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On the Origins of Life

Permanent Link: http://ufdc.ufl.edu/UFE0017564/00001

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Title: On the Origins of Life Prebiotic Carbohydrates, Primitive Catalytic Cycles and Engineering the Genetic Lexicon
Physical Description: 1 online resource (156 p.)
Language: english
Creator: Illangkoon, Heshan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: astrobiology, carbohydrate, dna, formaldehyde, glycolaldehyde, life, nucleosedes, of, origins, primordial, ribose, rna, soup, threose
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: ON THE ORIGINS OF LIFE: PREBIOTIC CARBOHYDRATES, PRIMITIVE CATALYTIC CYCLES & ENGINEERING THE GENETIC LEXICON By Heshan Indika Illangkoon Spring 2010 Chair: Steven A. Benner Major: Biochemistry Here we report experiments that establish xylulose and threose as stable products from formaldehyde and lower carbohydrates, such as glycolaldehyde and dihydroxyacetone, in the presence of borate. Xylulose is one of two diastereomeric five carbon carbohydrates known as 2-pentuloses, and threose is one of two diastereomeric four carbon tetroses. Arguments are presented that formaldehyde, glycolaldehyde and dihydroxyacetone are all likely to have been available on early Earth and therefore may have supported processes that created carbohydrates that could have been part of the first genetic material. Borate, weathered from igneous rock, is likely to have been available on early Earth. Borate may have also been available, as evaporite minerals, which result from evaporation of superfial bodies of water (e.g. colemanite, ulexite, and kernite); all of these minerals are found today in, for example, Death Valley. 1,2,4,5-Tetrahydroxy-3-pentanone was synthesized and shown to be an intermediate in the formation of 2-pentuloses, including xylulose and ribulose. Rate constants for the enolization of glycolaldehyde, glyceraldehyde, erythrulose, erythrose, and dihydroxyacetone, which are precursors, intermediates, and potential products in this putative prebiotic synthesis of carbohydrates, were estimated by NMR in the presence and absence of borate. Borate had only a modest impact on those rate constants for enolization of linear carbohydrates that presented 1,2-diol units, but had a dramatic impact on the rate constants for enolization of carbohydrates that could present the 1,2-diol in a cyclic form. In a separate line of work, experiments are directed towards the synthesis of a modified nucleoside heterocycle with an amine linker at the C5 position of cytidine. Molecular biology using the modified cytidine resulted in a successful PCR amplification.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Heshan Illangkoon.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Benner, Steven A.

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0017564:00001

Permanent Link: http://ufdc.ufl.edu/UFE0017564/00001

Material Information

Title: On the Origins of Life Prebiotic Carbohydrates, Primitive Catalytic Cycles and Engineering the Genetic Lexicon
Physical Description: 1 online resource (156 p.)
Language: english
Creator: Illangkoon, Heshan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: astrobiology, carbohydrate, dna, formaldehyde, glycolaldehyde, life, nucleosedes, of, origins, primordial, ribose, rna, soup, threose
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: ON THE ORIGINS OF LIFE: PREBIOTIC CARBOHYDRATES, PRIMITIVE CATALYTIC CYCLES & ENGINEERING THE GENETIC LEXICON By Heshan Indika Illangkoon Spring 2010 Chair: Steven A. Benner Major: Biochemistry Here we report experiments that establish xylulose and threose as stable products from formaldehyde and lower carbohydrates, such as glycolaldehyde and dihydroxyacetone, in the presence of borate. Xylulose is one of two diastereomeric five carbon carbohydrates known as 2-pentuloses, and threose is one of two diastereomeric four carbon tetroses. Arguments are presented that formaldehyde, glycolaldehyde and dihydroxyacetone are all likely to have been available on early Earth and therefore may have supported processes that created carbohydrates that could have been part of the first genetic material. Borate, weathered from igneous rock, is likely to have been available on early Earth. Borate may have also been available, as evaporite minerals, which result from evaporation of superfial bodies of water (e.g. colemanite, ulexite, and kernite); all of these minerals are found today in, for example, Death Valley. 1,2,4,5-Tetrahydroxy-3-pentanone was synthesized and shown to be an intermediate in the formation of 2-pentuloses, including xylulose and ribulose. Rate constants for the enolization of glycolaldehyde, glyceraldehyde, erythrulose, erythrose, and dihydroxyacetone, which are precursors, intermediates, and potential products in this putative prebiotic synthesis of carbohydrates, were estimated by NMR in the presence and absence of borate. Borate had only a modest impact on those rate constants for enolization of linear carbohydrates that presented 1,2-diol units, but had a dramatic impact on the rate constants for enolization of carbohydrates that could present the 1,2-diol in a cyclic form. In a separate line of work, experiments are directed towards the synthesis of a modified nucleoside heterocycle with an amine linker at the C5 position of cytidine. Molecular biology using the modified cytidine resulted in a successful PCR amplification.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Heshan Illangkoon.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Benner, Steven A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0017564:00001


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1 ON THE ORIGINS OF LIFE: THE PREB IOTIC SYNTHESIS OF CARBOHYDRATES, PRIMITIVE CATALYTIC CYCLES & E NGINEERING THE GENETIC LEXICON By HESHAN INDIKA ILLANGKOON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Heshan Indika Illangkoon

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3 To be is to do -Socrates To do is to be. -Sartre Do be do be do. -Sinatra To my dear parents Herath and Nandani I never had problems with my fellow scientists. Scientists are a friendly, atheistic, hard-working, beer-drinking lot w hose minds are preoccupied with sex, chess and baseball when they ar e not preoccupied with science. -Piscine Molitor Patel in Life of Pi by Yann Martel

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4 ACKNOWLEDGMENTS I thank my Advisor, Dr. Steven A. Benner, for the trust he has shown me throughout the years. I also thank the members of my comm ittee, Dr. Tom Lyons, Dr Nigel Richards, Dr. Aaron Aponick and Dr. David Hahn for their suppor t, input, helpful disc ussions and above all, patience. I also thank my friends and mentors Photon Rao, Abhijit Roychowdhury, Harch Ooi, Sangita Ghosh, Alonso Ricardo, Matt Carrigan, A. Michael Sismour, Cy nthia Hendrickson and Theodore Martinot who helped me through th e years. Photon, Abhijit and Theo were instrumental in teaching me the techniques a nd the art of organic ch emistry, while Cynthia taught me everything I needed to know about molecular biology. I am deeply indebted to them all. I also thank my parents, Herath and Na ndani Illangkoon, my aunt and uncle, Hema and Chandra Wijeratne, and finally my close frie nds John Brennan, Amanda Rowe, Cara Lubner, Kathryn Ranhorn, Jonny Gerstsen, Christos Lampropoulos, Robert o Laos, Nellie Eshleman, Ahu Demir, Sarah Luciano, Tammy Low and Joanna Fr ied, for their endless support and love. Last but not least I would like to thank Romaine Hugh es. Without her, our lives would have simply been chaos.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................7LIST OF FIGURES .........................................................................................................................8ABSTRACT ...................................................................................................................... .............11CHAPTER 1 PREBIOTIC CHEMISTRY .................................................................................................... 13Introduction .................................................................................................................. ...........13Early Earth ..............................................................................................................................17Prebiotic Organic Compounds ................................................................................................18Primordial Soup & The RNA World ...................................................................................... 182 THE PREBIOTIC SYNTHESIS OF SUGARS AND PRIMITIVE CATALYTIC CYCLES ........................................................................................................................ .........22The Geochemical Synthesis of Threose ................................................................................. 33Kinetics ...................................................................................................................... .............36Catalytic Cycles ......................................................................................................................43Discussion .................................................................................................................... ...........48Experimental .................................................................................................................. .........50Buffer and Stock Solution Preparations ..........................................................................50Kinetics ...................................................................................................................... ......533 2-DEOXYCYTIDINES CARRYING AM INO AND T HIOL FUNCTIONALITY: SYNTHESIS AND INCORPORATION BY VENT (EXO-) POLYMERASE .................... 54Experimental .................................................................................................................. .........63General Experimental: ..................................................................................................... 63General Procedure for the S ynthesis of the Linker: ........................................................ 64General Procedure for the 5-Tritylation Reaction: ......................................................... 65General Procedure for Sonogashira Coupling: ................................................................ 655'-O-4, 4'-dimethoxytrityl-5-(3''-Trifluor oacetamidobutynyl)-2'-deoxycytidine (10): .... 66General Procedure for Acetylation Reaction: .................................................................. 673'-O-4-N-Diacetyl-5'-O-4, 4'-dimethoxytrity l-5-(3''-Trifluoroacetamidobutynyl)2'deoxycytidine (13): ......................................................................................................683'-O-Acetyl-5-(3''-Trifluoroacetami dobutynyl)-5'-O-4,4'-dimethoxytrityl-2'deoxycytidine (15): ......................................................................................................69General Procedures for De-protection ............................................................................. 69Method A: ........................................................................................................................69

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6 3'-O-Acetyl-5-(1-butynyl)-2'-deoxycytidin e tert-B utyl Disulfide (16): .......................... 693'-O-Acetyl-5-(3''-Trifluoroacetami dobutynyl)-2'-deoxycytidine (17): .......................... 70General Procedure for triphosphate synthesis: ................................................................705-(3''-Trifluoroacetamidobutynyl)-2'-de oxycytidine-5'-triphosphate (19): ..................... 71General Experimental: Biochemical: .............................................................................. 71Acknowledgments ............................................................................................................... ...724 FUTURE WORK ................................................................................................................... .73APPENDIX NMR SPECTRA OF FURTHER EXPERIMENTS AND CARBOHYDRATES ............................................................................................................. 77LIST OF REFERENCES .............................................................................................................151BIOGRAPHICAL SKETCH .......................................................................................................156

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7 LIST OF TABLES Table page 2-1 Enolization rates of dihydroxyacetone, glyceralde hyde and erythrulose at 25 C with formaldehyde concentration of 167 mM. ........................................................................... 36

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8 LIST OF FIGURES Figure page 1-1 Pathways and structures pr oposed for HCN polym erization. ............................................ 141-2 The Whler synthesis of urea. ........................................................................................... 141-3 The Cannizaro reaction. .....................................................................................................151-4 Formaldehyde concentration dependence in the progress of the formose reaction.10,11 ....191-5 The affinity of borate to various carbohydrates, ribose is highest among the aldopentoses10, 11, 29. ........................................................................................................... 201-6 Crystal structure of borate co mplexed with 1,4-anhydroerythritol.30 ................................202-1 13C Spectrum of synthetic 1,2,4,5-tetrahydr oxy-3-pentanone standardized to methanol at 49.5 ppm. Spectrum file: 1,2,3,4-pentan-3-one.pdf ....................................... 232-2 Correlation experiment: Addition of pure 1,2,4,5-tetrahydroxy-3-pentanone to the mixture holding the reaction of erythrulose and formaldehyde. ........................................ 232-3 Reaction of dihydroxya cetone (DHA) with H13CHO without calcium. ............................ 252-4 Reaction of erythrulose with H13CHO. .............................................................................. 252-5 The influence of borate on the enolization of erythrulose and regioselectivity of reactions with HCHO.. .......................................................................................................262-6 13C NMR spectrum of arabinose (30 mg) in CBA buffer (1 mL), note the complexity. Spectrum file: arabinose_cba_meohref.pdf ....................................................................... 282-7 13C NMR spectrum of lyxose (30 mg) in CBA buffer (1 mL), note the complexity. Spectrum file: lyxose_cba_meohref.pdf ............................................................................282-8 13C NMR spectrum of xylose (30 mg) in CBA buffer (1 mL), note the complexity. Spectrum file: xylose_cba_meohref.pdf ............................................................................292-9 13C NMR spectrum of ribose (30 mg) in CBA buffer (1 mL), note the simplicity indicating a singular cyclic form, assigne d by NMR as the alpha furanose. Spectrum file: ribose_cba_meohref.pdf ............................................................................................. 292-10 13C NMR spectrum of ribulose (30 mg) in CBA buffer (1 mL), note the complexity. Spectrum file: ribulose_cba_meohref.pdf .......................................................................... 302-11 13C NMR spectrum of xylulose (30 mg) in CBA buffer (1 mL). Spectrum file: xylulose_cba_meohref.pdf ................................................................................................. 30

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9 2-12 Pathways of possible aldol/retro -aldol chem istry of carbohydrates .................................. 312-13 Cyclic forms of ribose (in its "alpha" epimer, where the OH hydroxyl group at C-1 is down; the "D" enantiomer is shown). ............................................................................342-14 NMR analysis of the reaction of glycolal dehyde in CBA buffer, forming threose, in the absence of HCHO. ....................................................................................................... 352-15 A) Phosphate backbone and heterocyclic base locations in a threose nucleic acid (TNA) model. B) Coordination of borate to the alpha anomer of threose. ........................ 362-16 The borate-compatible cycle for conversion of formaldehyde into pentoses, pentuloses and threose. Enolization ra tes of glyceraldehyde dihydroxyacetone and erythrulose in the pr esence of borate. ................................................................................382-17 Reaction of dihydroxyacetone (0.317 M) with formaldehyde (0.167 M) in the presence of borate (CBA buffer with a meas ured pH of 10.4 at 25 C) plotted against time. ......................................................................................................................... ..........402-18 Reaction of dihydroxyacetone (0.317 M) with formaldehyde (0.167 M) in the absence of borate (carbonate buffer with a measured pH of 10.4 at 25 C) plotted against time. ................................................................................................................. ......402-19 Reaction of erythrulose (0.317 M) with formaldehyde ( 0.167 M) in the presence of borate (CBA buffer with a measured pH of 10.4 at 25 C) plotted against time. .............. 412-20 Reaction of erythrulose (0.317 M) with formaldehyde ( 0.167 M) in the absence of borate (carbonate buffer with a measured pH of 10.4 at 25 C) plotted against time. ...... 412-21 Reaction of glyceraldeh yde (0.317 M) with formaldehyde (0.167 M) in the presence of borate (CBA buffer with a measured pH of 10.4 at 25 C) plotted against time. ......... 422-22 Reaction of glyceraldeh ydes (0.317 M) with formaldehyde (0.167 M) in the absence of borate (carbonate buffer with a measur ed pH of 10.4 at 25 C) plotted against time. ......................................................................................................................... ..........422-23 A segment of the abiotic metabolis m, the compounds in green are known prebiotically in either meteorites, via electrical discharge, photochemistry, or mineral-based processes on Earth, or in the interste llar nebula.. ....................................... 432-24 Synthesis of erythro branched pentose .............................................................................. 463-1 The Versant bDNA diagnostic test marketed by Bayer Diagnostics.46 .............................543-2 Synthesis of the trifluoroacetyl protected 1-aminobut-3-yne linker. ................................. 563-3a Synthesis of 3'-O-Acetyl-5'-O-(4,4'-D imethoxytrityl)-5-(1-butynyl)-2'-deoxycytidine tert-butyl disulfide ( 14). ..................................................................................................... 57

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10 3-3b Synthesis of 3'-O-Acetyl-5-(3''Trifluoroacetam idobutynyl)-5'-O-4,4'dimethoxytrityl-2'-deoxycytidine ( 15 ). .............................................................................. 573-4 Synthesis of 3'-O-Acetyl-5-(3''-Trif luoroacetamidobutynyl)-2'-deoxycytidine ( 17). ........593-5 Synthesis of 5-(3''-tert-butyl disulfid e 1-butynyl)-2'-deoxycytidine-5'-triphosphate ( 18) and 5-(3''-Trifluoroacetamidobutynyl)-2'-deoxycytidine-5'-triphosphate ( 19 ). .........603-6 Primer extension experiment using amino-functionalized 2-deoxycytidine triphosphate 19.. .................................................................................................................613-7 PCR amplification with 19 replacing dCTP. Agarose gel (2%) was used and stained with ethidium bromide.. ..................................................................................................... 624-1 The role of molybdenum in carbohydrate inte rconversion. ............................................... 734-2 A sample of DL-glyceraldehyde (A ldrich, 38 mg) in CBA buffer (0.30 mL) incubated for 5 minutes at 25 C. ....................................................................................... 744-3 The ketohexoses (psicose, fructose, sor bose & tagatose) are presumably formed by the reaction of either di hydroxyacetone or glyceral dehyde with themselves. ................... 754-4 A sample of dihydroxyacetone (Aldrich, 30 mg) in CBA buffer (0.50 mL) incubated for 3 hours at 25 C. ........................................................................................................... 75

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ON THE ORIGINS OF LIFE: PREBIOTIC CARBOHYDRATES, PRIMITIVE CATALYTIC CYCLES & ENGINEERING THE GENETIC LEXICON By Heshan Indika Illangkoon May 2010 Chair: Steven A. Benner Major: Chemistry Here we report experiments that establish xylulose and threose as stable products from formaldehyde and lower carbohydrates, such as gl ycolaldehyde and dihydroxyacetone, in the presence of borate. Xylulose is one of two di astereomeric five carbon carbohydrates known as 2pentuloses, and threose is one of two diastereomeric four carbon te troses. Arguments are presented that formaldehyde, glycolaldehyde and dihydroxyacetone are all likely to have been available on early Earth and therefore may have supported processes that created carbohydrates that could have been part of the first genetic material. Borate, weathered from igneous rock, is likely to have been available on early Earth. Borate may have also been available, as evaporite minerals, which result from evaporation of superf ial bodies of water (e.g. colemanite, ulexite, and kernite); all of these minerals are f ound today in, for example, Death Valley. 1,2,4,5Tetrahydroxy-3-pentanone was synthesized and shown to be an intermediate in the formation of 2-pentuloses, including xylulo se and ribulose. Rate consta nts for the enolization of glycolaldehyde, glyceraldehyde erythrulose, erythrose, and dihydroxyacetone, which are precursors, intermediates, and potential products in this putative prebiotic synthesis of carbohydrates, were estimated by NMR in the presence and absence of borate. Borate had only a modest impact on those rate c onstants for enolization of linear carbohydrates that presented 1,2-

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12 diol units, but had a dramatic impact on the rate constants for enolization of carbohydrates that could present the 1,2-diol in a cyclic form. In a separate line of work, experiments are directed towards the synthesis of a modified nucleoside heterocycle with an amine linker at the C5 position of cytidine. Molecular biology using the modified cytidine resulted in a successful PCR amplification.

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13 CHAPTER 1 PREBIOTIC CHEMISTRY Introduction All life, as we know it, contains genetic materi al in the form of nucleic acids, which are DNA and RNA. The field that considers the origin of life on Earth, therefore, must explain how nucleic acids arose at some point in the historical past, either on Earth or elsewhere. The field of astrobiology studies the origins, evolution and distribution of lif e on earth and in the universe. The study of the origins of life started long ago, though certain discoveries may not have been evident as important at the time they were made. One of the first reports is the polymerization of HCN, reported by Proust in 18061, 2. Further analysis of this polymer showed the presence of adenine, one of four RNA heterocycles. A recent investigation by Minard3 found the spontaneous polymerization products of HCN which gave a bl ack polyimine chain (Figure 11). This shows the formation of polymers from a common prebiotic precursor, and may form adenine. Soon after Prousts reactions came the eye-opening synthesis of urea by Whler4. A pioneer of organic chemistry, Wohler was attemp ting to form ammonium cyanate, and instead formed urea (Figure 1-2). Prior to this event, organic compounds were thought to have been created only by living organisms. This discov ery brought about a paradigm shift where once vitalism ruled. Whler demonstrated that organic molecules could be formed from inorganic materials. A little known fact, Whler also isolated many elements including aluminium, yttrium, beryllium, and titanium. Streckers synthesis of alanine from HCN followed in 18505, 6. Alanine was later discovered to be part of natural proteins. At this time, the fiel d began to make real progress

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14 Figure 1-1. Pathways and stru ctures proposed for HCN polym erization. A sample of HCN polymer may possess any or all of these st ructures including hybrids (multimers).7 Figure 1-2. The Whler s ynthesis of urea.

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15 towards a chemical understanding of life. In 1861, Butlerov reported his now famous formose experiment, where formaldehyde (HCHO) in hot (70 C or higher) solutions of calcium hydroxide (at pH 12.5) formed a sweet sugary substance8. In an incompletely understood first step, two molecules of HCHO are presumded to join to form a molecule of glycolaldehyde. Then under a proposal by Breslow, this glycolaldehyde then initiates a se ries of reaction cycles that, over time, fix more HCHO to give higher carbohydrates9. This is followed by a long induction period during which HCHO is consumed slowly to generate C4, C5, C6, C7, and some C8 and C9 carbohydrates. Then suddenly, the remaining HC HO is rapidly consumed, and the mixture begins to yellow10, 11. A side reaction in this experiment is the disproportionation of HCHO to form methanol and formic acid in a process no w known as the Cannizaro reaction (Figure 1-3). Figure 1-3. The Cannizaro reaction. In 1913, Lb formed glycine after passing an electric discharge through a mixture of carbon monoxide, ammonia and water vapor12. In the same year, Baudisch prepared uracil, a pyrimidine heterocycle found in RNA, by treating urea and malic aci d with fuming sulfuric acid in a process less likely to be c onsidered prebiotic on an early Ea rth but not entirely discounted. Modern theories on the origins of life came independently from Oparin in 1924 and Haldane in 192913. Although Haldanes work was published before Oparins Russian manuscript was translated into English, both suggested that a reducing primitive atmosphere coupled with cosmic rays, ultra-violet light and lightning could synthesize a variety of organic compounds. Haldane coined the term prebiotic soup in refe rence to this mixture. This process was shown

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16 experimentally in 1953 with Stanley Millers famous experiment combining methane, carbon dioxide and electrical discharg e simulating lighting. Millers experiment yielded amino acids, then widely accepted to be key building blocks for life14. There still remains a controversy among a small contingent of origins resear chers who question the novelty of Millers experiment, as Lb had conducted similar experiments years before Millers birth. Also in 1953, Watson and Crick proposed a structure for DNA15-17 and Frederick Sanger sequenced the amino acids of the protein insulin. Perhaps 1953 should be considered the dawn of the biotechnological era, as scientists made groundbreaking strides towards understanding chemical fundamentals of life, as we know it. Today, the dominant model for the first form of life holds that the first nucleic acid to arise was RNA (not DNA)18. Under this model, an early form of life on Earth used RNA to serve both genetic and catalytic roles (t he "RNA world hypothesis). R NA emerged from an abiotic environment to support the first self-sustaining ch emical system capable of Darwinian evolution (the "RNA-first" hypothesis). The central challe nge which has long faced this model is the apparent chemical complexity of RNA relative to the organic molecules th at were likely to have been present in the primordial soup. More recently, the RNA first hypothesis has been challenged by some who point out that certain components of RNA are not easily form ed under any conditions probable for an early Earth. The belief is that many of the intermed iates, including RNA itself, are sufficiently unstable and would not have survived long on an early Earth, includi ng the conditions where they were formed19. For example, adenine (a nucleobase within RNA) is formed from hydrogen cyanide polymerization1 and is well known to be formed ab iologically. The molecule hydrolyzes in water to give inosine and the carbohydrate ribo se, the "R" in RNA is also unstable.

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17 Current origins of life theory on a larger scale propose that a pool of prebiotically formed, catalytic, self-replicating RNA molecules (known as ribozymes) was subject to a selection pressure where the most fit molecules came to dominate the pool. This genetic material is subject to replication errors, which create slight variations from parent to offspring. Over time, this variation results in a progeny with a higher fitness relative to pre ceding generations. Given this model, the primary interest in origins of life research is to determine how the sugar and heterocycle components of the firs t molecule of RNA came to be. Our hypothesis is that sugars, specifically carbohydrates invo lved in primitive and modern genetic systems, formed prebiotically on an early earth. Further, we propose that borate played a central role in the reactivity and stability of the carbohydrate products. Early Earth The absence of a community-wide agreem ent concerning a "standard of proof" complicates studies regarding preb iotic relevance. When proposing a solution to this historical problem, a geological hypothesis describing the environment where the proposed prebiotic chemistry occurred is necessary. Though early models of Earth range widely in their proposed temper ature and mineralogy, the Hadean Earth almost certainly had igneous lava flows that carried olivine and tourmaline minerals20. In addition, pallasites (meteorites that carry olivines) and chondri tes (meteorites that carry organic species; for example, the Murchiso n meteorite) were almo st certainly delivering both to early Earth. Temperature ranges on an ea rly Earth may have varied from 0 C to the temperature of molten iron (~1538 C)21. This provides a vast chemical reaction landscape of possible conditions to be explored.

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18 Prebiotic Organic Compounds Microwave spectroscopy of in terstellar gas clouds confir m the presence of organics including glycolaldehyde, formaldehyde and more recently dihydroxyacetone.3 Meteorite bombardment would have delivered these mo lecules to an early Earth. Reactions of glycolaldehyde and formaldehyde on a prim itive Earth would provide a source for glyceraldehyde and erythrulos e (in an aldol reaction of gl ycolaldehyde and formaldehyde). Carbonaceous chondrites have been found to cont ain a variety of organic compounds, including amino acids.22 It is also well known that atmospheri c electrical discha rge yields HCHO and H2O2, while meteorites deliver a bundant (and stable) glycerol23. The availability of Fe(II) minerals and H2O2 give rise to dihydr oxyacetone from glycerol using Fenton chemistry.24, 25 These factors combined provide a rich pool of chemicals to consider when hypothesizing processes that may have occurred in the primordial soup. Primordial Soup & The RNA World The RNA first hypothesis is widely held as offering the most likely scenario for the emergence of life as we know it. In 1989, Tom C ech shared the Nobel Prize in chemistry with Sydney Altman for their disc overy of the catalytic pr operties of RNA molecules26-28. Catalytic RNA molecules are the key to the success of a RNA world. The RNA world theory presupposes the existence of a pre-biotic soup as the originator of the first library of life. To test this hypothesis, laboratories seek a series of stepwise reactions pr oviding support for the abiotic synthesis of carbohydrates in the prebiotic soup. Of primary intere st are sugars implicated in both modern and primitive genetic information systems. The central argument against the RNA world arising from the primordial soup involves the formation of problematic tar, which is a nearly useless byproduct from highly reactive species. The propensity of organic precursor s to life to form tar is an i ndication of the functionality and

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19 reactivity of these building blocks Apart from making a sticky me ss, the tar prevents in depth product analysis and leads to cr iticism of the viability of car bohydrates in the primordial soup19. Nowhere is this criticism more valid than when directed to the formose process. For example, an analysis performed by Ricardo dem onstrated the consumption of formaldehyde to give brown material (Figure 1-4)10, 11. Figure 1-4 shows the acceleration of formaldehyde consumption with increased concentrations of HCHO. In this rapid loss of HCHO, the mixture first yellowed, then turned brown, forming mixtur es that some suggest precludes the assumption that the formose process wa s important on early Earth. Figure 1-4. Formaldehyde concentration dependen ce in the progress of the formose reaction.10,11 Work by Ricardo shows the rapid deplet ion of formaldehyde at increasing concentrations during the formose reaction.

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20 Figure 1-5. The affinity of borate to vari ous carbohydrates, ribose is highest among the aldopentoses10, 11, 29. Figure 1-6. Crystal struct ure of borate complexed with 1,4-anhydroerythritol.30 In a paper published in 1995, Stan ley Miller states that the in stability of ribose precludes it from the primordial soup. However, in 2004, Ricardo et al. postulated that if a species could be found to bind the cis-diols of the formed sugars, this would in reduce th eir reactivity through the removal of the reacting carbonyl group, thus resulting in a carbohydrate which did not undergo uncontrolled further reaction to form tar10. This was achieved through th e use of borate. Borate

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21 has the ability to form a complex with cis-diols30, a motif ubiquitous in sugars (Figures 1-5 & 16). Additionally, the affinity of borate comp lexes varies among pentoses (Figure 1-5)10, 11, 29. In their first experiments, Ri cardo et al. acquired borate from colemanite, a calcium borate mineral found in the arid desert climates of today. They reported that borate did in fact complex with ribose, arabinose and other pentoses to form a stable complex. Furthermore, this stabilization was effective under conditions wher e the pentoses were formed from the C3 carbohydrate glyceraldehyde and the C2 glycolalde hyde. This dissertation carries this work further.

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22 CHAPTER 2 THE PREBIOTIC SYNTHESIS OF SUGARS AND PRIMITIVE CATALYTIC CYCLES When approaching the abiotic synthesis of genetically relevant carbohydrates, the challenge lies within the analysis and structure proof of the formed compounds. For this reason, reactions of one, two and three car bon building blocks in the pres ence of borate were examined separately. The reaction of dihydroxyacetone and formal dehyde was studied via the method as outlined in the experimental section (3 carbon unit, dihydroxyacetone + 1 carbon unit, formaldehyde 4 carbon unit, erythrulose). Next, th e reaction between erythrulose and formaldehyde (4 carbon unit, erythrulose + 1 carbon unit, formaldehyde 5 carbon unit, pentulose) was studied. In order to identify the r eaction products, a sample of authentic straight chain pentulose, 1,2,4,5-tetrahydroxy-3-pentulose was synthetically prepared. In its 13C NMR spectrum, major peaks superimposed with those from the prebiotic reaction of erythrulose and formaldehyde, confirming the formation of the st raight chain sugar, al ong with other species (Figure 2-1). In an effort to look solely at newly formed products, 13C labeled formaldehyde was used in reactions with dihydroxyacetone and erythrulose re spectively. These reactions were performed in a standard Carbonate Bor ic Acid (CBA) buffer Na2CO3 (1.18g, 11.1 mmol, Fisher) and H3BO3 (0.172g, 2.78 mmol, Fisher) was dissolved in D2O (10 mL, Cambridge Isotope Laboratories) resulting in a solution with a measured pH of 10.4 and a final boron concentration of 278 mM. The initial primary consumption of H13CHO to yield R13CH2OH products resonating in the 6062 ppm range was observed. A peak at 72.4 ppm was observed to rise over time (Figure 2-17 & Experimental section A) in these reactions. This peak continued to intensify as a manifold of peaks at 60-62 ppm continued to decrease, signaling a shift in the product ratios.

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23 Figure 2-1. 13C Spectrum of synthetic 1,2,4,5-tetrahydroxy-3-pentanone standardized to methanol at 49.5 ppm. Spectrum file: 1,2,3,4-pentan-3-one.pdf Figure 2-2. Correlation experiment: Addition of pure 1,2,4,5-tetrahydroxy-3-pentanone to the mixture holding the reaction of erythrulose and formaldehyde. This spectrum is standardized to methanol at 49.5 ppm. Spectrum file: pentulose_mixed_with_authentic_20060715.pdf

PAGE 24

24 A single sharp signal emerging at 72.4 ppm is a peak most likely characterized by a single product. Furthermore, NMR superimposition experiments that this peak came from C4 of xylulose. The resonance at 72.47 ppm had a barely split doublet structure (72.47 and 72.42) that was not clearly significant above the noise, but wa s reproducible. One possibi lity is that a single borate coordinates two chiral diols in a comp lex, creating diasteromers. Alternatively, this doubling could be assigned as a cyclic form w ith two anomers. The addition of an authentic sample of ribulose to this material did not re sult in any superimpositi ons, however. Addition to xylulose in the borate-carbonate buffer did. This is reminiscent of an acetoxy derivative of 3pentulose which was reported to yield, in acid (in the absence of borate), a mixture of xylulose and ribulose, where the compounds were a ssigned by thin layer chromatography. Amidst reports that calcium could poten tially influence reactions by catalyzing rearrangements as some molybdenum species are reported to do,31, 32 reactions were run both in the presence, and absence, of calcium (Fig. 2-3 & Fig. 2-4). In the absence of any measurable difference between the resulting reactions, we co ncluded that the presence of calcium did not affect the products of the reac tions. These experiments would b ecome the basis of a first look into the kinetics of the system.

