257 results on '"Anslyn EV"'
Search Results
2. Data-science-guided calibration curve prediction of an MLCT-based ee determination assay for chiral amines.
- Author
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Howard JR, Shuluk JR, Bhakare A, and Anslyn EV
- Abstract
Circular dichroism (CD) based enantiomeric excess (ee) determination assays are optical alternatives to chromatographic ee determination in high-throughput screening (HTS) applications. However, the implementation of these assays requires calibration experiments using enantioenriched materials. We present a data-driven approach that circumvents the need for chiral resolution and calibration experiments for an octahedral Fe(II) complex (1) used for the ee determination of α-chiral primary amines. By computationally parameterizing the imine ligands formed in the assay conditions, a model of the circular dichroism (CD) response of the Fe(II) assembly was developed. Using this model, calibration curves were generated for four analytes and compared to experimentally generated curves. In a single-blind ee determination study, the ee values of unknown samples were determined within 9% mean absolute error, which rivals the error using experimentally generated calibration curves., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.
- Published
- 2024
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3. A Method for Rigorously Selective Capture and Simultaneous Fluorescent Labeling of N-Terminal Glycine Peptides.
- Author
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Liu H, Deol H, Raeisbahrami A, Askari H, Wight CD, Lynch VM, and Anslyn EV
- Subjects
- Molecular Structure, Glycine chemistry, Fluorescent Dyes chemistry, Peptides chemistry
- Abstract
Although chemical methods for the selective derivatization of amino acid (AA) side chains in peptides and proteins are available, selective N-terminal labeling is challenging, especially for glycine, which has no side chain at the α-carbon position. We report here a double activation at glycine's α-methylene group that allows this AA to be differentiated from the other 19 AAs. A condensation reaction of dibenzoylmethane with glycine results in the formation of an imine, and subsequent tautomerization is followed by intramolecular cyclization, leading to the formation of a fluorescent pyrrole ring. Additionally, the approach exhibits compatibility with AAs possessing reactive side chains. Further, the method allows for selective pull-down assays of N-terminal glycine peptides from mixtures without prior knowledge of the N-terminal peptide distribution.
- Published
- 2024
- Full Text
- View/download PDF
4. Circular Dichroism Sensing: Strategies and Applications.
- Author
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Formen JSSK, Howard JR, Anslyn EV, and Wolf C
- Abstract
The analysis of the absolute configuration, enantiomeric composition, and concentration of chiral compounds are frequently encountered tasks across the chemical and health sciences. Chiroptical sensing methods can streamline this work and allow high-throughput screening with remarkable reduction of operational time and cost. During the last few years, significant methodological advances with innovative chirality sensing systems, the use of computer-generated calibration curves, machine learning assistance, and chemometric data processing, to name a few, have emerged and are now matched with commercially available multi-well plate CD readers. These developments have reframed the chirality sensing space and provide new opportunities that are of interest to a large group of chemists. This review will discuss chirality sensing strategies and applications with representative small-molecule CD sensors. Emphasis will be given to important milestones and recent advances that accelerate chiral compound analysis by outperforming traditional methods, conquer new directions, and pioneering efforts that lie at the forefront of chiroptical high-throughput screening developments. The goal is to provide the reader with a thorough understanding of the current state and a perspective of future directions of this rapidly emerging field., (© 2024 Wiley‐VCH GmbH.)
- Published
- 2024
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5. Tertiary Amine Coupling by Oxidation for Selective Labeling of Dimethyl Lysine Post-Translational Modifications.
- Author
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Emenike B, Czabala P, Farhi J, Swaminathan J, Anslyn EV, Spangle J, and Raj M
- Subjects
- Proteins chemistry, Amines, Aldehydes, Lysine chemistry, Protein Processing, Post-Translational
- Abstract
Lysine dimethylation (Kme
2 ) is a crucial post-translational modification (PTM) that regulates biological processes and is implicated in diseases. There is significant interest in globally identifying these methylation marks. Unfortunately, this remains challenging due to the lack of robust technologies for selectively labeling Kme2 . To address this, we present a chemical method named tertiary amine coupling by oxidation (TACO). This method selectively modifies Kme2 to aldehydes using Selectfluor and a base. The resulting aldehydes from Kme2 were then functionalized using reductive amination, thiolamine, and oxime chemistry. We successfully demonstrated the versatility of TACO in selectively labeling Kme2 peptides and proteins in complex cell lysate mixtures with varying payloads, including affinity tags and fluorophores. We further showed the application of TACO chemistry for the identification of Kme2 sites at a single-molecule level by fluorosequencing. We discovered novel 30 Kme2 sites, in addition to previously known 5 Kme2 sites, by proteomics analysis of TACO-modified nuclear extracts. Our work establishes a unique strategy for covalently modifying Kme2 , facilitating the global identification of low-abundance Kme2 -PTMs and their sites within complex cell lysate mixtures.- Published
- 2024
- Full Text
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6. Chemical and linguistic considerations for encoding Chinese characters: an embodiment using chain-end degradable sequence-defined oligourethanes created by consecutive solid phase click chemistry.
- Author
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Zhang L, Krause TB, Deol H, Pandey B, Xiao Q, Park HM, Iverson BL, Law D, and Anslyn EV
- Abstract
Sequence-defined polymers (SDPs) are currently being investigated for use as information storage media. As the number of monomers in the SDPs increases, with a corresponding increase in mathematical base, the use of tandem-MS for de novo sequencing becomes more challenging. In contrast, chain-end degradation routines are truly de novo , potentially allowing very large mathematical bases for encoding. While alphabetic scripts have a few dozen symbols, logographic scripts, such as Chinese, can have several thousand symbols. Using a new in situ consecutive click reaction approach on an oligourethane backbone for writing, and a previously reported chain-end degradation routine for reading, we encoded/decoded a confucius proverb written in Chinese characters using two encoding schemes: Unicode and Zhèng Mă. Unicode is an internationally standardized arbitrary string of hexadecimal (base-16) symbols which efficiently encodes uniquely identifiable symbols but requires complete fidelity of transmission, or context-based inferential strategies to be interpreted. The Zhèng Mă approach encodes with a base-26 system using the visual characteristics and internal composition of Chinese characters themselves, which leads to greater ambiguity of encoded strings, but more robust retrievability of information from partial or corrupted encodings. The application of information-encoded oligourethanes to two different encoding systems allowed us to establish their flexibility and versatility for data storage. We found the oligourethanes immensely adaptable to both encoding schemes for Chinese characters, and we highlight the expected tradeoff between the efficiency and uniqueness of Unicode encoding on the one hand, and the fidelity to a scripts' particular visual characteristics on the other., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2024
- Full Text
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7. Structural Control of Plasmon Resonance in Molecularly Linked Metal Oxide Nanocrystal Gel Assemblies.
- Author
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Kang J, Sherman ZM, Conrad DL, Crory HSN, Dominguez MN, Valenzuela SA, Anslyn EV, Truskett TM, and Milliron DJ
- Abstract
Nanocrystal gels exhibit collective optical phenomena based on interactions among their constituent building blocks. However, their inherently disordered structures have made it challenging to understand, predict, or design properties such as optical absorption spectra that are sensitive to the coupling between the plasmon resonances of the individual nanocrystals. Here, we bring indium tin oxide nanocrystal gels under chemical control and show that their infrared absorption can be predicted and systematically tuned by selecting the nanocrystal sizes and compositions and molecular structures of the link-mediating surface ligands. Thermoreversible assemblies with metal-terpyridine links form reproducible gel architectures, enabling us to derive a plasmon ruler that governs the spectral shifts upon gelation, predicated on the nanocrystal and ligand compositions. This empirical guide is validated using large-scale, many-bodied simulations to compute the optical spectra of gels with varied structural parameters. Based on the derived plasmon ruler, we design and demonstrate a nanocrystal mixture whose spectrum exhibits distinctive line narrowing upon assembly.
- Published
- 2023
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8. Peptide Macrocyclization Guided by Reversible Covalent Templating.
