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Enzymatic oxidations of thiophenes, including thiophene-containing drugs, are important for biodesulfurization of crude oil and drug metabolism of mono- and poly-cyclic thiophenes. Thiophene oxidative dearomatization pathways involve reactive metabolites, whose detection is important in the pharmaceutical industry, and are catalyzed by monooxygenase (sulfoxidation, epoxidation) and dioxygenase (sulfoxidation, dihydroxylation) enzymes. Sulfoxide and epoxide metabolites of thiophene substrates are often unstable, and, while cis-dihydrodiol metabolites are more stable, significant challenges are presented by both types of metabolite. Prediction of the structure, relative and absolute configuration, and enantiopurity of chiral metabolites obtained from thiophene enzymatic oxidation depends on the substrate, type of oxygenase selected, and molecular docking results. The racemization and dimerization of sulfoxides, cis/trans epimerization of dihydrodiol metabolites, and aromatization of epoxides are all factors associated with the mono- and di-oxygenase-catalyzed metabolism of thiophenes and thiophene-containing drugs and their applications in chemoenzymatic synthesis and medicine.

cis-Dihydrodiols, derived from monocyclic aromatic compounds, are valuable chiral pool intermediates for the synthesis of cyclic natural products. A drawback of this approach, to the synthesis of polycyclic secondary metabolites, is that additional rings must be annulated. To date, relatively few chiral natural products have been synthesized from polycyclic arene cis-dihydrodiols. Fungal metabolites, (-)-ribisins A and B, have now been obtained by functional group manipulation of a tricyclic arene metabolite, obtained from toluene dioxygenase-catalyzed regioselective and stereoselective cis-dihydroxylations of dibenzo[b,d]furan. The synthetic sequences were marginally shorter than the alternative routes, using monocyclic arene cis-dihydrodiols, and required no carbon-carbon bond-forming reactions.

Molecular docking studies of quinoline and 2-chloroquinoline substrates at the active site of toluene dioxygenase (TDO), were conducted using Autodock Vina, to identify novel edge-to-face interactions and to rationalize the observed stereoselective cis-dihydroxylation of carbocyclic rings and formation of isolable cis-dihydrodiol metabolites. These in silico docking results of quinoline and pyridine substrates, with TDO, also provided support for the postulated cis-dihydroxylation of electron-deficient pyridyl rings, to give transient cis-dihydrodiol intermediates and the derived hydroxyquinolines. 2-Chloroquinoline cis-dihydrodiol metabolites were used as precursors in the chemoenzymatic synthesis of enantiopure arene oxide and arene dioxide derivatives of quinoline, in the context of its possible mammalian metabolism and carcinogenicity.

Molecular docking studies of toluene dioxygenase (TDO) led to the prediction that angular and lateral cis‐dihydroxylation of tricyclic arene and heteroarene substrates could occur. Biotransformations of biphenylene, dibenzofuran, carbazole and dibenzothiophene, using P. putida UV4 whole cells expressing TDO, confirmed that both angular and lateral cis‐dihydroxylation occurred in the predicted regioselective and stereoselective manner. The TDO‐catalysed (P. putida UV4) biotransformation of dibenzofuran was optimized, to produce 1,2‐dihydrodibenzofuran‐1,2‐diol as the major metabolite (> 80% yield). 2‐Hydroxydibenzofuran, resulting from dehydration of 1,2‐dihydrodibenzofuran‐1,2‐diol, was also found to undergo cis‐ dihydroxylation to give a very minor cis‐dihydrodiol metabolite (< 2% yield). The enantiopurity (>98% ee) and (1R,2S) absolute configuration of the major dibenzofuran cis ‐dihydrodiol was rigorously established by formation of diMTPA ester derivatives and X‐ray crystallography of a diol epoxide derivative. The cis‐dihydrodiol metabolite of dibenzofuran has potential in the chemoenzymatic synthesis of natural products. dioxygenase‐catalysed cis‐dihydroxylation of substituted phenol and aniline substrates with Pseudomonas putida UV4, yielded arene cis‐dihydrodiol metabolites which tautomerised to the preferred cyclohex‐2‐en‐1‐one cis‐diols, as predicted by molecular docking studies. Further metabolism of cyclohex‐2‐en‐1‐one cis‐diols, under similar conditions, formed 4‐hydroxycyclohex‐2‐en‐1‐ones, as a new type of phenol metabolite

Enantiopure β-hydroxy sulfoxides and catechol sulfoxides were obtained, by chemoenzymatic synthesis, involving dioxygenase-catalysed benzylic hydroxylation or arene cis-dihydroxylation and cis-diol dehydrogenase-catalysed dehydrogenation. Absolute configurations of chiral hydroxy sulfoxides were determined by X-ray crystallography, ECD spectroscopy and stereochemical correlation. The application of a new range of β-hydroxy sulfoxides as chiral ligands was examined.

Benzene cis-diol dehydrogenase and naphthalene cis-diol dehydrogenase enzymes, expressed in Pseudomonas putida wild-type and Escherichia coli recombinant strains, were used to investigate regioselectivity and stereoselectivity during dehydrogenations of arene, cyclic alkane and cyclic alkene vicinal cis-diols. The dehydrogenase-catalysed production of enantiopure cis-diols, α-ketols and catechols, using benzene cis-diol dehydrogenase and naphthalene cis-diol dehydrogenase, involved both kinetic resolution and asymmetric synthesis methods. The chemoenzymatic production and applications of catechol bioproducts in synthesis were investigated. Fil: Boyd, Derek R.. The Queens University of Belfast; Irlanda Fil: Sharma, Narain D.. The Queens University of Belfast; Irlanda Fil: Berberian, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. The Queens University of Belfast; Irlanda Fil: Cleij, Marcel. The Queens University of Belfast; Irlanda Fil: Hardacre, Christopher. The Queens University of Belfast; Irlanda Fil: Ljubez, Vera. The Queens University of Belfast; Irlanda Fil: McConville, Gareth. The Queens University of Belfast; Irlanda Fil: Stevenson, Paul J.. The Queens University of Belfast; Irlanda Fil: Kulakov, Leonid A.. The Queens University of Belfast; Irlanda Fil: Allen, Christopher C. R.. The Queens University of Belfast; Irlanda

