NMR-spectroscopy. I: basics. Peter Schmieder

Transcription

1 NMR-spectroscopy I: basics

2 Why spectroscopy? 2/102 Why spectroscopy It is well established that all biological relevant processes take place via interactions of molecules, either small ones (metall ions, second messengers, drug-like molecules, lipids,hormones, proteins, DNA, RNA.) To fully understand those processes a knowledge of the 3Dstructures and the interactions of those key players is mandatory. Since molecules are to small to be observed directly they are investigated indirectly, e.g. via their interaction with electromagnetic radiation.

3 Why spectroscopy? 3/102 Interactions can be observed for almost every region of the electromagnetic spectrum. We are thus not observing the molecules directly but we observe phantoms or specters of molecules: spectrum (latin) = specter σκοπειν = scopein (greek) = to observe

4 Why spectroscopy? 4/102

5 Why spectroscopy? 5/102 The absorption of the electromagnetic radiation is a resonance phenomenon. Only certain types of radiation can be absorbed depending on the energy difference of the available states. Thus the famous equation: E = ћ

6 NMR-spectroscopy

7 General aspects of NMR-spectroscopy 7/102 Nuclear Magnetic Resonance NMR-spectroscopy detects the resonance of atomic nuclei with radio waves. The effect is only readily observable in a strong magnetic field. Each nucleus is observed separately and interactions between nuclei can be observed as well. The picture of a molecule provided by NMR thus corresponds well to the view of a chemist that is seeing molecules as atoms connected by bonds. In the areas of biochemistry and structural biology NMR yields information on structure, ligand-interaction and mobility necessarily at atomic resolution.

8 General aspects of NMR-spectroscopy 8/102 NMR as an analytic method during synthetic work 1 H-spectrum ppm 13 C-spectrum

9 General aspects of NMR-spectroscopy 9/102 Determination of the constitution of natural products

10 General aspects of NMR-spectroscopy 10/102 Determination of 3D structures of proteins Using NMR 3D structures of proteins can be determined either in solution of in the solid state. This is done indirectly by determining distances and angles within the molecule and then arranging the protein chain to fit all the data.

11 General aspects of NMR-spectroscopy 11/102 Detection of intermolecular interactions NMR can be used for the detection of protein-ligand interactions

12 General aspects of NMR-spectroscopy 12/102 Investigation of dynamic phaenomena Using NMR the mobility of proteins (and other molecules) can be investigated

13 General aspects of NMR-spectroscopy 13/102 Detection of processes in living cells (in-cell-nmr) Using in-cell-nmrspectroscopy it is possible to follow the modifications of proteins in living cells 15 1H N 15 N Ser (e.g. phosphorylation). Serine 1H 15 N 15 N 1H phospho-serine pser 1H

14 Basic aspects of NMR-spectroscopy

15 Basic aspects of NMR-spectroscopy 15/102 Prerequisite for NMR-spectroscopy is a nuclear spin that can be thought of as a mixture of a gyroscope and a little magnet + =

16 Basic aspects of NMR-spectroscopy 16/102 A gyroscope has an angular momentum that is firmly oriented in space

17 Basic aspects of NMR-spectroscopy 17/102 Orientation of the little nuclear magnet is prevented by its gyroscopic properties, the nucleus starts a precessional motion

18 Basic aspects of NMR-spectroscopy 18/102 The resonance frequency of the spins (here the proton spins) is determined by the strength of the magnetic field B 0 [Tesla] [MHz]

19 Basic aspects of NMR-spectroscopy 19/102 But we are dealing with quantum mechanical phaenomenon, in the case that we are interested in (high resolution NMR) there are two possible orientations ( and ) for the gyroscope/magnet=spin E = ћ B 0

20 Basic aspects of NMR-spectroscopy 20/102 We will then have a Boltzmann-distribution N /N = exp(- E/kT) = exp(- h B 0 / 2 kt) At 600 MHz frequency we get N /N = This extremely small difference is the reason for the low sensitivity of NMR spectroscopy

