2D 1H NMR Research Articles

Structure of a Sulfated Capsular Polysaccharide from the Marine Bacterium Cobetia marina KMM 1449 and a Genomic Insight into Its Biosynthesis.

Some marine and extremophilic microorganisms are capable of synthesizing sulfated polysaccharides with a unique structure. A number of studies indicate significant biological properties of individual sulfated polysaccharides, such as antiproliferative activity, which makes them a promising area for further research. In this study, the capsular polysaccharide (CPS) was obtained from the bacterium Cobetia marina KMM 1449, isolated from a marine sediment sample collected along the shore of the Sea of Japan. The CPS was isolated by saline solution, purified by a series of chromatographic procedures, and studied by chemical methods along with 1D and 2D 1H and 13C NMR spectroscopy. The following new structure of the CPS from C. marina KMM 1449 was established and consisted of sulfated and simultaneously phosphorylated disaccharide repeating units: →4)-α-L-Rhap2S-(1→3)-β-D-Manp6PGro-(1→. To elucidate the genetic basis of the CPS biosynthesis, the whole genomic sequence of C. marina KMM 1449 was obtained. The CPS biosynthetic gene cluster (BGC) of about 70 genes composes four regions encoding nucleotide sugar biosynthesis (dTDP-Rha and GDP-Man), assembly (GTs genes), translocation (ABC transporter genes), sulfation (PAPS biosynthesis and sulfotransferase genes) and lipid carrier biosynthesis (wcb operon). Comparative analysis of the CPS BGCs from available Cobetia genomes showed the presence of KMM 1449-like CPS BGC among strains of all three Cobetia species. The study of new natural sulfated polysaccharides, as well as the elucidation of the pathways of their biosynthesis, provides the basis for the development of potential anticancer drugs.

Nuclear Magnetic Resonance Study of Monoclonal Antibodies Near an Oil-Water Interface.

Monoclonal antibodies (mAb) represent an important class of biologic therapeutics that can treat a variety of diseases including cancer, autoimmune disorders or respiratory conditions (e.g. COVID-19). However, throughout their development, mAb are exposed to air-water or oil-water interfaces that may trigger mAb partial unfolding that can lead to the formation of proteinaceous aggregates. Using a combination of dynamic surface tensiometry and spatially resolved 1D 1H NMR spectroscopy, this study investigates if adsorption of a model IgG2a-κ mAb to the oil-water interface affects its structure. Localized NMR spectroscopy was performed using voxels of 375 µm, incrementally approaching the oil-water interface. Dynamic interfacial tension progressively decreases at the oil-water interface over time, confirming mAb adsorption to the interface. Localized NMR spectroscopy results indicate that, while the number of mAb-related chemical resonances and chemical shift frequencies remain unaffected, spectral line broadening is observed as voxels incrementally approach the oil-water interface. Moreover, the spin-spin (T2) relaxation of the mAb molecule was measured for a voxel centered at the interface and shown to be affected differentially across the mAb resonances, indicating a rotational restriction for mAb molecules due to presence of the interface. Finally, the apparent diffusion coefficient of the mAb for the voxel centered at the interface is lower than the bulk mAb. These results suggest that this specific mAb interacts with and may be in exchange with bulk mAb phase in the vicinity of the interface. As such, these localized NMR techniques offer the potential to probe and quantify alterations of mAb properties near interfacial layers.

Structural and genetic relationship of the O-antigens of the type strains <I>Azospirillum agricola</I> CC-HIH038 and <I>Azospirillum doebereinerae</I> GSF71

Lipopolysaccharide was isolated from cells of the type strain of rhizobacteria Azospirillum agricola CC-HIH038Т by phenol extraction. O-specific polysaccharide was obtained by mild acid hydrolysis of lipopolysaccharide followed by chromatographic fractionation. On the basis of monosaccharide analysis, including determination of absolute configurations, 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the O-specific polysaccharide repeating unit of A. agricola CC-HIH038T was elucidated: →3)-α-L-Rhap2Ac-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-β-D-GlcpNAc6Ac-(1→ , which is structurally related to A. doebereinerae GSF71T . Based on the analysis of full-genome sequencing data for strains A. agricola CC-HIH038T and A. doebereinerae GSF71T the O-specific polysaccharide biosynthesis loci were identified, which were characterized by a similar organization and a high level of gene homology, confirming the common structure of the O-antigens of these strains.

