Biomolecules & Spectroscopy
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University of Cape Town
CHEMICAL AND CONFORMATIONAL STUDIES OF BACTERIAL CELL SURFACE POLYSACCHARIDE REPEATING UNITS Zaheer Timol University of Cape Town Supervisors: Neil Ravenscroft, Michelle Kuttel and David Gammon A thesis presented for the degree of Master in Science University of Cape Town March 2017 The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Abstract Bacterial cell surface polysaccharides are primarily present as lipopolysaccharides or capsular polysac- charides. They are used by cells for both structure and function and have been shown to be a virulence factor of bacterial pathogens. Cell surface polysaccharides are widely utilised as antigenic components in vaccines and play an important role in the protection against numerous diseases including meningo- coccal disease and shigellosis. This study is composed of two parts: a computational section, which investigates the capsular polysaccharide (CPS) repeating unit (RU) conformations of meningococcal Y and W CPS vaccines and a second experimental component that involves synthetic studies toward the O-specific polysaccharide (O-SP) RU of Shigella sonnei. The CPS RU of MenY [!6)-a-D-Glc(1!4)-a-D-NeuNAc-(2!] and MenW [!6)-a-D-Gal(1!4)-a- D-NeuNAc-(2!] differ only in the orientation of the C-4 hydroxyl: equatorial in MenY and axial in MenW. -
Pentose PO4 Pathway, Fructose, Galactose Metabolism.Pptx
Pentose PO4 pathway, Fructose, galactose metabolism The Entner Doudoroff pathway begins with hexokinase producing Glucose 6 PO4 , but produce only one ATP. This pathway prevalent in anaerobes such as Pseudomonas, they doe not have a Phosphofructokinase. The pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) is a biochemical pathway parallel to glycolysis that generates NADPH and pentoses. While it does involve oxidation of glucose, its primary role is anabolic rather than catabolic. There are two distinct phases in the pathway. The first is the oxidative phase, in which NADPH is generated, and the second is the non-oxidative synthesis of 5-carbon sugars. For most organisms, the pentose phosphate pathway takes place in the cytosol. For each mole of glucose 6 PO4 metabolized to ribulose 5 PO4, 2 moles of NADPH are produced. 6-Phosphogluconate dh is not only an oxidation step but it’s also a decarboxylation reaction. The primary results of the pathway are: The generation of reducing equivalents, in the form of NADPH, used in reductive biosynthesis reactions within cells (e.g. fatty acid synthesis). Production of ribose-5-phosphate (R5P), used in the synthesis of nucleotides and nucleic acids. Production of erythrose-4-phosphate (E4P), used in the synthesis of aromatic amino acids. Transketolase and transaldolase reactions are similar in that they transfer between carbon chains, transketolases 2 carbon units or transaldolases 3 carbon units. Regulation; Glucose-6-phosphate dehydrogenase is the rate- controlling enzyme of this pathway. It is allosterically stimulated by NADP+. The ratio of NADPH:NADP+ is normally about 100:1 in liver cytosol. -
Redox and Complexation Chemistry of the Crvi/Crv–D-Galacturonic Acid System
Redox and complexation chemistry of the CrVI/CrV–D-galacturonic acid system Juan C. González,a Verónica Daier,a Silvia García,a Bernard A. Goodman,b Ana M. Atria,c Luis F. Sala*a and Sandra Signorella*a a Departamento de Química, Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, Suipacha 531, 2000, Rosario, Argentina. E-mail: [email protected]; [email protected] b Scottish Crop Research Institute, Invergowrie, Dundee, Scotland, UK DD2 5DA c Facultad de Ciencias Químicas y Farmacéuticas and CIMAT, Universidad de Chile, Casilla 233, Santiago, Chile The oxidation of D-galacturonic acid by CrVI yields the aldaric acid and CrIII as final products when a 30-times or higher excess of the uronic acid over CrVI is used. The redox reaction involves the formation of intermediate