VPB-112

BIOCHEMISTRY OF CARBOHYDRATES

I. Occurrence and general importance of carbohydrates

Occurrence:

Carbohydrates are widely distributed in plants and animals; they have important structural and metabolic roles.
The most abundant carbohydrate is cellulose, found in woody structures and fibers of plants. Starch is also
abundant and it is widespread in grains, tubers and roots. Cane sugar or sucrose present in the nectar of
flowers, in fruits and in the juices of various plants. Glucosides are constituent of plants and it is having
medicinal property. Simple sugars like glucose and fructose occur in small amounts and are widely distributed
in plants. Glucose is the most important carbohydrate; most dietary carbohydrate is absorbed into the
bloodstream as glucose, and other sugars are converted into glucose in the liver. Glucose is the major metabolic
fuel of mammals (except ruminants) and a universal fuel of the foetus. It is the precursor for synthesis of all the
other carbohydrates in the body, including glycogen for storage; ribose and deoxyribose in nucleic acids and
galactose in lactose of milk.

General Importance:
 Carbohydrates are the main source of energy in the body. Brain cells and RBC are almost totally
dependent on carbohydrates as energy source.

 Most of them are metabolic intermediates

 Ribose and deoxy ribose are parts of DNA and RNA

 Structural polysaccharide – cellulose (in plants and bacteria)

II. Chemical characteristics of carbohydrates

Contains carbon, hydrogen and oxygen. Their general molecular formula is Cn(H2O)n. Carbohydrates in
general are polyhydroxy aldehydes or ketones or compounds which yield these on hydrolysis. For eg: Glucose -
C6H12O6 (C6 (H2O) 6)

However, acetic acid (C2H4O2) & lactic acid (C3H6O3) are not considered as carbohydrates, while rhamnose
(C6H12O5) is a carbohydrate.

III. Classification of carbohydrates: (Saccharides derived from Greek word sakcharon, meaning sugar)

Carbohydrates are generally classified into two groups, simple and compound sugars
 Carbohydrates

Simple sugars Compound sugars

(Monosaccharides)

Oligosaccharides Polysaccharides
Aldoses ketoses Disaccharides Homopolysaccharides

Trisaccharides Hetreropolysaccharides

Tetrasaccharides

Simple sugars are those carbohydrates which cannot be hydrolysed into simpler compounds / smaller
units. eg. Glucose, fructose etc.

Compound sugars are those carbohydrates which are made up from two to a thousand or more molecules
of simple sugars into which they may be split by hydrolysis. eg. Sucrose, lactose, glycogen, starch etc.

Based on the number of potential sugar groups, simple sugars are also known as monosaccharides
(Monoses, monosaccharoses), contains only one actual or potential sugar group, cannot be hydrolysed into
smaller units. Based on the functional groups (aldo & keto group), monosaccharides are further divided into
aldoses and ketoses (Sugars having aldehyde group are called aldoses and those having keto group are called
ketoses). Monosaccharides are further classified into different groups, based on the number of carbon atoms
present.

Triose (C3), tetrose (C4), Pentose (C5), hexose (C6), heptose (C7) & so on.

Based on the number of potential sugar groups compound sugars are further classified into
oligosaccharides and polysaccharides. Oligosaccharides contain a few sugar units (2 to 10) or they are short
chains of monosaccharide units or residues joined by characteristic linkages called glycosidic bonds.
eg: Disaccharides – 2 sugar units (eg. sucrose, lactose)

Trisaccharides- 3 sugar units (eg: Gentianose, Raffinose)

Tetrasaccharides- 4 sugar units (eg: Stachyose, Scorodose)

Pentasaccharides - 5 sugar units (eg: Verbascose)

 Hexasaccharides- 6 sugar units

Hepta saccharides- 7 sugar units

Polysaccharides contain more than 10 sugar units. They are grouped into homopolysaccharide which
contain only one type of sugar unit (eg: glycogen) and heteropolysaccharide which contain different types of
sugar units (eg: gum acacia, agar etc).

Common monosaccharides:

Number of Generic name Aldoses Ketoses

carbon atoms
3 Triose Aldotriose Ketotriose
eg. glyceraldehyde eg. Dihydroxy acetone
4 Tetrose Aldotetrose Ketotetrose
eg. Erythrose eg. erythrulose
5 Pentose Aldopentose Keto pentose
eg. Arabinose eg:Xylulose
xylose Ribulose
Ribose
6 Hexose Aldohexose Ketohexose
eg. glucose eg. Fructose
Galactose
Mannose
7 Heptose Aldoheptose Ketoheptose
eg. glucoheptose eg. sedoheptulose

Structure of common monosaccharides

There are three representations of monosaccharide structure

In simple form, the structures of aldoses and ketoses are represented as straight (open) chain
projection formula.
Open chain projection formula for different aldoses and ketoses:
In aqueous solution, aldotetroses and all monosaccharides with 5 or more carbon atom occur predominantly as
cyclic or ring structures in which the carbonyl group has formed a covalent bond with the oxygen of hydroxyl
group along the chain. Ring structures represents hemiacetal or hemiketal form. The open chain projection
formula and hemiacetal ring structure of glucose was proposed by Emil Fischer in 1833 and hence called
Fischers formula. Fischer was awarded Nobel Prize in 1902.

