Synthesis
GENERAL METHODS: Suitable aldehydes 1j, 1n, or 1o (0.5 mmol), malonic acid (1.0 mmol), and piperidine (0.01 mmol) were dissolved in DMSO (500 mL), and the mixture was subsequently heated in a microwave reactor (60°C, 30 min). Upon completion of the reaction, the mixture was cooled to room temperature and ammonia solution (9.5 mL, 13% w/v, ~7 M, adjusted to pH 10.0 by slow addition of dry ice) was added, followed by addition of excess E. coli BL21(DE3) cells (400 mg wet cell paste) to catalyze the production of the target product, 3,4-difluoro-L-phenylalanine. The suspension was stirred at 37°C for several hours and the progress of the reaction was monitored by HPLC. To isolate the product, the reaction mixture was acidified to pH < 2.0 using aqueous sulfuric acid (20% w/v), followed by centrifugation (8000 rpm, 5 min) to remove the cells and the unconverted substrate precipitate. Dowex 50WX8 hydrogen-based resin (5.0 g) was loaded into a disposable plastic column and washed sequentially with deionized water (20 mL) and aqueous sulfuric acid solution (20 mL, 5% w/v). The supernatant after biotransformation was upsampled to the resin column at a flow rate of 1 mL/min. The resin bed was first rinsed with deionized water until the effluent was neutral, and then the product was eluted with aqueous ammonia solution (20 mL, 5% w/v). The product-containing fractions were combined and dried overnight in a centrifugal evaporator to give the corresponding L-phenylalanine derivatives L-3j, L-3n, or L-3o as white solids.4.7.1. (S)-2-Amino-3-(3,4-difluorophenyl)propionic acid (L-3j). Yield: 84 mg (84%), ee value 99% by HPLC.1H NMR (D2O/NaOH, 400 MHz), δ: 7.03-7.18 (m, 2H), 6.90-6.99 (m, 1H), 3.36-3.44 ( m, 1H), 2.81-2.90 (dd, J = 13.6, 6.0 Hz, 1H), 2.70-2.79 (dd, J = 13.6, 7.2 Hz, 1H).13C NMR (D2O/NaOH, 101 MHz), δ: 182.06, 150.94 ( dd, 1JCF = 243 Hz, 2JCF = 12 Hz), 150.09 (dd, 1JCF = 242 Hz, 2JCF = 13 Hz), 135.33 (dd, 3JCF = 5 Hz, 4JCF = 3 Hz), 125.51 (dd, 3JCF = 6 Hz, 4JCF = 3 Hz), 117.83 (d, 2JCF = 17 Hz), 116.97 (dd, 2JCF = 17 Hz), 57.33, 39.94. HRMS (ESI), m/z: calculated value 202.0680 [MH] , measured value 202.0689 [MH]
