TOCSY pulse

The Inverted Direct Response (IDR)-HSQC-TOCSY pulse sequence is shown in Fig. 18 [58]. The experiment begins with an HSQC segment... 

Fig. 10.18. IDR (Inverted Direct Response)—HSQC-TOCSY pulse sequence. The experiment first uses an HSQC sequence to label protons with the chemical shift of their directly bound carbons, followed by an isotropic mixing period that propagates magnetization to vicinal neighbor and more distant protons. The extent to which magnetization is propagated in the experiment is a function of both the size of the intervening vicinal coupling constants and the duration of the mixing period. After isotropic mixing, direct responses are inverted by the experiment and proton detection begins.

However, if side-chain carbon assignments are wanted, C(CC)(CO)NH experiments [33] that start directly with carbon magnetization and transfer it further to the amide proton for detection are available. If protonated substituents, for example methyl groups, have been introduced into the otherwise perdeuterated protein, the usual HC(C)(CO)NH-TOCSY pulse sequence can be used to obtain the proton chemical shifts. These protons can provide a small number of NOEs that, together with residual dipolar couplings and the secondary structure identification from chemical shifts, make the determination of the global fold of large proteins possible. 

The NOESY and TOCSY polarization transfers can also be arranged so that two NOESY steps are interrupted by one TOCSY transfer. This is useful for situations when a proton which is intended as a starting point for a ID TOCSY-NOESY experiment cannot be selectively excited, nevertheless it has a NOE contact to an isolated proton. The ID NOESY-TOCSY-NOESY sequence [72] (fig. 4(b)) is obtained by appending another NOESY step to the ID NOESY-TOCSY pulse sequence of fig. 1(c). The last NOESY step can be either selective or nonselective depending whether a selective 180° pulse is applied after the nonselective 90° pulse at the end of the TOCSY transfer. 

In this chapter, the discussion will be focused on the ID TOCSY (TO-tal Correlation SpectroscopY) [2] experiment, which, together with ID NOESY, is probably the most frequently and routinely used selective ID experiment for elucidating the spin-spin coupling network, and obtaining homonuclear coupling constants. We will first review the development of this technique and the essential features of the pulse sequence. In the second section, we will discuss the practical aspects of this experiment, including the choice of the selective (shaped) pulse, the phase difference of the hard and soft pulses, and the use of the z-filter. The application of the ID TOCSY pulse sequence will be illustrated by examples in oligosaccharides, peptides and mixtures in Section 3. Finally, modifications and extensions of the basic ID TOCSY experiment and their applications will be reviewed briefly in Section 4. 

The pulse sequence for ID TOCSY is a ID modification of the original TOCSY experiment [2] introduced by Braunschweiler and Ernst. The TOCSY experiment was also referred to as HOHAHA (which stands for HOmonuclear HArtman-HAhn) by Bax and Davis [3]. The ID TOCSY experiment was proposed by Bax and co-workers [4, 5], and by Kessler et al. [6]. The essential features of the pulse sequence involve the use of selective excitation of a desired resonance, followed by a homonu-clear Hartman-Hahn (or isotropic) mixing period [2, 7]. That is, the unit -Pnonsei - in the 2D TOCSY pulse sequence is replaced by Fsei -where P stands for a pulse (or pulses), ti is the evolution period in the 2D experiment and r is a fixed delay. 

Fig. 1. The ID TOCSY pulse sequence, (a) The Bax version and (b) The Kessler version. The phase 0 for the last 90° pulse and the receiver is rotated synchronously along the four...

In the previous sections, only the basic, non-gradient ID TOCSY pulse sequence, its experimental aspects and applications were described. In the following, the more recent modifications and extensions of the basic pulse sequence and their applicability to spectral assignments and structural elucidation will be briefly reviewed. Some of these more sophisticated techniques may not be as readily implementable as the basic ID TOCSY experiments, and thus have not yet found wide applications in routine practice. 

The ID TOCSY pulse sequence can be converted into a 2D experiment by inserting an incremented delay between the excitation pulse and the spinlock sequence as shown in the scheme below. In Check it 5.4.2.3 the 2D IR TOCSY experiment is calculated and the results compared with the corresponding IH COSY experiment without and with relay step to highlight the additional correlation peaks in the TOCSY spectrum. 

Fig, 8.12 Homonuclear TOCSY pulse sequence neighbor. The extent to which coherence will be [77-79]. Isotropic mixing is provided by a pulse transferred along a series of coupled, homo-... 

Fig. 20. (Top) SPIROE-TOCSY pulse sequence comprised of proton saturation, adabatic CHIRP transfer, and proton magnetization buildup during and TOCSY using the DIPSI-2 mixing sequence. (Bottom) 2D SPIROE-TOCSY performed in deuterated 1,1,2,2-tetra-chloroethane. Broken line indicates the diagonal. The inset shows the region of the camp-hanic ester protons. The total experiment time is only 4.5min. (Courtesy of Herve Desvaux. Reprinted from ref. 313 with permission. Copyright 2004, Elsevier SAS.)...

Fig. 7. HMQC-TOCSY pulse sequence described by Lerner and Bax (1986). Proton magnetization is manipulated and labeled with the chemical shift of the directly attached in a fashion identical to the HMQC experiment (see Fig. 1). After a refocusing period, A, proton magnetization is propagated from the directly attached proton to its vicinal neighbors using an MLEV-17-based isotropic mixing period. In the original work, the receiver was enabled and broadband heteronuclear decoupling initiated after a fixed delay, A = 1/2( Jch) to suppress the direct responses. Alternative considerations regarding direct responses in an HMQC-TOCSY spectrum are discussed in the text...

An experiment that actually preceded SIMBA, but which probably has somewhat lower utility, was the selective ID HMQC-TOCSY experiment described by the authors (Crouch et al. 1990c). The selective experiment differs from the HMQC-TOCSY pulse sequence only in the replacement of the final C 90° pulse by a selective pulse. Initially, we utilized a square pulse in this application but, obviously, the improvement in the SIMBA experiment by using a Gaussian 270° or an E-BURP-2 pulse would be realized in this experiment as well. To the best of our knowledge, there have been no applications of this experiment to alkaloids reported in the literature. 

Fig. 5 PANSY-TOCSY pulse sequence for simultaneous acquisition of two-dimensional H-C HETCOR and H-H TOCSY spectra...

An example of the Hadamard encoded PANSY-TOCSY pulse sequence is shown in Fig. 18. The conventional free evolution is replaced with a Hadamard encoding pulse as can be appreciated by comparing Figs. 17 and 5. The PANSY-TOCSY spectra of inosine recorded with Hadamard encoding in just 22 s are shown in Fig. 19. 

Fig. 18 PANSY-TOCSY pulse sequence with Hadamard encoding (to be compared with the conventional pulse sequence shown in Fig. 5)...

Figure 24 schematically illustrates the assembly of 2D-HMQC and 2D-COSY pulse sequences to form a 3D-HMQC-TOCSY pulse sequence the preparation period (relaxation delay and first pulse) in the COSY pulse sequence is replaced by the pulse sequence elements (excluding data acquisition) from the HMQC sequence to create the 3D pulse sequence on the bottom of the figure. To perform the 3D experiment, an array of ni2 FlDs is collected with 2 incremented this experiment is repeated nil times with ti incremented. The resulting 3D time-domain spectmm is Fourier transformed, first with respect to 3, then with respect to 2, and finally, with respect to 1 to produce the spectmm in Figure 24(c). 

In the first of these methods, the HCaCx-HH-TOCSY pulse sequence is used to perform sequential INEPT transfers as in the HCaCx experiment, as follows ... 

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