Where is my INEPT signal? - Proton Exchange Issues

INEPT and DEPT sequences are routinely used to enhance the NMR signals for low γ nuclides such as ¹⁵N or ¹³C.  The enhancement relies on polarization transfer between the protons J-coupled and the low γ nuclide.  The pulse sequences incorporate delays based on the reciprocal of the J-coupling constant between the protons and the low γ nuclide.  In the case of ¹⁵N INEPT, the enhancement for each scan can be as much as γ_H/γ_N (~ 10) compared to that from a conventional one-pulse sequence with inverse gated decoupling.  Furthermore, the recycle delay for the INEPT sequence depends on the the ¹H T₁ relaxation time rather than that of ¹⁵N.  ¹H T₁'s are typically an order of magnitude (or more) less than those of ¹⁵N so the recycle delays required for ¹⁵N INEPT spectra are at least ten (and possibly 100 times) shorter than those required for one-pulse data collection.  These two factors mean that the true time saving for a ¹⁵N INEPT measurement compared to a one-pulse ¹⁵N measurement can be on the order of 100 - 1000 times.  There are, however cases where ¹⁵N INEPT signals are attenuated or entirely nonexistent.  Attenuated ¹⁵N INEPT signals are observed when the protons (with short T₁) coupled to ¹⁵N exchange with those of water (longer T₁) on a time scale of seconds.*  The problem arises because of saturation transfer during the inverse gated decoupling used during the acquisition time. The partially saturated protons are unable to transfer as much polarization to the ¹⁵N as they would were they fully polarized. The problem can be reduced if a recycle delay much greater than the T₁ relaxation time of the water protons is employed.  If the protons bound to ¹⁵N undergo exchange with other labile protons at a rate fast with respect to the ¹H-¹⁵N J coupling interaction, polarization transfer from ¹H to ¹⁵N is not possible and a ¹⁵N INEPT signal cannot be observed.  This is demonstrated in the figure below.

Concentrated solutions of the methyl ester of anthranilic acid and anthranilic acid were prepared in DMSO-d₆.  The ¹⁵N NMR data were collected on a 600 MHz instrument with a cryoprobe.  The left-hand panel of the figure compares the ¹⁵N one-pulse spectrum with inverse gated decoupling (bottom) to the INEPT spectrum (top) for the methyl ester.  The spectra were collected with the same number of scans. For the methyl ester, the ¹⁵N bound protons do not exchange with any other labile protons.  The enhancement in the ¹⁵N INEPT spectrum is clear.  Similar spectra for anthranilic acid are shown on the right-hand side of the figure.  In anthranilic acid, the ¹⁵N bound -NH₂ protons undergo intramolecular exchange with the acid proton at a rate fast with respect to the one-bond ¹⁵N-¹H coupling constant (~90 Hz).  As a result, polarization transfer is not possible and no INEPT signal is observed.  The same is true for the meta- and para- isomers (data not shown). 

Thank you to Jin Hong for sharing her experience with collecting ¹⁵N INEPT data for anthranilic acid and Mojmir Suchy for kindly providing the samples.

* G.D. Henry and B.D. Sykes, J. Magn. Reson. B, 102, 193 (1993).

Exchange Effects in HSQC Spectra

The effects of chemical or dynamic exchange on NMR spectra are very well known.  Exchange is often studied by observing line shape changes as a function of temperature, by 2d EXSY, inversion transfer or saturation transfer methods.  Effects due to exchange can also be observed in ¹H - ¹³C HSQC spectra.  The HSQC method works by transferring ¹H magnetization to ¹³C magnetization via an INEPT transfer through the one-bond J coupling across the ¹H - ¹³C chemical bond.  The ¹³C magnetization evolves during the incremented delay, t1, of the 2D pulse sequence according to its chemical shift.  The ¹³C magnetization is then transferred back to ¹H magnetization where is observed during t2.  HSQC spectra thus exhibit cross peaks between ¹H resonances and the resonances of their attached carbons.  If there is exchange between nonequivalent carbon sites during t1, some ¹H resonances may appear to be correlated to two carbon resonances.  An example of this is shown in the figure below.

The ¹³C spectrum of cannabidiol has equally intense broad, resolved aromatic resonances for non-protonated carbons 2 and 6 (not shown) as well as for the protonated carbons 3 and 5.  The ¹H spectrum has broad resolved resonances for both aromatic protons.  This indicates that either the aromatic ring undergoes 180° flips about the 1 - 4 axis or it has two equally probable rotomers defined by a rotation about the 1 - 4 axis.  In either case, the dynamic exchange is slow enough on the NMR time scale to produce resolved resonances yet fast enough to cause significant line broadening.  For each of the two aromatic protons, the HSQC spectrum shows correlations to both C3 and C5; a strong correlation to the carbon to which it is chemically bonded and a weaker correlation to the carbon site in exchange with its attached carbon.          

