Purification and characterization of an extracellular α-l-arabinofuranosidase from Fusarium oxysporum

An α-l-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60°C. It hydrolyzed aryl α-l-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect between the α-l-arabinofuranosidase and an endo-(1 →4)-β-d-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan.     

Glycosylation of betulin acetates with glycals

Betulin 2-deoxy-α-d-, 2-deoxy-α-l-, and 2,6-dideoxy-α-l-arabino-hexopyranosides were synthesized by acid-catalyzed glycosylation (cationite in the H⁺ form, LiBr) of betulin 3- and 28-monoacetates with glycal acetates. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 531–534, March, 1998.     

Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

Preparation of optical active aliphatic <Emphasis Type="Italic">α</Emphasis>-amino acid from fatty acid: synthesis of <Emphasis Type="SmallCaps">l</Emphasis>-norvaline and <Emphasis Type="SmallCaps">d</Emphasis>-norvaline

A new synthesis of l-norvaline is described. Using valeric acid as the raw material, α-brominevaleryl chloride was achieved by acyl chlorination and α-position bromination in one-pot with the yield of 84.7%. The yields of the following ammoniation of α-brominevaleryl chloride and resolution of dl-norvaline were 88.7% and 26.7%, respectively. d-Norvaline was also obtained in a similar method from the waste resolution solution. Other optical active amino acids, valine, α-aminobutyric acid and alanine were also synthesized in similar ways.     

Structural modeling of glucanase–substrate complexes suggests a conserved tyrosine is involved in carbohydrate recognition in plant 1,3-1,4-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucanases

Glycosyl hydrolase family 16 (GHF16) truncated Fibrobacter succinogenes (TFs) and GHF17 barley 1,3-1,4-β-d-glucanases (β-glucanases) possess different structural folds, β-jellyroll and (β/α)₈, although they both catalyze the specific hydrolysis of β-1,4 glycosidic bonds adjacent to β-1,3 linkages in mixed β-1,3 and β-1,4 β-d-glucans or lichenan. Differences in the active site region residues of TFs β-glucanase and barley β-glucanase create binding site topographies that require different substrate conformations. In contrast to barley β-glucanase, TFs β-glucanase possesses a unique and compact active site. The structural analysis results suggest that the tyrosine residue, which is conserved in all known 1,3-1,4-β-d-glucanases, is involved in the recognition of mixed β-1,3 and β-1,4 linked polysaccharide.     

The synthesis ofN-acetyl-β-l-fucosamine-1-phosphate and uridine 5′-diphospho-N-acetyl-β-l-fucosamine

Uridine 5′-(2-acetamido-2,6-dideoxy-β-l-galactopyranosyl) diphosphate (uridine 5′-diphospho-N-acetyl-β-l-fucosamine) was synthesized. The key intermediate, 3,4-di-O-acetyl-2-azido-2,6-dideoxy-β-l-galactopyranosyl dibenzyl phosphate, was prepared by a previously unknown reaction of cesium dibenzyl phosphate with the corresponding α-glycosyl nitrate and was then converted into theN-acetylated glycosyl phosphate and nucleoside diphosphate sugarsvia 3,4-di-O-acetyl-2-amino-2,6-dideoxy-β-l-galactopyranosyl phosphate using mildN-acetylation andO-deacetylation as the last synthetic steps. Published inIzvestiya Akademii Nauk, Seriya Khimicheskaya, No. 11, pp. 1919–1923, November, 2000.     

Stereoselective synthesis of triterpene 2-deoxy-α-d-lyxo-hexopyranosides

2-Deoxy-α-d-lyxo-hexopyranosides of 18β-glycyrrhetic acid, its 11-deoxo derivative and allobetulin were synthesized by glycosylation of oleonane-type triterpene alcohols withd-galactal acetate in the presence ofN-iodosuccinimide followed by deiodination and deprotection. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 596–600. March, 1997.     

Combination of ionic liquid [bmim]PF<Subscript>6</Subscript> with boron trifluoride etherate as an efficient catalytic system for the glycosylation of α-tocopherol and naphthotocopherol

The use of a combination of ionic liquid [bmim]PF₆ with boron trifluoride etherate as the catalyst in the glycosylation of α-tocopherol and chromanol of vitamin K₁ (naphthotocopherol) allowed us to obtain β-glycosides in high yield when β-anomer of peracetylated D-glucose was used. In addition, usually inactive α-anomers of peracetylated D-glucopyranose and D-galactopyranose were involved in this reaction. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2401–2404, December, 2007.     

