Long-range Heteronuclear Correlation Research Articles

5-Chloro-6-oxo-6H-xantheno[4,3-d]thiazole-2-carbonitrile

Xanthones and benzothiazoles are important classes of heterocyclic compounds with versatile biological activities. Herein, we describe a straightforward and scalable synthesis of 5-chloro-6-oxo-6H-xantheno[4,3-d]thiazole-2-carbonitrile, a thiazole-fused xanthone, via a six-step approach, using Appel’s salt for the synthesis of the thiazole ring. The thiazole-fused xanthone was fully characterized employing 1H and 13C NMR spectra, using direct and long-range heteronuclear correlation experiments (HMBC and HMQC).

3,6-Dihydro-5H-pyrazolo [4′,3′:5,6]pyrano[3,4-b]indol-5-one

Pyrano [3,4-b]indol-1(9H)-ones and indolo [2,3-c]coumarins are important classes of heterocyclic compounds with versatile biological activities. Herein, we describe a straightforward and scalable synthesis of 3,6-dihydro-5H-pyrazolo [4′,3′:5,6]pyrano [3,4-b]indol-5-one, a pyrazolo-fused pyrano [3,4-b]indolone, via a three step approach including Fischer-indole synthesis and intramolecular esterification. The compound is fully characterized by means of 1H and 13C NMR spectra, using direct and long-range heteronuclear correlation experiments (HMBC and HMQC).

Selective Nuclear Magnetic Resonance Method for Enhancing Long-Range Heteronuclear Correlations in Solids.

The long-range heteronuclear correlation remains a significant challenge in solid-state nuclear magnetic resonance (NMR), which is critical in the structural elucidation of biomolecular, material, and pharmaceutical solids. We propose a selective NMR method, heteronuclear selective phase-optimized recoupling (hetSPR), to selectively enhance long-range correlations of interest by utilizing characteristic chemical shifts. Compared to conventional methods, hetSPR can selectively enhance desired heteronuclear correlations (e.g., 1H-13C and 1H-19F) by factors up to 5 and largely suppress the unwanted ones. The method proves useful by enhancing the long-range correlation from an intermolecular 1H-19F distance of 4.8 Å by a factor of 2.4 in a fluorinated pharmaceutical drug, bicalutamide, under fast magic-angle spinning. It does not use selective pulses and is thus user-friendly even for nonexperts. The new method is expected to boost solid-state NMR to elucidate the structures of various solids.

Digipregnosides A–C, three novel rearranged 11,12-secopregnane glycosides, and digipregnosides D and E, 12,20-epoxypregnane glycosides from the seeds of Digitalis purpurea

Three novel rearranged 11,12-secopregnane glycosides—named, digipregnosides A (1), B (2), and C (3)—and two novel 12,20-epoxypregnane glycosides—named, digipregnosides D (4) and E (5)—were isolated from the seeds of Digitalis purpurea. Their structures were determined based on extensive spectroscopic analysis, including long-range heteronuclear single quantum multiple bond correlation (HSQMBC) spectral data, and the results of hydrolytic cleavage. Compounds 1 and 4 showed tumor selective cytotoxicity against SBC-3 small cell lung carcinoma cells at a sample concentration of 0.001 μM and induced apoptotic cell death in SBC-3 cells with IC50 values of 1.3 ± 0.16 and 0.17 ± 0.072 μM respectively.

Identifying the Unknown Content of an Ancient Egyptian Sealed Alabaster Vase from Kha and Merit’s Tomb Using Multiple Techniques and Multicomponent Sample Analysis in an Interdisciplinary Applied Chemistry Course

This article highlights the multianalytical study of exuded liquid from an ancient Egyptian sealed alabaster vase by Master’s students in an applied chemistry for cultural heritage course. Master students are introduced to the field of Archaeometry that see the collaboration of experts in different areas of research such as conservators, curators of museums, physicists, chemists, etc. The sample is a residue exuded on the linen strip sealing an ancient Egyptian alabaster vase (inventory number S.8448) from the collection of the Museo Egizio in Turin (Italy). The students start to plan the noninvasive investigation by X-ray fluorescence (XRF), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDS) for the inorganic compounds characterization, followed by the extraction of the organic components (such as oils, fats, and waxes) to be analyzed by high-resolution 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and 2D NMR correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and long-range heteronuclear correlation (HMBC) techniques and by gas chromatography coupled with mass spectrometry (GC/MS). Reference standards, spectral databases, and published data on similar artifacts served as the basis for the interpretation of the instrumental results. The approach was introduced in the course of Applied Chemistry for Cultural Heritage for Master students in Archaeology (University of Palermo, Italy), where the need is to know how to approach the scientific investigation together with the conservation scientists and how to manage with a very low amount of sample. Pedagogically, the approach introduces students to the main techniques currently used in the field of Archaeometry while reinforcing fundamental concepts in sample collecting and multicomponent microsample analysis. This interdisciplinary approach provides a unique experience that demonstrates chemistry’s broad applicability outside of the traditional laboratory. Students are guided to identify the inorganic and organic components of the exudate liquid: the first one is ascribable to clay minerals iron oxides, which could impart the brown color to the sample; the second one is ascribed to triglycerides of various kinds, which probably comes from vegetable oil.

