Search

Biotransformation of 1,2- and 1,4-dihydronaphthalene substrates, using growing cultures of Pseudomonas putida UV4, resulted in dioxygenase-catalysed benzylic monohydroxylation, cis-tetrahydro diol and cis-dihydro diol formation, trihydroxylation and dehydrogenation. The arene hydrates, (R)-1,2-dihydronaphthalen-l-ol 5 and (R)-1,4-dihydronaphthalen-l-ol 7, were isolated as enantiopure metabolites while 1,2-dihydronaphthalen-2-ol 8 was found in almost racemic form. The structure, enantiopurity and absolute stereochemistry of these arene hydrates of naphthalene were confirmed by chemical synthesis. Deuterium labelling studies, and the use of enantiomerically pure arene hydrates 5 and 7 as substrates, were used to establish the metabolic pathways for the formation of (1R,2S)-1,2-dihydronaphthalene-l,2-diols 2, from both 1,2-dihydronaphthalene 3 and 1,4-dihydronaphthalene 6 substrates.

Substituted chiral thiophene 1-oxides and their cycloadducts of variable enantiopurity have been isolated as products of dioxygenase-catalysed sulfoxidation of the corresponding thiophenes using intact cells of Pseudomonas putida; thermal racemization (ΔG‡= 25.1 kcal mol–1) of the enantiopure metabolite (1R)-2-methylbenzo[b]thiophene 1-oxide has been observed.

Procaryotic (bacterial) dioxygenase-catalysed asymmetric dihydroxylation of chromene and 2,2-dimethylchromene to yield the (4S)-enantiomers of the corresponding cis-diols exclusively is reported. The epoxide, and derived cis- and trans-diol products from the previously reported eucaryotic (mammalian) metabolism of precocene-1 (7-methoxy-2,2-dimethylchromene), and the corresponding epoxide and diol derivatives of chromene and 2,2-dimethylchromene, have now been obtained in enantiopure form by chemical resolution of the corresponding bromohydrins using methoxy-(trifluoromethyl)phenylacetic acid (MTPA) or camphanate esters. The absolute configurations of the epoxides, cis- and trans-diols have been determined by chemical synthesis from, and stereochemical correlation with, the corresponding camphanate and MTPA esters. X-Ray crystal structure analysis has provided an unequivocal method for assignment of the absolute stereochemistry in each case.

A new range of heterocyclic ring cis/trans-dihydrodiol derivatives (1B, 3B–8B) obtained from metabolism of monocyclic (1A, 3A) and bicyclic heteroarenes (4A–8A) by Pseudomonas putida UV4, has been isolated and stereochemically assigned.

Selected strains of the bacterium Pseudomonas putida(previously shown to effect dioxygenase-catalysed asymmetric cis-dihydroxylation of alkenes) have been found to yield chiral sulfoxides from the corresponding sulfides with a strong preference for the (R)- or (S)-configurations but without evidence of sulfone formation; similar results obtained using an Escherichia coli clone (pKST11, containing the Tod C1 C2 B and A genes encoding toluene dioxygenase from P. putida NCIMB 11767) are again consistent with a stereoselective dioxygenase-catalysed sulfoxidation.

Enzyme-catalysed kinetic resolution and asymmetric dihydroxylation routes to enantiopure cis-diol metabolites of arenes and benzocycloalkenes of either absolute configuration have been developed using appropriate strains of the bacterium Pseudomonas putida.

Acid-catalysed dehydration of 3-substituted benzene cis-1,2-dihydrodiols exhibits a Hammett plot with ρ=–8.2, consistent with reaction via a benzenonium ion-like intermediate; however, correlation of +M resonance substituents such as Me and MeO by σp rather than σ+ constants indicates a marked imbalance between resonance and inductive stabilisation of the transition state.

Arene oxides of naphthalene, quinoline, triphenylene, benzo[e]pyrene, and dibenz[a,c]anthracene have been synthesised in enantiopure (1a–c) or racemic form (1d–f), free from oxepine isomers (3d–f) from cis-diol precursors (7a–f)via chloroacetate (9a–f) or dimesylate (13a) intermediates.

