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Item Open Access Unique Phosphorus-based Avenues for the Tuning of Functional Materials(American Chemical Society, 2023-03-07) Asok, Nayanthara; Gaffen, Joshua R.; Baumgartner, ThomasRecent ground-breaking advances in synthetic chemistry have transformed main-group molecules from simple lab curiosities into powerful materials for a range of applications in all realms of life. Electron-accepting or -deficient materials in particular, have been the focus for development since their generally limited availability and stability have been a key limiting factor towards establishing new practical applications. In addition to the general requirements for the design of these materials, a deeper understanding of their inherent electronics and molecular interactions is a requisite for the successful expansion of their utility. Previously, the incorporation of electron-deficient main-group elements, such as boron into a conjugated organic framework was considered an effective route toward the synthesis of high-performing electron-accepting materials. However, challenging conditions such as the need for bulky substituents for kinetic stabilization, air-free, and moisture-sensitive synthesis, and restricted storage abilities have led to the investigation of other elements across the periodic table to be used in a similar vein. Lately, heavier main-group elements such as Si, Ge, P, As, Sb, Bi, S, Se, and Te have also proven to be advantageous for electron-accepting materials as they exhibit polarizable molecular orbitals that are easily accessible to electrons or nucleophiles. This has laid the foundation for materials chemistry research on a variety of applications including, optoelectronic devices such as OLEDs, organic photovoltaics, energy storage such as batteries and capacitors, fluorescent sensors with both biological and physiological applications, organocatalysis and synthesis, and many more. Among the main group element-based materials, organophosphorus species are privileged as their frontier orbitals are easily altered by chemical modification or/and structural and geometrical manipulations at the phosphorus center itself, without the need for kinetic stabilization, or through electronic modification of the conjugated system. The five-membered phosphorus-based heterocycle, phosphole, is a particularly interesting motif in this context and extensive studies on the corresponding materials have uncovered the rich fundamentals of the σ*-π* interaction that imparts intriguing accepting properties, while sustaining morphological and physiological stability for utilization in real-life scenarios. Moreover, beyond the σ*-π* interaction in phospholes that is key to many of their acceptor properties as a material, the use of phosphorus also gives rise to easily accessible, low-lying antibonding orbitals. They pave the way toward Lewis acidic phosphorus species that, despite being considered as electron-rich species in general, open up several possibilities for intriguing chemical reactivity through hypervalency. Herein, we representatively discuss some recent advancements through the various approaches that leverage the unique structures and electronics of organophosphorus species toward the design of materials with outstanding electronic, chemical, and structural properties and reactivities for the functional material world.Item Open Access Aqueous Leaching of Ultrashort-Chain PFAS from (Fluoro)polymers: Targeted and Nontargeted Analysis(Amerian Chemical Society, 2024-02-19) Joudan, Shira; Gauthier, Jeremy; Mabury, Scott A.; Young, CoraFluoropolymers are a class of per- and polyfluoroalkyl substances (PFAS) defined as high molecular weight plastics containing only carbon-based backbones with F atoms directly attached. Here, we used targeted and nontargeted analytical methods to quantify the aqueous leaching of small-molecule PFAS from three types of fluoropolymer tubing material and three types of nonfluorinated polymer tubing material. C2–C4 perfluoroalkyl carboxylic acids (PFCAs) were quantified with ion chromatography–mass spectrometry, and C4–C9 PFCAs were quantified with liquid chromatography–tandem mass spectrometry. A new 19F nuclear magnetic resonance (NMR) method with lower detection limits provided an unbiased, nontargeted view of all fluorinated chemicals in the aqueous leachate. C2–C4 PFCAs had a higher concentration than longer-chain PFCAs. All tubing tested, including the nonfluorinated polymers, contained trifluoroacetic acid (C2 PFCA) concentrations above the blank. NMR identified additional fluorinated chemicals, especially in the nonfluorinated PEEK, a common replacement