- Author
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Maier JM, Valenzuela SA, van der Stok A, Menta AK, Shimizu Y, Ngo PH, Ellington AD, and Anslyn EV
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- Chemistry Techniques, Synthetic, Peptides chemistry, Nucleic Acids
- Abstract
The creation of complementary products via templating is a hallmark feature of nucleic acid replication. Outside of nucleic acid-like molecules, the templated synthesis of a hetero-complementary copy is still rare. Herein we describe one cycle of templated synthesis that creates homomeric macrocyclic peptides guided by linear instructing strands. This strategy utilizes hydrazone formation to pre-organize peptide oligomeric monomers along the template on a solid support resin, and microwave-assisted peptide synthesis to couple monomers and cyclize the strands. With a flexible templating strand, we can alter the size of the complementary macrocycle products by increasing the length and number of the binding peptide oligomers, showing the potential to precisely tune the size of macrocyclic products. For the smaller macrocyclic peptides, the products can be released via hydrolysis and characterized by ESI-MS., (© 2023 Wiley-VCH GmbH.)
- Published
- 2023
- Full Text
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9. Shear Thickening Behavior in Injectable Tetra-PEG Hydrogels Cross-Linked via Dynamic Thia-Michael Addition Bonds.
- Author
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Crowell AD, FitzSimons TM, Anslyn EV, Schultz KM, and Rosales AM
- Abstract
Injectable poly(ethylene glycol) (PEG)-based hydrogels were reversibly cross-linked through thia-conjugate addition bonds and demonstrated to shear thicken at low shear rates. Cross-linking bond exchange kinetics and dilute polymer concentrations were leveraged to tune hydrogel plateau moduli (from 60 to 650 Pa) and relaxation times (from 2 to 8 s). Under continuous flow shear rheometry, these properties affected the onset of shear thickening and the degree of shear thickening achieved before a flow instability occurred. The changes in viscosity were reversible whether the shear rate increased or decreased, suggesting that chain stretching drives this behavior. Given the relevance of dynamic PEG hydrogels under shear to biomedical applications, their injectability was investigated. Injection forces were found to increase with higher polymer concentrations and slower bond exchange kinetics. Altogether, these results characterize the nonlinear rheology of dilute, dynamic covalent tetra-PEG hydrogels and offer insight into the mechanism driving their shear thickening behavior.
- Published
- 2023
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10. Robust and scalable single-molecule protein sequencing with fluorosequencing.
- Author
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Mapes JH, Stover J, Stout HD, Folsom TM, Babcock E, Loudwig S, Martin C, Austin MJ, Tu F, Howdieshell CJ, Simpson ZB, Blom T, Weaver D, Winkler D, Vander Velden K, Ossareh PM, Beierle JM, Somekh T, Bardo AM, Anslyn EV, Marcotte EM, and Swaminathan J
- Abstract
The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequencing platform, based on determining the sequence positions of select amino acid types within peptides to enable their identification and quantification from a reference database. Here, we describe substantial improvements to fluorosequencing, including identifying fluorophores compatible with the sequencing chemistry, mitigating dye-dye interactions through the use of extended polyproline linkers, and developing an end-to-end workflow for sample preparation and sequencing. We demonstrate by fluorosequencing peptides in mixtures and identifying a target neoantigen from a database of decoy MHC peptides, highlighting the potential of the technology for high sensitivity clinical applications., Competing Interests: COMPETING INTERESTS E.M.M. and E.V.A. are co-founders and shareholders of Erisyon, Inc., and serve on the scientific advisory board. All other authors are affiliated with Erisyon, Inc., as employees or shareholders, and JS, AMB, HS, and CM additionally hold affiliation with UT Austin.
- Published
- 2023
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11. Remarkably Selective Binding, Behavior Modification, and Switchable Release of (Bipyridine) 3 Ru(II) vis-à-vis (Phenanthroline) 3 Ru(II) by Trimeric Cyclophanes in Water.
- Author
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Lin HY, Yao CY, Li J, Nimal Gunaratne HQ, Singh W, Huang M, Anslyn EV, and de Silva AP
- Abstract
A recurring dream of molecular recognition is to create receptors that distinguish between closely related targets with sufficient accuracy, especially in water. The more useful the targets, the more valuable the dream becomes. We now present multianionic trimeric cyclophane receptors with a remarkable ability to bind the iconic (bipyridine)
3 Ru(II) (with its huge range of applications) while rejecting the nearly equally iconic (phenanthroline)3 Ru(II). These receptors not only selectively capture (bipyridine)3 Ru(II) but also can be redox-switched to release the guest. 1D- and 2D(ROESY)-NMR spectroscopy, luminescence spectroscopy, and molecular modeling enabled this discovery. This outcome allows the control of these applications, e.g., as a photocatalyst or as a luminescent sensor, by selectively hiding or exposing (bipyridine)3 Ru(II). Overall, a 3D nanometric object is selected, picked-up, and dropped-off by a discrete molecular host. The multianionic receptors protect excited states of these metal complexes from phenolate quenchers so that the initial step in photocatalytic phenolate oxidation is retarded by nearly 2 orders of magnitude. This work opens the way for (bipyridine)3 Ru(II) to be manipulated in the presence of other functional nano-objects so that many of its applications can be commanded and controlled. We have a cyclophane-based toolkit that can emulate some aspects of proteins that selectively participate in cell signaling and metabolic pathways by changing shape upon environmental commands being received at a location remote from the active site., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)- Published
- 2023
- Full Text
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12. High-throughput determination of enantiopurity in atroposelective synthesis of aryl triazoles.
- Author
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Lim J, Guo M, Choi S, Miller SJ, and Anslyn EV
- Abstract
Atropisomeric scaffolds are a common design element found in pharmaceuticals, many deriving from an N-C axis of chirality. The handedness associated with atropisomeric drugs is oftentimes crucial for their efficacy and/or safety. With the increased use of high-throughput screening (HTS) for drug discovery, the need for rapid enantiomeric excess ( ee ) analysis is needed to keep up with the fast workflow. Here, we describe a circular dichroism (CD) based assay that could be applied to the ee determination of N-C axially chiral triazole derivatives. Analytical samples for CD were prepared from crude mixtures by three sequential steps: liquid-liquid extraction (LLE), a wash-elute, and complexation with Cu(ii) triflate. The initial ee measurement of five samples of atropisomer 2 was conducted by the use of a CD spectropolarimeter with a 6-position cell changer, resulting in errors of less than 1% ee . High-throughput ee determination was performed on a CD plate reader using a 96-well plate. A total of 28 atropisomeric samples (14 for 2 and 14 for 3) were screened for ee . The CD readings were completed in 60 seconds with average absolute errors of ±7.2% and 5.7% ee for 2 and 3, respectively., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2023
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13. Plasmonic Response of Complex Nanoparticle Assemblies.
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Sherman ZM, Kim K, Kang J, Roman BJ, Crory HSN, Conrad DL, Valenzuela SA, Lin E, Dominguez MN, Gibbs SL, Anslyn EV, Milliron DJ, and Truskett TM
- Abstract
Optical properties of nanoparticle assemblies reflect distinctive characteristics of their building blocks and spatial organization, giving rise to emergent phenomena. Integrated experimental and computational studies have established design principles connecting the structure to properties for assembled clusters and superlattices. However, conventional electromagnetic simulations are too computationally expensive to treat more complex assemblies. Here we establish a fast, materials agnostic method to simulate the optical response of large nanoparticle assemblies incorporating both structural and compositional complexity. This many-bodied, mutual polarization method resolves limitations of established approaches, achieving rapid, accurate convergence for configurations including thousands of nanoparticles, with some overlapping. We demonstrate these capabilities by reproducing experimental trends and uncovering far- and near-field mechanisms governing the optical response of plasmonic semiconductor nanocrystal assemblies including structurally complex gel networks and compositionally complex mixed binary superlattices. This broadly applicable framework will facilitate the design of complex, hierarchically structured, and dynamic assemblies for desired optical characteristics.
- Published
- 2023
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14. Determination of Enantiomeric Excess and Diastereomeric Excess via Optical Methods. Application to α-methyl-β-hydroxy-carboxylic acids.
