The following parameters can be extracted from 1-D spectra and contain useful information

Transcription

1 PARAMETER F NE-DIMENSINAL NMR SPECTRA: The following parameters can be extracted from 1-D spectra and contain useful information 1) chemical shifts (in ppm), 2) scalar coupling constants (in z) 3) line-widths (in z) 4) intensity of signals (integrals) 1) TE CEMICAL SIFT: The chemical shift is related to the resonance frequency of a particular nucleus. owever, instead of presenting the frequency itself, which depends on the strength of the static field, the chemical shift is usually given relative to a standard and normalized with respect to the transmitter frequency. This relative scale is field independent so that the values obtained in different laboratories or on different instruments can be compared: (X)= 10 6 ( ref )/ ref ( ref ) is the frequency of a standard, (e.g. TMS), usually but not always at 0 ppm. Since the obtained values are small, they are given in parts per million (ppm). The chemical shift of protons is mainly due to the diamagnetic contribution, which depends on the following three parameters: a) the electron density around the nucleus b) local anisotropy effects c) steric effects The electron cloud decreases the magnitude of the static field at the locus of the spin (the so-called shielding effect) so that the resulting effective field is weaker and hence the resonance frequency ( ) lower: 2 = eff eff = Bo - C Fig.1: The thickness of the lines is proportional to the strength of the magnetic field. A) Nucleus in the absence of elctrons. B) Nucleus surrounded by a spherical cloud. C) Electron distribution in a C- bond. Due to the similar electronegativities of carbon and hydrogen the electron cloud is a symmetric ellipsoid. D) Electron distribution about an fragment. Due to the much larger electronegativity of the oxygen the cloud is asymmetric with the effective field being much stronger at the proton site when compared to C).

2 2 ( is an atom-type dependent constant, the so-called gyromagnetic ratio, is the chemical shielding constant). Electronegative substituents reduce electron density at neighbouring protons, thereby reducing the shielding effect and leading to higher frequencies at neighbouring nuclei. The shielding gives rise to shifts with larger values in ppm. Methoxy protons resonate around 4 ppm whereas normal methyl protons are found close to 1 ppm. Some chemical bonds display pronounced anisotropy in their electron distribution. This means that the magnetic susceptibility of a proton can be strongly dependent on the orientation of surrounding protons. Such effects are particularly observed in the vicinity of -electrons. For examples, protons located over the center of an aromatic ring are deshielded whereas those outside the ring are shielded. The ring-current shifts contribute significantly to the chemical shift dispersion observed in spectra of proteins: Fig.2: Cones of anisotropy observed for double/triple bonds and aromatic systems (taken from [1])

3 3 Aliphat Me Aromat ppm Fig.3: Typical proton spectrum. Some easily recognizable protons have been annotated. Many functional groups are easily recognized from their typical chemical shifts. In general, more chemical shift dispersion is observed for molecules, which are functionalized in particular for those containing electron-withdrawing groups or aromatic moieties. In contrast, much signal overlap is found in pure hydrocarbon compounds. For such compounds 13 C spectra are highly valuable. Carbon chemical shifts are also easier to predict so that the proposed structures may also be better verified. Fig.4: 1 chemical shift regions for certain functional groups (taken from [2]).

4 4 The following table presents a rough overview of typical chemical shift ranges. Local anisotropy effect, however, may easily shift signals by more than 1 ppm. The total range encountered for protons is approx. 15 ppm (neglecting organometallic compounds). 2) SCALAR (SPIN-SPIN) CUPLINGS: Spins may interact via scalar or dipolar couplings. Scalar couplings are transmitted via electrons and therefore depend on the number of intervening bonds. To be effective s-electrons must be involved, because only these have non-zero probability to be located at the nucleus. The interaction with each local proton splits each line into a doublet. For weakly coupled systems the total number of lines can be calculated using the following formula: J ac J ac J ab J ab J ac J ac a a Fig.6: Peak patterns for a proton signal coupled to two other protons, with the two couplings being different in size (left) or identical (right). = 2 N where N is the number of coupled spin-1/2 nuclei. In case where the couplings are similar or identical certain lines overlap in the resulting multiplet and the total number of lines is: = N+1 where N is the number of coupling partners with identical couplings.

5 z Fig.7: Typical multiplet pattern in a 1-D spectrum. In the latter case some lines have larger intensities than others. The relative intensities of lines are given in the following table (which is constructed from the Pascal triangle): Number of coupled partners Total number of lines rel. Intensities I=1/2 I=1 0 1 (Singlet) (Doublet) 1:1 2 3 (Triplet) 1:2:1 3 4 (Quartet) 1:3:3:1 4 5 (Quintet) 1:4:6:4:1 5 6 (Sextet) 1:5:10:10:5:1 6 7 (Septet) 1:6:15:20:15:6:1 0 1 (Singlet) (Triplett) 1:1:1 2 5 (Quintet) 1:2:3:2:1 3 7 (Septet) 1:3:6:7:6:3:1 The magnitude of the scalar couplings largely depends on the number of intervening bonds, and mostly (but not always) decreases with increasing number of bonds. The number of intervening bonds is annotated as a superscript (e.g. 2 J or geminal, 3 J for vicinal couplings,etc.) 1 J >> 2 J > 3 J for J ( 1, 1 ) 1 J >> 3 J, 2 J for J ( 1, 13 C)

6 Based on coupling patterns ortho, meta- or para-disubstituted aromatic compounds are easily distinguished, because they contain signals with different multiplet patterns. The three-bond scalar couplings ( 3 J) depend on the dihedral angle about the central bond (Karplus relationship) and this fact is much used to determine stereochemistry. As shown in the figure below syn- or anti- conformations ( = 0 or 180 ) display large 3 J couplings and gauche conformations ( = ±60 ) small 3 J couplings. owever, this effect is only observable if the bonds are not freely rotating, otherwise the rotationally averaged values are observed. Averaging takes place over the gauche and trans conformations and usually leads to couplings of around 7 z. 6 3 J (z) b a /rad Fig.8: Karplus curve for vicinal 1, 1 couplings Rotation about single bonds is largely hindered (or impossible) in cyclic compounds. ence scalar couplings are very useful for determination of stereochemistry in sugars, in which axial-axial arrangements many be easily distinguished from axial-equatorial or equatorial-equatorial arrangements: 1a R R 2e a R' a b 2a b R' 3 Ja,e = 2-5 z 3 Ja,a = z 3 Je,e = 2-5 z 3 Ja,b 11 z 3 Ja,b z Fig.9: Vicinal couplings in rigid systems

7 7 Similarly, couplings are very useful to determine substitution at double bonds, with the trans arrangement usually giving rise to the larger coupling. It should be noted here that the magnitude of scalar couplings also depends on the substituents. Geminal couplings depend on the s-character of the involved bonds, and therefore are related to hybridization: 2 C z C=C z +2.5 z 120 Fig.10: Geminal couplings 4 J couplings are only observed when the intervening bonds are held constantly in a zig-zag arrangement, which is often the case in cyclic or double-bonded systems (they are small, mostly smaller than 1 z): 4e 2e Fig.11: Long-range ( 4 J) couplings 13 C nuclei are mostly coupled to protons (but not to other carbons for reasons of low 13 C abundance). Usually, these heteronuclear couplings are removed during signal acquisition by broadband decoupling. 1 J 13 C, 1 couplings depend on the hybridization: sp 3 : 125 z sp 2 : 167 z sp : 250 z Some 13 C experiments (e.g. DEPT) display information about the number of attached protons and provide very useful help for the assignment of the 13 C signals.

8 8 TE INTENSITY F SIGNALS: Definition: The integral of a signal reflects the intensity under the curve and is proportional to the number of protons contributing to that signal. They are usually plotted as numbers below the signal or as integral trails. Unfortunately, integrals can easily be substantially wrong. This is often the case when relaxation properties of the involved protons are different. Isolated protons (those far away from others) cannot efficiently relax via dipolar couplings and therefore have long T1 relaxation times. As a result they do not rapidly relax back into the equilibrium state, leading to partial saturation (and hence lower intensities). This problem may be circumvented (provided it has been recognized!) if the relaxation delay is set to longer values. Another type of protons, which notoriously display wrong values for the integrals are those, which are broad (either by exchange or by other mechanisms (paramagnetic relaxation) ppm Fig.17: 1 spectrum with integrals.

9 9 IGER RDER SPECTRA When the chemical shift difference of spins (in z) that are coupled to each other is not large with respect to their mutual scalar coupling constant ( a- b /Jab < 10) so-called higher-order spectra occur. In such systems the intensity of lines does not follow the normal rules and even more lines than usual may be encountered. In the case of two spins solely coupled to each other, the inner lines of the two doublets are larger than the outer lines; this effect is called the roof effect : AB-System J AB = 10 z ABX-System J AB = 10 z, J AX = 6 z, J BX = 4z z 3000 z z z 3000 z z z 3000 z z z 3000 z z Fig.12: Second order effects in dependence of the shift difference. For the simulations the following values have been used: A B /J=15(1), 3(2), 1(3) und 0(4). For the ABX spin system the X-part is shown separately. When a- b /J ab > 10 a two spin system is called an AX system, whereas if a- b /Jab < 10 it is an AB system and for a= b an A2 system is encountered. Scalar coupling constants cannot be simply read out of higher order spectra. In such cases spectra simulations are required in order to determine the true values.

10 10 TE NMENCALTURE F SPIN SYSTEMS : A spin system is a set of spins in a coupling network, in which no interruption of scalar coupling occurs. 3 C C 3 C 3 C 3 3 C Fig.13: Covalent structure of a steroid. The separate spin systems contained in the molecule are drawn in different colors. chemical equivalence: Atoms, which through symmetry operations can be transformed into each other are called chemically equivalent. For all spins, which are chemically non-equivalent different letters of the alphabet are used. The separation of the letters in the alphabet reflects the difference in chemical shift (AB vs. AX). The number of chemically equivalent nuclei is annotated as a subscript, e.g. A 2 X 3. isocronic nuclei: Nuclei, which by accidence or through chemical equivalence, have identical resonance frequencies are called isochronic. magnetic equivalence: nuclei, which have identical scalar coupling constants to all other spins within the molecule are called magnetically equivalent.