Protomix: a Python package for 1H-NMR metabolomics data preprocessing.

NMR-based metabolomics is a field driven by technological advancements, necessitating the use of advanced preprocessing tools. Despite this need, there is a remarkable scarcity of comprehensive and user-friendly preprocessing tools in Python. To bridge this gap, we have developed Protomix-a Python package designed for metabolomics research. Protomix offers a set of automated, efficient, and user-friendly signal-preprocessing steps, tailored to streamline and enhance the preprocessing phase in metabolomics studies. This package presents a comprehensive preprocessing pipeline compatible with various data analysis tools. It encompasses a suite of functionalities for data extraction, preprocessing, and interactive visualization. Additionally, it includes a tutorial in the form of a Python Jupyter notebook, specifically designed for the analysis of 1D 1H-NMR metabolomics data related to prostate cancer and benign prostatic hyperplasia. Protomix can be accessed at https://github.com/mzniber/protomix and https://protomix.readthedocs.io/en/latest/index.html.

Molecular insights into the interaction between cytochrome c and carbon nanomaterials

Carbon nanomaterials (CNMs) are a heterogeneous class of advanced materials. Their widespread use is associated with human safety concerns, which can be addressed by safe-by design strategies. This implies a deep knowledge of how physico-chemical properties drive biological effects. The ability of CNMs to interact with cytochrome c (cyt c), a heme-protein playing a key role in the respiratory chain, in apoptosis and in cellular redox homeostasis, has been reported in some studies. However, the consequences of this interaction on the cyt c functions are controversial. Here the mechanism of interaction of carbon nanoparticles (CNPs), chosen as model of redox-active CNMs, with cyt c has been studied with the aim to shed light into these discrepancies. The effect of CNPs on the redox state of cyt c was monitored by UV–vis spectroscopy and 1D 1H NMR, while the effect on the primary, secondary, and tertiary cyt c structure was investigated by FIA/LC-MS and Circular Dichroism (CD). Finally, the peroxidase activity of cyt c and the involvement of superoxide radicals was evaluated by EPR spectroscopy. We demonstrate the existence of two mechanisms, one leading to the suppression of the cyt c peroxidase activity following the NADH-independent reduction of the heme-iron, and the other resulting in the irreversible protein unfolding. Overall, the results suggest that these two processes might be independently modulated by redox and surface properties respectively.

First oxidative coupling of cyclazine heterocycle via regioselective dimerization of 1,2-dicarbomethoxy-3-phenylcycl[3.2.2]azine: Synthesis, theoretical aspects and physicochemical studies

The first covalently linked dimer has been prepared for cyclazine systems by regioselective oxidative homocoupling of 1,2-dicarbomethoxy-3-phenylcycl[3.2.2]azine. The regioselectivity of this reaction at 4-position, having been confirmed by X-ray diffraction analysis and NMR spectroscopy, has been also reliably predicted within the model of average local ionization energy on the molecular surface of the starting monomer at the BP86/def2-TZVP level of theory. Due to the planar π-π interaction of the cycl[3.2.2]azine subunits, the dimer is a green fluorophore (λem = 527 nm, toluene), characterized by a bathochromic shift of the main bands in the UV–vis and fluorescence spectra relative to the monomer by 41 and 71 nm, respectively. Furthermore, the dimer demonstrates an increased fluorescence quantum yield relative to the monomer (55 % vs. 35 % in toluene), and according to the data of X-ray diffraction analysis, DFT calculations and variable temperature 1D and 2D 1H NMR spectroscopy, is characterized by hindered rotation of the S1-state-involved cycl[3.2.2]azine cores along the C4–C4′ bond axis. Such prerequisites determine good application potential of the 4-4′ coupled cycl[3.2.2]azine derivatives as turn-on fluorescent, i.e. fluorogenic probes for advanced bioimaging in living systems. Finally, unlike the monomer, the dimer shows reversibility of both one- and two-electron reduction and oxidation processes, and therefore can become the basis of both n- and p-type semiconductors.

