Post on 25-Aug-2020
3. 4. 2016
Determination of nJ(CH) coupling constants
W. Bermel
9. 4. 2016
ENC workshop
Determination of nJ(CH) coupling constants
2
C. Thiele
3
Determination of nJ(CH) coupling constants
C H 3
C H 2
C H 2
C
C H
C H C
C H
C H
O H
O
N
C H 2
C H 2 C H 2
C H C N
C H
C
O
N H
C H
C O
C H 2
C H 2 C H 2
N H
C H C H
C H 3
C H 3
C
O N
C H 2
C H 2 C H 2
C H
O
C N H
C H
C H 2
C H O
C
N H
C H 3
C H C H
O C
N H
C H C H
O
C
C H 3
C H 2
C H 3
C H 3
C H 3
Pro(1) Pro(2)
Tyr(3)
Val(4)
Pro(5) Leu(6)
Ile(7)
Ile(8)
R.K. Konat, D.F. Mierke, H.Kessler, B. Kutscher, M. Bernd & R. Voegeli, Helv. Chim. Acta 76 , 1649 (1993)
(+)-Isopinocampheol ( (+)-IPC )
O H
Hymenistatin
20mM in DMSO 585mM in CDCl 3
HSQC
[ppm]
1.01.52.02.53.03.54.0[ppm]
20
25
30
35
40
45
50
55
60
65
70
12
3
4a4s
5
7a7s
89
10
Determination of nJ(CH) coupling constants
4
OH 1
2
3
4
5
6
7
9
8
10
Hetloc
Hecade
a/b HSQC-TOCSY
J-HMBC
Exside
HMBC-JRes
HSQMBC
SJS-HSQC
Splitting in F2
Splitting in F1
5
Determination of nJ(CH) coupling constants
Hetloc: x-filtered H,H TOCSY (F1 coupled)
13C X
H H
X
H
[ppm]
4.044.054.064.074.084.09[ppm]
1.00
1.05
1.10
1.15
1.20
1.25
1.001.051.101.151.201.25[ppm]
1J(CH) * cnst16nJ(CH)
H10/C10H3/C10
tm 80ms
expt47min
sign relative to 1J(CH) (positive)
6
Determination of nJ(CH) coupling constants
IPC
[ppm]
1.61.71.81.9[ppm]
1.65
1.70
1.75
1.80
1.85
1.90
1.95
2.00
2.05
1.60
1.61.71.81.9[ppm]1.61.71.81.9[ppm]1.61.71.81.9[ppm]1.61.71.81.9[ppm]
H4a
H2/H5
H1
cnst16 0.2 0.4 0.6 0.8 1.0
Hetloc: x-filtered H,H TOCSY (F1 coupled)
Determination of nJ(CH) coupling constants
7
Hetloc: x-filtered H,H TOCSY (F1 coupled)
OH1
2
3
4
5
6
7
9
8
10
2J(H3,C4)
2J(H3,C4)
3J(H3,C10)
- 1.62
- 1.63
+ 6.33
H2/H5 overlap
J(H1,H2) 2.0 Hz
(H4s)
(H4a)
* tm >= 80ms
3J(H3,C1)* + 0.91
4J(H3,C7)* - 0.32
4J(H3,C7)* - 0.26 (H7s)
(H7a)
digital resolution 0.16 Hz/pt
Hz
Hz
Hz
Hz
Hz
Hz
8
Determination of nJ(CH) coupling constants
Hecade: H,C HSQC-TOCSY (F1 coupled)
13C X
H H
X
H
4.044.054.064.074.084.09[ppm] 1.001.051.101.151.201.25[ppm]
1J(CH) * cnst16nJ(CH)
H10/C10H3/C10
tm 80ms
expt46min [ppm]
20.0
20.4
20.8
21.2
21.6
22.0
22.4
19.6
sign relative to 1J(CH) (positive)
9
Determination of nJ(CH) coupling constants
OH1
2
3
4
5
6
7
9
8
10
2J(H3,C4)
2J(H3,C2)
3J(H3,C5)
C1/C2 partial overlap
J(H1,H2) 2.0 Hz
