Synthesis and Characterization of Oligonucleotides Containing 5-Chlorocytosine

• Published in: Chemical research in toxicology 2004-09, Vol.17 (9), p.1236-1244
• Main Authors: Kang, Joseph I, Burdzy, Artur, Liu, Pingfang, Sowers, Lawrence C
• Format: Article
• Language: English
• Subjects: Cytosine - analogs & derivatives; Cytosine - chemistry; Deamination; Formates; Hydrolysis; Mutagens - chemical synthesis; Oligonucleotides - chemical synthesis; Oligonucleotides - chemistry; Uracil - analogs & derivatives; Uracil - chemical synthesis
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx0498962

Recent studies have shown that reactive chlorine species, derived from myeloperoxidase-mediated inflammation responses, can modify DNA bases, generating 5-chloropyrimidines. The chlorinated adducts could be mutagenic or perturb DNA−protein interactions; however, the biological significance of these adducts is as yet unknown. We report here a method for the synthesis of 5-chlorocytosine- (ClC-) containing oligonucleotides that will be used in subsequent biochemical and biophysical studies to determine the consequences of pyrimidine chlorination. The ClC-phosphoramidite synthon is obtained by chlorination of 2‘-deoxyuridine followed by conversion to the O 4-ethyl analogue. The amino group needed to form the corresponding cytosine derivative is added by displacement of the O 4-ethyl group during ammonia deprotection. A battery of methods, including mass spectrometry, has been used to characterize oligonucleotides containing ClC. Following oligonucleotide synthesis and deprotection, only trace amounts of the deamination product 5-chlorouracil can be detected by enzymatic cleavage of duplex oligonucleotides with the mispaired uracil glycosylase, MUG. In contrast to previous reports, we find that ClC is more stable in DNA than anticipated. Approximately 20% ClC is lost under standard formic acid hydrolysis conditions (88% formic acid, 140 °C, 30 min), while only 5% is recovered as 5-chlorouracil (ClU).