Chem 125 Lecture 65 4/6/08 Projected material This material is for the exclusive use of Chem 125 students at Yale and may not be copied or distributed further. It is not readily understood without reference to notes from the lecture.

Decoupling 13 C H 100 MHz25 MHz J ~ 125 Hz (in rotating frame)

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)

13 C NMR Assignments for Neotame Monohydrate Prof. Eric Munson, Kansas Univ. One peak per carbon, pretty well spread out

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

Double labeling introduction: Terpene Biogenesis * Sec pp *

Isopentenyl Pyrophosphate Dimethylallyl Pyrophosphate Adjacent unsaturation apparently speeds S N 2 Cl I benzyl 250 Cl allyl 90 Cl n-propyl k rel for rxn with I - in acetone [1] Cl

Isopentenyl Pyrophosphate Geranyl Pyrophosphate C5C5 C 10

Geranyl Pyrophosphate cis trans Neryl Pyrophosphate Limonene -H +  -Pinene -H + +H 2 O [Ox] Camphor "Terpene" essential oils C 10 Markovnikov anti-Markovnikov

Geranyl Pyrophosphate Farnesyl Pyrophosphate "head-to-head reductive dimerization" Squalene (shark liver oil) new bond C 15 “sesquiterpenes” C 30 “triterpenes”

+ Squalene H HO O Markovnikov Anti- Markovnikov Enzyme makes “O” selective among many trisubstituted alkene groups.

Squalene HO H H H CH 3 H H H + H3CH3C H3CH3C H3CH3C Lanosterol (source of cholesterol & steroid hormones) Not this time! (enzyme control) C 30 “triterpenes” 3°

Cute Story Is it True? (HDYK?)

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

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

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 Label Single Label Enrichment Enriched Peaks

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

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 the 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) Correlation both labeled, but not in the same molecule Few single presursor molecules have more than two labels, but most with one have two. These are both labeled, in the same molecule Confirms rearrangement scheme. Proves that they entered as a unit.

Dilute 13 C Double Labeling 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  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 by coupling through CH to R H H O N HR O N HR Off-diagonal peaks appear when “tickling” one signal on the diagonal enhances another.

End of Lecture 65 April 6, 2009