The authors thank the Department of Chemistry and Physics and the Creativity and Research Grant Program (Naik, Moehring) at Monmouth University for financial support of this work.

The authors have no conflicts of interest to disclose.

There are four items in the preparation of ReH7(PPh3)2 that can impact the quantity and purity of the material that is produced. First, the use of an ice bath during the first 15 min of the reaction is important to remove heat from the reaction that occurs between sodium borohydride and water. Higher initial temperatures lead to a decreased yield of the ReH7(PPh3)2 product due to formation of the thermal decomposition product Re2H8(PPh3)4. Second, the color of the reaction mixture is more important than the amount of time for the reaction. When the reaction mixture has gone to completion, the mixture will have a tan to orange color. Any shade of green in the reaction mixture indicates that the reaction must proceed further. If necessary, additional sodium borohydride can be added to the reaction mixture after 1.5 h in case the mixture still has a green color. Third, the washing step is crucial for ensuring a high purity product from the reaction. A thorough water wash ensures that inorganic products such as sodium chloride and sodium borate are washed away from the product. The ethyl ether washes are crucial for removing colored rhenium polyhydride impurities that are always produced in the reaction such as ReH5(PPh3)3 and Re2H8(PPh3)4. Finally, the tetrahydrofuran solvent must be peroxide free, which can be accomplished either by using freshly distilled solvent or by storing the solvent under an atmosphere of nitrogen.
For a complex of interest such as ReH5(PPh3)2(sec-butyl amine), which contains organic-type protons, hydride ligands, and diastereotopic phosphorus atoms, three different variable temperature series of experiments are informative: 1) a series of 1H NMR spectra, 2) a series of 1H-{31P} NMR spectra, and 3) a series of 31P-{1H} NMR spectra. Each of the three different spectra can be acquired sequentially at each temperature of interest. All the dynamic NMR spectra of interest for a complex can be collected on a single NMR sample. The two proton spectra can be measured with 32 K data points for a window of 24 ppm, at 400 MHz, centered at 0 ppm. The phosphorus spectrum can be measured with 32 K data points with a window of 100 ppm, at 162 MHz, centered at 20 ppm. Measuring spectra at temperatures separated by 10 K is usually sufficient for most applications, but increments of 5 K temperature differences obviously produce more data, which can be useful in providing data for an Eyring equation determination of activation parameters. A typical temperature series from room temperature down to 200 K, in increments of 10 K, requires at least consecutive 4 h on the spectrometer. The 4 h include: the time to set up the heat exchanger and bottled nitrogen for the temperature controller, time for setting up the three experiments that will be measured at each temperature, time to measure room temperature spectra and examine the quality of the sample, time to decrease the temperature in increments of 10 K and stabilize at each temperature, time to shim the sample at each temperature and measure the spectra of interest, and time to warm the sample and spectrometer back to room temperature in increments of 10 K with at least 2 min intervals to stabilize the instrument before again increasing the temperature. Obviously going to lower temperatures or decreasing the temperature increments to 5 K will increase the time required on the spectrometer.
The parameters used for each of the three NMR series in this investigation can be found in the supporting materials. While NMR parameters can be changed during a temperature series, it makes for better comparisons of spectra measured at different temperatures if the spectra are all measured with the same parameters. For ReH5(PPh3)2(sec-butyl amine) and similar complexes, the temperature series begins in the fast exchange domain. Resonances arising from exchanging nuclei appear as coalesced resonances. Typically, signal-to-noise ratio for the exchanging nuclei will be larger at room temperature and will reach a minimum at a temperature near the coalescence temperature. Due to the changing nature of signal-to-noise, it is best if the signal-to-noise ratio is much better than marginal for the room temperature spectra. Additionally, the acquisition window must be set large enough to include all the resonances that will occur in the slow exchange spectrum.
Complexes of the form ReH5(PPh3)2(amine) which include an unsymmetrically substituted aromatic amine such as 3-picoline exhibit E and Z isomers9,10. At lower temperatures where dynamic rearrangements are slowed, phosphorus resonances from both isomers can be observed. Coalescence of these resonances corresponds to observing an average signal from the two interconverting isomers. Since the free energy of the two isomers are not necessarily the same, the phosphorus resonances that arise from these isomers will not necessarily have the same intensities. The line shape fitting software allows for each phosphorus atom in the model to occur in different molecules with different populations. This feature of the line shape fitting software allows for the line shape fitting of 31P-{1H} NMR spectra that arise from samples that include E and Z isomers.
Line shape fitting of the hydride region of the 1H-{31P} NMR spectra can be challenging because the individual hydride ligands may participate in multiple dynamic processes. It can be helpful when a chiral center is present, such as occurs with ReH5(PPh3)2(sec-butyl amine), to compare rate constants for phosphorus atom rearrangement with rate constants for the hydride ligand rearrangements, in order to test whether a hydride ligand rearrangement and a phosphorus atom rearrangement are different manifestations of a single molecular rearrangement. Furthermore, proton exchanges, such as between a hydride ligand and an adventitious water proton (a common occurrence for rhenium polyhydride complexes)9,13,34 which move a hydride ligand beyond the inner coordination sphere of the metal center, should be readily apparent in line shape fitting as an inability to produce a good fit using models that include only intramolecular hydride ligand exchanges (Figure 9)13.
Rhenium polyhydride complexes serve as pre-catalysts for the transformation of small molecules23,38,39,40,41,42,43,44,45,46,47,48,49,50,51. The specific mechanisms for catalytic cycles, though, are generally not well understood. The low activation energy dynamic processes of such complexes essentially confound all of the atom resonances in room temperature NMR spectra, making the chemical properties of individual atoms in specific locations impossible to follow. Dynamic NMR spectroscopy can allow for the identification of some chemical properties of a specific hydride ligand9,13. Catalytic steps with activation energies within the range of 5 to 25 kcal/mol may be apparent with line shape fitting of dynamic NMR spectra of such catalytic systems. Dynamic NMR spectroscopy can also lead to an understanding of dynamic properties, which may lead to rational design of transition metal polyhydride complexes with restricted dynamic properties. Complexes with restricted dynamic properties should allow for room temperature NMR investigations of chemical properties of specific atoms in specific coordination sites and lead to insight into catalytic cycles that start with transition metal polyhydride complexes.