Hecade: H,C HSQC-TOCSY (F1 coupled)
4J(H3,C7)*
3J(H3,C10)
-2.19
-1.63
+ 1.72
- 0.28
+ 6.34
* tm >= 80ms
digital resolution 0.16 Hz/pt
Hz
Hz
Hz
Hz
Hz
3J(H3,C1)** + 0.97 Hz
** tm >= 80ms + td1 2k (NUS)
10
Determination of nJ(CH) coupling constants
Hecade: H,C HSQC-TOCSY(F1 coupled)
Hetloc: x-filtered H,H TOCSY(F1 coupled)
IPCH3/C10
Hymenistatin
Ile[8] Ha/Cg
td_effresolution[Hz/pt](LP + zf)
2k0.62
4k0.31
8k0.16
1k3.23
2k1.61
4k0.81
8k0.40
16k0.20
td_effresolution[Hz/pt](LP + zf)
2k0.62
4k0.31
8k0.16
1k3.23
2k1.61
4k0.81
8k0.40
16k0.20
11
Determination of nJ(CH) coupling constants
Hecade: H,C HSQC-TOCSY (F1 coupled)
IPC
4.044.064.08[ppm]
256 50%/512
tm 80ms
[ppm]
47.0
47.2
47.4
47.6
47.8
48.0
48.2
48.4
48.6
48.8
49.0
49.2
49.4
4.044.064.08[ppm] 4.044.064.08[ppm] 4.044.064.08[ppm]
25%/1k 12.5%/2kNUStd1
C1
C2
12
Determination of nJ(CH) coupling constants
Hecade: H,C HSQC-TOCSY (F1 coupled)
IPC
4.044.064.08[ppm]
[ppm]
20
25
30
35
40
45
50
4.044.064.08[ppm] 4.044.064.08[ppm] 4.044.064.08[ppm] 4.044.064.08[ppm]
tm 80ms40ms 52ms 60ms 100ms
C10
C7
C4
C5
C2/C1
Determination of nJ(CH) coupling constants
13
a/b H,C HSQC-TOCSY (1J-IPAP)
13C X
H H
X
H
4.044.054.064.074.084.09[ppm]
H3/C10
tm 52ms
expt 46min
[ppm]
20.5
21.0
21.5
[ppm]
20.5
21.0
21.5
nJ(CH)
sign relative to 1J(CH) (positive)
Determination of nJ(CH) coupling constants
14
OH1
2
3
4
5
6
7
9
8
10
2J(H3,C4)
2J(H3,C2)
3J(H3,C5)
C1/C2 partial overlap
J(H1,H2) 2.0 Hz
4J(H3,C7)
3J(H3,C10)
-2.22
-1.60
+ 1.72
- 0.29
+ 6.31
tm = 52ms
digital resolution 0.16 Hz/pt
Hz
Hz
Hz
Hz
Hz
a/b H,C HSQC-TOCSY (1J-IPAP)
Determination of nJ(CH) coupling constants
15
16
Determination of nJ(CH) coupling constants
J-HMBC: HMBC + J evolution
nJ(CH) * SCALEF
13C X
H H
X
H
20.0
21.0
22.0
23.0
24.0
4.044.054.064.074.084.09[ppm]
[ppm]
SCALEF always maximum according to td1 and cnst13
H3/C10
expt 1h2min
no sign information
17
Determination of nJ(CH) coupling constants
J-HMBC: HMBC + J evolution
OH1
2
3
4
5
6
7
9
8
10
C1/C2 partial overlap
2J(H3,C4)
3J(H3,C5)
4J(H3,C7)*
3J(H3,C10)
1.36
1.57
< 0.63
6.35
* cnst 13 = 0.5 Hz
digital resolution 0.25 Hz/pt
Hz
Hz
Hz
Hz
5J(H3,C9)* < 0.70 Hz
td1 = 256
18
Determination of nJ(CH) coupling constants
J-HMBC: HMBC + J evolution
resolution 2 * swh1
td1 * k
1
SCALEFswh1 =
inf1
SCALEF =2 * d6
td1 * inf1
2
