The conformational landscape of human transthyretin revealed by cryo-EM.

Transthyretin (TTR) is a natively tetrameric thyroxine transporter found in blood and cerebrospinal fluid whose misfolding and aggregation causes transthyretin amyloidosis. A rational drug design campaign identified the small molecule tafamidis (Vyndaqel/Vyndamax) as an effective stabilizer of the native TTR fold, and this aggregation inhibitor is regulatory agency-approved for the treatment of TTR amyloidosis. Despite 50 years of structural studies on TTR and this triumph of structure-based drug design, there remains a notable dearth of structural information available to understand ligand binding allostery and amyloidogenic TTR unfolding intermediates. We used single-particle cryo-electron microscopy (cryo-EM) to investigate the conformational landscape of this 55 kiloDalton tetramer in the absence and presence of one or two ligands, revealing inherent asymmetries in the tetrameric architecture and previously unobserved conformational states. These findings provide critical mechanistic insights into negatively cooperative ligand binding and the structural pathways responsible for TTR amyloidogenesis. This study underscores the capacity of cryo-EM to provide new insights into protein structures that have been historically considered too small to visualize and to identify pharmacological targets suppressed by the confines of the crystal lattice, opening uncharted territory in structure-based drug design.

Enantioselective Hydroalkenylation and Hydroalkynylation of Alkenes Enabled by a Transient Directing Group: Catalyst Generality through Rigidification.

The catalytic enantioselective synthesis of α-chiral alkenes and alkynes represents a powerful strategy for rapid generation of molecular complexity. Herein, we report a transient directing group (TDG) strategy to facilitate site-selective palladium-catalyzed reductive Heck-type hydroalkenylation and hydroalkynylation of alkenylaldehyes using alkenyl and alkynyl bromides, respectively, allowing for construction of a stereocenter at the δ-position with respect to the aldehyde. Computational studies reveal the dual beneficial roles of rigid TDGs, such as L-tert-leucine, in promoting TDG binding and inducing high levels of enantioselectivity in alkene insertion with a variety of migrating groups.

Structurally Diverse Bench-Stable Nickel(0) Pre-Catalysts: A Practical Toolkit for In Situ Ligation Protocols.

A flurry of recent research has centered on harnessing the power of nickel catalysis in organic synthesis. These efforts have been bolstered by contemporaneous development of well-defined nickel (pre)catalysts with diverse structure and reactivity. In this report, we present ten different bench-stable, 18-electron, formally zero-valent nickel-olefin complexes that are competent pre-catalysts in various reactions. Our investigation includes preparations of novel, bench-stable Ni(COD)(L) complexes (COD=1,5-cyclooctadiene), in which L=quinone, cyclopentadienone, thiophene-S-oxide, and fulvene. Characterization by NMR, IR, single-crystal X-ray diffraction, cyclic voltammetry, thermogravimetric analysis, and natural bond orbital analysis sheds light on the structure, bonding, and properties of these complexes. Applications in an assortment of nickel-catalyzed reactions underscore the complementary nature of the different pre-catalysts within this toolkit.

Ru-Catalyzed Enantioselective Hydrogenation of 2-Pyridyl-Substituted Alkenes and Substrate-Mediated H/D Exchange.

A highly efficient and enantioselective asymmetric hydrogenation catalyzed by Ru-DTBM-segphos is reported for a broad range of pyridine-pyrroline tri-substituted alkenes. Kinetic, spectroscopic, and computational studies suggest that addition of H2 is rate-determining and that alkene insertion is the enantio-determining step. These studies also reveal an intriguing Ru-catalyzed H/D exchange process that is facilitated by the substrate at room temperature and low pressure where hydrogenation activity is suppressed. These studies lead to a mechanistic proposal that further defines the roles of hydrogen gas, Ru-H species, and protic solvents in this catalytic system.

Three-Component Asymmetric Ni-Catalyzed 1,2-Dicarbofunctionalization of Unactivated Alkenes via Stereoselective Migratory Insertion.

