55.29, 37.86. IR (thin film): n = 3037, 2929, 2834, 1605, 1583,
1507, 1461, 1444, 1299, 1175, 1154, 1069, 1031, 980, 907, 840, 827,
791, 754, 726, 703, 682, 647 and 582 cm-1. HRMS: m/z [M+] calcd
for C40H34O4: 578.2452; found: 578.2449.
on a 1 micromole scale. A standard protocol for 2-cyanoethyl
phosphoramidites (0.1 M) was used, except that the coupling
was extended to 3 minutes. After the trityl-on synthesis, the resin
was incubated with conc. Aq. NH3 at 55 ◦C for 12 h and then
evaporated. The tritylated oligonucleotides were purified by C18
reverse phase HPLC (Merck LiChroCART 250 ¥ 4.6 mm, Puro-
spher STAR RP-18e) with 0.05 M aq. TEAA and MeCN as the
eluent (gradient: 15–80% MeCN in 20 min). The oligonucleotides
were then detritylated with 80% AcOH for 20 min, precipitated
with i-PrOH after addition of NaOAc, and again purified by
HPLC. In an alternative protocol, the tritylated oligonucleotides
were purified by Water Sep-Pak classic C18 cartridges. The trityl-
off oligonucleotides were purified by using a Waters Xterra column
(MS C18, 4.6 ¥ 50 mm) at 55 ◦C with 0.05 M aq. TEAA and MeCN
as the eluent. The identities of all oligonucleotides were confirmed
by MALDI–TOF MS.
Compound 5. To a solution of 3 (1.79 g, 3.09 mmol) and
N,N-diisopropylethylamine (3.09 mL, 16.4 mmol) in CH2Cl2
(52 mL) was added compound 4 (1.37mL, 6.18 mmol). After
2 h, the reaction mixture was poured into sat. aq. NaHCO3
(40 mL) and extracted with CH2Cl2 (3 ¥ 40 mL). The combined
organic extracts were dried over anhydrous Na2SO4, concentrated
in vacuo, and the resulting oil was purified by chromatography
over silica gel eluting with hexanes/ethyl acetate/triethylamine
(5:1:0.01), affording compound 5 as a light yellow foam (2.02 g,
84%). 31P NMR (121.5 MHz, CDCl3): d = 147.53, 147.16.
HRMS: m/z [M + H+] calcd for C49H52N2O5P1: 779.3608; found:
779.3602.
Compound 7. To a solution of 611 (300 mg, 1.33 mmol) in
THF (8 mL) at -78 ◦C was added slowly n-BuLi in hexane
(0.84 mL, 1.34 mmol). After stirring for 10 min at -78 ◦C,
BF3·Et2O (0.17 mL, 1.33 mmol) was added dropwise and the
stirring continued for another 5 min. The solution of compound
2 (498.6 mg, 1.33 mmol) in THF (5 mL) was then added slowly
Thermal denaturation. The melting studies were carried out in
1 cm path length quartz cells (total volume 325 mL; 200 mL sample
solutions were covered by mineral oil) on a Beckman 800 UV-VIS
spectrophotometer equipped with a thermo-programmer. Melting
curves were monitored at 260 nm with a heating rate of 1 ◦C/min.
Melting temperatures were calculated from the first derivatives of
the heating curves. Experiments were performed in duplicate and
mean values were taken.
◦
◦
at -78 C. Stirring was continued for another 15 min at -78 C,
then th◦e reaction was quenched with saturated NaHCO3 (50 mL)
at -78 C. The mixture was extracted with CH2Cl2 (3 ¥ 40 mL).
The combined organic layers were dried over anhydrous Na2SO4,
concentrated in vacuo, and the resulting red oil was purified
by chromatography over silica gel eluting with hexanes/ethyl
acetate/triethylamine (3:1:0.01), affording compound 7 as a light
Fluorescence measurements. The experiments were performed
in 96-well plates on a Molecular Devices SpectraMax M5 with
excitation at 315 nm and a cutoff filter at 325 nm. The ex-
periments with pyrene and pyrene acetylide-containing duplexes
were performed in 10 mM sodium phosphate, 100 mM NaCl,
pH 7.0, and the concentration of each GNA strand was 8 mM. For
copper sensor experiments, duplex D14 (2 mM of each strand)
was dissolved in 10 mM sodium phosphate, 50 mM NaCl,
ph 7.0.
1
yellow foam (0.55 g, 68%). H NMR (300 MHz, CDCl3): d =
8.44 (d, J = 9.1 Hz, 1H), 8.20 (m, 2H), 7.96–8.10 (m, 6H), 7.52
(m, 2H), 7.40 (m, 4H), 7.31 (m, 2H), 7.21 (m, 1H), 6.81 (d, J =
8.9 Hz, 4H), 4.19 (m, 1H), 3.67 (s, 6H), 3.48 (m, 2H), 2.98 (d, J =
6.3 Hz, 2H), 2.60 (d, J = 5.3 Hz, 1H). 13C NMR (75 MHz, CDCl3):
d = 158.46, 144.85, 135.84, 135.83, 131.78, 131.11, 130.95, 130.77,
130.05, 129.66, 128.08, 127.84, 127.78, 127.09, 126.79, 126.05,
125.42, 125.33, 125.29, 124.29, 124.26, 124.18, 118.05, 113.14,
91.53, 86.29, 81.76, 69.74, 66.15, 54.98, 25.34. IR (thin film):
3037, 2929, 2834, 1605, 1581, 1507, 1461, 1442, 1299, 1246, 1174,
1072, 1031, 905, 845, 826, 790, 754, 717, 701, 681, 613, 582 and
543 cm-1. HRMS: m/z [M+] calcd for C42H34O4: 602.2452; found:
602.2429.
Acknowledgements
We thank the Philipps-University Marburg for financial support
and Prof. Dr. Eric Meggers and Mark K. Schlegel for a generous
gift of the hydroxypyridone phosphoramidite and helpful discus-
sions.
References
Compound 8. To a solution of 7 (1.0 g, 1.66 mmol) and
N,N-diisopropylethylamine (1.66 mL, 8.83 mmol) in CH2Cl2
(27 mL) was added compound 4 (0.55 mL, 2.49 mmol). After
2 h, the reaction mixture was poured into sat. aq. NaHCO3
(30 mL) and extracted with CH2Cl2 (3 ¥ 30 mL). The combined
organic layers were evaporated and the residue was purified
by chromatography over silica gel eluting with hexanes/ethyl
acetate/triethylamine (3:1:0.01), affording 7 as a light yellow
foam (1.03 g, 77%). 31P NMR (161.9 MHz, CDCl3): d = 147.85,
147.80. HRMS: m/z [M + H+] calcd for C51H51N2O5P1: 803.3608;
found: 803.3570.
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This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 2297–2302 | 2301
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