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.     

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.     

HMBC‐1,n‐ADEQUATE spectra calculated from HMBC and 1,n‐ADEQUATE spectra

Unsymmetrical and generalized indirect covariance processing methods provide a means of mathematically combining pairs of 2D NMR spectra that share a common frequency domain to facilitate the extraction of correlation information. Previous reports have focused on the combination of HSQC spectra with 1,1‐, 1,n‐, and inverted ¹J_CC 1,n‐ADEQUATE spectra to afford carbon–carbon correlation spectra that allow the extraction of direct (¹J_CC), long‐range (ⁿJ_CC, where n ≥ 2), and ¹J_CC‐edited long‐range correlation data, respectively. Covariance processing of HMBC and 1,1‐ADEQUATE spectra has also recently been reported, allowing convenient, high‐sensitivity access to ⁿJ_CC correlation data equivalent to the much lower sensitivity n,1‐ADEQUATE experiment. Furthermore, HMBC‐1,1‐ADEQUATE correlations are observed in the F₁ frequency domain at the intrinsic chemical shift of the ¹³C resonance in question rather than at the double‐quantum frequency of the pair of correlated carbons, as visualized by the n,1, and m,n‐ADEQUATE experiments, greatly simplifying data interpretation. In an extension of previous work, the covariance processing of HMBC and 1,n‐ADEQUATE spectra is now reported. The resulting HMBC‐1,n‐ADEQUATE spectrum affords long‐range carbon–carbon correlation data equivalent to the very low sensitivity m,n‐ADEQUATE experiment. In addition to the significantly higher sensitivity of the covariance calculated spectrum, correlations in the HMBC‐1,n‐ADEQUATE spectrum are again detected at the intrinsic ¹³C chemical shifts of the correlated carbons rather than at the double‐quantum frequency of the pair of correlated carbons. HMBC‐1,n‐ADEQUATE spectra can provide correlations ranging from diagonal (⁰J_CC or diagonal correlations) to ⁴J_CC under normal circumstances to as much as ⁶J_CC in rare instances. The experiment affords the potential means of establishing the structures of severely proton‐deficient molecules. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

High resolution‐HMBC (HR‐HMBC), a new method for measuring heteronuclear long‐range coupling constants

A useful pulse sequence for measuring long‐range C? H coupling constants (J_{C? H}) named high resolution‐HMBC (HR‐HMBC) has been developed. In this pulse sequence, the J‐scaling pulse [(nt₁)/2? 180° (H/C) ? (nt₁)/2] is incorporated after the spin evolution period, and then followed by an ¹H 180° pulse to reverse the magnetization of J_{C? H} couplings. As a result, splittings of the cross peaks due to the long‐range J_{C? H} are realigned with separations of nJ_{C? H} along the F₁ dimension, and thus even the small long‐range J_{C? H} values can easily be determined. The efficiency of measuring the long‐range J_{C? H} using the proposed pulse sequences has been demonstrated in application to the complicated natural product, portmicin. Copyright © 2010 John Wiley & Sons, Ltd.     

D-HMBC versus LR-HSQMBC: Which experiment provides theoretically and experimentally the best results?

The long-range heteronuclear single quantum multiple bond correlation (LR-HSQMBC) experiment is the experiment of choice for visualizing heteronuclear long-range coupling interactions ⁿJ_CH across 4–6-bonds and is experimentally superior to the decoupled heteronuclear multiple-bond correlation (D-HMBC) experiment. Yet, the exact reasons have not been fully understood and established. On the basis of our recent investigation of the nonrefocused variants LR-HSQC and HMBC, we have extended a J_HH′-dedicated investigation to the D-HMBC and LR-HSQMBC experiments. Unlike the nonrefocused variants, the influence of homonuclear couplings J_HH′ on the intensity of long-range ⁿJ_CH cross-peaks is not easily predictable and may be summarized as follows: (a) irrespective of the magnitude and number of J_HH′ interactions long-range ⁿJ_CH cross-peaks are more intense in D-HMBC spectra as long as the evolution delay Δ is not too large, because in contrast to LR-HSQMBC no J_HH′-caused intensity zeroes will occur. (b) If J_HH′ is small and Δ large, the intensity of cross peaks in D-HMBC spectra may be weakened or may even vanish at Δ = (0.25+0.5k)/J_HH′, whereas for the LR-HSQMBC this unwanted effect occurs at Δ = k + 0.5/J_HH′. Consequently, when Δ is adjusted to visualize weak ⁿJ_CH long-range correlations, our findings corroborate that there are potentially more cross-peaks expected to show up in a LR-HSQMBC spectrum compared with a D-HMBC spectrum. This has been indeed noticed experimentally, even though the intensity of a many long-range ⁿJ_CH cross-peaks may still be higher in the spectra of the D-HMBC experiment correspondingly adjusted for detecting weak ⁿJ_CH correlations.     

