How to Analyze of 2D NMR Spectra ( small molecules) 2009. 12. 28 노 정 래

How to Analyze of 2D NMR Spectra ( small molecules)

2009. 12. 28

노 정 래

Pulse width(pw)

FT

1H

Conventional proton spectrum (1H NMR)

ppm2345678

ppm405060708090100110120130140150160170

1H and 13C NMR spectra (Chemical shifts, )

H

HO

H

NH

O

H

C

C

COCN

C

O

Carbon spectrum (13C NMR)

FT

1H 13C

CHCl3 (1% ) 13C1H 13C1H

13C

Bo

(Coupled carbon spectrum)

couplingJCH

Pulse width(pw)

13C

BB 13C1H

Carbon spectrum (13C NMR)(Decoupled carbon spectrum)

1H 13C

CHCl3 (1% )

13CH313CH2

13CH 13C

C C C C

H

H

H

H

H

H

Coupled Carbon

Decoupled Carbon Nuclear Overhauser Effect (nOe)

Proton-Proton coupling constant (JHH)

H

H

JHHJHH

JHH

Not through space, but through bonds

- Hybridization of the atoms- Bond lengths- Bond angles and dihedral angles- Substituent effects- The presence of neighboring -bonds

JHH [Hz] sign JCH [Hz] sign JCC [Hz] sign

1J 125 ~ 250 + 30 ~ 80 +

2J 0 ~ 30 -* -10 ~ 20 + / - < 20 + / -

3J 0 ~ 18 + 1 ~ 10 + 0 ~ 5 +

3+nJ 0 ~ 7 + / - < 1 + / - < 1 + / -

* Usually negative, but sometimes positive

Factors influencing scalar coupling

The order of magnitude and sign of scalar couplings

2-methylpent-1-en-3-ol

Connectivity of protons and carbons

OH

Men and their Partner (Direct Coupling)

Friendship of Partners (Indirect coupling)

Relation between Men and their friend’s partner (remote coupling)

• Direct Detection

New Techniques employed in modern NMR experiments

low sensitivity (low natural abundance)

long time for multidimensional NMR experiment

high sensitivity

short time for multidimensional NMR experiment

13C1H

• Indirect Detection (Inverse Detection)

13C1H

99%( inactive)

1% (active)

12C1H

13C1H

labeling(70% 13C)

natural (1% 13C)

H-C-Cl3

JCH=216 Hz

+ =

Comparison of spectrum using phase cycling and PFG

• Phase cycling ( it is necessary to select interested signals with several scans)

scan 1 scan 2 sum

• PFG ( select interested signals or eliminate the unwanted signals with one PFG pulse

One scan

In case of dense sample - gives spectrum in short time - gives clean spectrum

PFG

dz

Field strength

1H

x

y

Bo

x

y

Pulse Field Gradient (PFG)

PFG

x

y

x

y

x

y

Pulse Field Gradient (PFG)

Origin 2D Data

t J

t2

t1

t1

0 200 400 600 800 1000t2 pts

0.1220

0.1230

0.1240

0.1250

0.1260

0.1270

t1 sec

Production of 2D NMR spectrum

FT (t2)

t2t1

FT (t1)

t2

t1

1H

1H

F1

F2

Ha

Hb

HaHb

cross peak diagonal peaks

Ha / Hb

COSY spectrum

t1 t2

128 x 1024 (256 x 1024)256 x 1024 (512 x 1024)512 x 1024 (1024 x 2048)

90 90

COSY spectrum (COrrelation SpectroscopY)

C

Hb

C

Ha

2JHH

C Hb

Ha

3JHH

Ha Hb

4JHH1

23

4

t1

t2

= 0-30 Hz

= 0-18 Hz

FT(t1, t2)

C C C C C C

Ha Hb HcHd He

a b c ed

a

b

d

c

e

diagonal peak → cross peak → diagonal peakdiagonal peak → cross peak → diagonal peak

COSY spectrum and its Interpretation

a

b

d

c

e

Hb / Hc

C C C C C C

Ha Hb HcHd Hf

a bc fd

a

b

e

c

f

( )

diagonal peak → cross peak → diagonal peakdiagonal peak → cross peak → diagonal peak

He

COSY spectrum and its Interpretation

e

d

C C C C C C

Ha Hb HcHd He

a b/ec fd

a

b/e

d

c

f

( )

diagonal peak → cross peak → diagonal peakdiagonal peak → cross peak → diagonal peak

