DOCSLIB.ORG

Glycogen, Branched Polymer of Glucose Serves As a Major Repository of Carbon and Energy in Many Organisms Form Bacteria to Highe

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Glycogen, Branched Polymer of Glucose Serves As a Major Repository of Carbon and Energy in Many Organisms Form Bacteria to Highe STRUCTURE AND REGULATION OF YEAST GLYCOGEN SYNTHASE Sulochanadevi Baskaran Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology Indiana University August 2010 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Thomas D. Hurley, Ph.D., Chair Anna A. DePaoli-Roach, Ph.D. Doctoral Committee Millie M. Georgiadis, Ph.D. Peter J. Roach, Ph.D. May 19, 2010 William J. Sullivan Jr., Ph.D. ii Dedication I dedicate this work to my family, friends and mentors, for their support, encouragement, inspiration and guidance. iii Acknowledgements The work presented here is a result of the collaborative effort of many people and it is my pleasure to thank everyone who made this possible. First and foremost, I am indebted to my advisor, Dr. Thomas Hurley, for his invaluable scientific guidance, enthusiastic supervision and encouragement throughout my thesis research work. I am very grateful for his support, patience and his efforts in explaining and teaching me the concepts of macromolecular crystallography. I will always be thankful to him for teaching me the most important factor that is required for scientific research - perseverance. I would like to express my sincere gratitude to my committee members Drs. Peter Roach, Anna DePaoli-Roach, Millie Georgiadis and William Sullivan for their time, support, guidance and helpful suggestions. I am thankful to lab colleagues Dr. Samantha Perez-Miller, Dr. Heather Larson, Dr. Jianzhong Zhou, Dr. Lillian González-Segura, Ram Vanam, Jason Braid, Lili Zang, Bibek Parajuli, Dr. May Khanna and Dr. Ann Kimble-Hill for providing a stimulating and fun environment in the lab. I am especially grateful to Dr. Samantha Perez-Miller and Dr. Heather Larson for their friendship and support. I appreciate the help of my colleagues, Dr. Kristie Goodwin, Sarah Delaplane, Dr. Hongzhen He, Dr. Tsuyoshi Imasaki, Dr. Alexander Skurat, Dr. Jose Irimia-Dominguez, Vinnie Tagliabracci, Sixin Jiang, Cathy Meyer, Dyann Segvich and Chandra Karthik. I would like to extend my thanks to Drs. Yuichiro Takagi, Mark Goebl, Ronald Wek and Zhong-Yin Zhang for their encouragement and to the biochemistry office staff for their timely help and assistance. iv I wish to thank the beamline scientist at the SBC-CAT and GM/CA-CAT stations at the Advanced Photon Source, in particular Drs. Stephan Ginell, Norma Duke, Marianne Cuff, Nagarajan Venugopalan and Michael Becker for their scientific assistance. I would like to thank Dr. Lou Messerle for providing the tantalum cluster compound and Dr. Thomas Terwilliger for sending the scripts for partial model phasing. I am grateful to my teachers - Drs. Bhaskar-Rao, Sairam, Illango and Dharmalingam for encouraging me to pursue a career in scientific research. I would like to specifically thank Drs. Usha, Krishnaswamy and Mohammed Rafi for introducing me to the world of structural biology and macromolecular crystallography. I would like to thank my friends Dr. Shankar Varadarajan, Sowmya Chandrasekar, Dr. Raji Muthukrishanan, Sirisha Pochareddy, Dr. Judy Rose James and Dr. Aditi Bapat for their emotional support and helping me get through the difficult times. Finally I would like to express my sincere thanks to my family for everything they had provided me all through my life. v Abstract Sulochanadevi Baskaran STRUCTURE AND REGULATION OF YEAST GLYCOGEN SYNTHASE Glycogen is a major energy reserve in most eukaryotes and its rate of synthesis is controlled by glycogen synthase. The activity of eukaryotic glycogen synthase is regulated by the allosteric activator glucose-6-phosphate, which can overcome the inhibitory effects of phosphorylation. The effects of phosphorylation and glucose-6-phosphate on glycogen synthase are mediated by a cluster of six arginines located within a stretch of 12 amino acids near the C-terminus of the enzyme’s polypeptide chain. We studied isoform-2 of yeast glycogen synthase as a model to study the structural and molecular mechanisms that underlie the regulation of the eukaryotic enzymes and our primary tools of investigation were macromolecular X-ray crystallography, site-directed mutagenesis, intein- mediated peptide ligation and enzyme assays. We have solved the tetrameric structure of the yeast enzyme in two different activity states; the resting enzyme and the activated state when complexed with glucose-6-phosphate. Binding of glucose-6-phosphate to glycogen synthase induces large conformational changes that free the active site of the subunits to undergo conformational changes necessary to catalyze the reaction. Further, using site directed mutagenesis and intein-mediated peptide ligation to create specific phosphorylation states of the enzyme we were able to define specific roles for vi the arginine residues that mediate the regulatory