Structure Analysis of Polysacharides by NMR

8/4/2019 Structure Analysis of Polysacharides by NMR

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Structure analysis of polysaccharides byNMR

Lennart Kenne

Department of Chemistry, SLU, Swedish University of AgriculturalSciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden


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1. Structural information needed for carbohydrates

2. Information from NMR

3. Structure analysis by NMR

4. Modern NMR methods some applications

NMR Spectroscopy in Glycoscience


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Oligo- and polysaccharides

Structure Components

Linkages

Sequence

Conformation

Properties Interactions with solvent or other

molecules as proteins

Information on structure and properties

HO

O

H

H

HO

H

O

NHHH

OH

HO

O

H

H

OH

H

HH

H3C

O

H

O

H

HO

H

H

OHHOH

OHO

CH3


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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

H

O

H

HO

H

H

OHH

OH

OH

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


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FIDB0

NMR parameters

Information

Chemical shiftsChemical surrounding

Coupling constantsStereochemistry

IntensitiesNumber of atoms / molar ratio

Relaxation times T1 and T2Dynamic properties

NOE (nuclear Overhauser effect)Interatomic distances and dynamic properties


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OO

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

HH H

HO

H

For most

NMR experiments

OO

O

CH2OD

DO

DO

OD

CH3

OD

OD

H

H

H

H

H

H

HHH

DO

H

Sample dissolved in D2O

Deuterated solventgives no signal andlocks the frequencies

Substituted sugars other solvents


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HO

O

H

H

HO

H

O

NHHH

OH

HO

O

H

H

OH

H

HH

H3C

O

H

O

H

HO

H

H

OHHOH

OHO

CH3

StructureComponentsLinkages

SequenceConformation

Component

Which sugar

Anomeric configuration

Absolute configuration

Substituents


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O

OH

CH2OH

HO

HO

OHH

H

H

H

H

O

OH

CH2OH

HO

HO

OHH

H

H

H

H

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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O

OH

CH2OH

HO

HO

OHH

H

H

H

H

O

OH

CH2OHHO

HO

OHH

H

H

H

H

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

C b h d t / NMR


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Equatorial proton +0.6 ppm(axial proton)

OO

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

HHH

HO

H

d4.5 ppmd5 ppm

Chemical shifts - anomeric proton signals

Proton on a carbon linked to two oxygens

Carbohydrates / NMR


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OO

HO

CH2OH

HO

HO

OH

CH2OH

OH

OH

H

H

H

H

H

H

HHH

HO

H

A B

OH

Chemical shifts

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 3.90B 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

b-D-Glc b-D-Man


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180 degr

7-10 Hz

60 degr

1-4 Hz

O O

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

HH

H

H

H

H

H

H

HO

1.5 Hz

7.5 Hz

Coupling constants - 3JH,H


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OO

O

CH2OH

HO

HO

OH

CH2OH

OH

OH

H

H

H

H

H

H

HH

H

HO

H

A B


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

A 7.5 10 10 10 2 5 12B 1.5 3 10 10 2 5 12


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O

OH

CH2OH

HO

HO

OHH

H

H

H

H

O

OH

CH2OH

HO

HO

OHH

H

H

H

H

C-1 C-2 C-3 C-4 C-5

b-Glc 96.8 75.2 76.8 70.7 76.8

a-Glc 93.0 72.5 73.8 70.7 72.4Difference -3.8 -2.7 -3.0 0 -4.4


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O

OH

CH2OH

HO

HO

OHH

H

H

H

H

H

H

H

H

H

a

b -gauche effect = -4 ppm


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O

OH

CH2OH

HO

HO

OHH

H

H

H

H

O

OH

CH2OHHO

HO

OHH

H

H

H

H

C-1 C-2 C-3 C-4 C-5

b-Glc 96.8 75.2 76.8 70.7 76.8

b-Gal 97.4 73.0 73.8 69.7 75.9Difference +0.6 -2.2 -3.0 -1.0 -0.9


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OO

O

CH2OH

HO

HO

OH

CH3

OCH3

OH

H

H

H

H

H

H

HHH

HO

H

+ 8 ppm

Chemical shifts

Substituted carbon +4-10 ppm

(Depending onstereochemistry around theglycosidic bond)