CrIV and CrV species, with CrVI and the two intermediate species reacting with galacturonic acid at comparable rates. The rate of disappearance of CrVI, CrIV and CrV depends on pH and [substrate], and the slow reaction step of the CrVI to CrIII conversion depends on the reaction conditions. The EPR spectra show that five-coordinate oxo–CrV bischelates are formed at pH ≤ 5 with the uronic acid bound to CrV through the carboxylate and the -OH group of the furanose form or the ring oxygen of the pyranose form. Six-coordinated oxo–CrV monochelates are observed as minor species in addition to the major five- V VI coordinated oxo–Cr bischelates only for galacturonic acid : Cr ratio ≤ 10 : 1, in 0.25–0.50 M HClO4. At pH 7.5 the EPR spectra show the formation of a CrV complex where the vic-diol groups of Galur participate in the bonding to CrV. -
The Fischer Proof of the Structure of (+)-Glucose Started in 1888, 12
The Fischer proof of the structure of (+)-glucose Started in 1888, 12 years after the proposal that carbon was tetrahedral, and thus had stereoisomers. Tools: - melting points - optical rotation (determine whether a molecule is optically active) - chemical reactions Fischer knew: - (+)-glucose is an aldohexose. - Therefore, there are 4 stereocenters and 24 = 16 stereoisomers (8 D-sugars and 8 L-sugars) - At this time could not determine the actual configuration (D or L) of sugars - Fischer arbitrarily assigned D-glyceraldehyde the following structure. CHO H OH CH2OH - In 1951 Fischer was shown to have guessed correctly. CO2H CHO H OH HO H HO H CH2OH CO2H L-Tartaric acid L-glyceraldehyde Which of the 8 D-aldohexoses is (+)-glucose??? 1) Oxidation of (+)-glucose with nitric acid gives an aldaric acid, glucaric acid, that is optically active. Therefore (+)-glucose cannot have structures 1 or 7, which would give optically inactive aldaric acids. HNO3 (+)-glucose Glucaric acid Optically active CHO CO2H H OH H OH 1 H OH HNO3 H OH Mirror plane H OH H OH H OH H OH Since these aldaric CH2OH CO2H acids have mirror planes they are meso structures. CHO CO2H They are not optically H OH H OH active HO H HNO3 HO H 7 Mirror plane HO H HO H H OH H OH CH2OH CO2H 2) Ruff degradation of (+)-glucose gives (-)-arabinose. Oxidation of (-)-arabinose with nitric acid gives arabanaric acid, which is optically active. Therefore, (-)-arabinose cannot have structures 9 or 11, which would give optically inactive aldaric acids. If arabinose cannot be 9 or 11, (+)-glucose cannot be 2 (1 was already eliminated), 5 or 6, which would give 9 or 11 in a Ruff degradation. -
Carbohydrates: Structure and Function
CARBOHYDRATES: STRUCTURE AND FUNCTION Color index: . Very important . Extra Information. “ STOP SAYING I WISH, START SAYING I WILL” 435 Biochemistry Team *هذا العمل ﻻ يغني عن المصدر المذاكرة الرئيسي • The structure of carbohydrates of physiological significance. • The main role of carbohydrates in providing and storing of energy. • The structure and function of glycosaminoglycans. OBJECTIVES: 435 Biochemistry Team extra information that might help you 1-synovial fluid: - It is a viscous, non-Newtonian fluid found in the cavities of synovial joints. - the principal role of synovial fluid is to reduce friction between the articular cartilage of synovial joints during movement O 2- aldehyde = terminal carbonyl group (RCHO) R H 3- ketone = carbonyl group within (inside) the compound (RCOR’) 435 Biochemistry Team the most abundant organic molecules in nature (CH2O)n Carbohydrates Formula *hydrate of carbon* Function 1-provides important part of energy Diseases caused by disorders of in diet . 2-Acts as the storage form of energy carbohydrate metabolism in the body 3-structural component of cell membrane. 1-Diabetesmellitus. 2-Galactosemia. 3-Glycogen storage disease. 4-Lactoseintolerance. 435 Biochemistry Team Classification of carbohydrates monosaccharides disaccharides oligosaccharides polysaccharides simple sugar Two monosaccharides 3-10 sugar units units more than 10 sugar units Joining of 2 monosaccharides No. of carbon atoms Type of carbonyl by O-glycosidic bond: they contain group they contain - Maltose (α-1, 4)= glucose + glucose -Sucrose (α-1,2)= glucose + fructose - Lactose (β-1,4)= glucose+ galactose Homopolysaccharides Heteropolysaccharides Ketone or aldehyde Homo= same type of sugars Hetero= different types Ketose aldose of sugars branched unBranched -Example: - Contains: - Contains: Examples: aldehyde group glycosaminoglycans ketone group. -
