Carbohydrates – Characterization Overview
 Methods for Structure and Property Deduction





Chemical methods for functional group identification
Chemical methods for ring size identification
Methylation analysis
X-ray crystallography
Analytical
y
methods for
f characterization
 UV-Vis spectroscopy
 Circular dichroism spectroscopy
 NMR spectroscopy
 Mass spectrometry
1
Monosaccharides
 Mutarotation
M
i
CHO
H
HO
OH
H
H
OH
H
OH
CH2OH
D-glucose
-D-glucopyranose
(-D-glucose)
-D-glucopyranose
(-D-glucose)
Optical activity
[]D = +112.2
+112 2O
[]D = +18.7
+18 7O
At equilibrium …. []D = +52.7O (Not an average!
But weighted average; 63.6% of the -anomer)
2
Simple Tests for Reducing / Non-reducing Sugars
 Importance
I
t
off Reducing
R d i Sugars
S
 Case of hemoglobin (Hb)
Hb is glycated ….. HbA1c levels should be < 6.5%; Reaction of high glucose
with
ith NH2 group att the
th N-terminus
Nt
i
(Val)
(V l) off Hb;
Hb Non-enzymatic;
N
ti Reflects
R fl t
Glc levels for the past 3 months (typical life of erythrocytes)
 Can serve as sugar modifying handle
Label with chromogenic
g
ggroups
p or fluorophores
p
Preparation of sugar-labeled solid matrix
 Tollen’s Reagent
CHO
H
HO
OH
H
H
OH
H
OH
CH2OH
D Gl cose
D-Glucose
COOH
H
HO
Mild Oxidizing Agent H
e.g., Tollen’s reagent
H
(Ag+ / NH3 – H2O
OH
H
+
Metallic Silver
OH
OH
CH2OH
D-Gluconic
D Gl conic Acid
3
Simple Tests for Reducing / Non-reducing Sugars
 Benedict’s reagent
RCHO + 2Cu
RCHO
2C 2+ + 5HO
5HO‐
RCOO‐ + Cu
C 2O + 3H
O
3H2O
• Benedict's reagent
g is a solution of the citrate complex
p of CuSO4 in
water. Cu2+ is a weak oxidizing agent.
• A positive test is the formation of a red precipitate of Cu2O.
 Sugars that exhibit positive Tollen’s / Benedict’s tests
• Aldoses
• Ketoses, if they exhibit good keto-enol tautomerism
• Glycosides do not show positive test because they cannot exhibit
aldehyde  equlibrium
HOCH2
HO
HO
O
OH
OCH3
4
UV-Vis Spectroscopy of Carbohydrates




Glycans are UV-Vis transparent; groups include –OH, –CHO, –C=O, –
OCH3, –COOH, etc.
Some gglycans
y
have a conjugated
j g
double bond;; e.g.,
g , heparin
p
disaccharide. Such groups absorb at 232 nm, which allows
identification and quantitation of oligosaccharides
Have to be typically labeled for visualization
Use reducing end labels such as 2-aminopyridine (2-AP),
(2-AP) 2aminoacridone (AMAC), 7-amino-1,3-naphthalenedisulphonic acid
(AGA), p-aminobenzoic acid (PABA), 2-aminobenzamide (2ABA), 4aminobenzonitrile 8-aminonaphthalene-1,3,6-trisulfonic
aminobenzonitrile,
8 aminonaphthalene 1 3 6 trisulfonic acid (ANTS)
(ANTS),
1-phenyl-3-methyl-5-pyrazolone (PMP), 1-(4-methoxy)phenyl-3methyl-5-pyrazolne (PMPMP), 6-aminoquinoline (6-AQ), 1maltohepaosyl-1,5-diaminonaphthalene,
lt h
l 1 5 di i
hth l
8-aminopyrene-1,3,68
i
136
trisulfonate (APTS), etc.
5
UV-Vis Spectroscopy of Carbohydrates

Labeling reaction ... typically Schiff base formation followed by
reduction to a stable covalent bond
CH2OH
CH2OH
O
OH
OH
H
OH
OH
AcOH, 
RO
2-AP
OH
N
N
RO
OH
NaCNBH3
CH2OH
OH
H
OH
CH2OH
O
OH
RO
OH
N
OH
NH
RO
OH
reduced
d d Schiff
S hiff bbase
6
NMR Spectroscopy of Carbohydrates

Every polysaccharide has a unique 1D NMR spectrum, which
contains all of the information about the structure

Additional NMR experiments are performed to assign the resonances,
resonances
e.g., 1H,1H-COSY and 1H,1H-TOCSY for 1H resonances or 1H,13CHETCOR or HSQC for 13C resonances

Sequence determination is performed through experiments such as
NOESY (1H,1H through space correlations) or HMBC (3JCOCH)

Methods
M
th d ffor iinterpretation
t
t ti off 1H NMR spectrum
t
can save much
h ti
time
and effort
7
Basic Approach for NMR Structure Determination
1D 1H-NMR
+ 1D 13C-NMR
C NMR
2D 1H
H,1H
H-COSY
COSY
Taken from Desai et al. Carbohydr. Res. 1993, 241, 249-259.
8
Basic Approach for NMR Structure Determination
2D 1H,1H-NOESY
2D 1H,13C-HETCOR
9
Basic Principle
= Through-bond correlation
= Through-space correlation
= Anomeric proton

