds. Label was incorporated into C-2 of both 3 and 42 when [3-I3C]ornithine was fed. This unexpected parallel between clavam and clavu- lanic acid 3 biosynthesis was investigated further by feeding ~,~-[2,3-"C~]proclavaminic acid 212 (Scheme 12).222 The H OH ssp antibioticus TU 1718 / S.antibioticus 4ssp antibioticus TU 1718 NH* L-valine Scheme 12 labels were specifically incorporated into 42 and 3 indicating that the uptake of ornithine was in the same regiochemical sense as for clavulanic acid 3 that proclavaminic acid 169 was a common intermediate to both 42 and 3 and that the pathway for the two compounds was common up to pro- clavaminic acid. These workers speculated that the aldehyde 213 the enantiomer of the immediate precursor 173 of clavu- lanic acid may through decarboxylation and reduction to 215 provide a route to the clavams produced by S. cluvuligerus.222 A later p~blication,'~~ however reported that the decarboxy- lation of the natural immediate precursor 173 of 3 yielded only the aldehyde 209 with the 5R stereochemistry.213 H 1 H -$ $yo yo 0 0 214 215 Extending the discovery that 169 was a precursor of the clavam 42 to the S. antibioticus metabolites feeding 212 gave incorporation into both the hydroxyethyl clavam 46 and Baggalejl Brown Lind SclzoJield Clzemistr-v and biosynthesis of clavulanic acid and other clavams valclavam 48; L-[ 1 -13C]valine specifically labelled the valyl moiety of 48 as summarised in Scheme 12.222 Baldwin et al.223have studied the incorporation of a series of [U-I4C]-labelled compounds some of which were known precursors of proclavaminic acid 169 into 48 and 211 using S. antibioticus ssp. antibioticus Tu 1718.[U-14C]Arginine was incorporated into 211 to a greater extent than [U-'4C]ornithine consistent with the fact that ornithine had to be converted into arginine the point of entry to the pathway to 169. The lack of radiolabelling in 48/211 from [l-I4C]arginine argued for the same regiochemistry of utilisation as in the clavulanic acid 3 pathway. ~-[U-'~C]Lysine was not incorpor- ated into either 48 or 211 although the C-6 unit of both compounds is a masked P,G-dihydroxylysyl residue. There was a significant incorporation of ['4C-CH3]methionine into 211 suggesting that at least one of the carbon atoms in the six-carbon unit originated from the methyl group of (8-adenosylmethionine. [U- ''C]Glycine was not incorporated into either compound. [U-'4C]Glycerol ~-[U-'~C]lactate D-[U- 14C]lactate and [U-14C]pyruvate all known precursors of 169 were incorporated into 48 again suggesting a common pathway via proclavaminic acid 169.Cell-free with S. antibioticus ssp. antibioticus Tu1718 by the Oxford group demonstrated the presence of PAH (proclavaminic acid amidino hydrolase) and a clavaminate synthase. The last enzyme apparently a single isozyme was purified and N-terminal amino acid analysis showed higher homology to the CS1 enzyme of S. clavuligerus than to the CS2 isozyme. This result was confirmed by a subsequent report225 in which the new clavaminate synthase was assayed by the cyclisation of proclavaminic acid 169 to 38 and the hydroxylation of the natural clavulanate precursor 190.The evidence to date strongly suggests that the biosynthetic pathway to the clavams including valclavam and possibly the clavamycins is common with the clavulanic acid pathway at least as far as the clavaminic acid 38 stage. It is apparent that S. clavuligerus and the other clavulanic acid producers S. jumonjinensis66and S. katsur~hamanus,~~ possess the unique ability to effect the enantiomerisation of the clavam ring system from the 3S,5S to the 3R,5R stereochemistry of the clavaminic acid precursor 38 either at the oxidation stage or by the enzymatic isomerisation of the 3S,5S aldehyde 213. A full biosynthetic scheme which accounts for the generation of the clavams has been Acknowledgement. We acknowledge the efforts of all the scientists who have worked to make the clavulanic acid story so productive and fascinating.We apologise for any errors or omissions. Notes added in prooJ Evidence has been reported recently that (i) CS2 contains a single active site;226 (ii) the hydrogen at C-2 of glycerate is lost during the biosynthesis of 3;227(iii) pyruvate is the primary metabolic precursor of the p-lactam ring of 3;228(iv) clavaminic acid 38 is the branch point between the biosynthesis of 3 and the 5S-cla~ams.~~~ 9 References 1 R. Nagarajan L. D. Boeck R. L. Hamill C. E. Higgens M. M. Hoehn W. M. Stark and J. G. Whitney J. Am. Chem. Soc. 1971 93 2308. 2 A. G. Brown D. Butterworth M. Cole G. Hanscombe J. D. Hood C. Reading and G. N. Rolinson J. Antibiot. 1976,29 668. 3 Augmentin eds.G. N. Rolinson and A. Watson Excerpta Medica Amsterdam 1980. 4 Augmentin eds. D. A. 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ISSN:0265-0568    

