Chemistry 125: Lecture 61 March 26, 2010 NMR Spectroscopy Through-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 13 C H 100 MHz25 MHz J ~ 125 Hz (in frame rotating at 100 MHz) C 13 NMR spectrum irradiate H (100 MHz) and pulse (25MHz) to observe C 13 H average or H NMR spectrum C 13 up C 13 down C 12 H upH down

40 db (inverse log measure of rf power) CH 2 CH CH 3 C CH 2 CH CDCl 3 Observe 13 C while decoupling 1 H at various powers.

40 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

20 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

15 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

10 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

5 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

2 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

1 db (inverse log measure of rf power) CH 2 CH CH 3 C CH 2 CH CDCl 3 Observe 13 C while decoupling 1 H at various powers. NOE (Nuclear Overhauser Enhancement) RF excitation of a nucleus strengthens the signal from nearby nuclei. Bad for integration Good for determining structure (see below)

Precession Frequencies in Magnetic Field of ~23.5 kGauss MHz H1H1 F 19 P 31 C 13 O 17 1% 99.98% 6%

Proton-Decoupled 13 C NMR Assignments for the Artificial Sweetner Neotame Monohydrate Prof. Eric Munson, Kansas Univ. One peak per carbon, pretty well spread out Why no 13 C- 13 C splitting? Only 1% of 13 Cs have a 13 C neighbor in the same molecule. C=OC arom C-X e-neg C alkane

Power of Correlation: 2-D NMR Dilute 13 C Double Labeling 2-D Chromatography

Double Labeling Introduction: Lanosterol Biogenesis Cf. Frames 6-13 of Lecture 52 and Sec pp

+ Squalene H HO O

Squalene HO H H H CH 3 H H H + H3CH3C H3CH3C H3CH3C Lanosterol (source of cholesterol & steroid hormones)

Squalene HO H H H CH 3 H H H + H3CH3C H3CH3C H3CH3C Lanosterol (source of cholesterol & steroid hormones) 3° Cute Story Is it True? (Wait for NMR)

HO H H CH 3 H3CH3C H3CH3C H3CH3C Squalene Lanosterol 13 C Label Single Label Enrichment Enriched Peaks (100x stronger than natural-abundance peaks)

HO H H CH 3 H3CH3C H3CH3C H3CH3C Squalene Lanosterol 13 C Label Single Label Enrichment Enriched Peaks

HO H H CH 3 H3CH3C H3CH3C H3CH3C Squalene Lanosterol 13 C Double Label

Proves that they entered as a unit. The dilute double label experiment enhances the same C peaks as the single label experiments, but only 8 of them show spin-spin splitting (because their C-C bond stays intact). HO H H CH 3 H3CH3C H3CH3C H3CH3C 13 C Double Label Squalene DILUTE ! Double-Doublet (proton decoupled) 13 C- 13 C splitting (neighboring 13 Cs) Power of Correlation both labeled, but not in the same molecule Few single presursor molecules have any C 13 label, but those that are labeled have two C 13 s. These are both labeled, in the same molecule Strongly confirms the rearrangement scheme.

Dilute 13 C Double Labeling Power of Correlation: 2-D NMR 2-D Chromatography

1 H to 1 H Correlation by NOE (through-space magnetic interaction) protons in protein polymer 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 with amino acid by coupling through CH to R H H O N HR O N HR Less-congested, off-diagonal peaks appear when “tickling” one signal on the diagonal enhances another. diagonal shows normal spectrum heavily congesed by overlapping signals

1 H vs. 1 H Correlation in TIME 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 H3CH3C CH 3 H3CH3C H3CH3C H3CH3C + A B C D A D or 1-Anywhere?

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

2-D NMR Dilute 13 C Double Labeling Power of Correlation: 2-D Chromatography

Thin Layer Chromatography of partially purified extract of brown algae looking for ecdysteroids Developed with CHCl 3 : MeOH : C 6 H 6 (25 : 5 : 3) Developed with EtOAc : 96%EtOH : H 2 O (80 : 15 : 5) books/Milestones-TLC.pdf

Thin Layer Chromatography of partially purified extract of brown algae looking for ecdysteroids better resolution in 2 - D books/Milestones-TLC.pdf ("3-D" because ecdysone spots turn turquoise after vanillin/ H 2 SO 4 spray)

End of Lecture 61 March 26, 2010 Copyright © J. M. McBride Some rights reserved. Except for cited third-party materials, and those used by visiting speakers, all content is licensed under a Creative Commons License (Attribution-NonCommercial-ShareAlike 3.0).Creative Commons License (Attribution-NonCommercial-ShareAlike 3.0) Use of this content constitutes your acceptance of the noted license and the terms and conditions of use. Materials from Wikimedia Commons are denoted by the symbol. Third party materials may be subject to additional intellectual property notices, information, or restrictions. The following attribution may be used when reusing material that is not identified as third-party content: J. M. McBride, Chem 125. License: Creative Commons BY-NC-SA 3.0