Takehiro Ohta

Axial Ligand Effects on the Geometric and Electronic Structures of Nonheme Oxoiron(IV) Complexes

A series of complexes [Fe(IV)(O)(TMC)(X)](+) (where X = OH(-), CF3CO2(-), N3(-), NCS(-), NCO(-), and CN(-)) were obtained by treatment of the well-characterized nonheme oxoiron(IV) complex [Fe(IV)(O)(TMC)(NCMe)](2+) (TMC = tetramethylcyclam) with the appropriate NR4X salts. Because of the topology of the TMC macrocycle, the [Fe(IV)(O)(TMC)(X)](+) series represents an extensive collection of S = 1 oxoiron(IV) complexes that only differ with respect to the ligand trans to the oxo unit. Electronic absorption, Fe K-edge X-ray absorption, resonance Raman, and Mossbauer data collected for these complexes conclusively demonstrate that the characteristic spectroscopic features of the S = 1 Fe(IV)=O unit, namely, (i) the near-IR absorption properties, (ii) X-ray absorption pre-edge intensities, and (iii) quadrupole splitting parameters, are strongly dependent on the identity of the trans ligand. However, on the basis of extended X-ray absorption fine structure data, most [Fe(IV)(O)(TMC)(X)](+) species have Fe=O bond lengths similar to that of [Fe(IV)(O)(TMC)(NCMe)](2+) (1.66 +/- 0.02 A). The mechanisms by which the trans ligands perturb the Fe(IV)=O unit were probed using density functional theory (DFT) computations, yielding geometric and electronic structures in good agreement with our experimental data. These calculations revealed that the trans ligands modulate the energies of the Fe=O sigma- and pi-antibonding molecular orbitals, causing the observed spectroscopic changes. Time-dependent DFT methods were used to aid in the assignment of the intense near-UV absorption bands found for the oxoiron(IV) complexes with trans N3(-), NCS(-), and NCO(-) ligands as X(-)-to-Fe(IV)=O charge-transfer transitions, thereby rationalizing the resonance enhancement of the nu(Fe=O) mode upon excitation of these chromophores.

A functional nitric oxide reductase model

A functional heme/nonheme nitric oxide reductase (NOR) model is presented. The fully reduced diiron compound reacts with two equivalents of NO leading to the formation of one equivalent of N2O and the bis-ferric product. NO binds to both heme Fe and nonheme Fe complexes forming individual ferrous nitrosyl species. The mixed-valence species with an oxidized heme and a reduced nonheme FeB does not show NO reduction activity. These results are consistent with a so-called “trans” mechanism for the reduction of NO by bacterial NOR.

Intermediates Involved in the Two Electron Reduction of NO to N2O by a Functional Synthetic Model of Heme Containing Bacterial NO Reductase

Reaction of a functional biferrous heme/nonheme model complex at low temperature leads to the formation of a distal nonheme nitrosyl followed by a trans heme nonheme bis-nitrosyl intermediate. The EPR and Raman data on this intermediate indicate that the two nitrosyl centers are close. This complex gives off N2O and provides support for the trans mechanism proposed for NOR enzymes.

Formation of an end-on ferric peroxo intermediate upon one-electron reduction of a ferric superoxo heme.

The low-spin end-on ferric peroxo heme intermediate has been proposed as an alternative reactive intermediate involved in the catalytic cycles of enzymes such as nitric oxide synthase and cytochrome P450. This transient heme intermediate has never been captured using synthetic heme models. We demonstrate herein our success in the solution preparation of such an end-on ferric peroxo intermediate derived from a heme model, which features both a group hanging over the porphyrin macrocycle and a covalently appended axial imidazole ligand, through one-electron reduction of its ferric superoxo precursor. The obtained ferric peroxo intermediate was further transformed into the corresponding ferric hydroperoxo species upon protonation. This heme model compound provides a convenient system for sequential preparation of the important and biologically relevant superoxo/peroxo/hydroperoxo heme intermediates through an oxygenation/one-electron reduction/protonation process similar to the mechanisms used by enzyme systems.

Oxoferryl porphyrin/hydrogen peroxide system whose behavior is equivalent to hydroperoxoferric porphyrin.

The reaction between H(2)O(2) and a pyridine-coordinated ferric porphyrin encapsulated by a cyclodextrin dimer yielded a hydroperoxoferric porphyrin intermediate, PFe(III)-OOH, which rapidly decomposed to oxoferryl porphyrin (PFe(IV)═O). Upon reaction with H(2)O(2), PFe(IV)═O reverted to PFe(III)-OOH, which was converted to carbon monoxide-coordinated ferrous porphyrin under a CO atmosphere. PFe(IV)═O in the presence of excess H(2)O(2) behaves as PFe(III)-OOH.

Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis

Aldoxime dehydratase (OxdA), which is a unique heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. Unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron. Here, we determined the crystal structure of OxdA. We then constructed OxdA mutants in which each of the polar amino acids lying within ∼6 Å of the iron atom of the heme was converted to alanine. Among the purified mutant OxdAs, S219A had completely lost and R178A exhibited a reduction in the activity. Together with this finding, the crystal structural analysis of OxdA and spectroscopic and electrostatic potential analyses of the wild-type and mutant OxdAs suggest that S219 plays a key role in the catalysis, forming a hydrogen bond with the substrate. Based on the spatial arrangement of the OxdA active site and the results of a series of mutagenesis experiments, we propose the detailed catalytic mechanism of general aldoxime dehydratases: (i) S219 stabilizes the hydroxy group of the substrate to increase its basicity; (ii) H320 acts as an acid-base catalyst; and (iii) R178 stabilizes the heme, and would donate a proton to and accept one from H320.

