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Properties and Derivatives of 5-Hydroxyuridine a Component of Ribonucleic Acid

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Properties and Derivatives of 5-Hydroxyuridine a Component of Ribonucleic Acid AN ABSTRACT OF THE THESIS OF 0000SIDNFY goq-5X-P.1101-1-eliA .0.o forthe 00.01.11-soPs... in oMicrobiolocry December 19, 19690 PROPER TIES AND DERIVATIVES OF 5-1 IYDR OX YURIDINE A COMPONENT OF RIBONUCLEIC ACID 0 0 0 0 0 0 00 0 0 0000 n0 0 0 0 0 p 0 o000 u0 0 0 0000 00n00 0 r,0 0 000 0 0 0 0 0 0 0 D 0 0 0 0 0 Redacted for privacy Abstract approved: 0 0 000 0 0 0 0 0 0 0 0 Dr. Adam W. Lis This investigation involveda physico-chemical character- ization of 5-hydroxyuridineiB), its methylated derivatives, and related compounds.Dissociation constants, relation of optical rotation ( cotton effects ) to molecular conformation,the ability to bind divalent cations, and-transformations in alkaline ndia and toward various nucleophilcswere studied°It was shown that iB becomes unstable .toward nucleophilicattack subsequent to disSociation of the 5-hydroxyl proton.It was proven that the instability results from nucleophilic attack rather thanmolecular transformation as a consequence of proton removal.Alteration products formed include le(B-D-ribofuranosyl)-2-oxo-4-imid- a,..oline-1-( Irho\lii;Rhl l.1:4 lour other deriv:ilive!i Nyhich hay,' [a1. (1(!:(' hl))(' i;ii 1)11 Isotope dilution analysis for iB inyeast RNA revealed that the compound is unstable towardroutine RNA fractionation pro- cedures and cannot be isolated intact.The label in the analyses was associated with a spectrally unique complexwhich when hydrol- yzed yielded cytidine anda labeled derivative which appeared to be dihydrouracil bymass spectroscopic and paper chromatographic analysis.Calculations of isotope dilution, basedon a number of assumptions, revealed that fourper cent of the nucleosides pres- ent in ribosomal RNAare iB. It was concluded that themono anion of iB firmly bindsMg2+ and that the lattermay stabilize it to nucleophilic attack. iB can be modified to dihydro-compoundsand imidazoles spectrally resemb- ling dihydrouracil,as well as other derivatives, and exhibits mul- tiple modes of reactivity underRNA isolation, fractionation, and chemical hydrolytic conditions.Quantitative isolation of intact iB from biological materialwould not be possible above neutral pH or in the presence ofa reactive nucleophile.There is a far greater potential for possible loss of iBduring isolation than for its formation.Thus iB should be considereda natural com- ponent of yeast ribosomal RNA. Properties and Derivatives of 5-Hydroxyuridine A Component of Ribonucleic Acid by Sidney Joseph Hayes III A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 1971 APPROVED: Redacted for privacy Associate Professor of Microbiology, Research Director Redacted for privacy Professor of Microbiology in charge of major Redacted for privacy Chairman of Department of Microbiology Redacted for privacy Dean of Graduate School Date thesis is presented I Typed by Mrs. Aase Austad for Sidney J.Hayes ill A CKNOW LE DGEMENT This research was partially funded through a fellowship from the National Institutes of Health and was carried out at the University of Oregon Medical School (UOMS), Portland.I am especially indebted to Dr. K. S. Pitcher, Oregon State University (OSU) for coordin- ating this project and for his example and patient guidance through my various phases of graduate school; to the Oregon Graduate Center (OGC), Beaverton for use of their NMR, and mass spectrometers and for other innumerable privi- leges; to Dr. H. Kittinger, Oregon Regional Primate Center, Beaverton for several IR spectra and to Dr. T. King, OSU for the O.RD spectra; to Dr. D. Reed, OSU for advice on radiotracer methodology and, to Roman Bijan, UOMS who served as a tireless and invaluable sound- ing-board for my brainstorming; to Dr. Doyle Davos-, OGC for help, suggestions, and uncountable hours of concerned discussion and involvement; and to my sponsor Dr. Adam W. Lis, UOMS for support, direc- tion, and the independence to explore, relate,and build without intimi- dation or resistance, and for hi.s research enthusiasm. TABLE OF CONTENTS Page INTRODUCTION 1 5-Hydroxyuridine as a Potential Dynamic Cellular Metabolite 3 Reported Antimetabolic Activity of 5-Hydroxy- uridine Characteristics of 5-Hydroxyuridine asan 8 Antimetabolite Metabolism of 5-Hydroxyuridine 9 Comparison of Metabolism of 5-Hydroxyuridine- 11 2-14C and Uridine- 2-14C 15 Effect of Poly-5-hydroxyuridylic Acidon Amino Acid Incorporation Inhibition of Uridine 5'-triphosphate Incorpora- 16 tion into RNA by 5-Hydroxyuridine 5'-triphos- phate Consideration of the Chemistry of 5- Hydroxy- 17 pyrimidine s 19 Chemistry of Isobarbituric Acid 20 Synthesis and Stability of Isobarbituric Acidand Its N-1 Substituted