Application of a microcoil probe head in NMR analysis of chemicals related to the chemical weapons convention

A 1.7-mm microcoil probe head was tested in the analysis of organophosphorus compounds related to the Chemical Weapons Convention. The microcoil probe head demonstrated a high mass sensitivity in the detection of traces of organophosphorus compounds in samples. Methylphosphonic acid, the common secondary degradation product of sarin, soman, and VX, was detected at level 50 ng (0.52 nmol) from a 30-microL water sample using proton-observed experiments. Direct phosphorus observation of methylphosphonic acid with (31)P{(1)H} NMR experiment was feasible at the 400-ng (4.17 nmol) level. Application of the microcoil probe head in the spiked sample analysis was studied with a test water sample containing 2-10 microg/mL of three organophosphorus compounds. High-quality (1)H NMR, (31)P{(1)H} NMR, 2D (1)H-(31)P fast-HMQC, and 2D TOCSY spectra were obtained in 3 h from the concentrated 1.7-mm NMR sample prepared from 1 mL of the water solution. Furthermore, a 2D (1)H-(13)C fast-HMQC spectrum with sufficient quality was possible to measure in 5 h. The microcoil probe head demonstrated a considerable sensitivity improvement and reduction of measurement times for the NMR spectroscopy in identification of chemicals related to the Chemical Weapons Convention.     

Multivariate curve resolution applied to the analysis and resolution of two-dimensional [1H,15N] NMR reaction spectra

Multivariate curve resolution is proposed for the study of complex chemical reactions monitored by two-dimensional (2D) NMR spectroscopy. In particular, in this work, multivariate curve resolution is applied to the study of the reaction between (15)N-labeled cisplatin and the amino acid-nucleotide hybrid (Phac-Met-linker-p(5')dG). At several stages of the reaction, 2D [(1)H,(15)N] HSQC NMR spectra were acquired and stored in data matrices. In a first step, multivariate curve resolution was applied to analyze individually each one of these 2D spectra, allowing the resolution of the corresponding (1)H and (15)N one-dimensional correlation spectra. In a second step, the whole set of 2D spectra recorded along the reaction were simultaneously analyzed by multivariate curve resolution, allowing the resolution of the kinetic concentration profiles and of the pure 2D NMR spectra of each of the species detected along the reaction. Results finally obtained confirmed previously postulated reaction mechanisms involving the existence of two monofunctional adducts and of two bifunctional adducts, with the structure of one of them not completely resolved.     

Screening and identification of organophosphorus compounds related to the chemical weapons convention with 1D and 2D NMR spectroscopy

Two-dimensional 1H-31P Fast-HMQC was tested for determination of the presence in low concentrations of organophosphorus compounds related to the Chemical Weapons Convention. This method, based on inverse detection, demonstrated high sensitivity and selectivity. Background signals, such as solvent peaks, are suppressed with good efficiency, and organophosphorus compounds present at a concentration level 1-10 microg/mL can be detected within a few hours. In addition, phosphorus-selective one-dimensional 1H-31P HSQC-TOCSY was applied to produce a complete proton spectrum of selected organophosphorus compound from a sample containing intense background resonances. Application of the methods presented in this paper resulted in considerably improved performance of NMR spectroscopy as a complementary technique for screening as well as identification of chemical warfare agents in environmental samples.     

Trace level detection of compounds related to the chemical weapons convention by 1H-detected 13C NMR spectroscopy executed with a sensitivity-enhanced, cryogenic probehead