DOI:10.1039/NP9971400309

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Biosynthesis of fatty acids and related metabolites Bernard J. Rawlings Department of Chemistry University of Leicester University Road Leicester UK LEI 7RH Email bjr2@le. ac. uk Covering up to the end of 1994 1 Introduction Previous review 1995 12 1 This review summarises the literature up to the end of 1994 with particular emphasis on the period following on from 1 Introduction previous reviews in related series.” In general in prokaryotes 2 Eubacteria fatty acids are assembled by a series of discrete non-covalently 2.1 Gram negative bacteria attached enzymes and an acyl carrier protein (Type I1 syn- 2.1.1 Acetyl CoA carboxylase (ACC) thesis). In contrast eukaryotes use large multifunctional pro- 2.1.2 Acyl carrier protein (ACP) teins (Type I synthesis).For example fungal systems contain 2.1.3 Acyl CoA ACP transacylase (AT) two large multifunctional proteins each containing about half 2.1.4 3-Oxoacyl ACP synthase (KAS) the required activities in an hexameric arrangement and 2.1.5 3-Oxoacyl ACP reductase (KR) animals use a single multifunctional protein containing all the 2.1.6 (3R)-Hydroxyacyl ACP dehydrase (DH) required activities in a head-to-tail homodimeric arrangement 2.1.7 (3R)-Hydroxydecanoyl ACP dehydrase (HDDH) also referred to as Type I. However this convenient historical 2.1.8 Enoylacyl ACP reductase (ER) classification is becoming increasingly blurred by exceptions 2.1.9 Acyl ACP thioesterases (TE) 2.1.10 Cyclopropanated fatty acids in E.coli notably that several mycobacteria and a Brevibacterium use a and Euglena has both. The traditional concept 2.1.11 Acyl ACP synthase Type I ~ystem,~ of a single set of activities converting a ‘C2’starter unit plus 2.1.12 Other Gram negative bacteria malonyl extender units to ‘C,6’ or ‘C18’ fatty acids is also 2.2 Gram positive bacteria 2.2.1 Mycohacteriu and related bacteria becoming increasingly confused by the isolation of a number of different 3-oxoacyl synthases and other activities. Each of 2.2.2 Other Gram positive bacteria these are most active at different parts of the assembly process 3 Archaea 4 Protists notably ‘KAS 111’ which specifically assembles 3-oxobutanoyl 4.1 Protozoa ACP from acetyl CoA and malonyl ACP. The recent isolation 4.2 Eukaryotic algae of the genes and overexpression of enzymes responsible for polyketide antibiotic synthesis has demonstrated that there is 5 Fungi no direct involvement of FAS proteins in these processes 5.1 Unicellular fungi although the evolutionary connection remains a fascinating 5.2 Filamentous fungi area of study.6 Plants Recent highlights include the reassignment of the dehydra- 6.1 Lower plants tase domain in the mammalian fatty acid synthase multifunc- 6.2 Higher plants tional peptide by Joshi and Smith.4 A mammalian FAS has 7 Animals been functionally overexpressed in an insect cell culture using a 7.1 Invertebrates baculovirus ~ector.~ The E. coli FAS contains a new 3-oxoacyl 7.2 Vertebrates ACP synthetase KAS IV which is involved in the assembly of 8 Miscellaneous short chains up to C8e6 This may be only involved in lipoic acid 9 References biosynthesis or it may be an integral part of longer chain Isolated methyl carbon from acetate (not thought to be now part of an intact acetate unit) Isolated carbonyl carbon from acetate (not thought to be now part of an intact acetate unit) /.Acetate unit intact Butanoate unit intact A Propionate unit intact A/ 3-Methylbutanoate unit intact Intact acetate unit and intact C-0 bond from [l-13C 180]acetate A. Intact propionate unit and intact C-0 bond \/zo ./\. Intact malonate unit nIntact succinate unit Glycerol A Methyl from [I3CH3]methionine viaSAM *O From dioxygen in atmosphere as I8O2 *H Hydride from NAD(P)H Fig.1 Biosynthetic symbols used throughout this paper Rawlings Biosynthesis of fatty acids and related metabolites assembly. It is commonly believed that fatty acid building blocks are primarily glucose derived acetate and malonate with chain assembly by a ‘Claisen-like’ condensation. How- ever a report by Wagner and co-workers suggests that medium chain fatty acids in the Solanaceae are derived from amino acid starter units with an ‘a-ketoacid elongation’ pathway for the successive addition of C units quite different to the traditional ‘Claisen-like’ conden~ation.~ Ohlrogge has reported that the insertion of a medium chain thioesterase from the Californian bay tree into oil seed rape results in the production of large quantities of the industrially valuable dodecanoic acid (lauric acid) in the oil seed.8 Currently large quantities of dodecanoic acid are imported from tropical countries for use in soaps detergents and surfactants -a future productive use of European ‘set-aside’ land? This review is arranged using a more detailed taxonomic system than before that can also be used for related metabolites such as the polyketides and for future reviews in the area.’ A single set of biosynthetic symbols has been used as illustrated in Fig.1. The rapid advances in this area are dependent upon research carried out by a wide range of scientists from synthetic chemists to enzymologists microbiologists and molecular biologists and just as importantly upon communication between them.The interdisciplinary nature of the topic results in an extensive variety of nomenclature so a list of abbreviations used in this review follows ACC acetyl CoA carboxylase; ACP acyl carrier protein; AT acyl CoA ACP transacylase (acyl trans- ferase); BCCP biotin carboxyl carrier protein; FAS fatty acid synthase; HDDH (3R)-hydroxydecanoyl ACP dehydratase; KAS 3-oxoacyl ACP synthase (3-ketoacyl ACP synthase); kDa kilodalton; KR 3-oxoacyl ACP reductase (3-ketoacyl ACP reductase); M. Mycobacterium; MMCoA methylmalo- nyl CoA; MT malonyl CoA ACP transacylase; NAC N-acetyl cysteamine (N-acetyl2-aminoethanethiol); PHA polyhydroxy- alkanoate; PHB polyhydroxybutanoate; S. Streptomyces; SAM S-adenosylmethionine; TE acyl ACP thioesterase.Extensive general reviews covering general aspects of fatty acid biosynthesis are available. ‘‘-I8 2 Eubacteria The prokaryotic bacteria usually contain a Type I1 FAS comprising discrete enzymes and an ACP that assembles acetyl CoA and malonyl CoA into straight chain fatty acids primar- ily hexadecanoyl ACP (palmitoyl ACP) (9Z)-hexadec-9-enoyl ACP (palmitoleoyl ACP) and (1 1 Z)-octadec- 1 1 -enoyl ACP (cis-vaccenoyl ACP) which are then usually directly trans- esterified into glycerides without the intermediacy of the corresponding free carboxylic acids. (1 12)-Octadec-11 -enoyl ACP is formed by a separate membrane associated elongase system extending (9Z)-hexadec-9-enoyl ACP. Monounsatur- ation usually originates through the ‘anaerobic’ pathway which branches off the main pathway at C, through the action of (3R)-hydroxydecanoyl ACP dehydrase (HDDH) which can form (3Z)-dec-3-enoyl ACP chain extension then proceeding as for the saturated pathway.A few bacteria can introduce desaturation by the oxidative desaturation pathway along with limited further chain elongation particularly the cyano- bacteria and Bacillus. Bacteria usually regulate the fluidity of their membranes by varying the degree of unsaturation; when grown at low temperature they increase the level of unsatur- ation. Marine bacteria such as the cyanobacteria may need to introduce polyunsaturation due to the low temperature of water. An alternative method involves assembling fatty acids with branching near the methyl terminus.A wide range of bacteria including Streptomyces Bacillus Staphylococcus and Myxococcus assemble iso(o -2-methyl)-and (S)-anteiso (o-3-methyl)-alkanoic acids using starter units derived from valine leucine and isoleucine via their 2-0x0 acids and oxidat- ive decarboxylation to 2-methylpropanoyl CoA 3-methyl- butanoyl CoA and (25‘)-methylbutanoyl CoA replacing acetyl 336 Natural Product Reports 1997 CoA as starter unit. Alicyclobacillus lives at high temperatures (>60 “C) and assembles o-cyclohexanyl- and o-cycloheptanyl- alkanoic acids.’ A Pseudomonas sp. has been collected from antarctic soil that has 90% 13-methyltetradecanoate when grown at 5 ‘C.” The Cytophaga and Flexibacter genera of gliding bacteria produce sulfonolipids such as capnine 1 (2-amino-3-hydroxy- 15-methylhexadecane- 1-sulfonic acid) S03H that are essential for their gliding motility presumably assem- bled from the extension of 13-methyltetrade~anoate.~’ Bacteria can also contain low levels (<1%) of 2-and 3-hydroxyfatty acids.In some Pseudomonas and Serratia spp. the major component of the cell wall lipopolysaccharides are straight chain 3-hydroxy fatty acids. A major review by Kaneda on the occurrence of branched chain fatty acids in bacteria has appeared which also includes details of the occurrence of unusual fatty acids.21 The mycobacteria produce an extra-ordinary range and variety of fatty acids some as long as C9’ using a poorly understood mixture of Type I and Type I1 systems.Phylogenetic analysis (based upon ribosomal RNA sequences) of the bacteria suggests at least 12 different groups or phyla. For the purpose of this review one of these the Gram positive bacteria (containing an outer cell wall that reacts with the ‘Gram’ stain) will be considered separately and all the others loosely aggregated as Gram negative bacteria. These 11 remaining phyla include the purple bacteria (also called ‘proteobacteria’) [e.g. Escherichia Pseudomonas Salmonella and Myxococcus (the gliding bacteria)] the ‘purple sulfur bacteria’ (e.g Thiocystis) and the cyanobacteria (e.g. Anabaena) that are also called ‘blue-green algae’ and are not to be confused with the eukaryotic algae. The cyanobacteria contain chloroplasts and are thought to have been the first oxygen evolving phototrophic organisms probably respon-sible for oxygenating the Earth’s atmosphere.Phylogenetic studies also suggest a very close relationship between Gram positive bacteria the mitochondria purple bacteria chloroplasts and cyanobacteria. 2.1 Gram negative bacteria Escherichia coli is the most extensively studied Gram negative bacterium. It is a facultatively aerobic non-motile rod-forming enteric bacterium that inhabits the intestinal tract of mammals. The biosynthesis of fatty acids in E. coli is summarised in Scheme 1. 2.1.1 Acetyl CoA carboxylase (ACC) This enzyme converts acetyl CoA into malonyl CoA with retention of cryptic stereochemistry as demonstrated by the use of ‘chiral acetate’.22’ 23 This enzyme complex which has a major role in the regulation of fatty acid production contains a biotin prosthetic group attached via the &-amino group of a lysine residue.The hydrogencarbonate ion is first activated as carboxybiotin at the expense of an ATP before transfer to acetyl CoA to form malonyl CoA. The carboxylase from E. coli has been separated into three functional subunits the biotin carboxy carrier protein (BCCP) the biotin carboxylase and the transcarboxylase. Chapman-Smith et al. have recently over- expressed and fully biotinylated a biotin-domain peptide of 87 amino acids from the biotin carboxy carrier protein,24 which should now allow structural analysis of this region by NMR. The biotin domain shows considerable sequence homology to the lipoyl-attachment region of pyruvate dehydrogenase (that Malonyl CoA KAS Ill Malonyl ACP (3R)-Hydroxydecanoyl ACP (DH very slow) /HDDH\ 3 (3Z)-Decenoyl ACP =(2Q-Decenoyl ACP KAS Ior KAS II KR DH ER 16:1 9Z-ACP 16:O-ACP (Palmitoleoyl ACP) (Palmitoyl ACP) 18:1 11Z-ACP (cis-Vaccenoyl ACP) Diglycerides Triglycerides Scheme 1 Biosynthesis of fatty acids in E.coli oxidatively decarboxylates pyruvate to give acetyl CoA) in which the lipoyl cofactor is covalently attached to a specific lysine residue. The four Acc genes (accA,B,C,D) are located in three different positions on the E. coli genome (accA BC and D). Li and Cronan have reported a direct correlation between levels of transcription of the four ACC genes and the rate of cellular growth.In addition they report that both accA and accD are located in complex gene clusters the accA promoter being located within the gene that encodes the DNA poly- merase I11 catalytic subunit suggesting additional complex regulatory mechanism^.^' The molecular evolution of biotin- dependent carboxylases has recently been investigated by sequence comparison. Biotin carboxylase is homologous to carbamoyl phosphate synthetase and the biotin carboxy carrier protein homologous to lipoic acid-binding domain.26- 2.1.2 Acyl carrier protein (ACP) This small very soluble acidic (14 Glu 8 Asp isoelectric point at pH 4.2) protein of 77 amino acids (8847 Da) with a phosphopantetheine prosthetic group attached to a serine residue (Ser-36 part of a highly conserved Asp-Ser-Leu-Asp sequence) has been ~equenced,~’ extensively studied3’ and is commercially available.Attempts at its over- expression have been hindered by the small size of the genome and by cell death caused by high levels of expression of this protein. Use of a synthetic gene led to a 50-fold increase in production which unfortunately resulted in cell death.12 Virtually all of this ACP received the phosphopantetheine prosthetic group implying that the holo-ACP synthetase in E. coli is in functional excess. Revill and Leadlay have over-expressed (10 mg 1-’) a FAS ACP from the Gram positive Saccharopolyspora erythraea in E. coli. However this ACP was only partly in the holo form (70%) implying a certain specifi- city in the E.coli holo-ACP syntheta~e.~~ Whilst a preliminary report on the X-ray diffraction of E. coli ACP has been published,33 structural information is largely based upon extensive NMR and molecular modelling studies. The struc- ture has a high helical content with a putative hydrophobic groove or pocket suitable for binding a ‘growing fatty acyl chain’. However it is becoming increasingly evident that these early attempts at structure determination did not take into account the dynamic flexibility of the protein and the structure dependency on its environment such as pH or metal ion concentration. Kim and Prestegard have proposed two distinct conformers in dynamic equilibrium that results in a dramatic improvement in understanding NOE derived distance con- straint~.~~ Jones et al.have reported3’ a recent attempt to overexpress E. coli ACP using degenerate PCR. The resulting clone was inserted into overexpression systems but no overex- pression was obtained and sequencing revealed a series of mutations. Prestegard and co-~orkers~~ have investigated the kinetics and thermodynamics of ACP thermal denaturation using variable temperature and differential scanning calor- imetry and concluded that monovalent and divalent cations both stabilise ACP but in different ways. ACPs may simply act as ‘passive’ carriers of the ‘swinging arm’ that transfers the growing acyl chain from one catalytic activity to another or they may actively bind to the growing acyl chain controlling and regulating the product of the FAS complex.Early struc- tural studies on E. coli FAS ACP suggested the presence of a hydrophobic pocket or groove that may non-covalently bind to the growing fatty acyl chain. Prestegard and co-workers investigated this question by synthesising 6,6- and 13,13-difluorotetradecanoyl ACP. Examination by ‘”F NMR spectroscopy gave only a single resonance from the 13,13- difluorinated derivative but two resonances were seen from the 6,6-difluoro derivative suggesting that part of the hydrophobic chain was bound in the achiral environment of a protein binding pocket.37 5,5-Difluorohexanoyl ACP gave { I9F}-’H heteronuclear Overhauser enhancements to the methyl groups of Ile-54 and Ala-59.38 2.1.3 Acyl CoA ACP transacylase (AT) These enzymes catalyse the transthioesterification of acyl groups from ‘building block’ CoA thioesters onto the phos- phopantetheine thiol of the ACP or cysteine thiol of the 3-oxoacyl ACP synthase.The E. coli malonyl CoA ACP transacylase contains 358 amino acid residues and has recently been crystallised using the hanging drop method with a diffraction pattern of 0.2 nm resolution being obtained.39 2.1.4 3-Oxoacyl ACP synthase (KAS) Also called 3-ketoacyl ACP synthase this enzyme catalyses the ‘Claisen-like’ C-C bond formation with inversion of cryptic stere~chemistry.~~ Prior to 1975 this was believed to be a single enzyme of RMM 66 kDa which was used in each cycle,40 but Vagelos and co-workers then demonstrated that there were at least two forms (KAS I and II) both forms capable of participating in saturated fatty acid production but with KAS I (not KAS 11) also able to act on the unsaturated acyl chains in the anaerobic pathway to monounsaturated fatty acids.41.42 The activity of KAS I1 is strongly modulated by temperature thus influencing the level of monoun~aturation.~~ Mutants lacking KAS I are unable to produce monounsaturated fatty acids. Thiolactomycin 2 is an antibiotic that inhibits Type I1 FAS (not Type I) by inhibiting KAS.44 Overproduction of KAS I imparted thiolactomycin resistance to E. coli FAS both in vitro and in v~vo.~~ In 1989 Jackowski and Rock proposed that the first condensation that forms ‘3-oxobutanoate7 was Rawlings Biosynthesis of fatty acids and related metabolites Me 2 3 the rate limiting step in fatty acid biosynthesis and they located a separate enzyme 3-oxobutanoyl ACP synthase (also called KAS 111) that used acetyl CoA rather than acetyl ACP as the acetyl donor but still employed malonyl ACP as the source of mal~nate.,~ Cerulenin 3 a potent inhibitor of KAS I and TI did not inhibit this enzyme but KAS 111is sensitive to thiolac- tomycin 2.,' A JabJ gene encoded fourth enzyme 3-oxoacyl ACP synthase IV (KAS IV) has been reported that is ex- tremely sensitive to cerulenin 3 and is specific for short chain substrates.6 The gene encodes a protein of 413 amino acids (RMM 43 kDa) and it is proposed that it has a specialised function of supplying octanoic acid for lipoic acid biosynthesis.2.1.5 3-Oxoacyl ACP reductase (KR) This enzyme catalyses the NADH dependent reduction of 3-oxoacyl ACP to (3R)-hydroxyacyl ACP,4X the stereochemistry being established by examining the reverse reaction; only (3R)-hydroxyacyl ACPs were metaboli~ed.~~ The enzyme has a broad tolerance for differing chain lengths of C to c16 and prefers ACP thioesters though it will metabolise CoA thioesters slowly. 2.1.6 (3R)-Hydroxyacyl ACP dehydrase (DH) This enzyme has an absolute specificity for thioesters of ACP and does not metabolise CoA or pantetheine thi~esters.~' It has been shown that the R isomer of 3-hydroxybutanoyl ACP was dehydrated whilst the S isomer was not. The enzyme will accept chain lengths from C to c16 but interestingly shows a rate minima at Cl0 the point at which a separate enzyme (3R)-hydroxydecanoyl ACP dehydrase (HDDH) diverts material into the anaerobic unsaturated pathway (vide infra).The stereochemistry of hydrogen removal at C-2 has not been established in E. coli but has been elucidated in yeast as pro-2S (syn loss of ~ater).~' 2.1.7 (3R)-Hydroxydecanoyl ACP dehydrase (HDDH) This dehydrase catalyses the interconversion of (3R)-hydroxydecanoyl (2E)-dec-2-enoyl and (32)-dec-3-enoyl ACP thioesters (Scheme 1). This process is the critical branch point in the assembly of saturated or monounsaturated fatty acids. (2E)-Dec-2-enoyl ACP is reduced and chain extended resulting in saturated acyl chains whilst direct elongation of (32)- dec-3-enoyl ACP leads to the common monounsaturated acyl chains found in E.coli. This enzyme its inhibition and its stereochemical progress has been extensively studied by Schwab and co-workers and has been reviewed previously.2 In a recent report Schwab and co-workers treated "N-labelled dehydrase with the mechanism based alkynoic inhibitor S-(dec-3-ynoyl) N-acetylcysteamine [S-(dec-3-ynoyl) NAC] thioester. Chemical shift changes in the "N NMR spectra upon addition of the NAC thioester showed that the NC2 of His-70 becomes alkylated and is probably the locus of active site basicity in the normal reactions catalysed by the de- hydra~e.~~ Clustridium butyricum produces (72)-hexadec-7- enoate presumably via a modification of the above anaerobic route in which (3R)-hydroxydodecanoyl ACP is dehydratedl isomerised to (3Z)-dodec-3-enoyl ACP by the 'HDDH' followed by standard chain extension.53 2.1.8 Enoylacyl ACP reductase (ER) In E.coli the pro-4S hydrogen of NADPH is added to the si face of the double bond at C-3 and a solvent proton adds 338 Natural Product Reports 1997 suprafacially at C-2 re. The stereochemistry of ERs from different organisms vary and all four possible stereochemical combinations (C-2 silre C-3 silre) of the two hydrogen ad- ditions are now known.' In E. coli two activities have been partially purified by Weeks and Wakil both catalysing the reaction of (2Q-enoyl ACP thioesters. One is NADPH depen- dent and is most active with short chain derivatives and only accepts enoyl ACP derivatives whilst the second is NADH dependent and has a broader chain length tolerance and will accept both enoyl ACP and enoyl CoA derivative^.^^ Hogenauer and co-workers have recently purified from an overproducing strain of E.coli an NADH dependent enoyl ACP reductase that uses both (2E)-but-2-enoyl (crotonyl) ACP and (2E)-but-2-enoyl CoA (though more slowly) as substrates and the corresponding gene (fabl) shows sequence analogy with enoyl ACP reductases from Salmonella Anabaena and Brassica (mustard) sp. Complete inhibition of this NADH dependent enoyl reductase activity by diazaborines is lethal suggesting that the NADH dependent ER in E. coli cannot be fully replaced by the NADPH dependent ER.55 2.1.9 Acyl ACP thioesterases (TE) Thioesterases I and 11 of E.coli catalyse the hydrolytic cleavage of fatty acyl CoA thioesters in vitro 103-104 times faster than the corresponding fatty acyl ACP thi0este1-s.~~ TE I is a serine esterase that only hydrolyses long chain (Cl,-C18) thioesters whilst TE I1 is a tetramer with a broader substrate activity (C6-C18) and a different mechanism as it is insensitive to serine esterase inhibitors. Their role is unclear as they have no assigned role in E. coli lipid metabolism. Cho and Cronan report the cloning of the structural gene for E. coli TE I (te~A).~~ The sequence is the first reported for a non-vertebrate serine esterase and the enzyme showed an overall organisation similar to that of the evolutionarily distant TE I1 enzymes of birds and mammals.2.1.10 Cyclopropanated fatty acids in E. coli A major component of membrane phospholipids in E. coli is cyclopropanated fatty acids. A methylene group is transferred from SAM to the 2 double bond of the unsaturated fatty acid in membrane phospholipids by a soluble enzyme located in the cytoplasm. This interesting enzyme utilises a soluble reagent (SAM) an insoluble membrane component and unusually it is most active between the late log phase and early stationary growth phase. The gene has been cloned the protein over-expressed and Wang and Cronan have recently determined that one of the gene promoters depended upon the putative sigma 2.1.11 Acyl ACP synthase Exogenous free fatty acids supplied to the E.coli culture medium are converted to CoASH thioesters prior to transport into the cell. Ray and Cronan have isolated a calcium and magnesium requiring enzyme that converts long chain fatty acid (14:0) (16:0) (16:1 927 (18:1 112) CoA thioesters into the corresponding acyl ACPS.~~ It is instructive to consider the molecular basis of chain assembly in a bacterium such as E. coli. The conversion of butanoyl ACP to hexadecanoyl ACP involves 24 catalytic transformations plus at least 18 transthioesterifications. After cell breakage each enzyme can be isolated in a functionally active form and its activity towards substrates examined. However if each enzyme (AT KAS KR DH ER HDDH) and ACP were freely independent in the cell then 42 specific collisions in a specific sequence and orientation would have to occur.In addition the growing fatty acyl chain would need to be bound and then transferred to another binding site (e.g. from KR to DH to ER to ACP to KAS etc.) at least 30 times each dissociation and reassociation costing free energy. Pre- sumably in the cell all the required activities are fairly tightly associated with each other though not covalently bound this association being lost upon cell breakage. Traditionally the growing acyl chain is freely suspended at the end of a long ‘swinging arm’ the acyl carrier protein only acting as a ‘text book’ passive carrier protein with the ‘phosphopantetheine moiety acting as a swinging arm transporting acyl groups from one active site in the fatty acid synthase to another’.60 How- ever the NMR experiments of Prestegard and co-workers (vide supra) suggest the presence of a binding site on the ACP for the growing acyl chain.If the growing acyl chain were to remain at least partly bound in a groove on the surface of the ACP then the other enzymes such as KR DH and ER would only have to approach the ACP binding over the part containing the C-2 and C-3 portion of the acyl chain and deliver their catalytic activity (NADH His etc.) to the 3-0x0 3-hydroxy group etc. whilst the bulk of the acyl chain is still attached in the ACP’s groove. Stereochemistry could be determined by the binding of the growing acyl chain to the ACP. The ACP is thought to be very conformationally flexible having more than one NMR structure.The effect of binding a 3-OX0 acyl chain in this groove might effect a conformational change upon the ACP that encourages binding of the KR. Binding of a (3R)-hydroxy acyl chain might similarily attract a DH 2E unsatu- ration an ER and full saturation a KAS. For chain extension with malonate the growing acyl chain is transesterified onto the KAS and a malonyl CoA transesterified onto the ACP phosphopantetheine thiol. If the KAS and ACP are non-covalently bound at this time the hydrocarbon terminus of the growing acyl chain could still reside in the ACP groove whilst the other end is covalently attached to the cysteine thiol of a KAS. The ‘Claisen-like’ condensation could still occur and the KAS depart now allowing the approach of the KR.In this way the fatty terminus of the growing acyl chain could remain attached to the ACP groove during the entire process. A remaining question would be whether the growing acyl chain’s methyl terminus remains locked in place or whether it slides along the ACP groove as the acyl chain grows. However the methyl terminus might not be tightly bound at all as the E. coli FAS can assemble iso-fatty acids when supplied with 2-methylpropanoyl ACP (but not 2-methylpropanoyl COA),~’ and Bmillus subtilis can assemble a wide range of a-cycloalkyl fatty acids when cyclopropanyl- cyclobutanyl- cyclopentanyl- cyclohexanyl-or cycloheptanyl-carboxylic acids are added to the medium.62 2.1.12 Other Gram negative bacteria The acetyl CoA carboxylase (ACC) from Pseudomonas citronellolis has been examined.63 Best and Knauf has recently cloned and characterised a DNA fragment from Pseudornonas aeruginosa that complemented a lethal mutant of E.coli with a lethal mutation in the biotin carboxy carrier protein. Whilst E. coli only contains a single biotinylated protein P.aeruginosa contains at least three.64 Haselkorn and co-workers have cloned genes for the biotin carboxylase and biotin carboxy carrier protein from the cyano- bacterium an Anabaena sp. strain PCC 7120.65 The two genes are not linked unlike in E. coli and the sequence at the biotinylation site is Met-Lys-Leu not the usual Met-Lys-Met. A 62 desaturase from the cyanobacterium Synechocystis has been functionally overexpressed in Anabaena a cyanobacte- rium normally lacking in 62 desaturase that converts (18:2 92,122) (linoleic acid) to (1 8:3 62,92,122) (y-linolenic acid).66 The biosynthesis of the biopolymers polyhydroxyalkanoates (PHAs) was discussed extensively in the last review.’ A detailed paper has appeared on the biosynthesis of PHAs in Pseudomunus aeruginosa using “C-labelled precursors.When sodium acetate was the sole carbon source monomer units of even carbon numbers C to C, were obtained. In contrast when sodium propanoate was used as the sole carbon source all seven chain lengths from c6 to C, were obtained with the Rawlings Biosynthesis of fatty acids and related metabolites C- 1 of propanoate being selectively incorporated three carbons in from the methyl terminus of the C, C and C, chains.Propanoate is being converted into malonyl CoA and the original carbonyl carbon selectively eliminated as the malonate is loaded onto the FAS system for unlabelled two carbon extensions of the carbonyl labelled propanoate ‘starter’ unit.67 4-Hydroxyhexanoic acid has been identified as a constituent of PHAs when bacteria are supplied with 4-hydroxyhexanoic acid.68 PHA biosynthetic genes have been cloned from Thiocystis viol~cea,~~ an anoxygenic phototropic purple sulfur ba~terium.~’ The Gram positive bacteria also produce PHAs (vide infra). 2.2 Gram positive bacteria The Gram positive bacteria can be divided into those whose DNA has a low G+C content (Clostridium subdivision) and those with high G+ C content (Actinomycetes subdivision).The ‘Clostridium subdivision’ include the lactic acid bacteria (e.g. Lactobacillus) the ‘Gram positive cocci’ (e.g. Staphylococ- cus and Streptococcus) and ‘endospore’ formers (e.g. Bacillus and Clostridriurn). The ‘Actinomycetes subdivision’ which is of great interest to the natural product chemist can be split into major groups including The rod-shaped ‘Coryneforms’ (e.g. Corynebacteriurn Arthrobacter and Brevibucterium); the anaerobic ‘propionic acid bacteria’ (e.g. Propionibacterium and Eubacterium); and the filamentous ‘Actinomycetes’. The latter group are further split into several genera the actino- mycetes (e.g. Actinornyces); the mycobacteria (e.g. Mycobac- terium); the actinoplanes (e.g.Actinoplanes); the norcardias (e.g. Norcardia Saccharopolyspora and Rhodococcus); the streptomycetes (e.g. Streptomyces Streptoverticillium and Chairiia);and the micromonosporas (e.g. Micromonosporn and Thermoactinomyces). 2.2.1 Mycobacteria and other related bacteria Few afflictions have caused so much pain and anguish to more human beings than tuberculosis and leprosy both caused by the genus Mycobacteriurn. Many aspects of the myco- bacteria have been reviewed in three excellent volume^.^' A distinguishing feature of these bacteria (along with the Coryne-bacterium and Norcardia) is their cell wall comprised of very long chain and unusual fatty acids frequently as esters to trehalose containing carbohydrates.The extraordinary length and non-polar nature of these acids results in a very ‘waxy’ cell wall which is rather impermeable to most drugs and which is also responsible for these organisms’ ability to survive dor- mant in a host for many decades. Fatty acid biosynthesis in the mycobacteria and related bacteria is very complex difficult to investigate due to the complex nature of the products and slow growth rate of organisms and still not clearly understood. The acetyl CoA carboxylases have been examined. The enzyme from M smegmatis (formerly M. phlei) purifies as an aggregated complex which does not separate into its con- stituent enzymes7’ The ACCs from M. smegmatis are also able to carboxylate propanoyl CoA which the ACC from E. coli is unable to achieve.73 In M.smegmatis M. tuberculosis M. leprae and Coryne-bacterium diphtheriae there exists a Type I fatty acid synthase that does not require a discrete ACP and normally assembles hexadecanoic acid from acetyl and malonyl CoA. In the absence of solubilising fatty acid binding methylated carbo- hydrates the principle product is instead tetracosanoyl CoA thi~ester.~~.~~ The existence of Type 1 FAS is rare in the prokaryotes and probably reflects the evolutionarily advanced nature of these bacteria. Whilst a macrolide PKS from the prokaryotic Saccharopolyspora erythraea has recently been found to be Type I in a Type I1 ACP thought to be involved in fatty acid biosynthesis was isolated from the same organism.32 Three enzyme systems are known for fatty acid elongation in mycobacteria.The first employs a discrete Type I1 ACP similar to E. coli ACP in many regards except cSCoA 0 Eicosanoyl CoA + 4 x Methylmalonyl CoA (Mycocerosic acid synthase) COOH 4 Scheme 2 solubility and that it uses octadecanoyl CoA as a ‘starter’ unit elongating it to hexacosanoic acid.77 The second system is acetyl CoA dependent ‘discrete ACP’ non-requiring and the third being malonyl CoA dependent and ‘discrete ACP’ n~n-requiring.~~ A KAS79 and an NADPH dependent ERgO have been purified from M. smegmatis. Mycobacteria produce monounsaturated fatty acids such as (92)-octadecenoic acid (oleic acid) by both anaerobic and aerobic pathways. Cyclo- propanation or methylation of the double bond by SAM frequently occurs to give e.g.tuberculostearic acid (10R)-methyloctadecanoic acid.81 A synthesis of racemic tuberculo-stearic acid has recently been reported.82 M. tuberculosis produces mycocerosic acids (e.g. 2’4 6-trimethylhexacosanoic acid and 2,4,6,8-tetramethyl-octacosanoic acid 4)by the elongation of octadecanoic acid or eicosanoic acid thioester with three or four methylmalonyl CoAs using a Type I type system (Scheme 2).83 This intriguing system seems capable of specifically adding three or four branched extender units in a manner reminiscent of the water fowl uropygial (preening) glands. Mycocerosic acid synthase has recently been purified and is a multifunctional enzyme with high specificity for methylmalonyl CoA no thioesterase and an ACP domain that strongly binds the The gene has been cloned and sequenced (RMM 225 kDa) and a linear functional map proposed (KAS AT DH ER KR ACP no TE) the same order as that found for vertebrate FAS.X4 The DH domain could not be precisely located.The M. tuberculosis FAS has been purified and interestingly found to be a dimer with monomeric RMM ca. 500 kDa.74 This monomer may represent two fused FAS systems interestingly reminiscent of the streptomycete derived macrolide polyketide synthases. in which two or more synthase units can be fused together.’ Recent reports also suggest that the polyketide ‘double synthase unit proteins’ exist as dimers. Phenol phthiocerol 5 the main lipid component of the phenolic glycolipids such as ‘Mycoside B’ 6 comprises a II I OR OR OMe n= 16 or 18 5 Phenol phthiocerol R = H 7 Substance A R = CO(CHMeCH2)x(CH2)&H3 (x= 3 or 4; y= 16 or 18) Cell-free extracts I 6 Mycoside B (a phenolic glycolipid) (2-OMethylrhamnosyl phenolphthioceryl dimycocerosate) Scheme 3 tyrosine derived 4-hydroxybenzoate ‘starter unit’ with a methyl branched (from propanoate) hydroxylated side chain.85p87 Phenolic glycolipids consist of a phenol phthiocerol 340 Natural Product Reports 1997 core 5 esterified to two mycocerosic acids 4 and a sugar attached to the phenolic hydroxy group.Draper and co-workers have isolated two series of advanced intermediates (Substance A 7 and L) by adding radiolabelled propanoate to cell-free extracts of M.microti and identified them by mass spectrometry and NMR spectroscopy (Scheme 3).88 M. smegmatis (M. phlei) produces polyunsaturated fatty acids such as the phleic acids 8 which are very long (C35-C53) Z Me(CH2),(CH=CHCH2CH2),C0OH 8 Phleic acids (rn=14,n=4,5or6; m=12,n=5or6) fatty acids containing repeating (2a-but-2-ene moieties added to a normal length (C,6,,8)straight chain fatty acid ‘primer’. It has been proposed that this unusual repeating unit arises viu the incorporation of ‘crotonate’ unit^.