Chemical synthesis of the 5′-terminal part bearing cap structure of messenger RNA of cytoplasmic polyhedrosis virus (CPV): m⁷G⁵′ pppAmpG and m⁷G⁵pppAmpGpU

Abstract

The 5'-terminal structures of mRNA bearing the so-called 'cap' from cytoplasmic polyhedrosis virus (CPV), m7G5' pppAmpG and m7G5' pppAmpGpU, were first chemically synthesized. S,S-Di(4-methoxyphenyl) N6-benzoyl-2'-O-methyladenosine 5'-phosphorodithioate ((ArS) 2pAbmz) was prepared by phosphorylation of the 5'-hydroxyl group of N6-benzoyl-2'-O-methyladenosine with S,S-di(4-methoxyphenyl) phosphorodithioate by TPS. By the triester approach using (ArS) 2pAbmz as starting material, the protected dinucleotide and trinucleotide bearing 5'-phosphate group were synthesized. The protective groups of the dinucleotide and trinucleotide were removed to obtain pAmpG and pAmpGpU, respectively. By the reaction of a capping agent ((PhS) ppm7G) with pAmpG and pAmpGpU in the presence of silver nitrate or iodine. The 5'-terminal structure of the messenger RNA strand of CPV which was labelled isotopically, was confirmed completely as m7G5' pppAmGpU by cochromatography with the materials chemically synthesized here.

Preparation and Properties of mRNA 5-cap Structure

“…They can be overcome by performing the coupling reaction in the presence of imidazole that traps the metaphosphate ion, forming an imidazolide intermediate that acts as the actual capping agent [66]. The phenylthio and 4-methoxyphenylthio methods have also been applied to the synthesis of 5´-capped oligonucleotides [66,67]. Short capped oligonucleotides have been obtained in approximately 10 % yield by the reaction between the phenylthio [67] or 4-methoxy phenylthio [66] diphosphate reagents and a 5´-phosphorylated oligonucleotide in the presence of I 2 or AgNO 3 activators.…”

“…The phenylthio and 4-methoxyphenylthio methods have also been applied to the synthesis of 5´-capped oligonucleotides [66,67]. Short capped oligonucleotides have been obtained in approximately 10 % yield by the reaction between the phenylthio [67] or 4-methoxy phenylthio [66] diphosphate reagents and a 5´-phosphorylated oligonucleotide in the presence of I 2 or AgNO 3 activators. In the case of phenylthio coupling, both the diphosphate and 5-phosphorylated oligonucleotide were unprotected, and were introduced as tetrabutylammonium salts [67].…”

“…Short capped oligonucleotides have been obtained in approximately 10 % yield by the reaction between the phenylthio [67] or 4-methoxy phenylthio [66] diphosphate reagents and a 5´-phosphorylated oligonucleotide in the presence of I 2 or AgNO 3 activators. In the case of phenylthio coupling, both the diphosphate and 5-phosphorylated oligonucleotide were unprotected, and were introduced as tetrabutylammonium salts [67]. In contrast, the 4-methoxyphenylthio diphosphate and corresponding oligonucleotide reagents were both protected.…”

Synthesis ofN2,N2, 7-trimethylguanosine cap derivatives

“…Therefore, we employed an alternative way, i.e., the selective hydrolysis of 3 with triethylamine-pyridine-water (2: (17). Thus, compound 4 was obtained in 99% yield by evaporation followed by washing with ether to remove released benzenethiol. Treatment of 4 with 80% acetic acid gave S-phenyl N2,N2-dimethylguanosine 5'-phosphorothioate (5) in 88% yield.…”

“…In our laboratory, we have reported several methods for the chemical synthesis of 7-methylguanosine (m7G)-capped mRNAs (1)(2)(3)(4)(5)(6)(7)(8). Nakagawa (2) first reported a capping reagent, PhSppm7G, for the synthesis of m7G5'pppA and m7G5'pppG.…”