First Synthesis Of The Repeating Unit Of Vibrio Vulnificus MO6-24 Capsular Polysaccharide

Vibrio vulnificus,a halophilic Gram-negative bacterium,is ubiquitous in marine environment worldwide.It is highly pathogenic to humans and causes severe to life-threatening primary septicemia,necrotizing wound infection,and gastroenteritis through exposure to seawater,shellfish injury or ingestion of raw shellfish.The primary septicemia caused by V.vulnificus is rapidly progressing with the case-fatality rate exceeding 50%.The V.vulnificus infection cases have been growing year by year with the global climate warming and increasing of the offshore activities and susceptible populations such as patients with liver disease and diabetes.Consequently,the anti-V.vulnificus vaccine and rapid diagnostic are highly sought after.Like many other pathogenic bacteria,V.vulnificus expresses capsular polysaccharides(CPSs)as the major virulence factor,which could protect bacterium by conferring resistance to the bactericidal effects of serum and phagocytosis.V.vulnificus CPSs contain a variety of rare sugars that are virtually different from the host cell surface glycans,making them as the excellent haptens for the development of carbohydrate-based vaccines and pathogen detection strategies.Development of carbohydrate-based vaccines based on synthetic and structurally well-defined oligosaccharides has been demonstrated as a high effective strategy.Based on the above research backgroud,we here designed and synthesized a tetrasaccharide hapten corresponding to the repeating unit of V.vulnificus M06-24 CPS to facilitate the preparation of glycoconjugate vaccine.This text includes:In the first chapter,we briefly reviewed the epidemiology,pathogenic mechanism and typing of V.vulnificus,and systematically summaried the reported synthesis of similar oligosaccharide fragments.Accordingly,we proposed the research basis,objective,and specific experimental program.In the second chapter,the chemical synthesis of the tetrasaccharide repeating unit of V.vulnificus M06-24 CPS was introduced.The V.vulnificus M06-24 CPS repeating unit comprises a trisaccharide backbone,?3)-?-L-QuipNAc-(1?3)-?-D-GalpNAcA-(1?3)-?-L-QuipNAc-(1?,and a monosaccharide branch,a-L-QuipNAc,linked to the main chain 4'-O-position.Assembly of the target tetrasaccharide 2-1 was exceptionally synthetic challenging since all of its monosaccharide units were rare sugars and commercially unavailable.In addition,all the glycosyl linkages were 1,2-cis and their stereoselective formation needed to be judiciously controlled.Besides,assembly of a 3,4-branched a-D-GalN residue and its oxidation to a uronic acid were usually problematic.Initially,we attempted a[1+3]synthetic strategy to construct the desired tetrasaccharide skeleton,that was,to synthesize a linear trisaccharide first,and then install the branched structure.Since stereoselective formation of the a-L-QuiN glycosyl linkage was previously investigated only in a very few examples,we used 2-azido modified L-QuiN donors to study glycosylation conditions with an aim to establish a generally applicable method.The results showed that the 2-azido group functionalized L-QuiN thioglycoside donor produced the desired compound with high yield and excellent stereoselectivity in the presence of p-TolSCl/AgOTf at-40? in dichloromethane-ether(v:v=1:1)mixed solvent.A temporary TBS group was selected to protect the C6'-OH of trisaccharide 2-22 because TBS group could be easily removed after glycosylation to liberate the C6'-OH that could be used for subsequent oxidation reaction.However,glycosylation of receptor 2-4 with different glycosyl donors 2-3a-c by utilizing various promoters including p-TolSCl/AgOTf,DMTST,NIS/AgOTf,NIS/TfOH,and TMSOTf were all sluggish,and none of the desired tetrasaccharide was generated.In view of the steric hindrance of TBS,it was replaced by the relatively small Bz protecting group,and trisaccharide receptor 2-24 was accordingly prepared.Unfortunately,access to tetrasaccharide by using donor 2-3a-c and receptor 2-24 were infeasible again,and the desired 2-25 was generated at most in 20%yield.It was speculated that the sugar unit installed at the GalN?-anomeric position could present a steric crowding to force the C6' functionality to the top face of the GalN residue,thereby shielding the inherent weak nucleophilic C4'-OH of GalN.Subsequently,this influence aggravated the limitation of glycosylation reactions.In view of the above failure analysis,we employed an alternative[3+1]synthetic strategy to construct tetrasaccharide framework,that was,to preassemble the 3,4-branched trisaccharide first,then use it as donor to construct tetrasaccharide.To our delight,all of the glycosylation reactions involved this[3+1]strategy afforded the desired products in high yields and excellent to exclusive stereoselectivities by judiciously choosing glycosylation partners and reaction conditions.Finally,the fully protected tetrasaccharide intermediate was conterted to the target V.vulnificus M06-24 hapten through six-step reactions including debenzoylation,oxidation,benzylation,reduction of the azido groups,regioselective N-acetylation and hydrogenolysis.All of the intermediates and target compounds involved in this chapter were rigorously verified by 1D and 2D NMR and HR MS data.