Basic Two Dimensional NMR Spectroscopy

1. Basic Two Dimensional NMR Spectroscopy M. Manickam M. Manickam@ bham. ac. uk Semester- 1; Week-4 Second year: CHM2C3

2. First and Second Semester Basic of 2D NMR 1H-1H Correlation spectra 1H-13C Correlation spectra 13C DEPT Spectra HMBC, HSQC and NOESY Few examples 2h Workshop and Pro-Forma NOTE Assessment Given the expected compound-interpret the spectra Pro-forma- 1 week to hand in with usual penalties for late submission Haworth Room No 214 (administration office, between 12 noon and 2 pm)

3. Application of Organic Spectroscopy Why is it needed? What is it used for? Structure Determination Chemists synthesises new and known materials and they need to know the structure To characterise materials Is the structure as expected? Or is it different? Some interesting new reactions and materials have been discovered from unexpected results To enable the physical and chemical properties to be related to the structure. This facilitates the synthesis of better materials, for example, drugs, liquid crystals, pesticides, polymers etc Safety reasons dictate that the structure of a materials is known so that any hazards can be related to structure and so that a material can be safely used and disposed of The progress of reactions can be monitored by spectroscopy, usually NMR and this technique allows the perfect timing of reactions to provide optimum results and can prevent unwanted further reaction Reaction can be carried out in the NMR instrument to enable instant analysis of structure which allows the structure of intermediates to be determined and reaction mechanisms to be established The purity of materials can be determined by NMR, routine checking of structures

4. Why NMR To find out required and side products To analysis and confirm the natural products structures. We use a variety of spectroscopic techniques Mass spectroscopic: Gives a compound’s mass (little information) IR spectroscopic: Functional groups information UV spectroscopic: Chromospheres and conjugated systems NMR spectroscopic :Gives great detailed structural information and the most powerful spectroscopic method used by organic chemists

5. NMR: Nuclear Magnetic Resonance Basic principle • 1H NMR spectroscopy provides information about the environments of the H atoms in a molecule • It is based on the same principles as in 13C NMR spectroscopy • The 1H nucleus has nuclear spin 1/2, so when placed in a strong magnetic field, it can exist in higher or lower energy states.

6. NMR: Nuclear Magnetic Resonance • When the nucleus is irradiated, it absorbs radio frequency radiation, and nuclei in the lower energy spin states are promoted to higher energy spin states • There are important differences between 1H and 13C NMR spectroscopy: • The 1H atom has 99.98% abundance in naturally occurring H, so 1H NMR spectra can usually be measured by a single scan, so FT methods are only used in exceptional circumstances • As a result, the peaks are proportional to the number of H atoms that the peak represents - this is very valuable when analysing spectra.

7. Carbon 13 (13C) NMR Spectrum Basically the same in principle to proton NMR obviously, precessional frequencies of carbons are different to those of protons but this is no problem low sensitivity Major problem is that 13C is only 1.1% of carbon additionally the magnetic moment of 13C is 4x weaker than for 1H Overall 13C signals are 6000x weaker than 1H signals However, using pulse FT-NMR 30,000 pulses can be made reasonably quickly to give an excellent spectrum high resolution Useful advantage is that typically 13C signals are spread over 200  units and so there is less chance of coincidence-hence- 13CNMR is 20x more resolved than 1H NMR Chemical shift () values are determined in the same way as for proton signals-shielding and deshielding.

8. NMR Spectrum From each signal you should be able to obtain three pieces information: • From the Chemical shift, the environment of the proton-containing group; • From the integration, the relative number of protons in the proton- containing group; • From the splitting, the number of protons on an adjacent carbon atom.

9. 1 Dimensional NMR • These are the most essential NMR spectra • Spectra have one frequency axis and one intensity axis (see spectra) • 1H and 13C NMR spectra must contain all the required resonances for the expected compound. • 1H NMR: - Integration-all protons must be accounted for - Chemical shifts must be correct. - Protons on adjacent carbon atoms will couple to produce multiplets. • 13C NMR: number of peaks shows number of carbon atoms (accounting for overlap and equivalency) • Oxygen atoms, nitrogen atoms do not appear in NMR spectra but their presence is implied in the chemical shift