effects of phosphorylation and glucose-6-phosphate activation. Based on these studies, we propose a three state structural model for the regulation of the enzyme, which relate the observed conformational states to specific activity levels. In addition to these regulatory studies, we have also solved the structure of the enzyme complexed with UDP and with substrate analogs, which provide detailed insight into the catalytic mechanism of the enzyme and the ability of glycogen synthase to remain tightly bound to its substrate glycogen. Thomas D. Hurley, Ph.D., Chair . vii TABLE OF CONTENTS LIST OF TABLES ................................................................................................ xii LIST OF FIGURES ............................................................................................. xiii LIST OF ABBREVIATIONS ................................................................................ xvi INTRODUCTION .................................................................................................. 1 A. Glycogen ...................................................................................................... 1 1. Structure of glycogen ................................................................................. 1 2. Biosynthesis and degradation ................................................................... 2 3. Physiological role of glycogen in mammals ............................................... 5 4. Physiological role of glycogen in yeast ...................................................... 9 B. Glycogen synthase ..................................................................................... 11 1. Glycogen storage disease type-0 ............................................................ 11 2. Enzymatic activity of GS .......................................................................... 12 3. Catalytic mechanism of GS ..................................................................... 13 4. Influence of substrate on GS activity ....................................................... 16 5. Regulation of GS activity ......................................................................... 17 6. Structural classification of GS .................................................................. 24 C. Rationale and overview of the thesis research ........................................... 26 D. Theory of experimental methods used ....................................................... 27 1. Macromolecular x-ray crystallography ..................................................... 27 2. Intein mediated peptide ligation ............................................................... 38 METHODS.......................................................................................................... 40 A. Gsy2p wild-type and mutant expression constructs .................................... 40 1. Site-directed mutagenesis ....................................................................... 40 viii 2. Cloning of Gsy2p in the IMPACT vector .................................................. 41 B. Peptide synthesis ....................................................................................... 41 C. Expression and purification of Yeast Gsy2p ............................................... 42 1. Protein preparation from pET28A constructs ........................................... 42 2. Purification of Gsy2p core and semi-synthetic enzymes ......................... 43 D. Crystallization of Gsy2p .............................................................................. 45 1. R580A/R581A/R583A crystals................................................................. 45 2. R589A/R592A glucose-6-phosphate Co-crystals .................................... 46 3. Heavy atom and ligand soaks ................................................................. 47 E. Data collection, processing, structure solution and refinement ................... 48 1. Data collection ......................................................................................... 48 2. Structure solution, model building and refinement ................................... 49 F. Structure analysis ....................................................................................... 50 1. Protein surface analysis .......................................................................... 50 2. Domain rotation analysis ......................................................................... 51 G. GS activity measurement and data analysis .............................................. 51 1. Preparation
Load more

Recommended publications
  • Trimethylsilyl Trifluoromethanesulfonate-Mediated Additions to Acetals, Nitrones, and Aminals Chelsea Safran
