A. Marx et al.
RT. The reaction mixture was diluted with EtOAc (100 mL) and washed
with aqueous NaHCO3 (80%) and with brine. The organic layer was
dried over anhydrous magnesium sulfate and concentrated under reduced
pressure. The crude compound was purified by means of flash chroma-
tography on silica gel (5–6% acetone in CH2Cl2) to give compound 5 in a
quantitative yield. Rf =0.45 (5% acetone in CH2Cl2; highly fluorescent
when visualized with UV light); 1H NMR (CDCl3, 250 MHz): d=8.10 (s,
1H; triazole H-3), 8.07 (s, 1H; triazole H-5), 8.06 (s, 1H; H-6); 6.20 (t,
J=5.8 Hz, 1H; H-1’), 4.40–4.15 (m, 5H; H-5’, H-4’, H-3’), 2.80–2.53 (m,
5H; CH2CH2-Lev, H-2’), 2.44 (s, 3H; CH3-CO), 2.21–2.11 (m, 4H; CH3-
5, H-2’), 0.85 (s, 9H; (CH3)3C-Si), 0.04 (s, 3H; CH3-Si), 0.03 ppm (s, 3H;
CH3-Si); 13C NMR (CDCl3, 62.5 MHz): d=206.1, 172.3, 158.1, 153.7,
153.3, 146.3, 105.4, 96.0, 87.6, 85.4, 70.8, 62.9, 41.8, 37.7, 29.6, 27.7, 25.6,
17.8, 17.2, À4.8, À5.1 ppm; ESIMS: m/z: 528.2 [M+Na]+.
duplex, which eventually led to the formation of circular
bDNA.
Conclusion
In conclusion, we have demonstrated the synthesis of novel
asymmetric bDNA. Herein, the strategy depicted is based
on the employment of a nucleotide analogue that functions
as a branching point. By changing the protecting-group
strategy in comparison with known branching nucleotides
we were able to synthesize novel noncovalent and covalent
bDNA constructs that might be useful for DNA-based nano-
biotechnology.
Synthesis of 5’-O-levulinyl-3’-O-tert-butyldimethylsilyl-N4-(6-hydroxyhex-
yl)-5-methyl-2’-deoxycytidine (6): Compound
5
(2.64 g, 5.2 mmol,
1.0 equiv) in CH3CN (40 mL) was added dropwise at RT to a solution of
6-aminohexanol (5.5 g, 47 mmol, 9.0 equiv) in CH3CN (200 mL) and the
reaction mixture was stirred for 3 h. The reaction mixture was then dilut-
ed with EtOAc (300 mL) and washed with aqueous NaHCO3 (80%) and
with brine. The organic layer was dried over anhydrous magnesium sul-
fate and the solvent was removed under reduced pressure. The crude
compound was purified by means of flash column chromatography on
silica gel (4% MeOH in CH2Cl2) to yield compound 6 (2.56 g, 4.6 mmol,
88%). Rf =0.33 (4% methanol in CH2Cl2); 1H NMR (CDCl3, 250 MHz):
d=7.3 (s, 1H; H-6), 6.29 (t, J=6.3 Hz, 1H; H-1’), 5.1 (brs, 1H; H-
NCH2), 4.34–4.20 (m, 3H; H-5’, H-3’), 4.00 (q, J=4.1 Hz, 1H; H-4’), 3.61
(t, J=6.3 Hz, 2H; CH2-OH), 3.5 (brq, J=4.2 Hz, 2H; CH2-NH), 2.78 (t,
J=6.3 Hz, 2H; CH2-Lev), 2.61–2.55 (m, 2H; CH2-Lev), 2.41–2.35 (ddd,
J=4.8, 6.3, 11.3 Hz, 1H; H-2’), 2.18 (s, 3H; CH3-CO), 2.12–2.01 (m, 1H;
H-2’), 1.91 (s, 3H; CH3-5), 1.65–1.35 (m, 8H; CH2-N4-alkyl), 0.85 (s, 9H;
(CH3)3C-Si), 0.04 (s, 3H; CH3-Si), 0.03 ppm (s, 3H; CH3-Si); 13C NMR
(CDCl3, 62.5 MHz): d=206.3, 172.4, 162.9, 156.0, 136.4, 101.6, 85.8, 84.1,
71.4, 63.6, 62.6, 41.4, 40.8, 37.7, 32.4, 29.7, 29.1, 27.7, 26.4, 25.6, 25.2, 17.9,
13.2, À4.7, À5.0 ppm; ESIMS: m/z: 554.3 [M+1]+, 576.3 [M+Na]+; ele-
mental analysis calcd (%) for C27H47N3O7Si: C 58.56, H 8.55, N 7.59;
found: C 59.13, H 7.99, N 6.86.
