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.