Total Synthesis of Siomycin A: Completion of the Total Synthesis

The total synthesis of siomycin A ( 1 ), a representative compound of the thiostrepton family of peptide antibiotics, was achieved by incorporating the five synthetic segments A ( 2 ), B ( 3 ), C ( 4 ), D ( 5 ), and E ( 6 ). The dehydropiperidine segment A ( 2 ) was esterified with the dihydroquinoline segment C ( 4 ), and the subsequent coupling with the β‐phenylselenoalanine dipeptide segment D ( 5 ) at the segment C portion followed by lactamization between the segments A and D gave segment A‐C‐D ( 27 ). This was amidated with the pentapeptide segment B ( 3 ) at the segment A portion followed by one‐pot cyclization (between segments A and B) and elongation (with the β‐phenylselenoalanine dipeptide segment E ( 6 ) at the segment A portion), thus furnishing siomycin A ( 1 ).     

Molecular dynamics simulation study on stabilities and reactivities of NADH cytochrome B5 reductase

Binding free energies between coenzyme (FAD and NADH) and the apoenzyme of NADH-cytochrome b5 reductase (b5R) were estimated by applying the continuum Poisson-Boltzmann (PB) model to structures sampled from molecular dynamics simulations in explicit water molecules. Important residues for the enzymatic catalysis were clarified using a computational alanine scanning method. The binding free energies calculated by applying an alanine scanning method can successfully reproduce the trends of the measured steady-state enzymatic activities kcatNADH/KmNADH. Significant decreases in the binding free energy are expected when one of the four residues Arg91, Lys110, Ser127, and Thr181 is mutated into Ala. According to the results of the molecular dynamics simulation, Thr181 is considered to be one of the key residues that helps NADH to approach the isoalloxazine in FAD. Finally, we have constructed very simplified model systems and carried out density functional theory calculations using B3LYP/LANL2DZ//ROHF(or RHF)/LANL2DZ level of theory in order to elucidate a realistic and feasible mechanism of the hydride-ion transfer from NADH to FAD affected by HEME(Fe3+) as an electron acceptor. Our calculated results suggest that the electron and/or hydride-ion transfer reaction from NADH to FAD can be accelerated in the presence of HEME(Fe3+).     

Synthesis of Furoxans (1,2,5‐oxadiazole 2‐oxides) from Styrenes and Nitrosonium Tetrafluoroborate in Non‐Acidic Media and Mechanistic Study

Diverse furoxans (1,2,5‐oxadiazole 2‐oxides) were synthesized from the corresponding styrenes using nitrosonium tetrafluoroborate as the nitrosation reagent in pyridine (basic media) or dichloromethane (neutral media). Acid‐sensitive functional groups were tolerated under these conditions. The probable reaction mechanism was elucidated. The experimental results support an ionic reaction pathway in contrast to the conventional acidic conditions with a radical mechanism.     

Simple one-step extraction and cleanup by pressurized liquid extraction for gas chromatographic-mass spectrometric determination of pesticides in green leafy vegetables

A simple one-step extraction and cleanup using a pressurized liquid extraction method was developed for the gas chromatographic-mass spectrometric determination of pesticides in vegetables. The pressurized liquid extraction conditions were optimized and cleanup agents were evaluated. The investigated pesticides included six insecticides, chlorpyrifos methyl, pirimiphos-methyl, malathion, chlorpyrifos, O-ethyl O-4-nitrophenylphenyl phosphonothioate (EPN) and permethrins, a fungicide, isoprothiolane, and a herbicides, thiobencarb. The cleanup agent and a mixture of the vegetable and anhydrous sodium sulfate were separately packed in an extraction vessel. A transparent and colorless extract was obtained using graphitized carbon as the cleanup agent. The overall recoveries were 71-103% and the relative standard deviations ranged from 5.6 to 24%. The limit of detection values were 3-8 microg kg(-1). This method was successfully applied to green leafy vegetables.     

Yessotoxin analogues in several strains of Protoceratium reticulatum in Japan determined by liquid chromatography-hybrid triple quadrupole/linear ion trap mass spectrometry

Several strains of Protoceratium reticulatum, one of the dinoflagellates producing yessotoxins (YTXs), were collected from various shellfish producing areas in Japan. YTXs in the cultured strains were analyzed by liquid chromatography-mass spectrometry (LC-MS). Neutral loss scan monitoring, multiple reaction monitoring (MRM) for more than 20 YTX analogues, and full-scan MS/MS spectra obtained with a hybrid triple quadrupole/linear ion trap mass spectrometer showed that yessotoxin (YTX), 45,46,47-trinoryessotoxin (trinorYTX), 1-homoyessotoxin (homoYTX), and 45,46,47-trinor-1-homoyessotoxin (trinor-1-homoYTX) were the dominant toxins in these strains of P. reticulatum. Enone isomer of 42,43,44,45,46,47,55-heptanor-41-oxoyessotoxin (noroxoYTX enone) was also detected in some strains. Toxin profiles and contents were different among the strains. Some strains produced YTX, trinorYTX, 1-homoYTX, trinor-1-homoYTX, and noroxoYTX enone, whereas other strains produced only YTX or 1-homoYTX. This is the first identification of 1-homoYTX and noroxoYTX enone in P. reticulutum in Japan. Some strains did not produce any detectable YTX analogues.     

