Targeted projection NMR spectroscopy for unambiguous metabolic profiling of complex mixtures

Unambiguous identification of individual metabolites present in complex mixtures such as biofluids constitutes a crucial prerequisite for quantitative metabolomics, toward better understanding of biochemical processes in living systems. Increasing the dimensionality of a given NMR correlation experiment is the natural solution for resolving spectral overlap. However, in the context of metabolites, natural abundance acquisition of ¹H and ¹³C NMR data virtually excludes the use of higher dimensional NMR experiments (3D, 4D, etc.) that would require unrealistically long acquisition times. Here, we introduce projection NMR techniques for studies of complex mixtures, and we show how discrete sets of projection spectra from higher dimensional NMR experiments are obtained in a reasonable time frame, in order to capture essential information necessary to resolve assignment ambiguities caused by signal overlap in conventional 2D NMR spectra. We determine optimal projection angles where given metabolite resonances will have the least overlap, to obtain distinct metabolite assignment in complex mixtures. The method is demonstrated for a model mixture composition made of ornithine, putrescine and arginine for which acquisition of a single 2D projection of a 3D ¹H–¹³C TOCSY‐HSQC spectrum allows to disentangle the metabolite signals and to access to complete profiling of this model mixture in the targeted 2D projection plane. Copyright © 2010 John Wiley & Sons, Ltd.     

Study of near‐symmetric cyclodextrins by compressed sensing 2D NMR

The compressed sensing NMR (CS‐NMR) is an approach to processing of nonuniformly sampled NMR data. Its idea is to introduce minimal l_p‐norm (0 < p ≤ 1) constraint to a penalty function used in a reconstruction algorithm. Here, we demonstrate that 2D CS‐NMR spectra allow the full spectral assignment of near‐symmetric β‐cyclodextrin derivatives (mono‐modified at the C6 position). The application of CS‐NMR ensures experimental time saving and the resolution improvement, necessary because of very low chemical shift dispersion. In the overnight experimental time, the set of properly resolved 2D NMR spectra required for the unambiguous assignment of mono(6‐deoxy‐6‐(1‐1,2,3‐triazo‐4‐yl)‐1‐propane‐3‐O‐(phenyl)) β‐cyclodextrin was obtained. The highly resolved HSQC spectrum was reconstructed from 5.12% of the data. Moreover, reconstructed 2D HSQC–TOCSY spectrum yielded information about the correlations within one sugar unit, and 2D HSQC–NOESY technique allowed the sequential assignment of the glucosidic units. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of three saponins from a fraction using 1D DOSY as a solvent signal suppression tool. Agabrittonosides E–F. Furostane Saponins from Agave brittoniana Trel. spp. Brachypus

A careful NMR analysis, especially by 1D TOCSY and 1D ROESY, of a refined saponin fraction allowed us to determine the structure of three saponins from a polar extract of Agave brittoniana Trel. spp. Brachypus leaves. The use of 1D DOSY for the suppression of the solvent signal was useful to obtain the chemical shifts of anomeric signals. A full assignment of the ¹H and ¹³C spectral data for the new saponins, agabrittonosides E–F (1–2) and the well‐known Karatavioside C (3) and their methoxyl derivatives, is reported. The structures were established using a combination of 1D and 2D (¹H, ¹H‐COSY, TOCSY, ROESY, g‐HSQC, g‐HMBC and g‐HSQC‐TOCSY) NMR techniques and ESI–MS. In addition, the methoxylation of these furostane saponins in the presence of MeOH was studied. Copyright © 2010 John Wiley & Sons, Ltd.     

The assignment of the 1H and the 13C NMR chemical shifts of N-phenyl-2,4-dimethylbuta-1,3-diene-1,4-sultam

The assignment of the signals in the ¹³C and ¹H NMR spectra of N-phenyl-2,4-dimethylbuta-1,3-diene-1,4-sultam is difficult for the signal pairs C-2 and C-4, C-1 and C-3, (C-1)? H, (C-2)? CH₃ and (C-4)? CH₃. The ¹³C NMR spectrum recorded under gated decoupling conditions provide long-range couplings which make possible an unambiguous assignment of the ¹³C NMR signal pairs. Application of the ¹H CW off-resonance decoupling technique in recording the ¹³C NMR spectra enables the assignment information from the ¹³C NMR spectrum to be transferred to the ¹H NMR spectrum.     

