Methylation Products Research Articles

Analysis of histidine-tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry.

Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI). A protein standard, His-Ubq, and two recombinant proteins, His-SHAN and His-CS, expressed in Escherichia coli were immobilized on two immobilized metal affinity systems, Cu-nitriloacetic acid (Cu-NTA) and Ni-NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96-well plate form factor, or analyzed directly from immobilized metal affinity-coated microscope slides by DESI-MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis. Small proteins (His-Ubq) and medium proteins (His-SAHN) could readily be detected from 96-well plates by direct infusion ESI, or from microscope slides by DESI-MS after purification on surface from clarified E. coli cell lysate. Protein oxidation was observed for immobilized proteins on both Cu-NTA and Ni-NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His-SAHN and the methylation product of His-CS (theobromine to caffeine) were detected. The immobilization, purification, release and detection of His-tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI-MS or ambient DESI-MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.

Physicochemical property changes of Dendrobium officinale leaf polysaccharide LDOP-A and it promotes GLP-1 secretion in NCI-H716 cells by simulated saliva-gastrointestinal digestion.

A polysaccharide LDOP-A with a molecular weight of 9.9 kDa was isolated and purified from Dendrobium officinale leaves by membrane separation, cellulose column, and dextran gel column. The Smith degradable products, methylation products, and nuclear magnetic resonance analysis showed that LDOP-A may be composed of →4)-Glc-(1→, →3,6)-Man-(1→, and →6)-Glc-(1→sugar residues. In vitro, simulated digestion assays showed that LDOP-A could be partially digested in the stomach and small intestine, and produced a large amount of acetic acid and butyric acid during colonic fermentation. Further cell experiment results illustrated that LDOP-A-I (LDOP-A digested by gastrointestinal tract) could induce glucagon-like peptide-1 (GLP-1) secretion in NCI-H716 cells without showing any cytotoxicity.

Martin Silicates as Partners in Photoredox/Ni Dual Catalysis for the Installation of CH3, CH2D, CD2H, CD3and13CH3Groups onto (Hetero)Arenes

Martin Silicates as Partners in Photoredox/Ni Dual Catalysis for the Installation of CH₃, CH₂D, CD₂H, CD₃and¹³CH₃Groups onto (Hetero)Arenes

The flavonoid hesperidin methyl chalcone as a potential therapeutic agent for cancer therapy: Molecular docking, in vitro cytotoxicity, and in vivo antitumor activity

Hesperidin methyl chalcone (HMC) is a methylation product of the flavanone hesperidin, a flavonoid derived from citrus fruits. Many reports have emphasized the analgesic, anti-inflammatory and antioxidant properties of HMC. However, the anticancer potential of HMC has not been fully elucidated. The objective of this study was to assess the possible anticancer potential of HMC. MTT assay was carried out to assess the in vitro cytotoxicity of HMC against using A549 cancer cell line. In addition, the in vivo antitumor activity of HMC was screened against murine Ehrlich ascites carcinoma (EAC) model, in terms of tumor volume, tumor weight, life span, hematological and biochemical parameters, and compared with that of the anticancer agent, hesperetin. HMC was efficient to suppress the cell viability of A549 cancer cells with an IC50 value of 51.12 µM, which was comparable to that of the anticancer agent hesperetin (IC50 = 49.12 µM). Similarly, in Ehrlich ascites carcinoma model, HMC significantly inhibited the growth of Ehrlich ascites carcinoma with mutual increase in the life span of HMC-treated mice, compared to EAC control. Most importantly, HMC showed a good safety profile as evidenced by restoring hematological profile count of RBCs, WBCs, and hemoglobin to the normal levels. HMC also efficiently reduced the oxidative stress in EAC-bearing mice via increasing the levels of GSH, SOD and Catalase. Collectively, HMC exerted a potent anticancer activity and data presented in this work suggest the usefulness of HMC as a promising candidate in cancer therapy.

Acid-catalysed reactions of amines with dimethyl carbonate.

