Carbohydrates Lecture Notes PDF

Carbohydrates and glycobiology Marc A. Ilies, Ph. D. Lehninger - Chapter 7 (p243-280) [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas...

Carbohydrates and glycobiology Marc A. Ilies, Ph. D. Lehninger - Chapter 7 (p243-280) [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas ©MAIlies2024 1  Carbohydrates - Polyhydroxyaldehydes or polyhydroxyketones having the general formula (CH2O)n, with sometimes N, S, or P; can be linear or cyclic; also include the oligomers and polymers that can form these compounds upon hydrolysis - Also called saccharides (greek: sakcharon = sugar); all common ones have the name ending with the suffix “-ose” - Can be classified in - monosaccharides: single units of polyhydroxyaldehyde or ketone (e.g. glucose, fructose, erythrose, etc) - oligosaccharides: few units, linked via ether bonds (e.g. sucrose = disaccharide containing one unit of D-glucose and one unit of D-fructose) - polysaccharides (glycans): more than 20 monosaccharide units; some have more than 100.000 units; can be linear (e.g. amylose) or branched (e.g. amylopectin, glycogen) 2  Carbohydrates Roles: - energy generation and storage: oxidation of carbohydrates is the primary energy generating process in metabolism; - structural role in plants (cellulose), cell walls of bacteria, exoskeleton of insects; - inactive ingredients (excipients) in tablets, capsules, other pharmaceutical formulations - potent osmolites, contribute to osmotic pressure inside and outside the cells - recognition elements – immunity, immune response, cell identity and recognition 3  Monosaccharides: types and nomenclature - Two main types: polyhydroxyaldehydes (aldoses) and polyhydroxyketones (ketoses) - also stated is the number of C atoms in the molecule: no oxygen at - trioses position 2 - tetroses - pentoses - hexoxes 4  Monosaccharides: chirality H - Smallest monosaccharide: glyceraldehyde H2C C* CHO OHOH has two stereoisomers: - Most of natural sugars: D relative configuration 5 Note that its isomer, the ketotriose dihydroxyacetone, does not have stereoisomers  Monosaccharides: Fischer projection ► is a conventional 2D representation of the 3D molecular structure that shows the configuration of all chiral centers within a molecule 3D 2D Emil Fischer longest C-chain written vertically with the most highly oxidized C on top; horizontal lines = bonds projecting forward; vertical lines = bonds projecting to the rear ; C atoms at the intersection of the horizontal 1 1 and vertical lines are not shown; C atoms numbered in sequence from the top; 2 2 * * ► OH on most distant chiral carbon from the carbonyl D L group gives the relative configuration: 3 3 - on the right: D configuration; - on the left: L configuration. D-Glyceraldehyde L-Glyceraldehyde 6  Monosaccharide series: aldoses - most of natural sugars are of D relative configuration; - each chiral carbon generates two enatiomers; total number of stereoisomers for a sugar with n chiral carbons = 2n; only half of them will have the D-relative configuration ! 7 Chiral carbons in red  Monosaccharide series: ketoses 8 Chiral carbons in red  Monosaccharide series: epimers - Two sugars that differ only in the configuration of one chiral carbon atom: 9  Hexose O H Aldohexose O H 1 C C Epimers H C OH H C OH HO C H Asymmetric HO C H H C OH carbons=2,3,4,5 HO C H 2n=16 stereoisomers H C OH H C OH 6 CH2OH Penultimate CH2OH carbon D-glucose D-galactose CH2OH O H C Ketohexose C O L-galactose HO C H D-fructose HO C H Enantiomer H C OH H C OH of D-galactose H C OH Asymmetric H C OH HO C H carbons=3,4,5 CH2OH CH 2OH 10  Monosaccharides: cyclic structure - besides general properties of alcohols and carbonyl compounds, monosaccharides exhibit special properties due to the presence of both these functionalities in their molecules, simultaneously: 11  Monosaccharides: cyclic structure - a new chiral center is created after cyclization: - the two stereoisomers, which differ just on the stereochemistry at the hemiacetalic carbon, are called anomers 12 The