DOCSLIB.ORG

Copper-Free Click Chemistry-Mediated Approaches to Understanding

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Copper-Free Click Chemistry-Mediated Approaches to Understanding DEVELOPMENT OF NOVEL FLUORESCENCE AND NMR REAGENTS FOR MONITORING PROTEIN-PROTEIN INTERACTIONS BETWEEN SURVIVIN AND ITS PROTEIN BINDING PARTNERS By Stephanie Cara Bishop MA, University of Kansas, 2009 Submitted to the graduate degree program in Pharmacology, Toxicology and Therapeutics and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. _________________________________ Chairperson, Jed N. Lampe, Ph.D. _________________________________ Qi Chen, Ph.D. _________________________________ Mark Fisher, Ph.D. _________________________________ Bruno Hagenbuch, Ph.D. _________________________________ Greg Reed, Ph.D. Date Defended: 06/08/2015 The Dissertation Committee for Stephanie Cara Bishop certifies that this is the approved version of the following dissertation: DEVELOPMENT OF NOVEL FLUORESCENCE AND NMR REAGENTS FOR MONITORING PROTEIN-PROTEIN INTERACTIONS BETWEEN SURVIVIN AND ITS PROTEIN BINDING PARTNERS _________________________________ Chairperson, Jed N. Lampe, Ph.D. Date approved: 06/11/2015 ii Abstract Inhibition of protein-protein interactions is a promising therapeutic strategy for targeting non-enzymatic proteins. One strategy to inhibit protein-protein interactions is to design inhibitors specifically toward the protein-protein interaction interface. To achieve this objective using structure-aided design, knowledge of the structure of the interface of the protein-protein interaction is needed. Augmenting the utility of NMR to determine the structure of larger proteins, and of protein complexes, than is currently achieved with conventional approaches, expands structure-aided design and to evaluate protein-protein interactions for target proteins relevant to human disease such as cancer. A second approach to bring new therapies to the clinic is to identify lead molecules using high-throughput screening. High-throughput screening (HTS) offers the potential to more rapidly and less expensively identify promising molecules that may inhibit a specific protein-protein interaction. To improve efforts at structure-aided design, we hypothesize that we can expand the utility of fluorescence- and NMR-based approaches to monitor protein-protein interactions by incorporating fluorescent or paramagnetic labels, site-specifically, into a protein of interest. This technology will provide much more structural detail regarding the binding site of a candidate small molecule protein-protein interaction inhibitor. This structural information can then be used in downstream medicinal chemistry efforts to improve inhibitor affinity and specificity. Additionally, site-specific fluorescent and paramagnetic labels can be used in sensitive HTS assays for protein-protein interaction inhibitors. iii The lanthanide series of elements, which possess intriguing spectroscopic and paramagnetic properties, was chosen to accomplish these efforts. Lanthanides exhibit luminescence and have excitation and emission maxima unique from biological fluorophores, broad Stokes shifts, and narrow emission bands. In the context of NMR, some lanthanides impart long-range perturbations in the form of paramagnetic relaxation enhancements, pseudocontact chemical shifts, and residual dipolar couplings, which are used to facilitate protein structure determination. Site-specific labeling of the protein of interest was achieved using site-directed mutagenesis, incorporation of the unnatural amino acid paF, followed by copper-free click chemistry with a cyclooctyne-containing lanthanide chelator. Synthesis of the small-molecule lanthanide chelator amenable to copper-free click chemistry-mediated incorporation is described herein. To improve efforts at identification of candidate protein-protein interaction inhibitors, we hypothesized that a high-throughput fluorescence polarization screening assay could be established. Inhibitors identified in this assay would be further characterized using the lanthanide labeled Survivin protein. A recently-identified cancer target, the Survivin protein, was evaluated using the methodologies described herein. The results demonstrate we were able to fluorescently label the Survivin protein site- specifically using a click chemistry approach. In addition, preliminary