Refocused INEPT

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

INEPT Refocused INEPT Refocused INEPT Normal ... 

Figure 7 Relation of magnetization intensity and product of Jch and 6 for the INEPT refocusing period (see Equation (10)). The points where 5Jch is 1/3 and 2/3 are marked with dashed lines.

INEPT sequence. The delays t are set to 1/2Jhx required for the INEPT transfer and the delay set as necessary for the INEPT refocusing stage to enable the use of decoupling. The diffusion decoding gradient is applied as a bipolar pair during the first part of the INEPT transfer step. 

The INEPT experiment can be modified to allow the antiphase magnetization to be precessed for a further time period so that it comes into phase before data acquisition. The pulse sequence for the refocused INEPT experiment (Pegg et al., 1981b) is shown in Fig. 2.13. Another delay, A. is introduced and 180° pulses applied at the center of this delay simultaneously to both the H and the C nuclei. Decoupling during data acquisition allows the carbons to be recorded as singlets. The value of Z), is adjusted to enable the desired type of carbon atoms to be recorded. Thus, with D, set at V4J, the CH carbons are recorded at VsJ, the CH2 carbons are recorded and at VeJ, all protonated carbons are recorded. With D3 at %J, the CH and CH ( carbons appear out of phase from the CH2 carbons. 

What are the characteristic differences between basic INEPT and refocused INEPT experiments ... 

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. 

The first part, before the t period, of the experiment is identical to the HN(CO)CA-TROSY scheme. This step chooses solely the sequential pathway in an HN(CO)CA-TROSY manner. The chemical shift of the 13C nucleus is recorded during the t evolution period. The back-transfer route is, however, quite different. We transfer the desired coherence from 13C directly back to the 15N nucleus and remove the second 13C -> 13C INEPT step found in HN(CO)CA-TROSY and replace it with the HNCA like back-transfer step. The antiphase 2./N<> coupling then refocuses simultaneously with VNc during the 13C 15N back-INEPT step. Thus, the HN(CO)CANH-TROSY... 

The UV spectrum [/Imax 245, 296, 341, 352 (sh), and 368 nm] of TAN-999 (296) was very similar to that of (+)-staurosporine (295), indicating the presence of a similar indolo[2,3-fl]carbazole framework with a lactam function. This was also discernible from its IR spectrum with the presence of an NH band at v ax 3430 cm and an amide band at 1680 cm . The H-NMR spectrum of TAN-999 was also very similar to that of (+)-staurosporine, except for the presence of an additional methoxy group at S 3.95, resulting in the presence of a 1,2,4-trisubstituted aromatic ring instead of a 1,2-disubstituted aromatic ring. The location of this additional methoxy group was assigned to C-10, based on COSY, COSY, refocused INEPT, and... 

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. 

Polarization transfer, and its subsequent advantage of magnetization enhancement, has also been used. Thus 2D extension of refocused INEPT, with a selective... 

A refocused INEPT experiment100,104, also referred to as INEPTR105, which allows proton broad-band decoupling during acquisition, is performed as the following... 

To compensate for a broad range of coupling constants encountered within one sample, Wimperis and Bodenhausen115 developed Broadband-INEPT which includes six more pulses and four more delays, but the need for broadband-INEPT has apparently not yet been sufficiently felt in 29Si NMR just as better refocusing of INEPT CR116 went unnoticed in the organosilicon community. 

Non-refocused INEPT spectra can be used to determine both 29Si—XH and 1II—1II couplings (but not their signs) in a manner similar to that described earlier for SPT spectra. The INEPT spectra are measured with several r values chosen almost arbitrarily and then a simulation program119 is used to determine the exact values of the coupling constants120,121. 

FIGURE 16. 29Si INEPT spectra (not refocused) of bis(trimethylsilyl)adenine 11 measured without decoupling (bottom) and with selective decoupling of NH proton (top) during acquisition. The selective decoupling identifies the silicon with chemical shift S = 6.88 as Si—NH while that at iS — 14 77 js ]—silicon. Reproduced by permission of John Wiley Sons, Ltd... 

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