Fabrication of modified TiO2 nanoparticle carbon paste electrode for simultaneous determination of dopamine, uric acid, and l-cysteine

A carbon paste electrode, modified with 2, 2′-[1,7-hepthandiylbis(nitriloethylidyne)]-bis-hydroquinone and TiO₂ nanoparticles, was used for the simultaneous determination of dopamine (DA), uric acid (UA), and l-cysteine. The study was carried out by using cyclic voltammetry, chronoamperometry, and square wave voltammetry (SWV) techniques. Some kinetic parameters such as the electron transfer coefficient (α) and heterogeneous rate constant (k_s) were also determined for the DA oxidation. A dynamic range of 8.0–1400 μM, with the detection limit of 8.4 × 10⁻⁷ M for DA, was obtained using SWV (pH = 7.0). The prepared electrode was successfully applied for the determination of DA, UA, and l-cysteine in real samples.     

Determination of the Enantiomeric Purity of l-Carnitine and Acetyl-l-Carnitine by Chiral Liquid Chromatography

A chiral liquid chromatographic method for determination of the enantiomeric purity of both l-carnitine and acetyl-l-carnitine is described. Separation of the enantiomers of dl-carnitine and acetyl-dl-carnitine was achieved on a commercial chiral column (Chiralcel OD-R) after derivatization with (alpha-bromo)methyl phenyl ketone. Introduction of this lipophilic UV chromophoric group to the carnitine and acetylcarnitine molecules improved their retention, resolution, and UV detection. The mobile phase was 74:26 (v/v) 0.5 mol L^-1 sodium perchlorate–acetonitrile, pH 3.8, and the flow rate was 0.4 mL min^-1. Detection was performed at 235 nm. The method is selective and reliable for determination of the enantiomeric purity of bulk drug substances l-carnitine and acetyl-l-carnitine.     

An easy access to a 3,6-branched mannopentaoside bearing one terminal [1-<Superscript>13</Superscript>C]-labeled <Emphasis Type="SmallCaps">D</Emphasis>-mannopyranose residue

Methyl 2,4-di-O-benzoyl-α-D-mannopyranoside was used as a key intermediate in the synthesis of 3,6-branched mannopentaoside bearing one terminal D-[1-¹³C]mannopyranose residue, viz., methyl 6-O-[3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranosyl)-3-O-{α -D-[1-¹³C]mannopyranosyl}-α-D-mannopyranoside. Dedicated to Academician N. K. Kochetkov on the occasion of his 90th birthday. __________ Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1250–1255, May, 2005.     

The electrochemical polymerization of <Emphasis Type="Italic">o</Emphasis>-phenylenediamine on <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine functionalized glassy carbon electrode and its application

The polymerization of o-phenylenediamine (OPD) on l-tyrosine (Tyr) functionalized glassy carbon electrode (GCE) and its electro-catalytic oxidation towards ascorbic acid (AA) had been studied in this report. l-Tyrosine was first covalently grafted on GCE surface via electrochemical oxidation, which was followed by the electrochemical polymerization of OPD on the l-tyrosine functionalized GCE. Then, the poly(o-phenylenediamine)/l-tyrosine composite film modified GCE (POPD-Tyr/GCE) was obtained. X-ray photo-electron spectroscopy (XPS), field emission scanning electron microscope (SEM), and electrochemical techniques have been used to characterize the grafting of l-tyrosine and the polymerization and morphology of OPD film on GCE surface. Due to the doping of the carboxylic functionalities in l-tyrosine molecules, the POPD film showed good redox activity in neutral medium, and thus, the POPD-Tyr/GCE exhibited excellent electrocatalytic response to AA in 0.1 mol l⁻¹ phosphate buffer solution (PBS, pH 6.8). The anode peak potential of AA shifted from 0.58 V at GCE to 0.35 V at POPD-Tyr/GCE with a greatly enhanced current response. A linear calibration graph was obtained over the AA concentration range of 2.5 × 10⁻⁴–1.5 × 10^–3 mol l⁻¹ with a correlation coefficient of 0.9998. The detection limit (3δ) for AA was 9.2 × 10⁻⁵ mol l⁻¹. The modified electrode showed good stability and reproducibility and had been used for the determination of AA content in vitamin C tablet with satisfactory results.     

Amino acids in aqueous solution. Effect of molecular structure and temperature on thermodynamics of dissolution

The enthalpies of dissolution of glycine (Gly) and L-α-alanine (Ala) in water at 288.15–318.15 K were measured. The results were compared with the earlier obtained data for L-α-phenylalanine (Phe) and L-α-histidine (His). The standard enthalpies of dissolution (Δ_soln H ₀) and differences (ΔC _p ⁰ ) between the limiting partial molar heat capacity of the amino acids in solution and the heat capacity of the amino acids in the crystalline state were calculated in the temperature interval 273–373 K. Changes in the entropy of dissolution (ΔΔ_soln S ₀) and reduced Gibbs energy [Δ (Δ_soln G ₀/T)] in the temperature interval from 273 to 373 K were determined from the known thermodynamic relationships. The ΔC _p ⁰ value is negative for hydrophilic glycine and positive for other amino acids. The ΔΔ_soln S ⁰ values increase with an increase in the hydrophobicity of the amino acids. The Δ(Δ_soln G ₀/T) values become more negative in the order Ala, Phe, Gly, His. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 711–714, April, 2007.     

