Methylation Products Research Articles

Catalytic Synthesis of Methyl 9,10‐dihydroxystearate from Technical Feedstocks in Continuous Flow via Epoxidation and Hydrolysis

AbstractThe sequence of the homogeneously Ru‐catalyzed epoxidation of methyl oleate and acid‐catalyzed hydrolysis of the corresponding epoxide methyl 9,10‐epoxy stearate is successfully transferred from batch into flow mode, allowing for the continuous production of methyl 9,10‐dihydroxystearate. Thereby, methyl oleate is first converted up to 97% within 14 min at excellent selectivity in the epoxidation using aqueous hydrogen peroxide as the sole oxidant. In the subsequent hydrolysis, a residence time of 10 min is sufficient for quantitative conversion of the epoxide. The desired, pure vicinal diol is isolated upon crystallization from the crude reaction mixture in an integrated process starting from technical grade (91.5%) substrate. The isolated yield is increased upon the addition of water as a green antisolvent from 75% up to 97%. Finally, the concept is transferred to methyl oleate of even lower purity (76%), still obtaining an isolated yield of 66% of the vicinal diol. Thus, the integration of sequential epoxidation and hydrolysis into continuous flow and subsequent crystallization allows for high conversion and selectivities within a total residence time of 27 min, corresponding to a space–time yield of 190 g h−1 L−1 in the epoxidation and 164 g h−1 L−1 in the hydrolysis, respectively.Practical applications: The modular flow setup enables the targeted functionalization toward the epoxide intermediate or the vicinal diol. Both offer versatile applications for the production of polymers, surfactants, or toward further conversion as in oxidative cleavage starting from methyl oleate. The application of flow chemistry offers advantages for the safe handling of hydrogen peroxide even at high temperatures. With fats and oils being natural substances, oleochemicals such as fatty acid methyl esters are typically available in technical purity so that efficient strategies for the isolation of pure products are of need. Crystallization of the product is promising, as additional organic solvents are not required. Thus, using the difference in melting point and solubility behavior of the desired product compared to other compounds is a promising method for the applicability of renewable resource‐based substrate mixtures.

Statistical optimization for simultaneous removal of methyl red and production of fatty acid methyl\xa0esters using fresh alga Scenedesmus obliquus

Microalgae are a diverse group of microorganisms, the majority of which are photosynthetic in nature. Microalgae have different applications, the most important of which is the biological treatment of wastewater. Microalgae grow in various types of wastewater, such as wastewater polluted by Azo dyes, due to microalgae using wastewater as a culture medium, which contains many nutrients like nitrogen, phosphate, and carbon sources. Microalgae grow in various types of wastewater, such as wastewater polluted by Azo dyes, due to microalgae using wastewater as a culture medium, which contains many nutrients like nitrogen, phosphate, and carbon sources. So, microalgae are used for bioremediation of wastewater due to the efficiency of growing in wastewater and for the high production of lipids followed by trans-esterification to biodiesel. Face-centered central composite design (FCCCD) was used to determine the factors that have the most significant impact on the simultaneous decolorization of methyl red and lipid production by the fresh green alga Scenedesmus obliquus. The predicted results indicated that the alga decolorized 70.15% methyl red and produced 20.91% lipids by using 1 g/L nitrogen, an incubation time of 10 days, a pH of 8, and the concentration of methyl red is 17.65 mg/L. The dry biomasses of S. obliquus were also examined by SEM and FTIR before and after treatment with methyl red. SEM and FTIR showed that the properties of dry S. obliquus were altered after the biosorption of methyl red. According to GC–MS analysis of hexane extracts of S. obliquus, the lipid profile differed before and after methyl red decolorization. The results proved that it is possible to use S. obliquus to remove dyes and produce renewable fuels such as biodiesel. The novelty of this study is that this is the first time in which the effect of nitrogen concentrations in the medium used for algal growth on the removal of dye has been studied.