Using toluene dioxygenase as biocatalyst, enantiopure cis-dihydrodiol and cis-tetrahydrodiol metabolites, isolated as their ketone tautomers, were obtained from meta and ortho methoxyphenols. Although these isomeric phenol substrates are structurally similar, the major bioproducts from each of these biotransformations were found at different oxidation levels. The relatively stable cyclohexenone cis-diol metabolite from meta methoxyphenol was isolated, while the corresponding metabolite from ortho methoxyphenol was rapidly bioreduced to a cyclohexanone cis-diol. The chemistry of the 3-methoxycyclohexenone cis-diol product was investigated and elimination, aromatization, hydrogenation, regioselective O-exchange, Stork-Danheiser transposition and O-methylation reactions were observed. An offshoot of this technology provided a two-step chemoenzymatic synthesis, from meta methoxyphenol, of a recently reported chiral fungal metabolite; this synthesis also established the previously unassigned absolute configuration.

Toluene dioxygenase-catalysed cis-dihydroxylations of substituted aniline and phenol substrates, with a Pseudomonas putida UV4 mutant strain and an Escherichia coli pCL-4t recombinant strain, yielded identical arene cis-dihydrodiols, which were isolated as the preferred cyclohex-2-en-1-one cis-diol tautomers. These cis-diol metabolites were predicted by preliminary molecular docking studie, of anilines and phenols, at the active site of toluene dioxygenase Further biotransformations of cyclohex-2-en-1-one cis-diol and hydroquinone metabolites, using Pseudomonas putida UV4 whole cells, were found to yield 4-hydroxycyclohex-2-en-1-ones as a new type of phenol bioproduct. Multistep pathways, involving ene reductase and carbonyl reductase-catalysed reactions, were proposed to account for the production of 4-hydroxycyclohex-2-en-1-one metabolites. Evidence for the phenol hydrate tautomers of 4-hydroxycyclohex-2-en-1-one metabolites was shown by formation of the corresponding trimethysilyl ether derivatives.

The relative merits of the methods employed to determine enantiomeric excess (ee) values and absolute configurations of chiral arene and alkene cis-1,2-diol metabolites, including boronate formation, using racemic or enantiopure (+) and (-)-2-(1-methoxyethyl)phenylboronic acid (MEPBA), are discussed. Further applications of: 1) MEPBA derived boronates of chiral mono- and poly-cyclic arene cis-dihydrodiol, cyclohex-2-en-1-one cis-diol, heteroarene cis/trans-2,3-diol, and catechol metabolites in estimating their ee values, and 2) new chiral phenylboronic acids, 2-[1-methoxy-2,2-dimethylpropyl]phenyl boronic acid (MDPBA) and 2-[1-methoxy-1-phenylmethyl]phenyl boronic acid (MPPBA) and their advantages over MEPBA, as reagents for stereochemical analysis of arene and alkene cis-diol metabolites, are presented.

Enantioenriched and enantiopure thiosulfinates were obtained by asymmetric sulfoxidation of cyclic 1,2-disulfides, using chemical and enzymatic (peroxidase, monooxygenase, dioxygenase) oxidation methods and chiral stationary phase HPLC resolution of racemic thiosulfinates. Enantiomeric excess values, absolute configurations and configurational stabilities of chiral thiosulfinates were determined. Methyl phenyl sulfoxide, benzo[c]thiophene cis-4,5-dihydrodiol and 1,3-dihydrobenzo[c]thiophene derivatives were among unexpected types of metabolites isolated, when acyclic and cyclic 1,2-disulfide were used as substrates for Pseudomonas putida strains. Possible biosynthetic pathways are presented for the production of metabolites from 1,4-dihydrobenzo-2,3-dithiane, including a novel cis-dihydrodiol metabolite that was also derived from benzo[c]thiophene and 1,3-dihydrobenzo[c]thiophene.

Toluene dioxygenase-catalysed (TDO) oxidation converts substituted phenol substrates into catechols, hydroquinones, and chiral cyclohexenone cis -diol products. The ratio between the isolated products varied widely even between similar substrates, e.g. o -cresol, m -cresol and p -cresol. These differences are caused by different binding interactions within the active site of TDO. This study provides insight into the binding interactions by molecular docking using AutoDock tools. The nature of binding of phenolic substrates was of major interest, in order to explain the observed regio- and stereo-selectiviy of product formation. The ellipse-shaped binding pocket of TDO consists of a polar and a hydrophobic region, limiting the possible substrate orientations. The phenolic hydroxyl group was preferentially hydrogen bonded with Gln-215 and His-311 in the active site. In some cases, a hydrogen bond was formed with other amino acids, e.g. Asp-219 and Met-220, instead. The position and type of the substituent on the phenol ring influences the formation of transient intermediates, and thus the nature and stability of the major isolated product.

Acid-catalyzed dehydrations of substituted naphthalene-cis-1,2-dihydrodiols occur with loss of the 1- or 2-OH group to form 2- and 1-naphthols, respectively. Effects of substituents MeO, Me, H, F, Br, I, and CN at 3-, 6-, and 7-positions of the naphthalene ring are consistent with rate-determining formation of β-hydroxynaphthalenium ion (carbocation) intermediates. For reaction of the 1-hydroxyl group the 3-substituents are correlated by the Yukawa-Tsuno relationship with ρ = -4.7 and r = 0.25 or by σ(p) constants with ρ = -4.25; for reaction of the 2-hydroxyl group the 3-substituents are correlated by σ(m) constants with ρ = -8.1. The correlations for the 1-hydroxyl imply a surprisingly weak resonance interaction of +M substituents (MeO, Me) with a carbocation reaction center but are consistent with the corresponding correlation for acid-catalyzed dehydration of 3-substituted benzene-cis-1,2-dihydrodiols for which ρ = -6.9 and r = 0.43. Substituents at the 6- and 7-positions of the naphthalene rings by contrast are correlated by σ(+) with ρ = -3.2 for reaction of the 1-hydroxyl group and ρ = -2.7 for reaction of the 2-hydroxyl group. The unimpaired resonance implied by these substituent effects appears to be inconsistent with a previous explanation of the weak resonance of the 3-substituents in terms of imbalance of charge development and/or nonplanarity of the benzenium ring in the transition state. An alternative possibility is that the adjacent hydroxyl group interferes sterically with conjugation of +M substituents. "Hyperaromaticity" of the arenium ion intermediates does not appear to be a factor influencing this behavior.

