Diethyl Ethoxymethylenemalonate Research Articles

Ultrasound-assisted Synthesis of β-(N-arylamino)Acrylates Derivatives, Key Intermediates of Biopotent Compounds

Abstract: β-(N-arylamino)acrylates are building blocks of great industrial interest as they allow the obtention of a variety of heterocyclic substances, such as quinoline and pyridone derivatives. Therefore, the development of new methodologies for the synthesis of these substances is still of interest. In this work, fourteen β-(N-arylamino)acrylates were synthesized in a multigram scale from the reaction of mono- and di-substituted anilines with diethyl ethoxymethylenemalonate by sonochemistry (US) and the results compared to those obtained by conventional heating. The use of the US brought many benefits to these syntheses, including faster reactions and increased yields. The crystal structure of diethyl 2-(((2-chloro-5-nitrophenyl)amino)methylene)malonate is also reported.

The free amino acid profiling of milk- and cereal-based Kyrgyz ethnic fermented beverages and their contribution to taste formation

The present study aimed to determine the content of 18 free amino acids (FAA), including 10 essential amino acids, in traditional fermented beverages and their umami, sweet and bitter taste potential. The FAAs of traditional dairy and cereal-based fermented beverages were measured using high-performance liquid chromatography (HPLC) with a diode array detector and derivatization with diethyl ethoxymethylenemalonate (DEEMM). FAA content and taste attributes with a focus on umami, sweet and bitter were presented for koumiss, ayran, carbonated ayran, boza, boza with sea buckthorn, and maksym. The ratio of free essential amino acids (EFAA) to nonessential (NEFAA) was in the range of 0.41-1.12 except for ayran (0.28), indicating that each type of beverage has a rich source of their respective essential FAA profile. FAA represented 24.68% of the total protein, with the highest value for koumiss. The respective taste property contribution of bitter FAAs was high in koumiss (51%) and carbonated ayran (56%), whereas sweet-tasting FAAs were dominant in jarma (52%). The distribution of taste-active amino acids in the formation of the corresponding tastes was distinct.

Free Amino Acids and Biogenic Amines Profiling and Variation in Wild and Sub-Endemic Cardueae Species from Sardinia and Corse.

The cardueae are a common species in the Mediterranean area where they grow spontaneously and are traditionally employed as food and for health purposes. In this work, five Cardueae, including two sub-endemic species (four Carduus and three Ptilostemon casabonae (L.) Greuter samples from different locations) were collected from Sardinia and the Corse islands. All the considered plants are characteristic of the area, in particular the sub-endemic species C. cephalanthus and P. casabonae. This work aims to obtain, for the first time, the amino compounds profile (primary metabolites) of these little-studied species to detect for any similarities and differences among the different samples using statistical analyses. A recently developed method was employed, where diethyl ethoxymethylenemalonate (DEEMM) derivatives are detected in a neutral loss scan mode using high performance liquid chromatography in tandem with a mass spectrometry technique. In total, 42 amino compounds were detected, of which 33 were fully identified and semi-quantified. Overall, the results show that DEEMM-derivatized amino compounds are qualitatively similar among the considered samples. Nonetheless, a discrimination at the genus level is possible. This work adds more information regarding the phytochemical composition regarding the primary metabolites of the considered samples, their discriminations and the search for compounds with potential health benefits.

An Efficient Synthesis of Novel 4-Aryl-2-thioxo-3,4-dihydro-1H-pyrimido[1,2-a][1,3,5]triazin-6(2H)-ones and Their Antibacterial Activity

New substituted 4-aryl-8-methyl-2-thioxo-1,2,3,4-tetrahydro-6H-pyrimido[1,2-a][1,3,5]triazin-6-one 8a–b and ethyl 4-aryl-6-oxo-2-thioxo-1,3,4,6-tetrahydro-2H-pyrimido[1,2-a][1,3,5]triazine-7-carboxylate 8c–e were synthesized by the reaction of the corresponding 4-oxopyrimidin-2-ylthioureas with arylaldehydes. The formation of only one regioisomer was proven using complex spectral data and its structure was characterized. It was found that the interaction of 6-amino-4-phenyl-3,4-dihydro-1,3,5-triazine-2(1H)-thione with ethyl acetoacetate and diethyl ethoxymethylenemalonate leads to the formation of the same regioisomer. That is, changing the sequence of stages in this cascade process does not affect the structure of the final reaction product. All synthesized compounds exhibit antibacterial activity against E. coli and S. aureus cultures at a concentration (MIC) of 256 µg/mL.

