Spirodi(iminohydantoin) Products from Oxidation of 2′-Deoxyguanosine in the Presence of NH₄Cl in Nucleoside and Oligodeoxynucleotide Contexts

Abstract

Upon oxidation of the heterocyclic ring in 2′-deoxyguanosine (dG), the initial electrophilic intermediate displays a wide range of reactivities with nucleophiles leading to many downstream products. In the present study, the product profiles were mapped when aqueous solutions of dG were allowed to react with NH4Cl in the presence of the photooxidants riboflavin and Rose Bengal as well as the diffusible one-electron oxidant Na2IrCl6. Product characterization identified the 2′-deoxyribonucleosides of spiroiminodihydantoin, 5-guanidinohydantoin, and oxazolone resulting from H2O as the nucleophile. When NH3 was the nucleophile, a set of constitutional isomers that are diastereotopic were also observed, giving characteristic masses of dG + 31. ESI+-MS/MS of these NH3 adducts identified them to be spirocycles with substitution of either the C5 or C8 carbonyl with an amine. The NH3 adducts exhibit acid-catalyzed hydrolysis to spiroiminodihydantoin. Quantification of the NH3 and H2O adducts resulting from oxidation of dG in the nucleoside, single-stranded, and duplex oligodeoxynucleotide contexts were monitored allowing mechanisms for product formation to be proposed. These data also provide a cautionary note to those who purify their oligonucleotide samples with ammonium salts before oxidation because this will lead to unwanted side reactions in which ammonia participates in product formation.

Capturing Transient Endoperoxide in the Singlet Oxygen Oxidation of Guanine

“…It is necessary to examine similarities and differences between the 1 O 2 oxidation of free guanine and that of the guanosine nucleosides in DNA, so that we may extrapolate gas‐phase findings to solution models. Scheme outlines the oxidation products for dGuo+ 1 O 2 in aqueous solution ––. For clarity, we omitted products produced in low yields or those observed occasionally ,…”

“…The first step toward degradation of this highly active 4,8‐OO‐dGuo is rearrangement into 8‐OOHdGuo. Subsequent loss of a water molecule from 8‐OOHdGuo leads to an quinonoid intermediate, the C5=N7 bond of which is susceptible to nucleophilic attack –,. The addition of water to C5=N7 yields 5‐OH‐8‐oxodGuo, the fate of which depends on the reaction pH and temperature.…”

“…This process becomes feasible only in the presence of a reactive water molecule. Such a scenario is similar to the mechanism proposed for the oxidation of neutral guanosine by Burrows and co‐workers …”

Computational Study of Oxidation of Guanine by Singlet Oxygen (1Δg) and Formation of Guanine:Lysine Cross‐Links

“…A large number of experimental studies have investigated oxidative damage of isolated nucleobases, single‐stranded and double‐stranded DNA by singlet oxygen . It is widely accepted that the singlet oxygen addition to the guanine in aqueous solution results in the formation of a guanine endoperoxide intermediate (Scheme ).…”

“…Alternatively, reduction of 8‐OOH‐G produces the widely known guanine oxidation intermediate 8‐oxoG , which then can react with O 2 or undergo further oxidation to produce oxidized products such as Sp , guanidinohydantoin ( Gh ), and 2,5‐diamino‐4 H ‐imidazolone ( Iz ) . Different reaction conditions such as change in pH, presence of other nucleophiles and additional oxidizing agents along with the singlet oxygen can increase the diversity of the final products . Even solvent water can act as a nucleophile to react with oxidized guanine intermediates to produce Sp and other further oxidized products .…”