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Masterarbeit MASTERARBEIT Titel der Masterarbeit New Concepts in Isotopic Labeling of Proteins verfasst von Julia Schörghuber BSc angestrebter akademischer Grad Master of Science (MSc) Wien, August 2014 Studienkennzahl lt. Studienblatt: A 066 862 Studienrichtung lt. Studienblatt: Masterstudium Chemie UG2002 Betreut von: Univ.-Prof. Mag. Dr. Walther Schmid Things never quite work as you expect them to; this is one of the principal beauties of science. The Prestige i ACKNOWLEDGMENT First of all I want to express my sincere gratitude to my supervisor Roman for the continuous support of my master thesis and for his patience, motivation, enthusiasm, engagement and his invaluable guidance through the whole working process. Furthermore, I would like to thank Prof. Walther Schmid for the opportunity to be part of his working group and, moreover, to deal with this interesting topic. Besides, I would like to thank Prof. Robert Konrat for incorporating my precursors into small proteins. I want to thank the whole group who readily provided information and help any time; especially Manuel and Christoph for helping me interpreting NMR spectra. My sincere gratitude also goes to Tina who patiently revised and corrected my thesis to acquire an “English shape”. I am also grateful to my friends Julia and Johannes, who have gone with me through thick and thin and for always giving me words of encouragement and laughter. Everything is theoretically impossible, until it is done. Therefore, I owe my deepest gratitude to my boyfriend for having the courage to make us possible. At last I want to thank my family members, especially my parents and my sisters Pia and Lena, for encouraging and inspiring me to follow my dreams. Thank you for supporting me emotionally and financially throughout my life and my studies at university. ii ABSTRACT Isotope labeling is used to simplify complex NMR spectra of proteins by increasing the amount of structural information and facilitating signal assignment. Recently, we developed and tested various synthetic routes to selectively label small molecules; these are added as amino acid precursors to E. coli expression media of proteins. After successful 13C incorporation of the 1-13C-α-keto acid, we synthesized more elaborate side-chain labeled derivatives of indole as a precursor, featuring 13C isotope labeling in the five-membered ring and an isolated 13C-1H spin system of the six-membered ring in an otherwise deuterated background. Furthermore, we designed an economic and general reaction sequence applicable for several enantiopure amino acids such as Val, Phe, Leu, Tyr, Trp and potentially His; these could be used to enhance cell-free protein expression or solid phase synthesis. iii TABLE OF CONTENTS ACKNOWLEDGMENT ................................................................................................... II ABSTRACT ................................................................................................................... III TABLE OF CONTENTS ................................................................................................ IV 1. INTRODUCTION AND AIMS OF THE MASTER THESIS ..................................... 1 2. THEORETICAL BACKGROUND .......................................................................... 3 2.1. Metabolism of Specific Amino Acids: Val, Leu, Ile, Phe, Tyr, Trp and His .................................................... 3 2.1.1. Metabolism of Aromatic Amino Acids: Tyr, Phe, Trp and His ................................................................... 4 2.1.2. Metabolism of Aliphatic Amino Acids: Val, Leu and Ile ............................................................................ 9 2.2. Protein Determination ............................................................................................................................. 12 2.2.1. NMR Methods ......................................................................................................................................... 12 2.2.2. Cell-Based Protein Overexpression ......................................................................................................... 14 2.2.3. Cell-Free Methods .................................................................................................................................. 15 2.3. Labeling Strategies ................................................................................................................................... 16 2.3.1. Labeling of Branched-Chain Amino Acids ............................................................................................... 18 2.3.2. Labeling of Aromatic Amino Acids .......................................................................................................... 20 2.3.3. SAIL Strategy ........................................................................................................................................... 21 3. RESULTS AND DISCUSSION ............................................................................ 23 3.1. Synthesis of Enantiopure Amino Acids ..................................................................................................... 23 3.1.1. Synthesis of Labeled L-Valine .................................................................................................................. 26 3.1.2. Synthesis of Labeled L-Phenylalanine ..................................................................................................... 27 3.1.3. Synthesis of Unlabeled L-Leucine ............................................................................................................ 29 3.1.4. Synthesis of Unlabeled L-Tyrosine .......................................................................................................... 30 3.1.5. Synthesis of Unlabeled L-Tryptophan ..................................................................................................... 31 3.1.6. Synthesis of Unlabeled L-Histidine .......................................................................................................... 31 3.2. Trp-Precursors .......................................................................................................................................... 33 3.2.1. Synthesis of Indole-3-Pyruvate as a Trp-Precursor ................................................................................. 33 3.2.2. Synthesis of Indole as a Trp-Precursor .................................................................................................... 37 4. CONCLUSION AND OUTLOOK ......................................................................... 42 4.1. Conclusion ................................................................................................................................................ 42 4.2. Outlook .................................................................................................................................................... 43 iv 5. EXPERIMENTAL SECTION ................................................................................. 45 5.1. General Comments ...................................................................................................................................45 5.2. Synthetic Protocols of Enantiopure Amino Acids ......................................................................................46 5.3. Synthetic Protocols of Trp-precursors .......................................................................................................84 6. APPENDIX ......................................................................................................... 101 6.1. Spectra ................................................................................................................................................... 101 6.2. Abbreviations and Acronyms .................................................................................................................. 158 6.3. References .............................................................................................................................................. 161 6.4. Deutsche Zusammenfassung .................................................................................................................. 164 6.5. Curriculum Vitae ..................................................................................................................................... 165 v 0 1. INTRODUCTION AND AIMS OF THE MASTER THESIS Determination of protein structures is a useful target to analyze protein folding, dynamic properties and, furthermore, to shed light on their functions in organisms. The conformation of proteins provides information about cellular mechanisms and about their affinity to specific substrates. Therefore, diverse methods for three-dimensional protein determination are applicable; the most important methods include X-ray crystallography and specific NMR methods (NOESY, COSY, and TOCSY). X-ray crystallography enables determination of static high molecular weight proteins providing high accuracy. Complementary to this method, NMR spectroscopy has evolved into a new method to analyze proteins in aqueous solution and, consequently, protein folding and their dynamic properties providing three-dimensional information.1 A major disadvantage of this method is the complexity of resulting NMR spectra of large proteins (>100 kDa); this complexity derives from signal overlapping and line broadening, which causes limited signal assignment and difficult interpretation of these spectra.2
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