An asymmetric 1,2-dicarbofunctionalization of unactivated alkenes with aryl iodides and aryl/alkenylboronic esters under nickel/bioxazoline catalysis is disclosed. A wide array of aryl and alkenyl nucleophiles are tolerated, furnishing the products in good yield and with high enantioselectivity. In addition to terminal alkenes, 1,2-disubstituted internal alkenes participate in the reaction, establishing two contiguous stereocenters with high diastereoselectivity and moderate enantioselectivity. A combination of experimental and computational techniques shed light on the mechanism of the catalytic transformation, pointing to a closed-shell pathway with an enantiodetermining migratory insertion step, where stereoinduction arises from synergistic interactions between the sterically bulky achiral sulfonamide directing group and the hemilabile bidentate ligand.

Low-valent tungsten redox catalysis enables controlled isomerization and carbonylative functionalization of alkenes.

The controlled isomerization and functionalization of alkenes is a cornerstone achievement in organometallic catalysis that is now widely used throughout industry. In particular, the addition of CO and H2 to an alkene, also known as the oxo-process, is used in the production of linear aldehydes from crude alkene feedstocks. In these catalytic reactions, isomerization is governed by thermodynamics, giving rise to functionalization at the most stable alkylmetal species. Despite the ubiquitous industrial applications of tandem alkene isomerization/functionalization reactions, selective functionalization at internal positions has remained largely unexplored. Here we report that the simple W(0) precatalyst W(CO)6 catalyses the isomerization of alkenes to unactivated internal positions and subsequent hydrocarbonylation with CO. The six- to seven-coordinate geometry changes that are characteristic of the W(0)/W(II) redox cycle and the conformationally flexible directing group are key factors in allowing isomerization to take place over multiple positions and stop at a defined unactivated internal site that is primed for in situ functionalization.

Total Synthesis of Kibdelomycin.

A modular total synthesis of kibdelomycin is disclosed that should enable structure-activity relationship (SAR) studies of this interesting class of antibiotics. The route uses simple building blocks and addresses lingering questions about its structural assignment and relationship to amycolamicin, a recently described natural product reported to have a similar structure. Initial antibacterial assays reveal that both C-22 epimers (the N-glycosidic linkage) of the natural product have similar activity while structurally truncated analogs lose activity.

Convergent total synthesis of (+)-calcipotriol: A scalable, modular approach to vitamin D analogs.

A convergent approach for the total synthesis of calcipotriol (brand name: Dovonex), a proven vitamin D analog used for the treatment of psoriasis, and medicinally relevant synthetic analogs is described. A complete approach, not wedded to semisynthesis, toward both the A-ring and CD-ring is reported. From a retrosynthetic standpoint, hidden symmetry within the decorated A-ring is disclosed, which allowed for scalable quantities of this advanced intermediate. In addition, a radical retrosynthetic approach is described, which highlights an electrochemical reductive coupling as well as an intramolecular hydrogen atom transfer Giese addition to establish the 6,5-transcarbon skeleton found in the vitamin D family. Finally, a late-stage decarboxylative cross-coupling approach allowed for the facile preparation of various C20-arylated derivatives that show promising biological activity in an initial bioassay.

Kinetic and Thermodynamic Considerations in the Rh-Catalyzed Enantioselective Hydrogenation of 2-Pyridyl-Substituted Alkenes.

The mechanism of asymmetric hydrogenation of 2-pyridyl alkenes catalyzed by chiral Rh-phosphine complexes at ambient temperature is examined using kinetic, spectroscopic, and computational tools. The reaction proceeds with reversible substrate binding followed by rate-determining addition of hydrogen. Substrate binding occurs only through the pyridine nitrogen in contrast to other substrate classes exhibiting stronger substrate direction. The lack of influence of hydrogen pressure on the product enantiomeric excess suggests that a pre-equilibrium in substrate binding is maintained across the pressure range investigated. An off-cycle Rh-hydride species is implicated in the mild catalyst deactivation observed. In contrast to Ru-phosphine-catalyzed reactions of the same substrate class, the stereochemical outcome in this system correlates generally with the relative stability of the E and Z rotamers of the substrate.

