New Insights into the Binding Site and Affinity of the Interaction between Biotin and PAMAMs-NH2 via NMR Studies.

Abstract

Biotin-dendrimer conjugates (such as biotin-PAMAMs-NH2) are important macromolecules in the field of host-guest chemistry and widely used systems for delivery. The similar chemical structures of the inner and outer layers of PAMAM-NH2 make it difficult to illuminate the interaction and the binding affinity of biotin-PAMAMs-NH2. By utilizing NMR techniques including 1H NMR titration, CSSF-TOCSY, STDD methods, and 2D DOSY analysis, we demonstrate a method to sort out these interactions. The methylene protons of the inner and outer layers of PAMAM-NH2 are successfully identified and accurately positioned so that the carboxylic acid groups of biotins are having ionic interactions with the outermost amine groups of PAMAM-NH2. The inner PAMAM-NH2 is protonated when reaching the isoelectric point of PAMAM-NH2, increasing the hydrodynamic radius. On the basis of the NMR experiments, a model is proposed, where the carboxylic acid groups and heterocyclic skeleton of biotin arched over the outer layers of PAMAM-NH2 like a bridge. Furthermore, using STDD epitope mapping, the binding affinity between biotin and PAMAM-NH2 was quantified. The diffusion behavior of biotin-G5 PAMAM-NH2 complex is more complicated than that of biotin-G3 PAMAM-NH2 complex due to steric hindrance. The results provide a theoretical basis for understanding these complicated drug delivery systems.