DOCSLIB.ORG

Aldolases for Enzymatic Carboligation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Aldolases for Enzymatic Carboligation Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1318 Aldolases for Enzymatic Carboligation Directed Evolution and Enzyme Structure-Function Relationship Studies HUAN MA ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-554-9411-7 UPPSALA urn:nbn:se:uu:diva-266902 2015 Dissertation presented at Uppsala University to be publicly examined in B22, BMC, Husargatan3, Uppsala, Friday, 5 February 2016 at 13:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Prof. Dr. Wolf- Dieter Fessner (Organische Chemie, Technische Universität Darmstadt ). Abstract Ma, H. 2015. Aldolases for Enzymatic Carboligation. Directed Evolution and Enzyme Structure-Function Relationship Studies. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1318. 67 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9411-7. The research summarized in this thesis focuses on directed evolution and enzyme mechanism studies of two aldolases: 2-deoxyribose-5-phosphate aldolase (DERA) and fructose-6- phosphate aldolase (FSA). Aldolases are nature’s own catalysts for one of the most fundamental reactions in organic chemistry: the formation of new carbon-carbon bonds. In biological systems, aldol formation and cleavage reactions play central roles in sugar metabolism. In organic synthesis, aldolases attract great attention as environmentally friendly alternative for the synthesis of polyhydroxylated compounds in stereocontrolled manner. However, naturally occurring aldolases can hardly be used directly in organic synthesis mainly due to their narrow substrate scopes, especially phosphate dependency on substrate level. Semi-rational directed evolution was used in order to investigate the possibility of expanding the substrate scope of both DERA and FSA and to understand more about the relationship between protein structure and catalytic properties. The first two projects focus on the directed evolution of DERA and studies of the enzyme mechanism. The directed evolution project aims to alter the acceptor substrate preference from phosphorylated aldehydes to aryl-substituted aldehydes. Effort has been made to develop screening methods and screen for variants with desired properties. In the study of enzyme mechanism where enzyme steady state kinetic studies were combined with molecular dynamic simulations, we investigated the role of Ser238 and Ser239 in the phosphate binding site and the possible connection between enzyme dynamics and catalytic properties. The other two projects focus on the directed evolution of FSA and the development of a new screening assay facilitating screening for FSA variants with improved activity in catalyzing aldol reaction between phenylacetaldehyde and hydroxyacetone. The new assay is based on a coupled enzyme system using an engineered alcohol dehydrogenase, FucO DA1472, as reporting enzyme. The assay has been successfully used to identify a hit with 9-fold improvement in catalytic efficiency and to determine the steady state kinetic parameters of wild-type FSA as well as the mutants. The results from directed evolution illustrated the high degree malleability of FSA active site. This opens up possibilities to generate FSA variants which could utilize both aryl-substituted donor and acceptor substrates. Keywords: aldolase, 2-deoxyribose-5-phosphate aldolase, fructose-6-phosphate aldolase, directed evolution, enzyme dynamics Huan Ma, Department of Chemistry - BMC, Biochemistry, Box 576, Uppsala University, SE-75123 Uppsala, Sweden. © Huan Ma 2015 ISSN 1651-6214 ISBN 978-91-554-9411-7 urn:nbn:se:uu:diva-266902 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-266902) List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Ma, H., Szeler, K., Kamerlin, S. C. L. and Widersten, M. (2015) Linking Coupled Motions and Entropic Effects to the Catalytic Ac- tivity of 2-deoxyribose-5-phosphate Aldolase (DERA). Chem. Sci., in the press, DOI: 10.1039/C5SC03666F. II Ma, H., Enugala, T. R. and Widersten, M. (2015) A Microplate Format Assay for Real-Time Screening for New Aldolases that Ac- cept Aryl-Substituted Acceptor Substrate. ChemBioChem, in the press, DOI: 10.1002/cbic.201500466. Ma, H., Enugala, T. R. and Widersten, M. (2015) Directed Evolution of Fructose-6-Phosphate Aldolase Towards Improved Activity with Aryl-Substituted Acceptor Substrates. Manuscript. Reprints were made with permission from the respective publishers. Publication not included in this thesis IV Janfalk Carlsson, Å., Bauer, P., Ma, H. and Widersten, M. (2012) Obtaining Optical Purity for Product Diols in Enzyme-Catalyzed Epoxide Hydrolysis: Contributions from Changes in both Enantio- and Regioselectivity. Biochemistry, 51: 7627-7637. Contents Introduction ................................................................................................... 11 1. Biocatalysis and biocatalysts ................................................................ 11 2. Directed evolution ................................................................................ 13 3. Saturation mutagenesis and codon denegeracy .................................... 14 4. Molecular dynamics simulation ............................................................ 16 5. Enzyme dynamics and its potential link to enzyme catalysis ............... 17 6. Aldolases .............................................................................................. 18 6.1 Aldol reaction ................................................................................. 18 6.2 Aldolases ........................................................................................ 