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ISOLATION OF BIOLOGICALLY ACTIVE OLIGOSACCHARIDES FROM

MILK OF Ovies aries AND THEIR STRUCTURE ELUCIDATION

THESIS SUBMITTED

TO THE UNIVERSITY OF LUCKNOW

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

CHEMISTRY

By

ASHOK KUMAR RANJAN (M.Sc.)

DEPARTMENT OF CHEMISTRY UNIVERSITY OF LUCKNOW

LUCKNOW, UTTAR PRADESH (INDIA)

2015

Prof. R. N. Pathak Professor & Head

This is to certify that all necessary requirements for the submission of Ph.D.

thesis of Mr. Ashok Kumar Ranjan has been fully observed.

Pathak Department of Chemistry University of L

Email:

CERTIFICATE


thesis of Mr. Ashok Kumar Ranjan has been fully observed.

[Prof. R. N. Pathak

Department of Chemistry

University of Luckno

Department of Chemistry University of Lucknow

Lucknow 226007 INDIA

Email: [email protected]


[Prof. R. N. Pathak]

Head

Department of Chemistry

University of Lucknow

Lucknow.

Dr. Desh Deepak Phone: (R) 0522-2258879

M. Sc., Ph.D. (M) 09454856373 FNMRS, FISMS, FACCT, FISCB, FSCBS Email- [email protected]

Department of Chemistry University of Lucknow Lucknow-226007, India

Dated

CERTIFICATE

This is to certify that the work embodied in this thesis has been carried out by

Mr. ASHOK KUMAR RANJAN, M.Sc., under my supervision. He has fulfilled the

requirements for the degree of Doctor of Philosophy of Lucknow University,

regarding the nature and prescribed period of investigational work. The work

included in this thesis has not been submitted for any other degree and unless

otherwise stated, is all original.

Dr. Desh Deepak (Associate Prof.)

Department of Chemistry University of Lucknow,

Lucknow.

Dedicated

to

my Parents

CONTENTS

Page No.

ACKNOWLEDGEMENTS

ABBREVIATIONS i-iv

PREFATORY NOTE v-xix

CHAPTER-I INTRODUCTION 1-40

Importance of Carbohydrates and Oligosaccharides 1-6

Biological activity of Milk Oligosaccharides 6-12

Methods of Isolation of Milk Oligosaccharides 13-14

Purification of Milk Oligosaccharides 14-17

Structure Elucidation of Milk Oligosaccharides 18-40

CHAPTER-II ISOLATION 41-57

Isolation of Ovies aries Milk Oligosaccharides 41-44

Purification of Ovies aries Milk Oligosaccharides 45-47

Description of Isolated Oligosaccharides 48-57

CHAPTER-III RESULTS AND DISCUSSION 58-127

Structural Elucidation of Oligosaccharide-A (Capriose) 58-74

Structural Elucidation of Oligosaccharide-B (Viesose) 75-91

Structural Elucidation of Oligosaccharide-C (Ariesose) 92-109

Structural Elucidation of Oligosaccharide-D (Riesose) 110-127

CHAPTER-IV EXPERIMENTAL 128-134

BIBLIOGRAPHY 135-144

APPENDIX 145

ACKNOWLEDGMENT

First and foremost, I’d like to avail this opportunity to express my deep gratitude towards

my supervisor Dr. Desh Deepak, Associate Prof. Department of Chemistry,

University of Lucknow, Lucknow for his continuous support and guidance in my Ph.D.

dissertation and research work. If it was not for his patience, constant motivation, his un

bound zeal to do something new and his profound love and understanding of the subject,

this venture of mine would just not have been possible. It was completely blissful for me

to have found a counsellor in my guide whose suggestions to me are nothing less than

lessons for life.

Also, I am indebted to Prof. R. N. Pathak, Head, Department of Chemistry,

University of Lucknow, Lucknow for providing me with laboratory and library facilities

at whatever time I needed them and his support was something I could always fall back

on. I am grateful Prof. Naveen Khare, Department of Chemistry, University of Lucknow

for those meaningful discussions he would have with me at all points during the day and

his constructive criticism followed by excellent suggestions. My sincere gratitude goes to

Prof. Raja Roy, CBMR, SGPGI, Lucknow for providing me NMR facilities and fruitful

suggestions and discussions, whenever and where ever it was required. Without thanking

him this would totally be incomplete. I am also thankful to Dr. Sanjeev Kumar Shukla,

CDRI, Lucknow, for the successful running of my NMR spectra. I would like to thank

Dr. Sanjeev Kanaujia, CDRI, Lucknow for providing me Mass Spectrometry facilities. I

would like to thank Dr. Amit Srivastava for providing me HPLC facilities. My sincere

thanks go to Mr. Alok Shukla for his moral support. I am warmly thankful to Dr. (Mrs.)

Neelima Deepak for her blessings, love, affection and encouragement. It’s my deep

privilege to express gratitude to Prof. (Mrs.) Anakshi Khare, former Head, Department

of Chemistry, University of Lucknow, Lucknow.

I am grateful to my research colleague Mrs. Meenakshi Singh for her constant

support and helpful suggestions. I am also thankful to my research colleagues Ashish

Singh, Kuldeep Kumar, Gunjan, Mujeeb Khan, Pushpraj and Ranjeet . I would also

like to express my gratitude towards my seniors Dr. Narendra Mani Tripathi, Dr.

Aneesh Kumar, Dr. Mayank Agnihotri and Dr. R. K. Bajpayee for their valuable

suggestions and help.

I am extremely thankful to my friends Mr. Sheel Ratan, Mukul, Anil, and

Surjeet Singh, Ranvijay Singh, Shailendra Bharti, Ms. Stuti, Priydarshni, Ritu,

Shaheen, and Indu for their moral support.

In the end, a special mention of those people who put me in this place where I

could do my doctorate I, hence would like to express my deepest gratitude towards my

family, my parents in particular, Mr. Nanhu Ram and Mrs. Sharda Devi, without their

blessings and support this achievement was not possible. I am also thankful to my

brothers Mr. Pradeep Kr. Ranjan, Dr. Ajai Kr. Ranjan and Mr. Manoj Kr. Ranjan

and my most loving sister and her husband Mrs. Premlata Roshan and Mr. Satish

Roshan for their undying support and encouragement which helped me to do better every

day.

I wish to thanks to all those who helped me in one or other way.

Date Ashok Kumar Ranjan

i

ABBREVIATIONS USED

AcOH = Acetic acid

Ac2O = Acetic anhydride

Ag2CO3 = Silver carbonate

AgNO3 = Silver nitrate

AgOH = Silver hydroxide

Ag2S = Silver sulphide

Anhyd. = Anhydrous

Aq. = Aqeous

BaCO3 = Barium carbonate

BuOH = n-Butanol

Br2 = Bromine

C = Carbon

CC = Column chromatography

CDCl3 = Deuterated chloroform

CHCl3 = Chloroform

CH2Cl2 = Dichloromethane

CH3CHO = Acetaldehyde

CH3CN = Acetonitrile

C6H5CH3 = Toluene

conc. = Concentration

D = Doublet

Dd = Double doublet

D2O = Deuterated water

DTH = Delayed type hypersensitivity

2D

ESMS

=

=

Two dimensional

Electron Spray mass spectrometry

EtOH = Ethanol

Et2O = Diethyl ether = solvent ether

EtOAc = Ethyl acetate

ii

FABMS = Fast atom bombardment mass spectrometry

FeCl3 = Ferric chloride

Fe2(SO4)3 = Ferric sulphate

Fr.

Fuc

=

=

Fraction(s)

Fucose

G = Grams

Gal = Galactose

GalNAc = 2-Acetamido-2-deoxy-galactose

Glc = Glucose

GlcNAc = 2-Acetamido-2-deoxy-glucose

H = Hour

H = Hydrogen

HA = Haemagglutination titre

HCl = Hydrochloric acid

H2O = Water

HPLC = High performance liquid chromatography

H2S = Hydrogen sulphide

H2SO4 = Sulphuric acid

Hz = Hertz

i.p. = Intraperitoneal

J = Coupling constant

Kg = Kilogram

KOH = Potassium hydroxide

LND = Lacto-N-difucohexaose

LNF = Lacto-N-fucopentaose

LNH = Lacto-N-hexaose

LNT = Lacto-N-tetraose

LNnT = Lacto-N-neotetraose

Lt.

Lea

Leb

=

=

=

Liter

Lewis a

Lewis b

iii

Lex = Lewis x

M = Multiplet

MeOH = Methanol

Me2CO = Acetone

Mg = Milligram

MHz = Megahertz

Min = Minute

Ml = Milliliter

µl = Microlitre

MLR = Mixed lymphocyte reaction

MMI = Macrophage migration index

Mol. wt. = Molecular weight

Mp = Melting point

Mmp = Mixed melting point

N = Normal

NaBH4 = Sodium borohydride

Na2CO3 = Sodium carbonate

NaIO4 = Sodium metaperiodate

NaOH = Sodium hydroxide

NaOCH3 = Sodium methoxide

Na2SO4 = Sodium sulphate

NMR = Nuclear magnetic resonance spectroscopy

O = Oxygen

OAc = Acetyl

OH = Hydroxyl

ORD = Optical rotatory dispersion

Pb(OH)2 = Lead hydroxide

PC = Paper chromatography

PFC = Plaque-forming cells

P2O5 = Phosphorus penta-oxide

Pyr = Pyridine

iv

Q = Quartet

R P = Reverse phase

S = Singlet

S D = Standard deviation

SEM = Standard error of mean

SiO2 = Silica

SRBC = Sheep red blood cells

T = Triplet

TBA = Thiobarbituric acid

TDW = Triple distilled water

TLC = Thin layer chromatography

TMS = Tetra methyl silane

UV = Ultra violet

[α]D = Rotation/specific rotation

α = Alpha

β = Beta

δ = Delta

v

PREFATORY NOTE

The work presented in this thesis began in September 2011 under the able guidance of

Dr. Desh Deepak associate professor, Department of chemistry, University of Lucknow.

With a view to carry out a detailed chemical and physical investigation of milk

oligosaccharides isolated from Sheep milk (Oveis aries). The Sheep milk

oligosaccharides were isolated and purified by different chromatographic methods and

their structure were elucidated by chemical degradation, chemical transformation and

spectroscopic techniques (1H, 13C and 2D i.e. HSQC, HMBC, COSY and TOCSY NMR

and MASS spectrometry).

In the present scenario glyco compounds have established them self as important

therapeutic agents with milder or no side effects. Numbers of antibiotics, vaccines, anti

cancer agents, immunostimulant are in clinical trials having either carbohydrates or their

derivatives in their structure. Under the category of carbohydrates, oligosaccharides are

an important class of compounds. They are present in higher plants, fungi, algae, bacteria

and milk. The oligosaccharides isolated from these sources have showed varied

biological activities such anti-tumor, immunological, anticomplimentory, anti-cancer,

anti-inflammtroy, Anti-coagulant, hypoglycemic and anti-viral activities. In the recent

years milk has immerged as an important source of new and structurally complex

oligosaccharides which are promising therapeutic agents evaluated against dreaded

diseases like AIDS and cancer. In the ancient medicinal system like ayuerveda and

Unani, specific activities are reported for milks of various origin but at that time role of

oligosaccharides were not known. So the glycochemist has taken this field as a challenge

and isolated number of oligosaccharides from milk and studied their varied biological

activities. These milk oligosaccharides have been classified as acidic or sialylated

oligosaccharides and non-sialylated or normal oligosaccharides they are further classified

as fucosylated, branch chained and straight chained oligosaccharides. This classification

is based on either due to presence of specific moieties such as sialic acid (Neuraminic

acid), fucose, or on the basis of their structures i.e. the monosaccharides are present in

straight chain or they are having branching in them. Number of oligosaccharides have

been isolated from milk of Donkey, Mare, Buffalo, Camel, Cow, Chouri cow, Yak, Gaddi

vi

sheep etc. and their biological activities have been studied. As the medicinal importance

in the Sheep’s milk in ancient medicinal system was enormous and it has shown potent

activities against leukoderma, antimicrobial agents and enhance the organism’s natural

defenses against invading pathogens. Keeping in mind the medicinal importance of

Sheep milk and the role of oligosaccharides in it, in the present dissertation Sheep milk

was analyzed for its oligosaccharides contents. In the present work oligosaccharides were

isolated from Sheep’s milk by following established methods and combing the recent and

traditional chromatographic techniques. Further the stereospecific structures of these

oligosaccharides were established with help of recent two dimensional experiments of

NMR and MASS spectrometry and combining the data with traditional methods of

chemical degradation and chemical transformation.

The thesis entitled “ISOLATION OF BIOLOGICALLY ACTIVE

OLIGOSACCHARIDES FROM MILK OF Ovies aries AND THEIR STRUCTURE

ELUCIDATION” has been divided into four chapters:

CHAPTER I: INTRODUCTION

In this chapter a brief introduction of the biological significance of carbohydrates

and important drugs containing carbohydrate moieties are given. It also includes the

biological activities shown by naturally occurring oligosaccharides, especially those

found in milk of various origins and their biological importance. Various methods of

isolation of milk oligosaccharides mainly Modified method of Kobata and Ginsburg have

been described. This chapter includes different chromatographic techniques like Thin

Layer Chromatography, Column Chromatography, High Performance Liquid

Chromatography, Reverse Phase-HPLC etc. which have been used in the purification of

oligosaccharides. Various chemical degradation and transformation techniques such as

acid hydrolysis, acetylation/methylation etc have been discussed. Spectroscopic

techniques like NMR (1H, 13C and 2D NMR spectroscopy) and Mass spectrometry which

are used in the structure elucidation of milk oligosaccharides have also been discussed in

detail in this chapter.

vii

CHAPTER II: ISOLATION OF MILK OLIGOSACCHARIDES

This chapter describes in detail the methods of processing, isolation and

purification of ‘Sheep’ milk oligosaccharides. For the present chemical investigation of

milk oligosaccharide, Sheep milk was collected in bulk (10 lit.) and was processed by

‘modified method of Kobata and Ginsberg’ which afforded a crude mixture of

oligosaccharide containing normal and amino sugar in them. The lyophilized material

(crude milk oligosaccharide mixture) was purified on a Sephadex G-25 column which

separated glycoproteins and proteins from oligosaccharides. Oligosaccharide fractions so

obtained from Sephadex G-25 column were pooled and subjected to reverse phase HPLC

for establishing their homogeneity in crude oligosaccharide mixture. The peaks were

numbered in their increasing order of retention time i.e. 7.21 min (R1), 7.63 min (R2),

8.45 min (R3), 17.21 min (R4), 17.41 min (R5), 20.58 min (R6), 21.39 min (R7), 21.78 min

(R8), 24.69 min (R9), 25.42 min (R10), 25.97 min (R11), 27.51 min (R12). The HPLC

showed the presence of twelve oligosaccharide. Mixture of oligosaccharide was

acetylated by acetic anhydride and pyridine for getting their respective acetyl derivatives

which were resolved nicely on TLC and showed nine well resolved spots which were

designated as a, b, c, d, e, f, g, h and i. Repeated column chromatography of these

acetylated oligosaccharides mixture on silica gel, using different solvent systems

followed by their deacetylation afforded four novel oligosaccharides viz. A, B, C and D.

Table-1: Acetylated and Deacetylated oligosaccharides obtained from Sheep milk

Acetylated Compound Deacetylated Compound

Alphabetical name

Analytical notation

Quantity (mg) Alphabetical name

Analytical notation

Quantity (mg) Isolated by

CC Taken for

deacetylation

a ARSM-1A 112 40 A ARSM-1 35

b ARSM-2A 419 50 B ARSM-2 45

c ARSM-3A 46 32 C ARSM-3 24

d ARSM-4A 62 52 D ARSM-4 44

viii

The physico-chemical data of four novel oligosaccharides i.e. specific rotation, C, H

analysis 1H and 13C NMR is also been given in this chapter

Table-2: Description of isolated oligosaccharides from Sheep milk

A B C D

Analytical notation ARSMM-1 ARSM-2 ARSM-3 ARSM-4

Name of compound Capriose Viesose Ariesose Riesose

Physical state Syrupy Syrupy Syrupy Syrupy

��

+41.01o +72.02o +28.71o +113.88o

Mol. Formula C34H58O25N2 C34H58O26N2 C36H61O26N3 C40H68O31N2

ES mass (m/z) 894 910 951 1072

Phenol-sulphuric test*1 +ve +ve +ve +ve

Morgon-Elson test*2 +ve +ve +ve +ve

Thiobarbituric acid test*3 -ve -ve -ve -ve

Bromo cresol green test*4 -ve -ve -ve -ve

1. Test of normal sugar*. 2. Test of amino sugar* 3. Test of sialic acid*

4. Test of carboxylic acid*.

CHAPTER III: RESULTS AND DISCUSSION

This chapter includes discussion of the results of chemical and spectral studies

for structure elucidation of four novel oligosaccharides isolated from the Sheep milk

namely Capriose(A), Viesose(B), Ariesoe(C) and Riesoe(D). In this chapter the structure

elucidation of oligosaccharides were done with the help of chemical degradation,

chemical transformation and spectroscopic techniques like 1H, 13C, S.R.G. and 2D NMR

e.g. HOMOCOSY, TOCSY, HETROCOSY, HSQC, HMBC and mass spectrometric

techniques.

ix

STRUCTURAL CHARACTERIZATION OF MILK OLIGOSACCHRIDES

Number of oligosaccharides have been isolated from milk of various origin and these

oligosaccharides have shown varied biological activities viz. antitumor, anticancer,

immunostimulant etc. A survey of literature showed that they are constituted of some

common basic core units which were present in most of the milk oligosaccharides, they

are as follows -

Lactose :- Gal-(β1→4)-Glc

Lacto-N-tetraose (LNT):- Gal-(β 1→3)- GlcNAc-( β 1→3)- Gal-( β 1→4)-Glc

Lacto-N-neotetraose (LNnT):- Gal-(β 1→4) - GlcNAc-(β 1→3) - Gal-(β 1→4)-Glc

Lacto-N-hexaose (LNH):- Gal- β (1→4)-GlcNAc- (β 1→6) Gal- (β 1→4)-Glc Gal- (β 1→3)-GlcNAc- (β 1→3)

Lacto-N-neohexaose (LNneoH):- Gal- (β 1→4)-GlcNAc- (β 1→6) Gal- (β 1→4)-Glc Gal- (β 1→4)-GlcNAc- (β 1→3)

Para-Lacto-N-neohexaose (paraLNH):- Gal-(β1→4)-GlcNAc-(β1→3)-Gal-(β1→4)-GlcNAc-(β1→3)-Gal-(β1→4)-Glc

Para-Lacto-N-neohexaose (paraLNneoH):- Gal- (β 1→3)-GlcNAc-(β 1→3) - Gal-(β 1→4)-GlcNAc-(β 1→3) - Gal- (β 1→4)-Glc Lacto-N-octaose:- Gal-(β1→4)-GlcNAc (β1→3) Gal-(β1→4)-GlcNAc- (β1→6) Gal- (β 1→4)-Glc Gal- (β1→4)-GlcNAc- (β 1→3) Lacto-N-neooctaose:- Gal- (β1→3)-GlcNAc (β1→3) Gal- (β1→4)-GlcNAc- (β1→6) Gal- (β1→4)-Glc Gal- (β1→4)-GlcNAc- (β 1→3)

x

Iso-Lacto-N-octaose:- Gal- (β1→4)-GlcNAc (β1→3) Gal- (β1→4)-GlcNAc- (β1→6) Gal- (β 1→4)-Glc Gal- (β1→3)-GlcNAc- (β 1→3)

Para-Lacto-N-octaose:- Gal-(β1→3)-GlcNAc-(β1→3)-Gal-(β1→4)-GlcNAc-(β1→3)-Gal-(β1→4)-

GlcNAc-(β 1→3)- Gal-( β 1→4)-Glc

Lacto-N-decaose:- Gal- (β 1→4)-GlcNAc-(β 1→6) Gal- (β 1→4)-GlcNAc- (β 1→6) Gal- (β 1→4)-GlcNAc- (β 1→3) Gal- (β 1→4)-Glc Gal- (β 1→4)-GlcNAc- (β 1→3)

The previous workers have elucidated the structure of milk oligosaccharides by

chemical degradation, chemical transformation, structure reporter group theory and

spectroscopic techniques (NMR and Mass spectrometry). In the present study the

structures of four novel milk oligosaccharides were established by comparing the

chemical shift (1H and 13 C NMR) of anomeric proton and carbon resonance signals and

other important signals of unknown milk oligosaccharides with the chemical shifts of

known milk oligosaccharides. Simultaneously analogies between chemical shift of certain

‘structural reporter group resonances’ were used to make proton resonance

assignments as well as structural assignments of the oligosaccharides. All chemical shifts

of anomeric proton signals of milk oligosaccharides were further confirmed by 2D (1H-1H HOMOCOSY, TOCSY, HMBC and HSQC) NMR experiments, which were earlier

assigned with the help of 1H and 13C NMR data. Other techniques like deacetylation,

methylation, hydrolysis, chemical degradation and mass spectrometry were also used for

the structural elucidation of these novel oligosaccharide.

xi

COMPOUND-A CAPRIOSE

Compound A, C34H58O25N2, ��

� +41.01o gave positive Phenol-sulphuric acid test,

Fiegl test, and Morgan-Elson test, showing the presence of normal and amino sugars

moietie(s) in the compound-A. The HSQC spectrum of acetylated capriose showed the

presence of five cross peaks of six anomeric protons doublets and carbons in their

respective region at 6.37x89.68, 5.72x91.66, 5.40x91.66, 5.19x101.05, 4.48x101.05(2H),

suggesting the presence of six anomeric protons and carbon in it. Further the presence of

six anomeric signals were confirmed by presence of five 1H NMR doublets i.e. at δ

6.37(1H), 5.72(1H), 5.40(1H), 5.19(1H) 4.48(2H) in the acetylated spectrum of Capriose

in CDCl3 at 300 MHz. The presence of six anomeric carbons were also confirmed by the

presence of three anomeric carbon signals at δ 89.68(1C), 91.66(2C), 101.05(3C) in the 13C NMR spectrum of acetylated compound-A in CDCl3 at 300 MHz. The pentasccharide

nature of capriose was further supported by five anomeric proton doublets of five protons

δ 5.57(1H), 4.59(1H) 5.3 (1H), 4.35(2H) along with two methyl (NHCOCH3) signal at δ

1.94 (2NHAc) in the 300 MHz NMR spectrum of Capriose in D2O. These data suggested

that compound capriose may be a pentasccharides in its reducing form. In 1H NMR

spectrum of acetylated capriose out of 6 anomeric proton signals, signal at δ 6.37 and δ

5.72 were assigned for downfield shifted α and β anomeric protons at the reducing end

suggesting that was in its reducing form and suggested that compound-A ‘capriose’ may

be a pentasccharide in its reducing form. Further the ES mass spectrum of capriose

showed highest mass ion peaks at m/z 956 assigned to [M+Na+K]+ and m/z 933

assigned to [M+K]+, it also contain the molecular ion peak at m/z 894 confirming the

molecular weight as 894 which was in agreement with derived composition C34H58O25N2.

