| Pseudo-dynamic combinatorial chemistry (pDCC) combines the synthesis, screening and destruction of combinatorial libraries to kinetically resolve inhibitors based on their affinity for a target. In our proof-of-principle studies, a library of dipeptides was formed in the presence of a target, carbonic anhydrase (CA), and a destruction mechanism, a protease. Since the target and the protease were separated by a dialysis membrane, only the proportion of dipeptides that was not bound to the target was available for destruction and therefore, the rate of hydrolysis of the pseudo-dynamic combinatorial library (pDCL) could be correlated to the librarie's relative affinity for the target.;In order to study pDCC behavior a simplified pDCC mimic (pDCCm) was designed. In pDCCm, synthesis was replaced by a static library of compounds and destruction by dilution. The design of a pDCCm based kinetic model led to a pDCCm simulator (pDCCmSim). PDCCmSim confirmed the main advantage of pDCC over other receptor assisted combinatorial chemistry methods: selectivity is not limited. pDCCmSim also predicted that slight differences in the relative permeability of the pDCLs, the number of library members, their relative binding affinity and the experimental stoichiometry can influence the outcome of pDCC experiments dramatically. A set of guidelines that facilitate pDCL optimization is proposed.;The first set of proof-of-principle pDCC experiments were found to be flawed. Rather than reflecting the binding affinity of the library for the target, the final product distribution reflected the protease's substrate specificity. The main problems with these pDCLs were insufficient and imbalanced rate of destruction of the peptides in the absence of the target, pH drift, and insufficient permeability across 1000 MWCO cellulose ester membranes. A dipeptide amide based pDCL that was efficiently cleaved by thermolysin was designed. The new pDCL could be used in pH 7.5 75 mM HEPES, 16.6 mM CaCl 2 buffer, provided that the chambers were limited by 3500 MWCO membranes. A 4th generation pDCC experiment that evolved reflecting the library's affinity for the target was performed. |
