2'-Methylseleno-modified oligoribonucleotides for X-ray crystallography synthesized by the ACE RNA solid-phase approach

Abstract

Site-specifically modified 2'-methylseleno RNA represents a valuable derivative for phasing of X-ray crystallographic data. Several successful applications in three-dimensional structure determination of nucleic acids, such as the Diels-Alder ribozyme, have relied on this modification. Here, we introduce synthetic routes to 2'-methylseleno phosphoramidite building blocks of all four standard nucleosides, adenosine, cytidine, guanosine and uridine, that are tailored for 2'-O-bis(acetoxyethoxy)methyl (ACE) RNA solid-phase synthesis. We additionally report on their incorporation into oligoribonucleotides including deprotection and purification. The methodological expansion of 2'-methylseleno labeling via ACE RNA chemistry is a major step to make Se-RNA generally accessible and to receive broad dissemination of the Se-approach for crystallographic studies on RNA. Thus far, preparation of 2'-methylseleno-modified oligoribonucleotides has been restricted to the 2'-O-[(triisopropylsilyl)oxy]methyl (TOM) and 2'-O-tert-butyldimethylsilyl (TBDMS) RNA synthesis methods.

Figure 1.

Se-modified RNA for X-ray crystallography. (a) 2′-Methylseleno-modified RNA represents a highly requested derivative for RNA crystallography. (b) A single crystal with an anomalous scattering center such as selenium is required during X-ray structure determination using advanced techniques for phase determination, such as MAD, SAD (single-wavelength anomalous diffraction) or SIRAS (single isomorphous replacement with anomalous scattering). (c) Solid-phase synthesis of 2′-methylseleno RNA has been developed based on 5′-O-(4,4′-dimethoxytrityl) (DMT)-2′-O-silyl protected nucleoside building blocks. (d) Goal of the present study is the synthesis of 2′-methylseleno RNA by 2′-O-bis(acetoxyethoxy)methyl (ACE) RNA solid-phase synthesis. For this, novel building blocks and adaptation of the established ACE solid-phase synthesis cycle are required.

Scheme 1.

Synthesis of the 2′-methylseleno adenosine phosphoramidite A10 (a) i. 1.1 eq 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, in DMF/pyridine, room temperature, 2 h; ii. 2 eq chlorotrimethylsilane, room temperature, 2 h; iii. 1.1 eq acetyl chloride, room temperature, 1.5 h, 91%; (b) 1.1 eq p-toluenesulfonic acid monohydrate, molecular sieves, in dioxane, room temperature, 1.5 h, 78%; (c) 1.5 eq trifluoromethanesulfonyl chloride, 3 eq DMAP, in CH₂Cl₂, 0°C, 15 min; (d) 5 eq CF₃COO⁻K⁺, 1.5 eq (iPr)₂NEt, 2 eq 18-crown-6-ether, in toluene, 80°C, 16 h (67% over (c) and (d)); (e) 1.5 eq trifluoromethanesulfonyl chloride, 3 eq DMAP, in CH₂Cl₂, 0°C, 15 min; (f) 6 eq NaBH₄, 2 eq CH₃SeSeCH₃, in THF, room temperature, 30 min (59% over (e) and (f)); (g) 1 M TBAF, 0.5 M acetic acid, in THF, room temperature, 2 h, 94%; (h) 2 eq benzhydryloxy-bis(trimethylsilyloxy)chlorosilane, 3.4 eq (iPr)₂NH, in CH₂Cl₂/DMF, room temperature, 2 h, 72%; (i) 1.5 eq methyl-N,N-diisopropylchlorophosphoramidite, 10 eq EtNMe₂, in CH₂Cl₂, room temperature, 2 h, 70%; (DMAP 4-(dimethylamino)pyridine; TBAF tetrabutylammonium fluoride).

Scheme 2.

Synthesis of the 2′-methylseleno guanosine phosphoramidite G3; (a) 2 eq benzhydryloxy-bis(trimethylsilyloxy)chlorosilane, 4 eq (iPr)₂NH, in CH₂Cl₂/DMF, room temperature, 2 h, 46%; (b) 1.5 eq methyl-N,N-diisopropylchlorophosphoramidite, 3 eq EtNMe₂, in CH₂Cl₂, room temperature, 2 h, 80%.

Scheme 3.

Synthesis of the 2′-methylseleno cytidine phosphoramidite C4; (a) i. 1 M TBAF, 0.5 M acetic acid, in THF, room temperature, 18 h, ii. HCOOH, 2 min, 80%; (b) 2 eq benzhydryloxy-bis(trimethylsilyloxy)chlorosilane, 3.3 eq (iPr)₂NH, in CH₂Cl₂/DMF, room temperature, 2.5 h, 54%; (c) 1.5 eq methyl-N,N-diisopropylchlorophosphoramidite, 10 eq EtNMe₂, in CH₂Cl₂, room temperature, 2 h, 69%.

Scheme 4.

Synthesis of the 2′-methylseleno uridine phosphoramidite U4; (a) HCOOH, in CH₂Cl₂ (1/4 v/v), room temperature, 1 h, 99%; (b) 2 eq benzhydryloxy-bis(trimethylsilyloxy)chlorosilane, 3.4 eq (iPr)₂NH, in CH₂Cl₂/DMF, room temperature, 2 h, 73%; (c) 2.5 eq benzhydryloxy-bis(trimethylsilyloxy)chlorosilane, in pyridine, −15 °C, 4 h; (d) 6 eq NaBH₄, 2 eq CH₃SeSeCH₃, in THF, room temperature, 2 h (28% over (c) and (d)); (e) 1.5 eq methyl-N,N-diisopropylchlorophosphoramidite, 10 eq EtNMe₂, in CH₂Cl₂, room temperature, 2 h, 84%.

Figure 2.

HPLC-traces of deprotected 2′-methylseleno-modified RNA (anion exchange HPLC: Dionex DNAPac (4 × 250 mm), 80°C, 1 ml/min, 0–60% B in 45 min; A: 25 mM Tris–HCl, 6 M urea, pH 8.0; B: same as A + 0.5 M NaClO₄); (a) Crude and purified (inset) pentamers with 2′-methylseleno modifications at either A, C, G or U. (b) 2′-Methylseleno uridine-modified A/U-rich nonamer synthesized without (top) and with (bottom) threo-1,4-dimercapto-2,3-butanediol (DTT) treatment during solid-phase synthesis. Both nonamers were deprotected in the presence of DTT. (c) Non-modified nonamer as in (b) synthesized for reason of comparison. (d) Crude 30 nt RNA with two U_Se labels. Deprotection procedure includes four steps: 1. 0.39 M disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate, 150 mM DTT, DMF/H₂O (13/1), 20 min, room temperature; 2. 150 mM DTT in H₂O, 1–3 h, room temperature; 3. 40% CH₃NH₂ in H₂O, 150 mM DTT, 10 min, 60°C; 4. 0.1 M TEMED acetate buffer (pH 3.8), 150 mM DTT, 30 min, 60°C.

Figure 3.

Comparison of TOM versus ACE chemistry for the synthesis of 2′-methylseleno containing oligoribonucleotides, exemplified by the sequence rGCAGAGUUAAAUCUGU. HPLC-traces of crude, deprotected non-modified RNA (a) and of A_Se-modified RNA (b); anion exchange HPLC: Dionex DNAPac (4 × 250 mm), 80°C, 1 ml/min, 0-60% B in 45 min; A: 25 mM Tris–HCl, 6 M urea, pH 8.0; B: same as A + 0.5 M NaClO₄.