(BIOCHEM LAB) Carbohydrates
(BIOCHEM LAB) Carbohydrates
(BIOCHEM LAB) Carbohydrates
Functions
• Structural: cellulose, chitin, peptidoglycan
• Energy storage: starch, glycogen Fischer Projection
• Biologically active: - two-dimensional representation of the
o Transport: glycoproteins in plasma (transferring) stereochemistry of three-dimensional molecules
o Regulatory: glycoproteins like FSH, LH, TSH
O 1
o Catalytic: glycoproteins (ribonuclease, -amylase) H 1 CH2OH
C
o Immune response: Ig, interferons, Rh factors 2
2 C O
o Cell lubrication & supportive function: H C OH
3
sialoglycoproteins 3 HO C H
o Cell differentiation: ABO blood grouping HO C H
4
4
o Cell membrane, clotting factors, & protective H C OH H C OH
cellular coat proteins: glycocalyx, fibrinogen, 5 5
H C OH H C OH
prothrombin
6 6
CH2OH CH2OH
Structures
According to Number of Sugar Units D, L Notation
• If the –OH group on the next to the bottom carbon
▪ Monosaccharide atom points:
CH2OH CH2OH
• Right: D-isomer
O O
OH OH
▪ Disaccharide OH O OH
• Left: L-isomer
OH OH
▪
O
Polysaccharide O
OH
O
OH OH
O
OH
C O
H C OH
CH2OH CH2OH
Pentose
Hexose
Cyclic Structure Formation / Haworth Projection
HEMIACETALS / HEMIKETALS
- Hemi – “half”
- Formed by addition of alcohol (nucleophile) to the
Optical Isomers carbonyl (electrophile)
- also called stereoisomers - Responsible for the formation of the closed chain
- molecules that differ from each other only in configuration of saccharides
configuration of structures - Pyranose and furanose
6
- usual source of optical isomerism is a chiral carbon CH2OH 1
6
CH2OH O CH2OH
H 5 H
C O
2
Diastereomers OH H C H OH C
4 C H C 1
5
- stereoisomers that are non-superimposable, non- C C
3 C C2 H OH
OH OH 4 3
mirror image
H OH OH H
H
O H
O Hemiacetal Hemiketal
C C
H C OH HO C H
H C OH H C OH
CH2OH CH2OH
D-Erythrose D-Threose
Epimers
- diastereomers that differ from each other in
configuration at only one chiral carbon
O O
H H
C C
H C OH H C OH
H C OH HO C H
H C OH H C OH
CH2OH CH2OH
D-ribose D-xylose
1. Sugar Acids
• Aldonic Acid
Anomers
- stereisomers that differ in configuration at the
anomeric carbon
6
CH2OH 6 • Aldaric Acid
CH2OH
H 5 H H OH
C O 5
C O
OH H OH H
4 C H C 1 4 C C 1
H
3 C C2 C C2
OH OH OH 3 H
H OH H OH
2. Sugar Alcohol
a-D-glucose B-D-glucose • Alditol
Haworth Projection
- perspective representation of the cyclic forms of
sugars
Furanose – Furan
O
O O 3. Deoxysugars
C C
C C
Pyranose – Pyran
C O O
O
C C
C C
Chair Conformation
4. Amino Sugars
Derivatives of Monosaccharides
5. Sugar Phosphates
▪ Sucrose – Glucose + Fructose
Glycosidic Bonds
Lactose
DISACCHARIDES
- Linked by O-glycosidic bond
- Interaction between the anomeric carbon Maltose
(electrophile) and the lone pair e - (nucleophile)
from the primary alcohol
- Reducing disaccharide is produced if there is one
free anomeric carbon outside of the glycosidic
bond
Sucrose
Cellobiase
Lactose
OLIGOSACCHARIDE
- Carbohydrates that have 2-10 sugar molecules
- glycoproteins, glycolipids
POLYSACCHARIDES
- Carbohydrates that have more than 10 sugar
molecules
Important Disaccharides - Storage polysaccharides: cellulose, glycogen, starch
▪ Maltose – Glucose + Glucose
▪ Lactose – Glucose + Galactose • Glycogen and Starch
- Homopolysaccharides – hydrolysis will yield only
one kind of monosaccharide Glycosaminoglycans
- GLYCOGEN – energy storage liver / muscle - Type of polysaccharide based on repeating
- STARCH – energy storage in plants disaccharide units
- CELLULOSE – structural - One of the sugars is an amino sugar (either D-
• Overall non-reducing (contains a very small glucosamine or D-galactosamine) which carries a
concentration of anomeric carbons in the entire negative charge due to either sulfate or carboxylic
structure) group.
