Publications by authors named "Eric V Anslyn"

212 Publications

Efficient molecular encoding in multifunctional self-immolative urethanes.

Cell Rep Phys Sci 2021 Apr;2(4)

University of Texas at Austin, Austin, TX 78712, USA.

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 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.
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http://dx.doi.org/10.1016/j.xcrp.2021.100393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8573738PMC
April 2021

High-throughput screening of α-chiral-primary amines to determine yield and enantiomeric excess.

Tetrahedron 2021 Aug 5;94. Epub 2021 Jul 5.

Department of Chemistry, The University of Texas at Austin, Austin, TX 78712, USA.

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 () 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 determination using an EKKO CD plate-reader. With these two assays, both the 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.
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http://dx.doi.org/10.1016/j.tet.2021.132315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8568316PMC
August 2021

A Data-Driven Approach to the Development and Understanding of Chiroptical Sensors for Alcohols with Remote γ-Stereocenters.

J Am Chem Soc 2021 Nov 4;143(45):19187-19198. Epub 2021 Nov 4.

Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.

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.
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http://dx.doi.org/10.1021/jacs.1c09443DOI Listing
November 2021

Photoredox-Catalyzed Decarboxylative -Terminal Differentiation for Bulk- and Single-Molecule Proteomics.

ACS Chem Biol 2021 11 4;16(11):2595-2603. Epub 2021 Nov 4.

Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States.

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 -terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the - 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.
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http://dx.doi.org/10.1021/acschembio.1c00631DOI Listing
November 2021

The Evolution of Data-Driven Modeling in Organic Chemistry.

ACS Cent Sci 2021 Oct 19;7(10):1622-1637. Epub 2021 Oct 19.

Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States.

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.
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http://dx.doi.org/10.1021/acscentsci.1c00535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8554870PMC
October 2021

A self-degradable hydrogel sensor for a nerve agent tabun surrogate through a self-propagating cascade.

Cell Rep Phys Sci 2021 Sep 25;2(9). Epub 2021 Aug 25.

Department of Chemistry, University of Texas at Austin, Austin, TX 78712, USA.

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.
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http://dx.doi.org/10.1016/j.xcrp.2021.100552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8500376PMC
September 2021

Electrostatic and Covalent Assemblies of Anionic Hydrogel-Coated Gold Nanoshells for Detection of Dry Eye Biomarkers in Human Tears.

Nano Lett 2021 10 8;21(20):8734-8740. Epub 2021 Oct 8.

Institute for Biomaterials, Drug Delivery, and Regenerative Medicine, The University of Texas at Austin, Austin, Texas 78712, United States.

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.
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http://dx.doi.org/10.1021/acs.nanolett.1c02941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8588787PMC
October 2021

Boronic acid based dynamic click chemistry: recent advances and emergent applications.

Chem Sci 2020 Dec 17;12(5):1585-1599. Epub 2020 Dec 17.

Biomimetic Peptide Engineering Laboratory, Department of Chemistry, Indian Institute of Technology Ropar Punjab-781039 India

Recently, reversible click reactions have found numerous applications in chemical biology, supramolecular chemistry, and biomedical applications. Boronic acid (BA)-mediated -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.
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http://dx.doi.org/10.1039/d0sc05009aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179052PMC
December 2020

The emerging landscape of single-molecule protein sequencing technologies.

Nat Methods 2021 06 7;18(6):604-617. Epub 2021 Jun 7.

Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands.

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.
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http://dx.doi.org/10.1038/s41592-021-01143-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8223677PMC
June 2021

"Benchtop" Biaryl Coupling Using Pd/Cu Cocatalysis: Application to the Synthesis of Conjugated Polymers.

Org Lett 2021 Apr 30;23(8):2873-2877. Epub 2021 Mar 30.

Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States.

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.
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http://dx.doi.org/10.1021/acs.orglett.1c00479DOI Listing
April 2021

Combination of two analytical techniques improves wine classification by Vineyard, Region, and vintage.

Food Chem 2021 Aug 10;354:129531. Epub 2021 Mar 10.

Department of Chemistry, University of Nebraska-Lincoln, Lincoln NE 65888, United States; Nebraska Center for Integrated Biomolecular Communication, University of Nebraska-Lincoln, Lincoln NE 68588, United States. Electronic address:

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 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.
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http://dx.doi.org/10.1016/j.foodchem.2021.129531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8336071PMC
August 2021

A Colorimetric Method for Quantifying Cis and Trans Alkenes Using an Indicator Displacement Assay.

Angew Chem Int Ed Engl 2021 06 13;60(25):13819-13823. Epub 2021 May 13.

Department of Chemistry, University of Texas at Austin, 100 E 24th Street, Norman Hackerman Building Room 114A, Austin, TX, 78712, USA.

