Mechanistic Probes Research Articles

Chemoproteomic Profiling of Clickable Fumarate Probes for Target Identification and Mechanism of Action Studies.

Dimethyl fumarate (DMF) is an established oral therapy for multiple sclerosis worldwide. Although the clinical efficacy of these fumarate esters has been extensively investigated, the mode of action and pharmacokinetics of fumarates have not been fully elucidated due to their broad-spectrum reactivity and complex metabolism in vivo. To better understand the mechanism of action of DMF and its active metabolite, monomethyl fumarate (MMF), we designed and utilized clickable probes to visualize and enrich probe-modified proteins. We further perform quantitative chemoproteomics analysis for proteome-wide target identification and validate several unique and shared targets of DMF and MMF, which provide insight into the reactivity, selectivity, and target engagement of fumarates.

Tertiary Alcohols as Mechanistic Probes for Photocatalysis: the Gas-Phase Reaction of 2-Methyl-2-Pentanol on Titania P25 in a Microphotoreactor

Tertiary Alcohols as Mechanistic Probes for Photocatalysis: the Gas-Phase Reaction of 2-Methyl-2-Pentanol on Titania P25 in a Microphotoreactor

Perillaldehyde pretreatment alleviates cerebral ischemia-reperfusion injury by improving mitochondrial structure and function via the Nrf2/Keap1/Trx2 axis.

Perilladehyde, an extract of perillae in the Labiatae family, can produce significant anti-inflammatory and antioxidant effects. Although literature evidences the favorable effect of perillaldehyde on ischemic stroke, the exact mechanism remains blurred. This study attempted to explore the impact of perillaldehyde on cerebral ischemia-reperfusion injury and the related action mechanism. The rat tMCAO and neuronal OGD/R models were established to simulate cerebral ischemia-reperfusion injury. Lentiviruses were used to interfere with the expression of Nrf2 and Trx2 in neurons. The effects and action mechanisms of perillaldehyde were explored by various experimental methods, including chromatin immunoprecipitation assay, Western Blot, flow cytometry, dual-luciferase reporter gene assay, transmission electron microscopy, MRI, RNA-seq, and immunofluorescence staining. Perillaldehyde pretreatment effectively mitigated the tMCAO-induced brain injury in rats by reducing cerebral infarction, improving neuromotor function, and attenuating cell apoptosis in the ischemic penumbra. In vitro, perillaldehyde pretreatment alleviated cell death and excessive oxidative stress, preserved the mitochondrial membrane integrity, enhanced mitochondrial energy metabolism, and facilitated the restoration of mitochondrial ultrastructure after OGD/R. The mechanism probe revealed that perillaldehyde activated the Nrf2/Keap1/Trx2 signaling axis, thus promoting the transcription of Trx2 and improving mitochondrial structure and function. The aforementioned impacts of perillaldehyde were somewhat counteracted by disrupting the expression of Nrf2 and Trx2, suggesting that the neuroprotection of perillaldehyde partially involves the activation of the Nrf2/Keap1/Trx2 axis. This study firstly demonstrates the existence of the Nrf2/Keap1/Trx2 signaling axis in cerebral ischemia-reperfusion injury and evidences that perillaldehyde pretreatment can promote the restoration of neuronal mitochondrial structure and function by activating the Nrf2/Keap1/Trx2 axis after cerebral ischemia-reperfusion injury. These findings signify that perillaldehyde holds great promises for clinical management of ischemic stroke.

Photoredox-Catalyzed Decarboxylation of Oxetane-2-Carboxylic Acids and Unique Mechanistic Insights.

Oxetanes are valuable motifs in medicinal chemistry applications, with demonstrated potential to serve as bioisosteres for an array of functional groups. Through the visible-light-mediated photoredox hydrodecarboxylation of 2-aryl oxetane 2-carboxylic acids this work enables access to the products of a [2+2]-photocycloaddition between alkenes and aryl aldehydes without the challenges associated with a traditional UV-light-mediated Paternò-Büchi reaction. Investigation into the hydrodecarboxylation mechanism reveals substrate-dependent modes of initiation under the conditions reported herein. Divergence in diastereomeric outcome is observed, with mechanistic probes elucidating key hydrogen-bonding and steric interactions.

Nonequilibrium steady state full counting statistics in the noncrossing approximation.

