O-methyl Derivatives Research Articles

Biosynthesis of pterostilbene in Escherichia coli from resveratrol on macroporous adsorption resin using a two-step substrate addition strategy.

Pterostilbene (PST), a 3',5'-O-methylated derivative of resveratrol (RSV), is a potent natural antioxidant produced by some plants in trace amounts as defense compound. It exhibits various health-promoting activities, such as anticancer, antiviral, and antimicrobial effects. Large-scale biosynthesis of PST is crucial due to the challenges associated with extracting it from plants. This study aims to develop an efficient method for PST production using an engineered Escherichia coli strain by feeding RSV as a precursor. We introduced a two-step substrate addition strategy combined with immobilized RSV (IMRSV) on macroporous adsorption resin (MAR) to enhance PST production. Five MARs were selected for RSV immobilization, and the substrate addition strategy and fermentation parameters for PST synthesis were optimized. A maximum PST concentration of 403 ± 9 mg/L was achieved, representing a 239% increase over the control, which in a one-step addition of free RSV. The PST titer reached 395 ± 24 mg/L in a 3-L bioreactor. In conclusion, the combination of a two-step substrate addition system and IMRSV is a promising approach for the economical and industrial-scale production of PST.

Estimating the Bias of Model Compounds for the Determination of Species-Specific Protonation Constants.

The previously unknown extent of the goodness of using model compounds for the microspeciation of polyprotic systems was studied. Mirror-symmetric dibasic compounds and their monosubstituted derivatives were investigated to quantify how the derivatives are appropriate models of the minor microspecies to be mimicked in various microspeciation systems. The results were analyzed using statistical methods. It was found that the respective O-methyl and S-methyl derivatives of phenols and thiols as well as the methyl esters of carboxylic acids are sufficiently good derivatives for microspeciation. It was also found that the methyl esters are superior to the carboxylic amides for modeling the -COOH moiety.

Variations in microbial diversity and chemical components of raw dark tea under different relative humidity storage conditions.

Raw dark tea (RDT) usually needs to be stored for a long time to improve its quality under suitable relative humidity (RH). However, the impact of RH on tea quality is unclear. In this study, we investigated the metabolites and microbial diversity, and evaluated the sensory quality of RDT stored under three RH conditions (1%, 57%, and 88%). UHPLC-Q-TOF-MS analysis identified 144 metabolites, including catechins, flavonols, phenolic acids, amino acids, and organic acids. 57% RH led to higher levels of O-methylated catechin derivatives, polymerized catechins, and flavonols/flavones when compared to 1% and 88% RH. The best score in sensory evaluation was also obtained by 57% RH. Aspergillus, Gluconobacter, Kluyvera, and Pantoea were identified as the core functional microorganisms in RDT under different RH storage conditions. Overall, the findings provided new insights into the variation of microbial communities and chemical components under different RH storage conditions.

Characterization of two O-methyltransferases involved in the biosynthesis of O-methylated catechins in tea plant

Tea is known for having a high catechin content, with the main component being (−)-epigallocatechin gallate (EGCG), which has significant bioactivities, including potential anti-cancer and anti-inflammatory activity. The poor intestinal stability and permeability of EGCG, however, undermine these health-improving benefits. O-methylated EGCG derivatives, found in a few tea cultivars in low levels, have attracted considerable interest due to their increased bioavailability. Here, we identify two O-methyltransferases from tea plant: CsFAOMT1 that has a specific O-methyltransferase activity on the 3ʹʹ-position of EGCG to generate EGCG3′′Me, and CsFAOMT2 that predominantly catalyzes the formation of EGCG4″Me. In different tea tissues and germplasms, the transcript levels of CsFAOMT1 and CsFAOMT2 are strongly correlated with the amounts of EGCG3ʹʹMe and EGCG4ʹʹMe, respectively. Furthermore, the crystal structures of CsFAOMT1 and CsFAOMT2 reveal the key residues necessary for 3ʹʹ- and 4ʹʹ-O-methylation. These findings may provide guidance for the future development of tea cultivars with high O-methylated catechin content.