Petrorhagiosides A – D,New γ‐Pyrone Derivatives from Petrorhagia saxifraga Link

Four novel γ‐pyrone (=4H‐pyran‐4‐one) metabolites, petrorhagiosides A–D, along with four known analogs, have been isolated from the MeOH extract of Petrorhagia saxifraga, a perennial herbaceous plant typical of Mediterranean vegetation. The structures of the new compounds were established on the basis of extensive spectroscopic analyses including 1D‐ an 2D‐NMR (¹H,¹H‐DQ‐COSY, TOCSY, HSQC, CIGAR‐HMBC, and HSQC‐TOCSY) experiments.     

Interleaved Dual NMR Acquisition of Equivalent Transfer Pathways in TOCSY and HSQC Experiments

A dual NMR data acquisition strategy to handle and detect two active equivalent transfer pathways is presented and discussed. We illustrate the power of this time-efficient approach by collecting two different 2D spectra simultaneously in a single experiment: i) TOCSY or HSQC-TOCSY spectra with different mixing times, ii) F2-¹³C-coupled and decoupled HSQC spectra, iii) conventional and pure-shift HSQC spectra, or iv) complementary HSQC and HSQC-TOCSY spectra.     

Metabolic phenotyping of human plasma by 1H‐NMR at high and medium magnetic field strengths: a case study for lung cancer

Accurate identification and quantification of human plasma metabolites can be challenging in crowded regions of the NMR spectrum with severe signal overlap. Therefore, this study describes metabolite spiking experiments on the basis of which the NMR spectrum can be rationally segmented into well‐defined integration regions, and this for spectrometers having magnetic field strengths corresponding to ¹H resonance frequencies of 400 MHz and 900 MHz. Subsequently, the integration data of a case–control dataset of 69 lung cancer patients and 74 controls were used to train a multivariate statistical classification model for both field strengths. In this way, the advantages/disadvantages of high versus medium magnetic field strength were evaluated. The discriminative power obtained from the data collected at the two magnetic field strengths is rather similar, i.e. a sensitivity and specificity of respectively 90 and 97% for the 400 MHz data versus 88 and 96% for the 900 MHz data. This shows that a medium‐field NMR spectrometer (400–600 MHz) is already sufficient to perform clinical metabolomics. However, the improved spectral resolution (reduced signal overlap) and signal‐to‐noise ratio of 900 MHz spectra yield more integration regions that represent a single metabolite. This will simplify the unraveling and understanding of the related, disease disturbed, biochemical pathways. Copyright © 2017 John Wiley & Sons, Ltd.     

A conformational NMR analysis of methymycin aglycones: complete and unambiguous assignments of stereochemically diverse glycosylated methymycin analogs by 1D and 2D NMR techniques and molecular modeling

The ¹H and ¹³C NMR spectra of 10‐deoxymethynolide (1), 8.9‐dihydro‐10‐deoxymethynolide (2) and its glycosylated derivatives (3–9) were analyzed using gradient‐selected NMR techniques, including 1D TOCSY, gCOSY, 1D NOESY (DPFGSENOE), NOESY, gHMBC, gHSQC and gHSQC‐TOCSY. The NMR spectral parameters (chemical shifts and coupling constants) of 1–9 were determined by iterative analysis. For the first time, complete and unambiguous assignment of the ¹H NMR spectrum of 10‐deoxymethynolide (1) has been achieved in CDCl₃, CD₃OD and C₆D₆ solvents. The ¹H NMR spectrum of 8,9‐dihydro‐10‐deoxymethynolide (2) was recorded in CDCl₃, (CD₃)₂CO and CD₃OD solutions to determine the conformation. NMR‐based conformational analysis of 1 and 2 in conjugation with molecular modeling concluded that the 12‐membered ring of the macrolactones may predominantly exist in a single stable conformation in all solvents examined. In all cases, a change in solvent caused only small changes in chemical shifts and coupling constants, suggesting that all glycosylated methymycin analogs exist with similar conformations of the aglycone ring in solution. Copyright © 2013 John Wiley & Sons, Ltd.     

