title: Lipase-mediated stereoselective hydrolysis of stampidine and other phosphoramidate derivatives of stavudine.
abstract: Enzymatic hydrolysis of stampidine and other aryl phosphate derivatives of stavudine were investigated using the Candida Antarctica Type B lipase. Modeling studies and comparison of the hydrolysis rate constants revealed a chiral preference of the lipase active site for the putative S-stereoisomer. The in vitro anti-HIV activity of these compounds correlated with their susceptibility to lipase- (but not esterase-) mediated hydrolysis. We propose that stampidine undergoes rapid enzymatic hydrolysis in the presence of lipase according to the following biochemical pathway: During the first step, hydrolysis of the ester group results in the formation of carboxylic acid. Subsequent step involves an intramolecular cyclization at the phosphorous center with simultaneous elimination of the phenoxy group to form a cyclic intermediate. In the presence of water, this intermediate is converted into the active metabolite Ala-d4T-MP. We postulate that the lipase hydrolyzes the methyl ester group of the l-alanine side chain to form the cyclic intermediate in a stereoselective fashion. This hypothesis was supported by experimental data showing that chloroethyl substituted derivatives of stampidine, which possess a chloroethyl linker unit instead of a methyl ester side chain, were resistant to lipase-mediated hydrolysis, which excludes the possibility of a direct hydrolysis of stampidine at the phosphorous center. Thus, our model implies that the lipase-mediated formation of the cyclic intermediate is a key step in metabolism of stampidine and relies on the initial configuration of the stereoisomers.
authors.affiliation: Department of Chemistry, Pharmaceutical Sciences, Bioinformatics, Structural Biology, and Virology, Parker Hughes Institute, 2699 Patton Road, St. Paul, MN 55113, USA.
authors.forename: T K
authors.initials: TK
authors.lastname: Venkatachalam
authors.forename: P
authors.initials: P
authors.lastname: Samuel
authors.forename: G
authors.initials: G
authors.lastname: Li
authors.forename: S
authors.initials: S
authors.lastname: Qazi
authors.forename: C
authors.initials: C
authors.lastname: Mao
authors.forename: S
authors.initials: S
authors.lastname: Pendergrass
authors.forename: F M
authors.initials: FM
authors.lastname: Uckun
mesh_terms: D000577:Amides / Q000737:chemistry*; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D002175:Candida / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D054306:Dideoxynucleotides; D004950:Esterases / Q000378:metabolism; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D010756:Phosphoric Acids / Q000737:chemistry*; D011487:Protein Conformation; D018119:Stavudine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*; D013237:Stereoisomerism; D013938:Thymidine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000577:Amides; D000998:Antiviral Agents; D054306:Dideoxynucleotides; D010756:Phosphoric Acids; C472384:stampidine; D013938:Thymidine Monophosphate; C011067:phosphoramidic acid; D018119:Stavudine; D004950:Esterases; D008049:Lipase