J and RDC

The detection of ultra-long-range (⁴J_CH 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. The performance of the selHSQMBC experiment with respect to its broadband HSQMBC counterpart is evaluated. 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 ⁿJ_CH 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.

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.

Heteronuclear long‐range scalar coupling constants (ⁿJ_CH) are a valuable tool for solving problems in organic chemistry and are especially suited for stereochemical and configurational analyses of small molecules and natural products. This tutorial will focus on the step‐by‐step implementation of several 2D ¹H frequency selective HSQMBC experiments for the easy and accurate measurement of either the magnitude or both the magnitude and the sign of long‐range ⁿJ_CH couplings. The performance of these experiments will be showcased with several scenarios in a range of different experimental conditions.

The concepts of pure-shift NMR and time-shared NMR are merged in a single experiment. A ¹³C/¹⁵N time-shared version of the real-time BIRD-based broadband homodecoupled HSQC experiment is described. This time-efficient approach affords simultaneously ¹H-¹³C and ¹H-¹⁵N pure-shift HSQC spectra in a single acquisition, while achieving substantial gains in both sensitivity and spectral resolution. We also present a related ¹³C/¹⁵N-F2-coupled homodecoupled version of the CLIP-HSQC experiment for the simultaneous measurement of ¹J_CH and ¹J_NH from the simplified doublets observed along the direct dimension. Finally, a novel J-resolved HSQC experiment has been designed for the simple and automated determination of both ¹J_CH/¹J_NH from a 2D J-resolved spectrum.

The measurement of two-bond proton-proton coupling constants (²J_HH) in prochiral CH₂ groups from the F2 dimension of 2D spectra is not easy due to the usual presence of complex multiplet J patterns, line broadening effects and strong coupling artifacts. These drawbacks are particularly pronounced and frequent in AB spin systems, as those normally exhibited by the pair of diastereotopic CH₂ protons. Here, a novel ²J_HH-resolved HSQC experiment for the exclusive and accurate determination of the magnitude of ²J_HH from the doublet displayed along the highly-resolved indirect F1 dimension is described. A pragmatic ²J_HH NMR profile affords a fast overview of the full range of existing ²J_HH values. In addition, a ²J_HH/δ(¹³C)-scaled version proves to be an efficient solution when severe signal overlapping complicate a rigorous analysis. The performance of the method is compared with other current techniques and illustrated by the determination of challenging residual dipolar ²D_HH coupling constants of small molecules dissolved in weakly orienting media.

Selective refocusing (GSERF or the recent PSYCHEDELIC) experiments were originally designed to determine all proton–proton coupling constants (J_HH) for a selected proton resonance. They work for isolated signals on which selective excitation can be successfully applied but, as it happens in other selective experiments, fail for overlapped signals. To circumvent this limitation, a doubly‐selective TOCSY‐GSERF scheme is presented for the measurement of J_HH in protons resonating in crowded regions. This new experiment takes advantage of the editing features of an initial TOCSY transfer to uncover hidden resonances that become accessible to perform the subsequent frequency‐selective refocusing

A versatile ¹J_CH-resolved HSQC pulse scheme for the speedy, accurate and automated determination of one-bond proton-carbon coupling constants is reported. The implementation of a perfectBIRD element allows a straightforward measurement from the clean doublets obtained along the highly resolved F1 dimension, even for each individual ¹J_CHa and ¹J_CHb in diastereotopic H_aCH_b methylene groups. Real-time homodecoupling during acquisition and other alternatives to minimize accidental signal overlapping in overcrowded spectra are also discussed.

A user-friendly NMR interface for the visual and accurate determination of experimental one-bond proton-carbon coupling constants (¹J_CH) in small molecules is presented. This intuitive ¹J_CH profile correlates directly to δ(¹H), and ¹J_CH facilitates the rapid identification and assignment of ¹H signals belonging to key structural elements and functional groups. Illustrative examples are provided for some target molecules, including terminal alkynes, strained rings, electronegative substituents, or lone-pair-bearing heteronuclei.

