G.E. Martin/R.T. Williamson
(Merck & Co - USA)

Incorporating BIRD-based homodecoupling in the dual-optimized, inverted 1JCC 1,n-ADEQUATE experiment

1,n‐ADEQUATE is a powerful NMR technique for elucidating the structure of proton‐deficient small molecules that can help establish the carbon skeleton of a given molecule by providing long‐range three‐bond ¹³C─¹³C correlations. Care must be taken when using the experiment to identify the simultaneous presence of one‐bond ¹³C─¹³C correlations that are not filtered out, unlike the HMBC experiment that has a low‐pass J‐filter to filter ¹J_CH responses out. Dual‐optimized, inverted ¹J_CC 1,n‐ADEQUATE is an improved variant of the experiment that affords broadband inversion of direct responses, obviating the need to take additional steps to identify these correlations. Even though ADEQUATE experiments can now be acquired in a reasonable amount of experimental time if a cryogenic probe is available, low sensitivity is still the main impediment limiting the application of this elegant experiment. Here, we wish to report a further refinement that incorporates real‐time bilinear rotation decoupling‐based homodecoupling methodology into the dual‐optimized, inverted ¹J_CC 1,n‐ADEQUATE pulse sequence. Improved sensitivity and resolution are achieved by collapsing homonuclear proton–proton couplings from the observed multiplets for most spin systems. The application of the method is illustrated with several model compounds.

Selecting the most appropriate NMR experiment to access weak and/or very long-range heteronuclear correlations

Heteronuclear long-range NMR experiments are well established as essential NMR techniques for the structure elucidation of unknown natural products and small molecules. It is generally accepted that the absence of a given ⁿJ_XH correlation in an HMBC or HSQMBC spectrum would automatically place the proton at least four bonds away from the carbon in question. This assumption can, however, be misleading in the case of a mismatch between the actual coupling constant and the delay used to optimize the experiment, which can lead to structural misassignments. Another scenario arises when an investigator, for whatever reason, needs to have access to very long-range correlations to confirm or refute a structure. In such cases, a conventional HMBC experiment will most likely fail to provide the requisite correlation, regardless of the delay optimization. Two recent methods for visualizing extremely weak or very long-range connectivities are the LR-HSQMBC and the HSQMBC-TOCSY experiments. Although they are intended to provide similar structural information, they utilize different transfer mechanisms, which differentiates the experiments, making each better suited for specific classes of compounds. In this report we have sought to examine the considerations implicit in choosing the best experiment to access weak or very long-range correlations for different types of molecules.

Carbon Multiplicity-editing in Long-range Heteronuclear Correlation NMR Experiments: a Valuable Tool for the Structure Elucidation of Natural Products

A recently developed NMR method to simultaneously obtain both long-range heteronuclear correlations and carbon multiplicity information in a single experiment, ME-selHSQMBC, is demonstrated as a potentially useful technique for chemical shift assignment and structure elucidation of natural products presenting complicated NMR spectra. Carbon multiplicities, even for C/CH₂ and odd for CH/CH₃ resonances, can be distinguished directly from the relative positive/negative phase of cross-peaks. In addition, connectivity networks can be further extended by incorporating a TOCSY propagation step. Staurosporine (1) and sungucine (2) are utilized as model compounds to demonstrate these techniques.

Suppression of phase and amplitude J(HH) modulations in HSQC experiments

The amplitude and the phase of cross peaks in conventional 2D HSQC experiments are modulated by both proton–proton, J(HH), and proton–carbon, 1J(CH), coupling constants. It is shown by spectral simulation and experimentally that J(HH) interferences are suppressed in a novel perfect-HSQC pulse scheme that incorporates perfect-echo INEPT periods. The improved 2D spectra afford pure in-phase cross peaks with respect to 1J(CH) and J(HH), irrespective of the experiment delay optimization. In addition, peak volumes are not attenuated by the influence of J(HH), rendering practical issues such as phase correction, multiplet analysis, and signal integration more appropriate.

Pure In-Phase heteronuclear correlation NMR experiments

A general NMR approach to provide pure in‐phase (PIP) multiplets in heteronuclear correlation experiments is described. The implementation of a zero‐quantum filter efficiently suppresses any unwanted anti‐phase contributions that usually distort the multiplet pattern of cross‐peaks and can hamper their analysis. The clean pattern obtained in PIP‐HSQMBC experiments is suitable for a direct extraction of coupling constants in resolved signals, for a peak‐fitting process from a reference signal, and for the application of the IPAP technique in non‐resolved multiplets.

Implementing multiplicity editing in selective HSQMBC experiments

Even C/CH2 and odd CH/CH3 carbon-multiplicity information can be directly distinguished from the relative positive/negative phase of cross-peaks in a novel ME(Multiplicity-Edited)-selHSQMBC experiment. The method can be extended by a TOCSY propagation step, and it is fully compatible for the simultaneous and precise determination of long-range heteronuclear coupling constants. Broadband homonuclear decoupling techniques can also be incorporated to enhance sensitivity and signal resolution by effective collapse of J(HH) multiplets.