HMBC‐1,1‐ADEQUATE via generalized indirect covariance: a high sensitivity alternative to n,1‐ADEQUATE

1,1‐ADEQUATE and the related long‐range 1,n‐ and n,1‐ADEQUATE variants were developed to provide an unequivocal means of establishing ²J_CH and the equivalent of ⁿJ_CH correlations where n = 3,4. Whereas the 1,1‐ and 1,n‐ADEQUATE experiments have two simultaneous evolution periods that refocus the chemical shift and afford net single quantum evolution for the carbon spins, the n,1‐variant has a single evolution period that leaves the carbon spin to be observed at the double quantum frequency. The n,1‐ADEQUATE experiment begins with an HMBC‐type ⁿJ_CH magnetization transfer, which leads to inherently lower sensitivity than the 1,1‐ and 1,n‐ADEQUATE experiments that begin with a ¹J_CH transfer. These attributes, in tandem, serve to render the n,1‐ADEQUATE experiment less generally applicable and more difficult to interpret than the 1,n‐ADEQUATE experiment, which can in principle afford the same structural information. Unsymmetrical and generalized indirect covariance processing methods can complement and enhance the structural information encoded in combinations of experiments e.g. HSQC‐1,1‐ or ?1,n‐ADEQUATE. Another benefit is that covariance processing methods offer the possibility of mathematically combining a higher sensitivity 2D NMR spectrum with for example 1,1‐ or 1,n‐ADEQUATE to improve access to the information content of lower sensitivity congeners. The covariance spectrum also provides a significant enhancement in the F₁ digital resolution. The combination of HMBC and 1,1‐ADEQUATE spectra is shown here using strychnine as a model compound to derive structural information inherent to an n,1‐ADEQUATE spectrum with higher sensitivity and in a more convenient to interpret single quantum presentation. Copyright © 2012 John Wiley & Sons, Ltd.     

Inversion of 1JCC correlations in 1,n‐ADEQUATE spectra

ADEQUATE experiments provide an alternative to the more commonly employed GHMBC experiment for the establishment of long‐range heteronuclear connectivities. The 1,1‐ADEQUATE experiment allows the unequivocal identification of both protonated and non‐protonated carbon resonances adjacent to a protonated carbon. The 1,n‐ADEQUATE experiment establishes correlations via an initial ¹J_CH heteronuclear transfer followed by an ⁿJ_CC out‐and‐back transfer, most typically, via three carbon–carbon bonds. Hence, the 1,n‐ADEQUATE experiment allows the equivalent of ⁴J_CH heteronuclear correlations to be probed when they are not observed in a GHMBC spectrum. Aside from the lower sensitivity of the 1,n‐ADEQUATE experiment relative to GHMBC experiments, the interpretation of the former is also complicated by the ‘leakage’ of ¹J_CC correlations into the spectrum that must be identified. A method for the inversion of ¹J_CC correlations to facilitate the interpretation of 1,n‐ADEQUATE spectra is presented that allows a single experiment to be performed to access ¹J_CC and ⁿJ_CC correlation information. Copyright © 2012 John Wiley & Sons, Ltd.     

1JCC‐edited HSQC‐1,n‐ADEQUATE: a new paradigm for simultaneous direct and long‐range carbon–carbon correlation

Establishing the carbon skeleton of a molecule greatly facilitates the process of structure elucidation, leaving only heteroatoms to be inserted, heterocyclic rings to be closed, and stereochemical features to be defined. INADEQUATE, and more recently PANACEA, have been the only means of coming close to the goal of totally defining the carbon skeleton of a molecule. Unfortunately, the extremely low sensitivity and prodigious sample requirements of these experiments and the multiple receiver requirement for the latter experiment have severely restricted the usage of these experiments. Proton‐detected ADEQUATE experiments, in contrast, have considerably higher sensitivity and more modest sample requirements. By combining experiments such as 1,1‐ADEQUATE and 1,n‐ADEQUATE with higher sensitivity experiments such as GHSQC through covariance processing, sample requirements can be further reduced with a commensurate improvement in the s/n ratio and F₁ resolution of the covariance processed spectrum. We now wish to report the covariance processing of an inverted ¹J_CC 1,n‐ADEQUATE experiment with a non‐edited GHSQC spectrum to afford a spectrum that can trace the carbon skeleton of a molecule with the exception of correlations between quaternary carbons. Copyright © 2012 John Wiley & Sons, Ltd.     

