Generation of guanine–amino acid cross-links by a free radical combination mechanism

A direct method has been developed for the in vitro synthesis of stable DNA–protein cross-links (DPC's) between guanine and amino acids (lysine and arginine). This method employs the combination of guanine neutral radicals, G(–H)˙, and side-chain C-centered amino acid radicals. The latter were generated indirectly after first causing the selective photoionization of 2-aminopurine (2AP) embedded in the oligonucleotide, 5′-d(CC[2AP]TCGCTACC), by intense nanosecond 308 nm excimer laser pulses. The 2AP radical cation deprotonates rapidly to form the 2AP(–H)˙ neutral radical which, in turn, oxidizes the nearby guanine to form the neutral guanine G(–H)˙ radical, as described previously (Shafirovich et al., J. Phys. Chem. B, 2001, 105, 8431). In parallel, the hydrated electrons, generated by the photoionization of 2AP, are scavenged by nitrous oxide to generate hydroxyl radicals. In the presence of a large excess of the amino acids, the hydroxyl radicals oxidize the latter to produce C-centered amino acid radicals that combine with the G(–H)˙ radicals to form the guanine–amino acid cross-linked oligonucleotide product. Analogous products were generated by photoionizing the free nucleoside, 2′,3′,5′-tri-O-acetylguanosine, (tri-O-Ac-Guo), using intense nanosecond 266 nm Nd:YAG laser pulse irradiation. The guanine–amino acid cross-links thus produced site-specifically positioned either in oligonucleotides, or in the free nucleoside tri-O-Ac-Guo were isolated by HPLC methods and identified by high resolution LC-TOF/MS and LC-MS/MS methods. The possibility that analogous guanine–amino acid cross-linked products could be formed in vivo using single hit radical generation mechanisms during oxidative stress is discussed.