Special reactions of α,β-unsaturated ketones: Part II. Structure and stereochemistry of perhydro-5a,11a-epoxydibenz[b,f]oxepin-4a-ol,a bridged Diels–Alder dimer of 2-methylenecyclohexanone

The paper deals with the structure elucidation of (4aRS,5aRS,9aRS,11aRS)-perhydro-5a,11a-epoxydibenz[b,f]oxepin-4a-ol ( 6 ), which is unexpectedly formed from 2-dimethylaminomethylcyclohexanone hydrochloride ( 1 ·HCl) in the presence of phenylguanidine in boiling ethanol. In detail, two molecules of 2-methylenecyclohexanone (generated from 1 by elimination of dimethylamine) afforded via Diels–Alder reaction, subsequent addition of water and ring closure a product C₁₄H₂₂O₃, which finally proved to be the title compound. The structure of the tetracyclic hemiacetal 6 was deduced from one- and two-dimensional NMR experiments (1D TOCSY, DEPT-135, ¹H,¹H-COSY, gs-HSQC, gs-HMBC, gs-HSQC-TOCSY). In particular, the one-dimensional selective TOCSY and the gradient-selected HSQC-TOCSY, performed as a pseudo-3D experiment without the evolution period in the TOCSY part, proved to be very helpful for the unambiguous assignment of signals in overcrowded regions. NOE data confirmed the stereochemistry of epoxydibenzoxepinol 6 . Copyright © 2001 John Wiley & Sons, Ltd.     

Application of HPLC‐NMR for the Rapid Chemical Profiling of a Southern Australian Sponge,Dactylospongia sp.

Rapid chemical profiling of the antitumour active crude dichloromethane extract of the marine sponge, Dactylospongia sp. was undertaken. A combination of both offline (HPLC followed by NMR and MS) and on‐line (on‐flow and stop‐flow HPLC‐NMR) chemical profiling approaches was adopted to establish the exact nature of the major constituents present in the dichloromethane extract of this sponge. On‐flow HPLC‐NMR analysis was employed to initially identify components present in the dichloromethane extract, while stop‐flow HPLC‐NMR experiments were then conducted on the major component present, resulting in the partial identification of pentaprenylated p‐quinol ( 5 ). Subsequent off‐line RP semi‐preparative HPLC isolation of 5 followed by detailed spectroscopic analysis using NMR and MS permitted the complete structure to be established. This included the first complete carbon NMR chemical shift assignment of 5 based on the heteronuclear 2‐D NMR experiments, together with the first report of its antitumour activity. This study represents one of the few reports describing the application of HPLC‐NMR to chemically profile secondary metabolites from a marine organism.     

The 13C NMR spectrum of abietic acid and its methyl ester

The assignment of lines in the ¹³C NMR spectrum of abietic acid and its methyl ester have been made using carbon T₁ values and lanthanide induced shift effects. Structure–NMR parameter values are briefly discussed.     

A Comparison of the hyphenated experiments GHMQC-TOCSY and GHSQC-TOCSY

Heteronuclear shift correlations generally may be established using either the multiple quantum-based GHMQC experiment or, alternately, the single quantum-based GHSQC experiment. A scant few reports contained in the literature have compared results obtained with both types of sequences. The F₁ resolution of the GHMQC and GHSQC experiments are compared using the polynuclear heteroaromatic naphtho-[2′,1′:5,6]naphtho[2′,1′:4,5]thieno[2,3-c]quinoline. Even when augmented by linear prediction in F₁, the single quantum-based GHSQC sequence gives better F₁ resolution than its multiple quantum counterpart. To date, no studies have compared the hyphenated analogs of these experiments, GHMQC- and GHSQC- TOCSY. Similar conclusions to GHMQC/GHSQC are drawn for the comparison of GHMQC- and GHSQC- TOCSY experiments with inverted direct responses with, and without, linear prediction. The latter is recommended whenever there is congestion in both the F₂ and F₁ frequency domains.     

Metabolite assignment of ultrafiltered synovial fluid extracted from knee joints of reactive arthritis patients using high resolution NMR spectroscopy

Currently, there are no reliable biomarkers available that can aid early differential diagnosis of reactive arthritis (ReA) from other inflammatory joint diseases. Metabolic profiling of synovial fluid (SF)—obtained from joints affected in ReA—holds great promise in this regard and will further aid monitoring treatment and improving our understanding about disease mechanism. As a first step in this direction, we report here the metabolite specific assignment of ¹H and ¹³C resonances detected in the NMR spectra of SF samples extracted from human patients with established ReA. The metabolite characterization has been carried out on both normal and ultrafiltered (deproteinized) SF samples of eight ReA patients (n = 8) using high-resolution (800 MHz) ¹H and ¹H─¹³C NMR spectroscopy methods such as one-dimensional ¹H CPMG and two-dimensional J-resolved¹H NMR and homonuclear ¹H─¹H TOCSY and heteronuclear¹H─¹³C HSQC correlation spectra. Compared with normal SF samples, several distinctive ¹H NMR signals were identified and assigned to metabolites in the ¹H NMR spectra of ultrafiltered SF samples. Overall, we assigned 53 metabolites in normal filtered SF and 64 metabolites in filtered pooled SF sample compared with nonfiltered SF samples for which only 48 metabolites (including lipid/membrane metabolites as well) have been identified. The established NMR characterization of SF metabolites will serve to guide future metabolomics studies aiming to identify/evaluate the SF-based metabolic biomarkers of diagnostic/prognostic potential or seeking biochemical insights into disease mechanisms in a clinical perspective.     