Observation of potentially troublesome 2JCC correlations in 1,1‐ADEQUATE spectra

Despite the tremendous usage of HMBC to establish long‐range ¹H–¹³C and ¹H–¹⁵N heteronuclear correlations, an inherent drawback of the experiment is the indeterminate nature of the ⁿJ_XH correlations afforded by the experiment. A priori there is no reliable way of determining whether a given ⁿJ_CH correlation is, for example, via two‐, three‐, or sometimes even four‐bonds. This limitation of the HMBC experiment spurred the development of the ADEQUATE family of NMR experiments that rely on, in the case of 1,1‐ADEQUATE, an out‐and‐back transfer of magnetization via the ¹J_CC homonuclear coupling constant, which is significantly larger than ⁿJ_CC (where n = 2–4) couplings in most cases. Hence, the 1,1‐ADEQUATE experiment has generally been assumed to unequivocally provide the equivalent of ²J_CH correlations. The recent development of the 1,1‐ and 1,n‐HD‐ADEQUATE experiments that can provide homodecoupling for certain ¹J_CC and ⁿJ_CC correlations has increased the sensitivity of the ADEQUATE experiments significantly and can allow acquisition of these data in a fraction of the time required for the original iterations of this pulse sequence. With these gains in sensitivity, however, there occasionally come unanticipated consequences. We have observed that the collapse of proton multiplets, in addition to providing better s/n for the desired ¹J_CC correlations can facilitate the observation of typically weaker ²J_CC correlations across intervening carbonyl resonances in 1,1‐HD‐ADEQUATE spectra. Several examples are shown, with the results supported by the measurement of the ²J_CC coupling constants in question using J‐modulated‐HD‐ADEQUATE and DFT calculations. Copyright © 2016 John Wiley & Sons, Ltd.     

Additional pitfalls of using 1,1‐ADEQUATE for structure elucidation

1,1‐ADEQUATE is a powerful and robust NMR experiment to establish carbon–carbon connectivities using modest sample quantities when cryogenic probe technology is available. Yet potential pitfalls of applying this method are not widely appreciated, such as weak or missing ¹J_CC correlations in strongly coupled ¹³C‐¹³C AB spin systems and unusually large multi‐bond (ⁿJ_CC) correlations associated with particular functional groups. These large ⁿJ_CC correlations observed in 1,1‐ADEQUATE spectra could be mistaken for ¹J_CC correlations. Copyright © 2016 John Wiley & Sons, Ltd.     

Observation of untoward 3Jcc correlations in 1,1‐ADEQUATE spectra of pyrimidine analogs: Avoiding potential interpretation pitfalls

Recently, it has been reported that large ⁿJ_CC correlations can sometimes be observed in 1,1‐ADEQUATE spectra with significant intensity, which opens the possibility of structural misassignment. In this work, we have focused on pyrimidine‐based compounds, which exhibit multiple bond correlations in the 1,1‐ADEQUATE experiment as a consequence of ³J_CC coupling constants greater than 10 Hz. Results are supported by both the experimental measurement of ³J_CC coupling constants in question using J‐modulated‐ADEQUATE and density functional theory calculations.     

Posaconazole: Application of HSQC‐ADEQUATE from General Indirect Covariance Processing

Posaconazole is a structurally complex triazole antifungal agent that, by virtue of its structural complexity, provides a good test molecule for the evaluation of NMR structure elucidation methodologies. Although GHMBC and related long‐range ¹H–¹³C heteronuclear shift correlation techniques are extremely powerful, at the same time, when dealing with unknowns, they can be problematic in that there is no way to readily differentiate adjacent (² J_CH) correlations from longer range correlations, e.g., ³J_CH and ⁿJ_CH, n > 3. The 1,1‐ADEQUATE experiment, in contrast, provides unequivocal experimental access to adjacent carbon–carbon correlation information, albeit with a sensitivity penalty, as the experiment involves an adjacent ¹³C–¹³C out‐and‐back magnetization transfer. In part, the sensitivity penalty can be overcome by using unsymmetrical indirect covariance or general indirect covariance processing methods. The application of these methods through the coprocessing of multiplicity‐edited GHSQC and 1,1‐ADEQUATE data to generate an HSQC‐ADEQUATE correlation plot is demonstrated for posaconazole.     