Cellulose Examples:
• Keratan sulfates – found in connective tissues of
vertebrates; contains alternating units of D-galactose
and N-acetyl-D-glucosamine-6-sulfate.
• Chondroitin sulfate – major component of cartilage;
polymer of alternating units of D-glucuronate and N-
Starch acetyl-D-galactosamine-sulfate.
Amylopectin
• Hyaluronic acid – found in synovial fluid and vitreous
humor of the eye; heteropolymer of alternating units of
glucuronic acid and N-acetylglucosamine.
• Heparin – natural anti-coagulant, blocks blood
coagulation; polymer of D-iduronate-2-sulfate and N-
sulfo-D-glucosamine-6-sulfate.
a-Amylose
Proteoglycans
- Found in cartilage and connective tissues;
extremely high carbohydrate content
- Proteoglycan subunit is consists of
Chitin glycosaminoglycans (most often keratan and
chondroitin sulfate) covalently linked to protein
molecules called core protein.
- Each proteoglycan monomer is attached to
hyaluronic acid which is stabilized by linked protein.
Glycoproteins
- A antigen – α-D-N-acetylgalactosamine is found at
the non-reducing end of the oligosaccharide
- B antigen – α-D-galactose
Glycoconjugates - antigen – no A or B antigen
Peptidoglycans
- Found in bacterial cell wall; provides mechanical Nomenclature
strength to the cell wall. I. Oligosaccharide without a free hemiacetal group
- Polysaccharide crosslinks with peptide - is named as glycosyl glycoside.
- Peptidoglycan’s polysaccharide is repeating units of
N-acetylmuramic acid (NAM) and N-
acetylglucosamine (NAG) in β(1-4) glycosidic bonds.
- Tetramer are covalently bonded to NAM.
- Neighboring parallel peptidogylcan chains are
covalently croslinked through glycine pentamers.
- Cell walls in Gram-positive bacteria consist of a
cross-linked multilayered peptidoglycan, layered on
top of a lipid bilayer membrane, whereas the cell
walls in Gram-negative bacteria consist of a single
layer of peptidoglycan sandwiched between outer The parent structure (glycoside) is based on the following
and inner lipid bilayer membranes criteria:
1. The greatest number of occurrences of the most
preferred functional group: aldaric acid > uronic
acid/ketoaldonic acid/aldonic acid > dialdose >
ketoaldose/aldose > diketose > ketose
2. The parent with the greatest number of carbon atoms
in the chain, e.g. a heptose rather than a hexose.
3. The parent with the name that comes first in an
alphabetical listing based on:
a) the trivial name or the configurational prefix(es) of
the systematic name, e.g. allose rather than
glucose, a gluco rather than a gulo derivative;
b) the configurational symbol D rather than L ;
c) the anomeric symbol α rather than β.
IV. For branched oligosaccharides (i.e. trisaccharides)
- are named by enclosing the branches in square
II. Oligosaccharides with a free hemiacetal group
brackets.
- are depicted with reducing glycose residue on the
- In a branched chain, the longest chain is regarded
right and the nonreducing glycosyl group in the
as the parent. If two chains are of equal length the
left.
one with lower locants at the branch point is
preferred.
Isolation of Polysaccharides
Starch from Glycogen from
cassava chicken liver
Addition of H2O HOAc
EtOH
IODINE TEST
- A chemical test for starch by adding iodine solution
(yellow/brown)
- Positive result: presence of starch
- Product: iodine turns a blue/black color (starch-
iodine complex)
- It is possible to
distinguish starch
from glucose (and
other carbohydrates) using
this iodine solution test.
- Starch contain polymer
of α-amylose and
amylopectin which forms a
complex with iodine to give
the blue black colour.
ACIDIC HYDROLYSIS
- Complete hydrolysis of disacch/polysacch
- Product: monosaccharide
- Produces monosaccharides by breaking the
glycosidic links (ether bonds) between
monomer units in the structure of the
molecule.