Global hypomethylation pattern in systemic sclerosis: An application for absolute quantification of epigenetic DNA modification products by 2D-UPLC-MS/MS

BackgroundSystemic sclerosis (SSc) is a rare autoimmune disease characterized by progressive fibrosis of the skin and internal organs. Besides genetics risk factors, understanding the epigenetic modifications in SSc has been gaining acceleration in recent years. Epigenetic modifications are reversible and defined as druggable targets. In this context, it is highly important to present a systemic perspective to epigenetic modifications of SSc in terms of both pathogenesis and clinical utility. Material and methodsDNA samples from the whole blood specimens of the 41 SSc patients and 27 healthy controls (HCs) were obtained. Absolute quantification of 5-mC, 5-hmdC, 5-cadC, 5-fdC, and 5-hmdU as the DNA methylation and demethylation products were performed using 2D-UPLC-MS/MS. Demographic data and clinical scores were recorded in detail. Results5-hmdU was significantly higher in SSc patients while 5-hmdC was lower compared to the HCs (p < 0.01, p = 0.012 respectively). 5-cadC and 5-fdC had upward trend in SSc (p = 0.064; p = 0.066). These results support that SSc patients tend to have a global hypomethylation pattern. Clinical analyzes revealed that lung, gastrointestinal, joint, and vascular involvement of SSc is also associated with increased demethylation or decreased methylation profile. ConclusionWe performed absolute quantification of epigenetic DNA modification products in SSc for the first time. We demonstrated an upward trend in global hypomethylation in SSc. Furthermore, as a result of detailed clinical analyzes, the relationship between lung, GIS, and vascular involvement with epigenetic changes was shown. We believe that absolute quantification of DNA methylation and demethylation products with novel technologies can provide a deep understanding of disease pathogenesis and has the potential to mark an era for developing new therapeutic strategies.

Role of Fe2+-dependent Reaction in Biodecolorization of Methyl Orange by Brown-rot Fungus Fomitopsis pinicola

The involvement of Fenton reaction on biodegradation of methyl orange (MO) by brown-rot fungus Fomitopsis pinicola was investigated based on Fe2+-dependent reaction. The degradation of MO (final concentration 75 mg/L) was performed in mineral salt media with and without Fe2+ with incubation period at 0, 7, 14, 21, and 28 days. Degradation analysis was performed using UV-Vis Spectrophotometer and LC-TOF/MS. F. pinicola decolorized MO in a medium containing Fe2+ and a medium that lacked the mineral, at percentages of 89.47% and 80.08%, respectively. The optimum decolorization occurred after 28 days of incubation with the fungus on the presence of Fe2+, indicated that the presence of Fe2+ enhanced MO degradation with assumed to correlate with Fenton reaction. Two metabolites were detected through the LC-TOF/MS analysis, namely 4-(2-(4-(dimethyliminio)-2-hydroxycyclohexa-2,5-dien-1-ylidene) hydrazinyl) phenolate (m/z 258, RT: 1.28 min, compound 1) and 4-(2-(4-(dimethyliminio) cyclohexa-2,5-dien-1-ylidene) hydrazinyl) benzenesulfonate (m/z 391, RT: 2.70 min, compound 2). Compound 1 was a transformation product of hydroxylation and methylation, compound 2 was a product of dehydroxylation and desulfonation. This study indicated that the transformation of the metabolite structures was involved hydroxyl radical (OH.) and enzymatic mechanisms, which involved Fe2+-dependent reaction.

Role of Ester Sulfate and Organic Disulfide in Mercury Methylation in Peatland Soils.

