ISSN:0265-0568    

DOI:10.1039/a803hopy

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

The manumycin-group metabolites Isabel Sattler,a Ralf Thierickea and Axel Zeeckb aHans-Knöll-Institut für NaturstoV-Forschung, Beutenbergstraße 11, D-07745 Jena, Germany bInstitut für Organische Chemie, Universität Göttingen, Tammannstraße 2, D-37077 Göttingen, Germany Covering: up to June 1997 1 Introduction 1.1 General 1.2 Related classes of compounds 1.3 History and current status 2 Sources 3 Structures and characterization 3.1 Appearance and spectroscopic characterization 3.2 Structures 3.3 Stereochemistry 4 Biosynthesis 4.1 m-C7N Unit 4.2 Chains 4.3 Cyclohexane moiety 4.4 C5N Unit 4.5 Discussion 5 New analogues by manipulation of the biosynthetic pathways 5.1 Precursor-directed biosynthesis 5.2 Cultivation under increased oxygen partial pressure 6 Chemistry 6.1 Derivation 6.2 Degradation 6.3 Synthesis 7 Biological properties 7.1 Antibacterial and antifungal activity 7.2 Insecticidal activity 7.3 Enzyme inhibition 7.3.1 Inhibition of farnesyltransferase 7.3.2 Other 7.4 Cell and in vivo toxicity 8 References 1 Introduction 1.1 General The manumycin-group is a small and discrete class of metabolites that to date combines 23 secondary metabolites all of which are of microbial origin.The term ‘manumycin-group’ refers to its first reported member manumycin A 1 (formerly called manumycin) which was discovered by Zähner and co-workers in 1963.1 In 1973 Schröder and Zeeck established the novel structural type of manumycin A 1 (Fig. 1).2 The metabolite contains two unsaturated carbon chains (’upper’ and ‘lower’ chain) linked in meta-fashion to an unique multifunctional six-membered ring. This moiety is particulary distinctive due to its substitution pattern and is termed the m-C7N unit.3 A 2-amino-3-hydroxycyclopentenone moiety (C5N unit) is linked to the ‘lower’ chain. In general, the structural moieties of manumycin A 1, the m-C7N unit and the two carbon chains, are present in all members of this group thereby pointing out their close relationship.The most distinctive structural variations are found in the ‘upper’ side chain involving the pattern of methyl branches, the number and position of the double bonds and the chain length. Some compounds, like asukamycin 2, carry a terminal cyclohexane moiety in the ‘upper’ chain as an additional structural element.4 The central m-C7N unit varies among the manumycin-type compounds in its stereochemistry and the nature of the oxygen substituents. The characteristic oxirane as in manumycin A 1, may be ring-opened to give a hydroxyethylene moiety at C-5–C-6 (e.g.TMC-1A 3, B 4, C 5 and D 6; see Table 3). 5 The ‘lower’ triene chain is the best conserved structural feature within this class of compounds. In case of colabomycin A 76,7 and D 88 the ‘lower’ chain is extended to a tetraene and in antibiotic U-62162 99 we find an aliphatic four carbon chain. Finally, the C5N unit terminates the ‘lower’ chain in most of the metabolites, but is not strictly required.From detailed biosynthesis studies on manumycin A 1 and asukamycin 2 an almost complete picture of the metabolic origin of this group of secondary metabolites has been obtained.10 Most strikingly, the multifunctional m-C7N unit arises from a unique biosynthetic pathway with carbon building blocks from glycerol and tricarboxylic acid (TCA) cycle metabolism, as well as insertion of molecular oxygen and a nitrogen substituent from the cellular N-pool.In this review the manumycin-group metabolites are summarized in terms of their structure, chemistry, biosynthesis and H N Me HN O O O OH O O HO HN HN O O O OH O O HO Me Me Me 1 2 4 7 13 13¢ 1¢ m-C7N unit C5N unit 1� 2� ‘upper’ chain ‘lower’ chain 1 manumycin A 2 asukamycin Figure 1 Structural elements of the manumycin-group metabolites manumycin A 1 and asukamycin 2 Sattler et al.: The manumycin-group metabolites 221pharmacological potency. Investigations of the manumycins’ biosynthesis, in particular of the m-C7N unit, are treated in more detail.Furthermore, the manumycin-group metabolites provide an interesting example of biological structure modi- fication by precursor-directed biosynthesis.11 A new method to manipulate the secondary metabolite pattern of microorganisms, cultivation under increased oxygen partial pressure, has been applied to the manumycin A producing Streptomyces parvulus.12 At present, the manumycin-group metabolites are of particular interest because of inhibitory activity on Ras farnesyltransferase for which they also have shown an in vivo potential (manumycin A 1).13 A lot of academic and commercial research eVort is currently being put into farnesyltransferase as a molecular target for novel antitumor agents.In this first review article on the manumycin-group metabolites we cover the original literature up to mid-1997, starting from the first description of manumycin A in 1963. 1.2 Related classes of compounds Manumycin-group metabolites show structural relationships to other secondary metabolites.However, judged by biosynthetic origin as classification principle rather than plain structural similarities,14 the metabolites presented in this section are not manumycin-group metabolites. Originally, the manumycins had been considered as incomplete or broken chain analogues of ansamycin metabolites (e.g. ansatrienin A 10).15,16 This classification which followed structural examination was disproven by major biosynthetic diVerences in m-C7N synthesis and polyketide assembly.In the ansamycins the m-C7N unit is derived from an early branch of the shikimic acid pathway and the final metabolites carry only a single polyketide chain as opposed to two chains in manumycins (Fig. 2 and Scheme 1).17,18 There exists a number of small metabolites from Streptomyces spp. that show structural similarities to the oxygenated cyclohexenone of manumycin-type metabolites, but lack carbon chains and the C5N unit (Fig. 3). The antibiotics MM 14201 1119 and its acetylated enantiomer LL-C10037· 1220,21 exhibit a 2-amino-4-hydroxy-5,6-epoxycyclohex-2- enone ring but are lacking an exocyclic carbon substituent. HN R HN O O OH O HO HN R O O O HO HN R O O HO HN R O O O HO O O O 3 R = O HO HO O NH O HO HO O NH O 4 R = 5 R = 6 R = 7 R = 8 R = 9 R = CO2H HN O O Me HO Me OMe NH O O O O Me(CH2)5 NH O O O OH Me NH O O OH O OCOMe HO O O O HN O Me Me Me OMe OMe Me Me O O Me OH Me Me Me Me OH Me O O O HO 10 ansatrienin A 22 aranorosin 23 reductiomycin 24 bafilomycin B1 Figure 2 Selected metabolites with structural similarities to manumycin-group compounds 222 Natural Product Reports, 1998Biosynthetically, LL-C10037· 12 originates from the shikimic acid pathway with 3-hydroxyanthranilic acid as an intermediate.Remarkably, the epoxidation of MM 14201 11 and LL-C10037· 12 is achieved by complementary enzymes on identical substrates following a dioxygenase mechanism.22–24 An ortho-arrangement of an exocyclic carbon substituent neighbouring the oxirane (corresponding to the C-4 position in manumycins) and an amino substituent is present in enaminomycin B 13 while the carbon substitution is missing in enaminomycin C 14.25–27 Furthermore, 5,6-epoxy-1,4- benzoquinone structures with 2-amino-subsitution are present in enaminomycin A 15 (= I-85128 and antiphenicol29) and G7063-2 16.30,31 A meta-arrangement of a C7N unit occurs in the benzoquinone core of melemeleone A 17,32 and a para-arrangement in melemeleone B 1832 and epoxyquinomicin A 19 and B 20.33 The fungal polyketide metabolites of the epoxidone group,34–37 e.g.epoxydon 21, lack a nitrogen substituent on an epoxy cyclohexenone structure that is similar to the core of the manumycins.38,39 Aranorosin 2240–42 and the analogues aranorosinol A and B,43 which are all produced by Pseudoarachniotus species, carry structural similarities to manumycin-group metabolites in the 1-oxaspiro[4,5]decane ring system and the amide linked methyl-branched diene chain.We postulate that the spiro-ring system originates biosynthetically from tmino acid (L)- tyrosine while the diene chain is of polyketide origin. Finally, a number of secondary metabolites, e.g. reductiomycin 23,44,45 bafilomycin B1 24,46 the phoslactomycins,47,48 the depsipeptides of the enopeptin type49 and moenomycin A bear the same amide linked C5N unit as the manumycin-group metabolites.50–52 1.3 History and current status In 1963, the first member of the manumycin-group metabolites, manumycin A 1, was discovered by an antibacterial screening from a Streptomyces strain.1 Because this secondary metabolite was of a then unknown type of structure, elucidation by ‘classical’ degradation and derivation took almost a decade.2 During investigations of the configuration of 13,53 the constitution of a second compound of this remarkable structural class, asukamycin 2, was described in 1979.4,54 This was shortly followed by the discovery of the antibiotics U-62162 99 and U-56,407 2555,56 (see Table 3).Circular dichroism (CD) spectroscopy and the application of the exciton chirality method established the absolute con- figuration at C-4 in the m-C7N unit of asukamycin 2.54,57 In 1987, the absolute configuration of manumycin A 1 for all centers of chirality was described after detailed CD spectroscopical analysis of the parent molecule and several derivatives.53 Configuration of the oxirane was elucidated via epoxybenzoquinones which are obtained by oxidative degradation of 1.However, since the CD spectroscopic analysis is based on conformational hypotheses and is a comparative method, only a stereospecific total synthesis will provide the final proof for the stereochemical assignments. Recently, the first total synthesis of racemic alisamycin 26 was reported.58 The discovery of new manumycins received a boost in the late 1980s with 11 new metabolites reported up till 1995.Particulary fruitful for the growth of this class of compounds was 1996 in which 10 new manumycin-group metabolites were discovered. A heightened interest in manumycins is sparked by the discovery of novel biological activities and is indicated by the commercialization of manumycin A 1 for research purposes. In addition to growing pharmacologically motivated interest, a lot of questions about manumycin biosynthesis remain. Neither the biosynthetic enzymes, nor the genes of manumycin biosynthesis have been analyzed. 2 Sources All manumycin-group metabolites are produced by microorganisms which have been isolated from soil samples collected worldwide and are all taxonomically characterized as actinomycetes (genus: streptomycetes)—Gram positive, mycelial, sporulating bacteria. Most members of the manumycin-group metabolites were discovered by biological screening based on antibacterial activity, antitumor activity, inhibition of farnesyltransferase or inhibition of interleukin-1‚ converting enzyme (Table 1).A few manumycins were found through the non-target directed chemical screening approach.59 Usually, the manumycin-group producing organisms are cultivated in submersed cultures with complex nutrient sources (e.g. soybean meal, molasses or corn gluten meal) using procedures common for Streptomyces strains.The manumycin-type metabolites are produced during the stationary growth phase of the organisms and are found either in the culture filtrate (e.g. asukamycin 2,4 antibiotic U-62162 99), or in the mycelium (e.g. manumycin A 1,2,3 B 2760 and C 28, colabomycins A 76 and D 88). Isolation of the metabolites follows standard procedures which involve extraction with organic solvents and column chromatography on silica gel or RP-silica gel, and gel-permeation chromatography on Sephadex LH-20 (e.g.with methanol or chloroform). Asukamycin 24 and U-56,407 2556 were crystallized from antibiotic-enriched crude extracts; U-62162 99 was purified by counter-current distribution methods. Some caution has to be taken in handling since most of these compounds are chemically and photochemically labile. In a number of cases more than one antibiotic of the manumycin-group was discovered within a producing strain (Table 1, see Section 4.5).In addition, a few cometabolites are reported. Apart from the manumycins A 1, B 27, C 28 and D 29, Streptomyces parvulus (strain Tü 64) produces two red O O NH2 OH O O NHAc OH O O COOH HO O O COOH OH O O COOH O O O CONH2 O O O CH2OH OH Me NH2 NH2 NH2 NH2 O O OH HN R HO O R2 R1 O O CH2COMe Me Me Me O H 13 enaminomycin B 15 enaminomycin A 11 MM14201 12 LL-C10037a 17 melemeleone A R1 = H; R2 = NH(CH2)2SO3H 18 melemeleone B R1 = NH(CH2)2SO3H; R2 = H 16 G7063-2 21 epoxydon 14 enaminomycin C 19 epoxyquinomicin A R = Cl 20 epoxyquinomicin B R = H Figure 3 Metabolites with structural similarities to the m-C7N unit of manumycin-group compounds Sattler et al.: The manumycin-group metabolites 223antibiotic pigments,72 undecylprodigiosin 3073 and metacycloprodigiosin 31,74 as well as (L)-2,5-dihydrophenylalanin 32.75 Streptomyces verdensis (strain 360) produces U-56,407 2556 as well as the cyclic peptide globomycin.76,77 In the colabomycinproducing organism Streptomyces griseoflavus (strain Tü 2880) compound 2880-II 33 was discovered by chemical screening methods as an additional biosynthetically related metabolite.78 3 Structures and characterization 3.1 Appearance and spectroscopic characterization Usually, manumycin-type compounds appear as amorphous, yellowish powders and are instable under acidic or basic conditions and in light.The constitution of manumycin A 1 (C31H38N2O7) was elucidated by analysis of the entire natural product as well as degradation products (see Sections 6.1 and 6.2) and was later confirmed by detailed NMR spectroscopical analysis.Due to their close structural similarity there is a characteristic set of spectroscopic properties that facilitates structure elucidation of manumycin-type metabolites.53 For example, in the UV–VIS spectra typical absorption maxima between 250 and 280 nm and 300 to 350 nm provide information about the size of the olefinic chromophores. Moreover, the chiral arrangement of the two polyene amide chromophores gives rise to distinctive well-split Cotton eVects in the circular dichroism (CD) spectrum (see Section 3.3). 1H and 13C NMR signals of the m-C7N unit (Table 2) and the C5N unit contain characteristic structural information. For the C5N moiety, the C-2+ singlet at about ‰C 115 ppm is a reliable indicator, whereas tautomeric changes of the other carbons and the protons often show a line broadening that might even lead to signal disappearance.Manumycin-type compounds usually do not show molecular ion peaks by electron impact (EI) ionization, but field desorption (FD) or fast atom bombardement (FAB) mass spectrometry are convenient for observing the molecular ion or its mono-dehydrated derivative. The molecular weight of complete manumycin-group compounds varies between 501 (manumycin B 27) and 552 (manumycin D 29) and the molecular formulae with two nitrogen and seven oxygen atoms are particularly distinctive.Table 1 Producing organisms and the screening programs leading to manumycins Compound Producing organism Deposited as Screening program Ref. Manumycin A 1, B 27, C 28, D 29 Streptomyces parvulus (Tü 64) DSM 40722a Antibacterial screening, chemical screening 1,60 Manumycin A (=UCF1-C) 1, B (=UCF1-A) 27, C (=UCF1-B) 28 Streptomyces sp. (UOF1) FERM BP-2844b Inhibitor screening with farnesyltransferase 13,61 Manumycin E 38, F 42, G 39 Streptomyces sp.(WB-8376) ATCC 55484c Antibacterial screening 62,63 Asukamycin 2 Streptomyces nodosus subsp. asukaensis (AM-1042) FERM-P 3429b ATCC 29757c Antibacterial screening 4 Colabomycin A 7, D 8 Streptomyces griseoflavus (Tü 2880) — Chemical screening 6,8 U-62162 9 Streptomyces verdensis (Dietz, sp. n.; UC-8157) — Antibacterial screening 9 U-56,407 25 Streptomyces hagronensis (360; UC 5875) NRRL 8170d NRRL 15064d Antibacterial screening 55,56 Alisamycin 26 Streptomyces sp. (HIL Y-88,31582) DSM 5559a Antibacterial screening 64,65 Nisamycin (=antibiotic 106-B) 34 and alisamycin 26 Streptomyces sp.(K106) FERM P-12701b Antibacterial screening 66,67,68 El-1511-3 40, -5 37, manumycin G 39, ent-alisamycin 41, U-56,407 25 Streptomyces sp. (E-1511) FERM BP-4792b Inhibitor screening with interleukin-1‚ converting enzyme 69,70 El-1625-2 36, manumycin B 27, C 28 Streptomyces sp. (E-1625) FERM BP-4965b Inhibitor screening with interleukin-1‚ converting enzyme 69,70 TMC-1A 3, B 4, C 5, D 6, manumycin D (=TMC-1E) 29, A (=TMC-1F) 1, G (=TMC-1G) 39 Streptomyces sp.(A-230) FERM P-14460b Antitumor screening 5 Compound 1 35 Streptomyces parvullus (TA-8564) — Antibacterial screening 71 aDeutsche Sammlung von Mikroorganismen, Braunschweig, Germany. bFermentation Research Institute, Agency of Industrial Science and Technology, Ministry of Trade and Industry, Japan. cAmerican Type Culture Collection, USA. dNorthern Regional Research Laboratories, Agricultural Research Service, US Department of Agriculture, USA.NH NH N COOH HO O NH O OH OMe NH2 NH N NH OMe OMe C11H23 30 undecylprodigiosine 31 metacycloprodigiosine 32 (L)-2,5-dihydrophenylalanine 33 2880-II Me Figure 4 Cometabolites of the manumycin-group-producing organisms 224 Natural Product Reports, 19983.2 Structures The most characteristic structural element of manumycin-type compounds is the 2-amino-4-hydroxycyclohex-2-enone ring (m-C7N unit) located in the center of the molecule.Originally an oxirane at C-5/C-6 of the m-C7N unit was assumed characteristic for manumycins, but recently a couple of metabolites have been discovered with a hydroxyethylene unit at C-5/C-6 instead. This structural feature can be used to subdivide the manumycin-group compounds into type I (C-5/C-6 epoxide) and type II (hydroxyethylene at C-5/C-6) structures (Table 3). The type I compounds were further separated into type Ia with an olefinic ‘lower’ chain and type Ib exhibiting a saturated ‘lower’ chain.A nitrogen and a carbon substituent are located in a characteristic meta-arrangement at the C7N unit each of which are elongated by carbon chains. With the exception of U-62162 9 these ‘upper’ and ‘lower’ chains are at least of partial olefinic character. Limited structural variability is found for the ‘lower’ chain which usually is a triene amide chromophore in an all-E configuration. Only in case of colabomycin A 7 and D 8 is the ‘lower’ chain extended to an all-E tetraene chain.In most congeners the ‘lower’ chain is linked as an amide to a 2-amino- 3-hydroxycyclopent-2-enone moiety (C5N unit). In U-62162 9, U-56,407 25, nisamycin 34 and compound 1 35, the ‘lower’ chain is terminated by a carboxy group. The manumycin-type compounds get their individual character basically from the structure of the ‘upper’ carbon chain. There are 14 diVerent types within the 23 known metabolites. The chains vary within their overall length, the number and position of methyl branches and double bonds.A major source of structural variation derives from the biosynthetic starter unit for the polyketide assembly of the ‘upper’ chain which in the final metabolite ends up at the chain terminus (see Sections 4.2 and 4.3). Particulary noteworthy is the cyclohexane residue as in alisamycin 26, ent-alisamycin 41, asukamycin 2, manumycin F 42 and nisamycin 34. This structural element is otherwise only rarely found, e.g.in ansatrienin A 10,18,83 phoslactomycins47,48 or phospholine.84 3.3 Stereochemistry Stereochemical information of the manumycin-group compounds is based on circular dichroism (CD) spectra of the native metabolites and their degradation products53,57 as well as on detailed 1H NMR studies, the latter including chiral derivation reactions according to the Mosher method,5,85 nuclear Overhauser enhancement (NOE),5,8 and aromatic solvent induced shift (ASIS) eVects.60,86 The relative configuration of alisamycin 26 was independently confirmed by total synthesis.58 The available stereochemical information of the manumycin-type compounds is included in Table 3.The configuration of C-4 of the m-C7N Unit in asukaycin 254,57 and later in other manumycin-type metabolites7,53,62,66,70,79,82,87 was deduced from CD spectra via the exciton chirality method. It was found that this method can be applied to conjugated polyene and polyene amide systems in which the �]�* transitions are polarized along the long axes of the chromophores.57,88,89 In asukamycin 2 a positive Cotton eVect at the longer wavelength indicates a positive exciton chirality caused by the 4S configuration, which was later determined for most of the other manumycin-group metabolites.Remarkably, the negative CD couplets,59,79,82,87 e.g. observed in case of manumycin A 153 (in acetonitrile: [»]319="32 000, [»]282=+30 000, [»]259=+16 000), exhibit a significant lower intensity as e.g. the positive one of asukamycin 254,57 (in acetonitrile: [»]345=+72 000, [»]307= "98 000).The CD spectroscopical suitability of type II compounds is unclear. A disturbed CD spectrum of manumycin D 29 was related to conformational flexibility of the cyclohexenone ring that lacks the required chiral fixation of the chromophores. However, colabomycin D 8 yielded a CD spectrum with a perfect symmetry similar to that of colabomycin A 7.7,8 The absolute configuration of the oxirane moiety (C-5/C-6) in the type I m-C7N unit can be determined by CD spectroscopic analysis of the epoxybenzoquinones obtained by oxidative cleavage of the C-4–C-7 bond (see Section 6.2).53 This approach which was first applied to the C19- epoxybenzoquinone 43, and the C12-epoxybenzoquinone 44 obtained from manumycin A 1 is based on a comparison with the Cotton eVects of the structurally related antibiotic G7063-2 16, (")-terreic acid 45 and (")-terremutin 46 (Figs. 3 and 5).30,31,90 Analogous analysis indicated the same configuration for most type I manumycins, only nisamycin 34, alisamycin 26 and probably manumycin F 42 have the 5S,6R configuration. Recently, the well established method of chiral derivation by Mosher85 was applied to type II manumycins. Thus, in manumycin D 29 and in TMC-1 A 3, B 4, C 5 and D 6, the diastereotopic 1H NMR shift diVerences of H-6ax and H-6eq clearly indicated a 5R configuration for each metabolite.5 Table 2 13C and 1H NMRa data of the m-C7N unit of manumycin A 1 and D 29 1 2 3 4 5 6 7 8 1 manumycin A 29 manumycin D 1 2 3 4 5 6 7 O O HN HO HO O HN HO 8 Position Manumycin A 1 Manumycin D 29 13C NMR 1H NMR 13C NMR 1H NMR C-1 188.9 (s) 192.5 (s) C-2 127.9 (s) 131.7 (s) C-3 126.5 (s) 7.32 (d, J 2.5) 127.8 (s) 7.57 (d, J 1.5) C-4 71.2 (s) 73.3 (s) C-5 57.3(d) 3.72 (dd, J 2.5, 4.0) 71.8(d) 4.12 (m) C-6 52.8(d) 3.68 (d, J 4.0) 40.8 (t) 2.75 (dd, J 3.2, 16.2), 2.92 (dd, J 5.9, 16.2) C-7 136.5(d) 5.86 (m) 139.0(d) 6.06 (d, J 14.5) NH 7.9 (br, s) 7.70 (br, s) aJ Values are in Hz.Sattler et al.: The manumycin-group metabolites 225O O HN R1 HO R2 O O HN R1 HO R2 HO O HN R1 HO R2 CO2H CO2H R1 R2 Compound type Ia Ref. O O O O O O O O O O O O O OH HN O O OH HN O Stereochemistry and = Not determined. type Ia type Ib type II 4 6 5 7 4 12 4 5 5 6 6 7 7 12 12 O O OH HN O O O O O O O O O O O OH HN O O OH HN O CO2H O U-56, 407 25 type Ib U-62 162 9 type II colabomycin D 8 alisamycin 26 ent-alisamycin 41 asukamycin 2 colabomycin A 7 compound 1 35 El-1511-3 40 El-1511-5 37 El-1625-2 36 manumycin A 1 manumycin B 27 manumycin C 28 manumycin E 38 manumycin F 42 manumycin G 39 nisamycin 34 manumycin D 29 TMC-1A 3 TMC-1B 4 TMC-1C 5 TMC-1D 6 64,65,79,80 69 4,54,57,81 6,7 71 69,70,82 69,70,82 69,70,82 2,3,5,53,61,72 60,61,69 5,60,61,69 62,63 62,63 62,63,69 66,67,68,87 55,56,69 9 8 5,60 5 5 5 5 4 R,5 S,6 R 4 S,5 R,6 S 4 S,5 R,6 S 4 S,5 R,6 S nd a 4 S,5 R,6 S nd a 4 S,5 R 4 S,5 R 4 S,5 R,6 S 4 S,5 R,6 S 4 R,5 R,6 S 4 R,5 R,6 S,6¢ R 4 R,5 R,6 S 4 S,5 R,6 S 4 S, 5 and 6 nd a 4 S, possibly 5 S,6 R 4 S, 5 and 6 nd a 4 R,5 S,6 R 4 S,5 R 4 S,5 R 4 S,5 R 4 S,5 R Table 3 Structures of all manumycin-group metabolitesA characteristic ASIS eVect by [2H5]pyridine on the proton chemical shift of 3-H seemingly depends on the relative stereochemistry of the m-C7N unit.Compounds with a trans-arrangement of the oxygen functionalities at C-4 and C-5, like in manin A 1, show an ASIS (ƒ‰=‰[2H5]pyridine"‰[2H]chloroform) of 0.55 ppm whereas cisderivatives, like manumycin C 28, show a larger ASIS of 0.71–0.79 ppm.60 The study has recently been extended to 10 manumycin-type compounds and also applies to the type II compounds manumycin D 29 and colabomycin D 8.82 Only in case of EI-1625-2 36 there is some discrepancy with independently obtained stereochemical data.The configurations of double bonds in the unbranched polyene chains have readily been deduced by common NMR techniques like 3JH,H coupling constants of the vicinal protons and NOE measurements.The aromatic solvent [2H5]pyridine suppresses higher order couplings of the olefinic protons and simplifies spin patterns in the 1H NMR spectrum.81 In case of the branched ‘upper’ chain in manumycin A the configuration of the double bond between C-2* and C-3* was determined by the 3JC,H coupling constant between 3*-H and C-1* from partially decoupled 13C NMR spectra to be E.3 With the exception of colabomycin A 8 (Z configuration at C-6*)7 all double bonds in the manumycin-group metabolites possess E configuration.In manumycin A 1 the absolute configuration at the methyl branch C-6* of the ‘upper’ chain was elucidated via the degradation product (")-(2R)-2-methylhexanoic acid 47 and comparison of its optical rotation value to an authentic sample resulting in 6*R for 1 (Fig. 5).53 4 Biosynthesis The biosynthesis of manumycin A 1 and asukamycin 2 was extensively studied in the respective producing organisms Streptomyces parvulus (strain Tü 64) and Streptomyces nodosus subsp. asukaensis using radioactive and stable isotope tracer techniques.10 The biosynthesis of 1 and 2 was investigated in parallel because of the stereochemically divergent m-C7N units (C-4) and the diVerent nature of the ‘upper’ chain. Both metabolites bear biosynthetic building segments which originate from independent metabolic pathways.Manumycin A is built from four structural segments, the m-C7N unit, the C5N unit and two (‘upper’ and ‘lower’) polyene chains, whereas asukamycin contains the cyclohexane unit as an additional terminating moiety in the ‘upper’ polyene chain (see Fig. 1). 4.1 m-C7N Unit The six-membered ring (m-C7N unit) structurally resembles analogous units found in other antibiotics,91–94 like ansatrienin A 10,17,18,95 rifamycin B 48,96,97 mitomycin C 49,98,99 pactamycin 50,100 and acarbose 51.101 For the quinoid ansamycins 10 and 48 and the mitomycins (e.g. 49) it was demonstrated that the precursor of their m-C7N unit arises from a branch of the shikimate pathway.94 Although shikimate itself is not incorporated, the related 3-amino-5-hydroxybenzoic acid (AHBA)102,103 acts as a biosynthetic intermediate (Scheme 1). According to studies with cell-free extracts of the producing organisms rifamycin B 48 and ansatrienin A 10 are formed via 5-deoxy-5-amino-3-dehydroshikimic acid from phosphoenolpyruvate and erythrose-4-phosphate, and nitrogen insertion. 94,104 In the case of pactamycin 50 the m-C7N unit is built via 3-aminobenzoic acid (m-ABA) which also is a metabolite branching oV the shikimate pathway.100 A diVerent biosynthetic type of m-C7N unit is represented by the valienamine moiety 52 of the ·-glycosidase inhibitor acarbose 51. It originates from cyclization of heptulose phosphate which is formed via the pentose phosphate pathway (Scheme 2).101 Recently, Gould et al.reported that 4-hydroxy- 3-nitrosobenzamide and its ferrous chelate, produced by Streptomyces murayamaensis, are derived from succinate and, possibly, phosphoenol pyruvate proceeding through 3-amino- 4-hydroxybenzoic acid.105 The biosynthesis of the m-C7N unit in manumycin-group metabolites follows a pathway in which neither [7-13C]AHBA nor [7-13C]m-ABA are incorporated. Furthermore, incorporation of variously labelled acetates ruled out a valieneaminetype pathway like in acarbose 51.In both 1 and 2, labelled acetate is incorporated into the four-carbon segment C-4 to C-7 in a ‘tail-to-tail’ manner consistent with the TCAcycle intermediate succinate. Its role as a precursor was con- firmed by feeding of variously carbon-labelled succinates (Scheme 3).10,106 A key result in investigating the m-C7N biosynthesis of 1 and 2 was the incorporation of [U-13C3]glycerol, a characteristic intermediate of the triose pool (carbohydrate metabolism).The intact transfer of the C3-segment from glycerol into C-1 to C-3 of manumycin A 1 and asukamycin 2, was established by detailed NMR studies (proton-decoupled NMR, 13C NMR homodecoupling experiments and 2D INADEQUATE).10,106 Feeding experiments with chirally labelled glycerol were performed on asukamycin 2, in order to study the stereochemical orientation of whether the pro-R or the pro-S hydroxymethyl group of glycerol becomes C-1 in the m-C7N unit.81 The precursors (R)-[1,1-2H2]glycerol, (1R,2S)- and (1S,2S)-[1- 2H1]glycerol did not result in any label-incorporation into 2.These experiments indicate that none of the hydroxymethyl hydrogen atoms of glycerol are retained and that 3-H does not originate from glycerol. The label of the pro-S hydroxymethyl analogue (S)-[1-13C]glycerol, however, ends up in the carbonyl group (C-1) of the m-C7N unit of 2 (Scheme 4). The origin from a C3 (triose pool)- and C4 (TCA cycle)-intermediate established a new biosynthetic pathway for m-C7N units and made it a specific criterion for the classification of manumycin-group metabolites.Recently, Floss and co-workers reported the incorporation of labelled [7-13C,2-2H]-3-amino-4-hydroxybenzoic acid (3,4- AHBA) into manumycin A 1 and asukamycin 2.107 Thus, the aromatic intermediate supposedly acts as a starter unit for the ‘lower’ polyketide chain (Scheme 3). The assembly of the aromatic intermediate involves a complete loss of the hydroxymethyl protons of the C3 building block as can be seen from the feeding experiments with deuterium labelled glycerol.Remarkably, earlier feeding experiments with unlabelled 3,4- AHBA (5 mM) to Streptomyces parvulus (see Section 5.1) did not enhance the yield of manumycin A 1 compared to unfed cultures,108 however, small amounts of manumycin D 29, unidentified manumycin D derivatives and the red 2-amino-9- carboxyphenoxazinone 53 were detected as cometabolites O O O HN O Me Me HO Me O O O O HN Me Me OH O O OH Me O Me Me O O O OH Me O Me 44 43 45 46 47 Figure 5 Compounds that were relevant for the initial structure elucidation of manumycin 1 Sattler et al.: The manumycin-group metabolites 227(Scheme 5).The latter supposedly is a biotransformation product formed by oxidative dimerization and decarboxylation of 3,4-AHBA similar to a synthetic procedure.12 The origin of the nitrogen and oxygen atoms of the m-C7N unit in manumycin A 1 was studied by feeding experiments with 15N- and 18O-labelled precursors (Scheme 6).10,109 The incorporation of the 15N-label of [2-13C,15N]glycine into the m-C7N unit can be explained through glycine reductase mediated conversion of glycine into acetate by transfer of the 15N-label to the nitrogen pool. Subsequently, the nitrogen atom could be tranferred to the biosynthetic intermediate of the m-C7N unit via transamination.Cultivation under 18O2- enriched atmosphere showed incorporation of molecular oxygen into both the hydroxy group at C-4, and the oxirane. With the aromatic 3,4-AHBA as intermediate one can speculate whether the oxygen insertion in the m-C7N unit of 1 and 2 follows a mono- or a di-oxygenase mechanism.107 The action of a dioxygenase has been demonstrated in the case of the oxidation of vitamin K110,111 and during the biosynthesis of antibiotic LL-10037· 12 and MM 14201 11.24 A simultaneous insertion implies a cis-orientation of the neighbouring oxygens.COOH NH2 O O H2N Me O O COOH NH2 N Me O HN NH OH OCH2COOH OH Me O NH O NH2 O NH OMe O OMe O Me HO Me O HN Me O HO O Me Me OH Me OH Me MeO2C Me MeO Me O O NH2 HO CH2 Me OH NH O (Me)2N O O Me OH Me HO H 48 rifamycin B 49 mitomycin C 10 ansatrienin A 50 pactamycin Scheme 1 Biosynthesis of the m-C7N units in ansatrienin A 10, mitomycin C 49, rifamycin B 48 and pactamycin 50 from branched metabolites of the shikimate pathway HOCH2 NH2 HO HO HO HOCH2 HN HO HO HO O Me HO O HO O CH2OH HO O HO O CH2OH HO OH HO 52 valienamine 51 acarbose Scheme 2 Biosynthesis of the m-C7N unit in acarbose 51 derives from heptulose phosphate 228 Natural Product Reports, 1998Indeed, the cis-configuration of the oxygen functionalities at C-4 and C-5 is found in most manumycin-group compounds including those of type II (Table 3).In a small number of type I metabolites, in particular manumycin A 1 and B 27, the orientation of the oxygen substituents at C-4 and C-5 is trans.The trans-configuration could be achieved either by epimerization at C-4 during the late biosynthesis or by the involvement of two monooxygenases (Scheme 7). Oxygen insertion by Streptomyces parvulus (strain Tu 64) is particularly curious since this organism produces metabolites with both types of configuration. However, the exact mode of oxygen insertion in manumycins awaits elucidation.The involvement of mono- or di-oxygenases will possibly be determined in planned feeding experiments with 18O2/16O2-mixtures. 4.2 Chains Feeding experiments with labelled acetate, malonate and propionate showed the polyketide origin of the carbon chains in 1 and 2.10,112 The ¡®upper¡� side chain in manumycin A 1 is assembled by elongation of a starter acetyl CoA molecule (C-9* and C-10*) with one acetyl CoA building block (via malonyl CoA) and three propionyl CoA molecules (via methylmalonyl CoA) (Scheme 8).In asukamycin 2 a cyclohexane-containing moiety acts as polyketide starter of the ¡®upper¡� side chain (see Section 4.3) which is assembled from three malonyl CoA units. The triene chains (C-8 to C-13) are formed by the subsequent condensation of three malonyl CoA units with the m-C7N unit as polyketide starter unit. Feeding of [1-13C,18O2]acetate resulted in intact incorporation of the 13C.18O-moiety into the two amide carbonyl groups (C-13/O, and C-1*/O) of 1 which further supports the polyketide nature.109 As structural diversity of manumycin-group metabolites is based on variation of the ¡®upper¡� chain the biosynthesis of these moieties is of particular interest.Although there are only experimental results with manumycin A and asukamycin, one can postulate polyketide chain formation with acetyl CoA and malonyl CoA units for all metabolites. Obviously, there exist three diVerent kinds of starter units for the ¡®upper¡� side chains.Most common among manumycin-group metabolites is acetyl CoA which is used in manumycin A 1, B 27, C 28 and D 29, EI-1625-2 36, EI-1511-5 37, colabomycin A 7 and D 8 and TMC-1A 3, B 4, C 5 and D 6 (Table 3). The isopropyl type terminus in manumycin E 38 and G 39, EI-1511-3 40, U-56,407 25, and U62162 9 supposedly is derived from a branched amino acid which after deamination and decarboxylation may act as CoA-derivative for polyketide chain extension.Likewise in avermectin B1b biosynthesis (L)-valine ends up in the isopropyl polyketide starter moiety.113 It is notewothy that upon incorporation of the cyclohexane starter unit (see Section 4.3) as in asukamycin 2, alisamycin 26, nisamycin 34 and manumycin F 42, the chain is extended solely by acetate building blocks resulting in an unbranched ¡®upper¡� polyene chain. 4.3 Cyclohexane moiety Feeding of [U-13C3]glycerol to the asukamycin-producing organism demonstrated the origin of the cyclohexane moiety from the shikimate pathway.10,114 The shikimate-type labelling pattern of the cyclohexane and the adjacent C-7* (incorporation of intact glycerol into C-7*/C-8*/C-9* and C-11*/C-12*/C- 13* as well as a singly labelled carbon atom at C-10*) ruled out other biosynthetic pathways (Scheme 9). Feeding experiments with radioactive precursors revealed that the cyclohexane ring and the adjacent C-7* arise from cyclohexane carboxylic acid.Obviously, the cyclohexane carboxylic acid activated as CoA-derivative serves as starter for subsequent elongation on the polyketide pathway.Detailed studies about the biosynthesis of cyclohexane rings were reported for the ansatrienins18,95,104 produced by Streptomyces collinus Tu 1892 and for ¢�-cyclohexyl fatty acids from Alicyclobacillus CH2OH HOCH CH2OH COOH COOH COOH COOH OH NH2 O O HO HN O HO COOH NH2 NH2 COOH 1 4 6 7 1¡Ë m . C3 C4 + 1, 2 u m . . . . . . . . u u u * u u u * Scheme 3 Biosynthesis of the m-C7N unit in manumycin A 1 and asukamycin 2 HO OH O O OH HN HO H H HO OH HO H HO OH HO H HO OH HO H D D D H H D No incorporation of D No incorporation of D .. S S S R No incorporation of D S R Scheme 4 Biosynthesis of the m-C7N unit in asukamycin 2 O N O NH2 HO2C HO2C NH2 OH . 6H . CO2 2 x 53 carboxyphenoxazinone Scheme 5 Oxidative dimerization of 3,4-AHBA Me O OH COOH NH2 H N Me HN O O O OH O O HO Me Me Me u * 1 u u u n n n * * O2 Scheme 6 Origin of the heteroatoms in the biosynthesis of manumycin A 1 Sattler et al.: The manumycin-group metabolites 229acidocaldarius.104,115,116 Those studies involving 13C- and 2H-labelled samples of shikimic acid are providing insights into the stereospecific biosynthetic transformation of shikimate to cyclohexane carboxylic acid.The removal of the hydroxy functions of shikimate proceeds through a series of dehydrations and double bond reductions, the ring thereby never becoming aromatic. 4.4 C5N Unit The 2-amino-3-hydroxycyclopent-2-enone moiety (C5N unit) in manumycin-group metabolites which is also found in several other microbial metabolites (see Section 1.2), is biosynthesized from succinate (C4-segment) and a glycine (C1N) building block (Scheme 10).10,117 The incorporation pattern of [1-13C]- and [2-13C]-acetate showed that the biosynthetic precursor of the carbons C-1+, C-3+ , C-4+ , and C-5+ arises from two ‘tail-to-tail’ linked acetate units which corresponds with the incorporation of [1,4-13C2]- and [1,2-13C2]-succinate.The biosynthetic origin of C-2+ and the adjacent nitrogen atom (C1N-segment) was studied by feeding of labelled glycine (e.g. [2-13C,15N]glycine) which as expected was incorporated into C-2+/N of the C5N unit in manumycin A 1109 and asukamycin 2.117 These results are consistent with a biosynthetic origin of the C5N unit from 5-aminolevulinic acid that is formed from succinyl CoA and glycine by 5-aminolevulinate synthase.The carboxy group of glycine may be lost by decarboxylation during the pyridoxal phosphate (PLP) mediated reaction. From studies on other antibiotics containing analogous C5N units (e.g. reductiomycin 23118,119) this biosynthetic route seems to be common for this kind of moiety. A mechanism for the intramolecular cyclization of 5-aminolevulinate is postulated in the original literature.117 4.5 Discussion Apart from mechanistic aspects of the individual biosynthetic pathways as they have been discussed in the previous sections a broader view on manumycin biosynthesis might oVer new insights on secondary metabolite formation.The features highlighted in this section might stimulate future studies on the genetics and enzymology of this class of compounds. Preliminary hybridization studies with the polyketide synthase genes actI and actIII on the asukamycin producing Streptomyces nodosus indicate the accessibility of polyketide synthases in manumycin biosynthesis.120 Since biosynthetic genes are often clustered, an identification of polyketide synthase genes might also give access to the genes of the other pathways involved in manumycin biosynthesis, like the ones for the biosynthesis of the central m-C7N unit.For example, it would be interesting to get information about the oxygenating enzymes, especially about the control of stereospecificity that varies within the product mixture of manumycins A, B and C.An interesting feature of manumycin biosynthesis are the polyketide based structural variations of the ‘upper’ polyketide chain that can be found within single producing organisms (Table 1). The widest variability of the polyketide pattern is found in the Streptomyces strain that produces TMC-1 A 3, B 4, C 5, D A 1 and manumycin G 39. Here, variations in the nature of the incorporated building block—propionate or acetate—and in the stage of its processing—leading to an olefinic or aliphatic unit—are found at every step of the chain assembly.In contrast, the variability of the product mixture of EI-1511-3 40, -5 37, manumycin G 39, ent-alisamycin 41 and U-56,407 25 is mainly caused by a variation in the starter unit and two non-obligatory steps of acetate incorporation. A similar set of variations in the polyketide pattern can be found with the manumycins E 38, F 42 and G 39 that also show the alternative incorporation of a cyclohexyl group or a putative valine derived starter unit.The structural variability of manumycins A 1, B 27 and C 28 is particulary noteworthy since the same mixture can be found within two diVerent producing species. In Streptomyces parvulus (strain Tü 64), manumycins B 27 and C 28 are produced only in minor yields (1%) compared to 1 whereas a diVerent Streptomyces sp. (UOF1, FERM BP-2844) produces the three metabolites in nearly equal amounts. Looking at the biosynthesis of manumycins as a whole it would be interesting to find out how the diVerent biosynthetic pathways leading to manumycin group compounds get ‘orchestrated’.Other than in cases of additional ‘late’ or independent biosynthetic steps (e.g. oxygenations, glucosylations, methylations) the diVerent pathways towards manumycins are closely intertwined. OH HN O O HN O O OH HN O O O HN O HO O HN O HO O O HO ENZD O O O ENZD O HN O O O ENZD ENZM O O HN O O O O HN O HO O ENZM OH HN O O O ENZD 4 5 H+ 4 + 2H 5 a 4 b a, b a a, b type I metabolites ( e.g. 4 S,5 R,6 S as in 2) type II metabolites ( e.g. 4 S,5 R as in 29) type I metabolites ( e.g. 4 R,5 R,6 S as in 1 or 4 S,5 R,6 S as in 2) (a) (b) Scheme 7 Hypothetical oxygenation mechanisms for the conversion of 3,4-AHBA derived core into the m-C7N unit of manumycin-group metabolites: (a) dioxygenase mechanism (ref. 19); (b) mixed mechanism with a di- and a mono-oxygenase 230 Natural Product Reports, 19985 New analogues by manipulation of the biosynthetic pathways 5.1 Precursor-directed biosynthesis The production of new secondary metabolites by precursordirected biosynthesis is an eYcient approach for the derivation of lead structures from microbial sources.11 In certain cases added analogues of a ‘natural’ precursor are incorporated into the biosynthesis thereby yielding structurally modified metabolites.For manumycin-group metabolites precursor-directed biosynthesis was studied with Streptomyces parvulus (strain Tü 64) which resulted in diVerent analogues of manumycin A 1 with aromatic substitution of the m-C7N unit.121,122 Feeding of non-physiological high amounts of artificial precursors during the stationary growth phase allows the replacement of the ‘normal’ precursor of the m-C7N unit, thereby giving access to new products with some yields even higher than the native productivity (Fig. 6). The method was successfully transferred to the asukamycin- and the colabomycinproducing organisms.123 The discovery of new manumycin analogues resulted from feeding diVering amounts of 3-aminobenzoic acid (m-ABA) during investigations on the biosynthetic origin of the m-C7N unit of manumycin A 1.Up to levels of 2-4 mM m-ABA, manumycin A production was not aVected, whereas at concentrations of 7 mM the biosynthesis of 1 drastically decreased. Using even higher concentrations of the precursor, surprisingly, the aromatic manumycin analogue 64-mABA 54 showed up in the fermentation broth.The production of 54 reaches its maximum at a feeding level of 55 mM m-ABA (7.5 g l"1). Addition of [7-13C]m-ABA resulted in labelling of C-7 in 54 proving that in 64-mABA 54 the natural m-C7N unit is replaced by 3-aminobenzoic acid.121 The approach of replacing the natural m-C7N unit was extended to various other aromatic precursors which resulted in a series of derivatives of manumycin A 1 (Fig. 7). Although the genuine m-C7N unit is expected to possess an aminofunctionality in the meta-position to a free carboxy group, it is noteworthy that precursors with a very diVerent substitution pattern at the aromatic ring [e.g. 4-aminobenzoic acid (p-ABA)] are also incorporated by Streptomyces parvulus.122 It is also remarkable that bulky substituents next to the potential sites of chain attachment, as in 3,4-diaminobenzoic acid (3,4- DABA), do not prevent complete integration of the aromatic precursor.12,123 The production of the native metabolite manumycin A 1 usually is repressed upon precursor feeding in high concentration.There is an exception in the case of feeding 3-amino- 4-hydroxybenzoic acid (3,4-AHBA) which was later shown to be the natural precursor of the m-C7N unit (see Section 4.1). Feeding of 3,4-AHBA in high concentration results in a substantial production of the native metabolite in addition to the two products, 64-3,4AHBA1 60 and 64-3,4AHBA2 64.12,122,123 This incomplete turnover of the aromatic precursor suggests a limitation in the activity of the oxygenase reactions.It is obvious that added precursors which carry the amino- and carboxy-functionality in a meta-arrangement are much better incorporated into the biosynthetic machinery. However, 64-pABA2 62 which is extended with both polyketide chains in a para-configuration, provides a striking result.123 One can hypothesize that two enzymatic processes are essential for the incorporation of the m-C7N precursor.One of them, presumably by an amide synthase, connects the carboxylic terminus of the ‘upper’ chain to the amino group, and a second, possibly as part of a multienzyme complex, activates the carboxy group (CoA-transferase) for subsequent elongation via the polyketide pathway. Because precursor-directed metabolites are formed with diVerent levels of assembly (classes 1, 2 and 3), it is likely that the amide synthase and the CoA-transferase act independently with the amide synthase • • • • • • • • • • 7 4 1¢ 13 2� • •9¢ 4 7 13 1¢ 2� • • 1 2 HN HN O O O O O HO OH HN HN O O O O O HO OH 8¢ • • • • Me COOH • • COOH Me • • • • Scheme 8 Biosynthesis of the polyene chains in manumycin A 1 and asukamycin 2 COOH H2COH HCOH H2COH u 8¢ 2 • u 7¢ Scheme 9 Biosynthetic origin of the cyclohexane moiety in asukamycin 2 Me COOH H2C H2C COOH COOH COOH NH2 HOOC HN O OH O O H2N u • u 2� u u + u u 13¢ • • C4-segment C1N-segment p p p 1, 2 u Scheme 10 Biosynthesis of the C5N unit in manumycin A 1 and asukamycin 2 Sattler et al.: The manumycin-group metabolites 231being more sensitive to structural changes in the non-natural precursor.The polyketide chain extension of the activated m-C7N precursor is highly specific with respect to the termination of the chain length. Feeding of vanillic acid (4-hydroxy-3- methoxybenzoic acid) and ferulic acid (4-hydroxy-3- methoxycinnamic acid) both result in 64-Van 59 with the natural triene chain.The length of the triene chain seems to be under strict control either by the polyketide synthase itself, or by the second amide synthase which connects the activated triene carboxylic acid to the C5N unit. Since other manumycins that do not carry the C5N unit also contain a triene, chain length control during polyketide assembly seems to be more likely. In order to demonstrate the applicability of precursordirected biosynthesis to other manumycin-producing organisms, 3-aminobenzoic acid was used to prepare analogues of asukamycin and colabomycin (Fig. 6). While the feeding experiment with the colabomycin producer resulted in 2880- mABA 6612,123 which carries both side chains, the ‘upper’ chain is missing in the analogue of asukamycin, asuka-mABA 67.81 5.2 Cultivation under increased oxygen partial pressure It is well known that regulatory and toxic eVects of oxygen partial pressure aVect significantly microbial metabolism and growth.124–126 Besides distinct eVects on primary metabolismreversible reduction of cell growth up to 1200 mbar or enhanced production of organic acids, the secondary metabolism can also be aVected.127 For example, the production of the aminoglycoside antibiotic gentamycin by Micromonospora purpurea decreases continuously with increasing oxygen partial pressure. In case of the tetracycline/oxytetracycline production an increase of the oxygen partial pressure in diVerent Streptomyces strains results in a shift of the product mixture to the higher oxygenated compound oxytetracycline.124 Regarding the involvement of molecular oxygen in the biosynthesis of manumycin A 1 studies were initiated to manipulate the metabolite pattern of Streptomyces parvulus by an increased oxygen partial pressure.The experiments were performed by increasing the hydrostatic pressure (1 to 10 bar), as well as by raising the oxygen content of the aeration (20 to 100%). The identical results confirmed earlier findings that the mechanical eVect of increased pressure up to 10 bar is negligible.128 Manumycin A production decreases continuously with increasing oxygen partial pressure and is completely suppressed at pO2 1890 mbar.At the same time several new metabolites show up in diVering amounts in the range pO2 630–1680 mbar (Fig. 8). The structures of these new products, 64p-A 68, 64p-B 69 and 64p-C 70, represent the diVerent biosynthetic building blocks of the parent metabolite manumycin A 1. 64p-B 69 is the amide of the carboxylic acid of the original ‘upper’ chain, whereas 64p-A 68 is an analogous amide with a shorter chain length not otherwise found. The polyketide structure of 64p-A 68 is not assembled under regular cultivation conditions where manumycin A 1, B 27 and C 28 already exhibit variations of this polyketide chain. 64p-C 70 is the only aromatic manumycin analogue produced without feeding of an unnatural precursor.In order to further develop the approach of controlled metabolic engineering, cultivation under increased partial oxygen pressure was combined with precursor-directed biosynthesis. By feeding Streptomyces griseoflavus (Tü 2880) HN HN NH O HO O NH2 NH O HO O NH O HO O Me Me O Me Me Me O 54 64-mABA 66 2880-mABA NH2 COOH 67 asuka-mABA m-ABA (50mM) Streptomyces parvulus (Tü 64) Streptomyces nodosus ssp. asukaensis Figure 6 Precursor-directed biosynthesis with organisms producing manumycin-group metabolites using 3-aminobenzoic acid (m-ABA) 232 Natural Product Reports, 1998NH O HO O NH2 COOH NH2 OH NH O HO O COOH NH2 NH O HO O Me COOH Me NH2 NH O HO O OMe COOH OMe OMe NH O HO O OH COOH OH OH OH NH2 NH2 OMe OMe COOH OH COOH NH2 NH2 O HN NH2 OH COOH H N Me O Me Me Me H N Me O Me Me Me O HN NH2 COOH NH2 COOH NH2 COOH NH2 COOH NH2 NH2 H N Me O Me Me Me NH HO O O OH H N Me O Me Me Me NH HO O O H N Me O Me Me Me 64 64-3,4AHBA2 65 64-3,5DABA 62 64-pABA2 61 64-BZH 60 64-3,4AHBA1 63 64-3,4DABA NH HO O O class 3 55 64-pABA1 class 1 class 2 56 64-HBA NH2 OH H2N 57 64-3A4M 58 64-3A4MO 59 64-Van COOH NH2 NH H N Me OH O O O Me Me Me H2N 54 64-mABA Figure 7 Manumycin analogues obtained by precursor-directed biosynthesis with the manumycin-producing Streptomyces parvulus (strain Tü 64)3-aminobenzoic acid according to the high concentration method an unexpected six-fold increase in the yield of the known precursor-directed product 64-mABA 54 was observed at pO2 1260 mbar.Furthermore, the production of a new analogue, 64p-mABA 71, was initiated in which the original ‘upper’ chain is replaced by the shorter 2,4-dimethylhexa-2,4- dienecarboxylic acid moiety as found in 64p-A 68.12,123,128 This clearly demonstrates that the variation in polyketide assembly of the ‘upper’ chain depends on the oxygen concentration. Application of the method to the colabomycin (A 7 and D 8) producing Steptomyces griseoflavus did not result in a similar manipulation of polyketide metabolism,129 but since this strain does not show a variablility of polyketide assembly under regular conditions this might be not surprising.A striking result, however, was a nearly five-fold increase in colabomycin concentration in the mycelium of this strain at pO2 630 mbar compared to standard conditions. The newly formed metabolites allow further insights into the enzymes involved in manumycin biosynthesis. The decrease of manumycin production might suggest an overall inhibition of secondary metabolite formation by increased oxygen partial pressure.But the strong increase of production upon feeding of the m-C7N substitute suggests a specific eVect on the biosynthesis of the m-C7N unit, possibly caused by a kind of substrate inhibition of the oxygenases involved. An unexpected result is the specific eVect on the polyketide assembly of the ‘upper’ chain. The novel chain type of 64p-A 68 and 64p-mABA 71 obviously is caused by the omission of one propionate building block during polyketide biosynthesis.The altered metabolite pattern of Streptomyces parvulus demonstrates that cultivation under increased oxygen partial pressure is a novel approach to manipulate microbial secondary metabolism. Furthermore, cultivation under increased oxygen pressure can also be successfully combined with other methods like precursor-directed biosynthesis. 6 Chemistry 6.1 Derivation Although manumycin A 1 is chemically rather unstable a number of derivation reactions were carried out which retain the entire carbon skeleton of the parent antibiotic (Scheme 11).130 Reduction at the C-1 carbonyl group of 1 with sodium borohydride in aqueous methanol results in the dihydro derivative 72 (57% yield).This derivative rendered essential structural information about the linkage site of the ‘upper’ chain at C-2 of the cyclohexenone. The same reaction performed in dry methanol yielded the yellow deoxymanumycin 73 as the main product (60% yield).Here, the reduction of 1 with sodium borohydride at the C-1 carbonyl group was followed by elimination of water and regioselective opening of the epoxide. Deoxymanumycin 73 was also obtained in lower yields by treatment of 1 with potassium iodide in acetic acid. In addition, dideoxymanumycin 74 resulted in this reaction and became the main product using zinc dust in acetic acid.130 Compound 74 is identical to the precursor-directed product 64-3,4AHBA2 and was also identified as a fluorescent yellow ‘impurity’ in regular cultivations of Streptomyces parvulus which was diYcult to separate from 1.108 Catalytic hydrogenation led to three main products in varying yields depending on the reaction conditions.An important compound for the structure elucidation was decahydrodideoxymanumycin 75. Decahydrodeoxymanumycin 76 and tetradecahydromanumycin 77 were isolated as by-products of this reaction.Using ceric(IV) ammonium nitrate 1 was converted to the amide 78.131 Only the C5N unit is degraded under these conditions. Because of the new centers of chirality at C-2* and C-4* the reaction products were isolated as mixtures of diastereomers. Acetylation of manumycin 1 was carried out by using acetic acid anhydride–sodium acetate. The resulting manumycin diacetate 79 is unstable and turns into the monoacetate 80 by stirring a solution of 1 in chloroform for two days at room temperature.In order to determine the absolute stereochemistry by the method of Mosher, the (R)-(+)-, and (S)-(")-·-methoxy-·- (trifluoromethyl)phenylacetyl esters of the hydroxy group at C-5 of and TMC-1A 3, B 4, C 5 and D 6, and manumycin D 29 were prepared with 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC) in dry CH2Cl2 (see Section 3.3).5 With nisamycin 34, which is the analogue of alisamycin 26 missing the C5N unit, derivation was carried out at the free carboxylic acid terminus of the lower chain (Fig. 9).87 The amides 81–85 were prepared from 34 and the corresponding amines with cyanophosphonic acid diethyl ester (DEPC) and triethylamine (0 )C, THF, 1 h). 6.2 Degradation Degradation products obtained by chromic(VI) oxidation played a key role in the structure elucidation of manumycin A 1 (Scheme 12).2,3,53 Under mild oxidation conditions (75% aqueous acetic acid, room temperature, 3 to 6 h) the C-4/C-7 bond is selectively cleaved to result in the C19- epoxybenzoquinone 43.Increasing the concentration of chromic trioxide and longer reaction time leads to C12- epoxybenzoquinone 44, as well as (")-(2R)-2-methylhexanoic acid 47 formed by additional cleavage of the C-4* double bond. Formation of the respective epoxybenzoquinone for CD spectroscopy of the oxirane moiety was also performed with manumycin G 39,63 asukamycin 2,54 colabomycin A 7,7 alisamycin 26,79 ent-alisamycin 41,82 EI-1625-2 36,82 EI-1511-3 40,82 EI-1511-5 37,82 U-56,407 2582 and nisamycin 3487 (see Section 3.3).The degraded epoxybenzoquinone 86 was obtained in poor yield (1.3%) from colabomycin A 7. With all other manumycins, the ‘upper’ chain ends up in the benzoquinone derivative without any degradation, e.g. 87 obtained from asukamycin 2. Acetolysis (acetic anhydride, 150 )C, 3 to 5 h) sets free 2-acetamino-3-hydroxycyclopent-2-enone 88, the N-acetyl derivative of the C5N-moiety (Scheme 13).2,3 Using mild alkaline hydrolysis conditions (e.g. 0.1 M NaOH, 50 )C, 30 min) the ‘upper’ side chains of the manumycin-group Figure 8 Manumycin analogues obtained by cultivation under increased oxygen partial pressure 234 Natural Product Reports, 1998metabolites can be cleaved.3 In case of manumycin A 1 (E,E)-2,4,6-trimethyldeca-2,4-dienoic acid 89 was isolated in 88% yield. The methyl ester 90, prepared by methylation with diazomethane, as well as the amide 69, synthesized via its chloride, facilitated structure and stereochemistry elucidation of manumycin A 1.In case of alisamycin 26 the cyclohexanedienoic acid and its corresponding methyl ester were obtained following the same procedures.80 Formation of cyclohexanecarboxylic acid from asukamycin 2 and alisamycin 26 was achieved by nitric acid oxidation (70% nitric acid, 95 )C, 1 h).54,80 6.3 Synthesis Compared to other structurally outstanding natural products the manumycin-type compounds have not been the focus of HN O HN NH O HO O R OH HN NH O HO O R OH HN HN OH O R O HN OH HN OH HN OH HO HO O O HN OH O O O Me Me Me Me HO O HN HO O O NH2 O AcN OAc O O HN OAc O O HO HO NaBH4, aq. MeOH NaBH4, dry MeOH Potassium iodide, acetic acid Zn, acetic acid H2, cat. Pd/C, MeOH Ceric ammonium nitrate, acetonitrile Acetic anhydride, sodium acetate, rt, 65 h 1 manumycin A R = 72 73 73 74 74 73 75 76 77 78 79 80 CHCl3, rt + + 5 + 7 13 1¢ 2� + 1 4 HO 13¢ O 10¢ Scheme 11 Derivation of manumycin A 1 retaining the entire carbon skeleton Sattler et al.: The manumycin-group metabolites 235synthetic eVorts.However, recent findings on interesting biological activities have now sparked synthetic interest in this group of compounds (Scheme 14). Thus, recently the first total synthesis of a manuymcin-type compound, alisamycin 26, was reported by Taylor and co-workers.58 The racemic synthesis starts by preparing the core m-C7N unit of the final product.132,133 Two similar routes starting from dimethoxyaniline produce the enamine 91, or its N-protected analogue as a pivotal intermediate.In the next step the ‘upper’ chain was introduced by coupling the appropriate acid chloride. The olefinic chain was synthesized in a five carbon homologation procedure starting from the potassium salt 92 of glutaconaldehyde. 134,135 The siloxy dienal resulting from in situ silylation of 92 was coupled with cyclohexylmagnesium bromide to give adduct 93.Acidic hydrolysis of 93 resulted in the aldehyde of the thermodynamically preferred all-E dienal 94. Chlorite oxidation yielded the respective acid that could be transformed into its chloride by standard procedures. The attachment of the ‘lower’ chain was initiated by generating the stannane 95.58 In the coupling reaction towards 95 the (E)-2- tributylstannylethenyllithium also attacks the C-1 carbonyl with a slightly smaller preference (not shown).Both products were generated as single diastereomers of which the C-4 adduct yielded the cis-arrangement of the two oxygen substituents of the cyclohexene derivative. The assembly of alisamycin proceeded by Stille-type coupling with the bromide derivative of the ‘lower’ polyene chain. The synthetic strategy can be extended to other manumycin type-compounds or novel analogues by choice of the coupled organometallic derivative of the ‘upper’ chain. In addition to the cyclohexyl diene chain of alisamycin 26, the triene analogue of asukamycin 2 and manumycin F 42, and the tetraene chain of the colabomycins have already been prepared.Wipf and co-workers have also succeeded in synthesizing the structurally more complex methyl-branched side chain of manumycin A 1.136 The key step in preparing 89 was a zirconium to zinc transmetallation protocol which had been established for carbon–carbon bond formation (Scheme 15). Hydrozirconation of alkyne 96 and zinc-mediated addition to the chiral aldehyde 97 provided allylic alcohol 98 which could be converted to the dienoic acid 89.The reaction sequence also provided ready access to analogues of the ‘upper’ chain. Earlier synthetic approaches to manumycin-type compounds had targeted some of the less complex structurally related analogues, particularly those that resemble the m-C7N unit. The research groups of Wipf and Taylor have developed similar routes towards LL-C10037· 12 which both branched oV at the enamine 91 from the total synthesis of manumycintype compounds (Scheme 14).132,133 Finally, the synthesis of 2880-II 33 which had been isolated as a cometabolite from the colabomycin-producing Streptomyces griseoflavus (Tü 2880) was reported. 2880-II 33 comprises the C5N unit attached to ferulic acid.137 7 Biological properties Manumycin-group compounds show a variety of biological activities including antibiotic, cytotoxic and insecticidal properties as well as a number of enzyme inhibition activities.Out of all the biological activities, the recently discovered enzyme inhibition of Ras farnesyltransferase by manumycin A 1 is of particular interest. 7.1 Antibacterial and antifungal activity With the exception of TMC-1A 3, B 4, C 5 and D 6, and manumycin D 29 all tested manumycin-group metabolites inhibit the growth of Gram-positive bacteria, MIC (Ïg ml"1): e.g. asukamycin 2 against diVerent strains of Staphylococcus aureus (0.78–6.25), against Micrococcus flavus (1.56), against Bacillus subtilis (3.12–6.25);4 manumycins E 38, F 42 and G 39 against Staphylococcus aureus, S.epidermis and Bacillus subtilis: (0.3–8).62,63 In vivo application, however, towards the antibacterial activity in mice (experimentally infected with S. aureus) with U-62162 9 (8 mg kg"1)9 and U-56,407 25 (320 mg kg"1)56 did not induce any regression of the infection. The mode of the in vitro antibacterial activity remains unknown and due to its moderate size is not expected to be of any commercial interest. No activity against Gram-negative bacteria is observed with most of the manumycin-group metabolites (U-56,407 25, colabomycin A 7, manumycin A 1, alisamycin 26, nisamycin HN R O O O O HO HN Me HN HN Me Me Me HN HN OMe OH 34 Nisamycin 81 82 83 84 85 R Figure 9 Derivatives of nisamycin 34 O O O H N Me O O O O HN H O O O O HN O CrO3, 75% aq.acetic acid, 6 h, rt 1¢ O O O HN O O HO Me Me O Me Me Me Me Me O 1¢ 5¢ 6¢ 13¢ 10¢ 1 7 2 43 44 47 86 87 CrO3, acetic acid, H2O, 20 h, rt CrO3, 90% aq.acetic acid, 50 min, rt CrO3, 80% aq. acetic acid, 3 h, rt + Scheme 12 Degradation of manumycin A 1, colabomycin A 7 and asukamycin 2 by chromic acid oxidation 236 Natural Product Reports, 199834, and TMC-1A 3, B 4, C 5 and D 6). A minor activity was found for manumycins E 38, F 42 and G 39 against Escherichia coli (MIC=0.1 to 2.5 Ïg ml"1).62,63 In a small SAR study on antibacterial properties, derivatives 81–85 of nisamycin 34 were studied that carry diVerent hydrophobic residues instead of the C5N unit.87 An increase in hydrophobicity, e.g.the p-methoxybenzamide 85 vs. n-butylamide 81 led to an approx. 100-fold decrease in biological activity (see Section 6.1). Some of the mumycin-group metabolites exhibit antifungal activity (MIC in Ïg ml"1): e.g. against Candida albicans by manumycin A 1 (21),3 benzoquinone derivative of manumycin B 27 (10),61 alisamycin 26 (10),64 and benzoquinone derivative of manumycin C 28 (42).61 A weak antifungal activity against Trichophyton mentagrophytes at a concentration of 25 Ïg ml"1 was reported for asukamycin 2.4 Asukamycin was also found to possess anticoccidial activity when a concentration of 100 ppm was administrated to the diet of 4-day-old chicken infected with Eimeria tenella. 7.2 Insecticidal activity Manumycin A 1 was studied for its insecticidal eVects in laboratory and outdoor experiments with Lepidoptera and Coleoptera.3 The compound proved to be an insecticidal development restrictor. Promising eVects on the eggs and larvae of Pieris brassicae and Epilachna varivestis were achieved by means of low concentration aqueous solutions (0.05%).However, higher concentrations did not result in an eVective increase of activity. Compared to chitin synthase inhibitors the insecticidal eVect of manumycin A sets in slower. In addition, plant protection was observed by distinct repellent eVects on larvae who want to feed on the plants.H N Me HN O O O OH O O HO Me Me Me Me HN O OH O HO Me O Me Me Me R Me O Me Me Me 1 manumycin A 88 89 90 R = OMe 69 R = NH2 Acetic anhydride, 150 °C, 3–5 h 0.1 M NaOH, 50 °C Scheme 13 Degradation of manumycin A 1 OMe NH2 OMe MeO OMe NHBoc O MeO OMe NH2 O O O HN O O O O HN O O Cl O OHC O– TBDMSO CHO K+ –O NH Br O OH O HO SnBu3 Li SnBu3 1. Boc2O, THF, rt 2. C6H5I(OAc)2 MeOH, 0 °C 1. H2O2, K2CO3, THF–H2O, rt 2.BF3·OEt, CH2Cl2, rt 1. TBDMS-Cl, Et3N, DMAP, dry,THF, rt 2. c-C6H11MgBr, 0 °C 1. 3 M HCl, rt 1. NaClO2, Bu tOH, phosphate buffer 2. chloride formation 95, LiOBu t, THF THF, – 78 °C 92 12 LL-C10037a 93 94 91 95 26 rac-alisamycin cat. Pd0, DMF–THF, rt Scheme 14 Total synthesis of rac-alisamycin 26 Sattler et al.: The manumycin-group metabolites 237Manumycin A 1 is inactive against Tetranychus urticae and Myzus resicae. 7.3 Enzyme inhibition 7.3.1 Inhibition of farnesyltransferase In 1993, manumycins A 1, B 27 and C 28 (as UCF1-C, -A and -B) were discovered in a yeast based screening assay as inhibitors of Ras farnesyltransferase (FTase).13,61 Farnesyltransferase performs the essential step in Ras posttranslational processing by transferring a farnesyl residue from farnesyl pyrophosphate to a cysteine near the C-terminus of Ras.138,139 The proto-oncoproteine Ras is a central switch in cellular signal transduction and is in its oncogenic constitutively activated form a most prevalent factor in a number of solid tumors (e.g.pancreatic carcinomas 90%, colon 50%, lung 30%). Inhibiting Ras farnesylation is a suitable target to block abnormal cell growth and diVerentiation triggered by activated ras. Although farnesylation is taking place on a number of cellular proteins the eYcacy of this target for tumor therapy without significant toxicity has been shown in several animal models. Manumycins A 1, B 27 and C 28 have in vitro activities on FTase of IC50=5, 13 and 7 ÏM, respectively.13 As the structure of the branched ‘upper’ side chain suggests FTase inhibition by manumycin A 1 is competitive with respect to farnesylpyrophosphate.The possible role of the ‘upper’ side chain in enzyme binding is reflected in the lower inhibitory activities of manumycins E 38, F 42 and G 39 (IC50=ca. 100 ÏM) which all carry an ‘upper’ chain with less methyl branches.62 Additional proof for this assumption derives from a SAR study with the benzoquinones of manumycin A, B and C (see Section 6.2) whose lack of the ‘lower’ chain did not significantly aVect the inhibitory activity.61 A critical feature of a potent FTase inhibitor is its specificity for this particular enzyme since the closely related geranylgeranyltransferase I is of essential importance for cellular function.Manumycin A 1 exhibits a 400-fold specificity for FTase. On a cellular level, manumycin A 1 has been studied for its FTase inhibition in two diVerent yeast models, as well as in a Caenorhabditis elegans system.13,140 Furthermore, it was used as inhibitor to study farnesylation in plants.141 Manumycin A 1 and gliotoxin are the only natural products that have been shown to inhibit FTase in cellular models as well as in vivo.13,142,143 In a mouse model with an implanted ras dependent murine fibrosarcoma (K-BALB) manumycin A caused significant regression of the tumor.13 The required dosage (6.3 mg kg"1 per day for 5 days) is comparable to synthetic peptidomimetic inhibitors which have much better in vitro FTase inhibition activity. 7.3.2 Other Recently, manumycin-type compounds were detected in an in vitro screening as inhibitors of interleukin-1‚ converting enzyme (ICE). This biological activity might make them useful as lead structures for antiinflammatory agents.70,144 Interleukin-1 which is primarily secreted by activated monocytes or macrophages has been implicated in the pathogenesis of acute and chronic inflammation. The cytokine exists in two isoforms, IL-1· and IL-1‚, of which the active form of IL-1‚ has to be processed proteolytically by ICE from an inactive precursor. In the study that comprised EI-1511-3 40, EI-1511-5 37, EI-1625-2 36, U-56,407 25, ent-alisamycin 41 and manumycins A 1, B 27 and G 39, EI-1511-3 40 showed the best in vitro activity with IC50=90 nM.For all other compounds in this study submicromolar inhibitory activity was observed, and only manumycin A 1 was two orders of magnitude less active.The benzoquinone derivatives of some of the compounds (see Section 6.2) had similar activities as the parent compounds which indicates that the ‘lower’ chain is not necessary for ICE inhibition. The inhibitory specificity of the biological activity on the protease ICE was shown by the absence of any activity on the proteases cathepsin B and elastase by EI-1511-3 40, EI-1511-5 37 and EI-1625-2 36.The ICE inhibitory activity was also found in cellular assays. All eight manumycin-type compounds tested suppressed extracellular release of IL-1‚ with IC50s around 10 ÏM. At a level of 33 ÏM EI-1511-3 40, EI-1511-5 37 and EI-1625-2 36 completely inhibited IL-1‚ secretion from THP-1 cells while cell viability was over 90%. The results with the ICE protease relate to the first enzymatic activity that was described for manumycins which is the inhibition of human leukocyte elastase [polymorphonuclear (PMN) elastase] by manumycin A 1 (IC50=4.0 Ïg ml"1).144,145 A similar potency against this proteolytic enzyme was observed for the aromatic precursor-directed product 64-mABA (IC50=3.6 Ïg ml"1).145 The secretion of elastase by tumor cells plays an important role for invasive tumor growth. 7.4 Cell and in vivo toxicity A moderate but significant cytotoxicity of manumycin-type compounds has been observed. Colabomycin A 7 and manumycin A 1 showed cytotoxic eVects against murine L-1210 leukemia cells in both proliferation (IC50=4.6 and 3.1 Ïg ml"1, respectively) and stem cell assays (IC50=3.4 and 0.9 Ïg ml"1, respectively).3,6 Manumycins E 38, F 42 and G 39 displayed weak cytotoxicity on the human colon carcinoma cell line HCT-116 (IC50s=15.6 Ïg ml"1).62 A broader picture of the cytoxicity of manumycins can be gained from a study on eight diVerent tumor cell lines with TMC-1 A 3, B 4, C 5, D 6 and manumycins A 1, C 28 and D 29.5 Curiously, the type I compounds manumycin A 1 and C 28 which carry the reactive oxirane functionality showed only a slightly enhanced cytotoxicity compared to the type II compounds.Overall, the cytotoxicity on the tumor cell lines is of moderate size (IC50s between 0.2 and 30 Ïg ml"1). A few studies pointed to in vivo toxicity of manumycin-type compounds.13 Manumycin A 1 and asukamycin 2 showed a LD50 in mice by intraperitoneal injection of about 75 mg kg"1 and 48 mg kg"1, respectively.4 However, asukamycin had no eVect on mice up to 450 mg kg"1 when it was administered per os.The antibiotics U-62162 99 and U-56,407 2556 exhibited no toxic behaviour in mice at 8 mg kg"1 and 320 mg kg"1, respectively. Addition in proof The stereospecific total synthesis of ent-(+)-manumycin A has recently been achieved by Taylor and co-workers.146 The TBSO OHC Me TBSO Me Me Me Me Me Me Me HO Me Me Me Me O OH 96 98 89 1. Cp2Zr(H)Cl, CH2Cl2, rt 2. (Me)2Zn, – 60–0 °C 3. 1. TFAA, py, DMAP, 0 °C 2. TsOH, MeOH–Et2O 3. Dess–Martin periodinane, (Pri)2NEt, THF 4. NaClO2, NaH2PO4, THF–H2O, 2-methylbut-2-ene, rt 97 Scheme 15 Synthesis of 89, the carboxylic acid of the ‘upper’ chain of manumycin A 1 238 Natural Product Reports, 1998synthesized congener has a 4R,5S,6R configuration with syn arrangement of the hydroxy group at C-4 and the oxirane at C-5–C-6. Data comparison of the synthetic product with authentic (")-manumycin A 1 showed complete structural identity with opposite stereochemical data (optical rotation and CD spectra).Thus, the stereochemical assignment of C-4 of manumycin A 1, and possibly of manumycin B 27 and El-1625-2 36, should be revised as 4S-configuration (see Fig. 1 and Table 3). It can be concluded that the syn hydroxy epoxide structure is a specific criterion for manumycin-group metabolites. Obviously, the interpretation of CD spectra following the exciton chirality rule for the assignment of the C-4 con- figuration is ambiguous for some of the manumycins.Acknowledgements We wish to thank Professor R. J. K. 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Maruta and A. W. Burgess, Springer Verlag, Heidelberg, 1996. 139 M. Barbacid, Ann. Rev. Biochem., 1987, 56, 779. 140 M. Hara and M. Han, Proc. Natl. Acad. Sci. USA, 1995, 92, 3333. 141 D. Qian, D. Zhou, R. Ju, C. L. Cramer and Z. Yang, Plant Cell, 1996, 8, 2381. 142 T. Nagase, S. Kawata, S. Tamura, Y. Matsuda, Y. Inui, E. Yamasaki, H. Ishiguro, T. Ito and Y. Matsuzawa, Int. J. Cancer, 1996, 65, 620. 143 Y. Zou, I. Komuro, T. Yamazaki, R. Aikawa, S. Kudoh, I. Shiojima, Y. Hiroi, T. Mizumo and Y. Yazaki, J. Biol. Chem., 1996, 271, 33592. 144 T. Tanaka, E. Tsukuda, Y. Uosaki and Y. Matsuda, J. Antibiot., 1996, 49, 1085. 145 EP 318 845 A/1987. 146 L. Alcaraz, G. Macdonald, J. P. Ragot, N. Lewis and R. J. K. Taylor, J. Org. Chem., submitted. 240 Natural Product Reports, 1998

