Structural Characterization Of The Sulfated Polysaccharides And Oligosaccharides From Monostroma Angicava Kejllman

The polysaccharides from marine green algae are almost sulfatedpolysaccharides, and have various bioactivities such as anticoagulant, antioxidant,lipid-regulating effect, anti-virus, anti-tumors and immunomodulatory activities. TheStructural study of the sulfated polysaccharides will supply the theoreticalfoundations for the developments and applications of sulfated polysaccharides. Inthe present work, the sulfated polysaccharides from Monostroma angicava Kejllmanwere isolated, and their structural characterizations were investigated by acombination of chemical and spectroscopic methods. The research results were asfollows:The green alga Monostroma angicava Kejllman was extracted by cold and hotwater to obtain two polysaccharides CMK and HMK, their yields are27.28%and19.62%, respectively. The two polysaccharides CMK and HMK were furtherpurified on Q-Sepharose Fast Flow ion-exchange and Sephacryl S-400/HRsize-exclusion chromatography to generate three polysaccharides CMKS-1,CMKS-2and HMKS of uniform size and charge. The sulfated polysaccharidesCMKS-1, CMKS-2and HMKS are composed of about80%rhamnose residuestested by PMP pre-column high performance liquid chromatography. CMKS-1andCMKS-2have higher glucose content (~10%) than HMKS (~5%). The threepolysaccharides also contained minor amounts of glucuronic acid, galactose, xyloseand mannose residues. The total sugars content of CMKS-1, CMKS-2and HMKSwere54.21%,51.16%and54.33%respectively, the sulfate ester ontents are23.86%,22.52%,25.84%. The average molecular weights of CMKS-1, CMKS-2and HMKSwere estimated to be about283.8kDa,73.5kDa,926.5kDa, respectively.The linkage units of desulfated polysaccharide dsHMKS were detected by methylation analysis and detailed one-and two-dimensional nuclear magneticresonance spectroscopic analyses (1H NMR,13C NMR,1H-1H COSY,1H-13CHMQC,1H-13C HMBC,1H-1H NOESY). The results showed that dsHMKSmainly consisted of the disaccharide residues [â†'3)-α-L-Rhap(1â†'3)-α-L-Rhap(1â†'],[â†'2)-α-L-Rhap(1â†'3)-α-L-Rhap(1â†'] and [â†'2)-α-L-Rhap(1â†'2)-α-L-Rhap(1â†']linked with the disaccharide residues [α-L-Rhap(1â†'3)-α-L-Rhap(1â†'] or[α-L-Rhap(1â†'2)-α-L-Rhap(1â†']. The sulfated polysaccharides CMKS-1andCMKS-2were composed of the linkage units [â†'3)-α-L-Rhap(1â†'],[â†'2)-α-L-Rhap(1â†'],[â†'2,3)-α-L-Rhap(1â†'] and [α-L-Rhap(1â†'], and the sulfateesters are supposed to be mainly located at C-2of the [â†'3)-α-L-Rhap(1â†'] and C-3of the [â†'2)-α-L-Rhap(1â†'] and C-2/C-2/C-4of the residues [α-L-Rhap(1â†'].The twenty-seven sulfated polysaccharides with different molecular weightswere obtained under different trifluoroacetic acid concentration (0.01mol/L,0.05mol/L,0.1mol/L) and temperature (60℃,80℃,100℃) conditions, and themolecular weights were about from858.58kDa to1.28kDa measured by HPGPC.The low molecular weight sulfated polysaccharide QSA (16kDa) obtained byTFA hydrolysis of the sulfated polysaccharide HMKS consisted of the sugar linkageunits of [â†'3)-α-L-Rhap(1â†'] mainly, and [â†'3)-α-L-Rhap(2SO2-4)(1â†'],[â†'2)-α-L-Rhap(3SO2-4)(1â†'],[â†'2)-α-L-Rhap(1â†'],[â†'2,3)-α-L-Rhap(1â†'] andsmall amount [â†'2)-α-L-Rhap(4SO2-4)(1â†'], and the sulfate ester groups werelocated at C-2/C-3/C-4of the terminal residue [α-L-Rhap(1â†'] elucidated bymethylation analysis and1H NMR,13C NMR,1H-1H COSY,1H-13C HMQC,1H-13CHMBC.The structures of the sulfated oligosaccharides, whose degree of polymerizationare2~4gained from the polysaccharide HMKS hydrolysis with TFA, wereinvestigated by methylation analysis, electrospray ionization mass spectrometry(ESI-MS, ESI-CID-MS/MS). The results showed that the sulfate ester groups of theoligosaccharide are located at the C-3of sulfated mono-rhamnose. The structures of the sulfated disaccharides have isomers of α-L-Rhap(3SO4)-(1â†'3)-α-L-Rhap,α-L-Rhap(2SO4)-(1â†'3)-α-L-Rhap, α-L-Rhap(3SO4)-(1â†'4)-β-D-GlcAp, α-L-Rhap(2SO4)-(1â†'4)-β-D-GlcAp, β-D-GlcAp(4SO4)-(1â†'3)-α-L-Rhap, andβ-D-GlcAp(4SO4)-(1â†'2)-α-L-Rhap. The results indicated that the main chains ofthe sulfated polysaccharide HMKS are complicated. The structural characterizationof the sulfated polysaccharide HMKS were further confirmed.