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.     

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.     

Suppressing One‐Bond Correlations in HMBC Spectra: Improved Performance for the BIRD–HMBC Pulse Sequence

An improved version of the BIRD–HMBC experiment is proposed. In comparison to the original version, the filtering (suppression of ¹ J_CH signals) is accomplished using a double tuned G‐BIRD filter positioned in the middle of the long‐range correlations evolution period. Compensation of offset dependence by replacing the rectangular 180° pulses with the broadband inversion pulses (BIPs), with superior inversion performance and improved tolerance to B₁ field inhomogeneity, significantly improves the sensitivity of the original BIRD–HMBC experiment. For usual one‐bond coupling constants ranges (115–180 Hz), optimal results are easily obtained by adjusting the delays, δ, of the BIRD elements to an average J value. For larger ranges (e.g. 110–260 Hz), the use of a double tuned G‐BIRD filter allows excellent suppression degrees for all types of one‐bond constants present in a molecule, superior to the original scheme and other purging schemes. These attributes make the improved version of the BIRD–HMBC experiment a valuable and robust tool for rapid spectral analysis and rapid checks of molecular skeletons with a minimum spectrometer time. Copyright © 2009 John Wiley & Sons, Ltd.     

BASHD‐J‐resolved‐COSY: a new method for measuring proton‐proton spin coupling constants of multiplet signals

An effective pulse sequence for measuring H–H coupling constants, named BASHD‐J‐resolved‐COSY, has been developed. In the spin systems such as –CH_A–CH_B(CH₃)–CH_C–, a methine proton H_B splits into a multiplet owing to several vicinal couplings, resulting in attenuation of its cross‐peak intensity. Therefore, the measurements of ³J_H–H with respect to H_B are generally difficult in the E‐COSY‐type experiments. With the aim of accurate measurements of ³J_H‐H in such a spin system, we have developed a new pulse sequence, which selectively decouples the secondary methyl group. The proposed pulse sequence provides the simplified cross‐peak patterns, which are suitable for reliable measurements of ³J_H‐H in a complicated natural product. Copyright © 2012 John Wiley & Sons, Ltd.     

Conformation analysis of d‐glucaric acid in deuterium oxide by NMR based on its JHH and JCH coupling constants

d ‐Glucaric acid (GA) is an aldaric acid and consists of an asymmetric acyclic sugar backbone with a carboxyl group positioned at either end of its structure (i.e., the C1 and C6 positions). The purpose of this study was to conduct a conformation analysis of flexible GA as a solution in deuterium oxide by NMR spectroscopy, based on J‐resolved conformation analysis using proton–proton (³J_HH) and proton–carbon (²J_CH and ³J_CH) coupling constants, as well as nuclear overhauser effect spectroscopy (NOESY). The ²J_CH and ³J_CH coupling constants were measured using the J‐resolved heteronuclear multiple bond correlation (HMBC) NMR technique. NOESY correlation experiments indicated that H2 and H5 were in close proximity, despite the fact that these protons were separated by too large distance in the fully extended form of the chain structure to provide a NOESY correlation. The validities of the three possible conformers along the three different bonds (i.e., C2? C3, C3? C4, and C4? C5) were evaluated sequentially based on the J‐coupling values and the NOESY correlations. The results of these analyses suggested that there were three dominant conformers of GA, including conformer 1 , which was H2H3:gauche, H3H4:anti, and H4H5:gauche; conformer 2 , which was H2H3:gauche, H3H4:anti, and H4H5:anti; and conformer 3 , which was H2H3:gauche, H3H4: gauche, and H4H5:anti. These results also suggested that all three of these conformers exist in equilibrium with each other. Lastly, the results of the current study suggested that the conformational structures of GA in solution were ‘bent’ rather than being fully extended. Copyright © 2016 John Wiley & Sons, Ltd.     

A new technique for differentiating between 2J(C,H) and 3/4J(C,H) connectivities

We present a new pulse sequence that yields two simultaneously detected types of long‐range correlation spectra. The one spectrum is to show all ⁿJ(C,H) connectivities and the other is to show exclusively ²J(C,H) connectivities. The method is demonstrated by using strychnine as a test sample. A comparison with HMBC shows that the ²J(C,H)/ⁿJ(C,H) experiment supplies a ⁿJ(C,H) spectrum that is of equal standard with regard to sensitivity and spectral information. The additional ²J(C,H) spectrum allows the disentanglement of ²J(C,H) and ⁿJ(C,H) signals (n > 2) in HMBC type spectra, which greatly simplifies signal assignment and structure elucidation in general. Copyright © 2003 John Wiley & Sons, Ltd.     