Experimental Section
General: All synthetic reactions were performed under an inert atmos-
phere. Dry solvents were purchased from Fluka and were stored over
molecular sieves and used without further purification. Elemental analy-
sis was carried out by the microanalysis facility of the University of Kon-
stanz. NMR spectra were recorded by using Bruker AC 250 Cryospec
(1H: 250 MHz), Jeol JNA-LA-400 (1H: 400 MHz), and Bruker DRX 600
(1H: 600 MHz) spectrometers. Chemical shifts are given in parts per mil-
lion and tetramethyl silane was used as the external standard. Electro-
spray ionization ion trap (ESI-IT) mass spectra were recorded by using a
Bruker Daltonics esquire 3000+ instrument in positive or negative mode
with a flow rate of 3 mLminÀ1. DNA oligonucleotide synthesis was car-
ried out by using an Applied-Biosystems 392 DNA/RNA synthesizer. Re-
verse-phase HPLC was performed by using a Prominence HPLC (Shi-
madzu) instrument equipped with a Nucleosil-100-5 C18 column (250
4 mm, Macherey-Nagel). A binary gradient system (triethyl ammonium
acetate buffer (0.1m, pH 7.0)/CH3CN, 258C) was used.
Synthesis of 5’-O-levulinyl-3’-O-tert-butyldimethylsilyl-N4-[O-(4,4’-dime-
thoxytrityloxy)hexyl]-5-methyl-2’-deoxycytidine (7): Compound 6 (2.4 g,
4.33 mmol) was co-evaporated twice with dry pyridine (20 mL) and then
dissolved in dry pyridine (45 mL). 4,4’-Dimethoxytritylchloride (1.76 g,
5.2 mmol) and a catalytic amount of DMAP (0.05 g) were added to the
solution, which was stirred overnight at RT. The reaction mixture was
then quenched with dry methanol (0.9 mL, 26 mmol) before it was dilut-
ed with EtOAc (100 mL) and washed with aqueous NaHCO3 (80%) and
with brine. The organic layer was then dried over anhydrous magnesium
sulfate, the solvent was evaporated under reduced pressure, and the
crude compound was purified by means of flash column chromatography
on silica gel (75–80% EtOAc in petroleum ether, 1% NEt3) to give com-
pound 7 (3.02 g, 3.5 mmol, 80%). Rf =0.25 (80% EtOAc in petroleum
ether); 1H NMR (CDCl3, 250 MHz): d=7.43–7.17 (m, 10H; H-Ar, H-6),
6.79 (d, J=10.0 Hz, 4H; H-Ar), 6.29 (t, J=6.3 Hz, 1H; H-1’), 4.83 (t, J=
4.9 Hz, 1H; H-N4), 4.35–4.20 (m, 3H; H-5’, H-3’), 4.03–3.98 (m, 1H; H-
4’), 3.77 (s, 6H; CH3-O), 3.48 (q, J=5.9 Hz, 2H; CH2-NH), 3.01 (t, J=
6.5 Hz, 2H; CH2-ODMTr), 2.77 (t, J=6.3 Hz, 2H; CH2-Lev), 2.63–2.56
(m, 2H; CH2-Lev), 2.42 (ddd, J=5.0, 6.5, 11.3 Hz, 1H; H-2’), 2.18 (s, 3H;
CH3-CO), 2.12–2.02 (m, 1H; H-2’), 1.90 (s, 3H; CH3-5), 1.59–1.30 (m,
8H; CH2-N4-alkyl), 0.85 (s, 9H; (CH3)3C-Si), 0.04 (s, 3H; CH3-Si),
0.03 ppm (s, 3H, CH3-Si); 13C NMR (CDCl3, 150 MHz): d=209.1, 174.1,
164.7, 159.9, 158.5, 146.9, 137.8, 137.7, 131.1, 129.3, 128.6, 127.6, 113.9,
113.4, 105.2, 87.2, 87.0, 86.3, 73.6, 64.7, 64.3, 55.7, 42.0, 38.6, 31.0, 30.1,
29.6, 28.8, 27.8, 27.3, 26.2, 18.8, 13.4, À4.6, À4.8 ppm; ESIMS: m/z: 856.1
[M]+, 878.4 [M+Na]+; elemental analysis calcd (%) for C48H65N3O9Si: C
67.34, H 7.65, N 4.91; found: C 67.69, H 7.76, N 4.78.