18 6.3 Class I aldolases ............................................................................. 19 7. Aldolases studied in this thesis ............................................................. 20 7.1 2-Deoxyribose-5-phosphate aldolase: DERA ................................ 20 7.2 Fructose-6-phosphate aldolase: FSA ............................................. 23 Present Investigations .................................................................................... 25 Present Investigation I: Directed evolution of DERA- an attempt to expand the acceptor substrate scope to non-phosphorylated aromatic compounds. ............................................................................................... 26 1. Aim .................................................................................................. 26 2. The design of enzyme libraries ........................................................ 27 3. Screening assay using thin layer chromatography and 14C labeled acetaldehyde. ........................................................................................ 28 4. Screening results and hits sequence analysis ................................... 30 5. Conclusion and discussion ............................................................... 31 Present Investigation II: Enzyme kinetic study and molecular dynamics simulation on wild-type DERA and related mutants. ............................... 33 1. Aim .................................................................................................. 33 2. Choice of enzymes ........................................................................... 33 3. Role of phosphate binding in DERA ............................................... 34 4. Enzyme steady state kinetic analysis ............................................... 35 5. Molecular dynamics simulation ....................................................... 38 6. Conclusion ....................................................................................... 40 Present Investigation III: Development of screening assay for FSA variants with improved activity towards phenylacetaldehyde .................. 41 1. Aim .................................................................................................. 41 2. Development of a coupled enzyme assay for detecting the formation of phenylacetaldehyde ......................................................................... 41 3. Steady state kinetic study of FSA in aldol cleavage reaction .......... 42 4. Test screening on 96-well plate ....................................................... 43 5. Conclusion ....................................................................................... 45 Present Investigation IV: Directed evolution of FSA ............................... 46 1. Aim .................................................................................................. 46 2. Library design .................................................................................. 46 3. Library screening and identification of hits ..................................... 47 4. Steady state kinetic studies on first generation library hits .............. 49 5. Conclusion and future plan .............................................................. 49 Concluding Remarks and Future Perspectives .............................................. 51 Populärvetenskaplig Sammanfattning ........................................................... 52 Résumé .......................................................................................................... 55 论文摘要 ....................................................................................................... 58 Acknowledgements ......................................................................................
Load more

Recommended publications
  • 1. Nucleotides A. Pentose Sugars – 5-Carbon Sugar 1) Deoxyribose – in DNA 2) Ribose – in RNA B. Phosphate Group C. Nitroge
    1. Nucleotides a. Pentose sugars – 5-Carbon sugar 1) Deoxyribose – in DNA 2) Ribose – in RNA b. Phosphate group c. Nitrogenous bases 1) Purines a) Adenine b) Guanine 2) Pyrimidines a) Cytosine b) Thymine 2. Types of Nucleic Acids a. DNA 1) Locations 2) Functions b. RNA 1) Locations 2) Functions E. High Energy Biomolecules 1. Adenosine triphosphate a. Uses 1) Active transport 2) Movement 3) Biosynthesis reactions b. Regeneration 1) ADP + Pi + Energy → ATP 4. Classes of proteins a. Structural – ex. Collagen, keratin b. Transport – Hemoglobin, many β-globulins c. Contractile – Actin and Myosin of muscle tissue d. Regulatory - Hormones e. Immunologic - Antibodies f. Clotting – Thrombin and Fibrin g. Osmotic - Albumin h. Catalytic – Enzymes 1) Characteristics of enzymes • Proteins (most); ribonucleoproteins (few/ribozymes) • Act as organic catalysts • Lower the activation energy of reactions • Not changed by the reaction • Bind to their substrates o Lock-and-key model of enzyme activity o Induced-fit model • Highly specific • Named by adding -ase to substrate name; e.g., maltose/maltase • May require cofactors which may be: o Nonprotein metal ions such as copper, manganese, potassium, sodium o Small organic molecules known as coenzymes. The B vitamins like thiamine (B1) riboflavin (B2) and nicotinamide are precursors of coenzymes. • May require activation; e.g., pepsinogen pepsin in stomach chief cells 4. Factors Affecting Enzyme Action • pH o pepsin (stomach) @ pH = 2; trypsin (small int.) @ pH = 8 • Temperature o Denatured by high temp’s. • Enzyme inhibitors o Competitive inhibitors o Noncompetitive inhibitors • Effect of substrate concentration and reversible reactions and the Law of Mass D.
