Structure elucidation of colibactin and its DNA cross-links

Mengzhao Xue ¹ Chung Sub Kim ^1,2 Alan R. Healy ^1,2 Kevin M. Wernke ¹ Zhixun Wang ¹ Maddie C. Frischling ¹ Emilee E. Shine ^2,3 Weiwei Wang ^4,5 Jason M. Crawford ^1,2,3 Seth Herzon ^1,6

¹Department of Chemistry, Yale University, New Haven, CT, USA
²Chemical Biology Institute, Yale University, New Haven, CT, USA
³Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA
⁴Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, CT, USA
⁵W. M. Keck Biotechnology Resource Laboratory, Yale School of Medicine, New Haven, CT, USA
⁶Department of Pharmacology, Yale School of Medicine, New Haven, CT, USA

Colibactin is a genotoxic secondary metabolite whose biosynthetic pathway is encoded in the clb gene cluster harbored by certain strains of gut commensal E.coli. The existence of colibactin-producing bacteria correlates with the progression of colorectal cancer in humans. However, to date, colibactin has eluded isolation and conventional structural characterization. In vitro biochemical studies have shown that colibactin’s toxicity arises from generation of DNA interstrand cross-links. Genetic studies indicate that all biosynthetic enzymes in the clb pathway are required to biosynthesize colibactin. Previously, we prepared synthetic colibactin derivatives and established that they alkylate DNA via addition of a nucleotide to an electrophilic cyclopropane. We also characterized the first natural colibactin-(mono)adenine adduct derived from treatment of pUC19 DNA with clb⁺ E. coli. Herein, we used a combination of genetics, isotope labeling, tandem mass spectrometry, and chemical synthesis to deduce the structure of colibactin and the colibactin-(bis)adenine adduct derived from its DNA cross-link products.