N-acetylamino Sugars Research Articles

The allyl ether as a protecting group in carbohydrate chemistry. Part IV. The 2-methylallyl ether group

The O-2-methylallyl group, which is isomerised more slowly than the allyl group, is most suitable for use (in protection of OH) in the presence of the O-but-2-enyl group, since the latter can be eliminated without concomitant rearrangement of the 2-methylallyl group. 2-Methylallyl 2-benzamido-4,6-O-benzylidene-2-deoxy-β-D-gluco-and -galacto-pyranosides were prepared from the corresponding 3-O-but-2-enyl derivatives and these benzamido-derivatives were converted into the acetamido-derivatives by alkaline hydrolysis followed by acetylation. A convenient method for the conversion of an N-benzoyl amino-sugar into the corresponding N-acetyl amino-sugar by refluxing in acetic anhydride–glacial acetic acid is described. The benzylation of N-acetyl amino-sugars with benzyl chloride and sodium hydride is less satisfactory than the benzylation of the corresponding N-benzoyl amino-sugars, owing to extensive formation of N-benzylacetamido-derivatives.

Further studies on the specificity of the anti-A agglutinin from Helix pomatia

The following three findings were obtained by investigating the nature of the combining site of Helix anti-A: 1. (1) The agglutinin is absorbed specifically onto Sephadex G-200 as well as the immunoadsorbent (A-blood-groups substance) and released by N-acetyl-ated aminosugars and sugar compounds having α-(1→6) conformation as glucosyl and galactosyl terminals. 2. (2) Hemagglutination with human A and RDE-treated red cells can be inhibited by the presence of these sugars. 3. (3) These results indicate that the combining site of this agglutinin is directed not only to N-acetylated aminosugars, but also to a wide spectrum of glucose and galactose polymers of α-anomeric-(1→6) structure.

Reaction of sodium hypochlorite with amines and amides: A new method for quantitating polysaccharides containing hexosamines

The NaOCl-amylose-KI spectrophotometric method for determining free and N-acetylated amino sugars has been applied to the analysis of PS Y-6272, which is composed of N-acetylglucosamine and N-acetylglucosaminuronic acid and produced extracellularly by the black yeast strain NRRL Y-6272 (6). PS Y-6272, as well as other amino sugar-containing polysaccharides such as chondroitin sulfate A, colominic acid, heparin, and hyaluronic acid, can be assayed readily in the range of 0.04 to 0.30 μmole N without previous depolymerization. Studies of the effects of pH and reaction time and temperature, carried out on these glycosaminoglycans as well as on various glucosamine derivatives and oligosaccharides, indicate that the color yield is influenced by the identity of the amino sugars present as well as by the position and anomeric type of linkages. However, since the color yield accords well with the nitrogen content of a particular amino sugar-containing polysaccharide, unknown quantities in solutions or column effluents can be determined readily and accurately by reference to calibration curves of the individual polysaccharide.

Analysis of disaccharides as permethylated disaccharide alditols by gas—liquid chromatography—mass spectrometry

Gas-liquid chromatography (g.l.c.) and combined gas—liquid chromatography—mass spectrometry (g.l.c.—m.s.) have been used for the analysis of disaccharides containing simple hexoses and amino sugars. The disaccharides were reduced to the corresponding alditols with borodeuteride, methylated, and separated by g.l.c. The molecular weight of the monosaccharide units and the position of the glycosidic linkage could be determined on the basis of the mass spectra. Differentiation of (1→4)- and (1→3)-linked alditols was not possible without deuterium labelling, whereas (1→6)-linked alditols could be distinguished by the presence of a fragment containing carbon atoms 1–4 of the alditol moiety. The glycosidic linkage was cleaved by methanolysis after isolation of the disaccharide alditol methyl ethers by preparative g.l.c., and the products were identified by g.l.c.—m.s. Methylation (methyl iodide in the presence of methylsulphinyl carbanion) of disaccharide alditols containing N-acetylated amino sugars resulted in the formation of a derivative in which the nitrogen atom carried both a methyl and an acetyl substituent.