ISSN:0265-0568    

DOI:10.1039/a815221y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Isoflavonoids and related compounds Gerard M. Bolanda and Dervilla M. X. Donnellyb aDepartment of Chemistry, Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland bDepartment of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland Covering: January 1994 to December 1996 Previous review: 1995, 12, 321 1 Introduction 2 Isolation and identification 3 Isoflavones 4 Isoflavanones 5 Isoflavans 6 Pterocarpans 7 Rotenoids 8 Isoflavonoid oligomers 9 Miscellaneous structures 9.1 Coumaronochromones 9.2 Isoflav-3-enes 9.3 3-Arylcoumarins 9.4 Coumestans 9.5 2-Arylbenzofurans 10 References 1 Introduction This report is a review of the literature concerning isoflavonoid compounds published between January 1994 and December 1996.The format follows the pattern of its predecessor,1 with an emphasis on the isolation and synthesis of new members of the isoflavonoid family. Although the distribution of isoflavonoids among plants is relatively sparse they nevertheless form a large and very distinctive subclass of the flavonoid family with a wide variety of structural variations encountered in nature.Continued interest in the isolation of new isoflavonoids arises due to the diverse range of biological activities including antimicrobial, oestrogenic and insecticidal activities which they possess. The pharmacological properties of isoflavonoids have been reported, especially papers detailing the relationship between consumption of isoflavonoids and reduction in the incidence of cardiovascular disease and cancer but these will not be discussed in this report.It is interesting to note that there has been a slight decrease in the number of new iso- flavones isolated during this review period. While this may be a temporary slow down in productivity, it may also indicate that new compounds are becoming harder to find. It is significant that several new isoflavonoid oligomers are now reported (Section 8), as the existence of natural isoflavonoid oligomers has only recently been confirmed.2 These new compounds comprising of isoflavonoid units linked to other iso- flavonoid and neoflavonoid units complement the rare series of isoflavonoids linked to flavonoid, stilbene and phenylpropanoid units.In addition to this report two further papers detailing isoflavonoids from African plant species3 and the chemical and biological aspects of isoprenoid substituted isoflavonoids4 have been published. 2 Isolation and identification This section highlights developments in the isolation of iso- flavonoids from plant and food sources using high performance liquid chromatography (HPLC). The current use of soy food products in clinical and experimental studies to explore the relationship between soy consumption and the reduction of cancer risk has led to the determination of the quantitative and qualitative composition of isoflavones in selected soy foods.Barnes et al. have described for the first time the application of HPLC–mass spectrometry with heated nebulizer–atmospheric pressure chemical ionization (HN–APCI) and IonSpray interfaces to the analysis of isoflavones.5 The study showed that soybeans contained mostly isoflavone 6+-O-malonylglucoside (6-OMalGlc) conjugates with lesser quantities of the ‚- glucosides and only trace amounts of 6+-O-acetylglucoside conjugates. It was found that the isoflavone composition could be changed by variation of extraction temperatures, in particular isoflavone 6-OMalGlc conjugates were prone to thermal decarboxylation and de-esterification.A fast, sensitive, reliable and precise method for the diode array reversed-phase HPLC analysis of the most common isoflavonoids (daidzein, genistein, formononetin, biochanin A and coumestrol) in foods which allowed quantitation of phytoestrogen levels in legumes has been reported.6 The separation and identification of iso- flavone compounds from diVerent parts (stem, leaf, flower) of seven common Trifolium species by HPLC is of interest from a phytochemical point of view.7 Great diVerences, both quantitatively and qualitatively were found in isoflavone levels in the Trifolium genus and the data presented may be significant for the breeding of new Trifolium species with low isoflavone concentrations.The combination of HPLC with mass spectrometric detection, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) has proved to be a useful tool in analysing natural products. The combination of HPLC–APCI allows eYcient separation and identification of isoflavones with greater eYciency than HPLC (with UV detection) alone.8 Negative-ion APCI provides quality mass spectra and by variation of conditions, the collision-induced dissociations which are generated permits structure elucidation.Further information on isoflavone structure can be obtained by addition of deuterium oxide to the sample which induces peak shifts in the mass spectra and helps to identify the number of exchangeable hydrogen atoms in each molecule.The use of capillary electrophoresis (CE) in the determination of iso- flavones has also been described.9 In contrast to HPLC, the CE method is rapid, it does not require solvent gradient or elution and it does not consume organic solvents for elution. Capillary electrophoresis in combination with electrospray ionization–mass spectrometry (ESI–MS) has been shown to be a suitable technique for the determination of isoflavones.10 The use of CE–ESI–MS which permits the eYcient separation and identification of isoflavones with higher specificity than CE (with UV detection) alone, allows both the determination of the molecular mass of isoflavones and the recognition of various functional groups through analysis of several diagnostic fragment ions.Two dimensional NMR techniques including 1H–1H (COSY), 13C–1H (HETCOR), 13C–1H (COLOC) and NOESY are now routine methods for the structure elucidation of isoflavonoids.Application of 2D NMR techniques has led to the unambiguous 1H and 13C NMR assignments of the iso- flavonoids 5,7-dihydroxy-4*-methoxyisoflavone (biochanin A), 2*,5,7-trihydroxy-4*-methoxyisoflavone and 5,7-dihydroxy- 2*,4*-dimethoxyisoflavone, isolated from Virola caducifolia.11 Boland and Donnelly: Isoflavonoids and related compounds 241In isoflavones containing a 6- or 8-prenyl side chain, the location of the prenyl substituent can be deduced from the chemical shift of the 5-hydroxy proton.Fukai et al.12 have observed that the signal of the hydrogen bonded hydroxy proton appears further downfield (0.25–0.30 ppm) in 6- isoprenylated flavonoids compared with that of 6- non-substituted flavonoids having the same B and C rings. In contrast, the 5-OH signal of 8-isoprenylated flavonoids shows upfield shifts (ƒ 0.04–0.10 ppm) compared with that of flavonoids having the same B and C rings and no side chain.Hydroxylation at C-2* causes an upfield shift of the 5-OH signal arising due to a weak hydrogen bond between the carbonyl in the 4-position and the 2*-hydroxy group. The results of the study by Fukai et al. of substituent eVects on the chemical shift of the 5-OH proton signal in isoflavones are summarized in Table 1. These parameters are useful for the characterization of 6- or 8-prenylated isoflavones. In 5,7- dihydroxyisoflavanone derivatives, the determination of the position of a prenyl substituent on the A-ring cannot be achieved using 13C NMR, as the chemical shifts of the A-ring carbons of 6- and 8-prenylated isoflavanones are almost the same.The location of the prenyl substituent can however be deduced from the chemical shift of the 5-hydroxy proton. Analysis of the chemical shift of the 5-OH signal in the case of isoflavanone derivatives, indicates that prenylation of C-6 results in a downfield shift (ƒ 0.30 ppm) of the 5-OH proton whereas prenylation of C-8 results in a small upfield shift (ƒ 0.04 ppm).13 These trends follow those found in other prenylated flavonoids.Hydroxylation at C-2* of 3*- unsubstituted isoflavone derivatives causes an upfield shift (ƒ 0.26 ppm) of the 5-OH signal, the opposite is observed for 3*-unsubstituted isoflavanones where hydroxylation at C-2* causes a downfield shift (ƒ 0.12 ppm) of the 5-hydroxy group.Table 2 summarizes the substituent eVects on the 5-OH signal in isoflavanone derivatives. These parameters have been used in the structural assignment of the known isoflavanone, 3*-(„,„- dimethylallyl)kievitone from Glycyrrhiza glabra. Cyclization of an isoprenyl group onto a neighbouring phenol function results in the formation of either linear or angular isoflavonoids depending on the direction of ring fusion. A comparison of the chemical shifts of four known isoflavones [osajin, pomiferin (angular isoflavones), warangalone and aurisculasin (linear isoflavones)] has revealed that the signals of some protons are significantly characteristic.14 The signal of the H-2 proton and the prenyl methylene appear further downfield (ca. 0.1 ppm) in linear isoflavones compared with those of angular isoflavones. The signals of the protons peri to the free phenolic function of the four isomers (i.e. the prenyl CH2 for the angular isomers and the ‚-CH for the linear form) undergo a major paramagnetic shift in C5D5N.The direction of ring fusion can also be determined using UV spectroscopy. Angular isoflavones show a maximum absorption at a shorter wavelength and a shoulder at a higher wavelength with respect to their regioisomers, while addition of AlCl3 does not cause a significant bathochromic shift in the UV spectra of the angular isoflavones. The angular and linear arrangements can likewise be identified from their mass spectra.The linear isomer gives a base peak corresponding to loss of a methyl group, while the angular isomer gives a base peak corresponding to the fragment ion at m/z (M"C4H7)+2. Spectroscopic data (1H, 13C NMR, UV and MS) for the four isomers (osajin, pomiferin, warangalone and aurisculasin) were presented, which will aid future identification of isoprenoidsubstituted isoflavones fully substituted in the A-ring. 3 Isoflavones Isoflavones constitute the largest group of natural isoflavonoids with 66 new structures reported in the previous review1 and a further 46 new structures reported in the current paper.The occurrence of isoflavonoids in plants of traditional medicine has promoted the increasing interest in the search for biologically active constituents from both legume and nonlegume sources. Table 3 lists the structures and sources of new isoflavones both aglycones and glycosides, as well as known isoflavones that have been found in new sources.Unless otherwise stated all plants mentioned are members of the Leguminosae family. Several simple isoflavones lacking a prenyl substituent on either the A- or B-rings have been reported. Two isoflavones were isolated from the aerial parts of Celosia argentia, a species native to Taiwan. These were identified as 2*-hydroxy-5- methoxy-6,7-methylenedioxyisoflavone and its 2*-methoxy derivative.15 These known compounds have not previously been isolated from this species and are unusual in that iso- flavones with C-2* substituted B-rings and/or methylenedioxy substituted A-rings are very rare in nature.Two new methoxylated isoflavones 6,7,8,3*,4*,5*-hexamethoxy- and 7,8,3*,4*, 5*-pentamethoxy-isoflavone were isolated from the bark of Petalostemon purpureus, a species on which no previous phytochemical study has been conducted.16 A recent phytochemical investigation of the genus Garcinia, a species belonging to Clausiaceae, led to the isolation of the novel isoflavone nervosin from G.nervosa along with two known isoflavones irigenin and 7-methyltectorigenin, not previously reported from this species.17 Several non-legume sources are included in recent isolations. Isoflavones found in the family Iridaceae, in particular Iris species have a characteristic 5,6,7- trioxygenation pattern. New examples in this class include irisjaponins A and B from the aerial parts of Iris japonica,18 and nigricin and nigricanin from the rhizomes of I.nigricans, a species whose chemical constituents have not previously been investigated.19 The structures of these new isoflavones were established by spectroscopic and chemical methods. Iso- flavones with 5,6,7-trioxygenation patterns are also found in Belamcanda chinensis (Iridaceae) although the main diVerences Table 1 Substituent parameters (ppm) on the 5-hydroxy group of isoflavones (in [2H6]acetone) Substituent parameter Prenylation at C-6 (and 6,8-diprenylated) +0.28 Prenylation at C-8 "0.06 Methylation of 7-OH (6-prenylated isoflavone) "0.12 (8-prenylated isoflavone) +0.09 Hydroxylation at C-2* (3*-unsubstituted isoflavone) "0.26 (3*-alkenylated isoflavone) "0.52 Hydroxylation at C-3* +0.05 Methylation of 3*-OH or 4*-OH "0.02 Cyclization between 2*-OH and 3*-prenyl group "0.56 Table 2 Substituent parameters (ppm) on the 5-hydroxy group of isoflavanones (in [2H6]acetone) Substituent parameter Prenylation at C-6 +0.30 Prenylation at C-8 "0.04 Prenylation at C-3* (2*,4*-dihydroxylated derivative) "0.29 Hydroxylation at C-2* (3*-unsubstituted isoflavanone) +0.12 Methylation of 2*-OH or 4*-OH "0.05–0 Methylation of 7-OH (6,8-unsubstituted) "0.02 (6-prenylated) "0.16 Cyclization between 2*-OH and 3*-prenyl group +0.35 242 Natural Product Reports, 1998Table 3 Isoflavones isolated between 1994 and 1996 O O 2 3 1 4 1¢ 6 5 7 2¢ 8 4¢ 3¢ 5¢ 6¢ Plant sources Ref.Isoflavone 2*-Hydroxy-5-methoxy-6,7-methylenedioxy-a Celosia argentia, aerial parts (Amaranthaceae) 15 2*,5-Dimethoxy-6,7-methylenedioxy-a C.argentia, aerial parts (Amaranthaceae) 15 6,7,8,3*,4*,5*-Hexamethoxy- Petalostemon purpureus, bark 16 7,8,3*,4*,5*-Pentamethoxy- P. purpureus, bark 16 Nervosin (5,7,4*-trihydroxy-2*,3*,6*-trimethoxy-) Garcinia nervosa, leaves (Clausiaceae) 17 Irigenina (5,7,3*-trihydroxy-6,4*,5*-trimethoxy-) G. nervosa, leaves 17 7-Methyltectorigenina (5,4*-dihydroxy-6,7-dimethoxy-) G.nervosa, leaves 17 Irisjaponin A (5,7-dihydroxy-6,2*,3*,4*,5*-pentamethoxy-) Iris japonica, aerial parts (Iridaceae) 18 Irisjaponin B (5,7-dihydroxy-6,2*,3*,4*-tetramethoxy-) I. japonica, aerial parts 18 Nigricin (4*-hydroxy-5-methoxy-6,7-methylenedioxy-) I. nigricans, rhizomes (Iridaceae) 19 Nigricanin (4*-hydroxy-5,3*-dimethoxy-6,7-methylenedioxy-) I. nigricans, rhizomes 19 Isocladrastin (3*-hydroxy-6,7,4*-trimethoxy-) I. kashmiriana, rhizomes (Iridaceae) 20 Kashmigenin (4*-hydroxy-3*,5*-dimethoxy-6,7-methylenedioxy-) I.kashmiriana, rhizomes 20 2*-Methoxyformononetin (7-hydroxy-2*,4*-dimethoxy-) Eschscholtzia californica, whole plants (Papaveraceae) 21 2*,4*-Dihydroxy-7-methoxy- E. californica, whole plants 21 Gerontoisoflavone A (7,4*-dihydroxy-5,3*-dimethoxy-) Cudrania cochinchinensis var. gerontogea root wood, (Moraceae) 22 Aciceroneb (6-hydroxy-7-methoxy-3*,4*-methylenedioxy-) Astragalus cicer, roots 23 Erysenegalensein D 1 Erythrina senegalensein, stem bark 26 Erysenegalensein E 2 E.senegalensein, stem bark 26 Erysenegalensein F 3 E. senegalensein, stem bark 27 Erysenegalensein G 4 E. senegalensein, stem bark 27 Erysenegalensein H 5 E. senegalensein, stem bark 28 Erysenegalensein I 6 E. senegalensein, stem bark 28 Erysenegalensein K 7 E. senegalensein, stem bark 29 Erysenegalensein L 8 E. senegalensein, stem bark 30 Erysenegalensein M 9 E. senegalensein, stem bark 30 5-O-Methyl-4*-O-(3-methylbut-2-enyl) alpinumisoflavone 10 Milletia thonningii, root bark 31 Thonninginisoflavone 11 M.thonningii, root bark 31 Durallone 12 M. dura, seed pods 32 6-Demethyldurallone 13 M. dura, seed pods 32 Predurallone 14 M. dura, seed pods 32 Isoerythrinin A 4*-(3-methylbut-2-enyl) ether 15 M. dura, seed pods 32 Glyasperin N 16 Glycyrrhiza aspera, roots 33 Kanzonol K 17 G. uralensis, roots 12 Kanzonol L 18 G. uralensis, roots 12 Kanzonol T 19 G. glabra, roots 34 Eurycarpin A 20 G. eurycarpa, roots 35 Anagyroidisoflavone A 21 Laburnum anagyroides, pods 36 Anagyroidisoflavone B 22 L.anagyroides, pods 36 Laburnetin 23 L. anagyroides, pods 36 Alpinumisoflavonea 24 L. anagyroides, pods 36 Secundiflorol B 25 Sophora secundiflora, roots 37 Secundiflorol C 26 S. secundiflora, roots 37 Isolupalbigenin 27 Lupinus luteus, roots 38 Ormosidin 28 Ormosia monosperma, root bark 40 Eturunagarone 29 Derris scandens, stems 41 Eriosemaone D 30 Eriosema tuberosum, roots 42 Ficusin A 31 Ficus septica, root bark (Moraceae) 43 Ficusin B 32 F.septica, root bark 43 5,3*,4*,2*+-Tetrahydroxy-2+,2+-dimethylpyrano [5+,6+:7,8]-6-(3*+-methylbut-3*+-enyl-) 33 Maclura pomifera, fruit (Moraceae) 14 5-Hydroxy-8-methoxy-3*,4*-methylenedioxy 6+,6+-dimethylpyrano[2+,3+:7,6]- 34 Lonchocarpus subglaucescens, roots 44 6,8-Diprenylgenisteina Erythrina sigmoidea, root bark 46 Scandenonea E. sigmoidea, root bark 46 Neobavaisoflavonea E. sigmoidea, root bark 46 Glycosides Prunetin-4*-O-apiosyl(1]6)glucoside (coromandelin) Dalbergia coromandeliana, leaves (Fabaceae) 59 Genistein 5-methyl ether 4*-glucoside Cotoneaster simonsii, leaves (Rosaceae) 60 2*-Hydroxygenistein 6-C-·-L-rhamnosyl(1]2)glucoside (nodosin) Cassia nodosa, flowers 61 2*-Hydroxygenistein 8-C-glucoside C.siamea, leaves 62 5,6,6*-Trimethoxy-3*,4*-methylenedioxy isoflavone 7-O-(2+-p-coumaroylglucoside) 42 Trichosanthes anguina, seeds (Cucurbitaceae) 63 aNew source of known isoflavone.bPlant part was subjected to physiological stress. Boland and Donnelly: Isoflavonoids and related compounds 243between I. japonica and B. chinensis are the degree of oxygenation of the B-ring and variable O-substituents of 5,6,7-trihydroxy groups. Examination of the rhizomes of I. kashmiriana led to the isolation of two isomeric isoflavones isocladrastin and kashmigenin, in addition to the known junipegenin-B.20 These new isoflavones lack the characteristic 5,6,7-trioxygenated pattern found in Iris species.Two new isoflavones were isolated from whole plants of Eschscholtzia californica, the structures of which were determined as 2*-methoxyformononetin and 2*,4*-dihydroxy-7- methoxyisoflavone by spectroscopic methods.21 Both iso- flavones gave an identical compound on methylation. A novel methoxylated isoflavone named gerontoisoflavone A, together with 12 known flavonoids was isolated from the root wood of the medicinal plant Cudrania cochinchinensis var.gerontogea.22 The proposed structure was established by 2D NMR techniques. Many plants produce stress metabolites by treatment of the plant cells by abiotic elicitors of chemical or physical origin. These stress metabolites are thought to contribute as phytoalexins to the defence mechanism of the plants. A new isoflavone named acicerone, in addition to the pterocarpan maackiain and the isoflavone cajanin, was induced in root and leaflet tissues of Astragalus cicer by elicitation with the fungus Bipolaris zeicola or in leaflets by UV-C irradiation.23 Treatment of the cell suspension cultures of Pueraria lobata with yeast extract as an elicitor resulted in the rapid selective decline of the constitutive isoflavonoid conjugates followed by both reaccumulation of the conjugates and formation of the isoflavones daidzein and genistein.24 This observation is of importance in the understanding of the early defence response of P.lobata cells in a plant–pathogen interaction.Comparative studies between metabolite productions in cell culture and in whole plant of Maclura pomifera have been described.25 It was found that the isoflavones in cell cultures lacked the 3*,4*-dihydroxy substitution pattern found in fruits. The genus Erythrina (Leguminosae) is well known for its alkaloidal compounds. Chemical studies on the nonalkaloidal components of the medicinal plant E. senegalensis, a species widely distributed in the tropical and subtropical regions has revealed a variety of isoflavones and iso- flavanones.Erysenegalensein D 1 and erysenegalensein E 2 were isolated from the stem bark and their structures were determined by the usual spectroscopic and 2D NMR techniques. 26 Both isoflavones contain a chiral five-carbon hydroxylated unit linked to an aromatic ring although the stereochemistry at the chiral centre was not determined. Two novel epoxyisoflavones, erysenegalensein F 3 and erysenegalensein G 4 were also obtained from the stem bark of the same species.27 The structures were established by spectroscopic methods and confirmed by synthesis.Epoxidation of the co-occurring known compounds auriculatin and warangalone using meta-chloroperbenzoic acid in methylene chloride gave the corresponding epoxyisoflavones. The orientation of the dimethylchromene ring in 3 and 4 was established on the basis of comparison of the NMR spectra of the parent compound and its acylated derivative.Further examination of the stem bark led to the isolation of erysenegalensein H 5 and erysenegalensein I 6.28 The orientation of the dihydropyran ring in 6 was established as being linear as dehydration of 6 gave an identical compound to auriculatin. The furanoisoflavone, erysenegalensein K 7, was isolated from the stem bark in addition to a new 3- hydroxycoumaronochromone and five known pentacyclic triterpenes.29 The exact orientations of the A-ring substituents in 7 were established on the basis of 13C–1H (HETCOR and COLOC) NMR techniques.Two novel isoflavones erysenegalensein L 8 and erysenegalensein M 9 have been reported in addition to the known alkaloids erysodine, glucoerysodine and hypaphorine, isolated for the first time from E. senegalensis.30 The complete assignments of 1H and 13C NMR chemical shifts for these natural products were reported. The genus Milletia (Leguminosae) is noted for its insecticidal and piscicidal properties.The plant M. thonningii has O O O R OH OH O O O HO R OH OH OH 3 R = OH; Erysenegalensein F 4 R = H; Erysenegalensein G 1 R = OH; Erysenegalensein D 2 R = H; Erysenegalensein E O O OH OH OH O HO O O OH OH OH O HO O O OH OH OH O O O O R OH OH OH 7 Erysenegalensein K 6 Erysenegalensein I 5 Erysenegalensein H 8 R = OH; Erysenegalensein L 9 R = H; Erysenegalensein M 244 Natural Product Reports, 1998received little chemical attention apart from work done on the seeds.Examination of the petroleum extract of the root bark of M. thonningii led to the isolation of two new compounds 5-O-methyl-4*-O-(3-methylbut-2-enyl) alpinumisoflavone 10 and thonninginisoflavone 11.31 The isoflavone 11 was assigned the S-configuration, from the positive Cotton eVect at 480 nm obtained for the osmate ester–pyridine complex. The seed pods of M. dura yielded four novel isoflavones, durallone 12, 6-demethyldurallone 13, predurallone 14 and isoerythrinin-A 4*-(3-methylbut-2-enyl) ether 15.32 Durallone 12 was the major compound from the extract and is closely related to the known isoflavone durmillone which was isolated in the same investigation from the stem bark.The isoflavone 15 has a prenyl unit in the form of an ether substituent which is only occasionally found in isoflavones. The oxygenation pattern in 15 is the same as the known isoflavone erythrin-A which has the pyran ring substituent at C-6/C-7 and with a free phenol at C-4*.It is notable that the seed pods contain an entirely unique suite of isoflavonoids in comparison with the seeds as well as other parts of the plant that have been examined. New isoprenoidsubstituted isoflavones have been isolated from the roots of the Chinese licorices Glycyrrhiza aspera, G. uralensis, G. glabra and G. eurycarpa. The isoflavone glyasperin N 16 was isolated from G. aspera in addition to two new prenylated isoflavanones and a prenylated 3-arylcoumarin.33 The mass spectral breakdown patterns of some of these isoflavonoids were described.Two isoflavones, kanzonol K 17 and kanzonol L 18, were isolated from G. uralensis and their structures determined by detailed examination of the substituent eVects on the chemical shift of the 5-hydroxy proton,12 while kanzonol T 19 was isolated from G. glabra.34 It is interesting to note that the flavonoids obtained from Chinese licorice diVer from the phenolic compounds isolated from Russian and Spanish licorice.Three new isoflavones anagyroidisoflavone A 21, anagyroidisoflavone B 22 and laburnetin 23 each having a genistein-type oxygenation pattern were isolated from the pod extracts of Laburnum anagyroides in addition to the known isoflavone alpinumisoflavone 24 which has not been previously found in L. anagyroides.36 The novel isoflavone 22 with an epoxide side structure was only obtained in trace amounts. The isoflavones, secundiflorol B 25 and secundiflorol C 26 both containing the less common 1,1-dimethylallyl substituent attached to the B-ring were isolated from the roots of Sophora secundiflora and their structures were determined by 2D NMR techniques.37 Flavonoid compounds with a 2*,3*,4*-trioxygenated substitution on the B-ring, although common in leguminous plants such as Dalbergia are very rare in the genus Sophora.The known isoflavonoid secundifloran was also obtained from S. secundiflora and the 13C NMR spectrum which had not been previously assigned was reported.O O O O OMe O O OMe OMe O O O O O 10 5- O-Methyl-4¢- O-(3-methylbut-2-enyl)alpinumisoflavone 11 Thonninginisoflavone 15 Isoerythrinin-A 4¢-(3-methylbut-2-enyl) ether O O O OMe R OMe O O HO OMe MeO OMe 12 R = OMe; Durallone 13 R = OH; 6-Demethyldurallone 14 Predurallone O O HO OH OH O O O HO OH OH O O O MeO OH OH HO O O HO O OH HO HO O O HO OH OH 16 Glyasperin N 19 Kanzonol T 18 Kanzonol L 17 Kanzonol K 20 Eurycarpin A Boland and Donnelly: Isoflavonoids and related compounds 245Tahara et al.38 have discovered the first diprenylated (C-8/C- 3*) isoflavone (isolupalbigenin, 27) in yellow lupin, although compounds of this type are known to occur in white lupin (L.albus). The 2*-hydroxy derivative of isolupalbigenin has been previously reported in white lupin roots.39 The structure of isolupalbigenin was established from 1H NMR comparisons with lupalbigenin (6,3*-diprenylgenistein) and lupiwighteone (8-prenylgenistein) and the antifungal activity of various yellow lupin constituents was determined using Cladosporium herbarum as the test fungus.It was found that for isoflavones, the 6-prenyl and 3*-prenyl compounds were more fungitoxic than the 8-prenyl analogues and transformation of the prenyl group to a cyclized derivative greatly reduced or eliminated the fungitoxic eVects. Phytochemical studies on the phenolic compounds in the genus Ormosia have been rarely examined except for O.excelsa. Recently from the root bark of O. monosperma, ten isoflavonoids including a new isoflavone ormosidin 28 were isolated.40 Flavonoid compounds of this type containing C5 unit(s) have been frequently isolated from Sophora, Euchresta and Echinosophora species but their occurrence in Ormosia has not been previously reported. Additional prenylated iso- flavones which have been isolated during the course of this review include eturunagarone 29 from Derris scandens41 and eriosemaone D 30 which was fungitoxic against Cladosporium cucumerinum.42 Two isoflavones, ficusins A 31 and B 32, as well as the known compound genistein were isolated from Ficus septica Barm.F.143 These new compounds which are C-8-substituted genistein derivatives are unique isoflavones containing a cyclic-monoterpene-substituent. The structure of the known compound, maxima isoflavone J (7-O-„,„-dimethylallylformononetin) which had been earlier assigned on the basis of indirect evidence was confirmed by isolation of the pure compound from the roots of Tephrosia maxima.45 An X-ray crystallographic study of dalspinosin (5,7- dihydroxy-3*,4*,6-trimethoxyisoflavone) has confirmed the planarity of the fused ring system and the slight puckered arrangement of the 3-aryl ring, with the methoxy groups at C-6, C-3* and C-4* oriented slightly out of the plane of the rings to which they are attached.47 Analysis of the synthetic iso- flavone, ipriflavone (7-isopropoxyisoflavone) which is used in the treatment of bone loss in osteoporosis, indicated that the molecular structure was dominated by the dihedral angle of 50) between the planes of the aryl and benzopyran moieties.48 The synthesis of isoflavones can be broadly divided into three main synthetic pathways: the formylation of deoxybenzoins, the oxidative rearrangement of chalcones and flavanones, and the arylation of a preformed chromanone ring.Synthetic procedures using 2*-hydroxyphenyl benzyl ketones and 2*-hydroxychalcones have been reviewed.49 A series of isoflavones with 6,7-dimethoxy groups in the A-ring and diVerent substituents in the C-ring were prepared via deoxybenzoin intermediates.50 The deoxybenzoins were prepared in very good yields by condensation of 1,2,4-trimethoxybenzene with various phenylacetic acids in the presence of polyphosphoric acid (PPA) under modified Nencki’s conditions.It was expected that treatment of the deoxybenzoins with Vilsmeier–Haack reagent would formylate the active methylene and cyclize the deoxybenzoins with concomitant dealkylation, however only the ·-formyl deoxybenzoins were obtained.Cyclization to the corresponding isoflavone was achieved by treatment with pyridinium hydrochloride (Scheme 1). Two naturally occurring isoflavones; O O OH OH HO OH O O OH OH O O O OMe R HO OH OH O O OH OH O HO OMe O O OH OH O O 23 Laburnetin 24 Alpinumisoflavone 25 R = OH; Secundiflorol B 26 R = H; Secundiflorol C 22 Anagyroidisoflavone B 21 Anagyroidisoflavone A O O HO OH OH O O O OH OH OMe O O HO OH O O O HO O OH HO 27 Isolupalbigenin 29 Eturunagarone 28 Ormosidin 30 Eriosemaone D 246 Natural Product Reports, 19986,7,4*-trimethoxy- and 6,7,3*,4*-tetramethoxy-isoflavone have been synthesized by this method.Most frequently, isoflavone synthesis is achieved by oxidative rearrangement of the chalcone or flavanone skeleton. Chalcones are readily obtained by condensation of acetophenones and aromatic aldehydes and are thus much more accessible than deoxybenzoins particularly if complex substitution patterns are required.The thallium(III) nitrate (TTN) oxidation of 2*-hydroxychalcones in methanol or trimethylorthoformate is a convenient route for the synthesis of isoflavones and has been applied in the preparation of 7,8,4*-trihydroxy-6- methoxyisoflavone,51 5,6-dihydroxy-7-methoxyisoflavones52 O O O OH OH OH OH O O O O OH O OMe O O HO OH OH O O O OH OH HO 32 Ficusin B 31 Ficusin A 34 33 O O MeO OMe OMe MeO MeO MeO OMe OMe OMe HO2CCH2 OMe O MeO MeO OMe OMe OMe O MeO MeO OMe OMe CHO + i iii ii Scheme 1 Reagents and conditions: i, polyphosphoric acid, 40 )C; ii, POCl3, DMF, 55 )C; iii, pyridinium hydrochloride, pyridine, reflux O O BnO OBn OBn OBn I O O BnO OBn OBn OBn OH O O HO OH OH OH OH O O BzO OBz OBz OBz O O BzO OBz OBz OBz O O HO OH OH OH + i ii iii 5 steps iv 35 37 36 38 40 39 Scheme 2 Reagents and conditions: i, 2-methylbut-3-yn-2-ol, PdCl2, CuI, PPh3, Et3 N, DMF, 85 )C; ii, H2, Pd/C, MeOH; iii, C6H5COCl, then TsOH·H2O; iv, Hg(NO3)2, THF, then aq. NaOH, MeOH, 1,4-dioxane Boland and Donnelly: Isoflavonoids and related compounds 247and 4*,7-dihydroxy-6-methoxyisoflavone (glycitein).53 Prenylisoflavones are useful as precursors of pyrano- and furanoisoflavones, however direct prenylation of tetrahydroxyisoflavones can lead to problems of O- and di-alkylation, deprotection and lack of regioselectivity.The palladium catalysed coupling of an 8-iodoisoflavone with 2-methylbut- 3-yn-2-ol has been reported in the synthesis of 2,3- dehydrokievitone.54 The isoflavone 35 was prepared by the oxidative rearrangement of the corresponding chalcone with thallium(III) nitrate and subsequently coupled with the terminal alkyne in the presence of a palladium catalyst. Catalytic dehydrogenation of the resulting isoflavone 36 gave 2,3-dehydrokievitone hydrate 37.Benzoylation and dehydration of 37 gave the terminal and internal alkenes (38 and 39, respectively). These isomers were successfully separated by treatment with aqueous mercury(II) nitrate, which allowed isolation of 39. Removal of the protecting groups with aqueous alkali gave 2,3-dehydrokievitone 40 (Scheme 2). This general approach is suited to the synthesis of polyhydroxyprenylisoflavones found in nature. The use of organothallium(III) reagents in the synthesis of oxygen heterocycles has recently been reviewed.55 One of the major limitations of the TTN-mediated oxidative rearrangement of chalcones is the requirement for stoichiometric quantities of toxic thallium(III) salts.An alternative approach is the use of hypervalent iodine reagents in the oxidation of flavanones using either iodobenzene diacetate or [hydroxy(tosyloxy)iodo] benzene.56 This procedure is analogous to the biogenetictype aryl migration mechanism and provides isoflavones in high yields.The arylation of a preformed chromanone ring provides a useful synthetic pathway to many naturally occurring isoflavones. The synthesis of 2*-hydroxyisoflavones by arylation of 3-(allyloxycarbonyl)chroman-4-ones 41 with [4,5- dimethoxy-2-(methoxymethoxy)phenyl]lead(IV) triacetate has been described.57 Isoflavones containing a 2*-hydroxy substituent are key intermediates in the synthesis of pterocarpans and rotenoids, thus there is a need for general, eYcient and selective methods for their synthesis.The method described aVorded 2*-protected isoflavones and isoflavanones in high overall yields. Deprotection of the methoxymethyl group gave the 2*-hydroxyisoflavones in excellent yields (>90%), however the free isoflavanones could not be isolated under the same reaction conditions (Scheme 3). A rapid and eVective synthesis of the soybean isoflavonoids, daidzein, formononetin, genistein and biochanin A by cyclization of their corresponding ketones in a conventional microwave oven has been reported.58 This procedure has the advantages of reduced cost and time consumption.Furthermore the yields for the cyclization reaction using the microwave method were comparable or superior to literature values. The number of known isoflavonoid glycosides is extremely small compared with the vast range of flavonoid glycosides. New isoflavone glycosides are listed in Table 3, among which is coromandelin from the leaves of Dalbergia coromandeliana which was characterized as prunetin-4*-O-apiosyl(1]6) glucoside.59 This is the first reported occurrence of an apioglucoside of prunetin in nature and the second report of the occurrence of apioglucosides in the genus Dalbergia.Evidence from 13C NMR spectra indicated the glycosidic linkages had a ‚-configuration. Examination of Cotoneaster simonsii, a species on which no previous phytochemical or biological investigation has been reported, resulted in the isolation of a new isoflavone glycoside, genistein 5-methyl ether 4*- glucoside.60 While acylated isoflavone glycosides in which the acyl group is acetyl or malonyl are found in several species, cases where the acylating acid is p-coumaric acid are O O O O R1 R2 OCH2OMe (AcO)3Pb OMe OMe O O O O R1 R2 MeOCH2O OMe OMe O O R1 R2 OCH2OMe OMe OMe O O R1 R2 OCH2OMe OMe OMe O O R1 R2 OH OMe OMe ii + iii i iv 41a R1 = R2 = H b R1 = OMe; R2 = H c R1 = R2 = OMe Scheme 3 Reagents and conditions: i, pyridine, CHCl3, 40) C; ii, Pd(OAc)2, PPh3, HCO2H; Et3N, THF, room temp., 72 h; iii, Pd(OAc)2, DPPE, MeCN, reflux; iv, HCl, CH2Cl2–MeOH O O O O OMe O MeO O OH HO HO O O HO OMe 42 O O HO O MeO O O HO OH OMe O O HO OH OMe OMe 43 Sigmoidin H 45 Sigmoidin J 44 Sigmoidin I 248 Natural Product Reports, 1998relatively few. A new example of this class of compound 42, has recently been isolated from the seeds of Trichosanthes anguina.63 4 Isoflavanones Isoflavanones are considerably rarer than isoflavones, however several new structures have been reported (Table 4).A characteristic feature of the genus Erythrina is the widespread occurrence of prenylated flavanones, isoflavones and pterocarpans among the non-alkaloidal metabolites. The species E. sigmoidea is one of ten species growing in Cameroon, which has been used in traditional medicine to treat a variety of diseases especially microbial infection.Investigation of E. sigmoidea has resulted in the isolation of three new prenylated isoflavanones named sigmoidin H 43,46 sigmoidin I 44 and sigmoidin J 45.64,65 Further isoflavonoids isolated from this species included two novel coumestans (see Section 9.4), several known isoflavones and the known pterocarpan, phaseollidin. The 13C NMR structure of phaseollidin is reported for the first time.64 Examination of the root bark of E. eriotricha has yielded the isoflavanone, eriotrichin B 46, along with five known pterocarpans.66 These phenolic metabolites are responsible for the bulk of the antimicrobial activity of the extracts of this species.When tested against Staphylococcus aureus, it was found that the isoflavanone 46 was more potent than the cooccurring pterocarpans. Confirmation that the two isoprenyl groups were adjacent to the hydroxy group in the A-ring of 46, was obtained by cyclization of the isoflavanone Table 4 Isoflavanones isolated between 1994 and 1996 O O 2 3 1 4 1¢ 6 5 7 2¢ 8 4¢ 3¢ 5¢ 6¢ Plant sources Ref.Isoflavanone Sigmoidin H 43 Erythrina sigmoidea, root bark 46 Sigmoidin I 44 E. sigmoidea, root bark 64 Sigmoidin J 45 E. sigmoidea, root bark 65 Eriotrichin B 46 E. eriotricha, root bark 66 Erysenegalensein B 47 E. senegalensis, stem bark 67 Erysenegalensein C 48 E. senegalensis, stem bark 67 Eryvellutinone 49 E. vellutina Willd., stem bark (Fabaceae) 68 Glyasperin K 50 Glycyrrhiza aspera, roots 33 Glyasperin M 51 G.aspera, roots 33 Prostratol A 52 Sophora prostrata, roots 70 Prostratol B 53 S. prostrata, roots 70 Prostratol C 54 S. prostrata, roots 70 Tetrapterol A 55 S. tetraptera, roots 71 Tetrapterol C 56 S. tetraptera, roots 71 Tetrapterol D 57 S. tetraptera, roots 71 Tetrapterol E 58 S. tetraptera, roots 71 Secundiflorol A 59 S. secundiflora, roots 37 Ferreirinol 61 Swartzia polyphylla, heartwood 72 Dihydrolicoisoflavone 62 S. polyphylla, heartwood 73 Desmodianone A 63 Desmodium canum, roots 74 Desmodianone B 64 D.canum, roots 74 Desmodianone C 65 D. canum, roots 74 Glycosides Dalbergiodin 4*-Oglucoside 66 Ormosia monosperma, root bark 40 O O HO OH OH O O HO OH OH OH OH O O O OH OH OMe OH O O MeO OH OH 46 Eriotrichin B 47 Erysenegalensein B 48 Erysenegalensein C 49 Eryvellutinone O O MeO OMe HO OH O O HO O OH OH Me O O HO O HO OMe O O HO OR1 OH R2 R3 50 Glyasperin K 52 R1 = R3 = H; R2 = geranyl; Prostratol A 53 R1 = H; R2 = R3 = prenyl; Prostratol B 54 R1 = Me; R2 = prenyl; R3 = H; Prostratol C 51 Glyasperin M 55 Tetrapterol A Boland and Donnelly: Isoflavonoids and related compounds 249under acidic conditions to give two isomeric gemdimethylchroman derivatives.A novel isoflavanone, eryvellutinone 49, and a known flavanone were isolated from E. vellutina Willd.68 The flavanone was identified as 4*-Omethylsigmoidin, the structure of which was confirmed by 2D NMR experiments. Most isoflavanones are isolated in racemic form with optically active examples being the exception, since racemization may occur under relatively mild conditions.Glyasperin K 50, was obtained in an optically active form, but its circular dichroism (CD) spectrum showed no valuable Cotton eVect in the range of 330–350 nm due to the ease of racemization.33 The absolute configuration of glyasperin K was not established, although it is expected to have an S-configuration at C-3 by comparison with (")-ferreirin.The structure of glyasperin K is similar to glyasperin B, previously isolated from G. aspera,69 except for methylation at C-4*. Three new isoflavanones 52–54 have been found in the roots of Sophora prostrata.70 These prenylated isoflavanones lacking a hydroxy group at C-5 are very rare in nature and such isoflavanones having a geranyl group on the B-ring are known in a few instances only. Thus these particular isoflavanones are important chemical markers in the chemosystematics of the genus Sophora.A novel iso- flavanone tetrapterol A 55, and three further isoflavanones with a C-geranyl substituent on either the A- or B-ring were detected in S. tetraptera.71 The unusual side structure of tetrapterol A is derived from a geranyl group which is cyclized with the hydroxy group at C-4* through an exo-methine in the geranyl group and dehydrogenated to form a new aromatic ring. Biosynthetically, ring formation may be similar to that of cannabinol in Cannabis sativa or cyclization of the geranyl with the neighbouring hydroxy group through an endomethine of geranyl as found in lespedeol B.The formation of an aromatized ring from a geranyl group distinguishes S. tetraptera from other Sophora species (S. koreensis, S. tomentosa and S. chrysophylla). 3-Hydroxyisoflavanones are a relatively rare class of flavonoids, however two new examples have been recently reported. Secundiflorol A 59 from S. secundiflora contains the less common 1,1-dimethylallyl substituent in the 5*-position and a 2*,3*,4*-trioxygenated pattern in the B-ring.37 This type of oxygenation pattern is very rare in the genus Sophora.Another known 3-hydroxyisoflavanone secundifloran 60, was also isolated and the 13C NMR spectrum was reported for the first time. The other novel 3-hydroxyisoflavanone is ferreirinol 61, which was obtained in trace amounts from Swartzia polyphylla.72 Three new isoflavanones, desmodianones A, B and C were isolated from Desmodium canum.74 All three compounds were assigned a 3R configuration on the basis of their CD and optical rotatory dispersion (ORD) curves.Desmodianone A and B showed reproducible antimicrobial activity in vitro against Bacillus subtilis (9 IA-16), Staphylococcus aureus (IA-1), Mycobacterium smegmatis (IA-71) and Streptococcus faecalis (ATCC-6057). O O HO OH R3 R1 R2 R4 O O HO OMe OH HO OH O O HO OMe R HO OH OH O O HO OH HO OH 56 R1 = OH; R2 = geranyl; R3 = OMe; R4 = H; Tetrapterol C 57 R1 = OH; R2 = R3 = H; R4 = geranyl; Tetrapterol D 58 R1 = R2 = R3 = H; R4 = geranyl; Tetrapterol E 61 Ferreirinol 59 R = OH; Secundiflorol A 60 R = H; Secundifloran 62 Dihydrolicoisoflavone O O HO O Me OH HO O O R1O OH Me OH R3 HO R2 63 Desmodianone A O O HO OR OH 64 R1 = Me; R2 = prenyl; R3 = H; Desmodianone B 65 R1 = R2 = H; R3 = geranyl; Desmodianone C OH 66 R = Glc; Dalbergion 4¢- O-glucoside O HO OH OMe O HO OH MeO H O HO OH HO H O HO OH OH O HO OH OH CH2OH 67 Isomillinol-B 70 Tenuifolin A 71 Tenuifolin B; Kanzonol X 68 Neomillinol 69 Millinolol 250 Natural Product Reports, 1998Isoflavanone glycosides are extremely uncommon with only one O-glycoside previously reported.75 A new example has been isolated from the root bark of Ormosia monosperma and identified as dalbergiodin 4*-O-glucoside 66.40 Isoflavans Reduction of isoflavanones leads to isoflavans, many of which act as phytoalexins in legumes.Laevorotatory, dextrorotatory, and racemic isoflavans are known in nature (see Table 5) and the absolute configuration of these isoflavans is generally assigned by ORD measurement or CD curves.The species Millettia racemosa (Leguminosae) is known for its insecticidal and piscicidal activities. Three isoflavans including millinol-B have been previously reported from this species.76 A new isoflavan 67, isomeric with millinol-B except for methylation at C-4* has recently been described.77 Further examination of M.racemosa has resulted in the isolation of two novel isoflavans with unusual allyl substituents.78 The 1,2-dimethylallyl group in neomillinol 68 has to date not been encountered in other isoflavans while the presence of a CH2OH group as part of a prenyl group in millinolol 69 is the first reported occurrence of this type of substituent in an isoflavan. The absolute configuration of these isoflavans remains to be determined. Two isoflavans tenuifolin A 70 and tenuifolin B 71, were isolated from Maackia tenuifolia.79 The latter compound 71, has also been isolated from Glycyrrhiza glabra and was designated kanzonol X.80 The Glycyrrhiza species is a rich source of phenolics including flavonoids and isoflavonoids.Examination of G. inflata resulted in the isolation of three isoprenoid substituted isoflavans, a new 2-arylbenzofuran and two substituted dibenzoylmethanes. 81 The two isoflavans 73 and 74, are dipyranoisoflavans and structural isomers of hispaglabridin B.All three isoflavans possess a similar substitution pattern in the B-ring, the structure of which was elucidated by comparison with known isoflavans. Three formylated isoflavans have been identified in G. uralensis along with a new prenylated pterocarpan and a dihydropyranocoumarin which was isolated for the first time as a natural product.82 The structures of these isoflavans were established using HMBC and NOE measurements, however the presence of a formyl substituent in the A-ring made it diYcult to determine the absolute configuration of the compounds by their CD spectra due to the lack of suitable reference compounds. The absolute configuration of kanzonolM75 and N 76 was assigned to be 3R by comparison of their CD spectra with synthetic kanzonol M, obtained by formylation of kanzonol R 78.The stereochemistry of kanzonol O 77 remains to be established. The isoflavan 3*- methoxyglabridin previously isolated from G.glabra,83 has been re-isolated by Kinoshita et al. and the structure revised to 79 on the basis of NMR analysis.84 Several new examples of isoflavanquinones have been discovered. A study of two Egyptian species Astragalus alexandrinus and A. trigonus led to the isolation of the known isoflavan claussequinone 83 and a new isoflavanquinone named astragaluquinone 84 from A. alexandrinus, while examination of the roots of A. trigonus gave the isoflavan 81.85 Colutequinone 85 and the corresponding hydroquinone 82 were isolated from the root bark of Colutea arborescens.86 Table 5 Isoflavans isolated between 1994 and 1996 O 2 3 1 4 1¢ 6 5 7 2¢ 8 4¢ 3¢ 5¢ 6¢ Plant sources Chirality (Optical activity) Ref. Isoflavan Isomillinol B 67 Millettia racemosa, stem 3R (") 77 Neomillinol 68 M.racemosa, stem (") 78 Millinolol 69 M. racemosa, stem (") 78 Tenuifolin A 70 Maackia tenuifolia, roots 3R (") 79 Tenuifolin B 71 M. tenuifolia, roots 3R (+) 79 Glyinflanin I 72 Glycyrrhiza inflata, roots 3R (+) 81 Glyinflanin J 73 G.inflata, roots (+) 81 Glyinflanin K 74 G. inflata, roots (+) 81 Kanzonol M 75 G. uralensis, roots 3R 82 Kanzonol N 76 G. uralensis, roots 3R (") 82 Kanzonol O 77 G. uralensis, roots (") 82 Kanzonol R 78 G. glabra, roots 3R (") 82 Kanzonol X 71 G. glabra, roots 3R (+) 80 3*-Hydroxy-4-O-methylglabridinb 79 G. glabra, roots 3R (+) 84 8-Prenylphaseollinisoflavan 80 G. glabra, roots (") 84 8-Methoxyvestitol 81 Astragalus trigonus, roots (Fabaceae) 3R (") 85 Colutehydroquinone 82 Colutea arborescens, root bark 3R 86 Isoflavanquinones Claussequinonea 83 Astragalus alexandrinus, roots (Fabaceae) 3R 85 Astragaluquinone 84 A.alexandrinus, roots 3R (") 85 Colutequinone 85 Colutea arborescens, root bark 3R 86 Abruquinone D 86 Abrus precatorius, roots 87 Abruquinone E 87 A. precatorius, roots 87 Abruquinone F 88 A. precatorius, roots 97 aNew source of isoflavan. bRevised structure.Boland and Donnelly: Isoflavonoids and related compounds 251Oxidation of the hydroquinone to the quinone occurred on standing and may explain why no isoflavan with an o- or p-dihydroxylated B ring has been previously reported. It is interesting to note that the presence of both the quinone and hydroquinone in crude extracts established that the quinone is a genuine natural product and not an artefact of the isolation method. Five isoflavanquinones including three new examples were obtained from Abrus precatorius.87 The pharmacological activity of these compounds were evaluated and it was found that they possessed anti-inflammatory and anti-allergic eVects as well as inhibitory eVects on platelet aggregation.Catalytic hydrogenation of isoflavones or pterocarpans is the normal route to the synthesis of isoflavans. Reduction of phaseollin using liquid ammonia and lithium metal gave the unexpected product phaseollidin isoflavan, a presumed intermediate in the biosynthesis of pterocarpans.88 Previously reduction by this method resulted in the cleavage of the gemdimethyl chromene moiety.Most current synthetic routes fail to address the issue of stereocontrol at the stereogenic centres in the chiral non-planar isoflavonoids. Versteeg et al. have described the first and highly eYcient enantioselective route towards isoflavans with the potential for establishing chirality also at C-2 and C-4 of the 3-phenylchroman skeleton.89 The method involved the stereoselective ·-benzylation of phenylacetic acid derivatives obtained by reaction of phenylacetyl chlorides 91–93 with the chiral auxiliaries (4S,5R)-(+)- and (4R,5S)-(")-imidazolidin-2-ones (90a and 90b respectively). The N-acyl imidazolidinones 94–96 were alkylated in excellent yields with only one diastereomer being obtained.Reductive removal of the chiral auxiliary and cyclization under Mitsunobu conditions led to the isoflavans 106–108 in excellent enantiomeric excess and yield (Scheme 4). 6 Pterocarpans Pterocarpans contain a tetracyclic ring system derived from the basic isoflavonoid skeleton by an ether linkage between the C-4 and C-2* positions. Pterocarpans act as phytoalexins in leguminous plants and are produced following either fungal infection or abiotic elicitor treatment. Dextrorotatory, laevorotatory and racemic pterocarpans are known in nature with either a 6aR,11aR or 6aS,11aS configuration at the two chiral O HO OH O O O OH O O O OH O 72 Glyinflanin I 73 Glyinflanin J 74 Glyinflanin K O HO OR1 HO OMe R O HO OH O OMe CHO O O OMe O HO O OH OH OH 75 R = CHO; R1 = Me; Kanzonol M 76 R = CHO; R1 = H; Kanzonol N 78 R = H; R1 = Me; Kanzonol R 77 Kanzonol O 79 3¢-Hydroxy-4- O-methylglabridin 80 8-Prenylphaseollinisoflavan O HO OMe OMe OH H O MeO OMe H O O OH O MeO H O O OMe OMe O MeO H OH HO OMe OMe O R2 H OMe O O OMe R3 MeO R1 81 8-Methoxyvestitol 84 Astragaluquinone O HO 82 Colutehydroquinone 86 R1 = OMe; R2 = OH; R3 = H; Abruquinone D 87 R1 = R2 = R3 = OMe; Abruquinone E 88 R1 = OH; R2 = OMe; R3 = H; Abruquinone F H O O OMe 85 Colutequinone 83 Claussequinone 252 Natural Product Reports, 1998centres.New pterocarpans which have been characterized are presented in Table 6. Two new pterocarpans, prostratol D 109 and E 110 were obtained from Sophora prostrata and identified as derivatives of medicalpin.90 Both compounds had a 6aR,11aR absolute configuration as indicated by their laevorotatory optical activity.The known pterocarpans, maackiain, isoneorautenol, ficifolinol and erythrabyssin II were also isolated in the study. A novel pterocarpan, tetrapterol B 111 was obtained from the roots of Sophora tetraptera.71 This pterocarpan and the isoflavanone tetrapterol A 55, have a common characteristic partial structure in which a geranyl group is dehydrogenated and isomerized to form a new aromatic ring. Three pterocarpans were isolated from the flowers of Petalostemon purpureus including the new pterocarpan 3,4- dihydroxy-8,9-methylenedioxypterocarpan 112.91 The other constituents included (+)-maackiain 114, which has previously been isolated either as an inactive racemate or as the (")- isomer.All three dextro-pterocarpans have never been reported before as natural products. It appears that laevo-8,9- methylenedioxypterocarpan derivatives are more commonly distributed in plant species than their dextro enantiomers.Hydroxycristacarpone 116 isolated from Erythrina orientalis is a rare pterocarpan containing both a prenyl group and a p-quinol skeleton in the structure.92 It is the first example of this type of isoflavonoid from the genus Erythrina. To date, there have been few reports of 11b-hydroxydienones such as derivatives of phytoalexin pterocarpans which are produced by oxidative detoxification of microbial alteration. The known pterocarpan cristacarpin, was a cooccurring constituent of E.orientalis. The novel compound emoroidocarpan 117 isolated O Cl R1 R2 NX MeN Me Ph O N MeN Me Ph O O R2 R1 N MeN Me Ph O O R2 R1 R3O OR3 OH R2 R1 O R2 R1 91 R1 = R2 = H 92 R1 = OMe; R2 = H 93 R1 = H; R2 = OMe 89 X = H 90 X = Me3Si 94 R1 = R2 = H 95 R1 = OMe; R2 = H 96 R1 = H; R2 = OMe 100 R1 = R2 = H; R3 = MOM 101 R1 = OMe; R2 = H; R3 = MOM 102 R1 = H; R2 = OMe; R3 = MOM 97 R1 = R2 = H; R3 = MOM 98 R1 = OMe; R2 = H; R3 = MOM 99 R1 = H; R2 = OMe; R3 = MOM 103 R1 = R2 = R3 = H 104 R1 = OMe; R2 = R3 = H 105 R1 = R3 = H; R2 = OMe vi or vii iv ii 106 R1 = R2 = H 107 R1 = OMe; R2 = H 108 R1 = H; R2 = OMe 89 = a i and = b = and = v iii Scheme 4 Reagents and conditions: i, BuLi, Ph3CH (catalytic), THF, 0 )C; then Me3SiCl, "78 )C]room temp.; ii, tetrabutylammonium fluoride, MeCN, room temp.; iii, lithium isopropylcyclohexylamide, 2-O-methoxymethylbenzyl bromide, THF–CH2Cl2, "40 )C; iv, Li- AlH4, "24 )C]room temp.; v, 3 M HCl,MeOH, reflux; vi, BrPhSO2Cl, pyridine, CH2Cl2, room temp., then NaH (excess), 0 )C]room temp.; vii, PPh3, diethyl azodicarboxylate, THF, room temp.Table 6 Pterocarpans isolated between 1994 and 1996 O O 11 5 11a 10 1 2 3 4 6 6a 7 8 9 Pterocarpan Plant sources Chirality Ref. Prostratol D 109 Sophora prostrata, roots 6aR,11aR 90 Prostratol E 110 S. prostrata, roots 6aR,11aR 90 Tetrapterol B 111 S. tetraptera, roots 6aR,11aR 71 3,4-Dihydroxy-8,9-methylenedioxypterocarpan 112 Petalostemon purpureus, flowers 6aS,11aS 91 4-Hydroxy-3-methoxy-8,9-methylenedioxypterocarpan 113 P.purpureus, flowers 6aS,11aS 91 Maackiain 114 P. purpureus, flowers 6aS,11aS 91 Kanzonol P 115 Glycyrrhiza uralensis, roots 6aR,11aR 82 Hydroxycristacarpone 116 Erythrina orientalis, wood 6aS,11aS 92 Emoroidocarpan 117 Tephrosia emoroides, roots 93 Pterocarpina 118 T. maxima, roots 45 aNew source of pterocarpan. O HO O OMe R H H O HO O O H H Me 111 Tetrapterol B 109 R = g,g-dimethylallyl; Prostratol D 110 R = geranyl; Prostratol E Boland and Donnelly: Isoflavonoids and related compounds 253from Tephrosia emoroides is the first example of a pterocarpan with both dihydroisopropenylfuryl and methylenedioxy groups in the same molecule.93 One of the simplest synthetic routes to pterocarpans is the catalytic hydrogenolysis of 2*-hydroxyisoflavones, followed by treatment with acid.The synthesis of (&)-homopterocarpin via this route has been described.94 The required isoflavone was obtained by the oxidative rearrangement of a chalcone with hypervalent iodine.The Heck arylation of chromenes with lithium tetrachloropalladate as catalyst is another convenient method for the synthesis of natural pterocarpans. This approach was employed in the total synthesis of (&)-neorautenane 125 using the chemoselective coupling of the dipyranobenzene 123 and 2-chloromercurio-4,5- methylenedioxyphenol 124 as the key step (Scheme 5).95 Cycloaddition reactions of a 1,4-benzoquinone with a chromene in the presence of a Lewis acid has provided a further strategy to the pterocarpan skeleton.96 7 Rotenoids Rotenoids 126 are a class of isoflavonoids characterized by the inclusion of an extra carbon atom into a heterocyclic ring.Almost all the known rotenoids contain an isoprenoid substituent and are noted for their insecticidal, piscicidal and antiviral activities. They can be conveniently subdivided into three major types, rotenoids, 12a-hydroxyrotenoids and dehydrorotenoids depending on the oxidation level.The decline in the number of new rotenoid structures reported (see Table 7) is noteworthy. O R O H H R1 O O OH H H MeO MeO O O O O O OH HO H OMe 112 R = R1 = OH 113 R = OMe; R1 = OH 114 R = OH; R1 = H 115 Kanzonol P 116 Hydroxycristacarpone O O O O O O MeO O O O 117 Emoroidocarpan 118 Pterocarpin Table 7 Rotenoids isolated between 1994 and 1996 Plant sources Chirality Ref.Rotenoids 13·-Hydroxydeguelin 127 Mundulea sericea, bark 6aS,12aS 97 13·-Hydroxytephrosin 128 M. sericea, bark 6aS,12aS 97 Deguelina 129 Chadsia grevei, aerial parts (Papilionaceae) 98 Dehydrorotenoids 6-Ketodehydroamorphigenin 130 Dalbergia sissoides, stem bark 99 6-Oxo-6a,12a-dehydro-·-toxicarol 131 Derris oblonga, roots 101 aNew source of rotenoid. OH O OH HO O O O O OMe OMe O O OH OMe OMe O O OMe OMe O O O O HO ClHg O O O O O H H I OMe OMe 119 124 120 121 iv v vi 122 vii 125 i 123 ii, iii + Scheme 5 Reagents and conditions: i, 3-methylbut-2-enoic acid, MeSO3H, P2O5, 70 )C, 30 h; ii, NaH, DMF, 0 )C, 5 h; iii, toluene, 48 h; iv, NaBH4, EtOH, room temp., 24 h; v, p-TsOH, THF, reflux, 1.5 h; vi, p-TsOH, dioxane, reflux, 3 h; vii, Li2PdCl4, acetone, room temp., 24 h. 254 Natural Product Reports, 1998Two novel rotenoids, 13·-hydroxydeguelin 127 and 13·- hydroxytephrosin 128 were isolated from the bark of Mundulea sericea, along with the parent rotenoids deguelin and tephrosin.97 The new compounds had a cis B/C ring fusion and the absolute configuration was established as 6aS,12aS.The unambiguous 1H and 13C NMR data for the parent compounds was reported for the first time. These rotenoids exhibited potent inhibitory activity against phorbol ester-induced ornithine decarboxylase activity in cell culture. Deguelin 129, a known rotenoid has been isolated from the genus Chadsia, which is a new source of rotenoids in addition to common genera such as Derris, Tephrosia, Lonchocarpus, Milletia and Neorautanenia.98 From the stem-bark of Dalbergia sissoides, a new rotenoid 130 was isolated.99 This compound has not been previously reported as a natural product, although it has been obtained as an oxidation product of dehydroamorphigenin.100 A new rotenone derivative 131 was obtained from the roots of Derris oblonga, together with several known isoflavonoids including isoflavones, coumestans, pterocarpans and coumaronochromones. 101 The proposed structure was determined using spectroscopic and chemical evidence. A variety of synthetic approaches to the rotenoid system exist e.g. reactions of isoflavones with dimethyloxosulfonium methylide,102 Claisen rearrangement of aryl prop-2-ynyl ethers,103 acylation of enamines104 and intramolecular radical cyclization.105 However these strategies involve elaborate multi-step procedures from starting materials which are generally not readily available and which often result in low overall yields of the rotenoids.Recently a simple four-step O O 2 1 3 4 5 6 6a 7 8 9 10 11 12 12a 126 O O R1 R2 O Br R1 R2 O R1 R2 OMe O OMe O O O OMe H H R1 R2 O R1 R2 O OMe O H 132 R1R2 = –(CH2)5– + iv, v ii iii i 133 134 135 136 Scheme 6. Reagents and conditions: i, PBr3 , heat, 40 min; ii, BuLi, Et2O, N2, room temp., 45 min; iii, substituted benzonitrile, room temp., 1 h; iv, BCl3, CH2Cl2, N2, "10 )C, 1 h; v, NaOAc, EtOH, heat, N2, 3 h O O O OMe MeO H H O 137 Rotenone O MeO O OMe OR1 R1 O OR2 O H H H R3O OMe 138 R1 = R2 = H; R3 = Me; Hexaspermone A 139 R1 = R3 = H; R2 = Me; Hexaspermone B 140 R1 = R2 = R3 = H; Hexaspermone C Boland and Donnelly: Isoflavonoids and related compounds 255synthesis of a 6,6-disubstituted rotenoid starting from 2*-hydroxyacetophenone has been described.106 The key step involved lithiation of a 4-bromo-2H-chromene 133, followed by treatment with a substituted benzonitrile to give a 4-benzoyl-2,2-dimethyl-2H-chromene 134.Deprotection and base-promoted intramolecular ring closure gave the rotenoid 135 (Scheme 6). This procedure has also been successfully applied to the synthesis of the 5-thiorotenoid system. Structure–activity relationships for rotenone 137, the major biologically active component of Derris resin has been reported.107 Few rotenoid relatives in which the B/C ring system is altered are active and the cleavage of these rings results in loss of activity.Synthetic analogues in which the B/C ring system of rotenone had been replaced were prepared and tested for their ability to block NADH dehydrogenase. Results indicted that it was possible to replace rotenone rings B and C with retention of modest activity. Isoflavonoid oligomers In contrast to flavonoid oligomers which are a major group of flavonoid derivatives, similar oligomers of isoflavonoids are sporadic in nature. The first bi-isoflavonoid, a dimeric iso- flavan, was isolated from the heartwood of Dalbergia nitidula,2 further oligomers containing isoflavonoid–isoflavonoid, iso- flavonoid–flavonoid, isoflavonoid–stilbene and isoflavonoid– phenylpropanoid systems have been described.108 Three isoflavanone dimers (138–140) were isolated from Ouratea hexasperma together with the known 5,7,4*- trimethoxyisoflavone.109 The relative configuration of these three compounds was assigned as trans-isoflavanone–(2]2+)- trans-isoflavanone on the basis of coupling constants and comparison with alternative models.The first examples of isoflavonoid oligomers comprising pterocarpan–neoflavonoid and isoflavonoid–neoflavonoid constituent units from Dalbergia nitidula have been described.110 Daljanelins A–C 141–143, are comprised of a 3,9-dioxygenated pterocarpan nucleus substituted with the same structural unit at C-2, C-4 and C-8 respectively. These MeO MeOCH2O O O MeO MeOCH2O O OTBDMS O HO O OMe O R1O O OMe R2 O R1O O OMe O O OMe OCH2OMe 148 R1 = H; R2 = Br 149 R1 = CH2OMe; R2 = Br 150 R1 = CH2OMe; R2 = CO2Et 151 R1 = CH2OMe; R2 = CH2OH 152 R1 = CH2OMe; R2 = CH2Br 146 steps 145 i 147 153 ii–vi 146 + 152 vii 143 viii, ix Scheme 7.Reagents and conditions: i, Et3N, NaI, CH3CN, ButSiMe2Cl; ii, NBS in MeCO2Me; iii, NaH–THF, then ClCH2OMe; iv, BuLi– TMEDA, then ClCO2Et, then H2O; v, LiAlH4, THF; vi, 2,6- dimethylpyridine, LiBr, (CH3SO2)2O; vii, TASF–HMPA, then aq. NH4Cl; viii, PhMgBr, THF, then 3 M HCl, 0 )C; ix, 0.1 M HCl–MeOH, reflux O OH O O O OH O OH O O O OH H OH 154 Lupinalbin H 155 Erysenegalensein J 256 Natural Product Reports, 1998compounds not only represent the first neoflavonoid– pterocarpan dimers but also complement the rare series of neoflavonoids with the 3-arylbenzo[b]furan constitution.The absolute configuration of these natural products was established as 6aS,11aS by comparison with the known pterocarpan (+)-homopterocarpin and confirmed by synthesis of 143.Daljanelin D 144, is comprised of an isoflavan unit (3S)- vestitol, substituted at C-8 with the same neoflavonoid moiety as the three other oligomers. Compounds 142 and 144 complement the unique series of oligomeric flavonoids/isoflavonoids substituted at C-8 of the resorcinol-type ring of the chromane unit. The structure and stereochemistry of daljanelin C 143, were unambiguously confirmed by synthesis. The pterocarpan moiety 151 was obtained by bromination of (+)-medicarpin 147, followed by introduction of a methylene bridge at C-8 via reaction of the lithio derivative with ethyl chloroformate and subsequent reduction of the ethyl ester.The neoflavonoid precursor 145 was obtained from vanillin by literature procedures. The pterocarpan 151 was transformed into a highly labile benzyl bromide 152 and coupled with the stable silyl enol ether 146 to give the alkylated product 153 as a diastereomeric mixture. The final C6 fragment was introduced by Grignard reaction of 153 with phenylmagnesium bromide (Scheme 7). 9 Miscellaneous structures 9.1 Coumaronochromones The number of coumaronochromones that have been isolated in the past ten years has increased rapidly with 19 new compounds reported in the previous review alone.1 However since then there has been a decline in the number of new examples with just two reported in the present work. Various coumaronochromones are known to occur in white and yellow lupins.Lupinalbin H 154, isolated from Lupinus luteus,38 is the coumaronochromone derivative of the lineartype pyranoisoflavone, parvisoflavone B, which also occurs in white and yellow lupin roots. Erysenegalensein J 155, is a new 3-hydroxycoumaronochromone which may result from the addition of water to the double bond C-2–C-3 in ring-C of the coumaronochromone skeleton.29 The compound is optically active although the stereochemistry at the chiral centres remains to be determined. 9.2 Isoflav-3-enes Three new isoflav-3-enes, judaicin 156, judaicin 7-O-glucoside 157 and judaicin 7-O-(6+-O-malonyl)glucoside 158, in addition to several known pterocarpans have been isolated from the O HO MeO O O O O MeO O O O HO HO HO RO 156 Judaicin 157 R = H; Judaicin 7- O-glucoside 158 R = malonyl; Judaicin 7- O-(6�- O-malonyl)glucoside O HO OH O 159 Glabrene O HO HO O O MeO O O O OMe MeO OMe O O O OH HO 162 4,4¢-Di- O-methylscandenin 160 Glyasperin L 161 Kanzonol W OH OH HO O OR OH O O O OTs OMe O O O OTs OMe O O OH OTs OMe O O OH OMe O O O O OMe O OH O OMe O OH O R O O O 165 R = p-C6H4OMe O O (AcO)3Pb R = H R = Ts i, ii iii OMe (AcO)3Pb iv vii vi v R = Ts R = H viii x ix 164 R = 163a xi 163b Scheme 8.Reagents and conditions: i, 3-methylbut-2-enoic acid, PPA, dioxane, 60 )C, 3 h; ii, K2CO3, anhydrous acetone, reflux, 50 h; iii, TsCl, K2CO3, acetone, reflux, 2 h; iv, Me2SO4, K2CO3, acetone, reflux, 4h; v, K2CO3, MeOH, reflux, 2 h; vi, NaBH4, PdCl2, THF, H2O, 0–5 )C, 1.5 h; vii, TsOH, toluene, reflux, 20 min; viii, K2CO3, MeOH, reflux, 1.5 h; ix, NaH, Et2CO3, 45 )C, 30 min; x, 163a pyridine, CHCl3, 60 )C, 14 h; xi, 163b, pyridine, CHCl3, 60 )C, 14 h Boland and Donnelly: Isoflavoids and related compounds 257roots of Cicer judaicum.111 This is the first report of glycosylated forms of this uncommon isoflavonoid class.The structure of 156 is noteworthy, as it includes a methylenedioxy group not previously found in recorded examples of the isoflav-3-ene class.The occurrence of isoflav-3-enes and pterocarpans together in C. judaicum is of biogenetic interest as these classes of compounds share the same precursor, 2*-hydroxyisoflavanol. This report of an isoflav-3-ene and its glycosylated derivatives in wild Cicer does not support the published taxonomy of the species and demands a further systematic study of the genus. Glabrene, a known isoflav-3-ene from Glycyrrhiza glabra, has been re-isolated and the structure revised to that of 159, based on NOE experiments.80 A novel methodology for the introduction of substituents at the 2-position of the isoflav-3-ene nucleus involving Grignard reaction of the phenyl acetal of 3-arylcoumarins has been described.112 These isoflav-3-ene derivatives showed significant antiestrogenic activity. 9.3 3-Arylcoumarins Isoflav-3-en-2-ones are commonly referred to as 3-arylcoumarins and are subdivided into two groups, those lacking 4-oxygenation (3-arylcoumarins) and those with 4-oxygenation (3-aryl-4-hydroxycoumarins).Two new isoprenoid-substituted 3-arylcoumarins have been reported, glyasperin L 160, from Glycyrrhiza aspera,33 and kanzonol W 161, from G. glabra.80 3-Aryl-4-hydroxycoumarins are constituents of the genus Derris (Dalbergieae) and a new example 162, although known synthetically, has been reported for the first time as a natural product from D. scandens.41 Reported synthetic procedures for both 3-aryl- and 3-aryl- 4-hydroxy-coumarins have focused on the direct arylation of a coumarin ring using either palladium catalysed coupling113 or aryllead triacetates.114 The latter method has been successfully utilized in the synthesis of both robustin 164 and robustic acid 165 (Scheme 8). 9.4 Coumestans Coumestans represent the fully oxidized version of pterocarpans and the recently reported new examples have been isolated from the Cameroonian medicinal plant Erythrina sigmoidea.Both 4-hydroxycoumestrol 166,46 and sigmoidin K 16765 showed anti-bacterial activity. The known coumestan psoralidin 168 isolated from Psoralea corylifolia showed strong cytotoxicity against stomach cancer cell lines.115 9.5 2-Arylbenzofurans A wide variety of 2-arylbenzofuran structures are found in nature, however the compounds discussed here are only found in leguminous plants. Three new isoprenoid examples have been obtained from species of Glycyrrhiza.Glyinflanin H 169 was isolated from G. inflata81 and the remaining compounds 170, 171 from G. glabra.80 Compounds 169 and 170 are structural isomers. The cooccurrence of 2-arylbenzofurans with structurally related isoflavonoids suggests that the furan derivatives are derived from the isoflavonoids by loss of one carbon atom. 10 References 1 D. M. X. Donnelly and G. M. Boland, Nat. Prod. Rep., 1995, 12, 321. 2 P. M. Dewick, in The Flavonoids: Advances in Research since 1980, ed.J. B. Harborne, Chapman and Hall, London, 1988, p. 184. 3 N. A. M. Saleh, Phytochemistry, 1994, 36, 1109. 4 S. Tahara and R. K. 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Ebizuka, Phytochemistry, 1995, 38, 373. 25 G. Delle Monache, M. C. de Rosa, R. Scurria, A. Vitali, A. Cuteri, B. Monacelli, G. Pasqua and B. Botta, Phytochemistry, 1995, 39, 575. 26 J. Wandji, Z. T. Fomum, F. Tillequin, E. Seguin and M. Koch, Phytochemistry, 1994, 35, 245. 27 J. Wandji, Z.T. Fomum, F. Tillequin, G. Baudouin and M. Koch, Phytochemistry, 1994, 35, 1573. 28 J. Wandji, Z. T. Fomum, F. Tillequin, A. L. Skaltsounis and M. Koch, Planta Med., 1994, 60, 178. O HO O HO O O OH HO 169 Glyinflanin H 170 R = H; Kanzonol U 171 R = prenyl; Kanzonol V O HO O OH OH O O HO O OH O O O OH HO O 166 4-Hydroxycoumestrol 167 Sigmoidin H 168 Psoralidin 258 Natural Product Reports, 199829 J. Wandji, S. S. Awanchiri, Z. T. Fomum, F. Tillequin and S. Michel-Daniwicz, Phytochemistry, 1995, 38, 1309. 30 J. Wandji, S. S. Awanchiri, Z. T. Fomum, F. Tillequin and F. Libot, Phytochemistry, 1995, 39, 677. 31 W. A. Asomaning, C. Amoako, I. V. Oppong, W. R. Phillips, I. Addae-Mensah, E. Y. Osei-Twum, R. 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ISSN:0265-0568    