fluorescence results indicate that synthesis of a clickable lanthanide chelator was also successful. Finally, a high-throughput fluorescence polarization-based screening platform for analyzing Survivin protein-peptide interactions was evaluated. iv Taken together, the methodologies described herein attempt to augment efforts at structure-aided drug design as well as to produce new technologies to identify promising protein-protein interaction inhibitors, with the ultimate goal of improving our understanding of protein-protein interactions implicated in many human diseases, including cancer. v Dedication I dedicate my efforts described herein to my friend Bowen Pope (5/2/1979 – 10/24/2009), who continues to inspire me today. Bowen, I thank you for the time that we shared and have only the best memories of our childhood adventures together. The courage, endurance, positive attitude, and strength that you exhibited throughout your fight continue to impact my life. I am so blessed to have known you. vi Acknowledgments I am so grateful to my committee members Dr. Mark Fisher, Dr. Bruno Hagenbuch, Dr. Qi Chen, Dr. Greg Reed, and Dr. Jed Lampe for their countless hours dedicated to my professional development; your invaluable efforts have never gone unnoticed and I hope I have been able to convey my gratitude and appreciation for your suggestions and efforts over the last few years. Thank you so much. John Karanicolas, Ph.D.: I am immeasurably grateful to you for EVERY interaction we had and for your support of this project. I appreciated your patience, scientific insights, and the opportunity to work with you. Feng Li, Ph.D., thank you for performing numerous small-molecule mass spectrometry analyses; thank you for our discussions and for your efforts and time. I appreciated it very much. Antonio Artigues, Ph.D., and Maite Villar, Ph.D., performed whole-protein mass spectrometry analyses as well as tryptic digests on several samples; thank you for your time, efforts, and explanations of the data. I appreciate your efforts very much. Thank you so much Cody Tully, Rosa Meagher, Dorothy McGregor, Ann Hall, and Randy Stout for all of your time and help with so many tasks. Your efforts have been very much appreciated and have made my time here much easier. Thank you. I’m grateful to the following sources of financial support: the Madison and Lila Self Graduate Fellowship, the Environmental Toxicology Training Program 5T32ES007079-32, COBRE P20 RR021940-06, and R01 GM099959-01 (John Karanicolas, Ph.D.). vii I am indebted to my family for their endless support, in particular to my sister, Jessica, and her family, my parents Rodney Bishop, M.D. and Audrey Bishop, J.D., and my extended family. Jessi, I’ve been told that Barrett is well-adjusted and kind, which are undoubtedly reflections of the love and compassion you gave him. Thank you so much. You are a blessing in our lives and I am forever grateful. Thank you to my friends, in particular Ben Woolbright, Ph.D., Mitch McGill, Ph.D., Kelli Boxberger, and Brad Sullivan, Ph.D., for their unwavering support, encouragement, and advice. I enjoyed our science talks and learned a lot from each of you. Larry Long, Ph.D., I would not have made it to this stage without you. I cannot articulate how much you have helped me. I am so, so grateful for all of your time and efforts. Thank you so much. Thank you to my son, Barrett, for spontaneously sharing “I love you, Mommy”, and for bringing endless joy and purpose to my life. You are a light and a blessing and I love you so much. viii Table of Contents Page Abstract……………………………..…………………………………………………..iii Dedication………………………………………………………………………………vi Acknowledgments……………………...……………………………………………...vii Abbreviations…………………………..……...……………………………………….xi Chapter 1: Introduction……………………….….………………………..…………..1 Chapter 2: Materials and Methods……………………………………………….....21 Chapter 3: Utility of Copper-Free Click Chemistry to Site-Specifically Fluorescently Label the Survivin Protein for Protein-Protein Interaction Studies..……………..49 3.1 Introduction……..………………………………………………….……...50 3.2 Results and Discussion…………………………………………………..51 3.3 Conclusions………………………………………………………………..70 Chapter 4: Two-Step Aqueous Synthesis of a Clickable Lanthanide Chelator…82 4.1 Introduction……..…………………………………………………………83 4.2 Results and Discussion……..……………………………..