The reducing nature of compound was further confirmed by methylglycosylation

MeOH/H+ followed by its acid hydrolysis, which led to the isolation of α and β- methyl

glucosides along with Gal, GlcNac and FucNac, suggesting the presence of glucose at the

reducing end, for convenience all five monosaccharides were denoted as S-1, S-2, S-3, S-

4 and S-5. The monosaccharide constituents in compound-A were confirmed by its

killiani hydrolysis under strong acidic condition, followed by paper chromatography and

TLC. In this hydrolysis four spots were found identical with the authentic samples of Glc,

Gal, GlcNac and FucNac by co-chromatography. Thus the pentasaccharide contained

xii

four types of monosaccharides units i.e. Glc, Gal, GlcNac and FucNac. The positions of

glycosidation in the oligosaccharide were confirmed by position of anomeric signals,

Structure reporter group and comparing the signals in 1H and 13C NMR of acetylated and

deacetylated oligosaccharide. The glycosidic linkages in capriose were assigned by the

cross peaks for glycosidically linked carbons with their protons in the HSQC and HMBC

spectrum of acetylated Capriose. The values of glycosidic linkage in HSQC spectrum are

described as under. Cross peak of H-4 and C-4 of β-Glc (S-1) at 3.6x65.7 showed (1→4)

linkage between S-2 and S-1, cross peak of H-3 and C-3 of β-Glc (S-1) at 3.84x72.6

showed (1→3) linkage between S-3 and S-1, cross peak of H-2 and C-2 of β-Gal (S-2) at

δ 3.9 x70.09 showed (1→2) linkage between S-4 and S-2, cross peak of H-3 and C-3 of

β-Gal (S-2) at δ 4.01x70 showed (1→3) linkage between S-5 and S-2. On the basis of the

results obtained by comparison of chemical shifts of anomeric and ring protons and

carbons in 1H and 13C NMR of capriose and capriose acetate, results obtained from

chemical degradation, chemical transformation along with 2D NMR like COSY,

TOCSY, HSQC, HMBC experiments and mass spectrometry the structure of novel

oligosaccharide capriose was assigned as under-

CAPRIOSE

xiii

COMPOUND-B VIESOSE

Compound B, C34H58O26N2, ��

� +72.02o gave positive Phenol-sulphuric acid test, Fiegl

test and Morgan-Elson test showing the presence of normal and amino sugars moietie(s)

in the compound B. The HSQC spectrum of acetylated viesose showed the presence of

five cross peaks for six anomeric protons and six anomeric carbons in their respective

region at 6.22x89.10, 5.64x91.56, 4.976x91.56, 5.22x101.97, 4.56x100.84(2H),


six anomeric peaks of anomeric protons were separately confirmed by five NMR

doublets i.e. δ 6.22(1H), 5.64(1H), 5.22(1H), 4.97(1H), 4.56(2H) in the 1H NMR

spectrum of acetylated viesose in CDCl3 at 300 MHz. The presence of six anomeric

carbons were confirmed by the presence of four peaks at δ 89.10(1C), 91.56(2C),

100.97(1C), 100.84(2C) in 13C spectrum of viesose acetate in CDCl3 at 300 MHz. The

pentasccharide nature of viesose was further supported by the presence of five anomeric

proton doublets for six anomeric protons at δ 5.20(1H), 4.6(1H), 4.49(1H), 4.46(1H),

4.39(2H) in 1H NMR spectrum of viesose in D2O at 300 MHz. These data suggested that

viesose may be a pentasccharides in its reducing form. In 1H NMR spectrum of

acetylated viesose out of 6 anomeric proton signal, signal at δ 6.22 and 5.64 contained

downfield shifted α and β anomeric protons suggested that it was in its reducing form and

compound-B ‘viesose’ may be a pentasccharide in its reducing form. Further the ES mass

spectrum of viesose showed the highest mass ion peaks at m/z 972 assigned to

[M+Na+K]+ and m/z 949 assigned to [M+K]+, it also contain the molecular ion peak at

m/z 910 confirming the molecular weight as 910 which was in agreement of derived

composition i.e. C34H58O26N2. The reducing nature of compound-B viesose was

confirmed by its methylglycosylation MeOH/H+ followed by its acid hydrolysis, which

led to the isolation of α and β- methyl glucosides along with Gal, GlcNac and GalNac,

suggesting the presence of glucose at the reducing end, for convenience all five

monosaccharides were denoted as S-1, S-2, S-3, S-4 and S-5 respectively from the

reducing end. The monosaccharide constituents in compound-B were confirmed by its

killiani hydrolysis under strong acidic condition, followed by paper chromatography and

TLC. In this hydrolysis four spots were found identical with the authentic samples of Glc,

Gal, GlcNac and GalNac by co-chromatography. Thus the pentasaccharide contained four

xiv

types of monosaccharides units i.e. Glc, Gal, GlcNac and GalNac. The positions of


S.R.G. and comparing the signals in 1H and 13C NMR of acetylated and deacetylated

oligosaccharide.The glycosidic linkages in viesose were assigned by the cross peaks for

glycosidically linked carbons with their protons in HSQC and HMBC spectrum of

acetylated viesose. The values of glycosidic linkage in HSQC spectrum are described as

under. Cross peak of H-4 and C-4 of β-Glc(S-1) at (δ 3.6x72.50) showed 1→4 linkage of

S-2 and S-1 cross peak of H-3 and C-3 of β-Glc(S-1) at (δ 3.8x73.1) showed 1→3

linkage of S-3 and S-1. Cross peak of H-2 and C-2 of β-Gal(S-3) at (δ 3.72x72.40)

showed 1→2 linkage of S-4 and S-3, cross peak of H-3 and C-3 of β-Gal(S-3) at (δ

3.70x70.40) showed 1→3 linkage of S-5 and S-3. On the basis of the results obtained by

comparison of chemical shifts of anomeric and ring protons and carbons in 1H and 13C

NMR of viesose and viesose acetate, results obtained from chemical degradation,

chemical transformation along with 2D NMR like COSY, TOCSY, HSQC, HMBC

experiments and mass spectrometry the structure of novel oligosaccharide viesose was

assigned as under-

VIESOSE

xv

COMPOUND-C ARIESOSE

Compound C, C36H61O26N3, ��

� +28.71o gave positive Phenol-sulphuric acid test, Fiegl

test and Morgan-Elson test showing the presence of normal and amino sugars moietie(s)

in the compound C. The HSQC spectrum of acetylated Ariesose showed the presence of

six cross peaks of anomeric protons and carbons in their respective region at 6.23x89.13,

5.64x91.57, 4.58x101.88, 4.55x102.02, 4.49x100.92, 4.49x101.19 suggesting the

presence of six anomeric protons and carbons into it. Presence of five anomeric peaks for

six protons doublets were separately confirmed by 1H NMR of acetylated ariesose at 300

MHz i.e. δ 6.23(1H), 5.64(1H), 4.58(1H), 4.55(1H), 4.49(2H). The presence of six

carbons were also confirmed by the presence of six anomeric carbons peaks at δ

89.13(1C), 91.57(1C), 101.88(1C), 102.02(1C), 100.92(1C), 101.19(1C) in the acetylated

spectrum of Ariesose at 300 MHz. The pentasccharide nature of Ariesose was further

supported by five anomeric peaks for six protons doublets i.e. δ5.15 (1H), δ4.59 (1H),

δ4.47 (1H), δ4.44 (1H), 4.38 (2H) in 1H NMR spectrum of Ariesose in D2O at 300 MHz.

Further the anomeric proton singlet at δ6.23 and δ5.64 in the 1H NMR of acetylated

compound-C showed downfield shifted α and β anomeric protons showing its was in its

reducing form and suggested that compound Ariesose may be a pentasccharide in its

reducing form. Further the ES mass spectrum of Ariesose showed the highest mass ion

peaks at m/z 1013 assigned to [M+Na+K]+ and m/z 990 assigned to [M+K]+, it also

contain the moleculer ion peak at m/z 951 confirming the moleculer weight of ariesose

was 951 which was in agreement to derived composition i.e.C36H61O26N3. The reducing

nature of compound was further confirmed by methylglycosylation MeOH/H+ followed

by its acid hydrolysis, which led to isolation of α and β- methyl glucosides along with Gal

and GalNac suggesting the presence of glucose at the reducing end, for convenience all

five monosaccharides were denoted as S-1, S-2, S-3, S-4 and S-5. The monosaccharide

constituents in compound–C were confirmed by its killiani hydrolysis under strong acidic

condition, followed by paper chromatography and TLC. In this hydrolysis four spots

were found identical with the authentic samples of Glc, Gal, GlcNac and GalNac by co-

chromatography. Thus the pentasaccharide contained four types of monosaccharides units

i.e. Glc, Gal, GlcNac and GalNac. The 1H NMR also contain three methyl signals of

NHCOCH3 at δ 1.85 and δ 2.01(2NHAc) showing out of five three monosaccharides was

xvi

N-acetylated sugars. The positions of glycosidation in the oligosaccharide were

confirmed by position of anomeric signals, Structure reporter group and comparing the

signals in 1H and 13C NMR of acetylated and deacetylated oligosaccharide. The

glycosidic linkages in ariesose were assigned by the cross peaks for glycosidiccally

linked carbons with their protons in HSQC and HMBC spectrum of acetylated ariesose.

The values of glycosidic linkage in HSQC spectrum are described as under. Cross peak

of H-4 and C-4 of β-Glc (S-1) at (3.6x82) showed 1→4 linkage of S-2 and S-1 and cross

peak of H-3 and C-3 of β-Glc (S-1) at (3.83x73.50) showed 1→3 linkage between S-3

and S-1. Cross peak of H-3 and C-3 of β-GalNAc(S-3) at (3.85x73) showed 1→3 linkage

between S-4 and S-3. Cross peak of H-3 and C-3 of β-GalNAc(S-4) at (3.85x73) showed

1→3 linkage between S-5 and S-4. On the basis of the results obtained by comparison of

chemical shifts of anomeric and ring protons and carbons in 1H and 13C NMR of ariesose

and ariesose acetate, results obtained from chemical degradation, chemical

transformation along with 2D NMR like COSY, TOCSY, HSQC, HMBC experiments

and mass spectrometry the structure of novel oligosaccharide ariesose was assigned as

under-

ARIESOSE

xvii

COMPOUND-D RIESOSE

Compound-D, C40H68O31N2, ��

� +113o gave positive Phenol-sulphuric acid test, Fiegl

test and Morgan-Elson test showing the presence of normal and amino sugar moietie(s) in

the compound-D. The HSQC spectrum of acetylated riesose showed the presence of six

cross peaks for seven anomeric protons doublets and carbons in their respective region at

6.17x90.24, 5.37x90.12, 4.77x95.26, 4.59x101.90, 4.52x101.05, 4.52x100.96 suggesting

the presence of six anomeric protons and carbons in it. The presence of seven anomeric

proton doublets were confirmed by five anomeric signals in 1H NMR i.e. at δ 6.17,

5.37(2H), 4.77(1H), 4.59(1H), 4.52(2H) in the 1H NMR spectrum of riesose acetate in

CDCl3 at 300 MHz. The presence of seven anomeric carbons were confirmed by the

presence of six anomeric peak signals in 13C spectrum at δ 90.12(2C), 90.24(1C),

95.26(1C), 100.96(1C), 101.05(1C), 101.90(1C), of acetylated riesoses in CDCl3 at 300

MHz. These data suggested that compound riesose may be a Hexasccharide in its

reducing form. In 1H NMR spectrum of riesose acetate which contains seven anomeric

signals, out of which signal at δ 6.17 and 5.37 were assigned for downfield shifted α and

β anomeric protons of monosaccharide present at the reducing end suggested that

compound-D ‘riesose’ may be a hexasccharide in its reducing form. The hexaasccharide

nature of riesose was further supported by presence of five anomeric proton doublets for

six anomeric protons at δ 5.16(1H), 4.60 (1H), 4.52(1H), 4.39(2H), 4.21(1H) along with

two singlet at δ 1.94 and 1.86 for two methyl groups of NHCOCH3 at 300 MHz 1H NMR

spectrum of riesose in D2O. Further the ES mass spectrum of riesose showed the highest

mass ion peaks at m/z 1134 assigned to [M+Na+K]+ and m/z 1111 assigned to [M+K]+,

it also contain the molecular ion peak at m/z 1072 confirming the molecular weight as

1072 which was in agreement of derived composition C40H68O31N2. The reducing nature

of compound was further confirmed by methylglycosylation MeOH/H+ followed by its

acid hydrolysis, which led to isolation of α and β- methyl glucosides along with Gal,

GlcNac and GalNac, suggesting the presence of glucose at the reducing end, for

convenience all six monosaccharides denoted as S-1, S-2, S-3, S-4, S-5 and S-6. The

monosaccharide constituents in compound-D were confirmed by its killiani hydrolysis

under strong acidic condition, followed by paper chromatography and TLC. In this

hydrolysis four spots were found identical with the authentic samples of Glc, Gal,

xviii

GlcNac and GalNac by co-chromatography. Thus the hexasaccharide contained four

types of monosaccharides units i.e. Glc, Gal, GlcNac and GalNac. The positions of


S.R.G. and comparing the signals in 1H and 13C NMR of acetylated and deacetylated

oligosaccharide. The glycosidic linkages in riesose were assigned by the cross peaks for

glycosidically linked carbons with their protons in HSQC and HMBC spectrum of

acetylated riesose. The values of glycosidic linkage in HSQC spectrum are described as

under. Cross peak of H-4 and C-4 of β-Glc (S-1) at (3.8x76) showed 1→4 linkage of S-2

and S-1. Cross peak of H-2 and C-2 of βGal(S-2) at (3.80x72.3) showed 1→2 linkage

between S-3 and S-2 and cross peak of H-3 and C-3 of β-Gal(S-2) at (4.1x70) showed

1→3 linkage between S-4 and S-2. Cross peak of H-3 and C-3 of α-GlcNAc(S-3) at

(3.46x78) showed 1→3 linkage between S-6 and S-3. Cross peak of H-3 and C-3 of

βGal(S-4) at (4.2x70.5) showed 1→3 linkage between S-5 and S-4. On the basis of the

results obtained by comparison of chemical shifts of anomeric and ring protons and

carbons in 1H and 13C NMR of riesose and riesose acetate, results obtained from chemical

degradation, chemical transformation along with 2D NMR like COSY, TOCSY, HSQC,

HMBC experiments and mass spectrometry the structure of novel oligosaccharide riesose

was assigned as under-

RIESOSE

xix

CHAPTER-IV EXPERIMENTAL

This chapter deals with the experimental details of the chemical transformation

and chemical degradation. Various conditions adopted in acid hydrolysis and

deacetylation has been given in detail. Experimental conditions and details of different

instruments used are also been described.

BIBILIOGRAPHY

In this includes literature survey of important, recent and relevant references taken

from different journals and reference books. Survey of literature covers recent references

up to year 2015

CHAPTER I

INTRODUCTION

1

INTRODUCTION

Traditionally, carbohydrates were considered to be responsible for energy storage and as

skeletal components. However, this hypothesis was challenged in 1963 when a protein

was isolated from Canavalia ensiformis that demonstrated ability to bind to carbohydrates

on erythrocytes. In 1982 the first animal carbohydrate binding protein was identified, and

this sparked interest in the wider roles of carbohydrates and carbohydrate binding

proteins within biological systems. The carbohydrate binding proteins have been termed

lectins and it is now known that they are found in varying densities on all cell-surface

membranes1-4. The lectins interact specifically with oligosaccharides and glycoconjugates

(such as glycol-lipids and glycol-proteins) on surrounding cells via hydrogen bonding,

metal coordination, vanderwaals forces and hydrophobic interactions5,6. On the other

hand the molecular diversity reside in the carbohydrate structures is extremely high, it is

due to the occurrence of the two possible anomers and to the presence of four or five

attachment points per sugar unit with different stereochemistry, affording highly diverse

or complex linear or branched structures7. Due to their relevant biological role and

molecular diversity, carbohydrates are promising candidates for drug design and disease

treatment. It is believed that favorable interactions occur between the hydroxyl groups of

the carbohydrates and the amino acid functionalities of the proteins with recent advances

in analytical methods it is possible to deduce the structure of any complex carbohydrates

and hence identify new targets for glycobiology programmes8-13. Carbohydrate-based

therapeutics have received considerable attention in recent years. Diseases where

carbohydrate-based drugs are making an impact include cancer, diabetes, AIDS,

influenza, bacterial infections and rheumatoid arthritis14-22. Due to hydrophilic nature of

carbohydrates, they are generally located on the outside of cell membranes, so it is

postulated that the first contact that many cells have with each other will be via

interaction of the carbohydrates. They are therefore widely involved in cell-cell

recognition and cell-external agent interactions23. These interaction can initiate beneficial

biological events, such as fertilization, cell growth and differentiation (e.g., during

embryogenesis) and immune response, as well as detrimental disease processes, such as

inflammation, viral and bacterial infections and cancer metastasis. For example, a range

2

of tumor-associated carbohydrate antigens are known, including the O-linked glycan TN,

the carcinoma-associated Thomsen-Freidenreich T or TF antigen and sialyl TN or STN

antigen24-26. Carbohydrate antigens serve as diagnostic markers for specific tumor cells

and in some cases the presence of these antigens has been correlated with a more

aggressive disease state27. For example, the binding of TF to the asialylglycoprotien

receptor on hepatocytes is partly responsible for tumor cell metastasis in the liver. In

addition, many human pathogens, including the influenza virus, possess surface proteins

that complex with specific membrane-bound oligosaccharides on human cells28. Soluble

forms of human cell surfaces oligosaccharides components are being investigated and

developed for rational anti-infective drug design. The anti-infective carbohydrates and

their bio-mimetic can be administered in monomeric or multivalent form in solution, or

presented immobilized on accessible surfaces, to block or arrest the targeted adhesion

event. However, Anti-infective agents that are used clinically, include kanamycin used

when penicillin or other less toxic drugs cannot be used.

Fig- Kanamycin

An analogue of this, dibekacin, has anti-tuberculosis properties. Arbekacin an

aminoglycoside antibiotic that is currently on the market has antibacterial activity against

both Gram-positive and Gram-negative bacteria and is stable in the presence of

aminoglycoside-inactivating enzymes produced by methicillin-resistance

Statphylococcus aureus29. Carbohydrates vaccines are being analysed for their

effectiveness against various cancers and these rely on the generation of monoclonal

antibodies to tumor associated carbohydrate antigens30-33. The Sialyl TN glycopeptides is

found on the surface of several types of tumor cells, such as lung, breast, colon and

ovarian cancers. The usefulness of monoclonal antibodies to the globo H antigen which is

surface carbohydrate located on prostate, colon, Human breast and pancreatic tumor cells,

has also been probed34.

3

Fig- Globo-H

Saponins are found in various plant species and e.g. ginseng, liquorice, horse chestnuts,

ivy leaves, quillaia barks, primula roots, sarsaparilla roots and others have been used as

folk medicine35. The cardiac glycoside digoxin has been used for many years to treat

congestive heart failure, some recent studies showed that digoxin also has anti-cancer

activity and can be used as a novel cancer therapeutic agent36,37 antimicrobial, especially

antifungal, activities of many steroidal saponins have also been reported38-43.

Fig- Digoxin

Some saponins such as QS-21Aapi and QS-21Axyl has been used as the potent

immunoadjuvants for vaccine. Ginseng (Panax genus) family of Araliaceae has

produced more than 30 ginsenosides, considered to be the main active compounds in the

ginseng products44,45 which have Anti-inflammatry46, anticancer47,48, anti-diabetic49,50,

activites, and can prevent neurodegeneration51,52. OSW-1(A high potent anticancer

cholestane glycoside). OSW-1 and its analogues have been isolated from the bulbs of

Ornithogalum saundersiae, a perennial grown in southern Africa where it is cultivated as

a cut flower and garden plant53. These cholestane glycosides exhibited extremely potent

cytotoxicity against human promyelocytic leukemia HL-60 cells with IC50 between 0.1

and 0.3 nM. OSW-1, the major constituent, exhibited high potent activity against various

malignant tumor cells, including leukemia, mastrocarcinoma, lung adenocarcinoma,

pulmonary large cell carcinoma and pulmonary squamous

is 10-100 fold more potent than some well

such as mitomycin C, cisplatin, camptothecin, adriamycin and taxol

antibiotics such as erythromycin A,

midecamycin have sugar moieties in them and

Cytotoxic tetraene macrolide CE

antifungal drugs59,60.

teicoplanin, bleomycin and ristocetin etc., are very important antibiotics and some of

them have been considered as the last resort of treating multiple resistant infections

Iminosugars, also known as azasugars or polyhydroxylated alkaloids, are a family of

naturally occurring carbohydrate mimics. These sugars mimics

is replaced by nitrogen are classified into five structural classes: polyhydroxylated

piperidines, pyrrolidines, pyrrolizidines,

4

pulmonary large cell carcinoma and pulmonary squamous cell carcinoma. Its

100 fold more potent than some well-known anticancer agents in clinical use,

Fig- Gensenosides

such as mitomycin C, cisplatin, camptothecin, adriamycin and taxol

such as erythromycin A, oleandomycin, spiromycin, josamycin, tylosin and

have sugar moieties in them and have been successfully used

Fig- Erythromycin

ytotoxic tetraene macrolide CE-10858, are good candidates for broad

. Many of the glycosylated cyclic peptides, e.g.

planin, bleomycin and ristocetin etc., are very important antibiotics and some of

them have been considered as the last resort of treating multiple resistant infections


naturally occurring carbohydrate mimics. These sugars mimics in


piperidines, pyrrolidines, pyrrolizidines, indolizidines and nortropanes

cell carcinoma. Its cytotoxicity

anticancer agents in clinical use,

such as mitomycin C, cisplatin, camptothecin, adriamycin and taxol54,55. Many of

oleandomycin, spiromycin, josamycin, tylosin and

have been successfully used56,57.

, are good candidates for broad-spectrum

sylated cyclic peptides, e.g. Vancomycin,

planin, bleomycin and ristocetin etc., are very important antibiotics and some of

them have been considered as the last resort of treating multiple resistant infections61.


in which the ring oxygen


indolizidines and nortropanes62 are responsible

for treating diabetes, viral infections and

isolated from Streptomyces griseus

isolated from natural sources have been widely used as antibacterial agen

against mycobacterium tuberculosis.

Recently, acrabose miglitol,

inhibit catalytic RNAs in vitro as well as to interfere with HIV replication by disruption

of essential protein-RNA

and Gaucher disease for example,

Relenza and Tamiflu were developed in recent years as competitive inhibitors against

influenza viral neuraminidase

combating the recent flu pandemic and epidemics. Cancer chemotherapy and AIDS

realated Kaposi’s sarcoma treated with Doxorubicin

adriamycinone and a sugar dauno

reducing end of the molecule i.e. fondapaarinux

Fomivirsen69 used in treatment of cytomegalovirus retinitis (CMV) in immune

compromised patients including those with AI

5

diabetes, viral infections and cancers. Streptomycin

from Streptomyces griseus63. Streptomycin and many other

isolated from natural sources have been widely used as antibacterial agen

against mycobacterium tuberculosis.

Fig- Streptomycin

acrabose miglitol, voglibiose and aminoglycosides have been demonstrated to


RNA contacts64,65 and used to treat diabetes, viral infections, cancers,

and Gaucher disease for example,

Fig- Acrabose


influenza viral neuraminidase1-4 these two blockbuster “flu drugs” have important roles in

combating the recent flu pandemic and epidemics. Cancer chemotherapy and AIDS

realated Kaposi’s sarcoma treated with Doxorubicin66 i.e. a conjugat

adriamycinone and a sugar daunosamiine. A pentasaccharide with O

reducing end of the molecule i.e. fondapaarinux67,68 is used as an anticoagulant,

used in treatment of cytomegalovirus retinitis (CMV) in immune

compromised patients including those with AIDS also used as an anti

cancers. Streptomycin a amino glycoside

. Streptomycin and many other aminoglycosides

isolated from natural sources have been widely used as antibacterial agents, especially

aminoglycosides have been demonstrated to


to treat diabetes, viral infections, cancers,


these two blockbuster “flu drugs” have important roles in

combating the recent flu pandemic and epidemics. Cancer chemotherapy and AIDS-

i.e. a conjugate of an antracyclin

pentasaccharide with O-methyl group at the

is used as an anticoagulant,

used in treatment of cytomegalovirus retinitis (CMV) in immune-

DS also used as an anti-viral drugs anti-

6

microbial stubs and orphan drugs. Sodium ferric gluconate70 complex iron dextran and

iron sucrose are some of the potential carbohydrate associated iron drugs used for

maintenance of iron storage in human body. Sialylated oligosaccharides appear to be an

essential receptor component for many animal virus families, such as Newcastle disease

virus (paramyxovirus), cardiovirus (picornvirus) murine and primate polyoavirus

(papovavirus), rheovirus, Helicobacter pylori, Mycaplasma pneumonia, and

enterotoxigenic and uropathogenic71-75. The oligosaccharide-containing moiety of

saponin halotoxin, which is isolated from sea cucumber stichopus japonicas exhibits

interesting antifungal properties76. β-Glycans, a branched glucose polymer found in

mushroom, yeast, bran act as a potent regulator of the immune system and have shown

anti-tumor activity, it inhibits cancer cell growth and metastasis and prevents bacterial

infection77. Recent research in the area of carbohydrate food ingredients has shown the

efficiency of oligosaccharides when they are used as prebiotics or biopreservatives78.

Milk oligosaccharides-

Oligosaccharides are natural constituents of all bacteria, fungi, plants, placental mammal

and Bovine milk79. Oligosaccharides are present in milk either in the form of free

molecules or conjugated with other compounds (e.g., as lipid-or protein-conjugated);

some of them actually represent dissolved receptors which can bind pathogens and thus

prevent their binding to the respective target receptor in GIT mucosa, and the follow-up

initiation of infection. The oligosaccharides isolated from various milk sources are

categorized in two classes i.e. sialylated oligosaccharides and non-sialylated

oligosaccharides. The majority of oligosaccharides with function are fucosylated. They

are produced through the action of enzymes encoded by genes associated with expression

of the Lewis blood system antigens. Therefore the specific antimicrobial activity of milk

fucosylated oligosaccharides is directly linked with the maternal Lewis blood group type.

The specific oligosaccharides are thus one of main innate immunological mechanism of

human milk, through which the mother protects an infant against enteric as well as other

pathogens. The knowledge of the structure and function of these protective

oligosaccharides could becomes a basis for the development of new preventive and the

therapeutically drugs against GIT pathogens80, 81. Sialyllactose is the only oligosaccharide

that, as a monovalent sugar, potently inhibits initial bacterial adhesion by detaching cell-

7

bound bacteria from human gastrointestinal monolayers in vitro82. It worth noting that a

number of anti-infective agents occur naturally, such as in human breast milk which

contains numerous soluble oligosaccharides that provide newborn babies with a

mechanism for aborting infection processes83, 84. A prominent example is the ..Galβ1-

4GlcNAcβ1-3Galβ1…trisaccharide that has proposed as a receptor for adherence of

Streptococcus pneumonia to buccal epithelial cells. At corresponding concentrations,

sialylated milk oligosaccharides strongly inhibit binding of influenza A virus and S-

fimbriated enteropathogenic Escherichia coli to their respective host cells. The milk is

rich source of bioactive oligosaccharides containing number of their origin to which

mammal they belongs. The oligosaccharides isolated from different milk exhibit potent

biological activities such as anti-tumor, immunological, anti-complimentary, anti-cancer,

anti-inflammatory, anti-coagulant, hypoglycemic and antiviral activities85, 86. Medicinal

and pharmaceutical researchers have unrevealed the importance of these oligosaccharides

and the milk of various sources, which are either used in folk medicine, or their

importance is reported in ancient medicinal system (Ayurveda and Unani). Milk

oligosaccharides which have been tested for their varied biological activities as described

as under.

The Elephant milk oligosaccharides fraction contained a high ratio of sialyl

oligosaccharide; this may be significant with respect to the formation of brain

components, such as gangliosides of the suckling calves87. N-acetylneuraminlactose

sulphate may play an important role in the nutrition of the rat pups, which is the dominant

oligosaccharide in the Dog milk88. Buffalo Milk oligosaccharides have ability to

stimulate non-immunological resistance of the host against parasitic infections89. Donkey

milk oligosaccharides have ability to stimulate non-specific and specific immunological

resistance90. Goat milk oligosaccharides play an important roles in intestinal protection

and repair after damage caused by DSS (Dextron sodium sulphate)- induced colitis and

their implication in human intestinal inflammation91. Goat milk oligosaccharides have

anti-inflammatory effects in rats with trinitrobenzenesulfonic (T) acid induced colitis and

may be useful in the management of inflammatory bowel disease92. Cow milk

oligosaccharides reduce the adhesion of enterotoxic Eschererchia coli strains of the calf93.

Bovine milk oligosaccharides have several potentially important biological activities

8

including the prevention of the pathogen binding to the intestinal epithelial and as

nutrients for beneficial bacteria79. Mare’s milk has shown anti oxidant, lipid lowering

and post heparin lipolytic activity94. Camel’s milk oligosaccharides show potent activity

against gonorrhea septic and hysteric properties. Sheep milk aggravates hiccup and

dyspnoea. It also eliminates pitta, kapha and fat. It also contains fucose in its

oligosaccharides which causes various biological activities.

SOME PHYSIOLOGICAL FUNCTIONS OF MILK OLIGOSACCHARID ES

Milk Oligosaccharides as a Pre-biotic Properties

Breast-fed infant micro biota is rich in bifido bacteria. Herein, human milk

oligosaccharides (HMOS) have ability to promote the growth of bifido bacteria and to

acidify their environment95. Pre-biotic are non-digestible food which beneficially affect

the host by selectively affecting the growth and activity of bacteria in colon and thus

improve the health of host. The human intestine lacks enzymes able to hydrolyze β-

glycosidic linkage with exception of lactose. Thus milk oligosaccharides are considered

to be indigestible96, 97 which reach the colon and are utilized by health promoting colonic

bacteria and known as prebiotic.

