Carbohydrate Reading Module PDF

Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Carbohydrates Introduction Carbohydrates are the most abundant class of bioorganic molecules on ear...

Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Carbohydrates Introduction Carbohydrates are the most abundant class of bioorganic molecules on earth. Along with proteins and fats, carbohydrates are one of three main nutrients found in many foods. Many commonly encountered carbohydrates are polysaccharides, like glycogen, starch and cellulose. Our body breaks down starch and glycogen into glucose which can be used immediately as source of energy or stored in the liver and muscles for later use. Moreover, carbohydrates are metabolic precursors of virtually all other molecules, e.g. amino acids, fats, etc. Learning Objectives The student should be able to: 1. Relate the structures, properties, and reactivities to the functions of carbohydrates; 2. Perform isolation of glycogen from chicken liver; 3. Interpret results of carbohydrate qualitative tests; and 4. Estimate glucose content of commercially available drinks. *Objectives 2-4 are Laboratory Objectives Reference Textbooks: 1. Nelson, D., Cox, M. (2008). Lehninger Principles of Biochemistry, 5th Ed. New York: W.H. Freeman and Company. 2. Berg, J., Tymoczko, J., Stryer, L., Gatto, G. (2012). Biochemistry, 7th Ed. New York: W.H. Freeman and Company. 3. Voet, D. & Voet, J. (2011). Biochemistry. (4th ed). United States of America: John Wiley & Sons, Inc. 4. Campbell, M.K. & Farrell, S.O. (2012). Biochemistry. (7th ed). Belmont, CA, USA: Brooks/Cole Cengage Learning. Supplemental Readings: 1. Carbohydrates - Structure and Function (https://www.slideshare.net/ shainamavreenvillaroza/chem-45-biochemistry-carbohydrates) 2. Carbohydrates - Chemical Properties (slides 48-64) (https://www.slideshare.net/ ashokktt/carbohydrate-chemistry-37049261) I. Functions of Carbohydrates An energy storage; plants convert light energy into monosaccharides and starch which animals can use as energy source. Structure of organisms: cellulose, chitin and bacterial cell wall Component of nucleic acid: ribose, deoxyribose Carbohydrate “markers” on cell surfaces play key roles in cell-cell recognition processes Carbohydrates linked to lipid molecules (glycolipids) are common components of biological membranes Page 1 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Carbohydrates covalently linked to proteins (glycoproteins) are important components of cell walls and extracellular structures in plants, animals and bacteria Carbohydrates are the metabolic precursors of virtually all other molecules II. Structure, Nomenclature and Classification By definition, carbohydrates are polyhydroxy aldehydes and polyhydroxy ketones or compounds that produce such substances upon hydrolysis. The word carbohydrate originally referred to compounds of the general formula Cn(H2O)n. However, only the monosaccharides, known also as simple sugars follow this formula exactly. Oligosaccharides and polysaccharides have slightly different general formulas which are based on the monosaccharide units. Oligosaccharides are formed when a few (Greek oligos) monosaccharides are linked, and polysaccharides are formed when many (Greek polys) monosaccharides are bonded together. These carbohydrates are referred to as saccharides because of their sweet taste (latin, saccharum, meaning sugar). A. Classification of Carbohydrates According to Degree of Complexity a. Monosaccharides : glucose, fructose, mannose, galactose b. Disaccharides: sucrose, maltose, cellobiose c. Oligosaccharides: raffinose d. Polysaccharides: : starch, cellulose, glycogen Monosaccharides and disaccharides are called simple carbohydrates, and the oligosaccharides (2-10 sugar units) and polysaccharides (>10 sugar units) as complex carbohydrates. 1. Monosaccharide carbohydrate that cannot be hydrolyzed to a simpler carbohydrate general formula Cn(H2O)n, where n varies from 3 to 8 maybe an aldose, if containing an aldehyde group or a ketose, if containing a ketone group water soluble, white, crystalline solids classified according to the number of carbon atoms: triose (3C); tetrose (4C), pentose (5C), hexose (6C), and heptose (7C). hexoses are the most common of all monosaccharides Common Monosaccharides 1. Glucose - blood sugar - dextrose, grape sugar - most abundant monosaccharide in the body - provides a source of energy (ATP) to cells - from starch, legumes, cereal roots, animal tissues, liver - converted to other compounds in the body, i.e. glucose can be converted to some amino acids and fat for long term energy storage; stored as glycogen Page 2 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila 2. Fructose - levulose - sweetest monosaccharide - converted to glucose and used as energy source - naturally occurring monosaccharide; found primarily in honey, fruits and vegetables 3. Galactose - converted to glucose and used as energy source - components of glycolipids and glycoproteins - coupled with glucose to make lactose (milk sugar) 4. Mannose - converted to glucose in the body - found in certain bacteria, fungi and plant - used for treating carbohydrate-deficient glycoprotein syndrome, an inherited metabolic disorder. Relative Sweetness: Sucrose = 100 Galactose = 32 Invert sugar = 126 Glucose = 74 Lactose = 16 Fructose = 174 Maltose = 32 Convention of Representing Carbohydrates 1. Fischer Projection This convention is commonly used for representing open chain forms of carbohydrates. The carbon chain is vertical with the lowest numbered carbon at the top. Numbering follows the convention that the most oxidized end of the molecule has the lowest number. Common monosaccharides, like glucose, mannose, galactose and fructose follow the same formula, C6H12O6. However, they differ in their configuration or in their functional group. For example, glucose exists in two forms, D- glucose and L-glucose, the most naturally occurring of which is the D- isomer (common isomer for most naturally occurring monosaccharides). Glucose and fructose differ in the functional groups, glucose is an aldose and fructose is a ketose, thus, are functional isomers. The number of stereoisomers for a 6-C aldose which have 4 asymmetric centers is given by the formula, 2n, where n is the number of asymmetric centers. Thus, there are 16 possible sugar stereoisomers (8 D- sugars and 8 L-sugars). A comparison of the structures of D-glucose, L-glucose and D-fructose is shown in Figure 1. The D- and L- prefixes denote the position of the hydroxyl group attached to the highest-numbered chiral carbon ( the asymmetric C farthest from the aldehyde or keto group): D-, if OH is on the right side, and L- , if OH is on the left side. Terminologies to Remember about Stereochemistry of Carbohydrates