Studies on the enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester

The enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester, the key intermediate in the stereoselective synthesis of a statinanalogue, was studied with Hansenula anomala and Hansenula silvicola. Using whole cells of H. anomala gives complete conversion and a diastereomeric excess of 88% of the desired 3S, 4S statinanalogue. The strain contains two NADPH-dependent oxidoreductases, that can be separated by ion exchange chromatography or gelfiltration, yielding the 3S, 4S or 3R, 4S stereoisomers, respectively, with > 99% diastereomeric excess ( de). In the crude extract the 3S, 4S oxidoreductase is very unstable and could be purified with ⪡ 1% yield only. In contrast, H. silvicola, which gave poor conversions using whole cells, exhibited about 80-fold higher specific activity in the crude extract than H. anomala. The NADPH-dependent oxidoreductase was purified 317-fold in 12% yield. A single enzyme of 54 kDa reduces the substrate with 97.4% de. Besides the statinanalogue a wide range of other compounds could be reduced, most notably diones and chinones such as isatin or campherchinone. It was demonstrated that the enzymes often discussed for the reduction of β-ketoesters with yeast e.g. l-3-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35), the β-ketoreductase of the fatty acid synthase complex and also the 3-hydroxy-3-methyl glutaryl-CoA dehydrogenase (EC 1.1.1.34) are separated during the purification steps from the oxidoreductase acting on N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester. The physiological role of the new enzyme is still unknown.