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SHP2 is a tyrosine phosphatase that plays a regulatory role in multiple intracellular signaling cascades and is known to be oncogenic in certain contexts. In the absence of effectors, SHP2 adopts an autoinhibited conformation with its N-SH2 domain blocking the active site. Given the key role of N-SH2 in regulating SHP2, this domain has been extensively studied, often by X-ray crystallography. Using a combination of structural analyses and molecular dynamics (MD) simulations we show that the crystallographic environment can significantly influence the structure of the isolated N-SH2 domain, resulting in misleading interpretations. As an orthogonal method to X-ray crystallography, we use a combination of NMR spectroscopy and MD simulations to accurately determine the conformation of apo N-SH2 in solution. In contrast to earlier reports based on crystallographic data, our results indicate that apo N-SH2 in solution primarily adopts a conformation with a fully zipped central β-sheet, and that partial unzipping of this β-sheet is promoted by binding of either phosphopeptides or even phosphate/sulfate ions.

Abstract Precise, fast, and reliable identification and quantification of microplastic contamination are essential for determining their environmental concentrations for risk assessments. This study investigates the use of nuclear magnetic resonance (NMR) spectroscopy to quantify microplastics by analysing dilution series of polystyrene (PS), polyisoprene-cis (PI), polybutadiene-cis (PB), polylactic acid (PLA), polyvinyl chloride (PVC) and polyurethane (PU). Each polymer type was dissolved in a suitable solvent and an internal standard was utilized for quantification. Detection and quantification limits for each polymer type were established in two ways: (1) by using an equation based on proton signals and an internal standard with known concentration and (2) by using the LOQ based on the signal-to-noise ratio. Both data sets were compared and showed that using the internal standard (method 1) results in more accurate and lower concentration limits in the range of 0.2–8 µg mL−1 for all six polymer types, while the LOQ based on the SNR (method 2) gives consistently higher concentration limits (1–10 µg mL−1). The research shows the accuracy, efficacy, and reliability of quantitative NMR spectroscopy for polymer analysis in these concentration ranges compared to established quantifying methods, such as, PyGC/MS, FTIR, or Raman spectroscopy.

Abstract Background Comprehensive characterization of the metabolome in cerebrospinal fluid (CSF) and serum by Nuclear Magnetic Resonance (NMR) spectroscopy may identify biomarkers and contribute to the understanding of the pathophysiology of neurological diseases. Methods Metabolites were determined by NMR spectroscopy in stored CSF/serum samples of 20 patients with Parkinson’s disease, 25 patients with other neuro-degenerative diseases, 22 patients with cerebral ischemia, 48 patients with multiple sclerosis, and 58 control patients with normal CSF findings. The data set was analysed using descriptive and multivariate statistics, as well as machine learning models. Results CSF glucose and lactic acid measured by NMR spectroscopy and routine clinical chemistry showed a strong correlation between both methods (glucose, R2 = 0.87, n = 173; lactic acid, R2 = 0.74, n = 173). NMR spectroscopy detected a total of 99 metabolites; 51 in both, CSF and serum, 16 in CSF only, and 32 in serum only. CSF concentrations of some metabolites increased with age and/or decreasing blood–brain-barrier function. Metabolite detection rates were overall similar among the different disease groups. However, in two-group comparisons, absolute metabolite levels in CSF and serum discriminated between multiple sclerosis and neurodegenerative diseases (area under the curve (AUC) = 0.96), multiple sclerosis and Parkinson’s disease (AUC = 0.89), and Parkinson’s disease and control patients (AUC = 0.91), as demonstrated by random forest statistical models. Orthogonal partial least square discriminant analysis using absolute metabolite levels in CSF and serum furthermore permitted separation of Parkinson’s disease and neurodegenerative diseases. CSF propionic acid levels were about fourfold lower in Parkinson’s disease as compared to neurodegenerative diseases. Conclusions These findings outline the landscape of the CSF and serum metabolome in different categories of neurological diseases and identify age and blood–brain-barrier function as relevant co-factors for CSF levels of certain metabolites. Metabolome profiles as determined by NMR spectroscopy may potentially aid in differentiating groups of patients with different neurological diseases, including clinically meaningful differentiations, such as Parkinson’s disease from other neurodegenerative diseases.

Lignocellulosic biomass (LB) is promising feedstock for the production of various bio-based products. However, due to its heterogenous character, complex chemical structure and recalcitrance, it is necessary to know its structural composition in order to optimize pretreatment process and further (bio)conversion into bio-based products. Nuclear Magnetic Resonance (NMR) spectroscopy is a fast and reliable method that can provide advanced data on the molecular architecture and composition of lignocellulosic biomass. In this brief overview, characteristic examples of the use of high-resolution NMR spectroscopy for the investigation of various types of LB and their structural units are given and the main drawbacks and future perspectives are outlined.

Cellular production of tryptophan is metabolically expensive and tightly regulated. The small Bacillus subtilis zinc binding Anti-TRAP protein (AT), which is the product of the yczA/rtpA gene, is upregulated in response to accumulating levels of uncharged tRNATrp through a T-box antitermination mechanism. AT binds to the undecameric axially symmetric ring-shaped protein TRAP (trp RNA Binding Attenuation Protein), thereby preventing it from binding to the trp leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the trp operon. AT principally adopts two symmetric oligomeric states, a trimer (AT3) featuring three-fold axial symmetry or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form binds and inhibits TRAP. We apply native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) to monitor the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we use solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT3, while heteronuclear 15N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT3 and binding-inactive AT12, with implications for TRAP binding and inhibition.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous and synthetic ligands, including covalent antagonist inhibitors GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent inhibitors weaken—but do not prevent—the binding of other ligands via an allosteric mechanism, rather than direct ligand clashing, by shifting the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with orthosteric ligand binding. Crystal structures reveal different cobinding mechanisms including alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent inhibitor binding pose. Our findings highlight the significant flexibility of the PPARγ orthosteric pocket, its ability to accommodate multiple ligands, and demonstrate that GW9662 and T0070907 should not be used as chemical tools to inhibit ligand binding to PPARγ.

Abstract Proteins with intrinsically disordered regions (IDRs) often undergo phase separation to control their functions spatiotemporally. Changing the pH alters the protonation levels of charged sidechains, which in turn affects the attractive or repulsive force for phase separation. In a cell, the rupture of membrane‐bound compartments, such as lysosomes, creates an abrupt change in pH. However, how proteins’ phase separation reacts to different pH environments remains largely unexplored. Here, using extensive mutagenesis, NMR spectroscopy, and biophysical techniques, it is shown that the assembly of galectin‐3, a widely studied lysosomal damage marker, is driven by cation‐π interactions between positively charged residues in its folded domain with aromatic residues in the IDR in addition to π–π interaction between IDRs. It is also found that the sole two negatively charged residues in its IDR sense pH changes for tuning the condensation tendency. Also, these two residues may prevent this prion‐like IDR domain from forming rapid and extensive aggregates. These results demonstrate how cation‐π, π–π, and electrostatic interactions can regulate protein condensation between disordered and structured domains and highlight the importance of sparse negatively charged residues in prion‐like IDRs.

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined. In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase.