3J couplings may needed for conformational
assignment

Requires knowledge of saccharide residues

Additional experiments needed for larger structures
10
NMR Tools on the Internet
(Taken from a lecture on ‘Internet tools for the Interpretation of NMR spectra’
b
by
Dr. Roland Stenutz ([email protected]), University of Stockholm, Sweden)
Polysaccharide
y
structure:
Components
”type”
relative configuration
absolute configuration
ring size
Linkages
position
stereochemistry
Sequence
Hex or HexNAc
Glc or Man
D- or LD
L
-p or –f
4) or 6)
- or 4)Glc(4)Gal(
or
4)Gal(4)Glc(
NMR can be used to perform each of these steps
steps, except for the
determination of the absolute configuration
Most saccharides are typically present in one form in nature, which serves to identify the configuration
identify the configuration
11
Internet-based Approaches to NMR Analysis

comparison with a database (SugaBase)
simple and accurate but limited to known structures or substructures.

comparison with simulated NMR spectra (CASPER)
requires
q
information
f
about the components
p
and linkages
g to limit the
number of possible structures.

Just a few examples
p of tools available in polysaccharide
p y
analysis
y
Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz
([email protected]), University of Stockholm, Sweden)
12
SugaBase
http://www.boc.chem.uu.nl/sugabase/databases.html)
p
g
)
Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz
([email protected]), University of Stockholm, Sweden)
13
Sweet-DB

http://glycosciences.de hosts several tools and
databases for the carbohydrate chemist

Sweet-DB is an interface to CarbBank/SugaBase
Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz
([email protected]), University of Stockholm, Sweden)
14
Searching With NMR Data on Sugabase
Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz
([email protected]), University of Stockholm, Sweden)
15
Search Results
Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz
([email protected]), University of Stockholm, Sweden)
16
Best Search Result
Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz
([email protected]), University of Stockholm, Sweden)
17
CASPER
( p
(http://www.casper.organ.su.se)
p
g
)
Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz
([email protected]), University of Stockholm, Sweden)
18
CASPER
Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz
([email protected]), University of Stockholm, Sweden)
19
CASPER
Calculated shifts
Assignment
Experimental shifts
Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz
([email protected]), University of Stockholm, Sweden)
20
CASPER

CASPER also allows incorporation of glycosylation and
substituent shifts, positional shifts

Sequences
q
can also be derived using
g CASPER
Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz
([email protected]), University of Stockholm, Sweden)
21
Mass Spectrometry in the Structure Determination of Glycans

The premier structure determination tool today


Highly sensitivity (g quantities)
Highly information rich ... full sequence with detailed stereochemical
structure possible

Widely applicable ... N- & O-glycans, sialylated glycans, GAGs, etc.

May be used in tandem with chemical or enzymatic pre-treatment

Various MS technologies are available for ionization




MALDI ((Matrix assisted laser desorption
p
ionization))
ESI or nESI (Electrospray or nanoElectrospray ionization)
FAB (Fast atom bombardment)
Various approaches are available for structure elucidation



HR TOF (high resolution time of flight)
MS/MS or MS/MSn (tandem MS)
Ion Mobility
22
A Strategy for MS Sequencing of N-Glycans
Taken from Azadi
and Heiss. Methods
Mol. Biol. 2009, 534,
37 51
37-51.
23
Exoglycosidases in Sequencing of Glycans
A good ref: Kannicht et al. Methods Mol. Biol. 2008, 446, 255-266.
24
Permethylation/Peracetylation and Linkage Analysis

Permethylation advantages include easier determination of linkages and
branching

Allows simultaneous analysis of sialic acid and neutral glycans

Better ionization in MS chamber

Makes analysis possible with pM quantities of sample
CH3I, dry DMSO
CH2Cl2
2M TFA, >100 OC
1M NH4OH,
NaBD4
Ac2O, base, heat
25
The Mass Spectrometer




Molecules
l l can be
b imparted
i
d charge
h
(+ve
(
or –ve))
Charged molecules can be made to move (accelerate)
A moving object (e.g., a molecule) can be made to deflect
Deflection is related to mass
26
The Tandem Mass Spectrometer
Image from Access Science from McGraw Hill
27
Nomenclature of Product Ions



Fragment ions containing the non-reducing terminus are labeled A, B, C
Fragment ions containing the reducing terminus are labeled X, Y and Z
Subscripts 1, 2, 3, …. refer to the number of residue from either the reducing
or the non-reducing terminus

Subscript 0 refers to cleavage of the bond with the aglycon

Fragment labels A and X are reserved for intra-ring cleavage
Superscripts refer to intra-ring
intra ring bonds cleaved (clockwise numbering)


Products ions can be [M+nH]n+, [M-nH]n-, [M+nNa+mH](n+m)+,
Zaia, J., Mass Spectrom. Rev. 2004, 23, 161-227.
28
Formation of Product Ions
Zaia, J., Mass Spectrom. Rev. 2004, 23, 161-227.
29
Formation of Product Ions
Zaia, J., Mass Spectrom. Rev. 2004, 23, 161-227.
30
Formation of Product Ions
31
Glycan Characterization Using SimGlycanTM
Taken from Apte and Meitei, Methods Mol. Biol. 2010, 600, 269-281.
32