td1 * inf1 * k
td1 * inf1
2 * d6
1
k * d6 k
2 * cnst13
1
lp, zf: k = 4=
2
cnst13
d6 =1
cnst13 * 2
J-HMBC: HMBC + J evolution
20.021.0[ppm]
si 256 512 1k 2k 4k 8k 16k
cnst13 = 8Hz td1 = 256 SCALEF = 6
LP only
resolution (LP + zf) = 4Hz
H3/C10
20.021.0[ppm]20.021.0[ppm]20.021.0[ppm]20.021.0[ppm]20.021.0[ppm]20.021.0[ppm]
19
Determination of nJ(CH) coupling constants
J-HMBC: HMBC + J evolution
td1 256 512 1k128
cnst13 = 8Hz resolution (LP + zf) = 4Hz
H3/C10
20.021.0[ppm]20.021.0[ppm]20.021.0[ppm]20.021.0[ppm]
SCALEF 6 3 212
20
Determination of nJ(CH) coupling constants
J-HMBC: HMBC + J evolution
td1 = 256
H3/C10
20.021.0[ppm] 20.021.0[ppm]20.021.0[ppm] 20.021.0[ppm] 20.021.0[ppm]
cnst13 4 Hz 2 Hz 1 Hz8 Hz 0.5 Hz
SCALEF 12 24 486 98
21
Determination of nJ(CH) coupling constants
22
Determination of nJ(CH) coupling constants
J-HMBC: HMBC + J evolution
td1 = 256
cnst13
4 Hz
2 Hz
1 Hz
8 Hz
0.5 Hz
SCALEF
12
24
48
6
98
2J(H3,C4)2J(H3,C2) 3J(H3,C5) 4J(H3,C7) 3J(H3,C10)5J(H3,C9)
8.56 Hz
4.22 Hz
2.17 Hz
*
*
8.47 Hz
4.19 Hz
2.12 Hz
1.18 Hz
1.36 Hz
8.67 Hz
4.26 Hz
2.18 Hz
1.29 Hz
1.57 Hz
1.35 Hz
0.63 Hz
8.96 Hz
4.40 Hz
2.16 Hz
1.09 Hz
0.70 Hz
8.76 Hz
5.75 Hz
6.35 Hz
6.35 Hz
6.35 Hz
-2.19 Hz -1.63 Hz + 1.72 Hz - 0.28 Hz + 6.34 Hz
resol.
4 Hz/pt
2 Hz/pt
1 Hz/pt
0.5 Hz/pt
0.25 Hz/pt
n.a.Hecade
* C1/C2 overlap
**
**
**
** signal too weak
0.16 Hz/pt
1.55 Hz+ 1.56 Hz+
+td1 = 1K
J-HMBC: HMBC + J evolution
[ppm]
4.044.054.064.074.08[ppm]
20
25
30
35
40
45
50
4.044.054.064.074.08[ppm] 4.044.054.064.074.08[ppm] 4.044.054.064.074.08[ppm]
cnst13 = 8Hz resolution (LP + zf) = 4Hz
td(SCALEF) 128(12) 256(6) 512(3) 1k(2)
H3
C10
C9
C2
C5
C4
Determination of nJ(CH) coupling constants
23
J-HMBC: HMBC + J evolution
4.044.054.064.07[ppm]
cnst13(SCALEF) 8Hz(6) 4Hz(12) 2Hz(24) 1Hz(48)
H3
0.5Hz(98)
td1 = 256
[ppm]
20
25
30
35
40
45
50
4.044.054.064.07[ppm] 4.044.054.064.07[ppm] 4.044.054.064.07[ppm] 4.044.054.064.07[ppm]
C10
C9
C2
C5
C4
Determination of nJ(CH) coupling constants
24
25
Determination of nJ(CH) coupling constants
HSQMBC: long range HSQC
4.044.054.064.074.084.09[ppm]
H3/C10
expt 49min
[ppm]
20.5
21.0
21.5
13C X
H H
X
H
no sign information
OH1
2
3
4
5
6
7
9
8
102J(H3,C4)
2J(H3,C2)
3J(H3,C5)
C1/C2 partial overlap
4J(H3,C7)*
3J(H3,C10)
1.96
1.61
1.64
0.80
6.44
selective, cnst13 = 4 Hz
