NMR-based Method Research Articles

Alternative testing methods for skin sensitization: NMR spectroscopy for probing the reactivity and classification of potential skin sensitizers.

Evaluating consumer products for potentially harmful side effects of chemical ingredients is important for the protection of both the consumer and those involved in the manufacturing process. In order to assess the risk potential of chemicals, regulatory agencies have encouraged the development of several in silico, in vitro, and in chemico methods as alternatives to eliminate or minimize the use of animals. To add structural information to the existing in chemico methods, an NMR-based method is proposed for probing the reactivity and classification of the potential electrophiles (E) using a model thiol, DCYA, as a nucleophile. The major advantage of the NMR method is the quantitation of the actual adduct, DCYA-E. The degree of reaction is here provided as a direct measurement of adduct formation and/or electrophile depletion, in contrast to other in chemico assays, e.g., ADRA and DPRA, where the reactivity is inferred from the quantification of the test nucleophile depletion. Moreover, the developed NMR method should serve as a qualitative and quantitative tool in understanding the site of reaction and other structural information associated with test sensitizer. This is particularly valuable and advantageous over methods encouraged by regulatory agencies, which merely provide quantification of the reaction but lack any structural information. Several compounds with multiple reaction sites were successfully tested with the proposed NMR method. Otherwise, these compounds have proven to be a challenge to identify and classify using existing alternative methods.

Degree of reaction and phase content of silica-based one-part geopolymers investigated using chemical and NMR spectroscopic methods

One-part geopolymers were synthesized from two different silica materials (a silica-rich residue from chlorosilane production and a commercial microsilica) and sodium aluminate at three different SiO2/Al2O3 ratios and a nominal water/solids ratio of 0.5. The degree of reaction of the silica in the cured geopolymers (i.e. the fraction of silica dissolved to form aluminosilicates and minor products) was determined using two different methods: chemical attack with HCl to dissolve the reaction products and evaluation of peak areas of 29Si MAS NMR spectra. It was found that the degree of reaction of the silica decreases with increasing the silica content of the starting mix, and that it is almost constant after 1 day of curing and almost independent from the kind of starting silica. From the results of the NMR-based method, the mean SiO2/Al2O3 ratio of the reaction products (aluminosilicates and minor products) can be estimated to be ca. 2.0, nearly independent of the starting composition of the geopolymers. The dissolution method is biased, but of sufficient precision to be useful for following changes of the degree of reaction. Major crystalline phases in the cured geopolymers are zeolite A and/or hydrosodalite. Depending on the starting composition, the relative amounts of these zeolites vary; additionally, sodalite (only for the residue from chlorosilane production with >1 wt% Cl-), faujasite, and zeolite EMT can appear in the geopolymers. The 29Si and 27Al MAS NMR results indicate mainly Si(4Al) and Al(4Si) sites, in line with the presence of zeolite A, hydrosodalite, sodalite, and geopolymeric gel of comparatively low SiO2/Al2O3 ratio.

A Simple NMR-Based Method for Studying the Spatial Distribution of Linkers within Mixed-Linker Metal-Organic Frameworks.

The spatial distribution of different linkers within mixed-linker metal-organic frameworks crucially influences the properties of such materials. A simple and robust approach based on (1)H spin-diffusion magic-angle-spinning nuclear magnetic resonance measurements and modeling of spin-diffusion curves is presented; this approach facilitates the distinction between homogeneous and clustered distributions. The performance of the approach is demonstrated with an example of an aluminum-based metal-organic material DUT-5, which has framework consisting of biphenyl and bipyridyl dicarboxylic linkers. The distribution is shown to be homogeneous in this material. The approach could be applied to studying other spatially disordered crystalline materials.

Exploiting the Complementarity between Dereplication and Computer-Assisted Structure Elucidation for the Chemical Profiling of Natural Cosmetic Ingredients: Tephrosia purpurea as a Case Study.

