Periodate oxidation of 2-acetamido-2-deoxy-β-D-glucopyranosyl residues

Thirty-two substituted phenyl β-D-galactopyranosides were hydrolyzed in 0.1M aqueous hydrochloric acid at different temperatures. Rate coefficients and kinetic parameters were determined. Influences of the substituents were investigated by linear free-energy relationships and the Leffler—Exner isokinetic relation. Only electronic effects operate. Some ortho-substituents have a rather complex influence on the reaction.     

The structure of rooster-comb dermatan sulfate. Fragmentation of the polysaccharide chains by chondroitinase AC-II digestion,and by periodate oxidation,followed by alkali cleavage

The polysaccharide-chain fragments of rooster-comb dermatan sulfates (RC-20 and RC-30) were obtained by chondroitinase AC-II digestion and by periodate oxidation, followed by alkaline cleavage, and their structures analyzed both quantitatively and qualitatively. RC-20 having a lower d-glucuronic acid content (22.6%) is composed preponderantly of large clusters of N-acetyldermosine sulfate (M_r～17 600–41 000) at the nonreducing terminal, whereas RC-30, having a higher d-glucuronic acid content, (41.4%) is poor in this cluster. Both RC-20 and RC-30 have an N-acetyldermosine sulfate cluster (M_r 6500–7300) within the polysaccharide chains. Most N-acetylchondrosine sulfate units of RC-20 and RC-30 exist as clusters, the large clusters (M_r～17 600) being preponderant in RC-30; both RC-20 and RC-30 contain a large proportion of N-acetylchondrosine sulfate clusters (M_r 3500 and 9000) that corresponds to the uronic acid content. In RC-30, most N-acetyldermosine disulfate units (13.4%) are linked to N-acetylchondrosine sulfate units or clusters.     

Determination of the position of linkage of 2-acetamido-2deoxy-D-galactose and 2-acetamido-2-deoxy-D-glucose residues in oligosaccharides and glycoproteins. Synthesis of 2-acetamido-2deoxy-D-xylitol and 2-acetamido-2-deoxy-L-threitol

A method has been studied for the determination of the position of the linkage of the 2-acetamido-2-deoxy-D-galactose and 2-acetamido-2-deoxy-D-glucose residues in oligosaccharides and glycoproteins that is based on the borohydride reduction of the reducing terminal residues to the corresponding alditol derivatives periodate oxidation, borohydride reduction, hydrolysis (eventually followed by borohydride reduction), separation of the fragments as per-O-(trimethylsilyl) or per-O-(trifluoroacetyl) derivatives, and identification of the fragments as derivatives of 2-acetamido-2-deoxyglycerol, 2-acetamido-2-deoxy-L-threitol, 2-acetamido-2-deoxy-L-arabinitol, 2-acetamido-2-deoxy-D-xylitol, 2-acetamido-2-deoxy-D-galactitol, and 2-acetamido-2-deoxy-D-glucitol by gas-liquid chromatography-mass spectrometry. New syntheses for the standard compounds 2-acetamido-2-deoxy-L-threitol and 2-acetamido-2-deoxy-D-xylitol are described.     

Simple,rapid and micro high-pressure liquid chromatographic method for the simultaneous determination of tolbutamide and carboxy tolbutamide in plasma

A rapid high-pressure liquid chromatographic (HPLC) assay is described for the quantitative analysis of tolbutamide and its major metabolite, carboxy tolbutamide, in plasma. An aliquot (25–100 μl) of plasma was prepared for chromatography by deproteinization as follows. One volume of plasma and 2.5 volumes of acetonitrile were vortex mixed for a few seconds and then centrifuged for approx. 1 min. A 50-μl sample of the clear supernatant was injected into the chromatograph. A μBondapak C₁₂ reversed-phase column was used with a mobile phase of acetonitrile-0.05% phosphoric acid (45:55) at a flow-rate of 1.5 ml/min. The column effluent was monitored by a variable-wavelength UV detector set at 200 nm. Tolbutamide and its metabolite had retention times of 5.75 and 3.25 min, respectively. The procedure yields reproducible results with sensitivity adequate for routine clinical monitoring of plasma levels or for single-dose pharmacokinetic studies. A number of commonly used drugs do not interfere with the method. A single plasma sample can be analyzed in approx. 9 or 10 min.     

