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62
Pharmaceutical Chemistry of Natural Products
Structural elucidation
1. Molecular formula: Molecular formula of stachyose is found to be C
24
H
42
O
21
.
2. Enzymatic hydrolysis: On enzymatic hydrolysis, stachyose gives sucrose and raffi nose.
3. Structure of stachyose: Its exact structure has been established by methylation and
hydrolysis, when it gives 2,3,4,6-tetra-O-methyl-
D-galactose, 2,3,4-tri-O-methyl-D-
galactose, 2,3,4-tri-O-methyl-D-glucose and 1,3,4,6-tetra-O-methyl D-fructose. Thus the
structure of stachyose is as proposed as follows.
OH
H
HOH
1
2
3
4
5
CH OH
2
H
OH
H
O
1
23
4
5
6
H
O
1α
CH
2
H
OH
H
H
OH
OH
H
O
6
O
H
CH
2
H
OH
H
OH
H
H
OH
O
1
2
3
4
5
6
H
O
CH OH
2
O
H
OH
OH
H
H
CH OH
2
1
2
3
4
5
6
D
-Galactopyranose D-Galactopyranose D-Glucopyranose
D-Fructofuranose
POLYSACCHARIDES
The term polysaccharide is usually applied to polymers having more than 10 monomer units.
Polysaccharides usually contain 100–90,000 monosaccharide units. Polysaccharides are defi ned
as high molecular weight (16,000–14,000,000) carbohydrates obtained by condensation of
monosaccharides. They yield ultimately monosaccharides on complete hydrolysis. Some
polysaccharides are linear polymers, while others are highly branched. They are also called
glycans.
In living organisms, polysaccharides function as food reserves and structural elements. Plants
accumulate starch as their main food reserve, a material that is composed entirely of glucopyranose
units. The mammalian carbohydrate storage molecule is glycogen.
Classification and Nomenclature of Polysaccharides
Polysaccharides may be classifi ed into two groups according to their constituent sugars.
Homopolysaccharides
It contains only one kind of monosaccharide unit and they are also called homoglycans. Some
homopolysaccharides serve as storage forms of monosaccharides that are used as fuels (e.g. starch
and glycogen). Other homopolysaccharides, e.g. cellulose and chitin serve as structural elements
in plant cell walls and animal exoskeletons.
Carbohydrates
63
Biological importance
1. Inulin is used in physiological investigation for determination of the rate of glomerular
fi ltration rate.
2. Inulin has also been used for estimation of body water extracellular fl uid volume.
3. Dextrin solutions are often used as ‘mucilages’.
4. Dextran solutions having molecular weight of approximately 75,000 have been used as
plasma expander.
5. Agar is used as laxative in constipation.
6. Agar is used in agar plate for media culture.
Heteropolysaccharides
These are known as heteroglycans, and contain more than one kind of monosaccharide units
(e.g. α-heparin, chondroitin sulphate A and hyaluronic acid). Jeanloz coined the name
‘glycosaminoglycans’ to describe this group of substances. They are usually composed of amino
sugar and uronic acid units as the principal components.
Heteropolysaccharides provide extracellular support for organisms of all kingdoms. For example,
the rigid layer of the bacterial cell envelope (the peptidoglycan) is composed in part of a heteropoly-
saccharide built from two alternating monosaccharide units. In animal tissues, the extracellular space
is occupied by several types of heteropolysaccharides, which form a matrix that holds individual cells
together and provides protection, shape and support to cells, tissues and organs.
Biological importance
1. The invasive power of some pathogenic organisms may be increased because they secrete
hyaluronidase. In the testicular secretions, it may dissolve the viscid substances surrounding
the ova to permit penetration of spermatozoa.
2. Clinically, the enzyme is used to increase the effi ciency of absorption of solutions administered
by clysis.
Polysaccharides may also be classifi ed into two groups according to their biochemical importance.
1. Reserve polysaccharides: Those which contain reserve carbohydrates stored up by plants or
animals (e.g. glycogen, starch and inulin).
2. Skeletal polysaccharides: Those which serve as skeleton in plants and animals (e.g. cellulose).
Nomenclature
Earlier, polysaccharides were named on the basis of their source, which is not systemic. Now the
suffi x ‘an’ is used after the name of the sugar which is a monomeric unit, i.e. ‘ose’ is replaced by
an. For example, fructans are the polysaccharides containing fructose units. The general name for
polysaccharides is glycans.