PAGE 25

25 Figure 2-3. Reaction of dihydr oxyacetone (DHA) with H13CHO without calcium. Note the peak at 72.4 which was later tentatively assigned to xylulose by superimposition. Spectrum file: dha-100ul_h13cho50ul_cba_120106_t=54720.pdf Figure 2-4. Reaction of erythrulose with H13CHO. Note the peak at 72.4 which was later tentatively correlated to xylulose via superimposition. Spectrum file: erythrulose_100ul_cba_ 900ul_hcho50ul_nt=256.pdf

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26 Primitive Catalytic Cycles Breslow proposed that once even a single molecu le of glycolaldehyde is generated, catalytic cycles could set up an abiotic "metabolism" to fix HCHO.9 As the enolization DHA is compatible with borate, DHA can also catalyze such a metabolism, avoiding the need for the addition of a molecule of formaldehyde to itself to yield a molecule of glycolaldehyde. Further reaction of glyceraldehyde, glyc olaldehyde, as well as 1,2,4,5-tetr ahydroxypentan-3one leads to the pentoses. As stated earlier, another source for dihydroxya cetone is glycerol, which is abundant in meteorites. Glycerol can react vi a a Fenton style oxidation utilizing H2O2 from lightning, UV light and Fe(II) minerals to give dihydroxyacetone24, 25. Moreover Fe (II) is compatible with borate as precipitates are not formed. The presence of borate guides product formation as demonstrated in Figure 2-5. Figure 2-5. The influence of borate on the enoliz ation of erythrulose a nd regioselectivity of reactions with HCHO. Erythrulose can enoli ze to form two different products, the 1,2 or the 2,3 enol. Borate binds to the 3 and 4 hydroxyl groups eliminating 2,3 enolization. Further, the borate-mediated reaction adds formaldehyde to the more hindered center.

PAGE 27

27 There appears to be some thermodynamic control by borate on which cyclic forms of pentoses form. When ribose, xylul ose and ribulose are incubated in a borate buffer, their spectra display far fewer complexation products in compar ison to arabinose, xylose and lyxose. This is attributed to the thermodynamically favourable conformation of one borate-pentose complex, shifting the equilibrium to deplet e the others and generating one particular cycle in abundance (Figures 2-6 to 2-11). Two rules for the abiotic metabolism were esta blished: (a) retroenolization incorporating deuterium is negligible at pH 12.5 as long as th e concentration of HCHO is significant, and (b) retroaldol reactions that extrude HCHO are negl igible. The first was confirmed by showing that if the formose process is run in D2O, the C4, C5, C6, and C7 speci es incorporated negligible amounts of deuterium from solvent. The second is consistent with the general principle that retroaldol reactions are favored when they break sterically crowded bonds having (mostly or entirely) non-hydrogen bulky s ubstituents (Figure 2-12).

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28 Figure 2-6. 13C NMR spectrum of arabinose (30 mg) in CBA buffer (1 mL), note the complexity. Spectrum file: arabinose_cba_meohref.pdf Figure 2-7. 13C NMR spectrum of lyxose (30 mg) in CBA buffer (1 mL), note the complexity. Spectrum file: lyxose_cba_meohref.pdf

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29 Figure 2-8. 13C NMR spectrum of xylose (30 mg) in CBA buffer (1 mL), note the complexity. Spectrum file: xylose_cba_meohref.pdf Figure 2-9. 13C NMR spectrum of ribose (30 mg) in CBA buffer (1 mL), note the simplicity indicating a singular cyclic form, assigned by NMR as the alpha furanose. Spectrum file: ribose_cba_meohref.pdf

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30 Figure 2-10. 13C NMR spectrum of ribulose (30 mg) in CBA buffer (1 mL), note the complexity. Spectrum file: ribulose_cba_meohref.pdf Figure 2-11. 13C NMR spectrum of xylulose (30 mg) in CBA buffer (1 mL). Spectrum file: xylulose_cba_meohref.pdf

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31 Figure 2-12. Pathways of possible aldol/retro -aldol chemistry of carbohydrates. An abiotic "metabolism" that generates higher carbohydr ates via repetitive enolization of lower carbohydrates (top) followed by aldol addition of the resu lting enediol to formaldehyde (HCHO), followed by fragmentation of the carbohydrat es in a cyclic process. Compounds in green are indisputable prebiotic precursors because of either their established presence in meteorit es, their ready formation on Earth via atmospheric electrical discharge, photochemistry, or mineral processes, or their direct observation in interstellar nebulae by microwave spectro scopy. Compounds in blue neither enolize nor fragment (other than to give the same fragments from which they are formed). Bold arrows in black show aldol addition of HCHO to the primary, less hindered center of an enedio l; other black solid arrows indicate aldol addition to a more hindered center. Red dotted arrows show retroaldol fragmentation pathways that complete the cycle by cleaving the higher carbohydrate at the red, bold bond. Borate constrains this metabolism to occur only within the dotted box (magenta).

PAGE 32

32 HO OH H H HO H O H H HO OH H H HO H OH H HO OH H H OH OH H H HO H H HO H O H OH OH H H HO O H H HO H H HO CH2 HO OH H H HO CH2 O H H OH OH HO H H HO H OH OH OH H H HO H H HO H OH O OH H H H2C OH HO OH H H HO CH2 OH H OH HO H H HO CH2 O H OH OH HO O H H HO CH2 CH2 H OH OH H H HO OH HO H H HO CH2 OH OH OH OH H H HO H H HO H OH OH OH H H2C OH HO H H HO CH2 O HO CH2 OH OH H H OH HO H H HO CH2 HO O OH OH H H CH2 OH HO H H HO H OH O H OH H2C OH OH H H HO H H HO H OH HO O H H2C OH CH2 OH C4 C5 HO H H HO H OH OH OH H2C OH OH H H C7 C6HO H H HO CH2 HO OH OH OH H CH2 OH HO H H HO H OH O HO CH2 H2C OH OH H H OH HO H H HO H OH CH2 O H2C OH OH H H C8HO OH HO H H HO CH2 HO O OH H OH CH2 OH HO H H HO CH2 HO CH2 OH O H CH2 OH HO OH OH H H O H HO H OH H H HO OH H H OH H C2,3 H OH OH H OH HO O H H H H H OH O H H enolization aldol at primary C aldol at secondary C retroaldol bond broken in retroaldols O H HO HOH2C OH H H glycolaldehydeHCHO HCHOHO OH H H HO H H H glycerol formaldehydeH2O2Fe++ Fenton reaction HCHO HCHO HCHO HCHO dihydroxyacetone

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33 The Geochemical Synthesis of Threose In an effort to understand and distinguish known resonances in prebiotic reaction mixtures, attempts to eliminate known starting ma terials were made by incubating them alone in a Carbonate Boric Acid (CBA) buffer. While cer tain species, such as formaldehyde, were not expected to react with themselv es, the spectrum arising from incubation of glycolaldehyde was promising. A sample of glycolaldehyde (20 mg, Aldrich) was incubated in CBA buffer (1.0 mL) and an NMR spectrum was acquired. The methanol-standardized spectrum displayed four peaks with resonances at 103.28, 82.33, 76.61 and 71.61, where only two peaks for glycolaldehyde or its dimer were expected. These results showed that glycolaldehyde formed a four carbon species. This was determined to be threose by the superimpositi on of the four resonan ces on the four from authentic threose. Additionally, erythrose was not observed. We hypothesize that borate stabilizes the C1-hydroxy in a be ta orientation with the hydroxyl group at the C2 position (Fig. 2-13). Superimposition NMR correlation expe riments are pictured in Figure 2-14. Glycolaldehyde (20 mg, Aldric h) was allowed to react with itself in the absence on formaldehyde in CBA buffer (1 mL, 250 mM, pH 10.4) for 24 hours. Aliquots of this reaction were gradually added to a sample of authentic threose (17 mg, Fisher) dissolved in CBA buffer (1 mL, 142 mM, pH 10.4) in increasing ratios fro m 1:10 up to 10:1 and analyzed via NMR. The peaks in the sample (103, 82, 76 and 71 ppm) co rresponded directly to and superimposed upon the authentic threose peaks through out the concentration gradient.

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34 Figure 2-13. Cyclic forms of ribose (in its "alpha" epimer, where the OH hydroxyl group at C-1 is down; the "D" enantiomer is shown). 2'-Hydroxyerythrose (in both its alpha and beta form; the enantiomer shown is the one synthesized with a 13C label), and 2'hydroxymethyl threose (in both its alpha and beta form; the enantiomer shown is the one synthesized with a 13C label). The site of borate complexation is known by NMR spectrometry in ribose. The site of complexation of borate to the cyclic forms of the branched pentoses is not established experi mentally; experimental data suggests that the erythrose forms two borate complexes, while the threose forms only one. Compared to previous literature reports, th is reaction medium of a carbonate borate buffer offers a prebiotic route to sugars. Weber and Pizza rello reported that a peptide chain placed in a solution of glyceraldehyde demonstrated the synt hesis of threose and erythrose in an aqueous prebiotic context.33 There have been a few other repor ts of protein mediated carbohydrate synthesis such as one by Cordova which used N,N-dimethylformamide as a solvent.34, 35 Similarly Ibrahem et al used DMSO with a trace of water for their amino acid-catalyzed formation of carbohydrates.36 Both Albert Eschenmoser and Jack Szostak pr oposed threose to be a precursor to our modern genetic backbone and demonstrated its ability to form a stable and functional biopolymer as the backbone of an RNA-like genetic molecule37, 38. Additionally, Szostak demonstrated the ability DNA polym erases to elongate DNA primered strands of threose nucleic acids.39

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35 Figure 2-14. NMR analysis of the reaction of glyc olaldehyde in CBA buffer, forming threose, in the absence of HCHO. A 13C NMR spectrum of the reaction products of glycolaldehyde (250 mM) with itself. B 13C NMR spectrum of authentic threose (142 mM) in CBA buffer. C 13C NMR spectrum of samples A and B mixed together, here we show an equimolar mixture. D 13C NMR spectrum of authentic erythrose (167 mM) in CBA buffer. Signal at 49.500 ppm is reference CH3OH. Erythrose and threose do not undergo reaction in days in CBA buffer. Spectrum files: 070607_12C_GOL_cba_with_authentic_threose_added.pdf, 070523_erythrose_20mg_cba_1ml_nt=4096.pdf

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36 Figure 2-15. A) Phosphate backbone and heterocyc lic base locations in a threose nucleic acid (TNA) model. B) Coordination of borat e to the alpha anomer of threose. Kinetics A kinetics study of the rate of enolization of dihydroxy acetone, glyceraldehyde and erythrulose offered a more detail ed view of the borate-catalyzed cycle. Borate in CBA was found to slow the rate of enolizati on of erythrulose by a factor of 4.48, of dihydroxyacetone by a factor of 9.17, and glyceraldehyde by a factor of 3.33 (Table 2-1). Table 2-1. Enolization rates of dihydroxyacetone glyceraldehyde and erythrulose at 25 C with formaldehyde concentration of 167 mM. CBA Buffer pH=10.4 Slope of line [enolizable specie s] pH enoliz ation rate constant Dihydroxyacetone 1.0 x 10-2 min-1 0.317 M 10.4 0.0052 min-1 Glyceraldehyde 3.7 x 10-3 min-1 0.317 M 10.4 0.0018 min-1 Erythrulose 1.2 x 10-2 min-1 0.317 M 10.4 0.0063 min-1 Carbonate Buffer pH=10.4 Slope of line [enolizable species] pH enolization rate constant Dihydroxyacetone 9.5 x 10-2 min-1 0.317 M 10.4 0.0477 min-1 Glyceraldehyde 1.2 x 10-2 min-1 0.317 M 10.4 0.0060 min-1 Erythrulose 5.6 x 10-2 min-1 0.317 M 10.4 0.0282 min-1 When performing the kinetics, it was not neces sary to label the higher carbohydrates as 13C labeled HCHO was introduced into the medium at a concentration high enough to support a convenient rate. The concentration of enoliz able species must remain well below the

PAGE 37

37 concentration of borate. As a further complica tion, the product of the reaction is also an enolizing species, meaning that the rate of consumption of H13CHO as the reaction progresses need not slow down. Indeed, if the rate of enoli zation of the product is fa ster than the rate of enolization of the starting material, consumption of H13CHO may in fact speed up (Figure 2-16). The rate law could have the form velocity = k[enolizing species][H13CHO]. However, a classical experiment in physical organic chemistry, dating back to experiments in 1904 by Lapworth, showed that the rate of bromination of acetone was independent of the concentration of bromine40. This was the first evidence for a slow, rate limiting enolization of acetone as an obligatory step in the bromination reaction, and th e enol of acetone as an intermediate in the bromination reaction. Therefore, th e reaction process here is assu med to be rate limited by the enolization step and the rate law is expected to be of the form: Velocity = k[enolizing species]. To obtain rate constants in the standard CBA buffer, a known concentration of the enolizing species wa s incubated with H13CHO, and the initial rate was estimated by following the loss of label assigned to H13CHO relative to the intensity of the formate peak arising from the introduced formaldehyde. This ratio was converted to a logarithm, log ([H13CHO/H13COO-]) (now expected to be linear) normalized by divi sion against the first data point, and plotted against time (Figs. 2-17 to 2-22). This gives a "pse udo" rate constant that was converted to a first order rate constant by dividing by the c oncentration of enolizable species.

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38 HO C C C O H H HO H H H C C C HO OH OH H H enolization H C C O OH H H H C C OH OH H enolization HCHO H C C H OH O OH C C C OH H H OH H H OH C C C C C H H OH H HO O HO H H H OH C C C C HO H H OH H O H H OH C C C C HO H H OH H OH H OH C C C C O H H OH H C H HO OH H H HCHO enolization HCHO pentuloses boratestabilizes pentoses boratestabilizes H C C H OH O H C C OH OH H H threose boratestabilizes retroaldol fragmentation aldoladdition aldoladdition boratecontrols regiochemistry aldol addition aldol addition boratecontrols regiochemistry borateslows glycolaldehyde lookingforan electrophile lookingfora nucleophileC C C HO OH H O H H H erythrulose 0.0052min-10.0018min-10.0063min-1 Figure 2-16. The borate-compatible cycle for conversion of formaldehyde into pentoses, pentuloses and threose. Enolization ra tes of glyceraldehyde dihydroxyacetone and erythrulose in the presence of borate. The first order process for the consumption of H13CHO approximation is good only if [H13CHO] << [enolizable species], allowing the [enolizable sp ecies] to be assumed to remain constant. The slope of a line plotting [H13CHO] versus time is equal to k [enolizing species]. To compare these rate constants with rate constants obtained fo r various enolizing species however, the slope of the line, and the "pseudo first order" rate constant is converted to a real rate constant by dividing by the concentration of enolizable species. While peak intensities in 13C spectroscopy need not give precise values, these averaged over a dozen measurements proved not to create large variance when the data were plotted.

PAGE 39

39 In the presence of borate, the pseudo first or der rate constant for the reaction of DHA (317 mM, two-fold excess over HCHO) with H13CHO is measured to be 0.0052 min-1 (the error was calculated by comparing results using MeOH a nd formate as references, at pH 10.25, 25 C). This gives a second order rate constant of 0.016 M-1min-1. The loss of formaldehyde versus time plot was linear for up to 200 minutes (R2 0.98). The corresponding experiments for erythrulose s howed a slightly higher rate (pseudo first order rate constant of 0.0063 min-1) and giving a second order rate constant ([erythrulose] 317 mM) of 0.02 M-1min-1. In the absence of borate, the pseudo first order rate constant for the reaction of DHA (317 mM, two-fold excess over [HCHO]) with H13CHO is measured to be 0.0477 min-1 (error calculated by comparing results using MeOH and formate as references, at pH 10.25, 25 C). This gives a seco nd order rate constant of 0.150 M-1min-1. The loss of formaldehyde versus time plot wa s linear for up to 200 minutes (R2 0.98). The corresponding experiments for erythrulose showed a slower rate (pseudo first order rate constant of 0.0282 min1) and giving a second order ra te constant ([erythrulose] 317 mM) of 0.089 M-1min-1). Given that a "statistical factor" ope rates (erythrulose has one less primary enolizable hydrogen to abstract than DHA), and assuming that the reaction is under kineti c control (implying that the enolization at the primary center dominates the overall rate), this slower rate constant for the reaction of erythrulose with HCHO is entirely ex pected. This rate difference is not observed in the presence of borate. Though bor ate slows down the overall rate of enolization, borate also nearly equates the rates of formaldehyde consumption by dihydroxyacetone and erythrulose respectively. This fits the obser vation that borate favours the e nolization of erythrulose between carbons 2 & 3 over carbons 1 & 2.

PAGE 40

40 Time (minutes) 0 50 100150200250300 Log10(H13CHO/H13CHOO-) -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 y=-0.0103x+0.1603 R2=0.9802 Figure 2-17. Reaction of dihydr oxyacetone (0.317 M) with fo rmaldehyde (0.167 M) in the presence of borate (CBA buffer with a meas ured pH of 10.4 at 25 C) plotted against time. 4681012141618 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 Log10(H13CHO/H13CHOO-)Time (minutes) y=-0.0951x+0.4748 R2=0.9988 Figure 2-18. Reaction of dihydr oxyacetone (0.317 M) with fo rmaldehyde (0.167 M) in the absence of borate (carbonate buffer with a measured pH of 10.4 at 25 C) plotted against time.

PAGE 41

41 1416182022242628303234 -0.35 -0.30 -0.25 -0.20 -0.15 -0.10 Log10(H13CHO/H13CHOO-)Time (minutes) y=-0.0126x+0.0885 R2=0.9878 Figure 2-19. Reaction of erythrul ose (0.317 M) with formaldehyde (0.167 M) in the presence of borate (CBA buffer with a measured pH of 10.4 at 25 C) plotted against time. 4681012141618202224 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 Log10(H13CHO/H13CHOO-)Time (minutes) y=-0.0563x+0.2317 R2=0.9972 Figure 2-20. Reaction of erythr ulose (0.317 M) with formaldehyde (0.167 M) in the absence of borate (carbonate buffer with a measured pH of 10.4 at 25 C) plotted against time.

PAGE 42

42 1416182022242628303234 -0.04 -0.03 -0.02 -0.01 0.00 0.01 0.02 0.03 0.04 Log10(H13CHO/H13CHOO-)Time (minutes) y=-0.0036x+0.0893 R2=0.9519 Figure 2-21. Reaction of glyceral dehyde (0.317 M) with formalde hyde (0.167 M) in the presence of borate (CBA buffer with a measured pH of 10.4 at 25 C) plotted against time. Figure 2-22. Reaction of glycer aldehydes (0.317 M) with formal dehyde (0.167 M) in the absence of borate (carbonate buffer with a measured pH of 10.4 at 25 C) plotted against time.

PAGE 43

43 Catalytic Cycles A reaction cycle starting with DHA proved to be co mpatible with borate, and avoids the slow 1 carbon unit, formaldehyde + 1 carbon unit, formaldehyde 2 carbon unit, glycoaldehyde reaction. Glycerol from meteorites can form DHA via a Fenton oxidation utilizing H2O2 from lightning, UV light and Fe(II) minera ls. Additionally Fe(II) is comp atible with borate, as is H2O2. The presence of borate, in the end, can guide product formation (Figure 2-5). OH C C C O H H OH H H H C C C OH OH OH H H H C 13C O HO H H H C C OH HO H H C C H HO O HO C C C HO H H HO H H HO C C C C C H H HO H OH O OH H H H HO C C C C OH H H HO H O H H HO C C C C OH H H HO H OH H HO13C C C C O H H HO H13C H OH HO H H pentuloses, borate stabilizesxylose H C C H HO O HO C C HO H H H threose, borate stabilizes HO C C C OH H H O H H Erythrulose Dihydroxyacetone H13C HOH C C C OH O HOCH2H H OHH13CHO H13CHO xylulose H C C H HO O HO C C C HO H H HO H H H C C H HO O HO C C C HO H H HO H H H C C H HO O HO C C C HO H H HO H H lyxose arabinose ribose pentoses. borate stabilizesHO C C C C O H H HO H13C H OH HO H H HO C C C C C H H HO H OH O OH H H H ribulose threo erythro HO C C C OH H H H H HO H Glycerol Glycolaldehydeenolization enolization enolization aldol addition borate influences diastereoselectivity aldol addition aldol addition H2O2Fe++ branched pentosesretroaldol fragmentation slowed by borate complexation cannot enolize HO C C C C OH H H OH OH H H borate inhibits borate controls diastereoselectivity equilibrate in borate enolization aldol additionGlyceraldehydecannot enolize HO C C C C C H H HO H O H H OH H H xylulose Figure 2-23. A segment of the abiotic metabolism, the compounds in green are known prebiotically in either meteorites, via electrical discharge, photochemistry, or mineralbased processes on Earth, or in the interste llar nebula. Carbon atoms preceded by a superscript 13 have been 13C labeled in higher carbohydrat es prepared by laboratory chemical synthesis, to establish the movement of carbon through the pathway. Red, blue, and magenta colored carbons in the higher carbohydrates indi cate the position of label introduced from H13CHO in the immediately preceding aldol addition product.

PAGE 44

44 The sudden drop in formaldehyde concentra tion was hypothesized to stem from late retroaldol reactions undergone by C4, C5, C6, and C7 species that have such crowded bonds (red bonds in Fig. 2-12). Many of these retroal dols generate dihydroxyacetone. The rate of enolization of dihydroxyacetone was m easured through its reaction with H13CHO, and found to be 0.0103 min-1 at 25 C, pH 10.4 (CBA buffer), certainly competent at higher temperatures and pH to support the observed rapid co nsumption of HCHO in the crash. The formose process was repeated using a 2:1 mixture of H12CHO and H13CHO to help assign the products formed. After HCHO was consum ed, the characteristic "yellowing" of the formose product began. This mixture generated 13C-NMR resonances that were split (1:2:1) by 13C-13C coupling at 183.64, 69.46, and 21.04 ppm (assigned respectively as C-1, C-2, and C-3 of lactate), 182.68 and 62.70 (assigned respectively as C-1 and C-2 of glycolate), and 59.26 and 37.11 (assigned respectively as C-1 and C-3, and C2 of 1,3-propanediol). All of these products, formed only after HCHO was consumed, requiring enolization where the en ediol was not trapped by HCHO, but rather suffers beta-elimination a nd protonation. Further, they require Cannizzaro reactions41, as well as the well-known met hylglyoxal-lactate rearrangement42. All of these products are "metabolic" dead ends, as lactate, glycolate, and diols ar e essentially unreactive under a wide range of conditions, including these. A geological model where borate might be av ailable to stabilize C7, C6, C5, and C4 carbohydrates under conditions where they are formed was revisited. Serpentinization, a process of hydration and metamorphic transformation in ultramafic rock, spec ifically of olivinecontaining igneous rocks43 is well known to generate highly al kaline solutions having a pH >12. Such a high pH could not be maintainable unde r an atmosphere of carbon dioxide on primitive Earth, where CO2 would be absorbed to create a buffere d carbonate solution with a much lower

PAGE 45

45 pH. Attempts to run the formose reaction at pH 10 failed to yield detectable products. This is presumably due to the failure of the 1 carbon unit, formaldehyde + 1 carbon unit, formaldehyde 2 carbon unit, glyceraldehyde r eaction (which is remarkably slow even at pH 12.5). This inference was supported by the fact that addi ng small amounts of glycolaldehyde to HCHO in carbonate buffer at pH 10.4 generated formose be havior, with an induction period followed by rapid loss of HCHO. The inducti on period shortening as the con centration of glycolaldehyde increased. Small amounts of glycolaldehyde presumably could be obt ained prebiotically. While this allowed the cycles in figure 2-23 to begin even at lower pH, and diminished the amount of dead end products formed by the Cannizzaro reaction, they did not solve a second problem. This problem was encountered when bora te was introduced to st abilize the C7, C6, C5, and C4 products. Even at 6 mM, borate shut down the formation of higher carbohydrates from HCHO under formose conditions. Instead, the princi pal products were lactate, glycolate, and 1,3dihydroxypropane, with only two other additional compounds formed in assignable amounts. No 13C-resonances were observed at 100-115 ppm, which would represent the formation of aldopentoses or ketopentoses. Borate evidently s hut down the cycles that move HCHO to higher carbohydrates. Figure 2-12 was further studied wi th the goal of identifying small catalytic cycles that might be initiated by other precursors that woul d fix HCHO. As an alternative precursor, dihydroxyacetone was particularly attractive, no t only because it supported the consumption of HCHO, but also because it is available by the reaction of H2O2 (obtained by elec trical discharge through moist atmospheres) in the presence of fe rrous iron (indisputably present on early Earth) in a Fenton reaction with glycerol, which is well known to survive impact as a major (>100 ppm) component of the soluble frac tion of carbonaceous chondrites.

PAGE 46

46 As a model environment, sodium carbonate-bic arbonate buffer at pH 10.4 (1.1 M, essentially saturating, as expected under a CO2 atmosphere) was compared with the sodium carbonatebicarbonate buffer at pH 10.4 containing sodium bor ate at various concen trations up to 278 mM. The reaction of erythrulose with H13CHO was found to generate species that were tentatively assigned as the 2'-13C-2-hydroxymethyltetrose, a branched sugar arising from reaction of the 1,2-erythrulos e enediol with its more hindered center (Hyo-Joong Kim unpublished results). This assignment was confirmed by synthesizing both the erythro and threo diastereoisomers with label at both C-4 and C-2' (for the erythro diastereoisomer) and at C-2' (for the threo diastereoisomer), and demonstrating that the signals from the authentic materials superimposed on the signals arising in the reaction (Figure 2-24). A. 2'-13C-2-hydroxymethylerythrose B. 4-13C-2-hydroxymethylerythrose Figure 2-24. Synthesis of erythro branched pe ntose (A) and threo-branched pentose (B). Reagents and conditions: Scheme A: a. 2,2-dimethoxypropane, p-TsOH, acetone, 80%; b. 1. NaBH4, H2O. 2. NaIO4, AcOH, H2O, 71%; c. H13CHO, K2CO3, CH3OH, 65 C, 70%; d. Dowex 50Wx8 (H+), H2O, 80 C, 95%.Scheme B: a. 2,2dimethoxypropane, p-TsOH, acetone, 80%; b. 1. NaBH4, H2O. 2. NaIO4, AcOH, H2O, 70%; c. HCHO, K2CO3, CH3OH, 65 C, 55%; d. Dowex 50Wx8 (H+), H2O, 80 C, 95%. These compounds were synthesi zed by Hyo-Joong Kim at the Foundation for Applied Molecular Evolution.

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47 These results suggested that borate controls th e regiochemistry of tw o microscopic steps in the formose process starting with erythrulose. Firs t, borate appears to direct the enolization of erythrulose to give its 1,2-ened iol in preference to its 2,3-enedio l. This is interpreted as the consequence of borate coordinating to the C3 and C4 alcohol groups of erythrulose; the anionic nature of the borate complex is expected to suppress enolizati on at carbons 3 and 4. Once the 1,2-enediol is formed, however, the borate appeared to direct the incoming HCHO electrophile to the more hindered center carbon-2; the intrinsic regiochemistry in the absence of borate is expected to involve nucleophilic attack at carbon -1. Diastereocontrol of aldol additions by borate is well known in synthetic chemistry44, especially in non-aqueous me dia, but was unexpected in water, however. Relevant to the reactivity of the pentoses formed from H13CHO and erythrulose in the presence of borate, was the observation that these branched sugars had no enolizable proton alpha to the C=O aldehyde unit. Th ey were therefore stable agai nst enolization. Further, they formed cyclic hemiacetals via the reaction of their aldehyde units with the 4-hydroxyl group; hemiacetal formation appears to slow the enolizati on of sugars generally. Further, they appeared to be stabilized by borate, although the breadth of the 13C resonances of the borate complexes at pH 10.4 suggested that they were (at least) conformationally dynamic. They could, however, undergo slow retroaldol reaction to give glycolaldehyde and glyceraldehyde, the C2 and C3 species that have already been s hown to give pentoses and pentulos es in the presence of borate. The outcome of the reaction between glycol aldehyde and glyceraldehyde formed by the retroaldol reaction is also contro lled by borate. The initial products of the retroaldol reaction are glycolaldehyde and the 1,2-enediol of glyceraldehyde. If these reac t directly as the electrophile and the nucleophile respectively, the results are the corresponding pentuloses, ribulose and

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48 xylulose. If, however, the 1,2-en ediol of glyceraldehyde undergoe s a retroenolization to give glyceraldehyde, and glycolalde hyde undergoes an enolization to give the 1,2-enediol of glycolaldehyde, then glycolalde hyde and glyceraldehyde may enter the 2 + 3 = 5 reaction as the nucleophile and the electrophile resp ectively to give one or more of the four diastereoisomeric pentoses ribose, arabinos e, xylose and lyxose. Discussion These experiments establish many features of the reaction of formaldehyde (HCHO) and lower carbohydrates under conditio ns expected following the aqueous erosion of igneous rocks containing both olivine and tourmalines. All of these lead to stable pentoses. As with any prebiotic experiment, it is difficu lt to select environmental parame ters (temperature, pH, reaction time) that strike a defensible compromise betw een the need for reactions that proceed rapidly enough to measure in real time and outcomes that are likely to emerge after thousands or millions of years. Here, we have chosen a pH of 10.4 that is higher than that likely to be sustained (at least on the surf ace of the early Earth under a CO2 atmosphere) and at temperatures (65 C) that may be higher th an plausible on early Earth. The choice of harsher-than-perhapsplausible conditions strengthens, however, the inference that th ese processes generated stable borate-pentoses, including ribose, as organic minerals on early Ea rth. A previous study measured the stability of various borate-pentose complexes45. The relatively high affinity of ribose for borate suggested that after the pentose equilibr ium is achieved, borate-ribose would predominate as an organic mineral. A final parameter is, of course, the relative concentration of or ganic species. Excess HCHO (which is expected under current prebiotic models) drives the cycle in Figure 2-24 in a clockwise fashion. This may be an example of what is sought by individuals who advocate a "metabolism first" model for the formation of life, rather than a "genetics first" model. Here, the

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49 borate complexes of the branched sugars would accumulate as organic minerals, with their retroaldol products consuming HCHO. A kinetic s study of the enolization of dihydroxyacetone, glyceraldehyde and erythrulose foun d that borate slows the rate of enolization of erythrulose by a factor of 4.5, of dihydroxyacetone by a factor of 9.2, and glyceraldehyde by a factor of 3.3. Dihydroxyacetone was also found to enolize faster th an erythrulose in carbonate by a factor of 1.69, whereas in the presence of borate, erythrul ose enolizes by a fact or 1.2 faster than dihydroxyacetone. The preliminary establishment of kinetic rates for the cycle allows for future in-silico modeling of this system. The reaction of glycolaldehyde in the abse nce of HCHO, here again, demonstrates the ability of borate to control dias tereoselectivity. Incubation of glyc olaldehyde in carbonate-borate buffer generated just four detectab le signals, assigned as threose, not erythrose. These were also assigned by their superimposition on the signals aris ing from authentic threose. This result is more than remarkable, as threose (not erythrose), as noted above, is one of very few replacements for backbone ribose that support rule-based molecular recognition. The borate complex of threose as an organic mineral also proved to be very stable against base-catalyzed decomposition. It is remarkable that the two sugars (ribose and threose) that have emerged from synthetic biological experiments as being especially able to crea te useful backbones are also the two that emerge from borate-moderated formose processes. The concept of an organic mineral may pr ove to be useful in prebiotic chemistry generally. Although a few organic minerals are known on contemporary Earth, and one (mellite) has been proposed to be abundant on the su rface of Mars, organic minerals are generally consumed in the biosphere by the life that inhab its it. On a prebiotic Earth, of course, a wider range of organic minerals are expected. Organi c minerals involving borate and carbohydrates are

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50 reasonably soluble in water; however, if they were important on early Earth, and if high concentrations were needed, such organic minera ls would be useful onl y in evaporite basins. Such basins appear to be abundant on Mars, whether they could have existed on early Earth remains unknown. Experimental Buffer and Stock Solution Preparations Depolymerizaton of paraformaldehyde: 13C-labeled paraformaldehyde (1 g) Cambridge Isotope Laboratories, Andover MA) was suspended in D2O (4 mL) and sodium deuteroxide (NaOD, 1.0 mL, 10 M, Cambridge Isotope Laborato ries, in two 0.5 mL portions, the second 1 h after the first) at 4 C. The paraformaldehyde is depolymerized under these conditions to give H13CHO, half of which undergoes the Cannizzaro reaction to give labeled methanol (H3 13COH, set in all NMR experiments to 49.500 ppm) and labeled formate (H13COO-, 171.6 .02 ppm). These served as internal standards for both chem ical shift and (approximated) concentration of products. The material was stored at 10 C. The final concentration of total 13C was 3.22 M. A 13C NMR spectrum of H13CHO showed, in addition to the prominent peak at 82.30 .02 (assigned to the hydrate H2 13C(OH)2), small peaks at 90.18 (inten sity relative to 82.30 = 0.037 ) and 86.31 (intensity relative to 82.30 = 0.012) and 55.29 (intensi ty relative to 82.30 = 0.022). Buffer B: A sodium borate (anhydrous) and boric aci d buffer was prepared by mixing in D2O (Cambridge Isotope Laboratories, 10 mL), Na2B4O7 (Aldrich (anhydrous), 0.503 grams, 2.5 mmol contributing 250 mM to tota l borate concentration), and H3BO3 (61.4 mg, 1 mM, contributing 100 mM to total borate concen tration). The final pD of this was 9.53. CBA Buffer (Buffer C): CBA buffer was prepared by dissolving Na2CO3 (Fisher Scientific, 1.18 g, 11.1 mmol) and H3BO3 (Fisher Scientific, 0.172 g, 2.78 mmol) in 10 mL D2O. This buffer has a pH of 10.4 with a fina l borate concentration of 278 mM.

