Proton-Heteronucleus Correlation

COSY for other Nuclides. The basic COSY experiment can be carried out for any spin-5 nucleus that is 100% abundant. In addition to H- H, the procedure thus is applicable to (F,F-C0SY) and (P,P-COSY), respectively, in organofluorine and 

BIRD-HMQC. The most difficult aspect of implementing the HMQC experiment is the suppression of signals from protons attached to C (the center-band or single quantum coherences) in favor of the protons attached to C (the satellites or double quantum coherences). The use of pulse field gradients (PFG, Section 6-6) is the most effective technique, but relatively few spectrometers are equipped with the hardware required for their generation. Fortunately, there is an effective alternative for the suppression of center bands by means of the BIRD Bilinear Rotation Decoupling) sequence, which is outlined by the vector 

The overall experiment thus becomes BIRD-t-HMQC-DT, in which t is chosen to null the single quantum coherences and DT is the normal delay time between pulse repetitions, which includes the time taken to acquire the signal, as well as a recycle time during which the signal is regenerated through relaxation. The details of the experiment require 

The Heteronudear Single Quantum Correlation (HSQC) experiment is an alternative to HMQC that accomplishes a similar objective. The experiment generates, via an INEPT sequence, single quantum (or N) coherence, which evolves and then is transferred back to the proton frequency by a second INEPT sequence, this time in reverse. The main difference from the HMQC result is that HSQC spectra do not contain H- H couplings in the C ( Jj) dimension. As a result, HSQC cross peaks tend to have improved resolution over analogous HMQC cross peaks. HSQC is preferred when there is considerable spectral overlap. 

The principal disadvantage of the COLOC sequence lies in the fixed nature of the evolution period. In such a pulse sequence, C-H correlations are diminished or even absent when two- and three-bond H- C couplings are of a magnitude similar to that of H- H couplings within a molecular fragment. This situation occurs quite commonly (Chapter 4). 

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The most straightforward isotope-editing method for selecting protons bound to a heteronucleus and suppressing all others is the simple acquisition of a spectrum with an indirect heteronuclear dimension (in the literature the term isotope editing is often used as a synonym for these techniques). This can be accomplished by a simple 2D HMQC or HSQC shift correlation, or a more elaborate 3D technique including an additional NOESY or TOCSY step (3D X-edited NOESY/TOCSY etc.), or even 4D experiments with a second heteronuclear shift dimension [13, 14]. 

Over the last 10-15 years, multidimensional (3D and 4D) NMR spectroscopic and computational techniques have been developed to solve structures of larger proteins in solution. This technology has the advantage of resolving the severe overlap in 2D NMR spectra for proteins having >100 residues. Usually, multidimensional NMR experiments require isotopic labeling of some or all nuclei of interest. For example, signal overlap in a two-dimensional correlation spectrum can be resolved in the dimension of the heteronucleus—that is, N or attached to the protons of relevance. If fast relaxation is a problem,... 

N and 13C labeling of peptides facilitates the study of their molecular dynamics in solution by measurements of relaxation parameters (42,43). Heteronuclear relaxation times and heteronuclear NOEs are predominantly affected by the dipole-dipole interaction of the heteronucleus with the directly attached proton. Since the intemuclear (i.e., chemical bonding) distances are known from the molecular geometry, correlation times for overall and internal motions can be determined. 

A variety of methods have already been described in Chapter 4 that allow the editing of the ID spectrum of the heteronuclear spin, for example those based on spin-echoes or polarisation transfer, so providing valuable information on the numbers of attached protons. They do not, however, provide any direct evidence for which protons are attached to which heteronucleus (X-spin) in the molecule and for this heteronuclear 2D correlations are widely used to transfer previously established proton assignments onto the directly bonded heteronucleus or, on occasions, vice versa. This may then provide further evidence to support or reject the proposed structure as being correct, or may provide assignments that can be used as the basis of further investigations. In... 

The HMQC sequence aims to detect only those protons that are bond to a spin- A heteronucleus, or in other words only the satellites of the conventional proton spectrum. In the case of C, this means that only 1 in every 100 proton spins contribute to the 2D spectrum (the other 99 being attached to NMR inactive C) whilst for N with a natural abundance of a mere 0.37%, only 1 in 300 contribute. When the HMQC FID is recorded, all protons will induce a signal in the receiver on each scan and the unwanted resonances, which clearly represent the vast majority, must be removed with a suitable phase cycle if the correlation peaks are to be revealed (the notable exception to this is when pulsed field gradients are employed for signal selection, see Section 6.3.3 below). By inverting the first C pulse on alternate scans, the phase of the C satellites are themselves inverted whereas the C-bound protons remain unaffected (Fig. 6.5). Simultaneous inversion of the receiver will lead to cancellation of the unwanted resonances with corresponding addition of the desired satellites. This two step procedure is the fundamental phase-cycle of the HMQC experiment, as indicated in Fig. 6.3 above. 

The high crosspeak dispersion typically associated with heteronuclear shift correlation experiments alongside the lack of any requirement for well defined crosspeak fine structure means HMQC or HSQC experiments can be recorded with rather low digital resolution for routine applications, enhancing their time efficiency. An acquired digital resolution of 5 Hz/pt in the proton f2 dimension and only 50 Hz/pt in the heteronucleus fr dimension are generally sufficient to resolve correlations. Improved fi resolution can be achieved by linear... 

Heteronucleus-detected shift correlation experiments have now been largely supplanted by far more sensitive proton- or inverse -detected methods. The heteronu-cleus-detected experiments are now largely reserved, in laboratories with modem NMR spectrometers, for those occasions when very high digital resolution is needed in the carbon frequency domain because of high spectral congestion [109, 110]. The remainder of this section will focus on the now widely utihzed proton-detected heteronuclear shift correlation methods. 

The most sophisticated accordion-optimized long-range shift correlation experiment to be developed to date is the /, /-HMBC experiment [148]. For the first time in an inverse-detected experiment, it is possible to differentiate two-bond from three-bond long-range correlations to protonated carbon or nitrogen resonances. This capability was last available for protonated carbons via the heteronucleus-detected XCORFE sequence pioneered by Reynolds and co-workers in 1985 [130]. 

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