Studies on Steric and Electronic Control of 2‘−3‘ Phosphoryl Migration in 2‘-Phosphorylated Uridine Derivatives and Its Application to the Synthesis of 2‘-Phosphorylated Oligouridylates

Abstract

For the synthesis of 2'-phosphorylated oligouridylates by use of new phosphoramidite building units, several masked phosphoryl groups have been examined as 2'-phosphate precursors, which should not be migrated to the 3' position when the 3' hydroxy protecting group must be removed to introduce a phosphoramidite residue into the 3'-position. As a consequence, bis(2-cyano-1,1-dimethylethoxy)thiophosphoryl (BCMETP) was found to be the most suitable 2'-phosphate precursor. This thiophosphoryl group could be introduced into the 2'-hydroxyl of 3',5'-silylated uridine derivative 7 by phosphitylation with bis(2-cyano-1,1-dimethylethoxy)(diethylamino)phosphine followed by sulfurization. Treatment of the 2'-thiophosphorylated product 15 with (HF)(x)().Py in THF gave exclusively the 3',5'-unprotected uridine derivative 16a. Compound 16a was converted to the phosphoramidite unit 22 via a two-step reaction. This building block was used for the solution phase synthesis of U(2'-p)pU (29) and U(2'-ps)pU (30). Both the 2-cyano-1,1-dimethylethyl and 2-cyanoethyl groups were effectively removed from the fully protected derivative 25 by treatment with DBU in the presence of N,O-bis(trimethylsilyl)acetamide (BSA). The resulting 2'-thiophosphoryl group was successfully converted to a phosphoryl group by iodine treatment to give U(2'-p)pU (29). U(2'-ps)pU (30) was also synthesized by a modified procedure without the iodine treatment. Reaction of 29 with a new biotinylating reagent in aqueous solution in the presence of MgCl(2) gave a biotin-labeled product 35 having a pyrophosphate bridge at the 2' position. Reaction of 30 with monobromobimane gave the 2'-S-alkylated product 33 in aqueous solution. Application of the phosphoramidite unit 22 to the solid phase synthesis using aminopropyl CPG gel gave successfully [U(2'-p)p](n)()U (n = 1, 3, 5). It was found that stability of the succinate linker between the CPG and oligouridylates was unaffected by the treatment with DBU when BSA was present. Several enzymatic properties of the synthetic 2'-phosphorylated and 2'-thiophosphorylated oligouridylates are also described.

Postsynthetic On-Column 2′ Functionalization of RNA by Convenient Versatile Method

“…After the synthesis, the 5′- O -DMTr group of the terminal nucleoside is removed with the following acetylation of 5′ hydroxyl by acetic anhydride (as a standard capping procedure) to avoid the unwanted side reactions of the 5′ terminal hydroxyl. Then, to mitigate a possible 2′- to 3′ isomerization of a phosphotriester group [ 47 ], the 2-cyanoethyl-protecting groups are selectively removed by β-elimination induced by a non-nucleophilic strong organic base (2,3,4,6,7,8,9,10-octahydropyrimido(1,2-a)azepine, DBU) in the presence of a silylation agent ( N,O -bis(trimethylsilyl)acetamide, BSA) according to a described procedure [ 48 ]. After that, a removal of the temporary 2′- O - t BDMS group can be carried out by a reagent mixture containing the fluoride ion [ 49 ], leading to the support-bound 2′- OH /2′- O -TC RNA ( Figure 1 and Scheme S1 ).…”

“…Support-bound protected oligoribonucleotides (I) were treated by a mixture of N,O -bis(trimethylsilyl)acetamide in tetrahydrofuran (50% vol., 500 μL) for 30 min at room temperature and 2,3,4,6,7,8,9,10-octahydropyrimido(1,2- a )azepine (5% vol.) in the mixture of N,O -bis(trimethylsilyl)acetamide in tetrahydrofuran (50% vol., 500 μL) for 30 min at room temperature ( Scheme S1, steps a, b ) [ 48 ]. After the treatment, the support-bound silylated oligonucleotide (II) was sequentially washed by tetrahydrofuran and acetone and air-dried.…”

Novel Method for the Synthesis of 2′ -Phosphorylated Oligonucleotides

“…[4] The authors have had to address the 2 -3 -migration of the phosphotriester group and slow coupling of the 2 -phosphorylated 3 -phosphoramidites. Additionally, to introduce the 2 -phosphate group into any position within an oligonucleotide sequence by this method, all four of the corresponding phosphoramidites have to be prepared by a complex multi-step synthesis.…”

“…At the end of the assembly, the 5 -O-DMTr group is removed and the 5 -OH is capped by acetylation. Because in the presence of the free 2 -OH the vicinal phosphotriester migrates more easily and gives more chain cleavage than the corresponding phosphodiester, [5] [4] The 2 -OH group was then desilylated by triethylamine trihydrofluoride treatment [6] and phosphorylated with 2-[2-(4,4 -dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N ,Ndiisopropyl)-phosphoramidite [7] and 5-ethylthiotetrazole followed by aqueous iodine oxidation. The yield of the 2 -phosphorylation can be easily monitored by the DMTr cation release.…”

Nucleosides and oligonucleotides containing 2'-reactive groups: synthesis and applications

“…Studies by Sekine et al 148,149 describe the synthesis of nucleosides and oligonucleotides containing a 2 H -phosphate or a 2 H -thiophos-phate group. The 3 H -and 5 H -protected nucleosides 69a ± d were converted into the trivalent phosphorus derivatives 70a ± d; their subsequent oxidation with tert-butyl hydrogen peroxide or sulfur yielded 2 H -phosphates 71a ± d and thiophosphates 72a ± d, respectively.…”