Hemi acetal ring structure for glucose

Later in 1925, Sir Walter Haworth established cyclic structures for monosaccharides and suggested
monosaccharides having 5 or more carbons exist not as straight chain but as a 6 sided or five sided ring.
(Haworth projection formula). Six membered (six sided) ring forms of the sugars are called “pyranoses”
because pyran possesses the same ring of five carbons and an oxygen. Similarly, Haworth designated sugars
containing 5 membered or 5 sided rings as the “furanoses”, because furan contains the same ring. Sir Walter
Haworth got Nobel prize in1937 for his findings.

In solution β-D-glucopyranose is the predominantform (63%), α-D-glucopyranose 36% while 1% molecules are
in glucofuranose forms. For fructose, most stable form is furanose form.
Because of the tetrahedral nature of carbon bonds the cyclic forms of pyranose actually assumes chair or boat
confiormation, depending on the sugar. The chair form is more stable because of less steric hindrance as the
axial positions are occupied by hydrogen atoms.

Properties of monosaccharides

Hexoses and pentoses are the most important of the simple sugars. They are naturally occurring, well
crystallized solids, soluble in water and have more or less sweet taste. They all have the property of reducing
alkaline copper solutions and giving other reactions characteristic of free sugar group such as, reactions with
phenyl hydrazine, hydrogen cyanide and hydroxyl amine. Common aldohexoses are glucose, mannose and
galactose. Glucose is the most predominant sugar in the body. It is the major source of energy and is present in
blood. Mannose is the constituent of many glycoproteins, isolated from plant mannans, hence the name
mannose. Galactose is derived from greek word ‘gala’ meaning milk. Constituent of lactose (milk sugar) and
glycoprotein. It is epimerised to glucose in liver and then is utilized as fuel.

Features of monosaccharides

1. Stereoisomerism (space isomerism)

Compounds having same structural formula, but differ in the spatial configuration are called
stereoisomers, and the phenomenon is called stereoisomerism. Number of stereoisomers for a compound is
determined by counting the number of asymmetric carbon atoms. (Chiral centres) i.e.carbon atom with four
different atoms or groups.

Number of stereoisomers = 2n, n is the number of asymmetric carbon atoms

Glyceraldehyde is the simplest monosaccharide with one asymmetric carbon atom and it is considered as the
reference molecule to represent the structure of all carbohydrates.
eg: Glyceraldehyde, Number of stereoisomers = 21=2; Glucose, number of stereoisomers = 24=16

2. D & L isomerism

Penultimate carbon atom i.e. the carbon atom adjacent to the terminal primary alcohol group is
considered as the reference carbon atom, for naming mirror images. With reference to the penultimate carbon
atom the configuration of H and OH group is changed and two mirror images are produced called as D & L
forms. If the –OH group is on the right side it belongs to D series and if it is on left side it belongs to L series.
All monosaccharides can be considered as molecules derived from glyceraldehyde by successive addition of
carbon atoms. Therefore, penultimate carbon atom is the reference carbon atom for naming the mirror
images. This is also referred to as absolute configuration. These two forms are also called as stereo isomers. D
sugars are naturally occurring sugars and body can metabolize only D sugars.

Optical activity

The presence of asymmetric carbon atom causes optical activity. When a beam of plane polarized light
is passed through a solution of carbohydrates, it will rotate the light either to right or to left. Depending on the
rotation, molecules are called dextro rotatory (+) (d) or levorotatory (-) (l). Equimolar mixture of optical isomers
has no net rotation (racemic mixture). Compounds without chiral centres cannot rotate plane polarized light.
Heyrovsky was awarded Nobel Prize in 1924 for polarographic analysis of sugars.
3. Enantiomerism and diastero isomerism
Enantiomers

Stereoisomers which are mirror images of each other. They have identical chemical property but differ
in a characteristic physical property. The phenomenon is known as enantiomerism. eg: D & L glucose, they are
mirror images.

Diasteroisomers

Stereoisomers which are not mirror images of each other. The phenomenon is known as diastero
isomerism. eg: Aldohexoses are having 8 diesteroisomers with regard to the configuration to C2, C3 and C4.
Eight different monosaccharides, glucose, galactose, mannose, allose, altrose, gulose, idose and talose are
produced.

4. Epimerism

When sugars are different from one another, only in configuration with regard to a single carbon atom
(other than the reference carbon atom), they are called epimers.

eg. Glucose and mannose are epimeric pairs, differ only with respect to C2, mannose is the 2ndepimer of
glucose, galactose is 4thepimer of glucose but galactose and mannose are not epimers but diasteroisomers.

6. Anomerism

When D glucose is crystallized at room temperature and a fresh solution is prepared, its specific rotation
of plane polarized light is +1120, but after 12-18 hrs, it changes to +52.50. If initial crystallization is taking place
at 980C and then solubilised, the specific rotation is found to be +190, which also changes to +52.50, within a
few hours. The change in rotation with time is called mutarotation.
 This is due to the presence of 2 anomers i.e. α and β varieties for D glucose. These anomers are
produced by the spatial configuration with reference to the first carbon atom in aldoses and second carbon atom
in ketoses. So these carbon atoms are called anomeric carbon atoms. Thus α D glucose has a specific rotation of
1120 and β D glucose has +190. Both undergo mutarotation and at equilibrium 1/3rd molecules are α type and
2/3rd is β variety to get a specific rotation of +52.50.

Here, differences between α and β anomeric forms are dependent on 1st carbon atom only. For glucose,
there are 16 stereoisomers each of them will have 2 anomers, and hence there are a total of 32 isomers for
glucose.