- Author
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Moor SR, Howard JR, Herrera BT, McVeigh MS, Marini F, Keatinge-Clay AT, and Anslyn EV
- Abstract
Characterization of chiral molecules in solution is paramount for measuring reaction success. However, techniques to distinguish between chiral molecules containing more than one stereocenter through the use of optical techniques remains a challenge. Herein, we report a techique using a series of circular dichroism spectra to train multivariate regression models that are capable of predicting the complete speciation of 3-hydroxy-2-methylbutanoic acid stereoisomers. From this, it is possible to rapidly and accurately determine the enantiomeric excess and diastereomeric excess of the solution without the need for chiral chromatography., Competing Interests: Conflicts of interest There are no conflicts to declare.
- Published
- 2023
- Full Text
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15. Fluorescent probes for the detection of chemical warfare agents.
- Author
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Meng WQ, Sedgwick AC, Kwon N, Sun M, Xiao K, He XP, Anslyn EV, James TD, and Yoon J
- Subjects
- Humans, Fluorescent Dyes, Chemical Warfare Agents analysis
- Abstract
Chemical warfare agents (CWAs) are toxic chemicals that have been intentionally developed for targeted and deadly use on humans. Although intended for military targets, the use of CWAs more often than not results in mass civilian casualties. To prevent further atrocities from occurring during conflicts, a global ban was implemented through the chemical weapons convention, with the aim of eliminating the development, stockpiling, and use of CWAs. Unfortunately, because of their relatively low cost, ease of manufacture and effectiveness on mass populations, CWAs still exist in today's world. CWAs have been used in several recent terrorist-related incidents and conflicts ( e.g. , Syria). Therefore, they continue to remain serious threats to public health and safety and to global peace and stability. Analytical methods that can accurately detect CWAs are essential to global security measures and for forensic analysis. Small molecule fluorescent probes have emerged as attractive chemical tools for CWA detection, due to their simplicity, ease of use, excellent selectivity and high sensitivity, as well as their ability to be translated into handheld devices. This includes the ability to non-invasively image CWA distribution within living systems ( in vitro and in vivo ) to permit in-depth evaluation of their biological interactions and allow potential identification of therapeutic countermeasures. In this review, we provide an overview of the various reported fluorescent probes that have been designed for the detection of CWAs. The mechanism for CWA detection, change in optical output and application for each fluorescent probe are described in detail. The limitations and challenges of currently developed fluorescent probes are discussed providing insight into the future development of this research area. We hope the information provided in this review will give readers a clear understanding of how to design a fluorescent probe for the detection of a specific CWA. We anticipate that this will advance our security systems and provide new tools for environmental and toxicology monitoring.
- Published
- 2023
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16. Recent advances in fluorescent and colorimetric chemosensors for the detection of chemical warfare agents: a legacy of the 21st century.
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Kumar V, Kim H, Pandey B, James TD, Yoon J, and Anslyn EV
- Subjects
- Humans, Colorimetry, Coloring Agents, Chemical Warfare Agents analysis
- Abstract
Chemical warfare agents (CWAs) are among the most prominent threats to the human population, our peace, and social stability. Therefore, their detection and quantification are of utmost importance to ensure the security and protection of mankind. In recent years, significant developments have been made in supramolecular chemistry, analytical chemistry, and molecular sensors, which have improved our capability to detect CWAs. Fluorescent and colorimetric chemosensors are attractive tools that allow the selective, sensitive, cheap, portable, and real-time analysis of the potential presence of CWAs, where suitable combinations of selective recognition and transduction can be integrated. In this review, we provide a detailed discussion on recently reported molecular sensors with a specific focus on the sensing of each class of CWAs such as nerve agents, blister agents, blood agents, and other toxicants. We will also discuss the current technology used by military forces, and these discussions will include the type of instrumentation and established protocols. Finally, we will conclude this review with our outlook on the limitations and challenges in the area and summarize the potential of promising avenues for this field.
- Published
- 2023
- Full Text
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17. Modular mixing in plasmonic metal oxide nanocrystal gels with thermoreversible links.
- Author
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Kang J, Sherman ZM, Crory HSN, Conrad DL, Berry MW, Roman BJ, Anslyn EV, Truskett TM, and Milliron DJ
- Abstract
Gelation offers a powerful strategy to assemble plasmonic nanocrystal networks incorporating both the distinctive optical properties of constituent building blocks and customizable collective properties. Beyond what a single-component assembly can offer, the characteristics of nanocrystal networks can be tuned in a broader range when two or more components are intimately combined. Here, we demonstrate mixed nanocrystal gel networks using thermoresponsive metal-terpyridine links that enable rapid gel assembly and disassembly with thermal cycling. Plasmonic indium oxide nanocrystals with different sizes, doping concentrations, and shapes are reliably intermixed in linked gel assemblies, exhibiting collective infrared absorption that reflects the contributions of each component while also deviating systematically from a linear combination of the spectra for single-component gels. We extend a many-bodied, mutual polarization method to simulate the optical response of mixed nanocrystal gels, reproducing the experimental trends with no free parameters and revealing that spectral deviations originate from cross-coupling between nanocrystals with distinct plasmonic properties. Our thermoreversible linking strategy directs the assembly of mixed nanocrystal gels with continuously tunable far- and near-field optical properties that are distinct from those of the building blocks or mixed close-packed structures.
- Published
- 2023
- Full Text
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18. 11 B NMR Spectroscopy: Structural Analysis of the Acidity and Reactivity of Phenyl Boronic Acid-Diol Condensations.
- Author
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Valenzuela SA, Howard JR, Park HM, Darbha S, and Anslyn EV
- Subjects
- Magnetic Resonance Spectroscopy, Carbohydrates, Boronic Acids chemistry, Alcohols chemistry
- Abstract
Phenyl boronic acids are valuable for medical diagnostics and biochemistry studies due to their ability to readily bind with carbohydrates in water. Incorporated in carbohydrates are 1,2-diols, which react with boronic acids through a reversible covalent condensation pathway. A wide variety of boronic acids have been employed for diol binding with differing substitution of the phenyl ring, with the goals of simplifying their synthesis and altering their thermodynamics of complexation. One method for monitoring their p K
a 's and binding is11 B NMR spectroscopy. Herein, we report a comprehensive study employing11 B NMR spectroscopy to determine the p Ka of the most commonly used phenyl boronic acids and their binding with catechol or d , l -hydrobenzoin as prototypical diols. The chemical shift of the boronic acid transforming into the boronate ester was monitored at pHs ranging from 2 to 10. With each boronic acid, the results confirm (1) the necessity to use pHs above their p Ka 's to induce complexation, (2) that the p Ka 's change in the presence of diols, and (3) that11 B NMR spectroscopy is a particularly convenient tool for monitoring these interconnected acidity and binding phenomena.- Published
- 2022
- Full Text
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19. Conformational change in ricin toxin A-Chain: A critical factor for inhibitor binding to the secondary pocket.
- Author
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Goto M, Higashi S, Ohba T, Kawata R, Nagatsu K, Suzuki S, Anslyn EV, and Saito R
- Subjects
- Crystallography, X-Ray, Ribosomes metabolism, Ricin chemistry, Ricin metabolism, Ricin toxicity
- Abstract
Ricin toxin A-chain (RTA), a toxic protein from Ricinus communis, inactivates ribosomes to induce toxicity. The active site of RTA consists of two binding pockets. Many studies have focused on developing RTA inhibitors that can simultaneously bind to these critical pockets; however, almost all the inhibitors developed so far interact with only one pocket. In the present study, we discovered that pterin-7-carboxamides with aromatic l-amino acid pendants interacted with the active site of the enzyme in a 2-to-1 mode, where one inhibitor molecule bound to the primary pocket and the second one entered the secondary pocket in the active site of RTA. X-ray crystallographic analysis of inhibitor/RTA complexes revealed that the conformational changes of Tyr80 and Asn122 in RTA were critical for triggering the entry of inhibitor molecules into the secondary pocket of the RTA active site., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest associated with this manuscript., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
20. Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro.
