Derek R. Boyd

Aromatic dioxygenases: molecular biocatalysis and applications.

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.

Arene cis-dihydrodiol formation: from biology to application

The range of available arene dihydroxylating dioxygenase enzymes, their structure and mechanism, and recent examples of the application of arene cis-dihydrodiol bioproducts as chiral precursors in the synthesis of natural and unnatural products and chiral ligands are discussed.

Regioselectivity and enantioselectivity of naphthalene dioxygenase during arene cis-dihydroxylation: control by phenylalanine 352 in the alpha subunit.

The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the alpha subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl cis-3,4-dihydrodiol [77 and 60% (-)-(3S,4R), respectively] to that formed by wild-type NDO [>98% (+)-(3R,4S)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene cis-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoikuyae B8/36. A recombinant Escherichia coli strain expressing the F352V variant of NDO and the enantioselective toluene cis-dihydrodiol dehydrogenase from Pseudomonas putida F1 was used to produce enantiomerically pure (-)-biphenyl cis-(3S,4R)-dihydrodiol and (-)-phenanthrene cis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively.

Aerobic metabolism of 4-hydroxybenzoic acid in Archaea via an unusual pathway involving an intramolecular migration (NIH shift).

ABSTRACT 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.

Enantiomeric resolution of chiral sulfoxides on polysaccharide phases by HPLC

The enantiomeric resolution of chiral sulfoxides was investigated on amylose (S)-α-methylbenzyl carbamate phase coated on aminopropylated 7 μm silica with 500A diameter pores. This was shown to be very successful in the separation of alkyl/aryl, aryl/aryl, and non-aromatic sulfoxides. The effect of pore size using naked silica was also investigated, demonstrating that the pore size does not affect the resolution.

Absolute stereochemistry of the dihydroanthracene-cis- and trans-1,2-diols produced from anthracene by mammals and bacteria

(+)-1,2-Dihydroanthracene-trans- and -cis-1,2-diols have been isolated as major anthracene metabolites from rabbits and the bacterium Beijerinckia B-836, respectively. The absolute stereochemistry and optical purity of each diol has been related to that of 1,2,3,4-tetrahydroanthracen-2-ol. The latter was resolved via the diastereoisomeric (–)-methyloxyacetates, whose optical purity and absolute stereochemistry was determined by a range of techniques including kinetic resolution, asymmetric synthesis, and c.d. (exciton chirality method).

Chemoenzymatic synthesis of carbasugars (+)-pericosines A-C from diverse aromatic cis-dihydrodiol precursors.

cis-Dihydrocatechols, derived from biological cis-dihydroxylation of methyl benzoate, iodobenzene and benzonitrile, using the microorganism Pseudomonas putida UV4, were converted into pericosines A, C, and B, respectively. This approach constitutes the shortest syntheses, to date, of these important natural products with densely packed functionalities.

Dioxygenase-catalyzed cis-dihydroxylation of pyridine-ring systems

Toluene dioxygenase-catalyzed dihydroxylation, in the carbocyclic rings of quinoline, 2-chloroquinoline, 2-methoxyquinoline, and 3-bromoquinoline, was found to yield the corresponding enantiopure c...

Dioxygenase-catalysed oxidation of alkylaryl sulfides: sulfoxidation versus cis-dihydrodiol formation

Toluene- and naphthalene-dioxygenase-catalysed sulfoxidation of nine disubstituted methylphenyl sulfides, using whole cells of Pseudomonas putida, consistently gave the corresponding enantioenriched sulfoxides. Using the P. putida UV4 mutant strain, and these substrates, differing proportions of the corresponding cis-dihydrodiol sulfides were also isolated. Evidence was found for the concomitant dioxygenase-catalysed cis-dihydroxylation and sulfoxidation of methyl para-tolyl sulfide. A simultaneous stereoselective reductase-catalysed deoxygenation of (S)-methyl para-tolyl sulfoxide, led to an increase in the proportion of the corresponding cis-dihydrodiol sulfide. The enantiopurity values and absolute configurations of the corresponding cis-dihydrodiol metabolites from methyl ortho- and para-substituted phenyl sulfides were determined by different methods, including chemoenzymatic syntheses from the cis-dihydrodiol metabolites of para-substituted iodobenzenes. Further evidence was provided to support the validity of an empirical model to predict, (i) the stereochemistry of cis-dihydroxylation of para-substituted benzene substrates, and (ii) the regiochemistry of cis-dihydroxylation reactions of ortho-substituted benzenes, each using toluene dioxygenase as biocatalyst.

Structures and stereochemical assignments of some novel chiral synthons derived from the biotransformation of 2,3-dihydrobenzofuran and benzofuran by Pseudomonas putida

Abstract Metabolism of 2,3-dihydrobenzofuran using intact cells of Pseudomonas putida UV4 gave mainly (3S)-3-hydroxy-2,3-dihydrobenzofuran which was in turn oxidized to an unstable intermediate, (3S,4R,5S)-3,4,5-trihydroxy-2,3,4,5-tetrahydrobenzofuran. Spontaneous dehydration of this cis,cis-triol occurred in the carbocyclic ring to give (3S)-3,5-dihydroxy 2,3-dihydrobenzofuran and in the heterocyclic ring to yield (4R,5S)-cis-4,5-dihydroxy-4,5-dihydrobenzofuran. Bacterial metabolism of benzofuran was found to occur in the carbocyclic ring to form (6S,7S)-cis-6,7-dihydroxy-6,7-dihydrobenzofuran and its dehydration product, 6-hydroxybenzofuran. Dioxygenase-catalysed cis-dihydrodiol formation in the heterocyclic ring of benzofuran (to give cis-2,3-dihydroxy-2,3-dihydrobenzofuran as a transient intermediate) is proposed to account for the appearance of(1R)-1,2-dihydroxy-1-(2′-hydroxyphenyl)ethane as a major metabolite of benzofuran. cis-4,5-Dihydroxy-4,5-dihydrobenzofuran and cis-6,7-dihydroxy-6,7-dihydrobenzofuran are potentially valuable chiral synthons which can be added to the small pool of bicyclic cis-dihydrodiol metabolites currently available for synthesis.