Milk Oligosaccharides as Immunomodulatries

Milk oligosaccharides may play a physiological role in modulating cellular

adhesion in vivo. The human milk derived acidic oligosaccharide fraction is found to

enhance the production of certain cytokines after long-term exposure (20d) in vitro in the

CD4+ as well as in the CD8+ T-cell subfraction98. Significantly oligosaccharide isolated

from buffalo milk89 possesses high degree of immunostimulant activity and proposed to

be very helpful in cure of AIDS patient.

Role of Oligosaccharides in Brain Development

Oligosaccharides along with lactose and sialic acid play role in postnatal brain

development. Gangliosides are complex glycosphingolipids, which make up 10% of the

total lipid mass in the brain and contain different numbers of negatively charged sialic

acid moieties. Brain tissue is unique in that the quantity of lipid-bound sialic acid exceeds

that of the protein-bound fraction. Gangliosides are hybrid molecules composed of a

9

hydrophilic sialyl oligosaccharide and a hydrophobic ceramide portion that consists of

sphingosine and fatty acids99. Many newborn mammals undergo a period of rapid

postnatal brain development that requires large amount of glycolipid, which are

components of cell membranes of neurons and myelin. Sialic acid present in human milk

also contribute to the increased concentration of NeuAc, present in cerebral and

cerebellar glycoconjugates of breast fed and thus play an important role in the

development of the infant brain100,101.Since elephant milk contain sialylated

oligosaccharides, it plays a definite role in brain development87.

Effect of Milk Oligosaccharides on Mineral Absorption

Several studies in animals and humans have shown positive effects of non-

digestible oligosaccharides (NDO) on mineral absorption and metabolism and bone

composition and architecture. These include inulin, oligofructose, fructooligosaccharides,

galactooligosaccharides, soybean oligosaccharide, and also resistant starches, sugar

alcohols, and di-fructose anhydride102.

Milk Oligosaccharides as Tumor marker

1-Monoclonal antibodies of several tumor cell lines and carbohydrate antigens

have provided evidence that membrane glycoprotein or glycolipid which may function as

differentiation antigens or tumor-associate antigens occur as free oligosaccharide in

human milk. 2-Two newly isolated oligosaccharides B-1 and B-2 both have the sialyl Lea

and Lex or Le-1 structure respectively. 3-The sialyl- Le structure in glycolipid or

glycoprotein has been defined as gastrointestinal tumor associated antigen. These

structures have been found in mucin type glycoprotein and glycolipid in a variety of

human cancer. Oligosaccharides having the sialyl-Lea and difucosyl Le-Le structure also

occur in human milk and Lea-Lex structure exhibits high affinity to an antibody directed

to a human squamous lung carcinoma103-105.

Factors Affecting Biological Activities in Milk

There are number of factors due to which different milk oligosaccharides show

varied biological activities. Some of the important factors Affecting Biological Activity

in milk are summarized as follows.

10

1. Milk oligosaccharides are non-digested due to the presence of β-glycosidic

linkage. So this β-glycosidic linkage plays a important role for its pre-biotic

activity106, 107.

2. Oligosaccharide mimics containing galactose and fucose specifically label tumour

cell surfaces and inhibit cell adhesion to fibronectin108.

3. Supplementation of milk formula with galacto-oligosaccharides improves

intestinal micro-flora and fermentation in term infants109.

4. Galactose and sialic acid present in milk oligosaccharide are required for optimal

development of the infant’s brain110.

5. N-and O-linked oligosaccharide causes the release of histamine and other

mediators of the allergenic response which then lead to the development of

allergenic symptoms111.

6. Human milk oligosaccharide containing α1, 2-linked fucose inhibits the stable

toxin-producing Escherichia coli in vitro, and its toxin induced secretary diarrhea

in vitro and in vivo112, 113.

7. Glycoconjugate found in human milk also inhibits binding by Campylobacter

jejuni in vitro and in vivo and also inhibit binding by calciviruses in vitro112, 113.

8. Specific fucosyl oligosaccharides of human milk have been observed to inhibit

specific pathogens112, 113.

9. Some important enteric pathogens, for example- rotavirus, are inhibited by human

milk oligosaccharide, α1, 2-linked fucosylated oligosaccharide, probably in

conjugation with other families of oligosaccharide, constitute a powerful innate

immune system of human milk112, 113.

10. Presence of sialic acid in human milk serves as anti-inflammatory components

and reduces platelet-neutrophill complex formation leading to a decrease in

neutrophill B2 integrin expression114.

11

11. Sialylated human milk oligosaccharide also inhibits binding of pathogenic strains

of Escherichia coli and ulcer-causing human pathogen H. pylori114.

12. Neutral human milk oligosaccharide may protect the intestinal tract of neonates

from Vibriocholera114.

13. The ability of rotavirus to infect MA-104 cells in culture is inhibited by human

milk, and this inhibition is due to a mucin-associated 46kDa milk glycoprotein

named lactadherin. Lactadherin from human milk also inhibits rotavirus (EDIM

strain) gastroenteritis in mice115.

Effect of Constituent Monosaccharides and Linkages on Biological Activity of Milk

Oligosaccharides

1. Human milk oligosaccharides containing α1, 2-linked fucose inhibits the stable

toxin-producing Escherichia coli in vitro and its toxin induced secretary diarrhea

in vitro and in vivo116,71. Glycoconjugates found in human milk also inhibit

binding by Campylobacter jejuni in vitro and in vivo and also inhibit binding by

calci viruses in vitro. Some specific fucosyl oligosaccharides of human milk have

been observed to inhibit specific pathogens. Thus it can be concluded that the

family of α1, 2-linked fucosylated oligosaccharides, probably in conjugation with

other families of oligosaccharide, constitute a powerful innate system of human

milk116.

2. Due to the presence of sialic acid in human milk they serve as anti-inflammatory

components and reduce platelet-neutrophill complex formation leading to a

decrease in neutophill B2 integrin expression, while neutral milk oligosaccharide

fraction has no effect. Sialylated human milk oligosaccharides also inhibit binding

of pathogenic strains of Escherichia coli and ulcer causing human pathogen H.

pylori. On the other hand neutral human milk oligosaccharide may protect the

intestinal tract of neonates from Vibrio cholera118, 119.

3. Prebiotic is non-digestible food ingredients that beneficially affect the host by

selectively affecting the growth and activity of bacteria in colon that can improve

12

the host health. Milk oligosaccharides are non-digested due to the presence of β-

glycosidic linkage. So this β-glycosidic linkage plays an important role for

prebiotic activity104, 120 of milk oligosaccharides.

4- Infection by rotavirus is responsible for much of the diarrhea in infants around the

world. The ability of rotavirus to infect MA-104 cells in culture is inhibited by

human milk, and this inhibition is due to mucin-associated 46kDa milk

glycoprotein named lactadherin121. Furthermore after sialic acid is removed from

lactadherin, its ability to inhibit rotavirus is essentially lost, which suggests that

the glycon portion of the molecule is responsible for inhibition and specific

terminal sialic acid is required for inhibition.

5. N- and O-linked oligosaccharides cause the release of histamine and other

mediators of the allergic response which then lead to the development of

allergenic symptoms. Oligosaccharides mimics containing galactose and fucose

specifically label tumor cell surface and inhibit cell adhesion to fibronectin122.

6. Supplementation of milk formula with galacto-oligosaccharides improves

intestinal micro-flora and fermentation in infants. Galactose and sialic acid

present in milk oligosaccharide are required for optimal development of the

infant’s brain 123

PROCESSES FOR ISOLATION OF MILK OLIGOSACCHARIDES:-

More than 200 milk oligosaccharides have been isolated till date and

characterized by the various workers still new oligosaccharides are being isolated from

the milk of different species, due to qualitative variation which arise due to genetic

factors, which reflected in their biosynthesis. The pioneer workers have isolated number

of milk oligosaccharides from the milk of different origins by processing the milk by

various processing techniques which were by KOBATA AND GINSBURG124, 125

URASHIMA et.al.,126 SMITH et.al.,127 EGGE et.al.128 WEIRUSZESKI et.al.129. The

method which have been used in this dissertation is modified method of Kobata and

ginsburg.

13

MODIFIED METHOD OF KOBATA AND GINSBURG FOR ISOLATIO N OF

MILK OLIGOSACCHARIDES 90

In the modified method of Kobata and Ginsberg milk was preserved with equal

amount of ethanol until used. Milk was centrifuged for 20 minutes at 5000 rpm at -4oC.

The solidified lipid layer was removed by filtration through glass wool. Ethanol was

added to clear filtrate to final concentration of 68 % and the resulting solution was left

overnight at 0oC. The white precipitate formed was mainly of Lactose and protein, was

removed by centrifugation, and washed twice with 68% ethanol at 0oC. The supernatant

and washings were combined and concentrated under reduced pressure, and passed

through a column of fine grade G-25 sephadex that had been washed with triple distilled

water overnight. The column was eluted with triple distilled water and effluents were

collected in fractions and aliquots of each fraction were analyzed for oligosaccharides

and fraction containing oligosaccharide were used for further studies.

PURIFICATION OF MILK OLIGOSACCHARIDES

Chromatography plays a major role in purification and analysis of milk

oligosaccharides. Chromatographic separations can be carried out by using a variety of

supports, including immobilized silica on glass plates (thin layer chromatography),

volatile gases (gas chromatography), paper (paper chromatography), and liquids which

may incorporate hydrophilic, insoluble molecules (liquid chromatography). High-

performance liquid chromatography (HPLC) is the most frequently used techniques for

separation of oligosaccharides. High pH anion exchange chromatography with pulsed

amperometric detection (HPAEC-PAD) is a powerful method, which is widely used for

carbohydrate analysis. The details of various chromatographic techniques used in

separation of oligosaccharides are described as below-

Thin Layer Chromatography130-132

TLC is suitable for analysis of monosaccharide’s and oligosaccharides. In TLC

plates, the stationary phase is a powdered adsorbent. The adsorbent used for separation of

oligosaccharides are magnesium silicate, alumina, silica gel that is fixed to an aluminum,

glass, or plastic plate. The resolution of mixture of compounds depends on the suitable

solvent system (mobile phase). Starting from non-polar single solvent system to highly

14

polar three solvent systems are available for thin layer chromatography. With increase in

the polarity of the solvent system, all the components of the mixture move faster (and

vice versa with lowering the polarity). Although the TLC is limited to less polar

compounds and it is not very effective for the isolation of highly polar compounds like

oligosaccharides and other glycoconjugates etc, however it could be useful in isolation of

derivatized oligosaccharides.

Paper Chromatography133

Paper Chromatography is a traditional technique used for the separation and

qualitative determination of milk oligosaccharides. Paper chromatography is a partition

chromatography in which distribution takes place between a stationary sorbed liquid

phase and a mobile fluid in intimate contact with it. Separations in Paper

Chromatography occur because of differential migration velocities through the sorbent

layer in a fixed separation time. For purification of milk oligosaccharides descending

paper chromatography was performed with following solvents on Whatman filter paper -

Upper layer of ethyl acetate-pyridine-H2O (2:1:2)

Ethyl acetate-pyridine-acetic acid-H2O (5:5:1:3)

2-propanol-H2O (4:1)

1-butanol-pyridine-H2O (6:4:3)

Lower layer of phenol- formic acid-2-propanol-H2O (8:5:1:2)

Upper layer of pyridine -ethyl actate-H2O (1:3.6:1.15)

Phenol-H2O-conc.NH4OH (150:40:1)

Upper layer of ethylacetate- acetic acid-H2O (3:1:3)

Ethanol-1M ammonium acetate pH 7.8 (5:2)

Oligosaccharides were located with AgNO3 reagent, aniline oxalate reagent or

periodate-benzidine reagent. Oligosaccharides containing N-acetyl amino sugars were

located with Morgan reagent, while sialic acid containing oligosaccharides were

developed with Thiobarbituaric acid (TBA) reagent.

Column Chromatography134

Column chromatography plays a very important role in purification of derivatized

oligosaccharides. It consists of a column of particulate material such as silica or alumina

15

and a solvent pass through it at atmospheric or low pressure. The separation can be

liquid/solid (adsorption) or liquid/liquid (partition). The sample is dissolved in suitable

solvent and applied to the column. The solvent elutes the sample through the column,

allowing the components to separate based on adsorption (alumina, silica) or partition

(cellulose, diatomaceous earth). Traditionally, the solvent was non-polar and the surface

polar, although today there are wide ranges of packing including bonded phase systems.

Bonded phase systems usually utilize partition mechanisms rather than adsorption. The

solvent is usually changed stepwise, and fractions are collected according to the

separation required, with the eluted solvent usually monitored by TLC.

High Performance Liquid Chromatography 135

High Performance Liquid Chromatography (HPLC) is one mode of

chromatography, one of the most used analytical techniques. This chromatographic

process can be defined as separation technique involving mass-transfer between

stationary and mobile phase. HPLC utilises a liquid mobile phase to separate the

components of a mixture. The stationary phase can be a liquid or a solid phase. These

components are first dissolved in a solvent, and then forced to flow through a

chromatographic column under a high pressure. In the column, the mixture separates into

its components. The amount of resolution is important, and is dependent upon the extent

of interaction between the solute components and the stationary phase. The stationary

phase is defined as the immobile packing material in the column. The interaction of the

solute with mobile and stationary phases can be manipulated through different choices of

both solvents and stationary phases. As a result, HPLC acquires a high degree of

versatility not found in other chromatographic systems and it has the ability to easily

separate a wide variety of chemical mixtures.

In recent years, HPLC has become the most important technique for separation of

complex mixture of oligosaccharides. HPLC is a technique by which molecules in

solution are separated according to differences in their molecular size, ionic properties or

affinity for column packing material. The three main techniques used in HPLC are ion

exchange chromatography, reversed phase chromatography (RPC) and affinity

chromatography. In ion exchange chromatography bonded silica and bonded glass with

ionic groups on their surfaces are used as stationary phase separation media. Ionic solute

16

molecules are attracted to the stationary phase ionic groups of the opposite charge and

during elution the retarded substances are reversibly charged for ions of the same charge.

Anion exchange HPLC has been recently developed and has exceptional resolving power

for complex oligosaccharides. Such analysis is carried out at high pH coupled with pulsed

amperometric detection (PAD), allowing separation of oligosaccharides and

polysaccharides upto DP≥ 50. The separation depends on the molecular size, sugar

composition and type of linkages between the monosaccharides units. In RPC the mobile

phase is polar (aq. solutions) and stationary phase is non-polar. By far the most frequently

used systems for separation of oligosaccharides are those using chemically bonded phase

which fractionate materials on the basis of their relative affinities for mobile phase and

bonded phase. The most important columns are those containing the aminopropyl bonded

phase. HPLC has emerged as a popular alternative method to other conventional methods

for the isolation and purification of oligosaccharides because of its speed of performance,

wide applicability, sensitivity and high resolution. For HPLC purification, a judicious

selection of operating parameters is required for achieving the desired purity and yield.

The following sequence is followed for better resolution and yield-

1. Choice of solvent system136 – The separation of different compounds from a mixture

can be achieved by choosing the solvent system appropriately. The separation of

compounds depends upon the different chemical and physical properties of the solvent. In

certain cases, TLC analysis of the sample is used as a first indication of the correct

operating conditions, silica gel plates for normal-phase column and sialylated silica gel

plates for reversed-phase columns.

2. Optimization of analytical columns of small quantities137- In order to save time,

sample and solvent required in the HPLC system a preliminary analytical search is

necessary for the right choice of conditions.

3. Optimization of analytical HPLC separation aiming for small capacity factors- A

good analytical HPLC separation is usually a prerequisite for a successful preparative

operation. Relative intention (selectively, α) is a very important parameter in determining

possible sample size and it is necessary to maximize this value.

17

4. Scaling of preparative HPLC apparatus138- In many preparative HPLC examples,

the column is actually overloaded, nonlinear absorption isotherms are obtained and peaks

are not symmetrical. Scaling-up a successful analytical separation may cause problems

associated with the solubility of the sample.

Normal Phase HPLC139

The extremely hydrophilic nature of oligosaccharides in principle makes them

eminently suitable for normal phase chromatography. However most commonly used

normal phase matrix, silica has not proven to be very useful for oligosaccharide

separations, but much success has been achieved using hydrophilic bonded phases,

especially amino propyl silica. Oligosaccharides injected onto the column in a high

organic (generally acetonitrile) aqueous solvent, are eluted from the column by an

increasing aqueous concentration gradient.

Reverse Phase HPLC (RP-HPLC)140

Reverse phase chromatography is so named because it behaves in the opposite

way to normal phase chromatography. Reverse phase chromatography is a powerful

analytical tool and involves a hydrophobic, low polarity stationary phase, which is

chemically bonded to an inert solid such as silica. The separation is essentially an

extraction operation and is useful for separating non-volatile components. Although RP-

HPLC is the most widely used HPLC technique for organic compounds due to high

chromatographic efficiency and selectivity, which leads to very weak interactions with

the column matrix. However RP-HPLC can give good separation of oligosaccharides that

have been made more hydrophobic by derivitization. Per-O-methylated and per-O-

acetylated oligosaccharides are hydrophobic and give good RP-HPLC separation.

Acetylation has the advantages of increased UV detection sensitivity and the option to

remove the acetyl group at later stage. Using water as the mobile phase, more polar

oligosaccharides elute before the less polar molecule. The retention time of underivatized

sugar in reversed phase increases with molecular mass and it is also observed that the

retention time of branched oligosaccharides is shorter than those of the linear

counterparts141,142. Sumiyoshi W et al analyzed major neutral oligosaccharides in the milk

of sixteen Japanese women by RP-HPLC and found that in colostrums and mature milk

(30 days lactation), lacto-N-fucopentose (LNFP I) was the most abundant

18

oligosaccharide, followed by 2′-focosyl lactose (2′-FL) + lacto-N-difucotetraose

(LNDFT), LNFP II + lacto-N-difucohexose (LNDFH II), and 3-focosyllactose (3-FL)143.

STRUCTURE DETERMINATION OF OLIGOSACCHARIDES

After the isolation of oligosaccharides the most important task is to elucidation

and determined the three dimensional structure of oligosaccharide. It has created new

demands for analytical tools for structure elucidation of complex oligosaccharides

comprising composition, sequence, branching and linkage analysis, including

anomericity and finally also rings size and absolute configuration. The structural analysis

of the carbohydrate is usually carried out by a combination of chemical and enzymatic

and physico-chemical methods. Some frequently used techniques are periodate oxidation,

smith degradation, permethylation analysis144-146, acetolysis, alakanine hydrolysis and

acid hydrolysis involving mild and strong acid hydrolysis. i.e. killiani hydrolysis147 gives

us the information regarding the monosaccharides constituents of oligosaccharide while

the mild acid hydrolysis i.e. mannich hydrolysis148 gives us the information of sequence

of monosaccharide in oligosaccharides. All the modern structural methods for

oligosaccharides, structure determination are NMR (1D and 2D techniques) in

combination with Mass spectroscopy yields the most complete structure of

oligosaccharides, with or without prior structural knowledge.

NMR Spectroscopy.149-153

Today, NMR has become a sophisticated and powerful analytical technology that

has found a variety of applications in many disciplines of scientific research, medicine,

and various industries. Nuclear magnetic resonance (NMR) Spectroscopy is the study of

molecular structure through measurement of the interaction of an oscillating radio

frequency electromagnetic field with a collection of nuclei immersed in a strong external

magnetic field. These nuclei are parts of atoms that, in turn, are assemble into molecules.

An NMR spectrum, therefore, can provide detailed information about molecular

structure, dynamics etc that would be difficult to obtain by any other methods. The NMR

spectra of oligosaccharides gave important information about the protons and carbons

present in the oligosaccharides. In the proton spectra of oligosaccharide, most of the

signal occur in the bulk region (3.4-4.5), hence identification of each signal is a difficult

19

task. The dispersion of resonances in the carbon spectra is favorable, but the amount of

material needed to acquire such spectra is relatively high due to the low natural

abundance of 13C. However, advancements in instrument and Fourier transformation have

reduced the amount needed. In practical terms, about 1 mg of a pure oligosaccharide is

enough to perform a complete structural assignment by both 1H and 13C NMR

spectroscopy. When sample amounts are further limited, 1H NMR spectra can be

measured down to nanomole quantities. A typical NMR spectroscopy analysis of

oligosaccharide sample involves following steps-

1. Number of sugar residue- In the structural analysis of oligosaccharide, the

chemical shift of anomeric proton and anomeric carbon play an important role. The

integration of the anomeric resonances offers an initial estimate of the number of

different monosaccharide residues present. The anomeric proton resonances are found in

the shift range 4.4-5.5 ppm in 1H NMR. Additionally, the number of anomeric C-1

resonances present in a 13C NMR spectrum ranges between 90-110 confirms the number

of monosaccharide unit in the oligosaccharide molecule.

2. Constituent monosaccharides- After knowing the number of sugar residue in the

oligosaccharide, the next step is to know the constituent monosaccharides present in the

oligosaccharide. By combining the data of 1H and 13C anomeric chemical shifts one can

easily distinguish the monosaccharide present in the oligosaccharide. For sialic acid

moiety which do not have anomeric proton, characteristic signal of the H-3ax and H-3eq

proton are a good starting point for the assignment value. 2D homonuclear correlation

spectroscopy (COSY, HOHAHA), 13C NMR, 1H-1H TOCSY154,155 and 1H-13C correlation

spectroscopy156-190 are also useful in the identification of individual monosaccharide

residues.

3. Anomeric configuration- In oligosaccharide molecule normally α-anomer

resonates downfield compared to the β-anomer in D-pyranose in 4C1 conformation. If H-1

and the H-2 are both in an axial configuration in pyranose structure, a large coupling

constant (8-10 Hz) is observed, whereas if they are equatorial-axial, there is smaller

coupling constant (J1,2 ~ 4Hz), and for equatorial-equatorial oriented protons, even

smaller coupling constants are observed (<2Hz)160. The J value 6-9 Hz. shows the

20

presence of β-configuration where as J value 3-4 Hz. shows the presence of α-

configuration.

4. Linkages and sequence- The 1H and the 13C chemical shifts give an indication of

the linkage of complete oligosaccharide moiety. The effect of glycosylation depends on

the linkage type, and the changes in the chemical shift are in generally larger at the

glycosylation site than at neighboring positions. Primary information regarding the

glycosidic linkages is provided by comparison of 1H NMR chemical shift of methine

proton of natural oligosaccharide and acetylated oligosaccharide which generally shifted

about 1ppm downfield. HMBC and inter-residue NOEs experiment give information

about the glycosidic linkage. An effective way of knowing the glycosidic linkage

between two monosaccharide residues is by monitoring the nuclear overhauser effect

(NOE) from the signal for an anomeric group to the hydrogen of the substituted position

in the adjacent ring161, 162. However information obtained from COSY and TOCSY

experiments are also very useful.

5. Position of appended groups- A non-carbohydrate group like a methyl, acetyl,

sulfate, or a phosphate group shifts the proton and carbon where the appended group is

located. In the case of acetyl group attached, the proton corresponding to reducing sugar

downfield shifted by ~1.0-1.2 ppm whereas other protons downfield shifted by 0.1-0.2

ppm. In other cases downfield shifts ~ 0.2-0.5 ppm163 were observed in values for protons

where appended group is attached. This places these resonances in a less crowded area of

the spectra and helped in the identification of novel residues.

6. Structure Reporter Group- Since the NMR data of oligosaccharide are highly

complex, Vligenthart et.al. Introduced the “structural reporter group” (SRG)164-170

concept, which was based on signals outside the bulk region ( 3-4) in the 1H NMR

spectra of the oligosaccharide. This structural reporter group concept helped in the

identification of novel residues and characterization of oligosaccharides.

Different NMR Technique in Structure Elucidation of Milk Oligosaccharides

The structure of milk oligosaccharides has been determined by comparison of

NMR data of the new isolated compound with the reference data of different core units

21

found in milk oligosaccharides. While comparing the chemical shift values it is important

that the reference data is measured at the same temperature and the data are based on

same internal reference or one that can be correlated in a simple manner. Different NMR

experiments useful in structure elucidation of oligosaccharides are as below-

1H NMR Spectroscopy of Oligosaccharides

The 1H NMR is the most basic and important experiment of NMR series. It

provides maximum information regarding configuration and conformation of

monosaccharides present in the oligosaccharides. The limitation of resolution of

anomeric and ring protons is already surpassed by advances of Fourier transformation

and high frequency NMR spectrometers. The high resolution 1H NMR spectra give

valuable information about qualitative and quantitative aspects of the oligosaccharides

structure. The chemical shift of a particular anomeric proton and its splitting pattern gives

an idea of the monosaccharide units presents there in, simultaneously it also fixes the

configuration of sugar linkage and conformation of that monosaccharide unit. The proton

NMR spectroscopy of carbohydrates suffers from severe spectral overlap, because most

of the monomer residues differ only in their stereochemistry and their magnetic

properties are only little influenced by their position in chain. Since the chemical shift of

anomeric protons and methine protons of different sugars are confined to the region 4.3-

5.5 and 3.0-4.2 respectively hence it requires expert interpretation of spectra for

monosaccharide identification. The analysis of reducing oligosaccharides showed that the

anomeric configuration of the reducing end sugar also exerts its influence on the spectral

parameters of residues in its spatial neighborhood, being sometimes even the non-

reducing end sugar. To resolve the spectral complexities of oligosaccharides, Vligenthart

et.al. introduced the “structural reporter group” concept, which was based on signals

outside the bulk region (3-4) in the 1H NMR spectra of the oligosaccharide170. This

approach is used to identify individual sugars or sequence of residues. These structural

reporter groups include anomeric proton, equatorial protons, deoxy protons and that

distinct functional group such as amide group. 1H NMR gives anomeric protons at 4.3-5.9

ppm, methyl doublets of 6-deoxy sugars at 1.1-1.3 ppm, methyl singlet of acetamido

groups at 2.0-2.2 ppm and various others with distinctive chemical shift. In D-pyranoses

22

4C1 conformation the α-anomer resonates downfield in comparison to β-anomer. The

chemical shift value for α-anomer lied in the range 4.9-5.4 ppm and for β-anomer it lied

in the region 4.4-4.8 ppm. The α-anomeric doublet showed coupling constant J = 3-4 Hz

whereas the β-anomeric doublet showed J value of 6-9 Hz. All these values were

correlated with known structures to yield relevant information in terms of

monosaccharides units and their relative abundance. The structure of different linkages

can be defined in terms of. NMR parameters of their structural reporter groups. In case of

milk oligosaccharides the anomeric proton resonances are found in the chemical shift

range 4.3-5.5 ppm and the remaining ring proton resonance are found in the range 3.0-4.2

ppm. But in case of acetylated oligosaccharides acetyl groups induce a strong downfield

shift of proton which directly linked to acetylated carbons. Hence, the signals of methine

protons and methylene protons occur downfield in the region of 4.8-5.5 ppm and 4.0-4.8

ppm respectively. The resonances of protons linked to the non-acetylated carbons at the

site of glycosidic linkage and at the ring C-5 occur in the chemical shift range between

3.5 and 3.9 ppm.