Accurate and Efficient Structure Elucidation from Routine One-Dimensional NMR Spectra Using Multitask Machine Learning.

Rapid determination of molecular structures can greatly accelerate workflows across many chemical disciplines. However, elucidating structure using only one-dimensional (1D) NMR spectra, the most readily accessible data, remains an extremely challenging problem because of the combinatorial explosion of the number of possible molecules as the number of constituent atoms is increased. Here, we introduce a multitask machine learning framework that predicts the molecular structure (formula and connectivity) of an unknown compound solely based on its 1D 1H and/or 13C NMR spectra. First, we show how a transformer architecture can be constructed to efficiently solve the task, traditionally performed by chemists, of assembling large numbers of molecular fragments into molecular structures. Integrating this capability with a convolutional neural network, we build an end-to-end model for predicting structure from spectra that is fast and accurate. We demonstrate the effectiveness of this framework on molecules with up to 19 heavy (non-hydrogen) atoms, a size for which there are trillions of possible structures. Without relying on any prior chemical knowledge such as the molecular formula, we show that our approach predicts the exact molecule 69.6% of the time within the first 15 predictions, reducing the search space by up to 11 orders of magnitude.

Effect of bacterial dissociation on lipopolysaccharide structure: A study of O-polysaccharide from the marine bacterium Pseudoalteromonas agarivorans KMM 232 (O-form)

The lipopolysaccharide (LPS) was obtained from a bacterium Pseudoalteromonas agarivorans KMM 232 (O-form) isolated from a seawater sample collected at a depth of 500 m. The O-polysaccharide (OPS) was isolated by mild acid degradation of the LPS and studied by chemical methods along with 1D and 2D 1H and 13C NMR spectroscopy, including 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY and 1H,13C HSQC, and 1H,13C HMBC experiments. The following new structure of the OPS from P. agarivorans KMM 232 (O-form) containing 2-acetamido-2-deoxy-d-glucose (D-GlcNAc), d-glucose (D-Glc), d-glucuronic acid (D-GlcA), 4,6-O-[(R)-1-carboxyethylidene]-d-galactose [D-Galp4,6 (R-Pyr)] and two residues of d-galactose (D-Gal) was established:

Spectrochemical Insights into Aromatic Hydrocarbons in 1,2-Disubstituted Naphthalenes: Investigations Using 1D and 2D NMR Spectroscopy and X-Ray Crystallography Analysis

Naphthalene and its derivatives are often determined as markers of environmental pollution, although they are also recognized as crucial building blocks in many beneficial compounds. In the course of this study, a range of 1,2-disubstituted naphthalenes bearing nitro, amine, sulfonate, chlorine, and hydroxyl substituents were synthesized and characterized using several spectroscopic techniques, including FT-IR,1H NMR,13C NMR, DEPT-90, DEPT-135, DEPTq, elemental analysis, and high-resolution mass (HRMs). The NMR analysis of the aromatic hydrocarbon moiety in these compounds emphasized the impact of substituent variations on the appearance of 1H and 13C NMR signals. To resolve the issue of overlapping one-dimensional (1D) 1H NMR signals and determine the connectivity of hydrogen and carbon atoms from the aromatic naphtayl ring, we employed a “two-dimensional” experiment, using homonuclear correlation spectroscopy (2D 1H-1H COSY) and heteronuclear single quantum correlation (2D 1H - 13C HSQC) and heteronuclear multiple‐bond correlation spectroscopy (2D 1H - 13C HMBC). This work also investigated the correlation between 1H NMR signals and thin-layer chromatography (TLC) data. X-ray crystallography data validates that the presence of a specific group, not aligned coplanarly with the naphthyl ring, exerts a spatial influence on the manifestation of 1H NMR signals, alongside other electronic effects. This study will offer NMR data, providing insights into the potential applications and analyses of naphthalene derivatives across various theoretical and practical domains.

Synthesis, spectroscopic characterization, DFT calculations, in silico-ADMET and molecular docking analysis of novel quinoline-substituted 5H-chromeno [2,3-b] pyridine derivatives as antibacterial agents.

