BIOCHEMISTRY NOTES

DAB 2 (MOD 2)- TERM 2, 2022 (JUNE)

LESSON FOUR

MORE REACTIONS OF MONOSACCHARADES

1. Oxidation
a) In presence of bromine water (HOBr) monosaccharides get oxidised to
monocarboxylic acid known as aldonic acids

Bromine water does not react with ketoses, thus this reaction can be used to
differentiate an aldose from a ketose
In presence of strong oxidizing agents like HNO3 both aldehyde end and last OH
group get oxidized to form dicarboxylic acid i.e. aldaric.
b) Aldoses can be oxidised by; Fehling’s (alkaline solution of copper (II) ions
complexed with tartrate ions), tollen’s reagent (alkaline solution of Ag+ complexed by
ammonium ions) and benedict’s solution (alkaline solutions of copper (II) ions
complexed with citrate ions). Brown precipitate of copper (I) ions or silver (I) ions
inform of a mirror is formed. Ketoses reduce benedict’s, fehling’s and tollen’s, thus
they can’t be used to differentiate between ketoses and aldoses.
c) Oxidation by periodic acid- Like other compounds that contain two or more –OH
or =O groups on adjacent carbon atoms, carbohydrates undergo oxidative cleavage by
periodic acid, HIO4, as shown below;
i. When periodic acid is added to aldose or pyranoside (glycosides of pyranose) e.g.
glucopyranose or glucopyranoside oxidative cleavage will occur to give out
carboxylic acid and aldehydes.

ii. When periodic acid is added to ketose or furanoside (glycosides of furanose) e.g.
fructofuranose or fructofuranoside oxidative cleavage will occur to give out
carboxylic acid, aldehydes and liberate carbon dioxide

Thus the above two reactions can be used to distinguish between aldoses and ketoses,
pyranoside and furanoside .
Question
Compounds containing hydroxyl groups on adjacent carbon atoms undergo carbon-
carbon bond cleavage when treated with periodate ion (IO4¬). Explain how this
reaction can be used to distinguish between pyranosides and furanosides (4 mks)

2. Reduction- In presence of enzymes or reducing agents like sodium borohydride

(NaBH4) or sodium amalgam (Na/Hg), monosaccharides are converted to alcohols e.g.
glucose is converted into sorbital. Sorbital is used in crypreservation of tissues or
parasites for research.

Methanol can reduce carbohydrates to form glycosides. Glycosides are compounds

containing carbohydrates and non-carbohydrates residue in the same molecules e.g. β-
D-methyl glucoside or α-D-methyl glucoside are formed when methanol (CH3OH)
heated together with β-D-glucose or α- D-glucose respectively in presence of dry
hydrogen chloride gas. This process is called methylation of carbohydrates. Glycoside
bond is formed between carbon one and methyl carbon, marking carbon one to be less
electronegative (less oxidised) and thus lose its anomeric nature. In that case
glycosides formed don’t have anomeric carbon and thus they are non-reducing sugars
and don’t mutarotate (i.e.don’t rotate the polarized ligt) neither do they reduce
Tollens’ or Fehling’s reagents. Treatment of a methyl D- glucoside with methyl
 sulphate and sodium hydroxide brings about methylation of the four remaining OH
groups, and yields a methyl tetra-O-methyl-D-glucoside

3. Osozone formation (only in aldose )- When a monosaccharide(aldose and ketoses) is

heated together with excess phenyl hydrazine (NH2NH(C6H5)), NaCl and sodium acetate for
30-45 min, a precipitate of a compound called osazone is formed. Osazone contains 2 phenyl
hydrazine residue per molecule. Other products include aniline and ammonia.
D-Glucose Glucosazone