^^,^^ A possible unprecedented mechanism using (2O-pent-2-en- 1,Sdioic acid mono CoA thioester as an allylic homologue of malonyl CoA is outlined in Scheme 4. Me(CHp),COSEnz 4-PCOSEnz -co2 Me(CH2)nluSEnz COOH 8 Phleic acids +-Scheme 4 Mycolic acids are 2-alkyi-3-hydroxy fatty acids found in the norcardia corynebacteria and most mycobacteria having a general structure R2(CHOH)(CHR1)COOH with a variety of 0x0 epoxy cyclopropyl and methyl functionalities adorning the alkyl chains (e.g.9-12; m=lO 12 n=15 17 19). In the Corynebacterium and Norcardia mycolic acids usually contain Z Z Me(CH2)l7CH=CH(CH2),CH=CH(CH2),CH(OH)CH(COOH)(CH2)fle 9 a-Mycolates Me(CH2) 7CH&H(CH2)nCH(OH)CH(COOH)( CH2)fle 10 a’-Mycolates 0 /\ Me(CH& 7CHMeCH-CH(CH2),CH&H(CH2),CH(OH)CH(COOH)(CH2)fle trans 11 Epoxymycolates Me(CH~)l7CHMeCO(CH2),CH~CH(CH2)nCH(OH)CH(COOH)(CH~)fle 12 Ketomycolates 40-60 carbons but in the Mycobacterium there are usually 70-90 carbons.The side chain R’ is derived from a long chain saturated elongated fatty acid (24:0) and the main carbon chain R’ is derived from a very long fatty acid e.g. a meromycolic acid such as 13 or 14 with functionality such as unsaturation cyclopropanation methylation hydroxylation methoxylation or 0x0 functionality at intervals along the chain.”” ’)’A survey of structures in M. aurum has recently been published.’)3 In wax ester mycolates some ketonic car- bons have been enzymatically oxidised to an ester by a Baeyer-Villiger type process disrupting the carbon alkyl chain backbone. In 1984 Takayama reported the first cell-free system from the mycobacteria capable of producing long chain fatty acids such as the meromycolic acids 13 and 14 (Scheme 5).94 qCOOH (SZ)-Desaturase I NCOOH Elongation/desaturation Me(CH2)17CH=CH(CH2),COOH Z Z 14 Me(CHz)17CH=CH(CHz),CH=CH(CH2),COOH 1 13 $-9 a-Mycolates 10 a‘-Mycolates Scheme 5 However it was not known whether the very long chain meromycolic acids had been assembled via the repeated C addition to a C, 24 ‘starter unit’ or by the ‘head to tail’ joining of preformed C, 24 units.The first ‘linear’ route would involve an enormous number of enzymatic reactions the second route would involve the activation of the terminal methyl group followed by coupling with a ‘starter unit’. The regular spacing of functionality along the very long carbon chain favours the ‘head to tail’ pathway. However Lacave and co-workers found radioactivity from radioactive acetate mainly in the ‘extended’ regions of mycolic acid from M.uurum not in the ‘starter unit’ regi~n.”~. 96 Wheeler et ul. have found an insoluble extract from M. smegmutis that incorpor- ated radioactive acetate into mycolic acids. Addition of a labelled putative ‘primer’ unit (5Z)-tetracosenoic acid (24 1 52) 15 gave some incorporation suggesting that it may be the ‘starter unit’ for malonyl CoA additi~n.~’ The 52-desaturase that converts tetracosanoic acid to (5Z)-tetracosenoic acid is believed to be the main site of action of the antimycobacterial drug isoniazid 16. However it is not yet known how (24:O) is converted to (24 1 59 and what the carrier molecule of (24:O) is. Wheeler and co-workers synthesised the methyl ester of a cyclopropene analogue of (24:1 53 methyl (52)-5,6-methylene-5-tetraicosenoate 17 and found that it inhibited CONHN H2 16 COOMe 17 Ralttlings Biosjnthesis of futty acids and related metabolites mycolic acid synthesis.98 Presumably the mycolic acids are then assembled by a ‘Claisen-like’ condensation between the formal anion of the corresponding meromycolic acid thioester and an elongated fatty acid thioester perhaps as the malonyl analogue followed by 3-ox0 reduction.A soluble cell-free extract has been isolated from Corynebacterium diphtheriae that assembles (2R)-3-oxo-2-tetradecanyloctadecanoatethio-ester from two molecules of hexadecanoyl CoA presumably involving the condensation of a hexadecanoyl starter with a tetradecanylmalonyl extender unit (Scheme 6).Avidin inhib- ited this condensation suggesting the presence of a biotin dependent carboxylase. Sodium borohydride was then added as the cell-free extract was not capable of performing the 3-ox0 reduction to give corynemycolic acid 18 and its (3Q-epimer.’” 2.2.2 Other Gram positive bacteria Homogenous preparations of ACP have been obtained from Arthrobacter’”” and Clostridium butyricum. lo’ Cyclohexanecarboxylic acid is used as a starter unit in the biosynthesis of o-cyclohexanyl fatty acids found in thermo- acidophilic bacteria such as Alicyclobacillus acidocaldarius. A. acidocaldarius was formerly classified as ‘Bacillus’,however due to its high G+C content it has been placed in a new genus.Recent studies have shown that the cyclohexanecarboxy moiety is derived from shikimic acid 19 as outlined in the previous review (Scheme 7).’. Io3 A recent paper distin- guishes between two possible biosynthetic pathways to (3R,4R)-dihydroxycyclohexa-1,5-dienecarboxylic acid 20 an intermediate between shikimic acid and cyclohexanecarboxylic acid 21. The first pathway involves shikimic acid 19 (or its 3-phosphate derivative 22) undergoing trans-1,3-elimination of water (or phosphate) to give 20 directly in a manner analogous to the conversion of 5-enolpyruvylshikimate-3-phosphate 23 to chorismic acid 24. The second is the hydrolysis of chorismic acid with loss of pyruvate to give the dihydroxydienoic acid 20.’04Addition of [2-2H]shikimic acid to cultures of A.acido-caldarius singly enriched 1 1 -cyclohexanylundecanoic acid 25. However A. acidocaldarius is cultured at 50 “C at pH4 conditions in which chorismic acid 24 could be expected to non-enzymatically convert into dihydroxydienoic acid 20. Addition of glyphosphate (an inhibitor of enolpyruvylshikimic acid 3-phosphate synthase) did not affect the levels of cyclohexanyl fatty acid 25 produced again suggesting that chorismate was not an intermediate and that the former pathway is in operation. ’7 Reynolds and co-workers’ l2 have examined cell-free extracts from S. collinus that were able to convert cyclohexa- 1,5-dienecarbonyl CoA 26 to cyclohexanecarbonyl CoA 27. Purification gave an enzyme that was able to reduce both cyclohex- 1 -enecarbonyl CoA 28 to cyclohexanecarbonyl CoA 27 or cyclohexa-1 ,5-dienecarbonyl CoA 26 to cyclohex-2-enecarbonyl CoA 29 suggesting that this single enzyme may perform two steps on the biosynthetic pathway.This enoyl reductase transfers the pro-4S hydrogen from NADPH to the si face of the C-3 carbon. The other active enzyme component a A*,A’-cyclohexenecarbonylCoA isomerase converts 29 to 28. Addition of racemic [1-2H]cyclohex-2-enecarbonyl CoA to cell-free extracts gave (3S)-[3-2H]cyclohexanecarbonylCoA consistent with a single base catalysed 1,3-allylic suprafacial shift and a stereochemical preference for the 1s enantiomer of the racemic substrate. The cell-free extract was challenged with cyclohexa-1(6),3-30 and cyclohexa-l,3-dienecarbonylCoA 31 and both were only converted to cyclohex-3-enecarbonyl CoA 32 suggesting that neither of these dienoic acids were true intermediates (Scheme 8).An enoyl reductase that converted (2E)-butenoyl CoA to butanoyl CoA was purified from S. collinus and its stereochemistry compared with the above cyclohex-1-enecarbonyl CoA reductase. Both reductases use the pro-4S hydrogen of NADPH but the attack at C-3 is si by the cyclohex-1-enecarbonyl reductase and re by the (2E)- butenoyl CoA reductase. This is the first exception to the 341 COOH Carboxylase ? ;COOH *COSCoA Cell-free extract 3-Oxoacyl synthetase? \ /COO (2R)-3-0xo-2-tetradecanyloctadecanoic acid NaBH4 1 ICOOH 18 Scheme 6 COOH COOH COOH HO" OH OH OH OH 24 / OH OH 20 COOH / A n Alicyclobacillus acidocaldarius * )COOH 21 25 Scheme 7 COSCoA COSCoA COSCoA COSCoA The streptomycetes and related bacteria assemble fatty acids using a range of starter units resulting in a mixture of straight chain iso- and anteiso-fatty acids.21 The unusual starter units are obtained from the branched amino acids valine leucine 26 29 28 27 and isoleucine by transamination and decarboxylation.For c$ -0 -0 example analysis of the lipids from Saccharopolyspora COSCoA COSCoA COSCoA erythraea (formerly Streptornyces erythreus) show them to contain 14-methylpentadecanoic acid (isopalmitic acid 2-30 32 31 methylpropanoate starter; 3 YO) 13-methyltetradecanoic acid (3-methylbutanoate starter; 15%) and 14-methylhexadecanoic acid (2-methylbutanoate starter; 13%).lo5 The acetyl CoA Scheme 8 carboxylase (ACC) has been studied from Saccharopolyspora erythraea.'06 An ACP believed to be involved in FAS from Saccharopolyspora erythraea has been purified' O7 cloned and general pattern observed for enoyl thioester reductases in overexpressed in E. coli,'2 and has been obtained in 85% purity which transfer from thepro-4R hydrogen of the nucleotide is to on the large scale by freezing and thawing the heat induced the re face and vice versa. The evolutionary significance of short E. coli cells avoiding the need to disrupt the cells and the chain enoyl CoA reductases is discussed with the probability subsequent release of other intracellular components.'08 that they may be involved in the assembly of large primers Electrospray mass spectrometry (ESMS) has proved very able for malonyl CoA dependent FAS.The authors also reveal at determining the efficiency of phosphopantetheinylation of that preliminary results suggest the presence of a NADPH overexpressed acyl carrier proteins and the extent of synthetic dependent (2E)-hexenoyl CoA reductase in S. collinus. acylations of ~OZO-ACP.'~' The crude ACP was passed through 342 Natural Product Reports 1997 an anion exchange column and then loaded onto octyl-Sepharose CL-4B. Holo-ACP rapidly eluted whilst apo-ACP remained on the column providing the first large scale separ- ation of a holo-ACP from an apo-ACP which was confirmed by examination of the products by electrospray mass spec- trometry.ESMS has also been used by Staunton and co-workers to monitor the enzymatic assembly of growing fatty acyl chains on the overexpressed Saccharopolyspora erythraea ACP by the addition of cell-free extracts containing FAS and radiolabelled substrates.' lo The ACP was mainly derivatised to the C (methylbutanoate starter unit) with significant levels of methylbutanoate chain extended C , C, and C, acylated ACPs. Quite unexpectedly high levels of C acyl ACP were also observed even though no acetyl CoA had been added to the system. Staunton and co-workers explain this as caused by the reaction of malonyl CoA with free ACP to form malonyl ACP followed by decarboxylation to acetyl ACP and transfer of the acetyl group onto the KAS as starter unit.The interesting accumulation of C in the straight chain system suggests that the following cycle to C, is very slow. The Gram positive norcardia Rhodococcus ruber produces a 3-hydroxypentanoate (3-hydroxyvalerate) containing PHA polymer. A recent study added [2,3-13C2] and [1,4-"C,]succinic acid and examined the resulting monomeric 3-hydroxypentanoate (3HV) by NMR spectroscopy. The C,-C and C,-C fragments of 3HV were derived intact from carbons 2 and 3 of succinate and carbon 3 of 3HV was derived from a carboxy carbon of succinate. Methylmalonyl CoA mutase activity was detected and it was proposed that the known 'propionate pathway' in the Gram positive Propioni- bucteria was operative in R. ruber.l13 3 Archaea Archaeal membranes are comprised of terpene derived hydro- carbons linked to glycerol via ether linkages.The more chemically stable ether linkage presumably has survival value compared to an ester linkage when growing at high tempera- tures harsh pH or high saline conditions. Some archaea use dimeric hydrocarbons extending right across the 'bilayer' such as dibiphytanyldiglycerol tetraether found in Methanobacte- rium thermoautotrophicum in association with the monomeric analogue 2,3-diphytanyl-sn-glycerol. However small amounts of fatty acids have been found in the halophile Halobacterium cutirubrum and the above methanogen. Pugh and Kates have shown that the fatty acids do not occur in the purple mem- brane of H. cutiruhrum but occurred in significant quantities (over one molecule per protein molecule) in the red membrane only in the form of acylated protein.The major fatty acids were hexadecanoic and octadecanoic with small amounts of tetradecanoic and octadecenoic acids (not hexadecenoic acid as for E. coli) and their resistance to hydrolysis from protein suggest that they are amide linked. Discrete ACPs have been isolated from both archaea the ACP from H. cutirubrum being assayed with E. coli FAS. The FAS from M. thermautotrophi-cum was more active at 60 "C under nitrogen than at 37 "C and was dependent upon the addition of ACP but did not require acetyl CoA. NADPH was not required and indeed appeared inhibitory. In contrast the FAS from H. cutirurum appeared independent of the addition of ACP or NADPH.l14 These findings cause the authors to question the type of FAS present.The isolation and characteristics of the FAS strongly suggest a Type I1 system. The presence of 18:l fatty acid can be explained by a chain extension of 16:l by a KAS I1 as in E. coli. However the halophile's ACP appears very tightly bound and the synthase system must either have sufficient NADPH already bound or be NADPH independent. 4 Protists This section contains unicellular eukarotic microorganisms. Protozoa are unicellular eukaryotic microorganisms that lack Rawlings Biosynthcsis of fatty acids and related metabolites chlorophyll lack cell walls and are generally motile and include the flagellates (Mastigophora including Trypanosoma Giardiu and Leishmania) amoebae ciliates and sporozoans (e.g.Plasmodium responsible for malaria). Sponges can be considered as a colony of unicellular microorganisms. Algae are unicellular (or colonial e.g. seaweed) eukaryotic micro- organisms containing chlorophyll and carrying out oxygenic photosynthesis and possess cell walls. They include the green algae (Chlorophyta) brown algae (Phaeophyta) red algae (Rhodophyta) golden brown algae or diatoms (Chrysophyta) and dinoflagellates (Pyrrophyta). Most algae are immotile but a few such as the dinoflagellates euglenids and some green algae have flagella. Euglenids (e.g. Euglena) are unusual organisms resembling algae without cell walls but contain- ing a chloroplast. If this chloroplast is lost they become indistinguishable from a flagellated protozoan.A major series of reviews on marine natural product chem- istry appeared in Chemical Reviews during 1993 including microalgal metabolites the biosynthesis of marine natural products bioactive metabolites of symbiotic marine micro- organisms carbocyclic oxylipins of marine origin marine toxins marine invertebrate chemical defenses and marine haloperoxidases. l5-I2' The following reviews have recently appeared the biosynthesis of bioactive metabolites of marine blue-green green red and brown alga; marine sponges coelenterates and molluscs;'22 and the structure and bio- synthesis of marine algal oxylipins. 123 4.1 Protozoa Cyclopropane fatty acids such as cis-9,1 O-methano- octadecanoic acid (dihydrosterculic acid) 33 are found in the phospholipids of many plants bacteria and parasitic Previous studies have shown that sulfur sub- stituted derivatives of octadecanoic acid are potent inhibitors of dihydrosterculic acid biosynthesis in trypanosomatid protozoa.'25 A recent report describes the synthesis of bis(9- thiaoctadecanoy1)-containing phosphatidylethanolamines 34 and their inhibition of the cyclopropane fatty acid synthase from the parasitic protozoan Crithidiu fasciculata.126 The authors propose that the corresponding methylated sulfonium ACOOH 33 compound 35 binds tightly to the active site in place of the expected reactive intermediate 36. Garson et a/. have reported the isolation of two new brominated long chain fatty acids from the tropical marine sponge Amphimedon terpenensis (5E,92)-6-bromotetracosa-5,9-dienoic acid and (5E,92)-6-bromopentacosa-5,9-dienoic acid and on the biosynthetic implications.A subsequent paper confirms the location of these brominated fatty acids in sponge cells only not in eubacterial or cyanobacterial symbionts.128 The first report of in vitro biosynthesis of primary mamma- lian prostaglandins from coral extracts has appeared. Extracts from the White Sea soft coral Gersemia fruiticosa formed optically active prostaglandins PG D, E, FZa and 15-keto- F, from exogenous arachidonic acid. 129 4.2 Eukaryotic algae Marine algae contain homoallylic polyunsaturates as their major fatty acids. (20:4 52,82,112,142) (also written 20:4 n -6) occurs in brown and red algae (20:5 52,82,112,142 172) (also written 20:5 n -3) in diatoms and some brown and red algae (1 8:4) in a cryptomonas and some green and brown algae and (16:4) in some green algae.This high level of unsaturation is presumably to assist maintaining the fluidity of membranes in cold seawater. Fish can obtain their polyun- saturates from grazing algae. The biosynthesis of polyunsaturated fatty acids in Phaeodac-tylum tricornutum has been examined by Arao and co-workers by pulse labelling and 'chasing' with radioactive precursors. P. tricornutum is a unicellular silica-less marine diatom whose major fatty acid is 20:5 (n-3),that is reported to be assembled from 18:l (n-9) via a network pathway comprising four routes from 18:2 (n-6) two routes passing through (n-3) fatty acids one through (n-6) and one through both (n-3) and (n-6) fatty acids.'30 13' The actual pathway chosen may reflect whether a prokaryotic or eukaryotic pathway is in operation.It has long been known that marine algae produce a wide variety of polyunsaturated fatty acids but it has only recently been recognised that they are able to oxidise these compounds to a range of biochemically important compounds the oxy- lipins. Oxylipins are fatty acids oxidised by at least one step of mono- or di-oxygenase dependent oxidation. 13* Many of these oxidative pathways are analogous to those of the icosanoid pathway found in mammals producing compounds such as (12S)-hydroxyicosatetraenoic acid (12S-HETE) that can elicit physiological responses in mammalian tissues at extremely low concentrations and such algae may prove a useful source of these compounds.However the hydroperoxide precursor to 12s-HETE can also be metabolised by additional biosynthetic pathways without parallel in mammalian systems to a series of carbocyclic or rearranged products that are described by Gerwick and co-workers who propose that the constanolac- tones 37-40 are assembled in Constantinea simplex by the action of lipoxygenase on arachidonic acid 41 to give hepoxilin B 42 followed by dual ring closure (Scheme 9).123,133 134 Gerwick and co-workers have also investigated the biosyn- thesis of (1 3R)-hydroxyarachidonic acid (1 3R-HAA) 43. A cell-free homogenate of the red alga Lithothamnion corallioides was able to convert exogenous arachidonic acid 41 into 13R-HAA 43.Using "0-labelling Gerwick et a/. showed that unlike other similar oxylipins such as (52,82,11R,12E 142)- ll-hydroxyicosa-5,8,12,14-tetraenoic acid (1 1R-HETE) 44 the origin of the oxygen atom in the formation of 13R-HAA 43 was from water not from dioxygen despite dioxygen being required for the conversion and they proposed a mechanism involving water displacement of an enzyme-arachidonate complex (Scheme Gerwick and co-workers have examined the biosynthesis of conjugated triene containing fatty acids in the marine red alga Ptilota Jilicina and have purified a novel enzyme polyenoic acid is0mera~e.I~~ This enzyme was able to catalyse the formation of conjugated trienes from a variety of precursors the fastest reaction occurring in the transformation of (52,82,1 12,142,17Z)-icosa-5,8,11,14,17-pentaenoate45 to (52,7E,9E 142,17Z)-icosa-5,7,9,14,17-pentaenoate46.The reaction appears to be catalysed by a single enzyme with no requirement for oxygen. The enzyme intramolecularily trans- fers the bis-allylic pro-S hydrogen from the C-11 position of (62,92 12Z)-octadeca-6,9,12-trienoate(y-linoleate) to the C-13 position and the bis-allylic C-8 hydrogen is lost to the solvent (Scheme 11). When deuterium is placed at 8R,a kinetic isotope effect is seen suggesting the the second step is rate determining. A gene encoding acetyl CoA carboxylase (ACC) from a photosynthetic eukaryotic alga Cyclotella cryptica has been isolated and cloned.The predicted amino acid sequence shows high homology to animal and yeast ACCases with a Met-Lys- ZH 41 -HoccooH 42 /---l r dH0Ia b 37 Constanolactone A 39 Constanolactone F 38 C-9 epimer is constanolactone B 40 C-1 1 epimer is constanolactone G Scheme 9 344 Natural Product Reports 1997 CoA ACP transacylase and a cerulenin sensitive 3-oxoacyl COOH ACP synthase I (KAS I). Acetyl ACP supported fatty acid *OH biosynthesis as effectively as a mixture of ACP and acetyl 44 CoA. There was no evidence of the existence of a KAS 111 as for bacteria and higher plants. The possession of an acetyl Lipoxygenase *02 CoA ACP transacylase activity is unique amongst Type I1 FAS systems but is in common with the Type I yeast FAS.As /\/\COOH -411" Euglena appears to possess the only Type I1 FAS that uses acetyl ACP this further enhances the peculiar phylogenetic status of this organism that appears to show both animal and plant characteristics. * X-Enz(02) 5 Fungi #COOH In the past fungi have been considered along with the protists but are now considered separately alongside plants and ani- mals even though there are borderline examples of fungi that are hard to distinguish from the protists. t Acetyl CoA carboxylases from the fungi have all three 6H COOH activities on a single multifunctional polypeptide chain (yeast RMM 265 kDa) and may exist as a tetrameric complex (a4) with one covalently bound biotin per subunit.Kohlwein and 43 Scheme 10 co-workers have examined the factors that control the expres- sion of FAS3 gene from yeast that encodes the structural gene ACC1. Disruption of the gene prevented vegetative growth despite fatty acid supplements suggesting that ACC activity is essential for a process other than de novo fatty acid &COOH 17 14 - synthesis and that there is only a single functional copy of the 45 gene.14' 17 14 5 COOH 5.1 Unicellular fungi z In yeast and other fungi FAS activities are distributed between 46 1,3-single base allylic shift Enz-AH B-Enz Rate determining step H* Scheme 11 Met sequence in the biotin binding Lichtenthaler et a/. have examined the sensitivity of ACCases (and other biotin containing enzymes) from a wide range of plant sources to two herbicide classes the cyclohexane- 1,3-diones and the aryloxy- phenoxypropanoic acids and found a wide variation in sensi- tivity.'38 The KAS 111 gene from the chloroplasts of a red alga (Porphyra umbilicalisau) has been sequenced and found to have homology with KAS 111 gene from E.coli.'39 Euglena gracilis is a very primitive eukaryote whose evol- ution and chloroplast acquisition was independent of the higher plants. The organism is considered unique in possessing two de novo fatty acid synthases a Type I FAS in the cytosol and a Type I1 FAS in the chloroplast. Ernst-Fonberg and co-workers have examined the catalytic activity of the early steps in the Euglena Type I1 FAS.14' The enzymes involved in the first steps were an acetyl CoA ACP transacylase malonyl Rawlings Biosvnthesis of fatty acids and related metabolites two multifunctional polypeptide chains u and (3.The u-subunit contains the KAS ACP and KR activity whilst the (3-subunit contains the ER DH acetyl transferase and the malonyl/hexadecanoyl transacylases.'42-'44 The enzymatically active FAS is an a6P6complex (RMM 2.4 MDa) that electron micoscopy suggests comprises of six circular disk-like a-subunits arranged hexagonally in a plane around which six arch-like P-subunits are distributed three on either side in an alternate manner each linking two adjacent a-subunits. '45 Presumably a single u,(3 'synthase unit' assembles a whole hexadecanoate molecule though an interesting alternative would involve passing the growing acyl chain from one 'syn- thase unit' to another as it is assembled in a manner analogous to that of the macrolide antibiotic (e.g.erythromycin) syn- thases.' In yeast the two subunits are encoded for by two unlinked genes FAS2 and FAS1 transcription of which is not affected by fatty acid levels Wolf and co-workers find that unassembled a-subunits are short-lived and rapidly degraded. 146 Using targeted in vitro mutageneses Schweizer and co-workers located the phosphopantetheine binding serine residue of yeast FAS in the 3-oxoacyl reductase domain This area was originally assigned by Wakil and co-workers as belonging to the ACP domain who reported that the amino acid sequence at this putative ACP domain showed little homology to that of E.coli ACP but a proposed tertiary structure had remarkable similarity comprising four a-helices interrupted by three (3-turns with the Ser-180 located in a similar position on a p-turn to that of the E. coli Ser-36.143 Whilst mutations on nearby residues results in KR mutants S180G mutation resulted in a KAS defective mutant rather than a KR defective mutant.14' This presum- ably reflects the intimate contact and functional interplay between allelic domains and their effect upon each other's activities. Schweizer and co-workers suggest that there is no obvious distinct ACP domain in yeast FAS but that the binding and function of the Ser-180 attached phosphopantath- eine depends upon protein-protein interactions between widely separated (on the peptide sequence) parts of the a-subunit.'47 The yeast FAS was compared to the 6-methylsalicylic acid 14'(Ser-180) of the a-subunit.synthase (MSAS) from Penicillium patulum. The KR from both yeast FAS and P. patulum MSAS exhibited the same stereospecificity for the 4-H si hydrogen of NADPH but the FAS KR component reduced both 3-oxobutanoyl and triacetic acid ester model substrates whilst the PKS KR component was specific for the triacetyl model substrate. 147 The previous review' discusses the effect of the side chain of cerulenin 3 on its inhibition of a range of chloroplast FAS systems. The inhibition of yeast FAS by cerulenin and a wide range of analogues has been inve~tigated.'~~ The (7E,1OE) double bonds were both found to be crucial the corresponding (7E,A") isomer 47 was ten-fold less active.The six carbon homologue of cerulenin ( 12-hexanylcerulenin) 48 was 200 0 0 47 n 0 0 48 times less inhibitory. Omura and co-workers have isolated 12 cerulenin resistant yeast mutants and found that all the mutants mapped the FAS2 gene responsible for encoding the a-subunit. The FAS from one mutant was purified and found to be 30-fold resistant to cerulenin which was due to a single mutation G 1257s.149 The FAS from Candida albicans and two cerulenin resistant mutants has been purified and characterised (RMM a 195 kDa J3 210 kDa).'" 5.2 Filamentous fungi Fatty acid synthases have been isolated from Yarrowia lipoly- tica Neurospora crassa (bread mould) and Penicillium patulum all having two multifunctional domains and a structure consistent with that for yeast.'51 The biosynthesis of the lactone 6-pentyl-a-pyrone (6PP) 49 in Trichoderma harzianum has been investigated.6PP has a w 49 strong coconut aroma and is in great demand in the food industry as a flavour enhancer. Incubation with [U-'4C]linoleic acid gave a much higher incorporation than from radioactive mevalonate. 52 The proposed mechanism involves allylic per- oxidation of (92,12Z)-octadeca-9,12-dienoicacid (linoleic acid) reduction to (92,ll E)-13-hydroxyoctadeca-9,11 -dienoic acid followed by J3-oxidation to (32,5E)-7-hydroxydodeca-3,5-dienoic acid. An unusual isomerisation followed by further J3-oxidation results in (2E,4E)-5-hydroxydeca-2,4-dienoic acid which after EIZ isomerisation of the C-2 double bond can lactonise to 6PP.6 Plants Plants predominantly produce around six or seven fatty acids with chain lengths of c16 or C, and one to three double bonds occurring predominatly esterified to glycerol as digly- cerides in membranes and triglycerides in seed oils. Cutin which occurs on the surface of all land based plants is a 346 Natural Product Reports 1997 polymer formed by cross-linking of hydroxyfatty acids. Satu- rated and monounsaturated fatty acids are assembled in the plastids (e.g. chloroplast) whilst attached to an ACP in a Type I1 manner reminiscent of the prokaryotes. Plant 9E desaturase acts upon the ACP derivative of the saturated fatty acid and is a soluble enzyme (RMM 68 kDa) requiring oxygen NADH NADH-ferredoxin reductase and ferre-doxin.'53 Plastids have probably been acquired directly from prokaryotes; they still have their own genomic DNA and possess prokaryotic features. Plants do not assemble fatty acids in their cytoplasm. After release from the ACP by a TE they cross the plastid envelope membrane as the free acid whence they are thioesterified to CoA. Membrane bound enzymes in the endoplastic reticulum then catalyse further desaturation elongation or attachment to glycerol. Many plants contain high levels of homoallyic unsaturated fatty acids in their seeds such as (92,12Z)-octadeca-9,12-dienoic(linoleic) acid 50 (92,122,15Z)-octadeca-9,12,15-trienoic(a-linolenic) acid 51 and (62,92,122,15Z)-octadeca-6,9,12,15-tetraenoic (stearidonic) acid 52.These are derived from (927-b C-O O H 11 8 50 SCOOH -51 octadecanoic (oleic) acid 53 by the action of non-haem iron oxygen dependent desaturases that may be either cytosolic (eukaryotic endoplasmic reticulum bound) or in plastids (prokaryotic like). The conversion of homoallylic polyenes to conjugated polyenoic fatty acids in germinating marigold seeds (Calendula oficinalis) was examined by Crombie and H~lloway'~~ who showed that two hydrogens one from C-8 and one from C- 1 1 from (92,12Z)-octadeca-9,12-dienoic (lino-leic) acid 50 were lost in its conversion to (8E,lOE,12Z)-octadeca-8,10,12-trienoic(calendic) acid 54.There appeared to be no involvement of an oxygenated intermediate. This system may be related to that currently being studied by Gerwick and co-workers in the red algae.'36 (COOH 53 11 COOH * 54 6.1 Lower plants Polyunsaturated fatty acids notably arachidonic acid 41 are widespread in ferns and mosses and acetylenic fatty acids in the mosses and liverworts. The moss Ceratodon purpureus can accumulate acetylenic fatty acids in its protonema cell triacyl- glycerols in particular (9Z, 12Z)-octadeca-9,12-dien-6-ynoic acid (18:3 6A,92,122) (or 18:2A) 55 and (92,122,152)-octadeca-9,12,15-trien-6-ynoic acid (1 8:4 6A,92 122,1527 (or 18:3A) 56. Their biosynthesis was investigated by feeding radioactive precursors to triacyl glycerol accumulating cells.18:3A 56 was formed by a A1*desaturation of 18:2A 55 which was formed by a second desaturation of the A6 double bond of (18:3 62,92,122) (y-linolenate) 57 though it is not clear yet whether this occurs on the free acid or whilst as part of a triacylglycerol. ' 55 55 56 6.2 Higher plants The inhibition of de novo biosynthesis of fatty acids in higher plants has been reviewed. Spinach (Spinacia oleracea) ACP I has been overexpressed in E. coli and was fully in the holo-ACP form.'57 Incubation of this ACP with E. coli FAS gave high levels of hydroxyfatty acids suggesting that spinach 3-oxoacyl ACPs are poor substrates for E. coli DH. Using rotating frame nuclear Overhauser enhancement NMR spec- troscopy (ROESY) Kim and Prestegard have shown that the spinach ACP I exists in at least two conformationally discrete forms in slow e~change.'~' Jaworski et al.have substituted threonine and cysteine for the active serine (Ser-38) of spinach ACP I and found that it was no longer phosphopantetheinyl- ated and that it acted as a strong inhibitor of spinach holo-ACP synthase. The authors suggest that overexpression of these proteins in transgenic plants may be a useful mechanism for blocking ACP metabolism in vivo.'59 Some plants produce seed oils or 'storage oils' of unusual structure which includes variation in chain length or hydroxy epoxy acetylenic and cyclopropyl functionality many of these unusual fatty acids having a wide range of important industrial uses.In a review on how the genetic engineering of plant fatty acid metabolism can lead to the design of new plant products Ohlrogge describes how a dodecanyl (lauryl) ACP thioesterase from Umbellularia californica was inserted into oil seed rape that can now contain over 40% dodecanyl esters in its seeds. The current source of dodecanoic acid for the soap and detergent industry is coconut and palm kernels from the tropics and it is hoped that this will soon be an economic alternative to the import of these oik8 The same TE has been inserted into an E. coli strain that is deficient in fatty acid degradative ability. Dodecanoic acid normally a minor com- ponent of E. coli is secreted into the medium accumulating to ACP drop as FAS biosynthesis increases approximately six- fold.166 The activity of KAS I11 (cerulenin insensitive) from seeds of C.lanceolata was investigated by the addition of cerulenin 3 and radiolabelled acetate to the corresponding FAS. The major product was butanoyl ACP with hexanoyl and octanoyl ACP produced in very small portions. In the absence of cerulenin octanoyl and decanoyl ACPs were the major prod- uct~.