Alternative Method for G-to-G Cross-Link Formation
A R T I C L E S
organic extracts were dried over sodium sulfate. After filtration, the
solvent was removed in vacuo, and a yellow oil was obtained. Flash
chromatography with 3% ethyl acetate/hexane as the eluent afforded
392 mg of 8 as a white solid (61.7%). 1H NMR (500 MHz,
d6-DMSO): δ 8.59 (s, 1H), 7.52 (d, J ) 6.5 Hz, 2H), 7.43-7.33 (m,
3H), 5.92 (d, J ) 5.5 Hz, 1H), 5.59 (d, J ) 2.5 Hz, 2H), 4.88 (t, J )
5.0 Hz, 1H), 4.37 (t, J ) 3.5 Hz, 1H), 4.01-4.00 (m, 2H), 3.73 (dd, J
) 10.5, 7.5 Hz, 1H), 0.91 (s, 9H), 0.86 (s, 9H), 0.74 (s, 9H), 0.13 (s,
3H), 0.10 (s, 3H), 0.06 (d, J ) 3.0 Hz, 6H), -0.08 (s, 3H), -0.29 (s,
3H). 13C NMR (500 MHz, d6-DMSO): δ 159.8, 152.7, 143.2, 142.1,
135.4, 128.8, 128.5, 120.9, 88.1, 85.0, 73.9, 71.6, 69.1, 61.9, 25.74,
25.68, 25.4, 18.0, 17.7, 17.5, -4.6, -4.9, -5.4, -5.5, -5.6. ESI-
HRMS: m/e 801.28435 (M + Na+) (calcd 801.286881).
153.7, 153.4, 153.3, 140.7, 139.5, 136.23, 136.20, 128.7, 128.4, 128.3,
128.1, 128.0, 117.5, 117.2, 87.8, 85.4, 76.3, 73.0, 72.1, 71.7, 70.6, 68.41,
68.36, 62.6, 62.4, 26.1, 25.9, 25.7, 18.6, 18.3, 18.1, 17.9, -4.4, -4.6,
-4.7, -5.0, -5.1, -5.38, -5.40. ESI-HRMS: m/e 1150.58255 (M
+ Na+) (calcd 1150.580337).
2-N-[2-(2′-O-tert-Butyldimethylethylsilylacycloinosyl)]-5′-O,3′-
O,2′-O-tris (tert-butyldimethylethylsilyl)guanosine, 12. Compound
11 (427.3 mg, 0.378 mmol) was dissolved in 9.4 mL of THF, and 0.1
M tetrabutylammonium fluoride (TBAF) in THF solution (1.882 mL,
1.89 mmol) was added. The reaction solution was stirred for 1 h at 25
°C, then concentrated in vacuo, and the resulting residue was purified
by flash chromatography (silica gel, 8% methanol/methene chloride)
1
to give 30.7 mg product 8 (57.2%) as a white solid. H NMR (500
2′-O-(tert-Butyldimethylethylsilyl)acycloguanosine, 9. Acyclogua-
nosine (200 mg, 0.889 mmol) was added to a mixture of TBDMSCl
(294.8 mg, 1.956 mmol) and imidazole (266.19 mg, 3.91 mmol) in 4.5
mL of DMF. The resulting solution was stirred at room temperature
overnight. The solvent was removed, and the residue was purified by
flash chromatography with 7-10% MeOH/CH2Cl2 as eluent to give
332.4 mg of white product. Recrystallization from MeOH/H2O gave
MHz, d6-DMSO): δ 8.07 (s, 1H), 8.02 (s, 1H), 7.43 (d, J ) 7.5 Hz,
4H), 7.28-7.22 (m, J ) 6.1 Hz, 6H), 5.93 (d, J ) 6.5 Hz, 1H), 5.68
(d, J ) 3.0 Hz, 2H), 5.61 (d, J ) 3.5 Hz, 2H), 5.58 (d, J ) 3.0 Hz,
2H), 4.63 (t, J ) 5.2 Hz, 2H), 4.52 (s, 1H), 4.27 (q, J ) 2.1 Hz, 1H),
4.12 (d, J ) 2.5 Hz, 1H), 3.70 (d, J ) 2.8 Hz, 2H), 3.68 (d, J ) 3.0
Hz, 1H), 3.65 (s, 1H), 3.60 (d, J ) 3.0 Hz, 1H), 3.5 (s, 4H). 13C NMR
(500 MHz, d6-DMSO): δ 159.5, 153.0, 152.4, 140.9, 139.7, 135.6,
128.0, 127.9, 127.7, 127.6, 86.4, 77.5, 77.3, 77.0, 74.6, 72.6, 71.0, 70.4,
67.8, 60.4. ESI-HRMS: m/e 694.23341 (M + Na+) (calcd 694.234429).
