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Carbohydrates Lectures for Medical and Dentist Students 1 st Year Course, Spring Semester, 2009

Carbohydrates Lectures for Medical and Dentist Students 1 st Year Course, Spring Semester, 2009 Semmelweis University, Department of Medical Biochemistry Presented by dr. András Hrabák, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry. CARBOHYDRATES

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Carbohydrates Lectures for Medical and Dentist Students 1 st Year Course, Spring Semester, 2009

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  1. Carbohydrates Lectures for Medical and Dentist Students 1st Year Course, Spring Semester, 2009 Semmelweis University, Department of Medical Biochemistry Presented by dr. András Hrabák, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry

  2. CARBOHYDRATES What are carbohydrates ? - Polyhydroxy-oxo compounds Grouping principles: 1. According to the size of the molecules (i.e. the number of units) - monosaccharides, oligosaccharides, polysaccharides 2. According to the number of carbon atoms in monosaccharides - trioses, tetroses, pentoses, hexoses, heptoses etc. 3. According to the carbonyl group - aldoses (aldehyde group), ketoses (keto group) Significances: 1. Energy storage - homopolysaccharides 2. Structure material - heteropolysaccharides, cellulose 3. Intermediates in the metabolism - smaller sugars 4. Miscellaneous Structural characteristics: aldehyde group is always at the end of the molecule (C-1); keto group is theoretically positioned anywhere in the middle of the chain, however, in biologically important ketoses it is at C-2 position

  3. Monosaccharides: O = C - H CH2OH O = C - H O = C - H CH2OH      H - C - OH C = O H - C - OH HO - C - H C = O      CH2OH CH2OH H - C - OH H - C - OH H - C - OH    CH2OH CH2OH CH2OH D-glyceraldehyde dihydroxyacetone D-erythrose D-treose D-erythrulose aldotriose ketotriose aldotetroses ketotetrose Chirality and chiral centers in monosaccharides: The number of possible chiral isomers of a monosaccharide can be calculated by 2n where n is the number of chiral centers. A monosaccharide belongs to the D-series if the configuration of the chiral center closest to the primary alcoholic OH-group is identical to that of the chiral carbon of the D-glyceraldehyde.

  4. Explanation: if the groups written over the indicated chiral carbon are oxidized and eliminated as CO2, except the neighbouring, whose -OH is oxidized to aldehyde, the product is glyceralderhyde containing only one chiral carbon. Therefore, each bigger monosaccharide can be decomposed into D- or L- glyceraldehyde, whose chiral -OH group corresponds to the -OH neighbouring to the primary alcoholic group (or, generally to the OH in the largest distance to the aldehyde/keto group). R(S) nomenclature of monosaccharides: more complicated, not used frequently, because each chiral center must be characterized separately (too long names, etc.) Biologically important monosaccharides usually belong to D-series (exceptions: L-fucose, L-iduronate) Optical rotations: its direction is independent on the D- or L-configuration e.g. D-dlucose is dextrorotatory, D-fructose is levorotatory. Enantiomers: complete mirror images, in which each chiral centers are of different configurations, e.g. D- and L-glucose Diastereomers: partial mirror images in which the deciding OH-group is usually of D-configurations, but other hydroxyl groups may be in different positions.

  5. Biologically important pentoses and hexoses: Pentoses: O = C - H O = C - H CH2OH CH2OH     H- C - OH H - C - OH C = O C = O     H- C - OH HO- C - H H- C - OH HO- C - H     H- C - OH H- C - OH H- C - OH H- C - OH     CH2OH CH2OH CH2OH CH2OH D-ribose D-xylose D-ribulose D-xylulose Hexoses: O = C - H O = C - H O = C - H CH2OH     H- C - OH H - C - OH HO- C - H C = O Epimers:     sugar pairs in HO- C -H HO- C - H HO- C - H HO- C - H which only one     chiral center has H- C - OH HO- C - H H- C - OH H- C - OH different     configuration, H- C - OH H- C - OH H- C - OH H- C - OH e.g. glucose and     galactose CH2OH CH2OH CH2OH CH2OH D-glucose D-galactose D-mannose D-fructose

  6. Mutarotation, formation of cyclic monosaccharides (hemiacetals): It is based on the reaction of the aldehyde or keto group with a hydroxyl group appropriately positioned to form a five- or six-membered cyclohemiacetale or cyclohemiketale ring. Reaction type is an intramolecular nucleophilic addition. HO - C - H O = C - H H - C - OH    H - C - OH H - C - OH H - C - OH    HO - C - H O HO - C - H HO - C - H O    H - C - OH H - C - OH H - C - OH    H - C H - C - OH H - C    CH2OH CH2OH CH2OH  -D-glucose (67 %) open chain form  -D-glucose (33 %) Consequences: the appearance of a new chiral center at C-1 position and the existence of two new D-glucose isomers (anomers) which are in equilibrium with each other and with the open chain form. Optical rotation is changed during the process (e.g. dissolution of glucose in water), this is the explanation of the name „mutarotation”.