A convenient, straightforward, and effective one-step reaction for the synthesis of a three-component compound of biologically relevant novel 2,4-diamino-5-(8-hydroxyquinolin-7-yl)-5H-chromeno[2,3-b] pyridine-3-carbonitrile derivatives was designed and synthesized. The synthesis was developed by the reaction between salicylaldehyde 1, 8-hydroxyquinoline 2, 2-aminopropene-1,1,3-tricarbonitrile 3, and the catalytic amount of triethylamine in ethanol at 78°C. This methodology has many beneficial features, including the use of inexpensive and non-hazardous starting materials, single-flask reactions, optimized reaction conditions, the termination of intermediate isolation, easy workup, reducing organic waste products, being chromatography-free, and decreasing the reaction time along with quantitative yields with high functional group tolerance. A proposed mechanism with supporting experimental data is presented, including 1H NMR, 13C NMR, 2D NMR (HMBC, COSY, HSQC), mass, and IR spectroscopy, which are used to characterize the complete derivatives. All synthesized compounds were evaluated in vitro for their antibacterial activities against Bacillussubtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa bacterial strains via the agar-well diffusion method compared with the reference drug gentamicin. The data indicated that compounds 4A, 4F, 4G, 4J, and 4K consistently demonstrated strong antimicrobial activity against Gram-positive and Gram-negative bacteria. Furthermore, a molecular docking investigation was carried out to gain insight into the binding mode of the most promising compounds via the crystal structure of the S. aureus DNA gyrase complex with ciprofloxacin (PDB ID: 2XCT). Density functional theory (DFT) calculations were performed to determine the various molecular properties of the synthesized novel 2,4-diamino-5-(8-hydroxyquinolin-7-yl)-5H-chromeno [2,3-b] pyridine-3-carbonitrile derivatives (4A-4M). On the basis of the reactive sites explored by the molecular electrostatic potential maps, the antibacterial activities of the compounds were screened.

Non-Targeted Nuclear Magnetic Resonance Analysis for Food Authenticity: A Comparative Study on Tomato Samples.

Non-targeted NMR is widely accepted as a powerful and robust analytical tool for food control. Nevertheless, standardized procedures based on validated methods are still needed when a non-targeted approach is adopted. Interlaboratory comparisons carried out in recent years have demonstrated the statistical equivalence of spectra generated by different instruments when the sample was prepared by the same operator. The present study focused on assessing the reproducibility of NMR spectra of the same matrix when different operators performed individually both the sample preparation and the measurements using their spectrometer. For this purpose, two independent laboratories prepared 63 tomato samples according to a previously optimized procedure and recorded the corresponding 1D 1H NMR spectra. A classification model was built using the spectroscopic fingerprint data delivered by the two laboratories to assess the geographical origin of the tomato samples. The performance of the optimized statistical model was satisfactory, with a 97.62% correct sample classification rate. The results of this work support the suitability of NMR techniques in food control routines even when samples are prepared by different operators by using their equipment in independent laboratories.

Microencapsulation of the Biocide Benzisothiazolinone (BIT) by Inclusion in Methyl-β-cyclodextrin and Screening of Its Antibacterial and Ecotoxicity Properties.

The excessive use of biocides has considerable environmental and economic impacts; this is why new technologies have been sought to decrease the concentration levels applied in an effort to reduce the use of these substances. Microencapsulation using cyclodextrins has been widely used in the food and pharmaceutical industries as a way of reducing the concentrations of the active substance necessary to achieve a biological effect and/or eliminate its irritating or toxicological effects. In this study, the inclusion complexation behavior and binding ability of benzothiazolinone (BIT) with different β-cyclodextrins (β-CD, HP-β-CD, and Me-β-CD) was investigated. The intermolecular interactions were examined through UV and FTIR spectroscopy, DSC, 1D 1H NMR, and 2D ROESY. The highest stability constant was observed for the BIT/Me-β-CD inclusion complex (299.5 ± 2.9 M-1). Antibacterial activity was investigated against Staphylococcus aureus and Escherichia coli, and the results revealed that the BIT/Me-β-CD inclusion complex displays a higher antibacterial activity than BIT. The acute toxicity of the biocide and inclusion complex was also examined using the photobacterium Aliivibrio fischeri. Although BIT exhibited higher toxicity than the inclusion complex, further investigation is needed due to the quorum quenching effect of β-CDs. The data found suggest that BIT microencapsulation can increase its aqueous solubility and can be used as an effective tool to improve its chemical, biological, and ecotoxicological properties.