for fluoropolymers in laboratory chromatography systems. Overall, each fluoropolymer tested had different fingerprints of C2–C4 PFCAs, which may be related to their synthetic production such as processing aids, residuals, and inhibitors used; fluorinated chemicals were also identified from nonfluorinated polymers. The outcome of this work informs better trace analysis in the laboratory and presents an indication of how fluoropolymers and other plastics can be an emission source to the environment.Item Open Access Exploring controls on perfluorocarboxylic acid (PFCA) gas–particle partitioning using a model with observational constraints(Royal Society of Chemistry, 2022-09-05) Tao, Ye; VandenBoer, Trevor; Ye, RenXi; Young, CoraThe atmospheric fate of perfluorocarboxylic acids (PFCAs) has attracted much attention in recent decades due to the role of the atmosphere in global transport of these persistent chemicals. There is a gap in our understanding of gas–particle partitioning, limited by availability of reliable atmospheric measurements, partitioning properties, and models of gas–particle interactions. The gas–particle equilibrium phase partitioning of C2–C16 PFCAs in the atmosphere were modeled here by taking account of both deprotonation and phase partitioning equilibria among air, aerosol liquid water, and particulate water-insoluble organic matter using a range of available PFCA partitioning properties. We systematically varied water and organic matter content to simulate the full range of atmospheric conditions. Except in severe organic matter pollution episodes, shorter-chain PFCAs are predicted to mainly partition between air and aqueous phase, while for PFCAs with carbon chains longer than 12, organic matter is more likely to be the dominant particle phase reservoir. The model framework underestimated the particle fraction of C2–C8 PFCAs compared with several ambient observations, with larger discrepancies observed for longer-chain PFCAs. The discrepancy could result from externally mixed dust components, non-ideality of aerosol liquid water, surfactant descriptions at phase boundaries, and missed interactions between organic matter and charged PFCA molecules. Reliable measurements of ambient PFCAs with high time resolution and the measurement of uptake parameters by particle-relevant components will be beneficial to more reliable environmental fate modeling of ambient PFCAs.Item Open Access Reactive chlorine emissions from cleaning and reactive nitrogen chemistry in an indoor athletic facility(ACS Publications, 2022-11-03) Moravek, Alexander; VandenBoer, Trevor; Finewax, Zachary; Pagonis, Demetrios; Nault, Benjamin; Brown, Wyatt; DAY, DOUGLAS; HANDSCHY, ANNE; STARK, HARALD; Ziemann, Paul; Jimenez, Jose L; de Gouw, Joost; Young, CoraIndoor gas-phase radical sources are poorly understood but expected to be much different from outdoors. Several potential radical sources were measured in a windowless, light-emitting diode (LED)-lit room in a college athletic facility over a 2 week period. Alternating measurements between the room air and the supply air of the heating, ventilation, and air-conditioning system allowed an assessment of sources. Use of a chlorine-based cleaner was a source of several photolabile reactive chlorine compounds, including ClNO2 and Cl2. During cleaning events, photolysis rates for these two compounds were up to 0.0023 pptv min–1, acting as a source of chlorine atoms even in this low-light indoor environment. Unrelated to cleaning events, elevated ClNO2 was often observed during daytime and lost to ventilation. The nitrate radical (NO3), which is rapidly photolyzed outdoors during daytime, may persist in low-light indoor environments. With negligible photolysis, loss rates of NO3 indoors were dominated by bimolecular reactions. At times with high NO2 and O3 ventilated from outdoors, N2O5 was observed. Elevated ClNO2 measured concurrently suggests the formation through heterogeneous reactions, acting as an additional source of reactive chlorine within the athletic facility and outdoors.Item Open Access Near-source hypochlorous acid emissions from indoor bleach cleaning(Royal Society of Chemistry, 2023-01-05) Stubbs, Annastacia; Lao, Melodie; Wang, Chen; Abbatt, Jonathan; Hoffnagle, John; VandenBoer, Trevor; Kahan, TaraCleaning surfaces with sodium hypochlorite (NaOCl) bleach can lead to high levels of gaseous chlorine (Cl2) and hypochlorous acid (HOCl); these have high oxidative capacities and are linked to respiratory issues. We developed a novel spectral analysis procedure for a cavity ring-down spectroscopy (CRDS) hydrogen peroxide (H2O2) analyzer to enable time-resolved (3 s) HOCl