Stefan Roland Woroniecki

Isolation of two novel intracellular β-lactams and a novel dioxygenase cyclising enzyme from Streptomyces clavuligerus

Two novel β-lactams, one monocyclic, the other bicyclic, and a dioxygenase enzyme which converts the former to the latter, have been isolated from the mycelium of the clavulanic acid producing organism Streptomyces clavuligerus ATCC 27064.

Evidence that the immediate biosynthetic precursor of clavulanic acid is its N-aldehyde analogue

(3R, 5R) Clavulanate-9-aldehyde 1 has been detected in Streptomyces clavuligerus and an NADPH dependent dehydrogenase capable of reducing 1 to clavulanic acid 2 has been isolated from this organism.

Facile biocatalytic reduction of the carbon–carbon double bond of 5-benzylidenethiazolidine-2,4-diones. Synthesis of (±)-5-(4-{2-[methyl(2-pyridyl)amino]ethoxy}benzyl)thiazolidine-2,4-dione (BRL 49653), its (R)-(+)-enantiomer and analogues

A novel biotransformation system for the reduction of carbon-carbon double bonds in 5-benzylidenethiazolidine-2, 4-diones, to give the corresponding 5-benzylthiazolidine-2, 4-diones, using whole cells of red yeasts is described. These reduced compounds, which are recovered in good yield, are of potential use in the treatment of non-insulin dependent diabetes mellitus. The mild reaction conditions developed allow reduction of 5-benzylidenethiazolidine-2, 4-diones containing other functionalities which are not compatible with alternative reduction methods. The biocatalytic reduction is enantioselective and the synthesis of R-(+)-5-(4-{2-[methyl(2-pyridyl)amino]ethoxy}benzyl)thiazolidine-2, 4-dione by Rhodotorula rubra CBS 6469 and structure confirmation by X-ray crystallography is detailed. Optimisation of reaction conditions (including immobilisation) for these whole cell reduction systems is described.

Clavulanic acid biosynthesis; the final steps

The chemically unstable anabolite (3R,5R)-clavulanate-9-aldehyde 1 and an NADPH dependent dehydrogenase have been detected in the broth of Streptomyces clavuligerus. The purified enzyme was shown to make clavulanic acid by reduction of the aldehydic moiety of synthetic 1 to the allylic alcohol of clavulanic acid 2. A DNA sequence corresponding to the enzyme’s N-terminal amino acid sequence was located within the clavulanic acid biosynthetic gene cluster. We have named this novel enzyme clavulanic acid dehydrogenase (CAD). In an attempt to determine the origin of 1, fermentations of S. clavuligerus were fed ornithine labelled with stable isotopes in the carboxy group. The results of these experiments are discussed.

The roles of clavaminic acid and proclavaminic acid in clavulanic acid biosynthesis

Clavulanic acid samples isolated from fermentations of Streptomyces clavuligerus ATCC 27064 fed with 13C-clavaminic acid or 13C-proclavaminic acid were found to be appropriately labelled, indicating that the title compounds are biosynthetic precursors of clavulanic acid.

Synthesis of the novel monocyclic β-lactam proclavaminic acid

Proclavaminic acid has been chemically synthesised and one pure enantiomer separated which was shown to possess identical physicochemical and biochemical properties to the natural material isolated from Streptomyces clavuligerus.

A HIGHLY REGIOSELECTIVE ESTERASE FROM PENICILLIUM FREQUENTANS IMI 92265.

An esterase isolated from Penicillium frequentans IMI 92265 selectively hydrolyses the acetoxyethyl ester in preference to the acetoxymethyl esters in the triacetate substrate 1,4-diacetoxy-2-acetoxymethylbutane (1) which was converted to 4-acetoxy-3-acetoxymethylbutan-l-ol (2) in good yield. The enzyme gave no detectable hydrolysis of l,3-diacetoxy-2-acetoxymethylpropane (4). When immobilised to a cyanogen bromide activated Sepharose resin the enzyme was highly stable and showed no loss of regioselectivity in the hydrolysis of (1). A method is described for the elective isolation of microorganisms which have the ability to hydrolyse (1).

Microbial Stereoselective Epoxidation of 2,2-Dimethyl-6-Cyano-3-Chromene

The epoxidation of 2,2-dimethyl-6-cyano-3-chromene was achieved using a whole cell biotransformation. A number of microorganisms were studied for the required monooxygenase activity and a few were shown to exhibit high levels of enantioselectivity in producing the corresponding 3,4-(R,R) stereochemistry epoxide. This resulting enantiomerically pure (R,R) product is an intermediate to the (+)-isomer of the antihypertensive agent cromakalim (BRL 34915).