Antibacterial Properties of Phytochemicals Isolated from Leaves of Alstonia boonei and Aerial Parts of Ipomoea cairica

Objective The leaves of Alstonia boonei and aerial parts of Ipomoea cairica are used for treatment of microbial infections among other ailments in African traditional medicine. The aim of this study was to investigate the antimicrobial phytochemicals in A. boonei leaves and Ipomoea cairica aerial parts to validate their traditional use in Ugandan herbal medicine. Methods The plant materials were separately extracted using a dichloromethane/methanol (1:1) solvent system and subjected to repeated chromatographic separation to isolate pure compounds. The chemical structures of the isolated compounds were determined through 1H NMR, 13C NMR and 2D NMR (COSY, HSQC and HMBC). The antibacterial activity of the extracts and pure compounds were assessed using the agar well diffusion method. Results Chromatographic fractionation of the extracts yielded trans-fagaramide and a pentacyclic lupane-type triterpenoid, lupeol, from A. boonei, and friedelin from I. cairica. Trans-fagaramide was identified for the first time in the Alstonia genus while friedelin was identified for the first time in I. cairica. The isolated compounds demonstrated antibacterial activity, with trans-fagaramide showing a minimum inhibitory concentration (MIC) of 125 μg/mL against Pseudomonas aeruginosa and 250 μg/mL against Staphylococcus aureus, Salmonella typhi and Escherichia coli. Friedelin exhibited a MIC of 125 μg/mL against Escherichia coli and 250 μg/mL against Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella typhi. Conclusion The antibacterial activities observed in this study support the traditional use of A. boonei and I. cairica by indigenous communities in Uganda for treating microbial infections.

Parkinsonia praecox bark as a new source of antibacterial and anticancer compounds

IntroductionParkinsonia praecox is used in traditional medicine to treat various diseases; however, the antibacterial and anticancer properties from its bark have not been reported. Thus, this study aimed to evaluate the antibacterial activity, cytotoxicity and chemical study of methanolic extract of Parkinsonia praecox bark (PpBM). MethodsThe PpBM antibacterial activity was determined against pathogen strains using the broth microdilution method, the cytotoxic effect was assessed on cancerous (HepG2, SKOV-3 and HeLa) and non-cancerous (J774A.1 and HaCaT) cells using the crystal violet assay, the in vitro hemotoxicity was evaluated using the hemolysis assay on human erythrocytes and the identification of major compounds derived from PpBM was performed by 1H and 13C NMR and 2D NMR experiments. ResultsPpBM (2000 µg/mL) demonstrated antibacterial activity against Listeria monocytogenes, a potential breakthrough in the fight against this pathogen. PpBM decreased cell viability of HepG2 liver cancer cells (IC50, 104.56 µg/mL) with a selectivity index of 1.38 regarding J774A.1 macrophages and 2.04 to HaCaT keratinocytes, suggesting a selective anticancer potential. On the other hand, PpBM did not cause hemolysis to high concentrations (1000 µg/mL) or alterations in the morphology of human erythrocytes at doses lower than 200 µg/mL. For the first time, lupenone, germanicone, 3-oxo-oleanane-18-en-28-ol, and combretol were identified in P. praecox. PpBM showed antibacterial, selective anticancer, and non-hemotoxic properties. These activities may be related to the major compounds, such as lupenone. ConclusionP. praecox bark is a new option for obtaining bioactive compounds, further in vitro and in vivo studies are required to confirm their efficacy and safety.

LAMAIS: A library-aided approach for efficient 1D 1H NMR qualitative analysis in plant metabolomics