Anomeric carbon d 100-104 and 96-99 ppm

For mannoses almostno difference


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180 degr

7-10 Hz

60 degr

1-4 Hz

O O

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

HH

H

H

H

H

H

H

HO

1.5 Hz

7.5 Hz


For manno-configuration

1-2 Hz


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1JC,H = 170 Hz

Equatorial proton

O O

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

H H

H

H

HO

Coupling constants - 1JC,H - (Anomeric configuration)

1JC,H = 160 Hz

Axial proton


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HO

O

H

H

HO

H

O

NHHH

OH

HO

O

H

H

OH

H

HH

H3C

O

H

O

H

HO

H

H

OHHOH

OHO

CH3


Sequence

Linkage position

S b tit ti li k


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Substitution - linkages

O

OH

CH2OHHO

O

OHH

H

H

H

HO

H

CH2OH

HO

HO

OHH

H

H

H

O

OH

CH2OHHO

O

OHH

H

H

H

HO

H

HOH3C

H

H

H

HOH

HO

Glycosylation shifts

Dd-values

Substitution of a carbon = a b

+9 +9 -2.5


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HO

O

H

H

HO

H

O

NHHH

OH

HO

O

H

H

OH

H

H

H

H3C

O

H

O

H

HO

H

H

OHHOH

OHO

CH3


Sequence

Sequence of sugar residues

A

B

C


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O O

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

H

H

H

H

HO

Dipolar interactions - NOE - (Sequence information - interresidue

Connects the residues

NOESY or ROESY


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O O

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

H

H

H

HO

H

Dipolar interactions - NOE - (anomeric protons -intraresidue)

Through space short distances

NOESY or ROESY


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O O

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

H

H

H

HO

H

O O

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

H

H

HO

H

Three-bond coupling3JC,H - (Sequence information)

HMBC

Also 4-bond H,H-coupling


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1D-NMR of polysaccharides

Viscous solution = broad signals

Complex spectrum many overlapping signals

Following an enzymatic hydrolysis of three disaccharides1D-NMR


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Fuca12GalbOMe Fuca16GalbOMeFuca13GalbOMe

T = 0

T = 12 H

T = 24 H

a-L-Fucose

b-L-Fucose

Following an enzymatic hydrolysis of three disaccharidesand an a-L-fucosidase

1D NMR

Polysaccharide from an Plesiomonas LPS1D-NMR


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HO

O

H

HHN

H

HO

H

H3C

O

O

H

O

HHO

H

HNH

H

CH3

OH3C

OH

HO

H

H

HH

H

NHO

O

HO

HH

OCH3

Polysaccharide from an PlesiomonasLPS

corePS

1D NMR

T di i l NMR


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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

OO

O

CH2OH

HO

HO

OH

CH2OH

OH

OH

H

H

H

H

H

H

HH

H

HO

H

A B

H-1 H-2

T di i l NMR


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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)

OO

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

HHH

HO

H


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TOCSY

HO

O

H

H

HN

H

H

OH

H3C

OO

H

O

H

HO

H

H

NHH

CH3

O

H3C

OH

HOH

HH

HH

NH

O

O

HO

HH

O

CH3


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HMQC H,C-correlated

HO

O

H

H

HN

H

H

OH

H3C

O

O

H

O

H

HO

H

H

NHH

CH3

O

H3C

OH

HOH

HH

HH

NH

O

O

HO

HH

O

CH3


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NOESY


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HO

O

H

H

HN

H

H

OH

H3C

OO

H

O

H

HO

H

H

NHH

CH3

O

H3C

OH

HOH

HH

HH

NH

O

O

HO

HH

O

CH3

NOESY


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NMR spectroscopy Carbohydrates