2-N-(2-Acycloinosyl)guanosine, rG-to-aG. Ten percent palladium
on activated carbon catalyst (104.5 mg) was added to a solution of 12
(321.4 mg, 0.479 mmol) in methanol (104.5 mL). The system was
flushed with hydrogen gas three times before the reaction mixture was
hydrogenated for 24 h at 60 psi with stirring. After completion of the
reaction, the catalyst was removed by filtration through a pad of Celite.
The filtrate was concentrated in vacuo; the crude product was purified
by HPLC using analytical method A, and then desalted by HPLC
employing analytical method B to yield 110.2 mg (87%) of rG-to-aG
as a white solid. 1H NMR (500 MHz, d6-DMSO): δ 8.15 (s, 1H), 8.09
(s, 1H), 5.78 (d, J ) 5.5 Hz, 1H), 5.52 (s, 2H), 5.05 (s, 1H), 4.70 (s,
1H), 4.50 (s, 1H), 3.92 (q, J ) 4.5 Hz, 1H), 3.72 (d, J ) 10.0 Hz, 1H),
3.59 (t, J ) 4.5 Hz, 2H), 3.55 (dd, J ) 11.5, 3.5 Hz, 1H), 3.50 (s, 2H),
3.07 (q, J ) 7.0 Hz, 1H). 13C NMR (500 MHz, d6-DMSO): δ 149.0,
139.4, 137.8, 119.5, 119.0, 85.4, 73.7, 72.8, 71.0, 70.1, 61.4, 60.0. ESI-
HRMS: m/e 514.14206 (M + Na+) (calcd 514.140529).
rG-to-aG Formation by Guanosine Addition to Cyanoamine 4a.
1-[(2-Hydroxyethoxy)methyl]-5-cyanoamine-4-imidazolecarboxam-
ide (4a, 15 mg, 0.06 mmol) and guanosine (11 mg, 0.04 mmol) were
added to 250 µL of DMSO, and the solution was stirred at 80 °C for
2 h. Then, 10 µL of the resulting light-yellow solution were diluted
with 250 µL of DMSO for HPLC and LC-MS analyses using the
analytical system 3 (vide supra) and an injection volume of 10 µL.
The HPLC chromatogram of the reaction mixture showed the same
peak that was observed for synthetic rG-to-aG, and the retention time
was 16.0 min. To tune the mass spectrometer to optimal sensitivity,
the solution of synthetic rG-to-aG was diluted to 0.02 mM, and at
this concentration, the peaks of the synthetic cross-link and of the cross-
link formed by the rG addition also agreed with regard to their intensity.
1
263 mg of analytically pure product 9 in 87% yield. H NMR (500
MHz, d6-DMSO): δ 10.6 (s, 1H), 7.8 (s, 1H), 6.46 (s, 2H), 5.3 (s,
2H), 3.6 (t, J ) 4.5 Hz, 2H), 3.5 (t, J ) 5.0 Hz, 2H), 0.81 (s, 9H),
-0.023 (s, 6H). 13C NMR (500 MHz, d6-DMSO): δ 156.8, 153.8,
151.4, 137.6, 116.5, 72.1, 70.1, 61.8, 25.7, 17.9, -5.3. ESI-HRMS:
m/e 362.16214 (M + Na+) (calcd 362.161885).
2′-O-tert-Butyldimethylethylsilyl-6-O-benzylacycloguanosine, 10.