We examined the composition and spatial correlation of sulfur and mercury pools in peatland soil profiles by measuring sulfur speciation by 1s X-ray absorption near-edge structure spectrocopy and mercury concentrations by cold vapor atomic fluorescence spectroscopy. Also investigated were the methylation/demethylation rate constants and the presence of hgcAB genes with depth. Methylmercury (MeHg) concentration and organic disulfide were spatially correlated and had a significant positive correlation (p < 0.05). This finding is consistent with these species being products of dissimilatory sulfate reduction. Conversely, a significant negative correlation between organic monosulfides and MeHg was observed, which is consistent with a reduction in Hg(II) bioavailability via complexation reactions. Finally, a significant positive correlation between ester sulfate and instantaneous methylation rate constants was observed, which is consistent with ester sulfate being a substrate for mercury methylation via dissimilatory sulfate reduction. Our findings point to the importance of organic sulfur species in mercury methylation processes, as substrates and products, as well as potential inhibitors of Hg(II) bioavailability. For a peatland system with sub-μmol L-1 porewater concentrations of sulfate and hydrogen sulfide, our findings indicate that the solid-phase sulfur pools, which have a much larger sulfur concentration range, may be accessible to microbial activity or exchanging with the porewater.

Anti-inflammatory activity of flavonols via inhibiting MAPK and NF-κB signaling pathways in RAW264.7 macrophages

Fisetin (Fis), quercetin (Que), and myricetin (Myr) are flavonols with similar structure but different number of hydroxyl groups. The present research focused on the anti-inflammatory effect of these three flavonols in lipopolysaccharide-stimulated RAW264.7 cells. The number and site of hydroxyl group in flavonols obviously affected their anti-inflammation activity. These flavonols suppressed the overproduction of nitric oxide. Fis showed the best activity with an inhibition rate of 52% at 20 μM. Moreover, the flavonols reduced the levels of ROS, TNF-α, and IL-6. The mechanistic study showed that they inhibited the activation of NF-κB and MAPK pathways by suppressing the phosphorylation of IκBα, p65, JNK, ERK, p38, MEK, and reducing the nuclear translocation of NF-κB p65. In addition, the metabolism of the flavonols was examined. The results indicated that Fis was both methylated and glucuronidated. Que and Myr were mainly transformed into methylated products. This study highlights the anti-inflammatory activity of flavonols, particularly Fis, which has the potential for the prevention or treatment of inflammation as an adjuvant medicine or food additive.

Volatile composition changes of fruits in a biopolymer-coated polyethylene active packaging: Effects of modified atmosphere and packaging-shaped bacterial community

The potential impacts of active packaging on the volatile composition of fruits during preservations largely associated with consumers’ acceptance, yet was barely investigated. In this work, a biopolymer-coated polyethylene active film was developed and its effects on qualities and volatile compositions of fruits with different respiration rates were comprehensively investigated. Underlying reasons for the effect of active packaging on volatile composition change of fruits were elucidated through revealing ternary relationship among the packaging-shaped bacterial community, modified atmosphere and volatiles. Off-flavor production was reduced and more sesquiterpenes (α-cubebene, copaene, β-caryophyllene, α-caryophyllene, d-amorphene) were reserved for longan packaged with active film, due to its antimicrobial and moisture absorption ability, as leading contributors to the selective inhibition on spoilage bacteria genera including Zymobacter, Gluconobacter and Pantoea. Whereas volatile profile of strawberry with high respiration rate was more actively affected by the modified atmosphere induced by packaging, instead of the bacterial community, where hypoxia (<2% O2) led to the accumulation of ethyl esters (0.154–0.184 μg/g) that conferred off-flavor but decreased production of methyl (0.172–0.367 μg/g) and hexyl esters (1.26–1.89 × 10−2 μg/g) that represented typical fruity aroma. This work adds new knowledges on impacts of active packaging on the volatile profile of fruits and would provide guidance to smart design of active packaging for preservation of aromatic fruits.

Detection of MSH2 Gene Methylation in Extramammary Paget's Disease by Methylation-Sensitive High-Resolution Melting Analysis.