…………….86 4.3 Conclusions………………………………………………………………100 Chapter 5: A High-Throughput Fluorescence Polarization Small-Molecule Screening Assay for Identification of Inhibitors of the Survivin-histone-3 Protein-Peptide Interaction ...…………………………………………………………………………………........104 5.1 Introduction……..………………………………………………………..105 5.2 Results and Discussion……..……………….………………………....108 5.3 Conclusions…………………………………………………….............122 ix Chapter 6: Conclusions and Future Prospects.………………………………….123 References…………………………………………………………………………...146 x Abbreviations ACN: acetonitrile amu: atomic mass units ATP: adenosine triphosphate BGG: bovine gamma globulin BIR: baculoviral IAP repeat BLI: biolayer interferometry c-DTPA: cyclic diethylenetriaminepentaacetic acid (also known as DTPA dianhydride) CML: chronic myelogenous leukemia CPC: chromosomal passenger complex Da: Dalton DBCO: dibenzylcyclooctyne DBCO488: dibenzylcyclooctyne + fluorescein
Load more

Recommended publications
  • Read the Book of Abstracts
    2o21 ONLINE May 10- 12 2021 International Symposium on SupraBiomolecular Systems 2021 Table of Contents TBD 1 Prof. Andreas Herrmann TBD 2 Prof. Rachel O’Reilly Tackling challenges in nanomedicine with responsive supramolecular polymers and advanced mi- croscopy 3 Dr. Silvia Pujals, Mr. Edgar Fuentes, Dr. Lorenzo Albertazzi Water Soluble Nanotubular Architectures from Amphiphilic Dinucleobases 4 Dr. Fatima Aparicio, Ms. Paula Blue Chamorro, Dr. Raquel Chamorro, Prof. David Gonzalez-Rodriguez Glyconucleolipids as new drug delivery systems for Parkinson’s disease treatment 5 Mr. Anthony Cunha, Dr. Alexandra Gaubert, Prof. Philippe Barthélémy, Dr. Benjamin Dehay, Dr. Laurent Latxague Membraneless compartments based on intrinsically disordered proteins: from biology towards new pro- tein materials 6 Prof. Paolo Arosio Low Molecular Weight Oleogel formation via unique keto-enol-type nucleolipid supramolecular assembly 8 Mr. Arthur KLUFTS-EDEL, Ms. Bérangère Dessane, Dr. Aladin Hamoud, Dr. Geoffrey Prévot, Dr. Antoine Lo- quet, Dr. Brice Kauffmann, Prof. Philippe Barthélémy, Prof. Sylvie Crauste-Manciet, Dr. Valérie Desvergnes Multi-responsive supramolecular fibers from peptide-based amphiphiles 9 Mr. Edgar Fuentes, Dr. Marieke Gerth, Dr. Jose Augusto Berrocal, Dr. Carlo Matera, Prof. Pau Gorostiza, Prof. Ilja Voets, Dr. Silvia Pujals, Dr. Lorenzo Albertazzi Electrostatic Protein Assemblies Towards Biohybrid Photoactive Materials 10 Dr. Eduardo Anaya-Plaza, Prof. Mauri Kostiainen Exploring Polyoxometalates as Non-destructive Staining Agents for Contrast-Enhanced Microfocus Com- puted Tomography of Biological Tissues 11 Ms. Sarah Vangrunderbeeck, Mr. Sébastien De Bournonville, Mrs. Hong Giang T. Ly, Prof. Wim De Borggraeve, Prof. Tatjana Parac-Vogt, Prof. Greet Kerckhofs Hydrogels with Photo-Switchable Stiffness: A Tool to Mimic Extra Cellular Matrix 13 Prof.
    [Show full text]
  • Synthesis of Organometallic PNA Oligomers by Click Chemistry
    Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2008 1 Supporting Information: Synthesis of organometallic PNA oligomers by click chemistry Gilles Gasser, Nina Hüsken, S. David Köster and Nils Metzler-Nolte* Department of Inorganic Chemistry I – Bioinorganic Chemistry, Faculty of Chemistry and Biochemistry, Ruhr-University Bochum Universitätsstrasse 150; 44801 Bochum, Germany Fax: +49 (0)234 – 32 14378; E-Mail: [email protected] Table of contents I. Experimental Section a) Materials 2 b) Instrumentation and methods 2 c) Peptide Nucleic Acid synthesis 2 d) Synthesis, characterisation and NMR spectra of Fmoc-1-OtBu, Fmoc-1-OH, Fmoc-2-OtBu and 3 3 e) Characterisation of PNA1-4 10 f) Synthesis and characterisation of Fc-PNA1-3 and Fc2-PNA4 10 II. MALDI-TOF Spectra a) Figure 5: MALDI-TOF mass spectrum of Fc-PNA1 11 b) Figure 6: MALDI-TOF mass spectrum of Fc-PNA2 12 c) Figure 7: MALDI-TOF mass spectrum of Fc-PNA3 12 d) Figure 8: MALDI-TOF mass spectrum of Fc2-PNA4 13 III. References 13 Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2008 2 I. Experimental Section a) Materials. All chemicals were of reagent grade quality or better, obtained from commercial suppliers and used without further purification. Solvents were used as received or dried over 4 Å molecular sieves. b) Instrumentation and methods. 1H and 13C NMR spectra were recorded in deuterated solvents on a Bruker DRX 400 spectrometer at 30°C. The chemical shifts, δ, are reported in ppm (parts per million).