Highly effective acid-catalysed reactions of amines with dimethyl carbonate (DMC) have been conducted with significant yields and selectivity of carboxymethylation or methylation products. Lewis acids (FeCl3, ZnCl2, and AlCl3·6H2O), Brønsted acids (PTSA, acetic, and formic acids), and acids supported on silica (silica sulfuric and silica perchlorate) resulted in carboxymethylation of primary aliphatic amines with high conversions. It was found that the Lewis acid FeCl3 also promoted carboxymethylation of primary aromatic amines and secondary amines. At both 90 °C or an elevated temperature of 150 °C under pressure, AlCl3·6H2O demonstrated highly selective monomethylation of aromatic amines. In addition, both silica sulfuric acid and silica perchlorate at 90 °C exhibited no conversion for secondary amines but enhanced carboxymethylation with high conversions of 80.7-87.5% and selectivity of >99.00% at 150 °C in a pressure reactor. At 1.0 equivalent, both promoted excellent conversion and selectivity of primary aliphatic amines at 90 °C. In addition, they were easily recovered and reused for at least four additional reactions without significant loss of efficiency with consistent conversions and selectivity. Green metrics evaluation for the silica sulfuric acid-catalysed reaction highlighted the sustainability features of the process. Silica-supported catalysts are highly stable, making them ideal alternative catalysts for the methylation and carbonylation of various amines with DMC. Acid-catalysed DMC reactions of amines may expand the substrate scope and offer new opportunities for developing sustainable organic synthetic methodologies.

Elucidating the Kinetic Mechanism of Human METTL16.

Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3' triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 μM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 μM. Steady-state kinetic assays established a kcat of 0.07 min-1, and single-turnover assays established a kchem of 0.56 min-1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 μM and a kchem of 0.42 min-1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.

Characterization of ginsenoside structural isomers from mixtures using in situ methylation with direct analysis in real-time ionization tandem mass spectrometry

Characterization of structural isomers of bioactive molecules is important for recognizing their functions, but it has been challenging due to their highly similar structures. As the main bioactive constituents of Panax ginseng, ginsenosides have different structural isomers attributed to the aglycone structure and glycosylation sites as well as stereochemistry of sugar groups attached. This work demonstrated a simple and robust in situ methylation reaction with tetramethylammonium hydroxide (TMAH) using ambient ionization source of direct analysis in real time (DART) to characterize saponin structural isomers. The DART ion source provides favorable conditions to methylate hydroxyl groups of ginsenoside instantaneously with TMAH, and it can ionize the methylated products at the same time. Methylated ginsenoside stereoisomers even with subtle structure differences generated very different mass signals from full-scan MS and tandem MS. High-resolution mass spectrometry aided the assignment of molecular structures of the various precursor and fragment ions from different ginsenosides, which provided structural information for both the aglycone skeleton and the sugar moieties in ginsenosides. The presented method was successfully used for the identification of ginsenosides in Panax ginseng, and saponin isomers were characterized without the need for chromatographic separation and/or tedious offline sample pretreatment.

Pharmacokinetics and in vitro liver microsomal enzyme metabolism of Xylopic acid

Xylopic acid (XA) is a bioactive diterpene kaurene isolate of the Guinea pepper fruit, Xylopia aethiopica (Annonaceae) with numerous well-established biological effects. In this study, we aimed to fill certain scientific voids in terms of the scientific literature on XA, specifically, its pharmacokinetic (PK) parameters and in vitro liver microsomal enzyme metabolism. A new LC–MS/MS method was developed and validated for the determination of the plasma concentration–time profile of XA. The method was found to be accurate, precise, selective and repeatable with lowest limit of quantification (LLOQ) of 10 ng/mL and run time of 15 min. The maximum plasma concentration (Cmax), time at which maximum plasma concentration was attained (Tmax), half-life (t1/2), clearance (CL) and mean residence time (MRT) of XA were 167.03 ± 6.18 ng/mL; 10 h; 13.03 ± 7.33 h; 0.04 ± 0.01 mL/h/kg and 23.83 ± 11.02 h respectively. Six metabolites (M1–M6) were tentatively identified after XA was subjected to in vitro liver microsomal enzyme metabolism. The metabolites were the products of methylation (M1), glucuronidation (M2), deacetylation (M3), glucosylation (M4), hydroxylation and glutamic acid addition (M5) and glutathionylation (M6). The outcome of this study provides useful insights that could guide further research on XA.

Mechanism of S-Adenosyl-l-methionine C-Methylation by Cobalamin-dependent Radical S-Adenosyl-l-methionine Methylase in 1-Amino-2-methylcyclopropanecarboxylic Acid Biosynthesis.

