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Hydrolytically Stable Analogues of Sugar Phosphates and a Miniaturized in Situ Enzymatic Screen Xiang Fei University of Nebraska-Lincoln, [email protected]

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Hydrolytically Stable Analogues of Sugar Phosphates and a Miniaturized in Situ Enzymatic Screen Xiang Fei University of Nebraska-Lincoln, Xfei@Huskers.Unl.Edu University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Student Research Projects, Dissertations, and Chemistry, Department of Theses - Chemistry Department 11-2014 Hydrolytically Stable Analogues of Sugar Phosphates and a Miniaturized in Situ Enzymatic Screen Xiang Fei University of Nebraska-Lincoln, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/chemistrydiss Part of the Medicinal-Pharmaceutical Chemistry Commons, and the Organic Chemistry Commons Fei, Xiang, "Hydrolytically Stable Analogues of Sugar Phosphates and a Miniaturized in Situ Enzymatic Screen" (2014). Student Research Projects, Dissertations, and Theses - Chemistry Department. 50. http://digitalcommons.unl.edu/chemistrydiss/50 This Article is brought to you for free and open access by the Chemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Student Research Projects, Dissertations, and Theses - Chemistry Department by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Hydrolytically Stable Analogues of Sugar Phosphates and A Miniaturized In Situ Enzymatic Screen by Xiang Fei A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Chemistry Under the Supervision of Professor David B. Berkowitz Lincoln, Nebraska November, 2014 Hydrolytically Stable Analogues of Sugar Phosphates and A Miniaturized In Situ Enzymatic Screen Xiang Fei, Ph.D. University of Nebraska, 2014 Advisor: David. B. Berkowitz The glmS riboswitch undergoes self-cleavage upon binding its metabolic product GlcN6P, thereby providing a negative feedback mechanism limiting translation of the glmS protein when GlcN6P is abundant. As a first step toward the development of novel antimicrobials, we have synthesized a series of GlcN6P analogues bearing phosphatase- inert surrogates in place of the natural phosphate ester functionality. The self-cleavage assay identified two such compounds that display significant riboswitch actuator activity; namely those bearing a 6-phosphonomethyl group or a 6-O-malonyl ether. These two analogues exhibit a 22-fold and a 27-fold higher catalytic efficiency, respectively, than does glucosamine. Docking experiments were conducted to provide insight into the structural basis for SAR (Structure/Activity Relationship) seen across this battery of GlcN6P analogues and directions for future design of such small molecule actuators. M6P/IGF2R regulates intracellular sorting of lysosomal enzymes, as well as endocytosis of extracellular ligands. To explore the possibility of multivalent receptor- ligand interactions, we have utilized novel chemistries to synthesize “tailored” bivalent ligands. A “linker diversification” approach has been recently developed. It emanates from a monomer with a terminal azide. Five different chemistries were exploited to connect two monomers together, leading to five structurally and functionally distinct linkages. The assay showed that when the angles between two linking bonds are acute rather than obtuse, the corresponding ligands present higher binding affinity, suggesting the three dimensional shape of the ligand is crucial for achieving multivalency. The ISES technique has proven to be a useful technique for catalyst screening. In this procedure, an organic reaction product or byproduct diffuses into an aqueous layer, wherein an enzymatic transformation leads to signal that can be monitored by UV/vis spectroscopy. Herein, we describe proof of principle of a miniaturized ISES assay, in which volumes are significantly reduced by utilizing a quartz micromulticell. This miniaturized ISES platform is used to examine a 4 4 combinatorial library of salen ligands, that is derived from both oxa- and carbacyclic D-fructopyranosyl-1,2-diamines. The Co(III)-salen derived from 3’,5’-diiodo-salicylaldehyde and -D- carbafructopyranosyl-1,2-diamine shows the highest chiral bias. X-ray crystallographic analysis reveals important structural differences between the more selective carbofructopyranosyl-1,2-diamine-derived salens and their oxacyclic counterparts. Acknowledgements I am deeply grateful to my advisor, Professor David B. Berkowitz for his strong and enthusiastic mentorship. His constant encouragement and striving for excellence have helped me to grow as a scientist. I would like to thank other members of my committee: Professors Stephen G. DiMagno, Mark A. Griep, James M. Takacs and Richard G. MacDonald for their precious time and thoughtful suggestions, especially Professors Takacs and MacDonald for reading my thesis. The