title: Stereoselective determination of 2-benzamidomethyl-3-oxobutanoate and methyl-2-benzoylamide-3-hydroxybutanoate by chiral high-performance liquid chromatography in biotransformation.
abstract: (2S, 3R)-methyl-2-benzamidomethyl-3-hydroxybutyrate (MBHB) is a key intermediate in the synthesis of 4-aceoxyazetidinone, a building block for the production of penems and carbapenems. More attentions have been paid to screen biocatalysts achieving asymmetric preparation of (2S, 3R)-MBHB. In this study, an improved chiral high-performance liquid chromatographic (HPLC) method was developed for the stereoselective determination of 2-benzamidomethyl-3-oxobutanoate (BMOB) and MBHB, and further employed into the biotransformation of BMOB. Chiral separation was achieved within 12 min on Chiralpak AY-H column, which was faster and more suitable for screening biocatalysts exhibited reduction activity and (2S, 3R)-stereospecificity toward BMOB than on other columns. Ultimately, a new strain, Burkholderia gladioli ZJB-12126 capable of reducing BMOB to (2S, 3R)-MBHB was successfully isolated based on this newly constructed HPLC method. Samples were prepared by liquid-liquid extraction system using ethyl acetate as the extractor solvent. The extraction recoveries of BMOB and MBHB isomers ranged from 91.6 to 94.1% with relative standard deviation (RSD) below 10%. Linear calibration curves were obtained in the concentration range of 50-5000 μg/mL for both BMOB and MBHB isomers, respectively. Intra-day and inter-day precisions and accuracy were below 15% for all isomers evaluated by RSDs and relative errors (REs), respectively. This novel method was demonstrated to be suitable for assessing the biotransformation process of BMOB.
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification, Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification, Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China. Electronic address: zhengyg@zjut.edu.cn.
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification, Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Liu
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification, Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Li-Hui
authors.initials: LH
authors.lastname: Sun
mesh_terms: D001711:Biotransformation; D042725:Burkholderia gladioli / Q000737:chemistry / Q000378:metabolism*; D002087:Butyrates / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D013237:Stereoisomerism
chemical_list: D002087:Butyrates
keywords: 2-Benzamidomethyl-3-oxobutanoate; Biotransformation; Chiralpak AY-H; Methyl-2-benzoylamide-3-hydroxybutanoate.; Stereoselective determination