An in situ characterization method using liquid Nuclear Magnetic Resonance (NMR) spectroscopy has been developed to aid the preparation of highly reactive non-isocyanate polyurethanes (NIPUs) from cyclic carbonate aminolysis. Using this methodology, the aminolysis kinetics and the final polymer structure of a model NIPU obtained by reaction of a 5-membered bis-cyclic carbonate (5CC) and 1,4-diaminobutane have been fully investigated, as a function of the type and concentration of the aminolysis catalyst, and the reaction temperature. Several catalysts already reported in NIPUs syntheses, including 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), have been compared. The kinetics of the 5CC hydrolysis side reaction was also studied. With an activation energy of 29.7 kJ mol−1, TBD was clearly the most efficient catalyst used, allowing 5CC conversion ratio of up to 100 % using a concentration of 0.35 eq5CC. However, under these experiment conditions, TBD concentration also showed to have a non-negligible influence on the hydrolysis rate, representing between 6 and 14 % of the initial 5CC concentrations, at 353 K. Neither the catalyst or the temperature seemed to affect the polymer structure, with secondary hydroxyl-containing isomer proportions of (70 ± 6) %. Finally, this in situ NMR method is paving the way for rapid screening of innovative catalysts for sustainable NIPU synthesis.

How the sequences of intrinsically disordered proteins (IDPs) code for functions is still an enigma. Dynamics, in particular residue-specific dynamics, holds crucial clues. Enormous efforts have been spent to characterize residue-specific dynamics of IDPs, mainly through NMR spin relaxation experiments. Here, we present a sequence-based method, SeqDYN, for predicting residue-specific backbone dynamics of IDPs. SeqDYN employs a mathematical model with 21 parameters: one is a correlation length and 20 are the contributions of the amino acids to slow dynamics. Training on a set of 45 IDPs reveals aromatic, Arg, and long-branched aliphatic amino acids as the most active in slow dynamics whereas Gly and short polar amino acids as the least active. SeqDYN predictions not only provide an accurate and insightful characterization of sequence-dependent IDP dynamics but may also serve as indicators in a host of biophysical processes, including the propensities of IDP sequences to undergo phase separation.

Introduction. The structure of dihydroquercetin (DHQ) is characterized by two chiral centers at positions 2 and 3 of the benzopyran cycle, resulting in possible diastereomers: trans- and cis-isomers. Therefore, the development of methods for qualitative and quantitative control of DHQ diastereomers in analyzed samples is essential for patient safety management. Nuclear magnetic resonance (NMR) spectroscopy is one of the physicochemical methods that can be used for this purpose.Aim. The study objective was to accumulate the analytical and structural characteristics of cis-DHQ by NMR spectroscopy of the spheroidal form of this flavonoid (DHQs).Materials and Methods. 1D 1H, 1H,1H-COSY, 1H,1H-NOESY, and 1H,13C-HSQC NMR spectra were acquired at 298 K on an 800 MHz NMR spectrometer equipped with a TXI triple resonance probe. The number of scans was 32. The mixing time in the NOESY experiment was 400 ms. The 1H and 13C were analyzed using CcpNmr software. The dihedral angles were calculated by applying the Karplus equation.Results and discussion. In trans-DHQ, the chemical shift values for H2 and H3 are 4.93 ppm and 4.52 ppm, respectively, and in cis-DHQ they are 5.31 ppm and 4.20 ppm, respectively. The spin-spin coupling constants between H2 and H3 of trans- and cis-DHQ are 12.00 Hz and 2.40 Hz, respectively. Thus, the dihedral angles for the trans- and cis-isomers are 154° and 64°, respectively. We found that DHQs contains 12.5 % of the cis-isomer.Conclusion. Our experiments confirmed that NMR spectroscopy can discriminate between trans- and cis-DHQ based on the chemical shift values for the cross-peaks of H2 and H3. The second major finding was that this method can be considered as a more selective quantitative analysis than HPLC with UV detection without reference. One of the most important results of this study for drug development is the updated information on the structural parameters of DHQ diastereomers in the liquid phase.

The aim of the work was to review modern extraction, detection and quantification analytical methods of inositols and their derivatives. Methods. Inositols are extracted from vegetable raw materials by methods of liquid extraction, under pressure, microwave extraction and supercritical fluid extraction. Quantitatively analyzed by methods of gas and liquid chromatography with preliminary derivatization. The structure of inositols can be determined by the NMR spectroscopy. Results. Inositols and their derivatives are biologically active compounds, wich are involved in the egulation of the intracellular calcium level, the transmission of hormonal signals, the breakdown of fats and the reduction of cholesterol in the blood, the modulation of the neurotransmitters activity, etc. Inositols are used in the production of vitamin preparations. The main source for inositols extraction is vegetable raw material, namely alfalfa, as well as wheat sprouts, grapefruit, hazelnuts and others. In the paper, the methods of inositols extraction with organic and inorganic solvents, including the use of a Soxhlet apparatus, liquid extraction under pressure, microwave extraction and supercritical fluid extraction are considered. The procedure of preliminary sample preparation and polyols derivatization for their further separation and quantitative determination is described. Modern chromatographic methods of polyols identification and quantitative determination are analyzed. The possibility of using 1H, 13C and 31P NMR spectroscopy to identify the structure of inositols and their derivatives is described. Conclusions. Inositols are biologically active compounds of a wide spectrum of action, therefore there is an urgent need to develop biotechnological processes for their production and extraction from plant raw materials and microorganisms.

Abstract Fatty acid synthesis (FAS) is essential for the production of biological components such as cell membrane building blocks and metabolism-related compounds. There are two types of bacterial FAS: de novo FAS and FAS through the incorporation of external fatty acids. Enterococcus faecalis possesses two distinct acyl carrier proteins (ACPs), AcpA (EfAcpA) and AcpB (EfAcpB), which serve as cofactors in the two types of FAS. We previously showed through NMR spectroscopy that EfAcpA comprises only three long helices, while EfAcpB consists of four helices, including a short α3 helix, similar to other bacterial ACPs. An increase in melting temperature (Tm) from 64.0 to 76.1 °C confirmed that protein structural stability increased in the presence of calcium ions. Using NMR spectroscopy, two metal binding sites were identified in EfAcpA: site A was located at the start of the α2 helix while site B was situated near the α2 helix and α2α3 loop. To understand the importance of structural flexibility of EfAcpA in de novo FAS, we investigated its motional properties using backbone spin relaxation and molecular dynamics simulations. The α2α3 loop in EfAcpA displayed high flexibility, as indicated by low heteronuclear NOE values. The residues Val51, Glu54, and Gly58 exhibited significant R 2 values, likely due to the movement of this loop. EfAcpA created a novel cavity towards the α1α2 loop, in contrast to conventional cavity formation in most bacterial ACPs. This unique behavior was attributed to the flexibility exhibited by the α2α3 loop. The structural and motional characteristics of EfAcpA confirmed that its conformational plasticity is a crucial factor influencing acyl chain transfers in de novo FAS. Given the increasing antibiotic resistance observed for E. faecalis in clinical settings, the findings of this study may contribute to the development of more effective pathogen management strategies targeting FAS.