BackgroundOne-dimensional proton nuclear magnetic resonance (1D 1H NMR) spectroscopy is a non-destructive, non-targeted analytical technique providing both qualitative and quantitative insights, particularly beneficial for mixture analysis. However, the qualitative analysis of 1D 1H NMR spectra for mixture samples is laborious and time-consuming, involving extensive database searches and verification experiments like spiking. This process heavily relies on the analyst's expertise, leading to efficiency discrepancies. There is a pressing need for a reliable method to streamline operations and enhance the efficiency of qualitative analysis in complex mixtures. ResultsWe introduce a library-aided method for spectral profiling, named LAMAIS. This method achieves compound identification through similarity assessment between samples and template data, allowing rapid, automatic compound identification and full-spectrum peak assignment without the need for fitting. LAMAIS correctly identifies over 90 % of components in synthetic mixtures and more than 75 % in experimental mixtures, surpassing other representative methods with a higher F2 score. Our reference library, which currently includes 71 compounds, is tailored to capture the commonality of primary metabolites across diverse plant species. The analysis of real-world samples yielded encouraging results, underscoring LAMAIS's versatility as an auxiliary tool suitable for a variety of botanical sources. For analyst convenience, interactive graphics are utilized as the output format. SignificanceLAMAIS excels, demonstrating competitiveness and reliability. The approach minimizes repetitive tasks and sample wastage, improving the efficiency of 1D 1H NMR qualitative analysis. Constructing a reference library effectively preserves knowledge, mitigates reliance on human experience, and addresses gaps in the analysis of plant source samples.

Structure, Dynamics, and Reactivity of Encapsulated Molecules in Restricted Spaces: Arylazoisoxazoles within an Octa Acid Capsule.

In this study, a well-defined organic capsule assembled from two octa acid (OA) molecules acting as host and select arylazoisoxazoles (AAIO) acting as guests were employed to demonstrate that confined molecules have restricted freedom that translates into reaction selectivity in both ground and excited states. The behavior of these AAIO guests in confined capsules was found to be different from that found in both crystals, where there is very little freedom, and in isotropic solvents, where there is complete freedom. Through one-dimensional (1D) and two-dimensional (2D) 1H NMR spectroscopic experiments, we have established a relationship between structure, dynamics and reactivity of molecules confined in an OA capsule. Introduction of CF3 and CH3 substitution at the 4-position of the aryl group of AAIO reveals that in addition to space confinement, weak interactions between the guest and the OA capsule control the dynamics and reactivity of guest molecules. 1H NMR studies revealed that there is a temperature-dependence to guest molecules tumbling (180° rotation along the capsular short axis) within an OA capsule. While 1H NMR points to the occurrence of tumbling motion, MD simulations and simulation of the temperature-dependent NMR signals provide an insight into the mechanism of tumbling within OA capsules. Thermal and photochemical isomerization of AAIO were found to occur within an OA capsule just as in organic solvents. The observed selectivity noted during thermal and photo induced isomerization of OA encapsulated AAIOs can be qualitatively understood in terms of the well-known concepts due to Bell-Evans-Polanyi (BEP principle), Hammond and Zimmerman.

Ultraselective, Ultrahigh Resolution 1D TOCSY

AbstractSolution state 1H NMR spectroscopy provides valuable insights into molecular structure and conformation. However, when the spectrum exhibits severe signal overlap, it hampers the extraction of key structural information. Here, an ultraselective, ultrahigh resolution TOCSY method is introduced that greatly reduces spectral complexity, allowing the extraction of previously inaccessible spectral information. It combines the recently developed GEMSTONE excitation with homonuclear decoupling to provide highly simplified through‐bond correlation 1D 1H NMR spectra, showing all signals within the selected spin system as singlets. The new method can greatly facilitate the analysis of mixtures, as shown here for a mixture of Cinchona alkaloids (popular catalysts in asymmetric synthesis) and a mixture of glucocorticoids (used for treating conditions such as asthma).

Synthesis and toxicity of monothiooxalamides against human red blood cells, brine shrimp (Artemia salina), and fruit fly (Drosophilamelanogaster)

