Novel Disulfiram Derivatives as ALDH1a1-Selective Inhibitors

Aldehyde dehydrogenase-1a1 (ALDH1a1), the enzyme responsible for the oxidation of retinal into retinoic acid, represents a key therapeutic target for the treatment of debilitating disorders such as cancer, obesity, and inflammation. Drugs that can inhibit ALDH1a1 include disulfiram, an FDA-approved drug to treat chronic alcoholism. Disulfiram, by carbamylation of the catalytic cysteines, irreversibly inhibits ALDH1a1 and ALDH2. The latter is the isozyme responsible for important physiological processes such as the second stage of alcohol metabolism. Given the fact that ALDH1a1 has a larger substrate tunnel than that in ALDH2, replacing disulfiram ethyl groups with larger motifs will yield selective ALDH1a1 inhibitors. We report herein the synthesis of new inhibitors of ALDH1a1 where (hetero)aromatic rings were introduced into the structure of disulfiram. Most of the developed compounds retained the anti-ALDH1a1 activity of disulfiram; however, they were completely devoid of inhibitory activity against ALDH2.     

Synthesis, structure, and activity of supramolecular mimics for the active site and arg141 residue of copper, zinc-superoxide dismutase

Two supramolecular complexes, [Cu(L)(H2O)2(beta-CD)](ClO4)2.10.5H2O.CH3OH (1) and [Cu(L)(H2O)2(beta-GCD)](HClO4)(ClO4)2.10H2O (2) (L = 4-(4'-tert-butyl-benzyl)diethylenetriamine, beta-CD = beta-cyclodextrin, and beta-GCD = mono-6-deoxy-6-guanidinocycloheptaamylose cation), have been synthesized. The structure of 1 has been characterized by X-ray crystallography. The 4-tert-butyl-benzyl of [Cu(L)(H2O)2]2+ moiety in 1 as a guest inserts into the hydrophobic cavity of the beta-CD as a host along the primary hydroxyl side. On the basis of the structure data of 1, complex 2 was modeled, which showed that the distance between the Cu and C atom of the guanidinium is 5.2 A, comparable to the corresponding distance in bovine erythrocyte Cu, Zn-SOD (5.9 A) (SOD = superoxide dismutase). Apparent inclusion stability constants of the host and the guest were measured to be 0.66 (+/-0.01) x 104 and 1.15 (+/-0.03) x 104 M-1 for 1 and 2 respectively. The electronic absorption bands and electronic reflection bands of each complex are almost the same, indicating an identical structure of the complex in aqueous solution and in solid state. The two complexes showed quasi-reversible one-electron Cu(II)/Cu(I) redox waves with redox potentials of -0.345 and -0.338 V for 1 and 2, respectively. Their SOD-like activities (IC50) were measured to be 0.30 +/- 0.01 and 0.17 +/- 0.01 microM by xanthine/xanthine oxidase-NBT assay. The enhanced SOD activity of 2 by approximately 40% compared with 1 suggests that the guanidyl cation in the host of the supramolecular system of 2 can effectively mimic the side chain of Arg141 in the enzyme, which is known to be essential for high SOD activity possibly through steering of the superoxide substrate to and from the active copper ion.     

High-performance liquid chromatographic separation of noreximide and norendimide in bulk material, tablets and animal feed

Noreximide, a sedative, is generally contaminated to some extent with its endo-isomer, norendimide, which produces excitation. A high-performance liquid chromatographic assay was developed to separate and quantitate these compounds on a 5-microns Ultrasphere ODS column with methanol-water (20:30) as mobile phase and detection at 254 nm. Assay of mixtures of these compounds in bulk material and tablets utilized isoniazide as internal standard. Peak area ratios were linear (r = 0.9999) over 1.4-66.2 micrograms of injected noreximide and 0.2-8.4 micrograms of injected norendimide. Overall percent recovery from simulated tablets containing noreximide alone was 99.6 +/- 0.8% (S.D., n = 3). Overall percent recoveries (+/- S.D.) from tablets containing a mixture of these compounds were 98.9 +/- 0.5% and 102.3 +/- 1.1% for noreximide and norendimide, respectively (n = 3). Noreximide in animal feed for long-term pharmacological studies was isolated by ether extraction and after work up, subjected to the same procedure, except that theophylline was the internal standard. Peak area ratios were linear over 0.2-19.3 micrograms of injected noreximide (r = 0.9999). Overall percent recoveries (+/- S.D., n = 3) of noreximide from spiked animal feed were 97.4 +/- 1.4% and 99.0 +/- 0.5% at the 500- and 5000-ppm levels, respectively. Limits of detection at the 95% confidence level (0.01 a.u.f.s., 20-microliters sample volume injected) were 1.67 microgram/ml and 2.56 micrograms/ml of noreximide and norendimide, respectively, in the final test solution.     