Biotransformation of quinoline, isoquinoline, quinoxaline and quinazoline using growing cultures of Pseudomonas putida UV4 yielded cis-dihydro diols from the oxidation of the carbocyclic aromatic ring. Aromatic hydroxylation was observed in both carbocyclic and heterocyclic rings. Ring cleavage of the quinoline skeleton to yield anthranilic acid, and cis-diol formation (with alkene bond reduction) to yield cis-5, 6, 7, 8-tetrahydroquinazoline-5, 6-diol from quinazoline were observed. The cis-dihydro diol metabolites of quinoline (5, 6- and 7, 8-) and quinoxaline (5, 6-) were found to be optically pure, while metabolism of isoquinoline gave one homochiral (5, 6-) and one racemic (7, 8-)cis-dihydro diol product. The absolute configurations of the cis-dihydro diol metabolites have been determined using 1H NMR analyses, stereochemical correlations and X-ray crystallography methods.

The cis-dihydrodiol enantiomer preferentially formed, by dioxygenase-catalysed oxidation of 1,4-disubstituted benzene substrates in growing cultures of Pseudomonas putida UV4, is found to be largely controlled by the relative size of the substituents.

Benz[a]anthracene 3,4-oxide 2, when synthesised from optically pure dibromo ester precursors, was found to have racemized spontaneously and was accompanied by anthra[2,1-b]oxepine 14. Photoisomerization of benz[a]anthracene 3,4-oxide 2, triphenylene 1,2-oxide 4 and benz[a]anthracene 1,2-oxide 5 proceeds via an oxygen-walk mechanism to yield anthra[2,1-b]oxepine 14, phenanthro[10,9-b]oxepine 16 and anthra[2,1-f]oxepine 17 respectively.

New cis-diol metabolites of both aromatic and non-aromatic heterocyclic rings have been isolated from growing cultures of a mutant strain of the soil bacterium Pseudomonas putida(UV4) and stereochemically assigned; a novel heterocyclic cis-diol of benzothiophene, 2,3-dihydroxy-2,3-dihydrobenzothiophene is found to exist exclusively in the cis-configuration in water but equilibrates readily with the trans-isomer.

1,3-Benzodithiole 1-oxide, 5, has been resolved into enantiomers using chiral stationary phase high performance liquid chromatography. Alkylation of an excess (25%) of the late eluting (+)-(1R) enantiomer of the sulfoxide 5 under basic conditions yielded both cis and trans isomers of 2-methyl (6cis-1R,2S′,6trans-1R,2R), 2-ethyl (7cis-1R,2S′, 7trans-1R,2R) and 2-isopropyl (8cis-1R,2S′, 8trans-1R,2R)-benzodithiole 1-oxide (25% e.e.) and allowed a stereochemical correlation of absolute configuration between the sulfoxides 5–8.Treatment of racemic 1,3-benzodithiole 1-oxide (51S/51R) with potassium bis(trimethylsilyl)amide and (S)-(+)-1-iodo-2-methylbutane yielded four diastereoisomers of 2-(2′-methylbutyl)-1,3-benzodithiole 1-oxide (9cis-1R,2S,2′S′, 9cis-1S,2R,2′S′, 9trans-1S,2S,2′S, 9trans-1R,2R,2′S) which were separated by chiral stationary phase HPLC (CSP-HPLC). The most strongly retained diastereoisomer was analysed by X-ray crystallography and was assigned the (1S:2S:2′S) absolute configuration. Further alkylation of 9trans-1S,2S,2′S under similar conditions yielded 2-(2′S-methylbutyl)-2-(2″S-methylbutyl)-1,3·benzodithiole 1-oxide 10 of (1S,2′S,2″S) configuration exclusively. The same stereoisomer of the sulfoxide 10 was also obtained by dialkylation of the early eluting (–)-enantiomer of 1,3-benzodithiole 1-oxide (51S) using (+)-1-iodo-2-methylbutane, thus enabling the unequivocal establishment of the absolute configurations of the enantiomers of 1,3-benzodithiole 1-oxides listed in Table 1. A comparison of circular dichroism (CD) spectra for sulfoxides 5–9 indicates that this method may also be used to correlate absolute configurations of 2-alkyl substituted 1,3-benzodithiole 1-oxides.

Metabolism of bromobenzene and iodobenzene by growing cultures of Pseudomonas putida UV4 gave the corresponding cis-dihydrodiol products 1 and 2 in high yields; subsequent direct chemical substitution of the halogen atoms in these metabolites provided a new range of enantiomerically pure cis-dihydrodiols of known absolute configuration.

Bacterial metabolism of the dihydronaphthalenes 1 or 3 using cultures of Pseudomonas putida UV4 yielded the arene hydrates of naphthalene 2 or a mixture of 4 and 5 respectively; these arene hydrates have also been obtained in homochiral form either by synthesis from enantiomerically pure alcohols 10 and 12 and arene oxide 11 precursors or by chiral stationary phase HPLC separation of arene hydrate enantiomers.