Anomers are formed when carbonyl carbon and 5th carbon react and form an oxygen bridge which
makes the carbonyl carbon asymmetric. In the hemiacetal and hemiketal form, OH group of carbonyl carbon if
present in right it is α form and if it is on the left, it is β form.

Reactions of monosaccharides

1. Reaction with hydrazines to form hydrazones and osazones

Phenyl hydrazine and other substituted hydrazines react with the monosaccharides and other
carbohydrates containing a free sugar group to form hydrazones and osazones. All reducing sugars will form
osazones with excess phenyl hydrazine when kept at boiling temperature. Each sugar will have characteristic
crystal form of osazones. Hydrazones are water soluble but osazones are insoluble.
In case of glucose, fructose and mannose – produce needle shaped crystals arranged like sheaves of corn or a
broom. In case of maltose – sunflower shaped (petal shaped crystals) of maltosazone. In case of lactose –
Hedgehog shaped crystals or pincushion with pins or flower of touch-me-not-plant or cotton ball shape
(lactosazone).

Osazones are hydrolysed to the corresponding osones when treated with strong HCl. When osones are treated
with zinc and acetic acid, the aldehyde group is preferentially reduced to form the corresponding ketoses.

These reactions provide a method of converting an aldose into a ketose–for eg. Conversion of glucose to
fructose.
2. Reaction with hydrogen cyanide (HCN)

When cyanhydrins are hydrolysed the corresponding acids are formed.

The carboxyl groups of these acids may be reduced to aldehyde groups, forming aldotetroses.
D erythrose may be treated with HCN, and the process repeated to form the pentoses.

Reaction with HCN increases the chain length.

3. Reaction with hydroxyl amine to form oximes.

Hydroxyl amines condense with aldoses, to form oximes. Reaction with hydroxyl amine decreases the chain
length. The process cannot be applied to ketoses.

Glucose oxime is then treated with acetic anhydride, which removes a molecule of water and converts
oxime to a cyanhydrin or nitrile and also acetylates the hydroxyl group. The cyanhydrin is then treated with
ammoniacal silver nitrate solution which removes HCN to form an acetylated sugar with one carbon less than
the original, which can be decomposed into the sugar.

4. Reduction of monosaccharides to alcohols

Both aldoses and ketoses may be reduced to the corresponding polyhydroxy alcohols. This may be
accomplished with sodium amalgam or electrolytically or by hydrogen under high pressure in the presence of a
catalyst. Similarly galactose is reduced to dulcitol, erythrose to erythritol and ribose to ribitol.

5. Oxidation of monosaccharides to produce sugar acids

When aldoses are oxidised under proper conditions, the aldoses may form monobasic, aldonic acids or
dibasic saccharic acids or monobasic uronic acids containing the aldehyde group.

When aldoses are oxidised in presence of hypobromous acids, HOBr or Bromine water, it act as an
oxidising agent, and converts the aldehyde group to a carboxyl group and thereby forms the corresponding acid.
Oxidation of glucose forms gluconic acid. Similarly mannose gives mannonic acid, galactose gives galactonic
acid, arabinose gives arabonic acid. Ketoses are not readily oxidised by bromine water.
Adoses are readily oxidised to aldonic acids by alkaline iodine solution, but ketoses are not oxidised.

I2+2 NaOH NaOI +NaI +H2O

R-CHO +NaOI + NaOH R-COONa +NaI+H2O

When gluconic acid is heated water is readily lost, and it forms mixture of gamma (γ) and delta (δ) lactones.
Gamma (γ) and delta (δ) lactones are the most stable structure for lactone (i.e.5 and 6 membered lactones)
because of the minimal angle strain in the compound structure. β lactones exist, but can only be made by
special methods. α lactones can be seen on transient amount in mass spectrometry experiments.

Oxidation of aldoses with HNO3 under the proper conditions converts both aldehyde and primary alcoholic
groups to carboxyl groups forming dibasic sugar acids, the saccharic or aldaric acid.
When aldoses is oxidised in such a way that the primary alcoholic group is converted to carboxyl group without
oxidation of the aldehyde group, a uronic acid will be formed.

6. Action of alkalies (caramelisation) upon sugars.

 Sugars behave as very weak acids and form salts at high alkalinities. Monosaccharides, both aldoses
and ketoses, and compound carbohydrates containing a free sugar group tautomerise and form the enol salt in
alkaline solution.

The enol forms of the sugars are enediols, because two hydroxyl groups attached to the double bonded
carbon system. It will be noted that glucose, mannose and fructose form the same enediol and enediol salt
forms. These enediol salts are decomposed to the free enediol by acidification, the enediol tautomerises into all
three sugars as follows: D mannose, D glucose and D fructose.

This interconversion of related sugars by the action of dilute alkali is referred to as the Lobry de Bruyn-
van Ekenstein reaction, which is used for the synthesis of certain sugars.

When sugars with free sugar group are treated with strong alkali, enolisation occurs to form not only 1-2
but also 2-3 and 3-4 enediols. Carbohydrates such as sucrose, which do not contain a free sugar group are not
enolized by alkali and are relatively stable in alkaline solution.

When sugars containing a free sugar group are treated with alkali the solution turn yellow to reddish
brown as a result of the formation of complex resinous substance (Known as Moore’s test). The sugar enediols
exist in the cis and trans forms, around the double bond.

