BIOORGANIC MOLECULES IN THE COSMOS AND THE ORIGIN OF DARWINIAN MOLECULAR SYSTEMS By ALONSO RICARDO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004 This work is dedicated to my family. ACKNOWLEDGMENTS I will like to thank Dr. Steven Benner for the research opportunities and guidance that he has offered me. I also thank Dr. Matthew Carrigan for his help, friendship and for being an excellent lab partner. I am grateful to the people in the mass spectrometry laboratory at the University of Florida and to Dr. Maurice Swanson for providing laboratory space and collaboration . I also thank Fabianne for her unconditional love and support. Finally, I am forever grateful to my family for their constant support, care, and consideration that helped me to made this work possible. iii TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iii LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix ABSTRACT..................................................................................................................... xiii CHAPTER 1 ORIGINS....................................................................................................................1 Background and Significance.....................................................................................1 Prebiotic Chemistry.............................................................................................1 Sugars in the Prebiotic Environment ..................................................................3 RNA World and the Problem of Ribose Accumulation......................................6 The Role of Minerals on Ribose Formation and Accumulation .........................8 2 PREBIOTIC SYNTHESIS OF SUGARS..................................................................9 Introduction ................................................................................................................9 Boron Chemistry and Complexation Mechanism in Polyols............................13 pH dependence on the stability of boric acid esters and borate esters.......16 Differential coordination of borate to polyols. ..........................................17 Interaction of boron with carbohydrates : aldoses and ketoses..................19 Stabilization of pentoses towards decomposition in borate.......................23 Materials and Methods.............................................................................................23 Chemicals..........................................................................................................23 Enzymes ............................................................................................................24 Analytic Instrumentation...................................................................................24 Ultraviolet analysis (UV)...........................................................................24 Gas chromatography (GC).........................................................................24 Mass spectrometry (MS)............................................................................24 NMR Spectroscopy....................................................................................25 Synthetic Preparations.......................................................................................25 Synthesis of colemanite .............................................................................25 Synthesis of deuterated Colemanite...........................................................25 Synthesis of pentoses in the presence of colemanite .................................25 iv Synthesis of pentoses in the presence of calcium hydroxide.....................26 Derivatization of pentoses for gas chromatography analysis ....................26 Sugars Degradation Experiments......................................................................27 Sugar decomposition in the presence of calcium deuteroxide...................27 Sugar decomposition in calcium deuteroxide and colemanite...................27 Enzymatic Assays .............................................................................................28 Ribitol dehydrogenase assay......................................................................28 Cysteine-carbazole test ..............................................................................29 DIOS Analysis ..................................................................................................29 Preparation of PSi surfaces ........................................................................29 Competition Experiments .................................................................................30 1, 4-Anhydroerythritol (AET) vs Pentoses ................................................30 13C-Ribose vs Pentoses ..............................................................................30 Results ......................................................................................................................30 Synthetic Preparations.......................................................................................30 Synthesis of pentoses in the presence of colemanite .................................30 Synthesis of pentoses in the presence of calcium hydroxide.....................32 Sugar Degradation Experiments .......................................................................33 Sugar decomposition in the presence of Calcium Deuteroxide.................33 Sugar decomposition in the presence of Colemanite.................................34 Enzymatic Assays .............................................................................................35 DIOS Analysis: Competition Experiments. ......................................................36 1, 4-Anhydroerythritol (AET) vs Pentoses ................................................36 13C-Ribose vs Pentoses ..............................................................................36 Discussion ................................................................................................................39 3 CATALYSIS AND THE RNA WORLD ................................................................43 Introduction ..............................................................................................................43 Materials and Methods.............................................................................................45 Preparation of Precursor DNAzymes via PCR (Maniatis et al, 1982)..............45 Preparation of single-stranded DNAzymes.......................................................47 5’-End Labeling of DNA ..................................................................................48 DNAzyme Kinetic Assays ................................................................................49 Cloning and Sequencing DNAzymes................................................................49 In vitro Selection...............................................................................................50 Results ......................................................................................................................54 In vitro Selection...............................................................................................54 Cleavage of 614 does not go Completion .........................................................56 Inhibition by Incompletely Removed Complementary Strand .........................57 An approach to Chemical Equilibrium does not Account for the Plateau ........59 Testing if the Cleavage Products are Acting as Catalysts or Inhibitors............60 Improperly Folded ribose-614 Accounts for Part of the Plateau ......................61 Mutations Introduced into 614 during Cloning and Sequencing ......................63 Ribose-614 catalysis is not Mg++-dependent ...................................................66 Ribose-614 cleaves in trans ..............................................................................66 v Various ribose-containing substrates are cleaved by deoxyribose-614.............69 Competition Studies of Ribose-614 Cleavage ..................................................72 Saturation kinetics in trans cleavage by deoxyribose-614................................73 Compound deoxyribose-614 cleaves with multiple-turnovers..........................74 Catalytic power in trans is unaffected by annealing protocol...........................74 The commitment step for deoxyribose-614 cleavage........................................75 Dependence on temperature of deoxyribose-614 cleavage...............................77 Predictions of the energetically favored structure.............................................79 Discussion ................................................................................................................81 4 DETECTING ORGANIC MOLECULES ON MARS ............................................90 Introduction ..............................................................................................................90 Oxidation of Alkanes Under Martian Conditions .............................................93 Oxidation of Alkylbenzenes Under Martian Conditions