- Acidified water/acidic conditions
ENZYMATIC HYDROLYSIS
- Breaks specific bonds
- Salivary amylase will break down α-1,4 glycosidic
bonds
- Human beings can digest disaccharides and the BARFOED’S TEST
polysaccharide known as starch by hydrolysis - used to detect the differentiate reducing
using enzymes monosaccharides and disaccharides
- The ability of humans to digest the - Monosaccharides: ( < 3mins.)
disaccharide lactose, "milk sugar", decreases - Disaccharides (~10 mins)
as we develop due to a decline in the activity - Barfoed's reagent: a mixture of acetic acid and
of the enzyme lactase. copper(II) acetate, is combined with the test
o Lactose intolerance results from low levels solution and boiled.
of the enzyme lactose and hence - Positive result: presence of reducing sugar
incomplete hydrolysis of lactose. - Product: red copper(II) oxide precipitate
- Human beings cannot digest the
polysaccharide known as cellulose because we SELIWANOFF TEST
lack an appropriate enzyme to hydrolyse - a color reaction specific for ketoses.
cellulose. - Differentiation test for aldose (-) and ketose (+)
o Cellulose acts as dietary fibre in our diet. - Conc HCl: ketoses undergo dehydration to yield
furfural derivatives more rapidly than aldoses.
FEHLING’S TEST o The test reagent causes the dehydration of
- reduction test of carbohydrates ketohexoses to form 5-hydroxymethylfurfural.
- Fehling’s solution – contains blue alkaline cupric - These derivatives form complexes with resorcinol
hydroxide solution o 5-hydroxymethylfurfural reacts with resorcinol
o Contains copper (II) tartrate present in the test reagent to produce a red
- It is heated with reducing sugars gets reduced to product within two minutes
yellow or red cuprous oxide - Product: deep red color/cherry red solution
- Product: yellow or brownish-red colored - Aldohexoses reacts so more slowly to form the
precipitate same product
o helps in the detection of reducing sugars in the
test solution.
BENEDICT’S TEST
- Free aldehyde or keto group in the reducing sugars
reduce cupric hydroxide in alkaline medium
BIAL’S ORCINOL TEST
- Product: Brick red ppt cuprous oxide
- used to distinguish between pentoses and hexoses.
- Depending on the concentration of sugars, yellow
- They react with Bial’s reagent and are converted to
to green color is developed .
furfural
- All monosaccharides are reducing sugars as they
- Orcinol and furfural condense in the presence of
all have a free reactive carbonyl group.
ferric ion form a colored product.
- Some disaccharides, like maltose, have exposed
- Pentoses: green colour or bluish-green solution
carbonyl groups and are also reducing sugars, but
- Hexoses: muddy brown precipitate
less reactive than monosaccharides
- Oxidizing agent: Cu2+
- Sodium citrate used to keep Cu ions in solution
MUCIC ACID
- Used for the presence of galactose
- Conc. HNO3: is heated along with an aldose sugar to • The more –OH groups, the more polar
give a dicarboxylic acid. • glucose > maltose > dextrin in Rf value
- Nitric acid is able to oxidize the terminal groups of
aldoses, but leaves the secondary hydroxyl groups NELSON ASSAY
unchanged - process used to determine glucose
- The dicarboxylic acid formed from galactose is - Nelson-Somogyi Method
called mucic acid and is insoluble in cold aqueous - Is a widely used classical method for the
solution. quantitative determination of reducing sugars
- Visible result: broken glass-like - Demonstrates how much glucose is liberated from
crystals (galactaric acid) glycogen during enzymatic and acid hydrolysis
- HNO3 is a strong oxidizing agent - Characterization of carbohydrates was done by
computing the absorbance
- Can differentiate carbohydrates from each other as
well because it differ in their light absorbances and
values
PHENYLHYDRAZONE TEST
The ketoses and aldoses react with
phenylhydrazine to produce a phenylhydrazone
which further reacts with another two molecules of
phenylhydrazine to yield osazone.
o formyl functional group will get oxidized to
carboxylic acid
o Secondary alcohol will get oxidized to ketone
- phenylhydrazine is a strong oxidizing agent
- Phenylhydrazone + phenylhydrazine = osazone
- Visible Result: Yellow orange crystals (osazones)
- Glucose, fructose and mannose: needle-shaped
yellow osazone crystals
- Lactosazone: mushroom shaped crystals
- Maltose: flower-shaped crystals
THIN-LAYER CHROMATOGRAPHY
• Mobile phase:
• Para-anisaldehyde: visualizing agent
- Green colored spots – based on complex with the
sugar