Field-amplified on-line sample stacking for simultaneous enantioseparation and determination of some beta-blockers using capillary electrophoresis

A capillary electrophoresis method using carboxymethyl-beta-cyclodextrin as the chiral selector and mixture of methanol and ethanol as the organic additive was successfully developed for the simultaneous enantioseparation and determination of six beta-blockers, namely, carteolol, atenolol, sotalol, metoprolol, esmolol and propranolol in this paper. The most suitable running buffer for enantioseparation was found to be the solution of 20 mmol/L NaH(2)PO(4)-Na(2)HOP(4) (pH 5.5) containing 1.5% w/v carboxymethyl-beta-cyclodextrin, 5% v/v methanol and 5% v/v ethanol. Furthermore, field-amplified sample injection as an on-line sample stacking method was developed in order to increase the detection sensitivity. The experimental conditions for both simultaneous enantioseparation and the field-amplified sample injection method had been investigated in detail. Under the optimum conditions, the detection limits (defined as S/N=3) of this method were 0.01, 0.05, 0.05, 0.05, 0.05 and 0.5 microg /mL for (+/-) carteolol, (+/-) atenolol, (+/-) sotalol, (+/-) metoprolol, (+/-) esmolol and (+/-) propranolol, respectively, which were much lower than those of the conventional methods. The enhancement factors were greatly improved by 25-fold for the enantiomers of the beta-blockers except five-fold for (+/-) propranolol. Eventually, the proposed method has been applied for the analysis of human serum sample.     

A potentiometric and 51V NMR study of the aqueous H+/H2VO4-/H2O2/L-alpha-alanyl-L-histidine system

The speciation in the quaternary aqueous H+/H2VO4-/H2O2/L-alpha-alanyl-L-histidine (Ah) system has been determined from quantitative 51V NMR measurements and potentiometric data (glass electrode). The study was performed in 0.150 M Na(Cl) medium at 25 degrees C. Data were evaluated with the computer program LAKE, which is able to treat combined EMF and NMR data. The pKa values for Ah were determined as 8.06, 6.72 and 2.64. In the ternary H+/H2VO4-/Ah system, two complexes, (H+)p(H2VO4-)q(Ah)r, for which (p, q, r) values of (0, 1, 1) and (1, 1, 1) with log beta(0,1,1) = 2.52 +/- 0.03 and log beta(1,1,1) = 9.40 +/- 0.05 (pKa = 6.88), respectively, explain all data. The errors given are 3sigma. In the quaternary H+/H2VO4-/H2O2/Ah system, eight complexes were determined in addition to all binary and ternary complexes, four with a V/X/Ah ratio 1:1:1 and four with a ratio 1:2:1 (X = peroxo ligand). VX2Ah2- and VX2Ah- (pKa = 8.19) are the main complexes and predominate in the pH range 5 to 9. Three additional minor species have also been found but their compositions could not be determined owing to their small amounts. Equilibria are slow, significant decomposition of peroxide occurs only in acidic solutions. Data in the pH range 5 to 10 have been used for the LAKE calculations. Chemical shifts, compositions, and formation constants for the eight quaternary complexes are given, and equilibrium conditions are illustrated in distribution diagrams. Structural proposals for VX2Ah2- and VX2Ah- are made from 1H and 13C NMR measurements.     

