Structure Elucidation with Nuclear Magnetic Resonance (NMR)

(1)

Structure Elucidation with Nuclear

Magnetic Resonance (NMR)

(2)

NMR

spectrum

Structure

Chem. Shift

J Coupling

Peak Int.

NOE

Correlation

H-H，C-H

O

OH

O

CH

3

O

H

OH

CH

2

OH

COOCH

2

1

2

3

4

5

6

7

8

9

10

1''

2''

3''

4''

5''

6''

1'

2'

3'

4'

5'

6'

7''

Applications:

•

Sample quality control for Synthetic works.

•

Elucidation of chemical structures.

•

Getting functional group, bonding, dynamics, kinetics and

chemical exchange information of molecules.

•

3D structures of the molecules.

(3)

Connections through space

（

dipolar coupling

）

1D

、

2D NOESY

，

ROESY, HOESY(HSQC

-

NOESY)

usage

：

connecting spin systems

、

structure determination

Connections through bonds

（

spin

-

spin coupling

）

Homonuclear

：

1D

、

2D

1

H-

H

-

1

H COSY, DQF-

H COSY, DQF

-COSY, TOCSY

COSY, TOCSY

usage

：

spin system assignment

Heteronuclear

：

Direct (detect

13

C): APT, DEPT, HETCOR

C):

APT, DEPT, HETCOR

Inverse (detect

1

H): HMQC, HSQC, HMBC, HSQC

H):

HMQC, HSQC, HMBC, HSQC-

-COSY,

COSY,

HSQC

-

TOCSY, HMQC

-

TOCSY

usage

：

assigning heteronuclei

assigning

heteronuclei

、

connecting spin systems

Some common NMR experiments:

(4)

COSY spectrum is used for determining the connectivities

between protons on the basis of geminal and vicinal couplings.

Disadvantage: bulky dispersive diagonal peaks.

C

H

Vicinal

Geminal

2D COSY

—

Homonuclear

Shift CO

Shift

CO

rrelation S

rrelation

S

pectroscopY

pectroscop

Y

t

1

AQ

The basic COSY (x=45

°

or 90

°

) pulse sequence

90

°

x

°

(5)

ppm

1.0

1.5

2.0

2.5

3.0

3.5

ppm

1.0

1.5

2.0

2.5

3.0

3.5

Current Data Parameters

NAME butcosy

EXPNO 1

PROCNO 1

F2 - Acquisition Parameters

Date_ 20001102

Time 8.04

INSTRUM DRX500

PROBHD 5 mm TBI 1H/

PULPROG cosygp

TD 1024

SOLVENT CDCl3

NS 1

DS 16

SWH 2185.315 Hz

FIDRES 2.134096 Hz

AQ 0.2345700 sec

RG 40.3

DW 228.800 usec

DE 6.00 usec

TE 288.0 K

D0 0.00000300 sec

D1 1.60000002 sec

D13 0.00000300 sec

D16 0.00010000 sec

IN0 0.00045765 sec

============ CHANNEL f1 =============

NUC1 1H

P0 3.00 usec

P1 6.00 usec

PL1 -4.00 dB

SFO1 500.1310815 MHz

============ GRADIENT CHANNEL ========

GPNAM1 sine.100

GPNAM2 sine.100

GPX1 0.00 %

GPX2 0.00 %

GPY1 0.00 %

GPY2 0.00 %

GPZ1 10.00 %

GPZ2 10.00 %

P16 1000.00 usec

F1 - Acquisition parameters

ND0 1

TD 256

SFO1 500.1311 MHz

FIDRES 8.535453 Hz

SW 4.369 ppm

FnMODE undefined

F2 - Processing parameters

SI 2048

SF 500.1300144 MHz

WDW QSINE

SSB 0

LB 0.00 Hz

GB 0

PC 0.20

F1 - Processing parameters

SI 1024

MC2 QF

SF 500.1300144 MHz

WDW QSINE

SSB 0

LB 0.00 Hz

GB 0

1

2

3

4

1

2

3

4

HO

C

H

1

H

C

H

2

H

3

H

4

H

1

-

2

-

3

-

4

(6)

2D

DQF

－

COSY

(

D

ouble

-

Q

uantum

F

iltered

COSY

)

90

°

90

°

90

°

t

1

t

2

)

allow J coupling constant measurements

)

observing cross

-

peaks close to diagonal

)