Synthesis of methyl 6-(ammonium 2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate)-alpha-D-mannopyranoside and use of this compound for the determination of N-acetylglucosamine-1-phosphotransferase

Methyl 6-(ammonium 2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate)-alpha-D-mannopyranoside was synthesized and identified by 1H-n.m.r. and 13C-n.m.r. data, acid hydrolysis, and elemental analysis. It was utilized for the determination of UDP-N-acetylglucosamine-1-phosphotransferase in an assay procedure that employed methyl alpha-D-mannopyranoside as an acceptor. The assay product was identified and characterized by thin-layer chromatography with the title reference compound. The present technique does not require [32P]UDP-N-acetylglucosamine, but effectively uses commercially available UDP-[14C]GlcNAc.     

A convenient method for the synthesis of methyl 2-acetamido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-glucopyranoside

In the seeds of Tropaeolum and Tamarindus, which have been found to have the highest content of amyloids, these polysaccharides are associated with sucrose and O-d-galactosylsucroses of the raffinose-stachyose series. Balsamine seeds have a low content of amyloid and do not contain the aforementioned galactosides, which are otherwise widely distributed in plant seeds. They contain sucrose and a mono-O-D-galactosylsucrose that has been crystallized and identified as planteose. Planteose has previously been isolated from some Sympetalous plants. As far as we know, this is the first report of its occurrence in an Archichlamydeaeous plant seed.     

Analysis of the linkage positions in d-fructofuranosyl residues by the reductive-cleavage method

The suitability of the reductive-cleavage method for analysis of the linkage positions in d-fructofuranosyl residues of d-fructans was examined by using sucrose, chicory-root inulin, and Aerobacter levanicum levan as models. Permethylation, and reductive cleavage with triethylsilane in the presence of either boron trifluoride etherate or trimethylsilyl trifluoromethanesulfonate, gave the expected methylated derivatives of 2,5-anhydro-d-mannitol and 2,5-anhydro-d-glucitol. With either catalyst, nonreducing (terminal) d-fructofuranosyl groups and d-fructofuranosyl residues linked at O-1 gave derivatives having the manno configuration as the major product, whereas d-fructofuranosyl residues linked at O-6, and at both O-1 and O-6, gave derivatives having the gluco configuration as the major product. The independent synthesis, and n.m.r.- and mass-spectral characterization, of the methylated 2,5-anhydro-d-mannitol and 2,5-anhydro-d-glucitol derivatives formed from these residues by reductive cleavage are reported.     

Simple method for the determination of choline and acetylcholine by pyrolysis gas chromatography

An improved purification procedure is described for the simultaneous assay of endogenous choline and acetylcholine by pyrolysis gas chromatography, particularly for providing a simple and effective method for propionylation of choline in the presence of acetylcholine. The reaction was carried out in acetonitrile solution prepared by dissolving the evaporated residue of the supernatant of brain homogenate. Thus samples for propionylation were prepared without the use of ion-exchange chromatography.     

A specific micromethod for determination of acetyl residues in proteins

A method is described for the separation and concentration of rat liver lysosomes from mitochondria in a one-step procedure by zonal centrifugation. Some of the practical problems associated with the use of the B-XX1X rotor are discussed.     

Synthetic mucin fragments: methyl 3-O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl-beta-D- 3-O-(2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl- beta-D-glucopyranosyl)-beta-D-galactoyranoside. pyranosyl)-beta-D-galactopyranoside

Methyl 2,4,6-tri-O-benzyl-beta-D-galactopyranoside (5) was obtained crystalline by way of its 3-O-allyl derivative, which was in turn obtained by ring-opening of a presumed 3,4-O-stannylene derivative of methyl beta-D-galactopyranoside, followed by benzylation. Condensation of 5 with 2-methyl-(2-acetamido-3,4,6-tri-O-acetyl-1,2-dideoxy-beta-D-glucopyra no)-[2,1-d]-2-oxazoline in 1,2-dichloroethane in the presence of p-toluenesulfonic acid afforded the disaccharide derivative methyl 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2, 4,6-tri-O-benzyl-beta-D-galactopyranoside (6) Deacetylation of 6 in methanolic sodium methoxide afforded the disaccharide derivative 7, which was acetalated with alpha, alpha-dimethoxytoluene to afford the 4',6'-O-benzylidene acetal (10). Catalytic hydrogenolysis of the benzyl groups of 7 afforded the title disaccharide 8. Glycosylation of 10 with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in 1:1 benzene-nitromethane in the presence of mercuric cyanide gave the fully protected trisaccharide derivative 12. Systematic removal of the protecting groups of 12 then furnished the title trisaccharide 14. The structures of 5, 8, and 14 were all confirmed by 13C-n.m.r. spectroscopy. The 13C-n.m.r. chemical shifts for methyl alpha- and beta-D-galactopyranoside, and also those of their 3-O-allyl derivatives, are recorded, for the sake of comparison, in conjunction with those of compound 5.     

