Spin Saturation Transfer Difference NMR (SSTD NMR): A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

The overall goal of this experiment is to obtain the kinetic parameters and processes of mutual chemical exchange in small organic and organometallic molecules that are difficult to measure by traditional methods. This method can help to understand molecule dynamics of organic and organometallic molecules undergoing chemical exchange. For example rotational variance, conformational equilibrium, nitrogen inversion, lignin exchange, and tautomerization.

The main advantages of this technique are that there is no need to coalescence of the exchanging signals and that the rate constant and relaxation time are obtained in the same experiment. We first had the idea for this method when investigating the functional behavior of platinum ion complexes in solution and we realized the variable temperature align-ship analysis techniques were not suitable. For our experience in protein-lignin interactions by NMR we realized that in fact similar NMR approaches could be applied to study exchange processes in organic molecules and it turned out to be true.

Add five milligrams of nn dimethyacidamide to an NMR tube appropriate for low temperatures and dissolve in zero point six milliliters of duterated tolulene. TO setup the SSTD NMR experiments first insert the sample in the magnet. One of the key steps in assisting the NMR experiment is the temperature control so be sure to let the sample stabilize at the selected temperature for at least 20 minutes.

Once the sample is in the magnet, type EDTE in the command line. Change the temperature to the first selected temperature to carry out the experiment. Let the sample stabilize at the chosen temperature for at least 20 minutes.

Perform a one denominational proton NMR experiment on the sample as described in the text protocol then create a new data set of a proton NMR experiment by clicking on File, new and name the new experiment. In the new data set type RPAR in the command line. Select one of the STDDIFF parameter sets from the list.

To select the STDDIFF pulse sequence click the button with three dots in the pulse program line. Before carrying out the SSTD NMR experiment calibrate the proton 90 degree hard pulse P one. Type pulsecal in the command line and copy the value of the 90 degree pulse at the higher power which is the one that gives the shortest pulse.

Introduce the values for the calibrated hard pulse in the experiment with the command getprosol. Next set the length of the set pulse, type P13 and introduce a value of 50, 000 microseconds. Set the selective pulse shape by going to power and clicking the edit button next to shape then go to the shape pulsed 13 and choose the Gausian pulse.

Set the selective pulse power SP13 to 50 decibels. Type NS and set it to eight and then type DS and set it to four. To acquire the SSTD NMR experiment open the proton NMR experiment to check where the signal that will be irradiated is.

To do so search the experiment in the software browser right click in the data set and click display in a new window. The selectivity of the irradiation has to be precise to guarantee the success of the experiment. Ensure that no chemicals of correction is used or the irradiation frequency can be set incorrectly.

Move the cursor line to the center of the signal to irradiate and write down the chemical shift in parts per million. Select the spectral width that will be used in the experiment. In this case the signal to be irradiated is at 2.17 parts per million and the spectral width used is 1.46 parts per million.

Next go to the previously created SSTD NMR experiment create a list with the frequencies of irradiation by typing FQ2LIST in the command line, and selecting an existing list. Edit the list of irradiation frequencies as described in the text protocol. Save the list with a new name.

Then type FQ2LIST in the command line and select the list just created. To center the experiment on the signal under study type O1P and select the chemical shift in the signal that will be irradiated as the center of the experiment. Type SW to select the spectral width.

Choose the value for the inter-scan relaxation delay D1.Ensure that it is at least one to five times the value of the T1 of the slowest relaxing proton. Type D1 and set it to 40 seconds. Set the first value for the saturation time by typing D20 and setting it to 40 seconds.

Determine the receiver gain automatically by typing RGA. Create the next experiment by typing IEXPNO. Type D20 and select a saturation time of 20 seconds.

Then type RGA to automatically determine RG.Repeat the last step for D20 equals 10, five, 2.5, 1.25, 0.625, and 0.3 seconds. Once all the experiments are created open the first one and in the command line type multizg and specify the number of experiments. To process the data open the process number one from the set of experiments with the higher saturation time.

In the command line type LB and set the value to 1.5. In the command line type EFP and process FID number equals one in-process number equals two. Correct the phase of the experiment by clicking the interactive phase correction button and save it as a two G experiment then save and exit.

Type REP one in the command line to go to the process number one. In the command line type EFP and process FID number equals two, in-process number equals three. Next in the command line type point MD and then REP 2 to show a multiple display window with both process spectra two and three.

Click the button with the delta sign to calculate the difference spectra and save it in process number four. Then exit the multiple display window. Select and integration range from the signal on the left.

Always integrate the same region in process number three and process number four. Once integrated go to the integrals tab in each of the experiments and copy the absolute value of the integral. Repeat the procedure for the rest of the experiments with different saturation types.

To get the kinetic parameters first pull out the obtained SSTD parameter values versus the saturation time. Continue with data analysis as described in the text protocol. The hindered rotation around the amide bond differentiates both methyl groups into two signals in the proton NMR spectra.

Spin saturation of the methyl group at 2.17 parts per million leads to the disappearance it's signal in the proton NMR. Upon saturation of methyl group B at 2.17 parts per million transfer of saturation to methyl group A at 2.16 parts per million due to the internal rotation can be observed by a decrease in proton intensity in the signal at 2.61 parts per million. The SSTD parameter factor is calculated by dividing the value of the integral of methyl group A in the difference spectrum by the value of the integral of methyl group A in the original spectrum.

For a certain temperature the plot of the values of the SSTD parameter versus the saturation time gave exponential curves. Fitting of the curves gives the rate constant and the relaxation time of the proton of the measured signal. Plots of the SSTD parameter versus saturation time at different temperatures yielded corresponding rate constants and the relaxation times.

The Eyring equation was used to calculate the thermodynamic parameters. The values obtained with the SSTD NMR are an excellent agreement with the data reported using other techniques. Once mastered a set of experiments at a given temperature to get a value of rate constant and relaxation time can be done in about three hours if it is performed properly.

While attempting this procedure it's important to remember that the choice of solvents and temperatures is critical to obtain good results. Since the chemical exchange rates can vary significantly with these parameters. Following this procedure other processes like kinetic soft inter-molecular chemical exchange and the lignin exchange can be also studied and besides this method could be extended to deal with multi-site exchange and unequal populations provided a proper modification of the equation is made.

After watching this video you should have a good understanding of how to run a spin saturation transfer difference experiments and how to use magnetization transfer to obtain kinetic and thermodynamic parameters in molecules undergoing chemical exchange. Don't forget that working with chemicals can be extremely hazardous and precautions such as safety glasses and gloves should always be taken while performing the sample preparation on this procedure.