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Carbohydrates

CARBOHYDRATES are widely distributed both in animal and plant tissues.


They serve as important source of energy for vital activities.
Definition :
Carbohydrates are defined as polyhydroxy alcohols with aldehydes or
ketones and their derivates.
Ex: Glucose, sucrose, starch, inulin, heparin etc.
Classification:
Carbohydrates are divided into four major groups as
1)
2)
3)
4)

Monosaccharides
Disaccharides
Oligosaccharides
Polysaccharides

Monosaccharides:

They are often called Simple Sugars which cannot be hydrolyzed


further.
General formula is Cn(H2O)n
Monosaccharides are divided into different catogeries
(I)
Based on the functional groups.
(II)
Based on the number of carbon atoms.
I.
Based on functional groups :
Based on the functional groups, they are classified as aldoses and
ketoses.

Aldoses :
When the functional group in monosaccharides is an aldehyde ( CHO ),
they are referred to as aldoses. Ex : Glyceraldehyde, glucose.

Ketoses :
When the functional group in monosaccharides is an ketone ( C=0 ),

They are referred to as ketoses. Ex : Dihydroxyacetone, Fructose.

II Based on the number of carbon atoms :


Based on the number of carbon atoms, the monosaccharides are
regarded as trioses (3C),
Tetroses (4C), Pentoses (5C), Hexoses (6C) and Heptoses (7C).

These terms are used along with functional groups, while naming
monosaccharides.

Ex : Glucose : Aldohexose
Fructose : Ketohexose.

Carbon atoms
Trioses (3C)
Tetroses (4C)
Pentoses (5C)
Hexoses (6C)
Heptoses (7C)

Aldoses

Ketoses

Glyceraldehyde

Dihydroxyacetone

Erythrose
Ribose, xylose,
Arabinose
Glucose, Galactose,
Mannose
Glucoheptose,
Galactoheptose

Erythrulose
Rbulose, Xylulose
Fructose
Sedoheptulose

Chemical reactions of monosaccharides :


I)

Action of acid on carbohydrates :

H-C=O
H-C-OH
H-C-OH
H-C-OH
CH2OH

H-C=O
12 % HCl

C
3H2O

HC
HC
HC

Pentose Sugar

Furfural

H-C=O

H-C=O

H-C-OH

HO-C-H

HC

H-C-OH
O

HC

H-C-OH

CH2OH

CH2OH

D Glucose
furfuryl

Hydroxy Methyl

With strong mineral acids hexose sugars like glucose form hydroxy methyl
furfural and pentose sugars form furfural. This reaction forms the basis of
some colour tests ( Ex: Molisch test & bials orcinol test) for sugars as
aldehyde products of these reactions condense with certain organic phenols
to give characteristic coloured products.
Tetroses & trioses do not undergo this reaction, as they do not possess the
necessary 5 carbon atoms for furfural formation.

II ) Action of bases on carbohydrates :


H-C=O
HO-C-H

Mannose

R
H
H-C=O
H-C-OH
R
Glucose

H-C-OH

H-C-OH

C-OH

C=O

R
Enediol

R
Fructose

(Incomplete alcohol)

Glucose, fructose and mannose are interconvertible in weak alkaline


solutions such as Ca(OH)2 and Ba(OH)2 at low temperatures. This reaction is
known as Lobry De BRuyn Alberda Van Ekenstein Transformation. This
reaction is proposed in 1890, is of special significance as a similar reaction is
supposed to take place in human body also. The mechanism of this reaction
involves enolization, the migration of a proton from a carbon atom onto the
oxygen of an adjacent carboxyl group, resulting in the formation of an
unsaturated alcohol called enol.

Carbohydrates on Oxidation

Strong oxidants

CHO
(CHOH)4

COOH
+

3 [o]

(CHOH)4

CH2OH

(HNO3)

Glucose

nitric acid

H2O

COOH
Glutaric acid
(or) Saccharic acid

CH2OH
COOH

COOH
CH2OH

C=O
(CHOH)2 +

+
COOH

(CHOH)3
COOH
Fructose
acid
glycolic acid

[O]

(CHOH)3

COOH
trihydroxy
Glutaric acid

tartaric

II)

With mild oxidants :

CHO

COOH

(CHOH)4
CH2OH
Glucose

HOBr
bromine
water

(CHOH)4

HBr

CH2OH
Gluconic acid

With strong oxidants (like conc HNO3), both the aldehyde group (or ketone
group) and the primary alcohol group are oxidized to yield dicarboxylic
acids. With aldoses, acids with same number of carbon atoms are
obtained whereas ketoses react to produce acids with fewer number of
carbon atoms.
With mild oxidants (like HOBr), only the
aldehyde group is oxidized to produce monocarboxylic acids. Ketoses
however, do not respond to this reaction. Hence, this reaction can be used
to distinguish aldoses from ketoses.
III)

With metal hydroxides :

Metal hydroxides like Cu(OH)2, AgOH and Bi(OH)3 oxidize the free
aldehyde (or ketone) group of mutarotating mono and di saccharides and
at the same time reduce themselves to lower oxides or free metals.

