Altering the Localization of an Unpaired Spin in a Formal Ni(V) Species.

The participation of both ligand and the metal center in the redox events has been recognized as one of the ways to attain the formal high valent complexes for the late 3d metals, such as Ni and Cu. Such an approach has been employed successfully to stabilize a Ni(III) bisphenoxyl diradical species in which there exist an equilibrium between the ligand and the Ni localized resultant spin. The present work, however, broadens the scope of the previously reported three oxidized equivalent species by conveying the approaches that tend to affect the reported equilibrium in CH3 CN at 233 K. Various spectroscopic characterization revealed that employing exogenous N-donor ligands like 1-methyl imidazole and pyridine favors the formation of the Ni centered localized spin though axial binding. In contrast, due to its steric hinderance, quinoline favors an exclusive ligand localized radical species. DFT studies shed light on the novel intermediates' complex electronic structure. Further, the three oxidized equivalent species with the Ni centered spin was examined for its hydrogen atom abstraction ability stressing their key role in alike reactions.

Copper-Catalyzed Hydroamination: Enantioselective Addition of Pyrazoles to Cyclopropenes.

Chiral N-cyclopropyl pyrazoles and structurally related heterocycles are prepared using an earth-abundant copper catalyst under mild reaction conditions with high regio-, diastereo-, and enantiocontrol. The observed N2:N1 regioselectivity favors the more hindered nitrogen of the pyrazole. Experimental and DFT studies support a unique mechanism that features a five-centered aminocupration.

Copper-Phosphido Catalysis: Enantioselective Addition of Phosphines to Cyclopropenes.

We describe a copper catalyst that promotes the addition of phosphines to cyclopropenes at ambient temperature. A range of cyclopropylphosphines bearing different steric and electronic properties can now be accessed in high yields and enantioselectivities. Enrichment of phosphorus stereocenters is also demonstrated via a Dynamic Kinetic Asymmetric Transformation (DyKAT) process. A combined experimental and theoretical mechanistic study supports an elementary step featuring insertion of a CuI -phosphido into a carbon-carbon double bond. Density functional theory calculations reveal migratory insertion as the rate- and stereo-determining step, followed by a syn-protodemetalation.

Enzymatic Nitrogen Insertion into Unactivated C-H Bonds.

Selective functionalization of aliphatic C-H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C-H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on preoxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C-H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)-H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity toward a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity-determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.

Probing the Free Energy Landscape of Organophotoredox-Catalyzed Anti-Markovnikov Hydrofunctionalization of Alkenes.

Experimental 13C kinetic isotope effects (KIEs) provide unprecedented mechanistic insight into three intermolecular anti-Markovnikov alkene hydrofunctionalization reactions─hydroesterification, hydroamination, and hydroetherification─enabled by organophotoredox catalysis. All three reactions are found to proceed via initial oxidation of the model alkenes to form a radical cation intermediate, followed by sequential nucleophilic attack and hydrogen-atom transfer to deliver the hydrofunctionalized product. A normal 13C KIE on the olefinic carbon that undergoes nucleophilic attack provides qualitative evidence for rate-limiting nucleophilic attack in all three reactions. Comparison to predicted 13C KIE values obtained from density functional theory (DFT) calculations for this step reveals that alkene oxidation has partial rate-limiting influence in hydroesterification and hydroamination, while the nucleophilic attack is solely rate-limiting in the hydroetherification reaction. The basic additive (2,6-lutidine) activates the nucleophile via deprotonation and is an integral part of the transition state for nucleophilic attack on the radical cation, providing an important design principle for the development of asymmetric versions of these reactions. A more electron-rich pyridine base (2,6-dimethoxypyridine) exhibits considerable rate enhancements in both inter- and intramolecular hydrofunctionalization reactions.

Mechanism and Origin of Remote Stereocontrol in the Organocatalytic Enantioselective Formal C(sp2)-H Alkylation Using Nitroalkanes as Alkylating Agents.

