Published: November 12th, 2016
A detailed protocol describing the SSTD NMR method is presented here to help new users apply this new method to obtain the kinetic parameters of their own systems undergoing chemical exchange.
This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our group to study processes of mutual-site chemical exchange that are difficult to analyze by traditional methods. As the name suggests, this method combines the Spin Saturation Transfer method used for small molecules, with the Saturation Transfer Difference (STD) NMR method employed for the study of protein-ligand interactions, by measuring transient spin saturation transfer along increasing saturation times (build-up curves) in small organic and organometallic molecules undergoing chemical exchange.
Advantages of this method over existing ones are: there is no need to reach coalescence of the exchanging signals; the method can be applied as long as one signal of the exchanging sites is isolated; there is no need to measure T1 or reach steady state saturation; rate constant values are measured directly, and T1 values are obtained in the same experiment, using only one set of experiments.
To test the method, we have studied the dynamics of the hindered rotation of N,N-dimethylamides, for which much data is available for comparison. The thermodynamic parameters obtained using SSTD are very similar to the reported ones (spin-saturation transfer techniques and line-shape analysis). The method can be applied to more challenging substrates that cannot be studied by previous methods.
We envisage that the simple experimental set up and the wide applicability of the method to a great variety of substrates will make this a common technique amongst organic and organometallic chemists without extensive expertise in NMR.
Chemical exchange commonly refers to any intermolecular or intramolecular process in which a nucleus moves from one environment to another in which its NMR parameters (chemical shift, scalar coupling, dipolar coupling, relaxation rate) differ. There are numerous examples of chemical exchange in organic and organometallic molecules (e.g., rotational barriers in biaryls, ring flipping barriers and conformational equilibrium, nitrogen inversion, ligand binding, degenerate ligand exchange and tautomerization).1-3 The chemical exchange rate is related to the thermodynamics of the barrier of the exchange process, and therefore its study is of crucial importance to understand molecular dynamics of these systems.
The classic sign of dynamic exchange in NMR is a dramatic change in the line-shape of the NMR signals as the temperature changes. At low temperatures, the process is slow and two distinct chemical shifts are observed. At high temperatures, the two signals merge into one signal, which is known as "coalescence". At intermediate temperatures, the signals become very broad. This sensitivity of the NMR spectrum to chemical exchange makes NMR a very powerful method to study the dynamics of molecules in solution. Two methods have been mainly employed in the study of dynamic processes in solution: line-shape analysis,4-7 and spin saturation transfer experiments.8-9 Besides, it is also worth mentioning the inversion transfer method10 and the CIFIT program11 for the direct extraction of rate constants, that are a relatively efficient approach for exchange measurements in simple systems. Although these methods give very good results in most cases, they, however, have a number of drawbacks. The main disadvantage of the line-shape analysis is the high temperatures needed to reach coalescence in some samples.12 The main issues to consider when carrying out spin saturation transfer experiments are: the very long saturation times required to reach the steady state saturation transfer between the exchanging sites, and the need to determine the longitudinal relaxation time constant, T1, which can be difficult if there is overlap of different signals in the region of study.13
As part of our investigations in organometallic mechanisms,14-16 our group is studying the fluxional behavior of platinum-allene complexes in solution. This is a complex task that involves at least three different processes, one of them being the π-face exchange or rotation of the metal around one of the allene axis. We encountered that normal VT experiments and line-shape analysis techniques that have been employed before in similar systems,17-19 were not suitable in our study, due to a very slow rotation in our platinum-allene complex that made the coalescence temperature of the signals of interest higher than the temperature of decomposition of the complex.
In order to overcome this limitation, we developed and recently reported a new NMR protocol (SSTD NMR) to study processes of mutual-site chemical exchange.20 As the name suggests this method combines the Spin Saturation Transfer method used for small molecules, with the Saturation Transfer Difference NMR method employed for the study of protein-ligand interactions,21-24 by measuring transient spin saturation transfer along increasing saturation times (build-up curves) in small molecules undergoing chemical exchange.
With this new method (SSTD NMR) we have shown that we can obtain the kinetic parameters of intramolecular chemical exchange in small organic and organometallic molecules with some additional advantages over traditional approaches: coalescence of the signals is not needed, so a more flexible temperature range can be used in the study; signal overlap does not interfere, although at least one of the exchanging resonances should be isolated; there is no need to measure T1 or reach steady state saturation; rate constant values are measured directly and T1 values are obtained in the same experiment, using only one set of experiments. Another remarkable advantage of the SSTD NMR methodology is that, in contrast to lineshape analysis, the determination of the kinetics rate constants is not limited by the increase in coalescence temperatures associated with high magnetic fields. Thus, our methodology is then very well appropriated for both low and high magnetic fields. This article is intended to help new users apply this new method to their own systems undergoing chemical exchange, and describes sample preparation, experimental set up, data acquisition, and an example of data processing and analysis in a simple organic molecule.
Caution: Please consult all relevant material safety data sheets (MSDS) before use.