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51 Carbonate Solution: The carbonate solution was prepared by dissolving Na2CO3 (1.18 g) in D2O (Cambridge Isotope Laboratories, 10 mL). Carbonate Buffer: Carbonate Buffer with pH 10.4 was prep ared by adding a solution of sodium bicarbonate (1.11 M) to a solution of sodium car bonate (1.11 M) until the pH reached 10.4, the same as the pH of CBA buffer. Carbohydrate stock solutions: Dihydroxyacetone dimer (Aldrich, 3.0 g, 33.3 mmoles, 3.33 M) was dissolved in D2O (Cambridge Isotope Laboratories, 10 mL), and the solution was incubated at room temperature for four days. Separate experiments showed that this generated the monomer, with minimal forma tion of self-aldol products. General Experiemental Conditions Detailed experimental conditions not reported in this section can be found in Appendix A. Experimental A: Dihydroxyacetone and H13CHO in CBA buffer Dihydroxyacetone dimer (Aldrich, 3.3 M, 100 L) was dissolved in CBA buffer (1 mL). To this solution, H13CHO (Cambridge, 3.3 M, 50 L) was added. The mixture was agitated vigorously using a vortex stirrer. A portion of the reaction mixture (500 L ) was transferred to an NMR tube. Acquisition of an NMR spectrum began afte r four minutes at 25 C. Multiple time points were acquired. The following data represents the reaction composition after 9250 minutes (6.42 days). 13C NMR (D2O): 171.799, 167.313, 103.228, 73.990, 73.879, 72.460, 71.613, 71.564, 71.037, 70.038, 69.305, 69.214, 68.706, 68.058, 67.978, 66.986, 66.887, 65.445, 64.674, 64.346, 64.030, 63.801, 62.824, 61.775, 60.818, 59.334, 58.693, 58.510, 53.860, 49.962, 49.645, 49.500 (MeOH). Spectrum file: dha -100ul_h13cho-50ul_cba_120106_t=9250.pdf Experimental B: Erythrulose and H13CHO in CBA buffer

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52 Erythrulose (3.33 M, 100 L) was mi xed with CBA buffer (0.900 mL) and H13CHO (Cambridge Isotope Laboratories, 6 M, 50 L). The mixture was agitated vigorously using a vortex stirrer. A sample of the reaction mixture (500 L) was transferred to an NMR tube. Acquisition of an NMR spectrum began after four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.779, 166.698, 94.044, 72.461, 72.411, 69.325, 66.372, 64.671, 64.339, 63.839, 63.694, 63.450, 63.271, 63.149, 61.776, 49.500 (MeOH). Spectrum file: erythrulose_100ul_c ba_900ul_hcho50ul_nt=16_47sec_10spectra.pdf Experimental C: Glycolaldehyde in CBA buffer Glycolaldehyde dimer (20 mg) was dissolved in 1 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.042, 103.268, 82.318, 76.599, 71.598, 49.500 (MeOH). Spectrum file: 070515_12C-glycola ldehyde_20mg_cba_1ml_refmeoh.pdf Erythrulose in CBA buffer: Erythrulose (30 mg) was dissolved in CBA buffer (1 mL) and agitated vigorously using a vor tex stirrer. To this was added 10 L of a 10% MeOH in D2O solution as an internal reference. Acquisition of an NMR spectrum began within four minutes and ended after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.026, 129.955, 110.287, 106.045, 103.195, 98.942, 79.151, 76.557, 71.625, 66.903, 62.828, 61.787, 60.891, 49.500 (MeOH), 33.593. Spectrum file: 070510_erythrulose _28mg_cba_1ml_refmeoh.pdf

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53 Dihydroxyacetone with CBA buffer : Dihydroxyacetone dimer (18 mg, Aldrich) was dissolved in CBA buffer (1 mL). To this was added 10 L of 10% MeOH in D2O as an internal reference. The solution was agitated usi ng a hand vortexer and transferred to an NMR tube. Spectrum acquisition started within 4 minutes and ended after 4096 transi ents (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.904, 111.961, 110.920, 106.617, 85.522, 84.095, 83.012, 82.875, 79.220, 77.324, 75.470, 73.861, 73.170, 69.916, 64.358, 63.603, 62.706, 60.143, 58.911, 56.355, 49.500 (MeOH). Spectrum file: 070530_dha_cba_meohref.pdf Kinetics Kinetic runs: Runs were initiated by mixing CBA buffer (0.85 mL), carbohydrate solution (3.17 M, 0.1 mL, final concentration 317 mM) and H13CHO (3.33 M, 0.05 mL, final concentration 167 mM) The final pH was measured to be 10.4. Previous independent r uns with unadjusted carbonate buffer were run at pH 11.2. The reaction proceeded at room temperature with a half life measured in minutes, while the half life at pH 9.5 was measured in hours. Subsequently the pH of the carbonate solution was ad justed to 10.4 for kinetic runs. To determine whether calcium had any im pact on this rate, a solution of 1 M CaCl2 in H2O was treated with NaOH until its pH was 7.0. In an analogous run, including calcium at varying concentrations, no signifi cant deviations were found.

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54 CHAPTER 3 2-DEOXYCYTIDINES CARRYING AMINO AND THIOL FUNCTIONALITY: SYNTHESIS AND INCORPORATION BY VENT (EXO-) POLYMERASE The synthesis of 2-deoxycytidine nucleosides bearing amino and thiol groups appended to the 5-position of the nucleobase via a but ynyl linker is described. The corresponding triphosphates were then synthesized from the nuc leoside and incorporated into oligonucleotides by Vent (exo-) DNA polymerase. The ability of Vent (exo-) polymerase to amplify oligonucleotides containing thes e functionalized cytidine derivatives in a polymerase chain reaction (PCR) was demonstrated for the amine-functionalized derivative. Figure 3-1.The Versant bDNA diagnostic test marketed by Bayer Diagnostics.46 Deoxyribonucleosides carrying functionality ap pended at the 5-position of uracil were introduced three decades ago to complement th e functionality that DNA carries intrinsically.47, 48 Fluorescent appendages attached to 2,3-dideoxynucleotides have been the key to automated DNA sequencing.49 Functionalized appendages may also increase the power and versatility of nucleic acids as receptors, ligands, and catalysts.50 Modified standard nu cleotides have been incorporated into in vitro evolution experiments.51-56 The C5-position of the pyrimidine

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55 nucleobases is an appropriate pla ce to introduce functionality, as th e site lies in the major groove of the duplex, where appendages do not interfere with Watson-Cric k pairing. Appendages at this site are also well tolerated by RNA and DNA polymerases, which do not interact with the nucleobases in the major groove.57-60 Most of the previous work in this area has functionalized 2-de oxyuridine, although some work has also appended functionality through the 5-position of 2-d eoxycytidine analogues.61 We report here the synthesis of 2-deoxycytidin e nucleosides carrying am ino or thiol terminal functionality appended at position C5 via a f our-carbon alkynyl linker. We also report the successful incorporation of these as triphos phates into DNA using Vent (exo-) DNA polymerase and PCR amplification. Reports suggest that protected 2-deoxycytid ine nucleosides carrying a C5 acetylene group readily undergo undesi red cyclization.62 Anticipating this, we explored chemistry to attach substituents to the 5-position of an unprotected cytid ine nucleoside heterocy cle to circumvent any additional side products. Trifluoroacetyl protected 1-aminobut-3-yne was synthesized from but-3-yn-1-ol via a facile route adapted from the literature.63, 64 Mesylation of the free hydroxyl group of compound 1 allowed the conversion to the corres ponding azide in DMF using sodium amide. Reduction of the azide to the corres ponding amine was achieved via treatment with PPh3. Acidification go the reaction mixture, followed by th e extraction of side products yielded the free amine 5, isolated by the neutralizat ion of its acid salt. A fina l protection reaction with trifluoroacetic anhydride gave 6 in 51% yield (Figure 3-2).

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56 Figure 3-2. Synthesis of the trifluoroacetyl pr otected 1-aminobut-3-yne linker. Conditions: (a) MsCl/anhydrous ether/TEA/3 h/0C/ H2O; (b) NaN3/anhydrous DMF/3.5 h/67C/H2O; (c) PPh3/anhydrous ether/H2O/ 18 h/0 C to rt/10% HCl(aq); 10% NaOH (aq); (d) anhydrous MeOH/CF 3CO2CH3/18 h/0C to rt. Sonogashira coupling of the linkers with 5tritylated 5-iodo -2-deoxycytidine (8) in the presence of palladium(0) catalyst gave the desired products 9 and 10 (Figure 3-3). The 5tritylated nucleoside was used in preference to the unprotected 5-hydroxy nucleoside because of ease in handling with respect to monitoring and its greater solubility. This resulted in a significant conservation of solvents during colu mn chromatography purification. Substitution of the benzoyl-protecting group on 9 with a tert-butylthio group was achieved in a one-step reaction65 with di-t-butyl-1-(t-buty lthio)-1,2-hydrazine-d icarboxylate in th e presence of LiOH providing the more stable compound 11. Protection of 10 and 11 via treatment with acetic anhydride yielded mixtures of both mono ( 14, 15) and diacetylated products ( 13, 15) with the major products being the desired compounds 14 and 15. Attempts to selectively deacetylate 12 and 13 with an equimolar amount of the Lewis acid ZnBr2 gave the desired 14 and 15, together with the unreacted starting compound 12 and 13, in nearly equivalent amounts.

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57 Figure 3-3a. Synthesis of 3'-O-Acetyl-5'-O-(4,4'-Dime thoxytrityl)-5-(1-butynyl)-2'-deoxycytidine tertbutyl disulfide ( 14). Conditions: (a) DMTCl/TEA/DMAP/ Py anhydrous/6 h/0C to rt; (b) 2 /CuI/TEA /Pd(PPh3)4/DMF anhydrous/10-14 h/rt; (c) Di-t-butyl-1-(t-butylthio)-1,2hydrazine-dicarboxylate/LiOH/MeOH-THF/90 min anhydrous. (d) Py/DMAP/TEA/Ac2O/4 h/0C to rt. Figure 3-3b. Synthesis of 3'-O-Acetyl-5-(3''-Trifluoroacetamidobutynyl)-5'-O-4,4'-dimethoxytrityl-2'deoxycytidine ( 15). Conditions: (a) DMTCl/TEA/DMAP/ Py anhydrous/6 h/0C to rt; (b) 6 /CuI/TEA /Pd(PPh3)4/DMF anhydrous/10-14 h/rt; (c) Py/DMAP/TEA/Ac2O/4 h/0C to rt.

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58 Treatment with 2 equivalents of ZnBr2 yielded the N-4 deacetylat ed and deprotected 5-OH compounds 16 and 17 only. Compounds 16 and 17 were also obtained from 14 and 15 via the commercial deprotecting mixture (2.5% trichloroacetic acid in CH2Cl2) for solid phase oligonucleotide synthesis (Figure 3-4). Following literature procedure, triphosphate synthesis was carried out on compounds 16 and 17. Phosphorous oxychloride in trimethyl phosphate and a proton sponge gave the phosphorodichloridates as in termediates, which were converted in situ with pyrophosphate to th e corresponding triphosphate s. Deprotection via NH4OH hydrolysis gave the desired nucleotide triphosphates, 18 and 19 (Figure 3-5). Studies a ssessing the ability of the functionalized dCTP analogues ( 18 and 19) to serve as substrates for thermostable DNA polymerases under PCR conditions were then co nducted. Polymerases often behave differently in the presence of unnatural nucleosides.53 This prompted us to look at representatives from two evolutionary families of DNA polymerases66. Taq DNA polymerase from Thermus aquaticus (representing Family A) and Vent (exo-) DNA polymerase from Thermococcus litoralis (representing Family B) were examined as poten tial candidates. Primer extension experiments using PAGE purified 5-[ -32P]-radiolabeled primer (5GCG TAA TAC GAC TCA CTA TAG3) and template (5-GAC ACG CGC TAT AG T GAG TCG TAT TAC GC-3) were performed using primers oligos ordered from Integrated DNA Technologies, Coralville, IA. Taq polymerase did not incorporate 18 nor 19 with any acceptable efficiency (d ata not shown). Vent (exo-) DNA polymerase, on the other hand, successfu lly incorporated both triphosphates 18 and 19. Incorporation of 19 can be seen in Figure 3-6; Lane 4 opposite G in a single base extension. Upon the addition of a complete set of natu ral triphosphates excluding dCTP, the full-length extension product was obtained (Figure 3-6; Lane 5).

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59 Figure 3-4. Synthesis of 3'-O-Acetyl-5-(3''Trifluoroacetamidobutynyl)-2'-deoxycytidine ( 17) Conditions: (a) ZnBr2/MeOH/CHCl3/2 h/rt; (b) TCA in CH2Cl2/30 min/rt.

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60 Figure 3-5. Synthesis of 5-(3''-tert-butyl disu lfide 1-butynyl)-2'-deoxycyt idine-5'-triphosphate ( 18) and 5-(3''-Trifluoroacetamidobutynyl)-2'-deoxycytidine-5'-triphosphate ( 19 ). Conditions: (a) POCl3/(MeO)3P(O)/proton sponge/2.5 h/0 C; (b) Tri-nbutylammonium pyrophosphate/n-tributylam ine/DMF/TEAB/2 min/0 C to rt; (c) NH3/rt/18 h. Incorporation tria ls using analogue 18 with a protected thiol functional group, was also successful (data not shown). PCR amplification of an oligonucleotide replacing dCTP with compound 19 displaying a free amino group, using Vent (exo-) polymerase was also successful (Figure 3-7; Lane 6). The P CR experiment incorporated 22 and 31 functionalized cytidine analogues per strand in the forward and reverse r eactions respectively. Low concentrations of triphosphate (2 M) were used in the pr evious PCR amplification. Though compound 19 alone supported the synthesis of full-length products at such low concentr ations (Figure 3-7; lane 5), doubling the concentration of 19 increased the amount of full -length product formed. This

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61 suggested that 19 was able to support PCR amplification at concentrations similar dNTPs, though at a lower efficiency. In an experiment where a 1:1 mixture of 19 and dCTP was used, reduction in the amount of full-length product wa s observed relative to dCTP alone which is consistent with this conclusion. As a caveat, it is important to not e that though these results are consistent with, but do not absolutely prove, the presence of the functionalized cytidine derivative in the produc t. Two features of these results are particularly noteworthy. Figure 3-6. Primer extension experiment us ing amino-functionalized 2-deoxycytidine triphosphate 19. Denaturing 20% PAGE-urea gel was used. 5-Radiolabeled primer was visualized using a phosphoimager. A ll lanes contain annealed primer and template, buffer, and water. Lane 1: All natural dNTPs (0.1 mM each), no polymerase (negative control). Lane 2: Vent (e xo-) DNA polymerase and dCTP only showing pausing after incorporation of dC. Lane 3: Vent (exo-) polymerase and natural dNTPs showing formation of full-length product ( positive control). La ne 4: Vent (exo-) polymerase and 19 only, showing incorporation of a single 19 ; lower mobility due to positively charged functionality. Lane 5: Vent (exo-) polymerase and 19, dTTP, dGTP, dATP, expecting incorporation of three 19 s.

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62 First, in primer extension experi ments, DNA carrying 3 equivalents of 19 showed a double band of products. Polymerases frequently add a non-te mplated nucleotide to the product. We believe this is the simplest explanation for this observation.67 Second, although 19 supported the PCR amplification of DNA, the yield appeared to be lower than with standard dCTP. We considered the possibility that the positively charged amine linker group may disrupt the intercalation of the similarly cationic ethidium bromide. Figure 3-7. PCR amplification with 19 replacing dCTP. Agarose gel (2%) was used and stained with ethidium bromide. A total of 25 PCR cycles were run, with 2 min each incubation. Lane 1: Promega DNA ladder 25300 nts. Lane 2: Negative control, lacking polymerase. Lane 3: Positive control with standard dNTPs. Lane 4: Positive control including both 2-dCTP and 19 (1:1 ratio) and standard dNTPs. Lane 5: With 2 M 19, dATP, dGTP and dTTP. Lane 6: With 4 M 19, dATP, dGTP and dTTP. Template (100mer): 5-CGC ATT AT G CTG AGT GAT ATC TAT CCA GAC CTA GAA AGA GTG CAC TGA TGC TGT TCG AGC GCA CGG CCT CCA ACA TGC CGT CCA TGC ACC ACT A GA CCT C-3. Primer (24mer): 5-GAG GTC TAG TGG TGC ATG GAC GGC3. Reverse prim er (24mer): 5-CGC ATT ATG CTG AGT GAT ATC TAT-3. As the intensity of the band increases when the concentration of 19 is doubled, we we forced to reject this hypothesis. Rather, it seems the replacement of dCTP by 19 simply lowers the yield of full-length product. Accessibility to functionalized nucleoside de rivatives and polymerases that

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63 accept their triphosphates sufficiently well to support the polymerase chain reaction gives two new tools to those wishing to perform molecu lar biology with DNA cont aining thiol and amino functionalities. This is anothe r significant step toward the de velopment of a synthetic biology.68 Experimental General Experimental: Chemical reagents were purchased from Acros, Aldrich, Fischer, Berry & Associates. Reactions were carried under argon (Ar). TLCs were obtained on Whatman, silica gel plates (250 m Layer) and monitored with 254 nm fluorescence. Room temperature, where mentioned, range from 2224 C. Silica gel (230-425 mesh, Fischer) was used for column chromatography (CC). AG 1-X8 resin (Bio-Rad) was obtained as the Clform and converted to the HCO3 form by washing with 16 volumes of 1M NH4HCO3 solution, followed by de-ionized water and finally with 0.5 M NH4HCO3 solution; no Clwas detected. Ion-exchange chromatography was performed with DEAE Sephadex (Sigma) equilibrated in 0.2M (Et3NH) HCO3 (pH 7.0). NMR spectra was recorded on a Varian XL 300 spectrometer at 300 MHz, using TMS as an external reference for 1H and 13C while H3PO4 for 31P NMRs. UV spectra were measured on a Varian Cary 1 Bio spectrophotome ter. Mass spectrometry were recorded by the Spectroscopy Services of the University of Florida Chemistry Department; for LSIMS: Finnigan MAT-95Q apparatus, for HPLC/ESI-MS: Finnigan MAT (San Jose, CA) LCQ in electrospray ionization (ESI) mode and Beckman Instrument s (Fullerton, CA) System Gold, model 126 pump with Waters RP18 Symmetry Shield analyti cal column (2.1 x 150mm + guard). HPLC was performed using a Waters PrepLC 4000 System (1.2 mL Injection Loop) with a 486 tunable absorbance detector. Reversed phase HPLC separation was done using a Waters Prep Nova Pak HR C18 Preparative Column (6 M, 60, 25 x 100mm; WAT038510) with Waters Prep Nova Pak HR C18 Preparative Guard Column (6 M, 60, 25 x 10 mm; WAT038528).

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64 General Procedure for the Synthesis of the Linker: Buty-3-yne-1-methanesulfonate ( 3): Methanesulfonylchloride (34.37 g, 0.3 mol, 1.5 eq.) was added dropwise to a stirred solution of butyl-1-yne-3-ol 1 (14.0 g, 0.2 mol) and TEA (30.35 g, 0.3 mol, 1.5 eq.) in anhydrous ethyl ether (200 mL) at 0 C. After 3h, water (100 mL) was added to the S 2 reaction mixture. The organic layer was separated, washed with water (100 mL), dried with Na2SO4 and distilled in vacuo to yield 26.50 g (89.6 %) of 5 as dark yellow liquid. 1H NMR (CDCl3) : 2.10 (t, 1H, -CH), 2.64 2.69 (m, 2H, -CH2), 3.07 (s, 3H, -CH3), 4.31 (t, 2H, -CH2). 4-Azido-but-1-yne ( 4 ): Sodium azide (16.40 g, 0.25 mol) was added to a solution of the mesylate 3 (15.04 g, 0.1 mol) in anhydrous DMF (80 mL ). The mixture was stirred for 3.5 h at 67 C. The reaction mixture was poured over water (30 mL) and extracted with ethyl ether (3 x 100 mL). The solution was then dried over Na2SO4 and evaporated to yield 8.00 g (83.0%) of 4 as yellow liquid. 1H NMR (CDCl3) : 2.08 (t, 1H, -CH), 2.47 2.53 (m, 2H, -CH2), 4.40 (t, 2H, CH2). But-3-yne-1-amine ( 5): To a solution of azide 4 (8.00 g, 0.08 mol) in ethyl ether (50 mL) at 0 C, PPh3 (23.18 g, 0.08 mol) was added and allowe d to stir for 1.5 h. Water (3.0 mL) was then added to the reaction mixtur e and allowed to stir for anot her 16 h. The reaction mixture was poured over 10% aqueous HCl, extracted with et hyl ether (3 x 100 mL). The aqueous layer was made basic (pH = 9.0) with 10% aqueous NaOH a nd extracted with ethyl ether (5 x 100 mL). The solvent was dried over Na2SO4 and evaporated to yield 1.4 g (24.0 %) of 5 as light yellow liquid. 1H NMR (CDCl3) : 2.09 (t, 1H, -CH), 2.64 2.69 (m, 2H, -CH2), 2.84 (t, 2H, -CH2), 1.61 (bs, 2H, -NH2). N-But-3-ynyl-2,2,2-trifluoroacetamide ( 6): To a stirred solution of butyl-1-yne-3-amine 5 (1.4 g, 0.02 mol) in anhydrous MeOH (5.0 mL) at 0 C, was added methyl trifluoroacetate (2.45

PAGE 65

65 mL, 0.024 mol 1.2 eq.) drop wise and allowed to stir for next 14-16 h at room temperature. Thereafter, the solvent was evaporated and the re sidue was diluted with 10 mL chloroform and washed with aqueous NaHCO3 solution (2 x 10 mL) and water (1 x 10 mL). The organic layer was dried over Na2SO4 and evaporated to yield an orange residue which upon di stillation yielded 6 1.70 g (50.8 %), as light yellow liquid (s olidified at -20 C ). 1H NMR (CDCl3) : 2.09 (t, 1H, CH), 2.47 2.53 (m, 2H, -CH2), 3.53 (t, 2H, -CH2), 6.80 (bs, S3 1H, -NH). 13 C NMR (CDCl3) : 18.87 (-CH2), 21.98 (-CH2), 70.88 (-CH), 80.21 (-C), 114.03, 117.84, 120.89 (-CF3 ), 156.91 158.92 (-CO). Mass (m/z): 166 (M +1). 3) General Procedure for the 5-Tritylation Reaction: 5'-O-4,4'-dimethoxytrityl-5-iodo-2'deoxycytidine ( 8): 5-iodo-2'-deoxycytidine 7 (0.353 g, 10.0 mmol) was co-evaporated twice with pyridine and then dissolved in anhydrous pyridine (5.0 mL). To this solution at 0 C under Ar, Et3N (0.278 mL, 20.0 mmol, 2.0 eq.), DMAP ( 0.030 g, 2.5 mmol) and 4,4'-dimethoxytrityl chloride (0.677g, 20.0 mmol, 2.0 eq.) were added and stirred for first 15 min at 0 C and then at room temperature for 5 hours. The reaction mi xture was quenched with MeOH (2 mL), the solvent was evaporated and the residue was extracted with EtOAc, washed with aqueous NaHCO3 solution. Usual work-up yielded the semi-sol id residue which was purified by silica column chromatography (CHCl3: MeOH; 97:3 v/v) to give 0.260g (42 %) of, 8 (Rf. 6.5; CHCl3: MeOH; 82:18, v/v) as white solid. 1H NMR (DMSO-d6) : 2.03 2.28 (m, 2H, 2'), 3.15-3.20 (m, 2H, 5'), 3.74 (s, 6H, OMe), 3.893.90 (m, 1H, 4'), 4.18-4.22 (m, 1H, 3'), 5.26 (d, 1H, 3'-OH), 6.10 (t, 1H, 1'), 6.60 (bs, 1H, -NH), 6.89 (d, 4H, aromatic), 7.22 7.41 (m, 9H, aromatic), 7.85 (bs, 1H, NH), 7.96 (s, 1H, H-6). LRMS-FAB (NBA): 693.71 (M+) 4) General Procedure for Sonogashira Coupling: Compound 8 (0.353 g, 10.0 mmol) was dissolved in anhydrous DMF (10 mL) and degassed for 10minutes. Compound 2 (0.09 g, 10.0 m

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66 mol, 1.0 eq.) or 6 (0.173 g, 15.0 m mol, 1.5 eq.), TEA (0.276 mL, 20.0 mmol, 2.0 eq.), CuI (0.034 g, 2.0 mmol, 0.2 eq.) and (Ph3P)4Pd (0.155 g, 1.0 mmol, 0.1 eq.) were added to the reaction mixture with constant st irring at room temperature unde r Ar. After 18 h of stirring, the reaction mixture was diluted with EtOAc (3 mL), the solvent was removed in vacuo, the residue was washed with aqueous NaHCO3, extracted with EtOAc (3 x 25 mL), dried over Na2SO4 and evaporated to yield a semi-solid residue which was then purified by silica column chromatography. 5'-O-4, 4'-dimethoxytrityl-5-(4-benz oylthio-1-butynyl)-2'-deoxycytidine (9): Elution at: 96:4 (CHCl3: MeOH; v/v) to yield 0.254 g (42.0 %) as foamy yellow solid. Rf: 0.5 (88:12; CHCl3: MeOH, v/v). 1H NMR (CD3OD) : 2.12 2.24 (m, 1H, 2'), 2.51 (t, 2H, CH2), 2.71 2.80 (m, 1H, 2'), 2.94 -3.04 (m, 2H, -CH2), 3.24 3.38 (m, 2H, 5'), 3.75 (s, 6H, -OMe), 4.17 4.20 (m, 1H, 4'), 4.46 4.54 (m, 1H, 3'), 5.92 (bs, NH), 6.60 (t, 1H, 1'), 6.82 (d, 4H, aromatic), 7.18 7.36 (m, 12H, aromatic), 7.90 (d, 2H, ArH), 8.14 (d, 1H, H-6). 13C NMR (CD3OD) :21.2(3"), 27.8(4"), 36.1(2'), 55.1(OCH3), 63.7(5'), 72.1(3'), 77.5 (1"), 86.3(1'), 87.0(4'), 88.1(OC(PH3)3), 90.1(2"), 94.5(5), 127.0, 12128.0, 129.0, 130.2, 133.8, 135.8, 136.4(Ar), 143.6(6), 154.1(2), 158.2(4), 191.1(SCOPh). HRMS -FAB (NBA): m/z = 717.8134 (M+ + 1). 5'-O-4, 4'-dimethoxytrityl-5-(3''-Trifluoroacetamidobutynyl)-2'-deoxycytidine (10): Elution: 93:7; CHCl3: MeOH; (v/v) to yield compound 0.164 g (42.0 %) as foamy yellow solid. Rf: 0.6 (88:12; CHCl3: MeOH, v/v) 1H NMR (CD3OD) : 2.20 2.32 (m, 1H, 2'), 2.39 (t, 2H, CH2), 2.45 -2.53 (m, 1H, 2' ), 3.25 3.32 (m, 2H + 2H, 5' + -CH2), 3.75 (s, 6H, -OMe), 4.05 4.10 (m, 1H, 4'), 4.45 4.50 (m, 1H, 3'), 6.18 (t, 1H, 1'), 6.84 (d, 4H, aromatic), 7.18 7.36 (m, 9H, aromatic), 8.19 (d, 1H, H-6). HRMS-FAB (NBA): 694.2587 (M+)

