Total correlation spectroscopy experiments

The 2D homonuclear TOCSY (Total Correlation SpectroscopY) experiment... 

The interpretation of ROESY experiments is not devoid of complications and hence, if the molecular tumbling frequencies allow it, NOESY experiments are to be preferred. One of the complications of ROESY experiments is the possible appearance of cross-peaks originating from scalar coupling instead of cross-relaxation. These crosspeaks have the same origin as the ones that are observed in TOCSY (total-correlation spectroscopy) experiments and can be distinguished from the cross-relaxation peaks because they have opposite sign. However, TOCSY crosspeaks may cancel with NOESY peaks involving the same protons. 

A related experiment TOCSY (Total Correlation Spectroscopy) gives similar information and is relatively more sensitive than the REIAY. On the other hand, intensity of cross peak in a NOESY spectrum with a short mixing time is a measure of internuclear distance (less than 4A). It depends on the correlation time and varies as . It is positive for small molecules with short correlation time (o r <<1) and is negative for macromolecules with long correlation time (wr >>l) and goes through zero for molecules with 1 Relaxation effects should be taken into consideration for quantitative interpretation of NOE intensities, however. 

We now want to turn to another experiment which, we must make clear at the start, does not have any relationship in theory to NOE experiments. In fact the theory is so complicated that we shall not say anything about it at all, but just refer you to one of the books in the Appendix. We are including this experiment because of its unique advantages when the spectrum has overlapping multiplets. It is called TOCSY, which stands for Total Correlation SpectroscopY (it has a second, more amusing name HOHAHA, standing for HOmonuclear HArtmann-HAhn), and is of particular use when oligosaccharides or peptides are under study. 

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 most important two-dimensional NMR experiments for solving stmctural problems are COSY (Correlation SpectroscopY), NOESY (Nuclear Overhauser Enhancement SpectroscopY), HSC (Heteronuclear Shift Correlation) and TOCSY (Total Correlation SpectroscopY). Most modem high-held NMR spectrometers have the capability to routinely and automatically acquire COSY, NOESY, HSC and TOCSY spectra. 

H is particularly important in NMR experiments because of its high sensitivity and natural abundance. For macromolecules, 1H NMR spectra can become quite complicated. Even a small protein has hundreds of 1H atoms, typically resulting in a one-dimensional NMR spectrum too complex for analysis. Structural analysis of proteins became possible with the advent of two-dimensional NMR techniques (Fig. 3). These methods allow measurement of distance-dependent coupling of nuclear spins in nearby atoms through space (the nuclear Overhauser effect (NOE), in a method dubbed NOESY) or the coupling of nuclear spins in atoms connected by covalent bonds (total correlation spectroscopy, or TOCSY). 

The vast literature associated with flavanoid chemistry precludes a discussion here but two valuable reviews have been published. The first reviews a number of spectroscopic techniques used for flavonoid analysis, with a strong emphasis on NMR spectroscopy (plus also mass spectrometry, vibrational spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, X-ray crystallography, and circular dichrosim (CD)) . The second review deals with NMR methods that have been successful in the characterization of phenolic acids and flavonoids from plant extracts that have not been separated or isolated as single components. The emphasis of the article is 2-D NMR methodology and a variety of experiments such as total correlated spectroscopy (TOCSY), COSY, nuclear Overhauser enhancement spectroscopy (NOESY) and heteronuclear multiple quantum correlation (HMQC) are discussed . 

All the spectroscopic approaches applied for structural characterization of mixtures derive from methods originally developed for screening libraries for their biological activities. They include diffusion-ordered spectroscopy [15-18], relaxation-edited spectroscopy [19], isotope-filtered affinity NMR [20] and SAR-by-NMR [21]. These applications will be discussed in the last part of this chapter. As usually most of the components show very similar molecular weight, their spectroscopic parameters, such as relaxation rates or selfdiffusion coefficients, are not very different and application of these methodologies for chemical characterization is not straightforward. An exception is diffusion-edited spectroscopy, which can be a feasible way to analyze the structure of compounds within a mixture without the need of prior separation. This was the case for the analysis of a mixture of five esters (propyl acetate, butyl acetate, ethyl butyrate, isopropyl butyrate and butyl levulinate) [18]. By the combined use of diffusion-edited NMR and 2-D NMR methods such as Total Correlation Spectroscopy (TOCSY), it was possible to elucidate the structure of the components of this mixture. This strategy was called diffusion encoded spectroscopy DECODES. Another example of combination between diffusion-edited spectroscopy and traditional 2-D NMR experiment is the DOSY-NOESY experiment [22]. The use of these experiments have proven to be useful in the identification of compounds from small split and mix synthetic pools. 

