Lecture 13 General characteristic of the carbohydrates their

Lecture № 13 General characteristic of the carbohydrates, their sources of getting, properties, qualitative and quantitative analysis, storage and usage. Tannins. prepared: assist. Logoyda L. S.

Carbohydrates are carbon compounds that contain large quantities of hydroxyl groups. The presence of the hydroxyl groups allows carbohydrates to interact with the aqueous environment and to participate in hydrogen bonding. Carbohydrates can combine with lipid to form glycolipids or with protein to form glycoproteins. They have a wide range of functions including providing a significant fraction of the energy in the diet of most organism , acting as a storage form of energy in the body and serving as cell membrane components. Also carbohydrates serve as a structural component of many organisms including cell walls of bacteria. Carbohydrates serve as metabolic intermediate ( e. g Glucose 6 phosphate, fructose 1, 6 diphosphate). Ribose , deoxyribose play a major role in the synthesis DNA and RNA. all life activities are dependent upon carbohydrates. When insufficient carbohydrates are available from the diet, the body converts fat reserves to carbohydrates for its use, and amino acids are utilized as carbohydrates instead of being used to make body protein. Carbohydrates, along with proteins and fats, comprise the major components of living matter and are used for maintenance of cellular functional activities and as reserve and structural materials for cells

• Carbohydrates with an aldehyde group are called aldoses where • • • those with a keto group are called ketoses. For example , glyceraldehyde is an aldose, whereas, dihydroxyacetone is a ketose. Disaccharides: contain two monosaccharides units. Maltose , sucrose Oligosaccharides : contain from three to about 12 monosaccharides units. For example, Blood group antigens. Polysaccharides : contain more than 12 monosaccharides units and can be hundreds of sugar units in length. Starch , cellulose • All carbohydrates can be hydrolyzed (broken down) into two or • • more monosaccharides. For further understanding of these different classifications of carbohydrates, the monosaccharides and disaccharides can be grouped together and compared with the polysaccharides. This can be done because monosaccharides and disaccharides have certain things in common. They are both water soluble. In addition, they have a sweet taste and a crystalline structure.

Simple sugars, starches and cellulose are organic compounds that have the approximate formula C(H 2 O)n, which accounts for the name carbohydrate (or hydrate of carbon) that is usually applied to this group of compounds They are not truly hydrates of carbon but are polyhydroxy (alcohol) compounds that contai an aldehyde or ketone functional group. These functional groups give the carbohydrates some of their chemical properties that will be studied in this lab.

Monosaccharides Simple sugars & cannot be hydrolysed further. They are further classified on the basis of number of carbon atoms present as well as on the presence of functional groups.

Carbon atoms Examples Functional groups Trioses (3 carbon) Glyceraldehyde Dihydroxy acetone Aldehyde (aldotriose) Ketone (Ketotriose) Tetroses (4 carbon) Erythrose Aldehyde (aldotetrose) Pentoses (5 carbon Ribose Xylulose Aldehyde(Aldopentose) Ketone (Ketopentose) Hexoses (6 carbons) Glucose Galactose Fructose Aldehyde (Aldohexose) Ketone (Ketohexose)

Disaccharides. • Contain two molecules of same or different monosaccharide units. On hydrolysis they give two monosaccharide units. Monosaccharide units are joined by glycosidic bond.

Examples Product formed Upon hydrolysis Glycosidic Linkage Sources Maltose glucose + glucose α 1 -4 Malt Lactose galactose + glucose β 1 -4 Milk Sucrose glucose + Fructose Isomaltose glucose + glucose β 1 -2 α 1 -6 Sugar cane Digestion of amylopectin

Oligosaccharides Contain - molecules of monosaccharide units. E. g. Maltotriose. (Glucose + Glucose)

The D Aldose Family

Carbohydrates

Isomers and epimers • Compounds that have the same chemical formula but have • different structures are called isomers. For example Fructose, glucose , mannose and galactose are all isomers of each other having the same chemical formula C 6 H 12 O 6. If two monosaccharides differ in configuration around only one specific carbon atom , they are defined as epimers of each other. For example, glucose and galactose are C 4 epimers, their structures differ only in the position of the hydroxyl group at C 4 ( Note , the carbons in sugar are numbered beginning at the end that contain the aldehyde or ketone group. Glucose and mannose are C 2 epimers. However, galactose and mannose are not epimers they differ in the position of the hydroxyl group at two carbon 2 and 4 and therefore, defined only as isomers. • Enantiomers: A special type of isomerism is found in the pairs of structures that are mirror images of each other. These mirror images are called enantiomers. The two members of the pair are called as D and L sugars. The majority of the sugars in human are D sugars.