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67 General procedure for the replacem ent of the thiobenzoyl group : 5'-O-(4,4'Dimethoxytrityl)5-(1-but ynyl)-2-deoxycytidine te rt-butyl disulfide ( 11 ): To a stirred solution of 9 (0.113 g, 0.17 mmol) in THF/MeOH (both anhydrous ) (3:1, 4.0 mL), ditertbutyl 1-(tertbutylthio)-1,2-hydrazin e-dicarboxylate (0.07 g, 0.22 mmol) and LiOH. H2O (0.14g, 0.34 mmol, 1.5 eq.) were added quickly, causi ng the reaction mixture to temporarily turn lilac. The reaction mixture was stirred for 1 h 20 min, diluted with Et2O and washed with brine.S5 The lilac middle layer formed was kept aside, diluted with CH2Cl2 and washed with brine. Combined organic phases were dried over Na2SO4 and concentrated to yield the crude product, which was purified by chromatography on silica gel (CHCl3/CH3OH 97:3), to yield 11 (0.90 g 68%) as a yellow foam. 1H NMR (CDCl3) : 1.29 (s, 9H, tbutyl), 2.07 2.14 (m, 2H, 2'), 2.58 (t, 2H, -CH2), 2.71 -2.82 (m, 2H, -CH2), 3.29 3.40 (m, 2H, 5'), 3.7 7 (s, 6H, -OMe), 4.10 4.12 (m, 1H, 4'), 4.50 4.58 (m, 1H, 3'), 5.80 (bs, NH), 6.29 (t, 1H, 1'), 6.83 (d, 4H, aromatic), 7.19 7.40 (m, 9H, aromatic), 8.13 (d, 1H, H-6). 13C NMR (CD3OD) :21.2(3"), 27.8(4"), 30.1(C(CH3)3), 38.1(2'), 48.0(C(CH3)3), 57.1(OCH3), 63.7(5'), 72.4(3'), 77.5 (1"), 86.8(1'), 87.4(4'), 91.8(OC(PH3)3), 94.5(2"), 113.0(5), 127.0, 128.0, 130.2, 136.1(Ar), 144.2(6), 154.8(2), 164.8(4). HRMS-FAB (NBA): 702.8277 (M+) General Procedure for Acetylation Reaction: Compound 10 or 11 (1.0 mmol) was coevaporated with pyridine and then dissolved in anhydrous pyridine (2.0 mL), TEA (2.0 mmol, 2.0 eq.), DMAP (0.25 mmol, 0.25 eq.) and Ac2O (2.5 mmol, 2.5 eq.) were added to the reaction mixture and allowed to stir at 0C under Ar for 30 min. After 3 h stirring at room temperature, the reaction was quenched with MeOH (5 mL). The solvent was removed in vacuo, the residue obtained was washed with water and extracted with EtOAc (3 x 20 mL). Th e organics were dried

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68 over Na2SO4 and concentrated to afford a residu e which was purified by silica gel Column Chromatography to yield the di12, 13 and mono14, 15 acetylated products respectively. 3'-O-4-N-Diacetyl-5'-O-(4,4'-dimethoxytrityl )-5-(1-butynyl)-2'-deoxycytidine tert-butyl disulfide ( 12 ): Eluting at 98:2; CHCl3: MeOH; (v/v) and 9 (0.020 g; 54.80 % ) Rf: 0.8 (CHCl3: MeOH; 88: 12, v/v). 1H NMR (CDCl3) : 1.29 (s, 9H, tbutyl), 2.06 (s, 3H, -OCH3), 2.26 2.40 (m, 2H, 2'), 2.50 (t, S6 2H, -CH2), 2.69 (s, 3H, -NCH3), 2.78 -2.88 (m, 2H, -CH2), 3.36 3.80 (m, 2H, 5'), 3.76 (s, 6H, -OMe), 4.22 4.25 (m, 1H 4'), 5.35 5.42 (m, 1H, 3'), 6.30 (t, 1H, 1'), 6.83 (d, 4H, aromatic), 7.22 7.41 (m, 9H, aromatic), 8.35 (d, 1H, H-6). 13C NMR (CDCl3) : 21.2(C(CH3)), 23.5(3"), 26.8(4"), 30.1(C(CH3)3), 38.1(2'), 41.0(C(CH3)3), 57.1(OCH3), 63.7(5'), 72.0(3'), 75.5 (1"), 87.1(1'), 87.4(4'), 91.8(OC(PH3)3), 96.5(2"), 113.8(5), 127.0, 128.0, 129.2, 130.2, 136.1(Ar), 137.0(6), 143.5(6), 154.8(2), 158.3(4) 161.2(OCH3). HRMS-FAB (NBA): 785.9912(M+) 3'-O-4-N-Diacetyl-5'-O-4, 4'-dimethoxytrity l-5-(3''-Trifluoroacetamidobutynyl)2'deoxycytidine ( 13) : Eluting at 99:1; CHCl3: MeOH; (v/v) Rf: 0.8 (CHCl3: MeOH; 82: 18, v/v). 1H NMR (CDCl3) : 2.09 (s, 3H, -OCH3), 2.12 2.18 (m, 2H, 2'), 2.39 (t, 2H, -CH2), 2.98 (s, 3H, -NCH3), 3.22 3.37 (m, 2H + 2H, 5' + -CH2), 3.75 (s, 6H, -OMe), 4.21 4.26 (m, 1H, 4'), 5.31 5.36 (m, 1H, 3'), 6.25 (t, 1H, 1'), 6.80 (d, 4H, aromatic), 7.19 7.39 (m, 9H, aromatic), 8.35 (d, 1H, H-6). HRMS-FAB (NBA): 780.7981(M+) 3'-O-Acetyl-5'-O-(4,4'-Dimethoxytrityl)-5-(1 -butynyl)-2'-deoxycytidine tert-butyl disulfide ( 14 ) : Eluting at 96:4; CHCl3 : MeOH (v/v) Rf: 0.6 (CHCl3: MeOH; 88: 12, v/v). 1H NMR (CDCl3) : 1.30 (s, 9H, tbutyl), 2.05 (s, 3H, -OCH3), 2.20 2.31(m, 1H, 2'), 2.58 (t, 2H, CH2), 2.66-2.75(m, 1H, 2'), 3.34 3.41(m, 2H + 2H, 5' + -CH2), 3.79(s, 6H, -OMe), 4.18 4.21

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69 (m, 1H, 4'), 5.18 5.20 (m, 1H, 3'), 5.95 (bs, 1H ,-NH), 6.18(t, 1H, 1'), 6.84(d, 4H, aromatic), 7.20 7.43(m, 9H, aromatic), 8.18(d, 1H, H-6). HRMS-FAB (NBA): 743.9544 (M+) 3'-O-Acetyl-5-(3''-Trifluoroacetamidobutynyl)-5'-O-4,4'-dimethoxytrityl-2'-deoxycytidine ( 15) : Eluting at 98:2; CHCl3 : MeOH (v/v) Rf: 0.7(CHCl3 : MeOH; 82 : 18, v/v).S7 1H NMR (CDCl3) : 2.05(s, 3H, -OCH3), 2.20 2.31(m, 1H, 2'), 2.42(t, 2H, -CH2), 2.62-2.71(m, 1H, 2'), 3.24 3.40(m, 2H + 2H, 5' + -CH2), 3.78(s, 6H, -OMe), 4.16 4.19(m, 1H, 4'), 5.31 5.36(m, 1H, 3'), 6.09(bs, 1H,-NH), 6.30(t, 1H, 1'), 6.82(d, 4H, aromatic), 7.23 7.43(m, 9H, aromatic), 8.16(d, 1H, H-6). HRMS-FAB (NBA): 736.2674 (M+) General Procedures for De-protection Method A: To a stirred solution of compounds 12 or 13 (0.2 mmol) in anhydrous MeOH and CHCl3 (4:1; 2 mL) under Ar, was added ZnBr2 (0.4 mmol; 2.0 eq.) and allowed to stir for 2 hr at room temperature. The solvent was rem oved in vacuo and the residue was purified over silica gel CC to give compounds 14 or 15 respectively. Method B: To a stirred solution of compound 14 or 15 (0.5 mmol) in anhydrous CH2Cl2 (2 mL) under Ar, was added deprotecting solvent (2.0 mL) (trichloro acetic acid in dichloromethane). It was allowed to stir for 30 min at room temperature. Thereafter, a drop of TEA was added to the reaction mixture, the solvent removed in vacuo and the residue purified over CC to give compounds 16 or 17 respectively. 3'-O-Acetyl-5-(1-butynyl)-2'-deoxycy tidine tert-Butyl Disulfide ( 16) : Elution at 94:6; CHCl3: MeOH (v/v), gave compound 16 (0.012 g, 52.17%). Rf: 0.5 (CHCl3: MeOH; 82:18, v/v). 1H NMR (CDCl3) : 1.34 (s, 9H, tbutyl), 2.09 (s, 3H, -OCH3), 2.31 2.41 & 2.48 2.58 (m, 2H, 2'), 2.78 2.80 (m, 2H, -CH2 x 2), 3.89 3.96 (m, 2H, 5'), 4.10 4.15 (m, 1H, 4' ), 5.33 5.38 (m, 1H, 3' ), 6.08 (bs, 1H, NH), 6.19 (t, 1H, 1'), 7.98 (d, 1H, H-6). 13C NMR (CDCl3) : 20.2(3"), 21.0(CO(CH3)), 30.1(C(CH3)3), 38.1(4"), 38.5(2'), 48.2(C(CH3)3), 63.7(5'), 73.0(1''), 75.5 (2'),

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70 86.0(1'), 88.0(4'), 92.0(2"), 95.5(5), 136.0(6), 143.5(2), 154.1(2). LRMS-FAB (NBA): 442.15 (M+) 3'-O-Acetyl-5-(3''-Trifluoroacetamidobutynyl)-2'-deoxycytidine ( 17) : Elution at 96:4; CHCl3:MeOH (v/v), gave compound 17 (0.012 g, 52.17%). Rf: 0.5(CHCl3:MeOH; 82: 18, v/v). 1H NMR (CDCl3) : 2.07(s, 3H, -OCH3), 2.16 2.24 & 2.45 2.55( m,2H, 2' ), 2.70( t, 2H, -CH2 ), 3.52( t, 2H, -CH2), 3.79 3.81( m, 2H,5'), 4.10 4.15( m,1H, 4' ), 5.25 5.30( m, 1H, 3' ),6.22(t, 1H, 1'), 8.23(d, 1H, H-6). LRMS-FAB: 434.13 (M+). 13C NMR (CDCl3) : 19.8(3"), 22.5(CO(CH3)), 38.8(4"), 39.0(2'), 61.8(5'), 72.0(3'), 75.5(1'), 74.5 (1"), 86.0(1'), 87.5(4'), 92.7(2"), 95.5(5), 143.0(6), 155.8(2) 154.1(2), 168.0(4), 171.8(CO(CF3)), 172.0(CO(CH3)). General Procedure for triphosphate synthesis: The following precautions were taken: a) Compound 16 or 17 was co-evaporated with pyridine and dried over P2O5 overnight before the reaction. b) Tribuylammonium pyrophosphate in DMF (anhyd.) a nd n-tribuylamine were kept under Ar atmosphere over 4 molecular sieves (activated) for 48 h. Compound 16, 17 (0.18 mmole) was stirred in trimethylphosphate (0.70 mL ) with 1,8-bis (dimethylamino) naphthalene (proton sponge, 0.27 mmol) at 0 C for 5 min. Phosphorous oxychloride (0.27 mmol) was added and the mixture was allowed to stir at 0-4 C for 2.5 h. The solution of tri-n-butylammonium pyrophosphate (10.0 mmol), tri-n-butylamine (10.0 mmol) in anhyd. DMF (2.0 mL) was added quickly to the reaction mixture at 0 C. After 1 min, an aqueou s solution of triethylammonium bicarbonate (20 mL; 0.2 M) was added and allowed to stir for next 10 min. After evaporation (in vacuo), the residue was treated with aqueous a mmonia (2 mL), and stirred overnight at room temperature. Ammonia is removed in vacuo and the residue was subjected to the purification protocols which follow. Purificati on: This was carried out in two different steps, ion-exchange, followed by RP-HPLC. i) Ion-exchange: Th e crude sample, after the removal of NH4OH, was

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71 dissolved in water (MQ grade) and charged on a DEAE (diethylam inoethyl) cellulose (fast flow; fibrous form, 25.0 g in a 26.0 x 5.0 cm column wh ich was previously equilibrated in 0.2 M TEAB, pH 7.0). Starting from 0.2 M TEAB, the tr iphosphate was collected at 0.4 to 0.5 M. The fractions (UV active) were pool ed and lyophilized. ii) RP-HPLC (Carried out by Dr. A. P. Kamath): HPLC was done at a flow rate of 0.75 mL/ min. using 10mM (Et3NH)OAc (pH 7.0) (mobile phase A) and 20% MeCN in mobile phase A (mobile phase B) in the following linear gradient as: 95% A/ 5% B(10 min), 65% A/ 35% B(30 min), 100% B(10 min) and finally 95% A/ 5% B(10 min); retention time 24 min. The frac tions were pooled and lyophilized to yield the pure triphosphate. 5-(3''-tert-butyl disu lfide 1-butynyl)-2'-deoxycyt idine-5'-triphosphate ( 18 ): UV (water) max = 237, 297 nm. 31P NMR (D2O) : -9.80, -9.90 (d, 1P, J= 19.2 Hz, ), 10.04 10.06 (d, 1P, J= 19.2 Hz, ), 22.12 (t, 1P, J= 19.0 Hz, ). 1 R (D2O) : 1.34 (s, 9H, t-butyl), 2.18 2.20 (m, 1H, 2/), 2.22 2.35 (m, 1H, 2/), 2.75 (t, 2H, J= 4.6Hz, 2"), 2.80 (t, 2H, J= 4.6Hz, 1"), 4.02 4.08 (m, 2H, 5'), 4.42 4.49 (m, 2H, 4'), 4.46 4.54 (m, 2H, 3'), 6.12 (t, 1H, J= 6.6Hz, 1'), 7.90 (s, 1H, H-1). HPLC MS: 640 (M+H) 5-(3''-Trifluoroacetamidobutynyl)-2'-deoxycytidine-5'-triphosphate ( 19 ): UV (water) max = 234.5, 295.0 nm 31P NMR (D2O) : -9.09, -9.25 (d, 1P, J= 19.2 Hz, ), 10.42, 10.57 (d, 1P, J= 19.2 Hz, ), 21.75 (t, 1P, J= 19.0 Hz, ). 1 R (D2O) : 2.10 2.20 (m, 1H, 2/), 2.29 2.39 (m, 1H, 2/), 2.73 (t, 2H, J= 4.6Hz, 2"), 3.05 (t 2H, J= 4.6Hz, 1"), 3.68 3.72 (m, 2H, 5'), 4.08 4.11 (m, 2H, 4'), 4.31 4.40 (m, 2H, 3'), 6 .07 (t, 1H, J= 6.6Hz, 1'), 7.88 (s, 1H, H-1). HPLC MS: 535 (M+H) General Experimental: Biochemical: The primer (5-GCG TAA TAC GAC TCA CT A TAG ) and template (5-GAC ACG CGC TAT AGT GAG TCG TAT TAC GC) were pur chased from IDT. The primer was 5

PAGE 72

72 radiolabeled with 32P using T4 polynucleotide kinase (Promega) and Redivue [ 32P] ATP (Amersham Pharmacia Biotech). The radiolab eled primer was purified using QIAquick nucleotide removal kit (Qiagen). A solution of annealed primer was purified and template was prepared (9:1 ratio of unlabeled and labeled prim er with a 20% excess of template) by incubating at 95 C for 5 min then cooling slowly to room temperature. The concentration for the functionalized dCTPs were approximated assuming = 9000 M-1cm-1. Reactions were prepared [10 L, 2 M unlabeled primer, 0.2 M 32P labeled primer, 2.4 M template, 1X thermopol reaction buffer, 1 M dNTP(s), 2 units Vent (exo-) DNA polymerase (NEB)], incubated for 2 min at 72 C and quenched with 5 L quenching buffer (95% formamide, 40 mM EDTA, 0.05% xylene cyanol, 0.05% bromophenol blue). The reactions were analyzed by running 3 L of each on 20% denaturing polyacrylamide gels. The reac tion ran for 30 minutes at 37 C. Quantity Reagent 1 mL 1 nmol primer 1 mL 5 units T4 polynucleotide kinase 1 mL adenosine 5-[ -32P] triphosphate, triethyl ammonium salt 1 mL 10T T4 kinase buffer 6 mL water 10) Acknowledgments I am grateful to Dr. Jodie V. Johnson and th e personnel at the mass spectroscopy lab at the Department of Chemistry for acquisition of the ma ss spectra. Useful discussions with Drs. C. R. Geyer, T. R. Battersby, S. C. Jurczyk, and H. A. Held are acknowledged. Extensive help in the HPLC purification by Dr. A. P. Kamath is highly acknowledged. This work was partially supported by grants from the Department of Defense 6402-202-LO-B and the National Science Foundation (CHE-0213575).

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73 CHAPTER 4 FUTURE WORK Though significant progress towards understanding the origins of life is shown here, much work remains to be done. These include GC/MS analysis of the carbohydrate formation reactions, exploring phosphate-buffered reaction mediums and the inclusion of trace elements such as magnesium, manganese and other DNA f riendly minerals. GC/MS studies could help further confirm and determine low concentra tion carbohydrates. Phosphate buffered solutions may yield phosphorylated carbohydrate products, perh aps with the aid of other trace minerals. Molybdenum mineralogy may help us use dead end species, which do not have enolizable hydrogens or whose retro-aldols only yield their parent molecule s. The branched species do not release HCHO easily. This may not be entirely bad, as they are storehouses of relatively stable carbohydrates. The mineral molybdite (MoO3) can equilibrate these, in water (pH 4.2, 70 C) to linear carbohydrates via a Bilik st yle reaction (Fig. 4-1)32. As ribose and other pentoses are stabilized by borate under the conditions where they ar e formed, further analysis of the borate concentration landscape should also be pursued. Figure 4-1. The role of molybdenum in carbohydrate interconversion.

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74 In samples containing only glyceraldehyde in CBA buffer incubated for 5 minutes at 25 C, peaks at 103-111 ppm were observed. These were tentatively assigned, but not yet confirmed, to ketohexoses (Figure 4-2). This class of saccharides includes d-psicose, d-fructose, d-sorbose and d-tagatose (Figure 4-3). A similar patte rn is also observed when dihydroxyacetone is dissolved in CBA bu ffer (Figure 4-4). Figure 4-2. A sample of DL-glyceraldehyde (Aldrich, 38 mg) in CBA buffer (0.30 mL) incubated for 5 minutes at 25 C. Spectrum file: dl_glyceraldehyde_38mg_300ulcba_022507.pdf

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75 Figure 4-3. The ketohexoses (psico se, fructose, sorbose & tagatose) are presumably formed by the reaction of either di hydroxyacetone or glyceral dehyde with themselves. Figure 4-4. A sample of dihydroxya cetone (Aldrich, 30 mg) in CBA buffer (0.50 mL) incubated for 3 hours at 25 C. Spectrum file: 070530_dha_cba_meohref.pdf

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76 The prebiotic accumulation of mate rial into these six carbon suga rs is significant as they provide even more complex car bohydrates to the available preb iotic building blocks believed necessary for life to occur. For example, these carbohydrates could oligomerise to form structures, they could simply complex with various minerals and act as catalysts, or they could regulate the formation of furthe r carbohydrates by rest ricting their reaction environment. Here we have demonstrated significant progress into understanding the processes and products of the borate mediated pr ebiotic synthesis of sugars, formed as stable products from formaldehyde and lower carbohydrates such as glycolaldehyde and dihydroxyacetone. Further work in this area needs to tie up loose ends regarding the formation of nucleosides and oligomers. How the heterocycle, for example adenin e, becomes attached to the C-1 of ribose in a prebiotic environment is a key st ep in origins of life chemistry.

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77 APPENDIX A NMR SPECTRA OF FURTHER EX PERIMENTS AND CARBOHYDRATES The following compilation of reactions and their NMR signals are a provided as a supplement and reference for the reader. An electronic version of this is available. Reactions involving glycolaldehyde Glycolaldehyde in CBA Spectrum Name: 070510_12C-glycolald ehyde_20mg_cba_1ml_refmeoh.pdf Glycolaldehyde dimer (20 mg) was dissolved in 1 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hour s total acquisition at 25C). 13C NMR (D2O): 167.091, 103.272, 82.478, 82.231, 76.599, 71.602, 49.500 (MeOH). Four sharp signals were observe d. These signals superimposed on those arising from authentic threose [103.2, 82.3, 76.6, 71.6]. Formation of threose from glycolaldehyde Threose has the following peaks in CBA buffer: 103.279, 82.333, 76.607, 71.614. Glycolaldehyde in CBA Spectrum Name:070515_12C-glycolald ehyde_20mg_cba_1ml_refmeoh.pdf Glycolaldehyde dimer (20 mg) was dissolved in 1 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.042, 103.268, 82.318, 76.599, 71.598, 49.500 (MeOH).

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78 1-13C-Glycolaldehyde in D2O Spectrum Name: 070511_1-13C-glyco laldehyde_0.2ml_d2o_0.8ml_refmeoh.pdf 1-13C-Glycolaldehyde (0.200 mL) was mixed with 0.800 mL CBA buffer. The mixture was agitated vigorously using a vor tex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR sp ectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 205.104, 201.721, 181.850, 175.442, 139.961, 135.689, 130.024, 118.816, 110.314 ,109.615, 102.516, 99.827, 99.148, 97.763, 95.375, 93.056, 91.114, 90.649, 90.332, 78.339, 73.918, 72.834, 65.422, 65.113, 64.782, 54.646, 51.796, 49.500 (MeOH), 47.452, 36.393, 46.093, 45.273, 42.023, 35.393, 34.928, 30.263, 27.920, 25.052, 23.347, 18.658, 8.239 1-13C-Glycolaldehyde in CBA (t = 0) Spectrum Name: 070511_1-13C-glycolalde hyde_0.5ml_cba_0.5ml_refmeoh-166.889_t=0.pdf 1-13C-Glycolaldehyde (0.500 mL) was mixed with 0.500 mL CBA buffer. The mixture was agitated vigorously using a vor tex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR sp ectrum was begun within four minutes, and was completed after 512 transients (ca. 20 minutes total acquisition at 25C). 13C NMR (D2O): 177.536, 171.680, 166.889, 165.516, 143.684, 123.169, 105.988, 103.222, 98.286, 93.262, 93.094, 90.317, 87.773, 82.600, 82.054, 76.901, 76.653, 76.367, 69.344, 62.298, 60.822, 49.500 (MeOH), 31.159, 29.797

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79 1-13C-Glycolaldehyde in CBA (t = 1 hour) Spectrum Name: 070511_1-13C-glycolalde hyde_0.5ml_cba_0.5ml_refmeoh_1hourlater.pdf 1-13C-Glycolaldehyde (0.500 mL) was mixed with 0.500 mL CBA buffer. The mixture was agitated vigorously using a vor tex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR sp ectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.673, 166.889, 105.984, 103.222, 98.282, 93.266, 93.094, 90.378, 82.604, 82.268, 82.051, 76.308, 76.649, 76.359, 75.981, 75.390, 71.545, 71.156, 69.447, 69.332, 65.266, 49.500 (MeOH), 40.562, 39.399, 23.900 1-13C-Glycolaldehyde in CBA (t = 6 hours) Spectrum Name: 070511_1-13C-glycolalde hyde_0.5ml_cba_0.5ml_refmeoh_6hourslater.pdf 1-13C-Glycolaldehyde (0.500 mL) was mixed with 0.500 mL CBA buffer. The mixture was agitated vigorously using a vor tex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR sp ectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.885, 103.222, 98.278, 93.277, 92.377, 90.321, 82.604, 82.318, 82.051, 76.908, 76.653, 76.359, 75.985, 75.768, 75.413, 73.861, 71.545, 71.389, 71.156, 70.877, 69.363, 68.013, 65.117, 49.500 (MeOH), 40.566, 23.904

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80 1-13C-Glycolaldehyde in CBA (t = 8 hours) Spectrum Name: 070511_1-13C-glycolalde hyde_0.5ml_cba_0.5ml_refmeoh_8hourslater.pdf 1-13C-Glycolaldehyde (0.500 mL) was mixed with 0.500 mL CBA buffer. The mixture was agitated vigorously using a vor tex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR sp ectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.696, 171.673, 166.885, 103.371, 103.222, 98.283, 93.281, 90.344, 82.604, 82.321, 82.054, 76.908, 76.653, 76.359, 76.172, 75.985, 71.389, 71.137, 70.874, 69.676, 69.336, 68.020, 65.060, 49.500 (MeOH), 42.405, 40.566, 23.904 1-13C-Glycolaldehyde in CBA (t = 6 hours) Spectrum Name: 070515_1-13C-glyco laldehyde_0.5ml_cba_0.5ml.pdf 1-13C-Glycolaldehyde (0.500 mL) was mixed with 0.500 mL CBA buffer. The mixture was agitated vigorously using a vor tex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR sp ectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 182.068, 171.673, 166.862, 106.014, 103.359, 103.222, 103.058, 102.863, 100.678, 98.282, 82.604 82.321, 82.054, 81.440, 76.912, 76.653, 76.359, 76.187, 75.985, 75.772, 75.661, 75.413, 73.849, 71.549, 71.247, 72.167, 70.877, 70.641, 68.016, 66.899, 63.111, 62.909, 61.818, 49.500 (MeOH), 40.566, 23.904

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81 Glycolaldehyde + H13CHO Spectrum Name: 070617_gol_1.4mg_h13cho_6ul_cba_nomeoh.pdf Glycolaldehyde (1.4 mg) was dissolved in CBA (1 mL) buffer. To this was added 6 L of H13CHO (6.66 M) and agitated vigorously using a vortex stirrer. Acquisition of an NMR spectrum was begun within four minutes, and wa s completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.000 (carbonate), 82.095, 70.403, 63.240 Glycolaldehyde + H13CHO Spectrum Name: 070617_gol_1.8mg_h13cho_48ul_cba_nomeoh.pdf Glycolaldehyde was dissolved in 1 mL of CBA buffer. To this was added 48 L of H13CHO (6.66 M) and agitated vigorously using a vortex stirrer. Acquis ition of an NMR spectrum was begun within four minutes, and was completed af ter 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.056, 167.000 (carbonate), 105.099, 104.565, 89.187, 88.232, 86.449, 86.133, 85.025, 83.522, 83.248, 82.111, 81.030, 80.708, 80.574, 79.156, 77.767, 77.647, 73.759, 73.267, 68.965, 68.733, 65.252, 63.335, 62.647, 67.517 Spectrum Name: 071130_gol_8mg_ICN_cba_0.5ml_nomeoh_t=4min-1.5hr.pdf Glycolaldehyde (8 mg, 0.133 mm ol) was dissolved in CBA bu ffer (0.5 mL). The tube was shaken using a hand vortexer and was transferred to an NMR tube. To this was added 10 L of a 10% MeOH in D2O was added as a reference. The tube was maintained at 25 C and an NMR spectrum was acquired after 4 minutes.

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82 13C NMR (D2O): 167.198, 103.279, 93.033, 82.329, 76.607, 71.613, 69.477, 49.500 (MeOH). Threose can already be observed emerging at 103.2, 82.3, 76.6 and 71.6. Spectrum Name: 071130_gol_8mg_ICN_cba_0.5ml_nomeoh_t=1.5hr-3hr.pdf Glycolaldehyde (8 mg, 0.133 mm ol) was dissolved in CBA bu ffer (0.5 mL). The tube was shaken using a hand vortexer and was transferred to an NMR tube. To this was added 10 L of a 10% MeOH in D2O was added as a reference. The tube was maintained at 25 C and an NMR spectrum was acquired after 1.5 hours. 13C NMR (D2O): 167.205, 103.279, 93.048, 82.329, 76.603, 76.054, 75.752, 73.868, 71.613, 69.378, 49.500 (MeOH). Spectrum Name: 071130_gol_31mg_aldric h_cba_1.5ml_meohref_t=3hr-4.5hr.pdf Glycolaldehyde (8 mg, 0.133 mm ol) was dissolved in CBA bu ffer (0.5 mL). The tube was shaken using a hand vortexer and was transferred to an NMR tube. To this was added 10 L of a 10% MeOH in D2O was added as a reference. The tube was maintained at 25 C and an NMR spectrum was acquired after 3 hours. 13C NMR (D2O): 167.160, 103.279, 82.329, 76.607, 76.039, 75.753, 73.872, 71.610, 71.114, 68.036, 49.500 (MeOH). Spectrum Name: 071130_gol_31mg_aldrich_c ba_1.5ml_meohref_t=4.5hr-6hr.pdf Glycolaldehyde (8 mg, 0.133 mm ol) was dissolved in CBA bu ffer (0.5 mL). The tube was shaken using a hand vortexer and was transferred to an NMR tube. To this was added 10 L of a

PAGE 83

83 10% MeOH in D2O was added as a reference. The tube was maintained at 25 C and an NMR spectrum was acquired after 4.5 hours. 13C NMR (D2O): 167.163, 103.279, 82.329, 76.607, 71.613, 49.500 (MeOH). All four peaks correspond to the formation of threose, with carbonate at 167.16 and the spectrum referenced to methanol at 49.500. Spectrum Name: 070716_GOL_300ul_HCHO_3 5ul_SCBA_200ul_firstspectrumtaken070719.pdf A 0.110 M solution of 1-13C-glycolaldehyde (300 L, Omicron Biochemicals) and HCHO (35 L, Fisher) were mixed in 0.200 mL SCBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.634, 166.611, 111.332, 103.207, 98.271, 62.600, 82.314, 82.047, 77.011, 76.920, 76.664, 76.371, 75.981, 75.386, 73.845, 72.449, 72.163, 71.526, 71.160, 70.893, 67.997, 49.500 (MeOH), 40.555 Spectrum Name: 071115_gol_ald rich_25mg_cba_1ml_071113.pdf Glycolaldehyde (Aldrich, 25 mg) was dissolved in CBA buffer ( 1.0 mL) and agitated vigorously using a hand vortexer. A 0.5 mL aliquot was transferred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal reference. This tube was stored at 25 C and an NMR spectrum was acquired after 2 days. 13C NMR (D2O): 167.003, 103.264, 82.302, 76.596, 71.598, 49.500 (MeOH).

PAGE 84

84 This spectrum exhibited the expected peaks fo r the product threose after 2 days. Compare this with the spectrum from 071113_gol_aldrich_25mg_cba_1ml.pdf Spectrum Name: 071115_gol_34mg_carbonate_1ml.pdf Glycolaldehyde (Aldrich, 34 mg ) was dissolved in carbonate buffer (1.0 mL) and agitated vigorously using a hand vortexer. A 0.5 mL ali quot was transferred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal re ference. This tube was stored at 25 C and an NMR spectrum was acquired. The spectrum was referenced to carbonate. 13C NMR (D2O): 167.000, 114.812 (minor), 88.391, 73.590, 73.209, 69.226, 67.883, 60.658, 59.648, 49.500 (MeOH), 44.900, 32.346. All peaks in the above sp ectrum are minor except for 167.000 (carbonate), 73.590 and 73.209. The methanol peak is not resolved and remains in the noise. Reactions starting with glyceraldehyde 1,2,3-13C-glyceraldehyde + H13CHO. 2:1 Spectrum Name: 070331A_glyceraldehyde_H13CHO_start0130am040207end0730am_nt=8192.pdf CBA buffer (0.900 mL, pH 10.25) and 1,2,3-13C-glyceraldehyde (0.100 mL final concentration 11.1 mM) were mixed. Then H13CHO (3.5 L, 6.7 M final concentration 23.3 mM, giving a 2.1:1 ratio) was added and the mixture was agitated vigorously using a vort ex stirrer. NMR data acquisition was begun within four minutes, and wa s completed after 8192 transients (ca. 6 hours total acquisition at 25C). Three manifolds of signals at 62, 70 and 95110 ppm were observed, with some H13CHO remaining.