21 Basic aspects of NMR-spectroscopy 21/102 Without an external magnetic field all orientations are equal and the spins are randomly oriented

22 Basic aspects of NMR-spectroscopy 22/102 With an external magnetic field the resulting orientation yields a small magnetic moment, a small macroscopic magnet, the axis is called the z-axis

23 Basic aspects of NMR-spectroscopy 23/102 y z x Now we use electromagnetic radiation of the proper, the resonance frequency It is applied for a short amount of time ( sec to msec range) and is then called a pulse. It is short and strong exciting a wide range of frequencies at once p

24 Basic aspects of NMR-spectroscopy 24/102 right-hand-rule This pulse turns every spin and thus the net magnetic moment, the length of the pulse determines how far they go.

25 Basic aspects of NMR-spectroscopy 25/102 If give with correct length (a 90 pulse ) this results in a rotation of the magnetic moment into the x,y-plane, no z-magnetization is left. The pulse also forces the spins to precess with phase coherence after it stopped.

26 Basic aspects of NMR-spectroscopy 26/102 So after the pulse the precession continues and induces a current in the detection coil which is then digitized. The size of the time step determined what frequencies we can detect (Nyquist condition) f n = 1/(2 t)

27 Basic aspects of NMR-spectroscopy 27/102 A simple one-dimensional NMR spectrum is thus recorded with the scheme on the left and the acquisition of data is repeated to get better signal-to-noise

28 Basic aspects of NMR-spectroscopy 28/102 The detected time signal (the FID) is converted into a frequency spectrum by Fourier transform FT

29 Basic aspects of NMR-spectroscopy 29/102 A closer look to digitization and FT z y x 90 -x z y x 2000 Hz -500 Hz Hz y We record a spectrum with three lines using time steps of t = 166 sec, which means we can detect t = 0 sec x +/ Hz.

30 Basic aspects of NMR-spectroscopy 30/102 y y y y x x x x t = 0 sec t = 166 sec t = 332 sec t = 498 sec 1 Iy t [msec] 1 Ix That s how the data points look that we record

31 Basic aspects of NMR-spectroscopy 31/102 1 Iy t [msec] 1 Ix FT [Hz] [ppm]

32 Basic aspects of NMR-spectroscopy 32/102 The decay determines the shape of the peak, slow decay results in sharp lines, fast decay in broad lines. The decay is determined by the relaxation times

33 Basic aspects of NMR-spectroscopy 33/102 Magnetic properties of some NMR nuclei Kern I natürliche Häufigkeit gyromagnetisches Verhältnis 1 H 1/ % C % 0 13 C 1/ % N % N 1/ % F 1/2 100 % P 1/2 100 % Cd 1/ % -5.96

34 Basic aspects of NMR-spectroscopy 34/102 Sample changer Magnet Hardware cabinet Probe

35 Basic aspects of NMR-spectroscopy 35/102

36 NMR-Parameter

37 NMR-parameter 37/102 Chemical Shift The electrons around the nucleus shield it from the external magnetic field, the more electrons there are the less field reaches the nucleus B eff = (1 - ) B 0 = (1 - ) B 0 = ( ref ) / 0 x 10 6 = ( ref ) x 10 6

38 NMR-parameter 38/102 The values have no absolute meaning, they are given relative to a reference compound ArOH -CHO olefinic ROH RNH 2 aromatic CH -O n acetylenic CH nch -N CH 2 CH 3 -C n -CO CH -Ar 3 CH -C=C 3 TMS 1 ( H)/ppm

39 NMR-parameter 39/102 The chemical shift thus determines the position of the resonance line in the spectrum, and assignment means to determine which nucleus in the molecule gives rise to which line in the spectrum OHs ppm

40 NMR-parameter 40/102 But normally the electrons are not distributed spherically around the nuclei since the shape of most molecule is not a sphere. anisotropic electron distribution chemical shift anisotropy (CSA)

41 NMR-parameter 41/102 The result is a powder spectrum

42 NMR-parameter 42/102 solution In solution the rotation of the molecule takes care of that solid What can we do in the solid state?

