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) (RtV3; V = vancomycin)], binds an analogous t rivalent derivative of D-Ala-D-Ala, R'L-t'(3), (C6H3- 1,3,5-[(CONH)-H-epsil on(N-alpha-Ac)-L-Lys-D-Ala-D-Ala](3)) in water with a dissociation constant that is approximately 4 x 10(-17) M, as estimated by HPLC using a competit ive assay against N-alpha,N-epsilon-diacetyl-L-Lys-D-Ala-D-Ala (L). This bi nding is one of the tightest known for low molecular weight organic species . The dissociation of RtV3. R'L-t'(3) in the presence of an excess of L cou ld be followed by HPLC. The kinetics of dissociation are quite different fr om those of monovalent tight-binding systems such as avidin and biotin. In particular, the rate of dissociation of the aggregate RtV3.R'L-t'(3) is rap id 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 postulate d and discussed. Calorimetric measurements for the trivalent system indicat e that the enthalpy of association is similar to-40 kcal/mol, about three t imes that of V + L, and thus the entropy of association is similar to-18 kc al/mol, approximately 4.5 times that of V + L.