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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

November 2005

NMR Spectroscopy in Glycoscience


1. Structural information needed for carbohydrates

2. Information from NMR

3. Structure analysis by NMR

4. Modern NMR methods some applications

Oligo- and polysaccharides


Information on structure and properties
H OH

Structure
Components Linkages Sequence Conformation

H HO HO H H O

O H H NH O CH3 H HO O H H H H H HO H H OH H O OH OH H3C OH O

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%)

HO OH H H HO H H OH H
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O OH

Higher field higher energy more nuclei in the lower state 1 of 100,000

NMR parameters
Information

Chemical shifts
Chemical surrounding

Coupling constants
Stereochemistry

Intensities
Number of atoms / molar ratio

B0

FID

Relaxation times T1 and T2


Dynamic properties

NOE (nuclear Overhauser effect)


Interatomic distances and dynamic properties
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For most NMR experiments


H CH2OH HO HO H H H O HO O

H CH3 OH H H H O H OH

H OH

Sample dissolved in D2O

H CH2OD

H CH3
O

Deuterated solvent gives no signal and locks the frequencies

DO DO

H H

DO O H

OD

H H

H OD

H
OD

Substituted sugars other solvents


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Structure Components Linkages Sequence Conformation


HO HO

H OH H O H H H H O CH3 NH O HO O H H H H H HO H OH H O OH OH H3C OH O

Component Which sugar Anomeric configuration Absolute configuration Substituents

HO HO

H CH OH 2 H O OH H H OH H

HO HO

H CH OH 2 H O H H H OH OH

H-1
b-Glc a-Glc 4.64 5.23

H-2
3.25 3.54

H-3
3.50 3.72

H-4
3.42 3.42

H-5
3.46 3.84

Difference

+0.6

+0.3

+0.2

+0.4
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HO HO

H CH OH 2 H O OH H H OH H

HO CH OH 2 H O H HO H H OH H OH

H-1
b-Glc b-Gal 4.64 4.53

H-2
3.25 3.45

H-3
3.50 3.59

H-4
3.42 3.89

H-5
3.46 3.65

Difference

-0.1

+0.2

+0.1

+0.5

+0.2
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Carbohydrates / NMR

Chemical shifts
H CH2OH
HO HO

anomeric proton signals


H CH3

H H

HO O H

OH

H H

H OH

H
OH

Equatorial proton +0.6 ppm (axial proton)

d 4.5 ppm

d 5 ppm

Proton on a carbon linked to two oxygens

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Chemical shifts

A
b-D-Glc

B
b-D-Man

H CH2OH
HO HO

H CH2OH
O

HO OH HO H H

OH

H H

O OH

OH H

H H

H-1 H-2 H-3 H-4 H-5 H-6a H-6b A B


Diff 4.64 4.89 +0.2 3.25 3.95 +0.7 3.50 3.66 +0.2 3.42 3.60 +0.2 3.46 3.38 -0.1 3.72 3.75 0 3.90 3.91 0

12 Carbohydrate Research 188 (1989) 169-191

Coupling constants - 3JH,H


H CH2OH H O HO H HO H CH3 HO
O OH O

H H

H H
OH H

1.5 Hz

7.5 Hz

OH

H
H

180 degr 7-10 Hz

60 degr 1-4 Hz
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Coupling constants - 3JH,H A


H CH2OH
HO HO

B
H CH2OH

H H

HO O H

OH

H H

OH H

H
OH

H1,2 H2,3 H3,4 H4,5 H5,6a H5,6a H6a,b A B


7.5 1.5 10 3 10 10 10 10 2 2 5 5 12 12
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HO HO

H CH OH 2 H O OH H H OH H

HO HO

H CH OH 2 H O H H H OH OH

C-1
b-Glc a-Glc 96.8 93.0

C-2
75.2 72.5

C-3
76.8 73.8

C-4
70.7 70.7

C-5
76.8 72.4

Difference

-3.8

-2.7

-3.0

-4.4
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HO HO

H CH OH 2 H O H H H OH OH

H H

H H

a b

-gauche effect = -4 ppm

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HO HO

H CH OH 2 H O OH H H OH H

HO CH OH 2 H O H HO H OH H H

OH

C-1
b-Glc b-Gal 96.8 97.4

C-2
75.2 73.0

C-3
76.8 73.8

C-4
70.7 69.7

C-5
76.8 75.9

Difference

+0.6

-2.2

-3.0

-1.0 -0.9
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Chemical shifts
H CH2OH
HO HO