Inhibitory Potential of Quercetin Derivatives Isolated from the Aerial Parts of Siegesbeckia pubescens Makino against Bacterial Neuraminidase.

This study aimed to isolate bacterial neuraminidase (BNA) inhibitory O-methylated quercetin derivatives from the aerial parts of S. pubescens. All the isolated compounds were identified as O-methylated quercetin (1-4), which were exhibited to be noncompetitive inhibitors against BNA, with IC50 ranging from 14.0 to 84.1 μM. The responsible compounds (1-4) showed a significant correlation between BNA inhibitory effects and the number of O-methyl groups on quercetin; mono (1, IC50 = 14.0 μM) > di (2 and 3, IC50 = 24.3 and 25.8 μM) > tri (4, IC50 = 84.1 μM). In addition, the binding affinities between BNA and inhibitors (1-4) were also examined by fluorescence quenching effect with the related constants (KSV, KA, and n). The most active inhibitor 1 possessed a KSV with 0.0252 × 105 L mol-1. Furthermore, the relative distribution of BNA inhibitory O-methylated quercetins (1-4) in S. pubescens extract was evaluated using LC-Q-TOF/MS analysis.

Identification of an Optimal TLR8 Ligand by Alternating the Position of 2'-O-Ribose Methylation.

Recognition of RNA by receptors of the innate immune system is regulated by various posttranslational modifications. Different single 2′-O-ribose (2′-O-) methylations have been shown to convert TLR7/TLR8 ligands into specific TLR8 ligands, so we investigated whether the position of 2′-O-methylation is crucial for its function. To this end, we designed different 2′-O-methylated RNA oligoribonucleotides (ORN), investigating their immune activity in various cell systems and analyzing degradation under RNase T2 treatment. We found that the 18S rRNA-derived TLR7/8 ligand, RNA63, was differentially digested as a result of 2′-O-methylation, leading to variations in TLR8 and TLR7 inhibition. The suitability of certain 2′-O-methylated RNA63 derivatives as TLR8 agonists was further demonstrated by the fact that other RNA sequences were only weak TLR8 agonists. We were thus able to identify specific 2′-O-methylated RNA derivatives as optimal TLR8 ligands.

67-kDa Laminin Receptor-Mediated Cellular Sensing System of Green Tea Polyphenol EGCG and Functional Food Pairing.

The body is equipped with a “food factor-sensing system” that senses food factors, such as polyphenols, sulfur-containing compounds, and vitamins, taken into the body, and plays an essential role in manifesting their physiological effects. For example, (−)-epigallocatechin-3-O-gallate (EGCG), the representative catechin in green tea (Camellia sinensi L.), exerts various effects, including anti-cancer, anti-inflammatory, and anti-allergic effects, when sensed by the cell surficial protein 67-kDa laminin receptor (67LR). Here, we focus on three representative effects of EGCG and provide their specific signaling mechanisms, the 67LR-mediated EGCG-sensing systems. Various components present in foods, such as eriodictyol, hesperetin, sulfide, vitamin A, and fatty acids, have been found to act on the food factor-sensing system and affect the functionality of other foods/food factors, such as green tea extract, EGCG, or its O-methylated derivative at different experimental levels, i.e., in vitro, animal models, and/or clinical trials. These phenomena are observed by increasing or decreasing the activity or expression of EGCG-sensing-related molecules. Such functional interaction between food factors is called “functional food pairing”. In this review, we introduce examples of functional food pairings using EGCG.

