Studies on phytohemagglutinins XII. O-glycosyl polyacrylamide gels for affinity chromatography of phytohemagglutinins

Copolymerization of alkenyl O-glycosides with acrylamide and N,N′-methylene bisacrylamide produces neutral hydrophilic gels containing sugars having O-glycosidic links with the alkyl side chains of the polymer matrix. The preparation of these copolymers and their application as affinity adsorbents for the specific separation of phytohemagglutinins from other proteins is described.     

Polyacrylamide gels with modified cross-linkages

The retention of low molecular weight proteins during electrophoresis through gradient polyacrylamide gels was improved when a gradient of N,N′,N″-triallyl citric triamide (TACT) was superimposed on the gradient of acrylamide and N,N′-methylenebisacrylamide (MBA). Gels cross-linked only with N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) are soluble in dilute periodic acid or dilute aqueous solutions of bases. DHEBA cross-linked gradient gels have a smaller pore structure at high acrylamide concentrations and a more open structure at low acrylamide concentrations than gels cross-linked with MBA. Proteins labeled with tritium and carbon-14 were fractionated through DHEBA cross-linked gradient gels and the isotopes measured after solution of the gel with periodic acid. The mild solubilizing conditions enhanced isotope resolution. The characteristics of several cross-linking molecules are discussed and reasons advanced for the superiority of those with acrylamido end groups.     

Quantitative gas-liquid chromatography and mass spectrometry of the N(O)-perfluorobutyryl-O-isoamyl derivatives of amino acids.

A modification of a gas-liquid chromatographic method for quantitative analysis of amino acids as the N(O)-perfluorobutyryl-O-isoamyl derivatives is described. The modifications include changes in time and temperature for esterification, improved preparation of the esterification reagents and conducting the derivatizations in vacuo to obtain reproducible values for amino acids such as methionine and arginine. Mass spectral data are presented for all the derivatized amino acids.     

A facile synthesis of novel pyrazolopyrimidine thioglycosides as purine thioglycoside analogues

The easy, convenient and high yielding preparation of new thioglycosides incorporating mercaptopyrazolo[1,5-a]pyrimidine moieties from readily accessible starting materials has been reported. The main step of this protocol is the formation of 7-mercaptopyrazolo[1,5-a]pyrimidine-6-carbonitrile derivatives 4a-d by condensation of sodium 2-cyano-3-ethoxy-3-oxoprop-1-ene-1,1-bis(thiolate) 1 with 4-(aryldiazenyl)-1H-pyrazole-3,5-diamines 3a-d to form target compounds 4a-d, which coupled with tetra-O-acetyl-α-D-glycopyranosyl bromides 5a,b in the presence of basic medium to provide the corresponding product purine thioglycoside analogs 6a-h. Ammonolysis of the latter compounds 6a-d at ambient temperature for 10 minutes, led to the free glycoside derivatives 7a-h, which were obtained in approximately quantitative yields. Their structures were created based on the spectroscopic and elemental data.     

Novel dihydropyridine thioglycosides and their corresponding dehydrogenated forms as potent anti-hepatocellular carcinoma agents

A novel method for preparation of a new class of dihydropyridine thioglycosides and their corresponding dehydrogenated forms, via reaction of piperidinium salts of dihydropyridinethiones with 2,3,4,6-tetra-O-acetyl-α-D-gluco- and galactopyranosyl bromides has been studied. The evaluation of antiproliferative activity against HepG-2 cell lines (liver carcinoma cell lines) of the dihydropyridine thioglycosides and pyridine thioglycosides revealed that many of the thioglycosides have interesting antitumor activities specifically 5c, 5g, 5l, 5o, 5p, 7a, 7i, 7p, 8b, 8f, 8s, and 8v.     

A NEW CLASS OF BIHETEROCYCLIC THIOGLYCOSIDES FROM PYRIDINE-2-(1H)-THIONES

ABSTRACT

A reported method for preparation of a new class of biheterocyclic thioglycosides via reaction of pyridinethiones with 2,3,4,6-tetra- O -acetyl-α-D-gluco- and galactopyranosyl bromides has been studied.     

Alkaline urea solubilization, two-dimensional electrophoresis and lectin staining of mammalian cell plasma membrane and plant seed proteins

Plasma membranes, isolated from Chinese hamster ovary cells and seed proteins from Arachis hypogaea (L.) were analyzed by two-dimensional electrophoresis. Polypeptides were solubilized without employing sodium dodecyl sulfate (SDS), using in its place 5 mm K₂CO₃ and 9.5 m urea. After addition of dithiothreitol and the nonionic detergent Nonidet P-40, more than 95% of the total protein remained in the supernatant fraction after the preparation was centrifuged at 100,000 g. The solubilization was comparable to that achieved with boiling SDS solution. This soluble material could be used directly for either isoelectric focusing or nonequilibrium pH gradient electrophoresis in narrow bore, tubular, polyacrylamide gels crosslinked by means of N,N′-diallyltartardiamide. Up to 750 μg of protein could be analyzed in one such 3 mm gel. Electrophoresis in polyacrylamide slab gels containing SDS was used for separations in the second dimension. The method allows large amounts of both basic and acidic insoluble proteins to be solubilized and then analyzed without employing SDS as a solubilizing agent. Classes of glycoproteins on the gels were detected by incubating with small volumes of ¹²⁵I-lectins in heat-sealed plastic bags. CHO cells contain several high molecular weight acidic glycoproteins that bind wheat germ agglutinin, but which do not stain with Coomassie blue. Several of the storage polypeptides in peanut seeds were also shown to bind wheat germ agglutinin and are probably, therefore, glycoproteins containing N-acetyl d-glucosamine.     