PAGE 85

85 13C NMR (D2O): 171.767, 167.220, 105.502, 104.968, 104.789, 104.415, 104.262, 103.759, 103.438, 102.942, 102.359, 102.263, 101.832, 101.737, 100.814, 100.337, 99.490, 98.956, 98.891, 96.324, 95.813, 94.588, 82.305 (HCHO), 81.672, 81.145, 79.803, 77.060, 76.522, 75.976, 74.161, 73.642 73.043, 71.792, 71.300, 69.358, 67.123, 66.608, 65.787, 65.082, 64.387, 63.853, 63.102, 62.530, 61.923, 61.389, 58.936, 58.440, 49.217, 33.725, 31.104, 26.622, 26.527, 25.974, 25.878, 20.832, 15.602. The peaks at 74.161 and 69.358 correspond to C4 of th e erythro-branched pe ntose. A peak at 63.853 is interpreted as the C2 of the erythro-branched pentose 1,2,3-13C-Glyceraldehyde + HCHO. 2:1 Spectrum Name: 070331B_070410_glycer aldehyde_H12CHO_nt=4096_0300-0600.pdf CBA buffer (0.900 mL, pH 10.25) and 1,2,3-13C-glyceraldehyde (0.100 mL final concentration 11.1 mM). Then H12CHO (12 M, 2 L, final concentration 24 mM, giving a 2.2:1 ratio) was subsequently added and agitated vigorously us ing a vortex stirrer. Acquisition of an NMR spectrum was begun within four minutes, and wa s completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.767, 167.208, 105.506, 104.945, 104.781, 104.381, 104.262, 103.751, 103.400, 102.981, 102.343, 102.252, 101.726, 99.418, 98.887, 96.324, 95.809, 84.685, 84.300, 82.305 (HCHO), 81.355, 80.810, 79.867, 79.562, 77.991, 77.468, 77.125, 76.762, 76.213, 74.245, 72.753, 72.196, 71.365, 70.842, 68.484, 67.035, 66.623, 65.719, 65.005, 64.410, 63.872, 49.434, 26.527, 25.958, 25.859

PAGE 86

86 Spectrum Name: dl_glyceral dehyde_38mg_300ulcba_022507.pdf DL-Glyceraldehyde (38 mg, Aldrich) was dissolved in CBA buffer (0.3 mL). To this was added one drop of MeOH as an internal reference. The solution was agitated using a hand vortexer, transferred to an NMR tube and spec trum acquisition started in 4 minutes. 13C NMR (D2O): 166.851, 111.282, 110.928, 103.653, 100.998, 85.488, 84.061, 80.647, 80.078, 77.782, 76.741, 75.276, 71.629, 71.179, 69.867, 68.619, 64.412, 63.931, 62.573, 61.177, 60.204, 49.500 (MeOH). Spectrum Name: 071014_dl-12C-GLY _25mg_CBA_1ml_meohref.pdf DL-12C-Glyceraldehyde (Aldrich, 25 mg) was dissolv ed in CBA buffer (1.0 mL) in an eppendorf tube. An aliquot (0.5 mL) was tran sferred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal reference. Th is tube was maintained at 25 C and an NMR spectrum was acquired. 13C NMR (D2O): 167.084, 111.046, 100.949, 94.822, 92.137, 85.404, 84.240, 80.407, 77.256, 73.048, 71.656, 70.263, 68.600, 67.097, 64.213, 63.469, 62.588, 61.161, 49.500 (MeOH). Spectrum Name: 071014_d-12C-GLY _28mg_CBA_0.5ml_meohref.pdf D-12C-Glyceraldehyde (Fluka, 28 mg) was dissolved in CBA buffer (0.5 mL) in an eppendorf tube. The sample was transfe rred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal reference. This tube was maintained at 25 C and an NMR spectrum was acquired.

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87 13C NMR (D2O): 166.851, 110.977, 110.047, 103.294, 100.716, 94.837, 90.256, 85.675, 83.916, 80.632, 80.395, 80.113, 77.263, 76.596, 74.524, 72.819, 71.640, 71.110, 69.832, 67.116, 65.346, 64.633, 63.397, 62.554, 61.360, 61.142, 60.147, 49.500 (MeOH), 23.984 Spectrum Name: 071014_12C_GLY _25mg_CBA_1ml_nomeoh.pdf D-12C-Glyceraldehyde (Fluka, 25 mg) was dissolved in CBA buffer (0.5 mL) in an eppendorf tube. The sample was transfe rred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal reference. This tube was maintained at 25 C and an NMR spectrum was acquired. 13C NMR (D2O): 167.000, 110.894, 110.219, 110.203, 110.016, 100.850, 94.758, 83.852, 80.392, 77.183, 72.957, 68.513, 66.998, 64.118, 63.462, 62.344, 60.788, 49.500 (MeOH). Spectrum Name: 071015_d-gly_25mg_1-13c -gol_200ul_cba_1ml_meohref.pdf DL-12C-Glyceraldehyde (Aldrich, 25 mg) was dissolved in 1-13C-glycolaldehyde (Omicron Biochemicals, 200 l) in an eppendorf tube. To this wa s added CBA buffer (1.0 mL) and An aliquot (0.5 mL) was transfe rred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal reference. This tube was maintained at 25 C and an NMR spectrum was acquired. 13C NMR (D2O): 171.779, 166.950, 103.249, 102.471, 98.305, 95.036, 94.872, 94.647, 93.064, 90.225, 82.596, 82.043, 80.529, 76.981, 76.863, 76.779, 76.615, 76.279, 72.468, 72.178, 71.274, 71.179, 69.161, 64.778, 63.424, 49.500 (MeOH), 40.577, 23.965

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88 Spectrum Name: 071015_d-gly_25mg_113c-gol_200ul_cba_1ml_nomeoh.pdf DL-12C-Glyceraldehyde (Aldrich, 25 mg) was dissolved in 1-13C-glycolaldehyde (Omicron Biochemicals, 200 l) in an eppendorf tube. To this wa s added CBA buffer (1.0 mL) and An aliquot (0.5 mL) was transferred to an NMR tube. This tube wa s maintained at 25 C and an NMR spectrum was acquired. 13C NMR (D2O): 171.722, 166.950 (carbonate), 110.985, 103.233, 102.455, 100.922, 98.278, 97.290, 95.020, 93.067, 90.218, 83.332, 82.573, 82.035, 80.403, 76.962, 76.847, 76.760, 76.271, 72.453, 72.163, 71.259, 71.167, 70.099, 69.271, 68.589, 64.679, 63.534, 62.443, 40.566, 23.953 Spectrum Name: 071020_d-12c-gly_25mg_c ba_1ml_meohref_5dayslater.pdf DL-12C-Glyceraldehyde (Aldrich, 25 mg) was dissolv ed in CBA buffer (1.0 mL) in an eppendorf tube. An aliquot (0.5 mL) was tran sferred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal reference. Th is tube was maintained at 25 C and an NMR spectrum was acquired 5 days later. 13C NMR (D2O): 167.767, 110.966, 103.897, 100.891, 85.564, 84.061, 80.399, 77.717, 77.248, 75.798, 72.922, 71.637, 71.175, 70.214, 68.463, 66.735, 64.423, 63.740, 62.577, 61.135, 60.193, 49.500 (MeOH), 23.987 Spectrum Name: 071020_d-12c-gly_25mg_c ba_1ml_meohref_6dayslater_071014.pdf DL-12C-Glyceraldehyde (Aldrich, 25 mg) was dissolv ed in CBA buffer (1.0 mL) in an eppendorf tube. An aliquot (0.5 mL) was tran sferred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal reference. Th is tube was maintained at 25 C and an NMR spectrum was acquired 6 days later.

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89 13C NMR (D2O): 166.790, 110.989, 110.310, 103.916, 100.914, 85.419, 84.084, 80.418, 80.109, 77.740, 77.267, 75.821, 72.766, 71.659, 71.194, 69.836, 68.428, 66.754, 64.259, 63.725, 62.600, 61.135, 60.215, 49.500 (MeOH), 24.007 Reactions of glycolaldehyde and glyceraldehyde Glycolaldehyde dimer + Glyceral dehyde in CBA (t = 6 hours) Spectrum Name: 070519_GOL_40mg_GER_ 21mg_CBA_refmeoh_6hourslater_nt=8192.pdf Glycolaldehyde dimer (40 mg) and glyceraldehyde (21 mg) were dissolved in CBA buffer (1 mL) and agitated vigorously usi ng a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (c a. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.923, 166.374, 149.197, 125.042, 111.256, 103.253, 103.069, 102.902, 97.359, 85.179, 83.477, 83.336, 83.157, 82.638, 82.558, 82.474, 82.302, 79.933, 79.041, 77.813, 70.954, 76.752, 76.588, 76.359, 76.264, 76.161, 75.890, 73.704, 72.949, 72.449, 71.579, 71.522, 71.320, 69.786, 69.020, 68.680, 67.440, 66.964, 64.682, 64.351, 62.630, 60.894, 60.486, 60.189, 60.036, 58.709, 51.365, 49.500 (MeOH), 48.665, 34.154, 30.369, 22.504, 11.269 Glycolaldehyde dimer + Glyceral dehyde in CBA (t = 0-3 hours) Spectrum Name: 070519_GOL_40mg_GER_21mg_CBA_refmeoh_t=4.pdf Glycolaldehyde dimer (40 mg) and glyceraldehyde (21 mg) were dissolved in CBA buffer (1 mL) and agitated vigorously usi ng a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (c a. 3 hours total acquisition at 25C).

PAGE 90

90 13C NMR (D2O): 166.931, 103.249, 103.119, 102.947, 94.849, 93.029, 90.164, 82.302, 80.658, 76.954, 76.584, 76.378, 74.532, 73.700, 72.445, 71.583, 69.668, 64.597, 64.377, 63.446, 63.340, 62.569, 49.500 (MeOH). Spectrum Name: 070716_1-13C_GOL_280ul_0.11M_2-13CGLY_28ul_CBA_200ul_nomeohref.pdf A 0.110M solution of 1-13C-glycolaldehyde (280 L, Om icron Biochemicals) and a 0.110 M solution of 2-13C-glyceraldehyde (280 L, Omicron Bi ochemicals) was dissolved in 0.200 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. Acquisition of an NMR spectrum was begun within four minutes, and wa s completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.279, 166.301 (carbonate), 102.851, 97.926, 92.933, 90.061, 82.240, 81.951, 81.691, 80.161, 76.564, 76.309, 76.015, 75.630, 71.197, 69.072, 64.826 Spectrum Name: 070716_1-2-3-13C_GLY_60ul_0.11M_113C_GOL_200ul_0.11M_CBA_meohre.pdf A 0.110M solution of 1,2,3-13C-glyceraldehyde (60 L, Om icron Biochemicals) and 0.110 M solution of 1-13C-glycolaldehyde (200 L, Om icron Biochemicals) was dissolved in 0.500 mL CBA buffer. The mixture was agitated vigorously us ing a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acqui sition of an NMR spectrum was begun within four minutes, and was completed after 409 6 transients (ca. 3 hours total acquisition at 25C).

PAGE 91

91 13C NMR (D2O): 171.772, 167.126, 103.260, 98.316, 95.230, 94.673, 93.228, 90.968, 82.615, 82.066, 80.948, 80.494, 79.937, 76.893, 76.634, 76.344, 75.711, 69.325, 65.064, 63.885, 63.332, 49.500 (MeOH). Spectrum Name: 070716_2-13C-GLY_28ul1-13C -GOL_280ul_CBA_firstspectrum070719.pdf A 0.110M solution of 2-13C-glyceraldehyde (28 L, Omic ron Biochemicals) and a 0.110 M solution of 1-13C-glycolaldehyde (280 L, Omicron Bi ochemicals) was dissolved in 0.700 mL CBA buffer. The mixture was agitated vigorously us ing a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acqui sition of an NMR spectrum was begun within four minutes, and was completed after 409 6 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.634, 166.611, 111.332, 103.207, 98.271, 62.600, 81.314, 82.047, 77.011, 69.920, 76.664, 76.371, 75.981, 75.386, 73.845, 72.449, 72.163, 71.526, 71.160, 70.893, 67.997, 49.500 (MeOH), 40.555 Spectrum Name: 070716_2-13C_GLY_60ul_0.11M_1-13C_ GOL_200ul_0.11M_CBA_meohre.pdf A 0.110M solution of 2-13C-glyceraldehyde (60 L, Omic ron Biochemicals) and a 0.110 M solution of 1-13C-glycolaldehyde (200 L, Omicron Bi ochemicals) was dissolved in 0.740 mL CBA buffer. The mixture was agitated vigorously us ing a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acqui sition of an NMR spectrum was begun within four minutes, and was completed after 409 6 transients (ca. 3 hours total acquisition at 25C).

PAGE 92

92 13C NMR (D2O): 171.768, 167.129, 104.920, 104.378, 103.821, 103.260, 100.708, 98.316, 93.205, 90.496, 88.524, 82.615, 82.341, 82.066, 81.463, 80.494, 76.992, 76.893, 76.638, 76.344, 76.027, 71.595, 69.466, 68.280, 64.133, 62.790, 59.689, 58.094, 56.614, 49.500 (MeOH), 49.477 Spectrum Name: 070724_1-2-3-13C-GLY_280ul_1-13CGOL_280ul_SCBA_400ul_nomeoh.pdf A 0.110M solution of 1,2,3-13C-glyceraldehyde (280 L, Omic ron Biochemicals) and a 0.110 M solution of 1-13C-Glycolaldehyde (280 L, Omicron Bi ochemicals) was dissolved in 0.400 mL SCBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed af ter 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 180.171, 171.286, 170.668, 162.173, 105.632, 104.503, 103.980, 102.859, 99.715, 97.915, 96.595, 92.929, 92.868, 92.765, 90.133, 88.420, 80.310, 80.272, 80.188, 80.142, 80.070, 76.675, 76.328, 76.030, 75.759, 74.188, 62.782, 49.500 (MeOH), 40.187, 39.028, 23.510 Spectrum Name: 070724_1-2-3-13C-GLY _28ul_1-13C-GOL_280ul _SCBA_700ul_nomeoh.pdf A 0.110M solution of 1,2,3-13C-glyceraldehyde (28 L, Omic ron Biochemicals) and a 0.110 M solution of 1-13C-glycolaldehyde (280 L, Omicron Bi ochemicals) was dissolved in 0.700 mL SCBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed af ter 4096 transients (ca. 3 hours total acquisition at 25C).

PAGE 93

93 13C NMR (D2O): 171.340, 162.505, 102.851, 97.907, 92.143, 82.229, 81.680, 76.530, 76.271, 75.981, 75.633, 71.166, 68.290, 65.040, 62.721, 49.500 (MeOH), 40.161 Spectrum Name: 070724_2-13C-GLY_28ul _1-13C-GOL_280ul_SCBA_700ul_nomeoh.pdf A 0.110M solution of 2-13C-glyceraldehyde (28 L, Omicr on Biochemicals) and a 0.110 M solution of 1-13C-glycolaldehyde (280 L, Omicron Bi ochemicals) was mixed in 0.700 mL SCBA buffer. The mixture was agitated vigorously using a vortex stirrer. Acquisition of an NMR spectrum was begun within four minutes, and wa s completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.340 (carbonate), 162.520, 102.847, 97.907, 92.140, 91.136, 88.760, 82.229, 81.943, 81.676, 76.526, 76.271, 75.977, 75.622, 71.163, 68.167, 65.036, 64.025, 62.271, 54.527, 40.157, 38.990, 25.234, 23.514 Spectrum Name: 071117_from 071014_d-gly_28mg_cba_0.5ml.pdf D-Glyceraldehyde (Fluka, 28 mg) was dissolved in CBA buffer (0.5 mL) and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. The tube was kept in a car for 34 days, with temperatures fluctuating between 37-85 F Acquisition of an NMR spectrum (nt= 8192) was acquired 34 days later. 13C NMR (D2O): 187.685, 166.595, 111.229, 102.589, 97.191, 91.042, 88.642, 87.086, 83.878, 83.195, 80.616, 79.899, 77.488, 76.477, 74.520, 73.227, 72.346, 71.125, 69.760, 69.619, 67.994, 65.697, 63.851, 62.363, 61.497, 61.047, 60.124, 49.500 (MeOH).

PAGE 94

94 Spectrum Name: 071116_12c-d-gly_100ul_0.110 m_camh13cho_3.5ul_cba_900ul_nt=8192.pdf D-Glyceraldehyde (Fluka, 100 L, 0.110 M solution) was mixed with formaldehyde (Cambridge, 3.5 L, 6.6 M) in an eppendorf tube. To this was added CBA buffer (900 L) and a 0.5 mL aliquot was transferred to an NMR tube. An NM R spectrum was started after 4 minutes with an acquisition time of 6.5 hours (nt=8192). 13C NMR (D2O): 171.852, 167.320, 82.360, 76.397, 64.282, 63.652, 62.391, 53.985, 49.500 (MeOH). A peak at 62.391 was observed and is assigned to the branched te trose with the assumption that any erythrulose produced would be moved towa rds a pentose product. Accumulation of the branched tetrose is also favored as it has no e nolizable centers and is considered a dead end product. Formaldehyde is less than 2% consumed and appears at 82.360. Spectrum Name: 071117_24h_071116_12c-d-gly_100ul_0.110m_cam h13cho3.5ul_cba900ul.pdf Glyceraldehyde (Aldrich, 100 L, 0.110M) was dissolved in CBA buffer (1.0 mL) and to this was added H13CHO (Cambridge, 3.5 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 25 C. An aliquot (0.5 mL) was transferred to an NMR tube and an NMR spectrum was acquired. 13C NMR (D2O): 171.852, 167.312, 82.356, 74.002, 72.224, 71.644, 66.330, 65.525, 64.190, 63.805, 62.909, 58.766, 53.898, 50.271, 49.500 (MeOH). Reactions involving dihydroxyacetone Dihydroxyacetone in CBA

PAGE 95

95 Spectrum Name: 070530_dha_cba_meohref.pdf Dihydroxyacetone (90 mg) was dissolved in CBA buf fer (1 mL) and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.904, 111.961, 110.920, 85.522, 84.095, 83.012, 82.875, 79.220, 77.324, 75.470, 73.861, 73.170, 69.916, 64.358, 63.603, 62.706, 60.143, 58.911, 56.355, 49.500 (MeOH), 48.222, 42.676 Dihydroxyacetone + Glycolaldehyde in CBA Spectrum Name: 070530_dha_gol_cba_meohref.pdf Dihydroxyacetone (90 mg) was dissolved in CBA buf fer (1 mL) and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.782, 111.416, 111.290, 111.229, 103.028, 102.883, 83.309, 82.535, 82.390, 76.725, 76.626, 76.500, 76.329, 76.241, 76.134, 72.384, 71.583, 71.499, 71.240, 64.328, 49.500 (MeOH). Dihydroxyacetone + Glycolal dehyde in CBA no MeOH Spectrum Name: 070610_DHA_102mg_GOL_51mg_cba_sametime_nomeohref_ref-111.28.pdf Dihydroxyacetone dimer (Aldrich, 102 mg) and glyc olaldehyde (Aldrich, 51 mg) were dissolved in CBA buffer (1 mL) and agitated vigorously us ing a vortex stirrer. Acquisition of an NMR

PAGE 96

96 spectrum was begun within four minutes, and wa s completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.688 (carbonate), 111.280, 104.547, 104.055, 103.021, 98.714, 98.207, 84.428, 83.311, 83.257, 79.206, 78.539, 76.715, 76.620, 76.513, 73.221, 72.801, 72.439, 72.344, 71.054, 69.864, 68.620, 66.366, 64.325, 63.761, 62.803, 62.635, 61.701, 58.225, 54.426, 54.148 Spectrum Name: 070510_dha_18mg_cba_1ml_refmeoh.pdf Dihydroxyacetone dimer (18 mg, Aldrich) was disso lved in CBA buffer (1 mL). To this was added 10 L of 10% MeOH in D2O as an internal reference. Th e solution was agitated using a hand vortexer, transferred to an NMR tube a nd spectrum acquisition started in 4 minutes and continued for 3 hours (nt=4096). 13C NMR (D2O): 230.457, 226.028, 222.290, 217.117, 215.385, 212.468, 208.530, 203.163, 195.350, 193.355, 181.839, 167.061, 99.907, 93.487, 87.921, 85.450, 82.836, 80.231, 78.953, 77.923, 76.969, 74.551, 73.143, 70.389, 68.982, 66.800, 64.473, 62.333, 60.261, 58.339, 54.123, 52.502, 50.759, 49.500 (MeOH). Dihydroxyacetone + Glycolaldehyde in CBA Spectrum Name: 070610_DHA_104mg_GOL_67mg_CBA_sametime_refmeoh.pdf Dihydroxyacetone dimer (Aldrich, 104 mg) and Glyc olaldehyde (Aldrich, 67 mg) were dissolved in CBA buffer (1 mL) and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acqui sition of an NMR spectrum was begun within four minutes, and was completed after 409 6 transients (ca. 3 hours total acquisition at 25C).

PAGE 97

97 13C NMR (D2O): 166.664, 111.454, 111.405, 111.279, 104.061, 103.001, 102.341, 98.202, 92.572, 89.447, 87.574, 84.431, 83.309, 82.524, 80.868, 79.956, 77.908, 76.714, 76.619, 76.516, 76.317, 75.882, 73.616, 73.277, 72.434, 72.350, 71.823, 71.236, 70.904, 69.867, 68.631, 68.039, 67.101, 65.323, 65.033, 64.320, 63.584, 62.790, 61.718, 57.957, 56.080, 52.506, 49.916, 49.504 (MeOH), 49.412, 47.501, 46.399, 45.662, 44.652, 43.973 Dihydroxyacetone + Glycolaldehyde in CBA Spectrum Name: 070610_DHA_40mg_GOL_80mg_CBA_05302007.pdf Dihydroxyacetone dimer (Aldrich, 40 mg) and glyc olaldehyde (Aldrich, 80 mg) were dissolved in CBA buffer (1 mL) and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acqui sition of an NMR spectrum was begun within four minutes, and was completed after 409 6 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.595, 111.210, 83.165, 76.481, 73.178, 72.358, 71.007, 68.879, 68.615, 68.024, 65.724, 63.641, 62.470, 61.741, 49.500 (MeOH). Dihydroxyacetone + Glycolaldehyde in CBA Spectrum Name: 070610_DHA_80mg_GOL_42mg_CBA_sametime_refmeoh.pdf Dihydroxyacetone dimer (Aldrich, 80 mg) and glyc olaldehyde (Aldrich, 42 mg) were dissolved in CBA buffer (1 mL) and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acqui sition of an NMR spectrum was begun within four minutes, and was completed after 409 6 transients (ca. 3 hours total acquisition at 25C).

PAGE 98

98 13C NMR (D2O): 166.923, 111.431, 111.305, 83.332, 76.737.72.453, 65.346, 64.339, 49.504 (MeOH) Dihydroxyacetone + Glycolal dehyde in CBA no MeOH Spectrum Name: 070610_DHA_first_103mg_GOL_49mg_second_cba_nomeohref.pdf Dihydroxyacetone dimer (Aldrich, 103 mg) was disso lved in CBA buffer (1 mL) and 49 mg of glycolaldehyde was subsequently added and agitate d vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and wa s completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.020, 112.276, 111.444, 83.429, 83.154, 82.994, 76.765, 73.385, 72.546, 71.443, 70.192, 70.055, 64.539, 63.822, 62.971, 62.841, 61.891, 49.500 (MeOH). Dihydroxyacetone + Glycolal dehyde in CBA no MeOH Spectrum Name: 070611_DHA_30mg_GOL_106mg_nomeoh_cba.pdf Dihydroxyacetone dimer (Aldrich, 30 mg) and glyc olaldehyde (Aldrich, 1 06 mg) was dissolved in CBA buffer (1 mL) and agitated vigorously us ing a vortex stirrer. Acquisition of an NMR spectrum was begun within four minutes, and wa s completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.851 (carbonate), 111.244, 102.894, 90.149, 82.539, 76.332, 72.388, 71.499, 64.957, 64.339

PAGE 99

99 Dihydroxyacetone + Glycolaldehyde in CBA Spectrum Name: 070611_DHA_30mg_GOL_106mg_refmeoh_113c-xylose_added_nt=3072.pdf Dihydroxyacetone dimer (Aldrich, 30 mg) and glyc olaldehyde (Aldrich, 1 06 mg) was dissolved in CBA buffer (1 mL) and agitated vigorously us ing a vortex stirrer. To this was added 1-13Cxylose and 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and wa s completed after 3072 transients (ca. 2.5 hours total acquisition at 25C). 13C NMR (D2O): 177.166, 166.954, 150.745, 126.003, 111.279, 102.974, 102.757, 97.573, 97.271, 96.493, 92.813, 88.593, 86.140, 83.340, 82.566, 76.367, 76.165, 73.880, 72.411, 71.526, 68.699, 66.994, 65.781, 64.347, 49.500 (MeOH), 27.066 Dihydroxyacetone + Glycolaldehyde in CBA Spectrum Name: 070611_DHA_30mg_GOL_106mg_refmeoh_113c-xylulose_added_nt=3072.pdf Dihydroxyacetone dimer (Aldrich, 30 mg) and glyc olaldehyde (Aldrich, 1 06 mg) was dissolved in CBA buffer (1 mL)and agitated vigorously us ing a vortex stirrer. To this was added 1-13Cxylulose and 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and wa s completed after 3072 transients (ca. 2.5 hours total acquisition at 25C). 13C NMR (D2O): 166.908, 166.878, 111.706, 111.607, 110.993, 110.886, 102.921, 83.325, 82.547, 76.348, 76.153, 72.388, 71.507, 70.507, 69.634, 65.026, 64.694, 64.309, 63.973, 62.535, 61.757, 49.500 (MeOH).

PAGE 100

100 Dihydroxyacetone + Glycolaldehyde in CBA Spectrum Name: 070611_DHA_30mg_GOL_106mg_refmeoh_cba.pdf Dihydroxyacetone dimer (30 mg) and glycolaldehyde (106 mg) was dissolved in CBA buffer (1 mL) and agitated vigorously usi ng a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (c a. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.851, 111.347, 102.883, 90.149, 83.306, 82.543, 76.729, 76.336, 75.291, 73.647, 72.384, 71.503, 64.949, 64.335, 49.500 (MeOH). Dihydroxyacetone + Glycolal dehyde in CBA no MeOH Spectrum Name: 070617_dha_5.4mg_gol_100.6mg_cba_nomeoh.pdf Dihydroxyacetone dimer (Aldrich, 5.4 mg) a nd glycolaldehyde (Aldrich, 100.6 mg) was dissolved in CBA buffer (1 mL) a nd agitated vigorously using a vor tex stirrer. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.000 (carbonate), 149.313, 125.189, 112.123, 103.196, 103.029, 99.390, 97.160, 83.338, 82.684, 76.461, 76.281, 75.820, 76.265, 72.495, 71.614, 63.475, 62.633, 11.881 Spectrum Name: 20071307_DHA_30_GOL_103_carbonatebuffer.pdf Dihydroxyacetone dimer (Aldrich, 30 mg) and glyc olaldehyde (Aldrich, 1 03 mg) was dissolved in 1 mL of carbonate buffer. The mixture was agitat ed vigorously using a vortex stirrer. To this

PAGE 101

101 was added 10 L of a 10% MeOH in D2O as an internal reference. An NMR spectrum was acquired immediately. 13C NMR (D2O): 168.361, 100.227, 99.518, 98.442, 97.107, 84.244, 71.707, 78.602, 76.176, 75.627, 75.306, 74.707, 74.219, 72.407, 71.808, 70.935, 70.740, 69.695, 69.226, 66.037, 64.621, 64.080, 63.256, 62.287, 62.073, 61.570, 54.883, 49.500 (MeOH), 48.985, 48.501, 45.765, 40.593 Spectrum Name: 070617_october_sample_h13cho_dha_tetraborate.pdf Dihydroxyacetone dimer (Aldrich, 30 mg) was dissolv ed in 1 mL of CBA buffer. To this was added H13CHO (6 L, 6.66 M) and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. A sa mple prepared in October 2006 was analyzed by NMR. 13C NMR (D2O): 182.390, 171.681, 167.401, 106.454, 105.813, 105.119, 103.229, 102.161, 101.653, 101.006, 99.270, 88.513, 80.474, 79.419, 77.243, 76.174, 75.353, 74.612, 73.998, 73.503, 72.802, 72.402, 71.073, 70.692, 70.031, 69.210, 67.701, 67.080, 66.393, 65.898, 65.017, 64.570, 63.815, 63.441, 61.899, 61.271, 60.150, 59.509, 55.115, 49.500 (MeOH), 49.059, 45.841, 26.758, 26.298, 24.649, 23.921, 22.352, 20.729 Spectrum Name: 070702_DHA_31_GOL_95_CBA_1-13C-xylose_added_meohref.pdf Dihydroxyacetone dimer (Aldrich, 31 mg) and glyc olaldehyde (Aldrich, 95mg) were dissolved in CBA buffer (1 mL) and agitated vigorously us ing a vortex stirrer. To this was added 1-13Cxylose and 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and wa s completed after 4096 transients (ca. 3 hours total acquisition at 25C).

PAGE 102

102 13C NMR (D2O): 171.749, 166.797, 166.756, 111.435, 111.248, 103.047, 102.898, 97.248, 92.793, 83.321, 82.554, 76.748, 76.645, 76.538, 76.355, 76.264, 76.157, 74.211, 72.396, 71.503, 64.343, 63.385, 49.500 (MeOH). Spectrum Name: 070702_DHA_31_GOL_95_CBA_meohref.pdf Dihydroxyacetone dimer (Aldrich, 31 mg) and glyc olaldehyde (Aldrich, 95 mg) were dissolved in CBA buffer (1 mL) and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acqui sition of an NMR spectrum was begun within four minutes, and was completed after 409 6 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.759, 111.241, 103.043, 83.344, 82.547, 76.626, 76.519, 76.344, 72.403, 71.514, 64.335, 63.385, 63.080, 62.619, 49.500 (MeOH). Spectrum Name: 070713_DHA_30_GOL_100_meohborateremoval _immediate_white_nt=8192.pdf Dihydroxyacetone dimer (Aldrich, 30 mg) and glyc olaldehyde (Aldrich, 1 00 mg) was dissolved in CBA buffer (1 mL) and agitated vigorously usi ng a vortex stirrer. To remove the borate, 30 mL of MeOH was added and evaporated, at room temperature, from the mixture in three portions of 10 mL each. The remaining residu e was re-suspended in 1 mL of D2O. An NMR spectrum was acquired immediately. 13C NMR (D2O): 166.729, 103.066, 102.928, 83.245, 82.569, 76.554, 76.382, 76.214, 72.419, 71.499, 64.377, 63.416, 61.776, 49.500 (MeOH).

