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K. Motiram-Corral, P. Nolis, J. Saurí and T. Parella
J. Nat. Prod., 83, 1275-1282 (2020). DOI
LR-HSQMBC versus LR-selHSQMBC: Enhancing the observation of tiny long-range heteronuclear NMR correlations
The detection of ultra-long-range (4JCH and higher) heteronuclear connectivities can complement the conventional use of HMBC/HSQMBC data in structure elucidation NMR studies of proton-deficient natural products, where two-bond and three-bond correlations are usually observed. Despite its frequency-selectivity nature, selHSQMBC efficiently prevents any unwanted signal phase and intensity modulations due to passive proton–proton coupling constants typically involved in HSQMBC. As a result, selHSQMBC offers a significant sensitivity enhancement and provides pure in-phase multiplets, improving the detection levels for short- and long-range cross-peaks corresponding to small heteronuclear coupling values. This is particularly relevant for experiments optimized to small nJCH values (2–3 Hz), referred to as LR-selHSQMBC, where key cross-peaks that are not visible in the equivalent broadband LR-HSQMBC spectrum can become observable in optimum conditions.
K. Motiram-Corral, A.A. Souza, J. Saurí, P. Nolis and T. Parella
ChemPhysChem, 21, 280-283 (2020). DOI
LR-selHSQMBC: Simultaneous detection and quantification of very weak long-range heteronuclear NMR correlations
The optimum detection and accurate measurement of longer‐range (4J and higher) heteronuclear NMR correlations is described. The magnitude and/or the sign of a wide range of large and small long‐range couplings can be simultaneously determined for protonated and non‐protonated 13C and 15N nuclei using the LR‐selHSQMBC experiment.
J. Saurí, W. Bermel, T. Parella, R.T. Williamson and G.E. Martin
Magn. Reson. Chem., 56, 1029-1036 (2018). DOI
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 1JCH responses out. Dual‐optimized, inverted 1JCC 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 1JCC 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.
P. Nolis and T. Parella
Magn. Reson. Chem., 56, 976-982 (2018). DOI
Multiplicity-edited 1H-1H TOCSY experiment
A 1H‐1H total correlation spectroscopy (TOCSY) experiment incorporating 13C multiplicity information is proposed. In addition, broadband 1H homodecoupling in the indirect dimension can be implemented using a perfect BIRD module that affords exclusive 1H chemical shift evolution with full decoupling of all heteronuclear and homonuclear (including 2JHH) coupling constants. As a complement to the normal TOCSY and the recent PSYCHE‐TOCSY experiments, this novel multiplicity‐edited TOCSY experiment distinguishes between CH/CH3 (phased up) and CH2 (phased down) cross‐peaks, which facilitates resonance analysis and assignment.
J. Saurí, T. Parella, R.T. Williamson and G.E. Martin
Magn. Reson. Chem., 55, 191-197 (2017). DOI
Improving the Performance of J-modulated ADEQUATE Experiments Through Homonuclear Decoupling and Non-Uniform Sampling
Homonuclear 13C–13C couplings at natural abundance can be measured using the J‐modulated Adequate DoublE QUAntumTransfer Experiment (ADEQUATE) experiment. To somewhat ameliorate F1 digitization requirements, a scaling factor was incorporated into the original pulse sequence. Non‐uniform sampling provides an obvious avenue to further facilitate the acquisition of 1JCC and nJCC homonuclear coupling constant data. We introduce homonuclear decoupling (HD) analogous to that described for the 1,1‐HD‐ADEQUATE and 1,n‐HD‐ADEQUATE experiments and evaluate the combination of non‐uniform sampling and HD on the acquisition of both 1JCC and nJCC homonuclear 13C–13C coupling constants using ibuprofen as a model compound.
J. Saurí, Y. Liu, T. Parella, R.T. Williamson and G.E. Martin
J. Nat. Prod., 79, 1400-1406 (2016). DOI
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 nJXH 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.
J. Saurí., W. Bermel, A.V. Buevich, E.C. Sherer, L.A. Joyce, M.H.M. Sharaf, P.L. Schiff Jr., T. Parella, R.T. Williamson and G.E. Martin
Angew. Chem. Intl. Ed., 54, 10160-10164 (2015). DOI
Homodecoupled 1,1- and 1,n-ADEQUATE: Pivotal NMR Experiments for the Structure Revision of Cryptospirolepine
Cryptospirolepine is the most structurally complex alkaloid discovered and characterized thus far from any Cryptolepis specie. Characterization of several degradants of the original, sealed NMR sample a decade after the initial report called the validity of the originally proposed structure in question. We now report the development of improved, homodecoupled variants of the 1,1- and 1,n-ADEQUATE (HD-ADEQUATE) NMR experiments; utilization of these techniques was critical to successfully resolving long-standing structural questions associated with crytospirolepine.
