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82
Pharmaceutical Chemistry of Natural Products
STRUCTURAL DETERMINATION OF GLYCOSIDES
The structure of glycosides is fi rst determined by degradation studies and then it is confi rmed by
synthesis.
Degradation
1. The glycoside obtained upon extraction and isolation is hydrolysed with dilute acids to yield
sugar and aglycone parts. These are then separated by chromatographic method. After the
separation of glycone and aglycone parts, they are identifi ed and their structure is established
by the usual methods.
2. The confi guration of the glycoside linkage is determined by enzyme hydrolysis. The enzyme
emulsin hydrolyses the β-glycosides while maltase hydrolyses α-glycosides.
3. Specifi c enzymes are used to affect only partial hydrolysis of the glycoside, if the sugar unit
is disaccharide or trisaccharide, so as to identify the oligosaccharide.
Aglycone-sugar sugar
12
⫺ Aglycone-sugar sugar
12
⫺
Disaccharide
Aglycone-sugar sugar
12
⫹
Aglycone sugar⫹
1
4. If more than one hydroxyl groups are present, the hydroxyl group that is involved in the
glycosidic linkage should be established.
5. The ring size of the sugar is confi rmed by the usual method, i.e. complete methylation
followed by hydrolysis. Refer to page 32 under the heading of Determination of Size of the
Ring of sugars in Chapter 2.
Synthesis
The structure of a glycoside which was established by the degradation reactions is then confi rmed
by the synthesis.
The synthesis of a glycoside involves the reaction between an O-acetyl-α-glycosyl bromide
and an alcohol or phenol (i.e. the aglycone) in the presence of silver carbonate in anhydrous
benzene. O-Acetyl-α-glycosyl bromide is prepared by acetylating aldohexose in the presence of
zinc chloride to give α- or β-pentaacetate which is then treated with glacial acetic acid saturated
with hydrogen bromide to form α-acetobromohexose. During the glycoside synthesis, Walden
inversion takes place, and therefore, β-linked glycosides are produced. If the reaction is performed
in the presence of mercuric acetate or ferric chloride in place of silver carbonate, α-D-glycosides
are obtained.
Glycosides
83
GLYCOSIDES OF PHARMACEUTICAL IMPORTANCE
Salicin
O C
C
C
C
C
H
OHH
HO H
H
OH
H
O
HOH C
2
CH OH
2
3-(Hydroxymethyl)-7-(2-(hydroxymethyl)phenoxy)oxonane-4,5,6-triol
Salicin is a bitter glycoside present in most willow and poplar bark. It is an optically active solid drug
having levorotatory property. It was isolated in the nineteenth century from numerous species of
Salix (S. fraglis, S. purpurea) family Salicaceae. Salicin is oxidized to salicylic acid and hence it acts
an antipyretic and anti-infl ammatory agent. Interestingly, this willow bark was prescribed for pain
relief in the fi fth century AD.
Structural elucidation
1. Molecular formula: The molecular formula of salicin is found to be C
13
H
18
O
7
.
2. Hydrolysis: Salicin upon hydrolysis with emulsin yields 1 mole of D-glucose and 1 mole
of salicyl alcohol (saligenin). This reveals that the salicyl alcohol and D-glucose units are
linked via β-linkage in salicin.
OH
⫹
Emulsin
Salicin Salicyl alcohol
D-Glucose
CHO
13 18 7
CH OH
2
3. Now the question is how saligenin condensed with β-D-glucose? That is, whether the
alcoholic or the phenolic hydroxyl group of salicyl alcohol is linked with D-glucose. This is
answered by the following chemical reactions:
(a) The colour reaction of salicin with neutral ferric chloride indicates the absence of free
phenolic –OH in it.
(b) On mild oxidation with nitric acid followed by hydrolysis, salicin yields salicylaldehyde
(helicin). The formation of aldehyde reveals the presence of free alcoholic group
in salicin. Therefore, phenolic hydroxyl group must be involved in the β-glucosidic
linkage.
84
Pharmaceutical Chemistry of Natural Products
4. Synthesis: The above proposed structure of salicin is further confi rmed by the synthesis
Tetra-O-methyl-α-D-glucopyranosyl-1-bromide upon condensation with salicyl alcohol in
the presence of silver carbonate yields pentamethyl salicin, which is identical to that of the
product obtained from the natural salicin by methylation.