    University of Richmond UR Scholarship Repository Honors Theses Student Research 4-1-2013 Trimethylsilyl trifluoromethanesulfonate-mediated additions to acetals, nitrones, and aminals Chelsea Safran Follow this and additional works at: http://scholarship.richmond.edu/honors-theses Recommended Citation Safran, Chelsea, "Trimethylsilyl trifluoromethanesulfonate-mediated additions to acetals, nitrones, and aminals" (2013). Honors Theses. Paper 71. This Thesis is brought to you for free and open access by the Student Research at UR Scholarship Repository. It has been accepted for inclusion in Honors Theses by an authorized administrator of UR Scholarship Repository. For more information, please contact [email protected]. Trimethylsilyl trifluoromethanesulfonate-mediated additions to acetals, nitrones, and aminals By Chelsea Safran Honors Thesis In Program In Biochemistry and Molecular Biology University of Richmond Richmond, VA Spring 2012 Advisor: Dr. C. Wade Downey This thesis has been accepted as part of the honors requirements in the Program in Biochemistry and Molecular Biology ______________________________ _________________ (advisor signature) (date) ______________________________ _________________ (reader signature) (date) Table of Contents i. Acknowledgements ii ii. Abstract iii iii. Chapter I: Introduction 1-4 iv. Chapter II: Amides 4-15 v. Chapter III: I. Bisthione Synthesis 16-18 II. Reactions with other N,O-acetals 18-22 vi. Chapter IV: I. Additions to Nitrones 22-25 II. Future Work 25 vii. Chapter V: Experimental I. N,O-acetal Formation 25-28 II. Addition to Nitrones 28-29 viii. Chapter VI: References 30 i Acknowledgments I would like to acknowledge my research Dr. Wade Downey for all of his time and dedication to my research for the past two years.
    [Show full text]
  • Furanosyl Oxocarbenium Ion Conformational Energy Landscape
    DOI: 10.1002/chem.201900651 Full Paper & Stereoselectivity |Hot Paper| Furanosyl Oxocarbenium Ion Conformational Energy Landscape Maps as a Tool to Study the Glycosylation Stereoselectivity of 2-Azidofuranoses, 2-Fluorofuranoses and Methyl Furanosyl Uronates Stefan van der Vorm, Thomas Hansen, Erwin R. van Rijssel, Rolf Dekkers, Jerre M. Madern, Herman S. Overkleeft, Dmitri V. Filippov, Gijsbert A. van der Marel, and Jeroen D. C. Code*[a] Abstract: The 3D shape of glycosyl oxocarbenium ions de- ic furanoses by using a combined computational and experi- termines their stability and reactivity and the stereochemical mental approach. Surprisingly, all furanosyl donors studied course of SN1 reactions taking place on these reactive inter- react in a highly stereoselective manner to provide the 1,2- mediates is dictated by the conformation of these species. cis products, except for the reactions in the xylose series. The nature and configuration of functional groups on the The 1,2-cis selectivity for the ribo-, arabino- and lyxo-config- carbohydrate ring affect the stability of glycosyl oxocarbeni- ured furanosides can be traced back to the lowest-energy 3E um ions and control the overall shape of the cations. We or E3 conformers of the intermediate oxocarbenium ions. herein map the stereoelectronic substituent effects of the The lack of selectivity for the xylosyl donors is related to the C2-azide, C2-fluoride and C4-carboxylic acid ester on the sta- occurrence of oxocarbenium ions adopting other conforma- bility and reactivity of the complete suite of diastereoisomer- tions. Introduction and they may in fact outweigh steric effects. For example, pro- tonated iminosugars, that is, carbohydrates having the endocy- Stereoelectonic effects dictate the shape and behaviour of clic oxygen replaced by a nitrogen, may change their confor- molecules.
    [Show full text]
  • Discovery, Characterization, and Development of Small
    DISCOVERY, CHARACTERIZATION, AND DEVELOPMENT OF SMALL MOLECULE INHIBITORS OF GLYCOGEN SYNTHASE Buyun Tang Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology, Indiana University June 2020 Accepted by the Graduate Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Doctoral Committee ______________________________________ Thomas D. Hurley, Ph.D., Chair ______________________________________ Peter J. Roach, Ph.D. April 30, 2020 ______________________________________ Millie M. Georgiadis, Ph.D. ______________________________________ Steven M. Johnson, Ph.D. ______________________________________ Jeffrey S. Elmendorf, Ph.D. ii © 2020 Buyun Tang iii Dedication I dedicate this work to my beloved family, my grandparents, my parents, and my brother. iv Acknowledgement First and foremost, I would like to express my deepest gratitude to my thesis advisor, Dr. Thomas D. Hurley. I am grateful to have him as my research mentor over the past four years. He has continuously guided, supported, and inspired me during my study at IUSM. His passion for science and devotion of mentorship offered me the best training experience I could ever receive in graduate school. His easygoing personality reminded me of all the wonderful times inside and outside of the lab. I remember him instructing me handling crystal instrument hand by hand, flying with me to San Diego for a conference, and driving me to a farm restaurant at Illinois. Dr. Hurley is not only an outstanding research mentor, but also a trustful friend. I deeply thank him for all his time, patience, and encouragement along my graduate training.
