Chemistry of Bifunctional Photoprobes. 1. Perfluoroaryl Azido Functionalized Phosphorus Hydrazides as Novel Photoreactive Heterobifunctional Chelating Agents: High Efficiency Nitrene Insertion on Model Solvents and Proteins.

Synthesis and evaluation of a new class of photochemically activated heterobifunctional chelating agents for protein modification is described. Selective functionalization of perfluoroaryl azides by versatile phosphorus hydrazide ligating systems 2 and 3 for the complexation of transition metals and analogous radiometals form the basis for these new agents. The utility of the photogenerated precursors from these bifunctional agents to form covalent attachments is demonstrated through examination of C-H bond insertion on cyclohexane. Representative amide-coupled phosphorus hydrazides 5 and 6 provide >78% insertion of the probe into unactivated C-H bonds of cyclohexane with short photolysis times. Photoconjugation of the photoactivable heterobifunctional chelating agent 6 and its Pd metalated analog 7 with HSA is also evaluated. The uncomplexed chelate appears to add to HSA with high efficiency, consistent with the observed 82% bond insertion into model solvents. Covalent attachment of 7, evaluated through the use of (109)Pd, was estimated to be between 49% and 74% with the uncertainty arising because of prephotolysis association of the (109)Pd complex with HSA. The application of in situ (19)F NMR to distinguish between bond insertion and noninsertion processes is demonstrated. These results suggest that functionalized perfluoroaryl azido phosphorus hydrazides may find utility as heterobifunctional photolabeling agents for attaching radionuclides to proteins and antibodies.

Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum.

Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel ß-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved γ-core motif (GXCX(3-9)C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of ß2 and ß3 strands and the interposed loop. The γ-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their γ-core motifs. The MsDef1-γ4 variant in which the γ-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the γ-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic γ-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-γ4, MtDef4 and peptides derived from the γ-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the γ-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides.

Chemistry of bifunctional photoprobes. 6. Synthesis and characterization of high specific activity metalated photochemical probes: development of novel rhenium photoconjugates of human serum albumin and fab fragments.

Functionalization of perfluoro aryl azides by bifunctional chelating agents (BFCAs) capable of forming high specific activity complexes with (99m)Tc (for gamma-imaging) and (188)Re (for radiotherapy) is described. The synthesis of multidonor BFCAs containing N(2)S(2), N(4), and N(3)S donor groups containing imidazole, pyridine, and pyrazine functionalities that may be important for tuning the pharmacokinetic parameters is also described. Functionalization of perfluoro aryl azides at various sites on BFCAs yields novel bifunctional photolabile chelating agents (BFPCAs) that are useful for covalent attachment to biomolecules. A representative Re-BFPCA 8a in a model solvent, diethylamine, proceeded to give a high yield of intermolecular NH insertion product without the decomplexation of the metal ion from 8a. All products originated from the photolysis of 8a in diethylamine are characterized by analytical techniques, and a plausible mechanism of formation of different photolytic products is suggested. The high yield of intermolecular NH insertion of Re-BFPCA 8a is extended to labeling of human serum albumin (HSA) and Fab fragments under aqueous conditions. The photolabeling technology developed here offers a new way to attach diagnostically and therapeutically useful radiotracers (e.g., (99m)Tc, (188)Re) to Fab fragments for potential noninvasive imaging and therapy of cancer.

Synthesis and characterization of novel 99gTc(V) and Re(V) complexes with water-soluble tetraaza diamido dipyridino ligands: single-crystal X-ray structural investigations of mono- and dinuclear complexes.

Rhenium and technetium are known for their useful applications in nuclear medicine with similar properties. In this study, new diamido dipyridino (N(4)) water-soluble ligands (2-C(5)H(4)NCH(2)NHCO)(2)CH(2), 1 (L(1)H2), (2-C(5)H(4)NNHNHCO)(2)CH(2), 2, and [2-C(5)H(4)N(+)(O)(-)CH(2)NHCO](2)CH(2), 3, were synthesized. Reaction of L(1)H2 with ReOCl(3)(PPh(3))(2) resulted in the novel six-coordinated rhenium(V) complex, trans-ReO(L(1))(OEt), 4. The complex was characterized by spectroscopic methods, and its X-ray crystallographic analysis revealed that rhenium is coordinated to four nitrogen atoms of the ligand and to two oxygen atoms from the deprotonated ethanol and the oxo group respectively in a distorted octahedral geometry. In solution, complex 4 was transformed to a new complex 5, which was proved to be the dinuclear complex mu-oxo [ReO(L(1))](2)O. Reaction of 1 with [n-Bu(4)N][ReOCl(4)] resulted in the neutral complex 6, trans-[ReO(L(1))]Cl. Similarly, when ligand 1 was reacted with [n-Bu(4)N][(99g)TcOCl(4)], the neutral trans-[(99)TcO(L(1))]Cl complex 7 was formed, which upon dissolution transformed into a cationic complex 8, trans-[(99)TcO(L(1))(OH(2))](+)Cl(-). The single-crystal X-ray structure of 8 reveals that the coordination sphere about technetium is a distorted octahedron with four nitrogen atoms in the equitorial plane, while doubly bonded oxygen and coordinated water occupy the apical positions. Further dissolution of 8 resulted in the formation of dinuclear mu-oxo [TcO(L(1))](2)O, 9. This study shows that Tc and Re have similar metal core structures in solution for diamido dipyridino systems, besides similarity in geometrical structure, proved by the X-ray structures on the same ligands.