    [Show full text]
  • Multi-Enzymatic Cascades in the Synthesis of Modified Nucleosides
    biomolecules Article Multi-Enzymatic Cascades in the Synthesis of Modified Nucleosides: Comparison of the Thermophilic and Mesophilic Pathways Ilja V. Fateev , Maria A. Kostromina, Yuliya A. Abramchik, Barbara Z. Eletskaya , Olga O. Mikheeva, Dmitry D. Lukoshin, Evgeniy A. Zayats , Maria Ya. Berzina, Elena V. Dorofeeva, Alexander S. Paramonov , Alexey L. Kayushin, Irina D. Konstantinova * and Roman S. Esipov Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117997 GSP, B-437 Moscow, Russia; [email protected] (I.V.F.); [email protected] (M.A.K.); [email protected] (Y.A.A.); [email protected] (B.Z.E.); [email protected] (O.O.M.); [email protected] (D.D.L.); [email protected] (E.A.Z.); [email protected] (M.Y.B.); [email protected] (E.V.D.); [email protected] (A.S.P.); [email protected] (A.L.K.); [email protected] (R.S.E.) * Correspondence: [email protected]; Tel.: +7-905-791-1719 ! Abstract: A comparative study of the possibilities of using ribokinase phosphopentomutase ! nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Citation: Fateev, I.V.; Kostromina, Recombinant phosphopentomutase from Thermus thermophilus HB27 was obtained for the first time: M.A.; Abramchik, Y.A.; Eletskaya, a strain producing a soluble form of the enzyme was created, and a method for its isolation and B.Z.; Mikheeva, O.O.; Lukoshin, D.D.; chromatographic purification was developed. It was shown that cascade syntheses of modified nu- Zayats, E.A.; Berzina, M.Y..; cleosides can be carried out both by the mesophilic and thermophilic routes from D-pentoses: ribose, Dorofeeva, E.V.; Paramonov, A.S.; 2-deoxyribose, arabinose, xylose, and 2-deoxy-2-fluoroarabinose.
    [Show full text]
  • DNA Stands for Deoxyribose Nucleic Acid
    DNA and Protein Synthesis DNA • DNA stands for deoxyribose nucleic acid. • This chemical substance is found in the nucleus of all cells in all living organisms • DNA controls all the chemical changes which take place in cells • The kind of cell which is formed, (muscle, blood, nerve etc) is controlled by DNA Ribose is a sugar, like glucose, but with only five carbon atoms in its molecule. Deoxyribose is almost the same but lacks one oxygen atom. The nitrogen bases are: o ADENINE (A) o THYMINE (T) o CYTOSINE (C) o GUANINE (G) Nucleotides • A molecule of DNA is formed by millions of nucleotides joined together in a long chain. • DNA is a very large molecule made up of a long chain of sub-units. • The sub-units are called nucleotides. • Each nucleotide is made up of a sugar called deoxyribose, a phosphate group -PO4 and an organic (Nitrogen) base: A, T, C, G BASE PAIRING RULE amount of C= amount of G AND amount of A= amount of T • Adenine always pairs with thymine, and guanine always pairs with cytosine. DNA STRUCTURE • The nucleotide bases will point to the inside of the DNA molecule while the outside (backbone) of the DNA molecule will be made of the sugar and phosphate molecules. • When complete the DNA molecule forms a double helix (two spiral sides wrapped together). • The paired strands are coiled into a spiral called A DOUBLE HELIX. Genes • Each chromosome contains hundreds of genes. • Most of your characteristics: hair color, height, how things taste to a person, are determined by the kinds of proteins cells make (gene).
