Design, Synthesis, and Biochemical Evaluation of Alpha-Amanitin Derivatives Containing Analogs of the trans-Hydroxyproline Residue for Potential Use in Antibody-Drug Conjugates.

Alpha-amanitin, an extremely toxic bicyclic octapeptide extracted from the death-cap mushroom, Amanita phalloides, is a highly selective allosteric inhibitor of RNA polymerase II. Following on growing interest in using this toxin as a payload in antibody-drug conjugates, herein we report the synthesis and biochemical evaluation of several new derivatives of this toxin to probe the role of the trans-hydroxyproline (Hyp), which is known to be critical for toxicity. This structure activity relationship (SAR) study represents the first of its kind to use various Hyp-analogs to alter the conformational and H-bonding properties of Hyp in amanitin.

Scaling Amatoxin Synthesis with an Improved Route to (2S,3R,4R)-Dihydroxyisoleucine Exemplified by a Toxic, Clickable α-Amanitin Analogue.

Here we report a scalable synthesis of the key amino acid residue, (2S,3R,4R)-4,5-dihydroxyisoleucine (DHIle) in α-amanitin, that in turn enables the scalable synthesis of an equipotent analogue, Asn(N-ethylazide)-S,6'-dideoxy-α-amanitin, suitable for CuAAC conjugation to empower studies on therapeutic antibody-drug conjugates.

Site-Selective C-H Functionalization of (Hetero)Arenes via Transient, Non-Symmetric Iodanes.

A strategy for C-H functionalization of arenes and heteroarenes has been developed to allow site-selective incorporation of various anions, including Cl, Br, OMs, OTs, and OTf. This approach is enabled by in situ generation of reactive, non-symmetric iodanes by combining anions and bench-stable PhI(OAc)2. The utility of this mechanism is demonstrated via para-selective chlorination of medicinally relevant arenes, as well as site-selective C-H chlorination of heteroarenes. Spectroscopic, computational, and competition experiments describe the unique nature, reactivity, and selectivity of these transient, unsymmetrical iodanes.

Identification of a Small Molecule Anti-biofilm Agent Against Salmonella enterica.

Biofilm formation is a common strategy utilized by bacterial pathogens to establish persistence in a host niche. Salmonella enterica serovar Typhi, the etiological agent of Typhoid fever, relies on biofilm formation in the gallbladder to chronically colonize asymptomatic carriers, allowing for transmission to uninfected individuals. S. enterica serovar Typhimurium utilizes biofilms to achieve persistence in human and animal hosts, an issue of both clinical and agricultural importance. Here, we identify a compound that selectively inhibits biofilm formation in both S. Typhi and S. Typhimurium serovars at early stages of biofilm development with an EC50 of 21.0 and 7.4 µM, respectively. We find that this compound, T315, also reduces biofilm formation in Acinetobacter baumannii, a nosocomial and opportunistic pathogen with rising antibiotic resistance. T315 treatment in conjunction with sub-MIC dosing of ciprofloxacin further reduces S. enterica biofilm formation, demonstrating the potential of such combination therapies for therapeutic development. Through synthesis of two biotin-labeled T315 probes and subsequent pull-down and proteomics analysis, we identified a T315 binding target: WrbA, a flavin mononucleotide-dependent NADH:quinone oxidoreductase. Using a S. Typhimurium strain lacking WrbA we demonstrate that this factor contributes to endogenous S. enterica biofilm formation processes and is required for full T315 anti-biofilm activity. We suggest WrbA as a promising target for further development of anti-biofilm agents in Salmonella, with potential for use against additional bacterial pathogens. The development of anti-biofilm therapeutics will be essential to combat chronic carriage of Typhoid fever and thus accomplish a meaningful reduction of global disease burden.