43 NMR-parameter 43/102 If we rapidly interchange the x, y and z axis we should get the same averaging as we do in solution. We can achieve that by rotating around the diagonal of a cube. tan m = 2 m = That is known as the magic angle

44 NMR-parameter 44/102 So we do Magic Angle Spinning MAS Magic-Angle Spinning (MAS) drive bearing

45 NMR-parameter 45/102 Dipolar coupling This is interaction between the magnetic dipols of individual spins and works through space The size of the dipolar coupling depends on the distance of the spins (r jk ), the angle between the connecting line and the magnetic field ( jk ) and the gyromagnetic ratio of the nuclei involved ( j,k ) D ~ (3 cos 2 jk 1) j k r jk 3

46 NMR-parameter 46/102 Dipolar couplings can be quite large and can then distort the spectra so that they are useless. 1 H is particularly problematic. 5,000 Hz 40,000 Hz liquid-state NMR proton chemical shift (ppm)

47 NMR-parameter 47/102 0 π dθjk sinθ jk (3 cos 2 θ jk -1) = 0 In solution we again get a weighted average over all molecules and an averaging due to the rotation of the molecules and the coupling is invisible. It does, however, still have an effect by creating local fluctuating fields that help with the relaxation of the spins.

48 NMR-parameter 48/102 In the solid state we are rotating the sample to average the CSA. For the dipolar coupling that means that every connecting line between two nuclei is on average at an angle of with the magnetic field. D ~ (3 cos 2 jk 1) cos (54.73 ) = 1 / 3 D ~ (3 cos 2 (54.73 ) 1) = 0 The dipolar coupling is averaged to zero!

49 NMR-parameter 49/102 But the dipolar coupling carries distance and angle information, so we would like to measure it, this can be done by RF-pulses and is called recoupling i j D ~ (3 cos 2 jk 1) r 3 ij The most interesting distance information is long-range information, that is difficult to get because of dipolar truncation, small dipolar couplings can only be measured if large ones are absent, this can be solved by labeling.

50 NMR-parameter 50/102 How fast do we spin the rotor? As a rule of thumb we need to spin faster than the size of the interaction we want to remove. So it depends on the nucleus we detect. If we want to detect 1 H we have to cope with large dipolar couplings of approx. 100 khz. Diameter MAS frequency 7.0 mm < 8 khz 4.0 mm < 18 khz 3.2 mm < 23 khz 2.5 mm < 35 khz 1.3 mm < 70 khz 0.7 mm < 120 khz But we also have to consider the forces (e.g x g for khz) and the speed of the outer rotor surface, that should not exceed the speed of sound.

51 NMR-parameter 51/102 We also get a side effect, the spinning side bands Their position depends 10,000 Hz on spinning speed and spectrometer frequency 10,000 Hz MHz magnet 12,000 Hz * 30,000 25,000 20,000 15,000 10,000 5, MHz magnet 12,000 Hz

52 NMR-parameter 52/102 Relaxation Relaxation is the process during which the nuclei get rid of the energy transferred to the system by the RF pulse Contrary to other types of spectroscopy there are no ways to create the necessary fluctuating magnetic field, except the movement of the molecule itself. That is why NMR-states are fairly long-lived and allow for more complicated multidimensional experiments. And since the dynamics of the molecule is responsible for the relaxation of the spins we can learn something about the dynamics from the analysis of the relaxation times.

53 NMR-parameter 53/102 The two key players in creating those fluctuating fields are the dipole-dipole interaction and the CSA which can also interact with each other. And while they average out in sample as a whole they are still present locally for each molecule as a source of relaxation DD CSA

54 NMR-parameter 54/102 The molecules exhibit motions on many different time scales and there are internal as well as overall motions. The correlation time of a molecule is an important variable in the understanding of relaxation.