Some of the common 1H NMR structural reporter groups of milk

oligosaccharides have been summarized as below-197-201

Structure Reporter Groups

1. In the 1H NMR spectra the reducing Glc residue is characterized by the H-1

signals for it’s α and β anomers at δ5.221 (J1, 2 3.7 Hz) and δ 4.688 (J1, 2 8.0 Hz)

respectively with ratio of 7:10.

2. The 4-substituted reducing Glc shows anomeric signals for both the α- and the β-

anomeric at δ 5.22 and 4.66 ppm, with H-2 of the β-from in the range of δ3.2-3.3

ppm as triplet.

3. The 3,4-disubstituted reducing Glc shows anomeric signals from both the α- and

the β- anomeric at δ 5.22 and 4.66 ppm, with H-2 of the β-from at a typical

downfield shift above δ3.35 ppm.

23

4. The 3-substituted β-linked Gal shows signal for H-1 at 4.4 ppm and H-4 of β-

linked Gal showed at a typical downfield shift around δ 4.13-4.15 ppm due to

substitution at the 3-position.

5. The H-4 of (1→6) linked β-Gal appeared at δ 3.8-3.9 ppm and H-4 of (1→3)

linked β-Gal at δ 3.9-4.2 ppm.

6. Signal for H-1 of the un-substituted Gal residue appears around 4.44-4.47 ppm.

7. β-linked GlcNAc residues with anomeric signals appear at δ 4.6-4.7 ppm and

CH3 signals in the range of δ 2.02-2.08 ppm. H-1 of the (1→6) linked GlcNAc

appears at lower chemical shift value (δ 4.6 ppm.) than the (1→3) linked GlcNAc

residue (4.7ppm). A splitting of the anomeric doublets is due to the anomerization

of the reducing terminal.

8. The H-2 of β-GlcNAc appeared at 3.6-3.8 ppm and H-2 of β-GalNAc appeared at

3.8-4.2ppm.

9. Presence of anomeric signal with a integration of two proton at 4.44-4.6 ppm

suggest a LNT structure in which one β-Gal is attached to Glc by (1→4) linkage

while another β-Gal unit is attached to β-GlcNAc or β-Glc by (1→3) linkage i.e.

β-Gal(1→3) β-GlcNAc(1→3/6) β-Gal (1→4) Glc or β-Gal(1→3) β-Glc (1→3/6)

β-Gal(1→4) Glc moieties is present.

10. α-linked Gal residue appeared at δ4.94-5.2 ppm. The (1→4) linked α-Gal

residues showed anomeric signal at δ 5.02 ppm, (1→2) linked α-Gal residues

showed anomeric signal at δ 5.20 ppm and (1→3) linked α-Gal residues showed

anomeric signal between δ 5.02-5.20 ppm.

11. α-linked Fuc residues anomeric signals appeared at δ5.02-5.43 ppm. The presence

of fucose subunit could be inferred by the presence of CH3 doublet at δ 1.1-1.3,

H-5 at δ 4.2-4.9 and the anomeric doublet at δ5.02-5.4 ppm.

24

12. Generally (1→4) linked fucose occur near δ4.98ppm, (1→2) linked fucose occur

near δ5.38ppm and (1→3) linked fucose occur between the two.

13. The presence of sialic acid residue could be ascertained by the characteristic

resonances of H-3 axial and equatorial protons at 1.78 and 2.75 ppm respectively.

The location of Neu5Ac residue can be deduced as follows. (a) the signal for H-3a

and H-3e of Neu5Ac residue can be used to discriminate between (2-3) and (2-6)-

α-linkage to Gal. (b) for an α-Neu5Ac(2-3)-β-Gal-(1- sequence, the signal for H-3

of Gal residue is shifted downfield by 0.6 ppm of the ring protons.

14. The 3, 6-di-substituted β-linked Gal shows signal for H-1 at 4.4ppm and H-4 at a

typical downfield shift around 4.13-4.15 ppm, due to substitution in the 3-

position by a β -linked GlcNAc residue.

15. The location of Neu5Ac residue can be deduced as follows –

(a) The signal for H-3a and H-3e of the Neu5Ac residue can be used to

discriminate between (2-3) and (2-6)-α - linkages to Gal.

(b) For an α-Neu5Ac(2-3)- -Gal-1- sequence, the signal for H-3 of the Gal

residue is shifted down field of the ring protons by 0.6 ppm. Also, in a β-

GlcNAc-(1-3)-β-Gal-1- sequence, the signal for H-4 0f the Gal residue

appears at 4.15ppm.

16. The presence of α-Gal subunit could be inferred by the presence of the anomeric

doublet at 5.14-5.25ppm and H-4 doublet at 4.09-4.25.

17. Linkage of Gal(S4) to GlcNAc(S3) in LNT and LNeoT is confirmed by the

chemical shift value of Gal(S4).If its chemical shift value is exactly similar to that

of lactose′s Gal(S2) than it would be 1-4 linked to GlcNAc(S3)otherwise it would

be 1-3 linked to GlcNac(S3).

25

1H NMR data of Common Glycopyranoses Of oligosaccharides Found in Milk152

Sugar H-1 H-2 H-3 H-4 H-5 H-6 NHCOCH3

β-D-Glc

α-D-Glc

β -D-Gal

α -D-Gal

β -L-Fuc

α -L-Fuc

β - D-GlcNAc

α -D-GlcNAc

β -D- GalNAc

α -D-GalNAc

4.64

5.23

4.53

5.22

4.55

5.20

4.72

5.21

4.68

5.28

3.25

3.54

3.45

3.78

3.46

3.77

3.65

3.88

3.90

4.19

3.50

3.72

3.59

3.81

3.63

3.86

3.56

3.75

3.77

3.95

3.42

3.42

3.89

3.95

3.74

3.81

3.46

3.49

3.98

4.05

3.46

3.84

3.65

4.03

3.79

4.20

3.46

3.86

3.72

4.13

3.72,3.90

3.76,3.84

3.64,3.72

3.69,3.69

1.26

1.28

3.75,3.91

3.77,3.85

3.82,3.84

3.79,3.79

-

-

-

-

-

-

2.06

2.06

2.06

2.06

13C NMR Spectroscopy of Oligosaccharides171-173

The 13C NMR provides varied information regarding configuration and

conformation of monosaccharides present in the oligosaccharides. 13C NMR

Spectroscopy has enormous potential for carbohydrates and glycosides because its greater

chemical shift dispersion and lack of complexities arise from spin-spin coupling and

overlapping resonances with those arising from solvents. In contrast to rather crowded

and poorly resolved 1H NMR spectrum, the 13C NMR spectrum is usually well resolved

and has few overlapping lines and therefore is comparatively easy to interpret. The

anomeric signals for carbon appear in the region 90-110 ppm in the case of O-glycosides.

In the case of C-glycosides being monooxy-substituted appear in the chemical shift range

70-80 ppm. The appearance of anomeric resonances in a well separated chemical shift

range of 90-112 ppm help greatly in determining the number of O-linked

monosaccharides. The C-1 resonances of a reducing hexose absorbs at 5-10 ppm up field

relative to the chemical shift of C-1 glycosidic residue. The C-1 of reducing end residue

appears in the region 90-98 ppm and other non-reducing monosaccharide units appear at

98-112 ppm. The rest of methine and methylene resonances absorb between 51-86 ppm.

26

The appearance of methine resonances between 52-57 ppm174 is generally associated with

amino substituted carbon signals at an amino sugar residue. Low field absorption in the

region 169-178 ppm177 reflects the presence of a carboxylic group of hexapyranoic acids

or the carbonyl group of acetamido sugars. The presence of an acetaamido sugar may

further be complemented by the appearance of methyl resonances in the region 20-24

ppm. The spectral region between 57-64 ppm174 contains signals for all the un-substituted

hydroxy methylene resonances C-6, whereas methyl resonances of 6-deoxy sugars

generally appear in the region 16-19 ppm175, 176.

Since naturally occurring monosaccharides are generally hexoses or pentoses

therefore, each hexose and pentose unit introduces either six or five resonances,

respectively. Accordingly in a well-resolved 13C NMR spectrum, in most cases the

number of monosaccharide residues can be easily ascertained by simply dividing the total

number of signal absorbing between 60-85 ppm either by five or four or by combination

of both. In a hexose monosaccharide besides the anomeric signal it give rise to five

resonances whereas in case of 6-deoxy hexose and pentose it give rise to four resonances

in the above mentioned chemical shift range. The coupling pattern for GalNAc and

GlcNAc in 1H NMR is similar to Gal and Glc respectively but in 13C NMR an up-field

shift of δ C2α 55.4, δ C2 58ppm for GlcNAc and an upfield shift of δ C2α 51.4, δ C2β 54.9

ppm for GalNAc has been reported. In the chemical shift analogy method the chemical

shifts of carbon atoms in identical residues of similar oligosaccharide structure will be

influenced only by glycosylation shifts, primarily by the δ shift (approximately 8 ppm

downfield) for a substituted carbon atom and secondarily by the β shift (1-2 ppm up-

field) for those carbon atoms adjacent to the linkage position. The 13C chemical shift

reveals the anomeric configuration in a manner similar to the proton chemical shift but

most importantly the one bond 13C-1H coupling constant in pyranoses can be used to

determine the anomeric configuration. For D sugars in the 4C1 conformation a JC1, H1170

indicates an α-anomeric sugar whereas JC1, H1160 indicates an β-anomeric sugar

configuration177

27

13C NMR data of common glycopyranoses of oligosaccharide found in milk152

Sugar C-1 C-2 C-3 C-4 C-5 C-6 MeCONH

β-D-Glc

α -D-Glc

β -D-Gal

α -D-Gal

β -L-Fuc

α -L-Fuc

β -D-GlcNAc

α -D-GlcNAc

β -DGalNAc

α-DGalNAc

96.8

93.0

97.4

93.2

97.2

93.1

95.9

91.8

96.3

92.0

75.2

72.4

72.9

69.3

72.7

69.1

57.9

55.0

54.8

51.2

76.7

73.7

73.8

70.1

73.9

70.3

74.8

71.7

72.0

68.4

70.7

70.7

69.7

70.3

72.4

72.8

71.1

71.3

68.9

69.6

76.7

72.3

75.9

71.3

71.6

67.1

76.8

72.5

76.0

71.4

61.8

61.8

61.8

62.0

16.3

16.3

61.9

61.8

61.9

62.1

-

-

-

-

-

-

23.1,175.5

22.9,175.1

23.1,175.8

22.9,175.4

The presence of sialic acid residue could also be well determined by 13C NMR

spectroscopy. The anomeric signals (C-2) appear at δ 100-101 ppm while signal for –

COOH group appears at δ 174 ppm. The other characteristic signals of sialic acid are as

under-

N- ACETYL NEURAMINIC ACID (5-amino3, 5 dideoxy-D-glycero-D-galacto-2-nonulosonic acid)

C

C

C

C

COOH

C=O

C

=

H3C

OH

OH

OH

N

C

H

H

H

C

H

H

HO

HO

CO

H

HH

H

H

O

OHOH

COOHAcNH

HOHO

OH

28

13C and 1H NMR values of Sialic acid residue found in Milk178

Sialic acid residue 1H Chemical Shift 13C Chemical Shift

- Neuraminic- 5Ac 1 2

3ax

3eq

4

5

6

7

8

9

9’

C=O CH3

-

-

1.693-1.801

2.668-2.762

3.56-3.68

3.79-3.85

3.63-3.71

3.55-3.65

3.86-3.90

3.64

3.87-3.88

-

2.025-2.038

174.0-174.6

100.2-101.0

40.5-41.0

-

69.0-69.3

52.5-52.7

73.3-73.7

69.0-69.3

72.5-72.7

63.3-63.9

-

175.7-175.8

22.8-22.9

Two Dimensional NMR Spectroscopy164

Although the one dimensional NMR experiments provides enormous information

regarding the structure of oligosaccharide but due to overcrowding in anomeric and ring

proton chemical shift region it is not possible to perform unambiguous assignments of

anomeric and ring protons. Further this substantial overlapping could be well resolved by

the two dimensional NMR experiments. The structure elucidation of oligosaccharides is

the unambiguous assignment of the 1H resonances of individual sugar residues, is poorly

resolved in the region of ring protons i.e. from 3.2-4.2 ppm. For the structure

determination, it is important to observe the individual components of the overlapping

multiplets. The general approach is to assign an isolated resonance often an anomeric

proton (4.3-5.5 ppm) or the methyl resonance (1.2-1.4 ppm) in 6-deoxy sugars, then to

correlate spins in a step-wise manner around the spin system of the ring. However, spin

correlation can be done by one-dimensional difference decoupling experiments if only

few assignments are needed. These difficulties could be overcome by the use of modern

29

two-dimensional NMR experiments because they are more efficient for the simultaneous

determination of a large number of spin correlations. 2D NMR spectroscopy provides

actual, high quality and well interpretable data of the sugar molecule. Chemical shift

correlation maps obtained by 2D NMR experiments are found to be extremely useful in

the identification of components of oligosaccharides, without relying on the analogy with

any reference data. There are two fundamental types of 2D NMR spectroscopy. The first

is correlated spectroscopy, in which both frequency axis contain chemical shift

information and the other is J resolved spectroscopy in which one frequency axis contains

spin coupling (J) and other chemical shift (δ) information. Correlated 2D NMR

spectroscopy may be divided into two groups based on the mechanism of interaction

producing the observed signals. The first group includes COSY and its descendants,

HOHAHA, HETCOR and HMQC based on scalar coupling through coherent transfer of

transverse magnetization, and reveals through bond connectivity. The second group based

on dipole couplings through incoherent transfer of magnetization and provides

information through space connectivity, the NOESY experiment is representative of this

group. Salient features of two-dimensional NMR spectroscopy applied to

oligosaccharides are described as below-

Correlated Spectroscopy (COSY)179, 180

COSY technique is also known as JEENER experiment and is one of the

important 2D NMR spectroscopic methods for structure determination. 1H-1H COSY

(correlated Spectroscopy) is useful for determining signals which arise from neighboring

protons, especially when the multiplet overlap or there is extensive second order

coupling. A COSY spectrum yields through bond correlation via spin-spin coupling.

There are two types of correlation spectroscopy i.e. HOMOCOSY and HETEROCOSY.

In homonuclear shift correlation 2D experiments, the correlation is between similar

nuclei i.e. either 1H-1H or 13C-13C. The normal NMR spectra are plotted on a two

frequency axis and the conventional 1D spectrum appears along the diagonal. The clear

representation of 2D NMR spectrum is obtained as contour plots of mutual coupling

which exists between two nuclei (1H-1H, 13C-13C), cross peak appears at the chemical

shift coordinates (X, Y) and (Y, X). Identification of monosaccharide units is first

approached by analyzing the 1H homonuclear shift-correlation spectra. COSY spectra

30

contain information on spin coupling networks within the constituent’s residues of the

oligosaccharide through the observation of cross peaks. Assignment of this spectrum by

coupling-correlation requires an initial point for the identification of the individual spin

system of sugar rings. The most downfield 1H signals (anomeric) are always a convenient

starting point for the assignment. With in typical aldohexopyranosyl ring, the coupling

network is unidirectional i.e. H-1 couples to H-2; H-2 couples to H-1 and H-3, H-3 to H-

2 and H-4 and so on. However, the presence of no or small coupling between H-4 and H-

5 (J4,5=2-3 Hz) of galactopyranosyl residue and coupling between H-1 and H-2 in

mannopyranosyl residue prevents detection of cross peaks. COSY experiments and its

RELAY extensions give coupling pattern along with shift information, which allow each

monosaccharide residue to be identified and designated as α or β and also provide

information about sugar identity and substitution pattern. Sugar analysis indicated the

presence of 3Gal, 1Glc, 2GlcNAc and 3NeuAc residues, in the sialyl oligosaccharide

present in milk. Further, on the basis of the upfield resonances one can easily distinguish

the H-1 signal of Gal and GlcNAc. The H-1 signal of Glc-β was correlated with H-2

signal at δ 3.320 ppm. Gronberg et. al.166 used the COSY experiment in the structural

analysis of five new monosialylated oligosaccharides obtained from human milk.

Double Quantum Filtered Correlated Spectroscopy (DQF-COSY)181

This Technique was introduced by Rance et al. for better visualization of cross

peaks, which are close to diagonal axis.

DQF-COSY Spectra

31

It can be achieved by the introduction of a double quantum filter, which generates

a COSY spectrum having both cross peaks and a diagonal multiplet antiphase structure. It

provides a clear and accurate way of obtaining chemical shift values of coupled protons.

Furthermore, DQF-COSY spectra suppress the detection of spin-isolated protons (i.e.

uncoupled protons) such as those arising from solvent or isolated methyl groups. The

suppression of these signals is useful when they occurred in crowded regions of the

spectrum. H. Fiewer et. al. used the DQF-COSY experiment182 in the structural

elucidation of octasaccharide isolated from human milk.

Triple Quantum Filtered Correlated Spectroscopy (TQF-COSY)183

In some cases COSY employing a triple quantum filter can further edit COSY

spectra of oligosaccharides. This technique is applied often to assist in the detection and

assignment of H-6 and H-6′ protons of constituent aldohexopyranosyl rings containing an

exocyclic hydroxymethyl group (isolated hydroxymethyl groups, such as those appended

to C-2 of 2-ketoses, will not be detected by TQF-COSY).

Relay Correlation Spectroscopy (RELAY-COSY)184

In this type of COSY, the anomeric proton not only correlates with H-2 proton,

but also with other intra residue protons (H-3, H-4, H-5, H-6) in a well resolved region of

2D spectrum. The assignment of H-6 is less successful in this method.

In Adequate Spectroscopy (IN ADEQUATE)185

The two dimensional ‘incredible natural abundance double quantum transfer

experiment’ provides direct information on carbon bonding and thus can be used to trace

the entire carbon skeleton of the molecule.

Spin-Echo Correlation spectroscopy (SECSY)186

The method of two-dimensional homonuclear spin-echo J correlated

spectroscopy, developed by Ernst and coworkers. 2D spin-echo correlation spectroscopy

establishes scalar coupling (J) connectivities between peaks. The data are displayed as a

counter plot. By the use of 2D-SECSY the J connectivities of oligosaccharides can be

revealed. Thus by 2D-SECSY NMR the monosaccharide composition and anomeric

configurations of oligosaccharides can be obtained.

32

Total Correlation Spectroscopy (TOCSY)187,188

A recent 2D NMR experiment for identifying extended couplings is TOCSY

which is also known as HOHAHA (Homonuclear Hartman Hahn Spectroscopy). The

TOCSY experiment uses a specific series of pulses (referred to as an isotropic mixing

sequence), to mediate oscillatory magnetization transfer between all protons in a scalar

coupled network. Inter residue bond magnetization transfer cannot occur since the

glycosidic linkage between residues in an oligosaccharide is devoid of protons. Milk

oligosaccharides anomeric proton and other sugar proton give cross peak in the region of

TOCSY spectrum. This experiment is especially helpful in sugar for which similar

chemical shifts of methine protons leads to many instances of strong coupling or

intermediate coupling. Thus TOCSY can give total correlation of all protons in a chain

with each other and serve for the identification of single residue in oligosaccharides. H.

Kogelberg et.al. used the TOCSY technique in structural elucidation of oligosaccharide

isolated from human milk.

Nuclear Overhauser Effect Spectroscopy (NOESY)189,190

The principal use of NOSEY in oligosaccharide structure determination has been

in the assessment of molecular conformation (i.e. 3D structure). Cross peaks are observed

in 2D NOESY spectra between proton pairs that are close in space (typically less than

5Å). The intranuclear distance between H-1 and H-5 (and H-3) of -D-galactopyranosyl

residue is about 2.5 Å, and thus a strong cross peak is observed between these protons in

NOESY spectrum. In α configuration, only a nuclear overhauser effect between H-1 and

H-2 is observed. In general, 1, 3 diaxial and 1, 2-eq-ax proton pairs in pyranosyl rings

will produce intra residue NOESY cross peaks. However NOESY cross peaks may be

observed between linkage sites and O-glycoside conformation in oligosaccharides.

Vicinal proton coupling, which is very useful in proton assignments for individual

pyranoside rings, is less valuable for correlating monosaccharides residue to each other.

Since the protons across the glycosidic linkage are four bonds apart they do not show

scalar coupling and thus no correlation between individual spin systems could be

observed by COSY or HOHAHA. The presence of an intra residue NOE from the

anomeric proton of a particular sugar residue to protons of other sugar residue in case of

33

oligosaccharides, defines the glycosidic linkage between the two residues. This effect

depends on the local conformation about the glycosidic linkage. NOE depends not only

on the proximities of the protons but also on the correlation time of the molecule. Since

NOE also depends on the distances between protons, it is possible to determine inter

proton distances directly from NOE data.

NOE spectra

In practice semi selective excitation of one carbohydrate proton, combined with

multistep-relayed coherence transfer and the terminal NOE transefer has been used for

the sequential analysis of oligosaccharides. Assignment of the anomeric and some other

protons resonance may be made with the help of data from decoupling and NOE

experiments. In the structural determination of Lacto-N-hexose, selective decoupling

irradiation at each of three doublets assigned to the -galactose H-1 at 4.424, 4.453 and

4.504 ppm identified the resonance of the corresponding H-2 at 3.546, 3.489 and 3.496

ppm respectively. Selective decoupling irradiation of the narrow doublet at 4.145 ppm,

which is assigned to H-4 of a -galactose which is glycosylated at C-3 identifies the H-3

resonance of -galactose at 3.695 ppm. Decoupling at the Gal H-2 resonance at 3.546 ppm

identifies the same Gal H-3 resonance and thus completing the assignment of H-1, H-2,

H-3, and H-4 of the branching galactosyl residue. Thus an effective way of connecting

two monosaccharides residue is by monitoring the nuclear overhauser effect for an

anomeric reporter group to the hydrogen of the substituted position in the adjacent ring.

34

Rotating Frame Overhauser Enhancement spectroscopy (ROESY)191,192

The NOESY technique has the disadvantage that for molecules with a molecular

mass in the order of 1000 to 3000 the signal may disappear, since the NOE effect changes

its sign depending on the molecular correlation time. Due to the well known problem

involved with NOE measurements at medium field strength of medium-sized molecules,

a 2D NOE in a rotating frame (ROESY) can be of importance. In cases where attempts

to obtain reliable NOE crosspeaks are unsuccessful, a ROESY spectrum can show all

NOE crosspeaks defining interglycosidic linkage. Viscosity of the medium and

temperature are important in this type of experiment. H. Kogelberg et.al. used the

ROESY technique to elucidate the structure of undecasaccharide isolated from human

milk.

Hetronuclear Multiple Quantum Coherence Spectroscopy (HMQC) 193

The 2D hetrocosy experiment like HMQC plays a decisive role in spectral

assignments for small molecules in solution. It is a effective method for unambiguous

assignment of 1H and 13C chemical shifts and the C-H correlations in oligosaccharides for

deducing the structure of oligosaccharides. The main problem in this experiment is the

rejection of the signals which arise from proton that are not part of a hetronuclear coupled

spin system. These signals are usually cancelled by receiver and transmitter phase

attraction (phase cycling) and by application of pulsed field gradient. The second

problem associated with proton detected (inverse mode) hetronuclear shift correlation

experiment is the lack of resolution in the indirectly detected dimension F1. For a given

spectral width, an increase in F1 resolution requires an increase in the number of t1

increments thus F1 restricted 2D maps offer great help to insure a proper spectral

analysis.

HMQC Spectra

35

Heteronuclear single quantum coherence (HSQC)194

The HSQC experiment was proposed by Bodenhausen and Ruben and it is a

type of double INEPT experiment. This experiment correlates protons with their directly

attached hetro nuclei. HSQC experiment is useful in structural elucidation of

oligosaccharide since it provides direct information of protons attached to carbons. In the

HSQC experiment cross peak of those protons and carbons was observed which were one

bond apart i.e. HSQC experiment identifies proton nuclei with carbon nuclei that are

separated by one bond. Since only in very similar chemical environment it is possible that

two pairs of 1H and 13C shifts are identical hence generally only one peak appears for

each distant CH group in the molecule. Thus the Heteronuclear single quantum coherence

(HSQC) provides useful information about number of monosaccharide moieties present

in the oligosaccharide and therefore extensively used in structural elucidation of

oligosaccharides. Gronberg et. al used166 the HSQC experiment in the structural analysis

of five new monosialylated oligosaccharides obtained from human milk. For example, 1H-13C 2D HSQC NMR spectrum of n-butyric acid has been shown below highlighting

the cross peaks generated due to proton and carbon attached with one bonds.

HSQC Spectra

Heteronuclear multiple bond correlation (HMBC)195

Heteronuclear Multiple Bond Correlation is an experiment that identifies proton

nuclei with carbon nuclei that are separated by more than one bond. Thus by the use of

36

HMBC experiment the correlation of proton with adjacent carbon can be achieved and

this information is very useful in structural elucidation of oligosaccharides. Bendiak used

the peracetylation along with HMBC196,197 to separate free hydroxyl positions from

positions which were glycosidically linked. Bendiak used peracetylation of free hydroxyl

groups with 13C-carbonyl labeled acetic anhydride. The protons at acetyl-protected

position now show a three bond 13C-1H coupling and can be easily detected by HMBC

experiment and thus position of glycosidic linkage was confirmed. The sensitivity of

heteronuclear multiple bond correlation (HMBC) is increased by the use of 13C labeled

acetic anhydride, and the assignments can be readily identified.

HMBC spectra

Three Dimensional NMR Spectroscopy198

Hetronuclear 3D NMR spectroscopy is a useful method for resolving spectral

overlap in all frequency domains. This is important for assigning spectra and elucidating

the structures of complicated molecules. In addition, the increase in resolution afforded

by this technique help to automate peak-picking and assignment procedures and facilitate

the extraction of J couplings (HMQC-COSY) and quantitative NOE information

(HMQC-NOESY). By the application of homonuclear 3D NOE-HOHAHA and

hetronuclear 3D HMQC-NOE experiments in studies of complex oligosaccharides, NOEs

37

can be investigated which are hidden in conventional 2D NOE spectra. In the 3D NOE-

HOHAHA spectrum omega three cross sections were considered to be most suitable for

assignment of NOEs. In 3D HMQC-NOE spectroscopy the larger chemical shift

displacement of the carbon spectrum can be used to find NOEs hidden in the bulk region

of spectra.