Two-dimensional 1H-13C HSQC (heteronuclear single quantum correlation) and fast-HMQC (heteronuclear multiple quantum correlation) pulse sequences were implemented using a sensitivity-enhanced, cryogenic probehead for detecting compounds relevant to the Chemical Weapons Convention present in complex mixtures. The resulting methods demonstrated exceptional sensitivity for detecting the analytes at trace level concentrations. 1H-13C correlations of target analytes at < or = 25 microg/mL were easily detected in a sample where the 1H solvent signal was approximately 58,000-fold more intense than the analyte 1H signals. The problem of overlapping signals typically observed in conventional 1H spectroscopy was essentially eliminated, while 1H and 13C chemical shift information could be derived quickly and simultaneously from the resulting spectra. The fast-HMQC pulse sequences generated magnitude mode spectra suitable for detailed analysis in approximately 4.5 h and can be used in experiments to efficiently screen a large number of samples. The HSQC pulse sequences, on the other hand, required roughly twice the data acquisition time to produce suitable spectra. These spectra, however, were phase-sensitive, contained considerably more resolution in both dimensions, and proved to be superior for detecting analyte 1H-13C correlations. Furthermore, a HSQC spectrum collected with a multiplicity-edited pulse sequence provided additional structural information valuable for identifying target analytes. The HSQC pulse sequences are ideal for collecting high-quality data sets with overnight acquisitions and logically follow the use of fast-HMQC pulse sequences to rapidly screen samples for potential target analytes. Use of the pulse sequences considerably improves the performance of NMR spectroscopy as a complimentary technique for the screening, identification, and validation of chemical warfare agents and other small-molecule analytes present in complex mixtures and environmental samples.     

Measurement of absolute concentrations of individual compounds in metabolite mixtures by gradient-selective time-zero 1H-13C HSQC with two concentration references and fast maximum likelihood reconstruction analysis

Time-zero 2D (13)C HSQC (HSQC(0)) spectroscopy offers advantages over traditional 2D NMR for quantitative analysis of solutions containing a mixture of compounds because the signal intensities are directly proportional to the concentrations of the constituents. The HSQC(0) spectrum is derived from a series of spectra collected with increasing repetition times within the basic HSQC block by extrapolating the repetition time to zero. Here we present an alternative approach to data collection, gradient-selective time-zero (1)H-(13)C HSQC(0) in combination with fast maximum likelihood reconstruction (FMLR) data analysis and the use of two concentration references for absolute concentration determination. Gradient-selective data acquisition results in cleaner spectra, and NMR data can be acquired in both constant-time and non-constant-time mode. Semiautomatic data analysis is supported by the FMLR approach, which is used to deconvolute the spectra and extract peak volumes. The peak volumes obtained from this analysis are converted to absolute concentrations by reference to the peak volumes of two internal reference compounds of known concentration: DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) at the low concentration limit (which also serves as chemical shift reference) and MES (2-(N-morpholino)ethanesulfonic acid) at the high concentration limit. The linear relationship between peak volumes and concentration is better defined with two references than with one, and the measured absolute concentrations of individual compounds in the mixture are more accurate. We compare results from semiautomated gsHSQC(0) with those obtained by the original manual phase-cycled HSQC(0) approach. The new approach is suitable for automatic metabolite profiling by simultaneous quantification of multiple metabolites in a complex mixture.     

Hierarchical Alignment and Full Resolution Pattern Recognition of 2D NMR Spectra: Application to Nematode Chemical Ecology

Nuclear magnetic resonance (NMR) is the most widely used nondestructive technique in analytical chemistry. In recent years, it has been applied to metabolic profiling due to its high reproducibility, capacity for relative and absolute quantification, atomic resolution, and ability to detect a broad range of compounds in an untargeted manner. While one-dimensional (1D) (1)H NMR experiments are popular in metabolic profiling due to their simplicity and fast acquisition times, two-dimensional (2D) NMR spectra offer increased spectral resolution as well as atomic correlations, which aid in the assignment of known small molecules and the structural elucidation of novel compounds. Given the small number of statistical analysis methods for 2D NMR spectra, we developed a new approach for the analysis, information recovery, and display of 2D NMR spectral data. We present a native 2D peak alignment algorithm we term HATS, for hierarchical alignment of two-dimensional spectra, enabling pattern recognition (PR) using full-resolution spectra. Principle component analysis (PCA) and partial least squares (PLS) regression of full resolution total correlation spectroscopy (TOCSY) spectra greatly aid the assignment and interpretation of statistical pattern recognition results by producing back-scaled loading plots that look like traditional TOCSY spectra but incorporate qualitative and quantitative biological information of the resonances. The HATS-PR methodology is demonstrated here using multiple 2D TOCSY spectra of the exudates from two nematode species: Pristionchus pacificus and Panagrellus redivivus. We show the utility of this integrated approach with the rapid, semiautomated assignment of small molecules differentiating the two species and the identification of spectral regions suggesting the presence of species-specific compounds. These results demonstrate that the combination of 2D NMR spectra with full-resolution statistical analysis provides a platform for chemical and biological studies in cellular biochemistry, metabolomics, and chemical ecology.     

