Design and Synthesis of Modular Reagents for Chemical Biology
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DESIGN AND SYNTHESIS OF MODULAR REAGENTS FOR CHEMICAL BIOLOGY by BereketAb T. Mehari ____________________________ Copyright © Bereketab T. Mehari 2018 A Dissertation Submitted to the Faculty of the DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY WITH A MAJOR IN CHEMISTRY In the Graduate College THE UNIVERSITY OF ARIZONA 2018 1 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: BereketAb T. Mehari 3 ACKNOWLEDGEMENTS I would like to first extend my gratitude to my research advisor, Dr. John Jewett, for his guidance throughout my graduate career. It has been a great experience working under his mentorship. I would also like to thank my committee members for their advice in preparing for this work. I would like to thank my lab mates for both their chemistry input and moral support. I have made many friends in the department who have assisted me in becoming a better scientist. I would like to thank Dr. Robb Bagge and Dr. Phil Durlam for helping me as colleagues and their support as friends. I would also like to thank the department research facilities. I would also like to thank the teaching support stuff for doing all the behind the stage work that enables us to teach. I would like to thank my graduate coordinator Ms. Lori Boyd, for the continued support that she has provided me. Finally, I thank the department of chemistry and biochemistry for the opportunity to pursue my studies in this institution. 4 DEDICATION This dissertation is dedicated to my family. 5 Table of Contents List of Abbreviations ...................................................................................................................... 9 List of Schemes ............................................................................................................................. 11 List of Figures ............................................................................................................................... 14 Abstract ......................................................................................................................................... 16 Chapter 1 : Biomolecule labeling in chemical biology ................................................................. 17 1.1 Introduction ......................................................................................................................... 17 1.2 Site specific modification on native biomolecules .............................................................. 18 1.2.1 Tyrosine residues for selective bioconjugation ............................................................ 21 1.2.2 Masked diazoniums for bioconjugation ....................................................................... 28 1.3 Site specific unnatural modification for bioconjugation ..................................................... 31 1.3.1 Organic azides in bioortogonal chemistry .................................................................... 35 1.3.2 Staudinger reagents in bioconjugation ......................................................................... 38 Chapter 2 : Modular traceless Bertozzi-Staudinger reagents for biological labeling .................. 45 2.1 Introduction ......................................................................................................................... 45 2.2 Host-guest chemistry towards a modular Staudinger probe................................................ 51 2.3 A method to synthesize a modular traceless Bertozzi-Staudinger reagent ......................... 57 Chapter 3 : A bifunctionalized traceless Bertozzi-Staudinger-Triazabutadiene reagent .............. 65 3.1 Introduction ......................................................................................................................... 65 6 3.2. Synthesis of a bifunctionalized reagent .............................................................................. 68 3.3 A bifunctionalized reagent for modifying traceless Bertozzi-Staudinger and triazabutadiene reagents...................................................................................................................................... 74 Chapter 4 : Future directions......................................................................................................... 79 4.1 Modular traceless Bertozzi-Staudinger reagent .................................................................. 79 4.2 A bifunctionalized traceless Bertozzi-Staudinger-triazabutadiene reagent ........................ 83 Chapter 5 : Experimental Section ................................................................................................ 86 5.1. Experimental conditions and characterizations .................................................................. 86 5.1.1. Chlorodiphenylphosphineborane (134) ....................................................................... 87 5.1.2. 2-bromo-4-(5,5-dimethyl-1,3-dioxan-2-yl)phenol (139) ............................................ 87 5.1.3. 4-amino-2-bromophenol (142) .................................................................................... 88 5.1.4. 4-azido-2-bromophenol (143)...................................................................................... 89 5.2 General procedure for the synthesis of phosphine .............................................................. 89 5.2.1 (2-Hydroxyphenyl)diphenylphosphine borane (136) ................................................... 90 5.2.2. 4-(5,5-dimethyl-1,3-dioxan-2-yl)- (2-Hydroxyphenyl)diphenylphosphine Borane (140)....................................................................................................................................... 91 5.2.3. 4-azido-(2-Hydroxyphenyl)diphenylphosphine borane (144) ..................................... 92 5.3.1 Potassium (E)-4-((1,3-dimesityl-1,3-dihydro-2H-imidazol-2-ylidene)triaz-1-en-1- yl)benzoate (162) ................................................................................................................... 92 5.3.2. 2-(diphenylphosphanyl)phenyl 4-azidobenzoate borane (165) ................................... 93 7 5.4. 2-(diphenylphosphanyl)phenyl (E)-4-((1,3-dimesityl-1,3-dihydro-2H-imidazol-2- ylidene)triaz-1-en-1-yl)benzoate borane (163) ......................................................................... 94 5.5. General condition for the reaction of benzyl azide with phosphine borane ....................... 96 5.5.1. (E)-N-benzyl-4-((1,3-dimesityl-1,3-dihydro-2H-imidazol-2-ylidene)triaz-1-en-1- yl)benzamide (169) ................................................................................................................ 97 5.5.2. (E)-N-benzyl-4-((2-hydroxy-5-methylphenyl)diazenyl)benzamide (168) .................. 97 5.6. General condition for reaction of triazabutadiene with p-cresol ........................................ 98 5.6.1 2-(diphenylphosphanyl)phenyl (E)-4-((2-hydroxy-5-methylphenyl)diazenyl)benzoate borane (167) ........................................................................................................................... 99 5.6.2. (E)-N-benzyl-4-((2-hydroxy-5-methylphenyl)diazenyl)benzamide (168) ................ 100 Appendix A: Work done in collaboration ................................................................................... 101 A1. Collaboration with Jewett group members ....................................................................... 101 A2. Collaboration with other research groups ........................................................................ 104 Appendix B: Spectral data .......................................................................................................... 109 References ................................................................................................................................... 139 8 List of Abbreviations AIBN Azobisisobutyronitrile BARAC Biarylazacyclooctynone BH3 Borane BnN3 Benzyl azide CD Cyclodextrin Ch2Cl2 Dichloromethane CuAAC Copper-catalyzed azide-alkyne cycloaddition D2O Deuterium oxide DABCO 1,4-diazabicyclo[2.2.2]octane DCC N,N'-dicyclohexylcarbodiimide DIC N,N'-diisopropylcarbodiimide DIFO Difluorinatedcyclooctyne DIMAC Dimethoxyazacyclooctyne EDC (3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride Et2O Diethylether GFP Green fluorescent protein FRET Fluorescence resonance energy transfer HCl Hydrochloric acid 9 ICT Intramolecular charge transfer Ka Association constant t-BuOK Potassium tert-butoxide Na2S2O4 Sodium dithionate NaH Sodium hydride NaN3 Sodium azide NaNO2 Sodium nitrite NaOH Sodium hydroxide n-BuLi n-Butyllithium NH2OH·HCl Hydroxylammonium chloride NHC N-heterocyclic carbene NMR Nuclear magnetic resonance Pd(OAc)2 Palladium(II) acetate PEG Polyethylene glycol Rh2(AcO)4 Rhodium(II) acetate RT Room