Osazone reaction is useful when identifying the epimers. Epimers form the same osazone
which has the same melting point, same crystal structure, and same precipitation time and
height.
Properties of Monosaccharides
1 Solubility- Monosaccharide are soluble in water because of the hydrogen bonding
ability of the OH group, they are only slightly in alcohol (slightly soluble) and extremely
insoluble in non-polar solvents e.g. hydrocarbons.
2 Reducing sugars- All the sugars that contain free sugar group undergo enolization
and various other changes when placed in alkaline solution. The enediol forms of the
sugars are highly reactive and are easily oxidized by oxygen and other oxidising agents
and forms sugar acids. As a consequence they readily reduce oxidising ions such as Cu2+,
Ag+ e.g. This reducing action of sugars in alkaline solution is utilized for both qualitative
and quantative determination of sugars e.g. in clinical tests. Reagents containing Cu2+ions
are most commonly used. They are generally alkaline solution of copper (II) sulphate
either: (i) sodium potassium tartarate (Rochelle) and strong alkali (NaOH/KOH) as in
Fehling solution (ii) Sodium citrate and weak alkali sodium carbonate as in Benedict’s
qualitative reagent. Sodium citrate or Rochelle salt maintains the solubility by preventing
precipitation of copper (II) hydroxide and copper (II) carbonate by forming soluble,
slightly dissociable complexes, which dissociates sufficiently to provide supply of readily
available Cu2+ for oxidation. The alkali of reagent enolizes the sugars and thereby causes
them to be reducing agents. If a solution being tested is heated with one of the reagents,
the Cu2+ takes electrons from enediols, oxidise them to sugar acids, and are in turn
reduced to Cu+ ions. The Cu+ ions combine with the OH ions to form yellow copper (I)
oxide, which upon heating is converted to a brick red copper (I) oxide precipitate. The
appearance of a yellow to red precipitate indicates reduction and the quantity of sugar
present can be roughly estimated from colour and amount of precipitate. Most
dissacharides are reducing sugars because one of their two anomeric carbons is not the
glycosidic linkage. An exception is sucrose, which has both of its anomeric carbons in the
bond that links the two monosacchrides in the bond. Oxidation of D-glucose involves
conversion of the aldehyde group to a –COOH. All sugars containing a free hydroxyl
(OH) group on the anomeric carbon atom are called reducing sugars.
 3 Mutarotation- Direct determination of the configuration of anomeric form is
complicated by the fact equilibrium is set up between the anomers in solution. When
methyl α-D glucopyranoside is dissolved in water and its optical rotation measured by a
polarimeter the specific rotation does not vary with time. When crystals of ordinary D-
(+)-glucose of m.p 1460C are dissolved in water, the specific rotation gradually drops
from an initial +1120 to +52.70. On the other hand, when crystals of D-(+)-glucose of m.p.
1500C (obtained by crystallization at temperature above 980C) are dissolved in water,
specific rotation gradually rises from an initial +190 to +52.70, this changes occur over a
period of a few hours at room temperature. The form with the higher positive rotation is
called α-D-(+)-glucose and that with lower rotation β-D-(+)-glucose. Organic molecules
that change its optical rotation to a certain equilibrium due to change in its anomeric
configuration is called mutarotation.
4 Inter conversion of sugars- Hexose e.g. mannose , glucose and fructose are inter-
convertible in solution of weak alkalinity e.g. Ba (OH)2, Ca (OH)2. This is because they give
the same enediols form which tautomerism to all three sugars. This inter- conversion of
related sugars by action of dilute alkali is referred to as lobryde Bruyn- van ekenstein
reaction. Enediol formation in weak alkali is important for reduction action and also used in
the synthesis of other sugars.
B. OLIGOSACCHARIDES
These are carbohydrates which contains 3-10 monosaccharides linked by glycosidic
bond(s).
Disaccharides
When two monosaccharides are covalently bonded together by glycosidic linkages a
disaccharide is formed. Glycosidic bond is formed when the hydroxyl group on one of the
sugars reacts with the hydroxyl group on anomeric carbon on the second sugar releasing
water.
Biologically important disaccharides are sucrose, maltose, Lactose, cellobiose and
trehalose.
Maltose (malt sugar)
Found in barley, corn and germinating seeds. Maltose contains two D glucose residues (α-
D-glucose and α-D- glucose) joined by a glycosidic linkage between the carbonyl group
at the first carbon atom of the first glucose unit and OH at the fourth carbon atom of the
second glucose unit forming a α-(1-4) glycosidic linkage, water is released. Maltose is the
major degradative product of Starch and is formed when the enzyme amylase hydrolyzes
starch. Mailtose is also formed in one stage of the fermentation of starch to ethyl alcohol;
here hydrolysis is catalyzed by the enzyme diastase, which is present in malt (sprouted
barley). It is a product of the action of salivary amylase (ptyalin) and pancreatic amylase
(amylopsin) up on starch during the process of digestion. It is also formed as an
intermediate product in the acid hydrolysis of starch and is an important constituent of
corn syrups, which are prepared by partial hydrolysis of starch with dilute acids. Maltose
is hydrolyzed to two molecules of D- glucose by the intestinal enzyme maltase, which is
specific for the α-(1-4) glycosidic bond. It is a reducing agent due to presence of free
reactive anomeric OH group thus can react with phenyl hydrazine to form osazones . It
can confer optical activity by rotating plane polarised light and assume alpha and beta
anomers and thus it can mutarotate.
Cellobiose
Two D- glucose (β-D- glucose and β- D- glucose) are Linked by β (1-4) bond. It is a
reducing agent due to free anomeric OH thus it can react with phenylhydrazine to form
osazone and can mutarotate. Obtained by partial hydrolysis of cellulose.

Lactose
It is formed by the secretory cells of the mammary glands during lactation and occurs to
the extent of about 2-6 per cent in the milk. Lactose is a disaccharide of β-D galactose and
β-D- glucose which are linked by β-(1-4) glycosidic linkage. It is prepared commercially
from milk whey. It is hydrolyzed by acids and the specific enzyme lactose in to
constituent monosaccharides. Lactose acts as a reducing sugar since it has a free OH
group on anomeric carbon on second glucose, thus can mutarotate and forms osazones,
and is decomposed by alkali. It is found exclusively in milk of mammals (Milk sugar),
human milk differs from cow’s milk in containing, in addition to regular lactose, other
oligosaccharides such as L-fucosyl lactose.

Trehalose or mycose or tremalose

It is mainly found in fungi and insects and its made up two α-D- glucose joined by α-(1-1)
glycosidic bonds. It has no anomeric OH, thus it is non- reducing agent and don’t
mutarotate.
Sucrose
Sucrose is a disaccharide of α- D- glucose and α-D-fructose. It is obtained from cane
sugar and beetroots. It is also present in various fruits. Ripe fruits are rich in sucrose. It is
called table sugar. Hydrolysis of sucrose by dilute acid or enzyme invertase or sucrose
produces one molecule each of glucose and fructose. In contrast to other disaccharides
sucrose contains no free anomeric carbon atom. Since the anomeric carbons of both its
component monosaccharide units are linked to each other by α (1-2) glycosidic bond. For
this reason sucrose is non reducing sugar and doesn’t mutarotate, thus don’t react with
phenylhydrazine to form osazones. It is relatively stable towards the action of alkali.
When sucrose is heated to about 2000C, it loses water and forms a brown amorphous
mass called caramel. Sucrose is readily fermented by yeast which produces invertase that
split sucrose to glucose and fructose which are then fermented by zymase enzymes still
produced by yeast.