  7. Pyranose and furanose rings: stable steric structures are possible if the number of the ring atoms is 5 or 6. As pyrane is a six-membered ring containing one oxygen, while furane is similar with a five-membered ring, the cyclohemiacetale or cyclohemiketale sugar rings are called as „pyranose” (6-membered) or „furanose” (5-membered) structures. Pyranose is characteristic of aldohexoses while furanose is typically found in pentoses and ketohexoses. Anomers:Chiral isomers differing only in the position of carbonyl-derived hydroxyl group (glycosidic hydroxyl). If its position is identical to the D-configuration of the determining carbon of the projected formula, it is called as –anomer, while in the case of identity with L-configuration, it is –anomer. Anomers are in equilibrium and they can be transformed freely to each other, differently from other chiral isomers. Representation rules of cyclic sugars: 1.The oxygen atom of the hemiacetal/ketale ring is written into the upper right position of pyranose or in the uppest position of furanose rings. 2. Hydroxyl groups written on the right side of the carbon chain in open-chain model, have to be drawn below the ring plane. 3. The CH2OH group should be written over the ring plane in the case of D- sugars (and opposite for L-isomers).

  8. Hemiacetal ring structures of - and -D-glucose: -D-glucose -D-glucose The structures above cannot show the possible conformations. -D-glucopyranose (1-OH axial) -D-glucopyranose (1-OH equatorial) -D-glucopyranose is more stable (~63 %), because all of its hydroxyl groups are in equatorial position; equilibrium is shifted to right.

  9. Different endo-conformations of furanose rings in nucleic acids 2’-endo--D-deoxyribofuranose 3’-endo--D-ribofuranose 2’-endo--D-deoxyribofuranose is characteristic of B-DNA, 3’-endo--D-ribofuranose conformation is typical in A-DNA and in RNA.

  10. Reaction of carbohydrates I. 1. Reduction and oxidation 2. Ester formation 3. Ether formation 4. Glycoside formation 5. Isomerization 1a. Reduction of carbohydrates: aldehyde or keto group is reduced into primary or secondary alcoholic hydroxyl group, respectively. The product is called sugar alcohol (hexitol, pentitol). Glucose, or fructose are reduced to sorbitol. O = C - H CH2OH CH2OH    H - C - OH H - C - OH C = O    HO - C -H HO - C -H HO - C -H    H - C - OH H - C - OH H - C - OH    H - C - OH H - C - OH H - C - OH    CH2OH CH2OH CH2OH D-glucose D-sorbitol D-fructose

  11. Reactions of carbohydrates II. 1b. Oxidations of monosaccharides - primary alcoholic group is oxidized uronic acid - aldehyde group is oxidized aldonic acid - both terminal groups are oxidized aldaric acid O = C - H O = C - H COOH    H - C - OH H - C - OH H - C - OH    HO - C - H HO - C - H HO - C - H    H - C - OH H - C - OH H - C - OH    H - C - OH H - C - OH H - C - OH    COOH CH2OH CH2OH D-glucuronic acid D-glucose D-gluconic acid Significances: glucuronic acid is involved in biotransformations making compounds more hydrophilic by glucuronide formation. Phosphate ester of gluconic acid is an important intermediate of pentose phosphate cycle. Uronic acids are also building blocks of heteropolysaccharides.

  12. 2. Ester formation: alcoholic hydroxyl groups can react with acids forming esters. Important reactions: intramolecular lactone formation. 3. Ether formation: alcoholic groups can react with each other forming ethers. 4. Glycoside formation: involves the participation of glycosydic hydroxyl group. Glycosydic hydroxyl group is distiguished from other hydroxyl groups. They are derived from an aldehyde/keto group by the formation of an intramolecular hemiacetal or hemiketale in a reversible reaction. Therefore, glycosydic OH-group is a hidden aldehyde/keto group, which is more reactive compared to other hydroxyl groups. This group can form special glycosidic ethers (glycosides) or esters. Oligo- and polysaccharides are also glycosides. D-gluconic acid and its lactone form (on the right)

  13. Glycoside formation: e.g. –D-glucose reacting with another –D-glucose leads to the formation of maltose: Two glucose molecules react with each other; one of them (showing on the left side) participates in the reaction with its glycosidic –OH group, forming a glycosidic ether or glycoside. The other glucose (right side) participates with an ordinary secondary alcoholic hydroxyl group. The product is called glycoside, also considered as an acetale (from chemical aspect), which is a disaccharide, this one is called maltose. Polysaccharides are also formed via glycosidic bonds between monosaccharide units.