Reducing sugar + 2Cu2+


2Cu

Oxidized Sugar +

(blue)
Cupric ion (Cu++) is the most common oxidizing agent . It is the active
ingredient in fehlings, Benedicts and Barfoeds reagent.

Carbohydrates on reduction :
Sugars maybe reduced in various ways depending upon the type of
reducing agent used.
(a) With sodium amalgum: The monosaccharides are reduced to their
corresponding alcohols by treating thwm with reducing agents like Naamalgum. Thus glucose yields sorbitol (=glucitol), mannose yields

mannitol,galactose yields dulcitol, fructose yields a mixture of sorbitol


and mannitol and glyceraldehyde yields glycerol.
CHO
CH2OH
H-C-OH
C-OH
HO-C-H
+
C-H
H-C-OH
OH
H-C-OH
C-OH
CH2OH
CH2OH
Sorbitol (=glucitol)
CHO
CH2OH
HO-C-H
HO-C-H
HO-C-H
HO-C-H
H-C-OH
H-C-OH
H-C-OH
H-C-OH
CH2OH
CH2OH
Mannose
Mannitol

H2H
(Sodium Amulgum)

HOH-CH-

Glucose

+ 2H

Reaction with Phenyl hydrazene :


Reaction with phenyl hydrazene involves only 2 carbon atoms viz, the
carboxyl (i.e, the aldehyde or ketone group) and the adjacent one.
One mole of phenyl hydrazene reacts with one mole of aldose (or ketose) to
form a mole of hydrazone with second mole of phenyl hydrazine, the
hydrazone is oxidized to aldohydrazone and the phenylhydrazine itself is
reduced to aniline and ammonia. Finally, a third mole of phenyl hydrazine
reacts with aldohydrazone to produce osazone.

(1)H-C=O
H-C=N-NH-C6H5
H-C-OH +
H2N.NH.C6H5
C-OH
R
R

HH2O

Aldohexose
Phenylhydrazone

(2) H-C=N-NH.C6H5
NH.C6H5
H-C-OH
+ C6H5.NH2+NH3
R

H-C=NH2N-NH-C6H5

C=O
R

Aniline
Phenyl hydrazone
aldohydrzone
(3) H-C-N-NH.C6H5
NH-C6H5
C=O
C=N-NH-C6H5 + H2O
R

H-C-NH2N-NH-C6H5
H2O

Aldohydrazene
Osazone

Asymmetric carbon :
A carbon is said to be asymmetric carbon when
it is attached to four different atoms or groups. The number of asymmetric
carbon atoms (n) determines the possible isomers of a given compound,
which is equal to 2n.
For example glucose contains 4 asymmetrical carbons and thus has 16
isomers.

C1HO
H-C2-OH

( 1,2,4&5 are asymmetrical carbons in

glucose)
HO-C3-H
H-C4-OH
H-C5-OH
C6H2OH
Glucose

D & L isomers :
The D and L isomers are mirror images of each other. The spatial
orientation of H and
-OH on the carbon atom ( C5 for glucose ) that is adjacent to the terminal
primary alcohol carbon determines, whether the sugar is D or L isomer.
If the OH group is on the right side, it belongs to D series, and if on left,
it belongs to L series.

Note : Naturally occuring monosaccharides in the mammalian tissue are


mostly of D-configuration.

Optical activity of sugars :


Optical activity is the characteristic feature of compounds with asymmetrical
carbon atom, when a beam of polarized light is passed through a solution of
an optical isomer, it will be rotated either to the right or left.
The term dextrorotatory (+) and levorotatory (-) are used to compounds that
respectively rotate the plane of polarized light to the right or to the left.