Some available methods

1D NMR

2D NMRLC-NMR

HR-MAS

Saturation Transfer Difference NMR Spectroscopy

NMR imagingSolid-state NMR

LC-NMR Problems for


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HPLC 1

NMR

LC NMR Problems forstructural analysis

Solvent LC - NMRDifferent amountsTime for each compound1D 2D experiments

Structureinformation

LC SPE - NMR


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HPLC 1 SPE

SPE

SPE

SPE

SPE

LC SPE NMR

NMR

MS Advantages

Change solvent

Remove water

Several runs accumulate

Handling of compounds

Different scales

Manual or automation


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NMR analysis


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1D and 2D

0.1 - 1.5 mg

Multivariate data analysis structure analysis

Studies of carbohydrates by NMR


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Studies of carbohydrates by NMR

Carbohydrates in their natural environment The role of the hydroxyl groups

O

OH

H

HO

H

H

OHH

O

HO

H

O

H

HO

H

OOH

H

H

HO

Normally 1H NMR inD2O fast exchange

of hydroxyl protons

= not observed

but in 85% H2O /15% aceton-d6 theOH protons can beobserved

Sample preparationRemove ions that can


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Remove ions that canincrease the exchange

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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O

O

O

OH

OH

O

HOOH

OH

O

1'

4

OH

2'H

HH

H

g p

Detection of hydrogen bond interaction


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O

O

OMe

O

O

OH

OH

OH

OH

O

HOHO

OH

Me

OHHO

O

O

OMe

O

HOOH

OH

OHHO

HO

O

OH

OH

O

OH

HO

a-D-Galp

a-D-Glcp

a-D-Glcp

a-D-Galp

a-D-Glcp b-L-Fucp

5"

5"

2'

2'

Dd=- 0.852 ppm3JH,OH = 10.3 Hz

dd / dT = 4.8 ppb/K

Dd=- 1.438 ppm3JH,OH = 3.5 Hz

dd / dT = 5.5 ppb/K

Average values for other hydroxy protonsDd = 0.2 | ppm3JH,OH = 5.5 Hz

dd / dT =10 ppb/K

Hydrogen bondingChemical exchange

Chemical shift of the hydroxy proton of methanol as a function of the mole fraction of

methanol in water (), diethyl ether (), tetrahydrofuran () and dioxane ().


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3.1

3.3

3.5

3.7

3.9

4.1

4.3

4.5

4.7

4.9

5.1

5.3

5.5

0.0 0.2 0.4 0.6 0.8 1.0

Mole Fraction of Methanol

ChemicalShift(ppm)

HO

OH

H

HO

H

H

OHH

O

HO

CH2OH

O

OH

OH

H

H

H

H

H

H

H-O-Me

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

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

Structure analysis with two-dimensional NMR


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y

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)

OO

O

CH2OH

HO

HO

OH

CH3

OH

OH

H

H

H

H

H

H

HHH

HO

H

Molecules in dilute solutionsB0


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Results in high-resolution NMR spectra

Molecules in dilute solutionscan tumble and thus averageout several negative effects

as chemical shift anisotropyand dipolar couplings

HO

O

H

H

HO

H

H

OHH

OH

HO

5.5 5.0 4.5

B


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HO

OH

H

HO

H

H

OHH

OH

HO

B0

Too broad to be seen

BDifferent shielding effects


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HO

O

H

H

HO

H

H

OHH

OH

HO

B0

q

gdepending on thedifferent surroundings ofthe molecules

Chemical shift anisotropy

NO TUMBLING causes dipolar couplings and CSA

Very broad lines

Tilting the sample at 54.7 o and spinning at high speedovercome these problems (1-3cos2q)