~~~ KAS 111 has been purified from avocado with a subunit mass of 37 kDa the native enzyme being homo- dimeric. The enzyme is cerulenin insensitive and thiolacto- mycin 2 sensitive. The KAS I11 enzyme was separated from acetyl CoA ACP transacylase activity providing evidence for the first time that these two discrete activities exist in higher plants.16' The NADH specific ER from Brassica napus has been cry~tal1ised.l~~ A novel cDNA sequence encoding an TE from Cuphea lanceolata is described.I7' The biosynthesis of very long chain fatty acids in leek seedlings by acyl CoA elongase has been re~0rted.I~~ The soluble enzyme octadecanoyl (stearoyl) ACP 92 desaturase converts octadecanoyl ACP to (92)-octadecenoyl (oleoyl) ACP in the presence of oxygen NAD(P)H NAD(P)H- ferredoxin oxidoreductase and ferredoxin and is usually a 70 kDa homodimer.Sequence predictions for plant 92 desatu- rase enzymes show a very high degree of homology with each other but very little homology with the corresponding animal enzymes. The octadecanoyl ACP 92 desaturase from castor (Ricinus communis) has recently been expressed in E.coli and was shown to contain four iron atoms per homodirner.l7* Optical and Mossbauer spectroscopy was used to show that these iron atoms reside in a diiron-oxo cluster similar to those in hemerythrin ribonucleotide reductase ruberythrin purple acid phosphatase and methane monooxygenase hydroxylase and it is proposed that oxidative desaturation also involves a reactive high valent iron-oxo intermediate. Wada et al. has isolated thylakoid membranes from the photosynthetic prokarotic algae the cyanobacteria capable of desaturating the acyl groups in monogalactosyl diacylglycerol. The system requires the reduced form of ferredoxin.'73 (62)-Octadec-6-enoic (petroselinic) acid 58 is the 62 isomer of (92)-octadec-9-enoic (oleic) acid 53 and has a mp of 33 "C -GCOOH 58 -high levels and starving the cells of longer chain substrates for ;COOH membrane assembly.6o A clone has been isolated from Arabidopsis leaf cDNA library that encodes an 88 amino acid mature ACP with a 35 amino acid presequence that may target the ACP transport not into the chloroplast but into the pea mitochondria which were then able to both process the protein and to acylate it.16' Malonyl CoA ACP transacylase has been partially purified from seeds of Cuphea lanceolata 62 a commercial source of decanoic acid. After the initial discovery of plant KAS 111 by Jaworski et a1.'63 and Walsh et a1.,'64 KAS 111has now been located in a wide variety of plants. The gene coding for the 3-oxoacyl ACP synthase I11 from spinach (Spinacia oleracea) has been examined.The deduced amino acid sequence whilst homolo- gous to E. coli KAS 111 showed little homology with the other KAS I and I1 isoforms but interestingly showed homology to the plant chalcone synthases. 16' A second paper examined the relative contribution of acetyl CoA and acetyl ACP to lipid biosynthesis. Using mathematical models they found that KAS I11 activity is 40 times greater than KAS I activity and is the major conduit for lipid biosynthesis acetyl ACP being a less effective primer than acetyl CoA butanoyl ACP or hexanoyl ACP. They also observe that acetyl ACP levels accumulate in the dark and it becomes the major form of acyl ACP. After a few minutes in the light the levels of acetyl Rawlings Biosynthesis of fatty acids and related metabolites 59 20 degrees higher and thus may have potential as an ingredi- ent of unsaturated vegetable oils that are a solid at room temperature.The 36 kDa desaturase that forms petroselinic acid (1 8:1 62) in coriander has recently been over-expressed and found to be structurally related to the octadecanoyl ACP 92 desaturase. 174 Radioactive (1 6:O) or (1 8:O) free acids were incorporated into phospholipids but were not incorporated into petroselinic acid 58 suggesting that petroselinic acid is not formed by desaturation of a fatty acid bound to a glycero- lipid or by reactions involving CoA but was consistent with formation via an acyl ACP intermediate (Scheme 12).'75 A gene (fad7) that may encode a chloroplast a -3 desatu- rase of lipid linked (1 8:2) and (1 6:2) fatty acids in Arabidopsis thaliana has been 10cated.l~~~ Browse reports that a single nuclear mutation in the fad3 gene of A.thaliana resulted in depressed levels of (18:3) (linolenate) and elevated levels of (18:2) (linoleate) fatty acids in the endoplasmic reticu- lum suggesting that these mutants are deficient in (18:2) endoplasmic reticulum (eukaryotic pathway) desaturase activity. ' 766 Chloroplast lipids (prokaryotic pathway of desaturation) were largely unaffected by the mutation. A 62 The mechanism of the reaction of fatty acid hydroperoxides 0 with soybean peroxygenase a membrane bound ferrihemo- MecSACP protein has been investigated by Blee et al.The solubilised 42 Desaturase enzyme converted (92,l I E 13S 15.27 13-hydroperoxyoctadeca- SACP 9,11,15-trienoic acid (1 3-HPOT) 60 to the corresponding Me hydroxy compound (9Z 1lE 13x1 53-1 3-hydroxyoctadeca- C2-elongation 0 9,11,15-trienoic acid (1 3-HOT) 61 and a single epoxide regio- Petroselinoyl-ACP hydrolase isomer (92,ll E,139-1 5,16-cis-epoxy- 13-hydroxyoctadeca-9,- 11-dienoic acid 62,both of which are the expected products Me COOH from heterolytic cleavage of the 0-0 bond of 13-HPOT 60. No 58 products that would be expected from homolytic cleavage were Scheme 12 observed. ' desaturase from Brassica napus (rapeseed) has been function- ally overexpressed in a cyanobacterium.'77 A petunia has been COOH transformed with a yeast 9Z desaturase using Agrobacteriurn turnefaciens resulting in increased levels of monounsaturated 'OH 60 fatty acids.17' Recombinant octadecanoyl ACP 92 desaturase was found to desaturate (18:O) with V,,,IK of 100-fold larger COOH than for (16:0) but C-9 regiospecificity was maintained (16:O) OH 61 being converted to (16:1 92).'79 The seed oil of Thunbevgia data (Safflower) contains over 80% of the unusual fatty acid (6a-hexadec-6-enoic acid 59. A ferredoxin and dioxygen dependent soluble 62 desaturase was located whose preferred substrate was (1 6:O)-ACP and that could be functionally expressed in E. coli.'*' A recent paper by Wagner and co-workers' shows that C ,-C medium straight or branched chain acyl acids of The biosynthesis of dimethylated furan fatty acids 63 from a tomato C& straight chain of Petunia and C,-and C,-Saccharuin sp.(sugar cane) were discussed in the previous branched acyl acids of Nicotiana glutinosa arise from acetyl review. A new reportls2 demonstrates that labelled acetate is CoA elongation of amino acid derived 2-oxoacids without the only incorporated into the original fatty acid not into the involvement of FAS mediated reactions suggesting a much methyl substituents which were previously demonstrated to greater integration of amino acid and fatty acid metabolism originate from methionine. than previously supposed at least in the Solunaceae. The In plants over 600 different polyacetylenes have been char- resulting medium chain 2-oxoacid is converted to the acid by acterised many in the Asteraceae.The Asteraceae produce oxidative decarboxylation via a 2-oxoacid dehydrogenase. polyacetylenes in their roots in response to elicitor treatment Straight chain elongation by acetyl CoA can be achieved by from crude mycelial extracts of Pythiurn aphaiderrnaturn. A the '3-isopropylmalate synthase' reaction. The formation of a possible route to biosynthesise polyacetylenes involves lipooxy- branched is0 or anteiso product can be obtained by the use of genases however McKinley et al. has shown that addi-hydroxyethyl thiamine pyrophosphate as cosubstrate in the tion of lipoxygenase inhibitors does not affect the production 'acetolactate synthase' reaction (Scheme 13). of polyacetylenes. Alanine Threonine J i 0 i HO COOH MeACOOH Me LCOOH LMe/$,-coOH 0 OH 1 i-iii iv -ButanoylCoA MeqCOOH -Valine MeacooH Me iv\ i-iii i-iii 2-Methylpropanoyl CoA t iv 2-Oxohexanoic acid -Pentanoyl CoA I -Leucine I I 2-0x0-4-methylpentanoic acid i-iii i-iii i d 3-Methylbutanoyl CoA t IV I 2-Oxoheptanoic acid -Hexanoyl CoA 2-0x0-5-methylhexanoic acid i-iii i-iii i\ 4-Methylpentanoyl CoA I J 1 1 1 etc.etc. Scheme 13 a-Ketoacid pathway; i isopropylmalate synthase; ii isopropylmalate dehydratase; iii 3-isopropylmalate dehydrogenase; iv 2-oxoacid dehydrogenase; v acetolactate synthase 348 Natural Product Reports 1997 MeWMe Me(CH,l,,,A>L(CH2)nCOOH rn = 2 or 4; n = 8 or 10. 63 A A Me Me Me 7 Animals 7.1 Invertebrates Insect FAS have been purified and characterised from several species and are typical animal FAS homodimers of approx.500 kDa with the expected sequence kinetics cofactor and substrate specificity. '84 -'86 Insects produce a large number of unusual fatty acid derived compounds as pheromones etc. the biosynthesis of many has been covered in earlier reviews.'-2 Recent developments include the following. The main sex pheromone of the Egyptian army worm (Spodoptera littoralis) (9Z,1 lE)-tetradeca-9,1l-dien-l-ylacetate 64 is biosynthesised from hexadecanoic acid by chain shortening followed by 11E and 9Z desaturation reduction and acetylation. 187 A similar process gives (9a-tetradec-9-en- 1-yl acetatk 65 (Scheme 14). Gosalbo et al.have found that C-16 fatty acids with a cyclopropenyl group at C-10 -1 1 or -12 (such as 66) inhibited both the 112 desaturase of hexadecanoic acid and the 92 desaturase of (1 1E)-tetradec-1 1-enoic acid 67.'" Fabrias and co-workers have recently found that the C-14 fatty acid analogue 10,ll -methanotetradec- 10-enoic acid 68 inhib-itedlg9 the 9Z desaturase of (1 1E)-tetradec-1 1-enoic acid 67 but not the 1 1Zdesaturase of hexadecanoic acid. This suggests that the C-16 cyclopropenyl compound 66 was being P-oxidised to the corresponding C- 14 cyclopropenyl fatty acids such as 68 which would be the actual inhibitor of the 9Z desaturase (Scheme 15). This would be the first experimental demonstration of P-oxidation in the pheromone glands. A series of deuteriated and tritiated cyclopropene C, and C, fatty acids has recently been synthesised for affinity labelling of insect pheromone desaturases.190 A wide range of ;COOH (16:O) NCOOH (14:O) JI 11E Desaturase jCOOH 67 9Z Desaturase /-\COOH (14:2 9Z,11E) 1 OAc 1 64 COOH 66 I P-Oxidation 68 Scheme 15 specifically deuteriated mono- and di-unsaturated fatty acid alcohols (and acetates) has been synthesised for investigation into the biosynthesis of pheromones by Lepidoptera. 19' Saturated and unsaturated 2-ketones occur in the ejacu- latory bulbs of mature males of the mulleri subgroup of the Drosophila. The major aggregation pheromone of (the appro- priately named?) Drosophila buzzatii is ( 102)-heptadec-1O-en-2-one 69 with large quantities occurring in the ejaculatory bulb (690 ng per male) with smaller quantities of a second aggre- gation pheromone tridecan-2-one (230 ng per male) 70.'92A study by Skiba and Jackson using radiolabelled acetate has shown that aggregation pheromone biosynthesis is limited to tissues from the ejaculatory bulb and only in mature male flies greater than four days old.The microsomal fraction demonstrated the greatest biosynthetic activity. '93 In a recent study 94 microsomal fractions were incubated with [2H,,]dodecanoyl CoA and the products (acids as methyl esters) analysed by GCMS (SIM). Label was incorporated into tetradecanoate (14:O) and tridecan-2-one 70 but no label was observed in esterified unsaturated fatty acids or (102)-heptadec- 10-en-2-one 69 (Scheme 16).Microsomal fractions incubated with [2H27]tetradecanoyl CoA (myristoyl CoA) incorporated no deuterium into either tridecan-2-one 70 (102)-heptadec- 10-en-2-one 69 or unsaturated fatty acids though 7%) of the labelled tetradecanoate was elongated to labelled hexadecanoate. However 36% of label from (92)-[ 1,2-] 3C2] hexadec-9-enoyl (palmitoleoyl) CoA (1 6 1,9Z-CoA) 71 was incorporated into (1 OZ)-heptadec-lO-en-2-one69 though no label was incorporated from labelled (1 1 2)-octadec-1 1-enoyl CoA (cis-vaccenoyl CoA) 72. Avidin is an inhibitor of acetyl CoA carboxylase. Avidin treated microsomes were unable to convert acetyl CoA into either pheromone suggesting that the /COOH - (16:1 112) P-Oxidationi 4 C- O O H (14:1 92) Scheme 14 Biosynthesis of Egyptian army worm sex pheromones Rawlings Biosynthesis of fatty acids and related metabolites HexadecanoylCoA (Palmitoyl CoA) -COSCoA cCOSCoA COSCoA Tetradecanoyl CoA 3-OxotetradecanoylCoA (Myristoyl CoA) COSCoA 71 -COSCoA 72 Scheme 16 Biosynthesis of fly aggregation pheromones chain assembly is malonyl CoA dependent.Avidin treated microsomes were also unable to convert deuteriated dodecanoyl CoA (with acetyl CoA) into tridecan-2-one 70 or I3C-labelled hexadecanoyl CoA (with acetyl CoA) into (102)- heptadec-10-en-2-one 69 but were able to convert either upon addition of malonyl CoA to either system. All these obser- vations suggest that the pheromones are formed via malonate dependent elongation pathways rather than by degradation of longer chain precursors thus the C elongation enzymes are present in the ejaculatory bulbs.Male sap beetles produce a series of triene and tetraene hydrocarbon aggregation pheromones that attract beetles of both sexes such as (2E,4E,6E)-5-ethyl-3-methylnona-2,4,6-triene 73 (Carpophilus freemani) (2E,4E,6E,8E)-3,5-dimethyl-7-ethylundeca-2,4,6,8-tetraene74 (C. fueemani), (3E, 5E,7E)-6-ethyl-4-me thyldeca-3,5,7- triene 75 (C. freemani) (2E,4E,6E,8E)-3,5,7-trimethyldeca-2,4,6,8-tetraene 76 (C. freemani male only) and (2E,4E 6E 8E)-4,6,8- trime thyl- undeca-2,4,6,8-tetraene 77 (C. hemipterus male only).‘95 These hydrocarbons have conjugated polyene systems with alkyl TT 73 74 75 76 77 branches on alternate carbons.A recent study investigates their biosynthesis in Carpophilus freemani (Coleoptera Nitidulidae). This beetle produces a large amount of (2E,4E,6E)-5-ethyl-3-methylnona-2,4,6-triene 73. When fed on a diet containing organic acids (5% by wt) labelled with 2H or I3C enough labelled pheromone was emitted not only for examination by MS but also by NMR spectroscopy. The pheromone was collected by absorption onto porous polymer 350 Natural Product Reports 1997 which was then rinsed with hexane every two or three days. The experiments showed that the hydrocarbon was assembled from one acetate one propanoate and two butanoates with a decarboxylation. Two possible pathways are proposed.A putative triketide intermediate to chain extend a butanoate (or 2 x acetate) starter unit or more likely the decarboxylation of a ‘APBB’ tetraketide at the 3-0x0 stage (Scheme 17).’96 Two other beetle aggregation pheromones with related carbon skeletons are stegobinone 78 (Stegobium paniceum) and (2,3-czs)-serricorone 79 (Lasioderma serricorne) (both are Ano biidae) . 00 -CO2 WscoA- V) Scheme 17 0 0 78 79 Many insects use long chain (C2s-C3s)hydrocarbons in their cuticular lipids to help prevent dessication. In some species such as the housefly (Musca domestica Diptera:Muscidae) these compounds are also used in communication. Many of these hydrocarbons possess a single methyl branch. Recent studies confirm that propanoate via methylmalonyl CoA is inserted into the FAS at the appropriate point in the long chain assembly of the monornethylhexadecanoic or mono-methyloctadecanoic acids in both microsomal or soluble extracts.These acids would then be elongated by a fatty acyl COOH 13 Scheme 18 19'hydrocarbon. elongase system and then reductively decarbonylated to the The main female sex pheromone of the processionary moth (Thaumetopoea pityocampa) is (1 3Z)-hexadec- 13-en- 1 1 -ynyl acetate 80. Previous labelling experiments suggested that (1 12)-hexadec- 1l-enoic acid 81 was an intermediate formed from hexadecanoate by an 112 desat~rase.'~~ This acid could then either be converted into hexadec-1 1-ynoic acid 82 or into (1 12,13Z)-hexadeca-1 1,13-dienoic acid 83 before transforma- tion to (13Z)-hexadec-13-en-l l-ynoic acid 84 the presumed immediate precursor of the pheromone 80.In a recent study both potential intermediates were synthesised labelled with deuterium on their terminal carbon atoms. Incubation with pheromone glands revealed that hexadec-1 l-ynoic acid 82 was the immediate precursor of the enyne fatty acid 84 (Scheme 18).200 A recent in vitro study has confirmed that the regulatory effect of the pheromone biosynthesis activating neuropeptide (PBAN) is to regulate the final step in the biosynthesis of the sex pheromone of Bombyx mori i.e. the reduction of an acyl group to give (1OE,122)-hexadeca- 10,12-dien- 1-01.~'~ Studies on sex pheromone biosynthesis in the moth Mamestra hrassicae (Lepid0ptera:Noctuidae) suggest that PBAN follows a humoral route to its site of action rather than a neural route after being released from the brain.'7 202 The use of labelled precursors suggested that PBAN regulated an early step in the pathway contrary to earlier reports that it stimulated the All desa turase. Two reviews involving the biosynthesis of fatty acids in marine invertebrates has appeared marine invertebrate oxylipins2"' and the biosynthesis of secondary metabolites in marine molluscs. Other reviews included in the introduction to Section 4 include relevant material. 7.2 Vertebrates Animals usually produce free hexadecanoic acid except in mammary glands (shorter chain) or water fowl uropygial (preening sebaceous) glands which produce a specialised water repellant short chain polymethylated fatty acid.The ER and KR are both NADPH dependent and chain length specificity Rawlings Biosynthesis of fatty acids and related metabolites may be determined by the TE which has little affinity for C14 chain length and the KAS which has a slower rate for C14 and c16 chain length. There is no 'KAS III' i.e. the FAS takes C through to CI6 but there is some non-specifity propanoate or even phenylacetate can replace acetate as primer and under forcing conditions animal FAS will use methylmalonyl CoA instead of malonyl CoA as chain extender. Animals do not use the anaerobic pathway to obtain unsaturation but rely upon oxygen and NADH dependent desaturases which have been reviewed.204 In animals these desaturases can introduce double bonds at the A4 A5,A* and A9 positions but never beyond A' with the 2 configuration.In combination with C extension systems (elongases) that occur bound to the cytosolic side of the endoplasmic reticulum membranes (16:O) can be converted first to (161 92) 59 and then to (18:1 112) (cis-vaccenoic acid) and (18:O) to (20:3 52,82,112). However the inability of animal systems to desaturate closer to the methyl terminus than C-9 renders them unable to convert (16:O) into (18:2 92,122) (linoleic acid) 50 or (18:3 92,122,152) (a-linolenic acid) 51 thus both these polyunsatu- rated fatty acids are essential in the animal diet -why we should 'eat up our greens'.For instance (1 8:2 92,122) 50 can be extended by mammalian desaturases and elongases to (20:4 52,82,112,142) (arachidonic acid) 41 the precursor to the prostaglandins thromboxanes leukatrienes and prostacyclins. The animal FAS system is a homodimer of two polypeptide chains (2 x 272 kDa) each possessing all the catalytic activities (and the ACP domain) required for fatty acid assembly including TE activity.205 206 Several systems have now been sequenced and cloned including the FAS from chicken (Leghorn rooster) liver,207-209 goose2" and rat.211-213 A cDNA encoding the 2505-residue rat FAS has been expressed as a catalytically active protein in Spodoptera frugiperda cells using a baculo~irus.~~~ The recombinant FAS constituted 20% of the soluble cytoplasm protein was readily separated from the insect host's FAS and was indistinguishable from FAS isolated from rat liver.This suggested that the insect host cells had phosphopantetheinylated the rat FAS ACP's serines. This is in contrast to previous attempts to overexpress mammalian FAS ACPs in E. coli which only resulted in non-phosphopantetheinylated ACP. Mild proteolytic cleavage 35 1 NH? r NH2 15 nm ~ * GXGXXG NADH binding pocket Fig. 2 Model of animal FAS separates the individual multifunctional protein subunit into three domains; domain I containing KAS AT and DH activities; domain 11 containing ER KR and ACP activities; and domain 111 containing just the thioesterase activity.206,21 The active enzyme is considered a head-to-tail homodimer in which the KAS thiol from one subunit interacts with the DH ER KR and ACP activities from the second subunit and vice versa (Fig.2). (Recent unpublished work by Smith and co-workers using domain complementation studies in hetero- dimers suggest a possible revision of the accepted model. Whilst the DH may be adjacent to the AT on the polypeptide sequence the long interunit linker arms allows the spatial placement of the DH in the opposite active site region to the AT so that the KAS and AT from one subunit still cooperate with the DH ER KR ACP and TE activities of the second subunit. This may require a revision of the traditionally accepted two dimensional ‘flat’ model of the head-to-tail FAS homodimer possibly involving helical coiling of the two subunits.) When 1,3-dibromopropanone is added to the syn- thase the two subunits are now covalently linked via their spatially close (0.5 nm) ACP thiol and KAS cysteine thiol groups by the difunctional reagent to give a protein of doubled molecular mass (ca.550 kDa). Activities were orig- inally located by sequence homology more recently proteolytic mapping has allowed functional investigation of fragments. There is thought to be a catalytically inactive central ‘core region’ of the sequence that shows a strong tendency to dimerise and may be largely responsible for holding the two subunits together.206 Whilst no X-ray structures are available the overall topology has been examined by physical studies such as electron microscopy and small angle neutron-scattering studies which suggest an overall shape of two elongated subunits each composed of three major regions arranged head-to-tail with a central bulge.Subunit contact was closest in the central region with two 4nm diameter ‘holes’ near either end and overall dimensions 22 x 15 x 7 nm.215 Wakil and co-workers have investigated the overall topology of the chicken FAS and discuss possible topological arrange- ments consistent with the small angle neutron scattering and electron microscopic results.216 A model of animal FAS is shown in Fig. 2*15and a linear domain map of rat FAS in Fig. 3; in Fig. 2 the putative active site residues and NAD(P)H binding sites are highlighted. The core region and linker sequences have lower sequence homology with other mammals than the indicated domain sequences.Petithory and Smith2’ immobilised rat liver FAS (mono- meric or dimeric) onto a Sepharose matrix containing antibodies that recognise the thioesterase domain. Earlier investigations had suggested that the KR activity was lost or greatly reduced in the monomeric form.218 However Petithory and Smith found that the KR in both the dimer or monomer form were equally active in utilising 3-oxobutanoyl NAC thioester as a substrate but quite different in reducing 3-oxobutanoyl CoA. The kinetics suggested a single pathway for the monomer but two pathways for the dimer. This may result from the ability of this substrate to be presented to the enzyme both as a model substrate and as a covalently linked enzyme intermediate.This suggests that the transfer of sub- strate from the active site serine of the transferase domain to the 4‘-phosphopantetheine of the ACP is an inter-unit event and that it is this activity that is lost upon dissociation into monomers. The dehydrase domain has proved very difficult to map. Joshi and Smith have expressed a mutant (H878A) rat fatty acid synthase that retained all partial activities except dehy- drase forming 3-hydroxybutanoyl moieties from acetyl CoA malonyl CoA and NADPH.215 This confirms the location of the DH immediately adjacent to the AT domain not next to ER as has been previously ~uggested.~” This is also in agreement with the assignment of the single DH domain in the Saccharopolyspora erythraea erythromycin synthase gene.22o3 221 Joshi and Smith then propose a revised model of the ‘swinging arm’ hypothesis as the distances from the ACP to several active sites are up to 5 nm much greater than the length of the phosphopantetheine swinging arm (2 nm).They suggest that whilst the two subunits may be held together and stabilised by the central core regions the flexible inter-domain linkers allow ‘dynamic interactions’ between catalytic domains. Fkl AT DH ‘CORE‘ ER M KR ,&q :(proteolysis) (proteolysis) 1-406 KAS active residue Cys-161 428-815 AT active residue Ser-561 829-969 DH active residue His-878 1630-1850 ER GXGXXG NAD(P)H binding site at residues 1670-1675 1870-2100 KR GXGXXG NAD(P)H binding site at residues 1886-1891 21 14-2190 ACP active residue Ser-2151 2200-2505 TE active residue Ser-2302 Fig.3 Linear domain map of rat FAS 352 Natural Product Reports 1997 Rat mammary gland TE 11 that normally catalyses the chain termination and release of medium chain fatty acids has been expressed. TE I1 is not part of the FAS homodimer but interacts with FAS to hydrolyse fatty acyl thioesters as C, C, or C, acids before they are fully chain extended to C16. Site specific mutations suggested the presence of the same catalytic triad (Ser/His/Asp) as in FAS thioesterase (TE I). A hydrophobic C-terminus region appears to play an important role in stabilising the interaction with FAS. Removal of the terminal Leu residue obliterated TE I1 activity with FAS but did not affect reactivity with model substrates.222 FAS from the mammary glands of rat,223p225 rabbit,226 guinea pig227 and cow228.229 have been purified with expected RMM of 400-600 kDa except from the rabbit with a reported RMM of 910 kDa. The mammary synthases show a reduced K and increased V,, when butanoyl CoA is used as a primer rather than acetyl COA.,~' Butanoyl CoA may be synthesised from acetyl CoA by a series of discrete enzymes in the soluble fraction that are the reversal of the corre-sponding P-oxidation and then used as primer by mammary synthesis. Buckner and Kolattukudy have purified a dimeric FAS from the uropygial gland of goose (RMM 547 kDa).231 When supplied with acetyl CoA NADH and malonyl CoA or methylmalonyl CoA the major products were free hexa- decanoic acid and free 2,4,6,8-tetramethyldecanoicacid respectively.The authors conclude that there is little differ- ence in specificity between uropygial FAS and normal FAS and that the production of methyl branched fatty acids is dependent on the relative concentrations of malonyl and methylmalonyl CoA precursors. The thioesterase B from duck uropygial gland has been cloned and sequenced and shows little homology to the other two duck thioestera~es.~~ Expression of the gene is stimulated by estradiol treatment or under conditions of high peroxisome proliferation suggesting that TE B may play a role in the production of 3-hydroxy fatty acid duck pheromones.Desaturases achieve a chemically improbable regiospecific insertion of a double bond into an otherwise unactivated hydrocarbon chain. Microsomal fractions of rat liver able to convert octadecanoyl CoA into (9Z)-octadec-9-enoyl CoA were not inhibited by cyanide but were inhibited by carbon monoxide leading Oshino et ul. to suggest the involvement of the microsomal electron transport chain and cytochrome b,.233 Three components would be involved the desaturase the NADH-cytochrome b reductase and cytochrome b,. Octadecanoyl CoA 9Z desaturase was first purified (RMM 53 kDa) by Strittmater et ul. from rat liver and contained non-haem iron.234 James showed that it desaturated over 20 fatty acids at the C-9 suggesting that the 9Z desaturase contains a hydrophobic cleft of finite length holding the acyl chain such that C-9 and C-10 are in an eclipsed (gauche) conformation.All three components are thought to be in the endoplasmic reticulum membrane with all the required protein components exposed to the cyto- plasmic face. Membrane desaturases from mammals fungi insects higher plants and cyanobacteria all contain three conserved regions of amino acids containing eight histidines motifs that are also present in bacterial membrane bound enzymes alkane hydroxylase and xylene monooxygenase pre- sumably arising from a common ancestry. The rat 92 desatu- rase has been expressed in a yeast mutant deficient in its own 9Z desaturase. Site specific mutagenases were used to convert each His to Ala in turn but all eight mutations failed to complement the yeast mutant.Presumably all eight histidines are essential residues as ligands in a new type of diiron centre.236 The biosynthesis of prostaglandins and related compounds have been extensively covered in previous reviews.' Throm-boxane A and prostacyclin are formed by the enzymatic rearrangement of 9,ll -epidioxyprostaglandin H by throm- boxane and prostacyclin synthases respectively both of Rawlings Biosynthesis of fatty acids and related metabolites which are haemthiolate P-450 enzymes. The mechanistic aspects of these reactions has been recently reviewed by Ullrich and Br~gger.~~~ 8 Miscellaneous Autoregulatory factors and communication in the actinomyc- etes has recently been reviewed.238 A series of butanolactones can induce cytodifferentiation in many Streptomyces 85a-j.OXOH 6 85 R The biosynthesis of the butanolactone autoregulatory factor from Streptomyces virginiue Virginiae Butanolide A (VB A) 85a that induces cytodifferentiation including polyketide pro- duction has been reported by Yamada and co-w~rkers.~~~~~~' The carbon framework is assembled from a 3-methylbutanoate (isovalerate) unit two acetates and a three carbon unit derived from glycerol [Scheme 19(a)]. A probable mechanism of ring formation involves attack by the free hydroxy group of 1,3-dihydroxyacetone phosphate 86 onto a thioester of a [1,3-l3C2]Glycerol (4 0 c) ci *OH Cell-free extract YOH (4 0 -0 QJ-f m 0 0 *H 'OH 85a 2 x NADH* labile H t I 88 t Scheme 19 353 7-methyl-3-oxooctanoate 87 to give 88.Attack of a carbanion onto a C-2 carbonyl group of the dihydroxyacetone portion would give the butanolactone ring [Scheme 19(b)]. This has been investigated by the synthesis and incorporation of 3-hydroxy-2-oxopropanoyl 7-methyl-3-oxoheptanoate 88 into VB A by cell-free extracts of S. antibioticus demonstrating that it is a probable biosynthetic intermediate as earlier proposed. Two research groups have probed the origin of the car- bon atoms in biotin 89 with I3C-labelled precursors and rather confusingly used different numbering systems to label biotin. The origin of the carbon atoms of pimeloyl CoA (heptane- 1,7-dioic acid mono-CoA thioester) 90 the earliest known precursor in the biosynthesis of biotin has been investigated by Kajiwara and co-workers in a repressor mutant of E.coli carrying plasmids with a biotin operon that produces biotin and dethiabiotin 91 at high enough levels for NMR Labelled acetate was efficiently incorporated into C-1 to C-7 carbons but not into C-8 or C-9 C-1 being labelled by both acetate carbons and hydrogen carbonate (Scheme 20). 0 HO Pirnelic acid uOH-3ct 6OOH -ADO 89 A = Hydrogen carbonate or formate ADO -HOOCW COSCoA Hydrogen carbonate (A) i \ Citric acid cycle (mo) Scheme 20 [3-"C]Alanine was not only incorporated into C-9 but into alternate carbons C-2 C-4 and C-6. These results suggest that alanine can act as an alternative carbon source by efficient conversion into acetyl CoA (Scheme 21).Two possible path- 0 COOH - 0xCOSCoA O'COSCo A Citric acid cycle \. /* co2 Scheme 21 ways for the conversion of acetyl CoA into pimeloyl CoA are suggested. The first involves carboxylation of hexanoate by a hexanoyl (caproyl) CoA carboxylase. However free pimelic acid (heptane-l,7-dioic acid) is not incorporated into biotin. The second involves a malonyl CoA starter unit being extended by two malonates. As the carboxyl carbon of biotin is labelled by hydrogen carbonate and both acetate methyl and carbonyl carbons the source of this malonyl starter unit must be either hydrogen carbonate or the citric acid cycle. It is known from classical radiolabelling studies that the carboxylate carbon of malonyl CoA is at least partially derived from carbon dioxide/hydrogencarbonate however this raises the question of whether an alternate source is the citric acid cycle.Flint and co-workers have also added labelled acetate to the overproducing strain of E. ~oli.~~' Using the above numbering system showed that C-2 to C-7 was efficiently labelled by acetate but they could not detect incorporation at the carboxyl carbon C-1 perhaps due to much lower signal-to-noise ratio in the corresponding I3C NMR spectra than in the paper by Kajiwara and co-workers. Flint and co-workers also suggested the use of malonate as a starter unit but could not observe any malonate incorporation.Baxter et al. have investigated the mechanism of ureido ring formation by biotin synthetase by trapping the 8-aminocarbamate of (7R,8S)-diaminononanoate as its methyl ester 92 suggesting that the 8-amino group is converted into the intermediate carbamate 93 rather than the 7-amino derivative 94 prior to ureido ring formation to give dethio- biotin 91 (Scheme The final step in biotin biosynthesis the chemically intriguing insertion of the sulfur atom by biotin synthase has received recent attention. The most common hypothesis is the direct insertion of a sulfur into a C-H bond with a cyclisation analogous to that in penicillin biosynthesis but experimental evidence has been somewhat lacking. Biotin synthase from E. coli has now been overexpressed and crystallised in a form suitable for X-ray determinati~n.~~~ The gene for biotin synthase from yeast has been cloned and sequenced and functionally expressed in deficient E.coli strains.246 Marquet et ul. synthesised the possible thiol precursors 95 and 96 and fed them to E. coli auxotrophs or a strain of Bacillus sphuericus that efficiently converts dethio- biotin into biotin. Whilst the results were complex they suggested that the primary thiol 96 might be the main biosynthetic intermediate rather than 95 which was shown to lose its sulfur atom upon transformation to biotin (Scheme 22).247 Dehydrogenase enzymes specifically transfer one of the two prochiral hydrogens from NAD(P)H to the substrate. A-specific enzymes transfer the pro-R and B-specific enzymes the pro-S hydrogen.248 Benner and co-workers observed a correlation between the stereospecifity of the reaction and the equilibrium constant for the reaction.249 Almarsson and Bruice have used theoretical calculations to examine the factors affecting reactivity and stereospecifici ty .250 A model FAS/PKS system to mimic the biosynthesis of fatty acids and polyketides is being developed by Sun and Harrison.25'.252 The use of tetramethylglycoluril 97 as a template has allowed the repetitive Claisen-like conden-sation of acyl units to be achieved in a controlled manner with lithium tert-butoxide whilst the growing acyl chain remains attached to the template.The intermediate 3-oxoacyl chain 98 can be reduced to 3-hydroxyacyl chain 99 with NaBH,.Trifluoroacetic anhydride causes dehy-dration and L-Selectride the 'enoyl reduction' resulting in a saturated acyl chain 100 ready for another round of chain elongation. Chain extension of enoyl chains has also been achieved to give partly reduced chains 101 and 102 mimicking partly reduced polyketide biosynthesis (Scheme 354 Natural Product Reports I997 Me0 90 2=0 u H " 'WCOOHf 92 fet hiobiot in s y n t k Trapped at low temperature Lth diazomethane -0 0y0-H-\H Hgfi OR ;&H AH Md 'WCOOH Md s-CooH 94 0 /NH'NHg1 Biotin synthase NH'NH Biotin synthase -H++H :wH 9M4 '6 W C O O HQHmCOOH H -91 89 Biotin synthase tBiotin synthase (loss of sulfur) (retention of sulfur) L 96 Scheme 22 f Me Me I Me Me Me ,Me ii,iii o+N~N+o 1:;.N;N 100 OI Me 'H 101 102 Scheme 23 Reagents i Ac,O reflux; ii LDA AcC1; iii Bu'OLi; iv NaBH, MeOH; v TFAA Et,N; vi L-Selectride 9 References 7 A. B. Kroumova Z. Xie and G. J. Wagner Proc. Nutl. Acad. Sci. 1 D. O'Hagan Nat. Prod. Rep. 1995 12 1; 1993 10 593; 1992 9 USA 1994 91 11 437. 447. 8 J. B. Ohlrogge Plant Physiol. 1994 104 821. 2 T. J. Simpson Nat. Prod. Rep. 1991,8 573; 1987 4 339; 1985,2 9 T. D. Brock M. T. Madigan J. M. 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ISSN:0265-0568    