The oxidation of N-alkyl imines by m-chloroperoxybenzoic acid to yield nitrones was facilitated by (i) the presence of C-aryl substituents, (ii) steric inhibition of attack at the imino C-atom, (iii) electron donating para-substituents on the C-aryl substituent, (iv) non-hydroxylic solvents, (v) optimal conjugation between C-aryl substituents and the imino group, and (vi) less bulky N-alkyl groups.The oxidation of N-alkyl imines by dimethyldioxirane (DMD) in dichloromethane–acetone solution yielded nitrones without evidence of oxaziridine formation. The yields of isolated nitrones were higher for C,C-diaryl imines and for imines bearing less bulky N-alkyl substituents. N–H substituted ketimines were found to yield oximes after reaction with dimethyldioxirane.

Benz[a]anthracene 8,9-oxide, (5), a major initial metabolite of benz[a]anthracene, has been synthesised in optically pure form. The (–)-enantiomer has been unequivocally assigned as [8S,9R] by configurational correlation with (+)-trans-9S-bromo-8S-(menthyloxyacetoxy)-8,9,10,11-tetrahydrobenz[a]anthracene (1B) whose absolute stereochemistry has been determined by X-ray crystal-structure analysis.The major isolated mammalian liver metabolite of benz[a]anthracene, (–)-8,9-dihydroxy-8,9-dihydrobenz[a]-anthracene, (11), has [8R,9R] stereochemistry. It is deduced that the latter (–)-(8R,9R)-dihydrodiol is enzymatically derived from benz[a]anthracene (+)-(8R,9S)-oxide, and that it may be converted into trans-8R,9S-dihydroxy-10S,11R-epoxy-8,9,10,11-tetrahydrobenz[a]anthracene prior to being covalently bonded to cellular nucleic acids.

Quantum yields are reported for the formation of a dimeric adenine photoproduct, A=A, in adenine homopolymers and DNA irradiated at 254 nm. The A=A content of irradiated samples was assayed by using reversed-phase HPLC to isolate the 4,6-diamino-5-guanidinopyrimidine (DGPY) which is produced from A=A on acid hydrolysis. Acid hydrolysates derived from DNA radiolabelled with [ 14 C] 2′-deoxyadenosine were spiked with unlabelled DGPY before fractionation on HPLC and the recovered material was further purified by chromatography on Sephadex G-10 followed by co-crystallization with DGPY sulphate. Although A=A is formed with a relatively high quantum yield of 1.6 × 10 −3 mol einstein −1 in single-stranded poly(dA) the photoaddition reaction is strongly quenched in base-paired poly(dA)·poly(dT) and undetectable in poly(rA)·poly(dT). Respective quantum yields of 6 × 10 −5 and 9 × 10 −6 were estimated for the formation of A=A in single- and double-stranded E. coli DNA implying that the photoproduct has very limited biological significance. From studies with d(ApG), d(GpA), ApG, GpA, d(A) 20 and d(A 4 G) 4 it is concluded that adjacent guanine and adenine bases do not form a photoadduct analogous to A=A and also that guanine residues have no local or long-range quenching effect on photodimerization within A-A doublets.

A new type of dimeric adenine photoproduct has been isolated from d(ApA) irradiated at 254 nm in neutral aqueous solution. It is formed in comparable amounts to another, quite distinct, adenine photoproduct first described by Porschke (J. Am. Chem. Soc. (1973), 95, 8440-8446). Results from high resolution mass spectrometry and 1H NMR indicate that the new photoproduct comprises a mixture of two stereoisomers whose formation involves covalent coupling of the adenine bases in d(ApA) and concomitant incorporation of the elements of one molecule of water. The photoproduct is degraded specifically by acid to 4,6-diamino-5-guanidinopyrimidine (DGPY) whose identity has been confirmed by independent chemical synthesis. Formation of the new photoproduct in UV-irradiated d(pA)2 and poly(dA), but not poly(rA), has been demonstrated by assaying their acid hydrolysates for the presence of DGPY. The properties of the photoproduct are consistent with it being generated by the hydrolytic fission of an azetidine photoadduct in which the N(7) and C(8) atoms of the 5'-adenine in d(ApA) are linked respectively to the C(6) and C(5) positions of the 3'-adenine.

Peroxyacid oxidation of diimine (1) yielded only the trans isomer of dinitrone (2) which upon photoisomerization gave dioxaziridines (3a) and (3b) and amide (4) as the only isolable isomers; an X-ray crystallographic investigation of (1) shows a considerable geometry distortion.