Saturation Transfer and Exchange

Exchange processes that occur on the NMR time scale affect the NMR line shapes and can be studied by line shape analysis. If the exchange process is slow on the NMR time scale, one can employ EXSY or inversion transfer methods to study the exchange. An alternative to these is the saturation transfer technique. In this method, one of the slowly exchanging resonances (A) is saturated with low power CW irradiation and the effect on the intensity of the resonance of the exchange partner (B) is monitored. If there is exchange between A and B during the period of saturation some of the saturation from A will be transferred to B. The change in the intensity of B will depend on both the rate of exchange, k and the relaxation time of B, T_1B . If there is no nuclear Overhauser effects between A and B, then the rate of exchange is given by: where I_o is the intensity of B with no saturation of A, and I_∞ , is the intensity of B when A is saturated for an infinite time. The saturation transfer effect is useful for situations where the exchange is slow on the NMR time scale but faster than (or of the same order as) T_1B. An example using ³¹P NMR is illustrated in the figure below for a ruthenium phosphine complex which undergoes slow exchange between isomers with different modes of bonding. The ³¹P [¹H] NMR spectrum is shown in the upper right-hand panel of the figure. In this case, the P atoms of isomer A are chemical shift equivalent and give a singlet while those for isomer B are chemical shift nonequivalent and give an AB pattern. The spectrum of isomer B is shown in the lower panel of the figure as a function of saturation time of isomer A.



Many thanks to Carolyn Higman and Prof. Deryn Fogg for kindly allowing their data to be used in this post.

The NMR Time Scale

Many people use statements like, "It is fast on the NMR time scale" without really having an understanding of what the "NMR time scale" means. Assume we have a compound with two inter-convertible conformations, A and B, each of which gives a different NMR spectrum. If the interconversion between A and B is very slow then we expect to get a spectrum with both conformations A and B resolved. If the interconversion is very fast then we would get an unresolved spectrum which represents the average conformation of A and B. The interconversion between A and B is fast on the NMR time scale if it occurs at a rate much greater than the difference in frequency between A and B. Since the difference in frequency between A and B depends on the Larmor frequency of the nucleus being observed and the strength of the magnetic field, the "NMR time scale" depends on the particular experiment and the field strength. Therefore, when one uses the expression, "NMR time scale" one must qualify it with details of the measurement. An example of different NMR time scales is shown in the figure below. Here the ¹H (300 MHz) and ¹³C (75 MHz) NMR spectra of the N-methyl groups of N,N-dimethylacetamide are shown at various temperatures. The spectra were plotted on the same scale in Hz. In this molecule there is a hindered rotation about the central C-N bond. One can see that the rotation is fast on the ¹H NMR time scale before it is fast on the ¹³C NMR time scale because the difference in frequency between the methyl groups is smaller in the ¹H spectrum compared to the ¹³C spectrum.

What are Those Positive Peaks in My NOESY Spectrum?

Students will sometimes ask me, "What are those positive off-diagonal peaks in my NOESY spectrum?". Since the NOESY pulse sequence is exactly the same as the EXSY sequence, a NOESY spectrum will show all possible correlations due to cross relaxation. These correlations include those from NOE's, chemical exchange and conformational (or rotational) exchange. For small molecules, if one phases the diagonal responses such that they are positive, the NOE's correlations will be negative and the exchange correlations will be positive. The answer to the question is therefore that the positive peaks are due to exchange. The figure below shows an example where three different types of cross peaks are visible. The molecule in the figure has two distinct rotational conformations in slow exchange with one another and gives a proton spectrum with every resonance doubled. Each resonance for one rotational conformation gives a positive (black) cross peak correlating it to the corresponding resonance of the other conformation. There is also a positive cross peak correlating the -NH- proton in the molecule to the residual water in the DMSO-d6 solvent. These protons exchange chemically with one another. Finally, the negative (red) correlations in the figure are due to NOE's.
Thank you to Jean-Gregoire Roveda of Dr. Beauchemin's group for giving me permission to use his spectrum as an example.

2D EXSY

The 2D EXchange SpectroscopY (EXSY) technique is exactly the same as the same as the 2D NOESY technique. The pulse sequences are identical. The method provides off-diagonal responses for spins which exchange slowly with one another (either conformationally or chemically) and also between spins with NOE's. The EXSY method is useful for showing exchange when the rate of the exchange is greater than or of the same order as the T₁ relaxation rate (1/T₁) but less than the frequency difference between the two spins (in the absence of exchange). Depending on the experimental conditions, the responses due to exchange are often much more intense than those due to NOE's. The figure below shows the 300 MHz 2D ¹H EXSY spectrum of N,N-dimethylacetamide at room temperature. At this temperature, the molecule exhibits slow rotation about the (CH₃)₂N - C bond such that both methyl groups exchange with one another rotationally yet are distinct in the spectrum. This is evident by the cross peaks in the spectrum between the two methyl groups on the nitrogen. Note that the off diagonal peaks are of the same phase as the diagonal peaks.