quantification. We measured HOCl levels in a residential bathroom while disinfecting a bathtub and sink, with a focus on spatial and temporal trends to improve our understanding of exposure risks during bleach use. Very high (>10 ppmv) HOCl levels were detected near the bathtub, with lower levels detected further away. Hypochlorous acid concentrations plateaued in the room at a level that depended on distance from the bathtub. This steady-state concentration was maintained until the product was removed by rinsing. Mobile experiments with the analyzer inlet secured to the researcher's face were conducted to mimic potential human exposure to bleach emissions. The findings from mobile experiments were consistent with the spatial and temporal trends observed in the experiments with fixed inlet locations. This work provides insight on effective strategies to reduce exposure risk to emissions from bleach and other cleaning products.Item Open Access Elevated levels of chloramines and chlorine detected near an indoor sports complex(Royal Society of Chemistry, 2022-11-29) Angelucci, Andrea; Crilley, Leigh; Richardson, Rob; Valkenburg, Thalassa Sandra Eliza; Monks, Paul; Sommariva, Roberto; VandenBoer, TrevorChloramines (NH2Cl, NHCl2, and NCl3) are toxic compounds that can be created during the use of bleach-based disinfectants that contain hypochlorous acid (HOCl) and the hypochlorite ion (OCl−) as their active ingredients. Chloramines can then readily transfer from the aqueous-phase to the gas-phase. Atmospheric chemical ionization mass spectrometry using iodide adduct chemistry (I-CIMS) made observations across two periods (2014 and 2016) at an urban background site on the University of Leicester campus (Leicester, UK). Both monochloramine (NH2Cl) and molecular chlorine (Cl2) were detected and positively identified from calibrated mass spectra during both sampling periods and to our knowledge, this is the first detection of NH2Cl outdoors. Mixing ratios of NH2Cl reached up to 2.2 and 4.0 parts per billion by volume (ppbv), with median mixing ratios of 30 and 120 parts per trillion by volume (pptv) during the 2014 and 2016 sampling periods, respectively. Levels of Cl2 were observed to reach up to 220 and 320 pptv. Analysis of the NH2Cl and Cl2 data pointed to the same local source, a nearby indoor sports complex with a swimming pool and a cleaning product storage shed. No appreciable levels of NHCl2 and NCl3 were observed outdoors, suggesting the indoor pool was not likely to be the primary source of the observed ambient chloramines, as prior measurements made in indoor pool atmospheres indicate that NCl3 would be expected to dominate. Instead, these observations point to indoor cleaning and/or cleaning product emissions as the probable source of NH2Cl and Cl2 where the measured levels provide indirect evidence for substantial amounts transported from indoors to outdoors. Our upper estimate for total NH2Cl emissions from the University of Leicester indoor sports complexes scaled for similar sports complexes across the UK is 3.4 × 105 ± 1.1 × 105 μg h−1 and 0.0017 ± 0.00034 Gg yr−1, respectively. The Cl-equivalent emissions in HCl are only an order of magnitude less to those from hazardous waste incineration and iron and steel sinter production in the UK National Atmospheric Emissions Inventory (NAEI).Item Open Access An instrument to measure and speciate the total reactive nitrogen budget indoors: description and field measurements(Royal Society of Chemistry, 2023-01-26) Crilley, Leigh; Lao, Melodie; salehpoor, Leyla; VandenBoer, TrevorReactive nitrogen species (Nr), defined here as all N-containing compounds except N2 and N2O, have been shown to be important drivers for indoor air quality. Key Nr species include NOx (NO + NO2), HONO and NH3, which are known to have detrimental health effects. In addition, other Nr species that are not traditionally measured may be important chemical actors for indoor transformations (e.g. amines). Cooking and cleaning are significant sources of Nr, whose emission will vary depending on the type of activity and materials used. Here we present a novel instrument that measures the total gas-phase reactive nitrogen (tNr) budget and key species NOx, HONO, and NH3 to demonstrate its suitability for indoor air quality applications. The tNr levels were measured using a custom-built heated platinum (Pt) catalytic furnace to convert all Nr species to NOx, called the tNr oven. The measurement approach was validated through a series of control experiments, such that quantitative measurement and speciation of the total Nr budget are demonstrated. The optimum operating conditions of the tNr oven were found to be 800 °C