A novel approach to the protection of unstable fat-soluble vitamins, using retinol as an example, is presented in this work. This method is based on introducing vitamin A molecules into casein micelles. Protective properties of micellar casein towards different forms of retinol (native vitamin and palmitic acid ester) in vitro and in emulsion-type meat products are investigated. A technology of the introduction using micellar casein concentrate (MCC) has been developed. Conditions similar to those in which vitamin molecules can be in meat emulsions during heat treatment are simulated in vitro. The optimal time of “encapsulation” (2 hours) and the need for additional surfactant (tween-80) are identified. The use of the casein micelles protection made it possible to increase the number of retinol molecules that did not undergo decomposition under model conditions (in vitro) from ~30% to ~80%. Using the vitamin premix the degree of degradation of vitamin molecules does not exceed 4% after heat treatment. Data received allowed us to determine the efficiency of the protection properties of casein micelles for unstable vitamin A molecules.

Metabolomics has emerged as a critical field in understanding biological processes and disease mechanisms, necessitating advancements in analytical techniques to handle complex biological samples. This review explores the global landscape of metabolomics, with a focus on the use of spectroscopy and spectrometry. Techniques such as UV-Vis and Fourier Transform InfraRed (FTIR) spectroscopy offer fast and cost-effective metabolite tracing but are limited by their sensitivity, particularly for low-abundance metabolites. Nuclear Magnetic Resonance (NMR) spectroscopy, despite being less sensitive than mass spectrometry (MS), provides unparalleled structural information, distinguishing metabolites with similar mass-to-charge ratios. NMR's capability to detect metabolites in the 1-10 µM range highlights its effectiveness in metabolomics. This review categorizes advancements in these techniques, starting with global contributions to spectroscopy, followed by detailed discussions on FTIR strategies for metabolite tracing, and concluding with NMR's qualitative and quantitative applications in metabolomics and disease diagnostics. The review underscores the continuous development in sample preparation and data integration, enhancing the accuracy and applicability of these techniques, positioning NMR and FTIR as essential tools in modern metabolomic research.

Recurrent and metastatic breast cancer is frequently treatment resistant. A wealth of evidence suggests that reprogrammed lipid metabolism supports cancer recurrence. Overexpression of the RON and DEK oncoproteins in breast cancer is associated with poor outcome. Both proteins promote cancer metastasis in laboratory models, but their influence on lipid metabolite levels remain unknown. To measure RON- and DEK-dependent steady-state lipid metabolite levels, a nuclear magnetic resonance (NMR)-based approach was utilized. The observed differences identified a lipid metabolism-related gene expression signature that is prognostic of overall survival (OS), distant metastasis-free survival (DMFS), post-progression survival (PPS), and recurrence-free survival (RFS) in patients with breast cancer. RON loss led to decreased cholesterol and sphingomyelin levels, whereas DEK loss increased total fatty acid levels and decreased free glycerol levels. Lipid-related genes were then queried to define a signature that predicts poor outcomes for patients with breast cancer patients. Taken together, RON and DEK differentially regulate lipid metabolism in a manner that predicts and may promote breast cancer metastasis and recurrence.

Abstract Deciphering nature's remarkable way of encoding functions in its biominerals holds the potential to enable the rational development of nature‐inspired materials with tailored properties. However, the complex processes that convert solution‐state precursors into solid biomaterials remain largely unknown. In this study, an unconventional approach is presented to characterize these precursors for the diatom‐derived peptides R5 and synthetic Silaffin‐1A1 (synSil‐1A1). These molecules can form defined supramolecular assemblies in solution, which act as templates for solid silica structures. Using a tailored structural biology toolbox, the structure‐function relationships of these self‐assemblies are unveiled. NMR‐derived constraints are employed to enable a recently developed fractal‐cluster formalism and then reveal the architecture of the peptide assemblies in atomistic detail. Finally, by monitoring the self‐assembly activities during silica formation at simultaneous high temporal and residue resolution using real‐time spectroscopy, the mechanism is elucidated underlying template‐driven silica formation. Thus, it is demonstrated how to exercise morphology control over bioinorganic solids by manipulating the template architectures. It is found that the morphology of the templates is translated into the shape of bioinorganic particles via a mechanism that includes silica nucleation on the solution‐state complexes’ surfaces followed by complete surface coating and particle precipitation.

Abstract Pure shift NMR spectroscopy enables the robust probing on molecular structure and dynamics, benefiting from great resolution enhancements. Despite extensive application landscapes in various branches of chemistry, the long experimental times induced by the additional time dimension generally hinder its further developments and practical deployments, especially for multi‐dimensional pure shift NMR. Herein, this study proposes and implements the fast, reliable, and robust reconstruction for accelerated pure shift NMR spectroscopy with lightweight attention‐assisted deep neural network. This deep learning protocol allows one to regain high‐resolution signals and suppress undersampling artifacts, as well as furnish high‐fidelity signal intensities along with the accelerated pure shift acquisition, benefitting from the introduction of the attention mechanism to highlight the spectral feature and information of interest. Extensive results of simulated and experimental NMR data demonstrate that this attention‐assisted deep learning protocol enables the effective recovery of weak signals that are almost drown in the serious undersampling artifacts, and the distinction and recognition of close chemical shifts even though using merely 5.4% data, highlighting its huge potentials on fast pure shift NMR spectroscopy. As a result, this study affords a promising paradigm for the AI‐assisted NMR protocols toward broader applications in chemistry, biology, materials, and life sciences, and among others.

The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

Dihydroquercetin (DHQ) is a representative of flavonoids that is available on the market as a food supplement and registered as an active pharmaceutical ingredient. The structure of this compound is characterized by the presence of two chiral centers in positions 2 and 3 of the pyranone ring. Current regulatory documentation on DHQ lacks quantitative analysis of the stereoisomers of this flavanonol. This poses potential risks for consumers of DHQ-based dietary supplements and developers of new drugs. This review was conducted to systematize data on the pharmacology of DHQ stereoisomers and the possible methods of controlling them in promising chiral drugs. We found that relying on literature data of polarimetry for the identification of DHQ stereoisomers is currently impossible due to these heterogeneities. NMR spectroscopy allows to distinguishing between trans- and cis-DHQ using chemical shifts values. Only HPLC is currently characterized by sufficient enantioselectivity. Regarding pharmacology, the most active stereoisomer of DHQ should be identified, if the substituents in chiral centers both take part in binding with the biological target. The significant impact of stereochemical structure on the pharmacokinetics of DHQ isomers was reported. The question about these toxicity of these compounds remains open. The results of the conducted review of scientific literature indicate the necessity of revising the pharmacology of DHQ taking into account its stereoisomerism.

Abstract A library of novel bis-Schiff base derivatives based on thiobarbituric acid has been effectively synthesized by multi-step reactions as part of our ongoing pursuit of novel anti-diabetic agents. All these derivatives were subjected to in vitro α-glucosidase inhibitory potential testing after structural confirmation by modern spectroscopic techniques. Among them, compound 8 (IC50 = 0.10 ± 0.05 µM), and 9 (IC50 = 0.13 ± 0.03 µM) exhibited promising inhibitory activity better than the standard drug acarbose (IC50 = 0.27 ± 0.04 µM). Similarly, derivatives (5, 6, 7, 10 and 4) showed significant to good inhibitory activity in the range of IC50 values from 0.32 ± 0.03 to 0.52 ± 0.02 µM. These derivatives were docked with the target protein to elucidate their binding affinities and key interactions, providing additional insights into their inhibitory mechanisms. The chemical nature of these compounds were reveal by performing the density functional theory (DFT) calculation using hybrid B3LYP functional with 6-311++G(d,p) basis set. The presence of intramolecular H-bonding was explored by DFT-d3 and reduced density gradient (RGD) analysis. Furthermore, various reactivity parameters were explored by performing TD-DFT at CAM-B3LYP/6-311++G(d,p) method.