H CH3
O

H H

HO O H

OH

H H

H OH

H
OCH3

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


H CH2OH H O HO H HO H CH3 HO
O OH O

H H

H H
OH H

1.5 Hz

7.5 Hz

OH

H
H

For mannoconfiguration

1-2 Hz
H

180 degr 7-10 Hz

60 degr 1-4 Hz
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Coupling constants - 1JC,H - (Anomeric configuration)

H CH2OH
HO HO

H CH3
O

HO
O

OH

H H

H H H

H
OH

OH

1J

C,H = 160 Hz

1J

C,H

= 170 Hz

Axial proton

Equatorial proton

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Structure Components Linkages Sequence


HO HO

H OH H O H H H H O CH3 H NH O HO O H H H H H HO H H OH H O OH OH H3C OH O

Linkage position

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Substitution - linkages
H HO HO HO CH2OH H O H H H O H CH2OH H O H H OH H OH OH

Glycosylation shifts
Dd-values

H OH H HO O H HO C H3 H

HO H O

CH2OH H O H H H OH OH

Substitution of a carbon = a +9

b +9

-2.5
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Structure Components Linkages Sequence


HO HO

H OH

A
H O H H H NH O CH3 H HO O H H H H H HO H H OH H O OH OH H3C OH O

B C

Sequence of sugar residues

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Dipolar interactions - NOE - (Sequence information - interresidue)


Connects the residues

H CH2OH
HO HO

H CH3
O

HO
O

OH

H H

H H

H H
OH

OH

NOESY or ROESY
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Dipolar interactions - NOE - (anomeric protons -intraresidue)


Through space short distances

H CH2OH
HO HO

H CH3
O

HO
O

OH

H H

H H H

H
OH

OH

NOESY or ROESY
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Three-bond coupling 3JC,H - (Sequence information)

H CH2OH
HO HO

H CH3
O

HO
O

OH

H H

H H H

H
OH

OH

H H CH3
O

HMBC

H CH2OH
HO HO

HO
O

OH

H H

H H

H
OH

OH

Also 4-bond H,H-coupling


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1H

NMR spectrum of a polysaccharide can be divided into regions showing recognizable signals

characteristic for the sugar residues functional groups present

anomeric protons

CH3- groups ring protons

N- & O-acetyl groups

O HC O HC O HC
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1D-NMR of polysaccharides

Viscous solution = broad signals

Complex spectrum many overlapping signals

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1D-NMR Following an enzymatic hydrolysis of three disaccharides

and an a-L-fucosidase
Fuca12GalbOMe Fuca13GalbOMe

Fuca16GalbOMe

T=0
b-L-Fucose

a-L-Fucose

T = 12 H

T = 24 H

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1D-NMR

Polysaccharide from an Plesiomonas LPS


O H O CH3 H H H3C H H O H H HO H NH H CH3

HO H O H H H3C H HO HN H O H O OH

O HO

NH

O H H

PS

core
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Two-dimensional NMR
COSY
A
H CH2OH
HO HO

H-1/H-2

H-2/H-3
B
H CH2OH

H-3/H-4

H-4/H-5

H-5/H-6a,6b

H H

HO O H

OH

H H

OH H

H
OH

H-1

H-2

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Two-dimensional NMR
COSY TOCSY H-1 H-2 H-1 H-2 H-3 H-4 H-5 .....

NOESY

through space

HMQC

C-H

HMBC
H CH2OH
HO HO

C-C-H and/or C-X-C-H (2 or 3 bonds)


H CH3
O OH

H H

HO O H

H H

H OH

H
OH
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TOCSY

H H3C HO HN O H HO NH H O H HO H O H O

H3C

H H O H H HO H H O CH3 NH H O CH3 H O

H OH

H O

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HMQC H,C-correlated

H H3C HO HN O H HO NH H O H HO H O H O

H3C

H H O H H HO H H O CH3 NH H O CH3 H O

H OH

H O

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H H3C HO HN O H HO NH H O H HO H O H O