J(77Se,1H) and J(77Se,13C) couplings of seleno‐carbohydrates obtained by 77Se satellite 1D 13C spectroscopy and 77Se selective HR‐HMBC spectroscopy

Seleno‐carbohydrates are those in which the oxygen of the glycosidic bond or the hydroxyl group is artificially replaced with selenium. This substitution changes ¹H and ¹³C chemical shifts and produces spin coupling constants involving ⁷⁷Se. Coupling constants, such as ^2‐3J(⁷⁷Se, ¹H), are likely to be useful for conformational analyses of glycans because such couplings are never observed in natural glycans. Several papers have discussed the relationship between ^2‐3J(⁷⁷Se, ¹H) and conformation; however, only few reports describe ^1‐3J(⁷⁷Se, ¹³C), which could also be useful. Here, we obtain ⁷⁷Se coupling constants of seleno‐carbohydrates from ⁷⁷Se‐selective HR‐HMBC and ⁷⁷Se satellites in 1D ¹³C spectra and examine their conformations using the Newman projection scheme.     

Structural discrimination from in situ measurement of 1DCH and 2DHH residual dipolar coupling constants

A fast residual dipolar coupling constant‐assisted strategy involving the simultaneous determination of scalar and total coupling constants from a single ¹J_CH/²J_HH‐resolved NMR spectrum is reported. It is shown that the concerted use of the directly measured ¹D_CH (for all CH_n multiplicities) and ²D_HH residual dipolar couplings allows an on‐the‐fly assignment of diastereotopic CH₂ protons, as well as of an efficient discrimination between diastereoisomeric structures of strychnine which contains six stereocenters. Copyright © 2017 John Wiley & Sons, Ltd.     

Efficient measurement of the sign and the magnitude of long‐range proton‐carbon coupling constants from a spin‐state‐selective HSQMBC‐COSY experiment

A spin state‐selective Heteronuclear Single‐Quantum Multiple‐Bond Connectivities (HSQMBC‐COSY) experiment is proposed to measure the sign and the magnitude of long‐range proton‐carbon coupling constants (ⁿJ(CH); n > 1) either for protonated or for non‐protonated carbons in small molecules. The simple substitution of the selective 180° ¹H pulse in the original selHSQMBC pulse scheme by a hard one allows the simultaneous evolution of both proton‐proton and proton‐carbon coupling constants during the refocusing period and enables a final COSY transfer between coupled protons. The successful implementation of the IPAP principle leads to separate mixed‐phase α/β cross‐peaks from which ⁿJ(CH) values can be easily measured by analyzing their relative frequency displacements in the detected dimension. Copyright © 2012 John Wiley & Sons, Ltd.     

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

Site‐specific ¹³C isotope labeling is a useful approach that allows for the measurement of homonuclear ¹³C,¹³C 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 ¹³C,¹³C 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. Copyright © 2014 John Wiley & Sons, Ltd.     

How to measure long-range proton-carbon coupling constants from 1H-selective HSQMBC experiments

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.     

Selective J‐resolved‐HMQC‐1 and ‐2, new methods for measuring proton–proton coupling constants in strongly coupled spin systems

Efficient pulse sequences for measuring ¹H–¹H coupling constants (J_HH) in strongly coupled spin systems, named selective J‐resolved‐HMQC‐1 and ‐2, have been developed. In the strongly coupled spin systems such as ‐CH₂‐CH_A(OH)‐CH_B(OH)‐CH₂‐, measurements of ³J_HAHB are generally difficult owing to the complicated splitting caused by the adjacent CH₂ protons. For easier and accurate measurements of ³J_HAHB in such a spin system, a selective excitation pulse is incorporated into the J‐resolved HMQC pulse sequence. In the proposed methods, only two strongly coupled protons, H_A and H_B which are excited by a selective pulse, are observed as J‐resolved HMQC signals. The cross peaks of H_A and H_B appear as doublets owing to ³J_HAHB along the F₁ dimension in the selective J‐resolved HMQC‐1 and ‐2 experiments. The efficiency of the proposed pulse sequences has been demonstrated in application to the stereochemical studies of the complicated natural product, monazomycin. Copyright © 2011 John Wiley & Sons, Ltd.