C

Hf

Overlapped signals

COSY spectrum and its Interpretation

b c

b d

TOCSY (TOtal Correlation SpectroscopY)

d b c a

a

c

b

d

t1t2

128 x 1024 (256 x 1024)256 x 1024 (512 x 1024)512 x 1024 (1024 x 2048)

90 Mixing pulse60~80ms

C C C C

Ha Hb HcHd

3JHH3JHH

3JHH1

2

3

t1 t2

4

FT(t1, t2)

TOCSY spectrum

C C C C C C

Ha Hb HcHd He

a b/ec fd

a

b/e

d

c

f

C

Hf

d

a

c

b/e

f

TOCSY spectrum and its Interpretation

13C

CaCb

Ha

Hb

1H

Ca

Cb

13C

1H

HaHb

HMQC (Heteronuclear Multiple Quantum Correlation) / HSQC (Heteronuclear Single Quantum Correlation)

t1

t2

1/2JCH

128 x 1024 (256 x 1024)256 x 1024 (512 x 1024)

Pairing 1H and 13C Shifts by using the HSQC / HMQC spectrum

= 125-250 Hz1JCH

13C 1H

Ca Cb O

1Ha 1Hb

1

3

t1 t24

1JCH2

1JCH1JCH

FT(t1, t2)

HSQC spectrum

C C O C C

OH

H

H H H

H H

H

HMQC/HSQC spectrum and its Interpretation

abc d

a b c d

CH3, a

CH3, d

CH2, b

13C

1H

HMBC (Heteronuclear Multiple Bond Correlation)

t1

t2

128 x 1024 (256 x 1024)256 x 1024 (512 x 1024)

90 1801/2JCH

1/2nJCH

Assignment of Nonprotonated 13C’s on the basis of the HMBC spectrum

FT(t1, t2)

HMBC spectrum

Ca Cb O Cc Cd

OHa

Ha

Ha Hb Hd

Hb Hd

Hd

adbc

a

d

b

HMBC spectrum and its Interpretation

1H

13C

diagonal peak → cross peak → diagonal peakdiagonal peak → cross peak → diagonal peak

NOESY/ROESY( Nuclear Overhauser Effect Spectroscopy)

t1 t2

90 9090

t1t2

90 Mixing pulse350 ms

Mixing pulse200 ms

Determining Stereochemistry by using the NOESY / ROESY spectrum

C C C

HaHb Hc

NOE

t1

t2

NOE

H H

H

a b

c

2JHH

NOE / COSY

NOE

NOESY

ROESY

FT(t1, t2)

NOESY/ROESY spectrum

DEPT (Distortionless Enhancement by Polarization Transfer)

CH

CH2

CH3

1/2J1/2J 1/2J

x90

x180

x90

y180

y

H3C C

O

CH

CH3

CH2OH

220 200 180 160 140 120 100 80 60 40 20 0220 200 180 160 140 120 100 80 60 40 20 0

a

bcd

e

a

b c d e

13C spectrum

DEPT-135(CH + CH3 - CH2)

DEPT- 90 (CH)

DEPT- 45 (CH + CH2 + CH3)

DEPT spectra

4-hydroxy-3-methyl-2-butanone

13C spectrum

DEPT- 45

DEPT- 90

(DEPT- 45)-(DEPPT-135)

(DEPT- 45)+(DEPT-135)-(DEPT- 90) CH3

CH2

CH

All protonated

Pure subspectra

DEPT spectrum and its interpretation

I . Assignment of Resonances to Atoms Within a MoleculeI . Assignment of Resonances to Atoms Within a Molecule

Assigning 1H Resonances on the basis of Chemical Shifts

Assigning 1H Resonances on the basis of the COSY spectrum

Assigning 13C Resonances on the basis of Chemical Shifts

Pairing 1H and 13C Shifts by using the HSQC / HMQC spectrum

Assignment of Nonprotonated 13C’s on the basis of the HMBC spectrum

Determining Stereochemistry by using the NOESY / ROESY spectrum

II. Elucidation of Unknown Molecular StructuresII. Elucidation of Unknown Molecular Structures

Initial Inspection of the one-dimensional spectra : 1H and 13C

Establishment of connectivity between protons on the basis of the gCOSY spectrum

List the1H -13C data in tabular form

Pairing 1H and 13C Shifts by using the HSQC / HMQC spectrum

Assignment of Nonprotonated 13C’s on the basis of the HMBC spectrum

Determining Stereochemistry by using the NOESY / ROESY spectrum

C9H9ClO2 MW=184.62

* Problem 1 and 2 are selected from “Organic Structure Determination Using 2-D NMR Spectroscopy” - J. H. Simpson, 2008

