NMR in Chemistry and Biochemistry

1. NMR in Chemistry and Biochemistry ACCA Lecture Series on Spectroscopy September 29, 2009 Steve McKenna INEOS Technologies Naperville, IL

2. What do these have in common?

3. What do these have in common? They’re all the same technology: NMR.

4. What is NMR? • Nuclear Magnetic Resonance • A technology for chemical analysis and for imaging (“MRI”) • Uses magnetic fields and radio waves to determine molecular structure, the composition of mixtures, or the internal structure of objects—including people. • Spectroscopic side: • mostly used for organic compounds in solution: both small molecules and polymers, including biopolymers. • but there are inorganic and solid-state applications too. • intrinsically quantitative with no need for calibration. • Imaging side is one of the most powerful diagnostic tools in medicine, complementary with X-ray/CT techniques. • Huge body of active research, 4 Nobel Prizes since 1952.

5. How does it work? • Atomic nuclei of 1H, 13C, 19F, 29Si, 31P, and many other elements behave like tiny magnets. (They “spin”.) • Place them in a strong magnetic field and they line up with it, like a compass needle. • A pulse of radio frequency energy makes them flip over. • Tipped nuclei precess at a frequency which tells about their chemical environment. • A radio receiver and computer detect the precession and display it as a spectrum.

6. Gyroscope videos Gyroscopes precess with a torque is applied to them. Here the torque is provided by gravity.

7. Gyroscope videos The precession speed and even the direction can be calculated from the spinning speed and shape of the gyroscope, and the amount of torque.

8. Gyroscope videos The higher the torque (the higher the gravitational field), the faster the precession. Nuclear spins behave this way too. In NMR, we apply torque to them using a very large magnet. The larger the magnet, the faster the nuclei precess. Radio waves can also provide torque.

9. How does it work? (2) • Additional RF pulses or changes in magnetic field can manipulate nuclei into giving up even more information, such as their physical location (in imaging) or their neighbors’ identities (in spectroscopy). • Many variations of 1-D, 2-D, and higher dimensions. • Quantitative work normally uses only 1-D NMR. For identifying unknowns or determining structures, we often use 2-D experiments, sometimes 3-D or 4‑D. • Imaging is inherently a 2-D or 3-D NMR experiment. • NMR nomenclature ignores one dimension, the “y axis” or “intensity”. To a mathematician, a 1-D NMR spectrum is a 2-D graph, a 2-D spectrum is 3-D, etc.

10. 5mm 10mm 10mm 10mm 20mm NMR sample tubes Most NMR spectroscopy is done on solutions. The solvent is usually deuterated (for example D2O instead of H2O) to prevent the solvent’s H’s from overwhelming the sample’s H’s. The sample tube is like an ordinary test tube, but very high precision. Proton NMR is mostly done in 5 mm tubes. There are also 3 mm and 1.7 mm tubes for sample-limited applications. 10 mm tubes are used mostly for 13C NMR. 20 mm tubes are very uncommon. There are also flow-through instruments which don’t use tubes.

11. A 500 MHz NMR spectrometer • Sample changer robot. • Superconducting magnet. A giant thermos bottle with a coil of superconductor wire inside. Electric current circulates in the coil. As long as the coil is kept at liquid helium temperature, the current circulates forever. • Probe (detector), inserted at the bottom of the magnet, positioned at the center of the magnet coil. • Electronics console. • Vibration isolator. • Host computer (not shown): data processing and experiment programming.

12. NMR magnets NMR magnets have very strong, constant magnetic fields. Physicists measure magnetic field strength in Gauss or Tesla (1 T = 10000 G). NMR spectroscopists measure field strength by the NMR frequency of protons (1H) inside the magnet. NMR freqGauss or Tesla earth’s magnetic field (at equator) 2500 Hz 0.6 G refrigerator magnet 400-600 KHz 100-150 G hospital MRI scanner 65-130 MHz 1.5-3.0 T (15,000-30,000 G) research MRI scanner 400 MHz 9.4 T (94,000 G) NMR spectroscopy magnets: 300 MHz 7.0 T (70,000 G) 400 MHz 9.4 T (94,000 G) 500 MHz 11.7 T (117,000 G) 800 MHz 18.8 T (188,000 G) 1000 MHz 23.5 T (235,000 G) To put this in perspective, NMR magnets are stronger than the electromagnets used to move old cars around at the junkyard. 1st commercial 1000 MHz spectrometer was announced in 2009. Price: $16M.