Quantum transport is often characterized not just by mean observables like the particle or energy current but by their fluctuations and higher moments, which can act as detailed probes of the physical mechanisms at play. However, relatively few theoretical methods are able to access the full counting statistics (FCS) of transport processes through electronic junctions in strongly correlated regimes. While most experiments are concerned with steady state properties, most accurate theoretical methods rely on computationally expensive propagation from a tractable initial state. Here, we propose a simple approach for computing the FCS through a junction directly at the steady state, utilizing the propagator noncrossing approximation. Compared to time propagation, our method offers reduced computational cost at the same level of approximation, but the idea can also be used within other approximations or as a basis for numerically exact techniques. We demonstrate the method's capabilities by investigating the impact of lead dimensionality on electronic transport in the nonequilibrium Anderson impurity model at the onset of Kondo physics. Our results reveal a distinct signature of one dimensional leads in the noise and Fano factor not present for other dimensionalities, showing the potential of FCS measurements as a probe of the environment surrounding a quantum dot.

Organolanthanide‐Mediated Tandem Insertion/Cyclisation of Alkynylbenzonitriles with Secondary Amines Elucidated Through a Computational Approach

AbstractA detailed mechanistic probe of the organolanthanide‐mediated tandem insertion/annulation of alkynylbenzonitriles with secondary amines by an archetypical homoleptic lanthanum silylamide starting material is presented. An in‐depth computational scrutiny of alternatively plausible pathways for relevant productive steps and also performance‐degrading pathways identified the pathway likely traversed in productive catalysis. It entails the transformation of the starting material into various of silylamide/amide compounds, of which the lanthanum bis‐silylamide/amide is thermodynamically prevalent, capable of promoting the process. Benzonitrile insertion is irreversible to readily afford the lanthanum amidinate, which can adopt various easily interconvertible ligation pattern. The rather rapid protonolysis of the La─N imine linkage would lead to undesirable aminoamidines, but its net endergonicity renders this performance‐degrading avenue nonviable. Instead, the lanthanum amidinate is converted back into catalytically competent lanthanum bis‐silylamide/amide with the release of the observed aminoisoindole product. This transformation favors a stepwise insertative cyclisation/La─C alkenyl protonolysis sequence over an otherwise kinetically noncompetitive proton‐triggered stepwise N─C/C─H bond forming process. The operative insertative pathway comprises turnover‐limiting and irreversible insertion of the alkyne C≡C tether into the La─N amidinate linkage followed by La─C alkenyl aminolysis at the intervening lanthanum alkenylisoindinyl intermediate. The DFT‐assessed barrier for turnover‐limiting insertative N─C ring closure favorably compares with reported performance data.

A Tissue-Engineered Model of T-Cell–Mediated Oral Mucosal Inflammatory Disease

T-cell-mediated oral mucocutaneous inflammatory conditions, including oral lichen planus, are common, but development of new treatments aimed at relieving symptoms and controlling oral lichen planus progression is hampered by the lack of experimental models. In this study, we developed a tissue-engineered oral mucosal equivalent containing polarized T-cells to replicate oral lichen planus pathogenesis. Peripheral blood CD4+ and CD8+ T-cells were isolated, activated, and polarized into T helper 1 and cytotoxic T cells. Oral mucosal equivalents were constructed by culturing oral keratinocytes on an oral fibroblast-populated hydrogel to produce a stratified squamous epithelium. Oral mucosal equivalent stimulated with IFN-γ and TNF-α or medium from T helper 1 cells caused increased secretion of inflammatory cytokines and chemokines. A model of T-cell-mediated inflammatory disease was developed by combining oral mucosal equivalent on top of a Thelper 1 and cytotoxic T-cell-containing hydrogel, followed by epithelial stimulation with IFN-γ and TNF-α. T-cell recruitment toward the epithelium was associated with increased secretion of T-cell chemoattractants CCL5, CXCL9, and CXCL10. Histological assessment showed tissue damage associated with cleaved caspase-3 and altered laminin-5 expression. Treatment with inhibitors directed against Jak, KCa3.1 channels, or clobetasol in solution and through a mucoadhesive patch prevented cytokine and chemokine release and tissue damage. This disease model has potential to probe for mechanisms of pathogenesis or as a test platform for novel therapeutics or treatment modalities.

Stereodivergency in Copper-Catalyzed Borylative Difunctionalizations: The Impact of Boron Coordination.

