Studies On The Synthesis Of Nucleoside Polyphosphates And NDP-Sugars From Phosphoroazetidates

Nucleoside triphosphates and sugar nucleotides were important nucleotides,which not only play an important role in life activities,but also had been widely used in the biological and medical research.Natural nucleoside triphosphates are the source of cell energy and substrates for the biosynthesis of DNA and RNA.Modified nucleoside triphosphates have important applications in antiviral drug development and gene editing biotechnology.On the other hand,sugar nucleotides are an important substrates for the biosynthesis of oligosaccharides,glycolipids,and glycoproteins.Meanwhile,sugar nucleotides also have potential application in the enzymatic synthesis of oligosaccharides and the development and preparation of novel antibiotics.There are still some problems to be solved in the traditional chemical synthesis of nucleoside triphosphates and sugar nucleotides.For instance,in the synthesis of nucleoside triphosphates,the high reactivity of activated nucleoside monophosphate intermediates makes the coupling reaction difficult to control.The“P???-N activation strategy”reported by our research group in 2013 used 4,5-dicyanoimidazole?DCI?to activate the nucleoside phosphoropiperidates in the efficient synthesis of nucleoside triphosphates under mild reaction conditions.Although the"nucleoside phosphoropiperidate/DCI"system was successfully used for the synthesis of sugar nucleotides,the nucleophilic catalysis in the coupling reaction would lead to the generation of a large number of self-condensation byproducts due to the weak nucleophilicity of sugar monophosphate.This is also a problem in other methods of sugar nucleotides synthesis.Based on the“P???-N activation strategy”,the chapters 2–4 of this master?s thesis studied 1)a novel method for the synthesis of nucleoside triphosphate from the nucleoside phosphoroazetidate/TFA system,2)a novel method for the synthesis of sugar nucleotides from the nucleoside phosphoroazetidate/TFA system,3)a novel method for the synthesis of sugar nucleotides from sugar phosphorazetidate.The detailed research work were summarized as follows.The chapter 2 focused on the synthesis of nucleoside triphosphate from a new"P???-N activation system"--"nucleoside phosphoroazetidate/TFA".In this study,the optimization direction of"P???-N activation system"was clarified by combining the commom of crystal structure analysis of the reversible phosphoacylation intermediate catalyzed by creatine kinase and the reaction mechanism"P???-N activation".First,this study proved that the nucleophilic reagent involved in the reaction was highly protonated experimentally.The activator could be reduced from 6 eq to 2 eq by replacing the pyrophosphate of three tetrabutylammonium with the pyrophosphate of one tetrabutylammonium salt.In the reported studies on"P???-N activation system",there had no direct relationship between activation effect with the pK_a of activators in water.In this paper,the effect of a series of acidic activators with pK_a between 1.5 and8.5 in DMF/DMSO was tested.It was found that no matter whether the activators had nucleophilic conjugate base or not,the activators with pK_a between 3.45–4.5 had a good activation effect.Therefore,cheap and readily available TFA is an ideal activator.This study found that the reactivity of phosphoroazetidate with a smaller ring but greater ring strain was significantly better than phosphoropiperidate."Nucleoside phosphoroazetidate/TFA"system had shown extremely high efficiency and substrate versatility in the synthesis of a series of nucleoside triphosphate and nucleoside tetraphosphate.The nucleoside diphosphate was also successfully obtained by this system with monophosphate,indicating that the significant increase in the leaving ability of azetidine made the reaction no longer require nucleophilic catalysis of conjugate base.The chapter 3 focuses on the synthesis of sugar nucleotide from the new"P???-N activation system".As mentioned above,conjugate base nucleophilic catalysis is the main factor for the generation of dinucleoside diphosphate byproducts in the synthesis of sugar nucleotides.The"nucleoside phosphoroazetidate/TFA"system?only through the acid catalysis mechanism reaction?is very likely to solve the problem of the self-condensation of phosphoramidate.In this study,it was found that the"nucleoside phosphoroazetidate/TFA"system without nucleophilic catalytic mechanism significantly inhibited the generation of diphosphate byproducts in the coupling with monosaccharide phosphate.Subsequently,the dosage of TFA was further lowered to reduce the generation of byproducts and improve the yield of target products.Finally,7 natural/non-natural sugar nucleotides were efficiently synthesized using the newly optimized"P???-N activation system".Because the new method greatly reduced the generation of dinucleoside diphosphate and nucleoside monophosphate,this method greatly reduced the difficulty of product separation.The chapter 4 foucsed on a novel method for the synthesis of sugar nucleotides from sugar phosphoroazetidate.In the synthesis of sugar nucleotides,the"nucleoside phosphoroazetidate/TFA"system has solved the problem of nucleoside phosphoamidate self-condensation,but the poor availability,solubility and the anomeric configuration of most sugar monophosphates have limited the application of this method to some extent.In this chapter,we tried to synthesize of sugar nucleotides by coupling sugar phosphoroazetidate with nucleoside monophosphate for the first time.First,full-protected sugar phosphoroazetidates with defined anomeric configuration have been synthesized and isolated via isomerization reaction of sugar phosphites.Activated by TFA,almost all the deprotected sugar phosphoroazetidate produced sugar-1,2-cyclophosphate byproduct.Therefore,this study had to use glycosy-protected phosphoroazetidate to couple with nucleoside monophosphate.The results showed that the coupling reaction had high yield.The low polarity of the protective sugar nucleotides made the separation from nucleoside monophosphate much easier.However,the attempt to use CH₃ONa/CH₃OH and other methods to remove the protective groups of the sugar nucleotide led to the decomposition of a large portion of products.Finally,after many attempts,we found that Cs₂CO₃/CH₃OH could efficiently remove the protective groups of sugar nucleotides.On the basis of this new method,12 sugar nucleotides were synthesized.