A new family of monothiooxalamides derived from 2-aminobenzimidazole was synthesized, and their structures were confirmed by 1H and 13C one-dimensional and 2D NMR experiments (COSY, HSQC, and HMBC). The antioxidant capacity was evaluated by free radical scavenging assays: 1,1-diphenyl-2-picrylhydrazyl (DPPH•), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and the Fe(II) chelating ability. Our work group has previously reported the synthesis and antioxidant activity of monothiooxalamides derived from 2-aminopyridine (I). In this study, the in vitro hemolytic activity of compounds from the 2-aminopyridine (I) and 2-aminobenzimidazole (II) families was evaluated against human red blood cells (RBCs). The concentration at which monothiooxalamides showed no hemolytic activity was chosen to assess their ability to inhibit free radical-induced membrane damage in human RBCs, acute toxicity in brine shrimp, and in vivo toxicity against Drosophila melanogaster. Compounds with morpholine fragments (1g, 1h, 2g, and 2h) showed time- and concentration-dependent protective effects against radical-induced oxidative hemolysis. Moreover, they had the lowest acute toxicity in the brine shrimp lethality assay and a significant increase in chelating activity compared with the other molecules. In particular, monothiooxalamide 2g showed lower toxicity and can be considered for further biological screening and application trials.

Simulated LC-MS Data Set for Assessing the Metabolomics Data Processing Pipeline Implemented into MVAPACK.

Metabolomics commonly relies on using one-dimensional (1D) 1H NMR spectroscopy or liquid chromatography-mass spectrometry (LC-MS) to derive scientific insights from large collections of biological samples. NMR and MS approaches to metabolomics require, among other issues, a data processing pipeline. Quantitative assessment of the performance of these software platforms is challenged by a lack of standardized data sets with "known" outcomes. To resolve this issue, we created a novel simulated LC-MS data set with known peak locations and intensities, defined metabolite differences between groups (i.e., fold change > 2, coefficient of variation ≤ 25%), and different amounts of added Gaussian noise (0, 5, or 10%) and missing features (0, 10, or 20%). This data set was developed to improve benchmarking of existing LC-MS metabolomics software and to validate the updated version of our MVAPACK software, which added gas chromatography-MS and LC-MS functionality to its existing 1D and two-dimensional NMR data processing capabilities. We also included two experimental LC-MS data sets acquired from a standard mixture andMycobacterium smegmatiscell lysates since a simulated data set alone may not capture all the unique characteristics and variability of real spectra needed to assess software performance properly. Our simulated and experimental LC-MS data sets were processed with the MS-DIAL and XCMSOnline software packages and our MVAPACK toolkit to showcase the utility of our data sets to benchmark MVAPACK against community standards. Our results demonstrate the enhanced objectivity and clarity of software assessment that can be achieved when both simulated and experimental data are employed since distinctly different software performances were observed with the simulated and experimental LC-MS data sets. We also demonstrate that the performance of MVAPACK is equivalent to or exceeds existing LC-MS software programs while providing a single platform for processing and analyzing both NMR and MS data sets.

Isolated auto-citrullinated regions of PADI4 associate to the intact protein without altering their disordered conformation

PADI4 is one of the human isoforms of a group of enzymes intervening in the conversion of arginine to citrulline. It is involved in the development of several types of tumors, as well as other immunological illnesses, such as psoriasis, multiple sclerosis, or rheumatoid arthritis. PADI4 auto-citrullinates in several regions of its sequence, namely in correspondence of residues Arg205, Arg212, Arg218, and Arg383. We wanted to study whether the citrullinated moiety affects the conformation of nearby regions and its binding to intact PADI4. We designed two series of synthetic peptides comprising either the wild-type or the relative citrullinated versions of such regions – i.e., a first series of peptides comprising the first three arginines, and a second series comprising Arg383. We studied their conformational properties in isolation by using fluorescence, far-ultraviolet (UV) circular dichroism (CD), and 2D1H NMR. Furthermore, we characterized the binding of the wild-type and citrullinated peptides in the two series to the intact PADI4, by using isothermal titration calorimetry (ITC), fluorescence, and biolayer interferometry (BLI), as well as by molecular docking simulations. We observed that citrullination did not alter the local conformational propensities of the isolated peptides. Nevertheless, for all the peptides in the two series, citrullination slowed down the kinetic koff rates of the binding reaction to PADI4, probably due to differences in electrostatic effects compared to the presence of arginine. The affinities of PADI4 for unmodified peptides were slightly larger than those of the corresponding citrullinated ones in the two series, but they were all within the same range, indicating that there were no relevant variations in the thermodynamics of binding due to sequence effects. These results highlight details of the self-citrullination of PADI4 and, more generally, of possible auto-catalytic mechanisms taking place in vivo for other citrullinating enzymes or, alternatively, in proteins undergoing citrullination passively.