Comparison of the ionisation mode in the determination of free amino acids in beers by Liquid Chromatography tandem mass spectrometry

A comparison of positive and negative ionization modes in LC-ESI-MS/MS was carried out for the analysis of derivatized amino acids in 15 different beer samples. 22 free amino acids were derivatized using Diethyl ethoxymethylenemalonate (DEEMM) and their content was determined.When using the DEEMM as derivatization reagent the negative ionization mode provided analytical performance equal to or in some cases even superior to the positive ionization mode. For 6 amino acids (Thr, β-Ala, α-Ala, Met, Val and Orn) the negative mode led to lower LoQ values, while the positive mode offered lower LoQ values for 5 amino acids (Arg, Asp, Glu, GABA, and Pro). The remaining 11 amino acids showed similar LoQ values in both modes. Because of this, negative ionization mode allowed to detect and quantify amino acids such as: β-Alanine, threonine, and ornithine whose concentrations were low in most of the analysed samples. The relative standard deviation (RSD) for the results in both modes were similar. The method's linearity was determined to be in the range of 1 to 130 ppb with r2 > 0.99. Recoveries ranged from 93 to 112%. Negative mode was less affected by matrix effects the main effect was signal enhancement. In contrast, the positive ionization mode suffered from signal enhancement as well as signal suppression.

Derivatization-targeted analysis of amino compounds in plant extracts in neutral loss acquisition mode by liquid chromatography-tandem mass spectrometry

Amino compounds, such as amino acids and biogenic amines, are important metabolites that can be found in diverse natural matrices. The most common method for amino compound analysis nowadays is reversed-phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS). However, due to the polar and the basic nature of amines, their RPLC retention is often insufficient or peaks are tailing. Derivatization is a way to overcome the issue and in the present work amino compounds are derivatized with diethyl ethoxymethylenemalonate (DEEMM) and analyzed by a RPLC triple quadrupole MS system in neutral loss scan (NLS) mode (loss of 46). This allows to target all compounds in the sample that undergo derivatization with DEEMM, so that the amino compound profile of the sample is obtained. To the best of our knowledge, the NLS acquisition mode has never been employed to target amino compounds after DEEMM derivatization. In the first part of the study, eight amino acids (arginine, aspartic acid, threonine, proline, tyrosine, tryptophan, phenylalanine and isoleucine) were employed as model compounds for method optimization, with good results in terms of DEEMM derivatives detection and repeatability. The developed method was successfully applied to a complex extract from the plant species Carduus nutans subsp. macrocephalus (Desf.) Nyman, with 18 amino acids and 3 other amines being identified. The proposed approach could be employed for straightforward identification of known and unknown amino compounds in different types of matrices.

An Efficient Synthetic Method for the Synthesis of Novel Pyrimido[1,2-a]pyrimidine-3-carboxylates: Comparison of Microwave and Conventional Heating

A very simple and efficient procedure for the synthesis of novel pyrimido[1,2-a]pyrimidine derivatives is described. Thermal cyclization reactions of 2-aminopyrimidine and its substituted derivatives with diethyl ethoxymethylenemalonate (EMME) have been investigated. Conventional heating and microwave irradiation (MW) conditions were applied to enable the comparison of both techniques on the obtained products. Besides the effect of different reaction parameters and the substituents on the conversion reactions have been also investigated in detail. Structural analyses of all compounds synthesized with high yields have been carried out by suitable spectroscopic methods (FT-IR, NMR, mass spectroscopy, LC-MS TOF).