The radical S-adenosyl-l-methionine (SAM) methylase Orf29 catalyzes the C-methylation of SAM in the biosynthesis of 1-amino-2-methylcyclopropanecarboxylic acid. Here, we determined that the methylation product is (4″R)-4″-methyl-SAM. Furthermore, we found that the 5'-deoxyadenosyl radical generated by Orf29 abstracts the pro-R hydrogen atom from the C-4″ position of SAM to generate the radical intermediate, which reacts with methylcobalamin to give (4″R)-4″-methyl-SAM. Consequently, the Orf29-catalyzed C-methylation was confirmed to proceed with retention of configuration.

On the laminar flame propagation of C5H10O2 esters up to 10 atm: A comparative experimental and kinetic modeling study

Biodiesel is a family of renewable engine fuels with carbon-neutral nature. In this work, three C5H10O2 esters (methyl butanoate, methyl isobutanoate and ethyl propanoate), which can serve as model compounds of biodiesel and represent linear and branched methyl esters and linear ethyl esters, were investigated to characterize their laminar flame propagation characteristics up to 10 atm and unravel the effects of isomeric fuel structures. A high-pressure constant-volume cylindrical combustion vessel was used to achieve laminar burning velocity measurements at 1–10 atm, 423 K and equivalence ratios of 0.7–1.5, while comparative experimental work was performed on a heat flux burner at 1 atm, 393 K and equivalence ratios of 0.7–1.6 for methyl butanoate and ethyl propanoate. The laminar burning velocity generally decreases with increasing pressure and increases in the order of methyl isobutanoate, methyl butanoate and ethyl propanoate, which shows distinct fuel isomeric effects. A kinetic model of C5H10O2 esters was developed and validated against the new data in this work and previous data in literature. Modeling analyses were performed to provide insight into the fuel-specific flame chemistry of the three esters isomers. Remarkable differences in radical pools of three ester isomers are concluded to be responsible for the observed fuel isomeric effects on laminar flame propagation. The feature of two ethyl groups connected to the ester group in ethyl propanoate facilitates the ethyl production and inhibits the methyl and allyl production, making it propagate fastest among the three isomers. The branched structure feature of methyl isobutanoate with methyl and i-propyl groups connected to the ester group prevents the ethyl formation and results in considerable CH3 and allyl production, which decelerates its laminar flame propagation.

Establishing protocol for detecting methylation of Ubiquitin carboxyl terminal hydrolase 1 gene’s promoter in nasopharyngeal carcinoma

Background: The methylation of Ubiquitin Carboxyl Terminal Hydrolase 1 (UCHL1) gene’s promoter has been reported as the etiological factor of nasopharyngeal tumorigenesis. Purpose: This study is designed to establish a protocol for detecting methylation of UCHL1 gene’s promoter in nasopharyngeal carcinoma (NPC) in a Vietnamese population. Materials and methods: 10 samples of NPC biopsy tissues and 10 samples of non-cancerous swabs were collected from the local hospital. Chloroform/Phenol method and Nested-MSP assays were established to detect methylation of a target gene. Results: The isolated DNA reached purity and high concentration which were confirmed by the method of absorbance measurement at 260nm and 280nm. Additionally, the Nested-MSP products of methylation or unmethylation were analyzed and visualized in the agarose gel with the band of 169bps and 210bps, respectively. By sequencing, it was confirmed that the two sets of primer could distinguish the status of methylation and unmethylation of UCHL1 gene’s promoter. Conclusion: Our data suggested that the current protocol could successfully identify the status of methylation and/or unmethylation of UCHL1 gene’s promoter.

The new mechanism of cognitive decline induced by hypertension: High homocysteine-mediated aberrant DNA methylation.

The prevalence and severity of hypertension-induced cognitive impairment increase with the prolonging of hypertension. The mechanisms of cognitive impairment induced by hypertension primarily include cerebral blood flow perfusion imbalance, white and gray matter injury with blood-brain barrier disruption, neuroinflammation and amyloid-beta deposition, genetic polymorphisms and variants, and instability of blood pressure. High homocysteine (HHcy) is an independent risk factor for hypertension that also increases the risk of developing early cognitive impairment. Homocysteine (Hcy) levels increase in patients with cognitive impairment induced by hypertension. This review summarizes a new mechanism whereby HHcy-mediated aberrant DNA methylation and exacerbate hypertension. It involves changes in Hcy-dependent DNA methylation products, such as methionine adenosyltransferase, DNA methyltransferases, S-adenosylmethionine, S-adenosylhomocysteine, and methylenetetrahydrofolate reductase (MTHFR). The mechanism also involves DNA methylation changes in the genes of hypertension patients, such as brain-derived neurotrophic factor, apolipoprotein E4, and estrogen receptor alpha, which contribute to learning, memory, and attention deficits. Studies have shown that methionine (Met) induces hypertension in mice. Moreover, DNA hypermethylation leads to cognitive behavioral changes alongside oligodendroglial and/or myelin deficits in Met-induced mice. Taken together, these studies demonstrate that DNA methylation regulates cognitive dysfunction in patients with hypertension. A better understanding of the function and mechanism underlying the effect of Hcy-dependent DNA methylation on hypertension-induced cognitive impairment will be valuable for early diagnosis, interventions, and prevention of further cognitive defects induced by hypertension.