- Author
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Lee J, Coronado JN, Cho N, Lim J, Hosford BM, Seo S, Kim DS, Kofman C, Moore JS, Ellington AD, Anslyn EV, and Jewett MC
- Subjects
- Genetic Code, Peptides chemistry, Amino Acids metabolism, Protein Biosynthesis, Ribosomes metabolism, RNA, Transfer metabolism
- Abstract
The ribosome is a macromolecular machine that catalyzes the sequence-defined polymerization of L-α-amino acids into polypeptides. The catalysis of peptide bond formation between amino acid substrates is based on entropy trapping, wherein the adjacency of transfer RNA (tRNA)-coupled acyl bonds in the P-site and the α-amino groups in the A-site aligns the substrates for coupling. The plasticity of this catalytic mechanism has been observed in both remnants of the evolution of the genetic code and modern efforts to reprogram the genetic code (e.g., ribosomal incorporation of non-canonical amino acids, ribosomal ester formation). However, the limits of ribosome-mediated polymerization are underexplored. Here, rather than peptide bonds, we demonstrate ribosome-mediated polymerization of pyridazinone bonds via a cyclocondensation reaction between activated γ-keto and α-hydrazino ester monomers. In addition, we demonstrate the ribosome-catalyzed synthesis of peptide-hybrid oligomers composed of multiple sequence-defined alternating pyridazinone linkages. Our results highlight the plasticity of the ribosome's ancient bond-formation mechanism, expand the range of non-canonical polymeric backbones that can be synthesized by the ribosome, and open the door to new applications in synthetic biology., (© 2022. The Author(s).)
- Published
- 2022
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21. Data-Driven Prediction of Circular Dichroism-Based Calibration Curves for the Rapid Screening of Chiral Primary Amine Enantiomeric Excess Values.
- Author
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Howard JR, Bhakare A, Akhtar Z, Wolf C, and Anslyn EV
- Subjects
- Calibration, Circular Dichroism, Stereoisomerism, Amines
- Abstract
Here, we describe the prediction of the circular dichroism (CD) response of a three-component chiroptical sensor for enantiomeric excess ( ee ) determination of chiral amines using a multivariate fit to electronic and steric parameters. These computationally derived parameters can be computed for nearly any amine and correlate well with the CD response of the 12 amines comprising the training set. The resulting model was used to accurately predict the CD response of a test set of chiral amines. Theoretical calibration curves were then created and used to determine the ee of solutions of unknown ee . Using this method, the error in ee determination differed by less than 10% compared to experimentally generated calibration curves.
- Published
- 2022
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22. Molecular Encryption and Steganography Using Mixtures of Simultaneously Sequenced, Sequence-Defined Oligourethanes.
- Author
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Dahlhauser SD, Wight CD, Moor SR, Scanga RA, Ngo P, York JT, Vera MS, Blake KJ, Riddington IM, Reuther JF, and Anslyn EV
- Abstract
Molecular encoding in abiotic sequence-defined polymers (SDPs) has recently emerged as a versatile platform for information and data storage. However, the storage capacity of these sequence-defined polymers remains underwhelming compared to that of the information storing biopolymer DNA. In an effort to increase their information storage capacity, herein we describe the synthesis and simultaneous sequencing of eight sequence-defined 10-mer oligourethanes. Importantly, we demonstrate the use of different isotope labels, such as halogen tags, as a tool to deconvolute the complex sequence information found within a heterogeneous mixture of at least 96 unique molecules, with as little as four micromoles of total material. In doing so, relatively high-capacity data storage was achieved: 256 bits in this example, the most information stored in a single sample of abiotic SDPs without the use of long strands. Within the sequence information, a 256-bit cipher key was stored and retrieved. The key was used to encrypt and decrypt a plain text document containing The Wonderful Wizard of Oz . To validate this platform as a medium of molecular steganography and cryptography, the cipher key was hidden in the ink of a personal letter, mailed to a third party, extracted, sequenced, and deciphered successfully in the first try, thereby revealing the encrypted document., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)
- Published
- 2022
- Full Text
- View/download PDF
23. Chemical insights into flexizyme-mediated tRNA acylation.
- Author
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Coronado JN, Ngo P, Anslyn EV, and Ellington AD
- Subjects
- Acylation, Catalysis, Peptides metabolism, RNA, Transfer metabolism, RNA, Catalytic chemistry, Transfer RNA Aminoacylation
- Abstract
A critical step in repurposing the cellular translation machinery for the synthesis of polymeric products is the acylation of transfer RNA (tRNA) with unnatural monomers. Toward this goal, flexizymes, ribozymes capable of aminoacylation, have emerged as a uniquely adept tool for charging tRNA with ever increasingly diverse substrates. In this review, we present a library of monomer substrates that have been tested for tRNA acylation with the flexizyme system. From this mile-high view, we provide insights for understanding the chemical factors that influence flexizyme-mediated tRNA acylation. We conclude that flexizymes are primitive esterification catalysts that display a modest binding affinity to the monomer's aromatic recognition element. Together, these robust, yet flexible, flexizyme systems provide researchers with unprecedented access for preparing unnatural acyl-tRNA and the opportunity to repurpose the translation machinery for the synthesis of novel biologically derived structures beyond native proteins and peptides., Competing Interests: Declaration of interests J.N.C. and E.V.A. are co-inventors on a provisional patent (US Provisional Patent Serial No. 63/144,814) that incorporates technology described in this article., (Copyright © 2022 Elsevier Ltd. All rights reserved.)
- Published
- 2022
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24. Evaluating the Effect of Dye-Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides.
- Author
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Bachman JL, Wight CD, Bardo AM, Johnson AM, Pavlich CI, Boley AJ, Wagner HR, Swaminathan J, Iverson BL, Marcotte EM, and Anslyn EV
- Subjects
- Amino Acids, Azides chemistry, Ionophores, Peptides, Fluorescent Dyes chemistry, Xanthenes
- Abstract
A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG)
10 linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide-alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the single-molecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.- Published
- 2022
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25. Effect of pH on the Properties of Hydrogels Cross-Linked via Dynamic Thia-Michael Addition Bonds.
- Author
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FitzSimons TM, Anslyn EV, and Rosales AM
- Abstract
Hydrogels cross-linked with dynamic covalent bonds exhibit time-dependent properties, making them an advantageous platform for applications ranging from biomaterials to self-healing networks. However, the relationship between the cross-link exchange kinetics, material properties, and stability of these platforms is not fully understood, especially upon addition of external stimuli. In this work, pH was used as a handle to manipulate cross-link exchange kinetics and control the resulting hydrogel mechanics and stability in a physiologically relevant window. Poly(ethylene glycol)-based hydrogels were cross-linked with a reversible thia-Michael addition reaction in aqueous buffer between pH 3 and pH 7. The rate constants of bond exchange and equilibrium constants were determined for each pH value, and these data were correlated with the resulting mechanical profiles of the bulk hydrogels. With increasing pH, both the forward and the reverse rate constants increased, while the equilibrium constant decreased. These changes led to faster stress relaxation and less stiff hydrogels at more basic pH values. The elevated pH values also led to an increased mass loss and a faster rate of release of an encapsulated model bovine serum albumin fluorescent protein. The connection between the kinetics, mechanics, and molecular release profiles provides important insight into the structure-property relationships of dynamic covalent hydrogels, and this system offers a promising platform for controlled release between physiologically relevant pH values., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)
- Published
- 2022
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26. Multiplexing the Quantitation of MAP Kinase Activities Using Differential Sensing.
