Synthesis Of Monophosphoryl Lipid A Mimetics-Tn Carbohydrate Vaccine And Type Ia Group B Streptococcal Capsular Polysaccharide Repeat Unit Building Blocks

The study included two parts:1.Synthesis of mono-phosphate ester A-Tn antitumor vaccine;2.Synthesis of Type Ia group B streptococcal capsular polysaccharide repeat unit building blocks.Synthesis of monophosphoryl lipid A mimetics-Tn antitumor vaccine:Tumor is a common and frequently-occurring disease which threatens human health,and is the second cause of human death.The carbohydrate vaccine is the target of tumor-associated carbohydrate antigens for cancer therapy,which has high specificity,low side effects,and the potential to improve or cure the tumor in early stage.Tumor-associated carbohydrate antigens is a non-T cell-dependent antigen,which can only activate B cells,produce a low affinity IgM,without produce high affinity IgG.In order to active specific T cell immune response in vivo,the traditional strategy is to combine the glycoprotein vaccine with the carrier protein containing T cell epitopes,and then the glycoprotein vaccine enters the organism,it can be identified by the antigen-presenting cell(APCs)and presented to T cells to activate the T cells produce IgG antibodies,meantime B cells are induced to proliferate and produced memory B cells,thereby specific killing tumor cells.However,the glycoprotein vaccine has some disadvantages,such as uncertainty of the coupling sites of glycoprotein antigen and epitopes protein,unstable coupling ratio,complex composition and immune reaction of epitopes protein,which can suppress the immune response of tumor-associated carbohydrate antigens.To optimization the glycoprotein vaccine,the chemical synthesis of carbohydrate-based vaccines,which is a covalent connection of tumor-associated carbohydrate antigens and endogenous adjuvant,has the simple chemical structure,clear composition,immune reaction of carrier protein,which is the main direction of anti-tumor vaccine.It has been proved that agonist of toll-like receptor(TLR)is an endogenous adjuvant,the Chemical synthesis of carbohydrate-based vaccines with TLR adjuvant can be presented to the corresponding immune cells,resulting in the immune response to the tumor-associated carbohydrate antigens.Until now,the epitopes of endogenous adjuvants are mainly various subtypes of TLR agonists,and MPLA(Monophosphoryl lipid A)is one of the most important TLR agonists.Moreover,MPLA was used as a vaccine carrier and a variety of carbohydrate-based vaccines coupling into the two-component vaccine,it had been demonstrated the anti-tumor activity in clinical practice.Researchers found that the second generation of MPLA which named RC-529 had simpler structure and stronger immunogenicity.RC-529 as a vaccine adjuvant,has been elucidated the immunogenicity in the Phase ? clinical trial of recombinant HBV antigen.However,the research of RC-529 based carbohydrate vaccines have rarely been reported,therefore we tried to synthesize two-component carbohydrate vaccine which composed of the RC-529 and its derivatives and carbohydrate antigen Tn.Moreover,to test that if the RC-529-Tn can active the immune system and active the T cell to produce IgG antibody.We have designed and synthesized 4 kinds of RC-529 and its derivative-Tn carbohydrate vaccine,the structure were showed in figure 1.Chemical synthesis oftrisaccharide fragments of therepeat unit capsular polysaccharide from Streptococcus pneumoniae type IaB:Streptococcus agalactiae(GBS)is one of the major causes of bacterial sepsis and meningitis in neonates.10 serotypes have been identified according to differences of GBS surface capsule polysaccharide structure.Among them,GBS type Ia is associated with early-onset disease in 36%of newborns,Ia type B streptococcus capsular polysaccharide are Contains repeating pentasaccharide units(?-D-Neup5Ac-(2?3)-?-D-Galp-(1?4)-?-D-GlcpNAc(1?3)-?-D-Galp-(1?4)-?