DOI:10.1039/NP9971400335

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

The biosynthesis of plant alkaloids and nitrogenous microbial metabolites Richard B. Herbert School of' Chemistr-v University of Leeds Lee& UK LS2 9JT Covering 1995 Previous review 1996 13 45 1 Pyrrolidine and piperidine alkaloids 1.1 Tropane alkaloids 1.2 Anatoxins 1.3 Pinidine 1.4 Pyrrolizidine alkaloids 2 Benzylisoquinoline alkaloids 2. I General 2.2 Morphinan alkaloids 2.3 Protoberberine alkaloids 2.4 Colchicine 3 Tryptophan metabolites 3.1 Ergot alkaloids violacein and penitrems 3.2 Terpenoid indole alkaloids 3.3 Indolic phytoalexins 4 Other metabolites of the shikimate pathway 4.1 Acridone alkaloids and phenazines 4.2 Cyanogenic glycosides 4.3 Aphelandrine 5 P-Lactams 5.1 Clavulanic acid 5.2 Penicillins and cephalosporins 6 Nucleoside and aminoglycoside antibiotics 7 Miscellaneous metabolites 7.I Kinamycins 7.2 Berninamycins and pristinamycins (virginiamycins) 7.3 Lactacystin armentomycin and discorhabdins 8 References As always I aim to cover the literature under the above title comprehensively but I may miss some relevant work.I would be pleased to know of such omissions so that I can cover the material in next year's review. As before access to the litera- ture was obtained in substantial measure through the IS1 Data Service at Bath. Reference is made as usual to useful background material. ' -7 Alkaloids their biosynthesis including enzymology and exploitation have been the subject of a most interesting broad review -a good read and an excellent case for further work with these nitrogenous plant bases.8 1 Pyrrolidine and piperidine alkaloids 1.1 Tropane alkaloids Of recent times the deduced pathway to the tropane alkaloids e.g.hyoscyamine 3 has been revised to include 1 as an important new intermediate which is also pivotal to the biosynthesis of cocaine 5 and cuscohygrine 6 (ref. 5 p. 445). Evidence relating to hyoscyamine 3 and scopolamine 4 pre-viously available in a thesis (ref. 5 p. 445) has now been published in journal format.' The ester 2 which was fed as a racemate is an excellent precursor for scopolamine 4 in Datura innoxia; by contrast but in agreement with other results 13C-labelled hygrine 7 was a poor precursor for tropane alkaloids in this and other plants.Curiously it seems as if both the (R)-and the (9-isomer of 2 is used in the biosynthesis of 4 A> 0 -Ox ,,(,'OH Ph H 1 X=SCoA 3 4 2 X=OEt * = 13c NMe 0 5 (as hbelling of both C-2 and C-4 was observed with the latter slightly more enriched as well as a common label at C-3). This will hopefully be examined further. The tropic acid moiety 12 in e.g. hyoscyamine 3 originates from phenylalanine by way of phenyllactic acid 9 and littorine 11 acts as an intact precursor for hyoscyamine 3 (ref. 6 p. 45); the conversion of the phenyllactyl moiety of 11 into that of 12 clearly involves a most interesting mutase reaction.The results of two sets of experiments define (R)-D-phenyllactate 9 as the immediate mutase substrate with the (9-isomer being converted into it via phenylpyruvate 8 (Scheme I). Firstly the 8 9 X=H 10 X='H AH C02R AHR ,As Y,-H' Mutase Ph% -Ph*CO'R H* OH HO H' 12 R = Tropinyl 11 Littorine R = Tropinyl Scheme 1 deuterium label in (R)-~-3-phenyl[2-'~C,~H]lactic acid as 10 was incorporated largely intact in transformed root cultures of Datura stramonium whereas incorporation of I3C from the (9-isomer was attended by complete deuterium loss. lo Secondly," it was found in intact D.stramoniuni plants that with (R)-3-phenyl[l,3-"C,,l-'4C]lactic acid as 9 and the (S)-isomer the former was a more efficient precursor than the latter for hyoscyamine 3 and scopolamine 4.13C NMR spectra showed the specificity of the labelling and the 13C-'3C spin- spin coupling that was observed confirmed an intramolecular rearrangement. Herbert Tlzs biosynthesis of plant alkaloids and nitrogenous microbial metabolites 3 59 Littorine 11 0‘ Ph OH OH L J “OH1 I OH Ph/3’ 13 R = Tropinyl 14 Scheme 2 The tropane derivative 14 its 6P,7P-epoxide and 13 occur as minor bases in D. stramonium or Brugmansia candida x B. aurea. (R,S)-3-Phenyl[ 1,3-”C,]lactic acid as 9 has been found to be efficiently incorporated into the first two compounds with retention of both I3C labels.I2 On the other hand no label was incorporated from (R,S)-3-pheny1[2-13C,2-2H]lactate (similar results were obtained with the separate enantiomers”).Fur-ther labelled littorine 11 was an intact precursor. Thus it is C-2 of phenyllactate which is lost in the conversion to the phenylacetoxy moiety and free phenylactic acid does not appear to be involved. Moreover the results indicate that the biosynthesis of the phenylacetoxy and tropic acid moieties are closely linked. It is also clear from the results obtained that the biosynthesis of the hydroxyacetoxy moiety in 13 is also linked.l2 Here however deuterium was incorporated from compound 10.lo Following these results interrelated paths of biosynthesis have been very reasonably proposed in which a radical may form initially through removal of a hydrogen atom by cytochrome P450 catalysis from littorine 11 (Scheme 2).The stereochemistry associated with the mutase reactions is illustrated in Scheme 1. New results relate to the stereochemi- cal fate of the 3-pro4 proton in 9 upon migration to what becomes the C-3’ carbon in 12.l3 (R,S)-3-Phenyl[2-’H]lactic acid was incorporated into hyoscyamine 3 and the tropic acid hydroxymethyl group was reduced with addition of a deuterium atom. Analysis was then by the classical method involving chiral acetic acid. The tritium in 12 (~3) was found to occupy the 3‘-pro-S site. Thus rearrangement occurs with inversion of configuration on what becomes C-3‘ of 12; the carboxy group is known to migrate with retention of configu- ration (at C-2’). Tigloyl-CoA:pseudotropine acyl transferase which esterifies the 30-hydroxy group of pseudotropine 15 with tigloyl-CoA to give 3P-tigloyloxytropane 16 has been isolated from NR2 15 R’ = H; R2= Me 2 16 R’ = R2 = Me H 17 R‘ = R2=H transformed root cultures of D.stramonium purified to near homogeneity and ~haracterized.’~ Of a range of potential hydroxy-bearing substrates tested only pseudotropine 360 Natural Product Reports 1997 and 4-hydroxy- 1 -methylpiperidine were esterified; 3a-hydroxytropine and norpseudotropine 17 interestingly were not enzyme substrates.The specificity for the alcohol compo- nent is not matched in the acid; a wide range of aliphatic acyl-CoA thioesters were reactive. Acyltransferases involved in tropane alkaloid biosynthesis have been the subject of a short review.Resistance to p-fluorophenylalanine in cell and root cultures of Hyoscyamus muticus has been used to select for hyoscyamine and cinnamoyl putrescine overproduction. 1.2 Anatoxins The biosynthesis of anatoxin-a 20 has been examined in the cyanobacterium Anabaena jos-aquae with 13C-labelled gluta- mate and a~etate.’~ The results are illustrated in Scheme 3 NH2 -H02C pcHo H02C pCO2H NH2 H02CG 18 ‘0 20 19 Scheme 3 with 19 as a plausible intermediate formed from glutamic acid 18 and three acetate units. Significantly [l-’3C]glutamate has been shown to label C-5 of 20. Therefore the complete C carbon skeleton of the amino acid is used. This result is in conflict with the reported incorporation of the C diamine putrescine (ref.6 p. 46). The biosynthesis of anatoxin-a(s) 23 from arginine (ref. 4 p. 64) has been confirmed in an experiment with ~~-[3,3,4,4,5,5- 2H,]arginine as 21.’*Deuterium was present at C-4 and C-5 in +NH2 21 R=H 22 R=OH 23 but it was deduced to be absent from C-6. The absence of label at C-6 was suggested to be associated with the replace- ment of C-1 and C-2 of the amino acid 21 by the dimethyl- amino function of 23. 4-Hydroxyarginine 22 is a minor metabolite of A. jos-aquae and (2S,4S)-[3,3,4,5,5-2H,1-4-hydroxyarginine was incorporated into 23 giving a 2H NMR spectrum similar to that obtained with the 2H-arginine derived material i.e. label at C-4 and C-5 but not C-6. The presence of a deuterium label at C-5 means that this carbon cannot be oxidized to a ketone during biosynthesis; it is apparent that displacement of the hydroxy group in 22 during ring closure occurs with inversion of configuration.1.3 Pinidine As part of an investigation (ref. 5 p. 447) into the biosynthesis of pinidine 28 the production of the alkaloids 24 to 28 in relation to time from seeds to 28 day old seedlings of Pinus ponderosa has been studied.” The results are consistent with a enzymes which catalyse the conversion of (R)-reticuline 29 into salutaridine 30 (ref. 5 p. 448) and of two molecules of N-methylcoclaurine (31 and 32) into berbamunine 33 (ref. 5 25 p. 450) were isolated purified and characterized. They are P450-dependent oxidases reliant on oxygen and NADPH ?I24 I for activity.Now in signal work the cDNA encoding the P450-dependent oxidase berbamunine synthase has been heterologously expressed and further characterized.23 26 27 28 Scheme 4 putative biosynthetic route to 28 which is shown in Scheme 4. This route is strongly supported by the specific incorporation into 28 of 26 with a I3C label on C-10. For the early stages of pinidine biosynthesis see ref. 7 p. 122. 1.4 Pyrrolizidine alkaloids Recent notable work on the biosynthesis of pyrrolizidine alkaloids has been concerned with the key biosynthetic enzyme homospermidine synthase (ref. 6 p. 46). In new work the distribution biosynthesis and turnover of pyrrolizidine alka- loids in Cynoglossum oficinale has been studied.20 The most important finding was that both roots and shoots are able to produce alkaloids and that these organs have different patterns of production.”) 2 Benzylisoquinoline alkaloids 2.1 General The biosynthesis of benzylisoquinoline alkaloids has been authoritatively surveyed2’ under the title ‘Chasing the Enzymes of Alkaloid Biosynthesis’.The joining together of two benzene rings is an oft encoun- tered step in the biosynthesis of many secondary metabolites for which benzylisoquinoline alkaloids furnish pre-eminent examples. It was brilliantly recognized22 in 1957 that this joining together could be rationalized in terms of the pairing of two phenolate radicals. Down the years the hypothesis has stood the test of circumstantial evidence (see ref.7). Recently direct supporting evidence was obtained when the HOI-Ji HO T \ M e @Me Me0 Me0 OH 0 29 30 HO HO MeoqNMe + “^“q!Me MHOe o q M e MeoqNMe HO ”H 33 The enzyme was purified to homogeneity from Berberis stolonifera cell cultures and it was heterologously expressed in functional form in insect cell culture using a bacilovirus-based expression system (from 1 litre of culture the amount of enzyme obtained at one stage corresponded to the amount that would have required 18 000 litres of B. stolonijera cells). The expressed enzyme accepted electrons from B. stolonifera NADPH-cytochrome P450 reductase and catalysed the coup- ling of two molecules of (R)-N-methylcoclaurine 31 to give gauttegamerine or one molecule each of (R)-31 and (8-N-methylcoclaurine 32 to give berbamunine 33.Interestingly the proportions of the two coupled products formed from a mix of the N-methylcoclaurine enantiomers and overexpressed enzyme was different to that formed with berbamunine syn- thase isolated from B. stolonifera; the ratio of products also varied with reductase source. Apart from one other example P450-dependent proteins bind oxygen and function as monooxygenases. Berbamunine synthase acts of course as an oxidase and not as a mono- oxygenase and appropriately there is a bulky proline residue present in the protein replacing glycine-248 one of three residues deduced to be part of the oxygen-binding pocket of a well-studied bacterial monooxygenase P450,,,.Tyrosine and dopa decarboxylases catalyse the decarboxy- lation of tyrosine (to tyramine) and of dopa (to dopamine) and are thereby implicated in the biosynthesis of benzylisoquino- lines at an early stage. The decarboxylase gene family has been examined24 and found to exhibit differential and organ-specific expression. The organ-specific expression is developmentally regulated and a correlation with alkaloid accumulation could be drawn. Two of these decarboxylases have been heterologously expressed in Escherichia coli and partially ~haracterized.~~ (S)-Norcoclaurine 34 is the first benzylisoquinoline to be biosynthesized in plants and is the one from which all the other benzylisoquinolines derive. A specific 0-methylation” and HO 0 HO 34 R=H 36 35 R=Me 0 k C02Me 37 N-methylation then follow.A survey26 of cell cultures of plants known to produce benzylisoquinoline alkaloids revealed 0-and N-methyltransferase activity with norcoclaurine. Poor regard for stereochemistry was observed except with Dicentra spectabilis and Tinospora cordifolia (Menispermaceae) which showed stereospecificity for (8-norcoclaurine 34. The N-methyltransferase activity from the latter species has been purified and characterized. Of 15 compounds tested only Herbert The hiosynthesis of plant alkaloids and nitrogenous microbial metabolites (9-coclaurine 35 and (8-norcoclaurine 34 were substrates. Further screening showed that this stereospecific N-methyltransferase is confined to the Menispermaceae and to D.spectabilis.It has been suggested the cis-jasmonic acid 36 is an integral part of a general signal transduction system which regulates inducible defence genes in plants leading to the production of phytoalexins such as alkaloids (ref. 5 p. 449; ref. 6,p. 49). This has been supported in further work with Escholtzia cell cul- ture~.~~ Rapid but transient synthesis of jasmonic acid occurs in cell cultures after insect or microbial attack; the effect is highly specific and is not caused by environmental stress. Specific enzymes of the benzo[c]phenanthridine alkaloid pathway were shown to be induced by methyl jasmonate and its linoleic acid-derived precursor 12-oxophytodienoic acid 37. 2.2 Morphinan alkaloids Acetyl CoA:salutaridinol-7-O-acetyltransferase (SAT) is a highly specific enzyme from Papaver somniferum which cataly- ses the acetylation of the 7-hydroxy group in salutaridinol 38.At pH 8-9 this acetyl derivative 39 is converted into thebaine 40 (Scheme 5) (ref. 6 p. 47). Details of this work previously 30 I MeOa Meom / NMe CoASAc CoASH HO H 38 Spontaneous pH // AcO H 8-9 ’kpontaneous pH 6-7 Me0 Me0 40 c Me0 Me0 \ \ 41 42 Scheme 5 HOg NMe -HO*+ NMe NMe -NMe available in preliminary form,2x have now been published in full.29 Additionally it is reported that salutaridinol 7-0-acetate 39 at pH6-7 affords 42 which was identified as its sodium borohydride reduction product 41. This compound neo-dihydrothebaine has been identified as being present in Papaver bracteatum.It is reasonably suggested that the path shown in Scheme 5 may be general for dibenz[d,j”]azonine alkaloids (as 41). It all depends on a subtle change of pH which type of alkaloid is produced. In summary the biosynthesis of thebaine 40 involves 34+35+32+ +29+30+38+39. Beyond thebaine lie codeine 45 and morphine 48. Intermediates 362 Natural Product Reports 1997 between thebaine and codeine are neopinone 43 and codeinone 44. These two compounds exist in simple chemical equilibrium. A highly substrate-specific enzyme codeine:NADP oxido-reductase has been isolated3’ from P.somniferum cell suspen- sion cultures which converts codeinone 44 into codeine 45 (the previously unpublished results have been reviewed ref.5 p. 448). Interestingly two isozymes of the reductase were isolated from P. somniferum capsule tissue. An alternative pathway from thebaine to morphine 48 is deduced to involve oripavine 46 and morphinone 47. Since the codeinone reductase is active with 47 as well as 44 the enzyme can have a role in both pathways (Scheme 6). Finally it is to be noted that Thebaine 40 J/\ \ Me0 NMe Me0 43 46 ‘I NMe u 0 44 O 47 NADPH 1 Codeinone 1 Codeinone reductase NADPH reductase 1 J Meo$$ 9 NMe -H:%,NMe HO’ *w HO’ ,w 45 Codeine 48 Morphine Scheme 6 the P. somniferurn reductase catalyses similar reactions to mammalian and bacterial morphine dehydrogenases but the plant enzyme is kinetically very distinct.The uniquely defining step in the biosynthesis of morphine 48 is the conversion of (R)-reticuline 29 into salutaridine 30 which is catalysed by a very specific P450-dependent oxidase (Section 2.1). A similar enzyme has been identified in pig liver (ref. 3 p. 579) and it has now been purified to h~mogeneity.~’ It is absolutely specific for (R)-reticuline 29 salutaridine 30 being the product exactly as in the opium poppy. The pig enzyme also corresponds with the plant enzyme in other ways i.e. in the absorption at 450 nm of the reduced CO-complex and the dependence of the oxidase on 0 and NADPH. Enzyme activity was found in the livers of other mammals including humans. The fact that this crucial reaction can be catalysed in mammalian livers supports strongly the idea that the occurrence of morphine 48 in mammals is of naturally endogenous not dietary origin.It was further found that salutaridine synthesis only occurs in the liver and kidney of the pig not in the brain.31 The N-terminal sequence of the pig protein is known and isolation of the cDNA encoding this enzyme is under way. It will be most interesting to know how the pig and poppy sequences compare. Me0 IU -BBE HO attack on si face Hn u,,? H anti loss of H' and e- from N " OMe WH -0Me 50 -0Me 49 Canadine synthase I-non-stereospecific H loss 0 Me0 Scheme 7 2.3 Protoberberine alkaloids Aspects of the stereochemistry of reactions involving methyl groups which are associated with the biosynthesis of berberine 53 and jatrorhizine 54 have been very carefully examined.The rigorous results some of which have been published in prelimi- nary form (ref. 32; ref. I p. 188) are now available in full.33 In essence they are as follows. O-Methylation of norcoclaurine 34 to give 35 and subsequent N-methylation of this to give 32 occurs with orthodox inversion of configuration relative to 5'-adenosylmethionine. The berberine bridge enzyme (BBE) catalyses the oxidative cyclization of the N-methyl group of (9-reticuline 49 (an intermediate after 32) to give the berberine bridge C-8 of (9-scoulerine 50. Hydrogen removal from the N-methyl group of 49 occurs with a primary kinetic isotope effect (k,lk =4.0) and its replacement by the phenyl group takes place with inversion of stereochemistry (high or complete stereospecificity).The ensuing aromatization catalysed by (9-tetrahydroprotoberberine oxidase (STOX) involves nonstereo- specific hydrogen removaI from C-8 with little or no isotope effect. (The experiment with STOX was carried out directly on (9-scoulerine 50 to give 55; I have simply transferred the results to berberine biosynthesis in Scheme 7.) In the formation of the methylenedioxy group which occurs in the conversion of tetrahydrocolumbamine 51 into canadine 52 a hydrogen is removed from the appropriate methyl group with k,lk,>5 and replaced by the adjacent phenolic oxygen with apparent retention of configuration which is ac-companied by substantial racemization.The subsequent reduc- tive opening of the methylenedioxy group of berberine 53 to give the methoxy group of jatrorhizine 54 proceeds stereo- specifically apparently with inversion. It is notable that berberine bridge formation occurs with high (complete) stereo- specificity whilst methylenedioxy formation is accompanied by substantial racemization. This may simply be taken as a reflection of the difference in mechanisdenzyme type. The nature of BBE is discussed below and evidence for a two-step reaction has been deduced. Canadine synthase is a P450-dependent enzyme and so a radical rather than ionic inter- mediate is perhaps more likely; racemization may be simply associated with radical rotation prior to capture. A summary of the mechanistic conclusions is given in Scheme 7.BBE has been overexpressed in insect cell culture (ref. 6 p. 49). Further shows that the heterologously over-expressed enzyme contains one covalently bound FAD molecule per molecule of protein; the site of covalent attach- ment is histidine- 104. Nineteen benzylisoquinolines were tested as enzyme substrates; with five including the natural sub- strate (9-reticuline 49 a berberine bridge was made. It was concluded that successful substrates must have the (9-configuration and that there must be a hydroxy group next to the site of ring closure. (9-(N)-Methylcoclaurine 32 is a benzylisoquinoline which lacks such a hydroxy group so it does not ring-close. Interestingly it undergoes N-demethylation to give formaldehyde on incubation with BBE.This then provides good evidence for the immonium inter- mediate shown in Scheme 7 and that ring closure is a two step process. Three forms of tetrahydroberberine oxidase have been iso- lated35 from Thalictrum minus cell cultures which are able to convert canadine 52 into berberine 53; two are specific for the S-configuration of canadine 52 whereas the third will oxidize either enantiomer and appears to be a non-specific oxidase. Similarities to and differences from STOX from Berheris and Coptis species were noted. 2.4 Colchicine Among a variety of plant cultures screened for demethylase activity only one a culture of Colchicum variegatum was found which could demethylate c~lchicine.~~ A mixture of 2- and 3-demethylcolchicine was obtained.Two bacterial strains were found which converted colchicine into its 3-demethyl derivative exclusively. Herbert The biosynthesis of plant alkaloids und nitrogenous microbial metabolites 3 Tryptophan metabolites 3.1 Ergot alkaloids violacein and penitrems In the biosynthesis of ergot alkaloids7 (ref. 5 p. 453) in the fungus Claviceps purpurea the first pathway-specific step is catalysed by 4-(y,y-dimethylallyl)tryptophan synthase. The enzyme is a prenyltransferase and it catalyses the reaction of L-tryptophan 56 with dimethylallyl pyrophosphate (DMAPP) to give dimethylallyltryptophan 57. The enzyme has been H 58 miozH \ NH2 H H 56 57 purified from C. purpurea and characterized.Further results have allowed a mechanism of action to be proposed which is related to that of farnesyl disphosphate synthase (ref. 4 p. 58; for the outline mechanism see Scheme 3 in this reference). The cDNA of the synthase has been cloned and expressed in yeast.37 The gene has been sequenced; a putative prenyl diphosphate binding motif has been identified. As part of an investigation into the biosynthesis of violacein 58 in Chromobacterium violaceum mutants of this organism have been examined for the accumulation of possible inter-mediates (ref. 5 p. 454). Most recently cells of C. violaceum have been treated with diethyldithiocarbamate which is a known copper-enzyme inhibitor (copper ~helator).~~ Four new compounds have been isolated namely 59 60 61 and 62 which all have the same skeleton as 58.Consideration of the structures suggests that oxindole formation in the biosynthesis of 58 is a final step; 61 and 62 can be conceived to arise from 59 and 60 respectively by different oxindole formation. Unfortunately so far 59 and 60 have not been found to act as precursors for violacein 58 and its analogues. H H 59 R=H 61 R=H 60 R=OH 62 R=OH The penitrems are complex indole-diterpenoids. Aspects of their biosynthesis have been studied (ref. 6 p. 50). A new group of these alkaloids penitremones A-C have been isolated from a Penicillium species.39 3.2 Terpenoid indole alkaloids A detailed review of the regulation and biosynthesis of second-ary metabolites in cell and tissue cultures of Catharanthus roseus has been p~blished,~' it covers the literature from 1988 to 1993.Strictosidine synthase catalyses the stereospecific assembly of strictosidine from secologanin and tr~ptamine.~ Six isoforms of the enzyme have been isolated from C. roseus cell suspension cultures and purified4' (cf ref. 5 p. 453). They were all found to be kinetically indistinguishable. The possible limitation of the rate of alkaloid biosynthesis by low enzyme levels in two cell lines of Tabernaemontana divaricata has been in~estigated.~~ It was concluded from the results that alkaloid production in both cultures was limited 16 o-v, \ H \ C02Me 63 HOa-vl Me0n-v, ~ OMethyltransferase \ N H \ ~ N 16-Hydroxylase \ H \ C02Me C02Me 64 j Hydration ~ N-Methyltransferase \ H -../ a-p \ Me0 N 65 Me0 N 34 '\\ Me H '< HO C02Me HO COZMe 67 66 14-Hydroxylase P N 9 PN/\ a:!!,, \ Acetyltransferase ~ Me0 OH Me0 Me '.% HO C02Me HO C02Me 68 69 Vindoline Scheme 8 364 Natural Product Reports 1997 not by the enzyme activities but by the availability of terpenoid CHO 1 precursors.Tryptophan decarboxylase strictosidine synthase and isopentenyl pyrophosphate isomerase were not limiting but strictosidine glucosidase and geraniol 1 0-hydroxylase Ajma,ine 73 Peroxidase-H202 might have been. On the other hand addition of loganin to the cultures led to a marked increase in alkaloid accumulation. The effects have been studied43 of UV irradiation and of L li 2,3-dihydroxybenzoic acid on secondary metabolism includ- H ” CHOl ing enzyme activities in C.roseus cell suspension cultures. The biosynthesis of vindoline 69 in C. roseus has been deduced to be from tabersonine 63 as shown in Scheme 8. The pathway is importantly supported by the characterization of the N-and 0-methyltransferases the 4-hydroxylase and the acetyl tran~ferase.~~ The 16-hydroxylase has now been isolated from C. roseus leaves and ~haracterized,~~ whilst further work NADPH has been published46 on the 4-hydroxylase. NADP Redu ctase The 16-hydroxylase has an absolute requirement for 0 and 1 NADPH and is inhibited by CO clotrimazole miconazole H OH and cytochrome c.~’ The data clearly point to the 16- hydroxylase being a P450-dependent monooxygenase.The enzyme activity is tissue-specific (plant leaves) development- specific and light-regulated. This is similar to the last two enzymes (Scheme 8). Cell cultures of C. roseus do not carry out 75 the last three steps of vindoline biosynthesis but the 16-hydroxylase as well as the 0-methyltransferase were found to be present in the cultures. The 4-hydroxylase which catalyses the conversion of 67 into deacetylvindoline 68 is a 2-oxoglutarate-dependent dioxygen- ase (ref. 5 p. 453). It has been purified to apparent homo- geneity from C. roseus plants and further ~haracterized.~~ The \ sequence of binding to the enzyme is 2-oxoglutarate followed 74 by oxygen and 67 while 68 is the first product released Scheme 9 followed by CO and succinate.The enzyme was specific for C-4 of 67 and related alkaloids; tabersonine 63 was not a substrate and other compounds lacking the methoxy C-3 T he novel alkaloid rau macline 74 and relate d alka loids are hydroxy and N-methyl groups were poor substrates. formed dr novo when a jmaline 73 is admin istered to cell Vomilenine 71 is a key intermediate in the biosynthesis of ajmaline 73 in Rauwolfia serpent in^.^^ It leads to other alka- loids as well e.g. raucaffricine which is the glucoside 72. The susphas (not ultimpresent NAing specific to Rauwolfiate product of whiexclusively in R21 -hydroxyraumaclinension cultures of Rgiven results which shDPH-dependent and h cauwolfia cells. e 75 (also .serpentina. Cow that a cell­a cells) oxidizh is then redighly substrat The found in areful es the e specif wall-bound peroxidase uced by a redajminveajma reductase uctase is ic only accept- aline-fed ~tigation~~ line the 70 R=H I 73 Ajmaline I cultures) 77 natuknobios and perakine ral substrate for the ynthesis is illustrated in Scheme 9.respectively. It is nown normally produce 76; the reduction reductase but t clear which d alkaloid. Th aperakof 75 e prop ffords ine is and osed 74 and the only 76 is the course of 71 R=OH Pr oduction of ajmalicin e and catharan thine in root cultures 72 R =OGk of C. rose~s~~ and of ajm alicine in cell-s uspen sion c ultures of OAc this vincof V species5’ has been ininca minor.51 The relamine in a multiple sh vestigated as h ationship between cas the oot culture derived fr prod ell morphology uction of om hairy roots ’’ and C.r indole alkaloid produoseus has been examined.’ ction in cell-s uspens ion cu ltures of 76 R=CHO 77 R=CH20H 3.3 The Indolic phytoalexins biosynthesis of the indolic phyto alexins bras sinin 78 appropriate glucosyl transferase has been isolated from R. cyclcons iderable detail (ref. 6obrassinin 80 and spi p. 51; see Srobrassinin ha cheme s bee 4). A n eluc case has idated in serpentina cultures and characterized (ref. 3 p. 579). Now been made for the cycliza tion of 78 to 80 occ urring through vinorine hydroxylase which converts vinorine 70 into vomile- the naturally occurring methoxy deri vative 79. However nine 71 has been identified in microsomal preparations of usin g Pseudomonas cicho rr-inoculated r oots o f the Japanese R.serpentina cell-suspension cultures and ~haracterized.~~ It is strictly dependent on 0 and NADPH and is inhibited by typical cytochrome P4.50-dependent inhibitors (cJ similar properties for the 4-hydroxylase which converts 67 into 68). Nine cell cultures of six different plant families were analysed for the presence of the hydr~xylase.~’ The results indicate its specificity of action. It was found only in R. serpentina cultures 78 R=H 80 and this was the only culture making romilenine and vinorine. 79 R=OMe Herbert The biosynthesis of plant alkaloids and nitrogenous microbial metabolites radish 79 was found not to be labelled by deuteriated 78 when an excellent incorporation into 80 was recorded. Further it was not incorporated in deuteriated form into 80.53 These results argue strongly against the involvement of 79 in the biosynthesis of 80.Three new brassinin-related stress metabolites have been isolated from the Japanese radish.54 4 Other metabolites of the shikimate pathway Attention is drawn as previously to the reports by P. M. De~ick~~ which are a companion to my reviews. The shikimate pathway has been the subject of an overview56 and more specifically the molecular organization of the pathway in higher plants has been reviewed.57 Individual enzymes associated with the pathway have been surveyed namely anthranilate synthase5* and chorismate m~tase.~~ 4.1 Acridone alkaloids and phenazines N-Methylation of anthranilic acid 81 to give 82 is the first committed step in the biosynthesis of acridone alkaloid^,^ e.g.1,3-dihydroxy-N-rnethylacridone84 (Scheme lo). The next NHMe Me COSCoA 84 83 Scheme 10 i S-Adenosyl-L-methi0nine:anthranilicacid N-methyl-transferase; ii N-methylanthranilic acid CoA ligase; iii acridone synthase step is activation of N-methylanthranilic acid 82 as its CoA derivative 83. This is followed by condensation of 83 with three molecules of malonyl CoA which affords 84 (ref. 5 p. 455; see also ref. 2 p. 522; ref. 3 p. 582 and ref. 1 p. 193). The N-methyltransferase which catalyses the first step has been purified from cell cultures of Rutu gruveolens and char- acterized.60 The enzyme is remarkably specific for anthranilic acid. Acridone synthase which catalyses the conversion of 83 into 84 has been purified characterized cloned heterolo- gously expressed and its cDNA sequenced.61 Interestingly the synthase shows high homology with chalcone synthases.The effect has been studied of yeast elicitation on anthranilate synthase and chorismate mutase in cultures of R. gruveolens.62 There is ongoing research into the biosynthesis of microbial phenazines with current emphasis on the cDNA involved (ref. 5 p. 457; ref. 6 p. 53). New work on the latter which is concerned with the biosynthesis of phenazine- 1 -carboxylic acid 85 and 2-hydroxyphenazine-1 -carboxylic acid 86 in Pseudornonus uureofuciens has been reported.63 The DNA sequence of five contiguous open reading frames (ORFs) which encode for phenazine biosynthesis in P.uureo-85 R=H 86 R=OH 366 Natural Product Reports 1997 fuciens have been identified. The enzyme product of one of these which shows little similarity to known genes is respon- sible for the conversion of 85 into 86. Three ORFs were correlated with the shikimate pathway including anthranilate synthase and one had similarity with the DNA sequences of pyridoxamine oxidases. 4.2 Cyanogenic glycosides Cytochrome P45OtY,catalyses the conversion of tyrosine 87 into p-hydroxyphenylacetaldehyde oxime 90 in the biosyn- thesis of dhurrin 91 in Sorghum bicolor. This is a process which HO \ NH2 HO 87 88 90 89 J -c CN &O-GIuc HO 91 Scheme 11 consumes two molecules of 0,. N-Hydroxytyrosine 88 is the product of the first step.This compound then binds at the same catalytic site as 87.64The second oxidation is deduced to give 90 in steps which need not necessarily be enzyme catalysed (Scheme 11). The cDNA for P45OtY,has been sequenced and characterized.65 It has been found66 that a microsomal preparation from cassava will catalyse the conversion of the precursor valine 92 into linamarin 93; previously biosynthesis had only been H02C)(-x NH2 NC 0-GIUC 92 93 demonstrated in cassava leaves. An antibody raised against P45OtY (above) cross-reacted with a major polypeptide of similar molecular mass in cassava microsomes thus indicating similar enzyme-catalysed amino acid transformations (see Scheme 11) in the biosynthesis of linamarin 93 and dhurrin 91.4.3 Aphelandrine Aphelandrine 94 is constituted in Aphelundru tetrugona from polyamine sources and two units of cinnamic acid.67 Although it is a major constituent of A. tetrugonu 94 is rapidly degraded in macerated plant material. In macerated roots it has been shown6' that 94 first undergoes hydroxylation ortho to the existing hydroxy group then further oxidation to a quinone occurs followed by polymerization. 5 P-Lactams 5.1 Clavulanic acid In the course of the biosynthesis of clavulanic acid 99 the guanidino compound 95 is converted into claviminic acid 98 in OH 94 95 CS hydroxylation 1 c0,-cop-96 97 99 98 Scheme 12 four steps and these steps are catalysed by two enzymes. One is proclavaminate amidino hydrolase (PAH) which catalyses the conversion of 96 into 97.The second enzyme claviminate synthase (CS) is remarkable it catalyses three steps of hydroxylation oxidative cyclization and desaturation (Scheme 12). Both enzymes have been identified as present in the clavulanic acid gene cluster in Streptomyces clavuligerus. Fur-ther work on these enzymes has now been rep~rted.~’ 73 (for earlier work see references cited in these papers; and ref. 5 p. 458 and ref. 6 p. 52). PAH which is an amidino-hydrolase related protein has been overexpressed characterized and its DNA sequenced6’ (cf ref. 5 p. 458). The action of CS is dependent on that of PAH for it could be shown that only in the presence of PAH would the action of CS go beyond the initial hydroxylation.Two isozymes of CS are implicated in clavulanic acid biosyn- thesis. Studies with a S. clavuligerus mutant disrupted in cs2 (encodes CS2) has given results which show that both CSl and CS2 contribute to clavulanic acid biosynthesis but they are differently reg~lated.~‘’ Seven classes of S. clavuligerus mutants defective in clavu- lanic acid biosynthesis have been identified and they have been used to clone the cDNA in the gene cluster encoding eight biosynthetic enzymes including pah and at least one (cs2) of the two genes encoding claviminate synthase; the others remain ~nidentified.~~ CS is an Fe2’ 0 and a-ketoglutarate- dependent oxygenase. The two isozymes CS1 and CS2 have been overexpressed in Esclzerichia ~oli.~~ Evidence was obtained for the presence of histidine and cysteine residues in H COT-0 I H 100 101 proclavaminic acid 97 (ref.6 p. 54); this species does not produce clavulanic acid 99. The branchpoint to 99 100 and 101 reasonably lies at clavaminic acid 98 and indeed a clavami- nate synthase (CS3) has been isolated73 from S. antibioticus thus providing good evidence that clavams like 100 and 101 derive from clavaminic acid. The enzyme has been purified to homogeneity. It needs the same cofactors as CSl and CS2 and also catalyses three steps of oxidation (Scheme 12). CS3 is similar in physical and kinetic properties to CS1 and CS2; N-terminal sequences relate CS3 more closely to CSl than cs2. 5.2 Penicillins and cephalosporins P~blication~~ of the crystal structure of isopenicillin N syn- thase (IPNS) is an undoubted highlight of this reviewing year because this is the important enzyme which in the presence of 0 and Fe2+ catalyses the removal of four hydrogen atoms (H*) from the L,L,D-ACV tripeptide 102 to give isopenicillin N 103 and a molecule of water.7 And though an abundance of COnH 102 ACV lpNsL~:o H Ht;l H 3co -; y N n 3 0 COpH 103 lsopencillin N data was obtained for the enzyme the crystal structure has till now remained elusive.The structure was determined on the protein containing manganese instead of the natural metal ion Fe2’. The active site is unusually buried within a ‘jelly-roll’ motif and lined by hydrophobic residues. Ligands from the protein to the iron are deduced to be His214 Asp216 His270 and Gln330.Built upon this crystal structure and the many accreted pieces of evidence (ref. 6 p. 54; ref. 5 p. 459; ref. 4 p. 62; ref. 3 p. 584; ref. 2 p. 524; ref. 1 p. 196) a mechanism for the conversion of 102 into 103 has been proposed (Scheme 13). Sequence analyses indicated that IPNS 1-aminocyclopropane- 1 -carboxylic acid oxidase and many of the 2-0x0 acid-dependent oxygenases contain a conserved jelly-roll motif; together they constitute a new structural family of enzymes. The natural ACV 102 gives a single product on incubation with IPNS namely the penam isopenicillin N 103. With substrate analogues more than one product may be obtained including cephams. The product ratios obtained with four IPNS isozymes using 104 and 105 as substrates have been compared and carefully analysed in terms of structures within the enzyme active site.75 The analysis leads to the conclusion the proteins at or near the catalytic site and possibly involved that IPNS has evolved to control positively the reactions of in the binding of iron.high-energy enzyme-bound intermediates which are formed Clavams 100 and 101 are produced by Streptomyces anti- with the natural substrate 102 such that only a single product bioticus and derive via the advanced clavulanic acid precursor is formed i.e. 103. With unnatural substrates the control is Herbert The hiosynthesis of plant alkaloids and nitrogenous microbial metabolites P H+ RHN RHN H+ cop- c02- c02- cop- Hp0-Fe11-L2 H2OM I -H+ 7 c02- cop- cop- coz- R = L-6-(a-aminoadipoyl);L1,L2 L3 L4 = His214 Asp216 His270 Gln330 Scheme 13 104 C0pH C02H 105 relaxed to varying degrees which allows intrinsic chemical reactivity to take over resulting typically in the generation of multiple products.The synthesis of hydrophobic penicillins using acyl CoA 6-aminopenicillanic acid acyltransferase (AT) particularly coupled with acyl CoA ligases has been reviewed.76 It has been found77 that both of the C-5 epimers of the ACV analogue 106 are efficiently converted by IPNS into the penicillin analogues 107. It was hoped that this pair of compounds would provide 1IPNS C0pH 107 convenient access to 6-aminopenicillanic acid (also eventually analogues) for the preparation of penicillins with modified side chains.Unfortunately only low yielding conversions were achieved. Recombinant AT modified through site-directed muta-genesis of the cDNA has been used to study the effect on enzyme activity and proenzyme ~leavage.~' The overexpression has been engineered of IPNS and AT in Aspergillus nid~lans.'~ Studies at the genetic level with mutants in relation to the regulation of penicillin biosynthesis have been reported.*' Recombinant technology has been used to enhance the activity of a rate-limiting enzyme deacetoxycephalosporin synthase in cephalosporin C biosynthesis in Cephalosporium acremonium.*' 6 Nucleoside and aminoglycoside antibiotics The biosynthesis of the nikkomycins e.g.nikkomycin X 108 and the polyoxins e.g. polyoxin C 110 are closely related (ref. 4 p. 65). Nikkomycin X 108 along with nikkomycin Z 109 are metabolites of Streptomyces tendae. From the results of comparative experiments it was concluded that the quite unusual formylimidazolone moiety of 108 originates in OH OH 0 108 R=OHCT'*O 109 R= N I I H2N-w OH OH 110 111 368 Natural Product Reports 1997 histidine 111 and as would be expected the uridine portion of 109 has its provenance through uracLs2 This has been con- firmed in similar experiments using ~-[U-~~C]histidine and [2-14C]uracil.83 Distinctive support for the different origins of the two heterocyclic moieties is provided by the observation that a uracil auxotroph of S.tendae which normally just produces 108 switched almost completely to the production of 109 on supplementation with uracil. Further works3 has been concerned with attempting to unravel the mechanism whereby the side chain of histidine 111 becomes the aldehyde stump of 108. Of the normal metabolites of histidine histamine 112 was found not to be incorporated " N' H H 112 113 ?H H H 114 115 R=CHO 116 R=CH20H whilst a marginal low incorporation of urocanic acid 113 was recorded. The possibility was examined that a reverse aldol reaction on P-hydroxyhistidine 114 could provide the formylimidazolone fragment of 108. Although appropriate aldolase activity was found in S. tendae the level of conversion of either of the threo- or erythro-isomers of 114 into aldehyde product 115 was too low to be significant.In support 115 was an insignificant precursor (as was the corresponding alcohol 116). (w-S,P-S>-[U-'"C,~-'H]Histidine, as 111 and its similarly labelled (a-S,p-R)isomer were then used to probe the biosyn- thetic events (essentially of oxidation) which lead from 111 to 108. A good incorporation of tritium excluded an acid/ketone intermediate. Preferential retention was observed of the P-pro3 proton relative to a-pro-R proton from 111 but there was some loss of stereochemical integrity and the loss was variable. The results are in accord with an adventitious exchange process occurring during biosynthesis which is fol- lowed by a stereospecific removal of the P-pro-R proton.Genetic evidence (ref. 4 p. 65) indicates that 117 formed by OH C02H LN' I" H \ 117 118 transamination of 111 may be an intermediate in nikkomycin X biosynthesis; the chemical interconversion of 117 with its enol would explain the exchange process and loss of stereo- chemical integrity. It was suggesteds3 then that a later stereo- specific hydroxylation occurs (loss of 0-pro-R proton) and plausibly 118 may be the intermediate which then undergoes a reverse aldol reaction leading to nikkomycin X 108. The biosynthesis of carbocyclic nucleosides which includes that of aristeromycin 127 has been the subject of a review.84 In significant work it has been shown that neplanicin A 126 is an intermediate in the biosynthesis of aristeromycin 127 in Streptornyces citricolor (ref.5 p. 460; ref. 6 p. 56) (Scheme 14). It has now been shown that 121 isolated from a mutant of 119 120 121 R=H 122 R = PO3H2 I t OR HO*Ad HO OH f2,,opp I\ ,\ HO' OH HO' OH 127 126 123 R=H 124 R = PO3H2 Scheme 14 OH HOWNH2 HOW,*oH I\ ,\ ,I HO OH HO' OH HO' OH 128 129 130 S. citricolor is an efficient specific precursor for 126 and 127.85 The enone 120 was also identified as an intermediate (it supported the production of 126 and 127 in a mutant which did not otherwise produce these metabolites) whereas 128,129 130 and the C-7 epimer of 121 were not. Further confirma- tion of the role of neplanicin A 126 in the biosynthesis of aristeromycin 127 was obtaineds5 by the conversion of ~-[6-~H,]glucose,as 119 first into 126 then the conversion of this into 127; also the conversion of the labelled glucose directly into 127.The 126 was labelled at C-6' and both samples of aristeromycin 127 were labelled in the C-6' S position. Finally good evidence was obtained of the direct incorporation of adenine 125 into aristeromycin.8s The results when put together make a convincing case for the route shown in Scheme 14; the phosphates 122 123 and 124 are further possible intermediates. Overall the study is notable for the intelligent and cohesive experimental exploitation of S. citricolor mutants. OH OH 132 + f H* I NH2 133 Scheme 15 Herbert The hiosynthesis of plant alkaloids and nitrogenous microbial metabolites 369 2-Deoxystreptamine 133 is biosynthesized from 2-deoxy- scyllo-inosose 132 which in turn derives from D-glucose 119.Carbocycle formation involves glucose 6-phosphate 131 with NAD’-NADH as coenzyme. Important results show that during carbocycle formation the hydrogen atom at C-4 (in 119) is abstracted (by NAD’) then subsequently returned to the same molecule of now modified substrate by a mechanism (Scheme 15) which is deduced to resemble closely that of dehydroquinate synthase. Full details of the work previously published in part (ref. 5 p. 461) are now available in 7 Miscellaneous metabolites 7.1 Kinamycins The kinamycins are nitrogenous polyketide antibiotics. Recently their structures have been corrected they are 5-diazobenzo[b]fluorenes e.g.kinamycin D is 136 and not 134 135 136 R=H 137 R=Ac N-cyanobenzo[b]carbazoles.87The kinamycins are elaborated in Streptomyces rnuruyamaensis via 134 (ref. 32 p. 125). It has now been shown that the benzo[b]fluorene quinone 135 labelled with deuterium is incorporated satisfactorily into kinnamycins C 137 and D 136 in S. murayarnuensis; it is also present in the growing cultures.88 It was concluded that 135 or the corresponding dihydroquinone is involved normally in kinamycin biosynthesis. 7.2 Berninamycins and pristinamycins (virginiamycins) Berninamycin A 138 belongs to the thiopeptide group of antibiotics. Its biosynthesis has been studied in Streptornyces bernensis with 3C-labelled precursor^.^^ The results (Scheme 16) essentially confirm earlier ones with 14C-labelled com- pounds and the pattern of biosynthesis is similar to those of two other thiopeptide antibiotics nosiheptide and thiostrepton (ref.5 p. 462). It is manifest that the six dehydroalanine units of 138 are formed by dehydration of serine and this amino acid is also involved in oxazole and thiazole formation. Cysteine is from the results clearly the true precursor of part of the thiazole ring and threonine provides in part the oxazole moieties. The hydroxyvaline residue is formed from valine and not hydroxyvaline. Contrary to earlier conclusions lysine is not a precursor for the pyridine ring. The provenance of this residue is in two units of serine.It is suggested that this ring is formed in a penultimate biosynthetic step to give berninamy- cine B 139 from two dehydroalanine residues by a mechanism similar to that deduced for micrococcin nosiheptide and thiostrepton (see Scheme 20; ref. 5 p. 462). Hydroxylation of 139 would then give berninamycin A 138. A reasonable proposal89 for the biosynthesis of the berni- namycins involves non-ribosomal assembly of a polypeptide precursor like most polypeptide antibiotics,” followed by HzNPH H02C‘ H02C H2N%sH H2NH H02C OH t HN / NH 138 R=OH 139 R=H Scheme 16 oxidative cleavage of an amino acid residue from the C-terminus to give the amide residue. This is followed by appropriate serine dehydration and ring closure to give a precursor for berninamycin B 139 with oxazole and thiazole rings.Pristinamycin 11 (PII,) (virginiamycin M 1) 141 is consti- tuted from valine glycine proline and seven acetate units plus the methyl group of methionine. Results of experiments with 140 I PIIAsynthase reductase NADH o v 141 samples of proline and hydroxyproline indicate inter ah that PII 140 is a precursor for PII 141 and not vice versa; hydrogen loss from C-3 of the proline residue is stereospecific (ref. 32 p. 526; ref. 1 p. 203). In new work” high level conversion of PIIB 140 into PII 141 has been observed in cultures of Streptomyces pristinasespiralis (and some other Streptomyces species). The conversion requires NADH ribo- flavin 5’-phosphate (FMN) and molecular oxygen as cofactors.Two enzymes are associated with this conversion and both have been purified to homogeneity. The first is a NADH:FMN oxidoreductase (FMN reductase) which provides reduced FMN (FMNH,) for the second enzyme (PI1 synthase). It was 370 Natural Product Reports 1997 concluded from the data that the synthase catalyses a transient hydroxylation of PII 140 with molecular oxygen followed by a dehydration leading to PII 141. 7.3 Lactacystin armentomycin and discorhabdins Full details on the biosynthesis of lactacystin 142 (ref. 6 p. 55) have been p~blished.'~ This Streptomyces metabolite has its provenance in isobutyrate (and/or L-valine) L-leucine and L-cys teine. HO w -H HO ' 142 : C12CH-C-CO2-/ 144 \ 0 OH I1 I Me- C- C02- C12CH-C-CH2C02-/ co2-143 f Me-C-SCoA 1 145 hH3 C12CH-CH2-CH-CO2-3 21 146 Armentomycin 146 a chlorinated nonprotein amino acid is biosynthesized from two molecules of pyruvic acid 143 (almost equal labelling of C-1 and C-3 by [2-'3C]pyruvic acid).93 Acetate is an intact primary precursor for C-1 plus C-2.It is suggested that acetyl CoA 145 condenses with dichloropyruvic acid 144 leading to armentomycin. The intermediacy of 143 in biosynthesis affords a mechanistically acceptable substrate for chlorination not apparent in the saturated carbon chain of 146. ~-[U-'~C]phenylalanine has been foundg4 to label discorhab- din B 147 in Latrunculia species which is a marine sponge. 147 8 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. 5 R. B. Herbert Nat. Prod. Rep. 1995 12 445. 6 R. B. Herbert Nat. Prod. Rep. 1996 13 45. 7 R. B. Herbert The Biosynthesis of Secondary Metabolites 2nd edn. Chapman and Hall London 1989. 8 T. M. Kutchan The Plant Cell 1995 7 1059. 9 T. W. Abraham and E. Leete J. Am. Chem. Soc. 1995 117 8100. 10 N. C. J. E. Chesters. D. O'Hagan and R. J. Robins J. Chem. Soc. Chem. Commun. 1995 127. 11 M. Ansarin and J. G. Woolley J. Chem. Soc. Perkin Trans. 1 1995 487. 12 R. J. Robins N. C. J. E. Chesters D. O'Hagan A. J. Parr N. J. Walton and J. G. Woolley J.Chem. Soc. Perkin Trans. 1 1995 481. 13 N. C. J. E. Chesters D. O'Hagan R. J. Robins A. Kastelle and H. G. Floss J. Chem. Soc. Chem. Commun. 1995 129. 14 S. Rabot A. C. J. Peerless and R. J. Robins Phytochemistry 1995 39 315. 15 R. J. Robins P. Bachmann A. C. J. Peerless and S. Rabot Plant Cell Tissue Organ Cult. 1994 38 241. 16 F. Medina-Bolivar and H. E. Flores Plant Phystol. 1995 108 1553. 17 T. Hemscheidt J. Rapala K. Sivonen and 0. M. Skulberg J. Chem. Soc. Chem. Commun. 1995 1361. 18 T. Heimscheidt D. L. Burgoyne and R. E. Moore J. Chem. Soc. Chem. Commun. 1995 205. 19 J. N. Tawara F. R. Stermitz and A. V. Blokhin Phytochemistry 1995 39 705. 20 N. M. van Dam L. Witte C. Theuring and T. Hartmann Phytochemistry 1995 39 287.21 M. H. Zenk in Organic Reactivity Physical and Biological Aspects ed. B. T. Golding R. Griffin and H. Maskill The Royal Society of Chemistry London 1995. 22 D. H. R. Barton and T. Cohen in Festschrft Dr. A. Stoll. Birkhaiiser Basle 1957 p. 117. 23 P. F. X. Kraus and T. M. Kutchan Proc. Natl. Acad. Sri. USA 1995 92 2071. 24 P. J. Facchini and V. DeLuca The Plant Cell 1995 7 1811. 25 P. J. Facchini and V. DeLuca Phytochemistry 1995 38 11 19. 26 S. Loeffler B. Deus-Neumann and M. H. Zenk Phytochemistry 1995 38 1387. 27 S. Blechert W. Brodschelm S. Holder L. Kammerer T. M. Kutchan M. J. Mueller Z.-Q. Xia and M. H. Zenk Proc. Natl. Acad. Sci. USA 1995 92 4099. 28 R. Lenz and M. H. Zenk Tetrahedron Lett. 1994 35 3897. 29 R.Lenz and M. H. Zenk J. Biol. Chem. 1995 270 31 091. 30 R. Lenz and M. H. Zenk Tetrahedron Lett. 1995. 36 2449; R. Lenz and M. H. Zenk Eur. J. Biochem. 1995 233 132. 31 T. Amann P. H. Roos H. Huh and M. H. Zenk Heterocycles 1995 40 425. 32 R. B. Herbert Nat. Prod. Rep. 1990 7 11 1. 33 J. A. Bjorklund T. Frenzel M. Rueffer M. Kobayashi U. Mocek C. Fox J. M. Beale S. Groger M. H. Zenk and H. G. Floss J. Am. Chem. Soc. 1995 117 1533. 34 T. M. Kutchan and H. Dittrich J. Biol. Chem. 1995 270 24 475. 35 M. Hara H. Morio K. Yazaki S. Tanaka and M. Tabata Phytochemistry 1995 38 89. 36 A. Pouler E. Bombardelli C. Ponzone and M. H. Zenk J. Ferment. Bioeng. 1995 79 33. 37 H.-F. Tsai H. Wang J. C. Gebler C. D. Poulter and C. L. Schardl Biochem.Biophys. Res. Commun. 1995 216 1 19. 38 T. Hoshino T. Hayashi and T. Odajima J. Chem Soc. Perkin Trans. I 1995 1565. 39 J. T. Naik P. G. Mantle R. N. Sheppard and E. S. Waight J. Chem. Soc. Perkin Trans. 1 1995 1125. 40 P. R. H. Moreno R. van der Heijden and R. Verpoorte Plant Cell Tissue Organ Cult. 1995 42 1. 41 A. De Waal A. H. Meijer and R. Verpoorte Biochcm. J. 1995 306 571. 42 D. Dagnino J. Schripsema and R. Verpoorte Phytochemistry 1995 39 341. 43 P. R. H. Moreno R. van der Heijden and R. Verpoorte Hetero-cycles 1994 39 457. 44 R. B. Herbert in The Monoterpenoid Indole AIkaloidLy ed. J. E. Saxton supplement to vol. 25 part 4 of The Chemistry of Hetero-cyclic Compounds ed. E. C. Taylor John Wiley and Sons Chichester 1994 p.1. 45 B. St-Pierre and V. DeLuca Plant Phjisiol. 1995 109 131. 46 E. De Carolis and V. DeLuca Plant Cell Tissue Orgun Cult. 1994 38 281. 47 H. Falkenhagen and J. Stockigt Z. Naturforsch. Part C 1995 50 45. 48 P. Obitz S. Endreb and J. Stockigt Phytochemistry 1995 40 1407. 49 F. Vazquez-Flota 0.Moreno-Valenzuela M. L. Miranda-Ham J. Coello-Coello and V. M. Loyola-Vargas Plant Cell Tissue Organ Cult. 1994 38 273. Herbert The biosynthesis of plant alkaloids and nitropenou.7 mirrnhinl wwtnhnlitPr 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 J. E. Schlatmann E. Fonck H. J. G. ten Hoopen and J. J. Heijnen Plant Cell Rep.1994 14 157. N. Tanaka M. Takao and T. Matsumoto Plant Cell Tissue Organ Cult. 1995 38 61. S. W. Kim K. H. Jung S. S. Kwak and J. R. Liu Plant Cell Rep, 1994 14 23. K. Monde T. Tamura and M. Takasugi Phytochemistry 1995,39 587. K. Monde M. Takasugi and A. Shirata Phytochemistry 1995 39 581. P. M. Dewick Nut. Prod. Rep. 1995 12 579. K. M. Herrmann The Plant Cell 1995 7 907. J. Schmid and N. Amrhein Phytochemistry 1995 39 737. R. M. Romero M. F. Roberts and J. D. Phillipson Phyto-chemistry 1995 39 263. R. M. Romero M. F. Roberts and J. D. Phillipson Phyto-chemistry 1995 40 1015. W. Maier A. Baumert and D. Groger J. Plant Physiol. 1995 145 1. A. Baumert W. Maier D. Groger and R. Deutzmann 2. Natur-forsch.Part C 1994 49 26; Kr J. Junghanns R. E. Kneusel A. 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Busby J. Wimp and C. A. Townsend J. Biol. Chem. 1995 270 4262. J. W. Janc L. A. Egan and C. A. Townsend J. Biol. Chem. 1995 270 5399. P. L. Roach 1. J. Clifton V. Fulop K. Harlos G. J. Barton J. Hajdu I. Andersen C. J. Schofield and J. E. Baldwin Nature 1995 375 700. 75 J. M. Blackburn J. D. Sutherland and J. E. Baldwin Biochemistry 1995 34 7548. 76 J. M. Luengo J. Antibiot. 1995 48 1195. 77 J. E. Baldwin S. C. Davis A.K. Forrest and C. J. Schofield Bioorg. Med. Chem. Lett.. 1995 5 2507. 78 M. B. Tobin S. C. J. Cole J. R. Miller J. E. Baldwin and J. D. Sutherland Gene 1995 162 29. 79 J. M. Fernandez-Caiion and M. A. Peiialva MGG Mul. Gen. Genet. 1995 246 110. 80 B. Perez-Esteban E. Gomez-Pardo and M. A. Peiialva J. Bacte-rial. 1995 177 6069; A. A. Brakhage and J. Van den Brulle J. Bacteriol. 1995 177 6069; E. A. Espeso J. M. Fernandez- Caiion and M. A. Peiialva FEMS Microbiol. Lett. 1995 126 63. 81 J. Weil J. Miramonti and M. R. Ladisch Enzyme Microb. Technol. 1995 17 88. 82 R. M. Schmidt H. Pape and M. Z. Junack 2. Naturforsch. Part C 1986 41 135. 83 D. R. Evans R. B. Herbert S. Baumberg J. H. Cove E. A. Southey A. D. Buss M. J. Dawson D.Noble and B. A. M. Rudd Tetrahedron Lett. 1995 36 2351 84 G. N. Jenkins and N. J. Turner Chem. Soc. Rev. 1995 24 169. 85 J. M. Hill G. N. Jenkins C. P. Rush N. J. Turner A. J. Willetts A. D. Buss M. J. Dawson and B. A. M. Rudd J. Am. Chem. Soc. 1995 117 5391. 86 N. Yamauchi and K. Kakinuma J. Org. Chem. 1995 60 5614. 87 S. J. Gould N. Tamayo C. R. Melville and M. C. Cone J. Am. Chem. Soc. 1994 116 2207; S. Mithani G. Weeratunga N. J. Taylor and G. I. Dmitrienko J. Am. Chem. Soc. 1994 116 2209. 88 S. J. Gould and C. R. Melville Bioorg. Med. Chem. Lett. 1995 5 51. 89 R. C. M. Lau and K. L. Rinehart J. Am. Chem. Soc. 1995 117 7606. 90 K. Kurahashi in Antibiotics IV. Biosynthesis ed. J. W. Corcoran Springer-Verlag New York 1981 p. 325; H.Kleinkauf and H. von Dohren in Peptide Antibiotics Biosynthesis and Function de Gruyter Berlin 1982. 91 D. Thibault N. Ratet D. Bisch D. Faucher L. Debussche and F. Blanche J. Bacteriol. 1995 177 5199. 92 S. Takahashi K. Uchida A. Nakagawa Y. Miyake M. Kainosho K. Matsuzaki and S. Omura J. Antibiot. 1995 48 1015. 93 K. Liu R. L. White J.-Y. He and L. C. Vining J. Antibiot. 1995 48 347. 94 R. E. Lill D. A. Major J. W. Blunt M. H. G. Munro C. N. Battershill M. G. McLean and R. L. Baxter J. Nut. Prod. 1995 58. 306. 372 Natural Product Reports 1997