Enediol form is a powerful reducing agent as compared to sugar and they are highly reactive and are
easily oxidised by oxygen and other oxidising agents. This property is made use in the qualitative and
quantitative detection of reducing sugars in the solution. They readily reduces oxidising ions such as, Ag+,
Hg++, Bi+++, Cu++ and Fe(CN)6--- and the sugars are oxidised to complex mixtures of acids. Reagents
containing cu++ ions are most commonly used. These are generally alkaline solutions of cupric sulfate
containing sodium-potassium tartrate (rochelle salt) or sodium citrate. Sodium or KOH is used as alkalies in the
older reagents whereas more recent reagents use weaker alkalies such as sodium carbonate and sodium
bicarbonate.

The reagents commonly used to detect the presence of reducing agents in solution are Benedict’s
reagent, Fehling’s reagent, Nylander’s reagent.

Benedict’s reagent – sodium carbonate, sodium citrate, copper sulphate

Fehling’s reagent – potassium hydroxide, sodium potassium tartarate, copper sulphate

Nylnders regent – potassium hydroxide, sodium potassium tartarate, Bismuth sub nitrate

Sugar + alkali enediols

 Cu++ take electrons from the enediols and oxidize them to sugar acids and are in turn reduced to cuprous
ions (Cu+) which combine with hydroxyl ions to form yellow cuprous hydroxide, which upon heating is
converted to red cuprous oxide, Cu2O. The appearance of a yellow to red precipitate indicates reduction and the
quantity of sugar present is proportional to the colour produced. The process is known as caramelisation.

In quantitative determination, the amount of copper reduced is obtained by iodometric titration or colorimetric
methods; from this the amount of sugar is determined.

7. Action of acids upon carbohydrates

Monosaccharides when treated with concentrated H2SO4 undergo dehydration with the removal of 3
molecules of water. Therefore hexoses give hydroxy methyl furfural and pentoses give furfural. Ring forms
between 2nd and 5th. The furfural derivative can condense with phenolic compounds to give coloured products.
This forms the basis of Molisch’s test. To the carbohydrte solution, add a few drops of alpha naphthol solution
and pour pure concentrated H2SO4 through the sides of the tube. A pink or purple ring will develop. It is a
general test for carbohydrates.
8. Reactions of sugars due to hydroxyl groups

Monosaccharides with free reducing groups can condense with alcohol (R-OH) or phenol group in the
presence of hydrogen chloride as catalyst to form glycosides. Only the hydroxyl group at C-1 (glycosidic
hydroxyl) group of the sugar reacts under these conditions. Glycosides do not reduce alkaline copper solutions,
because the sugar group is combined. α glycosides are hydrolysed by maltase, an enzyme from yeast, while β
glycosides are hydrolysed by the enzyme emulsin, from bitter almonds.

Many glycosides occur in the roots, bark, and fruit and frequently the leaves of various plants. They are
usually well-crystallized, colorless, bitter solids, soluble in water and alcohol.

Glycosides contain both carbohydrate and non carbohydrate part which have medicinal properties.

Glycosides Sugar part aglycon (non Source

carbohydrate part
1. Phlortizin glucose Phloretin Rose bark
2. Digitonin Galactose, xylose Digitogenin leaves of fox glove
3. Indican glucose Indoxyl leaves of indigofera
4. Amygdalin glucose mandelonitrile bitter almond seeds

9. Formation of esters

Hydroxyl groups of sugars can be esterified to form sugar acetates, propionates, benzoates etc.
Generally accomplished by treating the sugar with the appropriate acid anhydride or chloride under the
proper conditions. Sugar phosphates are of great biological importance. Metabolism of sugars inside the body
starts with phosphorylation. Glucose-6-phosphate (Robinson ester) and glucose-1-phosphate (Cori ester) are
important intermediaries of glucose metabolism.

10. Fermentation of monosaccharides

Common monosaccharides, D glucose, D fructose, D mannose are readily fermented by ordinary yeast
and D galactose fermented by specially cultured yeasts

Other sugar derivatives of biological importance

1. Amino sugars: Amino groups may be substituted for various hydroxyl groups of sugars to give amino
sugars; mostly OH group of 2nd carbon atom is replaced with amino group.
 They give chemical reaction characteristic of the sugars; they form hydrazones but not osazones. Amino
sugars can also give reaction with acetic acid to form N- acetyl amino sugars. Glucosamine reacts with acetic
acid to form N- acetyl glucosamine (NAG or Glu Nac).

N- acetyl glucosamine (NAG or Glu Nac)

Glucosamine occurs widely in nature as a constituent of mucopolysaccharides and mucoproteins, such
as hyaluronic acid, heparin, etc. Chief constituent of cell wall of fungi and shells of crustaceae, where it occurs
as chitin. Chitin made up of many molecules of NAG. Glucosamine readily prepared by acid hydrolysis of
chitin, so glucosamine often called as chitosamine.

N-acetyl galactosamine is a constituent of sulfated mucopolysaccharides present in chondroproteins

found in cartilage, adult bone, cornea, skin, tendon, heart valves etc. because of its presence in chondroproteins,
galactosamine is also called chondrosamine (Gal Nac). Another derivative of N acetyl glucosamine is N-acetyl
muramic acid, formed by condensation of N-acetyl glucosamine with lactic acid. Another derivative – N acetyl
neuraminic acid (NANA) which is a condensation product of N- acetyl mannosamine and pyruvic acid. NANA
present in membrane of RBC and cell membrane, mucopolysaccharides and mucins.
2. Deoxy sugars

Deoxy sugars represent sugars in which the oxygen of a hydroxyl group has been removed leaving the
hydrogen.

