Department of Chemistry

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https://hdl.handle.net/10315/2685

Collection consists of research, scholarship and publications produced by graduate students and faculty members of the Department of Chemistry.

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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.
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, Derek
This 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.
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.
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.
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, Derek
PAI-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 3
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 E
Electrochemical 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.
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 E
The 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.
Open Access
Rapid characterization of folding and binding interactions with thermolabile ligands by DSC
(The Royal Society of Chemistry, 2016-10-23) Harkness V, Robert W; Slavkovic, Sladjana; Johnson, Philip E.; Mittermaier, Anthony K.
Differential scanning calorimetry (DSC) is a powerful technique for measuring tight biomolecular interactions. However, many pharma- ceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding inter- actions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Using experimental data for two DNA aptamers that bind to the thermolabile ligand cocaine, we present a new global fitting analysis that yields the complete set of folding and binding parameters for the initial and final forms of the ligand from a pair of DSC experiments, while accounting for the thermal conversion. Furthermore, we show that the rate constant for thermolabile ligand conversion may be obtained with only one additional DSC dataset.
Open Access
Visible Fluorescent Light-up Probe for DNA Three-Way Junctions Provides Host−Guest Biosensing Applications
(American Chemical Society, 2021-08-18) Van Riesen, Abigail J.; Le, Jennifer; Slavkovic, Sladjana; Churcher, Zachary R.; Shoara, Aron A.; Johnson, Philip E.; Manderville, Richard A.
DNA three-way junctions (3WJs) consist of a Y-shaped hydrophobic branch point connecting three double-stranded stems and are viewed as druggable targets for cancer treatment. They are also important building blocks for the construction of DNA nanostructures and serve as recognition elements for DNA aptasensors for a wide variety of diagnostic applications. However, visible fluorescent light-up probes for specific staining of DNA 3WJs are currently lacking. Herein, we report that a merocyanine containing the N-methylbenzothiazolium (Btz) acceptor vinyl linked to a 2-fluorophenolic (FPhO) donor (FPhOBtz) serves as a universal fluorescent turn-on dye for DNA 3WJs. Our evidence is based on a multifaceted approach to define the specificity and affinity of FPhOBtz for 3WJ DNA aptamers; the cocaine binding aptamer MN4, the cholic acid binding aptamer (CABA), and four steroid aptamers (DOGS.1, DISS.1, BES.1, DCAS.1). FPhOBtz exhibits impressive turn-on (up to 730-fold) fluorescence at 580 nm upon aptamer binding with low micromolar affinity. Direct FPhOBtz displacement from the 3WJ binding domain through competitive alkaloid and steroid binding provides immediate fluorescent read out for host−guest detection strategies in human blood serum in the low micromolar regime. Our results present the first visible light-up fluorescent probe for DNA 3WJ detection strategies.
Open Access
Comparison of the free and ligand-bound imino hydrogen exchange rates for the cocaine-binding aptamer
(Springer, 2017-05-05) Churcher, Zachary R; Neves, Miguel A. D.; Hunter, Howard N.; Johnson, Philip E.
Using NMR magnetization transfer experi- ments, the hydrogen exchange rate constants (kex) of the DNA imino protons in the cocaine-binding aptamer have been determined for the free, cocaine-bound, and quinine- bound states. The secondary structure of the cocaine- binding aptamer is composed of three stems built around a three-way junction. In the free aptamer the slowest exchanging imino protons are located in the middle of the stems. The highest kex values were found for a nucleotide in the GAA loop of stem 3 and for nucleotides at the end of the stems that form the three-way junction structure and in the tandem GA mismatch. Upon ligand binding, the kex values of nucleotides at the ligand binding site are reduced, indicating that these base pairs become more stable or less solvent accessible in the bound state. The imino proton kex values of nucleotides located away from the binding site are only minimally affected by ligand binding.
Open Access
Quantitative affinity electrophoresis of RNA–small molecule interactions by cross-linking the ligand to acrylamide
(Elsevier, 2013-08-06) Boodram, Sherry N; McCann, Lucas C; Organ, Michael G; Johnson, Philip E
We show that the affinity electrophoresis analysis of RNA–small molecule interactions can be made quantifiable by cross-linking the ligand to the gel matrix. Using an RNA–aminoglycoside model system to verify our method, we attached an acryloyl chloride molecule to the aminoglycosides paromomycin and neomycin B to synthesize an acrylamide–aminoglycoside monomer. This molecule was then used as a component in gel polymerization for affinity electrophoresis, covalently attaching an aminoglycoside molecule to the gel matrix. To test RNA binding to the cross-linked aminoglycosides, we used the amino- glycoside binding RNA molecule derived from thymidylate synthase messenger RNA (mRNA) that con- tains a C–C mismatch. Binding is indicated by the difference in RNA mobility between gels with cross- linked ligand, with ligand embedded during polymerization, and with no ligand present. Critically, the predicted straight line relationship between the reciprocal of the relative migration of the RNA and the ligand concentration is obtained when using cross-linked aminoglycosides, whereas a straight line is not obtained using embedded aminoglycosides. Average apparent dissociation constants are determined from the slope of the line from these plots. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand.