C
C
C
C
C
H
Br
H
H
H
H
⫹
OH
O
C
C
C
C
C
H
H
H
H
H
Pentamethyl salicin
O
O
HCO
3
OCH
3
OCH
3
CH OCH
23
HOH C
2
i) Ag CO
23
ii) CH ) SO(
32 4
H COH C
32
HCO
3
OCH
3
OCH
3
CH OCH
23
(CH ) SO
32 4
O
C
C
C
C
C
H
OH
H
OH
H
HO
H
H
O
Salicin
HOH C
2
CH OH
2
Ruberythric Acid
C
C
C
C
C
H
HO
H
H
CH
2
O
C
C
C
C
H
HO
H
O
OH
H
OH
H
O
OH
H
OH
H
O
O
OH
O
HC
2
1-Hydroxy-2-(5,6,7-trihydroxy-8-(2-((5,6,7-trihydroxyoxonan-4-yl)methoxy)ethyl)
oxonan-4-yloxy)anthracene-9,10-dione
Glycosides
85
It is obtained from madder root [Rubia tinctorum (Family: Rubiaceae)] as yellow prisms having silky
lustre. Originally the root was cultivated as a dyestuff, but later on many of the anthraquinone
glycosides were isolated from this, among which ruberythric acid is a chief constituent. It has a
faint taste, sparingly dissolves in cold, easily in hot water.
Structural elucidation
1. Molecular formula: The molecular formula of ruberythric acid is found to be C
25
H
26
O
13
.
2. Hydrolysis with emulsin: Ruberythric acid upon hydrolysis with emulsin, gives 1 mole
each of D-glucose, D-xylose and alizarin.
O
O
OH
OH
D-Glucose
D-Xylose
CHO 2HO
25 26 13 2
⫹ CH O +
6126
CH O
5105
⫹
Ruberythric
acid
Alizarin
3. Hydrolysis with enzyme of Primula offi cinalis: When hydrolysed with the enzyme of
Primula offi cinalis, alizarin and a disaccharide are obtained. By the usual standard method
it was found that the disaccharide is identifi ed as 6-O-β-D-xylopyranosyl-D-glucopyranose
(primeverose). The disaccharide on mild hydrolysis gives D-glucose and D-xylose. The
formation of primeverose as a hydrolysate indicates that alizarin is linked to the glucose
unit of the primeverose.
COOH
C
C
C
C
H
HO
H
H
CH
2
O
C
C
C
C
H
HO
H
O
OH
H
OH
H
O
OH
H
OH
HC
2
6-O- -D-Xylopyranosyl-D-glucopyranose
or
Primeverose

4. Now the question is to determine which hydroxyl group of alizarin is involved in the
β-glycoside formation.
Alizarin-1-methyl ether was obtained upon methylation and hydrolysis of ruberythric acid,
which reveals that the C
1
of alizarin is free and hence the primeverose molecule is linked
through C
2
of the alizarin molecule to give ruberythric acid.
5. Synthesis: The above structure established by degradation studies is confi rmed by the
synthesis.
86
Pharmaceutical Chemistry of Natural Products
O
O
OH
OH
Alizarin
O
OH
O
O
C
C
C
C
C
H
H
AcO
H
H
CH
2
O
C
C
C
C
H
AcO
H
O
OAc
H
OAc
H
O
OAc
OAc
H
HC
2
Br
Hexa-O-acetylprimeverosyl bromide
(i) Ag CO
(ii) NH
23
3
C
C
C
C
C
H
HO
H
H
CH
2
O
C
C
C
C
H
HO
H
O
OH
H
OH
H
O
OH
H
H
OH
HC
2
Ruberythric acid
+
Identification tests
1. It dissolves in boiling aqueous ferric chloride forming dark brown–red solution, which is
precipitated by alcohol.
2. It dissolves with gold–yellow colour in alcohol and ether.
Arbutin
3-(Hydroxymethyl)-7-(4-hydroxyphenoxy)oxonane-4,5,6-triol
O
C
C
C
C
C
H
HO
H
H
CH
2
OH
O
OH
OH
H
H
OH
Glycosides
87
It is obtained from bearberry (dried leaves of Aristostaphylos uvaursi, Family: Ericaceae). Arbutin is
a colourless crystalline compound (melting point: 187°C). It is optically active with levorotatory
property. It is used as a diuretic in urethritis.
Structural elucidation
1. Molecular formula: The molecular formula of arbutin is found to be C
12
H
16
O
7
.
2. Presence of phenolic group: Arbutin upon reaction with neutral ferric chloride gives
blue–violet colour, which indicates the presence of phenolic group in it.
3. Hydrolysis: Arbutin yields one molecule of D-glucose and one molecule of hydroquinone
upon hydrolysis with dilute hydrochloric acid or emulsin, which indicates that in arbutin
structure, D-glucose and hydroquinone are linked by means of β-linkage. Therefore, the
structure of arbutin is proposed to be I.