2 deoxy sugars give most of the reactions, though they cannot form osazones. Feulgen staining reaction for 2
deoxy sugar derivatives in tissues is based upon the reaction of 2 deoxy sugars with Schiff’s reagent.

3. Ascorbic acid/Vitamin C

It is a sugar derivative, present in plant and animal tissues, citrus juices, essential for prevention of
scurvy in man and some animals. Synthesized commercially in large amounts from L-sorbose. Stable in crystal
form, good reducing agent, oxidise immediately in solution.
Compound carbohydrates

Compound carbohydrates are derivatives of the monosaccharides in which monosaccharides are joined
together through glycosidic bonds. Divided into oligosaccharides (simple compound carbohydrates) and
polysaccharides (complex compound carbohydrates).

General properties of compound carbohydrates are,

 Do not crystallize, but are amorphous solids

 Most of them are tasteless, though dextrins are mildly sweet.
 Hydrolysed by hot dilute mineral acids or by specific glycosidase enzyme.

Sucrose – sucrase, invertase Lactose – lactase

Maltose – maltase Cellulose – cellulase

Starch, dextrins, glycogen – amylases or diastases

 Optically active

Oligosaccharides
Composed of disaccharides, trisaccharides and tetra saccharides

Disaccharides are reducing and non-reducing. Reducing sugars are those possessing free sugar group and give
other characteristic sugar reactions.

eg. Disaccharides Constituent monosaccharides

I. Reducing disaccharides

Maltose glucose, glucose

Lactose galactose, glucose

Cellobiose glucose, glucose

Gentiobiose glucose, glucose

Melibiose glucose, galactose

Turanose glucose, fructose

II. Non reducing disaccharides

Sucrose glucose, fructose

Trehalose glucose, glucose

Reducing disaccharides

1. Lactose - Milk sugar: Consists of galactose and glucose, linkage is β (1, 4) glycosidic linkage
Properties: Hydrolysed by acids and the specific enzyme, lactase into its constituent monosaccharides i.e.,
galactose and glucose. It is a reducing sugar, form osazones and oxime and is decomposed by alkali. Lactose
reacts with bromine water and gets oxidized to lactobionic acid. Here free -CHO group converted to free
-COOH group. Lactobionic acid hydrolysis into gluconic acid and galactose. Lactose is not fermented by yeast.

2. Maltose: Contains two glucose units in α (1, 4) glycosidic linkage. Important constituent of corn syrup and
commercial malt sugar. Formed by acid hydrolysis of starch or enzymatic hydrolysis.

Corn syrup formed by partial hydrolysis of starch with dilute acid. It contains maltose, glucose & dextrins. Malt
sugar formed by hydrolysis of starch with amylase and contains maltose and dextrins. During digestion, maltose
is product of action of salivary amylase (ptyalin) and pancreatic amylase (amylopsin) upon starch.

Properties: Free sugar group, gives other reactions characteristic of sugar group. Readily fermented by yeast

3. Isomaltose: It contains 2 glucose units in α (1, 6) linkage.

Obtained by partial hydrolysis of glycogen, amylopectins and
bacterial dextrans. It is hydrolyzed to glucose in the intestinal
tract by the enzyme oligo-1-6 – glucosidase.
4 Cellobiose: It contains 2 glucose units with β (1, 4) linkage. Incomplete hydrolysis of cellulose gives
cellobiose. Gives reaction characteristic of free sugar group.

5. Gentiobiose: It contains 2 glucose units with β (1, 6) linkage. Obtained by acid hydrolysis of trisaccharide
gentianose (glucose + glucose + fructose). It is β D glucopyranosyl 1, 6 glucopyranose.

6. Melibiose: Contains galactose and glucose in α (1, 6) linkage. It is α D galactopyranosyl 1, 6 glucopyranose.

Obtained from raffinose.

Raffinose

Invertase

{Galactose + glucose} + Fructose

Melibiose

II. Non Reducing disaccharides

1- Sucrose: Contains glucose and fructose in α1, β2 linkage. Main source is sugar cane, sorghum,
pineapple and other ripe fruits.
 Sucrose on hydrolysis with dilute acid and enzymes (sucrase or invertase) equimolar mixture of glucose
and fructose will be formed and that sugar turns to be reducing. The specific rotation of sucrose is + 66.5 0. On
hydrolysis, the specific rotation changes from +66.5° to -28.2° (i.e. from positive to negative) because D
fructose is more levorotatory than D glucose which is dextrorotatory. The process of change/inversion of the
sign of rotation in the rxn is called inversion. The mixture of glucose and fructose obtained is called invert
sugar. It is sweeter than sucrose. Honey contains large amounts of invert sugar.

Sucrose is non reducing sugar, so will not give reactions characteristic of sugar group. But it is readily
fermented by yeast. On heating sucrose melts at 1600 C and at 2000C it loses water and forms brown
amorphous mass called caramel.

2. Trehalose: Contains 2 glucose units, α 1-1 linkage (α D glucopyranosyl 1, 1 α D glucopyranoside). Widely

distributed in fungi (reserve food), non reducing, not hydrolysed by invertase, maltase or emulsin but
hydrolysed by enzyme trehalase found in fungi and yeast.