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls.

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[(3)H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods.

Effects of Glycine andd-Amino Acids on Growth of Various Microorganisms

Growth of various microorganisms in media containing high concentrations of glycine or d-amino acids was examined. Susceptibilities to glycine or d-amino acids differed among microorganisms, and the differences in susceptibility have no direct relation with Gram staining, morphological forms, and aerobic or anaerobic nature of the organisms. Certain glycine-resistant bacteria tested, which included Bacillus cereus, Staphylococcus aureus and Serratia marcescens, exhibited relatively high oxidative activities towards glycine. The inhibition of the growth of Escherichia coli by either glycine or d-amino acids, which included d-threonine, d-alanine and d-lysine, was reversed by l-alanine, partialy by l-serine, and not by l-lysine or l-threonine. These results suggest that the growth inhibition of microorganisms by d-amino acids was similar to that by glycine. The incorporation of l-alanine into E. coli cells which were preincubated with glycine was less than those of preincubated without glycine. Particularly, the incorporation into the cell wall fraction was most susceptible to glycine. An additive effect of penicillin and glycine was observed in the inhibition of cell wall biosynthesis as determined by the intracellular accumulation of N-acetylamino sugar compounds.

D-mannosaminuronic acid constituent of the K-7 antigen of Escherichia coli

The identification of d-mannosaminuronic acid (2-amino-2-deoxy-d-mannuronic acid) as one of the main constituents of an acidic polysaccharide material isolated from two Escherichia coli serotypes is described. This material carrying the specificity of the E. coli K7 antigen is isolated from serotypes O14:K7:H− and O7:K7:H4 by extraction of the bacterial dry mass–obtained by growth on solid medium–using the hot phenol-water procedure. Fractionation of the aqueous phase material, after removal of lipopolysaccharide by repeated ultracentrifugation, was achieved by a cetavlon precipitation technique. RNA, representing the bulk of the aqueous phase material, precipitates in the presence of cetavlon already at a NaCl molarity of 0.3 M whereas the K7 active material is only precipitable at a much lower NaCl molarity of 0.06. Examination of HCl-hydrolysates of this substance by high voltage electrophoresis in a non-complexing buffer of pH 5.3 revealed the presence of a positively charged component which was shown to be the lactone of an aminohexuronic acid. The amino sugars present in the K7 specific material were separated from uncharged hydrolysis products by passing the hydrolysate over a Dowex 50 column previously equilibrated with 0.1 N HCl. The amino sugar fraction obtained by elution with 0.5 N HCl consisted of three major components: (a) a hexosaminuronic acid, (b) the lactone of the hexosaminuronic acid and (c) d-glucosamine. (a) and (b) are interconvertible and give rise to the same product after N-acetylation. (a) Shows the characteristic ninhydrin staining of hexosaminuronic acids and does react in the Elson-Morgan reaction with a maximal absorption at 530–533 mμ, thus indicating a free 1-aldo-2-aminogroup and, in addition, an unsubstituted C-3 position. Chromatographic and electrophoretic data differed from those of two hexosaminuronic acid standards available (glucosaminuronic and galactosaminuronic acids) but were in agreement with data reported by Perkins [2] for mannosaminuronic acid isolated from a polysaccharide material of Micrococcus lysodeikticus. Since no reference material was available we tried to elucidate the structure of the hexosaminuronic acid by converting the previously esterified hexosaminuronic acid units of the polymer by NaBH4-reduction to the related hexos-amine units. Esterification was carried out with an aqueous solution of ethylene oxide at room temperature, and NaBH4-reduction in the presence of an excess of glycerol in order to maintain a pH of approximately 8. Examination of the reduced material revealed the presence of a newly formed hexosamine which subsequently was identified as d-mannosamine. The identification is based on the following results: a) identity of Rf- and MGlcn-values in several systems of the free and the N-acetylated amino sugar with authentic samples of d-mannosamine and N-acetyl-d-mannosamine; b) formation of a pentose with the Rf-value of arabinose after ninhydrin degradation; c) reactivity of the amino sugar with hexokinase and formation of a hexosamine-6-phosphate. This reaction in addition proves the d-configuration of the amino sugar. Quantitative analysis shows that d-mannosaminuronic acid forms at least 15–20% of the K7 specific material extracted from E. coli O14:K7, whereas d-glucosamine represents only a minor constituent (2–3%). Hitherto 2-amino-2-deoxy-mannuronic acid was only reported once [2] as a constituent of a cell wall polysaccharide from Micrococcus lysodeikticus. To our knowledge its occurence in the K7 specific material of E. coli is the first report from gramnegative bacteria.

