A preorganised amino acid derivative containing a cyclopropyl constraint was designed to orient an amino acid into its bound conformation. This constrained mimic was determined by ITC to be equally potent to the native Phe derivative. It was found that a more favorable enthalpy of binding was compensated by an equally unfavorable entropy compared to the native ligand. In order to properly ascertain the effects of the cyclopropane constraint, a flexible control containing the same number of heavy atoms was synthesized and tested, and it was found to be at least 200 fold less potent than the constrained analog. However, without structural data of the flexible control, it is difficult to infer if the differences in ligand binding affinity arose from the ligand constraint or some other unknown complexity to binding. We studied the thermodynamic and structural effects of modifying alkyl chains of n-alka(e)nol and phenylalka(e)nol binders to MUP-I by both the removal of a rotor via deletion of a methylene unit and restriction of a rotor via the installation of an internal olefin. In general, we observed that a similar thermodynamic signature accompanies modifications for both the n-alka(e)nol and phenylalka(e)nol ligands: A favorable T[delta][delta]Sºo̳b̳s̳ is compensated by an unfavorable T[delta][delta]Hºo̳b̳s̳ such that T[delta][delta]Gºo̳b̳s̳ for both removal of a methylene and insertion of an internal olefin are unfavorable and equipotent, respectively. The insertion of an internal olefin into an alkyl chain led to significantly more favorable entropies than does methylene removal, yet enthalpy-entropy compensation leads to nearly equipotent binding energetics. However, we did find a strong correlation between [delta]Ho̳b̳s̳° and buried apolar Connolly Surface Area (CSA). The intrinsic free energies of introducing an internal olefin into the n-alkanols and phenylalkanols differ markedly from the observed data. It was observed that intrinsic affinities are more favorable than the observable because a favorable T[delta][delta]S⁰i̳n̳t̳ dominates an unfavorable [delta][delta]Hºi̳n̳t̳. Also, we discovered that the intrinsic entropies of inserting an internal olefin are nearly double that of removing a methylene group, suggesting that the insertion of an internal olefin results in the restriction of more C-C rotors. We have shown through ITC analysis that the added substituents probed in this study provided binding increases to our Grb2 SH2 ligands as expected, but that the thermodynamic driving force of binding affinities depended greatly upon the specific nature and flexible mobility of the ligands in the binding pocket. Through a combination of X-ray and ITC studies it was shown that ligands containing rigid and aromatic functional groups bound with a higher [delta]H° than the more flexible alkyl ligands, and that this effect correlates well with more direct vdW contacts made in the pocket. Finally, we described a case study where a strict adherence to the Topliss operational schemes led to an expedient development of novel MUP-I binding analogs. The validity of the schemes was also depicted through the synthesis and testing of ligands that were correctly predicted to be weaker/equipotent to the starting ligand. Of important note is that the degree to which the schemes led to affinity boost depended greatly on the starting potency of the initial compound. / text

A series of phosphotripeptide ligands for the Grb2 SH2 domain was designed and synthesized, each of which derived from the minimal consensus sequence required for binding: Ac-pYXN. The binding affinity and related thermodynamic parameters were determined by isothermal titration calorimetry. Both the size and connectivity of the side-chain was varied. The consequences of incorporating α,α-disubstitution at the pY+1 residue on binding thermodynamics were evaluated, as were the effects of constraining the side-chains in a ring. The series was evaluated from a number of perspectives: (1) increasing size of the pY+1 residue by utilizing various amino acid types: monoalkyl, dialkyl, or cycloalkyl; (2) comparisons between ligands with the same number of carbons (scission control); and (3) by comparing ligands incorporating cyclic pY+1 residues with those incorporating α,α-dialkyl residues with one fewer methylene group (excision control). Inconsistencies in the thermodynamic consequence of constraining the backbone were observed within this set of ligands, which reveal the limitations of our understanding of protein-ligand interactions. Aspects of both the classical and non-classical hydrophobic effect were observed, but the occurance of one over the other could not be explained. / text

Conformationally constrained ligands and their flexible analogues were prepared as inhibitors of the Grb2 SH2 domain in order to study the structural and energetic effects of ligand preorganization in protein-ligand interactions. The compounds were prepared by using trans-cyclopropane-containing amino acid mimics, macrocyclization, or [alpha,alpha]-disubstituted amino acid residues. All trans-cyclopropane containing peptides were more potent than their corresponding succinate containing analogues due to an enthalpic advantage. Surprisingly, the binding of constrained peptides to the domain was entropically disfavored relative to their flexible controls. Effects of proton transfer and desolvation as being the source of the unprecedented entropic penalty for the constrained ligands relative to their respective controls were precluded, and X-ray crystallographic studies revealed that the binding conformations for the respective cyclopropane and succinate containing ligands were similar. This led us to believe that differential changes in protein dynamics may occur upon binding of the constrained and flexible ligands, which could contribute to the observed binding energetics. Two 23-membered macrocyclic ligands were slightly more potent than their corresponding linear controls. The amino acids used to link the N- and C-termini of the linear peptides to form the macrocycles were found to affect the energetics of binding. In one case, the 23-membered macrocycle was more potent than its control due to an entropic advantage, whereas the other 23-membered macrocycle was more potent than its control because it benefited from an enthalpic advantage. [alpha,alpha]-Disubstituted and [alpha]-monosubstituted residues that varied in hydrophobic character were incorporated into Grb2 SH2 domain binding tripeptides, and binding became more favorable as nonpolar surface area increased only for the set of tripeptides possessing cyclic [alpha,alpha]-disubstituted residues. The increase in affinity was due to an increasing enthalplic term, whereas the entropy of binding became less favorable. A total synthesis of (±)-lycopladine A was achieved in five steps from known compounds. The tricyclic core of the natural product was prepared utilizing a novel two-step sequence comprising a conjugate addition of a metalated picoline derivative followed by an intramolecular enolate arylation. It was demonstrated that the natural product existed in a solvent dependent equilibrium with its isomeric lactol. / text

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