TOCSY (total correlation spectroscopy) is an extension of the COSY experiment, in which the coherence transfer is not limited to a single jump from one proton to another via a J coupling. Instead, coherence is spread out over an entire spin system of coupled protons via multiple /-coupled jumps. For example, in a string of carbons CHa-CHb-CHc-CHd, coherence can be transferred by the TOCSY mixing sequence from Ha to Hc or from Ha to Hj. Thus, crosspeaks will be observed at F% = va and I = i b, vc or (Fig. B.5). 

Other strategies that show great promise in reducing NMR acquisition time utilise methods to obtain multiple sets of data from one experiment through a concept known as time-shared evolution. An example of this process that should find utility in natural products elucidation was demonstrated by a pulse sequence called CN-HMBC.93 Traditionally, a separate 13C-HMBC and 15N-HMBC were acquired independently. However, the CN-HMBC allows both 13C- and 15N-HMBC spectra to be obtained simultaneously. By acquiring both data sets simultaneously, an effective 50% time reduction can be achieved.93 This approach has also been demonstrated for a sensitivity-enhanced 2D HSQC-TOCSY (heteronuclear multiple bond correlation total correlation spectroscopy) and HSQMBC (heteronuclear single quantum... 

The value of COSY stems from its dependence on the presence of spin coupling between the nuclei involved in the correlation. As we have seen, such coupling for protons is usually limited to three or four chemical bonds, hence provides some specificity that is helpful for structure elucidation. On the other hand, useful complementary information can be obtained from longer range interactions among a set of coupled nuclei. The standard method for obtaining such information is described by two acronyms—TOCSY (for total correlated spectroscopy, which best describes the aim of the experiment) and HOHAHA (for fiomonu-clear Hartmann-Hahn, which better describes the mechanisms employed). 

Various authors have used different names for Hartmann-Hahn-type experiments that emphasize distinct experimental or theoretical aspects. For example, heteronuclear Hartmann-Hahn transfer in liquids has been called coherence transfer in the rotating frame (Muller and Ernst, 1979), J cross-polarization (JCP Chingas et al., 1981), heteronuclear crosspolarization (Ernst et al., 1991), HEHAHA (heteronuclear Hartmann-Hahn transfer Morris and Gibbs, 1991), and hetero TOCSY (total correlation spectroscopy Brown and Sanctuary, 1991). Homonuclear Hartmann-Hahn transfer has been referred to as TOCSY (Braunschweiler... 

Homonudear Hartmann-Hahn sequences with delays were developed for clean TOCSY experiments (see Section X.B). Examples are delayed MLEV-17 (Griesinger et al., 1988), delayed DIPSI-2 (Cavanagh and Ranee, 1992), and clean CITY (computer-improved total-correlation spectroscopy Briand and Ernst, 1991). The MGS sequences (Schwendinger et al., 1994) are examples of broadband heteronuclear Hartmann-Hahn mbdng sequences with delays and variable rf amplitudes. 

In this chapter multiple-pulse sequences for homonudear Hartmann-Hahn transfer are discussed. After a summary of broadband Hartmann-Hahn mixing sequences for total correlation spectroscopy (TOCSY), variants of these experiments that are compensated for crossrelaxation (clean TOCSY) are reviewed. Then, selective and semiselective homonudear Hartmann-Hahn sequences for tailored correlation spectroscopy (TACSY) are discussed. In contrast to TOCSY experiments, where Hartmann-Hahn transfer is allowed between all spins that are part of a coupling network, coherence transfer in TACSY experiments is restricted to selected subsets of spins. Finally, exclusive TACSY (E.TACSY) mixing sequences that not only restrict coherence transfer to a subset of spins, but also leave the polarization state of a second subset of spins untouched, are reviewed. 

Heteronuclear Hartmann-Hahn sequences also effect homonuclear Hartmann-Hahn transfer, resulting in (heteronuclear and homonuclear) total correlation spectroscopy (TOCSY Bearden and Brown, 1989 Zuiderweg, 1990 Brown and Sanctuary, 1991 Ernst et al., 1991). Simultaneous heteronuclear and homonuclear magnetization transfer can be beneficial in relayed transfer experiments (Gibbs and Morris, 1992 Tokles et al., 1992 Majumdar et al., 1993). However, as pointed out by Ernst et al. 

ROESY-TOCSY experiment Selective inverse detection of C-H correlation Scalar heteronuclear recoupled interaction by multiple pulse Simulation program one Selectively inverted soft PICSY Singular value decomposition Tailored correlation spectroscopy Triple-resonance J cross-polarization Total correlation spectroscopy TOCSY-ROESY experiment TOCSY without NOESY... 

The structures of the natural products, ceratospongamides from marine red alga (Rhodophyta) Ceratodictyon spongiosum, which each consist of two L-phenylalanine residues, one (L-isoleucine)-methyloxazoline residue, one L-proline residue, and one (L-proline)thiazole residue, were established through extensive NMR experiments, including heteronuclear multiple quantum correlation total correlated spectroscopy (HMQC-TOCSY), and... 

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