Background Extramammary Paget's disease (EMPD) is a rare skin tumor. Hypermethylation in the MSH2 promoter resulting in the downregulation of its protein expression shows a high detection rate in EMPD tumor tissue, which indicates that the methylation of MSH2 may play an important role in the pathogenesis of EMPD. Objective This study aims to establish a rapid analysis strategy based on the methylation-sensitive high-resolution melting curve (MS-HRM) to detect the methylation level of the MSH2 promoter. Methods With the use of universal methylated human DNA products, we established the MS-HRM standard curve to quantitatively detect the methylation level of the MSH2 promoter. Then, all 57 EMPD tumor DNA samples were analyzed. Pyrosequencing assay was also carried out to test the accuracy and efficacy of MS-HRM. Besides, a total of 54 human normal and other cancerous tissues were included in this study to test the reliability and versatility of the MS-HRM standard curve. Results In this study, by using the established MS-HRM, we found that 96.5% (55/57) EMPD tumor samples had varying methylation levels in the MSH2 promoter ranging from 0% to 30%. Then, the methylation data were compared to the results obtained from pyrosequencing, which showed a high correlation between these two techniques by Pearson's correlation (r = 0.9425) and Bland–Altman plots (mean difference = −0.1069) indicating that the methylation levels analyzed by MS-HRM were consistent with DNA pyrosequencing. Furthermore, in 23 normal and 31 other cancerous tissue samples, there were two colorectal cancer (CRC) tissues that tested MSH2 methylation positive (1% and 5%) which confirmed that our established MS-HRM can be widely applied to various types of samples. Conclusion MS-HRM standard curve can be used for the detection of the methylation level of MSH2 in EMPD tumor samples and other cancerous tissues potentially, which presents a promising candidate as a quantitative assay to analyze MSH2 promoter methylation in routine pathological procedure.

Increased symmetric dimethyl-arginine is a predictor factor of decreased platelet reactivity and increased bleeding risk in patients with acute coronary syndrome

Abstract Background One of the promising biomarkers in CVD are asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), which are products of L-arginine methylation and are both involved in endothelial dysfunction. ADMA, SDMA and L-homoarginine, have emerged as biomarkers linked to cardiovascular outcomes [1]. Purpose To investigate the association of SDMA with platelet reactivity and bleeding risk in patients with acute coronary syndrome (ACS) treated with potent P2Y12 inhibitors prasugrel and ticagrelor. Methods Our prospective observational study enrolled 292 patients with ACS undergoing percuteneus coronary intervention [2]. Plasma concentrations of SDMA were measured during the hospitalization for ACS. Impedance aggregometry was used. The primary study endpoint was the concentration of metabolites and platelet reactivity. The primary clinical outcome endpoint was the incidence of Thrombolysis in Myocardial Infarction (TIMI) bleeding events (major, minor and minimal). The efficacy endpoint was the composite of major adverse cardiac events (MACE: stent thrombosis, myocardial infarction, stroke and cardiac death). Results There was an inverse correlation between SDMA serum levels and platelet reactivity (r=−0.25; p&amp;lt;0.000). The ADP+PGE1-induced platelet reactivity was 33% lower among patients with the highest SDMA quartile (4th) as compared to those with the 1–3rd SDMA quartile (8 [0–29] vs 12 [0–126] U; p&amp;lt;0.001). The AA-induced platelet reactivity was 56% lower among patients with the highest SDMA quartile (4th) as compared to those with the 1–3rd SDMA quartile (4 [0–48] vs 9 [0–133]; p&amp;lt;0.001). In a multivariate model, the highest SDMA (4th) quartile was found to be an independent predictor of the lowest ADP+PGE1 and AA induced platelet aggregation (OR: 2.666, 95% CI [1.184–5.999], p=0.018). Conclusions Our study shows that high plasma concentration of SDMA, but not ADMA, is independently associated with low platelet reactivity to ADP and AA and is associated with major and minor bleeding events in patients with ACS on potent antiplatelet therapies. Therefore, SDMA might have a potential to be further evaluated as a blood biomarker for individualization of duration and potency of antiplatelet therapies in an ACS population at high risk of bleeding complications. Acknowledgment I-COMET research team Funding Acknowledgement Type of funding sources: None. Figure 1Table 1

Rapid, One-Step Synthesis of α-Ketoacetals via Electrophilic Etherification.

