CHAPTER-1
INTRODUCTION
1.1 Importance of the structure of polysaccharides and its application
In the seeds of many plants their exist giant molecules called
‘Polysaccharides’, ‘Gums’ or ‘Mucilages’ formed by the polymerization of
several monosaccharide units (usually ten to thousand). These giant molecules
are produced in the seeds of various plants by the tremendously complicated
process of photosynthesis and act as reserve or storage matter for cellular
reactions.
The past decade has seen a tremendous growth and expansion of the
knowledge of carbohydrate chemistry and biology including the knowledge
about polysaccharides, gums and mucilages. This has resulted in the discovery
of a wide ranging biological functions and activities of these compounds.
They can form gels, can interact with proteins forming glycoproteins, form
lipopolysaccharides with lipids, functions as enzymes and antibodies, possess
non-cytotoxic antitumour activity, can inhibit viruses, induce interferon
formation in cell cultures and can exist as plant lectins. Knowledge of the
structural features of these giant molecules as revealed by organic chemist can
help a great deal in understanding these biological processes and present a
model for the synthesis of biologically active carbohydrates.
Polysaccharides, comprising a large group of macromolecular
carbohydrates provide an extensive and ever increasing field of investigation
due to its molecular complexity and immense industrial applications.
Polysaccharides, as the name implies are polymerized saccharides, from the
macromolecular view point, they may be looked upon as the condensation
polymer consisting of monosaccharide residues with the elimination of water
molecules. Polysaccharides are the essential constituents of almost all living
organisms widely distributed in plant and animal kingdom¹.
The climatic condition of India provides large potential for production
of polysaccharides in abundance in the form of grains, mucilage’s, gums and
hemicelluloses along with proteineous substance.
In higher plants, polysaccharides are components of cell wall e.g.
cellulose, xylan, hemicelluloses, pectin and mannans. As major organic
skeletal substances of invertebrates, chitin is found in arthropods annelid and
molluscus Chondrotin sulphate, a mucopolysaccharide, is a major constituent
of cartilages. Mucotic sulphate occurs in gastric mucosa and in cornea.
Haperin and hyaluronic acid found in animal tissues and blood. They are also
most abundant in sea weeds, where they constitute approximately three
quarters of dry weight. A great variety of polysaccharides are also produced
by bacteria. These may be elaborated in the culture medium (extracellular
form) or to be present in capsules surrounding the cell.
Polysaccharide may be either a glycan or proteoglycan. “Glycan” are
condensation polymers in which ten or more units of monosaccharides or their
derivatives are glycosidically linked while “proteoglycans” are combination
products of glycan and protein. Based on the chemical composition and
structure, the glycans have been systematically classified into two groups:
Homoglycans and Hetroglycans. Homoglycans on hydrolysis yields only one
type of monosaccharide units e.g. xylan, glucan, mannam etc. Heteroglycans
on hydrolysis yields two or more than two types of monosaccharide moieties
e.g.
galactomannan,
glucomannan,
galactoglucomannan
etc.
The
polysaccharides have been classified in various ways but the most logical and
satisfactory classification is the one based on their chemical compositions and
structures. According to it they can be classified as follows:
PLANT POLYSACCHARIDE
HOMOGLYCAN
(Built up of a single monosaccharide repeating unit)
NITROGENOUS
POLYMERS
1) Glucosamine
2)Polymers
HETEROGLYCAN
(Built up of two or more
type of repeating units)
NEUTRAL
POLYMERS
1) Glucans(starch,cellulose)
2) Mannans
3) Galactans
4) Fructans(inulin)
5) Xylans
NITROGENOUS
1)Mucopolysaccharide
2)Glycoproteins
NEUTRAL
1)Hemicellulose
2)Arabinigalactans
3)Glucomannans
4)Galactomannans
ACIDIC
POLYMERS
1) Ploygalacturonic
acid (pectin)
2) Polymannuronic
acid
ACIDIC
1) Gum
2) Mucilage's
3) Alginates
(seaweeds)
The use of polysaccharides goes back to earliest times. From time
immemorial primitive people have used them as food. Even, now, one of the
important uses by man is as a component of food material, being used in
confectionary trade, bakery and in the preparation of peppermints and
beverages. In addition they are frequently employed in textile, paper, printing,
dyeing cosmetics and pharmaceutical industries.
When we come to plant seed polysaccharides they are classified as
according to their origin. They are mainly of four types-Plant seed
polysaccharides, tuber polysaccharides, exudates, gums and cell wall
polysaccharides. Out of the major carbohydrates making up the primary cell
wall are cellulose, hemicelluloses and pectin. Xyloglucan is a hemicellulose
found in the primary cell walls of many plant species. Xyloglucan is also
identified as seed storage polysaccharide as it has capability to mobilize into
the seed endosperm during seed germination².Therefore two different groups
of xyloglucans have been identified as seed wall and cell wall xyloglucans.
The most readily available group is that isolated from the seeds of various
dicotyledons as a resource for the embryo after germination The presence and
mobilization of xyloglucans following seed germination were first reported in
the 19th century for seeds of Impatiens balsamina, nasturtium(Tropaeolum
majus), and Cyclamen europaeum².The seeds of a few plants such as tamarind
(tamarindus indica L.), detarium senegalense, afzelia africana, and Jatoba
have abundant deposits of xyloglucan polysaccharide³. The tamarind seed is a
by-product of the tamarind industry and the fruit pulp is the chief souring
agent for curries, sauces and certain beverages⁴.
