CT-COSY experiments

Intensity-based methods. 1H— H RDCs have been obtained by analyzing the intensity ratios of the diagonal and cross-peaks in a series of 2D CT COSY spectra.177 This method can only be applied to resolved resonances, for example, those of anomeric protons.149 Similar limitations apply to /-modulated ID directed COSY,153,178 which uses selective 180° pulses to produce a series of ID spectra for each pair of coupled spins. This approach has recently been extended to include additional selection blocks yielding a versatile method for the measurement of coupling constants in compounds with severely overlapping proton resonances such as those found in carbohydrates.154 The problem of overlapping resonances can also be resolved by involving 13C nuclei, as demonstrated on natural abundance (13C COSMO HSQC)32 or uniformly 13C isotopically enriched carbohydrates (2D-HSQC-(sel C, sel H)-CT COSY experiment).73,158... 

A 2D-HSQC-carbon selective/proton selective-constant time COSY, 2D-HSQC-(sel C, sel H)-CT COSY experiment, which is applicable to uniformly C istopically enriched samples (U- C) of oligosaccharides or oligonucleotides, has been proposed by Martin-Pastor et al for the measurement of proton-proton RDC in crowded regions of 2D-spectra. In addition, a heteronuclear constant time-COSY experiment, C- C CT COSY, has been proposed for the measurement of one bond carbon-carbon RDC in these molecules. 

Fig. 7.3 Pul se sequence of the constant-time (ct) HNCA-COSY experiment. The passive spin is C. Obviously, C is affected by only one 180° pulse,...

Fig. 7.4 Schematic multiplet pattern of a C ,Hn correlation in the constant-time (ct) HNCA-COSY experiment leaving the C untouched. The 3J(Hn,C ) coupling can be derived from this experiment.

The pulse sequence which is used to record CH COSY involves the H- C polarisation transfer which is the basis of the DEPT sequence and which increases the sensitivity by a factor of up to four. Consequently, a CH COSY experiment does not require any more sample than a H broadband decoupled C NMR spectrum. The result is a two-dimensional CH correlation, in which the C shift is mapped on to the abscissa and the H shift is mapped on to the ordinate (or vice versa). The C and shifts of the Hand C nuclei which are bonded to one another are read as coordinates of the cross signal as shown in the CH COSY stacked plot (Fig. 2.14b) and the associated contour plots of the a-pinene (Fig. 2.14a and c). To evaluate them, one need only read off the coordinates of the correlation signals. In Fig. 2.14c, for example, the protons with shifts Sh = 1.16 (proton A) and 2.34 (proton B of an AB system) are bonded to the C atom at 5c = 31.5. Formula 1 shows all of the C77 connectivities (CT/bonds) of a-pinene which can be read from Fig. 2.14. 

To overcome the problem of low dispersion of sugar resonances in nucleotides Hu et al. proposed to incorporate HCN transfer into HCCH-type sequences resulting in 3D MQ-HCN-CCH-TOCSY and 3D MQ-HCN-CCH-COSY experiments. The coherence transfer pathway starts with HT-CT transfer followed by an out-and-back transfer to N1/N9 for indirect detection and subsequent transfer through network. As a result the CCH spectra are additionally resolved with chemical shift. During the C -N1/N9 transfer MQ HT/CT coherence is active to reduce the relaxation losses due to relaxation. The experiments were successfully applied to a 23-mer RNA aptamer. 

The application of three and more gradients is necessary if multiquantum states are to be suppressed as in the double-quantum filtered COSY experiment or if CT pathways in heteronuclear spin systems should be selected in sequences which include magnetization transfer steps. 

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