Simplifying LR-HSQC spectra using a triple-quantum filter: The LR-HTQC experiment.

Long-range heteronuclear single quantum correlation (LR-HSQC) experiments may be applied for detecting long-range correlations but suffer from two disadvantages, common to all heteronuclear long-range correlation experiments: (i) The information density in LR-HSQC spectra may be too high to be used directly without "filtering out" shorter range correlations, and (ii) often, substantial differences in intensity among cross peaks exist, potentially hampering the visualization of weak, often crucial cross peaks. In this contribution, we propose a modified LR-HSQC experiment, the LR-HTQC experiment (Long-Range Heteronuclear Triple Quantum Correlation) that partially solves the problems aforementioned. We show theoretically and experimentally that the LR-HTQC experiment removes the intense cross peaks of CH spin pairs, substantially reduces the medium intensity of cross peaks originating from CHH' spin systems, whereas the typically weak intensity of cross peaks of CHH'H″ and C(H)n, n > 3 spin systems is less affected. Consequently, the LR-HTQC experiment affords simplified long-range heteronuclear shift correlation spectra and scales down large intensity differences among different types of cross peaks, although a certain general reduction of signal intensities has to be accepted.

NMR characterization of rearranged staurosporine aglycone analogues from the marine sponge Damiria sp.

The indolocarbazole family of bisindole alkaloids is best known for the natural product staurosporine, a protein kinase C inhibitor that belongs to the indolo[2,3-a]carbazole structural class. A large number of other indolo[2,3-a]carbazoles have subsequently been isolated and identified, but other isomeric forms of indolocarbazole natural products have rarely been reported. An extract of the marine sponge Damiria sp., which represents an understudied genus, provided two novel alkaloids named damirines A (1) and B (2). Their structures were assigned by comprehensive NMR spectroscopic analyses, and for compound 2, this included application of the LR-HSQMBC pulse sequence, a long-range heteronuclear correlation experiment that has particular utility for defining proton-deficient scaffolds. The damirines represent a new hexacyclic carbon-nitrogen framework comprised of an indolo[3,2-a]carbazole fused with either an aminoimidazole or a imidazolone ring. Compound 1 showed selective cytotoxic properties toward six different cell lines in the NCI-60 cancer screen.

Why is HMBC superior to LR-HSQC? Influence of homonuclear couplings JHH' on the intensity of long-range correlations.

Long-range heteronuclear single quantum correlation (LR-HSQC) experiments may be applied as an alternative to heteronuclear multiple-bond correlation (HMBC) experiments for detecting long-range correlations but has never enjoyed popularity for that purpose. To the best of our knowledge, the exact reasons have not yet been fully established. For both experiments, it is widely accepted that the evolution of proton-proton homonuclear couplings JHH' during the polarization transfer delays Δ leads to significant losses, and that the intensity of the observable coherence is zero when JHH' matches the condition Δ=0.5/JHH' . Here, we analyze the influence of JHH' on the intensity of long-range correlations in HMBC and LR-HSQC spectra. We show that for both experiments long-range correlations will not be canceled because of homonuclear couplings JHH' . Our theoretical and experimental results definitely establish and validate the superiority of HMBC-based experiments among the family of heteronuclear long-range correlation experiments: (a) the overall cross peak's intensity is higher, and (b) in LR-HSQC experiments, the intensity of the long-range cross peaks is additionally influenced in an unwanted way by the magnitude and number of passive homonuclear proton-proton couplings JHH' .

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 (n)JXH 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.

Carbon Multiplicity Editing in Long-Range Heteronuclear Correlation NMR Experiments: A Valuable Tool for the Structure Elucidation of Natural Products.

A recently developed NMR method to simultaneously obtain both long-range heteronuclear correlations and carbon multiplicity information in a single experiment, ME-selHSQMBC, is demonstrated as a potentially useful technique for chemical shift assignment and structure elucidation of natural products presenting complicated NMR spectra. Carbon multiplicities, even for C/CH2 and odd for CH/CH3 resonances, can be distinguished directly from the relative positive/negative phase of cross-peaks. In addition, connectivity networks can be further extended by incorporating a TOCSY propagation step. Staurosporine (1) and sungucine (2) are utilized as model compounds to demonstrate these techniques.