DOI:10.1039/a815241y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Steroids: reactions and partial synthesis James R. Hanson School of Chemistry, Physics and Environmental Science, University of Sussex, Brighton, Sussex, UK BN1 9QJ Covering: 1996 Previous review: 1997, 14, 373 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 Amines 3.6 Rearrangements 4 Partial synthesis 4.1 Estranes 4.2 Androgens 4.3 Pregnanes 4.4 Cardenolides 4.5 Bile acids 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 1996 and follows a similar pattern to its predecessors.1 Interest has continued in the synthesis of potential inhibitors of aromatase and testosterone 5·-reductase.The diverse modes of action of progesterone and of its metabolites have been reviewed.2 Other articles have appeared on developments in brassinosteroid research3 and on methods for the stereoselective construction of the side chain of steroids.4 A useful ‘Handbook of Sterol Analysis’ has appeared.5 2 Spectroscopic and physical properties of steroids The shielding parameters of the ether C–O bond have been obtained6 from a study of the substituent induced shifts in the NMR spectra of the 4-oxa-5·- and 4-oxa-5‚-androstan-17- ones, 1 and 2.The 1H and 13C NMR spectra of a number of derivatives of androst-4-ene-3,17-dione have been assigned.7,8 1H–1H Long range couplings through four bonds in fused cyclopropanes have been examined9 in a study of the conformation of 17,18-cyclosteroids 3.Information on the stereochemistry of some (2-alkylmorpholinyl)-3·-hydroxy-5·- pregnanes has been obtained10 from their 13C NMR spectra. The 1H and 13C NMR spectra of some spiro cholestanes have been reported.11 A one- and two-dimensional study of the interaction of ursodeoxycholic acid 4 and ‚-cyclodextrin has been described.12 Evidence has been presented to show that the side chain of the bile acid enters the ‚-cyclodextrin cavity from the secondary hydroxy rim.The analysis of unsaturated C27 sterols from their 1H and 13C NMR spectra has been thoroughly examined13 in order to facilitate the characterization of these closely related sterols which are intermediates in the formation of cholesterol from lanosterol. Two-dimensional methods have been applied14 to the assignment of the structure of vitamin D analogues. The structure of some by-products obtained during the manufacture of betamethasone have been established15 by NMR and X-ray crystallographic methods.The fluorine NMR spectra of some fluorinated steroids have been reported.16 X-Ray crystallography has played an important part in many steroid investigations. The X-ray crystal structures of both the syn and anti benzyloxyiminoandrost-4-en-17‚-yl acetate 5 and of the parent compound, have been determined17 in connection with a study of their circular dichroism.The crystal structures of a number of intermediates in a synthesis of formestane 6, such as 3‚,4·-dihydroxy-5‚-androstan-17-one, have been established.18 The structures of some corticosteroid esters (e.g. 7) have also been reported,19,20 The X-ray structure of the extended molecule of 20-oxopregna-5-en-3‚-yl (4-allyloxybenzoate) 8 is of interest21 in connection with liquid crystal formation. Another interesting structure that has been reported22 is that of the lactone, ecdysantherin 9, which was obtained from Ecdysanthera rosea, a Vietnamese plant used as an antiinflammatory drug.X-Ray crystal structures have been reported for a 17‚-estradiol 3-benzoate,23 a fluorinated D-homoestrone 10,24 17·-benzyl- 17‚-hydroxy-16-hydroxyimino-3-methoxyestra-1,3,5(10)- triene,25 norethindrone acetate 11,26 a series of cholic acid O H H H H H H H H H O C Me O CO2H AcO OH H OH H 1 5a-H 2 5b-H 3 4 5 H H H H H H H H H N O CH2 O OH O O O OAc O O OMe Me OAc HO 5 6 7 Hanson: Steroids: reactions and partial synthesis 261derivatives,27–29 cholesteryl 3‚-toluene-p-sulfonate,30 a fungal ergostane 12,31 and some cholestane derivatives32–34 including a 3-stannylcholest-5-ene.35 An investigation of the gas phase photoelectron spectrum of dehydroepiandrosterone has shown36 that the lowest energy ionization event corresponds to the �-ionization energy closely followed by the carbonyl lone pair ionization. 3 Reactions 3.1 Alkenes Whereas peracids normally epoxidize ƒ5-steroids from the less hindered ·-face, the ‚-face epoxidation of steroidal 5-enes using potassium permanganate and various metal sulfates such as copper sulfate and ferric sulfate or some metal nitrates, has been described by several groups.37–39 The method has been extended40 to the preparation of 6‚-hydroxy-ƒ4-3-ketones.Chloral hydrate and hydrogen peroxide has also been shown41 to be an eVective epoxidizing agent for steroidal allylic and homoallylic alcohols.Steroidal olefins have been chlorinated (e.g. 13]14) by a mixture of manganese dioxide and acetyl chloride.42 The ozonolysis of cholesterol esters has been examined and the structures of some of the ozonides established.43 The allylic oxidation of ƒ5-steroids at C-7 (e.g. 13]15) has continued to attract attention. Pyridinium fluorochromate has been found to be an eVective reagent.44 A ruthenium trichloride catalysed oxidation using tert-butyl hydroperoxide in cyclohexane has been developed45 as a replacement for chromium based methodologies.A review of the use of organotin intermediates in the preparation of radiopharmaceuticals quotes46 the use of a number of steroidal vinyl stannanes. 3.2 Epoxides The use of the Dowex 50WX8 resin in the acid catalysed hydration of steroidal 5,6-epoxides has been examined.47 3.3 Alcohols The rapid acylation of alcohols using acid anhydrides in the presence of trimethylsilyl trifluoromethanesulfonate as a catalyst has been reported.48 Montmorillonite K10 in dichloromethane has been shown to catalyse49 the formation of 3‚,3‚*-disteryl ethers.Hindered 17‚-tertiary alcohols undergo dehydration accompanied by some rearrangement of the C-13 methyl group on treatment with tert-butyldimethylsilyl chloride.50 Inversion of steroidal secondary alcohols can be brought about51 by treatment of their chloromethanesulfonates with caesium acetate in the presence of 18-crown-6.The chloromethanesulfonate in the conversion of 16 to 17 reacted much more rapidly than the methanesulfonate. The stereoselective transesterification of steroidal side chain alcohols using vinyl acetate as the source of the acetate and Pseudomonas cepacia lipase as the catalyst, has been developed. 52 The hydrolysis of steroidal acetates using various lipases has been explored.53 The rearrangement of steroidal S-methyl xanthates by trimethylaluminium to form the S-methyl dithiocarbonates has provided54 a method for converting steroidal alcohols to thiols.The fluorination of the epimeric 3‚-acetoxy-7- hydroxyandrost-5-en-17-ones under various conditions using diethylaminosulfur trifluoride has been examined.55 The fluorination of 3‚-hydroxy steroids by the thermal decomposition of electrochemically generated alkoxytriphenylphosphonium tetrafluoroborates has been reported.56 Photolysis of steroidal nitrites such as cholestane-6‚-nitrite in the solid state has given57 the 6-ketone rather than a product of substitution of the methyl group.Several new variations of the Oppenauer oxidation have been described. Zirconium tert-butoxide with tert-butyl hydroperoxide58 or with chloral as the hydride acceptor59 have been examined. Tris(triphenylphosphine) ruthenium dichloride and potassium carbonate in refluxing acetone proved60 to be an eYcient system for the oxidation of 3‚-hydroxy-ƒ5-steroids 18 to the corresponding 4-en-3-ones 19.Pyridinium chlorochromate61 and pyridinium dichromate62 on the other hand are eYcient systems for H H H H H H H H OH H O O O C Me O O O O Me O MeO HN NO2 HO F H H H O OAc CºCH O 12 8 9 10 11 H H H AcO AcO Cl Cl 13 14 H H H AcO 15 H H H H O O SCH2Cl O O OAc 16 17 262 Natural Product Reports, 1998oxidation of the 3‚-hydroxy-ƒ5-steroids to ƒ4-3,6-diones. Selective oxidation of the C-12 alcohol of cholic acid amide 20 has been achieved63 using bromine via a neighbouring group participation of an N-bromoamide group in the side chain thus providing a route to chenodeoxycholic acid.A seven-centre fragmentation reaction has been observed64 in the reaction of some 3,7-dihydroxycholestane monomethanesulfonates with sodium tert-amylate. The reaction is exemplified by the cleavage of 21 to 22 and the formation of the rearrangement product 23. The Mitsunobu reaction of allylic alcohols with o-nitrobenzenesulfonyl hydrazone as the nucleophile proceeded65 with inversion and then on warming, the rearranged alkene was formed.The stereochemistry of the allylic alcohol determined the stereochemistry of the added hydrogen as in 24]25. 3.4 Carbonyl compounds A simple synthesis of tritiated steroidal alkenes involved66 the decomposition of the tosylhydrazone of a ketone to form a vinyllithium which was then quenched with tritiated water. Reduction of steroidal 17-ketones with sodium in deuterium oxide aVorded the [16,16,17-2H3]-17‚-alcohols.These were converted67 to their 17·-epimers without the loss of deuterium by treatment of their toluene-p-sulfonates with potassium nitrite. The selective reduction of the carbonyl group in organomercurial derivatives has been carried out68 in a steroid example 26 by first protecting the mercury by methylation. Subsequent deprotection permitted transformation of the organomercurial group. Magnesium in methanol has been used69 to reduce the conjugated double bond of an ·‚-unsaturated ester in the pregnane series as in 27]28.A copper on silica catalyst has been developed70 for the hydrogenation of unsaturated ketones to their saturated counterparts. The less hindered ƒ1-double bond of androsta-1,4-dien-3,17-dione 29 was preferentially reduced to give 19. The electrochemical deacetoxylation of 11-ketorockogenin diacetate 30 to 11-ketotigogenin 31 has been investigated.71 H H H H H H O O O HO 18 19 H H H CONH2 H HO OH HO 20 H H H H H H O MsO H OH OH 21 22 23 + H H H H H H HO H H 24 25 H H H CHO BrHg 26 H H H H H H HO HO CO2Et CO2Me H 27 28 H H H O H H H O O O O H AcO R 29 30 R = OAc 31 R = H Hanson: Steroids: reactions and partial synthesis 263The hydrolysis of ring A methoxy enol ethers by oxalic acid in the presence of silica gel has been developed72 as a selective method for converting steroidal 3-methoxy-2,5(10)-dienes 32 to the corresponding 5(10)-en-3-ones 33.The addition of perfluoroalkyllithium derivatives to the 3-position of silylated testosterone has aVorded 3-substituted perfluoroalkyl steroids which have been examined73 as inhibitors of glucose-6-phosphate dehydrogenase.The addition of ethynylcerium chloride to the 17-ketone of 13-ethyl-3- methoxygona-3,5-dien-17-one followed by acid hydrolysis formed an improvement in the synthesis of norgestrel.74 The dehydrogenation of steroidal ketones such as 19-norandrost-4-ene-3,17-dione with copper bromide has yielded75 estrone derivatives.The trichloromethyl cation has also been described76 as a dehydrogenating agent. The preparation of steroidal dioxolanes 34 by the fragmentation of peroxyhemiacetals has been reported.77 Dimeric steroidal 1,2,4,5-tetraoxanes have been prepared78 from the 3-ketone in the quest for compounds active against Plasmodium falciparum. The preparation of derivatives of steroidal oximes has continued to attract attention in the search for haptens for immunoassay.79–83 The groups were linked to steroids at C-3, C-7, C-11 and C-15. 3.5 Amines The preparation of a number of acylamido steroids has been reported.84 The use of enamides as inhibitors of testosterone 5·-reductase has led to the study85 of some reactions of enamides such as 35 with acetyl nitrate. 3.6 Rearrangements The substituent at position 6 in compounds of the Westphalen type 36 has been removed86 by WolV–Kishner reduction of the 6-ketone or by tributylstannane reduction of the 6‚-chloride.The backbone rearrangement of 5·- and 5‚-cholesta-6,8(14)- dienes 37 in the presence of toluene-p-sulfonic acid and acetic acid have been examined87 in connection with studies on the fate of steroids in fossils and their use as biomarkers. The rearrangements have been shown to aVord spirosteradienes such as 38. 4 Partial synthesis 4.1 Estranes Studies have continued with the object of defining the ring A requirements for binding to the estrogen receptor.These have involved88 the synthesis of a series of 2-hydroxyalkyl estradiols 39. These compounds were envisaged as being stable analogues of the estrogen metabolite, 2-hydroxyestradiol. The synthesis of the furanone 40 and its binding aYnity to the estrogen receptor have also been reported89 in this context. The oxidative transformations of 2-hydroxyestrone and the reactivity of the resultant 2,3-estronequinone 41 towards nucleophiles have been examined90 in the context of estrogen carcinogenicity and the known reactivity of 3,4-estronequinone 42 with DNA. The reactions of 42 with mimics of amino acid side chains have also been examined.91 Methods have been developed92 for the preparation of estrogen sulfamates on a large scale for the oral administration of estrogens.The synthesis and GC–MS properties of 6-alkylestradiols, e.g. 43, have been described.93 These are putative metabolites of 6-alkylandrostenedione aromatase inhibitors.Vinyl 6-sulfones and sulfoxides have been utilized94 as Michael acceptors for dimethylsulfoxonium methylide in the preparation of C-6–C-7 methylene bridged derivatives of H H H OH H H H OH MeO O 32 33 O H H O CO2Me H H PhSO2CH2 34 N H H H O H 35 H H H H OBz H OH BzO 36 37 38 H OH H H H O H H H O H H H O H H R HO O O O O O O 39 R = (CH2) nOH; n = 1–3 40 41 42 264 Natural Product Reports, 1998estradiol, e.g. 44. Improvements have been reported95 in the synthesis of 3,17‚-diacetoxyestra-1,3,5(10)-trien-6-one whilst the synthesis of the 6-oxa analogue of estrone has been described.96 The synthesis of ring B alkylated estratetraenes, including the propano-bridged derivative 45, have been reported97 in studies on the synthesis of estrogen receptor ligands.The acid-catalysed isomerization of estra-1,3,5(10), 8-tetraenes to the corresponding 9(11)-isomers has been examined.98 The synthesis of a series of 2,3,14‚,17·- polyhydroxylated estra-1,3,5(10)-trienes and further hydroxylation products, e.g. 46, have been reported99,100 as models for the Inagami–Tamura endogenous digitalis-like factor. These compounds were active at inducing contractile responses in the isolated rat aorta. The synthesis of 15,15-dialkylestradiols, e.g. 47, by the conjugate alkylation of ƒ15-17-ketones has been described101 in the context of the eVect of the modification of ring D on estrogenicity. The addition at C-15 takes place from the ‚-face of ring D.The synthesis and evaluation of the biological activity of some 16-[carbamoyl(bromomethyl)alkyl]estradiol derivatives, e.g. 48, has been reported.102,103 These compounds were designed to inhibit the 17‚-hydroxysteroid dehydrogenase which converts estrone to the more active estradiol and also to block the estrogen receptor. The E- and Z-phenylvinylestradiols have been prepared.104 The Z-isomer has a greater binding aYnity for the estrogen receptor and may be a useful probe for mapping the structure of the receptor.The estradiol derived enyne 49 has been prepared105 as an estramycin analogue and some ellipticine–estradiol conjugates, e.g. 50, have been evaluated106 for their ability to bind to the estrogen receptor and for their cytotoxicity to human cancer cells. 4.2 Androgens The control of the regioselectivity in the transition metal catalysed conjugate addition of alkylaluminium compounds and methyltitanium ate complexes to androsta-1,4,-diene-3,17- dione 29 has been examined.107 Catalytic amounts of copper(I) salts favour addition to C-1 rather than C-5 (97:3) whilst nickel acetylacetonate leads to a reversal of the regioselectivity in favour of C-5 (11:89).The conformations of ring A of neuromuscular blocking agents, e.g. 51, related to pancuronium have been studied108 by a combination of X-ray methods and molecular mechanics calculations. There has been considerable interest in testosterone 5·-reductase inhibitors. Evidence has been presented109 in support of a mechanism of action of finasteride 52 in terms of a C-2 NADP adduct.The synthesis and biological activity of (N-tert-butyl)-3-haloandrosta-3,5-diene-17‚-carboxamides 53 H OAc H H H OH H H HO HO 43 44 OH H OH OH H HO HO HO OH OH 45 46 H O H H H OH H H HO HO CH2Br (CH2)5CONBuMe 47 48 H OH H H HO H OH H H HO C CH2 CH2CºCH HN O N N H (CH2)6NHC O 49 50 H O H H AcO +N N+ C O H 51 N H C H H O H CONHBut H H Cl N H CONH H H O H O NHBut H CH Ph Ph H 52 54 53 Hanson: Steroids: reactions and partial synthesis 265as mimics of the intermediate enol form in the 5·-reductase transformation, has been described.110 The biological activity of a series of 4-aza-5·-androstane-17-carboxamides possessing aromatic rings on the carbamoyl moiety, e.g. 54, has been reported.111 New methods for the synthesis of hindered 17‚-amides have been reported112 using 2,4,6-triisopropylbenzenesulfonyl chloride as the condensing agent.The inhibitory activity of 11-substituted 4-aza-5·-androstanes113 and 4,17-diazasteroids114 against 5·-reductase have been evaluated. The 5·-reductase inhibitor, furosteride 55, has been synthesized in a labelled form from [20-14C]-pregnenolone.115 The preparation of some antiandrogenic C-17 spiro-2- oxasteroids containing substituents on ring B, e.g. 56, has been described.116 Aromatase is a cytochrome P450 enzyme involved in the biosynthesis of estrogens from androgen precursors.Interest in inhibiting this enzyme for the treatment of breast cancer has continued unabated. A new approach to the synthesis of 4-hydroxyandrost-4-ene-3,17-dione 6 has been reported.117 The synthesis has been described118 of a series of analogues of 19-[(methylthio)methyl]androstenedione e.g. 57, which bind to the haem iron of the cytochrome. 10‚-(1*-Azirinyl)estr-4-ene- 3,17-dione119 and 19-(cyclopropylamino)androst-4-ene-3,17- dione120 were synthesized as mechanism based inhibitors to target the same site.A series of 6·- and 6‚-alkylandrosta-1,4- diene-3,17-diones have been synthesized121 and evaluated as time-dependent inhibitors of aromatase. There was a greater flexibility in the length of the 6·-chain compared to the 6‚-series. The structure–activity relationships of a series of 6·- and 6‚-alkyl androst-4-en-3,17-diones, e.g. 58, have also been examined122 in this context. The bioconversion of 17‚-hydroxy-17·-methylandrosta-1,4- dien-3-one and androsta-1,4-diene-3,17-dione by cultures of the green alga, Scenedesmus quadricauda, involved hydration at C-5, 59, and rearrangement to form, for example, 60 and 61.123,124 The reduction of ƒ5-B-norandrostenes by diimide to aVord the antiandrogens, 17‚-hydroxy-B-nor-5·-androstan-3-one and its 17·-methyl analogue, has been described.125,126 Intramolecular nitrone 1,3-dipolar cycloadditions have been explored127 in the context of the cyclization of 19-nor-5,10- secosteroids, e.g. 62]63. The extent of intramolecular singlet and triplet energy transfer between the aryl moiety of 6‚-(dimethylphenylsiloxy)-5‚-androstanes and C-3 and C-17 ketones, has been examined.128 The selective hydrogenation of 17-substituted 13‚-ethyl-11‚- hydroxygona-4,9-dien-3-ones over a palladium on strontium carbonate catalyst poisoned with pyridine gave129 the corresponding 11‚-hydroxygon-4-en-3-ones without hydrogenolysis of the alcohol. A series of 11‚-aryl steroids, e.g. 64, have been synthesized130 to explore the 11‚-region in steroid:progesterone receptor interactions. Steric interactions between C-19 and the aryl residue bring about conformational changes in the steroid system which aVect the binding to the progesterone receptor. The synthesis of 3‚-acetoxy-15-oxoandrost-5-ene-17‚- benzoate and some 15‚-hydroxyandrostanes have been reported.131–133 The latter were of interest as GC–MS markers for the characterization of human steroidal metabolites.The 15‚-substituents were formed by conjugate addition to the ƒ15-17-ketones. The use of coupling reactions between 17-iodoandrost-16-enes and vinylstannanes together with a Diels–Alder reaction has aVorded134 pentacyclic steroids, N H CO H H O O H O H H O Me N C O NH Cl O H 56 55 O H O H O H H S 57 58 H H H H H H O O OH H OH H O OH HO OH 59 61 60 H H H H H H H O AcO O N Me AcO OAc OAc 63 62 H H N O OH 64 266 Natural Product Reports, 1998e.g. 65. A modification of the Mitsunobu protocol in which 4-nitrobenzoic acid was employed as the acidic component, has provided135 a better method for the synthesis of 17·- hydroxy steroids.Deuteriated glucuronides of testosterone and 17-epitestosterone have been prepared136 to facilitate their detection by mass spectrometry. Derivatives of testosterone bearing a 17‚-cyclopropyl ether, e.g. 66, have been synthesized137 as mechanism based inhibitors of the steroidal C-17(20)-lyase. The synthesis and antiinflammatory activity of 17‚-thio16·,17·-ketals has been examined.138 These compounds, e.g. 67, are of interest because of their high topical potency and their possible application in reducing lung inflammation as airway selective steroids in the treatment of asthma. The S-oxidative biodegradation of tipredane, 68, has been examined.139 Attack is faster on the 17‚-substituent with the formation of the sulfoxide. A series of 17‚-(hydrazonomethyl) steroids, e.g. 69, have been synthesized140 and examined for their ability to bind to the Na+, K+-ATPase receptor.An improved route for the preparation of 18-nor-17-ketosteroids has been described141 involving the cyclization of the ‘abnormal’ Beckmann rearrangement product of 17-oximes. 4.3 Pregnanes The discovery that the neuroactive steroid, 3·-hydroxy-5·- pregnan-20-one, is a potent allosteric modulator of the GABAA receptor has led to increased interest in the anaesthetic steroids. The eVect of methyl substitution at C-5 and C-10 on the GABAA receptor has been evaluated142 in a series of 5·- and 5‚-pregnanes.The eVect of varying the ring D structure by the introduction of a 16,17-double bond has also been examined.143 Photolysis of 3‚-acetoxy-5·-hydroxy-6‚- sulfanylpregnan-20-one, 70, with mercuric oxide–iodine gave144 the sulfenyl iodide and a ‚-fragmentation product, 71. 6-Oxa-5·-pregnane-3,20-dione, 72, has been synthesized145 by cyclization of a ring B seco iodo ketone. 11-Oxachlormadinone 73, has also been prepared146 and shown to be a powerful antiandrogenic agent. An alternative route for the conversion of 9·-hydroxyandrost-4-ene-3,17-dione to corticoids has been described.147 3‚,15‚,17·-Trihydroxypregn-5-en-20-one, 74, is a major 15‚-hydroxylated metabolite unique to the human perinatal period. The synthesis of a number of 15‚-hydroxypregnanes has been reported148–151 in order to provide marker samples for the GC–MS identification of these steroids and hence the detection of adrenal malfunction.A simple procedure for the preparation of ƒ16- corticosteroids has been described.152 The functionalization of C-18 by oxidation of 11‚-hydroxy steroids with phenyl iodosodiacetate and iodine has been reported.153 A synthetic approach to 12(13]18)-abeo-pregnanes by the base-catalysed rearrangement of 12,18-cyclopregnane-12,20-dione, 75]76, has been described.154 Details of an eYcient route for the construction of the cortical side chain have been given.155 The unusual product 77 has been obtained156 from dexamethasone using the methylating reagent, N,N-dimethylformamide dimethylacetal. The partial synthesis of some (20R)- and (20S)-11‚,20-dihydroxypregnanes has been described.157 21-Diazopregn-4-ene-3,20-dione and 18-diazomethyl-20- hydroxypregn-4-ene-3,18-dione have been synthesized158 as photoaYnity labelling reagents for the mineralcorticoid receptor.The synthesis of wortmannin 78, which is a specific inhibitor of Pi-3-kinases, has been achieved159 from H H H O O O H 65 H H H F H H F H H H OH H O O O O O O SMe SEt H N.N C NH2 NH2 F MeS H HO HO HO 67 68 69 66 H OH H O HO AcO H H O O AcO O H H H O H O O H H H O O OAc Cl SH S 70 71 72 73 H H H O HO OH OH 74 H H H O H H O O O O O O H 75 76 Hanson: Steroids: reactions and partial synthesis 267hydrocortisone.The introduction of the furan ring proved particularly troublesome. 4.4 Cardenolides Reductive amination of digoxigenone has been used160 to make 3·- and 3‚-aminodigoxigenin. The D-seco 79 and D-homo 80 digitalis derivatives have been synthesized and their binding to the Na+,K+-ATPase receptor has been examined. 161 Some of the D-seco derivatives showed a binding aYnity similar to that of digitoxigenin suggesting that parts of ring D may not be essential for recognition by the digitalis receptor.The eVect of modifications of the butenolide ring have also been examined.162 The synthesis of ‚-substituted butenolides by the palladium catalysed coupling of ƒ16-17- iodides and triflates with methyl 4-hydroxybut-2-enoate has been reported.163,164 An unusual oxidative transformation of the butenolide of gitoxin to form 81, which was mediated by tetra-n-butylammonium fluoride, has been reported.165 4.5 Bile acids The selective 3·-acetylation of cholic and deoxycholic acid can be carried out166 by transesterification using refluxing ethyl acetate in the presence of a catalytic amount of toluene-psulfonic acid and water.New dimers of the bile acids have been prepared167 by forming ester linkages between the C-3 hydroxy group and dicarboxylic acids of diVerent chain lengths. Analogues of the antimicrobial compound, squalamine, have been synthesized168 from hyodeoxycholic acid. The N-nitrosamides of 7‚-hydroxylated bile acid conjugates such as N-nitrosoglycoursodeoxycholic acid have been synthesized.169 These compounds are relatively stable and may enter the enterohepatic circulation.Their decomposition is similar to that of other N-nitrosamides which generate alkylating agents. A new enzymatic route for the synthesis of 12-ketoursodeoxycholic acid from cholic acid has been described.170 Chenodeoxycholic acid, 82, has the property of dissolving cholesterol gallstones and hence there has been interest in its synthesis from the more readily available cholic acid. The selective reduction of dehydrocholic acid to give 12-ketocholic acid and the WolV–Kishner reduction of the latter gave chenodeoxycholic acid.171 There has been considerable interest in the preparation of cholaphanes and other cyclic oligocholates with cavities of diVerent sizes.172 Examples include the cyclocholamides 83173 and 84,174 and the cholaphane 85.175 A number of further examples and new methods for the synthesis of these F H H CO O O H O O CO2Me O O MeO HO AcO 78 77 CHO H H H AcO H H H H HO O O O O O O H OH H H HO O O O OH 80 79 81 H H H H HO CO2H OH 82 OH OH OH HN HO HO O NH O O HN OH H H H H H H H HN H C O O O H H H C O O O C O O H H H H NH H HO HO HO OH CO CO O(CH2)4Me Me(CH2)4O 83 84 85 268 Natural Product Reports, 1998compounds have been reported.176–180 A family of bile acid molecular tweezers have been constructed from cholic acid linked to pyrene units.181 Chenodeoxycholic acid has been used182 as a scaVold for combinatorial synthesis.Attachment of a quinoline-3-carboxylate or acridine-9-carboxylate unit to position 24 of the steroid unit conferred activity against L1210 mouse leukaemia.183 4.6 Cholestanes The synthesis and X-ray crystal structure of the oxathiaphospholane derivative of cholesterol, 86, have been described.184 Compounds of this type have been used as reagents for introducing a cholesteryl moiety at the 5*-end of oligonucleotides.The reactions of the enamide, 4-azacholest- 5-en-3-one, and some relatives with electrophiles have been examined.185 The partial synthesis of the cytotoxic marine steroid, incrustasterol A, 87, has been reported.186 An enzyme system has been obtained187 from the marine gorgonian, Pseudopterogorgia americana, which will transform a variety of sterols into their 9(11)-secosteroid counterparts.A number of ergosterol derivatives have been prepared188 as inhibitors of the ƒ7-5-desaturase involved in ergosterol biosynthesis in the search for novel fungicides.Model studies have been reported189 in connection with a mechanism for the biogenesis of the ring D aromatic phytosteroids found in Nicotiana physaloides. The structural requirements for brassinosteroid activity have been examined.190 The synthesis and biological activity of the furostane 2·,3·,26-triol, 88, have been described,191 Although the compound did not exhibit plant growth promoting activity, it did reveal phytotoxicity. Some 3-deoxyecdysteroid analogues have been shown192 to possess some low moulting hormone activity. 3,24-Diepicastasterone, 89, a natural brassinosteroid with a ring A 2·,3‚-diol, has been synthesized193 from ergosterol. Brassinosteroid analogues containing an ester function, for example an isobutyrate, at C-20 of the pregnane skeleton have been synthesized194 and shown to possess some biological activity. The brassinolide intermediate 91 has been synthesized195 whilst the application of arsenic ylides has been explored196 in the construction of the side chain of castasterone.A short synthesis of (22E,24R)-5·-ergosta-2,22-dien- 6-one from ergosterol has been reported.197 Ergosterol also formed198 the starting material for syntheses of 24-epiteasterone 90, 24-epityphasterol 92, and their B-homo- 6a-oxalactone analogues. Sulfoxide chemistry has been exploited199 in the construction of the contiguous chiral centres of the brassinosteroid side chain. Improvements in the synthesis of brassinolide from stigmasterol have also been reported.200 The regioselective functionalization of the side chain of brassinosteroids by hydroxylation with methyl- (trifluoromethyl)dioxirane has been reported.201 Other synthetic studies on the construction of the sterol side chain which have been reported include the use of chlorosulfones,202 methods for the formation of 26,27-cyclopropylsterols,203 and the application of 22-cupriosteroids.204 Stereoselectivity in the epoxidation205 and hydroxylation206 of side chain double bonds has been examined. The results have been used in the elucidation of the structure of an ecdysteroid, gerardiasterone, 93,206 and in the preparation207 of possible intermediates in the formation of bile acids from cholesterol.The synthesis of deuterium labelled plant sterols208 and of 26,27-hexadeuteriocholesterol209 have been reported. The deuterium NMR spectra of oriented lipid bilayers derived from soyabean phosphatidylcholine and various 25-labelled plant sterols have revealed208 diVerences in orientation and mobility of the side chain between sterols.H H H O HO H HO AcO O P S S OAc O 86 87 H H H HO O H H H HO OH OH R H H H HO OH OH H H H HO O H H HO H OH O O 88 91 92 89 R = OH 90 R = H H OH HO OH OH HO H HO H OH O 93 Hanson: Steroids: reactions and partial synthesis 269Geodisterol, which was obtained210 from a marine sponge of the genus Geodia, has been shown to possess an aromatic ring A, 94.Reptansterone 95, was one of a number of ecdysteroids containing a ‰-lactone ring in the side chain which were isolated211 from Ajuga reptans. 3‚-Hydroxy-5·-cholest-8(14)-en-15-one is an important oxysterol in the regulation of cholesterol biosynthesis at the HMGCoA level. Its role and that of analogues has been reviewed212 and the preparation of a new analogue 96 has been described.213 11-Ketotigogenin cellobioside (pamaqueside) 97 obtained from hecogenin has been shown214 to be a potent inhibitor of cholesterol absorption in test animals.Studies on the use of silver ion HPLC in the separation of sterols have been reported215 and the method has been applied216 to an examination of the thermolability of cholesta-5,8-dien-3‚-ol and the formation of 19-norcholesta-5,7,9-trien-3‚-ol. 4.7 Vitamin D The ring opening of dehydrocholesterol to provitamin D has been studied217 by ultrafast UV spectroscopy with a time resolution of better than 300 femtoseconds.The kinetics of the [1,7a]-sigmatropic shift of vitamin D3 derivatives have been examined218 and correlated with the conformations of ring A. 25-Hydroxy-3-deoxy-2-oxavitamin D3 98 has been shown219 to be a potent inhibitor of 25-hydroxyvitamin D3 1·-hydroxylase. Some further fluoro derivatives of vitamin D have been prepared including the 19-fluoro220 and 24-difluoro221 analogues. The enantiospecific synthesis of vitamin D3 ring CD fragments have been discussed222 in the context of Diels–Alder approaches to the synthesis of this fragment.A number of other 1·,25-dihydroxyvitamin D analogues modified in the ring CD portion have been synthesized.223–226 The opening of a cyclopropane ring fused to ring A in some analogues of vitamin D3, e.g. 99, has been examined.227 The synthesis of some metabolites, e.g. 100, of 1·,25-dihydroxy-22-oxavitamin D3 has been reported.228 These compounds have antiproliferation activity towards human promyelocytic cells.Conformationally restricted analogues of 1·,25-dihydroxyvitamin D3 with extra methyl groups at C-22 aVecting the mobility of the side chain, have been synthesized229 in a study of the binding to the vitamin D receptor. The epoxidation of vitamin D by methyl(tri- fluoromethyl)dioxirane has been examined.230 4.8 Heterocyclic steroids The dimeric steroid–pyrazine marine alkaloids including the cephalostatins and ritterazines have been reviewed.231 15N NMR methods have been used232 to examine the orientation of the steroidal units about the pyrazine ring of ritterazine A.Syntheses of unsymmetrical bis-steroidal pyrazines, e.g. 101, some with antitumour activity, have been described.233,234 H H H HO OH OH H OH HO OH HO H HO O O O 94 95 H HO CF3 CF3 H F H OH O H O Me O O H H O O O HO HO OH O HO HO HO OH 96 97 OH O OH O OH HO OH OH HO H H H OH 98 99 100 N N O O O O H H HO OH CH2OH HO O H H H H H H 101 270 Natural Product Reports, 1998Syntheses of compounds with pyridine and pyrimidine rings fused to ring D of dehydroisoandrosterone have been reported.235,236 Some androstenes substituted at C-17 with triazole and imidazole rings, have been prepared237 as inhibitors of the 17·-hydroxylase and 17·,20-lyase.The coupling of purine bases to the carbonyl group at C-20 of the pregnanes has been reported.238 Steroidal selena-, tellura- and thialactones in the estrane series have been prepared.239 A review of steroidal oxazoles, oxazolines and oxazolidinones has appeared.240 5 References 1 J.R. 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Chem., 1996, 39, 4497. 227 M. M. Kabat, J. Kiegiel, N. Cohen, K. Toth, P. M. Wovkulich and M. R. Uskokovic, J. Org. Chem., 1996, 61, 118. 228 H. Watanabe, S. Hatakeyama, K. Tazumi, S. Takano, S. Masuda, T. Okano, T. Kobayashi and N. Kubodera, Chem. Pharm. Bull., 1996, 44, 2280. 229 K. Yamamoto, W. Y. Sun, M. Ohta, K. Hamada, H. F. DeLuca and S. Yamada, J. Med. Chem., 1996, 39, 2727. 230 R. Curci, A. Detomaso, M. E. Lattanzio and G. B. Carpenter, J. Am. Chem. Soc., 1996, 118, 11 089. 231 A. Ganesan, Angew. Chem., Int. Ed. Engl., 1996, 35, 611. 232 S. Fukuzawa, S. Matsunaga and N. Fusetani, Tetrahedron Lett., 1996, 37, 1447. 233 M. Droegemueller, R. Jautelat and E.Winterfeldt, Angew. Chem., Int. Ed. Engl., 1996, 35, 1572. 234 C. Guo, S. Bhandaru, P. L. Fuchs and R. M. Boyd, J. Am. Chem. Soc., 1996, 118, 10 672. 235 S. Ahmed and R. C. Boruah, Tetrahedron Lett., 1996, 37, 8231. 236 K. Rapole, A. H. Siddiqui, B. Dayal, A. K. Batta, S. J. Rao, P. Kumar and G. Salen, Synth. 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ISSN:0265-0568    

