Antiphase coherence

SELINQUATE (Berger, 1988) is the selective ID counterpart of the 2D INADEQUATE experiment (Bax et al., 1980). The pulse sequence is shown in Fig. 7.4. Double-quantum coherences (DQC) are first excited in the usual manner, and then a selective pulse is applied to only one nucleus. This converts the DQC related to this nucleus into antiphase magnetization, which is refocused during the detection period. The experiment has not been used widely because of its low sensitivity, but it can be employed to solve a specific problem from the connectivity information. 

The SELINCOR experiment is a selective ID inverse heteronuclear shift-correlation experiment i.e., ID H,C-COSYinverse experiment) (Berger, 1989). The last C pulse of the HMQC experiment is in this case substituted by a selective 90° Gaussian pulse. Thus the soft pulse is used for coherence transfer and not for excitation at the beginning of the sequence, as is usual for other pulse sequences. The BIRD pulse and the A-i delay are optimized to suppress protons bound to nuclei As is adjusted to correspond to the direct H,C couplings. The soft pulse at the end of the pulse sequence (Fig. 7.8) serves to transfer the heteronuclear double-quantum coherence into the antiphase magnetization of the protons attached to the selectively excited C nuclei. 

Fig. 10.14. Gradient-enhanced HMQC pulse sequence described in 1991 by Hurd and John derived from the earlier non-gradient experiment of Bax and Subramanian. For 1H-13C heteronuclear shift correlation, the gradient ratio, G1 G2 G3 should be 2 2 1 or a comparable ratio. The pulses sequence creates heteronuclear multiple quantum of orders zero and two with the application of the 90° 13C pulse. The multiple quantum coherence evolves during the first half of ti. The 180° proton pulse midway through the evolution period decouples proton chemical shift evolution and interchanges the zero and double quantum coherence terms. Antiphase proton magnetization is created by the second 90° 13C pulse that is refocused during the interval A prior to detection and the application of broadband X-decoupling.

The last 90° pulse on 13C acts as a purge pulse for the undesired dispersive magnetization.47,48 The function of the pulse is to convert any magnetization remaining antiphase with respect to the 13C spin into unobservable multiple-quantum coherence. This will provide cross peaks with pure lineshapes and with higher resolution, and consequently establishes reliable determination of coupling constants.47,48... 

The first part, before the t period, of the experiment is identical to the HN(CO)CA-TROSY scheme. This step chooses solely the sequential pathway in an HN(CO)CA-TROSY manner. The chemical shift of the 13C nucleus is recorded during the t evolution period. The back-transfer route is, however, quite different. We transfer the desired coherence from 13C directly back to the 15N nucleus and remove the second 13C -> 13C INEPT step found in HN(CO)CA-TROSY and replace it with the HNCA like back-transfer step. The antiphase 2./N<> coupling then refocuses simultaneously with VNc during the 13C 15N back-INEPT step. Thus, the HN(CO)CANH-TROSY... 

Although, the MP-HNCA-TROSY experiment alone can yield sequential assignment, it can be also used concomitantly with the HNCA-TROSY experiment. This strategy is explained later, but let us first focus on the coherence transfer efficiency of the MP-HNCA-TROSY experiment. The transfer functions for the antiphase experiment (the efficiency for the in-phase experiment is practically the same) are calculated according to Eqs. (10) and (11) for the intraresidual... 

The basis for the discrimination between protons directly bound to a specific NMR-active heteronucleus, 1H-X, and all others lies in the development of antiphase coherence of these protons with respect to the heteronucleus, under the influence of the scalar coupling Vh.x ... 

After a time x =1 /y, the cosine term will be zero, and the sine term unity, so that in-phase 1H coherence is completely converted into heteronuclear antiphase coherence, 2 Iy Sz. For protons not bound to an Ix spin, nothing happens (neglecting chemical shift evolution, other couplings, relaxation etc.), and they will stay at Ix coherence ... 

This scheme is applied in the so-called X-half-filter technique (Fig. 17.4a, c), with the only difference of an additional 90 (X) pulse with constant phase [16, 17]. Instead of generating heteronuclear multiple quantum coherence 2 Iy Sy, which cannot readily be detected (in case of selecting the 1H-X pairs), one now always ends up with proton antiphase coherence, but with the same phase alternation ... 

When the second 90° pulse is applied, the antiphase magnetization of the / and J spins is interchanged (Fig. 8.14B). During t2, the new antiphase coherence,... 

The coherence transfer provides cross peaks which are antiphase for the various 7//-split components. The antiphase nature of the cross peaks then leads to partial or total cancellation of the cross peaks themselves, especially if they are phased in the absorption mode. This behavior can be simulated (Fig. 8.15) using appropriate treatments of the time evolution of the spin system, for instance using the density matrix formalism [17,18]. It is quite common that signals in paramagnetic systems... 

Fig. 8.19. Vector representation of a H-13C HMQC experiment. The first 90° pulse along y rotates the equilibrium magnetization of the proton spin, /H, from the z axis to the x axis. After a time /d = 1/2/Hx, the antiphase coherence 2/J1/ t (see Appendix IX) is at its maximum. A 90° pulse on carbon along y then transforms the antiphase coherence into a MQ (multiple quantum) coherence (the 2/J1/ component is shown). During t the MQ evolves (with a 180 refocusing pulse on proton in the middle), until a further 90 pulse on carbon along x transforms the —2/ / component (shown at its maximum for clarity) into a 2/ /f antiphase coherence. After the time fd, in-phase coherence of the proton spin develops. The latter is detected during h. Its initial intensity is modulated by the carbon Larmor frequency during t (if proton refocusing has been used), thus originating a proton-carbon cross peak.

At this point the MQ coherence is let evolve for a variable time t. A refocusing 180° pulse on proton is applied in the middle of the evolution period, i.e. at t /2. A further 90° pulse on carbon converts the MQ coherence into an antiphase magnetization (Fig. 8.19), which is let evolve for another time t to fully develop the observable single quantum coherence on proton, which is then detected during the acquisition time t2. 

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