Hiromasa Kurosaki

An ultrasensitive and highly selective determination method for quinones by high-performance liquid chromatography with photochemically initiated luminol chemiluminescence

Quinones are a class of compounds of substantial toxicological and pharmacological interest. An ultrasensitive and highly selective chemiluminescence (CL) method was newly developed for the determination of quinones based on the utility of photochemically initiated luminol CL. The method involved ultraviolet (UV) irradiation of quinones to generate reactive oxygen species (ROS) through the unique photosensitization reaction accompanied with the photolytical generation of 3,6-dihydroxyphthalic acid (DHPA) from quinones. The photoproducts were detected by luminol CL reaction. Interestingly, it was noticed that DHPA had enhancement effect for the luminol CL. The generation of the enhancer (DHPA) in association with the oxidant (ROS) in the photochemical reaction greatly increases the sensitivity and selectivity of the proposed luminol CL method. In order to elucidate the type of ROS produced by the photosensitizaion reaction in relation to the proposed CL reaction, we investigated the quenching effect of selective ROS scavengers in the luminol CL. Although several ROS were generated, superoxide anion was the most effective ROS for the generated CL. Moreover, the enhancement mechanism of DHPA for luminol CL was confirmed. The enhancement was found to be through the formation of stabilized semiquinone anion radical that provided long-lived CL. The generation of the semiquinone radical was confirmed by electron spin resonance technique. Furthermore, we developed an HPLC method with on-line photochemical reaction followed by the proposed CL detection for the determination of four quinones. A luminol analogue, L-012, was used for its high sensitivity. The detection limits for quinones obtained with the proposed method (S/N=3) were in the range 1.5-24 fmol that were 10-1000 times more sensitive compared with the previous methods. Finally, the developed HPLC-CL system was successfully applied for the determination of quinones in airborne particulate samples collected at Nagasaki city.

Synthesis and characterization of a 1,8-difunctionalized dissymmetrical cyclam copper(II) complex bearing pyridylmethyl and N,N-dimethylcarbamoylmethyl groups

The new dissymmetrical ligand 1-(N,N-dimethylcarbamoylmethyl)-8-(2-pyridylmethyl)-4,11-dimethyl-1,4,8,11-tetraazacyclotetradecane, 5, was synthesized and the physicochemical properties of its copper(II) complex, [CuII(5)](ClO4)2·CH3CN, were determined. UV–Vis, IR, ESR experiments, and X-ray analysis of [CuII(5)](ClO4)2·CH3CN were carried out. The X-ray structure of [CuII(5)](ClO4)2·CH3CN revealed a distorted square pyramid with the four coordinating nitrogen atoms of the macrocycle lying in the basal position. Curiously, the fifth ligand is the N,N-dimethylcarbamoylmethyl group through the oxygen atom and not the pyridylmethyl moiety.

Structural Insights into the Subclass B3 Metallo-β-Lactamase, SMB-1, and the Mode of Inhibition by the Common Metallo-β-Lactamase Inhibitor, Mercaptoacetate

ABSTRACT A novel subclass B3 metallo-β-lactamase (MBL), SMB-1, recently identified from a Serratia marcescens clinical isolate, showed a higher hydrolytic activity against a wide range of β-lactams than did the other subclass B3 MBLs, i.e., BJP-1 and FEZ-1, from environmental bacteria. To identify the mechanism underlying the differences in substrate specificity among the subclass B3 MBLs, we determined the structure of SMB-1, using 1.6-Å diffraction data. Consequently, we found that SMB-1 reserves a space in the active site to accommodate β-lactam, even with a bulky R1 side chain, due to a loss of amino acid residues corresponding to F31 and L226 of BJP-1, which protrude into the active site to prevent β-lactam from binding. The protein also possesses a unique amino acid residue, Q157, which probably plays a role in recognition of β-lactams via the hydrogen bond interaction, which is missing in BJP-1 and FEZ-1, whose Km values for β-lactams are particularly high. In addition, we determined the mercaptoacetate (MCR)-complexed SMB-1 structure and revealed the mode of its inhibition by MCR: the thiolate group bridges to two zinc ions (Zn1 and Zn2). One of the carboxylate oxygen atoms of MCR makes contact with Zn2 and Ser221, and the other makes contact with T223 and a water molecule. Our results demonstrate the possibility that MCR could be a potent inhibitor for subclass B3 MBLs and that the screening technique using MCR as an inhibitor would be effective for detecting subclass B3 MBL producers.