Effects of the mobile phase on the chromatographic separation of l-lysine and 5-aminovaleric acid

5-Aminovaleric acid (5AVA), an attractive bio-based carbon-5 building block for polymer synthesis, is produced from l-lysine. However, their structural similarity makes separating 5AVA from l-lysine challenging. The simulated moving bed (SMB), continuous chromatographic separation process provides a solution with high productivity and low eluent consumption. Here, the effects of the mobile phase on the separation of 5AVA and l-lysine on a C18 column are reported. Pre-column derivatization using diethylethoxymethylene malonate enhanced the detection sensitivity and separation performance, which was strongly affected by the phosphate buffer pH. A 70/30 (v/v) mobile phase of 20 mM phosphate buffer at pH 6.8 and acetonitrile showed the best separation performance in isocratic mode. The selectivity at adsorption equilibrium was determined from pulse experiments, and the optimal operating conditions of the SMB process were determined from the equilibrium constants of both components. The feasibility of separating 5AVA and l-lysine by the SMB process was confirmed with purity and recovery of greater than 99%. The SMB process using pre-column derivatization can provide the competiveness of the bio-based chemical production because separation processes typically contribute to more than 70% of the total production cost.

Antimicrobial Properties of Some New Synthesized Benzothiazole Linked Carboxamide, Acetohydrazide, and Sulfonamide Systems

We report herein the interaction of diethylethoxymethylene malonate (1) with 2‐cyanomethylbenzothiazole (7) to give diethyl 2‐(2‐benzothiazole‐2‐(3H)‐ylidiene)‐2‐(cyano ethyl) malonate (8a) in excellent yield. Ethyl 4‐cyano‐1‐oxo‐1H‐benzo[4,5]thiazolo[3,2‐a]pyridine‐2‐carboxylate (9) was synthesized from 8a and subjected to react with hydrazine hydrate to give its corresponding acid hydrazide 10. Condensation of 10 with different acid anhydrides afforded the corresponding benzothiazolo pyridine carboxamide derivatives 11–15. In addition, we report a simple synthesis of N′‐(benzo[d]thiazol‐2‐yl)‐2‐((4‐ayl)amino)acetohydrazide derivative (17), which then reacted with different amines to give the corresponding acetohydrazide derivatives 19a–c. Moreover, compound 17 reacted with some sulfonamide derivatives to give the corresponding sulfonamide derivatives 20 and 22a,b.The newly synthesized compounds were established their structures on the bases of their correct analytical and spectral data and evaluated their antimicrobial activity. It was found that compounds 22a,b displayed the highest antimicrobial activity against the tested organisms.

Construction of Fully Substituted 2-Pyridone Derivatives via Four-Component Branched Domino Reaction Utilizing Microwave Irradiation.

Microwave irradiation, four-component branched domino reaction of methyl acetoacetate/2,4-pentanedione, diethyl malonate, triethyl orthoformate and amines offering an extremely efficient strategy for the construction of fully substituted 2-pyridone derivatives under sustainable conditions is established. This self-sorting branched domino transformation is proposed to proceed separate through N-nucleophilic addition and imine-enamine tautomerization/condensation reaction generated from enamino ester and diethyl ethoxymethylenemalonate, and then would be subjected to an aza-ene reaction and intramolecular cyclization mechanism to afford the 2-pyridones with only water and ethanol as byproducts. The simple experimental procedure, high bond-forming efficiency, step and atom economy, inexpensive readily available starting materials, moderate to excellent yields, and good functional group compatibility are other noteworthy advantages of this method.

Microwave‐Assisted, Solvent‐Free, Three‐Component Domino Protocol: Efficient Synthesis of Polysubstituted‐2‐Pyridone Derivatives

AbstractMicrowave assisted highly efficient synthesis of polysubstituted‐2‐pyridone derivatives via a three‐component domino reaction of 1,3‐dicarbonyl compounds, diethyl ethoxymethylenemalonate and amines is established. The main advantages of this protocol are short reaction time, good functional group compatibility, high bond‐forming efficiency, being solvent‐ free, availability of starting materials and simple experimental procedure. Furthermore, a postulated reaction pathway included imine‐enamine tautomerism/ condensation/ aza‐ene reaction/ intramolecular cyclization processes for this three‐component domino protocol is also discussed.

