Terminal amide functionality is prevalent in nature and significantly contributes to both biological activity and chemical stability of compounds. Terminal amidation is frequently utilized as a strategy in medicinal chemistry for lead modification to achieve improved bioactivity and bioavailability, thus this functionality is often present in drugs on the market (e.g., Rebetol®, Viberzi®, and Adlyxin®). Such wide occurrence and pharmaceutical application of terminal amidation drew our attention to thiotetroamide (TTM), a molecule being studied for its biosynthesis as part of our continuing efforts towards antibiotics discovery. TTM is the sole member that bears a terminal amide functionality within the thiolactomycin (TLM) class of antibiotics, and consequently exhibits enhanced bioactivity compared to other TLMs lacking terminal amidation. In addition, bioinformatics analyses suggested that the terminal amide formation in TTM involves a selective carboxylation of a saturated alkyl group, which is a challenging feat in synthetic chemistry and is also distinct from previously established enzymatic amidation processes. Therefore, the enhanced bioactivity and the unusual enzymology motivated the investigation of this unique tandem carboxylation-amidation enzymatic strategy to utilize it for the production of unnatural and potent TTM analogues. First, targeted in vivo gene disruption results supported the hypothesis that a unique enzyme pair consisting of a cytochrome P450 and an amidotransferase was involved in the biosynthesis of the terminal amide group. Next, in vitro enzymatic analysis showed that the recombinant cytochrome P450 selectively oxidized a terminal methyl group to ultimately form a carboxylic acid via a rare triple oxidation. This carboxylic acid was isolated and structurally characterized, and was subsequently investigated as a substrate for the specific amidotransferase, which successfully generated the final amide product. Furthermore, these two enzymes were assayed together and demonstrated the coupled carboxylation-amidation process. After the characterization of these two enzymes, structurally diverse TLM analogs are being used to probe the substrate specificity, which has so far resulted in the production of two unnatural TTM analogues with potentiated bioactivity. This suggested a potential of engineering this unique enzyme pair as a useful avenue to modify the drug-like properties of lead molecules in discovery programs.

Enzymatic tandem carboxylation-amidation as a bioactivity-potentiating strategy in the production of natural and unnatural thiolactomycin antibiotics

253rd National Meeting (2017)

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