Probing, Inhibition, and Crystallographic Characterization of Metallo‐β‐lactamase (IMP‐1) with Fluorescent Agents Containing Dansyl and Thiol Groups

The treatment of bacterial infections has advanced rapidly in recent years, primarily as the result of the development of multiple antibacterial agents. However, with the widespread use of such antibacterial agents, various pathogens have developed a resistance to antibiotics and are now a global problem as nosocomial pathogens in immunocompromised patients. One of the mechanisms of bacterial resistance is the production of enzymes that render drugs inactive. For example, b-lactamases, produced by various pathogenic bacteria, catalyze the hydrolysis of the b-lactam ring of b-lactam antibiotics. b-Lactamases can be classified into four molecular classes: A, B, C, and D. b-Lactamases in classes A, C, and D are serine enzymes with a serine residue at the active site, whereas class B b-lactamases require divalent metal ions such as Zn, and are referred to as the metallo-b-lactamases (MBLs). MBLs are able to hydrolyze nearly all b-lactam antibiotics, including carbapenems, and are also generally not susceptible to b-lactamase inhibitors such as clavulanic acid, tazobactam, and sulbactam, which are routinely used for clinical treatment. Among the most widely recognized MBLs, IMP-1 is extremely widespread. The resistance gene, blaIMP, is included in an integron structure that is present in the mobile plasmid as a cassette. Therefore, it is possible for the gene to horizontally propagate to other bacteria, and the isolation of IMP-1 has been reported in both Japan and Europe. The rampancy of pathogenic bacteria that retain IMP-1 is due to the fact that it efficiently hydrolyzes carbapenems, unlike the class A, C, and D b-lactamases. Thus, it is very important to confirm the production of IMP-1 in infectious bacteria for effective chemotherapy at the initial stages of a disease. We are currently in the process of developing reagents for IMP-1 detection based on the use of a fluorescent probe. We recently reported that thiocarboxylic acid derivatives inhibit IMP-1, reversibly or irreversibly. We have also reported on a fluorescent probe for detecting IMP-1, N-(5-(dimethylamino)1-naphthalenesulfonamidoethyl)-3-thiopropionamide, (DansylC2SH, Figure 1). [18] A compound containing a thiol group as a

Aurine tricarboxylic acid, a potent metal-chelating inhibitor of NFκB-DNA binding

The metal-interaction of aurine tricarboxylic acid (ATA) and its inhibitory effect on the DNA binding of NFkappaB were studied. Chemical speciation and spectroscopic studies have shown the strong interaction of ATA with metal ions present in the biological systems. EPR, FTIR and electronic spectral studies indicated the square planar structure of the metal-binding carboxylic and hydroxyl groups of ATA indicating the ground state 2B1g. Electrophoretic mobility shift assay using NFkappaB and 32P labeled DNA has shown that ATA was inhibitory against the DNA-NFkappaB binding at 30 microM. This activity was the strongest among the metal-chelating inhibitors of NFkappaB-DNA binding reported so far.

A model for ZnII-containing-β-lactamase: synthesis, X-ray crystal structure of a zinc(II) complex bearing thiol groupand hydrolysis of phosphate diester

A novel N3S1 typed tripodal ligand bearing an SH group, N-(mercaptoethyl)-di(2-pyridylmethyl)amine, DPASH, was prepared and its zinc(II) complex, [ZnII(DPAS)Cl], was structurally characterized by X-ray crystallography. [ZnII(DPAS)Cl] promoted hydrolysis of sodium bis(p-nitrophenyl)hydrogenphosphate), BNP-.

