Synthetic and computational investigation of neighboring group participation by a nucleophilic disulfide bond.

Disulfide bonds of 2-isocyanatophenyl methyl disulfide and 2-endo-isocyanato-6-endo-(methyldisulfanyl)bicyclo[2.2.1]heptane showed neighboring group participation in the formation of thiocarbamates. Natural Bond Orbital (NBO) analyses revealed that the unusual nucleophilicity requires a rigid through-space interaction between a lone pair of the disulfide bond and an antibonding orbital of isocyanate.

Increased Plasma Adenosine Concentration in the Subacute Phase May Contribute to Attenuation of Left Ventricular Dilation in the Chronic Phase in Patients With Acute Myocardial Infarction.

BACKGROUND: Changes in the plasma adenosine concentration and the effects on left ventricular (LV) function and remodeling in patients with acute myocardial infarction (AMI) remain unclear. Methods and Results: In 58 patients with AMI and 14 subjects without cardiac disease (controls), we measured the plasma adenosine concentration by LC-MS/MS. Blood samples were taken from the antecubital vein on days 0, 1, 7, and 14 after AMI, and from the controls on admission. Cardiac echocardiography was performed in the acute (within 7 days) and chronic (6 months) phases of AMI. There were no significant differences in the plasma adenosine concentrations among days 0 (211.5±150.2 nmol/L), 1 (192.7±141.3 nmol/L), 7 (218.8±154.1 nmol/L), and the controls (136.0±50.9 nmol/L). The plasma adenosine concentration increased significantly on day 14 (321.1±195.4 nmol/L) after AMI as compared with days 0, 1 and 7. AMI patients with a greater increase in the plasma adenosine concentration in the subacute phase showed an attenuation of LV dilation in the chronic phase. The plasma adenosine concentration in the acute phase did not affect the LV ejection fraction in the chronic phase. CONCLUSIONS: The plasma adenosine concentration significantly increased 14 days after AMI, which may contribute to attenuation of LV dilation in the chronic phase.

Feasibility of Trypsin Digestion as a Sample Preparation for Daptomycin Quantification in Murine Skeletal Muscles.

It is important to evaluate the amount of daptomycin (DAP) distributed to skeletal muscles to elucidate the mechanisms related to penetration and side effects, such as myopathies. However, no attempt has been made to measure DAP concentrations in skeletal muscles. The study's aim to investigate the feasibility of trypsin digestion, as a muscle sample preparation technique for the determination of DAP in murine skeletal muscle, was evaluated in conjunction with a conventional HPLC-UV analysis. Compared with trypsin digestion, DAP was less recovered from spiked skeletal muscle by the conventional extraction, including homogenization, centrifugation, and filtration, because of its incorporation into the muscle protein. On the other hand, a sample preparation technique involving enzymatic digestion employing trypsin fully recovered DAP from the spiked skeletal muscle. Based on the spike recovery assay results, we proposed an efficient muscle sample preparation method involving trypsin digestion. HPLC analysis in conjunction with the sample preparation method has successfully determined DAP concentrations of skeletal muscles collected from mice administrated subcutaneously with DAP. The proposed method is suitable for application to investigations that include animal experiments on drug migration into muscle and mechanism underlying skeletal muscle injury as a side reaction, such as myopathies, of DAP therapy.

Study on Redox Properties and Cytotoxicity of Anthraquinone Derivatives to Understand Antitumor Active Anthracycline Substances.

This study demonstrates the relation between the redox properties and cytotoxicity of anthraquinone derivatives with a hydroxyl and methoxy group. The redox behavior of the anthraquinone derivatives was initially observed via cyclic voltammetry and their characteristics were investigated using molecular orbital calculations. The cytotoxicity of the anthraquinone derivatives was then evaluated using human leukemia HL-60 and H2O2 resistant HP100 cells, and its correlation with the redox properties of these compounds was investigated. Therefore, it was suggested that the anthraquinone derivatives express cytotoxicity through H2O2 production, and that generation of the oxidized radical form influences their cytotoxicity.

