α-Amanitin, a highly toxic bicyclic octapeptide extracted from death-cap mushroom (Amanita phalloides), is one of the deadliest natural compounds known, with an LD50 of 50–100 μg/kg. It is a potent inhibitor of RNA polymerase II (Ki 1−10 nM), an enzyme critical for cellular function and survival, thereby affecting both dividing and quiescent cells. Due to its stability, potency, and unique mechanism, it shows promise as a payload for antibody-drug conjugates (ADCs) in cancer therapy. However, current extraction methods yield low amounts, underscoring the need for a synthetic production route.

The synthesis of di-hydroxy isoleucine (DHIle), a vital component, is particularly challenging. We successfully achieved a gram-scale synthesis of DHIle, streamlining the overall process and generating intermediates that were synthetically modified to produce 12 novel amanitin analogs. These analogs, compatible with solid-phase peptide synthesis, were utilized in SAR studies, revealing compounds with similar potency and selective toxicity toward HEK-293 cells. This work also introduced new bioconjugation handles for enhancing ADC applications with increased cytotoxicity and selectivity over liver-derived HepG2 cells, improving amanitin’s therapeutic index for cancer treatment. This work led to the development of new ADCs with efficacy comparable to those currently available on the market.