Structure and Metabolic‐Flow Analysis of Molecular Complexity in a 13C‐Labeled Tree by 2D and 3D NMR

Improved signal identification for biological small molecules (BSMs) in a mixture was demonstrated by using multidimensional NMR on samples from ¹³C‐enriched Rhododendron japonicum (59.5 atom%) cultivated in air containing ¹³C‐labeled carbon dioxide for 14 weeks. The resonance assignment of 386 carbon atoms and 380 hydrogen atoms in the mixture was achieved. 42 BSMs, including eight that were unlisted in the spectral databases, were identified. Comparisons between the experimental values and the ¹³C chemical shift values calculated by density functional theory supported the identifications of unlisted BSMs. Tracing the ¹³C/¹²C ratio by multidimensional NMR spectra revealed faster and slower turnover ratios of BSMs involved in central metabolism and those categorized as secondary metabolites, respectively. The identification of BSMs and subsequent flow analysis provided insight into the metabolic systems of the plant.     

Synthesis of new cyano-substituted analogues of Tröger's bases from bromo-derivatives. A stereochemical dependence of long-range (nJHH,n = 4, 5, and 6) proton–proton and proton–carbon (nJCH,n = 1, 2, 3, 4, and 5) coupling constants of these compounds

A free-catalyst microwave-assisted cyanation of brominated Tröger's base derivatives ( 2a - f ) is reported. The procedure is simple, efficient, and clean affording the nitrile compounds ( 3a - e, I ) in very good yields. Complete assignment of ¹H and ¹³C chemical shifts of 2a - f, I and 3a - d, I was achieved using gradient selected 1D nuclear magnetic resonance (NMR) techniques (1D zTOCSY, PSYCHE, DPFGSE NOE, and DEPT), homonuclear 2D NMR techniques (gCOSY and zTOCSY), and heteronuclear 2D NMR techniques (gHSQCAD/or pure-shift gHSQCAD, gHMBCAD, bsHSQCNOESY, and gHSQCAD-TOCSY) with adiabatic pulses. Determination of the long-range proton–proton coupling constants ⁿJ_HH (n = 4, 5, 6) was accomplished by simultaneous irradiation of two protons at appropriate power levels. In turn, determined coupling constants were tested by an iterative simulation program by calculating the ¹H NMR spectrum and comparing it to the experimental spectrum. The excitation-sculptured indirect-detection experiment (EXSIDE) and ¹H-¹⁵N CIGARAD-HMBC (constant time inverse-detection gradient accordion rescaled heteronuclear multiple bond correlation) were applied for determination of long-range carbon–proton coupling constants ⁿJ_CH (n = 2, 3, and 4) and for assignment of ¹⁵N chemical shift at natural abundance, respectively. DFT/B3LYP optimization studies were performed in order to determine the geometry of 2c using 6-31G(d,p), 6-311G(d,p), and 6–311 + G(d,p) basis sets. For calculation of ¹H and ¹³C chemical shifts, ⁿJ_HH (n = 2, 3, 4, 5, and 6), and ⁿJ_CH (n = 1, 2, 3, and 4) coupling constants, the GIAO method was employed at the B3LYP/6-31G(d,p), B3LYP/6-31+G(d,p), B3LYP/6-311+G(d,p), B3LYP/6-311++G(2d,2p), B3LYP/cc-pVTZ), and B3LYP/aug-cc-pVTZ) levels of theory. For the first time, a stereochemical dependence magnitude of the long-range ⁿJ_HH (n = 4, 5, and 6) and ⁿJ_CH (n = 1, 2, 3, 4, and 5) have been found in bromo-substituted analogues of Tröger's bases.     

