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Nucleic Acids Res. Feb 2008; 36(3): 970–983.
Published online Dec 20, 2007. doi:  10.1093/nar/gkm880
PMCID: PMC2241898

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.

INTRODUCTION

Selenium-labeled oligonucleotides have become recognized to represent useful derivatives for phasing of X-ray crystallographic data in nucleic acid structure analysis. Among various potential sites for modification with selenium, ribose 2′-methylseleno groups have attracted most attention so far. Since the pioneering work by Egli, Huang and coworkers, which led to successful multi-wavelength anomalous dispersion (MAD)-phasing of an A-form DNA duplex via 2′-methylseleno uridine (1–3), our laboratory elaborated advanced procedures for the preparation of 2′-methylseleno-modified RNA, site-specifically labeled at any of the four standard nucleosides, adenosine, cytidine, guanosine and uridine (Figure 1) (4–7). Thereby, the method we used for oligonucleotide synthesis relied on nucleoside phosphoramidites protected with the 2′-O-[(triisopropylsilyl)oxy]methyl (TOM) protecting group (TOM chemistry) (8–13). Importantly, the application of threo-1,4-dimercapto-2,3-butanediol (DTT) during all steps of RNA preparation, including the solid-phase synthesis cycle, was a major breakthrough for the high performance of the Se-approach (5). This resulted in the preparation of highly pure 2′-methylseleno modified RNAs with up to a hundred nucleotides, exemplified by the aptamer domain of the adenine riboswitch (5,14). Successful applications of the Se-derivatized RNAs in X-ray structure determination refer to the group I intron (15), to the Diels–Alder ribozyme (16), to a short RNA duplex that has been studied in context with the impact of 2′-methylseleno groups on crystallization behavior and crystal packing (7), and very recently, to HIV-1 genomic RNA dimerization initiation site (DIS) constructs bound to aminoglycoside antibiotics (17,18).

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 ...

In the present work, we report on preparation of oligoribonucleotides with site-specific 2′-methylseleno groups based on the 2′-O-bis(acetoxyethoxy)methyl (ACE) RNA solid-phase synthesis method. Nucleoside phosphoramidites providing a fluorine-labile silyl protecting group at the ribose 5′-OH and the acid-labile orthoester protecting group at the ribose 2′-OH were introduced for chemical RNA synthesis in the late nineties (19). Within a very short time, this innovative strategy turned out to be highly competitive to commonly used RNA synthesis methods based on 5′-O 4,4′-dimethoxytritylated (DMT) nucleoside building blocks and laid the basis for one of the largest custom RNA synthesis services today. In particular, the very good quality of ACE oligoribonucleotides that are commercially available contributed to the high reputation of the method. In research laboratories, usage of the ACE RNA approach has been limited (20–22) and is generally considered complex because of non-standard instrumentation, a long optimization period, and not least because of high costs when compared to the 5′-O-DMT methods. Under the aspect that our previously established concept of selenium-modified RNA for X-ray structure analysis would strongly benefit from compatibility with high-quality ACE RNA synthesis, we put great efforts into the adaptation of this approach for the preparation of 2′-methylseleno containing oligoribonucleotides. We show here that such derivatives are readily available via the novel nucleoside phosphoramidite building blocks and ACE RNA solid-phase synthesis procedures outlined subsequently.

MATERIALS AND METHODS

Synthesis of 2′-methylseleno modified nucleosides for ACE RNA synthesis

General

1H, 13C and 31P NMR spectra were recorded on a Bruker DRX 300 MHz, or Varian Unity 500 MHz instrument. The chemical shifts are reported relative to TMS and referenced to the residual proton signal of the deuterated solvents: CDCl3 (7.26 p.p.m.), d6-DMSO (2.49 p.p.m.) for 1H-NMR spectra; CDCl3 (77.0 p.p.m.) or d6-DMSO (39.5 p.p.m.) for 13C-NMR spectra. 31P-shifts are relative to external 85% phosphoric acid. 1H- and 13C-assignments were based on COSY and HSQC experiments. UV-spectra were recorded on a Varian Cary 100 spectrophotometer. Analytical thin-layer chromatography (TLC) was carried out on silica 60F-254 plates. Flash column chromatography was carried out on silica gel 60 (230–400 mesh or 70–230 mesh). All reactions were carried out under argon atmosphere. Chemical reagents and solvents were purchased from commercial suppliers and used without further purification. Benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (BzHCl) was obtained from Dharmacon. Organic solvents for reactions were dried overnight over freshly activated molecular sieves (4 Å).

Synthesis of 2′-methylseleno adenosine phosphoramidite (A10)

N6-Acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-O-(trimethylsilyl)adenosine (A2)

To a suspension of adenosine A1 (1.0 g; 3.742 mmol) in DMF (12 ml) and pyridine (12 ml), 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.3 g; 4.116 mmol) was added dropwise. The mixture was stirred for 2 h at room temperature, during which time it turned into a clear solution. Then, chlorotrimethylsilane (947 µl; 7.484 mmol) was added and stirring was continued for 2 h. The resulting white suspension was treated with acetyl chloride (292 µl; 4.116 mmol) and stirred for 1.5 h with occasional shaking. After completion of the reaction, the yellow solution was quenched by addition of 5% aqueous NaHCO3 and extracted with dichloromethane. The combined organic phases were washed with brine, dried over Na2SO4 and evaporated. The crude product was purified by column chromatography on SiO2 (CH2Cl2/CH3OH, 99.8/0.2 – 99/1 v/v). Yield: 2.127 g of A2 as white foam (91%). TLC (CH2Cl2/CH3OH, 94/6): Rf = 0.49; 1H-NMR (500 MHz, DMSO): δ 0.15 (s, 9H, ((CH3)3)Si); 1.03 (m, 28H, 2× ((CH3)2CH)2Si); 2.26 (s, 3H, COCH3); 3.95 (dd, J = 1.5, 8.0 Hz, 1H, H1-C(5′)); 4.07 (m, 1H, H-C(4′)); 4.13 (dd, J = 1.5, 8.0 Hz, 1H, H2-C(5′)); 4.72 (dd, J = 2.7, 5.4 Hz, 1H, H-C(3′)); 4.77 (d, J = 2.7 Hz, 1H, H-C(2′)); 5.97 (s, 1H, H-C(1′)); 8.47 (s, 1H, H-C(8)); 8.59 (s, 1H, H-C(2)); 10.72 (s, br, 1H, H-N6) p.p.m.; 13C-NMR (75 MHz, DMSO): δ 0.65 ((CH3)3Si); 12.76, 12.83, 13.17, 17.23, 17.34, 17.40, 17.52, 17.61, 17.76 (2× ((CH3)2CH)2Si); 24.79 (COCH3); 60.70 (C(5′)); 69.80 (C(3′)); 75.57 (C(2′)); 81.19 (C(4′)); 90.23 (C(1′)); 124.21; 142.38 (C(8)); 150.07, 151.36; 152.00 (C(2)); 169.25 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (ε) = 270 (16 000) nm (mol−1 dm3 cm−1); ESI-MS (m/z): [M+H]+ calcd for C27H49N5O6Si3, 624.96; found 624.19.

N6-Acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine (A3)

A mixture of p-toluenesulfonic acid monohydrate (671 mg; 3.526 mmol), dioxane (20 ml) and molecular sieves (1.5 g) was stirred for 2.5 h at room temperature. A solution of A2 (2.0 g; 3.205 mmol) in dioxane (10 ml) was added and stirring was continued for 1.5 h. The reaction mixture was then quenched by the addition of triethylamine (4.5 ml), evaporated and coevaporated with dichloromethane. The crude product was purified by column chromatography on SiO2 (CH2Cl2/CH3OH, 99.5/0.5 – 97/3 v/v). Yield: 1.384 g of A3 as white foam (78%). TLC (CH2Cl2/CH3OH, 94/6): Rf = 0.46; 1H-NMR (300 MHz, CDCl3): δ 1.09 (m, 28H, 2× ((CH3)2CH)2Si); 2.62 (s, 3H, COCH3); 3.41 (s, br, 1H, HO-C(2′)); 4.02–4.14 (m, 3H, H2-C(5′), H-C(4′)); 4.60 (m, 1H, H-C(2′)); 5.09 (m, 1H, H-C(3′)); 6.02 (d, J = 0.9 Hz, 1H, H-C(1′)); 8.18 (s, 1H, H-C(8)); 8.62 (s, 1H, H-C(2)); 9.09 (s, br, 1H, H-N6) p.p.m.; 13C-NMR (75 MHz, CDCl3): δ 12.63, 12.75, 13.02, 13.26, 16.88, 16.94, 16.96, 17.08, 17.23, 17.30, 17.39 (2× ((CH3)2CH)2Si); 25.64 (COCH3); 61.69 (C(5′)); 70.79 (C(3′)); 75.06 (C(2′)); 82.26 (C(4′)); 89.79 (C(1′)); 122.43; 141.96 (C(8)); 149.38, 150.47; 152.31 (C(2)); 170.58 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 271 (11 700) nm (mol−1 dm3 cm−1); ESI-MS (m/z): [M+H]+ calcd for C24H41N5O6Si2, 552.78; found 552.16.