PENTOSE PHOSPHATE PATHWAY — Restricted for Students Enrolled in MCB102, UC Berkeley, Spring 2008 ONLY
Metabolism Lecture 5 — PENTOSE PHOSPHATE PATHWAY — Restricted for students enrolled in MCB102, UC Berkeley, Spring 2008 ONLY Bryan Krantz: University of California, Berkeley MCB 102, Spring 2008, Metabolism Lecture 5 Reading: Ch. 14 of Principles of Biochemistry, “Glycolysis, Gluconeogenesis, & Pentose Phosphate Pathway.” PENTOSE PHOSPHATE PATHWAY This pathway produces ribose from glucose, and it also generates 2 NADPH. Two Phases: [1] Oxidative Phase & [2] Non-oxidative Phase + + Glucose 6-Phosphate + 2 NADP + H2O Ribose 5-Phosphate + 2 NADPH + CO2 + 2H ● What are pentoses? Why do we need them? ◦ DNA & RNA ◦ Cofactors in enzymes ● Where do we get them? Diet and from glucose (and other sugars) via the Pentose Phosphate Pathway. ● Is the Pentose Phosphate Pathway just about making ribose sugars from glucose? (1) Important for biosynthetic pathways using NADPH, and (2) a high cytosolic reducing potential from NADPH is sometimes required to advert oxidative damage by radicals, e.g., ● - ● O2 and H—O Metabolism Lecture 5 — PENTOSE PHOSPHATE PATHWAY — Restricted for students enrolled in MCB102, UC Berkeley, Spring 2008 ONLY Two Phases of the Pentose Pathway Metabolism Lecture 5 — PENTOSE PHOSPHATE PATHWAY — Restricted for students enrolled in MCB102, UC Berkeley, Spring 2008 ONLY NADPH vs. NADH Metabolism Lecture 5 — PENTOSE PHOSPHATE PATHWAY — Restricted for students enrolled in MCB102, UC Berkeley, Spring 2008 ONLY Oxidative Phase: Glucose-6-P Ribose-5-P Glucose 6-phosphate dehydrogenase. First enzymatic step in oxidative phase, converting NADP+ to NADPH. Glucose 6-phosphate + NADP+ 6-Phosphoglucono-δ-lactone + NADPH + H+ Mechanism. Oxidation reaction of C1 position. Hydride transfer to the NADP+, forming a lactone, which is an intra-molecular ester. -
Determination of Carbohydrates in Honey Manali Aggrawal, Jingli Hu and Jeff Rohrer, Thermo Fisher Scientific, Sunnyvale, CA
Determination of carbohydrates in honey Manali Aggrawal, Jingli Hu and Jeff Rohrer, Thermo Fisher Scientific, Sunnyvale, CA ABSTRACT RESULTS SAMPLE ANALYSIS METHOD ACCURACY Table 7. Adulteration parameters for HS6 adulterated with 10% SS1 through SS5. Purpose: To develop an HPAE-PAD method for the determination of carbohydrates in honey Honey sugar analysis Sample Recovery HS6 (Wild Mountain Honey) samples to evaluate their quality and to assess the possibility of adulteration. Separation Adulteration Honey sugars were separated using a Dionex CarboPac PA210-Fast-4μm column (150 × 4 mm) in Method accuracy was evaluated by measuring recoveries of 10 sugar standards spiked into honey Parameters 100% + 10% + 10% + 10% + 10% + 10% For this study, we purchased 12 commercial honey samples (Table 1) and analyzed them using Honey SS1 SS2 SS3 SS4 SS5 Methods: Separation of individual honey sugars was achieved on the recently introduced Thermo series with a Dionex CarboPac PA210 guard column (50 × 4 mm). The column selectivity allow samples. For spiking experiments, four honey samples were used (HS7–HS10) and spiked with a 10- HPAE-PAD. Figure 3 shows the representative chromatograms of 3 honey samples. For all 12 Glucose(G), mg/L 121 115 116 117 119 107 Scientific™ Dionex™ CarboPac™ PA210-Fast-4μm column. Carbohydrate detection was by pulsed carbohydrates to be separated with only a hydroxide eluent generated using an eluent generator. A sugar standard mix at two concentration levels. Figure 4 shows the representative chromatograms investigated honey samples, fructose and glucose (Peak 2 and Peak 3), were found to be the major Fructose(F), mg/L 127 115 115 116 126 116 amperometric detection (PAD) with a gold working electrode and, therefore, no sample derivatization solution of honey sugar standards was prepared and an aliquot (10 μL) of the solution was injected of unspiked and spiked honey sample HS7. -