55 NMR-parameter 55/102 Dipolar coupling is also responsible for the Nuclear Overhauser Enhancement (NOE) effect which results from what is called cross-relaxation. The NOE effect depends on the distance between the two spins that are involved in the cross relaxation and that distance can thus be derived from the measured intensity I NOE 1/r 6 r Because of the r 6 dependency only distances up to 500 pm can be observed

56 NMR-parameter 56/102 Scalar or J-coupling The electrons surrounding the nuclei do also establish an interaction between the nuclei

57 NMR-parameter 57/102 Scalar couplings are usually small compared to difference in chemical shift, his leads to a fine structure of the resonance lines RCH - CH 2 3 J = 8 Hz

58 NMR-parameter 58/102 We distinguish between an interaction between like nuclei (homonuclear J- coupling) and unlike nuclei (heteronuclear J-coupling) as well as between direct and long-range couplings. The number of bonds between the interacting nuclei is given as a superscript, the type of nuclei as a subscript.

59 NMR-parameter 59/102 Direct couplings via just one bond are one order of magnitude larger than those over more bonds, the latter carry structural information 1 J HH = 276 Hz 2 J HH = Hz 3 J HH = Hz 4 J HH = Hz 1 J HC = Hz 2 J HC = Hz 3 J HC = Hz 4 J HC = Hz 1 J HN = Hz 2 J HN = Hz 3 J HN = Hz 4 J HN = Hz

60 NMR-spectroscopy of proteins (1)

61 NMR-spectroscopy of proteins 61/102 The main problem obtaining assignments of NMR-spectra of proteins result from the fact that a protein is a polymer, i.e. a repetition of identical units

62 NMR-spectroscopy of proteins 62/102 (-)-Menthol ppm

63 NMR-spectroscopy of proteins 63/102 cyclic hexapeptid aromatic protons H N -protons H -protons aliphatic protons Indol-H N ppm

64 NMR-spectroscopy of proteins 64/102 1 H NMR-spectrum of a protein Aromaten aromatic Aromaten Aliphaten Aliphaten aliphatic H N H N H H CH CH 3 3

65 NMR-spectroscopy of proteins 65/102 Differences in the chemical shifts result from defined elements of secondary structure

66 NMR-spectroscopy of proteins 66/102 1 H NMR-spectrum of an unfolded protein ppm

67 DO2 HO2 NMR-spectroscopy of proteins 67/ C NMR-spectrum of a protein

68 DO2 HO2 NMR-spectroscopy of proteins 68/ N NMR-spectrum of a protein

69 DO2 HO2 NMR-spectroscopy of proteins 69/102 NMR-spectra of proteins are thus not that much different from spectra of small molecules and show similar features (chemical shift, J-coupling etc.). Aromaten Aliphaten H N H CH ppm Because of the large number of nuclei contributing to the spectrum, the fact that they are polymers and their size, one-dimensional spectra are not sufficient and we need to use multidimensional ones.

70 Multidimensional NMR-spectroscopy

71 Multidimensional NMR-spectroscopy 71/102 1D-NMR: 2 axes intensity vs. frequency 2D-NMR: 3 axes intensity vs. frequency (1) vs. frequency (2)

72 Multidimensional NMR-spectroscopy 72/102 The two most important advantages of multidimensional spectra are: Improved resolution: The signals are distributed over an area (2D) or a space (3D, nd) rather than a line (1D), that ensures an better separation of signals. Transfer of magnetization: The resulting signals indicate the interaction between nuclei. These can be interactions via J-coupling (i.e. bonds) or via NOE or dipolar coupling (i.e. through space). Taken together this enables an easier assignment and interpretation of spectra and a more straightforward extraction of information.