N6-Acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-O-(trifluoromethanesulfonyl)adenosine (A4)

To a solution of compound A3 (380 mg; 0.689 mmol) in CH2Cl2 (13 ml), 4-dimethylaminopyridine (253 mg; 2.067 mmol) was added at 0°C. The mixture was treated with trifluoromethanesulfonyl chloride (109 µl; 1.034 mmol) and stirred for 15 min at 0°C. The reaction mixture was then diluted with CH2Cl2, washed with 5% aqueous NaHCO3, dried over Na2SO4 and evaporated. The crude product can be used for the next step without further purification. For analysis, the product was purified by column chromatography on SiO2 (CH2Cl2/CH3OH, 100/0 – 99.3/0.7 v/v). Yield: 259 mg of A4 as white foam (55%). TLC (CH2Cl2/CH3OH, 94/6): Rf = 0.51; 1H-NMR (300 MHz, CDCl3): δ 1.08 (m, 28H, 2× ((CH3)2CH)2Si); 2.63 (s, 3H, COCH3); 4.05 (dd, J = 2.6, 13.4 Hz, 1H, H1-C(5′)); 4.12 (m, 1H, H-C(4′)); 4.20 (m, 1H, H2-C(5′)); 5.23 (dd, J = 4.8, 9.0 Hz, 1H, H-C(3′)); 5.77 (d, J = 4.5 Hz, 1H, H-C(2′)); 6.17 (s, 1H, H-C(1′)); 8.20 (s, 1H, H-C(8)); 8.61 (s, 1H, H-C(2)); 8.89 (s, br, 1H, H-N6) p.p.m.; 13C-NMR (75 MHz, CDCl3): δ 12.75, 12.80, 12.88, 13.22, 16.66, 16.73, 17.17, 17.21, 17.32 (2× ((CH3)2CH)2Si); 25.70 (COCH3), 59.53 (C(5′)); 68.16 (C(3′)); 77.19 (CF3); 81.63 (C(4′)); 87.05 (C(1′)); 87.99 (C(2′)); 122.29; 141.48 (C(8)); 149.54, 150.20; 152.67 (C(2)); 170.55 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 270 (16 100) nm (mol−1 dm3 cm−1); ESI-MS (m/z): [M+H]+ calcd for C25H40F3N5O8SSi2, 684.85; found 684.20.

N6-Acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)(β-D-arabinofuranosyl)adenine (A5)

To a solution of crude A4 (prepared from 744 mg of A3; 1.348 mmol) in toluene (36 ml), potassium trifluoroacetate (1.025 g; 6.740 mmol), 18-crown-6 (713 mg; 2.696 mmol) and N-ethyldiisopropylamine (346 µl; 2.022 mmol) were added. The mixture was stirred for 16 h at 80°C. After completion of the reaction, the mixture was filtrated over celite and the solvent was evaporated. The residue was then suspended in dichloromethane, and again filtrated over celite. The filtrate was washed with 5% aqueous NaHCO3, dried over Na2SO4 and evaporated. The crude product was purified by column chromatography on SiO2 (CH2Cl2/CH3OH, 100/0 – 97.5/2.5 v/v). Yield: 498 mg of A5 as slightly yellow foam (67% over two steps). TLC (CH2Cl2/CH3OH, 92/8): Rf = 0.48; 1H-NMR (300 MHz, CDCl3): δ 1.05 (m, 28H, 2× ((CH3)2CH)2Si); 2.50 (s, 3H, COCH3); 3.88 (m, 1H, H-C(4′)); 4.07 (m, 2H, H2-C(5′)); 4.26 (s, br, 1H, HO-C(2′)); 4.61 (t, J = 7.8 Hz, 1H, H-C(3′)); 4.67 (m, 1H, H-C(2′)); 6.28 (d, J = 6.0 Hz, 1H, H-C(1′)); 8.30 (s, 1H, H-C(8)); 8.53 (s, 1H, H-C(2)); 9.07 (s, br, 1H, H-N6) p.p.m.; 13C-NMR (75 MHz, CDCl3): δ 12.49, 12.94, 13.07, 13.48, 16.90, 16.94, 17.02, 17.30, 17.35, 17.46 (2× ((CH3)2CH)2Si); 25.52 (COCH3), 61.62 (C(5′)); 75.06 (C(3′)); 77.21 (C(2′)); 81.54 (C(4′)); 83.81 (C(1′)); 121.56; 142.82 (C(8)); 148.94, 150.98; 151.97 (C(2)); 170.69 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 271 (16 400) nm (mol−1 dm3 cm−1); ESI-MS (m/z): [M+H]+ calcd for C24H41N5O6Si2, 552.78; found 552.14.

N6-Acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-O-(trifluoromethanesulfonyl)(β-D-arabinofuranosyl)adenine (A6)

To a solution of compound A5 (49 mg; 0.089 mmol) in CH2Cl2 (1.7 ml), 4-dimethylaminopyridine (32 mg; 0.266 mmol) was added at 0°C. The mixture was treated with trifluoromethanesulfonyl chloride (14 µl; 0.133 mmol) and stirred for 15 min at 0°C. The reaction mixture was then diluted with CH2Cl2, washed with 5% aqueous NaHCO3, dried over Na2SO4 and evaporated. The crude product can be used for the next step without further purification. For analysis, the product was purified by column chromatography on SiO2 (CH2Cl2/CH3OH, 100/0 – 99.3/0.7 v/v). Yield: 31 mg of A6 as white foam (51%). TLC (CH2Cl2/CH3OH, 94/6): Rf = 0.54; 1H-NMR (300 MHz, CDCl3): δ 1.05 (m, 28H, 2× ((CH3)2CH)2Si); 2.64 (s, 3H, COCH3); 3.98 (m, 1H, H-C(4′)); 4.15 (m, 2H, H2-C(5′)); 5.39 (t, J = 7.2 Hz, 1H, H-C(3′)); 5.49 (t, J = 6.3 Hz, 1H, H-C(2′)); 6.43 (d, J = 6.0 Hz, 1H, H-C(1′)); 8.13 (s, 1H, H-C(8)); 8.66 (s, 1H, H-C(2)); 8.86 (s, br, 1H, H-N6) p.p.m.; 13C-NMR (75 MHz, CDCl3): δ 12.61-17.38 (2× ((CH3)2CH)2Si); 25.71 (COCH3); 61.99 (C(5′)); 74.06 (C(3′)); 77.19 (CF3); 81.00 (C(1′)); 81.23 (C(4′)); 88.27 (C(2′)); 121.84; 141.84 (C(8)); 149.50, 150.78; 152.55 (C(2)); 170.47 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 270 (20 400) nm (mol−1 dm3 cm−1); ESI-MS (m/z): [M+H]+ calcd for C25H40F3N5O8SSi2, 684.85; found 684.11.

N6-Acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-methylseleno-2′-deoxyadenosine (A7)

Sodium borohydride (204 mg; 5.418 mmol) was placed in a sealed 25 ml two-necked round-bottom flask, dried on high vacuum for 15 min to deplete oxygen, kept under argon and suspended in dry THF (7.3 ml). Dimethyldiselenide (174 µl; 1.806 mmol) was slowly injected to this suspension, followed by dropwise addition of anhydrous ethanol; 0.2 ml was required until gas bubbles started to occur in the yellow mixture. The solution was stirred at room temperature for 1.5 h and the almost colorless solution was injected into a solution of crude A6 (prepared from 498 mg of A5; 0.903 mmol) in dry THF (8.6 ml). The reaction mixture was stirred at room temperature for 30 min. Then, aqueous 0.2 M triethylammonium acetate buffer (15 ml, pH 7) was added, and the solution was reduced to half the volume by evaporation. Dichloromethane was added, and the organic layer was washed twice with 0.2 M triethylammonium acetate buffer and finally with saturated sodium chloride solution. The organic phase was dried over Na2SO4 and the solvent was evaporated. The crude product was purified by column chromatography on SiO2 (CH2Cl2/CH3OH, 99.8/0.2 – 98/2 v/v). Yield: 335 mg of A7 as white foam (59% over two steps). TLC (CH2Cl2/CH3OH, 94/6): Rf = 0.47; 1H-NMR (300 MHz, CDCl3): δ 1.01 (m, 28H, 2× ((CH3)2CH)2Si); 1.99 (s, 3H, SeCH3); 2.62 (s, 3H, COCH3); 4.09 (m, 3H, H2-C(5′) + H-C(2′)); 4.19 (m, 1H, H-C(4′)); 4.89 (t, J = 6.8 Hz, 1H, H-C(3′)); 6.31 (d, J = 3.9 Hz, 1H, H-C(1′)); 8.26 (s, 1H, H-C(8)); 8.66 (s, 1H, H-C(2)); 8.89 (s, br, 1H, H-N6) p.p.m.; 13C-NMR (75 MHz, CDCl3): δ 3.40 (SeCH3); 12.70, 12.95, 13.14, 13.47, 16.88, 16.98, 17.13, 17.28, 17.29, 17.34, 17.46 (2× ((CH3)2CH)2Si); 25.65 (COCH3); 47.12 (C(2′)); 61.73 (C(5′)); 71.47 (C(3′)); 84.68 (C(4′)); 90.14 (C(1′)); 122.36; 141.28 (C(8)); 149.25, 150.57; 152.37 (C(2)); 170.43 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 271 (17 300) nm (mol−1 dm3 cm−1); ESI-MS (m/z): [M+H]+ calcd for C25H43N5O5SeSi2, 629.77; found 629.98.