Enantiomers : mirror image molecules (D- and L-sugars are enantiomers) Diastereomers : non-superimposable; non-mirror image (e.g. galactose and mannose) Page 3 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 1. Comparison of the structures of D- and L- glucose and D-fructose. Epimers : differ in arrangement about one other chiral carbons (e.g. glucose and mannose ) D-glucose and D-mannose (shown below) are not superimposable on each other, and neither are they mirror images of each other. Such non-superimposable, non-mirror stereoisomers are called diastereomers. Diastereomers that differ from each other in the configuration at only one chiral carbon are called epimers. Figure 2. D-galactose and D-mannose as epimers of D-glucose D-glucose and D-mannose are examples of these diastereomers, that are called epimers. D-mannose and D-galactose are non-superimposable, and differ in the configuration of two chiral carbons; therefore, they are diastereomers that are not epimers. See Figures 3 and 4 (see next page) for the complete list of D-monosaccharides in Fischer projections. 2. Haworth Projection The Haworth representation is an attempt at a more realistic representation of the ring forms of the sugars in which the placement of substituent can be shown with respect to the plane of the paper with C-2 and C-3 nearest the observer. The substituents are positioned above and below this plane. Page 4 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 3. Fischer projections of aldoses. Figure 4. Fischer projections of ketoses. Like glucose, other sugars with five or six carbon atoms normally exist as cyclic molecules rather than in the open-chain form. The cyclization takes place as a result of interaction between the functional groups on distant carbons, such as C-1 and C-5 (in aldohexoses, e.g. glucose) to form a cyclic hemiacetal, and between C-2 and C-5 (in ketohexoses, e.g. fructose) to form a cyclic hemiketal. In either case, the carbonyl carbon becomes a new chiral center called the anomeric carbon. Page 5 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Cyclization of glucose produces a new asymmetric center at C1. The 2 stereoisomers formed, ⍺- and β- are called anomers. The designation β- means that -OH on the anomeric carbon is cis to the terminal -CH2OH; ⍺- means that it is trans. The Haworth structures of the ⍺- and β-anomers of glucose are shown below, as well as the Fischer formulas for comparison. Figure 5. Cyclization reaction of glucose forming pyranose rings. Adapted from Lehninger Principles of Biochemistry (4th ed) by Nelson, D.L. & Cox, M.M. (2004). Cyclization of fructose produces a new asymmetric center at C2, and two stereoisomers, ⍺- and β-anomers are formed. The Haworth structure of the -anomer (with -OH group at C2 trans to the terminal -CH2OH) is shown below: Figure 6. Cyclization of D-fructose form ⍺-D-Fructofuranose Comparison of the Fischer and Haworth Structures of Glucose and Fructose In the conversion from Fischer to Haworth, the groups oriented to the right in Fischer representations are set downwards in Haworth structures. Refer to Figure 7 on the next page. Page 6 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 7. Comparison of the Fischer and Haworth Projections. 3. Conformational Structures Haworth projection is often used as a simple representation of the ring forms of the sugars. However, because the bonds in sugars are single bonds that are flexible, the cyclic sugars can assume different conformations. For example, cyclic glucose can twist into alternative conformations called boat and chair conformations, or other orientations in between. These structures of common hexoses are based on computations of bond angles and orientation. The substituents are placed relative to a plane denoted by 4 atoms in the ring. Orientations of these substituents are either axial, i.e. perpendicular to the plane, or equatorial, i.e. parallel to the plane. Other orientations are in between these two and denoted quasi-axial or quasi-equatorial. The conformations of five-membered rings, however, are limited to two, envelope (E) and twist (T). The envelope conformation has four atoms in a plane while the twist form only has three. In the pyranose system, five conformers are possible: chair (C), boat (B), skew (S), half-chair (H) or envelope (E). In all cases there are four or more atoms that make up a plane. For more information on conformational analysis of hexoses and pentoses you may visit this link: https:// en.wikipedia.org/wiki/Carbohydrate_conformation The conformations are designated as follows (based on rules proposed by the British Carbohydrate Committee): 1. For six-membered rings, C = chair; B = boat; S or skew = twist boat 2. For five-membered rings, E = envelope, T = twist 3. The pyranoid ring is numbered clockwise starting with the ring oxygen which is numbered zero. Page 7 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila 4. A reference plane is chosen so that it contains four or three of the ring atoms. The reference plane is chosen so that the lowest numbered carbon atom in the ring is displaced from the plane 5. Ring atoms which lie above the reference plane (numbered clockwise) are written as superscripts and precede the letter, while ring atoms which lie below the reference plane are written as subscripts and follow the letter. Example: 4C1 conformation for glucose, in which C4 of the ring is above the plane, and C1 is below the plane. Ribose Conformation The conformations of ribose can be compared with those of the 5-membered cyclopentane’s envelope and the predominating twisted conformations. These two conformations, and those of ribose (a sugar in nucleic acids), are illustrated below. Ribose is the derivative of tetrahydrofuran with 4 atoms in the plane of the envelope form, (Figure b), and 3 atoms in the twisted conformation (Figure d). The strong electronegativity of oxygen forces the neighboring carbon atoms to be in one plane. The configurations of ribose in D- and L-ribose are also shown (Figure c). The twisted cyclic ribose (sugar-pucker conformation) assumes two dominant forms: (1). C3’-endo major and C2’-exo minor conformation and (2) C2’-endo major and C3’-exo minor forms found in A- and B-DNA, respectively (figure d). For details, visit this link, https://www.liebertpub.com/doi/pdfplus/ 10.1089/dna.2019.4943. Figure 8. Conformations of ribose. Adapted from https://www.liebertpub.com/doi/pdfplus/10.1089/ dna.2019.4943 Conformations of Pyranoses The Haworth representation suggests that monosaccharides are flat, but neither furanose nor pyranose rings are actually planar in their lowest energy confirmations. Pyranoses like the cyclohexane ring adopt a low-energy conformation that looks like a chair. Because of the tetrahedral nature of