Deoxyribonolactone formation in photoirradiation of 5-bromouracil-containing oligonucleotides by direct C1′ hydrogen abstraction

Article abstract of DOI:10.1016/S0040-4039(02)00231-9

Mechanistic studies on the formation of deoxyribonolactone by UV irradiation of hexamer d(GC1^BrUGC)₂ (ODN 2a) containing 1′-deuterio-2′-deoxyadenosine 1 was examined. The kinetic isotope effect (k_H/k_D) for the f

Full text of DOI:10.1016/S0040-4039(02)00231-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 2243–2245
Deoxyribonolactone formation in photoirradiation of
5-bromouracil-containing oligonucleotides by direct C1%
hydrogen abstraction
Kenzo Fujimoto, Yutaka Ikeda, Shigenori Ishihara and Isao Saito*
Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto 606-8501, Japan
Received 17 January 2002; revised 31 January 2002; accepted 1 February 2002
Abstract—Mechanistic studies on the formation of deoxyribonolactone by UV irradiation of hexamer d(GC1BrUGC)2 (ODN 2a)
containing 1%-deuterio-2%-deoxyadenosine 1 was examined. The kinetic isotope effect (kH/kD) for the formation of C1% oxidation
product 3 was 1.7 0.1. ESI-MS of d(pUGC) (7) obtained from the photoreaction of ODN 2a indicated that 1% deuterium was
abstracted by uracilyl-5-yl radical and incorporated into 7 in 96% yield. © 2002 Published by Elsevier Science Ltd.
plementary hexamer d(GC1BrUGC)2 (ODN 2a). Herein
The formation of deoxyribonolactone structure in
we report experimental evidence that the C1% oxidation
product results from direct C1% hydrogen abstraction in
photoirradiation of BrU-containing oligonucleotides.
DNA has been reported for C1% hydrogen abstraction
of deoxyribose by g and UV irradiation,1 cationic man-
ganese porphyrins2 and neocarzinostatin.3 We have
reported that photoiraddiation of BrU-containing DNA
hexamers induced C1% and C2%-oxidation at the 5% side
of BrU residue via hydrogen abstraction at both C1% and
C2% with a significant 5%-ABrU-3% sequence selectivity.4
Recently, Greenberg et al. proposed an intriguing
mechanism for C1% oxidation that is derived from C2%
hydrogen abstraction followed by 1,2 H shift in pho-
toirradiation of BrU-containing DNA (path b, Scheme
1).5a,b However, in our previous experiments on the
photoreaction of stereospecifically C2%a deuterated hex-
anucleotide, no suppression of the formation of C1%
oxidation product has been observed.4a To better
understand the mechanism of C1% oxidation, we now
examined a detailed analysis of the photoreaction of
1%-deuterio-2%-deoxyadenosine (1)-containing self-com-
Hexamer ODN 2a was synthesized by automated DNA
synthesizer using b-cyanoethylphosphoramidite of N-
benzoylated-1,6 which was synthesized in nine steps
from
D
-(+)-ribonolactone.7 1H NMR indicated that D
content in N-benzoylated-1 was more than 98%.8 Fig. 1
shows an HPLC profile of the photoirradiated mixture
of self-complementary ODN 2a, indicating that a
decreased amount of 3 as the C1% oxidation product
and an increased amount of 4 as C2% oxidation product
were produced in comparison with those of unlabeled
Scheme 1.
* Corresponding author. Fax: +81 75-753-5676; e-mail: saito@sbchem.kyoto-u.ac.jp
0040-4039/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0040-4039(02)00231-9

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K. Fujimoto et al. / Tetrahedron Letters 43 (2002) 2243–2245
Figure 1. HPLC profile of the photoirradiation of (a) ODN 2a and (b) ODN 2b.
Table 1. Product analysis and kinetic isotope effect (KIE) of photoirradiated ODN 2a and ODN 2b
Hexamer
Conversion (%)
Product (mM)
4 (2% ox)
Quantum yield (×10−4
)
kH/kD
4 (2% ox)
3 (1% ox)
3 (1% ox)
4 (2% ox)
3 (1% ox)
ODN 2a
ODN 2b
9.590.3
9.690.4
2.790.1
4.790.2
1.090.1
0.890.1
6.990.3
11.890.5
2.590.2
1.990.1
1.790.1
–
0.890.2
–
Scheme 2.
ODN 2b.9 Table 1 summarizes the product distribution
in the photoreaction of the hexanucleotides together
with the kinetic isotope effect (kH/kD) calculated based
on the quantum yields for the formation of 3 and 4.10
The kinetic isotope effects (KIE) for the formation of 3
and 4 by photoirradiation of ODN 2a as compared
with ODN 2b was found to be 1.7 0.1 and 0.8 0.1,
respectively. This result indicated that the formation of
3 occurs via a rate-limiting abstraction of C1% hydrogen
of deoxyribose by adjacent uracilyl-5-yl radical as
shown in Scheme 2. The magnitude of KIE (1.7 0.1)
for the formation of 3 was in the range (1.0ꢀ4.2)
previously observed for direct C1% H abstraction.5a,b,11
The inverse KIE (0.8 0.1) for the formation of 4 also
suggest the partitioning of C1% and C2% hydrogen
abstraction by uracilyl-5-yl radical as a common inter-
mediate in photoirradiation of BrU-containing DNA.
both ODN 2a and undeuterated ODN 2b, indicating
that the 1% D was abstracted by uracilyl-5-yl radical and
incorporated into 7 with 96% D content.13 This result
clearly indicated that 3 was caused from direct 1% D
abstraction by uracilyl-5-yl radical almost quantita-
tively. The formation of uracilyl hydroperoxide as pro-
posed by Greenberg et al.5b has not been observed in
our experiments.
In conclusion, by using ODN containing C1%-deuter-
ated deoxyadenosine, we have proved that C1% oxida-
tion product in the photoirradiation of BrU-containing
DNA is derived from direct 1% hydrogen abstraction by
uracilyl-5-yl radical.
Acknowledgements
In order to determine D content of 3, d(pDUGC)
fragment (7) was isolated by HPLC12 after heating 3
with conc. aqueous ammonia as shown in Fig. 2a and
subjected to ESI-MS. Fig. 2b shows the ESI-MS of
d(pUGC) fragments derived from the photoreactions of
This work was supported by a Grant-in-Aid for Scien-
tific Research from Ministry of Education and Science
and CREST, Japan Science and Technology
Corporation.

K. Fujimoto et al. / Tetrahedron Letters 43 (2002) 2243–2245
2245
Figure 2. (a) HPLC profile of a mixture of isolated C1% oxidation product 3 heated with conc. ammonia at 90°C for 10 min. (b)
ESI-MS spectrum of d(pUGC) fragments obtained from ODN 2a (dashed line) and ODN 2b (solid line).
the peak area corresponding to C1% H and uracil C6 H of
N-benzoylated-1 by H NMR spectrum.
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AODN 2b(462.5)
ꢀ
ꢁ
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×100
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(462.5)
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