Enantiopure arene cis-tetrahydrodiols of bromobenzene and iodobenzene have been obtained in good yields, from chemoselective hydrogenation (rhodium-graphite) of the corresponding cis-dihydrodiol metabolites. Palladium-catalysed substitution of the halogen, by hydrogen, boron, nitrogen and phosphorus nucleophiles, in the acetonide derivatives, has yielded highly functionalised products for application in synthesis with potential as scaffolds for chiral ligands.

Enantiopure cis-dihydro-1,2-diol metabolites, obtained from toluene dioxygenase-catalysed cis-dihydroxylation of six monosubstituted benzene substrates, have been converted to their corresponding cis-hexahydro-1,2-diol derivatives by catalytic hydrogenation via their cis-tetrahydro-1,2-diol intermediates. Optimal reaction conditions for total catalytic hydrogenation of the cis-dihydro-1,2-diols have been established using six heterogeneous catalysts. The relative and absolute configurations of the resulting benzene cis-hexahydro-1,2-diol products have been unequivocally established by X-ray crystallography and NMR spectroscopy. Methods have been developed to obtain enantiopure cis-hexahydro-1,2-diol diastereoisomers, to desymmetrise a meso-cis-hexahydro-1,2-diol and to synthesise 2-substituted cyclohexanols. The potential of these enantiopure cyclohexanols as chiral reagents was briefly evaluated through their application in the synthesis of two enantiomerically enriched phosphine oxides from the corresponding racemic phosphine precursors.

Absolute configurations of a number of cis-dihydrodiols (cis-1,2-dihydroxy-3,5-cyclohexadienes), synthetically useful products of TDO-catalyzed dihydroxylations of 1,2- and 1,3-disubstituted benzene derivatives, have been determined by a comparison of calculated and experimental CD spectra and optical rotations and by methods involving X-ray crystallography, 1H NMR spectra of diastereoisomeric derivatives, and by stereochemical correlations. The computations disclosed a significant effect of the substituents on conformational equilibria of cis-dihydrodiols and chiroptical properties of individual conformers. The assigned absolute configurations of cis-dihydrodiols have allowed the validity of a simple predictive model for TDO-catalyzed arene dihydroxylations to be extended. Chirality, 2008. © 2007 Wiley-Liss, Inc.

A series of ten cis-dihydrodiol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and 1H NMR spectroscopy. Absolute configurations of the majority of cis-dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis-dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples.

A series of cis-dihydrodiol metabolites, available from the bacterial dioxygenase-catalysed oxidation of monosubstituted benzene substrates using Pseudomonas putida UV4 , have been converted to the corresponding catechols using both a heterogeneous catalyst (Pd/C) and a naphthalene cis-diol dehydrogenase enzyme present in whole cells of the recombinant strain Escherichia coli DH5α(pUC129: nar B). A comparative study of the merits of both routes to 3-substituted catechols has been carried out and the two methods have been found to be complementary. A similarity in mechanism for catechol formation under both enzymatic and chemoenzymatic conditions, involving regioselective oxidation of the hydroxyl group at C-1, has been found using deuterium labelled toluene cis-dihydrodiols. The potential, of combining a biocatalytic step (dioxygenase-catalysed cis-dihydroxylation) with a chemocatalytic step (Pd/C-catalysed dehydrogenation), into a one-pot route to catechols, from the parent substituted benzene substrates, has been realised.

Factors that control the competition between toluene dioxgenase-catalysed arene cis-dihydroxylation and dehydrogenase-catalysed ketone reduction have been studied, using whole cells of Pseudomonas putida UV and three alkylaryl ketones. The triol metabolite, obtained from 2,2,2-trifluoroacetophenone, has been used in the synthesis of single enantiomer chiral phenols and benzylic alcohols. Potential applications of the methylether derivatives of the chiral phenols and benzylic alcohols, as resolving agents, have been found. Copyright © 2007 Society of Chemical Industry

Dioxygenase-catalysed asymmetric dihydroxylation, of a series of conjugated monoalkenes and polyenes, was found to yield the corresponding monols and 1,2-dihydrodiols. The diol metabolites were obtained from monosubstituted, gem-disubstituted, cis-disubstituted, and trisubstituted alkene substrates, using whole cells of Pseudomonas putida strains containing toluene and naphthalene dioxygenases. Dioxygenase selection and alkene type were established as important factors, in the preference for dioxygenase-catalysed 1,2-diHydroxylation of conjugated alkene or arene groups, and monohydroxylation at benzylic or allylic centres. Competition from allylic hydroxylation of methyl groups was observed only when naphthalene dioxygenase was used as biocatalyst. The structures, enantiomeric excess values and absolute configurations of the bioproducts, were determined by a combination of stereochemical correlation, spectroscopy (NMR and CD) and X-ray diffraction methods. cis-1,2-Diol metabolites from arenes, cyclic alkenes and dienes were generally observed to be enantiopure (>98% ee), while 1,2-diols from acyclic alkenes had lower enantiomeric excess values (

