General procedure for the synthesis of L-2,4-dichlorophenylalanine from 2,4-dichlorostyrenic acid: general method: suitable aldehydes (1j-1,1n 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 placed in a microwave reactor and heated (60 °C, 30 min ). Upon completion of the reaction, it 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 cubes) 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 L-2,4-dichlorophenylalanine. The suspension was stirred at 37°C for several hours and the reaction progress was monitored by HPLC. For product isolation, the reaction mixture was acidified to pH <2.0 with aqueous H2SO4 (20% w/v) and centrifuged (8000 rpm, 5 min) to remove the cells and unconverted substrate precipitate. Dowex 50WX8 hydrogen-based resin (5.0 g) was loaded in a disposable plastic column and washed sequentially with deionized water (20 mL) and H2SO4 aqueous 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 washed with deionized water until the effluent was neutral, and then the product was eluted with NH4OH aqueous solution (20 mL, 5% w/v). The product-containing eluates were combined and dried overnight in a centrifugal evaporator to give the L-phenylalanine derivative (L-3j-1, L-3n or L-3o) as a white solid.