Experimental 13C kinetic isotope effects (KIEs) and density functional theory (DFT) calculations are used to evaluate the mechanism and origin of enantioselectivity in the formal C(sp2)-H alkylative desymmetrization of cyclopentene-1,3-diones using nitroalkanes as the alkylating agent. An unusual combination of an inverse (∼0.980) and a normal (∼1.033) KIE is observed on the bond-forming carbon atoms of the cyclopentene-1,3-dione and nitroalkane, respectively. These data provide strong support for a mechanism involving reversible carbon-carbon bond formation followed by rate- and enantioselectivity-determining nitro group elimination. The theoretical free-energy profile and the predicted KIEs indicate that this elimination event occurs via an E1cB pathway. The origin of remote stereocontrol is evaluated by distortion-interaction and SAPT0 analyses of the E1cB transition states leading to both enantiomers.

Probing Catalyst Function - Electronic Modulation of Chiral Polyborate Anionic Catalysts.

Boroxinate complexes of VAPOL and VANOL are a chiral anionic platform that can serve as a versatile staging arena for asymmetric catalysis. The structural underpinning of the platform is a chiral polyborate core that covalently links together alcohols (or phenols) and vaulted biaryl ligands. The polyborate platform is assembled in situ by the substrate of the reaction, and thus a multiplex of chiral catalysts can be rapidly assembled from various alcohols (or phenols) and bis-phenol ligands for screening of catalyst activity. In the present study, variations in the steric and electronic properties of the phenol/alcohol component of the boroxinate catalyst are probed to reveal their effects on the asymmetric induction in the catalytic asymmetric aziridination reaction. A Hammett study is consistent with a mechanism in which the two substrates are hydrogen-bonded to the boroxinate core in the enantiogenic step. The results of the Hammett study are supported by a computational study in which it is found that the H-O distance of the protonated imine hydrogen bonded to the anionic boroxinate core decreases with an increase in the electron releasing ability of the phenol unit incorporated into the boroxinate. The results are not consistent with a mechanism in which the boroxinate catalyst functions as a Lewis acid and activates the imine by a Lewis acid/Lewis base interaction.

Biocatalytic, Intermolecular C-H Bond Functionalization for the Synthesis of Enantioenriched Amides.

Directed evolution of heme proteins has opened access to new-to-nature enzymatic activity that can be harnessed to tackle synthetic challenges. Among these, reactions resulting from active site iron-nitrenoid intermediates present a powerful strategy to forge C-N bonds with high site- and stereoselectivity. Here we report a biocatalytic, intermolecular benzylic C-H amidation reaction operating at mild and scalable conditions. With hydroxamate esters as nitrene precursors, feedstock aromatic compounds can be converted to chiral amides with excellent enantioselectivity (up to >99 % ee) and high yields (up to 87 %). Kinetic and computational analysis of the enzymatic reaction reveals rate-determining nitrenoid formation followed by stepwise hydrogen atom transfer-mediated C-H functionalization.

A General Approach to Convert Hemicyanine Dyes into Highly Optimized Photoacoustic Scaffolds for Analyte Sensing*.

Most photoacoustic (PA) imaging agents are based on the repurposing of existing fluorescent dye platforms that exhibit non-optimal properties for PA applications. Herein, we introduce PA-HD, a new dye scaffold optimized for PA probe development that features a 4.8-fold increase in sensitivity and a red-shift of the λabs from 690 nm to 745 nm to enable ratiometric imaging. Computational modeling was used to elucidate the origin of these enhanced properties. To demonstrate the generalizability of our remodeling efforts, we developed three probes for ß-galactosidase activity (PA-HD-Gal), nitroreductase activity (PA-HD-NTR), and H2 O2 (PA-HD-H2 O2 ). We generated two cancer models to evaluate PA-HD-Gal and PA-HD-NTR. We employed a murine model of Alzheimer's disease to test PA-HD-H2 O2 . There, we observed a PA signal increase at 735 nm of 1.79±0.20-fold relative to background, indicating the presence of oxidative stress. These results were confirmed via ratiometric calibration, which was not possible using the parent HD platform.

A Selenourea-Thiourea Brønsted Acid Catalyst Facilitates Asymmetric Conjugate Additions of Amines to α,ß-Unsaturated Esters.