73 Multidimensional NMR-spectroscopy 73/102 2D-NMR experiments contain two new elements: evolution time and mixing time preparation evolution mixing detection (t 1 ) (t 2 ) evolution time: creation of a further frequency axis by indirect detection mixing time: transfer of magnetization via spinspin interactions

74 Multidimensional NMR-spectroscopy 74/102 Evolution time Using the evolution time we create a further, indirect dimension. There still is only one real detection of signal, but the signal is modulated by a systematic change of a delay in the pulse-sequence. We thus need at least two pulses, this is the simplest NMR experiment, the COSY (COrrelation SpectroscopY)

75 Multidimensional NMR-spectroscopy 75/102 Evolution time We have discussed the acquisition of the signal in a 1D already including its digitization, we now just repeat this acquisition for every time value in the indirect dimension. As in direct detection these time values have to be multiples of a time value t that detemines the largest frequency we can detect.

76 Multidimensional NMR-spectroscopy 76/102 We again use our three lines to see what is going on in the simplest 2D-NMR-sequence possible. z y x 90 -x z y x 2000 Hz -500 Hz Hz 90 -x 90 Y k t k t 2 1

77 Multidimensional NMR-spectroscopy 77/102 The starting intensity in t 2 (k t 1 = t 1 ) depends on the delay k t 1

78 Multidimensional NMR-spectroscopy 78/102 y y y y x x x x t 1 = 0 sec t 1 = 166 sec t 1 = 332 sec t 1 = 498 sec t 1 = 498 sec t 1 = 332 sec t 1 = 166 sec t 1 = 0 sec [Hz] [ppm]

79 Multidimensional NMR-spectroscopy 79/102 Intensities along the artificial second time axis vary similar to those in real acquisition time 1 Iy t [msec] t 1 = 498 sec t 1 = 332 sec t 1 = 166 sec t 1 = 0 sec [Hz] [ppm]

80 Multidimensional NMR-spectroscopy 80/102 This results in a two-dimensional FID

81 Multidimensional NMR-spectroscopy 81/102 After a first FT we obtain an interferogram

82 Multidimensional NMR-spectroscopy 82/102 After a second FT we have the two-dimensional spectrum

83 Multidimensional NMR-spectroscopy 83/102 To analyze the spectra they are viewed as contourplots, in which intensities are display as contour levels

84 Multidimensional NMR-spectroscopy 84/102 Preparation Evolution Mixing Detection (t 1 ) (t 2 ) If there were only evolution and detection, we would detect the same frequency in both dimensions and not gain information or resolution. The critical element is the mixing time, during which a transfer of magnetization takes place. The mechanism used for the transfer (J-coupling, dipolar coupling, NOE, chemical exchange) is under full control of the operator that chooses the appropriate.

85 Multidimensional NMR-spectroscopy 85/102 Mixing time That means that we do not have only a 1 H or a 13 C, 15 N or 31 P spectrum but a large amount of different experiments that correlate different types of nuclei via a variety of interactions. They will be used to first obtain a reliable assignment for all resonance lines, different spectra provide different information that can be pieced together to obtain a resonance assignment. Once an assignment is obtained, that information can be further used to obtain information on the constitution, three-dimensional structure, the dynamics and intermolecular interactions of a molecule or a set of molecules. All information will inherently be at atomic resolution.

86 Multidimensional NMR-spectroscopy 86/102 We will take a closer look at 2D spectra of a small molecule. Here the resolution of 1D-spectra is usually good enough, for a full and unambiguous assignment 2D-spectra might still be necessary. O O Cinnamic acid ethyl ester Ethyl-3-phenylprop-2-enoat (IUPAC)

87 Multidimensional NMR-spectroscopy 87/102 Chemical shifts taken from a database are often not accurate enough ppm H O O H PPM

88 Multidimensional NMR-spectroscopy 88/ C is often much better than 1 H but may not be sufficient ppm O O PPM

89 Multidimensional NMR-spectroscopy 89/102 homonuclear spectra Here the transfer of magnetization takes place between nuclei of the same type (e.g. H and H or C and C). Both frequency axes then show the same type of chemical shift. If there is a transfer this results in two different chemical shifts in the two dimensions: Crosspeak If there is no transfer the chemical shift in both dimensions is identical: Diagonalpeak Diagonalpeak Crosspeak