N6-Acetyl-2′-methylseleno-2′-deoxyadenosine (A8)

Compound A7 (192 mg; 0.305 mmol) was dissolved in a mixture of 1 M tetrabutylammonium fluoride/0.5 M acetic acid in THF (1.3 ml). The solution was stirred for 2 h at room temperature and the reaction progress was monitored via TLC. Then, the solvent was evaporated and the residue dried under high vacuum. The crude product was purified by column chromatography on SiO2 (CH2Cl2/CH3OH, 100/0 – 97/3 v/v). Yield: 111 mg of A8 as white foam (94%). TLC (CH2Cl2/CH3OH, 90/10): Rf = 0.42; 1H-NMR (300 MHz, DMSO): δ 1.57 (s, 3H, SeCH3); 2.25 (s, 3H, COCH3); 3.58 (m, 1H, H1-C(5′)); 3.65 (m, 1H, H2-C(5′)); 4.00 (m, 1H, H-C(4′)); 4.18 (dd, J = 3.0, 9.0 Hz, 1H, H-C(2′)); 4.36 (m, 1H, H-C(3′)); 5.10 (t, J = 6.0 Hz, 1H, HO-C(5′)); 5.86 (d, J = 6.0 Hz, 1H, HO-C(3′)); 6.36 (d, J = 9.0 Hz, H-C(1′)); 8.66 (s, 1H, H-C(2)); 8.74 (s, 1H, H-C(8)); 10.68 (s, br, 1H, H-N6) p.p.m.; 13C-NMR (75 MHz, DMSO): δ 2.87 (SeCH3); 24.81 (COCH3); 46.73 (C(2′)); 62.14 (C(5′)); 73.21 (C(3′)); 87.98 (C(4′)); 89.90 (C(1′)); 124.00; 143.21 (C(8)); 150.17, 152.17; 152.29 (C(2)); 169.30 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 271 (15 000) nm (mol−1dm3cm−1); ESI-MS (m/z): [M+H]+ calcd for C13H17N5O4Se, 387.27; found 387.84.

N6-Acetyl-5′-O-[benzhydryloxy-bis(trimethylsilyloxy)silyl]-2′-methylseleno-2′-deoxyadenosine (A9)

Solution A: To a solution of compound A8 (60 mg; 0.155 mmol) in DMF (0.5 ml), N,N-diisopropylamine (22 µl; 0.155 mmol) was added and the mixture was cooled to 0°C. Solution B: N,N-diisopropylamine (53 µl; 0.372 mmol) was added dropwise to a solution of benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (132 mg; 0.310 mmol) in dichloromethane (0.3 ml) at 0°C. Solution B was added to solution A at 0°C in three portions (aliquots of 0.5/0.25/0.25 every 30 min) and the reaction progress was monitored by TLC. After 2 h, the reaction mixture was quenched by addition of 5% sodium bicarbonate solution and extracted with dichloromethane. The combined organic phases were washed with brine, dried over Na2SO4 and evaporated. The crude product was purified by column chromatography on SiO2 (hexane/ethyl acetate, 1/1 v/v). Yield: 87 mg of A9 as colorless oil (72%). TLC (ethyl acetate/hexane, 4/1): Rf = 0.46; 1H-NMR (300 MHz, CDCl3): δ 0.08, 0.10 (2s, 18 H, 2× (CH3)3Si); 1.84 (s, 3H, SeCH3); 2.66 (s, 3H, COCH3); 2.73 (m, 1H, HO-C(3′)); 3.82 (m, 2H, H2-C(5′)); 3.95 (dd, J = 5.1, 8.4 Hz, 1H, H-C(2′)); 4.18 (m, 1H, H-C(4′)); 4.28 (m, 1H, H-C(3′)); 5.94 (s, 1H, OCH(Ph)2); 6.26 (d, J = 9.0 Hz, 1H, H-C(1′)); 7.26–7.36 (m, 10H, H-C(ar)); 8.28 (s, 1H, H-C(8)); 8.66 (s, br, 2H, H-C(2) + H-N6) p.p.m.; 13C-NMR (75 MHz, CDCl3): δ 1.50 (2× (CH3)3Si); 4.23 (SeCH3); 25.68 (COCH3); 49.68 (C(2′)); 63.14 (C(5′)); 72.75 (C(3′)); 77.05 (OCH(Ph)2); 85.67 (C(4′)); 88.19 (C(1′)); 121.88; 126.31, 126.32, 127.33, 128.30 (C(ar)); 141.49 (C(8)); 143.83, 143.85, 149.13, 151.36; 152.36 (C(2)); 170.62 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 271 (17500) nm (mol−1 dm3 cm−1); HR-ESI-MS (m/z): [M+Na]+ calcd for C32H45N5O7SeSi3, 798.1689; found 798.1677.

N6-Acetyl-5′-O-[benzhydryloxy-bis(trimethylsilyloxy)silyl]-2′-methylseleno-2′-deoxyadenosine 3′-(methyl-N,N-diisopropyl)phosphoramidite (A10)

Compound A9 (139 mg; 0.179 mmol) was dissolved in a mixture of N-ethyldimethylamine (194 µl; 1.794 mmol) in dry dichloromethane (2.3 ml) under argon. After 15 min at room temperature, methyl-N,N-diisopropylchlorophosphoramidite (53 mg; 0.269 mmol) was slowly added and the solution was stirred at room temperature for 2 h. The reaction mixture was diluted with dichloromethane, washed with half-saturated sodium bicarbonate solution, dried over Na2SO4 and evaporated. The crude product was purified by column chromatography on SiO2 (ethyl acetate/hexane, 2/3 – 1/1 v/v (+0.5% Et3N)). Yield: 117 mg of A10 (mixture of diastereomers) as colorless oil (70%). TLC (ethyl acetate/hexane, 7/3): Rf = 0.47, 0.53; 1H-NMR (500 MHz, CDCl3): δ 0.07-0.09 (4s, 36 H, 4× (CH3)3Si); 1.19-1.27 (m, 24H, 2× ((CH3)2CH)2N); 1.52, 1.57 (2s, 6H, 2× SeCH3); 2.66 (s, 6H, 2× COCH3); 3.35 (d, J = 13.5 Hz, 3H, POCH3); 3.48 (d, J = 13.5 Hz, 3H, POCH3); 3.63 (m, 4H, 2× ((CH3)2CH)2N); 3.80–3.96 (m, 6H, 2× H-C(2′) + 2× H2-C(5′)); 4.31 (m, 2H, 2× H-C(4′)); 4.57 (dd, J = 5.4, 9.3 Hz, 1H, H-C(3′)); 4.66 (dd, J = 6.2, 11.0 Hz, 1H, H-C(3′)); 5.95, 5.96 (2s, 2H, 2× OCH(Ph)2); 6.45 (2d, J = 8.0, 8.5 Hz, 2H, 2× H-C(1′)); 7.19–7.37 (m, 20H, H-C(ar)); 8.27, 8.28 (2s, 2H, 2× H-C(8)); 8.53 (s, br, 2H, 2× H-N6); 8.66, 8.68 (2s, 2H, H-C(2)) p.p.m.; 31P-NMR (121 MHz, CDCl3): δ 150.3, 153.0 p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 271 (17 400) nm (mol−1 dm3 cm−1); HR-ESI-MS (m/z): [M+Na]+ calcd for C39H61N6O8PSeSi3, 959.2660; found 959.2701.

Synthesis of 2′-methylseleno guanosine phosphoramidite (G3)

N2-Acetyl-5′-O-[benzhydryloxy-bis(trimethylsilyloxy)silyl]-2′-methylseleno-2′-deoxyguanosine (G2)

Solution A: To a solution of compound G1 [reference (6)] (400 mg; 0.994 mmol) in THF (7.0 ml), N,N-diisopropylamine (179 µl; 1.99 mmol) was added and the mixture was cooled to 0°C. Solution B: N,N-diisopropylamine (179 µl; 1.99 mmol) was added dropwise to a solution of benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (846 mg; 1.99 mmol) in dichloromethane (5.0 ml) at 0°C. Solution B was added to solution A at 0°C in three portions (aliquots of 0.5/0.25/0.25 every 30 min) and the reaction progress was monitored by TLC. After 2 h, the reaction mixture was quenched by addition of 5% sodium bicarbonate solution and extracted with dichloromethane. The combined organic phases were washed with brine, dried over Na2SO4 and evaporated. The crude product was purified by column chromatography on SiO2 (CH2Cl2/acetone, 95/5 – 85/15 v/v). Yield: 358 mg of G2 as colorless oil (46%). TLC (CH2Cl2/acetone, 7/3): Rf = 0.71; 1H-NMR (300 MHz, CDCl3): δ 0.05–0.06 (2s, 18 H, 2× (CH3)3Si); 1.65 (s, 3H, SeCH3); 2.31 (s, 3H, COCH3); 3.57 (m, 1H, H-C(2′)); 3.67 (m, 1H, HO-C(3′)); 3.77 (m, 2H, H2-C(5′)); 4.15 (m, 1H, H-C(4′)); 4.31 (m, 1H, H-C(3′)); 5.92 (d, J = 9 Hz, 1H, H-C(1′)); 5.93 (s, 1H, OCH(Ph)2); 7.16-7.34 (m, 10H, H-C(ar)); 8.01 (s, 1H, H-C(8)); 10.49 (s, br, 1H, H-N2); 12.24 (s, br, 1H, H-N(1)) p.p.m.; 13C-NMR (75 MHz, CDCl3): δ 1.52 (2× (CH3)3Si); 3.63 (SeCH3); 24.28 (COCH3); 49.80 (C(2′)); 63.52 (C(5′)); 73.38 (C(3′)); 77.05 (OCH(Ph)2); 85.98 (C(4′)); 87.96 (C(1′)); 120.83, 126.25, 126.32, 127.33, 128.29 (C(ar)); 137.31 (C(8)); 143.72, 143.77, 147.79, 149.13 (C(ar)); 156.16 (C(6)); 172.95 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 256 (19 300) nm (mol−1 dm3 cm−1); HR-ESI-MS (m/z): [M+Na]+ calcd for C32H45N5O8SeSi3, 815.1656; found 815.1651.