Identification of 4-amino-4-deoxychorismate synthase as the molecular target for the antimicrobial action of (6s)-6-fluoroshikimate

(6S)-6-Fluoroshikimate has antimicrobial activity. The molecular basis of this effect had not been identified, but there was speculation that (6S)-6-fluoroshikimate is first converted in vivo into 2-fluorochorismate, which then could inhibit 4-amino-4-deoxychorismate synthase (ADCS). 2-Fluorochorismate was prepared from E-fluorophosphoenolpyruvate and erythose-4-phosphate by the sequential reactions of DAHP synthase, dehydroquinate synthase, dehydroquinase, shikimate dehydrogenase, EPSP synthase, and chorismate synthase. Inhibition studies on ADCS showed that it was inhibited rapidly and irreversibly by 2-fluorochorismate. Electrospray mass spectrometry of the inactivated enzyme showed an additional mass of 198 +/- 10 Da. A novel peptide of 1087.6 Da was identified in the HPLC trace for the tryptic digest of 2-fluorochorismate-inactivated ADCS. Sequencing of this peptide by MS/MS showed that the peptide corresponded to residues 272-279 with a modification of 206.1 Da on Lys-274. This observation is particularly exciting in the context of a recent proposal for the catalytic mechanism of ADCS.     

Calorimetric investigation of the interaction between lithium perfluorononanoate and poly(ethylene glycol) oligomers in water

The interaction of lithium perfluorononanoate (LiPFN) with poly(ethylene glycol) (PEG) molecules of different molecular weights (300 < MW < 20000 Da) has been investigated in water at 298.15 and 308.15 K by isothermal titration calorimetry (ITC). Density, viscosity, and conductivity measurements were also performed at 298.15 K. The aggregation process of this surfactant on the PEG polymeric chain was found to be very similar to that exhibited by cesium perfluorooctanoate (CsPFO) and appears to be consistent with the necklace model. ITC titrations indicated that a fully formed LiPFN micellar cluster can be wrapped by a PEG chain having a molecular weight (MW) of approximately 3200 Da, longer than that required by the shorter perfluorooctanoate (MW approximately 2600 Da), and also suggested a stepwise mechanism for the aggregation of successive micelles. Viscosity data indicate that the formation of polymer-surfactant complexes between PEG and LiPFN involves a conformational change of the polymer. The aggregation of preformed micelles of LiPFN or CsPFO or SDS on the PEG polymeric chain always gives rise to further stabilization.     

配合物[Ln(CH3COO)2(NO3)(bipy)]2的合成与晶体结构

合成了５种新型镧系四元混合阴离子配合物,用Ｘ射线四圆衍射仪测定了［Ｐｒ（ＣＨ３ＣＯＯ）２（ＮＯ３）（ｂｉｐｙ）］２的晶体结构,四个醋酸根呈两种配位方式,测定了铕和钆配合物的荧光和ＥＳＲ谱．     

High-frequency EPR study of a new mononuclear manganese(III) complex:. [(terpy)Mn(N3)3] (terpy = 2,2':6',2'-terpyridine)