Structural features of biologically active extracellular polysaccharide produced by green microalgae Dictyosphaerium chlorelloides

Ion-exchange chromatography of the biologically active extracellular biopolymer produced by D. chlorelloides yielded ten fractions differing in yield, protein content, monosaccharide composition and molecular weight distribution. Their sugar compositional analyses showed rhamnogalactans, substituted to different extent by mannose and glucose, as a dominant EPS component in all fractions (91 %) except one containing arabinogalactan (7 %). In highly branched rhamnogalactans the quantity of linear (1,3-; 1,4- and 1,6-linked) and branched β-D-galactose units (1,3,6-, 1,4,6- and 1,3,4,6-linked) was nearly equal. From various α-L-rhamnose linkages the 1,2,4-linkage was dominant. Data indicate a rhamnogalactan backbone of EPS, branched by terminal mannose and glucose units, and a lot of O-methylated derivatives of galactose residues (2-O-methyl, 2,3-O-dimethyl, 3-O-methyl and 6-O-methyl). In individual fractions their content and type varied. Detail study of the arabinogalactan showed that its backbone consists of 1,3-linked β-D-Galp units; some of them are branched through O-4 by 6-OMe-α-D-Galp- (1 → 2) -α-L-Araf side chain, other through O-6 by 3-OMe-β-D-Galp, 6-OMe-β-D-Galp, β-D-Galp and β-D-Galf.

Characterization and Structural Analysis of Emodin-O-Methyltransferase from Aspergillus terreus

All O-methylated derivatives of emodin, including physcion, questin, and 1-O-methylemodin, show potential antifungal activities. Notably, emodin and questin are two pivotal intermediates of geodin biosynthesis in Aspergillus terreus. Although most of the geodin biosynthetic steps have been investigated, the key O-methyltransferase (OMT) responsible for the O-methylation of emodin to generate questin has remained unidentified. Herein, through phylogenetic tree analysis and in vitro biochemical assays, the long-sought class II emodin-O-methyltransferase GedA has been functionally characterized. Additionally, the catalytic mechanism and key residues at the catalytic site of GedA were elucidated by enzyme-substrate-methyl donor analogue ternary complex crystal structure determination and site-directed mutagenesis. As we demonstrate, GedA adopts a typical general acid/base (E446/H373)-mediated transmethylation mechanism. In particular, residue D374 is also crucial for efficient catalysis through blocking the formation of intramolecular hydrogen bonds in emodin. This study will facilitate future engineering of GedA for the production of physcion or other site-specific O-methylated anthraquinone derivatives with potential applications as biopesticides.

Phenol group of terpenoids is crucial for antibacterial activity upon ion leakage.

Plant tissues are natural sources of bioactive molecules to control pathogens that cause infections in plants and humans, as they contain secondary metabolites with antibacterial activities. Especially, terpenoids are responsible for the protection of various plants against herbivores and pathogens. In this study, we studied the antibacterial activity of 12 different terpenoid or related structures in a comparative way and revealed that the phenolic terpenoids are superior to other substituted derivatives against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacteria. We also aimed to analyse effect of these compounds on the bacteria cell wall structure and maintenance of homeostasis. We observed an immediate loss of cell membrane integrity and ion leakage upon treatment of both Gram-negative and Gram-positive cells with phenolic terpenoids carvacrol and thymol. After prolonged treatment, leakage of genetic material and increase of membrane permeability for molecules were also observed. Hydroxyl group of the phenol moiety was essential for this effect, since neither the O-methyl derivatives nor the benzylic partners were as effective. We thus propose that perturbation of ion homeostasis upon increase in cell wall permeability is key for the action of terpenoids against bacteria cells.

METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection.

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N6-methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of the SARS-CoV-2 gene in regulating the host cell innate immune response. Our data show that the SARS-CoV-2 virus has m6A modifications that are enriched in the 3′ end of the viral genome. We find that depletion of the host cell m6A methyltransferase METTL3 decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances the downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in patients with severe coronavirus disease 2019 (COVID-19). These findings will aid in the understanding of COVID-19 pathogenesis and the design of future studies regulating innate immunity for COVID-19 treatment.