4. The above proposed structure of arbutin is confi rmed by its synthesis from tetra-O-acetyl-
α-D-glucopyranosyl bromide and hydroquinone monobenzoate in the presence of silver
oxide and quinoline.
H
H
AcO
H
Br
OAc
H
OAc
H
C
C
C
C
C
O
⫹
OH
C
C
C
C
C
H
OAc
H
AcO
H
H
OAc
H
O
NH
3
(Deacetylation and debenzoylation)
I
CH OAc
2
OCOC H
65
2,3,4,5,6-tetra-O-acetyl-a-
glucopyranosyle bromide
Hydroquinone
monobenzoate
Quinoline
Ag O
2
O
OCOC H
65
CH OAc
2
O
OR
C
C
C
C
C
H
OHH
HO H
H OH
H
O
CH OH
2
Arbutin: R H
Methylarbutin: R CH
⫽
⫽
3
88
Pharmaceutical Chemistry of Natural Products
Sinigrin (2-Propyl Glucosinolate)
CH CH.CH —C
22
⫽
S—C H O
6115
NSO K
3
⫺
⫹
It is a mustard oil glycoside and occurs in black mustard seed. It is an organic compound without
biological activity. They are precursors of fl avour compounds and have a wide range of biological
effects.
All glucosinolates occur in plant in conjunction with hydrolytic isoenzyme myrosinase, but are
located in separated cells. When the plant tissue has been crushed, they come together. Myrosinase
is found in seed, leaf, stem and roots of glucosinolate containing plants.
RC
S C H O
6115
Myrosinase
HSO X
4
⫺⫺
⫹
Oxazolidine 2-thione
amino,cyanoepithio
alkanes
R—C
NS
⫺⫹
R—S—C
N
RNC
S
Isothiocyanates Thiocyanates
Nitriles
+ C
H O
6126
D-glucose
N
OSO
3
X
+
RC
SH
N
OSO
3
X
+
Many glucosinolates give rise to stable isothiocyanates under neutral and alkaline conditions.
The bitterness has been attributed to the presence of sinigrin. Isothiocyanates are known to be
pungent. These hydrolysis products elicit a wide range of psychological changes in higher animals
and man. Some isothiocyanates have been shown to inhibit tumour formation and to increase the
activity of the key enzymes involved in cellular detoxifi cation systems.
Sinigrin and its derivatives have been used in ayurvedic formulations. It strengthens heart
and resists poison. It provokes urine and is used as a laxative to treat gout, sciatica pains, chronic
bronchitis, rheumatoid arthritis and neuralgia. It is also used as an antiseptic and deodorizer.
Structural elucidation
1. Molecular formula: The molecular formula of sinigrin was found to be C
10
H
16
NO
9
S
2
K.
2. Hydrolysis: The enzyme myrosin hydrolyses it and yields D-glucose, allyl isothiocyanate
and potassium hydrogen sulphate.
C
10
H
16
NO
9
S
2
K + H
2
O C
6
H
12
O
6
+ CH
2
= CH
2
NCS + KHSO
4
3. Degradation with sodium methoxide: Using sodium methoxide when sinigrin is
degraded, it yields thioglucose (C
6
H
11
O
5
SH) as one of the products. This reaction reveals
that the glucose unit is bonded to a sulphur atom in sinigrin.
Glycosides
89
4. In 1956, Ettlinger et al. proposed the structure as given below for sinigrin, whereby allyl
isothiocyanate is produced by rearrangement.
CH CH.CH —C
22
⫽
S—C H O
6115
NSO K
3
⫺
⫹
5. Glucose in sinigrin is found to be present as β-D-glucopyranoside.
6. In 1963, Waser et al. confi rmed the above structure for sinigrin by X-ray analysis and have
also shown that the sulphate and thioglucose groups are in the syn-confi guration.
Amygdalin (Mandelonitrile--gentiobiose)
2-Phenyl-2-(5,6,7-trihydroxy-8-(2-((5,6,7-trihydroxy-8-(hydroxy
methyl)oxonan-4-yl)methoxy)ethyl)oxonan-4-yloxy)acetonitrile
C
C
C
C
C
H
HO
H
H
CH
2
O
C
C
C
C
H
HO
H
H
O
OH
H
OH
H
O
OH
H
H
OH
C
CH OH
2
OHC
CN
CN
65
Amygdalin is one of the earliest known glycosides, occurs in bitter almonds (dried ripe seeds
of Prunus amygdalus, Family: Rosaceae) and was fi rst isolated in 1830. The carbohydrate present
in it is a gentiobiose (disaccharide). Used in control of epilepsy, it also possesses sedative and
demulcent property. It is one of the constituent in ayurvedic formulations such as Chyawanprash
and Dashmoolarishta.
