Protein Binding Chiral Discrimination of HPLC Stationary Phases Made with Whole, Fragmented, and Third Domain Turkey Ovomucoid

Abstract

Individual protein domains and two domains in combination were prepared by enzymatic and chemical cleavage of turkey ovomucoid followed by isolation and purification by size-exclusion and ion-exchange chromatography. Silica bonded-phase HPLC columns were made from either whole or isolated domains of turkey ovomucoid. The protein columns were tested for chiral recognition by their abilities to resolve enantiomers among a wide range of racemates. The columns made from whole turkey ovomucoid displayed chiral activity toward many racemates, where as a combination of the first and second domain resolved only a selected number of aromatic weak bases. The first and second domains independently gave no appreciable chiral activity. The turkey ovomucoid third domain exhibited enantioselective protein binding for fused-ring aromatic weak acids. Glycosylation of the third domain did not affect chiral recognition. Titration of the third domain with model compounds in conjunction with NMR measurements enabled the identification of the amino acids responsible for binding. Molecular modeling of the ligand-protein complexation provided insights into the ability of a protein surface to discriminate enantiomers on the basis of multiple intermolecular interactions.

Chiral separation by chromatographic and electromigration techniques. A Review

“…Phases based on ovomucoid from chicken egg white were developed by Miwa et al [206] and applied to the chiral separation of acidic, basic and neutral drug enantiomers [207]. Pinkerton et al [208] showed that only one of three domains in chicken and turkey ovomucoids is responsible for chiral recognition. Phases of this type showed enantioselectivity for benzodiazepines and 2-arylpropionic acid derivatives.…”

Survey and Trends in the Preparation of Chemically Bonded Silica Phases for Liquid Chromatographic Analysis

“…However, if the enantiomers form complexes with an enantiomeric reagent (a chiral selector), the resulting species may exhibit such large differences in binding properties that they can be separated in a conventional chromatographic system. Over the past decade many HPLC chiral stationary phases have been developed for the separation of enantiomers, with each phase employing its own unique separation strategy [108,109]. Since chiral recognition properties are highly specific, a large number of enantioselective phases has been designed to separate various chiral compounds.…”

Investigation of the stereoselectivity of an anti‐amino acid antibody using molecular modeling and ligand docking

“…9,20,21 More recently, molecular modeling has emerged as a powerful new method to investigate stereoselective protein-ligand interactions. In cases where structural information is available from X-ray crystallography or NMR studies, molecular modeling may be used to gain insight into a protein's ability to discriminate enantiomers, [22][23][24][25] or to predict the effect of structure-guided sitedirected mutagenesis in ligand docking and molecular dynamics experiments. 26,27 If structural information is not readily available, protein modeling can be combined with ligand docking and molecular dynamics to investigate experimentally observed stereoselectivity.…”