A reagent-controlled diastereodivergent copper-catalyzed borylative difunctionalization is reported. The formation of Lewis adducts that guide selectivity is commonly invoked in organic reaction mechanisms. Using density functional theory calculations, we identified BpinBdan as a sterically similar and less Lewis acidic alternative to B2pin2. Using a newly developed borylative aldol domino reaction as the proof-of-concept, we demonstrate a change in stereochemical outcome by a simple change of borylating reagent-B2pin2 affords the diastereomer associated with coordination control while BpinBdan overturns this mode of binding. We show that this strategy can be generalized to other scaffolds and, more importantly, that BpinBdan does not alter the diastereomeric outcome of the reaction when coordination is not involved. BpinBdan can be viewed as a mechanistic probe for coordination in future copper-catalyzed borylation reactions.

Gold(III) Auracycles Featuring C(sp3)-Au-C(sp2) Bonds: Synthesis and Mechanistic Insights into the Cycloauration Step.

The direct auration of arenes is a key step in numerous gold-catalyzed reactions. Although reported more than 100 years ago, understanding of its underlying mechanism has been hampered by the difficulties in the isolation of relevant intermediates given the propensity of gold(III) species to undergo reductive elimination. Here, we report the synthesis and isolation of a new family of intriguing zwitterionic [C(sp3)^C(sp2)]-auracyclopentanes, as well as of their alkyl-gold(III) precursors and demonstrate their value as mechanistic probes to study the C(sp2)-Au bond-forming event. Experimental investigations employing Kinetic Isotope Effects (KIE), Hammett plot, and Eyring analysis provided important insights into the formation of the auracycle. The data suggest a SEAr mechanism wherein the slowest step might be the π-coordination between the arene and the gold(III) center, en route to the Wheland intermediate. We also show that these auracyclopentanes can work as catalysts in several gold-promoted transformations.

Single-Plane Versus Synchronous Biplane Ultrasound Imaging During Vascular Access Procedures: Which Is Better and How Can We Improve?

Highlights Biplane ultrasound imaging reduces need for probe manipulation during procedure. Providers note clinical benefit of biplane ultrasound imaging for vascular access. Biplane disadvantages include probe size, imaging quality, size of the screen. Thorough didactic and practical education is essential for biplane success. Abstract Background: Use of ultrasound guidance for vascular access procedures is commonplace in inpatient and outpatient care settings. Standard ultrasound probes offer the operator a single-plane view, necessitating rotation of probe to attain dual complimentary views. This mechanical probe rotation increases technical difficulty of ultrasound use. The purpose of this study is to evaluate the rate of cannulation success and efficiency of a synchronous biplane ultrasound mode in ultrasound-guided arterial line placement as compared with a standard single-view ultrasound mode in the operating room setting. Methods: Patients scheduled for elective surgery in which a radial arterial catheter would be used for hemodynamic monitoring were approached for consent to this study. Patients were randomized to either undergo placement with single-plane view versus synchronous biplane view; outcomes were recorded. Providers were provided preprocedural ultrasound education as well as the option of a short hands-on experience; their level of experience was noted. Results: Placement time of a peripheral arterial catheter was longer and required more attempts to be successful using synchronous biplane imaging as compared with single-plane imaging across providers of all skill/experience levels. Subjectively, providers noted the benefit of synchronous biplane imaging in vascular access; however, disadvantages including probe size, quality of imaging, and size of the screen were noted. Discussion: Thorough education regarding the use and functionality of biplane synchronous imaging in vascular access is essential. Additional guided-practice time with experienced operators could also be helpful to overcome the challenges observed in this study.

Enhancing oil production via radical reactions during hydrothermal coliquefaction of biomass model compounds and plastics: A molecular dynamic simulation study

Recently, hydrothermal coliquefaction of biomass and plastic waste has attracted considerable research interest. However, there is a notable gap in understanding the fundamental reaction mechanisms between biomass and plastics during coliquefaction. This study focused on the coliquefaction of biomass model compounds and plastic polymers using ReaxFF molecular dynamics simulations under both subcritical and supercritical water conditions. Molecular-level tracking and probing of the reaction mechanisms between biomass model compounds and plastics were conducted to purposefully enhance oil production. The study observed related radical reactions between by-product molecules, with detailed mechanisms primarily involving (1) ▪OH radicals released by aqueous phase molecules from biomolecules, transferring as H2O molecules and facilitating plastic depolymerization, and (2) C1–C4 radicals in the gaseous phase, emitted from biomolecule and plastic, colliding and subsequently recombining to form oil molecules. Moreover, the yield of multiple products from various mixtures were evaluated by considering the key reaction parameters including reaction temperature and feedstock blended ratio. An exploration into the effect of coliquefaction on oil yield was conducted to precisely identify the optimal coliquefaction conditions. The positive effect of coliquefaction was more pronounced between biomass model compounds and aromatic polymers compared to aliphatic polymers. Analysis of reaction mechanisms and product outcomes has shown that hydrothermal coliquefaction is a viable approach to improving oil production from multi-source organic solid waste.

