Preparation Of Molecular Weight Controlled Chondroitin Sulfate Oligosaccharides Based On Biological Method And Its Fluorescent Labeling

Chondroitin sulfate(CS)is a sulfated glycosaminoglycans,which is involved in various biological functions such as cartilage regeneration,inflammatory response,nervous system development and damage repair.Its sugar chain is composed of 1,4-linked N-acetyl-β-d-galactosamine(β-D-Galnac)and 1,3-linkedβ-D-glucuronic acid(β-D-Gl CA)repeated disaccharide units.According to the different sulfate modification patterns on the disaccharide repeating unit,chondroitin sulfate is mainly divided into Chondroitin 4-Sulfate(CS-A),Chondroitin 6-Sulfate(CS-C),Chondroitin 2,6-Sulfate(CS-D),Chondroitin 4,6-Sulfate(CS-E),and several other subtypes.The number and sites of sulfate groups on the sugar chain play an important role in regulating the biological activity of chondroitin sulfate.Due to the sugar chain length,sulfation modification,isomerization modification and other factors,the structure of chondroitin sulfate is complex and the molecular weight is not uniform.Therefore,the research on the structure and neuromodulation of chondroitin sulfate oligosaccharides has gradually attracted people’s attention.At present,natural chondroitin sulfate is mainly derived from animal cartilage tissue,and it is difficult to obtain chondroitin sulfate oligosaccharide with controllable chemical structure and uniform molecular weight due to its complex configuration.The main metabolite of Escherichia coli K4 is fructosylated chondroitin,which can be used as a precursor of chondroitin sulfate.After defructose treatment,the same sugar chain structure chondroitin can be obtained.It is expected to be used as a raw material.The non-animal source chondroitin sulfate oligosaccharide and its derivatives are prepared.In this paper,two steps of enzymatic degradation and enzymatic sulfation were used to achieve chondroitin sulfate oligosaccharides with controllable chain size and sulfated sites,and further fluorescently labeled chondroitin tetrasaccharides.First,defructosylated K4(DK4)was degraded by chondroitin sulfate degrading enzyme to realize the preparation of chondroitin oligosaccharides;then,different sites were sulfated under the action of chondroitin sulfate sulfotransferase;Finally,the coumarin derivative fluorescent agent B was synthesized,and the reductive amination reaction with chondroitin tetrasaccharide was carried out to realize the fluorescent labeling of chondroitin oligosaccharide.The main findings are as follows:(1)K4 polysaccharide was obtained by fermentation of Escherichia coli K4.After purification and fructose removal,a higher purity DK4 was obtained.The M_w was 27.79 k Da and the PDI was 1.247 determined by GPC,and its structure was determined by infrared and nuclear magnetic resonance.The chondroitin sulfate-degrading enzymes AC and ABC were expressed in Escherichia coli at low temperature.Purified by nickel column and identified by SDS-PAGE,the purity was higher than 95%,and the molecular weights were about 75 k Da and100 k Da,respectively.Degradation was carried out with DK4 as the substrate,and the degradation conditions were optimized.Chondroitin tetrasaccharide,hexasaccharide and octasaccharide with controllable chain sizes were separated by Bio-Gel P-2 gel chromatography and high performance liquid chromatography.(2)By optimizing the insect cell baculovirus system to express chondroitin sulfate 4-O-sulfotransferase(CS4OST)and chondroitin sulfate 6-O-sulfotransferase(CS6OST),they were identified and measured by SDS-PAGE after purification.The molecular weights are approximately 48 k Da and 58 k Da,respectively.Using chondroitin oligosaccharide as substrate,3’-phosphate adenosine-5’-phosphoryl sulfate(PAPS)as sulfonic acid group donor,by optimizing the enzymatic reaction conditions of sulfate modification,resulting in CS-A and CS-C isoforms with controllable chain size and sulfation sites.(3)Using diethylamino-salicylaldehyde and ethyl nitroacetate as raw materials to synthesize fluorescent agent B,chondroitin tetrasaccharide and fluorescent agent B undergo reductive amination reaction to generate a tetrasaccharide fluorescent marker with a labeling rate of 3.64%.Characterized by fluorescence spectrophotometer,infrared and ultraviolet methods.The effects of p H,cytotoxicity and cellular uptake ability were investigated,and the results showed that the tetrasaccharide fluorescent label can exist stably in cells and be selectively taken up by cells.