Spectral Editing in the Third Dimension

The coherence pathway leading to NOESY cross peaks was illustrated in Eq. 11.88, where we showed that with appropriate phase cycling we had only a term ASy (with A measuring the extent of NOE polarization transfer). Double quantum and zero quantum coherences are then generated by the pathway 

FIGURE 12.12 (a) NOESY spectrum (500 MHz) of staphylococcal nuclease, uniformly enriched in 15N. (b) Two adjacent planes from a NOESY-HMQC experiment with staphylococcal nuclease, separated by 15N chemical shift difference of 0.9 ppm. Adapted from Marion et a/.118 

As we have seen, the DQC and ZQC evolve at frequencies f s flT. The 180° proton (I,S) pulse, which is a standard part of the HMQC sequence, refocuses proton chemical shifts that evolve during t2 and interchanges ZQC and DQC. The final 90° T (15N) pulse converts SxTy to SXTZ, and this antiphase 5 magnetization evolves during the final 1/2/ period to in-phase S magnetization, which is detected while T is broadband decoupled. 

Application of NOESY-HMQC to the nuclease sample replaces the 2D NOESY spectrum of Fig. 12.12a by a 3D spectrum that can be displayed in a cube but is more easily interpreted as a set of planes, as indicated schematically in Fig. 12.14. Two of the NOESY planes obtained in the nuclease experiment are illustrated in Fig. 12.126. Clearly, the 3D experiment is successful in editing the uninterpretable spectrum of Fig. 12.12a into manageable pieces. Moreover, each NOESY plane is labeled by the chemical shift of the 15N that is coupled to one of the protons, so additional useful information may be available if that 15N chemical shift can be related to structural features in the molecule. Note that a proton not coupled to any 15N generates an axial peak, rather than a cross peak, in the 3D spectrum, but phase cycling is used to remove axial peaks. Also, displays other 

FIGURE 12.13 Pulse sequence for the three-dimensional experiment NOESY-HMQC. The first three 90° H pulses constitute the usual NOESY sequence, with mixing time r.The 180° 15N pulse at time 3 removes the effect of H-15N couplings. The H and 15N pulses at times 7, 9, 11, and 13 constitute the HMQC sequence, with the pulse at time 7 serving as part of both sequences. 

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