Structural elucidation
1. Molecular formula: The molecular formula of amygdalin is found to be C
20
H
27
NO
11
.
2. Hydrolysis with dilute hydrochloric acid or emulsin: Amygdalin upon hydrolysis with
dilute hydrochloric acid or emulsin yields one molecule of benzaldehyde, one molecule of
hydrogen cyanide and two molecules of
D-glucose.
C H NO C H CHO + HCN + 2C H O
20
27 11 6 5 6 12 6
Dil. HCl
or Emulsin
3. Hydrolysis with zymase: The enzyme zymase hydrolyse amygdalin to yield 1 mole of
D-glucose and 1 mole of mandelonitrile-D-glucoside identical with prunasin, a naturally
occurring glucoside. This reaction reveals that the sugar component in the glycoside is
actually a disaccharide and thus the aglycone portion is mandelonitrile. The disaccharide
was found to be gentiobiose.
90
Pharmaceutical Chemistry of Natural Products
C H NO + C H O + C H CH(CN)OC H O
20 6 5
27 11 6 12 6 11 5
HO
26
Amygdalin Glucose Mandelonitrile- -glucosideD
Gentiobiose
CHOH
C
C
C
C
H
HO
H
H
CH
2
O
C
C
C
C
H
HO
H
H
O
OH
H
OH
H
O
OH
H
OH
C
CH OH
2
4. Synthesis: The above structure of amygdalin was confi rmed by synthesis as shown
below.
Ag O
2
( ) C H CHOHCOOC H
(Ethyl mandelate)
⫹
65 25
HO
(ii) Separation into diastereomers
by fractional crystallization
and the ( )-form treated with P O⫾
25
C
HC
C
C
C
H
H
CH
2
O
C
C
C
C
H
HO
H
H
O
OH
H
OH
H
O
OH
H
H
OH
C
CH OH
2
O
CH
65
CONH
2
(I) Ac O-Pyridine(to prepare
hepta-acetyl derivative)
2
NH —C H OH
325
AcO
C
HC
C
C
C
H
H
CH
2
O
C
C
C
C
H
AcO
H
H
O
OAc
H
OAc
H
O
OAc
H
H
OAc
C
CH OAc
2
O
CH
65
COOC
2
H
5
HCHC
HO
Gentiobiose
CHOH
C
C
C
C
H
H
H
CH
2
O
C
C
C
C
H
HO
H
H
O
OH
H
OH
H
O
OH
H
OH
C
CH OH
2
AcO
C
C
C
C
C
H
H
H
H
CH
2
O
C
C
C
C
H
AcO
H
H
O
OAc
H
OAc
H
O
OAc
Br
H
OAc
C
CH OAc
2
AC O—HBr
2
Hepta-acetyl-bromogentiobiose
Glycosides
91
PROBABLE QUESTIONS
1. Defi ne glycosides, classify with suitable examples and write a note on anthraquinone glycosides.
2. Write the structural elucidation of salicin.
3. Classify glycosides on the basis of their chemical structures with suitable examples.
4. Mention an example for C-glycosides with the chemical structure and how will you test them qualitatively?
5. Describe the general methods for structural determination of glycosides.
SUGGESTED READINGS
Cervoni, P, Crandall, DL and Chan, PS (1993). Kirk–Othmer Encyclopedia of Chemical Technology, 4th Ed., Vol 5. Wiley, New
York, pp. 251–257.
Conn, EE (1991). The metabolism of a natural product: Lessons learned from cyanogenic glycosides. Planta Med 57: S1–S9.
Deepak, D et al. (1996). Cardiac glycosides. Prog Chem Org Nat Prod 69: 71–155.
Hughes, MA (1999). Biosynthesis and Degradation of Cyanogenic Glycosides. Comprehensive Natural Products Chemistry, Vol
1. Elsevier, Amsterdam, pp. 881–895.
Evans, WC (2002). Trease and Evans Pharmacognosy, 15th Ed. Elsevier, Philadelphia, pp. 214.
AcO
C
HC
C
C
C
H
H
CH
2
O
C
C
C
C
H
AcO
H
H
O
OAc
H
OAc
H
O
OAc
H
H
OAc
C
CH OAc
2
O
CH
65
CN
NH —C H OH
325
HO
C
HC
C
C
C
H
H
CH
2
O
C
C
C
C
H
HO
H
H
O
OH
H
OH
H
O
OH
H
H
OH
C
CH OH
2
O
CH
65
CN
HC
HC
( )-Amygdalin⫹( )-Nitrile⫹