5Ј-6-Locked, 1,10-Phenanthroline-Containing Nucleoside
using ultrapure flash silica gel from Silicycle (40–63 µm, 60 Å).
Acetonitrile, dichloromethane and pyridine were freshly distilled
from calcium hydride prior to use. Anhydrous triethylamine was
purchased from Sigma Aldrich. DMSO was dried by vacuum distil-
lation and stored over activated molecular sieves (3 Å) under argon
until used. Liquid ammonia was dried by condensation over so-
dium followed by re-condensation into a pressure-reaction vial. All
moisture- and oxygen-sensitive reactions were carried out in oven-
or flame-dried glassware under argon. The phosphate buffer used
contained 10 m phosphate, 100 m NaCl, 0.1 m EDTA, pH =
7. NMR spectra were recorded with a Bruker Avance 400 spec-
trometer. 1H NMR chemical shifts are reported in reference to un-
deuterated residual solvent [D2O (δ = 4.60 ppm), CDCl3 (δ =
7.26 ppm), [D6]DMSO (δ = 2.50 ppm)]. 13C NMR chemical shifts
are reported in reference to undeuterated residual solvent [CDCl3
(δ = 77.0 ppm), [D6]DMSO (δ = 39.43 ppm)]. 31P NMR chemical
shifts were reported relative to 85% H3PO4 as an external standard.
MALDI-ToF mass spectra were recorded with a Bruker Autoflex
III by using 2,5-dihydroxybenzoic acid as the matrix. UV/Vis
spectra were recorded with a Perkin–Elmer Lambda 25 UV/Vis
spectrometer. Fluorescence spectra were recorded with a SPEX
FluoroMax spectrometer. CD spectra were recorded with a Jasco
J-810 spectropolarimeter. Tm values were recorded with a Perkin–
Elmer Lambda 25 UV/Vis spectrometer equipped with a PTP-1
Peltier Temperature Programmer. DNA duplexes were formed by
the following annealing protocol: 90 °C for 2 min, 60 °C for 5 min,
50 °C for 5 min, 40 °C for 5 min, 22 °C for 15 min.
4.42 (s, 1 H, O-H), 5.20 [d, J(H,H) = 4.3 Hz, 1 H, H3Ј], 6.03 [d,
3J(H,H) = 6.5 Hz, 1 H, H1Ј], 6.26 (s, 1 H, H6), 7.88–7.94 (m, 2 H,
Ar-H), 9.14 [dd, 3J(H,H) = 4.4, 3J(H,H) = 1.8 Hz, 1 H, Ar-H],
9.20–9.23 (m, 3 H, Ar-H), 11.23 (s, 1 H, NH) ppm. 13C NMR
(400 MHz, [D6]DMSO, 25 °C): δ = 45.68, 71.36, 72.20, 84.53,
87.96, 90.06, 123.86, 124.03, 125.49, 126.00, 131.22, 132.32, 133.73,
134.86, 138.49, 144.26, 145.20, 146.99, 148.71, 150.58, 151.85 ppm.
Phosphoramidite 6: Compound 1 (100 mg, 0.24 mmol) and diiso-
propylammonium tetrazolide (100 mg, 0.58 mmol) were suspended
in acetonitrile (10 mL) and the solvent removed in vacuo. After
performing this procedure twice, the residue was kept under vac-
uum for 12 h. The solid was dissolved in anhydrous DMSO (8 mL)
and NC(CH2)2OP[N(iPr)2]2 (0.200 mL, 0.62 mmol) added. The re-
action mixture was stirred for 1 h, and another aliquot each of
DIPAT and NC(CH2)2OP[N(iPr)2]2 were added. After stirring for
another 1 h, CH2Cl2 (20 mL) was added and the organic layer
washed with satd. NaHCO3(aq) (3ϫ20 mL) and brine (20 mL).
The organic phase was dried with anhydrous Na2SO4 and the sol-
vent removed in vacuo. The residue was dissolved in a minimum
amount of dry CH2Cl2 and precipitated with petroleum ether. Pre-
cipitation was repeated twice and the residue dried under vacuum,
to yield 6 (61 mg, 41%). 31P NMR (400 MHz, CDCl3, 25 °C): δ =
148.5, 148.9 ppm.
Oligonucleotide Synthesis: Oligonucleotides were synthesized on a
1000 nmol scale by using an ASM 800 DNA/RNA synthesizer with
the manufacturer’s standard protocols. Modified nucleotides were
introduced by pausing the synthesiser program after completion of
the prior cycle, removing the column from the synthesiser and run-
ning 200 µL of standard activator solution and 200 µL of a 0.1
solution of 6 in CH3CN back and forth through the column for
15 min. Then the column was re-mounted on the synthesiser to
complete the cycle. The oligonucleotides were cleaved from
the solid support, deprotected by using standard conditions
(concd. aq. NH3, 55 °C, 12 h) and purified by 20% denaturing
polyacrylamide gel electrophoresis. The modified oligonucleotides
5-Bromo-2Ј-deoxycytidine (3): A solution of Br2 (3 mL, 58.0 mmol)
in CCl4 (125 mL) was added slowly to a suspension of 2Ј-deoxycyti-
dine (2) (10.0 g, 44.0 mmol) in pyridine (165 mL). The resulting
mixture was stirred at 22 °C for 12 h. The solvent was removed
under reduced pressure and Na2CO3 (1.8 g) in water (150 mL)
added. The water was removed and compound 3 isolated from the
residue by flash column chromatography (CH2Cl2/MeOH, 80:20).
