D TOCSY

An even better scenario is to run a series of 1-D TOCSY experiments in which the mixing time is systematically increased while the proton being irradiated is kept constant. To illustrate these experiments, we irradiated the anomeric proton from the /3-anomer of the glucose ring in lactose at 4.67 ppm and run a series of experiments with mixing times ranging from 20 ms to 400 ms. The results of these experiments are shown in a series of stacked plots in Figure 5.26. 

FIGURE 5.26 Stacked plots of a series of 1-D TOCSY experiments on /3-lactose with increasing mixing times. See text for an explanation. A portion of the 1H NMR spectrum is reproduced for reference in the bottom plot. 

Correlations to the anomeric proton of galactose (4.46 ppm), however, only show four interactions along the carbon axis. This result is consistent with the 1-D TOCSY of the galactose anomeric proton shown in Figure 5.26, where we find that coherence transfer does not travel beyond H-4 (G4). All six correlations are found if we start at H-4 (G4, 3.93 ppm) instead. As an exercise, try a similar process by starting on the carbon axis and tracing horizontally to the left to find HMQC-TOCSY correlations to protons. The anomeric carbon resonances are the easiest to try, but it is worthwhile to try others as well. 

Given are the structure and H, 13C/DEPT, COSY, 1-D TOCSY, 2-D TOCSY, HMQC, HMQC-TOCSY, HMBC, and ROESY spectra for raffinose. Confirm the structure, assign all protons and carbons, and show as many correlations as possible. 

Study of the ascidian L. patella (order Enterogona, family Didemnidae) yielded two new closely related cyclic peptide alkaloids namely lissoclinamide 9 389 and lissoclinamide 10 390 <2000T8345>. Their structures were determined by a combination of 2-D NMR, selective 1-D TOCSY, MS, and series-wound electrospray ionization (ESI)-MS (MSn) techniques, and the assignment of absolute stereochemistry was achieved by the hydrolysis of lissoclinamides followed by chiral thin layer chromatography. In the case of lissoclinamide 9, 389, NOE-restrained molecular dynamics studies were also performed confirming the proposed stereochemistry. 

The total correlation spectroscopy (TOCSY) techniques, which come in both 1- and 2-D versions, offer an alternative to 1-D spin decoupling and COSY methods for establishing through-bond connectivities. The important difference between the two is that TOCSY methods allow easy identification of isolated spin systems. For example, using our trusty morpholine compound once more, you can see that it is possible to identify the -CH2-CH2- spin system between the nitrogen and the oxygen atoms, these hetero-atoms, effectively isolating the protons from all others in the molecule. 

Whereas spin decoupling, COSY and TOCSY techniques are used to establish connectivities between protons through bonds, techniques that make use of the nuclear Overhauser effect (NOE), such as 1-D NOE and NOESY, 1- and 2-D GOESY, 1- and 2-D ROESY, can establish connectivities through space. Before looking at these techniques in detail, it s worth spending a little time considering the NOE phenomenon itself - in a nonmathematical manner, of course ... 

By reversing the order of polarization transfer steps, NOE contacts of signals hidden under the bulk of other resonances can be established in a ID TOCSY-NOESY [39] experiment (fig. 1(d)). The necessary requirement is that an efficient TOCSY transfer can be made to a particular proton from... 

Additional reagents and equipment for preparation of anthocyanin NMR samples (see Support Protocol 1), recording the 1-D H NMR spectrum (see Support Protocol 2), and recording the 2-D H-13C HMBC, 2-D H-13C HSQC, 2-D H- H DQF-COSY, 2-D H- H TOCSY, 2-D H- H NOESY, and 1-D 13C CAPT spectrums (Braun et al., 1998)... 

Today, it is possible to make complete assignments of all proton and carbon atoms in the NMR spectra of most isolated anthocyanins. These assignments are normally based on chemical shifts (8) and coupling constants (J) observed in 1-D H and l3C NMR spectra (Fig. FI.4.2), combined with correlations observed as cross-peaks in various homo- and heteronu-clear 2-D NMR experiments (see below for details on COSY, TOCSY, HSQC, HMBC, NOESY, and ROESY). 

The appearance of a 2-D TOCSY experiment resembles in all aspects a COSY. The FI and F2 axes are for proton the diagonal contains 1-D information and even the cross peaks have the same appearance. The difference here is that the cross peaks in a COSY are due to coupled spins while the cross peaks in the TOCSY spectrum arise from relayed coherence transfer. For long mixing times in a TOCSY spectrum, all spins within a spin system appear to be coupled. To appreciate the advantages of TOCSY, we continue with the disaccharide lactose, which has three distinct (i.e., separate) spin systems. 

Both the 1-D and 2-D versions of TOCSY find wide application in deciphering overlapping signals that originate from different spin systems. The 1-D version is particularly exciting as it enables us to walk through a spin system as we systematically increase the mixing time. 

Make as many correlations as possible for lactose using the 2-D TOCSY found in Figure 5.25. Compare your results for the glucose residue to the results that were found in the 1-D HOHAHA in Figure 5.26. 

If one is truly pushing the limits of NMR detection or has only an extremely time-sensitive sample, yet another strategy is preferred. Here a 1-D proton spectrum may be all that is realistically attainable. Possible additional experiments include a 1-D version of TOCSY and a 1-D version of COSY, also known as a homonuclear decoupling experiment. This applies to sample quantities down to hundreds of nanograms, which is currently the practical limit of NMR detection using commercially available equipment. 

Our goal is to relay or to transfer magnetization beyond directly coupled spins, thus enabling us to see correlations among nuclei that arc not directly coupled but within the same spin system. The experiment is called TOCSY (Totally Correlated SpectroscopY) and we will consider both the 2-D and 1-D versions. The pulse sequence for a 2-D TOCSY resembles our prototype 2-D experiment but, instead of a second 7r/2 pulse, we insert a mixing period during which the... 

For example, the J-couplings, multiplicities, and integrals found in the 1-D spectrum are to be tmsted more than the relative intensities of some cross peaks in the 2-D H- H TOCSY spectrum. 

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