Studies On The Synthesis Of Cyclic Nucleotides And A Novel Alkylated CAMP

Natural cyclic nucleotides are a very special group of nucleoside monophosphates.Cyclic nucleotides function as secondary messengers and play a crucial role in the life processes of cell signaling.In biological and medical research,it is important to monitor the concentration and distribution of cyclic nucleotides in cells with high affinity antibodies in a real-time manner.But due to the fact that efficient chemical synthetic methods for cyclic nucleotide still lack,the anti-cAMP antibodies were induced by simply acylated cAMP hapten and their affinities still need to be improved.With the investigation of mechanism of the 5?-NMP DCC condensation method as entry point,the current thesis revealed the reaction path of the cyclization of 5?-NMP.On the basis of the mechanism,a novel cyclization method based on 3?-NMP substrate was established.The new cyclization method established provided an efficient access to the synthesis of 2?-O-alkylated cAMP hapten.The detailed research contents of the thesis are listed as follows.1)Studies on the reaction mechanism of 5?-NMP cyclization method: Since the reaction conditions feature high dilution and temperature,analytical HPLC tracing method was employed in my research.The intermediates,by-products and the effects of reaction conditions were determined via a series of real-time monitoring of reactions.Consequently,the reaction mechanism of cyclization was elucidated and the reaction conditions were optimized on the basis of the mechanism to improve reaction efficiency.2)Establishment of the novel and efficient 3?-NMP cyclization method: On the basis of the reaction mechanism of cyclization of 5?-NMP and analysis of structural rigidity characteristics of cNMPs,the novel 3?-NMP cyclization method was established by exchanging electrophilic and nucleophilic reaction sites.The new method significantly improved the reaction efficiency in terms of both reaction time and yield.The reaction mechanism of 3?-NMP cyclization was so elucidated to be different from the that of 5?-NMP cyclization method.In addition,condensing reagents and reaction concentrations were also optimized.The development of the efficient 3?-NMP cyclization method provided a foundation for synthesis of 2?-O-alkylated cAMP.3)Chemical synthesis of 2?-O-alkylated cAMP hapten: The 2?-OH group of adenosine was alkylated with N-(3-bromopropyl)phthalimide.After the 5?-OH was protected with DMT,adenosine 3?-H-phosphonate monoester was synthesized via phosphitylation,which was thus converted to silylated diester with BSA and oxidized with iodine to generate the 2?-O-alkylated 3?-AMP.The target 2?-O-alkylated cAMP was efficiently synthesized by the novel 3?-NMP cyclization method.Removal of the phthalimide and further acylation with succinic anhydride afforded the desired novel alkylated cAMP hapten in good yield.The hapten will be utilized for the generation of high-affinity monoclonal anti-cAMP antibodies.