Structure of metallo-β-lactamase IND-7 from a Chryseobacterium indologenes clinical isolate at 1.65-Å resolution

The X-ray crystal structure of metallo-beta-lactamase from Chryseobacterium indologenes IND-7 was determined at a resolution of 1.65 A. The overall structure adopted a four-layered alphabeta/betaalpha sandwich structure with a dinuclear zinc(II) active site, in which the zinc(II) ions were denoted as Zn1 and Zn2. The overall structure of IND-7 is analogous to those of subclass B1 metallo-beta-lactamases, as determined by X-ray crystallography. A significant structural difference, however, was observed in the dinuclear zinc(II) active site: the coordination geometry around Zn1 changed from tetrahedral, found in other subclass B1 metallo-beta-lactamases, to distorted trigonal bipyramidal, whereas that of Zn2 changed from trigonal bipyramidal to tetrahedral. Arg121(101), which is located in the vicinity of the dinuclear zinc(II) active site, may affect the binding affinity of Zn2 due to an electronic repulsion between the zinc(II) ion(s) and a positively charged guanidyl group of Arg121(101). Moreover, the hydrogen-bonding interaction of Arg121 with Ser71(53), which is conserved in IND-1, IND-3 and IND-5-IND-7, appeared to have important consequences for the binding affinity of Zn2 in conjunction with the above electrostatic effect.

Detection of a metallo-β-lactamase (IMP-1) by fluorescent probes having dansyl and thiol groups

A fluorescent probe for the detection of a metallo-beta-lactamase (IMP-1), N-[2-(5-dimethylaminonaphthalen-1-ylsulfonylamino)ethyl]-3- mercaptopropionamide (Dansyl-C2SH), 1, was designed based on combining the inhibitory function of mercaptocarboxylate and a fluorophore. The binding of 1 to IMP-1 was investigated by fluorescence spectroscopy. Compound 1 can act as fluorescent probe for detecting IMP-1 selectively.

Structure and O2 uptake properties of a novel nickel(II) complex of pyridyl-pendant dioxocyclam [1-(2-pyridyl)methyl-5,7-dioxo-1,4,8,11-tetraazacyclotetradecane]

Abstract Novel potentially five-coordinate pyridyl–pendant dioxocyclam [1-(2-pyridyl)methyl-5,7-dioxo-1,4,8,11-tetraazacyclotetradecane (H2L) and its homologs (6-methyl and 6,6-dimethyl derivatives)] have been synthesized to study nickel(II) complexation. A purple nickel(II) complex with a deprotonated amide (NiHL) was isolated from aqueous equimolar solution of H2L and Ni(ClO4)2. A yellow nickel(II) complex with two deprotonated amides (NiL) was crystallized from an H2O/CH3CN solution of H2L and Ni(OH)2. The X-ray crystal study of NiL showed a square-planar nickel(II) complex with the pyridyl–pendant remaining uncoordinated. It is concluded from the visible absorption and NMR study of NiL in aqueous solution that the four-coordinate NiL is in equilibrium with a five-coordinate square-pyramidal nickel(II) complex with the apical coordination of the pyridyl–pendant. A voltammetric study disclosed a low nickel(II/III) redox potential of +0.29 V vs SCE for NiL at pH 9.5 and 25  °C with 0.10 M Na2SO4. The nickel(II) complex NiL absorbed an equimolar amount of O2 at pH 9.5 and 25  °C, and the O2 was activated to cleave plasmid DNA.

DNA cleavage by pentadentate iron(II) complexes containing fluoro-substituted phenyl groups

Four pentadentate iron(II) complexes containing non- or fluoro-substituted phenyl group (2b-2e) were synthesized and cleaving activity of them to pUC19 DNA was evaluated in the presence of hydrogen peroxide. DNA cleavage activity increased with the number of substituted fluorine atoms on the phenyl group of 2b.