The decorticated flour, known as tamarind kernel powder (TKP), is a
major industrial product widely used as a sizing material in textile, paper, and
jute industries⁵. Compared to wood-based hemicelluloses, the extraction of
XG from seed powder is straightforward, also in a commercial perspective.
The decorticated seeds of tamarind contain a large proportion (ca. 60 %) of
xyloglucan polysaccharide⁶.It grows in more than 50 countries in the tropics
and subtropics, and produces brown pod-like fruits, which contain fruit pulp
and many hard-coated seeds⁷’⁸.
The polysaccharides present in the husk of the seeds of Plantago ovate
Forsk (Ispaghula husk) is widely used as a prophylactic in the treatment of
large bowel disorders⁹.Galactomannans have sometimes been used in binary
mixtures with other polysaccharides, such as with xanthan gum, agar and
kappa-carrageenan, to form gels with new properties¹⁰’¹¹.The three major
galactomannans of commercial importance in food and non-food industries
are guar (Cyamopsis tetragonolobus, M/G 1.5), tara (Caesalpinia spinosa,
M/G 3) and locust bean gums (Ceratonia siliqua, M/G 3.5)¹². The estimated
worldwide annual production of locust bean gum (LBG - E 410) alone is
15,000 tons, and current prices are from 12 to 22 euros/kg or more, depending
on the grade and supplier¹³.
The knowledge of medicinal value of plant gums and other
polysaccharides can be said to come from the ancient times. Cassia tora linn.
(Caesalpiniaceae) is a small annual herbs or undershrub growing as common
weed in Asian countries. It constitutes an Ayurvedic preparation
“Dadhughnavati” which is one of the successful antifungal formulations¹⁴.
Polysaccharides play an important role in the field of immunology.
Polysaccharides are true immunogens as they induce an immense response
and the generation of specific antibodies (Serum globulins).Recently, the use
of polysaccharides as antigens and immunogens has contributed greatly to the
classification and identification of bacteria to a better understanding of the
immune response, to the definition of the active site in antigen antibody
interaction, and to the detection and prevention of human disease caused by
invasive micro-organisms.
Polysaccharides obtained from the plants have an important role due to
their wide application hence their structure has always attracted the organic
chemist. All properties of polysaccharides are characterized by molecular
structure rather than sugar unit composition. Hence continuous investigation
on the structure of plant polysaccharide might lead in future the discovery of a
new group of physiological active compounds for combating the action of
various micro organisms and also provide a clue to the solution of the
fundamental problem of their biogenesis recent years. Due to the presence of
various derivable groups on molecular chains, polysaccharides can be easily
modified
chemically
and
biochemically
having
different
functional
properties¹⁵.
Due to this immense industrial importance, the structures of
polysaccharides are always a subject matter of keen interest. It has been
observed that the physical properties of polysaccharides like gel formation,
solubilities viscosities¹⁶etc. depend not so much on the actual building unit
(although this is an important consideration) as upon the overall fine
molecular architecture of the polysaccharide.It is interesting to note here that
the gelling mechanisms of pectins, isphagula husk (Plantago ovata),
caragenanas, etc. have been revealed the knowledge of their fine structures.
Even the fine structure of neutral polysaccharides like starch and cellulose
explain the wide difference in their properties although both contain the same
sugar unit i.e. glucose. Even the two fractions of starch i. e. amylose and
amylopectin differ significantly in their properties. Genetic modification of
starch crops has recently led to the development of starches with improved
and targeted functionality.Annual worldwide starch production is growing
year by year and thus created interest
in identifying new sources and
modifications or derivatives of this polysaccharide¹⁷.
During the past decade, many attempts have been reported to mimic
natural bionanocomposites by blending polysaccharide nanocrystals from
different sources with polymeric matrices¹⁸’¹⁹ .The resulting nanocomposite
materials display outstanding properties, in terms of both stiffness and thermal
stability. Formation of a rigid percolating network, resulting from strong
interactions between them was the basis of this phenomenon.
The research based on polysaccharides is being continued by National
Sugar Institute, Kanpur for last twenty years in order to elucidate their
structure and also to find their uses in various industrial applications.
1.2 Seed storage hemicellulosic polysaccharide- Occurrence
Xyloglucan is a hemicellulose found in the primary cell walls of dicots and
non-
graminaceous
monocots²⁰.
Xyloglucans,
or
more
generally
galactoxyloglucans, are only known hemicellulosic polysaccharides with the
main chain identical to that of cellulose, i.e. β-(1→4) linked D-glucan²¹’²².
Xyloglucan may account for up to 20% of the dry weight of the primary
wall. It is believed to function as a cementing material which contributes
crosslinks and rigidity to the cellulose framework. Xyloglucans are also
defined chemically as plant cell wall polysaccharides that have a backbone of
1, 4-linked β-D-pyranosyl residues in which O4 is in the equatorial orientation
(e.g. Glc, Man, and Xyl).
Hemicellulose is a very broad term that has been used historically to
describe various noncellulosic polysaccharides in plants. Different definitions
for hemicellulose have confused the matter since these have been isolated
from seeds²³⁻²⁵, plant cell walls²⁶’²⁷ and the extracellular media of suspension
cultured plant cells²⁸’²⁹.
Today, researchers have avoided the term, hemicellulose, and are
focusing on definitions based on structure or isolation techniques. Table 1.1
illustrates Aspinall's classification of plant cell wall polysaccharides by their
structural families³⁰.