Herein, we report a rapid, one-step synthesis of α-ketoacetals via electrophilic etherification of α-alkoxy enolates and monoperoxyacetals. Methyl, primary, and secondary α-ketoacetals were obtained in 44-63% yields from tetrahydropyranyl substrates; using methyl tetrahydropyranyl, alkyl tetrahydropyranyl, or methyl tetrahydrofuranyl peroxyacetals, however, methyl and primary products were isolated in 66-90% yields. The present method is applied to C-O bond formation at tertiary carbons, via alkyl and methyl peroxyacetals, with yields of 25-65%. Intermolecular "alkoxyl" transfer, from peroxyacetal to α-alkoxy enolate, relies heavily on decreased steric bulk surrounding the peroxide bond and site of etherification; additionally, we found the α-OCH3 group to be critical in ensuring product formation. α-Ketoacetals demonstrated excellent reactivity, as selective, nucleophilic attack at the unprotected carbonyl furnished α-hydroxy acetals in 80-100% yields; subsequent hydrolysis of the foregoing compounds provided α-hydroxy aldehydes in yields of 58-90%.

Methyl donor micronutrients, CD40-ligand methylation and disease activity in systemic lupus erythematosus: A cross-sectional association study.

ObjectiveHypomethylation of CD40-ligand (CD40L) in T-cells is associated with increased disease activity in systemic lupus erythematosus (SLE). We therefore investigated possible associations of dietary methyl donors and products with CD40L methylation status in SLE.MethodsFood frequency questionnaires were employed to calculate methyl donor micronutrients in 61 female SLE patients (age 45.7 ± 12.0 years, disease duration 16.2 ± 8.4 years) and compared to methylation levels of previously identified key DNA methylation sites (CpG17 and CpG22) within CD40L promotor of T-cells using quantitative DNA methylation analysis on the EpiTYPER mass spectrometry platform. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Linear regression modelling was used. P values were adjusted according to Benjamini & Hochberg.ResultsAmongst the micronutrients assessed (g per day), methionine and cysteine were associated with methylation of CpG17 (β = 5.0 (95%CI: 0.6-9.4), p = 0.04; and β = 2.4 (0.6-4.1), p = 0.02, respectively). Methionine, choline, and cysteine were additionally associated with the mean methylation of the entire CD40L (β = 9.5 (1.0-18.0), p = 0.04; β = 1.6 (0.4-3.0), p = 0.04; and β = 4.3 (0.9-7.7), p = 0.02, respectively). Associations of the SLEDAI with hypomethylation were confirmed for CpG17 (β=-32.6 (-60.6 to -4.6), p = 0.04) and CpG22 (β=-38.3 (-61.2 to -15.4), p = 0.004), but not the mean methylation of CD40L. Dietary products with the highest impact on methylation included meat, ice cream, white bread, and cooked potatoes.ConclusionsDietary methyl donors may influence DNA methylation levels and thereby disease activity in SLE.