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 E
The 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.
Open Access
Nanomolar binding affinity of quinine-based antimalarial compounds by the cocaine-binding aptamer
(Elsevier, 2018-09-17) Slavkovic, Sladjana; Churcher, Zachary R; Johnson, Philip E
An unusual feature of the cocaine-binding aptamer is that it binds quinine much tighter than the ligand it was selected for, cocaine. Here we expand the repertoire of ligands that this aptamer binds to include the quinine- based antimalarial compounds amodiaquine, mefloquine, chloroquine and primaquine. Using isothermal titra- tion calorimetry (ITC) we show that amodiaquine is bound by the cocaine-binding aptamer with an affinity of (7 ± 4) nM, one of the tightest aptamer-small molecule affinities currently known. Amodiaquine, mefloquine and chloroquine binding are driven by both a favorable entropy and enthalpy of binding, while primaquine, quinine and cocaine binding are enthalpy driven with unfavorable binding entropy. Using nuclear magnetic resonance (NMR) and ITC methods we show that these ligands compete for the same binding sites in the ap- tamer. Our identification of such a tight binding ligand for this aptamer should prove useful in developing new biosensor techniques and applications using the cocaine-binding aptamer as a model system.
Open Access
Development of a thermal-stable structure-switching cocaine-binding aptamer
(Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM), 2017-08-21) Shoara, Aron A; Reinstein, Oren; Borhani, Okty Abbasi; Martin, Taylor R; Slavkovic, Sladjana; Churcher, Zachary R; Johnson, Philip E
We have developed a new cocaine-binding aptamer variant that has a significantly higher melt tem- perature when bound to a ligand than the currently used sequence. Retained in this new construct is the ligand-induced structure-switching binding mechanism that is important in biosensing applications of the cocaine-binding aptamer. Isothermal titration calorimetry methods show that the binding affinity of this new sequence is slightly tighter than the existing cocaine-binding aptamer. The improved thermal performance, a Tm increase of 4 C for the cocaine-bound aptamer and 9 C for the quinine-bound aptamer, was achieved by optimizing the DNA sequence in stem 2 of the aptamer to have the highest stability based on the nearest neighbor thermodynamic parameters and confirmed by UV and fluores- cence spectroscopy. The sequences in stem 1 and stem 3 were unchanged in order to retain the structure switching and ligand binding functions. The more favorable thermal stability characteristics of the OR3 aptamer should make it a useful construct for sensing applications employing the cocaine-binding aptamer system.
Open Access
Structure–affinity relationship of the cocaine-binding aptamer with quinine derivatives
(Elsevier, 2015-03-06) Slavkovic, Sladjana; Altunisik, Merve; Reinstein, Oren; Johnson, Philip E
In addition to binding its target molecule, cocaine, the cocaine-binding aptamer tightly binds the alkaloid quinine. In order to understand better how the cocaine-binding aptamer interacts with quinine we have used isothermal titration calorimetry-based binding experiments to study the interaction of the cocaine-binding aptamer to a series of structural analogs of quinine. As a basis for comparison we also investigated the binding of the cocaine-binding aptamer to a set of cocaine metabolites. The bicyclic aromatic ring on quinine is essential for tight affinity by the cocaine-binding aptamer with 6-methoxyquinoline alone being sufficient for tight binding while the aliphatic portion of quinine, quinuclidine, does not show detectable binding. Compounds with three fused aromatic rings are not bound by the aptamer. Having a methoxy group at the 6-position of the bicyclic ring is important for binding as substituting it with a hydrogen, an alcohol or an amino group all result in lower binding affinity. For all ligands that bind, association is driven by a negative enthalpy compensated by unfavorable binding entropy.
Open Access
Designed Alteration of Binding Affinity in Structure-Switching Aptamers through the Use of Dangling Nucleotides
(ACS Publications, 2020-01-08) Slavkovic, Sladjana; Eisen, Sophie R.; Johnson, Philip E.
