118
P. Yu et al. / Bioorg. Med. Chem. 9 (2001) 107±119
III-1 (ꢁ5 mg) obtained from the above reactions was
dissolved in an aqueous NH4OH solution (28%, 2 mL)
and kept in a sealed vial at rt overnight. NH4OH was
removed under reduced pressure and then in vacuo and
the residue was applied on to a preparative reverse-
phase HPLC column using buer A with a gradient of B
from 2 to 20% in 55 min to aord desired III (3.5 mg).
5. Wagner, R. W.; Matteucci, M. D.; Grant, D.; Huang, T.;
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1
NMR: III is fully characterized by H, 13C and 31P 1-D
and 2-D correlation NMR spectra, such as DQF-COSY,
1
1
TOCSY, H±13C HMQC, HMBC, H±31P COSY, etc.,
and these results are reported in Table 3. ESI-MS calcd
for C40H55N13O19P2 1083.3, found 1083.3.
11. Barawkar, D. A.; Thomas, Y. K.; Bruice, T. W.; Bruice,
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Enzymatic Reactions of III
Phosphorylation of III
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In the presence of T4 polynucleotide kinase (10 u), III
(0.2 mmol) or CTTC (0.2 mmol, used in a reference
reaction) in a buer solution (0.1 mL, 70 mM Tris±HCl,
10 mM MgCl2, 100 mM KCl, 1 mM 2-mercaptoetha-
nol), pH 7.6, was incubated with ATP (3 mM) at 37 ꢂC.
The reaction was monitored by reverse-phase HPLC
using 50 mM triethylammonium acetate and CH3CN as
eluant. CTTC phosphorylation was complete within 4 h,
while 55% III was phosphorylated after 7 h of incuba-
tion. Additional enzyme (5 u) was added at this point
and HPLC pro®le indicated 99% phosphorylation after
total 17 h reaction.
14. De Mesmaeker, A.; Waldner, A.; Lebreton, J.; Homan,
P.; Fritsch, V.; Wolf, R. M.; Freier, S. M. Angew. Chem., Int.
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Ligation reaction: In the presence of T4 DNA ligase (5
u), the ¯anking strands [d(GCGTTTGCT) and d(pTT
CTTGCG)], and the template strand [d(CGCAAGAA-
GAAGAGCAAACGC)], pIII or pCTTC (50 mM) in a
buer solution (0.01 mL, 50 mM Tris±HCl, 10 mM
MgCl2, 1 mM ATP, 1 mM DTT, 5% (w/v) polyethylene
glycol-8000), pH 7.6, was incubated at 15 ꢂC. The reac-
tion was monitored using UV melting measurement (0±
80 ꢂC, Varian CAREY 3E) and denaturing poly-
acrylamide gel electrophoresis (20% acrylamide, Bis:
acrylamide 1:29, 100 mM Tris±HCl, pH 7.4, 4 ꢂC).
23. Ferrer, E.; Shevchenki, A.; Eritja, R. Bioorg. Med. Chem.
2000, 8, 291.
24. Uhlmann, E. Biol. Chem. 2000, 379, 1045.
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6228. (b) Gao, X.; Jes, P. W. J. Biomol. NMR 1994, 4, 17. (c)
Gao, X.; Jes, P. W. J. Biomol. NMR 1994, 4, 367. (d) Veal,
J. M.; Gao, X; Brown, F. K. J. Am. Chem. Soc. 1993, 115,
7139. (e) Rice, J. S.; Gao, X. Biochemistry 1997, 36, 399. (f)
Cross, C.; Rice, J. S.; Gao, X. Biochemistry 1997, 36, 4096.
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Acknowledgements
The authors thank Drs. Dawei Li for helping 2-D NMR
data collection and Dr. Jeerey S. Rice for proofreading
of the manuscript. The authors are also grateful to Dr.
Rich H. Griey (Isis Pharmaceutical, Inc.) for mass
analysis. The 600 MHz NMR spectrometer at the Uni-
versity of Houston was funded by the W. M. Keck
Foundation. This research is supported by Grants from
NIH and the Welch Foundation (E-1270).
26. Wang, W.; Liang, T. C.; Zheng, X.; Gao, X. Tetrahedron
Lett. 1995, 36, 1181.
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1994, 35, 5173.
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