1. Chemistry 125: Lecture 61 March 26, 2010 NMR Spectroscopy Decoupling & Correlation Preliminary This For copyright notice see final page of this file

2. 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

3. 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.

4. 40 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

5. 20 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

6. 15 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

7. 10 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

8. 5 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

9. 2 db (inverse log measure of rf power) Observe 13 C while decoupling 1 H at various powers.

10. 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)

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

12. 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

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

14. Double Labeling Introduction: Lanosterol Biogenesis Cf. Frames 6-13 of Lecture 52 and Sec. 12.13 pp. 554-562

15. + Squalene H + + + + + + HO O

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

17. 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)

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

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

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

21. Proves that they entered as a unit. The dilute double label experiment enhances the same 12 13 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.

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

23. 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

24. 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?

25. "3-D" Version of contour plot on previous slide

26. 2-D NMR Dilute 13 C Double Labeling Power of Correlation: 2-D Chromatography

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

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

29. Electrophilic Aromatic Substitution

30. H HH H H H D 2 SO 4 H HH H D H Sec. 16.4 H HH H H H D via A/D intermediate + D 2 SO 4 C6D6C6D6 etc. Observable! Or other electrophiles in place of D + e.g. NO 2 +, Br +, HOSO 2, R +, R-C=O R-C=O + HOSO 2 +

31. 1 H vs. 1 H correlation in time 0.3 sec 40°C CACA DBDB Remember

32. + + + SHMo2 (Simple H ü ckel Molecular Orbital Program) BenzenePentadienyl Cation addition converts  ring to  chain. H HH H H H D+D+ H HH H H H D + Locus of odd electron in radical, + charge (LUMO) in cation. - charge (HOMO) in anion, SOMO (nonbonding) Cf.

33. X NO 2 + + + + H O2NO2N X + + + + H O2NO2N X + + + H Substituent Effects on Rate X O2NO2N X O2NO2N XX NO 2 + + + (from HONO 2 /H 2 SO 4 ) X Relative Rate (overall) H[1] Cl0.03 NO 2 6  10 -8 CH 3 25 OH1000 (CH 3 ) 3 N + 1  10 -8  donation /  withdrawal  (or  ) e-donation eases formation of cation intermediates  (or  ) e-withdrawal retards formation of cation intermediates

34. Why is -NO 2 e-Withdrawing when -OH is e-Donating? -NO 2 6  10 -8  e-withdrawal retards formation of cation intermediates -OH1000  e-donation eases N OO O H pCpC  C=C  p O 0 High HOMO; Good Overlap with Phenyl Low OMO; Good Overlap with Phenyl (but poor E-match) High HOMO; Low LUMO; Good Overlap with Phenyl OH is a  Donor NO 2 is a  Acceptor No Overlap with Phenyl NO 2 is allylic (willing but not able)

35. Substituent Effects on Orientation (p. 763) X NO 2 + (from HONO 2 /H 2 SO 4 ) X NO 2 + + + + H O2NO2N X + + + + H H X + + + X- Relative Rate (per replaceable H) H-[1] Cl-0.00080.030.13 O 2 N- 9  10 -8 0.6  10 -8 0.03  10 -8 H 3 C-13946 (CH 3 ) 3 C-4672 Directing Activating Deactivating Act Deact  (CH 3 ) 3 N- 3  10 -8 0.6  10 -8 + _ orthometapara EtOC=O 0.0060.0006 - 0.003 o/po/p m o/po/p “e-donating” “e-withdrawing” (steric hindrance) ?

36. Preparation Problems NO 2 Cl NO 2 Cl 2 FeCl 3 NO 2 Cl Cl 2 FeCl 3 (Cl + FeCl 4 - ) NH 2 Cl HNO 3 H 2 SO 4 OH Cl “reduction” (Zn/HCl?) H2OH2O 100°C HONO (NaNO 2 /HCl) N2N2 Cl + diazonium salt Cl - or : Hal CN NO 2 H Cl

37. Yale ‘98S

38. End of Lecture 61 March 26, 2010 Copyright © J. M. McBride 2010. 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