HSQC-TOCSY Experiments Research Articles

The NOAH HSQC-COSY module revisited: A theoretical and practical comparison of pulse sequences

NMR supersequences, as exemplified by the NOAH (NMR by Ordered Acquisition using 1H detection) technique, are a powerful way of acquiring multiple 2D data sets in much shorter durations. This is accomplished through targeted excitation and detection of the magnetisation belonging to specific isotopologues (‘magnetisation pools’). Separately, the HSQC-COSY experiment has recently seen an increase in popularity due to the high signal dispersion in the indirect dimension and the removal of ambiguity traditionally associated with HSQC-TOCSY experiments. Here, we describe how the HSQC-COSY experiment can be integrated as a ‘module’ within NOAH supersequences. The benefits and drawbacks of several different pulse sequence implementations are discussed, with a particular focus on how sensitivities of other modules in the same supersequence are affected.

Sulfur-containing spiroketals from Breynia disticha and evaluations of their anti-inflammatory effect.

Breynia spp. are a key source of sulfur-containing spiroketal glycosides with potential anti-inflammatory activity. In this study, three new sulfur-containing spiroketals - breynin J (1), epibreynin J (2), and probreynogenin (3) - along with four known compounds - probreynin I (4), phyllaemblic acid (5), breynin B (6), and epibreynin B (7) - were isolated from the roots of Breynia disticha. The structures of compounds 1-7 were elucidated by extensive 1D and 2D NMR spectroscopic analyses, including 1D total correlation spectroscopy (TOCSY), HSQC, HMBC, double quantum-filtered (DQF)-COSY, heteronuclear two-bond correlation (H2BC), and HSQC-TOCSY experiments, as well as high-resolution electrospray ionization HRESIMS analysis, and quantum chemical electronic CD calculations. Furthermore, the absolute configurations of sugar residues were determined by derivatization of the hydrolysates with ʟ-cysteine methyl ester and o-tolyl isothiocyanate followed by HPLC analysis. The anti-inflammatory effects of the isolated compounds were evaluated based on the mRNA levels of proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells. Compounds 1, 2, 6, and 7 inhibited the increase in interleukin (IL)-1β and IL-6 mRNA levels stimulated by LPS. Moreover, the most potent compound 7 was found to significantly inhibit the production of IL-1β and IL-6 proteins, as revealed by the analysis of culture supernatants.

Structure of the O-specific polysaccharide of Ochrobactrum endophyticum KCTC 42485T containing 3-(3-hydroxy-2,3-dimethyl-5-oxoprolyl)amino-3,6-dideoxy-d-galactose

Ochrobactrum endophyticum (syn. Brucella endophytica) is an aerobic species of Alphaproteobacteria isolated from healthy roots of Glycyrrhiza uralensis. Here we report the structure of the O-specific polysaccharide obtained by mild acid hydrolysis of the lipopolysaccharide of the type strain KCTC 42485:→3)-α-l-FucpNAc-(1→3)-β-d-QuipNAc-(1→2)-β-d-Fucp3NAcyl-(1→where Acyl is 3-hydroxy-2,3-dimethyl-5-oxoprolyl. The structure was elucidated using chemical analyses along with 1H and 13C NMR spectroscopy (including 1H,1H COSY, TOCSY, ROESY and 1H,13C HSQC, HMBC, HSQC-TOCSY and HSQC-NOESY experiments). To our knowledge the OPS structure is novel and has not been previously published.

Three new compounds isolated from the bulbs of Fritillaria pallidiflora Schrenk and their anti-inflammatory activity

• Three new compounds were isolated from the bulbs of Fritillaria pallidiflora Schrenk. • Their structure were characterized by extensive 2D-NMR studies. • Their anti-inflammatory activities were also evaluated. Three new compounds, 17 β -cevanin-6-oxo-5 α ,20 β -diol yibeinine ( 1 ), 2-(tetrahydro-5-(2-hydroxyphenyl)-2H-pyran-3-yl) phenol ( 2 ), 1,3- O -diferuloyl-2-methoxypropane diol ( 3 ), as well as four known compounds ( 4 – 7 ), have been isolated from the ethanol extract of dried bulbs of Fritillaria pallidiflora Schrenk. All structures were determined based on their spectroscopic data (1D and 2D NMR (including 1 H NMR, 13 C NMR, 1 H- 1 H COSY, HMBC, HSQC, HSQC-TOCSY, and NOESY experiments), and MS). Biological evaluation showed that compounds 1 – 4 inhibited the production of nitric oxide (NO) in LPS-stimulated RAW 264.7 cells with IC 50 values of 18.0, 38.7, 29.5, and 47.1 μM, respectively. These results indicated that compound 1 has potential anti-inflammatory activity.