Stelleralides A-C, novel potent anti-HIV daphnane-type diterpenoids from Stellera chamaejasme L

Three novel 1-alkyldaphnane-type diterpenes, stelleralides A-C (4-6), and five known compounds were isolated from the roots of Stellera chamaejasme L. The structures of 4-6 were elucidated by extensive spectroscopic analyses. Several isolated compounds showed potent anti-HIV activity. Compound 4 showed extremely potent anti-HIV activity (EC(90) 0.40 nM) with the lowest cytotoxicity (IC(50) 4.3 μM) and appears to be a promising compound for development into anti-AIDS clinical trial candidates.     

MR signal change in venous thrombus relates organizing process and thrombolytic response in rabbit

Venous thrombus is subsequently organized and replaced by fibrous connective tissue. However, the sequential changes in venous thrombi are not reliably detected by current noninvasive diagnostic techniques. The purpose of this study is to reveal whether magnetic resonance (MR) can detect venous thrombus, define thrombus age and predict thrombolytic responses. Thrombus in the rabbit jugular vein was imaged with a 1.5-T MR system at 4 h and at 1, 2 and 4 weeks using three-dimensional (3D) fast asymmetric spin echo T2-weighted (T2W) and 3D-gradient echo T1-weighted (T1W) sequences. The jugular veins were histologically assessed at each time point. Magnetic resonance imaging (MRI) was also performed in vivo before and 30 min after tissue plasminogen activator (t-PA) administration. The thrombi in MRI were comparable in size to histological sections. The signal intensity (SI) of thrombi at 4 h was heterogeneously high or low on T2W or T1W images, respectively. The SI of thrombi on T2W images decreased time-dependently, but increased on T1W images at 1 and 2 weeks. Morphological analysis showed time-dependent decreases in erythrocyte, platelet and fibrin areas and time-dependent increases in smooth muscle cell, macrophage, collagen and iron areas. The t-PA administration significantly decreased thrombus volume at 4 h but not at 1, 2 and 4 weeks. Venous thrombosis can be reliably and noninvasively detected by MRI. Measurement of SI might support assessments of thrombus age and thrombolytic response.     

Toward a new approach for determination of solute's charge distribution to analyze interatomic electrostatic interactions in quantum mechanical/molecular mechanical simulations

We present an alternative approach to determine "density-dependent property"-derived charges for molecules in the condensed phase. In the case of a solution, it is essential to take into consideration the electron polarization of molecules in the active site of this system. The solute and solvent molecules in this site have to be described by a quantum mechanical technique and the others are allowed to be treated by a molecular mechanical method (QM/MM scheme). For calculations based on this scheme, using the forces and interaction energy as density-dependent property our charges from interaction energy and forces (CHIEF) approach can provide the atom-centered charges on the solute atoms. These charges reproduce well the electrostatic potentials around the solvent molecules and present properly the picture of the electron density of the QM subsystem in the solution system. Thus, the CHIEF charges can be considered as the atomic charges under the conditions of the QM/MM simulation, and then enable one to analyze electrostatic interactions between atoms in the QM and MM regions. This approach would give a view of the QM nuclei and electrons different from the conventional methods.     

In vitro angiogenesis assay for the study of cell-encapsulation therapy

Cell encapsulation within alginate beads has potential as a sustained release system for delivering therapeutic agents in vivo while protecting encapsulated cells from the immune system. There is, however, no in vitro model for cell-encapsulation therapy that provides a suitable platform for quantitative assessment of physiological responses to secreted factors. Here we introduce a new microfluidic system specifically designed to evaluate and quantify the pro-angiogenic potential of factors secreted from human fetal lung fibroblasts encapsulated in beads on an intact endothelial cell monolayer. We confirmed that cell-encapsulating beads induced an angiogenic response in vitro, demonstrated by a strong correlation between the encapsulated cell density in the beads and the length of the vascular lumen formed in vitro. Conditions established by in vitro tests were then further shown to exert a pro-angiogenic response in vivo using a subcutaneous mouse model, forming an extensive network of functional luminal structures perfused with red blood cells.