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Zeng L, Kaoud TS, Zamora-Olivares D, Bohanon AL, Li Y, Pridgen JR, Ekpo YE, Zhuang DL, Nye JR, Telles M, Winkler M, Rivera S, Marini F, Dalby KN, and Anslyn EV
- Subjects
- Humans, Peptides metabolism, Phosphorylation, Protein Kinases metabolism, p38 Mitogen-Activated Protein Kinases metabolism, MAP Kinase Signaling System physiology, Mitogen-Activated Protein Kinases metabolism
- Abstract
Protein kinases are therapeutic targets for many human diseases, but the lack of user-friendly quantitative assays limits the ability to follow the activities of numerous kinases at once (multiplexing). To develop such an assay, we report an array of sulfonamido-oxine (SOX)-labeled peptides showing cross-reactivity to different mitogen-activated protein kinases (MAPKs) for use in a differential sensing scheme. We first verified using linear discriminant analysis that the array could differentiate MAPK isoforms. Then, using principal component analysis, the array was optimized based on the discrimination imparted by each SOX-peptide. Next, the activity of individual MAPK families in ternary mixtures was quantified by support vector machine regression. Finally, we multiplexed the quantification of three MAPK families using partial least squares regression in A549 cell lysates, which has possible interference from other kinase classes. Thus, our method simultaneously quantifies the activity of multiple kinases. The technique could be applied to other protein kinase families and the monitoring of diseases.
- Published
- 2022
- Full Text
- View/download PDF
27. Assembling Inorganic Nanocrystal Gels.
- Author
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Green AM, Ofosu CK, Kang J, Anslyn EV, Truskett TM, and Milliron DJ
- Subjects
- Gels chemistry, Nanoparticles chemistry
- Abstract
Inorganic nanocrystal gels retain distinct properties of individual nanocrystals while offering tunable, network-structure-dependent characteristics. We review different mechanisms for assembling gels from colloidal nanocrystals including (1) controlled destabilization, (2) direct bridging, (3) depletion, as well as linking mediated by (4) coordination bonding or (5) dynamic covalent bonding, and we highlight how each impacts gel properties. These approaches use nanocrystal surface chemistry or the addition of small molecules to mediate inter-nanocrystal attractions. Each method offers advantages in terms of gel stability, reversibility, or tunability and presents new opportunities for the design of reconfigurable materials and fueled assemblies.
- Published
- 2022
- Full Text
- View/download PDF
28. Colorimetric quantification of linking in thermoreversible nanocrystal gel assemblies.
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Kang J, Valenzuela SA, Lin EY, Dominguez MN, Sherman ZM, Truskett TM, Anslyn EV, and Milliron DJ
- Abstract
Nanocrystal gels can be responsive, tunable materials, but designing their structure and properties is challenging. By using reversibly bonded molecular linkers, gelation can be realized under conditions predicted by thermodynamics. However, simulations have offered the only microscopic insights, with no experimental means to monitor linking leading to gelation. We introduce a metal coordination linkage with a distinct optical signature allowing us to quantify linking in situ and establish structural and thermodynamic bases for assembly. Because of coupling between linked indium tin oxide nanocrystals, their infrared absorption shifts abruptly at a chemically tunable gelation temperature. We quantify bonding spectroscopically and use molecular simulation to understand temperature-dependent bonding motifs, revealing that gel formation is governed by reaching a critical number of effective links that extend the nanocrystal network. Microscopic insights from our colorimetric linking chemistry enable switchable gels based on thermodynamic principles, opening the door to rational design of programmable nanocrystal networks.
- Published
- 2022
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29. Studies of Surface Preparation for the Fluorosequencing of Peptides.
- Author
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Hinson CM, Bardo AM, Shannon CE, Rivera S, Swaminathan J, Marcotte EM, and Anslyn EV
- Subjects
- Alkynes, Cycloaddition Reaction, Proteins, Azides, Peptides
- Abstract
Silica passivating agents have shown great success in minimizing nonspecific protein binding to glass surfaces for imaging and microscopy applications. Amine-derivatized surfaces are commonly used in conjugation with amide coupling agents to immobilize peptides/proteins through C-terminal or side-chain carboxylic acids. In the case of the single-molecule fluorosequencing of peptides, attachment occurs via the C-terminus and nonspecific surface binding has previously been a source of error in peptide identification. Here, we employ fluorosequencing as a high-throughput, single-molecule sensitivity assay to identify and quantify the extent of nonspecific binding of peptides to amine-derivatized surfaces. We show that there is little improvement when using common passivating agents in combination with the surface derivatizing agent 3-aminopropyl-triethoxysilane (APTES) to couple the peptides to the modified surface. Furthermore, many xanthene fluorophores have carboxylic acids in the appended phenyl ring at positions ortho and meta or ortho and para, and the literature shows that conjugation through the ortho position is not favored. Because xanthene-derived fluorophores are commonly used for single-molecule applications, we devised a novel assay to probe the conjugation of peptides via their fluorophores relative to their C-termini on silane-derivatized surfaces. We find significant attachment to the ortho position, which is a warning to those attempting to immobilize fluorophore-labeled peptides to silica surfaces via amide coupling agents. However, eliminating all amines on the surface by switching to 3-azidopropyl-triethoxysilane (AzTES) for coupling via copper-catalyzed azide-alkyne cycloaddition (CuAAC) and omitting additional passivation agents allowed us to achieve a high level of C-terminally bound peptides relative to nonspecifically or ortho-phenyl-bound, fluorophore-labeled peptides. This strategy substantially improves the specificity of peptide immobilization for single-molecule fluorosequencing experiments.
- Published
- 2021
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30. Photoredox-Catalyzed Decarboxylative C -Terminal Differentiation for Bulk- and Single-Molecule Proteomics.
- Author
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Zhang L, Floyd BM, Chilamari M, Mapes J, Swaminathan J, Bloom S, Marcotte EM, and Anslyn EV
- Subjects
- Alkylation, Amino Acids chemistry, Angiotensins chemistry, Catalysis, Decarboxylation, Humans, Oxidation-Reduction, Photochemical Processes, Peptides chemistry, Proteomics methods, Single Molecule Imaging methods
- Abstract
Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which labeling chemistry is designed. In practice, only two functional groups are present in every peptide fragment regardless of the protein cleavage site, namely, an N -terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N - and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative alkylation for peptide mass spectrometry and single-molecule protein sequencing that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino-acid residues for decarboxylative labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid-labeling technology in tandem to establish a new platform for fluorosequencing.
- Published
- 2021
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31. A Data-Driven Approach to the Development and Understanding of Chiroptical Sensors for Alcohols with Remote γ-Stereocenters.
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Dotson JJ, Anslyn EV, and Sigman MS
- Subjects
- Alcohols chemistry, Circular Dichroism, Density Functional Theory, Machine Learning, Models, Chemical, Molecular Structure, Pyridines chemistry, Quantitative Structure-Activity Relationship, Stereoisomerism, Workflow, Zinc chemistry, Alcohols analysis, Coordination Complexes chemistry
- Abstract
Dynamic covalent chemistry-based sensors have recently emerged as powerful tools to rapidly determine the enantiomeric excess of organic small molecules. While a bevy of sensors have been developed, those for flexible molecules with stereocenters remote to the functional group that binds the chiroptical sensor remain scarce. In this study, we develop an iterative, data-driven workflow to design and analyze a chiroptical sensor capable of assessing challenging acyclic γ-stereogenic alcohols. Following sensor optimization, the mechanism of sensing was probed with a combination of computational parametrization of the sensor molecules, statistical modeling, and high-level density functional theory (DFT) calculations. These were used to elucidate the mechanism of stereochemical recognition and revealed that competing attractive noncovalent interactions (NCIs) determine the overall performance of the sensor. It is anticipated that the data-driven workflows developed herein will be generally applicable to the development and understanding of dynamic covalent and supramolecular sensors.
- Published
- 2021
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32. The Evolution of Data-Driven Modeling in Organic Chemistry.
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Williams WL, Zeng L, Gensch T, Sigman MS, Doyle AG, and Anslyn EV
- Abstract
Organic chemistry is replete with complex relationships: for example, how a reactant's structure relates to the resulting product formed; how reaction conditions relate to yield; how a catalyst's structure relates to enantioselectivity. Questions like these are at the foundation of understanding reactivity and developing novel and improved reactions. An approach to probing these questions that is both longstanding and contemporary is data-driven modeling. Here, we provide a synopsis of the history of data-driven modeling in organic chemistry and the terms used to describe these endeavors. We include a timeline of the steps that led to its current state. The case studies included highlight how, as a community, we have advanced physical organic chemistry tools with the aid of computers and data to augment the intuition of expert chemists and to facilitate the prediction of structure-activity and structure-property relationships., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)
- Published
- 2021
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33. Electrostatic and Covalent Assemblies of Anionic Hydrogel-Coated Gold Nanoshells for Detection of Dry Eye Biomarkers in Human Tears.