5’-O-Levulinyl-3’-O-tert-butyldimethylsilylthymidine (4): A solution of 3’-
O-(tert-butyldimethylsilyl)thymidine (3) (5.53 g, 15.5 mmol, 1.0 equiv) in
dioxane (100 mL) was added to a suspension of 2-chloro-1-methylpyridi-
nium iodide (7.9 g, 31 mmol, 2.0 equiv) in CH3CN (100 mL). Subsequent-
ly, a solution of levulinic acid (7.2 g, 62 mmol, 4.0 equiv) and DABCO
(8.7 g, 77 mmol, 5.0 equiv) in dioxane (100 mL) was added dropwise to
the reaction mixture over a period of 1 h at RT. Stirring was continued
until the starting material had disappeared (7 h), as determined by TLC,
before the solvent was evaporated under reduced pressure. EtOAc was
added to the resulting residue, and the solution was washed with aqueous
NaHCO3 (80%) and with brine. The organic layer was dried over anhy-
drous magnesium sulfate and concentrated under reduced pressure. The
crude compound was purified by means of flash column chromatography
on silica gel (6% acetone in CH2Cl2) to give compound
4 (6.5 g,
13.9 mmol, 90%). Rf =0.25 (6% acetone in CH2Cl2); 1H NMR (CDCl3,
250 MHz): d=9.35 (s, 1H; H-N), 7.27 (s, 1H; H-6), 6.21 (t, J=6.5 Hz,
1H; H-1’), 4.33–4.12 (m, 3H; H-3’, H-5’), 3.97 (q, J=3.8 Hz, 1H; H-4’),
2.23 (m, 2H; CH2-Lev), 2.55–2.50 (m, 2H; CH2-Lev), 2.23 (ddd, J=3.8,
6.3, 10.3 Hz, 1H; H-2’), 2.12 (s, 3H; CH3-CO), 2.07–1.95 (m, 1H; H-2’),
1.86 (s, 3H; CH3-5), 0.8 (s, 9H; (CH3)3C-Si), 0.003 (s, 3H; CH3-Si),
À0.003 ppm (s, 3H; CH3-Si); 13C NMR (CDCl3, 62.5 MHz): d=206.2,
172.3, 163.8, 150.3, 135.2, 111.0, 84.9, 84.6, 71.6, 66.9, 63.3, 40.7, 37.7, 29.6,
27.7, 25.6, 17.8, 12.5, À4.8, À5.03 ppm; ESIMS: m/z: 477.2 [M+Na]+.
Synthesis of 4-(1,2,4-triazol-1-yl)-5’-O-levulinyl-3’-O-tert-butyldimethylsil-
yl-2’-deoxythymidine (5): POCl3 (4.12 g, 27 mmol, 3.5 equiv) was added
dropwise at 08C with vigorous stirring to a suspension of 1,2,4-triazole
(8.03 g, 116 mmol, 15.0 equiv) in CH3CN (135 mL). NEt3 (18 mL,
140 mmol, 18.0 equiv) was then added dropwise over a period of 15 min
and was stirred for an additional 30 min at 08C. Compound 4 (3.51 g,
7.7 mmol, 1.0 equiv) in CH3CN (35 mL) was added dropwise over a
period of 30 min to the resulting slurry, which was then stirred for 1 h at
Synthesis of 5’-O-levulinyl-N4-[O-(4,4’-dimethoxytrityl-6-oxy)hexyl]-5-
methyl-2’-deoxycytidine (8): Acetic acid (0.26 mL, 4.5 mmol) was added
to a solution of compound 7 (1.95 g, 2.3 mmol) in dry THF (30 mL)
before TBAF (1m in THF, 4.6 mL, 4.5 mmol) was added dropwise with
3562
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 3558 – 3564