The 13C-N.M.R. spectra of methyl (methyl O-methyl-α-d-glucopyranosid)uronates

¹³C-N.m.r. spectra of all of the methyl ethers of methyl (methyl α-d-glucopyranosid)uronate have been interpreted. The data can be used as an aid in the analysis of ¹³C-n.m.r. spectra of α-d-glucopyranosyluronic acid-containing polysaccharides.     

Bacteriophage degradation of Klebsiella K44 polysaccharide: an n.m.r. study and chemical proof of the position of the acetate group

Bacteriophages (phi) have been used to degrade polysaccharides into oligosaccharides containing one or more of their repeating units. The capsular polysaccharide from Klebsiella K44 contains an acetate group, and n.m.r. spectroscopy and chemical methods have been employed to prove its linkage to O-6 of the 4-linked glucose residue. Phage phi 44 was shown to be an alpha-glucosidase not influenced by the acetate moiety and thus able to depolymerize the polysaccharide into pentasaccharide repeating units, some of which contained acetate on O-6 of the reducing glucose residue. The two oligosaccharides were studied by 1H- and 13C-n.m.r. spectroscopy, and their spectra were compared with those of the native and the deacetylated polysaccharide. 13C-n.m.r. was a useful tool for locating the 6-linked acetate, the position of which was confirmed by the method of temporary protection using methyl vinyl ether. The importance of using bacteriophages to obtain oligosaccharides is highlighted by the better results obtained with the oligosaccharide in comparison to the polysaccharide, both in n.m.r. spectroscopy and the temporary protection method.     

A computer-assisted structural analysis of regular polysaccharides on the basis of 13C-n.m.r. data

A computerised approach to the structural analysis of unbranched regular polysaccharides is described, which is based on an evaluation of the 13C-n.m.r. spectra for all possible primary structures within the additive scheme starting from the chemical shifts of the 13C resonances of the constituent monosaccharides and the average values of the glycosylation effects. The analysis reveals a structure (or structures), the evaluated spectrum of which resembles most closely that observed. The approach has been verified by using a series of bacterial polysaccharides of known structure and, in combination with methylation analysis data, for the determination of the presently unknown structures of the O-specific polysaccharides from Salmonella arizonae O59 and O63, and Proteus hauseri O19.     

The 13C-N.M.R. spectra of disaccharides of D-glucose, D-galactose, and L-rhamnose as models for immunological polysaccharides.

Complete assignments of the 13C-n.m.r. spectra of disaccharides having beta-glycosidic linkages are presented and discussed. The disaccharides of D-glucose, D-galactose, L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose are model compounds for 13C-n.m.r. studies of immunological polysaccharides. Changing the nature of the reducing glucopyranose rings (D-glucose to L-rhamnose) has no important influence on the chemical shifts of the carbons of the non-reducing glucopyranose ring (D-glucose). The converse is also true: the chemical shifts of the carbons of the reducing glucopyranose ring (L-rhamnose) are not noticeably affected by a change of the non-reducing unit (D-glucose to D-galactose or 2-acetamido-2-deoxy-D-glucose).     

A bacteriophage-associated glycanase cleaving beta-pyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO)

A bacteriophage growing on Escherichia coli K13, K20, and K23 strains carries a glycanase that catalyzes the hydrolytic cleavage of the beta-ketopyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO) in the respective capsular polysaccharides. The main cleavage product of the K23 polysaccharide has been identified by 1H- and 13C-n.m.r. spectroscopy as beta beta Ribfl----7 beta KDOp2----3-beta Ribfl----7KDO. Cleavage of polysaccharides containing alpha-pyranosidic, or 5-substituted beta-pyranosidic KDO is not catalyzed by the enzyme.     

Evidence for a single type of linkage in a fructofuranan from Lolium perenne

On extraction with water, rye grass (Lolium perenne) yielded a mixture of polysaccharides. Fractionation thereof led to the isolation of two fructofuranans, one of which preponderated. Analysis of the main polysaccharide by ¹³C-n.m.r. spectroscopy indicated that it is composed of fructofuranosyl residues linked only (2 → 6), and terminated by a glucosyl group linked as in sucrose.     

