Synthesis and Characterization of Site‐Specific O 6 ‐Alkylguanine DNA‐Alkyl Transferase‐Oligonucleotide Crosslinks

O 6 ‐Alkylguanine DNA‐alkyltransferase (AGT), a DNA repair protein, can form crosslinks with DNA. The AGT‐DNA crosslinks are known to be mutagenic when AGT is heterologously expressed in Escherichia coli , as well as in mammalian cells. To understand the biological consequences, reliable access to AGT‐oligonucleotide crosslinks is needed. This article describes the synthesis and characterization of site‐specific AGT‐oligonucleotide crosslinks at the N2‐position of deoxyguanosine and N6‐position of deoxyadenosine. We developed a post‐oligomerization strategy for the synthesis of propargyl‐modified oligonucleotides. Copper‐catalyzed azide‐alkyne cycloaddition was used as a key step to obtain the iodoacetamide‐linked oligonucleotides, which serve as good electrophiles for the crosslinking reaction with cysteine‐145 of the active site of AGT. Trypsinization of AGT and hydrolysis of oligonucleotides, combined with analysis by liquid chromatography‐tandem mass spectrometry, was utilized to confirm the nucleobase‐adducted peptides. This method provides a useful strategy for the synthesis and characterization of site‐specific DNA‐protein crosslinks, which can be further used to understand proteolytic degradation–coupled DNA repair mechanisms. © 2019 by John Wiley & Sons, Inc.