| Cyclic dinucleotide is the second messenger molecules of microorganisms with the function to regulate the pathogenicity and morphology of pathogenic microbes.Cyclic dinucleotide could be recognized by human immune system to curb the attack from the pathogens.It is found that cyclic dinucleotide can stimulate the secretion of cytokines to kill cancer cells.In recent years,a unique cyclic dinucleotide was found to have a 2',5'-linkage,which is different from that found in microbes.The phosphodiester bond in cyclic dinucleotide is the easy target of ubiquitous phosphodiesterase in human body.The natural cyclic dinucleotide is difficult to be used as research tool and drug candidate because of its unstability,hydrophilicity and difficulty of preparation.Thus,the synthesis of cyclic dinucleotide and their analogues will lay the foundation for the subsequent study and discovery of biologically active cyclic dinucleotide analogues.On the basis of the reported literatures,we designed and optimized the chemical synthesis to 2',3'-c GAMP and its analogues from the corresponding phosphoramidite of nucleoside and their intermediates using guanosine and adenosine as raw materials.In chapter 1,the synthetic method and the application of 2',3 '-c GAMP and its analogues were reviewed.In chapter 2,startingfrom adenosine and guanosine,we have successfullysynthesized 2',3'-c GAMP through 9 step reactions.Firstly,the N6-amino of base,5'-hydroxyl groupand2'-hydroxygroup of adenosine were protected by benzoyl chloride,4,4-dimethoxytriphenylmethyl chlide and TBDMS-Cl respectively.Finally,the protected adenosine derivative reacted with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoramidite to give N-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-[(tert-butyl)dimethylsilyl]adenosine-3'-(2-cyanoethyl-N,N-diis opropyl)phosphoramidite(2.4),with a total yield of 26.2%.We used the same synthetic route to obtain N-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-[(tert-butyl)dimethylsilyl]guanosine-3'-(2-cyanoethyl-N,N-di isopropyl)phosphoramidite(2.8b)and N-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(tert-butyl)dimethylsilyl]guanosine-2'-(2-cyanoethyl-N,N-di isopropyl)phosphoramidite(2.8a)with a yield of 21.6% and 18.5% respectively.Using these intermediates as staring materials,we prepared 2',3'-c GAMP after hydrolysis,coupling,oxidation,cyclization,deprotection,and the yield is 38.5%.To reduce the influence of impurities on the yield of coupling and cyclization,we optimized the key reaction steps and purified the key intermediates by preparative chromatography,which greatly improved the reaction yield.In chapter 3,we used the protocol estabilished in the chapter 2 to synthesize 3',3'-c GAMP and c-di-GMP.Taking analogues 3',3'-c GAMP as an example,the phosphoramidite(2.4)as a starting material to get 3',3'-c GAMP,and the yield was 35.6%.Similarly,c-di-GMP was synthesized with phosphoramidite(2.8b)as a starting material with a yield of 35.8%.After optimization,the yield is 5% higher than the reported in literature.The structures of these compounds were confirmed by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry.In chapter 4,we explored the one-pot synthesis of 2',3'-c GAMP and 3',3'-c GAMP.Firstly,we used the N2-isobutyryl guanosine as a raw material,by reacting withtrimethylorthoacetate to get intermediate 2',3'-cyclic acid ester,which wasconverted to 2'-and 3'-acetylated guanosine mixturesby hydrolysis.Then throughthe protection of the hydroxyl group at the 5'position of the sugar ringwith DMT-Cl,the 2' or 3' position withlevulinic acid,and then a deprotection of the 5'position of ribose,we got the isomeric intermediates,followed by reacting with phosphoramidite(2.4)to give the dimer.Finally the macrocyclization of the dimers,which was expected to get 2',3'-c GAMP and 3',3'-c GAMP.But owing to the macrocyclization is not ideal,the protocol needed further optimization. |
PDF Full Text Request