First novel synthesis of triazole thioglycosides as ribavirin analogues

This study reports a novel and efficient method for the synthesis of the first reported novel class of triazole thioglycosides. These series of compounds were designed through the reaction of potassium cyanocarbonimidodithioate 2 with hydrazine derivatives 3a-d in EtOH at room temperature to give the corresponding potassium 5-amino-1H-1,2,4-triazole-3-thiolates 4a-d. The latter compounds were treated with tetra-O-acetyl-α-D-glucopyranosyl bromide 6a and tetra-O-acetyl-α-D-galactopyranosyl bromide 6b in DMF at room temperature to give in high yields the corresponding triazole thioglycosides 7a-h. Treatment of triazole salts 4a–d with hydrochloric acid afforded the corresponding 3-mercaptotriazoles 5a-d. Compounds 5a-d were then reacted with bromoperacetylated sugars 6a,b in sodium hydride-DMF at ambient temperature to afford the thioglycosyl compounds 7a-h. Ammonolysis of the triazole thioglycosides 7a-h afforded the corresponding free thioglycosides 8a-h. The scope and limitation of the method is demonstrated. The structure of the reaction products was confirmed on the basis of their elemental analysis and spectral data (IR, ¹H NMR, MS and ¹³C NMR).     

A water-soluble Mg2+-ATPase from erythrocyte membranes

A ouabain-insensitive ATPase activity associated with the water-soluble proteins of the human and bovine erythrocyte membrane is demonstrated by means of activity-staining in polyacrylamide gels. The ATPase activity from both sources had an absolute requirement for Mg²⁺, activity becoming easily detectable at 0.2 mM Mg²⁺. At low Mg²⁺ concentrations added Ca²⁺ appeared to decrease the intensity of the ATPase stain. The activity is unaffected by monovalent cations, does not hydrolyse p-nitrophenyl phosphate and is not inhibited by 2 : 4 dinitrophenol. The ATPase has an apparent molecular weight of approximately 100 000 as determined by electrophoresis in acrylamide gels containing dodecyl sulphate.     

The synthesis and decomposition of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-(N-nitroso)acetamido-α- and β-D-glucopyranose

The synthesis of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-(N-nitroso)acetamido-α- and β-D-glucopyranose is described. Decomposition of the α-nitrosoamide in chloroform containing 2% of ethanol at room temperature afforded β-D-glucopyranose pentaacetate and ethyl β-D-glucopyranoside tetraacetate as major products, the former predominating. Reaction in 1:5 (v/v) acetic acid—acetic anhydride containing sodium acetate also gave β-D-glucose pentaacetate as major product, together with 1,1,3,4,6-penta-O-acetyl-2,5-anhydro-D-mannose aldehydrol. Decompositions of both α and β-nitrosoamides in 1:1 (v/v) acetone—water gave mainly 3,4,6-tri-O-acetyl-2,5-anhydro-D-mannose and its aldehydrol form. The synthesis, from 2,5-anhydro-D-mannose, of four new derivatives is also reported.     

Ring opening of acylated β-d-arabinofuranose 1,2,5-orthobenzoates with nucleophiles allows access to novel selectively-protected arabinofuranose building blocks

β-d-Arabinofuranose 1,2,5-orthobenzoates with 3-O-acetyl, 3-O-benzoyl, and 3-O-chloroacetyl groups were prepared in an efficient manner starting from readily available crystalline methyl 2,3,5-tri-O-benzoyl-α-d-arabinofuranoside, and ring-opening reactions of these compounds with O- and S-nucleophiles were studied. Optimized conditions leading to the formation of the respective monosaccharide adducts (up to 96% isolated yields) and to α-(1→5)-linked disaccharide thioglycosides with 5′-OH unprotected (up to 30% isolated yields) were found. Basing on these results, a novel approach for effective differentiation of 3,5-diol system and 2-hydroxy group in arabinofuranose thioglycosides was proposed. The selectively protected derivatives prepared are valuable building blocks for the assembly of linear and branched oligoarabinofuranosides.     

The use of boric and benzeneboronic acids in the partial acetonation of monosaccharides

The preparation of mono-O-isopropylidene derivatives and mono-O-isopropylidene benzeneboronates of monosaccharides in one step is described, together with their p.m.r. and mass-spectral characteristics. In particular, the use of boric acid in the synthesis of the new acetal 1,2-O-isopropylidene-β-L-arabinopyranose (8) is described, together with improved procedures for the preparation of 2,3-O-isopropylidene-D-mannofuranose (5) and 3,4-O-isopropylidene-L-arabinopyranose (10). The use of boric acid in the partial hydrolysis of 1,2:3,4-di-O-isopropylidene-β-L-arabinopyranose to give the 1,2-acetal is reported.     

