Capillary zone electrophoresis of interconverting cis-trans conformers of peptidyl-proline dipeptides: estimation of the kinetic parameters

Peptides containing proline residues, except at the N-terminus, exist in cis and trans forms due to the rigidity of the peptidyl-proline bond. Computer-simulated and experimental electropherograms have been used to study the interplay of the electrophoretic migration and the kinetics of the cis-trans interconversion. The measure of the interference by the reaction is the dimensionless Damk?hler number (Da) which is the ratio of the migrant's residence time to the characteristic time of reaction in the capillary. The fraction of the trans conformer that is not separable with 90% purity is employed to quantify the extent of overlap between the concentration profiles of the two interconverting migrants and the effect of the various operational variables was examined by simulation. The deterioration of separation, as measured by the degree of overlap, is a quasi sigmoidal function of Da with the selectivity and the intrinsic efficiency of the system as the parameters. At sufficiently low temperatures the interference by reaction kinetics may vanish and the two conformers are separated. At high enough temperatures, when the rate of interconversion is very fast, the two conformers are not separated at all. A simple and rapid method is proposed for estimation of kinetic parameters for the cis-trans isomerization on the basis of data obtained with phenylalanyl-proline dipeptide. The procedure involves determination of the equilibrium constant by measuring equilibrium concentrations of the two conformers using nuclear magnetic resonance (NMR) or capillary zone electrophoresis (CZE) at very low temperature, correlation of the peak shapes in the electropherograms with the Da, and finally, evaluation of the forward rate constants from the assigned Da values in the domain: 0.01 < Da < 0.5. Separations using CZE were performed in the temperature range of 1-40 degrees C by using the Beckman P/ACE unit equipped with an auxiliary cooling system. The kinetic data thus obtained showed good agreement (average error less than 5%) with those measured by NMR.