Trisaccharides

I. Non reducing trisaccharides

1. Raffinose: Non reducing trisaccharide. Most important of trisaccharides contains galactose, glucose and
fructose. Concentrated in sugar beet molasses. Hydrolysis of raffinose with weak acids and sucrase may yield
Fructose and melibiose whereas hydrolysis with maltase yields galactose and sucrose. Fermented by yeast.
Hydrolysed by bacteria of GI tract of herbivores.

2. Gentianose: Non reducing trisaccharide. Contains glucose and fructose

II. Reducing trisaccharides

Mannotriose and robinose are examples for reducing trisaccharides.

Polysaccharides

Carbohydrates composed of 10 or more monosaccharide units are generally classified as polysaccharides

or glycans. Monoglycans, made up of single kind of monosaccharides. eg. Starch, glycogen, cellulose etc.
Heteroglycans, composed of two or more kinds of monosaccharides eg. Mucopolysaccharides.

Glucose polysaccharides are known as glucans similarly, fructose polysaccharides – fructans, mannose
polysaccharides – mannans etc.

Hydrolysed by group of enzymes called polysaccharidases

Monopolysaccharides – storage homopolysaccharides eg. Starch, glycogen

Structural homopolysaccharides eg. Cellulose, chitin

Heteropolysaccharides mainly provide extracellular support.

I. Storage forms of homopolysaccharides

1. Starch: Widespread as reserve carbohydrates in tubers, grains, seeds and many fruits and rhizomes. Contains
two types of glucose polymers amylose and amylopectin. Amylose – long unbranched chains of D glucose
residues connected by (α-1-4) linkages (soluble part). Amylopectin – high molecular weight and highly
branched, linkage are (α-1-4) in straight chain and (α-1-6) at branching points (occurring every 24 to 30
residues). Action of amylases upon starch

Salivary amylase (ptyalin) and pancreatic amylase (amylopsin) are α amylase, act randomly on α-1-4
glycosidic bonds to split starch to smaller units (dextrins) and finally to alpha maltose. α amylase first act upon
amylose to split the more central linkage forming a mixture of smaller dextrins but only a little maltose. As the
action proceeds dextrins converted to maltose.

β-Amylases are plant origin (almond, germinating seeds) forms β maltose. β-amylase hydrolyse amylose
completely to maltose. Splits maltose units from the ends of branches of amylopectin until its action is blocked
at 1,6 glycosidic linkages or branch points leaving branched large molecules called limit dextrins or residual
dextrins or grenz dextrins. α 1,6 linkage hydrolysed by 1,6 glycosidase enzyme from yeast, then β amylase
continues action. α amylase is dextrinogenic amylase whereas, β amylase is saccharogenic / maltogenic
amylase.

α amylases act on both raw and cooked starch, but β-amylases act only on cooked starch.

Hydrolysis of starch

Starch upon partial hydrolysis will be converted to dextrin, maltose and glucose. Starches with
unbranched chains (amyloses) give a blue colour with iodine whereas, those with branched chains
(amylopectins) give a violet to red violet colour. This is due to the formation of covalent bond between starch
and iodine molecule. On heating the colour disappears due to the breakage of this bond and on cooling the
colour reappears.

2. Dextrins: Polymer of glucose, produced by either partial hydrolysis of starch or as an intermediate in the
synthetic pathway of starch in plants.Soluble in water and not fermented by yeast. Used extensively as
adhesives and binders.

3. Dextrans: Polymer of glucose having 1-6, 1-4 and 1-3 linkages predominantly 1-6. Formed from various
strains of leuconostoc mesenteroides and by some other microorganisms grown in sucrose solution. It forms a
ropy slimy material and is having a molecular weight of 40 lakhs.

Important in medicine, preparations of hydrolysed dextrans with a molecular weight of approximately

76,000 is used as plasma volume extenders for treatment of shock. Native dextran is unsuitable for this due to
its high molecular weight and bigger particle size. The beneficial properties of dextran are low osmotic
pressure, increased viscosity and slow utilisation.
4. Glycogen: Animal starch, found in liver and muscle

In liver, 7% of wet weight of liver is glycogen and in Muscle = 0.5% wet weight of muscle is glycogen

Polymer of D glucose, chemically more like amylopectin. Highly branched. Branching occurs at every 8-12
glucose residues. α1,4 and α1,6 linkage.

glycogen + Iodine gives mahagony red / Reddish brown colour

Isolation from tissues – extract obtained by boiling the minced tissue with H2O. So that glycogen is released
into the medium and forms opalescent solution, from which it can be precipitated with alcohol. It is not a
complete extraction.

Complete extraction of glycogen is done by Pfluger’s method. Here treat the minced tissue with hot 60% KOH
solution. KOH will disintegrate the tissue leaving glycogen in solution. Precipitate the glycogen with alcohol.
Centrifuge the solution containing the precipitate. Pour off the supernatant & wash the precipitate with alcoholic
KOH solution and then with alcohol. Quantisation of glycogen present can be done by acid hydrolysis of
glycogen & estimation of glucose.

Glycogen broken down to glucose when there is a decline in glucose content in the blood. Liver
glycogen is broken down to glucose and passed into the blood stream for use by the tissues. Muscle glycogen is
the source of energy for muscle contraction.