90 Multidimensional NMR-spectroscopy 90/102 The diagonal peaks correspond to the 1D spectrum and thus carry no new information. The cross peaks, however, provide very important information: behind every line in the spectrum is a nucleus, depending on the type of spectrum we recorded, e.g. a proton ( 1 H). A cross peak now indicates an interaction between two nuclei and since we choose the experiment we also know what interaction it is. If we choose a 1 H-COSY then we know the interacting peaks are not more than three bonds apart in the molecule. H (ppm) Transfer H 1 H 2 H (ppm)

91 Multidimensional NMR-spectroscopy 91/102 In a COSY the ethyl group can be ppm easily identified, but not the aromatic and olefinic protons H H H O O 1 2 H H H H 3 ppm H O O 1.30 H ppm ppm

92 Multidimensional NMR-spectroscopy 92/102 heteronuclear spectra Here the transfer takes place between different types of nuclei und thus both axes exhibit different chemical shifts. If there is no transfer then there will be no peak, but if there is, the peak appears at the intersection of the chemical shifts of the two nuclei involved.

93 Multidimensional NMR-spectroscopy 93/102 HMQC = Heteronuclear MultipleQuantum Correlation X The signal indicates a direct bond between the proton and the carbon (or other heteronculeus) H The coupling itself is not visible in the spectrum due to decoupling, manages by the application of additional pulses during the pulse sequence

94 Multidimensional NMR-spectroscopy 94/102 In the HMQC of our small molecule we obtain two signal for ethyl group and 5 for the aromatic/olefinic proton-carbon pairs ppm ppm O O H H H H H H H O O % assignment is still not achieved, even though we gained information by spreding ppm out the signals in two dimension ppm

95 Multidimensional NMR-spectroscopy 95/102 If we combine the two spectra we have so far, the signal of the olefinic and aromatic resonances can be seperated H H O H H O H O H O H H H H H H H H ppm ppm O O H O O H ppm ppm

96 Multidimensional NMR-spectroscopy 96/102 To complete the full and unambiguous assignment we need another 2D spectrum the HMBC (Heteronuclear Multi Bond Correlation) that will give correlations via multiple bonds using the smaller long-range couplings. In general 2 J und 3 J couplings are large enough to give correlations, but there size may vary and sometimes even nuclei more than three bonds apart may give rise to correlation signals. Signals originated from the larger direct coupling are redundant (since we have the HMQC already) and are best suppressed.

97 Multidimensional NMR-spectroscopy 97/102 HMBC = Heteronuclear MultipleBond Correlation Signals thus indicate a correlation via 2 or 3 bonds, the suppression of the direct signals from the HMQC is often not completely possible. HMQC Transfer via 1 J HX X Transfer via n J HX HMBC = via 1 J HX H H

98 Multidimensional NMR-spectroscopy 98/102 In the HMBC the signals from the ethyl group are again easily found an assigned as well as the signal for the CO. ppm H H O H H H H H H O CH 3 80 H The CO does not have a proton attached so it did not show up before O O ppm

99 Multidimensional NMR-spectroscopy 99/102 An assignment of the other carbons needs a close look at HMBC and HMQC ppm H H O H H H H H H O CH H O O green = HMQC, red= not suppressed signals ppm

100 Multidimensional NMR-spectroscopy 100/102 If we increment more than one delay in the pulse-sequence independently we obtain several indirect dimensions, 3D, 4D, 5D. 3D-NMR

101 Multidimensional NMR-spectroscopy 101/102 3D-NMR Analysis of the data still takes place on the level of 2Ds but with better resolution A problem of higher dimensionality is the time required to record the spectra: 1D with 2048 datapoints: 1 min 2D with 512 x 2048 datapoints: 8.5 hours 3D with 128 x 128 x 2048 datapoints: 11 days, 9 hours 4D with 32 x 32 x 32 x 2048 datapoints: 22 days, 18 hours

102 102/102 That s it