- Author
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Wechsler ME, Jocelyn Dang HKH, Simmonds SP, Bahrami K, Wyse JM, Dahlhauser SD, Reuther JF, VandeWalle AN, Anslyn EV, and Peppas NA
- Subjects
- Biomarkers, Gold, Humans, Hydrogels, Static Electricity, Dry Eye Syndromes, Nanoshells
- Abstract
Although dry eye is highly prevalent, many challenges exist in diagnosing the symptom and related diseases. For this reason, anionic hydrogel-coated gold nanoshells (AuNSs) were used in the development of a label-free biosensor for detection of high isoelectric point tear biomarkers associated with dry eye. A custom, aldehyde-functionalized oligo(ethylene glycol)acrylate (Al-OEGA) was included in the hydrogel coating to enhance protein recognition through the formation of dynamic covalent (DC) imine bonds with solvent-accessible lysine residues present on the surface of select tear proteins. Our results demonstrated that hydrogel-coated AuNSs, composed of monomers that form ionic and DC bonds with select tear proteins, greatly enhance protein recognition due to changes in the maximum localized surface plasmon resonance wavelength exhibited by AuNSs in noncompetitive and competitive environments. Validation of the developed biosensor in commercially available pooled human tears revealed the potential for clinical translation to establish a method for dry eye diagnosis.
- Published
- 2021
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34. A self-degradable hydrogel sensor for a nerve agent tabun surrogate through a self-propagating cascade.
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Lee DH, Valenzuela SA, Dominguez MN, Otsuka M, Milliron DJ, and Anslyn EV
- Abstract
Nerve agents that irreversibly deactivate the enzyme acetylcholinesterase are extremely toxic weapons of mass destruction. Thus, developing methods to detect these lethal agents is important. To create an optical sensor for a surrogate of the nerve agent tabun, as well as a physical barrier that dissolves in response to this analyte, we devise a network hydrogel that decomposes via a self-propagating cascade. A Meldrums acid-derived linker is incorporated into a hydrogel that undergoes a declick reaction in response to thiols, thereby breaking network connections, which releases more thiols, propagating the response throughout the gel. A combination of chemical reactions triggered by the addition of the tabun mimic initiates the cascade. The dissolving barrier is used to release dyes, as well as nanocrystals that undergo a spontaneous aggregation. Thus, this sensing system for tabun generates a physical response and the delivery of chemical agents in response to an initial trigger., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.
- Published
- 2021
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35. Chemically Triggered Click and Declick Reactions: Application in Synthesis and Degradation of Thermosetting Plastics.
- Author
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Wu T, Liang T, Hu W, Du M, Zhang S, Zhang Y, Anslyn EV, and Sun X
- Abstract
In this Letter, we report that two amines can be coupled together rapidly and quantitatively through amine-thiol scrambling using a bisvinylogous thioester conjugate acceptor under mild conditions. The resulting bisvinylogous amide conjugate acceptors can be decoupled via an ethylene diamine-induced cyclization. Four representative conjugate acceptors have been utilized in the couple-decouple reactions, which were monitored and characterized by nuclear magnetic resonance, high-resolution mass spectrometry, and UV-vis spectroscopy. Further, we applied these small-molecule-based "click-declick" reactions to polymer synthesis and degradation. Highly cross-linked polymers, i.e., plastics, were quantitatively synthesized by simple reactions between commercial tris(2-aminoethyl)amine and the conjugate acceptors without solvent and any initiator or catalyst through ball milling within 60 min. Significantly, these thermosetting plastics can be degraded within 3-24 h via addition of ethylene diamine. The multiple architectures, application to plastics synthesis, and chemically triggered clean degradation to the thermosets at mild conditions with little input of energy herald a new generation of "intelligent" materials.
- Published
- 2021
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- View/download PDF
36. Combination of two analytical techniques improves wine classification by Vineyard, Region, and vintage.
- Author
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Crook AA, Zamora-Olivares D, Bhinderwala F, Woods J, Winkler M, Rivera S, Shannon CE, Wagner HR, Zhuang DL, Lynch JE, Berryhill NR, Runnebaum RC, Anslyn EV, and Powers R
- Subjects
- Farms, Humans, Magnetic Resonance Spectroscopy, Phenols analysis, Smell, Taste, Vitis chemistry, Wine analysis
- Abstract
Three important wine parameters: vineyard, region, and vintage year, were evaluated using fifteen Vitis vinifera L. 'Pinot noir' wines derived from the same scion clone (Pinot noir 667). These wines were produced from two vintage years (2015 and 2016) and eight different regions along the Pacific Coast of the United States. We successfully improved the classification of the selected Pinot noir wines by combining an untargeted 1D
1 H NMR analysis with a targeted peptide based differential sensing array. NMR spectroscopy was used to evaluate the chemical fingerprint of the wines, whereas the peptide-based sensing array is known to mimic the senses of taste, smell, and palate texture by characterizing the phenolic profile. Multivariate and univariate statistical analyses of the combined NMR and differential sensing array dataset classified the genetically identical Pinot noir wines on the basis of distinctive metabolic signatures associated with the region of growth, vineyard, and vintage year., (Copyright © 2021 Elsevier Ltd. All rights reserved.)- Published
- 2021
- Full Text
- View/download PDF
37. High-throughput screening of α-chiral-primary amines to determine yield and enantiomeric excess.
- Author
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Moor SR, Howard JR, Herrera BT, and Anslyn EV
- Abstract
A novel screening protocol was developed using a combination of a fluorescent indicator displacement assay and a circular dichroism (CD) active Fe(II) complex to determine concentration and enantiomeric excess ( ee ) of α-chiral amines, respectively. The analyte concentration is quantified with a pre-formed non-fluorescent imine, where transimination with the chiral amine results in displacement of the fluorophore 2-naphthylamine. After discerning the concentration of amine via fluorescence in a wellplate reader, the analyte is then incorporated into a three-component octahedral Fe(II) assembly for ee determination using an EKKO CD plate-reader. With these two assays, both the ee and yield of asymmetric transformations of 192 samples could be determined with acceptable errors in under fifteen minutes (not counting the preparation time). This combined speed and accuracy provides an attractive solution to overcoming analytical bottlenecks when creating α-chiral amines., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
- Published
- 2021
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38. A Colorimetric Method for Quantifying Cis and Trans Alkenes Using an Indicator Displacement Assay.
- Author
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Valenzuela SA, Crory HSN, Yao CY, Howard JR, Saucedo G, de Silva AP, and Anslyn EV
- Subjects
- Molecular Structure, Stereoisomerism, Alkenes chemistry, Colorimetry
- Abstract
A colorimetric indicator displacement assay (IDA) amenable to high-throughput experimentation was developed to determine the percentage of cis and trans alkenes. Using 96-well plates two steps are performed: a reaction plate for dihydroxylation of the alkenes followed by an IDA screening plate consisting of an indicator and a boronic acid. The dihydroxylation generates either erythro or threo vicinal diols from cis or trans alkenes, depending upon their syn- or anti-addition mechanisms. Threo diols preferentially associate with the boronic acid due to the creation of more stable boronate esters, thus displacing the indicator to a greater extent. The generality of the protocol was demonstrated using seven sets of cis and trans alkenes. Blind mixtures of cis and trans alkenes were made, resulting in an average error of ±2 % in the percentage of cis or trans alkenes, and implementing E
2 and Wittig reactions gave errors of ±3 %. Furthermore, we developed variants of the IDA for which the color may be tuned to optimize the response for the human eye., (© 2021 Wiley-VCH GmbH.)- Published
- 2021
- Full Text
- View/download PDF
39. The emerging landscape of single-molecule protein sequencing technologies.
- Author
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Alfaro JA, Bohländer P, Dai M, Filius M, Howard CJ, van Kooten XF, Ohayon S, Pomorski A, Schmid S, Aksimentiev A, Anslyn EV, Bedran G, Cao C, Chinappi M, Coyaud E, Dekker C, Dittmar G, Drachman N, Eelkema R, Goodlett D, Hentz S, Kalathiya U, Kelleher NL, Kelly RT, Kelman Z, Kim SH, Kuster B, Rodriguez-Larrea D, Lindsay S, Maglia G, Marcotte EM, Marino JP, Masselon C, Mayer M, Samaras P, Sarthak K, Sepiashvili L, Stein D, Wanunu M, Wilhelm M, Yin P, Meller A, and Joo C
- Subjects
- Mass Spectrometry methods, Nanotechnology, Proteins chemistry, Proteomics methods, Sequence Analysis, RNA methods, Single-Cell Analysis methods, Sequence Analysis, Protein methods, Single Molecule Imaging methods
- Abstract
Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.