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 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.
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http://dx.doi.org/10.1002/anie.202101004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184614PMC
June 2021

Effects of linker flexibility on phase behavior and structure of linked colloidal gels.

J Chem Phys 2021 Feb;154(7):074901

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78712, USA.

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.
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http://dx.doi.org/10.1063/5.0038672DOI Listing
February 2021

Ribosome-mediated incorporation of fluorescent amino acids into peptides in vitro.

Chem Commun (Camb) 2021 Mar 16;57(21):2661-2664. Epub 2021 Feb 16.

Department of Chemical and Biological Engineering and Center for Synthetic Biology, Northwestern University, Evanston, IL 60208, USA.

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.
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http://dx.doi.org/10.1039/d0cc07740bDOI Listing
March 2021

Colloidal Nanocrystal Gels from Thermodynamic Principles.

Acc Chem Res 2021 02 3;54(4):798-807. Epub 2021 Feb 3.

McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E Dean Keeton Street, Austin, Texas 78712, United States.

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.
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http://dx.doi.org/10.1021/acs.accounts.0c00796DOI Listing
February 2021

Indicator displacement assays (IDAs): the past, present and future.

Chem Soc Rev 2021 Jan 10;50(1):9-38. Epub 2020 Nov 10.

Department of Chemistry, The University of Texas at Austin, 105 East 24th Street A5300, Austin, Texas 78712-1224, USA.

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.
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http://dx.doi.org/10.1039/c9cs00538bDOI Listing
January 2021

Capture and Release of Protein-Nanoparticle Conjugates by Reversible Covalent Molecular Linkers.

Bioconjug Chem 2020 09 14;31(9):2191-2200. Epub 2020 Aug 14.

Charles Stark Draper Laboratory Incorpation, 555 Technology Square, Cambridge, Massachusetts 02139, United States.

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.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00372DOI Listing
September 2020

High-Throughput Determination of Enantiopurity by Microplate Circular Dichroism.

J Org Chem 2020 08 5;85(16):10858-10864. Epub 2020 Aug 5.

Department of Chemistry, Georgetown University, 37th and O Streets, Washington, DC 20057, United States.

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.
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http://dx.doi.org/10.1021/acs.joc.0c01395DOI Listing
August 2020

Next-Generation TLC: A Quantitative Platform for Parallel Spotting and Imaging.

J Org Chem 2020 08 6;85(15):9447-9453. Epub 2020 Jul 6.

Center for Systems and Synthetic Biology/Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States.

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 () 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.
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http://dx.doi.org/10.1021/acs.joc.0c00349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7748073PMC
August 2020

Nanogel receptors for high isoelectric point protein detection: influence of electrostatic and covalent polymer-protein interactions.

Chem Commun (Camb) 2020 Jun 4;56(45):6141-6144. Epub 2020 May 4.

Institute for Biomaterials, Drug Delivery, and Regenerative Medicine, The University of Texas at Austin, Austin, TX, USA.

An aldehyde acrylate-based functional monomer was incorporated into poly(N-isopropylacrylamide-co-methacrylic acid) nanogels for use as protein receptors. The aldehyde component forms dynamic imines with surface exposed lysine residues, while carboxylic acid/carboxylate moieties form electrostatic interactions with high isoelectric point proteins. Together, these interactions effect protein adsorption and recognition.
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http://dx.doi.org/10.1039/d0cc02200dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377432PMC
June 2020

Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics.

ACS Chem Biol 2020 06 14;15(6):1401-1407. Epub 2020 May 14.

Department of Chemistry, University of Texas at Austin, 100 E. 24th Street, Austin, Texas 78712, United States.

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.
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http://dx.doi.org/10.1021/acschembio.0c00040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765702PMC
June 2020

Chemically Triggered Synthesis, Remodeling, and Degradation of Soft Materials.

J Am Chem Soc 2020 02 17;142(8):3913-3922. Epub 2020 Feb 17.

Department of Chemistry/McKetta Department of Chemical Engineering , University of Texas at Austin , Austin , Texas 78712 , United States.

Polymer topology dictates dynamic and mechanical properties of materials. For most polymers, topology is a static characteristic. In this article, we present a strategy to chemically trigger dynamic topology changes in polymers in response to a specific chemical stimulus. Starting with a dimerized PEG and hydrophobic linear materials, a lightly cross-linked polymer, and a cross-linked hydrogel, transformations into an amphiphilic linear polymer, lightly cross-linked and linear random copolymers, a cross-linked polymer, and three different hydrogel matrices were achieved via two controllable cross-linking reactions: reversible conjugate additions and thiol-disulfide exchange. Significantly, all the polymers, before or after topological changes, can be triggered to degrade into thiol- or amine-terminated small molecules. The controllable transformations of polymeric morphologies and their degradation herald a new generation of smart materials.
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http://dx.doi.org/10.1021/jacs.9b12122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8574170PMC
February 2020

Sequencing of Sequence-Defined Oligourethanes via Controlled Self-Immolation.

