Carboxylic reducing

Glycosyl-linkages were determined by GC-EIMS of the partially methylated alditol acetates. RG-II samples (2 mg) were methylated using sodium methyl sulfmyl carbanion and methyl iodide in dimethyl sulfoxide [24] followed by reduction of the uronosyl groups with lithium triethylborodeuteride (Superdeuteride , Aldrich) [23,25]. Methylated and carboxyl-reduced samples were then submitted to acid hydrolysis, NaBIlt reduction and acetylation, partially methylated alditol acetates being analysed by EIMS on two fused-silica capillary columns (DB-1 and DB-225) [20]. 

Carboxyl redution. A sample of pennethylated PI (5 mg) was carboxyl-reduced by a modification of the method described by Lindberg and Lbnngren [9], as follows. The methylated fraction was solubilized and added a mixture of LiAlH4 (25 mg) in THF (5 mL) at 20 °C for 4 h. and refluxed during 1 h. The excess of reagent was destroyed with ethyl acetate (5-6 drops) and water (10 drops) and the pH of the mixture adjusted to neutrality with acetic acid. The insoluble residue was removed by centrifugation. The reduced fi action was precipitated with EtOH. The reaction was monitored by l.r. specroscopy. Hydrolysis products were analysed by GC-MS as methyl alditol acetates... 

The identification of L-iduronic acid as the major glycuronic acid constituent of heparin proved to be a much slower process than the identification of the amino sugar residue. Although this compound was detected in acid hydrolyzates of heparin116117 and heparin oligosaccharides,118 its yield was usually poor, because of the drastic conditions used for the acid hydrolysis (which are known to lead to extensive destruction of uronic acid).119120 Also, L-iduronic acid escaped detection as L-idose in the hydrolyzates of carboxyl-reduced heparin, probably because L-idose is readily converted into 1,6-anhydro-L-idose under the usual hydrolytic conditions. 

Examination of early -n.m.r. spectra of heparin and of chemically modified heparins121 prompted a reinvestigation of N,0-desulfated, carboxyl-reduced heparin, leading to the isolation of substantial amounts of L-iditol pentaacetate.121,122 In addition, improved conditions for the acid hydrolysis of heparin and carboxyl-reduced heparin gave increased recoveries of L-iduronic acid and 1,6-anhydro-L-idose, respectively.123 These findings confirmed the L-enantiomeric designation of the iduronic acid, and established that it is the main uronic acid in heparin. 

Both positional linkages (uronic acid to hexosamine and hexosamine to uronic acid) were established as being (1 — 4) by structural analysis of the previously mentioned (see Section IV), crystalline disaccharides containing D-glucuronic acid, isolated from an acid hydrolyzate of carboxyl-reduced heparin.128-129 Further evidence was obtained from the structure of the D-glucuronic acid-containing counterpart of disaccharides 6 and 8, obtained as minor products from pig-mucosal heparin following nitrous acid deamination,1110 136-138 and acid hydrolysis followed by N-acetylation,130 respectively. 

Whereas the configuration at C-l of the amino sugar units in heparin was established as a-D by early studies, especially the structural characterization129 of crystalline disaccharide 9, the configuration of the D-glucuronic acid has been the subject of controversy. It was originally presumed to be a-D, on the basis of the structure proposed for a disaccharide obtained, in addition to 9, from desulfated, carboxyl-reduced heparin.129... 

The work on the first sulfated oligosaccharides was based on investigations of the polymeric heparinoids derivatives of heparin had been prepared to obtain information on the structural requirements of heparin for SMC antiproliferative activity. A clue for further studies was brought about by carboxyl-reduced sulfated heparin (CRS-heparin, 9) in which all carboxyl groups were reduced and... 

While the size requirement for SMC antiproliferative activity had been investigated for heparin (cf. Sect. 2.2), a similar study on fractions of highly active heparan sulfate has not been carried out. After the discovery of active sulfated tetrasaccharides it seemed possible that relatively small heparan sulfate substructures with the antiproliferative activity of heparin could be identified. Model heparan sulfate oligosaccharides were devised in which the W-sulfate groups were replaced by 0-sulfates and the glucuronic acids were replaced by glucoses, form y carboxyl-reduced and sulfated glucuronic acids - in parallel to CRS-heparin (see above). With this approach Ae heparan sulfate backbone... 

TABLE 2.1 Methylation analysis of intact, desulfated, and desulfated/carboxyl-reduced fractions prepared from the sea cucumber 5. japonicas GAG... 

After hydrolysis of carboxyl-reduced and partially desulfated heparin with hydrochloric acid, Wolfrom and coworkers117 isolated a disaccharide that was identified118 as 2-amino-2-deoxy-4-0-a-D-glucopyran-osyl-D-glucopyranose ( maltosamine ) hydrochloride, which gave a crystalline N-acetyl derivative (50). This structure was subsequently... 

In the literature, there are several examples of partial hydrolysis of polysaccharides or glycoconjugates containing 2-amino-2-deoxyhex-ose residues. One example is the hydrolysis of carboxyl-reduced, N deacetylated chondroitin, with the formation of 4-0-(2-amino-2-deoxy-/3-D-galactopyranosyl)-D-glucose.51 Another is the isolation of 6-0-(2-amino-2-deoxy-/8-D-galactopyranosyl)-D-galactose after N-de-acetylation and partial hydrolysis of the pentasaccharide (19) obtained... 

Oxidation of the carboxyl-reduced and acetylated Pneumococcus type 2 capsular polysaccharide revealed that only one L-rhamnose residue in the hexasaccharide repeating-unit, later demonstrated to have the structure 60, was oxidized and, consequently, /3-L-linked.156 Replacement of 2,3,6-tri-O-methyl-D-glucose in the methylation analysis of the original polysaccharide by 2,3,4,6-tetra-O-methyl-D-glucose in that of the oxidized polysaccharide established that this L-rhamnose residue is linked to 0-4 of a D-glucose residue. The analysis also showed that it was an L-rhamnose residue in the chain (and not the branching L-rhamnose residue) that was /3-linked. 

It has been stated that (-F)-tartaric acid is fully described" by the D-config-uration because, if either asymmetric carbon is reduced to a CH2 group, d -2-hydroxy-butanedioic acid is formed. Which configuration would be assigned to (+)-tartaric acid, if either one of the carboxyls were reduced to a methyl group, the hydroxyl next to the remaining carboxyl reduced to CH2, and the product compared to D- and l-3-hydroxybutanoic acids ... 

Figure 1. Methylation linkage analysis of PSL-I by GC/MS total ion chromatogram of partially methylated alditol and myoinositol acetates (PMAA) from PSL-I carboxyl-reduced trisaccharide by gas chromatography /mass spectrometry in...

Major Oligosaccharides Prepared from the Carboxyl-Reduced Concentrate of Glycophosphoceramides of Tobacco Leaves... 

For the remaining components in the concentrate, Hsieh (22) prepared a mixture of oligosaccharides from the carboxyl-reduced... 

Acid treatment of the carboxyl-reduced derivative of /V-acetylneuraminic acid yields the 2,7-anhydride of the corresponding non-2-ulopyranose.268... 

When the monosaccharide was not available directly after hydrolysis or solvolysis of the polysaccharide, a monomeric derivative for MS analysis could be obtained by a combination of chemical modifications and cleavages. Thus, in order to obtain an open-chain derivative of pseudaminic acid, OPS of S. boydii type 7 (Fig. 4, structure 12) was carboxyl-reduced, hydrolyzed under mild acid conditions, the products were carbonyl-reduced, solvolyzed with anhydrous hydrogen fluoride, and methylated.9,24... 

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