| Influenza is an acute infectious respiratory disease caused by the segmented negativesense single-stranded RNA enveloped virus influenza virus of the family Orthomyxoviridae,with annual seasonal epidemics or local outbreaks worldwide.Influenza causes a significant number of deaths globally each year.The highly pathogenic avian influenza virus is spreading globally,causing a large number of deaths in wild birds and significant economic losses in the poultry industry.Influenza viruses have established interconnected spill-over and transmission networks among various species,including birds,poultry,livestock,and humans,posing a threat to global public health security.Due to phenomena such as antigenic drift and antigenic shift,influenza viruses undergo rapid mutation.The development of new strategies for the prevention and treatment of influenza viruses is urgently needed.With the continuous advancement of glycobiology research,the glycochemical biology in influenza has gained significant attention.The binding of influenza viruses to receptors mediated by hemagglutinin is a critical step in the influenza virus lifecycle.There are two natural receptor terminal structures recognized by hemagglutinin: α-2,3-sialyl galactose(Neu5Ac-α-2,3-Gal)and α-2,6-sialyl galactose(Neu5Ac-α-2,6-Gal).Sialylated lactose structures can also serve as recognition and catalytic sites for influenza virus neuraminidase.This provides a natural affinity glycan structure for the design of anti-influenza oligosaccharide molecules in this study.Chitooligosaccharides have been widely studied due to its high biosafety,good biocompatibility,bioidentifiability and good biodegradability.Studies have shown that chitooligosaccharides exhibit good anti-influenza virus activity when covalently modified or non-covalently adsorbed.However,due to the difficulty in obtaining homogenous mono-chitooligosaccharides,studies use complex mixtures of multiple components,making it challenging to establish a clear structure-activity relationship.In this study,a series of chitooligosaccharides grafts against influenza viruses were constructed based on oligosaccharide compounds with a degree of polymerization ranging from 2 to 6,designated as compounds 1 to 5,as the active structural basis.Lactosamine,which is a widely present basic disaccharide structure in organisms,was used for the construction of antiinfluenza virus active molecules.In this study,the sialylated galactose affinity center was grafted with a mono-chitooligosaccharides active center,and the lactosamine structure was formed in situ.The sialyl lactosamine grafted chitooligosaccharides were designed,and the structure-activity relationship of chitooligosaccharides grafts against influenza was elucidated.In this study,the concept of organic synthesis was applied to enzymatic catalysis.Utilizing nine sugar-processing enzymes,different combinations of enzymes were employed to target specific glycosidic bonds,resulting in the construction of six enzyme modules corresponding to different types of glycosidic bonds.Through the combination of these enzyme modules,the efficient enzymatic synthesis of structurally defined sialyl lactosamine grafted chitooligosaccharides was achieved.This study first addressed the preparation of homogeneous chitooligosaccharides(Chitobiose,Chitotriose,Chitotetraose,Chitopentaose,and Chitohexaose)from chitosan.Chitosan with a deacetylation degree of 92% was enzymatically degraded by the glycosidase Csn W2 to yield products with degrees of polymerization ranging from 2 to 6.Subsequently,selective full acetylation of the amino group at the C2 position was performed to transform the multi-component mixture of oligosaccharides into a mixture of five components.The acetylated products were then purified by molecular exclusion chromatography,and DEAE ion exchange chromatography to obtain the homogeneous mono-chitooligosaccharides.The enzymatic modular synthesis of lactosamine-grafted chitooligosaccharideswas achieved through a one-pot three-enzyme β-1,4-galactosidase module(EM1).By introducingβ-1,4-galactose at the non-reducing end of the homogeneous chitooligosaccharides,the monolactosamine-grafted chitooligosaccharides 6～10 was synthesized at a yield of more than80%.Then,by β-1,3-N-acetylglucosagenesis module(EM2),the non-reducing end of monolactosamine-grafted