Endogenous Adenosine May Be Related to Left Ventricular Dysfunction, Dilation, and Wall Thinning in Patients With Heart Disease.

BACKGROUND: The role of endogenous adenosine in cardiac patients is still unclear, so we investigated the relationship between the plasma adenosine concentration and left ventricular (LV) function, LV dilation and LV wall thinning in cardiac patients.Methods and Results:In 97 cardiac patients, with angina pectoris, old myocardial infarction, dilated or hypertrophic cardiomyopathy, and valvular heart disease, plasma adenosine concentrations were measured using the LC-MS/MS system, and the LV function, LV end-diastolic dimension (LVDd), LV posterior wall thickness (LVPWth), and interventricular septum thickness (IVSth) were assessed by echocardiography. The plasma adenosine concentration was significantly higher in patients with a LV ejection fraction (EF), an indicator of the LV systolic function, <47% compared with those with LVEF ≥47% (P=0.027). There was no difference between the plasma adenosine concentration and E/e', an indicator of LV diastolic function. The plasma adenosine concentration was significantly higher in patients with LVDd ≥50 mm than in those with LVDd <50 mm (P=0.030). The plasma adenosine concentration was inversely correlated with IVSth (P=0.003) and LVPWth (P=0.0007). The plasma adenosine concentration was significantly higher in patients with IVSth <8 mm than in those with IVSth ≥8 mm (P=0.015), and was significantly higher in patients with LVPWth <8 mm than in those with LVPWth ≥8 mm (P=0.020). CONCLUSIONS: Endogenous adenosine may be related to LV dysfunction, dilation, and wall thinning in cardiac patients.

Considerations about the Use of a Loading Dose of Daptomycin in a Neutropenic Murine Thigh Infection Model with Methicillin-Resistant Staphylococcus aureus Infection.

Previous clinical studies have showed the clinical benefits of the initiation of treatment with a daptomycin (DAP) loading dose, but only a few studies have evaluated its antimicrobial benefits. We evaluated the efficacy of a DAP loading dose against methicillin-resistant Staphylococcus aureus (MRSA) infections in a neutropenic murine thigh infection model. Three MRSA isolates (DAP MIC: 0.5, 1, and 2 mg/L) were tested. Four DAP regimens simulating human concentration-time profiles, i.e., (i) day 1: 8 mg/kg and day 2: 6 mg/kg, (ii) days 1 and 2: 6 mg/kg/day, (iii) day 1: 8 mg/kg and day 2: 4 mg/kg, and (iv) days 1 and 2: 4 mg/kg/day, were administered to the mice. Efficacy was calculated as the change in bacterial density. DAP loading-dose regimen iii showed greater antimicrobial activity against MRSA with MIC 1 mg/L than nonloading regimen iv (-3.10 ± 0.63 vs. -0.71 ± 0.34 log10 CFU; p < 0.01). Loading-dose regimen iii achieved greater log10 CFU changes than nonloading regimen ii, while the total DAP dose for 2 days was the same (-3.10 ± 0.63 vs. -1.46 ± 0.48 log10 CFU; p < 0.05). DAP loading-dose regimen iii showed enhanced antimicrobial activity against MRSA with DAP MIC 0.5 mg/L when compared with nonloading regimen iv. However, loading-dose regimens i and iii did not reduce bacterial density for MRSA with DAP MIC 2 mg/L. Our data suggest that a DAP loading-dose regimen would be an advantageous procedure for patients infected with MRSA with DAP MIC ≤1 mg/L.

Anti-Oncogenic gem-Dihydroperoxides Induce Apoptosis in Cancer Cells by Trapping Reactive Oxygen Species.