carbon bonds, pyranose sugars actually assume a "chair" or "boat" configuration. The C2-C3-C5-O square forms a plane whereas the C1 and C4 atoms take up a higher or lower position, thus yielding the two possible chairs 1C4 and 4C1 (Figure 9). The representation below reflects the chair configuration (4C1) of the Page 8 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila glucopyranose ring more accurately than the Haworth projection. Other possible conformation of pyranoses are shown in Figure 10. Figure 9. Chair conformations of D-glucose Figure 10. Other possible conformation for pyranoses. The relative stabilities of these various conformations depend on the stereochemical interactions between the substituents on the ring. Close proximity of the flagpole hydrogens in the boat conformation results in steric strain. This, and the torsional strain associated with eclipsed bonds at the four carbon atoms that form the side of the boat conformer, make the boat conformation less stable than the chair conformer. The ring substituents on the chair conformer fall into two geometrical classes: axial and equatorial. Hydroxyl groups in pyranoses tend to adopt equatorial position, a conformation that makes hydroxyl groups farther from each other (depending on the sugar), and minimizes electrostatic repulsions (repulsions between the electrons of oxygen in the -OH groups). Although D-glucose has a strong preference for the 4C1 chair conformation, this is not true for all monosaccharides. For example, L-iduronic acid (an epimer of glucuronic acid) is more stable in the 1C4 configuration. In fact, L-iduronic acid is a special case, because the 1C4 configuration is in equilibrium with the so-called skew-boat shape (2S0), which favors the formation of hinges in linear glycan chains. F o r d e t a i l s , v i s i t t h i s l i n k , h t t p s : / / w w w. p h y s i o l. u z h. c h / d a m / jcr:00000000-06c2-5b85-0000-00002a863784/Structures.pdf Supplemental Readings: Conformations of the Pyranoid Sugars: - https://nvlpubs.nist.gov/nistpubs/jres/64A/jresv64An2p171_A1b.pdf Properties of Monossacharides 1. Optical Activity As a consequence of its asymmetric center, monosaccharides exhibit optical activity. The isomer that rotates plane-polarized light in the counterclockwise direction is termed levorotatory, designated l, or (-), while the isomer that rotates plane-polarized Page 9 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila light in the clockwise direction is designated d, or (+), and is dextrorotatory. Optical activity is used in the sugar industry to measure the sugar concentration of syrup, and generally in chemistry to measure the concentration or enantiomeric ratio of chiral molecules in solution. 2. Mutarotation Mutarotation is the change in optical rotation observed when pure ⍺- or β-anomers are dissolved in water (or other solvents). Due to ring-chain tautomerism, the ⍺- and β- anomers slowly interconvert until equilibrium is established. The ⍺-anomer of D-glucose has a specific rotation of +112.2 degrees in water, and the β-anomer, +18.7 degrees. When either anomer is dissolved in water, the value of the specific rotation changes over time, eventually reaching the same value of +52.5°. This behavior is called mutarotation (literally means, “change in rotation). Figure 11. Specific rotations of the different forms of D-glucose. At equilibrium, the aqueous solution of glucose is consists of 36% ⍺-D-glucose, 64% β-D-glucose, and traces of the linear form. See Figure 12 for details. Figure 12. Interconversion between the ⍺- and β-anomers of D-glucose. Adapted from https:// www.masterorganicchemistry.com/2017/08/17/mutarotation/ Page 10 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila 3. Oxidation A. Oxidation of Monosaccharides Monosaccharides in solution have small amounts of the open chain form present which can be oxidized to carboxylic acid by oxidizing agent such as Benedict’s. The oxidation is due to the presence of aldehyde group. Oxidation at C-1 produces aldonic acid. This reaction is the basis of Benedict’s, Barfoed’s, and Fehling’s tests. The brick red precipitate formed, which is cuprous oxide (Cu2O) is due to the reduction of Cu2+ to Cu+ by the sugar, thus, the sugar is called reducing agent. Figure 13. Oxidation reactions with weak oxidizing agents. Oxidation of the hydroxymethyl group (C-6 in hexoses), results in the formation of uronic acids, e.g. glucose glucuronic acid; galactose galacturonic acid. Uronic acids are biologically important active substances found in mucins and other glycosaminoglycans. Figure 14. Formation of Glucuronic acid via enzymatic oxidation. Other aldohexoses are also oxidized by HNO3, but the aldaric acids products are soluble in water. Refer to Figure 15. Oxidation of a cyclic hemiacetal gives a lactone. Refer to Figure 16. Page 11 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 15. Formation of glucaric acid. Figure 16. Lactone formation from the oxidation of a cyclic hemiacetal. B. Oxidation of Disaccharides Compared with monosaccharides, disaccharides are generally much weaker reducing agents due to the presence of non-reducing component (sugar component with anomeric carbon involved in glycosidic linkage). A disaccharide with a free hemiacetal end is a reducing sugar because of the presence of a free anomeric aldehyde carbonyl or potential aldehyde group. Shown below is a disaccharide with a non-reducing monosaccharide (left monosaccharide) and reducing end (right monosaccharide). The reducing end has a potential aldehyde group. The ring can open to yield free C=O at anomeric carbon. Figure 17. Reducing vs. non-reducing ends. Because most polysaccharides, e.g. starch, glycogen and cellulose have only one reducing end (the rest of the monosaccharide components are linked in glycosidic bonds), they do not have the ability to reduce the oxidizing agents added to them. Page 12 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila 4. Reduction The carbonyl group of a monosaccharide can be reduced to an hydroxyl group by a variety of reducing agents, such as NaBH4, and H2 in the presence of a metal catalyst. Reduction of the C=O group of a monosaccharide gives a polyhydroxy compound called an alditol (sugar alcohol). For more details visit this link: https://www.clutchprep.com/organic- chemistry/monosaccharides-reduction-alditols/12033/learn/14210 Reduction of D-fructose with sodium borohydride yields a mixture of two alditols due to C2 racemization.. One of these isomers has the same configuration as the alditol of glucose, glucitol. The other is the same as the alditol of mannose, mannitol. Figure 18. Reduction of D-fructose forming D-glucitol and D- mannitol. Reduction of aldoses produces only one alditol, e.g. glucose to glucitol (sorbitol) with