The isolation and structural characterization of [(terpy)Mn(III)(N3)3], complex 1, is reported (terpy = 2,2':6',2' '-terpyridine). Complex 1, a product of the reaction between the mixed-valence dimer [(terpy)(H2O)Mn(III)(O)2Mn(IV)(OH2)(terpy)](NO3)3 and NaN3, crystallizes in a triclinic system, space group P1, a = 8.480(1) A, b = 8.9007(2) A, c = 12.109(2) A, alpha = 93.79(1) degrees, beta = 103.17(1) degrees, gamma = 103.11(1) degrees, and Z = 2. Complex 1 exhibits a Jahn-Teller distortion of the octahedron characteristic of a six-coordinated high-spin Mn(III). A vibrational spectroscopic study was performed. The nu(asym)(N3) mode of complex 1 appears in the IR as a strong band at 2035 cm(-1) with a less intense feature at 2072 cm(-1), and in the FT-Raman as a strong band at 2071 cm(-1) with a weaker broad band at 2046 cm(-1). The electronic properties of complex 1 were investigated using a high-field and high-frequency EPR study (190-475 GHz). The different spin Hamiltonian parameters have been determined (D = -3.29 (+/-0.01) cm(-1), E = 0.48 (+/-0.01) cm(-1), E '= 0.53 (+/-0.01) cm(-1), g(x) = 2.00 (+/-0.005), g(y) = 1.98 (+/-0.005), g(z) = 2.01 (+/-0.005)). These parameters are in agreement with the geometry of complex 1 observed in the crystal structure, a D < 0 related to the elongated distortion, and a value of E/D close to 0.2 as expected from the highly distorted octahedron. The two values of the E-parameter are explained by the presence of two slightly different structural forms of complex 1 in the crystal lattice. A second hypothesis was explored to explain the experimental data. The calculation for the simulation was done taking into account that the g and D tensors are not collinear due to the low symmetry of complex 1. In that case, the spin Hamiltonian parameters found are D = -3.29 (+/-0.01) cm(-1), E = 0.51 (+/-0.01) cm(-1), g(x) = 2.00 (+/-0.005), g(y) = 1.98 (+/-0.005), and g(z) = 2.01 (+/-0.005).     

Observation of kinetic and structural scalings during slow coalescence of nanobubbles in an aqueous solution

The addition of salt can induce the slow coalescence of nanobubbles (approximately 100 nm) in an aqueous solution of alpha-cyclodextrin (alpha-CD). A combination of static and dynamic laser light scattering was used to follow the coalescence. Our results reveal that its kinetic and structural properties follow some scaling laws; namely, the average size () of the nanobubbles is related to their average mass () and the coalescence time (t) as approximately (d(f)) and approximately t(gamma) with two salt-concentration-dependent scaling exponents (d(f) and gamma). For a lower sodium chloride concentration (C(NaCl) = 40 mM), gamma = 0.13 +/- 0.01 and d(f) = 1.71 +/- 0.02. The increase of C(NaCl) to 80 mM results in gamma = 0.32 +/- 0.01 and d(f) = 1.99 +/- 0.01. The whole process has two main stages: the aggregation and the coalescence. At the lower C(NaCl), the process essentially stops in the aggregation stage with some limited coalescence. At higher C(NaCl), coalescence occurs after the aggregation and results in large bubbles.     

DFT based Monte Carlo simulations of poly(9,9-dialkylfluorene-2,7-diyl) polymers in solution

The dilute solution properties of poly(9,9-dihexylfluorene-2,7-diyl) (PDHF) were studied by coupled SEC/light scattering and MALDI-TOF over a large molecular weight (MW) span ranging from PDHF oligomers (1-8-mer) to high MW polymer. The results were compared with Monte Carlo simulations based on realistic PDHF models obtained from X-ray data and density functional theory (DFT) calculations and with a DFT based Kratky-Porod-Benoit-Doty (KPBD) worm-like chain. The simulations called "selective random walk" (SRW) and the corresponding "selective self-avoiding random walk" (SSAW) explicitly take into account the rotationally labile bonds between the fluorene units in that four distinct torsion angles (+/-37.5 and +/-143 degrees) between the units are chosen randomly. The simulations better account than the KPBD model for the experimental data obtained by us and others for various poly(9,9-dialkylfluorene-2,7-diyl) polymers but still give somewhat larger values for the radii of gyration and hydrodynamic volumes. The torsion angle selectivity of the SRW and SSAW simulations predict long chain sections punctuated by sudden sharp loops.     

Synthesis and Crystal Structure of (E)-4-(Benzyloxy)-2-(cinnamoyloxy)-N,N,N-trimethyl-4-oxobutan-1-aminium Chloride as a Double-prodrug

A novel hydrochloride quaternary ammonium salt (E)-4-(benzyloxy)-2-(cinnamo- yloxy)-N,N,N-trimethyl-4-oxobutan-1-aminium chloride (C23H29NO4Cl2, Mr = 454.37) has been synthesized via the sequence of acetylation and esterification by using L-carnitine (L-4-N-trimethy- lammonium-3-hydroxybutyric acid, LC) and cinnamic acid as the starting materials, and its crystal structure was determined by single-crystal X-ray diffraction method. The crystal belongs to monoclinic, space group P212121 with a = 10.1670(4), b = 10.4488(4), c = 22.9795(11) ?, V = 2441.18(18) ?3, Z = 4, Dc = 1.236 g/cm3, μ(MoKα) = 0.293 mm?1, F(000) = 960, Flack factor = –0.01(11), the final R = 0.0489 and wR = 0.1550 for 3350 observed reflections (I > 2σ(I)) and R = 0.0953 for all 5648 unique reflections. The crystal structure involves a conjugated system which shows a reverse olefin structure.     

