The TOCSY experiment

TOCSY is a rotating frame experiment designed to detect scalar connectivities over a large range of Ju values, especially useful for small (ca. 1 Hz) J/j values. The most common pulse sequence is shown in Fig. 8.2F [22], The spin lock is achieved by applying a train of relatively high power pulses (the 

MLEV17 sequence being one of the most used sequences [23]) in such a way as to continuously refocus the chemical shift evolution of the various signals in the xy plane. Analogously to ROESY experiments, the magnetization during the spin-lock (mixing) time disappears with T p (i.e. essentially Tj, see Section 3.4). It follows that coherence transfer in the xy plane, which is built up with a sin(7T J/jt) function, also decreases with time constant p p — p[p + p p)/2  

A TOCSY experiment on a paramagnetic molecule is reported in Fig. 8.7 for the 5Cl-Ni-SAL-MeDPT complex [5]. Cross peaks between signals with linewidths of the order of 100 Hz were easily detected. In particular, the couplings of each aromatic proton with its neighbors are evident. TOCSY cross peaks between signals with similarly broad lines can also be detected in proteins (see Fig. 8.18) [24]. 

In TOCSY experiments, the problem of overheating the sample is more serious than in ROESY experiments because of the large irradiation energy required by the spin-lock pulse. Each individual component of the pulse train must have enough power to irradiate the whole spectral window of interest. Spin-lock sequences different from the MLEV17 sequence, that may alleviate the heating problem, 

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Total correlation spectroscopy (TOCSY) is similar to the COSY sequence in that it allows observation of contiguous spin systems [35]. However, the TOCSY experiment additionally will allow observation of up to about six coupled spins simultaneously (contiguous spin system). The basic sequence is similar to the COSY sequence with the exception of the last pulse, which is a spin-lock pulse train. The spin lock can be thought of as a number of homonuclear spin echoes placed very close to one another. The number of spin echoes is dependent on the amount of time one wants to apply the spin lock (typically 60 msec for small molecules). This sequence is extremely useful in the identification of spin systems. The TOCSY sequence can also be coupled to a hetero-nuclear correlation experiment as described later in this chapter. 

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. 

As an illustration of the use of the half-filter element for the measurement of small coupling constants, consider the TOCSY experiment... 

The purpose of the C(o i)-half-filter is to start the TOCSY experiment only with the magnetization of protons bound to C. No further C pulses are applied after the start of the evolution time t. For the description of the multiplet fine-structure of the resulting cross-peaks, it is instructive to consider a 3-spin system with the operators H, and C denoting the spins of two protons and one carbon. Starting from antiphase magnetiza-... 

Fig. 3.23 The 2D spectrum of peracetylated glucose from a 2D TOCSY experiment. The same sample has been used and the expansion is the same as for the 2D phase-sensitive, DQ-filtered COSY spectrum (Fig. 3.21). Note the additional cross peaks obtained with the TOCSY experiment.

Any one proton behaves as if it is coupled to all protons of the spin system as it is coupled to at least one member of the group and all the other protons are strongly coupled to each other. This way of understanding the TOCSY scheme comes closest to the actual theoretical explanation, so hopefully it will give you a feeling for what goes on in the TOCSY experiment. 

Up to a 90° phase shift in both frequency dimensions (which can be easily corrected for), the two resulting spectra are almost identical. Because the noise of the two spectra is uncorrelated (Cavanagh and Ranee, 1990b), it is increased only by a factor of yfl if the two spectra are added, whereas the signal is increased by a factor of 2. Overall, a sensitivity improvement of /2 can be achieved in the PEP version of the TOCSY experiment, relative to experiments where one of the two transverse magnetization components is simply eliminated. 

TOCSY (Total Correlation Spectroscopy) is another important homonuclear 2D correlation experiment where correlations arise due to the presence of homonuclear scalar coupling.In the standard COSY experiment, crosspeaks appear for spins in which the scalar coupling occurs over typically two to four bonds. In the TOCSY experiment crosspeaks can appear for spins separated by many more bonds as long as they are part of a contiguous network of coupled spins. The correlations are effected by the application of a series of low-power rf pulses termed the spin-lock. The duration of the spin-lock period determines the extent to which the correlations are propagated through the spin system. The TOCSY experiment is a useful complement to the COSY methods for the elucidation of complex structures. 

As mentioned in Section 7-7b, the ID version of the TOCSY experiment is especially useful for larger molecules that possess complicated and overlapping spin systems. The 2D TOCSY spectra of classes of molecules such as oligosaccharides can be very difficult to interpret. ID TOCSY experiments, however, permit the mapping of entire spin systems when the chemical shift of just one member of the system is distinct. An example is the anomeric (H-1) protons of oligosaccharides, which are situated at higher frequency (down-field) from the carbinol protons. 