ß-Amino esters are obtained with high levels of enantioselectivity via the conjugate addition of cyclic amines to unactivated α,ß-unsaturated esters. A related strategy enables the kinetic resolution of racemic cyclic 2-arylamines, using benzyl acrylate as the resolving agent. Reactions are facilitated by an unprecedented selenourea-thiourea organocatalyst. As elucidated by DFT calculations and 13C kinetic isotope effect studies, the rate-limiting and enantiodetermining step of the reaction is the protonation of a zwitterionic intermediate by the catalyst. This represents a rare case in which a thiourea compound functions as an asymmetric Brønsted acid catalyst.

Transition state analysis of an enantioselective Michael addition by a bifunctional thiourea organocatalyst.

The mechanism of the enantioselective Michael addition of diethyl malonate to trans-ß-nitrostyrene catalyzed by a tertiary amine thiourea organocatalyst is explored using experimental 13C kinetic isotope effects and density functional theory calculations. Large primary 13C KIEs on the bond-forming carbon atoms of both reactants suggest that carbon-carbon bond formation is the rate-determining step in the catalytic cycle. This work resolves conflicting mechanistic pictures that have emerged from prior experimental and computational studies.

Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase.

Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg2+-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-ß-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisGS is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisGS, but steady-state kinetics and 31P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg2+ by Mn2+ enhances catalysis by HisGS but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an SN2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn2+ increasing HisGS reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisGS's catalytic rate, but decreases the hetero-octamer's, indicating that chemistry and product release are rate-limiting for HisGS and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisGS. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisGS, accelerating chemistry beyond the product release rate.

Asymmetric Induction via a Helically Chiral Anion: Enantioselective Pentacarboxycyclopentadiene Brønsted Acid-Catalyzed Inverse-Electron-Demand Diels-Alder Cycloaddition of Oxocarbenium Ions.

An enantioselective catalytic inverse-electron-demand Diels-Alder reaction of salicylaldehyde acetal-derived oxocarbenium ions and vinyl ethers to generate 2,4-dioxychromanes is described. Chiral pentacarboxycyclopentadiene (PCCP) acids are found to be effective for a variety of substrates. Computational and X-ray crystallographic analyses support the unique hypothesis that an anion with point-chirality-induced helical chirality dictates the absolute sense of stereochemistry in this reaction.

13 C Kinetic Isotope Effects as a Quantitative Probe To Distinguish between Enol and Enamine Mechanisms in Aminocatalysis.

A combination of experimental 13 C kinetic isotope effects (KIEs) and high-level density functional theory (DFT) calculations is used to distinguish between "enamine" and "enol" mechanisms in the Michael addition of acetone to trans-ß-nitrostyrene catalyzed by Jacobsen's primary amine thiourea catalyst. In light of the recent findings that the widely used 18 O-incorporation probe for these mechanisms is flawed, the results described in this communication demonstrate an alternative probe to distinguish between these pathways. A key advantage of this probe is that quantitative mechanistic information is obtained without modifying experimental conditions. This approach is expected to find application in resolving mechanistic debates, while providing valuable information about the key transition state of organocatalyzed reactions involving the α-functionalization of carbonyls.

Formation of hydrazones and stabilized boron-nitrogen heterocycles in aqueous solution from carbohydrazides and ortho-formylphenylboronic acids.

A recent addition to the suite of fast bioorthogonal reactions combines hydrazines and hydroxylamines with ortho-carbonyl substituted phenylboronic acids. Carbohydrazides are easily incorporated into biomolecules, making them appealing substrates in these reactions. Here we show that simple alkyl carbohydrazides form a single product with ortho-formylphenylboronic acid in an organic solvent and in the solid state. The solution structures of the products formed from the carbohydrazides in buffered aqueous solution, however, are markedly different from those identified in the organic solvent and solid state. The reactants form a mixture of hydrazone and heterocyclic products, the relative composition of which varies with pH. The observed reversibility of bioconjugates using carbohydrazide can thus be explained by the reversibility of the boron-nitrogen bond in the heterocycle. In contrast, the inclusion of an α-amine into the carbohydrazide substrate yields a single product in which both nitrogens are bonded to boron. These tricyclic structures are the same in organic solvent, solid state and aqueous solution from pH 4 to pH 9. Bioconjugates formed with α-amino carbohydrazides are stable to SDS-PAGE, while those formed with simple alkyl carbohydrazides are not. We propose that the inclusion of an intramolecular stabilizing ligand into a carbohydrazide substrate is a generally applicable principle that may be exploited to form boronic acid-based bioconjugates with a defined structure and resistance to hydrolysis.