Absolute configurations and conformations of selected cis-1,2-dihydrodiols, isolated from bacterial enzyme-catalyzed arene dihydroxylation, have been examined by comparison of experimental and DFT-calculated CD spectra and confrontation with the results of X-ray diffraction studies in the crystalline phase. The equilibrium between the diene P and M conformers in cis-dihydrodiols is strongly dependent on the intramolecular OH-OH, OH-pi, and OH-F hydrogen bonding pattern and is crucial in determining the sign and magnitude of the long-wavelength diene pi-pi transition Cotton effect. The differences originate from a dominant contribution of either P-helical (1b, X=Me) or M-helical conformers (1d, X=F), or are due to M and P low-energy conformers, both contributing a positive rotational strength (1c, X=Br). Computations show that cis-dihydrodiol 1e (X=CF3) has only one M conformer stabilized by an intramolecular O-H...F hydrogen bond. cis-Dihydrodiol 1f (X=CN) shows a Cotton effect of the sign opposite to the sense of helicity of the dominating conformer. The results of the computations highlight the inadequacy of the Diene Helicity Rule and the Allylic Chirality Rule to correlate observed Cotton effects with dihydrodiol absolute configuration. A reliable model is presented to predict the absolute configuration of substituted benzene dihydrodiol derivatives from CD spectra, based on the confrontation of DFT-computed and experimental CD spectra. For 3-alkyl derivatives, a simple noncomputational model is offered, which is based on the contributions of the allylic hydroxy groups and the diene core in P and M conformers.

The kinetic resolution of racemic sulfoxides by dimethyl sulfoxide (DMSO) reductases was investigated with a range of microorganisms. Three bacterial isolates (provisionally identified as Citrobacter braakii, Klebsiella sp. and Serratia sp.) expressing DMSO reductase activity were isolated from environmental samples by anaerobic enrichment with DMSO as terminal electron acceptor. The organisms reduced a diverse range of racemic sulfoxides to yield either residual enantiomer depending upon the strain used. C. braakii DMSO-11 exhibited wide substrate specificity that included dialkyl, diaryl and alkylaryl sulfoxides, and was unique in its ability to reduce the thiosulfinate 1,4-dihydrobenzo-2, 3-dithian-2-oxide. DMSO reductase was purified from the periplasmic fraction of C. braakii DMSO-11 and was used to demonstrate unequivocally that the DMSO reductase was responsible for enantiospecific reductive resolution of racemic sulfoxides.

Toluene- and naphthalene-dioxygenase-catalysed oxidation of six bicyclic disulfide substrates, using whole cells of Pseudomonas putida , gave the corresponding monosulfoxides with high ee values and enantiocomplementarity, in most cases. Two alcohol-sulfoxide diastereoisomers, formed from the reaction of the ( R )-1,3-benzodithiole-1-oxide metabolite with n -butyllithium and benzaldehyde, were separated and stereochemically assigned. Treatment, of enantiopure (1 R ,3 R )-benzo-1,3-dithiole-1,3-dioxide, obtained by chemoenzymatic synthesis, with alkyllithium reagents, resulted in a novel ring-opening reaction which proceeded with inversion of configuration to yield a series of acyclic disulfoxides.

Toluene dioxygenase (TDO)-catalysed sulfoxidation, using Pseudomonas putida UV4, was observed for the thiophene substrates 1A–1N. The unstable thiophene oxide metabolites, 6A–6G, 6K–6N, spontaneously dimerised yielding the corresponding racemic disulfoxide cycloadducts 7A–7G, 7K–7N. Dimeric or crossed [4 + 2] cycloaddition products, derived from the thiophene oxide intermediates 6A and 6D or 6B and 6D, were found when mixtures of thiophene substrates 1A and 1D or 1B and 1D were biotransformed. The thiophene sulfoxide metabolite 6B was also trapped as cycloadducts 17 or 18 using stable dienophiles. Preferential dioxygenase-catalysed oxidation of the substituent on the thiophene ring, including exocyclic sulfoxidation (1H–1J) and cis-dihydroxylation of a phenyl substituent (1G and 1N), was also observed. An enzyme-catalysed deoxygenation of a sulfoxide in P. putida UV4 was noticed when racemic disulfoxide cycloadducts 7A, 7B and 7K were converted to the corresponding enantioenriched monosulfoxides 8A, 8B and 8Kvia a kinetic resolution process. The parent thiophene 1A and the 3-substituted thiophenes 1K–1N were also found to undergo ring dihydroxylation yielding the cis/trans-dihydrodiol metabolites 9A and 9K–9N. Evidence is provided for a dehydrogenase-catalysed desaturation of a heterocyclic dihydrodiol (9Kcis/9Ktrans) to yield the corresponding 2,3-dihydroxythiophene (24) as its preferred thiolactone tautomer (23). A simple model to allow prediction of the structure of metabolites, formed from TDO-catalysed bacterial oxidation of thiophene substrates 1, is presented.

Several potential approaches to the enzyme-catalysed synthesis of arene trans -diols have been examined including epoxidation/hydrolysis, bis-benzylic hydroxylation, cis -dihydroxylation/alcohol dehydrogenation/ketone reduction, cis -dihydroxylation/ cis – trans isomerisation and multi-enzyme synthesis of trans -dihydrodiol secondary metabolites from primary metabolites. The lack of general applicability of these enzymatic methods has led to the development of several chemoenzymatic routes for the synthesis of a series of trans -dihydrodiols from the readily available cis -dihydrodiol precursors. Partial hydrogenation of cis -dihydrodiol metabolites to yield the corresponding cis -tetrahydrodiols followed by a regioselective Mitsunobu inversion process gave trans -tetrahydrodiols that were in turn converted to trans -dihydrodiols. The formation of anti-benzene dioxides or iron tricarbonyl complexes from the corresponding cis -dihydrodiol precursors provided shorter and more convenient chemoenzymatic routes to trans -dihydrodiols. The application of cis -dihydrodiol metabolites of polycyclic azaarenes in the synthesis of the corresponding arene oxides followed by chemical hydrolysis provides a convenient route to trans -dihydrodiols.

