MT6310 Biochem MONOSACCHARIDES

3rd Shifting • Building blocks of carbohydrates

Carbohydrates: Structure, Function and Nomenclature • Two types:
o Aldose – polyhydroxy aldehyde
Carbohydrates – the most abundant organic compounds o Ketose – polyhydroxy ketone
found in nature
- a biological compound containing carbon, Glyceraldehyde
hydrogen, and oxygen that is an important
source of food and energy
- the formula of most of these compounds may be
expressed as CnH2nOn

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

CH2OH CH2OH CH2OH CH2OH

O O O
• The D form is usually the isomer found in nature.
O
OH OH OH OH

▪
O
Polysaccharide O
OH
O
OH OH
O
OH

• Enantiomers – stereoisomers that are mirror images

but not superimposable
According to Functional Group
- farthest carbon from the aldehyde determines D/L
Aldose Ketose
designation (penultimate carbon)
O
CH2OH
CH

C O
H C OH

CH2OH CH2OH

According to Number of Carbons

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

Mutarotation – small amount of open chain is in equilibrium

with the cyclic forms
- The most stable form of glucose is β-D-glucose
-D-glucose D-glucose (open) β-D-glucose
(36%) (trace) (64%)

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

Haworth to Chair Conformation

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
- GLYCOGEN – energy storage liver / muscle
- STARCH – energy storage in plants
- CELLULOSE – structural
• Overall non-reducing (contains a very small
concentration of anomeric carbons in the entire
structure)
Cellulose
Starch
Amylopectin
a-Amylose
Chitin
Glycoconjugates
Peptidoglycans
- Found in bacterial cell wall; provides mechanical
strength to the cell wall.
- Polysaccharide crosslinks with peptide
- 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
and inner lipid bilayer membranes
Glycosaminoglycans
- Type of polysaccharide based on repeating
disaccharide units
- One of the sugars is an amino sugar (either D-
glucosamine or D-galactosamine) which carries a
negative charge due to either sulfate or carboxylic
group.
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-
acetyl-D-galactosamine-sulfate.
• 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.
Proteoglycans
- Found in cartilage and connective tissues;
extremely high carbohydrate content
- Proteoglycan subunit is consists of
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
- antigen – no A or B antigen
Nomenclature
I. Oligosaccharide without a free hemiacetal group
- is named as glycosyl glycoside.
The parent structure (glycoside) is based on the following
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.

III. For higher oligosaccharides (i.e. trisaccharides)

- they are named as glycosylglycosyl glycosides or
glycosyl glycosylglycosides (with nonreducing end)
or glycosyl[glycosyl]nglycose (with reducing end).
- Alternatively, a sequential (end-to-end) naming
approach may be used.
Use of symbols for defining oligosaccharide structures
• Oligosaccharide and polysaccharide structures occur
not only in free form but often as parts of glycopeptides
or glycoproteins or of glycolipids.
• It can be cumbersome to designate their structures by
using the previous naming of oligosaccharides.
• The use of three-letter symbols for monosaccharide
residues is therefore recommended. With appropriate
locants and anomeric descriptors, long sequences can
thus be adequately described in abbreviated form.

• In extended form, the branch connection is explicitly

depicted.

• In short notation, it is derived by omitting:

- locants of anomeric carbon atoms,
 - the parentheses around the locants of the linkage, MT6310 Biochem
and hypens 3rd Shifting
- Branches can be indicated on the same by using Experiment 5: Carbohydrates
enclosing marks (parentheses, square
brackets,etc.) • General formula: Cn(H2O)n
Glcα4(Glcα6)Glc • Examples: sugar, starch, fibers
• “Go” foods – primary source of energy
• In biochemistry, they are known as “saccharides”

Isolation of Polysaccharides
Starch from Glycogen from
cassava chicken liver
Addition of H2O HOAc
EtOH

• For homopolysaccharide, replace the ending -ose by

MOLISCH TEST
an –an, such as (1→4)-α-D-glucan. - Common test for all carbohydrates larger than
• For heteropolysaccharide, named as glyco-glycan. All tetroses (pentoses and hexoses) if it will be
glycose residues should be cited alphabetically. dehydrated by H2SO4 to produce an aldehyde
- Aldehydes: furfural or hydroxymethylfurfural
- These aldehydes will condense with a-naphthol
(phenolic structure)
- Product: purple color at the interface

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
polysaccharide known as starch by hydrolysis
using enzymes
- The ability of humans to digest the
disaccharide lactose, "milk sugar", decreases
as we develop due to a decline in the activity
of the enzyme lactase.
o Lactose intolerance results from low levels
of the enzyme lactose and hence
incomplete hydrolysis of lactose.
- Human beings cannot digest the
polysaccharide known as cellulose because we
lack an appropriate enzyme to hydrolyse
cellulose.
o Cellulose acts as dietary fibre in our diet.
FEHLING’S TEST
- reduction test of carbohydrates
- Fehling’s solution – contains blue alkaline cupric
hydroxide solution
o Contains copper (II) tartrate
- It is heated with reducing sugars gets reduced to
yellow or red cuprous oxide
- Product: yellow or brownish-red colored
precipitate
o helps in the detection of reducing sugars in the
test solution.
BARFOED’S TEST
- used to detect the differentiate reducing
monosaccharides and disaccharides
- Monosaccharides: ( < 3mins.)
- Disaccharides (~10 mins)
- Barfoed's reagent: a mixture of acetic acid and
copper(II) acetate, is combined with the test
solution and boiled.
- Positive result: presence of reducing sugar
- Product: red copper(II) oxide precipitate
SELIWANOFF TEST
- a color reaction specific for ketoses.
- Differentiation test for aldose (-) and ketose (+)
- Conc HCl: ketoses undergo dehydration to yield
furfural derivatives more rapidly than aldoses.
o The test reagent causes the dehydration of
ketohexoses to form 5-hydroxymethylfurfural.
- These derivatives form complexes with resorcinol
o 5-hydroxymethylfurfural reacts with resorcinol
present in the test reagent to produce a red
product within two minutes
- Product: deep red color/cherry red solution
- Aldohexoses reacts so more slowly to form the
same product

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