    [Show full text]
  • Professor of Chemistry
    Bruce C. Gibb FRSC Professor of Chemistry Department of Chemistry Tulane University New Orleans, LA 70118, USA Tel: (504) 862 8136 E-mail: [email protected] Website: http://www.gibbgroup.org Twitter: @brucecgibb Research Interests: Aqueous supramolecular chemistry: understanding how molecules interact in water: from specific ion- pairing and the hydrophobic effect, to protein aggregation pertinent to neurodegenerative disorders. Our research has primarily focused on: 1) novel hosts designed to probe the hydrophobic, Hofmeister, and Reverse Hofmeister effects, and; 2) designing supramolecular capsules as yocto-liter reaction vessels and separators. Current efforts to probe the hydrophobic and Hofmeister effects include studies of the supramolecular properties of proteins. Professional Positions: Visiting Professor, Wuhan University of Science and Technology as a Chair Professor of Chutian Scholars Program (2015-2018) Professor of Chemistry, Tulane University, New Orleans, USA (2012-present). University Research Professor, University of New Orleans, USA (2007-2011). Professor of Chemistry, University of New Orleans, USA (2005-2007). Associate Professor of Chemistry, University of New Orleans, USA (2002-2005). Assistant Professor of Chemistry, University of New Orleans, USA, (1996-2002). Education: Postdoctoral Work Department of Chemistry, New York University. Synthesis of Carbonic Anhydrase (CA) mimics with Advisor: Prof. J. W. Canary, (1994-1996). Department of Chemistry, University of British Columbia, Canada De Novo Protein development. Advisor: Prof. J. C. Sherman (1993-1994). Ph.D. Robert Gordon’s University, Aberdeen, UK. Synthesis and Structural Examination of 3a,5-cyclo-5a- Androstane Steroids. Advisors: Dr. Philip J. Cox and Dr. Steven MacManus (1987-92) . B.Sc. with Honors in Physical Sciences Robert Gordon’s University, Aberdeen, UK.
    [Show full text]
  • Comparative Analysis of Click Chemistry Mediated Activity-Based Protein Profiling in Cell Lysates
    Molecules 2013, 18, 12599-12608; doi:10.3390/molecules181012599 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Communication Comparative Analysis of Click Chemistry Mediated Activity-Based Protein Profiling in Cell Lysates Yinliang Yang, Xiaomeng Yang and Steven H. L. Verhelst * Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany; E-Mails: [email protected] (Y.Y.); [email protected] (X.Y.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +49-8161-713505; Fax: +49-8161-714404. Received: 26 June 2013; in revised form: 7 August 2013 / Accepted: 26 September 2013 / Published: 11 October 2013 Abstract: Activity-based protein profiling uses chemical probes that covalently attach to active enzyme targets. Probes with conventional tags have disadvantages, such as limited cell permeability or steric hindrance around the reactive group. A tandem labeling strategy with click chemistry is now widely used to study enzyme targets in situ and in vivo. Herein, the probes are reacted in live cells, whereas the ensuing detection by click chemistry takes place in cell lysates. We here make a comparison of the efficiency of the activity-based tandem labeling strategy by using Cu(I)-catalyzed and strain-promoted click chemistry, different ligands and different lysis conditions. Keywords: activity-based probes; cathepsins; click chemistry; proteases; protein modification 1. Introduction Within chemical biology, site specific protein modification by covalent small molecule probes is a powerful and often used technique to interrogate biomolecular interactions and protein function. The introduction of covalent small molecule probes can be based on different types of chemistries: probes may be incorporated by the use of exogenous or endogenous enzymes [1,2], or they can contain an intrinsic reactivity such as an electrophile or photocrosslinker that by itself forms a covalent bond to the target proteins [3–5].