Organic gem-dihydroperoxides (DHPs) and their derived peroxides have attracted a great deal of attention as potential anti-cancer agents. However, the precise mechanism of their inhibitory effect on tumors is unknown. To determine the mechanism of the inhibitory effects of DHPs, we examined the effects of DHPs on leukemia K562 cells. As a result, certain DHPs used in this study exhibited growth-inhibitory activity according to a clear structure-activity relationship. The most potent DHP, 12AC3O, induced apoptosis in K562 cells, but not in peripheral blood monocytes (PBMCs) or fibroblast cells. 12AC3O induced apoptosis through the intrinsic mitochondrial pathway and thereafter through the extrinsic pathway. The activity of the former pathway was partly attenuated by a JNK inhibitor. Interestingly, 12AC3O induced apoptosis by trapping a large amount of ROS, leading to an extremely lower intracellular ROS level compared with that in the cells in the steady-state condition. These results suggest that an appropriate level of intracellular ROS was necessary for the maintenance of cancer cell growth. DHPs may have a potential to be a novel anti-cancer agent with minimum adverse effects on normal cells.

Importance of Proton-Coupled Electron Transfer from Natural Phenolic Compounds in Superoxide Scavenging.

The superoxide O2(·-) scavenging reaction of (+)-catechin (Cat), quercetin (Que), rutin, and α-tocopherol (α-TOH) as natural phenolic compounds is investigated on the basis of electrochemical and ESR spectral measurements with the aid of density functional theory (DFT) calculations. Reversibility of the O2/O2(·-) redox couple is significantly affected by the presence of the phenolic compounds. The catechol moiety of Cat, Que, and rutin plays an essential role in concerted proton-coupled electron transfer (PCET) to HO2(·) derived from O2(·-) to give H2O2 and the corresponding o-benzoquinone radical anions. On the other hand, the presence of α-TOH causes sequential electron and proton transfers to HO2(·) to give H2O2 and the α-tocopheroxyl radical. These electron transfers in the presence of the phenolic compounds are inferred from the ESR spectral measurements. The DFT calculation results suggest that the O2(·-) scavenging reaction of the natural phenolic compounds proceeds efficiently with the one-step concerted PCET or sequential PCET mechanism.

Electrochemical analysis in a liposome suspension using lapachol as a hydrophobic electro active species.

This study demonstrated that the electro-chemical analysis of hydrophobic quinones can be performed in liposome suspension systems. We prepared and analyzed liposome suspensions containing lapachol, which is a quinone-based anti-tumor activity compound. In this suspension system, a simple one redox couple of lapachol is observed. These results are quite different from those obtained in organic solvents. In addition, the pH dependence of redox behaviors of lapachol could be observed in multilamellar vesicle (MLV) suspension system. This MLV suspension system method may approximate the electrochemical behavior of hydrophobic compounds in aqueous conditions. A benefit of this liposome suspension system for electrochemical analysis is that it enables to observe water-insoluble compounds without using organic solvents.

Concerted Two-Proton-Coupled Electron Transfer from Piceatannol to Electrogenerated Superoxide in N,N-Dimethylformamide.

The reactivity of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,2-diol (piceatannol) toward electrochemically generated superoxide radical anion (O2 •-) was investigated using electrochemistry and in situ controlled-potential electrolytic electron spin resonance (ESR) measurements in N,N-dimethylformamide with density functional theory (DFT) calculations. The quasireversible cyclic voltammogram of dioxygen/O2 •-, modified in the presence of piceatannol, indicated that the electrogenerated O2 •- was scavenged by piceatannol via proton-coupled electron transfer. Differences in the reactivities of piceatannol and 5-[(E)-2-(4-hydroxyphenyl)ethen-1-yl]benzene-1,3-diol (trans-resveratrol) toward O2 •-, originating from the presence of the benzene-1,2-diol (catechol) moiety, were observed in the voltammograms and ESR measurements. The electrochemical and computational results show that the reaction mechanism is a concerted two-proton-coupled electron transfer (2PCET) via the catechol moiety of piceatannol. The stilbene moiety of piceatannol kinetically promotes 2PCET via its catechol moiety. These findings indicate that piceatannol is a better O2 •- scavenger than catechol and trans-resveratrol.