13. Inside an NMR magnet • Outer shell • Vacuum and aluminized mylar insulation • Liquid nitrogen (77K) • Vacuum and more insulation • Liquid helium (4K) • Outer surface of the magnet coil • Part of the coil cut away to show the superconducting wire This was a 360 MHz magnet. It was about 2 meters tall on its legs, but the superconducting coil inside was less than 0.5 meter high. A magnet like this can go 7-14 days between liquid N2 refills and 2-3 months between liquid He refills.

14. A bad day in an imaging lab

15. Proton NMR spectrum of ethylbenzene

16. Chemical shift Electron cloud shields the nucleus from some of the external magnetic field. The amount of shielding depends on the chemical environment of the atom: how many substituents, what kind, etc. More shielded nuclei have lower NMR frequencies. increasing shielding increasing NMR frequency increasing chemical shift • In NMR spectra, frequency increases from right to left, not left to right. • Sometimes we talk about frequency in units of Hz. Other times we call it “chemical shift”, and we use units of “ppm”. • “Chemical shift” is more convenient for talking about chemical environments, because it doesn’t change when we use a larger or smaller magnet. • Larger magnets spread peaks out: higher field = fewer overlaps. • Overlaps are a big problem in identifying unknowns and proving structures.

17. Coupling Nuclei know about their neighbors. Nearby nuclei are little magnets too, and they can feel each other. This is called “coupling”. There are several kinds. For NMR in liquids, the most important coupling is called “J-coupling”. It travels only through bonds (farther apart = more bonds = smaller coupling). Also very sensitive to bond angles. It causes splitting into “multiplets”: n neighbors = n+1 peaks. Coupling is very important in deciding which atoms are next to which other atoms—the basis of determining molecular structure. There are other kinds of coupling. Some of them travel through space, not bonds—so they can tell who’s nearby, even if the molecule loops back on itself and the neighbor is many bonds away. Large biomolecules like proteins and DNA/RNA do this.

18. Example of coupling: 15N NMR of Pyridine JNHo = 10.9 Hz JNHm = 1.5 Hz JNHp = 0.27 Hz 15N linewidth = 0.07 Hz 15N T1 = 57 sec 1H T1 = 9 sec

19. NMR for structure determination • For pure compounds (the easiest case), NMR provides a wealth of information on chemical structure. • 1-D NMR (1H, 13C, 29Si, 31P, etc.) shows chemical environment. • For small molecules, this is often good enough. • For bigger molecules, it provides a starting point. • 2-D NMR experiments show the relationships between atoms. This is very valuable in determining structure. • Some 2-D experiments show connections by J-coupling. For 1H, they determine nearest neighbors ~2-4+ bonds apart. • Other J-coupling experiments show connections between 1H and other nuclei like 13C and 15N. Some show 1-bond, others show 2-4 bond connections. • Some experiments look for coupling through space and don’t depend on bonds. Range is short (~4-5 Å). • J-couplings trace backbone, through-space couplings show molecular folding. • There are 3-D and 4-D experiments which combine all of these functions.

20. NMR for quantitative measurements • Inherently quantitative on a mole% basis. No need for calibration from standards. No other molecular spectroscopy technique can do this. • Mole% can usually (not always) be converted easily to wt%. • Quantitation by proton NMR takes minutes. By 13C, can take hours. Choice depends on application. • Reputation for low sensitivity--but not always justified. • 13C NMR usually down to about 1%. • 1H NMR down to a few ppm (0.0001%) in favorable applications.

21. Maleic acid hydrogenationan industrial example featuring quantitation and water suppression • High pressure hydrogenation reaction with a supported noble metal catalyst. • Reaction medium is mostly water, with varying levels of at least 13 known organics.

22. A problem: working in water • All nuclei give NMR signals proportional to their concentrations. • Concentration of hydrogen: • H2O: 110 M • 1% maleic: 0.2 M • 0.006% maleic: 0.001 M • NMR signal mustn’t overload the receiver (makes the entire spectrum unusable). • So turn down receiver sensitivity to keep H2O from overloading. Result: lower sensitivity for organics. • Also, very strong signals become artificially broad (“radiation damping”). They obliterate smaller signals nearby. • The strategy: avoid exciting the water signal in the first place. This is called water suppression.

23. Water suppression by presaturation • Irradiate sample with weak RF (105 x weaker than normal) at same frequency as H2O peak. • Weak RF field has a narrow range of effect, about 1 ppm. • After ~50 sec of irradiation, H2O has absorbed energy and become saturated; no more magnetization left to excite. • Then proceed with the NMR experiment as usual.

24. Results of water suppression • Aqueous sample diluted 1:2 with D2O. • Spectrometer does a prescan to locate H2O peak, presaturates the H2O, then acquires the final spectrum. • Automated data acquisition, 15 minutes per sample. • 13 species measured. • Smallest species measured: around 0.006 wt%.