DOI:10.1039/a815261y

出版商:RSC    

年代:1998

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 LE1 7RH E-mail: bjr2@le.ac.uk Coverage: 1995 and 1996 Previous review: 1997, 14, 335 1 Introduction 1.1 Summary of recent highlights 2 Eubacteria 2.1 Gram negative bacteria 2.1.1 Escherichia coli 2.1.1.1 Acyl carrier protein (ACP) and holo-ACP synthase (ACPS) 2.1.1.2 Transacylases 2.1.1.3 3-Oxoacyl ACP synthases (KAS) 2.1.1.4 (3R)-Hydroxyacyl ACP dehydrase (DH) and (3R)- hydroxydecanoyl ACP dehydrase (HDDH) 2.1.2 Other Gram negative bacteria 2.2 Gram positive bacteria 2.2.1 Brevibacteria 2.2.2 Mycobacteria 2.2.3 Bacillus 2.2.4 Streptomycetes 2.2.5 Other Gram positive bacteria 3 Archaea 4 Protoctista 5 Plants 5.1 Acetyl CoA carboxylase (ACC) 5.2 3-Oxoacyl ACP synthase (KAS) 5.3 Enoyl reductase (ER) 5.4 Desaturases 5.5 Thioesterase (TE) 5.6 Polyhydroxyalkanoates 5.7 Thienes and furans 5.8 Hydrocarbons 5.9 Esters and lactones 6 Fungi 6.1 Fatty acid synthase 6.2 Elongases 6.3 Desaturases 7 Animalia 7.1 Cnidaria 7.2 Mollusca 7.3 Arthropoda 7.3.1 Chelicerata 7.3.2 Crustaceae 7.3.3 Uniramia 7.3.3.1 Insecta 7.3.3.2 Dictyoptera 7.3.3.3 Hemiptera 7.3.3.4 Lepidoptera 7.3.3.5 Diptera 7.3.3.6 Hymenoptera 7.4 Chordata 8 References 1 Introduction This review summarises the literature during 1995 and 1996, and is organised as in the previous review in the series,1 and should be read in conjunction with that review and similar ones involving polyketide biosynthesis2 and others involving fatty acid biosynthesis.3,4 The review is arranged in a taxonomical order loosely based upon Five kingdoms by Margulis and Schwartz,5 and Biology of Microorganisms by Brock, Madigan, Martinko and Parker,6 with archaea inserted between prokaryotes and eukaryotes as a sixth kingdom.The protoctista kingdom is defined by exclusion as eukaryotes that are not animal, plant or fungi. Fungal systems are considered after plants due to their closer similarity with animals in their assembly of many natural products such as Type I FAS.A consistent set of biosynthetic symbols are used in this series of reviews, as illustrated in Fig. 1. Abbreviations used in this review: aa, amino acids; ACC, acetyl CoA carboxylase; ACP, acyl carrier protein; ACPS, holo-acyl carrier protein synthase; AT, acyl CoA ACP transacylase (acyl transferase); B., Bacillus; BCCP, biotin carboxyl carrier protein; E., Escherichia; ESMS, electrospray mass spectrometry; 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, methylmalonyl CoA; MPT, malonyl/palmitoyl CoA ACP transacylase; MT, malonyl CoA ACP transacylase; MS, mass spectrometry; NAC, N-acetyl cysteamine (N-acetyl 2-aminoethanethiol); ORF, open reading frame; PHA, polyhydroxyalkanoate; PHB, polyhydroxybutanoate; S., Streptomyces; SAM, S-adenosyl methionine; TE, acyl ACP thioesterase.Figure 1 Biosynthetic symbols used throughout this paper Rawlings: Biosynthesis of fatty acids and related metabolites 2751.1 Summary of recent highlights The references, compound structures and details for the following recent highlights are to be found in the main text: The E. coli acyl carrier protein synthetase has been purified, overexpressed and compared with other phosphopantetheinylation proteins.The X-ray structure of malonyl CoA ACP transacylase from E. coli has been obtained. The enzyme preparation previously claimed as KAS IV, is now thought to have been a mixture of other KAS proteins, mainly KAS III. KAS III has now been overexpressed. HDDH, previously thought to only act upon C10 substrates, has been found to have C10 substrate specificity in the isomerisation reaction, but has now been found to act as a dehydrase to a wide range of saturated 3-hydroxyacyl ACPs.A crystal structure suggests that the mechanism is ‘two base’, not ‘one base’ as previously suggested. The NADH dependent enoyl reductase has been found to reduce all substrates on the FAS cycle, but no evidence could be found for a second, NADPH dependent ER, as previously reported. A crystal structure has been obtained for Vibrio harveyi tetradecanoyl ACP thioesterase, along with a reassignment of the catalytically active serine.There has been a series of reports on the mode of action of isoniazid, an antimycobacterial drug that is thought to act by binding to an enoyl reductase (InhA). A crystal structure of the InhA– NADH complex has been obtained, whilst other reports suggest that InhA reduces very long chain dienes such as (2E,5Z)-tetracosa-2,5-dienoyl CoA to (5Z)-tetracos-5-enoyl ACP. Pathogenic mycobacteria possess cyclopropanated long chain fatty acids which are resistant to attack by macrophages.The genes responsible for cyclopropanation have been located. The FAS ACP in S. coelicolor does not eYciently replace the PKS ACPs in polyketide assembly, but a common malonyl transacylase might be responsible for charging both the FAS and PKS systems with malonate. Whilst certain FAS and PKS proteins appear interchangeable, it is becoming clear that PKSs have evolved horizontally between the various streptomycetes, and not from individual FASs. A seed expressed ACP from Brassica campestris and KAS III from B.napus have been sequenced and cloned, whilst the X-ray crystal structure of enoyl reductase from B. napus has been obtained. Castor seed octadecanoyl ACP 9Z desaturase crystal structure has two iron atoms in distorted octahedral coordination geometry coordinated to aspartates and histidines, and a gap for a dioxygen molecule. A linear substrate channel is bent near the di-iron cluster to favour formation of the 9Z product. The thioesterase from the Californian bay tree, when overexpressed in E. coli, produces dodecanoic acid, and only very low levels of tetradecanoate.A triple mutation changes the selectivity of this dodecanoyl ACP thioesterase into exclusively that of a tetradecanoyl ACP thioesterase, resulting in the exclusive formation of tetradecanoate. Bacterial genes responsible for assembling PHB have been inserted into the cotton plant by particle bombardment. The resulting cotton fibre contained PHB giving it a higher heat capacity and improved insulating characteristics.The enzymes in pine trees that produce heptane could be inserted into bioengineered fermentative microorganisms to produce a renewable source of hydrocarbon fuel. It has been shown that heptane is biosynthesised directly from C8 precursors, rather than from the degradation of longer chain fatty acids, as is the case for some other plant hydrocarbons. A much improved model of the yeast fatty acid synthase ·6‚6 complex has been obtained from electron microscopy, with 12 funnel shaped openings for substrate entry and product exit, with active sites on the inside cavity of a barrel like structure with 32 pt symmetry.There was also a high degree of negative cooperativity between the acyl binding sites, to avoid substrate swamping the sites needed to bind later intermediates. An investigation of the mechanism of the fungal 9Z desaturases appears to rule out the classical proposals that involved synchronous removal of the two hydrogens, instead most likely would be the rate determining abstraction of a hydrogen radical from C-9, which then collapses to the alkene.Aspergillus nidulans produces the polyketide aflatoxin B1 that has a hexanoate starter unit. It has now been shown that there is a FAS system dedicated to production of hexanoate, whose genes are close to the PKS cluster. Gene deletion prevents aflatoxin production, but otherwise grows normally using the main FAS system.The production of polyunsaturated fatty acids in brine shrimps has been examined by feeding radiolabelled precursor soaked defatted rice bran to starving shrimps. Insect cuticular lipids contain long chain hydrocarbons formed by loss of the carboxy carbon. Recent work suggested this process was decarbonylation of the corresponding aldehyde. However, NADPH, dioxygen and microsomal preparations from the house fly have now been shown to convert (15Z)-tetracos-15-enal (C24) to (9Z)-tricos-9-ene and carbon dioxide in a cytochrome P450 type reaction.The substrate aldehydic proton was transferred to (9Z)-tricos-9- ene. Human FAS has been isolated and characterised from a hepatoma cell line, and like the rat FAS but unlike the chicken FAS, it prefers butanoyl CoA to acetyl CoA as a starter unit. The human FAS was only partially phosphopantetheinylated, and in this way, may be postranslationally regulated. Human FAS from brain only has a low sequence homology to human hepatoma FAS, has been expressed in E.coli. Domain I contained KAS, AT and DH activity, agreeing with the recent reassignment of animal FAS DH to domain I from domain II. The in vitro complementation of inactive mutants has been used to probe the interunit cooperation between the two halves of the antiparallel homodimeric animal FAS. A single mutation can prevent fatty acid assembly at both active sites. However, subunit exchange enables assembly of catalytically active fatty acid synthase heterodimers from pairs of inactive homodimers carrying mutations in diVerent functional domains.Thus, two mutations located on domains that normally cooperate with each other on an intersubunit basis may now be capable of fatty acid synthesis. Mixing a KAS mutant homodimer and ACP mutant homodimer under ‘exchange’ conditions restored approx. 50% activity. The ‚-oxidation of (5Z)-fatty acids has been extensively investigated, and found to proceed via a series of unexpected intermediates in which (2E,5Z)-dienoyl CoA is isomerised to (3E,5Z)- and (3Z,5Z)-dienoyl CoAs, which are then isomerised to (2E,4E)-dienoyl CoA and reduced to (3Z)-enoyl CoA, before conversion by an ‘HDDH’ like enzyme to (2E)-enoyl CoA for standard ‚-oxidation.The mammalian conversion of 20:5 (n"3) to 22:6 (n"3) has been unexpectedly found to proceed via 24:6 (n"3) followed by one round of ‚-oxidation to 22:6 (n"3), with transfer of substrates between peroxisomes and microsomes.The selective accumulation of 22:6 (n"3) in our membranes has been found to rely upon the remarkable selectivity of the 2,4-dienoyl reductase between (2E,4Z,8Z . . .) and (2E,4Z,7Z . . .) substrates. 2 Eubacteria 2.1 Gram negative bacteria 2.1.1 Escherichia coli Rock and Cronan have published an extensive review of fatty acid biosynthesis in E. coli and its role as a model for Type II FAS.7 This includes an extensive discussion about regulation by acyl ACPs.There is a cluster of genes at 24 min involved in FAS biosynthesis, plsX (involved in phospholipid synthesis), fabH (KAS III), fabD (MT), fabG (KR), acpP (ACP) and fabF (KAS II). Zhang and Cronan have examined the transcription of these genes using polar allele duplication.8 They were able to identify the ACP promoter, deletion of which abolished ACP expression. However, the overall transcription of these genes is 276 Natural Product Reports, 1998very complex, with multiple promotors (allowing modulation of relative expression), and each gene shares at least one transcript with another gene (allowing coordination of expression).Regulation of E. coli FAS has been reviewed.9 2.1.1.1 Acyl carrier protein (ACP) and holo-ACP synthase (ACPS) NMR spectroscopic studies by Kim and Prestegard have suggested that the ACP has at least two distinct solution conformations.10 Jackowski and Rock have reported a novel form of ACP with increased electrophoretic mobility isolated from fabF mutants, called fast migrating ACP (F-ACP).As they were unable to detect any alteration in the amino acid sequence, they suggested that the altered mobility was due to an unidentified post-translational modification.11 Keating and Cronan have reinvestigated the mutant and have found that Val-43 of the ACP has been substituted by Ile, resulting in a more compact structure at high pH.12 Val-43 lies in a region of conformational flexibility, and the V43I substitution may be aVecting the equilibrium between the two conformational states, shifting it towards the more compact of the two conformers.This was confirmed by introducing the V43I mutation into a synthetic acpP gene, overexpression resulted in F-ACP. At pH 7, this F-ACP behaved similarly to wild type on gel filtration, but at pH 9 it eluted much more slowly. F-ACP was phosphopantetheinylated at a similar rate to wild type ACP, and functions eYciently in vivo.ACP is produced as the inactive apo-ACP, which is then post-translationally modified by E. coli holo-ACP synthase (ACPS) by phosphopantetheinylation of the Ser-36 hydroxy group in a Mg2+ dependent reaction. Whilst E. coli ACPS13 and spinach ACPS14 have been partially purified, little is understood about this process. Lambalot and Walsh have N-terminally sequenced ACPS that had been purified over 70 000-fold, and identified a corresponding gene, dpj, in the pdxJ (pyridoxal) operon, cloning and overexpression of which now allows the preparation of ACPS in milligram quantities as a homodimer of 28 kDa.15 The dpj gene contains rare codons characteristic of low expression in E.coli, correlating with the observation that even at wild type 70 000-fold purification, the ACPS sample was not homogenous. In a major paper on phosphopantetheinyl transferases, Walsh and co-workers propose a new designation of dpj as the acpS gene, report marginal sequence similarity with the carboxy terminal region of five fungal fatty acid synthases (FAS2), and propose that the phosphopantetheinylation activities were subsumed as a domain in these multifunctional proteins.16 Sequence similarity was also obtained for other bacterial proteins that are thought to be involved in non-ribosomal peptide synthesis in Bacillus brevis and B.subtilis, and for a yeast sequence believed to be involved in lysine biosynthesis. Hill et al.have reported the overexpression, purification and characterisation of ACP and two mutants.17 Cronan and co-workers have reported that unmodified E. coli ACP, apo-ACP, is toxic to E. coli through inhibition of lipid metabolism, and acts as a potent in vitro inhibitor of sn-glycerol-3-phosphate acyltransferase.18 Levels of apo-ACP are very low in wild-type cells under normal culture conditions, and overexpression of ACP from multicopy plasmids strongly inhibits growth, as levels of ACPS are not high enough to rapidly convert the apo-ACP into holo-ACP.Whilst high levels of apo-ACP accumulated in mutants lacking holo-ACP synthase (ACPS), Cronan and co-workers observe that apo-ACP levels did not accumulate in cells starved of ‚-alanine, a precursor of CoASH.19 Initial suggestions were that the synthesis of ACP was coupled to the availability of the prosthetic group donor, CoASH. However, Cronan and co-workers found that addition of glutamate restored ACP synthesis.Glutamate, aspartate and their amidated forms, constitute one-third of the amino acid residues in E. coli ACP and any decrease in activity of the tricarboxylic acid cycle (TCA) would aVect ACP synthesis more severely than the production of other, less acidic, proteins. As acetyl CoA is required for entry of carbon into the tricarboxylic acid cycle, Cronan and co-workers proposed that it was the lack of availability of these acidic amino acids that was preventing transcription of acpP or translation of its mRNA.Northern analysis showed no decrease in levels of acpP mRNA, strongly suggesting that it was translation being aVected by low glutamate levels. 2.1.1.2 Transacylases Derewenda and co-workers have obtained a crystal structure of E. coli malonyl CoA ACP transacylase (FabD MT) at 0.15 nm resolution.20 This soluble cytoplasmic enzyme transfers the malonyl moiety from malonyl CoA to the thiol of ACP, forming malonyl ACP, and is highly specific towards malonyl CoA as substrate, not participating in the priming of ACP with acetyl CoA (unlike Type I FAS).The 32 kDa protein has an ·/‚ architecture, but with a unique fold. The active site serine (Ser-92) is located in a gorge between the two subdomains at the centre of a GHSLG sequence that compares with the GXSXG sequence found in most acyl hydrolases such as chymotrypsin. Other acyl hydrolases use the well-known Ser–His–Asp(Glu) catalytic triad, MT has a Ser–His diad stabilised by the main chain carbonyl of Gln-251.A major diVerence between MT and other hitherto characterised hydrolases is that the intermediate acyl–enzyme complex is stable in aqueous solution, only the specific thiol can cause deacylation. The acyl–enzyme complex must prevent access by a suitably activated hydrolytic water molecule, yet provide access for the thiol acceptor. 2.1.1.3 3-Oxoacyl ACP synthases (KAS) The 3-oxoacyl ACP synthases (KAS, ‚-keto synthases, condensing enzymes) catalyse the Claisen like carbon–carbon bond forming step.KAS I catalyses condensations up to 3-oxotetradecanoyl ACP, whilst KAS II is specific for long chain acyl ACPs, in particular, C14 to C16. KAS III specifically catalyses the first condensation in Type II systems, that between acetyl CoA and malonyl ACP to form 3-oxobutanoyl ACP. As it is this reaction that initiates FAS in Type II systems, it may play a crucial role in regulation of fatty acid biosynthesis.Heath and Rock have obtained and purified E. coli FabH KAS III, and found its activity to be suppressed by acyl ACPs (>C12), their potency increasing with chain length up to C20, and proposes that this is one of several routes through which acyl ACPs regulate FAS.21 KAS III had a Km of 40 ÏM for acetyl CoA and 5 ÏM for malonyl ACP, and would not utilise butanoyl CoA or hexanoyl ACP. Stuitje and co-workers have overexpressed this KAS III in a KAS III mutant of E.coli, and in oil seed rape (Brassica napus).22 Overproduction in E. coli resulted in an increase in 14:0 and a decrease in 18:1 (11Z) (cis-vaccenate) levels as well as an arrest in cell growth of membrane lipids. Overexpression in the seed plastids decreased the 18:1 (11Z) content of the storage lipids, but increased levels of 18:2 and 18:3. The authors suggest that FAS is not regulated by one rate limiting enzyme such as ACC, but that regulation is shared by a number of enzymes.22 Siggaard-Anderson et al.recently reported locating a new gene, fabJ, and suggested that it may code for a new KAS, KAS IV, and proposed that it was specific for the synthesis of short chain acids, possibly for lipoic acid (C8) biosynthesis.23 Cronan and co-workers have been attempting to obtain FabF KAS II, but have been hindered by the extreme instability of clones carrying the fabF gene, but have been able to report that the sequence of fabF is identical to that reported for fabJ.24 Cronan suggests that the enzyme preparation studied by Siggaard-Anderson may have consisted largely of KAS III (along with some KAS I/KAS II), which would have had a similar substrate specificity to that reported for ‘KAS IV’ and similar insensitivity towards cerulenin.Unfortunately, it seems that it is still not possible to overexpress fabF and overproduce KAS II. Rawlings: Biosynthesis of fatty acids and related metabolites 2772.1.1.4 (3R)-Hydroxyacyl ACP dehydrase (DH) and (3R)- hydroxydecanoyl ACP dehydrase (HDDH) The E.coli fabZ gene codes for what has been referred to as the DH, whilst the fabA gene codes for what has been referred to as the HDDH. Classical studies by Morisaki and Bloch using 3-hydroxyacyl-N-acetyl cysteamine substrate analogues and allenic inhibitors showed that the fabA product, HDDH, was highly selective for such substrate analogues containing between C9 and C11, suggesting that HDDH provided a specific route into the monounsaturated fatty acids through the ‘anaerobic pathway’.25 Heath and Rock have reexamined the roles of DH and HDDH in FAS by purifying both these proteins and five other FAS proteins to reconstitute cycles of FAS in vitro, and by using ACP thioesters as substrates.26 Surprisingly, they found that both HDDH and DH acted eYciently to dehydrate 3-hydroxybutanoyl ACP to (2E)-but- 2-enoyl ACP.This led the authors to examine a wide range of enzymes with a wide range of substrates.The FabG KR eYciently reduced every 3-oxoacyl ACP substrate, whether short or long chain saturated, or unsaturated. The FabI NADH dependent ER functions in every cycle in the pathway as recently reported.27 Heath and Rock could find no evidence of the NADPH dependent ER as reported by Weeks and Wakil.28 Both the FabZ DH and FabA HDDH eYciently dehydrated all chain lengths (C4 to C16) of 3-hydroxyacyl ACP (assayed by saturated acyl ACP formation when coincubated with FabI ER).However, FabZ DH was most active on short chain lengths, and FabA HDDH was most active on medium chain lengths, with a maximum at C10. Both DH and HDDH had low activity for C14 and C16 substrates. However, a diVerent situation was found when the isomerisation product series were used as sustrate. For example, only FabZ DH, and not FabA HDDH, was able to dehydrate (9Z)-3- hydroxyhexadec-9-enoyl ACP, thus the HDDH was unable to participate as a dehydrase in the chain elongation of the unsaturated fatty acids.Heath and Rock confirmed that FabZ DH did not have isomerase activity, and then went on to show that FabB KAS I was required to be present for FabA HDDH to form isomerisation products, the cis mono-unsaturated acyl ACPs.26 Assays containing HDDH, KAS I and KR converted 3-hydroxydecanoyl ACP to (5Z)-3-hydroxydodec-5-enoyl ACP and (2E,5Z)-dodeca-2,5-dienoyl ACP. Addition of cerulenin,1 which inhibits KAS I, led to only dehydration occurring.Schwab et al. have previously shown that at equilibrium, HDDH gives a ratio of 3-hydroxy:2E:3Z products of 75:22:3.29 Thus, these results of Heath and Rock are explained by KAS I having an extremely high aYnity for (3Z)-dec-3-enoyl ACP, pulling this equilibrium in the direction of unsaturated fatty acid formation. It is interesting that HDDH appears so substrate specific for isomerisation, yet is so non-specific for dehydration (Scheme 1).Schwab, Smith and co-workers have obtained crystal structures at 0.2 nm resolution for both ‘free’ FabA HDDH and the covalent complex of HDDH with the suicide inhibitor dec-3-ynoyl-N-acetylcysteamine.30 The HDDH dimer consists of two 171 residue subunits, each made up of a seven stranded antiparallel ‚-sheet ‘bun’ wrapping itself almost completely around an extremely hydrophobic five turn ·-helical ‘sausage’ in an appropriately named ‘hot dog’ fold.Only two of the 19 residues comprising the central helix are polar, and these face the active site. Each active site is at the subunit interfaces. There is a linear hydrophobic tunnel shaped pocket resembling a worm hole suitable for the fatty acid portion of the substrate. The ‘hole’ starts on the subunit interface as a polar region suitable for binding the polar ACP end of the substrate, and then burrows into the hydrophobic core of the protein between the ·-helix and the inner surface of the ‚-sheet (Fig. 2). The suicide inhibitor fills this worm hole so that the catalytic site is sequestered from the bulk solvent. The catalytically active His-70 is located half-way along this binding tunnel, and belongs to the subunit that makes up the polar half of the binding site. There is a rare non-proline cis peptide bond between His-70 and Phe-71, which is associated with the active site. Asp-84* (from the other subunit) was located near the C-3/C-4 region of the suicide substrate, suggesting a role in the Acetyl CoA FabH KAS III 3-Oxobutanoyl ACP FabZ DH or FabA HDDH FabI ER Butanoyl ACP Malonyl CoA Malonyl ACP ACC CO2 etc FabB KAS I or FabF KAS II FabG KR FabZ DH or FabA HDDH FabI ER Octanoyl ACP FabB KAS I or FabF KAS II FabG KR (3 R)-Hydroxydecanoyl ACP FabI ER Decanoyl ACP Hexanoyl ACP FabB KAS I or FabF KAS II FabZ DH or FabA HDDH FabI ER Dodecanoyl ACP FabB KAS I or FabF KAS II FabG KR (3 R)-Hydroxytetradecanoyl ACP FabI ER FabZ DH or FabA HDDH FabB KAS I or FabF KAS II FabG KR Tetradecanoyl ACP FabZ DH or FabA HDDH Hexadecanoyl ACP FabI ER FabZ DH or FabA HDDH FabZ DH or FabA HDDH FabG KR FabI ER FabG KR ACPSH FabA HDDH (3 Z)-Dec-3-enoyl ACP FabB KAS I (5 Z)-Dodec-5-enoyl ACP [Palmitoyl ACP] FabB KAS I or FabF KAS II (2 E)-Dec-2-enoyl ACP FabG KR FabZ DH FabZ DH FabB KAS I FabB KAS I (9 Z)-Hexadec-9-enoyl ACP FabI ER FabG KR FabI ER FabZ DH (11 Z)-Octadec-11-enoyl A CP Elongases [ cis-Vaccenoyl ACP] LIPID A Cyclopropanated fatty acyl ACPs (both slow) (7 Z)-Tetradec-7-enoyl ACP FabI ER Lipoic acid FabG KR (both slow) Diglycerides and Triglycerides FabD MT Scheme 1 Biosynthesis of fatty acids in E.coli S O OH Me A C P Polar Polar H70 G79 C80 D84¢ Subunit interface Subunit 1 Subunit 2 2 nm 0.6 nm + – + + + + + – – – – – Figure 2 Model of E. coli HDDH active site 278 Natural Product Reports, 1998reaction. For many years, HDDH has been thought to have a ‘one-base’ mechanism.31 Schwab et al. now propose that both H70 and D84* are involved in a ‘two-base’ mechanism (Scheme 2).Schwab et al. compared FabA HDDH with FabZ DH and suggest that the major active site diVerence is the replacement of D84* with glutamate, and that this alteration results in the carboxylate no longer being correctly positioned to remove the C-4 hydrogen.30 A mutant unable to transfer the long acyl chain from ACP to sn-glycerol-3-phosphate led to an accumulation of long chain acyl ACP which resulted in the inhibition of de novo fatty acid biosynthesis.32 Whilst acetyl CoA carboxylase (ACC) activity remained high, levels of malonyl CoA did not accumulate due to the stimulation by long chain acyl ACPs of FabB KAS I and FabF KAS II to decarboxylate malonyl ACP to acetyl ACP which is then converted to acetyl CoA by the transacylase activity of FabH KAS III.Cells treated with cerulenin, that inhibits KAS I and KAS II, massively accumulated malonyl CoA.Heath and Rock examined the regulatory eVect of long chain acyl ACP on fatty acid biosynthesis, 33 and found that hexadecanoyl ACP attenuated the action of both FabH KAS III and FabI ER in the first cycle. As there is now reported to be only a single ER for all of FAS in E. coli, any attenuation of its activity would eVectively down regulate every cycle in the pathway. ER is inhibited by diazaborines (only in the presence of NAD+), whose action is thought to prevent lipopolysaccharide biosynthesis and retard cell growth.34 Rice and co-workers have obtained the X-ray structure of ER as a complex with NAD+, with and without diazaborine.35 Voelker and Davies have found that expression of plant medium chain (C12) thioesterase in E.coli considerably increased FAS activity.36 Ohlrogge et al. have observed that acyl ACP levels and ACC expression are altered by this expression of a medium chain TE, and suggest that levels of long chain acyl ACP have a considerable repressive eVect on FAS activity.37 Lipid A, as part of a lipopolysaccharide, is a major component of the outer membrane monolayer of Gram negative bacteria.UDP-N-acetylglucosamine 3-O-acyltransferase (LpxA), that catalyses the transfer of (3R)-hydroxytetradecanoyl moiety from its ACP to the 3*-OH of UDP-Nacetylglucosamine, has been cloned and an X-ray structure obtained by Raetz and Roderick.38 2.1.2 Other Gram negative bacteria Infection of the roots of leguminous plants by certain Gram negative bacteria of the Rhizobium genus leads to the formation of root nodules which are able to ‘fix’ dinitrogen.A cluster of ten genes in Rhizobium spp., the nod genes, are responsible for directing nodulation. Leguminous plants are unusual in that their roots secrete large amounts of certain flavonoids, which have been found to induce expression of the nod genes in nearby rhizobium bacteria, and thus initiate nodulation. In Rhizobium leguminosarum bv.viciae (host plant Vicia sativa), NodE appears homologous with KASs and NodF with ACPs. Expression of NodE and NodF proteins are required for the synthesis of (2E,4E,6E,11Z)-octadeca- 2,4,6,11-tetraenoic acid 1, required as a structural element in conferring host-specificity in lipochitin oligosaccharide signals. 39 Despite a consensus sequence with E. coli ACP (acpP) of LGXDSL around the presumed site of prosthetic group attachment in NodF, the sequence homology elsewhere is quite low.Prestegard and co-workers have examined by NMR spectroscopy the secondary structure of NodF and find only three ·-helices.40 However, NOE studies suggest close correlation between long range contacts between the three long helices of both NodF and E. coli AcpP. There is evidence for at least two other ACPs in the rhizobium, suggesting that minor structural features in the NodF ACP may be responsible for assembling the signal molecule 1. Lipid As of Rhizobium spp.are acylated with 27-hydroxyoctacosanoic acid (C28) 2 on the 3-hydroxy group, a fatty acid not found in E. coli. Raetz and co-workers have located the ACP in Rhizobium meliloti as a 92 aa protein containing the DSLD consensus sequence, but very little (<25%) consensus with any other ACP, and has been designated AcpXL.41 Mass spectral analysis of the isolated AcpXL protein suggested that the major acylation species was 27-hydroxyoctacosanoic acid, with small levels of corresponding (¢-1)-hydroxylated C26, C24, C22, and C20 species. This ACP may have a specialised function in acylating the 3-hydroxy fatty acids in lipid As of the rhizobium.An extract, containing soluble and membrane components, transfers 2 to unacylated Lipid A (Scheme 3) to give 3. Raetz and co-workers speculate that this rhizobium FAS uses 3-hydroxybutanoate as a starter unit and cannot dehydrate the 3-hydroxybutanoyl moiety. In the bioluminescent bacterium Vibrio harveyi, ACP is involved in supplying tetradecanoic acid, a precursor of S O O NH O H HN N H H70 O D84' O H N H N H S O O NH O H HN N H70 O D84' O– H N H N H C6H13 H C6H13 H H S O O NH O H HN N H70 O D84' OH H N H N H C6H13 H G79 C80 C80 C80 + Scheme 2 Me CO2H 18 11 1 Rawlings: Biosynthesis of fatty acids and related metabolites 279tetradecanal, the luciferase substrate.Shen and Byers have cloned the KR ( fabG), ACP (acpP) and KAS II ( fabF) regions, and purified the ACP.42 The ACP has 79% homology to E.coli ACP, 76 aa (cf. 77 aa), and an isoelectric point of pH 3.99 (cf. pH 4.2). Both ACPs have identical sequences between residues 31 and 71, thus obtaining the full structure would allow the eVect of amino acid sequence diVerences at discrete regions to be examined, as well as the eVect of these changes upon interactions with other FAS components. Meighen, Derewenda and co-workers have obtained a crystal structure of tetradecanoyl ACP thioesterase from Vibrio harveyi and found a lipase-like catalytic triad Ser-114– His-241–Asp-211.43 Ser-77 was previously considered to be the catalytically active serine.44 Whilst S77G mutation did not significantly alter the structure, it did significantly reduce the activity.The authors propose that the substrate lies between the catalytic triad and a ‘cap’. The overall three-dimensional structure is that of an ·/‚ hydrolase, however the nucleophilic elbow GXSXG consensus sequence has the two Gs replaced by an A and an S.43 A Gram negative bacterium isolated from marine sediments, Malonomonas rura, is able to grow on malonate which is decarboxylated to acetate using a sodium ion dependent, ACP and biotin containing malonate decarboxylase.Dimroth and co-workers report that the prosthetic group of the ACP is 2*-(5+-phosphoribosyl)-3*-dephosphocoenzyme A, previously found as a prosthetic group in citrate lyase from Klebsiella pneumoniae.45 However, the site of attachment has not yet been determined.The malonyl ACP then transfers its ‘CO2’ to biotin, giving carboxylated biotin and acetyl ACP, the acetyl group of which is then exchanged with a new malonate by a specific transferase. Malonate decarboxylases have also been recently located in Acinobacter calcoaceticus46 and from Klebsiella pneumoniae.47 A series of reviews has appeared on the biosynthesis of polyhydroxyalkanoates: ‘The biosynthesis of poly(3- hydroxybutanoate-co-3-hydroxypentanoate) in Rhodococcus ruber’;48 ‘Formation of novel poly(hydroxyalkanoates) from long chain fatty acids’;49 ‘Methylobacterium rhodesianum MB 126 possesses two stereospecific crotonyl CoA hydratases’;50 and ‘Isolation and purification of granule associated proteins relevant for poly(3-hydroxybutyric acid) biosynthesis from methylotrophic bacteria relying on the serine pathway’.51 Park and co-workers have examined the formation of poly- (3R)-hydroxybutanoate (PHB) by Alcaligenes eutrophus.52 As NADPH is a cosubstrate for 3-oxobutanoyl CoA reductase, Park and co-workers have examined, in particular, the relationship between NADH/NADPH levels and PHB production.Davies and co-workers have cloned and characterised the PHA biosynthetic gene cluster from an Acinetobacter sp. and found three clustered genes; phaAAC (3-oxothiolase), phaBAC (3-oxobutanoyl CoA reductase) and phaCAC (PHA synthase), and an unidentified ORF coding for a 13 kDa protein.53 The entire pha locus when transformed into E.coli, accumulated PHA. Davies and co-workers have demonstrated increased transcription under conditions of phosphate starvation. Steinbüchel and co-workers have examined metabolic pathways to PHAs in Alcaligenes eutrophus when grown on 4-hydroxybutanoate, and found no evidence for the direct conversion of 4-hydroxybutanoate into 3-hydroxybutanoate, and suggest conversion into succinate (via succinate semialdehyde) and degradation via the citric acid cycle.54 Doi and co-workers have examined the biosynthesis of polyester blends by a Pseudomonas sp.from alkanoic acids.55 2.2 Gram positive bacteria 2.2.1 Brevibacteria Brevibacterium ammoniagenes produces both saturated and monounsaturated fatty acids using a Type I FAS first purified by Kawaguchi and co-workers as an ·6 homomultimer (2 MDa).56 Unlike the eukaryotic Type I FAS, this FAS contains an HDDH domain to insert the unsaturation.57 Schweizer and co-workers recently sequenced a fas gene from B.ammoniagenes that was homologous with yeast FAS with a domain order corresponding to head-to-tail fusion of the two yeast FAS subunits.58 However, gene disruption did not produce any FAS defective mutants. Schweizer and co-workers have now located and sequenced a second fas gene. FasA disruptants required (9Z)-octadec-9-enoic acid for growth, whilst fasB disruptants exhibited no fatty acid requirement. However, the location of the fasB ‘HDDH’ domain is unclear, as there is no obvious sequence homology with the E.coli HDDH fabA gene.59 2.2.2 Mycobacteria A major review ‘The envelope of mycobacteria’ by Brennan and Nikaido has been published, which not only reviews the structures present but also their biosynthesis.60 The fatty acid synthase from Mycobacterium tuberculosis var. bovis BCG has a bimodal product distribution, not only assembling hexadecanoate from acetate and malonate, but also chain elongation to hexacosanoate (C26), resulting in a bimodal product distribution and allowing it to utilise the host’s fatty acids.This Type I FAS has been isolated and purified, and unlike vertebrate FAS, requires both NADH and NADPH, and can accept as substrates CoA thioesters of C2 to C8, or C16 and C18 fatty acids.61 Fernandes and Kolattukudy have cloned and sequenced the entire fas gene coding for a protein containing 2797 aa (301 kDa).62 Analysis of the sequence suggests the domains in the following order: acyl transferase (AT), ER, malonyl/palmitoyl transferase (MPT), ACP, KR, KAS, this order resembling a head-to-tail fusion of the two yeast FAS ·- and ‚-subunits.The FAS showed greatest homology (56%) with Brevibacterium ammoniagenes FAS which also requires both NADH and NADPH, but only assembles hexadecanoate. The MPT domain had high homology with the corresponding region in B. ammoniagenes. This may be because both organisms release hexadecanoate via transfer to CoA, whilst in animals hydrolytic cleavage occurs.There is also high homology of the N-terminal acyl transferase domain and nearby ER domain with those in other Type I FAS. Whilst there is homology between the putative DH site and that in B. ammoniagenes, there is little homology with DHs in either E. coli, yeast FAS2 or rat FAS. There are two putative KR sites, this may reflect the need for both NADH and NADPH, and this may allow the FAS the flexibility to perform its dual chain length role.The single KAS is located at the C-terminus. Mycolic acids are 2-alkyl-3-hydroxy fatty acids found in the norcardia, corynebacteria and most mycobacteria, having a general structure R2(CHOH)(CHR1)COOH, with a variety of oxo-, epoxy-, cyclopropyl- and methyl functionalities adorning the alkyl chains.1 Isoniazid 4 has been the drug of choice to Me OH O HN O O Me O Me OH HO O 28 2 16 28 3 Extracts of Rhizobium leguminosarum Scheme 3 280 Natural Product Reports, 1998combat mycobacterial infection for over 45 years and is believed to act by inhibiting the biosynthesis of mycolic acids.The protein product of inhA (M. tuberculosis) is thought to be the primary target of action, and shows homology with ER from Brassica napus and EnvM from E. coli that can reduce (2E)-but-2-enoyl ACP.63 Baker has found that both E. coli EnvM ER, ER from Brassica napus, and InhA from M. tuberculosis, lack the YXXXK motif generally found in dehydrogenases, the tyrosine (Y) being replaced by methionine (M).As the tyrosine’s phenolic group, assisted by the protonated lysine (K), is thought to catalyse hydride transfer, this suggests novel aspects of catalysis for these ERs.64 Blanchard, Sacchettini and co-workers have overexpressed InhA in E. coli and showed that it eYciently catalysed the reduction of (2E)-oct-2-enoyl ACP using NADH.65 However, neither isoniazid, or the closely related ethionamide bound to InhA suggesting that both drugs are metabolically activated prior to binding.They have obtained a crystal structure of the InhA–NADH complex (both wild type and S94A inactive mutant). Interestingly, the wild type enzyme and S94A mutant had similar Km and Vmax values for (2E)-oct-2-enoyl ACP, but the mutant had a five-fold larger Km value for NADH. The authors discuss the disruption of the hydrogen bonding to NADH in the mutant and how this could lead to the design of better analogues of isoniazid.In a separate paper, Blanchard and co-workers show that the preferred primers for InhA are C16- to C24-enoyl ACPs, and that the 4S hydrogen of NADH is transferred.66 No reaction could be observed with (2E)-but- 2-enoyl CoA (crotonyl CoA, in contrast to most ERs), and the Km value for (2E)-oct-2-enoyl ACP was 100 times that for (2E)-hexadec-2-enoyl ACP. Blanchard and co-workers propose a mechanism in which the protonated lysine hydrogen bonds to the enolate oxyanion.Schultz and co-workers have suggested that isoniazid is first activated by mycobacterial catalase-peroxidase, and then covalently reacts with Cys-243.67 Blanchard and co-workers added activated isoniazid to C243S mutant in the presence of NADH, but did not observe any reduction in enzyme inactivation, demonstrating that Cys-243 is not the residue modified by activated isoniazid.68 Wheeler and Anderson have used crude cell preparations from M.aurum to suggest that it is the elongase ‘enoyl ACP reductases’ that reduce (2E,5Z)-long chain alka-2,5-dienyl ACPs, that are inhibited by activated isoniazid. The authors observed that isoniazid treated cells accumulate 24:0 and (5Z)-tetracos-5-enoyl ACP 5, and were unable to convert exogenous 5 to mycolic acids. This suggests that isoniazid 4 acts after insertion of the 5Z double bond.69 However, the insertion of the 5Z double bond remains a mystery as no evidence of a 5Z desaturase has been detected, nor any enzyme analogous to the E.coli anaerobic desaturation using an ‘HDDH’ analogue in which (2E)-docos-2-enoyl ACP would be isomerised to (3Z)-docos-3-enoyl ACP followed by elongation to 5 (Scheme 4). These elongases apparently convert ACP thioesters, in contrast to other elongases which operate on CoA thioesters. Pathogenic mycobacteria, such as M. tuberculosis, have a high proportion of bis-cyclopropanated fatty acids (·-mycolic acids) such as 2-docosyl-21,22:33,34-dimethano-3-hydroxydipentacontanoate 6 in their cell-wall.The cyclopropanation appears to raise the phase transition of the membrane, contributing to its structural integrity. Barry and co-workers have located the genes in M. tuberculosis that are responsible for the cyclopropanation of the distal (cma1) and proximal double bond (cma2) to form the dicyclopropanated ‘·’ mycolic acids.70 These genes appear restricted to pathogenic mycobacteria. Coexpression of both cma1 and cma2 in nonpathogenic M.smegmatis resulted in cyclopropanation of both its mycolic acid’s double bonds, producing a biscyclopropanated fatty acid similar to those produced by M. tuberculosis. Barry and co-workers found that a distal double bond in the major mycolic acid from M. smegmatis was cyclopropanated when he transformed a cosmid containing cma1 from M. tuberculosis to M. smegmatis that encoded for a protein with homology to the E. coli cyclopropane fatty acid synthase.71 The modified M.smegmatis now had considerably increased resistance to hydrogen peroxide, and would thus be more resistant to the action of macrophages that produce reactive oxygen species to cross-link and degrade double bond containing mycolic acids. A plausible biosynthesis of 6 could be the assembly of (5Z)-tetracos-5-enoyl ACP 5 by either the action of a putative 5Z desaturase or HDDH (vide supra), followed by isoniazid sensitive elongation to (13Z)-ditriacont- 13-enoyl ACP 7, cyclopropanation by Cma2 followed by further elongation, desaturation and cyclopropanation by Cma1 to give the meromycolic acid 19:20,31:32- dimethanopentacontanoyl ACP 8. A Claisen like condensation with tetracosanoyl CoA (or docosylmalonyl CoA) would give 6.ORFs close to cma1 and cma2 were found to have homology with actIII (a KR from S. cinnamonensis) and with the HDDH gene from Candida tropicalis. M. tuberculosis, in addition to ·-mycolates, also produces lesser quantities of methoxymycolates in which a methoxy group (adjacent to a methyl group) replaces the distal cyclopropane of the ·-mycolates.Barry and co-workers have cloned and sequenced a cluster of genes (mma1-4) coding for four highly homologous methyl transferases (Mma1-4). When the cluster was transformed into M. smegmatis, it conferred the ability to produce methoxymycolates from the diunsaturated fatty acids present.72 Expression of each gene individually showed that Mma4 was responsible for the SAM dependent conversion of the distal Z alkene into a secondary alcohol with a vicinal methyl branch; Mma3 O-methylated this alcohol; Mma2 cyclopropanates the proximal double bond, and is thought to prefer cyclopropanating oxygenated precursor fatty acids, whilst Cma2 (from cma2, vide supra) prefers a cyclopropanated precursor fatty acid.Expression of Mma1 in M. smegmatis gave no observable phenotype. Barry and co-workers propose that each of these Mma mediated reactions proceeds via a common cationic intermediate that can also account for mycolic acids possessing E double bonds adjacent to a methyl branch (Scheme 5).They also propose a sequence for the assembly of methoxylated and cyclopropanated mycolic acids (Scheme 6). 2.2.3 Bacillus Cronan and co-workers have obtained and sequenced a gene cluster responsible for FAS in Bacillus subtilis that encodes for a PlsX protein (of unknown function, but complements E.coli plsX mutant); a thermosensitive malonyl CoA ACP transacylase ( fabD); KR ( fabG); and part of the ACP (acpP).73 There was no gene coding for KAS III ( fabH) which is located between plsX and fabD on the E. coli genome and would assemble straight chain butanoyl ACP (over 90% of fatty acids in B. subtilis are branched and the corresponding primers are formed from branched chain amino acids). This is the first report of PlsX in Gram positive bacteria.Cronan was unable to isolate intact or overexpress the bacillus acpP gene in E. coli, but has isolated, purified and sequenced the ACP from B. subtilis. It had high homology (>60%) with E. coli ACP, was phosphopantetheinylated by E. coli ACPS, but was unusual in that most of the protein retained the initiating methionine residue. The biosynthesis of biotin in bacillus has been examined. Ploux and Marquet have improved the overexpression in E. coli of 8-amino-7-oxononanoate synthase from B.sphaericus, N CONHNH2 4 Rawlings: Biosynthesis of fatty acids and related metabolites 281which catalyses the pyridoxal phosphate (PyP) dependent condensation of L-alanine with heptane-1,7-dioic acid mono- CoA (pimeloyl CoA). They showed that the mechanism first involved abstraction of the alanine C-2 proton from the alanine-PyP aldimine. This carbanion then attacked the thioester, followed by decarboxylation, in common with other 2-oxoamine synthases such as 5-aminolevulinate synthase.74 Pero and co-workers have cloned, sequenced and characterised a cluster of genes involved in biotin synthesis from B.subtilis.75 2.2.4 Streptomycetes Streptomyces coelicolor A3(2) produces two aromatic polyketides: the blue pigment actinorhodin and a grey spore pigment of unknown structure, for both of which PKS gene clusters have been located. Revill et al. have located a third Me Me Me Me Me Me Me COSCoA Me Elongase ER 24 OH 16:0 ACP O 32 CO2H 26 Me 26 5 7 7 2 13 19 31 50 24 21 33 52 1 2 3 22¢ 22:1 (2 E) 20:0 5 Z Desaturase ? 'HDDH' (Isoniazid) 22:1 (3 Z) 24:0 (CO2H)? Elongase Cyclopropanation ( cma2) Elongation Desaturation Cyclopropanation ( cma1) Claisen condensation ACP O ACP O ACP O ACP O ACP O 32 13 1¢ 6 5 7 8 Scheme 4 Me Me OH Me + ‡ Scheme 5 282 Natural Product Reports, 1998ACP, presumed to be involved in fatty acid biosynthesis, whose gene has been tentatively labelled acpP, which was near to fabD (MT) and fabH (KAS III),76 and whose protein sequence was more similar to the E.coli FAS ACP (AcpP) than to S. coelicolor PKS ACP. The S. coelicolor ACP (acpP) was overexpressed in E. coli and isolated as the acylated holo-form, strongly suggesting that it is indeed part of a FAS. Overexpression of either actinorhodin PKS ACP or grey pigment PKS ACP in E. coli did not result in acylated holo-ACP.77 Revill et al. substituted acpP for actI in S. coelicolor that codes for the Type II PKS ACP involved in actinorhodin biosynthesis, and found that actinorhodin formation was now extremely slow, suggesting that these FAS and PKS ACPs are not interchangeable.76 As both pigments are only biosynthesised at specific times during the life of the organism, this non-interchangeability would prevent the FAS and PKS proteins from interfering with each other.However, Revill et al. have found that the S. coelicolor MT (FabD) may be responsible for charging both the FAS ACP (AcpP) and both the PKS ACPs, and suggests that this MT provides a link between fatty acid biosynthesis and polyketide biosynthesis in the same organism.78 Hutchinson and co-workers have sequenced the fab gene cluster in the tetracenomycin C (Tcm) producing S.glaucesens and found the following order of activities: fabD (MT), fabH (KAS III), fabC (ACP) and fabB (KAS I).79 The ACP (FabC) was overexpressed in E. coli and found to be entirely in the holo-form. This, along with the similarity of the cluster with that for E. coli, strongly implies that this is indeed the FAS cluster.However, MT (FabD) has been shown to transfer malonate to E. coli ACP (AcpP), S. glaucesens ACP (FabC) and the TcmM ACP (part of the Type II tetracenomycin PKS).80 This implies that the S. glaucesens MT (FabD) transfers malonate in both fatty acid and polyketide biosynthesis, as for S. coelicolor (vide supra). It is also interesting that the S. glaucesens FAS and PKS gene clusters have very diVerent organisations, whilst the organisation of aromatic PKS gene clusters in diVerent streptomycetes is very similar, strongly suggestive of a single origin for the PKS gene cluster, followed by horizontal transmission amongst the streptomycetes. Sherman and co-workers have compared KAS sequences from the PKS gene clusters from a wide variety of streptomycetes, the E.coli FabB KAS I and the rat FAS KAS domain. 