11 11 Chemically equivalent nuclei need not be magnetically equivalent in cases where isochronic nuclei are not magnetically equivalent, an additional dash is used, e.g. AA instead of A 2. b b a S a Fig.14: Thiophene molecule In thiophene the protons a und a' are chemically equivalent because they may be transformed into each other (the molecule has a C 2 axis). But because the coupling constant of J (a, b) is not equal to the coupling J (a', b) the two spins are magnetically non equivalent and hence the spin system is called AA BB. omo-, enantio- and diastereotopic spins Two groups or nuclei X within a -CX 2 -R fragment may be homotopic, enantiotopic or diastereotopic. In order to test which of the three possibilities is correct for the group in question X is replaced by a new group T: X T and T X: omotopic spins: If X T and T X are identical molecules, the two groups are homotopic. omotopic protons are chemically equivalent and will only lead to a single signal. Enantiotopic spins: In case X and T X form a pair of enantiomers, the two groups are enantiotopic. Such groups will give a single signal in a non-chiral environment but may by resolved into the separate signals by using chiral solvents or chiral shift reagents.

12 12 Diastereotopic spins: Where X T and T X are diastereomers, the groups are called diastereotopic. Diastereotopic groups are chemically inequivalent but may still (by accidence) be isochronic. Whether one or two signals are observed is most often related to the distance from the chiral centre.

13 13 General literature: [1] M. esse,. Meier, B. Zeeh, Spektroskopische Methoden in der organischen Chemie, Verlag Thieme. Good overview over the most common spectroscopic methods (IR, NMR, UV, MS) with a very useful chapter on NMR. [2]. Friebolin, Ein- und zweidimensionale NMR Spektroskopie, Verlag Chemie, Simple explanation in german language. [3] T.D.W. Claridge, igh-resolution NMR Techniques in rganic Chemistry, Pergamon Press ne of the best books in my view. Contains a lot of practical tips and also some descriptions of the hardware. [4] J.K.M. Sanders, B.K. unter, Modern NMR Spectroscopy, xford University Press, [5]. Günter, NMR-Spektroskopie, Verlag Thieme, [6] S. Braun,.-. Kalinowski, S. Berger: 150 and more Basic NMR Experiments, VC-Wiley Practical introduction into many of the commonly used experiments. Very useful for those, who run their own spectra. 2-Dimensional NMR spectroscopy [7] W.R. Croasmun, R.M.K. Carlson, Two-Dimensional NMR Spectroscopy, Applications for Chemists and Biochemists, Verlag Chemie, Expensive, but useful guide for 2D NMR (see the NMR library). Special books [8].. Kalinowski, S. Berger, S. Braun, 13 C NMR Spektroskopie contains an enormous amount of 13 C data. [9] D. Neuhaus, M. Williamson, The Nuclear verhauser Effect in Structural and Conformational Analysis, Verlag Chemie, The best book about the NE. [10] K. Wüthrich, NMR of Proteins and Nucleic Acids, Wiley, Bible for Protein/Peptide and DNA/RNA NMR. [11] G.C.K. Roberts, NMR of Macromolecules, A practical Approach, IRL Press, A bit more practically oriented. [12] J. Cavanagh, W.J. Fairbrother, A.G. Palmer III, N.J. Skelton, Protein NMR Spectroscopy, Academic Press [13]J. Mason, Multinuclear NMR, Plenum Press, Bible for all other nuclei ( e.g. 11 B, 15 N...) [14] M.. Levitt, Spin Dynamics, Wiley 2001 Very good book for a deeper understanding of theory (my favorite book)

14 13 C-NMR-Spectroscopy 1 13 C is the only NMR-active isotope of carbon. Unfortunately, its natural abundance is low (1.1%). In addition the gyromagnetic ratio of carbon is about one fourth of that of protons resulting in further loss of sensitivity. Therefore, 13 C NMR is inherently much less sensitive than proton NMR and usually much larger quantities (or much longer measuring times) are needed. Exercise: Please classify the relative sensitivities of the following nuclei (high, medium, small, inactive) nucleus Spin 1/2 > 1/2 0 natural abundance N (%) γ (10 7 rad s -1 T -1 ) sensitivity C C F P B B N N Pt

15 2 Due to the scalar couplings between 13 C and (mainly the directly attached) protons, rather complicated spectra can result (see Fig. 1). Although analysis of the multiplicities would, in principle, allow the determination of the number of attached protons and would thus enable us to distinguish between C, C 2 and C 3 groups, the couplings would degrade signal intensity enormously: ppm Fig.1: 13 C-spectrum (non 1 -decoupled) of 10% ethylene benzene in CDCl 3 The recording of so called DEPT spectra, in which the carbon lines are proton decoupled, but which by their signal phase still encode the number of attached protons is much more sensitive. For 13 C natural abundance molecules 13 C, 13 C-couplings are usually not visible in the 1D- 13 C- spectra. Mostly, carbon spectra are recorded in fully proton-decoupled mode. Thereby signal intensity is increased because the lines collapse, and signal dispersion is increased (because the number of lines is reduced). In addition, proton-decoupling gives rise to the NE (nuclear verhauser effect), which may result in increases in signal intensities of up to 200%. The gain in intensity due to the hetereonuclear NE depends on the distance separation to the next proton and yields significant values only for directly attached protons ppm Fig.2: 13 C-spectrum (CPD-decoupled) of 10% ethylene benzene in CDCl 3 owever, not only the differing extent to which signal intensities are influenced by the NE contribute to the vastly different signal intensities observed in 13 C spectra, as illustrated in Fig. 2. Signal intensities are even more strongly influenced by the T1 relaxation rates of the protons, which in turn depend (in first approximation) on the distance to the nearest protons. Quaternary

16 3 carbons, for example as well as carbonyl carbons, relax very slowly. Considering that pulse repetition rates are usually chosen in the range of 1-4 seconds, these carbon resonances (for which T1 values can easily reach 20 seconds or longer) remain in a partially saturated state, leading to lower signal intensities. Therefore, 13 C spectra are usually not integrated. In cases were 13 C spectra need to be integrated, e.g. when working with compound mixtures, socalled inverse-gated spectra may be recorded. Two measures are taken to re-establish proper signal intensities: The relaxation delay is set to sufficiently long values (20 to 60 seconds) and proton decoupling is only turned on during signal acquisition but not during the relaxation delay. Since the NE buildup takes some time (usually during the relaxation delay) but decoupling takes place instantaneously, reasonable signal intensities are restored with perfectly decoupled signals (see Fig. 3). Relaxation During a 1D 13 C-NMR-experiment equilibrium populations of α and β states are disturbed. Longitudinal relaxation (T1 relaxation) will re-establish the equilibrium population of the levels according to the Boltzman distribution. The time constant of this first-order process is called T1. Since the two states are characterized by different energies, this process is enthalpic. Many interactions will contribute to T1 relaxation, with the dipole-dipole interaction between the 13 C nucleus and its attached proton being the most important source. Where no proton is directly attached (because dipole-dipole interaction depends on d -6 ), other mechanisms such as chemical shift anisotropy (CSA, the chemical shift depends on the orientation of the molecule with respect to the static field and hence fluctuates due to (Brownian) motion in solution). The following figure presents typical values of T1 in a small organic molecule. bviously, a wide range of values is observed. The T1 values depend on 1) vicinity of protons, 2) molecular weight (tumbling time), segmental mobility etc. Example of some T1 values (in seconds): C 9.3 C Br-C 2 -C 2 -(C 2 ) 5 -C 2 -C 2 -C

17 4 Figure 3 depicts 13 C{ 1 }-spectra of phenyl ethylene, recorded at different pulse repetition rates as well as with an inverse-gated experiment. Experiments 1 to 3 demonstrate the influence of choosing different relaxation delays on the signal intensities. The signal-to-noise ratio is always determined on the ortho C atom (128 ppm). bviously, signal intensity dramatically decreases when reducing the relaxation delay, especially for quaternary carbons. It is important to note that even with relaxation delays as long as 64 seconds signal intensities are still not identical but vary due to the different amounts of NE the carbons receive. 1 pulse repetition rate 2s Si/No=94 2 pulse repetition rate 8s Si/No=311 3 pulse repetition rate 64s Si/No=458 4 pulse repetition rate 64s Si/No= ppm Fig.3: C{ 1 }-Spectra of 10% ethylene benzene in CDCl 3, recorded with various repetition rates. Spectrum 4 is recorded with the inverse-gated sequence. Attention: Routine carbon spectra on the sample changer are not optimized for slowly relaxing nuclei! Therefore quaternary carbons may easily be missed. If you think some signals are missing, the relaxation delay (usually called d1 on Bruker instruments) can be extended.