anomers of glucose and fructose Haworth perspective formulas 13  H C O Anomeric 1 6CH2OH 6 CH2OH H C OH 2 H 5 OH H H 5 O H HO 3C H H H 4 OH H O 4 OH H 1 H C OH 1 4 OH 3 2 OH 3 OH H C OH 2 5 H OH H OH CH2OH -D-Glucopyranose Intramolecular nucleophilic cycloaddition H CH2OH OH O OH C O O H R O C H H R'' Pyran OH H + R'' H OH R OH Hemiacetal H OH -D-Glucopyranose 14  1 CH Anomeric 2OH 6 CH OH 1 CH OH 2 2 2C O O HO 3C H H OH H 4C OH H OH H 5 C OH OH H 6 CH2OH -D-Fructofuranose 6 CH2OH R' O OH OH O 5 C O H OH 2 R O C R' Furan H 4 R'' 3 CH2OH R'' 1 + OH H R OH Hemiketal -D-Fructofuranose 15  Monosaccharides: mutarotation Open Chain -D-glucopyranose -D-glucopyranose H C O CH2OH 6 CH 2OH 1 H C OH H O OH H 5 O H 2 H HO C H H 4 1 3 OH H OH H OH H C OH H OH 3 2 4 OH H C OH H OH 5 H OH + 112.20 6CH 2OH + 18.70 36.4% 0.1% 63.5 % At equilibrium optical rotation is + 52.70 - the interconversion of the two anomers via the acyclic form is called MUTAROTATION 16  Conformational formulas of pyranoses - the pyranose ring is not flat but 3D - a conformational equilibrium exists between two possible chair forms: ecuatorial-ecuatorial axial- axial (no steric hindrance) steric hindrance - when substituents are in CH2OH equatorial positions, they are e HOH2C OH a a O less hindered, therefore e HOHO O OH e more stable: e OH OH a OH OH a a preferred chair conformation 17 of α-D-glucopyranose  Combined effect of conformation and configuration - anomer stability for D-glucopyranose: -D-glucopyranose -D-glucopyranose H CH2OH O H CH2OH O H H H HO H HO OH OH OH H HO HO OH H H H 1,2 dipolar all groups are equatorial repulsions MORE STABLE MORE STABLE  and  are different configurations (not conformations) they are diastereomers and can only be inter-converted by bond cleavage. 18 19  Reducing properties of monosaccharides - copper is reduced from +2 to +1 thus glucose is a reducing sugar - this is the Fehling’s reaction and the basis of Clinitab® test for measuring urine glucose levels - reducing properties are displayed only if the sugar can adopt an acyclic form; for 20 cyclic forms the glycosidic OH must be free!!!  Glucose measurements in diagnosis and treatment of diabetes - self-monitoring of blood or urine glucose level (Dextrostix, Clinistix, Diastix, Tes Tape, bG Chemstrips, One Touch System ): Glucose Oxidase method glucose oxidase D-Glucose + O2 D-Glucono-δ-lactone + H2O2 peroxidase H2O2 + dianisidine oxidized dianisidine + H2O (colorless) (colored) Hexokinase method 21 Disaccharides contain a glycosidic bond Reducing end - from partial hydrolysis of starch by a-amylases (malting & mashing of barley) 22  Disaccharides: structure and properties - lactis = milk, ose = sugar; Reducing - excipient in pharm tablets one free anomeric OH - broken down by lactase (β-D-galactosidase) - lactose intolerance - table sugar, saccharose Non-reducing - main circulating sugar in anomeric OHs are linked plants (cane, sugar beet) - high energy → obesity, diabetes - in insects - strongly retains water → Non-reducing anhydrobiosis, cosmetics anomeric OHs are linked - cleaved by trehalase (yeasts, etc) liberating glucose 23 24  6 CH2 OH 6 CH2 OH OH H O H H O Reducing Sugar H5 H5 4 1 4 free Anomeric carbon OH H OH H OH O H 3 2 3 2 H OH H OH 6 CH2 OH 6 CH2 OH H O H H OH O H5 H5 4 H 1 4 H 1 OH OH OH O H 3 2 3 2 H OH H OH To be a reducing sugar mutarotation must be possible !! 25  Oligosaccharides Cyclodextrins - cyclic oligomers of of D-glucose, linked α1→4, with different sizes; - most common n = 6, 7, 8 (α, β, γ-cyclodextrins), also hydroxypropyl version of β-cyclodextrin - toroidal structures, with a hydrophilic exterior and a relatively hydrophobic internal compartment, in which lipophilic drugs can be encapsulated (host-guest complexes): - nontoxic (GRAS status) , can be biodegraded to maltose and /or glucose; used in pharmaceutical formulations for delivery of hydrophobic drugs, excipients, cosmetics, etc Polysaccharides: constitution and topology 27  Fuel-storage homopolysaccharides Starch: amylose + amylopectin Amylose - linear polymer