A novel approach for the fast and efficient structural discrimination of molecules containing multiple stereochemical centers is described. A robust J-resolved HSQC experiment affording highly resolved ¹J_CH/¹T_CH splittings along the indirect dimension and homodecoupled ¹H signals in the detected dimension is proposed. The experiment enables in situ distinction of both isotropic and anisotropic components of molecules dissolved in compressed PMMA gels, allowing a rapid and direct one-shot determination of accurate residual dipolar coupling constants from a single NMR spectrum.

The presence of a highly abundant passive nucleus (Z = ¹⁹ F or ³¹P) allows the simultaneous determination of the magnitude and the sign of up to three different heteronuclear coupling constants from each individual cross‐peak observed in a 2D ¹H‐X selHSQMBC spectrum. Whereas J(HZ) and J(XZ) coupling constants are measured from E.COSY multiplet patterns, J(XH) is independently extracted from the complementary IPAP pattern generated along the detected F2 dimension. The incorporation of an extended TOCSY transfer allows the extraction of a complete set of all these heteronuclear coupling constants and their signs for an entire ¹H subspin system. ¹H‐X/¹H‐Y time‐shared versions are also proposed for the simultaneous measurement of five different couplings (J(XH), J(YH), J(XZ), J(YZ), and J(ZH)) for multiple signals in a single NMR experiment.

The C–H_A cross-peak corresponding to a diastereotopic CH_AH_B methylene spin system exhibits a characteristic 1:0:1 multiplet pattern along the indirect dimension of a ω₁-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(CH_A) and ¹J(CH_B) along the indirect dimension of a 2D spectrum as well as to the magnitude and the sign of the geminal ²J(H_AH_B) 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.

An NMR method to enhance the sensitivity and resolution in band-selective long-range heteronuclear correlation spectra is proposed. The excellent in-phase nature of the seIHSQMBC experiment allows that homonuclear and/or heteronuclear decoupling can be achieved in the detected dimension of a 2D multiple-bond correlation map, obtaining simplified cross-peaks without their characteristic fine J multiplet structure. The experimental result is a resolution improvement while the highest sensitivity is also achieved. Specifically, it is shown that the H-1-homodecoupled band-selective (HOBS) HSQMBC experiment represents a new way to measure heteronuclear coupling constants from the simplified in-phase doublets generated along the detected dimension.

Two complementary 1D NMR approaches for the fast and easy determination of the magnitude and the sign of heteronuclear J(XH) coupling constants are proposed: The Up&Down technique relies on the direct analysis of anti-phase multiplets whereas the Left&Right technique is based on the relative displacement between separate IPAP components.

A long-range optimized P.E.HSQC experiment, named P.E.HSQMBC, is proposed for the simultaneous measurement of a complete set of homonuclear and heteronuclear coupling constants from a single 2D cross-peak. The sign and the magnitude of proton–proton coupling constants are measured along the direct dimension from the relative E.COSY-type multiplet pattern displacement due to the passive one-bond coupling constant splitting generated in the indirect dimension. On the other hand, long-range proton–carbon coupling constants are independently determined in the detected dimension from a traditional fitting analysis of antiphase multiplet patterns or, more conveniently, from the IPAP multiplet displacement obtained from extended HSQMBC experiments.

Improved pulsed‐field gradient echo methods are presented and discussed for the direct selective excitation of the ¹³C‐satellite lines in overcrowded ¹H NMR spectra of small molecules. Sensitivity enhancements in ¹³C spin‐state selection can be achieved by combining multiple‐proton‐frequency excitation and Hadamard phase encoding. Several satellite‐selective (SATSEL) NMR experiments are proposed and exemplified by measuring the sign and the magnitude of small, long‐range proton–carbon coupling constants for ¹H resonances showing several levels of signal overlapping.

Two novel HSQC-IPAP approaches are proposed to achieve α/β spin-state editing simultaneously for ¹³C and ¹⁵N in a single NMR experiment. The pulse schemes are based on a time-shared (TS) 2D ¹H,¹³C/¹H,¹⁵N-HSQC correlation experiment that combines concatenated echo elements for simultaneous J(CH) and J(NH) coupling constants evolution, TS evolution of ¹³C and ¹⁵N chemical shifts in the indirect dimension and heteronuclear α/β-spin-state selection by means of the IPAP principle. Heteronuclear α/β-editing for all CH _n (n = 1–3) and NH _n (1–2) multiplicities can be achieved in the detected F2 dimension of a single TS-HSQC-F2-IPAP experiment. On the other hand, an alternative TS-HSQC-F1-IPAP experiment is also proposed to achieve α/β-editing in the indirect F1 dimension. Experimental and simulated data is provided to evaluate these principles in terms of sensitivity and performance simultaneously on backbone and side-chain CH, CH₂, CH₃, NH, and NH₂ spin systems in uniformly ¹³C/¹⁵N-labeled proteins and in small natural-abundance peptides.