The aqueous-ethanolic extract of Tephrosia purpurea seeds is currently exploited in the cosmetic industry as a natural ingredient of skin lotions. The aim of this study was to chemically characterize this ingredient by combining centrifugal partition extraction (CPE) as a fractionation tool with two complementary identification approaches involving dereplication and computer-assisted structure elucidation. Following two rapid fractionations of the crude extract (2 g), seven major compounds namely, caffeic acid, quercetin-3-O-rutinoside, ethyl galactoside, ciceritol, stachyose, saccharose, and citric acid, were unambiguously identified within the CPE-generated simplified mixtures by a recently developed (13)C NMR-based dereplication method. The structures of four additional compounds, patuletin-3-O-rutinoside, kaempferol-3-O-rutinoside, guaiacylglycerol 8-vanillic acid ether, and 2-methyl-2-glucopyranosyloxypropanoic acid, were automatically elucidated by using the Logic for Structure Determination program based on the interpretation of 2D NMR (HSQC, HMBC, and COSY) connectivity data. As more than 80% of the crude extract mass was characterized without need for tedious and labor-intensive multistep purification procedures, the identification tools involved in this work constitute a promising strategy for an efficient and time-saving chemical profiling of natural extracts.

Solution-state NMR structure of the putative morphogene protein BolA (PFE0790c) from Plasmodium falciparum.

Protozoa of the genus Plasmodium are responsible for malaria, which is perhaps the most important parasitic disease to infect mankind. The emergence of Plasmodium strains resistant to current therapeutics and prophylactics makes the development of new treatment strategies urgent. Among the potential targets for new antimalarial drugs is the BolA-like protein PFE0790c from Plasmodium falciparum (Pf-BolA). While the function of BolA is unknown, it has been linked to cell morphology by regulating transcription in response to stress. Using an NMR-based method, an ensemble of 20 structures of Pf-BolA was determined and deposited in the PDB (PDB entry 2kdn). The overall topology of the Pf-BolA structure, α1-β1-β2-η1-α2/η2-β3-α3, with the β-strands forming a mixed β-sheet, is similar to the fold observed in other BolA structures. A helix-turn-helix motif similar to the class II KH fold associated with nucleic acid-binding proteins is present, but contains an FXGXXXL signature sequence that differs from the GXXG signature sequence present in class II KH folds, suggesting that the BolA family of proteins may use a novel protein-nucleic acid interface. A well conserved arginine residue, Arg50, hypothesized to play a role in governing the formation of the C-terminal α-helix in the BolA family of proteins, is too distant to form polar contacts with any side chains in this α-helix in Pf-BolA, suggesting that this conserved arginine may only serve a role in guiding the orientation of this C-terminal helix in some BolA proteins. A survey of BolA structures suggests that the C-terminal helix may not have a functional role and that the third helix (α2/η2) has a `kink' that appears to be conserved among the BolA protein structures. Circular dichroism spectroscopy shows that Pf-BolA is fairly robust, partially unfolding when heated to 353 K and refolding upon cooling to 298 K.

Application of data mining tools for classification of protein structural class from residue based averaged NMR chemical shifts

The number of protein sequences deriving from genome sequencing projects is outpacing our knowledge about the function of these proteins. With the gap between experimentally characterized and uncharacterized proteins continuing to widen, it is necessary to develop new computational methods and tools for protein structural information that is directly related to function. Nuclear magnetic resonance (NMR) provides powerful means to determine three-dimensional structures of proteins in the solution state. However, translation of the NMR spectral parameters to even low-resolution structural information such as protein class requires multiple time consuming steps. In this paper, we present an unorthodox method to predict the protein structural class directly by using the residue's averaged chemical shifts (ACS) based on machine learning algorithms. Experimental chemical shift information from 1491 proteins obtained from Biological Magnetic Resonance Bank (BMRB) and their respective protein structural classes derived from structural classification of proteins (SCOP) were used to construct a data set with 119 attributes and 5 different classes. Twenty four different classification schemes were evaluated using several performance measures. Overall the residue based ACS values can predict the protein structural classes with 80% accuracy measured by Matthew correlation coefficient. Specifically protein classes defined by mixed αβ or small proteins are classified with >90% correlation. Our results indicate that this NMR-based method can be utilized as a low-resolution tool for protein structural class identification without any prior chemical shift assignments.

