Development of transition state analogue inhibitors for <Emphasis Type="Italic">N</Emphasis>-acetylglycosyltransferases bearing <Emphasis Type="SmallCaps">d</Emphasis>psico- or <Emphasis Type="SmallCaps">d</Emphasis>tagatofuranose scaffolds

New potential transition state analogue inhibitors for N-acetylglucosyltransferases (GnTs) were synthesised. These compounds based on psico- and tagatofuranose (structure) scaffold contained a 2-thiophenyl-1-O-diethylphosphate moiety mimicking the proposed model of the transition state of the enzymatic reaction catalysed by N-acetylglucosyltransferases. The synthesised compounds as well as their precursors were fully characterised by NMR, optical rotation and mass techniques. Anomeric configuration of tagatofuranose derivatives was confirmed by X-ray crystallography. Two types of potential human glycosyltransferase (GnTs) inhibitors representing donor UDP-GlcNAc, assigned for biological assays on human GnTs, were prepared.     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-(2-chloroethyl)glycine and -<Emphasis Type="SmallCaps">DL</Emphasis>-alanine esters

Two methods for the synthesis of N-(2-chloroethyl)glycine and-DL-alanine esters are proposed: 1) reductive amination of the C=O group of glyoxilic or pyruvic acids upon treatment with 2-chloroethylamine and sodium cyanoborohydride in methanol and 2) alkylation of 2-chloroethylamine with α-haloalkanoic acid esters in K₂CO₃-MeCN two-phase system. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2372–2374, December, 2007.     

Determination of <Emphasis Type="SmallCaps">D</Emphasis>- and <Emphasis Type="SmallCaps">L</Emphasis>-amino acids produced by cyanobacteria using gas chromatography on Chirasil-Val after derivatization with pentafluoropropyl chloroformate

A rapid and simple method was developed for the determination of free amino acids (AAs) released from cyanobacteria. The procedure involves trapping of AAs from the centrifuged cyanobacterial culture fluid on a cation-exchange resin, their release together with the resin by direct treatment with the reaction medium, followed by immediate derivatization with a corresponding chloroformate. The extractive alkylation transfers the analytes into an organic phase, an aliquot of which is subjected to GC analysis. Identification and quantification of AAs was performed by GC/MS and GC/FID, respectively, using propyl chloroformate (PCF) as the derivatization reagent. For chiral analysis, the cyanobacteria extracts were treated with 2,2,3,3,3-pentafluoropropyl chloroformate (PFPCF) to create more volatile analytes. Separation of the AA enantiomers was accomplished on a Chirasil-Val capillary column and the D/L enantiomeric ratios were determined. AAs of cyanobacteria are considered to be important for the assessment of energy flow in an aquatic food web, nutrition value of cyanobacteria in a food web and for cell–cell communication within cyanobacteria. The highest levels of AAs were found in the summer period at the beginning of the season (July). In the September and October samples, the amount of AAs was lower, the number of D-AAs decreased and the D/L ratio was higher than in the July sample. Based on the obtained results it can be assumed that young populations excrete AAs in higher concentrations and a different composition compared to actively growing populations. Figure PFPCF derivatization scheme     

Oral <Emphasis Type="Italic">L</Emphasis>-Carnitine Supplementation and Exercise Metabolism

Summary. Oral L-carnitine supplementation is frequently reported to have beneficial effects on exercise capacity in clinical populations and has been considered as a potential ergogenic aid for endurance athletes. However, this latter view is largely unsubstantiated possibly due to many experimental studies being poorly controlled or difficult to compare. The potential for oral L-carnitine supplementation to influence skeletal muscle carnitine content has been questioned and there are several key factors identified that may explain variations between study outcomes. Recent more well controlled research suggests some potential for L-carnitine to act as a key regulator of cellular stress, possibly through an impact on the integration of carbohydrate and lipid metabolism, and this work should be followed up in future by well controlled studies in both athlete and clinical subject groups.     

New tin(<Emphasis Type="SmallCaps">iv</Emphasis>) <Emphasis Type="Italic">o</Emphasis>-iminosemiquinone complexes

New tin(iv) mono- and bis-o-iminosemiquinone complexes were obtained by the exchange reaction of radical anion lithium salt of 4,6-di-tert-butyl-N-(2,6-diisopropylphenyl)-o-imino-benzoquinone with tin(iv) organochlorides. The compounds synthesized were characterized by EPR spectroscopy and X-ray diffraction analysis. Substituents on the tin atom were found to affect stability of paramagnetic metal derivatives formed.     

Optical enzyme sensor for determining <Emphasis Type="SmallCaps">l</Emphasis>-lysine content using <Emphasis Type="SmallCaps">l</Emphasis>-lysine oxidase from the rockfish <Emphasis Type="BoldItalic">Sebastes schlegeli</Emphasis>

l-Lysine (l-Lys) in living bodies is critical for metabolism; therefore, determination of its levels in food is important. Most enzymatic methods for l-Lys analysis are performed using l-lysine oxidase (LyOx), but commercially manufactured LyOx is generally not highly selective for l-Lys among amino acids. We previously isolated LyOx as an antibacterial protein secreted from the skin of the rockfish Sebastes schlegeli. In the present study, we developed an optical enzyme sensor system for rapid and continuous determination of l-Lys using this LyOx. The system comprised an immobilized LyOx membrane, an optical oxygen probe, a flow system, and a personal computer. The amount of l-Lys was detected as a decrease in the oxygen concentration due to the LyOx reaction. The specificity of the sensor was examined against various amino acids. The sensor response was specific for l-Lys. Good reproducibility was obtained in 58 assays. The response of the sensor using commercially prepared LyOx was unstable compared with the response using LyOx isolated in our laboratory. Our sensor system could be used for 5 weeks without our having to change the enzyme membrane. The calibration curve for a standard l-Lys solution was linear from 0.1 to 3.0 mmol L⁻¹. One assay could be completed within 2 min. The sensor was applied to determine the l-Lys content in food samples such as bonito cooking water and scallop hepatopancreas. The values obtained using the sensor and conventional high-performance liquid chromatography methods were well correlated.     

Bioconversion of <Emphasis Type="SmallCaps">d</Emphasis>-glucose into <Emphasis Type="SmallCaps">d</Emphasis>-glucosone by Glucose 2-oxidase from <Emphasis Type="Italic">Coriolus versicolor</Emphasis> at Moderate Pressures

Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of d-glucose at carbon 2 in the presence of molecular O₂ producing d-glucosone (2-keto-glucose and d-arabino-2-hexosulose) and H₂O₂. It was used to convert d-glucose into d-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of d-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H₂O₂ acted as inhibitor for this reaction. The rate of bioconversion of d-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO₂ at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55°C) and pH (5.0) of d-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E _a) was 32.08 kJ mol⁻¹ and kinetic parameters (V _max, K _m, K _cat and K _cat/K _m) for this bioconversion were 8.8 U mg⁻¹ protein, 2.95 mM, 30.81 s⁻¹ and 10,444.06 s⁻¹ M⁻¹, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of d-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.     

Protolytic properties of <Emphasis Type="Italic">DL</Emphasis>-alanyl-<Emphasis Type="Italic">DL</Emphasis>-methionine and its complexation in aqueous solutions and micellar solutions

Dissociation constants of DL-alanyl-DL-methionine have been determined in water and micellar solutions of surfactants (anionic sodium n-dodecyl sulfate, cationic cetylpyridinium chloride, and nonionic Brij 35). It has been established that CuA⁺ and CuH_–1A complexes are formed in water and micellar solutions of sodium n-dodecyl sulfate, while CuA⁺, CuH_–1A, and Cu_–2A^– complexes are formed in micellar solutions of cetylpyridinium chloride and Brij 35. Stability of the complexes depends on micelle surface charge and degrees of binding of individual chemical forms by a micellar pseudophase.     

Extracellular Production and Characterization of Two <Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Asparaginases

l-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four l-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.     

Preparation of nanoparticle colloids of methoxy poly(ethylene glycol)-<Emphasis Type="Italic">b</Emphasis>-poly(<Emphasis Type="SmallCaps">D</Emphasis>,<Emphasis Type="SmallCaps">L</Emphasis>-lactide): effects of surfactant and organic solvent

Nanoparticle colloids of methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (MPEG-b-PDLL) diblock copolymer were prepared by a modified spontaneous emulsification solvent diffusion method using acetone/ethanol as the mixture organic solvents. The MPEG-b-PDLL was synthesized by ring-opening polymerization of D,L-lactide using stannous octoate and MPEG with molecular weight of 5,000 g/mol as the initiating system. The MPEG-b-PDLL obtained was an amorphous polymer with molecular weight of 73,600 g/mol. Influences of acetone/ethanol (v/v) ratios and Tween 80 surfactant concentrations on characteristics of the colloidal nanoparticles were investigated and discussed. Light-scattering analysis showed that average diameters of the surfactant-free colloidal nanoparticles were in the range of 86–124 nm. The nanoparticle sizes decreased as the ethanol ratio increased. The Tween 80 did not show the significant effect on the nanoparticle sizes. Scanning electron micrographs of dried nanoparticles that demonstrated the aggregation of most particles suggested they were the soft nanoparticles. However, the dried nanoparticle morphology can be observed from scanning electron microscopy as having a spherical shape and smooth surfaces.     

Determination of serum <Emphasis Type="SmallCaps">d</Emphasis>-lactic and <Emphasis Type="SmallCaps">l</Emphasis>-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization

d-Lactic and l-lactic acids were simultaneously determined by means of a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. As a fluorescence reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was employed for the fluorescence derivatization of lactic acid. The proposed HPLC system adopted both octylsilica (Cadenza CD-C8) and amylose-based chiral columns (CHIRALPAK AD-RH), which proved to give a sufficient enantiomeric separation of the lactic acid derivatives with a separation factor () of 1.32 and a resolution (R_s) of 1.98. Moreover, the features of the first elution of d-lactic acid peak in the proposed HPLC were convenient for the determination of trace amount of serum d-lactic acid, which is known to increase under diabetes. Intra-day and inter-day accuracies were in the range of 90.5–101.2 and 89.0–100.7%, and the intra-day and inter-day precisions were 0.3–1.2 and 0.4–4.8%, respectively. The proposed method was applied to determine d-lactic and l-lactic acids in human serum of normal subjects and diabetic patients, showing that both d-lactic and l-lactic acid concentrations were significantly increased in the serum of diabetic patients (n=31) as compared with normal subjects (n=21). This fact was found for the first time owing to the development of the proposed HPLC method which is able to determine d-lactic and l-lactic acid simultaneously. Finally, serum d-lactic acid concentrations determined by the proposed HPLC method were compared with those from a reported enzymatic assay, and the smaller p value between normal subjects and diabetic patients was shown by the proposed HPLC method.     

Thermal properties of amphiphilic biodegradable triblock copolymer of <Emphasis Type="SmallCaps">l</Emphasis>,<Emphasis Type="SmallCaps">l</Emphasis>-lactide and ethylene glycol

Samples of poly(l,l-lactide)-block-poly(ethylene glycol)-block-poly(l,l-lactide) (PLLA-PEG-PLLA) were synthesized from l,l-lactide polymerization using stannous 2-ethylhexanoate, Sn(Oct)₂ as initiator and di-hydroxy-terminated poly(ethylene glycol) (PEG) (M _n  = 4000 g mol⁻¹) as co-initiator. The chemical linkage between the PEG segment and the PLA segments was characterized by Fourier transform infrared spectroscopy (FTIR). Thermogravimetry analysis (TG) revealed the copolymers composition and was capable to show the deleterious effect of an excess of Sn(Oct)₂ in the polymer thermal stability, while Differential Scanning Calorimetry (DSC) allowed the observation of the miscibility between the PLLA and PEG segments in the different copolymers.     

Ferrocene-containing tri- and tetranuclear cyclic copper(<Emphasis Type="SmallCaps">i</Emphasis>) and silver(<Emphasis Type="SmallCaps">i</Emphasis>) pyrazolates

New tri- and tetranuclear macrocyclic silver(i) and copper(i) 3-ferrocenyl-5-(trifluoromethyl)pyrazolates were prepared: [{(3-((η⁵-C₅H₄)Fe(η⁵-C₅H₅))-5-(CF₃)-Pz}M]₃ (M = Cu (1), Ag (2)) and [{(3-(( η⁵-C₅H₄)Fe(η⁵-C₅H₅))-5-(CF₃)-Pz}Cu]₄ (3). The structures of compounds were established by X-ray diffraction analysis. In the crystalline state, a planar trinuclear silver-containing macrocycliс pyrazolate and a saddle-shaped tetranuclear copper-containing macrocycle are formed. The introduction of a bulky substituent, ferrocene, into the pyrazole ligand results in complete shielding of the acidic metal sites, which precludes the coordination of base molecules.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Low-temperature thermodynamic properties of <Emphasis Type="SmallCaps">dl</Emphasis>-cysteine

Heat capacity C _p(T) of the crystalline dl-cysteine was measured on heating the system from 6 to 309 K by adiabatic calorimetry; thermodynamic functions were calculated based on these data smoothed in the temperature range 6–273.15 K. The values of heat capacity, entropy, and enthalpy at 273.15 K were equal to 142.4, 153.3, and 213.80 J K⁻¹ mol⁻¹, respectively. At about 300 K, a heat capacity peak was observed, which was interpreted as an evidence of a first-order phase transition. The enthalpy and the entropy of the transition are equal, respectively, to 2300 ± 50 and 7.6 ± 0.1 J K⁻¹ mol⁻¹.     

<Emphasis Type="SmallCaps">d</Emphasis>-Amino Acids in Animal Peptides

Summary. Secreted peptides from diverse sources have been found to contain a d-amino acid. From the sequence of cloned mRNAs coding for the precursors of such peptides it could be deduced that in all cases tested so far the d-amino acid in the final product is derived from the corresponding l-amino acid present in the primary product of translation. Enzymes catalyzing such an l- to d-isomerization in peptide linkage have been isolated from the venom of a spider and the skin secretions of frogs. Even though these are completely different proteins, the reaction mechanism is the same, namely a de-protonation/re-protonation of the α-carbon of an amino acid with concomitant inversion of the chirality. Sequences potentially coding for homologues of the frog enzyme are present in the genome of different vertebrate species.     

Low-temperature thermodynamic properties of <Emphasis Type="Italic">L</Emphasis>-cysteine

Heat capacity C _p(T) of the orthorhombic polymorph of L-cysteine was measured in the temperature range 6–300 K by adiabatic calorimetry; thermodynamic functions were calculated based on these measurements. At 298.15 K the values of heat capacity, C _p; entropy, S _m⁰(T)-S _m⁰(0); difference in the enthalpy, H _m⁰(T)-H _m⁰(0), are equal, respectively, to 144.6±0.3 J K⁻¹ mol⁻¹, 169.0±0.4 J K⁻¹ mol⁻¹ and 24960±50 J mol⁻¹. An anomaly of heat capacity near 70 K was registered as a small, 3–5% height, diffuse ‘jump’ accompanied by the substantial increase in the thermal relaxation time. The shape of the anomaly is sensitive to thermal pre-history of the sample.     

Coordination polymers based on zinc(<Emphasis Type="SmallCaps">ii</Emphasis>) and manganese(<Emphasis Type="SmallCaps">ii</Emphasis>) with 1,4-cyclohexanedicarboxylic acid

Three new metal-organic coordination polymers were obtained namely, [Mn₃(chdc)₃-(NMP)₂(DMF)₂] (1, chdc^2– is trans-1,4-cyclohexanedicarboxylate, NMP is N-methylpyrrolidone, DMF is N,N-dimethylformamide), [Zn₃(chdc)₃(NMP)₂]?2NMP (2), and [Zn₃(chdc)₃(ur)-(DMF)_0.5]?DMF (3, ur is the urotropine). The crystal structures of polymers 1, 2, and 3 were determined by single-crystal X-ray crystallography. All three compounds were found to contain a trinuclear secondary building unit {M₃(OOC)₆}. Coordination polymers 1 and 2 have a layered structure, while polymer 3 has a three-dimensional coordination framework with isolated pores formed due to the presence of urotropine bridging molecules. Compounds 1 and 3 were characterized by IR spectroscopy, thermogravimetric and elemental analysis data, powder X-ray diffraction. Compound 3 was also characterized by UV-Vis diffuse reflectance spectrum.     

Temperature- and pressure-responsive properties of <Emphasis Type="SmallCaps">l</Emphasis>- and<Emphasis Type="SmallCaps"> dl</Emphasis>-forms of poly(<Emphasis Type="Italic">N</Emphasis>-(1-hydroxymethyl)propylmethacrylamide) in aqueous solutions

The structural transition of the l- and dl forms of poly(N-(1- hydroxymethyl)propylmethacrylamide (PHMPMA) in aqueous solution was studied by measuring the pressure dependence of the apparent scattering intensity, differential scanning calorimetry (DSC), and circular dichroism (CD). The thermodynamic implications of the results are discussed in relation to the chiral structure of the side chain, and differences in the thermal and barometric transitions. T-P diagrams of the transition showed characteristic ellipsoid features. Antagonism of the temperature and pressure effects was observed only for P(dl-HMPMA). For P(l-HMPMA), the transition temperature (T _tr) decreased with increasing pressure, and the highest T _tr was observed at atmospheric pressure (0.1 MPa). For both polymers, the highest P _trs were observed at the lowest temperatures. The l polymer showed a specific negative peak in its CD spectrum at around 220 nm in the lower temperature region and the temperature dependence was reproduced by a single-step transition, with the midpoint corresponding to the T _tr obtained from the scattering measurements. Coupled with the results from the DSC, the different behavior between the P(l-HMPMA) and P(dl-HMPMA) could be explained in terms of the chain states before and after the transition. The cooperative factors derived from the DSC measurement revealed that about 4 to 5 polymers of the present size were necessary to perform a thermal transition for P(l-HMPMA), and that P(dl-HMPMA) underwent its transition as an almost single molecular event.This revised version was published online in June 2005 with correction to the article category.     

Mechanochemical synthesis of platinum(<Emphasis Type="SmallCaps">IV</Emphasis>) complexes with <Emphasis Type="Italic">N</Emphasis>-heterocyclic carbenes

A new method for the synthesis of complexes Pt^IV(NHC)X₄L (NHC is N-heterocyclic carbene of imidazole or benzimidazole series; X = Cl, Br; L is N-coordinated pyridine or NHC) based on mechanochemical oxidation of complexes Pt^II(NHC)X₂L with dichloroiodobenzene (PhICl₂) or pyridinium hydrobromide perbromide (PyHBr₃) was proposed. Mechanochemical activation led to reduction in the synthesis time and increase in the selectivity of halogenation and yields of the target Pt^IV complexes (74–98%) as compared to the reaction in solutions.