Reactivity of trans-Resveratrol toward Electrogenerated Superoxide in N,N-Dimethylformamide.

The reactivity of 5-[(E)-2-(4-hydroxyphenyl)ethen-1-yl]benzene-1,3-diol (trans-resveratrol) and related compounds toward electrogenerated superoxide radical anion (O2•-) were investigated using electrochemistry, in situ electrolytic electron spin resonance, and in situ electrolytic ultraviolet-visible spectral measurements, in N,N-dimethylformamide (DMF) with the aid of density functional theory (DFT) calculations. The quasi-reversible cyclic voltammogram of dioxygen/O2•- was modified by the presence of trans-resveratrol, suggesting that the electrogenerated O2•- was scavenged by trans-resveratrol through proton-coupled electron transfer (PCET) via three phenolic hydroxy groups (OH) on the stilbene moiety. The reactivity of trans-resveratrol toward O2•- characterized by the OHs was experimentally confirmed in comparative analyses using some related compounds, pinosylvin, pterostilbene, p-coumaric acid, and so on, in DMF. The electrochemical and DFT results suggested that a concerted PCET mechanism via 4'OH of trans-resveratrol proceeds, where the coplanarity of the two phenolic rings in the stilbene moiety linked by an ethylene bridge is essential for a successful O2•- scavenging.

Cilostazol protects the heart against ischaemia reperfusion injury in a rabbit model of myocardial infarction: focus on adenosine, nitric oxide and mitochondrial ATP-sensitive potassium channels.

1. The present study examined whether or not cilostazol reduces the myocardial infarct size, and investigated its mechanism in a rabbit model of myocardial infarction. 2. Japanese white rabbits underwent 30 min of coronary occlusion, followed by 48 h of reperfusion. Cilostazol (1 and 5 mg/kg) or vehicle was given intravenously 5 min before ischaemia. 8-p-sulfophenyl theophylline (8SPT; an adenosine receptor blocker, 7.5 mg/kg), Nω-nitro-L-arginine methylester (l-NAME; an NOS inhibitor, 10 mg/kg) or 5-hydroxydecanoic acid sodium salt (5-HD; a mitochondrial ATP-sensitive potassium (KATP) channel blocker, 5 mg/kg) was given intravenously 5 min before cilostazol injection. Infarct size was determined as a percentage of the risk area. 3. The myocardial interstitial levels of adenosine and nitrogen oxide (NOx) during ischaemia and reperfusion, and the intensity of myocardial dihydroethidium staining were determined. 4. Infarct size was significantly reduced in the cilostazol 1 mg/kg (38.4% (2.9%)) and cilostazol 5 mg/kg (30.7% (4.7%)) groups compared with that in the control group (46.5% (4.2%)). The infarct size-reducing effect of cilostazol was completely abolished by 8SPT (46.6% (3.5%)), L-NAME (49.0% (5.5%)), or 5HD (48.5% (5.1%)). 8SPT, L-NAME or 5HD alone did not affect the infarct size. Cilostazol treatment significantly increased myocardial levels of adenosine and NOx during ischaemia, and attenuated the intensity of dihydroethidium staining during reperfusion. 5. These findings show that cilostazol reduces the myocardial infarct size by increasing adenosine and NOx levels, attenuating superoxide production and opening the mitochondrial KATP channels. Cilostazol might provide a new strategy for the treatment of coronary heart disease.

[Basic Study on Organic Electron Transfer Reactions by Electrochemistry Combined with Quantum Chemical Calculations].