10. General regions of Chemical Shifts

11. 13C Chemical Shifts

12. 1H-1H COSY (Correlation Spectroscopy) 2-D NMR spectra have two frequency axes and one intensity axis. The most common 2-D spectra involve 1H-1H shift correlation; they identify protons that are coupled (i.e., that split each other’s signal). This is called 1H-1H shift- correlated spectroscopy, which is known by the acronym COSY. • 1H-1H correlation spectra • 2-D dimensional plot with 1H spectrum along each axis and on the diagonal • Protons coupling to one another produce off diagonal correlations • This allows assignment of proton groups that are connected in the molecule • Shows connectivity in the compound

13. COSY spectrum of Ethyl Vinyl Ether Fig: 2 Contour plot Fig:1 Stack plot y x

14. 1H-1H COSY Spectrum of Ethyl Vinyl Ether Fig:1. It looks like a mountain range viewed from the air because intensity is the third axis. These “mountain-like” spectra (known as stack plots) are not the spectra actually used to identify a compound. Instead, the compound is identified using a contour plot Fig:2, where each mountain in Fig:1 is represented by a large dot (as if its top had been cut off). The two mountains shown in Fig:1 correspond to the dots labelled B and C in Fig: 2 Fig:2, the usual one-dimensional 1H NMR spectrum is plotted on both the x- and y- axes. To analyze the spectrum, a diagonal line is drawn through the dots that bisect the spectrum.

15. 1H-1H COSY Spectrum of Ethyl Vinyl Ether The dots that are not on the diagonal (A, B, C) are called cross peaks. Cross peaks indicate pairs of protons that are coupled. For example, if we start at the cross peak labeled A and draw a straight line parallel to the y-axis back to the diagonal, we hit the dot on the diagonal at ~ 1.1 ppm produced by the Ha protons If we next go back to A and draw a straight line parallel to the x-axis back to the diagonal, we hit the dot on the diagonal at ~ 3.8 ppm produced by the Hb protons. This means that the Ha and Hb protons are coupled.

16. 1H-1H COSY Spectrum of Ethyl Vinyl Ether If we then go to the cross peak labelled B and draw two perpendicular line back to the diagonal, we see that the Hc and He protons are coupled; the cross peak labelled C shows that the Hd and He protons are coupled. Notice that we used only cross peaks below the diagonal; the cross peaks above the diagonal give the same information. Notice also that there is no cross peak due to the coupling of Hc and Hd, consistent with the absence of coupling for two protons bonded to an sp2 carbon.

17. HETCOR Spectrum or (1H- 13C COSY) 2-D NMR spectra that show 13C-1H shift correlation are called HETCOR from heteronuclear correlation) spectra. HETCOR spectra indicate coupling between protons and the carbon to which they are attached. Example: 2-methyl-3-pentanone The 13C NMR spectrum is shown on the x-axis and the 1H NMR spectrum is shown on the y-axis. The cross peaks in a HETCOR spectrum identify which hydrogens are attached to which carbons. For example, cross peak A indicates that the hydrogens that shows a signal at ~ 0.9 ppm in the 1H NMR are bonded to the carbon that shows a signal at ~ 6 ppm in the 13CNMR spectrum. Cross peak C shows that the hydrogens that show a signal at ~ 2.5 ppm are bonded to the carbon that shows a signal at ~ 34pp

18. HETCOR spectrum of 2-methyl-3-pentanone CH3 CH CH2 CH3 x y

19. DEPT 13C NMR SPECTRA 13C DEPT spectra enable different carbon (CH3, CH2, CH, and quaternary) Types to be identified DEPT 90: only CH peaks visible? DEPT 135: -CH2 peaks negative -CH and CH3 peaks positive PENDANT: -CH2 and quaternary peaks negative -CH3 and CH peaks positive

20. DEPT 13C NMR SPECTRA • DEPT: stands for distortionless enhancement by polarization transfer. • This technique to distinguish among CH3, CH2, and CH group • It is now much more widely used than proton coupling to determine the number of hydrogens attached to a carbon. • DEPT 13C spectrum does not show a signal for a carbon that is not attached to a hydrogen. • For example: 13C NMR spectrum of 2-butanone shows 4 signals because it has 4 nonequivalent carbons, whereas the DEPT 13C NMR of 2-butanone shows only three signals because the carbonyl carbon is not bonded to a hydrogen, so it will not produce a signal. 4 3 2 1 Normal 13C NMR gives 4 signals CH3-CH2-CO-CH3 DEPT 13C NMR gives 3 signals