25. Hydrogenation kinetics from NMRmeasured in water using presaturation

26. Ethylidene norbornenean example of structure determination using 1-D and 2-D NMR • An important industrial chemical, an ingredient in “EPDM” synthetic rubbers • Used to make rubber sheeting for roof membranes and pond liners; also hoses, washers, and door weatherstripping • Easy to make (1 step from butadiene and cyclopentadiene) • Comes as a mixture of two cis/trans isomers, “E” and “Z”

27. Ethylidene norbornene: proton NMR

28. Ethylidene norbornene: 13C NMR

29. 1-D NMR is not enough This is a very typical case. There is a lot of information in the 1-D NMR spectra, but sometimes not enough to determine the structure right away. We might be able to solve it eventually by painstaking comparison vs. model compounds. But NMR can quickly provide more information to make the job easier. • “Multipulse” 1-D techniques like APT, DEPT, and NOE difference provide limited amounts of extra information: “multiplicity” (CH3/CH2/CH/C), nearest neighbors of 1 proton, etc. • 2-D and higher dimensions provide large amounts of information at once, filtered in useful ways. For example, one 2-D experiment can show all of the C-H connections in the molecule. • Hundreds of experiments to choose from, tailored for different situations and different types of information.

30. Ethylidene norbornene: APT • Easy 13C experiment. • Result is like a 13C, except CH and CH3 peaks are upside down. C, CH2 CH, CH3

31. Ethylidene norbornene: DEPT • More complicated 1-D experiment, but very common. • “Edited” version shown here gives subspectra for CH, CH2, CH3. • Quaternary “C” give no signal in DEPT. • DEPT has higher signal/noise than ordinary 13C. • Nearly all 29Si spectra are acquired using DEPT. CH3 only CH2 only CH only all CHx

32. Ethylidene norbornene: COSY • Easy 1H-1H experiment. • Symmetrical 2-D map which aligns with 1-D 1H spectrum on each side. • Every H is represented by a spot on the diagonal. • Connections shown by off-diagonal peaks (square patterns). • Connections are caused by J-couplings: 2-4 bonds.

33. Ethylidene norbornene: HMQC • Moderately difficult experiment, requires special hardware which is now becoming common. • 2-D map which aligns with 1H on one side, 13C (or 15N etc.) on other side. • More sensitive than 1-D 13C etc. experiments. • Spots show connections by 1-bond J-couplings. • Many related experiments use 1- or 2-4-bond J-couplings.

34. Summary of structure determination • Gather evidence from several information-rich NMR experiments. • Typical workup for a small molecule might be: • 1-D 1H • 1-D 13C (if enough material; if not, infer 13C from 2-D experiments) • possible 13C APT or DEPT • COSY for H-H connections • HMQC (or HSQC, HETCOR) for 1-bond C-H connections • HMBC (or variations) for long-range C-H connections • possibly other experiments as appropriate • Combine with any info available from other techniques • Still requires a lot of deduction, but much easier than trying to solve structure from chemical shifts alone.

35. NMR of proteins • NMR is one of the most powerful tools for studying proteins. • Can determine both amino acid sequence and 3-D structure (folding) of proteins • Competes with X-ray diffraction. But XRD determines structures of solid, crystallized protein while NMR works in solution (more realistic). • Can measure interaction between proteins and drug candidate molecules. • Can measure internal motions in proteins. • Very challenging: proteins can have hundreds or thousands of H’s and C’s, and often only available in tiny quantities. • Not just proteins: structures of other biopolymers like nucleic acids and carbohydrates can be determined by NMR. NMR also used to detect molecules in biofluids (blood, urine, etc.) for metabolic studies.

36. Typical plan for NMR structure determination of a protein • First, develop a way to produce and purify the protein (generally using tailored bacteria etc.). • Make “labeled” samples of the protein: uniformly enriched in 13C, 15N, or both. • Use 2/3/4-D NMR experiments to trace the protein backbone (NH-C-CO). • Use other 2/3/4-D experiments to identify side chains. • Use NOESY to measure distances between H’s. • Sometimes also bond angles from other NMR experiments. • Combine distance/angle restraints in an optimization program – generates final 3-D structure.