Problem 1

1H NMR

The carbon signal at 157.0 ppm is lost

13C NMR

HMQC

1H COSY

NOESY

C7H12O2

Problem 2

1H NMR

13C NMR

HSQC

1H COSY

HMBC

10.88.06

(d, 7.7)

7.37(d, 8.0)

7.24(dd, 8.0, 7.4)

7.04(dd, 7.7, 7.4)

3.64

2.33 2.26

Problem 3

1H spectrum

x x x

13C spectrum

70%M-hsqc.esp

120 110 100 90 80 70 60 50 40 30 20F2 Chemical Shift (ppm)

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

F1

Ch

em

ica

l S

hift (p

pm

)

HSQC

70%M-cosy.esp

8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9F2 Chemical Shift (ppm)

6.8

6.9

7.0

7.1

7.2

7.3

7.4

7.5

7.6

7.7

7.8

7.9

8.0

8.1

8.2

F1

Che

mic

al S

hift

(pp

m)

COSY Spectrum8.06 7.37 7.24 7.04

70%M-hmbc.esp

150 145 140 135 130 125 120 115 110 105 100 95F2 Chemical Shift (ppm)

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

F1

Ch

em

ica

l S

hift (p

pm

)

HMBC Spectrum

70%M-hmbc.esp

145 140 135 130 125 120 115 110 105F2 Chemical Shift (ppm)

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.9

3.0

3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.8

F1 C

hem

ical S

hift (ppm

)

Expanded HMBC Spectrum

12

7.0

10

8.5

13

8.3

13

6.9

70%M-hmbc.esp

140 135 130 125 120 115 110F2 Chemical Shift (ppm)

6.9

7.0

7.1

7.2

7.3

7.4

7.5

7.6

7.7

7.8

7.9

8.0

8.1

8.2

F1 C

hem

ical S

hift (ppm

)

Expanded HMBC Spectrum

13

9.5

12

3.8

12

2.8

12

1.9

11

8.0

11

0.2

70%M-hmbc.esp

140 135 130 125 120 115 110F2 Chemical Shift (ppm)

10.75

10.76

10.77

10.78

10.79

10.80

10.81

10.82

10.83

10.84

10.85

10.86

10.87

F1 C

hem

ical S

hift (ppm

)

Expanded HMBC Spectrum

10

9.6

12

2.8

13

6.9

13

9.5

문제 풀이

C9H9ClO2 MW=184.62

Problem 1

1H NMR

Degrees of unsaturation = 9 – 9/2 + 1-1/2 =10

7.22

6.85

4.20 3.903.34

2.90 2.75

* The carbon signal at 157.0 ppm is lost

13C NMR

HMQC 129.4 115.9

69.0 50.0 44.6

7.22

6.85

4.20

3.90

3.34

2.90

2.75

129.4 - 7.22115.9 – 6.85 69.0 – 3.90 / 4.20 50.0 – 3.34 44.6 – 2.75 / 2.90

1H COSY

XH

HH

H

129.4 - 7.22115.9 – 6.85 69.0 – 3.90 / 4.20 50.0 – 3.34 44.6 – 2.75 / 2.90

7.22

6.85

3.904.20

3.34

2.752.90

O

NOESY

H

HH

H 7.22

6.853.904.20

2.752.90

O3.34

O

C9H9ClO2

O

Cl

O

Cl

O

C9H9ClO2

C9H9ClO2

C7H12O2

Problem 2

1H NMR

6.15

4.90

4.683.79 3.50

2.07 1.86

1.76

1.16Degrees of unsaturation = 7 – 12/2 + 1 =2

13C NMR

HSQC

140.4 101.1 63.5

26.8

16.3

15.0

96.7

6.15

4.90

4.68

3.793.50

2.071.86

1.76

1.16

140.4 – 6.15101.1 – 4.68 96.7 – 4.90 63.5 – 3.50 / 3.79 26.8 – 1.76 (CH2) 16.3 – 1.86 /2.07 15.0 – 1.16 (CH3)