Catalytic Enantioselective Synthesis of Lactams through Formal [4+2] Cycloaddition of Imines with Homophthalic Anhydride.

An amide-thiourea compound, operating through a novel ion pairing mechanism, is an efficient organocatalyst for the asymmetric reaction of homophthalic anhydride with imines. N-aryl and N-alkyl imines readily undergo formal [4+2] cycloaddition to provide lactams with high levels of enantio- and diastereoselectivity. The nature of the key chiral ion pair intermediate was elucidated by DFT calculations.

A Designed Approach to Enantiodivergent Enamine Catalysis.

The rational design and implementation of enantiodivergent enamine catalysis is reported. A simple secondary amine catalyst, 2-methyl-l-proline, and its tetrabutylammonium salt function as an enantiodivergent catalyst pair delivering the enantiomers of α-functionalized aldehyde products in excellent enantioselectivities. This novel concept of designed enantiodivergence is applied to the enantioselective α-amination, aldol, and α-aminoxylation/α-hydroxyamination reactions of aldehydes.

Isotope Effects Reveal the Mechanism of Enamine Formation in l-Proline-Catalyzed α-Amination of Aldehydes.

The mechanism of l-proline-catalyzed α-amination of 3-phenylpropionaldehyde was studied using a combination of experimental kinetic isotope effects (KIEs) and theoretical calculations. Observation of a significant carbonyl (13)C KIE and a large primary α-deuterium KIE support rate-determining enamine formation. Theoretical predictions of KIEs exclude the widely accepted mechanism of enamine formation via intramolecular deprotonation of an iminium carboxylate intermediate. An E2 elimination mechanism catalyzed by a bifunctional base that directly forms an N-protonated enamine species from an oxazolidinone intermediate accounts for the experimental KIEs. These findings provide the first experimental picture of the transition-state geometry of enamine formation and clarify the role of oxazolidinones as nonparasitic intermediates in proline catalysis.

Transition states of native and drug-resistant HIV-1 protease are the same.

HIV-1 protease is an important target for the treatment of HIV/AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease transition state has not been previously resolved using these methods. We have measured primary (14)C and (15)N KIEs and secondary (3)H and (18)O KIEs for native and multidrug-resistant HIV-1 protease (I84V). We observed (14)C KIEs ((14)V/K) of 1.029 ± 0.003 and 1.025 ± 0.005, (15)N KIEs ((15)V/K) of 0.987 ± 0.004 and 0.989 ± 0.003, (18)O KIEs ((18)V/K) of 0.999 ± 0.003 and 0.993 ± 0.003, and (3)H KIEs ((3)V/K) KIEs of 0.968 ± 0.001 and 0.976 ± 0.001 for the native and I84V enzyme, respectively. The chemical reaction involves nucleophilic water attack at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage. A transition structure consistent with the KIE values involves proton transfer from the active site Asp-125 (1.32 Å) with partial hydrogen bond formation to the accepting nitrogen (1.20 Å) and partial bond loss from the carbonyl carbon to the amide leaving group (1.52 Å). The KIEs measured for the native and I84V enzyme indicate nearly identical transition states, implying that a true transition-state analogue should be effective against both enzymes.

Stereospecific Nickel-Catalyzed Cross-Electrophile Coupling Reaction of Alkyl Mesylates and Allylic Difluorides to Access Enantioenriched Vinyl Fluoride-Substituted Cyclopropanes.

Cross-electrophile coupling reactions involving direct C-O bond activation of unactivated alkyl sulfonates or C-F bond activation of allylic gem-difluorides remain challenging. Herein, we report a nickel-catalyzed cross-electrophile coupling reaction between alkyl mesylates and allylic gem-difluorides to synthesize enantioenriched vinyl fluoride-substituted cyclopropane products. These complex products are interesting building blocks with applications in medicinal chemistry. Density functional theory (DFT) calculations demonstrate that there are two competing pathways for this reaction, both of which initiate by coordination of the electron-deficient olefin to the low-valent nickel catalyst. Subsequently, the reaction can proceed by oxidative addition of the C-F bond of the allylic gem-difluoride moiety or by directed polar oxidative addition of the alkyl mesylate C-O bond.