Biofunctionalization of aqueous dispersed, alumina membrane-templated polymer nanorods for use in enzymatic chemiluminescence assays

The noncovalent immobilization of alkaline phosphatase (ALP) onto aqueous dispersed nylon 6 nanorods ( approximately 310 nm mean diameter; approximately 6 microm mean length) prepared by anodic aluminum oxide (AAO) membrane templating was studied. Using multi-stacked layer-by-layer (LBL) assembly with the cationic quaternary ammonium polymer Sapphire II , the amount of ALP enzyme loaded onto the polymer nanostructures was found to be 115+/-7 microg mg(-1) nanorod. The biofunctionalized nanorods were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD . The results indicate that the kinetic parameters, K(m) and V(max), for the catalytic activity of the nanostructure-bound ALP enzyme are different from those of soluble ('free') ALP. While the K(m) value was measured to be 156 microM for free ALP, the apparent K(m) value determined for the LBL-immobilized ALP is approximately 20% lower (122 microM). Furthermore, despite the relatively high enzyme loading capacity of the nanorods, the specific activity of the bound ALP enzyme was found to be almost nine times lower than that measured for free ALP. Finally, additional experiments revealed that the catalytic activities of both free ALP and nanorod-conjugated ALP are affected similarly by changes in pH, with optimal performance levels occurring under conditions of pH 9.5. To the best of our knowledge, this study represents the first report examining the preparation of aqueous dispersed, AAO-templated polymer nanorods for potential application as enzyme scaffolds in chemiluminescent-based assay systems.     

Ligand-localized electron trapping at sensitized semiconductor interfaces

Nanocrystalline (anatase), mesoporous TiO2 thin films were derivatized with [Ru(bpy)2(deebq)](PF6)2 or [Os(bpy)2(deebq)](PF6)2, where bpy is 2,2'-bipyridine and deebq is 4,4'-diethylester-2,2'-biquinoline. Both compounds bind to the nanocrystalline TiO2 films with typical limiting surface coverages of 7 (+/-2) x 10-8 mol/cm2. Electrochemical measurements show that the first reduction of these compounds (-0.60 V vs SCE) occurs prior to TiO2 reduction. Steady-state illumination in the presence of the sacrificial electron donor triethylamine leads to the appearance of the reduced compound, MII(deebq-)(bpy)2+/TiO2. Neither the photoluminescent excited states or the reduced forms of these compounds inject electrons efficiently into TiO2. Transient absorption measurements after a approximately 10-ns laser pulse, reveal greater than 80% MLCT excited states and a smaller fraction of extremely long-lived charge-separated state intermediates assigned to equal concentrations of MII(deebq-)(bpy)2+/TiO2 and MIII(deebq)(bpy)23+/TiO2. The results are consistent with a mechanism of ultrafast electron injection followed by ligand-localized trapping on a second compound. The quantum yield for formation of the charge-separated states (phiCSS) is excitation wavelength dependent. With 417 nm excitation, phiCSS(417) = 0.14 +/- 0.03, and this decreases with 532.5 nm excitation, phiCSS(532.5) = 0.08 +/- 0.03, and 683 nm excitation for M = Os, phiCSS(683) = 0.05 +/- 0.01. Electron transfer to yield ground-state products, MII(deebq-)(bpy)2+/TiO2 + MIII(deebq)(bpy)23+/TiO2 --> 2 MII(deebq)(bpy)22+/TiO2, occurs with a driving force of 2.05 eV for Ru/TiO2 and 1.64 eV for Os/TiO2. The dynamics of this process were quantified on a millisecond time scale and were found to follow second-order kinetics. The intermediates are sufficiently long-lived that continued pulsed excitation at 10 Hz leads to high concentrations and the formation of transient images on the semiconductor surface that are easily observed by the naked eye.     