    [Show full text]
  • Increased Glycosidic Bond Stabilities in 4-C-Hydroxymethyl Linked Disaccharides ⇑ Gour Chand Daskhan, Narayanaswamy Jayaraman
    Carbohydrate Research 346 (2011) 2394–2400 Contents lists available at SciVerse ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres Increased glycosidic bond stabilities in 4-C-hydroxymethyl linked disaccharides ⇑ Gour Chand Daskhan, Narayanaswamy Jayaraman Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India article info abstract Article history: Three new hydroxymethyl-linked non-natural disaccharide analogues, containing an additional methy- Received 25 May 2011 lene group in between the glycosidic linkage, were synthesized by utilizing 4-C-hydroxymethyl-a-D- Received in revised form 24 August 2011 glucopyranoside as the glycosyl donor. A kinetic study was undertaken to assess the hydrolytic stabilities Accepted 25 August 2011 of these new disaccharide analogues toward acid-catalyzed hydrolysis, at 60 °C and 70 °C. The studies Available online 2 September 2011 showed that the disaccharide analogues were stable, by an order of magnitude, than naturally-occurring disaccharides, such as, cellobiose, lactose, and maltose. The first order rate constants were lower than Keywords: that of methyl glycosides and the trend of hydrolysis rate constants followed that of naturally-occurring Acid hydrolysis disaccharides. -Anomer showed faster hydrolysis than the b-anomer and the presence of axial hydroxyl Carbohydrates a Disaccharide analogues group also led to faster hydrolysis among the disaccharide analogues. Energy minimized structures, Glycosidic bond derived through molecular modeling, showed that dihedral angles around the glycosidic bond in disac- Kinetics charide analogues were nearly similar to that of naturally-occurring disaccharides. Oxocarbenium ion Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction determined to be important, prior to heterolysis of C1–O1 bond.
    [Show full text]
  • Characterization of Glycosyl Dioxolenium Ions and Their Role in Glycosylation Reactions
    ARTICLE https://doi.org/10.1038/s41467-020-16362-x OPEN Characterization of glycosyl dioxolenium ions and their role in glycosylation reactions Thomas Hansen 1,4, Hidde Elferink2,4, Jacob M. A. van Hengst1, Kas J. Houthuijs 2, Wouter A. Remmerswaal 1, Alexandra Kromm2, Giel Berden 3, Stefan van der Vorm 1, Anouk M. Rijs 3, Hermen S. Overkleeft1, Dmitri V. Filippov1, Floris P. J. T. Rutjes2, Gijsbert A. van der Marel1, Jonathan Martens3, ✉ ✉ ✉ Jos Oomens 3 , Jeroen D. C. Codée 1 & Thomas J. Boltje 2 1234567890():,; Controlling the chemical glycosylation reaction remains the major challenge in the synthesis of oligosaccharides. Though 1,2-trans glycosidic linkages can be installed using neighboring group participation, the construction of 1,2-cis linkages is difficult and has no general solution. Long-range participation (LRP) by distal acyl groups may steer the stereoselectivity, but contradictory results have been reported on the role and strength of this stereoelectronic effect. It has been exceedingly difficult to study the bridging dioxolenium ion intermediates because of their high reactivity and fleeting nature. Here we report an integrated approach, using infrared ion spectroscopy, DFT computations, and a systematic series of glycosylation reactions to probe these ions in detail. Our study reveals how distal acyl groups can play a decisive role in shaping the stereochemical outcome of a glycosylation reaction, and opens new avenues to exploit these species in the assembly of oligosaccharides and glycoconju- gates to fuel biological research. 1 Leiden University, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC Leiden, The Netherlands. 2 Radboud University Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
