The EXSY experiment

It has been mentioned in Section 7.3, and it was implicit all over Chapter 7, that a finite time is required to achieve selective saturation or inversion of a signal by a soft pulse, during which time polarization starts to be exchanged, causing non-linearity of the response (see also Section 9.3). It should be stressed that this is not the case in all common 2D experiments based on non-selective pulses, which have durations of the order of microseconds instead of milliseconds, as required for selectivity. Selectivity in 2D experiments is intrinsic because of the double frequency labeling along f and /2. 

We recall here that the phases of the pulses in Fig. 8.2A sequence, as well as of all other pulse sequences shown in Fig. 8.2 and described later in this chapter, must be properly cycled to achieve selection of the desired connectivities and suppression of artifacts and of other connectivities due to different types of interactions. The criteria to choose the appropriate phase cycling do not depend on the presence of a paramagnetic center in the molecule, and the reader should refer to the many publications on multidimensional NMR for details. 

However, if the aim is only that of measuring xm, it is more straightforward to perform a single steady state ID experiment, as explained in Section 4.3.4. An EXSY experiment is shown in Fig. 8.5, relative to the complex praseodymium di-ethylenetriaminepentaacetate (Pr(DTPA)2-) [3,4]. The complex undergoes chemical exchange between two conformational isomers. The T values of the signals are around 30 ms. 

EXSY cross peaks are also obtained in TOCSY experiments (see later) because scalar interactions in the rotating frame are not separable from exchange interactions [7]. An EXSY experiment, performed using a TOCSY sequence (see Section 8.6) is reported in Fig. 8.7 relative to the complex 5Cl-Ni-SAL-MeDPT [5]. This complex, as shown in Fig. 8.8, displays a chemical equilibrium in which the two salicylaldiminate moieties exchange their non-equivalent positions [8]. It is interesting to learn that such complex interconversion occurs with times of the order of the spin-lock time (20 ms) or shorter. 

As EXSY cross peak intensities can be as strong as the diagonal peak intensities in favorable cases, EXSY experiments can be performed with relative ease also on nuclei other than protons. 

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Determination of reaction mechanisms by combining the observed intermediates in a catalytic cycle. To do this, it is often necessary to measure under different conditions - that is, variable temperature NMR. The use of high-pressure NMR cells is crucial in order to measure under the real catalytic conditions. The EXSY experiment helps to unravel exchange pathways, both intra-and intermolecular. 

Quantitation and Interpretation of 2D EXSY Spectra. Quantification of the EXSY experiment is accomplished through a series of... 

The change from cosine to sine modulation in the EXSY experiment can be though of as a phase shift of the signal in tl. Mathematically, such a phase shifted cosine wave is written as cos(f2jtj +(/)), where (f) is the phase shift in radians. This expression can be expanded using the well known formula cos( + B) = cos A cos B - sin A sin B to give... 

In words, a cosine wave, phase shifted by n 2 radians (90°) is the same thing as a sine wave. Thus, in the EXSY experiment the effect of changing the phase of the first pulse by 90° can be described as a phase shift of the signal by 90°. 

The two frequency axes may consist of a diverse assortment of pairs of fundamental NMR parameters. Examples might include chemical shift on one axis and a frequency axis for scalar couplings on the second as in the 2D /-resolved NMR experiments. Both axes may be proton chemical shift, in which responses may be correlated by scalar (/) couphng as in the COSY experiment [46—48], by dipolar relaxation pathways as in the NOESY [35, 36, 49—51] and ROESY [35, 36, 52, 53] experiments, or by chemical exchange pathways as in the EXSY experiment [54—59]. Other examples may involve chemical shift on one axis and a multiple quantum frequency on the second axis. Examples here would include proton double [60 62] and zero quantum spectroscopy [63—67], C—INADEQUATE [68, 69], etc. The available axes in a 2D NMR experiment may also be used for hetero-nuclear chemical shift correlation, e.g. H—or H— N, where the respective nucHde pairs are correlated via their one-bond ( /xh) or multiple bond ("/xh) hetero-nuclear couphngs [14, 16, 17, 23—27, 29—31, 70—72]. 

Fig. 8.4. The first EXSY experiment on a paramagnetic system [1] the 300 MHz spectrum, taken with mixing time of 50 ms, shows species in chemical exchange belonging to two different redox states of a cytochrome d, a protein containing four low spin hemes. The signals marked M1-M7 represent various heme methyl groups. EXSY cross peaks are observed between M, of two species containing two (II) or three (III) oxidized hemes, respectively.

With the use of a 2D EXSY experiment on a [La(TTHA)]3 sample, an exchange between the diastereotopic protons in the terminal acetate groups has been observed. The activation energy of this process is relatively high (Ea = 69.6 kj) [48], which can be accounted for by a mechanism via decoordination of a terminal N(CH2COO)2 moiety, followed by inversion and recoordination. 

Qualitative 2D EXSY Spectroscopy of Vanadium(V) Complexes. The 2D EXSY experiment is similar to the 2D nuclear Over-hauser effect (NOESY) spectroscopy experiment) used to estimate in-... 

Figure 11 shows the contour plots of the 2D 51V EXSY spectrum of a two-site system (40 mM total vanadate at pH 10.9) in which V exchanges with V2. Quantification of the EXSY spectrum and calculation of the error propagated to the rate constants from the integration precision gives a 25% error on the rate constant. The results (both the rate constants and the errors) correspond nicely to the results obtained from a ID magnetization transfer experiment on the same sample (27). The EXSY spectrum of a sample containing 12.5 mM total vanadate at 1.0... 

Applications of NMR spectroscopy to structural, thermodynamic, and dynamic processes have been described. A brief discussion of the types of problems appropriate for study by this technique has been included. H and 13C NMR spectroscopy has been applied to define the ligand coordination in complexes. These experiments, combined with 170-labeling experiments, allowed deduction of the coordination number of the vanadium atom. Integration of NMR spectra allowed measurement of the formation constants and equilibrium constants. 2D 13C and 51V EXSY experiments were used in a qualitative and quantitative manner to examine intra- and intermolecular dynamic processes, of which several examples are discussed. The interpretation of the rate matrix and its relationship to the chemical processes under examination were also described. 2D EXSY spectroscopy has great potential as a tool with which to probe mechanisms in complex reactions however, such uses often requires estimation of errors. The major source of error in 2D 51V EXSY NMR studies on a two- and four-site vanadate system were found to be baseline distortion and the errors were estimated. Our results suggest... 

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