81 The PKS KAS sequences have a high homology to each other, with considerable divergence from the E.coli FabB KAS. The two FAS sequences are highly divergent in comparison. The authors report that insertion of a plasmid carrying E. coli fabB (KAS I) was able to complement an S. coelicolor actI ORF 1 KAS deficient mutant under certain conditions. Hopwood and co-workers have reported that Saccharopolyspora erythraea FAS ACP can complement S. coelicolor actinorhodin ACP deficient mutant,82 again demonstrating that certain FAS proteins can substitute for certain PKS proteins.The streptomycetes and related bacteria (especially Bacillus spp.) assemble fatty acids using a range of starter units resulting in a mixture of straight chain, iso- and anteiso-fatty acids such as 13-methylpentadecanoate 9.83 The unusual branched starter units are obtained from the branched amino acids L-valine 10, L-leucine 11 and L-isoleucine 12 via their corresponding catabolites 2-methylpropanoyl CoA 13, 3-methylbutanoyl CoA 14 and 2-methylbutanoyl CoA 15.Deuteriated 2-methylpropanoyl CoA has been specifically incorporated into 14-methylpentadecanoate (isopalmitate) 16 from S. fradiae.84 However, specific deuterium labelling was also observed in hexadecanoate (palmitate) 17 suggesting that 2-methylpropanoyl CoA was being isomerised to butanoyl CoA 18 as previously proposed by Robinson and co-workers in S. cinnamonensis.85 Reynolds and co-workers have shown that the straight chain fatty acids can be assembled in S.collinus, S. cinnamonensis and Saccharopolyspora erythraea using butanoate as a starter unit,86 as has previously been demonstrated in bacillus.87 Reynolds and co-workers have also incorporated hexanoate, but largely after degradation to butanoyl CoA.86 L-Valine has been shown to be a source of butanoyl CoA building block, via the B12-mediated isomerisation of 2-methylpropanoyl CoA to butanoyl CoA, however Reynolds and co-workers have found that only ca. 14% of the butanoyl CoA is derived from valine via 2-methylpropanoyl CoA. An alternative source of butanoyl CoA starter units is the condensation of two molecules of acetyl CoA, as has previously been observed in streptomycete secondary metabolite biosynthesis, mammalian mammary glands88 and Euglena gracilis.89 Reynolds and co-workers have purified an NADPH dependent (2E)-but-2-enoyl CoA reductase (Ccr) from S. collinus, overexpressed it in E. coli and found evidence for other ccr genes in S.cinnamonensis, S. lividans and S. hygroscopicus.90 The enzyme was highly specific, not reacting with prop-2-enoyl CoA, (2E)-pent-2-enoyl CoA, (2E)-but-2- enoyl NAC thioester or pantetheine thioester, and was strongly inhibited by long straight chain CoA thioesters, but far less so by branched long chain thioesters. The sequence was compared with that of cyclohex-1-enylcarbonyl CoA reductase also from S. collinus that is involved in assembling the cyclohexanylcarbonyl CoA starter for assembly of the polyketide ansatrienin,1 and no homology was observed, consistent with the suggestion that many secondary metabolic enzymes have travelled horizontally between the streptomycetes, rather than evolved within a species alongside their corresponding FAS enzymes.The observations of Reynolds and co-workers are consistent with Scheme 7. Robinson and co-workers have examined the reversible interconversion of 2-methylpropanoyl CoA and butanoyl CoA by partially purified 2-methylpropanoyl (isobutyryl) mutase of S.cinnamonensis.91 In this reaction, there is a vicinal interchange of the C-1 carbonyl group and hydrogen group (Scheme 8). Robinson and co-workers, using gradient enhanced inverse detected heteronuclear 2D 1H-13C correlation NMR spectroscopy, observed that (2S)-[3-13C]-2- methylpropanoyl CoA 19 was predominantly converted into [2-13C]butanoyl CoA 20, whilst (2R)-[3-13C]-2-methylpropanoyl CoA 21 was predominantly converted into [4-13C]butanoyl CoA 22.However, there was some isotopomeric products being formed, at less than ten-fold rate (Scheme 9). [2-2H]-2-Methylpropanoyl CoA 23 was incubated for several hours with enzyme to give a 60:40 mixture of (3R)-[3-2H1]butanoyl CoA 24 and (3S)-[3-2H1]butanoyl CoA 25 (Scheme 10). However, the high proportion of unexpected (3S)-isotopomer 25 may be due to a kinetic isotope eVect aVecting the reverse reaction, discriminating against the conversion of the (3S)-isomer back to 23.A comparison is made with methylmalonyl CoA mutase, previously examined by Rétey and co-workers.92 COSCoA Me COSCoA Me Me OH COSCoA Me Me OMe COSCoA Me Me OMe MMAS-4 MMAS-3 MMAS-2 Scheme 6 Rawlings: Biosynthesis of fatty acids and related metabolites 283The structure of a polycyclopropanated compound isolated from Streptomyces sp. UC 11136 that inhibited cholesteryl ester transfer protein has been shown by Kuo et al.to be N-(2-methylpropanyl)-(2E,14E)-all trans-4,5:6,7:8,9:10,11:12, 13:16,17-hexamethanooctadeca-2,14-dienamide 26, though the full stereostructure remains unclear.93 The backbone originates from acetate and the extra cyclopropyl carbons originate from methionine, and a biosynthetic scheme from the linear polyene (2E,4E,6E,8E,10E,12E,14E,16E)-octadeca-2,4,6,8,10,12,14,16- octaenoic acid 27 was proposed (Scheme 11). Me COSCoA Me COSCoA O Me COSCoA OH Me COSCoA Me COSCoA Me COSCoA Me Me Me COSCoA O Me Me NH2 CO2H CO2H Me Me COSCoA Me Me COSCoA Me Me COSCoA Me NH2 CO2H Me Me Me COSCoA Me NH2 CO2H Me COSCoA Me Me Me Me Me Me COSCoA Me Me Me COSCoA CO2H SACP O 4 6 (Exogenous) L-Valine L-Leucine L-Isoleucine Hexadecanoate 14-Methylpentandecanoate 13-Methylpentadecanoate (isopalmitate) (palmitate) 2-Methylpropanoyl mutase B12 (Exogenous) (anteisopalmitate) Crotonyl CoA reductase 13-Methyltetradecanoate (Isopentadecanoate) 10 11 12 13 (major route) 14 15 16 17 18 9 Scheme 7 H* SCoA O Me Me H* SCoA O Me H H H mutase Scheme 8 H3 12C COSCoA 13CH3 H Me COSCoA H3 13C COSCoA 12CH3 H Me COSCoA 2-methylpropanoyl mutase • 2-methylpropanoyl mutase • slow slow 19 20 • • 21 22 Scheme 9 Me COSCoA Me D Me COSCoA Me COSCoA D H D H R S Ratio of 24:25 = 60 : 40 mutase 24 25 23 NH O (one possible stereoisomer) 26 Scheme 11 Scheme 10 284 Natural Product Reports, 1998A review on fluorinated natural products has appeared.94 Bacteria or fungi that can utilise fluoroacetate as a source of acetate contain enzymes that convert fluoroacetate to hydroxyacetate.95 Harper and co-workers have examined the biosynthesis of fluoroacetate and 4-fluorothreonine by S.cattleya NRRL 8057, and suggest that hydroxyacetate is the precursor of fluoroacetate.96 2.2.5 Other Gram positive bacteria The thermoacidophilic Alicyclobacillus genus produce ¢-cycloalkylated fatty acids presumably to increase the density of the cell membrane as they live in hot acidic environments.The membrane of Alicyclobacillus heptanicus has three major components after saponification: 11-cycloheptanylundecanoic acid 28, 13-cycloheptanyltridecanoic acid 29 and 11- cycloheptanyl-2-hydroxyundecanoic acid 30. [1-13C]Cycloheptanecarboxylic acid 31 was incorporated into 28 and 30 suggesting that 31 (presumably as the CoA thioester), was the ‘starter unit’. Moore and Floss have identified three minor lipid components by MS as 9-cycloheptanylnonanoic acid 32, 10-cycloheptanyldecanoate 33, and 13-cycloheptanyl-2- hydroxytridecanoate 34.97 Addition of 13C-labelled 28 or 30 to growing whole cell cultures of A.heptanicus gave labelled 33 but no label in 29. Floss suggests that 28 is ·-hydroxylated to 30, which is then subject to dehydrogenation and oxidative decarboxylation to give 33. A similar sequence has been found in the protozoan Tetrahymena pyriformis where 2-hydroxyhexadecanoic acid is converted to pentadecanoic acid.98 The absence of 13C-label in 29 suggests that the ·-dehydrogenation and oxidative decarboxylation of labelled 28 is eYcient and occurs at the free acid level, rather than as a thioester in which case chain extension of 28 to 29 would also be observed (Scheme 12). 3 Archaea Corcelli et al. have examined the acylation of halorhodopsin in Halobacterium halobium and shows the acylation to be highly specific, rather than a random association during solubilisation, and that bis-hexadecanoylation strongly aVected photoreactivity.99 4 Protoctista This kingdom can be defined by exclusion, containing all eukaryotic microorganisms not included in animals (those that develop from a blastula), plants (that develop from an embryo) or fungi (that develop from spores and lack undulipodia); some phylum of the protoctista have traditionally been classi- fied as ‘single celled animals’ or ‘fungi’.This new enlarged ‘kingdom’ has been recently recommended by Margulis and Schwartz5 as one of ‘five kingdoms’ (though the archaea now need to be added as a sixth). This avoids having to classify large organisms such as giant kelp and seaweed or those that can be multicellular amongst ‘protists’ which are associated with ‘single-celled’ organisms.Also included are the oomycetes and myxomycetes (traditionally classified with fungi). The marine brown algae such as Ectocarpus siliculosus (laminariales: phaeophyta) use cyclohepta-1,4-dienes as pheromones, and it has been proposed that they are assembled from the spontaneous Cope rearrangements of divinylcyclopropanes. 100 Boland and co-workers have recently shown that female gametes of brown algae use C20 fatty acids as precursors to these pheromones,101 and propose a biosynthesis of ectocarpene 35 from (5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11, 14,17-pentaenoic acid 36, via (5Z,7E,11Z,14Z,17Z)-9- hydroperoxyicosa-5,7,11,14,17-pentaenoic acid 37, in which (5Z,7E)-9-oxonona-5,7-dienoic acid 38 is a by-product (Scheme 13).102 Boland and co-workers examined the activation energies of these Cope rearrangements at ambient Mediterranean or Arctic temperatures, and found half-lives of ca. 20 min at Mediterranean temperatures, and over 1 h at Arctic temperatures, and suggests that it may be the immediate precursor (3R,4S,5E,7Z)-3,4-methanodeca-1,5,7-triene 39 that is the actual pheromone, not the cyclised product 35. To examine this possibility, a thermostable analogue of 39, (3R,4S,5E,7Z)-3,4-methano-10-oxaundeca-1,5,7-triene 40, was synthesised and found to have high biological activity. Whilst land based pheromones can be transient due to their volatility, this slow degradation through a Cope rearrangement appears an eloquent method for obtaining transient character in a marine environment.Graber and Gerwick have proposed a biosynthesis for agardhilactone 41, an oxylipid isolated from the marine red alga Agardhiella subulata, involving the 8-lipoxygenation CO2H CO2H CO2H CO2H CO2H OH OH CO2H CO2H MINOR 4 x Acetate 5 x Acetate 6 x Acetate • • • • • MAJOR MAJOR MAJOR Efficient dehydrogenation Oxidative decarboxylation MINOR MINOR a-Hydroxylation 28 31 29 30 33 34 32 Scheme 12 Rawlings: Biosynthesis of fatty acids and related metabolites 285of 36 to form (5Z,8S,9E,11Z,14Z,17Z)-8-hydroperoxyicosa- 5,9,11,14,17-pentaenoic acid 42 (Scheme 14).103 The (¢"3) hydroxy group in 41 may be introduced by (¢"3) lipoxygenation.The eukaryotic algae can produce a wide variety of C20- and C22-polyunsaturated fatty acids (which can be further metabolised to ‘oxylipins’ and icosanoids), whilst the prokaryotic cyanobacteria (blue-green algae) are restricted to C14 to C18. A novel heptaenoic acid, (4Z,7Z,9E,11E,13Z,16Z,19Z)-docosa- 4,7,9,11,13,16,19-heptaenoic acid (stellaheptaenoic acid) 43 has been isolated by Gerwick and co-workers from the marine green alga Anadyomene stellata. A chloroplast preparation produced increased levels of 43 when 22:6 (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid 44 or 22:4 (7Z,10Z,13Z,16Z)-docosa-7,10,13,16- tetraenoic acid 45 was added (Scheme 15).104 This interesting conversion of a Z double bond into a conjugated E,E-diene resembles the reverse of the 2,4-dienoyl CoA reductase discussed in the biosynthesis of animal long chain polyunsaturated fatty acids or the ‚-oxidation of (5Z)-fatty acids (Section 7.4). The authors report six fatty acids were converted by the chloroplasts into various tetraene conjugated fatty acids.Polyunsaturated fatty acids such as 20:5 (n"3) 36 are currently being examined pharmacologically. For economic production, comprehensive understanding of the biosynthetic pathways will be required to employ genetic methodology. Cohen and co-workers added exogenous fatty acids to the microalga Porphyridium cruentrum to increase levels of intermediates along the pathway to 20:5 (n"3).Cohen found that the main pathway was from (9Z,12Z)-octadeca-9,12-dienoic acid [18:2 (n"6), linoleic acid] as shown in Scheme 16 via the so-called ‘n"6’ pathway.105 Intermediates along the ‘n"3’ pathway, such as 18:4 (n"3) were observed to inhibit the f6 desaturase, and could regulate the production of C20 and C22 fatty acids. 5 Plants Harwood has reviewed recent advances in the biosynthesis of plant fatty acids,106 and Ohlrogge and Browse have reviewed lipid biosynthesis in plants.107 Me CO2H Me CO2H O OH H Me OHC CO2H Me 5 8 11 14 17 20 17 14 11 20 5 7 Enzyme 39 38 5 7 35 36 + Cope rearrangement 37 8-Lipoxygenase Scheme 13 O Me 40 10 3 4 Me CO2H Me OOH CO2H HO2C Me O 5 O O Me OH 8 11 O H 14 17 20 36 H 11 14 17 20 8 + 41 42 5 Scheme 14 Me Chloroplast Chloroplast 22 CO2H 19 16 13 10 7 4 Me 16 13 CO2H 10 Me 22 7 22 19 16 CO2H 13 4 7 9 11 43 44 45 Scheme 15 18:2 ( n–6) 18:3 ( n–6) 20:3 ( n–6) 20:4 ( n–6) 18:3 ( n–3) 18:4 ( n–3) 20:4 ( n–3) 20:5 ( n–3) (' n–6 pathway') (' n–3 pathway') D6 Desaturase D5 Desaturase D6 Desaturase Elongation Elongation (w-3) Desaturase 36 D5 Desaturase Scheme 16 286 Natural Product Reports, 1998Sparace and co-workers have found that pea (Pisum sativum) root plastids are able to convert glucose, pyruvate, glucose-6-phosphate, malate or acetate into fatty acids, pyruvate being the preferred precursor.Thus the entire pathway, from glucose through to glycerolipids is operating in pea root plastids.108 5.1 Acetyl CoA carboxylase (ACC) In plants, the major site of fatty acid synthesis is in the prokaryotic like plastids, there is no evidence of de novo fatty acid synthesis in the cytosol.The C16-fatty acids are then exported to the eukaryotic like cytosol for elongation by malonyl CoA. Thus malonyl CoA is required in both the plastids and cytosol. Sasaki et al. reviews current knowledge of plant acetyl CoA carboxylases (ACCs).109 In this review ACCs comprising of separate activities on separate polypeptide chains will be classed as ‘Type II ACC’, and those with activities on a single multifunctional polypeptide ‘Type I ACC’, by analogy with ‘FAS’ systems.Early attempts to isolate plant ACCs suggested that they were comprised of separate proteins (biotin carboxylase, biotin and two carboxyl transferase components), similar to prokaryotic systems.110 However, more recent attempts have only been able to isolate ACC as a multifunctional single peptide, the earlier separate proteins found in chloroplasts presumed to be degradation products.However, complete chloroplast genome sequencing has located a gene similar to the E. coli accD gene (transcarboxylase ‚ subunit) in all plants so far examined except those of the gramineae (grasses, wheat, barley, etc.). No homologues of other E. coli ACC genes have been identified in the sequenced cloroplast genomes. Thus, in many plants, these three genes (accA,B,C) may have been transferred to the nuclear genome, and in the gramineae, some nuclear DNA may have been transferred, resulting in fatty acid synthesis with a combination of prokaryotic and eukaryotic characteristics. In the cytosol, ‘eukaryotic’ ACC is abundant, especially in tissues where flavanoids and cuticular waxes are formed.Interestingly, ACC activity increases with UV radiation, presumably to regulate production of the UV protective compounds such as the flavanoids.Dicotyledons have a prokaryotic plastidial ACC and an extraplastidial eukaryotic multifunctional ACC. The graminae only have the multifunctional enzyme type, in both the plastids and elsewhere.111 Understanding the developmentally regulated synthesis of storage lipids in seed tissue of oil seeds is of considerable commercial importance. A seed expressed ACP from Brassica compestris has been sequenced and cloned.112 Transcripts from the ACPSF1 gene could be detected in developing seeds, but not in leaves. 5.2 3-Oxoacyl ACP synthase (KAS) Cerulenin is a potent inhibitor of de novo synthesis of fatty acids by inhibiting most KAS I.Iwasaki and co-workers have found that the C18 homologue of cerulenin (cerulenin 18)1 had little inhibitory eVect on yeast FAS, presumably because the KAS active site could not accommodate the extended side chain.113 Schneider and Cassagne have found that whilst cerulenin 18 had no eVect upon de novo fatty acid synthesis in leek seedlings (Allium porrum) it strongly inhibited the synthesis of very long chain fatty acids, presumably by the specific inhibition of KAS components of elongases known to be present in leek seedling endomembranes.114 KAS III catalyses the condensation between acetyl CoA and malonyl ACP to form 3-oxobutanoyl ACP.Spener and co-workers have found that acyl ACPs, in particular, decanoyl ACP, inhibit the activity of KAS III from seeds of Cuphea lanceolata, that normally produces triglycerides comprising mostly of decanoate.115 Inhibition by decanoyl ACP was found to be non-competitive with relation to malonyl ACP, and uncompetitive with relation to acetyl CoA.Thus the formation of medium chain fatty acids is not only dependent upon the presence of a medium chain thioesterase, but also on feed-back regulation of KAS III. The E. coli KAS III (FabH) has been overexpressed in Brassica napus seeds (oil seed rape).22 The elevated KAS III levels resulted in an increase in tetradecanoate levels, and a large decrease in (9Z)-octadec-9- enoate levels in lipids. 5.3 Enoyl reductase (ER) RaVerty and Rice have obtained the X-ray crystal structure of the ER in Brassica napus seeds (oil seed rape).116 5.4 Desaturases Plant desaturases are soluble non-membrane bound enzymes found in the plastids that introduce a double bond into a saturated acyl ACP. Desaturases in the animals and fungi, and subsequent desaturations in plants are mediated by membrane bound proteins.Both soluble and membrane bound ƒ9 desaturases contain a non-haem di-iron oxo cluster. Fox et al. have classified the homodimeric octadecanoyl 9Z desaturase from castor oil plant (Ricinus communis) as Class II (characterised by two EXXH sequences) based upon sequence homology with ribonucleotide reductases, bacterial hydrocarbon hydroxylases (e.g. methane monooxygenase) and ruberythrin.117 Mössbauer and resonance Raman spectroscopy demonstrates the presence of Ï-oxo bridging in the oxidised diferric state with an Fe–O–Fe angle of 123).The di-iron centres have no intersubunit interaction, being separated by over 2.3 nm. Lindqvist et al. have obtained the crystal structure of the a octadecanoyl ACP 9Z desaturase from castor seed at 0.24 nm resolution, thought to be in its reduced form (diferrous) due to photochemical reduction due to the X-ray irradiation leading to loss of Ï-oxo bridge and ligand rearrangement.118 The dimeric protein has 363 aa per subunit, eleven ·-helices per subunit, and a pair of iron atoms per subunit separated by 0.42 nm.The iron atoms have a distorted octahedral coordination geometry with one ligand unoccupied. E143 (Asp-143) and E229 act as bridging ligands, E105 is a bidentate ligand to one iron and E196 is a bidentate ligand to the second iron. A nitrogen from H146 ligates to one iron, and from H232 to the second. This is thought to be the reduced diferrous form of the enzyme that would result from in vivo interaction with ferredoxin.The oxidised version may have Ï-oxo bridging, with a closer Fe–Fe distance. The di-iron centre is buried in the interior of the protein, and there is a very deep narrow channel extends from the surface into the protein and past the di-iron centre. Interestingly, the channel is bent at the di-iron cluster, which would favour binding of the bent ‘Z’ product, (9Z)- octadec-9-enoyl ACP (Scheme 17). Insertion of octadecanoic acid into this pocket places C-9 carbon about 0.55 nm from one of the iron atoms.Presumably, in the active enzyme, this gap will be occupied by a dioxygen molecule bound to one or both iron atoms. This gap may initially be filled by a water molecule that provides the O atom to bridge the two Fe atoms. A possible overall sequence is summarised in Scheme 17. Ricinoleic acid, (9Z,12R)-12-hydroxyoctadec-9-enoic acid is formed by the direct hydroxylation of (9Z)-octadec-9-enoic acid, the labile, NADH and dioxygen requiring, endoplasmic reticulum associated, 12-hydroxylase has not been purified. Somerville and co-workers have obtained a gene from Ricinus communis coding for the 12-hydroxylase (RMM 44.4 kDa) that has a 67% homology with the microsomal (."6)- desaturase from Arabidopsis, suggesting that both enzymes have a common ancestor.119 Cahoon et al.have overexpressed hexadecanoyl ACP ƒ6 desaturase from Thunbergia alata (black eyed Susan vine) in E.coli and obtained ƒ6-hexadecenoic acid and ƒ8-octadecenoic acid. Coexpression of a plant ferredoxin doubled the production of ƒ6-hexadecenoic acid. This suggests that the plant aerobic acyl ACP desaturation system can be superimposed on the anaerobic pathway in E. coli.120 Rawlings: Biosynthesis of fatty acids and related metabolites 287Pest (e.g. aphids, spider mites) resistance in the geranium (Pelargonium xhortorum) is linked with high levels of anacardic acids possessing (¢"5)-unsaturation in the glandular trichome exudate.This unsaturation allows the geranium’s sticky exudate to adhere to the insects exoskeleton, enhancing the pests exposure to the anacardic acids which aVect enzymatic steps in the pest’s reproduction. Hexadecanoic acid 46 and octadecanoic acid 47 have been shown to be precursors of the saturated C22 and C24 anacardic acids 48 and 49, respectively. 121 Tetradecanoic acid 50 was also incorporated into the unsaturated (¢"5)-anacardic acids 51 and 52, suggesting the presence of a f5 desaturase, rather than desaturation of saturated anacardic acids 48 and 49.Schultz et al. show that expression of tetradecanoyl ACP f9 desaturase is required for production of (¢"5)-anacardic acids and for acquisition of the pest resistant phenotype. Thus desaturation of tetradecanoate 50 to (5Z)-tetradec-5-enoyl ACP 53 followed by fatty acid elongation and polyketide extension/aromatisation results in 51 and 52 (Scheme 18).122 5.5 Thioesterase (TE) Acyl ACP thioesterases have a chain length determining role in de novo fatty acid synthesis, converting an ACP thioester to the corresponding free acid.When 12:0 ACP TE (Uc FatB1) from the Californian bay tree (Umbellularia californica: Lauraceae) is expressed in the developing oil seed of arabidopsis, the bay enzyme redirects the arabidopsis FAS from long chain to 12:0 production.123,124 Plant engineering, such as the high level expression of Uc FatB1 in Brassica oil seeds to enable the commercial production of ‘Laurate Canola’ has been reviewed.125 Ohlrogge and co-workers have overexpressed in E.coli a long chain thioesterase from Arabidopsis thaliana that hydrolyses 14:0, 16:0, 16:1 (9Z) and 18:1 (11Z).126 Voelker and co-workers have found a 16:0 ACP TE from Cuphea hookeriana (Lythraceae) that had sequence similarity to medium chain acyl ACP TE, and based upon sequence homologies, suggests two families, each derived from one of two gene classes, FatA that encodes 18:1 ACP TE, and FatB encoding saturated acyl ACP TEs.127 Voelker and co-workers propose that medium chain acyl ACP TEs have evolved O Fe O O Fe O O O O O N N N N Me COS-ACP H H O Fe O O Fe O O O O O N N N N O O• O O Fe O O Fe O O O O O N N N N O E143 E105 E229 H146 E196 H232 O2 E196 E229 9 10 18 E105 H146 1 0.42 nm Bent hydrophobic pocket Gap for dioxygen binding 3+ 3+ H232 E196 E229 E105 H146 E143 3+ 3+ –CH2CH2– Ferredoxin –CH=CH– + 2H2O H2O H232 E143 Scheme 17 Me Me CO2H CO2H Me Me OH OH HO2C HO2C Me CO2H COS-ACP Me OH OH HO2C HO2C Me Me 16 18 14 14 14:0-ACP D9 Desaturase Pest susceptible plants 49 48 51 52 53 50 46 47 Pest susceptible plants Pest resistant plants Scheme 18 288 Natural Product Reports, 1998independently several times from 16:0 ACP TE, that is ubiquitous in higher plants.127 For example, the genus Cuphea is the only genus amongst the lythraceae that produces medium chain fatty acids, and the medium chain TE must have evolved from the corresponding 16:0 ACP TE within the last seven million years, the fossil record ‘lifetime’ of the Cuphea.Voelker extensively discusses the evolutionary origin of plant TEs. The substrate specificity of a plant TE has been modified by protein engineering. When 12:0 ACP TE (Uc FatB1) from the Californian bay tree (U. californica) is expressed in E. coli, Brassica napus or Arabidopsis thaliana, dodecanoate and only a small amount of tetradecanoate is accumulated.A TE (Cc FatB1) from camphor (Cinnamomum camphorum) which mainly hydrolyses tetradecanoyl ACP when expressed in E. coli, has 92% sequence homology with the 12:0 producing bay tree TE. Using the Cc FatB1 sequence as a guide, Yuan et al. changed several amino acids in Uc FatB1 using site specific mutations.128 A double mutant (M197R/R199H) changes the Uc FatB1 so that it produces equal amounts of 12:0 and 14:0. A triple mutant (M197R/R199H/T231K) changes the Uc FatB1 into exclusively a 14:0 ACP TE.Plant acyl ACP thioesterases employ a Cys residue in contrast to most bacterial and mammalian TEs which have a Ser in the active site. Yuan et al. have substituted conserved Cys and His in 12:0 ACP TE (Uc FatB1) by alanine scanning mutagenises and found that only substitution of C320A or H285A completely inactivated the enzyme, identifying these as the two crucial residues in the active site.129 The serine analogue, the C320S mutant retained 60% activity. Whilst a crystal structure from the 14:0 ACP TE from the bioluminescent bacterium Vibrio harveyi has been obtained,130 and a typical catalytic triad (Ser–His–Asp) obtained no such structure is yet available for a plant acyl ACP TE. Plant TEs are highly conserved, even between FatA and FatB classes, and Yuan proposes the two active site motifs NQ(K)HN(S)N and YRR(K)ECG(Q/T).However, the third component of the catalytic triad is not yet clear.The seed oil of Cuphea palustris has a bimodal acyl chain length distribution, comprising mainly of tetradecanoate and octanoate. Dehesh et al. have isolated two genes Cp FatB1 and Cp FatB2 which when expressed in E. coli produce 8:0/10:0 ACP and 14:0/16:0 ACP, respectively.131 As the triglycerides contain both 8:0 and 14:0, both acyl ACP TEs must be produced at the same time in the same cells, suggesting that both TEs act together in the same FAS system.Liu and Post-Beittenmiller have purified an 18:0 ACP TE from leek epidermal extracts.132 The enzyme was ten-fold less reactive with 16:0 ACP or 18:1 ACP or 18:0 CoA. Leek epidermal tissue is solely responsible for producing epicuticular (surface) wax, which is mainly a C31-ketone assembled from elongation of 18:0 CoA, and can comprise 15% of total leaf lipid. 5.6 Polyhydroxyalkanoates Bacteria such as Alcaligenes eutrophus produce poly-(3R)- hydroxybutanoate (PHB) from acetyl CoA using a 3-oxothiolase (PhbA), an NADPH dependent 3-oxobutanoyl CoA reductase (PhbB), and polyhydroxyacid synthase (PhbC).Somerville and co-workers have transformed each of these genes into Arabidopsis thaliana (cruciferae, ‘Thale cress’) and the plants accumulated PHB up to 14% by dry weight as granules within the plastids, with no deleterious eVect on growth.133 John and Keller have transformed phbB and phbC into the cotton plant (Gossypium hirsutum) by particle bombardment, and found that the resulting cotton fibre contained PHB, giving the cotton fibre improved insulating characteristics with a higher heat capacity.134 5.7 Thienes and furans Croes and co-workers have examined the biosynthesis of bithienyls in hairy root cultures of French marigold (Tagetes patula: asteraceae), in particular, the biosynthesis of 5-(but- 3-en-1-ynyl)-2,2*-bithienyl 59, 5-(but-3-en-1-ynyl)-5*-methyl- 2,2*-bithienyl 56, 5-(but-3-en-1-ynyl)-5*-hydroxymethyl- 2,2*-bithienyl 57, 5-(acetoxymethyl)-5*-(but-3-en-1-ynyl)-2,2*- bithienyl 58, 5-(4-hydroxybut-1-ynyl)-2,2*-bithienyl 60, 5-(4- acetoxybut-1-ynyl)-2,2*-bithienyl 61, 5-(3,4-dihydroxybut-1- ynyl)-2,2*-bithienyl 62 and 5-(3,4-diacetoxybut-1-ynyl)-2,2*- bithienyl 63.135 Bohlmann and Hinz suggested that they all arose via trideca-3,5,7,9,11-pentayn-1-ene 54.136 Addition of sulfur to the 5,7-diyne moiety gives the monothiophene 2-(but- 3-en-1-ynyl)-5-(penta-1,3-diynyl)thiophene 55, which has been isolated from several plants.Further addition of sulfur to the remaining diyne moiety results in 56 which Christensen and Lam have suggested was oxidatively demethylated to 59.137 However, by feeding a series of 35S-labelled intermediates, and examining the fate of the labels, Croes and co-workers could find no evidence for the conversion of the bithienyl 56 to 59, and instead suggests that the demethylation of 55 occurs before the second thienylation.135 There was no evidence for the conversion of 56, 57 or 58 into any of 59, 60, 61, 62 or 63 (Scheme 19).No label from 55 could be found in the ·-terthienyl metabolite 2,2*:5*,2+-terthienyl 64. Croes and co-workers have also examined the elicitation of thiophene production in Tagetes patula by extracts of Fusarium oxysporum.138 Spiteller and co-workers have shown that furan fatty acids (F-acids) from Saccharum spp. are assembled from an acetate derived fatty acid chain, dioxygen derived oxygen and methionine derived methyl groups.139–141 Naturally occurring F-acids possess either a propanyl or pentanyl side chain, and are usually either unmethylated, such as 7-(5-pentylfuran-2- yl)heptanoic acid 65, monomethylated at the ‘3’ position of the furan ring such as 7-(3-methyl-5-pentylfuran-2-yl)heptanoic acid 66, or dimethylated, such as 7-(3,4-dimethyl-5- pentylfuran-2-yl)heptanoic acid 67.To probe whether unmethylated F-acid could act as a substrate for the methyltransferases, Spiteller and co-workers added the unnatural synthetic butyl homologue, 7-(5-butylfuran-2-yl)heptanoic acid 68, to suspension cultures of Saccharum sp. and examined for methylation by mass spectrometry.They unexpectedly obtained the F-acid methylated only at the 4-position, 7-(5- butyl-4-methylfuran-2-yl)heptanoic acid 69, with very little evidence of dimethylation to give 7-(5-butyl-3,4- dimethylfuran-2-yl)heptanoic acid 70 (Scheme 21).142 The authors suggest that the methylation process may be more complicated than first proposed, with one possibility being monomethylation of the aliphatic precursor, or the first methylation being combined with furan formation via a multienzyme complex (reminiscent of SAM methylated polyketides?), such that free 65 is not a precursor of 66 (Scheme 20). 5.8 Hydrocarbons Most plant volatiles are terpenoid in origin, however some are straight chain hydrocarbons.For example, developing peanuts evolve pentane which is derived from the lipoxygenase catalysed degradation of (9Z,12Z)-octadeca-9,12-dienoic acid (linoleic acid).143 The genus Pinus produces short chain (C7–C11) alkanes in oleoresin, a sticky mixture of volatile alkanes or monoterpenes with non-volatile diterpenes, produced as defence against insect or pathogen attack. In Pinus jeVreyi (JeVrey pine) the volatile component is over 95% short chain alkanes.144 These hydrocarbon mixtures have excellent combustion properties and understanding their biosynthesis could lead to bioengineered fermentative organisms that produce renewable hydrocarbon fuel.Savage, Hristova and Croteau have found that the KAS inhibitor cerulenin1 inhibited the production of C18 fatty acids in xylem sections of Pinus jeVreyi but not the conversion of radiolabelled acetyl CoA into C8-acyl groups and heptane 71.145 Longer chain acyl derivatives were not incorporated into 71, suggesting the direct Rawlings: Biosynthesis of fatty acids and related metabolites 289involvement of a C8 thioester rather than the degradation of longer acyl units as in the case of peanut volatiles vide supra.These results also presumably imply that cerulenin has a much greater inhibitory eVect on the C10–C18 fatty acid assembly than the short chain assembly. 2-Sulfanylethanol (‚- mercaptoethanol) inhibited heptane production in the xylem sections, resulting in a build up of octanal 72 and octanol 73.Radiolabelled octanal was converted into octanol and heptane. It is thought that octanol is reversibly formed from octanal as a shunt metabolite. Clarification is still required on the nature of the malonate extension unit, the conversion of octanoyl CoA 74 to octanal 72, and whether 72 is decarbonylated or oxidatively decarboxylated to give heptane (Scheme 22). 5.9 Esters and lactones Schöttler and Boland have investigated the biosynthesis of dodecano-4-lactone 75 in ripening strawberries (Fragaria ananassa) and peaches (Prunus persica).146 Lactones such as 75 are flavour constituents thought to be derived from (9Z)- octadec-9-enoic acid 76 by oxidation of the double bond and three rounds of ‚-oxidation in an unknown sequence (Scheme 23).Addition of the deuterium labelled nor-analogues of precursors [8,8-2H2]-9,10-epoxyheptadecanoic acid 77 and methyl [8,8-2H2]-9,10-dihydroxyheptadecanoate (methyl ester of 78) to ripening fruits resulted in the emission of [2-2H1]undecano-4-lactone 79, with loss of a single deuterium atom.146 The enantiopurity of the product 79 was found to depend upon the precursor added.Addition of the epoxide 77 or its trans-epoxide regioisomer resulted in (4R)-undecano-4- lactone 79 with high ee (ca. 70–90%) However, addition of later intermediates resulted in a product of lower enantiopurity. Addition of 78 gave 79 (60–70% ee) and addition of [2,2-2H2]-3,4-epoxyundecanoic acid 80 gave 79 (35–50% ee), suggesting that the sequence of reactions was epoxide hydrolysis of 77 to 78 followed by three rounds of ‚-oxidation to [2,2-2H2]-3,4-dihydroxyundecanoic acid 81.Lactonisation Me S Me S S S S S S S S S Me S S HO S S AcO OH S S OAc S S OH OH S S OAc OAC 1 13 54 56 57 58 59 60 61 62 63 55 64 Scheme 19 O CO2H Me Me O CO2H 4 Me 1 Me 7 Me 5 O 1 CO2H 7 1 7 Me Acetate, methionine, dioxygen 5 5 ? 65 66 67 Scheme 20 O CO2H Me O CO2H Me Me O CO2H Me Me Me 4 3 7 1 3 7 7 4 4 4 1 68 69 70 1 Scheme 21 290 Natural Product Reports, 1998gives [2,2-2H2]-3-hydroxyundecano-4-lactone 82, loss of HOD gives [2-2H]undec-2-eno-4-lactone 83 and ‘enoyl reduction’ gives the product lactone 79 (Scheme 24).Rowan et al. have investigated the biosynthesis of (2S)- methylbutanoate esters in apples (Malus domesticus).147 These esters occur widely as volatiles emitted from fruit as components of aroma and flavour, and also possibly acting as insect attractants.A wide variety of deuteriated precursors were added, and it was confirmed that (2S)-isoleucine was the precursor of (2S)-methylbutanoate and 2-methylbutanol. Red Delicious apples, but not Granny Smiths, also produced (2E)-2-methylbut-2-enyl esters from (2S)-isoleucine. 6 Fungi Pyruvate (2-oxopropanoate) can be converted to acetyl CoA either directly by the action of pyruvate dehydrogenase (PDH) or, under aerobic, glucose limited conditions, by the so-called ‘PDH bypass’ which involves pyruvate decarboxylase, ethanal dehydrogenase and acetyl CoA synthetase.Two ACS genes code for an active acetyl CoA synthetase protein. Van den Berg and Steensma found that disruption of ACS1 gene (acetyl CoA synthetase 1 gene) caused no major change in phenotype, but disruption of ACS2 caused inability to grow on glucose, but retained ability to grow on ethanol or acetate.148 The authors suggest that ACS1 is glucose repressible, but is induced by ethanol, ethanal or acetate, and that PDH mediated production of acetyl CoA might only be occurring in the mitochondria, where there might be no means of exporting the acetyl CoA to the cytoplasm. The ACS1–ACS2 double mutant was not viable. 6.1 Fatty acid synthase The yeast (Saccharomyces cerevisiae) FAS consists of six copies each of an ·-subunit, containing the KAS, ACP and KR activity) and ‚-subunit (containing the ER, DH, and Me Me Me OH SCoA H O O Me Me Acetate + Malonate 8 8 8 7 Me(CH2)16CO2H Cerulenin insensitive Cerulenin 71 b-Mercaptoethanol 73 72 74 – CO or – CO2 ? Scheme 22 CO2H Me O O Me 18 18 76 Epoxidation, hydrolysis 3 x b-Oxidation 75 Scheme 23 CO2H Me O D D CO2H D D Me OH OH O O Me HO D D O O Me D O O Me D 17 11 17 11 11 77 CO2H Me O 82 78 D D 79 83 11 CO2H D D 3 x b-Oxidation Epoxide hydrolase Me 11 OH OH Epoxide hydrolase 3 x b-Oxidation 81 80 4 Scheme 24 Rawlings: Biosynthesis of fatty acids and related metabolites 291transferase/transacylases), with overall Mr ca. 2.4 MDa. Early electron microscopic investigations suggested a complex of six circular disks in a planar hexagonal arrangement around which six arch-like ‚-subunits are distributed, three on either side in an alternate manner, each linking two adjacent ·-subunits.149 Six moles of enzyme can be assembled per synthase complex, suggesting six equivalent centres of fatty acid assembly.150 Stoops and co-workers have now reported the 2.5 nm resolution structure of this synthase that was computed from electron microscopy of stain images.151 The resulting barrel like structure with 32 point group symmetry has 12 openings of ca. 2 nm diameter, presumably for substrate entry and product exit for each of the six active sites. These funnel shaped openings in the surface lead to a solvent filled cavity lined with 42 active sites. The individual ·-subunits have an ‘H’ shape and are close to the barrel’s equator, related to each other by 180) rotation perpendicular to the equator, so they are arranged in pairs head-to-tail (Fig. 3). A pair of ·-subunits has an ‘N’ shape, and interface on the two-fold axis. Part of each subunit forms part of the central axis of the barrel (that extend from pole-to-pole, Fig. 4) with an interior cavity as illustrated (Fig. 3). The ‚-subunits are much more flexible, three on either side of the equator, forming the ‘triangular ends’ of the barrel, with finger-like extensions linking the part of the ·-subunit below the equator to the triangle on the north side of the equator or vice versa (Fig. 5). The polar caps, each formed from part of three ‚-subunits, have a distinct triangular shape, each vertex having a small finger-like projection in the plane of the triangle (Fig. 6). The 12 funnel-like openings (ca. 2 nm diameter) are above and below the ·-subunits, the approximate locations of those on the front surface are illustrated (Fig. 6). The four acyl binding sites in yeast FAS have been mapped as follows: ·-Ser-180 (ACPSH), ·-Cys-1305 (KAS), ‚-Ser-819 (acetyltransferase, OHAc), ‚-Ser-5421 (malonyl/palmitoyl transferase, OHmal). Schweizer and co-workers have used targeted in vitro mutagenesis to replace each of these amino acids by non-acylable aa.152 The mutants were expressed in FAS deletants, and the resulting purified proteins were added to radiolabelled acetyl and/or malonyl CoA.Malonate could only be transferred to the ACP thiol exclusively via ‚-Ser-5421 (OHmal), not via the acetyltransferase hydroxy group (‚-Ser-819, OHAc).However, acetate could be transferred by either hydroxyl transferase to the ACP thiol and ultimately on to the KAS thiol (Scheme 25). Thus the ‘malonyl’ transferase is really an acetyl/malonyl/palmitoyl transferase. Acetylation of the KAS thiol was dependent on the presence of the ACP thiol, but acetylation or malonation of the ACP thiol was unaVected by the presence or absence of the KAS thiol.Schweizer and co-workers also observed a pronounced negative cooperativity between the equivalent sites in the hexameric ·6‚6 FAS complex. Of the 12 sites that could bind malonate, only two to three malonates could readily be bound. Only six or seven of the 24 acetate binding sites could be loaded even at high acetate concentration. Whilst this ‘static’ enzyme could only be partially loaded, complete acylation of all 12 thiol sites is observed during active FAS assembly.This negative cooperative aYnity towards Above equator Below equator a a a a a a Central axle Figure 3 Equatorial slice showing ·-subunits, with three slightly above and three slightly below the equator; also shows cavity within the complex Figure 4 Triangular north and south poles, with central axis and linkers to each of six equatorial ·-subunits a a a a a North pole South pole b b b b b b a Figure 5 Side view of FAS complex showing arrangement of ·- and ‚-subunits (hexagonal arrangement of ·-subunits in plane for clarity) a b a a a a b b b b b a X X X X X Approx location of entrance tunnel or exit on front surface X Figure 6 Model showing finger-like extensions in plane of north and south pole triangles, and the approximate location of the entrance and exit holes on near side 292 Natural Product Reports, 1998acetate and malonate could be to ensure that suYcient binding sites are reserved for acylating later intermediates in the FAS cycle, rather than swamping them with substrates.This negative cooperativity would be more readily achieved with the fungal hexamer than with the mammalian homodimer, where no such negative cooperativity has been observed. The FAS2 gene from Candida albicans that encodes for the FAS ·-subunit has been analysed and overexpressed by Southard and Cihlar.153 Aspergillus nidulans produces the polyketide aflatoxin B1, one of the most carcinogenic natural products known.The polyketide synthase uses the unusual starter unit, hexanoate. Keller and co-workers have shown that there are two distinct FAS systems in A. nidulans, one for primary fatty acid metabolism and one (sFAS) for secondary metabolism. sFAS mutants cannot produce the polyketide (unless supplemented with hexanoic acid) but otherwise grow normally. Genes coding for a FAS homologue have recently been located near the PKS cluster.154 This is the first characterised example of a FAS dedicated to the assembly of a polyketide and of a fungal FAS producing hexanoate. It is interesting to speculate how the fungal FAS can be modified to only perform two cycles of assembly.155 6.2 Elongases Fatty acid elongase enzyme complexes can elongate tetradecanoate or hexadecanoate.In yeast there are thought to be two systems, an endoplasmic reticulum associated system that can elongate 12:0 and 14:0 to 16:0 and 18:0, and a mitochondrial membrane system capable of elongating 18:0 up to 24:0.Yeast FAS defective mutants are normally able to grow when supplied with 14:0. Toke and Martin have isolated FAS defective mutants unable to grow on tetradecanoate, yet still able to transport fatty acids when supplied, and was thus considered as elongation mutants (ELO).156 The mutants, though unable to grow on 14:0, grew on 16:0 and were able to elongate 16:0, suggesting to the authors that ELO1 gene product is specific for elongating 14:0 to 16:0.Cloning and sequencing suggests that the ‘ELO1’ gene codes for a 310 aa membrane bound protein with five bilayer spanning sequences, and an NADPH binding site. Intriguingly, the coding sequence also contained HXXHH motifs in the membrane portion, reminiscent of non-haem dioxy iron cluster proteins found in fatty acid desaturases and ribonucleotide reductase. 