with a sampling flow rate of 630 cubic centimetres per minute (ccm). Oxidized nitrogen species are known to be quantitatively converted under these conditions. Here, the efficiency of the tNr oven to convert reduced Nr species to NOx was found to reach a maximum at 800 °C, with 103 ± 13% conversion for NH3 and 79–106% for selected relevant amines. The observed variability in the conversion efficiency of reduced Nr species demonstrates the importance of catalyst temperature characterization for the tNr oven. The instrument was deployed successfully in a commercial kitchen, a complex indoor environment with periods of rapidly changing levels, and shown to be able to reliably measure the tNr budget during periods of longer-lived oscillations (>20 min), typical of indoor spaces. The measured NOx, HONO and basic Nr (NH3 and amines) were unable to account for all the measured tNr, pointing to a substantial missing fraction (on average 18%) in the kitchen. Overall, the tNr instrument will allow for detailed survey(s) of the key gaseous Nr species across multiple locations and may also identify missing Nr fractions, making this platform capable of stimulating more in-depth analysis in indoor atmospheres.Item Open Access A Rapid Derivatization for Quantitation of Perfluorinated Car-boxylic Acids from Aqueous Matrices by Gas Chromatography-Mass Spectrometry(ACS Publications, 2023-05-03) Ye, RenXi; Di Lorenzo, Robert A.; Clouthier, Jessica; Young, Cora; VandenBoer, TrevorUltrashort chain perfluorocarboxylic acids (PFCAs) are receiving more attention due to their ever-increasing presence in the environment. Methods have been established for the analysis of short and long-chain PFCAs, while robust quantitation of ultrashort chain species are scarce. Here we develop a novel derivatization method using diphenyl diazomethane for quantitation of C2-C14 PFCAs in aqueous matrices. The method is highlighted by rapid completion of derivatization (<1 min), and retention and separation of ultrashort chain (C2/C3) PFCA derivatives using H2 carrier gas (R > 1.5). A weak anion solid phase extraction procedure for analyte recovery from representative aqueous samples was developed and validated by spike and recovery from ultrapure water, synthetic ocean water, and simulated denuder extracts used for collecting gaseous PFCAs. Recoveries for C2-C9 PFCAs ranged from 83-130 % for the majority of analytes and matrices. The instrument detection limits (IDLs) range from 8-220 fg per injection, and method detection limits (MDLs) range from 0.06-14.6 pg/mL for 500 mL aqueous samples, which are within an order of magnitude to conventional LC-MS/MS methods. The method was applied to the analysis of real samples of tap water, rainwater, ocean water, and annular denuder extracts. The overall method provides a cost-effective alternative to conventional LC-MS/MS methods, overcoming the typical GC-MS drawbacks of high detection limits and long sample preparation times, while being able to simultaneously analyze the complete spectrum of environmentally relevant PFCAs.Item Open Access Suppressor and calibration standard limitations in cation chromatography of ammonium and 10 alkylamines in atmospheric samples(Royal Society of Chemistry, 2023-07-13) Salehpoor, Leyla; VandenBoer, TrevorAmmonia (NH3) and alkylamines are ubiquitous in the atmosphere and have been suggested to play important global roles through new particle formation and aerosol growth. In this work, we optimized an ion-chromatographic (IC) method to separate and quantify the ten most abundant atmospheric alkylamines with high selectivity and separation efficiency, using 4 μm packed columns and resin-based suppressors, alongside stabilizing amine standards. Modern resin suppressors operating on a gradient elution program affected the linear response of this IC technique. Calibration statistical analyses found a loss of analytes in these cation-exchange devices. Suppressor operational longevity was optimized by using a stepped current and an external water supply, which improved precision, accuracy, and LODs compared to other suppression modes. When this new method was applied to real samples, amines were found ubiquitously in size-resolved marine aerosol samples; monopropylamine, isomonopropylamine, and monobutylamine were detected and quantified, which have not been reported before. The molar ratio of the sum of aminium to ammonium ranged from 0.02 to 0.2, showcasing the application of the developed method towards studying the diversity and importance of alkylamines in coastal marine particle composition. The new analytical method also found NH3 present in a suite of new homes with a mean mixing ratio of 25 ± 15 ppbv; a common level reached between homes across the study during the first year of occupation which can then be transported outdoors.Item Open Access Disentangling the Conformational Space and Structural Preferences of Tetrahydrofurfuryl Alcohol Using Rotational Spectroscopy and Computational Chemistry(2024-04-18) Dias de Paiva Silva, Weslley Guilherme; van Wijngaarden, JenniferThe influence of the hydroxymethyl (CH2OH) group on the tetrahydrofuran (THF) ring structure was investigated by disentangling the gas phase conformational landscape of the sugar analogue tetrahydrofurfuryl alcohol (THFA). By combining rotational spectroscopy (6–20 GHz) and quantum chemical calculations, transitions corresponding to two stable conformers of THFA and their 13C isotopologues were observed and assigned in the rotational spectrum. The positions of the C atoms were precisely determined to unambiguously distinguish between nearly isoenergetic pairs of conformers that differ in their ring configurations: envelope (E) versus twist (T). The rotational spectrum confirms that the E ring geometry is favoured when the CH2OH fragment lies gauche (−) to the THF backbone (OCCO ~−60°) whereas the T form is more stable for the gauche (+) alignment of the substituent (OCCO ~+60°). The observed spectral intensities suggest that conformational relaxation of the THF geometry (E↔T) to the more stable form readily occurs within the pairs of g− and g+ conformers which is consistent with the low barriers (1.5–1.7 kJ mol−1) for conversion determined via transition state calculations. Insights into the intramolecular hydrogen bonding and other weak interactions stabilizing the lowest energy structures of THFA were derived and rationalized using non-covalent interaction analyses.Item Open Access Exploring the conformational landscape, hydrogen bonding, and internal dynamics in the diallyl ether and diallyl sulfide monohydrates(2024-01-23) Dias de Paiva Silva, Weslley Guilherme; Poonia, Tamanna; van Wijngaarden, JenniferThe conformational spaces of the diallyl ether (DAE) and diallyl sulfide (DAS) monohydrates were explored using rotational spectroscopy from 6-19 GHz. Calculations at the B3LYP-D3(BJ)/aug-cc-pVTZ level suggested significant differences in their conformational behaviour, with DAE-w exhibiting 22 unique conformers and DAS-w featuring three stable structures within 6 kJ mol-1, however, only transitions from the lowest energy conformer of each were experimentally observed. Spectral analysis confirmed that binding with water does not alter the conformational preference for the lowest energy structure of the monomers but it does influence the relative stabilities of all other conformers, particularly in the case of DAE. Non-covalent interaction (NCI) and quantum theory of atoms in molecules (QTAIM) analyses showed that the observed conformer for each complex is stabilized by two intermolecular hydrogen bonds (HBs), where water primarily interacts with the central oxygen or sulfur atom of the diallyl compounds, along with secondary interactions involving the allyl groups. The nature of these interactions was further elucidated using symmetry-adapted perturbation theory (SAPT) which suggests that the primary HB interaction with S in DAS is weaker and more dispersive in nature compared to the primary HB in DAE. This supports the experimental observation of a tunneling splitting exclusively in the rotational spectrum of DAS-w as the weaker contact allows water to undergo internal motions within the complex as shown based on calculated transition state structures for possible tunneling pathways.Item Open Access Exploring the Distinct Conformational Preferences of Allyl Ethyl Ether and Allyl Ethyl Sulfide using Rotational Spectroscopy and Computational Chemistry(2023-06-08) Poonia, Tamanna; van Wijngaarden, JenniferThe conformational energy landscapes of allyl ethyl ether (AEE) and allyl ethyl sulfide (AES) were investigated using Fourier transform microwave spectroscopy in the frequency range of 5-23 GHz aided by density functional theory (DFT) B3LYP-D3(BJ)/aug-cc-pVTZ calculations. The latter predicted highly competitive equilibria for both species including 14 unique conformers of AEE and 12 for the sulfur analog AES within 14 kJ mol-1. The experimental rotational spectrum of AEE was dominated by transitions arising from its three lowest energy conformers which differ in the arrangement of the allyl side chain while in AES, transitions due to the two most stable forms, distinct in the orientation of the ethyl group, were observed. Splitting patterns attributed to methyl internal rotation were analyzed for AEE conformers I and II, and the corresponding