Cardiac arrhythmias are the most common pathologies of the cardiovascular system. Allapinin® and Allaforte® from “Pharmcenter VILAR” are effective IC-class antiarrhythmic agents. The main component of these drugs is a pharmaceutical substance with INN: lappaconitine hydrobromide, which in addition to lappaconitine hydrobromide itself, contains impurities of other diterpene alkaloids. This work is devoted to a detailed analysis of the alkaloid composition of a new pharmaceutical substance isolated from roots and rhizomes, as well as from the aerial part of plants of the genus Aconite (monkshood, wolfsbane) of the Ranunculaceae family (buttercups) using chromato-mass spectrometry and NMR spectroscopy. In addition, an assessment was made of the quantitative ratios of alkaloids in several samples of pharmaceutical substances isolated from different batches of medicinal plant raw materials.

Abstract Quantitative 1H-NMR became an increasingly important issue in pharmaceutical analytical chemistry. This study used NMR spectroscopy to assay the bronchodilator drug terbutaline sulfate and its pro-drug bambuterol hydrochloride in pure form and pharmaceutical preparations. The technique proceeded using deuterium oxide (D2O) as an 1H-NMR solvent and phloroglucinol anhydrous as an internal standard (IS). Comparatively, to the phloroglucinol signal at 5.9 ppm, the resulting quantitative signals of the studied drugs were corrected. The terbutaline singlet signal at 6.3 ppm was chosen for quantification, while the bambuterol quantitative singlet signal was at 2.9 ppm. The two drugs were rectilinear over the concentration range of 1.0–16.0 mg/mL. LOD values were 0.19 and 0.21 mg/mL while LOQ values were 0.58 and 0.64 mg/mL for terbutaline and bambuterol respectively. The developed method has been validated according to the International Conference of Harmonization (ICH) regarding linearity, accuracy, precision, specificity, and robustness. A greenness profile assessment was applied, and the method proved to be green. The method enables the assay of the two drugs in pure drug and pharmaceutical preparations. The method also enables the assay of the two drugs in the presence of each other; thus, it is considered a stability-indicating method where terbutaline is an acid degradation product of bambuterol.

Background: One defining feature of various aggressive cancers, including glioblastoma multiforme (GBM), is glycolysis upregulation, making its inhibition a promising therapeutic approach. One promising compound is 2-deoxy-d-glucose (2-DG), a d-glucose analog with high clinical potential due to its ability to inhibit glycolysis. Upon uptake, 2-DG is phosphorylated by hexokinase to 2-DG-6-phosphate, which inhibits hexokinase and downstream glycolytic enzymes. Unfortunately, therapeutic use of 2-DG is limited by poor pharmacokinetics, suppressing its efficacy. Methods: To address these issues, we synthesized novel halogenated 2-DG analogs (2-FG, 2,2-diFG, 2-CG, and 2-BG) and evaluated their glycolytic inhibition in GBM cells. Our in vitro and computational studies suggest that these derivatives modulate hexokinase activity differently. Results: Fluorinated compounds show the most potent cytotoxic effects, indicated by the lowest IC50 values. These effects were more pronounced in hypoxic conditions. 19F NMR experiments and molecular docking confirmed that fluorinated derivatives bind hexokinase comparably to glucose. Enzymatic assays demonstrated that all halogenated derivatives are more effective HKII inhibitors than 2-DG, particularly through their 6-phosphates. By modifying the C-2 position with halogens, these compounds may overcome the poor pharmacokinetics of 2-DG. The modifications seem to enhance the stability and uptake of the compounds, making them effective at lower doses and over prolonged periods. Conclusions: This research has the potential to reshape the treatment landscape for GBM and possibly other cancers by offering a more targeted, effective, and metabolically focused therapeutic approach. The application of halogenated 2-DG analogs represents a promising advancement in cancer metabolism-targeted therapies, with the potential to overcome current treatment limitations.

The quinoline ring is found in many biologically active natural alkaloids and is still being highly exploited by researchers due to its numerous potential applications in fields ranging from pharmacology to material science. During our synthetic attempts for new quinoline-4-carboxylic acids, using an extended version of the Doebner reaction, a new puzzling compound emerged when para-iodine aniline was reacted with salicylaldehyde and pyruvic acid in acetic acid as a reaction medium. The chemical structure of this new compound was established based on the information obtained from 1D and 2D NMR experiments (1H-, 13C-, and 15N-NMR), corroborated with MS spectrometry and IR spectroscopy. The photophysical properties (UV–vis and fluorescence) were also investigated. The proposed structure contains as the main elements a 1,4-dioxane-2,5-dione core symmetrically substituted with a propylidene chain that has attached to it a salicylaldehyde fragment and a pyrrole-2-one ring containing two 4-iodophenyl fragments. The isolation of this compound, reported here for the first time, is direct evidence that unexpected compounds can emerge from “classical” synthetic pathways when the right components are combined.

Abstract Quadruplex‐duplex (QD) junctions, which represent unique structural motifs of both biological and technological significance, have been shown to constitute high‐affinity binding sites for various ligands. A QD hybrid construct based on a human telomeric sequence, which harbors a duplex stem‐loop in place of a short lateral loop, is structurally characterized by NMR. It folds into two major species with a (3+1) hybrid and a chair‐type (2+2) antiparallel quadruplex domain coexisting in a K+ buffer solution. The antiparallel species is stabilized by an unusual capping structure involving a thymine and protonated adenine base AH+ of the lateral loop facing the hairpin duplex to form a T·AH+·G·C quartet with the interfacial G·C base pair at neutral pH. Addition and binding of Phen‐DC3 to the QD hybrid mixture by its partial intercalation at corresponding QD junctions leads to a topological transition with exclusive formation of the (3+1) hybrid fold. In agreement with the available experimental data, such an unprecedented discrimination of QD junctions by a ligand can be rationalized following an induced fit mechanism.

NMR spectroscopy has played a pivotal role in fragment-based drug discovery by coupling detection of weak ligand-target binding with structural mapping of the binding site. Fragment-based screening by NMR has been successfully applied to many soluble protein targets, but only to a limited number of membrane proteins, despite the fact that many drug targets are membrane proteins. This is partly because of difficulties preparing membrane proteins for NMR—especially human membrane proteins—and because of the inherent complexity associated with solution NMR spectroscopy on membrane protein samples, which require the inclusion of membrane-mimetic agents such as micelles, nanodiscs, or bicelles. Here, we developed a generalizable protocol for fragment-based screening of membrane proteins using NMR. We employed two human membrane protein targets, both in fully protonated detergent micelles: the single-pass C-terminal domain of the amyloid precursor protein, C99, and the tetraspan peripheral myelin protein 22 (PMP22). For both we determined the optimal NMR acquisition parameters, protein concentration, protein-to-micelle ratio, and upper limit to the concentration of D6-DMSO in screening samples. Furthermore, we conducted preliminary screens of a plate-format molecular fragment mixture library using our optimized conditions and were able to identify hit compounds that selectively bound to the respective target proteins. It is hoped that the approaches presented here will be useful in complementing existing methods for discovering lead compounds that target membrane proteins.