ISSN:0265-0568    

DOI:10.1039/NP9971400359

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Steroids reactions and partial synthesis James R. Hanson School oj’Molecular Sciences University of Sussex Brighton Sussex UK BNl 9QJ Covering 1995 Previous review 1996 13 227 1 Introduction 2 Spectroscopic and physical properties of steroids 3 Reactions 3.1 Alkenes 3.2 Epoxides 3.3 Alcohols 3.4 Carbonyl compounds 3.5 Remote functionalization and photochemical reactions 3.6 Rearrangements 4 Partial synthesis 4.1 Estranes 4.2 Androgens 4.3 Pregnanes 4.4 Bile acids 4.5 Digitalis and related steroids 4.6 Cholestanes 4.7 Vitamin D 4.8 Heterocyclic steroids 5 References 1 Introduction This review covers the literature published between January and December 1995 and follows a similar pattern to its predecessors.’ There has continued to be considerable interest in the synthesis of potential inhibitors of aromatase and testosterone 5a-reductase. Interest has also been focused on the role of 15-oxosterols in the regulation of cholesterol biosynthesis by HMG CoA reductase and on the partial synthesis of the dimeric cephalostatins. Although it is outside the scope of this review it is worth noting that there has been a substantial effort on the total synthesis of analogues of vitamin D whilst some new insight has been obtained into the biological cyclization of squalene epoxide. A book devoted to methods of steroid analysis has appeared.2 2 Spectroscopic and physical properties of steroids The predictive value of additivity relationships has been explored3 in the I3C NMR spectra of polyhydroxysteroids.The I3C NMR chemical shift data for steroidal sapogenins has been re~iewed.~ The influence of 17a-substituents on the I3C chemical shifts of estradiols has been e~amined.~ Effects on the resonances of C-12-C-18 were observed. The conformation of rings A and B of steroids possessing a cylcopropane ring has been studied6 by NMR methods and some significant modifi- cation to the conformation has been observed. The structure of 20P-hydroxyecdysone isolated from Tinospora cordifolia was established by detailed NMR studies in this ~eries.~ The 13C NMR spectrum of cholest-4-en-3,6-dione has been examined’ in detail.The configuration of a series of 2-amino-3-hydroxypregnan-20-ones has been correlated’ with their 3C NMR data. The full ‘H and 13C NMR spectra of pregne- nolone and some relatives have been assigned.” Proton-proton distances obtained from X-ray studies for some pregnanes have been compared” with those derived from NMR studies. Some I3C NMR investigations on fluorinated cortical steroids have been reported.I2 A complete proton Hanson Steroids reactions and partial synthesis assignment for cholesteryl acetate has been obtainedI3 using novel NMR techniques. Molecular motions in solid estradiol have been studied14 by NMR methods. Tandem mass spectrometry has been used15 to differentiate between polyhydroxylated steroids. The origin of fragment ions in the mass spectrum of ergosterol has been studied16 by deuteriation experiments.Characteristic fragments have been observed in the mass spectra of solanidine N-oxidesI7 whilst tandem mass spectrometry has been applied to the characteri- zation of ecdysteroids conjugated with phosphoric acid. ’’ The circular dichroism of some cisoid a,P-unsaturated ketones has been investigated.” Studies have also been reported on a series of cholestenopyrimidines such as 1.20,21 The pyrimidines were prepared from the relevant hydroxy- methylene ketones by condensation with bases such as guanidine. 1 LC‘ 0 / 3 4 The crystal structures of a number of steroids have been reported including three different crystalline forms of dehydro- epiandrosterone,22 some prednisolone derivative^,^^ the re-arrangement product 2,24tetrahydroc~rtisol,~~ doisynolic acid 326 and ethyl 21 -acetoxy- 1 1 P-hydroxy- 1,4-pregnadien-3-0ne 16a,l7a-orthoacetate.*’ The X-ray crystal structure of the oxime 4 showed2’ that it possessed the unusual anti-structure.The X-ray structure of the unusual spirobicyclo-3,l,O-hexane 5 has been rep~rted.~’ The stereochemistry of a series of inter- mediates involved in the synthesis of mifepristone (RU 486) 6 has been confirmed30 by X-ray methods. The butynoate 7 the structure of which was established3 by X-ray crystallography was the unexpected product of the addition of the lithium salt of propyne to the llfi-hydroxy-3,17-ketone8. The X-ray crystal structures of the C-5 epimers of the oxadiazoles 9 have 373 0 OH OH 5 Ho 10 11 0 9 7 R=MeC=C-C-8 R=H been determined32 in connection with studies on their biologi- cal activity.Androst-4-en-3,17-dione forms high melting 1 :1 complexes with a-and P-naphthol which have been studied33 by X-ray methods. The structure and crystal packing of cholest-5-en-3P-y14'-vinylbenzoate has been examined34 in the context of its liquid crystal properties. The value of supercritical fluid chromatography in the separation and purification of steroids such as cyproterone acetate has been demon~trated.~~? 36 The stereochemistry of the interactions involved in steroid:cyclophane complexes has been in~estigated.~~ 3 Reactions 3.1 Alkenes The stereochemistry of hydroboration of an androst-4-ene is normally determined by the presence and stereochemistry of the C-10 methyl group.However an allylic 3-hydroxy group has been shown3* to direct hydroboration to the anti-face and in the case of a 3a-hydroxy group this effect opposes the normal hydroboration from the a-face and leads to the 4P-alcohol (10-1 1). The anomalous ozonolysis of allylic alco- hols which leads to the cleavage not only of the double bond but also of the adjacent single bond has been studied with some steroidal examples such as 12.39 Condensation of the product afforded-the unsaturated ketone 13. The oxidation of 3-hydroxy-A5-steroids with the chromium trioxide:pyrazole complex to form the A4-3,6-dione4' and the allylic acetoxyla- tion of androst-5-en- 17-one41 have been further examined.The epoxidation of various A4- A5-and A'-steroids by the biphasic reagent potassium permanganate:copper sulfate has been shown42,43 to take place predominantly from the p-face. Ruthenium tetroxide has also been shown to act as an epoxidizing agent in this series.44 The catalytic oxidation of steroidal olefins by metalloporphyrin-molecular oxygen sys tems has been investigated .45 17-Iodo-A' 6-steroids are readily available and a series of palladium-catalysed organometallic coupling reactions based on these derivatives have been Thus the iodoolefin 14 on treatment with carbon monoxide in aqueous DMF in the presence of palladium(o) gave the 17-carboxylic acid 15 whilst treatment with vinyltributylstannane led to 374 Natural Product Reports 1997 0 H 13 COpH H 15 H 14 H 16 the diene 16.The regeneration of steroidal 5,7-dienes from their 4-phenyl- 1,2,4-triazoline-3,5-dione adducts using diiso- butylaluminium hydride has been described.49 Under different conditions steroidal dienes can be reduced by this reagent.50 3.2 Epoxides The methanolysis of the epimeric C-4 and C-5 epoxides catalysed by tetracyanoethylene has been shown to proceed in a trans manner.51 The effect of different groups at C-3 on the cleavage reactions of SP,6P-steroidal epoxides with lithium bromide and lithium chloride has been examined,52 whilst a series of anilino-steroids have been obtained53 from 5a,6a- epoxides by cleavage with aniline and boron trifluoride etherate.3.3 Alcohols The methylation of allylic alcohols including the cholest-4-en- 3-01s with methanol and a cis-dichlorobis(tripheny1phosphine) platinum(I1)-tin dichloride catalyst have been described.54 The reaction proceeded with predominant retention of configura-tion. The microwave induced esterification and hydrolysis of bile acid derivatives has been reported.55 Bis(tributy1tin)oxide has been used56 for the selective mild deprotection of steroid esters. Enzymatic methods for hydrolysing 3P-acetates using the lipase from Candida cylindracea have also been described.57 The cleavage of tetrahydropyranyl ethers and cyclic acetals of carbonyl compounds has been reported5' in the steroid series using copper(I1) sulfate adsorbed on silica gel.The Mitsunobu acylation of cholestan-3a and -3P-alcohols to give the epimeric esters has been shown59 to proceed more efficiently using N,N,N',N'-tetramethylazodicarboxamide-tributylphosphine rather than the conventional diethyl azodicarboxylate-triphenylphospine reagent. Alkoxymethyl- carbonates have been prepared6' from steroidal alcohols using chloromethyl alkyl ethers and silver carbonate. The combination of n-perfluorobutanesulfonyl fluoride with DBU has been shown6' to efficiently convert steroidal alcohols such as cholestan-3P-01 to the epimeric fluoride. The reaction of steroidal C-17 acetylenic alcohols with diethylamidosulfur trifluoride furnished62 the C-17a-flUOrO derivatives rather than the C-17P-fluorides.Perfluoro-cis-2,3-dialkyloxaziridines have been shownh3 to be selective reagents for the oxidative cleavage of the methyl ethers of steroidal secondary alcohols to form the corresponding ketones. 3.4 Carbonyl compounds The factors governing the reduction of 1 -0xosteroids have been thoroughly reviewed.64 A new variation of the Meerwein- Ponndorf-Verley reduction using zirconium tetra-tert-butoxide and 1 -(4-dimethylaminophenyl)ethanol as the hydro- gen donor has been explored65 with some steroid examples. The reaction proceeds efficiently at room temperature. Semi- empirical calculations have shown that there is little difference in the heats of formation of 3-ketals and 17-ketals of steroidal 3,17-dione~.~~ Selectivity in favour of the 3-ketal has been achieved by working at low temperature or by using chloro- trimethylsilane as a catalyst.67 The y-fluorination of a,P-unsaturated ketones such as cholest-4-en-3-one has been reported6' using the reaction of the potassium dienoxyborate with N-fluorobenzene-sulfonimide.The preparation of 4-fluoro-A4-3-keto steroids has been described69 by a method involving the treatment of the 4-trimethylstannyl-A4-3-keto steroid with caesium fluor- oxysulfate. Modifications of the Knoevenagel methodology using antimony chemistry have facilitated the preparation of alkylidenemalonates from steroid ketones for use in chola- phane ~ynthesis.~' Improved methods for the alkylation at C-16 of 3-methoxyestrone have been de~cribed.~' Nickel acetylacetonate has been to be an efficient catalyst for the 1,4-addition of trimethylaluminium to hindered unsatu- rated ketones including androstenedione.A transformation of (2)-and (E)-5,10-secosteroidal a,P-unsaturated oximes to the corresponding 1,3-dioximes has been reported.73 3.5 Remote functionalization and photochemical reactions Remote intramolecular free radical functionalization has played an important role in steroid chemistry and a useful review of recent developments has appeared.74 Alkoxyl radi- cals formed by photolysis of the hypoiodite of Sa-cholestan- 7a-yl 4-(a-hydroxyphenylmethyl)phenylacetate17 have been to abstract hydrogen from C-25 of the cholestane side chain to give the macrocyclic lactone 18.Reductive cleavage of the benzyl ether and hydrolysis afforded Sa-cholestane- 7a,25-diol. Irradiation at 266 nm of (173ethylidene-3a-(dimethylphenylsiloxy)-5a-androstan-6- and -1 1 -ones (e.g. 19) has been shown76,77 to lead to 2-E isomerization of the alkene. The presence of the carbonyl group was essential for the isomerization. Fluorescence spectroscopy confirmed that there was efficient intramolecular energy transfer from the dimethylphenylsiloxy chromophore to the carbonyl group which then acted as a singlet-triplet switch to produce the alkene isomerization. The structure of the product obtained by irradiation of 5-0x0-5,lO-secocholest-1 (lO)-en-3a-y1 acetate 20 has been revised7' to 21. Alkoxyl radical induced transannular Hunson Steroids reactions and partial synthesis 1 rI1 .''n HH AcO IJ Me0 0 \OH 21 22 23 cyclizations of 5,10-secocholest-l(1 O)-en-5-ones (e.g. 22-23) have been examined.79 Photolysis of the lactol 24 in the presence of (diacetoxyiodo)benzene lead tetraacetate or mercuric oxide and iodine and oxygen has been shown" to generate an alkoxyl radical which underwent fragmentation to form the P-peroxylactone 25. On heating in acetic acid the 6,9-dioxocyclodec-1 -enyl derivative 27 which had been obtained by the mercuric oxide; iodone oxidation of the 8,14-secoandrostane 26 gave*' the spiro-y-lactone 28. OH 24 25 375 4 Partial synthesis 4.1 Estranes In order to define the structural parameters associated with the AcO&-*cow antitubulin activity and cytotoxicity of 2-methoxyestradiol a OH 26 27 Aco+oAc 0 28 + Me&0 FCl 0 30 0 0 S 31 ‘ 32 3.6 Rearrangements The formation of methoxylactones in the Baeyer-Villiger re-arrangement of the a-substituted-p-spirocyclobutanone(29-30) in basic methanolic hydrogen peroxide has been exam- ined.82 3-Thioxoandrosta- 1,4-dien- 17-one 31 has been showns3 to undergo an acid-catalysed dienone:phenol type of rearrange- ment to afford 1-mercapto-4-methylestra-1,3,5( 10)-trien- 17- one 32 and its dithio dimer.The selectivity of the reactions of this unsaturated thioketone towards various nitrogen nucleo- philes has been in~estigated.~~ The migration of a silyl group from oxygen to carbon has been noted in the steroid series.85 OR OBn 33 R=H 34 37 R = THP OH 0 38 39 376 Natural Product Reports 1997 mammalian metabolite of estradiol a number of analogues have been synthesized.86 2-Ethoxy- and 2-[(E)prop-1-enyllestradiol were more potent than 2-methoxy-estradiol.The C-2 phenol was obtained from estradiol 33 by formylation followed by Baeyer-Villiger oxidation of the benzyl derivative 34 to afford 35. The estrogen-adenine adduct 36 has been obtained87 by the electroreductive coupling of estrone-3,4-o- quinone and adenine. 6-Oxoestradiols have been prepareds8 by the metallation of protected estradiols (e.g. 37) at C-6 using lithium diisopropyl- amide:potassium I 1 -dimethylpropoxide quenching the metal- lated derivative with trimethylborate and oxidation with hydrogen peroxide to afford 38.Further oxidation with sodium hypochlorite and deprotection gave 39. 6,6-Dimethylestradiol has been obtained.89 The Beckmann fragmentation of syn-3-methoxy-6a 17p-dihydroxyestra- 1,3,5(1O)-trien-7-one oxime 40 with thionyl chloride has been examined” and shown to afford the 6-oxo-6,7-seco-7-nitrile 42. A plausible mechanism might involve the extrusion of SO from a cyclic sulfite 41. An improved synthesis of the 17-ethylene ketal of 11-0xoestrone-3-acetate has been reported” involving the oxidation of estrone with DDQ to form 9(11)dehydroestrone. Hydroboration of the 17-ketal and oxidation then gave the required ketone. A series of 1 1 P-ethyl- and 11 P-methoxy- estradiol derivatives containing 17a-substituents have been prepared as probes for the estrogen receptor and their binding affinity has been evaluated.92p94 The stereoselective synthesis of 12a-amino- and 12a-aminomethyl-estrogens (e.g.43) has been achieved95 by oxidative nucleophilic substitution using DDQ as the oxidant and trimethylsilylcyanide or azide as the nucleophile. The 9( 11) double bond is formed by dehydrogena- tion and then a carbocation is formed at C-12 which is discharged by attack of a nucleophile from the less-hindered a-face. 8a,14a-and 14a 1 5a-methylene bridged estradiol derivatives (e.g. 44) have been synthesi~ed.~~. 97 The stereo- control in the Simmons-Smith cyclopropanation was obtained by using the 17a-alcohols.The synthesis of some 15a-hydroxyestrogen-15-N-acetylglucosaminides has been reported.98 The inhibition of human placental estradiol 17P-hydroxy steroid dehydrogenase the enzyme that converts estrone to estradiol has attracted some interest. The synthesis of some 16-(bromoalky1)estradiols 4599 and the spiro-lactone 48”’ have been reported in this 35 36 OH / 0Y0 0 40 41 42 Me HN OH OH OAc &Me3 43 44 OH 0 47 40 context. The spiro-lactones were prepared by the addition of allylmagnesium bromide to a protected estrone 46 hydro-boration of 47 and oxidation then afforded 48. 19-Nor- 17P-hydroxy- 17a-trifluoromethyl-A4-estren-3-one and its A439-and A419.''-analogues have been synthesized'" by trifluoromethylation of 19-nor-3,3-dimethoxy-A5(10)-estren-17-one with trimethylsilyltrifluoromethane.These compounds showed high affinity for the progesterone receptor. The epimerization of 3-methoxyestra-l,3,5( 10),14-tretraen-17P-o1 has been investigatedIo2 in order to obtain a high yield of the 17a-alcohol to direct addition to the AI4 double bond (vide supra). The synthesis of 17a-cyanomethyl-l7P-hydroxy-(14a 1Sa-'HH)estra-4,9-dien-3-one has been reported."' Rhenium carbonyl complexes of estradiol derivatives (e.g. 49) which have a surprisingly high affinity for the estradiol recep- tor have been synthesizedIo4 as potential imaging agents. A molecular modelling analysis of the various conformations of diethylstilbestrol and their comparison with estradiol has been made'05 in the light of the binding of diethylstilbestrol to the oestrogen receptor.4.2 Androgens Syntheses of some C-3 diacylglyceryl sulfides sulfones,'06 some alkynyl sulfonylmethyl derivativesIo7 and phospha-tides'" of dehydroisoandrosterone as potential inhibitors of glucose-6-phosphate dehydrogenase have been reported. Some 3-keto-4a,Sa-epoxysteroids bearing electron-withdrawing substituents at C-2 for example 50 have been reportedIo9 to be inhibitors of HIV-replication. The steroids may exist in their enolic form. Further neuromuscular block- Hanson Steroids reactions and partial synthesis 50 OH HO I H CH20H 52 ing agents such as 51 containing 3-and 17-ammonium substituents have been described.' lo The synthesis of ['3C6]testosterone multiply labelled on ring A has been pub- lished.' ' ' The synthesis of some 4-hydroxymethyl steroid analogues such as 52 of the diterpenoid tumour inhibitor aphidicolin 53 has been reported.'12 The inhibition of aromatase has continued to attract con- siderable attention.The conformational analysis of steroids with a 4-ene or a 2,4-diene that are competitive inhibitors of aromatase has been examined'I3 in the light of their role. The dibromination and dehydrobromination of 17P-acetoxy-1a-methyl-5a-androstan-3-one to afford atamestane has been exarnined.'l4 The synthesis and evaluation of a series of 4-substituted androst-4-en-3,17-dione derivatives (4.g. 54) as 0 0 R OH 56 R = OH. Me 57 aromatase inhibitors afforded' I5 results consistent with the presence of a hydrophobic pocket in the active site around the 4a-region.Some 4-hydroxy- and 4-methyl- 1 S-oxaandrost-4- en-3-ones 56 have been prepared' l6 from the diterpenoid sandaracopimaric acid 55. The synthesis of 4a and 4P,17- dihydroxyandrost-1 -en-3-one 57 has also been reported.' I7 Further studies on 6-alkylandrost-4-en-3,17-diones (e.g. 58) have revealed a marked increase in the affinity for aromatase upto n-pentyl suggesting the formation of a stable enzyme- inhibitor complex in the hydrophobic binding pocket. Several steroidal [4,6-b,c]benzothiazepines(e.g. 59) have been pre- pared'Ig as aromatase inhibitors. Although a series of 6,7- aziridines proved to be poor inhibitors 7a-acetoxy-6P-azidoandrost-4-en-3,17-dione 60 was a potent inhibitor.'*' A number of 7a- and 7P-alkyl and aryl androst-4-en-3,17-diones 0 0 0 60 61 R = (CH*),Ph n= 1-3 have been prepared.'21 122 The derivatives 61 were effective inhibitors. The preparation of 7a- and 7P-(4-hydroxyphenyl) androst-5-ene-3p 17P-diacetate has also been reported. 123 The 6P-hydroxy derivatives of some anabolic steroids such as 17a-methyltestosterone have been synthesi~ed'~~ by photo- chemical oxidation of their trimethylsilyldienol ethers to aid their detection as human metabolites. The synthesis of C-6 fluoroandrogens such as 6a-fluorotestosterone and their evaluation as binding agents for androgen receptors in prostate tumours has been reported.125 1 1 P-Fluoro-Sa- dihydrotestosterone 64 and its 19-nor analogue has also been prepared'26 in an investigation of their possible application when labelled with I'F as androgen receptor based imaging agents for prostate cancers.The key step in the synthesis involved the halofluorination of the 9(11)alkene 62 with 1,3- dibromo-5,5-dimethylhydantoin and hydrogen fluoride in pyridine followed by reductive debromination with tributyltin hydride. 62 63 I 64 The excessive production of Sa-dihydrotestosterone is the major cause of many androgen-related disorders. The bio- chemical studies on testosterone 5a-reductase and the inhibi- tion of this enzyme has been reviewed.127 The synthesis and biological activity of a further series of 17P-(N-alkyl/ arylformamido)-4-methyl-4-aza-3-oxo-5a-androstanes 65 as inhibitors of this enzyme has been reported.128 The dehydro- genation of 3-keto-4-aza steroids with DDQ to form the 378 Natural Product Reports 1997 I Me OH 65 R = alkyl/aryl 66 I Me 67 R = OH halogen 1,5-dienes has been examined.129 A series of 17P-carboxamides (e.g. 66) have been prepared.I3' These compounds showed a moderate inhibition of human type I1 5a-reductase activity and relatively potent antiandrogenic activity. The antiandrogenic activity of some y-hydroxy and y-haloalkynyl azaandrostanes (e.g. 67) has been described.I3l Syntheses of labelled 17-epitestosterone have been reported. 132 133 The S,2 displace-ment of the 17P-toluene-p-sulfonate by potassium nitrite afforded a method of preparing the 17a-alcohol.2-Carboxy- ethyloxime derivatives of some androstan- 17-ones have been prepared134 and the 17E configuration of the oximes has been established by NMR methods. The structural features in a series of pyridyl derivatives (e.g. 68) that are required for the inhibition of human testicular 17a-hydroxylase and C,7:20 lyase in the possible treatment of hormone dependent prostatic carcinoma have been examined.'35 The synthesis and in vitro degradation of testosterone lipid conjugates has been reported. 136 The preparation and X-ray crystal structures of 14,15-secoandrost-4-en-3-ones (e.g. 69) possessing an acetyl- enic unit at C-17 has been reported.'37*13* These com-pounds are reported to be inactivators of 3a-hydroxysteroid dehydrogenases.68 69 4.3 Pregnanes A novel synthesis of 4-amino-(20S)hydroxymethylpregn-4-en-3-one 70 which is a potent 5a-reductase and 17,20-lyase inhibitor has been described.139 The sequence involved nitra- tion of a A4-3-oxo steroid. 6a-['8F]Fluoroprogesterone has been ~ynthesized'~' for use in binding studies to the progester- one receptor. The procedure involved halofluorination of pregnenolone oxidation of the 3-alcohol elimination of HBr and epimerization of the fluorine. 6-0xa-5P-pregnane-3,20- dione 75 has been preparedI4l from pregnenolone. Oxidation of the trans-diol 71 with iodine and mercuric oxide gave the 6,19-ether which was in turn cleaved to afford the lactol 73 and thence the secoiodo ketone 74.The oxa steroid 75 was obtained by reduction and deoxygenation of C-19. The Me &OH / AcO& 0 HO OH NH7 70 71 I Me 73 72 Me Me CHOAc AcOJ.-J+:'H CH2I -74 synthesis of 20-oxopregnacalciferol analogues (e.g. 76) has been and their binding to the progesterone recep- tor has been examined. The photochemical hypoiodination of cortisol acetonide gave'43 a mixture of 18-iodocortisol aceto- nide and the 11b,19-oxido derivative from which the cortisol analogue 77 was obtained. The preparation of some 14p-H antiprogestins such as 78 has been reported'44 in an attempt to separate antiprogestational and antiglucocorticoid activity. Some I 5P-hydroxysteroids are markers for adrenal disorders in newborn infants.3a 1 5P 17a-Trihydroxy-Sa-pregnan-20-one 79 and its 5p-isomer have been prepared145 to aid the identification of abnormal metabolites. The synthesis of some 18-vinyl steroids such as 80 has been re~0rted.I~~ These compounds are of interest in the study of the conversion of 1 I-deoxycorticosterone to aldosterone. Some steroid [16a,17a-4-3'-carboethoxyisoxazolines (e.g. 81) have been prepared'47 Me co FH20H 76 77 Me OH OH H 78 79 Hanson Steroids reactions and partial synthesis as novel antiinflammatory agents. The heterocyclic ring was added by a 1,3-dipolar cycloaddition of Et0,CCNO to a 16-ene. These adducts have also been used to prepare 16-cyano steroids and thence the 16a-methoxycarbonyl derivatives.14' Some modifications of 3 P-acetoxy- 14p 17a-pregn-5-en-2O-one leading to the ester 82 have been de~cribed.'~~ Syntheses of 21-hydroxy- 1 1,19-0xidopregn-4-ene-3,20-dione and 2 1-hydroxy-6,19-oxidopregn-4-ene-3,20-dione have been rep~rted'~' in which the hydroxylation at C-21 was effected using iodoso- benzene. A number of des-A analogues (e.g. 83) of 3a-hydroxy-5P-pregnan-2O-onehave been prepared in the search for neuroactive steroidal analogues of the modulator of GABA receptor function. 15' 152 Some pentacyclic pregnanes containing an additional cyclohexane ring fused at C-16 and C-17 on ring D (e.g. 84) have been shown to exhibit antiprogestational effects.'53 154 Me 0 80 R = COCHzCH2 v 81 Me Jo2Et 82 R = COCH~CH~COZH 83 Me co 84 4.4 Bile acids The conversion of methyl cholate to its 3P-epimer by the Mitsunobu reaction and to a 3P-aminocholic acid by displace- ment of the toluene-p-sulfonate with azide has been reported.'55 3a,7a 12a 19-Tetrahydroxy-5P-cholan-24-oic acid 85 has been ~ynthesized'~~ and identified in human neonatal urine.The inclusion compounds derived from cholic acids have continued to attract interest with a number of crystal structures being described.' 57-1 59 The interaction between methyl cholate and acetonitrile has been examined in detail. 159 The synthesis and reactions of some macrocyclic derivatives involving cholic acid have been reported.160-'62 spectroscopic studies were used in an investigation of the interaction between anisole as a guest molecule and macrolide hosts based on 3a-hydroxy-5~-cholan-24-oic acid.An unusual oxidation of bromide to bromine by a strained diazenedicar- boxylate in this series has been noted.I6' Bile acids have been used as chiral auxiliaries in the synthesis of chiral lac tone^'^^ and in the Diels-Alder reaction of acryl- ate^.'^^ Some chiral Troeger's base analogues (e.g. 86) have 86 n=1,2 been prepared.165 The structure of a symmetrical (n=1) deriva- tive was confirmed by X-ray crystallography. The cholic acid skeleton has been modified'66 to provide a scaffold for the creation of combinatorial libraries. The synthesis and DNA binding properties of C-3 C-12 and C-24 substituted amino steroids derived from cholic acids has been re~0rted.l~~ Various cholic acid amides have also been described.168 The synthesis of 245-squalamine 87 from OS03H 87 #HA NH3 -03so-\ 88 3P-hydroxy-A5-cholenic acid and of some relatives as anti- infective agents has attracted attention. 169 I7O A readily avail- able 'reverse' analogue 88 possessed an antibiotic activity similar to that of squalamine against a broad spectrum of microorganisms. A series of heterocyclic rings have been atta~hed'~' to the cholic acid side chain to exploit the carrier properties of the steroid ring system. 4.5 Digitalis and related steroids An interesting review has appeared'72 of the extensive work in Berlin on digitalis and its application in the treatment of heart disease.A synthesis of digitoxigenin 93 from 3~-acetoxyandrost-5-en-17-one 89 has been reported. 173 The key steps involved the synthesis of the diepoxide 90 its 380 Natural Product Reports 1997 ++ AcO THPO 89 90 1 4 OH THPo &OH H 91 OH HO THPO H H 93 92 94 95 AcO H 96 conversion to the pregnane 91 and the formation of the butenolide 92 by the Wittig Leaction with (triphenylphos- phorany1idene)ketene (Ph3P'-C= C =0). An efficient pro- cedure for the preparation of 17a-amino derivatives 94 has been described'74 based on the thermolysis of 17P-azidocarbonyloxymethyl derivatives 95 to afford 96. The use of enzymatic methods of ester hydrolysis has permitted175 the selective hydrolysis of some haptens containing a carboxylic acid ester as a chain attached to C-3.Radiolabelled derivatives of ouabain have been prepared'76 for possible application as myocardial imaging agents.Some bufo toxin homologues have been obtained177 from scillarenin for study as cardiac agents. 4.6 Cholestanes Several new analogues (e.g. 97) of the anti-HIV agent cosalane 98 have been prepared'78 and shown to possess potentially useful antiviral activity. 7a-Hydroxycholest-4-en-3-one is an ? OH > OH C02H / CI C02H H O 101 OH 97 I? Ho2c*c' HO .Me H 102 \ Ho2cm HO li CI no intermediate in the biosynthetic conversion of cholesterol to the bile acids. A convenient route for its preparation involved'79 the hydroxypropyl-P-cyclodextrin facilitated cholesterol oxidase enzymatic oxidation of 3p,7a-dihydroxycholest-Sene to the ring A unsaturated ketone.Cholesteryl esters such as the crown ether 99 have been used in chiral recognition studies.'*' The hydroboration of the C-3 cyclic thioacetal 100 of cholest-4-en-3-one has been shown'" to produce some cleavage of the C-S bond. L S 100 The use of the cholesterol framework has been explored'" 183 as a carrier for anticancer drugs such as N,N'-bis(2-chloroethyl)-N'-nitrosourea based on the fact that many tumours have a high LDL requirement. Some cholesterol- baccatin analogues (e.g. 101) have been synthe~ized.'~~ A critical evaluation has appearedlS6 of the role of various oxysterols in the regulation of cholesterol biosynthesis by interaction with HMG CoA reductase.15-Keto sterols such as 3P-hydroxy-Sa-cholest-8( 14)-en- 15-one play an important role in regulating the cholesteryl ester transfer protein and hence the bioavailability of cholesterol. The synthesis of a number of compounds in this series has been rep~rted."~~ The heptafluoro-7a-methyl derivative 102 lowered the level of Hanson Steroids reactions and partial synthesis HMG CoA reductase activity and showed a significant hypo- cholesterolemic effect in rats. 3a-Hydroxy-4a-allylcholestane has been shown's9 to be a potential hypocholesterolemic agent by stimulating the expression of LDL-receptors and the clearance of plasma LDLXholesterol. There has been significant effort at modifying the sterol side chain.Cholesterol analogues with a diacetylenic moiety in their side chain 103 have been examined'" as potentially HO 103 0 106 photopolymerizable lipids. Some cis-and trans-fused ditetra- hydropyrans (e.g. 105) have been prepared'" by the reduction of the methanesulfonate 104. Other studies have involved sulfoxide chemistry' 92 in the preparation of 3a,7a-di- and 381 synthesis of substituted sterol side chains. The asymmetric synthesis of (24Rjhydroxycholesterol has been rep~rted'~' and a squalamine desulfate analogue have been synthesized from stigmasterol.198 The synthesis of the brassinosteroid phytohormones has been reviewed.199 Epimeric 2,3-epoxybrassinosteroids includ-ing secasterone 107 have been prepared.200Some new methods for the construction of the brassinolide side chain have been explored.201,202 Aspects of ecdysteroid chemistry have been explored including the selective acetylation HO PH of 20-hydroxyecdysone 108,203 the preparation of 25-deoxyecdysone from e~dysone,~'~ and of shidasterone 109 by a method which established its configuration at C-ZL205 The partial synthesis of marine sterols has also attracted interest.Clionasterol has been transformed into some ring B hydroxylated derivatives.206The aragusterols (e.g. llOj which HO IH OH 108 110 H II 0 possess a cyclopropane ring in the side chain and show 109 antiturnour activity and the isocalysterols which also contain a cyclopropene moiety in the side chain have both been synthesized.207,208 The highly degraded marine sterol (17Rj-3a,7a,12a-trihydroxy-5P-cholestan-26-oic acids which are 17-methylincisterol 111has been synthesized from vitamin D,.intermediates in cholic acid biosynthesis. The 24-0x0-The significant effort which has been at the CoA derivative 106 synthesis of the cephalostatins has culminated in a biomimetic 3a,7a,12a-trihydroxy-5~-cholestan-26-oyl and synthesis of cephalostatin 7 114 cephalostatin 12 and rittera-has also been synthesized.193 n-Ally1 zirc~nocene'~~ isoxazoline chemistry'95 96 have been explored in the zine K.214These bis-steroidal pyrazines show potent activity in AcOV 113 X = OH;Y = H (for north) X = H;Y = OH (for south) 114 382 Natural Product Reports 1997 a wide range of NCI cancer screens.The individual ‘north’ and ‘south’ halves were synthesized from the readily available hecogenin acetate via the pentacylic aldehyde 112 and the intermediate 113 obtained by the addition of methylallylstan- nane. These were then converted to their 2a-azido-3-ketones which were reduced to the corresponding amino ketones dimerized and oxidized to form the separable mixture of pyrazines. Structural work on the diacholestanes 115 of geochemical significance has 4.7 Vitamin D Recent studies on the chemistry and the conformation of vitamin D derivatives has been Geviewed2l6 in the context of their biological activity. A great deal of work has been devoted to the total synthesis of vitamin D and to many analogues containing variants in the side chain such as fluoro alkyl and alkynyl groups and in the ring system such as aromatic analogues.However total synthesis is outside the scope of this article. Synthetic work in the area has been reviewed.217 Some further studies on the photochemistry of vitamin D have also been described.218 la-Hydroxyprovitamin D has been shown2I9 to undergo a new photochemical isomerization cascade initiated by the cleavage of the C-1-C-10 bond. The pre-vitamin D,:vitamin D equilibrium has been shown220 to be affected by the nature of the substituent at C-3. The photoisomerization of a 5,7-diene possessing an 11 p-19-oxide bridge has been examined221 and the product shown to be the stereoisomeric 9p 1Oa-compound. 25-Hydroxyvitamin D 3-and 5-monoglucuronides have been prepared.222 Amino- propylation of vitamin D hydroxy groups has been examined in order to provide anchoring groups for affinity studies with receptor proteins.223 4.8 Heterocylic steroids The partial synthesis of aza analogues of steroids from natu- rally occurring steroid precursors has been reviewed.224 4-Aza steroids are of interest as 5a-reductase inhibitors225 and this activity has been found226 with 6-aza derivatives including 6-azacholest-4-en-3-one 116 as well.The selective reduction of ring fused 4-aza-5-enes to cis-and trans-fused lactams has been described.227 A number of novel azasteroids bearing nitrogen at the ring junctions have been synthesized.228 229 Estrone has been con- verted,,’ via cleavage of a 9,l l-double bond to 1l-azaestrone.The modification of ring D to form lactams has been described.231-232 Heterocyclic rings including triazoles and pyrimidines have been fused to ring D.233-236 A number of Hanson Steroids reactions and partial synthesis 17-azasteroids including 17-azacho1estero1 have been ~repared.~~~-~~’ The steroid framework has also been used as a carrier for 5 References 1 J. 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ISSN:0265-0568    