Comparison of physico-chemical pretreatment methods to seawater reverse osmosis: Detailed analyses of molecular weight distribution of organic matter in initial stage

In desalination, effective pretreatment is the key to reduce membrane fouling that occurs during the seawater reverse osmosis (SWRO) process. However, it is difficult to compare the flux decline after different pretreatments using a small-scale reverse osmosis filtration unit. In this study, we successfully evaluated the effect of pretreatment on SWRO in terms of molecular weight distribution (MWD) of seawater organic matter (SWOM) after 20 h of SWRO operation. Microfiltration (MF), ultrafiltration (UF), ferric chloride (FeCl₃) flocculation and powdered activated carbon (PAC) adsorption, were used as pretreatment. The effluents and the retentates after each pretreatment and 20 h of SWRO operation were characterized in terms of MWD.Although the normalized flux of SWRO showed similar flux decline (J/J₀ = 0.17) with/without pretreatment, SWOM concentration in the retentates after different pretreatments was different in quantity and it increased linearly with time. The slope of the SWOM increase was 0.110, 0.096, 0.077 and 0.059 after MF, FeCl₃ flocculation, UF and PAC adsorption pretreatments, respectively. MW peaks for the seawater used in this study consisted of 1200 Da (biopolymers), 950 Da (fulvic acids), 650 Da (hydrolysates of humic substances), 250 Da (low MW acids) and 90 Da (low MW neutrals and amphiphilics). FeCl₃ flocculation preferentially removed 1200 Da (biopolymers), while PAC adsorption mostly removed 950 Da (fulvic acids). UF and NF removed only a marginal amount of relatively large organics, while RO removed the majority of organics. The intensity of 1200, 950, 650 and 250 Da MW in the RO retentates increased with the RO operation time. The organics of MW around 1200 Da (biopolymers) had a relatively low rate of increase with time compared with those of lower MW. This suggests that the SWOM of 1200 Da MW was preferentially retained on the membrane surface.     

Gas-phase oxidation of Cm+ and Cm2+ --thermodynamics of neutral and ionized CmO

Fourier transform ion cyclotron resonance mass spectrometry was employed to study the products and kinetics of gas-phase reactions of Cm (+) and Cm (2+); parallel studies were carried out with La (+/2+), Gd (+/2+) and Lu (+/2+). Reactions with oxygen-donor molecules provided estimates for the bond dissociation energies, D[M (+)-O] (M = Cm, Gd, Lu). The first ionization energy, IE[CmO], was obtained from the reactivity of CmO (+) with dienes, and the second ionization energies, IE[MO (+)] (M = Cm, La, Gd, Lu), from the rates of electron-transfer reactions from neutrals to the MO (2+) ions. The following thermodynamic quantities for curium oxide molecules were obtained: IE[CmO] = 6.4 +/- 0.2 eV; IE[CmO (+)] = 15.8 +/- 0.4 eV; D[Cm-O] = 710 +/- 45 kJ mol (-1); D[Cm (+)-O] = 670 +/- 40 kJ mol (-1); and D[Cm (2+)-O] = 342 +/- 55 kJ mol (-1). Estimates for the M (2+)-O bond energies for M = Cm, La, Gd, and Lu are all intermediate between D[N 2-O] and D[OC-O] - that is, 167 kJ mol (-1) < D[M (2+)-O] < 532 kJ mol (-1) - such that the four MO (2+) ions fulfill the thermodynamic requirement for catalytic oxygen-atom transport from N2O to CO. It was demonstrated that the kinetics are also favorable and that the CmO (2+), LaO (2+), GdO (2+), and LuO (2+) dipositive ions each catalyze the gas-phase oxidation of CO to CO2 by N2O. The CmO 2 (+) ion appeared during the reaction of Cm (+) with O 2 when the intermediate, CmO (+), was not collisionally cooled - although its formation is kinetically and/or thermodynamically unfavorable, CmO 2 (+) is a stable species.     