- Published
- 2021
- Full Text
- View/download PDF
40. Efficient molecular encoding in multifunctional self-immolative urethanes.
- Author
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Dahlhauser SD, Moor SR, Vera MS, York JT, Ngo P, Boley AJ, Coronado JN, Simpson ZB, and Anslyn EV
- Abstract
Molecular encoding in sequence-defined polymers shows promise as a new paradigm for data storage. Here, we report what is, to our knowledge, the first use of self-immolative oligourethanes for storing and reading encoded information. As a proof of principle, we describe how a text passage from Jane Austen's Mansfield Park was encoded in sequence-defined oligourethanes and reconstructed via self-immolative sequencing. We develop Mol.E-coder, a software tool that uses a Huffman encoding scheme to convert the character table to hexadecimal. The oligourethanes are then generated by a high-throughput parallel synthesis. Sequencing of the oligourethanes by self-immolation is done concurrently in a parallel fashion, and the liquid chromatography-mass spectrometry (LC-MS) information decoded by our Mol.E-decoder software. The passage is capable of being reproduced wholly intact by a third-party, without any purifications or the use of tandem MS (MS/MS), despite multiple rounds of compression, encoding, and synthesis., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.
- Published
- 2021
- Full Text
- View/download PDF
41. "Benchtop" Biaryl Coupling Using Pd/Cu Cocatalysis: Application to the Synthesis of Conjugated Polymers.
- Author
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Minus MB, Moor SR, Pary FF, Nirmani LPT, Chwatko M, Okeke B, Singleton JE, Nelson TL, Lynd NA, and Anslyn EV
- Abstract
Typically, Suzuki couplings used in polymerizations are performed at raised temperatures in inert atmospheres. As a result, the synthesis of aromatic materials that utilize this chemistry often demands expensive and specialized equipment on an industrial scale. Herein, we describe a bimetallic methodology that exploits the distinct reactivities of palladium and copper to perform high yielding aryl-aryl dimerizations and polymerizations that can be performed on a benchtop under ambient conditions. These couplings are facile and can be performed by simple mixing in the open vessel. To demonstrate the utility of this method in the context of polymer synthesis: polyfluorene, polycarbazole, polysilafluorene, and poly(6,12-dihydro-dithienoindacenodithiophene) were created at ambient temperature and open to air.
- Published
- 2021
- Full Text
- View/download PDF
42. Ribosome-mediated incorporation of fluorescent amino acids into peptides in vitro.
- Author
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Lee J, Schwarz KJ, Yu H, Krüger A, Anslyn EV, Ellington AD, Moore JS, and Jewett MC
- Subjects
- Acylation, Amino Acids chemistry, Genetic Code, RNA, Catalytic chemistry, RNA, Transfer chemistry, RNA, Transfer genetics, Amino Acids genetics, Fluorescent Dyes chemistry, Peptides chemical synthesis, Protein Engineering methods, Ribosomes genetics
- Abstract
We report the design, chemical synthesis, and flexizyme-catalyzed transfer RNA (tRNA) acylation of a variety of fluorescent amino acids (FAAs). The fluorescent groups include pyrene, coumarin, nitrobenzoxadiazole, and fluorescein variants. We further demonstrate site-specific incorporation of the FAAs into peptides by the ribosome in vitro through genetic code reprogramming.
- Published
- 2021
- Full Text
- View/download PDF
43. Effects of linker flexibility on phase behavior and structure of linked colloidal gels.
- Author
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Howard MP, Sherman ZM, Sreenivasan AN, Valenzuela SA, Anslyn EV, Milliron DJ, and Truskett TM
- Abstract
Colloidal nanocrystal gels can be assembled using a difunctional "linker" molecule to mediate bonding between nanocrystals. The conditions for gelation and the structure of the gel are controlled macroscopically by the linker concentration and microscopically by the linker's molecular characteristics. Here, we demonstrate using a toy model for a colloid-linker mixture that linker flexibility plays a key role in determining both phase behavior and the structure of the mixture. We fix the linker length and systematically vary its bending stiffness to span the flexible, semiflexible, and rigid regimes. At fixed linker concentration, flexible-linker and rigid-linker mixtures phase separate at low colloid volume fractions, in agreement with predictions of first-order thermodynamic perturbation theory, but the semiflexible-linker mixtures do not. We correlate and attribute this qualitatively different behavior to undesirable "loop" linking motifs that are predicted to be more prevalent for linkers with end-to-end distances commensurate with the locations of chemical bonding sites on the colloids. Linker flexibility also influences the spacing between linked colloids, suggesting strategies to design gels with desired phase behavior, structure, and, by extension, structure-dependent properties.
- Published
- 2021
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44. Colloidal Nanocrystal Gels from Thermodynamic Principles.
- Author
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Sherman ZM, Green AM, Howard MP, Anslyn EV, Truskett TM, and Milliron DJ
- Abstract
Gels assembled from solvent-dispersed nanocrystals are of interest for functional materials because they promise the opportunity to retain distinctive properties of individual nanocrystals combined with tunable, structure-dependent collective behavior. By incorporating stimuli-responsive components, these materials could also be dynamically reconfigured between structurally distinct states. However, nanocrystal gels have so far been formed mostly through irreversible aggregation, which has limited the realization of these possibilities. Meanwhile, gelation strategies for larger colloidal microparticles have been developed using reversible physical or chemical interactions. These approaches have enabled the experimental navigation of theoretically predicted phase diagrams, helping to establish an understanding of how thermodynamic behavior can guide gel formation in these materials. However, the translation of these principles to the nanoscale poses both practical and fundamental challenges. The molecules guiding assembly can no longer be safely assumed to be vanishingly small compared to the particles nor large compared to the solvent.In this Account, we discuss recent progress toward the assembly of tunable nanocrystal gels using two strategies guided by equilibrium considerations: (1) reversible chemical bonding between functionalized nanocrystals and difunctional linker molecules and (2) nonspecific, polymer-induced depletion attractions. The effective nanocrystal attractions, mediated in both approaches by a secondary molecule, compete against stabilizing repulsions to promote reversible assembly. The structure and properties of the nanocrystal gels are controlled microscopically by the design of the secondary molecule and macroscopically by its concentration. This mode of control is compelling because it largely decouples nanocrystal synthesis and functionalization from the design of interactions that drive assembly. Statistical thermodynamic theory and computer simulation have been applied to simple models that describe the bonding motifs in these assembling systems, furnish predictions for conditions under which gelation is likely to occur, and suggest strategies for tuning and disassembling the gel networks. Insights from these models have guided experimental realizations of reversible gels with optical properties in the infrared range that are sensitive to the gel structure. This process avoids time-consuming and costly trial-and-error experimental investigations to accelerate the development of nanocrystal gel assemblies.These advances highlight the need to better understand interactions between nanocrystals, how interactions give rise to gel structure, and properties that emerge. Such an understanding could suggest new approaches for creating stimuli-responsive and dissipative assembled materials whose properties are tunable on demand through directed reconfiguration of the underlying gel microstructure. It may also make nanocrystal gels amenable to computationally guided design using inverse methods to rapidly optimize experimental parameters for targeted functionalities.
- Published
- 2021
- Full Text
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45. Indicator displacement assays (IDAs): the past, present and future.