NaBH4. Figure 19. Reduction of D-glucose forming sorbitol. Alditols are linear molecules that cannot cyclize in the manner of aldoses. Sugar alcohols such as sorbitol, D-xylitol and D-mannitol are used as sweeteners in many sugar- free products such as diet drinks and sugarless gums. Page 13 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila 5. Exhaustive Methylation The reaction with alcohols affects only the anomeric carbon. The other hydroxyl groups, however, can also be methylated using dimethyl sulfate or excess methyl iodide. The addition of methyl (CH3-) to all the free hydroxyl groups found in the sugar is called exhaustive methylation, a technique used to determine glycosidic linkages, and the component monosaccharides. Exhaustive methylation of trehalose (systematic name, ⍺-D- glucopyranosyl-⍺-D-glucopyranoside) using dimethyl sulfate followed by hydrolysis gives equimolar amount of 2,3,4,6-tetra-O-methyl-D-glucopyranose. Figure 20. Exhaustive methylation of trehalose. Adapted from https://www.bartleby.com/questions-and-answers/ trehalose-is-a-disaccharide-that-can-be-obtained-from-fungi-sea-urchins-and-insects.-acid-hydrolysis/ f0866d0d-2017-4856-ba42-bce4cc255c28 6. Formation of Derivatives A. Esterification: Reaction due to alcohol group Phosphoric esters are particularly important in the metabolism of sugars to provide energy. The first step in the utilization of glucose as source of energy is phosphorylation to glucose-6-phosphate. Phosphorylation involves transfer of a phosphate group from ATP as shown below: Figure 21. Formation of a phosphoric ester. Adapted from Biochemistry (7th ed) by Campbell, M.K. & Farrell, S.O. (2012). B. Glycoside Formation A glycoside is a carbohydrate in which the -OH of the anomeric carbon is replaced by -OR; those derived from furanoses are furanosides; and those from pyranoses are Page 14 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila pyranosides. In the following reaction (See Figure 22), methanol reacts with the anomeric OH on glucose to form methyl glucoside (methyl-⍺-D-glucopyranose). Figure 22. Glycoside formation. Adapted from Biochemistry (7th ed) by Campbell, M.K. & Farrell, S.O. (2012). Carbohydrate acetals, are generally called glycosides. - acetal of glucose ⟶ glucoside - acetal of mannose ⟶ mannoside - ketal of fructose ⟶ fructoside A hemiacetal or hemiketal can react with non-carbohydrate unit such as alcohol, glycerol, a sterol or a phenol to give a glycoside. The absence of aldehyde group or potential aldehyde group (e.g. methyl-⍺-D-glucopyranose) makes glycosides non- reducing, and therefore cannot react with the oxidizing agents such as Tollen’s, Benedict’s, and Fehling’s reagents. In acidic solutions, glycosides undergo hydrolysis to produce sugar and alcohol. C. Formation of Amino Sugars In an amino sugar, like glucosamine, an amino group substitutes for a hydroxyl commonly attached to C-2. In some amino sugars, the amino group is acetylated. Amino sugars are frequently found in glycoproteins found in the cell membrane, and proteoglycans. Figure 23. Structures of glucosamine and N- acetylglucosamine. Other examples of amino sugars are shown in Figure 24.: Page 15 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 24. Other examples of amino sugars. D. Formation of Sugar Sulfates Some polysaccharides contain monosaccharide units with sulfates, at C-2, C-4 and/ or C-6. These polysaccharides are found mostly in the proteoglycans of the extracellular matrix. These include chondroitin sulfates, keratin sulfates, dermatan sulfates and heparin. The sulfate groups which are negatively charged at physiological pH impart negative charge to these compounds. Figure 25. Structure of Chondroitin-4-sulfate. Adapted from Lehninger Principles of Biochemistry (4th ed) by Nelson, D.L. & Cox, M.M. (2004). E. Formation of N-acetylneuraminate N-acetylneuraminic acid, also called sialic acid is often found as a terminal residue of oligosaccharide chains of glycoproteins. Sialic acid imparts negative charge to glycoproteins, because its carboxyl group dissociates with a release of proton at physiological pH. Figure 26. Structure of N-acetylnueraminate also known as sialic acid. Page 16 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila 2. Disaccharide How are disaccharides and polysaccharides formed from monosaccharides? Sugars are readily joined together in cells. Two monosaccharides can react to form a disaccharide. The anomeric hydroxyl and a hydroxyl of another sugar can join together, with removal of water to form a glycosidic bond (the bond from the anomeric carbon to the -OR group). This reaction is also the basis for the formation of polysaccharides and oligosaccharides. Figure 27. Formation of glycosidic bond. The formation of disaccharides and longer saccharides is an example of dehydration (an elimination) reaction. Some of the disaccharides are repeating units of the high molecular weight polysaccharides. Common digestible disaccharides include maltose, lactose and sucrose. Only monosaccharides can be absorbed from the digestive tract into the blood, therefore these disaccharides must first be digested into their monosaccharide units. In the small intestine, are specific enzymes for each of these disaccharides: maltase to digest maltose, lactase to digest lactose, and sucrase to digest sucrose. Figure 28. Structures of the three most common digestible disaccharides: maltose, lactose, and sucrose. Page 17 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Below in Table 1 is a comparison of the characteristics of the three common disaccharides: Table 1. Comparison of the three common disaccharides. Name Composition Characteristics Maltose Glucose + Glucose Found in germinating seeds, and beer Lactose Glucose + Galactose Milk sugar; found in dairy products Table sugar; most common dietary disaccharide; Sucrose Glucose + Fructose naturally found in beet and cane sugar, brown sugar, and honey Glycosidic linkages can take various forms; the anomeric carbon of one sugar to any of the -OH groups of another sugar to form an ⍺- or β-glycosidic linkage. Shown below are three of the many disaccharides that can be formed from two glucose molecules. Figure 29. Various forms of glycosidic linkages. 