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.

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.

[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.

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).

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.

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.

[Improvement of the Non-aqueous Titration Method in the Japanese Pharmacopoeia for a Purpose of Advancement in Experimental Safety Using Alternative Solvents].

In this study, we attempted to improve the non-aqueous titration method using N,N-dimethylformamide (DMF) in the Japanese Pharmacopoeia seventeenth edition (JP XVII) for advancement in experimental safety. As an alternative solvent for DMF, we demonstrate titrations using dimethyl sulfoxide (DMSO), which has similar properties as and much higher safety than DMF. Five drugs (i.e., acetohexamide, glibenclamide, sulfamethoxazole, tranilast, and furosemide) listed in JP XVII use DMF as a solvent for titrations with sodium hydroxide standard solution. For these drugs, we examined whether DMF can be replaced with DMSO in quantitative analyses. As a result, a quantification similar to that of the Pharmacopoeia protocol is possible by simply replacing DMF with DMSO or using a mixed solvent of DMSO and water.

Preparation of Cyclic-1,N2-propano-2'-deoxyguanosine-d7 as an Internal Standard for ESI-MS/MS Determination of DNA Damage from Acetaldehyde.

Cyclic-1,N2-propano-2'-deoxyguanosine-d7 (CPr-dG-d7) was prepared as an isotopic internal standard (IS) for electrospray ionization tandem mass spectrometry (ESI-MS/MS) quantification of CPr-dG in DNA as a candidate cancer risk marker of acetaldehyde intake, mainly from drinking. The deuterated compound was reasonably synthesized from acetaldehyde-d4 and 2'-deoxyguanosine in deuterium oxide (D2O), preventing the deuterium atoms of acetaldehyde-d4 from being substituted by hydrogen atoms, which occurred seriously in aqueous synthesis media via keto-enol tautomerism. Furthermore, another deuterium atom was added from D2O to form CPr-dG-d7. After four weeks of storage in H2O at 10°C, CPr-dG-d7 was found to be sufficiently stable for practical use. The calibration curve of CPr-dG by using a hydrophilic interaction chromatography-ESI-MS/MS system with CPr-dG-d7 as the IS showed sufficient linearity from 1.0 × 10-10 to 4.0 × 10-9 M with r2 = 0.998.

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.

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.

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.

Evaluation of Type-A Endonucleases for the Quantitative Analysis of DNA Damage due to Exposure to Acetaldehyde Using Capillary Electrophoresis.

The substrate selectivities of three endonucleases were studied quantitatively using capillary zone electrophoresis to find one giving N2-ethyl(Et)-2'-deoxyguanosine-5'-monophosphate (5'-dGMP) and cyclic 1,N2-propano(CPr)-5'-dGMP from DNAs damaged by acetaldehyde (AA). Six 2'-deoxyribonucleoside-5'-monophosphates to be quantified in the hydrolysis solutions of DNAs, namely, Et-5'-dGMP, CPr-5'-dGMP, and four authentic ones, were completely separated using a 100 mM borate running buffer solution having an optimized pH of 9.67. Using the present method, nuclease reactions of nuclease S1 (NS1), nuclease P1 (NP1), and nuclease Bal 31 to 2'-deoxyribonucleoside-5'-monophosphates from damaged Calf thymus (CT-) DNAs were monitored. The CT-DNAs were prepared by treatment with AA to generate Et-guanine or CPr-guanine internally. Bal 31 hydrolyzed the damaged CT-DNAs to yield Et-5'-dGMP and CPr-5'-dGMP quantitatively. The two 5'-dGMP adducts were not detected in the hydrolysis solutions using NS1 or NP1. Bal 31 can be a suitable nuclease for analyzing DNA damages caused by AA.

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.

Organic dye-catalyzed radical ring expansion reaction.

Herein, we reported an attractive method for synthesizing medium-sized rings that are catalyzed by erythrosine B under fluorescent light irradiation. This synthetic approach featured mild conditions, a facile procedure, a broad substrate scope, and moderate-to-good yields.

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.

Progress in a selective method for the determination of the acetaldehyde-derived DNA adducts by using HILIC-ESI-MS/MS.

Acetaldehyde (AA), which is present in tobacco smoke, automobile exhaust gases and alcohol beverage, is a mutagen and carcinogen. AA reacts with 2'-deoxyguanosine (dG) in DNA to form N2-ethyl-dG (EtdG) and cyclic, 1, N2-propano-dG (CPrdG), which are considered to have a critical role in carcinogenesis induced by AA. In this study, we have developed a highly sensitive method for the quantitation of the two AA-derived DNA adducts by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in which hydrophilic interaction chromatography (HILIC) employing mobile phases of high organic solvent concentration was selected to improve the ionization efficiency in the ESI process. Fourteen times and 11 times larger peak areas for EtdG and CPrdG, respectively, in HILIC-ESI-MS/MS were obtained compared with those in reversed phase (RP)-LC-ESI-MS/MS. Furthermore, 6.9 times (for EtdG) and 2.4 times (for CPrdG) larger peak areas were also obtained as additional enhancement by varying additive compounds in the HILIC mobile phases from ammonium acetate to ammonium bicarbonate. In total, the enhancements in detected MS signal intensities by exchanging from the RP-LC system to the HILIC system are 97 times for EtdG and 26 times for CPrdG, respectively. Three commercially available HILIC columns with different polar functional groups were examined and sufficient separation between normal 2'-deoxynucleosides and the AA-derived DNA adducts was achieved by a carbamoyl-bonded HILIC column. Finally, we applied the established method to quantify EtdG and CPrdG in the damaged calf thymus DNA.