NMR spectroscopy is commonly used to characterize dynamic processes that occur within or between molecules. For many simple intramolecular rearrangements, estimation of &#916;G&#8225; is as straight-forward as measuring the frequency difference, &#916;&#957;, between two resonances at the slow exchange limit and determining the coalescence temperature for those same resonances (Figure 1)1. The relationship,
&#916;G&#8225; = 4.575 x 10-3 kcal/mol x Tc [9.972 + log (Tc/&#916;&#957;)]
where Tc is the coalescence temperature for a pair of resonances that represent the slow exchange form of a dynamic sample, can be used to solve for the free energy of activation for such a dynamic rearrangement. More complex dynamic systems require line shape fitting of dynamic NMR spectra or another NMR technique such as two-dimensional exchange spectroscopy (2D-EXSY) or two-dimensional rotating-frame Overhauser effect spectroscopy (2D-ROESY) to estimate activation parameters.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64160/64160fig01.jpg" /> 
Figure 1: NMR spectra for a d8-toluene solution of ReH5(PPh3)2(sec-butyl amine) at two temperatures. The frequency difference between the two slow exchange doublets (lower trace, 117.8 Hz) and a coalescence temperature of 250 K (upper trace) correspond to an energy barrier (&#916;G&#8225;) of 11.8 kcal/mol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/64160fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Line shape fitting of dynamic NMR spectra is a common technique that has long been used for the estimation of activation parameters that describe dynamic rearrangements for substances with an activation energy of approximately 5 to 25 kcal/mol2,3,4,5. Determination of the energy barriers to proton exchange between water and amine molecules6, the energy barrier to rotation about the C-N bond in dimethylformamide7, or the general size of organic moieties8 are only a few examples of the many properties that have been assessed through line shape fitting of dynamic NMR spectra. This manuscript demonstrates the use of line shape fitting to characterize the intermolecular and intramolecular dynamic processes that occur for the complex ReH5(PPh3)2(sec-butyl amine). The goals of this and similar line shape fitting NMR experiments are to: 1) characterize all NMR observable intramolecular dynamic atom exchange processes if present, 2) identify and characterize NMR observable intramolecular dynamic atom exchange processes if present, 3) identify correlated intramolecular atom exchanges that occur for, in this example, both hydrogen and phosphorus atoms, and 4) for the example presented here, compare two published models for the dynamic processes that occur in the complex ReH5(PPh3)2(sec-butyl amine).
Eight-coordinate rhenium(V) polyhydride systems are complex dynamic systems in which the ligands participate in multiple dynamic processes and the phosphorus atoms can participate in a single dynamic process that is a second aspect of a hydride ligand exchange process9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26, 
27,28,29. Eight-coordinate, pseudododecahedral, rhenium(V) polyhydride complexes adopt a molecular geometry (Figure 2), which can be described as a pair of orthogonal trapezoids of ligands17,26. The vertices on the long edges of the trapezoids are commonly labelled as B sites and, in rhenium polyhydride complexes, are usually the sites occupied by neutral two-electron donor ligands such as tertiary phosphines or amine ligands. The vertices on the short edges of the trapezoids are commonly labelled as A sites and are typically occupied by anionic, two-electron donor, hydride ligands. The room temperature NMR spectra of rhenium(V) polyhydride complexes are, typically, deceptively simple due to the several dynamic processes that occur in room temperature solutions.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64160/64160fig02.jpg" /> 
Figure 2: A dodecahedral coordination set (left) and the complex ReH5(PPh3)2(sec-butyl amine) from the same perspective (right). The red-colored sites represent coordination sites that form a vertical trapezoid, and the blue-colored sites represent coordination sites that form a horizontal trapezoid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/64160fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Complexes of the form ReH5(PPh3)2(amine) are the most thoroughly studied class of rhenium polyhydride complexes with respect to dynamic processes9,10,12,13,16,30,31. Three dynamic processes (Figure 3) have been identified for ReH5(PPh3)2(amine) complexes: 1) a proton exchange between the sole B site hydride ligand and a proton from a water molecule (adventitious or intentional)9,13, 2) a turnstile exchange of a pair of A site hydride ligands with an adjacent B site hydride ligand9,11,13,30,31, and 3) a steric inversion (or pseudorotation) that manifests itself as a pairwise exchange of the A site hydride ligands and a pairwise movement of the B site atoms to the opposite side of the rhenium center (as depicted in Figure 4)4,5,6,8,26,27. The movement of B site atoms to the opposite side of rhenium is observable by dynamic NMR spectroscopy as: 1) a process that makes the inequivalent 3 and 5 protons of N = pyridine equivalent at room temperature10,30,31, 2) a process that causes the E and Z isomers of N = unsymmetrically substituted aromatic amine ligands to undergo fast exchange at room temperature9,10,13,30,31, or 3) a process that causes a fast exchange of the steric perspectives of a diastereotopic pair of phosphorus atoms with respect to a chiral center located on the amine ligand9,30,31. The previously unreported chiral complex ReH5(PPh3)2(sec-butyl amine) provides an opportunity to generally describe the methods that can be used to identify and characterize the dynamic rearrangements of rhenium polyhydride complexes.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64160/64160fig03.jpg" /> 
Figure 3: Representations of the dynamic processes that are observed by NMR spectroscopy for solutions of ReH5(PPh3)2(sec-butyl amine). Representation A depicts the exchange of a single proton of adventitious water for the unique B-site hydride ligand. Representation B depicts the turnstile exchange of three adjacent hydride ligands, two of which reside in A site while the third is the unique B site hydride ligand. Representation C depicts both the pairwise exchange of A site hydride ligands as well as the steric inversion of the phosphorus atoms with respect to the chiral amine ligand (N*). It should be noted that the A site hydride ligand pairwise exchange does not require a shift of the A site hydride ligands to the opposite side of the rhenium center. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/64160fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For chemical systems such as rhenium polyhydride complexes, which exhibit a complex set of dynamic processes, line shape fitting of dynamic NMR spectra is the most used NMR technique to characterize the processes9,11,13,16,21,29. Two-dimensional EXSY9,32 or 2D-ROESY11 are alternative dynamic NMR techniques that can also be used to quantitatively characterize the dynamic processes. Two-dimensional EXSY spectra are typically measured in the slow exchange temperature domain; two-dimensional ROESY spectra are typically measured in the fast exchange temperature domain. Both two-dimensional techniques may require considerable time in the spectrometer for data acquisition, in that each of the techniques is acquiring a much larger data set, at a given temperature, than the one-dimensional data sets needed for line shape fitting analysis. Simple dynamic processes that are well understood, such as the dynamic exchange of the two methyl groups of dimethylformamide, can be readily characterized by any of the three NMR techniques. More complex systems, such as ReH5(PPh3)2(sec-butyl amine), in which individual hydride ligands participate in multiple dynamic processes, or systems that are not necessarily well understood, such as a novel transition metal polyhydride complex which may or may not exchange protons between a hydride ligand and adventitious water, are more easily quantitatively characterized by the line shape fitting NMR method than by the two-dimensional NMR methods. Unlike the two-dimensional NMR methods, the line shape fitting method provides an easily interpretable visualization of the match between a tested model and the experimental data as well as visual evidence of an exchange that moves a hydride ligand beyond the inner coordination sphere of rhenium. Based upon peak heights and peak shapes in slow exchange spectra, even a complex dynamic system such as ReH5(PPh3)2(sec-butyl amine) can lead to an easily tested initial set of exchange models. Additionally, when multiple theoretical models have been reported for a molecular transformation, line shape fitting of dynamic NMR spectra can allow for a visual comparison of each model versus observed spectra.
Beyond the three NMR techniques mentioned above, isotopic substitution NMR experiments involving D2O or HD have been used to qualitatively demonstrate intermolecular exchange of atoms for complex rhenium polyhydride systems, but have not been used for quantitative characterizations9,33,34,35. Theoretical calculations present an additional method for characterizing the dynamic processes of complex dynamic systems30,31,36. Theoretical calculations have the advantage over line shape fitting in that they can be used to differentiate between possibilities that cannot be distinguished by line shape fitting analysis. For example, theoretical calculations have been used to describe an exchange that involves three adjacent hydride ligands on certain rhenium(V) complexes as a turnstile exchange of all three hydride ligands, rather than an alternating pair of pairwise exchanges with each pairwise exchange including a unique hydride ligand and one of two chemically equivalent hydride ligands30,31. The results of theoretical calculations are typically compared to experimentally observed quantitative characterizations from one of the three NMR techniques mentioned above as a check on the validity of the calculated results.
Line shape fitting of dynamic NMR spectra takes advantage of the change in the appearance of NMR spectra that occurs when NMR-active nuclei move between different chemical environments during an NMR measurement. Slow exchange NMR spectra (spectra with independent Lorentzian resonances for each set of exchanging nuclei) occur at temperatures where the frequency difference between resonances for nuclei that exchange is large compared with the rate of exchange of the nuclei37. Fast exchange NMR spectra (spectra with a single Lorentzian resonance for exchanging nuclei) occur at temperatures where the rate of exchange of the nuclei is much greater than the frequency difference between the slow exchange resonances37. Intermediate exchange rates occur for temperatures between the slow exchange temperature domain and the fast exchange temperature domain37. If the fundamental parameters of Larmor frequency, chemical shift of the exchanging nuclei, coupling constants (if any) for the exchanging nuclei, and relative populations of each nucleus type are known, rate constants for putative exchanges between nuclei can be determined by comparing simulated spectra to observed spectra at several intermediate temperatures. Good fits for simulations at several temperatures result in temperature and rate constant data that can be used with the Eyring equation to estimate activation parameters for the putative exchange(s). Results from the method have been found to be both accurate and reproducible.

