Biosynthesis of Largimycins in Involves Transient β-Alkylation and Cryptic Halogenation Steps Unprecedented in the Leinamycin Family.

Largimycins A1 and A2 are key members of a recently identified family of hybrid nonribosomal peptide polyketides belonging to the scarcely represented group of antitumor leinamycins. They are encoded by the gene cluster of . This cluster contains a halogenase gene and two sets of genes for the biosynthesis and incorporation of β branches at C3 and C9. Noticeably, largimycins A1 and A2 are nonhalogenated compounds and only contain a β branch at C3. By generating mutants in those genes and characterizing chemically their accumulated compounds, we could confirm the existence of a chlorination step at C19, the introduction of an acetyl-derived olefinic exomethylene group at C9, and a propionyl-derived β branch at C3 in the biosynthesis pathway. Since the olefinic exomethylene group and the chlorine atom are absent in the final products, those biosynthetic steps can be considered cryptic in the overall pathway but essential to generating keto and epoxide functionalities at C9 and C18/C19, respectively. We propose that chlorination at C19 is utilized as an activation strategy that creates the precursor halohydrin to finally yield the epoxy functionality at C18/C19. This represents a novel strategy to create such functionalities and extends the small number of natural product biosynthetic pathways that include a cryptic chlorination step.