Synthesis and properties of an acetaldehyde-derived oligonucleotide interstrand cross-link.

Acetaldehyde (AA), occurring widely in the human environment, is a mutagen and carcinogen. AA can react with DNA to form AA-DNA adducts. Several types of adducts, including an interstrand cross-link 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-(N2-deoxyguanosyl)-6-methylpyrimido[1,2-a]purine-10(3H)one (7), have been previously characterized by our laboratory. We hypothesize that cross-link 7 may be involved in determining the mutagenic and carcinogenic properties of AA. To address this question, the double-stranded oligonucleotide 13, bearing cross-link 7, was synthesized in a sequence appropriate for mutagenicity studies in human cells. Oligonucleotide 9, containing 2-fluoro-O6-(trimethylsilylethyl)deoxyinosine (dIno), was reacted with 4-amino-1,2-pentanediol, followed by treatment with NaIO4. The resulting oligonucleotide 11 containing the 1,N2-propano-deoxyguanosine (dGuo) 5 was incubated with the complementary oligonucleotide 12 to give the desired cross-link 13, which was characterized by negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) and enzymatic hydrolysis to cross-link 7. The formation of cross-link 13 at 5'-CpG-3' was confirmed by incubation of 11 with [15N5]12 containing a 5'-Cp[15N5]G-3' sequence. The formation of cross-link 13 was reversible. Therefore, oligonucleotide 24 containing the irreversible analogue of cross-link 7, 1,3-bis(2'-deoxyguanos-N2-yl)butane, was synthesized for use as a control in the mutagenicity studies. Oligonucleotide 21 was reacted with 1,3-diaminobutane dihydrochloride, followed by incubation with the complementary oligonucleotide 23, to give 24. To determine the optimum distance and orientation for cross-link formation, six oligonucleotides, containing 5 at the i + 1, i + 2, and i + 3 or the i - 1, i - 2, and i - 3 positions relative to dGuo in the complementary strand, were 5'-end labeled with [gamma-32P]ATP, followed by incubation of each labeled oligonulceotide with its complementary strand and then analysis by denaturing polyacrylamide gel electrophoresis. Only the oligonucleotide containing 5'-Cp5-3' formed the cross-link with the complementary 5'-CpG-3' sequence. The results of this study confirm the structure of an AA-derived DNA cross-link, supply characterized cross-link-containing oligonucleotides for mutagenicity studies, and provide information on the optimum distance and orientation for cross-link formation.