<table><tbody><tr><td>Bruker Avance II 400 MHz NMR spectrometer</td><td>Bruker Biospin</td><td></td><td>The instrument includes a two channel probe (1H and X) with the X channel tunable from 162 MHz to 10 Mhz. The instrument is also VT capable with a dewar and heat exchanger for VT work.</td></tr><tr><td>d&lt;sub&gt;8&lt;/sub&gt;-toluene</td><td>MilliporeSigma</td><td>434388</td><td></td></tr><tr><td>Powerstat variable transformer</td><td>Powerstat</td><td></td><td></td></tr><tr><td>sec-butyl amine</td><td>MilliporeSigma</td><td>B89000</td><td></td></tr><tr><td>Sodium borohydride</td><td>MilliporeSigma</td><td>452882</td><td></td></tr><tr><td>Tetrahydrofuran</td><td>MilliporeSigma</td><td>186562</td><td></td></tr><tr><td>Thermowell C3AM 100 mL</td><td>Thermowell</td><td></td><td></td></tr><tr><td>Topspin 3.0 or 4.1.4 with dNMR</td><td>Bruker Biospin</td><td></td><td>Data was acquired with Topspin version 3.0 and data handling was performed on a second computer that was running Topspin version 4.1.4..</td></tr><tr><td>Trichlorooxobis(triphenylphosphine) rhenium(V)</td><td>MilliporeSigma</td><td>370193</td><td></td></tr><tr><td>Vacuubrand PC3000 vacuum pump with a CVC 3000 controller</td><td>Vacuubrand</td><td></td><td></td></tr></tbody></table>

