Clean PDI-1 SQ: Suppression of HSQC artifacts in 2D proton-detected INADEQUATE spectra by pulse sequence redesign.

Proton-detected INADEQUATE NMR experiments are widely used for structure elucidation of small molecules, in particular the implementations that display 13C single-quantum rather than double-quantum frequencies in the indirect dimension of 2D spectra. But unfortunately, such spectra in addition to the desired 1H-13C two-bond correlations also contain HSQC artifacts of comparable magnitude. The redesigned versatile experiment presented in this paper requires no compromise based on different 13C multiplicities and suppresses the HSQC artifacts that are a source of possible spectral misinterpretation. Demonstration of the new method is shown by applications to typical small molecules of different complexity.

Origin and remedy for HSQC artifacts in proton-detected INADEQUATE spectra.

NMR pulse sequences visualizing 1JCC and nJCC bond connectivity via an intermediate state of 13C-13C double-quantum coherence and 1H detection are an indispensable tool to solve small-molecule structures at the natural abundance level of 13C. A longstanding issue with these experiments set up to display 2D spectra with single-quantum frequencies is that in addition to the 1H-13C-13C correlations of interest, appearance of HSQC-type artifacts can complicate analysis and obscure JCC connectivities. The origin of these artifacts is described and remedies for their suppression are introduced. They include refocusing of 1JCH couplings prior to creation of 13C-13C double-quantum coherence, which is known to enhance sensitivity by reducing loss into zero-quantum coherence for pairs of two protonated 13C.

Four-in-one: HSQC, HSQC-TOCSY (or H2BC), TOCSY, and enhanced HMBC spectra integrated into a single NO Relaxation Delay (NORD) NMR experiment.

The NMR pulse sequence design strategy of NORD (NO Relaxation Delay) is extended to design of two new three-module experiments, NORD {HMBC}-{HSQC-TOCSY}-{TOCSY} and NORD {HMBC}-{2BOB}-{TOCSY}, each delivering four spectra - HMBC, HSQC, TOCSY, and either HSQC-TOCSY or H2BC. Compared to individual recording of these spectra particularly the sensitivity of the least sensitive module, HMBC, is enhanced by designing the homonuclear TOCSY module to allow buildup of magnetization pertinent to HMBC during its execution. Effectively, the sensitivity of the heteronuclear modules is boosted at the expense of the inherently much higher TOCSY sensitivity, thus resulting in a significant saving in spectrometer time.

Synergy and sensitivity-balance in concatenating experiments in NO relaxation delay NMR (NORD).

The NMR experiment design strategy of NO Relaxation Delay (NORD), introduced mostly as an idealized theoretical approach, is extended and put to practical use by considering synergy and sensitivity-balance in concatenation of experiments. It is illustrated by a novel experiment, NORD {HMBC}-{HSQC}-{TOCSY}, where magnetization of non-13C attached protons effectively is channeled from the TOCSY spectrum toward primarily the least sensitive spectrum of HMBC. The experiment is expected to find its place as a full-package NMR method for metabolomics, carbohydrates, peptides and small-molecules in general.

NORD: NO Relaxation Delay NMR Spectroscopy.

The novel concept of NORD (NO relaxation delay) NMR spectroscopy is introduced. The idea is to design concatenated experiments in a way that the magnetization used in the first relaxes toward equilibrium during the second and vice versa, thus saving instrument time. Applications include complete well-resolved 1 H-1 H and 1 H-13 C one-bond and long-range correlation maps of an 80 mM solution of a trisaccharide recorded in less than two minutes and hydrocortisone with extensive spectral overlap.

Double and adiabatic BANGO for concatenating two NMR experiments relying on the same pool of magnetization.

It is shown how the same pool of magnetization can be tapped twice in two different concatenations of three experiments into a single pulse sequence with only one relaxation delay. This is accomplished by using the BANGO pulse sequence element twice for independent rotations of 1H magnetization attached or not attached to 13C and it includes a refinement of BANGO with an adiabatic 13C inversion pulse resulting in improved tolerance to a spread in 1JCH coupling constants that translates directly into improved sensitivity of the modular experiment relying on 1H magnetization attached to 13C. The two new pulse sequences are SEA XLOC-HMBC-H2OBC/2BOB and SEA XLOC(ZQ)-SEA XLOC(2Q)-H2OBC/2BOB which both represent a rapid route to complete heteronuclear one-bond and long-range JCH correlation maps for small molecules, as is demonstrated on ibuprofen and prednisolone.

BANGO SEA XLOC/HMBC-H2OBC: complete heteronuclear correlation within minutes from one NMR pulse sequence.

Novel NMR experiments, BANGO SEA XLOC-H2OBC or BANGO HMBC-H2OBC, can deliver complete heteronuclear correlation maps on a time scale of minutes for small molecules. By way of example, it is demonstrated that all intra- and inter-residue 1H and 13C correlations and assignments of a trisaccharide are obtained in 20 or 5 minutes of instrument time without or with 25% NUS, respectively.