Epimers :

If two monosaccharides differ from each other in their configuration


around a single specific carbon (other than anomeric) atom, they are refrred
to as epimers to each other.
Ex :
H-C=O

H-C=O

H-C-OH

H-C-OH

HO-C-H

HO-C-H

HO-C-H
H-C-OH

H-C=O
HO-C-H
HO-C-H

H-C-OH

H-C-OH

H-C-OH

H-C-OH

CH2OH

CH2OH

D-galactose

D-glucose

Glucose and galactose


Are C4 isomers

CH2OH
D-Mannose
Glucose and mannose are
C2 isomers

Hemiacetal :
The hydroxyl group of monosaccharides can react with its own aldehyde
functional group to form hemiacetal.
H

OR2

R1-C

R2-OH

R1 C-H

OH

Aldehyde

Alcohol

Hemiacetal

The aldehyde group of glucose at C1 reacts with alcohol group at C5 to form


two types of cyclic hemiacetals namely & .
H

OH
C

H-C-OH

HO

H
C

H-C-OH

HO-C-H

HO-C-H

H-C-OH

H-C-OH

H-C

H-C

CH2OH

CH2OH

-D-Glucose

-D-Glucose

Anomers :
The & cyclic forms of D-glucose are known as anomers. They differ from
each other in the configuration only around C1 known as anomeric carbon
(Hemi acetal carbon).
anomer OH group held by anomeric carbon is on the the opposite side of
group CH2OH of sugar ring and vice versa.
Note : Anomers differ in certain physical and chemical properties.

Mutarotation :
It is defined as change in specicfic optical representing the interconversion of
and forms of D-glucose to an equilibrium mixture. It is explained by the
& form of glucose which requires the presence of asymmetric carbon C1 . In
mutarotation, hemi acetal ring is opened and reformed with change of
position of H and OH.
For example when D-glucose is dissolved in water its specicfic rotation is
112.2o, when ammonia is passed, the value is 52.5. If it is recrystallized from
boiling pyridine, the rotation is 18.7 and finally 52.5o.

D glucose
+ 112.2o

Disaccharides :

Equilibrium
mixture +52.7o

D glucose
+18.7o

Disaccharides are composed of two monosaccharide units linked by a


glycosidic linkage. Physiologically important disaccharides are maltose,
lactose and sacrose.
Maltose (Malt sugar) :

Doesnt occur in the body.


Sources of it are germinating cerals and malt. It is the intermediate
product in the breakdown of starch by amylase in the alimentary canal.
It is hydrolyzed to glucose by the enzyme maltse and the products are
absored.
It has one free aldehyde group and hence shows mutarotation and the
final roatation of the solution is +130o. It can exist in or forms.
It ca reduce fehlings and benedicts solutions since it is a reducing
sugar but cannot reduce barfoeds solution.
It forms an osazone with phenyl hydrazene.
Structure of Maltose is

Glucose

Glucose

( D-glucosyl (14) D-glucose)

(Free aldehyde group resent on C1 of secon glucose makes it as reducing


sugar)

General formula (C12H22O11)

Lactose (Mlik sugar) :

It is present in milk and formed in the lactating mammary gland it may


occur in urine during pregnancy.
It is hydrolysed to glucose and galactose by the enzyme lactose in the
elementary canal and the products are absorbed.
It has free aldehyde group and hence shows mutarotation and the final
constant specific rotation of the solution is +55.2o.
It can exist in or forms.
Since it is a reducing sugar, it can reduce fehlings and benedicts
solution. It cannot reduce barfoeds solution.
It forms osazone with phenylhydrazene

Galactose

glucose

Lactose (-D-galactosyl(14) -D-glucose)


Sucrose (cane sugar) :

It doesnt exist in the body but occurs in the sugar cane, pine apple,
carrot roots, sweet potato and honey.
It is hydrolysed to glucose and fructose by the enzyme invertase in the
alimentary canal. The product of hyrolysis are observed.
It has no free aldehyde group or keto group because the inkage is
between the aldehyde group of glucose and keto group of fructose.
Hence it is non reducing sugar.
It doesnt exhibit mutarotation and cannot exist in or forms.
Since it is a non-reducing sugar, it doesnt reduce fehlings solution and
benedicts solution.
It cannot reduce barfoeds solution.
It cannot form osazone with phenyl hydrazene.
The specific rotation of sucrose is +66.5o.During hydrolysis this solution
changes to -19.5o.

Since the laevo rotation of fructose is greater than dextro roatation of


glucose. The product of hydrolysis is refrred as invert sugar.