6.3 Desaturases Plant ƒ9 desaturases are well characterised soluble enzymes, but mammalian and fungal ƒ9 desaturases are membrane bound, less well characterised, dioxygen dependent, non-haem iron-containing enzymes.Fungal ƒ9 desaturase157 is known to abstract the proR hydrogens at C-9 and C-10 in the conversion of 16:0 to 16:1 (9Z) or 18:0 CoA 84 to 18:1 (9Z) CoA 85 (Scheme 26),158as is found in the alga Chlorella vulgaris,159 the bacterium Corynebacterium diphtheria160 and pig liver.161–163 There is now a recognition of mechanistic similarities between desaturases and cytochrome P450 monooxygenases, many of which can act in a dehydrogenation process.Buist and Marecak have shown that the ƒ9 desaturases can act as regioand enantio-selective oxygenating agents by adding thiaoctadecanoates to Saccharomyces cerevisiae and obtaining the corresponding (S)-oxides with high ee.164 The 9-thia fatty acids were consistently oxidised faster than the corresponding 10-thia analogues, suggesting that the iron–oxo species is located closest to C-9, abstracting the corresponding hydrogen atom in the rate determining step.The 8-thia- and 11-thia-octadecanoates were not oxidised. Stereochemical analysis of the chiral sulfoxide product 86 was eVected by feeding [10,10-2H2]-9-thiaoctadecanoate 87, allowing NMR spectroscopic analysis of the 8-methylene group as an ABXY multiplet. Addition of three equivalents of (S)-(+)-2-methoxy- 2-phenylacetic acid shifted downfield one of the diastereotopic C-8 protons by 0.15 ppm, allowing an estimation of enantiopurity of 86 greater than 96% ee.Similar analysis on related synthetic sulfoxides allowed determination of absolute stereochemistry as R. This agreed with a Pirkle type complexation model analysis of the deshielding eVect of aromatic shielding on the chemical shift of C-13 in the 13C NMR spectrum.165 Analysis of the chiral sulfoxide 88 obtained from feeding [11,11-2H2]-10-thiaoctadecanoate 89 also gave R stereochemistry, with 90% ee, but in only half the yield of feeding 87 (Scheme 26) The stereochemistry of the desaturation and thiaoctadecanoate oxidation can be correlated (Scheme 27).A recent paper modifies this experiment so that it can examine purified enzyme systems using micromolar OHAc OHMal KAS-SH ACP-SH KAS-SH Malonyl CoA OHAc Acetyl CoA ACP-SH OHMal Scheme 25 Me SCoA O HR HR SCoA O Me Me S OMe O Me S OMe O Me S OMe O Me S OMe O O O D D D D D D D D S CO2 H F3C S CO2 H F3C O 18 9 10 9 10 18 9 D9 Desaturase – 2 x H R 18 18 10 18 9 18 87 10 89 86 88 Yeast whole cells Yeast 84 85 91 90 Purified 'desaturase' :: : whole cells : : : Scheme 26 Rawlings: Biosynthesis of fatty acids and related metabolites 293amounts of substrate.166 Buist et al.used terminal substituted (4-trifluoromethylphenyl)thiafatty acids allowing routine assay by 19F NMR spectroscopy for desaturase activity. The trifluoromethyl moiety in 9-thia-11-(4-trifluoromethylphenyl) undecanoate 90 was found to be suYciently far removed not to aVect the formation of sulfoxide 91 (Scheme 26).Buist and co-workers have also investigated the order of C–H bond breaking during desaturation by simultaneously adding methyl [9,9-2H2]-7-thiaoctadecanoate and methyl [10,10-2H2]-7-thiaoctadecanoate to growing yeast. Methyl 7-thiaoctadecanoate is smoothly converted to (9Z)-7- thiaoctadec-9-enoate. A primary isotope eVect of ca.7 was observed at C-9, whilst C–H bond breaking at C-10 was essentially insensitive to deuterium substitution.167 These kinetic isotope observations are not consistent with the classical mechanistic proposals involving synchronous removal of the two hydrogens.The first step in the mechanism would now be expected to be the rate determining abstraction of a hydrogen radical from C-9 of 84 to give 92, which could then collapse to the alkene either directly (path a) or via a cation (path b), or via an intermediate hydroxy species (path c, Scheme 28). The mechanism has been further probed by Buist et al. who have incubated fluorinated fatty acids with Saccharomyces cerevisiae in an attempt to detect the intermediacy of any hydroxy species.168 Racemic 9- or 10- fluorinated octadecanoic acid methyl esters were incubated with whole cell Saccharomyces cerevisiae.The enantiomers with fluorine in the 9R or 10R positions are presumably inactive, thus any fluorinated products are due only to the corresponding S isomers, methyl (9S)-9-fluorooctadecanoate 94 and methyl (10S)-10-fluorooctadecanoate 98.After methylation, the only fluorinated products obtained were methyl (9Z)-9-fluorooctadec-9-enoate acid 93 and methyl (9Z)-10-fluorooctadec-10-enoate 97, respectively. There was no evidence for the formation of the intermediate fluorohydrins methyl (9S)-9-fluoro-9-hydroxyoctadecanoate 95 or methyl (10S)-9-fluoro-10-hydroxyoctadecanoate 99. Fluorohydrin 95 would be expected to lead to the formation of the corresponding ketone, methyl 9-oxooctadecanoate 96, or the corresponding 9-hydroxy reduction product.Buist et al. only observed a single fluoroalkenoate for each substrate, and saw no evidence of fluorohydrins, alcohols or any ketone (Scheme 29), excluding any desaturase pathway involving a hydroxylated intermediate (such as path c, Scheme 28). (4R)-Decanolactone („-decanolactone) 100 is a key aroma component in many fruits (see Section 5), the enantiopurity being a characteristic of its fruit of origin. The lactone 100 is also produced by many microorganisms including the yeast Sporobolomyces odorus which produces only the R form (>98% ee). The industrial preparation of 100 is based upon the bioconversion of (9Z,12R)-12-hydroxyoctadec-9-enoic acid (ricinoleic acid) 101 in many species, including Sporobolomyces odorus.Radioactive 10:0 is eYciently incorporated into 100, however radioactive 12:0 was not, so 10:0 must be directly incorporated rather than via chain degradation.Radioactive 12:0 was however incorporated into (6Z)-dodec-6-eno-4- lactone 102. (9Z,12Z)-[9,10-2H2]Octadeca-9,12-dienoic acid (linoleic acid) 103 was not incorporated into lactone 100, but label was eYciently incorporated into (6Z)-dodec-6-eno- 4-lactone 102. HaVner and Tressl have added (9Z)-[9,10- 2H2]octadec-9-enoic acid 104 to Sporobolomyces odorus and obtained (4R)-[2-2H2]decano-4-lactone 105 along with (4S,6Z)-[4,5-2H2]dodec-6-eno-4-lactone 106 and (4S)-[4,5- 2H2]dodecano-4-lactone 107.The authors propose a biosynthetic scheme (Scheme 30), with separate pathways for each lactone from (9Z)-octadec-9-enoic acid, consistent with the observed deuterium labelling from 104 and selective incorporation of label from 103.169 Lactone 100 can be generated from 76 without the intermediacy of linoleic acid by 12Rhydroxylation to 101 followed by using a fatty acid degradation pathway (Scheme 31) also used to generate long chain mammalian polyunsaturated fatty acids and more fully discussed in Section 7.4.Several steps in Scheme 30 are yet to be supported by experimental evidence, such as the intermediacy of 109 and 110. The characteristic flavour of edible mushrooms is largely due to oct-1-en-3-ol 111 and oct-1-en-3-one. As these compounds are normally only produced in the fruiting bodies, their biosynthesis has been hard to examine. However, Dosoretz and co-workers have found that the addition of soybean flour or soybean oil to submerged cultures of Pleurotus pulmonarius enhances levels of 111 and its equimolarily-produced cometabolite 10-oxodec-8-enoic acid 112 production to levels higher than in fruiting bodies.170 Another metabolite, (9Z,11E)-13-hydroperoxy-octadeca-9,11- dienoic acid 113, was produced in even larger amounts.Dosoretz and co-workers have suggested that increased levels Scheme 27 Compounds shown as enzyme thioesters, not CoA or methyl esters, as previously HR HR (CH2)7COSEnz Me(CH2)7 H H Fe O HR (CH2)7COSEnz Me(CH2)7 H H Fe OH HR (CH2)7COSEnz Me(CH2)7 H H Fe OH (CH2)7COSEnz Me(CH2)7 H H Fe HOH OH HR (CH2)7COSEnz Me(CH2)7 H H Fe 5+ 9 10 9 10 9 10 9 10 9 10 84 85 a c b 92 : : : + + • • Scheme 28 294 Natural Product Reports, 1998of (9Z,12Z)-octadeca-9,12-dienoic acid (linoleate) hydroperoxidation takes place in parallel to formation of 111 and 112 in distinct biosynthetic pathways (Scheme 32).Fumonisins are mycotoxins produced by Fusarium moniliforme that inhibit the conversion of sphinganine to dihydroceremides in mammals.This fungus is prevalent on maize and other commercial cereals, the resulting toxins causing neurodegenerative disorders in farm animals, especially horses, by aVecting lipid biosynthesis. Carman and co-workers have found that fumonisin B1 115 inhibits ceramide synthases in yeast, resulting in a build up of sphingoid bases such as sphinganine 116 and phytosphingosine 117.171 7 Animalia The animalia kingdom consists of at least 33 phyla and can be divided into the subkingdom parazoa (no shape or tissues organised into organs) represented by two phyla; placozoa and porifera (sponges), and the subkingdom eumetazoa (31 phyla).This subkingdom is split into two branches, the radially symmetrical organisms (radiata) such as the cnidaria (coelenterates or corals), and bilaterally symmetrical organisms (29 phyla). Most animal phyla are marine dwelling (many in shallow waters), with two ‘truly land dwelling’ phyla; arthropoda and chordata.The vast majority of animal species are invertebrate except for those in the craniata, a subphylum of the chordata. The evolution of natural products within this kingdom may be confused by diVerent phyla evolving from diVerent protoctist phyla. 7.1 Cnidaria Lipoxygenases are non-haem iron dioxygenases that specifically hydroperoxidise polyunsaturated fatty acids. Whilst lipoxygenases that result in (S)-hydroperoxides occur widely in plants and mammals, and are well characterised, (R)-hydroperoxides are only known in marine invertebrates.Brash et al. have purified and cloned (but not overexpressed) the (8R)-lipoxygenase from the coral Plexaura homomalla that is involved in prostaglandin biosynthesis.172 There was considerable sequence homology to plant and animal (S)- lipoxygenases, with the three iron binding histidine residues Me OMe O F Me OMe O F Me OMe O F Me OMe O F OH 18 18 94 9 10 18 18 OMe O 95 99 Me F 9 OMe O Whole yeast 18 Me Whole yeast 9 F 9 18 93 HO Me OMe 9 O 10 98 9 18 96 O 97 Scheme 29 Me CO2H D D CO2H D D Me CO2H Me D D OH Me CO2H D O(O)H D O O Me O O Me O O Me Me CO2H D D HO D D D D D (99% ee) (59% ee) 18 84% ee) 12 9 9 18 18 9 18 10 8 10 4 12 4 6 4 12 18 9 106 103 (10 1 D = H) 109 110 (100 D = H) 107 (102 D = H) 12-Hydroxylase b-Oxidation b-Oxidation b-Oxidation Isomerisation 104 105 (76 D = H) 108 Scheme 30 Rawlings: Biosynthesis of fatty acids and related metabolites 295conserved but the fourth binding residue, the C-terminal isoleucine (carboxy group as ligand), replaced by threonine. Brash et al.suggest that the basis for the stereochemistry lies in substrate binding, and that there may be a connection between 8R oxygenation and 12S oxygenation, and vice versa. As the (R)-lipoxygenases are in the same gene family as the (S)- lipoxygenases, Brash et al. suggest that they may be more widely distributed than just the ‘invertebrate animals’. 7.2 Mollusca The mollusc phylum can be divided into eight classes, including the cephalopoda (octopus/squid), gastropoda (slugs and snails) and pelecypoda (bivalves such as oysters, clams and mussels). The fatty acid and sterol content of three species of slugs and three species of snails has been compared with marine molluscs, some diVerences being accounted for by the diVerence in a land or marine based diet. (3Z,6Z,9Z,12Z,15Z,18Z)- Docosa-3,6,9,12,15,18-hexaenoic acid [22:6 (n"3)] was absent in snails and slugs, suggesting that they cannot convert 22:5 (n"6) (present) to 22:6 (n"3).173 Graziani and Anderson have incorporated [1,2-13C2]-acetate into the diacylguanidine metabolite triophamine 118 produced by the dorid nudibranch (marine shell-less snails) Triopha catalinae (‘clown nudibranch’).174 The incorporation demonstrates de novo biosynthesis, rather than dietary consumption, and that the ten acyl units are all acetate derived.It is interesting to speculate whether butanoate can be incorporated as these metabolites are somewhat reminiscent of hydrocarbon aggregation pheromones produced by male sap beetles that have been shown to incorporate butanoate.1 7.3 Arthropoda The arthropod phylum can be divided into three subphyla; the chelicerata, which includes the class arachnida containing scorpions, daddy long legs, spiders, mites and ticks (acarina); the crustacea, containing water fleas, barnacles and the order decapoda that contains shrimps, lobsters crayfish and crabs; and the uniramia, that can be split into five classes, including those for the millipedes, centipedes and insects (insecta).OH O Me D D OH O Me D D OH Me D D OH SCoA O Me D D OH SCoA O Me OH SCoA O D D Me OH SCoA O D D Me OH SCoA O D D Me OH SCoA O D O O D Me 18 18 14 14 14 14 14 10 10 4 105 104 108 (12 R)-Hydroxylase 2 x b-Oxidation D3,D2-Enoyl CoA isomerase D3,5,D2,4-Dienoyl CoA isomerase 2,4-Dienoyl CoA reductase D3,D2-Enoyl CoA isomerase 2 x b-Oxidation Scheme 31 CO2H Me CO2H Me OOH CO2H OHC Me OH or 8 111 113 114 112 + 9 18 12 18 9 11 13 Scheme 32 Me Me NH3 + OH OH OR Me OR Me OH Me OH NH3 + OH R R = COCH2CH(CO2H)CH2CO2H 116 R = H 20 18 115 117 R = OH Me NH N O Me Me Me NH2 Me Me O NH N O NH2 O • 118 • • • • • • • • • 296 Natural Product Reports, 19987.3.1 Chelicerata Most mature arthropods are distinguished by having segmented bodies split into three parts; head, chest and abdomen, but in the chelicerata the first two are combined into a single part.Prostaglandins (PGs) and other icosanoids have been found in a large number of invertebrates, though their physiology is relatively little understood. Whilst in mammals PG biosynthesis is only associated with microsomal fractions, in some invertebrates it may also be associated with soluble fractions. Stanley-Samuelson and co-workers have investigated the localisation of PG biosynthesis in the Lone Star tick Amblyomma americanum.175 Microsomal and cytosolic fractions were both capable of PG biosynthesis, that can be inhibited by indomethacin, a potent inhibitor of mammalian cyclooxygenases. Bowman et al.have investigated the biosynthesis of PGs in the saliva of the Lone Star tick.176 These blood feeding ixodid ticks remain attached to their hosts for many days, and they need to prevent immune or inflammatory responses by the host, thus they introduce PG2s into their host via their saliva.The salivary glands contain high levels of arachidonic acid, which is obtained entirely from hosts as they themselves cannot biosynthesise it. Due to their very short half-life, PGs are usually autocoids, acting as local hormones where they are produced. However, whilst the salivary gland has been found to be a potent producer of PG2s, no PG synthase activity has previously been detected. Bowman et al. have found that dopamine stimulated the conversion of radioactive arachidonic acid into PG2s by salivary glands, and they suggest that this PG synthetase is very diVerent from other PG synthetases. 7.3.2 Crustaceae The ability of the brine shrimp, Artemia sp., to biosynthesise de novo (¢"3) and (¢"6) fatty acids has been examined by Ito and Simpson.177 It is now possible to cultivate Artemia spp. under axenic conditions using inorganic mercury to disinfect cysts, thus removing the possibility that biosynthetic conversions are being carried out by microorganisms associated with the shrimp.Radiolabelled (9Z,12Z)-octadeca-9,12-dienoic acid was added to defatted micronised rice bran and fed to starving shrimps under axenic conditions. Radioactivity was incorporated into (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid [18:3 (¢"3)], (6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoic acid [18:4 (¢"3)] and (5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17- pentaenoic acid [20:5 (¢"3)], with similar incorporation levels to shrimps cultivated under xenic conditions. 7.3.3 Uniramia 7.3.3.1 Insecta The class insecta can be divided into over 25 orders, including orders that are primitive wingless insects such as the collembola (springtails) and thysanura (silverfish); those orders in which their is no pupal stage and the young are nymphs, such as odonata (dragonflies), dictyoptera (cockroaches) and hemiptera (true bugs); and those orders with complete metamorphosis whose young are larval, such as lepidoptera (butterflies and moths), diptera (true flies), hymenoptera (bees, wasps, ants) and coleoptera (beetles). 7.3.3.2 Dictyoptera Insect methyl branched fatty acids were first described in 1992 along with a microsomal synthetase capable of their synthesis. 178 They are thought to be precursors of branched long chain hydrocarbons, such as 3,11-dimethylnonacosane 119, and components of sex pheromones. The German cockroach (Blattella germanica) produces both straight chain fatty acids and methyl branched fatty acids, which are thought to be precursors of both long chain (C27–C31) hydrocarbons and the major sex pheromone 3,11-dimethylnonacosan-2-one 120, as the methyl branching patterns are related.179 This would require the addition in a specific order of two MMCoAs and four malonyl CoAs to octadecanoic acid 47 to give 4,12- dimethyl-3-oxotricontanoyl CoA 121 (Scheme 33).Whilst the goose uropygial (preening) gland exclusively uses MMCoA as an extender unit to form 2,4,6,8-tetramethyldecanoic acid, this occurs as only MMCoA, not malonyl CoA is present in this gland.When supplied with malonyl CoA, the uropygial FAS produces straight chain fatty acid.180 In the case of the integument (outer parts/skin, not body fat) enriched tissues of the cockroach, one or more MMCoAs can be introduced at specific points along the growing chain, requiring a more sophisticated ‘intelligent’ system that knows when to insert MMCoA.It is interesting to speculate whether the insertion of the second MMCoA, forming 121, triggers ‘FAS catalysed’ decarboxylation to 120, terminating elongation. There are two FAS systems from integument enriched tissue, neither yet purified, a soluble and a microsomal FAS system, both of which can incorporate radiolabelled MMCoA into methyl branched fatty acids.178 Blomquist and co-workers have shown that the soluble FAS system has almost no activity with MMCoA (in the absence of malonyl CoA).However, the microsomal FAS activity was high, and was able to catalyse fatty acid synthesis in the absence of any malonyl CoA. However, MMCoA could bind to both enzymes and MMCoA Me Me Me Me Me CO2H Me COSCoA Me Me Me Me Me Me O 29 3 O 11 47 121 120 2 x Methylmalonyl CoA 4 x Malonyl CoA 119 3 11 18 30 29 Scheme 33 Rawlings: Biosynthesis of fatty acids and related metabolites 297acted as a competitive inhibitor against malonyl CoA with both systems.The microsomal FAS has parabolic inhibition, consistent with multiple reversibly connected points of inhibitor addition. Blomquist and co-workers conclude that the major diVerence between the two FAS systems is in their ability to turn over enzyme bound methylmalonyl substrate, rather than in the binding of MMCoA to the enzyme.181 It is interesting to speculate whether these systems more closely resemble macrolide synthases with a separate catalytic activity (with full reduction) for each cycle, than traditional eukaryotic FAS (with repetitive turnover by a single set of catalytic activities).A recent paper by Schal and co-workers demonstrates that the biosynthesis of hydrocarbons and methyl ketone hormones such as 119 and 120 in females of B. germanica occurs only in the abdominal integument, and locates a high density lipophorin in the haemolymph that transports them to other tissues such as the ovaries and body fat.182 7.3.3.3 Hemiptera Juarez et al.have investigated the two FASs (soluble and microsomal) present in eggs of Triatoma infestans.183 Methyl branched C16 and C18 fatty acids are thought to be present at levels below 0.7% in egg fatty acids. When incubated with acetyl CoA or malonyl CoA, both FASs gave hexadecanoic acid. However, the microsomal FAS was able to incorporate MMCoA in the absence of malonyl CoA, whilst the soluble FAS was not able. In the presence of both MMCoA and malonyl CoA, both produce a mixture of hexadecanoic acid and methyltetradecanoic acid, the position of the methyl branch not known or specified. 7.3.3.4 Lepidoptera In Lepidoptera, most pheromone biosynthesis involves fatty acid biosynthesis (16:0) followed by desaturation, chain shortening/elongation, followed by reduction to the alcohol, acetylation and/or oxidation. Desaturation usually occurs using a ƒ11 desaturase, for example, the silkworm moth, Bombyx mori produces a ƒ10–ƒ12 system from a ƒ11 fatty acid followed by an isomerase.184,185 DiVerent strains of the European Corn Borer Ostrinia nubilalis (Pyralidae) use diVerent blends of (11E)-tetradec-11- enyl acetate 122 and (11Z)-tetradec-11-enyl acetate 123 as their sex pheromone, despite possessing the same ratio of acid precursors (11E)- 124 and (11Z)-tetradec-11-enoic acids 125.Löfstedt and co-workers have shown that the ratio of pheromone components is controlled by the specificity of each strain’s reductase system that converts the acids 124 and 125 to alcohols (11E)-tetradec-11-enol 126 and (11Z)-tetra-dec- 11-enol 127, respectively.186 Löfstedt and co-workers then examined the substrate specificity of the acetyl transferase that acetylates 126 or 127 and found that it had a very low substrate specificity, and would convert a wide range of substrates, thus having no control over product 122:123 ratio (Scheme 34).187 Foster and Roelofs have shown that deuteriated 14:0, 16:0 and 18:0 [but not 12:0 or 18:1 (9Z)] fatty acids are all converted into (5Z)-tetradec-5-enyl acetate, the sex pheromone of the tortricid moth Ctenopseustis herana (Tortricidae).188 The highest incorporation was from 14:0, consistent with chain shortening to 14:0, followed by the novel biosynthetic route for a moth sex pheromone of 14:0 ƒ5 desaturation.The biosynthesis of the sex pheromone of the Egyptian armyworm Spodoptera littoralis, (9Z ,11E)-tetradeca-9,11- dienyl acetate, is thought to involve chain shortening of 16:0 to 14:0.A series of monofluorinated, alkynyl and cyclopropanated analogues of 16:0 have previously been found to inhibit its biosynthesis.1 Guerro and co-workers have prepared 2,2-, 3,3- and 4,4-difluorohexanoic acid, and found that only the 2,2- and 3,3-derivatives acted as an inhibitor.189 The female tobacco hornworm moth Manduca sexta (Sphingidae) produces a blend of unsaturated C16-aldehydes as its sex pheromone (aldehyde analogues of 128, 129, 130, 131, 132 and 133).Tumlinson and co-workers have investigated the conversion of deuteriated hexadecanoic acid and some later intermediates by the female’s sex pheromone glands, and proposed a biosynthetic scheme (Scheme 35).190 Hexadecanoic acid 46 was converted into all the pheromones. The conversion into both (11E)-hexadec-11-enoic acid 128 and (11Z)-hexadec- 11-enoic acid 129 suggests the presence of 11E and 11Z desaturases. Labelled (11E)-hexadec-11-enoic acid 128 is reduced to the corresponding aldehyde pheromone, but is not converted to the aldehydes of 130, 131, 132 or 133.However, 129 was converted into the aldehydes of (10E,12E)- hexadeca-10,12-dienoic acid 130, (10E,12Z)-hexadeca-10,12- dienoic acid 131, (10E,12E,14E)-hexadeca-10,12,14-trienoic acid 132 and (10E,12E,14Z)-hexadeca-10,12,14-trienoic acid 133. Presumably both trienes are formed via the corresponding dienes (dotted arrows in Scheme 35), however the mechanism is not yet known. 7.3.3.5 Diptera The insect cuticular lipids prevent desiccation and are usually complex mixtures of long straight chain, methyl branched and unsaturated hydrocarbons, that in some species can also act as semiochemicals.191 The C18 fatty acids are chain extended and then converted to very long chain hydrocarbons with loss of the carboxy carbon.Recent work has suggested that the very long chain fatty acid CoAs are reduced using NADPH to aldehydes and then decarbonylated to the hydrocarbon and carbon monoxide under anaerobic conditions without any cofactors (Scheme 36).For example, (9Z)-octadec-9-enoic acid 76 can be elongated to (19Z)-octacos-19-enoic acid 134, reduced by NADPH to (19Z)-octacos-19-enal 135 and decarbonylated to (9Z)-heptacos-9-ene 136 (Scheme 36). Yoder et al. have reported that addition of [1-14C]octadecanal to the fleshfly Sarcophaga crassipalpis (Calliphoridae) under anaerobic conditions with no cofactors gave 14CO which was trapped as a rhodium(I) complex.192 Dennis and Kolattukudy had previously reported the decarbonylation of aldehydes to hydrocarbons in plants, animals and microorganisms.193 However, Blomquist and co-workers have found that microsomal preparations from the house fly Musca domestica (Muscidae) converted (15Z)-tetracos-15-enal 137 to (9Z)-tricos-9-ene 138 (the major component of the female housefly sex pheromone) and carbon dioxide, using NADPH and dioxygen in a cytochrome P450 type reaction which was inhibited by the addition 127 R = H Me OR OR Me 16:0 14:0 14:1 (11 E) 14:1 (11 Z) 14 14 125 126 R = H 124 11 11 123 R = Ac 122 R = Ac Scheme 34 298 Natural Product Reports, 1998of carbon monoxide or by the addition of antibodies to other fly cytochrome P450 reductases (Scheme 36).194 Mpuru et al.have shown that [1-14C]octadecanal was converted to radioactive carbon dioxide and heptadecane by microsomes from the house fly (Musca domestica), the blowfly (Phormia regina), the German cockroach (Blattella germanica), the house cricket (Acheta domesticus), the Mormon cricket (Anabrus simplex) and the dampwood termite (Zootermopsis nevadensis), as well as the fleshfly (Sarcophaga crassipalpis).195 Most cytochrome P450s, such as hepatic microsomal hydroxylases, do not catalyse reactions when NADPH and dioxygen are substituted for by hydrogen peroxide, but these do, suggesting they have an atypical catalytic scheme.The microsomal preparations converted [2,2-2H2]- or [3,3-2H2]-tetracosanoyl CoA (C24) into dideuteriated tricosane (C23), showing that ·,‚-unsaturated intermediates were not involved. [1-2H]Tetracos-15-enal was converted into deuteriated (9Z)-tricos-9-ene, showing that the aldehydic proton was transferred to the chain shortened hydrocarbon. Reitz and co-workers propose a cytochrome P450 based mechanism in which the iron oxene 139 abstracts an electron from the carbonyl before attacking the C-1 forming a thiyl-iron-hemiacetal diradical 140.After C–C bond homolysis, the resulting alkyl radical 141 then abstracts the methanoyl hydrogen from 142 to give hydrocarbon 143 and carbon dioxide (Scheme 37).196 Alternatively, direct aldehyde radical abstraction by 139 on the aldehyde substrate would form 141 and 142. Scheme 37 illustrates the fate of deuterium labels to be in accordance with the above experiments. Male and previtellogenic female house flies produce (9Z)-heptacos- 9-ene (C27) 136, whilst vitellogenic (capable of producing eggs) females produce (9Z)-tricos-9-ene (C23) 138, the extent of the elongation of acyl CoAs by elongases being presumably regulated by ecdysteroids produced by the mature ovaries (Scheme 36).197 Reitz and co-workers have investigated the chain length dependency and house fly sex dependency of the oxidative decarbonylation, and conclude that acyl CoA elongation was the main endocrine regulated step in hydrocarbon synthesis.198 Presumably, the reductive and oxidative decarbonylation enzymes work well converting either 134 into 136 or (15Z)-tetracos-15-enoyl CoA 144 to (9Z)-tricos-9-ene 138.Presumably, elongation of 144 to 134 only occurs in males and previtellogenic females. 7.3.3.6 Hymenoptera Slessor and co-workers have investigated the caste selective biosynthesis of pheromones in the mandibular glands of the 16 16 16 11 11 12 10 12 10 14 12 Me CO2H Me CO2H CO2H Me CO2H Me Me CO2H Me CO2H CO2H Me 16 16 16 10 10 12 14 46 128 129 130 131 132 133 11 E Desaturase 11 Z Desaturase Scheme 35 Me CO2H Me COSCoA Me CHO Me Me Me COSCoA Me CHO Me Me 18 24 9 15 24 1 15 9 23 28 19 28 19 1 27 9 Acyl CoA elongases Elongases (only in males and previtellogenic females) NADPH NADPH/O2/P450 CO2? 76 138 144 137 134 135 136 NADPH NADPH/O2/P450 CO2? Scheme 36 Rawlings: Biosynthesis of fatty acids and related metabolites 299Honeybee Apis mellifera (Apidae).199 Queen bees produce (2E)-9-hydroxydec-2-enoic acid 149 along with other (¢"1)- functionalised fatty acids [e.g. (2E)-9-oxodec-2-enoic acid 150] as their predominant pheromones, which are powerful attractants towards (female) worker bees, and may also serve to assert her reproductive dominance over them.The worker bees produce acids functionalised at the ¢-position, such as (2E)- 10-hydroxydec-2-enoic acid 146 and (2E)-9-carboxynon-2- enoic acid 147. Slessor and co-workers have shown that both sets of compounds are produced preferentially from octadecanoic acid 47 rather that from 16:0 or 10:0.199 They then demonstrate the selective incorporation of deuterium labelled 18-hydroxyoctadecanoic acid 145 into the ¢- functionalised series and labelled 17-hydroxyoctadecanoic acid 148 into (¢"1)-series, conversions that were both inhibited by the ‚-oxidation inhibitor 2-fluorooctadecanoic acid (Scheme 38).200 Slessor and co-workers have found quantities of both series in both queen and worker bees, and suggest that both castes have the two pathways, but diVer in their pathway selection/activity.It is interesting to speculate on the hydroxylation of octadecanoic acid 47. This may be an opportunity to probe the remarkable stereo- and regio-chemical selectivity of P450s. Is both the C-17 and C-18 hydroxylation performed by a single enzyme, other factors (allosteric?) aVecting regiospecificity, from almost exclusively one to almost exclusively the other regioisomer? More likely would be a single progenitor gene has duplicated and one copy slightly mutated, such that either gene can be caste selectively expressed as required to perform either hydroxylation. This situation would enable comparison of the active sites to reveal factors aVecting regiospecificity of the hydroxylation.The stereochemistry of 148 has not been determined. 7.4 Chordata The chordate phylum, characterised by a single dorsal nerve chord, includes vertebrates such as mammals, birds, fish, amphibians and reptiles, but also a few invertebrates such as lampreys.The chordates can be classified into four subphyla, including the brainless ‘tunicates and ascidians’ (tunicata) and ‘lancelots’ (cephalochordata). The other two subphyla are both ‘craniates’; the ‘lampreys’ (agnatha, with brain but no jaws) and the main subphylum, the ‘jawed chordates’ (gnathostomes). The gnathostomes can be divided into two superclasses, the fish (pisces; sharks, rays and bony fish) and four limbed animals (tetrapoda) which can be divided into four classes; the amphibians (amphibia), reptiles (reptilia), birds (aves) and mammals (mammalia).The animal FAS is a head-to-tail homodimer of a multifunctional protein that contains all the required activities to convert acetyl CoA and malonyl CoA into free hexadecanoic acid. Early studies found that human FAS activity was much S Fe OH S Fe O C D O RCD2CD2 S Fe O C O D CD2CD2R S Fe O C O D CD2CD2R S Fe O CH O DCD2CD2R S Fe RCD2CD2CDO 3+ + 2+ 4+ 4+ 3+ + + 2- CO2 + – + Direct aldehyde radical abstraction 3+ Thiyl-iron-hemiacetal diradical 'Perferryliron oxene' – 141 139 140 142 143 • • • • • • • • • Scheme 37 Me CO2H CO2H Me CO2H OH HO HO2C CO2H Me CO2H O CO2H Me CO2H OH HO 18 47 148 145 149 146 150 147 b-Oxidation P450 P450 18 17 9 9 10 (Female worker bee) (Queen bee) 10 b-Oxidation Scheme 38 300 Natural Product Reports, 1998lower than in other animals, hindering purification and suggesting strong repression of lipogenesis in humans.In humans, de novo fatty acid synthesis mainly occurs in the liver, so Wakil and co-workers isolated and characterised human FAS from a hepatoma cell line.201 The human FAS product was over 90% hexadecanoic acid (14:0 4%, 18:0 6%), whilst the corresponding FAS from chicken produced only ca. 60–70% 16:0 (14:0, 5% and 18:0 15–20%). Chicken FAS prefers acetyl CoA as a substrate to butanoyl CoA, however, the human FAS acyl substrate specifi- city is similar to that for rat FAS in that it prefers butanoyl CoA (4 ÏM) to acetyl CoA (8 ÏM).The human FAS can eYciently substitute 3-oxobutanoyl CoA for acetyl CoA, with no lag in NADPH oxidation, suggesting that the conversion to butanoyl thioester is fast. The human FAS may intercept and use 3-oxobutanoyl CoA as a physiologically significant substrate. The specific activity of the purified human FAS, as judged by the oxidation of NADPH, was only one-third that found for chicken liver FAS, yet most partial activities were similar, except for the KAS which was three times lower, the DH which was three times higher and the ER which was twenty times higher in human FAS.Wakil and co-workers found that there was only 0.42 mol of pantetheine per mol human enzyme (0.96 in chicken), and suggested that human FAS may be regulated post-translationally. The human hepatoma FAS contained very low sequence homology to brain FAS of only 15% in the interdomain linker regions, and included a 53 bp insertion in the sequence. AT KAS DH Central core ER KR ACP TE KAS AT DH Central core ER KR ACP TE AT KAS DH Central core ER KR ACP TE KAS AT DH Central core ER KR ACP TE AT KAS DH Central core ER KR ACP TE KAS AT DH Central core ER KR ACP TE AT KAS DH Central core ER KR ACP TE KAS AT DH Central core ER KR ACP TE AT KAS DH Central core ER KR ACP TE KAS AT DH Central core ER KR ACP TE AT KAS DH Central core ER KR ACP TE KAS AT DH Central core ER KR ACP TE AT KAS DH Central core ER KR ACP TE KAS AT DH Central core ER KR ACP TE Active Active Inactive Inactive Inactive KAS–/KAS– ACP–/ACP– Inactive Separate into monomers, mix and redimerise ACP–/KAS– KAS–/ACP– ACP–/ACP– KAS–/KAS– Model of native homodimeric FAS Scheme 39 Rawlings: Biosynthesis of fatty acids and related metabolites 301In a more recent paper, Wakil and co-workers clone and express human FAS from brain in E.coli as a maltose fusion protein using recombinant PCR to join overlapping DNA fragments without a unique restriction site, and found the active domains to be comparable to the enzyme recently purified from a hepatoma cell line (vide supra).202 Domain I was separately expressed and showed KAS, AT and DH activity, whilst domain II and III, when expressed together showed ER, KR, ACP and TE activity. This agreed with the reassignment of the rat FAS DH active site to domain I (conserved motif: HXXXGXXXXP)203 from earlier suggestions that it was present in domain II.204 The location of all seven catalytic activities on the mammalian FAS peptide chain is now established, however, their spatial arrangement to each other in the homodimer remains unknown, except for ACP and KAS domains.Smith and co-workers have developed methodology to probe the interdomain and active site communication in rat FAS homodimer by in vivo complementation of inactive mutants.205 A series of inactive mutant FAS homodimers were formed, that were altered in either the KAS (C161T or K326A) or ACP domains (S2151A) and expressed in insect cells using a baculovirus expression system. Animal FAS are cold-labile homodimeric proteins that at low temperatures and low ionic strengths slowly dissociate into two monomers.When a KAS mutant homodimer and ACP mutant homodimer were mixed at 20 )C, no FAS activity reappeared, even after six days, suggesting there was no ‘spontaneous’ dissociation/ reassociation of the dimers occurring.This would have resulted in randomisation of the mutants, and partial regaining of activity as KAS–ACP heterodimers would have half activity of the native enzyme. Smith and co-workers have developed a procedure to rapidly obtain monomeric FAS during purifi- cation. Use of 50 mM Tris buVer on certain anion exchange columns led to elution of high levels of monomer, which can easily be randomly reassociated with a second diVerent monomeric mutant.Using this methodology, Smith and co-workers have confirmed that the KAS and ACP mutations complement each other (Scheme 39). In a control experiment, the two KAS mutations (C161T and K326A) failed to complement each other. In theory, complementary mutants should restore 50% of the original activity. The situation is more complicated because somewhat surprisingly, some mutants dissociate far more readily than others, and there may also be diVerences in rate of reassociation of the two (KAS–KAS or ACP–ACP) homodimers versus the (KAS–ACP) heterodimer and even that the functional activity of one active site may aVect the activity of the second.Despite these complications, this methodology can now be extended to investigate the interaction of all the other active sites. It would be interesting to speculate whether monomers from two closely related species can dimerise into active FAS.Rangan and Smith have also overexpressed residues 429–815 of rat FAS in E. coli and obtained catalytically active malonyl/acetyl transferase indistinguishable from that Me CO2H Me CO2H CO2H Me CO2H Me Me CO2H 18 9 9 12 18 5 14 151 152 114 153 5 Z Desaturase 8 5 14 76 Scheme 40 Me COSCoA Me COSCoA Me Me Me COSCoA Me COSCoA COSCoA COSCoA b-Oxidation Me COSCoA 154 Me 155 COSCoA 158 Me 161 Me COSCoA 162 160 COSCoA 159 Me COSCoA Me COSCoA 163 164 165 157 Further b-oxidation 156 5 5 2 5 5 3 5 3 5 3 10 2 3 5 2 4 2 3 5 10 10 10 '5-Reductase' b-Oxidation Scheme 41 302 Natural Product Reports, 1998obtained from native enzyme through limited proteolysis.206 The kinetic constants were so similar, it seems likely that the transferase has assumed the correct folding conformation.H683A mutation reduced activity by 99.95%, presumably it is this His that accepts a proton from the active site Ser as part of the catalytic triad. The DNA flanking the 5*-region of both human and chicken FAS have been found to contain promoters whose interaction with hormones such as thyroid (chicken) or insulin (human) regulate transcription and translation.207,208 In vertebrate retina, many signal transduction proteins are N-acylated with 14:0 151 or 12:0 fatty acids and the very unusual (5Z)-tetradec-5-enoic acid [14:1 (n"9)] 152 and (5Z,8Z)-tetradeca-5,8-dienoic acid [14:2 (n"6)] 153 (double bond geometry not specified in paper), which otherwise normally only occurs in marine mammals and an Asian plant, Evodia rutaecarpa.Anderson and co-workers have found that frog retina outer rod segments converted radiolabelled oleic acid 18:1 (n"9) 76 to 152 and 18:2 (n"6) 114 to 153, presumably by two rounds of ‚-oxidation, and that 18:3 (n"3) or 14:0 151 were not incorporated into either 14:1 (n"9) 152 or 14:2 (n"6) 153 (Scheme 40).209 There does not appear to be a 5Z desaturase operative in this system. The ‚-oxidation of ƒ5-fatty acids, or any fatty acid with an odd numbered double bond, in the mitochondria and liver has stimulated great interest, and considerable discussion.This had been thought to occur exclusively by an NADPH independent pathway involving hydration of the corresponding (2E,5Z)-fatty acid, oxidation, chain shortening and ƒ3,ƒ2 isomerisation. However, Tserng and Jin reported in 1991 that eVective ‚-oxidation of (5Z)-enoyl CoAs required NADPH, with direct reduction (‘5-reductase’) of the 5Z double bond as illustrated by the direct reduction of (5Z)-dec-5-enoyl CoA 154 or (5E)-dec-5-enoyl CoA 155 to decanoyl CoA 156, which could then be converted to (2E)-dec-2-enoyl CoA 157 and so on by routine ‚-oxidation (Scheme 41).210 In 1992, Schulz and co-workers were unable to detect this 5-reductase, and instead showed that the reduction proceeded via the corresponding (2E,5Z)-dienoyl CoA and suggested the following sequence: ƒ5-enoyl CoA to (2E,ƒ5)-dienoyl to 3,5-dienoyl to (2E,4E)- dienoyl to 3-enoyl CoA, by enzymes in either the mitochondria or peroxisomes.211 Tserng and co-workers purified the ƒ3,ƒ5- (2E,4E)-dienoyl CoA isomerase from rat liver mitochondria as a tetramer (200 kDa) of subunit mass 55 kDa.212 Schulz and co-workers have also purified this enzyme from the same source, reporting a mass of 126 kDa (32 kDa subunits), and could find no activity in the peroxisomes.213 In 1995 Tserng and co-workers report on the stereochemical requirement of substrates for the enzymes in this sequence.214 (2E,5Z)-Deca- 2,5-dienoyl CoA 158 was isomerised to both (3E,5Z)-deca- 3,5-dienoyl CoA 159 and (3Z,5Z)-deca-3,5-dienoyl CoA 160 by the ƒ2,ƒ3-enoyl CoA isomerase, the relative ratio depending upon the pH of the incubation medium.(2E,5E)- Deca-2,5-dienoyl CoA 161 gave both (3Z,5E)-deca-3,5-dienoyl CoA 162 and (3E,5E)-deca-3,5-dienoyl CoA 163. Dienoyl CoAs 159, 160 and 162 were all substrates for ƒ3,ƒ5-(2E,5E)- dienoyl CoA isomerase, all three substrates being converted to the same product, (2E,4E)-deca-2,4-dienoyl CoA 164, but (3E,5E)-deca-3,5-dienoyl CoA 163 did not act as a substrate (Scheme 41).(2E,4E)-Deca-2,4-dienoyl CoA 164 was then reduced to (3E)-dec-3-enoyl CoA 165 which could then be isomerised by an ‘HDDH’ like enzyme to 157 for routine ‚-oxidation. The ‚-oxidation of polyunsaturated fatty acids would require these enzymes for every odd numbered double bond. In 1995 Sprecher and co-workers showed that the peroxisomal ‚-oxidation of (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoic acid (arachidonic acid) 165 required both ƒ3,5,ƒ2,4-dienoyl CoA isomerase and 2,4-dienoyl CoA reductase, and suggested that the so called ‘trifunctional’ enzyme (analogous to the E.coli ‘HDDH’) converts the 3-enoyl CoA 166 to 2-enoyl CoA 167.215 After several routine ‚-oxidation steps, the resulting (2E,4Z,7Z,10Z)-hexadeca-2,4,7,10-tetraenoyl CoA 168 can be further chain shortened using a similar sequence to that used for (2E,4Z,8Z,11Z,14Z)-icosa-2,4,8,11,14-pentaenoyl CoA 169, and so on.The overall scheme is illustrated in Scheme 42, all steps occurring in the peroxisome.215 Long chain polyunsaturated fatty acid biosynthesis has been generally considered to occur using elongases in the COSCoA Me COSCoA Me COSCoA Me Me COSCoA 20 Me COSCoA 14 5 20:4 ( n–6) 20 14 Me 11 COSCoA 8 5 20 14 11 Me 11 COSCoA 8 8 5 20 14 11 4 3 20 14 Me 8 COSCoA 2 OH 11 8 20 14 8 18 12 Me 11 COSCoA 9 6 Fatty acid oxidase enzymes 2,4-Dienoyl CoA reductase NADPH dependent 20 Me D2,D3-Enoyl CoA isomerase COSCoA D3,5,D2,4-Dienoyl CoA isomerase 14 8 'Trifunctional enzyme' 'Trifunctional enzyme' Fatty acid oxidase enzymes 16 10 7 4 Fatty acid oxidase enzymes 14 8 11 5 and so on Peroxisome 3 2 165 169 167 168 18:3 ( n–6) 16:3 ( n–6) 14:2 ( n–6) As above for 169 166 Scheme 42 Rawlings: Biosynthesis of fatty acids and related metabolites 303endoplasmic reticulum (microsomes), whilst ‚-oxidation of long chain saturated fatty acids to C16/18 fatty acids occurs in the peroxisomes, after which they are transferred to the mitochondria for completion of the ‚-oxidation.Sprecher and co-workers have investigated the conversion of (7Z,10Z,13Z,16Z)-docosa-7,10,13,16-tetraenoic acid [22:4 (n"6)] 170 to (4Z,7Z,10Z,13Z,16Z)-docosa-4,7,10,13,16- pentenoic [22:5 (n"6)] 171 by rat liver microsomes.216 This has previously been considered to occur through a 4Z desaturase.Under standard conditions to detect a 4Z desaturase, no such enzyme could be detected. However, upon addition of malonyl CoA and NADPH, 22:4 (n"6) 170 was elongated to (9Z,12Z,15Z,18Z)-tetracosa-9,12,15,18- tetraenoic acid 24:4 (n"6) 172 which was then desaturated (by a 6Z desaturase) to (6Z,9Z,12Z,15Z,18Z)-tetracosa- 6,9,12,15,18-pentaenoic acid 24:5 (n"6) 173, which can be ‚-oxidised by rat hepatocytes to 22:5 (n"6) 171 (Scheme 43). Thus, the apparently simple ƒ4-desaturation of 170 to 171 involves chain extension by an elongase, ƒ6 desaturation, transfer to hepatocytes and ‚-oxidation. In a subsequent paper, Sprecher and co-workers then examined the regulation of (4Z,7Z,10Z,13Z,16Z,19Z)-docosa- 4,7,10,13,16,19-hexaenoyl CoA [22:6 (n"3)] 174 formation, the main polyunsaturated component of the membrane lipids.217 Sprecher proposes a scheme for the conversion of 20:5 (5Z,8Z,11Z,14Z,17Z) CoA 175 to 174 via 24:6 (6Z,9Z,12Z,5Z,18Z,21Z) CoA 176 in which substrates are elongated to 24:6 (n"3) 176 in the microsomes, and then transferred to peroxisomes for one round of ‚-oxidation and transfer back to a microsome as 22:6 (n"3) 174 (Scheme 44).This scheme relies upon slow peroxisomal oxidation and rapid microsomal esterification of 22:6 (n"3) 174, and slow microsomal esterification of 24:6 (n"3) 176 and 24:5 (n"3) 177. ‚-Oxidation of 24:6 (n"3) 176, 24:5 (n"3) 177 and 22:5 (n"3) 178 only requires standard ‚-oxidation enzymes.However, ‚-oxidation of 22:6 (n"3) 174 or 20:5 (n"3) 175 would require enzymes of the NADPH independent pathway (Scheme 41). Addition of 22:6 (n"3) 176 to peroxisomes (in the absence of microsomes) led to the accumulation of (2E,4Z,7Z,10Z,13Z,16Z,19Z)-docosa-2,4,7,10,13,16,19- heptaenoyl CoA 179, suggesting that 179 was not a substrate for the 2,4-dienoyl CoA reductase. However, addition of 20:5 (n"3) 175 to peroxisomes led to ‚-oxidation, implying that (2E,4Z,8Z,11Z,14Z,17Z)-icosa-2,4,8,11,14,17-hexaenoyl CoA 180 was a substrate for the 2,4-dienoyl CoA reductase This remarkable subtle selectivity of the 2,4-dienoyl CoA reductase between (2E,4Z,8Z .. .) and (2E,4Z,7Z . . .) substrates is presumably the basis for the selective accumulation of 22:6 (n"3) 174 in the membranes (Scheme 45). Naval and co-workers have examined desaturase activity in human cell lines and find evidence of two 6Z desaturases involved in 22:6 (n"3) 174 biosynthesis in human cells.218,219 Earlier reports failed to locate ƒ6 desaturase activity in K562 human leukaemia cells.220 172 24:5 ( n–6) 173 b-Oxidation in hepatocytes Malonyl CoA NADPH D6 Desaturase D4 Desaturase COSCoA 22 16 Microsomes 16 13 7 22 COSCoA 10 7 15 12 4 24 18 9 COSCoA Microsomes 12 9 24 18 15 COSCoA 6 13 10 Me Me Me Me (peroxisomal) 22:4 ( n–6) 170 22:5 ( n–6) 171 24:4 ( n–6) Scheme 43 Microsome/endoplasmic reticulum Peroxisomes 24:5 ( n–3)-CoA 24:6 ( n–3)-CoA 22:6 ( n–3)-CoA 22:5 ( n–3)-CoA 20:5 ( n–3)-CoA Acetyl CoA Acetyl CoA Acetyl CoA FAS 6 Z Desaturase FAS slow slow Dietary linolenate (18:3) Elongases and desturases in the ER NADPH Independent b-oxidation b-Oxidation b-Oxidation 22:7 2 E ( n–3) NADPH Independent b-oxidation b-Oxidation 175 176 177 178 174 179 [ via 20:6 2 E ( n–3)-CoA] 180 fast 2,4-dienoyl CoA reductase 22:6 3 Z ( n–3)-CoA Esterification into membrane phospholipids Scheme 44 304 Natural Product Reports, 1998The first step in the conversion of arachidonic acid 165 to leukotrienes is catalysed by 5-lipoxygenase.Two new series of orally active potent inhibitors of this enzyme have been reported by Crawley and Briggs.221 Fatty acid amides have recently been isolated from the cerebrospinal fluid of sleep deprived cats.222,223 Intraventricular injection of nanomole quantities of (9Z)-octadec-9-enamide induces sleep. The formation of N-acylglycine in mammals by an acyl CoA:glycine N-acyl transferase in mammals is well known.224 Peptidyl glycine ·-amidating enzyme, converts C-terminal glycine peptides into chain shortened ·-amidated peptides by oxidative cleavage.Merkler et al. report that this enzyme can convert N-tetradecanoylglycine 181 into tetradecanamide 182, and that a diverse range of N-fatty acyl glycines, synthesised in the liver and circulating into the blood are there converted into the corresponding amides (Scheme 46).225 A major iodolipid found in the thyroid is 2-iodohexadecanal 183.Boeynaems and co-workers have incubated phosphatidylcholine containing plasmenylcholine 184 with lactoperoxidase, radio-iodine and hydrogen peroxide, and obtained radioactive 2-iodohexadecanal 185. A scheme is proposed in which a positive iodine species reacts with the vinyl ether of plasmenylcholine (Scheme 47).226 Banerjee and co-workers have purified human liver MMCoA mutase from Saccharomyces cerevisiae in active homodimeric form, that interconverts MMCoA 186 and succinyl CoA 187 (Scheme 48).Banerjee and co-workers have examined its inhibition by 2,2-ethanomalonyl CoA 188, which Me COSCoA Me COSCoA 176 DIGLYCERIDES D6-Desaturase 24 21 Me COSCoA 16 13 10 7 b-Oxidation b-Oxidation b-Oxidation 2,4-Dienoyl CoA reductase NADPH independent b-oxidation 20 17 11 8 5 20:5 ( n–3) (Not a substrate for 2,4-dienoyl CoA reductase) 22:5 ( n–3) 24:5 ( n–3) 177 Me COSCoA 178 175 Me Peroxisome COSCoA 22 4 7 10 13 16 19 24 18 15 12 21 9 6 1.Transfer to ER 22:6 ( n–3) 24:6 ( n–3) Further b-oxidation Me 174 179 20 17 14 8 2 COSCoA 11 20:5 ( n–3) 2. Esterification 18 15 12 9 22 19 NADPH independent b-oxidation 14 Me 22 4 7 10 13 16 19 2 (Accumulates in absence of ER) 180 COSCoA 4 22:7(2 E,4 E) ( n–3) Scheme 45 R SCoA O H2N CO2 H R N H CO2H O R NH2 O H CO2 H O 182 R = Me(CH2)13 + Ascorbate/O2 Semihydroascorbate + 181 R = Me(CH2)13 Scheme 46 O O O P O O– O NH3 + O R OH O O P O O– O NH3 + O R OHC I Me Me I Me HO I+ 183 184 185 [H] Scheme 47 S-CoA HO O O O S-CoA HO O HO Me S-CoA O O HO O 186 S-CoA O 189 187 188 Scheme 48 Rawlings: Biosynthesis of fatty acids and related metabolites 305is an analogue of the transition state 189.The mixed-type kinetics observed implied reversible binding to both enzyme and enzyme–substrate complexes, with the two non-equivalent active sites interacting.227 8 References 1 B.J. Rawlings, Nat. Prod. Rep., 1997, 14, 335. 2 B. J. Rawlings, Nat. Prod. Rep., 1997, 14, 523. 3 D. O’Hagan, Nat. Prod. Rep., 1995, 12, 1; 1993, 10, 593; 1992, 9, 447. 4 T. J. Simpson, Nat. Prod. 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ISSN:0265-0568    