The lipopolysaccharides of Pseudomonas solanacearum i Pseudomonas cichorii

Structure analysis by the methods of methylation, 1H- and 13C-n. m. r. spectroscopy has shown that O-specific polysaccharides of typical strains of Pseudomonas solanacearum (biovar I) and P. cichorii are identical by their structure and constructed of branched pentasaccharide repeating links which include three residues of rhamnose (one of them is in the branching node), one residue of beta-xylose (it occupies terminal position) and one reside of N-acetyl-beta-glucosamine. The other strain of P. solanacearum of biovar I and two strains belonging to biovars III and IV also produce structure-similar O-specific polysaccharides, constructed of linear tetrasaccharide repeating links which include three residues of alpha-L-rhamnose and one residue of N-acetyl-alpha-D-glucosamine.     

Cryoenzymology of trypsin. 13C-n.m.r. detection of an acyl-trypsin intermediate in the trypsin-catalysed hydrolysis of a highly specific substrate at subzero temperature

The kinetics of the trypsin-catalysed hydrolysis of the highly specific substrate N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester were studied under cryoenzymological conditions by 13C-n.m.r. spectroscopy at pH approx. 3.0. The kinetics of this reaction are shown to be in agreement with similar studies made with the use of u.v.-visible-absorption-spectrophotometric techniques. A combination of 13C-n.m.r. spectroscopy and cryoenzymology has for the first time detected an acyl-trypsin intermediate in the hydrolysis of this highly specific substrate. The advantages and difficulties of using 13C-n.m.r. spectroscopy coupled with cryoenzymology in the detection and characterization of enzyme-substrate intermediates are discussed.     

A 13C-N.M.R.-Spectral study of a gel-forming,branched (1→3)-β-d-glucan, (lentinan) from lentinus edodes, and its acid-degraded fractions. Structure,and dependence of conformation on the molecular weight

The structure and conformation of lentinan, an anti-tumor, branched (1→3)-β-d-glucan from Lentinus edodes, and its acid-degraded, lower molecular-weight fractions have been investigated by ¹³C-n.m.r. spectroscopy. It is found that their ¹³C-n.m.r. spectra are considerably changed, depending on the molecular weight. The conformational behavior as studied by ¹³C-n.m.r. spectroscopy is consistent with that revealed by a study of the shift in the absorption maximum of Congo Red complexed with lentinan and its acid-degraded fractions. It is found that the water-soluble fraction II (mol. wt. 3,640) gives rise to well-resolved ¹³C-n.m.r. spectra; the ¹³C-signals are assigned to (1→3)-β-d-glucan and branch points at C-6. The branched structure is also confirmed by examination of the ¹³C-n.m.r. spectra of the compounds in dimethyl sulfoxide. For the gel state of the fractions of higher molecular-weight, lentinan (mol. wt. 1,000,000) and fraction IV (mol. wt. 16,200), however, ¹³C-n.m.r. spectra of considerably attenuated signal-amplitude are observed. The fact that the ¹³C-signals of the β-d-(1→3)-linked main chain and side chains are completely suppressed is explained as a result of immobilization caused by their taking an ordered conformation. The ¹³C-resonances observed in the gel state, which are assigned to β-d-(1→6)-linkages, are unequivocally assigned to the side chains (of disordered conformation). Finally, the ordered conformation of both the β-d-(1→3)-linked main chain and side chains is identified as the single-helix conformation, which tends to form multiple helixes as junction zones for gel structure.     

Structural features of a water-soluble arabinoxylan from the endosperm of wheat.

A cold-water-soluble wheat-endosperm arabinoxylan consisting of a backbone of (1----4)-linked beta-D-xylopyranosyl residues that are variously unsubstituted, and 3- or 2,3-substituted with single alpha-L-arabinofuranosyl groups, was subjected to 1H-n.m.r. spectroscopy. The results of 2D homonuclear Hartmann-Hahn and 1D 1H-n.m.r. spectroscopy allowed the identification of 3- and 2,3-substituted xylose residues, each with adjacent unsubstituted xylose residues, and also substituted xylose residues with a substituted xylose residue as a neighbour. The 1H-n.m.r. data were correlated with 13C-n.m.r. data by means of a 13C-1H 2D proton-detected heteronuclear multiple-quantum correlation experiment, which showed that only different types of branching (i.e., 3- and 2,3-) can be identified by the 13C-n.m.r. data.     

Chondroitinase ABC digestion of dermatan sulphate. N.m.r. spectroscopic characterization of the oligo- and poly-saccharides.