Conventional and microwave prompted synthesis, antioxidant, anticholinesterase activity screening and molecular docking studies of new quinolone-triazole hybrids

The synthesis of ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylates (4, 5) was performed via the reaction of corresponding anilines with diethyl ethoxymethylenemalonate under conventional and also microwave promoted conditions. The treatment of 4 and 5 afforded the corresponding hydrazides (6 and 7). These hydrazides were converted to the corresponding carbo(thio)amides (9a-f and 10a-e) which were then subjected to an intramolecular cyclisation leading to the formation of quinolone-triazole hybrids (11a-f and 12a-e). The newly synthesized compounds were screened for their biological activities such as antioxidant capacity (AC) and acetylcholinesterase Activity. Inhibition of cholinesterases is an effective method to curb Alzheimer’s disease, a progressive and fatal neurological disorder. A series of some novel quinolonederivatives were designed, synthesized, and their inhibitory effects on AChE were evaluated. We obtained our compounds and determined their anticholinesterase activities according to the Ellman’s method. 9b and 10c showed the best AChE inhibition with 0.48 ± 0.02 and 0.52 ± 0.07, respectively. Docking studies were performed for the most active compounds (9b, 10c) and interaction modes with enzyme active sites were determined. As a result of these studies, a strong interaction between these compounds and the active sites of AChE enzyme was revealed.

Quantification of methionine and selenomethionine in biological samples using multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS)

Quantification of selenated amino-acids currently relies on methods employing inductively coupled plasma mass spectrometry (ICP-MS). Although very accurate, these methods do not allow the simultaneous determination of standard amino-acids, hampering the comparison of the content of selenated versus non-selenated species such as methionine (Met) and selenomethionine (SeMet). This paper reports two approaches for the simultaneous quantification of Met and SeMet. In the first approach, standard enzymatic hydrolysis employing Protease XIV was applied for the preparation of samples. The second approach utilized methanesulfonic acid (MA) for the hydrolysis of samples, either in a reflux system or in a microwave oven, followed by derivatization with diethyl ethoxymethylenemalonate. The prepared samples were then analyzed by multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS). Both approaches provided platforms for the accurate determination of selenium/sulfur substitution rate in Met. Moreover the second approach also provided accurate simultaneous quantification of Met and SeMet with a low limit of detection, low limit of quantification and wide linearity range, comparable to the commonly used gas chromatography mass spectrometry (GC–MS) method or ICP-MS. The novel method was validated using certified reference material in conjunction with the GC-MS reference method.

Development of a precolumn derivatization HPLC method with diode-array detection for the determination of amino sugars in peat and soil humic acids.

The work is focused on the development of a high-performance liquid chromatography method with diode-array detection for the separation and quantitation of the three most abundant amino sugars; d-glucosamine, d-galactosamine, and d-mannosamine. The high-performance liquid chromatography separation was carried out by reversed-phase chromatography on Chromolith Performance RP-18e monolithic column after acid hydrolysis (5M HCl) and precolumn derivatization of samples using diethyl ethoxymethylenemalonate. Gradient elution and a mobile phase composed of ammonium formate buffer solution (10mmol/L, pH 3.60) and methanol with flow rate of 1.0mL/min were used. The monitoring wavelength was set at 280nm. The limits of detection and quantitation for analytes ranged from 0.017 to 0.122mg/L and from 0.057 to 0.407mg/L, respectively. The proposed method was successfully applied for the determination of amino sugars in samples of humic acids isolated from different soils and peat.

Free amino acids, biogenic amines, and ammonium profiling in tobacco from different geographical origins using microwave-assisted extraction followed by ultra high performance liquid chromatography.