Excessive exposure to light is a global issue. Artificial light pollution has been shown to disrupt the body's natural circadian rhythm. To investigate the impacts of light on metabolism, we studied Sprague-Dawley rats chronically exposed to red or blue light during daytime or nighttime. Rats in the experimental group were exposed to extended light for 4 hours during daytime or nighttime to simulate the effects of excessive light usage. Strikingly, we found systemic metabolic alterations only induced by blue light during daytime. Furthermore, we conducted metabolomic analyses of the cerebrospinal fluid, serum, heart, liver, spleen, adrenal, cerebellum, pituitary, prostate, spermatophore, hypothalamus and kidney from rats in the control and blue light exposure during daytime. Significant changes in metabolites have been observed in cerebrospinal fluid, serum, hypothalamus and kidney of rats exposed to blue light during daytime. Metabolic alterations observed in rats encompassing pyruvate metabolism, glutathione metabolism homocysteine degradation, phosphatidylethanolamine biosynthesis, and phospholipid biosynthesis, exhibit analogous patterns to those inherent in specific physiological processes, notably neurodevelopment, cellular injury, oxidative stress, and autophagic pathways. Our study provides insights into tissue-specific metabolic changes in rats exposed to blue light during the daytime and may help explain potential mechanisms of photopathogenesis.

Polysaccharide derivatives with two types of functionalities were synthesized; reactive groups for click-chemistry approaches and groups that can tune the water solubility of the products. Xylan phenylcarbonates (XPCs) and cellulose phenylcarbonates (CPCs) with degrees of substitution (DS) ranging from 0.6 to 3.0 were prepared as starting materials. In a modular synthesis approach, these carbonate derivatives were converted with different amines to obtain functional xylan carbamates (XCs) and cellulose carbamates (CCs) with (i) reactive alkynyl- or furfuryl groups and / or (ii) hydrophilic hydroxyalkyl or tertiary amino groups. Both types of functionalities were introduced separately or simultaneously in a convenient one-pot-approach. Mixed polysaccharide carbamates with low, intermediate, and high DS values (0.6 to 2.5) were successfully prepared and characterized. The water solubility of the carbamates and mixed carbamates was tuned by variation of the type and DS of the two hydrophilic substituents. Surprisingly, some of the products showed lower or upper critical solution temperatures (LCSTs, UCSTs). Thus, the thermoreversible phase transition in aqueous solutions was studied. The products obtained in this work are very promising for the preparation of hydrogels through selective covalent crosslinking as well as for the fabrication of thermoresponsive biomaterials.

Abstract Here, an in vitro characterization of a family of prazole derivatives that covalently bind to the C73 site on Tsg101 and assay their ability to inhibit viral particle production is presented. Structurally, increased steric bulk on the 4‐pyridyl of the prazole expands the prazole site on the UEV domain toward the β‐hairpin in the Ub‐binding site and is coupled to increased inhibition of virus‐like particle production in HIV‐1. Increased bulk also increased toxicity, which is alleviated by increasing flexibility. Further, the formation of a novel secondary Tsg101 adduct for several of the tested compounds and the commercial drug lansoprazole. The secondary adduct involved the loss of the 4‐pyridyl substituent to form an irreversible species, with implications for increasing the half‐life of the active species or its specificity toward Tsg101 UEV. It is also determined that sulfide derivatives display effective viral inhibition, presumably through cellular sulfoxidation, allowing for delayed conversion within the cellular environment, and identify SARS‐COV‐2 as a target of prazole inhibition. These results open multiple avenues for the design of prazole derivatives for antiviral applications.

In the context of our ongoing studies on chromane derivatives as inhibitors of the salicylate synthase from M. tuberculosis, we isolated a new, unexpected compound from the cyclization of 3-(propargyloxy)-5-benzyloxy-benzoic acid methyl ester. Its molecular structure was elucidated by means of 1D and 2D NMR analyses, FT-IR, ESI-MS, and HRMS.

Abstract Erionite (ERI) zeolite has recently attracted considerable attention for its application prospect in the selective catalytic reduction of NOx with NH3 (NH3‐SCR), provided that the high‐silica (Si/Al > 5.5) analog with improved hydrothermal stability can be facilely synthesized. In this work, ERI zeolites with different Si/Al ratios (4.6, 6.4, and 9.1) are synthesized through an ultrafast route, and in particular, a high‐silica ERI zeolite with a Si/Al ratio of 9.1 is obtained by using faujasite (FAU) as a starting material. The solid‐state 29Si MAS NMR spectroscopic study in combination with a computational simulation allows for figuring out the atomic configurations of the Al species in the three ERI zeolites. It is revealed that the ERI zeolite with the highest Si/Al ratio (ERI‐9.1, where the number indicates the Si/Al ratio) exhibits a biased Al occupancy at T1 site, which is possibly due to the presence of a higher fraction of the residual potassium cations in the can cages. In contrast, the Al siting in ERI‐4.6 and ERI‐6.4 proves to be relatively random.

Recently, we could show that scallops show limitations of muscular performance like a reduced force under ocean warming and acidification. However, the underlying mechanisms at the cellular level are not completely understood. Metabolomics has become a valuable tool to evaluate the responses of marine organisms to various stressors. In the present study we therefore used a semi-targeted, multi tissue NMR based metabolomic approach to analyze metabolite patterns in the Atlantic king scallop, Pecten maximus, that were long-term acclimated to different end of century conditions of ocean warming (OW), ocean acidification (OA) and their combination (OWA). We investigated tissue specific metabolic profiles and metabolite concentrations in frozen tissues from gills, mantle and phasic and tonic adductor muscle of P. maximus under present conditions using 1H-HR-MAS NMR spectroscopy. A set of 33 metabolites revealed a clear tissue-specific pattern which can be attributed to the individual functions of the respective tissue type. We then evaluated the impact of OW, OA and OWA on the metabolic profiles of the different tissues. OW was the main driver of the changes in metabolites. In particular, energy-related metabolites seem to play an important role in the physiological response of scallops to OW and OWA. In combination with pathway analysis and network exploration we propose a possible correlation between metabolic changes in the adductor muscle and limited swimming performance of P. maximus under future climate. While the metabolic response of the phasic muscle seems to mainly depend on net consumption of energy related metabolites such as ATP and phospho-L-arginine, the tonic muscle seems to rely on metabolizing specific amino acids and beta-oxidation to account for the elevated energetic requirements under ocean warming and acidification.