Local Alteration of Ionic Strength in a Nucleosome Core Particle and Its Effect on N7-Methyl-2'-deoxyguanosine Depurination.

Positively charged N-terminal histone tails play important roles in maintaining the nucleosome (and chromatin) structure and function. Charge alteration, including those imposed by post-translational modifications, impacts chromatin dynamics, protein binding, and the fate of DNA damage. There is evidence that N-terminal histone tails affect the local ionic environment within a nucleosome core particle (NCP), but this phenomenon is not well understood. Determining the modulation of the local ionic environment within an NCP by histone tails could help uncover the underlying mechanisms of their functions and effects. Utilizing bottom-up syntheses of NCPs containing wild-type or mutated histones and a fluorescent probe that is sensitive to the local ionic environment, we show that interaction with positively charged N-terminal tails increases the local ionic strength near nucleosomal DNA. The effect is diminished by replacing positively charged residues with neutral ones or deleting a tail in its entirety. Replacing the fluorescent probe with the major DNA methylation product, N7-methyl-2'-deoxyguanosine (MdG), revealed changes in the depurination rate constant varying inversely with local ionic strength. These data indicate that the MdG hydrolysis rates depend on and also inform on local ionic strength in an NCP. Overall, histone tail charge contributes to the complexity of the NCP structure and function by modulating the local ionic strength.

Investigation of photochemical behavior of furan derivatives containing an allomaltol fragment

The photochemical behavior of terarylenes containing furan and allomaltol fragments was studied. It was demonstrated that starting 2-aminofurans containing 3-hydroxypyran-4-one moiety did not undergo any transformations under UV-irradiation. We supposed that all photoreactions are blocked due to the presence of amino group in the structure of the studied terarylenes. Wherein, the conversion of the amino group into pyrrole unit in the considered compounds allows one to unlock both 6π-electrocyclization of the 1,3,5-hexatriene system and ESIPT-promoted processes. At the same time, subsequent methylation of the hydroxyl group of allomaltol fragment leads to regiospecific photocyclization of terarylene core due to the suppression of ESIPT-induced reactions. Thus, UV-irradiation of methylated pyrrole-containing products results in 6π-electrocyclization of the 1,3,5-hexatriene system with the formation of polyaromatic compounds. Alternatively, ESIPT-promoted contraction of allomaltol moiety was observed in the cases when aforementioned 6π-electrocyclization is impossible. The structures of two synthesized products were established by X-ray diffraction.

2D imaging of absolute methyl concentrations in nanosecond pulsed plasma by photo-fragmentation laser-induced fluorescence

The methyl radical plays a central role in plasma-assisted hydrocarbon chemistry but is challenging to detect due to its high reactivity and strongly pre-dissociative electronically excited states. We report the development of a photo-fragmentation laser-induced fluorescence (PF-LIF) diagnostic for quantitative 2D imaging of methyl profiles in a plasma. This technique provides temporally and spatially resolved measurements of local methyl distributions, including in near-surface regions that are important for plasma-surface interactions such as plasma-assisted catalysis. The technique relies on photo-dissociation of methyl by the fifth harmonic of a Nd:YAG laser at 212.8 nm to produce CH fragments. These photofragments are then detected with LIF imaging by exciting a transition in the B-X(0, 0) band of CH with a second laser at 390 nm. Fluorescence from the overlapping A-X(0, 0), A-X(1, 1), and B-X(0, 1) bands of CH is detected near 430 nm with the A-state populated by collisional B-A electronic energy transfer. This non-resonant detection scheme enables interrogation close to a surface. The PF-LIF diagnostic is calibrated by producing a known amount of methyl through photo-dissociation of acetone vapor in a calibration gas mixture. We demonstrate PF-LIF imaging of methyl production in methane-containing nanosecond pulsed plasmas impinging on dielectric surfaces. Absolute calibration of the diagnostic is demonstrated in a diffuse, plane-to-plane discharge. Measured profiles show a relatively uniform distribution of up to 30 ppm of methyl. Relative methyl measurements in a filamentary plane-to-plane discharge and a plasma jet reveal highly localized intense production of methyl. The utility of the PF-LIF technique is further demonstrated by combining methyl measurements with formaldehyde LIF imaging to capture spatiotemporal correlations between methyl and formaldehyde, which is an important intermediate species in plasma-assisted oxidative coupling of methane.