Calculation and Control of Solid-Liquid Extraction with Characteristic Function

The kinetics of extraction of practically interesting vegetable raw materials: carsil (Silibium marianum L.), amorpha (Amorpha fructicosa L.), and coriander (Coriandrum sativum L.), was studied experimentally. The results obtained were represented as a characteristic function. The optimal conditions of the extraction process were studied.     

3,6‐Di­hydroxy‐2‐(11‐phenyl­undecan­oyl)­cyclo­hex‐2‐en‐1‐one from Virola venosa bark

The structure of the title compound, C₂₃H₃₂O₄, an aryl­alkanone isolated from the petroleum ether fraction of the ethanol extract of the bark of Virola venosa, has been established by NMR spectroscopy and, for the first time, by X‐ray structure analysis. Two independent mol­ecules of the same enantiomer are present in the unit cell. Both mol­ecules exhibit an intramolecular hydrogen bond, which can be correlated with a rare signal observed at 18.28 p.p.m. in the ¹H NMR spectrum. The packing, in space group P1, is determined by a pseudo‐center of symmetry leading to a short intermolecular contact, which is present in one mol­ecule but does not occur in the other. As a consequence, the O—C—C—O torsion angles [−16.9 (3) and −12.7 (3)°] through the ketone and its adjacent hydroxy group are significantly different in the two mol­ecules.     

Application of HR‐MAS NMR in the solid‐phase synthesis of a glycopeptide using Sieber amide resin

The solid‐phase synthesis (SPS) of a structurally complex glycopeptide, using Sieber amide resin, was monitored by high resolution magic angle spinning NMR, demonstrating the further application of this technique. A synthetic peptidoglycan derivative, a precursor of a biologically active PGN, known to be involved in the cellular recognition, was prepared by SPS. The synthesis involved the preparation of an N‐alloc glucosamine moiety and the synthesis of a simple amino acid sequence L ‐Ala‐D ‐Glu‐L ‐Lys‐D ‐Ala‐D ‐Ala. Last step consisted the coupling, on solid‐phase, of the protected muramyl unit to the peptide chain. Proton spectra with good suppression of the polystyrene signals in swollen resin samples were obtained in DMF‐d₇ as a solvent and by using a nonselective 1D TOCSY/DIPSI‐2 scheme, thus allowing to follow the SPS without losses of compound and cleavage from the resin. The assignment of the proton spectra of the resin‐bound amino acid sequence and of the bound glycopeptide was achieved through the combination of MAS COSY, TOCSY and NOESY. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of the attached proton test technique in 31P NMR spectroscopy

Intensity modulated ³¹P NMR spectra were obtained using the pulse sequence published by Patt and Shoolery. This attached proton test (APT) technique for signal assignment could be applied to systems with long-range heteronuclear couplings in P? O? C? H fragments. In a model system derived from the alcoholysis of P₄S₁₀ the six reaction products were assigned to the six signals in the ³¹P NMR spectrum.     

Comprehensive 1H and 13C NMR assignment of 13 protobassic acid saponins

This study aimed to carry out complete ¹H and ¹³C NMR assignment of 13 protobassic acid saponins, including arganins A–C ( 1 – 3 ) and F ( 4 ), butyrosides B–D ( 5 – 7 ), tieghemelin ( 8 ), 3′-O-glucosyl-arganin C ( 9 ), Mi-saponins A–C ( 10 – 12 ), and mimusopsin ( 13 ), recorded in methanol-d₄. This was accomplished by the analysis of high-resolution one-dimensional (1D) NMR (¹H and ¹³C), two-dimensional (2D) NMR (¹H–¹H COSY, HSQC, and HMBC), and selectively excited 1D TOCSY spectra. Before this study, ¹H and ¹³C NMR data of arganins A–C ( 1 – 3 ) and F ( 4 ) were partially assigned. Our effort leads to their complete assignment, especially the glycon residue, and revises some reported data. Some revisions of the ¹H and ¹³C NMR data in the glycon part of butyroside C ( 6 ), tieghemelin ( 8 ), Mi-saponin A ( 10 ), and mimusopsin ( 13 ) were made. Those data of butyrosides B and D ( 5 & 7 ) and Mi-saponin B ( 11 ), which had not been recorded in methanol-d₄, are provided. In addition, the ¹H and ¹³C NMR data of Mi-saponin C ( 12 ) are reported for the first time. These data, being recorded in methanol-d₄, should be more friendly for use as a reference for identifying the related triterpenoid saponins.