line shape analysis of dynamic nmr spectra for characterizing coordination sphere rearrangements at a chiral rhenium polyhydride complex

Dynamic solution nuclear magnetic resonance (NMR) spectroscopy is the typical method of characterizing the dynamic rearrangements of atoms within the coordination sphere for transition metal polyhydride complexes. Line shape fitting of the dynamic NMR spectra can lead to estimates for the activation parameters of the dynamic rearrangement processes. A combination of dynamic 31P-{1H} NMR spectroscopy of metal-bound phosphorus atoms with dynamic 1H-{31P} NMR spectroscopy of hydride ligands may identify hydride ligand rearrangements that occur in conjunction with a phosphorus atom rearrangement. For molecules that exhibit such a coupled pair of rearrangements, dynamic NMR spectroscopy can be used to test theoretical models for the ligand rearrangements. Dynamic 1H-{31P} NMR spectroscopy and line shape fitting can also identify the presence of an exchange process that moves a specific hydride ligand beyond the metal&#39;s inner coordination sphere through a proton exchange with a solvent molecule such as adventitious water. The preparation of a new compound, ReH5(PPh3)2(sec-butyl amine), that exemplifies multiple dynamic rearrangement processes is presented along with line shape fitting of dynamic NMR spectra of the complex. Line shape fitting results can be analyzed by the Eyring equation to estimate the activation parameters for the identified dynamic processes.