Quinoline‑ and Benzoselenazole-Derived Unsymmetrical Squaraine Cyanine Dyes: Design, Synthesis, Photophysicochemical Features and Light-Triggerable Antiproliferative Effects against Breast Cancer Cell Lines.

Photodynamic therapy is an innovative treatment approach broadly directed towards oncological diseases. Its applicability and efficiency are closely related to the interaction of three main components, namely a photosensitizer, light and molecular triplet oxygen, which should drive cell death. Recently, several studies have demonstrated that squaraine cyanine dyes have a set of photophysical and photochemical properties that have made of these compounds’ potential photosensitizers for this therapeutic modality. In the present research work, we describe the synthesis and characterization of four quinoline- and benzoselenazole-derived unsymmetrical squaraine cyanine dyes. Except for the precursor of aminosquaraine dyes, i.e., O-methylated derivative, all dyes were evaluated for their behavior and absorption capacity in different organic and aqueous solvents, their ability to form singlet oxygen, their light-stability, and in vitro phototherapeutic effects against two human breast cancer cell cultures (BT-474 and MCF-7). Regardless of the nature of the used solvents, the synthesized dyes showed intense absorption in the red and near-infrared spectral regions, despite the formation of aggregates in aqueous media. Dyes showed high light-stability against light exposure. Despite the low ability to produce singlet oxygen, aminosquaraine dyes demonstrated worthy in vitro phototherapeutic activity.

Lysophosphatidylcholine Containing Anisic Acid Is Able to Stimulate Insulin Secretion Targeting G Protein Coupled Receptors.

Diabetes mellitus is a worldwide health problem with high rates of mortality and morbidity. Management of diabetes mellitus by dietary components is achievable especially at the initial stage of the disease. Several studies confirmed the antidiabetic activities of simple phenolic acids and lysophosphatidylcholine (LPC). The main goal of this study was to identify new potential insulin secretion modulators obtained by combining the structures of two natural compounds, namely O-methyl derivatives of phenolic acids and phospholipids. LPC and phosphatidylcholine bearing methoxylated aromatic carboxylic acids were tested as potential agents able to improve glucose-stimulated insulin secretion (GSIS) and intracellular calcium mobilization in MIN6 β pancreatic cell line. Our results show that LPC with covalently bonded molecule of p-anisic acid at the sn-1 position was able to induce GSIS and intracellular calcium flux. Notably, 1-anisoyl-2-hydroxy-sn-glycero-3-phosphocholine did not affect the viability of MIN6 cells, suggesting its potential safe use. Furthermore, we have shown that three G protein coupled receptors, namely GPR40, GPR55, and GPR119, are targeted by this LPC derivative.

Firefly luciferin precursor 2-cyano-6-hydroxybenzothiazole: Fluorescence à la carte controlled by solvent and acidity

2-Cyano-6-hydroxybenzothiazole (CBTOH) is a key intermediate in the biosynthesis of D-luciferin, also used for its chemical synthesis and as luciferin bioluminescence precursor for chemical analysis. We characterized CBTOH and its O-methylated derivative by UV–vis absorption, by steady-state and time-resolved fluorescence spectroscopy, and by density functional theory (DFT) quantum mechanical calculations. Both the acidity of the hydroxyl group and the basicity of the thiazole nitrogen strongly increase upon electronic excitation, triggering solvent- and acidity-dependent deprotonation and protonation processes, with concomitant changes in the fluorescence properties. DFT and time-dependent DFT calculations revealed the existence of a charge displacement that takes place in the excited state from the phenol moiety to the cyanothiazole group for the neutral and the cationic forms of CBTOH. This study reveals CBTOH as the first member of a new class of small dyes with strong photoacid and photobase character, its rich excited-state behavior allowing modulation of its fluorescence properties through solvent and acidity changes. These features make CBTOH a potentially useful fluorescent probe.

