Preparation of (R)- and (S)-N-protected 3-hydroxypyrrolidines by hydroxylation with Sphingomonas sp HXN-200, a highly active, regio- and stereoselective, and easy to handle biocatalyst

Hydroxylation of N-benzylpyrrolidine 8 with resting cells of Sphingomonas s p. HXN-200 gave N-benzyl-3-hydroxypyrrolidine 15 in 53% ee (S) with an acti vity of 5.8 U/g CDW. By changing the "docking/protecting group" in pyrrolid ines, hydroxylation activity and enantioselectivity were further improved a nd the enantiocomplementary formation of 3-hydroxypyrrolidines was achieved : hydroxylation of N-benzoyl-, N-benzyloxycarbonyl-, N-phenoxycarbonyl-, an d N-tert-butoxycarbonyl-pyrrolidines 9-12 gave the corresponding 3-hydroxyp yrrolidines 16-19 in ee of 52% (R), 75% (R), 39% (S), and 23% (R), respecti vely, with an activity of 2.2, 16, 14, and 24 U/g CDW, respectively. Simple crystallizations increased the ee of 16-18 to 95% (R), 98% (R), and 96% (S ), respectively. Hydroxylation of pyrrolidines 8-12 with soluble cell-free extracts of Sphingomonas sp. HXN-200 and equimolar NADH gave 3-hydroxypyrro lidines 15-19 in nearly the same ee as the products generated by whole cell transformation, suggesting that this strain possesses a novel soluble alka ne monooxygenase. Cells of Sphingomonas sp. HXN-200 were produced in large amounts and could be stored at -80 degreesC for 2 years without significant loss of activity. The frozen cells can be thawed and resuspended for biohy droxylation, providing a highly active and easy to handle biocatalyst for t he regio- and stereoselective hydroxylation of nonactivated carbon atoms. T hese cells were used to prepare 1.0-3.2 g (66.4-93.5% yield) of 3-hydroxypy rrolidines 16-19 by hydroxylation of pyrrolidines 9-12 on 0.9-2 L scale. Pr eparative hydroxylation was also achieved with growing cells as biocatalyst s; hydroxylation of pyrrolidine 11 on 1 L scale gave 1.970 g (79.7% yield) of 3-hydroxypyrrolidine 18.