  14. Reactions of sugars with acids and bases 1. Strong acids cause the dehydration of pentoses to furfural and that of hexoses to hydroxymethylfurfural: 2. In basic environment, monosaccharides may be isomerized. During this process enolate anion is formed by proton movement: H - C = O | HO - C - H mannose | R H | H - C = O H - C - OH H - C - OH | || | H - C - OH C - OH C = O glucose | | | fructose R R ene-diol R

  15. Deoxy sugars: H - C = O H - C = O H - C = O    CH2 HO - C - H CH2    H - C - OH H - C - OH HO - C - H    H - C - OH H - C - OH H - C - OH    CH2OH HO - C - H H - C - OH   CH3 CH2OH 2-deoxy-D-ribose L-fucose 2-deoxy-D-glucose Amino sugars (in natural amino sugars, amino group is found in position 2) H - C = O H - C = O H - C = O    H - C - NH2 H - C - NH2 NH2 - C - H    HO - C - H HO - C - H HO - C - H    H - C - OH HO - C - H H - C - OH    H - C - OH H - C - OH H - C - OH    CH2OH CH2OH CH2OH D-glucosamine D-galactosamine D-mannosamine

  16. N-acetylated sugar derivatives: H - C = O H - C = O COOH    H - C - NH - CO-CH3 H - C - NH - CO - CH3 C = O    HO - C - H CH3 - CH - O - C - H CH2     H - C - OH HOOC H - C - OH CH3 H - C - OH     H - C - OH H - C - OH CO - NH - C- H    CH2OH CH2OH OH - C - H N-acetyl-D-glucosamine N-acetyl-D-muramic acid  Muramic acid: N-acetyl-D-glucosamine bearing lactic acid H - C - OH by an ether bond at C-3 position  Sialic acid: N-acetyl-mannosamine connected to pyruvic acid H - C - OH sialic acid at C-1 position  Function of deoxy and amino sugars: CH2OH Deoxyribose is a component of DNA, fucose is found in the carbohydrate moieties of glycoproteins; 2-deoxy-D-glucose is used in research. Acetylated amino sugars are the components of heteropolysaccharides,glycoproteins, blood group and histocompatibility antigens

  17. Functions of sugar alcohols and acids: Ribitol is a component of riboflavin (vitamin B2). Sorbitol is used as a sweeting agent instead of glucose or sucrose (diabetes!) D-glucuronic acid is important in biotransformation and together with other uronic acids are components of mucopolysaccharides. L-ascorbic acid is Vitamin C. D-glyceric acid is important in glycolysis and its bis-phosphate ester is a regulator of oxygen binding of hemoglobin. Sugar phosphates (phosphate esters) H - C = O CH2 - O- PO3H2 COOH COOH     H - C - OH C = O H - C - OH H - C - PO3H2     HO - C - H HO - C - H CH2- O-PO3H2 CH2 - O - PO3H2   3-phosphoglycerate 2,3-bisphosphoglycerate H - C - OH H - C - OH   Functions of phosphate esters: H - C - OH H - C - OH important intermediates of glycolysis and   other processes of carbohydrate metabolism CH2 - O - PO3H2 CH2 - O - PO3H2 glucose-6-phosphate fructose-1,6-bisphosphate

  18. Sugar alcohols: CH2OH CH2OH CH2OH CH2OH | | | | H - C - OH H - C- OH H - C - OH H - C - OH | | | | H - C - OH HO - C - H H - C - OH CH2OH | | | H - C - OH H - C - OH CH2OH | | CH2OH H - C - OH | CH2OH D-ribitol D-sorbitol D-erythritol glycerol Uronic and aldonic acids: H - C = O O = C COOH | | | H - C - OH HO - C H - C - OH | || O | HO - C - H HO - C CH2OH | | H - C - OH H - C | | H - C - OH HO - C - H | | COOH CH2OH D-glucuronic acid L-ascorbic acid (lactone) D-glyceric acid

  19. Disaccharides I. They are composed of monosaccharides by glycosidic bondings containing 2-10 monosaccharide units maltose cellobiose Maltose and cellobiose are reducing disaccharides composed of two -D-glucoses and -D-glucoses, respectively, via 1-4 glycosidic bonds.