3. Polysaccharide Carbohydrates exist in nature mostly as polysaccharides, such as cellulose, chitin, glycogen, starch, and hyaluronic acid. These compounds are high molecular weight Page 18 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila polymers composed of monosaccharide- or disaccharide-repeating units linked together by glycosidic bonds. The main functions of polysaccharides are structural support, energy storage, and cellular communication. Classification: I. Homoglycans – homopolysaccharides containing only one type of monosaccharide, e.g. Glucans – polymer containing glucoses II. Heteroglycans – heteropolysaccharides containing residues of more than one type of monosaccharide. Glucans are polysaccharides made up of glucose as the only monosaccharide constituent, e.g. amylose and amylopectin (components of starch), glycogen, dextran and cellulose. Other classes of homopolysaccharides are the homopolyaminosaccharides such as chitin and homopolyuronosaccharides which include alginic and pectin. The mucopolysaccharides or glycosaminoglycans and glycosaminoglucuronoglycans are examples of heteropolysaccharides. These substances are found in connective tissues, bacterial cell walls, eyeball synovial fluids, cornea and in the circulatory system in humans. The most abundant of the mucopolysaccharides are hyaluronic acid, chondroitin sulfates, keratin sulfates, dermatan sulfates and heparin. I. Homopolysaccharides A. Storage Polysaccharide Organisms store carbohydrates in the form of polysaccharides, to lower the osmotic pressure. Since osmotic pressure depends on the number of molecules, the osmotic pressure is greatly reduced by formation of a few polysaccharide molecules. 1. Starch Starch is the most important source of carbohydrate in the human diet and accounts for more than 50% of our carbohydrate intake. Plants are able to synthesize glucose using light energy gathered in photosynthesis, and the excess glucose beyond the plant’s immediate energy needs, is stored as starch in different plant parts, including roots and seeds. Starch is stored in globular structures called starch granules. It consists of two types of polysaccharides: amylose (10-30%) and amylopectin (70-90%). Both polymers are consist of -D-glucose units in the 4C1 conformation. a. Amylose - linear chain of D-glucose in (14) linkages; adopts helical conformation - its repeating disaccharide unit is maltose (D-glucopyranosyl α(1→4)-D- glucopyranoside. Figure 30. Repeating structure of amylose. Page 19 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 30. Spiral structure of amylose. Adapted from https://chem.libretexts.org/ Courses/Sacramento_City_College/SCC%3A_Chem_309_- _General_Organic_and_Biochemistry_(Bennett)/Text/14%3A_Carbohydrates/ 14.7%3A_Polysaccharides. Amylose adopts a helical conformation with 6 glucose units per turn, stabilized by hydrogen bonding. When coiled, it can accommodate iodine molecule that gives characteristic blue-violet color. The blue-violet color is due to the formation of amylose- iodine complex. This color test is sensitive enough to detect even minute amounts of starch in solution. b. Amylopectin - component of starch, other than amylose - highly branched chain of glucose units; with (1→4) and (1→6) linkages shown below: Figure 31. Repeating structure of amylopectin. Amylopectin molecule may contain many thousands of glucose units with branch points occurring about every 25-30 units. This branching is determined by branching enzymes which leave each chain with up to 30 glucose residues. Due to branching, the helical structure of amylopectin is disrupted, so instead of the deep blue-violet color amylose gives with iodine, amylopectin produces a less intense reddish brown. Page 20 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Partial hydrolysis of starch produces polysaccharides of intermediate sizes, called dextrins. Dextrins have characteristic stickiness when wet, that is why they are used as adhesives, as binders to hold pills and tablets together, and as pastes. They are more easily digested than starch and are therefore used extensively in the commercial preparation of infant foods. The complete hydrolysis of starch yields, in successive stages, glucose: starch → dextrins → maltose → glucose In the human body, several enzymes known collectively as amylases degrade starch sequentially into usable glucose units. 2. Glycogen Glycogen is the storage form of glucose in animals. In humans, glycogen is made from excess glucose and stored primarily in the cells of the liver and skeletal muscle. It has a structure similar to amylopectin, but is more extensively branched (with more ⍺(1⟶6) linkages), and compact than starch. Its highly branched structure permits rapid glucose release especially when glucose availability is limiting. e.g., in muscle during exercise. Glycogen is found in the form of granules in the cytoplasm in many cell types. It exists in cells as spherical or globular structure. The structure of glycogen and the two types of linkages are shown below: Schematic two-dimensional cross- sectional view of glycogen: a core protein of glycogenin is surrounded by vranches of glucose units. The entire globular granule may contain around 30,000 glucose units. Figure 32. Repeating structure of glycogen (top). Cross-sectional view of glycogen (bottom), adapted from Haggstrom, M. (2014). Glycogen structure. WikiJournal of Medicine, 1(2), 8. Page 21 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Laboratory Activity: Experiment 7: Isolation of Glycogen from Chicken Liver B. Structural Polysaccharide 1. Cellulose Cellulose, a major structural component of plant cell walls is consists of long linear chains of glucose joined in β(1⟶4) linkages. The glucose units in cellulose are linked in a linear fashion, as shown in the drawing below. Figure 33. Repeating structure of cellulose. The beta-glycoside bonds permit these chains to stretch out, and this conformation is stabilized by intramolecular hydrogen bonds. A parallel orientation of adjacent chains is also favored by intermolecular hydrogen bonds. Stability is achieved by having each glucose unit flipped or rotated 180° relative to the preceding and succeeding monosaccharide unit. The result is almost fully extended molecule. For the arrangement of fibrils, microfibrils, and cellulose in cell walls, visit this link, http://impas- itsb.blogspot.com/2013/06/historical-development-of-cellulose.html. Molecular Structure of Cellulose In the cellulose chain, the glucose units are in 6-membered rings, called pyranoses. They are joined by single oxygen atoms (acetal linkages) between the C1 of one pyranose ring and the C-4 of the next ring. The spatial arrangement, or stereochemistry, of these acetal linkages is very important. The pyranose rings of the cellulose molecule have all of the groups larger than hydrogen sticking out from the periphery of the rings (equatorial positions). The stereochemistry at carbons 2, 3, 4 and 5 of the glucose molecule are fixed; but when glucose forms a pyranose ring, the hydroxyl at C-5 can approach the carbonyl at C-1 from either side, resulting in two different conformations at C-1. In plant cell walls, approximately 36 individual cellulose molecule chains connect with each other through hydrogen bonding to form larger units known as elementary