digital resolution 0.16 Hz/pt
Hz
Hz
Hz
Hz
Hz
HSQMBC: long range HSQC
4J(H3,C6)* 1.12 Hz
5J(H3,C9)* 1.08 Hz
* from antiphase
Determination of nJ(CH) coupling constants
26
HSQMBC: long range HSQC
transfer amplitude
sin ( p | k J CH | D ) * P cos( p | n J HH | D )
BIRD r,X removes 1J peaks
CPMG (XY16)
selective
remove JHH contributions
IPAP two separate spectra
k = 1, n
27
Determination of nJ(CH) coupling constants
cnst13 = 4 Hz
HSQMBC: long range HSQC
digital resolution 0.16 Hz/pt
4.044.054.064.074.08[ppm] 4.044.054.064.074.08[ppm] 4.044.054.064.074.08[ppm] 4.044.054.064.074.08[ppm]
1D BIRD CPMG selective
H3C10
H3C4
28
Determination of nJ(CH) coupling constants
HSQMBC: long range HSQC - IPAP
4.044.054.064.074.084.09[ppm]
H3/C10
expt 49min
13C X
H H
X
H
[ppm]
20.5
21.0
21.5
[ppm]
20.5
21.0
21.5
nJ(CH)
no sign information
Determination of nJ(CH) coupling constants
29
cnst13 = 4 Hz
HSQMBC: long range HSQC
digital resolution 0.16 Hz/pt
4.044.054.064.074.08[ppm] 4.044.054.064.074.08[ppm] 4.044.054.064.074.08[ppm]
1D BIRD - IPAP selective - IPAP
H3C10
H3C4
30
Determination of nJ(CH) coupling constants
31
Determination of nJ(CH) coupling constants
OH1
2
3
4
5
6
7
9
8
10
2J(H3,C4)
2J(H3,C2)
3J(H3,C5)
C1/C2 partial overlap
4J(H3,C7)
3J(H3,C10)
2.16
1.71
1.84
1.13
6.29
cnst13 = 4 Hz
digital resolution 0.16 Hz/pt
Hz
Hz
Hz
Hz
Hz*
HSQMBC: long range HSQC - IPAP
4J(H3,C6) 1.45 Hz*
5J(H3,C9) 1.23 Hz
* poor signal-to-noise
2.29
1.71
1.78
6.34
Hz
Hz
Hz
Hz
Hz*
Hz*
1.23 Hz
BIRD selective
32
Determination of nJ(CH) coupling constants
HSQMBC: long range HSQC - COSY/TOCSY IPAP
13C X
H H
X
H
X
H
13C X
H H
X
H
X
H
TOCSY
COSY
13C X
H H
X
H
X
H
4.044.054.064.074.084.09[ppm] 1.001.051.101.151.201.25[ppm]
H10/C10
tm 52ms
expt 6h25min
H3/C10
[ppm]
20.5
21.0
21.5
[ppm]
20.5
21.0
21.5
nJ(CH)
HSQMBC: long range HSQC - COSY/TOCSY IPAP
13C X
H H
X
H
X
H
33
Determination of nJ(CH) coupling constants
OH1
2
3
4
5
6
7
9
8
10
2J(H3,C4)
2J(H3,C2)
3J(H3,C5)
C1/C2 partial overlap
4J(H3,C7)
3J(H3,C10)
-2.12
-1.63
+1.72
-0.38
+6.34
digital resolution 0.16 Hz/pt
HSQMBC: long range HSQC - IPAP
4J(H3,C6) 0.33
5J(H3,C9) 1.15
COSY [Hz] TOCSY [Hz]
3J(H3,C1) +1.13
-2.19
-1.64
+1.72
0.31
+6.34
0.58
1.00
+0.84 H7s, H10
H3, H7s, H10
H3, H7s
H3, H7s
H1
H3,H1
H7s, H2/5
H3, H2/5, H1
H3, H4a
H4, H7s
H2/5