A novel haloarchaeal strain, Haloarcula sp. strain D1, grew aerobically on 4-hydroxybenzoic acid (4HBA) as a sole carbon and energy source and is the first member of the domain Archaea reported to do so. Unusually, D1 metabolized 4HBA via gentisic acid rather than via protocatechuic acid, hydroquinone, or catechol. Gentisate was detected in 4HBA-grown cultures, and gentisate 1,2-dioxygenase activity was induced in 4HBA-grown cells. Stoichiometric accumulation of gentisate from 4HBA was demonstrated in 4HBA-grown cell suspensions containing 2,2′-dipyridyl (which strongly inhibits gentisate 1,2-dioxygenase). To establish whether initial 1-hydroxylation of 4HBA with concomitant 1,2-carboxyl group migration to yield gentisate occurred, 2,6-dideutero-4HBA was synthesized and used as a substrate. Deuterated gentisate was recovered from cell suspensions and identified as 3-deutero-gentisate, using gas chromatography-mass spectrometry and proton nuclear magnetic resonance spectroscopy. This structural isomer would be expected only if a 1,2-carboxyl group migration had taken place, and it provides compelling evidence that the 4HBA pathway in Haloarcula sp. strain D1 involves a hydroxylation-induced intramolecular migration. To our knowledge, this is the first report of a pathway which involves such a transformation (called an NIH shift) in the domain Archaea .

Toluene dioxygenase-catalyzed dihydroxylation, in the carbocyclic rings of quinoline, 2-chloroquinoline, 2-methoxyquinoline, and 3-bromoquinoline, was found to yield the corresponding enantiopure cis-5,6- and -7,8-dihy dro diol metabolites using whole cells of Pseudomonas putida UV4. cis-Dihydroxylation at the 3,4-bond of 2-chloroquino line, 2-methoxyquinoline, and 2-quinolone was also found to yield the heterocyclic cis-dihydrodiol metabolite, (+)-cis-(3S,4S)-3,4-dihydroxy-3,4-dihydro-2-quinolone. Heterocyclic cis-dihydrodiol metabolites, resulting from dihydroxylation at the 5,6- and 3,4-bonds of 1-methyl 2-pyridone, were isolated from bacteria containing toluene, naphthalene, and biphenyl dioxygenases. The enantiomeric excess (ee) values (>98%) and the absolute configurations of the carbocyclic cis-dihydrodiol metabolites of quinoline substrates (benzylic R) and of the heterocyclic cis-diols from quinoline, 2-quinolone, and 2-pyridone substrates (allylic S) were found to be in accord with earlier models for dioxygenase-catalyzed cis-dihydroxylation of carbocyclic arenes. Evidence favouring the dioxygenase-catalyzed cis-dihydroxylation of pyridine-ring systems is presented.Key words: dioxygenases; cis-dihydroxylation, pyridines, 2-pyridones, absolute configurations.

The enantiopure (1 S, 2 S )- cis -dihydrodiol metabolites 2B - 5B have been obtained in low yield from the corresponding monosubstituted halobenzene substrates 2A - 5A, using a wild-type strain of Pseudomonas putida (ML2) containing benzene dioxygenase (BDO). Benzene cis -dihydrodiol dehydrogenase (BCD) from P. putida ML2 and naphthalene cis -dihydrodiol dehydrogenase (NCD) from P. putida 8859 were purified and used in a comparative study of the stereoselective biotransformation of cis -dihydrodiol enantiomers 2B - 5B. The BCD and NCD enzymes were found to accept cis -dihydrodiol enantiomers of monosubstituted benzene cis -dihydrodiol substrates 2B - 5B of opposite absolute configuration. The acyclic alkene 1,2-diols 10 - 17 were also found to be acceptable substrates for BCD.

Monocyclic allylic cis-diols (I) react with sulfuryl chloride at 0 °C regio- and stereoselectively to give 2-chloro-1-sulfochloridates (II), which can be hydrolyzed to yield the corresponding trans-chlorohydrins (III).

Aromatic dioxygenases have been found to catalyse single and tandem oxidation reactions of conjugated polyenes. Rational selection and design of dioxygenases, allied to substrate shape, size and substitution pattern, has been used to control regiochemistry and stereochemistry during the oxygenation process. The resulting enantiopure bioproducts have been increasingly utilised as precursors for new and alternative routes in chiral synthesis.

Dibenz[a,h]anthracene 3,4-oxide 5ARS, synthesised from the enantiopure dibromoMTPA precursor 9ARRS*, was found to have totally racemized and to be accompanied by benz[5,6]anthra[1,2-b]oxepine 11A. Phenanthrene 3,4-oxide 5BRS, obtained from the enantiopure bacterial metabolite cis-3,4-dihydroxy-3,4-dihydrophenanthrene 12B by a modified synthetic approach involving the chlorohydrin ester 16B, was observed to racemize spontaneously at ambient temperature. Dibenz[a,h]anthracene 3,4-oxide 5ARS/5ASR, phenanthrene 3,4-oxide 5BRS/5BSR, triphenylene 1,2-oxide 5CRS/5CSR, and dibenz[a,c]anthracene 1,2-oxide 5DRS/5DSR, obtained from the corresponding racemic cis-tetrahydrodiol precursors 14A–14D by the new method, were obtained without any evidence of the formation of benz[5,6]anthra[1,2-b]oxepine 11A, naphth[1,2-b]oxepine 11B, phenanthro[10,9-b]oxepine 11C, or benz[3,4]anthra[1,2-b]oxepine 11D isomers respectively. The total racemization of arene oxide 5ARS and formation of oxepine 11A from the bromoMTPA precursor 8ARRS* are in accord with earlier PMO predictions based on resonance energy considerations. Photoisomerization of arene oxides 5ARS/5ASR, 5CRS/5CSR, and 5DRS/5DSR was found to yield the corresponding oxepines 11A, 11C, and 11D.