of D-glucose, linked α1→4: - cleaved by a-amylases into maltose units; - curved polysaccharide chain due to α1→4 linkages; intra-chain H-bonding generates a helical structure: - I2 molecules can insert inside the helical structure forming an inclusion complex (dark blue)  Fuel-storage homopolysaccharides Amylopectin and Glycogen: - branched polymers of D-glucose, linked α1→4 and α1→6: The cluster structure of starch granules (amylose + amylopectin) - Starch (amylose + amylopectin) and glycogen are storage polysaccharides in plant and animal cells: - starch is used as an excipient primarily in oral solid-dosage formulations (tablets and 29 capsules) as a binder, diluent, and disintegrant  Cellulose - polymer of D-glucose, linked β1→4: - excellent mechanical properties and water insolubility due to intra-chain (a) and inter- chain (b) H bonds; forms very tough fibers (c): - hydrophilic, biodegradable; (c) 30 - OH groups can be derivatized by acetylation, carboxymethylation, etc to modify base properties (lipophilicity); used in pharmaceutical formulations as adsorbent, glidant, suspending agent, tablet and capsule diluent, tablet disintegrant  Chitin - polymer of NAG (N-acetylglucosamine), linked β1→4: - another way to draw it (see structural resemblance with cellulose!) - more than 1 bn tons/year synthesized in biosphere - used, together with its deacetylated polymer chitusan, as pharmaceutical excipient 31  Glycoconjugates: peptidoglycans - peptidoglycan gives bacterial anvelopes their strength and rigidity: II Gram-Positive bacteria PEPTIDOGLYCAN II STRUCTURE: Fig 20-31 32  N-Acetylgucosamine CH2OH (β1→4) CH2 OH N-Acetylmuramic acid (NAG) H O (NAM) O O H H H H O H H O OH H NH C CH3 H NHCOCH3 H O O H3C CH C Peptidoglycans of NH (Gly)5 Gram Positive bacteria L-Ala Isoglutamate L-Lys (β1→4) CH2 OH CH2 OH D-Ala H O O O Gly H OH O O H H NH C CH3 Gly Pentaglycine link H NH COCH3 H O O H3C CH C Gly NH Gly L-Ala Isoglutamate Gly L-Lys D-Ala 33  CH2 OH Peptidoglycans of CH2OH H O Gram Negative H H O H O H O O OH H H bacteria H NH C CH3 H NHCOCH3 H O O CH2 OH H3C CH C CH2OH NH H O O H H H O H L-Ala O H H O OH Isoglutamate H H NH C CH3 L-Lys NHCOCH3 H O O D-Ala H3C CH C NH L-Ala Isoglutamate Direct links between D-Ala L-Lys and Lysine (no pentaglycine) D-Ala 34  Synthesis of bacterial peptidoglycan - multistep pathway consisting of 8 major processes: - polysaccharide chain cross-linking (step 8) inhibited by penicillins thus weakening the bacterial cell wall Fig 20-32 35  Penicillins: general structure and representatives - general structure of penicillins: - representatives: CH Ampicillin NH2 HO CH Amoxicillin NH2 Box 20-1 36  Penicillins: mechanism of action - Penicillins and β-lactam antibiotics irreversibly bind a serine residue into the active site of bacterial transpeptidase, inactivating it:  defective bacterial wall  bacteria explodes under osmotic shock 37  Penicillins: mechanism of action Bacteria (E. coli) bursts upon treatment with ampicillin Bacteria (E. Coli) in the presence (left) and absence (right) of pennicilin 38  Penicillins: mechanism of inactivation - constant use of antibiotics placed evolutionary pressure on pathogenic bacteria, which evolved to express β-lactamases – enzymes that cleave β-lactam antibiotics rendering them inactive: Overcoming bacterial resistance: use a suicide → inhibitor of -lactamase (clavulanic acid) to preserve the potency of an antibiotic: 39 eg Augmentin® (Amoxicillin + Clavulanic Acid)  Glucosaminoglycans - linear heteropolysaccharides composed of repeating disaccharide units: Hyalos = glass (Gr.) Sinovial joints, Vitreous humor of the eye Extracellular matrix of cartilage and tendons Chondros = cartilage (Gr.) Cartilage, tendons, ligaments Walls of the aorta Keras = horn (Gr.) Cornea, cartilage, bone Horns, hairs, hoofs, nails, claws 40  Heparin and congeners Heparin - fractionated form of heparan sulfate, used as anticoagulant - highest negative charge density of any known biological macromolecule - average MW=15,000 LMWH (low molecular weight heparin) - average