    [Show full text]
  • Sufex-Enabled, Agnostic Discovery of Covalent Inhibitors of Human Neutrophil Elastase
    SuFEx-enabled, agnostic discovery of covalent inhibitors of human neutrophil elastase Qinheng Zhenga,1, Jordan L. Woehlb,1, Seiya Kitamurab, Diogo Santos-Martinsc, Christopher J. Smedleyd, Gencheng Lia, Stefano Forlic, John E. Mosesd, Dennis W. Wolanb,2, and K. Barry Sharplessa,2 aDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; bDepartment of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037; cDepartment of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; and dLa Trobe Institute for Molecular Science, La Trobe University, Bundoora, Melbourne, VIC 3086, Australia Contributed by K. Barry Sharpless, July 23, 2019 (sent for review July 9, 2019; reviewed by Peng R. Chen and Lei Wang) Sulfur fluoride exchange (SuFEx) has emerged as the new gener- unique ensemble of factors between the naturally folded protein ation of click chemistry. We report here a SuFEx-enabled, agnostic and its correct partner probe’s latent S–F electrophilic site. approach for the discovery and optimization of covalent inhibi- When the perfect S–F probe for a given protein encounters the tors of human neutrophil elastase (hNE). Evaluation of our ever- denatured form of the latter, there is no detectable reaction (24, growing collection of SuFExable compounds toward various bio- 33). In fact, in our experience with many S–F capture probes and logical assays unexpectedly revealed a selective and covalent hNE various denatured proteins, even including entire denatured inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization proteomes, is that misfolded proteins simply do not react with of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl S–F electrophiles at all (24).
    [Show full text]
  • Applications of Click Chemistry Themed Issue
    This article was published as part of the Applications of click chemistry themed issue Guest editors Professors M.G. Finn and Valery Fokin Please take a look at the issue 4 table of contents to access other reviews in this themed issue CRITICAL REVIEW www.rsc.org/csr | Chemical Society Reviews Click chemistry with DNAw Afaf H. El-Sagheerab and Tom Brown*a Received 1st September 2009 First published as an Advance Article on the web 9th February 2010 DOI: 10.1039/b901971p The advent of click chemistry has led to an influx of new ideas in the nucleic acids field. The copper catalysed alkyne–azide cycloaddition (CuAAC) reaction is the method of choice for DNA click chemistry due to its remarkable efficiency. It has been used to label oligonucleotides with fluorescent dyes, sugars, peptides and other reporter groups, to cyclise DNA, to synthesise DNA catenanes, to join oligonucleotides to PNA, and to produce analogues of DNA with modified nucleobases and backbones. In this critical review we describe some of the pioneering work that has been carried out in this area (78 references). Introduction Azides and unactivated alkynes are almost entirely un- reactive towards the functional groups normally encountered Click chemistry was developed to provide a simple method to join in nature; they react only with each other. together organic molecules in high yields under mild conditions The triazole unit is extremely stable, and is not toxic. 3 and in the presence of a diverse range of functional groups. The In this review we describe the use of click chemistry across best example of this new class of extremely efficient chemical the nucleic acids field, focusing on synthetic strategies and I reactionsistheCu catalysed [3+2] azide–alkyne cycloaddition briefly describing some important practical applications.
    [Show full text]
  • PEO/PEG, Click Chemistry, Hydralink
    H10E Innovative conjugation & labeling solutions / biochemistry methods and reagents from Interchim CHEMISTRIES | FUNCTIONALIZATION | NANOMATERIALS | LABELING ● Hydrazone Chemistry reagents (HyNic/4FB) (PH) • Bio-Orthogonal • Controllable activation: UV-traceable • Stable activation: flexible Uses HyNic ligators and aldehyde ligators, to activate i.e. amines (SANH), or nucleic acids (with amidite-Alkynes). HyNic and Aldehyde groups react to form a stable hydrazine bond, in bioorthogonal and highly specific way. ● Standard Click Chemistry reagents (PH) • Chemoselective Uses alkyne ligators and azide ligators, to activate i.e. amines (ZL5530 & ZL5540), or nucleic acids (with amidite-Alkynes). Alkyne and Azide react in presence of Copper(II)- TBTA complex FY2780 , in bioorthogonal and highly specific way ● Copper-free Click Chemistry reagents (DBCO reagents) (PH) • DBCO reagents make easier Click reactions • no more need catalyzer! • no toxic by-products Use cyclooctynes ligators (DBCO) and azide ligators, to activate i.e. amines or other biomolecules. DBCO and azide partners react directly (SPAAC reaction), in bioorthogonal and highly specific way. ● Readilink Rapid Protein Labeling Kits (Buccutite based) (PH) • Click-chemistry like base technology (use Buccutite-FOL/MTA reagents) • Selective – easy (no final desalting step) 1+1/2hr procedure • Available with mFluor dyes for FCM, iFLuor dyes, APC, APC-Cy5.5 Tandem, PE-Cy5 Tandem, PE-Cy5.5 Tandem, PE-Texas Red Tandem, trFluor™ Eu & Tb, HRP, Proteins conjugation. Use Baccutite FOL/MTA reagents as partners for a highly specific reaction. Also ReadiLink Kits using SE method, available with more labels ● Staudinger Chemistry (TriArylPhosphine reagents) (PH) • Chemoselective • not toxic • ideal for In-vivo and metabolic labelings Use an appropriate substituted triaryl phosphine and an azide to form an amide bond.