Characterization of Mononuclear Non-heme Iron(III)-Superoxo Complex with a Five-Azole Ligand Set†

Reaction of O2 with a high-spin mononuclear iron(II) complex supported by a five-azole donor set yields the corresponding mononuclear non-heme iron(III)-superoxo species, which was characterized by UV/Vis spectroscopy and resonance Raman spectroscopy. (1)H NMR analysis reveals diamagnetic nature of the superoxo complex arising from antiferromagnetic coupling between the spins on the low-spin iron(III) and superoxide. This superoxo species reacts with H-atom donating reagents to give a low-spin iron(III)-hydroperoxo species showing characteristic UV/Vis, resonance Raman, and EPR spectra.

Long-term Vaccination with Multiple Peptides Derived from Cancer-Testis Antigens Can Maintain a Specific T-cell Response and Achieve Disease Stability in Advanced Biliary Tract Cancer

Purpose: The prognosis of patients with advanced biliary tract cancer (BTC) is extremely poor and there are only a few standard treatments. We conducted a phase I trial to investigate the safety, immune response, and antitumor effect of vaccination with four peptides derived from cancer-testis antigens, with a focus on their fluctuations during long-term vaccination until the disease had progressed. Experimental Design: Nine patients with advanced BTC who had unresectable tumors and were refractory to standard chemotherapy were enrolled. HLA-A*2402–restricted epitope peptides, lymphocyte antigen 6 complex locus K, TTK protein kinase, insulin-like growth factor-II mRNA-binding protein 3, and DEP domain containing 1 were vaccinated subcutaneously once a week at doses of 0.5, 1, or 2 mg and continued until disease progression. The adverse events were assessed by Common Terminology Criteria for Adverse Events and the immune response was monitored by an enzyme-linked immunospot assay or by flow cytometry. The clinical effects observed were tumor response, progression-free survival (PFS), and overall survival (OS). Results: Four-peptide vaccination was well tolerated. No grade 3 or 4 adverse events were observed. Peptide-specific T-cell immune responses were observed in seven of nine patients and clinical responses were observed in six of nine patients. The median PFS and OS were 156 and 380 days. The injection site reaction and CTL induction seemed to be prognostic factors of both PFS and OS. Conclusions: Four-peptide vaccination was well tolerated and seemed to provide some clinical benefit to some patients. These immunologic and clinical responses were maintained over the long term through continuous vaccinations. Clin Cancer Res; 19(8); 2224–31. ©2013 AACR.

A vinylogue of bis-fused tetrathiafulvalene: novel π-electron framework for two-dimensional organic metals

Several derivatives of 2-(1,3-dithiol-2-ylidene)-5-(2-ethanediylidene-1,3-dithiole)-1,3,4,6-tetrathiapentalene (1Aa), DTEDT, which is a vinylogue of bis-fused TTF (BDT–TTP) containing one 2,2′-ethanediylidenebis(1,3-dithiole)(2), have been synthesized. The cyclic voltammograms of the DTEDTs consist of four pairs of single-electron redox waves. Comparison of the first oxidation potentials (E1) reveals that donating ability of 1Aa is rather weaker than those of 2 and TTF, in spite of the extension of the apparent π-conjugation. Substituent effects on the E1 values suggest that the first oneelectron oxidation occurs mainly in the vinylogous TTF moiety. The smaller E2–E1 value of 1Aa compared with those of BDT–TTP, TTF and 2 suggests a decrease in the on-site coulombic repulsion in the dication. An X-ray structure analysis of the neutral 4,5-dimethyl-4′,5′-bis(methylthio) derivatives (1Be) reveals that the crystal has a so-called ‘κ-type array’ of molecules. An MNDO molecular orbital calculation indicates that the sulfur atoms at the termini of the vinylogous 1,3-dithiole rings are out of phase with the other sulfur atoms in the HOMO. The present donors have produced many molecular complexes exhibiting high conductivity. In particular, all of the radical-cation salts based on 1Aa obtained so far exhibit metallic conducting behaviour down to 1.4–4.2 K, regardless of the counter-anions.

Heme environment in aldoxime dehydratase involved in carbon–nitrogen triple bond synthesis

Resonance Raman spectra have been measured to characterize the heme environment in aldoxime dehydratase (OxdA), a novel hemoprotein, which catalyzes the dehydration of aldoxime into nitrile. The spectra showed that the ferric heme in the enzyme is six‐coordinate low spin, whereas the ferrous heme is five‐coordinate high spin. We assign a prominent vibration that occurs at 226 cm−1 in the ferrous enzyme to the Fe‐proximal histidine stretching vibration. In the CO‐bound form of OxdA, the correlation between the Fe–CO stretching (512 cm−1) and C–O stretching (1950 cm−1) frequencies also supports our assignment of proximal histidine coordination.