Process Research Of Enzymatic Synthesis Of Caffeic Acid Phenethyl Ester In Non-aqueous System

In recent years, caffeic acid phenethyl ester (CAPE) is a lead compound of naturaldrug with the significant anti-tumor activity. Enzymatic synthesis of CAPE has manyadvantages, such as mild reaction conditions and environmentally friendly. However,there are several bottleneck factors in the procedure, such as using organic solvents asthe reaction medium, the relatively low yield and long time-consuming. Therefore,enzymatic synthesis of CAPE in non-aqueous phase analyzed by LC-MS analysis wasadopted. Ionic liquids (ILs) were used as the reaction medium instead of traditionalorganic solvents, and then the yield of CAPE increased from3.06%to63.75%. Whentransesterification was used as a reaction type, replacing traditional esterification, theyield could be furtherly increased to66.40%. Specific results were as follows:(1) Qualitative and quantitative methods used for enzymatic synthesis of CAPE innon-aqueous was established. LC-MS was used for qualitative analysis. LC-MSconditions were as follows: LC separation was performed on a Kromasil C18column(150mm×4.6mm;5μm). The mobile phase was isocratic elution. The elution programwas methanol-water (65:35, v/v). The wavelength range of detection was from200to400nm. The flow rate was1.0ml/min for LC and detection with the column kept at40°C. The electrospray ionization (ESI) was performed using nitrogen to assistnebulisation. Selected ion monitoring (SIM) with negative ion mode, capillary voltage at1.6KV, and temperature of curved desolvation line and heat block both set at200°Cwere used. Quantitative analysis was used by HPLC. HPLC-UV conditions were asfollows: an HC-C18column (250mm×4.6mm;5μm) was used, and it was maintainedat30°C. The mobile phase consisted of acetonitrile-deionized water-acetic acid(50:50:0.5, v/v/v), at a flow rate of1.0mL/min, and detection wavenumber was325nm.The sample size is20μL. The regression equations of standard curves of CA and CAPEin non-aqueous system were Y=1.03×108X－26610.10(r=0.9999) and Y=6.25×107X＋28920.15(r=0.9999), respectively.(2) Lipase-catalyzed esterification synthesis process of CAPE using caffeic acid(CA) and phenethanol (PE) in organic solvents was established. Isooctane and Novozym435were selected as the suitable reaction media and biocatalyst, respectively. Theoptimal reaction temperature was80°C. The highest yield of CAPE was only3.06%after72h. The results indicated that it was unsuitable for practical application that enzymatic synthesis of CAPE in organic solvents.(3) Enzymatic esterification synthesis process of CAPE using CA and PE in ionicliquids was established.[Emim][Tf2N] and Novozym435were selected as the suitablereaction media and biocatalyst, respectively. ILs containing weakly coordinating anions,including [BF4]-,[PF6]-and [Tf2N]-, and cations with adequate alkyl chain length, suchas [Emim]+,[Bmim]+and [TOMA]+, were beneficial to the enzymatic synthesis. Theoptimal parameters were as follows by response surface methodology (RSM): reactiontemperature,84.0°C; mass ratio of Novozym435to CA,14:1; and molar ratio of PE toCA,16:1. CA conversion and CAPE yield reached98.76%and63.75%after48h,respectively. The results indicated that enzymatic synthesis of CAPE in ionic liquids issuperior to synthesis in organic solvents.(4) Lipase-catalyzed transesterification synthesis process of CAPE using methylcaffeate (MC) and PE in ionic liquids was firstly established. The results indicated that[Bmim][Tf2N] and Novozym435were selected as the suitable reaction media andcatalyst, respectively. When the time of transesterification reaction was24h, CAPEyield achieved66.40%, under the optimal condition as follows: temperature90°C, molarratio of PE to MC20:1，mass ratio of Novozym435to MC10:1.The novel lipase-catalyzed transesterification process of CAPE in ionic liquids hashigher CAPE yield in a shorter reaction time, which significantly increase the productyield and reaction efficiency,and it provided a new kind of strategy and method for thesynthesis of natural products.