Effets of Glycine and D-Amino Acids on Growth of Various Microorganisms

Growth of various microorganisms in media containing high concentrations of glycine or D-amino acids was examined. Susceptibilities to glycine or D-amino acids differed among microorganisms, and the differences in susceptibility have no direct relation with Gram staining, morphological forms, and aerobic or anaerobic nature of the organisms. Certain glycine-resistant bacteria tested, which included Bacillus cereus, Staphylococcus aureus and Serratia marcescens, exhibited relatively high oxidative activities towards glycine. The inhibition of the growth of Escherichia coli by either glycine or D-amino acids, which included D-threonine, D-alanine and D-lysine, was reversed by L-alanine, partialy by L-serine, and not by L-lysine or L-threonine. These results suggest that the growth inhibi-tion of microorganisms by D-amino acids was similar to that by glycine. The incorpora-tion of L-alanine into E. coli cells which were preincubated with glycine was less than those of preincubated without glycine. Particularly, the incorporation into the cell wall fraction was most susceptible to glycine. An additive effect of penicillin and glycine was observed in the inhibition of cell wall biosynthesis as determined by the intracellular accumulation of N-acetylamino sugar compounds.

2-amino-2,6-dideoxy-L-mannose (L-rhamnosamine) isolated from the lipopolysaccharide of Escherichia coli 03:K2ab(L):H2.

From the lipopolysaccharide of Escherichia coli U41/14 (serotype 03:K2ab(L):H2) an unusual amino sugar was isolated. By sequential oxidation with periodate and hypoiodite and by oxidation with periodate, followed by detection of acetaldehyde formed, the amino sugar was characterized as a 2-amino-dideoxyaldose. The hydrochloride of the amino sugar, in comparison to those of 2-amino-2,6-dideoxy-D-glucose, 2-amino-2,6-dideoxy-L-galactose, 2-amino-2,6-dideoxy-D-mannose, 2-amino-2,6-dideoxy-D-allose, and 2-amino-2,6-dideoxy-D-gluose, was studied with aid of ion exchange column chromatography. The N-acetylated amino sugar was compared with the N-acetylated reference amino sugars with regard to their relative mobilities in paper chromatography. The amino sugar alcohols and reference amino sugar alcohols were studied using paper electrophoresis in molybdate buffer. On the basis of the results obtained the unknown amino sugar was identified as 2-amino-2,6-dideoxymannose (rhamnosamine). Its optical rotation showed it to be of the L-configuration. This is the first report on the natural occurrence of L-rhamnosamine.

Purification and properties of lysozyme produced by Staphylococcus aureus.

A method based on cold ethyl alcohol fractionation at different pH levels and ionic strengths and on gel filtration on a Sephadex G-200 column was used to concentrate and purify lysozyme from the culture supernatant fluid of Staphylococcus aureus strain 524. The final, nondialyzable product exhibited a 163-fold rise in specific activity over that of the starting material. Staphylococcal lysozyme is a glycosidase which splits N-acetylamino sugars from the susceptible substrate. Staphylococcal lysozyme was shown to be similar to egg white lysozyme in its optimal temperature for reaction, optimal pH, activation by NaCl and Ca(++) ions, inhibition by sodium citrate and ethylenediaminetetraacetate, and inactivation by Cu(++) ions and sodium dodecyl sulfate. It differs from the egg white lysozyme in its temperature susceptibility range (staphylococcal lysozyme is inactivated at 56 C). It acts on whole cells and cell walls of Micrococcus lysodeikticus, murein from S. aureus 524, and cell walls of S. epidermidis Zak. The last substrate was not susceptible to the action of egg white lysozyme in the test system used. The mechanism of action of staphylococcal lysozyme seems to be analogous to that of egg white lysozyme; however, the biological specificity of the two enzymes may be different.