The solid-state structure of a symmetrical carbohydrazone, namely 1,5-bis(2-hydroxy-3-methoxybenzylidene)carbonohydrazide was determined by X-ray single crystal diffraction method. Compound 1 crystallizes in the monoclinic space group P21/n with unit cell parameters a= 10.1198(6), b= 22.7847(11), c= 15.1738(10) Å, β= 100.458(6)°, Z= 4, V= 3440.6(3) Å3, R1= 0.0540. Crystal structure of 1 is defined by two crystallographic independent molecules, which are bonded via N–H···O hydrogen bond. The organic molecules are as keto tautomers with respect to the carbamide fragment, and adopt the anti conformation. 1D and 2D NMR experiments have argued on the presence of the title compound in DMSO-d6 solution mostly as keto tautomer in syn conformation, and enol-imino form when considering o-vanillin residue.

In this study, we assessed the quantity, strength, and acidity of zeolite composites comprising Silicalite-1 grown on ZSM-5 crystals using a combination of infrared (IR) and solid-state nuclear magnetic resonance (NMR) spectroscopy. The composites were created through the direct growth of Silicalite-1 crystals on ZSM-5 (P_ZSM-5), either with or without the organic structure-directing agent (OSDA) introduced into the ZSM-5 channels (samples: H_ZSM-5_Sil1 and TPA_ZSM-5_Sil1). The results revealed that Silicalite-1 grew differently when the ZSM-5 core was in the H+ form (empty pores) compared to when the OSDA was still present in the sample. This distinction was evident in the textural properties, with a decrease in the micropore surface area and an increase in the external surface area in the H_ZSM-5_Sil1 compared to the parent sample. The TPA_ZSM-5_Sil1 composite exhibited characteristics similar to the parent zeolite. These findings were further supported by 29Si NMR, which revealed a comparable local order for the parent (P_ZSM-5) and TPA_ZSM-5_Sil1 samples, along with a broadening of the Q4 peak for the H_ZSM-5_Sil1 composite. Additionally, the acid sites were preserved in the TPA_ZSM-5_Sil1 composite, while in the H+-form core, the concentration of Brønsted acid sites significantly decreased. This reduction in isolated Brønsted acid sites was further corroborated by 1H NMR.

The strong associations between the serum levels of adiponectin and the lipoprotein subclasses observed in healthy subjects are much weaker in patients with metabolic syndrome (MS). However, the impact of sex on these associations remained unexplored. Therefore, in the present study, we examined associations between adiponectin and the lipoprotein subclasses, analyzed by nuclear magnetic resonance spectroscopy, separately in healthy females and males, as well as in females and males with MS. We observed negative correlations between adiponectin and VLDL, IDL, and small-dense LDL in healthy males, but neither in healthy females nor in females or males with MS. Additionally, adiponectin was positively correlated with some HDL subclasses in healthy males and females with MS, but not in healthy females or males with MS. Adjusting for age and either body mass index, waist circumference, C-reactive protein, or interleukin-6 weakened the associations between adiponectin and VLDL and IDL but not small-dense LDL. The adjustment weakened the associations between adiponectin and HDL in healthy males but not in females with MS. Based on our results, we conclude that sex and the presence of MS are strong determinants of the associations between adiponectin and serum lipoproteins and that the complex regulatory network comprising adiponectin and other molecular players involved in the regulation of lipoprotein metabolism is primarily operative in healthy males and females with MS.

Folic acid (FA) is an essential vitamin involved in crucial metabolic processes, while copper(II) ions play significant roles in various biological functions. This study aims to investigate the interaction between FA and Cu2+ using 1H and 13C NMR spectroscopy under different pH levels and concentrations. The research employed detailed NMR analysis to explore how Cu2+ binds to FA, focusing on changes in chemical shifts, diffusion coefficients, and copper-induced paramagnetic effects. The key findings reveal that Cu2+ predominantly coordinates with the pteridine ring (PTE) of FA, with minimal involvement from the glutamic acid (Glu) moiety. The interaction is strongly concentration-dependent: at lower FA concentrations, Cu2+ binds effectively to the PTE ring, while at higher concentrations, intermolecular interactions among FA molecules hinder copper binding. The study also observed pronounced paramagnetic effects on the PTE and p-aminobenzoic acid protons, with negligible effects on Glu signals. These results provide new insights into the structural characteristics of FA-Cu2+ complexes, contributing to a better understanding of their biochemical interactions and implications for folate metabolism.

Ultrasmall silver nanoparticles (2 nm) were prepared by reduction with sodium borohydride (NaBH4) and stabilized by the ligand glutathione (a tripeptide: glycine–cysteine–glutamic acid). NMR spectroscopy and optical spectroscopy (UV and fluorescence) revealed that these particles initially consist of silver nanoparticles and fluorescing silver nanoclusters, both stabilized by glutathione. Over time, the silver nanoclusters disappear and only the silver nanoparticles remain. Furthermore, the capping ligand glutathione eliminates hydrogen sulfide (H2S) from the central cysteine and is released from the nanoparticle surface as tripeptide glycine–dehydroalanine–glutamic acid. Hydrogen sulfide reacts with the silver core to form silver sulfide. After four weeks in dispersion at 4 °C, this process is completed. These processes cannot be detected by transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), or differential centrifugal sedimentation (DCS) as these methods cannot resolve the mixture of nanoparticles and nanoclusters or the nature of the nanoparticle core. X-ray photoelectron spectroscopy showed the mostly oxidized state of the silver nanoparticle core, Ag(+I), both in freshly prepared and in aged silver nanoparticles. These results demonstrate that ultrasmall nanoparticles can undergo unnoticed changes that considerably affect their chemical, physical, and biological properties. In particular, freshly prepared ultrasmall silver nanoparticles are much more toxic against cells and bacteria than aged particles because of the presence of the silver clusters.

Biliverdin IXβ reductase (BLVRB) has emerged as a promising therapeutic target for thrombocytopenia due to its involvement in reactive oxygen species (ROS) mechanisms. During the pursuit of inhibitors targeting BLVRB, olsalazine (OSA) became apparent as one of the most potent candidates. However, the direct application of OSA as a BLVRB inhibitor faces challenges, as it is prone to degradation into 5-aminosalicylic acid through cleavage of the diazenyl bond by abundant azoreductase (AzoR) enzymes in gut microbiota and eukaryotic cells. To overcome this obstacle, we devised olsalkene (OSK), an inhibitor where the diazenyl bond in OSA has been substituted with an alkene bond. OSK not only matches the efficacy of OSA but also demonstrates improved stability against degradation by AzoR, presenting a promising solution to this limitation. Furthermore, we have found that both OSK and OSA inhibit BLVRB, regardless of the presence of nicotinamide adenine dinucleotide phosphate, unlike other known inhibitors. This discovery opens new avenues for investigating the roles of BLVRB in blood disorders, including thrombocytopenia.