The resolution of arene oxides, diols and other derivatives of bromohydrins has been markedly improved by the use of bromohydrin esters of (−)-(S)-a-methoxy-a-trifluoromethyl- phenylacetic acid (MTPA).

Photoisomerization of the N,N′-dioxides of two N,N′-dialkyl-2,2,4,4-tetramethylcyclobutane-1,3-di-imines occurs in a stepwise manner via the analogous mono-oxaziridine-mononitrone, dioxaziridine (and/or monoamide-mononitrone), and monoamide-mono-oxaziridine intermediates en route to the diamide products. The relative configurations of all products have been deduced by n.m.r. methods (chemical shift non-equivalence and n.O.e.d. spectroscopy). The stereochemistry of the photoisomerization reactions can be rationalized in terms of steric and stereoelectronic effects.

Steric and mesomeric effects have a marked influence upon the formation of oxaziridine (normal pathway) or nitrone (abnormal pathway) products from the imine-peroxyacid reaction; n.m.r. studies of the thermal isomerization of oxaziridines to nitrones provide evidence of a pseudo-abnormal oxidation pathway.

The geminal α-methytene protons in 3-ethylnaphthalene-l,2-oxide show chemical shift nonequivalence in 1 H NMR spectra recorded at ambient temperature consistent with a high barrier to enantiomerization via the orepine tautomer. The methylene protons in α-benzoyloxytoluene-2,3-oxide which are a singlet at ambient temperature split into an AB system at -135 °C and there is evidence of ca . 5% of the oxepine isomer. Lineshape analysis of the coalescing arene oxide AB Signals provides the activation parameters for the valence isomerization/degenerate racemization process: ΔGXXX = 7.6 kcal mol -1 , ΔHXXX = 8.6 kcal mol -l , ΔSXXX= 5.8 cal K -l mol -1 .

The principal oxidative metabolites formed from benz[a]anthracene (BA) by the rat liver microsomal monooxygenase system are the 5,6- and 8,9-arene oxides. In order to determine the enantiomeric composition and absolute configuration of these metabolically formed arene oxides, an HPLC procedure has been developed to separate the six isomeric glutathione conjugates obtained synthetically from the individual enantiomeric arene oxides. Both (+)- and (−)-BA 5,6-oxide gave the two possible positional isomers, but only one positional isomer was formed in each case from (+)- and (−)-BA 8,9-oxide. When [ 14 C]-BA was incubated with a highly purified and reconstituted monooxygenase system containing cytochrome P-450c, and glutathione was allowed to react with the arene oxides formed, radio-active adducts were formed predominantly (>97%) from the (+)-(5S,6R) and (+)-(8R,9S) enantiomers. The present results are in accord with theoretical predictions of the steric requirements of the catalytic binding site of cytochrome P-450c.

Acetone photosensitisation of 2'-deoxyguanosine in deaerated aqueous solution gives 8-(2,3,4-trihydroxybutyl)guanine as a major photoproduct. Its structure and that of its tetraacetate have been determined primarily by high resolution 1H NMR and mass spectrometry; a di-isopropylidene derivative has also been prepared. Mechanistic aspects of this novel photochemical transformation are discussed, particularly in relation to the alkaline cleavage of acetone photosensitised DNA at the sites of guanine bases.

Metabolism of the bicyclic alkenes 1,2-dihydronaphthalene, indene, and 1,2-benzocyclohepta-1,3-diene by a mutant strain of Pseudomonas putida yields benzylic monols (exclusively with R configuration) as major metabolites and vicinal cis diols as minor products having an excess of the S configuration at the benzylic position.

N-Unsubstituted diaryl, alkyl aryl and dialkyl ketimines are prepared in good yield at ambient temperature by the reaction of KOBut on oxaziridines having an α-hydrogen atom on the N-alkyl substituent; the mechanism and stereochemistry of the reaction are discussed.

(+)-trans-2-Bromo-1-hydroxyindan (5) has been resolved into itsenantiomersby preparative h.p.l.c. orfractional crystallization of the bromo-menthyloxy-acetoxydiastereoisomers (6a) and (6b). The bromohydrin (5) or bromo-esters (6a, 6b, and 7) have in turn been converted into (–)-(1R,2S)-1,2-epoxyindan (2), (–)(1R,2R)-trans-1-acetoxy-2-bromoindan (7), (+)-(1S)-indanol (8), (–)-(1R,2R)-trans-indan-1,2-diol (3), and (–)-(1S,2R)-cis-indan-1,2-diol (4). The optical purity of the alcohol products (3), (4), and (8) was determined by n.m.r. and h.p.l.c. analysis of their 2-methoxy-2-phenyl-2-trifluoromethylacetates. The previously unknown optical purity of the chiral alcohols (3), (4), (5), and (8), which had been isolated in earlier mammalian and microbial metabolism studies, has also been deduced.