PAGE 103

103 Spectrum Name: 070713_DHA_30_GOL_100_meohborateremoval _overnight_yellow_nt=8192.pdf Dihydroxyacetone dimer (Aldrich, 30 mg) and glyc olaldehyde (Aldrich, 1 00 mg) was dissolved in CBA buffer (1 mL) and agitated vigorously usi ng a vortex stirrer. To remove the borate, 30 mL of MeOH was added and evaporated at 50 C from the mixture in three portions of 10 mL each. The remaining residue was kept under vacuum for 24 hours and subsequently re-suspended in 1 mL of D2O. An NMR spectrum was acquired immediately. 13C NMR (D2O): 168.178, 160.663, 111.378, 103.279, 103.115, 86.109, 83.515, 82.905, 76.775, 76.561, 76.474, 72.518, 71.610, 64.652, 60.124, 57.633, 55.874, 55.493, 54.814, 53.524, 52.689, 51.625, 50.564, 50.488, 50.236, 49.500 (MeOH), 48.752, 48.440, 47.375, 46.315, 44.446, 43.126, 38.880, 37.472 Spectrum Name: 070716_1-13C_GOL_280ul_0.11M_DHA_28ul_0.11M_CBA_500ul_refmeoh.pdf A 0.110M solution of 1-13C-glycolaldehyde (280 L, Omic ron Biochemicals) and a 0.110 M solution of dihydroxyacetone (28 L)in D2O was dissolved in 0.500 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.730, 167.000, 110.268, 103.245, 98.305, 93.216, 90.431, 82.611, 82.062, 76.905, 76.649, 76.355, 71.141, 70.176, 69.317, 49.500 (MeOH), 40.585, 13.497

PAGE 104

104 Dihydroxyacetone and H13CHO in CBA buffer Dihydroxyacetone dimer (Aldrich, 3.3 M, 100 L) was dissolved in CBA buffer (1 mL). To this solution was added H13CHO (Cambridge, 3.3 M, 50 L) was added and agitated vigorously using a vortex stirrer. A portion of the reaction mixture (500 L ) was transferred to an NMR tube and acquisition of an NMR spectrum began within four minutesat at 25C. Spectrum Name: dha-100ul_h13cho-50ul_9cba_120106_1955-2003.pdf 13C NMR (D2O): 171.811, 167, 90.087, 89.626, 83.732, 82.981, 82.309, 81.611, 80.902, 65.399, 64.064, 63.465, 62.859, 55.264, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=15.pdf 13C NMR (D2O): 171.811, 167, 90.087, 89.626, 83.732, 82.981, 82.309, 81.611, 80.902, 65.399, 64.064, 63.465, 62.859, 55.264, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=23.pdf 13C NMR (D2O): 171.371, 166.965, 89.651, 89.178, 88.007, 83.292, 82.545, 81.869, 81.179, 80.462, 68.465, 64.963, 63.777, 63.006, 62.434, 54.824, 49.068, 48.915 MeOH ref corr. to 49.500. Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=32.pdf 13C NMR (D2O): 171.803, 167, 90.083, 83.728, 82.977, 82.305, 81.604, 65.331, 64.251, 63.427, 62.862, 62.687, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=41.pdf 13C NMR (D2O): 171.807, 167.394, 90.083,88.447, 83.709, 82.977, 82.305, 81.607, 80.894, 71.617, 69.942, 69.187, 68.924, 68.642, 67.036, 66.421, 65.453, 64.232, 63.423, 62.855, 62.679, 61.710, 55.275, 53.860, 49.653, 49.500 (MeOH), 49.347, 49.035

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105 Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=51.pdf 13C NMR (D2O): 171.807, 167.390, 90.106, 82.305, 71.613, 69.206, 68.691, 67.013, 66.429, 65.445, 64.224, 63.423, 62.859, 62.668, 61.703, 53.864, 49.653, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=61.pdf 13C NMR (D2O): 171.803, 167.386, 90, 73.341, 71.609, 69.206, 68.630, 67.051, 66.391, 65.174, 64.232, 63.419, 62.851, 61.707, 49.500 (MeOH), 49.344 Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=71.pdf 13C NMR (D2O): 171.803, 167.386, 82.302, 73.395, 71.609, 71.041, 69.199, 68.672, 66.990, 66.410, 65.445, 64.217, 63.427, 62.862, 62.679, 61.733, 56.126, 54.539, 53.856, 53.658, 80.545, 49.694, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=81.pdf 13C NMR (D2O): 171.807, 167.386, 82.302, 73.326, 71.613, 69.199, 68.661, 67.715, 67.032, 66.406, 65.441, 65.186, 64.205, 63.423, 62.847, 62.679, 61.714, 53.856, 49.649, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=91.pdf 13C NMR (D2O): 171.803, 167.386, 82.302, 73.665, 72.891, 72.460, 71.930, 71.609, 70.606, 69.187, 68.672, 66.990, 66.402, 65.453, 64.194, 63.419, 62.840, 61.710, 54.871, 53.856, 54.472, 50.988, 50.187, 49.946, 49.500 (MeOH), 49344, 48.741, 48.005 Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=101.pdf 13C NMR (D2O): 171.811, 167.390, 82.305, 73.871, 72.494, 71.613, 69.195, 68.676, 66.994, 66.399, 65.460, 64.266, 63.419, 62.851, 62.660, 61.730, 58.480, 53.860, 49.962, 49.500 (MeOH), 47.997

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106 Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=111.pdf 13C NMR (D2O): 171.811, 167.390, 82.305, 73.894, 73.467, 72.471, 71.613, 69.195, 68.642, 66.982, 66.429, 65.445, 64.201, 63.423, 62.840, 62.687, 61.714, 58.563, 53.868, 49.500 (MeOH), 47.997 Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=121.pdf 13C NMR (D2O): 171.807, 167.386, 82.302, 73.860, 73.326, 72.475, 71.609, 71.022, 69.187, 68.706, 66.986, 66.402, 65.445, 65.125, 64.293, 63.427, 62.851, 61.730, 53.586, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=131.pdf 13C NMR (D2O): 171.807, 167.386, 82.302, 73.406, 72.487, 71.609, 69.206, 68.649, 66.978, 66.406, 65.456, 64.674, 64.240, 63.426, 62.851, 62.695, 61.688, 53.860, 49.649, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=14310.pdf 13C NMR (D2O): 171.811, 167.310, 103.240, 73.826, 72.460, 71.613, 71.564, 71.052, 70.160, 69.214, 68.684, 68.062, 66.986, 66.891, 65.449, 65.201, 64.674, 64.350, 64.056, 63.805, 62.824, 61.779, 59.334, 58.693, 53.864, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=180.pdf 13C NMR (D2O): 171.379, 166.958, 72.039, 71.181, 68.759, 68.248, 66.554, 65.978, 65.020, 64.223, 63.876, 62.980, 62.411, 62.228, 61.282, 58.066, 53.432, 49.500 (MeOH), 49.220, 49.068, 48.919

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107 Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=25935.pdf 13C NMR (D2O): 171.811, 167.275, 110.423, 103.232, 73.807, 72.456 ,71.613, 71.564, 69.969, 69.218, 68.661, 66.982, 65.445, 64.659, 64.335, 64.059, 63.805, 62.843, 61.775, 59.330, 58.945, 58.693, 53.860, 49.653, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=54720.pdf 13C NMR (D2O): 171.811, 167.230, 73.494, 73.192, 72.460, 71.617, 71.571, 69.298, 69.221, 68.726, 66.986, 66.887, 65.445, 64.667, 64.030, 63.797, 62.821, 61.779, 57.334, 53.864, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=9240.pdf 13C NMR (D2O): 171.815, 167.329, 102.3, 72.464, 71.613, 69.206, 66.986, 66.887, 65.441, 65.132, 64.663, 64.327, 64.060, 63.793, 62.637, 61.775, 49.500 (MeOH). Spectrum Name: dha-100ul_h13cho-50ul_cba_120106_t=9250.pdf 13C NMR (D2O): 171.799, 167.313, 103.228, 73.990, 73.879, 72.460, 71.613, 71.564, 71.037, 70.038, 69.305, 69.214, 68.706, 68.058, 67.978, 66.986, 66.887, 65.445, 64.674, 64.346, 64.030, 63.801, 62.824, 61.775, 60.818, 59.334, 58.693, 58.510, 53.860, 49.962, 49.645, 49.500 (MeOH). Spectrum Name: dha_100ul_carbonate_900ul_nt=16_47sec-each_10spectra.pdf Dihydroxyacetone dimer (Aldrich, 3.33 M, 100 L) wa s mixed in 1 mL of carbonate buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 10 spectra @ nt=16

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108 13C NMR (D2O): 171.195, 82.235, 64.808, 64.526, 64.515, 64.312, 63.424, 63.046, 62.684, 62.310, 49.500 (MeOH). Reactions involving erythrulose Erythrulose in CBA Spectrum Name: 070510_erythrulose_28mg_cba_1ml_refmeoh.pdf Erythrulose (28 mg) was disso lved in 1 mL of CBA buffe r. 10 L of a 10% MeOH in D2O was added as a reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acqui sition at 25C). 13C NMR (D2O): 167.026, 129.955, 110.287, 106.045 103.195, 98.942, 79.151, 76.557, 71.625, 66.903, 62.828, 61.787, 60.891, 49.500 (MeOH), 33.593 Erythrulose in CBA Spectrum Name: 070530_erythrulose_cba_meohref.pdf Erythrulose (30 mg) was dissolved in CBA buffer (1 mL) and agita ted vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 213.348, 180.431, 178.776, 171.600, 164.245, 110.535, 103.493, 103.054, 99.682, 98.824, 98.446, 85.285, 85.072, 84.095, 82.852, 82.138, 81.757, 81.150, 82.254, 79.575, 76.893, 76.256, 75.436, 74.665, 74.070, 73.754, 73.376, 73.250, 72.991, 72.773, 72.380, 72.285, 72.041, 71.072, 70.801, 70.622, 70.111, 68.859, 68.074, 65.941, 65.655, 64.614, 64.454, 63.336, 63.286, 63.145, 63.054, 62.729, 62.348, 62.088, 61.718, 61.463, 61.329, 60.990, 60.444, 59.529, 59.353, 59.311, 59.021, 57.686, 54.947, 49.500 (MeOH), 40.463, 33.852, 10.297

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109 Spectrum Name: erythrulose_100ul_carbona te_900ul_nt=256_725sec-each_12spectra.pdf 100 L of a 3.33 M solution of erythrulose was mi xed with 0.900 mL of carbonate buffer and 50 L of H13CHO from Cambridge Isotope Laboratories and was agitated vigorously using a vortex stirrer. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25 C). 12 spectra @ nt=256 13C NMR (D2O): 171.787, 82.306, 76.382, 69.920, 71.991, 69.920, 98.531, 68.242, 67.299, 66.361, 65.308, 64.656, 64.511, 64.331, 64.087, 63.691, 63.588, 63.321, 63.588, 63.321, 63.050, 62.668, 62.603, 61.844, 60.955, 59.716, 58.598, 54.722, 51.117, 49.500 (MeOH). Spectrum Name: erythrulose_100ul_carbonate _900ul_hcho50ul_nt=16_47s ec_10spectra.pdf 100 L of a 3.33 M solution of erythrulose was mi xed with 0.900 mL of carbonate buffer and 50 L of H13CHO from Cambridge Isotope Laboratories and was agitated vigorously using a vortex stirrer. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 10 spectra @ nt=16 13C NMR (D2O): 171.779, 82.314, 65.304, 64.515, 64.331, 63.584, 63.321, 63.050, 62.676, 49.500 (MeOH). Reactions involving erythrose Spectrum Name: 070523_erythro se_20mg_cba_1ml_nt=4096.pdf Erythrose (20 mg) was dissolved in CBA buffer (1 mL)and agita ted vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C).

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110 13C NMR (D2O): 167.034, 103.428, 103.203, 102.883, 82.466, 76.027, 75.734, 73.864, 71.343, 71.118, 68.039, 67.444, 49.500 (MeOH). Spectrum Name: 1-13c-erythrose_h13cho_noboron.pdf 1-13C-Erythrose (100 L, 0.085 M, Omicron Biochemicals) wa s dissolved in carbonate buffer (1.18 g / 10 mL, 0.9 mL). A NMR spectrum was acquired after four minutes. Two spectra were taken on December 23, 2006 (immediately) and January 9, 2007. 13C NMR (D2O): 171.104, 167.000 (carbonate) Spectrum Name: 1-13c-erythro se_h13cho_withboron_15min_24h_18days.pdf 1-13C-Erythrose (100 L, 0.085 M, Omicron Biochemicals) was dissolved in CBA buffer (0.9 mL). One drop of MeOH was added as an intern al standard. A NMR spec trum was acquired after four minutes. 15 minutes 13C NMR (D2O): 171.830, 167.695, 103.438, 103.259, 82.366, 49.500 (MeOH). 24 hours 13C NMR (D2O): 171.830, 167.703, 106.459, 103.438, 103.240, 82.366, 66.090, 64.579, 64.327, 63.831, 63.458, 63.095, 49.500 (MeOH). 18 days 13C NMR (D2O): 171.834, 167.710, 106.452, 103.240, 82.363, 65.826, 64.331, 63.839, 63.469, 63.065, 49.500 (MeOH).

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111 Spectrum Name: 2-13c-erythrose _h13cho_noboron_60min_24hours_17days.pdf 1-13C-Erythrose (100 L, 0.099 M, Omicron Biochemicals) wa s dissolved in carbonate buffer (0.9 mL). One drop of MeOH was added as an in ternal standard. A NMR spectrum was acquired after four minutes. 60 minutes 13C NMR (D2O): 171.799, 167.642, 110.419, 63.084, 61.791, 49.500 (MeOH), 47.303, 44.953 24 hours 13C NMR (D2O): 171.799, 167.642, 61.787, 49.500 (MeOH). 17 days 13C NMR (D2O): more peaks around 60-65 ppm similar to 24 hours Spectrum Name: 2-13c-erythrose _h13cho_withboron_35min_36hours_18days.pdf 1-13C-Erythrose (100 L, 0.099 M, Omicron Biochemicals) was dissolved in CBA buffer (0.9 mL). One drop of MeOH was added as an intern al standard. A NMR spec trum was acquired after four minutes. 35 minutes 13C NMR (D2O): 171.386, 167.244, 82.060, 81.923, 75.305, 49.056 note methanol standardization correction is necessary to 49.500 ppm. 36 hours 13C NMR (D2O): 171.830, 167.321, 82.504, 82.366, 75.752, 72.464, 65.372, 63.923, 63.511, 63.149, 49.500 (MeOH), 49.366 18 days 13C NMR (D2O): 171.838, 167.325, 82.366, 63.877, 49.500 (MeOH).

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112 Spectrum Name: 071118_1-13c-erythrose _300ul_cba_900ul_camh13cho50ul_65degC_48h.pdf In an eppendorf tube, 1-13C-erythrose (Omicron Biochemicals, 300 L, 0.085 M) was mixed with CBA buffer (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 65 C for 48 hours. An aliquot (0.5 mL) was transferred to an NMR tube and an NMR spectrum was acquired. 13C NMR (D2O): 171.776, 166.973, 105.301, 104.790, 90.138, 82.371, 81.879, 81.257, 80.387, 72.396, 68.928, 68.104, 67.841, 67.719, 67.467,67.135, 66.288, 66.010, 65.514, 65.293, 65.026, 64.587, 64.187, 63.801, 63.546, 63.286, 63.034, 62.771, 62.409, 62.172, 61.818, 61.444, 61.005, 60.662, 53.856, 50.213, 49.500 (MeOH). Spectrum Name: 071118_2-13c-eryth rose_300ul_h12cho_50ul_50mmcba_900ul.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with Buffer B (900 L) and H12CHO (Fisher, 50 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 25 C. An aliquot (0.5 mL) was transferred to an NMR tube and a spectrum was acquired after 4 minutes. 13C NMR (D2O): 168.506, 82.352, 75.692, 49.500 (MeOH). Spectrum Name: 071118_2-13c-erythrose_h13cho_cba_65degC_2days.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with CBA buffer (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 65 C for 48 hours. An an aliquot (0.5 mL) was transferred to an NMR tube and an NMR spectrum was acquired.

PAGE 113

113 13C NMR (D2O): 171.722, 166.988, 106.880, 90.134, 86.609, 84.667, 82.367, 80.990, 79.388, 77.969, 76.123, 73.471, 72.380, 69.321, 67.418, 66.296, 64.862, 63.515, 61.814, 60.944, 59.433, 55.459, 53.853, 52.479, 51.487, 49.500 (MeOH), 46.521, 43.541, 39.567 Spectrum Name: 071118_2-13c-erythrose _300ul_h12cho_50ul_50mmcba_900ul_19hours.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with Buffer B (900 L) and H12CHO (Fisher, 50 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 25 C. An aliquot (0.5 mL) was transferred to an NMR tube and a spectrum was acquire d after 19 hours. 13C NMR (D2O): 221.988, 168.484, 107.086, 102.696, 90.138, 88.959, 84.683, 82.352, 80.662, 76.001, 75.859, 75.768, 75.692, 73.864, 70.862, 68.055, 66.639, 55.164, 55.504, 55.237, 49.500 (MeOH). Spectrum Name: 071118_2-13c-erythrose_300ul_h12cho_50ul_50mmcba900ul_65degC_24h.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with Buffer B (900 L) and H12CHO (Fisher, 50 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 65 C. An aliquot (0.5 mL) was transferred to an NMR tube and a spectrum was acquire d after 24 hours. 13C NMR (D2O): 171.722, 167.316, 90.092, 82.333, 81.765, 81.242, 81.059, 80.796, 80.529, 79.674, 78.812, 77.950, 76.676, 76.329, 76.191, 75.577, 74.398, 73.803, 72.522, 72.068, 71.827, 71.026, 70.809, 66.670, 65.083, 62.767, 55.233, 49.500 (MeOH).

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114 Spectrum Name: 071118_2-13c-eryth rose_300ul_h12cho_50ul_cba_900ul.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with CBA buffer (900 L) and H12CHO (Fisher, 50 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 25 C. An a liquot (0.5 mL) was transf erred to an NMR tube and an NMR spectrum was acquired after four minutes. 13C NMR (D2O): 167.122, 90.126, 82.489, 82.379, 75.718, 74.944, 73.876, 49.500 (MeOH). Spectrum Name: 071118_2-13c-erythro se_300ul_h12cho_50ul_cba_900ul_13hours.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with CBA buffer (900 L) and H12CHO (Fisher, 50 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 25 C for 13 hours. An aliquot (0.5 mL) was transferred to an NMR tube and an NMR spectrum was acquired. 13C NMR (D2O): 167.122, 113.560, 103.558, 97.450, 94.578, 94.513, 94.383, 90.126, 84.362, 82.493, 82.379, 80.696, 79.399, 75.718, 74.944, 73.861, 68.036, 67.318, 55.520, 55.253, 49.500 (MeOH). Spectrum Name: 071118_2-13c-erythro se_300ul_h12cho_50ul_cba_900ul_65degC_24h.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with CBA buffer (900 L) and H12CHO (Fisher, 50 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 65 C for 1 day. An aliquot (0.5 mL) was transferred to an NMR tube and an NMR spectrum was acquired. 13C NMR (D2O): 171.741, 166.385, 110.217, 94.380, 90.119, 86.903, 85.121, 85.018, 84.366, 84.023, 82.371, 80.643, 79.396, 78.701, 77.965, 70.813, 49.500 (MeOH).

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115 Spectrum Name: 071118_2-13c-erythrose_h13cho_cba_65degC_2days.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with CBA buffer (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 65 C for 2 days. An aliquot (0.5 mL) was transferred to an NMR tube and an NMR spectrum was acquired. 13C NMR (D2O): 171.772, 166.988, 90.134, 86.609, 84.667, 82.367, 80.990, 79.388, 77.969, 76.123, 73.471, 72.380, 69.321, 67.418, 66.296, 64.862, 63.515, 61.814, 60.944, 59.433, 55.459, 55.853, 52.479, 51.487, 49.500 (MeOH). Spectrum Name: 071118_1-13c-erythrose_300ul_cba_900ul_camh13cho_50ul_65deg_2days.pdf In an eppendorf tube, 1-13C-erythrose (Omicron Biochemicals, 300 L, 0.085 M) was mixed with CBA buffer (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer and was maintained at 65 C for 2 days. A 0.5 mL aliquot was transferred to an NMR tube and an NMR spectrum was acquired. 13C NMR (D2O): 171.776, 166.973, 105.301, 104.790, 90.138, 82.371, 81.879, 81.257, 80.387, 72.396, 98.928, 68.104, 67.841, 67.719, 67.467, 67.135, 66.288, 66.010, 65.514, 62.293, 65.026, 64.587, 64.187, 63.801, 63.546, 63.286, 63.034, 62.771, 62.409, 62.172, 61.818, 61.444, 61.005, 60.662, 55.241, 53.856, 50.213, 49.500 (MeOH).

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116 Spectrum Name: 071127_1-13c-erythro se_300ul_50mmboron_cba_900ul_cam50ul_071113.pdf In an eppendorf tube, 1-13C-erythrose (Omicron Biochemicals, 300 L, 0.085 M) was mixed with 50 mM CBA buffer (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer and an aliquot (0.5 mL) was transfe rred to an NMR tube. The tube was maintained at 25 C for 2 days and an NMR spectrum was acquired. 13C NMR (D2O): 171.825, 167.549, 110.306, 82.348, 81.276, 80.723, 79.354, 78.793, 78.324, 75.726, 73.716, 68.089, 67.486, 66.327, 64.793, 63.561, 63.313, 63.027, 62.764, 62.157, 61.802, 61.428, 59.437, 53.879, 51.842, 51.236, 50.187, 49.500 (MeOH), 43.541, 23.995, 20.219, 10.903 Spectrum Name: 071127_2-13c-erythro se_300ul_50mmboron_cba_900ul_cam50ul_071113.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with 50 mM CBA buffer (five fold dilution CBA, 900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer a nd an aliquot (0.5 mL) was transferred to an NMR tube. The tube was maintained at 25 C for 14 days and an NMR spectrum was acquired. 13C NMR (D2O): 181.755, 181.118, 180.378, 171.825, 167.549, 82.341, 79.453, 77.992, 67.795, 67.414, 66.304, 64.808, 63.561, 63.252, 63.023, 62.153, 61.799, 61.497, 61.425, 61.425, 61.211, 59.334, 53.879, 49.500 (MeOH), 48.771, 23.999 Spectrum Name: 071115_1-13c_erythrose_cba_071113.pdf In an eppendorf tube, 1-13C-erythrose (Omicron Biochemicals, 300 L, 0.085 M) was mixed with CBA buffer (900 L). The tube was shaken using a hand vortexer. A 0.5 mL aliquot was transferred to an NMR tube. To this t ube was added 10 L of a 10% MeOH in D2O as an internal

PAGE 117

117 reference. This tube was stored at 25 C a nd an NMR spectrum was acquired after 2 days. The sample is referenced to carbonate (167.000). 13C NMR (D2O): 169.949, 167.000 (carbonate), 74.666, 72.793, 72.278, 69.879, 69.718, 69.592, 69.375, 68.894, 59.915 The sample is referenced to carbonate (167.000) as the methanol is not resolved over the noise. Major peaks appear at 74.666, 72.793, 72.278, 69.879, 69.375 and 59.915. Spectrum Name: 071115_E2B_2-13c-erythrose_061223.pdf A sample from December 23rd 2006 labeled E2B was found and a spectrum was acquired on November 11th 2007. The sample was prepared as follows: In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with Buffer C (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer. A 0.5 mL aliquot was transferred to an NMR tube. This tube was stored at 50 C for 18 days, then at 25 C for the remainder of time. 13C NMR (D2O): 171.882, 167.316, 113.552, 84.667, 84.008, 82.356, 76.832, 73.399, 72.029, 71.049, 70.317, 68.699, 67.288, 65.304, 63.778, 63.431, 63.061, 61.806, 49.500 (MeOH). A new peak is observed at 113.552 ppm and re mains unassigned. Formaldehyde is faintly present at 82.356 ppm. A broad range of peaks are present: 84-86, 74, 72, 70, 62-68 ppm. Spectrum Name: 071113_1-13c-erythro se_300ul_cba_900ul_hcho_cam_50ul.pdf In an eppendorf tube, 1-13C-erythrose (Omicron Biochemicals, 300 L, 0.085 M) was mixed with CBA (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand

PAGE 118

118 vortexer. A 0.5 mL aliquot was tran sferred to an NMR tube. This tube was maintained at 25 C and a NMR spectrum was acquired. 13C NMR (D2O): 171.764, 167.194, 106.445, 103.417, 103.241, 97.817, 90.130, 88.330, 84.355, 83.554, 82.894, 82.371, 81.837, 81.223, 80.387, 79.392, 76.416, 55.264, 55.237, 53.849, 49.500 (MeOH). A peak at 103.417 is assigned to 1-13C-erythrose. A signal appearing at 90.130 ppm is believed to be the adduct of methanol with formaldehyde. The formalde hyde signal is observed at 82.371 ppm. A moderate size peak is observed at 55.264 and 55.237 ppm. Spectrum Name: 071113_1-13c-erythrose_300ul_50mmboronCBA_900ul_h13cho_cam_50ul.pdf In an eppendorf tube, 1-13C-erythrose (Omicron Biochemicals, 300 L, 0.085 M) was mixed with CBA buffer B (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer. A 0.5 mL aliquot was tr ansferred to an NMR tube. This tube was maintained at 25 C and a NMR spectrum was acquired. 13C NMR (D2O): 171.745, 168.571, 106.438, 103.409, 94.879, 92.278, 90.138, 88.307, 84.332, 83.474, 83.218, 82.348, 76.390, 72.419, 69.687, 64.328, 63.942, 63.820, 63.454, 62.996, 55.245, 55.218, 49.500 (MeOH), 46.521 A peak at 103.409 is assigned to 1-13C-Erythrose. A signal appear ing at 90.138 ppmis thought to be the adduct of methanol with formaldehyde The formaldehyde signal is observed at 82.348 ppm. A moderate size peak is observed at 55.245 and 55.218 ppm. Small peaks are seen at 106.438, 88.307, 76.390, 72.419, 69.687, 64.328, 63.942, 63.820, 63.454, 62.996, and 46.521 ppm.

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119 Spectrum Name: 1-erythrose_100ul_c ba_900ul_50ulhcho_nt=16_47sec_14spectra.pdf 1-13C-Erythrose (100 L, 0.120 M, Omicron Bioche micals) was mixed with 0.9 mL of CBA buffer and 50 L of H13CHO and was agitated vigorously usi ng a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was comple ted after 4096 transien ts (ca. 3 hours total acquisition at 25C). 14 spectra @ nt=16 13C NMR (D2O): 171.799, 82.386, 49.500 (MeOH). Spectrum Name: 1-erythrose_300ul_c ba_900ul_50ulhcho_nt=16_47sec_1-10_11-17_725s.pdf A 0.120 M solution of 1-13C-erythrose (300L) was mixed w ith 0.9 mL of CBA buffer and 50 L of H13CHO and was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisiti on of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 h ours total acquisition at 25C). Started at 21:52 PM, spectra, 17 in a row, 1-10 spectra @ nt=16, 11-17, spectra @ nt=256 13C NMR (D2O): 171.199, 167, 103, 90, 82.379, 49.500 (MeOH). Spectrum Name: 071115_1-13c-eryth rose_carbonate_refmeoh_071113.pdf In an eppendorf tube, 1-13C-erythrose (Omicron Biochemicals, 300 L, 0.085 M) was mixed with carbonate buffer (900 L). The tube was sh aken using a hand vortexer. A 0.5 mL aliquot was transferred to an NMR tube. To this tube was added 10 L of a 10% MeOH in D2O as an internal reference. This tube was stored at 25 C and an NMR spectrum was acquired after 2 days.

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120 13C NMR (D2O): 171.779, 168.835, 142.399, 76.458, 74.116, 71.732, 71.553, 71.347, 71.057, 68.375, 61.837, 49.500 (MeOH). We must note that the original erythrose degradation was performed in Buffer C. Spectrum Name: 071115_2-13c-erythro se_300ul_cba_900ul_cam_h13cho_50ul_071113.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with CBA buffer (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer. A 0.5 mL aliquot was transferred to an NMR tube. This tube was stored at 25 C and an NMR spectrum was acquired after 2 days. 13C NMR (D2O): 171.802, 167.213, 110.367, 90.126, 83.409, 82.493, 82.363, 81.776, 81.331, 75.726, 55.275, 53.883, 49.500 (MeOH). A peak at 75.726 2-13C-Erythrose. A signal appearing at 90.126 is thought to be the adduct of methanol with formaldehyde. The formaldehyde signal is observed at 82.363 ppm. Small yet significant peaks are seen at 110.367, 55.275 and 53.883 ppm. Spectrum Name: 071115_1-13c-erythrose_300ul_50mmc ba900ul_cam_h13cho_50ul_071113.pdf In an eppendorf tube, 1-13C-Erythrose (Omicron Biochemicals, 300 L, 0.085 M) was mixed with CBA buffer B (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer. A 0.5 mL a liquot was transferred to an NMR tube. This tube was stored at 25 C and a NMR spectrum was acquired after 2 days.

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121 13C NMR (D2O): 171.783, 168.567, 106.442, 104.786, 103.420, 90.126, 84.320, 83.351, 82.913, 82.333, 81.734, 81.295, 78.785, 72.411, 69.882, 69.699, 69.493, 64.969, 64.686, 64.343, 64.011, 63.778, 63.469, 62.939, 55.260, 50.210, 49.500 (MeOH), 48.745 A peak at 103.420 ppm is assigned to 1-13C-erythrose. A signal appearing at 90.126 is thought to be the adduct of methanol with formaldehyde The formaldehyde signal is observed at 82.333 ppm. Two small peaks are seen at 69.882 and 55.260 ppm as well as a broad range from 62-65 ppm with approximately the same intensity. Spectrum Name: 071115_1-13c-erythro se_300ul_cba_900ul_cam_h13cho_50ul_071113.pdf In an eppendorf tube, 1-13C-erythrose (Omicron Biochemicals, 300 L, 0.085 M) was mixed with CBA buffer (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer. A 0.5 mL aliquot was transferred to an NMR tube. This tube was stored at 25 C and a NMR spectrum was acquired after 2 days. 13C NMR (D2O): 171.810, 167.221, 106.453, 103.432, 103.245, 97.798, 90.130, 83.405, 82.363, 76.405, 72.430, 55.279, 55.253, 49.500 (MeOH). Peaks at 103.432 and 103.245 are assigned to 1-13C-erythrose. A signal appearing at 90.130 ppm is thought to be the adduct of methanol with formaldehyde. The formaldehyde signal is observed at 82.363 ppm. Small yet significant p eaks are seen at 106.453, 55.279 and 55.253 ppm. Spectrum Name:071115_2-13c-erythrose _300ul_50mmcba900ul_cam_h13cho_50ul_65c12h.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with CBA buffer B (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer and stored at 65 C. A 0.5 mL aliquot was transferred to an NMR tube and an NMR spectrum was acquired after 12 hours.