Structure elucidation of a proton-deficient natural product using LR-HSQMBC supported by DFT calculations

A severely proton-deficient marine alkaloid (C28H21N7O2) was intractable to structural characterization using only conventional 1D and 2D NMR techniques including HMBC data, which generally provides primarily two- and three-bond correlations. A sensitive, complementary approach to obtain very long-range (≥ 4 J CH) heteronuclear correlations is afforded by the recently reported LR-HSQMBC experiment. An INEPT pulse sequence element inserted after t1 evolution, converts anti-phase long-range heteronuclear coupling information back into in-phase magnetization thereby alleviating cancelation of responses emanating from small (< 2 Hz) scalar couplings. The role of the LR-HSQMBC experiment in the determination of this natural product structure will be illustrated.

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 1–2Hz 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. A comparative 1H–13C study is performed using strychnine as a model compound and several examples are also provided including 1H–15N applications.

The impact of LR-HSQMBC very long-range heteronuclear correlation data on computer-assisted structure elucidation

The impact of LR-HSQMBC very long-range (n)JCH heteronuclear shift correlation data as a supplement to HMBC data as input for the computer-assisted structure elucidation program, Structure Elucidator(®), is assessed for the first time. The severely proton-deficient xanthone antibiotic cervinomycin A2 and the alkaloid staurosporine were employed as a model compounds.

Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of (1)H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond (1)H-(15)N or (1)H-(13)C couplings. However, by filtering against protons directly bonded to (13)C or (15)N nuclei, signals from slowly-exchanging hydroxyls can be observed in the (1)H-NMR spectrum of a uniformly (13)C/(15)N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with (13)C/(15)N-serine or (13)C/(15)N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via (3)JC-OH couplings in long-range (13)C-HSQC spectra. These scalar couplings (~5-7 Hz) were measured in a sample of uniformly (13)C/(15)N-labeled BcX using a quantitative (13)C/(15)N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a (13)C/(15)N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.

Implementing homo- and heterodecoupling in region-selective HSQMBC experiments

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 selHSQMBC 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 1H-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.

Broadband homodecoupled heteronuclear multiple bond correlation spectroscopy

A general concept for removing proton–proton scalar J couplings in 2D NMR spectroscopy is proposed. The idea is based on introducing an additional J resolved dimension into the pulse sequence of a conventional 2D experiment to design a pseudo 3D NMR experiment. The practical demonstration is exemplified on the widely used gradient coherence selected heteronuclear long-range correlation spectroscopy (HMBC). We refer to this type of pulse sequence as tilt HMBC experiment. For every 13C chemical shift evolution increment, a homonuclear J resolved experiment is recorded. The long-range defocusing delay of the HMBC pulse sequence is exploited to implement this building block. The J resolved evolution period is incremented in a way very similar to ACCORDION spectroscopy to accommodate the buildup of heteronuclear long-range antiphase magnetisation as well. After Fourier transformation in all dimensions the spectra are tilted in the J resolved dimension. Finally, a projection along the J resolved dimension is calculated leading to almost disappearance of proton–proton spin multiplicities in the 2D tilt HMBC spectrum. The tilt HMBC experiment combines sensitivity with simple experimental setup and can be recorded with short recycle delays, when combined with Ernst angle excitation. The recorded spectra display singlet proton signals for long-range correlation peaks making an unambiguous signal assignment much easier. In addition to the new experiment a simple processing technique is applied to efficiently suppress the noise originating from forward linear prediction in the indirect evolution dimensions. In case of issues with fast repetition times, probe heating and RF power handling most of the RF pulses can be replaced by broadband, frequency swept pulses operating at much lower power.

Disentangling diffusion information of individual components in a mixture with a 3D COMPACT-IDOSY NMR experiment

A new 3D diffusion-ordered heteronuclear NMR experiment COMPACT-IDOSY (cross-polarization optimized multisite polarized accelerated time internally encoded diffusion ordered spectroscopy) has been designed and experimentally implemented on a mixture of flavonoids rutin and quercetin. The pulse sequence uses a cross-polarization mixing period and diffusion encoding gradients internally incorporated into the coherence transfer interval of a long-range heteronuclear correlation experiment. Substantial reduction in experimental time, good sensitivity and excellent resolution of signal overlap lead to the accurate determination of translational diffusion coefficients of individual components in the mixture.

Long‐Range 1H—15N Heteronuclear Shift Correlation

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Homonuclear long‐range correlation spectra from HMBC experiments by covariance processing

We present a new application of covariance nuclear magnetic resonance processing based on 1H--13C-HMBC experiments which provides an effective way for establishing indirect 1H--1H and 13C--13C nuclear spin connectivity at natural isotope abundance. The method, which identifies correlated spin networks in terms of covariance between one-dimensional traces from a single decoupled HMBC experiment, derives 13C--13C as well as 1H--1H spin connectivity maps from the two-dimensional frequency domain heteronuclear long-range correlation data matrix. The potential and limitations of this novel covariance NMR application are demonstrated on two compounds: eugenyl-beta-D-glucopyranoside and an emodin-derivative.

Synthesis and NMR Spectroscopic Characterization of Some Fluoro‐2H‐1‐benzopyran Derivatives.

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