Resolution Enhancement in SEA XLOC for Heteronuclear NMR Long-Range Correlation.

It is shown how the resolution in SEA XLOC NMR spectra for distinguishing between heteronuclear two- and three-bond correlations for all 13C multiplicities can be improved by a modified experiment delivering absorptive profiles in the indirect dimension. The method is demonstrated with applications to ibuprofen and strychnine.

Distinguishing between two- and three-bond correlations for all 13C multiplicities in heteronuclear NMR spectroscopy.

A novel two-dimensional method, SEA XLOC, for distinguishing between two- and three-bond correlations in heteronuclear NMR spectroscopy is introduced and demonstrated on ibuprofen and by a complete set of correlations with a simple and most complex quaternary 13C multiplet in strychnine.

2BOB - extracting an H2BC and an HSQC-type spectrum from the same data set, and H2OBC - a fast experiment delineating the protonated 13 C backbone.

Two new related methods, 2BOB and H2OBC, for tracking out the backbone of protonated 13 C nuclei are presented. 2BOB extracts an H2BC and an HSQC-type spectrum from one and the same data set, and the combined information of these two spectra tracks out the molecular backbone. The faster method, H2OBC, typically requiring only a few minutes of instrument time, yields a single spectrum with distinct and different phases imposed on the H2BC and one-bond peaks thus obviating the need to separate them in the absence of complicating spectral overlap. Copyright © 2017 John Wiley & Sons, Ltd.

S3 HMBC hetero: Spin-State-Selective HMBC for accurate measurement of long-range heteronuclear coupling constants.

A novel method, Spin-State-Selective (S3) HMBC hetero, for accurate measurement of heteronuclear coupling constants is introduced. The method extends the S3 HMBC technique for measurement of homonuclear coupling constants by appending a pulse sequence element that interchanges the polarization in 13C-1H methine pairs. This amounts to converting the spin-state selectivity from 1H spin states to 13C spin states in the spectra of long-range coupled 1H spins, allowing convenient measurement of heteronuclear coupling constants similar to other S3 or E.COSY-type methods. As usual in this type of techniques, the accuracy of coupling constant measurement is independent of the size of the coupling constant of interest. The merits of the new method are demonstrated by application to vinyl acetate, the alkaloid strychnine, and the carbohydrate methyl ß-maltoside.

S(3) HMBC: Spin-State-Selective HMBC for accurate measurement of homonuclear coupling constants. Application to strychnine yielding thirteen hitherto unreported J(HH).

A novel method, Spin-State-Selective (S(3)) HMBC, for accurate measurement of homonuclear coupling constants is introduced. As characteristic for S(3) techniques, S(3) HMBC yields independent subspectra corresponding to particular passive spin states and thus allows determination of coupling constants between detected spins and homonuclear coupling partners along with relative signs. In the presented S(3) HMBC experiment, spin-state selection occurs via large one-bond coupling constants ensuring high editing accuracy and unequivocal sign determination of the homonuclear long-range relative to the associated one-bond coupling constant. The sensitivity of the new experiment is comparable to that of regular edited HMBC and the accuracy of the J/RDC measurement is as usual for E.COSY and S(3)-type experiments independent of the size of the homonuclear coupling constant of interest. The merits of the method are demonstrated by an application to strychnine where thirteen J(HH) coupling constants not previously reported could be measured.

Suppressing one-bond homonuclear 13C,13C scalar couplings in the J-HMBC NMR experiment: application to 13C site-specifically labeled oligosaccharides.

Site-specific (13)C isotope labeling is a useful approach that allows for the measurement of homonuclear (13)C,(13)C coupling constants. For three site-specifically labeled oligosaccharides, it is demonstrated that using the J-HMBC experiment for measuring heteronuclear long-range coupling constants is problematical for the carbons adjacent to the spin label. By incorporating either a selective inversion pulse or a constant-time element in the pulse sequence, the interference from one-bond (13)C,(13)C scalar couplings is suppressed, allowing the coupling constants of interest to be measured without complications. Experimental spectra are compared with spectra of a nonlabeled compound as well as with simulated spectra. The work extends the use of the J-HMBC experiments to site-specifically labeled molecules, thereby increasing the number of coupling constants that can be obtained from a single preparation of a molecule.

Recent progress in heteronuclear long-range NMR of complex carbohydrates: 3D H2BC and clean HMBC.