    [Show full text]
  • Controlling the Stereoselectivity of Glycosylation Via Solvent Effects
    Canadian Journal of Chemistry Controlling the Stereoselectivity of Glycosylation via Solvent Effects Journal: Canadian Journal of Chemistry Manuscript ID cjc-2016-0417.R1 Manuscript Type: Invited Review Date Submitted by the Author: 16-Sep-2016 Complete List of Authors: Kafle, Arjun; University of New Mexico Liu, Jun; University of New Mexico Cui, Lina; University of New Mexico, Chemistry and Chemical Biology; University Draftof New Mexico, UNM Comprehensive Cancer Center Glycosylation, Stereoselectivity, Synthesis, Solvent effect, Carbohydrate Keyword: chemistry https://mc06.manuscriptcentral.com/cjc-pubs Page 1 of 29 Canadian Journal of Chemistry Controlling the Stereoselectivity of Glycosylation via Solvent Effects Arjun Kafle, Jun Liu, and Lina Cui* Address: Department of Chemistry and Chemical Biology, UNM Comprehensive Cancer Center, University of New Mexico, Albuquerque, NM 87131, U.S.A. Corresponding author: e-mail: [email protected]; Tel: 505-277-6519; Fax: 505-277-2609 Invited Review Dedicated to Prof. David R. Bundle onDraft the occasion of his retirement (Special Issue for Prof. Bundle) 1 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 2 of 29 Abstract: This review covers a special topic in carbohydrate chemistry – solvent effects on the stereoselectivity of glycosylation reactions. Obtaining highly stereoselective glycosidic linkages is one of the most challenging tasks in organic synthesis, as it is affected by various controlling factors. One of the least understood factors is the effect of solvents. We have described the known solvent effects while providing both general rules and specific examples. We hope this review will not only help fellow researchers understand the known aspects of solvent effects and use that in their experiments, moreover we expect more studies on this topic will be started and continued to expand our understanding of the mechanistic aspects of solvent effects in glycosylation reactions.
    [Show full text]
  • 1 General Aspects of the Glycosidic Bond Formation Alexei V
    j1 1 General Aspects of the Glycosidic Bond Formation Alexei V. Demchenko 1.1 Introduction Since the first attempts at the turn of the twentieth century, enormous progress has been made in the area of the chemical synthesis of O-glycosides. However, it was only in the past two decades that the scientificworldhadwitnessedadramatic improvement the methods used for chemical glycosylation. The development of new classes of glycosyl donors has not only allowed accessing novel types of glycosidic linkages but also led to the discovery of rapid and convergent strategies for expeditious oligosaccharide synthesis. This chapter summarizes major prin- ciples of the glycosidic bond formation and strategies to obtain certain classes of compounds, ranging from glycosides of uncommon sugars to complex oligosac- charide sequences. 1.2 Major Types of O-Glycosidic Linkages There are two major types of O-glycosides, which are, depending on nomen- clature, most commonly defined as a-andb-, or 1,2-cis and 1,2-trans glycosides. The 1,2-cis glycosyl residues, a-glycosides for D-glucose, D-galactose, L-fucose, D-xylose or b-glycosides for D-mannose, L-arabinose, as well as their 1,2-trans counter- parts (b-glycosides for D-glucose, D-galactose, a-glycosides for D-mannose,etc.),are equally important components in a variety of natural compounds. Representative examples of common glycosides are shown in Figure 1.1. Some other types of glycosides, in particular 2-deoxyglycosides and sialosides, can be defined neither as 1,2-cis nor as 1,2-trans derivatives, yet are important targets because of their com- mon occurrence as components of many classes of natural glycostructures.