Toluene dioxygenase (TDO)-catalysed monooxygenation of methylsulfanylmethyl phenyl sulfide 1 and methylsulfanylmethyl 2-pyridyl sulfide 4, using whole cells of Pseudomonas putida UV4, occurred exclusively at the alkyl aryl sulfur centre to yield the alkyl aryl sulfoxides 2 and 5 respectively. These sulfoxides, accompanied by the dialkyl sulfoxides 3 and 6, were also obtained from naphthalene dioxygenase (NDO)-catalysed sulfoxidation of thioacetals 1 and 4 using intact cells of P. putida NCIMB 8859. Enzymatic oxidation of methyl benzyl sulfide 7, 2-phenyl-1,3-dithiane 19, and 2-phenyl-1,3-dithiolane 23, using TDO, gave the corresponding dialkyl sulfoxides 8, 20 and 24 as minor bioproducts. TDO-catalysed dioxygenation of the alkyl benzyl sulfides 7,15 and 17 and the thioacetals 19 and 23, with P. putida UV4, yielded the corresponding enantiopurecis-dihydrodiols 9, 16, 18, 21 and 25 as major metabolites and cis-dihydrodiol sulfoxides 14, 22 and 26 as minor metabolites, resulting from a tandem trioxygenation of substrates 7, 19 and 23 respectively. Chemical oxidation, of the enantiopure cis-dihydrodiol sulfides 9, 16, 18 and 21 with dimethyldioxirane (DMD), gave separable mixtures of the corresponding pairs of cis-dihydrodiol sulfoxide diastereoisomers 14 and 27, 28 and 29, 30 and 31, 22 and 32. While dialkyl sulfoxide bioproducts 3, 6, 20 and 24 were of variable enantiopurity (27-≥98% ee), alkyl aryl monosulfoxides 2 and 5, cis-dihydrodiols 9, 16, 18, 21 and 25 and cis-dihydrodiol sulfoxide bioproducts 14, 22 and 26 were all single enantiomers (≥98% ee). The absolute configurations of the products, obtained from enzyme-catalysed (TDO and NDO) and chemical (DMD) oxidation methods, were determined by stereochemical correlation, circular dichroism, and X-ray crystallographic methods.

Bacterial dioxygenase-catalysed cis-dihydroxylation of the tetracyclic arenes benzo[c]phenanthrene 2, and the isosteric compounds benzo[b]naphtho[1,2-d]furan 8, and benzo[b]naphtho[1,2-d]thiophene 9, has been found to occur exclusively at fjord-region bonds. The resulting cis-dihydrodiols 7, 10 and 11 were found to be enantiopure and of similar absolute configuration. cis-Dihydroxylation was also observed in the pseudo-fjord region of the 8,9,10,11-tetrahydro-precursors (12 and 13) of benzo[b]naphtho[1,2-d]furan 8, and benzo[b]naphtho[1,2-d]thiophene 9, to yield the corresponding enantiopure hexahydro cis-diols 14 and 15. A novel tandem cis-dihydroxylation and bis-desaturation of the tetrahydro-substrate, tetrahydrobenzo[b]naphtho[1,2-d]thiophene 13, catalysed by biphenyl dioxygenase, was found to yield the fjord-region cis-dihydrodiol 17 of benzo[b]naphtho[1,2-d]thiophene 9.

The chiral quinoline alkaloids platydesmine 3, platydesmine methosalt 4 and edulinine 9, have been synthesised in enantiopure form via asymmetric dihydroxylation of the achiral alkaloid atanine 1. Chromatographic separation of MTPA diastereoisomers 20 formed from racemic bromohydrin derivatives of atanine 1 was a key step in the synthesis of geibalansine 7, edulinine 9, ribalinine 10, Ψ-ribalinine 11 and araliopsine 12 as single enantiomers. The absolute configurations of (+)-platydesmine methosalt 4 and (−)-Ψ-ribalinine 11 were unequivocally determined by X-ray crystallography while stereochemical correlation and circular dichroism spectroscopy methods were used to assign absolute configurations to platydesmine 3, geibalansine 7, ribalinine 10, araliopsine 12 and edulinine 9. Possible errors which earlier led to the incorrect assignment of absolute configurations of the quinoline alkaloids platydesmine 3, platydesmine methosalt 4, edulinine 9, araliopsine 12 and other related chiral quinoline alkaloids are discussed.

Asymmetric cis-dihydroxylation to yield diols 2A–2G and sequential benzylic monohydroxylation–cis-dihydroxylation to yield triols 4A–4G (trihydroxylation), occurred during biotransformation of a series of monosubstituted alkylbenzene substrates 1A–1G using toluene dioxygenase, a biocatalyst present in Pseudomonas putida UV4. Dioxygenase-catalysed cis-dihydroxylation of the R and S benzylic alcohol enantiomers 3B–3D, 3B′–3D′ gave the corresponding enantiopure triols 4B–4D, 4B′–4D′. Biotransformation of substrates 1J–1L yielded cis-diols 2J–2L and a minor triol metabolite 4A. Benzylic alcohols 3J–3L were postulated as unstable intermediates yielding triol 4Avia benzaldehyde 5 and benzyl alcohol 3A intermediates. cis-Dihydroxylation of monosubstituted benzylic substrates containing bulky groups (1H, 1I) or 1,4-dialkyl-substituted benzene substrates (10A–10C) gave the corresponding cis-dihydrodiol metabolites (2H, 2I, 11A–11C) exclusively. The cis-diols 2A–2L, 11A–11C and triols 4A–4F, 4B′–4D′ were stereochemically assigned as single enantiomers of 1S,2R-configuration based on NMR and CD spectroscopy. The absolute configurations of the exocylic chiral centres in the triol bioproducts 4A–4F, 4B′–4D′ were established by stereochemical correlation and aromatisation/hydrogenation to yield the corresponding enantiopure phenolic benzylic alcohols having similar CD spectra.