34
Determination of nJ(CH) coupling constants
HSQMBC: long range HSQC - selective
3.954.004.054.104.15[ppm]
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85[ppm] [ppm]
3.94.04.14.24.34.44.54.6[ppm]
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
IPC H3 Hymenistatin Ha region
35
Determination of nJ(CH) coupling constants
EXSIDE: selective long range HSQC + J-evolution
4.044.054.064.074.084.09[ppm]
H3/C10
expt 1h7min
13C X
H H
X
H
nJ(CH) * SCALEF
20.0
21.0
22.0
23.0
24.0
[ppm]
no sign information
36
Determination of nJ(CH) coupling constants
OH1
2
3
4
5
6
7
9
8
10
2J(H3,C4)
2J(H3,C2)
3J(H3,C5)
C1/C2 partial overlap
4J(H3,C7)
3J(H3,C10)
cnst16 = 80, td1 = 512, cnst13 = 2 Hz
digital resolution 0.15 Hz/pt
4J(H3,C6)
5J(H3,C9)
* smaller than n1/2
2.20
1.51
1.61
6.33
Hz
Hz
Hz
Hz
Hz*
Hz*
0.52 Hz
EXSIDE: selective long range HSQC + J-evolution
3J(H3,C1) 0.51 Hz
< 0.38
< 0.38
37
Determination of nJ(CH) coupling constants
EXSIDE: selective long range HSQC + J-evolution
20253035404550[ppm] 15
C1
C2 C4C5
C6
C7
C8
C9
C10
H3
cnst16resol.
1200.20 Hz/pt
2000.12 Hz/pt
1600.15 Hz/pt
td1 = 256
cnst16resol.
cnst16resol.
Determination of nJ(CH) coupling constants
38
EXSIDE: selective long range HSQC + J-evolution
20253035404550[ppm] 15
C1
C2 C4C5
C6
C7
C8
C9
C10H3
cnst16resol.
600.20 Hz/pt
1000.12 Hz/pt
800.15 Hz/pt
td1 = 512
cnst16resol.
cnst16resol.
39
Determination of nJ(CH) coupling constants
EXSIDE: selective long range HSQC + J-evolution
20253035404550[ppm] 15
C1
C2 C4C5
C6
C7
C8
C9 C10
H3
cnst16td1
200256
resol. 0.12 Hz/pt
100512
501k
cnst16td1
cnst16td1
40
Determination of nJ(CH) coupling constants
EXSIDE: selective long range HSQC + J-evolution
20253035404550[ppm] 15
C1
C2 C4C5
C6
C7
C8
C9 C10
H3
cnst16td1
2525%/2k
resol. 0.12 Hz/pt
5050%/1k
501k
cnst16td1
cnst16td1
41
Determination of nJ(CH) coupling constants
SJS-HSQC: HSQC + J-evolution and E.COSY
13C X
H H
X
H
3J(H3C10) * cnst16
H10/C10 H4s/C4[ppm]
1.101.111.121.131.141.151.16[ppm]
20.2
20.4
20.6
20.8
21.0
21.2
21.4
21.6
21.8
22.0
22.2
20.0
[ppm]
2.472.482.492.502.512.522.53[ppm]
38.4
38.6
38.8
39.0
39.2
39.4
39.6
39.8
40.0
40.2
40.4
38.2
2J(H3C4) * cnst16
3J(H3H4s)
3J(H3H4s) and 2J(H3C4) have opposite signcnst16 = 30, td1 = 512
42
Determination of nJ(CH) coupling constants
OH1
2
3
4
5
6
7
9
8
10