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122 13C NMR (D2O): 171.783, 168.312, 83.077, 77.957, 77.599, 73.325, 72.430, 67.421, 66.574, 65.834, 65.075, 64.930, 64.682, 64.011, 63.820, 62.890, 62.111, 61.760, 61.383, 50.198, 49.500 (MeOH). A peak corresponding to 2-13C-erythrose has disappeared (~ 75 ppm). A signal appearing at 90.126 is thought to be the adduct of methanol with formaldehyde. The formaldehyde signal is no longer observed (82.363). Many small yet si gnificant peaks are observed at 72.430 and 61.760. Multiplexes are seen at 82, 78 and 62-68 ppm. Spectrum Name: 071115_2-13c-erythrose_300ul_50mmc ba900ul_cam_h13cho_50ul_071113.pdf In an eppendorf tube, 2-13C-erythrose (Omicron Biochemicals, 300 L, 0.099 M) was mixed with CBA buffer B (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer. A 0.5 mL a liquot was transferred to an NMR tube. This tube was stored at 25 C and an NMR spectrum was acquired after 2 days. 13C NMR (D2O): 171.779, 168.564, 90.126, 83.859, 82.333, 81.734, 81.284, 75.703, 72.476, 69.699, 66.590, 64.698, 64.331, 64.045, 63.725, 63.439, 63.225, 62.897, 62.645, 61.760, 55.256, 49.500 (MeOH). A peak at 75.703 2-13C-erythrose. A signal appearing at 90.126 is though t to be the adduct of methanol with formaldehyde. The formalde hyde signal is observed at 82.333. Small yet significant peaks are seen at 69.699, 64.698, 64.331, 63.725, 55.256.

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123 Spectrum Name: 071115_1-13c-erythrose_300ul_50mmcba 900ul_cam_h13cho_50ul_65c12h.pdf In an eppendorf tube, 1-13C-erythrose (Omicron Biochemicals, 300 L, 0.085 M) was mixed with CBA buffer B (900 L) and H13CHO (Cambridge, 50 L, 6.6 M). The tube was shaken using a hand vortexer and stored at 65 C. A 0.5 mL aliquot was transferred to an NMR tube and an NMR spectrum was acquired after 12 hours. 13C NMR (D2O): 171.783, 168.339, 83.325, 67.410, 64.942, 62.993, 62.111, 61.760, 61.387, 60.612, 49.500 (MeOH). A peak corresponding to 1-13C-erythrose has disappeared (~103 ppm). The signal appearing at 90.126 is thought to be the adduct of methanol with formaldehyde is no longer observed. The Formaldehyde signal is barely observed (82.325). Many small yet significant peaks are observed at 72.430 and 61.760. Multiplexes are seen at 80, 74 and 60-67 ppm. Reactions involving Threose Threose in CBA Spectrum Name: 070523_threose_17mg_cba_1ml_nt=4096.pdf Threose (17 mg) was dissolved in CBA buffer (1 mL)and agitated vigo rously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.198, 103.279, 82.333, 76.607, 71.614, 49.500 (MeOH).

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124 Spectrum Name: 071115_13c_l-threose_cba.pdf L-Threose (Aldrich, 18 mg) was dissolved in CB A buffer (1.0 mL) and agitated vigorously using a hand vortexer. A 0.5 mL aliquot was transferred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal reference. 13C NMR (D2O): 167.175, 103.275, 82.318, 76.603, 71.610, 65.182 (minor), 49.500 (MeOH). This spectrum exhibits the expected peaks for threose. A minor peak at 65.182 is present and unassigned. Reactions involving pentoses Ribose Spectrum Name: ribose_cba_meohref.pdf Ribose (30 mg, Fluka, final concentration 200 mM ) was dissolved in CBA buffer ([B]=278 mM; 1 mL). To this was added 10 L of 10% MeOH in D2O as an internal reference. The solution was agitated using a hand vorte xer, transferred to an NMR tube and spectrum acquisition started in 4 minutes. 13C NMR (D2O): 103.771, 102.482, 102.246, 102.211, 97.538, 87.834, 83.115, 82.756, 79.480, 79.167, 79.018, 77.595, 76.699, 76.527, 76.306, 71.675, 71.602, 71.392, 63.710, 62.317, 61.150, 60.967, 60.921, 49.500 (MeOH). Interpretation is that the multiple peaks come from ribose and borate complexes that are 1:1 and 2:1.

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125 For the following ribose spectra UL-13C-D-Ribose (15 mg, Omicron Biochemicals) was dissolved in CBA buffer (1 mL). As an internal standard, 35 l of H13CHO was evaporated, dissolved in 35 L of D2O, and added to the ribose solution. NMR spectra were acquired over a time period of five months. Spectrum Name: 120906_13C_RIBOSE_5C_1655-1705.pdf 13C NMR (D2O): 171.811, 167.123, 104.136, 103.515, 102.710, 102.466, 102.244, 102.000, 101.207, 97.297, 96.191, 94.737, 88.359, 88.146, 87.840, 87.608, 87.314, 87.112, 83.618, 83.259, 82.839, 82.641, 82.317, 80.020, 79.708, 79.475, 79.174, 78.926, 78.609, 78.457, 78.006, 77.514, 77.175, 77.015, 76.786, 76.523, 76.290, 76.038, 75.802, 73.429, 72.906, 72.079, 71.884, 71.560, 71.354, 71.041, 70.839, 69.965, 68.973, 68.146, 65.376, 63.976, 63.755, 63.427, 63.210, 62.550, 61.997, 61.417, 61.196, 60.845, 60.627, 49.500 (MeOH). Spectrum Name: 120906_ul5_13c_ribose_1655-1705.pdf 13C NMR (D2O): 171.811, 167.123, 104.136, 103.515, 102.710, 102.466, 102.244, 102.000, 101.207, 97.297, 96.191, 94.737, 88.359, 88.146, 87.840, 87.608, 87.314, 87.112, 83.618, 83.259, 82.839, 82.641, 82.317, 80.020, 79.708, 79.475, 79.174, 78.926, 78.609, 78.457, 78.006, 77.514, 77.175, 77.015, 76.786, 76.523, 76.290, 76.038, 75.802, 73.429, 72.906, 72.079, 71.884, 71.560, 71.354, 71.041, 70.839, 69.965, 68.973, 68.146, 65.376, 63.976, 63.755, 63.427, 63.210, 62.550, 61.997, 61.417, 61.196, 60.845, 60.627, 49.500 (MeOH). Spectrum Name: 121006_ul_13c_ribose_1640.pdf 13C NMR (D2O): 171.811, 167.115, 104.121, 103.507, 102.710, 102.450, 102.240, 102.004, 101.207, 97.758, 97.255, 94.127, 88.359, 87.844, 87.627, 87.306, 87.116, 83.610, 83.244, 82.839, 82.633, 82.313, 80.020, 79.704, 79.475, 79.155, 78.922, 78.609, 78.457, 78.025, 77.510, 77.175, 77.015, 76.782, 76.519, 76.290, 76.038, 75.802, 73.406, 72.906, 72.079, 71.869, 71.556,

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126 71.358, 71.041, 70.835, 69.500, 68.989, 68.119, 65.376, 63.976, 63.740, 63.423, 62.546, 61.970, 61.413, 61.192, 60.845, 60.623, 49.500 (MeOH). Spectrum Name: 010807_ul_13c_ribose_1110.pdf 13C NMR (D2O): 171.811, 102.706, 102.240 ,79.471, 78.922, 76.736, 76.286, 75.802 ,72.067, 71.548, 71.369, 71.626, 71.022, 70.759, 69.496, 68.981, 69.409, 61.180, 60.845, 60.616, 49.500 (MeOH). Spectrum Name: 022207_ul_13c_ribose.pdf 13C NMR (D2O): 171.802, 112.236, 111.832, 111.702, 111.508, 111.366, 111.218, 110.989, 110.657, 110.489, 104.107, 103.481, 102.707, 102.440, 102.242, 101.982, 101.807, 88.352, 87.826, 87.582, 87.300, 83.618, 83.229, 82.596, 82.192, 81.932, 81.509, 80.571, 80.029, 79.487, 78.934, 78.694, 78.461, 78.244, 77.957, 77.416, 77.214, 76.866, 76.722, 76.371, 76.302, 76.233, 75.843, 75.806, 74.566, 73.323, 73.235, 73.014, 72.838, 72.705, 72.525, 72.194, 72.068, 71.907, 71.770, 71.549, 71.400, 71.263, 71.026, 70.900, 70.759, 69.500, 69.306, 68.985, 68.463, 67.097, 67.006, 66.712, 66.647, 66.430, 66.128, 65.419, 64.991, 64.690, 64.324, 63.981, 63.702, 63.443, 62.577, 62.138, 62.020, 61.840, 61.410, 61.192, 60.852, 60.626, 59.571, 59.079, 49.500 (MeOH). Spectrum Name: 070702_d-ribose_cba_ samplefrom070411_81dayslater.pdf D-ribose (30 mg, Aldrich) was dissolved in 1.00 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 179.370, 165.404, 76.053, 73.211, 72.418, 70.930, 63.400. No reference.

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127 Spectrum Name: 071019_ul-13c-d-ri bose_7mg_cba_1ml_meohref.pdf D-UL-13C-Ribose (Omicron Biochemicals, 7 mg) was dissolved in CBA buffer (1.0 mL) in an eppendorf tube. An aliquot (0.5 mL) was transferred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal reference. This tube was maintained at 25 C and an NMR spectrum was acquired. 13C NMR (D2O): 167.297, 104.271, 103.684, 102.722, 102.253, 97.306, 96.737, 96.268, 88.410, 87.891, 87.372, 83.355, 82.939, 82.756, 82.341, 80.036, 79.487, 78.938, 78.476, 77.671, 76.813, 76.321, 75.829, 73.403, 72.934, 72.110, 71.587, 71.068, 69.096, 68.589, 68.089, 63.969, 63.431, 62.558, 61.993, 61.436, 60.868, 49.500 (MeOH). Spectrum Name: 20071307_d-ribose_47mg_carbonatebuffer_refmeoh.pdf D-ribose (47 mg, Aldrich) was dissolved in 1.0 mL of carbonate buffer and was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 168.770, 100.319, 94.818, 94.494, 83.031, 71.610, 71.171, 70.843, 70.610, 69.535, 69.310, 67.719, 67.650, 64.129, 63.481, 62.596, 49.500 (MeOH). Spectrum Name: 071020_ul-13c-d-r ibose_7mg_cba_1ml_nomeoh.pdf D-UL-13C-Ribose (Omicron Biochemicals, 7 mg) was dissolved in CBA buffer (1.0 mL) in an eppendorf tube. An aliquot (0.5 mL) was transferre d to an NMR tube. This tube was maintained at 25 C and an NMR spectrum was acquired.

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128 13C NMR (D2O): 167.000 (carbonate), 103.962, 103.379, 102.418, 101.952, 101.151, 96.943, 96.459, 95.936, 88.116, 87.807, 87.601, 87.052, 83.062, 82.631, 82.005, 79.735, 79.186, 78.637, 78.183, 77.389, 76.516, 76.020, 75.532, 73.083, 72.690, 71.809, 71.286, 70.767, 69.199, 68.795, 68.185, 67.849, 63.668, 63.123, 62.253, 61.684, 61.135, 60.567. Arabinose Spectrum Name: arabinose_cba_meohref.pdf D-arabinose (30 mg, Aldrich) was dissolved in CBA buffer (1 mL). To this was added 10 L of 10% MeOH in D2O as an internal reference. The solu tion was agitated usin g a hand vortexer, transferred to an NMR tube and spec trum acquisition started in 4 minutes. 13C NMR (D2O): 104.050, 103.920, 103.539, 97.370, 92.260, 95.047, 94.628, 93.106, 86.544, 86.197, 85.739, 85.556, 85.194, 83.672, 83.393, 83.206, 83.073, 78.831, 77.256, 76.798, 75.165, 72.968, 72.361, 72.197, 71.957, 71.408, 71.072, 70.919, 70.519, 70.298, 70.179, 69.867, 69.027, 68.653, 68.570, 68.447, 66.975, 65.789, 63.961, 62.279, 62.916, 62.691, 62.607, 49.500 (MeOH). Spectrum Name: 070626_2-13C-arabi nose_100ul_CBA_500ul_refmeoh.pdf A 0.120M solution of 2-13C-arabinose (100L) was dissolv ed in 0.500 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.309, 110.329, 83.329, 80.231, 75.127, 72.235, 70.233, 68.493, 49.500 (MeOH).

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129 Spectrum Name: 070626_5-13C-arabi nose_100ul_CBA_500ul_refmeoh.pdf A 0.120M solution of 5-13C-arabinose (100L) was dissolv ed in 0.500 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.312, 95.089, 94.769, 78.457, 72.258, 70.546, 68.768, 67.009, 66.086, 64.099, 63.321, 62.878, 52.807, 49.500 (MeOH). Spectrum Name: 070626_UL-ARABI NOSE_100ul_D2O_500ul_refmeoh.pdf A 0.120M solution of UL-13C-arabinose (100L) was dissolved in 0.500 mL D2O and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 101.956, 101.372, 98.068, 97.725, 97.630, 97.546, 97.244, 97.161, 97.065, 96.058, 95.482, 93.495, 93.441, 93.350, 93.258, 93.083, 92.097, 81.141, 83.577, 83.016, 82.043, 81.471, 76.855, 76.630, 76.161, 74.746, 73.731, 73.635, 73.204, 73.113, 72.930, 72.819, 72.644, 72.358, 71.839, 70.019, 69.600, 69.493, 69.409, 69.355, 69.207, 69.161, 69.107, 69.073, 68.993, 68.924, 68.837, 68.726, 68.612, 67.219, 67.006, 66.719, 63.213, 63.202, 62.973, 62.859, 61.997, 61.425, 49.500 (MeOH). Spectrum Name: 070626_ul_arabi nose_50ul_cba_950ul_refmeoh.pdf A 0.120M solution of UL-13C-arabinose (50L) was dissolved in 0.950 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH

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130 in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 206.802, 167.000, 161.179, 94.906, 94.441, 71.866, 71.324, 70.618, 70.145, 69.592, 68.539, 68.002, 67.582, 63.149, 62.611, 49.500 (MeOH), 40.174 Xylose Spectrum Name: xylose_cba_meohref.pdf Xylose (30 mg, Aldrich) was dissolved in CBA buffer (1 mL). To this was added 10 L of 10% MeOH in D2O as an internal reference. The solu tion was agitated using a hand vortexer, transferred to an NMR tube and spec trum acquisition started in 4 minutes. 13C NMR (D2O): 111.344, 103.569, 103.028, 97.279, 92.816, 83.523, 83.157, 79.918, 77.568, 76.958, 76.73, 76.664, 76.430, 76.310, 75.077, 74.551, 73.307, 72.461, 71.949, 69.897, 49.794, 65.781, 71.570, 61.398, 61.066, 60.730, 60.227, 59.582, 57.656, 54.310, 49.500 (MeOH). 1-13C-Xylose in CBA Spectrum Name: 070610_1-13C-xylose_50ul_0.120M_cba_950ul.pdf 1-13C-xylose (50 L, 0.120 M) was dissolved in CBA buffer (1 mL) and agitated vigorously using a vortex stirrer. To this was added a sa mple of authentic threose and 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.370, 103.249, 49.500 (MeOH).

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131 Spectrum Name: 070626_1-2-13C-xylose_100ul_CBA_500ul_refmeoh.pdf A 0.120M solution of 1,2-13C-xylose (100L) was dissolved in 0.500 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.305, 103.489 103.024, 83.622, 83.161, 49.500 (MeOH). Spectrum Name: 070626_1-2-13C-xylose_100ul_D2O_500ul_refmeoh.pdf A 0.120M solution of 1,2-13C-xylose (100L) was dissolved in 0.500 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 97.557, 96.947, 96.390, 95.822, 93.167, 92.552, 74.959, 74.349, 72.392, 71.782, 69.874, 65.823, 49.500 (MeOH). Spectrum Name: 070626_2-13C-xylose_100ul_CBA_500ul_refmeoh.pdf A 0.120M solution of 2-13C-xylose (100L) was dissolved in 0.500 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.305, 103.489, 103.024, 83.622, 83.161, 49.500 (MeOH). Spectrum Name: 070626_2-13C-xylose_100ul_D2O_500ul_refmeoh.pdf

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132 A 0.120M solution of 2-13C-xylose (100L) was dissolved in 0.500 mL D2O and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 97.550, 96.939, 89.066, 81.219, 77.774, 76.794, 76.218, 75.402, 74.665, 74.364, 73.868, 73.231, 72.094, 69.874, 68.815, 61.558, 55.020, 54.207, 49.500 (MeOH). Spectrum Name: 070626_UL-xylose_100ul_CBA_500ul_refmeoh.pdf A 0.120M solution of UL-13C-xylose (100L) was dissolved in 0.500 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.305, 103.485, 103.028, 83.912, 83.336, 82.871, 77.820, 77.324, 76.767, 75.562, 75.058, 74.574, 61.161, 60.635, 49.500 (MeOH). Spectrum Name: 070626_ulxylose_100ul_d2o_500ul_refmeoh.pdf A 0.120M solution of UL-13C-xylose (100L) was dissolved in 0.500 mL D2O and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 97.584, 97.519, 96.985, 96.920, 93.163, 92.568, 76.969, 76.500, 76.462, 76.016, 75.951, 75.188, 74.677, 74.589, 74.547, 74.078, 74.032, 73.502, 73.006, 72.609, 72.544,

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133 72.016, 72.010, 71.961, 71.511, 71.461, 70.538, 70.485, 70.385, 70.343, 70.023, 69.985, 69.859, 69.538, 69.474, 69.355, 69.310, 66.067, 65.537, 61.802, 61.284, 49.500 (MeOH). Spectrum Name: 070702_xylose_cba_samplefrom070411_81dayslater.pdf D-xylose (30 mg) was dissolved in CBA buffer (1 mL) and agita ted vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.855, 122.715, 111.282, 83.241, 83.142, 76.962, 76.504, 72.464, 63.965, 49.500 (MeOH). Spectrum Name: 20071407_d-xylose_cba_20070411.pdf To a sample, from a reaction prepared on 04/11/ 2007, of d-xylose (30 mg) in CBA buffer (1 mL) was added 10 L of a 10% MeOH in D2O as an internal reference. A NMR spectrum was acquired three days later on 07/14/2007. 13C NMR (D2O): 166.717, 111.309, 83.271, 83.130, 76.954, 76.508, 72.464, 49.500 (MeOH). Spectrum Name: 071127_2-13c-xylose_2-13c-x ylulose_enzyme_50ul_cba_450ul_meohref.pdf In an eppendorf tube, 2-13C-xylose (500 L, 0.120 M) was added to a resin bound enzyme. An aliquot (50 L) was dissolved in CBA buffer (450 L ). To this tube was added10 L of a 10% MeOH in D2O as a reference. Acquisition of an NM R spectrum was begun after four minutes, and was completed after 4096 transients (c a. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.259, 111.435, 83.386, 74.623, 49.500 (MeOH).

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134 Lyxose Spectrum Name: lyxose_cba_meohref.pdf Lyxose (30 mg, Aldrich) was dissolved in CBA buffer (1 mL). To this was added 10 L of 10% MeOH in D2O as an internal reference. The solu tion was agitated using a hand vortexer, transferred to an NMR tube and spec trum acquisition started in 4 minutes. 13C NMR (D2O): 111.351, 102.516, 102.375, 97.790, 97.206, 96.443, 95.230, 94.712, 83.153, 82.726, 81.715, 81.463, 80.201, 78.972, 76.958, 76.847, 76.767, 75.199, 73.658, 72.472, 70.614, 68.833, 68.447, 68.180, 64.854, 64.686, 61.181, 61.017, 49.500 (MeOH). 1-13C-Lyxose in CBA Spectrum Name: 070611_1-13c-lyx ose_cba_refmeoh_nt=1024.pdf 1-13C-Lyxose (50L, 0.120M) was dissolved in CB A buffer (1 mL) and agitated vigorously using a vortex stirrer. To this was added a sa mple of authentic threose and 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.385, 102.574, 99.934, 97.790, 96.359, 95.188, 49.500 (MeOH).

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135 Ribulose Spectrum Name: 121106_ribulose_ cba_start1900-1930.pdf nomeoh Ribulose (Fluka, 30.5 mg) was dissolved in CBA (0.9 mL) D2O (0.1 mL). Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 178.005, 167.000 (carbonate), 139.291, 111.082, 76.655, 71.178, 69.209, 66.966, 64.609 Spectrum Name: 121106_ribul ose_cba_start1930-0200.pdf 13C NMR (D2O): 167.000 (carbonate), 111.631, 111.090, 104.994, 82.003, 76.869, 76.659, 76.339, 75.145, 73.699, 72.723, 71.353, 71.189, 69.217, 68.671, 66.963, 64.620, 64.338, 64.113 Spectrum Name: 121206_ribulose_cba_1015.pdf 13C NMR (D2O): 167.000 (carbonate), 111.082, 104.971, 81.988, 76.655, 76.331, 75.141, 72.719, 71.330, 71.181, 69.209, 68.687, 66.921, 64.609, 64.296, 61.588, 61.042, 60.726, 55.584 Spectrum Name: ribulose_cba_start121106_050407.pdf To the sample started on 12/11/06 was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.799, 166.694, 111.096, 105.072, 82.066, 79.945, 76.928, 76.706, 76.420, 75.238, 73.902, 72.808, 72.483, 72.430, 71.270, 70.065, 69.298, 68.779, 65.697, 63.824, 61.791, 61.142, 59.342, 53.856, 49.500 (MeOH). Xylulose Spectrum Name: xylulose_CBA_030207.pdf

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136 Xylulose (20 mg) was dissolved in CBA buffer (1 mL)and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 166.980, 111.532, 111.407, 83.322, 83.185, 76.977, 76.532, 72.490, 64.704, 64.274, 49.500 (MeOH). 1,2-13C-Xylulose in CBA Spectrum Name: 070610_1-2-13C -xylulose_cba_05302007.pdf 2048 1,2-13C-Xylulose (50 L, 0.120 M) was dissolved in CBA buffer (1 mL) and agitated vigorously using a vortex stirrer. To this was added a sa mple of authentic threose and 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.316, 111.752, 111.035, 65.068, 64.343, 49.500 (MeOH). Spectrum Name: 1-2-13c-xylulose_cba_refmeoh.pdf A 0.120 M solution of 1,2-13C-Xylulose (50 L) was mixed with 0.950 mL CBA buffer. The mixture was agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.316, 111.790, 111.073, 65.072, 64.354, 49.500 (MeOH).

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137 Spectrum Name: xylulose_CBA_start 1500-033107-scanned-033107-2245-0445_nt=8192.pdf 30 mg of d-xylulose was dissolved in 1.0 mL of CBA buffer and wa s agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 167.000, 111.470, 110.753, 106.553, 106.358, 105.939, 102.685, 83.150, 83.092, 82.661, 76.920, 76.665, 76.428, 72.430, 72.213, 71.934, 64.759, 64.454, 64.347, 64.042, 63.737, 63.630, 49.500 (MeOH). 1,2,4,5-tetrahydroxypentan-3-one (3-pentulose) Spectrum Name: pentulose_d2o_030807_eureka.pdf 1,2,3,4-tetrahydroxy-pentan-3-one (3 0 mg) was dissolved in D2O (1 mL). One drop of MeOH was added as an internal standard and a spectrum was acquired after four minutes. 13C NMR (D2O): 222.522, 212.226, 81.799, 76.336, 74.421, 73.483, 73.346, 73.037, 72.399, 71.335, 70.958, 63.374, 63.149, 59.861, 59.075, 49.500 (MeOH), 48.802, 47.741, 43.187, 42.866, 26.799, 25.159, 19.471, 19.318 Spectrum Name: 1,2,3,4-pentan-3-one.pdf 1,2,3,4-tetrahydroxy-pentan-3-one (3 0 mg) was dissolved in D2O (1 mL) and a spectrum was acquired after four minutes. 13C NMR (D2O): 211.818, 75.923, 62.959. No reference.

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138 Spectrum Name: 20071307_pentulose _15mg_200ul_d2o_h13cho_30ul_800ul_cba_nt=256.pdf 3-Pentulose (15 mg) was dissolved in 200 L of D2O. To this was added 30 L of H13CHO (6.66 M), 0.800 mL of CBA buffer and was agitated vigor ously using a vortex s tirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was comple ted after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.787, 168.770, 82.329, 65.335, 64.732, 64.362, 64.087, 63.786, 63.538, 63.065, 62.588, 62.527, 61.989, 61.722, 61.325, 61.036, 49.500 (MeOH). Spectrum Name: 20071307_pentulose _15mg_200ul_d2o_h13cho_30ul_800ul_cba_nt=8192.pdf 3-Pentulose (15 mg) was dissolved in 200 L of D2O. To this was added 30 L of H13CHO (6.66 M), 0.800 mL of CBA buffer and was agitated vigor ously using a vortex s tirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was comple ted after 4096 transien ts (ca. 3 hours total acquisition at 25C). 13C NMR (D2O): 171.787, 168.770, 82.329, 65.335, 64.732, 64.362, 64.087, 63.786, 63.538, 63.065, 62.588, 62.527, 61.989, 61.722, 61.325, 61.036, 49.500 (MeOH). Branched pentose Branched pentose in bora te (0.250 M, t = 0 hours) Spectrum Name: 070518_branched_pent ose_borate-only_refmeoh.pdf Erythro-branched pentose (30 mg) was dissolved in 1 mL of 0.250 M Sodium tetra-borate in D2O and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in

PAGE 139

139 D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25 C). 13C NMR (D2O): 104.664, 104.603, 104.466, 104.366, 98.953, 98.870, 90.046,85.396, 85.232, 84.572, 84.439, 84.294, 84.023, 82.371, 78.167, 78.007, 77.771, 73.750, 71.253, 71.183, 71.011, 70.187, 69.271, 68.863, 68.787, 68.749, 64.072, 63.931, 63.805, 63.412, 63.366, 63.199, 63.004, 49.500 (MeOH), 48.970, 48.680, 48.394 Branched pentose in bora te (0.250 M, t = 13 hours) Spectrum Name: 070519_branched_pentose_bo rate-only_refmeoh_13ho urslater_nt=8192.pdf Erythro-branched pentose (30 mg) was dissolved in 1 mL of 0.250 M Sodium tetra-borate in D2O and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (ca. 3 hours total acquisition at 25 C). 13C NMR (D2O): 104.366, 104.247, 104.186, 104.049, 103.946, 98.537, 98.449, 89.629, 84.983, 84.815, 84.270, 84.155, 84.026, 83.873, 83.605, 81.954, 77.747, 77.590, 73.337, 70.877, 70.758, 70.667, 70.594, 70.152, 69.770, 68.851, 68.443, 68.370, 68.328, 63.652, 63.510, 63.388, 62.992, 62.950, 62.782, 62.583, 49.500 (MeOH), 49.083, 48.553, 48.263, 47.977 Branched pentose in CBA Spectrum Name: 070523_branched_pent ose_30mg_cba_1ml_nt=8192.pdf Erythro-branched pentose (30 mg) was dissolved in CBA buffer (1 mL) and agitated vigorously using a vortex stirrer. To this was added 10 L of a 10% MeOH in D2O as an internal reference.

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140 Acquisition of an NMR spectrum was begun within four minutes, and was completed after 8192 transients (ca. 6 hours total acquisition at 25C). 13C NMR (D2O): 105.255, 104.778, 104.645, 104.462, 104.355, 98.747, 85.404, 84.687, 84.587, 84.370, 84.027, 82.306, 80.132, 77.366, 73.986, 73.685, 71.347, 71.236, 71.175, 70.916, 69.199, 68.722, 64.446, 64.236, 63.816, 63.420, 49.500 (MeOH). Spectrum Name: 071119_ery-branched-pentose_cba_refmeoh_nt=8192.pdf Erythro-branched-pentose (25 m g, 0.167 mmol) was dissolved in CBA buffer (1 mL). The tube was shaken using a hand vortexer and an aliquo t (0.5 mL) was transferred to an NMR tube. To this was added 10 L of a 10% MeOH in D2O was added as a reference. The tube was maintained at 25 C for 2 days and an NMR spectrum was acquired. 13C NMR (D2O): 167.259, 105.335, 104.813, 85.125, 84.397, 82.341, 80.689, 80.223, 74.223, 74.021, 71.213, 69.230, 64.644, 63.851, 49.500 (MeOH). Sharp singlets are seen at 104.813, 84.397, 82.341, 74.021, 71.213, 69.230 and 63.851. Hexoses Spectrum Name: 071014_d-12C-GLUCO SE_50mg_CBA_1ml_meohref.pdf Glucose (Aldrich, 50 mg) was dissolved in CBA (1.0 mL) in an eppendorf tube. An aliquot (0.5 mL) was transferred to an NMR tube and 10 L of a 10% MeOH in D2O solution was added as an internal reference. This tube was maintain ed at 25 C and an NMR spectrum was acquired. 13C NMR (D2O): 103.920, 103.218, 103.058, 96.539, 92.659, 82.753, 78.743, 78.137, 77.671, 76.493, 76.157, 75.997, 75.821, 74.658, 73.174, 71.980, 70.080, 69.104, 64.507, 64.217, 61.200, 61.051, 49.500 (MeOH).

