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

Abstract

Hydroxylation of N-benzylpyrrolidine 8 with resting cells of Sphingomonas sp. HXN-200 gave N-benzyl-3-hydroxypyrrolidine 15 in 53% ee (S) with an activity of 5.8 U/g CDW. By changing the "docking/protecting group" in pyrrolidines, hydroxylation activity and enantioselectivity were further improved and the enantiocomplementary formation of 3-hydroxypyrrolidines was achieved: hydroxylation of N-benzoyl-, N-benzyloxycarbonyl-, N-phenoxycarbonyl-, and N-tert-butoxycarbonyl-pyrrolidines 9-12 gave the corresponding 3-hydroxypyrrolidines 16-19 in ee of 52% (R), 75% (R), 39% (S), and 23% (R), respectively, 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-hydroxypyrrolidines 15-19 in nearly the same ee as the products generated by whole cell transformation, suggesting that this strain possesses a novel soluble alkane monooxygenase. Cells of Sphingomonas sp. HXN-200 were produced in large amounts and could be stored at -80 degrees C for 2 years without significant loss of activity. The frozen cells can be thawed and resuspended for biohydroxylation, providing a highly active and easy to handle biocatalyst for the regio- and stereoselective hydroxylation of nonactivated carbon atoms. These cells were used to prepare 1.0-3.2 g (66.4-93.5% yield) of 3-hydroxypyrrolidines 16-19 by hydroxylation of pyrrolidines 9-12 on 0.9-2 L scale. Preparative hydroxylation was also achieved with growing cells as biocatalysts; hydroxylation of pyrrolidine 11 on 1 L scale gave 1.970 g (79.7% yield) of 3-hydroxypyrrolidine 18.

Achieving Regio‐ and Enantioselectivity of P450‐Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure‐Guided Directed Evolution

“…It should be noted that WT P450pyr was known to be completely regioselective at the inactivated 3 position (53 % ee ( S )), with no reaction occurring at the activated N‐benzylic position where the CH bond strength is expected to be considerably lower 26. 27 Specific positioning of a substrate in the binding pocket of P450 enzymes, whatever the underlying factors may be, determines regioselectivity. In the case of P450pyr acting as a catalyst in the 3‐selective hydroxylation of N ‐benzyl pyrrolidine, reversal of enantioselectivity of up to 83 % ee had previously been achieved by utilizing iterative saturation mutagenesis (ISM) 4c.…”

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

“…Previous studies also demonstrated that Sphingomonas sp. HXN-200 bacteria contain hydrocarbon-degrading enzymes [17,18]. The results of these studies suggest that Sphingomonas sp.…”

High‐Throughput Method for Determining the Enantioselectivity of Enzyme‐Catalyzed Hydroxylations Based on Mass Spectrometry

“…To further demonstrate the generality of this method, enantioselective biohydroxylation of N ‐benzylpyrrolidine ( 7 ) at the 3‐position was selected as another example. This represents an enantioselective hydroxylation of a nonactivated carbon atom, a symmetric substrate with equal CH bonds at the 3‐ and 4‐positions, and a useful transformation to prepare the corresponding product ( S )‐ 8 or ( R )‐ 8 , which is a key synthetic intermediate for several pharmaceuticals 20…”

“…At the beginning, Sphingomonas sp. HXN‐200, a known biocatalyst for the hydroxylation of non‐activated carbon atoms,20 was selected as the catalyst. Biohydroxylation was performed with 5 m M ( R )‐ 9 or ( S )‐ 9 in a cell suspension (5.5 g cdw L −1 ) in 50 m M potassium phosphate buffer (5 mL, pH 7.5) containing glucose 2 % (w/v) at 30 °C for 1 h. Samples were taken directly from the aqueous biotransformation mixtures and analyzed by LC/MS; typical chromatograms are shown in Figure 2.…”