The characterizations of both rhenium polyhydride products described in this manuscript are best accomplished by 1H-{31P} and 31P-{1H} NMR spectroscopy. In a room temperature d6-benzene solution, the hydride ligand resonance of ReH7(PPh3)2 appears as a binomial triplet at &#948; = -4.2 ppm with 2JPH = 18 Hz by 1H NMR spectroscopy (Supplementary Figure 2). The same d6-benzene solution will exhibit a singlet resonance at &#948; = 31.4 ppm by 31P-{1H} NMR (Supplementary Figure 3). In a d8-toluene solution, the hydride ligand 1H-{31P} NMR resonance of ReH5(PPh3)2(sec-butyl amine) appears as a broad singlet at &#948; = -4.83 ppm (Supplementary Figure 4). The same d8-toluene solution will exhibit a singlet resonance at &#948; = 47.3 by 31P-{1H} NMR spectroscopy (Supplementary Figure 5). Common impurities that can occur for either sample are ReH5(PPh3)3 (&#948;hydride = -4.73; 2JPH = 18.8 Hz, quartet; &#948;phosphorus = 34.16 measured in d8-toluene) and Re2H8(PPh3)4 (&#948;hydride = -4.93; 2JPH = 9.3 Hz, pentet; &#948;phosphorus = 42.79 measured in d6-benzene).
Line shape fitting is generally straight-forward for dynamic 31P-{1H} NMR spectra of rhenium polyhydride complexes that do not exhibit E and Z isomers10. The best fit simulations and 31P-{1H} NMR spectra for the complex ReH5(PPh3)2(sec-butyl amine) for several temperatures are shown in Figure 5. Only one model is needed to exchange phosphorus atoms on such complexes. When the phosphorus nuclei exhibit spin-spin coupling, as is the case with the complex ReH5(PPh3)2(sec-butyl amine), that coupling must be included in the model for good results. To simulate 31P-{1H} NMR spectra measured at the coalescence temperature and above, the temperature dependence of the chemical shift difference between the two resonances must be tracked and used to estimate the chemical shifts of the nuclei at the coalescence temperature and above (Figure 6). Additionally, NMR spectra measured at temperatures near the freezing point of the solvent may exhibit broadening of resonances due to increased solvent viscosity and precipitation of the analyte. Spectra that exhibit such resonance broadening should not be included in determination of the rate constants that are subsequently used in Eyring plot determinations.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64160/64160fig05.jpg" /> 
Figure 5. The 31P-{1H} NMR spectra (black traces) and best fit simulations (red traces) for a d8-toluene solution of ReH5(PPh3)2(sec-butyl amine). The black traces show the coalescence of the two resonances that arise from the diastereotopic phosphorus atoms into a single resonance at higher temperatures. The red traces show a good match of the simulated spectra that arise from line shape fitting and the observed data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/64160fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64160/64160fig06.jpg" /> 
Figure 6. A plot of the temperature dependence of the difference in chemical shifts between the two 31P-{1H} resonances. An extrapolation of this line allows for estimation of the chemical shifts of the individual resonances at higher temperatures. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/64160fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Line shape fitting of the hydride region of dynamic 1H-{31P} NMR spectra is more challenging than line shape fitting for phosphorus resonances. Line shape fitting of hydride resonances requires more nuclei and more exchange models. Common hydride ligand exchange models that have been used for rhenium(V) polyhydride complexes include: 1) exchange between a pair of adjacent hydride ligands16, 2) a turnstile exchange of three adjacent hydride ligands9,11,13,30,31, 3) exchange between a specific hydride ligand and a proton from water9,13, and 4) pairwise exchange of the A site hydride ligands on one side of rhenium with the A site hydride ligands on the other side of rhenium9,13,31. The latter exchange has been reported as a second aspect of the associated interconversion of E and Z phosphorus resonances or with the steric inversion of diastereotopic phosphorus resonances13. As such, the activation parameters and the rate constants for the latter hydride ligand exchange (if it occurs) should reflect the same values for the associated dynamic phosphorus process.
Line shape fitting can be used to test theoretical models of hydride ligand exchanges13. As with the phosphorus resonances mentioned above, the temperature dependence of the hydride resonances that will be modelled has to be determined so that chemical shifts can be adjusted for temperature drift. Figure 7 shows the temperature dependence that was observed for the hydride resonances of an ReH5(PPh3)2(sec-butyl amine) sample in d8-toluene as well as the best linear fit equations for that drift. The models for line shape fitting of 1H-{31P} NMR spectra used chemical shifts that were calculated for each resonance even when the resonance frequency could be determined directly from the spectrum. Chemical shifts of the hydride resonances were not treated as variable when line shape fitting the hydride region of dynamic 1H-{31P} NMR spectra. Figure 8 compares the results of line shape fitting, based on a pairwise exchange of A site hydride ligands, a turnstile exchange of three adjacent hydride ligands, and a proton exchange between one proton of water and hydride ligand H4, with the observed hydride region of a series of 1H-{31P} NMR spectra collected from 225 K to 240 K.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64160/64160fig07.jpg" /> 
Figure 7. Best fit lines for the temperature dependence of each 1H-{31P} NMR hydride resonance. The chemical shifts calculated from the best linear fits were used in the models for line shape fitting of the observed spectra. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/64160fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64160/64160fig08.jpg" /> 
Figure 8. The hydride region of 1H-{31P} NMR spectra (black traces) and best fit simulations (red traces) for a solution of ReH5(PPh3)2(sec-butyl amine). The spectra were measured on a d8-toluene solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/64160fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 9 displays best fits for two models of hydride ligand exchange for ReH5(PPh3)2(amine) complexes in the hydride region of the 225 K 1H-{31P} NMR spectrum for a sample of ReH5(PPh3)2(sec-butyl amine) in d8-toluene. The line shape fits are based upon theoretical models of hydride ligand exchange for the compound ReH5(PPh3)2(pyridine)30,31. Two aspects of the best fit spectra are important. First, the blue traces represent the best fits of the spectrum line shape based entirely on the reported exchange models. The blue traces indicate that a proton exchange between a specific hydride ligand and a proton from beyond the inner coordination sphere is missing. For this example, ReH5(PPh3)2(sec-butyl amine) complex, the missing exchange includes a proton from adventitious water along with the unique B site hydride ligand. Second, the red traces indicate that when a proton exchange with water is included with either theoretical model, a good line shape may or may not be obtained. For the complex ReH5(PPh3)2(sec-butyl amine), Model A generates the better fit for the observed spectrum. A comparison of rate constants for the steric inversion of diastereotopic phosphorus atoms with the rate constants for an associated hydride ligand rearrangement in each model also favors Model A over Model B (Table 1).