V3 barriers were determined to be 12.172(55) kJ mol-1 and 12.373(32) kJ mol-1, respectively The experimental ground state geometries of both AEE and AES were derived using the observed rotational spectra of the 13C and 34S isotopic species and are highly dependent on the electronic properties of the linking chalcogen (oxygen versus sulfur). The observed structures are consistent with a decrease in hybridization in the bridging atom from oxygen to sulfur. The molecular-level phenomena that drive the conformational preferences are rationalized through natural bond orbital (NBO) and non-covalent interaction (NCI) analyses. These shows that interactions involving the lone pairs on the chalcogen atom with the organic side chains favour distinct geometries and energy orderings for the conformers of AEE and AES.Item Open Access A Unique Conformational Distortion Mechanism Drives Lipocalin 2 Binding to Bacterial Siderophores(ACS Publications, 2019-10-15) Huang, Xiaojing; Slavkovic, Sladjana; Song, Erfei; Botta, Amy; Mehrazma, Banafsheh; Lento, Cristina; Johnson, Philip E.; Sweeney, Gary; Wilson, Derek J.Lcn2 is a host defense protein induced via the innate immune response to sequester iron-loaded bacterial siderophores. However, excess or prolonged elevation of Lcn2 levels can induce adverse cellular effects, including oxidative stress and inflammation. In this work, we use Hydrogen−Deuterium eXchange (HDX) and Isothermal Titration Calorimetry (ITC) to characterize the binding interaction between Lcn2 and siderophores enterobactin and 2,3-DHBA, in the presence and absence of iron. Our results indicate a rare “Type II” interaction in which binding of siderophores drives the protein conformational equilibrium toward an unfolded state. Linking our molecular model to cellular assays, we demonstrate that this “distorted binding mode” facilitates a deleterious cellular accumulation of reactive oxygen species that could represent the molecular origin of Lcn2 pathology. These results add important insights into mechanisms of Lcn2 action and have implications in Lcn2-mediated effects including inflammation.Item Open Access Proline/Alanine Tails as Ionization Enhancement Tags in Native Mass Spectrometry(2023-05-12) Abdalla, Marwa; Lento, Cristina; Jiang, Guifeng; Power, Austin; Wilson, DerekThis study aimed to study the impact of Proline/Alanine tag on l-asparaginase protein dynamics. Our current analysis demonstrates that the PA tail fragment did not change the core protein dynamics using TRESI HDX MS to provide identical stability with untagged l-asparaginase. Novel Aspects In this work, we propose PASylation ® as a new tool for ionization enhancement through chain ejection.Item Open Access BF3‑Catalyzed Intramolecular Fluorocarbamoylation of Alkynes via Halide Recycling(American Chemical Society, 2023-05-12) McKnight, E. Ali; Arora, Ramon; Pradhan, Ekadashi; Fujisato, Yuriko H.; Ajayi, Ayonitemi, J.; Lautens, Mark; Zeng, Tao; Le, Christine M.A BF3-catalyzed atom-economical fluorocarbamoylation reaction of alkyne-tethered carbamoyl fluorides is reported. The catalyst acts as both a fluoride source and Lewis acid activator, thereby enabling the formal insertion of alkynes into strong C–F bonds through a halide recycling mechanism. The developed method provides access to 3-(fluoromethylene) oxindoles and γ-lactams with excellent stereoselectivity, including fluorinated derivatives of known protein kinase inhibitors. Experimental and computational studies support a stepwise mechanism for the fluorocarbamoylation reaction involving a turnover-limiting cyclization step, followed by internal fluoride transfer from a BF3-coordinated carbamoyl adduct. For methylene oxindoles, a thermodynamically driven Z–E isomerization is facilitated by a transition state with aromatic character. In contrast, this aromatic stabilization is not relevant for γ-lactams, which results in a higher barrier for isomerization and the exclusive formation of the Z-isomer.Item Open Access Synthesis of Carbamoyl Fluorides Using a Difluorophosgene Surrogate Derived from Difluorocarbene and Pyridine N-Oxides(American Chemical Society, 2022-08-16) Cadwallader, Dusty; Tiburcio, Tristan R.; Cieszynski, George A.; Le, Christine M.We report a method for the synthesis of carbamoyl fluorides from secondary amines using bench-stable, inexpensive, and readily accessible starting materials that, when combined, yield a surrogate for toxic difluorophosgene (COF2) gas. In contrast to state-of-the-art methods for the synthesis of carbamoyl fluorides, our protocol does not require the use of pre-functionalized substrates, the preparation of light-, temperature-, and/or moisture-sensitive