Direct strand cleavage via furanyladenine formation in anaerobic photoirradiation of 5-bromouracil-containing oligonucleotides

Article abstract of DOI:10.1016/S0040-4039(00)01026-1

The mechanism of direct strand cleavage induced by anaerobic irradiation of 5-bromouracil ((Br)U)-containing DNA was investigated. The formation of an oligonucleotide fragment containing 3'-furanyladenine end like 5 was observed as a major photoproduct for the direct strand breaks. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01026-1

Tetrahedron Letters 41 (2000) 6455±6459
Direct strand cleavage via furanyladenine formation in
anaerobic photoirradiation of 5-bromouracil-containing
oligonucleotides
Kenzo Fujimoto, Yutaka Ikeda and Isao Saito*
Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto 606-8501, Japan
Received 26 May 2000; revised 12 June 2000; accepted 16 June 2000
Abstract
The mechanism of direct strand cleavage induced by anaerobic irradiation of 5-bromouracil (BrU)-
containing DNA was investigated. The formation of an oligonucleotide fragment containing 30-fur-
anyladenine end like 5 was observed as a major photoproduct for the direct strand breaks. # 2000 Elsevier
Science Ltd. All rights reserved.
Photoreactions of 5-bromouracil (BrU)-containing polynucleotides have been investigated from
several interesting aspects such as for mapping protein±nucleic acid interactions,1 and the
mechanism of photoreaction of BrU in DNA oligomers has been thoroughly investigated.2
Recently, Greenberg et al. proposed an intriguing mechanism for direct strand breaks caused by
C20 hydrogen abstraction by uracil-5-yl-radical with putative formation of 30-keto-20-deoxy-
nucleotide 1 in anaerobic photolysis of duplex DNA containing BrU (path a, Scheme 1).3
However, 1 has been known to be unstable in aqueous solution even at 0ꢀC and gradually
decomposed to adenine and a furanone analogue.4 We now report here that oligonucleotide
fragment 2 containing furanyladenine at the 30 end is the major isolable photoproduct in direct
strand cleavage induced by anaerobic photoirradiation of 50-ABrU-30-containing oligonucleotides
(path b, Scheme 1).
Fig. 1 shows a HPLC pro®le of the anaerobic photoirradiation of duplex d(GTAC-
GABrUGCACA) (3)/d(TGTGCATCGTAC) (4), showing the formation of 7 as the C10 oxidation
product.5 The peak eluted at 14.3 min was pUGCACA 6 originated from direct strand
cleavage.6 Enzymatic digestion of the peak eluted at 28 min corresponding to 5 indicated the
formation of dA, dG, dC, dT and furanyladenine 8 in a ratio of 1:2:1:1:1 (Fig. 2).7 The structure
of 8 was con®rmed by direct comparison of the HPLC retention time and the UV spectrum
* Corresponding author. Fax: +81 75-753-5676; e-mail: saito@sbchem.kyoto-u.ac.jp
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01026-1

6456
Scheme 1.
Figure 1. HPLC pro®le of UV-irradiated ODN 3/ODN 4 under anaerobic conditions
Figure 2. HPLC analysis of the enzymatic digestion of isolated 5

6457
obtained by photodiodarray assay with those of authentic sample prepared according to the
known method.8 ESI-TOF mass of the fragment 5 also supported the structure (Scheme 2).9
Scheme 2.
The product yields as determined by HPLC are summarized in Table 1. The equimolar
formation of pUGCACA 6 and 5 without piperidine treatment clearly indicates the direct strand
cleavage shown in path b of Scheme 1, although the major product of the photoreaction was C10
oxidation product 7.
Table 1
Product analysis of anaerobic photoirradiation of ODN 3/ODN 4
PAGE analysis of the photoirradiation of 32P-50-end-labeled ODN 3 in the presence of ODN 4
was shown in Fig. 3.10,11 In lane 3, a new band resulting from direct strand breaks of ODN 3
appeared near the 0dBrU site as reported by Greenberg et al.3 This band comigrated with the band
of authentic 32P-5 -end-labeled 5 (lane 4). This observation led us to con®rm that the product
derived from the direct strand break was 5. The mechanism of the formation of 5 was not clear at
present. However, C20 hydrogen abstraction by the resulting uracil-5-yl-radical from photoinduced
SET from A to BrU would explain the formation of furanyladenine-containing fragment 5
(Scheme 3). To our knowledge, this is the ®rst example for the direct strand cleavage that

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Figure 3. PAGE analysis of UV-irradiated 50-32P-end-labeled ODN 3/ODN 4. Lane 1, G+A sequencing reaction; lane
2, unphotolyzed; lane 3, anaerobic photolysis; lane 4, 50-32P-end-labeled 5 prepared independently
Scheme 3.
produces furan-containing termini. The formation of 30-keto-20-deoxyadenosine-containing frag-
ment as proposed by Greenberg et al.3a has not been observed in our experiments.
In conclusion, we have observed for the ®rst time the formation of a furanyladenine-containing
oligodeoxynucleotide fragment produced by the direct strand cleavage induced by photoirradiation
of BrU-containing DNA.
Acknowledgements
We thank CREST, Japan Science and Technology Corporation for funding.
References
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6459
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5. Each of the reaction mixtures containing 0.1 mM (strand conc.) of an oligonucleotide in 50 mM sodium
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6. ESI-TOFF MS: calcd for pUGCACA 6 (C57H72N23O36P6) (M H^) 1840.2935; found 1840.2773.
7. The photoproduct (retention time at 28 min) was collected by HPLC, and the residue was dissolved in water and
then subjected to enzymatic digestion with s.v. PDE (0.3 unit/mL) and calf intestine AP (100 unit/mL) at 37ꢀC for
2 h. The mixture was subjected to HPLC analysis.
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9. ESI-TOFF MS: calcd for fragment 5 (C59H70N25O32P5) (M-H^) 1794.3228; found 1794.467.
10. The reaction mixture (total volume 20 mL) containing ODN 4 (10 mM, strand conc.) and ODN 3 (ca. 2Â104 cpm,
0.1±0.3 mM) in 50 mM sodium cacodylate bu€er (pH 7.0) was irradiated with a transilluminator (302 nm) at 0ꢀC.
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