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/64160/64160fig09.jpg" /> 
Figure 9. A comparison of two models for the rearrangement of hydride ligands at ReH5(PPh3)2(amine) complexes without proton exchange. Both models were tested with the inclusion of an exchange of a specific hydride ligand with a proton from water (red traces) and without such a proton exchange (blue traces). The black traces are the measured 1H-{31P} NMR spectrum of ReH5(PPh3)2(sec-butyl amine) at 225 K. The model used to produce the A traces includes a pairwise exchange of A site hydride ligands. The model used to produce the pair of B traces includes a basal turnstile exchange of hydride ligands. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/64160fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Temperature (K)</td>
			<td>k Steric inv. (Hz)</td>
			<td>k Pairwise (Hz)</td>
			<td>k Basal (Hz)</td>
		</tr>
		<tr>
			<td>225</td>
			<td>94.5</td>
			<td>88.2</td>
			<td>6.6</td>
		</tr>
		<tr>
			<td>230</td>
			<td>131.3</td>
			<td>151.3</td>
			<td>28.4</td>
		</tr>
		<tr>
			<td>235</td>
			<td>236</td>
			<td>219.3</td>
			<td>46.1</td>
		</tr>
		<tr>
			<td>240</td>
			<td>376.4</td>
			<td>324.2</td>
			<td>66.4</td>
		</tr>
	</tbody>
</table>
Table 1. A comparison of rate constants for phosphorus atom steric inversion with pairwise exchange of the A site hydride ligands and with the basal turnstile exchange of hydride ligands. All simulations of hydride resonances included an exchange of protons between adventitious water and the unique B site hydride ligand.
Activation parameters for each modelled dynamic process of Model A can be estimated from Eyring plots (Figure 10 and Figure 11, Supplementary Figure 6, and Supplementary Figure 7). Eyring plots of dynamic 31P-{1H} rate constants have the advantage over Eyring plots of dynamic 1H-{31P} rate constants in that only one model is needed to describe phosphorus atom exchanges. Having a single model for phosphorus atom exchange means that there is no confounding of the phosphorus atom exchange results, unlike hydride ligand exchanges which have multiple exchange models that involve the same atoms. Dynamic 31P-{1H} NMR data is also generally available for a larger range of temperatures than for dynamic 1H-{31P} NMR data which means more data points for the Eyring plot.
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/64160/64160fig10.jpg" /> 
Figure 10. Eyring plot from the line shape fitting of 31P-{1H} NMR spectra for a d8-toluene solution of ReH5(PPh3)2(sec-butyl amine). The trend line shows that the rate constants that arise from line shape fitting of the 31P-{1H} NMR spectra at several temperatures fit the Eyring equation well. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/64160fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/64160/64160fig11.jpg" /> 
Figure 11. Eyring plot from the pairwise exchange of A site hydride ligands. The data arises from line shape fitting of 1H-{31P} NMR spectra measured on a d8-toluene solution of ReH5(PPh3)2(sec-butyl amine). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/64160fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: An example of the end point color for the reaction of ReOCl3(PPh3)2 with sodium borohydride to form ReH7(PPh3)2. The color of the reaction, as shown in the Figure, is the best indication that the reaction between ReOCl3(PPh3)2 and sodium borohydride, in tetrahydrofuran and water, has gone to completion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/Supplementary Figure 1 Moehring final.jpg" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2. The 1H NMR hydride resonance for a sample of ReH7(PPh3)2 dissolved in d6-benzene. The 1H NMR spectrum of a sample can be used to readily identify the product of a reaction as a genuine sample of ReH7(PPh3)2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/Supplementary Figure 2 Moehring final.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3. The 31P-{1H} NMR spectrum of a sample of ReH7(PPh3)2 dissolved in d6-benzene. The 31P-{1H} NMR spectrum can be used to qualitatively characterize a sample of ReH7(PPh3)2, and such a spectrum provides a convenient check for impurities in the sample. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/Supplementary Figure 3 Moehring final.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4. The room temperature 1H NMR hydride resonance for a sample of ReH5(PPh3)2(sec-butyl amine) dissolved in d8-toluene. The small spike on the upfield shoulder of the peak is due to an impurity of Re2H8(PPh3)4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/Supplementary Figure 4 Moehring final.PDF" target="_blank">Please click here to download this File.</a>
Supplementary Figure 5. The 31P-{1H} NMR spectrum of a sample of ReH5(PPh3)2(sec-butyl amine) dissolved in d8-toluene. The 31P-{1H} NMR spectrum of a sample can be used to qualitatively identify a sample of ReH5(PPh3)2(sec-butyl amine) and to check for impurities. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/Supplementary Figure 5 Moehring final.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 6. Eyring plot from the turnstile exchange of two A site hydride ligands with an adjacent B site hydride ligand. The data arises from line shape fitting of 1H-{31P} NMR spectra measured on a d8-toluene solution of ReH5(PPh3)2(sec-butyl amine). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/Supplementary Figure 6 Moehring final.png" target="_blank">Please click here to download this File.</a>
Supplementary Figure 7. Eyring plot from the exchange of protons between adventitious water and the unique B site hydride ligand. The data arises from line shape fitting of 1H-{31P} NMR spectra measured on a d8-toluene solution of ReH5(PPh3)2(sec-butyl amine). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/Supplementary Figure 7 Moehring final.png" target="_blank">Please click here to download this File.</a>
Supplementary Table 1. NMR experiment parameters. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64160/Supplmentary NMR Experiment Settings final.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Line Shape Analysis of Dynamic NMR Spectra for Characterizing Coordination Sphere Rearrangements at a Chiral Rhenium Polyhydride Complex at JoVE.com

