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Attempts to quantify binding interactions of noncovalent complexes in aqueous solution have been stymied by complications arising from enthalpy-entropy compensation and cooperativity. We have extended work detailing the relationship between noncovalent structure and free energy of binding to include the roles of enthalpy and entropy of association. On the basis of van't Hoff measurements of the dimerization of vancomycin type antibiotics, we demonstrate that positive cooperativity manifests itself in a more favorable enthalpy of association and a partially compensating less favorable entropy of association. Finally, we extend these results to rationalize thermodynamic observations in unrelated systems.

Wild-type p53 plays a crucial role in the prevention of cancer. Since dysfunction of p53 can be caused by increased levels of the protein MDM2, small molecules which antagonize the interaction between these two proteins have potential in cancer therapy. The discovery and structure determination of a fungal metabolite, chlorofusin, which antagonizes the p53/MDM2 interaction are reported.

The factors that give rise to binding enhancements when a strongly dimerizing vancomycin-group antibiotic (chloroeremomycin) binds to a model cell surface of vancomycin-resistant enterococci (VRE) have been semiquantitated. The model cell surface is comprised of vesicles to which have been anchored cell wall precursor analogues of vancomycin-resistant bacteria (which terminate in -D-lactate) via a hydrophobic docosanoyl (C-22) chain. Using H-1 and F-19 NMR spectroscopy, a large binding enhancement at the model cell surface (compared to the binding of an analogous ligand in free solution) has been observed. This enhancement can be partitioned into two distinct factors: a simple concentrating factor arising from an-increase in local concentration of ligand when it is located at the vesicle surface and a factor arising from the cooperative interaction of species mutually bound to the membrane surface. The overall enhancement to binding at a surface compared to binding in free solution was found to be a factor of 10(2)-10(3). In contrast, no significant surface binding enhancement was observed for the weakly dimerizing antibiotic vancomycin.

The mode of action of a semisynthetic glycopeptide active against vancomycin-resistant bacteria has been investigated. It is shown that the antibiotic, biphenylchloroeremomycin or LY307599, dimerizes strongly and anchors to membranes. It is hypothesized that these two locating devices, previously identified by us when acting separately, might combine to give enhanced binding at a cell surface. This hypothesis is tested experimentally by showing that glycopeptides can bind cell-wall precursor analogues from resistant bacteria (terminating in D--lactate) in a similar manner to those from susceptible bacteria (terminating in D--alanine) and by using model cell surfaces where the benefits of dimerization can be expressed and studied. These model systems use vesicles to represent the cell. membrane, to which cell wall analogues are anchored via a docosanoyl chain, so mimicking the arrangement encountered at the cell surface. Using H-1 NMR spectroscopy, we demonstrate enhanced binding due to dimerization and propose that this enhancement will act cooperatively with membrane anchoring in biphenylchloroeremomycin.

Evidence is presented for the formation or dimers in aqueous solutions or the glycopeptide antibiotics cremomycin, A82846B, vancomycin, and eremomycin-Ψ. The dimerization constant K dim is determined by 1 H NMR spectroscopy for the last two compounds and also for the related compound ristocetin-Ψ, for which dimerization has previously been reported in mixed solvents. Values or K dim are obtained for these compounds over a range or temperatures, and thus ΔH dim and ΔS dim are calculated

Molecules in solution have degrees of freedom of overall translation and rotation, as well as internal rotations about single bonds, that become highly constrained when two molecules associate to form a stable complex. We have considered the thermodynamics of fusion and sublimation of hydrocarbons and polar organic molecules as instructive models for features of complex dissociation. The entropy of fusion within homologous series of alkanes, alkyl carboxylic acids, and 2-methyl ketones provides an estimate of the entropic cost of restricting a rotor, within a hydrocarbon chain, as -1.6 to -3.6 kJ mol -1 (TΔS at 300 K) upon crystallization

