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 HETEROGLYCAN

(Built up of a single mono- (Built up of two or more

saccharide repeating unit) type of repeating units)

NITROGENOUS NEUTRAL ACIDIC

POLYMERS POLYMERS POLYMERS

1) Glucosamine 1) Glucans(starch,cellulose) 1) Ploygalacturonic

2)Polymers 2) Mannans acid (pectin)

3) Galactans

4) Fructans(inulin) 2) Polymannuronic

5) Xylans acid

NITROGENOUS NEUTRAL ACIDIC

1)Mucopolysaccharide 1)Hemicellulose 1) Gum

2)Glycoproteins 2)Arabinigalactans 2) Mucilage's

3)Glucomannans 3) Alginates

4)Galactomannans (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 β-(1→4)

Callose β-(1→3)

Cereal β-D-glucans β-(1→3) (1→4)

Xyloglucan 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.

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.

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

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 cell-

wall 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

O

OH

HO

OOH

OH

O

O

O

OH

OH

OH

OH

OH

OH

OO

OOH

O

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)-β-

D-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

4 Glcp-(1,4) Glcp-(1,4) Glcp-(1,4) Glcp-(1β β β β

6 6 6

α-Xylp α-Xylp α -Xylp

2

β-Galp

2

α Fucp

4 Glcp-(1,4) Glcp-(1,4) Glcp-(1,4) Glcp-(1β β β β

6 6 6

α-Xylp α-Xylp α -Xylp

2 2

β-Galpβ-Galp

2

α Fucp

X X XF G L F G

4 Glcp-(1,4) Glcp-(1,4) Glcp-(1,4) Glcp-(1β β β β

XX X X G

6 6 6

α-Xylp α-Xylp α -Xylp

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, orchid-

like 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

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

Raw MaterialChemicalComposition

CrudePolysaccharide

Monosaccharide

Composition

Methylation

Analysis/GC-MS

PurifiedPolysaccharide

Specific

Degradation

Oligosaccharide Mixtures

Purification

Isolation ofOligosaccharides

Isolated

Oligosaccharides

MS, FABMALDI,ESI

H, C and2D NMR

Extraction

¹ ¹³

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 more

carefully.Moreover hydrolysis would be done by TFA method.

1.4.2 Methanolysis

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

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

MeO

H

MeO

H

H

H

O

OO

O

OOO

OO

O

OO

O

H

H

MeO

H

H H

H

H

MeO

H

H

OH

OH

OMe

OMe

OMe

MeO

MeO OMe

HH

H

H

HH

OMe

OMeMeO MeO

MeOMeOHO

O

H H

H

HH

H

HH

H

H

H

O

MeO

MeO

H

H

H

OMe

OMe

OMe

OOO

MeO MeO

OMe

OMe

HHH

HH

HH

H H

n

OMe

H

Methanolysis

no free hydroxyl group formed

Methanolysis

Formation of two free hydroxyl group

C

C

B

B

A

A

B

OMe

OMe

H H

a b

n

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

.

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

O

O

OH

HO

OH

H

H

OH

CH2

CH2

O

H

OOH OH

H H

O

OH

CH2

H

OH OH

HH

O

O

H

O

OH

H

CH2

CH2

O

H

O

H H

O

CH2

H

HH

O

O

H

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

CH O3

CH O3 CH O3

H

H

CH

D

OAc

OCH3

H CO3

H

H

H

H

H CO3

OAc

CH OCH2 3

CH

D

OAc

OCH3

H CO3

H

H

H CO3 OAc

CH O2

CH

D

OAc

OCH3

H CO3

H

H

H CO3

OAc

CH OCH2 3

OAc

H CO3 H CO3

cA

OAc

Methylation

Hydrolysis

Deuterised Reduction

Acetylation

(a)

(b) ©

(a)

(b)©

(a) (b) ©

m

m

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 acid-

labile 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 and D-galactofuranose residues can be

selectively hydrolyzed under mild acidic conditions after

methylation. The D-galactofuranosyl linkage is 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

O

HO

CH OH2

H

H

H

H H

H

H

HHH

H

H

H

H

H

OH

OH

O

CH OH2

O

OH

OH O

O

CH OH2

OH

OH

OH

O O

OO

CH OH2 CH OH2CH OH2

H

H H

H

H HH

ΙCΙΙO

ΙCΙΙO

CΙΙO

CΙΙO

Ι

C=O

ΙH

CΙΙO

H―C3

H H H

=O

OH

+n

n

Periodate Oxidation

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 multi-

dimensional 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

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

6

5

4

3

2

1

0

H - 1 of α-gly, (5.1-5.8ppm)

H - 1 of ppm)β-gly,(4.3-4.8

-O-CH(3.0-3.8ppm)

H2- H6(3.2-4.5ppm)

OIIC–O–CH (1.8-2.2ppm)

C–CH (1.1ppm)

ppm

3

3

3

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 β-

configuration.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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