-D-Glcp-(1?.Because of the three-dimensional selectivity and poor yield of sialic acid,chemical synthesis of Ia type B streptococcus capsular polysaccharide are Contains repeating pentasaccharide units is difficult.Therefore,we prepared to use a combination of chemical synthesis and enzymatic synthesis to synthesize the repeating unit.We used chemically synthesized tri-sugar compounds as starting materials(Fig 2),and then two-step enzymatic grafting to efficiently obtain five-sugar repeating units.The five-sugar repeating units provided a material basis for the activity evaluation of Ia type B streptococcus capsular polysaccharide.I mainly contributed to build a block of tri-sugar compounds.Experimental design and synthesis route:1.Synthesis of Monophosphate A Conjugation of Tn Antitumor Vaccine1.1 Total synthesis of tumor-associated Tn antigen:Using N-acetyl-D-Galactosam-ine as starting material,in the presence of 2-chloroethanol and acetyl chloride at 50?to obtain compound H-1 with alpha configuration,using pyridine as Fully acetylation with acetic anhydride under solvent and catalyst gave compound H-2,which was then reacted with sodium azide to give compound H-3.Removal of the 3,4,6 acetyl group with sodium methoxide gave compound Tn.1.2 Synthesis of two component vaccines1.2.1 Chemical synthesis of 3-R-tetradecanoyloxytetradecanoic acid and its derivatives in RC-529:Preparation of reagents and R-epichlorohydrin using 1-bromodecane as starting material An important intermediate,2-undecyl-(2R)-oxirane,is obtained under the catalysis of cuprous iodide,followed by three steps to obtain a fatty acid compound.1.2.2 Synthetic synthesis of RC-529 and its derivatives and tumor saccharide antigen Tn two-component vaccine:RC-529 is based on D-glucosamine hydrochloride as its starting material,and its reactivity varies according to the hydroxyl group on glucose.Following a rational protection strategy and route design,four sets of RC-529 derivatives were obtained by an 18-step reaction and then the RC-529 derivative was conjugated to Tn.The propargyl group of the RC-529 derivative and the azido group on the antigen Tn were conjugated with a triazolyl "click" reaction catalyzed by cuprous iodide and N,N-diisopropylethylamine.Removal of the protecting group yields four groups of two component vaccines(MLL-2-67)6-0-{[1-(2-deoxy-acetyl-?-D-galactoside)ethyl-5-methylene]-1,2,3-triazolyl}-1-O-{2-[3-R-(tetradecyloxy)tetradecyla mido]ethoxy}-4-O-phosphate-2-Deoxy-[3-R-(tetradecanoyloxy)tetradecylamido]-3-O-[3-R-(tetradecanoyloxy)tetradec-ylamido]-beta-D-glucoside,(MLL-2-68)6-O-{[1-(2-deoxy-a cetyl-?-D-galactoside)et-hyl-5-methylene]-1,2,3-triazolyl}-1-O-{2-[3-R-(tetradecanoylox y)tetradecylamido]ethoxy}-4-O-phosphate-2-deoxy-[3-R-(tetradecanoyloxy)tetradecylam ido]-3-O-[3-R-(tetradecanoyloxy)tetradecylamido]-beta-D-glucoseGlycosides,(MLL-2-69)6-O-{[1-(2-deoxy-acetyl-?-D-galactoside)ethyl-5-methylene]-1,2,3-triazolyl}1-O-{2-[3-R-(tetradecanoyloxy)tetradecylamido]ethoxy}-4-O-phosphate-2-deoxy-[3-R-(Tetradecyl oxy)tetradecylamido]-3-O-[3-R-(tetradecanoyloxy)tetradecylamido]-?-D-glucoside,(ML L-2-70)6-O-{[1-(2-deoxy-acetyl-?-D-galactoside)ethyl-5-methylene]-1,2,3-triazolyl}-1-{2-[3-R-(Dodecanoy loxy)dodecanamido]ethoxy}-4-O-phosphate-2-deoxy-[3-R-(tetradeca noyloxy)tetradecaneamideBase]-3-O-[3-R-(tetradecanoy loxy)tetradecy lamido]-[beta]-D-glucoside.2.Chemical synthesis of trisaccharide fragment candidate MLL-159:First,three monosaccharide compounds(MLL-13,MLL-86,MLL-149)were synthesized and then glycosylated in turn.The reaction yields the target trisaccharide compound.The specific experimental procedure was:the glycosylation substitution reaction of MLL-13 and MLL-86 in the presence of N-Iodosuccinimide,trifluoromethanesulfonic acid,and molecular sieve gave the disaccharide MLL-89 to cyano Sodium borohydride as a catalyst,with 4-methyl orange as an indicator under the action of etheric ether of hydrochloric acid,selective multiple 4-ether bond to give compound MLL-95,then MLL-149 in N-Iodosuccinimide,The glycosylation displacement reaction occurs under the presence of trifluoromethanesulfonic acid and molecular sieves to give the trisaccharide compound MLL-151.The acetyl,benzoyl,and phthalyl protecting groups are removed by refluxing with ethylenediamine to remove the solvent.The benzoic acid and water were removed at 50?.to remove the 4,6-benzylidene benzoate,and then acetic anhydride was used as a solvent to acetylate all the hydroxyl groups.Then,the acetyl group of the compound was deprotected with a mixture of sodium methoxide and methanol.Finally,the protective benzyl group is deprotected by the catalytic action of hydrogen and palladium carbon,and the azide group is reduced to an amino group to obtain the target product MLL-159.The synthesized candidates were identified by nuclear magnetic resonance spectroscopy and mass spectrometry.The structures of the synthesized compounds were identified by means of structural analysis methods such as nuclear magnetic resonance and mass spectrometry.