Mass Spectroscopy

The structural analysis of the carbohydrate is usually carried out by a combination

of chemical and enzymatic methods .The structure determination of oligosaccharides is a

difficult task because of their presence in meager quantity and low resolution on

chromatography. In earlier days the structural studies of oligosaccharides depend upon

paper chromatography, sequential exoglucosidase digestion and quantitative methylation

analysis etc. Some frequently used techniques is periodate oxidation, Smith degradation,

permethylation analysis, acetolysis, alkaline degradation (β- elimination), and sequential

degradation with glycosidases. In recent years with the advent of modern

chromatographic techniques (HPLC) and recent physicochemical techniques like NMR 1H, 13C and 2D NMR and Mass spectroscopy FAB, MALDI and ES-MS, most of the

problems that are unattended previously seem to be resolved. Despite the high degree of

sophistication reached by these methods, it is evident that still some uncertainty remains

even with regard to the monosaccharide composition. Of all the modern structural

methods for oligosaccharides/oligoglycosides, NMR (lD & 2D techniques) in

combination with Mass spectrometry (FAB, MALDI & ES) yields the complete

stereoscopic structure of oligosaccharides/oligoglycosides, with or without prior

structural knowledge. Besides the NMR, the Mass spectrometry is the most basic and a

developed technique which is used in the structural elucidation of glycol compounds i.e.

glycosides, glycoprotein and oligosaccharide. In the present study we have used the Mass

spectrometry as a bench tool in the structural elucidation of various glycol-compounds.

The history of Mass spectrometry and various techniques used in structure determination

of oligosaccharides are as under.

Various methods of mass spectrometry were used in the structural determination

of natural products which are as follows-

38

1- Electron Ionisation (EI), 2- Chemical Ionisation (CI), 3- Field desorption (FD)

field desorption ionization, 4- Plasma desorption ionization, 5- Fast Atom Bombardment

(FAB), 6- Laser desorption (LD)/ionization, 7- Matrix-assisted Laser

Desorption/Ionization (MALDI), 8- Atmospheric Pressure Chemical Ionization (APCI),

9-Electrospray Ionization (ESI) and 10- Nanospray Ionization.

In initial stages it was only the electron impact mass spectroscopy199. Which was

being used in the structure elucidation of oligosaccharides but its limitation was that only

the lower fragments were observed200 .Further after the innovation of field desorption

mass spectrometry this problem was solved but the information obtained from FD-MS

was limited to higher fragments only and at that time chemists were coupling the results

of EI-MS and FD-MS for complete interpretation of mass spectrometry data. But a major

revolutionizing breakthrough was achieved with the development of soft or cold

ionization techniques (FAB-MS, MALDI-MS, thermo-spray and electro-spray MS) that

allowed the ionization and desorption of large polar compounds like intact proteins,

glycoconjugates and nucleic acids without prior evaporization from liquid or solid

masses,[hence expanded the utility of MS for analysis of large biopolymers. The

sensitivity is often in the picomol range or better. While MS methods can provide very

accurate masses of molecular or fragment ions. The details of various mass techniques

are described as under.

Electron Ionisation (EI)201

In Electron ionization technique the analyte must be vaporized; this is usually

achieved by heating the probe tip containing a droplet of the analytic in solution. If the

sample is thermally unstable, this will often be the first cause of sample fragmentation.

Once in the gas-phase, the analyte passes into an EI chamber. Where it interacts with a

homogeneous beam of electrons typically at 70 electron volts energy. The electron beam

is produced by a filament (rhenium or tungsten wire) and steered across the source

chamber to the electron trap. A fixed magnet is placed, with opposite poles slightly off-

axis, across the chamber to create a spiral in the electron beam. This is to increase the

chance of interactions between the beam and the analytic gas. There are no actual

39

collisions between analytic molecules and electrons ionization is caused by electron

ejection from the analytic or by analytic decomposition.

Fast Atom Bombardment (FAB)

The development of fast particle desorption culminate with the development of

FAB by Michael Barber in the early 1980's202. The techniques of FAB and LSIMS are

very similar in concept and design as they both involve the bombardment of a solid spot

of the analyte/matrix mixture on the end of a sample probe by a fast particle beam. The

matrix (a small organic species like glycerol or 3-nitro benzyl alcohol) is used to keep a

homogenous sample surface. The particle beam is incident onto the surface of the

analyte/matrix spot, where it transfers its energy bringing about localized collisions and

disruptions. Some species are ejected from the surface as secondary ions by this process.

These ions are then extracted and focused before passing to the mass analyser. The

polarity of ions produced depends on the source potentials. In FAB, the particle beam is a

neutral inert gas (Ar or Xe) at 4-10 keV and in LSIMS; the particle beam is ions (usually

Cs+) at 2-30 keV. Both methods are comparatively 'soft' ionization methods very little

residual energy is possessed by the ions after desorption making them particularly suited

to the analysis of low volatility analytes. FAB-MS 203,204,205 was used for elucidating the

structure of lactose derived oligosaccharide from Goat’s milk.

Mass Fragmentation of Oligosaccharides

Mass spectrometry plays an important role in the structure elucidation of natural

products particularly in the field of oligosaccharides. With the advent of new inlet

technique and specific studies on volatile derivative, researcher has put another step to

overcome the difficulties which were initially limited due to relatively low volatility of

these compounds. In initial stages it was only the electron impact mass spectroscopy

which was being used in the structure elucidation of oligosaccharides but its limitations

was that only the lower fragments were observed. Further after the innovation of Field-

desorption mass spectrometry this problem was solved but the information obtained from

FD-MS was limited to the higher fragments only and at that time chemists were coupling

the results of EI-MS201 and FD-MS for complete interpretation of mass spectrometry

data. Later the FAB-MS technique was introduced which gave complete information

40

regarding the lower and higher mass fragments into one spectrum. In FAB-MS203, 204, 205

an abundant molecular ion, or its protonated species (M+H)+ or a cationic species

(M+Na)+, (M+K)+ is obtained. It play decisive role in the structure elucidation of milk

oligosaccharides. Recently it has been seen that the FAB-MS not only fixe the molecular

weight of the oligosaccharide but also ascertain the sequence of the monosaccharide

units. The molecular ion (M+) fragments into the fragment units which were formed by

the decomposition pathway in which repeated H transfer in the oligosaccharide is

accompanied by the elimination of terminal sugars less water, such fragmentation goes

on until the monosaccharide is left (Scheme 1). Negative ion fast-atom bombardment

mass spectroscopy has been important tools in the structure elucidation of milk

oligosaccharides and the result have also been found to be comparable with the proposed

structure, based on the results obtained from high resolution NMR spectroscopy.

Negative ion FABMS of milk oligosaccharides gave molecular ions [M-H]+. FABMS has

also been found to be very useful in assigning the monosaccharide sequence and some

linkage positions. By FAB-MS203, 204, 205 we can identify the presence of acetamido

monosaccharides, fucosylated and sialyalated branching. Besides the routine losses of

H2O, OH, CH2OH etc. was also observed.

Scheme 1

H – Transfer in oligosaccharide and elimination of monosaccharide from non-

reducing end

O O O ORS4 S3 S2HO

H

S1

O O OR

S4

S3 S2

HO H

S1

O OR

S3

S2HO

H

S1

OR

S2HO H

S1

HO

HO

HO

ORH

HO S1

CHAPTER II

ISOLATION

41

ISOLATION

Sheep (Ovis aries) are quadrupedal, ruminant mammals typically kept as livestock.

sheep are members of the order Artiodactyla. Although the name "sheep" applies to

many species in the genus Ovis, in everyday usage it almost always refers to Ovis

aries. Numbering a little over one billion, domestic sheep are also the most numerous

species of sheep. An adult female sheep is referred to as a ewe an intact male as a ram

or occasionally a tup, a castrated male as a wether, and a younger sheep as a lamb.

Classification (Zoological description)

Kingdom- Animalia

Phylum- Chordata

Subphylum- Vertebrata

Class- Mammalia

Order- Artiodactyla

Family- Bovidae

Subfamily- Caprinae

Genus- Ovis

Species- O. aries

Fig- SHEEP

42

Description

Depending on breed, sheep show a range of heights and weights. Their rate of growth

and mature weight is a heritable trait that is often selected for in breeding. Ewes

typically weigh between 45 to 100 kilograms, and rams between 45 to160 kilograms.

When all deciduous teeth have erupted, the sheep has 20 teeth. Mature sheep have 32

teeth. As with other ruminants, the front teeth in the lower jaw bite against a hard,

toothless pad in the upper jaw. These are used to pick off vegetation, and then the rear

teeth grind it before it is swallowed. There are eight lower front teeth in ruminants,

but there is some disagreement as to whether these are eight incisors, or six incisors

and two incisor-shaped canines. This means that the dental formula for sheep is either

0.0.3.3/4.0.3.3 or 0.0.3.3/3.1.3.3 there is a large diastema between the incisors and the

molars. Sheep have a gestation period of about five months, and normal labour takes

one to three hours. Although some breeds regularly throw larger litters of lambs, most

produce single or twin lambs.

Properties of sheep milk

Sheep milk is delicious and healthy alternative to cow’s milk it is particularly

popular among those with lactose intolerance because of sheep milk’s low lactose

properties. Nearly 75% of the world’s population is considered to have a lactose

allergy or “lactose intolerant” those with a lactose allergy have difficulty digesting

cow’s milk causing symptoms such as gas and diarrhoea. The fats in sheep milk are

mono-saturated and poly-unsaturated, which are both having healthy essential fats.

Sheep milk also contains medium high chain triglycerides that help the body in

reducing high cholesterol levels. Sheep milk is white in colour as compared with Cow

milk which is yellowish due to the presence of carotene. The gross composition of

Goat and Sheep milk is similar, but sheep milk contains more fat, solids, non-fat,

proteins, caseins, whey-proteins and total ash as compared with goat milk. These

differences make the rennet coagulation time for sheep milk shorter and curd firmer

owing to the differences in the caseins. Solids in sheep milk range from 15 to 20%

and proteins are between 5 to 6%. There are many significant differences in the amino

acids of goat and sheep milk proteins and also in the relative proportions of the

various milk proteins and their genetic polymorphism. K-casein has been isolated and

characterized from goat. K-casein has been isolated and characterized from goat milk

and sheep milk and both were similar to cow K-casein in many respect.

43

Constituents unit Cow Goat Water Buffalo Sheep

Water g 87.8 88.9 81.1 83.0 Protein g 3.2 3.1 4.5 5.4 Fat g 3.9 3.5 8.0 6.0 Carbohydrate g 4.8 4.4 4.9 5.1 Energy kcal 66 60 110 95 kJ 275 253 463 396 Sugars (Lactose) g 4.8 4.4 5.1 4.9 Fatty Acids:

Saturated g 2.4 2.3 4.2 3.8 Mono-unsaturated g 1.1 0.8 1.7 1.5 Polyunsaturated g 0.1 0.1 0.2 0.3 Cholesterol mg 14 10 8 11 Calcium IU 120 100 195 170

Table-1: Comparison of sheep milk and other animals milk properties.

ISOLATION OF SHEEP MILK OLIGOSACCHARIDES BY MODIFIED

METHOD OF KOBATA AND GINSBURG

10 litter of sheep milk was collected in 38 days in normal milking condition from a

single domestic sheep from district Ghazipur, (Vill-Gausabad) Uttar Pradesh, India.

After milking the milk was fixed immediately by addition of equal amount of ethanol.

The preserved milk was taken in to the laboratory for further experiments. It was

filtered then it was centrifuged on C-25 centrifuging machine at 5500 rpm at -4oC,

after centrifugation the solidified layer was removed by filtration through glass wool.

After filtaration lipid layer was discarded and supernatant was precipitated by

addition of 68% ethanol and separated by centrifugation and after removing protein

and lactose supernatant was filtered through a micro filter (0.2 µ) to remove

remaining lactose. It was then lyophilized (mixture of oligosaccharides). Lyophilized

material was then fractionated on a sephadex G-25 column, eluted with triple distilled

water at flow rate 3ml/min. Fractions were analyzed for sugars by phenol-sulphuric

acid reagent.

44

Isolation of Milk oligosaccharides by modified method of Kobata and Ginsburg

Sheep Milk

Carbohydrate containing fractions (Fractions were pooled, lyophilized and analyzed by HPLC)

Deacetylation

Analytical HPLC The carbohydrate fractions were eluted with TDW (containing 0.1%TFA & CH3CN) at a flow rate 1ml/min., to check homogeneity of the oligosaccharide mixture. The elution was monitored by UV absorbance at 220 nm.

Chemical transformation Oligosaccharide mixture was acetylated with Ac2O and pyridine converting free sugar into non-polar acetyl derivatives which were resolved nicely on TLC and were separated by column chromatography over silica gel which resulted in the isolation of chromatographically pure compounds

The chromatographically pure acetylated Milk oligosaccharides were deacetylated by dissolving then in acetone & NH4OH and left overnight. Ammonia was removed under reduced pressure and the compound was washed with CHCl3 and was finally freeze dried giving the deacetylated milk oligosaccharides.

Lipid Layer (Discarded)

Was precipitated by addition of 68% ethanol and separated by centrifugation

Supernatant Was filtered through a micro filter (0.2µ) to remove remaining lactose. It was then lyophilized

Supernatant

Protein and Lactose Residue (Discarded)

Lyophilized material (Mixture of oligosaccharides)

Was then fractionated on a sephadex G-25 column, eluted with triple distilled water at flow rate 3ml/min. Fractions were analyzed for sugars by phenol-sulphuric acid reagent

Equal amount of ethanol was added and filtered followed by centrifugation (5500 rpm) at -4oC, and filtered through a loosely packed glass wool

45

Sephadex G-25 Gel filtration of Sheep milk Oligosaccharide Mixture

The repeated gel filtration was performed by Sephadex G-25 chromatography

of crude Sheep milk oligosaccharide mixture. Sheep milk oligosaccharide mixture

was packed in a column (1.6 x 40 cm) (void volume = 25 ml) equilibrated with glass

triple distilled water (TDW) and it was left for 10-12 hrs to settle down. The material

was applied on a Sephadex G-25 column and was eluted for separation of protein and

glycoprotein from oligosaccharide (low molecular weight component). Presence of

neutral sugars was monitored in all eluted fractions by phenol-sulphuric acid test. In

this U.V. monitored Sephadex G-25 chromatography of Sheep milk oligosaccharide

mixture showed four peaks i.e. I, II, III and IV. A substantial amount of proteins,

glycoproteins and serum albumin were eluted in the void volume which was

confirmed by positive coloration with p-dimethylaminobenzaldehyde reagent and

phenol-sulphuric acid reagent. Fractions under peaks II and III gave a positive phenol-

sulphuric acid test for sugars which showed the presence of oligosaccharide mixture

in Sheep milk. These fractions (peak II and III) were pooled and lyophilized.

Graph-1: Spehadex G-25 chromatography of Sheep Milk Oligosaccharides

Detected by Phenol Sulphuric Acid Method. Elution was Made with TDW

0.1 -

0.0 -

Vo

IV I

II

III

10 30 50 70 90 110

Fraction Numbers

Absorbance 280 nm

46

Table-2: 12.09 gm of Sheep Milk Oligosaccharide Mixutre Chromatographed

Over Sephadex G-25 Chromatography

FRACTION NO. SOLVENT COMPOUND

(grams)

PHENOL-H2SO4 TEST FOR SUGAR

FURTHER INVESTIGATION

1-31 32-45 46-61 62-84 85-106

Glass triple distilled water ,, ,, ,, ,,

2.4 1.0 4.5 3.6 1.3

-ve

-ve +ve +ve -ve

- - purified by column chromatography after acetylation

HPLC analysis of total milk oligosaccharide mixture

HPLC finger print profile was established for Milk oligosaccharide using a

Waters model (Water Corp, Milford, USA), equipped with a pump (Waters 515) with

a chromatopak column RP – 18 (250 x 4.6 mm, i.d., 5 µm pore size) and a waters

autosampler, detection was at 310 nm using 2996 PDA detector. 20 µl of 1 mg/ml

concentration of oligosaccharide material was injected. Elution was carried out at a

flow rate of 1.5 ml/min with water : phosphoric acid (99.7 : 0.3 v/v) as solvent A and

acetonitrile : water : phosphoric acid (80.8:19:0.2 v/v) as solvent B using a gradient

elution in 0-5 min. of 88 to 85% A, 5-15 min. with 85 to 70% of A, 15-20 min. with

70 to 50% A and 20-25 min. with 50 to 30% of A and then isocratic up to 30 min.

with 30% A.

Graph-2: HPLC chromatogram of Sheep milk oligosaccharides

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

47

Table-3: HPLC Table of crude Sheep milk oligosaccharides

S.No. Retention Time % compound

1. 7.21 11.11

2. 7.63 12.07

3. 8.45 5.29

4. 17.21 8.81

5. 17.41 3.32

6. 20.58 20.92

7. 21.39 10.79

8. 21.78 3.21

9. 24.69 12.33

10. 25.42 8.99

11. 25.97 3.22

12. 27.51 2.88

Acetylation of Oligosaccharide mixture

9.48 gm Oligosaccharides mixture was acetylated with pyridine and acetic anhydride

at 60oC and solution was stirred overnight. The mixture was evaporated under water

bath and reduced pressure and viscous residue was taken in CHCl3 (200 ml) and

washed twice in ice cold water, evaporated to dryness yielding the acetylated mixture.

The acetylation converted the free sugars into their non polar acetyl derivatives which

were resolved nicely on TLC, giving 9 spots i.e. a, b, c, d, e, f, g, h and i of which six

compounds were finally separated by column chromatography over silica gel using

varying proportions of hexane, chloroform and methanol as eluants.

CHCl3: MeOH (98:2) CHCl3: MeOH (95:5)

TLC of acetylated oligosaccharides mixture at different polarity proportions

48

PURIFICATION OF ACETYLATED MILK OLIGOSACCHARIDES ON SILICA GEL COLUMN

Column Chromatography -1

Separation of the acetylated product (6.0 g.) was carried out over silica gel (200 g.)

using varying proportion of Hex: CHCl3, CHCl3, CHCl3: MeOH as eluants, collecting

fraction of 200 ml each. All these fractions were checked on TLC and those showing

similar spots were taken together for further investigations. The chromatographic

details are given in the table No-1 as below.

Table 1

6.0 g Mixture Chromatographed Over 200 g SiO2

Fraction No.

Solvent Eluted Residue Amorphous

Spots on TLC

Further Investigation

1-25 CHCl3 500 mg Streaking -

26-48

CHCl3:MeOH

99.9 : 0.1

400 mg

streaking -

49-83 CHCl3:MeOH

99.5 : 0.5

300 mg a with streaking

-

84-103 CHCl3:MeOH 99 : 1

180 mg a and b CC-2

104-137 CHCl3:MeOH

98 : 2

1.270 mg b, c, d, e CC-3, CC-4

137-147

CHCl3:MeOH

95: 5

1.883 mg

CC-5

148-157

CHCl3:MeOH

95 : 5

400 mg

f with streaking

-

158-170

CHCl3:MeOH

90 : 10

700 mg

washing

-

49


The combined fractions of 84-103 (180 mg) from chromatography 1

containing a and b were chromatographed over 20 g silica gel. The elution was carried

out using Hex: CHCl3, CHCl3 and CHCl3: MeOH as eluant in different proportion and

collecting fraction of 20 ml each. Details are given in table No – 2.

Table 2

Fractions No. 48-83 (180 mg) Mixture Chromatographed Over 20 g SiO2

Fraction No.

Solvent Eluted Residue Amorphous

Spots on TLC


1-10 CHCl3 Streaking

11-22 CHCl3:MeOH

99.9 : 0.1

23-46 CHCl3:MeOH

99.8:0.2

112 mg a Physico-chemical investigation

47-57 CHCl3:MeOH

99.5:0.5

5 mg a with streaking

58-67 CHCl3:MeOH

99: 1

30 mg streaking

68-80 CHCl3:MeOH

98 : 2

10 mg streaking

64-69

CHCl3:MeOH

95 : 5

10 mg washing

50


The combined fractions of 104-137 (1.270 g) from chromatography 1

containing d, c, d and e were chromatographed over 120 g silica gel. The elution was

carried out using Hex: CHCl3, CHCl3 and CHCl3: MeOH as eluant in different

proportion and collecting fraction of 120 ml each. Details are given in table No – 3.

Table 3

Fractions No. 104-137 (1.270 g) Mixture Chromatographed over 120 g SiO2

Fraction No. Solvent

Eluted Residue Amorphous

Spots on TLC


1-12 CHCl3 - - -

13-22

CHCl3:MeOH

99.9 : 0.1

12 mg

a with streaking

-

23-35

CHCl3:MeOH

99.8 : 0.2

5 mg

b with streaking

-

36-50 CHCl3:MeOH

99.5 : 0.5

419 mg b Physio-chemical investigation

51-65

CHCl3:MeOH

99: 1

20 mg

b with streaking

-

66-75 CHCl3:MeOH

98 : 2

409 mg

C and d CC-4

76-100 CHCl3:MeOH

95 : 5

22 mg

Streaking -

101-105 CHCl3:MeOH

90 : 10

- washing -

.

51


The combined fractions of 66-75 (409 mg) from chromatography 3 containing

c and d were chromatographed over 50 g silica gel. The elution was carried out using

Hex: CHCl3, CHCl3 and CHCl3: MeOH as eluant in different proportion and

collecting fraction of 50 ml each. Details are given in table No – 4.

Table 4

Fractions No. 66-75 (409 mg) Mixture Chromatographed Over 50 gm SiO2



Spots on TLC


1-15 Hex : CHCl3

25 : 75

65 mg -

16-35 CHCl3 95 mg -

35-50 CHCl3:MeOH

99.8 : 0.2

88 mg -

51-60 CHCl3:MeOH

99.5 : 0.5

10 mg -

61-75

CHCl3:MeOH

99: 1

46 mg

c

Physico-chemical investigation

76-85 CHCl3:MeOH

99 : 1

32mg

c with streaking

86-105 CHCl3:MeOH

98 : 2

62 mg d Physici-chemical investigation

106-115 CHCl3:MeOH

95 : 5

5 mg - -

116-120 CHCl3:MeOH

90 : 10

10 mg Washing -

52


The combined fractions of 137-147 (1.883 gm) from chromatography 1

containing e and f were chromatographed over 100 gm silica gel. The elution was

carried out using Hex: CHCl3, CHCl3 and CHCl3: MeOH as eluant in different

proportion and collecting fraction of 100 ml each. Details are given in table No – 5

Table 5

Fractions No.137-147 (1.883 gm) Mixture Chromatographed Over 100 g SiO2



Spots on TLC


1-15 CHCl3 16 mg -

16-30 CHCl3:MeOH

99.9 : 0.1

105 mg

31-80 CHCl3:MeOH

99.5 : 0.5

300 mg Streaking

81-120 CHCl3:MeOH

99 : 1

426 mg -

121-140 CHCl3:MeOH

98 : 2

17 mg

e Physio-chemical investigation

141-160

CHCl3:MeOH

95 : 5

80 mg

f Physio-chemical investigation

161-180 CHCl3:MeOH

90 : 10

324 mg washing

53

DEACETYLATION OF ISOLATED COMPOUNDS

The acetylated compounds a, b, c and d were obtained from column

chromatography. On deacetylation they gave native oligosaccharides A, B, C, and D

respectively.

Table 8

Acetylated and Deacetylated Oligosaccharides Obtained from Sheep Milk

Acetylated Compound Deacetylated Compound

Alphabetical name

Analytical notation

Quantity (mg) Alphabetical name

Analytical notation

Quantity (mg) Obtained by

column taken for deacetylation

a ARSM-1A 112 40 A ARSM-1 35

b ARSM-2A 419 50 B ARSM-2 45

c ARSM-3A 46 32 C ARSM-3 24

d ARSM-4A 62 52 D ARSM-4 44

DESCRIPTION OF ISOLATED COMPOUNDS

Compound A (ARSM-1) CAPRIOSE:

Compound a (112 mg) was obtained from fraction 23-46 of column

chromatography 2. On deacetylation of 40 mg of substance a with NH3/acetone, it

afforded substance A (35.0 mg) ��

+41.01o (c 1% H2O). For experimental analysis,

this compound was dried over P2O5 at 100oC and 0.1 mm pressure for 8 hr. It gave

positive Phenol-sulphuric acid test, Feigl test and Morgan-Elson test.

C34H58O25N2 %C %H %N

Calculated 45.63 6.48 3.13

Found 45.64 6.49 3.13 1H NMR of Comound-A Capriose Acetate in CDCl3 at 300 MHz

6.37[d, 1H, J=3.0 Hz, α-Glc(S-1) H-1], 5.72[d, 1H, J=8.0Hz, β-Glc(S-1) H-1], 5.19

[d, 1H, J=8.7Hz, β-Gal(S-2) H-1)], 5.4 [d, 1H, J=3.0Hz, α-FucNAc(S-3) H-1], 4.48

[d, 2H, J=7.2, β-Gal(S-4), H-1, β-GalNAc(S-5) H-1], 4.12[d, 1H, βGal(S-2), H-1],

3.6[m, 1H, βGlc(S-1), H-4], 1.23[d, 3H, αFucNAc(S-3), H-6].

54

13C NMR of Comound-A Capriose Acetate in CDCl3 at 300 MHz

89.68[1C, α-Glc(S-1) C-1], 91.66[2C, β-Glc(S-1) & α-FucNAc(S-3), C-1],

101.05[3C, β-Gal(S-2), β-Gal (S-4) & β-GalNAc(S-5) C-1]. 1H NMR of Comound-A Capriose in D2O at 300 MHz

5.57[d, 1H, J=3.0Hz, α-Glc(S-1) H-1], 4.64[d,1H, J=8.0Hz, β-Glc(S-1) H-1], 5.3[d,

1H, J=3.0Hz, α-FucNAc(S-3), H-1], 4.35[d,1H, J=8.0Hz, β-Gal(S-4) H-1 and β-

GalNac(S-5)], 1.91[S, 3H, 2(NHCOCH3), β-GalNAc(S-5)], 1.2[d, 3H, α-FucNAc(S-

3)].