fibrils, which are packed into larger microfibrils with 5-50 nm in diameter and several micrometers in length (https://www.intechopen.com/books/nanocrystals-synthesis- characterization-and-applications/recent-development-in-applications-of-cellulose- nanocrystals-for-advanced-polymer-based-nanocomposit). These microfibrils have disordered (amorphous) regions and highly ordered (crystalline) regions as shown in the illustration below. In the crystalline regions, cellulose chains are closely packed together by a strong and highly intricate intra- and intermolecular hydrogen-bond network , while the amorphous domains are loosely packed. The alternate flipping of glucose in the Page 22 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila cellulose molecule promotes these intra-chain and inter-chain H-bonds and van der Waals interactions among cellulose molecules, and this explains why cellulose chains are straight and rigid, and packed well in a crystalline arrangement in thick bundles, called microfibrils. When subjected to mechanical shearing and treatment with chemicals or enzymes, the loose amorphous regions of cellulose microfibrils are selectively hydrolyzed because they are more susceptible to be attacked in contrast to crystalline regions. The two distinct regions in cellulose, crystalline and amorphous are differentiated below: Figure 34. Amorphous and crystalline regions in celllulose microfibrils. Adapted from https:// www.intechopen.com/books/nanocrystals-synthesis-characterization-and-applications/ recent-development-in-applications-of-cellulose-nanocrystals-for-advanced-polymer- based-nanocomposit Cellulose – Not a Human Nutrient Enzymes that hydrolyze ⍺-linkages in starch cannot hydrolyze the β-linkages in cellulose. Only a few organisms possess cellulase, the enzyme that hydrolyzes β(1⟶4) linkages in cellulose. Ruminants (cows, goats) and termites have within their digestive tracts microorganisms that produce cellulase. All wood fungi, likewise, have cellulase. Since human’s digestive juices lack the enzyme cellulase, cellulose cannot be digested and absorbed. Cellulose simply passes through the digestive tract as insoluble fiber. The cellulose fiber, however, functions by: facilitating the smooth passage of food through the digestive tract by stimulating mucus secretion; decreasing absorption of glucose and cholesterol; by increasing the bulk of stool, thus, preventing constipation. 2. Chitin Chitin is the major structural component of the exoskeletons of invertebrates, such as insects and crustaceans. It also occurs in cell walls of algae, fungi, and yeasts. This earth’s second most abundant carbohydrate polymer (after cellulose) is composed of units of N-acetyl-β-D-glucosamine joined by β-1,4-glycosidic bonds. The repeating unit is shown in Figure 35.The structural feature of chitin is similar to that of cellulose, but the hydroxyl group at C-2 of its glucoses is replaced by an acetylamido group. Page 23 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 35. Repeating structure of chitin. Chitin-based coatings can extend the shelf life of fruits. It is also used to make a strong and flexible surgical thread that decomposes after the wound or incision heals. 3. Alginic acid Alginic acid is a linear polymer made up of D-mannuronic acid, joined by β-1⟶4 linkages. It is the main structural polysaccharide of brown algae, and is used mainly as a food additive. C. Other homopolysaccharides 1. Pectic acid (a homopolyuronosaccharide) This polygalacturonan polysaccharide occurs mainly in cell walls and intercellular layers of all plant tissues. It is a linear structure made up of galacturonic acid linked in ⍺(1⟶4) bonds. 2. Dextran Dextran is a group of glucans made up of glucose units joined in ⍺(1⟶6) with cross linkages. It is produced by lactic acid bacteria (Lactobacillaceae) from sucrose. It is used as food additive or as a matrix in protein separation technique such as gel filtration chromatography. In the oral cavity, dextran forms a net-like structure that forms the matrix for plaques. The structure favors bacterial aggregation that produces acids destroying the enamel of teeth. 3. Inulin Inulin is a polymer of D-fructose (fructosans), joined in β(1⟶2) glycosidic linkage. It occurs in bulbs of onion and garlic, but not utilized by the body. Instead, it is used for assessing kidney function through measurement of glomerular filtration rate (GFR). II. Heteropolysaccharide A. Glycosaminoglycan Glycosaminoglycans or mucopolysaccharides are long linear polysaccharides consisting of repeating disaccharides in which one of the monosaccharide units is an amino Page 24 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila sugar and one (or both) of the monosaccharide units contains at least one negatively charged sulfate or carboxyl group (e.g. D-glucuronic). The amino sugar is always either N- acetylglucosamine or N-acetylgalactosamine. In some glycosaminoglycans, one or more of the hydroxyls of the amino sugar are esterified with sulfate. The glycosaminoglycans are the main constituents of the mucins, connective tissues and the extracellular matrix. They are normally found linked with proteins as a proteoglycan structure. The structures of the repeating disaccharide units of glycosaminoglycans are shown below: Figure 36. Structures of glycosaminoglycans. Adapted from Lehninger Principles of Biochemistry (4th ed) by Nelson, D.L. & Cox, M.M. (2004). 1. Hyaluronic acid contains alternating residues of D-glucuronic acid and N-acetylglucosamine, joined in β(1⟶3) & β(1⟶4) glycosidic lnkages. forms clear, highly viscous solution that serves as lubricants in the synovial fluid of joints and give the vitreous humor of the vertebrate eye its jelly-like consistency an essential component of the extracellular matrix of cartilage and tendons; contributes to their tensile strength and elasticity Consist of as many as 25,000 disaccharide units, with molecular weights of up to 107 2. Heparin has the highest net negative charge of the glycosaminoglycans shown above. a natural anticoagulant substance. binds strongly to antithrombin III (a protein involved in terminating the clotting process) and inhibits blood clotting. Page 25 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila 3. Chondroitins and keratan sulfate found in tendons, cartilage, and other connective tissue 4. Dermatan sulfate As its name implies, is a component of the extracellular matrix of skin B. Proteoglycans Proteoglycans are macromolecules on the cell surface or extracellular matrix in which one or more glycosaminoglycan chains ((≈95%) are joined covalently to a membrane protein or a secreted protein. They serve the following functions: act as lubricants; give tensile strength and elasticity to soft tissues; act as shock absorber of hard