chemicals, or the application of explosive fluorinating reagents.Item Open Access Pinpointing The Structural Dynamics of Plasminogen Activator Inhibitor 1 binding to Heparin using Hydrogen/Deuterium Exchange Mass Spectrometry(ScienceOpen Posters, 2023-05-17) Abdalla, Marwa; Anacleto, Joseph; Wilson, DerekPAI-1 is a key player to regulate the activation of fibrinolysis, with broad influence effects on inflammation, hemostasis, tissue remodeling, and wound healing 1 The binding of endogenous cofactor vitronectin to PAI 1 helps to extend PAI 1 half life and delay its latency transition thus controlling the stability of the active form 1 Previous studies have suggested that low molecular weight heparin alters the levels of circulating PAI 1 and enhances endogenous fibrinolysis However, the intrinsic dynamics of this binding are not completely understood 2 Our findings reveal that Low Molecular Weight Heparin n ( may contribute to the localization of PAI 1 at specific sites, hence involved in the regulation of plasminogen activation and its functional stability Hydrogen-Deuterium Exchange ( coupled to MS is widely used to study protein dynamics Continuous time resolved Electrospray ionization TRESI HDX MS technique is used to characterize protein structural transitions in relatively ordered regions of proteins 3Item Open Access Redox Reporter - Ligand Competition to Support Signaling in the Cocaine-Binding Electrochemical Aptamer-Based Biosensor(Wiley-VCH GmbH, 2023-05-05) Dauphin-Ducharme, Philippe; Churcher, Zachary R; Shoara, Aron A; Rahbarimehr, Erfan; Slavkovic, Sladjana; Fontaine, Nicolas; Boisvert, Olivia; Johnson, Philip EElectrochemical aptamer-based (E-AB) biosensors have demonstrated capabilities in monitoring molecules directly in undiluted complex matrices and in the body with the hopes of addressing personalized medicine challenges. This sensing platform relies on an electrode-bound, redox- reporter-modified aptamer. The electrochemical signal is thought to originate from the aptamer undergoing a binding- induced conformational change capable of moving the redox reporter closer to the electrode surface. While this is the generally accepted mechanism, it is notable that there is limited evidence demonstrating conformational change or distance-dependent change in electron transfer rates in E-AB sensors. In response, we investigate here the signal transduction of the well-studied cocaine-binding aptamer with different analytical methods and found that this sensor relies on a redox-reporter - ligand competition mechanism rather than a ligand-induced structure formation mechanism. Our results show that the covalently bound redox reporter, methylene blue, binds at or near the ligand binding site on the aptamer resulting in a folded conformation of the cocaine-binding aptamer. Addition of ligand then competes with the redox reporter for binding, altering its electron transfer rate. While we show this for the cocaine-binding aptamer, given the prevalence of methylene blue in E-AB sensors, a similar competition-based may occur in other systems.Item Open Access Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an Off-Target Ligand(American Chemical Society, 2013-10-31) Reinstein, Oren; Yoo, Mina; Han, Chris; Palmo, Tsering; Beckham, Simone A; Wilce, Matthew CJ; Johnson, Philip EThe cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30−40 times stronger than it is for cocaine. Competitive binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.Item Open Access Analysis of the role played by ligand-induced folding of the cocaine-binding aptamer in the photochrome aptamer switch assay(Elsevier, 2020-04-11) Shoara, Aron A.; Churcher, Zachary R; Steele, Terry W.J.; Johnson, Philip EThe Photochrome Aptamer Switch Assay (PHASA) relies on ligand binding by an aptamer to alter the local environment of a stilbene compound covalently attached to the 5’ end of the aptamer. We used the PHASA with both structure switching and non-structure switching versions of the cocaine-binding aptamer. We show that the largest change in fluorescence intensity and the lowest concentration limit of detection (CLooD) is obtained using the structure-switching cocaine-binding aptamer. Fluorescence anisotropy measurements were used to quantify the affinity of the conjugated aptamer to cocaine. We also used thermal melt analysis and Nuclear Magnetic Resonance (NMR) spectroscopy to show that the addition of the stilbene to the aptamer increases the melt temperature of the cocaine-bound structure-switching aptamer by (6.4 ± 0.3) °C compared to the unconjugated aptamer while the free form of the structure-switching aptamer-stilbene conjugate remains unfolded.