(7)

ppm

3.6

3.8

4.0

4.2

4.4

4.6

4.8

5.0

5.2

5.4

5.6

5.8

6.0

6.2

ppm

3.6

3.8

4.0

4.2

4.4

4.6

4.8

5.0

5.2

5.4

5.6

5.8

6.0

6.2

Current Data Parameters NAME gbc35dqf EXPNO 2 PROCNO 1 F2 - Acquisition Parameters Date_ 20001103 Time 4.01 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG cosydfetgp.2 TD 4096 SOLVENT Pyr NS 8 DS 16 SWH 3822.630 Hz FIDRES 0.933259 Hz AQ 0.5359376 sec RG 512 DW 130.800 usec DE 6.00 usec TE 288.0 K D0 0.00000300 sec D1 1.60000002 sec D11 0.03000000 sec D16 0.00010000 sec DELTA 0.00110300 sec DELTA1 0.00110800 sec IN0 0.00026161 sec l3 256

============ CHANNEL f1 ============= NUC1 1H

P1 5.80 usec P2 11.60 usec PL1 -4.00 dB SFO1 500.1317609 MHz

============ GRADIENT CHANNEL ======== GPNAM1 sine.100

GPNAM2 sine.100 GPNAM3 sine.100 GPX1 0.00 % GPX2 0.00 % GPX3 0.00 % GPY1 0.00 % GPY2 0.00 % GPY3 0.00 % GPZ1 30.00 % GPZ2 10.00 % GPZ3 50.00 % P16 1000.00 usec F1 - Acquisition parameters ND0 1 TD 512 SFO1 500.1318 MHz FIDRES 7.465717 Hz SW 7.643 ppm FnMODE undefined F2 - Processing parameters SI 4096 SF 500.1300038 MHz WDW SINE SSB 2 LB 0.00 Hz GB 0 PC 0.20 F1 - Processing parameters SI 1024 MC2 echo-antiecho SF 500.1300038 MHz WDW SINE SSB 2 LB 0.00 Hz GB 0

2D Gradient DQF

(8)

3

(9)

TOCSY

(TO

(

TO

tal C

tal

C

orrelation S

orrelation

S

pectroscopY

pectroscop

Y

) or

HOHAHA

HOHAHA(

(Ho

Homonuclear

monuclear Ha

Hartman

rtman

-Ha

-

Ha

hn Spectroscopy)

t

1

τ

MLEV17

τ

AQ

Pulse sequence for a TOCSY spectrum.

Different mixing time gives different degree of relay of

correlation. At small mixing time, TOCSY spectrum is similar to

COSY spectrum. At long mixing time, gives total correlation.

HO

C

H

1

H

C

H

2

H

3

H

4

H

HO

C

H

1

H

C

H

2

H

3

H

4

H

HO

C

H

1

H

C

H

2

H

3

H

4

H

COSY

RL

-

COSY

TOCSY

mixing time

(10)

ppm

1.0

1.5

2.0

2.5

3.0

3.5

ppm

1.0

1.5

2.0

2.5

3.0

3.5

Current Data Parameters NAME buttoc10 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001102 Time 4.14 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG mlevetgp TD 1024 SOLVENT CDCl3 NS 8 DS 16 SWH 2185.315 Hz FIDRES 2.134096 Hz AQ 0.2345700 sec RG 16 DW 228.800 usec DE 6.00 usec TE 300.0 K D0 0.00000300 sec D1 1.79999995 sec D9 0.01000000 sec D11 0.03000000 sec D16 0.00010000 sec DELTA 0.00109943 sec DELTA1 0.00110800 sec FACTOR1 0 IN0 0.00045765 sec l1 0 l3 128 SCALEF 6

============ CHANNEL f1 ============= NUC1 1H

P1 5.60 usec P2 11.20 usec P5 19.33 usec P6 29.00 usec P7 58.00 usec P17 2500.00 usec PL1 -4.00 dB PL10 10.00 dB SFO1 500.1310815 MHz ============ GRADIENT CHANNEL ======== GPNAM1 sine.100