DOI:10.1039/a815275y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

The dioxygenase-catalysed formation of vicinal cis-diols Derek R. Boyd and Gary N. Sheldrake School of Chemistry, David Keir Building, The Queen’s University of Belfast, Belfast, UK BT9 5AG Covering: up to 1997 1 Introduction 1.1 Earlier reviews 1.2 The role of mono- and di-oxygenase enzymes in arene metabolism 1.3 Types of arene dioxygenase biocatalysts 1.4 Types of dioxygenase-catalysed oxidations 1.5 Syntheses using arene cis-dihydrodiols 1.6 Industrial applications and potential 2 Structure and stereochemistry of cis-diols 2.1 Classification of substrates 2.2 Regioselectivity 2.3 Stereoselectivity 3 Enzymatic and chemoenzymatic routes to unusual cis-dihydrodiol regioisomers, enantiomers and other arene metabolites 4 Conclusions 1.Introduction 1.1 Earlier reviews This report on the current status of dioxygenase-catalysed oxidation of mono- and poly-cyclic aromatic ring systems to yield cis-dihydrodiols and derivatives provides both an update on earlier work1–3 and complements the recent review4 by Gibson and co-workers.The latter article focused on naphthalene dioxygenase as a biocatalyst for a range of diVerent types of oxidations including cis-dihydroxylation of polycyclic arene systems. The current report will concentrate on the wider range of arene cis-dihydrodiols obtained from arene substrates and mainly toluene (TDO) and, to a lesser extent, biphenyl (BPDO) and naphthalene (NDO) dioxygenases. Particular emphasis will be placed on regioselectivity and stereoselectivity of dihydroxylation observed during formation of the cis-diol metabolites of mono- and poly-cyclic aromatic and heteroaromatic ring systems. Less emphasis will be placed on the cis-dihydrodiol metabolites of benzoic acids obtained using benzoate dioxygenases (BZDO) which have been previously reviewed.5 Discussion of the reactions of the cis-dihydrodiol metabolites will be restricted to their stability and use in the synthesis of other cis-dihydrodiol isomers (enantiomers and regioisomers) not readily available by direct cisdihydroxylation.The related chemoenzymatic synthesis of other types of arene metabolites, e.g. phenols, arene oxides and arene trans-dihydrodiols, which are diYcult to obtain in acceptable yields by enzyme-catalysed oxidation of arenes in eucaryotic systems, will also be discussed. Many of the latter aspects have not previously been included in earlier reviews of the value of arene cis-dihydrodiols in organic synthesis.5–8 1.2 The role of mono- and di-oxygenase enzymes in arene metabolism The metabolism of aromatic rings in animals, plants and fungi (eucaryotes) has been widely assumed to involve monooxygenase-catalysed oxidation to yield phenols via an arene oxide intermediate9,10 The conclusion that arene oxide metabolites are involved has generally been inferred indirectly from studies of the migration and retention of 2H- or 3H-label during aromatic hydroxylation (the NIH shift).More recent studies have however shown that the NIH shift can occur via the intermediacy of either an arene oxide, the derived transdihydrodiol, (Scheme 1, Path A) or an arene cis-dihydrodiol (Scheme 1, Path B),11 and thus the assumption of a role for arene oxides in the metabolism of arenes to phenols based solely upon this indirect type of evidence must be treated with caution. Direct evidence in favour of arene oxide intermediates is however available from the isolation of arene oxide metabolites of methyl benzoate,12 naphthalene13 and quinoline.14 Dioxygenase-catalysed oxidation of arenes generally occurs in bacterial systems (procaryotes) to yield vicinal cisdihydrodiols as the initial bioproducts from an asymmetric dihydroxylation process.In this context, the isolation of the achiral vicinal cis-dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenzene (benzene cis-glycol), as a metabolite of benzene by Gibson et al.using a strain of the soil bacterium Pseudomonas putida proved to be a milestone in the establishment of this new class of metabolites.15 Over the intervening years it has been recognised that some dioxygenase enzymes, e.g. TDO, show a remarkably broad substrate specificity and this has led to a steady growth in the number of reports of cis-dihydrodiol metabolites in the literature. An earlier attempt to compile a list of known arene cis-dihydrodiol metabolites16 showed that by 1990 more than one hundred examples had been reported and this number is currently in excess of three hundred. A significant proportion of these cis-dihydrodiols have either been proposed as transient intermediates, or their presence has been based upon rather tentative structural evidence.Furthermore, in many cases both their enantiopurities and their relative/absolute configurations have been assumed without rigorous application of stereochemical analytical methods.Unfortunately, insuYcient space is available within this report to include all examples of the latter types and the present review will concentrate mainly on arene cis-dihydrodiols obtained from TDO-, NDO- and BPDO-catalysed oxidations whose structure, enantiopurity and absolute configuration have been firmly established. The dioxygenase-catalysed formation of cis-dihydrodiol metabolites and the monooxygenase/epoxide hydrolasecatalysed formation of trans-dihydrodiol metabolites of arenes O OH OH OH OH OH OH Path B Dioxygenase/O2 + H2O Epoxide hydrolase – 2H cis-Dihydrodiol dehydrogenase – H2O – 2H Arene Arene cis-dihydrodiol Arene oxide Catechol Arene trans-dihydrodiol Phenol OH Path A Mono- – H2O trans-Dihydrodiol dehydrogenase oxygenase/O2 Scheme 1 Boyd and Sheldrake: The dioxygenase-catalysed formation of vicinal cis-diols 309can be distinguished by the origin of their oxygen atoms.Thus, both oxygen atoms in a cis-diol are derived from atmospheric dioxygen in contrast to a trans-diol in which one oxygen atom is derived from dioxygen and one from water.Furthermore, the dioxygenase system will catalyse the oxidation of an arene bond to yield an arene cis-dihydrodiol whereas the monooxygenase system will give an epoxide, i.e. arene oxide, as the initial metabolite. Regioselectivity could, in principle, occur both in enzymecatalysed epoxidation (Scheme 2) and cis-dihydroxylation (Scheme 3) of the 1,2-, 2,3- and 3,4-bonds of a monosubstituted arene.In practice, the arene oxide metabolites have mostly proved to be too unstable to be isolated and their presence has been inferred from the formation of the corresponding ortho-, meta- or para-phenols. Use of 2H-labelled monosubstituted arenes (e.g. chlorobenzene and bromobenzene as substrates) and analysis of the derived phenol metabolites has also provided indirect evidence of 2,3-oxide formation during ortho-hydroxylation processes in fungi.17 As discussed earlier the isolation of the relatively stable 1,2-arene oxide (oxepine) metabolite of methyl benzoate from the wood-rotting fungus Phellinus tremulae provides unequivocal evidence of epoxidation at a 1,2-bond12 (Scheme 4).In general, however, evidence of regioselectivity during monooxygenasecatalysed epoxidation of arenes remains elusive due to the low yield and instability of the arene oxide intermediates. Prior to the studies of Gibson et al. in 1968,15 and the availability of suitable mutant strains, regioselectivity during dioxygenase-catalysed cis-dihydroxylation of monosubstituted arenes also proved diYcult to establish due to the transient nature of the arene cis-dihydrodiol intermediates.Thus, wildtype bacterial strains capable of using arenes as a carbon source generally contain cis-diol dehydrogenase enzymes which rapidly catalyse dehydrogenation of the initial cis-diol metabolites to yield the corresponding catechols (Scheme 1, path B) and which in turn undergo further dioxygenase-catalysed oxidation to yield ring-opened products.The availability of mutant strains of bacteria lacking a cis-diol dehydrogenase enzyme has allowed regioselectivity studies to be carried out by accumulation of the cis-dihydroxylation products of monosubstituted arenes. The majority of arene substituents appear to ‘direct’ the TDOcatalysed cis-dihydroxylation process exclusively to the 2,3- bond in monosubstituted arenes (Scheme 3).The carboxy group present in benzoic acids has, in some bacterial species, been found to direct the cis-dihydroxylation to the 1,2-bond yielding an ipso-cis-dihydrodiol.18,19 To date, little evidence of cisdihydrodiol formation at the 3,4-bond of a monosubstituted benzene substrate has been reported (Scheme 3). Monooxygenase-catalysed oxidations include alkene and arene epoxidation, aliphatic and aromatic hydroxylation, heteroatom oxygenation, ketone oxidation, dealkylation and deamination.Dioxygenase-catalysed oxidations include many of the latter types of oxidation (with the exception of epoxidation and ketone oxidation) but, uniquely, also catalyse alkene and arene cis-dihydroxylation (cf. Section 1.4). 1.3 Types of arene dioxygenase biocatalysts Dioxygenases responsible for the formation of cis-dihydrodiols from arene substrates appear to be most prevalent in procaryotic organisms. They have generally been found to contain multicomponent enzyme systems involving several proteins, non-haem iron and require NADH.1–4 To date dioxygenasecontaining organisms have been grown on a range of carbon sources and these enzymes have been classified accordingly, e.g.benzene (BDO), toluene (TDO), naphthalene (NDO), benzoate (BZDO) and biphenyl (BPDO). More than twelve types of multicomponent ring-hydroxylating dioxygenases have been identified and purified.20 The vast majority of reported biotransformations however appear to have been catalysed by TDO, NDO, BPDO and BZDO enzymes and this will be reflected in the cis-dihydrodiol bioproducts discussed herein.It is important to specify the particular mutant strain being used since they may provide diVerent types of dioxygenase and thus diVerent bioproducts. TDO-catalysed cis-dihydroxylation in cultures of Pseudomonas putida appears to be the least substrate-specific type of arene biotransformation. While some mutant strains (e.g. 39 D1,15 and UV421) of Pseudomonas putida provide a source of TDO, other mutants provide BZDOs (e.g. JT103,22 B13,23 mt-223 and BG119) or NDO (9816/11).4 Mutant strains of a Beijerinckia species (B8/36,24 recently reclassified as Sphingomonas yanoikuyae B8/3625) and Alcaligenes eutrophus (335 strain B9)18 have been found to contain BPDO and BZDO, respectively. All of these mutant strains have the cis-dihydrodiol dehydrogenase enzyme blocked and thus the initial cis-dihydrodiol metabolite will accumulate. The genes encoding the TDO components in both inducible strain 39D and constitutive strain UV4 have been cloned and expressed in Escherichia coli (pDTG 601)26 and (pKST 11),27 respectively.Similarly the genes encoding for the NDO components in Pseudomonas species 9816-4 have also been cloned and expressed in a strain of E. coli (pDTG 141).28 It is diYcult to provide definitive information on the most appropriate type of dioxygenase for a particular type of biotransformation.In general terms however the TDO system is most suitable for cis-dihydroxylation of substituted benzene substrates and bicyclic arenes, NDO is particularly useful for bi- and tri-cyclic arenes, BPDO is more appropriate for larger R R R R R O O O 1 2 3 4 5 6 1 2 3 4 5 6 1 2 2 3 3 4 Monooxygenase O2 3,4-bond oxidation 1,2-bond oxidation or or 2,3-bond oxidation Scheme 2 R R R R R OH OH OH OH OH OH 1 2 3 4 5 6 1 2 3 4 5 6 1 2 Dioxygenase O2 2 3 3 4 1,2-bond oxidation 2,3-bond oxidation or or 3,4-bond oxidation Scheme 3 MeO2C O CO2Me O CO2Me OH CO2Me Phellinus tremulae Scheme 4 310 Natural Product Reports, 1998(3–5 membered) polycyclic aromatic hydrocarbons (PAHs) and BZDO is required for benzoic acid substrates. 1.4 Types of dioxygenase-catalysed oxidations Using wild-type, mutant and recombinant strains of bacteria or the purified dioxygenase enzyme it has been shown that a remarkably wide range of oxidations occur.1–5 Thus monohydroxylations have been observed at methyl and methylene groups which are activated by neighbouring phenyl rings (benzylic hydroxylation), vinyl groups (allylic hydroxylation), carbonyl groups (keto alcohol formation), oxygen atoms (O-dealkylation), sulfur atoms (S-dealkylation) and nitrogen atoms (N-dealkylation).Dihydroxylation may occur either by sequential monohydroxylation at two benzylic centres, or by concomitant attack of an alkene or an arene by both atoms of a dioxygen molecule.A combination of monohydroxylation and cis-dihydroxylation (triol formation) and of two cisdihydroxylation steps (tetraol formation) has also been catalysed by the dioxygenase system. Desaturation reactions of both dihydro- and tetrahydro-arene rings to yield arenes, often followed by cis-dihydroxylation have also been catalysed by dioxygenases. Sulfur atom oxygenation has been observed in dialkyl-, alkylaryl-, diaryl-sulfides, thioacetals and thiophenes to yield the corresponding sulfoxides.While the focus of this report will remain on cis-dihydroxylation, this pathway can also be combined with other dioxygenase-catalysed oxidation pathways using a single substrate. 1.5 Syntheses using arene cis-dihydrodiols The synthetic chemistry exploiting the new chiral centres created in the cis-dihydroxylation of arenes was reviewed extensively during the early part of this decade5–7 and more recently by Hudlicky and Thorpe.8 This last review in particular illustrates how the complexity of natural product syntheses approached using these compounds has developed over the last decade and indicates how the area may continue to develop into the future.Synthetic aspirations have progressed from rapid, stereoselective preparations of cyclitols, such as pinitol29a and conduritol F 1,29b through relatively simple bicyclic alkaloids, such as (")-trihydroxyheliotridine 2 and its enantiomer,30 to the total synthesis of more challenging polycyclic natural products, such as pancratistatin 3.31 Reported within the last 18 months (to March 1997) have been several syntheses of 6-‚-hydroxyshikimic acid 4,32 enantiopure cisdecalins, e.g. 5,33 trans-dihydrodiols34a,34b and arene oxides,34a of the type shown in Schemes 1 and 2, and approaches to the morphine skeleton35 all of which have started with the cisdihydroxylation of simple mono- or di-substituted benzenes. Apart from the imaginative use of available cis-dihydrodiol metabolites as starting points for synthetic schemes, there have been a number of significant milestone advances which have contributed to the successful development of synthetic chemistry using these compounds.Firstly, a number of groups around the world, using a variety of organisms, have demonstrated the diversity of substrate range of the dioxygenase enzymes. The structures of a large number of arene cisdihydrodiols have been published and this subsection of the chiral pool now has a rich array of highly functionalised, enantiopure starting materials for the synthetic chemist to utilise.General methods for the unambiguous assignment of the absolute stereochemistry and enantiomeric purity of the cis-diols have been crucial in this respect and these are discussed at greater length in Section 2.3. The list of successful dioxygenase-catalysed arene cis-dihydroxylations continues to grow both through probing the limits of the substrate ranges of known organisms and also by the discovery and development of new mutant and recombinant organisms with diVerent dioxygenase enzyme systems.A second advance widened the range of available cis-diols further still by simple synthetic transformations to give compounds which are not readily accessible by direct biotransformation. For example, the alkylthio-substituted cis-dihydrodiols have been prepared36,102 by substitution of the corresponding iodo-compound.By contrast, direct biotransformation of the corresponding alkyl phenyl sulfides gives a very poor yield of the cis-diols with sulfoxidation occurring in preference (Scheme 5). A third major synthetic advance came with the application of enantiodivergence8 which, in certain cases, has overcome the problem that only one enantiomer of the bioproduct is available in most cases. The preparation of D- and L-erythrose derivatives (6, Scheme 6) by Hudlicky et al.37 was the first example of an enantiodivergent synthesis starting from a cis-dihydrodiol and there have been several more since, including the synthesis of both enantiomers of pinitol (7, Scheme 7).38 In an alternative approach to enantiocomplementarity, Boyd et al.39 demonstrated that both enantiomers of a range of monosubstituted cis-dihydrodiols 8 could be prepared (Scheme 8).The ‘normal’ enantiomers 8a were obtained by biotransformation of the parent monosubstituted benzene in the usual way.The enantiomers were prepared from the partially racemic iodo cis-dihydrodiols 9 which were obtained by biotransformation of the corresponding 1,4-disubstituted benzenes. A chemoenzymatic sequence of hydrogenolysis to remove the iodine and kinetic resolution of the mixture of enantiomers of the monosubstituted diols using the diol NH O O N HO OH OH OH H OH HO OH OH OH OH HO HO HO OH OH OH O CO2H O OH OH 1 2 3 4 5 SR I OH OH SR OH OH RS– O2/ P.putida UV4 <1% Scheme 5 Cl OH OH O O O OH 6 O O O HO Scheme 6 Boyd and Sheldrake: The dioxygenase-catalysed formation of vicinal cis-diols 311dehydrogenase enzyme present in a wild-type strain of P.putida allowed the isolation of the ‘unnatural’ isomers (8b) for the first time. Apart from total syntheses of increasingly complex natural products, the unique combination of stereochemistry and functionality of the arene cis-dihydrodiols has facilitated the synthesis of metabolites from the eucaryotic monooxygenase series which have been diYcult to isolate directly or prepare by other means.Thus, syntheses of trans-dihydrodiols and arene oxides have been reported from cis-dihydrodiols of mono- or di-substituted benzenes (cf. Section 3). With the range of cis-dihydroxylation reactions now extending to larger heterocyclic substrates (Section 2), eYcient routes to larger alkaloids, sugars and other natural product targets will undoubtedly become more accessible. 1.6 Industrial applications and potential In the mid-1980s, when biologically-derived arene cisdihydrodiols first became commercially available, there was an air of anticipation and expectation about the synthetic potential of these compounds and the ‘obvious’ benefits to be derived from the predictable and controlled preparation of a range of functionalised molecules containing two new chiral centres from simple and cheap aromatic precursors.Indeed, the previous section illustrates the increasing scope and complexity of synthetic schemes based on arene cis-dihydrodiol synthons from academic laboratories around the world.It was surely only a matter of time before the development of a multi-tonne pharmaceutical or agrochemical product based on cis-dihydrodiol technology. However, in spite of the fact that the technology to produce cis-dihydrodiols by whole cell biotransformation of arenes on multi-kilogram and even multi-tonne scale has been available to several companies for well over ten years, the world demand for most of these bioproducts has, at best, not exceeded a few tens of kilograms.To date, the only commercially manufactured products which are known to incorporate an arene cis-diol derivative are the three illustrated in Fig. 1. The preparation of polyphenylene from benzene cisdihydrodiol was first reported in 198321 and has been reviewed extensively elsewhere.40,41 The ‘green’ manufacture of indigo via the cis-dihydrodiol of indole using a recombinant strain of E.coli containing NDO cloned from a P. putida species caused much discussion and general interest when it was reported in 1995.42 Most recently, the Merck HIV protease inhibitor indinavir has been produced using a cis-dihydroxylation of indene as one approach to the synthesis of the aminoindanol intermediate.8,43,132 The reasons for this slow development of commercial exploitation are not entirely clear. There has certainly been a significant amount of patent activity in the area over the last decade as the various interested parties seek to protect the technology in anticipation of the ‘big’ product and in response to the rapid developments in the utilisation of these compounds in academic groups.There has been a considerable volume of published work over the years on the enhancement of the manufacturing process for arene cis-dihydrodiols from optimisation of the fermentation and biotransformation conditions to the extraction and isolation of the bioproducts.Many improvements of the biocatalyst performance have been discussed in depth elsewhere.1–4,20 One of the enduring problems of scale-up of these biotransformation processes is the high water solubility of the bioproducts coupled with (in many cases) their inherent instability. This adds an extra dimension to the process design which is often the slowest and least eYcient part of the overall process. The conventional extraction method involves centrifugation of the biotransformation liquor to remove cell debris followed by repeated liquid–liquid extraction with an organic solvent.In most cases, eYcient extraction of the cis-diol requires at least the addition of an inorganic salt to increase the ionic strength of the aqueous liquor and may also require preconcentration of the solution by evaporation under reduced pressure. In all cases, careful control of the pH and temperature of the aqueous solution is required to minimise losses through thermal or acid-catalysed decomposition.This extraction process is wasteful of both inorganic and organic materials, even with an eYcient recycle of the solvent, and OH HO OH OH OH MeO Br OH OH OH HO OMe OH OH HO 7 Scheme 7 X X OH OH X OH OH X OH OH X OH OH I I 8a 8b 9 H2/cat. cis-Diol dehydrogenase TDO + 8b Scheme 8 NH N N N NH HN O O O N H Ph HN O OH OH OH OH OH OH OH OH n Polyphenylene Indigo Indinavir Fig. 1 312 Natural Product Reports, 1998often results in a product solution which requires further purification to remove polymeric and phenolic by-products.More eYcient alternatives to this conventional approach have been examined and some of this development work has been published in the patent and journal literature. The selective removal of the cis-diols onto a solid medium followed by elution has been a common theme. Various activated carbons have been employed, most recently the commercially available Ambersorb XEN-575 (Rohm & Haas).44 Continuous extraction via two-phase biotransformations is an attractive option both from a point of view of isolation of the product but also as a potential method for prolonging the activity of the catalyst since many cis-diols are toxic to the organisms.There has been some recent success45 and this is still an active area of research and development. An alternative method for the selective removal of the cis-diol from the biotransformation medium without the use of solvents was disclosed by ICI in 199046 and exploits the poor aqueous solubility of cyclic areneboronate esters of the cisdiols at neutral pH which allows these esters to be precipitated directly from clarified biotransformation media.The problems of eYcient recovery of the cis-diols from the esters and recycling of the areneboronic acid are still the subject of research. The principle of aYnity through molecular recognition in extraction processes, particularly when the product is of high added value, is likely to become more attractive as the legislative and economic drive for cleaner downstream processing of biotransformation processes continues. 2 Structure and stereochemistry of cis-diols 2.1 Classification of substrates Since the last comprehensive reviews in the area, the remarkable diversity of acceptable substrates for dioxygenase enzymes has been exploited leading to the reports of hundreds of new diol metabolites.This section aims to provide, as far as possible, a comprehensive report of diols which have been isolated and characterised (at least to the extent of rigorous assignment of absolute configuration and preferably with an optical rotation and an estimate of enantiomeric excess). Generally, reports have not been included where cis-diols have been only postulated as metabolic intermediates, or where no supporting evidence for the proposed structure has been given.Many references18,22,47–71 give considerable experimental and analytical data on the isolation and characterisation of a diverse range of cis-dihydrodiols but do not include an assignment of absolute configuration. The authors have recently become aware of another review article in preparation72 which includes structures from many of these references and is thus complementary to this work. By far the largest number of reported substrates have been monocyclic aromatic compounds with varying degrees of substitution.In the case of monosubstituted benzene rings (see Table 1), several of the early generalisations made about the cis-dihydrodiol formation have been found to be still valid and have fostered much speculation on the nature of the active sites of the dioxygenase enzymes with the most extensive studies of R OH OH R 98% ee except 38 Table 1 2,3-cis-Dihydrodiols from monosubstituted benzenes Cpd R [·]D Enzyme Cpd R [·]D Enzyme 10 H achiral15 TDO 40 (CH2)2SCN +92 (MeOH)107 TDO 11 D "9 (MeOH)102 TDO 41 (CH2)2SPh +81 (CHCl3)107 TDO 12 CH3 +26 (MeOH)92,104 TDO 42 (CH2)2NCS +162 (MeOH)107 TDO 13 CH2CH3 +40 (MeOH)103,105 TDO 43 (CH2)3Br +88 (MeOH)103 TDO 14 (CH2)2CH3 +65 (MeOH)103 TDO 44 CH2CN +13 (MeOH)108 TDO 15 (CH2)3CH3 +87 (MeOH)103 TDO 45 CH2OAc +104 (MeOH)102 TDO 16 (CH2)4CH3 +113 (MeOH)103 TDO 46 Fb "33 (CHCl3)92,102 TDO 17 CH(CH3)2 +17 (MeOH)103 TDO 47 Cl +36 (CHCl3)92,102,113 TDO 18 CH(CH3)CH2CH3 +27 (MeOH)103,106 TDO 48 Br +20 (MeOH)92,102 113 TDO 19 CH2CH(CH3)2 +94 (MeOH)103 TDO 49 I +41 (MeOH)92,102,113 TDO 20 CH2C(CH3)3 +152 (MeOH)103 TDO 50 OCH3 +44 (CHCl3)92,102 TDO 21 C(CH3)3 +28 (MeOH)103 TDO 51 OCH2CH3 +51 (CHCl3)118 TDO 22 CH=CH2 +115 (MeOH)102,114,115 TDO 52 CN +201 (MeOH)102 TDO 23 CH2CH=CH2 +16 (MeOH)102 TDO 53 CF3 "63 (MeOH)102 TDO 24 C(CH3)=CH2 +87 (MeOH)111 TDO 54 COCH3 +98 (MeOH)102,105 TDO 25 CH=CHCH3 +128 (MeOH)111 TDO 55 SCH3 +37 (MeOH)109,27 TDO 26 CH=CHCH3 (E) +78 (MeOH)111 TDO 56 SCH2CH3 +60 (CHCl3)102 TDO 27 C=CH +194 (MeOH)102,112,114 TDO 57 SPh +20 (MeOH)102 TDO 28 CH2OH +35 (MeOH)103 TDO 58 SCH(CH3)2 +50 (CHCl3)102 TDO 29 CH(OH)CH3 a +54 (MeOH)103,105 TDO 59 SC(CH3)3 +160 (CHCl3)102 TDO 30 CH(OH)CH2CH3 a +24 (MeOH)103 TDO 60 S(O)CH3 +77 (CHCl3)27 TDO 31 CH(OH)(CH2)2CH3 a +23 (MeOH)103 TDO 61 CH[-S(CH2)3S-] +66 (CHCl3)27 TDO 32 CO2Me +59 (CH2Cl2)32a TDO 62 Ph +252 (MeOH)109,110 TDO 33 CH[-O(CH2)2O-] +70 (THF)117 TDO 63 4-H3CPh "16 (CHCl3)110 TDO 34 CH2OCH3 +78 (MeOH)108 TDO 64 4-ClPh +1256 (MeOH)110 TDO 35 (CH2)2Br +90 (CHCl3)103 TDO 65 2,3,5,6-(CH3)4Ph "39 (MeOH)110 TDO 36 (CH2)2CN +97 (MeOH)107 TDO 66 1-Naphthyl +125 (MeOH)110 TDO 37 (CH2)2OAc +41 (CHCl3)107 TDO 67 2-(H3CO)Ph +160 (CHCl3)116 TDO 38 (CH2)2OH +45 (MeOH)107 TDO 68 2,3-(H3CO)2Ph +63 (CHCl3)116 TDO 39 (CH2)2N3 +93 (MeOH)107 TDO aR configuration at benzylic position. b60–80% ee.Boyd and Sheldrake: The dioxygenase-catalysed formation of vicinal cis-diols 313substrate range having been carried out for TDO-containing organisms.Charged or very polar substituents usually render a compound resistant towards dioxygenase-catalysed dihydroxylation. Thus, benzoic acids, benzenesulfonates, sulfoxides, sulfones and simple anilines and phenols have not been reported as successful substrates for TDO systems and such compounds are generally metabolised via diVerent pathways. Nitrobenzene was also thought to belong to this group until relatively recently but there is now direct evidence of the formation of nitrobenzene cis-dihydrodiol with a number of TDO-containing organisms.47,48 However, in spite of these exceptions, the dioxygenase enzymes are capable of accepting arene substrates with a remarkably wide range of substituent types; from electron-donating groups (e.g.methoxy) to strongly electron-withdrawing groups (e.g. acetyl). Benzoic acids, as mentioned earlier, are metabolised to ipso-cis-diols but only by organisms containing a BZDO enzyme for which a benzoic acid functionality is a prerequisite.Unsurprisingly, the extent of metabolism decreases with an increasing number of substituents on the benzene ring (Table 2). This is particularly pronounced when several electron-withdrawing substituents (e.g. Cl) are present, reducing the electron density of the reacting C–C bond. In addition, there is now considerable evidence that two substituents in a 1,3-arrangement are likely to have an increased inhibitory eVect on the rate and extent of ring dihydroxylation compared with the same substituents positioned 1,2 or 1,4 (the so-called ‘meta eVect’).The regioand stereo-chemical consequences of dihydroxylation of multiply substituted benzene rings are discussed in the following sections. There is a size limitation on substrate acceptability for TDO. Thus, cis-dihydrodiols of alkylbenzenes with substituents from R1 OH OH R1 R2 R3 R4 R2 R3 R4 Table 2 cis-Dihydrodiols from di- and poly-substituted benzenes Cpd R1 R2 R3 R4 [·]D % ee Enzyme 69 Me F H H "32 (CHCl3) >98123 TDO 70 F F H H" 92 (MeOH) 64108 TDO 71 Cl F H H "13 (MeOH) >98123 TDO 72 Br F H H +6 (MeOH) >98123 TDO 73 I F H H +20 (MeOH) >9839 TDO 74 CN F H H +80 (MeOH) >98123 TDO 75 I Cl H H +81 (MeOH) >9839 TDO 76 I Br H H +75 (MeOH) >9839 TDO 77 I Me H H +48 (MeOH) >9839 TDO 78 (CH2)2Br Br H H +72 (MeOH) >95125 TDO 79 CH=CH2 Br H H nra 91120 TDO 80 CH=CH2 Cl H H nra >98119 TDO 81 CF3 F H H "65 (MeOH) >98123,124 TDO 82 Cl Cl H H +65 (MeOH) >9874 TDO 83 F H F H unstable 56108 TDO 84 Me H F H +118 (MeOH) >98123 TDO 85 Cl H F H +61 (MeOH) nr9,123 TDO 86 Br H F H +61 (MeOH) nr9,123 TDO 87 I H F H +74 (MeOH) >98123 TDO 88 I H Br H +28 (MeOH) >9839 TDO 89 CF3 H F H "26 (MeOH) >98123 TDO 90 CH=CH2 H Cl H nra 54119 TDO 91 Cl H Cl H unstable >9874 TDO 92 F H H F achiral —46 TDO 93 Cl H H Cl achiral —126 b 94 I H H F +62 (MeOH) 8875 TDO 95 I H H Cl +6 (MeOH) nr9,75 TDO 96 I H H Br +5 (MeOH) nr9,75 TDO 97 I H H Me +3 (MeOH) 8039 TDO +4 (MeOH) >9839 TDOc 98 Br H H Me "9 (MeOH) 3775 TDO 99 Cl H H Me "12 (MeOH) 1575 TDO 100 Me H H F +132 (MeOH) 8675 TDO 101 Me H H Me achiral —122 TDO 102 Cl H H F +54 (MeOH) 60123 TDO 103 Br H H F +34 (MeOH) 56123 TDO 104 I H H F +62 (MeOH) 8875 TDO 105 CH=CH2 H H Cl nra 15119 TDO 106 CF3 H H I "56 (MeOH) >9875 TDO 107 CF3 H H Me "119 (MeOH) >9875 TDO 108 CF3 H H F "35 >98123,124 TDO 109 CN H H F +69 nr9,123 TDO 110 CO2H H H Br +35 >98121 JT107 anr=not reported. bUnspecified dioxygenase.cE. coli JM109 (pDTG601). 314 Natural Product Reports, 1998methyl to n- or iso-butyl have been isolated with reasonable yields but tert-butylbenzene and n-pentylbenzene are poor substrates and the cis-dihydrodiol metabolites of higher homologues are unknown. Both naphthalene and biphenyl are good substrates for TDO enzymes, perhaps indicating that flat, rigid molecules fit better into the active site than molecules with bulky, flexible, substituents.For larger PAHs, both NDO and BPDO are capable of catalysing dihydroxylation, with only the latter enzyme capable of metabolising tetracyclic or larger examples (Table 3). By contrast, the NDO enzyme appears to be generally incapable of catalysing the cis-dihydroxylation of monocyclic aromatic rings.The 1,2- (or ipso) cisdihydroxylation of substituted benzoic acids has been studied extensively4,49–54 but relatively few of the product diols have fully assigned absolute stereochemistries. Dihydroxylation of di- and tri-cyclic substrates at substituted or angular arene bonds has yielded a series of cis-monohydrodiols (132–136, Table 4). The metabolism of heterocyclic compounds (Table 5) has been studied using most of the available dioxygenase systems. In general, when presented with a choice of carbocyclic or heterocyclic aromatic rings, the dioxygenase enzymes will normally give metabolites with a cis-diol in the carbocyclic ring.Dioxygenation in the heterocyclic rings of quinoline and isoquinoline was implied by the isolation of phenolic metabolites thought to have arisen by dehydration of a cisdihydrodiol. 73 To date, however, the only reported cisdihydrodiol of a pyridine ring has been the diol amide (143, Table 5) isolated from the metabolism of 2-chloroquinoline with P.putida UV4.88 The metabolism of benzofurans and benzothiophenes does lead to dihydroxylation of the heterocyclic ring in addition to diol formation in the benzene ring (see Section 2.2).84,85 More recently, attention has turned to the dihydroxylation of non-aromatic double bonds. Much of this work is still in progress and most published results in this area have been with benzocycloalkene or analogous substrates (Table 6). Opposite stereochemical preferences were found for alkene cisdihydroxylations of indene, 1,2-dihydronaphthalene and 1,2- benzocyclohepta-1,3-diene with TDO and NDO enzymes (see Section 2.3). 2.2 Regioselectivity The combination of a low degree of substrate specificity combined with a high degree of regioselectivity is a characteristic feature of dioxygenase-catalysed cis-dihydroxylation of monosubstituted benzene substrates. Thus, to date more than 50 cis-dihydrodiols have been formed as a result of cisdihydroxylation at the 2,3-bond of monosubstituted benzene substrates using TDO.The BZDO-catalysed dihydroxylation of benzoic acid provides the sole example of attack at a 1,2-bond in a monosubstituted benzene substrate and no examples of dioxygenase-catalysed cis-dihydroxylation at a 3,4-bond have been found (Scheme 3). The TDO system has thus generally been found to yield cis-dihydrodiols as a result of oxidation at an unsubstituted arene 2,3-bond. By contrast, the benzoate dioxygenase system will prefer to attack arene 1,2-bonds bearing a carboxy substituent.With the exception of biphenyl,4 the NDO system has not been reported to catalyse the cis-dihydroxylation of monocyclic arenes at any bond. As previously discussed in Section 2.1, although showing a wide range of substrate acceptability, the TDO-containing mutant strains appear reluctant to dihydroxylate substrates containing some types of substituents, e.g. hydroxy, amino, carboxy, nitro, sulfonic acid and sulfone groups, directly linked to the benzene ring.The polarity and/or size of such substituents may be important factors in substrate acceptability. Furthermore, the ability of the dioxygenase system to catalyse monohydroxylation, sulfoxidation, dealkylation and desaturation of substituents can often result in both concomitant oxidation of the substituent and cis-dihydroxylation of the benzene ring. Regioselectivity during TDO-catalysed cis-dihydroxylation of disubstituted benzene substrates is restricted to unsubstituted arene bonds.Similarly a strong preference for oxidation of carboxy-substituted bonds is shown during BZDOcatalysed oxidations of benzoic acids. Thus for paradisubstituted benzene substrates (Scheme 9), TDO systems will catalyse attack at bond b (R,R*|CO2H) and BZDO will favour dihydroxylation at bond a (R=CO2H). Studies with TDO suggest that the size of spherically symmetrical substituents (determined from the Taft, E*s, or Charton, Ì, steric parameters) will have a predictable eVect on regioselectivity.75 With ortho-disubstituted benzene substrates (Scheme 10), TDO-catalysed oxidation will occur at bonds b or d with a preference being shown for b where substituent R was larger than R* (and for d where R* was larger than R).39,75 Thus the directing eVect of the substituent will decrease across the series in the sequence CF3>I>Br>Cl~Me>F>H.Where both substituents are of similar size, e.g.Me and Cl, two cisdihydrodiols are formed due to oxidation at both bonds b and d. With non-symmetrical groups, e.g. cyano-, vinyl-, alkoxyand alkylthio-, these appear to have a relatively strong directing eVect compared with the symmetrical substituents.74 However, predictions of regioselectivity are more diYcult with the latter category of substituents due to: (i) uncertainty about the eVective size of substituent in diVerent conformations, (ii) product instability and (iii) further dioxygenase-catalysed oxidation of the substituent. Oxidations of ortho-disubstituted benzene substrates using BZDO will again occur preferentially at a bond bearing a carboxy group.51 Regioselectivity found when metadisubstituted benzene substrates are dihydroxylated using TDO is similar to that for ortho-disubstituted benzene substrates; i.e.a preference for attack at bonds b or c with a preference for bond b when R is larger (see Scheme 13).39,75 When the R substituent in a meta-disubstituted benzene substrate is a carboxy group then cis-dihydroxylation will occur at both bonds a and f in the presence of BZDO.51 A similar pattern of regioselectivity was observed in the oxidation of trisubstituted benzene substrates using TDO and BZDO.Regioselectivity has been observed during dioxygenasecatalysed cis-dihydroxylation of polycyclic arenes and heteroarenes. When linear carbocyclic aromatic substrates e.g. naphthalene76,77 (TDO, NDO) and anthracene78,79 (NDO, BPDO) are examined, dihydroxylation will occur exclusively at the 1,2- (non-K region) bond (Table 3).For angular carbocyclic aromatic substrates e.g. phenanthrene78,80 (NDO, BPDO), chrysene81 (BPDO), benz[a]anthracene82,83 (BPDO) and benzo[a]pyrene83 (BPDO), a strong preference for dihydroxylation at the bay-region bond is observed with a minor proportion of oxidation occurring at non-K positions (Table 3). It is noteworthy that little evidence has been obtained for attack at the region of highest electron density in the angular arenes i.e.the K-region with either the NDO or BPDO enzymes. The presence of one or more heteroatoms in the arene ring has been found to exert a strong influence on the regioselectivity of dioxygenase-catalysed dihydroxylation. A range of carbocyclic ring cis-dihydrodiols has been found using the bicyclic heteroarenes quinoline,73 isoquinoline,73 quinoxaline, 73 quinazoline,73 benzothiophene,84 2-methylbenzothiophene, 84,85 benzofuran,86 and 2-methylbenzofuran85,86 as R R¢ R R¢ OH OH R R¢ OH OH R R¢ b a a c c b c a b a b c a b para-Disubstituted benzenes or O2 Dioxygenase Scheme 9 Boyd and Sheldrake: The dioxygenase-catalysed formation of vicinal cis-diols 315substrates with TDO (Table 5).This preference for the formation of carbocyclic cis-dihydrodiols is also observed in the tricyclic heteroarene substrates dibenzothiophene4,87 (TDO, NDO), dibenzofuran4,74,111 (TDO,NDO) and acridine74 (BPDO) (Table 5).cis-Dihydroxylation of the carbocyclic arene ring is generally preferred over the heterocyclic ring. However, where no carbocyclic alternative exists, dihydroxylation will occur in the heteroarene ring itself. Thus with thiophene85 (TDO) and 3-methylthiophene85 (TDO) oxidation has in each case yielded a cis-dihydrodiol which, being a hemithioacetal, can interconvert spontaneously in solution to yield a trans-dihydrodiol via an acyclic aldehyde intermediate (Scheme 11, X=S).cis/trans-Diol metabolites have been isolated after dihydroxylation in the unsubstituted heterocyclic rings of benzothiophene84,85 (TDO) and benzofuran85 (TDO). Furthermore TDO-catalysed dihydroxylation also occurs at the substituted 2,3-bond of 2-methylbenzothiophene85 and 2-methylbenzofuran85 (Table 5). Evidence of oxidative attack in the azaheterocyclic rings of quinoline (TDO), isoquinoline (TDO), quinoxaline and quinazoline (TDO) is based solely on the isolation of the corresponding phenols.73 It is thus assumed that where cis-dihydrodiol metabolites of six-membered Table 3 cis-Dihydrodiols from polycyclic aromatic hydrocarbons Substrate Product [·]D % ee Enzyme Substrate Product [·]D % ee Enzyme Br Br Me Me Me Br Br Br OH OH OH OH OH OH OH OH OH OH OH OH OH HO OH OH 111 112 113 114 115 116 120 121 MeO MeO OH OH OH OH MeO HO OH MeO 117 118 119 +218 (MeOH) +28 (CHCl3) +84 (MeOH) +255 (MeOH) nra nr +247 (MeOH) +166 (MeOH) "225 (MeOH) +255 (MeOH) +35 (MeOH) >98 >98129 >98129 >98129 nr130 nr130 >98134 >98134 >98134 >9879 >9878,80 TDO81 NDO76 NDO NDO NDO NDO NDO TDO, NDO, BPDO TDO NDO BPDO TDO NDO BPDO 123 124 125 126 127 128 129 130 122 OH OH OH OH OH OH OH OH OH OH OH HO HO OH OH HO OH OH +81 (THF) "37.2 (THF) +361 (THF) +112 (MeOH) +80 (CHCl3) "16 (CHCl3) nr +95 (MeOH) +186 (MeOH) >9882 >9882 >9882 >9881 >9874,83 >9889 >98131 >98131 >98131 BPDO BPDO BPDO BPDO BPDO TDO NDO NDO NDO anr=not reported. 316 Natural Product Reports, 1998azaheterocyclic rings are formed they are unstable and will readily dehydrate to the corresponding phenols. Stronger evidence of cis-dihydroxylation in a pyridine ring was obtained when the cis-dihydrodiol of quinol-2-one was isolated as a minor product from TDO-catalysed oxidation of 2- chloroquinoline88 (Table 5). The latter cis-dihydrodiol could be formed as a hydrolysis product of the initial unstable cis-dihydrodiol metabolite of 2-chloroquinoline or as a metabolite of quinol-2-one obtained by hydrolysis of 2-chloroquinoline.88 Bicyclic and tricyclic substrates containing fused benzene and non-aromatic ring systems (benzocyclobutene,89,90 TDO; fluorene,4,91,127 NDO; 9,10-dihydroanthracene,101 NDO; 9,10- dihydrophenanthrene,101 NDO), show a similar pattern of regioselectivity to that found in ortho-disubstituted benzene substrates during dioxygenase-catalysed oxidation to yield relatively unstable cis-dihydrodiols (Table 3).A diVerent pattern of regioselectivity i.e. ipso-oxidation at a substituted angular bond has however been observed when dioxygenase enzymes are used with the substrates benzocyclobutene89,90 (TDO), benzocyclobutenol89,90 (TDO), biphenylene89,90 (TDO) and fluorene91,127 (unspecified dioxygenase type) to yield the corresponding remarkably stable cis-monohydrodiols (Table 4). 