Nucleic Acids Research, 2012, Vol. 40, No. 8 3735
chromatography (10% EtOAc in petroleum ether) to yield
compounds 3a–c as white solids. 3a: yield 64%. H NMR
Compounds 6a–c. A solution of compounds 5a–c
(0.2 mmol) in EtOH (5 ml) was treated with CsF
(0.303 g, 2.0 mmol) and stirred at 85ꢀC for 3 days. The
solvent was evaporated and the crude product was
purified by flash column chromatography [4% MeOH in
CH2Cl2, containing 1% NH3 (aq)] to give cyclized com-
1
(CDCl3): d 7.76 (s, 1H, CH), 7.20 (s, 2H, 2Ar-CH), 4.39–
4.19 (m, 1H, CH), 3.46 (s, 3H, CH3), 2.99–2.83 (m, 1H),
1.29 (d, J = 6.8 Hz, 12H, 4CH3), 1.25 (d, J = 6.9 Hz, 6H,
CH3). 13C NMR (CDCl3): d 163.06, 154.90, 151.68,
150.07, 130.41, 124.27, 86.29, 38.67, 34.42, 29.80, 24.65,
23.59. HR-APCI-MS: m/z 493.0737 [M+Na]+ calculated
1
pounds 6a–c as yellowish solids. 6a: yield 40%. H NMR
(CDCl3): d 7.42 (s, 1H, CH), 6.74 (s, 1H, Ar-CH), 6.44
(s, 1H, Ar-CH), 3.33 (s, 3H, CH3), 1.43 (s, 6H, 2 CH3),
1.39 (s, 6H, 2CH3). 13C NMR (CDCl3): d 154.82, 154.73,
144.87, 144.42, 142.18, 128.02, 126.30, 125.90, 111.84,
108.16, 62.89, 62.77, 37.40, 31.95, 31.87. HR-APCI-MS:
m/z 313.1658 [M+H]+ calculated for C17H20N4O2
312.1586. 6b: yield 45%. 1H NMR (CDCl3): d 7.54
(s, 1H, CH), 6.76 (s, 1H, Ar-CH), 6.42 (s, 1H, Ar-CH),
3.74 (q, J = 7.2 Hz, 2H, CH2), 1.43 (s, 6H, 2CH3), 1.39
(s, 6H, 2CH3), 1.31 (t, J = 7.2 Hz, 3H, CH3). 13C NMR
(CDCl3): d 154.61, 154.08, 144.53, 144.22, 142.00, 127.94,
126.13, 124.63, 111.79, 107.85, 62.74, 62.62, 44.82, 31.78,
31.70, 14.24. HR-APCI-MS: m/z 327.1791 [M+H]+
1
for C20H27BrN2O4S 470.0875. 3b: yield 44%. H NMR
(CDCl3): d 7.74 (s, 1H, CH), 7.20 (s, 2H, 2Ar-CH),
4.38–4.21 (m, 2H, 2CH), 3.85 (q, J = 7.2 Hz, 2H, CH2),
2.90 (dd, J = 13.8, 6.9 Hz, 1H, CH), 1.34 (t, J = 7.2 Hz,
3H, CH3), 1.30 (d, J = 6.8 Hz, 12H, 4CH3), 1.25
(d, J = 6.9 Hz, 6H, 2CH3). 13C NMR (CDCl3): d 162.83,
154.88, 153.03, 151.67, 149.06, 130.44, 124.27, 86.39,
46.66, 34.42, 29.80, 24.66, 23.59, 14.32. HR-APCI-MS:
m/z 507.0904 [M+Na]+ calculated for C21H29BrN2O4S
1
484.1031. 3c: yield 48%. H NMR (CDCl3): d 7.72 (s,
1H, CH), 7.20 (s, 2H, 2Ar-CH), 4.38–4.19 (m, 2H,
2CH), 3.80–3.68 (m, 2H, CH2), 2.90 (dt, J = 13.8,
6.9 Hz, 1H, CH), 1.75 (dd, J = 14.9, 7.4 Hz, 2H, CH2),
1.29 (d, J = 6.7 Hz, 12H, 4CH3), 1.25 (d, J = 6.9 Hz,
6H, 2CH3), 0.93 (t, J = 7.4 Hz, 3H, CH3). 13C NMR
1
calculated for C18H22N4O2 326.1743. 6c: yield 44%. H
NMR (CDCl3): d 7.49 (s, 1H, CH), 6.73 (s, 1H, Ar-CH),
6.43 (s, 1H, Ar-CH), 3.69 – 3.62 (m, 2H, CH2), 1.74 (dd,
J = 14.5, 7.3 Hz, 2H, CH2), 1.42 (s, 6H, 2CH3), 1.38
(s, 6H, 2CH3), 0.97 (t, J = 7.4 Hz, 3H, CH3). 13C NMR
(CDCl3): d 154.50, 154.21, 144.60, 144.26, 142.02, 127.73,
126.20, 125.10, 111.83, 107.87, 62.71, 62.59, 51.33, 31.78,
31.72, 22.20, 11.02. HR-APCI-MS: m/z 341.1973 [M+H]+
calculated for C19H24N4O2 340.1899.
