Study On Kinetic Resolution Of Halomandelic Acid Enantiomers By Enzymatic Transesterification

As a very important chiral intermediate,halogenated tonsillic acid is widely used in pharmaceutical production,fine organic synthesis,pesticide chemistry and other fields,and the demand for optically pure halomandelic acid is increasing.Therefore,it is of great research and economic value to develop efficient and environmentally friendly separation methods.In this thiese,biocatalysts were used to realize the efficient resolution of five halomandelic acid enantiomers:2-fluoromandelic acid,3-fluoromandelic acid,4-fluoromandelic acid,4-chloromandelic acid and 4-bromomandelic acid.The stereoselective lipase catalyzed transesterification of enantiomers of 2-fluoromandelic acid,3-fluoromandelic acid and 4-fluoromandelic acid with acyl donors in organic phase was studied.The reaction systems of lipase catalyzed resolution of enantiomers of 2-fluoromandelic acid,3-fluoromandelic acid and 4-fluoromandelic acid were constructed by examining the experimental parameters of enzyme type,acyl donor,solvent,temperature,enzyme dosage and time.The optimum resolution conditions of 2-fluoromandelic acid were as follows:10 mmol/L2-fluoromandelic acid and 60 mmol/L vinyl propionate reacted in 2 m L methyl tert-butyl ether with an enzyme amount of 50 mg at a temperature of 55°C for 9 hours;The optimum reaction conditions of 3-fluoromandelic acid were as follows:the concentration of 10 mmol/L 3-fluoromandelic acid and 60 mmol/L vinyl propionate were reacted for 3.5 h under the conditions of 2 ml1,2-dichloroethane,enzyme dosage of 45 mg and reaction temperature of 65℃;The optimum reaction conditions of 4-fluoromandelic acid were as follows:Under the conditions of 60 mmol/L vinyl acetate as acyl donor,substrate concentration of 10 mmol/L,2 m L methyl tert-butyl ether,enzyme content of 45 mg,reaction temperature of 55℃,reaction 3.5 h.Under the optimum conditions,50.05%,49.81%and 49.84%conversion（c）and 97.23%,97%and 98%enantiomeric excess（eep）were obtained by transesterification of 2-fluoromandelic acid,3-fluoromandelic acid and 4-fluoromandelic acid with acyl donors,respectively.Based on the three resolution systems,the enzyme-catalyzed resolution of three mandelic acids with different substituted positions was studied.The results showed that the para-substituted mandelic acid had the fastest reaction rate,which may be attributed to the steric hindrance effect and electronic effect.The immobilized enzyme PCL@Ui O-67（Zr）was prepared by covalent bonding method using Pseudomonas cepacia lipase（PCL）as biocatalyst and Ui O-67（Zr）as immobilization carrier.It is demonstrated by characterization that PCL was immobilized on the surface of Ui O-67（Zr）.PCL@Ui O-67（Zr）was used to catalyze the resolution of 4-chloromandelic acid enantiomers in organic phase.The optimum reaction conditions for the application of PCL@Ui O-67（Zr）in the catalytic resolution of 4-chloromandelic acid were as follows:in 2 ml methyl tert butyl ether,the substrate concentration of 4-chloromandelic acid was 20 mmol/L,and the transesterification reaction was carried out with 120 mmol/L vinyl acetate at 55℃and the amount of PCL@Ui O-67（Zr）was 30 mg.After 18 hours of reaction,47.67%of the total conversion and 97.1%of the enantiomeric excess of the product were obtained,respectively.The activity decreased to half of the initial activity after 5 cycles.A zirconium based metal organic framework material[NH₂-Ui O-66（Zr）]was synthesized and used as an immobilization carrier to covalently immobilize Pseudomonas fluorescens lipase（PFL）on NH₂-Ui O-66（Zr）.It was confirmed that the lipase was immobilized on the carrier surface by characterization.The reaction conditions such as solvent,temperature,enzyme dosage and reaction time on the resolution of 4-bromomandelic acid enantiomers when using PFL@NH₂-Ui O-66（Zr）as catalyst.By comparing the catalytic performance of free PFL and PFL@NH₂-Ui O-66（Zr）,it was found that immobilized enzyme PFL@NH₂-Ui O-66（Zr）had better solvent and temperature tolerance and better catalytic activity than free PFL.The optimum reaction conditions of immobilized enzyme catalytic reaction were as follows:when the substrate concentration was 20mmol/L of 4-bromomandelic acid and the concentration of vinyl acetate was 120 mmol/L,in 2 ml methyl tert butyl ether,the temperature was 55℃,the amount of enzyme was 15 mg,and after 7hours of reaction,the total conversion and product enantiomeric excess reached 48.9%and98.1%,respectively,while the total conversion of free PFL-catalyzed reaction with the same enzyme content was only 15%.Moreover,the immobilized enzyme has good recycling performance,and its activity has not decreased significantly after repeated use for three times.