18 DEPT135-, DEPT90-experiments 5 For reasons of sensitivity carbon spectra are usually recorded in the fully proton decoupled mode. The disadvantage is that information about the number of attached protons is lost. DEPT spectra are a very valuable source of such information, which is very useful for signal assignment. In addition, the polarization transfer in DEPT spectra leads to considerably higher signal intensities. In the DEPT experiment the proton polarization (the population difference between α and β levels) is transferred via the large 1 J C, -couplings onto the carbons. In addition, DEPT experiments allow us to edit signal phases according to the number of directly attached protons. In the DEPT-135 experiments, methyl and methine signals have positive and methylene protons negative phases (or vice versa), whereas the quaternary carbons are completely missing. Where the magnitude of the 1 J 1, 13C -coupling constants differ significantly from standard values of 140 z (e.g. for alkynes, aldehydes, aromatic systems) DEPT spectra may actually deliver false information about the number of attached protons: DEPT135 C 3 C C 2 DEPT90 C q C{ } ppm Fig.4: 13 C{ 1 } and DEPT-spectrum of 10% ethylene benzene in CDCl 3

19 13 C-Chemical Shifts 6 The total carbon chemical shift range comprises about 250 ppm and is about 20 times larger than that of protons. Considering that signals usually appear as singlets signal dispersion is very good and signal overlap is rarely encountered. Range of 13 C chemical shifts: [ppm] δ Shielding frequencies resonance frequency B eff γ ν = 2π ' ν ( B 0 B ) = 2π old-fashioned expressions: low-field modern expression: deshielded high-field shielded 13 C-chemical shifts are largely determined by the paramagnetic contribution in contrast to proton chemical shifts, which are governed by the diamagnetic contribution. The former depends on the excitation energy for electrons ( E = E M E LUM ).. This is the reason why the carbon chemical shifts depend on the hybridization of the carbon atoms: δ (sp 3 ) < δ (sp) < δ (sp 2 ) 3 C C 3 C C C 2 C 2 6 ppm 72 ppm 123 ppm

20 7 Fig.5: Ranges of 13 C- chemical shifts grouped according to functional groups Carbon chemical shifts almost completely depend on the neighboring groups and local anisotropy effects have negligible influence. In contrast, stereochemistry has a strong impact on the values: 15.7 ppm C ppm C 3 trans-9-methylene decalin cis-9-methylene decalin Extensive 13 C chemical shift databases have been build up and quite often compounds can be determined from the carbon chemical shifts using expert systems. In addition carbon chemical shifts are more easily calculated than proton chemical shifts.

21 8 Influences of substituents on the 13 C-chemical shifts The introduction of functional groups into unsubstituted alkanes generally leads to a shielding at the α-position and to deshielding at the γ-position, whereas the influence at the δ and ε positions are usually negligible. The deshielding effect increases with increasing electronegativity of the substitutents. The following example demonstrates the effect of introduction of a hydroxyl group: C 3 -C 2 -C 2 -C 2 -C C 2 -C 2 -C 2 -C 2 -C When comparing pentane, 2-methylpentane and 2-dimethyl pentane it becomes obvious that increasing alkylation leads to deshielding at C1, C2 and C3: C C C C C C C C C Molecule symmetry and the number of signals in the 13 C{ 1 }-spectrum The symmetry of molecules reduces the number of signals. Any carbons that can be transformed into another by a symmetry operation (rotation, translation, reflection) will give rise to only a single line in the spectrum. Br Br Br Cl Cl Cl 6 signals 6 signals 4 signals

22 9 Increment-method Using increments, chemical shifts of carbon spectra can be estimated quite reliably with accuracies of about +/- 5ppm. Provided the substituent effects are additive the following general formula may be used to calculate the shifts: δ i = B + A k n k + S iα δ i = chemical shift of the carbon in question B = basis value (depends on the compound class, e.g. the value for aromatic carbons in benzene) A = substituent increment n = number of substituents S = steric or electronic correction factor Increment systems for alkanes According to Grant and Paul, substitution of a hydrogen atom by a methyl group leads to a lowfield shift of the attached carbon by approx. 9 ppm and for the β-carbon to a low-field shift of about the same extent. Assuming free rotation about C-C bonds leads to an up-field shift of approx. 2.5 ppm.

23 steric correction factors substituent increments 10 observed 13 C-resonance primary secondary tertiary quaternary C α, highest substituted neighboring C-atom C 3 C 2 C C A α = +9.1ppm A β = +9.4ppm A γ = -2.5ppm A δ = + 0.3pp example: Calculation of the 13 C-shifts of 2,2-dimethyl pentane and comparison with experimental data: 1 3 C 3 C C C C Basis value α β γ δ steric Calc. Exp. value No. B correction * * *9.1 +4* * * *

24 Double bond equivalents (DBE) 11 Provided data from elementary analysis, and hence the composition is known, the double bond equivalent allows us to estimate the number of double bonds or ring systems in the molecule. For example, a DBE of 6 indicates that the molecule has 6 rings, double bonds or combination of rings and double bonds. It is not possible to distinguish double bonds from rings. For molecules that only contain C,,, N, S and halogens, the following (shortened) rules may be used to estimate the DBE: 1. and S are removed from the sum formula 2. halogens are replaced by hydrogens 3. trivalent nitrogen is replaced by C 4. the resulting hydrocarbon C n x is compared to the corresponding saturated hydrocarbon molecular formula C n 2n+2. The DBE is calculated from the following formula: DBE = (2n + 2) x 2 Example: N F SF: C 9 12 SF: C 5 4 FN rule 1 rule 2 C 9 12 rule 3 C 6 6 DBE = 4 DBE = 4

25 12 13 C,X-Spin,Spin-Couplings 13 C, 1 -Couplings 1 J C, -Coupling Constants: Scalar couplings are transmitted via Fermi-contact interaction, which requires electrons to have non-vanishing probability at the nucleus. Therefore hybridization (percentage of s-orbitals!) strongly influences the 1 J C, -coupling constants. As a rule of thumb the scalar coupling constant can be estimated by multiplying the s-percentage with 500 (e.g. for sp 3 : 0.25*500=125z). Another factor contributing to the magnitude of the scalar couplings are inductive effects from substituents. 1 J-coupling constants in z C 4 Cl C 3 - C 3 -Li Cl C Cl J C, -coupling constants (geminal couplings) The magnitude of the geminal coupling constants depends strongly on the system and usually takes values in the range 0 to 20 z (triple-bonds: approx. 50 z). The magnitude of the geminal coupling constant therefore reveals little structural information. 3 J C, - coupling constants (vicinal couplings) The magnitude of the vicinal coupling constants assumes values in the range 0 to 16 z. It is strongly influenced by the dihedral angle (similar to the proton, proton couplings), the C-C bond length, the bond angle and the electronegativity of the substituents. The dependence of the vicinal coupling on the magnitude of the involved dihedral angle is exploited in NMR of carbohydrates, proteins and nucleic acids. 3+n J C, - coupling constants (long-range couplings) 13 C, 1 -coupling constants between proton and carbon nuclei separated by more than 3 bonds are usually very small. owever, in conjugated π-systems, values are a little larger so that these

26 13 couplings can be observed. Long-range couplings are not really important for structure elucidation. It is important to note that the magnitude of the scalar couplings is often not sufficient to distinguish two- and three bond C, scalar couplings.

27 13 C, 13 C couplings 14 1 J C,C -coupling constants 13 C-couplings cannot usually be observed due to the low natural abundance of 13 C (1.1%). The so-called satellite lines are 200 times smaller than the centre lines. Carbon-Carbon couplings are of course observable in isotopically enriched molecules; this fact is exploited in protein NMR. The magnitude of the 1 J C,C -coupling constant depends on the hybridization of the involved nuclei. Substitutent effects are largely limited to the couplings of the attached carbon (see Fig.6). C N 2 C 33 C C C 61 C 59.5 C 56.5 C C C C Br C 70 C C C C 3 Fig.6 13 C, 13 C-coupling constants of various molecules [in z] 13 C, 31 P-couplings Since phosphorus is 100% comprised of the spin-1/2 isotope 31 P, 13 C, 31 P-couplings will appear as doublets in proton decoupled carbon spectra. The magnitude of the one-bond 31 P, 13 C-couplings vary dramatically with values observed in the range between 53 and 476 z. Interestingly, 13 C, 31 P- 1 J-coupling constants may also take up values about 0 z, and may therefore assume values otherwise observed for geminal or vicinal couplings. As a consequence, multiplicities cannot be easily used to determine by how many bonds the carbon and phosphorus nuclei are separated. ( 3 C-C 2 -C 2 -C 2 ) 3 P C 3 -P()-(-C 2 -C 3 ) 2 1 J C,P = J C,P =+12 3 J C,P =+13 1 J C,P = J C,P = J C,P = J C,P = 0

28 13 C, 19 F -couplings 15 Like phosphorus, fluorine entirely exists in the form of the 19 F isotope, and therefore gives rise to line splittings in carbon spectra similar to 31 P. The magnitude of the coupling decreases with increasing number of separating bonds. 1 J C,F -coupling constants are relatively large ( z). In aliphatic systems, 13 C, 19 F-couplings may not be observed over 4 bonds, but in conjugated π-systems couplings over up to 8 bonds have been found: 4 1 J C2,F = 238z J C3,F = 37z 6 N 2 F 3 J C4,F = 7.7z, 3 J C6,F = 14.5z 4 J C5,F = 4.2z C2 C6 C4 C5 C ppm ppm 141 ppm 121 ppm ppm Fig. 7: Proton decoupled 13 C- spectrum of 2-fluoropyridine

29 1 1D vs. 2D NMR spectra (general definitions) A one dimensional NMR spectra has two dimensions: The x axis corresponds to the frequency axis (the chemical shifts in ppm) and the Y axis corresponds to the intensity (see the following figure). Intensity ppm increasing frequencies In contrast, a 2D NMR spectrum contains two frequency axes. Intensities present the third axis and are therefore usually displayed as contour plots (similar to the presentation used in geographical maps). F2 F1 Figure: Two different presentations of a 2D spectrum: Stacked plot (left), contour plot (right) [taken from: Derome, A.E., Modern NMR Techniques for Chemistry Research] Definition: The horizontal axis is defined as F2 (direct dimension) and the vertical axis as F1 (indirect dimension). This definition is valid for Bruker spectrometers, Varian actually uses it the other way around. If both dimensions contain chemical shifts, the experiment is called shift-correlated 2D NMR, if one dimension denotes scalar couplings, the spectra are called J-resolved.