Line Shape Analysis of Dynamic NMR Spectra for Characterizing Coordination Sphere Rearrangements at a Chiral Rhenium Polyhydride Complex

Line shape analysis of NMR spectra collected over a range of temperatures serves as a guide for the rearrangement of inner coordination-sphere atoms at a chiral, eight-coordinate, rhenium(V) polyhydride complex, ReH5(PPh3)2(sec-butyl amine). Line shape analysis is also used to determine the activation parameters &#916;H&#8225;, &#916;S&#8225;, and &#916;G&#8225; for those atom rearrangements.

Dynamic solution nuclear magnetic resonance (NMR) spectroscopy is the typical method of characterizing the dynamic rearrangements of atoms within the ...

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Chemistry

2.7K Views.  Monmouth University. Line shape analysis of NMR spectra collected over a range of temperatures serves as a guide for the rearrangement of inner coordination-sphere atoms at a chiral, eight-coordinate, rhenium(V) polyhydride complex, ReH5(PPh3)2(sec-butyl amine). Line shape analysis is also used to determine the activation parameters ΔH‡, ΔS‡, and ΔG‡ for those atom rearrangements.

Video: Line Shape Analysis of Dynamic NMR Spectra for Characterizing Coordination Sphere Rearrangements at a Chiral Rhenium Polyhydride Complex

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Biochemistry

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-&#954;3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-&#954;3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-&#954;3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-&#954;3P{CH3CO(CH2)2CO2H}-&#954;2O](PF6) (6).

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru&#8211;N-triphos complex with levulinic acid is described.

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the &#181;s-ms Timescale

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the &#181;s-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a&#160;15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the &#181;s-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the  &#181;s-ms Timescale

Here, a detailed description of the protocol implemented in the laboratory for acquisition and analysis of 15N relaxation dispersion profiles by solution NMR spectroscopy is provided.

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important ...

Vibrational Spectra of a N719-Chromophore/Titania Interface from Empirical-Potential Molecular-Dynamics Simulation, Solvated by a Room Temperature Ionic Liquid

The accurate molecular simulation prediction of vibrational spectra, and other structural, energetic and spectral characteristics, of photo-active metal-oxide surfaces in contact with light-absorbing dyes is an ongoing thorny and elusive challenge in physical chemistry. With this in mind, a molecular-dynamics (MD) simulation were performed by using optimized empirical potentials for a well-representative and prototypical dye-sensitized solar cell (DSC) solvated by a widely-studied room temperature ionic liquid (RTIL), in the guise of a [bmim]+[NTf2]- RTIL solvating an N719-sensitizing dye adsorbed onto 101 anatase-titania. In doing so, important insights were gleaned into how using a RTIL as the electrolytic hole acceptor modulates the dynamical and vibrational properties of a N719 dye, estimating the spectra for the DSC photo-active interface via Fourier transformation of mass-weighted velocity autocorrelation functions from MD. The acquired vibrational spectra were compared with the experiment spectra and those sampled from ab initio molecular dynamics (AIMD); in particular, various empirical-potential spectra generated from MD provide insight into how partial-charge charge parameterization of the ionic liquid affects vibrational spectra prediction. In any event, careful fitting of empirical force-field models has been shown to be an effective tool in handling DSC vibrational properties, when validated by AIMD and an experiment.

