Enzymatic kinetic resolution of piperidine atropisomers: synthesis of a key intermediate of the farnesyl protein transferase inhibitor, SCH66336.

The resolution of secondary amines via enzyme-catalyzed acylation is a relatively rare process. The kinetic resolution of a series of intermediates of SCH66336 (1), by either enzymatic acylation of the pendant piperidine (4, 5) or hydrolysis of the corresponding carbamate 3, was investigated. In the case of 4, the molecule exists as a pair of enantiomers due to atropisomerism about the exocyclic double bond. The enzymatic acylation of (+/-)-4 was optimized in terms of acylating agent, solvent, and moisture content. The use of lipase, Toyobo LIP-300, and trifluoroethyl isobutyrate as acylating agent resulted in isobutyrylation of the (+)-enantiomer, which is easily separated from the unwanted (-)-4. Hydrolysis of the isobutyramide 6c yielded the desired (+)-4 in high enantiomeric excess. (-)-4 may be recovered from the resolution step, racemized, and resubjected to enzymatic acylation to increase material throughput.

Enzymatic glucuronidation of a novel cholesterol absorption inhibitor, Sch 58235.

A glucuronide of a novel cholesterol absorption inhibitor was synthesized on a 200-mg scale in one step via bovine liver glucuronyltransferase-catalyzed coupling of the glucuronyl moiety of UDP-glucuronic acid with the phenolic hydroxyl of Sch 58235. It was shown that the product yield is limited by the hydrolysis of UDP-glucuronic acid by impurities present in the commercial microsomal preparation of the transferase. This detrimental effect of UDPGluA hydrolysis could be diminished by the presence of high concentration of glucuronlytransferase. Optimization of reaction conditions and purification procedure resulted in a process that proceeded with 95% conversion and 88% isolated product yield. The 13C6-glucuronide of Sch 58235 was prepared with the help of a cascade of eight enzymes operating concurrently in one pot.

Altered Cro repressors from engineered mutagenesis of a synthetic cro gene.

A portion of the gene coding for the Cro repressor protein of bacteriophage lambda has been chemically synthesized, incorporating base pair changes that generate restriction endonuclease sites without altering the amino acid coding sequence. These restriction endonuclease sites were used to remove small segments of the synthetic cro gene and the segments were replaced with duplexes carrying desired mutations. Altered Cro proteins produced by mutants constructed in this manner were then assayed for binding to lambda operator OR3 in vivo. Mutations directed into the region of the cro gene encoding the alpha-3 helix produced altered Cro proteins with a range of affinities for operator DNA. These changes suggest which amino acids play an important role in Cro-OR3 complex formation.