Although regioselective removal of 6-O-sulfate groups of heparin has been undertaken by several researchers, complete 6-O-desulfation with little side reaction has not been attained successfully. In this work, a modified method with a certain silylating reagent, N-methyl-N-(trimethylsilyl)trifluoroacetamide, has been established to produce completely 6-O-desulfated heparin with few other chemical changes. The degrees of 6-O-desulfation were estimated by means of chemical disaccharide analyses and/or 13 C NMR spectra. Although the completely 6-O-desulfated heparin lost about 20% of 2-O-sulfate groups, any other chemical changes and depolymerization were not detected. The completely 6-O-desulfated heparin displayed strong inhibition of COS-1 cell adhesion to basic fibroblast growth factor (bFGF)-coated well in a dose-dependent manner, as was clarified by the competitive cell-adhesion assay. Furthermore, the completely 6-O-desulfated heparin was shown to promote in vitro A31 fibroblast proliferation in a dose-dependent manner in the presence of bFGF. These results suggest that signal transduction through bFGF/bFGF receptor in A31 cells occurs in the absence of 6-O-sulfate groups in heparin. The involvement of 6-O-sulfate group(s) of heparin/heparan sulfate in the promotion of bFGF mitogenic activity was reported by several groups. This discrepancy between our results and those of other groups would be due to the differences in molecular size of heparin/heparan sulfate derivatives and/or cell species used for the assay.
Heparin and heparan sulfate (HS)1 are known as glycosaminoglycan (GAG) components of extracellular matrix-forming connective tissues of animals. The former GAG, heparin, is exclusively distributed in mast cells, and the latter GAG, HS, is widely distributed in animal tissues. With the accumulation of the information concerning biological roles of heparin and HS, it has been revealed that their biological functions mostly depend upon interaction between polysaccharides and physiologically active molecules, although the biological roles of heparin and HS are highly diverged. For instance, they interact with lipoprotein lipase (1, 2), anti-thrombin III (3, 4), basic fibroblast growth factor (bFGF) (5-8), etc. Furthermore, minimum structures of heparin and/or HS necessary for binding with antithrombin III and/or bFGF have been determined (9, 12). Chemical modification of heparin has been undertaken by several researchers, focusing on the elucidation of the mechanism underlying interaction between heparin and the physiologically active molecules as described above. Specific removal of major sulfate groups of heparin such as 2-O-sulfate, 6-Osulfate, and N-sulfate groups would be useful in order to clarify the backbone structures of oligosaccharides bearing specific array of sulfate groups responsible for the interactions with physiologically active molecules. For instance, selective removal of 6-O-sulfate groups from glucosamine residues of heparin is of great importance in order to evaluate the involvemen...