and brittle structures, such as bone; provide passageways in extracellular matrix; and help in disposing of unwanted cellular artifacts as well as pathogens. Figure 37. Structure of a proteoglycan from an integral protein (left). Proteoglycan structure from the extracellular matrix (right). Adapted from Lehninger Principles of Biochemistry (4th ed) by Nelson, D.L. & Cox, M.M. (2004). The above structure is a very flexible or loose structure due to the fact that covalent linkage is only between proteins and the mucopolysaccharides. The mucopolysaccharides are autonomous of each other, interacting only via noncovalent bonds. These are weaker interactions that can readily form, break, then reform again. Thus, because the interactions are weak, the molecules are allowed to expand to variable sizes. When water binds to the hydroxyl, sulfates and carboxyl groups, the structure increases in size several fold, sometimes reaching 500X its original size. Because mucopolysaccharides are heavily negatively charged due to the presence of sulfates and carboxyl groups, they could easily bind positively charged groups in proteins. Membrane proteins and surface antigens in viruses and bacteria, as well as hydrolytic products of antigen-processing T cells (APC) can Page 26 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila therefore interact with the mucopolysaccharides facilitating their expulsion from tissues. It is thus one of the host’s responses and defense to infection. Below is a proteoglycan structure that shows the trisaccharide bridge, a typical trisaccharide linker (blue) that connects a glycosaminoglycan (e.g. chondroitin sulfate) to a serine residue (pink) in the core protein. The xylose residue at the reducing end of the linker is joined by its anomeric carbon to the hydroxyl of serine residue. In proteoglycans, as well as glycoproteins, the sugars are attached to proteins via serine or threonine, and to a lesser extent, tyrosine (O-linked), or lysine, asparagine and glutamine (N-linked) Figure 38. Trisaccharide bridge in a proteoglycan. Adapted from Lehninger Principles of Biochemistry (4th ed) by Nelson, D.L. & Cox, M.M. (2004). C. Peptidoglycans Bacteria use cell wall to protect their cellular contents. Cell wall helps by providing protection from osmotic lysis, toxic chemicals, and pathogens. Normally, bacteria exhibit high internal osmotic pressures, and frequently encounter variable, often hypotonic exterior conditions that lead to their swelling. Swelling is prevented (or in some cases shrinkage due to variations in solution osmotic strength) through their rigid cell walls. Peptidoglycans are the major components of bacterial cell wall. They are peptide joined heteroglycan chains composed of alternating GlcNAc and N-acetylmuramic acid (MurNAc) connected by β-1,4 linkages. The peptide subunits usually contain four alternating L- and D-amino acids, connected to the glycan stands via the lactyl groups of the N-acetylmuramic acid residues. Cross- linking of the glycan strands generally occurs between the carboxyl group of D-Ala at position 4 and the amino group of the lysine at position 3, either directly or through a short peptide bridge. The GlcNAc (β-1⟶4) MurNAc strands are covalently connected by a pentaglycine bridge through the epsilon amino group of the tetrapeptide Lys on one strand and the D-Ala of a tetrapeptide on another strand. Below is a pictorial presentation of peptidoglycan structure. Page 27 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 39. Structure of a typical peptidoglycan. https:// www.pnas.org/content/105/30/10348/tab-figures-data Figure 40. Pictorial representation of the peptidoglycan structure. https:// www.creative-proteomics.com/services/peptidoglycan-structure- analysis.htm Gram (+) bacteria have a thick cell wall, approximately 2 nm, with multiple layers of peptidoglycan, surrounding the bacterial plasma membrane, whereas, Gram (-) bacteria have a thin cell wall, consisting of single layer of peptidoglycan sandwiched between the inner and outer lipid bilayer membranes. Below is a comparison of the cell envelopes of gram-positive and gram-negative bacteria. Figure 41. Structures of the cell walls of Gram (+) and Gram (-) bacteria. Adapted from CNX OpenStax/Wikimedia Commons/CC BY-SA 4.0 The peptidoglycan layer is responsible for reacting with crystal violet giving the characteristic blue violet color with gram stain of gram (+) bacteria. Gram (-) bacteria have a protective lipopolysaccharide layer which does not dissolve hydrophilic dyes such as crystal violet, thus, preventing it from interacting with the peptidoglycan layer of the bacterial cell wall. Page 28 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila The peptidoglycan layers of many gram-positive bacteria are densely functionalized with anionic glycopolymers called wall teichoic acids (WTAs). Teichoic acid is often attached to the C6 of MurNAc. It is a polymer of glycerol or ribitol to which alternative GlcNAc and D- Ala are linked to the middle C of the glycerol. Multiple glycerols are linked through phosphodiester bonds. Teichoic acids often make up 50% of the dry weight of the cell wall, and present a foreign (or antigenic) surface to infected hosts. These often serve as receptors for viruses that infect bacteria (called bacteriophages). Below is the structure of teichoic acid. Figure 42. Representative chemical structure of wall teichoic acids. Adapted from Swoboda, J. G., Campbell, J., Meredith, T. C., & Walker, S. (2010). Wall teichoic acid function, biosynthesis, and inhibition. Chembiochem, 11(1), 35-45. Figure 43. Structure of bacterial lipopolysaccharides. Adapted from Lehninger Principles of Biochemistry (4th ed) by Nelson, D.L. & Cox, M.M. (2004). Page 29 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila D. Lipopolysaccharide in the Outer Membrane of Gram-negative Bacteria The outer membrane of Gram (-) bacteria is consists of a lipid group (anchored in the outer membrane) joined to a polysaccharide made up of long chains with many different and characteristic repeating structures. These many different unique units determine the antigenicity of the bacteria; that is, animal immune systems recognize them as foreign substances and raise antibodies against them. As a group, these antigenic determinants are called the O antigens. See Figure 43. E. Glycoproteins Glycoproteins contain carbohydrate residues in addition to the polypeptide chain. Some of the most important examples of glycoproteins are involved in the immune response, for example, antibodies, which bind to and immobilize antigens (the substances attacking the organism). Carbohydrates also play an important role as antigenic determinants, e.g. blood group antigenic determinants, A and B. Known glycoproteins also include