Dermatan sulphates, in which iduronate was the predominant uronate constituent, were partially digested by chondroitinase ABC to produce oligosaccharides of the following structure: delta UA-[GalNAc(4SO3)-IdoA]mGalNAc(4SO3) [where m = 0-5, delta UA represents beta-D-gluco-4-enepyranosyluronate, IdoA represents alpha-L-iduronate and GalNAc(4SO3) represents 2-acetamido-2-deoxy-beta-D-galactose 4-O-sulphate], which were fractionated by gel-permeation chromatography and examined by 100 MHz 13C-n.m.r. and 400/500 MHz 1H-n.m.r. spectroscopy. Experimental conditions were established for the removal of non-reducing terminal unsaturated uronate residues by treatment with HgCL2, and reducing terminal N-acetylgalactosamine residues of the oligosaccharides were reduced with alkaline borohydride. These modifications were shown by 13C-n.m.r. spectroscopy to have proceeded to completion. Assignments of both 13C-n.m.r. and 1H-n.m.r. resonances are reported for the GalNAc(4SO3)-IdoA repeat sequence in the oligosaccharides as well as for the terminal residues resulting from enzyme digestion and subsequent modifications. A full analysis of a trisaccharide derived from dermatan sulphate led to the amendment of published 13C-n.m.r. chemical-shift assignments for the polymer.     

Structural analysis of the specific capsular polysaccharide of Rhodococcus equi serotype 2

The specific capsular polysaccharide produced by Rhodococcus equi serotype 2 is a high-molecular-weight acidic polymer composed of D-glucose, D-mannose, D-glucuronic acid and 3-O-[(S)-1-carboxyethyl]-L-rhamnose in equimolar proportions. Structural analysis, employing a combination of chemical and n.m.r. techniques, established that the polysaccharide is composed of linear repeating tetrasaccharide units. (formula; see text) in which the beta-D-mannose residues carry O-acetyl groups at O-2 and O-3 to the extent of 1.7 mol equivalents. Unequivocal determination of the absolute chirality of the 3-O-[(S)-1-carboxyethyl]-alpha-L-rhamnose residues was achieved by chemical correlation with an authentic synthetic sample. The 1H and 13C-n.m.r. resonances of the native and O-deacetylated serotype 2 polysaccharides were fully assigned by homo- and heteronuclear chemical-shift correlation methods.     

A 13C-n.m.r. investigation of ionizations within a trypsin-inhibitor complex. Evidence that the pKa of histidine-57 is raised by interaction with the hemiketal oxyanion.

The 13C-n.m.r. titration shifts of the alpha-methylene group of N-alkylated imidazoles are shown to be a sensitive probe of the ionization of the imidazolium ion. The 13C-n.m.r. titration shifts of both the intact and denatured/autolysed 2-13C- and 1-13C-enriched trypsin-7-amino-3-benzyloxycarbonylamino-1-chloroheptan-2-one (Z-Lys-CH2Cl) complexes are compared. The titration shift for the denatured/autolysed complex confirms that this ionization is due to deprotonation of the N-alkylated imidazolium ring of histidine-57. In the intact trypsin-inhibitor complex the titration shift due to the 1-13C-enriched carbon is anomalous. We conclude that this titration shift cannot arise solely from the ionization of the imidazolium ion of histidine-57 and that the pKa of the imidazolium ion of histidine-57 is raised in the trypsin-inhibitor complex. The relevance of these studies to the mechanism of action of the serine proteinases is discussed.     

Substituent distribution on O,N-carboxymethylchitosans by 1H and 13C n.m.r.

The use of the n.m.r. method in the investigation of chitosan carboxymethylation was evaluated. It seems to be the most effective technique to determine concurrently the degree and the position of substitution of the carboxymethylated chitosan derivatives. The 13C-n.m.r., by the DEPT method, 1H-1H and 1H-13C-n.m.r. correlations give much valuable information from the chemical shifts of the complex carboxymethylchitosan spectra. The relative reactivity of the functional groups of chitosan towards carboxymethylation was also determined assuming a higher reactivity of the C-6 position.     

Structural investigation of the capsular polysaccharide of Klebsiella serotype K 49

The structure of the capsular polysaccharide from Klebsiella type K 49 was investigated by 1H- and 13C-n.m.r. spectroscopy of the original, carboxyl-reduced, and Smith-degraded polysaccharides. Methylation of the original K 49 and derivatives showed that the polysaccharide consists of a tetrasaccharide repeating-unit having D-galacturonic as a single lateral substituent. All of the sugars have the alpha-D-configuration. This conclusion is in agreement with measurements of spin-lattice relaxation-times for the anomeric proton. O-Acetyl groups are located on galacturonic acid, but do not occupy a unique position. (Formula: see text).     

The use of 13C-n.m.r. spectroscopy to monitor alginate biosynthesis in mucoid Pseudomonas aeruginosa.

