Lifetimes and Reaction Pathways of Guanine Radical Cations and Neutral Guanine Radicals in an Oligonucleotide in Aqueous Solutions

摘要

The exposure of guanine in the oligonucleotide 5'-d(TCGCT) to one-electron oxidants leads initially to the formation of the guanine radical cation G~(·+), its deptotonation product G(-H)~·, and, ultimately, various two- and four-electron oxidation products via pathways that depend on the oxidants and reaction conditions. We utilized single or successive multiple laser pulses (308 nm, 1 Hz rate) to generate the oxidants CO_3~(·-) and SO_4~(·-) (via the photolysis of S_2O_8~(2-) in aqueous solutions in the presence and absence of bicarbonate, respectively) at concentrations/pulse that were ~20-fold lower than the concentration of 5'-d(TCGCT). Time-resolved absorption spectroscopy measurements following single-pulse excitation show that the G~(·+) radical (pK_a = 3.9) can be observed only at low pH and is hydrated within 3 ms at pH 2.5, thus forming the two-electron oxidation product 8-oxo-7,8-dihydroguanosine (8-oxoG). At neutral pH, and single pulse excitation, the principal reactive intermediate is G(-H)~·, which, at best, reacts only slowly with H_2O and lives for ~70 ms in the absence of oxidants/other radicals to form base sequence-dependent intrastrand cross-links via the nucleophilic addition of N3-thymidine to C8-guanine (5'-G~*CT~* and 5'-T~*CG~*). Alternatively, G(-H)~· can be oxidized further by reaction with CO_3~(·-), generating the two-electron oxidation products 8-oxoG (C8 addition) and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih, by C5 addition). The four-electron oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), appear only after a second (or more) laser pulse. The levels of all products, except 8-oxoG, which remains at a low constant value, increase with the number of laser pulses.