structural proteins, enzymes, membrane receptors, and transport proteins. The oligosaccharide components of glycoproteins exhibit great variability in sugar sequence and composition. Proteins with identical amino acid sequence but different oligosaccharide composition are called glycoforms. In cell membranes, glycoproteins are concentrated mainly at the outer half of the membrane bilayer where they comprise the glycocalyx of the cell. Glycosylation can affect the physical and chemical properties of proteins, altering solubility, mass, and electrical charge. Carbohydrate moieties have been shown to stabilize protein conformations and protect proteins against proteolysis. How are the oligosaccharides joined to proteins in the glycoproteins? The carbohydrate units, mostly N-acetylglucosamine and N-acetylgalactosamine, are covalently linked to polypeptide chains via the hydroxyl groups of serine, threonine or hydroxylysine residues (in O-linked saccharides); or via the amide N of Asn (N-linked), as shown in Figure 44. Figure 44. O- and N-linkage for protein connections. Page 30 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila O-linked oligosaccharides have a glycosidic bond to the hydroxyl group of serine (or threonine) residues. N-linked oligosaccharides have N-glycosyl bond to the amide nitrogen of asparagine residue. 1. O-linked Saccharides Often found in cell surface glycoproteins and in mucins Mucins – large glycoproteins that coat and protect mucous membranes in the respiratory and gastrointestinal tracts in the body A unique family of O-linked glycoproteins permits fish to live in the icy seawater of the Arctic and Antartic regions (-1.90℃) 2. N-linked Saccharides Found in many different proteins, including immunoglobulins G and M, ribonuclease B, ovalbumin and peptide hormones The Blood Group Antigens Oligosaccharide portions of glycoproteins act as antigenic determinants. Among the first antigenic determinants discovered were the blood group substances. In the ABO system, individuals are classified according to four blood types: A, B, AB, and O. Below are the structures of blood-group antigenic determinants: Figure 45. Structures of the ABO blood groups. At the cellular level, the biochemical basis for specific blood type is a group of relatively small membrane-bound carbohydrates (oligosaccharides), called blood group antigens, denoted ABO antigens. These oligosaccharides are found on membrane surfaces of red blood cells and are either O-linked to proteins or covalently bonded to lipids forming glycolipids. They elicit an antibody response causing agglutination of the blood of individuals who do not have the specific oligosaccharide in their erythrocyte membrane. For example, an individual whose erythrocyte contains the oligosaccharide, denoted A antigen Page 31 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila will elicit an immunologic response to the blood group type B which will not be recognized as self. Type O antigen will not elicit antibody reaction in individuals with type A and B surface antigens since the 4-saccharide cluster is common to all three antigens. Exercises 1. Indicate whether each of the following pairs of sugars consists of anomers, epimers, or an aldose-ketose pair: A. D-glyceraldehyde and dihydroxyacetone B. D-glucose and D-mannose C. D-glucose and D-fructose D. ⍺-D-glucose and β-D-glucose E. D-ribose and D-ribulose F. D-galactose and D-glucose 2. Draw the open chain and two anomers of D-galactopyranose, and predict the most stable form. Explain your answer. 3. Identify the following four sugars: 4. Why is mannose most frequently found as ⍺-anomer? 5. How many different oligosaccharides can be made by linking one glucose, one mannose, and one galactose? Assume that each sugar is in its pyranose form. Compare this number with the number of tripeptides that can be made from three different amino acids. 6. What structural property of a sugar enables it to reduce oxidizing agents? Based on this property, determine which of the following are reducing sugars? A. Ribofuranose C. Lactose E. Sucrose B. Methylglucose D. Amylose F. Cellulose 7. Use the following figure of raffinose to answer the following questions: Page 32 of 33 Module on Carbohydrates CHEM 41 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila A. Is raffinose a reducing sugar? Explain. B. What are the monosaccharides that compose raffinose? C. β-galactosidase is an enzyme that will remove galactose residues from an oligossaccharide. What are the products of β-galactosidase treatment of raffinose? 8. What products can be formed from exhaustive methylation of the β-anomer of lactose? 9. A trisaccharide obtained from the partial hydrolysis of amylopectin showed two glycosidic linkages. A. Draw the structure of the trisaccharide B. If the trisaccharide is to be exhaustively methylated and subsequently hydrolyzed with an acid, how many different methylated products will be obtained? Draw their structures. C. Can an aqueous solution of the trisaccharide precipitate Cu as Cu2O? If it can, encircle the potential carbonyl carbon(s) in the trisaccharide. D. Can the trisaccharide exist in different anomeric forms? If yes, draw their structures. 10. After exhaustive methylation and hydrolysis, a polysaccharide yielded equimolar amounts of 2,3,4-tri-O-methylglucose and 2,3,6-tri-O-methylglucose. The polysaccharide has one reducing terminus. Draw the structure of the polysaccharide. 11. Carbohydrates in the membranes are in the form of glycolipids and glycoproteins. Given the following peptide, attach glucose residues to the peptide shown below: M-R-V-I-L-S-C-K-R-V-M-C-N-E-H-G-G-A-T-H 12. Discuss the relationship of the structure of each of the following carbohydrates to the function indicated. A. Hyaluronic acid is a major component of the synovial fluid which lubricates joints B. Cellulose is a major component of the rigid cell walls of plants C. Amylose and amylopectin are storage forms of D-glucose in plants D. Glycogen is the storage polysaccharide in animal cells 13. Why is type AB blood considered a universal acceptor (i.e. does not elicit antibody reaction with blood group types A,B, and O? References: 1. Principles of Biochemistry, Lehninger 2. Biochemistry, Stryer 3. Biochemistry, Campbell and Farrell 4. https://www.slideshare.net/shainamavreenvillaroza/chem-45-biochemistry-carbohydrates 5. https://www.slideshare.net/ashokktt/carbohydrate-chemistry-37049261 6. https://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/carbhyd.htm 7. http://www.allometric.com/tom/courses/protected/MCB6/ch10/10-20.jpg 8. https://www.intechopen.com/books/nanocrystals-synthesis-characterization-and- applications/recent-development-in-applications-of-cellulose-nanocrystals-for-advanced- polymer-based-nanocomposit Page 33 of 33