30 2 Diagonal-, cross peaks In a [ 1, 1 ]-CSY-experiment both frequency axes denote proton chemical shifts. Peaks in 2D spectra will connect nuclei, which are correlated in one way (usually either by scalar or dipolar couplings). Cross peaks correlate spins with different frequencies: ν1 ν2 In homonuclear spectra (those, which contain similar nuclei (e.g. both proton frequencies) in the two frequency dimensions), peaks are symmetric with respect to the diagonal of the spectrum. The diagonal peaks correlate identical spins, and are therefore of little analytical use. ν1 = ν2 The diagonal in some way represents the 1D spectrum. Each CSY-spectrum contains duplicated sets of cross peaks due to its symmetry. The peaks (ν1, ν2, cross peak) (ν1, ν1; diagonal peak), (ν2, ν1; cross peak) and (ν2, ν2; diagonal peak) form the corners of a square. All peaks with ν1 = ν1 form the anti-diagonal. ν 2 ν 1 F1 ν 1 ( ν 1, ν 2 ) (ν 1, ν 1 ) ν 2 ( ν 2, ν 2 ) (ν 2, ν 1 ) Diagonal: ν 1 = ν 1 F2 Anti-Diagonal: ν 1 = -ν 1

31 3 The principle of 2D NMR spectroscopy In a standard 1D proton experiment, acquisition of the signal starts (almost) immediately after the excitation radiofrequency pulse. But how are frequencies encoded in a 2D experiment? In principle, all 2D experiments are designed according to the same principle: They consist of a series of 1D experiments, in which a single delay has been altered in length. The building blocks of 2D experiments are: preparation, evolution, mixing and detection. Both evolution and detection are time periods, called t 1 and t 2, during which chemical shift and scalar couplings evolve. Therefore, signal intensities and phase are variables of Int = f (t1, t2) t 1 Fig: Working principle of a 2D CSY experiment [taken from: van de Ven, F.J.M., Multidimensional NMR in Liquids] During the preparation period the system is prepared, the magnetization is usually prepared along a transverse axis (x or y). During the evolution time t 1, magnetization evolves with chemical shift and/or scalar couplings. During the mixing period coherences are transferred from one spin (the one that is frequency encoded in F1) to another spin (the one that is detected during t 2 ). If the two spins are different, such a transfer will give rise to cross peaks, otherwise it will yield a diagonal peak. The different 2D experiments differ by which mechanism (e.g. scalar coupling 1 or dipolar coupling 2 ) magnetization is transferred. The signal is finally detected during the detection (acquisition) period. During recording of a 2D experiment the same NMR experiment is repeated over and over again, simply setting the evolution time to another value from 1D spectrum to 1D spectrum. The increment t1, that is added to the evolution time from experiment to experiment, depends on the spectral width in the indirect dimension. 1 the dipolar coupling, often called dipole-dipole coupling, is mediated via the dipole moment of the spins via space. Its size depends on the orientation of the molecule with respect to the static field. 2 the scalar coupling, often called spin-spin-couplung, is mediated via electrons through bonds. Its size is independent of the orientation of the molecule with respect to the static field. Scalar couplings give rise to multiplet patterns of the signals.

32 4 ow many 1D experiments need to be recorded for the complete 2D spectrum? For a typical 2D [ 1, 1 ]-CSY spectrum usually a series of 512 1D spectra is recorded. The 1D spectra contain resonances at identical frequency, but the amplitudes (intensities) of the signals are modulated (vary) from experiment to experiment. A Fourier transformation along the direct frequency dimension F2 results in a set of 1D spectra containing all chemical shifts and couplings, which are active during the acquisition period t 2. Because the signals physically give rise to a signal in the detection coil this dimension is called the direct dimension. nly so-called single-quantum frequencies can be recorded, because only these will result in a signal in the coil. Int (t 1,t 2 ) FT Int (t 1,ν 2 ) Figure: Schematic representation of a set of free induction decays (FIDs) (left) subject to the first fourier transformation. [taken from: van de Ven, F.J.M., Multidimensional NMR in Liquids] The modulation of the amplitude of the signals in the different 1D spectra is due to evolution of chemical shifts and scalar couplings during the evolution time t 1. A second Fourier transformation is performed in the orthogonal dimension (along t 1 ), and data points correspond to different FIDs. Int (t 1,ν 2 ) FT Int (ν 1, ν 2 ) Since the frequencies are derived from the amplitude modulation of the signals indirectly, the F1 frequency dimension is called the indirect dimension. The second FT therefore yields the full spectrum with two frequency dimensions:

33 5 Figure: FT along t 1 will yield the full 2D spectrum. Cross peaks may be displayed either as cross peaks with a contour plot (c) or a stacked plot (a,b) [taken from: van de Ven, F.J.M., Multidimensional NMR in Liquids] Depending on whether only scalar couplings or scalar couplings and chemical shifts were active during t 1, a J-resolved or a shift-correlated spectrum will result. In a CSY experiment, chemical shifts are active during t 1 and t 2, and coherence transfer takes place via scalar couplings. ow much time and how much disk space are required for recording a 2D experiment? The NMR signal (the FID) is recorded in stroboscopic fashion; single data points, separated in time, are measured. Resolution gets better when more data points are recorded. igh-resolution 1D proton spectra typically contain (32K) data points corresponding to 128 kilobyte disk space. The resolution, assuming a spectral width of z, is then 0.4z per data point. In order to yield the same resolution in both dimensions in the 2D spectrum, 32768*32768 (2 GB) need to be recorded. Even if only a single scan per increment would be used (which is usually not sufficient), the whole experiment would last almost 2 days. In order to reduce disk space requirements and, nowadays more important, in order to save measuring time, 2D data sets are usually recorded with reduced resolution. For a typical [ 1, 1 ]-CSY experiment 512 FIDs with 2048 data points each are recorded. The total disk space requirements are then 2 MB, and the measuring time would last for 18 min.

34 6 Projections Along the edges of 2D contour plots the one-dimensional spectra may be plotted. Either internal projections (generated by projecting all signals onto one axis) or external projection (by plotting the separately recorded 1D spectrum along the axes) may be chosen. The following figure displays a 2D [ 1, 13 C]-SQC-spectrum. In these experiments proton frequencies are recorded in F2 and carbon frequencies in F1 in order to correlate protons with their directly attached carbon nuclei. 13C NMR Spektrum as external projection 1 NMR Spektrum as external projection ppm ppm F1 internal projection internal projection F2 Internal and external projections are both plotted along the spectrum. Since the internal projections are generated from the 2D spectrum, which is recorded with reduced resolution, internal projections have lower resolution. This is obvious from the two signals at 6.6 and 6.8 ppm, for which the small couplings are not resolved in the internal projections. Similarly, the very close signals at 111 and 112 ppm are not fully resolved in the internal projection. Two-dimensional spectra have much lower resolution than their 1-dimensional counterparts. owever, since signals are dispersed in two dimensions, signal overlap in the 2D spectrum is actually much smaller. Therefore, apart from the fact that 2D spectra display correlations between signals, they also allow to better extract the chemical shifts from the better-dispersed signals.

35 2D Experiments 7 The [ 1, 1 ]-CSY-experiment The CSY (correlated spectroscopy) experiment correlates nuclei via their scalar couplings. Chemical shifts are displayed along both frequency dimensions. In contrast to the TCSY experiment, correlations will only appear between protons that possess a resolved coupling to each other. ppm ppm Fig: Expansion of the region displaying correlations between aromatic protons in the 2D [ 1, 1 ]-CSY spectrum of Melatonin Active vs. passive couplings Cross peaks in the CSY spectra display a characteristic fine structure, which reflects the scalar couplings. Active couplings are those that give rise to the cross peaks; if the cross peak is observed at the frequencies (ν a, ν b ) then the J a,b coupling is the active coupling. Active couplings are in anti-phase; the corresponding peak components display opposite phase. Couplings to all other nuclei are called passive couplings and display in-phase splittings: J J ppm anti-phase doublet ppm inphase doublet The cross peak pattern, shown in the figure above, arises only for correlations between nuclei that possess no further scalar couplings. The separation of the multiplet components is given by J A,B.

36 8 J(A,B) ppm F1 J(A,B) ppm F2 Fig.: Cross peak of a 2-spin system in the CSY-spectrum For the following spin system consisting of a linear chain of three protons, in which C is coupled to A and B coupled to A spin-system: C B A the cross peak (ν A, ν B ) would be as illustrated in the following figure: J(A,B) J(A,C) ppm F1 J(A,B) ppm F2 Fig.: Cross peak of the three-spin system in the CSY The active coupling J A,B leads to the anti-phase splitting. Due to the passive coupling J A,C an additional in-phase splitting occurs. The distance separation of the in-phase components therefore allows, in principle, to extract the passive coupling J A,C (however, partial signal cancellation leads to wrong values for small couplings; these are better extracted from ECSY spectra).

37 9 Artefacts in CSY spectra t1-noise is noise strips running parallel to ppm 2 the frequency axes. They mostly originate from instrumental instabilities, with temperature instabilities are being the most serious source. 4 Since the noise is proportional to the signal height, they are most prominent for strong 6 signals, e.g. singlet methyl groups or other sharp lines. T1-noise always degrades spectrum quality but becomes particularly annoying when cross peaks with small ppm Fig.:Example for t1-noise in a CSY-Spectrum intensities should be interpreted. If the chosen relaxation delay is too short, so-called rapid-scanning artefacts are observed. They occur at the double-quantum frequencies (the sum of the frequencies of the coupled nuclei) and lead to a second diagonal, twice as steep. They can (and should!) be easily recognized by the fact that they occur at positions at which no signals are found in the 1D spectrum. Fig.: taken from: Cavanagh, J. et al. Protein Spectroscopy The anti-phase character of CSY cross peaks leads to cancellation of signal intensities for small couplings. It is important to note that the resolution in the two frequency dimensions is

38 usually very different. Therefore, the two symmetry-related peaks may not both be observed, but only one of them may occur: 10 Fig.: [taken from: Cavanagh, J. et al. Protein Spectroscopy]

39 11 The [ 1, 1 ]-TCSY-experiment Similar to the CSY, the TCSY is a homonuclear, shift-correlated 2D NMR experiment, in which coherence transfer takes place via scalar couplings. Cross-peaks contain both passive and active couplings in-phase. In contrast to the CSY, correlation between a spin and all other spins from the same spin system 3 may be observed. For example, correlations from the amide proton will, under favorable conditions, include all side-chain protons from the same amino acid (e.g. for lysine). Another example would be correlations from the anomeric proton in sugars, which could display correlations to all other protons from the same sugar unit. The strength of the experiment lies in the fact that one resolved (non-overlapped) resonance (e.g. the anomeric proton or the amide proton) may be sufficient to determine, which spins are part of the same spin system, even if parts of the spin systems heavily overlap with other spin systems. By proper choice of the length of the mixing time (e.g. 10 ms), coherence transfer can be limited to just vicinal correlations, thus resulting in a CSY-like spectrum, or to correlations between all members of the spin system (e.g. for 80 ms). Because the multiplet components are in-phase, signal cancellation does not occur even if line-widths become larger, and hence a short mixing time TCSY is preferable to a CSY for larger molecules. mixing time 15ms mixing time 100ms ppm ppm ppm ppm Fig.: Expansion of the region displaying aromatic correlations of Melatonin, for different settings of the mixing time. 3 Definition of a spinsystem: Spins, which belong to the same coupling network are part of the same spin system.

