Chemo-enzymatic Synthesis Of The Chiral Intermediate Of Pregabalin

Pregabalin, i.e.,（S）-3-aminomethyl-5-methylhexanioc acid, is a gamma-aminobutyric acid（GABA） analogue receptor agonist that was developed by Pfizer for the treatment ofneuropathic pain, anxiety disorders, and partial-onset epilepsy. The chemo-enzymatic method,which integrates traditional chemistry with modern biocatalysis, has become an increasinglyattractive approach to prepare chiral intermediate of Pregabalin.（S）-3-cyano-5-methylhexanol, which is a key chiral intermediate for the preparation ofPregabalin, could be gained by the region-and stereo-specific nitrilase-catalyzed hydrolysisof isobutylsuccinonitrile （IBSN）. In this study, firstly, isobutylsuccinonitrile （IBSN）, thesubstrate of nitrilase-catalyzed reaction, was synthesized by three-step reaction ofknoevenagel condensation, cyano-addition and decarboxylation reaction, withisovaleraldehyde and ethyl cyanacetate as starting materials. The influences of molar ratioof raw materials, reaction temperature as well as reaction time, on the yields of theseintermediates were investigated. After optimization, the overall yield of the three-step reactionwas increased from62.3%to81.4%. The structures of the intermediates were characterizedand confirmed by¹H NMR,¹³C NMR, FT-IR and MS.Secondly, using racemic isobutylsuccinonitrile （IBSN） as the substrate, nitrilase-producingbacterium Pseudomonas8was screened by CoCl2chromogenic method, phenol-hypochloritechromogenic method and chiral gas chromatography with good enantioselectivity ofhydrolyzing IBSN to form （S）-3-cyano-5-methylhexanol. The structure of（S）-3-cyano-5-methylhexanol was characterized and confirmed by¹H NMR,¹³C NMR,HMQC, FT-IR and MS.Finally, effects of temperature, time, pH, substrate concentration, cell concentration,detergents and lacquer solvent on the nitrilase-catalyzed hydrolysis of IBSN by resting cellsof Pseudomonas8was investigated. The results showed that the optimal reaction conditionswere as follows:20mM substrate concentration,2.0g/L （DCW） cell concentration,30℃, pH7.0,12h,5%（w/v） TritonX-100. After optimization,（S）-3-cyano-5-methylhexanol wassuccessfully prepared with conversion of57%and eepvalue of67%.