C8953
NMR strukturní analýza
semináˇr
TOCSY, ROESY, Introduction to heteronuclear correlations
Jan Novotný
176003@is.muni.cz
April 12, 2017
TOCSY & ROESY
spin lock - isotropic mixing
� series of 180 ◦ pulses
� various pulse schemes (MLEV-17,
DIPSI,. . . )
� precession around BSpinlock < B0 →
“locking" spins in transversal plane
(xy)
� TOCSY: lower power, offset in the
center of spectrum
� ROESY: higher power, offset on edge
� crosstalk (ROE in TOCSY, J in
ROESY)
acq
t1 t2
90
P E M D
tmix
Spin Lock
TOCSY (TOtal Correlation SpectroscopY)
HOHAHA (HOmonuclear HArtmann-HAhn)
correlation based on J, like COSY
� correlate mutually all protons within a spin system
� τmix ≈ 20 − 120 ms
� intensity depends on τmix and J value
HA
JAB
⇐⇒ HB
JBC
⇐⇒ HC
JCD
⇐⇒ HD
Biotin - COSY vs. ROESY
(G. T. Crisp and Yu-Lin Jiang, 2001)
N1H
N3H
H3a
H6a
H6x
H6y
H4 H5'
H4'
H3'
H2'
O
NN
HH
S
HH
COOH
N1H
N3H
H3a
H6a
H6x
H6y
H4 H5'
H4'
H3'
H2'
O
NN
HH
S
HH
COOH
Biotin - COSY vs. ROESY
(G. T. Crisp and Yu-Lin Jiang, 2001)
N1H
N3H
H3a
H6a
H6x
H6y
H4 H5'
H4'
H3'
H2'
O
NN
HH
S
HH
COOH
N3HN1H H6a H3a H4 H5' H4'
H2'
H3'H6y H6x
N1H
N3H
H3a
H6a
H6x
H6y
H4 H5'
H4'
H3'
H2'
O
NN
HH
S
HH
COOH
N3HN1H H6a H3a H4 H5' H4'
H2'
H3'H6y H6x
HMQC (Heteronuclear Multiple Quantum Correlation)
HSQC (Heteronuclear Single Quantum Correlation)
correlate 1
H-X (X=13
C, 15
N,...) based on
1
JHX
HMQC (a)
+ more robust experiment
+ change of parameters - HMBC
- lower sensitivity and worse resolution
HSQC (b)
+ better resolution, sensitivity
+ part of more complex mutlidimensional
experiments
- less robust
H-1
X
acq
dec
90
90 90
180
1/2J 1/2J
t1
(a)
H-1
X
acq
dec
t1
d d d d
d = 1/4J
(b)
Practical notes 1H-X HSQC
� resolution of overlaps
� routine experiments to control biomolecular sample
� easy identification of geminal protons
� indirect determination of protons bonded to NMR inactive heteroatom
� heteronuclear correlation ⇒ no diagonal crosspeak, no symmetry
� X decoupled during acquisition ⇒ singlet crosspeak
HMBC(Heteronuclear Multiple-Bond Correlation)
heteronuclear correlation based on long-range H-X spin-spin
interaction( n
JHX , n>1 )
� utilizes polarization transfer from H through 2-5 bonds on heteroatom ( 13C, 15N )
� allows to detect quaternary heteroatoms (Cq) or connect signals among isolated
spin systems
HMBC
correct settings of d1, d2 fo evolution of J-coupling necessary
� d1=1/2∗1JC−H - (120-180 Hz)
� d2=1/2∗2−5JC−H - (3-12 Hz)
1
H
X
acq
t1/2 t1/2
90 90
90 180
d1 d2
90
1H-13C HMBC
H4-C4
H2-C2
H10-C10
H9-C9
H7-C7
H8-C8
H7-C9
H7-C8
H7-C10
H9-C10
H2-C7
H4-C6
H2-C6
H4-C1 H7-C1
H7-C2
H9-C1
