Total Synthesis Of Cell Surface Carbohydrate Antigens Of Two Gut Pathogenic Bacteria

The intestinal microbiota has been associated with many diseases,such as obesity,inflammatory diseases and neurodevelopmental disorders related behavioral and physiological abnormalities.The surface of bacterial pathogens is covered with a series of dense polysaccharides,which is not only the unique characteristics of specific strains,but also the unique characteristics of different serotypes of bacteria.Bacterial surface polysaccharide antigen can induce specific immune response.Oligosaccharides and glycoconjugates synthesized by chemical methods are widely used in new drug development and vaccine development of infectious diseases.In this paper,the chemical synthesis and immunology of Clostridium bolteae capsular octadecasaccharide and the chemical synthesis of O-antigen trisaccharide of Vibrio cholerae O100 were studied.1.Total synthesis of C.bolteae capsular octadecasaccharide and its fragmentsThe C.bolteae capsular polysaccharide（CPS）consists of a[→3)-α-D-Manp-（1→4）-β-D-Rhap-（1→]disaccharide repeating unit with an average length of 18 sugar residues.To create vaccines against C.bolteae,it is important to identify exact protective epitopes of the immunologically active CPS.Here,a series of C.bolteae CPS glycans,up to an octadecasaccharide,was prepared.Four mannosides and one rhamnoside were successfully synthesized starting from common D-mannose.Using diacetone-D-glucose as the raw material,D-quinovoside was prepared in 10 steps and 33%yield.The C.bolteae CPS contain a large number of 1,2-cis-β-rhamnoside bonds,thus introduction of an effective method to install challenging stereoselectiveβ-D-rhamnosidic linkages was key to the total synthesis of C.bolteae octadecasaccharide.Firstly,we hypothesized that the O4 fluorenylmethyloxycarbonyl（Fmoc）group in D-rhamnoside may enhance theβ-selectivity by remote participation.Unfortunately,preactivation of D-rhamnoside with trifluoromethanesulfonic anhydride（Tf₂O）and 1-benzenesulfinyl piperidine（BSP）in the presence of 2,4,6-tri-tert-butylpyrimidine（TTBP）and subsequent addition of the acceptor led exclusively toα-linked disaccharide.The next attempt at the synthesis ofβ-D-rhamnoside utilizes the Crich’sβ-mannosylation approach（thioglycoside/BSP/Tf₂O glycosylation with conformationally-locked D-mannose donor）and subsequent C6 deoxygenation.The fully benzylatedβ-disaccharide was obtained in 41%total yield over 5 steps.Furthermore,the 1,2-cis-β-disaccharide can be achieved without conformation locking effect by using 1,2-trans-β-glycosylation and C2 epimerization strategy.On the other hand,C2 epimerization ofβ-D-quinovoside toβ-D-rhamnoside was efficiently achieved by Swern oxidation and borohydride reduction,but not nucleophilic substitution of triflate.With the building block in hand,the synthesis of the C.bolteae CPS began.Disaccharide,tetrasaccharide,all the way to octadecasaccharide were synthesized by[1（+2）_n+1]coupling strategy.The reliable method to install challengingβ-D-rhamnosides orβ-D-mannosides will serve well for the syntheses of other complex glycans containing such linkages.2.Antigenicity evaluation of synthetic glycan fragments using glycan microarraysThe glycotope of C.bolteae CPS that can be recognized by antibodies was mapped by glycan microarrays.The synthetic glycans of different length（2-mer,4-mer,6-mer,8-mer,10-mer,12-mer,14-mer,16-mer,18-mer）and isolated C.bolteae CPS were covalently attached on microarrays to screen polyclonal rabbit sera raised against inactivated C.bolteae bacteria.Tetrasaccharide exhibited the strongest interaction with rabbit Ig G antibodies among all synthetic glycans.Therefore,tetrasaccharide may be key motif for designing glycoconjugate vaccines against C.bolteae.It provides a theoretical basis for the exploration of immune epitopes of C.bolteae capsular polysaccharide and the development of glycoconjugate vaccine of C.bolteae.3.Total synthesis of Vibrio cholerae O100 O-antigen trisaccharide derivativesThe Vibrio cholerae O100 O-antigen consists of a[→3）-β-D-Quip N-Ac4N（d Hh）-（1→3）-α-D-Fucp4N（RHb）-（1→3）-α-L-Fucp NAc-(1→]trisaccharide repeating unit containing two rarely occurred amino sugar derivatives.In view of the undetermined absolute configuration of the two chiral centers of 3,5-dihydroxyhexanoyl group,four different structures of target trisaccharides with different configuration of 3,5-dihydroxyhexanoyl group were designed.We hope to build a biological immune strategy to determine the absolute configuration of 3,5-dihydroxyhexanoyl group.Using D-Fucose as starting material,D-quinovosamine building block was successfully synthesized in 23%yield over 10 steps.D-fucosamine building block was synthesized in 6%yield over 10 steps.The 1,2-cis-α-L-fucosidic bond was stereoselectively synthesized depended on a nonparticipating C2 azide group of trifluoroacetimide ester donor and the solvent effect of ether.Further,L-fucosamine building block was obtained in 9%overall yield.The 1,2-cis-α-D-fucosidic bond was synthesized stereoselectively with D-fucosamine block as donor and L-fucosamine block as receptor by solvent effect of ether.The orthogonally protected trisaccharide intermediate was synthesized using the D-quinovosamine as donor and disaacharide as acceptor.The rare 3,5-dihydroxyhexanoyl group was successfully introduced under mild reaction with（3R,5R）-3,5-di-O-benzylhexanoyl chloride and triethylamine.The synthesis of the trisaccharide fragment provides a valuable reference for the total synthesis of other complex amino sugars,and provides a theoretical basis for the development of carbohydrate vaccine against Vibrio cholerae O100 serotype.