    [Show full text]
  • The Influence of Acceptor Nucleophilicity on the Glycosylation Reaction Mechanism
    Chemical Science View Article Online EDGE ARTICLE View Journal | View Issue The influence of acceptor nucleophilicity on the glycosylation reaction mechanism† Cite this: Chem. Sci.,2017,8, 1867 S. van der Vorm, T. Hansen, H. S. Overkleeft, G. A. van der Marel and J. D. C. Codee´ * A set of model nucleophiles of gradually changing nucleophilicity is used to probe the glycosylation reaction mechanism. Glycosylations of ethanol-based acceptors, bearing varying amounts of fluorine atoms, report on the dependency of the stereochemistry in condensation reactions on the nucleophilicity of the acceptor. Three different glycosylation systems were scrutinized, that differ in the reaction mechanism, that – putatively – prevails during the coupling reaction. It is revealed that the stereoselectivity in glycosylations of benzylidene protected glucose donors are very susceptible to acceptor nucleophilicity whereas condensations of benzylidene mannose and mannuronic acid donors represent more robust glycosylation systems in terms of diastereoselectivity. The change in stereoselectivity with decreasing acceptor nucleophilicity is related to a change in reaction mechanism Received 17th October 2016 shifting from the SN2 side to the SN1 side of the reactivity spectrum. Carbohydrate acceptors are Creative Commons Attribution 3.0 Unported Licence. Accepted 8th November 2016 examined and the reactivity–selectivity profile of these nucleophiles mirrored those of the model DOI: 10.1039/c6sc04638j acceptors studied. The set of model ethanol acceptors thus provides a simple and effective “toolbox” to www.rsc.org/chemicalscience investigate glycosylation reaction mechanisms and report on the robustness of glycosylation protocols. Introduction are displaced in a reaction mechanism having an associative SN2- character, while the oxocarbenium ion-like intermediates are The connection of two carbohydrate building blocks to construct engaged in an SN1-like reaction.
    [Show full text]
  • Predictive Model for Oxidative C−H Bond Functionalization Reactivity with 2,3-Dichloro-5,6-Dicyano-1,4-Benzoquinone (DDQ)
    Predictive Model for Oxidative C!H Bond Functionalization Reactivity with 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) Cristian A. Morales-Rivera, Paul E. Floreancig*, and Peng Liu* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States ABSTRACT: 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) is a highly effective reagent for promoting C!H bond functionalization. The oxidative cleavage of benzylic and allylic C−H bonds using DDQ can be coupled with an intra- or intermolecular nucleophilic addition to generate new carbon–carbon or carbon–heteroatom bonds in a wide range of substrates. The factors that control the reactivity of these reactions are well defined experimentally but the mechanistic details and the role of substituents in promoting the transformations have not been firmly established. Herein, we report a detailed computational study on the mechanism and substituent effects for DDQ-mediated oxidative C−H cleavage reactions in a variety of substrates. DFT calculations show that these reactions proceed through a hydride transfer within a charge transfer complex. Reactivity is dictated by the stability of the carbocation intermediate, the degree of charge transfer in the transition states, and, in certain cases, secondary orbital interactions between the π orbital of the forming cation and the LUMO of DDQ. A linear free energy relationship was established to offer a predictive model for reactivity of different types of C−H bonds based on the electronic properties of the substrate. 1. Introduction DDQ-mediated C–H functionalization has been performed on Carbon–hydrogen bond functionalization reactions can greatly a wide variety of substrates, with specific examples being illustrated facilitate chemical synthesis due to their capability to increase in Scheme 2.
    [Show full text]
  • Template for Electronic Submission to ACS Journals
    Max-Planck-Institut für Kohlenforschung This is the peer-reviewed version of the following article: Nature Chemistry 2018, 10, 888-894, which has been published in final form at: https://www.nature.com/articles/s41557-018-0065-0. This article may be used for non-commercial purposes. DOI: 10.1038/s41557-018-0065-0 Approaching Sub-ppm Level Asymmetric Organocatalysis of a Highly Challenging and Scalable Carbon–Carbon Bond Forming Reaction Han Yong Bae,a Denis Höfler,a Philip S. J. Kaib,a Pinar Kasaplar,a Chandra Kanta De,a Arno Döhring,a Sunggi Lee,a Karl Kaupmees,b Ivo Leitob and Benjamin Lista* aMax-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Mülheim an der Ruhr, Germany. bUniversity of Tartu, Institute of Chemistry, Ravila 14a, Tartu 50411, Estonia. *email: [email protected] Abstract: The chemical synthesis of organic molecules involves, at its very essence, the creation of carbon–carbon bonds. In this context, the aldol reaction is among the most important synthetic methods, and a wide variety of catalytic and stereoselective versions have been reported. However, aldolizations yielding tertiary aldols, which result from the reaction of an enolate with a ketone, are challenging and only a few catalytic asymmetric Mukaiyama aldol reactions with ketones as electrophiles have been described. These methods typically require relatively high catalyst loadings, deliver substandard enantioselectivity or need special reagents or additives. We now report extremely potent catalysts that readily enable the reaction of silyl ketene acetals with a diverse set of ketones to furnish the corresponding tertiary aldol products in excellent yields and enantioselectivities.
