ABNOH-Linked Nucleotides and DNA for Bioconjugation and Cross-linking with Tryptophan-Containing Peptides and Proteins

Abstract

Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC reaction of ABNOH-PEG₄-N₃ with 5-ethynyl-dUMP or -dUTP. The modified triphosphate was used as substrate for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The keto-ABNO radical reacted with tryptophan (Trp) and Trp-containing peptides to form a stable tricyclic fused hexahydropyrrolo-indole conjugates. Similarly modified ABNOH-linked nucleotides reacted with Trp-containing peptides to form a stable conjugate in the presence but surprisingly even in the absence of NaNO₂ (presumably through activation by O₂). The reactive ABNOH-modified DNA probe was used for bioconjugations and crosslinking with Trp-containing peptides or proteins.

Keywords: Bioconjugations; N-oxyl radicals; Nucleotides; Oligonucleotides; Peptides.