A sensitive method for the determination of cytolytic activity

A new method is described for the determination of the cytolytic activity of extremely low levels of stable as well as very labile cytotoxins. The method involves the application of the cytotoxin to a column of immunobilized erythrocytes or other suitable cells and a continuous monitoring of the column eluate for the presence of hemoglobin or other cell constituents. The cytotoxic activity of horseradish peroxidase at concentrations as low as 10^?12, m can be measured with this technique. The column hemolytic assay is compared with a static (batch) hemolytic assay with respect to sensitivity and reproducibility. Furthermore, a method is described to determine the true rates of lysis, i.e., the number of cells lysed per minute.     

A rapid,sensitive method for accurate determination of the lecithin/sphingomyelin ratio by thin-layer chromatography and reflectance spectrofluorometry

A highly reproducible thin-layer chromatographic procedure has been developed for accurate determination of the lecithin/sphingomyelin ratio. Two interfering compounds, phosphatidyl inositol and phosphatidyl serine, have been investigated and eliminated by adsorption onto DEAE-cellulose. A uniform fluorescence staining procedure employing 2′,7′-dichlorofluorescein has been developed. Accurate quantitation was performed by direct measurement of the reflected fluorescence intensity of the lecithin and sphingomyelin fluorophore spots with a spectrofluorometer equipped with a thin-layer scanning attachment. Stability and reproducibility studies are reported.     

A general and sensitive chemical method for sequencing the glycosyl residues of complex carbohydrates

This paper describes a new glycosyl-sequencing method. This method was made possible by the ability to fractionate complex mixtures of peralkylated oligosaccharides by reversed-phase, high-pressure liquid chromatography. The fractionation ability of the reversed-phase system allows the isolation and subsequent unambiguous identification by g.l.c.-m.s. of disaccharides, almost all trisaccharides, and, in some cases, tetrasaccharides generated by successive partial acid hydrolysis, reduction, and ethylation of a permethylated, complex carbohydrate. As these small oligosaccharides overlap within the unhydrolyzed, complex carbohydrate, the oligosaccharide sequences may be pieced together, and, with the glycosyl-linkage composition of the intact complex carbohydrate, can be used to determine the glycosyl sequence of the complex carbohydrate. The details of the sequencing method are illustrated by the elucidation of the glycosyl sequences of three complex carbohydrates. These examples demonstrate the wide variety of complex carbohydrates whose structures can be ascertained by the new sequencing technique. Two of the examples are the commercially available polysaccharides, lichenan and xanthan, whose structures have already been reported. The other example is a nonasaccharide derived from xyloglucan, a structural polymer of plant cell-walls. The glycosyl residues of the complex carbohydrates studied include hexosyl, deoxyhexosyl, pentosyl, glycosyluronic, and pyruvic acetal-substituted hexosyl residues. It will be demonstrated that the new glycosyl-sequencing technique is not compromise by the presence, in the carbohydrate to be analyzed, of glycosyl linkages possessing very different acid labilities. Two major advantages of this sequencing technique are that it is relatively rapid and that it requires only milligram quantities of carbohydrate.     

A sensitive method for determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine in human plasma by high-pressure liquid chromatography

Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts.     

Colorimetric determination of arginine residues in proteins by p-nitrophenylglyoxal

A new method is presented for the colorimetric determination of arginine residues in proteins. Under mildly alkaline conditions, p-nitrophenylglyoxal reacted with arginine to produce a stable colored solution in the presence of 0.15 m sodium ascorbate. Complete color development was obtained after 30 min at pH 9.0 and 30°C. The color produced at 475 nm obeyed Beer's law in the range 0.03–0.33 mm arginine. This color reaction was used to determine the number of arginine residues in several proteins of known arginine content. Best results were obtained when the protein samples were digested with a mixture of trypsin and subtilisin prior to assaying. The arginine contents obtained by this method agreed well with either the published values or with the results of amino acid analysis.     

Prevention of overoxidation in periodate oxidation of reducing oligosaccharides by their conversion into 1,5-anhydroalditol derivatives

A procedure for the conversion of reducing oligosaccharides into their 1,5-anhydroalditol derivatives was devised to prevent overoxidation during periodate oxidation. Gas-chromatographic analysis of the aldehydes in the products of complete oxidation of the resultant 1,5-anhydroalditol derivatives by the dithioacetal method^1b indicated that new types of dialdehyde characteristic of the linkage-types were formed, together with ordinary simple aldehydes. A number of d-gluco-oligosaccharides having various types of interglycosidic linkage were examined by this method. The results were consistent with expectations.