NEW INSIGHTS INTO BIOMOLECULE POLYMERIZATION UNDER PLAUSIBLE PRIMORDIAL EARTH CONDITIONS: IMPLICATIONS FOR THE ORIGIN OF LIFE A Dissertation Presented to The Academic Faculty By Eric T. Parker In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy in Chemistry Georgia Institute of Technology [August, 2016] Copyright © Eric Thomas Parker 2016 NEW INSIGHTS INTO BIOMOLECULE POLYMERIZATION UNDER PLAUSIBLE PRIMORDIAL EARTH CONDITIONS: IMPLICATIONS FOR THE ORIGIN OF LIFE Approved by: Dr. Facundo M. Fernández Dr. Thomas Orlando School of Chemistry and Biochemistry School of Chemistry and Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Eric Gaucher Dr. Loren Williams School of Biology School of Chemistry and Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Nicholas Hud School of Chemistry and Biochemistry Date Approved: June 21, 2016 Georgia Institute of Technology To my parents, Tom and Carol, and my sisters, Amy and Kristen, for supporting and motivating me throughout my pursuit of a Ph.D. ACKNOWLEDGEMENTS My sincere gratitude to my advisor, Dr. Facundo Fernández, for his guidance during this dissertation. I am appreciative of my committee members, Dr. Eric Gaucher, Dr. Nicholas Hud, Dr. Thomas Orlando, and Dr. Loren Williams for shaping this thesis. Many thanks to the Center for Chemical Evolution, the primary funding source for this dissertation. As the director of the center, Dr. Nicholas Hud deserves significant credit for helping secure the funds necessary for this thesis research, and my stipend. I would like to give a special thank you to Dr. Jeffrey Bada, who has been a tremendous mentor, collaborator, and friend. From my time as an undergraduate, to the completion of my Ph.D., his leadership and advice has been inspirational and invaluable. Dr. Jim Cleaves is a great colleague who has improved my understanding of, and appreciation for, our work. Dr. Daniel Glavin and Dr. Jason Dworkin provided great leadership and teachings about handling precious samples and making careful analytical measurements. I am grateful for the analytical expertise shared by Dr. Aaron Burton, and the organic expertise of Dr. Charles Liotta and Dr. Ramanarayanan Krishnamurthy. I owe a debt of gratitude to Dr. María Eugenia Monge, whose sage analytical advice and friendship is greatly appreciated. Dr. David Gaul, Dr. Jennifer Pittman, Dr. Jay Forsythe, Dr. Martin Paine, Dr. Matthew Bernier, Dr. Anyin Li, Dr. Molly Soper, and Dr. Chaevien Clendinen have provided important guidance based on their graduate careers. I am thankful for the support of my fellow graduate students, Dr. Christina Jones, Dr. Rachel Bennett, Joel Keelor, Adam Kaylor, Ezequiel Morzan, Xiaoling Zang, Scott Hogan, Danning Huang, Jaslynn Murphy, and Stephen Zambrzycki. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ………………………………………………………….iv LIST OF TABLES …………………………………………………………………..x LIST OF FIGURES …………………………………………………………………xii LIST OF ABBREVIATIONS ……………………………………………….xviii SUMMARY ……………………………………………………………………….xxiv CHAPTER 1: INTRODUCTION …………………………………………………..1 1.1 Abstract …………………………………………………………………..1 1.2 The Importance of Studying the Origin of Life …………………………..2 1.3 Early Abiotic Synthesis of Molecules Important for Life …………..3 1.3.1 The Abiotic Synthesis of Nucleobases …………………………………..4 1.3.2 The Abiotic Synthesis of Urea …………………………………………..5 1.3.3 The Abiotic Synthesis of AAs …………………………………………..6 1.3.4 The Abiotic Synthesis of Sugars …………………………………..6 1.4 Possible Primordial Earth Environmental Scenarios …………………..7 1.5 The Miller-Urey (MU) Experiments …………………………………10 1.5.1 The Classic MU Experiment …………………………………………11 1.5.2 The Miller Volcanic Spark Discharge Experiment …………………13 1.5.3 The Silent Electric Discharge Experiment …………………………15 1.6 The Evolution of Analytical Chemistry used to Study the Origin of Life …………………………………………………………………………16 1.6.1 Paper Chromatography with Ninhydrin Detection …………………16 1.6.2 High Performance Liquid Chromatography-Fluorescence Detection …21 1.6.3 Ultra Performance Liquid Chromatography-Mass Spectrometry …23 1.7 Analysis of Previously Reported and Unreported Miller ED Experiments using Modern Analytical Techniques …………………………………26 1.7.1 Re-analysis of the Classic Miller-Urey Experiment …………………27 1.7.2 Re-analysis of the Miller Volcanic Spark Discharge Experiment …29 1.7.3 Re-analysis of the Silent Electric Discharge Experiment …………30 1.7.4 The Hydrogen Sulfide Experiment …………………………………32 1.8 References …………………………………………………………37 v CHAPTER 2: CONDUCTING MILLER-UREY EXPERIMENTS …………………44 2.1 Abstract …………………………………………………………………44 2.2 Introduction …………………………………………………………45 2.3 Protocol …………………………………………………………………49 2.3.1 Setting Up a Manifold/Vacuum System …………………………49 2.3.2 Preparation of Reaction Flask …………………………………………51 2.3.3 Introduction of Gaseous NH3 ………..