Table 1.1: Aspinall's classification of plant cell wall polysaccharides by
structural family.
Glucans
Cellulose
Callose
Cereal β-D-glucans
Xyloglucan
β-(1→4)
β-(1→3)
β-(1→3) (1→4)
β-(1→4) and branches
Xyloglucan is also identified as seed storage polysaccharide as it has
capability to mobilize into the seed endosperm during seed germination.
Therefore these are also known as cell wall seed storage (CWSPs), These are
commonly found in immature and storage tissues of many plant species.
Originally they were termed amyloids because of their starch-like response to
iodine³¹. The term amyloid obviously a misnomer was designated in view of
characteristic
colour
blue,
iodine
staining
properties
exhibited
by
xyloglucans³².
The presence and mobilization of xyloglucans following seed
germination were first reported in the 19th century for seeds of Impatiens
balsamina, Nasturtium (Tropaeolum majus), and Cyclamen europaeum.These
xyloglucans act as a reserve food supply for the developing seed and are
generally composed of the D-sugars of glucose, xylose, and galactose linked
in almost identical patterns as in Table 1.2. It has been shown that these
polymers are composed of a β-(l→4)-linked glucan backbone that is
substituted (C-6) with a limited number of xylosyl residues, either singly, or
with a terminal galactosyl residue (C-1 to C-2). Two different groups of
xyloglucans have been identified as seed wall and cell wall xyloglucans. The
most readily available group is that isolated from the seeds of various
dicotyledons as a resource for the embryo after germination³³’³⁴.
Table 1.2. Normalized sugar compositions of some seed xyloglucans.
Xyloglucan
Normalised Sugar Composition
Glu
Gal
Xyl
Tamarindus Indica
4
3
1.3
Tropaeoleum majus
4
2.7
1.3
Brassica campestris
4
1.5
0.8
Annona muricata
4
1
1
Histochemical studies have shown that, in general, 'amyloids' in plant seeds
occur as thickenings of the cell walls. Their disappearance during germination
and the very large deposits which occur in some seeds (40-50% of
Tamarindus seeds); suggest the role of energy reserves. Early microscopic
studies suggested that amyloids disappear from seeds after germination and it
has been assumed that they are reserve polysaccharides. Surprisingly, this was
confirmed only in the course of an investigation of xyloglucan metabolism in
the cotyledons of the Nasturtium seed (T. majus L.) after germination³⁵.
The amyloid of the Nasturtium seed was shown to be mobilized
completely following germination. It constituted 30% of the total substrate
reserves utilized by the seed, and must therefore be classified as a major cellwall storage polysaccharide³⁷.The reserve function of xyloglucan in
cotyledons has been demonstrated for seeds of Nasturtium³⁶, Tamarindus
indica³⁸, Copaifera langsdorffii³⁹ and Hymenaea courbaril⁴⁰. Among these,
xyloglucan derived from tamarind seed was highly studied for different
applications.
The basic structure of storage xyloglucans is similar to the primary wall
xyloglucans. They have a backbone composed of β-(1→4)-linked glucan with
regular branching with α-(1→6)-linked xylosyl residues that can be branched
further with β-(1→2)-linked galactosyl residues. Except for the absence of
terminal fucosyl units α-L-(1→2)-linked to the branching β-D-galactosyl
residues as shown in Figure 1.1. There is a remarkable similarity between
seed reserve xyloglucan and structural xyloglucan from primary cell walls of
dicotyledonous tissues⁴¹. It is known that the distribution of side chain
residues is different in the xyloglucans extracted from different species⁴². Up
to 75% of these residues are substituted at O-6 with mono-, di-, or triglycosyl
side chains.
Figure 1.1 Structure of xyloglucan
HO
OH
OH
O
O
O
OH
OH
OH
O
OH
O
O
OH
O
OH
O
OH
O
OH
This is the common structure for all the xyloglucans, but additional residues
are attached to xylose, depending on the source of xyloglucan. This variation
of the structure dominates the detail of the functionality and physicochemical
properties. For instance, the galactose substituted to the xylose dominates the
water solubility in the case of xyloglucan extracted from tamarind seed.
General structure of tamarind xyloglucan consists of a β (1→4) glucan
backbone variously substituted with xylosyl and galactosyl residues⁴³.
To help describe the structures of xyloglucans, S.C.Frydeveloped an
unambiguous nomenclature with the letters G, X, S, L, and F referring to the
following structures⁴⁴: “G = unsubstituted β-D-Glcp; X = α-D-Xylp-(1→6)-βD-Glcp; S and L = X with α-L-Araf-(1→2)- and β-D-Galp-(1→2)- attached,
respectively; and F = L with α-L-Fucp-(1→2)- attached”. It is elaborately
described in Appendix – I. The typical structures of the subunits in these
xyloglucans are shown below in Table -1.3
Table 1.3 Single letter nomenclature of xyloglucan
α Fucp
α Fucp
2
β-Galp
2
β-Galp
α-Xylp
2
α-Xylp
2
α-Xylp
6
6
6
β-Galp
α-Xylp
α-Xylp
2
α-Xylp
6
6
6
4β Glcp-(1,4) βGlcp-(1,4) βGlcp-(1,4) βGlcp-(1
X
X
4β Glcp-(1,4) βGlcp-(1,4) βGlcp-(1,4) βGlcp-(1
X
G
F
α-Xylp
α-Xylp
L
α-Xylp
6
6
6
4β Glcp-(1,4) βGlcp-(1,4) βGlcp-(1,4) βGlcp-(1
X
X
X
G
F
G
1.3 Morphology of Bauhnia Malabarica tree
Malabar Bauhinia is a small or moderate sized deciduous tree belongs to
family Fabaceae- Caesalpinaceae (Gulmohar family) ⁴⁵.Its local name is
Alibangbang while trade name is Malabar Orchid and known as ‘Kachnar’ in
India. It is distributed in India to Indo-China, Pakistan Java and Timor,
Southeast Asia, northern Australasia; in secondary forests. Bauhinis is a genus
of more than 200 species. The genus was named after the Bauhin brothers,
Swiss-French botanists. Specimen height is 6-10 meters with trunk bole erect
as shown in the following Figure 1.2.
