Ruthenium(II) Complexes of 2-Aryl-1,10-phenanthrolines: Synthesis, Structure, and Photophysical Properties.

The synthesis, characterization, and photochemical investigation of a series of Ru(II) complexes having 2-phenyl- and 2,9-diphenyl substituted phenanthroline ligands are reported. Structural characterization of some of the complexes revealed that the phenyl substituents of the phenanthroline ligand are oriented nearly perpendicular to the phenanthroline ring and pi-stack with adjacent coordinated 2,2'-bipyridyl ligands. Most of the complexes are nonluminescent at room temperature, and temperature-dependent luminescence studies suggest nonradiative relaxation in solution is dominated by rapid thermally activated internal conversion from the initially populated (3)MLCT state to a ligand field (LF) state which decays rapidly to the ground state. The photochemical lability of the complexes was investigated, and it was found that, while the complexes efficiently populate the substitutionally labile LF state, yields for ligand loss are less than expected on the basis of comparison to closely related complexes lacking the phenyl groups which are capable of pi-stacking interactions.

Molecular targeting of inhibitor of apoptosis proteins based on small molecule mimics of natural binding partners.

An assay based on a solvent-sensitive fluorogenic dye molecule, badan, is used to test the binding affinity of a library of tetrapeptide molecules for the BIR3 (baculovirus IAP repeat) domain of XIAP (X-linked inhibitor of apoptosis protein). The fluorophore is attached to a tetrapeptide, Ala-Val-Pro-Cys-NH(2), through a thiol linkage and, upon binding to XIAP, undergoes a solvatochromic shift in fluorescence emission. When a molecule (e.g., a natural protein known to bind to XIAP or a tetrapeptide mimic) displaces the dye, the emission shifts back to the spectrum observed in water. As emission intensity is related to the binding of the tetrapeptide, the intensity can be used to determine the equilibrium constant, K, for the displacement of the dye by the tetrapeptide. The results permit residue-specific analysis of the interaction. Furthermore, we show that hydrophobic effects in the fourth position are general and can effectively increase overall affinity.

A high-throughput screen for identification of molecular mimics of Smac/DIABLO utilizing a fluorescence polarization assay.

Resistance to apoptosis is afforded by inhibitor of apoptosis proteins (IAPs) which bind to and inhibit the caspases responsible for cleavage of substrates leading to apoptotic cell death. Smac (or DIABLO), a proapoptotic protein released from the mitochondrial intermembrane space into the cytosol, promotes apoptosis by binding to IAPs, thus reversing their inhibitory effects on caspases. We have developed a high-throughput fluorescence polarization assay utilizing a fluorescein-labeled peptide similar to the "IAP binding" domain of Smac N terminus complexed with the BIR3 domain of X-linked IAP (XIAP) to identify small-molecule mimics of the action of Smac. The IC(50)s of peptides and a tetrapeptidomimetic homologous to the N terminus of Smac demonstrated the specificity and utility of this assay. We have screened the National Cancer Institute "Training Set" of 230 compounds, with well-defined biological actions, and the "Diversity Set" of 2000 chemically diverse structures for compounds which significantly reduced fluorescence polarization. Highly fluorescing or fluorescence-quenching compounds (false positives) were distinguished from those which interfered with Smac peptide binding to the XIAP-BIR3 in a dose-dependent manner (true positives). This robust assay offers potential for high-throughput screening discovery of novel compounds simulating the action of Smac/DIABLO.