Preparation Of Optically Pure (R)-2-Cl-MA Via Biotransformation By Nitrilase

(R)-o-Chloromandelic acid is widely used in pharmaceutical production,fine chemical products,asymmetric synthesis,optical resolution and other fields.(R)-o-Cl-MA primary use is employed to synthesize the clopidogrel,a thienopyridine-class antiplatelet agent used to inhibit blood clots,safely and efficiently,and important optically active agents.Bio-transformation using nitrilasecould convert(R,S)-o-chloromandelonitrile to(R)-o-chloromandelic acid under mild conditions with high stereoselectivity and without cofactor regeneration compared with traditional methods.O-chlorobutyronitrile(2-Cl-MN)racemate as substrate,which could theoretically provide 100% yield of 2-Cl-MA due to the efficient in situ racemization of residual(S)-o-chloromandelonitrile.At present,the nitrilase is not tolerant to the substrate 2-Cl-MN and the selectivity is not strong.In this paper,we use the method of genome digging to select a nitrile hydrolysis derived from Labrenzia aggregata from NCBI database Enzyme LaN.In this research,theLabrenzia aggregata nitrilase(LaN)was used as catalyst,andthe o-Chloromandelonitrile(o-Cl-MN)as substrate.The recombinant expressing LaN reaction speed reached 2.53 μmol/min/mg at 37 ℃ and pH 8.0 under the conditions of pH 8.0.In practical applications,o-chloromethillonitrile was converted to(R)-2-Cl-MA directly using Escherichia coli whole cells of LaN.The use of "batch feeding reaction method" to improve the reaction system substrate concentration,and to a large extent reduce the cost of production The apparent concentration of o-Cl-MN in reaction system has reached 425mM(based on the total volume of reaction),finally,the yield and enantiomer excess against(R)-o-Cl-MA were 86.4% and 99.9% after recrystallization in toluene.Provide a practical way of large-scale production of(R)-2-Cl-MA.