1H-13C HMBC
H4-C4
H2-C2
H10-C10
H9-C9
H7-C7
H8-C8
H7-C9
H7-C8
H7-C10
H9-C10
H2-C7
H4-C6
H2-C6
H4-C1 H7-C1
H7-C2
H9-C1
1H-13C HMBC
H7-C9
H7-C8
H7-C10
H9-C10
H2-C7
H4-C6
H2-C6
H4-C1 H7-C1
H7-C2
H9-C1
H4-C4
H2-C2
H10-C10
H9-C9
H7-C7
H8-C8
1H-13C HMBC
H4-C4
H2-C2
H10-C10
H9-C9
H7-C7
H8-C8
H7-C9
H7-C8
H7-C10
H9-C10
H2-C7
H4-C6
H2-C6
H4-C1 H7-C1
H7-C2
H9-C1
Colchicine 1D-1H
Colchicine - 1H-13C HSQC
8
8
6
6
4
4
2
2
δ2 - 1H (ppm)
120 120
100 100
80 80
60 60
40 40
20 20δ1-13C(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
δ1-1
H(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
δ1-1
H(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''
Colchicine - 1H-13C HSQC
8
8
6
6
4
4
2
2
δ2 - 1H (ppm)
120 120
100 100
80 80
60 60
40 40
20 20δ1-13C(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
H16-C16
H12-C12
H13-C13
H1-C1
H9-C9
H22-C22
H8'/''-C8
H7'/''-C7
H19-C19
H21-C21
H20-C20
H23-C23
Riboflavine: DQF-COSY
8
8
6
6
4
4
2
2
δ2 - 1H (ppm)
8 8
7 7
6 6
5 5
4 4
3 3
δ1-1
H(ppm)
5a
9a
6
9
7
8
4a
10a
N
5
N
10
NH
3
2
4
N
1
13
14
15
16
17
OH
OH
OH
OH
O
O
CH3
11
CH3
12
Riboflavine: 1H-13C HSQC
8
8
6
6
4
4
2
2
δ2 - 1
H (ppm)
120 120
100 100
80 80
60 60
40 40
20 20
δ1-13C(ppm)
5a
9a
6
9
7
8
4a
10a
N
5
N
10
NH
3
2
4
N
1
13
14
15
16
17
OH
OH
OH
OH
O
O
CH3
11
CH3
12
Riboflavine: NOESY
8
8
6
6
4
4
2
2
δ2 - 1
H (ppm)
8 8
7 7
6 6
5 5
4 4
3 3
δ1-1
H(ppm)
5a
9a
6
9
7
8
4a
10a
N
5
N
10
NH
3
2
4
N
1
13
14
15
16
17
OH
OH
OH
OH
O
O
CH3
11
CH3
12
Riboflavine: 1H-13C HMBC
8
8
7
7
6
6
5
5
4
4
3
3
δ2 - 1H (ppm)
150 150
100 100
50 50
δ1-13
C(ppm)
5a
9a
6
9
7
8
4a
10a
N
5
N
10
NH
3
2
4
N
1
13
14
15
16
17
OH
OH
OH
OH
O
O
CH3
11
CH3
12
Riboflavine: 1H-13C HMBC + HSQC
8
8
7
7
6
6
5
5
4
4
3
3
δ2 - 1H (ppm)
150 150
100 100
50 50
δ1-13
C(ppm)
5a
9a
6
9
7
8
4a
10a
N
5
N
10
NH
3
2
4
N
1
13
14
15
16
17
OH
OH
OH
OH
O
O
CH3
11
CH3
12
Riboflavine: DQF-COSY
8
8
6
6
4
4
2
2
δ2 - 1H (ppm)
8 8
7 7
6 6
5 5
4 4
3 3
δ1-1
H(ppm)
5a
9a
6
9
7
8
4a
10a
N
5
N
10
NH
3
2
4
N
1
13
14
15
16
17
OH
OH
OH
OH
O
O
CH3
11
CH3
12
Me12
H6
H9
Me11
H13''
H13'
H14
H15
H17'
H16, H17''
Riboflavine: NOESY
8
8
6
6
4
4
2
2
δ2 - 1
H (ppm)
8 8
7 7
6 6
5 5
4 4
3 3
δ1-1
H(ppm)
5a
9a
6
9
7
8
4a
10a
N
5
N
10
NH
3
2
4
N
1
13
14
15
16
17
OH
OH
OH
OH
O
O
CH3
11
CH3
12
H6
H9
H13''
H13'
H14
H14-H13''
H14-H13''
H15-H13'
H15
H16, H17', H17''
H9-H13'' H9-H13'
H9-H14
H11
H12
H9-H12
H6-H11
Riboflavine: 1H-13C HSQC
8
8
6
6
4
4
2
2
δ2 - 1
H (ppm)
120 120
100 100
80 80
60 60
40 40
20 20
δ1-13C(ppm)
5a
9a
6
9
7
8
4a
10a
N
5
N
10
NH
3
2
4
N
1
13
14
15
16
17
OH
OH
OH
OH
O
O
CH3
11
CH3
12
Next session:
Midterm test + Complex exercises