The association of thyroid function with essential and non-essential amino acids is understudied, despite their common metabolic roles. Thus, our aim was to evaluate the association of thyroid function with the levels of branched-chain amino acids (BCAAs—leucine, isoleucine, and valine) and of alanine in the general population. We utilized data from the São Paulo research center of ELSA-Brasil, a longitudinal population-based cohort study. Thyroid parameters included thyroid stimulating hormone (TSH), free T4 and free T3 levels, and the FT4:FT3 ratio. BCAAs and alanine were analyzed on a fully automated NMR platform. The current analysis included euthyroid participants and participants with subclinical hyperthyroidism and hypothyroidism. We used Pearson’s coefficient to quantify the correlation between thyroid-related parameters and amino acids. Linear regression models were performed to analyze whether thyroid parameters were associated with BCAAs and alanine levels. We included 4098 participants (51.3 ± 9.0 years old, 51.5% women) in this study. In the most adjusted model, higher levels of TSH were associated with higher levels of alanine, FT4 levels were inversely associated with isoleucine levels, FT3 levels were statistically significant and positively associated with valine and leucine, and the T3:T4 ratio was positively associated with all amino acids. We observed that subclinical hypothyroidism was positively associated with isoleucine and alanine levels in all models, even after full adjustment. Our findings highlight the association of subclinical hypothyroidism and thyroid-related parameters (including TSH, free T4, free T3, and FT4:FT3 ratio) with BCAAs and alanine. Further studies are needed to explore the mechanisms underlying this association. These insights contribute to our understanding of the influence of thyroid-related parameters on BCAA and alanine metabolism.

As obesity develops, metabolic changes increase the risk of non-communicable diseases such as type 2 diabetes (T2D). Weight loss is crucial for improving health in T2D and cardiometabolic conditions. However, weight loss rates vary between individuals, even with identical diets or energy restrictions, highlighting the need to identify markers or predictors of weight loss success to enhance intervention outcomes. Using nuclear magnetic resonance (NMR) spectroscopy-based metabolomics, we investigated the change in serum polar metabolites in 28 women with overweight or obesity and prediabetes who completed an 8-week low-energy diet (LED) as part of the PREVIEW (PREVention of diabetes through lifestyle intervention and population studies in Europe and around the World) clinical trial. We aimed to characterize the metabolic shift in substrate oxidation under fixed energy intake (~4 MJ/day) and its relation to weight loss success. Nine of the thirty-four serum metabolites identified significantly changed during the LED phase: 3-hydroxybutyrate, O-acetylcarnitine, 2-hydroxybutyrate, mannose, dimethyl sulfone and isobutyrate increased, whilst choline, creatine and tyrosine decreased. These results confirmed a shift towards lipid oxidation, but no metabolites predicted the response to the LED-induced weight loss. Further studies in larger populations are required to validate these metabolites as biomarkers of diet exposure.

A novel tridentate ligand featuring an acridine core and pyrazole rings, namely 2,7- di-tert-butyl-4,5-di(pyrazol-1-yl)acridine, L, was designed and used to create two ruthenium(II) complexes: [RuL2](PF6)2 and [Ru(tpy)L](PF6)2. Surprisingly, the ligand adopted different coordination modes in the complexes: facial coordination for the homoleptic complex and meridional coordination for the heteroleptic complex. The electronic absorption and electrochemical properties were evaluated. Although both complexes exhibited favorable electronic properties for luminescence, neither emitted light at room temperature nor at 77 K. This study highlights the complex interplay between ligand design, coordination mode, and luminescence in ruthenium(II) complexes.

The data on the synthesis of N-aminomorpholine hydrazones are presented. It is shown that the interaction of N-aminomorpholine with functionally substituted benzaldehydes and 4-pyridinaldehyde in isopropyl alcohol leads to the formation of corresponding hydrazones. The structure of the synthesized compounds was studied by 1H and 13C NMR spectroscopy methods, including the COSY (1H-1H), HMQC (1H-13C) and HMBC (1H-13C) methodologies. The values of chemical shifts, multiplicity, and integral intensity of 1H and 13C signals in one-dimensional NMR spectra were determined. The COSY (1H-1H), HMQC (1H-13C), and HMBC (1H-13C) results revealed homo- and heteronuclear interactions, confirming the structure of the studied compounds. The antiviral, cytotoxic, and antimicrobial activity of some synthesized hydrazones were investigated. It is shown that 2-((morpholinoimino)methyl)benzoic acid has a pronounced viral inhibitory property, comparable in its activity to commercial drugs Tamiflu and Remantadine. A docking study was performed using the influenza virus protein models (1930 Swine H1 Hemagglutinin and Neuraminidase of 1918 H1N1 strain). The potential binding sites that are complementary with 2-((morpholinoimino)methyl)benzoic acid were found.

Fifteen type I terpene synthase homologs from diverse actinobacteria that were selected based on a phylogenetic analysis of more than 4000 amino acid sequences were investigated for their products. For four enzymes with functions not previously reported from bacterial terpene synthases the products were isolated and their structures were elucidated by NMR spectroscopy, resulting in the discovery of the first terpene synthases for (+)-δ-cadinol and (+)-α-cadinene, besides the first two bacterial (−)-amorpha-4,11-diene synthases. For other terpene synthases with functions reported from bacteria before the products were identified by GC–MS. The characterised enzymes include a new epi-isozizaene synthase with monoterpene synthase side activity, a 7-epi-α-eudesmol synthase that also produces hedycaryol and germacrene A, and four more sesquiterpene synthases that produce mixtures of hedycaryol and germacrene A. Three phylogenetically related enzymes were in one case not expressed and in two cases inactive, suggesting pseudogenisation in the respective branch of the phylogenetic tree. Furthermore, a diterpene synthase for allokutznerene and a sesterterpene synthase for sesterviolene were identified.

Abstract Background Endophytic Aspergillus species produce countless valuable bioactive secondary metabolites. In the current study, Aspergillus flavus an endophyte from the soft coral Sarcophyton ehrenbergi was chemically explored and the extracted phytoconstituents were subsequently evaluated for antimicrobial activity. This is accomplished by employing nuclear magnetic resonance (NMR) spectroscopy and computational techniques. Additionally, An in vitro anticancer analysis of A. flavus total extract against breast cancer cells (MCF-7) was investigated. Result Six compounds were separated from the crude alcohol extract of the endophytic Aspergillus flavus out of which anhydro-mevalonolactone was reported for the first time. The anti-fungal and anti-Helicobacter pylori properties of two distinct compounds (Scopularides A and B) were assessed. Additionally, computational research was done to identify the binding mechanisms for all compounds. Both the compounds were found to be active against H. pylori with minimum inhibitory concentration (MIC) values ranging from 7.81 to 15.63 µg/ mL as compared with clarithromycin 1.95 µg/ mL. Scopularides A was potent against both Candida albicans and Aspergillus niger with MIC values ranging from 3.9 to 31.25 µg/ mL, while scopularides B only inhibits Candida albicans with MIC value of 15.63 µg/ mL and weak inhibitory activity against A. niger (MIC = 125 µg/ mL). Furthermore, cytotoxic activity showed a significant effect (IC50: 30.46 mg/mL) against MCF-7 cells. Conclusion Our findings report that cytotoxic activity and molecular docking support the antimicrobial activity of Aspergillus flavus, which could be a promising alternative source as a potential antimicrobial agent.