    [Show full text]
  • New Deoxyribozymes for the Native Ligation of RNA
    molecules Article New Deoxyribozymes for the Native Ligation of RNA Carolin P. M. Scheitl 1, Sandra Lange 2 and Claudia Höbartner 1,* 1 Institute of Organic Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany; [email protected] 2 Agricultural Center, BASF SE, Speyerer Str 2, 67117 Limburgerhof, Germany; [email protected] * Correspondence: [email protected] Academic Editors: Michael Smietana, Stellios Arseniyadis and Sabine Müller Received: 29 July 2020; Accepted: 10 August 2020; Published: 11 August 2020 Abstract: Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalyzing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 50-adenylated RNA (50-AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N40 random DNA library and were shown to catalyze the intermolecular linear RNA-RNA ligation via the formation of a native 30-50-phosphodiester linkage. The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 50-adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations. Keywords: RNA ligation; DNA catalysis; in vitro selection; deoxyribozyme 1. Introduction DNA is best known in the form of the famous Watson-Crick double helix of two antiparallel phosphodiester single strands held together by hydrogen bonding and stacking interactions of complementary A–T and G–C base pairs.
    [Show full text]
  • Click Chemistry: New Protocol for the Labeling and Modification of Biomolecules
    Click Chemistry: new protocol for the labeling and modification of biomolecules Click Chemistry is a reaction between azide and alkyne yielding covalent product - 1,5-disubstituted 1,2,3-triazole. This process is also known as CuAAC - Cu catalyzed alkyne azide cycloaddition. Click Chemistry is based on copper catalysis. The catalyst is often introduced as Cu-TBTA complex. Among the vast variety of organic reactions, Click Chemistry has been selected as a conjugation chemistry reaction because of several advantages. • It is very selective. Click Chemistry reaction takes place only between azide and alkyne components. It is does not interfere with most any other organic groups present in DNA and proteins being labeled, such as amino and carboxy groups. • There are no azides and alkynes in native biomolecules. These groups should be specially introduced into DNA and proteins. Alkyne-containing DNA can be prepared with alkyne phosphoramidite during standard oligo synthesis. Proteins labeled with azide and alkyne can be made using azide activated ester and alkyne activated ester. • Click Chemistry takes place in water. Aqueous DMSO, DMF, acetonitrile, alcohols, or pure water and buffers can be used for the reaction. The reaction is biocompatible and can take place in living cells. • Reaction is quick and quantitative. Click Chemistry is a tool that allows preparation of nanomols of conjugates in diluted solutions. • The reaction is pH-insensitive. Unlike reaction of NHS esters with amines, and some other conjugation chemistries, there is no need to control pH in reaction mixture. There is no need to add any special buffer, acid or base - Click Chemistry works well in pH interval of 4-11.