Lignosulfonate Microcapsules for Delivery and Controlled Release of Thymol and Derivatives.

Thymol and the corresponding brominated derivatives constitute important biological active molecules as antibacterial, antioxidant, antifungal, and antiparasitic agents. However, their application is often limited, because their pronounced fragrance, their poor solubility in water, and their high volatility. The encapsulation of different thymol derivatives into biocompatible lignin-microcapsules is presented as a synergy-delivering remedy. The adoption of lignosulfonate as an encapsulating material possessing relevant antioxidant activity, as well as general biocompatibility allows for the development of new materials that are suitable for the application in various fields, especially cosmesis. To this purpose, lignin microcapsules containing thymol, 4-bromothymol, 2,4-dibromothymol, and the corresponding O-methylated derivatives have been efficiently prepared through a sustainable ultrasonication procedure. Actives could be efficiently encapsulated with efficiencies of up to 50%. To evaluate the applicability of such systems for topical purposes, controlled release experiments have been performed in acetate buffer at pH 5.4, to simulate skin pH: all of the capsules show a slow release of actives, which is strongly determined by their inherent lipophilicity.

Synthesis and Biological Evaluation of Polyfluoroalkylated Antipyrines and their Isomeric O-Methylpyrazoles.

Formally belonging to the non-steroidal anti-inflammatory drug class pyrazolones have long been used in medical practices. Our goal is to synthesize N-methylated 1-aryl-3-polyfluoroalkylpyrazolones as fluorinated analogs of antipyrine, their isomeric O-methylated derivatives resembling celecoxib structure and evaluate biological activities of obtained compounds. In vitro (permeability) and in vivo (anti-inflammatory and analgesic activities, acute toxicity, hyperalgesia, antipyretic activity, "open field" test) experiments. To suggest the mechanism of biological activity, molecular docking of the synthesized compounds was carried out into the tyrosine site of COX-1/2. We developed the convenient methods for regioselective methylation of 1-aryl-3- polyfluoroalkylpyrazol-5-ols leading to the synthesis N-methylpyrazolones and O-methylpyrazoles as antipyrine and celecoxib analogs respectively. For the first time, the biological properties of new derivatives were investigated in vitro and in vivo. The trifluoromethyl antipyrine represents a valuable starting point in design of the lead series for discovery new antipyretic analgesics with anti-inflammatory properties.

Petunidin, a B-ring 5'-O-Methylated Derivative of Delphinidin, Stimulates Osteoblastogenesis and Reduces sRANKL-Induced Bone Loss.

Several lines of evidence suggest that oxidative stress is one of the key pathogenic mechanisms of osteoporosis. We aimed to elucidate the bone protective effects of petunidin, one of the most common anthocyanidins, considering its potent antioxidative activity. Petunidin (>5 μg/mL) significantly inhibited osteoclastogenesis and downregulated c-fos, Nfatc1, Mmp9, Ctsk, and Dc-stamp mRNA expression in RAW264.7 cells. Conversely, petunidin (>16 μg/mL) stimulated mineralized matrix formation and gene expression of Bmp2 and Ocn, whereas it suppressed Mmp13, Mmp2, and Mmp9 mRNA expression and proteolytic activities of MMP13 and MMP9 in MC3T3-E1 cells. Micro-CT and bone histomorphometry analyses of sRANKL-induced osteopenic C57BL/6J mice showed that daily oral administration of petunidin (7.5 mg/kg/day) increased bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), the ratio of osteoid volume to tissue volume (OV/TV), osteoid thickness (O.Th), the ratio of osteoid surface to bone surface (OS/BS), the ratio of osteoblast surface to bone surface (Ob.S/BS), and the number of osteoblast per unit of bone surface (N.Ob/BS), and decreased trabecular separation (Tb.Sp), the ratio of eroded surface to bone surface (ES/BS), the ratio of osteoclast surface to bone surface (Oc.S/BS), and number of osteoclast per unit of bone surface (N.Oc/BS), compared to untreated mice. Furthermore, histological sections of the femurs showed that oral administration of petunidin to sRANKL-induced osteopenic mice increased the size of osteoblasts located along the bone surface and the volume of osteoid was consistent with the in vitro osteoblast differentiation and MMP inhibition. These results suggest that petunidin is a promising natural agent to improve sRANKL-induced osteopenia in mice through increased osteoid formation, reflecting accelerated osteoblastogenesis, concomitant with suppressed bone resorption.