N2-Acetyl-5′-O-[benzhydryloxy-bis(trimethylsilyloxy)silyl]-2′-methylseleno-2′-deoxyguanosine 3′-(methyl-N,N-diisopropyl)phosphoramidite (G3)

Compound G2 (179 mg; 0.226 mmol) was dissolved in a mixture of N-ethyldimethylamine (74 µl; 0.680 mmol) in dry dichloromethane (5.0 ml) under argon. After 15 min at room temperature, methyl-N,N-diisopropylchlorophosphoramidite (67 mg; 0.340 mmol) was slowly added and the solution was stirred at room temperature for 2 h. The reaction mixture was diluted with dichloromethane, washed with half-saturated sodium bicarbonate solution, dried over Na2SO4 and evaporated. The crude product was purified by column chromatography on SiO2 (CH2Cl2/acetone, 100/0 – 95/5 v/v). Yield: 172 mg of G3 (mixture of diastereomers) as colorless oil (80%). TLC (CH2Cl2/acetone, 8/2): Rf = 0.48, 0.63; 1H-NMR (500 MHz, CDCl3): δ 0.07 (m, 36 H, 4× (CH3)3Si); 1.18-1.27 (m, 24H, 2× ((CH3)2CH)2N); 1.46, 1.50 (2s, 6H, 2× SeCH3); 2.14, 2.15 (2s, 6H, 2× COCH3); 3.35 (d, J = 13.3 Hz, 3H, POCH3); 3.47 (d, J = 13.2 Hz, 3H, POCH3); 3.63 (m, 4H, 2× ((CH3)2CH)2N); 3.76 (m, 4H, 2× H2-C(5′)); 3.87 (m, 2H, 2× H-C(2′)); 4.25, 4.29 (2m, 2H, 2× H-C(4′)); 4.45, 4.65 (2m, 2H, H-C(3′)); 5.97, 5.98 (2s, 2H, 2× OCH(Ph)2); 6.10, 6.15 (2d, J = 9.6, 9.6 Hz, 2H, 2× H-C(1′)); 7.19-7.34 (m, 20H, H-C(ar)); 7.92 (s, 2H, 2× H-C(8)); 8.46 (s, br, 2H, 2× H-N2); 11.85 (s, br, 2H, 2× H-N(1)) p.p.m.; 31P-NMR (121 MHz, CDCl3): δ 150.5, 152.8 p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 257 (20 800) nm (mol−1dm3cm−1); HR-ESI-MS (m/z): [M+H]+ calcd for C39H61N6O9PSeSi3, 953.2790; found 953.2807.

Synthesis of 2′-methylseleno cytidine phosphoramidite (C4)

N4-Acetyl-2′-methylseleno-2′-deoxycytidine (C2)

N4-Acetyl-3′-O-tert-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-methylseleno-2′-deoxycytidine C1 [reference (4)] (824 mg; 1.055 mmol) was dissolved in a mixture of 1.0 M tetrabutylammonium fluoride/0.5 M acetic acid in THF (3.0 ml). The solution was stirred for 18 h at room temperature, and the reaction progress was monitored via TLC. Then, the solvent was evaporated and the residue partitioned between CH2Cl2 and water. The organic layer was concentrated in vacuum and then detritylation was initiated by addition of 4.0 ml of formic acid. The reaction was complete after 2 min. CH3OH was added followed by evaporation and coevaporation with CH3OH and toluene for several times. For workup, the crude product was partitioned between water and CH2Cl2. The aqueous layer was evaporated and dried under high vacuum. Yield: 305 mg of C2 as white powder (80%). TLC (CH2Cl2/CH3OH, 90/10): Rf = 0.38; 1H-NMR (300 MHz, DMSO): δ 1.89 (s, 3H, SeCH3); 2.10 (s, 3H, COCH3); 3.56 (m, 1H, H-C(2′)); 3.58 (m, 1H, H1-C(5′)); 3.64 (m, 1H, H2-C(5′)); 4.18 (m, 1H, H-C(4′)); 4.22 (m, 1H, H-C(3′)); 5.17 (s, 1H, HO-C(5′)); 5.78 (s, 1H, HO-C(3′)); 6.25 (d, J = 7.8 Hz, 1H, H-C(1′)); 7.20 (d, J = 7.8 Hz, 1H, H-C(5)); 8.34 (d, J = 7.8 Hz, 1H, H-C(6)); 10.92 (s, br, 1H, H-N4) p.p.m.; 13C-NMR (75 MHz, DMSO): δ 2.33 (SeCH3); 24.25 (COCH3); 47.55 (C(2′)); 60.70 (C(5′)); 71.22 (C(3′)); 86.41 (C(4′)); 89.97 (C(1′)); 95.66 (C(5)); 145.30 (C(6)); 154.61, 162.28; 170.96 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 262 (12 900) nm (mol−1 dm3 cm−1); ESI-MS (m/z): [M+H]+ calcd for C12H17N3O5Se, 363.14; found 363.03.

N4-Acetyl-5′-O-[benzhydryloxy-bis(trimethylsilyloxy)silyl]-2′-methylseleno-2′-deoxycytidine (C3)

Solution A: To a solution of compound C2 (80 mg; 0.221 mmol) in DMF (1.0 ml), N,N-diisopropylamine (31 µl; 0.221 mmol) was added and the mixture was cooled to 0°C. Solution B: N,N-diisopropylamine (70 µl; 0.497 mmol) was added dropwise to a solution of benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (188 mg; 0.442 mmol) in dichloromethane (1.0 ml) at 0°C. Solution B was added to solution A at 0°C in three portions (aliquots of 0.5/0.25/0.25 every 30 min) and the reaction progress was monitored by TLC. After 2 h, the reaction mixture was quenched by addition of 5% sodium bicarbonate solution and extracted with dichloromethane. The combined organic phases were washed with brine, dried over Na2SO4 and evaporated. The crude product was purified by column chromatography on SiO2 (CH2Cl2/acetone, 1/0 – 1/1 v/v). Yield: 90 mg of C3 as colorless oil (54%). TLC (CH2Cl2/acetone, 3/7): Rf = 0.65; 1H-NMR (300 MHz, CDCl3): δ 0.12–0.24 (2s, 18 H, 2× (CH3)3Si); 2.14 (s, 3H, SeCH3); 2.22 (s, 3H, COCH3); 2.61 (m, 1H, HO-C(3′)); 3.38 (m, 1H, H-C(2′)); 3.78 (m, 1H, H1-C(5′)); 3.91 (m, 1H, H2-C(5′)); 4.07 (m, 1H, H-C(4′)); 4.10 (m, 1H, H-C(3′)); 5.96 (s, 1H, OCH(Ph)2); 6.35 (d, J = 6.8 Hz, 1H, H-C(1′)); 7.19–7.39 (m, 10H, H-C(ar)); 7.35 (d, J = 7.5 Hz, 1H, H-C(5)); 8.26 (d, J = 7.5 Hz, 1H, H-C(6)); 8.37 (s, br, 1H, H-N4) p.p.m.; 13C-NMR (75 MHz, CDCl3): δ 1.54 (2× (CH3)3Si); 4.36 (SeCH3); 24.87 (COCH3); 51.29 (C(2′)); 62.55 (C(5′)); 70.90 (C(3′)); 77.09 (OCH(Ph)2); 85.20 (C(4′)); 89.01 (C(1′)); 97.29 (C(5)); 126.21, 127.37, 128.33, 143.72 (C(ar)); 144.38 (C(6)); 155.36, 162.96 (C(ar)); 170.97 (COCH3) p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 263 (12 500) nm (mol−1 dm3 cm−1); HR-ESI-MS (m/z): [M+Na]+ calcd for C31H45N3O8SeSi3, 775.1595; found 775.1606.

N4-Acetyl-5′-O-[benzhydryloxy-bis(trimethylsilyloxy)silyl]-2′-methylseleno-2′-deoxycytidine 3′-(methyl-N,N-diisopropyl)phosphoramidite (C4)

Compound C3 (280 mg; 0.373 mmol) was dissolved in a mixture of N-ethyldimethylamine (404 µl; 3.729 mmol) in dry dichloromethane (5.0 ml) under argon. After 15 min at room temperature, methyl-N,N-diisopropylchlorophosphoramidite (111 mg; 0.559 mmol) was slowly added and the solution was stirred at room temperature for 2 h. The reaction mixture was diluted with dichloromethane, washed with half-saturated sodium bicarbonate solution, dried over Na2SO4 and evaporated. The crude product was purified by column chromatography on SiO2 (CH2Cl2/acetone, 99/1–93/7 v/v). Yield: 235 mg of C4 (mixture of diastereomers) as colorless oil (69%). TLC (CH2Cl2/acetone, 7/3): Rf = 0.37, 0.52; 1H-NMR (500 MHz, CDCl3): δ 0.06-0.09 (4s, 36H, 4× (CH3)3Si); 1.14–1.27 (m, 24H, 2× ((CH3)2CH)2N); 1.96, 2.07 (2s, 6H, 2× SeCH3); 2.24 (s, 6H, 2× COCH3); 3.26, 3.49 (2d, 2H, 2× H-C(2′)); 3.31 (d, J = 13.5 Hz, 3H, POCH3); 3.44 (d, J = 13.5 Hz, 3H, POCH3); 3.60 (m, 4H, 2× ((CH3)2CH)2N); 3.79–3.96 (m, 4H, 2× H2-C(5′)); 4.23 (m, 2H, 2× H-C(4′)); 4.46 (m, 2H, H-C(3′)); 5.93 (s, 2H, 2× OCH(Ph)2); 6.43, 6.49 (2d, J = 5.5, 7.5 Hz, 2H, 2× H-C(1′)); 7.18–7.35 (m, 22H, H-C(ar), H-C(5)); 8.15, 8.25 (2d, J = 7.8, 7.3 Hz, 2H, 2× H-C(6)); 8.96, 9.05 (2s, br, 2H, 2× H-N4) p.p.m.; 31P-NMR (121 MHz, CDCl3): δ 150.6, 152.7 p.p.m.; UV/Vis (MeOH): λmax (epsilon) = 264 (12 800) nm (mol−1 dm3 cm−1); HR-ESI-MS (m/z): [M+Na]+ calcd for C38H61N4O9PSeSi3, 936.2567; found 936.2587.