Compound 10 was prepared in analogy to the preparation of 7,
employing 9 (172 mg, 0.505 mmol), triphenylphosphine (266 mg,
1.0147 mmol), dry dioxane (8.7 mL), benzyl alcohol (105 µL, 1.0147
mmol), and diethylazodicarboxylate (464 µL, 1.0147 mmol). The crude
product was purified by preparative TLC (1.5:4 hexane/ethyl acetate)
1
to give 124.5 mg of white solid 10 (60% yield). H NMR (500 MHz,
d6-DMSO): δ 7.71 (s, 1H), 7.48 (d, J ) 7.1 Hz, 2H), 7.33-7.24 (m,
3H), 5.56 (s, 2H), 5.49 (s, 2H), 4.92 (br, 2H), 3.71 (t, J ) 4.7 Hz, 2H),
3.57 (t, J ) 5.3 Hz, 2H), 0.86 (s, 9H), 0.027 (s, 6H). 13C NMR (500
MHz, d6-DMSO): δ 161.2, 159.5, 154.3, 139.4, 136.4, 129.3, 128.4,
128.2, 128.0, 115.5, 72.9, 70.8, 68.1, 62.5, 25.9, 18.3, -5.3. ESI-
HRMS: m/e 452.20677 (M + Na+) (calcd 452.208835).
2-N-[2-(2′-O-tert-Butyldimethylethylsilyl-6-O-benzylacycloinosyl)]-
5′-O,3′-O,2′-O-tris(tert-butyldimethylethylsilyl)-6-O-benzylgua-
nosine, 11. In a dry flask, amine 10 (226.5 mg, 0.528 mmol), cesium
carbonate (240.85 mg, 0.739 mmol, 1.4 equiv), palladium acetate (11.85
mg, 0.0528 mmol, 0.1 equiv), BINAP (49.32 mg, 0.0792 mmol, 0.15
equiv), bromide 8 (494.2 mg, 0.634 mmol, 1.2 equiv), and toluene (4.6
mL) were mixed under argon at room temperature for 30 min and then
heated to 90 °C for 16 h. After the completion of the reaction, the
reaction solution was diluted with ethyl acetate. The precipitate was
removed by centrifugation, and the supernatant liquid was concentrated
in vacuo. The residue was purified by flash chromatography (silica
gel, 20-22% ethyl acetate/hexane) and afforded product 11 as a light-
1
yellow oil (427.3 mg, 71.8% yield). H NMR (500 MHz, CDCl3): δ
Acknowledgment. We are grateful for support by NIH Grant
GM61027.
7.88 (s, 1H), 7.78 (s, 1H), 7.50 (m, 4H), 7.33-7.27 (m, 6H), 6.05 (d,
J ) 5.0 Hz, 1H), 5.77 (s, 2H), 5.68 (dd, J ) 47, 12.5 Hz, 3H), 5.57
(dd, J ) 13, 10.5 Hz, 2H), 4.52 (t, J ) 4.5 Hz, 1H), 4.29 (t, J ) 3.5
Hz, 1H), 4.08 (q, J ) 2.5 Hz, 1H), 3.97 (dd, J ) 11.5, 4.0 Hz, 1H),
3.77 (dd, J ) 11.5, 2.5 Hz, 1H), 3.62 (t, J ) 4.5 Hz, 2H), 3.52 (t, J )
5.5 Hz, 2H), 0.92 (d, J ) 3.0 Hz, 18H), 0.82 (s, 9H), 0.78 (s, 9H),
0.11 (d, J ) 3.5 Hz, 6H), 0.01 (s, 6H), -0.02 (s, 6H), -0.05 (s, 3H),
-0.23 (s, 3H). 13C NMR (500 MHz, d6-DMSO): δ 160.7, 160.6, 153.8,
Supporting Information Available: 1H and 13C NMR spectra
of 7-12 and rG-to-aG, and the results of the computational
study. This material is available free of charge via the Internet
JA045108J
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J. AM. CHEM. SOC. VOL. 127, NO. 3, 2005 887