Regioselective introduction of N-acetyl-d-glucosamine and d-glucosamine branches at C-6 of chitin and chitosan has been accomplished. N-Phthaloylchitosan was used as an organosoluble key intermediate for a series of controlled modification reactions, and the derived 3-O-acetyl-2-N-phthaloylchitosan and 3-O-acetyl-2-N-phthaloyl-6-O-trimethylsilylchitosan proved to be suitable acceptors for glycosylation with an oxazoline from glucosamine. The resulting branched product had a substitution degree up to 0.63. It was deprotected to give branched chitosan, which was subsequently N-acetylated to afford branched chitin. The introduction of such branches markedly improved the affinity for solvents, and the product with protecting groups was soluble in common organic solvents in sharp contrast to the insoluble chitin and chitosan. Furthermore, both the branched chitin and chitosan were readily soluble in neutral water and showed high moisture absorption and retention. The branched chitin was degraded with lysozyme, and the susceptibility decreased with increasing extent of branching. The branched chitosan was characterized by marked antimicrobial activity and aggregation ability for bovine serum albumin.

In view of the interesting properties of branched polysaccharides occurring in nature, biological activities of nonnatural branched chitins having beta-1,6-N-acetyl-D-glucosamine branches on the poly(beta-1,4-N-acetyl-D-glucosamine) backbone have been studied. The immunostimulatory activities of the branched chitins were determined and compared with those of lentinan, a beta-1,3-D-glucan having beta-1,6-D-glucose branches, using the mouse macrophagelike cell line RAW264.7 in vitro. The secretions of the tumor necrosis factor and nitric oxide proved to be significantly higher with the branched chitins than with lentinan. Moreover, when interferon-gamma was used in conjunction with the branched chitins on macrophage treatment, a marked augmentation of nitric oxide production was observed. These results are interpreted as the direct stimulation of macrophages by the branched chitins, and the distinctive activities suggest the possibility of developing new types of polysaccharide antitumor agents.

N-Acetyl-D-glucosamine and D-glucosamine branches were introduced into chitin and chitosan, respectively, by a series of regioselective modification reactions based on N-phthaloyl-chitosan. Unlike insoluble chitin and chitosan, the resulting nonnatural branched amino polysaccharides showed a remarkable water solubility. Moreover, the branched chitosan exhibited significant antimicrobial activity.