- Author
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Sedgwick AC, Brewster JT, Wu T, Feng X, Bull SD, Qian X, Sessler JL, James TD, Anslyn EV, and Sun X
- Abstract
Indicator displacement assays (IDAs) offer a unique and innovative approach to molecular sensing. IDAs can facilitate the detection of a range of biologically/environmentally important species, provide a method for the detection of complex analytes or for the determination and discrimination of unknown sample mixtures. These attributes often cannot be achieved by traditional molecular sensors i.e. reaction-based sensors/chemosensors. The IDA pioneers Inouye, Shinkai, and Anslyn inspired researchers worldwide to develop various extensions of this idea. Since their early work, the field of indicator displacement assays has expanded to include: enantioselective indicator displacement assays (eIDAs), fluorescent indicator displacement assays (FIDAs), reaction-based indicator displacement assays (RIAs), DimerDye disassembly assays (DDAs), intramolecular indicator displacement assays (IIDAs), allosteric indicator displacement assay (AIDAs), mechanically controlled indicator displacement assays (MC-IDAs), and quencher displacement assays (QDAs). The simplicity of these IDAs, coupled with low cost, high sensitivity, and ability to carry out high-throughput automation analysis (i.e., sensing arrays) has led to their ubiquitous use in molecular sensing, alongside the other common approaches such as reaction-based sensors and chemosensors. In this review, we highlight the various design strategies that have been used to develop an IDA, including the design strategies for the newly reported extensions to these systems. To achieve this, we have divided this review into sections based on the target analyte, the importance of each analyte and then the reported IDA system is discussed. In addition, each section includes details on the benefit of the IDAs and perceived limitations for each system. We conclude this Tutorial Review by highlighting the current challenges associated with the development of new IDAs and suggest potential future avenues of research.
- Published
- 2021
- Full Text
- View/download PDF
46. Boronic acid based dynamic click chemistry: recent advances and emergent applications.
- Author
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Chatterjee S, Anslyn EV, and Bandyopadhyay A
- Abstract
Recently, reversible click reactions have found numerous applications in chemical biology, supramolecular chemistry, and biomedical applications. Boronic acid (BA)-mediated cis -diol conjugation is one of the best-studied reactions among them. An excellent understanding of the chemical properties and biocompatibility of BA-based compounds has inspired the exploration of novel chemistries using boron to fuel emergent sciences. This topical review focuses on the recent progress of iminoboronate and salicylhydroxamic-boronate constituted reversible click chemistries in the past decade. We highlight the mechanism of reversible kinetics and its applications in chemical biology, medicinal chemistry, biomedical devices, and material chemistry. This article also emphasizes the fundamental reactivity of these two conjugate chemistries with assorted nucleophiles at variable pHs, which is of utmost importance to any stimuli-responsive biological and material chemistry explorations., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)
- Published
- 2020
- Full Text
- View/download PDF
47. Capture and Release of Protein-Nanoparticle Conjugates by Reversible Covalent Molecular Linkers.
- Author
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Moran IW, Sprachman MM, Bachman JL, Dahlhauser SD, Anslyn EV, and Carter DJD
- Subjects
- Gold chemistry, Polyethylene Glycols chemistry, Surface Properties, Biotin chemistry, Hydrazones chemistry, Nanoparticles chemistry, Streptavidin chemistry
- Abstract
A hybrid approach to covalently detachable molecules for nanoparticle capture and release from several custom-functionalized surfaces is described. This new surface chemistry capability provides a means for reversible binding of functionalized nanoparticles without relying on costly nucleic acid-based complexation. A new surface linker motif was devised wherein custom molecules were synthesized with components for surface anchoring, cleavage, and target capture through biotin-streptavidin binding. All capture-and-release chemistry is performed using physiological conditions (aqueous, pH 7). Covalent cleavage of linker molecules was achieved through incorporation of a tunable orthogonal reversible covalent (TORC) hydrazone functional group which underwent exchange with a competitive hydrazide aided by an aniline catalyst. The influence of the linker architecture on hydrazone exchange and nanoparticle release was probed by altering the distance between hydrazone and biotin groups using different length PEG spacers. Cleavable linkers were used to functionalize microwells, magnetic separation beads, and gold-coated glass surfaces. Upon functionalization, all surface types bound streptavidin and conjugated nanoparticles regardless of the linker structure. Conversely, the extent of hydrazone exchange as well as release of nanoparticles were influenced both by the hydrazone surface density and the linker molecular structure.
- Published
- 2020
- Full Text
- View/download PDF
48. High-Throughput Determination of Enantiopurity by Microplate Circular Dichroism.
- Author
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Pilicer SL, Dragna JM, Garland A, Welch CJ, Anslyn EV, and Wolf C
- Abstract
Methods for the rapid determination of enantiomeric excess (ee) in asymmetric synthetic methodology development are increasingly in demand as high-throughput experimentation protocols in academia and industry are adopted. Optical approaches have been reported, many of which rely on the use of chemical derivatization or molecular assemblies, resulting in UV/vis, fluorescence, or circular dichroism (CD) signals that report the ee values. While UV/vis and fluorescence approaches benefit from readily available 96- and 384-well plate readers, until recently, no CD plate readers existed. Herein, we report the utility of using the EKKO CD plate reader to analyze a chlorocoumarin amine derivatization methodology for the ee determination of a diverse set of chiral amines with an error margin within ±7%. Linear calibration curves of ee versus CD responses for each amine were obtained, the minimum detectable and quantifiable ee values were calculated, the technique was applied to an asymmetric hydrogenation, and various interferents expected to be present in crude samples are explored. The technique described herein is found to be suitable for high-throughput experimentation that requires a parallel and rapid ee determination step.
- Published
- 2020
- Full Text
- View/download PDF
49. Next-Generation TLC: A Quantitative Platform for Parallel Spotting and Imaging.
- Author
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Boulgakov AA, Moor SR, Jo HH, Metola P, Joyce LA, Marcotte EM, Welch CJ, and Anslyn EV
- Subjects
- Chromatography, Thin Layer, Ultraviolet Rays
- Abstract
A high-throughput screening approach for simultaneous analysis and quantification of the percent conversion of up to 48 reactions has been developed using a thin-layer chromatography (TLC) imaging method. As a test-bed reaction, we monitored 48 thiol conjugate additions to a Meldrum's acid derivative ( 1 ) in parallel using TLC. The TLC elutions were imaged using a cell phone and a LEGO brick-constructed UV/vis light box. Further, a spotting device was constructed from LEGO bricks that allows simple transfer of the samples from a well-plate to the TLC plate. Using software that was developed to detect "blobs" and report their intensity, we were able to quantitatively determine the extent of completion of the 48 reactions with one analysis.
- Published
- 2020
- Full Text
- View/download PDF
50. Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics.
- Author
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Howard CJ, Floyd BM, Bardo AM, Swaminathan J, Marcotte EM, and Anslyn EV
- Subjects
- Aldehydes chemistry, Amino Acid Sequence, HEK293 Cells, Humans, Mass Spectrometry methods, Peptides analysis, Proteolysis, Proteome analysis, Proteome isolation & purification, Sequence Analysis, Protein methods, Peptides isolation & purification, Proteomics methods, Solid Phase Extraction methods
- Abstract
The field of proteomics has expanded recently with more sensitive techniques for the bulk measurement of peptides as well as single-molecule techniques. One limiting factor for some of these methods is the need for multiple chemical derivatizations and highly pure proteins free of contaminants. We demonstrate a solid-phase capture-release strategy suitable for the proteolysis, purification, and subsequent chemical modification of peptides. We use this resin on an HEK293T cell lysate and perform one-pot proteolysis, capture, and derivatization to survey peptide capture biases from over 40 000 unique peptides from a cellular proteome. We also show that this capture can be reversed in a traceless manner, such that it is amenable for single-molecule proteomics techniques. With this technique, we perform a fluorescent labeling and C-terminal derivatization on a peptide and subject it to fluorosequencing, demonstrating that washing the resin is sufficient to remove excess dyes and other reagents prior to single-molecule protein sequencing.
- Published
- 2020
- Full Text
- View/download PDF
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