40 12 The [ 1, 1 ]-NESY-experiment The NESY is a homonuclear, shift correlated experiment, in which cross peaks result from dipolar interactions between spins. Dipolar couplings result from through-space interactions and only depend on the distance of the spins, but not on the number of intervening bonds. They are observable for nuclei separated by up to 4-5 Å. The efficiency of the NE transfer additionally depends on the motional properties of the molecule, and NEs are generally stronger for larger molecules. ppm 4 ppm ppm ppm Fig.: NESY-spectrum of Melatonin with expansion. Gray peaks: positive signals, black peaks negative phase. The underlying effect of the NESY is the nuclear verhauser effect. The NE describes a phenomenon whereby a non-equilibrium population of α- and β-states relaxes back to its equilibrium value, such that populations of energy levels of other spins (and hence their signal intensities) are changed. The sign of the NE (increase or decrease of signal intensity) depends on the tumbling properties and is positive for small and negative for large molecules. For intermediate-size molecules the NE may actually be small or close to zero: Fig.: Dependency of the proton, proton NE on the molecular reorientation time τ c [taken from: Neuhaus, D., Williamson, M., The NE in Structural and Conformational Analysis]

41 13 Because the sign of the NE depends on the molecular reorientation time τ c of the molecule, peaks in the NESY may be positive (large molecules) or negative (small molecules). The reorientation time is largely influenced by the viscosity of the solvent. Even smaller molecules therefore tend to behave like large molecules when measured in DMS. A dramatic influence on motional properties is also seen by the temperature: As a rule of thumb, changing the temperature by 20 degrees corresponds to the same change in motional properties, as would be observed upon doubling the molecular weight. The following table describes the behavior of molecules of different size in NESY experiments: Phase of the diagonal peaks Phase of the cross peaks Small molecules in low-viscosity solvents positive negative Medium-sized molecules positive Very weak signals (positive or negative) Large molecules, viscous solvents positive positive Artifacts in the NESY EXSY-(exchange)-peaks: They often display large intensities, possess the same phase as the diagonal peaks, and are often also observed for very short mixing times. Typical examples are exchange peaks between amide protons, or sugar hydroxyl protons, and the water signal. ppm ppm Fig.: NESY-spectrum containing exchange peaks. CSY-peaks (anti-phase CSY-type peaks; zero-quantum interference peaks): They are observed between protons that display both dipolar and scalar couplings (e.g. for geminal protons), and for shorter mixing times. Due to the different relaxation properties of protons in larger molecules, they disappear in NESY spectra of proteins. These peaks are manifested by their CSY-type typical anti-phase peak pattern. When overlapped with genuine NESY signals they lead to partial cancellation of NE cross peak intensity resulting in titled peaks. t 1 -noise und rapid scanning artifacts (see remarks for the CSY-experiment).

42 14 The [ 1, 1 ]-RESY-experiment The RESY (rotating frame NE experiment, sometimes also called CAMELSPIN) experiment is like the NESY, a 2D homonuclear shift-correlated experiment, in which coherence transfer is achieved through dipolar couplings for protons separated by less than 5Å. The NE transfer takes place in a rotating frame leading to a different dependence of the sign of the RE on the motional properties of the molecule. Effectively, the RE is always positive (leading to negative cross peaks for positively phased diagonal peaks). The RE buildup is twice as fast as the NE buildup. During the mixing time T 1ρ relaxation takes place, which is similar in size to T 2, and therefore the use of the RSY is limited to smaller or medium-size molecules. ppm ppm Fig.: RESY-spectrum of a peptide; grey peaks = positive signals, black peaks = negative signals Artefacts in the RESY TCSY-Peaks, (in-phase, positive), observed for geminal protons, whose chemical shift difference is small spin-diffusion peaks (RE-RE relay peaks) (in-phase, positive) TCSY-RESY transfer Peaks (in-phase, negative) exchange peaks (positive) It can be seen that almost all artifacts can be readily recognized from the different sign of the peaks.

43 15 The [X, X]-EXSY-experiment The EXSY experiment is a homonuclear, shift correlated experiment, in which coherence transfer takes place through chemical or conformational exchange. In fact, the pulse sequence is the same as the one used for the NESY. Because exchange is usually faster than the NE buildup, shorter mixing times may be used for the EXSY. From recording a series of EXSY spectra with different mixing times, exchange kinetics may be deduced. N N ppm 2 4 ppm ppm ppm Fig.: [ 1, 1 ]-EXSY-spectrum displaying exchange between the two rotamers in the figure on top. Artefacts in EXSY spectra: NESY-Peaks

44 16 The [ 13 C, 13 C]-INADEQUATE-experiment Like the CSY experiment the INADEQUATE provides spectra of the homonuclear, shiftcorrelated type. INEADEQUATE experiments are mainly used for 13 C, 13 C correlation spectra of natural abundance 13 C molecules. The INADEQUATE contains a very efficient filter to suppress 13 C signals of 13 C, 12 C isotopomers. This filter selects for 13 C, 13 C double quantum coherences, which can only be formed by a pair of coupled 13 C nuclei. In F1 the double quantum frequencies are recorded, and hence the cross peaks have the following coordinates: F2: ν(a), F1: ν(a)+ ν(b). Because 13 C, 13 C isotopomers are very rare (0.01*0.01=0.0001) in 13 C natural abundance molecules, extremely concentrated samples are required. The experiment is very powerful, and very useful for highly substituted compounds, in which proton density is low. The following figure displays an expansion of an INADEQUATE experiment recorded on melatonin: C9 C7/C8 C12 C10 C5 C11 C6 Melatonin 3 C N 7 N C C 3 ppm ppm Fig.: Expansion of a 2D INADEQUATE spectrum recorded on melatonin Cross peaks are spilt into doublets by the one-bond C-C coupling. The two coupled resonances can be recognized as two separate peaks at a common frequency in F1 (on a horizontal line). Sometimes one of the two peaks is missing due to low signal-to-noise. The coupled partner can be easily calculated, because its frequency plus the frequency of the coupling partner must add up to the F1 frequency.

45 17 The [ 1, 13 C]-SQC-experiment The SQC experiment is the most popular heteronuclear shift correlation experiment. Nuclei, usually separated by one bond are correlated via their scalar couplings. In [ 13 C, 1 [-SQC spectra, no correlations to quarternary carbons are observed. Since the one-bond proton-carbon or proton-nitrogen couplings are large and rather uniform, these spectra are quite sensitive. ppm ppm Fig.: Expansion of a [ 13 C, 1 [-SQC recorded on Melatonin Artefacts in the SQC Folding in F1 If the spectral width of the indirect dimension is chosen too small (this is sometimes done on purpose) folding (or aliasing) of signals occurs. Whereas folded signals in the direct dimension are usually strongly attenuated by audio filters (and do not occur at all in the oversampling mode), folding in F1 gives signals with full intensity at erroneous positions in the spectrum. Depending on the quadrature detection mode in F1 (real or complex acquisition) signals may be folded about the near or the distant edge: Fig.: Folding in data sets with real (left) or complex (right) acquisition.

46 18 The [ 1, 13 C]-MBC-experiment The MBC experiment like the SQC gives heteronuclear shift-correlation spectra. In contrast to the SQC, coherences are transferred through the much smaller long-range couplings. The long-range couplings span rather a wide-range. The 1, 13 C 3 J coupling, for example, displays a Karplus-type dependence on the dihedral angle. Therefore, some couplings may be close to zero, and such correlations will then of course be absent from the spectrum. Depending on the system under study, the 2 J or the 3 J coupling may be larger, so that these spectra contain much ambiguity. Nevertheless, the MBC is a very useful experiment, because it contains correlations to quarternary carbons. ppm ppm Fig.: Expansion of the [ 13 C, 1 ]- MBC-spectrum of Melatonin Artefacts MBC spectra often contain correlations due to 1 J C, -couplings. Since MBC spectra are not usually decoupled during acquisition, these couplings will show up as rather larger (e.g. 200 z) doublets. The MBC contains a filter for such correlations, which however fails to work when the one-bond couplings differ significantly from standard values (e.g. from 140z, aromatic carbons). ppm 25 1J(C,) coupling ppm Fig.: MBC-spectrum displaying correlations due to the 1 J C, couplings axial peaks (artifacts which can be found on a horizontal line along the center frequency) folded signal similar to the situation encountered for SQC spectra

47 Summary 1 chemical shift of protons, integrals, coupling constants 13 C{ 1 } chemical shift of 13 C; couplings to nmr-active nuclei like 31 P, 19 F, will be observed ( except to 1 ) DEPT135 distinguish between von C; C 2 und C/C 3 DEPT90 distinguish between C and C 3 (only C are observable) 1, 1 -CSY scalar couplings between protons ( 2 J, 3 J) C J C TCSY Determination of protons which belong to the same spin system. C J C J C SQC Which proton is directly bonded to which heteronucleus ( 13 C, 15 N). J J J C C C MBC 2 J-, 3 J- or 4 J- C-couplings C J C C NESY RESY Correlations between protons separated by less than 5Ǻ. No matter how many bonds are in between. C NE/RE C INADEQUATE 1 J- 13 C- 13 C-couplings will be observed. Determination of the 13 C skeleton. Especially useful for highly substituted compounds. C J C SQC-TCSY Additional to the one-bond C-correlation also the neigbouring protons of C x - groups are observed. This can also be N- or -protons. Very useful experiment when the proton shift dispersion is small. C C C

