| Microbial infection is the biggest enemy threatening human life and health,and the number one killer that leads to human death.Fortunately,the British microbiologist Alexander Fleming discovered the first antibiotic-penicillin in 1928.With the development and application of antibiotics by mankind,mankind has a powerful weapon that can fight against death.However,the adaptability of bacteria is far beyond imagination.Excessive use of antibiotics will cause bacterial resistance,thus making human beings face a huge threat of"super bacterial infection".Thus,the development of new antibacterial drugs is urgently in need.Sugars,nucleic acids,proteins,and lipids are four important biological molecules involved in life activities,which are widely distributed in animals,plants,microorganisms,and viruses.Among them,carbohydrates exist in large quantities in nature,and they were initially regarded as a nutrient essential to the human body,providing energy for living organisms to maintain life activities.With the advancement in the field of life sciences,chemists and biologists have gradually discovered that carbohydrates in organisms play a variety of roles,and participate in various physiological processes of cells in various forms,including intercellular recognition,cell migration,and adhesion,etc.Sugars mainly exist in the form of polysaccharides or glycoconjugates and play an important role in life activities.In recent years,with the gradual deepening of people’s understanding of the role of glycosylation in living organisms and the improvement of the level of sugar analysis,the research on sugar substances is becoming another new frontier in the field of life science.At the same time,the concept of carbohydrate medicine arose.There are many types of sugars and their conjugates present on the surface of bacteria,which have certain immunogenicity.Importantly,the glycosylation motifs of sugars on the surface of bacteria exhibit high conservation in the evolution of bacteria.Therefore,the glycoconjugate vaccine is one of the key points in the development of new vaccines.Most carbohydrate antigens induce immunoglobulin antibody M(IgM)rather than immunoglobulin antibody G(IgG),while lymphoid T cell-mediated immune response depends on IgG response.Therefore,sugar molecules can only induce weaker biological immune responses and do not have long-term memory.This problem seriously hinders the development of carbohydrate vaccines.Fortunately,scientists have discovered that after conjugating sugar chain molecules with some immunogenic carriers,sugar antigens can induce T cells to produce effective immune responses.The glycoconjugate vaccines currently used clinically are mostly prepared from the extraction of polysaccharide antigens from bacterial cells.However,these extracted polysaccharides have microscopic heterogeneity,making them not pure enough.This problem has brought great influence and restriction on the quality and safety of vaccines.To overcome this,the strategy developed by scientists is to obtain a bacterial polysaccharide with a well-defined structure and a uniform composition through synthesis,and then couple it with a carrier to form a glycoconjugate vaccine.To achieve this goal,we need to develop methodologies and synthetic strategies that can efficiently synthesize oligosaccharide fragments of bacterial surface polysaccharide antigens and their derivatives,and then study the immunological properties of the sugar complexes prepared therefrom.Cronobacter turicensis belongs to Cronobacter spp.,which is a Gram-negative pathogen.Recent studies have shown that Cronobacterium is an opportunistic pathogen that exists widely in food and the environment.Under normal circumstances,it will not cause harm to human health but can infect people of different ages with low immunity,such as infants and young children.It can cause necrotizing enterocolitis,meningitis,and sepsis,and bring serious neurological sequelae,which seriously threaten human life and health,especially to the infected newborns(mortality rate up to 40-80%).In recent years,most of the Cronobacter infections are related to infant formula milk powder;thus,formula milk powder is considered to be the main channel that causes infants and young children to be infected by Cronobacter.Therefore,the development of effective prevention and treatment methods for infections caused by Cronobacter is of great significance.Cell surface lipopolysaccharides are the main components of the cell wall of gram-negative bacteria.They play an important role in the host-bacterial interaction and the regulation of host immune response.They are considered to be ideal target antigens for the development of antibacterial carbohydrate vaccines.In this subject,we designed and synthesized lipopolysaccharide O-antigen tetrasaccharide repeat fragment derivatives of Cronobacter turicensis type 5 through chemical methods.This thesis mainly contains the following three chapters:The first chapter briefly introduces the development status of glycoconjugate vaccines,the structural characteristics and immune properties of Gram-negative bacteria lipopolysaccharides,the synthesis and application of O-polysaccharides,and the hazards,classification,and research progress of Cronobacter spp.The second chapter introduces the chemical synthesis of tetrasaccharide repeat fragment derivatives of Cronobacter turicensis type 5 lipopolysaccharide O-antigen.The synthesis of the 1,2-cis-rhamnosidic bond,1,2-trans-glucosamidic bonds,and 1,2-cis-glycosidic bond was efficiently constructed by using H-bond mediated aglycone delivery effects,neighboring group participation,and solvent effect.Therefore,we successfully prepared the target tetrasaccharide compound CT-1.First,the donor CT-6 was prepared from L-rhamnose;and both the glucosamine acceptor CT-7a equipped with azidoethyl chain and the glucosamine trichloroacetimide ester donor CT-4 were prepared from D-glucosamine hydrochloride;Glucothioside donor CT-2 was prepared from D-glucopyranose.Then,the donor CT-6 and the acceptor CT-7a are dissolved in the non-polar solvent 1,2-dichloroethane with NIS and TfOH under the condition of-30℃,complete the preparation of the disaccharide compound CT-5a by the glycosylation reaction which using H-bond mediated aglycone delivery effect.Subsequently,the 2-picolinyl group at the 3-O position of rhamnose was selectively removed by copper acetate to obtain the receptor CT-26a.Thereafter,the coupling reaction of compound CT-26a and imidate donor CT-4 were carried out in dichloromethane with a catalytic amount of TfOH under the condition of-78℃,providing the stereospecific trisaccharide compound CT-3a.Next,using iridium complex for selectively removing the allyl group at the 2-O position of the rhamnosyl residue to obtain the trisaccharide acceptor CT-27a.Then,the mixture of trisaccharide compound CT-27a and glucosyl thioglycoside donor CT-2 in co-solvent composed of dichloromethane and ethyl ether with NIS and trifluoromethanesulfonic acid under the condition of-100℃ to complete the fully protected tetrasaccharide CT-28a.Finally,the compound CT-28a was subjected to global deprotection of all acyl groups,selective acetylation of amino groups,followed by hydrogenolysis of all benzyl groups and reduction of azide groups to yield the final target product CT-1,whose structure was fully confirmed by NMR and MS analysis. |
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