Polysaccharides of Pharmaceutical Importance
Starch
(Synonyms: amylum, amido, amidon, amilo)
Starch is the most important source of carbohydrates in the human diet. It occurs as a reserve food
material of plants, i.e. it is the form in which glucose is stored. The latter is mobilized for metabolic
uses by enzymatic breakdown. The different sources of starch are grains of maize (Zea mays, Linn,
64
Pharmaceutical Chemistry of Natural Products
Gramineae/Poaceae), rice (Oryza sativa, Linn, Gramineae/Poaceae), wheat (Triticum aestivum, Linn,
Gramineae/Poaceae), tubers of potato (Solanum tuberosum, Solanaceae) and arrow root (Maranta
arundinacea, Marantaceae).
It is a very fi ne powder, white or slightly yellowish powder or irregular, angular white masses
readily reducible to powder, which cracks when pressed between the fi ngers. It is odourless and
tasteless. In addition to its use as a source of heat and energy to body it is also used for the
manufacture of glucose, dextrin. It is also used in oral solid dosage formulations as an excipient
(as a binder, diluent and disintegrant).
Starch contains chemically two different polysaccharides, amylose [α-amylose (about 25%)]
and amylopectin [β-amylose (about 75%)]. Amylose is insoluble in water and amylopectin is water
soluble, but swells in water and is responsible for the gelatinizing property of the starch. Soluble
starch is obtained by partial acid hydrolysis and is completely soluble in hot water. α-Amylose is
insoluble in water and gives blue colour. Amylopectin is soluble in water and gives red to violet
colour with iodine. The blue colour is due to the entrapping of iodine molecules between the
chains of α-amylose. As the blue colour of ordinary starch is entirely due to the adsorptive power
of the α-amylose present, this property is used to estimate the proportion of amylose in starch.
Chemical tests
1. Boil about 10 gm of starch with 50 mL of water and cool. All starches produce thin and
cloudy mucilage except potato starch which gives thick and more transparent mucilage.
2. The above prepared mucilage upon addition of 0.05 mL of iodine turns deep blue. The blue
colour disappears on warming and reappears on cooling.
3. On water bath heat the starch solution with dilute hydrochloric acid for 10 min. Neutralize
the excess of acid then by adding 2–3 drops of sodium hydroxide solution. To this mixture
add 2 mL of Fehling’s solution and heat it on water bath for 15 min. A brick red-coloured
precipitate is formed.
Structural elucidation
1. Molecular formula: Molecular formula of starch is found to be (C
6
H
10
O
5
)
n
.
2. Presence of two structurally different polysaccharides (amylose and amylopectin):
The starch solution when treated with water at 70°C, the water soluble fraction diffuses
out from starch. When n-butanol is added to the hot solution of starch and the mixture
is allowed to cool to room temperature, the α-amylose fraction is precipitated as complex
with n-butanol. The β-amylose fraction (amylopectin) remains in the mother liquor from
which it can be precipitated by adding methanol. α-Amylose is insoluble in water and
amylopectin is soluble in water. This proves that the starch consists of two structurally
different polysaccharides.
3. ␣-Amylose/A-fraction/amylose and -amylose/B-fraction/amylopectin: Most
starches consist of nearly 20% amylose and 80% amylopectin.
To establish the structure of starch, let us discuss the constitution of amylose and amylopectin.
Constitution of ␣-amylose
1. Molecular formula: Molecular formula of amylose is found to be (C
6
H
10
O
5
)
n
.
2. Hydrolysis:
(a) Acid hydrolysis: Hydrolysis of amylose with acids yields D-glucose in quantitative
manner. This indicates that amylase is composed of only D-glucose units.
Carbohydrates
65
(b) Enzymatic hydrolysis: Enzymatic hydrolysis of amylose by diastase yields the quantitative
yield of maltose. As maltose is 4-O-α-D-glucopyranosyl-D-glucopyranose, all the glucose
units in starch are linked through C
1
and C
4
. Therefore, the following structure is
proposed for amylose, which explains the hydrolysis products.
O
H
OH
H
H H
OH
H
O
O
H
OH
H
H H
OH
H
O
CH OH
2
O
H
OH
H
H
OH
H
O
O
H
OH
H
OH
H
O
H
OH
H
O
H
OH
H
O
H
OH
H
OH
H
H
OH
H
O
H
OH
H
H
OH
H
O
OH
O
H
OH
H
OH
H
OH
H
H, OH
Diastase
H
+
C
1
Starch
C
1
C
4
CH OH
2
CH OH
2
CH OH
2
CH OH
2
HH
O
HH
O
C
4
CH OH
2
CH OH
2
HO
2
HO
2
CH OH
2
Linear chain nature of amylose: Hydrolysis of the fully methylated derivative to 2,3,6-tri-
O-methyl-D-glucopyranose and the acetolysis of its methylated derivative to malto-triose and
malto-tetrose derivatives confi rms the structure of amylose. Moreover, the percentage (0.32%)
of 2,3,4,6-tetra-O-methyl-D-glucopyranose reveals that amylose has a chain length of 300–350
glucose units. As no dimethyl glucose is obtained, the chain must be linear and not branched.
