100
R. O. Schonleber et al. / Bioorg. Med. Chem. 10 (2002) 97–101
¨
(122 MHz, CDCl3) d ꢁ9.5 (t, J=6.2 Hz); MS (FAB): m/
z (%) 439 (50) [MH]+; MS (FAB,+KCl): m/z (%) 478
(23) [M+K]+, 439 (44) [MH]+; elemental analysis calcd
(%) for C22H34NO6P (439.49) C 60.12, H 7.80, N 3.19,
O 21.84; found C 59.93, H 7.79, N 3.01, O 21.56.
(34) [M+Na]+; MS (ESI, —): m/z (%) 631 (100)
[MꢁH]ꢁ, 1263 (5) [M2ꢁH]ꢁ; elemental analysis calcd
(%) for C23H30N4O13P2ꢀ2H2O (668.49) C 41.33, H
5.13, N 8.38; found C 41.20, H 5.17, N 8.26.
Photolysis and quantum yield measurements
(7-Diethylaminocoumarin-4-yl)methyl phosphate (6). A
solution of the di-tert-butyl protected phosphate 5 (150
mg, 341 mmol) in anhydrous CH2Cl2 (2 mL) was cooled
to 4 ꢂC. Trifluoroacetic acid (214 mg, 144 mL, 1.88
mmol) was added under stirring and the mixture was
agitated for 6 h. The mixture was washed with hexane
(3ꢀ2 mL) and concentrated in vacuo (for this com-
pound no accurate combustion analysis or HR-MS
could be obtained), yielding 106 mg (324 mmol, 95%):
Rf 0 (Et2O); 1H NMR (500 MHz, D2O) d 7.80 (d,
J=8.7 Hz, 1H), 7.43 (d, J=2.3 Hz, 1H), 7.35 (dd,
J=2.3, 8.7 Hz, 1H), 6.59 (t, J=1.4 Hz, 1H), 5.08 (dd,
J=1.5, 7.4 Hz, 2H), 3.58 (q, J=7.2 Hz, 4H), 1.05 (t,
J=7.2 Hz, 6H); 13C NMR (126 MHz, D2O) d 164.5,
154.9, 153.7 (d, J=7.1 Hz), 144.2, 126.8, 116.2, 114.9,
110.6, 107.2, 63.3 (d, J=3.8 Hz), 51.3, 11.0; 31P NMR
(203 MHz, D2O) d 0.7 (t, J=7.3 Hz); MS (ESI, +): m/z
(%) 328 (85) [MH]+, 655 (100) [M2H]+; MS (ESI, —):
m/z (%) 326 (21) [MꢁH]ꢁ, 653 (100) [M2ꢁH]ꢁ.
The UV-spectra were recorded with a U-3410 spectro-
photometer (Hitachi, Japan). Photolysis was carried out
using a high-pressure mercurylamp (HBO 500, Oriel)
with controlled light intensityand metal interference
filter of 365, 405 and 436 nm (Schott, Germany). For
quantum yield determination the irradiated solutions
were analyzed using HPLC and, additional, using the
matrix analysis14 of the irradiation spectra. The results
of both methods were identical. The photochemical
quantum yield was defined as the ratio of caged mole-
cules converted to the amount of photons absorbed
using the potassium ferrioxalate actinometer.15 Further
9c
details are described bySchade et al.
Fluorescence measurements
The fluorescence spectra were measured using a MPF-
2A fluorescence spectrophotometer (Hitachi-Perkin–
Elmer). In the case of the photosensitive caged com-
pounds the excitation intensitywas verylow to
minimize photolysis. The excitation wavelength was
P2-(7-Diethylaminocoumarin-4-yl)methyl cytidin 50-di-
phosphate (1). The free acid of CMP (441 mg, 1.36
mmol) was dried over P2O5 in vacuo (3ꢀ10ꢁ2 mbar) for
2 h at 50 ꢂC. The resulting white powder was dissolved
in anhydrous DMF (1.4 mL), treated with tri-n-octyla-
mine (333 mL, 1.36 mmol) for a few min at 100 ꢂC and
allowed to cool to room temperature. Carbonyldiimi-
dazole (441 mg, 2.72 mmol) was added and the mixture
was stirred overnight at room temperature. The phos-
phate 6 (106 mg, 324 mmol) was dissolved in MeOH/
EtOH (1:1, 3.4 mL) and tri-n-butylamine (81 mL, 340
mmol) was added. After evaporation of the solvent in
vacuo and coevaporation with anhydrous pyridine (2ꢀ2
mL), the residue was dissolved in anhydrous DMF,
combined with the imidazolide solution and stirred for
11 days at room temperature. The mixture was con-
centrated in vacuo, dissolved in H2O and purified first
bypreparative HPLC, that was performed with a
Waters HPLC system using a Lichrospher 100 RP 18
(310ꢀ16 mm, 5 mm) column from Knauer and UV
detection (254ꢀ400 nm). The flow was 8 mL/min and
the gradient used for elution is as follows: 0–60 min, 10–
35% acetonitrile in 0.1 M sodium acetate buffer (pH 5).
