Structure analysis of polysaccharides by NMR

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Structure analysis of polysaccharides by NMR

• Lennart Kenne
• Department of Chemistry, SLU, Swedish University
  of Agricultural Sciences, P.O. Box 7015, SE-750
  07 Uppsala, Sweden

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NMR Spectroscopy in Glycoscience

• Structural information needed for carbohydrates
• Information from NMR
• Structure analysis by NMR
• Modern NMR methods some applications

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Oligo- and polysaccharides
Information on structure and properties

• Structure
• Components
• Linkages
• Sequence
• Conformation
• Properties
• Interactions with solvent or other molecules as
  proteins

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• Studies of carbohydrates
• Isolated material
• Carbohydrates on solids
• Carbohydrates in their natural environment

When can NMR be used?
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The basic NMR experiment
1H (100) and 13C (1.1)
Higher field higher energy more nuclei in the
lower state 1 of 100,000
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NMR parameters Information
Chemical shifts Chemical surrounding Coupling
constants Stereochemistry Intensities Number of
atoms / molar ratio Relaxation times T1 and
T2 Dynamic properties NOE (nuclear Overhauser
effect) Interatomic distances and dynamic
properties
FID
B0
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For most NMR experiments
Sample dissolved in D2O
Deuterated solvent gives no signal and locks the
frequencies
Substituted sugars other solvents
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Structure Components Linkages Sequence Conformatio
n
Component Which sugar Anomeric configuration Absol
ute configuration Substituents
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H-1 H-2 H-3 H-4 H-5 b-Glc 4.64 3.25 3.50 3.42 3
.46 a-Glc 5.23 3.54 3.72 3.42 3.84 Difference 0.
6 0.3 0.2 0 0.4
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H-1 H-2 H-3 H-4 H-5 b-Glc 4.64 3.25 3.50 3.42 3
.46 b-Gal 4.53 3.45 3.59 3.89 3.65 Difference -0.
1 0.2 0.1 0.5 0.2
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Carbohydrates / NMR
Chemical shifts - anomeric proton signals
d 5 ppm
Equatorial proton 0.6 ppm (axial proton)
d 4.5 ppm
Proton on a carbon linked to two oxygens
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Chemical shifts
b-D-Glc
b-D-Man
H-1 H-2 H-3 H-4 H-5 H-6a H-6b A
4.64 3.25 3.50 3.42 3.46 3.72 3.90 B 4.89 3.95 3.
66 3.60 3.38 3.75 3.91 Diff 0.2 0.7 0.2 0.2 -
0.1 0 0
Carbohydrate Research 188 (1989) 169-191
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Coupling constants - 3JH,H
1.5 Hz
7.5 Hz
180 degr 7-10 Hz
60 degr 1-4 Hz
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Coupling constants - 3JH,H
H1,2 H2,3 H3,4 H4,5 H5,6a H5,6a H6a,b A
7.5 10 10 10 2 5 12 B 1.5 3 10 10 2 5 12
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C-1 C-2 C-3 C-4 C-5 b-Glc 96.8 75.2 76.8 70.7 7
6.8 a-Glc 93.0 72.5 73.8 70.7 72.4 Difference -3.
8 -2.7 -3.0 0 -4.4
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g-gauche effect -4 ppm
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C-1 C-2 C-3 C-4 C-5 b-Glc 96.8 75.2 76.8 70.7 7
6.8 b-Gal 97.4 73.0 73.8 69.7 75.9 Difference 0.
6 -2.2 -3.0 -1.0 -0.9
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Chemical shifts
Anomeric carbon d 100-104 and 96-99 ppm
For mannoses almost no difference
8 ppm
Substituted carbon 4-10 ppm (Depending on
stereochemistry around the glycosidic bond)
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Coupling constants - 3JH,H
1.5 Hz
7.5 Hz
For manno- configuration 1-2 Hz
180 degr 7-10 Hz
60 degr 1-4 Hz
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Coupling constants - 1JC,H - (Anomeric
configuration)
1JC,H 170 Hz Equatorial proton
1JC,H 160 Hz Axial proton
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Structure Components Linkages Sequence
Linkage position
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Substitution - linkages
Glycosylation shifts Dd-values
Substitution of a carbon a b g 9 9 -2.5
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Structure Components Linkages Sequence
A
B
C
Sequence of sugar residues
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Dipolar interactions - NOE - (Sequence
information - interresidue) Connects the residues
NOESY or ROESY
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Dipolar interactions - NOE - (anomeric protons
-intraresidue) Through space short distances
NOESY or ROESY
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Three-bond coupling 3JC,H - (Sequence
information)
HMBC
Also 4-bond H,H-coupling
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• 1H NMR spectrum of a polysaccharide can be
  divided into regionsshowing recognizable signals
• characteristic for the sugar residues
• functional groups present

