Table 3. One-Pot Three-Step Chemoenzymatic Cascade Synthesis of Pharmaceuticals 5a–d^a.

^aMichael-type addition of 1 to 2a–d catalyzed by 4-OT L8Y/M45Y/F50A to form S-3a and R-3b–d, followed by aldehyde oxidation catalyzed by PRO-ALDH(003), using PRO-NOX(009) for cofactor recycling, to form S-4a and R-4b–d, followed by nitro reduction catalyzed by nickel boride to form S-5a and R-5b–d. The reaction mixtures consisted of 50 mM 1, 3 mM 2a or 4 mM 2b–d in 100 mM sodium phosphate buffer pH 7.3 and 10% (v/v) ethanol. Five mol % of 4-OT (compared to concentration of nitroalkene) was used; PRO-ALDH(003) was added to a final concentration of 0.5 mg/mL; PRO-NOX(009) was added to a final concentration of 1 mg/mL; 0.5 mM of NAD⁺ was added; 40 mM of NiCl₂ and 40 mM of NaBH₄ were used. Because of the poor stability of nickel boride in aqueous buffer, a 10-fold excess of this reagent (40 mM versus 3–4 mM 2a–d) is required to achieve high conversion.

^bMonitored by UV spectroscopy.

^cMonitored by HPLC.

^dMonitored by TLC.

^ePurified by cation exchange chromatography.

^fProducts 5a–d were derivatized using N_α-(2,4-dinitro-5-fluorophenyl)-l-valinamide, and the e.r. of the corresponding diastereomers was determined by HPLC with an achiral stationary phase. Notably, the e.r. of product 5a is likely to be 99:1; however, because a minor contaminant from the derivatizing agent has the same retention time as the R enantiomer of product 5a, the e.r. for product 5a is cautiously reported as 98:2, consistent with the observed peaks in the HPLC chromatogram.