Table 2. One-Pot Two-Step Biocatalytic Enantioselective Synthesis of γ-Nitrobutyric Acids 4a–d^a.

^aMichael-type addition of 1 to 2a–d catalyzed by 4-OT L8Y/M45Y/F50A or 4-OT A33D to form either S-3a and R-3b–d or R-3a and S-3b–d respectively, followed by aldehyde oxidation catalyzed by PRO-ALDH(003), using PRO-NOX(009) for cofactor recycling, to form either S-4a and R-4b–d or R-4a and S-4b–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 nitroalkene) was used, except for entry 6 (1.5 mol %) and entry 7 (3 mol %). 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. The concentration NAD⁺ was 8 mM (4a) or 10 mM (4b–d). The amounts of applied cofactor were adjusted such that short reaction times and high conversions were achieved.

^bMonitored by UV spectroscopy.

^cMonitored by HPLC.

^dProducts 4a–d were esterified and the e.r. was determined by GC or HPLC with a chiral stationary phase.