Nonracemic Dimethylphenyl Glycerol Ethers in the Synthesis of Physiologically Active Aminopropanols

Six regioisomeric nonracemic dimethylphenyl glycerol ethers were synthesized by asymmetric dihydroxylation of the corresponding allyl dimethylphenyl ethers. The enantioselectivity of the reaction with o-methyl derivatives was lower (down to 34% ee) than with m-methylphenyl ethers (up to 86% ee). Enantiomeric 3-(3,4-dimethylphenoxy)propane-1,2-diols were used to obtain enantiomerically pure physiologically active amino alcohols and their derivatives.

Microbial Biosynthesis of Antibacterial Chrysoeriol in Recombinant Escherichia coli and Bioactivity Assessment

Various flavonoid derivatives including methoxylated flavones display remarkable biological activities. Chrysoeriol is a methoxylated flavone of great scientific interest because of its promising anti-microbial activities against various Gram-negative and Gram-positive bacteria. Sustainable production of such compounds is therefore of pronounced interest to biotechnologists in the pharmaceutical and nutraceutical industries. Here, we used a sugar O-methyltransferase enzyme from a spinosyn biosynthesis gene cluster of Saccharopolyspora spinosa to regioselectively produce chrysoeriol (15% conversion of luteolin; 30 µM) in a microbial host. The biosynthesized chrysoeriol was structurally characterized using high-resolution mass spectrometry and various nuclear magnetic resonance analyses. Moreover, the molecule was investigated against 17 superbugs, including thirteen Gram-positive and four Gram-negative pathogens, for anti-microbial effects. Chrysoeriol exhibited antimicrobial activity against nine pathogens in a disc diffusion assay at the concentration of 40 µg per disc. It has minimum inhibitory concentration (MIC) values of 1.25 µg/mL against a methicillin-resistant Staphylococcus aureus 3640 (MRSA) for which the parent luteolin has an MIC value of sixteen-fold higher concentration (i.e., 20 µg/mL). Similarly, chrysoeriol showed better anti-microbial activity (~1.7-fold lower MIC value) than luteolin against Proteus hauseri, a Gram-negative pathogen. In contrast, a luteolin 4′-O-methylated derivative, diosmetin, did not exhibit any anti-microbial activities against any tested pathogen.

Synthesis and biological activity of methylated derivatives of the Pseudomonas metabolites HHQ, HQNO and PQS.

Selectively methylated analogues of naturally occurring 2-heptyl-4(1H)-quinolones, which are alkaloids common within the Rutaceae family and moreover are associated with quorum sensing and virulence of the human pathogen Pseudomonas aeruginosa, have been prepared. While the synthesis by direct methylation was successful for 3-unsubstituted 2-heptyl-4(1H)-quinolones, methylated derivatives of the Pseudomonas quinolone signal (PQS) were synthesized from 3-iodinated quinolones by methylation and iodine–metal exchange/oxidation. The two N- and O-methylated derivatives of the PQS showed strong quorum sensing activity comparable to that of PQS itself. Staphylococcus aureus, another pathogenic bacterium often co-occurring with P. aeruginosa especially in the lung of cystic fibrosis patients, was inhibited in planktonic growth and cellular respiration by the 4-O-methylated derivatives of HQNO and HHQ, respectively.