GPNAM2 sine.100 GPX1 0.00 % GPX2 0.00 % GPY1 0.00 % GPY2 0.00 % GPZ1 30.00 % GPZ2 30.00 % P16 1000.00 usec F1 - Acquisition parameters ND0 1 TD 256 SFO1 500.1311 MHz FIDRES 8.535453 Hz SW 4.369 ppm FnMODE undefined F2 - Processing parameters SI 1024 SF 500.1300144 MHz WDW SINE SSB 3 LB 0.00 Hz GB 0 PC 0.20 F1 - Processing parameters SI 1024 MC2 echo-antiecho SF 500.1300144 MHz WDW SINE SSB 3 LB 0.00 Hz GB 0

HO

C

H

1

H

C

H

2

H

3

H

4

H

2D Gradient TOCSY,

mixing time=10

ms

Same as COSY spectrum

1

2

3

4

1

2

3

4

1

-

2

-

3

(11)

ppm

1.0

1.5

2.0

2.5

3.0

3.5

ppm

1.0

1.5

2.0

2.5

3.0

3.5

Current Data Parameters NAME buttoc20 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001102 Time 5.28 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG mlevetgp TD 1024 SOLVENT CDCl3 NS 8 DS 16 SWH 2185.315 Hz FIDRES 2.134096 Hz AQ 0.2345700 sec RG 16 DW 228.800 usec DE 6.00 usec TE 300.0 K D0 0.00000300 sec D1 1.79999995 sec D9 0.02000000 sec D11 0.03000000 sec D16 0.00010000 sec DELTA 0.00109943 sec DELTA1 0.00110800 sec FACTOR1 1 IN0 0.00045765 sec l1 6 l3 128 SCALEF 6

============ CHANNEL f1 ============= NUC1 1H

2D Gradient TOCSY,

mixing time=20

ms

1

2

3

4

1

2

3

4

1

-

2

-

3

-

4

1

-

3

2

-

4

Same as RL

－

COSY

HO

C

H

1

H

C

(12)

ppm

1.0

1.5

2.0

2.5

3.0

3.5

ppm

1.0

1.5

2.0

2.5

3.0

3.5

Current Data Parameters NAME buttoc50 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001101 Time 16.43 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG mlevetgp TD 1024 SOLVENT CDCl3 NS 8 DS 16 SWH 2185.315 Hz FIDRES 2.134096 Hz AQ 0.2343412 sec RG 18 DW 228.800 usec DE 6.00 usec TE 300.0 K D0 0.00000300 sec D1 1.79999995 sec D9 0.05000000 sec D11 0.03000000 sec D16 0.00010000 sec DELTA 0.00109943 sec DELTA1 0.00110800 sec FACTOR1 4 IN0 0.00045765 sec l1 24 l3 128 SCALEF 6

============ CHANNEL f1 ============= NUC1 1H

GPNAM2 sine.100 GPX1 0.00 % GPX2 0.00 % GPY1 0.00 % GPY2 0.00 % GPZ1 30.00 % GPZ2 30.00 % P16 1000.00 usec F1 - Acquisition parameters ND0 1 TD 256 SFO1 500.1311 MHz FIDRES 8.535453 Hz SW 4.369 ppm F2 - Processing parameters SI 1024 SF 500.1300144 MHz WDW QSINE SSB 4 LB 0.00 Hz GB 0 PC 0.20 F1 - Processing parameters SI 1024 MC2 echo-antiecho SF 500.1300144 MHz WDW QSINE SSB 4 LB 0.00 Hz GB 0

2D Gradient TOCSY,

mixing time=50

ms

1

2

3

4

1

2

3

4

1

-

2

-

3

-

4

1

-

3

2

-

4

1

-

4

HO

C

H

1

H

C

(13)

DEPT

:

D

istortionless

E

nhancement by

P

olarization

T

ransfer

Heteronuclear expt.

Detection:

13

C

Distinguish

CH, CH2, CH

3

By suitable combination of

θ

=45, 90 & 135 spectra

All CH’s

Only CH

CH & CH

3

up

(14)

HETCOR

(

Het

eronuclear

chemical shift

cor

relation,

1

H

-

13

C COSY)

13

C

1

H

t

1

Δ

1

Δ

2

The standard pulse sequence for

13

C

-

detected

1

H

-

13

C chemical shift correlation.

