Location of O-acetyl groups in S-657 using the reductive-cleavage method.

A two-step procedure is described in this paper to identify the position of O-acetyl groups in S-657 polysaccharide. Reductive-cleavage experiments performed on the fully methylated (base-catalyzed) polysaccharide, followed by acetylation of anhydroalditols, identified individual sugar residues and their position of linkage. In a second experiment, the polysaccharide was methylated under neutral conditions leaving native acetate groups intact. Reductive cleavage of the neutral methylated polysaccharide using CF3SO3SiMe3 as a catalyst, followed by acetylation in situ, identified sugar residues containing native acetate groups and established their position of substitution. Using this two-step procedure of analysis, S-657 polysaccharide is shown to contain O-acetyl groups on the 2-position and the 2,6-positions of 3-linked glucopyranosyl residues.

Analysis of Macrocystis pyrifera and Pseudomonas aeruginosa alginic acids by the reductive-cleavage method.

Permethylated alginic acids comprised of 4-linked D-mannopyranosyluronic acid and 4-linked L-gulopyranosyluronic acid residues undergo reductive cleavage to yield, after acetylation, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-D-mannonate (2b) and methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-D-gluconate (3b) as major products. Small amounts (ca. 13%) of ring-contracted products, namely methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-D-mannonate (9) and methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-D-gluconate (10), were also observed in these experiments. These results are in marked contrast to previous results on the reductive cleavage of 4-linked D-glucopyranosyluronic acid residues, wherein the ring-contracted product was formed exclusively. Formation of the ring-contracted products could be completely eliminated by reduction (LiAlH4) of ester groups in the permethylated alginic acid prior to reductive cleavage. In the latter experiments, 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-mannitol (5b) and 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-L-gulitol (6b) were the sole products of reductive cleavage of the 4-linked ManA and 4-linked GulA residues, respectively. However, in the previous experiments it was noted that low yields of permethylated alginic acids were obtained and that extensive depolymerization occurred under methylation conditions. Depolymerization could be avoided and higher yields of permethylated polysaccharides could be obtained, by reduction of the carboxyl groups of the alginic acids prior to methylation. Reductive cleavage of the latter polysaccharides yielded the products expected from 4-linked D-mannopyranosyl and 4-linked L-gulopyranosyl residues, namely 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-D-mannitol (13b) and 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-L-gulitol (14b), respectively. Using the latter analytical strategy, it was established that the Macrocystis pyrifera alginate was comprised of 60% 4-linked ManA and 40% 4-linked GulA residues, whereas the Pseudomonas aeruginosa alginate was comprised of 80% 4-linked ManA and 20% 4-linked GulA residues.

Analysis of positions of substitution of O-carboxymethyl groups in partially O-carboxymethylated cellulose by the reductive-cleavage method.

A method is described for the analysis of positions of substitution of O-carboxylated groups in commercial samples of O-carboxymethylcellulose. Sequential permethylation of the polymer and reductive cleavage gives eight products, which are analyzed as their O-acetyl derivatives by gas-liquid chromatography.

Analysis of a pyruvic acid acetal-containing polysaccharide by the reductive-cleavage method.

The applicability of the reductive-cleavage method to the analysis of polysaccharides bearing pyruvic acid acetals has been demonstrated. Direct reductive cleavage of fully methylated gum xanthan yielded the expected products, including 1,5-anhydro-4,6-O-[(S)-1-methoxycarbonylethylidene]-2,3-di-O-methy l-D- mannitol. The latter product was not observed when reductive cleavage was performed subsequent to reduction of ester groups in the fully methylated polysaccharide and mild hydrolysis to remove pyruvic acid acetal substituents. Instead, the latter experiment yielded 1,5-anhydro-2,3-di-O-methyl-D-mannitol, establishing the presence in the polysaccharide of terminal (nonreducing) D-mannopyranosyl groups bearing 4,6-O-(1-carboxyethylidene) substituents. The products of reductive cleavage were characterized, where appropriate, by comparison of the gas chromatographic retention times and chemical ionization- and electron ionization-mass spectra of their acetates to those of authentic standards. Alternatively, the products of reductive cleavage could be characterized without resort to comparison with authentic standards by analysis of the 1H-n.m.r. spectra of their benzoates, which were obtained in pure form by high-performance liquid chromatography. By either method of product characterization, this two-step procedure of analysis reveals the presence of pyruvic-acetal residues in polysaccharides and establishes both the identity of the sugar residue to which they are attached and their positions of attachment.

Synthesis and mass spectra of 4-O-acetyl-1,5-anhydro-2,3,6-tri-O -(methoxycarbonylmethyl)-D-glucitol and the positional isomers of 4- O-acetyl-1,5-anhydro-di-O-(methoxycarbonylmethyl)-O-methyl-D-glucitol and 4-O-acetyl-1,5-anhydro-O-(methoxycarbonylmethyl)-di-O-methyl-D-glucitol .