Protein-carbohydrate interaction: V. Further inhibition studies directed toward defining the stereochemical requirements of the reactive sites of concanavalin A

Inhibition studies on the concanavalin A-dextran interaction have been extended using as inhibitors the members of the maltose-, isomaltose-, and methyl α-isomaltoside series, amino and N-acetyl amino sugars, and a number of oligosaccharides containing more than one linkage type. The interaction of the plant protein at the terminal chain ends of the α-linked glucans and mannans has been essentially confirmed, and the different characteristic inhibition activities of the linkage types is discussed in terms of steric effects. A possible application of the inhibition technique towards structural analysis of carbohydrate molecules is suggested by the authors.

An automated method for the determination of N-acetylamino sugars

The p-dimethylaminobenzaldehyde method for the determination of N-acetylhexosamines has been automated. Under the conditions of the automated procedure, N-acetylgalactosamine and N-acetylmannosamine give the same color yields and give 30% of the color produced by N-acetylglucosamine. The method, as described, can be used for the determination of 5–100 μg of N-acetylglucosamine per milliliter of sample and 10–200 μg of N-acetylgalactosamine and N-acetylmannosamine per milliliter of sample.

Quantitative analysis of carbohydrates using gas-liquid chromatography

Sugars can be analyzed quantitatively as their trimethylsilyl derivatives using gas-liquid chromatography. Standardization of polyethylene glycol succinate columns with α-methylgalactopyranoside as the standard gave a quantitative correlation between GLC relative peak areas and relative molar compositions of sugar standard solutions for: the pentose, ribose; the deoxyhexose, fucose; and the hexoses, glucose, galactose, and mannose. Quantitative data were also obtained for N-acetylamino sugars. Mixtures of sugars were analyzed by using the standardization graphs, and the amounts determined were accurate within 10%. This use of GLC will make it easier for the biochemist to attack problems in carbohydrate chemistry by enabling quantitative work to be done readily on mixtures heretofore difficult to analyze.

ACTION OF QUINACILLIN ON STAPHYLOCOCCUS AUREUS.

QUINACILLIN, the disodium salt of 3-carboxy-2-quinoxalinylpenicillin, has recently been described1,2 and has, for a penicillin, unusual properties. The striking feature is the pattern of the antibacterial spectrum which shows that quinacillin is significantly active (0.15–0.62 µg/ml., 0.32–1.35 µM) against Staphylococcus aureus (Oxford strain) but that a 10–100-fold increase in concentration is necessary before activity against other Gram-positive organisms can be demonstrated. It seemed profitable, therefore, to compare the action, if any, of quinacillin on causing the intracellular accumulation of N-acetylamino sugars and to compare this quantitatively with benzyl-penicillin as evidence of a typical penicillin-like mode of action3,4. Staphylococcus aureus was growrn for 18 h in a medium containing yeast extract 5 g peptone, 5 g glucose, 2 g K2HPO4, 1 g water to 1 1. In the middle of the logarithmic growth the penicillin was added and after 90 min the intracellular acetylaminosugar was determined colorimetrically5. Results are shown in Fig. 1.

A STUDY OF URINARY AND SERUM LYSOZYME IN PATIENTS WITH RENAL DISEASE.

LYSOZYME, a bacteriolytic enzyme discovered in 1922 by Sir Alexander Fleming,2 is found in a number of biologic fluids. The enzyme is a basic protein of low molecular weight that lyses susceptible bacteria by a reaction with cell-wall mucopolysaccharides releasing N-acetyl amino sugars and N-acetyl amino sugar-peptide complexes.3 Kidney contains more lysozyme activity than any other mammalian tissue,4 but little, if any, lysozyme activity is found in normal urine.5 , 6 Lysozymuria has been reported in some children with nephrotic syndrome7 and in some adults with renal disease,6 but no definitive evidence of the source of urinary lysozyme is available.In the . . .

