Animesh Goswami

Synthesis of Biologically Active Piperidine Metabolites of Clopidogrel: Determination of Structure and Analyte Development

Clopidogrel is a prodrug anticoagulant with active metabolites that irreversibly inhibit the platelet surface GPCR P2Y12 and thus inhibit platelet activation. However, gaining an understanding of patient response has been limited due to imprecise understanding of metabolite activity and stereochemistry, and a lack of acceptable analytes for quantifying in vivo metabolite formation. Methods for the production of all bioactive metabolites of clopidogrel, their stereochemical assignment, and the development of stable analytes via three conceptually orthogonal routes are disclosed.

Microbiological reductions of enantiomeric 2-oxo-1,4-cineoles

Abstract A selection of bacteria, fungi and yeasts known to achieve ketone reduction reactions were assessed for their abilities to transform enantiomeric 2-oxo-1,4-cineoles to their respective alcohol products. Microbiological reduction of the (−)- ketone isomer led to the formation of a preponderance of the corresponding endo-alcohol isomer with all organisms, indicating that ‘re’-face reductase specificity predominates. The (+)- ketone isomer gave mixed results . Curvularia lunata formed a preponderance of the endo-alcohol isomer, indicating the presence of ‘si’-face specific reductases in this organism, while Penicillium frequentans gave a higher proportion of the exo-alcohol isomer, indicating the presence of ‘re’-face specific reductases. Kinetic studies showed that constant ratios of exo/endo-alcohol products were obtained when (+)- 2-oxo-1,4-cineole is reduced by C. lunata, but that the ratios of alcohol isomers changed with time when reductions were catalyzed by P. frequentans.

Biotechnology Based Process for Production of a Disulfide-Bridged Peptide

A disulfide-bridged peptide drug development candidate contained two oligopeptide chains with 11 and 12 natural amino acids joined by a disulfide bond at the N-terminal end. An efficient biotechnology based process for the production of the disulfide-bridged peptide was developed. Initially, the two individual oligopeptide chains were prepared separately by designing different fusion proteins and expressing them in recombinant E. coli. Enzymatic or chemical cleavage of the two fusion proteins provided the two individual oligopeptide chains which could be conjugated via disulfide bond by conventional chemical reaction to the disulfide-bridged peptide. A novel heterodimeric system to bring the two oligopeptide chains closer and induce disulfide bond formation was designed by taking advantage of the self-assembly of a leucine zipper system. The heterodimeric approach involved designing fusion proteins with the acidic and basic components of the leucine zipper, additional amino acids to optimize interaction between the individual chains, specific cleavage sites, specific tag to ensure separation, and two individual oligopeptide chains. Computer modeling was used to identify the nature and number of amino acid residue to be inserted between the leucine zipper and oligopeptides for optimum interaction. Cloning and expression in rec E. coli, fermentation, followed by cell disruption resulted in the formation of heterodimeric protein with the interchain disulfide bond. Separation of the desired heterodimeric protein, followed by specific cleavage at methionine by cyanogen bromide provided the disulfide-bridged peptide.