48 Chirality and NMR Enantiomeric molecules in principle do not differ in their physical properties unless they are placed in a chiral environment. In optical spectroscopy, linearlypolarized light is used to determine the rotation angle in order to determine purity of optically active compounds. Protons attached to chiral centers will give rise to separate signals only if the molecule is placed in a non-chiral environment. Protons at prochiral centers (e.g. methylen protons) may be enantiotopic or diastereotopic. Enantiotopic protons can be converted into each other by a drehspiegel operation and will give rise to a single peak; they are isochronous. To reveal whether protons are enantiotopic or diasterotopic one proton may be substituted by a (so far non-existing group) X. If the resulting molecules are diastereoisomers, the protons are called diastereotopic. They may than (but not necessarily) resonate at different frequencies (e.g. methylen protons at -positions of amino acids). Whether or not two separate signals are observed often depends on the spatial separation to the nearest chiral center. In the case a pair of enantiomers is formed the protons are called enantiotopic. Diastereotopic protons can only be found in molecules that contain at least one chiral center. Enantiotopic protons (belonging to the R- and S-form) can only be distinguished in the presence of a chiral environment. Such a chiral environment may for example be a chiral solvent but could also be a chiral molecule that complexes to the molecule of interest: chiral solvents CF 3 N 2 2,2,2-Trifluoro-1-phenylethanol 1-Phenylethylamine Since these solvents are non-deuterated another (external) substance must be added for enabling locking on deuterium.

49 chiral reagents ( chiral shift reagents) Lanthanide shift reagents contain unpaired electrons, which by interaction of the electrons of the metal with the protons of the substance of interest lead to (large) changes in the resonance frequency (paramagnetic contribution to the chemical shift). Pr 3+ : igh-field shift of the signals ( ) Eu 3+ : Low-field shift of the signals ( ) By adding lanthanide shift reagents highly overlapped (crowded) regions of the spectrum can be better dispersed. Commonly used shift reagents and their properties: Fig. 1 taken from:.günther, NMR-Spectroscopy Chiral shift reagents form diastereotopic complexes with the compounds, which differ in their physical properties. Some lanthanide shift reagents contain chiral ligands and the resulting complexes with chiral molecules are diastereotopic. Provided sufficient chiral lanthanide shift reagent has been added the enantiotopic protons are shifted into opposite directions until they are (completely) resolved. But because shift reagents contain paramagnetic material proton-electron dipolar relaxation will lead to (significant) signal broadening. Therefore it is highly

50 recommended to add only little quantities until the signal separation of R- and S- signals is sufficient. A chiral shift reagent that does not induce line-broadening is the Pirkle reagent. It is commercially available in both forms (R(-) and S(+)). CF 3 Pirkle s reagent 1-(9-Antryl)-2,2,2-trifluorethanol Determination of optical purity in compound 2a using Pirkle s reagent Si C 3 (2a) 1. Initially, a 1 NMR-spectrum of racemic (2a) is recorded. (Lower trace spectrum in Fig. 2) The methylen protons attached to Si display a singlet signal ( =0.25ppm). The methoxy group shows up as a singlet ( =3.3ppm). 2. Thereafter another 1 NMR Spectrum is recorded after 20mg of Pirkl s reagent was added. (middle-trace spectrum in Fig. 2 ) The methylgroups at Si are still not completely resolved ( =0.25ppm). The methoxy protons are clearly separated and can be integrated. ( =3.3ppm). 3. After adding another 20mg of Pirkle s reagent another 1 NMR-spectrum is measured. (bottom-trace spectrum in Fig. 2 ) Even now the methyl protons at Si are not fully separated. The methoxy protons are even better separated ( =3.3ppm). In order to determine the optical purity it is sufficient that one set of signals is sufficiently well separated (in our case the methoxy protons).

51 Racemat +40mg Reagens ppm 0.25 ppm Racemat +20mg Reagens ppm 0.25 ppm Racemat ppm 0.25 ppm ppm Fig. 2 : 1 NMR-Spectrum of (2a) Determination of optical purity of alcohols and amines

52 Mosher s reagent Mosher s reagent enables the determination of absolute stereochemistry of secondary alcohols or amines. Preparation: Reaction: All proton chemical shifts should be assigned in the molecule. Take two samples of the molecule of interest and treat them with either R(-)-Mosher s reagent or S(+)-Mosher s reagent. Mosher s reagent: 2-Methoxy-2-(trifluoromethyl)-2-phenylaceticacid chloride Cl Cl F R(-)-MTPA-Cl 3 C C 3 S(+)-MTPA-Cl 3 C CF SFr./500mg SFr./500mg store at -18 C store at -18 C Measurement: Record 1-D proton spectra of both reaction products. The method relies on the large contribution of the ring current from the phenyl moiety of the reagent to the chemical shifts of the methylene protons. The magnitude of the effect is proportional to the distance of the methylene protons to the MTPA-moiety. Never use benzene-d6 or pyridine-d5 as the solvent (for obvious reasons!). Interpretation: Compute the differences in chemical shift for the methylene protons ( = S R ) (in z) between the R- or S-MTPA-esters. For all protons on one side of the stereocenter > 0 and on the other side < 0. The absolute stereochemistry at the chiral center can then be extracted by using the following picture: 1 ' MTPA 1 2 ' <0 C >0 2 3 ' 3

53 Referencing of NMR-Spectra In order to compare NMR spectra recorded at different places spectra need to be referenced correctly. Moreover, the exact conditions under which samples were prepared (p, salt content etc.) and recorded (temperature) should be described and general standards have to be used. Standards may be directly added to the sample or given as an external reference. In the latter, the standard is filled into a small capillary, which is placed inside the tube. Unfortunately, the external reference does not experience identical conditions of susceptibility, p, temperature or p, and therefore internal standards are usually preferable. An ideal standard should not interfere (react!) with the sample. The signal ideally is a singlet, which resonates outside the region (e.g. tetramethylsilane, TMS), in which the signals commonly occur. In addition, temperature and p sensitivity must be small and known. Frequently used chemicals for referencing: TMS Tetrametylsilane 1 : = 0 ppm 13 C: = 0 ppm 3 C C 3 Si C 3 C 3 Cyclosilane-d 18 1 : = ppm D 3 C CD 3 Si D 3 C Si Si CD 3 D3 C CD 3 DSS 2,2-Dimethyl-2-silapentane-sulfonic acid sodium salt 3-Trimethylsilyl-1-propanesulfonic acid sodium salt 1 : = 0 ppm 3 C C 3 Si C 3 Na S TSP 3-(Trimethylsilyl)-propionic acid Sodium salt 1 : = 0 ppm 3 C C 3 Si 13 C: = 1.7 ppm C 3 Na Dioxane 1 : = 3.75 ppm 13 C: = 67.4 ppm

54 Calibration of proton spectra TMS TSP DSS Cyclosilan-d 18 Dioxan =0 ppm =0 ppm =0 ppm =0.327 ppm =3.75 ppm Be careful when referencing measurements in water or methanol: shifts are p and temperature dependend! Measurements in water: TMS is not water insoluble and therefore TSP is mostly used. The resonance frequency of TSP is p dependent. TSP may also interact with hydrophobic parts of the molecule, and the chemical shift will then be the population-weighted average, which of course depends on the concentration ( 0 ppm). Another often-used possibility is to use the water signal for referencing. The water frequency is highly temperature and weakly p dependent (0.02 ppm / p-unit). Provided the exact temperature in the sample is known (which may not be trivial! Some experiments do deliver a considerable amount of heating, e.g. the TCSY) the chemical shift of the water is calculated from the following formula: ( 2 ) = 7.83 T 96.9 [ppm] T = measuring temp. in Kelvin at p = 5.5 Measurements in organic solvents: In most organic solvents TMS is used as an internal standard. It is added in small amounts (!) (5 drops of TMS to 30 ml solvent, one may also use a pipet and suck some TMS from the gas phase and add it to the NMR sample, never directly add the TMS liquid!). Because TMS is highly volatile it is better substituted by Cyclosiland 18, whose boiling point is 208 C, for high-temperature measurements. A less precise method is to use the solvent signal for referencing.

55 Calibration of 13 C-spectra TMS = 0 ppm TSP Dioxan =1.7 ppm = 67.4 ppm p dependent 13 C-spectra are usually referenced to the solvent line resulting in uncertainties as large as 1 ppm! In the case of CDCl 3 the solvent 13 C signal occurs between 77.4 and 76.5 ppm, depending on the concentration and type of the solute. Calibration of 15 N-spectra It is confusing that two major standards are nowadays used for referencing of 15 N spectra. Whereas inorganic or organic molecules are usually referenced with respect to C 3 N 2 or N 4 Cl in bio-nmr applications shifts are presented relative to N 3. The two scales differ by a considerable amount: Scale : N 4 Cl C 3 N 2 N 3 N 4 Cl : [ppm] m N 4 Cl in 2 ; saturated C 3 N 2 : [ppm] m C 3 N 2 in 2 N 4 N 3 : [ppm] N 4 N 3 saturated in 2 (C 3 N 2 ) = (N 4 Cl) 352.9[ppm] = (N 3 ) [ppm] Calibration of 31 P-spectra 31 P-NMR-spectra are referenced against 85% phosphoric acid (added as an external standard in a capillary). 3 P 4 (85% in 2 ) = 0 ppm Calibration of 17 -spectra 2 is added as an external standard for 17 -NMR-spectroscopy: 2 = 0 ppm

56 Be careful: Sometimes C 3 N 2 - or (C 3 ) 2 C is used for referencing! The resulting scales are very different: 17 ( 2 ) = 17 (C 3 N 2 ) [ppm] = 17 ((C 3 ) 2 C) [ppm] Calibration of 19 F-spectra Mostly CFCl 3 is used as an external standard in 19 F NMR spectroscopy: CFCl 3 = 0 ppm Unfortunately 19 F chemical shifts are highly solvent dependent and hence the exact conditions of measurement must be presented! Fig. 19 F NMR spectrum of CFCl 3 in CDCl ppm The fine structure of the 19 F-signal of CFCl 3 is due to the different isotopes of chlorine: 35 Cl and 37 Cl. For referencing F of CF 35 Cl 2 37 Cl is set to 0 ppm. Another, less frequently used standard is C 6 F 6. Calibration of 29 Si-spectra For 29 Si NMR TMS is used as an internal standard: TMS = 0 In case the 29 Si resonance of TMS overlaps with signals from the compound of interest a spectrum is measured without standard, after which one drop of TMS is added and another spectrum is taken.