………………………………..52 2.3.4 Introduction of CH4 …………………………………………………53 2.3.5 Introduction of Further Gases (e.g., N2) …………………………54 2.3.6 Beginning the Experiment …………………………………………55 2.3.7 End of Experiment …………………………………………………55 2.3.8 Collecting Liquid Sample …………………………………………56 2.3.9 Cleaning the Apparatus …………………………………………………56 2.3.10 Sample Analysis …………………………………………………57 2.4 Representative Results …………………………………………………57 2.5 Discussion …………………………………………………………60 2.5.1 Introduction of Gaseous NH3 …………………………………………64 2.5.2 Introduction of CH4 …………………………………………………64 2.5.3 Introduction of N2 …………………………………………………65 2.6 List of Materials Used …………………………………………………70 2.7 Acknowledgements …………………………………………………71 2.8 References …………………………………………………………71 CHAPTER 3: AMINO ACIDS GENERATED FROM HYDRATED TITAN THOLINS: COMPARISON WITH MILLER-UREY ELECTRIC DISCHARGE PRODUCTS …73 3.1 Abstract …………………………………………………………………73 3.2 Introduction …………………………………………………………74 3.3 Materials and Methods …………………………………………………79 3.3.1 Chemicals and Reagents …………………………………………79 3.3.2 Titan Tholins …………………………………………………………79 3.3.3 MU ED Polymers …………………………………………………80 3.3.4 Standard Analysis …………………………………………………80 3.3.5 UPLC-FD/TOF-MS Analysis …………………………………………82 3.4 Results and Discussion …………………………………………………82 3.5 Conclusions …………………………………………………………94 3.6 Acknowledgements …………………………………………………95 3.7 References …………………………………………………………………95 vi CHAPTER 4: A PLAUSIBLE SIMULTANEOUS SYNTHESIS OF AMINO ACIDS AND SIMPLE PEPTIDES ON THE PRIMORDIAL EARTH ………………..101 4.1 Abstract ………………………………………………………………..101 4.2 Introduction ………………………………………………………..102 4.3 Materials and Methods ………………………………………………..105 4.3.1 Chemicals and Reagents ………………………………………..105 4.3.2 Sample Discovery and Experimental Setup ………………………..107 4.3.3 Original Miller Cyanamide Sample Analyses ………………………..108 4.3.4 Repeated Cyanamide Experiment Design ………………………..112 4.3.5 Repeated Cyanamide Experiment Sample Analyses ………………..116 4.3.6 Heating Experiment Design ………………………………………..117 4.3.7 Heating Experiment Sample Analyses ………………………………..118 4.4 Results and Discussion ………………………………………………..118 4.4.1 AAs in the Original Miller Cyanamide Samples ………………..119 4.4.2 Dipeptides in the Original Miller Cyanamide Samples ………………..121 4.4.3 Dipeptides Produced by the Repeated Cyanamide Experiment ..124 4.4.4 Polymerization Chemistry Observed in Heating Experiments ………..127 4.5 Conclusions ………………………………………………………..133 4.6 Acknowledgements ………………………………………………..134 4.7 References ………………………………………………………..134 CHAPTER 5: QUANTITATION OF -HYDROXY ACIDS IN COMPLEX PREBIOTIC MIXTURES VIA LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY ………………………………………………………………..139 5.1 Abstract ………………………………………………………………..139 5.2 Introduction ………………………………………………………..140 5.3 Materials and Methods ………………………………………………..142 5.3.1 Chemicals and Reagents ………………………………………..142 5.3.2 ED Experiment #1 ………………………………………………..143 5.3.3 ED Experiment #2 ………………………………………………..143 5.3.4 ED Experiment #3 ………………………………………………..144 5.3.5 ED Experiment #4 ………………………………………………..145 5.3.6 ED Sample Analysis ………………………………………………..145 5.3.7 Data Analysis ………………………………………………………..149 5.4 Results and Discussion ………………………………………………..151 5.4.1 Analytical Figures of Merit ………………………………………..154 5.4.2 Detection of AHAs in ED Experiments ………………………..157 5.4.3 Complimentary Detection of AAs in ED Experiments ………..164 5.4.4 Comparison to the Murchison Meteorite ………………………..165 5.5 Conclusions ………………………………………………………..166 vii 5.6 Acknowledgements ………………………………………………..167 5.7 References ………………………………………………………..167 CHAPTER 6: ANALYSIS OF DIDEPSIPEPTIDES IN PREBIOTIC MIXTURES VIA TIME-NESTED ANALYTICAL TECHNIQUES ………………………………..172 6.1 Abstract ………………………………………………………………..172 6.2 Introduction ………………………………………………………..173 6.3 Materials and Methods ………………………………………………..176 6.3.1 Chemicals and Reagents ………………………………………..176 6.3.2 Environmental Cycling Experiments with Model Mixtures ………..178 6.3.3 ED Experiment #1 ………………………………………………..178 6.3.4 ED Experiment #2 ………………………………………………..178 6.3.5 ED Experiment #3 ………………………………………………..179 6.3.6 ED Experiment #4 ………………………………………………..179 6.3.7 Environmental Cycling of ED Samples ………………………..179 6.3.8 Acid-Vapor Hydrolysis Experiments ………………………………..180 6.3.9 Mobility Survey via UPLC-Q-TWIMS-TOF-MS ………………..181 6.4 Results and Discussion ………………………………………………..184 6.4.1 Environmental Cycling Experiments with Model Mixtures ………..184 6.4.2 Environmental Cycling of ED Mixtures ………………………..200 6.4.3 Acid-Vapor Hydrolysis Experiments ……..…………………………209 6.5 Conclusions ………………………………………………………..213 6.6 Acknowledgements ………………………………………………..215 6.7 References ………………………………………………………..215 CHAPTER 7: CONCLUSIONS ………………………………………………..220