Polysaccharides methylated, hydrolysis

A, Hydrolysis of naturally-occurring polysaccharide B, inethylation of aldobiouronio acid isolated after graded hydrolysis of polysaccharide C, hydrolysis of methylated polysaccharide D, methylation of partially methylated aldobiouronic acid isolated by method C. 

In most cases, uronic acids are liberated from acidic polysaccharides by hydrolysis leading to irreproducible concomitant formation of lactones. Several methods to circumvent this problem have been published describing conversion of the uronic acid into methyl esters followed by reduction with borohydride or borodeuteride reagents and subsequent hydrolysis and GC-MS detection [129]. Other techniques are based on the liberation and quantification of carbon dioxide. Direct determination of uronic acid residues in hydrolyzates has frequently been performed according to colorimetric assays, which are rather insensitive and have thus mostly been replaced by high-performance anion exchange chromatography (HP-AEC) methods [130-132]. 

The research that the Aspinall group produced in Edinburgh over the period 1948-1967 helped substantially in taking the science of plant polysaccharide structure from its infancy to a moderately developed state. While a great deal of the information gathered was obtained by methylation/hydrolysis analysis, Gerald was always aware of the need to find and use other methods that would contribute to refining structures. He therefore made extensive use of such methods as partial hydrolysis and periodate oxidation, when appropriate. 

Methylation Analysis. Methylation analysis, as described previously, involves complete methylation of a polysaccharide, the hydrolysis of the methylated product to a mixture of partially methylated monosaccharides, the reduction of the methylated monosaccharides, the acetylation of the reduced products and, finally, analysis of the partially methylated alditol acetates by GLC and MS. Methylation analysis gives information on the carbons... 

Pneumococcal Sill polysaccharide has been treated with a water-soluble carbodi-imide and then reduced with sodium borohydride the i.r. spectrum of the reduced polysaccharide showed negligible absorption attributable to the presence of carboxy-groups. The reduced material furnished 1,4,5-tri-O-acetyl-2,3,6-tri-0-methyl- and 1,3,5-tri-0-acetyl-2,4,6-tri-0-methyl-D-glucitol following methylation, hydrolysis, further reduction, and acetylation, compatible with the presence of equal proportions of (1 3)- and (1 -> 4)-linkages. The reduced... 

Glycogen differs only slightly from amylopectin, and studies such as methylation, hydrolysis, and periodate oxidation yield closely similar results for the two polysaccharides. These do establish, however, that there are... 

In 1947, L-rhamnose was first recognized by Stacey as a constituent of Pneumococcus Type II specific polysaccharide. This finding was confirmed, in 1952, by Kabat et al. and in 1955 again by Stacey when 2,4- and 2,5-di-O-methyl-L-rhamnose were synthesized and the former was shown to be identical with a di-O-methylrhamnose, obtained by hydrolysis of the methylated polysaccharide. This result indicated a pyranose ring structure for the rhamnose units in the polysaccharide. Announcement of the identification of D-arabinofuranose as a constituent of a polysaccharide from M. tuberculosis aroused considerable interest. The L-enantiomer had been found extensively in polysaccharides, but reports of the natural occurrence of D-arabinose had been comparatively rare. To have available reference compounds for comparison with degradation products of polysaccharides, syntheses of derivatives (particularly methyl ethers) of both d- and L-arabinose were reported in 1947. 

The floss silk from Chorisia speciosa furnished a polysaccharide with a main chain of (1 -> 4) linked P-Xylp substituted at 0-2 by 5 % of uronic acid. The xylan structure also was interposed with a-Rhap units in small amounts. The defatted seeds furnished on aqueous extraction a major fraction, ((9-acetyl, 10 % and protein, 45 %) wich was hydrolysed and analysed by p.c. and GLC, showing Rha (20 %), Ara (16 %), Gal (64 %) and also uronic acids (45 %). Partial hydrolysis gave rise to a polysaccharide free of arabinose, with 46 % of uronic acids. Methylation analysis (GLC -MS) indicated a chain of (1 4) - linked Gal/ (42 % of 2,3,6-Me3-Gal). 