Bark is rough brown, peeling in linear flakes, fibrous, red inside.
Leaves are broader than long, 1.5-4 inches long, 2-5 inches broad, divided
through 1/3 of the length, 7-9 nerved, slightly heart-shaped at base, rigidly
leathery, glaucous and smooth beneath. Seeds are 20-30.This tree is drought
tolerant, evergreen, grassfire tolerant and nitrogen fixing. Bark, leaves,
flowers are used for various reasons⁴⁶.
True Bauhinia malabarica is an esteemed tree by plant hobbyists and
farm owners due to its edible tart leaves. Chopped leaves, particularly the
shoots and tips, are used to flavor all sorts of “Sinigang”. In recent years,
interest in these plants has increased considerably throughout the world.
Figure 1.2: Tree of Malabarica bauhinia
Leaves are sour, commonly used as flavoring for meat and fish. It is excellent
source of calcium and iron. The species share the 'butterfly' configuration of
the leaves as shown in Figure 1.3.
Figure: 1.3 Butterfly shape of malabarica’s leaf.
Bauhinia is also known as Mountain Ebony, purple orchid tree or simply
orchid trees are grown as avenue trees for their colorful and ornate, orchidlike flowers are white and rather large. Pods are long, narrow, and flattened,
20 to 30 centimeters by 1.5 to 2.5 centimeters. Flowers are borne in stalk less
racemes in leaf axils, 1.5-2 inches long, often 2-3 together. Male and female
flowers are usually on different stems. The five-petaled flowers are 7.5-12.5
cm diameter, generally in shades of red, pink, purple, orange, or yellow, and
are often fragrant. The tree begins flowering in late winter and often continues
to flower into early summer. Depending on the species, bauhinia flowers are
usually in magenta, mauve, pink or white hues with crimson high lights as
shown in followingc Figure 1.4 and Figure 1.5⁴⁷.
Figure 1.4: Yellow colored Bauhinia flower in Hyderabad, India
Figure 1.5: Bauhinia malabarica Roxb. - Purple Orchid Tree flowers in
Hyderabad, India
Plants of the genus Bauhinia (Fabaceae), commonly known as cow's-paw or
cow's hoof, are widely distributed in most tropical countries and have been
used frequently in folk medicine to treat different kinds of pathologies,
particularly diabetes, infections, as well as pain and inflammation. In recent
years, interest in these plants has increased considerably throughout the world.
The biological properties of different phytopreparations and pure metabolites
have been investigated in numerous experimental in vivo and in vitro models.
Although some contradicting evidence has been documented, in general, the
results support the reported therapeutic properties, indicating that they are
mainly due to the presence of flavonoids. This review summarizes the recent
chemical and biological knowledge of plants of the genus Bauhinia⁴⁸.
1.4 Seed xyloglucans- Methods of structural elucidation
Since structural analysis of polysaccharides is a complex and demanding task,
a good strategy is necessary before starting any experiments. Following
Figure 1.6 summarizes the necessary steps frequently used for elucidating a
detailed structure of a polysaccharide. A polysaccharide extracted from plant
materials or food products is usually purified before being subjected to
structural analysis The first step of characterizing a polysaccharide is the
determination of its purity, which is reflected by its chemical composition,
including total sugar content, levels of uronic acid, proteins, ash, and
moisture.
Figure 1.6: Strategy and methods for structure analysis of polysaccharide
Chemical
Composition
Raw Material
Extraction
Purification
Crude
Polysaccharide
Purified
Polysaccharide
Monosaccharide
Composition
Methylation
Analysis/GC-MS
Specific
Oligosaccharide Mixtures
Degradation
MS, FAB
MALDI,ESI
Isolation of
Oligosaccharides
Isolated
Oligosaccharides
¹H,¹³C and
2D NMR
During the last three decades, rapid development of modern
technologies including fast atom bombardment mass spectrometry (FAB-MS),
matrix-assistant laser desorption ionization (MALDI-MS) , electrospray
ionization (ESI-MS) spectrometry ,one and two- (multi)-dimensional NMR
spectroscopy have been developed. These modern techniques and
methodologies have been shown to be extremely powerful for solving the
structural problems of polysaccharides. In addition, numerous highly specific
and purified enzymes have become readily available. All of these advances in
science and technology have made the structural analysis of polysaccharide an
interesting task.
1.4.1 Complete acid hydrolysis
The most widely applied methods for the quantification of
polysaccharides prerequisite to break down these polymers into
monosaccharides are known as hydrolysis. The resulting individual
sugars (glucose, xylose, mannose, galactose and arabinose) can then
be determined in different ways, for example, by HPLC or after
derivatization by GC. The hydrolysis of polysaccharides can be
performed mostly in the presence of mineral acids, and 72% sulfuric
acid is generally used. Since hydrolysis is associated with some
unavoidable side reactions, mass losses occur and therefore the
reaction conditions should be selected carefully. On the other side,
the neutralization of the hydrolyzates may further decrease the yield
of sugars and therefore the losses are accounted for either by
correction factors for each of the 5 sugars or by the treatment of
sugar standards throughout the procedure in exactly the sameway.