This review summarizes the results of the author's basic research on organic electrochemistry conducted at Gifu Pharmaceutical University over about 40 years. After completing graduate school, the author became a research associate in Prof. Tanekazu Kubota's laboratory and started research on molecular spectroscopy in 1983. After Prof. Kubota retired in 1989, the author continued investigations in the field of organic electrochemistry as an independent researcher. At that time, a research environment in which ab initio molecular orbital calculations can be used as an analytical tool for experimental research was developed, and the author commenced research on organic electrochemistry combined with quantum chemical calculations as a lifework. The author's research topics were basic research on the molecular theory of redox potentials of organic molecules, molecular design of functional molecules, intermolecular interactions of organic molecules involving electron transfers and electron transfer systems composed of bioactive quinones, and analytical application research based on the basic electrochemistry. In this review article, the essence of the research results is introduced while reflecting on the existing situation at the time of the research. The author concludes the review by expressing gratitude to all colleagues for supporting the research in the author's laboratory.

Electrochemical and Mechanistic Study of Reactivities of α-, ß-, γ-, and δ-Tocopherol toward Electrogenerated Superoxide in N,N-Dimethylformamide through Proton-Coupled Electron Transfer.

Scavenging of superoxide radical anion (O2•-) by tocopherols (TOH) and related compounds was investigated on the basis of cyclic voltammetry and in situ electrolytic electron spin resonance spectrum in N,N-dimethylformamide (DMF) with the aid of density functional theory (DFT) calculations. Quasi-reversible dioxygen/O2•- redox was modified by the presence of TOH, suggesting that the electrogenerated O2•- was scavenged by α-, ß-, γ-TOH through proton-coupled electron transfer (PCET), but not by δ-TOH. The reactivities of α-, ß-, γ-, and δ-TOH toward O2•- characterized by the methyl group on the 6-chromanol ring was experimentally confirmed, where the methyl group promotes the PCET mechanism. Furthermore, comparative analyses using some related compounds suggested that the para-oxygen-atom in the 6-chromanol ring is required for a successful electron transfer (ET) to O2•- through the PCET. The electrochemical and DFT results in dehydrated DMF suggested that the PCET mechanism involves the preceding proton transfer (PT) forming a hydroperoxyl radical, followed by a PCET (intermolecular ET-PT). The O2•- scavenging by TOH proceeds efficiently along the PCET mechanism involving one ET and two PTs.

Highly Sensitive Fluorescence Detection of Daptomycin in Murine Samples through Derivatization with 2,3-Naphthalenedialdehyde.

A highly sensitive high-performance liquid chromatography method has been developed using the pre-column fluorescent derivatization of daptomycin (DAP) through cyclization of the amino group of ornithine with 2,3-naphthalenedialdehyde. With the proposed method, the limits of detection and quantification of DAP in murine serum were 8 and 3 nmol/L, respectively, and the calibration curve was linear across the examined dynamic range from 8 nmol/L to 1 µmol/L (n = 8, r = 0.9986). This method is suitable for animal experiments examining the side effects of DAP therapy using mice as a simple method with quantification to the order of 10 nmol/L.

Complementary Effect of Intra- and Intermolecular Hydrogen Bonds on Electron Transfer in ß-Hydroxy-Anthraquinone Derivatives.

We have carried out an electrochemical and theoretical study on the relationship between electron transfer (ET) and hydrogen bonding in 11 different 9,10-anthraquinone (AQ) derivatives, including ß-hydroxy AQs and their methoxylated analogs, in the presence of hydrogen donors in acetonitrile (ACN). The complementary effects of the intra- and intermolecular hydrogen bonds (HBs) on ET were studied by analyzing the complex formation constants derived from the intermolecular HB. Our results revealed that the inductive effect of the ß substituent that indirectly controls the charge distribution on the AQ carbonyl oxygen, and steric hindrance at the ß position, affect the complex formation constants. Furthermore, the analysis of ET in different isomeric dihydroxy AQs suggested that the position of the hydroxy groups affects the charge distribution and stabilizes structures through conjugation of the quinone moiety including the ipso ring, controlling the intra- and intermolecular HBs complementarily.

Effect of High Blood Glucose Level on the Antimicrobial Activity of Daptomycin against Staphylococcus aureus in Streptozotocin-Induced Diabetic Mice.

