Revealing Equilibrium and Rate Constants of Weak and Fast Noncovalent Interactions

摘要

Rate and equilibrium constants of weak noncovalentnmolecular interactions are extremely difficult to measure.nHere, we introduced a homogeneous approach callednequilibrium capillary electrophoresis of equilibrium mixturesn(ECEEM) to determine kon, koff, and Kd of weak (Kd > 1 μM)nand fast kinetics (relaxation time, τ < 0.1 s) in quasi-equilibriumnfor multiple unlabeled ligands simultaneously in one microreactor.nConceptually, an equilibrium mixture (EM) of a ligandn(L), target (T), and a complex (C) is prepared. The mixture isnintroduced into the beginning of a capillary reactor with aspectnratio >1000 filled with T. Afterward, differential mobility of L, T, and C along the reactor is induced by an electric field. Thencombination of differential mobility of reactants and their interactions leads to a change of the EM peak shape. This change is anfunction of rate constants, so the rate and equilibrium constants can be directly determined from the analysis of the EM peak shapen(width and symmetry) and propagation pattern along the reactor. We proved experimentally the use of ECEEM for multiplexndetermination of kinetic parameters describing weak (3mM> Kd > 80 μM) and fast (0.25 sgτg0.9 ms) noncovalent interactionsnbetween four small molecule drugs (ibuprofen, S-flurbiprofen, salicylic acid and phenylbutazone) and R- and β-cyclodextrins. Thenaffinity of the drugs was significantly higher for β-cyclodextrin than R-cyclodextrin and mostly determined by the rate constant ofncomplex formation.