Synthetic biological approaches,
such as site-directed biosynthesis,
have contributed to the expansion of the chemical space of natural
products, making possible the biosynthesis of unnatural metabolites
that otherwise would be difficult to access. Such methods may allow
the incorporation of fluorine, an atom rarely found in nature, into
complex secondary metabolites. Organofluorine compounds and secondary
metabolites have both played pivotal roles in the development of drugs;
however, their discovery and development are often via nonintersecting
tracks. In this context, we used the biosynthetic machinery of Trichoderma arundinaceum (strain MSX70741) to incorporate
a fluorine atom into peptaibol-type molecules in a site-selective
manner. Thus, fermentation of strain MSX70741 in media containing ortho- and meta-F-phenylalanine resulted
in the biosynthesis of two new fluorine-containing alamethicin F50
derivatives. The fluorinated products were characterized using spectroscopic
(1D and 2D NMR, including 19F) and spectrometric (HRESIMS/MSn) methods, and their absolute configurations were established
by Marfey’s analysis. Fluorine-containing alamethicin F50 derivatives
exhibited potency analogous to the nonfluorinated parent when evaluated
against a panel of human cancer cell lines. Importantly, the biosynthesis
of fluorinated alamethicin F50 derivatives by strain MSX70741 was
monitored in situ using a droplet–liquid microjunction–surface
sampling probe coupled to a hyphenated system.