AQ

t

2

1

H decoupling

Removing J

CH

splittings

*

But Inverse experiment has the following Advantages:

•increase sensitivity of detecting the less sensitive nuclei

(15)

HSQC

(H

(

H

eteronuclear S

eteronuclear

S

ingle Q

ingle

Q

uantum C

uantum

C

oherence)

AQ

1

H

13

C

Δ

/2

Δ

/2

GARP-1

t

1

Δ

/2

Δ

/2

Pulse sequence for

1

H-detected

1

H-

13

C

correlation through single-quantum coherence

•

HSQC can give considerably better

13

C resolution and sensitivity

than HMQC for CH

2

groups of natural products

•

HMQC has simpler pulse sequence and it is more robust and easier

to perform.

13

C decoupling

Removing J

CH

splittings

HMQC

(H

(

H

eteronuclear M

eteronuclear

M

ultiple Q

ultiple

Q

uantum C

uantum

C

oherence)

(16)

ppm

1.0

1.5

2.0

2.5

3.0

3.5

ppm

10

15

20

25

30

35

40

45

50

55

60

65

Current Data Parameters NAME buthsqc EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001102 Time 10.31 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG invietgpsi TD 1024 SOLVENT CDCl3 NS 1 DS 16 SWH 2185.315 Hz FIDRES 2.134096 Hz AQ 0.2345700 sec RG 2298.8 DW 228.800 usec DE 6.00 usec TE 300.0 K D0 0.00000300 sec D1 2.00000000 sec D4 0.00170000 sec D11 0.03000000 sec D13 0.00000300 sec D16 0.00010000 sec D24 0.00090000 sec DELTA 0.00116720 sec DELTA1 0.00110700 sec IN0 0.00003600 sec l3 256

============ CHANNEL f1 ============= NUC1 1H

P1 5.60 usec P2 11.20 usec P28 2500.00 usec PL1 -4.00 dB SFO1 500.1310815 MHz ============ CHANNEL f2 ============= CPDPRG2 garp

NUC2 13C P3 17.70 usec P4 35.40 usec PCPD2 89.00 usec PL2 -1.00 dB PL12 13.00 dB SFO2 125.7633722 MHz ============ GRADIENT CHANNEL ======== GPNAM1 sine.100

HO

C

H

1

H

C

H

2

H

3

H

4

H

1

2

3

4

1

2

3

4

HSQC spectrum: H

HSQC spectrum: H-

-C correlated

C correlated

(HMQC and HETCOR spectra look similar)

C-dimension

H

(17)

HSQC

-

TOCSY

AQ

1

H

13

C

Δ

/2

Δ

/2

GARP-1

t

1

Δ

/2

Δ

/2

Pulse sequence for

1

H-

H

-detected HSQC

detected HSQC-

-TOCSY experiment

TOCSY experiment

through single

-

quantum coherence.

Longer mixing time gives longer extend of spin

-

spin

correlations.

SLy

MLEV17

SLy

mixing time

(18)

ppm

1.0

1.5

2.0

2.5

3.0

3.5

ppm

10

15

20

25

30

35

40

45

50

55

60

65

Current Data Parameters NAME buthsto10 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001101 Time 18.31 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG invietgsml TD 1024 SOLVENT CDCl3 NS 4 DS 16 SWH 2185.315 Hz FIDRES 2.134096 Hz AQ 0.2343412 sec RG 2048 DW 228.800 usec DE 6.00 usec TE 300.0 K CNST0 2.0000000 D0 0.00000300 sec D1 1.79999995 sec D4 0.00170000 sec D11 0.03000000 sec D12 0.00002000 sec D13 0.00000300 sec D16 0.00010000 sec d20 0.00111720 sec d21 0.00110700 sec IN0 0.00003600 sec L1 3 l3 128

============ CHANNEL f1 ============= NUC1 1H

P1 5.60 usec P2 11.20 usec P5 19.34 usec P6 29.00 usec P7 58.00 usec P17 2500.00 usec P28 2000.00 usec PL1 -4.00 dB PL10 10.00 dB SFO1 500.1310815 MHz ============ CHANNEL f2 ============= CPDPRG2 garp

NUC2 13C P3 15.70 usec P4 31.40 usec PCPD2 89.00 usec PL2 -2.00 dB PL12 13.00 dB SFO2 125.7633722 MHz ============ GRADIENT CHANNEL ======== P16 1000.00 usec F1 - Acquisition parameters ND0 2 TD 256 SFO1 125.7634 MHz FIDRES 54.253471 Hz SW 110.437 ppm F2 - Processing parameters SI 1024 SF 500.1300144 MHz WDW SINE SSB 2 LB 0.00 Hz GB 0 PC 0.20 F1 - Processing parameters SI 512 MC2 echo-antiecho SF 125.7577961 MHz WDW SINE SSB 2 LB 0.00 Hz GB 0

HO

C

H

1

H

C

H

2

H

3

H

4

H

Same as

Same as HSQC

HSQC

－

COSY

spectrum

Gradient HSQC

Gradient HSQC-

-TOCSY(mixing time=10ms)

TOCSY(mixing time=10ms)

1

2

3

4

1

2

3

4

1

-

1

-

2

-

1

H

-

dimension

(19)

ppm

1.0

1.5

2.0

2.5

3.0

3.5

ppm

10

15

20

25

30

35

40

45

50

55

60

65

Current Data Parameters NAME buthsto20 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001101 Time 19.08 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG invietgsml TD 1024 SOLVENT CDCl3 NS 4 DS 16 SWH 2185.315 Hz FIDRES 2.134096 Hz AQ 0.2343412 sec RG 2048 DW 228.800 usec DE 6.00 usec TE 300.0 K CNST0 2.0000000 D0 0.00000300 sec D1 1.79999995 sec D4 0.00170000 sec D11 0.03000000 sec D12 0.00002000 sec D13 0.00000300 sec D16 0.00010000 sec d20 0.00111720 sec d21 0.00110700 sec IN0 0.00003600 sec L1 8 l3 128

============ CHANNEL f1 ============= NUC1 1H

1

2

3

4

1

2

3

4

HO

C

H

1

H

C

H

2

H

3

H

4

H

Gradient HSQC

Gradient HSQC-

1

-

2

-

1

-

1

-

3

-

1

C-dimension

H

(20)

ppm

1.0

1.5

2.0

2.5

3.0

3.5

ppm

10

15

20

25

30

35

40

45

50

55

60

65

Current Data Parameters NAME buthsto80 EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001101 Time 19.45 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG invietgsml TD 1024 SOLVENT CDCl3 NS 4 DS 16 SWH 2185.315 Hz FIDRES 2.134096 Hz AQ 0.2343412 sec RG 2048 DW 228.800 usec DE 6.00 usec TE 300.0 K CNST0 2.0000000 D0 0.00000300 sec D1 1.79999995 sec D4 0.00170000 sec D11 0.03000000 sec D12 0.00002000 sec D13 0.00000300 sec D16 0.00010000 sec d20 0.00111720 sec d21 0.00110700 sec IN0 0.00003600 sec L1 40 l3 128

============ CHANNEL f1 ============= NUC1 1H

HO

C

H

1

H

C

H

2

H

3

H

4

H

1

2

3

4

1

2

3

4

Gradient HSQC

Gradient HSQC-

1

-

2

-

1

-

1

-

3

-

1

-

4

-

1

C-dimension

H

(21)

HMBC

(H

(

H

eteronuclear M

eteronuclear

M

ultiple

-B

-

B

ond C

ond

C

orrelation Spectroscopy)

13

C

1

H

t

1

AQ

C

2

, C

3

and C

4

: Quaternary or protonated carbons X: O, N

C

1

C

2

C

3

C

4

H

1

C

1

X

C

2

C

3

H

1

Pulse sequence for HMBC

(22)

ppm

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

ppm

30

40

50

60

70

80

90

100

110

120

130

140

150

160

Current Data Parameters NAME cfyhmbc EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001103 Time 7.36 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG inv4gplrnd TD 1024 SOLVENT CDCl3 NS 4 DS 16 SWH 3443.526 Hz FIDRES 3.362818 Hz AQ 0.1488800 sec RG 11585.2 DW 145.200 usec DE 6.00 usec TE 300.0 K D0 0.00000300 sec D1 1.50000000 sec D6 0.07000000 sec D13 0.00000300 sec D16 0.00050000 sec IN0 0.00002380 sec ============ CHANNEL f1 ============= NUC1 1H

P1 6.00 usec P2 12.00 usec PL1 -4.00 dB SFO1 500.1326379 MHz ============ CHANNEL f2 ============= NUC2 13C

P3 15.70 usec PL2 -2.00 dB SFO2 125.7693867 MHz ============ GRADIENT CHANNEL ======== GPNAM1 sine.100