水産動物のキチン分解酵素に関する研究-II

In the previous paper, 1) by the present authors it was reported that the chitinolytic enzyme was present in the stomach of Japanese sea-bass, Lateolabrax japonicus and suggested that the enzyme was inherent in the fish itself. The present study was carried out in order to ascertain whether the chitinolytic enzyme was present in the liver or not. In order to determine chitinolytic system, water extracts of the liver were incubated with a chitin suspension, then chitobiose and N-acetylamino sugar liberated were determined. The amount of N-acetylamino sugar was measured by the method as described by REISSIG et al.4) Chitobiose was estimated from the amount of N-acetylamino sugar produced by the addition of β-glucosidase.5) Results obtained are as follows. 1) The chitinolytic enzyme is present in the liver of Japanese sea-bass. 2) Substances which are formed by the action of the chitinolytic enzyme may be N-acetylglucosamine and N, N'-diacetylchitobiose (Fig. 1), and the enzyme may consist of chitinase and chitobiase. 3) The optimum pH value of chitinase action is about 4 and the optimum temperature is at 40 ?? 50°C. The optimum pH value of chitobiase is also about 4 (Fig. 3, Fig. 4 and Fig. 6).

Time course of accumulation of UDP- N-acetylamino sugar derivatives in Staphylococcus aureus

Under incubation conditions including deprivation of nitrogen sources, deprivation of lysine, inhibition by oxamycin and inhibition by penicillin, the accumulation of UDP- N-acetylamino sugar derivatives in Staphylococcus aureus cells, with time, has been studied. UDP- N-acetylglucosamine and UDP- N-acetylglucosamine-3- O-lactic ether accumulated in cells which were incubated in amino acid-free medium. During the initial phase of incubation there was a rapid rise in the appearance of UDP- N-acetylglucosamine followed later by a decrease of this nucleotide; concomitant accumulation of UDP- N-acetylglucosamine-3- O-lactic ether and UDP- N-acetylglucosamine-3-O-lactyl-peptides was observed under every condition which induced UDP- N-acetylglycosamine-3- O-lactyl-peptide accumulation. The relationship of the observed accumulation to the pathway of cell-wall mucopeptide synthesis is discussed.

Thymidine diphosphate N-acetylamino sugar compounds from [formula omitted] strains

Thymidine diphosphate N-acetylamino sugar compounds from [formula omitted] strains

Studies of the bacterial cell wall VI. Wall composition and sensitivity to lysozyme

All of the isolated walls of the Gram-positive bacteria investigated showed some degree of susceptibility to digestion with egg-white lysozyme; the % Turbidity reduction varied from 9% for Staphylococcus aureus walls to 98% for the walls of Micrococcus lysodeikticus. Although all of the walls contained muramic acid and glucosamine, the sensitivity to lysozyme was not correlated with the total amino sugar contents. The nature of the monosaccharide constituents and the relative proportions of amino acids have been determined. Walls of a number of organisms contain peptides of fairly similar composition. Several unidentified compounds have been detected in walls of various species. Only Lactobacillus arabinosus walls showed an increased sensitivity to lysozyme after removal of O-ester groups or the teichoic acid. Apart from substituent groups, one likely explanation of differences in sensitivity to lysozyme seems to be the proportion of N-acetylamino sugars linked in a specific manner.

Uridine diphosphate N-acetylamino sugar compounds from Staphylococcus aureus strain 209P. I. Amino acid constituents

1. 1. The components of UDP- N-acetylamino sugar compounds accumulated in normal and in penicillin-treated Staphylococcus aureus strain 209P cells were investigated. 2. 2. In these compounds, two additional amino acids, aspartic acid and glycine, were found besides alanine, glutamic acid, and lysine, already known.