H H

1H COSY

6.15

4.90

4.68

3.79

3.50

1.861.76

1.16

2.07

140.4 – 6.15101.1 – 4.68 96.7 – 4.90 63.5 – 3.50 / 3.79 26.8 – 1.76 (CH2) 16.3 – 1.86 / 2.07 15.0 – 1.16 (CH3)

6.15 4.68

1.862.07

1.76

4.90 O1.16

3.503.79

HMBC

C7H12O2 H H6.15 4.68

1.862.07

1.76

4.90 O1.16

3.503.79

140.4 – 6.15101.1 – 4.68 96.7 – 4.90 63.5 – 3.50 / 3.79 26.8 – 1.76 (CH2) 16.3 – 1.86 / 2.07 15.0 – 1.16 (CH3)

OH

H

O

Degrees of unsaturation = 2

10.8

8.06(d, 7.7)

7.37(d, 8.0)

7.24(dd, 8.0, 7.4)

7.04(dd, 7.7, 7.4)

3.64

2.33 2.26

Problem

1H spectrum

MW = 241C15H15NO2

Degrees of unsaturation = 15 -15/2 + 1 +1/2 = 9

x x x

13C spectrum

70%M-hsqc.esp

120 110 100 90 80 70 60 50 40 30 20F2 Chemical Shift (ppm)

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

F1

Ch

em

ica

l S

hift (p

pm

)

HSQC Spectrum

142.6

139.5

138.3

136.9

127.0

123.8 - 7.24

122.8

121.9 - 8.06

118.0 - 7.04110.2 - 7.37109.6108.5 60.6 - 3.64 (CH3) 13.4 - 2.33 (CH3) 12.5 - 2.26 (CH3)

70%M-cosy.esp

8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9F2 Chemical Shift (ppm)

6.8

6.9

7.0

7.1

7.2

7.3

7.4

7.5

7.6

7.7

7.8

7.9

8.0

8.1

8.2F

1 C

hem

ical

Shi

ft (p

pm

)

COSY Spectrum

H

H

H

H

8.06 7.37 7.24 7.04

8.06

7.04

7.24

7.37

70%M-hmbc.esp

150 145 140 135 130 125 120 115 110 105 100 95F2 Chemical Shift (ppm)

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

F1

Ch

em

ica

l S

hift (p

pm

)

HMBC Spectrum

70%M-hmbc.esp

145 140 135 130 125 120 115 110 105F2 Chemical Shift (ppm)

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.9

3.0

3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.8

F1 C

hem

ical S

hift (ppm

)

X

CH3

O

CH3

CH3

Expanded HMBC Spectrum

12

7.0

10

8.5

13

8.3

13

6.9

138.3

108.5

127.0

136.9

142.6

139.5

138.3

136.9

127.0

123.8 - 7.24

122.8

121.9 - 8.06

118.0 - 7.04110.2 - 7.37109.6108.5 60.6 - 3.64 (CH3) 13.4 - 2.33 (CH3) 12.5 - 2.26 (CH3)

70%M-hmbc.esp

140 135 130 125 120 115 110F2 Chemical Shift (ppm)

6.9

7.0

7.1

7.2

7.3

7.4

7.5

7.6

7.7

7.8

7.9

8.0

8.1

8.2

F1 C

hem

ical S

hift (ppm

)

H

H

H

H

Expanded HMBC Spectrum

8.06 / 121.9

7.04 /118.0

7.24 / 123.8

7.37 / 110.2

139.5

122.8

13

9.5

12

3.8

12

2.8

12

1.9

11

8.0

11

0.2

8.06

7.37

7.24

7.04

70%M-hmbc.esp

140 135 130 125 120 115 110F2 Chemical Shift (ppm)

10.75

10.76

10.77

10.78

10.79

10.80

10.81

10.82

10.83

10.84

10.85

10.86

10.87

F1 C

hem

ical S

hift (ppm

)

Expanded HMBC Spectrum

10

9.6

12

2.8

13

6.9

13

9.5

H

H

H

H

139.5

122.8

X

CH3

O

CH3

CH3

136.9

109.5

NH

10.2

10.2

C15H15NO2

Degrees of unsaturation = 9

NH

OH

OCH3MW = 241C15H15NO2