Organophosphorus chemical agents are included in the 1st List of the Annex on Chemicals of the Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on Their Destruction (Chemical Weapons Convention, CWC). For the purposes of verification of compliance with the provisions of the CWC, special methods, which are considered the most informative at determining the retrospective effects of organophosphorus toxicants on the body, are necessary. Typical long-lived biomarkers of organophosphate toxic agents are tyrosine phosphorylation products, the presence of which in biomedical samples clearly indicates the exposure to sarin, soman, tabun and V-series agents. We have elaborated methods for the synthesis and isolation of tyrosine adducts derivatives of methylphosphonic and phosphoric acids, used as reference samples. The synthesis scheme included the consecutive protection of carboxyl and amino groups of tyrosine, its O-phosphorylation by the corresponding alkylphosphonates and phosphates, the removal of protective groups with the release of corresponding O-phosphorylated tyrosine adducts. Their purification from impurities was carried out, using column chromatography (SiO2, eluent: dichloromethane/ethyl acetate 1:1). The purity of the obtained products was more than 90 %, so it was possible to involve them in further transformations with the use of catalyst without the threat of its «poisoning». Benzyl and carboxybenzyl protection of phosphorylated L-tyrosines (12–17) was removed by means of catalytic hydrogenation by molecular hydrogen under atmospheric pressure. Target adducts of phosphorylated reagents and L-tyrosin were obtained (63–82 %) in form of crystal white substances, readily soluble in water and ethanol, and poorly – in dichloromethane and acetonitrile.

Contrary to the 4′- and 5′-nitro- and aminofluoresceins, the corresponding 3′-derivatives are practically unexplored. In this paper, we describe the synthesis and spectral properties of 3′-nitrofluorescein and 3′-aminofluorescein, as well as their methyl esters. Among other methods, X-ray analysis, 13C NMR spectroscopy, and ESI mass spectrometry made it possible to establish the molecular structure of the target compounds as well as intermediates and by-products. Some unexpected products, though in small amounts, were revealed within the course of study. Whereas the fluorescence of the double-charged R2− ion of 3′-nitrofluorescein in both aqueous and organic solvents is weak, the R2− anion of 3′-aminofluorescein in a non-hydrogen bonding donor solvent, but not in water, exhibits intensive fluorescence, analogous to the case of 4′- and 5′-aminofluoresceins. Interestingly, the λmax values in water of the R2− ions bearing an NO2 group in the 3′- and 6′-positions are 7 to 10 nm higher than those of the 4′- and 5′-nitro derivatives. The difference was also observed in dimethyl sulfoxide. This correlates with the angles between the xanthene and phthalic planes of the dyes. The dye 3′-aminofluorescein could be used as a fluorescent indicator sensitive to hydrogen bonding ability of the solvent. It could also serve as a platform for synthesizing fluorescent molecular probes for biochemical research, analogous to the very popular application of 4′- and 5′-amino derivatives.

Gentamicin sulfate is a broad-spectrum bactericidal aminoglycoside antibiotic produced by Micromonospora purpurea and used in clinical practice as an active substance containing a mixture of gentamicins C1 and C2. Gentamicin content is a standardised parameter controlled as part of a pharmaceutical product review. Currently, the quality control of the active substance gentamicin sulfate involves the use of high-performance liquid chromatography (HPLC) for the indirect identification of the component composition by comparison with a reference standard and for its quantification by calibration against the reference standard.The aim of the study was to develop a procedure for identifying and quantifying the components of the active substance gentamicin sulfate using nuclear magnetic resonance (NMR) spectroscopy that would not require reference standards.Materials and methods. The authors studied a sample of the active substance gentamicin sulfate using an Agilent DD2 NMR spectrometer operating at 600 MHz to record NMR spectra and an Agilent 1200 HPLC system equipped with a multiple wavelength detector (MWD) to obtain chromatograms.Results. Having determined the spectral properties of the C1, C1A, C2, C2A and C2B gentamicins comprising the active substance, the authors observed characteristic signals of the same type that corresponded to each of the gentamicins and did not overlap with other signals in the 1H and 13C spectra. These characteristic signals provided spectral markers indicative of the gentamicins of interest in the sample, with normalised integrated intensities equal to the mole fractions of the analytes. The authors compared NMR and HPLC measurements of the active substance composition.Conclusions. The authors developed a procedure for identifying and quantifying the constituents of the active substance gentamicin sulfate by 1H and 13C NMR spectroscopy, which requires less effort than HPLC procedures and no reference standards for the intended applications.

The series of double network (DN) hydrogels, based on poly(N,N′-diethylacrylamide) (PDEAAm), polyacrylamide (PAAm), or poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) were synthesized with various mass ratios of components and their thermal response was investigated. The formation of DN structure in studied hydrogels results in significant increase in Young’s modulus. PDEAAm/PAMPS hydrogels exhibited rather high swelling ratio and consequently their temperature sensitivity was not detected. DN hydrogels PDEAAm/PAAm and PDEAAm/PDEAAm displayed thermo-responsive behavior, and the dependence of transition parameters on the feed molar concentration of acrylamide and N,N′-diethylacrylamide monomers, respectively, was determined through deswelling, NMR, and Differential scanning calorimetry (DSC) experiments. A two-state process model was employed to describe the phase transition of hydrogels. By utilizing a modified van’t Hoff equation with data from deswelling, NMR, and DSC experiments, we were able to obtain thermodynamic parameters of the transition and determine the size of the cooperative domains consisting of polymer units and water molecules.

IntroductionThe development of agriculture in terms of sustainability and low environmental impact is, at present, a great challenge, mainly in underdeveloped and marginal geographical areas. The Salvia rosmarinus “Eretto Liguria” ecotype is widespread in Liguria (Northwest Italy), and farmers commonly use it by for cuttings and for marketing. In the present study, this ecotype was characterized in comparison with other cultivars from the same geographical region and Campania (Southern Italy), with a view to application and registration processes for the designation of protected geographical indications. Moreover, the possibility of using the resulting biomass after removing cuttings or fronds as a source of extracts and pure compounds to be used as phytosanitary products in organic farming was evaluated. Specifically, the potential of rosemary extracts and pure compounds to prevent soft rot damage was then tested.MethodsA targeted NMR metabolomic approach was employed, followed by multivariate analysis, to characterize the rosemary accessions. Bacterial soft rot assay and disk diffusion test were carried out to evaluate the activity of extracts and isolated compounds against Pectobacterium carotovorum subsp. carotovorum. Enzymatic assay was performed to measure the in vitro inhibition of the pectinase activity produced by the selected pathogen. Molecular docking simulations were used to explore the possible interaction of the selected compounds with the pectinase enzymes. Results and DiscussionThe targeted metabolomic analysis highlighted those different geographical locations can influence the composition and abundance of bioactive metabolites in rosemary extracts. At the same time, genetic factors are important when a single geographical area is considered. Self-organizing maps (SOMs) showed that the accessions of “Eretto Liguria” appeared well characterized when compared to the others and had a good content in specialized metabolites, particularly carnosic acid. Soft rotting Enterobacteriaceae belonging to the Pectobacterium genus represent a serious problem in potato culture. Even though rosemary methanolic extracts showed a low antibacterial activity against a strain of Pectobacterium carotovorum subsp. carotovorum in the disk diffusion test, they showed ability in reducing the soft rot damage induced by the bacterium on potato tissue. 7-O-methylrosmanol, carnosol and isorosmanol appeared to be the most active components. In silico studies indicated that these abietane diterpenoids may interact with P. carotovorum subsp. carotovorum pectate lyase 1 and endo-polygalacturonase, thus highlighting these rosemary components as starting points for the development of agents able to prevent soft rot progression.