Bodiniosides S-Y, Seven New Triterpenoid Saponins from Elsholtzia bodinieri and Their Anti-Influenza Activities.

Investigation of the n-BuOH extract of the aerial parts of Elsholtzia bodinieri led to the isolation of seven new triterpenoid saponins, Bodiniosides S–Y (1–7, resp.). Their strictures were elucidated on the basis of spectroscopic techniques, including HSQC, HSBC, and HSQC–TOCSY experiments, together with acid hydrolysis and GC analysis. The anti-influenza activities of compounds 1–7 were evaluated against A/WSN/33/2009 (H1N1) virus in MDCK cells. The results showed that compounds 2 and 5 exhibited moderate anti-influenza activities against A/WSN/33/2009 (H1N1), with inhibition rates of 35.33% and 24.08%, respectively.

Long-range proton–carbon coupling constants for the determination of the stereochemical structure of organic compounds.

A new methods for elucidating stereochemistry of organic compounds was developed on the basis of long-range proton–carbon coupling constants (2,3JC,H) and interpreting spin-coupling constants (3JH,H). Reaction of compound containing pyrin ring with nucleophile reagent was done to open the ring. HSQC-TOCSY experiments one of the new NMR spectroscopy method used to measure this values of spin-coupling constants and elucidating the stereochemistry of the product.

The unique structure of bacterial polysaccharides - Immunochemical studies on the O-antigen of Proteus penneri 4034-85 clinical strain classified into a new O83 Proteus serogroup

The serological classification scheme of the opportunistic Proteus bacilli includes a number of Proteus penneri strains. The tested P. penneri 4034-85 strain turned out to be serologically distinguished in ELISA and Western blotting. The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of this strain and studied by sugar and methylation analyses and dephosphorylation along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, HMBC, and HSQC-TOCSY experiments, The O-polysaccharide was found to have a linear repeating unit containing glycerol 1-phosphate and two residues each of Gal and GlcNAc. The following O-polysaccharide structure was established, which, to our knowledge, is unique among known bacterial polysaccharide structures:→4)-β-d-GlcpNAc-(1→3)-α-d-Galp-(1→3)-β-d-GlcpNAc-(1→2)-β-d-Galp-(1→3)-Gro-1-P-(O→.

Phosphorus NMR and Its Application to Metabolomics.

Stable isotopes are routinely employed by NMR metabolomics to highlight specific metabolic processes and to monitor pathway flux. 13C-carbon and 15N-nitrogen labeled nutrients are convenient sources of isotope tracers and are commonly added as supplements to a variety of biological systems ranging from cell cultures to animal models. Unlike 13C and 15N, 31P-phosphorus is a naturally abundant and NMR active isotope that does not require an external supplemental source. To date, 31P NMR has seen limited usage in metabolomics because of a lack of reference spectra, difficulties in sample preparation, and an absence of two-dimensional (2D) NMR experiments, but 31P NMR has the potential of expanding the coverage of the metabolome by detecting phosphorus-containing metabolites. Phosphorylated metabolites regulate key cellular processes, serve as a surrogate for intracellular pH conditions, and provide a measure of a cell's metabolic energy and redox state, among other processes. Thus, incorporating 31P NMR into a metabolomics investigation will enable the detection of these key cellular processes. To facilitate the application of 31P NMR in metabolomics, we present a unified protocol that allows for the simultaneous and efficient detection of 1H-, 13C-, 15N-, and 31P-labeled metabolites. The protocol includes the application of a 2D 1H-31P HSQC-TOCSY experiment to detect 31P-labeled metabolites from heterogeneous biological mixtures, methods for sample preparation to detect 1H-, 13C-, 15N-, and 31P-labeled metabolites from a single NMR sample, and a data set of one-dimensional (1D) 31P NMR and 2D 1H-31P HSQC-TOCSY spectra of 38 common phosphorus-containing metabolites to assist in metabolite assignments.