Detection of the 1H and 15N NMR resonances of sulfamate groups in aqueous solution: a new tool for heparin and heparan sulfate characterization

Sulfamate (NHSO(3)(-)) groups are critically important structural elements of the glycosaminoglycans heparin and heparan sulfate (HS). Experimental conditions are presented for detection of the sulfamate (1)H NMR resonances in aqueous solution. NMR spectra reported for N-sulfoglucosamine (GlcNS) and the synthetic pentasaccharide drug fondaparinux demonstrate the broad utility of the sulfamate group (1)H chemical shifts to reflect differences in molecular structure. The sulfamate protons also provide an efficient route for detection of (15)N chemical shifts through proton-nitrogen correlations measured with the heteronuclear single quantum coherence (HSQC) experiment. The HSQC spectra of GlcNS, fondaparinux, and the low-molecular weight heparin enoxaparin illustrate the power of the (1)H and (15)N chemical shifts of the sulfamate NH groups for the structural characterization of heparin and HS.     

Exploring the analytical potential of NMR spectroscopy in chiral anisotropic media for the study of the natural abundance deuterium distribution in organic molecules

The deuterium/hydrogen (D/H)(i) ratio measurement by quantitative (2)H NMR spectroscopy is a method of choice for the analysis of kinetic isotopic effects associated with enzyme-catalyzed reactions during a biosynthetic pathway. However, the efficiency of the current isotropic (2)H-[(1)H] NMR can be limited by the rather small chemical shift dispersion of deuterium nuclei. In addition, this method does not allow the enantiotopic deuterons in prochiral molecules to be spectrally discriminated, hence precluding the quantification of isotopic fractionation on methylene prostereogenic sites. In this work, we explore another analytical strategy able to circumvent these disadvantages. This approach is based on the use of natural abundance (2)H 2D NMR experiments on solutes embedded in polypeptidic, chiral liquid crystalline solvent. Thus, we show that NMR in these oriented phases is a powerful way to separate deuterium signals on the basis of the quadrupolar interactions, providing a promising alternative to overcrowded (2)H NMR spectra obtained in liquid state. To illustrate our purpose, we have experimentally investigated the case of 1,1'-bis(phenylthio)hexane derived by cleavage from methyl linoleate of safflower. The (2)H NMR results in chiral liquid crystals are presented and discussed. We show, for the first time, that (D/H)(pro-R) and (D/H)(pro-S) can be measured at the same methylene position of a fatty acid chain.     

邻巯基氧化吡啶双齿配位的过渡金属(Ni,Pd)化合物NMR研究

本文报道了化合物 Ni(mpo)_2(1)和 Pd(mpo)_2(2)的~1H,~(13)C NMR 的化学位移和偶合常数 J_(H-H)并借助同核和异核二维相关谱,进行分析,归属了~1H,~(13)C NMR 谱线。简单讨论了这两个非对称结构化合物在溶液中的稳定性。     

苯乙烯乙烯/丁烯嵌段共聚物的二维核磁方法

利用二维核磁方法对苯乙烯-乙烯/丁烯嵌段共聚物的链段微结构进行了定性。利用普通一维1H和13C核磁得到了共聚物分子的基本结构信息;利用1H-1H-COSY二维核磁确定了共聚物中同核氢质子之间的偶合关系;利用13C-1H-HSQC和13C-1H-HMBC二维核磁确定了共聚物异核碳氢原子的单键相关性,明确了直接相连接的C-H化学位移,为微结构的确定提供了依据。归属了苯乙烯嵌段结构中的亚甲基和次甲基的化学位移;同时归属了乙烯-丁烯嵌段链段结构中的甲基、亚甲基和次甲基的化学位移,并确定了重复单元的链段结构状态。二维核磁方法可以得到很多在普通一维核磁中难以解析的信息。二维核磁是对苯乙烯-乙烯/丁烯嵌段共聚物链结构定性的有效手段。     