Anomeric carbon • Formation of a ring results in the creation of an anomeric carbon at C 1 of an aldose or C 2 of a ketose. These structures are called the α andβ configuration of the sugar. For example α-D glucose and β-D-glucose. These two sugars are both glucose but they are anomers of each other. Enzymes are able to distinguish between these two structures and use one or the other preferentially. For example glycogen is synthesised from α-D –glucosepyranose whereas, cellulose is synthesised from β-D-glucopyranose. The cyclic α and β anomers of a sugar in solution are in equilibrium with each other and can be spontaneously interconverted in a process called mutarotation.

Optical activity The compounds having asymmetric carbon atoms can rotate the beam of plane polarized light and are said to be optically active. An isomer which can rotate the plane of polarized light to the right is called as dextrorotatory and is designated as (d) or (+) Example: D- (d)-glucose or it is also known as dextrose. While the isomer which rotates the plane of polarized light to left is known as levorotatory, and is identified as (l) or ( -). Example: D-(l)-fructose.

A levorotatory (–) substance rotates polarized light to the left. [E. g. , l-glucose; (-)-glucose] A dextrorotatory (+) substanc rotates polarized light to the right. [E. g. , dglucose; (+)-glucose] Molecules which rotate the plane of of polarized light are optically active. Most biologically important molecules are chiral, and hence are optically active. Often, living systems contain only one of all of the possible stereochemical forms of a compound. In some cases, one form of a molecule is beneficial, and the enantiomer is a poison (e. g. , thalidomide).

Polarimetry monochromator light source polarizer sample cell

Glucose cyclic formed by reaction of CHO with -OH on C 5.

Glucose • Ribose and deoxyribose

Anomers =>

Mutarotation

Reducing Sugars • If the oxygen on the anomeric carbon of a sugar is not attached to any other structure that sugar is a reducing sugar. A reducing sugar can react with chemical reagents ( Benedicts solution ) and reducing the reactive component with the anomeric carbon becoming oxidized ( Note only oxygen on the anomeric carbon determines if the sugar is reducing or non-reducing.

• Glucose : • This monosaccharide is the most important carbohydrate in human • • • nutrition because it is the one that the body fuses directly to supply its energy needs. Glucose is formed from the hydrolysis of di- and polysaccharides, including starch, dextrin, maltose, sucrose and lactose; from the monosaccharide fructose largely during absorption; and from both fructose and galactose in the liver during metabolism. Glucose is the carbohydrate found in the bloodstream, and it provides an immediate source of energy for the body's cells and tissues. Glucose is also formed when stored body carbohydrate (glycogen) is broken down for use. Fructose : Fructose, a monosaccharide, is very similar to another monosaccharide, galactose. These two simple sugars share the same chemical formula; however, the arrangements of their chemical groups along the chemical chain differ. Fructose is the sweetest of all the sugars and is found in fruits, vegetables and the nectar of flowers, as well as molasses and honey. In humans, fructose is produced during the hydrolysis of the disaccharide, sucrose.

• Galactose differs from the other simple sugars, glucose and fructose, in that it does not occur free in nature. It is produced in the body in the digestion of lactose, a disaccharide.

Disaccharides . The linkage of two monosaccharides to form disaccharides involves • • • a glycosidic bond by dehydration. Several physiogically important disaccharides are sucrose, lactose and maltose. Sucrose prevalent in sugar cane and sugar beets, is composed of glucose and fructose through an a-(1, 2)β -glycosidic bond. Lactose is found exclusively in the milk of mammals and consists of galactose and glucose in a β -(1, 4) glycosidic bond. This disaccharide is found only in milk. Human milk contains about 4. 8 g per 100 ml and cow's milk contains approximately 6. 8 g per 100 ml. When lactose is hydrolyzed it yields one unit of the monosaccharide glucose and one unit of the monosaccharide galactose. The enzyme lactase is needed to digest lactose. • Maltose : This involved C 1 and C 4 , this special bond is called 1 -4 glycosidic bond. Maltose occurs in the body as an intermediate product of starch digestion. (Starch is a polysaccharide. ) When maltose is hydrolyzed, it yields two molecules of glucose.