Derivatives 23 EXPERIMENTAL METHODS AND MATERIALS 27 Paper Chromatography 27 Preparation of paper Photography of chromatograms 27 Solvents used in paper chromatography 27 Elution of samples from chromatograms 28 Electrophoresis 28 Reagents 29 Column Chromatography - Fractionation 29 Technique s 32 Chromatography on Dowex 32 Celite partition chromatography 33 (1. )General 34 (2.)Solvent systems 34 (3.)Hydrolysis 34 (4.) Primary fractionation 35 (5.)Subfractionation columns Ultraviolet Absorption Measurements 36 Spectral Titrations 37 Calculation of pKa 37 Ultraviolet Absorption Measurements for 38 Chemical Stability Tests Solutions and Incubation Procedure for 39 Chemical Stability Tests Cultures, Culture Medium and Incubation 40 Procedure 40 Page Extraction of Ribonucleic Acid fromYeast Methods of Analysis of Radioactivity 41 Paper chromatograms and electro- 43 phoretograms Column fractions 43 Synthesis of Labeled 5-Hydroxyuridine 44 Qualitative Chemical Spray Tests 45 Sugar test 46 Pyrimidine reduction test 46 Ninhydrin test 47 Melting points 47 Methods of Spectrometric OrganicAnalysis 48 Mass spectroscopy 48 Infrared spectroscopy 48 Nuclear magnetic resonance spectro- 49 scopy 50 RESULTS 51 Characterization of Absorbance andIoni- zation Characteristicsof 5-Hydroxy- uridine Preparation, Absorbance, and Character- 51 istics of Methylated Derivatives of 5- Hydroxyuridine Characterization of Absorbance and Ioni- 53 zation Characteristics of Isobarbituric Acid (5- Hydroxyuracil) Characterization of Absorbance and Ion- 59 ization Characteristics of 5-Hydroxy- 2' deoxy- uridine Optical Rotatory Dispersion Curves for 60 5- Hydroxyuridine Binding of Magnesium Cations with 5- 62 Elydroxyuridine Analysis of Ribonucleic Acids 64 Yeast Nucleotide Pool Analysis 65 Column Fractionation Procedures and 66 Results Dowex columns 68 Celite columns 68 Celite Fractionation of Commercial 68 Uridine Fractionation of Yeast Ribosomal RNA 70 Methods of Examination of the CeliteColumn 71 Labeled Water-Wash Peak Paper Chromatographic Analysis ofthe 72 Labeled Wash Concentrate 72 Page Analysis of the LabeledWash Concen- trate from PartitionChromatography Purification of LabeledConstituents 74 from the Labeled WashConcentrate Acid Hydrolysis ofCompound A 75 Electrophoresis of Compound 77 A 78 Paper Chromatographyof Compound A Cleavage Products of 79 Compound A 80 Alteration of 5-Hydroxyuridineand the Formation of CompoundsA and B Stability of 5-Hydroxyuridine 90 A generalsurvey of the stability 94 of 5-Hydroxyuridineto common laboratory reagents The effect of heat,pH, and cations 94 on the stability of 5-hydroxyuridine Stability of 5-Hydroxyuridinein Acid 94 Stability of 5-Hydroxyuridinein Alkali 96 Stability of 5-Hydroxyuridinein NH3 and 97 Amine s Stability of 5-hydroxyuridinein NH3 99 Stability of 5-hydroxyuridine 99 in piperidine 100 Synthesis of CompoundH28 Synthesis of CompoundP 101 Stability of 5-Hydroxyuridinein Hydroxy- 10Z lamine Nature of ChemicalReactivity of 5-Hydroxy- 104 uridine in Alkali Reaction of Ammoniaand Cytidine with 107 Derivatives of 5-Hydroxyuridineand with Related Compounds Stability of MethylatedDerivatives of 108 5-Hydroxyuridine in Alkali Comparison of the AlkalineHydrolysis 112 Products of Uridine,5-Hydroxyuridine and Dihydrouridine Chemical AlterationProducts of 5- 116 Hydroxyuridine 120 DISCUSSION 126 5-Hydroxyuridineas an Antimetabolite: A Reevaluation 126 The Binding of5-Hydroxyuridine and Magnesium Cations 132 Mutagenisity and the IonicCharacter of 5-Hydroxyuridine A Hypothesison the Bathochromic Shift 133 of Solutions of Ionized 5-Hydroxyuridine 137 Page Modifications of 5-HydroxyuridineUnder Standard RNA Chemical Hydrolyticand Fractionation Procedures 143 Studies on the Stability and Modification of 5-Hydroxyuridine 156 Products Formed in the Reaction of Alkali with 5- Hydroxyuridine 160 Consideration of the Effect of Alkalion 5- Hydroxyuridine 162 Nature of the Chemical Reactivity of 5- Hydroxyuridine in Alkali 165 5-Hydroxyuridine as a Component of RNA 174 SUMMARY 1 81 BIBLIOGRAPHY 2 84 LIST OF FIGURES Page 1. Absorbance spectra of 5-hydroxyuridine. 1 86 2. Absorbance spectra of 5-hydroxyuridine pH 2. 975- 9. 500. 1 87 3,Absorbance spectra of 5-hydroxyuridine pH 9.500- 11. 000. 188 4, Absorbance spectra of 5-hydroxyuridine pH 10.750- 12. 7 50. 1 89 5. Chromatogram showing production of methylated derivatives a through e with time. -OHUR equals 5-hydroxyuridine. 190 6.Absorbance spectra of "methylated" derivativea, conformationally altered 5-hydroxyuridine. 191 7.Absorbance spectra of methylated derivative b, 5-methoxyuridine. 192 8. Absorbance spectra of methylated derivativec, 3- methyl -5- hydroxyuridine. 193 9.Absorbance spectra of methylated derivative d, 3-methyl- 5- met hoxyuridine 194 10. Absorbance spectra of methylated derivativee, it 2' - me thoxy- 3- methyl-
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