The fraction with the retention time of 23.2 min was
collected, lyophilized and the product was finally pur-
ified on Dowex 50W-X5 (H+) to give 72.6 mg of 1 as
l
exc=375–390 nm. The fluorescence quantum yields
were determined at 298 K bythe relative method 16 using
quinine sulfate as a standard. The time-resolved fluor-
escence decaymeasurements were performed using the
pulse sampling method.17
Acknowledgements
We thank Dr. S. Helm, D. Geissler and B. Dekowski for
technical assistance. We are grateful to Professor R.
Schmidt, Universityof Frankfurt/Main, for the time-
resolved fluorescence measurements. This work was
supported bythe Swiss National Science Foundation
and bythe Deutsche Forschungsgemeinschaft (DFG);
R.O.S. thanks the Amt fur Ausbildungsbeitrage des
Kantons Basel-Landschaft for financial support.
References and Notes
1. (a) Stubbe, J. Adv. Enzymol. Relat. Areas Mol. Biol. 1990,
63, 349. (b) Stubbe, J.; van der Donk, W. A. Chem. Biol. 1995,
2, 793.
2. (a) Nordlung, P.; Eklund, H. J. Mol. Biol. 1993, 232, 123.
(b) Uhlin, U.; Eklund, H. Nature 1994, 370, 533.
3. (a) Recent reviews on RNR: Sjoberg, B. M. In Structure
and Bonding, Metal Sites in Proteins and Models/Iron Centers;
Sadler, P. J., Ed.; Springer: Berlin, 1997; Vol. 88, p 139; (b)
Stubbe, J.; van der Donk, W. Chem. Rev. 1998, 98, 705.
4. Petersson, L.; Graslund, A.; Ehrenberg, A.; Sjoberg, B.-M.;
Reichard, P. J. Biol. Chem. 1980, 255, 6706.
5. (a) Tanner, J. W.; Thomas, D. D.; Goldman, Y. E. J. Mol.
Biol. 1992, 223, 185. (b) Berger, C. L.; Craik, J. S.; Trentham,
D. R.; Corrie, J. E. T.; Goldman, Y. E. Biophys. J. 1995, 68,
1
dihydrate (109 mmol, 34%): H NMR (600 MHz, D2O)
d 8.14 (d, J=7.9 Hz, 1H), 7.85 (d, J=8.7 Hz, 1H), 7.45
(s, 1H), 7.38 (d, J=8.7 Hz, 1H), 6.64 (s, 1H), 6.20 (d,
J=7.9 Hz, 1H), 5.73 (d, J=3.3 Hz, 1H), 5.27 (s, 2H),
4.34 (d, J=11.7 Hz, 1H), 4.28 (m, 1H), 4.25 (m, 1H),
4.24 (m, 1H), 4.18 (d, J=11.7 Hz, 1H), 3.68 (q, J=7.1
Hz, 4H), 1.18 (t, J=7.1 Hz, 6H); 13C NMR (101 MHz,
D2O) d 163.5, 159.4, 154.3, 152.9, 148.5, 144.3, 142.3,
127.0, 117.4, 116.4, 111.8, 109.0, 95.4, 90.0, 83.3, 74.7,
69.1, 64.5, 63.7, 52.4, 10.5; 31P NMR (243 MHz, D2O) d
ꢁ14.5; MS (ESI, +): m/z (%): 633 (100) [MH]+, 655