Nigel I. Bowers

Stereoselective dioxygenase-catalysed benzylic hydroxylation at prochiral methylene groups in the chemoenzymatic synthesis of enantiopure vicinal aminoindanols

Abstract 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.

Stereochemical and mechanistic aspects of dioxygenase-catalysed benzylic hydroxylation of indene and chromane substrates

Toluene dioxygenase (TDO)-catalysed benzylic hydroxylation of indene substrates (8, 16 and 17), using whole cell cultures of Pseudomonas putida UV4, was found to yield inden-1-ol (14 and 22) and indan-1-one bioproducts (15 and 23). The formation of these bioproducts is consistent with the involvement of carbon-centred radical intermediates. TDO-catalysed oxidation of indenes 8 and 16 also gave cis-diols 13 and 18 respectively. TDO and naphthalene dioxygenase (NDO), used as both whole-cell preparations and as purified enzymes, were found to catalyse the benzylic hydroxylation of chromane 30, deuteriated (+/-)-chromane 30D and enantiomers (4S)-30D and (4R)-30D to yield (4R)- and (4S)-chroman-4-ols 31/31D respectively. The mechanism of benzylic hydroxylation of chromane 30/30D involves the stereoselective abstraction of a pro-R (with TDO) or a pro-S (with NDO) hydrogen atom at C-4 and a marked preference for retention of configuration.

Stereoselective cis-dihydroxylation of azulene and related non-aromatic polyenes

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

Chemoenzymatic synthesis of the carbasugars carba-β-L-galactopyranose, carba-β-L-talopyranose and carba-α-L-talopyranose from methyl benzoate

The cis-dihydrodiol metabolite from methyl benzoate has been used as a synthetic precursor of carba-beta-L-galactopyranose, carba-beta-L-talopyranose and carba-alpha-L-talopyranose. The structures and absolute configurations of these carbasugars were determined by a combination of NMR spectroscopy, stereochemical correlation and X-ray crystallography.

Enantioselective toluene dioxygenase catalysed di- and tri-hydroxylation of monosubstituted benzenes

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.

STEREOSELECTIVE BENZYLIC HYDROXYLATION OF 2-SUBSTITUTED INDANES USING TOLUENE DIOXYGENASE AS BIOCATALYST

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.

The potential role of cis -dihydrodiol intermediates in bacterial aromatic hydroxylation and the NIH Shift

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.