Design, Synthesis, and Characterization of a High-Affinity Trivalent System Derived from Vancomycin and l-Lys-d-Ala-d-Ala

A trivalent derivative of vancomycin, tris(vancomycin carboxamide), [C6H3-1,3,5-(CONHC6H4-4-CH2NHCOV)3 (Rt V 3; V = vancomycin)], binds an analogous trivalent derivative of d-Ala-d-Ala, R‘t L‘3, (C6H3-1,3,5-[CONεH(N α-Ac)-l-Lys-d-Ala-d-Ala]3) in water with a dissociation constant that is approximately 4 × 10-17 M, as estimated by HPLC using a competitive assay against N α,ε-diacetyl-l-Lys-d-Ala-d-Ala (L). This binding is one of the tightest known for low molecular weight organic species. The dissociation of Rt V 3·R‘t L‘3 in the presence of an excess of L could be followed by HPLC. The kinetics of dissociation are quite different from those of monovalent tight-binding systems such as avidin and biotin. In particular, the rate of dissociation of the aggregate Rt V 3·R‘t L‘3 is rapid in the presence of monovalent L at concentrations greater than the value of the dissociation constant for the complex of L with V; by contrast, the rate of dissociation of biotin·avidin is independent of the concentration of biotin. Two mechanisms by which the dissociation may occur are postulated and discussed. Calorimetric measurements for the trivalent system indicate that the enthalpy of association is ∼−40 kcal/mol, about three times that of V + L, and thus the entropy of association is ∼−18 kcal/mol, approximately 4.5 times that of V + L.