Profile of urinary amino acids and their post-translational modifications (PTM) including advanced glycation end-products (AGEs) of lysine, arginine and cysteine in lean and obese ZSF1 rats

Heart failure with preserved ejection fraction (HFpEF) is associated with high mortality and has an increasing prevalence associated with the demographic change and limited therapeutic options. Underlying mechanisms are largely elusive and need to be explored to identify specific biomarkers and new targets, which mirror disease progression and intervention success. Obese ZSF1 (O-ZSF1) rats are a useful animal model, as they spontaneously develop hypertension, hyperlipidemia and glucose intolerance and finally HFpEF. The urinary profile of amino acids and their metabolites of post-translational modifications (PTM), including the advanced glycation end-products (AGEs) of lysine, arginine and cysteine, are poorly investigated in HFpEF and ZSF1 rats. The aim of the present study was to characterize the status of free amino acids and their metabolites of PTM and glycation in lean ZSF1 (L-ZSF1) and O-ZSF1 rats in urine aiming to find possible effects of glucose on the excretion of native and modified amino acids. In the urine of twelve L-ZSF1 and twelve O-ZFS1 rats collected at the age of 20 weeks, we measured the concentration of native and modified amino acids by reliable previously validated stable-isotope dilution gas chromatography-mass spectrometry (GC–MS) approaches. Serum glucose was 1.39-fold higher in the O-ZSF1 rats, while urinary creatinine concentration was 2.5-fold lower in the O-ZSF1 rats. We observed many differences in urinary amino acids excretion between L-ZSF1 and O-ZSF1 rats. The creatinine-corrected homoarginine excretion was twofold lower in the O-ZSF1 rats. We also observed distinct associations between the concentrations of serum glucose and urinary amino acids including their PTM and AGE metabolites in the L-ZSF1 and O-ZSF1 rats. Our study shows that PTM metabolites and AGEs are consistently lower in the L-ZSF1 than in the O-ZSF1 rats. Serum malondialdehyde (MDA) concentration was higher in the O-ZSF1 rats. These results suggest that hyperglycemia, hyperlipidemia and elevated oxidative stress in the O-ZSF1 rats favor PTM methylation of arginine and lysine and the glycation of lysine and cysteine. The area under the receiver operation characteristic (ROC) curve values were 0.996 for serum glucose, 0.951 for urinary creatinine, 0.939 for serum MDA, 0.885 for Nε-carboxyethyl-lysine, 0.830 for carboxyethyl-cysteine, and 0.792 for monomethyl-lysine. Non-invasive measurement of methylation and glycation products of arginine, lysine and cysteine residues in proteins in urine of L-ZSF1 and O-ZSF1 rats may be useful in studying pathophysiology and pharmacology of HFpEF.

Asymmetric reduction of 2-chloro-3-oxo-ester into enantiomerically high pure diltiazem precursor by a Candida ketoreductase

• A highly stereoselective reductase towards the 2-chloro-3-oxo-ester identified. • Highest space-time yield (46 g L −1 d −1 ) reported in bio-reduction of 1a . • Nearly enantiopure (2 R ,3 S )-MPGM obtained through chemo-enzymatic synthesis. Methyl (2 R ,3 S )-3-(4-methoxyphenyl)glycidate [(2 R ,3 S )-MPGM] is an advanced chiral synthon for the synthesis of the cardiovascular drug diltiazem. It can be easily accessed by cyclizing the reduction products of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate ( 1a ). Herein, we report an identified carbonyl reductase ( Cp KR) from Candida parapsilosis that displayed an excellent stereoselectivity toward the keto substituent at the C 3 -position of the 2-chloro-3-oxo-ester 1a . The engineered Escherichia coli cells harboring Cp KR gene were directly applied for the asymmetric reduction of keto ester 1a with a space-time yield of 46 g L −1 d −1 , which represents the highest productivity in bio-reduction of 1a reported so far. The isolated chiral alcohol products were then applied to the chemical synthesis of (2 R ,3 S )-MPGM in 99% ee and a total yield of 76% in the two-step chemo-enzymatic reactions, which far exceeded the maximum theoretical yield (50%) of the existing industrial process based on a lipase-catalyzed resolution of racemic MPGM. This work provides a promising eco-friendly and cost-effective route toward industrial synthesis of pharmaceutically relevant diltiazem.

Isolation, separation, identification, and quantification of bioactive methylated flavone regioisomers by UHPLC-MS/MS.

