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J = 2.5 Hz, 1 H, 8-H), 6.08 (s, 1 H, 3-H), 5.50 (t, J = 7.5 Hz, 1 H, OH), Si[CH(CH3)2]3} ppm. 13C NMR (125.8 MHz, [D6]DMSO): δ = 163.1,
4.66 (dd, J = 3.5, 1.0 Hz, 2 H, CH2OH), 3.41 (q, J = 7.0 Hz, 4 H, 161.3, 158.6, 156.2, 155.4, 152.8, 150.8, 145.2, 140.5, 135.9, 135.8,
CH2CH3), 1.11 (t, J = 7.0 Hz, 6 H, CH2CH3) ppm. 13C NMR (125.8 MHz,
[D6]DMSO): δ = 161.2, 156.9, 155.6, 150.2, 125.1, 108.5, 105.7, 103.9,
96.8, 59.0, 43.9, 12.3 ppm. MS (ESI): m/z = 248.2 [M + H+]. HRMS
130.2, 128.4, 128.2, 127.2, 125.9, 113.7, 109.2, 105.9, 104.9, 97.3, 95.3,
88.9, 88.2, 86.3, 83.1, 78.6, 74.8, 69.2, 63.6, 60.3, 60.2, 55.5, 45.1, 45.0,
44.5, 41.2, 32.9, 29.1, 21.24, 21.16, 18.24, 18.16, 14.6, 12.8, 11.8 ppm.
(MALDI): calcd. for
248.12883.
C14H18NO3 [M
+
H+]: 248.12812; found MS (MALDI): m/z = 1138.56 [M + Na+]. HRMS (MALDI): calcd. for
C63H85N5O11SiNa [M + Na+]: 1138.59071; found 1138.58911.
tert-Butyl-1-{[7-(diethylamino)-2-oxo-2H-chromen-4-yl]meth- Cytidine Phosphoramidite with Protected Spin Label (3): To a
oxy}-2,2,6,6-tetramethylpiperidin-4-ylcarbamate (9): tert-Butyl-
solution of 2′-O-TOM-5′-O-DMT-U° 12 (3.67 g, 3.29 mmol,
[4-(2,2,6,6-tetramethyl)piperidinyl-N-oxyl]carbamate[20] (3.84 g, 1.00 equiv.) in CH2Cl2 (60 mL), Et3N (2.31 mL, 16.44 mmol,
14.18 mmol, 1.00 equiv.), bromomethylcoumarin 8[6] (5.28 g, 5.00 equiv.) and N,N-diisopropylaminocyanoethylphosphoramidic
17.02 mmol, 1.20 equiv.), copper powder (1.35 g, 21.23 mmol,
chloride (1.39 g, 5.89 mmol, 2.00 equiv.) were added. The reaction
1.50 equiv.), Cu(OTf)2 (0.26 g, 0.71 mmol, 0.05 equiv.) and 4,4′-di- mixture was stirred for 21 h at ambient temperature. Subsequently,
methyl-2,2′-bipyridyl (0.39 g, 2.13 mmol, 0.15 equiv.) were sus- conc. NaHCO3 solution was added and stirred for 5 min at ambient
pended in toluene (140 mL). The suspension was degassed, put
under argon and heated to reflux for 16 h. The suspension was
filtered through silica gel and the residue was eluted with CH2Cl2.
