C8953
NMR strukturní analýza
semináˇr
Introduction to heteronuclear correlations
Jan Novotný
novotnyjan@mail.muni.cz
March 25, 2020
Polarization transfer
bigger population difference of 1H nucleus is transfered via J-coupling to less
sensitive nucleus X (13C, 15N)
fundamental building block of heteronucler correlation experiments: in 2D-HX
experiment each crosspeak manifests interaction of H and X nucleus coupled
trough bonds
Task: Draw the evolution of magnetization during basic INEPT pulse
sequence. Consider C-H interacting pair.
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
2D spectrum with X= 13C or 15N chemical shift in indirect dimension (f1) and 1H in
direct dimension (f2)
heteronuclear correlation ⇒ no diagonal crosspeak, no symmetry
X decoupled during acquisition ⇒ singlet crosspeak
resolution of overlapped 1H signals
routine experiments to control biomolecular sample (in proteins each aminoacid is
represented by N-H crosspeak)
easy identiﬁcation of geminal diastereotopic protons (CH2)
indirect determination of protons bonded to NMR inactive heteroatom
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
small nJHX means longer evolution of polarisation transfer - sensitivity drops
because of relaxation
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)
in real HMBC spectrum, single bond (HSQC-like) correlations might be partially
visible as dublets
1
H
X
acq
t1/2 t1/2
90 90
90 180
d1 d2
90
1H-13C HMBC + 1H-13C HSQC
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 + 1H-13C HSQC
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 + 1H-13C HSQC
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 + 1H-13C HSQC
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
Folic Acid: 1H 1D
N
N
N
N
O
NH2
N
H
O
O
O
–
O
–
O
NH
1
2
3
4
5
6
7
8
9
10 11 12
13
14
15
16
17
18
19
20
21
22
23
2425
Folic Acid: 13C 1D
N
N
N
N
O
NH2
N
H
O
O
O
–
O
–
O
NH
1
2
3
4
5
6
7
8
9
10 11 12
13
14
15
16
17
18
19
20
21
22
23
2425
Folic Acid: 1H-15N HMBC
8
8
6
6
4
4
2
2
δ2 - 1
H (ppm)
300 300
250 250
200 200
150 150
100 100
δ1-15
N(ppm)
N
N
N
N
O
NH2
N
H
O
O
O
–
O
–
O
NH
1
2
3
4
5
6
7
8
9
10 11 12
13
14
15
16
17
18
19
20
21
22
23
2425
Folic Acid: 1H-13C HMBC
8
8
6
6
4
4
2
2
δ2 - 1
H (ppm)
150 150
100 100
50 50
δ1-13
C(ppm)
N
N
N
N
O
NH2
N
H
O
O
O
–
O
–
O
NH
1
2
3
4
5
6
7
8
9
10 11 12
13
14
15
16
17
18
19
20
21
22
23
2425
Folic Acid: 1H a 13C 1D
N
N
N
N
O
NH2
N
H
O
O
O
–
O
–
O
NH
1
2
3
4
5
6
7
8
9
10 11 12
13
14
15
16
17
18
19
20
21
22
23
2425
H23'H23''
H24
H21
H11
H8
H15,17
H14,18
C23
C24
C11
C21C14,18
C13
C15,17
C5
C8,9
C16
C4
C2
C25
C22
C19
C6
Folic Acid: 1H-15N HMBC
8
8
6
6
4
4
2
2
δ2 - 1
H (ppm)
300 300
250 250
200 200
150 150
100 100
δ1-15
N(ppm)
N
N
N
N
O
NH2
N
H
O
O
O
–
O
–
O
NH
1
2
3
4
5
6
7
8
9
10 11 12
13
14
15
16
17
18
19
20
21
22
23
2425
N20-H23'
N20-H23''
N20-H21
N12-H21
N12-H14,18N12-H15,17
N7-H8
N10-H8
N7-H11
N10-H11
Folic Acid: 1H-13C HMBC
8
8
6
6
4
4
2
2
δ2 - 1
H (ppm)
150 150
100 100
50 50
δ1-13
C(ppm)
C25-H23'C25-H23''
C25-H24
C22-H23'
C22-H23''
C21-H23'
C21-H23''
C21-H24 C24-H23'
C24-H23''
C24-H24
C23-H23''
C23-H23'
C23-H21
C24-H21
C21-H21
C11-H11
C11-H8
C22-H21
C19-H21
C5-H8
C8-H8
C9-H8
C8-H11
C9-H11
C4-H8
C5-H11
C4-H11
C16-H11
C6-H8 C19-H15,17 C19-H14,18
C16-H15,17
C15,17-H15,17
C13-H14,18
C14,18-H14,18
C14,18-H15,17
N
N
N
N
O
NH2
N
H
O
O
O
–
O
–
O
NH
1
2
3
4
5
6
7
8
9
10 11 12
13
14
15
16
17
18
19
20
21
22
23
2425
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
δ1-13C(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: DQF-COSY
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: NOESY
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
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
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
C11-H11
C12-H12
C13-H17''
C17-H17'
C14-H17''C16-H16
C15-H15
C17-H17''
C15-H14
C11-H6
C12-H9
C9-H6
C9-H9
C6-H6C6-H9
H12-C9 H11-C9
H12-C6 H11-C6
H12-C7 H11-C7
H12-C8 H11-C8
C7-H6
C8-H9
C9a-H13'
C5a-H9
C5a-H6
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
δ1-13C(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
C11-H11
C12-H12
C13-H17''
C17-H17'
C14-H17''
C16-H16
C15-H15
C17-H17''
C15-H14
C11-H6
C12-H9
H12-C9 H11-C9
H12-C6 H11-C6
H12-C7 H11-C7
H12-C8 H11-C8
C9a-H13'
C9-H6
C9-H9
C6-H6C6-H9
C7-H6
C8-H9
C5a-H9
C5a-H6
C14-H14
C13-H13'C13-H13''
Next session:
Complex exercises