Synthesis of 2′-methylseleno uridine phosphoramidite (U4)

2′-Methylseleno-2′-deoxyuridine (U2)

A solution of 5′-O-(4,4′-dimethoxytrityl)-2′-methylseleno-2′-deoxyuridine U1 [reference (4)] (600 mg; 0.962 mmol) in dichloromethane/formic acid (14.4 ml; 5/1 v/v) was stirred at room temperature for 1 h. After addition of methanol (5 ml), the solvents were evaporated at reduced pressure. The residue was coevaporated several times with methanol and toluene until a clear syrup was obtained which was dissolved in dichloromethane/water. Extraction of the product into water, evaporation to dryness and coevaporation with methanol yielded 306 mg of U2 as white foam (99%). TLC (CH2Cl2/MeOH, 88/12): Rf = 0.47; 1H-NMR (300 MHz, DMSO): δ 1.87 (s, 3H, SeCH3); 3.51 (dd, J = 5.6, 8.6 Hz, 1H, H-C(2′)); 3.58 (m, 2H, H2-C(5′)); 3.88 (m, 1H, H-C(4′)); 4.21 (m, 1H, H-C(3′)); 5.09 (s, br, 1H, HO-C(5′)); 5.71 (d, J = 9.0 Hz, 1H, H-C(5)); 5.75 (m, 1H, HO-C(3′)); 6.20 (d, J = 9.0 Hz, 1H, H-C(1′)); 7.86 (d, J = 9.0 Hz, 1H, H-C(6)); 11.36 (s, br, 1H, H-N(3)) p.p.m.; 13C-NMR (75 MHz, DMSO): δ 2.93 (SeCH3); 46.80 (C(2′)); 61.81 (C(5′)); 72.81 (C(3′)); 87.04 (C(4′)); 89.07 (C(1′)); 102.76 (C(5)); 140.91 (C(6)); 151.12 (C(2)); 163.36 (C(4)) p.p.m.; UV/Vis: λmax (epsilon) = 260 (8700) nm (mol−1 dm3 cm−1); ESI-MS (m/z): [M-H] calcd for C10H14N2O5Se, 320.19; found 320.94.

5′-O-[Benzhydryloxy-bis(trimethylsilyloxy)silyl]-2′-methylseleno-2′-deoxyuridine (U3)

Preparation starting with U2

Solution A: To a solution of compound U2 (299 mg; 0.933 mmol) in DMF (2 ml), N,N-diisopropylamine (132 µl; 0.933 mmol) was added and the mixture was cooled to 0°C. Solution B: N,N-diisopropylamine (317 µl; 2.237 mmol) was added dropwise to a solution of benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (793 mg; 1.866 mmol) in dichloromethane (1.8 ml) at 0°C. Solution B was added to solution A at 0°C in three portions (aliquots of 0.5/0.25/0.25 every 30 min) and the reaction progress was monitored by TLC. After 2 h, the reaction mixture was quenched by addition of 5% sodium bicarbonate solution and extracted with dichloromethane. The combined organic phases were washed with brine, dried over Na2SO4 and evaporated. The crude product was purified by column chromatography on SiO2 (hexane/ethyl acetate, 4/1 – 1/1 v/v). Yield: 481 mg of U3 as white foam (73%).

Preparation starting with U6

Sodium borohydride (18 mg; 0.476 mmol) was placed in a sealed 25 ml two-necked round-bottom flask, dried on high vacuum for 15 min to deplete oxygen, kept under argon, and suspended in dry THF (0.6 ml). Dimethyldiselenide (15 µl; 0.159 mmol) was slowly injected to this suspension, followed by dropwise addition of anhydrous ethanol; 25 µl was required until gas bubbles started to occur in the yellow mixture. The solution was stirred at room temperature for 1 h, and the almost colorless solution was injected into a solution of U6 (48 mg; 0.078 mmol) in dry THF (0.8 ml). The reaction mixture was stirred at room temperature for 2 h. Then, aqueous 0.1 M triethylammonium acetate buffer (5 ml, pH 7) was added, and the organic solvent was removed by evaporation. Water was added, and the solution extracted with dichloromethane. The organic phase was dried over Na2SO4, and the solvent was evaporated. The crude product was purified by column chromatography on SiO2 (CH2Cl2/CH3OH, 99/1 – 99/2 v/v). Yield: 33 mg of U3 as white foam (59%).

TLC (ethyl acetate/hexane, 1/1): Rf = 0.48; 1H-NMR (300 MHz, CDCl3): δ 0.11, 0.12 (2s, 18 H, 2× (CH3)3Si); 2.03 (s, 3H, SeCH3); 2.66 (m, 1H, HO-C(3′)); 3.29 (dd, J = 4.8, 8.4 Hz, 1H, H-C(2′)); 3.86 (m, 2H, H2-C(5′)); 4.15 (m, 2H, H-C(3′), H-C(4′)); 5.59 (d, J = 8.1 Hz, 1H, H-C(5)); 5.95 (s, 1H, OCH(Ph)2); 6.19 (d, J = 8.4 Hz, 1H, H-C(1′)); 7.31 (m, 10H, H-C(ar)); 7.77 (d, J = 8.1 Hz, 1H, H-C(6)); 8.27 (s, br, 1H, H-N(3)) p.p.m.; 13C-NMR (75 MHz, CDCl3): δ 1.54 (2× (CH3)3Si); 4.22 (SeCH3); 50.08 (C(2′)); 63.26 (C(5′)); 71.97 (C(3′/4′)); 77.13 (OCH(Ph)2); 85.11 (C(3′/4′)); 87.15 (C(1′)); 103.01 (C(5)); 126.23, 126.28, 127.50, 128.38 (C(ar)); 139.58 (C(6)); 143.64, 143.67 (C(ar)); 150.39 (C(2)); 162.80 (C(4)) p.p.m.; UV/Vis: λmax (epsilon) = 260 (7800) nm (mol−1 dm3 cm−1); HR-ESI-MS (m/z): [M+Na]+ calcd for C29H42N2O8SeSi3, 733.1311; found 733.1321.

5′-O-[Benzhydryloxy-bis(trimethylsilyloxy)silyl]-2′-methylseleno-2′-deoxyuridine 3′-(methyl-N,N-diisopropyl)phosphoramidite (U4)

Compound U3 (106 mg; 0.150 mmol) was dissolved in a mixture of N-ethyldimethylamine (162 µl; 1.50 mmol) in dry dichloromethane (2 ml) under argon. After 15 min at room temperature, methyl-N,N-diisopropylchlorophosphoramidite (45 mg; 0.225 mmol) was slowly added and the solution was stirred at room temperature for 2 h. The reaction was quenched by the addition of methanol (0.1 ml). The reaction mixture was diluted with dichloromethane, extracted with saturated sodium bicarbonate solution, dried over Na2SO4 and the solvent was evaporated. The crude product was purified by column chromatography on SiO2 (ethyl acetate/hexane, 2/8–3/7 v/v (+0.5% NEt3)). Yield: 107 mg of U4 (mixture of diastereomers) as thick, colorless oil (84%). TLC (ethyl acetate/hexane, 3/7): Rf = 0.50; 1H-NMR (500 MHz, CDCl3): δ 0.07, 0.08, 0.09, 1.10 (4s, 36 H, 4× (CH3)3Si); 1.17–1.29 (m, 24H, 2× ((CH3)2CH)2N); 1.92, 1.96 (2s, 6H, 2× SeCH3); 3.17 (m, 1H, H-C(2′)); 3.34 (m, 4H, H-C(2′), POCH3); 3.49 (d, J = 13.1 Hz, 3H, POCH3); 3.66 (m, 4H, 2× ((CH3)2CH)2N); 3.83 (m, 4H, 2× H2-C(5′)); 4.23 (m, 2H, 2× H-C(4′)); 4.47, 4.55 (2m, 2H, 2× H-C(3′)); 5.50 (m, 2H, 2× H-C(5)); 5.93 (s, 2H, 2× OCH(Ph)2); 6.38 (2d, J = 9 Hz, 2H, 2× H-C(1′)); 7.22-7.32 (m, 20H, H-C(ar)); 7.70, 7.74 (2d, J = 8.2 Hz, 2H, 2× H-C(6)); 8.61 (s, br, 2H, 2× H-N(3)) p.p.m.; 31P-NMR (121 MHz, CDCl3): δ 150.3, 153.0 p.p.m.; UV/Vis: λmax (epsilon) = 260 (8400) nm (mol−1dm3cm−1); HR-ESI-MS (m/z): [M+Na]+ calcd for C36H58N3O9PSeSi3, 894.2282; found 894.2310.

5′-O-[Benzhydryloxy-bis(trimethylsilyloxy)silyl]- 2,2′-anhydro uridine (U6)

Solution A: A solution of 2,2′-anhydro uridine U5 [reference (4)] (25 mg; 0.111 mmol) in pyridine (2.5 ml) was cooled to −15°C. Solution B: Benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (118 mg; 0.277 mmol) was added to dichloromethane (1.06 ml) at −15°C. Solution B was added to solution A at −15°C in 5 portions (aliquots of 0.5 over 3.5 h) and the reaction progress was monitored by TLC. After 4 h, the reaction mixture was quenched by addition of 5% citric acid solution and extracted with dichloromethane. The combined organic phases were washed with water and 5% sodium bicarbonate solution, dried over Na2SO4 and evaporated. The crude product was purified by column chromatography on SiO2 (CH2Cl2/CH3OH, 99/1 – 93/7 v/v). Yield: 32 mg of U6 as white foam (47%). TLC (CH2Cl2/CH3OH, 92/8): Rf = 0.47; 1H-NMR (300 MHz, CDCl3): δ −0.02, −0.01 (2s, 18H, 2× (CH3)3Si); 3.42 (m, 2H, H2-C(5′)); 4.04 (m, 1H, H-C(4′)); 4.36 (m, 1H, H-C(3′)); 4.58 (s, br, 1H, HO-C(3′)); 5.25 (m, 1H, H-C(2′)); 5.84 (s, 1H, OCH(Ph)2); 5.93 (d, J = 7.44 Hz, 1H, H-C(5)); 6.04 (d, J = 5.73 Hz, 1H, H-C(1′)); 7.21 (m, 11H, H-C(6), H-C(ar)) p.p.m.; 13C-NMR (75 MHz, CDCl3): δ 1.57 (2× (CH3)3Si); 62.40 (C(5′)); 75.58 (C(3′)); 77.01 (OCH(Ph)2); 87.45 (C(4′)); 89.40 (C(2′)); 89.75 (C(1′)); 110.25 (C(5)); 126.59, 127.40, 128.40 (C(ar)); 135.33 (C(6)); 144.07, 144.09 (C(ar)); 159.94 (C(2)); 172.46 (C(4)) p.p.m.; UV/Vis: λ260 (epsilon) = 260 (9800) nm (mol−1 dm3 cm−1); ESI-MS (m/z): [M+H]+ calcd for C28H38N2O8Si3, 615.88; found 615.29.

