C8953 NMR strukturní analýza seminář
TOCSY & NOESY
Jan Novotný
176003@is.muni.cz
March 28, 2018
TOCSY (TOtal Correlation Spectroscopy)
spin lock - isotropic mixing
► series of short 180° pulses
► "lock-in" of spins in transversal plane
► higher power in case of TOCSY, offset set into the middle (on resonance)
► smaller power in case of ROESY, offset set into the edge (off resonance)
► crosstalk (ROE transfer in TOCSY, J-coupling in ROESY)
TOCSY (TOtal Correlation Spectroscopy)
correlation based on j
► mutual correlation of all protons in one spin system
► rmix « 20 - 120ms
► crosspeak intensity depends on rmix a J value
u   Jabj I I    Jbc - -    Jcq - -
NAD+: TOCSY (40ms)
82 - 1H (ppm)
NAD+: TOCSY (40ms), detail of aromatics
9.0 8.5 82 - 1H (ppm)
NAD+: TOCSY (40ms), detail of aromatics
9.5
9.0
8.5
8.0
8.0-
8.5-
E
CL CL
9.0-
Nic H6-H5 Nic H4-H5
Nic H2-H5
Nic H6-H4
Nic H2-H4
\ I
Nic H2-H6
\
0
■8.0
9.5
9.0
!
Ade H2
Ade H8
/
■8.5
v Nic
0=
O
*NH2
8
ľ
0=
OH OH
10
NH2
■9.0
7     r J6
Ade N~~ü^^n 8<fT í
60
'X5' „  9^  4 ^N' -o^   I 3
ľ
'S'] [2' OH OH
8.5
8.0
52 - 1H (ppm)
NAD+: TOCSY (40ms), detail of aliphatics
6.5
4.5-
E 5.0-
Cl
CL
5.5-
6.0-
6.5
6.0
6.0
5.5
5.0
5.5 5.0 52 - 1H (ppm)
4.0
■4.5
0-Nic
4
5 ^^3.
*NH2
8
o       31I2'
■5.0
OH OH
10
NH2
Ade 0=P-0
L O
ľ     c    16
■5.5
4'
9? N
ľ
3| |2-OH OH
■6.0
0 »
4.5
4.0
□ ť3? - =
NAD+: TOCSY (40ms), detail of aliphatics
52 - 1H (ppm)
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 A
dAIa dt
= ~Pl(Iz ~
-s°z)
°is = W2-W0
90 90 90
tl       ■ tmix ■ t2
acq
NOE vs. size of a molecule
Correlation time rc
► cc;0tc < 1 <a> u0j < 1 <a> u0 < f (small molecules < 1 kDa)
►
►
fast molecular motion, (3(3 —> aa
dominates     W2 > W0 positive NOE
crosspeaks have opposite phase relative to diagonal
^orc > 1 (large molecules > 1 kDa)
► slow molecular motion, a/3 —> /3a
dominates     W0 > W2
► negative NOE
► crosspeaks have the same phase
uoqtc « 1 (cca 1 kDa)
► NOE^O - no crosspeaks
► ROESY
noe
0.5
-0.5
-1
NOE W2_W°
NOE
Application of NOESY
Mixing time rmjX
► small molecules rm\x « 500 - 800 ms
► biomolecules rmiv « 50 — 300 ms
approximative determination of interatomic distatces (< 5
► at short rmix
► TiiX Iji
0.2        0.4        0.6        0.8 1
^mix
NOE differential experiment
PROBLEM 4
NOE- Difference Spectroscopy
Figure 4.1 shows the 'H NMR and a 'H NOE difference spectrum of a 3-indolylacetic acid derivative 13 bearing a methoxy group at the benzenoic ring.
9
I I     I-1     (■   1 "I-1     I —I   T    I     I     I    -|     I     I     I     r   I     I-T—1-1 -|     I     I     I    T-|     I     I     ,-1" I     I-1     I     I    I I
7.0 6.5 6.0 5.5 5.0 4.5 4.0 5.5
Fig. 4.1. 400 MHz 'H NMR spectrum of 13 in a mixture of CDCI, and CD3OD. a Full spectrum; b expanded section of the aromatic proton signals; c 'H NOE difference spectrum, same section as in b, irradiation position at 8 = 3.64.