H3C

H H O

HMBC
O H H HO H H O CH3 NH H CH3 H O H

OH

H O

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NOESY
H H3C HO HN O H HO NH H H H O H O H OH H HO H O H O H3C H H O H H HO H O CH3 NH H O CH3 H O

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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

HPLC 1

NMR

Structure information

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LC SPE - NMR
MS
Advantages

Change solvent
HPLC 1 SPE SPE SPE SPE SPE

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
HO H H O O O HO H H OH H H HO H H HO H O H

= not observed but in 85% H2O / 15% aceton-d6 the OH protons can be observed
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OH

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.
H O H O H
2' OH O HO OH OH OH O 1' 4 OH O

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Detection of hydrogen bond interaction

a-D-Glcp
HO O 5" HO HO

OH OH

OH OH

b-L-Fucp

Me
HO OH O OH O 5" HO OH O

a-D-Galp
OH O

a-D-Galp
O

HO

2'
O

OH O

HO

2'
O

OH

O OH OMe

a-D-Glcp

OH OMe

a-D-Glcp

Dd = - 0.852 ppm 3 JH,OH = 10.3 Hz dd / dT = 4.8 ppb/K

Dd = - 1.438 ppm 3 JH,OH = 3.5 Hz dd / dT = 5.5 ppb/K

Average values for other hydroxy protons Dd = 0.2 | ppm 3 JH,OH = 5.5 Hz dd / dT =10 ppb/K

Hydrogen bonding Chemical exchange


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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 ().
HO H H H O H O O HO H H OH H H CH2OH H H OH H OH

H-O-Me
5.5 5.3 5.1 4.9 4.7 4.5 4.3 4.1 3.9

HO

O(3)H of Me a-D-Galp

Chemical Shift (ppm)

O(3)H of Me a-D-Galp
3.7 3.5 3.3 3.1 0.0 0.2 0.4 0.6 0.8 1.0 Mole Fraction of Methanol

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Structure analysis with two-dimensional NMR


COSY TOCSY H-1 H-2 H-1 H-2 H-3 H-4 H-5 .....

NOESY

through space

HMQC

C-H

HMBC
H CH2OH
HO HO

C-C-H and/or C-X-C-H (2 or 3 bonds)


H CH3
O OH

H H

HO O H

H H

H OH

H
OH
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B0
HO H H O OH HO HO H H OH H

Molecules in dilute solutions can tumble and thus average out several negative effects as chemical shift anisotropy and dipolar couplings

5.5

5.0

4.5
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Results in high-resolution NMR spectra

B0
HO H H O OH HO HO H H OH H

Too broad to be seen

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B0

Different shielding effects depending on the different surroundings of the molecules


Chemical shift anisotropy

HO H H O OH

HO HO H H

OH H

Very broad lines

NO TUMBLING causes dipolar couplings and CSA 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
rotor cap

air
rotor spacer sealing screw

Spinning at magic angle removes effects of dipolar interactions and chemical shift anisotropy

Q
spinning 2-15 kHz

sample in D2O ( 10-30 ml) rotor

improved linewidths

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 n=500

n=1

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T2-filter (t-180-t)n

Artefacts generated by the filter

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
Trehalose Glycerol Gln Glu

Arabitol

Ethanol

O2-limitations
Trehalose Arabitol Glycerol

61 Exo- and intra-cellular metabolites compared by GC and HR-MAS NMR

HR-MAS - a non-destructive method?


HO O HO O HO HO HO O OH O HO OH O HO OH

HO O HO O HO HO O HO HO OH O HO OH HO O

O CH2OH

Increased amounts of microthecin after 16 h of spinning of alga (>5000 Hz)

OH O

microthecin

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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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Normal

Irradiation

B0

Irradiation/saturation

OH
a-L-fucosidase

HN

OH

CH2OH

OH

1-Deoxymannojirimycin (DMJ)

- inhibitor

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Normal spectrum

Saturation
Difference spectrum

OH

OH
a-L-fucosidase

HN

OH

HN

OH

H2C OH

OH

H2C OH

OH
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1-Deoxymannojirimycin (DMJ)

NMR imaging

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Bruker Daltonics ESI-Ion Trap MS (esquire3000plus and esquire2000), APEX Fourier Transform Mass Spectrometer (FTMS), BioTOF II Time-of-Flight mass spectrometer.

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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

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