Succinate- and cyclopropane-derived phosphotyrosine (pY) replacements were incorporated into a series of Grb2 SH2 binding ligands wherein the pY+1 residue was varied to determine explicitly how variations in ligand preorganization affect binding energetics and structure. The complexes of these ligands with the Grb2 SH2 domain were examined in a series of thermodynamic and structural investigations using isothermal titration calorimetry and X-ray crystallography. The binding enthalpies for all ligands were favorable, and although binding entropies for all ligands having a hydrophobic residue at the pY+1 site were favorable, binding entropies for those having a hydrophilic residue at this site were unfavorable. Preorganized ligands generally bound with more favorable Gibbs energies than their flexible controls, but this increased affinity was the consequence of relatively more favorable binding enthalpies. Unexpectedly, binding entropies of the constrained ligands were uniformly disfavored relative to their flexible controls, demonstrating that the widely held belief that ligand preorganization should result in an entropic advantage is not necessarily true. Crystallographic studies of complexes of several flexible and constrained ligands having the same amino acid at the pY+1 position revealed extensive similarities, but there were some notable differences. There are a greater number of direct polar contacts in complexes of the constrained ligands that correlate qualitatively with their more favorable binding enthalpies and Gibbs energies. There are more single water-mediated contacts between the domain and the flexible ligand of each pair; although fixing water molecules at a protein-ligand interface is commonly viewed as entropically unfavorable, entropies for forming these complexes are favored relative to those of their constrained counterparts. Crystallographic b-factors in the complexes of constrained ligands are greater than those of their flexible counterparts, an observation that seems inconsistent with our finding that entropies for forming complexes of flexible ligands are relatively more favorable. This systematic study highlights the profound challenges and complexities associated with predicting how structural changes in a ligand will affect enthalpies, entropies, and structure in protein-ligand interactions.

A concise second-generation total synthesis of the fungal-derived alkaloid (+)-gliocladin C (11), in ten steps and 11% overall yield from isatin, is reported. In addition, the ETP natural product (+)-gliocladine C (6) is prepared in six steps and 29% yield from the di-(tert-butoxycarbonyl) precursor of 11. The total synthesis of (+)-gliocladine C (6) constitutes the first total synthesis of an ETP natural product containing a hydroxyl substituent adjacent to a quaternary carbon stereocenter in the pyrrolidine ring.

A prevailing hypothesis in the field of molecular recognition in chemistry and biology is that the preorganization of flexible hosts and their guests in a manner corresponding to the three-

The thermodynamic and structural effects of macrocyclization as a tactic for stabilizing the biologically-active conformation of Grb2 SH2 binding peptides were investigated using isothermal titration calorimetry and x-ray crystallography. 23-Membered macrocycles containing the sequence pYVN were slightly more potent than their linear controls; however, preorganization did not necessarily eventuate in a more favorable binding entropy. Structures of complexes of macrocycle 7 and its acyclic control 8 are similar except for differences in relative orientations of corresponding atoms in the linking moieties of 7 and 8. There are no differences in the number of direct or water-mediated protein-ligand contacts that might account for the less favorable binding enthalpy of 7; however, an intramolecular hydrogen bond between the pY and pY+3 residues in 8 that is absent in 7 may be a factor. These studies highlight the difficulties associated with correlating energetics and structure in protein-ligand interactions.

A common strategy for preparing tryptophan-derived epidithiodioxopiperazine (ETP) natural product containing a hydroxyl substituent adjacent to a quaternary carbon stereocenter is reported. This strategy is exemplified by enantioselective total syntheses of four heptacyclic ETP natural products — gliocladine C (6), leptosin D (7), T988C (8), and bionectin A (9)—starting with the di-(tertbutoxycarbonyl) derivative 17 of the trioxopiperazine natural product gliocladin C, which is readily available by enantioselective chemical synthesis. In addition, total syntheses of the enantiomer of gliocladine C (ent-6) and gliocladin A (11), the di(methylthio) congener of bionectin A, are reported. These syntheses illustrate a synthetic strategy wherein diversity in the dioxopiperazine unit of ETP natural products is introduced at a late stage in a synthetic sequence. In vitro cytotoxicity of compounds in this series against invasive human prostrate (DU145) and melanoma (A2058) cancer cell lines is described and compared to that of chaetocin A (4).