ES Mass

879 [894-CH3], 862[879-OH], 833[862-CHO], 747[833-NHCOCH3], 691[894-S-5],

633[691-NHCOCH3], 616[633-OH], 585[616-CH2OH], 567[585-H2O], 674[691-

OH], 657[674-OH], 641[674-H2O, CH3], 609[641-CH2OH, H+], 529[691-S4],

342[488-S3], 324[342-H2O], 180[342-S2].

Compound B (ARSM-2) VIESOSE:

Compound b (419 mg) was obtained from fraction 36-50 of column chromatography

3. On deacetylation of 50 mg of substance b with NH3 / acetone it afforded substance

B (45.0 mg) ��

+72.02o (c 1% H2O). For experimental analysis, this compound was

dried over P2O5 at 100oC and 0.1 mm pressure for 8 hr. It gave positive Phenol-

sulphuric acid test, Feigl test and Morgan-Elson test.

C34H58O26N2 %C %H %N

Calculated 44.84 6.38 3.07

Found 44.83 6.39 3.08 1H NMR of Compound-B Viesose in CDCl3 at 300 MHz

δ 6.22[d, 1H, J= 3.0 Hz, α-Glc(S-1) H-1], 5.64[d,1H, J= 8.4 Hz, β-Glc(S-1) H-1 5.22

[d,1H, J = 8.0 Hz, β-GlcNAc(S-5), H-1)], 4.97[d, 1H, J=9.6 Hz, β-Gal(S-3) H-1],

4.56[d, 2H, J = 8.0 Hz, β-Gal(S-2), H-1, β-GalNAc(S-4) H-1], 4.01[d, 1H, βGal(S-3),

H-3], 3.8[m, 1H, βGlc(S-1), H-3], 3.72[d, 1H, βGal(S-3), H-2], 3.6[d, 1H, βGlc(S-1),

H-4]. 13C NMR of Compound-B Viesose in CDCl3 at 300 MHz

89.10[1C, α-Glc (S-1) C-1], 91.56[2C, β-Glc (S-1) C-1, β-Gal(S-3), C-1)], 101.97[1C,

β-GlcNAc (S-5) C-1], 101.84[2C, β-Gal(S-2), C-1, β-GalNAc (S-4) C-1].

55

1H NMR of Compound-B Viesose in D2O at 300 MHz

δ 5.2[d, 1H, J= 3.0 Hz, α-Glc(S-1) H-1], 4.6[d,1H, J=7.8 Hz, β-Glc(S-1), H-1)],

4.49[d, 1H, β-Glc(S-3), H-1)], 4.46[d, 1H, β-Gal(S-2), H-1)], 4.39[d, 2H, β-

GalNAc(S-4), H-1 and β-Gal (S-5) H-1], 1.94[s,3H, NHCOCH3 β-GalNAc(S-4)],

1.85[s, 3H, NHCOCH3, βGlcNAc(S-5)].

ES Mass

850[910-CH2OHCHO], 819[850-CH2OH], 761[819-NHCOCH3], 743[761-H2O],

726[743-OH], 707[H2O, H+], 879[910-CH2OH], 861[879-H2O], 821[879-

NHCOCH3], 789[821-CH3, OH], 803[821-H2O], 689[707-H2O], 672[689-OH],

614[672-NHCOCH3], 597[614-OH], 566[597-CH2OH], 535[566-CH2OH], 517[535-

H2O], 504[707-S-4], 342[504-S-3],180[342-S-2].

Compound-C (ARSM-3) ARIESOSE:

Compound-c (46 mg) was obtained from fraction 61-75 of column chromatography-4.

On deacetylation of 32 mg of substance c with NH3/ acetone it afforded substance C

(24.0 mg) ��


dried over P2O5 at 100oC and 0.1 mm pressure for 8 hr. It gave positive Phenol-

sulphuric acid test, Feigl test, Morgan-Elson test.

C36H61O26N3 % C % H % N

Calculated 45.43 6.42 4.41

Found 45.42 6.43 4.41 1H NMR of Compound-C Ariesose acetate in CDCl3 at 300 MHz

6.23[d, 1H, J=3.3Hz, α-Glc(S-1) H-1)], 5.64[d, 1H, J=8.4Hz, β-Glc(S-1) H-1], 4.58

[d, 1H, J=7.5 Hz, β-Gal(S-2), H-1)], 4.55[d, 1H, J=7.8Hz, β-GalNAc (S-5) H-1],

4.49[d, 2H, J= 8.0 Hz, β-GalNAc(S-3), H-1, GalNAc(S-4), H-1], 3.88[m, 2H,

βGalNAc(S-3, S-4), H-3], 3.83[d, 1H, βGlc(S-1), H-3], 3.6[d, 1H, βGlc(S-1), H-4]. 13C NMR of Compound-C Ariesose acetate in CDCl3 at 300 MHz

89.13[1C, α-Glc(S-1), C-1], 91.57[1C, β-Glc(S-1), C-1], 101.88[1C, β-Glc(S-2), C-

1], 102.02[1C, β-GalNAc(S-5), C-1], 100.92[1C, β-GalNAc(S-3), C-1], 101.19[1C, β-

GalNAc(S-4), C-1].

56

1H NMR of Compound-C Ariesose in D2O at 300 MHz

5.15[d, 1H, J=3.3 Hz, α-Glc(S1), H-1], 4.59[d, 1H, J=7.8Hz, β-Glc(S1), H-1], 4.47 [d,

1H, J= 7.5 Hz, β-Gal(S-2), H-1], 4.45[d, 1H, J=7.8 Hz, β-Gal(S-5), H-1], 4.38[d, 1H,

J= 8.0 Hz, β-GalNAc(S-3), H-1 and β-GalNAc(S-4), H-1].

ES Mass:

892 [951- CH2OHCHO (59)], 848 [892-CH3CO,H (44),], 893 [951-NHCOCH3 (58)],

916 [951- H2O, OH (35)], 730 [748- H2O (18), 687 [730-CH3O (43)], 686 [687- H

(1)], 668 [686-H2O (18)], 510 [545-H2O, OH (35)], 467 [510-CH3CO (43)], 487[545-

NHCOCH3 (58)], 405 [465-CH2OHCHO (60)].

Compound -D (ARSM-4) RIESOSE

Compound d (62 mg) was obtained from fraction 86-105 of column chromatography

4. On deacetylation of 52 mg of substance d with NH3 / acetone it afforded substance

D (44 mg) ��


dried over P2O5 at 100o C and 0.1 mm ��

+28.71ofor 8 hrs. It gave positive Phenol-

sulphuric acid test, Feigl test, Morgan-Elson test.

C40H68O31N2 %C %H %N

Calculated 44.84 6.38 3.08

Found 44.83 6.37 3.08 1H NMR of Compound-D Riesose acetate in CDCl3 at 300 MHz

δ 6.17[d, 1H, J=3.0, αGlc (S-1)], 5.37[d, 2H, βGlc (S-1) H-1 and αGlcNAc(S-3), H-

1)], 4.77[d, 1H, J=7.2 Hz, βGalNAc (S-5) H-1], 4.59 [d, 1H, J= 8.4 Hz, βGal(S-6),

H-1] 4.52[d, 2H, J=7.5 Hz, βGal(S-4), H-1 and βGal(S-2), H-1], 4.2[d, 1H, βGal(S-2),

H-2], 4.1[d, 1H, βGal(S-4), H-3], 3.8[d, 1H, βGlc(S-1), H-4, βGal(S-2), H-3], 3.46[d,

1H, βGalNAc(S-3), H-3]. 13C NMR of Compound-D Riesose acetate in CDCl3 at 300 MHz

90.24[ 1C, αGlc (S-1) C-1], 90.12[1C, βGlc (S-1) C-1], 90.12[1C, αGlcNAc (S-3) C-

1], 95.26[1C, βGalNAc (S-5), C-1)], 101.90[1C, β-Gal (S-6), C-1)], 101.05[1C, βGal

(S-4) C-1], 100.96[1C, βGal(S-2) C-1]. 1H NMR of Compound-D Riesose in D2O at 300 MHz

δ 5.16[d, 1H, J=3.0Hz, α-Glc(S-1), H-1], 4.60[d, 2H, J= 7.8 Hz, βGlc(S-1), H-1,

βGlcNAc(S-3), H-1], 4.52[d, 2H, J=7.8 Hz, β-Gal(S-4), β-Gal(S-6), H-1], 4.39[d,

2H, J=7.5 Hz, β-GalNAc(S-5), H-1)].

57

ES Mass

1014[1072-NHCOCH3], 1055[1072-OH], 1024[1055-CH2OH], 989[1014-OH],

956[989-CH2OH, 2H+], 910[1072-S-6], 892[910-H2O], 875[892-OH], 826[875-

H2O,CH2OH], 789[826-2H2O, H+], 750[826-CH2OCHO, OH], 709[CH2OH, H2O],

545[707-S-4], 527[545-H2O], 487[545-NHCOCH3], 427[487-CH2OHCHO],

342[545-S-3], 180[342-S-2].

Table: Description of isolated oligosaccharides from SHEEP milk

A B C D

Analytical notation ARSMM-1 ARSM-2 ARSM-3 ARSM-4

Name of compound Capriose Viesose Ariesose Riesose

Physical state Syrupy Syrupy Syrupy Syrupy

��

+41.01o +72.02o +28.71o +113.88o

Mol. Formula C34H58O25N2 C34H58O26N2 C36H61O26N3 C40H68O31N2

ES mass (m/z) 894 910 951 1072

Phenol-sulphuric test*1 +ve +ve +ve +ve

Morgon-Elson test*2 +ve +ve +ve +ve

Thiobarbituric acid test*3 -ve -ve -ve -ve

Bromo cresol green test*4 -ve -ve -ve -ve

*1 Test of normal sugar.

*2 Test of amino sugar

*3 Test of sialic acid

*4 Test of carboxylic acid.

CHAPTER III

RESULTS AND DISCUSSION

58

RESULTS AND DISCUSSION

COMPOUND-A CAPRIOSE

Compound A, C34H58O25N2, ��

� +41.01o gave positive Phenol-sulphuric acid test206,

Fiegl test207 and Morgan-Elson test208 showing the presence of normal and amino sugars

moietie(s) in the compound A. The HSQC spectrum of acetylated capriose showed the

presence of five cross peaks of six anomeric protons doublets and carbons in their

respective region at 6.37x89.68, 5.72x91.66, 5.40x91.66, 5.19x101.05, 4.48x101.05(2H),


six anomeric peaks of protons were separately confirmed by five 1H NMR doublets at δ

6.37(1H), 5.72(1H), 5.40(1H), 5.19(1H) 4.48(2H) in the acetylated spectrum of Capriose

in CDCl3 at 300 MHz. The presence of six anomeric carbons were confirmed by the

presence of three peaks at δ 89.68(1C), 91.66(2C), 101.05(3C) in the 13C NMR spectrum

of acetylated compound-A in CDCl3 at 300 MHz. These data suggested that compound

capriose may be a pentasccharides in its reducing form. In 1H NMR spectrum of

acetylated capriose out of 6 anomeric proton signals, signal at δ 6.37 and δ 5.72 were

assigned for downfield shifted α and β anomeric protons at the reducing end suggesting

that was in its reducing form and suggested that compound-A ‘capriose’ may be a

pentasccharide in reducing form. Further the ES mass spectrum of capriose showed the

highest mass ion peaks at m/z 956 assigned to [M+Na+K]+ and m/z 933 assigned to

[M+K] +, it also contain the molecular ion peak at m/z 894 confirming the molecular

weight as 894 which was in agreement with derived composition C34H58O25N2. The

reducing nature of compound-A was further confirmed by its methylglycosylation


glucosides, suggesting the presence of glucose at the reducing end, for convenience all

five monosaccharides were denoted as S-1, S-2, S-3, S-4 and S-5. The monosaccharides

constituents in compound-A were confirmed by its killiani hydrolysis147 under strong

acidic condition, followed by paper chromatography and TLC. In this hydrolysis four

spots were found identical with the authentic samples of Glc, Gal, GlcNac and FucNac by

co-chromatography. Thus the pentasaccharide contained four types of monosaccharides

units i.e. Glc, Gal, GlcNac and FucNac. The pentasccharide nature of capriose was

60

further supported by five anomeric proton doublets of five protons δ 5.57(1H), 4.59(1H)

5.3 (1H), 4.35(2H) along with two methyl (NHCOCH3) signal at δ 1.94 (2NHAc) in the

300 MHz NMR spectrum of capriose in D2O. Further the presence of two anomeric

protons signals at δ 5.57 (J=4.0 Hz) and δ 4.59 (J=8.0 Hz) in the 1H NMR spectrum of

capriose in D2O at 300 MHz were assigned for α and β anomers of glucose (S-1),

confirming the presence of Glc(S-1) at the reducing end209, 210 in compound-A. The

anomeric proton doublet for Gal(S-2) could not be identified in 1H NMR of capriose as it

was merged with the signal of D2O however it was present at δ 5.19 (J=8.7 Hz) in the 1H

NMR of capriose in CDCl3 at 300 MHz. In addition to above signals presence of a triplet

at δ 3.21 which was assigned H-2 of βGlc(S-1) along with earlier described anomeric

proton doublets at δ 5.54 and 4.59 for α and β glucose (Structure reporter group)211,212

suggested the presence of lactose type213 structure i.e. β-Gal(1-4)→Glc) linkage at the

reducing end of capriose. The anomeric signal at δ 5.72 assigned to β-Glc(S-1) gave three

cross peaks at δ 5.72x5.12, 5.12x3.84 and 3.84x3.6 in the TOCSY spectrum of capriose

acetate in CDCl3 at 300 MHz. Which was later assigned for H-2, H-3 and H-4

respectively by COSY spectrum. The chemical shift of cross peak of H-3 and H-4 at δ

3.84 and 3.60 respectively confirmed that H-3 and H-4 of S-1 was linked glycosidicaly

by the next monosaccharide unit. Since H-4 of S-1 was already assigned for linkage with

Gal(S-2) and hence only H-3 position was left for glycosidic linkage by the next

monosaccharide unit. The next anomeric proton doublet which appeared at δ 5.4

(J=3.0Hz) along with a singlet of amide methyl at δ 1.94 and a secondary methyl doublet

at δ 1.12 was due to the presence of α-FucNAc moiety in the 1H NMR of capriose acetate

in CDCl3 at 300 MHz . As already suggested by the COSY spectrum of capriose acetate

that position 3 and 4 of Glc(S-1) were vacant for glycosidic linkages and position 4 was

already occupied by Gal(S-2), so FucNAc must be linked to H-3 of S-1. This linkage was

further supported by the presence of 1H NMR signal of acetylated capriose in which the

signal for H-3 of S-1 appeared at δ 3.84 confirming the 1→3 linkage between S-3 and S-

1. Smaller coupling constant (J=3.0 Hz) of anomeric proton at δ 5.4 confirmed α

glycosidic linkage in it. Further the anomeric proton signal of β-Gal (S-2) at δ 5.192

showed two consequent complementary signals in the linkage region at δ 4.1 and 3.86 in

the TOCSY spectrum of capriose acetate in CDCl3 at 300 MHz. Later these signals were

61

Fig- HSQC spectrum of Capriose acetate in CDCl3 at 300 MHz

62

OAcO

OAc

NHAc

S-5

AcO

O

O

OAc

S-2

AcO

O

OAc

S-4

OAc O

AcO

OAc

O

OAc

S-1OO OAc

OAc

O

OAc

H3C

AcO

NHAcS-3

63

OAcO

OAc

NHAc

S-5

AcO

O

O

OAc

S-2

AcO

O

OAc

S-4

OAc O

AcO

OAc

O

OAc

S-1OO OAc

OAc

O

OAc

H3C

AcO

NHAcS-3

64

OHO

OH

NHAc

S-5

HO

O

O

OH

S-2

HO

O

OH

S-4

OH O

HO

OH

O

OH

S-1OO OH

OH

O

OH

H3C

HO

NHAcS-3

65

OA cO

OAc

NHAc

S-5

AcO

O

O

OAc

S-2

AcO

O

OAc

S-4

OAc O

AcO

OAc

O

OAc

S-1OO OAc

OAc

O

OAc

H3C

AcO

NHAcS-3

66

OAcO

OAc

NHAc

S-5

AcO

O

O

OAc

S-2

AcO

O

OAc

S-4

OAc O

AcO

OAc

O

OAc

S-1OO OAc

OAc

O

OAc

H3C

AcO

NHAcS-3

67

Fig-HMBC spectrum of capriose acetate in CDCl3 at 300 MHz

OAcO

OAc

NHAc

S-5

AcO

O

O

OAc

S-2

AcO

O

OAc

S-4

OAc O

AcO

OAc

O

OAc

S-1OO OAc

OAc

O

OAc

H3C

AcO

NHAcS-3

68

Table-1: 1H NMR values of CAPRIOSE in D2O at 300 MHz

identified as H-2 and H-3 of β-Gal (S-2) by the COSY spectrum of capriose aceteate

suggesting that H-2 and H-3 of S-2 were available for glycosidic linkage by the next

monosaccharide units. Another anomeric proton signal which appeared as doublet at δ

4.48 (J=7.2 Hz) along with a singlet of amide methyl was due to the presence of β-

GalNAc moiety. The anomeric proton signal present at δ 4.48 has its complementary

signal at 101.05 in HSQC spectrum of capriose acetate. The anomeric carbon further

gave cross peak at 3.86 in HMBC spectrum of capriose acetate confirming the 1→3

linkage between S-5 and S-2. The large coupling constant of anomeric signal (S-5) with J

value 7.2 Hz confirmed the β-configuration of the β-GalNAc (S-5). The absence of

methine protons in linkage region of β-GalNAc (S-5) confirm that β-GalNAc (S-5) was

present at non-reducing end, which was confirmed by the TOCSY and COSY

experiments of capriose acetate in CDCl3 at 300 MHz. Since it was ascertained by the

COSY and TOCSY spectrum of capriose acetate that the position of H-2 and H-3 of

Gal(S-2) were available for glycosidic linkages and position H-3 of Gal(S-2) was already

linked with β-GalNAc (S-5), the left over H-2 position of Gal (S-2) must be linked with

β-Gal (S-4) which was further confirmed by the appearance of H-2 signal of S-2 at δ 4.1

in 1H NMR of capriose acetate at 300 MHz. The next anomeric proton signal which

appeared at δ 4.88 (J=7.2 Hz) was due to the presence of β-Gal (S-4) moiety. Further the

presence of a double doublet at δ 4.1 in the 1H NMR of capriose acetate which has its

complimentary signal at δ 70 in the HSQC spectrum of capriose acetate suggested that

the H-2 of S-2 was vacant for glycosidic linkage and was glycosidicaly linked to S-4.

The large coupling constant of anomeric signal of (S-4) with J value 7.2 Hz confirmed

Moieties 1H NMR (δ) Coupling cons. (J)

α-Glc (S-1) β-Glc (S-1)

β-Gal (S-2)

β-FucNAc (S-3) β -Gal (S-4) β-GalNAc (S-5)

5.57 4.59 n.d. 5.3 4.35 4.35

4.0 Hz 8.0 Hz n.d.

3.0 Hz 8.0 Hz 8.0 Hz

69

the β 1→2 glycosidic linkage between S-4 and S-2. None of the methine proton of S-4

was present in any cross peak into linkage region in the TOCSY spectrum of capriose

acetate so it was confirm that S-4 was present at non reducing end and none of its OH

group was available for glycosidic linkage. All the 1H NMR assignments for ring protons

of monosaccharide units of capriose were confirmed by HOMOCOSY214, 215 and

TOCSY216 experiments. The positions of glycosidation in the oligosaccharide were

confirmed by position of anomeric signals, S.R.G. and comparing the signals in 1H and 13C NMR of acetylated and deacetylated oligosaccharide. The glycosidic linkages in

capriose were assigned by the cross peaks for glycosidically linked carbons with their

protons in the HSQC217, 218 spectrum of acetylated Capriose. The values of these cross

peaks appeared as- β-Glc (S-1) H-4 and C-4 at 3.6x65.7 showed (1→4) linkage between

S-2 and S-1, β-Glc (S-1) H-3 and C-3 at 3.84x72.6 showed (1→3) linkage between S-3

and S-1, β-Gal (S-2) H-2 and C-2 at δ 3.9 x70.09 showed (1→2) linkage between S-4

and S-2, β-Gal (S-2) H-3 and C-3 at δ 4.01x70 showed (1→3) linkage between S-5 and

S-2. All signals obtained in 1H and 13C NMR of compound Capriose were in conformity

with the assigned structure and their position were confirmed by 2D 1H-1H COSY,

TOCSY and HSQC experiments. Thus based on the pattern of chemical shifts of 1H, 13C,

COSY, TOCSY and HSQC NMR experiments it was interpreted that the compound was

a pentasaccharide having structure as-

The Electronspray Mass Spectrometry data of Capriose not only confirmed the

derived structure but also supported the sequence of monosaccharide in Capriose. The

highest mass ion peaks were recorded at m/z 956 and 933 which were due to [M+Na+K]

and [M+K] respectively. It also contains the molecular ion peak at m/z 894 confirming

the molecular weight of Capriose as 894 and was in agreement with its molecular

formula. Further the mass fragments were formed by repeated H transfer in the

oligosaccharide and was accompanied by the elimination of terminal sugar less water.

74

The pentasaccharide m/z 894 (I) fragmented to give mass ion at m/z 691(II) [894-S5], this

fragment was arised due to the loss of terminal β-GlcNAc(S5) moiety from

pentasaccharide indicating the presence of β-GlcNAc(S5) at the non -reducing end. It

further fragmented to give mass ion peak at m/z 488 (III) [894-S4] which was due to loss

of β-Gal (S-4) moiety from tetrasaccharide. This fragment of 488 further fragmented to

give mass ion peak at m/z 342 (IV) [488-S3] which was due to loss of α-Fuc (S3) moiety

from the trisaccharide. This disaccharide unit again fragmented to give mass ion peak at

m/z 180(V) [342-S2], which was due to loss of Gal (S2) moiety from disaccharide. These

four mass ion peak II, III, IV and V were appeared due to the consequent loss of S-5, S-4,

S-3 and S2 from original molecule. The mass spectrum also contain the mass ion peak at

are m/z 366, 539, and 529 correspond to the mass ion fragment A, B, C, Which confirm

the position of S1, S2, S3 ,S4 and S5. The other fragmentation pathway in ES Mass spectrum

of compound A m/z 894 shows the mass ion peak at 879 [894-CH3], 862[879-OH],

833[862-CHO], 747[833-NHCOCH3], 691[894-S-5], 633[691-NHCOCH3], 616[633-

OH], 585[616-CH2OH], 567[585-H2O], 674[691-OH], 657[674-OH], 641[674-H2O,

CH3], 609[641-CH2OH, H+], 529[691-S4], 342[488-S3], 324[342-H2O], 180[342-S2].

Based on result obtained from chemical degradation/acid hydrolysis, Chemical

transformation, Electro spray mass spectrometry and 1H, 13C NMR and HOMOCOSY,

TOCSY, HMBC and HSQC 2D NMR techniques of acetylated Capriose and Capriose

the structure and sequence of isolated Novel oligosaccharide Capriose structure was

deduced as-

CAPRIOSE

75

COMPOUND-B VIESOSE

Compound B, C34H58O26N2, ��



moietie(s) in the compound C. The HSQC spectrum of acetylated viesose showed the

presence of five cross peaks for six anomeric protons and six anomeric carbons in their

respective region at 6.22x89.10, 5.64x91.56, 4.976x91.56, 5.22x101.97,

4.56x100.84(2H), suggesting the presence of six anomeric doublet and carbon in it.

Further the presence of six anomeric peaks of anomeric protons were confirmed by

presence of five doublets at δ 6.22(1H), 5.64(1H), 5.22(1H), 4.97(1H), 4.56(2H) in the 1H

NMR spectrum of acetylated viesose in CDCl3 at 300 MHz. The presence of six

anomeric carbons were confirmed by the presence of four signals of anomeric carbons at

δ 89.10(1C), 91.56(2C), 100.97(1C), 100.84(2C) in 13C spectrum of viesose acetate in

CDCl3 at 300 MHz. These data suggested that viesose may be a pentasccharides in its

reducing form. In 1H NMR spectrum of acetylated viesose out of 6 anomeric proton

signal, signal at δ 6.22 and 5.64 contained downfield shifted α and β anomeric protons

suggested that it was in its reducing form and compound-B ‘viesose’ may be a

pentasccharide in its reducing form. Further the ES mass spectrum of viesose showed the

highest mass ion peaks at m/z 972 assigned to [M+Na+K]+ and m/z 949 assigned to

[M+K] +, it also contain the molecular ion peak at m/z 910 confirming the molecular

weight as 910 which was in agreement of derived composition i.e. C34H58O26N2. The

reducing nature of compound-B viesose was confirmed by its methylglycosylation


glucosides, suggesting the presence of glucose at the reducing end, for convenience all

five monosaccharides were denoted as S-1, S-2, S-3, S-4 and S-5 respectively from the

reducing end. The monosaccharides constituents in compound-B were confirmed by its

killiani hydrolysis147 under strong acidic condition, followed by paper chromatography

and TLC. In this hydrolysis four spots were found identical with the authentic samples of

Glc, Gal, GlcNac and GalNac by co-chromatography. Thus the pentasaccharide

contained four types of monosaccharides units i.e. Glc, Gal, GlcNac and GalNac. The

pentasccharide nature of viesose was further supported by the presence of five anomeric

proton doublets for six anomeric protons at δ 5.20(1H), 4.6(1H), 4.49(1H), 4.46(1H),

77

4.39(2H) in 1H NMR spectrum of viesose in D2O at 300 MHz, it also contain a singlet of

six protons for (NHCOCH3) group at δ 1.94 also supported by its 13C NMR in D2O which

contain two peaks for carbonyl (C=O) of NHCOCH3 at δ 173.67 and 177.39 confirming

the presence of the NHCOCH3 group in Compound-B. Presence of two anomeric protons

signals at δ 5.20 (J=3.0 Hz) and δ 4.6 (J=7.8 Hz) in the 1H NMR spectrum of viesose in

D2O at 300 MHz were assigned for α and β anomers of glucose (S-1), confirming the

presence of Glc(S-1) at the reducing end209, 210 in compound-B. Further the presence of

another anomeric doublet at δ4.56 (J=8.0 Hz) showed the presence of β-Gal residue as

the next monosaccharide. In addition to above signals presence of a triplet at δ 3.20 which

was assigned for H-2 of βGlc(S-1) along with earlier described anomeric proton doublets

at δ 5.2 and 4.6 for α and β glucose (Structure reporter group)211, 212 suggested the

presence of lactose213 type structure i.e. β-Gal(1-4)→Glc) linkage at the reducing end of

viesose. The anomeric proton doublet at δ 5.64 assigned to β-Glc(S-1) in the TOCSY

spectrum of viesose acetate gave three cross peaks i.e. at δ 3.6, 3.80 and 5.02 out of

which signals present at δ 3.6 and δ 3.80 were predicted for the linkage region which was

later assigned as H-4 and H-3 respectively of β-Glc(S-1) by the COSY spectrum of

viesose actetate confirming that H-4 and H-3 of β-Glc (S-1) was involved in glycosidic

linkage. Therefore it was confirmed that H-4 and H-3 β-Glc(S-1) were linked with next

monosaccharide units. As described earlier the presence of lactose type structure i.e. β-

Gal (S-2) was glycosidicaly linked by 1→4 linkage with the reducing Glc and it was

confirmed by the chemical shift of H-4 of S-1 at δ 3.60 in 1H NMR of acetylated viesose.