Polysaccharides can also be hydrolyzed in the presence of
trifluoroacetic acid (TFA). The TFA method has been introduced
with 2 major advantages: causing smaller losses and omitting the
step of neutralization since the TFA can be removed by
evaporation⁴⁹.
The conditions of TFA hydrolysis should be varied depending on
the nature and composition of lignocellulosic material and, in case
of incomplete hydrolysis, a pretreatment step is necessary⁵⁰. In this
study, the technique of two-step hydrolysis with sulfuric acid is
somewhat changed with the aim of enabling complete hydrolysis
under atmospheric pressure. With exact temperature control and
without
pressure,
hydrolysis
could
be
carried
out
carefully.Moreover hydrolysis would be done by TFA method.
1.4.2 Methanolysis
more
Methanolysis breaks the glycosidic linkages of permethylated
polysaccharides by introducing a methyl group and consequently
forms methyl glycosides. Glycosidic bonds differ in their
susceptibility to methanolysis. The rate of reaction is dependent on
the anomeric configuration, position of the glycosidic linkage, and
the identity of the monosaccharide. Therefore, monitoring products
of a time course methanolysis of permethylated polysaccharides is
useful for determining sequences, branching patterns, and location
of substituents.The permethylated polysaccharides are gradually
degraded by acid catalyzed methanolysis and gives methyl
glycosides at the released reducing terminal. A free hydroxyl group
will be formed from released each glycosidic oxygen and each
hydrolyzed substituent⁵¹.
Sequence and branching information can be derived from the
number of free hydroxyl groups produced. If hydrolytic removal of
one or more residues occurs without the generation of a free
hydroxyl group, the removed residues must be at the reducing end of
the intact oligosaccharide as in Figure1.7. In a case where the
methanolysis process produced two free hydroxyl groups, two
different branches must have been simultaneously hydrolyzed.
(Figure 3b)
Figure 1.7 Methanolysis of polysaccharide (a)Methanolysis of
permethylated Polysaccharide with no free hydroxyl group formed
(b)Release of free hydroxyl group formed
MeO OMe
H
O
H
MeO
H
OMe
H
H
H OMe
O
H
OMe
O
O
O
MeO
O
H
H
H
H OMe
MeO
H
MeO
H
H
C
O
O MeO
OH
H
B
H
OMe
H
H
H
A
n
Methanolysis
no free hydroxyl group formed
MeO
H OH
OMe
O
O
HO
H
H
MeO
H
OMe
H
MeO
H
B
O
O
H
OMe
H
H
H
H
A
OMe
O
O
O
H
H
H
H
H
H
MeO
MeO
O
OMe
O
MeO
H OMe
MeO
H
H
Methanolysis
Formation of two free hydroxyl group
OMe
MeO
H
H
C
H
H
B
H
a
n
b
1.4.3 Methylation Analysis
Methylation analysis has been used to determine the structure of
carbohydrate for over a century and it is still the most powerful
method in carbohydrate structural analysis. Methylation of
polysaccharide followed by acid hydrolysis of the methylated
product, reduction, acetylation and identification of the resulting
partially methylated alditol acetates by GLC and or GC-MS helps to
reveal the point of linkages between the various monosaccharide
units. A current methylation analysis consists of two steps:
 Chemical derivatization.
 Gas-liquid chromatography–mass spectroscopy. (GC-MS)
The derivatization of a polysaccharide for methylation analysis
includes conversion of all free hydroxyl groups into methoxyls
followed by acid hydrolysis. Acidic hydrolysis of the resulting
poly-methyl-ethers only cleaves the inter-glycosidic linkages and
leaves the methyl-ether bonds intact. The hydrolyzed monomers
are reduced and acetylated to give volatile products, i.e., partially
methylated alditol acetate, which can be identified and
quantitatively
determined
by
gas-liquid
chromatography
equipped with a mass spectroscopic detector (GC-MS). Figure
1.8 depicts the procedures and chemical reactions involved in
methylation analysis. The substitution pattern of the O -acetyl
groups on the PMAA reflects the linkage patterns and ring sizes
of the corresponding sugar in the original polymer.
Figure 1.8 Illustration of chemical reactions in methylation analysis
OH
CH2
O
HO
OH
OH
H
H
OH
O
C H2
CH2
H
OH
O
H
O
OH
(a)
OH
O
H
H
O
H
H
OH
O
H
H
(b)
©
m
Methylation
OCH3
CH2
O
CH3 O
OCH3
OCH3
H
H
OCH3
O
CH2
CH2
O
(a)
O
H
H
CH3 O
OCH3
H
O
H
OCH3
O
H
CH3 O
O
H
H
©
Hydrolysis
(b)
H
m
Deuterised Reduction
Acetylation
D
H
C
H
D
D
OAc
H
OAc
C
H
C
OA c
OCH3
H3CO
OCH3
H3CO
H3CO
H
H3CO
H
H3CO
H
H3CO
H
H3CO
OAc
H3CO
OAc
H
OAc
H
OAc
H
OAc
CH2 OCH3
(a)
OCH3
CH2 OAc
(b)
CH2 OCH3
©
.