Glucose

Fructose

Sucrose ( D glucosyl (12) D-fructose)

Oligosaccharides :
In Greek : oligo few.
Oligosaccharides contain 2-10 monosaccharide molecules which are
liberated on hydrolysis.
Based on the no. of monosaccharide units present, oligosaccharides are
further sub divided to disaccharides, trisaccharides etc.
Ex : of Trisaccharides : raffinose ( Galactose + glucose+ fructose)
Tetrasaccharides : stachyose (2galactose+glucose+fructose)
Pentasaccharides : verbascose (3galactose+glucose+fructose)

PolysacchaRides :

Polysaccharides (or simple glucans) consists of repeating units of


monosaccharides or their derivatives held together by glycosidic
bonds. They are primarily concerned with two important functionsstructural and storage of energy.
Polysaccharides are linear as wel as branched polymer.
The occurance of branches in polysaccharides is due to the fact that
glycosidic linkage can be formed at anyone of the hydroxyl group of a
monosaccharide.
Polysaccharides are of two types :

Homopolysaccharide : which on hydrolysis yield only single type of


monosaccharide.
They are named on the basis of nature of monosaccharide unit.

Thus glucans are polymers of glucose, whereas fructosans are


polymers of
Fructose.

Heteropolysaccharide : On hydrolysis yield a mixture of a few


monosaccharides or their derivates.

Homopolysaccharides :
Starch :

Starch is the carbohydrate reserve of plants which is the most


important dietary source for higher animals, including man.
High content of starch is found in cereals, roots, tubers, vegetables etc.
Starch is a homopolymer compund of D-glucose units held by
glycosidic bond.
It is known as glucosan or glucan.
Starch consists of two polysaccharide components water soluble
amylose (15-20%) and a water insoluble (80-85%).
Chemically, amylose is a long unbranched chain with 200-1000 D
glucose units held by (14) glycosidic linkages.
Amylopectin, on the other hand is a branched chain with (16)
glycosidic bonds at the branching points and (14) linkages
everywhere else.
Starches are hydrolyzed by amylase to liberate dextrins & finally
maltose and glucose units.
Amylose acts specifically on (14) glycosidic bonds.

Amylose

Amylopectin
Dextrins :

Dextrins are breakdown products of starch by the enzyme amylase or


dilute acids.
Starch is sequentially hydrolyzed through different dextrins and finally
to maltose and glucose.
The various intermediates are soluble starch, amylodextrin (violet),
erythrodextrin (red) and achrodextrin (no color).

Inulin :

Inulin is a polymer of fructose i.e, fructosan.


It occurs in dahlia bulbs, garlic, onion etc.
It has low MW (around 5000) polysaccharide.
Easily soluble in water.
Inulin is not utilized by the body.
It is used for assessing kidney function through measurement of
glomerular filteration rate (GFR).

Glycogen :

Glycogen is the carbohydrate reserve in animals, hence often referred


to as animal starch.
It is present in high concentration in liver followed by muscle, brain etc.
Also found in plants that do not possess chlorophyll (Ex:yeast and
fungi)
Structure of glycogen is similar to that of amylopectin with more
number of branches.
Glucose is the repeating unit in glucose joined together by (14)
glycosidic bonds and (16) glycosidic bonds at branching points.
MW (up to 1X108) and the number of glucose units upto 25000 vary in
glycogen depending on the source glycogen is obtained.

General structure of glycogen


Cellulose :

Cellulose occurs exclusively in plants.


Most abundant organic substance in plant kingdom.
Predominant constituent of plant cell wall.
Totally absent in animal body.
Composed of D glucose units linked by (14) glycosidic bonds.
Cannot be digested by mammals including man due to lack of cellulose
enzyme that cleaves glycosidic bonds.
Certain ruminants and herbivorous animals contain microorganisms in the
gut which produces enzymes that can cleave glycosidic bonds.
Hydrolysis of cellulose yields a disaccharide, cellobiose, followed by D
glucose.
Is a major constituent of fibre, the non digestable carbohydrate.
The functions of dietary fibre include decreasing the absorption of glucose &
cholestrol from the intestine besides increasing bulk of feces.

-D-Glucose
-D-Glucose

-D-Glucose

Chitin :

Chitin is composed of N-acetyl D glucosamine units held together by -

(14) glycosidic bonds.


It is structural polysaccharide found in the exoskeleton of some invertebrates.
Ex: Insects, crustaceans.

Heteropolysaccharides :
When the polysaccharides are composed of different types of sugars or
their derivates, they are referred to as heteropolysaccharides or
heteroglycans.
Mucopolysaccharides :
These are heteroglycans made up of repeating units of sugar derivates,
namely amino sugars and uronic acids.

More commonly known as glycosaminoglycans (GAG).


Acetylated amino groups, beside sulfate and carboxyl groups are
generally present in GAG structure.
Mucoproteins may contain upto 95% carbohydrate and 5% protein.
Mucopolysaccharides are essential components of tissue structure.

Important mucopolysaccharides include include hyaluronic acid,


chondroitin 4 sulfate, heparin, dermatan sulfate and keratan sulfate.