J Am Chem Soc 2020 02 31;142(6):2744-2749. Epub 2020 Jan 31.

Department of Chemistry , The University of Texas at Austin , Austin , Texas 78712 , United States.

Sequence-defined polymers show promise for biomimetics, self-assembly, catalysis, and information storage, wherein the primary structure begets complex chemical processes. Here we report the solution-phase and the high-yielding solid-phase syntheses of discrete oligourethanes and methods for their self-immolative sequencing, resulting in rapid and robust characterization of this class of oligomers and polymers, without the use of MS/MS. Crucial to the sequencing is the inherent reactivity of the terminal alcohol to "unzip" the oligomers, in a controlled and iterative fashion, releasing each monomer as a 2-oxazolidinone. By monitoring the self-immolation reaction via LC/MS, an applied algorithm rapidly produces the sequence of the oligourethane. Not only does this process provide characterization of structurally complex molecules, it works as a reader of molecular information.
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http://dx.doi.org/10.1021/jacs.9b12818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8573737PMC
February 2020

2-Amino-3'-dialkylaminobiphenyl-based fluorescent intracellular probes for nitric oxide surrogate NO.

Chem Sci 2020 Jan 2;11(5):1394-1403. Epub 2020 Jan 2.

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology Meilong Road 130 Shanghai 200237 China

Fluorescent probes for nitric oxide (NO), or more frequently for its oxidized surrogate dinitrogen trioxide (NO), have enabled scientists to study the contributions of this signaling molecule to many physiological processes. Seeking to improve upon limitations of other probes, we have developed a family of fluorescent probes based on a 2-amino-3'-dialkylaminobiphenyl core. This core condenses with NO to form benzo[]cinnoline structures, incorporating the analyte into the newly formed fluorophore, which results in product fluorescence with virtually no background contribution from the initial probe. We varied the substituents in the core in order to optimize both the reactivity of the probes with NO and their cinnoline products' fluorescence wavelengths and brightness. The top candidates were then applied to cultured cells to verify that they could respond to NO within cellular milieus, and the top performer, NO, was compared with a "gold standard" commercial probe, DAF-FM, in a macrophage-derived cell line, RAW 264.7, stimulated to produce NO. NO demonstrated similar or better sensitivity and higher selectivity for NO than DAF, making it an attractive potential alternative for NO tracking in various applications.
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http://dx.doi.org/10.1039/c9sc04304gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8148321PMC
January 2020

Synthesis of Carboxy ATTO 647N Using Redox Cycling for Xanthone Access.

Org Lett 2020 01 11;22(2):381-385. Epub 2019 Dec 11.

Department of Chemistry , The University of Texas at Austin , Austin , Texas 78712 , United States.

A synthesis of the carbopyronine dye Carboxy ATTO 647N from simple materials is reported. This route proceeds in 11 forward steps from 3-bromoaniline with the key xanthone intermediate formed using a new oxidation methodology. The step utilizes an oxidation cycle with base, water, iodine, and more than doubles the yield of the standard permanganate oxidation methodology, accessing gram-scale quantities of this late-stage product. From this, Carboxy ATTO 647N was prepared in only four additional steps. This facile route to a complex fluorophore is expected to enable further studies in fluorescence imaging.
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http://dx.doi.org/10.1021/acs.orglett.9b03981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549415PMC
January 2020

Reengineering a Reversible Covalent-Bonding Assembly to Optically Detect ee in β-Chiral Primary Alcohols.

Chem 2019 Dec 31;5(12):3196-3206. Epub 2019 Oct 31.

Department of Chemistry, University of Texas, Austin, TX 78712, USA.

The use of parallel synthesis protocols for asymmetric reaction discovery has increased the need for new methods to rapidly determine enantiomeric excess (ee) values. Most chirality sensing is performed on stereocenters that are α (i.e., proximal) to the target functional group. Finding a general approach to detect more distant point chirality would increase the substrate scope of such assays. Herein, we demonstrate a design principle to "reach out" to more distant stereocenters, in this case β-chirality in primary alcohols. Therefore, we see the design principles established in this work as a step forward in sensing distant point chirality and, eventually, multi-stereocenter relationships.
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http://dx.doi.org/10.1016/j.chempr.2019.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774303PMC
December 2019

Mechanistic studies of a "Declick" reaction.

Chem Sci 2019 Oct 13;10(38):8817-8824. Epub 2019 Aug 13.