Sucrose =>

Lactose =>

Maltose =>

Polysaccharides • Most of the carbohydrates found in nature occur in the form of high molecular weight polymers called polysaccharides. The building blocks used to generate polysaccharides can be varied; however, the predominant monosaccharide found in polysaccharides is D-glucose. When polysaccharides are composed of a single monosaccharide building block, they are termed homopolysaccharides. Polysaccharides composed of more than one type of monosaccharide are termed heteropolysaccharides. Many polysaccharides unlike sugars are insoluble in water. Dietary fiber include polysacchaides and oligosaccharides that are resistant to digestion and absorption in the human small intestine but which are completely or partially fermented by microorganisms in the large intestine. •

• Glycogen : • Glycogen is the major form of stored carbohydrate in animals. This • • molecule is a homopolymer of glucose in a-(1, 4) linkage; it is also highly branched, with a-(1, 6) branch linkages occurring every 8 -10 residues. Glycogen is a very compact. This compactness allows large amounts of carbon energy to be stored in a small volume. Glycogen is the reserve carbohydrate in humans. Glycogen is very similar to amylopectin, having a high molecular weight and branched-chain structures made up of thousands of glucose molecules. The main difference between glycogen and amylopectin is that glycogen has more and shorter branches, resulting in a more compact shape. Glycogen is stored primarily in the liver and muscles of animals. About two -thirds of total body glycogen is stored in the muscles and about one-third is stored in the liver. Starch is the major form of stored carbohydrate in plant cells. Its structure is identical to glycogen, except for a much lower degree of branching (about every 20 -30 residues). Unbranched starch is called amylose; branched starch is called amylopectin

• Amylose : Molecules consist of 200 - 20, 000 glucose units which • • • form helix as a result of the bond angles between the glucose units. ( α- 1, 4 glycosidic linkage). Amylopectin : Differs from amylose is being highly branched. Short side chains of about 30 glucose units are attached with α 1 -6 linkage approximately every 20 - 30 glucose unit along the chain. Amylopectin molecules may contain up to 2 million glucose units. Dextran : Is a polysaccharides similar to amylopectin but the main chains are formed by α 1 -6 glucosidic linkages and the side branches are attached by α 1 -3 or α 1 -4 linkages. Dextran is an oral bacterial product that adheres to the teeth , creating a film called plague. It is used commercially as food additives. Cellulose : Is composed of chains of D-glucose unit joined by β 1 -4 glycosidic linkages. The chains are linear unbranched. It is a structural polysaccharides of plant cells. Like starch and glycogen, cellulose is composed of thousands of glucose molecules. It is the structural constituent of the cell walls of plants. Cellulose is, therefore, the most abundant naturally-occurring organic substance. It is characterized by its insolubility and its physical rigidity. This polysaccharide can be digested by cows, sheep, horses, etc. , as these animals have bacteria in their rumens (stomachs) whose enzyme systems break down cellulose molecules. Humans do not have the enzyme needed to digest cellulose, so it is passed through the digestive tract unchanged.

Amylose =>

Amylopectin

Cellulose =>

• • • Amino sugars Glucosamine, Galactosamine Sugar acids Ascorbic acid, Glucuronic acid Sugar alcohol D-Sorbitol from D-glucose D- Mannitol from D- Mannose D- Dulcitol from D- Galactose • Glycoprotein Component of cell wall and membrane • Blood group antigens: Specific oligosaccharides bound to proteins , lipids on membrane surfaces.

• Disease Conditions Related To Carbohydrate Consumption 1. Lactose intolerance 2. Galactosemia 3. Dental caries 4. Diabetes mellitus 5. Hypoglycemia.

Functions of carbohydrates • 1. Most abundant dietary source of energy • • (4 Cal/g) 2. They are precursors for many organic compounds (fats, amino acids) 3. Carbohydrates (glycoprotein, glycolipids) participate in the structure of cell membrane and cellular functions 4. Structural components of many organisms. These include the fibers (cellulose) of plant, exoskeleton of some insects and the cell wall of microorganisms. 5. Serve as the storage form of energy (glycogen) to meet the immediate energy demands of the body.

Qualitative Tests for Carbohydrates Reducing sugars are usually detected with Benedict's reagent, which contains Cu 2+ ions in alkaline solution with sodium citrate added to keep the cupric ions in solution. The alkaline conditions of this test causes isomeric transformation of ketoses to aldoses, resulting in all monosaccharides and most disaccharides reducing the blue Cu 2+ ion to cuprous oxide (Cu 2 O), a brick red-orange precipitate. This solution has been used in clinical laboratories for testing urine.