The biphenyl dioxygenase-catalyzed asymmetric mono-cis-dihydroxylation of the tetracyclic arenes chrysene 1A, benzo[c]phenanthridine 1B, and benzo[b]naphtho[2,1-d]thiophene 1C, has been observed to occur exclusively at the bay or pseudo-bay region using the bacterium Sphingomonas yanoikuyae B8/36. The mono-cis-dihydrodiol derivatives 2A and 2C, obtained from chrysene 1A by oxidation at the 3,4-bond (2A) and benzo[b]naphtho[2,1-d]thiophene 1C by oxidation at the 1,2-bond (2C), respectively, have been observed to undergo a further dioxygenase-catalyzed asymmetric cis-dihydroxylation at a second bay or pseudo-bay region bond to yield the corresponding bis-cis-dihydrodiols (cis-tetraols) 4A and 4C, the first members of a new class of microbial metabolites in the polycyclic arene series. The enantiopurities and absolute configurations of the new mono-cis-dihydrodiols 2B, 2C, and 3B were determined by 1H NMR analyses of the corresponding (R)- and (S)-2-(1-methoxyethyl)benzeneboronate (MPBA) ester derivatives. T...

The biotransformation of the polycyclic aromatic hydrocarbons (PAHs) naphthalene and phenanthrene was investigated by using two dioxygenase-expressing bacteria, Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae B8/36, under conditions which facilitate mass-transfer limited substrate oxidation. Both of these strains are mutants that accumulate cis-dihydrodiol metabolites under the reaction conditions used. The effects of the nonpolar solvent 2,2,4,4,6,8,8-heptamethylnonane (HMN) and the nonionic surfactant Triton X-100 on the rate of accumulation of these metabolites were determined. HMN increased the rate of accumulation of metabolites for both microorganisms, with both substrates. The enhancement effect was most noticeable with phenanthrene, which has a lower aqueous solubility than naphthalene. Triton X-100 increased the rate of oxidation of the PAHs with strain 9816/11 with the effect being most noticeable when phenanthrene was used as a substrate. However, the surfactant inhibited the biotransformation of both naphthalene and phenanthrene with strain B8/36 under the same conditions. The observation that a nonionic surfactant could have such contrasting effects on PAH oxidation by different bacteria, which are known to be important for the degradation of these compounds in the environment, may explain why previous research on the application of the surfactants to PAH bioremediation has yielded inconclusive results. The surfactant inhibited growth of the wild-type strain S. yanoikuyae B1 on aromatic compounds but did not inhibit B8/36 dioxygenase enzyme activity in vitro. Polycyclic aromatic hydrocarbons (PAHs) are a major cause of concern as anthropogenic pollutants in the environment. They arise from diverse sources, including petrochemical products and the combustion of fossil fuels (3). Concern arises for two reasons, first because many are recalcitrant, and second because of the health hazards associated with these compounds. Many, such as benzo[a]pyrene, chrysene, and benz [a]anthracene, are also carcinogens in animals. One approach that has been considered for enhancing PAH bioremediation in contaminated soils is the application of nonionic surfactants (26). The theoretical justification for this solution is based upon two hypotheses, first that surfactant micelles may sequester PAHs which are sorbed to the soil matrix, and second that the surfactant micelles may increase the concentration of PAHs in the aqueous phase because the PAHs are more soluble in the micelles. Where the rate of PAH degradation is limited by mass transfer from the solid phase to the aqueous phase, the PAH oxidation rates by microorgan

Indane, 1A, and a series of 2-substituted indane substrates, 1B–1D, 1G, 1I–1L, were found to undergo benzylic monohydroxylation catalysed by toluene dioxygenase, present in the intact cells of Pseudomonas putida UV 4, to yield enantiopure cis-indan-1-ols, 2A–2D, 2G, 2I–2L of the same absolute configuration at C-1 as major bioproducts. Enantiopure trans-indan-1-ols 6B, 6C, and 6G were also obtained as minor metabolites. Evidence of further sequential benzylic hydroxylation (bis-hydroxylation) was found only with substrates 2A, 1C, 1D and 1L to yield the corresponding enantiopure trans-1,3-diols, 3A, 3C, 3D and 3L. Minor enzyme-catalysed processes also observed include benzylic alcohol oxidation to ketones (4A, 5A, 4B, 4L, 5L), ketone reduction to benzylic alcohol 6A, ester hydrolysis to indan-2-ol 1B, and cis-dihydroxylation of indan-1-ol 6A to triol 7. The enantiopurities and absolute configurations of bioproducts have been determined using MTPA ester formation, circular dichroism spectroscopy and stereochemical correlation methods.The contribution of asymmetric oxidation and kinetic resolution to the production of bioproducts of high ee (>98%), and the metabolic sequence involved in their biotransformation by P. putida UV4 is discussed. Enantiocomplementarity was found during the benzylic hydroxylation of indan-2-ol 1B, using toluene dioxygenase and naphthalene dioxygenase, when both single enantiomers of the metabolites 2B, 4B and 6B of opposite configurations were obtained.

Dioxygenase-catalysed dihydroxylation of azulene and related non-aromatic polyenes has been found to yield enantiopure chiral cis -diols of synthetic potential.

A series of alkyl aryl sulfides were metabolised, using selected strains of the soil bacterium Pseudomonas putida containing either toluene dioxygenase (TDO) or naphthalene dioxygenase (NDO), to give chiral sulfoxides. Alkyl aryl sulfoxides 2a–2k, 4a–4j and 4l, having enantiomeric excess (ee) values of >90%, were obtained by use of the appropriate strain of P. putida (UV4 or NCIMB 8859). Enantiocomplementarity was observed for the formation of sulfoxides 2a, 2b, 2d, 2j, 4a, 4b and 4d, with TDO-catalysed (UV4) oxidation favouring the (R) enantiomer and NDO-catalysed oxidation (NCIMB 8859) the (S) enantiomer. Evidence of involvement of the TDO enzyme was obtained using a recombinant strain of Escherichia coli (pKST 11). The marked degree of stereoselectivity appears to be mainly due to enzyme-catalysed asymmetric sulfoxidation, however the possibility of a minor contribution from kinetic resolution, in some cases, cannot be excluded.

