Stereoselective Synthesis Of (S)-3-cyano-5-methylhexanoic Acid By Nitrilase

Pregabalin[(S)-3-aminomethyl-5-methylhexanoic acid,PGB],is the 3-isobutyl substituted analogue of the neurotransmitter inhibitor?-aminobutyric acid(GABA).PGB was first applied in the treatment of peripheral neuropathic pain and refractory partial-onset seizures,then currently for other indications.It is reported that PGB has a promising market with sales reaching nearly $4.59 billion in 2013.Researches demonstrated that the pain suppressing effect of PGB(S)-(+)-enantiomer is ten times of the(R)-(-)-enantiomer.Therefore,the synthesis of optically pure(S)-(+)-enantiomer is important to improve drug efficacy,reduce the dosage and reduce the side effects of diastereomers.Nitrilase(EC3.5.5.1)is one of the important nitrile-converting enzymes that convert nitriles to the corresponding acids and ammonia in one step.The enzymatic process is preferable due to its mild reaction conditions,high efficiencies and selectivities.It complied with the direction of atom economy and green chemistry.The regio-and stereo-specific synthesis of(S)-3-cyano-5-methylhexanoic acid from isobutylsuccinonitrile(IBSN)by nitrilase and then prepare PGB through hydrogenation,which exhibits the highest atom economy than other chemistry-enzymatic processes,was developed.And the difficulty is to obtain a nitrilase with high activity and selectivity towards IBSN.In this study,the nitrilase from Alcaligenes faecalis ZJUTB10 was modified at the gene level.Based on homologous modeling and docking,four loci of the nitrilase(Y296,A197,1289 and L175)were selected.But the selected mutations failed to improve the selectivity of the nitrilase significantly.Our lab has acquired some new stains which produce nitrilase.Therefore,we screened the nitrilase from nitrilase library of our lab futher.And Arabidopsis thaliana nitrilase(NIT)was screened as the best biocatalyst for enantioselective synthesis of(S)-3-cyano-5-methylhexanoic acid.Under the condition of 30?and pH 7.0,after 4 hour-reaction with 150 mM IBSN.the ee value and the conversion reached 96%and 42%,respectively.The reaction conditions of NIT were optimized by single factor method.The reaction parameters including temperature,pH,substrate concentrations and metal ions were investigated.The optimum temperature,pH,substrate concentration and reaction time were 30?,8.0,250 mM and 5 h,respectively.Under above conditions,43%yield and 95%e.e.were achieved.We studied the fermentation conditions of E.coli BL21/pET28b-NIT in 500 L fermenter.E.coli BL21/pET28b-NIT entered logarithmic growth phase after 3 hours,and decline phase 10.5 later.Under the optimized conditions,after 8 hour-reaction with 450 mM IBSN,the conversion and e.e.reached 42%and 95%,respectivily.After purification,the purity and e.e.of(S)-3-cyano-5-methylhexanoic acid reached 97%and 98%.