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 8,604,000 B2 De Kort Et Al
    USOO8604000B2 (12) United States Patent (10) Patent No.: US 8,604,000 B2 de Kort et al. (45) Date of Patent: Dec. 10, 2013 (54) PALATABLE NUTRITIONAL COMPOSITION 2011/0027391 A1 2/2011 De Kort et al. COMPRISING ANUCLEOTDE AND/ORA 2013, OO12469 A1 1/2013 De Kort et al. NUCLEOSDE AND A TASTE MASKING 2013, OO18012 A1 1/2013 Hageman et al. AGENT FOREIGN PATENT DOCUMENTS (75) Inventors: Esther Jacqueline de Kort, Wageningen EP O 175468 A2 3, 1986 (NL); Martine Groenendijk, EP 1216 041 B1 6, 2002 EP 1282 365 B1 2, 2003 Barendrecht (NL); Patrick Joseph EP 1656 839 A1 5, 2006 Gerardus Hendrikus Kamphuis, EP 1666 092 A2 6, 2006 Utrecht (NL) EP 18OO 675 A1 6, 2007 JP 64-080250 A 3, 1989 (73) Assignee: N.V. Nutricia, Zoetermeer (NL) JP 06-237734. A 8, 1994 JP 10-004918 A 1, 1998 JP 10-136937 A 5, 1998 (*) Notice: Subject to any disclaimer, the term of this JP 11-071274. A 3, 1999 patent is extended or adjusted under 35 WO WO-0038829 A1 T 2000 U.S.C. 154(b) by 213 days. WO WO-01 (32034 A1 5, 2001 WO WO-02/088159 A1 11, 2002 WO WO-02/096464 A1 12/2002 (21) Appl. No.: 12/809,431 WO WO-03 (013276 A1 2, 2003 WO WO-03/041701 A2 5, 2003 (22) PCT Filed: Dec. 22, 2008 WO WO-2005/039597 A2 5, 2005 WO WO-2006/031683 A2 3, 2006 (86). PCT No.: PCT/NL2O08/050843 WO WO-2006,118665 A2 11/2006 WO WO-2006, 127620 A2 11/2006 S371 (c)(1), WO WO-2007/001883 A2 1, 2007 (2), (4) Date: Dec.
    [Show full text]
  • Nucleosides & Nucleotides
    Nucleosides & Nucleotides Biochemistry Fundamentals > Genetic Information > Genetic Information NUCLEOSIDE AND NUCLEOTIDES SUMMARY NUCLEOSIDES  • Comprise a sugar and a base NUCLEOTIDES  • Phosphorylated nucleosides (at least one phosphorus group) • Link in chains to form polymers called nucleic acids (i.e. DNA and RNA) N-BETA-GLYCOSIDIC BOND  • Links nitrogenous base to sugar in nucleotides and nucleosides • Purines: C1 of sugar bonds with N9 of base • Pyrimidines: C1 of sugar bonds with N1 of base PHOSPHOESTER BOND • Links C3 or C5 hydroxyl group of sugar to phosphate NITROGENOUS BASES  • Adenine • Guanine • Cytosine • Thymine (DNA) 1 / 8 • Uracil (RNA) NUCLEOSIDES • =sugar + base • Adenosine • Guanosine • Cytidine • Thymidine • Uridine NUCLEOTIDE MONOPHOSPHATES – ADD SUFFIX 'SYLATE' • = nucleoside + 1 phosphate group • Adenylate • Guanylate • Cytidylate • Thymidylate • Uridylate Add prefix 'deoxy' when the ribose is a deoxyribose: lacks a hydroxyl group at C2. • Thymine only exists in DNA (deoxy prefix unnecessary for this reason) • Uracil only exists in RNA NUCLEIC ACIDS (DNA AND RNA)  • Phosphodiester bonds: a phosphate group attached to C5 of one sugar bonds with - OH group on C3 of next sugar • Nucleotide monomers of nucleic acids exist as triphosphates • Nucleotide polymers (i.e. nucleic acids) are monophosphates • 5' end is free phosphate group attached to C5 • 3' end is free -OH group attached to C3 2 / 8 FULL-LENGTH TEXT • Here we will learn about learn about nucleoside and nucleotide structure, and how they create the backbones of nucleic acids (DNA and RNA). • Start a table, so we can address key features of nucleosides and nucleotides. • Denote that nucleosides comprise a sugar and a base.