21. DEPT 13C NMR Spectra of Ipsenol In CDCl3 at 75.6 MHz: Subspeectrum A, CH up. Subspectrum B, CH3 and CH up, CH2 down. The conventional 13C NMR spectrum is at the bottom. 3X CH- 3X CH, 2X CH3 4X CH2

22. Other Types Of 1H-13C COSY 1. HMBC: 1H-13C several bond correlation 2. HSQC: 1H-13C carbon and protons direct correlation 3. HMQC: correlation between protons and other nuclei such as 13C or 15N

23. HMBC SPECTRUM (Hetronuclear Multiple-Bond CH Correlation) • This is a 2D experiment used to correlate, or connect, 1H and 13C peaks for atoms separated by multiple bonds (usually 2 or 3). • The coordinates of each peak seen in the contour plot are the 1H and 13C chemical shifts. This is extremely useful for making assignments and mapping out covalent structure. Points • Heteronuclear Multiple Bond Correlation • 13C-1H Correlations over Several Bonds • Typically over 2 or 3 bonds can be seen. • Possible because of sensitivity of the powerful magnets of today's NMR spectrometers. • Can be used to establish connectivity across barriers such as O atoms or quaternary carbon atoms 1H-C-13C (Two-bond) 1H-C-C-13C (Three- bond)

24. HMBC of Codeine H-8 H-9 C-2 C-3 C-4 C-6 C-1 H-9 to aromatic carbons C-1, C-3 and C-4 ( both are three bond coupling H-8 to aromatic carbons C-1 and C-6 ( both are three bond coupling

25. HSQC (Heteronuclear Single-Quantum Coherence) 13C-1H correlation spectra 2 Dimensional plot -1H spectrum on one axis, 13C on the other Shows Correlations between carbons and protons directly attached to one another allows further connectivity within the molecule to be established

26. Nuclear Overhauser (NOESY) Spectrometry Proximity Through Space A proton that is close in space to the irradiated proton is affected by the NOE whether or not it is coupled to the irradiated proton; if it is coupled, it remains at least partially coupled because the irradiation is week in comparison with that used for a decoupling experiment. NOESY for very large molecules, ROESY for mid-size molecules These spectra are used to locate protons that are close together in space Can be a 1D or 2D NOESY technique nOe is a through space effect It has nothing to do with connectivity in the molecule

27. Nuclear Overhauser Enhancement Applications • Elucidation of molecular constitution and conformation Is used to solve geometric problems within a molecule Relative stereochemistry can be seen Regiochemistry can be seen Powerful technique for 3D study of proteins and other macromolecules • Aiding assignments • Investigating molecular motions

28. Nuclear Overhauser Enhancement Which methyl signal belongs to which group? +45% +18% +17% - 2% - 4% Dimethylformamide semiclathrate 3-methylcrotonic acid two Me groups are non- equivalent owing to hindered rotation about the C-N bond. Both Me signals are therefore found At  2.79 and  2.94, together with a singlet at 8.0 for the formyl proton. If one now saturates the Me signal at  2.94, the intensity of the formyl proton signal increases by 18%. When instead the other methyl signal is saturated, a decrease of 2% is observed.

29. Nuclear Overhauser Effect Difference (NOESY) Spectrometry, 1H 1H Proximity Through Space readily available 3 2 natural product 1 NOE difference spectrometry determined the substitution pattern of a natural product, whose structure was either 1 or 2. NOE difference spectrometry of compound 3 will help us to settle the final structure

30. NOE difference spectrometry for Compound 3 3 Irradiation of the 5-Me group resulted in enhancement of both H-4 and H-6, whereas irradiation of the 3-Me group enhanced only H-4; the assignments of these entities to the absorption peaks is now clear.

31. 2-D 13C-13C Correlations: INADEQUATE Spectra (Incredible Natural Abundance DoublEQUAntum Transfer Experiment) 2-D INADEQUATE provides direct carbon connectivities enabling us to sketch the carbon sheleton unambiguously 2-D INADEQUATE has very limited applicability because of its extremely low sensitivity

32. Further Reading • Spectroscopic Methods in Organic Chemistry (fifth edition): Dudley H. Williams and Ian Fleming • Spectrometric Identification of Organic compounds (six edition): Robert M. Silverstein and Francis X. Webster • Basic One- and Two- Dimensional NMR Spectroscopy : Horst Friebolin