Methylated flavones, commonly found in many plants of the Brassicaceae family, have potent antioxidant and anticancer activity with diverse therapeutic potential. However, the specific regioisomers of methylated flavones can have significantly different biochemical and potentially therapeutic properties as shown by various bioassays but analytically differentiating these compounds has been technically challenging and rarely reported. In this study, we demonstrate differentiation and identification of selected bioactive methylated flavone regioisomers, namely 5,7,3'-trihydroxy-4'-methoxyflavone, and 5,7,4'-trihydroxy-3'-methoxyflavone extracted from Coronopus didymus, a member of the Brassicaceae family, using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS/MS). Characteristic MS/MS product ions produced from neutral loss of carbon monoxide, and a methyl radical from the [M-H]- ion, exhibited differential relative abundances attributed to different structural stabilities under the same activation and collision-induced dissociation conditions. MS/MS also provided structural information which was sufficient to differentiate the methylated regioisomers and determine the position of the methyl group based on interpretation of their respective fragmentation patterns. Quantification showed 5,7,4'-trihydroxy-3'-methoxyflavone was at least 1.60mg per 10g plant material in C. didymus extracts. This study demonstrates a straightforward and novel approach to rapidly differentiate, identify and quantify regio-isomeric methylated flavone natural products using reversed-phase UPLC-MS/MS.

Synthesis of Methylcellulose Using Dimethyl Carbonate with Conventional and Green Methods

Synthesis of methylcellulose (MC) using dimethyl carbonate (DMC) with conventional and green methods has been conducted. The cellulose used in this synthesis was obtained from sago waste, which contains 86.67% cellulose. All the methylation products were analyzed using IR and 1H-NMR spectrometers. The determination of cellulose content was done using TAPPI T-203 method, while the degree of substitution (DS) was based on the Zeisel Method. Conventional synthesis of MC was performed using Na2HPO4 (12:1), DMC and NaCl 5% (w/v) (volume ratio = 10:10) at 90 °C. The reaction time was varied (2 to 5 hours); the optimum condition was reached in 3 hours and gave the DS of 1.028. Green synthesis of MC was conducted by employing microwave and sonochemistry techniques. Synthesis MC with the microwave was carried out at the power of 400 W for 200 seconds and gave the DS of 1.036. Synthesis MC with sonochemistry (the variation time 1 to 3 hours) was conducted at the optimum time of 2 hours and gave the DS of 0.646. The DMC volume was also varied (17.5; 20; 22.5; 25; and 30 mL), where the volume ratio of 1:24 gave the highest DS of 1.177. The microwave technique is the best method for synthesis MC compared to conventional and sonochemistry techniques.

Direct N1-Selective Alkylation of Hydantoins Using Potassium Bases.

Hydantoins, including the antiepileptic drug phenytoin, contain an amide nitrogen and an imide nitrogen, both of which can be alkylated. However, due to the higher acidity of its proton, N3 can be more easily alkylated than N1 under basic conditions. In this study, we explored methods for direct N1-selective methylation of phenytoin and found that conditions using potassium bases [potassium tert-butoxide (tBuOK) and potassium hexamethyldisilazide (KHMDS)] in tetrahydrofuran (THF) gave N1-monomethylated phenytoin in good yield. The applicable scope of this reaction system was found to include various hydantoins and alkyl halides. To explore the function of methylated hydantoins, the effects of a series of methylated phenytoins on P-glycoprotein were examined, but none of methylated products showed inhibitory activity toward rhodamine 123 efflux by P-glycoprotein.