Measurement of long‐range heteronuclear coupling constants using the peak intensity in classical 1D HMBC spectra

In this contribution, we show that the magnitude of heteronuclear long‐range coupling constants can be directly extracted from the classical 1D HMBC spectra, as all multiplet lines of a cross‐peak always and exclusively vanish for the condition Δ = k/ⁿJ_CH. To the best of our knowledge, this feature of the classical HMBC has not yet been noticed and exploited. This condition holds true, irrespective of the magnitude and numbers of additional active and passive homonuclear ⁿJ_HH′ couplings. Alternatively, the ⁿJ_CH value may also be evaluated by fitting the peak's intensity in the individual spectra to its simple sin(πⁿJ_CHΔ)exp(−Δ/T_2eff) dependence. Compared to the previously proposed J‐HMBC sequences that also use the variation of the cross‐peak's intensity for extracting the coupling constants, the classical HMBC pulse sequence is significantly more sensitive.     

BASHD‐J‐resolved‐HMBC,an efficient method for measuring proton–proton and heteronuclear long‐range coupling constants

Natural products often possess various spin systems consisting of a methine group directly bonded to a methyl group (e.g. –CH_a–CH_b(CH₃)–CH_c–). The methine proton H_b splits into a broadened multiplet by coupling with several vicinal protons, rendering analysis difficult of ⁿJ_C–H with respect to H_b in the J‐resolved HMBC‐1. In purpose of the reliable and easy measurements of ⁿJ_C–H and ⁿJ_H–H in the aforesaid spin system, we have developed a new technique, named BASHD‐J‐resolved‐HMBC. This method incorporates band selective homo decoupled pulse and J‐scaling pulse into HMBC. In this method, high resolution cross peaks can be observed along the F₁ axis by J‐scaling pulse, and band selective homo decoupled pulse simplified multiplet signals. Determinations of ⁿJ_C–H and ⁿJ_H–H of multiplet signals can easily be performed using the proposed pulse sequence. Copyright © 2013 John Wiley & Sons, Ltd.     

Selective J‐resolved HMBC,an efficient method for measuring heteronuclear long‐range coupling constants

An efficient pulse sequence for measuring long‐range C? H coupling constants (J_{C? H}) named selective J‐resolved HMBC has been developed by replacing a ¹H 180° pulse with a selective ¹H 180° pulse and the HMBC pulse scheme with the constant time (CT) HMBC employed in the J‐resolved HMBC pulse sequence that we reported previously. The novel pulse sequence providing only long‐range J_{C? H} cross peaks for easy and accurate analysis enables to overcome disadvantages of the previous HMBC‐based pulse sequences (J‐resolved HMBC‐1) along with maintaining high sensitivity. The efficiency of measuring long‐range J_{C? H} using the proposed pulse sequence has been demonstrated in applications to the complicated natural products, portmicin and monazomycin. Copyright © 2009 John Wiley & Sons, Ltd.     

Coniothyrione: anatomy of a structure revision

Coniothyrione is a xanthone‐derived antibiotic reported several years ago by researchers at Merck & Co. Inc. Revision of the position of the chloro substitution was recently proposed on the basis of empirical reinterpretation of the carbon chemical shift data and a hypothetical biosynthetic argument without the acquisition of any new spectral data to support the postulated change in substituent location. The originally published HMBC data lead to an equivocal assignment of the structure and do not provide a solid basis of support for either structure. Neural network ¹³C chemical shift calculations and density functional theory calculations also led to undifferentiated structures. Definitive confirmation of the structure of coniothyrione based on the acquisition and interpretation of 1,1‐ADEQUATE and inverted ¹J_CC 1,n‐ADEQUATE data is now reported. Copyright © 2013 John Wiley & Sons, Ltd.     