ACE RNA solid-phase synthesis of 2′-methylseleno nucleoside containing RNAs

2′-O-ACE standard nucleoside phosphoramidites and the corresponding solid-phase supports were obtained from Dharmacon. Oligoribonucleotides containing 2′-methylseleno nucleosides were synthesized on a slightly modified Pharmacia instrumentation (Gene Assembler Plus with a bypassed UV detection unit) following modified DNA/RNA standard methods containing an additional cycle step of treatment with DTT; desilylation (0.55 min): 1.1 M HF/2.9 M Et3N/DMF; coupling (3.0 min): phosphoramidites/acetonitrile (0.1 M × 130 µl) were activated by benzylthiotetrazole/acetonitrile (0.3 M × 360 µl); capping (3 × 0.4 min): A: Ac2O/sym-collidine/acetonitrile (20/30/50), B: 4-(dimethylamino)pyridine/acetonitrile (0.5 M), A/B = 1/1; oxidation (1.0 min): I2 (10 mM) in acetonitrile/sym-collidine/H2O (10/1/5); DTT treatment (2.0 min): DTT (100 mM) in ethanol/H2O (2/3). A ready-to-use synthesis method file is provided in the Supplementary Data available online. Solutions of standard amidites, tetrazole solutions and acetonitrile were dried over activated molecular sieves overnight. Solutions of 2′-methylseleno nucleoside phosphoramidites were only dried for 4–6 h over activated molecular sieves before consumption.

Deprotection and purification of 2′-ACE-protected RNAs with 2′-methylseleno modifications

After strand assembly, methyl groups were removed from the phosphate backbone of the RNA attached at the solid support by treatment with disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMF (0.39 M, 2.0 ml) and DTT in H2O (2 M, 150 µl; final DTT concentration 150 mM) for 20 min at room temperature, followed by filtration of the beads. Then, the beads were removed from the column and additionally treated with DTT in H2O (150 mM, 200 µl) for 1–3 h at room temperature in a 1.5 ml vial. Cleavage from the solid support and deprotection of acyl groups was performed by MeNH2 in H2O (40%, 0.74 ml) and DTT in H2O (2 M, 60 µl; final DTT concentration 150 mM). The mixture was heated to 60°C and held at this temperature for 10 min. Then, the solution was evaporated to dryness, and removal of 2′-O-orthoesters was accomplished by treatment with N,N,N′,N′-tetramethylethylenediamine (TEMED) acetate buffer (100 mM, 1 mL, pH 3.8) for 30 min at 60°C. The mixture was evaporated to dryness, the residue dissolved in H2O (0.7 ml), and the solution loaded on a size exclusion column (Amersham HiPrep 26/10 Desalting; 2.6 × 10 cm; Sephadex G25). The crude RNA was eluted with H2O and dried.

Analysis of crude RNA products after deprotection was performed by anion-exchange chromatography on a Dionex DNAPac100 column (4 × 250 mm) at 80°C. Flow rate: 1 ml/min; eluant A: 25 mM Tris–HCl (pH 8.0), 6 M urea; eluant B: 25 mM Tris–HCl (pH 8.0), 0.5 M NaClO4, 6 M urea; gradient: 0–60% B in A within 45 min; UV-detection at 265 nm. Crude RNA products were purified on a semi-preparative Dionex DNAPac100 column (9 × 250 mm). Flow rate: 2 ml/min; gradient: Δ5–10% B in A within 20 min. Fractions containing RNA were loaded on a C18 SepPak cartridge (Waters/Millipore), washed with 0.1–0.2 M (Et3NH)HCO3, H2O, and eluted with H2O/CH3CN (6/4). RNA fractions were lyophilized.

Mass spectrometry of 2′-methylseleno group containing RNAs

All experiments were performed on a Finnigan LCQ Advantage MAX ion trap instrumentation connected to an Amersham Ettan micro LC system. RNAs were analyzed in the negative-ion mode with a potential of −4 kV applied to the spray needle. LC: Sample (250 pmol RNA dissolved in 30 µl of 20 mM EDTA solution; average injection volume: 25–30 µl); column (XTerra®MS, C18 2.5 µm; 1.0 × 50 mm) at 21°C; flow rate: 30 µl/min; eluant A: 8.6 mM TEA, 100 mM 1,1,1,3,3,3-hexafluoroisopropanol in H2O (pH 8.0); eluant B: methanol; gradient: 0–100% B in A within 30 min; UV-detection at 254 nm. Prior to each injection, column equilibration was performed by eluting buffer A for 30 min at a flow rate of 30 µl/min.

RESULTS AND DISCUSSION

2′-O-ACE RNA synthesis method

The ACE method for chemical synthesis of RNA was designed under the aspect that mildly acidic aqueous conditions are most desirable for the final 2′-O deprotection of the synthesized RNA (19). The loss of orthogonality in combination with the classic 5′-O-DMT group was an obstacle to using a mildly acid-labile 2′-O protecting group and thus, the concept was achieved based on the fluoride labile 5′-O-bis(trimethylsilyloxy)cyclododecyloxysilyl ether (DOD), together with the 2′-O-bis(2-acetoxyethoxy)methyl (ACE) orthoester. The 3′-OH group was derivatized as methyl-N,N-diisopropylphosphoramidite, since the cyanoethyl group turned out to be unstable with fluoride reagents. After oligonucleotide assembly, the phosphate methyl protecting groups were removed with disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMF. Then, basic conditions (40% aqueous MeNH2) caused oligonucleotide cleavage from the solid support, along with the removal of the acyl protecting groups at the exocyclic amino groups and, importantly, of the acetyl groups at the 2′-orthoesters. The resulting 2′-O-bis(2-hydroxyethoxy)methyl orthoesters being 10 times more acid labile than prior to the removal of the acetyl groups, therefore required very mild acidic conditions (pH 3.8, 30 min, 60°C) for the final deprotection step (9,19,23–25). For preparation of Se-modified RNA oligonucleotides based on the ACE method, we have elaborated the syntheses of appropriate nucleoside phosphoramidites as described subsequently.

Synthesis of 2′-methylseleno adenosine phosphoramidite

Our route began with the simultaneous protection of the 3′- and 5′-hydroxyl groups of commercially available adenosine A1 using 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl2), followed by protection of the 2′-hydroxyl group as trimethylsilyl ether and reaction with acetyl chloride to furnish the N6-acetyl adenosine derivative A2 (Scheme 1). Then, the trimethylsilyl group was cleaved by p-toluenesulfonic acid (26). Triflation of the ribose 2′-OH of compound A3 gave intermediate A4 which was converted into arabino nucleoside A5 in diastereoselective manner by treatment with potassium trifluoroacetate and 18-crown-6-ether. After triflation of the arabinose 2′-OH, compound A6 was reacted with sodium methyl selenide, producing key diastereomer A7 in high yields. Deprotection of the TIPDS moiety proceeded straightforward using tetrabutylammonium fluoride (TBAF) and acetic acid. Derivative A8 was transformed regioselectively into the 5′-O protected analog A9 by using benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (BzHCl). Conversion into the corresponding phosphoramidite A10 was achieved in good yields by reaction with methyl-N,N-diisopropylchlorophosphoramidite. In principle, intermediate A8 would be alternatively accessible via N6-acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-{[(triisopropylsilyl)oxy]methyl} (TOM) adenosine which accumulates as regioisomeric by-product during the synthesis of standard 2′-O-TOM adenosine phosphoramidite (8). With this precursor, introduction of the selenium moiety proceeds in four steps along the lines described in reference (5), followed by cleavage of the DMT group to provide intermediate A8. Such a route, however, would involve ten steps in total starting from adenosine, and overall yields would significantly suffer from the nearly 1:1 ratio of 2′- and 3′-O-TOM regioisomers obtained in the third step of synthesis (8). Therefore, we decided to follow the strategy depicted in Scheme 1; our route provides phosphoramidite A10 in a 13% overall yield in nine steps with seven chromatographic purifications; in total, 0.5 g of A10 was prepared in the course of this study.

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 ...

Synthesis of 2′-methylseleno guanosine phosphoramidite

For the guanosine building block, we expanded a previously developed route to obtain N2-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-methylseleno-2′-deoxyguanosine phosphoramidite (6). Briefly, commercially available 9-[β-d-arabinofuranosyl]guanine was reacted with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl2) to protect the 3′- and 5′-hydroxyl groups simultaneously. Then, the 2′-hydroxyl group together with the exocyclic N2-amino group were acetylated, followed by protection of the guanine lactam moiety with a O6-(4-nitrophenyl)ethyl (NPE) group introduced under Mitsunobu conditions (6). Release of the 2′-OH, transformation into the desired 2′-methylseleno moiety, and simultaneous deprotection of the NPE and TIPDS groups yielded derivative G1 (Scheme 2). This compound was then transformed regioselectively into the 5′-O protected analog G2 by using benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (BzHCl). Conversion into the corresponding phosphoramidite G3 was achieved in good yields by reaction with methyl-N,N-diisopropylchlorophosphoramidite. Our route provides phosphoramidite G3 in a 4% overall yield in nine steps with six chromatographic purifications; in total, 0.2 g of G3 was prepared in the course of this study.