The biosynthesis of alginate by a mucoid strain of Pseudomonas aeruginosa, isolated from a cystic-fibrosis patient, was monitored by using 13C-n.m.r. spectroscopy of bacterial cultures incubated with 1-13C- or 2-13C-enriched fructose. When 1-13C- or 2-13C-enriched fructose was used as the precursor of alginate, enrichment with 13C in the constituent uronic acid monomers of the polysaccharide could only be detected in C-1 or C-2 respectively, indicating that alginate is synthesized in Ps. aeruginosa directly from fructose, with the hexose molecule being retained intact; this rules out the involvement of C3 intermediates, which occurs when glucose is the alginate precursor. The absence of detectable poly-L-gluluronate block sequences from the alginate of Ps. aeruginosa was confirmed, and it was shown that there is no modification of the arrangement of the constituent uronic acids between polymerization to form alginate and the appearance of the mature alginate in the extracellular medium. The 13C-n.m.r. data also provided independent evidence for acetylation on D-mannuronate residues and for the ratio of D-mannuronate to L-guluronate residues in newly synthesized alginate, which had previously been determined only for material secreted from bacteria into the extracellular medium.     

An improved synthesis of 1,6-anhydro-2,4-dideoxy-β-D-threo-hexopyranose

Dextran fractions from NRRL strains Leuconostoc mesenteroides B-1299 and B-1399, and the native, structurally homogeneous dextrans from L. mesenteroides. B-640, B-1396, B-1422, and B-1424, were examined by ¹³C-n.m.r. spectroscopy at 34 and at 90°, and by g.l.c.-m.s. The ¹³C-n.m.r. data indicate that the dextrans of this series branch exclusively through α-d-(1→2)-linkages, and differ from one another only in degree of linearity. Diagnostic, ¹³C-n.m.r resonances, correlating with 2,6-di-O-substituted α-d-glucosyl residues at branch points, have chemical shifts that are independent of the degree of linearity of the dextran. The intensities of these diagnostic resonances from branching residues, compared to the resonances associated with linear dextran (low degree of branching), are generally proportional to the degree of branching established by methylation-fragmentation analysis. The validity of assignment of the diagnostic, ¹³C-n. m.r. resonances is substantiated by a critical review of methods previously used to provide structural information on dextrans having α-d-(1→2)-linkages, and by evaluation of the corresponding results on the basis of the ultimate standard-methylation structural analysis.     

The structure of the biological repeating unit of the O-antigen of Hafnia alvei O39.

On mild acid-catalysed degradation of the lipopolysaccharide from Hafnia alvei O39 followed by gel filtration of Sephadex G-50, the O-specific polysaccharide and three oligosaccharides were obtained, which represent the core substituted with 0-2 O-antigen repeating-units. On the basis of sugar and methylation analyses, 13C-n.m.r. data, solvolysis of the polysaccharide with anhydrous hydrogen fluoride, and computer-assisted 13C-n.m.r. analysis of the Smith-degraded polysaccharide, it was concluded that the biological repeating unit of the O39 antigen was Formula; see text     

1H- and 13C-N.M.R. spectra of eledoisin and intermediate oligopeptides.

The 1H- and 13C-n.m.r. spectra of eledoisin and minor oligopeptides were measured and assigned. The proton spectra were interpreted on the basis of homonuclear decoupling, chemical shift criteria and spectra simulation. The information obtained was used in the assignment of the 13C spectrum via heteronuclear 1H-13C. The steric arrangement of proline residue was deduced from the 13C spectrum. Moreover the similarity of the 13C spectrum of eledoisin with that of component oligopeptides suggests that no considerable conformational change occurs in the undecapeptide relative to the component fragments.     

4-O-(1-carboxyethyl)-L-rhamnose, a second unique acidic sugar found in an extracellular polysaccharide from Butyrivibrio fibrisolvens strain 49.

Butyrivibrio fibrisolvens strain 49 excretes a polysaccharide that contains D-glucose, D-galactose, 4-O-(1-carboxyethyl)-D-galactose, and an acidic component of previously unknown structure. We report here the identity of the unknown as 4-O-(1-carboxyethyl)-L-rhamnose. The structure of this previously unknown compound was deduced from (1) comprehensive electron-impact and chemical-ionization mass-spectroscopic studies of differentially labelled derivatives prepared from the unknown, (2) 13C-n.m.r. and 1H-n.m.r. studies of purified neutral sugars derived from the unknown and (3) chemical degradation experiments.