Modulation of fragmental charge transfer via hydrogen bonds. Direct measurement of electronic contributions.

Hydrogen bonds play an important role in an overwhelming variety of fields from biology to surface and supramolecular chemistry. The term "hydrogen bond" refers to a wide range of interactions with various covalent and polar contributions. In particular, hydrogen bonds have an important role in the folding and packing of peptides and nucleic acids. Recent studies also point to the importance of hydrogen bonding in the context of second-shell interactions, in metal binding and selectivity in metalloproteins, and in controlling the dynamics of membrane proteins. In this study, we demonstrate and quantify the modulation of fragmental charge transfer from hydrogen-bonded ligands to a metal center, by employing our recently introduced molecular potentiometer. The molecular details that affect this type of fragmental charge transfer are presented and a path for transferring chemical information is demonstrated. We found that H-bond interactions in the extended positions of axial ligands provide an effective means of modulating the amount of fragmental charge transfer to a metal center, thereby dramatically influencing the electronic properties of the ligand, the binding affinity, and the binding of additional ligands. The magnitude of fragmental charge-transfer modulation induced by a single ligand-solvent H-bond interaction is comparable to those induced by covalent substitution, although H-bond enthalpy is only on the order of several kilojoules per mole. Importantly, we find a significant change in the ligand electronic properties, even for weak C-H...O=C H-bond formation, where the bond enthalpy is substantially lower than for conventional H-bond interactions. The excess fragmental charge transferred to the metal center, deduced from the spectroscopic measurements, correlates well with the computationally determined values. Our findings underscore the importance of second-shell interactions in the active sites of enzymes, beyond the structural and electrostatic importance that is widely recognized today.

Novel water-soluble bacteriochlorophyll derivatives for vascular-targeted photodynamic therapy: synthesis, solubility, phototoxicity and the effect of serum proteins.

New negatively charged water-soluble bacteriochlorophyll (Bchl) derivatives were developed in our laboratory for vascular-targeted photodynamic therapy (VTP). Here we focused on the synthesis, characterization and interaction of the new candidates with serum proteins and particularly on the effect of serum albumin on the photocytotoxicity of WST11, a representative compound of the new derivatives. Using several approaches, we found that aminolysis of the isocyclic ring with negatively charged residues markedly increases the hydrophilicity of the Bchl sensitizers, decreases their self-association constant and selectively increases their affinity to serum albumin, compared with other serum proteins. The photocytotoxicity of the new candidates in endothelial cell culture largely depends on the concentration of the serum albumin. Importantly, after incubation with physiological concentrations of serum albumin (500-600 microM), WST11 was found to be poorly photocytotoxic (>80% endothelial cell survival in cell cultures). However, in a recent publication (Mazor, O. et al. [2005] Photochem. Photobiol. 81, 342-351) we showed that VTP of M2R melanoma xenografts with a similar WST11 concentration resulted in approximately 100% tumor flattening and >70% cure rate. We therefore propose that the two studies collectively suggest that the antitumor activity of WST11 and probably of other similar candidates does not depend on direct photointoxication of individual endothelial cells but on the vascular tissue response to the VTP insult.

WST11, a novel water-soluble bacteriochlorophyll derivative; cellular uptake, pharmacokinetics, biodistribution and vascular-targeted photodynamic activity using melanoma tumors as a model.

WST11 is a novel negatively charged water-soluble palladium-bacteriochlorophyll derivative that was developed for vascular-targeted photodynamic therapy (VTP) in our laboratory. The in vitro results suggest that WST11 cellular uptake, clearance and phototoxicity are mediated by serum albumin trafficking. In vivo, WST11 was found to clear rapidly from the circulation (t1/2=1.65 min) after intravenous bolus injection in the mouse, whereas a longer clearance time (t1/2=7.5 min) was noted in rats after 20 min of infusion. The biodistribution of WST11 in mouse tissues indicates hepatic clearance (t1/2=20 min), with minor (kidney, lung and spleen) or no intermediary accumulation in other tissues. As soon as 1 h after injection, WST11 had nearly cleared from the body of the mouse, except for a temporal accumulation in the lungs from which it cleared within 40 min. On the basis of these results, we set the VTP protocol for a short illumination period (5 min), delivered immediately after WST11 injection. On subjecting M2R melanoma xenografts to WST11-VTP, we achieved 100% tumor flattening at all doses and a 70% cure with 9 mg/kg and a light exposure dose of 100 mW/cm2. These results provide direct evidence that WST11 is an effective agent for VTP and provide guidelines for further development of new candidates.