However, this method gives no information on sequences or the
anomeric configuration of the glycosidic linkages. In addition, this
method cannot distinguish whether an alditol is derived from a 4– O
–linked aldopyranose or the corresponding 5– O –linked
aldofuranose. These drawbacks can be overcome by a method called
reductive cleavage, which is described later.Experimentally; the
reaction of converting the hydroxyl groups into methoxyls requires
an alkaline environment and methyl group provider silver oxide–
methyl iodide⁵² and sodium hydroxide–methyl sulphate⁵³ were used
in the past. More recently a simpler procedure using dry powdered
sodium hydroxide and methyl iodide has been adapted. More
recently a simpler procedure using dry powdered sodium hydroxide
and methyl iodide has been adapted.This method has been modified
by using a sodium hydroxide suspension in dry DMSO ⁵⁴.
The partially methylated alditols are then acetylated with acetic
anhydride to give partially methylated alditol acetates, which are
analyzed by GC-MS. The congruence of retention time and mass
spectrum of each PMAA with those of known standards is used to
identify the monosaccharide unit and its linkage pattern while the
area or height of the chromatographic peak is used for
quantification.
1.4.4 Partial acid hydrolysis
Partial acid hydrolysis of the polysaccharides followed by
isolation and characterization of degraded polyschharides or
oligosaccharides gives detailed information about the sequence and
anomeric configuration in addition to providing information of
linkage types. In case of linear polysaccharides containing uniform
linkages and assuming that all glycosidic bonds are equally
suscepltible to hydrolysis, partial hydrolysis leads to the information
of
polymer
homologous
series
of
polysaccharides⁵⁵
(di,tri,tetra,penta,hexasaccharides).
Similarly for multi linkage types of polysaccharides, given the
susceptibilities to hydrolysis of different linkages are approximately
equal a representative selection of all the possible oligosaccharides
from different regions is liberated⁵⁶.However, rates of hydrolysis of
different glycosidic linkages are in fact sufficiently different so that
all the possible oligosaccharides are not liberated in a single step.
Partial degradation of polysaccharides by acid hydrolysis is based on
the fact that some glycosidic linkages are more labile to acid than
others. If a polysaccharide contains only a limited number of acidlabile glycosidic linkages, a partial hydrolysis will afford a mixture
of monosaccharides and oligosaccharides. Partial acid hydrolysis of
fully methylated polysaccharides often furnishes useful information
on the positions at which the oligosaccharides were linked in the
original polysaccharides For example, a polysaccharide consisting of
D-galactopyranose
selectively
and
hydrolyzed
methylation.
The
D-galactofuranose
under
mild
D-galactofuranosyl
residues
acidic
can
conditions
linkage
is
be
after
preferably
hydrolyzed under mild acid conditions and the resultant products are
reduced with borodeuteride and remethylated with trideuteriomethyl
iodide. A trideuteriomethyl group at O-3 in the D-galactopyranose
residue can be determined by mass spectrometry. Likewise the
locations of the three trideuteriomethyl groups on the reducing end
unit can also be identified by MS. The combination of the structural
information on the disaccharide derivative and the mild acid
hydrolysis method provides meaningful information leading to the
structure of the repeating unit.
1.4.5 Enzymatic hydrolysis
Two classes of enzymes are known to be involved in the
metabolism of xyloglucans⁵⁷. One is endo-1, 4-β-glucanase.The
enzyme is a hydrolase capable of cleaving 1, 4-β-glucosyl linkages
in
several
glucans,
including
xyloglucans
and
carboxymethylcellulose (CMC), and has also been termed a
cellulase because of its potential activity towards cellulose. The
enzyme showed xyloglucan-hydrolase activity but no cellulose
activity. The physiological role of this enzyme has not been
reported. The other is endo-xyloglucan transferase (EXGT) or
xyloglucan endotransglycosylase (XET) that catalyzes molecular
grafting between xyloglucan molecules and can thereby mediate an
interchange
between
xyloglucan
cross-links
in
the
framework⁵⁸.However; EXGTs do not degrade xyloglucans in the
absence of xyloglucan oligosaccharides.
The structural characterization of xyloglucan is greatly facilitated
by its endoglucanase –catalyzed fragmentation into well defined
subunit oligosaccharides. Normally, we treat the oligosaccharide
fragments with sodium borohydried, converting them into the
corresponding
oligoglycosyl
alditols.
These
derivatives
are
advantageous in that they adopt a single form, in contrast to native
oligosaccharides, whose reducing glucose residues can adopt either
α-configuration
or
β-configuration,
which
are
in
dynamic
equilibrium.
1.4.6 Periodate oxidation
Oxidation with periodate ion, resulting in 1, 2- glycol cleavage is
one of the most widely used reaction in the structural elucidation of
polysaccharides since it furnishes information regarding the point of
linkage.The reaction mixture can be analysed by titrimetric methods
to determine periodate consumed, by acid – base titration to measure
formic acid liberated, and by various colorimetric methods for
formaldehyde produced. In addition, the presence of sugar residues
that are substituted in a manner no diol groups susceptible to
oxidation may be ascertained by the liberation of the unchanged
sugar after hydrolysis.
Non ideal behavior of polysaccharides during periodate oxidation
arises from both oxidation and under oxidation.Overoxidation is
frequently encountered when oxidation gives rise to tartonic acid
half aldihyde derivatives, from hexuronic acid end groups or to
tartron
dialdehyde
derivative
from
hexafuranosides
or
heptapyranosides.
