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proline cyanomethyl ester (12) in 50 µL of DMF was stirred
at room temperature for 4 h. A 10-µL aliquot of the mixture
was diluted with 90 µL of 1:1 CH3CN – 50 mM NH4OAc,
pH 4.5. Ten µL of the diluted aliquot was analyzed by
HPLC on a C18 reversed phase column (250 × 10 mm). The
column was washed with 1→63% CH3CN in 50 mM
NH4OAc, pH 4.5, over a period of 45 min at a flow rate of
3.5 mL/min (monitoring at 260 nm). The reaction mixture
was diluted to a total volume of 400 µL of 1:1 CH3CN –
50 mM NH4OAc, pH 4.5, and purified using the same
semipreparative C18 reversed phase column, affording β-
nitroveratryl
N-(6-nitroveratryloxycarbonyl)-(2S,3R)-car-
boxyproline pdCpA ester (13) as a pale yellow foam: yield
0.5 mg (14%). Mass spectrum (FAB) m/z: 1212.257 (M+H)+
(C44H52N11O26P2 requires 1212.2560).
Synthesis of misacylated tRNAs
Ligation reactions were carried out in 50 µL of 50 mM Na
Hepes buffer, pH 7.5, containing 5 µL (~0.2 nmol) of
tRNA(CUA)COH, 0.5 A260 unit (~20 nmol) of an aminoacyl-
pdCpA, 0.5 mM ATP, 15 mM MgCl2, 10% dimethyl-
sulfoxide, and 100 units of T4 RNA ligase. Reaction mix-
tures were incubated at 37°C for 25 min and then quenched
by the addition of 5 µL of 3 M Na acetate, pH 4.5. The
aminoacylated tRNA (I) was precipitated with 2.5 volumes
of ethanol, collected by centrifugation, washed with 70%
ethanol, and dried. The product was redissolved in 1 mM
KOAc to a final concentration of 1 µg/µL and then irradi-
ated with a 500 W mercury–xenon lamp using Pyrex and
water filters. The protected aminoacyl tRNA (I) was cooled
in an ice bath during irradiation, which was typically carried
out for 5 min for amino acid derivatives containing an
NVOC group and a nitroveratryl ester. The deprotected
aminoacyl-tRNAs (II) were used in in vitro suppression ex-
periments soon after deprotection.
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Benson, C.A. Altenbach, K. Hideg, W.L. Hubbell, and P.G.
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M.E. Saks, C.G. Labarca, S.K. Silverman, W. Zhong, J.
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(1997).
Acknowledgment
This work was supported by Research Grant CA77359
from the National Cancer Institute.
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