NOESY zero mixing time

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. 

NOESY was performed at 32 °C on a VXR-500 spectrometer operating at a proton frequency of 500 MHz. The protein was dissolved in 20 mM sodium phosphate, pH 7.5 (direct pH meter reading), 100 mM NaCl, 5 mM DTT in D2O. The protein concentration was 2 mM. The data was acquired in the hypercomplex mode with a mixing time of 150 ms (Jeener et al., 1979 Macura Ernst,1980). The spectral width was 7200 Hz in both dimensions. 2048 complex points in the t2 dimension and 256 complex points in the tl dimension were acquired. 96 transients were collected for each FID. Data processing was performed on a Sun Sparc 10 station using VNMR software from Varian. The time domain data were zero-filled once and multiplied by shifted sinebell or Gaussian functions before Fourier transformation in both dimensions. Chemical shifts were referenced to internal sodium 3-(trimethylsilyl)-propionate-2,2,3,3-d4. 

Figure 3. TOCSY and NOESY contour plots are shown for Ppep-4 for the amide/aromatic region in Hj0(90%)/Dj0(10%) (left) and for the aH region in DjO (right). Ppep-4 peptide concentration was 20 mg/mL in 20 mM potassium phosphate, pH 6.3, 40°C. The NOESY mixing time was 0.2 s, and the TOCSY spin iock time was 40 ms. The data were zero-fiiied to 1024 in t1. The raw data were multiplied by a 30° shifted sine-squared function in t1 and t2 prior to Fourier transformation.

Figure 8.33. The 2D NOESY spectrum of the terpene andrographolide 8.12 in DMSO at 25°C. The spectrum was recorded with a 600 ms mixing time and a recovery delay of 1.5 s. 2K data points were collected for 512 increments of 16 scans, using TPPI fi quadrature detection. Data were processed with a squared cosine-bell window in both dimensions with a single zero-fill in f[.

These purging pulses can be used to generate pure z-magnetization without contamination from zero-quantum coherence by following them with a 90°(y) pulse, as is shown in the NOESY sequence (b). Zero-quantum coherences present during the mixing time of a NOESY experiment give rise to troublesome dispersive contributions in the spectra, which can be eliminated by the use of this sequence. 

A more direct approach to the elimination of zero-quantum interference is via the use of swept-frequency inversion pulse applied in the presence of a field gradient, which destroys the zero-quantum contributions in a single transient. The application of this has already been described in Section 5.7.3 with a view to obtaining pure lineshapes in TOCSY spectra, and the operation of the filter is itself described in Section 10.6 and so will not be explored here. The implementation of this in the 2D NOESY sequence is illustrated in Fig. 8.37 and requires the incorporation of the filter within the usual mixing time, followed by a purging gradient. The effective suppression of zero-quantum interference is illustrated in the high-resolution NOESY spectra of Fig. 8.38 in which spectrum (a) shows clear anti-phase... 

As for the 2D NOESY data described above, spectra are vulnerable to interference from undesirable zero-quantum contributions between coupled spins this is apparent, for example, in trace (c) of Fig. 8.42 between H4 and H5. This can be especially problematic when shorter mixing times are employed, as in the generation of NOE build-up curves [35], and again the inclusion of the swept-frequency/gradient zero-quantum filter should prove beneficial. The complete selective ID NOESY sequence incorporating this and employing the optimised DPFGSE selection procedure is presented in Fig. 8.43, and an illustration of the improvements provided by the filter may be seen in Fig. 8.44 where the removal of unwelcome anti-phase dispersive contributions between coupled spins is apparent in (b). 

NOESY sequence incorporating zero-quantum suppression. The mixing time contains non-selective 180° nulling pulses bracketed by opposing purging gradients spaced throughout as shown, followed by the ZQ filter. 

To establish interpulse delays for two-dimensional NMR experiments, it is frequently convenient to run a very quick proton Tj relaxation measurement. Given the sensitivity of modem spectrometers, this can usually be done with only a single or a few transients for each of the r values in the series, and typically requires 10 min or less. By visual inspection, the Tj relaxation time can be estimated from the r value at which response intensity is zero. A knowledge of the Tj relaxation time is also useful for establishing mixing times for NOESY and ROESY experiments. 

---