    [Show full text]
  • Part 1 in Our Series of Carbohydrate Lectures. in This Section, You Will Learn About Monosaccharide Structure
    Welcome to Part 1 in our series of Carbohydrate lectures. In this section, you will learn about monosaccharide structure. The building blocks of larger carbohydrate polymers. 1 First, let’s review why learning about carbohydrates is important. Carbohydrates are used by biological systems as fuels and energy resources. Carbohydrates typically provide quick energy and are one of the primary energy storage forms in animals. Carbohydrates also provide the precursors to other major macromolecules within the body, including the deoxyribose and ribose required for nucleic acid biosynthesis. Carbohydrates can also provide structural support and cushioning/shock absorption, as well as cell‐cell communication, identification, and signaling. 2 Carbohydrates, as their name implies, are water hydrates of carbon, and they all have the same basic core formula (CH2O)n and are always found in the ratio of 1 carbon to 2 hydrogens to 1 oxygen (1:2:1) making them easy to identify from their molecular formula. 3 Carbohydrates can be divided into subcategories based on their complexity. The simplest carbohydrates are the monosaccharides which are the simple sugars required for the biosynthesis of all the other carbohydrate types. Disaccharides consist of two monosaccharides that have been joined together by a covalent bond called the glycosidic bond. Oligosaccharides are polymers that consist of a few monosaccharides covalently linked together, and Polysaccharides are large polymers that contain hundreds to thousands of monosaccharide units all joined together by glycosidic bonds. The remainder of this lecture will focus on monosaccharides 4 Monosaccharides all have alcohol functional groups associated with them. In addition they also have one additional functional group, either an aldehyde or a ketone.
    [Show full text]
  • 8| Nucleotides and Nucleic Acids
    8| Nucleotides and Nucleic Acids © 2013 W. H. Freeman and Company CHAPTER 8 Nucleotides and Nucleic Acids Key topics: – Biological function of nucleotides and nucleic acids – Structures of common nucleotides – Structure of double‐stranded DNA – Structures of ribonucleic acids – Denaturation and annealing of DNA – Chemistry of nucleic acids; mutagenesis Functions of Nucleotides and Nucleic Acids • Nucleotide Functions: – Energy for metabolism (ATP) – Enzyme cofactors (NAD+) –Signal transduction (cAMP) • Nucleic Acid Functions: – Storage of genetic info (DNA) – Transmission of genetic info (mRNA) –Processing of genetic information (ribozymes) –Protein synthesis (tRNA and rRNA) Nucleotides and Nucleosides • Nucleotide = – Nitrogeneous base –Pentose – Phosphate • Nucleoside = – Nitrogeneous base –Pentose • Nucleobase = – Nitrogeneous base Phosphate Group •Negatively charged at neutral pH • Typically attached to 5’ position – Nucleic acids are built using 5’‐triphosphates •ATP, GTP, TTP, CTP – Nucleic acids contain one phosphate moiety per nucleotide •May be attached to other positions Other Nucleotides: Monophosphate Group in Different Positions Pentose in Nucleotides • ‐D‐ribofuranose in RNA • ‐2’‐deoxy‐D‐ribofuranose in DNA •Different puckered conformations of the sugar ring are possible Nucleobases •Derivatives of pyrimidine or purine • Nitrogen‐containing heteroaromatic molecules •Planar or almost planar structures •Absorb UV light around 250–270 nm Pyrimidine Bases • Cytosine is found in both DNA and RNA •Thymineis found only in DNA
    [Show full text]
  • Nucleotides and Nucleic Acids
    CHAPTER 8 Nucleotides and Nucleic Acids Functions of Nucleotides and Nucleic Acids • Nucleotide Functions: – Energy for metabolism (ATP) – Enzyme cofactors (NAD+) – Signal transduction (cAMP) • Nucleic Acid Functions: – Storage of genetic info (DNA) – Transmission of genetic info (mRNA) – Processing of genetic information (ribozymes) – Protein synthesis (tRNA and rRNA) Nucleotides and Nucleosides • Nucleotide = – Nitrogeneous base – Pentose – Phosphate • Nucleoside = – Nitrogeneous base – Pentose • Nucleobase = – Nitrogeneous base Phosphate Group • Negatively charged at neutral pH • Typically attached to 5’ position – Nucleic acids are built using 5’- triphosphates • ATP, GTP, TTP, CTP – Nucleic acids contain one phosphate moiety per nucleotide • May be attached to other positions Other