57 Referencing without standard (indirect calibration) Some nuclei are so insensitive that internal standards yield insufficient signal-tonoise. In these cases the chemical shift scale of the heteronucleus may be computed from the proton scale using the following formula: X o = o X X denotes the tabulated standard values of resonance frequencies of X-nuclei. Therein is 100 Mz, 0 X the frequency of 0 ppm for the X nucleus and 0 the frequency of 0 ppm 1. The ratio of frequencies depends on the nature of the used proton standard: 13 C 15 N (rel. to N 3 ) TMS DSS TSP Fig: Taken from J.Cavanagh et al., Protein NMR Spectroscopy Indirect referencing is more precise than the use of external standards! Closing remarks: The following rules should be obeyed when publishing chemical shifts: - Don t define your own standards or own rules, because comparing your data to those taken by others will be difficult (or impossible). - Always note which signal has been used for referencing. - When using indirect referencing exactly report how this was achieved. - Always add: Temperature, p- (for measurements in water), concentration, referencing mode.

58 1 Terpenes Natural products which are produced biosynthetically from activated isoprene (isopentenylpdiphosphate or dimethylallyldiphosphate, see Fig. 1) are called terpenes. 4 2 PP 4 2 PP isopentenyldiphosphate 5 dimethylallyldiphosphate Two or more of these isoprene units are coupled to each other in a head-to-tail fashion resulting in molecules whose number of carbons can be divided by 5. Upon coupling the double bond is shifted from position 3,4 to 2, PP 4 2 PP C 2 PP 5 5 geranyldiphosphate owever, secondary modifications, occurring during ring-closures for example, may decrease the number of carbon atoms. ther modifications such as methylation may also increase the number of carbon atoms. Terpenes are classified according to: monoterpenes = C 10 skeleton sesquiterpenes = C 15 skeleton (very abundant) diterpene = C 20 skeleton (total number large, but may be rare in certain plants) sesterterpenes = C 25 skeleton (very rare) triterpenes = C 30 skeleton ((very)abundant) steroids = C 27 skeleton (some of them ubiquitous, others rare)

59 2 tetraterpenes= C 40 skeleton The following figures display representative compounds for all these classes. Monoterpenes: C 2 geraniol thymol (-)-menthol (-)-α-phellandrene camphor 1,8-cineol (=eucalyptol)

60 3 Sesquiterpenes: Up to 70 different subclasses of sequiterpenes are known today. Sesquiterpenes are encountered in form of their open-chain and cyclic compounds. They are often derivatized by oxidation involving one or more carbon atoms resulting in the corresponding alcohols, ketons, aldehydes, carbonic acids and lactones. germacrane caryophyllane bisabolane eudesmane cadinane guajane

61 4 Diterpenes: The majority of diterpenes is bi- or tricyclic, but open-chain as well as tetracyclic compounds are also known. Similarly to the sesquiterpenes, oxidation reactions result in various carbonyl derivatives labdan clerodan abietan kauran

62 5 Triterpenes: Triterpenes are structurally rather diverse. Steroids belong to this class of terpenes, and theses have been described in great detail before dammaran-type oleanan-type (most abundant) ursan-type

63 friedelan-type lupan-type Due to the immense structural diversity of terpenes it is difficult to describe a generally valid route for their unambiguous identification. Some hints, nevertheless, are given here: 1.) Terpenes are usually found in the lipophilic extract (dichloromethane, ethyl acetate). This is not true for glycosides or other highly functionalized molecules. 2.) The carbon and proton spectra are dominated by many signals from C, C 2 and C 3 groups, which occasionally leads to spectra highly crowded between 0.9 and 2.0 ppm. 3.) Signals due to methyl groups are dispersed over the range from (~0.7 to 2.0 ppm) Successful identification of terpenes requires the use of 2D NMR experiments. Due to the small signal dispersion, carbon editing (SQC, MBC) is very useful. For the same reason an SQC-TCSY is preferable to the CSY experiment.

64 NMR of carbohydrates 1 Saccharides General remarks: This introduction is very brief. For an excellent and more detailed description of the topic see for example: T. Lindhorst: Essentials of carbohydrate chemistry and biochemistry, 2 nd edition, Wiley Carbohydrates are polyhydroxycarbonyl compounds with the general formula: C n ( 2 ) n. According to whether only a single, a few or many monomeric units are linked, the resulting molecules are called mono-, oligo- or polysaccharides. Most naturally occurring sugars are optically active. Monosaccharides are further classified according to the number of carbon atoms into trioses (3C), tetroses (4), pentoses (5C) and hexoses (6C). Naturally occurring monosccharides are usually pentoses [C( 2 )] 5 or hexoses [C( 2 )] 6. Monosaccharides preferably exist as cyclic hemiactelas or hemiketals. The most-simple precursors are 2,3-dihydroxypropanal (glycerinaldehyde) and 1,3-dihydroxypropanon. Sugars derived from the aldehyde are referred to as aldoses and those derived from the ketone as ketoses. Forming the intramolecular hemiacetals or hemiketals usually yields five-membered (furanoses) or sixmembered rings (pyranoses). C 2 D-(-)-Glucose C 2 neues Ciralitätszentrum C 2 neues Ciralitätszentrum C D-(+)-Glucofuranose weniger stabil D-(+)-Glucopyranose stabiler Examples for pentoses: Arabinose, Xylose, Ribose Examples for hexoses: Glucose, Mannose, Fructose, Sorbose

65 NMR of carbohydrates 2 Aldoses: C C C C C 2 C C C C C 2 C C C C C 2 C C C C C C 2 C-1, neues Chiralitätszentrum, anomeres C-Atom C 2 Arabinose Xylose Ribose Glucose!-D-Glucopyranose Ketoses: C 2 C C C C 2 C 2 C C C C C 2 C-1, anomeres C-Atom C 2 Xylulose Fructose!-D-Fructopyranose A new stereocenter is created while the hemiacetal / hemiketal ring closure occurs, leading to the formation of two possible diastereoisomers. The molecule with S-configuration at C-1 is called the α -form, the one with R-configuration is called the β-form. It is actually easier to remember the definition when drawing the molecule in the chair ( 4 C 1 )-conformation (C4- corner pointing up, C-1 corner pointing down): 4 C C C C 2 1!-D-Glucose ( 1 C 4 )!-D-Glucose ( 4 C 1 ) "-D-Glucose ( 4 C 1 ) In the α -form the hydroxl-group is placed axial and in the β-form it is placed equatorial. The two diasteromers derived from different configuration at C-1 are also called anomers, and carbon-1 is referred to as the anomeric carbon. Conformations of pyranoses and furanoses The tetrahydropyran six-membered ring represents a cyclohexane-type skeleton, which usually has a chair-like conformation. The lowest-energy conformation corresponds to the one in which the most bulky substituents are positioned equatorially. The situation is much less clear for furanoses. Furanose stereochemistry is of great importance in nucleic acid chemistry and biology. Five-membered rings can adopt conformations in which all

66 NMR of carbohydrates 3 atoms are in one plane, but unfavorable 1,2 ecliptic interactions are usually avoided by folding up one corner to adopt an envelope-like conformation (ring pucker). In contrast to the situation encountered in six-membered rings, the so-called twist conformation is also reasonably low in energy. It is therefore very difficult to predict conformations of five-membered rings. Any assumptions about which NEs should be and which should not be observable have therefore to be made with much care. Structural data derived from crystallography may help to predict conformations. If no such information is available, optimization of energy by molecular mechanics programs may help, but these methods tend not to be sufficiently precise. chair boat twisted boat Tetrahydropyran E 2 Me 1 2 C Tetrahydrofuran T 2 2 C C(C 3 ) 2 E-2 denotes that carbon 2 is placed at the corner, which is bent out of the plane formed by the remaining four atoms. Similarly, 3 T 2 indicates that in the twisted form C-3 is pointing up and C-2 is pointing down with respect to the plane of the remaining three atoms. Which atom is displaced from the plane is difficult to predict and depends on the nature of all substituents. Therefore great care is required when predicting the ring pucker. Mutarotation: Stereochemical analysis is complicated by the fact that in solution α- and β-pyranoses interconvert via the open-chain form (base- and acid-catalysis).

67 NMR of carbohydrates 4 C C C 2 C C 2 C C C 2!-D-(+)-Glucopyranose Aldehydform "-D-(+)-Glucopyranose 36.4% 0.003% 63.6% Which of the two anomeric forms predominates in equilibrium depends on the solvent. In apolar solvent the α -form and in protic solvents the β-form is favored.