    [Show full text]
  • An Oxocarbenium-Ion Intermediate of a Ribozyme Reaction Indicated by Kinetic Isotope Effects
    An oxocarbenium-ion intermediate of a ribozyme reaction indicated by kinetic isotope effects Peter J. Unrau†‡ and David P. Bartel‡§ †Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6; and §Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142 Edited by Thomas R. Cech, Howard Hughes Medical Institute, Chevy Chase, MD, and approved October 14, 2003 (received for review May 23, 2003) Many of the enzymes that catalyze reactions at nucleotide glyco- take place through a very weakly concerted oxocarbenium-like sidic linkages proceed through either a reactive oxocarbenium-ion transition state (5), a ribozyme, in analogy with protein enzymes, intermediate or a transition state with considerable oxocarbenium could employ at least two distinct catalytic strategies. One would be character. To investigate how an RNA active site deals with the to promote the oxocarbenium-ion character of the uncatalyzed catalytic challenge of nucleotide synthesis, we probed the transi- transition state while simultaneously limiting access of water to this tion state of a ribozyme able to promote the formation of a reactive state (Fig. 1a). Alternatively, a ribozyme might promote a pyrimidine nucleotide. Primary and secondary kinetic isotope ef- concerted mechanism in which the attack of the 4SUra and the fects indicate that this ribozyme stabilizes a highly dissociative departure of the pyrophosphate are made more synchronous reaction with considerable sp2 hybridization and negligible bond (Fig. 1b). The relative accessibility of these catalytic strategies for order between the departing pyrophosphate leaving group and emergent ribozymes is difficult to assess based on previous studies, the anomeric carbon.
    [Show full text]
  • Preparation of Α-Acetoxy Ethers by the Reductive Acetylation of Esters
    DOI:10.15227/orgsyn.089.0143 Discussion Addendum for: Preparation of -Acetoxy Ethers by the Reductive Acetylation of Esters: endo-1-Bornyloxyethyl Acetate 1. DIBALH CH O H3C 3 CH CH Cl , –78 °C, 45 min H C 3 CH3 O 2 2 3 CH3 O CH3 2. pyr, DMAP, Ac O O CH 2 3 O CH –78 to 0 °C, 12 h 3 Prepared by Nicholas Sizemore and Scott D. Rychnovsky.*1 Original Article: Kopecky, D. J.; Rychnovsky, S. D. Org. Synth. 2003, 80, 177. Reductive acetylation has proven to be a powerful methodology primarily because its products are excellent substrates for carbon-carbon bond forming reactions. Since 2003, the Organic Syntheses article and the primary references have been cited over 100 times. This discussion addendum aims to provide a brief overview of recent developments in this methodology. Improvements and variations of -acetoxy ether formation, as well as the synthetic utility of -acetoxy ethers will be discussed. Recent applications in the context of the total synthesis of complex natural products are also included. Improvements and Variations of -Acetoxy Ether Formation The original reductive acetylation protocol involves the addition of diisobutylaluminum hydride (DIBALH) as a 1.0 M solution in hexanes to the ester at –78 °C. The resulting aluminum alkoxide is then treated with pyridine, 4-dimethylaminopyridine (DMAP), and acetic anhydride (Ac2O) sequentially. The internal temperature during these additions should not exceed –72 °C in order to maximize the yield and prevent undesired side reactions.2 While this method is general, recent developments include the use of asymmetric electrophiles in order to generate more complex acyl ethers and the extension of reductive acetylation to lactams and imides.
    [Show full text]