DNA Damage Induced via 5,6-Dihydrothymid-5-yl in Single-Stranded Oligonucleotides

Abstract

5,6-Dihydrothymid-5-yl (4) is generated via Norrish type I cleavage of isopropyl ketone 7. Ketone 7 was site specifically incorporated into chemically synthesized polythymidylates and an oligonucleotide containing all four native deoxyribonucleotides. No damage is induced in oligonucleotides containing 7 upon photolysis under anaerobic conditions. In the presence of O2, strand breaks and alkaline labile lesions are formed at the original site of 7, and at nucleotides adjacent to the 5‘-phosphate of 7. Kinetic isotope effect experiments reveal that direct strand scission at the thymidine adjacent to the 5‘-phosphate of 4 arises from C1‘ hydrogen atom abstraction. The observed KIE (∼3.9) is attributed to hydrogen atom abstraction from C1‘ by the peroxyl radical 35 derived from 4. Enzymatic end group analysis and measurement of free base release are consistent with a process involving C1‘ hydrogen atom abstraction. Cleavage experiments carried out in the presence of t-BuOH (1.05 M) and NaN3 (10 mM) indicate that damage does not result from hydroxyl radical, but that 1O2 is responsible for a significant amount of the observed strand damage.

Reactivity of Nucleic Acid Radicals

“…Formal hydrogen atom addition adducts of thymidine ( 80 ) and 2′-deoxyuridine ( 83 ) have been produced using these types of precursors, as has the C5-thymidine HO• adduct ( 76 , Scheme 35 ). 46,233,242–251 In each instance, the chemical integrity of the photochemical precursor with respect to generating the respective radical is characterized at the nucleoside level using standard product analysis conditions.…”

“…243 Strand scission is reduced approximately fourfold when the 5′-adjacent nucleotide is deuterated at the C1′-position. 242 Deuteration of the C2′-, or C4′-positions has no effect suggesting that peroxyl radical 86 selectively abstracted the C1′-hydrogen atom from the 5′-adjacent nucleotide (Scheme 36 ). Additional support for internucleotidyl C1′-hydrogen atom abstraction was obtained when the tandem lesion containing 2-deoxyribonolactone ( 15 ) was observed in low yield when 80 was generated from 82 in a dinucleotide under aerobic conditions (Scheme 36 ).…”

Pyrimidine nucleobase radical reactivity in DNA and RNA

“…The C5-thymidine HO• adduct (1 from 5, Scheme 2) has been studied using this approach, as have a number of formal hydrogen atom addition adducts of pyrimidine 2′-deoxy- and ribonucleosides. 15–26 Norrish Type I cleavage of ketones (7, 10, 12, 14) has been the most common strategy (Scheme 3), although aryl sulfides (5) and phenyl selenides (8) have also been used (Schemes 2, 3). In several investigations the formal hydrogen atom adducts were generated.…”

“…In the absence of O 2 1, 6, or 9 do not lead to direct strand breaks or alkali-labile lesions, consistent with the ≥19 kcal/mol barrier predicted for hydrogen atom abstraction. 15, 17, 19, 20, 22, 24, 51, 52 Direct strand breaks were detected at the 5′-adjacent nucleotide when the 5,6-dihydrothymidin-5-yl radical (6) was produced under aerobic conditions. 15, 17 However, the direct strand break yield is very low.…”

“…In contrast, deuteration of the C1′-position of the 5′-adjacent nucleotide reduced strand scission ~4-fold. 15 The observed direct strand scission could have been an artifact of sample handling, as C1′-oxidation typically produces alkali-labile 2-deoxyribonolactone (20). 55–57 Product analysis of photolysis of a dinucleotide (8) provided additional support for internucleotidyl C1′-hydrogen atom abstraction by 19.…”