I. Structural homopolysaccharides

They are cellulose, chitin and inulin

1. Inulin : It is a homopolysaccharide made up of D fructose. Linkage is β 1,2 present in tubers of chicory,

Jerusalem artichoke, dahlia, bulb of onion, garlic etc. It is a white crystalline powder, soluble in hot water, but
slightly soluble in cold water. Inulin does not give any colour with iodine. Not hydrolysed by amylase but split
by inulinase. Apparently it is not a branched structure, have molecular weight of 5000. Cannot be digested in
the intestinal tract due to β linkages. Molecular weight is low, easily passed through renal tubule, so used for
studying glomerular filtration rates. It is a commercial source of fructose.

2. Cellulose: Present in the cell wall of plants – stalk, trunk, stem and all fibrous tissue. Most abundant organic
material in the nature water insoluble.

Cotton contains 97-99% cellulose, Wood- 41-53%, Cereal straws- 30-43%

Contains glucose molecule in β 1-4 linkage, only linear chain. No reaction with iodine. Hydrolysis with strong
acid yield glucose. Partial hydrolysis give rise to cellulodextrins, cellobiose and glucose. Digested by cellulase
producing bacteria. Also digested by termites (by tryconympha – parasitic microorganism present in termites
which produce cellulase).

Cellulase is absent in animal digestive juices. Herbivorous animals utilise cellulose as food by virtue of
action of gastrointestinal bacteria and fungi which split it into glucose and other utilisable products.

Industrially cellulose is important in manufacture of nitrocellulose which is used in the manufacture of

explosives and celluloid. Cellulose acetate used in making photographic film, and rayon.

3. Chitin: Polymer of N-acetyl glucosamine with β 1-4 linkage. Linear chain structural material of arthropoda,
exoskeleton of crustaceans, insects etc. Hydrolysis yield glucosamine and acetic acid.

Heteropolysaccharides

Have more than one type of monosaccharide units.

1. Agar: Certain marine red algae, including some of the sea weeds, have cell walls that contain agar, a
mixture of sulfated heteropolysaccharides made up of D-galactose and an L-galactose derivative ether-linked
between C-3 and C-6. The two major components of agar are the unbranched polymer agarose and a branched
component, agaropectin. The remarkable gel-forming property of agarose makes it useful in the biochemistry
laboratory. Agarose gels are used as inert supports for the electrophoretic separation of nucleic acids, an
essential part of the DNA sequencing process. Agar cannot be utilized by bacteria, so used as supporting agent
in bacterial culture media. Another commercial use of agar is for the capsules in which some vitamins and drugs
are packaged; the dried agar material dissolves readily in the stomach and is metabolically inert.

2. Gum acacia: Polysaccharide of hexoses and pentoses. Used as gum or adhesive.

3. Gum Arabic: Polysaccharide of galactose, arabinose, rhamnose and glucuronic acid. Used in preparation of
pharmaceuticals, confectionaries and as adhesives.

4. Pectin: Methyl ester of pectic acid (polymer of galactose and galacturonic acid). Mainly seen in pulp of
citrus fruits, apple, carrot etc.

5. Mucopolysaccharides / glycosaminoglycans:

The extracellular space in the tissues of multicellular animals is filled with a gel-like material, the
extracellular matrix, which holds the cells together and provides a porous pathway for the diffusion of nutrients
and oxygen to individual cells. The extracellular matrix is composed of an interlocking meshwork of
heteropolysaccharides and certain fibrous proteins These heteropolysaccharides, the glycosaminoglycans, are a
family of linear polymers composed of repeating disaccharide units. One of the two monosaccharides is always
either N-acetylglucosamine or N-acetyl- galactosamine; the other is in most cases a uronic acid, usually D -
glucuronic or L -iduronic acid. In some glycosaminoglycans, one or more of the hydroxyls of the amino
sugar are esterified with sulfate.

Functions

Contribute to the viscous lubricating property. Found in extra cellular matrix, plays important role in mediating
cell to cell interaction and it stabilize and support cellular and fibrous components of tissue. It helps to maintain
water and salt balance of the body.

Relation between Structure and function

The combination of sulfate groups and the carboxylate groups of the uronic acid residues makes
glycosaminoglycans a very highly negative charged polar molecule at physiological pH. Due to large number of
–ve charge, GAG chains tend to be extended in solution and repel each other. A shell of H2O molecule is
present around each molecule. When these chains are brought together, they slip apart each other. Thus it is
producing slippery consistency of mucous secretion and synovial fluid. On compression of solution of GAG the
H2O molecule squeezes out and GAG is forced to occupy a smaller volume. But on release of compression it
springs back to the original volume due to the repulsive –ve charges. GAG are linear chains. Examples are the
following,

1. Hyaluronic acid

The glycosaminoglycan hyaluronic acid contains alternating residues of D-glucuronic acid and N-
acetylglucosamine. With up to 50,000 repeats of the basic disaccharide unit, they form clear, highly viscous
solutions that serve as lubricants in the synovial fluid of joints and give the vitreous humor of the vertebrate eye
its jelly-like consistency. Hyaluronate is also an essential component of the extracellular matrix of cartilage and
tendons, to which it contributes tensile strength and elasticity as a result of its strong interactions with other
components of the matrix. Hyaluronidase, an enzyme secreted by some pathogenic bacteria, can hydrolyze the
glycosidic linkages of hyaluronate, rendering tissues more susceptible to bacterial invasion. In many organisms,
a similar enzyme in sperm hydrolyzes an outer glycosaminoglycan coat around the ovum, allowing sperm
penetration.

