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
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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
This is to certify that all necessary requirements for the submission of Ph.D.
thesis of Mr. Ashok Kumar Ranjan has been fully observed.
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),
suggesting the presence of six anomeric protons and carbon in it. Further the presence of
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
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 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
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
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
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),
suggesting the presence of six anomeric protons and carbon in it. Further the presence of
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
MeOH/H+ followed by its acid hydrolysis, which led to the 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
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
59
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.
70
71
72
73
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],
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
under strong acidic condition, followed by paper chromatography and TLC. In this
111
112
hydrolysis four spots were found identical with the authentic samples of Glc, Gal,
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
113
114
115
116
117
118
119
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
Moieties 1H NMR (δ) Coupling cons. (J)
α-Glc (S-1) β-Glc (S-1)
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
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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
122
123
124
125
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
oligosaccharide and was accompanied by the elimination of terminal sugar less water.
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
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spectrum of compound D m/z 1072 shows the mass ion peak at 1014[1072-NHCOCH3],