Peroxidative Depolymerization Of Fucosylated Glycosaminoglycan: Bond-cleavage Pattern And Anticoagulant Activities Of Oligosaccharides

Polysaccharides are functional biomacromolecules widespread in nature,and the structure elucidation is of great significance in the study of their biological functions.One of the effective strategies to study their structure and structure-activity relationship was to produce oligosaccharides from polysaccharides.Peroxidative depolymerization is a general chemical depolymerization method of polysaccharides,which is widely used in the structure elucidation and structure-activity relationship research of complex polysaccharides.Peroxidative depolymerization can cleave both glycosidic bonds and C-C bonds containing ortho-dihydroxy sugar rings.The newly formed reducing end after cleavage can be further oxidized and degraded.Therefore,peroxidative depolymerization products usually contain complex terminal structures.The complex chemical structures bring great technical challenges to the separation and purification,structural elucidation and analysis of the depolymerization mechanism of the depolymerized products.Fucosylated glycosaminoglycans（FG）from echinoderms have complex and special chemical structures and a wide range of physiological activities.For the chemical and pharmacological studies of FG,the peroxidative depolymerization method was one of the most widely used methods,but the peroxidative depolymerization cleavage of polysaccharides and the chemical structure characteristics of the depolymerization products,especially the reducing end and non-reducing end structure characteristics,are still rarely reported.In this paper,the highly regular structure of FG（FG from Stichopus variegatus,Sv FG）was depolymerized by Cu²⁺-catalyzed peroxidative depolymerization.The terminal structure of oligosaccharides from depolymerization products elucidate the glycosidic bond cleavage pattern during peroxidative depolymerization.Meanwhile,the peroxidative depolymerization process of FG（Hf FG）containing Fuc_3S4S side chain reported previously was compared to analyze the effect of sulfated fucosyl side chain on glycosidic bond cleavage and depolymerization kinetics.The main methods and results of this paper are as follows:（1）Separation and purification,structure analysis and reaction rule analysis of per-oxidative depolymerized products of Sv FG.A series of oligosaccharides were purified from the Sv FG peroxidative depolymerized product d Sv FG by GPC and SAX-HPLC,and their structures were analyzed by 1D/2D NMR and MS.The chemical structures of the resulting oligosaccharides are as follows.F1:α/β-L-Fuc_2S4SF2:α/β-D-Gal NAc_4S6SF3:Gal A-NAc_4S6SF4-a:2-O-(L-Fuc_2S4S-α1)-3-O-(D-Gal NAc_4S6S-β1)-2,3,4-trihydroxy-glutaric acid（TGA）F4-b:2-O-(L-Fuc_2S4S-α1)-3-O-(D-Gal NAc_4S6S-β1)-2,3-dihydroxy-butanedioic acid（DBA）F4-c:2-O-(L-Fuc_2S4S-α1)-3-O-(D-Gal NAc_4S6S-β1)-DBAF5-a:β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-D-Gal A-NAc_4S6SF6-a:β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,2]-DBAF7-a:[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-D-Gal A-NAc_4S6SF7-b:β-D-Gal NAc_4S6S-1,3-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-α/β-D-Gal NAc_4S6SF8-a:[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-{β-D-Gal NAc_4S6S-β1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-}₂-D-Gal A-NAc_4S6SF8-b:β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-{β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-}₂-D-Gal A-NAc_4S6SF8-c:β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,2]-DBAF8-d:[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-{β-D-Gal NAc_4S6S-β1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-β1,3-}₂-Gal NAc_4S6SF8-e:β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-{β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-}₂-α/β-D-Gal NAc_4S6SF9-a:[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-{β-D-Gal NAc_4S6S-1,4-[α-L-Fuc_2S4S-1,3]-β-D-Glc A-1,3-}₂-D-Gal A-NAc_4S6S（1）The peroxidative depolymerization of Sv FG has glycosidic bond cleavage selectivity,which the Glc A-β1,3-Gal NAc_4S6S glycosidic bond could be preferentially cleaved.（2）The reducing end Glc A of the depolymerized product formed by the cleavage of Glc A-β1,3-Gal NAc_4S6S glycosidic bond can undergo further oxidative degradation,and this oxidative degradation process may be accompanied by cleavage of the side chain glycosidic bond.Oxidative degradation of reducing end Glc A is the main reason for the complex and diverse depolymerization products.（2）Discussion on the mechanism of glycosidic bond selectivity during peroxidative depolymerization of Sv FG.FG contains three types of glycosidic bonds,D-Glc A-β1-,D-Gal NAc-β1-and L-Fuc-α1-,respectively.To compare the sensitivity of hexuronic acid,hexosamine,and deoxyhexose glycosidic bonds to peroxidative depolymerization conditions,polymannuronic acid（PMan A,[-4-Man A-β1-]_n）,poly N-acetyl glucosamine（PGlc NAc,[-3-Glc NAc-β1-]_n）and polyfucose(PFuc S,[-3-L-Fuc_2S-α1-]_n)were used for model reactions.The results showed that the depolymerization rates of the three polysaccharides were significantly different under the same peroxidative depolymerization conditions,k PMan A>k PFuc S>k PGlc NAc.It can be seen that there are significant differences in the sensitivity of different types of glycosidic bonds to peroxidative depolymerization,with hexuronic acid>deoxyhexose>N-acetyl hexosamine.This should be the main reason for glycosidic bond cleavage selectivity in the peroxidative depolymerization of FG.（3）The model reaction of Glc A oxidative degradation.In this paper,the oxidative degradation products of glucuronic acid（D-Glc A）were studied and analyzed with the sulfated fucose(L-Fuc_2S4S)as the control,and the characteristics of the oxidative degradation of Glc A at the reducing end of the depolymerized products were further studied.The results showed that under the same peroxidation conditions,the structures of L-Fuc_2S4S showed no changes significantly,while D-Glc A could undergo obvious oxidative cleavage.This result is consistent with the results of the oligosaccharides obtained by the peroxidative depolymerization of FG,and the reducing end glucuronic acid formed by depolymerization is prone to further oxidation.The L-Fuc-α1-glycosidic bond could be cleaved due to the sequential C-C bond cleavage of D-Glc A to yieldα-L-Fuc_2S4S residues.（4）The effect of side chain structure on the kinetics of FG peroxidative depoly-merization.To analyze the effect of different fucosyl side chains on the kinetics of FG peroxidative depolymerization,the kinetics of peroxidative depolymerization of Hf FG(side chain is Fuc_3S4S)and Sv FG(side chain is Fuc_2S4S)were further analyzed.The results showed that the peroxidative depolymerization of FG followed first-order kinetics,and the rate of peroxidative depolymerization of Hf FG branched with Fuc_3S4S is faster than that of Sv FG,indicating that the sulfated types of the fucose side chains of FG can affect the rate of peroxidative depolymerization.（5）Structure-activity relationship analysis of oligosaccharides from peroxidative depolymerized of Sv FG.The anticoagulant activity of oligosaccharides purified by peroxidative depolymerization was reported for the first time and compared with the anti-i Xase activity of oligosaccharides with the same chain length and similar structure.The oxidation of Glc A to saccharic acid in FG oligosaccharides could enhance its i Xase inhibitory activity.This paper expands the knowledge on the peroxidative depolymerization characteristics of complex glycosaminoglycans,and the resulting oligosaccharides with novel structural sequences can be further used for pharmacological activity evaluation,and for the application of chemical depolymerization methods of polysaccharides and structure-activity relationship research provides scientific information.