Chromatographic evaluation of structure selective and enantioselective retention of amines and acids on cellobiohydrolase I wild type and its mutant D214N

The mechanisms of structure selective and enantioselective retentions of am ines and acids on two chiral stationary phases based on wild type cellobioh ydrolase I (CBH I) and its mutant D214N have been investigated, All the ami no alcohols tested had an enantioselective site that overlaps with the cata lytically active site of BH I, whereas the enantioselectivity of prilocaine was not affected by the mutation. The hydroxyl group of the amino alcohols did not seem to be an important contributor to the total binding strength whereas a bromo substituent in the aromatic ring promotes a high enantiosel ectivity (alpha=7.05), Interestingly, the chiral recognition site of the ac id warfarin overlaps with the binding site of the amino alcohols. Di-p-tolu oyltartaric acid and dibenzoyltartaric acid were strongly retained probably due to electrostatic attraction, but no enantioselectivity was observed. T he difference in retention characteristics for the amino alcohols on the tw o stationary phases was strongly pi-I-dependent, A change in elution order of different amino alcohols occurred when changing the pH from 5.0 to 7.0. The difference between the two phases was lower at low pH, The retention ti mes could also be affected by ionic strength and by use of cellobiose as a mobile phase additive but no indication of ion-pair retention of the amines was observed, when adding hexanesulphonate as counter ion to the mobile ph ase. The temperature dependence of the retention of the enantiomers of prop ranolol at pH 7.0 on the mutant D214N was similar to what was earlier obser ved on the wild type CBH I at lower pH. (C) 1999 Elsevier Science B.V. AU r ights reserved.