Towards the Discrimination of Carboxylates by Hydrogen‐Bond Donor Anion Receptors

The binding constants (log Kass ) of small synthetic receptor molecules based on indolocarbazole, carbazole, indole, urea and some others, as well as their combinations were measured for small carboxylate anions of different basicity, hydrophilicity and steric demands, that is, trimethylacetate, acetate, benzoate and lactate, in 0.5 % H2 O/[D6 ]DMSO by using the relative NMR-based measurement method. As a result, four separate binding affinity scales (ladders) including thirty-eight receptors were obtained with the scales anchored to indolocarbazole. The results indicate that the binding strength is largely, but not fully, determined by the strength of the primary hydrogen-bonding interaction. The latter in turn is largely determined by the basicity of the anion. The higher is the basicity of the anion the stronger in general is the binding, leading to the approximate order of increasing binding strength, lactate<benzoate<acetate≤trimethylacetate, which holds with all investigated receptors. Nevertheless, there are a number of occasions when the binding order changes with changing of the carboxylate anion, sometimes quite substantially. Principal component analysis (PCA) reveals that this is primarily connected to preferential binding of trimethylacetate, supposedly caused by an additional hydrophobic/solvophobic interaction. These findings enable making better predictions, which receptor framework or cavity is best suited for carboxylate anions in receptor design.

Identification of new hit scaffolds by INPHARMA-guided virtual screening

INPHARMA, an NMR-based method that determines ligand binding poses, allows finding novel scaffolds as inhibitors of a target protein.

NMR fingerprints, an integrated approach to uncover the unique components of the drug-like natural product metabolome of termite gut-associated Streptomyces species

A new NMR-based method to access to the unique components of the drug-like natural product metabolome of termite-gut associatedStreptomyces strainshas been developed.

Simple and validated quantitative ¹H NMR method for the determination of methylation, acetylation, and feruloylation degree of pectin.

The knowledge of pectin esterification degree is of primary importance to predict gelling and other properties of pectin from different sources. This paper reports the development of a simple and rapid (1)H NMR-based method for the simultaneous quantitative determination of methylation, acetylation, and feruloylation degree of pectin isolated from various food sources. Pectin esters are hydrolyzed in NaOH/D2O, and the obtained methanol, acetic acid, and ferulic acid are directly measured by (1)H NMR. High accuracy, repeatability, and reproducibility of the method were obtained, and the analysis time is reduced as compared to conventional chromatography- or titration-based methods.

Metabolic discrimination of Swertia mussotii and Swertia chirayita known as “Zangyinchen” in traditional Tibetan medicine by 1H NMR-based metabolomics

Swertia mussotii Franch. and Swertia chirayita Buch.–Ham. have been commonly used under the same name “Zangyinchen” for the treatment of liver and gallbladder diseases in traditional Tibetan medicine. Detailed characterization and comparison of the complete set of metabolites of these two species are critical for their objective identification and quality control. In this study, a rapid, simple and comprehensive 1H NMR-based metabolomics method was first developed to differentiate the two species. A broad range of metabolites, including iridoid glycosides, xanthones, triterpenoids, flavonoids, carbohydrates, and amino acids, were identified. Statistical analysis showed evident differences between the two species, and the major markers responsible for the differences were screened. In addition, quantitative 1H NMR method (qHNMR) was used for the target analysis of the discriminating metabolites. The results showed that S. mussotii had significantly higher contents of gentiopicrin, isoorientin, glucose, loganic acid, and choline, whereas S. chirayita exhibited higher levels of swertiamarin, oleanolic acid, valine, and fatty acids. These findings indicate that 1H NMR-based metabolomics is a reliable and effective method for the metabolic profiling and discrimination of the two Swertia species, and can be used to verify the genuine origin of Zangyinchen.

13C NMR analysis of 3,6-dihydro-2H-pyrans: assignment of remote stereochemistry using axial shielding effects.

The rational analysis of (13)C NMR axial shielding effects has enabled the assignment of remote relative stereochemistry in 3,6-oxygen-substituted 3,6-dihydro-2H-pyrans. Comparison of the (13)C NMR shifts of equivalent centers in cis- and trans-substituted 3,6-dihydro-2H-pyrans allows the relative configuration at the C3 and C6 positions to be defined in diastereoisomeric mixtures. Density functional calculations were used to validate this method and assess the conformational bias present in the ring system. Ultimately, the coupling of computational chemistry with this (13)C NMR-based method provided a reliable and convenient method for stereochemical assignment of a single diastereomer. This approach provides a facile and complementary alternative to the practices previously employed for determining the relative configuration in 3,6-dihydro-2H-pyrans.