Global analysis of qualitative and quantitative metabolism of Notoginsenoside R1 in rat liver-brain-gut axis based on LC-IT-TOF/MS combing mMDF strategy

The metabolism study of active components for traditional Chinese medicine (TCM) in target organs is conducive to clarify the authentic active ingredients. Notoginsenoside R1 (NG-R1), one of the bioactive components of Panax notoginsenoside (PNS), is commonly acknowledged as the characteristic marker of PNS. However, the metabolism of NG-R1 in target organs has not been clarified yet due to the lack of robust technique and approach. The present study aimed to develop a reliable and efficient strategy and technology for revealing the qualitative and quantitative metabolism of active components of TCMs in target organs, and to clarify the biotransformation of NG-R1 in liver-brain-intestinal axis. The metabolic transformation of NG-R1 in the brain gut axis was investigated in the in vitro incubation system of fresh rat brain, liver homogenate, and intestinal flora. To quickly lock the target metabolites, we set the mass defect filter (MDF) in different ranges to screen metabolites with different molecular weight (MW). This strategy was defined as multi-stage MDF (mMDF). In addition, we performed relative quantitative analysis on all metabolites according to the peak area acquired by LC-IT-TOF/MS to overcome the challenge that metabolites are difficult to be quantified due to the lack of standards. When MDF was set at 0.50 to 0.65 to screen metabolites with MW of 900 to 1200 Da, 6 novel metabolites were quickly found, and then identified as glucuronic acid binding, oxidation, dehydrogenation, methylation and hydrogenation products according to their LC and MS characteristics. When setting MDF at 0.42 - 0.52, 6 metabolites with MW of 600 to 900 Da were effectively screened and identified as Rg1, NG-R2, Rh1, Rg1+CH2+2H and Rg1+CH2. To screen the metabolites with MW of 300 to 600 Da, MDF was set at 0.25 - 0.42, and 4 novel metabolites were screened rapidly. The results of quantitative metabolism suggested that intestinal flora was the main metabolic site of NG-R1 in rat, and more than 60% of NG-R1 was converted to Rg1 by deglycosylation in the intestinal flora. The mMDF strategy can significantly improve the research efficiency of qualitative metabolism of saponins. Although NG-R1 could be transformed into a variety of metabolites in rat liver and brain homogenate, it still existed mainly in prototype form. In the rat flora, NG-R1 mainly existed in the form of deglycosylated metabolite Rg1.

Metabolism, tissue distribution and excretion of taxifolin in rat.

The metabolism, tissue distribution and excretion of taxifolin in rat after oral administration of taxifolin encapsulated zein-caseinate Nanoparticles (TZP) were studied. The isomerization of taxifolin in rat small intestine and colon was found. Besides isomers, 16 metabolites of taxifolin were identified in rat feces, plasma and urine by UPLC-QTOF-MS. In colon, taxifolin underwent the metabolism of hydration, dehydration and ring-fission through the gut microflora. The main metabolites of taxifolin found in plasma and urine were its sulfated, glucuronidated, and/or methylated products. The dynamic variation of taxifolin and its metabolites in tissues and urine were quantified by UPLC-QqQ-MS/MS. Taxifolin and its metabolites could be quickly absorbed and distributed in the tissues, and relatively low concentrations were found in the heart and brain. The feces excretion of taxifolin was determined by HPLC. The total excretion during 24h was 2.83±0.80% to its given does, and the maximum excretion was found during 8-10h post administration. Compared with feces, the excretion of taxifolin and its metabolites in urine was much faster, and the total excretion was 1.96±0.23% during 12h.

The Interpretation of the Role of a Polyketide Synthase ClPKS18 in the Pathogenicity of Curvularia lunata.