Daptomycin is active against Staphylococcus aureus including methicillin-resistant S. aureus (MRSA), demonstrating efficacy in the treatment of infections in diabetic patients. However, daptomycin degrades in 5% glucose solution, and data on the efficacy of daptomycin in hyperglycemic patients are limited. Therefore, we investigated the effect of high levels of blood glucose on the efficacy and concentration of daptomycin. The efficacy of simulated human exposure to daptomycin against S. aureus was compared in a neutropenic murine thigh model, with and without hyperglycemia. A clinically isolated MRSA strain and S. aureus ATCC25923 standard strain were used. Daptomycin concentrations, in the serum and at the infected site, were preliminarily analyzed using the high-performance liquid chromatography assay. Even in hyperglycemic mice, the mean concentration of daptomycin in hyperglycemic mice was equivalent to that in untreated mice within the physiological blood glucose levels. Additionally, the efficacy of daptomycin against MRSA was equal to that observed in the untreated and hyperglycemic mice. Based on similar studies using S. aureus ATCC25923, the efficacy in hyperglycemic mice was equal to or greater than that observed in untreated mice. In conclusion, daptomycin is an alternative therapeutic option in diabetic mice with serious staphylococcal infections, regardless of blood glucose control in this animal model.

Preparation of N2-Ethyl-2'-deoxyguanosine-d4 as an Internal Standard for the Electrospray Ionization-Tandem Mass Spectrometric Determination of DNA Damage by Acetaldehyde.

The deuteration of N2-ethyl-2'-deoxyguanosine (Et-dG), which is a DNA adduct generated from acetaldehyde, was studied by the addition reaction of acetaldehyde-d4 to 2'-deoxyguanosine (dG) in deuterium oxide (D2O), with the aim to obtain an isotope internal standard for the liquid chromatography/tandem mass spectrometry (LC/MS/MS) quantitation of Et-dG. The replacement of the dG C-8 hydrogen atom by a deuteron atom took place at 50°C in D2O and afforded a mixture of Et-dG-d4 and Et-dG-d5. Et-dG-d4, which was stable in aqueous solutions, was prepared by incubating the mixture in H2O at 60°C for 48 h. The calibration curve was obtained by multiple reaction monitoring (MRM) measurements using a hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometric (HILIC/ESI-MS/MS) system between the Et-dG concentration, ranging from 1.0 × 10-10 to 4.0 × 10-9 M in the sample solutions, and the relative peak areas of Et-dG (m/z: 296.1 → 180.1) to the value of Et-dG-d4 (m/z: 300.2 → 184.2), with an internal standard showing good linearity (R2 = 0.995, n = 5).

Cross-link dimer formation of the acetaldehyde-derived cyclic 1,N(2)-Propano-2'-deoxyguanosine adduct using electrochemical oxidation.

The electrochemically oxidative lesion of the acetaldehyde-derived cyclic propano adduct 2 of 2'-deoxyguanosine 1 was identified as the cross-linked dimer 4 of adduct 2. Cross-link formation is explained by the nucleophilic preference of the exocyclic amino group in 2 to the carbocation 3 electrogenerated by 1-proton and 2-electron transfers. Dimer formation was also detected in duplex DNA during exposure to acetaldehyde followed by electrochemical oxidation. The dimer has been deduced to be an intrastrand cross-link generated specifically in the G-G sequence in duplex DNA, which is expected to contribute to acetaldehyde-mediated genotoxicity.

Synthesis of cis-ß-Amidevinyl Benziodoxolones from the Ethynyl Benziodoxolone-Chloroform Complex and Sulfonamides.

The synthesis of cis-ß-amidevinyl benziodoxolones from the ethynyl benziodoxolone-chloroform complex and sulfonamides is reported. Evidence indicates that highly reactive unsubstituted ethynyl benziodoxolone undergoes Michael addition of sulfonamides, including sterically demanding acyclic amino acid derivatives. The synthesis of selectively deuterated cis-ß-amidevinyl benziodoxolones and investigation of ethynyl-λ3-iodane reactivity are also described.