There are many reasons behind the incomplete oxidation of sugar
moieties. Most commonly, hexacetal formation between aldehyde
fragments in oxidatively cleaved residues and hydroxyl group is
adjacent but not yet oxidized residues protects the latter unit from
oxidation⁵⁹.
The highest degree of incomplete oxidation occurs primarily in
4-linked polysaccharides whose sugars residues do not carry primary
hydroxyl groups at C-6. Another reason is the hydrogen bonding
between one of a pair of hydroxyl group, normally susceptible to
oxidation.
Polysaccharides containing free hydroxyl groups have the
potential to react with oxidation reagents. The oxidation reaction
could be used to elucidate structural information of polysaccharides.
For example, vicinyl-glycols (two neighboring hydroxyl groups) can
react with periodic acid or its salts to form two aldehydic groups
upon the cleavage of the carbon chain. For polysaccharides, sugar
units with different linkage patterns will vary significantly in the
way they react with periodates. For example, nonreducing end sugar
residue and/or 1 → 6-linked nonterminal residues have three
adjacent hydroxyl groups; double cleavages will occur and the
reaction consumes two molar equivalents of periodate and gives one
molecular equivalent of formic acid . Non terminal units, such as 1,
2- or 1, 4-linked residues will consume one equivalent of periodate
without formation of formic acid. Sugar units that do not have
adjacent hydroxyl groups, such as 1, 3-linked residues or branched
at C-2 or C-4 positions will not be affected by this reaction. A
quantitative determination of periodate consumed and the formic
acid formed, combined with the information on the sugar units
surviving the oxidation reaction, will provide clues to the nature of
the glycosidic linkage and other structural features of the
polysaccharides.Periodate oxidation can be used to estimate the
degree of polymerization of linear 1 → 4-linked polysaccharides.
Each 1 → 4-linked polymer chain will release three formic acid
equivalents after the oxidation reaction: one from the nonreducing
end and two from the reducing end as shown in Figure 1.9.
Figure 1.9: Periodate oxidation of 1→4 linked polysaccharides
CH2 OH
CH2 OH
O
H
H
CH2 OH
O
H
H
H
H
OH
O
HO
OH
H
H
OH
O
H
H
H
OH
H
OH
O
H
H
OH
H
OH
n
Periodate Oxidation
CH2 OH
H
Ι
C
ΙΙ
O
CH 2 OH
O
H
H
Ι
C
ΙΙ
O
H
H
O
H
H
H
H
O
H
C
ΙΙ
O
C
ΙΙ
O
O
CH2 OH
Ι
C=O
Ι
H
C
ΙΙ
O
n
+ O
3 H―C
=
OH
Experimentally, the polysaccharide is oxidized in a dilute solution of
sodium periodate at lower temperatures (i.e. 4 °C). The amount of
formic acid produced and periodate consumed are determined at
time intervals. A constant value of two consecutive measurements of
formic acid and/or periodate indicates the end of the reaction. The
periodate concentration can be measured by titrimetric or
spectrophotometric methods.
1.4.7 Smith degradation
Smith Degradation is a method of selective degradation by
oxidation⁶⁰.The combination of periodates oxidation, reduction, and
mild acid hydrolysis is known as the Smith Degradation. It involves
a controlled acid hydrolysis of the reduced (by sodium borohydride)
oxidized polysaccharide in which hydrolysis of acyclic acetals from
cleaved sugar units occurs with periodate- resistant sugar residues,
the degradation may result in the formation of an isolated unit of low
molecular weight in which the sugar residues are present as simple
glycosides of fragments such as glycerols as tetritols which can be
analysed by GLC.
The success of Smith degradation depends not only on ensuring
that all the potential vulnerable diol and triol groups have been
oxidized but also upon the selectivity in the acid hydrolysis step.
The consequent characterization of the resulting surviving
monosaccharides and/or oligosaccharides will shed light on the fine
structure of the original polysaccharide. The formation of erythritol
suggests that the polysaccharide contains an adjacent (1→4)-linked
D-glucose unit, which was later confirmed by 2D NMR
spectroscopy and specific enzyme hydrolysis.
This controlled
degradation may lead to the formation of a glycolaldehyde acetal of
2-O-β-D-glucopyranosyl- D-erythritol, which should be considered
when determining the amount of D-glucopyranosyl-D-erythritol
produced by the reaction.
1.4.8 Application to mass spectroscopy
Mass spectroscopy has become an important and versatile
technique in the structural elucidation of complex polysaccharides
particularly when it is used in conjunction with gas liquid
chromatography⁶¹.
The combined GC-MS is a major tool in the characterization of
mono and disaccharides derivatives. Most underivatised mono and
disaccharides are nonvolatile and thermally unstable and are,
therefore, unsuitable for GC-MS analysis. For GC-MS ,the sugars
must be stable volatile derivatives.In terms of thermal stability and
ease of interpretation of spectra , permethylated alditol acetates
(formed
on
reduction
of
permethylated
and
hydrilysed
polysaccharides with sodium borohydried followed by acetylation)
are the most widely used derivatives for the characterization of
methylated sugars by mass spectroscopy.
With the adoption of GC-MS for the analysis of methylated sugar
derivatives it is now possible to determine the glycosidic linkages
between sugars in a polysaccharide without the preparative scale
isolation of methylated sugars from their mixtures. The resolving
power of GC coupled with the fragmentation pattern of mass
spectrometer helps in the identification of the various glycosidic
linkages in a polysaccharide. This is because the GLC retention time
and the mass spectrometric fragmentation pattern are characterstics
of the substitution patterns (acetoxy and methoxy groups) in
partially methylated alditol acetates derivatives.