Barfoed's solution contains cupric ions in an acidic medium. The milder condition allows oxidation of monosaccharides but does not oxidize disaccharides. If the time of heating is carefully controlled, disaccharides do not react while reducing monosaccharides give the positive result (red Cu 2 O precipitate). Ketoses do not isomerize with this reagent. Carbohydrates are dehydrated in the presence of nonoxidizing acids to form furfural and hydroxymethylfurfural.

Seliwanoff's reagent contains resorcinol in 6 M hydrochloric acid. Hexoses undergo dehydration when heated in this reagent to form hydroxymethylfurfural, that condenses with resorcinol to give a red product. Ketohexoses (such as fructose) and disaccharides containing a ketohexose (such as sucrose) form a cherryred condensation product. Other sugars may produce yellow to faint pink colors.

Bial's reagent contains orcinol (5 - methylresorcinol) in concentrated HCl with a small amount of Fe. Cl 3 catalyst. Pentoses are converted to furfural by this reagent, which form a bluegreen color with orcinol. This test is used to distinguish pentoses from hexoses.

Iodine forms a deep blue color in the presence of starch. Potassium iodide is added to the reagent solution in order to make the iodine more soluble in water. Some forms of starch may yield a greenish color. Simple carbohydrates (mono- and disaccharides) and cellulose do not cause any change in the orangebrown color of the iodine reagent.

Glucose anhydrous Appearance. The crystalline powder of white color with sweet taste. Solubility. Easily soluble in water R, moderately soluble in 96% alcohol R.

IDENTIFICATION 1. 2. TLC Reaction with reagents Feling. 0. 1 g of the substance is dissolved in 10 ml of water R, 3 ml solution of copper tartratic R is added and it is heated; red sediment is formed:

To the 0, 02 g of the substance are added a few crystals of resorcinol R, 1 -2 ml of dilute hydrochloric acid R and it is heated till boiling; there appears pink color. 4. To 0, 01 g of the substance is added 0. 01 g thymol R, 5 -6 drops of sulphate acid R and R 1 -2 drops of water R; there appears dark red color. 3.

TEST ON PURITY • Irrelevant sugars, soluble starch, dextrins. 1. 0 g of the substance is dissolved by boiling in 30 ml of alcohol (90% v / v) R then it is cooled; the solution must remain transparent.

QUANTITATIVE DETERMINATION • State Pharmacopoeia of Ukraine does not provide quantitative determination of glucose in the substance. • Iodometry, the reverse titration Approximately 0. 1 g of substance (exact batch), is placed in a flask capacity 250 ml, it is dissolved in 10 ml of water R. It is added 20. 0 ml of 0. 05 M solution of iodine, 10. 0 ml of 1% solution of sodium hydroxide R and left for 15 min. Then the solution is acidified by 10 ml dilute acid sulphate R and titrated by 0. 1 M solution of sodium thiosulfate (indicator - starch solution R). In parallels a control experiment is conducted.

• I 2 + 2 Na. OH → Na. I + Na. IO + H 2 O; • Na. IO + Na. I + H 2 SO 4 → I 2 + Na 2 SO 4 + H 2 O; • I 2 + 2 Na 2 S 2 O 3 → 2 Na. I + Na 2 S 4 O 6. • Em = М. м. /2

STORAGE In tightly closed container. APPLICATION During various diseases of heart, liver, at shock treatment, collapse, as a source of nutrition, which is easily assimilated by organism and improves the functions of different organs.

• Tannins can be modified to change their solubility properties or to eliminate the reactive phenolic functional groups. The modified tannins do not retain the characteristic chemical or biological reactivities of native tannins.

Acetylation • Puts an acetyl group on each hydroxyl group of the starting material. Polarity of the tannin is diminished, and it is insoluble in aqueous solvents. Slowly drip a mixture of 5 m. L pyridine and 5 m. L fresh acetic anhydride into a flask containing 2 g tannic acid. Pour the solution into water; a solid should form. The solid is washed with dilute acetic acid (to remove the pyridine) and then with water. It can be freeze dried. Its IR spectrum shows loss of the phenolic OH group.

Methylation • Converts each hydroxyl group to its methyl ester. Polarity of the tannin is diminished and its solubility altered. We have not attempted this procedure. To methylate tannin, mix it with excess methyl iodide, reagent acetone and solid potassium carbonate. Reflux the mixture overnight and purify the product.