NOE differential experiment
PROBLEM 4
NOE- Difference Spectroscopy
Figure 4.1 shows the *H NMR and a 'H NOE difference spectrum of a 3-indoly[acetic acid derivative 13 bearing a methoxy group at the benzenoic ring.
9
I   I   I   I   ■   I   I   ■   ■   I   I   I   I   '   ■   I   ■   ■   '   I   I   ■   '   I   ■   I   i   I   '   I   I   '   I   I   I   I ■'   '   ■   ■   I ' 7.0 6.5 6.0 5.5 5.0 A.5 4.0 5.5
Fig. 4.1. 400 MHz 'H NMR spectrum of 13 in a mixture of CDCI3 and CD3OD. a Full spectrum; b expanded section of the aromatic proton signals; c 'H NOE difference spectrum, same section as in b, irradiation position at 5 = 3.64.
NOESY - Palmatine
NOESY - Palmatine
H,C r=
H,C—O
PALOMe2-Hl        PALH5-H4 PALH5-H6
r
x
PALOMe9-H8
x \
PALOMelO-Hll
*\
> PALH6-H8
X
I
PALOMe3-H4 ^
X
E
q. q.
I
6-
PALH1-H13
X
I
I
xx^
PALH12-H13
PALH12-H11
X
x
x x
6 5 52 - 1H (ppm)
m
CD
1.000
1.125
b
ro
m w m
z
m
CD
CO Ol
1.060 0.978
D
ro
O
b
G) (O
G) CO
OJ O)
b)
a> in
V.
Ca) O
1.111 -o
*— "D
3
3.482
4.244
0.932 3.682
ro
In
CO
ro b
2.340
Q]
Ol
3.518
"O "O
3
P
9
NOESY - Eserine in DMSO
11
I—
H 13 M 14 O
H3C'V
O
82 - -"-H (ppm)
NOESY - Eserine in DMSO
H15-H16 1
H9-H17 H12-H19
H11-H12
H 13 M 14 O
H3C'V
0 ,1
82 - -"-H (ppm)
< □ ►  < rS1 ►  < e ►  < e ►      e     0 0,0
-O "b O    =     <=>■<=> <^><\n>
1.125
u b
3.482
4.244
0.932. 3.682,
2.340
3.518
(ixidd) HT-2g S'Z O'E
■o
T3
3
9UU9S3 - ASaON
NOESY - Eserine
4.0
3.5
3.0
2.5
2.0
1.5
i-
H5-H17 H2"-H3V
H3"-H3'
H5-H3"
1=1
\
■1.5
.H5-H18 H2"-H2'
H19-H18
"H5-H2"
H3'-H17 I
■2.0
H2'-H3"
■2.5
H5-H19
■3.0
H
13 QH3C N 14 O
17 3
H3C15Y
Ns
1 CHo
^^7"N   H   18 3
■3.5
■4.0
3.5
3.0 2.5 82 - -"-H (ppm)
2.0
1.5
Colchicine - DQF-COSY
6 4 82 - 1H (ppm)
Colchicine - DQF-COSY
2
4 H17-H9
6
8
H,C.18;0
/3 _ H3C-O P
21 H,C
'22
13 O H3C23
H12-H3
H9-H8' *
H7'-H8"'. > »,
H7"-H7
0» 'i
6 4 82 - 1H (ppm)
it *
ti
■8
Colchicine - NOESY
E
Q. Q.
CO
6 4 ô2 - 1H (ppm)
Colchicine - NOESY
E
Q. Q.
to
8-
8
i i
H17-H19 H9-H8" H1-H7"
H12-H22
H13-H2::
H17-H16 .
•
H1-H20
8
-ft
H3C.%0
819 r
■8
6 4 ô2 - 1H (ppm)
Next session:
Midterm Test, Heteronuclear correlations