The new NMR experiments 3D H2BC and clean HMBC are explored for challenging applications to a complex carbohydrate at natural abundance of (13)C. The 3D H2BC experiment is crucial for sequential assignment as it yields heteronuclear one- and two-bond together with COSY correlations for the (1)H spins, all in a single spectrum with good resolution and non-informative diagonal-type peaks suppressed. Clean HMBC is a remedy for the ubiquitous problem of strong coupling induced one-bond correlation artifacts in HMBC spectra of carbohydrates. Both experiments work well for one of the largest carbohydrates whose structure has been determined by NMR, not least due to the enhanced resolution offered by the third dimension in 3D H2BC and the improved spectral quality due to artifact suppression in clean HMBC. Hence these new experiments set the scene to take advantage of the sensitivity boost achieved by the latest generation of cold probes for NMR structure determination of even larger and more complex carbohydrates in solution.

3D H2BC: a novel experiment for small-molecule and biomolecular NMR at natural isotopic abundance.

3D H2BC is introduced for heteronuclear assignment on natural abundance samples even for biomolecules up to at least 10 kDa in low millimolar concentrations as an overnight experiment using the latest generation of cryogenically cooled probes. The short pulse sequence duration of H2BC is maintained in the 3D version due to multiple use of the constant-time delay. Applications ranging from a small lipid to a non-recombinant protein demonstrate the merits of 3D H2BC and the ease of obtaining assignments in chains of protonated carbons.

Adiabatic low-pass J filters for artifact suppression in heteronuclear NMR.

NMR artifact purging: Modern NMR experiments depend on efficient coherence transfer pathways for their sensitivity and on suppression of undesired pathways leading to artifacts for their spectral clarity. A novel robust adiabatic element suppresses hard-to-get-at artifacts (see picture).

Clean HMBC: suppression of strong-coupling induced artifacts in HMBC spectra.

A new experiment, clean HMBC, is introduced for suppression of strong-coupling induced artifacts in HMBC spectra. The culprits of these artifacts are an inherent shortcoming of low-pass J filters in the presence of strong coupling and the (1)H pi pulse in the middle of the evolution period aimed at suppressing evolution under heteronuclear J couplings and (1)H chemical shifts. A pi pulse causes coherence transfer in strongly coupled spin systems and, as is well known in e.g., homonuclear J spectra, this leads to peaks that would not be there in the absence of strong coupling. Similar artifacts occur in HMBC spectra, but they have apparently been overlooked, presumably because they have been assigned to inefficiency of low-pass J filters or not noticed because of a coarse digital resolution in the spectra. Clean HMBC is the HMBC technique of choice for molecules notorious for strong coupling among protons, such as carbohydrates, and the new technique is demonstrated on D-mannose. Finally, a fundamental difference between HMBC and H2BC explains why strong-coupling artifacts are much less of a problem in the latter type of spectra.

HAT HMBC: a hybrid of H2BC and HMBC overcoming shortcomings of both.

A new 2D NMR experiment, HAT HMBC, that is a hybrid of H2BC and HMBC aims at establishing two-bond correlations absent in H2BC spectra because of vanishing (3)J(HH) coupling constants. The basic idea is to create an additional pi phase difference in the multiplet structure in HMBC peaks with respect to the (n+1)J(HH) coupling constant between the proton(s) attached to a (13)C and a (1)H separated by n bonds. Thus HMBC peaks associated with small J(HH) will be the most attenuated in a HAT HMBC spectrum in comparison to a regular HMBC spectrum, i.e. peaks associated with (n+1)J(HH) and (n)J(CH) will for n>2 usually be strongly attenuated. The HAT HMBC pulse sequences contain the same number of pulses as regular HMBC and are only a few milliseconds longer.

2CALIS doubling the sensitivity of CALIS for calibration of the rf field strength for indirectly observed nuclei.

A new set of pulse sequences, 2CALIS, that exhibit double sensitivity of the recent CALIS pulse sequences for accurate calibration of the rf field strength for an indirectly observed spin is introduced. The sensitivity gain is a result of not forming heteronuclear coherence transfer gradient echoes although they are excellent for artifact suppression. It is, however, demonstrated that the scheme in 2CALIS for suppression of non (13)C-attached proton magnetization is adequate for calibration of the (13)C rf field strength even on natural abundance samples. A 2CALIS version with Watergate applicable to biomolecules in aqueous solution is also presented and demonstrated both in (13)C natural abundance and on a (13)C, (15)N enriched protein sample.

Improved multiplicity-editing of HMBC NMR spectra.

A new improved multiplicity-edited HMBC experiment is introduced that leads to better J cross-talk suppression in the even (i.e. C + CH2 groups) and odd (i.e. CH + CH3 groups) subspectra. By combining data recorded with three different pulse sequences J cross-talk becomes a second-order effect in Delta1J, i.e. the deviation of an actual 1J coupling constant from the value 1J0 used in setting delays tau = (1J0)(-1/2), which is adequate for most applications. As for the original multiplicity-edited HMBC experiment, the improved experiment can be performed with a single excitation delay or implemented in a broadband version similar to broadband HMBC.