chitooligosaccharide compounds 6～8 was ligated to obtain oligosaccharide compounds 11～13 with a yield higher than 85%.Compounds 11～13 was then catalytically linked by EM1 β-1,4-galactose to obtain bilactosamine-grafted chitooligosaccharide derivatives 16～18 with a yield more than 93%.Selective sialylation modification was performed on the non-reducing end galactose of Lactosamine-grafted chitooligosaccharides.Compounds 6 to 10 and 16 to 18 were subjected to a one-pot two-enzyme α-2,6-sialyltransferase module(EM3)catalysis to synthesize α-2,6-sialyl lactosamine-grafted chitooligosaccharides,resulting in compounds 19 to 26 with a yield more than 91%.Then by α-2,3-sialylase module(EM4),the grafted chitooligosaccharide compounds 27～34 was synthesized with a yield of more than 65%.In order to improve the solubility of the bilactosamine-grafted copolymer,based on the above four enzyme modules,combined with the sequential one-pot enzymatic synthesis strategy,the combined enzyme assembly modules EM5 and EM6 were constructed,and the further sialylation of the second lactosamine structure on chitopentaose and chitohexaose were realized.In the case of activated sugars synthesized by individual enzymatic method as donors,the nonaccharide(35)and decasaccharide compound(36)were obtained successively catalyzed by three glycosyltransferases sequential,HpLgt A,Nm Lgt B and Pd2,6ST A200Y/S232 Y with yields of 69% and 75%,respectively.The three glycosyltransferases of HpLgt A,Nm Lgt B and Pm ST3Δ35 were successively catalyzed sequential to obtain α-2,3-sialyl lactosamine-grafted chitooligosaccharide nonaccharide compound(37)and decasaccharide compound(38)with a total yield of 72% and 73%,respectively.CPE inhibition assays were conducted on MDCK cells to evaluate the inhibitory effects of compounds 6-10 and 16-38 against the virulent strain vir09.Ribavirin was used as a positive control,while α-2,3-sialylated lactosamine and α-2,6-sialylated lactosamine,known for their potent anti-influenza virus potential,served as negative controls.The results showed that MDCK cells exposed to α-2,6-sialylated lactosamine-grafted oligosaccharide compounds(19-26,35,and 36)exhibited significantly higher cell protection rates compared to the negative control group.Based on the precise structures of the five series of chitooligosaccharide grafts,an analysis of the structure-activity relationship against influenza virus was performed.It was found that sialic acid is an essential active moiety,and its configuration is correlated with the subtype selectivity of the virus.The oligosaccharide chain with a degree of polymerization ranging from 6 to 10 showed better activity,with the heptasaccharide exhibiting the highest activity.The activity of heptaose is the highest in sialylated monolactosamine grafted chitooligosaccharides,and the grafting activity of monolactosamine was higher than that of bilactosamine grafting.The trend of increasing activity with increasing polymerization degree was observed in the chitooligosaccharides sstructural units,with the chitopentaose-grafted compounds displaying the highest activity and the chitobiose-grafted compounds showing the most broad-spectrum potential.The results of molecular docking showed that the compound had strong binding to HA/NA at the same time has better anti influenza effect than the compound had strong binding to one of them.In conclusion,this study successfully constructed an efficient enzyme modular assembly strategy,which realized the efficient enzymatic synthesis of lactosamine grafted chitooligosaccharides and sialyl lactosamine grafted chitooligosaccharides.The antiviral activity of the synthesized chitooligosaccharides grafted derivatives against the vir09 strain was evaluated,and a corresponding structure-activity relationship between the anti-influenza virus and the chitooligosaccharides grafted derivatives was elucidated,providing new ideas and meaningful reference for the design and development of oligosaccharide-based molecules for the prevention and treatment of influenza viruses.Some of the chitooligosaccharids grafts exhibited higher antiviral activity than the negative control group,indicating their potential as promising lead compounds for the development of antiviral drugs and influenza vaccines. |
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