Patent ID: 9605261
Date: 2017-03-28
CPC Classifications: C07H,C12N

Claim:
1. A method for the synthesis of an RNA oligonucleotide, comprising a structure represented by the following formula: wherein: B, B′ or B″ is a member selected from the group consisting of 9-adeninyl, 1-cytosinyl, 9-guaninyl, 1-uracilyl, 9-inosinyl, 5-methyl-1-cytosinyl, 5-methyl-1-uracilyl, 5-fluoro-1-uracilyl, 7-deaza-9-adeninyl, and 5-fluoro-1-cytosinyl; n is an integer between 0 and 100; L is a nucleoside, a non-nucleoside ligand selected from the group consisting of cholesterol attached via a linker or via a spacer, biotin, ethylene glycol, glycerol, a polyethylene glycol, a hexaethylene glycol, an amino linker, a disulfide linker, a peptide linker, a polypeptide linker, a protein, a fluorophore, a quencher dye, one or more 2′,5′-linked deoxynucleoside units, one or more 2′,5′-linked ribonucleoside unit, and one or more 2′,5′-linked deoxyribose units, wherein L is attached at the 3′-end of the RNA nucleotide through an intervening phosphate; and the RNA oligonucleotide is synthesized by a process in a direction from the 5′-end to the 3′-end, and the process comprises the steps of: (a) taking a nucleoside solid support represented by Formula 2: wherein: M is a hydrogen radical or a linker; W is selected from the group consisting of an oxygen diradical, an N—H diradical, and a fluorine radical, and R is selected with the proviso that: B is selected from the group consisting of nucleoside base radicals consisting of 9-(N B is a modified nucleoside base radical selected from the group consisting of 1-(N Z is a protecting group selected from the group consisting of dimethoxy triphenyl (DMT), monomethoxy triphenyl (MMT) and trimethoxy triphenyl (TMT); (b) placing a phosphoramidite represented by Formula 1 in an appropriate container component of an oligonucleotide synthesizer; wherein Y is an oxygen atom or a sulfur atom; W is selected from the group consisting of an oxygen diradical, an N—H diradical, and a fluorine radical; and R B is selected from the group consisting of nucleoside base radicals consisting of 9-(N B is a modified nucleoside base radical selected from the group consisting of 1-(N Z is a protecting group selected from the group consisting of dimethoxy triphenyl (DMT), monomethoxy triphenyl (MMT) and trimethoxy triphenyl (TMT); R R R B is hydrogen or a nucleobase-derived substituent moiety which is optionally functionalized at each primary amine with an amine protecting group; (c) removing the protecting group Z from the nucleoside-derived solid support represented by Formula 2 to form a nucleoside with an active OH group at 3′; (d) performing the process of RNA synthesis by coupling the nucleoside with the active OH group at 3′ of step (c) and the phosphoramidite of Formula 1 in the oligonucleotide synthesizer to result in an oligonucleotide having at least one protecting group; (e) providing a phosphoramidite with an L group; (f) adding the phosphoramidite with the L group at the end of the oligonucleotide to result in an oligonucleotide having the L group and contacting the product of L group attachment with an oxidizing agent in sufficient quantity to convert all of the P(III) linkages to P(V) linkages; (g) detaching the oligonucleotide having the L group from the solid support; (h) removing the at least one protecting group from the oligonucleotide; (i) removing a silyl protecting group to result in the oligonucleotide; (j) precipitating the oligonucleotide; and (k) analyzing the oligonucleotide for purity, and the RNA oligonucleotide is essentially free of M+1 species as impurity according to an electropherogram.