(Invited) Transport Limits for Zinc Aqueous Electrolyte Batteries: Investigation over Multiple Length Scales

Large scale grid level storage would benefit from low cost environmentally sustainable batteries. A system class under investigation is rechargeable zinc anode batteries utilizing aqueous electrolytes. These systems hold promise due to their potential for safe low-cost scalable energy storage. Consideration of the systems over various length scales is warranted due to the complexity of the possible reactions and pernicious parasitic processes. Further, the diversity and intricacies of these battery systems inherently reduce the probability of optimization experiments alone resulting in marketable products.Many of the fundamental issues influencing ion and electron transport and electron transfer and how the transport phenomena evolve under flux are not yet fully undefined. More complete understanding demands multiple characterization approaches used in concert, to tie together information gathered at the local or atomic level and the mesoscale with systems level performance from electrochemical impedance spectroscopy (EIS), galvanostatic cycling (GC), and related techniques. This presentation will provide insight into probes of the reaction mechanisms, evolution of zinc interfaces, methods to determine limitations of cathode function at high rate, and impact of the findings on the electrochemical behavior of the batteries.

Effects of Fuel Cell Operating Conditions on Electrochemical Pressure Impedance Spectroscopy (EPIS) Diagnostics

Fuel cells are versatile, low-emission alternative energy sources, but their optimization and failure analyses are complex due to their black-box nature. Conventional in-situ diagnostic techniques such as electrochemical impedance spectroscopy (EIS), are widely used to deconvolute transient processes within operating fuel cells. EIS is invaluable to understanding some of these processes, but other processes within the cell, such as mass transport and individual water fluxes, get overshadowed and are hard to detect.In an effort to provide information that is currently unattainable with conventional fuel cell diagnostic techniques, this project focuses on the further development of electrochemical pressure impedance spectroscopy (EPIS). EPIS is based on a pressure alternating frequency response analysis (pFRA). This diagnostic method is similar to EIS, except that it implements mechanical perturbations in the form of gas pressure oscillations, rather than voltage or current oscillations, to achieve an electrochemical response. Since EIS is based on electrical perturbations, the results are primarily based on the transport of electrons and provides electron-based performance metrics, including charge transfer, adsorption, diffusion, and double-layer behaviors. Conversely, the mechanical perturbations in EPIS enable the investigation of non-electron-based mechanisms, such as flow and gas transport resistances.This project builds from the work of Zang et al., who developed an experimental setup for EPIS and showed that pressure perturbations affect local reaction rates and transport phenomena within the cell, providing a sinusoidal voltage response.1 This study focused on systematically analyzing the effects of varying fuel cell operating conditions to better understand the benefits and limitations of EPIS as a diagnostic technique. The EPIS response and subsequent flow resistances and gas transport resistances were monitored to understand the effects of variations in the cathode inlet relative humidity, the cell inlet temperatures, and the oxygen stoichiometry (stoic). The goal is to improve the metrics by which fuel cells are measured by providing new methods to assist in the real-time probing of processes and failure mechanisms within operating cells. 1 Zhang, Q., Homayouni, H., Gates, B. D., Eikerling, M. H., & Niroumand, A. M. (2022). Electrochemical Pressure Impedance Spectroscopy for Polymer Electrolyte Fuel Cells via Back-Pressure Control. Journal of The Electrochemical Society, 169(4), 044510.