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141 Spectrum Name: 071014_d-12C_GLUCOSE_50mg_D2O_1ml_nomeoh.pdf Glucose (Aldrich, 50 mg) was dissolved in D2O (1.0 mL) in an eppendorf tube. An aliquot (0.5 mL) was transferred to an NMR tube. This tube was maintained at 25 C and an NMR spectrum was acquired. 13C NMR (D2O): 96.057, 92.243, 76.091, 75.908, 74.287, 72.914, 71.632, 71.582, 69.797, 69.751, 60.905, 60.745. No reference. Formaldehyde in CBA buffer saturated with sodiumtetraborate Spectrum Name: 070724_H13CHO_SCBA_1ml_nomeoh.pdf A 6.66 M solution of H13CHO (30 L) was mixed with 1.00 mL SCBA buffer. The mixture was agitated vigorously using a vortex stirrer. Acquisition of an NMR spectrum was begun within four minutes, and was completed after 4096 transients (c a. 3 hours total acquisition at 25 C). 13C NMR (D2O): 171.378, 162.673, 92.578, 87.272, 86.189, 85.143, 84.079, 83.736, 83.515, 83.160, 82.473, 81.954, 81.390, 80.718, 80.341, 80.142, 79.830, 79.437, 79.009, 78.769, 77.407, 75.584, 71.334, 49.080. No reference. For the following six spectra (070331A) 1,2,3-13C-glyceraldehyde (0.1 mL, fi nal concentration 11.1 mM, 11.1 micromoles, Omicron Biochemicals) was mixed with H13CHO (0.0035 mL, 6.66 M, [H13CHO] = 23.3 mM final concentration 2.1:1 ratio) in CBA buffer (0.9 mL). Spectrum Name: 070331A_glyceraldehyde_H13CHO_040507_1015am.pdf 13C NMR (D2O): 171.752, 167.204 (carbonate), 82.305 (HCHO), 71.784, 65.734, 65.028, 63.842, 62.499

PAGE 142

142 070331A_glyceraldehyde_H13CHO_040907_nt=256.pdf 13C NMR (D2O): 171.767, 167.223 (carbonate), 102.374, 101.775, 98.903, 95.832, 82.305 (HCHO), 82.099, 79.989, 76.518, 74.050, 73.596, 72.547, 71.738, 71.265, 70.811, 66.959, 66.611, 65.768, 65.036, 64.262, 63.014, 61.874, 61.385, 58.883, 58.387 Spectrum Name: 070331A_glyceral dehyde_H13CHO_041007_nt=4096_0000-0300.pdf 13C NMR (D2O): 171.767, 167.216 (carbonate), 105.502, 104.964, 104.396, 104.270, 103.755, 103.396, 102.931, 102.256, 101.722, 100.905, 99.490, 99.421, 98.887, 96.301, 95.813, 82.305 (HCHO), 81.642, 81.172, 79.360, 77.361, 77.010, 76.518, 75.973, 74.164, 73.657, 73.081, 72.372, 71.796, 71.300, 69.385, 68.843, 67.065, 66.608, 65.787, 65.013, 63.869, 63.056, 62.465, 62.122, 61.393, 60.798, 58.887, 58.391, 26.618, 25.977 Spectrum Name: 070331A_glyceraldehyde_H13CHO_041507.pdf 13C NMR (D2O): 171.759, 170.512, 167.201 (carbonate), 112.845, 111.380, 104.968, 104.274, 103.434, 102.244, 101.813, 99.410, 98.887, 96.232, 95.828, 85.479, 82.305 (HCHO), 79.837, 76.564, 74.157, 73.657, 72.639, 71.796, 71.262, 69.373, 67.058, 66.608, 65.723, 65.009, 64.056, 61.393, 60.317, 58.871, 58.395, 28.041, 26.542, 25.962, 24.635, 23.162 Spectrum Name: 070331A_glyceralde hyde_H13CHO_CBA_nt=256_start2215-2230.pdf 13C NMR (D2O): 167.227 (carbonate), 151.995, 104.411, 102.359, 101.741, 99.978, 94.520, 82.305 (HCHO), 80.432, 77.373, 73.364, 73.325, 71.414, 65.715, 63.834, 63.110, 62.534, 60.378, 52.810 Spectrum Name: 070331A_glyceraldehyde _H13CHO_CBA_t=2_1300_ref=co3-167.pdf 13C NMR (D2O): 167.000 (carbonate), 102.139, 94.857, 94.319, 82.074, 80.685, 80.136, 79.709, 76.260 73.594, 63.569, 63.008

PAGE 143

143 Spectrum Name: 070331_glyceraldehyde_H13CHO_start0130am040207end0730am_nt=8192.pdf 13C NMR (D2O): 171.767, 167.220 (carbonate), 105.502, 104.968, 104.789, 104.415, 104.262, 103.759, 103.438, 102.942, 102.359, 102.263, 101.832, 101.737, 100.814, 100.337, 99.490, 98.956, 98.891, 96.324, 95.813, 94.588, 84.014, 82.305 (HCHO), 81.672, 81.145, 79.803, 77.060, 76.522, 75.976, 74.161, 73.642, 73.043, 71.792, 71.300, 69.358, 67.123, 66.608, 65.787, 65.082, 64.387, 63.853, 63.102, 62.530, 61.923, 61.389, 58.936, 58.440, 26.622, 26.527, 25.974, 25.878 Experiment 070331A spectra U-13C-glyceraldehyde (0.1 mL, final concentrat ion 11.1 mM, 11.1 micromoles) was mixed with H13CHO (0.0035 mL, 6.66 M, [H13CHO] = 23.3 mM final con centration 2.1:1 ratio) and dissolved in CBA buffer (0.9 mL). Spectrum Name: 070331A_U13C-glyceraldehyde_H13CHO_ start0130am040207end0730am.pdf 13C NMR (D2O): 171.767, 167.220 (carbonate), 105.502, 104.968, 104.789, 104.415, 104.262, 103.759, 103.438, 102.942, 102.359, 102.263, 101.832, 101.737, 100.814, 100.337, 99.490, 98.956, 98.891, 96.324, 95.813, 94.588, 84.014, 82.305 (HCHO), 81.672, 81.145, 79.803, 77.060, 76.522, 75.976, 74.161, 73.642, 73.043, 71.792, 71.300, 69.358, 67.123, 66.608, 65.787, 65.082, 64.387, 64.853, 63.102, 62.530, 61.923, 61.389, 58.936, 58.440, 26.622, 26.527, 25.974, 25.878

PAGE 144

144 Spectrum Name: 070331A_U13C-glyceraldehyde_ref H13CHO-82.350_nt=2048_040107-1300.pdf 13C NMR (D2O): 171.763, 167.223 (carbonate), 104.976, 104.415, 103.751, 103.442, 102.363, 102.263, 101.925, 100.505, 99.494, 96.347, 95.801, 94.558, 83.991, 82.305 (HCHO), 80.478, 74.164, 73.627, 71.784, 71.296, 65.787, 65.066, 64.449, 63.823, 63.106, 62.556, 26.538, 25.974 070331A_U-glyceraldehyde_H13C HO_CBA_t=2_1300_refco3_-167.pdf 13C NMR (D2O): 167.000 (carbonate), 102.139, 94.857, 94.319, 82.074, 80.685, 80.136, 79.709, 76.260, 73.594, 64.319, 63.569, 63.008 Spectrum Name: 070427_repeatof070331A_withMeOHref.pdf U-13C-glyceraldehyde (0.1 mL, fi nal concentration 11.1 mM, 11.1 micromoles) was mixed with H13CHO (0.0035 mL, 6.66 M, [H13CHO] = 23.3 mM final concentration 2.1:1 ratio) and disso lved in CBA buffer (0.9 mL). To this was added 10ul of a 10% MeOH in D2O solution as an internal reference. 13C NMR (D2O): 171.821, 167.248, 113.552, 112.919, 106.796, 106.102, 105.557, 105.030, 104.321, 103.821, 103.043, 102.337, 101.803, 99.549, 99.484, 99.018, 98.953, 98.328, 95.879, 90.142, 85.064, 82.371, 81.696, 81.162, 80.693, 77.076, 76.580, 76.050, 74.215, 73.708, 73.182, 72.419, 71.877, 71.362, 70.996, 70.427, 69.455, 67.124, 66.662, 65.850, 65.785, 65.483, 65.072, 64.057, 63.080, 62.180, 61.455, 59.578, 58.945, 58.464, 49.500 (MeOH), 26.684, 26.028 Spectrum Name: 070427_repeatof070331A_withMolybdicac id_13mg_nomeoh_refhcho82.37.pdf U-13C-glyceraldehyde (0.1 mL, fi nal concentration 11.1 mM, 11.1 micromoles) was mixed with H13CHO (0.0035 mL, 6.66 M, [H13CHO] = 23.3 mM final

PAGE 145

145 concentration 2.1:1 ratio) and disso lved in CBA buffer (0.9 mL). To this was added 13 mg of molybdic acid (Aldrich). 13C NMR (D2O): 171.810, 165.844, 137.157, 136.879, 136.402, 136.082, 118.755, 118.534, 117.970, 117.611, 114.220, 113.522, 113.289, 112.927, 106.686, 106.072, 105.511, 104.981, 104.267, 103.447, 102.986, 99.518, 99.453, 98.923, 96.829, 95.802, 84.992, 82.371 (HCHO), 81.669, 81.135, 80.597, 77.069, 76.569, 76.031, 74.761, 74.208, 73.681, 73.139, 72.792, 72.346, 71.835, 71.293, 70.946, 70.420, 68.720, 67.112, 66.658, 66.319, 66.208, 65.762, 65.049,63.080, 62.191, 61.463, 61.062, 60.868, 59.582, 59.079, 58.922, 58.434, 54.165, 50.492, 50.213, 50.148, 50.042, 49.931 Experiment 070331B spectra Spectrum Name: 070331B_glyceraldehyde_H12CHO _start0730am040207end1330pmnt=8192.pdf 1,2,3-13C-glyceraldehyde (0.1 mL, fi nal concentration 11.1 mM, 11.1 micromoles) was mixed with HCHO (0.002 mL, 12 M, [HCHO] = 24 mM final concentration 2.2:1 ratio) and dissolved in CBA buffer (0.9 mL) 13C NMR (D2O): 171.763, 167.216, 105.502, 104.949, 104.789, 104.400, 104.262, 103.755, 103.412, 102.939, 102.363 102.267, 101.829, 101.737, 100.833, 100.333, 99.486, 99.418, 98.952, 96.328, 95.798, 94.569, 82.305 (HCHO), 79.528, 77.937, 77.434, 76.766, 76.213, 75.763, 74.760, 74.145, 72.745, 72.211, 70.838, 70.155, 68.481, 65.719, 65.002, 64.395, 63.796, 73.014, 61.389, 49.438, 26.618, 26.523, 25.966, 25.867

PAGE 146

146 Experiment 070331D spectra 1-13C-glycolaldehyde (0.1 mL, final concentrat ion 11.6 mM, 11.6 micromoles) was mixed with H13CHO (Aldrich-Isotec, 0.0054 mL, 6.66 M, [H13CHO] = 35.6 mM final concentration, 3.1:1 ratio) and the mixture was di ssolved in CBA buffer (0.9 mL) Spectrum Name: 070331D_glycolalde hyde_H13CHO_ref=hcho_82.305_nt=256_t=2.pdf 13C NMR (D2O): 167.253, 98.262, 93.059, 82.305 070331D_glycolaldehyde_H13CHO_ref=hcho_82.305_nt=256_t=17.pdf 13C NMR (D2O): 171.767, 167.231, 98.273, 97.220, 92.975, 89.938, 82.305 (HCHO), 80.440, 73.939, 63.533, 53.081, 9.906 Spectrum Name: 070331D_glycolalde hyde_H13CHO_ref=hcho_82.305_nt=256_t=32.pdf 13C NMR (D2O): 167.231, 98.266, 93.005, 92.002, 91.132, 82.305 (HCHO). Spectrum Name: 070331D_glycolalde hyde_H13CHO_ref=hcho_82.305_nt=256_t=47.pdf 13C NMR (D2O): 167.235, 93.120, 82.305 (HCHO). Spectrum Name: 070331D_glycolalde hyde_H13CHO_ref=hcho_82.305_nt=256_t=62.pdf 13C NMR (D2O): 191.954, 185.877, 176.077, 171.763, 167.231, 138.373, 98.269, 93.131, 90.571, 82.305 (HCHO), 63.495 Spectrum Name: 070331D_glycolalde hyde_H13CHO_ref=hcho_82.305_nt=256_t=77.pdf 13C NMR (D2O): 171.767, 167.231, 161.391, 103.225, 98.262, 96.538, 92.998, 91.636, 90.655, 82.305 (HCHO), 69.137, 63.480, 11.138 Spectrum Name: 070331D_glyc olaldehyde_H13CHO_040207_1400-2000.pdf 13C NMR (D2O): 171.767, 167.223, 103.213, 100.650, 100.192, 99.250, 98.788, 98.266, 96.034, 94.806, 93.165, 85.521, 82.553, 82.305 (HCHO), 82.007, 81.412, 79.661, 76.816,

PAGE 147

147 76.560, 76.263, 74.161, 71.803, 71.551, 71.288, 65.459, 64.147, 63.728, 63.438, 63.090, 62.820, 62.537, 59.200, 58.643, 58.395, 56.171, 53.218, 40.542, 36.186, 26.676, 26.298 Spectrum Name: 070331D_glycolaldehyde_H13CHO_040507_1045am.pdf 13C NMR (D2O): 219.652, 171.748, 167.212, 167.067, 129.401, 116.748, 110.198, 103.217, 98.262, 82.305 (HCHO), 76.813, 76.545, 71.563, 71.139, 65.387, 63.865, 63.209, 26.195. Spectrum Name: 070331D_glycol aldehyde_H13CHO_040907_nt=256.pdf 13C NMR (D2O): 171.622, 171.428, 166.877, 150.149, 102.874, 110.683, 109.863, 106.410, 102.874, 97.911, 95.622, 93.227, 88.024, 85.323, 84.270, 82.546, 82.305 (HCHO), 81.954, 71.594, 71.407, 70.930, 63.777, 63.114, 62.454. Spectrum Name: 070331D_glycolal dehyde_H13CHO_041007_nt=4096_0600-0900.pdf 13C NMR (D2O): 171.763, 167.216, 103.217, 102.393, 102.096, 100.257, 99.227, 98.266, 96.053, 82.305 (HCHO), 82.007, 76.816, 76.556, 76.266, 73.920, 73.657, 72.375, 71.796, 71.559, 71.300, 70.258, 67.065, 66.604, 65.372, 65.306, 64.132, 63.026, 61.855, 58.883, 58.650, 58.391, 40.546 Spectrum Name: 070331D_glycolalde hyde_H13CHO_nt=2048_040107_start_1500-1630.pdf 13C NMR (D2O): 171.767, 167.227, 103.217, 98.262, 94.859, 82.305 (HCHO), 82.007, 76.819, 76.556, 76.266, 71.551, 65.513, 63.506, 62.827

PAGE 148

148 3-Pentulose Synthetic Procedure O O 1 3,3-Dimethoxypentane (1) To a stirred soln. of the 3-pentanone (2 mol), trimethyl orthoformate (5 mol), and MeOH (700 ml), TsOH (500 mg) was a dded at r.t. After 4 days, the mixture was poured onto ice, worked up with Et2O, and washed with sat. NaHCO3 soln. and brine, then distilled. 115 g (63%). B.p. 120 124_. 1H-NMR: 0.82 (t, 6 H); 1.60 (q, 4 H); 3.15 (s, 6 H). 13C NMR (D2O): 123.3(3), 51.2(OCH3), 31.3(2,4), 8.1(1,5) OH O O O O 2 (2 S ,4S )-1,2:4,5-DiO -(3,3-pentylidene)arabitol (2). To a refluxing suspension of L-arabitol (5.0 g, 32.84 mmol) in 3,3-dimethoxypentane (19.6 g, .148 mol) and THF (50 mL) was added CSA (2.3 g, 9.9 mmol) and the reaction was stirred at reflux for 5 min. Triethylamine (10 mL) was added to the refluxing reaction, a nd the mixture was concentrated in vacuo and loaded directly onto a silica gel column (hexane/ethyl acetate 8:2) yielding a colorless oil 7 (7.3 g, 77%). 13C NMR (D2O): 123.1(3), 76.1(2,4), 69.2(3), 66.4( 1,5), 31.2(2,4), 7.9(1,5)

PAGE 149

149 O O O O O 3 (2 S ,4S )-1,2:4,5-Bis(3,3-pentylidenedioxy)-3-pentanone (3). A suspension of SO3 pyridine (5.1 g, 32.4 mmol) in CH2Cl2 (20 mL) was dissolved in DMSO (40 mL) and Et3N (5 mL, 36.11 mmol). This solution was immediately added dropwise to a stirred solution of 2 (3.0 g, 10.4 mmol) in CH2Cl2 (20 mL) and DMSO (35 mL) at -5 C.The reaction mixture was stirred at 0 C for 6 h. The reaction mixture was poured into a solution of saturated aqueous NH4Cl:water:Et2O:pentane (1:1:1:1, 100 mL), and the a queous phase was extracted with an Et2O:pentane mixture (1:1, 3 x 50 mL). The combin ed organic phases were dried over anhydrous Na2SO4. After the solvent was removed in vacuo the obtained pale yellow oil was purified by column chromatography (hexane/ethyl acetate 9: 1) to yield a colorl ess oil (483 mg, 99%). 13C NMR (D2O): 212.4(3), 124.1(3), 75.2(2,4), 65.7(1,5), 32.1(2,4), 8.2(1,5) O OH OH HO OH 4 (2S,4S )-1,2,4,5-tetrahydroxy-3-pentanone (4). To a solution of 11 (1.09g, 3.8 mmol) in methanol (15 mL) and H2O (0.3 mL) was added CSA (111 mg, 0.48 mmol). The mixture was stirred at room temperature for 48 h. Solid NaHCO3 (120 mg) was added, and the reaction was stirred for 15 min. The suspension was di rectly loaded onto a silica gel column

PAGE 150

150 (CH2Cl2/methanol 95:5). Elution wi th an 8:2 mixture of CH2Cl2/methanol yielded 4 as a colorless oil (513 mg, 90%). 13C NMR (D2O): 212.2 (3), 76.2(2,4), 63.3(1,5)

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151 LIST OF REFERENCES 1. Proust, J. L., Ann. Chim. Physique 1806, 60, 233. 2. Proust, J. L., Gehlens J. Chem. Physik 1807, 3, 384. 3. Hudson, R., Moore, M., Session 16. Extrater restrial organic chemistrybiological, prebiological, and abiological. Astrobiology 2008, 8, 362-371. 4. Whler, F., Ueber knstliche bildung de s harnstoffs. Ann. Phys. Chem. 1828, 37, 330334. 5. Strecker, A., Ueber die knstliche bil dung der milchsure und einen neuen, dem glycocoll homologen. Ann. Chem. Pharm. 1850, 75, 27-45. 6. Strecker, A., Ueber einen neuen aus al dehyd ammoniak und blausure entstehenden krper. Ann. Chem. Pharm. 1853, 91, 349-351. 7. Matthews, C. N., Minard, R.D., Hydrogen cyanide polymers connect cosmochemistry and biochemistry. Proc. IAU Symp. 2008, 251, 453-459. 8. Butlerov, A., Formation synthtique dune substance sucre. Comptes Rend. Acad. Sci. 1861, 53, 145-147. 9. Breslow, R., On the mechanism of the formose reaction. Tetrahedron Lett. 1959, 1, 2226. 10. Ricardo, A., Bioorganic molecules in the cosm os and the origin of darwinian molecular systems. Ph.D. Dissertation, University of Florida, 2004. 11. Ricardo, A., Carrigan, M., Olcott, A. N. Benner, S. A., Borate minerals stabilize ribose. Science 2004, 303, 196. 12. Lb, W., Ueber das verhalten des formamid s unter der wirkung der stillen entlandung. Ein beitrag zur frage der stickstoff-a ssimilation. Deut. Chem. Gese. 1913, 46, 684-697. 13. Oparin, A. I., The Origin of Life. Moscow: Moscow Work er publisher, 1924. 14. Miller, S. L., A production of amino aci ds under possible primitive earth conditions. Science 1953, 117, 528-529. 15. Watson, J. D., Crick, F. H., Genetical impli cations of the struct ure of deoxyribonucleic acid Nature 1953, 171, 964-967. 16. Watson, J. D., Crick, F. H., A structure fo r deoxyribose nucleic acid. Nature 1953, 171, 737-738. 17. Watson, J. D., Crick, F. H., The structure of DNA. Cold Spring Harb. Symp. Quant. Biol. 1953, 18, 123-131.

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152 18. Gilbert, W., Origin of life: The RNA world. Nature 1986, 319, 618-618. 19. Larralde, R., Robertson, M. P., Miller, S., Rates of decomposition of ribose and other sugars: implications for chemical evolu tion. Proc. Nat. Acad. Sci. USA 1995, 92, 81588160. 20. Moody, J. B., Serpentinization: a review. Lithos 1976, 9, 125-138. 21. Holm, N. G., Dumont, M., Ivarsson, M. ; Konn, C., Alkaline fluid circulation in ultramafic rocks and formation of nucleotide constituents: a hypothesis. Geochem. Trans. 2006, 7, 1-7. 22. Sephton, M., Organic compounds in carbona ceous meteorites. Nat. Prod. Rep. 2002, 19, 292-311. 23. Liang, M. C., Kopp, R. E., Kirschvink, J. L., Yung, Y. L., Production of hydrogen 561635-3778peroxide in the atmosphere of a s nowball earth and the origin of oxygenic photosynthesis. Proc. Nat. Acad. Sci. USA 2006, 103, 18896-18899. 24. Fenton, H. J., A new synthesis in the s ugar group. J. Chem. Soc. Trans. 1897, 71, 375383. 25. Fenton, H. J., Jones, H. O., The oxidation of organic acids in th e presence of iron. J. Chem. Soc. Trans. 1900, 77, 69-76. 26. Cech, T. R., Self-splicing RNA: implica tions for evolution. Int. Rev. Cytol. 1985, 93, 322. 27. Cech, T. R., RNA as an enzyme. Sci. Am. 1986, 255, November, 64-75. 28. Cech, T. R., A model for RNA catalysed repl ication of RNA. Proc. Nat. Acad. Sci. USA 1986, 83, 4360-4363. 29. Ricardo, A., Carrigan, M. A., Tipton, J. D., Powell, D. H., Benner, S. A., 2Hydroxymethylboronate as a reag ent to detect car bohydrates: Application to the analysis of the formose reaction. J. Org. Chem. 2006, 71, 9503-9505. 30. Benner, K., Klufers, P., A combined X-ray and NMR study of borate esters of furanoidic cis-1,2-diols. Carb. Res. 2000, 327, 287-292. 31. Hricoviniova-Bilikova Z., Petr usova R., Serianni A.S., Petr us L., Stereospecific molybic acid-catalyzed isomerization of 2-hexuloses to branched-chain aldoses. Carb.Res. 1999, 319, 38-46. 32. Petrus, L., Hricoviniova Z., The B ilik reaction. Top. Curr. Chem. 2001, 215, 15-41. 33. Pizzarello, S. Weber, A., Prebiotic Amino Ac ids as Asymmetric Catalysts. Science 2004, 303, 1151.

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153 34. Chowdari, N. S., Ramachary, D. B., Crdova, A., Barbas III, C. F., Proline-catalyzed asymmetric assembly reactions: enzyme-lik e assembly of carbohydrates and polyketides from three aldehyde substrates. Tetrahedron Lett. 2002, 43, 9591-9595. 35. Cordova, A., Notz, W., Barbas III, C. F., Direct organocatalytic aldol reactions in buffered aqueous media. Chem. Comm. 2002, 24, 3024-3025. 36. Ibrahem, I., Zou, W., Xu, Y., Crdova A., Amino acid-catalyzed asymmetric carbohydrate formation: Organocatalytic one-s tep denovo synthesis of keto and amino sugars. Adv. Syn. & Catal. 2006, 348, 211-222. 37. Schoning, K., Scholz, P., Guntha, S., Wu, X., Krishnamurthy, R., Eschenmoser, A., Chemical etiology of nucleic acid st ructure: The alpha-threofuranosyl-(3' 2') oligonucleotide system. Science 2000, 290, 1347-1351. 38. Horhota, A., Zou, K., Ichida, J.K., Yu, B., McLaughlin, L.W., Szostak, J.W., Chaput J.C., Kinetic analysis of an efficient DNA-depe ndent TNA polymerase. J. Am. Chem. Soc. 2005, 127, 7427-7434. 39. Chaput, J. C., Ichida, J.K., Szostak, J. W., DNA polymerase-mediated DNA synthesis on a TNA template J. Am. Chem. Soc. 2003, 125, 856-857. 40. Lapworth, A., The action of halogens on compounds containing the carbonyl group J. Chem. Soc. 1904, 85, 30. 41. S. Cannizzaro, Ueber den der benzosure entsprechenden alkohol. Ann. der Chem. und Pharm. 1853, 88, 129-130. 42. Inoue, Y., Kimura, A., Poole, R. K., Methyl glyoxal and regulation of its metabolism in microorganisms. Adv. Micro. Phys. 1995, 37, 177-227. 43. Kawakami, T., Tourmaline breakdown in the migmatite zone of the Ryoke metamorphic belt, SW Japan. J. Metamorph. Geol. 2001, 19, 61-75. 44. Hoffmann, R. W., Niel, G., Schlapbach, A., St ereocontrol in allylbor ation reactions. Pure & Appl. Chem. 1990, 62, 1993-1998. 45. Li, Q., Ricardo, A., Benner, S. A., Wi nefordner, J. D., Powell, D. H., Desorption/ionization on porous silicon mass spectrometry studies on pentose-borate complexes. Anal. Chem. 2005, 77, 4503-4508. 46. Benner, S. A., Sismour, A. M., Synt hetic biology. Nat. Rev. Gen. 2005, 6, 533-543. 47. Bergstrom, D. E., Ruth, J.L., Synthesis of C-5 substituted pyrimidine nucleosides via organopalladium intermediates. J. Am. Chem. Soc. 1976, 98, 1587-1589.

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154 48. Langer, P. R., Waldrop, A. A., Ward, D. C., Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Nat. Acad. Sci. USA 1981, 78, 6633-6637. 49. Prober, J. M., Trainor, G. L., Dam, R. J ., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A., Baumei ster, K., A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 1987, 238, 336-41. 50. Benner, S. A., Allemann, R. K., Ellington, A. D., Ge, L., Glasfeld, A., Leanz, G. F., Krauch, T., MacPherson, L. J., Moroney, S. E., Piccrilli, A. J., Weinhold, E., Natural selection, protein engineeri ng, and the last riboorganism: Rational model building in biochemistry Cold Spring Harb. Symp. Quant. Biol. 1987, 52, 53-63. 51. Vaught, J. D., Dewey, T., Eaton, B. E., T7 R NA polymerase transcription with 5-position modified UTP derivatives. J. Am. Chem. Soc. 2004, 126, 11231-7. 52. Latham, J. A., Johnson, R., Toole, J. J., Th e application of a modified nucleotide in aptamer selection: novel thrombin aptamers containing 5-(1-pen tynyl)-2'-deoxyuridine. Nuc. Acids Res. 1994, 22, 2817-22. 53. Battersby, T. R., Ang, D. N., Burgstaller, P., Jurczyk, S. C., Bowser, M. T., Buchanan, D. D., Kennedy, R. T., Benner, S. A., Quantitati ve analysis of receptors for adenosine nucleotides obtained via in vitro selection from a library incorporating a cationic nucleotide analog. J. Am. Chem. Soc. 1999, 121, 9781-9. 54. Ang, D. N., An alternative to the origins of life thories: Amino acid-like DNA molecules capable of improved catalysis. Ph.D. Disse rtation, University of Florida, 1999. 55. Perrin, D. M., Garestier, T., Helene, C., Expa nding the catalytic repert oire of nucleic acid catalysts: simultaneous incor poration of two modified de oxyribonucleoside triphosphates bearing ammonium and imidazolyl f unctionalities. Nucl. Nucl. 1999, 18, 377-91. 56. Tarasow, T. M., Tarasow, S. L., Ea ton, B. E., RNA-catal ysed carbon-carbon bond formation. Nature 1997, 389, 54-7. 57. Ahmadian, M., Zhang, P., Bergstrom, D. E., A comparative study of the thermal stability of oligodeoxyribonucleotides containing 5-subs tituted 2'-deoxyuridines. Nuc. Acids Res. 1998, 26, 3127-35. 58. De Clercq, E., Descamps, J., Balzarini, J., Giziewicz, J., Barr, P. J., Robins, M. J., Nucleic acid related compounds. 40. Synthesis and biological activitie s of 5-alkynyluracil nucleosides. J. Med. Chem. 1983, 26, 661-6. 59. Barnes, T. W., Turner, D. H., Long-range c ooperativity in molecu lar recognition of RNA by oligodeoxynucleotides with multiple C5-(1-propynyl) pyrimidines. J. Am. Chem. Soc. 2001, 123, 4107-18.

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155 60. Lutz, S., Burgstaller, P., Benner, S. A., An in vitro screening technique for DNA polymerases that can incorporate modified nucleotides. Pseudo-thymidine as a substrate for thermostable polymerases. Nuc. Acids Res. 1999, 27, 2792-8. 61. Kuwahara, M., Takahata, Y., Shoji, A., Ozak i, A. N., Ozaki, H., Sawai, H., Substrate properties of C5-substituted pyrimidin e 2'-deoxynucleoside 5'-triphosphates for thermostable DNA polymerases during PCR. Bioorg. Med. Chem. Lett. 2003, 13, 37358. 62. Matulic-Adamic, J., Daniher, A. T., Ka rpeisky, A., Haeberli, P., Sweedler, D., Beigelman, L., Functionalized nucleoside 5'-tri phosphates for in vitro selection of new catalytic ribonucleic acids. Bi oorg. Med. Chem. Lett. 2000, 10, 1299-302. 63. Taylor, E. C., Gillespie, P., Patel, M., Novel 5-desmethylene analogs of 5,10-dideaza5,6,7,8-tetrahydrofolic acid as potential anticancer agents. J. Org. Chem. 1992, 57, 32183225. 64. Taylor, E. C., Macor, J. E., Pont, J. L ., Intramolecular dielsalder reactions of 1,2,4triazines: A general synthesis of furo[2,3-]pyridines, 2,3-dihydropyrano[2,3-]pyridines, and pyrrolo[2,3-]pyridines. Tetrahedron 1987, 43, 5145-5158. 65. Held, H. A., Roychowdhury, A., Benner, S. A., C-5 modified nucleosides: direct insertion of alkynyl-thio functi onality in pyrimidines. Nucleos. Nucleot. Nucl. Acids 2003, 22 (4), 391-404. 66. Horlacher, J., Hottiger, M., Podust, V. N., Hubscher, U., Benner, S. A., Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with nonstandard hydrogen bonding patterns. Proc. Na tl. Acad. Sci. USA 1995, 92, 6329-33. 67. Hu, G., DNA polymerase-cataly zed addition of nontemplated ex tra nucleotides to the 3' end of a DNA fragment. DNA Cell Biol. 1993, 12, 763-70. 68. Benner, S. A., Synthetic biol ogy: Act natural. Nature 2003, 421, 118.

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156 BIOGRAPHICAL SKETCH Heshan Illangkoon was born and raised in Montreal, Canada. His parents Herath & Nandani Illangkoon both immigrated from Sri Lanka in the 1970s. There he attended the French parochial school cole Georges P. Vanier. Shortly before his eleventh birthday his parents moved to West Palm Beach, Florida. There he attended Pahokee elementary school (6th grade), Pahokee Jr./Sr. High School (7th grade), Wellington Landings Middle School (8th grade). In 1994, Heshan was chosen to attend Suncoast Community High Sc hool in Riviera Beach, Fl orida, ranked the 3rd best high school by U.S. News and World report. In 1998, he entered the University of Florida and performed undergraduate research with Dr. Steven A. Benner and graduated in December 2002. He continued working for the Benner Labo ratories and in September 2003 entered the University of Florida chemistry graduate program. During his stint he was deeply involved with issues of shared governance and University ma nagement and was elected to the student body government as a graduate senator, and re-elected two additional time s. He has served as chairman of the allocations sta nding committee and as chairman of the graduate and professional student caucus. He was also appointed as the vice -chair of the senate graduate issues committee and liaison to the faculty senate. He was also nominated to the college of liberal arts and sciences finance committee and the presidency of the grad uate student council, both of which he humbly declined. In the Spring of 2009, Heshan, a co-f ounder of the of the progress party, was the partys vice-presidential candida te for University of Florida student government elections. To date, Heshan holds the title of be ing the longest serving Senator in University of Florida Student Government history. An avid photographer, he is also the offici al photographer for the Graham Center for Public Service. After graduating Hesh an plans to travel to Tanzania and will be involved with an NGO. In the future, Heshan plan s to pursue a law degree, a career with NASA, and hopes to culminate as a statesman and public servant, ideally, a U.S. Senator or Ambassador.