Control of redox transitions and oxygen species binding in Mn centers by biologically significant ligands; model studies with [Mn]-bacteriochlorophyll a.

Mn-superoxide dismutase (Mn-SOD), which protects the cell from the toxic potential of superoxide radicals (O(2)(-*)), is the only type of SOD which resides in eukaryotic mitochondria. Up-to-date, the exact catalytic mechanism of the enzyme and the relationship between substrate moieties and the ligands within the active site microenvironment are still not resolved. Here, we set out to explore the possible involvement of hydroperoxyl radicals ((*)OOH) in the catalytic dismutaion by following the interplay of Mn(III)/Mn(II) redox transitions, ligands binding, and evolution or consumption of superoxide radical, using a new model system. The model system encompassed an Mn atom chelated by a bacteriochlorophyll allomer macrocycle (BChl) in aerated aprotic media that contain residual water. The redox states of the Mn ion were monitored by the Q(y) electronic transitions at 774 and 825 nm for [Mn(II)]- and [Mn(III)]-BChl, respectively (Geskes, C.; Hartwich, G.; Scheer, H.; Mantele, W.; Heinze, J. J. Am. Chem. Soc. 1995, 117, 7776) and confirmed by electron spin resonance spectroscopy. Evolution of (*)OOH radicals was monitored by the ESR spin-trap technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The experimental data suggest that the [Mn]-BChl forms a (HO(-))[Mn(III)]-BChl(OOH) complex upon solvation. Spectrophotometeric titrations with tetrabutylamonnium acetate (TBAA) and 1-methylimidazole (1-MeIm) together with ESI-MS measurements indicated the formation of a 1:1 complex with [Mn]-BChl for both ligands. The coordination of ligands at low concentrations to [Mn(III)]-BChl induced a release of a (*)OOH radical and a [Mn(III)]-BChl --> [Mn(II)]-BChl transition at higher concentrations. The estimated equilibrium constants for the total redox reaction ( )()are 1.9 x 10(4) +/- 1 x 10(3) M(-)(1) and 12.3 +/- 0.6 M(-)(1) for TBAA and 1-MeIm, respectively. The profound difference between the equilibrium constants agrees with the suggested key role of the ligand's basicity in the process. A direct interaction of superoxide radicals with [Mn(III)]-BChl in a KO(2) acetonitrile (AN) solution also resulted in [Mn(III)]-BChl --> [Mn(II)]-BChl transition. Cumulatively, our data show that the Mn(III) center encourages the protonation of the O(2)(-)(*) radical in an aprotic environment containing residual water molecules, while promoting its oxidation in the presence of basic ligands. Similar coordination and stabilization of the (*)OOH radical by the Mn center may be key steps in the enzymatic dismutation of superoxide radicals by Mn-SOD.

Local photodynamic therapy (PDT) of rat C6 glioma xenografts with Pd-bacteriopheophorbide leads to decreased metastases and increase of animal cure compared with surgery.

Photodynamic therapy (PDT), locally applied to solid C6 rat glioma tumors in the foot of CD1 nude mice, eradicated the primary tumor and also decreased the rate of groin and lung metastases. Pd-Bacteriopheophorbide (Pd-Bpheid), a novel photosensitizer synthesized in our laboratory, was used in our study. The primary lesion in the hind leg was treated by an i.v. injection of 5 mg/kg of Pd-Bpheid and immediate illumination (650-800 nm, 360 J/cm(2)). This protocol and the surgical amputation of the leg were compared for local and metastasis responses. Following PDT, hemorrhage, inflammation with tumor necrosis and flattening were observed and histologically verified in the photodynamically treated tumor. Whereas local tumor control rates were up to 64% following PDT, in surgically treated animals, local tumor control was absolute. The rates of metastases in the groin and the lungs were at least 12-fold lower in the photodynamically treated animals compared with untreated or surgery-treated groups. The overall cure rates after PDT or surgery were 36% and 6%, respectively, at 8 weeks. These findings suggest that local PDT with Pd-Bpheid, which acts primarily on the tumor vasculature, efficiently eradicates the solid C6 tumors. In addition, the local PDT of the primary lesion has beneficial therapeutic effects on remote C6 metastasis, which is not obtained with surgery. It is therefore suggested, that although surgery is highly efficient for the immediate removal of the primary tumor, it lacks such systemic, therapeutic effects on distant metastases. Pd-Bpheid-PDT may thus offer a potentially superior curative therapy for C6 glioma tumors in the limb by eradicating the target tumor and by reducing the rate of metastasis in the groin and lung, possibly due to innate immunity.