1. NMR Sample Preparation
2. NMR Experimental Setup25
3. NMR Data Acquisition and Processing25
4. Data Analysis30
The SSTD NMR technique was applied for the calculation of the kinetic parameters in the rotation of the amide bond of N,N-dimethylacetamide.21 This is a simple example for which extensive data for comparison can be found in the literature.31
The hindered rotation around the amide bond, due to the partial double bond character in the resonance form, differentiates both methyl groups into two signals in the 1H-NMR spectra (2.61 and 2.17 ppm at 22.5 °C). Spin saturation of the signal of the methyl group at 2.17 ppm (MeB) leads to the disappearance of its signal in the 1H NMR. Upon saturation of MeB, transfer of saturation to the other methyl group (MeA) due to the internal rotation process can be observed by a decrease in 1H intensity in the signal at 2.61 ppm. The magnitude of this decrease will depend on the saturation time. Figure 15 shows the 1H NMR spectra of the N,N-dimethylacetamide at 22.5 °C, and the expansions show the spectra without (a) and with (b) saturation of the methyl group at 2.17 ppm, as well as the difference spectrum (c), used to calculate the values of ηSSTD. The ηSSTD factor is calculated dividing the value of the integral of MeA in the SSTD NMR spectrum (c) by the value of the integral of the MeA in spectra (a), as explained in the protocol. The obtained values of ηSSTD for each saturation time at different temperatures are gathered in Table 1. The plot of the obtained values of ηSSTD versus the saturation time gave exponential curves in which a plateau was reached at higher saturation times. For a certain temperature, the exponential fit of the curve permits the calculation of the rate constant (k) and the relaxation time of the 1H of the measured signal (T1A) (Figure 16). Figure 17 shows all the obtained curves along with the k and T1A values obtained in the fits.
Finally, the plot of ln(k/T) versus 1/T and the fit to the Eyring equation (Figure 18) were used to calculate the enthalpy and entropy of activation. The determined activation parameters are shown in Table 2, together with the previously reported parameters calculated using different methodologies.
As can be observed in Table 2, the values of the activation parameters obtained with the Spin Saturation Transfer Difference technique (SSTD NMR) are in excellent agreement with the data previously reported using other techniques, such as SST NMR or line shape analysis. The wide range of values reported for ΔS‡ is due to the difficulty in the measurement of this parameter with NMR techniques.31 As for the rest of the activation parameters, the values obtained with our method are not only really similar to the ones already reported but also more accurate, since our errors (SD) are smaller in all the cases.
Figure 1: List of experiments after typing rpar. The figure shows the different parameter sets among which STDDIFFESGP should be selected. Please click here to view a larger version of this figure.
Figure 2: Acquisition parameters. The button highlighted in a red square leads to a list of the different pulse programs. Please click here to view a larger version of this figure.
Figure 3: List of pulse programs. The figure shows the selected pulse program in the experiment (STDDIFF). Please click here to view a larger version of this figure.
Figure 4: Pop-up window appeared after the 90° pulse calibration. The figure shows the values of the calibrated 90° pulse at different power levels. Please click here to view a larger version of this figure.
Figure 5: Screenshot of the command line. The figure shows how to introduce the value for the calibrated hard pulse. Please click here to view a larger version of this figure.
Figure 6: Value for the length of the shaped pulse. The figure shows how to introduce the value for the length of the shaped pulse. Please click here to view a larger version of this figure.
Figure 7: Acquisition parameters. The figure shows the power parameters. Please click here to view a larger version of this figure.
Figure 8: Parameters for the shaped pulse. The values for the shaped pulse will be introduced in line 13. Please click here to view a larger version of this figure.
Figure 9: List of irradiation frequencies. The figure includes the following data in the 3 first rows: Row 1. P (indicates that the following data is in ppm); Row 2. Frequency of the signal to be irradiated in ppm, as measured in 3.1.1; Row 3.40 ppm (a frequency that is far from the 1H signals of the compound so the irradiation in that frequency does not affect the spectra). Please click here to view a larger version of this figure.
Figure 10: Processing of the first FID. The figure shows the pop up window that appears after typing efp. Please click here to view a larger version of this figure.
Figure 11: Phase correction. Screenshot showing the window for the manual phase correction. Please click here to view a larger version of this figure.
Figure 12: Processing of the second FID. The figure shows the pop up window that appears after typing efp. Please click here to view a larger version of this figure.
Figure 13: Multiple display of spectra 2 and 3. The button highlighted in a red square is the one to calculate the difference spectra. Please click here to view a larger version of this figure.
Figure 14: Integrals tab. The figure shows the values of the absolute and relative integrals. Please click here to view a larger version of this figure.
Figure 15: Structure and 1H NMR spectra of N,N-dimethylacetamide at 22.5 °C in toluene-d8. (a) 1H NMR expansion of the region from 2.13 to 2.66 ppm before irradiation. (b) Expansion of the same region after irradiation of the methyl group at 2.17 ppm. (c) Difference spectrum [(a)-(b)]. Please click here to view a larger version of this figure.