Nucleotides: Monophosphate Group in Different Positions Pentose in Nucleotides • -D-ribofuranose in RNA • -2’-deoxy-D-ribofuranose in DNA • Different puckered conformations of the sugar ring are possible Purine Bases • Adenine and guanine are found in both RNA and DNA • Also good H-bond donors and acceptors • Adenine pKa at N1 is 3.8 • Guanine pKa at N7 is 2.4 • Neutral molecules at pH 7 • Derivatives of pyrimidine or purine • Nitrogen-containing heteroaromatic molecules • Planar or almost planar structures • Absorb UV light around 250–270 nm Pyrimidine Bases • Cytosine is found in both DNA and RNA • Thymine is found only in DNA • Uracil is found only in RNA • All are good H-bond donors and acceptors • Cytosine pKa at N3 is 4.5 • Thymine pKa at N3 is 9.5
    [Show full text]
  • Deoxyribose and Deoxysugar Derivatives from Photoprocessed Astrophysical Ice Analogues and Comparison to Meteorites
    ARTICLE https://doi.org/10.1038/s41467-018-07693-x OPEN Deoxyribose and deoxysugar derivatives from photoprocessed astrophysical ice analogues and comparison to meteorites Michel Nuevo 1,2, George Cooper3 & Scott A. Sandford 1 Sugars and their derivatives are essential to all terrestrial life. Their presence in meteorites, together with amino acids, nucleobases, amphiphiles, and other compounds of biological 1234567890():,; importance, may have contributed to the inventory of organics that played a role in the emergence of life on Earth. Sugars, including ribose (the sugar of RNA), and other sugar derivatives have been identified in laboratory experiments simulating photoprocessing of ices under astrophysical conditions. In this work, we report the detection of 2-deoxyribose (the sugar of DNA) and several deoxysugar derivatives in residues produced from the ultraviolet irradiation of ice mixtures consisting of H2O and CH3OH. The detection of deoxysugar derivatives adds to the inventory of compounds of biological interest that can form under astrophysical conditions and puts constraints on their abiotic formation pathway. Finally, we report that some of the deoxysugar derivatives found in our residues are also newly identified in carbonaceous meteorites. 1 NASA Ames Research Center, MS 245-6, Moffett Field, CA 94035, USA. 2 BAER Institute, NASA Research Park, MS 18-4, Moffett Field, CA 94035, USA. 3 NASA Ames Research Center, MS 239-4, Moffett Field, CA 94035, USA. Correspondence and requests for materials should be addressed to M.N. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:5276 | https://doi.org/10.1038/s41467-018-07693-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-018-07693-x ugars (monosaccharides) and their derivatives are ubiqui- contamination.
    [Show full text]
  • Studies on the Prebiotic Origin of 2-Deoxy-D-Ribose
    Studies on the Prebiotic Origin of 2-Deoxy-D-ribose Andrew Mark Steer Doctor of Philosophy University of York Chemistry August 2017 Abstract DNA is an important biological structure necessary for cell proliferation. The origins of cell- like structures and the building blocks of DNA are therefore also of great concern. As of yet the prebiotic origin of 2-deoxy-D-ribose, the sugar of DNA, has no satisfactory explanation. This research attempts to provide a possible explanation to the chemical origin of 2-deoxy- D-ribose via an aldol reaction between acetaldehyde 1 and D-glyceraldehyde D-2 (Error! Reference source not found.). The sugar mixture is trapped with N,N-diphenylhydrazine 3 for ease of purification and characterisation. The reaction is promoted by amino acids, amino esters and amino nitriles consistently giving selectivities in favour of 2-deoxy-D- ribose. This is the first example of an amino nitrile promoted reaction. Potential prebiotic synthesis of 2-deoxy-D-ribose and subsequent trapping with N,N-diphenyl hydrazine 3. The research is developed further by exploring the formation of 2-deoxy-D-ribose in a “protocell” environment – a primitive cell. Here we suggest that primitive cells may have been simple hydrogel systems. A discussion of the characterisation and catalytic ability of small peptide-based supramolecular structures is included. ii Contents Abstract ............................................................................................................................ ii Contents .........................................................................................................................