A Novel Synthesis of Sex Pheromone from the Longicorn Beetle (Psacothea hilaris)

An asymmetric synthesis of (8Z,21R)-21-methylpentatriacont-8-ene, the sex pheromone of the yellow-spotted longicorn beetle Psacothea hilaris, has been achieved using Evan’s induction as the key step. Based on the asymmetric methylation product of chiral (R)-4-benzyl-1,3-oxazolidin-2-one, the carbon chain of the target molecule was assembled through a C5+C12+C11+C8 sequence. (2R)-4-(Benzyloxy)-2-methylbutan-1-ol, which can be obtained from γ-lactone following Evan’s protocol, was connected to a C12 alkyl group. The chiral methyl group remained the key moiety (97% ee). After another Wittig reaction and catalytic hydrogenation step, the designed key intermediate (13R)-13-methylheptacosan-1-ol was obtained. Finally, after oxidation and Wittig reaction, the synthesis of the target molecule was completed in 10 linear steps with an ultra-high overall yield of 36.2%.

Clues to the non-carcinogenicity of certain N-Nitroso compounds: Role of alkylated DNA bases

N-Nitroso compounds (NOC) are known for the carcinogenicity of most members. However, 13% of 332 NOC reviewed in 1984 were found to be non-carcinogenic. The non-carcinogenicity of all N-nitrosamines with even one tertiary alkyl group is notable. Clues to the lack of carcinogenicity include (a) inability to generate the reactive ultimate carcinogen which alkylates DNA bases, and (b) inability of the alkylated DNA base to mispair during DNA replication. This DFT study probes a three-stage process for the induction of mutations, including (a) N-deprotonation of O-alkylated DNA bases formed by attack of the carcinogen, (b) adoption of a conformer by the O-alkylated base conducive to mutagenic base mispairing, and (c) creation of the base mismatch involving the O-alkylated base. These three criteria are applied to the products of methylation, ethylation, isopropylation and tert-butylation at the N7-G, O6-G and O4-T sites. The N-deprotonation criterion differentiates the non-mutagenic N7-alkylguanines from the promutagenic O6-alkylguanines and O4-alkylthymines. All the O-alkylated bases except O4-tert-butylthymine are predicted as capable of adopting a conformer conducive to successful mispairing. O4-tert-butylthymine is predicted as incapable of creating a base mismatch by H-bonding with guanine, pointing to the non-mutagenic effects of tert-butylation of the O4-T site. By extrapolating to all tertiary alkyl groups, this explains why tert-alkylating N-nitrosamines are carcinogenically inactive. These results also highlight the carcinogenic role of alkylation at the O4-T site rather than at the O6-G site.

Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

Biochemical Characterization of a Flavonoid O-methyltransferase from Perilla Leaves and Its Application in 7-Methoxyflavonoid Production.

Methylation is a common structural modification that can alter and improve the biological activities of natural compounds. O-Methyltransferases (OMTs) catalyze the methylation of a wide array of secondary metabolites, including flavonoids, and are potentially useful tools for the biotechnological production of valuable natural products. An OMT gene (PfOMT3) was isolated from perilla leaves as a putative flavonoid OMT (FOMT). Phylogenetic analysis and sequence comparisons showed that PfOMT3 is a class II OMT. Recombinant PfOMT3 catalyzed the methylation of flavonoid substrates, whereas no methylated product was detected in PfOMT3 reactions with phenylpropanoid substrates. Structural analyses of the methylation products revealed that PfOMT3 regiospecifically transfers a methyl group to the 7-OH of flavonoids. These results indicate that PfOMT3 is an FOMT that catalyzes the 7-O-methylation of flavonoids. PfOMT3 methylated diverse flavonoids regardless of their backbone structure. Chrysin, naringenin and apigenin were found to be the preferred substrates of PfOMT3. Recombinant PfOMT3 showed moderate OMT activity toward eriodictyol, luteolin and kaempferol. To assess the biotechnological potential of PfOMT3, the biotransformation of flavonoids was performed using PfOMT3-transformed Escherichia coli. Naringenin and kaempferol were successfully bioconverted to the 7-methylated products sakuranetin and rhamnocitrin, respectively, by E. coli harboring PfOMT3.