title: Synthesis of oligonucleotides containing two putatively mutagenic DNA lesions: 5-hydroxy-2'-deoxyuridine and 5-hydroxy-2'-deoxycytidine.
abstract: Spontaneous oxidative DNA damage occurs as a consequence of aerobic metabolism, lipid peroxidation, immune responses, ionizing radiation, and some chemical oxidants. These processes yield a vast array of oxidized DNA bases and sugars. The existence of significant steady-state levels of oxidized DNA bases in the genome suggests that these lesions are not completely repaired on a biologically relevant time scale and thus may contribute to mutagenesis. In particular, studies have shown that the steady-state levels of 5-hydroxy-2'-deoxycytidine (dC5-OH) and its deamination product, 5-hydroxy-2'-deoxyuridine (dU5-OH), are similar to those found for 7,8-dihydro-8-oxoguanosine, a known highly mutagenic lesion formed by oxidation of guanosine. Structural and biological properties of dC5-OH and dU5-OH have been constrained by the lack of synthetic methodology for oligonucleotides containing these modified bases. A method is described here for the solid-phase synthesis of oligonucleotides containing dC5-OH and dU5-OH. Preparation of each of the required phosphoramidites involved the selective protection of the base 5-hydroxyl group over the deoxyribose 5'- and 3'-hydroxyl groups. The base composition and the incorporation of the adducts into synthetic heptanucleotides were confirmed after purification of the modified oligonucleotides by enzymatic digestion and HPLC analysis. Mass spectrometric analysis of the oligonucleotide products by electrospray MS and GC/MS further confirmed their composition. Most significantly, deamination of the dC5-OH oligomer to a putative dU5-OH product during solid-phase DNA synthesis or oligonucleotide deprotection was not detected by any analytical technique employed.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Morningstar
authors.forename: D A
authors.initials: DA
authors.lastname: Kreutzer
authors.forename: J M
authors.initials: JM
authors.lastname: Essigmann
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D008401:Gas Chromatography-Mass Spectrometry; D016296:Mutagenesis; D009841:Oligonucleotides / Q000138:chemical synthesis*; D009943:Organophosphorus Compounds / Q000737:chemistry
chemical_list: D009841:Oligonucleotides; D009943:Organophosphorus Compounds; D003841:Deoxycytidine; C074088:5-hydroxy-2'-deoxyuridine; C074071:5-hydroxy-2'-deoxycytidine; D003857:Deoxyuridine