A significant challenge toward studies of the human microbiota involves establishing causal links between bacterial metabolites and human health and disease states. Certain strains of commensal Escherichia coli harbor the 54-kb clb gene cluster which codes for small molecules named precolibactins and colibactins. Several studies suggest colibactins are genotoxins and support a role for clb metabolites in colorectal cancer formation. Significant advances toward elucidating the structures and biosynthesis of the precolibactins and colibactins have been made using genetic approaches, but their full structures remain unknown. In this Perspective we describe recent synthetic efforts that have leveraged biosynthetic advances and shed light on the mechanism of action of clb metabolites. These studies indicate that deletion of the colibactin peptidase ClbP, a modification introduced to promote accumulation of precolibactins, leads to the production of non-genotoxic pyridone-based isolates derived from the diversion of linear biosynthetic intermediates toward alternative cyclization pathways. Furthermore, these studies suggest the active genotoxins (colibactins) are unsaturated imines that are potent DNA damaging agents, thereby confirming an earlier mechanism of action hypothesis. Although these imines have very recently been detected in bacterial extracts, they have to date confounded isolation. As the power of "meta-omics" approaches to natural products discovery further advance, we anticipate that chemical synthetic and biosynthetic studies will become increasingly interdependent.