Comprehensive chemical derivatization for gas chromatography-mass spectrometry-based multi-targeted profiling of the major phytohormones

In the present investigation we report selection of the N-methyl-N-(tert.-butydimethylsilyl)trifluoroacetamide (MTBSTFA) reagent as the most comprehensive derivatization protocol among 17 tested reactions covering trifluoroacetylation, pentafluorobenzylation, methylations, and trimethylsilylations. MTBSTFA allowed easy and robust tert.-butyldimethylsilyl derivatization of 1-aminocyclopropane-1-carboxylic acid, indole-3-acetic acid, (+/-)-jasmonic acid, salicylic acid, (+/-)-abscisic acid, meta-topolin, and trans-zeatin. Detection limits as analysed by selected ion monitoring quadrupole GC-MS were 0.2, 0.01, 1.0, 0.02, 0.3, 0.3, and 0.9 pmol of injected substance, respectively. Analysis of gibberellic acid A3, trans-zeatin riboside and (+/-)-abscisic acid-beta-D-glucopyranosyl ester was best when coupled by splitting extracts and trimethysilylation. The MTBSTFA derivatization protocol was optimised, and validated. The preparation was insensitive to 2% residual water and to < or = 1 day storage at room temperature. The final scheme was highly reproducible and successfully applied to extracts from approximately 300 mg (fresh mass) of tobacco (Nicotiana tabacum) root and Arabidopsis thaliana seedling.     

Domino double Michael-Claisen cyclizations: a powerful general tool for introducing quaternary stereocenters at C4 of cyclohexane-1,3-diones and total synthesis of diverse families of sterically congested alkaloids

Reactions of substituted acetone derivatives with acrylic acid esters (>200 mol %) in the presence of t-BuOK (200 mol %) in t-BuOH-THF (1:1 by volume) turned out to proceed as a cascade process consisting of the first Michael addition, the second Michael addition, and the last Claisen reaction to afford 4,4-disubstituted cyclohexane-1,3-diones. Only more substituted enolates play the role of a Michael donor in this cascade process, and therefore the ketone took up two alkoxycarbonylethyl groups on the same carbon bearing more substituents. Such intermediates were followed by intramolecular Claisen reactions leading to cyclohexane-1,3-diones bearing quaternary stereogenic centers at C(4), which bears an alkoxycarbonylethyl group and the substituent of the starting acetone derivatives. Thus-obtained 4,4-disubstituted cyclohexane-1,3-diones were successfully employed for total syntheses of intricate alkaloids of biological interest such as (+)-aspidospermidine, (+/-)-galanthamine, (+/-)-lycoramine, and (+/-)-mesembrine, all featuring quaternary stereogenic centers. DFT calculations provided us with clear-cut explanations for the observed chemoselectivity of the cascade process involving ketone-based enolates under thermodynamically controlled conditions.     

Resolution of (R,S)-(+/-)-glycidyl butyrate with immobilized Y-11 Trichosporon capitatum lipase in gelatin-containing microemulsion-based organogels

An extracellular lipase produced by the strain of Y-11 Trichosporon capitatum was purified to homogeneity by ammonium sulfate precipitation and four chromatographic steps. The purified lipase showed enhanced activity when it was immobilized in gelatin-containing microemulsion-based organogels. Furthermore, the resolution of (R,S)-(+/-)-glycidyl butyrate by this immobilized lipase gave a product of (S)-(-)-glycidol with approximately 98% ee (E=96). By using chiral HPLC separation, (S)-(-)-glycidol was obtained in enantiopure form. Scaled-up reaction in 2l shake flask was also performed and the repeated use of 15 times of immobilized Y-11 T. capitatum lipase resulted in little loss in its activity (4.8%).     

Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine

The excitation of pBr322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width approximately 140 fs, 76 MHz pulse repetition, average power output focused through 10x microscope objective is approximately 1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 microM of the intercalator proflavine (PF), both direct single- and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 +/- 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 +/- 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (approximately 400-500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.