DOI:10.1039/NP9971400373

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

P-Phenylethylamines and the isoquinoline alkaloids ~~ Kenneth W. Bentley Marrview Tillybirkoch Midmar Aberdeenshire UK AB.51 7PS Covering July 1995 to June 1996 Previous review 1996 13 127 1 p-Phen ylethylamines 2 Isoquinolines 3 Naphthylisoquinolines 4 Benzylisoquinolines 5 Bisbenzylisoquinolines 6 Cularines 7 Pavines and isopavines 8 Benzop yrrocolines 9 Berberines and tetrahydroberberines 10 Secoberberines 11 Pro topines 12 Ph thalide-isoquinolines 13 Spiro benzylisoquinolines 14 Other modified berberines 15 Emetine and related alkaloids 16 Benzophenan thridines 17 Aporphinoid alkaloids 17.1 Proaporphines 17.2 Aporphines 17.3 Dimeric aporphines 17.4 Phenanthrenes 17.5 Oxoaporphines 17.6 Dioxoaporphines 17.7 Aristolochic acids and aristolactams 17.8 Azafluoranthenes and related alkaloids 18 Alkaloids of the morphine group 19 Phenet hylisoquinolines 20 Colchicine 21 Erythrina alkaloids 21.1 Homoerythrina alkaloids 21.2 Cephalotaxine and related alkaloids 22 Other isoquinoline alkaloids 1 P-Phenylethylamines Methods of estimating ephedrine and pseudoephedrine in body fluids have been reported.',* Ab initio calculations of vibra- tional circular dichroism spectra of the 1 S,2R configurations of ephedrine pseudoephedrine norephedrine norpseudo-ephedrine N-methylephedrine and N-methylpseudoephedrine have given values that are in good agreement with observations of the spectra of these corn pound^.^,^ Ephedrine 1 has been condensed with formaldehyde propionaldehyde butyralde- hyde isobutyraldehyde and acetone to give the oxazolidines 2a 2b 2c 2d and 2e respectively all of which have been shown to stimulate locomotor a~tivity.~ Ephedrine has been converted into thioephedrine 3 which has been oxidised to di(thioephedrine) 4 and the effects of these compounds on the reaction of diethylzinc with benzaldehyde have been studied.6 The physiological and pharmacological effects of e~hedrine,~' norpseudoephedrine" and hordenine' ' have been studied.Mescaline has been demethylated to the amine 5 which has been shown to act as a substrate for the enzyme microsomal aldehyde oxidase.I2 Bentley P-Phenylethylamines and the isoquinoline alkaloids o&R2 OH 1 2a R1 =R2=H 2b R' = H; R2 = Et 2~ R'=H; R2=Bu 2d R'=H; R2=Bui 2e R1= R2= Me HO OH 4 5 2 Isoquinolines Simple isoquinoline and tetrahydroisoquinoline alkaloids have been isolated from the following plant species Berberis turcomanica' corypalline Berberis virget~rum'~ noroxyhydrastinine Dactylocapnos torulosa' hydrastinine chloride Hernandia sonora16 backebergine and oxyhydrastinine.Schiff bases and 3,4-dihydroisoquinolines react readily with acid chlorides and the products react with Grignard reagents to give tertiary amides. In this way 6,7-dimethoxy-3,4-dihydroisoquinoline has been converted into the amide 6a Me0 Me0 Me CH~OH 6a R=C02Et 7 6b R=Me 6~ R=H which has been reduced by lithium aluminium hydride to racemic carnegine 6b.I7 A synthesis of (+)-(9-N-methylcalycotomine 7 in a high degree of optical purity has been achieved by the Pictet-Spengler condensation of 3,4-dihydroxy-phenylethylamine with (2R)-N-glyoxyloylbornane- 2,lO-sultam followed by O-methylation of the product.l8 Solid-phase Bischler-Napieralsky cyclisations of p-phenylethylamides 8 linked as carboxylic esters to supports have been achieved and the products 9 have been reduced and cleaved from the support by reduction with sodium cyanoborohydride to give tetrahydroisoquinolines 8 (R =Me) giving carnegine 6b.l9 Treatment of the chiral dihydroisoqui- nolinium salt 10 with methylmagnesium iodide has afforded 96% of the (9-tetrahydroisoquinoline 11 with less than 4% of its R isomer and reduction of these which are derivatives Me0 Me0 Meo* CI- Me0 Q0 OJ Q0R2 I OR’ 15 16a R1R2=CH2 16b R1 = R2 = Me MeO A n MeO.A A Ph-0 Ph-0 10 11 A H Y ~ of hydrazine has yielded (S)-and (R)-salsolidine The biological effects of (+)-and (-)-salsolinol have been studied.2’ Although bisbenzylisoquinoline alkaloids are widely distrib- uted and numerous the first bisisoquinoline alkaloids have been reported during the period of this review. The isomeric alkaloids turcberine l2I3 and turconidine 13,22which presum- ably arise from alternative oxidative couplings of derivatives of 6,7-dihydroxytetrahydroisoquinoline,have been isolated from Berberis turcomanica. Their structures have been determined from their NMR spectra and from their fission by sodium and liquid ammonia to 6,7-dimethoxytetrahydroisoquinolineand the tetrahydroisoquinolines 14a and 14b respectively.The conformational dynamics of the two alkaloids have been studied using NMR spectroscopy.23 17 OMe OH @Me OMe Me 19 I OMe Ph’ 18 Meo9(7Me/N OMe Me 20 ,CH20H MeowNMeMe0 M e o q M eMe0 MeowNMe OMe Me OMe Me 21 22 O Me0 ENMe Me0omMe 12 13 R’omNMe R20 14a R1 = H; R2 = Me 14b R1 = Me; R2 = H l-Aryl-3,4-dihydroisoquinolinium salts of general structure 15 are reduced by sodium trisacylborohydrides to give the tetrahydroisoquinolines with limited enantiomeric excess. Norcryptostyline-I 16a obtained in this way when treated with camphorsulfonyl chloride afforded the (1R)-N-camphorsulfonyl amide in high yield.Condensation of cam- phorsulfonylhomoveratrylamine with piperonal has afforded (R)-and (5‘)-N-camphorsulfonylnorcryptostyline in a 4:3 ratio.24 The dihydroisoquinolinium salt 17 on treatment with hydride reducing agents afforded a highly selective yield of the (8)-tetrahydroisoquinoline 18 and this was reduced to ( +)-(R)-norcryptostyline-I1 16b which was N-methylated to ( -)-(R)-cryptostyline-11.l9 This process is mechanistically identical with that involved in the synthesis of (9-salsolidine 6c from 10. 3 Naphthylisoquinolines The new 5-(4’-naphthy1)isoquinoline alkaloid 4‘-0-demethylancistrocladine 19 has been isolated from Ancistro-cladus tectorius together with the three simple isoquinolines 388 Natural Product Reports I997 20 21 and 22 of a type not previously encountered which represent 0-methylated forms of putative intermediates in the biosynthesis of the naphthylisoquinoline alkaloids.25 Such isoquinolines which are derived from acetate units are biogenetically distinct from those covered in Section 2.Reaction of the Grignard reagent 23 with the chiral naph- thyloxazolines 24a and 24b has yielded products containing an 80% excess of the R atropisomer of 25a and a 92% excess of 25b. Treatment of 25b with trifluoroacetic acid and acetic anhydride afforded 26 which was converted through 27a and 27b into 27c and since this was previously converted into ( -)-0-methylancistrocladine these reactions constitute a new formal synthesis of this alkaloid.26 The predominant forma- tion of the R rotamer in this process has been attributed to approach of the Grignard reagent on the least hindered side of 24 with subsequent collapse of the intermediate 28.In similar manner the R form of 29 has been obtained in 91% excess in an approach to the synthesis of dioncophylline C.27 Sharpless asymmetric epoxidation of the olefin 30 has given the epoxide 31 and this has been converted through the amine 32 into the rotamers 0-trimethylkorupensamines A and B.28 A review of the chemistry of the alkaloids of this group has been published.29 4 Benzylisoquinolines 1 -Benzylisoquinoline alkaloids have been isolated from the following plant species OMe OMe Annona spinescens3' II reticuline MgBr 0 Berberis heterobotrys3 reticuline q> OMeR N l Berberis integerrima32 armepavine and reticuline OMe R* Berberis turcomanica' 23 24a R1 = Ph; R2 = CH20Me armepavine 24b R1 = H R2 = Pri Glaucium fluvum reticuline OMe OMe OMe OMe Litseu g~rciae~~ II reticuline Neetandra salici$olia3 coclaurine N-methylcoclaurine juziphine norjuziphine and reticuline Neolitsea villo~a~~ coclaurine juziphine and norjuziphine Ocotea atirrensi~~~ OMe OMe norarmepavine Ocotea veUo~iana~~ 25a R1 = Ph; R2 = CH20Me 26 reticuline 25b R1 = H; R2 = Pri Zanthoxylum ch~ylbeurn~~ OMe OMe oblongine and tembetarine OMe OMe Zanthoxylum us~mbarense~~ oblongine and tembetarine.Syntheses of the isomeric alkaloids annonelliptine 33a and R thalmeline 33b by the Bischler-Napieralsky route has con- firmed their structures.39 N-Trifluoroacetylnorcodamine has Meo*ol Br OMe Me0 27a R=C02H 28 27b R=CH20H 27~R=CH3 OR1 MeOQQ NCOCF3 PhAO I OMe Meo*NMeR20 0 9OMe HopcocF3 OH OMe 33a R1 = Me; R2 = H 34 33b R1 = H; Rz = Me OMe Me0 Meoa R' Me0:,pr;\cocF.OMe OMe OMe OMe Me0 k2 0 35a R1 = H; R2 =OMe 36 Me Me 35b R1 = OMe; R2 = H OMe OMe been oxidised by lead tetraacetate in the presence of (+)-(S)-30 31 2-phenylpropionic acid to give easily separable salts of the diastereoisomers of the hemiquinone 34 which in trifluoro- OMe OMe II acetic acid at 5 "C gave ( -j-(Rj and (+>-(Is? forms of N-trifluoroacetylnorwilsonirine 35a and N-trifluoro-acetylnordomesticine 35b.With trifluoroacetic acid in acetonitrile at -30 "C the norsebiferine derivative 36 was also obtained .40 The physiological effects of papa~erine,~' higenamine,42 atrac~rium~~~~ have been and of analogues of atrac~rium~~~~~ studied. OMe 2-Benzylisoquinolines all of which are new alkaloids have 32 been isolated from the following plant species Bentley 8-Pht.nylethylarnines and the isoquinoline alkaloids 389 Berber is in tegerr ima32 Berberis integerrima6= intebrimine 37a and intebrinine 37b oxyacanthine Bereberis num~laris~~*~~ Berberis numularis62 bernumine 38a bernumidine 38b and bernumicine 38c. oxyacanthine Berberis p~irettii~~ RiO MeomN, Me0 R20 Q-OR' The structures of intebrimine and intebrinine have been confirmed by syntheses of the alkaloids from salsolidine 6c by reductive condensation with veratric aldehyde and piperonal re~pectively.~' Bernumine bernumidine and bernumicine are the first N-benzyl- 1-methyltetrahydroisoquinolinesto be discovered.5 Bisbenzylisoquinolines Bisbenzylisoquinolines have been isolated from the follow- ing plant species the six marked with asterisks being new alkaloids Anisocycla jolly an^^^^^^ homoaromoline limacine limacine 2'P-N-oxide 2-norlimacine 2'-norlimacine limacusine 2'P-N-oxide* 39 0-methylpunjabine secohomoaromoline* 40 secojollya-nine* 41 1,2-dehydrotelobine trilobine and isotrilobine Berberis uem~lans~~ berbamine isotetrandrine and oxyacanthine Berberis amuvensis6' oxyacan t hine Berberis candidula5' berbamine isotetrandrine and oxyacanthine Berberis glauca6' berbamine isothalicberine 0-methylisothalicberine and 7-0-demethylisothalicberine Berberis heterobotrys3 ' berbamine obaberine and oxyacanthine berbamine isotetrandrine and oxyacanthine Berberis pr~inosa~~ berbamine isotetrandrine and oxyacanthine Berberis sibiri~a~~ berbamine and oxyacanthine Berberis turcomunica' berbamine and 0-methylthaliberine Berberis v~lgaris~~-~~ bargustanhe* 42 berbamine berbamunine and oxyacan- thine hernandiu sonora16 malekulatine Laurella ~empervirens~~ secotetrandrine* 44 Nectandru ~aliclfolia~~ costaricine" 43 Paraquilegiu anemonoideP8 fangchinoline and 0,O-dimethylcurine.Berbamine has been shown to be partially converted into its C-1 epimer penduline in methanolic solution when exposed to air a process that has been suggested to involve the C-1 free radical.69 The pharmacological and physiological effects of berb-amine,70,71 ~epharanthine,~~ dauris~line,~~~~~ da~ricine,~~-~~ ~baberine,~~ i~otetrandrine,~' tetrandrine,75*76,80-94 dimethyl-and have gri~abine,~' anti~quine~~ tub~curarine~~-~~been studied.6 Cularines Cularidine has been isolated from Ceratocapnos hetero~arpa.'~ The cis-and trans-N-oxides of cularidine have been prepared their structures being distinguished by their NMR spectra; only the trans form 45 has the configuration to permit Cope degradation to the olefin 46 on pyr~lysis.'~ The low energy ultraviolet transitions of sarcophylline have been ~tudied.~' Reaction of the lithium salt 47 with the halide 48 has afforded the thioketal 49a which was carefully hydrolysed to the phenol 49b.Oxidation of this with copper oxide in pyridine followed by hydrolysis of the thioketal gave the ketones 50a and 50b. These were converted through the oxime ethers such as 51 and the amine 52a into the amino ketal 53 cyclisation of which in acid afforded hydroxycularine 54 OMe 390 Natural Product Reports 1997 g::: -HO' OH \/ Me0 Me0 OMe Me0 OMe Me0 Me0 45 46 56 57 oJ)JJOMe OMe OMe OMe 58 47 48 0 Me0 &OH Me0 Me0 lfoH Me0 OMe Me0 R2 Me0 OMe Me0 OMe 49a R=CH20Me 50a R1 = H; R2= OMe 59 60 49b R=H 50b R1 = OMe; R2 = H Me0 OMe Me0 &,N-OMe Meo&R Meo%o \ OMe OMe Og OMe OMe /\ Me0 OMe Me0 OMe 61 62 51 52a R=NHMe 52b R=OH Me0Gs Me0 OMe NMe M e 0 2 Me 09OMe / \ Meo%Me OMe Meo%qMe0\/OMe Me0 OMe 53 54 55 which was oxidised to dioxocularine 55.The preferred approach to 53 however involved reduction of the ketone 50a to the alcohol 52b followed by esterification with toluene-p-sulfonic acid and reaction of the ester with N-methylaminoacetal.'OO Using this general approach the dehydrocularine analogue 56 was prepared and converted into 57,'"' but the product differed in properties from linaresine to which the structure 57 has been assigned."' It has been claimed that linaresine and dihydrolinaresine are identical to rugosinone 58 and dihydrorugosinone,'02 though this is diffi- cult to reconcile with the reported spectroscopic characteristics originally recorded for these alkaloids.In a novel synthesis of the rearranged cularine alkaloid clavizepine the ketone 50a has been oxidised with selenium dioxide to the diketone 59 which was reduced to the cis-diol Ben tley :p-Phen y lethylam ines and the isoquinoline alkaloids OMe OMe 63 64 60. Pinacol-pinacolone rearrangement of this diol afforded the aldehyde 61 which was converted through the alcohol into the amino acetal62. Cyclisation of this yielded the benzazepine 63 which was reduced hydrolysed and methylated to give clavizepine 64. The physiological effects of cularine have been studied.79 7 Pavines and isopavines The pavine alkaloid caryachine has been shown to have useful activity against cardiac arrhythmias.Io4 8 Benzopyrrocolines Two new benzopyrrocoline alkaloids have been isolated from Litsea cubeba namely litcubine and litcubinine which have been assigned the structures 65a and 65b on the basis of their 391 Berberis iliensis' ' berberine berberrubine and trans-N-methylcorypalmine spectra the assignment of absolute stereochemistry being based on the identity of their methyl ethers 65c with the tetramethyl ether of the product (which was assumed to have the structure 65d) of the oxidation of (5')-laudanosoline. lo' This assignment appears to have been made in ignorance of previously reported work106-'08 that clearly demonstrates that inversion at C-1 occurs during the oxidation of (S-laudanosoline and that the product of the oxidation is the mirror image of 65d.Litcubine and litcubinine clearly have structures that are enantiomeric with 65a and 65b. 9 Berberines and tetrahydroberberines Alkaloids of the berberine group have been isolated from the following plant species the nine marked with asterisks being new alkaloids Acangelisia gusanlung' O9 gusanlung D* 66 8-oxoberberine 67c &oxothalifendine* 67a and 8-oxoberberrubine* 67b Annona spinescen~~~ pessoine* 68a and spinosine* 68b Aristolochia arcuata" the betaine 69a* or its isomer 69b and its glucoside Berberis aem~lans~~ berberine columbamine jatrorrhizine and palmatine Berberis amurensis60 berberrubine and pseudopalmatine Berberis candidul~~~ berberine columbamine and jatrorrhizine Berberis heterobotry~~' berberine and jatrorrhizine Berberis heteropoda' 11*112 N-methyldihydroberberine chloride* 70 8-oxoberber-rubine and the dimeric base berpodine" 71 chloride Berberis integerrimd2 berberine Berberis numularis62 berberine Berberis p~irettii~~ berberine columbamine jatrorrhizine and palmatine Berberis pr~inosa~~ berberine columbamine jatrorrhizine and palmatine Berberis sibiricd3 berberine berberrubine 8-oxoberberine 8-oxoberber-rubine and palmatine Berberis turcomanica'3 berberine columbamine and jatrorrhizine Berberis virget~rum'~ berberine and jatrorrhizine Berberis v~lgaris~~,~~ berberine jatrorrhizine and palmatine Chelidonum majus' l4 berberine and coptisine Dact~ylocapnos torulosa' cis-N-methylstylopine chloride Enantia chlorantha" ' jatrorrhizine and palmatine Glaucium arabicum' l6 cis-N-methylcanadine chloride Meconopsis quintuplinerva' l7 mequinine" 72 Zanthoxylum ~haylbeurn~~ cis-N-methylcanadine chloride jatrorrhizine and palma- tine Zanthoxylum usambarense38 cis-N-methylcanadine chloride.Berberine has also been obtained from cultures of callus cells of Berberis pruinosa.'" Gusanlung D 66 is only the second alkaloid of this group bharatamine being the first to be discovered that has no substituent in ring D. The crystal structure of trans-N-['3C]methylcorysamine iodide has been studied confirming the 7S,13S,14s stereo-chemistry shown in 73.II9 The dihydroisoquinoline-boron tribromide complex 74 has been reacted with the lithium salt 75 to give the tetracyclic lactam 76 which on heating with Raney nickel has afforded a mixture of the lactams 8-oxostylopine 77 and 8-oxodihydrocoptisine 78.This on reduction with lithium 0 OR1 OH -0 / \ I R2 OR2 OH R' 66 67a Ri = Me; R2 = H 68a R=H 69a R1 = H; R2 = OH 67b Ri = H; R2 = Me 68b R=Me 69b R1 =OH; R2 = H 67c R' = R2 = Me OMe Me0 OMe OMe OMe HO OMe OMe OMe 70 71 72 392 Natural Product Reports 1997 10 Secoberberines Secoberberine alkaloids have been isolated from the follow- ing plant species the two marked with asterisks being new alkaloids Acungelisis gusunlung 'O9 gusanlung C* 85 Berberis virgetorum'4 73 74 oxohydrastine* 86 Corydalis dec~rnbens'~~ egenine 87 Hypecoum leptoc~rpum'~~ corydamine <SO) Hypecoum procumbens' 49 hypecorinine 88a and 8-oxohypecorinine 88b.\o 75 76 (0 TNyO HO OMe UO) 0 85 86 77 78 Me0W N y O OH OMe ? CN Ph / Ph 79 80 Me0 Me0 81 82 Me0 HO OMe 83a X =O 84 83b X=H.H aluminium hydride yielded stylopine (tetrahydrocoptisine) and dihydrocoptisine respective1y.l2' The Reissert compound 79 has been condensed with the bromo ester 80 in the presence of base to give 81 and elimination of benzoyl cyanide from this gave the isoquinoline 82. Catalytic reduction of this afforded the phenolic lactam 83a which yielded bharatamine 83b on reduction with lithium aluminium hydride.l2I Xylopinine has been synthesised by the Bischler-Napieralsky cyclisation of the lactam 84 followed by reduction of the product.'22 A review of the alkaloids of this group has been p~blished.'~~ The pharmacological and physiological effects of berberine I 24-34 tetrahydroberberine '35,136 palmatine 133 tetrahydropalmatine 35,1 377139 7-chlorobenzyltetrahydro-0-J 87 88a X=H,H 88b X=O Oxohydrastine 86 could equally well be assigned to the phthalide-isoquinoline group.Gusanlung C 85 lacks the 3-hydroxy group of dopamine assumed to be mechanistically required for the closure of the isoquinoline ring in alkaloids derived from benzylisoquinolines and from berberine.11 Protopines Alkaloids related to protopine have been isolated from the following plant species the two marked with asterisks being new alkaloids Corydalis de~umbens'~~ pro to pine Gluucium urabicum' l6 allocryptopine 13-0xoal1ocryp topine and pro to pine Gluucium cornicul~tum'~~ protopine and dihydroprotopine* 89 Gluucium~?avum~~ pro t opine Hypecoum lepto~urpum'~"'~' allocryptopine leptocarpine" 90 and protopine Hypecoum pro~umbens'~~ allocryptopine cryptopine and protopine. Leptocarpine 90 which is 8-oxoprotopine is the first lactam of this group to be discovered. Its structure was deduced from palmatine,'40.'4' 7-bromoethoxybenzyltetrahydropalmatine,'42 Do) UO) and 12-chloroscoulerine,143 ~tepholidine'~~-'~~~tylopine'~~ 0 0 have been studied.89 90 Bentley P-Phenylethylamines and the isoquinoline alkaloids 393 its spectra and confirmed by reduction to dihydroprotopine 89 which is the first alcohol of the series to be isolated from natural sources. 12 Phthalide-isoquinolines Adlumidine has been isolated from Glaucium jlav~m.~’ Adlu-midine and corlumidine have been isolated from Corydalis decumbens,14’ as has the fully aromatic alkaloid decumbenine C,Is2 the previously proposed structure for which15’ has been confirmed by detailed studies of its NMR and mass spectra. This alkaloid when first isolated was given the name hypecoumine. 54 13 Spirobenzylisoquinolines The new alkaloid isohyperectine 91 which is a diastereoisomer of the previously known hyperectine 92 has been isolated from Hypecoum erectum lS5 and together with hyperectine from Hypecoum leptocarpum .14’ 91 92 N-Acyl-3,4-dihydroxyphenylethylamines and their ethers have been found to undergo Pictet-Spengler cyclisation with arylalkyl aldehydes and ketones to give 1-benzyltetrahydro-isoquinolines.In this way N-acetylhomoveratrylamine reacts with phenylacetaldehyde and with phenylacetone to give 93a and 93b respectively and N-methanesulfonylhomo-veratrylamine reacts with indan-l,2,3-trione to give the spiro compound 94.156This approach has not yet been applied to *Me Me0 Me0 0 94 93a R=H 93b R=Me \ \ OMe OMe 95a R’ =Me; R* = OH 96a R=Me 95b R‘ = CHZPh; R2 =OH 96b R=Ph R = C6H4N02 95C R’ = CH2Ph; R2 = CH20H 96~ 96d R = 3-indolyl the synthesis of an alkaloid of the group.A series of spiro bases including 95a-c and 96a-d has been synthesised and the members tested for antibacterial activity. 157 394 Natural Product Reports 1997 t1- HN OMe HN OMe OMe 0 OMe 97 98 OMe 0 OMe 99 14 Other modified berberines A novel alkaloid dactyline 99 has been isolated from Dactylocapnos torulosa. This could possibly arise from oxo- hydrastine 86 by reaction with ammonia to give the carbinola- mide 97 followed by rearrangement to give 98 quaternisation and reduction. Two syntheses of lennoxamine 107 which have some simi- larities have been reported. In the first of these the lactone 100 was reduced with zinc and acetic acid with loss of chlorine to give the olefinic acid 101.Conversion of this into the sub- stituted amide 102 followed by successive treatments with triethyloxonium tetrafluoroborate butyllithium and trimethyl- silyl chloride resulted in the loss of hydrogen chloride and the formation of the trimethylsilylacetylene imidate 103 which was hydrolysed to the amide 104. Under the influence of a .o C13C%OMe \ eO\ M e OMe OMe 100 101 d 5 ”:-N+OEt HN-0 “+OMe TMS-AOMe CI -0Me UOMe 1 02 103 rPh rPh 9 0 w 104 105 107 -0Me palladium catalyst saturated with hydrogen this amide reacted with 3,4-methylenedioxyiodobenzeneto give 106 presumably by reductive cyclisation of the intermediate 105. Cyclisation of 106 in acid followed by catalytic reduction then afforded lennoxamine 107.'58 In the second synthesis the acetylenic amide 108 was cyclised by tributyltin hydride in the presence of aluminium borohydride to give the olefin 109 which was further cyclised by potassium tert-butoxide to 110 reduc-tive removal of the trimethylsilyl group from which yielded lennoxamine 107.159 0 0 TMS 108 (-0Bu' OMe OMe OMe 109 110 15 Emetine and related alkaloids Alkaloids related to emetine have been isolated from the following plant species the seven marked with asterisks being new alkaloids Alangium bussyanum 6o 9 (or 10)-0-demethylprotoemetinol,tubulosine O-methyl- tubulosine 9-0-demethyltubulosine deoxytubulosine and 0-demeth yldeoxytubulosine Alangium lamarckii' 61-162 isoalangiside 0-methylisoalangiside 2-0-methyl-3-0-demethylalangiside 2'-0-trans-feruloylalangiside* 11 la 2'-0-trans-feruloyl-O-demethylalangiside*11 lb 2'-0-trans-feruloyl-2-0-methyl-3-0-demethylalangiside* 11 lc 2'-0-trans-sinapoylalangiside*llld 2'-O-trans-sinapoyl-3-0-demethylalangiside* 11 le 2'-0-trans-sinapoyl-2-0-methyl-3-0-demethylalangiside*lllf and 2'-0-[4-( 1,3- hydroxypropyloxy-3-0-methyl)cinnamoyl]alangiside* 11 lg neoalangiside and 0-demethylneoalangiside.R20 OH Bentley p-Phenylethylarnines and the isoquinoline alkaloids Pictet-Spengler cyclisation of 3,4-dihydroxyphenylalanine 112 with secologanin 113 has been shown to give a highly stereoselective and regioselective yield of neoipecoside 114.163 HG,-oG1uc HOP CHO -(H \o HO NH2 Me02C 112 113 HO ,OGluc 114 Following studies on simpler models the chiral formamidine 115 has been reacted with cis- 1-chloro-4-benzyloxybut-2-ene to give a good stereochemical excess of the (19-tetrahydro- isoquinoline 116 which was converted through the secondary base into the tertiary amine 117.Cyclisation of this in the presence of an iron(m) complex afforded the benzoquinoxaline 118 which yielded homoprotoemetinol 119 on hydrolysis and reduction. Me0 Me0 Me0 0-Ph I Butoy 115 116 Me0 H, 9\\ 0-Ph 117 I 0-Ph OH 118 119 16 Benzophenanthridines Benzophenanthridine alkaloids have been isolated from the following plant species the three marked with asterisks being new alkaloids Chelidonum majus' l4 chelerythrine chelidonine and sanguinarine 395 Corydalis de~urnbens'~~ decumbenine B* 120 Hypecoum leptocarpus' 51 dihydrosanguinarine 6-acetonyldihydrosanguinarine and 6-methox ydihydrosanguinarine Zanthoxylum simuluns' 65 simulansamide* 121 Zanthoxylum ~sambarense~~ chelerythrine nitidine and usambanoline" 122.0 \ /N Me0 OH 'CHO L O OMe 120 121 Me0 CI-OMe 122 The production of alkaloids of the group during the growth of Chelidunum mujus has been studied.'66 It has long been thought that basification of sanguinarine salts 123 affords the carbinolamine 124 but it has been shown that this affords the dimeric ether 125 when basification is effected by sodium carbonate and the dimeric amine 126 when ammonia is used.These presumably arise from the initial production of 124 or the related 6-amino compound followed by the reaction of this with the remaining quaternary salt. The amine 124 must be involved in the process since 6-oxosanguinarine 127 is formed from it by disproportionation during ba~ification.'~"~~~ The formation of 125 and 126 has 0 0 LO OH 123 124 125 126 127 been studied by pulsed field gradient NMR spectroscopy. Similar reactions have been observed during the basification of salts of chelerythrine.I6' Macarpine has been synthesised from sesamol methyl ether. Friedel-Crafts reaction of this ether with 3,4-methylenedioxyphenylacetyl chloride afforded the ketone 128 which was converted into the hydroxy acid 129 by the Reformatsky reaction.Dehydration catalytic reduction and cyclodehydration of this yielded the tetralone 130 which was dehydrogenated to the phenol 131a. Conversion of this through 131b and 131c into the amide 131d followed by Bischler-Napieralsky ring closure yielded macarpine 132 confirming the placement of the methoxy groups in this alkaloid. I7O 0y L-0 L-0 128 129 0 OR2 LO 130 131a R1=R2=H 131b R' = NOp; R2 = H 131~ R' = NH2; R2= H 131d R1 = NMeCHO; R2 = Me OMe &O)/ I 0 o+NMeCi-L-0 132 The benzofuran 133 has been oxidised with osmium tetrox- ide to the diol 134 and further oxidation of this with periodic acid yielded the aldehyde 135a which was converted through 135b into 135c.This has been cyclised by phosphoryl chloride to isofagaridine (decarine) 136 but a better yield of this alkaloid was achieved by photolytic N-deformylation of 13% with concomitant cyclisation. A similar result was achieved by the hydrolysis of the amide 13% with aqueous potassium hydroxide but the yields obtained via this route were poor.I7' The olefinic ester 137 has been cyclised over palladium to the enol lactone 138 which reacted with methylamine to give the lactam 139. This ketal was cyclised through 140 to oxonitidine 141 which has been independently converted into nitidine 142 and this constitutes a new synthesis of this alkaloid.'72 Simulansamide has been shown to inhibit the aggregation of platelets.16' The physiological effects of chelerythrine,17' ~anguinarine'~',' 74 and a total alkaloid extract of CheZidonurn majus175have been studied. 17 Aporphinoid alkaloids 17.1 Proaporphines Proaporphine alkaloids have been isolated from the following plant species 396 Natural Product Reports 1997 17.2 Aporphines Aporphine alkaloids have been isolated from the follow-ing plant species the six marked with asterisks being new alkaloids co Ph <O' Ph Me 133 134 a0) / I 0 RIOVc~ONYHO HO CI-OR' OMe 135a R' = CH2Ph; R2 = AC 136 135b R1 = CH2Ph; R2 = Me 13% R' = H; R2 = Me M\e O e ) M e\ O p I ) Me0 Me0 C02Me 0 137 138 Me0 e O e ) M Me0 0 0 139 140 0 141 142 Berberis sibiricd' pronuciferine Merendera robusta' 76 robustamine 143a Nectandra ~aliclfolia~~ glaziovine.Robustamine is a new alkaloid and has been assigned the structure 143a which is epimeric with that of misramine 143b previously isolated from Roemeria hybrida on spectroscopic evidence. Bentley p-Phenylethylarnines and the isoquinoline alkaloids A con itum callibo tryod4 magnoflorine Aconitum Jirm~m~~ corytuberine Aconitum vulpari~~~ magnoflorine Actaea ~picata~~ magnoflorine Anisocycla jollyana5' remrefidine Annona reticulata' 77 asimilobine corydine norcorydine glaucine and xylopine Annonu senegalensi.~'~~.'~' anonaine boldine isoboldine isocorydine and roemerine Berberis amurensis6' amurenine* 144 Berberis heterobotrys3' isocorydine and thalicmidine Berberis iliensis' l3 magnoflorine Berberis integerrimd2 glaucine magnoflorine and thalicmidine Berber is n um ularis62 glaucine magnoflorine and thalicmidine Berberis turcomanica' ' glaucine and corydine Berberis v~lguris~~,~~ isocorydine magnoflorine and thalicmidine Corydalis marchalliana' " bulbocapnine and glaucine Desmos longijolia' ' discretamine and xylopine Glaucium corniculatum'49 corydine N-methylcorydine chloride corydine N-oxide and isocorydine N-oxide Glaucium jh~rn~~ isoboldine isocorydine glaucine and N-methyl-lauro te tanine Hernandia nymphaeifolia' 82 N-formylnorhernangerine* 145 N-formylnornantenine N-formylovigerine* 146a and N-hydroxyovigerine* 146b Hernandia sonoral corytuberine dehydrohernandaline" 147 N-methyl-hernangerine chloride 7-formyldehydronornantenine* 148 ovigerine and N-formyldehydroovigerine* 149 Litsea garci~e'~ actinodaphnine boldine isodomesticine and laurolitsine Mugnolia kobd4 magnoflorine Magnolia ~oulangeana~~ magnoflorine Magnolia spec io~a~~ magnoflorine Nectandra ramonensis36 boldine Nectandra ~alicifolia~~ boldine isoboldine corydine laurolitsine laurotetantine N-methyllaurotetanine norisocorydine and norpur-pureine Ocotea gorne~ii~~ preocoteine Ocotea me~iana~~ nandigerine Ocotea veUosi~na'~ corydine isocorydine dicentrine nordicentrine glaucine leucoxylonine ocoteine ocotominarisine and predicentrine Paraquilegia anemonoides6' magnoflorine Me0 Me0 (OFCHO 0 Me0 HO HO 144 145 Me0 Me0 CHO of$ OMe OMe 146a R=CHO 147 146b R= OH Me0 0 148 149 Telitoxicum krukovii' 83 N-fonnylnoranonaine N-formylnornuciferine and N-formyldehydronornuciferine Zanthoxylum chaylbeum3' magnoflorine Zanthoxylum ~sambarense~~ magnoflorine.It has been found that when N-cyanomethyl quaternary salts of 6-hydroxytetrahydroisoquinolines are heated with sodium methoxide in methanol 5-methoxymethyl-8-hydroxytetrahydroisoquinolines are formed. This has been explained as involving ring opening of the quaternary salt 150 to 151 followed by reaction with methoxide ion to give the Me HomN/Me Me0 Me '1 Me0&NMe I CN OH 154 155 compound resisted cyclisation under the same conditions.Following this process (5')-boldine 156 has been converted into the quaternary salt 157 and this on treatment with sodium methoxide was converted through 158 into the phenanthrene alkaloid litebamine 159.184 " Hop!N Me0 F\M "He Me0 \ / / Me0 Me0 OH OH 156 157 Me Me0 Me0 Me0 OH OH 158 159 Treatment of a mixture of homoveratrylamine 160 and cyclohexenylacetic acid 161 with trifluoroacetic acid led to cyclodehydration to the amino ketone 162 rather than to the amide and 162 has been further cyclised to the enamine 163. Dehydrogenation of this over palladium afforded de-hydronornuciferine 164 which was catalytically reduced to nornuciferine. 85 P-H OMe Me0 b'rmyea Me0TN<" MeO-CN CN 150 151 cOMe cOMe Me0@NMe Me0 OH 152 153 anion 152 which is in turn cyclised with the loss of cyanide ion to give 153.This view is supported by the fact that the phenol 154 obtained by the Emde reduction of 150 with sodium borohydride is cyclised by methoxide ion to 155 whereas the product of reduction of the isomeric 7-hydroxy-6-methoxy-398 Natural Product Reports 1997 U Me0 160 161 162 Me0 Me0 \ 1/64 MeoFH 163 2'-Bromo-N-methoxycarbonylnorcodamine165a has been photochemically cyclised to N-methoxycarbonyl-0,O-dimethylnorcorytuberine 166 in the presence of potassium phenanthrenes of structures 171a and 171b and their mono- and di-alkyl ethers has been prepared from boldine and the use HO HO MeoqNC02Me MeowNC02Me of the compounds as cardiac anti-arrhythmic agents has been patented.21 Me0 Me0 RouMeou 165a R=Me 166 165b R=H tert-butoxide and ammonia.'86 As reported in Section 4 N-trifluoroacetylnorcodamine has been converted into the R and S forms of the aporphines 35a and 35b.40 The pharmacological and physiological effects of anon-aine,67.187of boldine,'88 dicentrine,"' gla~cine,''~ N-ethoxy- carbonylnorglaucine '' isocorydine,'78 magnoline,192 nantenine,' 90 n~rstephalgine,~~ dehydro-r~emerine,'~~,'~~ roemerine,'87 xylopine'" and aporn~rphine'~~-~'~ have been studied.17.3 Dimeric aporphines A new 7,7'-dehydroaporphine dimer dactyline 167 has been isolated from Dactylocapnos torulosa.l5 This alkaloid is in a higher state of oxidation than and has a different pattern of substitution from previously identified 7,7'-dimers. 167 17.4 Phenanthrenes The preparation of litebamine 159 from boldine 156 is dis- cussed in Section 17.2. Elimination of hydrogen bromide from the bromo olefin 168 by treatment with butyllithium in tetra- hydrofuran has resulted in internal Diels-Alder addition of the derived aryne to the olefin with fission of a C-N bond to give a mixture of the atherosperminine derivative 169 and the isomeric dihydrophenanthrene 170.211A series of Me0 Me0 168 169 HO Me0 MeoKNHCO$3 Me0 Me0 OH 170 171a R=CH2Ph 171b R =Alkyl c1-C~ Bentley P-Phenylethylamines and the isoquinoline alkaloids 17.5 Oxoaporphines Oxoaporphine alkaloids have been isolated from the following plant species the three marked with asterisks being new alkaloids Annona retic~lata'~~ liriodenine oxonantenine and oxoxylopine Annona senegalensis' 79 liriodenine Artabotrys zeylanicus2' atherospermidine Desmos longifolia'" atherospermidine lanuginosine and liriodenine Hernandia nympaeifolia" hernanymphine" 173 oxohernangine* 172a and oxohern- angerine" 172b Hernandia sonora16 atheroline and hernandonine.172a R1= R2= Me 173 172b R1R2=CH2 OMe I 174 175 Hernanymphine 173 is the third aporphinoid alkaloid to be assigned a structure bearing only one oxygen substituent in positions 1 2 and 3 the others being the dioxoaporphine 174 and the dehydroaporphine sinomendine 175.21 The physiological effects of liri~denine'~~ and of oxo-glaucine216 have been studied.17.6 Dioxoaporphines The new alkaloid N-methoxynorcepharadione B (artabotrine) 176a has been isolated from Artabotrys ~eylanicus~'~ and norcepharadione B 176b 7-chloronorcepharadione B and the new alkaloid telikovinone 177 have been isolated from Teli- toxicum kr~kovii.''~ The last named alkaloid presumably arises from the interaction of ammonia with the dioxoaporphine 178a. This has not so far been identified as a natural pro- duct but its 0,N-dimethyl derivative 178b is the alkaloid ouregidione. Artabotrine has been found to be active against P388 le~kaemia.~' V M e o g M e0ONH2 O Me0A Me0 Me0 \ \ / / 176a R=OMe 177 176b R=H 178a R=H 178b R=Me 17.7 Aristolochic acids and aristolactams Aristolochic acids and aristolactams have been isolated from the following plant species the two marked with asterisks being new alkaloids Aristolochia cinnabarina21 7.21 aristolochic acid aristolochic acids I1 and 111 tuberosi- none and the N-glucosides of aristolactams I and IIIa and tuberosinone Aristolochia manchuren~is~' aristolochic acids I 11 IIIa IV and IVa and aristolochic acid B-I1 methyl ester* 179 Aristolochia tub$oraZ2' aristolochic acids I 11 IIIa and VIIa aristolactams I and I1 and 9-hydroxyaristolactam I* 180.In contrast to the behaviour of 168 the bromo olefins 181a I CHO 183 184 185 OMe MeopN Me0 MeO* \ /N Me0 MeO& J 186 187 OMe Me0 Me09NS02T~~ MeO& CN 1 88 OMe OMe Me0 MeoQ Me0 -Me02C 189 190 and 181b have been cyclised by strong base at room tempera- The isoquinoline aldehyde 184 when treated with the ture through the related arynes to cepharanone B 182a and lithium salt 185 afforded the dithioketal 186 which was 7-methoxyaristolactam B-I11 182b.2' oxidised by mercuric chloride in anhydrous methanol to the ester 187.Treatment of this with potassium cyanide and toluene-p-sulfonyl chloride yielded the Reissert compound 188 which was cyclised by potassium tert-butoxide with aromati- Me0 sation to the enamine 189 and this being the enamine of a Meop P-keto acid was hydrolysed and decarboxylated to the ketone 190.When this was reacted with the methoxymethylene- malonic acid derivative 191 the a-pyrone 192 was formed. This reacted with the cyclopropenone ketal 193 to give the Diels- Alder adduct 194 which collapsed to the tropone 195a in / OMe ethanolic hydrogen chloride. Treatment of the tropone with 1 79 180 hydrazine afforded the aminotropone 195b which was hydro- MeRPHlysed to the alkaloid grandirubrine 195c and methylation of this with diazomethane gave a mixture of imerrubrine 195d and isoimerrubrine 196.222 Me0 18 Alkaloids of the morphine group \ Alkaloids related to morphine have been isolated from the / following plant species the five marked with asterisks being RQ! Me0 new alkaloids R R 181a R=H 182a R = H 181b R=OMe 182b R =OMe 17.8 Azafluoranthenes and related alkaloids The new alkaloid pareitropone 183 has been isolated from Cissampelos pareira.221 This represents a new structural type being the first tropone rather than tropolone discovered among the isoquinoline alkaloids.It has been shown to have potent cyto toxic activity .22 400 Natural Product Reports 1997 Cissampelos sympodiali~~~~ milonine* 197 Cocculus trilob~s~~~ isosinococculine* 198 Nectandra salicifo lia3 sebiferine Stephania ~epharantha~'~ aknadicine aknadinine aknadilactam cephakicine" 199a alkaloid FK3000 199b cepharamine cepharasine* 200 cephatonine* 201 14-episinomenine sinoacutine and tannagine Meo+ox 0 0 191 193 OMe 202 203 MeO- \ Meoy,,%) Meo*NMe0 '0 192 H' Ho 194 Me Me 204 Br 205a R=H 205b R=OMe 205~R=CI OMe OMe 205d R=Br Me0 R 0 195a R=H 195b R=NH2 196 4-N 195~R =OH Ph Me0 195d R=OMe 206a R=OMe 207a R= H 206b R=H 207b R=Ac 206~R =CI OMe 206d R= Br Me0 Me0" P N M'H e MeonRO Ph0 0 197 198 199a R=Me Meo@o "Me 0 H,' \ Me0 OMe 200 Stephania sutchuenensis226 199b R=H ?H Ph Ph 208a R= H 209a R = H 208b R=Ac 209b R=Ac -.- - -NMe addition of the dienophile on the least-hindered face of the / OMe diene.Addition of the same dienophile to the conformationally OMe more open p-dihydrothebaine 207a and its acetyl ester 207b however affords mixtures in which the major products are the 201 em-etheno-adducts 208a and 208b and the 1-substituted com- pounds 209a and 209b.The thebaine adduct 206a is decom- posed to its components by a retro-Diels-Alder reaction in polar aprotic solvents in the presence of bases with low aknadinine 1-nitroaknadinine and sinococculine. nucleophilic character.229 The Diels-Alder adduct 210a of 6a,7a-Methylenedihydrothebaine 202 has been prepared N-cyclopropylmethylnorthebaine and cyclopropenone has in high yield by treating thebaine with diazomethane in been C-methylated in the presence of strong base through the the presence of copper(I1) trifl~oromethanesulfonate.~~~two enol forms without inversion to give 210b and 2lO~.~~" a-Chlorocodide 203 and bromocodide 204 readily form n-complexes with iron tricarbonyl and pentacarbonyl and with molybdenum tricarbonyl which can be dehydrohalogenated to the n-diene complexes of 6-demethoxythebaine 205a.228 Thebaine 205b N-formylnorthebaine 6-demethoxythebaine 205a 6-chloro-20% and 6-bromo-6-demethoxythebaine 205d undergo Diels-Alder reaction with 4-phenyl-4H- 1,2,4- trazoline-3,4-dione to give the endo-etheno adducts 206a its N-formylnor-analogue 206b 206c and 206d respectively by Bentley p-Phenylethylarnines and the isoguinoline alkaloids The 5,7-bridged adduct 211 has been prepared for studies of opiate receptor binding.231 0-Benzylsinomenine 212 has been reduced to the 6P-alcohol 213a which has been converted through 213b and 213c into the 6a-selenide 214.Oxidation of this with hydrogen peroxide first gave the diene 215 and then the hydroxy enone 216 which was further oxidised by cobalt chloride and dimethyl sulfoxide to 217a.The enol ether of this 217b was catalytically reduced to the dihydroketone the lithium enolate of which was 40 1 HoQ pi‘Me 210a R1 = R2 = H 21 1 210b Ri =Me; R2 = H 210c R’ = R2= Me Ph NMe HOV OMe OMe 21 2 213a R = Me 213b R=H 213~R = C02CH2Ph ph) ‘NBoc 02N6 T e OMe 214 21 5 Me0 ‘NBoc OR 0 21 6 217a R=H 217b R=Me oxidised to a mixture of 218a and 218b. Of these 218a was reduced to a mixture of sinococculine 219 and 7-episino- cocculine 220 and reduction of 218b afforded the C-6 epimers of these. The 6P-selenide isomeric with 214 obtained from the 6a-alcohol on treatment with hydrogen peroxide afforded a mixture of 215 and the enone 222 presumably formed by a sigmatropic rearrangement of the intermediate selenoxide to 221 before further oxidation.232 Hofmann degradation of sebiferine methiodide has yielded the phenanthrene 224 presumably by elimination of dimethyl- aminoethanol from 223.233 7,7-Diphenyldihydrocodeinone 225a is demethylated to 225b in 10min by boron tribromide in dichloromethane but after 30min the main product is the rearranged alcohol 226 and after 2 h is the bromo olefin 227.In this process it must be the axial phenyl group that migrates in 225 since a similar rearrangement is observed with 228a but not with 22813 in which the methyl group occupies the axial position. 7-Phenyldihydrocodeinone the stable isomer of which has the phenyl group in the equatorial position 228c is stable to this rearrangement but over four days is converted into 7-phenylmorphine 229 in the absence of air which oxidises the product to the ketone 7-phenylm0rphinone.~~~ 402 Natural Product Reports 1997 OH 220 Me0 Me0 Ph@o .’NBoc I Ph OMe Ar OMe 221 222 ?Me OMe NMe2 Me0 Me0 0) OH 223 224 HO -Br Ph 225a R=Me 226 225b R=H Meo* @ op0.p 0. NMe NMe NMe \ / Ph Br 0 HO‘. Ph Ri R2 Ph 227 228a R1 =Me; R2= Ph 229 228b R’ = Ph; R2 = Me 228~R’ = Ph; R2 = H Details of the preparation of the following principally for pharmacological studies have been reported 3-0-ethylmorphine-6-gl~curonide,~~~ tritium-labelled 6P-amino-deoxymorphine and 6P-amino- 14-hydro~ymorphine,~~~ the amide 230,2377-thiocyanato-6-demethoxythebaine231,238de-rivatives of 14-hydroxydihydromorphinone of structure 232,239 3-deoxy-14-methoxy-N-phenethylnordihydromorphinone,240 4,5-deoxy- 14-hydroxydihydromorphinone and its deriva-tive~,~~’ derivative the 10-oxo-6J3-thiodihydrodeoxymorphine 233,242 7-ben~ylidenenaltrexone~~’ (also labelled with tri-ti~m~~~) the dithiobases 234 and 235,246the nalb~phine,~~~ dimeric base 236,247‘251-labelled naltrind~le,~~~ derivatives of HO %\Me R’ Q.AJ s kCF3 AcSM A N=N C=N=S 0 231 232 233 234 230 R1 = H or OH R2 = Me or But R3 = H Me or Et R4 = CH&H=CH* or CH~-<] I 235 237a X=NH 238 237b X=O 237~X=S and of its analogues 237b 237c and on the cardiovascular sy~tem,~”~~~~ naltrindole 237a24’*250 on the gastrointestinal 238,250.25 et~rphine,’~~ tritium-labelled on the brain,302-305 on the liver,’06 on re~piration,~’~ I b~prenorphine,~~~ b~prenorphine’~~.~~~ ‘251-labelled on locomotor activity,308 on immune respon~es,~~)’-”~ and dihydr~morphine,’~~ on 2-fluoroapocodeine lymphocyte^,^ ’63317 on macro phage^,"*-^^' on the production 19-0-alkyl ethers of dipren~rphine,’~~ Methods of estimation of of inter leu kin^'^^,^^^ and of tumour necrosis on and 2-fl~oroapomorphine.’~~ and m~rphine,~~~.~”of morphine 3-glucuronide and 6-phagocytosis,322 on killer cell cytotoxicity,323 on neuro-on glucuronide’60 have been described.transmitter sy~tems,~’~p’’~ motor”’ and sensory3” 333 Preferential 8,2’-coupling of 2’-bromo-N-methoxy-neurones on trigeminal ganglia,’34 on hippocampal field carbonylnorreticuline 165b to give N-methoxycarbonyl-potentials,335 on the spinal cord,336,337 on responses to 342 on thyroid343 and norsalutaridine 239 has been accomplished in the presence of stress,338 on the new-b~rn,~’’ palladium chloride triphenylphosphine potassium carbonate function on the synthesis of RNA345and of gene proteins,346 and dimethylformamide at 140 “C.lg6 on the binding of DNA to proteins,347 on the release of brad~kinin,’~~ dynorphins and enkephalin~,’~~-~~’ of of cat echo la mine^,^^^-^^^ of hi~tamine,’~~,~~~ of y-aminobutyric acid,353,358 of acetyl~holine,~~’.~~~ of prolac- of o~ytocin,~~’ tin,362 of prostagladin E,,”’ and of vasopressin,’6’ on the Meon on HO activity of nitric oxide ~ynthetase,’~~ the somatostatin receptor in breast cancer,364 on Friend retrovirus infection,36s on taste perception,366 and on the effects of A review of the clinical use of morphine has been The morphine antagonist proper tie^^^'-^^^ and the pharma- Me0Tco2Me 0 c~kinetics’~~ of naloxone have been studied as have the effects of this compound on behavio~r,~~~ on anaphylaxis,377 on 239 anxiety,378 on respirati~n,~~’.~~~ on vaso-vagal responses,381 on sensory”’ and motor383 neurones on pr~ritus,~“,’~~ on the kidney,386 on the release of acetylcholine,360 of adreno-The analgesic effects of morphine and in com- corticotropic hormone387 and of ~~rti~~~ter~ne,~*~ and on bination with dextromethorphan,280 with clonidine281*282 and the effects of barbiturate^,'^^ of and of with scopolamine,283 the cataleptic effect of the alkaloid,284 the apomorphine.388 binding of morphine to analgesic receptors,285 and the phar- The pharmacological and physiological effects of the follow- macokinetics of the alkaloid after have been ing have also been studied 6-0-acetylmorphine,3s” 3-0-acetyl- studied as have the effects of morphine on behavio~r,~’~-~’~ morphine 3-gluc~ronide,~~”~~~~~’~ morphine-6-~ulfate,~’~ -393 Bentley P-Phenylethylarnines and the isoguinoline alkaloids 403 morphine 6-glucuronide,286~2883389~393395 dihydr~codeine,~~~,~~~ 3-O-ethylrn0rphine,~'~ dihydro-Me0 HAc HAc :::q morphin~ne,~~~ 5-methyldihydromorphinone (rnetop~n),~"~ naltrex~ne,~~~'~~~~'~ nalo~onazine,~'~ methylnaltre~one,~~' Me0 6-N-cinnamoyl and 6-N-(p-nitrocinnamoyl)-naltrexamine,4'6 Me0 0 R nalbufine,354.384,417.418 nal~~lefene,~~' naltrindole and its oxygen and sulfur analogue^,^' 9423 the 6-and 8-pyridinyl- R 0 dithio-compounds 234 and 235,246 n~rbinaltorphimine,~~~ 244a R=OMe 245a R=NMe2 dihydroet~rphine,~~~?~~~ et~rphine,~~~ b~prenorphine,~~~~~~244b R=OPri 244~R=OBU 245b R=Ns aknadinine,226 l-nitroaknadinine,226 and sinococculine.226 19 Phenethylisoquinolines Four new homoaporphine alkaloids have been identified.(+)-Kreysigine N-oxide 240a and (+)-szovitsamine N-oxide 240b have been isolated from Colchicum vit~hiz~~" and mero- bustine 241a and merobustinine 242 have been isolated from Merendera robust^.^^' Me0 RO Me0 I Me0 Me0 240a R1= H; R2 = Me 241a R=H 240b R1= Me; R2 = H 241b R=Me HO Me0 Me0 Me0 Me0 OMe 242 243 Kreysigine 241b has been obtained in 71% yield by the oxidation of 7-O-benzyl-3'-0-methylautumnaline243 with ferric chloride in dichloromethane followed by reductive deben~ylation.~~' 20 Colchicine Colchicine 2-O-demethyldemecolcine cornigerir and 3-0-demethylcornigerine have been isolated from Colchicum speciosumU2 and speciosine and its methyl ether (which is a new alkaloid) have been isolated together with P-lumidemecolcine and P-lumicornigerine from Colchicum rit~hii.~~~ Colchicine 244a has been transesterified by heating with isopropanol and butanol in the presence of the corresponding titanium or zirconium alkoxides to give 244b and 244c,443and colchiceine 244d has been converted by borontrifluoride ether- ate followed by dimethylamine or piperidine into mixtures of 244e and 245a and of 244f and 245b.4444-Formylcolchicine 246a on treatment with tributoxyzirconium methyl has afforded a mixture of the epimeric alcohols 246b and 246c both of which yielded 4-acetylcolchicine 246c on oxidation with phosgene and dimethyls~lfoxide.~~' Colchicine labelled at C-9 and C-10 with "C and I3C has been prepared.446 The 'H 244d R=OH 244e R=NMe2 244f R = N s Meom OMe 246a R=CHO 246b R = CH(0H)Me 246c R=COMe 21 Evythvina alkaloids 21.1 Homoerythrina alkaloids Comosivine dyshomoerythrine 2,7-dihydrohomoerysotrine holidinine 3-epi- 12-hydroxyschelhammericine and lenticellar- ine have been isolated from Lagarostrobos c~lensoi.~~~ The keto ester 248 prepared as previously reported from 247 and 2-trimethylsilyloxybutadiene,has been reduced and hydrolysed to the hydroxy ketone 249 the methansulfonyl ester of which in base afforded the cyclopropane derivative 0 0 (%: OTMS 0 247 248 (Oa$o 0 <so 'OH C02Me C02Me 0 249 250 250.Treatment of this with phenylselenyl chloride and boron trifluoride followed by oxidation with mercuric perchlorate gave the keto ketal 251 which was reduced to the alcohol and the methyldithiocarbonate of this on treatment with tributyltin hydride yielded the enone 252a.This was hydrolysed and decarboxylated to 252b which was isomerised to the. P,y-unsaturated ketone 253 by calcium chloride in dimethyl- sulfoxide. Reduction of this with tetra-tert-butylammonium borohydride afforded a 1% mixture of the alcohols 254a and 254b whereas reduction with sodium borohydride gave a 5:1 mixture of the same compounds. O-Methylation of these and I3CNMR spectra of thiocolchicine have been analy~ed.~~~ A process for the radioimmuno-assay of thiocolchicoside has alcohols and reduction of the lactams with lithium aluminium been hydride afforded 3-epischelhammericine 255a and schelham- The biological effects of colchicine have been studied.449456 mericine 255b.458 404 Natural Product Reports 1997 251 252a R=C02Me 252b R=H 253 255a R1= OMe; R2= H 256a R1=OH; R2= H 255b R1 = H; R2= OMe 256b R1= H; R2= OH 257a R1 =OH; R2= H 258a R1=OH R2 = H 257b R'=H R2=OH 258b R' = H; R2= OH Reduction of the a,P-unsaturated ketone 252b with the same two reagents afforded similar proportions of the epimeric alcohols 256a and 256b together with the saturated ketones 257a and 257b.The mixtures were most conveniently separated after U-methylation and the methyl ethers were reduced with lithium aluminium hydride to comosivine 258a dihydroschel-hammeridine 258b and their dihydro-deri~atives.~~~ 21.2 Cephalotaxine and related alkaloids The clinical response to homoharringtonine of patients in the late chronic phase of myeloid le~kaemia,~~' and the response to the alkaloid alone and in combination with daunorubicine of sufferers from acute nonlymphocytic le~kaemia~~' have been studied.22 Other isoquinoline alkaloids Jamtinine has been isolated from Cocculus hirs~tus.~~~ The new alkaloid leptocarpine 259 isolated from Hypecoum leptocar-pus,'48is not an isoquinoline but since it occurs together with protopine allocryptopine the secoberberine corydamine and e 0 259 OMe the spirobenzylisoquinoline hyperectine and isohyperectine it is likely derived from an isoquinoline probably related to berberine. 23 References 1 M. 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ISSN:0265-0568    

