Designer tetrazine probes for chemical labeling of isocyanide-containing natural products

The identification of novel antibacterial natural products (NPs) is paramount in confronting the global threat of antimicrobial resistance. New NPs with novel core structures and mechanisms of action (MoA) have become significantly more difficult to discover, as modern methods remain largely similar to those employed for decades. An alternative approach to classical bioactivity-guided isolation of NPs is chemical reactivity-guided isolation of NPs. Here, the reactivity of isocyanides with tetrazines via a [4+1] click reaction was exploited to establish a protocol for the detection and isolation of novel isocyanide-containing NPs using carefully designed tetrazine-based probes and contemporary metabolomics methods. Notably, these chemical probes can be used for the direct detection and isolation of primary, secondary, and tertiary isocyanides. Proof-of-concept was achieved, with the help of genome-mining, through the discovery of hazimycin in Kitasatospora purpeofusca. For this underexplored antibacterial agent, we further report on the biosynthesis, total synthesis, structure-activity relationship, metal-binding properties and antibacterial mechanism.