37. Protein NMR Experiments Most are 3-D NMR experiments which take advantage of 1H, 13C, and 15N. There are dozens of different possibilities. Each starts with one kind of magnetization (often the H of NH groups) and follows J-couplings to transfer it from one nucleus to the next. The specific path chosen determines the kind of information we can learn. 1H-15N HSQC HNCO HNCA HN(CO)CA HN(CA)CO CBCA(CO)NH For a good tutorial on protein NMR experiments: http://www.protein-nmr.org.uk

38. Interpreting 3-D NMR experiments “3-dimensional” representation looks nice, but really isn’t very interpretable. To look at 3-D and 4-D data, we select a particular plane of interest; for example, the 13C/1H plane which has 15N chemical shift = 115 ppm. Then we display the plane as a 2-D spectrum. This is one plane from an HNCA experiment. It contains the signals from all of the amino acid residues which have a particular 15N chemical shift. The resolution is poor in the 15N dimension, so 8-10 N’s overlap at the same 15N shift and are all represented here. Each amino acid residue gives a vertical pair of peaks at the 1H chemical shift of its NH. The stronger peak is the C of the same amino acid as the NH; the weaker one is the previous amino acid. 13C 1H

39. Distance Measurement and Structure Refinement To measure distance, we use a 2-D experiment called NOESY. It looks like a COSY, but the off-diagonal peaks show H’s that are near each other through space, not through bonds. The intensity of the peak tells how far apart the two H’s are. It falls off very quickly (1/r6), so it’s only good to about 4 Å. Proteins have lots of close contacts between H’s. Here two parallel protein sheets are held together by hydrogen bonds, and arrows who the pairs of H’s which would give “NOE’s”. The spectroscopist will often measure hundreds of peak intensities in the NOESY of a protein. These are translated into distance restraints and fed into a program which finds the best structure. Here the computer has overlaid the 20 best NOE structures it came up with. Note how some regions are better defined than others.

40. Magnetic Resonance Imaging • MRI is a 2-D or 3-D 1H NMR experiment in which the axes show physical location instead of chemical shift. • Intensity is due to number of H’s at that location (but this can be modified in various ways). • Magnets used for people are low in field (by NMR standards) but large in size. Chemical shift resolution is low to nonexistent. • All of the H’s at each location contribute to the same pixel of the image. Since the human body is ~60% water, an MRI is mostly an image of the water in the body. • This is why MRI is good for imaging “wet” parts of the body: brains, muscles, internal organs, etc. • X-ray/CT works by X-ray absorption (elements with high atomic number = more absorption), so it’s better for “hard” parts: bones etc. • But there is overlap between the techniques.

41. Using Field Gradients to Create an Image In normal NMR spectroscopy, we apply a magnetic field which is constant across the whole sample. All of the protons in the sample resonate at the same frequency, so we get one peak (except for chemical shift etc.). In imaging, we apply a gradient: the magnetic field increases as you go from one side of the sample to the other. Protons on the left side resonate at a lower frequency than protons on the right side. This encodes position information into the spectrum. This produces a 1-dimensional image, showing where the protons are, left to right across the sample. If you apply two different gradients, first left to right, and then top to bottom, you can produce a 2-dimensional image. It’s really a 2-D NMR spectrum.

42. selective pulse here Slice Selection Imaging would ordinarily be a “3-D NMR” experiment: x,y,z of position vs. NMR intensity. This would require a huge amount of data which would take a long time to acquire. Usually a “slice” through the subject is sufficient anyway. So we select just the H’s in that slice. This gets rid of one dimension, and we can image the other two dimensions at high resolution. We apply a field gradient along the subject. Here the subject’s feet experience a lower magnetic field than the head. Then we apply a “selective pulse”, a shaped RF pulse which tips only the H’s at a particular frequency. We can choose the frequency to select any slice we want. Then we turn off the selection gradient and continue with the rest of the imaging sequence. Only the H’s in the slice we selected will contribute to the image. T2-weighted image of human brain, slice at eye level

43. Contrast Contrast is the difference between light and dark parts of the image (more and less intense signal in the NMR spectrum). We can choose different NMR experiments to “weight” NMR properties in different ways, to highlight different features in the subject. T1-weighted image: water is dark, fats are bright, different tissues are in between. The image can be enhanced by injecting a contrast agent (often a salt of Gd3+) which makes some tissues brighter. Spin-density image: weighted by number of H’s. Very little contrast for brains, but good for water and fatty tissue (lots of H) vs. protein (less H). T2-weighted image: tends to highlight water in mobile vs. restricted environments.

44. MRI for Detection of Flowing Liquids Different types of MRI experiments are set up to detect flowing liquids, such as blood. The liquid can be tagged using a chemical marker injected upstream, or the MRI can tag the flow directly by tipping spins in different areas and watching how they move. note right lung is missing left lung heart liver aorta Doctors call this “MRA”, Magnetic Resonance Angiography. Engineers use it to study fluid flow in real-world devices.

45. Chemical Shift Images of Rat Brain “axial” cross sections through brain seen from the front Normal “anatomical” image. Tumor shows up in this chemical shift image as a region rich in lactate. brain tumor Chemical shift images. Each shows the position and concentration of a particular brain chemical.