The solvent was removed under reduced pressure. Purification by
silica gel chromatography (CH2Cl2/EtOAc, 10:1) gave the title com-
pound 9 (6.30 g, 89 %) as a light-yellow solid. Rf = 0.44 (CH2Cl2/
EtOAc, 10:1). 1H NMR (500 MHz, CDCl3): δ = 7.24 (d, J = 9.0 Hz, 1 H,
5-H), 6.55 (dd, J = 9.0, 2.5 Hz, 1 H, 6-H), 6.51 (d, J = 2.5 Hz, 8-H),
6.29 (s, 1 H, 3-H), 4.95 (s, 2 H, OCH2), 4.28 (br. s, 1 H, NHCH), 3.84
(br. s, 1 H, CHNH), 3.41 (q, J = 7.0 Hz, 4 H, CH2), 1.86 (d, J = 11.5 Hz,
temperature. The organic layer was separated and the aqueous
layer was extracted with CH2Cl2. The combined organic layers were
dried with MgSO4 and solvent was removed under reduced pres-
sure. Purification by silica gel chromatography (EtOAc/c-hexane/
Et3N 70:30:1) gave the title compound 3 (4.10 g, 95 %) as a light-
yellow foam. Rf = 0.48, 0.62 (EtOAc/c-hexane, 7:3). 1H NMR
(500 MHz, [D6]DMSO): δ = 7.68–7.62 (m, 2 H, NH, 6-H), 7.43–7.37
(m, 3 H, Ar-H), 7.33–7.24 (m, 7 H, Ar-H), 6.90–6.86 (m, 4 H, Ar-H),
6.68 (dd, J = 9.0, 2.0 Hz, 1 H, 6-H), 6.53 (d, J = 2.1 Hz, 1 H, 8-H), 6.08
(s, 1 H, 3-H), 5.95–5.93 (m, 1 H, 1′H), 5.55 (d, J = 7.4 Hz, 1 H, 5-H),
2 H, CHH), 1.46 (s, 9 H, tBu), 1.34 (t, J = 12.5 Hz, 2 H, CHH), 1.28– 5.00–4.93 (m, 4 H, OCH2O, NOCH2), 4.37–4.18 (m, 3 H, CHNH, 2′H, 3′
1.26 (m, 6 H, CH3), 1.22–1.19 (m, 12 H, CH3, CH2CH3) ppm. 13C NMR H), 4.10–4.05 (m, 1 H, 4′H), 3.81–3.77 (m, 1 H, POCHH), 3.74, 3.73 (s,
(125.8 MHz, CDCl3): δ = 162.3, 156.1, 155.2, 151.7, 150.4, 124.4,
6 H, OCH3), 3.65–3.47 [m, 3 H, NCH(CH3)2, POCHH], 3.42 (q, J =
108.4, 106.3, 105.9, 97.8, 74.5, 60.4, 46.0, 44.7, 42.0, 32.8, 28.4, 20.9, 6.4 Hz, 4 H, CH2CH3), 3.37–3.32 (m, 1 H, 5′H), 3.25–3.17 (m, 1 H, 5′′
12.4 ppm. MS (ESI): m/z = 503.5 [M + H+]. HRMS (MALDI): calcd. for
H), 2.76–2.73 (m, 1 H, CHHCN), 2.65–2.57 (m, 1 H, CHHCN), 1.85–
1.72 (m, 2 H, CHHCH), 1.39 (q, J = 12.5 Hz, 2 H, CHHCH), 1.22, 1.20
(2 × s, 12 H, CH3), 1.13–1.01 [m, 18 H, CH2CH3, NCH(CH3)2], 0.99–
0.93 {m, 21 H, Si[CH(CH3)2]3} ppm. 13C NMR (125.8 MHz, [D6]DMSO):
δ = 162.6, 160.8, 158.2, 155.8, 154.8, 152.3, 150.3, 144.6, 140.2, 135.2,
135.1, 129.7, 127.8, 127.7, 126.8, 125.5, 118.8, 118.7, 113.2, 108.7,
105.4, 104.5, 96.9, 95.1, 88.7, 88.5, 86.1, 86.0, 82.0, 81.8, 77.1, 74.3,
70.5, 63.0, 59.8, 58.7, 58.6, 58.1, 58.0, 55.0, 44.6, 44.5, 44.0, 42.5, 42.4,
40.7, 32.5, 24.22, 24.16, 20.8, 20.7, 19.7, 17.7, 14.1, 12.3, 11.4 ppm.
31P NMR (202.5 MHz, [D6]DMSO): δ = 149.1, 148.8 ppm. MS (MALDI):
m/z = 1338.56 [M + Na+]; calcd. for C72H102N7NaO12PSi [M + Na+]
1338.70.
C28H43N3O5K [M + K+]: 540.28343; found 540.28460.
2′-O-TOM-5′-O-DMT-U° (12): To a solution of 2′-O-TOM-5′-O-DMT-
uridine 11[14] (0.37 g, 0.50 mmol, 1.00 equiv.) in CH2Cl2 (20 mL), Et3N
(0.63 mL, 4.50 mmol, 9.00 equiv.) and DMAP (0.009 g, 0.08 mmol,
0.15 equiv.) were added. The reaction mixture was cooled to 0 °C,
treated with TPS-Cl (0.20 g, 0.66 mmol, 1.32 equiv.) and stirred for
10 min at 0 °C. The mixture was warmed to ambient temperature
and stirred for 20 h. The reaction was quenched with conc. NaHCO3
solution. The organic layer was separated and the aqueous layer
was extracted with CH2Cl2. The combined organic layers were dried
with MgSO4 and the solvent was removed under reduced pressure.