Hyphenation of capillary HPLC to microcoil (1)H NMR spectroscopy for the determination of tocopherol homologues

Highly selective reversed phases (C(30) phases) are self-packed in 250 microm inner diameter fused-silica capillaries and employed for capillary HPLC separation of shape-constrained natural compounds (tocopherol homologues, vitamin E). Miniaturized hyphenated systems such as capillary HPLC-ESI-MS (positive ionization mode) and, with special emphasis, continuous-flow capillary HPLC- NMR are used for structural determination of the separated compounds. Despite the small amount of sample available (1.33 microg of each tocopherol), the authors have been able to monitor the capillary HPLC separation under continuous-flow (1)H NMR conditions, thus allowing an immediate peak identification. Further structural assignment was carried out in the stopped-flow NMR mode as shown, for example, by a 2D (1)H,(1)H COSY NMR spectrum of alpha-tocopherol. We demonstrate in this paper the considerable potential of hyphenated capillary separations coupled to MS and NMR for the investigation of restricted amounts of sample.     

Magic angle spinning NMR and 1H-31P heteronuclear statistical total correlation spectroscopy of intact human gut biopsies

Previously we have demonstrated the use of 1H magic angle spinning (MAS) NMR spectroscopy for the topographical variations in functional metabolic signatures of intact human intestinal biopsy samples. Here we have analyzed a series of MAS 1H NMR spectra (spin-echo, one-dimensional, and diffusion-edited) and 31P-{1H} spectra and focused on analyzing the enhancement of information recovery by use of the statistical total correlation spectroscopy (STOCSY) method. We have applied a heterospectroscopic cross-examination performed on the same samples and between 1H and 31P-{1H} spectra (heteronuclear STOCSY) to recover latent metabolic information. We show that heterospectroscopic correlation can give new information on the molecular compartmentation of metabolites in intact tissues, including the statistical "isolation" of a phospholipid/triglyceride vesicle pool in intact tissue. The application of 31P-1H HET-STOCSY allowed the cross-assignment of major 31P signals to their equivalent 1H NMR spectra, e.g., for phosphorylcholine and phosphorylethanolamine. We also show pathway correlations, e.g., the ascorbate-glutathione pathway, in the STOCSY analysis of intact tissue spectra. These 31P-1H HET-STOCSY spectra also showed different topographical regions, particular for minor signals in different tissue microenvironments. This approach could be extended to allow the detection of altered distributions within metabolic subcompartments as well as conventional metabonomics concentration-based diagnostics.     

Assessment of lisdexamfetamine dimesylate stability and identification of its degradation product by NMR spectroscopy

Lisdexamfetamine dimesylate (LDX), a long-acting prodrug stimulant indicated for the treatment of the attention-deficit/hyperactivity disorder (ADHD), was subjected to forced degradation studies by acid and alkaline hydrolysis and the degradation profile was studied. To obtain between 10–30% of degraded product, acid and alkaline conditions were assessed with solutions of 0.01?M, 0.1?M, 0.5?M, and 1?M of DCl and NaOD. These solutions were analyzed through 1?H NMR spectra. Acid hydrolysis produced no degradation in 0.01?M and 0.1?M DCl and 4.38%, 9.69%, and 17.75% of degradation LDX, respectively, in 0.5?M, 1?M (4h) and 1?M (4?+?12?h) DCl. And alkaline hydrolysis produced no degradation in 0.01?M and 0.1?M DCl and a degradation LDX extension of 8.5%, 14.30%, and 22.91%, respectively, in 0.5?M, 1?M (4h) and 1?M (4?+?12?h) NaOD. LDX solutions subjected to 1?M (4?+?12?h) acid and alkaline hydrolysis were evaluated by NMR spectra (1?H NMR, 13?C NMR, HSQC and HMBC). LDX degradation product (DP) was identified and its structure elucidated as a diastereoisomer of LDX: (2R)-2,6-diamino-N-[(2S)-1-phenylpropan-2-yl] hexanamide without their physical separation.     

