SUMMARY A desire to better understand the role of voltagegated sodium channels (NaVs) in signal conduction and their dysregulation in specific disease states motivates the development of high precision tools for their study. Nature has evolved a collection of small molecule agents, including the shellfish poison (+)-saxitoxin, that bind to the extracellular pore of select NaV isoforms. As described in this report, de novo chemical synthesis has enabled the preparation of fluorescently labeled derivatives of (+)-saxitoxin, STX-Cy5, and STX-DCDHF, which display reversible binding to NaVs in live cells. Electrophysiology and confocal fluorescence microscopy studies confirm that these STX-based dyes function as potent and selective NaV labels. The utility of these probes is underscored in single-molecule and super-resolution imaging experiments, which reveal NaV distributions well beyond the optical diffraction limit in subcellular features such as neuritic spines and filopodia.

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.DOI: http://dx.doi.org/10.7554/eLife.25174.001

An enantioselective gram-scale synthesis of a key dihydroindolizine intermediate for the preparation of myrmicarin alkaloids is described. Key transformations in this convergent approach include a stereospecific palladium-catalyzed N-vinylation of a pyrrole with a vinyl triflate, a copper-catalyzed enantioselective conjugate reduction of a β-pyrrolyl enoate, and a regioselective Friedel-Crafts reaction. The synthesis of optically active and isomerically pure samples of (4aR)-myrmicarins 215A, 215B, and 217 in addition to their respective C4a-epimers is presented. The myrmicarins are a family of structurally fascinating alkaloids isolated from the poison gland secretions of the African ant species Myrmicaria opaciventris (Figure 1). 1 Despite significant isolation and purification challenges due to air and temperature sensitivity, elegant spectroscopic studies have revealed their molecular structures. 2 The pyrroloindolizine core of myrmicarins 215A (1), 215B (2), and 217 (3) is a common structural motif within many of these alkaloids. Within the family, only the absolute stereochemistry of myrmicarin 237A (4) has been secured through an enantioselective synthesis. 1a,3 Interestingly, the conversion of an unsaturated derivative of 4 to myrmicarin 217 (3) suggests the possible biogenesis of other myrmicarins from simpler indolizine derivatives. 1b,4 The synthesis of (R)-myrmicarin 217 (3) and (R)-myrmicarin 215 as a mixture of olefin isomers has been reported starting with D-glutamic acid. 5 The intriguing molecular structures of these poisonous alkaloids combined with challenges associated with their sensitivity provide an exciting arena to test and discover new methodologies for organic synthesis. Herein we describe a convergent synthesis of all naturally occurring tricyclic myrmicarin alkaloids employing an efficient approach to a pivotal optically active dihydroindolizine intermediate. The first preparation of isomerically pure samples of myrmicarins 215A and 215B is discussed. Key steps of the synthesis include an efficient palladium-catalyzed fragment coupling reaction, a copper catalyzed asymmetric conjugate reduction and a regioselective Friedel-Crafts reaction. We envisioned utilization of the optically active dihydroindolizine 7 as a key intermediate for the preparation of myrmicarin alkaloids (Scheme 1). A regioselective Friedel-Crafts reaction of the pyrrole ring (C7a-alkylation) upon Brönsted-acid activation of the dimethoxyacetal 8 and elimination of methanol was expected to afford the bicyclic vinyl pyrrole 7. We planned to use a metal-catalyzed enantioselective conjugate reduction of the β-pyrrolyl enoate 9 to introduce the C4a-stereochemistry. 6,7 A convergent synthesis of the pyrrolylenoate 9 was envisioned via a metal-catalyzed union of pyrrole 11 and readily available Z-vinyl triflate 10 (Scheme 1). The synthesis of the required β-pyrrolylenoate 9 began with the Claisen condensation of the lithium enolate 12 and methyl 4-(dimethoxy)-butyrate (13) 8 to give the β-ketoester 14 (Scheme 2). The lit...

A stereospecific palladium catalyzed N-vinylation of azaheterocycles with vinyl triflates is described. Cyclic and acyclic vinyl triflates along with non-nucleophilic azaheterocycles were found to be substrates for this palladium catalyzed synthesis of N-vinyl pyrrole and indole derivatives.

Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease. Due to the non-covalent nature of their interactions and their unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. In this manuscript, we present a structurally precise chemoproteomics probe for the biologically active molecule 20( S )-hydroxycholesterol (20( S )-OHC) and provide a map of its proteome-wide targets in the membranes of living cells. Our target catalogue consolidates diverse OHC ontologies and demonstrates that OHC-interacting proteins cluster with specific processes in immune response and cancer. Competition experiments reveal that 20( S )-OHC is a chemo-, regio-, and stereoselective ligand for the protein Tmem97 (the σ2 receptor), enabling us to reconstruct the 20( S )-OHC:Tmem97 binding site. Our results demonstrate that multiplexed, quantitative analysis of cellular target engagement can expose new dimensions of metabolite activity and identify actionable targets for molecular therapy.