Inter-residual Hydrogen Bonding in Carbohydrates Unraveled by NMR Spectroscopy and Molecular Dynamics Simulations.

Carbohydrates, also known as glycans in biological systems, are omnipresent in nature where they as glycoconjugates occur as oligo- and polysaccharides linked to lipids and proteins. Their three-dimensional structure is defined by two or three torsion angles at each glycosidic linkage. In addition, transglycosidic hydrogen bonding between sugar residues may be important. Herein we investigate the presence of these inter-residue interactions by NMR spectroscopy in D2 O/[D6 ]DMSO (70:30) or D2 O and by molecular dynamics (MD) simulations with explicit water as solvent for disaccharides with structural elements α-d-Manp-(1→2)-d-Manp, β-d-GlcpNAc-(1→2)-d-Manp, and α-d-Glcp-(1→4)-β-d-Glcp, all of which have been suggested to exhibit inter-residue hydrogen bonding. For the disaccharide β-d-GlcpNAc-(1→2)-β-d-Manp-OMe, the large extent of O5'⋅⋅⋅HO3 hydrogen bonding as seen from the MD simulation is implicitly supported by the 1 H NMR chemical shift and 3 JHO3,H3 value of the hydroxy proton. In the case of α-d-Glcp-(1→4)-β-d-Glcp-OMe, the existence of a transglycosidic hydrogen bond O2'⋅⋅⋅HO3 was proven by the presence of a cross-peak in 1 H,13 C HSQC-TOCSY experiments as a result of direct TOCSY transfer between HO3 of the reducing end residue and H2' (detected at C2') of the terminal residue. The occurrence of inter-residue hydrogen bonding, albeit transient, is judged important for the stabilization of three-dimensional structures, which may be essential in maintaining a conformational state for carbohydrate-protein interactions of glycans to take place in biologically important environments.

ASAP-HSQC-TOCSY for fast spin system identification and extraction of long-range couplings.

Based on Ernst-angle-type excitation and Acceleration by Sharing Adjacent Polarization (ASAP), a fast HSQC-TOCSY experiment is introduced. In the approach, the DIPSI-2 isotropic mixing period of the ASAP-HSQC is simply shifted, which provides a TOCSY period without additional application of rf-energy. The ASAP-HSQC-TOCSY allows the acquisition of a conventional 2D in about 30 s. Alternatively, it allows the acquisition of highly carbon-resolved spectra (several Hz digital resolution) on the order of minutes. An ASAP-HSQC-TOCSY-IPAP variant, finally, allows the sign-sensitive extraction of heteronuclear long-range coupling constants from a pair of highly resolved spectra in less than an hour. Pulse sequences, several example spectra, and a discussion of results are given.

(3, 2)D 1H, 13C BIRDr,X-HSQC-TOCSY for NMR structure elucidation of mixtures: application to complex carbohydrates

Overlap of NMR signals is the major cause of difficulties associated with NMR structure elucidation of molecules contained in complex mixtures. A 2D homonuclear correlation spectroscopy in particular suffers from low dispersion of 1H chemical shifts; larger dispersion of 13C chemical shifts is often used to reduce this overlap, while still providing the proton–proton correlation information e.g. in the form of a 2D 1H, 13C HSQC-TOCSY experiment. For this methodology to work, 13C chemical shift must be resolved. In case of 13C chemical shifts overlap, 1H chemical shifts can be used to achieve the desired resolution. The proposed (3, 2)D 1H, 13C BIRDr,X-HSQC-TOCSY experiment achieves this while preserving singlet character of cross peaks in the F1 dimension. The required high-resolution in the 13C dimension is thus retained, while the cross peak overlap occurring in a regular HSQC-TOCSY experiment is eliminated. The method is illustrated on the analysis of a complex carbohydrate mixture obtained by depolymerisation of a fucosylated chondroitin sulfate isolated from the body wall of the sea cucumber Holothuria forskali.