Carbohydrates Adapted from Pellar
Carbohydrates Adapted from Pellar OBJECTIVE: To learn about carbohydrates and their reactivities BACKGROUND: Carbohydrates are a major food source, with most dietary guidelines recommending that 45-65% of daily calories come from carbohydrates. Rice, potatoes, bread, pasta and candy are all high in carbohydrates, specifically starches and sugars. These compounds are just a few examples of carbohydrates. Other carbohydrates include fibers such as cellulose and pectins. In addition to serving as the primary source of energy for the body, sugars play a number of other key roles in biological processes, such as forming part of the backbone of DNA structure, affecting cell-to-cell communication, nerve and brain cell function, and some disease pathways. Carbohydrates are defined as polyhydroxy aldehydes or polyhydroxy ketones, or compounds that break down into these substances. They can be categorized according to the number of carbons in the structure and whether a ketone or an aldehyde group is present. Glucose, for example, is an aldohexose because it contains six carbons and an aldehyde functional group. Similarly, fructose would be classified as a ketohexose. glucose fructose A more general classification scheme exists where carbohydrates are broken down into the groups monosaccharides, disaccharides, and polysaccharides. Monosaccharides are often referred to as simple sugars. These compounds cannot be broken down into smaller sugars by acid hydrolysis. Glucose, fructose and ribose are examples of monosaccharides. Monosaccharides exist mostly as cyclic structures containing hemiacetal or hemiketal groups. These structures in solution are in equilibrium with the corresponding open-chain structures bearing aldehyde or ketone functional groups. The chemical linkage of two monosaccharides forms disaccharides. -
Carbohydrates: Occurrence, Structures and Chemistry
Carbohydrates: Occurrence, Structures and Chemistry FRIEDER W. LICHTENTHALER, Clemens-Schopf-Institut€ fur€ Organische Chemie und Biochemie, Technische Universit€at Darmstadt, Darmstadt, Germany 1. Introduction..................... 1 6.3. Isomerization .................. 17 2. Monosaccharides ................. 2 6.4. Decomposition ................. 18 2.1. Structure and Configuration ...... 2 7. Reactions at the Carbonyl Group . 18 2.2. Ring Forms of Sugars: Cyclic 7.1. Glycosides .................... 18 Hemiacetals ................... 3 7.2. Thioacetals and Thioglycosides .... 19 2.3. Conformation of Pyranoses and 7.3. Glycosylamines, Hydrazones, and Furanoses..................... 4 Osazones ..................... 19 2.4. Structural Variations of 7.4. Chain Extension................ 20 Monosaccharides ............... 6 7.5. Chain Degradation. ........... 21 3. Oligosaccharides ................. 7 7.6. Reductions to Alditols ........... 21 3.1. Common Disaccharides .......... 7 7.7. Oxidation .................... 23 3.2. Cyclodextrins .................. 10 8. Reactions at the Hydroxyl Groups. 23 4. Polysaccharides ................. 11 8.1. Ethers ....................... 23 5. Nomenclature .................. 15 8.2. Esters of Inorganic Acids......... 24 6. General Reactions . ............ 16 8.3. Esters of Organic Acids .......... 25 6.1. Hydrolysis .................... 16 8.4. Acylated Glycosyl Halides ........ 25 6.2. Dehydration ................... 16 8.5. Acetals ....................... 26 1. Introduction replacement of one or more hydroxyl group (s) by a hydrogen atom, an amino group, a thiol Terrestrial biomass constitutes a multifaceted group, or similar heteroatomic groups. A simi- conglomeration of low and high molecular mass larly broad meaning applies to the word ‘sugar’, products, exemplified by sugars, hydroxy and which is often used as a synonym for amino acids, lipids, and biopolymers such as ‘monosaccharide’, but may also be applied to cellulose, hemicelluloses, chitin, starch, lignin simple compounds containing more than one and proteins. -