HSQC-ADEQUATE: an investigation of data requirements

Utilizing ¹³C‐¹³C connectivity networks for the assembly of carbon skeletons from HSQC‐ADEQUATE spectra was recently reported. HSQC‐ADEQUATE data retain the resonance multiplicity information of the multiplicity‐edited GHSQC spectrum and afford a significant improvement in the signal‐to‐noise (s/n) ratio relative to the 1,1‐ADEQUATE data used in the calculation of the HSQC‐ADEQUATE spectrum by unsymmetrical indirect covariance (UIC) processing methods. The initial investigation into the computation of HSQC‐ADEQUATE correlation plots utilized overnight acquisition of the 1,1‐ADEQUATE data used for the calculation. In this communication, we report the results of an investigation of the reduction in acquisition time for the 1,1‐ADEQUATE data to take advantage of the s/n gain during the UIC processing to afford the final HSQC‐ADEQUATE correlation plot. Data acquisition times for the 1,1‐ADEQUATE spectrum can be reduced to as little as a few hours, while retaining excellent s/n ratios and all responses contained in spectra computed from overnight data acquisitions. Concatenation of multiplicity‐edited GHSQC and 1,1‐ADEQUATE data also allows the interrogation of submilligram samples with 1,1‐ADEQUATE data when using spectrometers equipped with 1.7‐mm Micro CryoProbes ?. Copyright © 2011 John Wiley & Sons, Ltd.     

Selective COSY‐J‐resolved‐HMBC,a new method for improving sensitivity of cross peaks of methine proton signals attached to a methyl group

Improved pulse sequences for measuring long‐range C‐H coupling constants (ⁿJ_C‐H), named selective COSY‐J‐resolved HMBC‐1 and ?2, have been developed. In the spin systems, such as ‐CH_C‐CH_A(CH₃)‐CH_B‐, a methine proton H_A splits into a multiplet owing to several vicinal couplings with protons, resulting in attenuation of its cross‐peak intensity. Therefore, the measurements of ⁿJ_C‐H with H_A are generally difficult in the J‐resolved HMBC or selective J‐resolved HMBC spectrum. With the aim of accurate measurements of ⁿJ_C‐H in such a spin system, we have developed new pulse sequences, which transfer the magnetization of a methyl group to its adjacent methine proton. The proposed pulse sequences successfully enable to enhance the sensitivity of H_A cross peak in comparison with the selective J‐resolved HMBC pulse sequence. Copyright © 2012 John Wiley & Sons, Ltd.     

On the interference of J(HH) modulation in HSQMBC‐IPAP and HMBC‐IPAP experiments

The effects of phase modulation due to homonuclear proton–proton coupling constants in HSQMBC‐IPAP and HMBC‐IPAP experiments are experimentally evaluated. We show that accurate values of small proton–carbon coupling constants, ⁿJ_CH, can be extracted even for phase‐distorted cross‐peaks obtained from a selHSQMBC experiment applied simultaneously on two mutually J‐coupled protons. On the other hand, an assessment of the reliability of ⁿJ_CH measurement from distorted cross‐peaks obtained in broadband IPAP versions of equivalent HMBC and HSQMBC experiments is also presented. Finally, we show that HMBC‐COSY experiments could be an excellent complement to HMBC for the measurement of small ⁿJ_CH values. Copyright © 2013 John Wiley & Sons, Ltd.     

Unsymmetrical covariance processing of COSY or TOCSY and HSQC NMR data to obtain the equivalent of HSQC‐COSY or HSQC‐TOCSY spectra

Artifacts observed in the indirect covariance NMR spectrum of HSQC‐TOCSY data have recently been analyzed and a method for their elimination proposed. More recently, unsymmetrical covariance processing has been applied HSQC and HMBC spectral data to afford long‐range carbon‐carbon correlation information equivalent to that obtained from n, 1‐, 1, n‐ and m,n‐ADEQUATE spectra. We now wish to describe the results obtained through the application of unsymmetrical covariance processing of HSQC and COSY or TOCSY data, which affords the equivalent of HSQC‐COSY and HSQC‐TOCSY data in a fraction of the time required to record these spectra directly and with considerably higher sensitivity.     

19F-detected dual-optimized inverted 1JCC 1,n-ADEQUATE

Modification of the recently reported ¹⁹F-detected 1,1-ADEQUATE experiment that incorporates dual-optimization to selectively invert a wide range of ¹J_CC correlations in a 1,n-ADEQUATE experiment is reported. Parameters for the dual-optimization segment of the pulse sequence were modified to accommodate the increased size of ¹J_CC homonuclear coupling constants of poly- and perfluorinated molecules relative to protonated molecules to allow broadband inversion of the ¹J_CC correlations. The observation and utility of isotope shifts are reported for the first time for 1,1- and 1,n-ADEQUATE correlations.     