Quantification of high-resolution (1)H NMR spectra from rat brain extracts

Extracting quantitative information about absolute concentrations from high-resolution (1)H NMR spectra of complex mixtures such as brain extracts remains challenging. Partial overlap of resonances complicates integration, whereas simple line fitting algorithms cannot accommodate the spectral complexity of coupled spin systems. Here, it is shown that high-resolution (1)H NMR spectra of rat brain extracts from 11 distinct brain regions can be reproducibly quantified using a basis set of 29 compounds. The basis set is simulated with the density matrix formalism using complete prior knowledge of chemical shifts and scalar couplings. A crucial aspect to obtain reproducible results was the inclusion of a line shape distortion common among all 73 resonances of the 29 compounds. All metabolites could be quantified with <10% and <3% inter- and intrasubject variation, respectively.     

1H and 13C nuclear magnetic resonance studies of the hindered phencyclone adducts of some smaller branched N-alkyl maleimides: rigorous aryl proton assignments with high-resolution two-dimensional (COSY45) spectroscopy, and anisotropic shielding effects and ab initio geometry optimizations

Phencyclone, 1, a potent Diels-Alder diene, reacts with a series of N-alkylmaleimides, 2, to form hindered adducts, 3. The 300 MHz 1H and 75 MHz 13C NMR studies of these adducts at ambient temperatures have demonstrated slow rotations on the nuclear magnetic resonance (NMR) timescales for the unsubstituted bridgehead phenyl groups, and have revealed substantial magnetic anisotropic shielding effects in the 1H spectra of the N-alkyl groups of the adducts. The selected N-alkyl groups for the target compounds emphasized smaller branched alkyls, including C3 (isopropyl, a); C4 (isobutyl, b; and t-butyl, c); C5 (n-pentyl, d; isopentyl [isoamyl], e; 1-ethylpropyl, f; t-amyl, g;) and a related C8 isomer (1,1,3,3-tetramethylbutyl ["t-octyl"], h). The straight-chain n-pentyl analog was included as a reference. This present work on the branched N-alkylmaleimide adducts appreciably extends our earlier compilation on the N-n-alkylmaleimide adducts. Key methods for proton assignments included "high-resolution" 1H-1H chemical shift correlation spectroscopy, COSY45. 13C NMR of the adducts, 3, verified the expected number of aryl carbons for slow exchange limit (SEL) spectra of the bridgehead phenyl groups. The synthetic routes involved reaction of the corresponding amines, 4, with maleic anhydride to give the N-alkylmaleamic acids, 5, which underwent cyclodehydration to form the maleimides, 2. Magnetic anisotropic shielding magnitudes for alkyl group protons in the adducts were calculated relative to corresponding proton chemical shifts in the maleimides. Geometry optimizations for the above adducts (and for the N-n-butylmaleimide adduct) were performed at the Hartree-Fock level with the 6-31G* basis set. The existence of different contributing conformers for the adducts is discussed with respect to their calculated energies and implications regarding experimentally observed anisotropic shielding magnitudes.     

Heteronuclear 1H-31P statistical total correlation NMR spectroscopy of intact liver for metabolic biomarker assignment: application to galactosamine-induced hepatotoxicity