NMR Method for Simultaneous Host–Guest Binding Constant Measurement

An NMR-based relative binding affinity measurement method has been developed in which differences in the binding affinities of different hosts toward a particular guest (ΔlogK(ass) values) are measured in the same solution. As an advancement, the method allows the simultaneous determination of several ΔlogK(ass) values in a single run. As a proof of principle, the method was used to measure binding affinity differences of a number of indolocarbazole- and urea-based synthetic receptors toward acetate ion in DMSO-d6/H2O (99.5%:0.5% m/m). As a result, a binding affinity scale containing 33 receptors and spanning 2.32 log units with excellent self-consistency (consistency standard deviation = 0.01 log unit) was created. Together with the very good agreement of the results with those obtained by UV-vis spectrophotometry, this demonstrates the high accuracy of the method and the fact that the NMR and UV-vis techniques can be used interchangeably (in spite of the very different concentrations used in these techniques). Additionally, it was found for symmetrical receptor molecules from the same compound family that there is a correlation between the acetate binding affinity of a receptor and the (15)N chemical shift of the nitrogen atoms of its binding centers.

Pore size distribution of bioresorbable films using a 3-D diffusion NMR method

Pore size distribution (PSD) within porous biomaterials is an important microstructural feature for assessing their biocompatibility, longevity and drug release kinetics. Scanning electron microscopy (SEM) is the most common method used to obtain the PSD of soft biomaterials. The method is highly invasive and user dependent, since it requires fracturing of the sample and then considers only the small portion that the user had acquired in the image. In the current study we present a novel nuclear magnetic resonance (NMR) method as an alternative method for estimation of PSD in soft porous materials. This noninvasive 3-D diffusion NMR method considers the entire volume of the specimen and eliminates the user’s need to choose a specific field of view. Moreover, NMR does not involve exposure to ionizing radiation and can potentially have preclinical and clinical uses. The method was applied on four porous 50/50 poly(dl-lactic-co-glycolic acid) bioresorbable films with different porosities, which were created using the freeze-drying of inverted emulsions technique. We show that the proposed NMR method is able to address the main limitations associated with SEM-based PSD estimations by being non-destructive, depicting the full volume of the specimens and not being dependent on the magnification factor. Upon comparison, both methods yielded a similar PSD in the smaller pore size range (1–25μm), while the NMR-based method provided additional information on the larger pores (25–50μm).

Probing the Intracellular Glutathione Redox Potential by In‐Cell NMR Spectroscopy

Non-invasive and real-time analysis of cellular redox processes has been greatly hampered by lack of suitable measurement techniques. Here we describe an in-cell nuclear magnetic resonance (NMR) based method for measuring the intracellular glutathione redox potential by direct and quantitative measurement of isotopically labeled glutathione introduced exogenously into living yeast. By using this approach, perturbations in the cellular glutathione redox homeostasis were also monitored as yeast cells were subjected to oxidative stress.

Complementarity between in Silico and Biophysical Screening Approaches in Fragment-Based Lead Discovery against the A2AAdenosine Receptor

Fragment-based lead discovery (FBLD) is becoming an increasingly important method in drug development. We have explored the potential to complement NMR-based biophysical screening of chemical libraries with molecular docking in FBLD against the A(2A) adenosine receptor (A(2A)AR), a drug target for inflammation and Parkinson's disease. Prior to an NMR-based screen of a fragment library against the A(2A)AR, molecular docking against a crystal structure was used to rank the same set of molecules by their predicted affinities. Molecular docking was able to predict four out of the five orthosteric ligands discovered by NMR among the top 5% of the ranked library, suggesting that structure-based methods could be used to prioritize among primary hits from biophysical screens. In addition, three fragments that were top-ranked by molecular docking, but had not been picked up by the NMR-based method, were demonstrated to be A(2A)AR ligands. While biophysical approaches for fragment screening are typically limited to a few thousand compounds, the docking screen was extended to include 328,000 commercially available fragments. Twenty-two top-ranked compounds were tested in radioligand binding assays, and 14 of these were A(2A)AR ligands with K(i) values ranging from 2 to 240 μM. Optimization of fragments was guided by molecular dynamics simulations and free energy calculations. The results illuminate strengths and weaknesses of molecular docking and demonstrate that this method can serve as a valuable complementary tool to biophysical screening in FBLD.