Compound 3 was collected as a white solid (9.72 g, 72%). 1H NMR
(400 MHz, D2O, 25 °C): δ = 2.20–2.27 (m, 1, H2Ј), 2.36–2.42 (m, were characterized by MALDI-ToF mass spectrometry: 5Ј-
1 H, H2ЈЈ), 3.70 [dd, 3J(H,H) = 12.6, 3J(H,H) = 4.9 Hz, 1 H, H5Ј], d(1GC CTC GCA TCG TG) (X) calcd. 4487, found 4487; 5Ј-
3
3
3.79 [dd, J(H,H) = 12.6, J(H,H) = 3.5 Hz, 1 H, H5ЈЈ], 3.97–4.00 d(1AC CTC GCA TCG TG) (Y) calcd. 4471, found 4471.
(m, 1 H, H4Ј), 4.34–4.38 (m, 1 H, H3Ј), 6.13 [dd, 3J(H,H) = 6.4 Hz,
UV/Vis, Fluorescence and CD Studies: Samples of nucleoside 1 for
UV/Vis and fluorescence measurements were prepared by dissolv-
ing 1 in DMSO and diluting the samples with DMSO until the
absorbance at 377 nm was ca. 0.05. Fluorescence spectra were re-
1 H, H1Ј], 8.15 (s, 1 H, H6) ppm. 13C NMR (400 MHz, D2O,
25 °C): δ = 39.58, 60.85, 70.03, 86.48, 86.76, 88.74, 142.27, 156.02,
162.70 ppm.
corded with excitation at 365.5 nm and quantum yields determined
by using anthracene in EtOH as a standard (ΦF = 0.27). Samples
of oligonucleotides for UV/Vis, fluorescence and CD measurements
were prepared by dissolving 3 nmol of each strand in 100 µL of
phosphate buffer, annealing in the case of duplex samples and di-
luting to the desired final concentration with phosphate buffer.
5-Amino-2Ј-deoxycytidine (4): 5-Bromo-2Ј-deoxycytidine (3)
(3.00 g, 9.80 mmol) was stirred in a pressure-reaction vial in dried
NH3(l) at 70 °C for 5 d. The vial was subsequently cooled in liquid
N2, opened and the cooling bath removed to allow the ammonia
to evaporate. The product was purified by flash column chromatog-
raphy (CH2Cl2/MeOH/Et3N, 59:40:1) to give compound 4 as a
pale-yellow solid (0.90 g, 38%). 1H NMR (400 MHz, D2O, 25 °C):
δ = 2.19–2.26 (m, 1 H, H2Ј), 2.35–2.41 (m, 1 H, H2ЈЈ), 3.69 [dd,
Tm Measurements: Samples for Tm measurements were prepared by
annealing 3 nmol of each strand in 100 µL of phosphate buffer and
diluting to 1000 µL with the same buffer. Samples were heated at
3
3
3J(H,H) = 12.5, J(H,H) = 4.9 Hz, 1 H, H5Ј], 3.79 [dd, J(H,H) =
12.6, 3J(H,H) = 3.5 Hz, 1 H, H5ЈЈ], 3.96–4.00 (m, 1 H, H4Ј), 4.33– a rate of 1 °C/min.
4.37 (m, 1 H, H3Ј), 6.12 (dd, J1 = J2 = 6.4 Hz, 1 H, H1Ј), 8.13 (s,
Molecular Modelling: Model helix structures were generated by
1 H, H6) ppm.
using the program Hyperchem 8.0. A B-DNA helix was generated
with standard parameters and the model nucleoside built into the
structure. The modified base pair, along with the flanking base pair,
were then minimized by using the Amber force-field, while keeping
the rest of the helix frozen.
Nucleoside 1: 5-Amino-2Ј-deoxycytidine (4) (500 mg, 2.07 mmol)
and 1,10-phenanthroline-5,6-dione (5) (435 mg, 2.07 mmol) were
dissolved in 70% ethanol (8.25 mL) and heated in a closed vial at
95 °C for 12 h. The solution was allowed to cool to 22 °C and the
precipitate collected, washed with cold 70% ethanol and dried to
Supporting Information (see footnote on the first page of this arti-
cle): CD spectra for duplexes GI, GII, AI and AII. Fluorescence
1
give compound 1 as a pale-yellow solid (620 mg, 72%). H NMR
1
(400 MHz, [D6]DMSO, 25 °C): δ = 2.20–2.26 (m, 1 H, H2Ј), 2.52–
spectra for oligonucleotides X and Y and helixes GII and AII. H
2.58 (m, 1 H, H2ЈЈ), 4.09 (s, 2 H, H5Ј), 4.29–4.32 (m, 1 H, H4Ј), and 13C NMR spectra of nucleoside 1.
Eur. J. Org. Chem. 2010, 4713–4718
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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