Phase modulated 2D HSQC-TOCSY for unambiguous assignment of overlapping spin systems

We present a new method that allows one to unambiguously resolve overlapping spin systems often encountered in biomolecular systems such as peptides and proteins or in samples containing a mixture of different molecules such as in metabolomics. We address this problem using the recently proposed phase modulation approach. By evolving the 1H chemical shifts in a conventional two dimensional (2D) HSQC-TOCSY experiment for a fixed delay period, the phase/intensity of set of cross peaks belonging to one spin system are modulated differentially relative to those of its overlapping counterpart, resulting in their discrimination and recognition. The method thus accelerates the process of identification and resonance assignment of individual compounds in complex mixtures. This approach facilitated the assignment of molecules in the embryo culture medium used in human assisted reproductive technology.

Exploring the use of Generalized Indirect Covariance to reconstruct pure shift NMR spectra: Current Pros and Cons

The current Pros and Cons of a processing protocol to generate pure chemical shift NMR spectra using Generalized Indirect Covariance are presented and discussed. The transformation of any standard 2D homonuclear and heteronuclear spectrum to its pure shift counterpart by using a reference DIAG spectrum is described. Reconstructed pure shift NMR spectra of NOESY, HSQC, HSQC-TOCSY and HSQMBC experiments are reported for the target molecule strychnine.

Five New Oleanane Triterpenoid Saponins from the Aerial Parts ofElsholtzia bodinieri

Five new oleanane triterpenoid saponins, bodiniosides H – L (1 – 5, resp.), were isolated from the aerial parts of Elsholtzia bodinieri. Their structures were elucidated on the basis of spectroscopic techniques, including HSQC, HMBC, and HSQC-TOCSY experiments, together with acid hydrolysis and GC analysis.

Structural studies of O-polysaccharide isolated from Cronobacter sakazakii Sequence Type 12 from a case of neonatal necrotizing enterocolitis

The O-polysaccharide (OPS) of Cronobacter sakazakii NTU 696 (Sequence Type 12) from a case of neonatal necrotizing enterocolitis was isolated from the polysaccharide fraction obtained after lipopolysaccharide (LPS) hydrolysis. Purified OPS was analyzed by NMR spectroscopy (1H, COSY, TOCSY, NOESY, HSQC, HSQC-TOCSY and HMBC experiments) and chemical methods. Obtained monosaccharide derivatives analyzed by gas chromatography and gas chromatography-mass spectrometry allowed the identification of six sugar components.Performed experiments enabled to establish a structure of the OPS repeating unit of C. sakazakii NTU 696, as:

1H and 13C chemical shift assignment of the monomers that comprise carboxymethyl cellulose

1H and 13C chemical shift assignment of the anhydroglucose units (AGUs) that comprise sodium carboxymethyl cellulose (CMC) with a degree of substitution (DS) of 0.70 was performed using 2D NMR spectra obtained from COSY, TOCSY, HSQC, and HSQC–TOCSY experiments. In the 2D COSY and TOCSY spectra, there are eight correlation networks for the H1 to H6′ atoms of AGU; each 1H resonance of the eight AGUs was assigned from the changes in the intensities of the resonances with mixing times during the TOCSY experiments. The ring 13C resonances were assigned via analysis of HSQC–TOCSY spectra and confirmed via the HSQC spectra. Comparison of the shift data enabled the assignment of the eight AGUs as two types of both 2,3,6-tri-O- and 3,6-di-O-carboxymethylated, one type each of 2,6-di-O-, 2-mono-O-, and 6-mono-O-carboxymethylated AGU, and unsubstituted AGU. The obtained shift data will also be useful for characterization of the substitution distribution of other cellulose derivatives.

Characterizing the microstructure of heparin and heparan sulfate using N-sulfoglucosamine 1H and 15N NMR chemical shift analysis.