The probe of current conduction mechanisms, interface states, and the forward bias intersection point of the al/Al2O3/Ge/p-Si heterostructures depending on temperature

The current conduction mechanisms (CCMs), temperature-sensitivities (S), energy-dependent interface traps (Nss), and origin of the intersection points in the forward bias (IF−VF) plots of the Al/Al2O3/Ge/p-Si heterostructure were investigated in wide temperature range of 90–420 K. Firstly, main electrical parameters, including reverse-saturation current (Io), ideality factor (n), zero-bias barrier-height (ΦBo), and series-resistance (Rs) values, were extracted for each temperature. The lnIF-VF curves illustrate two distinct linear regimes at low and intermediate bias voltages. Despite the observed decline in n values as temperature rises, the corresponding ΦBo values exhibit an upward trend. The conventional Richardson plots deviated from linearity at low temperatures, and the Richardson-constants (A*) value obtained from its linear part is quite lower than its theoretical value. Hence, ΦBo−q/2 kT, ΦBo−n, and n(kT)/q−(kT/q) correlations were plotted to seek indications of the Gaussian distribution (GD) of barrier heights (BHs) and tunneling mechanism. Temperate sensitivity (S = dV/dT) for 0.01, 0.10, 0.50, and 1 μA was found as 2.30, 2.33, 2.34, and 2.35 mV/K, which indicated that the fabricated Al/Al₂O₃/Ge/p-Si heterostructure is highly sensitive to temperature, rendering it suitable for use in temperature sensor applications. The observed crossing point at about 2.4V was explained by an increase in the apparent BH with temperature and the presence of Rs.

Depth filtration of dredged sediments via reduced iron driving persulfate sodium activation: Perspectives from technical viability, potential mechanism, and prospect assessment

The existing single-use conditioning agents for dredged sediments (DS) treatment were confronted with problems such as high cost, insufficient efficiency, or potential toxicity. The study, for the first time, utilized a heterogeneous process of reduced iron (RI)/Persulfate sodium (PS) to deeply filter DS and further uncovered the potential mechanism as well as raised the application prospect. The results showed that after 120 mg/g TSS RI with 40 mg/g TSS PS treatment, the water content (WC) of DS dropped from 75.4% (Blank) to 41.6%, which is obviously lower than that of traditional flocculant-treated DS reported. Mechanistic probe indicated that the strengthened filterability of DS is likely evoked by the sulfate radical (SO4●-)/hydroxyl radical (•OH) with the ferrous/ferric substances generated from RI/PS process. SO4●-/•OH damaged extracellular polymeric substances (EPS) along with their major ingredients and possibly disturbed cellulate physiology of DS, which further promoted the dissociation/liberation of bound-water and strengthened a trend toward fluidization but declined the hydrophilicity and stickiness of DS. Moreover, SO4●-/•OH may evoke the proteins conformation to unfold the hydrophobic spots, promoting the escape of untied water also. By extruding the electric-double layers and lowing the negative charge, Ferrous and/or ferric substances promotes the re-aggregation of disrupted DS particles, which potentially leads to the formation of dense & stable flocs through interaction of ferric substances with carboxyl and hydroxyl functional groups in DS, thus facilitating bound-water removal. The findings here not only deepen our understandings upon mechanisms of heterogeneous AOPs conditioning DS filterability but also offer a promising approach for DS treatment in the fields of solid waste management and water environment remediation.

Error-controlled adaptive machining of aeronautical cabin structures by laser triangulation on-machine measurement.

Laser Triangulation On-Machine Measurement (LTOMM) is being implemented increasingly to inspect aeronautical components accurately and efficiently, with its enhanced application in adaptive machining. This work proposes an error compensation and controlling method for measuring the typical features of steps, holes, and freeform surfaces to improve accuracy. Then, the global path to inspect the cabin's structures is planned by introducing optimization algorithms, thus providing an appropriate sequence to shorten the traveling length. After these, the test piece was designed, measured, and manufactured using the adaptive machining process that integrates the LTOMM. The results show that the measurement errors of steps, holes, and freeform surfaces are +0.0092, -0.006, and +0.0406mm, respectively, and further reduced to +0.0013, -0.0019, and +0.0083mm after error controlling. The cabin's freeform surface was fabricated with the maximum positive and minimum negative errors of +0.184 and -0.123mm, which is evaluated by the mechanical probe. The measured data-driven machining process can guarantee that the error satisfies the required tolerance, promoting the application of the LTOMM process in aeronautical intelligent manufacturing.

Quantifying spatial peat depth with seismic micronodes and the implications for carbon stock estimates

Peatlands are a major store of soil carbon, due to their high concentration of carbon-rich decayed plant material. Consequently, accurate assessment of peat volumes is important for determining land-use carbon budgets, especially in the Northern hemisphere. Determination of carbon stocks at the scale of individual peat sites has principally relied on either mechanical probing or electromagnetic geophysical methods. In this study, we investigated the use of seismic nodal instrumentation for quantifying peat depth. We used Stryde™ nodes for a deployment at the Whixall moss in Shropshire, England. We measured seismic arrival times from peat-bottom reflections, as well as dispersive surface waves to invert for a model of variable peat depth along a linear cross-section. The use of very small seismic nodes (micronodes) allows for particularly rapid deployment on challenging terrain.

Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family.

The engineering of efficient enzymes for large-scale production of industrially relevant compounds is a challenging task. Utilizing rational protein design, which relies on a comprehensive understanding of mechanistic information, holds significant promise for achieving success in this endeavor. Pre-steady-state kinetic measurements, obtained either through fast-mixing techniques or photoswitchable substrates, provide crucial mechanistic insights. The latter approach not only furnishes mechanistic clarity but also affords real-time structural elucidation of reaction intermediates via time-resolved femtosecond crystallography. Unfortunately, only a limited number of such valuable mechanistic probes are available. To address this gap, we applied a multidisciplinary approach, including computational analysis, chemical synthesis, physicochemical property screening, and enzyme kinetics to identify promising candidates for photoswitchable probes. We demonstrate the approach by designing an azobenzene-based photoswitchable substrate tailored for haloalkane dehalogenases, a prototypic class of enzymes pivotal in developing computational tools for rational protein design. The probe was subjected to steady-state and pre-steady-state kinetic analysis, which revealed new insights about the catalytic behavior of the model biocatalysts. We employed laser-triggered Z-to-E azobenzene photoswitching to generate the productive isomer in situ, opening avenues for advanced mechanistic studies using time-resolved femtosecond crystallography. Our results not only pave the way for the mechanistic understanding of this model enzyme family, incorporating both kinetic and structural dimensions, but also propose a systematic approach to the rational design of photoswitchable enzymatic substrates.

Promising anti-proliferative indolic benzenesulfonamides alter mechanisms with sulfonamide nitrogen substituents

Agents that cause apoptotic cell death by interfering with tubulin dynamics, such as vinblastine and paclitaxel, are an important class of chemotherapeutics. Unfortunately, these compounds are substrates for multidrug resistance (MDR) pumps, allowing cancer cells to gain resistance to these chemotherapeutics. The indolesulfonamide family of tubulin inhibitors are not excluded by MDR pumps and have a promising activity profile, although their high lipophilicity is a pharmacokinetic limitation for their clinical use. Here we present a new family of N-indolyl-3,4,5-trimethoxybenzenesulfonamide derivatives with modifications on the indole system at positions 1 and 3 and on the sulfonamide nitrogen. We synthesized and screened against HeLa cells 34 novel indolic benzenesulfonamides. The most potent derivatives (1.7–109 nM) were tested against a broad panel of cancer cell lines, which revealed that substituted benzenesulfonamides analogs had highest potency. Importantly, these compounds were only moderately toxic to non-tumorigenic cells, suggesting the presence of a therapeutic index. Consistent with known clinical anti-tubulin agents, these compounds arrested the cell cycle at G2/M phase. Mechanistically, they induced apoptosis via caspase 3/7 activation, which occurred during M arrest. The substituents on the sulfonamide nitrogen appeared to determine different mechanistic results and cell fates. These results suggest that the compounds act differently depending on the bridge substituents, thus making them very interesting as mechanistic probes as well as potential drugs for further development.

Resolving Conflicting Mechanisms for Photoredox Allylic sp3-CH Arylation Using Deuterium-Labeling and Isotope Effects.

Two conflicting mechanisms have emerged for the direct arylation of allylic C-H bonds enabled by the combined use of thiol and photoredox catalysis. In the original report (Nature, 2015, 519, 74-77), a radical coupling step-between a radical anion of an arene and an allylic radical-is proposed to be the key C-C bond-forming step. A recent mechanistic study (J. Org. Chem. 2022, 87, 223-230) has suggested that the C-C bond formation occurs via radical anion capture by the olefin followed by an H atom transfer (HAT) event to deliver the allylic C-H arylation product. Utilizing cyclohexene-4,4,5,5-d 4 as a mechanistic probe to distinguish between otherwise indistinguishable regioisomeric allylic C-H arylation products in the reaction of cyclohexene and dicyanobenzene, we establish that the radical anion capture-HAT mechanism is not operative. Furthermore, experimental k H/k D studies and DFT calculations lend strong support to the radical coupling mechanism proceeding via irreversible HAT to form the allylic radical of cyclohexene, followed by regioselectivity-determining radical coupling (for unsymmetrical olefins) and facile decyanation.