Enzymatic Resolution Of 2-aminobutyramide For Chiral Intermediates Of Levetiracetam By Amidase

Levetiracetam is a new kind of anti-epileptic drugs.Due to its unique mechanism of action,working quickly,good curative effect,good tolerance and security on epilepsy,it becomes a wildly used anti-epileptic drug.Levetiracetam has a promising market with sales reaching nearly € 1.03 billion in 2007.(S)-2-aminobutyramide is the key intermediate of Levetiracetam.However,the traditional chemical synthetic method uses chiral catalyst or split reagent,whose synthetic route is long,and the atom economy is low.Therefore,developing an efficient biocatalytic process for preparing key intermediates of Levetiracetam.Herein the enantioselective hydrolysis of 2-aminobutyramide by amidase,which exhibits higher atom economy than the chemical synthetic method,was developed.It laid the groundwork for the synthesis of chiral intermediates of Levetiracetam with high activity and environmental friendliness.Firstly,a TLC and a HPLC analysis method of substrate and product were established.Substrate and product as well as their enantiomorphism isomers were determined successfully.And a recombinant strain Dt-Ami 6with stereoselective catalytic ability was selected after screening in our collection to prepare(S)-2-aminobutyramide.The catalytic conditions,including temperature,p H,metal ions,two phase system,organic cosolvent and substrate concentration for hydrolysis of 2-aminobutyramide were investigated.The results indicated that the optimum temperature,substrate concentration,cell concentration and reaction time were 40 ?,1 mol/L,1.6 g DCW/L,and 40 min,respectively and the solvent was deionized water.In optimal conditions,41.7 % yield and 94.0 % ee were achieved.Finally,the extraction conditions of(S)-2-aminobutyramide was studied.The results indicated that the optimum pH,temperature,substrate concentration and extraction times were 9.5,40 ?,no more than 0.5 mol/L and 4,respectively.In that conditions,the extraction rate and purity of(S)-2-aminobutyramide were 85.6 %,93 %,respectively.The total yield of(S)-2-aminobutyramide was 35.7 %.The structure of the product was further confirmed by FT-IR,LC-MS and NMR spectra.