Oxygen independent DNA interstrand cross-link formation by a nucleotide radical

Abstract

A 5-(2'-Deoxyuridinyl)methyl radical (1) was independently generated from three photochemical precursors and is the first example of a DNA radical that forms interstrand cross-links. Oxygen labeling experiments support generation of 1 by all precursors. Interstrand cross-links are produced upon irradiation of DNA containing any of the precursors. Cross-linking occurs via reaction with the opposing 2'-deoxyadenosine and is independent of O(2). The independence of cross-link formation on O(2) is explained by kinetic analysis, which shows that the radical reacts reversibly with O(2). Examination of the effects of glutathione on cross-link formation under anaerobic conditions suggests that adoption of the syn-conformation by 1 is the rate-limiting step in the process. Interstrand cross-link formation is reversible in the presence of a good nucleophile. The stability of the interstrand cross-link suggests that the isolated molecule is a rearrangement product of that formed in solution. The rearrangement is a consequence of the isolation procedure but also occurs slowly in solution. Oxygen independent cross-link formation may be useful for the purposeful damage of DNA in hypoxic tumor cells, where O(2) is deficient.

Scheme 1

Generation of, and Interstrand Cross-Link Formation by the 5-(2′-Deoxyuridinyl)methyl Radical (1)

Scheme 2

Trapping Products Produced from the 5-(2′-Deoxyuridinyl)methyl Radical (1)

Scheme 3

Reversible Trapping of the 5-(2′-Deoxyuridinyl)methyl Radical (1) by O₂

Scheme 4

Interstrand Cross-Link Formation via Methide (20) Formation

Scheme 5

Detecting Reversibility of Interstrand Cross-Linking via Trapping with Azide

Scheme 6

Competition between ISC Formation and GSH Trapping of 1

Scheme 7

ISC Formation via Rotation about the Glycosidic Bond in the 5-(2′-Deoxyuridinyl)methyl Radical (1)

Figure 1

Product dependence of monomeric 5-(2′-deoxyuridinyl)methyl radical (1) derived from 12 under aerobic conditions as a function of GSH concentration.

Figure 2

Stability of ISC from 14 in the absence and presence of NaN₃ (0.3 M) or GSH (5 mM). (A) Photochemically generated ISC. (B) NaIO₄ generated ISC. No additive (■), GSH (▲), NaN₃ ([unk]).

Figure 3

Dependence of the observed rate constant for the disappearance of ISC (k_Obs) on NaN₃ concentration.

Figure 4

ISC stability to heating (90 °C, 30 min) upon incubation in aqueous buffer.

Figure 5

Ratio of ssDNA:ISC produced from photolysis of 14 as a function of GSH concentration.