Department of Chemistry , University of Texas at Austin , Austin , Texas 78712 , USA . Email:

A kinetic analysis of a "declick" reaction is described. Compound , previously reported to couple an amine and a thiol ( "click") under mild aqueous conditions to create , undergoes release of the unaltered coupling partners upon triggering with dithiothreitol (). In the study reported herein various aniline derivatives possessing para-electron donating and withdrawing groups were used as the amines. UV/vis spectroscopy of the declick reaction shows time-dependent spectra lacking isosbestic points, implying a multi-step mechanism. Global data fitting using numerical integration of rate equations and singular value decomposition afforded the spectra and time-dependence of each species, as well as rate constants for each step. The kinetic analysis reveals a multi-step process with an intermediate where both thiols of have added prior to expulsion of the aniline leaving group, followed by rearrangement to the final product. Hammett plots show a negative rho value on two of the steps, indicating positive charge building ( reduction of a negative charge) in the step leading to the intermediate and its rate-determining breakdown. Overall, the kinetic study reported herein gives a complete mechanistic picture of the declick reaction.
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http://dx.doi.org/10.1039/c9sc00690gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849640PMC
October 2019

Quantification of ERK Kinase Activity in Biological Samples Using Differential Sensing.

ACS Chem Biol 2020 01 16;15(1):83-92. Epub 2019 Dec 16.

Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy , The University of Texas at Austin , Austin , Texas 78712 , United States.

The understanding of complex biological systems requires an ability to evaluate interacting networks of genes, proteins, and cellular reactions. Enabling technologies that support the rapid quantification of these networks will facilitate the development of biological models and help to identify treatment targets and to assess treatment plans. The biochemical process of protein phosphorylation, which underlies almost all aspects of cell signaling, is typically evaluated by immunoblotting procedures (Western blot) or more recently proteomics procedures, which provide qualitative estimates of the concentration of proteins and their modifications in cells. However, protein modifications are difficult to correlate with activity, and while immunoblotting and proteomics approaches have the potential to be quantitative, they require a complex series of steps that diminish reproducibility. Here, a complementary approach is presented that allows for the rapid quantification of a protein kinase activity in cell lysates and tumor samples. Using the activity of cellular ERK (extracellular signal-regulated kinase) as a test case, an array sensing approach that utilizes a library of differential peptide-based biosensors and chemometric tools was used to rapidly quantify nanograms of active ERK in micrograms of unfractionated cell lysates and tumor extracts. This approach has the potential both for high-throughput and for quantifying the activities of multiple protein kinases in a single biological sample. The critical advantages of this differential sensing approach over others are that it removes the need for the addition of exogenous inhibitors to suppress the activities of major off-target kinases and allows us to quantitate the amount of active kinase in tested samples rather than measuring the changes in its activity upon induction or inhibition.
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http://dx.doi.org/10.1021/acschembio.9b00580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085986PMC
January 2020

Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo.

Nat Commun 2019 11 19;10(1):5232. Epub 2019 Nov 19.

Division of Chemical Biology and Medicinal Chemistry, The University of Texas at Austin, Austin, TX, 78712, USA.

Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK. The DRS is a conserved region that lies distal to the active site and mediates ERK-protein interactions. We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo. BI-78D3 does not covalently modify p38MAPK, JNK or ERK5. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation. These studies provide the basis for designing modulators of protein-protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers.
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http://dx.doi.org/10.1038/s41467-019-12996-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863825PMC
November 2019

Expanding the limits of the second genetic code with ribozymes.

Nat Commun 2019 11 8;10(1):5097. Epub 2019 Nov 8.

Department of Chemical and Biological Engineering, Northwestern University, Evanston, 60208, IL, USA.

The site-specific incorporation of noncanonical monomers into polypeptides through genetic code reprogramming permits synthesis of bio-based products that extend beyond natural limits. To better enable such efforts, flexizymes (transfer RNA (tRNA) synthetase-like ribozymes that recognize synthetic leaving groups) have been used to expand the scope of chemical substrates for ribosome-directed polymerization. The development of design rules for flexizyme-catalyzed acylation should allow scalable and rational expansion of genetic code reprogramming. Here we report the systematic synthesis of 37 substrates based on 4 chemically diverse scaffolds (phenylalanine, benzoic acid, heteroaromatic, and aliphatic monomers) with different electronic and steric factors. Of these substrates, 32 were acylated onto tRNA and incorporated into peptides by in vitro translation. Based on the design rules derived from this expanded alphabet, we successfully predicted the acylation of 6 additional monomers that could uniquely be incorporated into peptides and direct N-terminal incorporation of an aldehyde group for orthogonal bioconjugation reactions.
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http://dx.doi.org/10.1038/s41467-019-12916-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6841967PMC
November 2019
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