This work describes a rapid, stable, and accurate method for determining the free amino acids, biogenic amines, and ammonium in tobacco. The target analytes were extracted with microwave-assisted extraction and then derivatized with diethyl ethoxymethylenemalonate, followed by ultra high performance liquid chromatography analysis. The experimental design used to optimize the microwave-assisted extraction conditions showed that the optimal extraction time was 10min with a temperature of 60°C. The stability of aminoenone derivatives was improved by keeping the pH near 9.0, and there was no obvious degradation during the 80°C heating and room temperature storage. Under optimal conditions, this method showed good linearity (R2 > 0.999) and sensitivity (limits of detection 0.010-0.081 μg/mL). The extraction recoveries were between 88.4 and 106.5%, while the repeatability and reproducibility ranged from 0.48 to 5.12% and from 1.56 to 6.52%, respectively. The newly developed method was employed to analyze the tobacco from different geographical origins. Principal component analysis showed that four geographical origins of tobacco could be clearly distinguished and that each had their characteristic components. The proposed method also showed great potential for further investigations on nitrogen metabolism in plants.

Comparison of amino acid derivatization reagents for liquid chromatography atmospheric pressure chemical ionization mass spectrometric analysis of seven amino acids in tea extract

The applicability of five amino acid derivatization reagents was tested for liquid chromatography atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). The aim of the study was to investigate the matrix effects (ME) during LC-APCI-MS analysis. The derivatization reagents were diethyl ethoxymethylenemalonate (DEEMM), dibenzyl ethoxymethylene malonate (DBEMM), dansyl chloride (DNS), 9-fluorenylmethyl chloroformate (FMOC-Cl) and 2,5-dioxopyrrolidin-1-yl N-tri(pyrrolidino)phosphoranylideneamino carbamate (FOSF).APCI-MS parameters were optimized and the suitability of methanol and acetonitrile as eluent component was assessed. For the evaluation of ME, post-column infusion for some DEEMM and FMOC-derivatives and sample dilution approach for seven amino acids in green tea extract with DEEMM, DBEMM, DNS and FMOC-Cl were used. The measurements with electrospray ionization (ESI) were carried out using the same chromatographic conditions for comparing ME in ESI and APCI.The method development showed that acetonitrile was suitable for APCI ionization. From the derivatization reagents, FOSF was not suitable for APCI analysis. In general, APCI showed less ME compared to ESI in case of all the derivatization reagents. Although the derivatives of DEEMM and DNS exhibited the least ME in APCI, some amino acids could not be analyzed. ME was evaluated for DBEMM derivatives with both ESI and APCI for the first time, and despite some ME, the sensitivity of DBEMM allowed elimination of ME with sample dilution. Analysis of seven amino acids in green tea extract was carried out and concentrations obtained with ESI and APCI agreed (differences were not statistically significant).

Dependence of matrix effect on ionization polarity during LC-ESI-MS analysis of derivatized amino acids in some natural samples.

Matrix effect, the influence of co-eluting components on the ionization efficiency of the analyte, affects the trueness and precision of the LC-ESI-MS analysis. Derivatization can reduce or eliminate matrix effect, for example, diethyl ethoxymethylenemalonate (DEEMM) derivatives have shown less matrix effect compared to other derivatives. Moreover, the use of negative ion mode can further reduce matrix effect. In order to investigate the combination of derivatization and different ionization modes, an LC-ESI-MS/MS method using alternating positive/negative ion mode was developed and validated. The analyses in positive and negative ion modes had comparable limit of quantitation values. The influence of ESI polarity on matrix effect was investigated during the analysis of 22 DEEMM-derivatized amino acids in herbal extracts and honeys. Sample dilution approach was used for the evaluation of the presence of matrix effect. Altogether, 4 honeys and 11 herbal extracts were analyzed, and the concentrations of 22 amino acids in the samples are presented. In the positive ion mode, matrix effect was observed for several amino acid derivatives and the matrix effect was stronger in honey samples compared to the herbal extracts. The negative ion mode was free from matrix effect, with only few exceptions in honeys (average relative standard deviation over all analytes and matrices was 8%; SD = 7%). The matrix effect was eliminated in the positive ion mode by sample dilution and agreement between concentrations from the two ion modes was achieved for most amino acids. In conclusion, it was shown that the combination of derivatization and negative ion mode can be a powerful tool for minimizing matrix effect in more complicated applications.