A dye-sensitized solar cell was solvated by RTILs; using optimized empirical potentials, a molecular dynamics simulation was applied to compute vibrational properties. The obtained vibrational spectra were compared with experiment and ab initio molecular dynamics; various empirical potential spectra show how partial-charge charge parameterization of the ionic liquid affects vibrational spectra prediction.

The accurate molecular simulation prediction of vibrational spectra, and other structural, energetic and spectral characteristics, of photo-active ...

Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1&#961; relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing.
The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1&#961; experiments with both isotopically labeled and unlabeled RNA samples.

We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R1&#961; relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...

High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible manner. Hydrostatic pressure drives thermodynamical equilibria toward the state(s) with the lower molar volume. Increasing pressure offers, therefore, the opportunities to finely tune the stability of globular proteins and the oligomerization equilibria of protein complexes. High-pressure NMR experiments allow a detailed characterization of the factors governing the stability of globular proteins, their folding mechanisms, and oligomerization mechanisms by combining the fine stability tuning ability of pressure perturbation and the site resolution offered by solution NMR spectroscopy. Here we present a protocol to probe the local folding stability of a protein via a set of 2D 1H-15N experiments recorded from 1 bar to 2.5 kbar. The steps required for the acquisition and analysis of such experiments are illustrated with data acquired on the RRM2 domain of hnRNPA1.

We provide a detailed description of the steps required to assemble a high-pressure cell, set up and record high-pressure NMR experiments, and finally analyze both peak intensity and chemical shift changes under pressure. These experiments can provide valuable insights into the folding pathways and structural stability of proteins.

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible ...

&sup1;H NMR of Conformationally Flexible Molecules: Temporal Resolution

At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the axial and equatorial protons, resulting in a single peak at &#948; 1.4.
Similarly, NMR averages all the conformations of any molecule with rapid conformational equilibria. For example, the CH3 protons in bromoethane have a single resonance and coupling constant for splitting by the CH2 protons due to the rapid internal rotation of the carbon-carbon bond. Lastly, besides conformational equilibria, the time-averaging effect of NMR spectroscopy is also observed in some chemical reactions.

At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately ...

Education

JoVE Core

Analytical Chemistry

Unit 1

Advanced Nuclear Magnetic Resonance Spectroscopy

KEY TERMS AND CONCEPTS

&sup1;H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR

The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one proton, the remaining eleven protons are replaced by deuterium. The deuterium substitution avoids the possible peak splitting caused by the spin-spin coupling between the adjacent protons. The remaining proton flips between the axial and equatorial positions.
<img alt="Figure1" src="/files/ftp_upload/13016/13016-PT_Figure_1.svg" style="text-align: center; height: 480px;" />
Figure 1. The temperature-dependent proton NMR spectra of cyclohexane.
Figure 1 depicts the proton NMR spectrum of the deuterium substituted sample, recorded at various temperatures. At room temperature, the rapid ring flipping of cyclohexane results in a single sharp peak. As the temperature is lowered to &#8722;60&#176;C, the rate of chair-chair interconversion reduces, resulting in the broadening of the peak.
Further lowering of the temperature broadens the peak, forming a saddle that splits into two peaks. On reaching a temperature lower than &#8722;89&#176;C, two sharp, well-resolved peaks corresponding to the equatorial and axial protons are formed. The peaks corresponding to the equatorial and axial protons occur at &#948; 1.62 and 1.14, respectively. At such low temperatures, the rate of chair-chair interconversion is reduced significantly, enabling the detection of the two distinct sets of protons on the NMR time scale.

The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one ...

Line Shape Analysis of Dynamic NMR Spectra for Characterizing Coordination Sphere Rearrangements at a Chiral Rhenium Polyhydride Complex

Summary

Explore More Videos

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D ¹H NMR

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphos^Ph Complexes

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

¹⁵N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the µs-ms Timescale

Vibrational Spectra of a N719-Chromophore/Titania Interface from Empirical-Potential Molecular-Dynamics Simulation, Solvated by a Room Temperature Ionic Liquid

Practical Aspects of Sample Preparation and Setup of ¹H R_1ρ Relaxation Dispersion Experiments of RNA

High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States

¹H NMR of Conformationally Flexible Molecules: Temporal Resolution

¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR