Chemistry 125: Lecture 61March 26, 2010

NMR SpectroscopyThrough-Space Coupling, Decoupling & Correlation

This

For copyright notice see final page of this file

A spin = 1/2 nucleus is on perfectly frictionless bearings in a molecule,

oriented by the magnetic field.

Proton Decoupling

13C H

100 MHz25 MHz

J ~ 125 Hz

(in frame rotating at 100 MHz)

C13 NMR spectrum

irradiate H (100 MHz) and pulse (25MHz)

to observe C13

H average

oror

H NMR spectrum

C13 up C13 downC12

H up H down

40 db (inverse log measure of rf power)

CH2

CHCH3

C

CH2CH

CDCl3

Observe 13C while decoupling 1H at various powers.

40 db (inverse log measure of rf power)

Observe 13C while decoupling 1H at various powers.

20 db (inverse log measure of rf power)

Observe 13C while decoupling 1H at various powers.

15 db (inverse log measure of rf power)

Observe 13C while decoupling 1H at various powers.

10 db (inverse log measure of rf power)

Observe 13C while decoupling 1H at various powers.

5 db (inverse log measure of rf power)

Observe 13C while decoupling 1H at various powers.

2 db (inverse log measure of rf power)

Observe 13C while decoupling 1H at various powers.

1 db (inverse log measure of rf power)

CH2

CHCH3

C

CH2CH

CDCl3

Observe 13C while decoupling 1H at various powers.

NOE(Nuclear Overhauser

Enhancement)

RF excitation of a nucleus strengthens

the signal from nearby nuclei.

Bad for integrationGood for

determining structure

(see below)

Precession Frequencies in Magnetic Field

of ~23.5 kGauss MHz

H1

F19

P31

C13

O17

1%

99.98%

6%

Proton-Decoupled 13C NMR Assignments forthe Artificial Sweetner Neotame Monohydrate

Prof. Eric Munson, Kansas Univ.

ON

O

O N O

O

1 3

14

15

18-20

16 17HH

13

7-12

6

542

- +H

One peak per carbon, pretty

well spread out

Why no 13C-13C splitting?

Only 1% of 13Cs have a 13C neighbor

in the same molecule.C=O Carom

C-Xe-neg

Calkane

Power of Correlation:

2-D NMR

Dilute 13C Double Labeling

2-D Chromatography

Double Labeling

Introduction:Lanosterol Biogenesis

Cf. Frames 6-13 of Lecture 52and Sec. 12.13 pp. 554-562

+

Squalene

H +

+

+

+

+

+

HOO

Squalene

+

+

+

+

+

HO

HH

H

CH3

HH

CH3

H+

CH3H3C

H3C CH3

CH3

CH3

CH3H3C

Lanosterol(source of cholesterol& steroid hormones)

Squalene

+

+

+

+

+

HO

HH

H

CH3

HH

CH3

H+

CH3H3C

H3C CH3

CH3

CH3

CH3H3C

Lanosterol(source of cholesterol& steroid hormones)

3°

3°

3°

3°3°

Cute StoryIs it True?

(Wait for NMR)

HO

HH

CH3H3C

H3C CH3

CH3

CH3

CH3H3C

Squalene

Lanosterol

OPP

13C Label

Single Label Enrichment

Enriched Peaks

(100x stronger than natural-abundance

peaks)

HO

HH

CH3H3C

H3C CH3

CH3

CH3

CH3H3C

Squalene

Lanosterol

OPP

13C Label

Single Label Enrichment

Enriched Peaks

HO

HH

CH3H3C

H3C CH3

CH3

CH3

CH3H3C

Squalene

Lanosterol

OPP

13C Double Label

Proves that they entered as a unit.

The dilute double label experiment enhances the same 12 13C peaks as the single label experiments, but only 8 of them show spin-spin splitting (because their C-C bond stays intact).

HO

HH

CH3H3C

H3C CH3

CH3

CH3

CH3H3C

13C Double Label

Squalene

OPP

DILUTE !

Double-Doublet (proton decoupled)13C-13C splitting (neighboring 13Cs)

Power of Correlation

both labeled, but notin the same molecule

Few single presursor molecules have any C13 label, but thosethat are labeled have two C13s.

These are both labeled, in the same molecule

Strongly confirms the rearrangement scheme.

Dilute 13C Double Labeling

Power of Correlation:

2-D NMR

2-D Chromatography

1H to 1HCorrelation by NOE

(through-spacemagnetic interaction)

protonsin proteinpolymer

proximity(< 6Å) of

With Molecular Mechanics Constraints gives 3-D Structure (without crystal!)

NH at 7.25

is within 6Å of NHs at 8.9, 8.3, 8.25, 7.7

Narrow range; mostly HN-C=O protons

Identify NH withamino acid by

coupling throughCH to R

H

HO

NHR O

NHR

Less-congested, off-diagonal peaks appear when “tickling” one signal on the diagonal enhances another.

diago

nal s

hows n

orm

al sp

ectru

m

heav

ily co

nges

ed b

y ove

rlapp

ing si

gnals

H3C CH3

H3C

H3C

CH3

CH3

CH3

+

A

C C

B

D

B

1H vs. 1H Correlation

in TIME

H3C CH3

H3C

H3C

CH3

CH3

CH3

+

CH3

CH3

H3C

H3C

CH3

CH3

CH3

+

A

A

C C

B

D

B

C

C

B

D

B

0.3 sec 40°C

(ppm)

Note: ppm scale is slanted and "wackbards". The protons in methyls C and D are near + charge (see resonance

structures), thus deshielded from lack of electron density, and appear furthest to right - at highest .

(Range of peaks is 150 Hz in 60 MHz spectrometer = 2.5 ppm.)

+

+

+

+

+

+

++

+ +

+

C A

D B

B C

Methide Shift: 1-2 (as shown)

“2-Dimensional”NMR

H3C CH3

H3C

CH3

CH3

H3C CH3

H3C

CH3

CH3

CH3

+

A B

C D

A D

or 1-Anywhere?

"3-D" Version of contour plot on previous slide

2-D NMR

Dilute 13C Double Labeling

Power of Correlation:

2-D Chromatography

Thin LayerChromatography

of partially purified extractof brown algae

looking for ecdysteroids

Developed with CHCl3 : MeOH : C6H6 (25 : 5 : 3)

Developed withEtOAc : 96%EtOH : H2O (80 : 15 : 5)

http://www.chromsource.com/books/Milestones-TLC.pdf

Thin LayerChromatography

of partially purified extractof brown algae

looking for ecdysteroids

better resolution

in 2 - D

http://www.chromsource.com/books/Milestones-TLC.pdf

("3-D" becauseecdysone spots turn turquoise

after vanillin/ H2SO4 spray)

End of Lecture 61March 26, 2010

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