Generation of DNA interstrand cross-links by post-synthetic reductive amination

Abstract

DNA interstrand cross-links (ICLs) are the clinically most relevant adducts formed by many antitumor agents. To facilitate the study of biological responses triggered by ICLs, we developed a new approach toward the synthesis of mimics of nitrogen mustard ICLs. 7-Deazaguanine residues bearing acetaldehyde groups were incorporated into complementary strands of DNA and cross-link formation induced by double reductive amination. Our strategy enables the synthesis of major groove cross-links in high yields and purity.

Figure 1

Structures of NM ICLs 1 and strategy for the synthesis of NM ICL analog 2. NM ICLs (1) connect two complementary DNA strands by connecting two guanine bases through the N(7) positions. Our target NM ICL mimic 2 has the nitrogen at the 7 positions replaced with carbon to render the glycosidic bond of the ICL stable for synthesis and functional studies (X = amine-containing compound). We envisioned that ICL 2 could be obtained via double reductive amination of an aldehyde 3 with an appropriate amine, XH. 3 in turn could be derived from phosphoramidite 4, where the aldehyde is masked as a protected diol.

Scheme 1

Synthesis of the ICL precursor phosphoramidite 4

Figure 2

Synthesis of DNA ICLs through reductive amination. A. Phosphoramidite 4 was incorporated into complementary strands of DNA and deprotected (10a-d). Following annealing and oxidization with NaIO₄ to aldehydes 11a-d, reaction with ammonium choride, methylamine, hydrazine, ethylene diamine or dimethylethylene diamine followed by reduction with NaBH₃CN was attempted to generate ICLs 12. B. Structures of the target ICLs 12a-h. C. Denaturing PAGE analysis of the reductive amination reaction of the two aldheyde containing 20-mers. The amine group X resulting from the reaction, as well as presence or absence of NaIO₄ and NaBH₃CN in the reaction mixture are indicated above the gel. The positions of marker 20-mer (for ssDNA) and 40-mer (for ICL containing DNA) oligomers are indicated. The gel was stained with methylene blue.

Figure 3

Structure of B-from DNA highlighting the distance linked by a NM ICL. The distance between the two crosslinked N7 atoms of guanine is marked by a red dotted line. The nominal distance of an amine, hydrazine and ethylene diamine ICLs are indicated in the right panel.