  20. Disaccharides II. Lactose (milk sugar) and saccharose (sucrose, cane sugar): lactose saccharose (sucrose) Lactose is a reducing disaccharide composed of a -D-galactose and a -D-glucose via 1,4-glycosidic bond. Sucrose is a non-reducing di- saccharide composed of an -D-glucose and a -D-fructose via a 1,2- glycosidic bond.

  21. Disaccharides III. Reducing and non-reducing disaccharides: If a saccharide contains a free aldehyde or glycosidic (hidden aldehyde) group, it can reduce various reactants, e.g. Cu2+ or Ag+- ions. Sugars lacking these free aldehyde or glycosidic hydroxyl groups fail to reduce these ions. In non-reducing disaccharides, their glycosidic bonding has been formed with the participation of both glycosidic hydroxyl groups. Consequently, non-reducing disaccharides nor did show mutarotation, also requiring the presence of free aldehyde (or glycosidic -OH) group. Biological importance of disaccharides: Lactose: the most abundant disaccharide in the milk. Sucrose (saccharose): the most important sugar in nutrition in the civilized world. Maltose and cellobiose are structural units and degradation products of starch and cellulose, respectively.

  22. Polysaccharides (glycans) Polysaccharides contain more than 10 monosaccharide units linked by glycosidic bonds. They may be homopolysaccharides composed of one type of monosaccharides or hetero- polysaccharides composed of more than one type of monosaccharides and their units are di- or oligosaccharides. Biologically important homopolysaccharides: Name Monosaccharide unit Linkage Found in Starch -D-glucose amylose -1,4 plants amylopectine -1,4; -1,6 plants Glycogene -D-glucose -1,4; -1,6 liver, muscle Cellulose -D-glucose -1,4 plants Inulin -D-fructose -1,6 plants Dextrane -D-glucose -1,6 bacteria Significance: Starch and glycogen are energy stores in plants and animals, respectively, degraded by amylase and phosphorylase into glucose and glucose-1-phosphate units, respectively. The -1,6-bonding is splitted by -1,6-glycosidases. Cellulose is the most important structural polysaccharide in the plant cell wall. It is degraded by cellulase produced by bacteria and snails only. Inulin is used to determine the blood volume, dextrane is used for gel filtration after sulfation with H2SO4.

  23. Amylose - disaccharide unit of -D-glucoses, 1,4-bonding Amylopectin and glycogen - tetrasaccharide unit of -D- glucoses including a branching point, 1,4 and 1,6-bonding Cellulose - trisaccharide unit of -D-glucoses, 1,4-bonding

  24. Important heteropolysaccharides Name Major monosaccharides Linkage kDa size Found in Hyaluronic acid D-glucuronic acid and -1,3; -1,4 3-8000 synovial fluid N-acetyl-D-glucosamine cartilage, skin Chondroitin D-glucuronic acid and -1,3; -1,4 5-50 cornea, bone N-acetyl-D-galactosamine vascular wall, skin - sulfate A 4-sulfate ester - sulfate C 6-sulfate ester Dermatan sulfate L-iduronic acid and -1,3; -1,4 15-40 skin, heart N-acetyl-D-galactosamine 4-sulfate ester vascular wall Keratan sulfate D-galactose and -1,3; -1,4 4-20 cartilage N-acetyl-D-glucosamine 4-sulfate ester cornea Heparin (sulfate) L-iduronic acid -1,4 6-25 cartilage N-acetyl-D-glucosamine 2-sulfate ester heart , muscle D-glucuronic acid (6-sulfate ester) mast cell, liver Bacterial cell wall N-acetyl-muramic acid -1,4 bacterial polysaccharide N-acetyl-D-glucosamine cell wall

  25. Heteropolysaccharides I. Hyaluronic acid - disaccharide unit, -1,3-bonding in the unit Chondroitin-4(6)-sulfate - disaccharide unit, -1,3-bonding in the unit

  26. Dermatan sulfate - -1,3-bonding

  27. Keratan sulfate I; in keratan sulfate II N-acetyl D-galactosamine is instead of D-galactose - disaccharide unit, -1,3-bonding