The linear structure of the amylose is indicted by the high positive rotation; it is also further
indicated by the fact that amylose acetate forms fi lms and fi bres like cellulose. The chain length
(300–350) of glucose is confi rmed by the molecular weight determination by mass spectra.
Constitution of amylopectin
1. Molecular formula: Molecular formula of amylopectin is found to be (C
6
H
10
O
5
)
n
.
2. Hydrolysis:
(a) Acid hydrolysis: Amylopectin upon acid hydrolysis yields D-glucose units quantitatively;
it indicates that it is also composed of D-glucose units.
(b) Enzymatic hydrolysis: Hydrolysis of amylopectin by β-amylase yields 55% of maltose
and high molecular weight compound called limit dextrin. This reveals that amylopectin
has some other bonds (which are not attacked by β-amylase) in addition to 1,5-linkages
(which are attacked by δ-amylase).
(c) Hydrolysis of methylated amylopectin: Upon hydrolysis, fully methylated amylopectin
gives the following:
(i) 90% of 2,3,6 tri-O-methyl glucose;
(ii) 4% of 2,3,4,6-tetra-O-methyl glucose;
(iii) 4% of 2,3-di-O-methyl glucose.
66
Pharmaceutical Chemistry of Natural Products
This reaction leads to the following important conclusions:
1. Chain length of branches: The yield (4%) of tetra-O-methyl glucose reveals that the
average chain length of branches is about 25 glucose units.
2. Number of branches:
The formation of 2,3-dimethyl glucose suggests that amylopectin is a branched polymer. The
branch chain is linked through C
6
of glucose unit.
The branched structure is further indicated by the fact that osmotic pressure measurements of
amylopectin affords a molecular weight of about 500,000, which corresponds to nearly 3000
glucose units.
The total number of glucose units (3000) and the average number of glucose units (25) in
chain length of amylopectin further indicate that amylopectin has nearly 120 branches.
Nature of linkage: The formation of 2,3-di-O-methyl glucose reveals the presence of branched
structure of amylopectin. This also indicates that the branch chain is linked through C
6
carbon
atom.
The branched structure of amylopectin is supported by the following evidences:
1. Amylopectin acetate does not form fi bres, a characteristic feature of linear molecules
(unlike cellulose and amylase acetates).
2. Upon hydrolysis with β-amylase, amylopectin gives only 55% of maltose. Thus, there are
block points which are not attacked by this enzyme. These block points occur at the branch
points.
3. As the tetramethyl glucose and the dimethyl glucose are formed almost in equal amounts
there would be one branch point for each end group.
4. Partial enzymatic hydrolysis of amylopectin also yields a small amount of isomaltose (1,6-
α-linked diglucose) in addition to the normal product maltose (1,4-α-linked diglucose),
which indicates that C
1
of one glucose unit and C
6
of the other glucose unit are involved in
branching, i.e. branching involves 1,6-linkages.
O
H
OH
H
OH
H
H
O
H
OH
H
H
O
H
OH
H
OH
H, OH
Maltose
O
H
OH
H
OH
H
H
OH
H
O
O
CH
2
H
OH
H
OH
H
H
OH
H, OH
Isomaltose
CH
2
OH CH
2
OH
CH
2
OH
Carbohydrates
67
5. The 1,6-linkages are further confi rmed by periodate oxidation method.
6. From the foregoing points, structure of amylopectin may be represented as follows:
Amylopectin
O
H
OH
H
OH
H
H
OH
H
CH
2
OH
O
H
OH
H
H
H
OH
H
O
O
H
OH
H
H
H
OH
H
O
O
H
OH
H
H
O
H
OH
H
O
O
O
H
OH
H
H
H
OH
H
O
O
H
OH
H
H
H
OH
H
O
CH
2
OH CH
2
OH
CH
2
CH
2
OH CH
2
OH
7. Origin points: The points of origin of the branches are still not settled in amylopectin.
Various structures have been suggested and the two most acceptable structures are described
below:
Haworth laminate structure: According to this, every chain is composed of nearly 24–25 glucose
units and the branch arises from the middle of the chain.
Thus, β-amylase hydrolyses only half of the total glucose units of amylopectin to form maltose
and the residue as limit dextrin. Because after this the 1,6-linkages start to which β-amylase is
not effective. Using end group assay it has been observed that dextrin has a unit chain length of
11–12 glucose units to support the above explanation.