J. Saurí, N. Marcó, R.T Williamson, G.E Martin and T. Parella
J. Magn. Reson., 258, 25-32 (2015).DOI
Extending long-range heteronuclear NMR connectivities by HSQMBC-COSY and HSQMBC-TOCSY experiments
The detection of long-range heteronuclear correlations presenting J(CH) coupling values smaller than 1–2 Hz is a challenge in the structural analysis of small molecules and natural products. HSQMBC-COSY and HSQMBC-TOCSY pulse schemes are evaluated as complementary NMR methods to standard HMBC/HSQMBC experiments. Incorporation of an additional J(HH) transfer step in the basic HSQMBC pulse scheme can favor the sensitive observation of traditionally missing or very weak correlations and, in addition, facilitates the detection of a significant number of still longer-range connectivities to both protonated and non-protonated carbons under optimum sensitivity conditions.
J. Saurí, E. Sistaré, R.T Williamson, G.E Martin and T. Parella
J. Magn. Reson., 252, 170-175(2015).DOI
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.
L. Castañar, E. Sistaré, A. Virgili, R.T. Williamson and T. Parella
Magn. Reson. Chem,, 53, 115 (2015). DOI
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.
L. Castañar, J. Saurí, R. T. Williamson, A. Virgili and T. Parella
Angew. Chem. Intl. Ed., 53, 8379-8382 (2014). DOI.
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.
R.T. Williamson, A. Buevich, G.E. Martin and T. Parella
J. Org. Chem., 79, 3887-3894 (2014). DOI
LR-HSQMBC: A sensitivity NMR technique to probe very long-range heteronuclear coupling pathways
HMBC is one of the most often used and vital NMR experiments for the structure elucidation of organic and inorganic molecules. We have developed a new, high sensitivity NMR pulse sequence that overcomes the typical 2,3JCH limitation of HMBC by extending the visualization of long-range correlation data to 4-, 5-, and even 6-bond long-range nJCH heteronuclear couplings. This technique should prove to be an effective experiment to complement HMBC for probing the structure of proton-deficient molecules. The LR-HSQMBC NMR experiment can, in effect, extend the range of HMBC to provide data similar to that afforded by 1,n-ADEQUATE even in sample-limited situations. This is accomplished by optimizing responses for very small nJCH coupings as opposed to relying on the markedly less sensitive detection of long-range coupled 13C–13C homonuclear pairs at natural abundance. DFT calculations were employed to determine whether the very long-range correlations observed for cervinomycin A2 were reasonable on the basis of the calculated long-range couplings.
L. Castañar, P. Nolis, A. Virgili and T. Parella
Chem. Eur. J., 19, 15472-15475 (2013). DOI
Simultaneous multi-slice excitation in spatially encoded NMR Experiments
A novel strategy to enhance the experimental sensitivity in spatially encoded NMR experiments has been developed. The use of a multiple‐frequency modulated pulse applied simultaneously to an encoding gradient can afford a substantial sensitivity gain with respect to single‐slice selected experiments.
J. Saurí, L. Castañar, P. Nolis, A. Virgili and T. Parella
J. Magn. Reson., 242, 33 (2014). DOI
Straightforward measurement of individual 1J(CH) and 2J(HH) in diastereotopic CH2 groups
The C–HA cross-peak corresponding to a diastereotopic CHAHB methylene spin system exhibits a characteristic 1:0:1 multiplet pattern along the indirect dimension of a x1-coupled HSQC spectrum. It is shown here that the use of the initial 13C Boltzmann polarization instead of the regular INEPT-based 1H Boltzmann polarization makes visible the central lines of this multiplet pattern. A spin-state-selective method is proposed for the efficient measurement of both 1J(CHA) and 1J(CHB) along the indirect dimension of a 2D spectrum as well as to the magnitude and the sign of the geminal 2J(HAHB) coupling constant from the straightforward analysis of a single four-component E.COSY cross-peak. Additionally, the extraction of 1J(CH) values for CH and CH3 multiplicities can be also performed from the same spectrum. The success of the method is also illustrated for the determination of residual dipolar 1D(CH) and 2D(HH) coupling constants in a small molecule weakly aligned in a PMMA swollen gel.
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