CH3- groups
anomeric protons
ring protons
N- O-acetyl groups
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1D-NMR of polysaccharides
Viscous solution broad signals
Complex spectrum many overlapping signals
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Following an enzymatic hydrolysis of three
disaccharides and an a-L-fucosidase
1D-NMR
Fuca1?2GalbOMe
Fuca1?6GalbOMe
Fuca1?3GalbOMe
T 0
b-L-Fucose
a-L-Fucose
T 12 H
T 24 H
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Polysaccharide from an Plesiomonas LPS
1D-NMR
core
PS
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Two-dimensional NMR COSY H-1/H-2 H-2/H-3
H-3/H-4 H-4/H-5 H-5/H-6a,6b
H-1
H-2
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Two-dimensional NMR COSY H-1 H-2 TOCSY H-1
H-2 H-3 H-4 H-5 ..... NOESY through
space HMQC C-H HMBC C-C-H and/or C-X-C-H (2
or 3 bonds)
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TOCSY
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HMQC H,C-correlated
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HMBC
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NOESY
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• NMR spectroscopy Carbohydrates
• Some available methods
• 1D NMR
• 2D NMR
• LC-NMR
• HR-MAS
• Saturation Transfer Difference NMR Spectroscopy
• NMR imaging
• Solid-state NMR

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LC-NMR
Problems for structural analysis Solvent LC -
NMR Different amounts Time for each compound 1D
2D experiments
NMR
Structure information
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LC SPE - NMR
MS
Advantages Change solvent Remove water Several
runs accumulate Handling of compounds Different
scales Manual or automation
NMR
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NMR analysis 1D and 2D
0.1 - 1.5 mg
Multivariate data analysis structure analysis
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• Studies of carbohydrates by NMR
• Carbohydrates in their natural environment
• The role of the hydroxyl groups

Normally 1H NMR in D2O fast exchange of
hydroxyl protons not observed but in 85 H2O /
15 aceton-d6 the OH protons can be observed
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Sample preparation Remove ions that can increase
the exchange
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Expanded region of the 2D DQF-COSY spectrum (85
H2O/15 (CD3)2CO, -10 C) of maltose, showing the
scalar connectivities between OH and CH protons.
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Chemical shift of the hydroxy proton of methanol
as a function of the mole fraction of methanol in
water (?), diethyl ether (?), tetrahydrofuran (?)
and dioxane (?).
H-O-Me
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Structure analysis with two-dimensional
NMR COSY H-1 H-2 TOCSY H-1 H-2 H-3
H-4 H-5 ..... NOESY through
space HMQC C-H HMBC C-C-H and/or C-X-C-H (2
or 3 bonds)
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Molecules in dilute solutions can tumble and thus
average out several negative effects as chemical
shift anisotropy and dipolar couplings
Results in high-resolution NMR spectra
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Tilting the sample at 54.7 o and spinning at high
speed overcome these problems (1-3cos2q)
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HR-MAS NMR - High-Resolution Magic-Angle-Spinning
NMR
B0
Spinning at magic angle removes effects of
dipolar interactions and chemical shift anisotropy
rotor cap
air
rotor spacer
sealing screw
sample in D2O ( 10-30 ml)
Q
spinning 2-15 kHz
improved linewidths
rotor
Q 54.7 ("magic angle")

• Analysis of small molecules or biopolymers that
  are mobile in the cells or in a semi-solid
  systems.

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T2-filter CPMG pulse sequence - (t-180-t)n
t 387 ms
n500
n1
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T2-filter
Artefacts generated by the filter
(t-180-t)n
multiplets
Intensity differences
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DQF-COSY 5000 Hz Ca 1 mg alga (dry weight)
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Relay-COSY 5000 Hz Ca 1 mg alga (dry weight)
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TOCSY 5000 Hz Ca 1 mg alga (dry weight)
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HMQC 14600 Hz Ca 1 mg alga (dry weight)
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Pichia anomala 5000 Hz
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Studies of the metabolism

• Pichia anomala inhibit the growth of mold in
  stored cereals.
• How will oxygen limitation influence the
  metabolism?

• Extract or analyse intact cells?
• NMR needs normally a homogenious sample in
  solution.

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Pichia anomala living cells
Control
O2-limitations
Exo- and intra-cellular metabolites compared by
GC and HR-MAS NMR
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HR-MAS - a non-destructive method?
Increased amounts of microthecin after 16 h of
spinning of alga (gt5000 Hz)
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Gracilariopsis lemaneiformis 5-15 kHz, over
night 1 mg alga (dry weight)
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Characterization of Ligand Binding by Saturation
Transfer Difference NMR Spectroscopy
The difference between a saturation transfer
spectrum and a normal NMR spectrum provides a new
and fast method (saturation transfer difference
(STD) NMR spectroscopy) to screen compound
libraries for binding activity to proteins. STD
NMR spectroscopy of mixtures of potential ligands
with as little as 1 nmol of protein yields 1D and
2D NMR spectra that exclusively show signals from
molecules with binding affinity. In addition, the
ligands binding epitope is easily identified
because ligand residues in direct contact to the
protein show much stronger STD signals.
Moriz Mayer and Bernd Meyer
Angew. Chem. Int. Ed. 1999, 38, No. 12 1784-1788
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a-L-fucosidase
1-Deoxymannojirimycin (DMJ) - inhibitor
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Normal spectrum
Saturation
Difference spectrum
a-L-fucosidase
1-Deoxymannojirimycin (DMJ)
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NMR imaging
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World's Largest, Most Powerful NMR
Spectrometer The Department of Energy's Pacific
Northwest National Laboratory celebrated the
arrival of the world's largest,
highest-performance nuclear magnetic resonance
spectrometera first-of-its-kind 900 megahertz
(MHz) wide-bore system developed by Oxford
Instruments and Varian Inc.
A powerful magnet developed for chemical,
biological and materials research was lifted by a
crane into DOE Office of Science's William R.
Wiley Environmental Molecular Sciences Laboratory