Most early publications on bacterial polysaccharides were concerned with impure products and poorly-described organisms. Many more recent papers are of limited value also, due to low yields, lack of characterization of products and arbitrary interpretations of data. Low yields of methylated polysaccharides may be due to degradation of the bacterial polysaccharide during methylation, or to degradation of the hydrolytic products of the methylated polysaccharide (to form methyl levulinate, etc.46). The great importance of (a) complete methylation of polysaccharide products prior to structural determination by hydrolysis and (6) quantitative identification of the hydrolytic products, has been emphasized previously. Other difficulties in end group analysis have been discussed recently.7... 

In a recent paper650 the relationships between Rhizobium radicicolum polysaccharides and those of pneumococcus are indicated by crossprecipitin reactions of the former with Types III and VI anti-pneumococcus horse sera. In addition, hydrolysis of the methylated polysaccharide of Rhizobium radicicolum yields equal amounts of 2,3,6-trimethyl-D-glucose, 2,3-dimethyl-D-glucose and 2,3-dimethyl-D-glucuronic acid.65 1 The minimum trisaccharide repeating unit consists, in part, of cello-biuronic acid, and may be represented ... 

Reeves and Goebel72 have shown that hydrolysis of the reduced methylated capsular polysaccharide of Type III pneumococcus yields 2,3,6-trimethyl-D-glucose and the anomeric forms of methyl 2,4-dimethyl-D-glucoside. The cellobiuronic acid units in the polysaccharide are thus linked through position 3 of the D-glucuronic acid residue, probably by /3-D-linkages. That is, the polysaccharide contains alternate 1,3-and l,4-/3-D-linkages. 

Yeast Insoluble Polysaccharide. The structure of an insoluble polysaccharide from the yeast Saccharomyces cerevisiae was investigated by Zechmeister and Toth,90a and also by Hassid, Joslyn and McCready.904 The isolation904 of 2,4,6-trimethyl-D-glucose as the sole product of the hydrolysis of the methylated polysaccharide indicated a chain of gluco-pyranose units joined by 1,3-glucosidic linkages. 

A large number of polyfructosans that have been reported from time to time by different authors have been investigated by Schlubach and his associates. In order to obtain polysaccharides of constant optical rotation, 100 to 300 precipitations from aqueous solution by the addition of alcohol were necessary. Fifty to 150 precipitations from chloroform solution with petroleum ether were required for purification of the acetate derivatives. These were methylated according to the procedure of Haworth and Straight,24 and upon hydrolysis partially methylated fructoses were obtained. 

Secalin. Secalin has been isolated from the stems of unripe rye.46,68 Schlubach and Bandmann69 studied its structure. The great difficulty they encountered in obtaining the polysaccharide and its acetate in homogeneous form made the determination of physical properties uncertain. However, by hydrolysis of the methyl derivative, they obtained, after separation by means of the /3-naphthoates, tetramethyl-, trimethyl-, and dimethyl-D-fructoses in the proportions of 1 2 1. The trimethyl-D-fructose was identified as 1,3,4-trimethyl-D-fructofura-nose by its melting point and specific rotation. 

Schlubach, Knoop and Liu85 investigated the structure of irisin by hydrolysis of the methylated polysaccharide. The acetyl derivative was converted to methylirisin by one treatment with dimethyl sulfate and sodium hydroxide in acetone solution. It was obtained as a white powder having [a]D20 = — 63.2° (c = 2.14, chloroform), m. p. 188-190°. 

Chemolysis of ester bonds is performed by hydrolysis or methanolysis. Acidic metha-nolysis, for 24 h at 80 °C, cleaves ester bonds by transesterification, obtaining the fatty acid methyl esters (FAMEs), and has been used to simultaneously study oils, waxes, tannins, resins and polysaccharides in samples collected from embalming materials from Egyptian mummies [17]. Transesterification with trimethyl sulfonium hydroxide in methanol is also used [33,34],... 

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