As part of our ongoing development of methods for enhanced biomarker information recovery from spectroscopic data we present the first example of a new hetero-nuclear statistical total correlation spectroscopy (HET-STOCSY) approach applied to intact tissue samples collected as part of a toxicological study. One-dimensional 1H and 31P-{1H} magic angle spinning (MAS) NMR spectra of intact liver samples after galactosamine (galN) treatment to rats and after cotreatment of galN plus uridine were collected at 275 K. Individual samples were also followed by 1H and 31P-{1H} MAS NMR through time generating time dependent modulations in metabolite signatures relating to toxicity. High-resolution 1H NMR spectra of urine and plasma and clinical chemical data were also collected to establish a biological framework in which to place these novel statistical heterospectroscopic data. In HET-STOCSY, calculation of the covariance between the 31P-{1H} and 1H NMR signals of phosphorus containing metabolites allows their molecular connectivities to be established and the construction of virtual two-dimensional heteronuclear correlation spectra that connect all protons on the molecule to the heteroatom. We show how HET-STOCSY applied to MAS NMR spectra of liver samples can be used to augment biomarker detection. This approach is generic and can be applied to correlate the covarying signals for any spin-active nuclei where there is parallel or serial collection of data.     

Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra

One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) spectroscopy is used extensively for high-throughput analysis of metabolites in biological fluids and tissue extracts. Typically, such spectra are treated as multivariate statistical objects rather than as collections of quantifiable metabolites. We report here a two-dimensional (2D) (1)H-(13)C NMR strategy (fast metabolite quantification, FMQ, by NMR) for identifying and quantifying the approximately 40 most abundant metabolites in biological samples. To validate this technique, we prepared mixtures of synthetic compounds and extracts from Arabidopsis thaliana, Saccharomyces cerevisiae, and Medicago sativa. We show that accurate (technical error 2.7%) molar concentrations can be determined in 12 min using our quantitative 2D (1)H-(13)C NMR strategy. In contrast, traditional 1D (1)H NMR analysis resulted in 16.2% technical error under nearly ideal conditions. We propose FMQ by NMR as a practical alternative to 1D (1)H NMR for metabolomics studies in which 50-mg (extract dry weight) samples can be obtained.     

Deconvolution of chemical mixtures with high complexity by NMR consensus trace clustering

Identification and quantification of analytes in complex solution-state mixtures are critical procedures in many areas of chemistry, biology, and molecular medicine. Nuclear magnetic resonance (NMR) is a unique tool for this purpose providing a wealth of atomic-detail information without requiring extensive fractionation of the samples. We present three new multidimensional-NMR based approaches that are geared toward the analysis of mixtures with high complexity at natural (13)C abundance, including approaches that are encountered in metabolomics. Common to all three approaches is the concept of the extraction of one-dimensional (1D) consensus spectral traces or 2D consensus planes followed by clustering, which significantly improves the capability to identify mixture components that are affected by strong spectral overlap. The methods are demonstrated for covariance (1)H-(1)H TOCSY and (13)C-(1)H HSQC-TOCSY spectra and triple-rank correlation spectra constructed from pairs of (13)C-(1)H HSQC and (13)C-(1)H HSQC-TOCSY spectra. All methods are first demonstrated for an eight-compound metabolite model mixture before being applied to an extract from E. coli cell lysate.     

含手性膦配体MBPA和邻巯基吡啶配体PyS的过渡金属钯(Ⅱ)化合物的2D NMR研究

利用一维和二维NMR技术,对含手性膦配体MBPA和邻巯基吡啶配体PyS的过渡金属Pd(Ⅱ)化合物Pd(3-MBPA)(PyS)Cl)(1),Pd(2-MBPA)(PyS)Cl)(2)。进行~1H和~(13)C NMR谱分析,归属了所有的~1H和~(13)CNMR谱线,并对配体在配位前后的化学位移的变化作了简单讨论。     

N—(对硝基苯基)—N’—(甲氧基羰基)硫脲及其铜(Ⅰ)配合物的NMR研究

利用~1H—~1H COSY,HMQC等2D NMR技术对一种新的配体N—(对硝基苯基)—N’—(甲氧基羰基)硫脲(H_2pmt(Ⅰ)进行~1H、~(13)C NMR谱数据分析与归属;对于它与Cu~+离子配位的化合物Cu(H_2pmt)_2Cl(2)也作了~1H、~(13)C NMR的测定,归属了它们的所有谱线,对于它们的化学位移与配位行为作了简单讨论。