Professional Documents
Culture Documents
Lennart Kenne
Department of Chemistry, SLU, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden
November 2005
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
3
Studies of carbohydrates
Isolated material Carbohydrates on solids Carbohydrates in their natural environment
When can NMR be used?
4
The basic
NMR experiment
1H
HO OH H H HO H H OH H
5
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
H CH3 OH H H H O H OH
H OH
H CH2OD
H CH3
O
DO DO
H H
DO O H
OD
H H
H OD
H
OD
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
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
9
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
10
Carbohydrates / NMR
Chemical shifts
H CH2OH
HO HO
H H
HO O H
OH
H H
H OH
H
OH
d 4.5 ppm
d 5 ppm
11
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 H
H H
OH H
1.5 Hz
7.5 Hz
OH
H
H
60 degr 1-4 Hz
13
B
H CH2OH
H H
HO O H
OH
H H
OH H
H
OH
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
15
HO HO
H CH OH 2 H O H H H OH OH
H H
H H
a b
16
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
17
Chemical shifts
H CH2OH
HO HO
H CH3
O
H H
HO O H
OH
H H
H OH
H
OCH3
+ 8 ppm
Substituted carbon +4-10 ppm (Depending on stereochemistry around the glycosidic bond)
18
H H
H H
OH H
1.5 Hz
7.5 Hz
OH
H
H
For mannoconfiguration
1-2 Hz
H
60 degr 1-4 Hz
19
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
20
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
21
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
22
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
23
H CH2OH
HO HO
H CH3
O
HO
O
OH
H H
H H
H H
OH
OH
NOESY or ROESY
24
H CH2OH
HO HO
H CH3
O
HO
O
OH
H H
H H H
H
OH
OH
NOESY or ROESY
25
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
1H
NMR spectrum of a polysaccharide can be divided into regions showing recognizable signals
anomeric protons
O HC O HC O HC
27
1D-NMR of polysaccharides
28
and an a-L-fucosidase
Fuca12GalbOMe Fuca13GalbOMe
Fuca16GalbOMe
T=0
b-L-Fucose
a-L-Fucose
T = 12 H
T = 24 H
29
1D-NMR
HO H O H H H3C H HO HN H O H O OH
O HO
NH
O H H
PS
core
30
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
31
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
H H
HO O H
H H
H OH
H
OH
32
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
33
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
34
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
35
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
36
LC-NMR
HPLC 1
NMR
Structure information
38
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
39
40
41
NMR analysis
1D and 2D
0.1 - 1.5 mg
42
= not observed but in 85% H2O / 15% aceton-d6 the OH protons can be observed
43
OH
44
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
45
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
Average values for other hydroxy protons Dd = 0.2 | ppm 3 JH,OH = 5.5 Hz dd / dT =10 ppb/K
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
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
47
NOESY
through space
HMQC
C-H
HMBC
H CH2OH
HO HO
H H
HO O H
H H
H OH
H
OH
48
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
49
B0
HO H H O OH HO HO H H OH H
50
B0
HO H H O OH
HO HO H H
OH H
NO TUMBLING causes dipolar couplings and CSA Tilting the sample at 54.7 o and spinning at high speed overcome these problems (1-3cos2q)
51
air
rotor spacer sealing screw
Spinning at magic angle removes effects of dipolar interactions and chemical shift anisotropy
Q
spinning 2-15 kHz
improved linewidths
- Analysis of small molecules or biopolymers that are mobile in the cells or in a semi-solid systems.
52
n=1
53
T2-filter (t-180-t)n
multiplets
Intensity differences
54
55
56
57
58
Pichia anomala
5000 Hz
59
metabolism?
- Extract or analyse intact cells? - NMR needs normally a homogenious sample in solution.
60
Arabitol
Ethanol
O2-limitations
Trehalose Arabitol Glycerol
HO O HO O HO HO O HO HO OH O HO OH HO O
O CH2OH
OH O
microthecin
62
63
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.
Normal
Irradiation
B0
Irradiation/saturation
OH
a-L-fucosidase
HN
OH
CH2OH
OH
1-Deoxymannojirimycin (DMJ)
- inhibitor
65
Normal spectrum
Saturation
Difference spectrum
OH
OH
a-L-fucosidase
HN
OH
HN
OH
H2C OH
OH
H2C OH
OH
66
1-Deoxymannojirimycin (DMJ)
NMR imaging
67
68
Bruker Daltonics ESI-Ion Trap MS (esquire3000plus and esquire2000), APEX Fourier Transform Mass Spectrometer (FTMS), BioTOF II Time-of-Flight mass spectrometer.
69
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
70