ADEQUATE CR: 13C connectivity mapping in indirect detection mode with composite refocusing

We report a novel rare spin correlation experiment termed ADEQUATE with composite refocusing (CR), which is the ¹H‐detected version of 2D INADEQUATE CR. ADEQUATE CR begins with a polarization transfer from protons to the attached carbon, followed by ¹³C–¹³C double‐quantum (DQ) preparation. Unlike the ADEQUATE class of experiments, ¹³C DQ coherence is converted after evolution to single‐quantum single transitions (SQ‐STs) by CR. ¹³C SQ‐ST is then transferred back to the coupled protons by a coherence order selective reconversion. The present sequence produces partial transition selectivity in the ¹H dimension as does ¹H Indirect detected ¹³C Low‐Abundance Single‐transition correlation Spectroscopy (HICLASS), thereby mitigating the reduction in sensitivity enhancement because of the presence of homonuclear proton couplings. However, unlike HICLASS (which is an experiment that involves SQ‐TS evolution), no homonuclear zero quantum mixing is required on the ¹³C channel in the present experiment. Experimental results are demonstrated on a variety of samples, establishing the efficiency of the proposed method. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

NMR spectral properties of the tetramantanes – nanometer‐sized diamondoids

Tetramantanes, and all diamondoid hydrocarbons, possess carbon frameworks that are superimposable upon the cubic diamond lattice. This characteristic is invaluable in assigning their ¹H and ¹³C NMR spectra because it translates into repeating structural features, such as diamond‐cage isobutyl moieties with distinctively complex methine to methylene signatures in COSY and HMBC data, connected to variable, but systematic linkages of methine and quaternary carbons. In all tetramantane C₂₂H₂₈ isomers, diamond‐lattice structures result in long‐range ⁴J_HH, W‐coupling in COSY data, except where negated by symmetry; there are two highly symmetrical and one chiral tetramantane (showing seven ⁴J_HH). Isobutyl‐cage methines of lower diamondoids and tetramantanes are the most shielded resonances in their ¹³C spectra (<29.5 ppm). The isobutyl methylenes are bonded to additional methines and at least one quaternary carbon in the tetramantanes. W‐couplings between these methines and methylenes clarify spin‐network interconnections and detailed surface hydrogen stereochemistry. Vicinal couplings of the isobutyl methylenes reveal positions of the quaternary carbons: HMBC data then tie the more remote spin systems together. Diamondoid ¹³C NMR chemical shifts are largely determined by α and β effects, however γ‐shielding effects are important in [123]tetramantane. ¹H NMR chemical shifts generally correlate with numbers of 1,3‐diaxial H–H interactions. Tight van der Waals contacts within [123]tetramantane's molecular groove, however, form improper hydrogen bonds, deshielding hydrogen nuclei inside the groove, while shielding those outside, indicated by Δδ of 1.47 ppm for geminal hydrogens bonded to C‐3,21 . These findings should be valuable in future NMR studies of diamondoids/nanodiamonds of increasing size. Copyright © 2015 John Wiley & Sons, Ltd.     

HSQC-1,n-ADEQUATE: a new approach to long-range 13C-13C correlation by covariance processing

Long-range, two-dimensional heteronuclear shift correlation NMR methods play a pivotal role in the assembly of novel molecular structures. The well-established GHMBC method is a high-sensitivity mainstay technique, affording connectivity information via (n)J(CH) coupling pathways. Unfortunately, there is no simple way of determining the value of n and hence no way of differentiating two-bond from three- and occasionally four-bond correlations. Three-bond correlations, however, generally predominate. Recent work has shown that the unsymmetrical indirect covariance or generalized indirect covariance processing of multiplicity edited GHSQC and 1,1-ADEQUATE spectra provides high-sensitivity access to a (13)C-(13) C connectivity map in the form of an HSQC-1,1-ADEQUATE spectrum. Covariance processing of these data allows the 1,1-ADEQUATE connectivity information to be exploited with the inherent sensitivity of the GHSQC spectrum rather than the intrinsically lower sensitivity of the 1,1-ADEQUATE spectrum itself. Data acquisition times and/or sample size can be substantially reduced when covariance processing is to be employed. In an extension of that work, 1,n-ADEQUATE spectra can likewise be subjected to covariance processing to afford high-sensitivity access to the equivalent of (4)J(CH) GHMBC connectivity information. The method is illustrated using strychnine as a model compound.