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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