Synthesis of Oligonucleotides Containing Two Putatively Mutagenic DNA Lesions: 5-Hydroxy-2'-deoxyuridine and 5-Hydroxy-2'-deoxycytidine

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• 作者：Marshall L. Morningstar ; Deborah A. Kreutzer ; and John M. Essigmann
• 刊名：Chemical Research in Toxicology
• 出版年：1997
• 出版时间：December 1997
• 年：1997
• 卷：10
• 期：12
• 页码：1345 - 1350
• 全文大小：206K
• 年卷期：v.10,no.12(December 1997)
• ISSN：1520-5010

文摘

Spontaneous oxidative DNA damage occurs as aconsequence of aerobic metabolism, lipidperoxidation, immune responses, ionizing radiation, and some chemicaloxidants. Theseprocesses yield a vast array of oxidized DNA bases and sugars. Theexistence of significantsteady-state levels of oxidized DNA bases in the genome suggests thatthese lesions are notcompletely repaired on a biologically relevant time scale and thus maycontribute tomutagenesis. In particular, studies have shown that thesteady-state levels of 5-hydroxy-2'-deoxycytidine (dC^5-OH) and itsdeamination product, 5-hydroxy-2'-deoxyuridine(dU^5-OH), aresimilar to those found for 7,8-dihydro-8-oxoguanosine, a known highlymutagenic lesion formedby oxidation of guanosine. Structural and biological properties ofdC^5-OH anddU^5-OH havebeen constrained by the lack of synthetic methodology foroligonucleotides containing thesemodified bases. A method is described here for the solid-phasesynthesis of oligonucleotidescontaining dC^5-OH anddU^5-OH. Preparation of eachof the required phosphoramidites involvedthe selective protection of the base 5-hydroxyl group over thedeoxyribose 5'- and 3'-hydroxylgroups. The base composition and the incorporation of the adductsinto synthetic heptanucleotides were confirmed after purification of the modifiedoligonucleotides by enzymatic digestionand HPLC analysis. Mass spectrometric analysis of theoligonucleotide products by electrosprayMS and GC/MS further confirmed their composition. Mostsignificantly, deamination of thedC^5-OH oligomer to a putativedU^5-OH product during solid-phaseDNA synthesis or oligonucleotide deprotection was not detected by any analytical techniqueemployed.