The main limitation of the use of partially methylated alditol
acetates for the characterization of methylated sugars lies in the
structural symmetry, which may exist when the primary hydroxyl
group (O-5 in the pentoses) and O-6 in hexoses is not etherified.This
difficulty, can be overcome by reduction of the sugars with sodium
borodeuteride which introduces deuterium at C-1. These will create
an isotopic shift in primary fragmentation of MS.
1.4.9 Nuclear magnetic resonance spectroscopy
NMR Spectroscopy has become an important tool in the structural
elucidation of complex polysaccharides as it has a number of
characteristics that makes it more advantageous over other physical
methods.NMR spectroscopy is non-destructive and the material can
be recovered back. It can provide detailed structural information of
carbohydrates,
including
identification
of
monosaccharide
composition, elucidation of α- or β-anomeric configurations,
establishment of linkage patterns, and sequences of the sugar units
in oligosaccharides and/or polysaccharides. The most useful
information from ¹H spectra is obtained from the anomeric regions
of protons. These signals are well separated (in both ¹H and¹³C
NMR) from those produced by other nuclei. This greatly helps in
determining the number of different monosaccharide residues in
polysaccharides and also in estimating their relative proportion.
Although NMR spectroscopy provide valuable information about
the structure of polysaccharides, it is not always easy to interpret the
spectra due to poor resolution of signals. Unfortunately, the signals
of carbohydrates in NMR spectra are frequently crowded in a
narrow region, especially for the ¹H NMR spectrum, mostly between
3 to 5 ppm. As a result, the interpretation of ¹H NMR spectra
becomes difficult if a polysaccharide contains many similar sugar
residues. The most recent development in two and multidimensional NMR techniques has significantly improved the
resolution and sensitivity of NMR spectroscopy. For example,
homonuclear correlated spectra are extremely useful for assigning
¹H resonances while the complete assignment of ¹³C- resonances is
achieved by ¹H-¹³C heteronuclear correlation. Long range correlation
techniques, such as nuclear Overhauser enhancement NOE and
heteronuclear multiple bond correlation HMBC , are most useful in
providing sequence information of polysaccharides However, high
frequency instruments (360 Mhz) now available are capable of
resolving all the protons into singlets and multiplets. FT mode is
also advantageous for ¹H spectra. As polysaccharisdes are generally
soluble in water , solutions are prepared in deuterium oxide.The
preparation of solution of a polysaccharide requires prior exchange
treatment with deuterium oxide of high isotopic content (preferably
99.96%). Neverthless, a strong peak due to residual water (HOD
signal) is often obtained whose chemical shift at room temperature
(δ=4.8) obscures the vitally important anomeric region. However, at
a higher temperature (70-80°C) signal shifts upfield (~δ=4.5),
thereby exposing the anomeric region of the spectrum. The anomeric
protons are then easily distinguished.
Other factor that complicate the acquisition of ¹H NMR spectra
of polysaccharides are interference by exchangeable protons (O-H,
N-H) and line broadening of signals in aqueous solution. However
this can be overcome by recording the spectra in deuterated dimethyl
sulphoxide (DMSO-d⁶). Because of low exchange rates in this
medium, hydroxyl and amino proton resonance are clearly observed
in the spectra and provide valuable structural information⁶². It is
much easier to assess the degree of molecular complexity (which is
a measure of different kinds of sugars residues and their ratios ) of a
polysaccharide from ¹³C NMR spectra rather than the ¹H NMR
spectra because the signals in the former are much better dispersed
than the signals of latter. Owing to the low natural abundance
(1.1%) and low sensitivity of the¹³C nucleus the ¹³C spectra are
always recorded in the FT mode.
The chemical shifts of different protons in carbohydrates are
assigned in Figure 1.10. ¹H NMR signals are much more sensitive
than ¹³C signals due to their natural abundance. As a result high ¹H
NMR signals can be used for quantitative purposes in some
applications. However, most of the proton signals fall within a 2
ppm chemical shift range (3 to 5 ppm), which results in substantial
overlap of the signal.
Figure 1.10: Illustration of chemical shifts of carbohydrates in ¹ H
NMR spectroscopy
6
H - 1 of α-gly, (5.1-5.8ppm)
5
H - 1 of β-gly,(4.3-4.8 ppm)
4
p pm
H2- H6
(3.2-4.5ppm)
-O-CH 3
(3.0-3.8ppm)
3
2
1
O
II
C–O–CH 3(1.8-2.2ppm)
C–CH 3 (1.1ppm)
0
Although ¹³C-NMR has a much weaker signal, it has significant
advantages over ¹H- NMR spectroscopy in the analysis of
polysaccharides because the chemical shifts in ¹³C-NMR spectrum
are spread out over a broader range. This broad distribution of
signals helps to overcome the severe overlapping problems
associated with the proton spectrum. In a ¹³C spectrum, signals from
the anomeric carbons appear in the 90 to 110 ppm whereas the non
anomeric carbons are between 60 to 85 ppm.
For NMR spectrum, the xyloglucans are first converted into
oligosaccharides by enzymatic hydrolysis. Their derivatisation into
oligoglycosyl alditols make easy interpretation as they are converted
into single anomeric form either α- configuration or βconfiguration.The oligoglycosyl alditols thus are much easier to
isolate and have simpler NMR spectra
than the parent
oligosaccharides because of the absence of reducing end effects.
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