Heparin and heparan sulfate (HS) are members of a biologically important group of highly anionic linear polysaccharides called glycosaminoglycans (GAGs). Because of their structural complexity, the molecular-level characterization of heparin and HS continues to be a challenge. The work presented herein describes an emerging approach for the analysis of unfractionated and low molecular weight heparins, as well as porcine and human-derived HS. This approach utilizes the untapped potential of (15)N NMR to characterize these preparations through detection of the NH resonances of N-sulfo-glucosamine residues. The sulfamate group (1)H and (15)N chemical shifts of six GAG microenvironments were assigned based on the critical comparison of selectively modified heparin derivatives, NMR measurements for a library of heparin-derived oligosaccharide standards, and an in-depth NMR analysis of the low molecular weight heparin enoxaparin through systematic investigation of the chemical exchange properties of NH resonances and residue-specific assignments using the [(1)H,(15)N] HSQC-TOCSY experiment. The sulfamate microenvironments characterized in this study include GlcNS(6S)-UA(2S), ΔUA(2S)-GlcNS(6S), GlcNS(3S)(6S)-UA(2S), GlcNS-UA, GlcNS(6S)-red(α), and 1,6-anhydro GlcNS demonstrating the utility of [(1)H,(15)N] HSQC NMR spectra to provide a spectroscopic fingerprint reflecting the composition of intact GAGs and low molecular weight heparin preparations.

Structures of a unique O-polysaccharide of Edwardsiella tarda PCM 1153 containing an amide of galacturonic acid with 2-aminopropane-1,3-diol and an abequose-containing O-polysaccharide shared by E. tarda PCM 1145, PCM 1151 and PCM 1158

Lipopolysaccharides of four strains of Edwardsiella tarda were degraded by mild acid hydrolysis, and the released O-polysaccharides were isolated by GPC and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, 1H, 13C HMBC, HSQC and HSQC-TOCSY experiments. The O-polysaccharide from E. tarda PCM 1153 was found to contain d-GalA, d-GlcNAc, d-Gal and 2-amino-1,3-propanediol (GroN). In the tetrasaccharide repeating unit, GroN is amide-linked to one of the GalA residues, and Gal is non-stoichiometrically 2- or 3-O-acetylated (∼45% at each position):▪Three other E. tarda strains examined (PCM 1145, PCM 1151 and PCM 1158) share the following O-polysaccharide structure:▪where Abe indicates 3,6-dideoxy-d-xylo-hexose (abequose). This structure resembles those of Citrobacter freundii O22 (PCM 1555) and Salmonella enterica O4. In accordance with the structural data, SDS–PAGE and immunoblotting of the lipopolysaccharides with anti-C. freundii O22 serum demonstrated that the O-antigens of the three E. tarda strains are serologically identical to each other and to the O-antigens of C. freundii O22 and S. enterica O4.

A Definitive NMR Solution for a Simple and Accurate Measurement of the Magnitude and the Sign of Small Heteronuclear Coupling Constants on Protonated and Non‐Protonated Carbon Atoms

Simply successful: a proton-selective HSQMBC-TOCSY experiment can be used to measure small proton-carbon ((n)J(CH); n>1) coupling constants on both protonated and non-protonated carbon atoms. The method combines in a single pulse scheme all the benefits of the widely used HSQMBC and HSQC-TOCSY experiments. The magnitude and the sign of (n)J(CH) can be determined simply with excellent accuracy.

A closer look at the nitrogen next door: 1H–15N NMR methods for glycosaminoglycan structural characterization

Recently, experimental conditions were presented for the detection of the N-sulfoglucosamine (GlcNS) NHSO3- or sulfamate 1H and 15N NMR resonances of the pharmaceutically and biologically important glycosaminoglycan (GAG) heparin in aqueous solution. In the present work, we explore further the applicability of nitrogen-bound proton detection to provide structural information for GAGs. Compared to the detection of 15N chemical shifts of aminosugars through long-range couplings using the IMPACT–HNMBC pulse sequence, the more sensitive two-dimensional 1H–15N HSQC–TOCSY experiments provided additional structural data. The IMPACT–HNMBC experiment remains a powerful tool as demonstrated by the spectrum measured for the unsubstituted amine of 3-O-sulfoglucosamine (GlcN(3S)), which cannot be observed with the 1H–15N HSQC–TOCSY experiment due to the fast exchange of the amino group protons with solvent. The 1H–15N HSQC–TOCSY NMR spectrum reported for the mixture of model compounds GlcNS and N-acetylglucosamine (GlcNAc) demonstrate the broad utility of this approach. Measurements for the synthetic pentasaccharide drug Arixtra® (Fondaparinux sodium) in aqueous solution illustrate the power of this NMR pulse sequence for structural characterization of highly similar N-sulfoglucosamine residues in GAG-derived oligosaccharides.