work presented in this dissertation would have not been possible without the help and collaboration of the following friends: Dr. Kannan R. Karukurichi, Dr. Weijun Shen, Dr. Sangeeta Dey, Dr. Sylvain Broussy, Dr. Pulakesh Maity, David L. Nelson, Kaushik Panigrahi, Dr. Guillaume Malik, Professor Richard G. MacDonald, Dr. Christopher M. Connelly, Megan E. Zavorka, Professor Juliane K. Soukup and Thomas Holmes. I want to convey my gratitude to other members of the Berkowitz group for being wonderful colleagues. I was very fortunate to have my family around during the time in Lincoln. I want to thank my wife Gonghua, for always being supportive and encouraging along the way. I want to acknowledge my parents, especially my mom for spending a great deal of time with us, and taking care of our sons Ben and Jerry. I also want to thank the boys for all the joys they have brought to the family. xi List of Abbreviations AA Amino Acid Ac Acyl ADH Alcohol Dehydrogenase aq Aqueous APPA 2-Amino-5-Phosphono-3-Pentenoic Acid Ar Aryl Bn Benzyl Boc tert-Butyloxycarbonyl Bu Butyl Calcd. Calculated CD-MPR Cation Dependent Mannose 6-Phosphate Receptor CI-MPR Cation Independent Mannose 6-Phosphate Receptor CM Cross Metathesis cod Cyclooctadiene Cy Cyclohexyl DCE Dichloroethane DCM Dichloromethane de Diastereomeric excess DMAP 4-N,N-Dimethylaminopyridine DMF N,N-Dimethylformamide xii DMSO Dimethyl Sulfoxide DTBMP 2,6-Di-tert-butyl-4-methylpyridine FPP Farnesyl Pyrophosphate ee Enantiomeric Excess ESI Electron Spray Ionization Equiv. Equivalents F6P Fructose 6-Phosphate G6P Glucose 6-Phosphate GlcN6P Glucosamine 6-Phosphate GlcNAc N-Acetyl Glucosamine GlmS Glucosamine 6-Phosphate Synthase hGUS Human β-Glucuronidase HMDS Hexamethyldisilazane HMPA Hexamethylphosphoramide HPLC High Performance Liquid Chromatography HRMS High Resolution Mass Spectrometry Hz Hertz IDCP Iodonium Di-sym-Collidine Perchlorate IGF Insulin Like Growth Factor J Coupling Constant KRED Ketoreductase LDA Lithium diisopropylamide M6P Mannose 6-Phosphate xiii M Molarity MD Molecular Dynamics Me Methyl MeOH Methanol min Minute MRH Mannose 6-Phosphate Receptor Homology MS Mass Spectrometry N Normality PEG Polyethylene Glycol PMP Pentamannosyl Phosphate Pyr Pyridine rt Room Temperature SES 2-Trimethylsilylethanesulfonyl TBAF Tetrabutylammonium fluoride Tf Trifluoromethanesulfonyl TGN Trans Golgi Network THF Tetrahydrofuran TLC Thin Layer Chomatography TBS tert-Butyldimethylsilyl TBDPS tert-Butyldiphenylsilyl TM Transition Metal Ts 4-Toluenesulfonyl i Table of Contents Table of Figures v Table of Tables x List of Abbreviations xi Chapter 1. Overview of Phosphonates in Bioorganic Chemistry I. Naturally Occurring Phosphonates 1 A. Fosfomycin/Fosphonomycin 3 B. 2-Amino-5-Phosphono-3-Pentenoic Acid (APPA) and APPA- Containing Peptides: Plumbemycins and Rhizocticins 4 C. FR-900098 and Fosmidomycin 5 D. Latest Discoveries 6 II. Synthetic phosphonates 8 A. Glyphosate/Roundup 8 B. Bisphosphonates 9 C. Acyclic Nucleoside Phosphonates (ANPs) 12 III. Sugar Phosphonate in Bioorganic Chemistry 15 IV. References 21 Chapter 2. Phosphatase-Inert Actuators for the glmS Riboswitch I. Introduction 35 A. Background of Riboswitches 35 ii B. Background of the glmS riboswitch 40 C. GlmS riboswitch as an antimicrobial target 44 II. Results and Discussion 48 A. Synthesis of GlcN6P Analogues 48 B. Self-Cleavage Assays with the glmS Riboswitch 56 C. Molecular Modelling and pKa Titration 62 III. Future directions 65 IV. Experimental Section 66 A. Organic Synthesis 66 B. Self-Cleavage Assay 100 C. pKa Titration Experiments 101 D. Molecular Docking 101 V. References 104 VI. NMR Spectra 113 Chapter 3. Hydrolytically-Stable Bivalent Ligands for M6P/IGF2R I. Introduction 125 A. Background of M6P/IGF2R 125 B. Previous Work on Multivalent Ligands for M6P/IGF2R 134 II. Results and Discussion 137 A. Monovalent ligands and cross-metathesis approach 137 B. Ligand Diversification at the Linking Stage 141 C. Binding Affinity Assay 150 iii D. PEG Based Bivalent Ligands 152 III. Experimental Section 158 A. Organic Synthesis 158 B. Binding Affinity Assay 185 IV. References 187 V. NMR Spectra 204 Chapter 4. Carbafructopyranosyl 1,2-Diamines as New Chiral Scaffolds I. Introduction 248 A. Chiral Pool in Asymmetric Catalysis 248 B. A Chiral Pool Derived Salen Ligand Library 250 C. Novel Screening Methods in Combinatorial Catalysis 252 D. In Situ Enzymatic Screening 256 II. Results and Discussion 259 A. Synthesis of the Oxa- and Carbafructopyranose Derived Diamines 259 B. A New Miniaturized ISES 262 C. Preserved Enantioswitch and Boosted Enantioselectivity 266 D. Comparison to A Directed Evolution Based Approach 268 E. X-Ray Structures and Transition State Model of HKR 269 F. Streamlined Synthesis of the D-Carbafructopyranosyl-1,2- Diamines 274 III. Experimental Section
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