Total Synthesis and Structure Determination of Mutanolins Detected in a Human Oral Microbiome Symbiont

Abstract Mutanolins are macrocyclic lipopeptides produced in the human oral microbiome by Streptococcus mutans UA159, an etiological agent of caries. Putative structures have been published based on MS/MS analysis of bacterial extracts. Accordingly, the configuration of the natural products was not determined, and the constitutional assignment was not secured spectroscopically. Mutanolins share a common lipidated γ-amino acid fragment that incorporates a β-S-linked cysteine, which had been reported to be elongated at its N-terminus with Glu or Glu–Gly. Herein, we present streamlined total syntheses to elucidate the structures of mutanolins A–F. Regio- and diastereoselective sulfenylchlorinations were key to efficiently access the variable exocyclic peptidic side chains of these natural products. All four C24/C25 diastereomers of mutanolin A were synthesized. Coinjection experiments (UHPLC) of synthetic standards with crude extracts of S. mutans UA159 led to the configurational assignment of mutanolin A as C24(R)C25(R). Mutanolin B was synthesized and assigned as C24(R)C25(R) by analogy to mutanolin A. We then prepared nominal mutanolins C–F; however, the observed MS/MS fragmentation patterns of synthetic nominal mutanolins C–F did not match the reported data. Based on biosynthetic considerations, we speculated that all mutanolins are derived from intermediates in the glutathione biosynthesis. Accordingly, our proposed structures of mutanolins C–F were synthesized and characterized by NMR spectroscopy. The revised assignment was validated by MS/MS and coinjection experiments. By securing the structures of these metabolites, our work provides a starting point for exploring the biological function of the mutanolins as secondary metabolites within the human oral microbiome.