Mild and Chemoselective Phosphorylation of Alcohols Using a Ψ-Reagent.

An operationally simple, scalable, and chemoselective method for the direct phosphorylation of alcohols using a P(V)-approach based on the Ψ-reagent platform is disclosed. The method features a broad substrate scope of utility in both simple and complex settings and provides access to valuable phosphorylated alcohols that would be otherwise difficult to obtain.

Use of Crystallography and Molecular Modeling for the Inhibition of the Botulinum Neurotoxin A Protease.

Botulinum neurotoxins (BoNTs) are extremely toxic and have been deemed a Tier 1 potential bioterrorism agent. The most potent and persistent of the BoNTs is the "A" serotype, with strategies to counter its etiology focused on designing small-molecule inhibitors of its light chain (LC), a zinc-dependent metalloprotease. The successful structure-based drug design of inhibitors has been confounded as the LC is highly flexible with significant morphological changes occurring upon inhibitor binding. To achieve greater success, previous and new cocrystal structures were evaluated from the standpoint of inhibitor enantioselectivity and their effect on active-site morphology. Based upon these structural insights, we designed inhibitors that were predicted to take advantage of π-π stacking interactions present in a cryptic hydrophobic subpocket. Structure-activity relationships were defined, and X-ray crystal structures and docking models were examined to rationalize the observed potency differences between inhibitors.

Stereoselective Synthesis of ß-Branched Aromatic α-Amino Acids by Biocatalytic Dynamic Kinetic Resolution*.

ß-Branched noncanonical amino acids are valuable molecules in modern drug development efforts. However, they are still challenging to prepare due to the need to set multiple stereocenters in a stereoselective fashion, and contemporary methods for the synthesis of such compounds often rely on the use of rare-transition-metal catalysts with designer ligands. Herein, we report a highly diastereo- and enantioselective biocatalytic transamination method to prepare a broad range of aromatic ß-branched α-amino acids. Mechanistic studies show that the transformation proceeds through dynamic kinetic resolution that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction was applied to the synthesis of several sp3 -rich cyclic fragments and the first total synthesis of jomthonic acid A.

A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria.

Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium Bacillus subtilis with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect B. subtilis growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.

Selenomethionine as an expressible handle for bioconjugations.

Site-selective chemical bioconjugation reactions are enabling tools for the chemical biologist. Guided by a careful study of the selenomethionine (SeM) benzylation, we have refined the reaction to meet the requirements of practical protein bioconjugation. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, is selectively labile to glutathione, and embodies a broadly tunable cleavage profile. Specifically, a 4-bromomethylphenylacetyl (BrMePAA) linker has been applied for efficient conjugation of complex organic molecules to SeM-containing proteins. This expansion of the bioconjugation toolkit has broad potential in the development of chemically enhanced proteins.

Mechanistic study of enantioselective Pd-catalyzed C(sp3)-H activation of thioethers involving two distinct stereomodels.

Enantioselective C(sp3)-H activation has gained considerable attention from the synthetic chemistry community. Despite the intense interest in these reactions, the mechanisms responsible for enantioselection are still vague. In the course of the development of aryl thioether-directed C(sp3)-H arylation, we noticed extreme variation in sensitivity of two substrate classes to substituent effects of ligands and directing groups: whereas 3-pentyl sulfides (prochiral α-center) responded positively to substitution on ligands and directing groups, isobutyl sulfides (prochiral ß-center) were entirely insensitive. Quantitative structure selectivity relationship (QSSR) analyses of directing group and ligand substitution and the development of a new class of mono-N-acetyl protected amino anilamide (MPAAn) ligands led to high enantiomeric ratios (up to 99:1) for thioether-directed C(sp3)-H arylation. Key to the realization of this method was the exploitation of transient chirality at sulfur, which relays stereochemical information from the ligand backbone to enantiotopic carbons of the substrate in a rate- and enantio-determining cyclometallation deprotonation. The absolute stereochemistry of the products for these two substrates were revealed to be opposite. DFT evaluation of all possible diastereomeric transition states confirmed initial premises that guided rational ligand and directing group design. The implications of this study will assist in the further development of enantioselective C(sp3)-H activation, namely by highlighting the non-innocence of directing groups, distal steric influences, and the delicate interplay between steric Pauli repulsion and London dispersion in enantioinduction.