HR-MAS NMR -High-Resolution Magic-Angle-Spinning NMR


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- Analysis of small molecules or biopolymers that

are mobile in the cells or in a semi-solid systems.

air

Q = 54.7 ("magic angle")

Q

spinning2-15 kHz

sample in D2O( 10-30 ml)

rotor

sealing screw

rotor spacer

rotor capB0 Spinning at magic angle

removes effects of dipolarinteractions and chemicalshift anisotropy

improved linewidths

T2-filter CPMG pulse sequence - (t-180-t)n


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t =387 ms

n=1

n=500

Artefacts generated by the filterT2-filter

( 180 )


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Artefacts generated by the filter(t-180-t)n

multipletsIntensity differences

DQF-COSY


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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)

TOCSY


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TOCSY

5000 Hz

Ca 1 mg alga

(dry weight)

HMQC


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HMQC

14600 Hz

Ca 1 mg alga

(dry weight)

Pichia anomala


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5000 Hz

Studies of the metabolism


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Pichia anomalainhibit 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.

Pichia anomala living cells


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g

Control

O2-limitations

TrehaloseArabitol

GlnGlu

Ethanol

Glycerol

Trehalose

ArabitolGlycerol

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

HR MAS a non destructive method?


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HR-MAS - a non-destructive method?

HO

O

HOOH

HO

O

O

HOOH

HO

O

O

HOOH

HO

HO

O

HOO

HO

HO

O

HOOH

HO

O

O

HOOH

HO

O

O

CH2OH

OH

microthecin

Increased amounts ofmicrothecin after 16 hof spinning of alga(>5000 Hz)

Gracilariopsis lemaneiformis


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5-15 kHz, over night

1 mg alga (dry weight)

Characterization of Ligand Binding by SaturationT f Diff NMR S t


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Transfer Difference NMR Spectroscopy

Angew. Chem. Int. Ed. 1999, 38, No. 12 1784-1788

The difference between a saturation transfer spectrum and a normal NMRspectrum 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 frommolecules with binding affinity. In addition, the ligands binding epitope is easilyidentified because ligand residues in direct contact to the protein show muchstronger STD signals.

Moriz Mayer and Bernd Meyer

B

Normal Irradiation


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a-L-fucosidaseHN OH

OH

CH2OHOH

1-Deoxymannojirimycin (DMJ) - inhibitor

Irradiation/saturationB0

Normal spectrum


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a-L-fucosidaseHN OH

OH

H2C

OH

OH

HN OH

OH

H2C

OH

OH

Saturation

1-Deoxymannojirimycin (DMJ)

Difference spectrum

NMR imaging
http://images.google.se/imgres?imgurl=www.physics.brocku.ca/~edik/MRI/pax1018.gif&imgrefurl=http://www.physics.brocku.ca/~edik/MRI/fruits.html&h=384&w=381&sz=106&tbnid=oAxdreAMgOEJ:&tbnh=118&tbnw=118&prev=/images%3Fq%3DNMR%2Bimaging%26start%3D20%26hl%3Dsv%26lr%3D%26ie%3DUTF-8%26sa%3DN


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http://images.google.se/imgres?imgurl=www.physics.brocku.ca/~edik/MRI/pax1018.gif&imgrefurl=http://www.physics.brocku.ca/~edik/MRI/fruits.html&h=384&w=381&sz=106&tbnid=oAxdreAMgOEJ:&tbnh=118&tbnw=118&prev=/images%3Fq%3DNMR%2Bimaging%26start%3D20%26hl%3Dsv%26lr%3D%26ie%3DUTF-8%26sa%3DNhttp://www.chemistry.mcmaster.ca/facilities/nmr/images/600-1a-512ds.jpg


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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.
http://www.chemistry.mcmaster.ca/facilities/nmr/images/700b-512.jpghttp://www.chemistry.mcmaster.ca/facilities/nmr/images/600-1a-512ds.jpg


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Structure Analysis of Polysacharides by NMR

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