Identification and characterization of a novel cross-link lesion in d(CpC) upon 365-nm irradiation in the presence of 2-methyl-1,4-naphthoquinone.

We report the isolation and characterization for the first time of a cross-link lesion between two adjacent cytosines from the 2-methyl-1,4-naphthoquinone (menadione)-sensitized 365-nm irradiation of d(CpC). Electrospray ionization mass spectrometry (ESI-MS), tandem MS and (1)H NMR results indicate that the cross-link occurs between the C5 carbon atom of one cytosine and the N(4) nitrogen atom of the other cytosine. Furthermore, we synthesized d(CpC) with a (15)N being incorporated on the amino group of either of the two cytosines. We then irradiated the two (15)N-labeled dinucleoside monophosphates, isolated the cross-link products and characterized them by MS and multi-stage tandem MS. The latter results established unambiguously that the N(4) nitrogen atom of the 3'-nucleobase is involved in the covalent bond formation between the two cytosines. This, in combination with two-dimensional nuclear Overhauser effect spectroscopy (NOESY) results, demonstrates that the cross-link arises from the formation of a covalent bond between the C5 carbon atom of the 5' cytosine and the N(4) nitrogen atom of the 3' cytosine. We also show that the solution pH has a significant effect on the formation of the cross-link lesion, which supports that the deprotonation at the exocyclic amino group of cytosine cation radical is essential for the formation of the cross-link lesion.