A new one-shot NMR experiment (CN-HMBC) is proposed for the simultaneous acquisition of 2D ¹H,¹³C and ¹H,¹⁵N HMBC spectra. Important sensitivity enhancements (up to 41% simultaneously for both ¹³C and ¹⁵N) or time savings (about 50%) can be achieved when compared to the separate acquisition of individual HMBC spectra. The experiment is highly recommended for the complete structural analysis and simultaneous chemical shift assignments of protonated and nonprotonated ¹³C and ¹⁵N resonances in nitrogen-containing organic compounds.

Most conventional heteronuclear spin-state-selective (S³) NMR experiments only work for a specific multiplicity, typically IS spin systems. Here, we introduce a general and efficient IPAP strategy to achieve S³ editing simultaneously for all multiplicities in the acquisition dimension of the HSQC experiment. Complementary in-phase (HSQC-IP) and anti-phase (HSQC-AP) data are separately recorded with a simple phase exchange of two 90° proton pulses involved in the mixing process of the F2-coupled sensitivity-improved HSQC pulse sequence. Additive and subtractive linear combination of these IP/AP data generates simplified F2-α/β-spin-edited HSQC subspectra for all IS, I₂S, and I₃S spin systems and combines enhanced and optimized sensitivity with excellent tolerance to unwanted cross-talk contributions over a considerable range of coupling constants. Practical aspects such as pulse phase settings, transfer efficiency dependence, inter-pulse delay optimization, and percentage of cross-talk are theoretically analyzed and discussed as a function of each I_nS multiplicity. Particular emphasis on the features associated to spin-editing in diastereotopic I₂S spin systems and application to the measurement of long-range proton–carbon coupling constants are also provided.

New NMR pulse schemes completely driven under homonuclear and heteronuclear cross-polarization conditions are proposed for the study and the measurement of coupling constants in symmetrical molecules in solution. The appropriate superimposition of independent magnetization components can afford several spin-selective multiplet patterns that are suitable for the determination of the magnitude and the sign of proton-proton and proton-carbon coupling constants with optimum sensitivity levels. A detailed product operator formalism analysis for the proposed doubly selective 1D and nonselective 2D HCP-TOCSY versions is provided and experimental verification for the configurational analysis of symmetric olefinic systems having chemical equivalence is demonstrated.

Simple modifications of the sensitivity-improved HSQC-TOCSY pulse sequence are proposed for the easy determination of the sign and the magnitude of homonuclear and heteronuclear coupling constants. Whereas in well-resolved regions, a clean two-component E.COSY-like pattern allows a direct measurement from a single 2D spectrum, separate acquisition of equivalent single-component TROSY/anti-TROSY spectra becomes highly interesting when spectral crowding complicates the spectral analysis. It is also demonstrated that an additional restricted planar mixing element after the isotropic TOCSY process completely retains all spin-editing features and permits the accurate measurement of the sign and the size of the corresponding homonuclear proton-proton coupling constants. Among others, the proposed techniques are particularly suited for molecules presenting a great number of CH and NH spin systems. Examples and practical details of the implementation of these techniques on standard carbohydrates and peptides at C-13 and N-15 natural abundance are provided.

An isotope-filtered selective refocusing (IFSERF) experiment is presented for the sensitive and precise measurement of the proton–proton coupling constant between chemically equivalent protons. The 2D NMR method combines an initial doubly selective isotope filter based on heteronuclear cross-polarization followed by a selective J-resolved block. The coupling topologies obtained from several 2D variants of the IFSERF experiment are described for the simultaneous measurement of both proton–proton and proton–carbon coupling constants in the involved AA′XX′ spin system. Application on the determination of the relative configuration of double bonds in symmetrical molecules is illustrated.