Crosspeak fine structure

Thus Fourier transformation in F will yield two peaks a positive peak at F = 2a — nJ and a negative peak at F = 2a + nJ. This is an antiphase doublet in F centered at frequency 2a and separated by 2nJ rad or / Hz. So we have a crosspeak that is an antiphase doublet in F2 (—2IyI observed in the FID) and an antiphase doublet in F (sin( 2aH)sin(7r/fi)), with both doublets showing a separation of /ab- This is the crosspeak fine structure shown in Figure 9.29. 

What about the COSY-35 experiment (Fig. 9.34) We can now show with product operators why it simplifies the crosspeak fine structure. Consider again the AMX spin system of a peptide residue in D2O ND-CHa-CH H -Y. For the crosspeaks shown in Figure 9.33 (left) let s focus on the lower one F = Hp/F2 = Hq, (H -> Hq, coherence transfer). We start the t period with -ly and write down the terms that result from J coupling, keeping in mind that there are two / couplings affecting H Jap and Jppr. 

A simple variant of the COSY experiment is COSY-35 (sometimes called COSY-45), in which the second 90° pulse is reduced from a 90° pulse to a 35° or 45° pulse (Fig. B.3). The result is that the fine structure of crosspeaks is simplified, with half the number of peaks within the crosspeak. This makes it much easier to sort out the coupling patterns in both dimensions and to measure couplings (active and passive) from the crosspeak fine structure. A more important variant of the COSY experiment is the DQF (double-quantum filtered)— COSY (Fig. B.4), which adds a short delay and a third 90° pulse. The INEPT transfer is divided into two steps antiphase I spin SQC to I,S DQC, and I,S DQC to antiphase S spin SQC. The filter enforces the DQC state during the short delay between the second and third pulses either by phase cycling or with gradients. DQF-COSY spectra have better phase characteristics and weaker diagonal peaks than a simple COSY, so this has become the standard COSY experiment. 

The spectrum of Fig. 5.11 also reveals within each diagonal and crosspeak fine structure that is equivalent to the structure seen within the one-dimensional multiplets. Whether such fine structure is always resolved depends on the experimental settings used in the acquisition of the data. Whilst in the illustrative... 

Absolute-value (magnitudemode) COSY-90 Simple and robust, magnitude processing well suited to automated operation. Phase-twisted lineshapes produce poor resolution which require strong resolution enhancement functions. Crosspeak fine structure not usually apparent. 

Phase-sensitive COSY-90 High-resolution display due to absorptive lineshapes. Crosspeak fine structure apparent J measurement possible. Diagonal peaks have dispersive lineshapes which may interfere with neighbouring crosspeaks. Requires high digital resolution to reveal multiplet structures. 

TOCSY Provides multistep (relayed) transfers to overcome ambiguities arising from crosspeak overlap. High sensitivity. In-phase lineshapes can provide correlations even in the presence of broad resonances. Number of transfer steps associated with each crosspeak not known, a priori. In-phase lineshapes tend to mask crosspeak fine-structure and may preclude J-measurement. 

The high crosspeak dispersion typically associated with heteronuclear shift correlation experiments alongside the lack of any requirement for well defined crosspeak fine structure means HMQC or HSQC experiments can be recorded with rather low digital resolution for routine applications, enhancing their time efficiency. An acquired digital resolution of 5 Hz/pt in the proton f2 dimension and only 50 Hz/pt in the heteronucleus fr dimension are generally sufficient to resolve correlations. Improved fi resolution can be achieved by linear... 

For identifying new natural products, TOCSY spectra are often less straightforward to analyze than COSY spectra, because a TOCSY crosspeak does not necessarily indicate that two protons are coupled with each other - the presence of a crosspeak only shows that two protons are part of the same spin system. Furthermore, TOCSY spectra may be significantly more crowded than COSY spectra, and TOCSY crosspeak intensity is often difficult to correlate with structural properties. Finally, the fine structure of TOCSY crosspeaks is much less amenable to detailed analysis than dqfCOSY crosspeaks as described below, the antiphase dqfCOSY crosspeaks contain information on proton multiplicities and scalar coupling constants, which TOCSY crosspeaks cannot provide. 

Figure 4 Part of the dqfCOSY spectrum of the ascaroside (11), a component of the Caenorhabditis elegans dauer pheromone. The fine structure of the four shown crosspeaks permits accurate determination and assignments of the geminal and all vicinal coupling constants of the two methylene protons (red).43...

Previous sections have already made the case for acquiring COSY data such that it may be presented in the phase-sensitive mode. The pure-absorption lineshapes associated with this provide the highest possible resolution and allow one to extract information from the fine-structure within crosspeak multiplets. However, it was also pointed out that the basic COSY-90 sequence suffers from one serious drawback in that diagonal peaks possess dispersion-mode lineshapes when crosspeaks are phased into pure absorption-mode. The broad tails associated with these can mask crosspeaks that fall close to the diagonal, so there is potential for useful information to be lost. The presence of dispersive contributions to the diagonal may be (largely) overcome by the use of the double-quantum filtered variant of COSY [37], and for this reason DQF-COSY is the experiment of choice for recording phase-sensitive COSY data. 

Figure 5.51. A region of the DQF-COSY spectrum of Andrographolide 5.5. The data were collected under conditions of high f2 resolution (1.7 Hz/pt) to reveal the coupling fine structure within the crosspeaks.

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... 

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