Syntheses of sulfated glycopolymers and analyses of their BACE-1 inhibitory activity

The glycopolymers for glycosaminoglycan mimic were synthesized, and the inhibitory effects of Alzheimer’s β-secretase (BACE-1) were examined. The regio-selective sulfation was conducted on N-acetyl glucosamine (GlcNAc), and the acrylamide derivatives were synthesized with the consequent sulfated GlcNAc. The glycopolymers were synthesized with acrylamide using radical initiator. The glycopolymer with sulfated GlcNAc showed the strong inhibitory effect on BACE-1, and the inhibitory effects were dependent on the sulfation positions. Especially, glycopolymers carrying 3,4,6-O-sulfo-GlcNAc showed the strong inhibitory effect. The docking simulation suggested that glycopolymers bind to the active site of BACE-1.     

Blocksynthese von O-glycopeptiden und anderen T-antigen strukturen

Under the conditions of in situ anomerisation, the 2-azido-4,6-di-O-benzoyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-galactopyranosyl bromide reacted directly and with high selectivity with the reactive hydroxyl groups of l-serine and l-threonine derivatives to form α-glycosidically-linked products. Thus, the glycopeptides containing l-serine and l-threonine are more accessible. The disaccharide block could also be coupled with other reactive hydroxyl compounds to give compounds that contain the T-receptor.     

Acetonation of 2-(acylamino)-2-deoxy-d-glucoses

2-Acetamido-2-deoxy-d-glucose and 2-(benzyloxycarbonylamino)-2-deoxy-d-glucose were each treated with 2,2-dimethoxypropane in N,N-dimethylformamide containing a trace of p-toluenesulfonic acid. The new 5,6-O-isopropylidene derivatives 2-acetamido-2-deoxy-5,6-O-isopropylidene-d-glucofuranose, 2-acetamido-1,4-anhydro-2-deoxy-5,6-O-isopropylidene-d-arabino-hex-1-enitol, 2-acetamido-2-deoxy-3,4:-5,6-di-O-isopropylidene-aldehydo-d-glucose-dimethyl acetal, and 2-(benzyloxycarbonylamino)-2-deoxy-5,6-O-isopropylidene-d-glucofuranose were isolated. The formation of these furanoid acetals may be important in ascertaining the mechanism of this unique acetonation accompanied by glycosidation.     

The isolation, characterization and structure of uroterpenol, a monoterpene from human urine

Human urine is shown to contain, in a conjugated form, a compound of molecular formula C₁₀H₁₈O₂, which is named uroterpenol. Details are reported of the isolation procedure, the preparation of derivatives and the chemical and physical properties, together with a proof that the compound is p-menth-1-ene-8,9-diol.     

A method for the quantitative determination of protein incorporated in solubilized polyacrylamide gels

Dense and light polyacrylamide gels containing N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) or N,N′-diallyltartardiamide (DATD) as the crosslinker have been tested for their solubilization properties. Both types of gel can be dissolved in 10 mm periodic acid. The time and temperature required for complete dissolution of slabs of DHEBA crosslinked gels (12 h at 50°C), however, greatly exceed those required for dissolving slabs of DATD-polyacrylamide gel (0.5 h at 22°C). Bovine serum albumin kept under the respective dissolving conditions gave a lower response in the Lowry protein assay in instances where hot periodic acid had been used. Nearly independent of the type and concentration of their constituents, the different dissolved polyacrylamide gels interfere slightly with the Lowry assay by causing some “aspecific” color development. A method is outlined enabling a reliable quantitative determination of protein incorporated in DHEBA or DATD crosslinked polyacrylamide gels.     

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.     

Ruthenium(II) arene complexes containing four- and five-membered monoanionic O,O-chelate rings

Optimization of the design of half-sandwich organometallic Ru^II arene complexes as anticancer agents depends on control of ligand exchange reactions. We have studied the aqueous chemistry of complexes containing O,O-chelate rings. The presence of the four-membered O,O-chelate ring from acetate (AcO) in [η⁶-p-cymene)Ru(AcO)Cl] was confirmed by X-ray crystallography, but in solution the acetate ligand was labile and the hydroxo-bridged dimer [((η⁶-p-cymene)Ru)₂(μ-OH)₃]⁺ readily formed. The dimer was relatively unreactive towards 9-ethyl guanine. The tropolonato (trop) complex [(η⁶-p-cymene)Ru(trop)Cl] was stable in aqueous media and the X-ray crystal structure of the aqua adduct [(η⁶-p-cymene)Ru(trop)(H₂O)]CF₃SO₃, containing a five-membered O,O-chelate ring from trop, was determined. [(η⁶-p-cymene)Ru(trop)Cl] reacted with guanosine to form N7 adducts and with adenosine to form both N7 and N1 adducts. Competitive reactions with guanosine and adenosine gave rise to guanosine:adenosine adducts in a ca. 1.3:1 mol ratio.