    [Show full text]
  • Deoxyribose-5-Phosphate Aldolase As a Synthetic Catalyst'
    J. Am. Chem. SOC.1990, 112, 2013-2014 2013 chemical shift for the iminosilaacyl carbon at 6 299.07.3a3b Ad- Deoxyribose-5-phosphateAldolase as a Synthetic dition of 1 equiv of CN(XyI) to a benzene solution of 4, or reaction Catalyst' of 1 with 2 equiv of CN(Xyl), results in formation of a blue complex 5. The combustion analysis of isolated 5 is consistent Carlos F. Barbas, 111, Yi-Fong Wang, and Chi-Huey Wong* with a 1:2 adduct of 1 with isocyanide, Cp,Sc(CN(Xyl)),Si- (SiMe3)3. However, 'H NMR data for 5 indicate a complex Department of Chemistry structure and the presence of three inequivalent SiMe3 groups in The Research Institute of Scripps Clinic a 1 :1: 1 ratioss Formation of X-ray-quality, blue crystals from La Jolla, California 92037 diethvl ether allowed comdete characterization of this comwund. Received November 29, 1989 Tie crystal structure7'(Figure 1) shows that 5 is the pioduct of an isocyanide-coupling reaction that results in further rear- Enzyme-catalyzed stereocontrolled aldol condensations are rangements. The structure drawn in Scheme I reflects the ob- valuable in organic synthesis, particularly in the synthesis of served structural parameters. The chelate ring of 5 is derived from carbohydrates and related s~bstances?~~We report here an initial the two isoc anide groups and contains a Sc( 1)-N( 1) single bond study on the synthetic utility of a bacterial 2-deoxyribose-5- (2.133 (7) i),a longer (dative) Sc(l)-N(2) bond (2.324 (8) A), phosphate aldolase (DERA, EC 4.1.2.4) overexpressed in Es- and a C=C double bond (C(l)-C(2) = 1.375 (12) A).
    [Show full text]
  • Carbohydrates Hydrates of Carbon: General Formula Cn(H2O)N Plants
    Chapter 25: Carbohydrates hydrates of carbon: general formula Cn(H2O)n Plants: photosynthesis hν 6 CO2 + H2O C6H12O6 + 6 O2 Polymers: large molecules made up of repeating smaller units (monomer) Biopolymers: Monomer units: carbohydrates (chapter 25) monosaccharides peptides and proteins (chapter 26) amino acids nucleic acids (chapter 28) nucleotides 315 25.1 Classification of Carbohydrates: I. Number of carbohydrate units monosaccharides: one carbohydrate unit (simple carbohydrates) disaccharides: two carbohydrate units (complex carbohydrates) trisaccharides: three carbohydrate units polysaccharides: many carbohydrate units CHO H OH HO HO HO H HO HO O HO O glucose H OH HO HO OH HO H OH OH CH2OH HO HO HO O HO O HO HO O HO HO O HO HO HO O O O O O HO HO O HO HO HO O O HO HO HO galactose OH + glucose O glucose = lactose polymer = amylose or cellulose 316 160 II. Position of carbonyl group at C1, carbonyl is an aldehyde: aldose at any other carbon, carbonyl is a ketone: ketose III. Number of carbons three carbons: triose six carbons: hexose four carbons: tetrose seven carbons: heptose five carbons: pentose etc. IV. Cyclic form (chapter 25.5) CHO CHO CHO CHO CH2OH H OH HO H H OH H OH O CH2OH H OH H OH HO H HO H CH2OH H OH H OH H OH CH2OH H OH H OH CH OH 2 CH2OH glyceraldehyde threose ribose glucose fructose (triose) (tetrose) (pentose) (hexose) (hexose) 317 (aldohexose) (ketohexose) 25.2: Depicting carbohydrates stereochemistry: Fischer Projections: representation of a three-dimensional molecule as a flat structure.
    [Show full text]