Toluene dioxygenase-catalysed oxidation of mono-substituted benzene substrates (R = F, Cl, Br, I, Me, Et, CH2OAc, CHCH2, CCH, CF3, CN, OMe, OEt, SMe) in growing cultures of Pseudomonas putida UV4 yielded the corresponding cis-dihydrodiol metabolites. Palladium-catalysed cross-coupling of cis-(1S,2S)-1,2-dihydroxy-3-iodocyclohexa-3,5-diene with a range of tributyltin compounds provided a chemoenzymatic route to a further series of cis-dihydrodiol derivatives of monosubstituted benzenes (R = D, CH2CHCH2, Bun, SEt, SPri, SBut, SPh, SC6H4Me-4). The enantiopurities and absolute configurations of the cis-dihydrodiols, obtained by both enzymatic and chemoenzymatic routes, were determined by several new methods including 1H NMR spectroscopic analysis of the bis-MTPA esters of the 4-phenyl-1,2,4-triazoline-3,5-dione cycloadducts, X-ray crystallography, circular dichroism spectroscopy and stereochemical correlation.

Specifically deuteriated samples of toluene, anisole, chlorobenzene, α,α,α-trifluoromethylbenzene, naphthalene and quinoline have been synthesised and used as substrates for dioxygenase-catalysed asymmetric dihydroxylation studies to yield the corresponding cis-dihydrodiols as major bioproducts. Phenols were also detected as minor metabolites in some cases. Dehydration of the deuterium-labelled cis-dihydrodiol metabolites, under thermal conditions, in all cases, resulted in phenol formation accompanied by the NIH Shift. A comparison of NIH Shift results, obtained when phenols are produced by aromatisation of chemically synthesised deuteriated arene cis- and trans-dihydrodiols (dehydration) and arene oxides (isomerisation), suggests that this phenomenon may be associated with both monooxygenase- and dioxygenase-catalysed aromatic hydroxylations.

Monocyclic allylic cis-1,2-diols reacted with sulfuryl chloride at 0 °C in a regio- and stereo-selective manner to give 2-chloro-1-sulfochloridates, which were hydrolysed to yield the corresponding trans-1,2-chlorohydrins. At −78 °C, with very slow addition of sulfuryl chloride, cyclic sulfates were formed in good yields, proved to be very reactive with nucleophiles and rapidly decomposed on attempted storage. Reaction of a cyclic sulfate with sodium azide yielded a trans-azidohydrin without evidence of allylic rearrangement occurring. An enantiopure bicyclic cis-1,2-diol reacted with sulfuryl chloride to give, exclusively, a trans-1,2-dichloride enantiomer with retention of configuration at the benzylic centre and inversion at the non-benzylic centre; a mechanism is presented to rationalise the observation.

Biotransformation of the environmental pollutant chrysene 1 by resting cells of a mutant strain (B8/36) of the soil bacterium Sphingomonas yanoikuyae produces (+)-cis-3,4-dihydroxy-3,4-dihydrochrysene 4 which has been assigned (3S,4R) absolute configuration by stereochemical correlation with (-)-(3S,4R)-cis-3,4- dihydroxy-1,2,3,4-tetrahydrochrysene 6. Both cis-3,4-diol 6 and cis-1,2-dihydroxy-1,2,3,4- tetrahydrochrysene 12 are obtained in enantiopure form after chromatographic separation of the individual bis(2-methoxy-2-phenyl-2-trifluoromethylacetyl) (bis-MTPA) diastereoisomers of compound 6 and the MTPA diastereoisomers of bromohydrin 19, respectively, followed by hydrolysis. A new general synthetic route to cis-dihydrodiols, from the corresponding cis-tetrahydrodiol cyclic carbonates, is used to obtain both racemic and enantiopure forms of the bay-region diol 4, and the non-bay region diol 5. 1 H NMR and CD spectra of the cis- and trans-dihydrodiols of chrysene are described.

The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.

A mutant strain (UV4) of the soil bacterium Pseudomonas putida, containing toluene dioxygenase, has been used in the metabolic oxidation of 1,2-dihydrobenzocyclobutene 12 and the related substrates 1,2-dihydrobenzocyclobuten-1-ol 13 and biphenylene 33. Stable angular cis-monohydrodiol metabolites (1R,2S)-bicyclo[4.2.0]octa-3,5-diene-1,2-diol 7, (1S,2S,8S)-bicyclo[4.2.0]octa-3,5-diene- 1,2,8-triol 8 and biphenylene-cis-1,8b-diol 9, isolated from each of these substrates, have been structurally and stereochemically assigned. The structure, enantiopurity and absolute configuration of the other cis-diol metabolites, (2R,3S)-bicyclo[4.2.0]octa-1(6),4-diene-2,3-diol 14 and cis-1,2-dihydroxy-1,2-dihydrobenzocyclobutene 16, and the benzylic oxidation bioproducts, 1,2-dihydrobenzocyclobuten-1-ol 13, 1,2-dihydrobenzocyclobuten-1-one 15 and 2-hydroxy-1,2-dihydrobenzocyclobuten-1-one 17, obtained from 1,2-dihydrobenzocyclobutene and 1,2-dihydrobenzocyclobuten-1-ol, have been determined with the aid of chiral stationary-phase HPLC, NMR and CD spectroscopy, and stereochemical correlation. X-Ray crystallographic methods have been used in the determination of absolute configuration of the di-camphanates 27 (from diol 7) and 32 (from diol 9), and the di-MTPA ester 29 (from diol 14) of the corresponding cis-diol metabolites. The metabolic sequence involved in the formation of bioproducts derived from 1,2-dihydrobenzocyclobutene 12 has been investigated.

Enantiopure benzylic alcohols containing two stereogenic centres in a cis -relationship result from stereoselective monohydroxylation of achiral 2-substituted indans in cultures of Pseudomonas putida UV4 and are used in the chemoenzymatic synthesis of both cis - and trans -aminoindanol enantiomers.