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, ...

Synthesis of 2′-methylseleno cytidine phosphoramidite

For synthesis of the Se-modified cytidine building block, we relied on a previously developed route to obtain N4-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-methylseleno-2′-deoxycytidine phosphoramidite (4). The strategy involved transformation of the nucleobase from the readily available 3′-O-tert-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-methylseleno-2′-deoxyuridine (4) into the corresponding cytidine derivative C1 (Scheme 3). After deprotection, compound C2 was reacted regioselectively with benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (BzHCl) to furnish the 5′-O protected analog C3. Conversion into the corresponding phosphoramidite C4 was achieved in good yields by reaction with methyl-N,N-diisopropylchlorophosphoramidite. Our route provides phosphoramidite C4 in a 10% overall yield in ten steps with eight chromatographic purifications; in total, 0.4 g of C4 was prepared in the course of this study. We note that an alternative pathway starting from cytidine via the N4-acetylated, 3′,5′-O TIPDS protected analog suffered from very poor yields during triflation reactions that were required for inversion of the configuration at C2′ and introduction of the methylseleno group. This pathway was therefore not further continued.

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, ...

Synthesis of 2′-methylseleno uridine phosphoramidite

Synthesis of the Se-modified uridine building block refers to a previously developed route to obtain 5′-O-(4,4′-dimethoxytrityl)-2′-methylseleno-2′-deoxyuridine phosphoramidite (4,27). Thereby, uridine was transformed into 2,2′-anhydro uridine; subsequent protection with DMT at the 5′-OH, followed by introduction of the 2′-methylseleno group with sodium methylselenide gave precursor U1 (Scheme 4). After release of the DMT group, compound U2 was transformed regioselectively into the 5′-O-benzhydryloxy-bis(trimethylsilyloxy)silyl protected analog U3 by using benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (BzHCl). Conversion into the corresponding phosphoramidite U4 was achieved in good yields by reaction with methyl-N,N-diisopropylchlorophosphoramidite. Our route provides phosphoramidite U4 in a 28% overall yield in six steps with four chromatographic purifications. Alternatively, intermediate U3 was accessed from 2,2′-anhydro uridine U5 by direct introduction of the BzH group at the 5′-OH to give U6, and subsequent introduction of the 2′-methylseleno group. However, the yields over both steps were only about 30%, therefore we preferred the route via the tritylated derivative U1; in total, 0.5 g of U4 was prepared in the course of this study.

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, ...

Chemical synthesis of Se-containing RNA using the ACE method

The preparation of RNA with 2′-methylseleno modified nucleosides relied on the 2′-O-ACE RNA synthesis method for strand assembly (9,19,23–25). We used an automated solid-phase synthesizer of the type Pharmacia Gene Assembler Plus where we bypassed the UV detection unit to avoid damage of the flow cell during treatment with HF/TEA solution. The solid-phase synthesis cycle was programmed according to a general description in reference (23). An optimized protocol is provided in the Supplementary Data available online. Therein, the cycle is substantially changed from standard ACE RNA synthesis by an additional step, treating the oligonucleotide chain on the solid support with threo-1,4-dimercapto-2,3-butanediol (DTT) after the capping–oxidation–capping operation. With these protocols, the novel 2′-methylseleno-modified phosphoramidites A10, C4, G3 and U4 were successfully incorporated into oligoribonucleotides (Figure 2, Table 1). The repeated exposure of the growing chain to DTT is a requirement for the reliable synthesis of RNAs (>20 nt) containing multiple Se-labels and was previously found to be advantageous for Se-containing RNAs prepared by the 2′-O-TOM approach (5,6). Deprotection of the methyl groups from the phosphate backbone, cleavage from the solid support and deprotection of acyl groups from 2′-methylseleno-modified RNAs were performed in the presence of DTT as well, added in millimolar amounts to the deprotection solutions of disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMF, of CH3NH2 in H2O and of the N,N,N′,N′-tetramethylethylenediamine (TEMED) acetate buffer (pH 3.8). After deprotection, DTT and other low molecular weight components were removed by size exclusion chromatography. RNAs were then purified by anion-exchange chromatography under strong denaturating conditions (6 M urea, 80°C; Figure 2). The molecular weights of the purified RNAs were confirmed by liquid chromatography (LC) electrospray-ionization (ESI) mass spectrometry (MS). Several oligoribonucleotides containing any of the four nucleosides with a 2′-methylseleno moiety were prepared. Their sequences S1S11 are listed in Table 1. Sequences S7S10 represent self-complementary 16 nt RNAs, that contain two isolated G–A mispairs. Crystal structures of the non-modified duplex and of a GSe containing analog have been recently solved in cooperation with A. Serganov to rationalize the influence of 2′-methylseleno groups on crystallization and crystal packing (6). Sequence S6 represents the minimal binding motif of the tandem zinc finger domain of protein TIS11d (28). This protein binds to the class II AU-rich element in the 3′-untranslated region of target mRNAs and promotes their deadenylation and degradation.

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 ...
Table 1.
RNAs with 2′-methylseleno modifications prepared by ACE RNA solid-phase synthesisa

Se-RNA preparation with 2′-O-TOM- versus 2′-O-ACE RNA chemistry

For reason of comparison, we synthesized the non-modified oligoribonucleotide 5′-rGCA GAG UUA AAU CUG U-3′ and the 2′-methylseleno-modified analog 5′-rGCASe GAG UUA AAU CUG U-3′ with both, the TOM and the ACE solid-phase synthesis methods. From the HPLC profiles of the crude deprotected oligos (Figure 3), it is obvious that the novel access for 2′-methylseleno-containing RNA is of very high quality and can compete with the established Se-TOM approach. We note that for the Se-ACE method, complete deprotection of acyl groups and of 2′-O protecting groups is much faster (10 min plus 30 min) when compared to the corresponding deprotection procedures (5 h plus overnight) required for Se-TOM chemistry. Taken together, our goal to transfer the Se-approach to the well-reputed ACE RNA synthesis has been satisfactorily achieved.

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 ASe-modified RNA (b); anion ...

RNA crystallography

For X-ray structure analysis, we consider RNA with covalent 2′-methylseleno groups best applicable for sizes up to about 80 nt. Se-RNA of this dimension can be readily obtained by solid-phase synthesis in combination with enzymatic ligation procedures (4,29,30). For RNAs up to about 35 nt, the Se-approach is in competition with 5-iodo and 5-bromo pyrimidine derivatization (31–37). We render the Se-approach superior since all four 2′-methylseleno nucleoside phosphoramidites are available, and therefore a great flexibility for adequate positioning within the RNA target is attained. This is important since the Se-labels should always be placed in double helical regions of a complex fold to minimize structural perturbance (5). In addition, 5-halogen pyrimidine derivatives are highly photo-reactive species (38–40). Inherent radiation damage of 5-halogen-modified nucleic acids during MAD data collection has been reported as a limitation (35). For medium-size RNA (up to 100 nt), the Se-approach competes with heavy metal ion derivatization (41–44). Search for a suitable heavy atom is a time-consuming process which requires soaking of the RNA crystals with dozens of compounds at various concentrations, therefore demanding many reasonably good crystals. This can be a serious obstacle, as had been encountered for the Diels–Alder ribozyme where the Se-approach finally delivered the key derivative to enable structure determination (16). Moreover, we have recently shown that 2′-methylseleno-modified model duplexes gave crystals in many more buffer conditions compared to their unmodified counterparts, and thus Se-modifications hold promise to actively support the crystallization process (6).

CONCLUSION

In the present study, we have shown that highly requested, 2′-methylseleno-functionalized RNA that represents a key derivative for RNA crystallography, is readily accessible by the ACE synthesis protocols elaborated here. The study further shows that the methodological transfer to ACE-based synthesis is not only feasible but highly satisfying since the quality of the modified Se-RNAs can well compete with the quality of TOM-made Se-RNA. We are convinced that this new access to 2′-methylseleno RNA will contribute to a fast dissemination of the Se-approach for RNA X-ray structure analysis.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

[Supplementary Data]

ACKNOWLEDGEMENTS

We thank the Austrian Science Fund FWF (P17864) and the bm:bwk (Gen-AU programme; project ‘Non-coding RNAs’ No. P7260-012-011) for funding. Dr Thomas Müller (University of Innsbruck) is acknowledged for HR ESI mass spectra. We are grateful to Dr Philipp Hattwiger (Alnylam Europe AG, Kulmbach) for a generous gift of ACE nucleoside building blocks. Funding to pay the Open Access publication charges for this article was provided by Austrian Science Fund (FWF).

Conflict of interest statement. None declared.

