Study On Kinetic Resolution Of Hydroxy Acid Enantiomers By Enzymatic Transesterification

Hydroxy acids and their derivatives are very important chiral intermediates,which are widely used in the fields of pharmaceutical production,fine organic synthesis,cosmetics production and food processing.As the market demand for chiral drugs increases,drugs synthesized using hydroxy acids as intermediates are also increasing year by year,and many hydroxy acids in the market exist in racemic form.Therefore,the separation and purification of hydroxy acid enantiomers is of great scientific significance and economic value.In this paper,the resolution of three enantiomersof2-chloro-?-hydroxyphenylaceticacid,2-methoxy-?-hydroxyphenylacetic acid and 4-fluoro-?-hydroxyphenylacetic acid were investigated by lipase-catalyzed transesterification in organic solvents.Optically pure?S?-2-chloro-?-hydroxyphenylacetic acid and?R?-2-chloro-?-hydroxyphenylacetic acid were prepared by enzymatic irreversible transesterification of 2-chloro-?-hydroxyphenylacetic acid enantiomer?Cl MA?with vinyl acetate in methyl tert-butyl ether,using Pseudomonas fluorescens lipase as the biocatalyst.The influence of various reaction conditions on entioselectivity and catalytic activity were studied for enzymatic transesterification reaction,including temperature,water content,substrate ratio,enzyme loading,and reaction time.Based on homogeneous reaction and Ping-Pong bi-bi mechanism,a quantitative model was constructed to simulate and optimize the reaction process.Through simulation and optimization,the best reaction conditions were obtained,including water content of0.10%?v/v?,temperature of 50?,substrate ratio of 6:1,enzyme loading of 25mg/m L and reaction time of 24 h.Under the optimal conditions,excellent results were obtained with the high conversion of the?R?-2-Cl MA?c _R?98.85%?and large enantiomeric excess?ee _S?98.15%?.The predicted values were in good agreement with the experimental data,indicating that the model was a powerful tool for optimizing the enantioselective transesterification separation of enantiomers.The reactive system was constructed for enzyme-catalyzed transesterification of4-methoxy-?-hydroxyphenylacetic acid enantiomers?4-MMA?,screening Pseudomonas fluorescens lipase as the best lipase,vinyl acetate?VA?as the best acyl donor and methyl tert-butyl ether?MTBE?as the best organic solvent.The effects of temperature,substrate ratio,enzyme loading and reaction time on resolution effect were investigated.Based on the Ping-Pong bi-bi mechanism with4-methoxy-?-hydroxyphenylacetic acid inhibition,kinetic models of?R?-enantiomer and?S?-enantiomer were established.Kinetic parameters were obtained by fitting time-concentration curves at different initial concentrations of4-methoxy-?-hydroxyphenylacetic acid.The average relative error was less than 3.0%,indicating the model predictions are in good agreement with the experimental data.Additionally,kinetic models are used to predict the effects of enzyme loading,initial concentration of vinyl acetate and reaction time on enantiomeric excess?ee _S?and conversion?c?.The optimal process parameters were obtained through simulation and optimization,including 50°C of temperature,300 mmol/L of vinyl acetate concentration,17.5 mg/m L of enzyme loading and 13 h of reaction time.Under optimal conditions,excellent results with high 4-MMA conversion?50.2%?and substrate enantiomeric excess?98.6%?were obtained.The immobilized lipase[PEG-PFL@NH₂-MIL-53?Fe?]was firstly prepared by using NH₂-MIL-53?Fe?metal-organic frameworks as carrier to covalently immobilize pseudomonas fluorescens lipase of PEG modification.PEG-PFL@NH₂-MIL-53?Fe?as an high efficient biocatalyst was used for the lipase-catalyzed resolution of4-fluoro-?-hydroxyphenylaceticacidenantiomerstoprepare?R?-4-fluoro-?-hydroxyphenylacetic acid in methyl tert-butyl ether.Compare with the catalytic performances of free PFL and PEG-PFL@NH₂-MIL-53?Fe?,PEG-PFL@NH₂-MIL-53?Fe?possessed better catalytic performance.Meanwhile,the effects of temperature,substrate ratio,amount of enzyme and reaction time on conversion and enantiomeric excess were investigated for the lipase-catalyzed transesterification reaction.The optimal reaction conditions were obtained by optimizing the reaction conditions,including 50?of temperature,300 mmol/L of the concentration of vinyl acetate,60 mg of immobilized enzyme loading and 12 h of the reaction time.Under these reaction conditions,the total conversion and enantiomeric excess of substrate were 49.6%and 97.6%,respectively.