2J(H3,C4)
2J(H3,C2)
3J(H3,C5)
3J(H3,C10)
cnst16 = 120, td1 = 512
digital resolution 0.10 Hz/pt
5J(H3,C9)
* from line broadening
-2.16
1.71
6.29
Hz
Hz
Hz
Hz
3J(H3,C1)
SJS-HSQC: HSQC + J-evolution and E.COSY
(H4s)
(H4a)
2J(H3,C4) -1.62 Hz
Hz0.82
Hz0.87
decoupling during acquisition
-1.63
4J(H3,C7) Hz* < 0.2
expt 1h19min
43
Determination of nJ(CH) coupling constants
HMBC-JRes: HMBC + J-resolved
4.044.054.064.074.084.09[ppm] 1.001.051.101.151.201.25[ppm]
nJ(CH)
H3/C10
expt52min [ppm]
18.0
19.0
20.0
21.0
22.0
23.0
24.0
sequence I sequence II
H3/C10
13C X
H H
X
H
magnitude
no sign information
44
Determination of nJ(CH) coupling constants
OH1
2
3
4
5
6
7
9
8
10
2J(H3,C4)
2J(H3,C2)
3J(H3,C5)
C1/C2 partial overlap
3J(H3,C10)
cnst16 = 25, cnst13 = 4 Hz
digital resolution 0.16 Hz/pt
5J(H3,C9)
2.31
1.69
1.70
6.21
Hz
Hz
Hz
Hz
1.02 Hz
HMBC-JRes: HMBC + J-resolved
C4/C6 partial overlap
signal too weak
45
Determination of nJ(CH) coupling constants
HMBC-JRes: HMBC + J-resolved
4.044.054.064.074.08[ppm]
H3/C10
[ppm]
18.0
19.0
20.0
21.0
22.0
23.0
24.0
sequence II
4.044.054.064.074.08[ppm] 4.044.054.064.074.08[ppm]
cnst16 25 20 15
magnitude 46
Determination of nJ(CH) coupling constants
Hetloc
2J(H3,C4) 3J(H3,C10)
- 1.62 + 6.33
3J(H3,C1)
+ 0.91
4J(H3,C7)
- 0.29
Hz 2J(H3,C2) 3J(H3,C5)
Hecade -2.19 -1.63 + 1.72 - 0.28 + 6.34+ 0.97
J-HMBC 1.36 1.57 < 0.63 6.35
5J(H3,C9)
< 0.70
4J(H3,C6)
-2.22 -1.60 + 1.72 - 0.29 + 6.31a/b HSQC- TOCSY
1.96 1.61 1.64 0.80 6.44HSQMBC sel.
1.12 1.08
2.16 1.71 1.84 1.13 6.29HSQMBC- sel. IPAP
1.45 1.23
-2.12 -1.63 +1.72 -0.38 +6.34HSQMBC- TOCSY
0.33 1.15+1.13
2.20 1.51 1.61 6.330.52EXSIDE 0.51 < 0.38 < 0.38
2.31 1.69 1.70 6.211.02HMBC-JRes
SJS-HSQC -2.16 1.71 6.290.82 0.87-1.63 < 0.2
47
Determination of nJ(CH) coupling constants
48
Determination of nJ(CH) coupling constants
Hetloc
NS
Hecade
J-HMBC
a/b HSQC- TOCSY
HSQMBC sel.
HSQMBC- sel. IPAP
HSQMBC-TOCSY
EXSIDE
HMBC-JRes
SJS-HSQC
8
4
4
2
4
2
16
2
4
2
128
256
256
512
256
512
512
512
256
512
47min
46min
1h2min
46min
49min*
49min*
6h35min*
1h7min*
52min
1h19min*
295
267
123
263
194
271
64
312
152
658+
1
1
25
120
0.5
4
4
4
2
4
td1 expt S/N cnst16 cnst13
+ at C10 (CH3)
-COSY 266 4
H3
H2
* selective pulse signal: 4.1-4.0, noise: 3.7-2.9
49
Thank
You
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