2. Chondroitin sulphate

Composed of disaccharide units of D glucuronic acid and N acetyl galactosamine. 2 types are there.
Chondridin 4 SO4 and Chondridin 6 SO4. It is depend on sulphation of carbon atoms in galactosamine. It
contributes to the tensile strength of cartilage, tendons, ligaments, and the walls of the aorta.
3. Dermatan sulphate

It is present in skin. Composed of L iduronate and N acetylgalactosamine. L-iduronate is the 5th carbon
epimer of glucuronate. Dermatan sulfate contributes to the pliability of skin and is also present in blood vessels
and heart valves.

4. Keratan sulphate

It has β-1, 4 linked galactose and N acetyl glucosamine. Keratan sulfates have no uronic acid and their sulfate
content is variable. Mostly 6th carbon of glucosamine is sulphated. Keratin sulphate also contains small amount
of fucose, mannose and sialic acid. This is the most heterogenous of the major GAG. They are present in
cornea, cartilage, bone, and a variety of horny structures formed of dead cells: horn, hair, hoofs, nails, and
claws.

5. Heparin

Polymer of D iduronic acid and D glucosamine. It is highly sulphated, in each disaccharide unit an
average of 25 sulphate radicals are present. Linkage is α -1, 4. Heparin is a natural anticoagulant made in mast
cells (a type of leukocyte) and released into the blood, where it inhibits blood coagulation by binding to the
protein antithrombin. Heparin binding causes antithrombin to bind to and inhibit thrombin, a protease essential
to blood clotting. The interaction is strongly electrostatic; heparin has the highest negative charge density of any
known biological macromolecule. Purified heparin is routinely added to blood samples obtained for clinical
analysis, and to blood donated for transfusion, to prevent clotting.

6. Bacterial cell wall polysaccharide

Bacterial cell wall has peptidoglycan framework or murien framework. The rigid component of bacterial
cell walls is a heteropolymer of alternating (ᵝ-1, 4)-linked N-acetylglucosamine and N-acetylmuramic acid
residues with several D amino acids. The linear polymers lie side by side in the cell wall, crosslinked by short
peptides, the exact structure of which depends on the bacterial species. Gram-positive bacteria have a
pentaglycine chain in the cross-link. Gram-negative bacteria, such as E. coli, lack the pentaglycine; instead, the
terminal D-Ala residue of one tetrapeptide is attached directly to a neighboring tetrapeptide through either
L-Lys or a lysine-like amino acid, diaminopimelic acid. The peptide cross-links weld the polysaccharide chains
into a strong sheath that envelops the entire cell and prevents cellular swelling and lysis due to the osmotic entry
of water. The enzyme lysozyme kills bacteria by hydrolyzing the (ᵝ-1, 4) - glycosidic bond between N-
acetylglucosamine and N-acetylmuramic acid). Lysozyme is notably present in tears, presumably as a defense
against bacterial infections of the eye. It is also produced by certain bacterial viruses to ensure their release from
the host bacterial cell, an essential step of the viral infection cycle. Penicillin and related antibiotics kill bacteria
by preventing synthesis of the cross-links, leaving the cell wall too weak to resist osmotic lysis.

Gram positive bacterial cell well contains teichoic acid which is a complex of ribitol or glycerol and
phosphate ester linked to alanine and N acetyl glucosamine. The bacterial cell wall polysaccharide and teichoic
acid are often responsible for the immunological specificity of bacteria.

Kiliani reaction: To find out the presence of deoxy sugars. Deoxy sugars are treated with glacial acetic acid
containing ferrous salt and sulphuric acid. Then a blue colour is developed if it is deoxy sugar.

Proteoglycans

Proteoglycans are macromolecules of the cell surface or extracellular matrix in which one or more
glycosaminoglycan chains are joined covalently to a membrane protein or a secreted protein. The
glycosaminoglycan moiety commonly forms the greater fraction (by mass) of the proteoglycan molecule (only
5% proteins and 95% carbohydrates), dominates the structure, and is often the main site of biological activity.
Polysaccharides will be in linear chain.

Proteoglycans are major components of connective tissue such as cartilage, in which their many
noncovalent interactions with other proteoglycans, proteins, and glycosaminoglycans provide strength and
resilience. Extracellular matrix is rich in proteoglycans.

A proteoglycan monomer is formed by covalent attachment of proteins with any GAG except hyaluronic
acid. The monomer looks like a bottle brush. The proteoglycan aggregation of proteoglycan monomers with
hyaluronic acid, the association is not covalent but by ionic interactions between core proteins and hyaluronic
acid. The association is stabilised by additional small proteins called link proteins.

Glycoproteins

Glycoproteins have one or several oligosaccharides of varying complexity joined covalently to a protein.
They are found on the outer face of the plasma membrane, in the extracellular matrix, and in the blood. Inside
cells they are found in specific organelles such as Golgi complexes, secretory granules, and lysosomes. Chains
are short and often branched as against the long linear chains in proteoglycans. The carbohydrate content varies.
Role of oligosaccharides:

1. To stabilize proteins and aid in protein folding

2. Participate in molecular targeting and cell recognition.

 Almost all of the globular proteins present in human plasma are glycoproteins except albumin. Enzymes
and hormones are also glycoproteins.

Glycolipids are membrane lipids in which the hydrophilic head groups are oligosaccharides, which, as in
glycoproteins, act as specific sites for recognition by carbohydrate-binding proteins.