The ability to change binding affinity in a controlled fashion is a key step in the rational design of biomolecules in general and functional nucleic acids in particular. Here, we use dangling nucleotides to alter the binding affinity of structure-switching aptamers. Dangling nucleotides can stabilize or destabilize a nucleic acid structure with a known ΔG°37. When the dangling nucleotide stabilizes the structure, less free energy from ligand binding is needed to fold the molecule and hence the ligand is observed to bind tighter than in the absence of the unpaired nucleotide. For a destabilizing dangling nucleotide, the opposite occurs, and the observed binding is weaker. We demonstrate this concept using both the cocaine-binding aptamer and the ATP-binding aptamer systems. We find that for both aptamers there is a direct, but different, relationship between the predicted stabilization and the change in the observed binding free energy.
Open Access
Optimizing Stem Length To Improve Ligand Selectivity in a Structure-Switching Cocaine-Binding Aptamer
(ACS Publications, 2017-09-20) Neves, Miguel A.D.; Shoara, Aron A.; Reinstein, Oren; Martin, Taylor R.; Borhani, Okty Abbasi; Johnson, Philip E.
Understanding how aptamer structure and function are related is crucial in the design and development of aptamer-based biosensors. We have analyzed a series of cocaine-binding aptamers with different lengths of their stem 1 in order to understand the role that this stem plays in the ligand-induced structure-switching binding mechanism utilized in many of the sensor applications of this aptamer. In the cocaine-binding aptamer, the length of stem 1 controls whether the structure-switching binding mechanism for this aptamer occurs or not. We varied the length of stem 1 from being one to seven base pairs long and found that the structural transition from unfolded to folded in the unbound aptamer is when the aptamer elongates from 3 to 4 base pairs in stem 1. We then used this knowledge to achieve new binding selectivity of this aptamer for quinine over cocaine by using an aptamer with a stem 1 two base pairs long. This selectivity is achieved by means of the greater affinity quinine has for the aptamer compared with cocaine. Quinine provides enough free energy to both fold and bind the 2-base pair-long aptamer while cocaine does not. This tuning of binding selectivity of an aptamer by reducing its stability is likely a general mechanism that could be used to tune aptamer specificity for tighter binding ligands.
Open Access
Insufficient evidence for the existence of natural trifluoroacetic acid
(Environmental Science: Processes & Impacts, 2021-10-01) Joudan, Shira; De Silva, Amila O.; Young, Cora
Trifluoroacetic acid (TFA) is a persistent and mobile pollutant that is present ubiquitously in the environment. As a result of a few studies reporting its presence in pre-industrial samples and a purported unaccounted source, TFA is often claimed to exist naturally. Here, we examine the evidence for natural TFA by: (i) critically evaluating measurements of TFA in pre-industrial samples; (ii) examining the likelihood of TFA formation by hypothesized mechanisms; (iii) exploring other potential TFA sources to the deep ocean; and (iv) examining global budgets of TFA. We conclude that the presence of TFA in the deep ocean and lack of closed TFA budget is not sufficient evidence that TFA occurs naturally, especially without a reasonable mechanism of formation. We argue the paradigm of natural TFA should no longer be carried forward.
Open Access
Structure-reactivity studies on hypervalent square-pyramidal dithieno[3,2-b:2’,3’-d]phospholes
(Royal Society of Chemistry, 2021-02-14) Asok, Nayanthara; Gaffen, Joshua R.; Pradhan, Ekadashi; Zeng, Tao; Baumgartner, Thomas
A series of neutral pentacoordinate dithieno[3,2-b:2’,3’-d]phosphole compounds were synthesized by [4+1] cycloaddition with o-quinones. Counter to the expected trigonal bipyramidal geometry, the luminescent hypervalent dithienophospholes exhibit square pyramidal geometry with inherently Lewis acidic phosphorus center that is stabilized via supramolecular π-stacking interactions in the solid state and in solution. Due to their Lewis-acid character, the compounds react with nucleophiles, suggesting their potential as mediator in organic transformations. The new species thus present an intriguing structural plaform for the design of neutral P(V) Lewis acids with useful reactivities.
Open Access
Fluorometry studies of aptamers that bind intrinsically fluorescent ligands: techniques, obstacles and optimizations
(2022-09-26) Shoara, Aron A; Johnson, Philip E
Intrinsic fluorescence analysis is a sensitive technique to gain insight about aptamer-small molecule interactions.Employing fluorescence properties of the ligand, the binding affinities of the aptamer-ligand complex can be quantified in the nM to mM range with great accuracy and precision. Here, we present a detection method for aptamer-ligand binding analysis that is based on the inherent fluorescence of the ligand. Further, we discuss how to optimize and resolve some common experimental challenges.