2.3 Stereoselectivity Major advances in the study of stereoselectivity of dioxygenase-catalysed cis-dihydroxylation of arenes have resulted from the recent development of generally applicable methods for the determination of enantiopurity. The instability of many cis-dihydrodiols precluded the direct formation of diastereoisomeric esters using chiral acids, e.g. R- or S-·- methoxy-·-(trifluoromethyl)phenylacetic acid (MTPA). The formation of syn-cycloadducts between the cis-dihydrodiol metabolite and 4-phenyl-1,2,4-triazoline-3,5-dione followed by conversion to the corresponding diMTPA esters and 1H NMR analysis provided the first generally applicable method for enantiopurity determination (Scheme 12, A).92,102 Further advantages of this method include the formation of crystalline products which allow absolute configurations to be confirmed by X-ray crystallography, good resolution of signals using 19F NMR analysis and ready availability of both R and S enantiomers of MTPA.A more recent method for enantiopurity determination of cis-dihydrodiols involves the direct formation of diastereoisomeric boronate esters using R- and S-2-(1- methoxyethyl)benzeneboronic acid (MPB) followed by 1H NMR analysis (Scheme 12, B).93 Additional attractive features of this method are the smaller quantity of cis-dihydrodiols required and the rapid rate of reaction which allows less stable metabolites to be analysed under milder conditions. The most sensitive and direct method for enantiopurity determination of cis-dihydrodiols involves the use of chiral stationary phasehigh pressure liquid chromatography (CSP-HPLC).While the latter method is clearly preferred it suVers from the disadvantage of requiring both enantiomers to be available in order to demonstrate their separation. Since the majority of cisdihydrodiol metabolites are rather diYcult to obtain in racemic form and appear to be enantiomerically homogeneous, the CSP-HPLC analysis method75 is generally used in a complementary manner with the other methods.92,93,102 Techniques used for the determination of absolute configuration of cis-dihydrodiols have included X-ray crystallography of crystalline diols85,128 and derivatives, stereochemical correlation with compounds of known configuration involving chemical conversion and synthesis of cis-diols,86 circular dichroism, 81 and more empirical methods depending upon trends in the NMR spectra of diastereoisomeric derivatives. 92,93 Since the recent methods for enantiopurity determination have generally only been applied to the cis-dihydrodiol metabolites derived from TDO, NDO and BPDO-catalysed oxidations, discussions of stereoselectivity will be confined to the latter enzymes. Prior to the availability of generally applicable methods for determination of enantiopurity,92,93,102 it had been assumed that all cis-dihydrodiols obtained by TDO-catalysed oxidation of monosubstituted arenes were formed as single enantiomers.It is now accepted that, with the exception of fluorobenzene which yielded a cis-dihydrodiol of lower enantiomeric excess (60–80%), all cis-dihydrodiol metabolites of monosubstituted benzene substrates obtained using TDO are essentially single enantiomers of the absolute configuration shown (Fig. 2). One factor in the reduced stereoselectivity observed with fluorobenzene as substrate may be the relatively small size of the fluorine atom which allows more flexibility of the substrate within the active site of the TDO enzyme.Similarly, where cis-dihydrodiol metabolites of ortho- and meta-disubstituted benzene substrates have been obtained using TDO and analysed stereochemically, they appear to be enantiopure with the Table 4 cis-Monohydrodiol formation at substituted aromatic bonds Substrate Product [·]D % ee Enzyme 132 133 134 135 136 131 CO2H OH HO2C OH OH OH OH OH OH OH OH OH OH OH O OH OH "106 (EtOH) >98128 BZDO "166 (CHCl3) >9890 TDO "197 (CHCl3) >9890 TDO +429 (MeOH) >9890 TDO nra >9893 b +132 (MeOH) >98127 c anr=not reported.bCarbazole dioxygenase. cDioxygenase-containing organism grown on fluorene. R R HO HO R R¢ R R¢ R¢ R¢ R OH R¢ OH HO HO e d a b c a b c a b d e d ortho-Disubstituted benzenes O2 Dioxygenase or or Scheme 10 Boyd and Sheldrake: The dioxygenase-catalysed formation of vicinal cis-diols 317Table 5 cis-Dihydrodiols from heterocyclic arenes Substrate Product [·]D % ee Enzyme Substrate Product [·]D % ee Enzyme N N N N N N N N N N N N N NH O Cl Cl Cl OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH N N N O N N N O O OH O OH OH OH OH OH OH OH OH OH 138 139 140 141 142 149 148 147 146 137 145 144 143 +220 (THF) >9873 TDO S O O S O O S O OH OH OH OH OH OH HO OH OH OH S S S S S S S S S OH OH OH OH OH OH OH OH OH HO 150 152 151 153 154 159 155 157 158 156 +201 (CHCl3 )a 8085 TDO +45 (MeOH) >9873 TDO nrb +217 (CHCl3) >954 >98111 NDO TDO +163 (MeOH/ THF) >9873 TDO nrb >95111 NDO ca. 0 <273 TDO +98 (CHCl3) >9884 TDO +140 (MeOH) >98117 TDO +117 (CHCl3) >9884 TDO +148 >98117 TDO +125 (MeOH) >9885 TDO +6 (MeOH) >98117 TDO +1 (CHCl3)a 985 TDO +210 (MeOH) >9873 TDO +231 (MeOH) >9887 BPDO impure >9873 TDO "4 (CHCl3) a 4385 TDO +23 (MeOH) >9874 BPDO 15 (CHCl3)a 4885 TDO "35 (MeOH) >98133 TDO +16 (MeOH) >9885 TDO "34 (CHCl3)b 5585 TDO aCharacterised as benzeneboronate ester.bnr=Not reported. 318 Natural Product Reports, 1998exception in each case of the difluorobenzene substrates.74,108 The lower ee values obtained for the cis-dihydrodiol metabolites of ortho-difluoro- (64%) and meta-difluoro-benzene (56%) (70 and 83, Table 2) may again be a result of the reduced steric requirements of the fluorine atoms. The absolute configuration of the preferred cis-dihydrodiol enantiomer from TDOcatalysed oxidation of mono- and di- (ortho- and meta- )substituted benzene substrates (R>R*) is shown in Figs. 2 and 3(a),(b). cis-Dihydroxylation at unsubstituted bonds in the arene rings of benzocyclobutene89,90 (TDO), fluorene4,91,127 (NDO), 9,10-dihydroanthracene4 (NDO) and 9,10- dihydrophenanthrene4 (NDO) was in each case found to yield a single enantiomer of identical absolute configuration to that of cis-dihydrodiol metabolites of ortho-disubstituted benzene substrates [Fig. 3(a)]. Dioxygenase-catalysed cis-dihydroxylation of paradisubstituted benzene substrates does give cis-dihydrodiols with a wide range of enantiopurity.75 The relative sizes of the spherically symmetrical para-substituents (CF3>I>Br> Cl~Me>F>H) as given by the Charton and Hammett steric parameters appeared to provide an empirical method for both the prediction of regioselectivity (Section 2.2) and facial selectivity during TDO-catalysed cis-dihydroxylation of benzene substrates.The general structure shown in Fig. 3(c) thus indicates that the preference for a particular enantiomer will be related to the diVerential in size between the large (R) and small (R*) groups. As previously found during regioselectivity studies (Section 2.2), the non-symmetrical cyano-, vinyl-, alkoxy- and alkylthio-groups again appear to be dominant in determining facial selectivity, i.e. equivalent to a large directing substituent (R).74 The level of confidence which may be placed on the predictive model for TDO-catalysed cisdihydroxylation of para-disubstituted benzenes [Fig. 3(c)] will again be higher for spherically symmetrical than for nonsymmetrical groups for reasons stated earlier. The availability of both enantiomers of the cis-dihydrodiol metabolites of para-disubstituted benzenes has proved useful in evaluating the diVerent methods of enantiopurity determination,75,92,93 as precursors for kinetic resolution studies,94 and for the chemoenzymatic synthesis of diVerent arene cis-dihydrodiol enantiomers and regioisomers.39 The regioselectivity and stereoselectivity of dioxygenase-catalysed cis-dihydroxylations Table 6 Dioxygenase-catalysed cis-dihydroxylation in non-aromatic rings Substrate Product [·]D % ee Enzyme S O O S O O OH OH OH OH HO OH OH OH OH OH OH OH OH OH OH OH HO OH 160 161 162 163 164 165 166 167 168 "11 (CHCl3) "15 (CHCl3) nra 2084 28140 95134 TDO TDO TDO +40 (CHCl3) +41 (CHCl3) 86137 8594,111 NDO NDO +39 (CHCl3) >98135 TDO "36 (CHCl3) >98137 NDO +25 (CHCl3) >98138,139 TDO "21 (MeOH) >98139 NDO BPDO +64 (THF) >98136 TDO "15 (CHCl3) >98136 TDO +53 (CHCl3) >9884 TDO anr=not reported. Boyd and Sheldrake: The dioxygenase-catalysed formation of vicinal cis-diols 319at unsubstituted bonds of monosubstituted arenes shown in Schemes 9, 10 and 13 and Figs. 1–3 can be combined and generalised as shown in Fig. 4. The cis-dihydrodiol metabolites obtained using either TDO-, NDO- or BPDO-catalysed oxidation of all the polycyclic aromatic hydrocarbons (PAHs) were found to have an identical configuration as shown in Fig. 5 and Table 2.81,82 Similar results were observed for the majority of the cisdihydrodiol metabolites of the aza-PAHs quinoline (5,6-, 7,8-; TDO),73 isoquinoline (5,6-;TDO),73 quinoxaline (5,6-; TDO)73 and acridine (5,6-; BPDO)74 (Table 5). The 7,8-cis-dihydrodiol derivative of isoquinoline, formed using TDO, proved exceptional in being essentially racemic.A lower degree of stereoselectivity was, however, more common among members of the thia-PAH and oxa-PAH series. Thus although the carbocyclic cis-dihydrodiols from dibenzothiophene,4,87 dibenzofuran, 4,74,111 benzothiophene,84,85 2-methylbenzothiophene85 and benzofuran85 appeared to be enantiopure and of identical absolute configuration to that shown in Fig. 5, the heterocyclic cis-dihydrodiols from thiophene,85 3-methylthiophene,85 benzothiophene,85 benzofuran,85 2-methylbenzothiophene85 and 2-methylbenzofuran85 were generally of lower enantiopurity which varied between diVerent biotransformations (Table 5).The preferred absolute configuration for the heterocyclic cis/trans-dihydrodiols is as indicated in Fig. 6, X=O,S. Although a systematic study of the dioxygenase-catalysed formation of heterocyclic cis-dihydrodiols has not yet been X X R R¢ R¢ R XH R R¢ X R R¢ OH OH OH OH O OH O2 Dioxygenase Scheme 11 N N N O B O R R O O R OMe Me OMTPA OMTPA OH OH H B A Scheme 12 R OH OH Fig. 2 Preferred absolute configuration of cis-dihydrodiol metabolites of monosubstituted benzenes R OH OH R OH OH R OH OH R¢ R¢ R¢ R > R¢ R > R¢ R > R¢ ( a) ( b) ( c) Fig. 3 Preferred absolute configuration of cis-dihydrodiol metabolites of disubstituted benzenes R R¢ R R¢ R R¢ R OH R¢ R R¢ R OH R¢ OH HO HO HO HO HO e e d f a b c d f a b c a c b f or meta-Disubstituted benzenes or or O2 Dioxygenase Scheme 13 L L L OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH L S S S S S S S S O2 TDO, NDO, S BPDO Minor O2 TDO, NDO, CF3 > I > Br > Cl = Me > F > H S BPDO Major Fig. 4 Preferred regio- and stereo-isomers of mono- and poly-cyclic arene cis-dihydrodiols OH OH Fig. 5 Preferred absolute configuration of carbocyclic cis-dihydrodiol derivatives of polycyclic heteroarenes X OH OH X OH OH Fig. 6 Epimerisation of heterocyclic cis- and trans-dihydrodiol metabolites from five-membered heteroarenes 320 Natural Product Reports, 1998reported, the possibility of partial racemisation via the openchain aldehyde form (Scheme 11, X=S), and preferential destruction of one enantiomer, cannot be excluded.It is noteworthy that the preferred absolute configurations of all the enantiopure cis-dihydrodiols formed using TDO, NDO and BPDO [Figs. 2, 3(a),(b), 5, 6 ] show a striking similarity. To date no examples of enantiopure cis-dihydrodiols of opposite chirality have been reported by dioxygenase-catalysed arene dihydroxylation of unsubstituted arene bonds. The cis-monohydrodiol metabolites of benzocyclobutene (132, TDO), benzocyclobutanol (133, TDO) and biphenylene (134, TDO) were found to be enantiopure and of the same absolute configuration (Fig. 7 and Table 4).89,90 A recent biotransformation of benzocyclobutene in the presence of a carbazole dioxygenase has however provided the first example of enantiocomplementarity during enzyme-catalysed cis-dihydroxylation.93 Thus, the cis-monohydrodiol 135 was obtained as an enantiopure sample of opposite absolute configuration to that formed using TDO (134, Table 4). 3. Enzymatic and chemoenzymatic routes to unusual cis-dihydrodiol regioisomers, enantiomers and other arene metabolites The diol metabolites from the TDO-catalysed asymmetric cis-dihydroxylation of monosubstituted benzenes, ortho- or meta-disubstituted benzenes, PAHs and aza-PAHs have generally been isolated as single enantiomers of the indicated absolute configuration [Figs. 2, 3(a),(b), 5]. Exceptions include the cis-dihydrodiol metabolites of fluorobenzene,92 1,2- difluorobenzene,108 1,3-difluorobenzene,108 and isoquinoline (7,8-bond)73 where both enantiomers were present. The opposite or ‘abnormal’ enantiomer has been obtained using an enzyme-catalysed kinetic resolution process.94 It is generally assumed that the cis-diol dehydrogenase enzyme present within a bacterial species is designed to accept the cis-dihydrodiol enantiomer initially produced by enzymecatalysed asymmetric dihydroxylation within the same organism.Thus when the mixture of cis-dihydrodiol enantiomers (60–80% ee), produced by metabolism of fluorobenzene using the TDO mutant strain (P. putida UV4, lacking the cis-dioldehydrogenase), 92,102 was added as a substrate for the wild-type species NCIMB 8859 of P. putida (containing a cis-diol-dehydrogenase), the normal enantiomer was selectively removed leaving the ‘abnormal’ enantiomer.94 A similar enzyme-catalysed kinetic resolution process using P.putida NCIMB 8859 was again employed to obtain exclusively the ‘abnormal’ cis-dihydrodiol enantiomers of toluene, chloro-, bromo- and iodo-benzene from enantiomeric mixtures obtained by selective catalytic hydrogenolysis of an iodine atom from the corresponding series of para-substituted iodobenzene cis-dihydrodiols (Scheme 8). The kinetic resolution of a racemic mixture of naphthalene cis-dihydrodiol enantiomers (available by chemical synthesis) to yield the ‘abnormal’ enantiomer was also achieved by addition as substrate to wild-type cultures of P.putida (NCIMB 11767 and 8859).94 Substitution of an iodine atom has proved to be of considerable value in the synthesis of a range of new cis-dihydrodiols which are not readily available by direct dioxygenase-catalysed asymmetric dihydroxylation. Direct replacement of the iodine atom has thus yielded cis-dihydrodiols bearing nitrile, alkyl, vinyl, allyl, alkynyl, sulfide, sulfoxide and sulfone groups.36,102 Substitution of both iodine and bromine substituents in the protected cis-dihydrodiols has also been used to yield cisdihydrodiols bearing aryl and other groups.95,117 More than 20 examples of new cis-dihydrodiol derivatives of monosubstituted benzenes have been produced by this chemoenzymatic route.Using a further series of iodobenzene substrates containing substituents at ortho- and meta-positions with the TDOcontaining mutant strain P.putida UV4, the corresponding enantiopure cis-dihydrodiols were obtained. Using the catalytic hydrogenolysis procedure it was possible to remove the iodine atom yielding a new range of 3,4-cisdihydrodiols in either enantiomeric form and of absolute configuration specified (Scheme 14).39 This particular type of cis-dihydrodiol regioisomer is currently unavailable by direct enzyme-catalysed asymmetric dihydroxylation. While the majority of cis-dihydrodiol metabolites are generally suYciently stable to be isolated, in some cases they have been found to decompose spontaneously during the biotransformation or purification procedure.Kinetic studies on the aromatisation of a series of cis-dihydrodiol metabolites of monosubstituted benzene substrates have shown a remarkable diVerence in stabilities.96 Piperidinobenzene provides a rare example of an amino-substituted benzene whose cisdihydrodiol metabolite has been detected.74 While it was suYciently stable to survive the biotransformation and extraction procedure unfortunately it decomposed during attempted purification. A rate diVerence of 107 was observed between the least (ethoxybenzene) and most stable (methyl phenyl sulfone) isolated cis-dihydrodiols of the monosubstituted benzene series.96 Electron donating groups, e.g.amino or alkoxy, were found to destabilise the cis-dihydrodiol and phenols are frequently observed as metabolites.Similarly several of the cis-diols resulting from oxidation in the carbocyclic rings of bicyclic aza- or oxa-arene substrates were found to be very unstable and the corresponding phenols were major metabolites, e.g. benzofuran (4,5- and 6,7-),86 quinoline (7,8-)73 and isoquinoline (5,6- and 6,7-).73 The isolation of aromatic hydroxylation products from arenes in bacterial systems containing dioxygenases can thus be accounted for by spontaneous dehydration of unstable cis-dihydrodiols to yield phenols.A further mechanism involves cis-diol dehydrogenase-catalysed aromatisation. The resulting catechols have been observed both as minor bioproducts when wild-type organisms are used in arene biotransformations or as major metabolites when mutant strains (lacking the catechol dioxygenase) or isolated dehydrogenases are used. While arene cis-dihydrodiols, phenols and catechols appear to be the major bioproducts from dioxygenase-catalysed oxidation of arene rings, arene oxide, arene hydrate and arene trans-dihydrodiol derivatives have also been postulated to occur as minor or transient metabolites during both microbial and animal biotransformations. In view of the availability of cis-dihydrodiols in multi-gram quantities from dioxygenasecatalysed oxidation of arenes, chemoenzymatic routes to the OH OH Fig. 7 Preferred absolute configuration of angular cis-monohydrodiols obtained using TDO H2/Pd/C I OH OH X OH OH X I OH OH X H2/Pd/C OH OH X Scheme 14 Boyd and Sheldrake: The dioxygenase-catalysed formation of vicinal cis-diols 321other types of arene metabolites have recently been developed.Arene oxides, as shown in Schemes 1 and 2, play an important role in eucaryotic metabolism and are the initial products of monooxygenase-catalysed arene epoxidation. Both monooxygenase and dioxygenase enzymes are present in bacteria, e.g. P. putida. Recently, evidence has been found which is consistent with either cis-dihydroxylation or epoxidation (trans-diol formation) occurring during biotransformation of an alkene substrate using a Rhodococcus species according to the choice of inducing agent.97 To date, no unequivocal evidence of the formation of both arene oxides and arene cis-dihydrodiols from a common substrate and organism is available.Selective reduction of the unsubstituted alkene double bond of the cis-dihydrodiols of fluoro-, chloro-, bromoand iodo-benzene, naphthalene and quinoline (5,6- and 7,8-) to yield the corresponding cis-tetrahydrodiols is a key step in the synthesis of arene oxide34a,98 (Scheme 15), arene transdihydrodiol (Scheme 16) and arene hydrate (Scheme 17) metabolites.The conversion of the cis-dihydrodiols to transbromoacetates or -chloroacetates, followed by base-catalysed cyclisation to the corresponding tetrahydroepoxides and a benzylic bromination–dehydrobromination sequence gave the corresponding arene oxides of fluorobenzene, chlorobenzene, bromobenzene, iodobenzene, naphthalene and quinoline (5,6- and 7,8-) (Scheme 15).This route appears to be generally applicable to the spontaneously racemising substituted benzene oxides and the configurationally stable arene oxides of polycyclic arenes, e.g. naphthalene and quinoline.98 Application of the Mitsunobu inversion procedure to the allylic hydroxy group of the cis-tetrahydrodiol derivatives from fluorobenzene, chlorobenzene, bromobenzene and iodobenzene yielded the corresponding trans-tetrahydrodiols which were in turn converted to the trans-dihydrodiols by a bromination–dehydrobromination procedure (Scheme 16).34 The enantiopure trans-dihydrodiols obtained from this procedure were much more stable than either the corresponding arene oxides or cis-dihydrodiols.An alternative synthetic route to trans-dihydrodiols, involving thermal cycloaddition and reversion steps, has also been reported.34b Evidence for the formation of an unstable arene hydrate derivative during metabolism of acetophenone by P.putida UV4 was obtained by conversion to the corresponding tricarbonyliron complex.99 While examples of arene hydrates of naphthalene and phenanthrene have been isolated from dioxygenase-catalysed oxidation of 1,2- and 1,4-dihydronaphthalene100 and 9,10-dihydrophenanthrene,101 respectively, further evidence for the direct formation of arene hydrates from substituted benzene substrates has to date been unavailable. Chemoenzymatic synthetic routes to arene hydrates from the arene oxide and tetrahydroepoxide derivatives of monosubstituted benzenes have however been developed.Treatment of the arene oxides of fluoro- and chloro-benzene substrates with lithium aluminium hydride gave the corresponding arene hydrates in racemic form. Enantiopure forms of these arene hydrates were obtained by treatment of the tetrahydroepoxides with methyl lithium (Scheme 17).74 Conclusions The primary focus of this report has been on the remarkable ability of bacterial oxygenases to catalyse the cisdihydroxylation of a diverse range of arenes and alkenes to yield a single enantiomer.Although the examples shown in Tables 1–6 (ca. 160) have generally been selected for their stability and their rigorously established structures, enantiopurity and absolute configurations, we are aware of a large number of cis-dihydrodiols which have been reported47–71 but which do not meet these requirements and thus have been excluded (ca. 60). Furthermore, when the substantial number of new and unreported cis-diol metabolites from these (ca. 40) and other laboratories (Hudlicky, Gibson and others) are combined, the total number of cis-diols produced by dioxygenase biocatalysis is probably in excess of 300. Based upon the increasing level of interest shown in such enantiopure bioproducts as chiral synthons, it is now certain that these versatile new additions to the chiral pool will continue to stimulate and occupy the interests of synthetic chemists and biochemists well into the next millenium. 4 References 1 D. T. Gibson and P. J. Chapman, Critical Reviews in Microbiology, 1971, 199. R = F, Cl, Br, I; R¢ = H R = I; R¢ = F, Cl, Br R,R¢ = CH=CH–CH=CH R,R¢ = CH=CH–CH=N R,R¢ = N=CH–CH=CH R OH OH R¢ R OH OH R¢ R Br OAc R¢ R O R¢ R O R¢ R O R¢ Br H2 Rh/Al2O3 AcOCMe2COBr NaOMe DBU NBS Scheme 15 R R¢ OH OH R R¢ OCOC6H4NO2-4 OH R R¢ OH OH R R¢ OAc OAc R R¢ OAc OAc R R¢ OH OH Br EtO2CN=NCO2Et PPh3 4-O2NC6H4CO2H i.Li2CO3/LiCl ii. K2CO3 NBS Ac2O AcOCMe2COBr R = Cl, Br, I; R¢ = H R = I; R¢ = Br Scheme 16 R O R R O OH MeLi LiAlH4 R = F, Cl Scheme 17 322 Natural Product Reports, 19982 D. T. Gibson and V. Subramanian, in Microbial Degradation of Organic Compounds, ed. D. T. Gibson, Marcel Dekker Inc., New York, 1984, p. 181. 3 D. T. Gibson, G. J. Zylstra, S. Chauhan, in Pseudomonas: Biotransformations, Pathenogenesis and Evolving Biochemistry, ed.S. Silver, A. M. Charkraberthy, B. Iglewski and S. Kaplan, American Society for Microbiology, 1990, ch. 13, p. 121. 4 S. M. Resnick, K. Lee and D. T. Gibson, J. Ind. Microbiol., 1996, 17, 438. 5 D. A. Widdowson and D. W. Ribbons, Janssen Chim. Acta, 1990, 8, 3. 6 H. A. J. Carless, Tetrahedron: Asymmetry, 1992, 3, 795. 7 G. N. Sheldrake, in Chirality in Industry: The Commercial Manufacture and Applications of Optically Active Compounds, ed.A. N. Collins, G. N. Sheldrake and J. Crosby, J. Wiley and Sons, Chichester, 1992, Ch. 6. 8 (a) S. M. Brown and T. Hudlicky, in Organic Synthesis: Theory and Applications, JAI Press Inc., Greenwich, CT, 1993, p. 113; (b) T. Hudlicky and J. W. Reed, in Advances in Asymmetric Synthesis, ed. A. Hassner, JAI, Greenwich, CT, 1995, vol. 1, p. 271; (c) T. Hudlicky and A. J. Thorpe, Chem. Commun., 1996, 1993; (d) T. Hudlicky, D. A. Entwhistle, K. K. Pitzer and A. J.Thorpe, Chem. Rev., 1996, 96, 1195. 9 D. R. Boyd and D. M. Jerina, in Small Ring Heterocycles. Part III, in The Chemistry of Heterocyclic Compounds, ed. A. Hassner, Interscience, New York, 1985, vol. 45, p. 197 and references therein. 10 D. R. Boyd and N. D. Sharma, Chem. Soc. Rev., 1996, 289. 11 S. A. Barr, D. R. Boyd, N. D. Sharma, L. Hamilton, R. A. S. McMordie and H. Dalton, J. Chem. Soc., Chem. Commun., 1994, 1921 and references therein. 12 W. A. Ayer and E. Z. Cruz, Tetrahedron Lett., 1993, 34, 1589. 13 D. M. Jerina, J. W. Daly, B. Witkop, P. Zaltzman-Nirenberg and S. Udenfriend, Biochemistry, 1970, 9, 147. 14 S. K. Agarwal, D. R. Boyd, H. P. Porter, W.B. Jennings, S. J. Grossman and D. M. Jerina, Tetrahedron Lett., 1986, 27, 4253. 15 D. T. Gibson, J. R. Koch and R. E. Kallio, Biochemistry, 1968, 7, 2653. 16 R. A. S. McMordie, PhD. Thesis, The Queen’s University of Belfast, 1990. 17 B. J. Auret, S. K. Balani, D. R. Boyd, R. M. E. Greene and G. A.Berchtold, J. Chem. Soc., Perkin Trans. 1, 1984, 265. 18 A. M. Reiner and G. D. Hegeman, Biochemistry, 1971, 10, 2530. 19 G. M. Whited, W. R. McCombie, L. D. Kwart and D. T. Gibson, J. Bacteriol., 1986, 166, 1028. 20 C. S. Butler and J. R. Mason, Adv. Microb. Physiol., 1997, 38, 48. 21 D. G. Ballard, A. Courtis, J. M. Shirley and S. C. Taylor, J. Chem. Soc., Chem. Commun., 1983, 954. 22 J. T. Rossiter, S. R. Williams, A. E. G. Cass and D. W. Ribbons, Tetrahedron Lett., 1987, 28, 5173. 23 W. Reineke and H. J. Knackmuss, Biochim. Biophys. Acta, 1978, 542, 412. 24 D. T. Gibson, R. L. Roberts, M. C. Wells and V. M. Kobal, Biochem. Biophys. Res. Commun., 1973, 50, 211. 25 A. A. Khan, R.-F. Wang, W.-W. Cao, W. Franklin and C.E. Cerniglia, Int. J. Sys. Bacteriol., 1996, 46, 466. 26 G. J. Zylstra and D. T. Gibson, J. Biol. Chem., 1989, 264, 14 940. 27 C. C. R. Allen, D. R. Boyd, H. Dalton, N. D. Sharma, S. A. Haughey, R. A. S. McMordie, B. T. McMurray, G. N. Sheldrake and K.Sproule, Chem. Commun., 1995, 119. 28 W-C. Suen and D. T. Gibson, Gene, 1994, 143, 67. 29 (a) S. V. Ley and F. Sternfield, Tetrahedron Lett., 1987, 28, 225; (b) S. V. Ley and A. J. Redgrave, Synlett., 1990, 393. 30 T. Hudlicky, H. Luna, J. D. Price and F. Rulin, J. Org. Chem., 1990, 55, 4683. 31 T. Hudlicky, X. Tian, K. Ko�nigsberger, R. Maurya, J. Rouden and B. Fan, J. Am. Chem. Soc., 1996, 118, 10 752. 32 (a) A. J. Blacker, J. R. Booth, G. M. Davies and J. K. Sutherland, J.Chem. Soc., Perkin Trans. 1, 1995, 2861; (b) H. A. Carless and Y. Dove, Tetrahedron: Asymmetry, 1996, 7, 649; (c) C. H. Tran, D. H. G. Crout, W. Errington and G. M. Whited, Tetrahedron: Asymmetry, 1996, 7, 691. 33 M. G. Banwell and J. R. Dupuche, Chem. Commun., 1996, 869. 34 (a) D. R. Boyd, N. D. Sharma, H. Dalton and D. A. Clarke, Chem. Commun., 1996, 45; (b) B. P. McKibben, G. S. Barnosky and T. Hudlicky, Synlett, 1995, 806. 35 G. Butora, T. Hudlicky, S. P. Earnley, A.G. Gum, M. R. Stabile and K. Abboud, Tetrahedron Lett., 1996, 37, 8155. 36 D. R. Boyd, M. V. Hand, N. D. Sharma, J. Chima, H. Dalton and G. N. Sheldrake, J. Chem. Soc., Chem. Commun., 1991, 1630. 37 T. Hudlicky, H. Luna, J. D. Price and F. Rulin, Tetrahedron Lett., 1989, 30, 4053. 38 (a) T. Hudlicky, R. Fan, T. Tsunoda, H. Luna, C. Andersen and J. D. Price, Isr. J. Chem., 1991, 31, 229; (b) T. Hudlicky, R. Fan, T. Tsunoda, H. Luna and J. D. Price, J. Am. Chem. Soc., 1990, 112, 9439. 39 D. R. Boyd, N. D. Sharma, S. A. Barr, H. Dalton, J. Chima, G. Whited and R. Seemayer, J. Am. Chem. Soc., 1994, 116, 1147. 40 See also D. G. H. Ballard, A. Courtis, A. Shirley and S. C. Taylor, Macromolecules, 1988, 21, 294. 41 D. G. H. Ballard, A. J. Blacker and S. C. Taylor, Plast. Microbes, ed. D. P. Mobley, Hanser, Munich, 1994, pp. 139–168. 42 A. Berry, S. Battist, S. Peck, S. Power and W. Weyler, 2nd Biomass Conf. Am.; Energy, Environ., Agric. Ind., National Renewable Energy Laboratory, Golden (USA), 1995, pp. 1121– 1129; see also B. D. Ensley, B. J. Ratzkin, T. D. Osslund, M. J. Simon, L. P. Wackett and D. T. Gibson, Science, 1983, 222, 167. 43 I. W. Davies, C. H. Senanayake, L. Castonguay, R. D. Larsen, T. R. Verhoven and P. J. Reider, Tetrahedron Lett., 1995, 36, 7619: Chem. Eng. News, 1996, 74, 6. 44 A. A. Garcia, D. H. Kim, G. Whited, L. Kwart, W. Anthony and C. Downie, Isol. Purif., 1994, 2, 19. 45 A. M. Collins, J. M. Woodley and J.M. Liddell, J. Ind. Microbiol., 1995, 14, 382. 46 A. B. Herbert, G. N. Sheldrake, P. J. Somers and J. A. Meredith, EP Appl. 379 300 A2/1990 (to ICI Plc). 47 K-H. Jung, J-Y. Lee and H-S. Kim, Biotechnol. Bioeng., 1995, 48, 625. 48 B. E. Haigler and J. C. Spain, Appl. Environ. Microbiol., 1991, 57, 3156. 49 J. J. DeFrank and D. W. Ribbons, J. Bacteriol., 1977, 129, 1356 50 H. J. Knackmuss, Chem. Ztg., 1975, 99, 213 51 W. Reineke, W. Otting and H. J. Knackmuss, Tetrahedron, 1978, 34, 1707. 52 W.Reineke and H. J. Knackmuss, Biochim. Biophys Acta, 1978, 542, 412. 53 E. Dorn, M. Hellwig, W. Reineke and H. J. Knackmuss, Arch. Microbiol., 1974, 99, 61. 54 J. Hartmann, W. Reineke and H. J. Knackmuss, Appl. Environ. Microbiol., 1979, 37, 421. 55 T. Hudlicky, E. E. Boros and C. H. Boros, Synlett, 1992, 391. 56 H. J. Knackmuss, W. Beckmann and W. Otting, Angew. Chem., Int. Ed. Engl., 1976, 15, 549. 57 M. J. Schocken and D. T. Gibson, Appl. Environ. Microbiol., 1976, 48, 10. 58 G. Bestetti, E. Galli, C. Benigni, F. Orsini and F. Pellizzone, Appl. Microbiol. Biotechnol., 1989, 30, 252. 59 A. M. Warhurst, K. F. Clarke, R. A. Hill, R. A. Holt and C. A. Fewson, Appl. Environ. Microbiol., 1994, 60, 1137. 60 B. Morawski, R. W. Eaton, J. T. Rossiter, S. Guoping, H. Griengl and D. W. Ribbons, J. Bacteriol., 1997, 179, 115. 61 B. E. Haigler, S. F. Nishino and J. C. Spain, Appl. Environ. Microbiol., 1988, 54, 294. 62 R. E. Martin, P. B. Baker and D.W. Ribbons, Biocatalysis, 1987, 1, 37. 63 E. Spiess, C. Sommer and H. Gorisch, Appl. Environ. Microbiol., 1995, 61, 3884. 64 N. C. Bruce and R. B. Cain, FEMS Microbiol. Lett., 1988, 50, 233. 65 K. H. Engesser, V. Strubel, K. Christoglou, P. Fischer and H. G. Rast, FEMS Microbiol. Lett., 1989, 65, 205. 66 H. R. Schlafli, M. A. Weiss, T. Lessinger and A. M. Cook, J. Bacteriol., 1994, 176, 6644. 67 J. Zeyer, P. R. Lehrbach and K. N. Timmis, Appl. Environ. Microbiol., 1985, 50, 1409. 68 B. E. Haigler and J. C. Spain, Appl. Environ. Microbiol., 1989, 55, 372. 69 J. D. Haddock, J. R. Horton and D. T. Gibson, J. Bacteriol., 1995, 177, 20. 70 P. Sander, R-M. Wittich, P. Fortnagel, H. Wilkes and W. Francke, Appl. Environ. Microbiol., 1991, 57, 1430. 71 J. A. M. de Bont, M. J. A. W. Vorage, S. Hartmans and W. J. J. van den Tweel, Appl. Environ. Microbiol., 1986, 52, 677. 72 T. Hudlicky, G. Seoane, D. Gonzalez and D. T. Gibn, Aldrichimica Acta, in preparation. 73 D. R. Boyd, N. D. Sharma, M. R. J. Dorrity, M. V. Hand, R. A. S. McMordie, J. F. Malone, H. Dalton, J. Chima and G. N. Sheldrake, J. Chem. Soc., Perkin Trans. 1, 1993, 1065. 74 D. R. Boyd, H. Dalton, G. N. Sheldrake et al., unpublished results. Boyd and Sheldrake: The dioxygenase-catalysed formation of vicinal cis-diols 32375 D. R. Boyd, N. D. Sharma, M. V. Hand, M. R. Groocock, N. A. Kerley, H. Dalton, J. Chima and G. N. Sheldrake, Chem. Commun., 1993, 974. 76 A. M. JeVrey, H.J. C. Yeh, D. M. Jerina, T. R. Patel, J. F. Davey and D. T. Gibson, Biochemistry, 1975, 14, 575. 77 D. R. Boyd, R. A. S. McMordie, H. P. Porter, H. Dalton, R. O. Jenkins and O. W. Howarth, J. Chem. Soc., Chem. Commun., 1987, 1722. 78 D. M. Jerina, H. Selander, H. Yagi, M. C. Wells, J. F. Davey, V. Mahadevan and D. T. Gibson, J. Am. Chem. Soc., 1976, 98, 5988. 79 M. N. Akhtar, D. R. Boyd, N. J. Thompson, M. Koreeda, D. T. Gibson, V. Mahadevan and D. M. Jerina, J. Chem.Soc., Perkin Trans. 1, 1975, 2506. 80 M. Koreeda, M. N. Akhtar, D. R. Boyd, J. D. Neill, D. T. Gibson and D. M. Jerina, J. Org. Chem., 1978, 43, 1023. 81 D. R. Boyd, N. D. Sharma, R. Agarwal, S. M. Resnick, M. J. Schoken, D. T. Gibson, J. M. Sayer, H. Yagi and D. M. Jerina, J. Chem. Soc., Perkin Trans. 1, 1997, 1715. 82 D. M. Jerina, P. J. van Bladeren, H. Yagi, D. T. Gibson, V. Mahadevan, A. S. Neese, M. Koreeda, N. D. Sharma and D. R. Boyd, J. Org. Chem., 1984, 49, 3621. 83 D.T. Gibson, V. Mahadevan, D. M. Jerina, H. Yagi and H. J. C. Yeh, Science, 1975, 189, 295. 84 D. R. Boyd, N. D. Sharma, R. Boyle, B. T. McMurray, T. A. Evans, J. F. Malone, J. Chima, H. Dalton and G. N. Sheldrake, J. Chem. Soc., Chem. Commun., 1993, 49. 85 D. R. Boyd, N. D. Sharma, I. N. Brannigan, D. A. Clarke, H. Dalton, S. A. Haughey and J. F. Malone, Chem. Commun., 1996, 2361. 86 D. R. Boyd, N. D. Sharma, R. Boyle, J. F. Malone, J. Chima and H. Dalton, Tetrahedron: Asymmetry, 1993, 4, 1307. 87 A. J. Laborde and D. T. Gibson, Appl. Environ. Microbiol., 1977, 34, 783. 88 D. R. Boyd, N. D. Sharma, J. G. Carroll, J. F. Malone, D. G. Mackerracker and C. C. R. Allen, Chem. Commun., 1998, 683. 89 D. R. Boyd, N. D. Sharma, P. J. Stevenson, J. Chima, D. J. Gray and H. Dalton, Tetrahedron Lett., 1991, 32, 3887. 90 D. R. Boyd, N. D. Sharma, T. A. Evans, M. Groocock, J. F. Malone, P. J. Stevenson and H. Dalton, J. Chem Soc., Perkin Trans. 1, 1997, 1879. 91 S.T. Trenz, K. H. Engesser, P. Fischer and H.-J. Knackmuss, J. Bacteriol., 1994, 789. 92 D. R. Boyd, M. R. J. Dorrity, M. V. Hand, J. F. Malone, N. D. Sharma, H. Dalton, D. J. Gray and G. N. Sheldrake, J. Am. Chem. Soc., 1991, 113, 666. 93 S. M. Resnick, D. S. Torok and D. T. Gibson, J. Org. Chem., 1995, 60, 3546. 94 C. C. R. Allen, D. R. Boyd, H. Dalton, N. D. Sharma, I. N. Brannigan, N. A. Kerley, G. N. Sheldrake and S. C. Taylor, J. Chem. Soc., Chem. Commun., 1995, 117. 95 S.V. Ley, A. J. Redgrave, S. C. Taylor, S. Ahmed and D. W. Ribbons, Synlett, 1991, 741. 96 D. R. Boyd, A. J. Blacker, B. Byrne, H. Dalton, M. V. Hand, S. Kelly, R. A. More O’Ferrall, S. N. Rao, N. D. Sharma and G. N. Sheldrake, Chem. Commun., 1994, 313. 97 C. C. R. Allen, D. R. Boyd, E. M. Gribbon, L. A. Kulakov, M. J. Larkin, K. A. Reid and N. D. Sharma, Appl. Environ. Microbiol., 1997, 63, 15. 98 D. R. Boyd, N. D. Sharma, R. Agarwal, N. A. Kerley, R. A. S. McMordie, A. Smith, H.Dalton, A. J. Blacker and G. N. Sheldrake, Chem. Commun., 1994, 1693. 99 P. W. Howard, G. R. Stephenson and S. C. Taylor, Chem. Commun., 1990, 1182. 100 R. Agarwal, D. R. Boyd, N. D. Sharma, H. Dalton, N. A. Kerley, R. A. S. McMordie, G. N. Sheldrake and P. Williams, J. Chem. Soc., Perkin Trans. 1, 1996, 67. 101 S. M. Resnick and D. T. Gibson, Appl. Environ. Microbiol., 1996, 63, 3355. 102 D. R. Boyd, N. D. Sharma, B. Byrne, M. V. Hand, J. F. Malone, G. N. Sheldrake, A.J. Blacker and H. Dalton, J. Chem. Soc., Perkin Trans. 1, 1998, in the press. 103 D. R. Boyd, N. D. Sharma, J. DuVy, J. Harrison and H. Dalton, unpublished results. 104 D. T. Gibson, M. Hensley, H. Yoshioka and T. J. Mabry, Biochemistry, 1970, 9, 1626. 105 D. T. Gibson, B. Gschwendt, W. K. Yeh and V. M. Kobal, Biochemistry, 1973, 12, 1520. 106 G. Baaggi, D. Castelani, E. Galli and V. Treccani, Biochem. J., 1972, 126, 1091. 107 T. Hudlicky, M. A. Eudoma and G. Butra, J. Chem. Soc., Perkin Trans. 1, 1996, 2187. 108 N. Bowers, Ph.D. Thesis, The Queen’s University of Belfast, 1997. 109 D. T. Gibson, R. L. Roberts, M. C. Wells and V. M. Kobal, Biochem. Biophys. Res. Commun., 1973, 50, 211. 110 B. Byrne, PhD Thesis, The Queen’s University of Belfast, 1995. 111 I. Brannigan, PhD Thesis, The Queen’s University of Belfast, 1997. 112 M. R. Stabile, T. Hudlicky and M. L. Meisels, Tetrahedron: Asymmetry, 1995, 6, 537. 113 D. T. Gibson, J. R. Koch, C. L. Schuld and R. E. Kallio, Biochemistry, 1968, 7, 3795. 114 T. Hudlicky, H. Luna, G. Barbieri and L. D. Kwart, J. Am. Chem. Soc., 1988, 110, 4735. 115 T. Hudlicky, G. Seoane and T. Pettus, J. Org. Chem., 1989, 54, 4239. 116 D. Gonzalez, V. Schapiro, G. Seoane and T. Hudlicky, Tetrahedron: Asymmetry, 1997, 8, 975. 117 T. Hudlicky and E. E. Boros, Tetrahedron: Asymmetry, 1992, 3, 217. 118 S. T. Astley, M. Meyer and G. R. Stephenson, Tetrahedron Lett., 1993, 34, 2035. 119 T. Hudlicky, E. E. Boros and C. H. Boros, Tetrahedron: Asymmetry, 1993, 4, 1365. 120 K. Konigsberger and T. Hudlicky, Tetrahedron: Asymmetry, 1993, 4, 2474. 121 S. J. C. Taylor, D. W. Ribbons, A. M. Z. Slawin, D. A. Widdowson and D. J.Williams, Tetrahedron Lett., 1987, 28, 6391. 122 D. T. Gibson, V. Mahadevan and J. F. Davey, J. Bacteriol., 1974, 119, 930. 123 N. Kerley, PhD Thesis, The Queen’s University of Belfast, 1994. 124 J. A. Schofield, EP 252 568/1988 (to Shell Internationale). 125 M. R. Stabile, T. Hudlicky, M. L. Meisels, G. Butora, A. G. Gum, S. P. Fearnley, A. J. Thorpe and M. R. Ellis, Chirality, 1995, 7, 556. 126 N. H. Kirsch and H-J. Stan, J. Chromatogr., A, 1994, 684, 277. See also ref. 63 and (a) J. C. Spain and S. F. Nishino, Appl. Environ. Microbiol., 1987, 53, 1010; (b) J. C. Spain and D. T. Gibson, Appl. Environ. Microbiol., 1988, 54, 1399. 127 S. A. Selifonov, M. Grifoll, J. E. Guist and P. J. Chapman, Biochem. Biophys. Res. Commun., 1993, 193, 67. 128 G. N. Jenkins, D. W. Ribbons, D. A. Widdowson, A. M. Z. Slavin and D. J. Williams, J. Chem Soc., Perkin Trans. 1, 1995, 2647. 129 T. Hudlicky, M. A. A. Endoma and Gabor Butora, Tetrahedron: Asymmetry, 1996, 61. 130 M. E. Deluca and T. Hudlicky, Tetrahedron Lett., 1990, 31, 13. 131 S. M. Resnick and D. T. Gibson, Appl. Environ. Microbiol., 1996, 62, 3355. 132 N. Connors, R. Prevoznak, M. Chartrans, J. Reddy, R. Singhai, Z. Patel, O. Olewinski, P. Salmon, J. Wilson and R. Greasham, J. Ind. Microbiol. Biotechnol., 1997, 18, 353. 133 D. R. Boyd, N. D. Sharma, R. Boyle, R. A. S. McMordie, J. Chima and H. Dalton, Tetrahedron Lett., 1992, 33, 1241. 134 G. M. Whited, J. C. Downie, T. Hudlicky, S. P. Fearnley, J. C. Dudding, H. F. Olivo and D. Parker, Bioorg. Med. Chem., 1994, 2, 727. 135 D. R. Boyd, N. D. Sharma, N. A. Kerley, R. A. S. McMordie, G. N. Sheldrake, P. Williams and H. Dalton, J. Chem. Soc., Perkin Trans. 1, 1995, 67. 136 D. R. Boyd, N. D. Sharma, R. Boyle, T. A. Evans, J. F. Malone, K. M. McCombe, H. Dalton and J. Chima, J. Chem. Soc., Perkin Trans. 1, 1996, 1757. 137 D. T. Gibson, S. M. Resnick, K. Lee, J. M. Brand, D. S. Torok, L. P. Wackett, M. J. Schocken and B. E. Haigler, J. Bacteriol., 1995, 177, 2615. 138 D. R. Boyd, M. R. J. Dorrity, J. F. Malone, R. A. S. McMordie, N. D. Sharma, H. Dalton and P. Williams, J. Chem. Soc., Perkin Trans. 1, 1990, 489. 139 S. M. Resnick and D. T. Gibson, Appl. Environ. Microbiol., 1996, 62, 1364. 140 L. P. Wackett, L. D. Kwart and D. T. Gibson, Biochemistry, 1988, 27, 1360. 324 Natural Product Reports, 1998

ISSN:0265-0568    

DOI:10.1039/a815309y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Book review The Systematic Identification of Organic Compounds. Seventh Edition Ralph L. Shriner Christine K. F. Hermann Terence C. Morrill David Y. Curtin and Reynold C. Fuson. John Wiley and Sons New York 1997 669 pp. Price £27.50. ISBN 0 471 59748 1. Many of the methods for the successful identification of natural products have their foundations in the principles of qualitative organic analysis. Shriner and Fuson’s Systematic Identification of Organic Compounds is a well-established textbook of qualitative organic analysis which first appeared over sixty years ago. This is the seventh edition. In this edition it is claimed that the spectroscopy sections have been modernized and treated separately from the chemical methods. Although this is a general textbook written for undergraduate qualitative organic analysis courses there is much that could help the postgraduate student starting on the identification of natural products.Many of the separation techniques described in Chapters 2 and 10 may be applied as much to the separation and characterization of natural products as to other classes of organic compounds. The chapter on spectroscopic methods covers the foundations of mass spectrometry nuclear magnetic resonance infrared and ultraviolet spectroscopy and contains many of the correlation tables that are used in structure elucidation. However a student involved in natural product work would need a much more detailed coverage of NMR spectroscopy. For example there is no discussion of the use of coupling constants to obtain stereochemical information via a Karplus equation nor does the discussion of 13C NMR deal with the DEPT spectrum for determining the number of hydrogen atoms attached to a carbon.The older oV-resonance decoupling method for distinguishing between methyl methylene and methine carbons is described. Other useful NMR techniques such as NOE spin decoupling and COSY methods are also not described. Chapters 7 and 8 which form the major part of the book are concerned with the chemical tests for functional groups and give details for the preparation of derivatives. However there are many useful colour tests and chromatographic spray reagents for the detection of natural products such as alkaloids which are not included.With an increasing emphasis on X-ray crystallographic methods of structure determination methods for the preparation of crystalline derivatives may have an additional significance for the natural product chemist. However the scale on which these are carried out would need modification for most natural product applications. Natural product chemists rarely have the luxury of plenty of material with which to work. A useful additional feature for each of the preparations is a description of the way in which the residues from each experiment can be cleared up. Chapter 9 is a collection of problems combining spectroscopic and chemical information. The book concludes with a guide to the literature. The appendix contains a useful set of tables including solvents for chromatography and extraction and details of drying agents. There is an extensive list of the melting points of derivatives of organic compounds. However apart from amino acids and some sugars only a few common natural products figure in these tables. Whilst this book is not aimed specifically at the natural product chemist it will be useful in the laboratory as a source of general spectroscopic information and data on organic compounds. James R. Hanson University of Sussex Brighton UK 325

ISSN:0265-0568    

DOI:10.1039/a815325y

出版商:RSC    

年代:1998

数据来源: RSC