Any bimolecular association is entropically unfavorable because of degrees of freedom of translation and rotation lost when two molecules come together to form a complex. For a ligand of molecular weight 200, the formation of a «rigid» dimer (one in which there is no residual relative motion of the associating components A and B in the complex A.B) opposes binding by ca. 10 -9 to 10 -10 M -1 in binding constant

An expression is presented for the estimation of approximate binding constants for bimolecular associations in solution. The consequences of the approach have been examined for the bimolecular association of two peptide components in aqueous solution: specifically for the binding of two vancomycin group antibiotics, vancomycin itself and ristocetin A, to the peptide cell wall analogue ^V-Ac-D-AIa-D-AIa and related ligands. Uncertainties in the treatment are relatively large, but the physical insights gained into the binding process (in part with the aid of calorimetric data obtained by others) are enlightening. We conclude that for amide-amide hydrogen bond formation in aqueous solution at room temperature, the intrinsic binding energy is ca. 24 kJ mol"1 (an intrinsic binding constant of ca. 104); this process is almost completely driven by a favorable entropy change associated with the release of water molecules from the amide NH and CO groups involved in hydrogen bond formation. The bimolecular association of N-Ac-D-AIa-D-Ala with ristocetin A has a remarkably small entropy change at 298 K (TAS = 3 ± 1.5 kJ mol"1)- We conclude that the release of water from polar and hydrocarbon groups involved in the binding almost exactly compensates for (i) the unfavorable entropy change due to the freezing out of four rotors of /V-Ac-D-AIa-D-AIa upon binding and (ii) the unfavorable entropy change of a bimolecular association. A crude quantitation of these effects is presented. We also present an estimate of the increase in translational plus rotational free energy, as a function of the ligand mass, occurring when a ligand binds to a larger receptor. This quantity, fundamental to all binding processes, is relatively insensitive to the shape of the ligand. Extension of the approach will allow, in those cases where there is good complementarity between ligand and receptor, the prediction of approximate peptide-peptide binding constants in aqueous solution.

Two models (A and B) have been proposed to account for decreased downfield chemical shifts of a proton bound by noncovalent interactions at a ligand/antibiotic interface as the number of ligand/antibiotic interactions is decreased. In model A, the proton involved in the noncovalent bond suffers a smaller downfield shift because the bond is, with a relatively large probability, broken, and not because it is longer. In model B, the proton involved in the noncovalent bond suffers a smaller downfield shift because the bond is longer, and not because it is, with a relatively large probability, broken. We show that model A cannot account for the chemical shift changes. Model B accounts for the process of positively cooperative binding, in which noncovalent bonds are reduced in length and thereby increase the stability of the organized state.

Changes in the relative populations of the monomer and asymmetric dimer forms of ristocetin A, upon binding of two molecules of ligand, suggest that ligand binding is negatively cooperative with respect to dimerization. However, strong hydrogen bonds formed in the binding sites of the ligands are reinforced in the dimer relative to the monomer, and the barrier to dissociation of the dimer is increased upon binding of the ligands. It is concluded that the interactions which are common in the binding of both ligands are made with positive cooperativity with respect to those involved in dimerization. The conclusions are relevant to the binding of ligands to proteins, where ligand binding energy can be derived from stabilization of the protein in its ligand-bound form.

Glycopeptide antibiotics of the vancomycin group bind to bacterial cell wall analogue precursors, and typically also form dimers. We have studied the interplay between these two sets of noncovalent bonds formed at separate interfaces. Indole-2-carboxylic acid (L) forms a set of hydrogen bonds to the glycopeptide antibiotic chloroeremomycin (CE) that are analogous to those formed by N-Ac-D-Ala. The ligand/CE dimer interactions (in L/CE/CE/L) are shown to occur with positive cooperativity and structural tightening at the dimer interface. From theoretical considerations and from other data, it is inferred, but not proven, that in the exercise of positive cooperativity, the interface that will be tightened to the greatest degree is the one that lies in the shallowest free energy well.