Figure 16: Example of the plot of ηSSTD and its exponential fit at 278 K. Reproduced from the supporting information of reference21 with permission from the Royal Society of Chemistry. Please click here to view a larger version of this figure.
Figure 17: Plots of ηSSTD vs. saturation time at different temperatures. The figure shows the plot for N,N-dimethylacetamide and the table with the obtained rates constants and relaxation times. Please click here to view a larger version of this figure.
Figure 18: Eyring plot. The figure shows the plot for N,N-dimethylacetamide. Please click here to view a larger version of this figure.
|(T = 278 K)
|(T = 283 K)
|(T = 285.5 K)
|(T = 288 K)
|(T = 290.5 K)
|(T = 293 K)
|(T = 295.5 K)
Table 1: Values of ηSSTD. The table shows the values obtained at different saturation times for N,N-dimethylacetamide in the range of temperatures 278-295.5 K.
|Ea 298 (KJ mol-1)
|79.7 ± 0.1
|73.1 ± 1.4
|70.5 ± 1.7
|82.0 ± 1.3
|79.5 ± 0.4
|ΔH‡ (KJ mol-1)
|77.2 ± 0.1
|70.6 ± 1.4
|79.5 ± 0.4
|76.6 ± 0.4
|ΔS‡ (J mol-1 K-1)
|11.5 ± 0.4
|-10.5 ± 5.0
|-15.0 ± 5.1
|13 ± 8
|3 ± 4
|ΔG‡298 (KJ mol-1)
|73.8 ± 0.1
|73.7 ± 2.0
|75.3 ± 0.4
|75.7 ± 0.4
Table 2: Activation parameters. The table shows the activation parameters for the internal rotation of N,N-dimethylacetamide obtained by the SSTD NMR method compared with the same parameters obtained using different NMR methods for analysis.4,5,6 Errors in this table refer to standard deviation errors (SD). (Reproduced from reference21 with permission from the Royal Society of Chemistry).
One of the more obvious advantages of this methodology is that the rate constants and the relaxation time for a given temperature can be obtained with a single set of experiments, with a robust pulse sequence (the same used for STD experiments to study protein-ligand interactions, which is typically found within the available set of experiments from the spectrometer manufacturer). This simplifies the experimental setup since there is no need to measure T1 or reach steady state saturation. Besides, it is remarkable that this method does not depend on the magnet strength, as coalescence methods. On the other hand, the main limitation is that this technique cannot be applied to chemical exchange processes too fast or too slow, which would depend on the temperature range of the NMR machine or the solvents used.
This new technique for the calculation of kinetic parameters can be applied to a great variety of substrates and its applicability has already been demonstrated with some interesting molecules.21 The kinetic parameters of the 4-N,N-dimethylamido[2.2]paracyclophane, a challenging substrate in which the signal of one of the methyl groups of interest is overlapped with other signals from the molecule, were successfully calculated using SSTD NMR. Interestingly, this methodology can be applied as long as one of the signals of study is isolated. SSTD NMR is also a useful protocol for the calculation of kinetic parameters in molecules in which the coalescence temperature is so high that the molecule decomposes before reaching it. This is the case with PtCl2(dimethylallene)(pyridine), in which the methodology was successfully applied without the need of reaching coalescence. The choice of solvents and temperatures is critical to obtain good results, since the chemical exchange rates can vary significantly with these parameters. Moreover, in addition to the criteria in a normal NMR experiment, key steps in a SSTD NMR experiment are the selectivity of the irradiation as well as the temperature control. Both factors have to be precise to guarantee the success of the experiment.
The representative results presented here are for the kinetics of intramolecular chemical exchange, but the technique can also be applied to study the kinetics of intermolecular chemical exchange and also ligand exchange, common processes in the dynamic behavior of transition metal complexes.
Finally, providing a proper modification of the equations is made,32 this method could be extended to deal with multi-site exchange and unequal populations, as it has been done in former double resonance experiments,8-9 increasing the usefulness of this technique for the study of chemical exchange processes in challenging compounds.
The authors declare that they have no competing financial interests.
Funding by the University of East Anglia, the EPSRC (EP/L012855/1) and the EU (H2020-MSCA-IF-2014-EF-ST-658172) is gratefully acknowledged (MTQ).
|Flammable and toxic
|500MHz 7" Select Series NMR Tubes
|TopSpin program, Bruker Corp., http://www.bruker.com/products/mr/nmr/nmr-software/software/topspin/ (2015).
|Origin 6.0 software, OriginLab Corp., http://originlab.com.
|Bruker Avance III 500 MHz fitted with 5mm broadband observed BBFOplus Z-gradient SmartProbeTM probe
|Bruker Corp., http://www.bruker.com
|Bruker Avance I 500 MHz Inverse Triple Resonance NMR spectrometer fitted with a 5mm TXI Z-gradient probe
|Bruker Corp., http://www.bruker.com
|Ceramic Spinner standardbore shimsystems (5 mm)
|Bruker Corp., http://www.bruker.com
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