Yeast reductase reductions of alpha-keto-beta-lactams; chemo- and enzymatic synthesis of 3-hydroxy-beta-lactams

3-Hydroxy substituted beta-lactams are convenient precursors of beta-amino-alpha-hydroxy esters that constitute key fragments of many biologically important natural products, including analogues of the anticancer drug TaxolRTM. Numerous studies have shown that modifications of the TaxolRTM side chain can improve biological activity and other physicochemical properties, making the drug more effective. A series of 3-acetoxy-4-substituted-beta-lactams were synthesized by cycloadditions of imines and ketenes (Staudinger reaction). Modifications of the reaction conditions allowed the stereoselective formation of cis beta-lactams. The corresponding enantiopure 3-hydroxy-beta-lactams were obtained in subsequent biotransformation steps through lipase resolutions of 3-acetoxy-beta-lactams and reductase-catalyzed reductions of alpha-keto-beta-lactams.;Among reductases studied, only aldose keto reductase Ara1p was found to be enantioselective, even though it did not discriminate between R and S configurations of the racemic ketone. Further investigation of this strain in the fermentation of alpha-keto-beta-lactams with resting cells showed synthetic utility in the preparation of single trans isomers of the corresponding 3-hydroxy-beta-lactams.;The absolute configurations of all 3-hydroxy-beta-lactams were established by X-ray crystallography and a new "mix and shake" 1H NMR methodology. Five of the 3-hydroxy-beta-lactams were transformed to C-13 side chain analogues and were submitted for attachment to baccatin III and biological testing.;Baker's yeast has been widely used for asymmetric reductions of ketones to alcohols. However, the presence of multiple enzymes with overlapping substrate specificities but different stereoselectivities was believed to diminish optical purity of products in reductions of alpha- and beta-ketoesters. This study was designed to evaluate enantio- and stereoselectivity of yeast reductases vis a vis alpha-keto-beta-lactams. Several recombinant yeast strains with overexpressed or knocked-out native reductases as well as recombinant Escherichia coli overexpressing yeast reductases were assessed. These experiments was conducted on a number of beta-lactams substituted with alkyl, aryl, and polar groups and demonstrated that the lack of selectivity was often linked to the inherent low enantioselectivity of the enzyme, rather than the competition between enzymes for the same substrate.