Heteronuclear experiment

In homonuclear-shift-correlated experiments, the Ft domain corresponds to the nucleus under observation in heteronuclear-shift-correlated experiments. Ft relates to the unobserved or decoupled nucleus. It is therefore necessary to set the spectral width SW, after considering the ID spectrum of the nucleus corresponding to the Ft domain. In 2D /-resolved spectra, the value of SW depends on the magnitude of the coupling constants and the type of experiment. In both homonuclear and heteronuclear experiments, the size of the largest multiplet structure, in hertz, determines... 

If phase-sensitive spectra are not required, then magnitude-mode Pico) (or absolute-mode ) spectra may be recorded by combining the real and imaginary data points. These produce only positive signals and do not require phase correction. Since this procedure gives the best signal-to-noise ratio, it has found wide use. In heteronuclear experiments, in which the dynamic range tends to be low, the power-mode spectrum maybe preferred, since the S/N ratio is squared and a better line shape is obtained so that wider window functions can be applied. 

We have concentrated on the proton-proton, homonuclear NOE experiments in this section but the potential use of analogous heteronuclear experiments should not be overlooked, if you have the appropriate hardware available to you. The 19F- 1H NOE experiment, for example, can be very useful in certain situations as demonstrated in the following example. You have one of two possible positional isomers (Structure 8.6). 

In addition to well-resolved one-dimensional (ID) 1H and 13C spectra, which are usually sufficient for monitoring synthetic steps, HR-MAS techniques can be applied to two-dimensional (2D) homonuclear and heteronuclear experiments, which allow a wealth of structural information to be obtained. H,13C HMQC (heteronuclear multiple quantum coherence) spectra are particularly useful in the analysis of solid support-bound oligosaccharides, since the anomeric protons exhibit characteristic resonances. Such a spectrum of a polymer-bound trisaccharide glycal is shown in Figure 8.4. 

Strongly overlapping multiplets may be resolved by two-dimensional J,<5-spectros-copy2" 11G 118, where the first frequency domain (F,) contains coupling and the second frequency domain (F2) chemical shift information. The spectrum in Figure 2 (homonuclear [JH H, 6 ( H)]) demonstrates the use of this technique by showing unperturbed multiplets for ll signals. Second-order effects are principally not eliminated. Heteronuclear experiments [7uc,<5(13C)] are also common. 

This protocol describes how a 1-D H NMR spectrum is recorded. This type of spectrum may provide useful information about the identity of the aglycone, number of attached sugar units, and presence or absence of acyl moieties. This procedure may also be used as the basis for recording t3C NMR spectra, and for initial preparations of 2-D homo- and heteronuclear experiments. See Basic Protocol for materials. 

Fig. 8.2. Some of the most common 2D pulse sequences that can be employed using a proper choice of parameters to record 2D spectra of paramagnetic molecules (A) NOESY, (B) ROESY, (C) COSY, (D) ISECR COSY, (E) zero-quantum (double quantum) COSY, (F) TOCSY, (G) HMQC, (H) HSQC. Sequences (A), (B) and (F) are also used to obtain EXSY spectra. SL indicates a soft spin-lock sequence, while MLEV17 indicates a train of spin-locking hard pulses that optimizes the development of J/j coupling. In the reverse heteronuclear experiment (G) the upper and lower levels refer to H and heteronucleus, respectively. The phase cycles are not indicated. For clarity of discussion, all initial pulses can be thought to be applied along the y axis, in such a way that the coherence after the first 90° pulse is always along x. ...

For heteronuclear experiments, changing the phase of a pulse will only affect that part of the coherence that is sensitive to the pulse. For example, in the stepwise INEPT with 2IZSZ as in intermediate step in coherence transfer... 

ID ll spectrum, and crosspeaks are arranged symmetrically around the diagonal. There is only one radio frequency channel in a homonuclear experiment, the H channel, so the center of the spectral window (set by the exact frequency of pulses and of the reference frequency in the receiver) is the same in If and F (Varian tof, Bruker ol). The spectral widths should be set to the same value in both dimensions, leading to a square data matrix. Heteronuclear experiments have no diagonal, and two separate radio frequency channels are used (transmitter for F2, decoupler for F ) with two independently set spectral windows (Varian tof and dof, sw, and swl, Bruker ol and o2, sw(If), and sw(I )). Heteronuclear experiments can be further subdivided into direct (HETCOR) and inverse (HSQC, HMQC, HMBC) experiments. Direct experiments detect the X nucleus (e.g., 13C) in the directly detected dimension (Ff) using a direct probe (13C coil on the inside, closest to the sample, H coil on the outside), and inverse experiments detect XH in the To dimension using an inverse probe (XH coil on the inside, 13C coil outside). 

For non-deuterated complexes, NMR experiments were performed on either Vaiian Unity600 or Varian Unity+500 spectrometers. The 600 MHz instrument was equipped with a triple resonance probe and a PTS synthesizer as a pseudo fourth channel. The 500 MHz spectrometer was a four channel instrument with a triple resonance probe with an actively shielded pulsed field gradient coil. All experiments were performed at 37 C. The heteronuclear experiments shown in Figure 1 were performed as described in Zhang et al (11) and Revington et al (24). For all 3D ex riments 32 transients were required for sufficient signal to noise. This necessitated the use of fewer increments and the use of linear prediction (25)... 

Table 2 Calculated signal-to-noise ratios of different heteronuclear experiments [27]...

Figure 15. Pulse sequence for a conventional separated local field (SLF) heteronuclear experiment.

Figure 13(b) shows a JH—15N HSQC spectrum acquired from 0.5 mmol l-1 sample of a 41-residue peptide toxin from the spider Agelena orientalis. The toxin was produced recombinantly and uniformly labeled with 15N. This HSQC spectrum was collected in 30 min, compared with the 12 h required to acquire a natural abundance spectrum from an unlabeled sample of equivalent concentration (see Figure 11). The HSQC, together with the related heteronuclear multiple quantum coherence (HMQC)54 experiment, forms the cornerstone of a wide range of 2D, 3D, and 4D experiments that are designed to facilitate sequence-specific resonance assignment and determination of protein structure. Note that the HSQC technique is the technique of choice for correlation of H and 15N shifts due to generally narrower linewidths in the 15N dimension.55,56 Furthermore, because these and most of the other heteronuclear experiments described below are designed to observe amide protons, the sample must be in H20 (rather than D20). Consequently, a means of suppressing the H20 resonance is required (for details see Section 9.09.2.6).

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