2.1. General procedure for lipase-catalyzed acylation of Andrographolide

In general experimental conditions, 0.1 mmol andrographolide, 1.0 mmol vinyl esters, and 5 mg enzyme were dissolved in 3 mL of acetone/ethyl acetate / vinyl acetate / methanol / tetrahydrofuran and incubated in a shaking incubator, as reported earlier [1], [2]. After a specific time of interval, aliquots were drawn from the reaction mixture, filtered through 0.45 µm syringe filter and diluted with methanol for HPLC analysis. The initial water activity (a_w) of the reaction (the lipase with the substrates) was controlled in a different saturated salt solution at 25 °C by gaseous equilibrium in separate closed containers as reported earlier [3], [4]. Reaction with the predetermined condition without enzyme served as a negative control, which did not detect the acylated product. All experiments were performed in triplicate, and the average results with standard deviation are reported. For characterization of product formed in a general experiment, 0.1 mmol andrographolide, 1 mmol vinyl esters dissolved in acetone and 90 mg of Amano lipase AK from Pseudomonas fluorescens was incubated at 55 °C in a shaking incubator set at 100 rpm for 5 h. The reaction mixture was filtered to get rid of lipase and filtrate was concentrated under vacuum. The obtained residue was further purified by silica gel column chromatography with acetone-petroleum ether mixture as solvent system.