O
O
O
O
O
1212
1212
1212
1212
12
12
12
12
12
= Non reducing groups or end groups giving 2,3,4,6-tetramethyl glucopyranoses
= Chains of α-1,4 linked glucopyranoses giving 2,3,6-trimethyl glucopyranoses
= Reducing end groups
= -1,6-Linkages giving 2,3-dimethyl glucopyranosesβ
O
Amylopectin
Maltose +
Limit dextrin
β–amylase
68
Pharmaceutical Chemistry of Natural Products
Meyer random structure: According to this, amylopectin is a multibranched structure and
which explains the behaviour of amylases on amylopectin in a better way.
O
O
O
OOO
OO
O
Enzymatic hydrolysis of starch: By attacking the 1,4-linkages of nonreducing end, β-amylase
hydrolyses the amylopectin of the outer chains to about 60% maltose and the residual
polysaccharide is known as limit dextrin. But the enzyme α-amylase attacks 1,4 glycosidic linkages
at random, i.e. indiscriminately in both inner and outer chains of amylopectin.
Lastly, the enzyme isoamylase from yeasts or R-enzyme from higher plants attacks the 1,6-
glycosidic linkages only. The hydrolytic scheme of starch and its reaction with iodine are shown
below:
Course of Hydrolysis: Reaction with Iodine
Starch Blue
Soluble starch Blue
Amylodextrin Purple
Erythrodextrin Red
Achrodextrin Colourless
Maltose
Cellulose
It is reputedly the most abundant organic material on earth, being the main constituent in plant
cell walls. It is composed of glucopyranose units linked β1→4 in a linear chain.
Carbohydrates
69
PROBABLE QUESTIONS
1. Defi ne and classify carbohydrates with suitable examples. Enumerate the various chemical reactions exhibited by the
reducing sugars and explain any two of them.
2.
Explain Kiliani’s synthesis of a higher monosaccharide from the lower one.
3. How is the cyclic nature of glucose established?
4. Write in detail about the chemistry and structure of glucose.
5. How many isomers of
D-glucose are possible? Draw their confi gurational formulae and explain the methods used to
determine the size.
6. How will you determine the structure of disaccharides?
7. Defi ne and classify polysaccharides and write their biological importance.
8. Write in brief about the structural elucidation of starch.
9. Explain the steps in the development of complete structure of
D-glucose.
10. Briefl y describe the methods for determination of ring structure of hexose sugar.
11. Describe the preparation of glucosazones.
12. Defi ne mutarotation and explain it with chemical reaction.
13. Describe the experimental evidence for the ring structure of glucose.
14. Write the general methods used in establishing the cyclic structure of hexose.
15. How is the confi guration of monosaccharide determined?
16. Mention some examples for disaccharides and describe the structural determination of any one of the disaccharides.
17. Write in detail about the following reactions:
a. Acetylation of hexose
b. Preparation of osazone
18. What are the different disaccharides? Describe the structural elucidation of maltose.
19. Describe the confi gurational formula of fructose. Indicate the evidence leading to the ring structure of fructose.
20. How will you prove the structure of sucrose?
21. Write in detail the general methods for synthesis of glycosides.
22. Outline the constitution of starch.
23. How are amylose and amylopectin separated from natural starch? Describe the constitution of amylose and
amylopectin.
24. Write a brief note on the following:
a. Epimerization
b. Photosynthesis of carbohydrates
c. Mutarotation
25. Answer any three of the following:
a. Why glucose and mannose form the same osazone?
b. How will you convert
D(+)-glucose into mannose?
70
Pharmaceutical Chemistry of Natural Products
c. What are the stages involved in the conversion of glucose to arabinose?
d. How can you show whether a given hexose is pyranose or furanose?
e. How will you prove that fructose is a 2-ketohexose and not a 3-ketohexose?
26. Write equations to show how
D(+)-glucose can be converted into 2,3,4,6-tetramethyl-D-glucose.
27. Describe the importance of methylation and periodate oxidation in the structural determination of carbohydrate
chemistry.
28. Describe the evidence in support of the accepted structure of maltose.
29. What is the other name for sucrose? Explain why this name has been given.
30. Write the various methods adopted for synthesis of monosaccharides by descending in sugar series.
31. Write a note on confi guration of monosaccharides.
32. Write the various methods adopted for synthesis of monosaccharides by ascending in sugar series.
33. Enumerate the methods adopted for the determination of the ring size in sugars and explain any one of them with suitable
example.
34. How will you prove that glucose exists as ring structure and not as open chain structure?
35. How will you classify disaccharides? Mention few disaccharides and write the biological importance of disaccharides.
36. Explain the following with suitable examples:
a. Kiliani’s synthesis
b. Osazone reaction
c. Epimerization
d. Mutarotation
e. Invert sugar
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Bernfeld, P (ed) (1967). Biogenesis of Natural Compounds, 2nd Ed. Pergamon, Oxford.
Bourne, EJ and Finch, P (1970). Polysaccharides—enzymic synthesis and degradation. R Inst Chem Rev 3 (1), 45–60.
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