The 1→4 linkage between S-1 and S-2 was further confirmed by HMBC spectrum of

viesose acetate at 300 MHz which contain the cross peak signal of H-4 of Glc(S-1) and

C-1 of Gal(S-2) at δ 3.60x101.84. Since the anomeric proton of β-Gal(S-2) was appeared

at δ 4.56 in the 1H NMR of viesose acetate at 300 MHz and this anomeric signal in its

TOCSY spectrum does not gave any cross peak with methine proton in linkage region

confirmed that β-Gal(S-2) was present at non-reducing end and none of its OH group was

glycosidically linked. Since H-4 of S-1was already assigned for linkage with Gal(S-2)

and hence only H-3 position was Left for glycosidic linkage by the next monosaccharide

unit. The next anomeric proton signal which appeared at δ 4.97 (J=9.6 Hz) in 1H NMR of

compound viesose in CDCl3 was assigned for β-Gal(S-3).

78

Fig- HSQC Spectrum of Viesose in CDCl3 at 300 MHz

O

O

OOAc

OAc

O

AcO

AcOOAc

OAcO

O

O

S-1S-2

S-3

O

AcO

AcO

NHAc

S-4

O

AcO

AcONHAc

S-5

OAcOAc

OAc

OAc

OAc

79

O

O

OOAc

OAc

O

AcO

AcOOAc

OAcO

O

O

S-1S-2

S-3

O

AcO

AcO

NHAc

S-4

O

AcO

AcONHAc

S-5

OAcOAc

OAc

OAc

OAc

80

O

O

OOAc

OAc

O

AcO

AcOOAc

OAcO

O

O

S-1S-2

S-3

O

AcO

AcO

NHAc

S-4

O

AcO

AcONHAc

S-5

OAcOAc

OAc

OAc

OAc

81

Table-2: 1H NMR values of VIESOSE in D2O at 300 MHz

The coupling constant of anomeric signal β-Gal (S-3) with larger value of 9.0 Hz showed

the β configuration of the β-Gal(S-3). This linkage was further confirmed by the presence

of 1H NMR of acetylated viesose in which the signal for H-3 of S-1 was appeared at δ

3.80 confirming the 1→3 linkage between S-1 and S-3. Further the anomeric proton

signal of β-Gal(S-3) at δ 4.97 in the 1H NMR of viesose acetate in CDCl3 showed three

consequent complementary signals at δ 3.72, 4.01 and 4.35 respectively in TOCSY

spectrum of viesose acetate, out of which two signals at δ 3.72 and δ 4.01were assigned

for the linkage by the next monosaccharide units. Later These signals were identified as

H-2 and H-3 of β-Gal(S-3) by the COSY spectrum of viesose acetate showing that two

OH groups of β-Gal(S-3) were available for glycosidic linkage by the next

monosaccharide units. The next anomeric proton which appeared at δ5.22 (J=8.0 Hz)

along with a singlet of amide methyl at δ 1.9 was due to presence of β-GlcNAc moiety in

the pentasaccharide was identified as β-GlcNAc (S-5). The anomeric proton signal of S-5

at δ 5.22 gave its complimentary signal at δ 101.97 in the HSQC spectrum which further

gave cross peak with 1H NMR signal at 4.10 in the HMBC spectrum of viesose acetate

suggesting the 1→3 linkage between S-5 and S-3. The position of linkage H-3 of S-3 was

confirmed by the H-3 signal at δ 4.01 in the 1H NMR spectrum of compound viesose.

Since the anomeric proton of β-GlcNAc(S-5) at δ 5.22 in TOCSY spectrum of compound

viesose acetate does not show any methine proton signal in linkage region confirming

that β-GlcNAc(S-5) was present at non-reducing end and none of its OH group was

available for glycosidic linkage. The next anomeric proton signal which appeared as a

doublet at δ 4.56 (J=8.0 Hz) in pentasaccharide was due to the presence of β-GalNAc



β-Gal (S-2)

β-Gal (S-3) β -GalNAc (S-4) β-GlcNAc (S-5)

5.2 4.6 4.39 4.46 4.39 4.49

3.0 Hz 7.8 Hz 8.0 Hz. 9.0 Hz 8.0 Hz 8.0 Hz

82

O

O

OOAc

OAc

O

AcO

AcOOAc

OAcO

O

O

S-1S-2

S-3

O

AcO

AcO

NHAc

S-4

O

AcO

AcONHAc

S-5

OAcOAc

OAc

OAc

OAc

83

O

O

OOA c

O A c

O

A cO

A cOO A c

OA cO

O

O

S-1S-2

S-3

O

A cO

A cO

N H A c

S-4

O

A cO

A cON HA c

S-5

O A cOA c

O A c

OA c

O A c

84

O

O

OOH

OH

O

HO

HOOH

OHO

O

O

S-1S-2

S-3

O

HO

HO

NHAc

S-4

O

HO

HONHAc

S-5

OHOH

OH

OH

OH

85

Fig-HMBC Spectrum of Viesose acetate in CDCl3 at 300 MHz

O

O

OOAc

OAc

O

AcO

AcOOAc

OAcO

O

O

S-1S-2

S-3

O

AcO

AcO

NHAc

S-4

O

AcO

AcONHAc

S-5

OAcOAc

OAc

OAc

OAc

86

moiety (S-4). As already suggested by TOCSY spectrum of viesose that position 2 and 3

of Gal(S-3) were vacant for glycosidic linkages and position 3 was already occupied by

GlcNAc(S-5), and leftover position of H-2 of Gal (S-3) may be linked with β-GalNAc(S-

4). The linkage between S-4 and S-3 was further supported by the presence of 1H NMR

signal of acetylated viesose in which the signal for H-2 of S-3 appeared at δ 3.72

confirming the 1→2 linkage between S-4 and S-3. The position of GalNAc(S-4) was also

confirmed at non-reducing end by TOCSY spectrum of viesose in which the anomeric

proton signal of GalNAc (S-4) at δ 4.56 does not contain any of its ring protons in

glycosidic region. The pentasaccharide nature of compound viesose was further

confirmed by the spectral studies of acetylated derivative of this compound. The

heteronuclear single quantum coherence (HSQC)217, 218 spectrum of acetylated compound

confirmed the position of glycosidic linkages by cross peaks of β-Glc(S-1) H-4 and C-4

at (δ 3.6x72.50) showed 1→4 linkage of S-2 and S-1 and β-Glc(S-1) H-3 and C-3 at (δ

3.8x73.1) showed 1→3 linkage of S-3 and S-1i.e. its H-4 and H-3 positions of Glc(S-1)

was involved in linkage. β-Gal(S-3) H-2 and C-2 at (δ 3.72x72.40) showed 1→2 linkage

of S-4 and S-3, β-Gal(S-3) H-3 and C-3 showed 1→3 linkage of S-5 and S-3. These

chemical shifts obtained from cross peaks of HSQC were consistent with the COSY and

TOCSY. On the basis of 1H NMR assignments for ring protons of monosaccharide units

of compound-B were confirmed by HOMOCOSY214, 215 and TOCSY216 experiments it

was interpreted that the compound-B ‘viesose’ was pentasaccharide having the structure:

The Electronspray Mass spectrometry data of Compound-B not only confirmed

the derived structure but also supported the sequence of monosaccharide units in

compound-B. The highest mass ion peaks were recorded at m/z 972 and 949 which were

due to [M+Na+K] and [M+K] respectively. It also contains the molecular ion peak at m/z

910 confirming the molecular weight of compound-B viesose as 910 and was agreement

with its molecular formula i.e. C34H58O26N2. Further the mass fragments were formed by

repeated H transfer in the oligosaccharide and were accompanied by the elimination by-

91

repeated sugar less water. The pentasaccharide m/z 910 (I) fragmented to give mass ion

at m/z 707 (II) [910-S-5], this fragment was arises due to the loss of terminal βGlcNAc

(S-5) moiety from pentasaccharide indicating the presence of βGlcNAc (S-5) at non-

reducing end. It was further fragmented to give mass ion peak at m/z 504 (III) [707-S-4]

which was due to loss of βGalNAc S-4 moiety from tetrasaccharide. This fragment of

504 further fragmented to give mass ion peak at m/z 342 (IV) [504-S3] which was due to

loss of βGal (S-3) moiety from the trisaccharide, which further fragmented to give mass

ion peak at m/z 180 [342-S-2] which was due to loss of βGal S-2 moiety from

disaccharide. These Four mass ion peak II, III, IV, V were appeared due to the

consequent loss of S-5, S-4, S-3 and S-2 from original molecule. The mass spectrum also

contain the mass ion peak at are m/z 342, 545, 586 and 707 correspond to the mass ion

fragments A, B, C and D which confirm the position of S-1, S-2, S-3, S-4 and S-5. The

other fragmentation pathway in ES Mass spectrum of compound-B m/z 910 shows the

mass ion peak 850[910-CH2OHCHO], 819[850-CH2OH], 761[819-NHCOCH3],

743[761-H2O], 726[743-OH], 707[H2O, H+], 879[910-CH2OH], 861[879-H2O], 821[879-

NHCOCH3], 789[821-CH3, OH], 803[821-H2O], 689[707-H2O], 672[689-OH], 614[672-

NHCOCH3], 597[614-OH], 566[597-CH2OH], 535[566-CH2OH], 517[535-H2O],

504[707-S-4], 342[504-S-3],180[342-S-2]. Based on result obtained from chemical

degradation/acid hydrolysis, chemical transformation, electro spray mass spectrometry

and 1H, 13C and 2D NMR techniques of acetylated viesose and viesose the structure and

sequence of isolated novel oligosaccharide viesose was deduced as-

VIESOSE

92

COMPOUND-C ARIESOSE

Compound C, C36H61O26N3, ��



moietie(s) in the compound C. The HSQC spectrum of acetylated Ariesose showed the

presence of six cross peaks of anomeric protons and carbons in their respective region at

6.23x89.13, 5.64x91.57, 4.58x101.88, 4.55x102.02, 4.49x100.92, 4.49x101.19

suggesting the presence of six anomeric protons and carbons into it. Presence of five

anomeric peaks for six protons doublets were separately confirmed by 1H NMR of

acetylated ariesose at 300 MHz i.e. δ 6.23(1H), 5.64(1H), 4.58(1H), 4.55(1H), 4.49(2H).

The presence of six carbons were also confirmed by the presence of six anomeric carbons

peaks at δ 89.13(1C), 91.57(1C), 101.88(1C), 102.02(1C), 100.92(1C), 101.19(1C) in the

acetylated spectrum of Ariesose at 300 MHz. Further the anomeric proton singlet at δ6.23

and δ5.64 in the 1H NMR of acetylated compound-C showed downfield shifted α and β

anomeric protons showing its was in its reducing form and suggested that compound

Ariesose may be a pentasccharide in its reducing form. Further the ES mass spectrum of

Ariesose showed the highest mass ion peaks at m/z 1013 assigned to [M+Na+K]+ and

m/z 990 assigned to [M+K]+, it also contain the moleculer ion peak at m/z 951

confirming the moleculer weight of ariesose was 951 which was in agreement to derived

composition i.e.C36H61O26N3. The reducing nature of compound was further confirmed

by methylglycosylation MeOH/H+ followed by its acid hydrolysis, which led to isolation

of α and β- methyl glucosides, suggesting the presence of glucose at the reducing end, for

convenience all five monosaccharides were denoted as S-1, S-2, S-3, S-4 and S-5. The

monosaccharides constitute in compound–C were confirmed by its killiani147 hydrolysis



GlcNac and GalNac by co-chromatography. Thus the pentasaccharide contained four

types of monosaccharides units i.e. Glc, Gal, GlcNac and GalNac. The pentasccharide

nature of Ariesose was further supported by five anomeric peaks for six protons doublets

i.e. δ5.15 (1H), δ4.59 (1H), δ4.47 (1H), δ4.44 (1H), 4.38 (2H) in 1H NMR spectrum of

Ariesose in D2O at 300 MHz. The 1H NMR also contain three methyl signals of

NHCOCH3 at δ 1.85 and δ 2.01(2NHAc) showing out of five

94

three monosaccharides was N-acetylated sugars. Further the presence of two anomeric

protons signals at δ 5.15 (J=3.3 Hz) and δ 4.59 (J=7.8 Hz) in the 1H NMR spectrum of

Ariesose in D2O at 300 MHz were assigned for α and β anomers of glucose (S-1),

confirming the presence of Glc(S-1) at the reducing end209, 210 in compound-C ariesose.

Further the presence of another anomeric doublet at δ4.58 (J=7.5 Hz) suggested the

presence of β-Gal residue as the next monosaccharide unit. In addition to above signals

presence of a triplet at δ 3.20 which was assigned for H-2 of βGlc(S-1) along with earlier

described anomeric proton doublets at δ 5.15 and 4.59 for α and β glucose (Structure

reporter group)211, 212 suggested the presence of lactose213 type structure i.e. β-Gal(1-

4)→Glc linkage at the reducing end of ariesose. The anomeric proton doublet present at δ

5.64 assigned to β-Glc(S-1) in the TOCSY spectrum of ariesose acetate gave three cross

peaks at δ 3.6, 3.84 and 5.02 respectively out of which signal present at δ 3.6 and δ 3.84

were suggested in linkage region which was further assigned as H-4 and H-3 of β-Glc(S-

1) by the COSY spectrum showing that H-4 and H-3 of β-Glc (S-1) were available for

glycosidic linkage by next monosaccharide units. The earlier suggested 1→4 linkage

between Glc(S-1) and Gal(S-2) was further confirmed by HMBC spectrum at 300 MHz

which contain the cross peak signal of H-4 of S1 and C-1of β-Gal(S-2) at δ 3.6x101.88.

As suggested by the TOCSY spectra ariesose acetate that anomeric proton present at δ

4.58 assigned to βGal(S2) does not showed any cross peak in the linkage region

confirming that β-Gal(S-2) was present at non-reducing end and none of its OH group

were available for glycosidic linkage. Since the S-1 has its vacant positions i.e. H-3 and

H-4 and it was confirmed the H-4 was linked with Gal(S-2) therefore the H-3 of S-1 was

vacant and was available for glycosidic linkage by the next monosaccharide unit. The

next anomeric proton signal which appeared at δ 4.49 (J=8.0 Hz) in CDCl3 along with a

singlet of amide methyl (-NHCOCH3) at δ 1.85 in the 1H NMR of Ariesose in D2O was

assigned as β-GalNAc(S-3). The coupling constant of anomeric signal β-GalNAc (S-3)

with larger value of 8.0 Hz showed that β configuration of the β-GalNAc(S-3). The

linkage between S-1 and S-3 was ascertained by the presence of 1H NMR signal present

in acetylated Ariesose in which the signal for H-3 of S-1 appeared at δ3.84 which was

confirmed by the TOCSY and COSY spectrum. The 1→3 linkage between S-1 and S-3

95

Table-1: 1H NMR values of ARIESOSE in D2O at 300 MHz

was further confirmed by HMBC spectrum in acetylated Ariesose from cross peak signal

of β-Glc(S-1) H-3 and β-GalNAc(S-3) C-1 at δ3.83x100.92 at 300 MHz. Further the

anomeric proton signal of β-GalNAc(S-3) at δ 4.49 in the 1H NMR of acetate in CDCl3

was showed two consequent complementary signals in the linkage region at δ 4.14 and δ

3.85 in TOCSY spectrum out of which signal at δ 4.14 was assigned to H-2 of S-3 in

NHCOCH3 while the signal of 3.85 was assigned to glycosidic position of (S-3), showing

that one OH groups of β-GalNAc(S-3) was available for Glycosidic linkage. This signal

was identified for H-3 of β-GalNAc(S-3) by the COSY spectrum of Ariesose acetate,

suggesting that he H-3 postion of β-GalNAc(S-3) was linked with next monosaccharide

units. The next anomeric proton which also appeared at δ4.49 (J=8.0 Hz) along with a

singlet of amide methyl (NHCOCH3) at δ 2.01 in the 1H NMR of Ariesose was due to the

presence of β-GalNAc(S-4). The coupling constant of anomeric signal β-GalNAc (S-4)

with larger value of 8.0 Hz showing that β configuration of the β-GalNAc(S-4). The

linkage between S-3 and S-4 was ascertained by the presence of 1H-NMR signal in

acetylated Ariesose spectrum in which the signal for H-3 of S-3which was the only

position vacant at S3 appeared at 3.85 which was confirmed by the TOCSY and COSY in

CDCl3 at 300 MHz. The 1→3 linkage between S-3 and S-4 was further confirmed by the

cross peak of β-GalNAc(S-3)H-3 and β-GalNAc(S-4)C-1 at 3.851x101.19 in HMBC

spectrum of acetylated Ariesose at 300 MHz. The anomeric proton signal of β-

GalNAc(S4) which was also at δ 4.49 in the 1H NMR of acetate in CDCl3 was showed

two consequent complementary signals in the linkage region at δ 4.14 and δ 3.85 in

TOCSY spectrum out of which signal at δ 4.14 was assigned to H-2 of S-3 while the


α-Glc (S-1)

β-Glc (S-1)

β-Gal (S-2)

β-GalNAc (S-3) β -GalNAc (S-4) β-GlcNAc (S-5)

5.15 4.59 4.47 4.45 4.38 4.38

3.3 Hz 7.8 Hz 7.5 Hz. 7.8 Hz 8.0 Hz 8.0 Hz

96

signal of 3.85 was assigned to glycosidic linkage of Areisose showing that one OH

groups of β-GalNAc(S-4) was available for Glycosidic linkage. This signal was further

identified for H-3 of β-GalNAc(S-4) by the COSY spectrum of Ariesose acetate in CDCl3

at 300 MHz. The H-3 postion of β-GalNAc(S-4) linked with next monosaccharide unit.

Another anomeric proton signal which appeared as a doublet at δ 4.55 (J=7.8 Hz) in 1H

NMR spectrum of Ariesose in CDCl3 along with singlet of amide methyl (NHCOCH3) at

δ 2.001was due to β-GalNAc(S-5). The1→3 linkage between S-4 and S-5 Further

confirmed by the cross peak signal of β-GalNAc(S-4)H-3 and β-GalNAc(S-5)C-1 at δ

3.85x102.02 by HMBC spectrum of acetylated ariesose at 300 MHz. Further the absence

of all the methine protons in linkage region of β-GalNAc(S-5) confirmed that β-

GalNAc(S-5) was present at non-reducing end and none of the OH group was available

for glycosidic linkage, which was confirmed by TOCSY and COSY in acetylated

ariesose at 300 MHz. All the 1H NMR assignments for ring protons of monosaccharide

units of Ariesose were confirmed by HOMOCOSY214, 215 and TOCSY216 experiments.

The positions of glycosidation in the oligosaccharide were confirmed by position of

anomeric signals, S.R.G. and comparing the signals in 1H and 13C NMR of acetylated and

deacetylated oligosaccharide. The glycosidic linkages in Ariesose were assigned by the

cross peaks for glycosidically linked carbons with their protons in the HSQC217, 218

spectrum of acetylated Ariesose. The values of these cross peaks appeared as- β-Glc (S-

1) H-4 and C-4 at (3.6x82) showed 1→4 linkage of S-2 and S-1 and β-Glc (S-1) H-3 and

C-3 at (3.83x73.50) showed 1→3 linkage i.e. its H-4 and H-3 positions of Glc(S-1) was

involved in linkage region. β-GalNAc(S-3) H-3 and C-3 at (3.85x73) showed 1→3

linkage between S-4 and S-3 i.e. its H-3 position of β-GalNAc(S-3) was involved in

linkage region. β-GalNAc(S-4) H-3 and C-3 at (3.85x73) showed 1→3 linkage between

S-5 and S-4 i.e. its H-3 position of β-GalNAc(S-4) was involved in linkage region. On

the basis of above data, it was interpreted that the compound-C was pentasaccharide

having the structure:

97

Fig-HSQC Spectrum of Ariesose acetate in CDCl3 at 300 MHz

OO O

OAcO OAc

OAcOAcOAc

AcO

OAc

O

OAcAcO

O

NHAc

O

OAcAcO

O

NHAc

O

OAcAcO

AcO

NHAc

S-1S-2

S-3S-4S-5

98

OO O

OAcO OAc

OAcOAcOAc

AcO

OAc

O

OAcAcO

O

NHAc

O

OAcAcO

O

NHAc

O

OAcAcO

AcO

NHAc

S-1S-2

S-3S-4S-5

99

OO O

OAcO OAc

OAcOAcOAc

AcO

OAc

O

OAcAcO

O

NHAc

O

OAcAcO

O

NHAc

O

OAcAcO

AcO

NHAc

S-1S-2

S-3S-4S-5

100

OO O

OHO OH

OHOHOH

HO

OH

O

OHHO

O

NHAc

O

OHHO

O

NHAc

O

OHHO

HO

NHAc

S-1S-2

S-3S-4S-5

101

OO O

OAcO OAc

OAcOAcOAc

AcO

OAc

O

OAcAcO

O

NHAc

O

OAcAcO

O

NHAc

O

OAcAcO

AcO

NHAc

S-1S-2

S-3S-4S-5

102

OO O

OAcO OAc

OAcOAcOAc

AcO

OAc

O

OAcAcO

O

NHAc

O

OAcAcO

O

NHAc

O

OAcAcO

AcO

NHAc

S-1S-2

S-3S-4S-5

103

OO O

OAcO OAc

OAcOAcOAc

AcO

OAc

O

OAcAcO

O

NHAc

O

OAcAcO

O

NHAc

O

OAcAcO

AcO

NHAc

S-1S-2

S-3S-4S-5

108

The Electronspray Mass Spectrometry data of Ariesose not only confirmed the

derived structure but also supported the sequence of monosaccharide in Ariesose. The

highest mass ion peaks were recorded at m/z 1013 assigned to [M+Na+K]+ and m/z 974

assigned to [M+Na]+, it also contain the molecular ion peak at m/z 951 confirming the

molecular weight as 951 which was in agreement with its molecular formula

C36H61O26N3. The mass fragments were formed by repeated H transfer in the


The pentasaccharide m/z 951 (I) fragmented to give mass ion at m/z 748 (II) [951-(S-5)],

this fragment was arised due to the loss of β-GalNAc (S-5) moiety from pentasaccharide.

After this, it (II) fragmented to give mass ion peak at m/z 545(III) [748-(S-4)] which was

due to the loss of β-GalNAc (S-4) moiety from tetrasaccharide. This trisaccharide of m/z

545 is then fragmented to give mass ion peak at m/z 342 (IV) [545-(S-3)] which was a

disaccharide(IV), due to loss of β-GalNAc (S-3) moiety. This disaccharide unit again

fragmented to give monosaccharide mass ion peak at m/z 180 [342- (S-2)] which was due

to loss of β-Gal (S-2). These four mass ion peak II, III, IV, and V, were appeared due to

the consequent loss of S-5, S-4, S-3, and S-2 from original molecule. The mass spectrum

also contain the mass ion peak at are m/z 342, 546, and 629 correspond to the mass ion

fragment A, B, C, Which confirm the position of S1, S2, S3 ,S4 and S5.The other

fragmentation pathway in ES Mass spectrum of compound C m/z 951 shows the mass

ion peak at 892 [951- CH2OHCHO (59)], 848 [892-CH3CO,H (44),], 893 [951-

NHCOCH3 (58)], 916 [951- H2O, OH (35)], 730 [748- H2O (18), 687 [730-CH3O (43)],

686 [687- H (1)], 668 [686-H2O (18)], 510 [545-H2O, OH (35)], 467 [510-CH3CO (43)],

487[545-NHCOCH3 (58)], 405 [465-CH2OHCHO (60)]. Based on result obtained from

chemical degradation/acid hydrolysis, Chemical transformation, Electro spray mass

spectrometry and 1H, 13C NMR and HOMOCOSY, TOCSY and HSQC 2D NMR

techniques of acetylated and Deacetylated Ariesose the structure and sequence of isolated

Novel oligosaccharide molecule was deduced as-

109

ARIESOSE

110

COMPOUND-D RIESOSE

Compound-D, C40H68O31N2, ��

� +113o gave positive Phenol-sulphuric acid test206,

Fiegl test207 and Morgan-Elson test208 showing the presence of normal and amino sugar

moietie(s) in the compound-D. The HSQC spectrum of acetylated riesose showed the

presence of six cross peaks for seven anomeric protons doublets and carbons in their

respective region at 6.17x90.24, 5.37x90.12, 4.77x95.26, 4.59x101.90, 4.52x101.05,

4.52x100.96 suggesting the presence of six anomeric protons and carbons in it. The

presence of seven anomeric proton doublets were confirmed by five anomeric signals in 1H NMR i.e. at δ 6.17(1H), 5.37(2H), 4.77(1H), 4.59(1H), 4.52(2H) in the 1H NMR

spectrum of riesose acetate in CDCl3 at 300 MHz. The presence of seven anomeric

carbons were confirmed by the presence of six anomeric signals in 13C spectrum at δ

90.12(2C), 90.24(1C), 95.26(1C), 100.96(1C), 101.05(1C), 101.90(1C), of acetylated

riesose in CDCl3 at 300 MHz. These data suggested that compound riesose may be a

Hexasccharide in its reducing form. In 1H NMR spectrum of riesose acetate which

contains seven anomeric signals, out of which signal at δ 6.17 and 5.37 were assigned for

downfield shifted α and β anomeric protons of monosaccharide present at the reducing

end suggested that compound-D ‘riesose’ may be a hexasccharide in its reducing form.