REFERENCES

1. Du Q, Carrasco N, Teplova M, Wilds CJ, Egli M, Huang Z. Internal derivatization of oligonucleotides with selenium for X-ray crystallography using MAD. J. Am. Chem. Soc. 2002;124:24–25. [PubMed]
2. Teplova M, Wilds CJ, Wawrzak Z, Tereshko V, Du Q, Carrasco N, Huang Z, Egli M. Covalent incorporation of selenium into oligonucleotides for X-ray crystal structure determination via MAD: proof of principle. Biochimie. 2002;84:849–858. [PubMed]
3. Pallan PS, Egli M. Selenium modification of nucleic acids: preparation of oligonucleotides with incorporated 2′-SeMe-uridine for crystallographic phasing of nucleic acid structures. Nat. Protocols. 2007;2:647–651. [PubMed]
4. Höbartner C, Micura R. Chemical synthesis of selenium-modified oligoribonucleotides and their enzymatic ligation leading to an U6 snRNA stem-loop segment. J. Am. Chem. Soc. 2004;126:1141–1149. [PubMed]
5. Höbartner C, Rieder R, Kreutz C, Puffer B, Lang K, Polonskaia A, Serganov A, Micura R. Syntheses of RNAs with up to 100 nucleotides containing site-specific 2′-methylseleno labels for use in X-ray crystallography. J. Am. Chem. Soc. 2005;127:12035–12045. [PubMed]
6. Moroder H, Kreutz C, Lang K, Serganov A, Micura R. Synthesis, oxidation behavior, crystallization and structure of 2′-methylseleno guanosine containing RNAs. J. Am. Chem. Soc. 2006;128:9909–9918. [PubMed]
7. Micura R, Höbartner C, Rieder R, Kreutz C, Puffer B, Lang K, Moroder H. Preparation of 2′-deoxy-2′-methylseleno-modified RNA. Curr. Prot. Nucleic Acid Chem. 2006;1:C1.15.1.–C1.15.34.
8. Pitsch S, Weiss PA, Jenny L, Stutz A, Wu X. Reliable chemical synthesis of oligoribonucleotides (RNA) with 2′-O-[(triisopropyl)oxy]methyl (2′-O-tom)-protected phosphoramidites. Helv. Chim. Acta. 2001;84:3773–3795.
9. Micura R. Small interfering RNAs and their chemical synthesis. Angew. Chem. Int. Ed. Engl. 2002;41:2265–2269. [PubMed]
10. Höbartner C, Kreutz C, Flecker E, Ottenschläger E, Pils W, Grubmayr K, Micura R. The synthesis of 2′-O-[(triisopropylsilyl)-oxy]-methyl (TOM) phosphoramidites of methylated ribonucleosides (m1G, m2G, m22G, m1I, m3U, m4C, m6A, m62A) for use in automated RNA solid-phase synthesis. Monatsh. Chem. 2003;134:851–873.
11. Höbartner C, Mittendorfer H, Breuker K, Micura R. Triggering of RNA secondary structures by a functionalized nucleobase. Angew. Chem. Int. Ed. Engl. 2004;43:3922–3925. [PubMed]
12. Kreutz C, Kählig H, Konrat R, Micura R. A general approach for the identification of site-specific RNA binders by 19F NMR spectroscopy: proof of concept. Angew. Chem. Int. Ed. Engl. 2006;45:3450–3453. [PubMed]
13. Höbartner C, Micura R. Bistable secondary structures of small RNAs and their structural probing by comparative imino proton NMR spectroscopy. J. Mol. Biol. 2003;45:3450–3453. [PubMed]
14. Serganov A, Yuan YR, Pikovskaya O, Polonskaia A, Malinina L, Phan AT, Höbartner C, Micura R, Breaker RR, et al. Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem. Biol. 2004;11:1729–1741. [PubMed]
15. Adams PL, Stahley MR, Kosek AB, Wang J, Strobel SA. Crystal structure of a self-splicing group I intron with both exons. Nature. 2004;430:45–50. [PubMed]
16. Serganov A, Keiper S, Malinina L, Tereshko V, Skripkin E, Höbartner C, Polonskaia A, Phan AT, Wombacher R, et al. Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation. Nat. Struct. Mol. Biol. 2005;12:218–224. [PubMed]
17. Ennifar E, Dumas P. Polymorphism of bulged-out residues in HIV-1 RNA DIS kissing complex and structure comparison with solution studies. J. Mol. Biol. 2006;356:771–782. [PubMed]
18. Ennifar E, Paillart JC, Bodlenner A, Walter P, Weibel JM, Aubertin AM, Pale P, Dumas P, Marquet R. Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell. Nucleic Acids Res. 2006;34:2328–2339. [PMC free article] [PubMed]
19. Scaringe SA, Wincott FA, Caruthers MH. Novel RNA synthesis method using 5′-O-silyl-2′-O-orthoester protecting groups. J. Am. Chem. Soc. 1998;120:11820–11821.
20. Piton N, Mu Y, Stock G, Schiemann O, Prisner T, Engels JW. Base-specific spin-labeling of RNA for structure determination. Nucleic Acids Res. 2007;35:3128–3143. [PMC free article] [PubMed]
21. Sumita M, Desaulniers J-P, Chang Y-C, Chui HM-P, Clos LII, Chow CS. Effects of nucleotide substitution and modification on the stability and structure of helix 69 from 28S rRNA. RNA. 2005;11:1420–1429. [PMC free article] [PubMed]
22. Desaulniers J-P, Chui HM-P, Chow CS. Solution conformations of two naturally occurring RNA nucleosides: 3-methyluridine and 3-methylpseudouridine. Bioorg. Med. Chem. 2005;13:6777–6781. [PubMed]
23. Scaringe SA. Advanced 5′-silyl-2′-orthoester approach to RNA oligonucleotide synthesis. Methods Enzymol. 2000;317:3–18. [PubMed]
24. Scaringe SA. RNA oligonucleotide synthesis via 5′-silyl-2′-orthoester chemistry. Methods. 2001;23:206–217. [PubMed]
25. Hartsel SA, Kitchen DE, Scaringe SA, Marshall WS. RNA oligonucleotide synthesis via 5′-silyl-2′-orthoester chemistry. Methods Mol. Biol. 2005;288:33–50. [PubMed]
26. Bain JD, Wacker DA, Kuo EE, Lyttle MH, Chamberlin AR. Preparation of chemically misacylated semisynthetic nonsense suppressor tRNAs employed in biosynthetic incorporation of non-natural residues into proteins. J. Org. Chem. 1991;56:4615–4625.
27. Salon Z, Chen G, Portilla Y, Germann MW, Huang Z. Synthesis of a 2′-Se-uridine phosphoramidite and its incorporation into oligonucleotides for structural study. Org. Lett. 2005;7:5645–5648. [PMC free article] [PubMed]
28. Hudson BP, Martinez-Yamout MA, Dyson HJ, Wright PE. Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d. Nat. Struct. Mol. Biol. 2004;11:257–264. [PubMed]
29. Rieder R, Lang K, Graber D, Micura R. Ligand-induced folding of the adenosine deaminase A-riboswitch and implications on riboswitch translational control. Chembiochem. 2007;8:896–902. [PubMed]
30. Lang K, Rieder R, Micura R. Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach. Nucleic Acids Res. 2007;35:5370–5378. [PMC free article] [PubMed]
31. Shui X, Peek ME, Lipscomb LA, Gao Q, Ogata C, Roques BP, Garbay-Jaureguiberry C, Wilkinson AP, Williams ID. Effects of cationic charge on three-dimensional structures of intercalative complexes: structure of a bis-intercalated DNA complex solved by MAD phasing. Curr. Med. Chem. 2000;7:59–71. [PubMed]
32. Deng J, Xiong Y, Sundaralingam M. X-ray analysis of an RNA tetraplex (UGGGGU)4 with divalent Sr2+ ions at subatomic resolution (0.61 Å) Proc. Natl Acad. Sci. USA. 2001;98:13665–13670. [PMC free article] [PubMed]
33. Wing R, Drew H, Takano T, Broka C, Tanaka S, Itakura K, Dickerson RE. Crystal structure analysis of a complete turn of B-DNA. Nature. 1980;287:755–758. [PubMed]
34. Zhang L, Doudna JA. Structural insights into group II intron catalysis and branch-site selection. Science. 2002;295:2084–2088. [PubMed]
35. Ennifar E, Carpentier P, Ferrer JL, Walter P, Dumas P. X-ray-induced debromination of nucleic acids at the Br K absorption edge and implications for MAD phasing. Acta Crystallogr. D Biol. Crystallogr. 2002;58:1262–1268. [PubMed]
36. Nowakowski J, Shim PJ, Stout D, Joyce GF. Alternative conformations of a nucleic acid four-way junction. J. Mol. Biol. 2000;300:93–102. [PubMed]
37. Correll CC, Freeborn B, Moore PB, Steitz TA. Metals, motifs, and recognition in the crystal structure of a 5S rRNA domain. Cell. 1997;91:705–712. [PubMed]
38. Gott JM, Wu H, Koch TH, Uhlenbeck OC. A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA. Biochemistry. 1991;30:6290–6295. [PubMed]
39. Xu Y, Sugiyama H. Highly efficient photochemical 2′-deoxyribonolactone formation at the diagonal loop of a 5-iodouracil-containing antiparallel G-quartet. J. Am. Chem. Soc. 2004;126:6274–6279. [PubMed]
40. Zeng Y, Wang Y. Facile formation of an intrastrand cross-link lesion between cytosine and guanine upon pyrex-filtered UV light irradiation of d((Br)CG) and duplex DNA containing 5-bromocytosine. J. Am. Chem. Soc. 2004;126:6552–6553. [PubMed]
41. Golden BL. Heavy atom derivatives of RNA. Methods Enzymol. 2000;317:124–132. [PubMed]
42. Ennifar E, Walter P, Dumas P. An efficient method for solving RNA structures: MAD phasing by replacing magnesium with zinc. Acta Cryst. D Biol. Crystallogr. 2001;57:330–332. [PubMed]
43. Francois B, Lescoute-Phillips A, Werner A, Masquida B. Preparation and handling of RNA crystals. In: Hartmann RK, Bindereif A, Schön A, Westhof E, editors. Handbook of RNA Biochemistry. I. Wiley-VCH; 2005. pp. 438–452.
44. Ke A, Doudna JA. Crystallization of RNA and RNA-protein complexes. Methods. 2004;34:408–414. [PubMed]

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