68 NMR of carbohydrates 5 ligo- und Polysaccharides ligosaccharides consist of 2 to 6 monosaccharide units, whereas polysaccharides contain more than 6 units. Upon forming the glycosidic bond two monomeric units are linked by forming the mixed acetal. Therein, the glycosidic bond is chemically more inert and mutarotation is no longer possible. examples: Disaccharides (C ) : Saccharose, Maltose, Lactose,... C 2 C 2 C 2 C 2!-D-Glucopyranose "-D-Frucofuranose Saccharose C 2 D-Glucose D-Glucose Maltose ("-Form) C 2 C 2 D-Galactose D-Glucose Lactose (!-Form) Trisaccharides (C ) : Raffinose C 2 C 2 C 2 C 2 Raffinose D-Galactose D-Glucose D-Fructose

69 NMR of carbohydrates 6 Polysaccharides: e.g. Cellulose C 2 C 2 C 2 C 2 Amylopectin:. C 2 Verzweigungspunkt. C 2 C 2

70 NMR of carbohydrates 7 Amylose: C 2. C-1,! C-4 C 2 Maltoseeinheit C 2 Aminodeoxycarbohydrates ften, hydroxyl groups are replaced by amino groups. If the nitrogen is bound to the anomeric carbon they are called glycosamines, otherwise they are called aminodeoxysugars. C 2 C 2 2 N N 2!-D-Glucosamin oder 2-Amino-2-desoxy-D-glucopyranose!-D-Glucopyranosamin Deoxycarbohydrates In deoxycarbohydrates one (or more) hydroxyl groups are replaced by hydrogens. A famous example is the deoxy-d-ribofuranose, which forms an important part of DNA. C 2 C 3 2-Desoxy-D-ribofuranose!-D-Rhamnose

71 NMR of carbohydrates 8 NMR-spectroscopy of carbohydrates The most useful 2D NMR experiments are listed here: DQF-CSY: elps to establish neighboring connectivities in the sugar rings via 1, 1 scalar couplings. From the appearance of cross peaks, the magnitude of the scalar coupling and thereby the relative stereochemistry can be deduced. TCSY: SQC: MBC: Determination of the monosaccharide spin-systems. The experiment is especially useful for oligosaccharides, because it allows assignment of protons to the different monosaccharide units. Signal dispersion is usually best in the the anomeric proton region. Delivers 13 C information about the attached carbons. Is very useful to identify anomeric protons/carbons, and to distinguish C from C 2 moieties. ne of the two experiments for establishing the linkage between monosaccharide units via the glycosidic bond. Sometimes, the corresponding dihedral angle is close to 90, and therefore the corresponding cross peaks may be missing. C 2 C * C-4* C 2 SQC-TCSY: NESY/RESY: This experiment is extremely useful, because chemical shifts of sugar ring protons are very similar, and cross peaks required for establishing neighboring connectivities may be close to the diagonal in the CSY. The better dispersion in the carbon spectrum is a great help in alleviating these problems. An SQC is also required to distinguish one-bond from two- and three-bond correlations. Unfortunately, larger amounts of substance are required. Experiments to establish stereochemistry and the location of the glycosidic linkage. In our experience the RESY/NESY are much less useful for establishing relative stereochemistry when compared to other classes of compounds, especially in the case of pyranoses. Part of the problem is that the ring conformation may not be known exactly. Never ever establish stereochemistry based upon observation of NE between protons attached to neighboring carbons, only use 1,3 (diaxial) NEs! C 2 C 2 NE

72 NMR of carbohydrates 9 Useful NEs to identify carbohydrate units: nce again: 1.2 NEs will usually always be observable, because these distances tend to be smaller than 4 Å in any case. It may also be that the ring conformation significantly deviates from the standard chair/envelope forms, so that the distances may not be so different between the cisand trans-proton.

73 NMR of carbohydrates 10 Typical ranges for 1 - and 13 C-chemical shifts of standard carbohydrates: 1 : anomeric protons ppm ring protons ppm 13 C : anomeric carbons ppm hexoses: δ C1 > 90ppm aldose (pyranose) δ C2 > 90ppm ketose (furanose) ring carbons ppm aldose: a single C 2 in the range ppm ketose: two C 2 in the range ppm

74 NMR of carbohydrates 11 Shift differences (monosaccharide - oligosaccharide): 1 : anomeric protons: - (0.2 to 0.6) ppm vicinal to the linkage site: - (0.03 to 0.31) ppm 13 C : anomeric carbons: + (4 to 10) ppm adjacent positions: - approx. 1 ppm

75 NMR of carbohydrates 12 Typical values for 3 J( 1, 1 )- und 1 J-( 13 C, 1 )- scalar coupling constants in carbohydrates: 3 J, : ax, ax : 7-9 z for -C-C- approx. 180 eq, ax : 2-4 z for -C-C- approx. 60 eq, eq : 2-4 z for -C-C- approx J C, : z for anomeric carbons 1 J C, : 145 z for all other carbons Identification of hydroxyl protons in 1D spectra: /D-exchange: temperature: RESY/NESY: If the sample is dissolved in D 2 or d 4 -methanol the signals due to hydroxyl protons will disappear. Although some stereochemical information seems to be lost by this at first glance, complementary information can be obtained from the carbon-bound protons. Spectra are significantly less complicated in fullydeuterated protic solvents (due to the absence of hydroxl protons), and cross peak patterns in the CSY are also much simplified, which helps a lot in the stereochemical analysis. When recording spectra in DMS, addition of one drop of deuterated water causes the hydroxyl resonances to disappear. ydroxyl protons may also be identified by comparing spectra recorded with and without water suppression, because saturation transfer will lead to signal attenuation in the presaturation experiment. Increasing the temperature leads to broadening of the hydroxyl resonances (due to accelerated exchange). Usually, the carbon-bound protons sharpen up when the temperature is increased. bservation of exchange peaks between hydroxyl protons and the water signal for spectra recorded in 90% 2 /D 2. Such water exchange-mediated peaks may also be found between hydroxyl protons in the TCSY spectra. Identification of stereochemistry at the anomeric center: α -form β-form proton chemical shift (-1) ppm ppm carbon chemical shift (C-1) ppm ppm scalar coupling constants ( 3 J (-1,-2) ) 1-4 z 6-8 z 1 J C-1,-1 coupling constants 170 z 160 z

76 NMR of carbohydrates C-atoms bearing hydroxyl groups can be identified from /D-isotope shifts in 13 C-spectra In order to measure the isotope shift, a spectrum of the sugar in a non-protic solvent (e.g. DMS) is recorded. After addition of a drop of D 2 the spectrum is re-measured and the isotope shift calculated according to: Δ (ppm) = δ 13 C(DMS) - δ 13 C(DMS+D 2 ) In general, large isotope shifts are observed for those carbons which have hydroxyl groups directly attached. This method requires the exchange with the water protons to be slow, because the lifetime of the deuterium is very short otherwise (and hence the magnitude of the isotope shift is small)!

77 NMR of carbohydrates 14 Strategy for structure determination of carbohydrates Determine the number of monosaccharide units 1. Count the number of signals in the 13 C NMR spectrum. Determine the number of C 2 - groups in the range between ppm. 2. Determine the number of anomeric protons and carbons ( ppm) using 1 -, 13 C-, DEPT90- und SQC-spectra. 3. Correlate protons signals to their directly attached carbon resonances by using the SQC spectrum. 4. Determine the number and type of spin systems from the TCSY. In particular, traces along the positions of the anomeric protons (in which the proton spectra are best resolved) are very helpful. For larger carbohydrates the SQC-TCSY is an enormously helpful experiment to resolve possible overlap by exploiting dispersion of the carbon frequencies. Determine the relative configuration Assign hydroxyl groups to axial or equatorial positions. This may be done by using information from the scalar couplings, which may be read out of the CSY spectrum. Alternatively, 1,3 NEs (diaxial correlations) may be used. Determine the glycosidic linkage sites This can be done from NEs/REs or from correlations in the long-range proton carbon correlation experiment (MBC). Tip: It has already been mentioned that recording spectra in fully deuterated, protic solvents leads to rapid exchange of all hydroxyl protons by deuterium. This leads to a remarkable simplification of the spectra. Since stereochemical information derived from hydroxyl protons and the corresponding carbon-bound protons is complementary, the loss of information content is small and is easily compensated by the fact that the spectra are much easier to interpret. In addition, cross peaks pattern in the DQF-CSY are simplified, so that stereochemistry can be read off much more easily. Determining relative stereochemistry of the hydroxyl groups from CSY cross peaks: CSY cross peaks display both active (anti-phase) and passive (in-phase) couplings. 1,2 diaxial protons will display large couplings, whereas axial-equatorial or equatorial-equatorial couplings are small. ow this can be exploited in monosaccharide units is shown in the following figure: Quite often it is sufficient to recognize whether the couplings are large or small. An instructive example is given for the cross peaks C3(F2)-C2(F1) of β-mannose. In F2, the in-phase coupling is large, whereas the anti-phase coupling is small. Thereby it is clear that protons at C-3 and C4 must both be axial (because the passive coupling is large). The proton at C2 must be equatorial, because the active coupling is small. If both the passive and the active couplings are large and similar, certain multiplet components cancel each other (e.g. as seen in β-glucose). The advantage of reading out scalar couplings from the CSY is that this method can still be used when signal overlap in the 1D spectrum prevents extraction of the coupling constants.

78 NMR of carbohydrates 15

79 Peptide/Protein NMR Peptides Peptides are macromolecules which are built up of amino acids, connected through the formation of a peptide bond. In nature all peptides exists in the L-configuration (at least in eukaryotes). R Cα C N The amide bond is almost exclusively found in the trans configuration, although for Xxx-Pro the bond may exist in the cis configuration to a considerable extent. Therefore, short, Pro-containing peptides often display a (minor) second set of signals due to the cis conformer. Shorter, non-cyclic peptides are rarely structured and hence are described as random coils. Longer peptides are mostly (but not always!) structured. Their secondary structure is classified according to the dihedral angles, which are defined as follows: ω ψ φ χ α α i = 0 for Ci Ci cis to Ni+ 1 Ci+ 1 α i = 0 for Ci Ni trans to Ci i α i = 0 for Ci Ci trans to Ni i 1 α β γ i = 0 for Ci Ni cis to Ci i 1

80 Peptide/Protein NMR Secondary structures: The three major classes of secondary structure are as follows: the α-helix Left: Schematic Drawing of the α-helix. Middle: Backbone presentation with direction of dipole moment. Right: Structure of the 434 repressor. the parallel β-sheet the antiparallel β-sheet 2