Plant pathogenic fungus Curvularia lunata (Wakker) Boedijn causes leaf spot diseases in several plants such as Oryza sativa, Sorghum bicolor (L.) Moench, and Capsicum frutescens. It has been spread worldwide, specifically in maize-growing regions. The polyketide synthase (PKS) plays a significant role in secondary metabolite production and its effect on virulence. The Clpks18 of C. lunata strongly correlated with its pathogenicity. The role of Clpks18 gene on the pathogenic activity of C. lunata remains unclear. Hence, in this study, we analyzed the importance of Clpks18 gene on the hyphae and conidial melanization and on the sporulation and hyphal growth. The deletion of Clpks18 gene reduced the production of methyl 5-(hydroxymethyl)furan-2-carboxylate toxin. The virulence of ΔClpks18 mutant was significantly reduced compared with the wild type. The metabolomics data revealed that (R)-(-)-mellein was a vital factor in the virulence of C. lunata. The (R)-(-)-mellein and the toxin produced by C. lunata were detected in the maize leaves during its infestation. In addition, the metabolomic analysis showed that the Clpks18 gene influences glycerolipid, non-ribosomal peptide biosynthesis, and its metabolism. This study demonstrates that the Clpks18 gene is important for the pathogenicity of C. lunata by influencing the complex metabolic network.

Pharmacokinetics, Prostate Distribution and Metabolic Characteristics of Four Representative Flavones after Oral Administration of the Aerial Part of Glycyrrhiza uralensis in Rats.

(1) Background: The aerial part of G. uralensis had pharmacological effects against chronic non-bacterial prostatitis (CNP), and flavonoids are the main efficacy components. The purpose of this study was to obtain the pharmacokinetics, prostate distribution and metabolic characteristics of some flavonoids in rats. (2) Methods: The prototype flavones and the metabolites of four representative flavonoids, namely puerarin, luteolin, kaempferol and pinocembrin in plasma, prostate, urine and feces of rats were analyzed by UPLC-Q-Exactive Orbitrap-MS. In addition, the pharmacokinetic parameters in plasma and distribution of prostate of four components were analyzed by HPLC-MS/MS. (3) Results: In total, 22, 17, 22 and 11 prototype flavones were detected in the prostate, plasma, urine and feces, respectively. The metabolites of puerarin in the prostate are hydrolysis and glucose-conjugated products, the metabolites of kaempferol and luteolin in the prostate are methylation and glucuronidation, and the metabolites of pinocembrin in the prostate are naringenin, oxidation, sulfation, methylation and glucuronidation products. The t1/2 of puerarin, luteolin, kaempferol and pinocembrin was 6.43 ± 0.20, 31.08 ± 1.17, 18.98 ± 1.46 and 13.18 ± 0.72 h, respectively. The concentrations of the four flavonoids in prostate were ranked as kaempferol > pinocembrin > luteolin > puerarin. (4) Conclusions: Methylation and glucuronidation metabolites were the main metabolites detected in the prostate. A sensitive and validated HPLC–MS/MS method for simultaneous determination of puerarin, luteolin, kaempferol and pinocembrin in rat plasma and prostate was described, and it was successfully applied to the pharmacokinetic and prostate distribution studies.

Tuning the Properties of 5-Azido and 5-Nitramino-tetrazoles by Diverse Functionalization - General Concepts for Future Energetic Materials.

5‐Azido and 5‐nitraminotetrazole backbones are established heterocyclic motifs in the research field of energetic materials synthesis. Despite the high energy content of the compounds, the problem with many derivatives is that their sensitivities are far too high. Functionalization of one of the ring nitrogen atoms is the aim of this study to adjust the sensitivity by inserting nitratoethyl, azidoethyl and methyl groups. In this context, derivatives of 2‐(2‐azidoethyl)‐5‐nitraminotetrazoles (2, 2 a–2 d), as well as 1‐nitrato and 1‐azidoethyl substituted 5‐azidotetrazole (7 and 10) and the methylation products of 5‐azidotetrazole (5‐azido‐1‐methyl‐tetrazole, 11 and 5‐azido‐2‐methyl‐tetrazole, 12) were prepared. The obtained nitrogen‐rich compounds were extensively characterized through multinuclear NMR spectroscopy and IR spectroscopy. The structural confinement was checked by X‐ray diffraction experiments. The pure samples (verified by elemental analysis) were investigated regarding their behavior toward friction, impact (BAM methods) and electrostatic discharge as well as heating (DTA and DSC). For all metal‐free compounds the detonation properties were computed with the EXPLO5 code using their density and heat of formation, calculated based on CBS‐4 M level of theory.