Hyaluronic acid :

It is an important GAG found in the ground substance of synovial fluid


of joints and vitreous humor of eyes.
It is also present as a ground substance in connective tissues and
forms a gel around the ovum.
Serves as a lubricant and shock absorbant in joints.
Hyaluronic acid is composed of alternate units of D-glucuronic acid and
N acetyl D glucosamine.
The two molecules form disaccharide units held together by (13)
glycosidic bond.
Contains about 250-25000 disaccharide units (held by 14bonds) with a
MW upto 4 million.
Hyaluronidase is an enzyme that breaks (14 linkages) hyaluronic acid and
other GAG.

Chondroitin Sulfate :

Chondroitin sulfate is a major constituent of various mammalian


tissues (bone, cartilage, tendons, heart vavles, skin, cornea etc).
Consists of repeating disaccharide units composed of D-glucuronic acid
and N-acetyl D galactosamine 4 sulfate.
Chondroitin 6 Sulfate is also present in many tissues (sulfate group is
present on C6 instead of C4)

Heparin :
Heparin is an anticoagulant (prevents blood clotting) that occurs in
blood, lung, liver, kidney, spleen etc.
Heparin helps in the release of the enzyme lipoprotein lipase which
causes clearing the turbidity of lipenic plasma.
Composed of alternating units of N-sulfo D glucosamine 6 sulfate &
glucuronate 2 sulfate.

D glucuronate
2 sulfate

N sulfoglucosamine
6 sulfate

Amino Acid Sequencing


Fredrick Sanger determined the first known protein sequence of bovine
insulin in 1953, establishing that proteins have unique covalent structues.
Knowledge of a proteins amino acid sequence is pre requisite for
determining its 3D structure and is essential for understanding its
molecular mechanism of action. Many inherited diseases are caused by
mutations that result in an amino acid change in a protein. Thus amino
acids analysis can assist in the development of diagnostic tests and
effective therapies.
Sangers determination of the sequence of insulins 51 residues
took about 10 years and required 100g of protein.
The protein must be broken down into
fragments small enough to be indiviually sequenced and the primary
structure of the intact protein is then reconstructred from the sequenced
of overlapping fragments.
Four methods are employed to sequence the protein. They are as follows,
(1)Sangers method
(2)DANSYL chloride method (5-dimethyl amino-1-naphthalene sulfonyl)
(3)Edmans method
(4)Carboxypeptidase method
Sangers, DANSYL cloride and Edmands method are the methods used to
identify N terminal of the amino acid and do the sequencing. Whereas
carboxypeptidase method identify the c terminal of amino acid & do the
sequencing.

I)

Sangers method :

Sanger used the reagent 2,4-dinitrofluorobenzene, which forms a


yellow dinitrophenyl (DNP) derivative at terminal amino groups without
breaking any peptide bonds.

DANSYL Chloride Method :

The fluorescent compound 5-dimethyl amino 1 naphthalene sulfonyl chloride


(Dansyl chloride) reacts with primary amines to yield dansylated polypeptides. The
treatment of a dansylated polypeptide. The treatment of a dansylated polypeptides.
The treatment of a densylated polypeptide with aqueos acid at high temperature
hydrolyzes its peptide bonds. This liberates the dansylated N-terminal residue,
which can be separated by chromatography

N(CH3)2
R1 O
+

R2 O

R3 O

H 2N-CH-C-NH-CH-C-NH-CH-C-OH- - - - (poly peptide)

O=S=O
OH -

5 methyl amino-1
Naphthalene sulfonyl

HCL

Chloride (dansyl chloride)

N(CH3)2

SO2 R1 O

R2 O

R3 O

NH-CH-C-NH-CH-C-NH-CH-C-OH

H 2O
H+

Dansyl polypeptide

N(CH3)2
R2
+ H 3N-CH-COOH
SO2
NH-CH-C-OH
Edmans method :

R3
+ H3N-CH-COOH (Free amino acids)

Edmans degradation named after its inventor, Pehr Edman.

Phenyl Isothiocyanate (PITC) also known as edmans reagent reacts with Nterminal amino group of a polypeptide under mildly alkaline conditions to
form a phenylthiocarbonyl (PTC) adduct. This product is treated with
anhydrous trifluoro acetic acid which cleaves the N-terminal residue as a
thiazolinone derivative but doesnt hydrolyze other peptide bonds
Edmans reaction therefore releases the N-terminal amino acid residue but
leaves the rest of the poly peptide chain.

Carboxypeptidase Method :
This method identifies the C terminal of the polypeptide and sequences it.