The general procedure for the synthesis of O-methyl DL-serine from 2-amino-3-methoxypropionamide was as follows: in small-scale experiments, ACL racemase (Locus, E01594) and PepB (Locus, D84499) were added to an aqueous solution containing α-amino-β-methoxypropionamide. The reaction mixture (20 mL) contained 2 mM pyridoxal 5'-phosphate (PLP), 0.1 M potassium phosphate buffer (KPB, pH 9.0) and 0.5 mol/L α-amino-β-methoxypropionamide. Subsequently, ACL racemization enzyme (3.0 mg) and PepB (1.2 mg) were added and incubated for 18 h at 38°C. The reaction progress was monitored by timed sampling. D/L-α-amino-β-methoxyalanamide and O-methyl-L-serine were determined by HPLC equipped with a Crownpak CR(+) column at a flow rate of 0.5 mL/min using 50 mM HClO4 as solvent system, and the absorbance of the eluate was monitored at 210 nm. After the reaction was completed, O-methyl-L-serine was obtained by deproteinization with trichloroacetic acid and separation by Dowex-X8 (H+) column chromatography. The isolated O-methyl-L-serine (7.98 mmol, 950 mg) was recrystallized by water-methanol-isopropanol-ether solvent mixture to obtain white crystals with more than 99.9% enol purity. The product has a melting point of 211-213 °C (decomposition) and a specific rotation of 1/25D (c 1, 6 mol/L HCl).1H NMR (400 MHz, D2O) data: δ 3.24 (s, 3H), 3.78-3.81 (m, 1H), 3.83 (dd, J = 3.5, 9.8 Hz, 1H), 4.01 (dd, J = 6.1, 9.8 Hz, 1H).13C NMR (100 MHz, D2O) data: 177.3 (CO), 76.5 (CH2), 60.2 (CH), 56.7 (OCH3). Elemental analysis (C4H9NO3) calculated values: C, 40.32; H, 7.58; N, 11.75. measured values: C, 40.35; H, 7.54; N, 11.77. ESI-MS (m/z): 120.1230 [M + H]+.