The first step in the assigmnent scheme would be to determine the chemical shifts of the protons contained in each residue. This collection of intra-residue chemical shifts is called a spin system. Spin systems are usually defined using a TOCSY experiment (total correlation spectroscopy) (Bax and Davis, 1985). In the TOCSY experiment magnetization is passed from one spin to another by scalar coupling in much the same way a baton is passed in a relay race (see Figure 3.8, top). While the magnetization exists on a spin it is possible to record the chemical shift of that particular spin. Under favorable conditions, the chemical shifts of all of the spins in an amino acid can be obtained. 

The TOCSY experiments provide the chemical shifts of the aliphatic protons. Depending on the residue type, it is usually possible to assign a spin system to a class of amino acids. In favorable cases, it may be possible to uniquely identify the residue type of a spin system. For example, residues like Asn, Asp, Glu, and Gin are easily distinguished from hydrophobic residues based on characteristic proton chemical shifts. A number of residues, such as Ser, Ala, and Thr, can be uniquely assigned from their characteristic proton shifts. Note that if any of these residues... 

Before moving on we briefly examine the key characteristics of the different experiments and consider why these might be of interest to the research chemist. Table 5.5 summarises the most significant attributes, some of which have already been introduced whilst others are expanded in the sections that follow. Whilst the TOCSY experiment is not strictly a member of the COSY family, its information content is so closely related to that of COSY it has also been included in the table. 

It has also been mentioned that TOCSY results in the net transfer of in-phase magnetisation, meaning the cancellation effects from antiphase multiplet fine-structure associated with COSY are not a feature of TOCSY. Such cancellation can be problematic for molecules that posses large natural linewidths, for example (bio)-polymers, but may also prevent the observation of COSY peaks in the spectra of small molecules that have complex multiplet structures which may cancel under conditions of poor digital resolution. In these cases the TOCSY experiment may be viewed as the more sensitive option because of the greater crosspeak intensities. The lack of antiphase structure also means spectra... 

There are essentially two approaches based on composite-pulse methods in widespread use for the practical implementation of the TOCSY experiment (Fig. 5.68). The first of these [51] (Fig. 5.68a) is based on the so-called MLEV-17 spin-lock, in which an even number of cycles through the MLEV-17 sequence are used to produce the desired total mixing period. To ensure the collection of absorption-mode data, only magnetisation along a single axis should be retained, so it is necessary to eliminate magnetisation not parallel to this before or after the transfer sequence. In this implementation, this is achieved by the use of trim-pulses applied for 2-3 ms along the chosen axis. 

The first of these arises when the long spin-lock pulse acts in an analogous fashion to the last 90" pulse of the COSY experiment so causing coherence transfer between J-coupled spins. The resulting peaks display the usual antiphase COSY peak stmcture and tend to be weak so are of least concern. A far greater problem arises from TOCSY transfers which arise because the spin-lock period in ROESY is similar to that used in the TOCSY experiment (Section 5.7). This may, therefore, also induce coherent transfers between J-coupled spins when these experience similar rf fields, that is, when the Hartmann-Hahn matching condition is satisfied. Since the ROESY spin-lock is not modulated (i.e. not a composite pulse sequence), this match is restricted to mutually coupled spins with similar chemical shift offsets or to those with equal but opposite... 

The ability to spin-lock magnetisation along a predefined axis plays an essential role in the TOCSY experiment, and Section 5.7.1 has already introduced the idea that a single continuous pulse or a series of closely spaced... 

Homonuclear correlation experiments are not just restricted to the standard COSY experiment, but also include the TOCSY and INADEQUATE experiments. The separation of TOCSY and INADEQUATE from the homonuclear COSY experiment is based on the different coherence evolution and transfer processes involved. Thus the TOCSY experiment is based on cross-polarization in contrast to the polarization transfer used in the homonuclear COSY experiments. INADEQUATE experiments are characterized by the double quantum state of two scalar-coupled nuclei during the tl period such that the second dimension (fl) is scaled into a double quantum frequency. Nevertheless these experiments can all be considered together because they are based on homonuclear scalar coupling and the fl and f2 dimension of the corresponding 2D spectra are related to the same nucleus. 

The HOHAHA or TOCSY experiment [5.150, 5.151] has proved a popular alternative in many applications to the main homonuclear correlation experiment for sensitive nuclei, the basic COSY experiment. Both the HOHAHA and the TOCSY experiment are based on the principal of isotropic mixing but differ in the type of spinlock sequence used. Nevertheless they may be considered together and for convenience in the following discussion the expression TOCSY experiment will be used for both sequences. The TOCSY experiment uses cross polarization for the coherence... 

Note depending upon the speed of the computer the calculation of the TOCSY experiment can take more than 8 minutes. 

The advantages of the TOCSY experiment have led to the implementation of spinlock sequences in heteronuclear correlation experiments to detect additional homonuclear coupling interaction. In the l C, IR TOCSY-HMQC experiment in addition to cross peaks from one-bond connectivities between a DC and a IR nucleus there are correlation peaks to protons which are coupled to the primary proton by homonuclear H, H coupling. The advantage of this method is that it allows the differentiate between overlapped iR signals of different spin systems provided that the spin systems differ by the carbon shift of at least one carbon atom. This approach is shown by the simulations in the Check it 5.4.2.4. 

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