MW=4,500 41 42 Oligosaccharide Links in Glycoproteins 43  Other glycoconjugates - saccharides conjugated with steroids: Digoxin O (Digitalis) O OH CH3 12 CH3 H3 C H3 C H3 C Lactone O O O OH 3 HO O O O HO HO HO Steroid Tri-digitoxose Aglycone - cardiac-active glycoside extracted from foxglove - in the treatment of heart conditions (heart failure, atrial fibrillations) - toxic at high doses 44 Lipopolysaccharides - On the surface of gram-negative bacteria - Recognized by vertebrate immune systems; trigger very vigorous immune responses in bacterial infections 45  Oligosaccharide recognition: lectins - Lectins are proteins which bind carbohydrates with high affinity and specificity; oligosaccharides often H-bond to specific areas in the lectin. 46 Lectin/Oligosaccharide recognition in inflammation Selectins: a group of lectins in cell membranes which are involved in processes of cell recognition and adhesion including the movement of T-lymphocytes from blood to tissue during infection or inflammation. Integrins: lectins that can serve as “receptors”, conveying information across plasma membranes - important role in inflammation, blood coagulation via platelet aggregation, interaction between cells and extracellular matrix, etc - an integrin blocker Eptifibatide (Integrilin®) has utility in preventing platelet aggregation and blood clots in order to reduce the risk of acute cardiac ischemic events (together with aspirin, heparin). It is approved for use during surgical installation of coronary stents among other uses. 47 47  Lectin/Oligosaccharide recognition in inflammation - leukocyte rolling and migration to an inflammation site is modulated by selectins (located on endothelial cell wall) and integrins (located on the surface of leukocytes) that recognize specific glycoproteins: 48 http://www.dnatube.com/video/2508/Interaction-of-Leukocyte-Rolling Lectin/oligosaccharide recognition in inflammation - gram negative Helicobacter pylori colony can cause inflammation of stomach lining, being associated with gastro-duodenal ulcers and stomach cancer - the bacterium adheres to the gastric surface through the interaction of a surface lectin and a blood group antigen (glycoconjugated) of the gastric epithelium Note the helical shape of the bacterium 49 gastric epithelium cell Oligosaccharides in cell adhesion and recognition (overview) 50 51  Goals and Objectives Upon completion of this series of lectures at minimum you should be able to answer the following: ►What are carbohydrates and which roles do they play in living organisms ? ►How are carbohydrates classified and which are the main structural features of each class? ►What kind of isomers we can distinguish for monosaccharides in terms of configuration and conformation? How are they generated? Can they interconvert? ►How is the cyclic structure of monosaccharides generated, what are anomers and how can they interconvert? ►What classes of sugar derivatives can derive from hexoses and which are their structural and functional roles? ►What are the redox properties of monosaccharides, and how are they exploited for sugar dosing/monitoring? ►Which are the main structural characteristics of disaccharides, polysaccharides and what specific properties and physiologic roles do they display/possess? ►Which are the main classes of glycoconjugates and what specific properties and physiologic roles do they display/possess? 52  Diseases, Drugs, Diagnostics ►Diseases: diabetes, bacterial infections, heart conditions (heart failure, atrial fibrillations, acute cardiac ischemic events), thrombosis, viral infections, gastro- duodenal ulcers, stomach cancer ►Drugs: penicillins (G, V, Methicillin, Amoxicillin, Ampicillin), combos penicillin/- lactamase inhibitor (Amoxicillin/Clavulanic Acid = Augmentin®), Digoxin (Digitalis), Eptifibatide (Integrilin®), heparin and low molecular weight heparin ►Diagnostics: Clinitab tablets, testers for self-monitoring of blood or urine glucose level (Dextrostix, Clinistix, Diastix, Tes-Tape, bG Chemstrips, One Touch System) 53