The hexaasccharide nature of riesose was further supported by presence of five anomeric

proton doublets for six anomeric protons at δ 5.16(1H), 4.60 (1H), 4.52(1H), 4.39(2H),

4.21(1H) along with two singlet at δ 1.94 and 1.86 for two methyl groups of NHCOCH3

at 300 MHz 1H NMR spectrum of riesose in D2O suggested that out of six

monosccharides two monosaccharides having N-acetyl groups. Further the ES mass

spectrum of riesose showed the highest mass ion peaks at m/z 1134 assigned to

[M+Na+K]+ and m/z 1111 assigned to [M+K]+, it also contain the molecular ion peak at

m/z 1072 confirming the molecular weight as 1072 which was in agreement of derived

composition C40H68O31N2. The reducing nature of compound was further confirmed by

methylglycosylation MeOH/H+ followed by its acid hydrolysis, which led to isolation of

α and β- methyl glucosides, suggesting the presence of glucose at the reducing end209, 210,

for convenience all six monosaccharides denoted as S-1, S-2, S-3, S-4, S-5 and S-6. The

monosaccharides constitutes in compound were confirmed by its killiani hydrolysis147


112


GlcNac and GalNac by co-chromatography. Thus the hexasaccharide contained four

types of monosaccharides units i.e. Glc, Gal, GlcNac and GalNac. Further the presence

of two anomeric protons signals at δ 5.16 (J=3.0 Hz) and δ 4.60 (J=8.1 Hz) in the 1H

NMR spectrum of riesose in D2O at 300 MHz were assigned for α and β anomers of

glucose (S-1), confirming the presence of Glc(S-1) at the reducing end in compound-D

riesose. Further the presence of another anomeric doublet at δ 4.52 (J=7.5 Hz) suggested

the presence of β-Gal residue as the next monosaccharide unit. In addition to above

signals presence of a triplet at δ 3.20 which was assigned for H-2 of Glc(S-1) along with

earlier described anomeric proton doublets at δ 5.16 and 4.60 for α and β glucose

(Structure reporter group)211, 212 suggested the presence of lactose213 type structure i.e. β-

Gal(1-4)→Glc linkage at the reducing end of riesose. Further in the 1H NMR of riesose

acetate the anomeric proton signal at δ 6.17 assigned to Glc(S-1) gave one cross peaks at

δ 3.80 in the TOCSY spectrum of riesose acetate in CDCl3 at 300 MHz. This signal was

further identified as H-4 of β-Glc(S-1) by the COSY spectrum of riesose acetate showed

that H-4 of β-Glc(S-1) was available for glycosidic linkage by the next monosaccharide

unit. The earlier suggested 1→4 linkage between Glc(S-1) and Gal(S-2) by SRG was

further confirmed by the cross peak signal of H-4 of Glc(S-1) and C-1 of βGal(S-2) at

δ3.8x100.96 in HMBC spectrum at 300 MHz. The coupling constant of anomeric signal

at δ 4.52 for βGal(S-2) with J value of 7.5 Hz confirmed the β configuration of the

βGal(S-2) moiety and hence β 1→4 glycosidic linkage between S-2 and S-1 was

confirmed. Further the anomeric proton signal of βGal(S-2) at δ 4.52 in the 1H NMR of

riesose acetate in CDCl3 showed two consequent complementary signals in the linkage

region at δ 3.8 and 4.2 in the TOCSY spectrum of riesose acetate showing that two OH

groups of βGal(S-2) were available for glycosidic linkage. These signals were identified

for H-2 and H-3 respectively of βGal(S-2) by the COSY spectrum of riesose acetate

suggesting that H-2 and H-3 of βGal(S-2) were available for glycosidic linkages by the

next monosaccharide units. The next anomeric proton signal which appeared as a doublet

at δ 4.52 (J=7.5) in the 1H NMR spectrum of acetylated riesose was assigned to βGal(S-

4). The appearance of H-3 signal of S-2 at δ 4.2 in the 1H NMR of riesose acetate was

120

Table-4: 1H NMR values of RIESOSE in D2O at 300 MHz

suggested that Gal(S-2) may be linked to βGal(S-4). The anomeric proton signal present

at δ 4.52 has its complimentary carbon signal at δ 101.05 in HSQC spectrum of riesose

acetate. This anomeric carbon further gave cross peak at δ 4.2 of S-2 (H-3) in HMBC

spectrum of riesose acetate confirming the 1→3 linkage between S-4 and S-2. 1→3

linkage between S-4 and S-2 was also supported by TOCSY and COSY spectrum of

compound riesose acetate. The large coupling constant of βGal(S-4) of J=7.5 Hz

confirmed the β1→3 glycosidic linkage between S-4 and S-2. Since, the H-2 and H-3

position of S-2 were available for glycosidic linkage and position H-3 of βGal(S-2) was

already linked with βGal(S-4), the leftover position H-2 of βGal(S-2) must be linked by

next monosaccharide unit. The next anomeric proton signal which appeared at δ 5.37

along with a singlet of amide methyl at δ 1.86 was due to the presence of αGlcNAc(S-3)

moiety. Since the signal for H-2 of S-2 appeared at δ 3.8 in the 1H NMR spectrum of

riesose acetate was suggested that GlcNAc(S-3) may be linked to H-2 of S-2, which was

further supported by TOCSY and COSY spectrum of acetylated riesose. The coupling

constant of anomeric signal of (S-3) with smaller J value (0-2 Hz) confirmed the α

configuration of GlcNAc(S-3) moiety confirming α1→2 glycosidic linkage between S-3

and S-2. Since in the TOCSY spectrum of riesose acetate the anomeric proton of

αGlcNAc(S-3) at 5.37 showed four cross peak signals at δ 3.46, 4.21, 4.5 and 5.0

respectively and out of which only one position at δ 3.46 of αGlcNAc(S-3) was available

for glycosidic linkage. Later this signal of δ 3.46 was ascertained as H-3 of αGlcNAc(S-

3) by COSY spectrum of riesose acetate showing that H-3 of S-3 was available for



β-Gal (S-2) α-GalNAc (S-3) β -Gal (S-4)

β-GalNAc (S-5) β -Gal (S-6)

5.16 4.60 4.52 n.d. 4.21 4.39 4.39

3.3 Hz 7.8 Hz 7.5 Hz.

n.d. 7.8 Hz 8.0 Hz 8.0 Hz

121

glycosidically linked with next monosaccharide unit. The next anomeric proton signal

which appeared at δ 4.59 (J=8.4) assigned for βGal(S-6). Since the signal for H-3 of S-3

appeared at δ 3.46 in the 1H NMR spectrum of riesose acetate supported that 1→3

linkage between S-6 and S-3 which was further confirmed by TOCSY and COSY

spectrum of acetylated riesose. The large coupling constant of βGal(S-6) of J=8.4 Hz

confirmed the β-glycosidic linkage between βGal(S-6) and GlcNAc(S-3). The critical

studies of TOCSY spectrum of riesose acetate revealed that anomeric signal for βGal(S-

6) at δ 4.59 not give any cross peak in the linkage region have confirming the presence of

S-6 at non-reducing end. Since the presence of Gal(S-4) was confirmed the presence of

anomeric signal at δ 4.52 in 1H NMR of riesose acetate. The anomeric signal at δ 4.52

gave one cross peak at δ 4.1 in linkage region in TOCSY spectrum which was later

assigned as H-3 of Gal(S-4) in COSY spectrum of riesose acetate, which was available

for glycosidic linkage with next monosaccharide unit. The next anomeric proton signal

which appeared at δ 4.77 (J=7.2 Hz) along with a singlet of amide methyl at δ 1.94 was

due to presence of βGalNAc moiety in the hexasaccharide was assigned as βGalNAc(S-

5). The linkage between S-5 and S-4 was supported by presence of H-3 signal of S-4 at δ

3.8 in 1H NMR of acetylated riesose. The large coupling constant J=7.2 Hz confirmed the

β1→3 glycosidic linkage between S-5 and S-4. Since the anomeric proton of

βGalNAc(S-5) at δ 4.77 does not show any methine proton signal in linkage in its

TOCSY region confirming that βGalNAc(S-5) was present at non-reducing end and none

of its OH group was available for glycosidic linkage. All the 1H NMR assignments for

ring protons of monosaccharide units of riesose were confirmed by HOMOCOSY214, 215

and TOCSY216 experiments. The positions of glycosidation in the oligosaccharide were

confirmed by position of anomeric signals, S.R.G. and comparing the signals in 1H and 13C NMR of acetylated and deacetylated oligosaccharide. The glycosidic linkages in

riesose were assigned by the cross peaks for glycosidically linked carbons with their

protons in the HSQC217, 218 spectrum of acetylated riesose. The values of these cross

peaks appeared as- β-Glc (S-1) H-4 and C-4 at (3.8x76) showed 1→4 linkage of S-2 and

S-1 i.e. its H-4 positions of Glc(S-1) was involved in linkage region. βGal(S-2) H-2 and

C-2 at (3.80x72.3) showed 1→2 linkage between S-3 and S-2 and β-Gal(S-2) H-3 and C-

3 at (4.1x70) showed 1→3 linkage between S-4 and S-2 i.e. its H-3 and H-2 position of

126

β-Gal(S-2) was involved in linkage region. β-GlcNAc(S-3) H-3 and C-3 at (3.46x78)

showed 1→3 linkage between S-6 and S-3 i.e. its H-3 position of β-GlcNAc(S-3) was

involved in linkage region. βGal(S-4) H-3 and C-3 at (4.2x70.5) showed 1→3 linkage

between S-5 and S-4 i.e. its H-3 position of Gal(S-4) was involved in linkage region. On

the basis of above data, it was interpreted that the compound-D riesose was

hexasaccharide having the structure:

The Electronspray Mass Spectrometry data of riesose not only confirmed the

derived structure but also supported the sequence of monosaccharide in riesose. The

highest mass ion peaks were recorded at m/z 1134 and 1111 which were due to

[M+Na+K] and [M+K] respectively. It also contains the molecular ion peak at m/z 1072

confirming the molecular weight of riesose as 1072 and was in agreement with its

molecular formula. Further the mass fragments were formed by repeated H transfer in the


The hexasaccharide m/z 1072 (I) fragmented to give mass ion at m/z 910(II) [1072-S-6],

this fragment was arised due to the loss of terminal β-Gal(S-6) moiety from

hexasaccharide indicating the presence of β-Gal (S-6) at the non -reducing end. It was

further fragmented to give mass ion peak at m/z 707 (III) [910-S-5] which was due to loss

of β-GalNAc (S-5) moiety from pentasaccharide. This fragment of 707 further

fragmented to give mass ion peak at m/z 545 (IV) [707-S-4] which was due to loss of

βGal (S-4) moiety from the tetrasaccharide. This trisaccahride unit again fragmented to

give mass ion peak at m/z 342(V)[545-S-3], due to los of αGlcNAc (S-3) moiety. This

disaccharide unit again fragmented to give mass ion peak at m/z 180(VI) [342-S-2],

which was due to loss of Gal (S-2) moiety from disaccharide. These four mass ion peak

II, III, IV, V and VI were appeared due to the consequent loss of S-6, S-5, S-4, S-3 and S-

2 from original molecule. The mass spectrum also contain the mass ion peak at are m/z

504, 545, and 707 correspond to the mass ion fragment A, B, C, Which confirm the

position of S-1, S-2, S-3, S-4, S-5 and S-6 The other fragmentation pathway in ES Mass

127

spectrum of compound D m/z 1072 shows the mass ion peak at 1014[1072-NHCOCH3],

1055[1072-OH], 1024[1055-CH2OH], 989[1014-OH], 956[989-CH2OH, 2H+], 910[1072-

S-6], 892[910-H2O], 875[892-OH], 826[875-H2O,CH2OH], 789[826-2H2O, H+],

750[826-CH2OCHO, OH], 709[CH2OH, H2O], 545[707-S-4], 527[545-H2O], 487[545-

NHCOCH3], 427[487-CH2OHCHO], 342[545-S-3], 180[342-S-2]. Based on result

obtained from chemical degradation/acid hydrolysis, Chemical transformation, Electro

spray mass spectrometry and 1H, 13C NMR and HOMOCOSY, TOCSY, HMBC and

HSQC 2D NMR technique of acetylated riesose and riesose the structure and sequence of

isolated Novel oligosaccharide molecule riesose was deduced as-

RIESOSE

CHAPTER IV

EXPERIMENTAL

128

EXPERIMENTAL

General Procedure

The sugars were visualized on TLC with 30% aqueous H2SO4 reagent and on paper

chromatography sugars were visualized with acetyl acetone and p-dimethyl amino

benzaldehyde reagents. The absorbent for TLC was silica gel G (SRL) and CC silica

gel (SRL, 60-120 mesh). Freeze drying of the compound was done with the help of

CT 60e (HETO) Lyophylizer and centrifuged by a cooling centrifuge Remi

instruments C-25 at 5500 rpm. To check the homogeneity of the compounds reverse

phase HPLC system was used equipped with Perkin Elmer 250 solvent delivering

system, 235 diode array detector and G.P. 100 printer plotter. Authentic samples of

glucosamine, galactosamine, galactose, glucose, fucose and sialic acid were

purchased from Aldrich Chemicals. The 1H and 13C NMR spectra of oligosaccharides

were recorded in CDCl3 and the spectra of deacetylated oligosaccharides were

recorded in D2O at 25oC on a Bruker AM 300 NMR spectrometer. The electro spray

mass spectra were recorded on a MICROMASS QUATTRO II triple quadruple mass

spectrometer. The optical rotations of oligosaccharides were measured with AA-5

series automatic apolarimeter in 1 cm tube. The sample (dissolved in suitable solvents

such as methanol/acetonitrile/water) was introduced into the ESI source through a

syringe pump at the rate 5µl per min. The ESI capillary was set at 3.5 KV and the

cone voltage was 40 V.

Chromatography

Following chromatographic techniques have been used for the isolation and

identification of the oligosaccharides

Paper Chromatography (PC)

Paper chromatography was performed on whatman paper No.1 by the use of a

three solvent system of toluene, butanaol and water and spots were detected by

appropriate reagent for specific moieties.

Thin Layer Chromatography (TLC)

The glass plates coated with slurry of silica gel G (SRL) in distilled water were used

which were dried at room temperature for about 24 hrs and activated at 100-110oC.

129

Phenol-sulphuric acid test206 for sugars

To each fraction (0.1ml) distilled water (1ml), 5% phenol (1ml) and conc.

H2SO4 (25 ml) was added and the mixture was vigorously shaken. After 30 min the

carbohydrate rich fractions showed yellow colour.

Feigl test207 for sugars

The test sample (0.1 mg) was placed in a micro-crucible and one drop of

syrupy phosphoric acid was added. The crucible was covered with filter paper

moistened with 10% solution of aniline in AcOH (10%). A small watch glass was

used as a paper weight. The bottom of the crucible was cautiously heated for 30-60

min with micro burner, avoiding excess heating. A pink to red colour was imparted on

filter paper for normal sugars and 6-deoxy sugars exhibited brown coloration. The 2-

deoxy sugars did not give any colour under this test.

P-Dimethyl amino benzaldehyde test219 for proteins

The presence of protein in a sample was tested by p-dimethyl amino

benzaldehyde test. The sample (solid or in solution) was mixed in a micro-crucible

with several drops of saturated glacial acetic acid solution of p-

dimethylaminobenzaldehyde and one drop of fuming hydrochloric acid. A violet

colour indicated the presence of proteins.

Partridge reagent220 for sugars

Freshly distilled aniline (0.93 g) in water saturated with butanol (100 ml) was

mixed with phthalic acid (1.66 g) and was shaken till dissolve. The reagent was

sprayed on the paper on which a spot of test sample was applied and then heated at

100-118oC for 3-5 min. A pink-brown colour indicated the presence of normal

hexoses.

Morgan-Elson test208 for sugars

Acetyl-acetone reagent I: Solution A- 0.5 ml of acetyl-acetone was dissolved in

butanol (50 ml). Solution B- 50% (w/v) aq. KOH (5 ml) was dissolved in ethanol

(20ml). For formation of reagent I, 10 ml of Solution A was added in 0.5 ml of

solution B.

130

p-Dimethyl amino benzaldehyde reagent II: 1 gm of p-dimethyl amino

benzaldehyde was dissolved in the mixture of 30 ml of ethanol and 30 ml of conc.

HCl and solution was then diluted with 180 ml of distilled butanol to get reagent II.

Chromatograms were sprayed with reagent I and heated in the oven for 5 min

at 105°C. The dry paper strips were then sprayed with reagent II and returned to the

oven for 5 min at 90°C. The appearance of purple-violet colour indicated the presence

of amino sugars.

Thiobarbituric acid assay (Warren assay)221 for sialic acid

Reagent A: 1.07 g of sodium metaperiodate was dissolved in 1.0 ml of water. It was

added in 14.5 ml of concentrated ortho phosphoric acid and water was added to make

it 25 ml.

Reagent B: 10 g of sodium arsenite and 0.71 g of sodium sulphate was dissolved in

0.1M sulphuric acid (made by diluting 0.57 ml concentrated sulphuric acid with 100

ml of water) to a total volume of 10 ml.

Reagent C: 0.12 g of thiobarbituric acid and 142 g of sodium sulphate was dissolved

in water and the volume was made up to 20ml.

In the sample (0.05µ mole in a volume of 0.2 ml) 0.1 ml of reagent A was

added. The tube was shaken and allowed to stand at room temperature for 20 minutes.

1 ml of reagent B was then added and the tube was shaken until a yellow-brown

colour disappears. 3 ml of reagent C was added in the tube with shaking; it was

capped with a glass bead and then heated in a vigorously boiling water bath for 15

minutes. The tube was then removed and placed in cold water for 5 minutes. During

cooling the colour faded and the solution becomes cloudy. From this solution, 1 ml

was transferred to another tube which contained 1 ml of cyclohexanone. The tube was

shaken and then centrifuged for 3 minutes. The clear upper cyclohexanone phase was

red and the colour was more intense than it was when in water.

Bromocresol green test222

Spraying reagent Bromocresol green (0.04 g) was dissolved in ethanol (96%,

100 ml) Drops of 0.1 N NaOH were added until a blue coloration just appeared. A

spot of the test sample when applied on a paper moistened with this reagent (blue)

give a yellow coloration which indicated the presence of a carboxylic group.

131

Acetylation of oligosaccharide mixture

9.48 gm of pooled fractions (peak II and III) which gave positive phenol-

sulphuric acid test were acetylated with pyridine (9.50 ml) and acetic anhydride (9.50

ml) at 60°C and the solution was stirred overnight. The mixture was evaporated under

reduced pressure and the viscous residue was taken in CHCl3 (250 ml) and it was

washed in sequence with 2N-HCl (1 x 25 ml), ice cold 2N-NaHCO3 (2 x 25 ml) and

finally with H2O (2 x 25 ml). The organic layer was dried over anhydrous Na2SO4,

filtered and evaporated to dryness yielding the acetylated mixture (9.49 gm).

Deacetylation of compound ‘a’

Compound ‘a’ (112 mg) was obtained from column chromatography 2 of

acetylated oligosaccharide mixture. 40 mg of compound-a was dissolved in acetone (3

ml) and 3.2 ml of NH3 was added in it and was left overnight in a stoppered

hydrolysis flask. After 24 hrs ammonia was removed under reduced pressure and the

compound was washed with (3 x 5 ml) CHCl3 (to remove acetamide) and the water

layer was finally freeze dried giving the deacetylated oligosaccharide A (35 mg).

Methyl glycosidation/Acid hydrolysis of compound A

Compound A (8 mg) was ref1uxed with absolute MeOH (2 ml) at 70°C for 18

hrs in the presence of cation exchange IR-120 (H) resin. The reaction mixture was

filtered while hot and filtrate was concentrated. In the solution of methylglycoside of

A, 1, 4-dioxane (1 ml), and 0.1N H2S04 (1 ml) was added and the solution was

warmed for 30 minutes at 50oC. The hydrolysis was complete after 24 hrs. The

hydrolysate was neutralized with freshly prepared BaCO3 filtered and concentrated

under reduced pressure to afford α-and β-methylglucosides along with the Glc,

GalNAc and GlcNAc. Their identification was confirmed by comparison with

authentic samples (TLC, PC).

Kiliani hydrolysis of compound A

Compound A (5 mg) was dissolved in 2 ml Kiliani mixture (AcOH-H2O-HCI, 7:11:2) and

heated at 100oC for 1 h followed by evaporation under reduced pressure. It was dissolved in 2

ml of H2O and extracted twice with 3 ml CHCl3. The aqueous residual solution was made

neutral by addition of 1-2 drops of 2N NaOH and was evaporated under reduced pressure to

132

afford glucose, GalNAc and GlcNAc on comparison with authentic samples of glucose,

GalNAc and GlcNAc.

Deacetylation of compound ‘b’

Compound ‘b’ (419 mg) was obtained from column chromatography 3 of

acetylated oligosaccharide mixture. 50 mg of compound b was dissolved in acetone (3



compound was washed thrice with CHCl3 (5 ml) (to remove acetamide) and the water

layer was finally freeze dried giving the deacetylated oligosaccharide B (45 mg).

Methyl glycosidation/Acid hydrolysis of compound B

Compound B (8 mg) was ref1uxed with absolute MeOH (2 ml) at 70°C for 18

h in the presence of cation exchange IR-l20 (H) resin. The reaction mixture was


B, 1, 4-dioxane (1 ml), and 0.1N H2S04 (1 ml) was added and the solution was



under reduced pressure to afford α-and β-methylglucosides along with the Glc, Gal,

GalNAc and GlcNAc. Their identification was confirmed by comparison with

authentic samples (TLC, PC).

Kiliani hydrolysis of compound B

Compound B (5 mg) was dissolved in 2 ml Kiliani mixture (AcOH-H2O-HCI,

7:11:2) and heated at 100oC for 1 h followed by evaporation under reduced pressure.

It was dissolved in 2 ml of H2O and extracted twice with 3 ml CHCl3. The aqueous

residual solution was made neutral by addition of 1-2 drops of 2N NaOH and was

evaporated under reduced pressure to afford Glc, Gal, GalNAc and GlcNAc on

comparison with authentic samples of Glc, Gal, GalNAc and GlcNAc.

Deacetylation of compound ‘c’

Compound ‘c’ (46 mg) was obtained from column chromatography 6 of

acetylated oligosaccharide mixture. 32 mg of compound C was dissolved in acetone

(2 ml) and 3 ml of NH3 was added in it and was left overnight in a stoppered


133

compound was washed thrice with CHCl3 (5 ml) (to remove acetamide) and water

layer was finally freeze dried giving the deacetylated oligosaccharide C (24 mg).

Methyl glycosidation/Acid hydrolysis of compound C

Compound C (8 mg) was ref1uxed with absolute MeOH (2 ml) at 70°C for 18

h in the presence of cation exchange IR-120 (H) resin. The reaction mixture was


C, 1, 4-dioxane (1 ml), and 0.1N H2S04 (1 ml) was added and the solution was



under reduced pressure to afford α-and β-methylglucosides along with the Glc, Gal

and GlcNAc. Their identification was confirmed by comparison with authentic

samples (TLC, PC).

Kiliani hydrolysis of compound C

Compound C (5 mg) was dissolved in 2 ml Kiliani mixture (AcOH-H2O-HCI,

7:11:2) and heated at 100oC for 1 h followed by evaporation under reduced pressure.



evaporated under reduced pressure to afford glucose, Gal and GlcNAc on comparison

with authentic samples of glucose, Gal and GlcNAc.

Deacetylation of compound‘d’

Compound‘d’ (62 mg) was obtained from column chromatography 4 of

acetylated oligosaccharide mixture. 52 mg of compound d was dissolved in acetone (3



compound was washed thrice with CHCl3 (5 ml) (to remove acetamide) and water

layer was finally freeze dried giving the deacetylated oligosaccharide D (44 mg).

Methyl glycosidation/Acid hydrolysis of compound D

Compound D (8 mg) was ref1uxed with absolute MeOH (2 ml) at 70°C for 18

h in the presence of cation exchange IR-120 (H) resin. The reaction mixture was


D, 1, 4-dioxane (1 ml), and 0.1N H2S04 (1 ml) was added and the solution was

134

warmed for 30 minutes at 50oC. The hydrolysis was complete after 24 h. The


under reduced pressure to afford α-and β-methylglucosides along with the Glc, Gal

and GlcNAc. Their identification was confirmed by comparison with authentic

samples (TLC, PC).

Kiliani hydrolysis of compound D

Compound D (5 mg) was dissolved in 2 ml Kiliani mixture (AcOH-H2O-HCI,

7:11:2) and heated at 100oC for 1 hrs followed by evaporation under reduced pressure.



evaporated under reduced pressure to afford glucose, Gal and GlcNAc on comparison

with authentic samples of glucose, Gal and GlcNAc.

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135

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APPENDIX-I

List of Publications /seminars

Publications:

1. Ashok Kr. Ranjan and Desh Deepak, Isolation and purification of sheep milk

oligosaccharide as therapeutic agents, Journal of biological and chemical research. 2015,

32(2), 455-465.

2. Ramendra S Rathore, Ashok Kumar Ranjan, Desh Deepak, Anakshi Khare, Ragini

Sahai, V. M. L. Srivastava., Isolation of Novel Hexa and Heptasaccharide from

Immunostimulant Oligosaccharide Fraction of Donkey’s Milk., Journal of molecular

structure. (communicated)

Seminars/Conferences

1. Poster presented in National seminar on “Newer trends in physico-chemical techniques”

at Chemistry department, university of Lucknow, Lucknow on 7th Aug. 2013. Published

in proceeding on p. no. 23

“Isolation of novel compounds from oligosaccharide fraction of Chauri Cow milk by

physico-chemical techniques”

2. Poster presented in “international carbohydrate symposium” at Indian institute of science,

Bangalore on 12th -17th Jan. 2014. Published in proceeding on p. no. 265

“Isolation of two novel oligosaccharides from yak milk”

3. Poster presented in International seminar on “recent advances in analytical science” at

BHUIIT, Varanasi on 27th -29th March, 2014. Published in proceeding on p. no. 87

“Structure elucidation of milk oligosaccharide by MASS spectrometry”

4. Poster presented in National seminar on “Trends in carbohydrate” at (IISER) Mohali on

29th -31st Dec. 2014. Published in proceeding on p. no. 106

“ Identification of novel Sheep milk oligosaccharide by NMR”

5. Poster presented in “Indian science congress” at Mumbai University, Mumbai on 3rd to

7th Jan. 2015. Published in proceeding on p. no. 136

“Novel oligosaccharide isolated from Sheep’s milk”

isolation of biologically active …shodhganga.inflibnet.ac.in/bitstream/10603/70755/1/ashok thesis...

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