Benz[a]anthracene 1,2-oxide (1,2-epoxy-1,2-dihydrobenz[a]anthracene), a minor metabolite of benz[a]anthracene in hepatic systems, has been synthesized both from racemic precursors and from separated diastereoisomeric bromohydrin esters whose absolute stereochemistry has been determined by n.m.r. methods. The benz[a]anthracene 1,2-oxide obtained was racemic as previously predicted from PMO calculations.

The synthesis of a new range of oxaziridines (18)–(23), (30)–(35) by peracid oxidation of diaryl ketimines and aryl aldimines is reported. Relatively stable NH ketimine products (24)–(27) have been isolated from base-catalysed decomposition of the oxaziridines (18)–(20), (30)–(35) and a primary kinetic isotope effect (ca. kH/kD 6.0) was observed during decomposition of the oxaziridine trans-(31). The trans-oxaziridines (31)–(35) were found to decompose at a faster rate than the corresponding cis isomers. The relative rates of base-catalysed decomposition of oxaziridine stereoisomers are consistent with a mechanism involving an α-C–H proton abstraction mechanism.

(+)-Benz[a]anthracene 10,11-oxide (9) has been obtained in optically pure form by a four-step synthesis from (–)-trans-(10R,11R)-10-bromo-11-(menthyloxyacetoxy)-8,9,10,11-tetrahydrobenz[a]anthracene (1A) which was separated from diastereoisomer (1B) by recrystallization. The absolute configuration of (+)-(9) was established as (10S,11R) by application of the exciton-chirality method to the circular dichroism curve of the benzoate (+)-(6) and by a correlation of stereochemistry between (+)-(6),(–)-(4), and (+)-(9). (+)-Benz[a]anthracene 5,6-oxide (15) has been synthesised in 30% optical yield using partially resolved (+)-cis-5,6-dihydroxy-5,6-dihydrobenz[a]anthracene (11) obtainable by chromatographic resolution of the (–)-cis-5,6-dimenthyloxyacetoxy-5,6-dihydrobenz[a]anthracene diasteroisomers (12A) and (12B). Both arene oxides, (+)-(9) and (+)-(15), showed configurational stability at ambient temperature in accord with perturbation molecular orbital calculations.

trans-Bromohydrins of 1,2,3,4-tetrahydronaphthalene (1a), 1,2,3,4-tetrahydroanthracene (1b), and 1,2,3,4-tetrahydrophenanthrene (1c) and (1d) were isolated in optically pure forms by reductive cleavage of the corresponding 2-methoxy-2-phenyl-2-trifluoromethylacetate (MTPA) diastereoisomers (2a–d) which had been separated by preparative h.p.l.c. Using reactions with established mechanisms and stereochemistry, the configurations at the chiral centres in the bromohydrins (1) were correlated with those in the tetrahydroepoxides (5), the arene oxides (7), and cis-(3) and trans-(4) tetrahydrodiols. An X-ray structure analysis of the 1,2,3,4-tetrahydroanthracene bromo-MTPA ester (+)-(2bE) indicated the trans equatorial quasi-equatorial conformation and confirmed the absolute stereochemistry of the MTPA group [derived from (–)-acid] as (S).

The synthesis of arene oxides and oxepines, and the unexpectedly facile rearrangement of a range of non-K- and bay-region arene oxides of polycyclic aromatic hydrocarbons to isolable isomeric oxepines via a photochemical oxygen walk pathway is reported; oxepine formation can be predicted from PMO calculations which indicate the associated loss of resonance energy during the oxygen walk process.

A range of oxaziridines containing N-methylene substituents has been synthesized by peracid oxidation of the corresponding fluorenylidene N-alkylamines. Spontaneous and tertiary amine base-catalysed decomposition of the oxaziridines into unstable NH aldimines and derivatives was observed. Acrylaldehyde, 2-methylacrylaldehyde, and benzaldehyde NH imines have been identified as initial products from decomposition of the corresponding oxaziridines. 2,4,6-Trialkylhexahydro-1,3,5-triazines, N,N′-dialkylidene-1,1 -diaminoalkanes, N,N′-diarylidene-1,1 -diaminoalkanes, and N-iso-butylidene-2-methylpropenylamine were among the isolated products formed via the undetected alkyl aldehyde NH imines resulting from oxaziridine decomposition.