  28. Heparin; disaccharide unit, -1,4,-bonding

  29. Glycoproteins and proteoglycans Covalent conjugates of proteins and carbohydrates. In glycoproteins, the protein part is bigger, while in proteoglycans the size of the poly- saccharide is definitive. They are different in certain aspects: Proteoglycan Glycoprotein Found in cartilage, bone membranes, body fluids carbohydrate glycosaminoglycan oligosaccharide monosaccharide units/molecule > 50 < 25 repeating unit disaccharide no branching no yes hexuronic acid found not found The carbohydrates are linked to protein via special amino acid side chains. The most frequent glycopeptide bonds are the N-glycosidic bond via asparagine side chains and the O-glycosidic bonds via serine or threonine residues. The most frequent sugars in glycoproteins are mannose, glucose, galactose, N-acetylated hexosamines, sialic acid, L-fucose. Proteoglycans contain heteropolysaccharides.

  30. Glycoproteins and proteoglycans II. In the figure below the two specific glycopeptide bonds are shown. In bacterial cell walls, where the muramic acid has a lactic side chain which can be esterified, a tetrapeptide is linked to this part of the molecule (L-Ala-D-Glu-L-Lys-D-Ala) and the peptidoglycan chains are linked to each other by pentaglycine bridges between L-Lys -NH2 side chains and D-Ala COOH terminals (by amide bondings)

  31. Glycolipids and lipopolysaccharides Covalent conjugates of lipids and carbohydrates. The most known example is the ABO blood group system where a penta- or hexa-saccharide unit is linked to the sphingosine part of a ceramide and the specificity is determined by the composition of the carbohydrate moiety. Lipopolysaccharides (LPS) are known as bacterial endotoxins. They may cause serious septic shock because LPS induces the inducible nitric oxide synthase enzyme in various cells resulting in a high NO level causing a dramatic decrease of blood pressure leading to death. LPS is also involved in the initiation of inflammatory responses. Salmonella LPS lipid A

  32. Blood group antigens; AB0 system, 0 antigen right down

  33. Analysis of carbohydrates • Classical protocol: • Isolation and purification • Polysaccharide or not ? – positive Lugol reaction indicates starch or glycogen • If not, disaccharide or not ? – positive Barfoed probe suggests monosaccharide or • reducing disaccharide. Maltose and lactose are distinguished by their different • fermentation. • If monosaccharide, pentose or hexose – pentose is detected by Bial-orcin reaction • For hexose, aldose, or ketose – Seliwanoff-reaction positivity indicates fructose • Aldohexose – glucose and galactose are fermented differently • More detailed analysis is possible using more sophisticated chemical methods • including various hydrolytic processes, methylation, osazone formation. • Recent protocol: Gas chromatography, HPLC and the study of various bondings by • spectroscopic methods. These are chemical methods. • Blood glucose content is a very important indicator of metabolic homeostasis. It can • be measured by a kit based on the specific oxidation of glucose by glucose oxidase • enzyme. This reaction requires FAD coenzyme, which is reoxidized forming peroxide. • Peroxide is removed by peroxidase enzyme, using a chromofor substrate, finally the • oxidation of the substrate is accompanied by a color formation measured by spectro- • photometer. Using a glucose standard with known glucose concentration, the glucose • content of blood samples can be calculated. Normal range of blood sugar content is • 3.3-5.5 mM. It is strictly regulated by various hormones (details in 2nd year)

  34. Role of a hexose phosphate in the regulated reaction of glycolysis PFK-I is inhibited allosterically by ATP, which is the end product of glycolysis, activated by ADP and AMP. It is also activated by fructose- 2,6-bisphosphate, produced by PFK-II.

  35. Role of a pentose phosphates in the catabolism of glucose in liver This is a reaction of pentose phosphate pathway, an alternative glucose catabolic route, contributing to NADPH and pentose synthesis.

  36. Role of a sugar phosphate in the synthesis of a sugar nucleotide

  37. ATTENTION! The material of carbohydrate lectures will be presented onto the website of the Department of Medical Biochemistry. You may try to read it and to save it onto your own pendrive after searching the following website: www.biokemia.sote.hu, for students Medical Chemistry II. authorized pages, username: file; Password: open2; In the case of any troubles write an e-mail to Dr. István Léránt to the address of li@puskin.sote.hu Filename: carblec.ppt Software needed: Office/PowerPoint Recommended material: Lehninger book, list of carbohydrate structures created by Dr. Zsolt Rónai. Have a good learning, dr. A. Hrabák

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