Enantioselective hydrolysis of some 2-aryloxyalkanoic acid methyl esters and isosteric analogues using a penicillin G acylase-based HPLC monolithic silica column.
A technique based on liquid chromatography has been developed to facilitate studies of enantioselectivity in penicillin G acylase (PGA)-catalyzed hydrolysis of some 2-aryloxyalkanoic acid methyl esters and isosteric analogues. PGA was covalently immobilized on an aminopropyl monolithic silica support to create an immobilized HPLC-enzyme reactor. Two sets of experimental data were drawn to calculate the enantioselectivity (E) of the kinetically controlled enantiomer-differentiating reaction, the degree of substrate conversion and the enantiomeric excess of the product. The developed enzymatic reactor was coupled through a switching valve to an achiral analytical column for separation and quantitation of the hydrolysis products. The enantiomeric excess was determined off-line on a PGA-chiral stationary phase. In this way, highly precise E values were determined. A computational study related to the hydrolysis of the considered racemic esters was also carried out in order to unambiguously clarify both the substrate specificity and the enantioselectivity displayed by PGA.
Massolini
Gabriella
G
Dipartimento di Chimica Farmaceutica, Universita' di Pavia, Via Taramani 12, 1-27100 Pavia, Italy. g.massolini@unipv.it
Calleri
Enrica
E
Lavecchia
Antonio
A
Loiodice
Fulvio
F
Lubda
Dieter
D
Temporini
Caterina
C
Fracchiolla
Giuseppe
G
Tortorella
Paolo
P
Novellino
Ettore
E
Caccialanza
Gabriele
G
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D004952:Esters / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D010399:Penicillin Amidase / Q000737:chemistry* / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D004952:Esters; D010399:Penicillin Amidase