GPNAM2 sine.100 GPNAM3 sine.100 GPX1 0.00 % GPX2 0.00 % GPX3 0.00 % GPY1 0.00 % GPY2 0.00 % GPY3 0.00 % GPZ1 50.00 % GPZ2 30.00 % GPZ3 40.10 % P16 1000.00 usec F1 - Acquisition parameters ND0 2 TD 256 SFO1 125.7694 MHz FIDRES 82.064079 Hz SW 167.039 ppm FnMODE undefined F2 - Processing parameters SI 1024 SF 500.1300144 MHz WDW QSINE SSB 0 LB 0.00 Hz GB 0 PC 0.20 F1 - Processing parameters SI 512 MC2 QF SF 125.7577961 MHz WDW QSINE SSB 0 LB 0.00 Hz GB 0

N

O

CH

3

CH

3

H

3

C

H

1

3

5

2

8

7

6

4

Gradient HMBC

1

2

3

5

1

4

3

2

5

6

7

8

3

-

4

3

-

5

2

-

6

2

-

7

1

-

7

1

-

8

H

-

dimension

(23)

2D

2D NOESY

NOESY

(N

(

N

uclear O

uclear

O

verhauser E

verhauser

E

nhancements S

nhancements

S

pectroscopY

pectroscop

Y

)

t

1

τ

m

AQ

The NOESY pulse sequence.

—

C

— ~ —

C

—

Ha

Hb

V

∝

C*

r

-6

, r

<

0.5nm

r

<

0.5nm

For resonance assignment, chemical structure elucidation &

3D structure determination

t

(24)

ppm

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

ppm

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Current Data Parameters NAME cfynoesy EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001103 Time 8.53 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG noesygptp TD 1024 SOLVENT CDCl3 NS 8 DS 16 SWH 3443.526 Hz FIDRES 3.362818 Hz AQ 0.1488800 sec RG 64 DW 145.200 usec DE 6.00 usec TE 288.0 K D0 0.00000300 sec D1 1.60000002 sec D8 0.40000001 sec D16 0.00010000 sec d20 0.19890000 sec IN0 0.00014520 sec

============ CHANNEL f1 ============= NUC1 1H

P1 6.00 usec P2 12.00 usec PL1 -4.00 dB SFO1 500.1326379 MHz

============ GRADIENT CHANNEL ======== GPNAM1 sine.100

GPNAM2 sine.100 GPX1 0.00 % GPX2 0.00 % GPY1 0.00 % GPY2 0.00 % GPZ1 40.00 % GPZ2 -40.00 % P16 1000.00 usec F1 - Acquisition parameters ND0 2 TD 256 SFO1 500.1326 MHz FIDRES 13.451274 Hz SW 6.885 ppm FnMODE undefined F2 - Processing parameters SI 1024 SF 500.1300144 MHz WDW SINE SSB 2 LB 0.00 Hz GB 0 PC 0.20 F1 - Processing parameters SI 1024 MC2 TPPI SF 500.1300144 MHz WDW SINE SSB 2 LB 0.00 Hz GB 0

N

O

CH

3

CH

3

H

3

C

H

1

3

5

2

8

7

6

4

3

(25)

NOESY

gives sequential

assignment of peptides

2

3

1

2

3

CH

2

C

H

2

N

C

H

N

O

H

C

H

CH

2

O

N

H

C

CH

2

C

H

OH

O

1

3

2

(26)

Spin-lock

90

°

t

1

t

2

mixing time

2D ROESY pulse program

For small molecule NOE can be very small or zero

，

ROESY can be used in place of NOESY experiment. ROE

(27)

Structure Determination Procedures

¾

1D

1

H &

13

C & DEPT (+MS

、

IR

，

basic chemical structure or

functional groups information

）

¾

Establish

13

C

-

1

H connections by thru bond J

CH

couplings

HMQC

、

HSQC

、

HSQC

-

TOCSY experiments

¾

Establish

1

H

-

1

H connection (spin systems or partial pieces

）

Decoupled

1

H, 1D TOCSY, 2D

1

H

-

1

H COSY, TOCSY

expts

.

（

usually starts with well

-

resolved

1

H signals

）

¾

Long range connections

（

connecting spin systems & assigning

quaternary carbon

）

1D NOESY & 2D HMBC, NOESY, ROESY experiments

¾

3D structure or conformation determination

1D NOESY & 2D NOESY, ROESY, (HSQC)