Enhanced Green Fluorescent Protein Transgenic Expression In Vivo Is Not Biologically Inert

Enhanced green fluorescent protein (EGFP) is a widely used biological reporter. However, the effects of EGFP expression in vivo are still unclear. To investigate the effects of EGFP transgenic expression in vivo, we employed an NMR-based metabonomics method to analyze the metabonome of EGFP transgenic mice. The results show that the metabonomes of urine, liver, and kidney of the EGFP transgenic mice are different from their wild-type counterparts. The EGFP mice expressed high levels of urinary 3-ureidopropionate, which is due to the down-regulated transcriptional level of β-ureidopropionase. The expression of EGFP in vivo also affects other metabolic pathways, including nucleic acid metabolism, energy utilization, and amino acids catabolism. These findings indicate that EGFP transgenic expression is not as inert as has been considered. Our investigation provides a holistic view on the effect of EGFP expression in vivo, which is useful when EGFP is employed as a functional biological indicator. Our work also highlights the potential of a metabonomics strategy in studying the association between molecular phenotypes and gene function.

Fast and Robust Method To Determine Phenoyl and Acetyl Esters of Polysaccharides by Quantitative 1H NMR

The acetyl (AcE), feruloyl (FE), and p-coumaroyl (pCE) ester contents of different cereal and grass polysaccharides were determined by a quantitative ¹H NMR-based method. The repeatability and the robustness of the method were demonstrated by analyzing different plant polysaccharide preparations. Good sensitivity and selectivity for AcE, FE, and pCE were observed. Moreover, an optimized and easy sample preparation allowed for simultaneous quantification of AcE, FE, and pCE. The method is suitable for high-throughput analysis, and it is a good alternative for currently used analytical procedures. A comparison of the method presented to a conventional HPLC-based method showed that the results obtained are in good agreement, whereas the combination of the optimized sample preparation and analysis by the ¹H NMR-based methodology results in significantly reduced analysis time.

Fast Screening of Dendrimer-Binding Compounds by Diffusion NMR Techniques

High-throughput screening of dendrimer-binding drugs is essential for the design and optimization of dendrimer-based drug delivery systems. In this study, pulsed gradient spin echo (PGSE) NMR was used for fast screening dendrimer-binding compounds using common amino acids as a screening pool. Diffusion coefficients of the amino acids before and after the addition of poly(propylene imine) (PPI) dendrimer were used to rank the binding affinities of the amino acids to the dendrimer. Among the common amino acids, cysteine, glutamic acid, aspartic acid, and tryptophan show strong binding affinity to PPI dendrimer. Tryptophan forms inclusion complexes with PPI dendrimer through hydrophobic interactions, while other amino acids mainly bind with PPI dendrimer via ionic and hydrogen-bond interactions. The PGSE NMR-based screening method provides new insights into high-throughput screening of dendrimer-binding compounds and should facilitate the study of dendrimer-based host-guest systems.

Plasma Metabonomics as a Novel Diagnostic Approach for Major Depressive Disorder

Major depressive disorder (MDD) is a socially detrimental psychiatric disorder, contributing to increased healthcare expenditures and suicide rates. However, no empirical laboratory-based tests are available to support the diagnosis of MDD. In this study, a NMR-based plasma metabonomic method for the diagnosis of MDD was tested. Proton nuclear magnetic resonance ((1)H NMR) spectra of plasma sampled from first-episode drug-naı̈ve depressed patients (n = 58) and healthy controls (n = 42) were recorded and analyzed by orthogonal partial least-squares discriminant analysis (OPLS-DA). The OPLS-DA score plots of the spectra demonstrated that the depressed patient group was significantly distinguishable from the healthy control group. Moreover, the method accurately diagnosed blinded samples (n = 26) in an independent replication cohort with a sensitivity and specificity of 92.8% and 83.3%, respectively. Taken together, NMR-based plasma metabonomics may offer an accurate empirical laboratory-based method applicable to the diagnosis of MDD.