C8953
NMR strukturní analýza
semináˇr
COSY,NOESY
Jan Novotný
176003@is.muni.cz
March 18, 2020
NOESY - introduction
Nuclear Overhauser effect
dipol-dipol interaction
magnetisation transfer TROUGH
SPACE as a consequence of
cross-relaxation
NOESY
correlates nuclei if their distance is
smaller than 5 Å
acq
t1 t2
90 9090
P E M D
tmix
NOE vs. size of a molecule
Correlation time τc
ω0τc < 1 ⇔ ω0
1
f
< 1 ⇔ ω0 < f (small
molecules 1 kDa)
fast molecular motion, ββ → αα
dominates ⇒ W2 > W0
positive NOE
crosspeaks have opposite phase
relative to diagonal
ω0τc > 1 (large molecules 1 kDa)
slow molecular motion, αβ → βα
dominates ⇒ W0 > W2
negative NOE
crosspeaks have the same phase
ω0τc ≈ 1 (cca 1 kDa)
NOE≈0 - no crosspeaks
ROESY
Application of NOESY
Mixing time τmix
small molecules τmix ≈ 500 − 800 ms
biomolecules τmix ≈ 50 − 300 ms
approximative determination of interatomic distatces (< 5 Å)
at short τmix
rij≈A×Iij
0.2 0.4 0.6 0.8 1
Intensity
τmix
NOE differential experiment
NOE differential experiment
NOESY - Palmatine
9
9
8
8
7
7
6
6
5
5
4
4
3
3
δ2 - 1H (ppm)
9 9
8 8
7 7
6 6
5 5
4 4
3 3
δ1-1H(ppm)
4
1
3
2
6
N
+
5
8
13
9
10
1112
O
O
CH3
CH3
O
O
CH3
CH3
NOESY - Palmatine
9
9
8
8
7
7
6
6
5
5
4
4
3
3
δ2 - 1H (ppm)
9 9
8 8
7 7
6 6
5 5
4 4
3 3
δ1-1H(ppm)
4
1
3
2
6
N
+
5
8
13
9
10
1112
O
O
CH3
CH3
O
O
CH3
CH3
Eserine 1H
NOESY - Eserine
6
6
4
4
2
2
δ2 - 1
H (ppm)
7 7
6 6
5 5
4 4
3 3
2 2
δ1-1
H(ppm)
NOESY - Eserine
6
6
4
4
2
2
δ2 - 1
H (ppm)
7 7
6 6
5 5
4 4
3 3
2 2
δ1-1
H(ppm)
H15-H16
H12-H19
H11-H12
H9-H17
NOESY - Eserine
4.0
4.0
3.5
3.5
3.0
3.0
2.5
2.5
2.0
2.0
1.5
1.5
δ2 - 1
H (ppm)
4.0 4.0
3.5 3.5
3.0 3.0
2.5 2.5
2.0 2.0
1.5 1.5δ1-1
H(ppm)
NOESY - Eserine
4.0
4.0
3.5
3.5
3.0
3.0
2.5
2.5
2.0
2.0
1.5
1.5
δ2 - 1
H (ppm)
4.0 4.0
3.5 3.5
3.0 3.0
2.5 2.5
2.0 2.0
1.5 1.5δ1-1
H(ppm)
H5-H17
H2'-H3''
H2''-H3'
H2''-H2'
H3''-H3'
H3'-H17
H19-H18
H5-H19
H5-H18
H5-H3''
H5-H2''
ROESY - GSSG
8
8
6
6
4
4
2
2
δ2 - 1H (ppm)
8 8
6 6
4 4
2 2
δ1-1
H(ppm)
ROESY - GSSG
8
8
6
6
4
4
2
2
δ2 - 1H (ppm)
8 8
6 6
4 4
2 2δ1-1
H(ppm)
CysHa-GlyNH
GlyHa-NH
CysHa-NH
CysHb’-NH
GluHc-CysNH
GluHc-Ha
GluHb-Ha GluHb-Hc
GluHc-Hb
GluHa-Hb
GluHa-Hc
CysHb’’-Hb’
CysHb’-Hb’’
CysHb’’-Ha
CysHb’-Ha
CysHa-Hb’
CysHa-Hb’’
CysNH-Hb’
CysNH-Ha
GlyNH-CysHa
GlyNH-Ha
CysNH-GluHc
GluHb
GluHc
CysHb’
CysHb’’
GluHa
GlyHa
CysHa
GlyNH
CysNH
GluHb
GluHc
CysHb’
CysHb’’
GluHa
GlyHa
CysHa
GlyNH
CysNH
Colchicine 1D-1H
Colchicine - DQF-COSY
8
8
6
6
4
4
2
2
δ2 - 1H (ppm)
8 8
6 6
4 4
2 2
δ1-1H(ppm)
54
6
3
1
2
11
10 16
14
15
7
8
9 NH
17
18CH3
19
O
O
O O
CH3
22
CH3
21
CH3
20
O
CH3 23
O
12
13
Colchicine - DQF-COSY
8
8
6
6
4
4
2
2
δ2 - 1H (ppm)
8 8
6 6
4 4
2 2
δ1-1H(ppm)
54
6
3
1
2
11
10 16
14
15
7
8
9 NH
17
18CH3
19
O
O
O O
CH3
22
CH3
21
CH3
20
O
CH3 23
O
12
13
H17-H9
H9-H8'
H7''-H7'
H7'-H8''
H17-H9
H12-H3
Colchicine - NOESY
8
8
6
6
4
4
2
2
δ2 - 1H (ppm)
8 8
6 6
4 4
2 2
δ1-1H(ppm)
54
6
3
1
2
11
10 16
14
15
7
8
9 NH
17
18CH3
19
O
O
O O
CH3
22
CH3
21
CH3
20
O
CH3 23
O
12
13
Colchicine - NOESY
8
8
6
6
4
4
2
2
δ2 - 1H (ppm)
8 8
6 6
4 4
2 2
δ1-1H(ppm)
54
6
3
1
2
11
10 16
14
15
7
8
9 NH
17
18CH3
19
O
O
O O
CH3
22
CH3
21
CH3
20
O
CH3 23
O
12
13
H17-H16
H17-H19
H1-H20
H1-H7''
H13-H23
H12-H22
H21
H9-H8''
Next session:
? Heteronuclear correlation