2.6 General experimental procedure for the domino Knoevenagel reaction (1a-k→3a–k)
A 25 mL round-bottomed flask was charged with NerA (3 mg mL-1), GDH (1.5 mg mL-1), NAD+ (1 mM), deionized water (10 mL), to which the aldehydes (50 mM) and 1, 3-dicarbonyl compound (100 mM) were introduced. The resulting mixture was stirred for the specified amount of time at 25 ºC. After completion, the product was extracted into EtOAc (4×10 mL), The combined organic layers were dried over anhydrous Na2SO4 and evaporated under a vacuum. The residue was purified by flash column chromatography with petroleum ether/ethyl acetate to afford the desired products.