Enzymatic Resolution Of 3-cyano-5-methylhexanoic Acid Ethyl Ester For Chiral Intermediates Of Pregabalin

Pregabalin is the 3-isobutyl substituted analogue of the inhibitory neurotransmitter y-aminobutyric acid.Due to its good curative effect on neuropathic pain and epilepsy,Pregabalin was recommended as first-line treatment option by some international guidances such as I ASP,EFNS and NICE.Pregabalin is one of the fastest growing and block-buster drugs in the market with sales reaching nearly $4.15 billion in 2012.The most successful scalable chemoenzymatic strategy for Pregabalin involves a lipolase-catalyzed resolution of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester to produce the desired(S)-mono acid enantiomer.However,a closer look at the chemoenzymatic process made us realize that the atom economy of the strategy was still not satisfactory.Herein our aim was to develop an efficient biocatalytic process for preparing key intermediates of Pregabalin.Therefore,the enantioselective hydrolysis of(R,S)-3-cyano-5-methylhexanoic acid ethyl ester by lipases or esterases,which exhibits higher atom economy than the second-generation chemoenzymatic route,was developed.Eight commercially available lipases were screened for their capacity for the kinetic resolution of(R,S)-3-cyano-5-methylhexanoic acid ethyl ester.Immobilized lipase PS from Pseudomonas cepacia was screened and shown to be the best commercialized biocatalyst for the enantioselective hydrolysis of racemic 3-cyano-5-methylhexanoic acid ethyl ester.The optimum temperature and pH for the biocatalytic process were 35? and 6.0,respectively.Lipase PS IM exhibited a strong tolerance toward high substrate concentrations of up to 2.0 M.(S)-3-cyano-5-methylhexanoic acid ethyl ester was produced in 0.89 M(162.9 g/L),99.2%ee,and 44.5%yield.Then strain ZJB-09277,capable of S-enantioselective hydrolysis of(R,S)-3-cyano-5-methylhexanoic acid ethyl ester,was isolated from soil samples.Based on morphology,physiological characteristics,16S rDNA analysis and phylogenetic tree analysis,the isolate was designated as Arthrobacter sp.ZJB-09277.The substrate preference of enzyme from Arthrobacter sp.ZJB-09277 which investigated with various p-nitrophenyl esters suggested that the enzyme involved in the enantioselective hydrolysis of(R,S)-3-cyano-5-methylhexanoic acid ethyl ester was an esterase.The medium components and cultivation conditions of Arthrobacter sp.ZJB-09277 were optimized by single factor method.The optimal medium composition was as follows(g/L):sodium citrate 15,yeast extraction 30,MgSO4·7H2O 0.5,NaCl 1.0,CUCl2 0.02,FeSO4·7H2O 0.01,Na2HPO4 1.0,KH2PO4 1.0 The satisfactory fermentation conditions for cell growth and formation of esterase were as follows:medium volumetric ratio,20%(v/v);inoculum volume,2.0%(v/v);initial pH value,7.0;30?;150 rpm.Under these conditions,9.1 g/L biomass and 8.7 U/L specific activity were achieved.Racemic 3-cyano-5-methylhexanoic acid esters were synthesized with different alkyl chains in order to study the effect of the alcohol moiety on the enantioselectivity and activity of Arthrobacter sp.ZJB-09277 esterase.The enantioselectivity and activity of the esterase can be remarkably influenced by steric hinderance effect.Under optimal temperature 35?,after 18 hour-reaction with 100 mM racemic 3-cyano-5-methylhexanoic acid ethyl ester in reaction media consists of sodium phosphate buffer(100 mM,pH 8.0)and 50%dimethyl sulfoxide,the ee value of(S)-acid enantiomer reached 95.1%,and the conversion and E were 44.6%and 80,respectively.Furthermore,the resting cell activity was remained after 120 hours exposed to 50%dimethyl sulfoxide,suggesting it exhibited rather good stability and tolerance toward dimethyl sulfoxide.