Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine “signature” of probe 7 constitutes an additional advantage resulting in a more confident adduct–amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (−)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

Scheme 1. Convergent synthesis of 14 and 15,precursors to the antibiotic( + +)-monensin (16). The fragment coupling products 14 and 15 were prepared by astereoselective aldol addition employing the aldehydes 12 a or 12 b as electrophile, and the magnesiumenolates derived from the methyl ketones 13 a or 13 b as nucleophile. Bn = benzyl, brsm = based on recovered starting material, TES = triethylsilyl.

The antiproliferative antimicrobial fungal metabolites known as the myrocins have been proposed to cross-link DNA by double nucleotide addition. However, the nature of the DNA-reactive species is ambiguous, as myrocins have been isolated as functionally distinct 5-hydroxy-γ-lactone and diosphenol isomers. Based on literature precedent, we hypothesized that the diosphenol 7 (assigned here the trivial name myrocin G) is the biologically active form of the representative isolate (+)-myrocin C (1). To probe this, we developed a short enantioselective route to 7. A powerful fragment-coupling reaction that forms the central ring of the target in 38% yield and in a single step was developed. In support of our hypothesis, 7 was efficiently transformed to the bis(sulfide) 6, a product previously isolated from reactions of 1 with excess benzenethiol. This work provides the first direct access to the diosphenol 7, sets the stage for elucidating the mode of interaction of the myrocins with DNA, and provides a foundation for the synthesis of other pimarane diterpenes. Efforts to elucidate the mechanism of action of natural products are complicated when the metabolite can adopt two or more functionally distinct forms. This issue is exemplified by the antiproliferative antimicrobial metabolites myrocins C (1) 1 and B (2), 2,3 fungal isolates that contain a sensitive 5-hydroxy-γ-lactone residue (Scheme 1A, blue in 1 and 2). The literature indicates 4 this substructure undergoes facile ring opening to the corresponding diosphenol under mildly acidic or basic conditions, raising uncertainty about its fidelity under biological conditions. Consistent with this, the diosphenol isomer of 2, (−)-myrocin A (3), has been identified in fungal cultures. 5 Following their landmark total synthesis of (±)-myrocin C (1), 6 Chu-Moyer and Danishefsky disclosed that treatment of synthetic (±)-1 with excess thiophenol and triethylamine generated the bis(sulfide) 6 (63%, Scheme 1B). 7 The mechanism for formation of 6 was *

Rational assembly of small molecule libraries for purposes of drug discovery requires an efficient approach in which the synthesis of bioactive compounds is enabled so that numerous structurally related compounds of a similar basic formulation can be derived. Here, we describe (4 + 3) and (3 + 2) indole annulation strategies that quickly generate complex indole heterocycle libraries that contain novel cyclohepta- and cyclopenta[b]indoles, respectively. Screening of one such library comprised of these indoles identifies JWU-A021 to be an especially potent stimulator of glucagon-like peptide-1 (GLP-1) secretion in vitro. Surprisingly, JWU-A021 is also a potent stimulator of Ca2+ influx through TRPA1 cation channels (EC50 ca. 200 nM), thereby explaining its ability to stimulate GLP-1 release. Of additional importance, the available evidence indicates that JWU-A021 is one of the most potent non-electrophilic TRPA-1 channel agonists yet to be reported in the literature.

We describe an enantioselective total synthesis of the nonahydroxylated sesquiterpenoid euonyminol, the dihydro-βagarofuran nucleus of the macrocyclic terpenoid alkaloids known as the cathedulins. Key features of the synthetic sequence include a highly diastereoselective intramolecular alkene oxyalkylation to establish the C10 quaternary center, an intramolecular aldol− dehydration to access the tricyclic scaffold of the target, a tandem lactonization−epoxide opening to form the trans-C2−C3 vicinal diol residue, and a late-stage diastereoselective α-ketol rearrangement. The synthesis provides the first synthetic access to enantioenriched euonyminol and establishes a platform to synthesize the cathedulins.