instrumentation used. gradient strength ratio 80 : 20.1, g1 with sign alternation according to echo/anti-echo mode initial value for ¢; evolution: 3 μs initial value for f, evolution: 3 μs increment for f, evolution: 1/[2-sw1] increment for f, evolution: 1/[2-sw2] ¹³C decoupler pulse for GARP decoupling [70 μs, 11.5 dB] ¹⁵N decoupler pulse for GARP decoupling [200 μs, 19 dB] ds: 16

5. Processing

Apply zero-filling to 128 real data points in F and to 256 real data points in F; to obtain a matrix of 1024x128x256 real data points. This would result in a huge file of processed real data. Since only the aliphatic proton region is of interest, use strip transformation in F3; (600 points). Application of forward linear prediction (20 coefficients) for both F, and F, results in somewhat better resolved 3D spectra. Use Gaussian multiplication [gb = 0.1, lb = –3 Hz] in F; and a 7/2-shifted squared sinusoidal window in the other dimensions. For the data file described, extensive experimentation with different window functions may be worthwhile. Phase correction and base-line correction may be necessary for all dimensions. Further details are very dependent on the particular software you use to process such a 3D data file. Reference the ¹³C dimension (F2) using the =-scale procedure described in the introduction to this chapter using the DSS signal set to 6, = 0.

6. Result

The figures show results from the 3D spectrum obtained on a DRX-600 spectrometer with a multinuclear inverse z-gradient triple resonance probe-head (fixed third channel on ¹³C). To demonstrate the large amount of information, we first show in a the projection along the 1,3 planes of the cuboid. The assignment of all these peaks is difficult for the reasons already discussed in Experiment 15.17. As an example for NOEs starting from an H, proton, we show in b the H,H strip of the amino acid phenylalanine F-4 taken from the corresponding plane at & = 55.3. As an example of NOEs starting from B-protons, we show a strip for valine V-26 in c. Figure b shows the diagonal peak for F-4 at 6, = 5.63 and trivial NOEs to the same-group NH proton at 5, = 8.62 and to the aromatic ortho protons at 6; = 7.1, as well as to the B-protons at Ou = 3.03 and 2.85. There is an NOE peak to an NH at dj = 9.3, which belongs to the following amino acid valine V-5. However, there are also additional NOE cross signals at dy = 4.94, 1.08 and 0.71, which have to be identified in an iterative structural calculation. In Figure ¢ we find the diagonal peak of the valine V-26 B-proton at dj = 2.33 taken from the plane at & = 30.9. There are NOE cross-peaks to the same-group NH proton at &; = 8.13 and to the following amino acid lysine K-27 at dj; = 8.59.

There are additional trivial NOEs to the same-group H, proton and to the same-group methy! protons.