Refocusing delay

The DEPT experiment (Doddrell elal, 1982) involves a similar polarization transfer as the INEPT experiment, except it has the advantage that all the C signals are in phase at the start of acquisition so there is no need for an extra refocusing delay as in the refocused INEPT experiment. Coupled DEPT spectra, if recorded, would therefore retain the familiar phasing and multiplet structures (1 1 for doublets, 1 2 1 for triplets, etc.). Moreover, DEPT experiments do not require as accurate a setting of delays between pulses as do INEPT experiments. 

The basic INEPT spectrum cannot be recorded with broad-band proton decoupling, since the components of multiplets have antiphase disposition. With an appropriate increase in delay time, the antiphase components of the multiplets appear in phase. In the refocussed INEPT experiment, a suitable refocusing delay is therefore introduced that allows the C spin multiplet components to get back into phase. The pulse sequences and the resulting spectra of podophyllotoxin (Problem 2.21) from the two experiments are given below ... 

Both experiments are based on polarization transfer from sensitive nuclei to insensitive nuclei, and therefore the mjyor portions of their pulse sequences are common. The INEPT experiment, without refocusing and decoupling, however, yields spectra with distorted" multiplets. For instance, the two lines of a doublet appear in antiphase with respect one another. Similarly, the central line of a triplet may be too small to be visible, while the outer two lines of the triplet will be antiphase to one another. Introducing a variable refocusing delay A and broadband decoupling in the INEPT sequence can convert this experiment into a more useful one. 

Experimental approaches to direct characterization of the conformational exchange motions in proteins have been suggested earlier [67-69]. The most recent methods [66, 70-73] are based on a relaxation-compensated version of CPMG that alleviates the previous restriction on the duration of the refocusing delay due to evolution of magnetization from scalar couplings and dipole-dipole cross-correlations. 

In both the one-dimensional carbon- or proton-detected INEPT polarization transfer experiment and the two-dimensional proton-detected experiment, one needs to optimize the polarization transfer delay with an estimate of the coupling constant. In addition, the refocusing delay. A, must be set separately for an IS and IS2 spin system. For an IS spin system, the optimum delay is A = (l/4)Jis, whereas for an IS2 system the delay should be set to A = (l/8)Jis to get maximum polarization transfer. If one is interested in, for instance, the determination of relaxation parameters for carbons carrying two protons as well as for CH carbons, this requires separate experiments. 

Various NMR pulse sequences contain certain building blocks like BIRD, INEPT, refocusing delay etc., that perform some specific functions. A building block that serves... 

The width of the final1H pulse in the DEPT sequence ( ) affects the intensity and phase (positive or negative) of the 13C peaks in a way very similar to the length of the refocusing delay A in refocused INEPT, according to the relation = nJA. The intensities are shown below for the three types of carbon in DEPT ... 

These editing factors are the same we observe for the refocused INEPT experiment, if we replace with r/A, where A is variable refocusing delay. 

The strategy is quite simple after the mixing step, the cosine term is refocused as before with a spin echo of duration 1/(27) whereas the sine term sits out the refocusing delay on the sidelines because ZQC and DQC do not undergo 7-coupling evolution with respect to the active coupling ( ch)-... 

Then the cosine term is flipped to the z axis where it sits out another 1/(27) refocusing delay while the sine term, converted by the same pulse to antiphase coherence (completing the coherence transfer), is refocused to in-phase lH coherence ... 

Once we have diagramed the desired coherence order pathway, it is easy to add gradients to select that pathway. One simple solution is to use the 3,4,5 relationship of a right triangle 3x3 + 4x4 = 5x5. Put a gradient in the first half of t of relative amplitude G = 5, another in the second half with amplitude G2 = 3, and a third in the refocusing delay with amplitude G3 = 4. As the coherence order p is 5, —3, and —4, respectively, during these three periods, we have a total twist of ... 

The refocusing delay just before acquisition has been eliminated, so the peaks in F2 will be antiphase doublets separated by the long-range/cH (2-15 Hz). This splitting is in addition to any H splitting pattern already present in the ID proton spectrum (Fig. 11.11). The second 13 C 90° pulse is phase cycled as before to make sure that only H magnetization... 

Thus we cannot escape the depressing reality that 7 2 will get shorter and linewidth will get bigger as we increase the size of the protein studied. The reduced T2 is not only a problem for linewidth, but also causes loss of sensitivity as coherence decays during the defocusing and refocusing delays (1/(2J)) required for INEPT transfer in our 2D experiments. The only ray of hope comes in the form of a new technique called TROSY (transverse relaxation optimized spectroscopy), which takes advantage of the cancellation of dipole-dipole relaxation by CSA relaxation to get an effectively much longer 7 2 value we will briefly discuss TROSY at the end of this chapter. 

Similar editing techniques may also be applied to more complex spin systems as outlined for an olefinic doublet in Figure 6. Figures 6(c) and 6(d) illustrate the phase distortion from pure absorptive lineshapes that result from a mismatch between the refocusing delay and the value of V(" Sn, H) on which the editing is based. 

Figure 5 ID spectral editing of the proton spectrum of r-Bu2SnCl2 in dimethyl sulphoxide solution using H-" Sn HMQC spectroscopy (a) no refocusing delay (b) with refocusing delay (c) with refocusing delay and " Sn decoupling...

Figure 26 Spectra obtained at 500.13 MHz from three variants of the ID proton detected H- Sn HMQC for the hydroxylic proton of compound 3. The spectra a-d were recorded in one run without reshimming. The experiments were performed with a delay of 260 ms fitting the evolution of the long-range V(" Sn, H) couplings without refocusing dday (b), with refocusing delay (c) and with refocusing delay and " Sn decoupling (d). The standard ID proton spectrum (a) is plotted at the top for comparison. (Reproduced with permission from ref. 16)...

Since the time A of HMBC is so much larger than that of either HMQC or HSQC ( 60 ms vs. 3.6 ms), the refocusing delays with accompanying C decoupling of the latter experiments are not used, for reasons of sensitivity. Too much signal would be lost through relaxation processes in the approximately 60 ms that it would take the antiphase H vectors to refocus prior to detection (Section 6-2). 

Fig. 3. Basic pulse sequences for 2D- X,"T H correlations. Tbe same notation as in Hg. 1 is used. Minimum phase cycles for selection of correlation signals are given, more elaborate schemes for quadrature detection in FI and phase-sensitive spectra may be applied following standard rules. (a) HETCOR (without 180° pulses)/INEPT (with 180° pulses), the refocusing delays A are optional in both experiments setting the mixing pulses 8 to 45°/135° instead of 90° allows to determine coupling signs in ABX-type spectra. (b), HSQC. (c), HMQC the refocusing delay A2 is optional.

Figure 4.29. Editing with INEPT can be achieved with a judicious choice of refocusing delay, A2. A total delay of 1/2Jch retains CH resonances but eliminates CH2 (and CH3) resonances.

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