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.     

Stereoselective synthesis of 2,6-dideoxy-α-l-arabino-hexopyranoside of glycyrrhetic acid in the presence of iodine-containing promoters

Glycosylation of methyl glycyrrhetate withl-rhamnal acetate in the presence of iodine-containing promoters and subsequent hydrogenolysis yield 2,6-dideoxy--l-arabino-hexopyranoside of glycyrrhetic acid, an analog of glycyrrhizic acid, the natural glycoside of licorice root extract.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2993–2996, December, 1996.     

Determination and Pharmacokinetic Study of Calycosin-7-O-β-d-glucoside in Rat Plasma After Intravenous Administration of Aidi Lyophilizer

Aidi injection is a clinical medicine used in China for the treatment of cancer. Calycosin-7-O-β-d-glucoside is the main effective components of the formulas. In this study, a high performance liquid chromatographic (LC) method was developed to quantify calycosin-7-O-β-d-glucoside in rat plasma using a liquid–liquid extraction and ultraviolet (UV) absorbance detection. LC analysis was performed on a Diamonsil C₁₈ column (200 × 4.6 mm i.d., 5 μm particle size) with isocratic mobile phase consisting of acetonitrile–0.05% phosphoric acid (19.5:80.5, v/v) of a flow rate of 1.0 mL min⁻¹. The linear range was 0.11–17.6 μg mL⁻¹ and the low quantification limit was 0.11 μg mL⁻¹ (S/N = 10). The intra- and inter-day relative standard deviations (RSD) in the measurement of quality control (QC) samples 0.11, 0.22, 1.32 and 8.80 μg mL⁻¹ ranged from 4.1 to 6.3 and 4.3 to 6.2%, respectively. The accuracy was from −6.7 to 4.3% in terms of relative error (RE). Calycosin-7-O-β-d-glucoside was stable in storage at −20 °C for 2 weeks and stable after three freeze–thaw cycles in rat plasma. This method was validated for specificity, accuracy, precision and was successfully applied to pharmacokinetic study of calycosin-7-O-β-d-glucoside in rat plasma after intravenous administration of Aidi lyophilizer.     

Chiral discrimination ofN-(dansyl)-dl-amino acids on human serum albumin stationary phase: Effect of a mobile phase modifier

Summary The effect of perchlorate anion as mobile phase modifier on the separation factor, α, forN-(dansyl)-dl-norvaline andN-(dansyl)-dl-tryptophan on a human serum albumin (HSA) column was studied by varying the concentration,c, of the chaotropic agent and the column temperatureT. Gibbs-Helmholtz parameters Δ(ΔH) and Δ(ΔS) between thed andl enantiomers were determined from linear van't Hoff plots of lnα against 1/T. Thermodynamic results indicated that the enhancement of the separation factor observed asc was increased was enthalpically controlled owing to stereoselective H-bonding interactions. Such behavior was used to optimize the chromatographic conditions for separation ofN-(dansyl)-amino acids on HSA.     

Synthesis of chiral organophosphorous ligands based on transformations of methyl 3,4-O-isopropylidene-l-threonate

Chiral organophosphorous compounds which are of interest as ligands for metallocomplex catalysts for enantioselective transformations were synthesized from methyl 3,4-O-isopropylidene-l-threonate. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp 393–396, February, 1997.     

Conformational phase diagram of poly(l-lysine) in sodium alkanesulfonate aqueous solutions at various temperatures

The conformation of poly(l-lysine) (PLL) was investigated in sodium alkanesulfonate C_nSO₃Na (n=8, 7) at various temperatures by circular dichroism spectrum measurements. C₈SO₃Na induced a double-step conformational change from a coil, to a β-sheet, and then to an α-helix, in which C₇SO₃Na induced a single-step coil-to-helix conformational change. Binding isotherms of C₈SO₃Na by PLL were constructed from the potentiometry of equilibrium concentration of the surfactant using a surfactant ion-selective electrode. The curves indicated the cooperative binding characteristic and were analyzed by a linear lattice model using the Bethe approximation. The thermodynamic parameters obtained from the model revealed that the binding of C₈SO₃Na by PLL was an entropy-driven process. The conformational change was observed at nearly full binding, presumably due to the surfactant clustering of the ordered conformation.