Alkoxy base-mediated selective synthesis and new rearrangements of 1,2,4-triazolodipyrimidinones

A versatile approach for the synthesis of [1,2,4]triazolodipyrimidinones with various annulations of the triazole and pyrimidine rings was developed. The isomeric triazolodipyrimidinones were obtained by the stepwise condensation of partially hydrogenated [1,2,4]triazolo[1,5-a]pyrimidin-2-amines with β-ketoesters or diethyl ethoxymethylenemalonate, alkoxy base-mediated cyclization of the enamines, and subsequent cascade rearrangement of the 10-oxo-[1,2,4]triazolo[1,5-a:4,3-a′]dipyrimidines.

Downstream processing of serinol from a glycerol-based fermentation broth and transfer to other amine containing molecules.

A possible application of glycerol, which is produced in large amounts as a by-product from the biodiesel industry, is its fermentation to serinol (2-amino-1,3-propanediol), a glycerol derivative. The downstream processing of this glycerol-based fermentation broth was investigated. The challenge of the isolation of serinol was the complex media and the solubility of the desired substance in aqueous media. In this study, the isolation of serinol was investigated by an appropriate reversible derivatization method. Serinol was isolated by protecting the amino group with diethyl ethoxymethylenemalonate directly in the aqueous phase, followed by extraction of the 2,2-bis(ethoxycarbonyl)vinyl-serinol (BECV-serinol) with ethyl acetate resulting in an isolated yield of 63%. We demonstrate the possibility of isolation of a hydroscopic amino alcohol from the fermentation broth and the comparison of the products in water as well as the cleavage of the 2,2-bis(ethoxycarbonyl)vinyl group (BECV group). The procedure can also be used for other amino group containing molecules, such as serine, glucosamine, hexylamine and amino methyl laureate.

Effect of Gluconic Acid Submerged Fermentation of Strawberry Purée on Amino Acids and Biogenic Amines Profile

Gluconic fermentation of fruit substrates is an innovative strategy to elaborate nonalcoholic beverages which has not been studied so far. Futhermore, it prevents food waste of perishable products like strawberries. Amino acids, biogenic amines and ammonium ion were evaluated after the fermentation of strawberry purée using Gluconobacter japonicus in submerged culture. The process was monitored with the determination of pH, total acidity, glucose, fructose and gluconic acid concentrations. The determination of nitrogen compounds was accomplished using a HPLC–UV method involving diethyl ethoxymethylenemalonate (DEEMM) as the derivatization agent. Most amino acids did not present significant changes, except for lysine and histidine, which increased their concentration in the final product. However, the principal component analysis was able to group the initial and final samples in different sets using the amino acids concentration as variables. Biogenic amines were not detected in the final samples, thus ensuring the safety of this product for human consumption. Practical Applications Because of the high level of competitiveness, the success of a new product requires the use of a raw material different from the traditionally used ones, and the application of innovative techniques of production. Strawberry is a fruit prized for its aroma and flavor. In addition, Spain is the fifth strawberry producer in the world, and consequently there are surpluses of second quality strawberries. This second quality fruit is generally discarded, involving large economic losses. These facts make strawberry a good candidate to be used as raw material for the production of fruit derived products. Hence, gluconic acid fermentation is an innovative way of transforming surplus strawberries into beverages which do not present glucose, as it is transformed into gluconic acid whilst keeping fructose as sweetener. Hence, the final product could be the principal substract to elaborate a new beverage which could be consumed by diabetics. Gluconic acid submerged fermentations of strawberry purée were carried out with Gluconobacter japonicus. Additionally, the amount of biogenic amines was assessed for food safety purposes.