    [Show full text]
  • Catalog & Reference Manual
    2019/20 Catalog & Reference Manual Reagents for TCO-Tetrazine Ligation Next Generatin Azide Probes DBCO Reagents for Cu-free Click Chemistry Metabolic Labeling Reagents Click Chemistry Azide/Alkyne Reagents Cleavable Click Chemistry Biotin Probes Fluorogenic Azides Probes Biotin/Streptavidin-Free Enrichment Kits and Media Contents Bioorthogonal Ligation Chemistry Overview 6 Trifunctional Click Chemistry Biotin Probes 38 Non-cleavable 38 TCO-Tetrazine Ligation 10 Cleavable 39 TCO Reagents 12 Tetrazine Selection Guide 14 Cleavable Click Chemistry Biotin Probes 40 Tetrazine Reagents 16 DADPS Biotin Probes 40 Fluorescent Tetrazines 19 Photocleavable Biotin Probes 41 Dde Biotin Probes 42 DBCO Reagents 20 Diazo Biotin Probes 43 Labeling Reagents and Building Blocks 21 Biotinylation Reagents 24 Click Chemistry Auxiliary Reagents 44 Fluorescent Reagents 25 Metabolic Labeling Reagents 47 Termanial Alkynes 27 Enrichment Media and Kits 52 Labeling Reagents and Building Blocks 27 Non-cleavable Enrichment Kits 53 Biotinylation Reagents 28 Cleavable Enrichment Kits and Media 54 Fluorescent Reagents 29 Click Functionalized Agarose, magnetic 55 Azides 30 Tetrazine/TCO Modified Media 57 Labeling Reagents and Building Blocks 30 Credit Card Order Form 58 Biotinylation Reagents 31 Fluorescent Reagents 32 Fluorogenic Azide Probes 34 Fluorescent Picolyl Azides 35 3 General Information: If you have a technical question about a product you received or have seen in the catalog, please send an e – mail to [email protected] or call us at (480) – 584 – 3340. Material Safety Data Sheets MSDS are available upon request. We can also fax or e – mail a copy. Please mention your request on the order form if needed. Product Analysis Purity is assayed by HPLC, LC/MS, TLC, and/or NMR.
    [Show full text]
  • Applications of Click Chemistry in Drug Discovery
    APPLICATIONS OF CLICK CHEMISTRY IN DRUG DISCOVERY, BIOCONJUGATION AND MATERIAL SCIENCE by XINGHAI NING (Under the Direction of Geert-Jan Boons) ABSTRACT Click chemistry is a chemical philosophy introduced by Sharpless in 2001 and describes chemistry tailored to generate substances quickly and reliably by joining small units together. This dissertation has focused on the applications of click chemistry in three most relevant categories including drug discovery, bioconjugation, and materials science. The first application is developing new anti-influenza drugs by using click chemistry. We designed the novel anti-influenza reagents based on the Altermune method, which would stimulate the immune system to attack influenza viruses using a Linker Molecule(LM), which can recognize the viruses and also be recognized by immune system. The LM then redirects the immune response to the influenza viruses. The second application is developing a new bioconjugation strategy basing on the “click philosophy”. We developed a new click reagent, 4- dibenzocyclooctynol, which reacts exceptionally fast with azido compounds to give stable triazoles in the absence of any catalyst. A biotin-modified derivative is ideally suited for visualizing and tracking glycoconjugates of living cells that are metabolically labeled with azido- containing monosaccharides. In addition, we also developed a novel quantitative isotopic and chemoenzymatic tagging (QUIC-Tag) containing 4-dibenzocyclooctynol for monitoring the dynamics sialylation in vivo using quantitative mass spectrometry-based proteomics. Because sialic acid is a terminal glycan residue with a notably increased expression in cancers, this method will help to delineate the molecular basis for aberrant glycosylation in cancer and could ultimately be applied for diagnostic and therapeutic.
    [Show full text]
  • Click Chemistry Reagents
    Click Chemistry Reagents Table 1. Contents and storage information. Material Amount Storage Stability • ≤–20˚C Alkyne or azide-containing click chemistry Varies, see product When stored as directed the • Desiccate reagent label product is stable for 6–12 months. • Protect from light Approximate fluorescence excitation/emission maxima: See Table 2. Introduction Click chemistry describes a class of chemical reactions that use bio-orthogonal or biologically unique moities to label and detect a molecule of interest using a two-step procedure.1-4 The two-step click reaction involves a copper-catalyzed triazole formation from an azide and an alkyne (Figure 1). The azide and alkyne moieties can be used interchangeably; either one can be used to tag the molecule of interest, while the other is used for subsequent detection. The azides and alkynes are biologically unique, inert, stable, and extremely small (Figure 2). Click chemistry can be used when methods such as direct labeling or the use of antibodies are not applicable or efficient. The click chemistry label is small enough that tagged molecules (e.g., nucleotides5, sugars6, and amino acids7) are acceptable substrates for the enzymes that assemble these building blocks into biopolymers. The small size of click detection molecules allows them to easily penetrate complex samples, including intact, supercoiled DNA, with only mild permeabilization required. The characteristics of click reactions include: • Efficiency—the reaction between the detection moieties is complete in less than 1 hour and does not require extreme temperatures or solvents. • Stability—the reaction product contains an irreversible, covalent bond. • Biologically inert—the components of the reaction do not undergo any side reactions.
    [Show full text]