Reductive cleavage of fully methylated, partially O-carboxymethylated cellulose had previously been shown to produce 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-, -2-O-(methoxycarbonylmethyl)-3,6-di-O-methyl-, -3-O-(methoxycarbonylmethyl)-2,6-di-O-methyl-, -6-O-(methoxycarbonylmethyl)-2,3-di-O-methyl-, -2,3-di-O-(methoxycarbonylmethyl)-6-O-methyl-, -2,6-di-O-(methoxycarbonylmethyl)-3-O-methyl-, -3,6-di-O-(methoxycarbonylmethyl)-2-O-methyl-, and -2,3,6-tri-O-(methoxycarbonylmethyl)-D-glucitol. Described herein is the independent synthesis of these derivatives, except for the first, which had been reported. In addition, their 1H-n.m.r. spectra, chemical-ionization (NH3) mass spectra, and electronionization mass spectra are tabulated.

Model studies on the analysis of pyruvic acid acetal-containing polysaccharides by the reductive-cleavage method.

The 4,6-O-(1-methoxycarbonylethylidene), -(hydroxyisopropylidene), and -(methoxyisopropylidene) acetals of methyl 2,3-di-O-methyl-alpha-D-glucopyranoside were subjected to reductive cleavage in the presence of triethylsilane and trimethylsilyl methanesulfonate-boron trifluoride etherate (Me3SiOMs-BF3.Et2O), BF3.Et2O, or trimethylsilyl trifluoromethanesulfonate (Me3SiOSO2CF3) and the mole fractions of products were determined as a function of reaction time. The 4,6-(1-methoxycarbonylethylidene) acetal was quite stable to reductive-cleavage conditions but isomerization of the initial R,S mixture of diastereomers to the more-stable S diastereoisomer was noted. In addition, a slow, regiospecific, reductive ring-opening of the acetal was observed to give 6-O-[1-(methoxycarbonyl)ethyl] derivatives. The 4,6-(hydroxyisopropylidene) acetal was very unstable under reductive-cleavage conditions. Both Me3SiOMs-BF3.Et2O and Me3SiOSO2CF3 catalyzed complete removal of the group, via the intermediate 6-[1-(hydroxymethyl)ethyl] ether, but BF3.Et2O gave a mixture of products. The 4,6-(methoxyisopropylidene) acetal was also very labile under reductive-cleavage conditions; Me3SiOMs-BF3.Et2O catalyzed complete removal of the acetal, via the intermediate 6-[1-(methoxymethyl)ethyl]ether, but the intermediate ether was quite stable in the presence of either BF3.Et2O or Me3SiOSO2CF3. It is concluded from these studies that polysaccharides bearing 4,6-O-(1-carboxyethylidene) substituents can be analyzed directly by sequential permethylation and reductive cleavage. It is proposed that the identity of the substituted monomer and the positions of substitution of the acetal can be determined by sequential permethylation, ester reduction, and reductive cleavage.

Synthesis and mass spectra of 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-ethyl-D-glucitol and the positional isomers of 4-O-acetyl-1,5-anhydro-di-O-ethyl-O- methyl-D-glucitol and 4-O-acetyl-1,5-anhydro-O-ethyl-di-O-methyl-D-glucitol.

Reductive cleavage of fully methylated, partially O-ethylated cellulose or fully ethylated, partially O-methylated cellulose and subsequent acetylation had previously been shown to produce 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-, -6- O-ethyl-2,3-di-O-methyl-, -3-O-ethyl-2,6-di-O-methyl-, -2-O-ethyl-3,6-di-O- methyl-, -2,3-di-O-ethyl-6-O-methyl-, -2,6-di-O-ethyl-3-O-methyl-, -3,6-di-O- ethyl-2-O-methyl-, and -2,3,6-tri-O-ethyl-D-glucitol. Described herein is the independent synthesis of these derivatives, except for the first (which had been reported); and their 1H-n.m.r. spectra, chemical-ionization (NH3) mass spectra, and electron-impact mass spectra are tabulated.

Analysis of positions of substitution of O-methyl or O-ethyl groups in partially methylated or ethylated cellulose by the reductive-cleavage method.

A method is described for the analysis of positions of substitution of O-methyl and O-ethyl groups in commercial samples of O-methylcellulose and O-ethylcellulose, respectively. The method requires perethylation of O-methyl-cellulose and permethylation of O-ethylcellulose. Subsequent reductive cleavage of both polymers gives the same eight products, which are analyzed as their O-acetyl derivatives by gas-liquid chromatography.