Correction to "Synthetic Elaboration of Native DNA by RASS (SENDR)".

[This corrects the article DOI: 10.1021/acscentsci.0c00680.].

Synthetic Elaboration of Native DNA by RASS (SENDR).

Controlled site-specific bioconjugation through chemical methods to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this conjugation through the tactical combination of two recently developed technologies: one for the manipulation of DNA in organic media and another for the chemoselective labeling of alcohols. Reversible adsorption of solid support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent reaction with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3' or 5' alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs.

Structural basis for the stabilization of amyloidogenic immunoglobulin light chains by hydantoins.

Misfolding and aggregation of immunoglobulin light chains (LCs) leads to the degeneration of post-mitotic tissue in the disease immunoglobulin LC amyloidosis (AL). We previously reported the discovery of small molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which slow or stop the LC aggregation cascade at the outset. A predominant structural category of kinetic stabilizers emerging from the high-throughput screen are coumarins substituted at the 7-position, which bind at the interface between the two variable domains of the light chain dimer. Here, we report the binding mode of another, more polar, LC kinetic stabilizer chemotype, 3,5-substituted hydantoins. Computational docking, solution nuclear magnetic resonance experiments, and x-ray crystallography show that the aromatic substructure emerging from the hydantoin 3-position occupies the same LC binding site as the coumarin ring. Notably, the hydantoin ring extends beyond the binding site mapped out by the coumarin hits. The hydantoin ring makes hydrogen bonds with both LC monomers simultaneously. The alkyl substructure at the hydantoin 5-position partially occupies a novel binding pocket proximal to the pocket occupied by the coumarin substructure. Overall, the hydantoin structural data suggest that a larger area of the LC variable-domain-variable-domain dimer interface is amenable to small molecule binding than previously demonstrated, which should facilitate development of more potent full-length LC kinetic stabilizers.

Semisynthesis of a Bacterium with Non-canonical Cell-Wall Cross-Links.

The cell wall is an elaborate framework of peptidoglycan that serves to protect the bacterium against osmotic challenge. This exoskeleton is composed of repeating saccharides covalently cross-linked by peptide stems. The general structure of the cell wall is widely conserved across diverse Gram-negative bacteria. To begin to explore the biological consequence of introducing non-canonical cross-links into the cell wall of Escherichia coli, we generated a bacterium where up to 31% of the cell-wall cross-links are formed by a non-enzymatic reaction between a sulfonyl fluoride and an amino group. Bacteria with these non-canonical cell-wall cross-links achieve a high optical density in culture, divide and elongate successfully, and display no loss of outer membrane integrity. This work represents a first step in the design of bacteria with non-canonical "synthetic" cell walls.

Cascade CuH-Catalysed Conversion of Alkynes to Enantioenriched 1,1-Disubstituted Products.

Enantioenriched α-aminoboronic acids play a unique role in medicinal chemistry and have emerged as privileged pharmacophores in proteasome inhibitors. Additionally, they represent synthetically useful chiral building blocks in organic synthesis. Recently, CuH-catalyzed asymmetric alkene hydrofunctionalization has become a powerful tool to construct stereogenic carbon centers. In contrast, applying CuH cascade catalysis to achieve reductive 1,1-difunctionalization of alkynes remains an important, but largely unaddressed, synthetic challenge. Herein, we report an efficient strategy to synthesize α-aminoboronates via CuH-catalyzed hydroboration/hydroamination cascade of readily available alkynes. Notably, this transformation selectively delivers the desired 1,1-heterodifunctionalized product in favor of alternative homodifunctionalized, 1,2-heterodifunctionalized, or reductively monofunctionalized byproducts, thereby offering rapid access to these privileged scaffolds with high chemo-, regio- and enantioselectivity.