DOI:10.1039/NP9971400387

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Recent progress in the chemistry of non-monoterpenoid indole alkaloids Masataka Ihara and Keiichiro Fukumoto Pharmaceutical Institute Tohoku University Aobayama Sendai 980-77 Japan Covering July 1995 to June 1996 Trikentrin B 5 has been synthesised enantioselectively (Scheme Previous reviews 1996 13 241 l)." The chiral sulfoxide 6 was converted into the allene 7 which was subjected to an intramolecular Diels-Alder reac-1 Introduction tion. The indole derivative 8 obtained after aromatisation was 2 Simple alkaloids transformed into cis-trikentrin B 5. 2.1 Non- tryp tamines The structures of clausenol 9 and clausenine 10 isolated 2.2 Non-isoprenoid tryptamines from an alcoholic extract of the stem bark of Clausena anisata 3 Isoprenoid indole alkaloids have been established from physical and chemical evidence and 3.1 Ergot alkaloids ~ynthesis.~ Clausenol 9 was found to be active against Gram- 4 Bisindole alkaloids positive bacteria and fungi.Clausena Zansium root bark gave 5 References the new carbazole alkaloid 11.6A novel neuronal cell protect- ing substance lavanduquinocin 12 has been isolated from 1 Introduction Streptomyces uiridochromogenes 2942-SVS3.7 The structures of One chapter in Volume 47 of The Alkaloids is concerned with murrayamines D 13 E 14 I 15 K 16 J 17 N 18 M 19 0 20 non-iridoid bisindole alkaloids.' Reviews of marine natural and P 21 isolated from Murraya euchrestifoliu have been products2" and antiinsectan natural products2b including a determined by spectral analyses.'-'' number of indole alkaloids have been published.The free radical scavenger carazostatin 22 and the marine alkaloid hyellazole 23 have been synthesised by way of an allene-mediated electrocyclic reaction (Scheme 2). ' ' The prop- 2 Simple alkaloids 2-ynylindole 25 derived form 2-formyl-3-iodoindole 24 was 2.1 Non-tryptamines Four indole derivatives 14 have been isolated from Xeno-rhabdus hovienii A2. Compounds 1 and 2 showed antibiotic activity against Cryptococcus neoformans while compounds 3 and 4 showed activity against Phytophthora infest an^.^ cis-MeoQ-lMe HoayqcHo OH R 0 0 11 1 9 Clausenol R =OH 10 Clausenine R=OMe H H 1 R=Ac 2 R=Ac 3 R=H 4 R=H hyqo ..,,\OH Me ,OTr 12 Lavanduquinocin 7 Men = do-I i 160°C A ii Mn02 13 Murrayamine D 14 Murrayamine E OTr I R OAc 5 cis-Trikentrin B 8 R = Pivaloyl 15 Murrayamine I R = OH Scheme 1 16 Murrayamine K R = H Ihara and Fukurnoto Recent progress in the chemistry of non-monoterpenoid indole alkaloids 413 28 29 R=C7H15or Ph 17 MurrayamineJ R=H 18 MurrayamineN R = OMe 19 MurrayamineM 20 Murrayamine0 R1 =Me; R2=OH 21 MurrayamineP R' =OH; R2 = Me a H O PhSO; OMOM 24 25 QyqoEt- Me QyQEt PhS02 OMOM PhS02 OMOM J 27 L 26 t 22 Carazostatin Ri = C7HI5; R2 = H 23 Hyellazole R1 = Ph; R2 = Me Scheme 2 treated with potassium tert-butoxide in hot tert-butanol to give the carbazole 27 via the allene intermediate 26.The product 27 was transformed into alkaloids 22 and 23 through Suzuki cross-coupling reaction.' Carazostatin 22 and hyellazole 23 have been further synthesised by iron-mediated cyclisation. l2 Reaction of iron complex salt 28 with arylamines 29 resulted in electrophilic aromatic substitution to afford the iron com-plex 30 (Scheme 3). Oxidation of 30 with commercial man-genese dioxide followed by further oxidation with activated manganese dioxide yielded the 4b,8a-dihydrocarbazol-3-ones 31. Both alkaloids 2213 and 2314 were prepared from 31. 414 Natural Product Reports 1997 Carbazomycin B 32 an inhibitor of 5-lipoxygenase was pre-pared through Diels-Alder reaction of the vinylindole 33 with dimethyl acetylenedicarboxylate (Scheme 4).*' ~~f0c02Et C02M: OMe TMe OC02Et Heat C02Me C02Et Et02C C02Me 33 1 1 ?H Qy.d$:""' Me Me 32 Carbazomycin B Scheme 4 Almazole D 34 which shows potent antibacterial activity has been isolated from a delesseriacean seaweed collected near Dakar.I6 Neocryptolepine 35 has been isolated from the root bark extract of the African medicinal plant Cryptolepis s~nguinolenta.~Full details regarding the synthesis of grossularines-1 36 as well as -2 37 have been reported." Treatment of the amido ketone 38 with sodium hydride provided the tetracyclic compound 39 which was converted into hydrocryptolepine 40 and cryptolepine 41 (Scheme 5).l9 Epoxide opening on reaction with indole is accelerated under high pressure conditions in the presence of a catalytic amount of ytterbium(II1) triflate.The procedure was applied to the enantioselective synthesis of diolmycin A2 42 from 43 (Scheme 6).20 ( f)-Mitomycin K 44 was synthesised in 13 steps from commercially available 2,5-dirnethylani~ole.~~ One of the key steps is the oxidation of azidomitosene 45" with (hexamethyl-phosphoramido)oxodiperoxomolybdenium(vr) to give 46 9 34 Almazole D Me 35 Neocryptolepine NMe2 Nd 36 Grossularine-1 R = H 37 Grossularine-2 R = H O O R PhCOHN NaH ---D a-@ PhS02 0 0 38 39 R=COPh 40 Hydroxycryptolepine R=Me 1 41 Cryptolepine Scheme 5 i@ H Yb(OTf)3 10 kbar * ii H2 Pd(OH)2-C 43 I OH moH together with the stereoisomer (Scheme 7). Various 6-demethylmitomysins and 6-demethyl-6-halomitomysins were prepared and evaluated for antitumour activitie~.~~ TBSO MoOS-HM PA MeOH Me N3 TBSO "'0 Ms 45 TBS? -TBS~ L-/ NMe 'OMS 46 44 Mitomycin K Scheme 7 2.2 Non-isoprenoid tryptamines Stellarines A 47 and B 48 have been isolated from Stellaria dichotoma var.lan~eolata.~~ N9-Substituted P-carboline alka- loids didemnolines A 49 B 50 C 51 and D 52 have been isolated from an ascidian of the genus Didemnum collected near the island of Rota Northern Nariana Islands.25 Cl-Substituted P-carboline alkaloid hyrtiomanzamine 53,was found in the marine sponge Hyrtios erecta collected in the Red sea,26 and showed immunosuppressive activity in the B lymphocytes reaction assay. O:QCONHR H 0 AMe 47 Stellarine A R = H C02Me 48 Stellarine B R = MeS y!) II 0 49 Didemnoline A R = Br 51 Didemnoline C R = Br 50 Didemnoline B R = H 52 Didemnoline D R = H 53 Hyrtiomanzamine Bauerine B 5427 and eudistomins D 5528 and T 5629 were H synthesised by a convergent method which involved metalla- 42 Diolrnycin A2 tion hetero-ring cross-coupling and cyclisation.Further Scheme 6 methodologies for the synthesis of P-carbolines have been lhara and Fukumoto Recent progress in the chemistry of non-monoterpenoid indole alkaloids Br X 54 Bauerine B 55 Eudistomin D 56 Eudistomin T 57 developed3' 31 and a naturally occurring furyl-substituted p-carboline 57 has been ~ynthesised.~~ The Pictet-Spengler condensation is very effective for the construction of a-carbolines and the reaction has been reviewed from a new point of view.33 Asymmetric Pictet- Spengler reactions have been successfully carried out by several The Pictet-Spengler reaction utilising tryptophan linked to the Kaiser oxime resin was applied for synthesis of P-carboline libraries.38 Reaction of the P-carboline 58 with methyl iodide followed by reduction with sodium .i Me1 QTQNMe ii NaBH4 HH d' T Y \ '"*' Boc Ph Boc Ph 58 59 Scheme 8 borohydride afforded diastereoselectively the tetrahydro-p- carboline 59 (Scheme 8).39 Catalytic asymrnetic hydrogenation of imines 60 giving (R)-isomers 61 was achieved in a highly enantioselective manner in the presence of ruthenium(I1) catalyst 62 formic acid and triethylamine in dimethylformamide (Scheme 9).4" HI R 60 R=Me and Ph 62 Scheme 9 Eudistomins 63 shows strong; antiviral activity against v -Herpes simplex virus (HSV-1) and activities against certain types of tumours in vivo.It has been shown that 12-carbaeudistomin analogues have similar a~tivities.~' Nucleophilic addition of the chiral enolate 65 with the chiral iminium ion 64 took place with total stereochemical control (Scheme I O).42 The product 66 possesses the appropriate stereochemistry for the synthesis of eudistomins. 416 Natural Product Reports 1997 63 Eudistomins h p o y NONaP h I 64 66 Scheme 10 ( -)-Horsfiline 67 was synthesised by the Tc-facial diastereo- selective 1,3-dipolar cycloaddition of N-methylazomethine ~lide.~~ Nauclefine 68 angustine 69 dihydroangustine 70 and naucletine 71 were prepared by the enamide cyclisation followed by palladium(0)-catalysed reactions.44 MeO~,,,pMe QyJ II \ N O H R \N 67 Horsfiline 68 Nauclefine R = H 69 Angustine R = CH = CH2 70 Dihydroangustine R = Et 71 Naucletine R = Ac 72 1-Methoxyrutaecarpine 75 Elaeocarpidine R = OMe R = P-H 73 Rutaecarpine 76 Epielaeocarpidine R=H R = a-H 74 1-Hydroxyrutaecarpine R=OH I-Methoxyrutaecarpine 72 has been isolated together with known alkaloids rutaecarpine 73 and 1-hydroxyrutaecarpine 74 from Zunthoxylum integrif~liolum.~~ 1-Hydroxyrutae-carpine 74 exhibits antiplatelet activity.45 It was shown that rutaecarpine 73 produces a full nitric oxide-dependent va~odilatation.~~ Two known alkaloids elaeocarpidine 75 and epielaeocarpidine 76 were obtained from leaves of Pelargonium species.47 Cytotoxic peptides hemiasterlins A 77 and B 78 and heptapeptide segetaline E 85 has been obtained from the criamides A 79 and B 80 have been isolated from the marine seeds of F segetali~.~' The elucidation of the stereostructure sponge Cymbastela species4' A cyclopeptide alkaloid mauri- and conformational analysis of lyciumin A 86 isolated from tine J 81 was found in the root bark of Zizyphus ma~ritiana.~~ Lycium chinense have been carried Microviridins B 87 The structure was established by comparison with amphibine and C 88 have been obtained from the freshwater blue-green E 82.Cyclic peptides segetalins A 83 and B 84 isolated from alga Microcystis aeruginosa (NIES-298)53 and it has been A cyclic shown that they potently inhibit elastase. Vaccaria segetalis show an estrogen-like a~tivity.~' R 77 Hemiasterlin A R = Me 78 Hemiasterlin B R = H R 79 Criamide A R = H 80 CriamideB R=Me R I 81 M auritine J R = H 82 Amphibine E R = Me 83 Segetalin A H II NH H 85 Segetalin E I 0 0 86 Lyciumin A A novel class of cytochalasans penochalasins A 89 B 90 and C 91 have been isolated from a strain of Penicillium species originally separated from the marine alga Entero- morpha intestinali~.~~ Different conformations of penochalasin A 89 in CDC1 and [2H,]pyridine were determined.All the compounds exhibited potent cytotoxicity against cultured P 388 cells. A stereoselective synthesis of optically active 2-bromo-5- hydroxytryptophan 92 which is present in two cyclopeptides konbamide and jaspamide was achieved via a regiospecific bromination pr~cedure.~~Optically active 6-chloro-5- hydroxytryptophan 93 which is present in the cyclic hexa- peptide keramamide A has also been enantioselectively ~ythesised.~~ Two diastereoisomers of konbamide 94 have been prepared but the natural konbamide was not identical with synthetic The total synthesis of nephilatoxin-7 (NPTX-7) 95 a neurotoxin of Joro spider (Nephila clavata) was achieved by employing an azide ~trategy.~' 0 Two diketopiperazines maremycins A 96 and B 97 have been found in the culture broth of the marine Streptomyces species B 9173.59 From an unidentified Penicillium species a known diketopiperazine 98 which is a plant growth regulator has been isolated.The absolute configuration was deter-mined from its CD spectrum.60 An N-carboxyindole alkaloid pallidin 99 has been obtained from the sponge Rhaphisia 84 Segetalin B pallida."' The structure was elucidated by spectroscopic Ihara and Fukumoto Recent progress in the chemistry of non-monoterpenoid indole alkaloids C02H 87 Microviridin B /,,,,,,,A 0 89 Penochalasin A 0 90 Penochalasin B 0 91 Penochalasin C methods. Three diketopiperazines tryprostatins A 100 and B 101 and demethoxyfumitremorgin C-102 have been isolated together with known diketopiperazines from the fermentation 418 Natural Product Reports.1997 88 Microviridin C 92 93 94 Konbamide broth of Aspergillus fumigatus BM 939.62 They showed an inhibitory activity on the cell cycle progression in mice. Seven fumiquinazolines (FQs) A 103 B 104 C 108 D 109 E 105 F 106 and G 107 have been obtained from a strain of A. fumigatus originally separated from the marine fish Pseudo-labrus japoni~us.~~ All the compounds showed moderate cyto- toxicity against cultured P 388 cells. Full details regarding a stereocontrolled synthesis of (+)-paraherquamide B 110 have been published.64 Two canthin-6-one alkaloids bruceollines C 111 and G 112 have been isolated from the root bark and root wood of Brucea mollis var. tokinensis together with several known canthin- 6-one alkaloids carboline alkaloids and quas~inoids.~~ The carbocyclic analogue 114 of canthin-6-one has been prepared by the intramolecular Diels-Alder reaction of the acetylene 113 in refluxing sulfolane (Scheme 11).66 95 Nephilatoxin-7 cJT$r/~H~.H,c C02H 0 Me 0 SMe 96 Maremycin A R = a-OH 99 Pallidin 97 Maremycin 6 R = P-OH 100 Tryprostatin A R = OMe 102 DemethoxyfumitremorginC 101 Tryprostatin B R = H Yo \ Me 0 / 0 110 Paraherquamide B 0 R3 R' 103 FumiquinazolineA 106 Fumiquinazoline F Ri =Me; R2= H R1=Me; R2 = H 104 Fumiquinazoline B 107 Fumiquinazoline G R1 = H;R2 = Me R1 = H; R2= Me 0 105 Fumiquinazoline E 111 Bruceolline C R1 = R3 = OMe; R2 = OH Ri = Me R2 = OMe 112 Bruceolline G R1 = R2 = H; R3 = 0-Glu aTy ___) 1 Sulfolane 0u oT\ 113 108 Fumiquinazoline C 109 Fumiqunazoline D a-2 Tricyclic marine alkaloids isobatzelline C 115 makalu-vamine A 116 batzelline C 117 and damirone A 118 have been synthesised starting with both an indole derivative 11967and a 0 quinoline derivative 12068(Scheme 12).Four halogenated indole-imidazole alkaloids securamines A 121 B 122 C 123 and D 124 were isolated from the marine Scheme 11 114 Ihara and Fukumoto Recent progress in the chemistry of non-monoterpenoid indole ulkuloids 419 N/\ 119 115 lsobatzelline C R = CI 116 Makaluvamine A N+ R=H f Me0 Me0 120 Me 8 117 Batzelline C R = CI 118 Damirone A R=H Scheme 12 bryozoan SecurlJustva secuvifyurzs.69It has been shown that securamines A 121 and B 122 are in equilibrium with two macrocyclic alkaloids securines A 125 and B 126 respectively (Scheme 13).R R* CI NLBr H 0 121 Securamine A 123 Securamine C R=H R = Br 122 Securamine B 124 Securamine D R = Br R=H ti Br 125 Securine A R=H 126 Securine B R = Br Scheme 13 Full details about an asymmetric synthesis of ( -)-eserethole 12770 and a formal synthesis of ( f)-physostigmine 12S7' respectively have been recorded. ( f)-Deoxyeseroline 129 has been synthesised via a 3,3-sigmatropic rearrangement (Scheme 14).72Thermolysis of the bis-enamine 130 yielded the tricyclic compound 132 in a moderate yield through the ring closure of the rearranged product 131.420 Natural Product Reports 1997 IHI Me Me 127 Eserethole R = Et 128 Physostigmine R = CONHMe C02Me I MeN / -C02Me 3 Me 130 131 C02Me IHI HH I Me Me Me 129 Deoxyeseroline 132 Scheme 14 Indole derivatives have been sythesised by a titanium-induced reductive 0x0 amide coupling reaction. Reaction of the 0x0 amide 133 with titanium-graphite gave the cyclised product which was converted into the indole alkaloid indolo- pyridocoline 134 (Scheme 15).73 Secofascaplysin 135 was prepared by the same strategy. n e01Ti-graphite "Yo \ (TiCI3-C8K) ____) NH 135 Secofascaplysin 134 lndolopyridocoline Scheme 15 The stereostructure of a new manzamine congener man- zamine L 136 isolated from Amphimedon species has been elucidated by spectroscopic studies including CD spectra.74 Synthesis of manzamine alkaloids is currently under way and several synthetic approaches have been p~blished.~~-~~ 136 Manzamine L 3 Isoprenoid indole alkaloids An indole-diterpenoid alkaloid paspaline B 137 was isolated from Penicillium paxilli together with known alkaloids paspaline and 12-de~oxypaxilline.~~ Eight indole diterpenoids terpendoles E 138 F 139 G 140 H 141 I 142 J 143 K 144 and L 145.were found in the culture broth of Albophoma yamanashiensis using a different production medium.80 Ter- pendoles J 143 K 144 and L 145 showed moderate inhibition of acetyl-CoA cholesterol acyltransferase (ACAT) activity in rat liver microsomes while terpendoles E-I (138-142) showed weak activity.Paxinorol 146 a modified indole-diterpenoid which is believed to be derived from paxilline 147 has been detected with the immunoblot technique coupled to TLC analysis (TLC-ELISAgram) as a trace contaminant of a paxilline-con taining extract from the fungus Penicilluim paxilIi. ' The F-G-H ring lactone precursor 152 of penitrem D 148 has been synthesised in a stereocontrolled manner (Scheme 16).82 The symmetrical ketone 149 was enantioselectively con- verted into chiral enone 150 which was transformed through L-i,+OH H 0 148 Penitrem D the introduction of the vicinal quaternary methyl groups into 151. The putative intermediate 152 was prepared from 151 by a conventional procedure.For the purpose of the synthesis of radarin A 153 a cytotoxic alkaloid the construction of the tricyclic diterpene part 157 has been carried out with high stereoselection utilising an intramolecular Diels-Alder reaction (Scheme 17).*' The known bicyclic enone 154 was stereo-and site-selectively converted into the nitrile 155. Heating 156 derived from 155 provided the tricyclic compound 157 as a single stereoisomer. 3.1 Ergot alkaloids An oxaspiro dimer 159 has been obtained by the biotransfor- mation of lysergene 158 with an Euphorbia calyptrata suspen-sion cell culture (Scheme 18).84 Both possible 6-N-oxides 160 and 161 of agroclavine and elymoclavine have been prepared by hydrogen peroxide oxidation. The half chair conformation of the D ring was determined.85 4 Bisindole alkaloids A P-carboline dimer 162 which was a known synthetic com- pound has been isolated from a Didernnum species of ascidian H H OH OH 137 Paspaline B 138 Terpendole E R = Me 139 Terpendole F R = CH20H 141 Terpendole H 140 Terpendole G R = CHO R H H s OH 142 Terpendole I R = H Me 144 Terpendole K R = H 6,7-dehydro 146 Paxinorol 143 Terpendole J R = &Me Me 145 Terpendole L R = &Me 6,7-dihydro H OH 147 Paxilline lhara and Fukumoto Recent progress in the chemistry of non-monoterpenoid indole alkaloids 42 1 i ii BnO% BnO-Qo 0 -1 49 150 111 ivi...154 155 v vi 1 SPh SPh vi vii I vii-ix- viii-x -. 0 156 -ido I 151 xi-xiii 1.SPhMeo2chHI rNH 0 158 Lysergene 159 153 Radarin A Scheme 18 collected from the Great Barrier Reef in Australia.86 Alangio- bussine 163 and alangiobussinine 164 have been isolated from the leaves of Alangium bussyanum along with tubulo- sine 9-desmethyltubulosine deoxydesmethyltubulosine 0-methyltubulosine deoxytubulosine and 9-or 10-desmethylprot~emetinol.~~A bis-indole alkaloid 165 has been found in the roots of Antirhea Zucida together with R N,N-dimethyltryptamine and 6-methoxy-2-methyl-1,2,3,4- tetrahydro-p-carboline.88 Three antioxidative serotonin derivatives 166-168 have been isolated from safflower oil cake (Carthamus tin~torius).~~The antioxidative activities of these compounds were measured 160 Agroclavine N-oxide R=H by a ferric thiocyanate method and an a,a-diphenyl-b-picrylhydrazyl (DPPH) method; the compounds showed 161 Elymoclavine N-oxide R=OH 422 Natural Product Reports 1997 m \ / 162 163 Alangiobussine 3,4-dihydro 164 Alangiobussinine 3,4-dehydro eoaTflNr e H H 165 potent antioxidative activity.Biscryptolepine 169 has been found in the root bark extract of Cryptolepis sanguinolenta along with neocryptolepine 35.17 The ascidian Polycarpa auratu collected in Chuuk Federated States of Micronesia yielded N,N-didesmethylgrossularine-1170 whose structure was determined by X-ray crystallography .90 Cyclic peptide antibiotics A21459A 171 and A21459B 172 are produced by a number of the genus Actinoplunes species." They are homodetic cyclic peptides constituted from eight amino acid units.Cysteine and alanine condense to form a thiazole moiety according to the biosynthesis of thiazole-containing antibiotics. These compounds inhibited bacterial protein synthesis and showed selective antimicrobial activity H 171 A21459A R=Et 172 AP!i159R R = Me 166 R' = R2 = H 167 R1 =OMe; R2 = H 168 R1 = R2= OMe Me Me 169 Biscryptolepine 170 N,N-Didesmethylgrossularine-1 Ph 0 173 Kapakahine B I 174 Himadatin 175 Kauluamine Ihara and Fukumoto Recent progress in t@ chemistry of non-monoterpenoid indole alkaloids against clostridia mycoplasma and some Gram-negative bacteria. A cyclic hexapeptide kapakahine B 173 was isolated from the sponge Cribrochalina olemda collected at Pohnpei Federated States of Mi~ronesia.~~ The unique structure pos- sessing a-carboline ring system was established by spectral analysis.The compound showed moderate bioactivity against P 388 murine leukaemia cells. The structure of the antitumour antibiotic himastatin 174 isolated from Streptomyces hygroscopicus has been deter- mined using a combination of spectroscopic and chemical degradation technique^.^^ Himastatin is a dimeric cyclo- hexadepsipeptide joined through a biphenyl linkage between two oxidised tryptophan units. Unsymmetrical manzamine dimer kauluamine 175 has been found in an Indonesian sponge Prianos species.94 The complicated structure of kauluamine 175 was established by spectroscopy. Compound 175 showed moderate immuno- suppressive activity in the mixed lymphoma reaction and was inactive in cytotoxicity and antiviral assays.The natural dimeric carbazole alkaloid bismurrayaquinone A 176 has been synthesised by oxidative coupling of the monomer 177 followed by a further oxidation (Scheme 19).95 Bipolaramide 178 has been prepared from (2S)-2,3-dihydroindole-2-carboxylic acid in three steps (Scheme 20).96 The structure of isoeudistomin U isolated from the marine ascidian Lissoclinum fragile has been revised to 179 as a result of synthetic Bis-indole alkaloid yuehchukene 180 exhibits strong antiimplantation activity in rats mice and pigs. oyqMe OH 177 Bu'202 I 0 I 176 BismurrayaquinoneA Scheme 19 A I 178 Bipolaramide Scheme 20 Reagents i DCC; ii TTFA; 111 CuSO 424 Natural Product Reports 1997 Owing to its potential biological activity several analogues have been ~ynthesised.~~-~~ A review about staurosporine 181 which possesses import- ant biological activities such as potent protein kinase C H 179 lsoeudistominU 180 Yuehchukene H Me07 NHMe 181 Staurosporine H MeO2CVNvGO2Me 182 Lycogalic acid dimethyl ester A R' = R2 = H 183 Lycogalic acid dimethyl ester 6 R' = OH; R2 = H 184 Tjipanazole D 185 Fascaplysin inhibition has been published by Omura et ~1.'~~ Full details of the first total syntheses of staurosporine 181 and its enanti- omer by Danishefsky et al.lo' have been reported.'0' Minor metabolites of staurosporine produced by a Streptomyces longisporofavus have been studied.'02 From the slime mould Lycogala epidendrum the known lycoglic acid dimethyl eaters A 182 and B 183 have been ~btained."~ The known tjipanazole D 184 isolated from Fischerella ambigua shows moderate antibacterial activities. 'O4 The known fascaplysin 185 was isolated from the sponge Fascaplysinopsis species together with a new sesterterpene palauolol. '05 Owing to the pharmacological activities as well as the unique structure of staurosporine much effort has gone into the synthesis of staurosporine analogues. '06-' The simple synthesis of 2,2'-biindole 188 was carried out through the cyclisation of the 1,3-diacetyIene 187. Treatment of 187 pre-pared form N-ethoxycarbonyl-2-iodoaniline186 with sodium ethoxide gave 2,2'-biindole 188 in an excellent yield (Scheme 21)."* i-iii NH HT NHC02Et C02Et C02Et 186 187 J 188 Scheme 21 Reagents i =-TMS PdCl,(PPh,), CuI Et,N; ii K,CO,; iii PdCI,(PPh,) CuI Et,N DMF 0,; iv NaOEt The assembly of the indolo[2,3-a]carbazole ring system was accomplished by palladium(0)-catalysed polyannulation.l3 Reaction of the 1,3-diacetylene 189 with N-benzyl-3,4-dibromomaleimide in the presence of potassium carbonate and a catalytic amount of tetrakis(tripheny1phosphine) palladium(o) provided the indolo[2,3-a]carbozole 190 in a moderate yield (Scheme 22). U-D NH HN I I COCF3 COCF3 189 CPh OYNFO I I HH 190 Scheme 22 The synthesis of the mould alkaloid arcyroxocin A 191 has been communicated.I4 The acid catalysed oxidative cyclisa- tion of the bis-indolylmaleimide derivative 192 afforded the eight membered compound 193 in a good yield (Scheme 23).Il4 i ii -I Boc Boc 192 1 iii Me odNko iv-vi t- 191 Arcyroxocin A 193 Scheme 23 Reagents i 4-(tetrahydropyranyloxy)indolylmagnesium bromide; ii Amberlite' 15; iii DDQ PPTS; iv 180 "C; v 10% KOH then 2 M HCl; vi (TMS),NH MeOH The control of dissymmetry was solved by glycosylation of 2,2'-indolylindolines. Treatment of the 2,2'-indolylindoline 195 obtained by the dimerisation of indole-3-acetic acid methyl ester 194 with trifluoroacetic acid with D-glucose in hot ethanol yielded a diastereoisomeric mixture of 196 (Scheme 24).Oxidation of 196 with DDQ gave the 2,2'-biindole 197.Il5 194 Me02C/ / Me02C' 195 Me02C \ Me02C/ 196 1 iii H I Glu 197 Scheme 24 Reagents i TFA; ii glucose; iii DDQ The above methodology was applied to the simple synthesis of (+)-tjipanazole F2 198 (Scheme 25).II6 Thus cyclisation of 199 followed by bromination produced the bromide 200. Ihara and Fukumoto Recent progress in the chemistry of non-monoterpenoid indole alkaloids 199 200 1 iii OH OH 198 Tjipanazole F2 201 Scheme 25 Reagents i TFA; ii NBS; iii xylose; iv DDQ; v CuC1 DMF Glycosylation of 200 gave 201 as a diastereoisomeric mixture. Without separation 201 was converted into (+)-tjipanazole F2 198 through an oxidation and halogen exchange reaction.K252a 202 the potent inhibitor of protein kinases has been synthesised via an acid-catalysed bis-glycosidation reaction by two groups.117' '18 Coupling of the diazolactam 203 and 2,2'- biindole 188 gave the hexacyclic compound 204 (Scheme 26). Treatment of the product 204 with the furanose component 205 which was prepared via tandem [3,3]-[ 1,2] rearrange- ment in the presence of camphorsfulfonic acid followed by deprotection produced (+)-K252a 202.'l7 DMB H H 203 204 H 205 ~ 1 ii TFA thioanisole Me0 DMB = fvleO&Ct+-202 K252a Scheme 26 A ring expansion of the K252a analogue 206 to the pyrano- sylated indoiocarbazole 208 was carried out in three steps via compound 2O7.ll9On the other hand treatment of 208 with copper(1) chloride in methanol caused oxidation followed by benzilic acid rearrangement of 209 to afford 206 (Scheme 27).I2O These conversions would suggest a possible biosyn- thetic link between the furanosylated and pyranosylated indolocarbazoles.Reaction of the known oxime TAN-1030A 426 Natural Product Reports 1997 $\ I( $\ Me02C OH OHC OH 206 207 iii 1 0 209 208 Scheme 27 Reagents i LiBH,; ii DCC DMSO; iii BF,*OEt,; iv CuCl MeOH 210 with sulfuric acid gave rise to Beckmann type rearrange- ment to yield the compound 211 closely related to K252a 202 (Scheme 28).12' H H ("0 oo HO" ' 210 TAN-1 030A 21 1 Scheme 28 Full details concerning the synthesis of (+)-duocarmyin SA 212 have been published'22 and a number of duocarmycin derivatives have been synthesised in order to improve the an ti tumour activity.'23-1 25 Enantioselective total synthesis of (+)-duocarmycin A 213 was achieved by Boger and his co- workers.'26 The olefin 214 was transformed into the optically Me02C 0 212 Duocarmycin SA 0 OMe OMe n U 213 Duocarmycin A OTs 9- i ii NC OBn NHCOPh NC OBn NHCOPh 214 iii-ivI duocarmycin A 213. Syntheses of chiral precursors of duocarmycins and CC-1065 have also been studied.I2" 128 Sch 52900 218 and Sch 52901 219 two inhibitors of c-fus proto-oncogene induction were isolated from the fermentation broth of the fungal culture (SCF-1168) Gliocladium species together with the known verticillin The spectral analyses established that both compounds 218 and 219 were closely related to the verticillin family of diketopiperazines.It has been suggested that this class of compound exerts antitumour activity by blocking a signal transduction pathway. The trimeric pyrrolidinoindoline alkaloid ( -)-idiospermuline 220 has been found in a methanol extract QTBS from the seeds of Idiuspermun australiense along with two known dimeric alkaloids (+)-calycanthine and ( -)- chimonanthine. I 30 The structure of idiospermuline 220 was determined by spectroscopic methods and the absolute NaH H configuration by an X-ray crystallographic study of its BocN NcvNBoc trimethiodide. OBn OTBS 215 n 0 0 Boc OBn 216 vii-ix OTBS 213 Duocarmycin A Boc OBn 217 Scheme 29 Reagents i ADH (DHQD),-PHAL; ii TBSOTf amine; iii NaH; iv H,NNH, EtOH; v Boc,O; vi TFA; vii LDA; viii LiOMe; ix TsOH active compound 215 (Scheme 29).After the introduction of oxazolidinone treatment of 216 under thermodynamically controlled reaction conditions provided diastereoselectively the tricyclic compound having the desired stereochemistry. The ketone 217 thus obtained was converted into (+)-Me Me IHI &a NiN Me lcll. . I, Me Me 218 Sch 52900 220 ldiospermuline R = CH(0H)Me 219 Sch52901 R = Et 5 References 1 J. Sapi and G. Massiot in The Alkaloids eds. A. Brossi and G. A. Cordell Academic Press New York 1995,47 Ch. 3 pp. 173-226. 2 (a)G. M. Koning and A. D. Wright Pluntu Med.1996 62 193; (6) J. B. Gloer Acc. Chem. Rex 1995 28 343. 3 J. Li G. Chen J. M. Webster and E. Czyzewska J. Nut. Prod. 1995 58 1081. 4 M. Lee I. Ikeda T. Kawabe S. Mori and K. Kanematsu J. Org. Chem. 1996 61 3406. 5 A. 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ISSN:0265-0568    

DOI:10.1039/NP9971400413

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Book review Analysis of sterols L. John Goad and Toshihiro Akihisa. Blackie Academic and Professional London 1997 pp. xvii 437. Price E89 ISBN 0 7514 0230 3 The sterols are an important ubiquitous group of natural products which have been studied throughout the history of bioorganic chemistry. This book presents the reader with a description of a representative range of techniques that are involved in the various phases of the analysis and identification of sterols. The opening chapter includes a description of sterol nomen- clature and an outline of sterol biosynthesis. The latter is helpful as an aid to recognizing which sterols might co-occur. The next chapters describe the extraction and preliminary separation procedures as applied to sterols.These chapters include a series of flow charts outlining the key steps and they contain useful tables of chromatographic solvent systems. Chapters 4 and 5 give details of HPLC and GC methods of sterol analysis including the description of columns and the tabulation of retention times for a very large number of sterols. The subsequent chapters deal with various spectroscopic methods as applied to the identification of sterols. After a description of IR and UV methods there is a full chapter describing the mass spectral fragmentation patterns of sterols. These are particularly valuable in locating double bonds and additional alkyl groups on the sterol ring system and in the side chain. There are three chapters devoted to the 'H and I3C NMR spectra of sterols and associated techniques including two-dimensional NMR methods.These chapters include tabu- lations of characteristic chemical shift data. There is a short chapter on the X-ray crystallography of sterols. The last chapter presents information on relatively readily available sources and separation procedures for a number of the more common sterols which are useful as standards. The appendices contain a lengthy list of the trivial and corresponding system- atic names for sterols the structures of some common penta- cyclic triterpenes found along with the sterols and the physical data of a large number of sterols and triterpenes. The final appendix describes quantitative methods for the analysis of sterols. Each chapter contains a good list of references includ- ing some for 1994 and 1995 and the book is well indexed. This is a well-presented book for those involved in the analysis of sterols and in the study of their biochemistry and it can be recommended as a useful textbook in this field. Jurnes R. Hanson University of Sussex Bvighton UK 431

ISSN:0265-0568    

DOI:10.1039/NP9971400431

出版商:RSC    

年代:1997

数据来源: RSC