The residue was dissolved in DMF and DIPEA (0.22 mL, 1.30 mmol,
Recovery of Spin Labels, EPR Spectroscopy: Buffer systems for
2.60 equiv.) and 4-[(4-amino-2,2,6,6-tetramethylpiperidin-1-yl- deprotection: 10 m
M
phosphate buffer (NaH2PO4/Na2HPO4) for
oxy)methyl]-7-(diethylamino)-2H-chromen-2-one 10[6] (0.26 g,
0.65 mmol, 1.30 equiv.) were added. The reaction mixture was
stirred for 8 h at 85 °C, cooled to ambient temperature and stirred
for 14 h. Subsequently the reaction was quenched with conc.
NaHCO3 solution, the organic layer was separated and the aqueous
layer was extracted with CH2Cl2. The combined organic layers were
dried with MgSO4 and the solvent was removed under reduced
pressure. Purification by silica gel chromatography (EtOAc/c-hex-
pH 4.6, 7.4, and 8.0; 50 m carbonate buffer (NaHCO3/K2CO3) for
M
pH 8.5 and 9.0. Directly after irradiation, 20 μL of the aliquots were
filled in quartz EPR tubes of 1 mm inner diameter. Continuous wave
(cw) EPR measurements were performed at X-band frequency
(9.54 GHz) with a Bruker E500 spectrometer equipped with a TE102
cavity. Experimental parameters: 100 kHz modulation frequency,
0.1 mT modulation amplitude, 0.2 mW microwave power, 40.96 ms
time constant, 40.96 ms conversion time, 1024 points, 7 mT sweep
ane/Et3N, 60:40:1) gave the title compound 12 (0.35 g, 64 %) as a width, 20 scans. For PELDOR measurement 20 μL of sample volume
light-yellow foam. Rf
=
0.22 (EtOAc/c-hexane, 3:2). 1H NMR
with 80 % buffer (pH 8.5)/20 % d8-glycerol was transferred into
1.6 mm outer diameter quartz EPR tubes (Suprasil, WilmadLabGlass)
directly after irradiation and annealing. Pulsed experiments at Q-
band frequencies (33.7 GHz) were performed with an ELEXSYS
(500 MHz, [D6]DMSO): δ = 7.64 (d, J = 7.6 Hz, 1 H, NH), 7.60 (d, J =
7.6 Hz, 1 H, 6-H), 7.43 (d, J = 9.1 Hz, 1 H, 6-H), 7.40–7.38 (m, 2 H,
Ar-H), 7.32 (t, J = 7.6 Hz, 2 H, Ar-H), 7.27–7.24 (m, 5 H, Ar-H), 6.90
(dd, J = 8.9, 1.6 Hz, 4 H, Ar-H), 6.68 (dd, J = 9.1, 2.4 Hz, 1 H, 6-H),
6.54 (d, J = 2.4 Hz, 1 H, 8-H), 6.08 (s, 1 H, 3-H), 5.94 (d, J = 4.3 Hz, 1
H, 1′H), 5.54 (d, J = 7.5 Hz, 1 H, 5-H), 5.06 (d, J = 6.1 Hz, 1 H, 3′OH),
5.01–4.96 (m, 4 H, OCH2O, NOCH2), 4.27–4.21 (m, 1 H, CHNH), 4.19–
SuperQ-FT accessory unit,
a continuous-flow helium cryostat
(CF935, Oxford Instruments), a temperature control system (ITC 502,
Oxford Instruments), and a Bruker AmpQ 10 W amplifier with a
Bruker EN5107D2 cavity at 50 K. Pulse lengths were 32 ns (π/2 and
4.13 (m, 2 H, 2′H, 3′H), 3.97–3.94 (m, 1 H, 4′H), 3.74 (s, 6 H, OCH3), π) for the observer pulses and 20 ns (π) for the pump pulse. The
3.43 (q, J = 6.8 Hz, 4 H, CH2CH3), 3.27–3.20 (m, 2 H, 5′H, 5′′H), 1.81– pump pulse frequency was set to the maximum of the echo-de-
1.79 (m, 2 H, CHHCH), 1.43–1.35 (m, 2 H, CHHCH), 1.23 (s, 6 H, CH3), tected field swept spectrum and the observer pulses were set
1.20 (s, 6 H, CH3), 1.12 (t, J = 7.0 Hz, 6 H, CH3), 0.99–0.93 {m, 21 H,
70 MHz lower. For PELDOR experiments, the dead-time free four-
Eur. J. Org. Chem. 0000, 0–0
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