4202-B: Nucleic Acids and Carbohydrates L-4 1 Deoxy Sugars
4202-B: Nucleic acids and Carbohydrates L-4 Deoxy sugars In these sugars one of the OH groups is replaced by a hydrogen. 2-Deoxyribose (oxygen missing at C-2 position) is an important example of a deoxy sugar. It is important component of DNA, and lack of C-2 hydroxyl provide additional stability to it as compared to RNA as no intramolecular nuclephilic attack on phosphate chain can occur. Amino sugars In amino sugars one of the OH groups is replaced by an amino group. These molecules allow proteins and sugars to combine and produce structures of remarkable variety and beauty. The most common amino sugars are N-acetyl glucosamine and N-acetyl galactosamine, which differ only in stereochemistry. The hard outer skeletons of insects and crustaceans contain chitin, a polymer very like cellulose but made of N-acetyl glucosamine instead of glucose itself. It coils up in a similar way and provides the toughness of crab shells and beetle cases. Some important antibiotics contain amino sugars. For example, the three subunits of the antibiotic gentamicin are deoxyamino sugars (the middle subunit is missing the ring oxygen). N-Acetyl glucosamine N-Acetyl galactosamine Gentamicin, an antibiotic Cell membranes must not be so impermeable as they need to allow the passage of water and complex molecules. These membranes contain glycoproteins—proteins with amino sugar residues attached to asparagine, serine, or threonine in the protein. The attachment is at the anomeric position so that these compounds are O- or N-glycosides of the amino sugars. The structure below shows N-acetyl galactosamine attached to an asparagine residue as an N-glycoside. -
Structures of Branched Dextrins Produced by Saccharifying A-Amylase of Bacillus Subtilis
/. Biochem., 72, 1219-1226 (1972) Structures of Branched Dextrins Produced by Saccharifying a-Amylase of Bacillus subtilis Kimio UMEKI and Takehiko YAMAMOTO Downloaded from https://academic.oup.com/jb/article/72/5/1219/878303 by guest on 29 September 2021 The Faculty of Science, Osaka City University, Osaka Received for publication, May 25, 1972 The branched dextrins produced from waxy rice starch ^-limit dextrin by Bacillus subtilis' saccharifying a-amylse [EC 3.2.1.1] were isolated by the multiple develop- ment paper chromatography and their chemical structures were investigated. The analyses using £-amylase [EC 3.2.1. 2], glucoamylase [EC 3.2.1.3], pullulanase, and pullulan a-1,4-glucoside hydrolase revealed that they were doubly branched dextrins with the following structures: e'-a-^'-a-glucosylmaltosylJ-maltotriose, 68-a-, 65-a- diglucosylmaltopentaose, and 68-a-{6*-a-glucosylmaltotriosyl)-maltotriose. The results were discussed in connection with the interior structure of /3-limit dextrin. Only a few papers have so far been published from each other (8, 9). on the action of a-amylase [EC 3.2.1.1] on The saccharifying a-amylase is character- branched substrates (1-3). French and his istic in that it hydrolyzes starch to produce coworkers have reported that the porcine reducing sugars of about twice as much as pancreatic a-amylase produces various branched those produced by the liquefying a-amylase dextrins as the limit dextrins (4). Our pre- and none of the hydrolysis products by the vious paper (5) has also shown that a-amylase saccharifying a-amylase are attacked by the of starch liquefying type from Bacillus subtilis liquefying a-amylase. -
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.