Abstract
Late-stage functionalization of natural products offers an elegant route to create novel entities in a relevant biological target space. In this context, enzymes capable of halogenating sp3 carbons with high stereo- and regiocontrol under benign conditions have attracted particular attention. Enabled by a combination of smart library design and machine learning, we engineer the iron/α-ketoglutarate dependent halogenase WelO5* for the late-stage functionalization of the complex and chemically difficult to derivatize macrolides soraphen A and C, potent anti-fungal agents. While the wild type enzyme WelO5* does not accept the macrolide substrates, our engineering strategy leads to active halogenase variants and improves upon their apparent kcat and total turnover number by more than 90-fold and 300-fold, respectively. Notably, our machine-learning guided engineering approach is capable of predicting more active variants and allows us to switch the regio-selectivity of the halogenases facilitating the targeted analysis of the derivatized macrolides’ structure-function activity in biological assays.
Highlights
Late-stage functionalization of natural products offers an elegant route to create novel entities in a relevant biological target space
While soraphen A can be accessed through an optimized bioprocess[47,51], its penultimate biosynthetic congener soraphen C is a much less explored member of the soraphen family and very difficult to isolate in sufficient amounts from fermentation despite its value as a chemical probe[52]
Our most active engineered variant WelO5* VLA catalyzes the halogenation of the macrolide 1 to yield product 1a with an apparent kcat value and a total turnover number which mirror the activity of wild type aliphatic halogenases for their natural substrate[12] highlighting the malleability of WelO5*’s active site and underlining the effectiveness of our engineering strategy
Summary
Late-stage functionalization of natural products offers an elegant route to create novel entities in a relevant biological target space. In this context, enzymes capable of halogenating sp[3] carbons with high stereo- and regiocontrol under benign conditions have attracted particular attention. Late-stage functionalization (LSF) of C–H bonds offers direct access to new analogs of a lead structure In this way, LSF constitutes a valuable tool to investigate structure-activity relationships of small molecules, especially natural products[4], and supports the optimization of on-target potency, selectivity, and absorption-distribution-metabolism-excretion (ADME) properties while helping to improve physical properties such as solubility and stability. A handful of αKGHs have been described: The carrierprotein dependent halogenases BarB1 and BarB215, SyrB216, CytC317, CmaB18, HctB19, CurA20 and the synthetically more interesting freestanding halogenases WelO521, WelO5*22, WiWelO1523, AmbO524, the BesD25 family, the recently identified plant halogenases SaDAH and McDAH26 as well as the halogenase AdeV27, which acts on nucleotide substrates
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