(CDCl3):
d 162.79, 154.88, 153.12, 151.68, 149.51,
130.41, 124.27, 86.14, 53.17, 34.41, 29.79, 24.66, 23.58,
22.18, 11.05. HR-APCI-MS: m/z 521.1047 [M+Na]+
calculated for C22H31BrN2O4S 498.1188.
Compounds 5a–c. A solution of compounds 3a–c
(0.50 mmol) in CH2Cl2 (10 ml) was treated sequentially
with amino phenol 4 (27) (0.124 g, 0.6 mmol) followed
by Et3N (0.14 ml, 1.0 mmol). The resulting reaction
mixture was stirred at 25ꢀC for 48 h in the dark, the
solvent removed in vacuo and the oily residue was
triturated with CH3CN (2 ml). The precipitated product
was filtered and dried in vacuo and used for the next
step without further purification. 5a: yield 80%. 1H
NMR (95 : 5 CDCl3 : CD3OD): d 8.47 (s, 1H, CH), 7.96
(s, 1H, NH), 7.62 (s, 1H, Ar-CH), 6.58 (s, 1H, Ar-CH),
3.37 (s, 3H, CH3), 1.69 (s, 6H, 2CH3), 1.61 (s, 5H, 2CH3).
13C NMR (95 : 5 CDCl3 : CD3OD): d 148.04, 145.98,
129.67, 113.31, 106.54, 96.60, 68.61, 67.80, 37.90, 29.59,
28.43. HR-APCI-MS: m/z 393.0896 [M+H]+ calculated
for C17H21BrN4O2 392.0848. 5 b: yield 75%. 1H NMR
(95 : 5 CDCl3 : CD3OD): d 7.97 (s, 1H, CH), 7.56 (s, 1H,
Ar-CH), 6.59 (s, 1H, Ar-CH), 3.80 (dd, J = 14.0, 6.9 Hz,
2H, CH2), 1.46 (s, 6H, 2CH3), 1.40 (s, 6H, 2CH3), 1.29 (t,
J = 7.0 Hz, 3H, CH3). 13C NMR (95 : 5 CDCl3 : CD3OD):
d 157.16, 155.43, 147.69, 144.73, 138.56, 126.13, 114.44,
108.66, 88.72, 64.15, 63.78, 45.84, 30.77, 30.64, 29.69,
14.41. HR-APCI-MS: m/z 407.1052 [M+H]+ calculated
for C18H23BrN4O2 406.1004. 5 c: yield 78%. 1H NMR
(95 : 5 CDCl3 : CD3OD): d 8.06 (s, 1H, CH), 7.54 (s, 1H,
Ar-CH), 6.61 (s, 1H, Ar-CH), 3.75 – 3.68 (m, 2H, CH2),
1.71 (dd, J = 14.7, 7.3 Hz, 2H, CH2), 1.54 (s, 6H, 2CH3),
1.47 (s, 6H, 2CH3), 0.91 (t, J = 7.4 Hz, 3H, CH3). 13C
NMR (95 : 5 CDCl3 : CD3OD): d 157.13, 155.58, 147.90,
145.22, 126.57, 114.30, 108.42, 100.06, 88.51, 52.37, 30.26,
30.14, 22.36, 10.90. HR-APCI-MS: m/z 421.1199 [M+H]+
calculated for C18H23BrN4O2 420.1161.
Spin labels 7a–c. To a solution of compounds 6a–c
(0.033 mmol) in CH2Cl2 (10 ml) was added a solution of
mCPBA (8.6 mg, 0.05 mmol) in CH2Cl2 (2 ml) at 0ꢀC. The
resulting reaction mixture was stirred for 6 h at 0ꢀC and
concentrated in vacuo. The residue was purified by column
chromatography (2% MeOH in CH2Cl2) to afford spin
labels 7a–c as pale yellow solids. 7a: yield 60%. 1H
NMR (CDCl3): d 9.17 (s, 1H), 8.61 (bs, 1H), 3.23 (s,
1H). HR-APCI-MS: m/z 328.1534 [M+H]+ calculated
for C17H19N4O3 327.1457. 7 b: yield 58%. 1H NMR
(CDCl3): d 6.70 (bs, 1H), 3.90 (bs, 2H, CH2), 1.40 (bs,
6H). HR-APCI-MS: m/z 342.1686 [M+H]+ calculated
for C18H21N4O3 341.1614. 7c: yield 65%. 1H NMR
(CDCl3): d 6.55 (s, 1H), 3.75 (s, 4H), 1.91–1.82 (m, 2H),
1.26 (s, 7H), 0.92–0.74 (m, 10H). HR-APCI-MS: m/z
356.1859 [M+H]+ calculated for C19H23N4O3 355.1843.
Note: due to the paramagnetic nature of nitroxides the
NMR spectra of these compounds shows significant
broadening of the signals for which reason some peaks,
particularly of nuclei close to the radical, are not seen in
the spectra.
RESULTS AND DISCUSSION
Effect of flanking sequences on spin label binding
Abasic sites in nucleic acids have been used as receptors
for binding small organic ligands (31–36). A major factor
that governs the binding of ligands to abasic sites is
hydrogen bonding with the orphan base on the comple-
mentary strand, opposite to the abasic site (33–36).