Mild Alkaline Conditions Research Articles

A carbamoyl-protective group for tyrosine that facilitates purification of hydrophobic synthetic peptides

The Boc- N-methyl- N-[2-(methylamino)ethyl]carbamoyl group (Boc-Nmec) is reported as a new side chain-protective group for tyrosine in Fmoc solid-phase peptide synthesis. Tyrosine is incorporated into the peptide as Fmoc-Tyr(Boc-Nmec)-OH by standard coupling methods. During the cleavage of the peptide from the resin with TFA the Boc group is simultaneously cleaved while the cationic N-methyl- N-[2-(methylamino)ethyl]carbamoyl group remains attached to the tyrosine residue, thereby increasing the solubility of the peptide. After purification of the peptide, the Nmec protective group can be cleaved under neutral or mild alkaline conditions via an intramolecular cyclization reaction.

Ring Opening of 2‐(Benzylamino)‐2H‐1,4‐benzoxazin‐3(4H)‐ones and 2‐Bromo‐2H‐1,4‐benzoxazin‐3(4H)‐ones

AbstractSubstituted 2‐(benzylamino)‐2H‐1,4‐benzoxazin‐3(4H)‐ones are unstable under alkaline and acidic conditions, undergoing opening of the benzoxazinone ring. 2‐Bromo‐2H‐1,4‐benzoxazin‐3(4H)‐ones show similar degradation under alkaline conditions, while replacement of Br at C(2) to give 2‐hydroxy‐2H‐1,4‐benzoxazin‐3(4H)‐ones was observed only under mild alkaline conditions. Mechanisms of ring opening and degradation to 2‐aminophenol derivatives are proposed.

Templated Synthesis of Electroactive Periodic Mesoporous Organosilica Bridged with Oligoaniline

The synthesis and characterization of novel electroactive periodic mesoporous organosilica (PMO) are reported. The silsesquioxane precursor, N,N'-bis(4'-(3-triethoxysilylpropylureido)phenyl)-1,4-quinonene-diimine (TSUPQD), was prepared from the emeraldine base of amino-capped aniline trimer (EBAT) using a one-step coupling reaction and was used as an organic silicon source in the co-condensation with tetraethyl orthosilicate (TEOS) in proper ratios. By means of a hydrothermal sol-gel approach with the cationic surfactant cetyltrimethyl-ammonium bromide (CTAB) as the structure-directing template and acetone as the co-solvent for the dissolution of TSUPQD, a series of novel MCM-41 type siliceous materials (TSU-PMOs) were successfully prepared under mild alkaline conditions. The resultant mesoporous organosilica were characterized by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetry, X-ray diffraction, nitrogen sorption, and transmission electron microscopy (TEM) and showed that this series of TSU-PMOs exhibited hexagonally patterned mesostructures with pore diameters of 2.1-2.8 nm. Although the structural regularity and pore parameters gradually deteriorated with increasing loading of organic bridges, the electrochemical behavior of TSU-PMOs monitored by cyclic voltammetry demonstrated greater electroactivities for samples with higher concentration of the incorporated TSU units.

Nanoparticles formed by complexation of poly-gamma-glutamic acid with lead ions

The present investigation describes the preparation and characterization of novel biodegradable nanoparticles based on complexation of poly-gamma-glutamic acid (γ-PGA) with bivalent lead ion. The prepared nano-systems were stable in aqueous media at low pH, neutral and mild alkaline conditions. The particle size and the size of the complexes were identified by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that the size of the complexes depended on the pH and concentrations of γ-PGA and lead ions. Particle sizes measured by TEM revealed that at low concentrations, nanosized particles were formed, however, at high concentrations of γ-PGA and lead ions, the formation of large aggregates with a broad size distribution was promoted. The size of individual particles was in the range of 40–100 nm measured by TEM. The results from the DLS measurements showed that the low and high pH values in mixtures with high concentrations of γ-PGA and Pb 2+ ions favored the growth of large complexes. The γ-PGA nanoparticles, composed of a biodegradable biomaterial with high flocculating and heavy metal binding activity, may be useful for various water treatment applications.

Lignin modification in the initial phase of softwood kraft pulp delignification with polyoxometalates (POMs)

Abstract Commercial softwood kraft pulp with kappa number 30.5 (KP30.5) was delignified with polyoxometalates (POM, Na5(+2)[SiV1(-0.1)MoW10(+0.1)O40]), and POM-treated kraft pulp of kappa number 23.6 was obtained (KPPOM,23.6). Residual lignin from pulps was isolated by mild acid hydrolysis and characterized by analytical and spectral methods to gain insight into lignin reactions taking place during the initial delignification phase. Lignin from POM-delignified pulp was isolated in lower yield. Comparative analysis of residual lignins (RL-KP30.5, RL-KPPOM,23.6) showed that POM leads to products enriched in carbonyl/carboxyl groups and carbohydrates. POM lignins have a lower molecular mass and a lower content of phenolic hydroxyl and methoxyl groups. Based on these results and FTIR spectra, we suggest that aromatic ring cleavage and quinone formation occur during POM delignification. The degree of lignin-cellulose association increases after POM delignification. Lignin-cellulose association was found to be partially unstable under mild alkaline conditions, as residual lignin isolated after alkaline extraction of KPPOM,23.6 pulp (RL-KPPOM/NaOH) exhibited lower glucose content, higher Klason lignin content, and less extraneous material.

The Transcriptional Response of the Yeast Na+-ATPase ENA1 Gene to Alkaline Stress Involves Three Main Signaling Pathways

Adaptive response of the yeast Saccharomyces cerevisiae to environmental alkalinization results in remodeling of gene expression. A key target is the gene ENA1, encoding a Na(+)-ATPase, whose induction by alkaline pH has been shown to involve calcineurin and the Rim101/Nrg1 pathway. Previous functional analysis of the ENA1 promoter revealed a calcineurin-independent pH responsive region (ARR2, 83 nucleotides). We restrict here this response to a small (42 nucleotides) ARR2 5.-region, named MCIR (minimum calcineurin independent response), which contains a MIG element, able to bind Mig1,2 repressors. High pH-induced response driven from this region was largely abolished in snf1 cells and moderately reduced in a rim101 strain. Cells lacking Mig1 or Mig2 repressors had a near wild type response, but the double mutant presented a high level of expression upon alkaline stress. Deletion of NRG1 (but not of NRG2) resulted in increased expression. Induction from the MCIR region was marginal in a quadruple mutant lacking Nrg1,2 and Mig1,2 repressors. In vitro band shift experiments demonstrated binding of Nrg1 to the 5. end of the ARR2 region. Furthermore, we show that Nrg1 binds in vivo around the MCIR region under standard growth conditions, and that binding is largely abolished after high pH stress. Therefore, the calcineurin-independent response of the ENA1 gene is under the regulation of Rim101 (through Nrg1) and Snf1 (through Nrg1 and Mig2). Accordingly, induction by alkaline stress of the entire ENA1 promoter in a snf1 rim101 mutant in the presence of the calcineurin inhibitor FK506 is completely abolished. Thus, the transcriptional response to alkaline stress of the ENA1 gene integrates three different signaling pathways.

Determination of nitrofurans in animal feeds by liquid chromatography-UV photodiode array detection and liquid chromatography-ionspray tandem mass spectrometry

Within the EU, the use of nitrofurans is prohibited in food production animals. For this reason detection of these compounds in feedingstuffs, at whatever limit, constitutes an offence under EU legislation. This detection generally involves the use of analytical methods with limits of quantification lowers than 1mgkg−1. These procedures are unsuitable for the detection and confirmation of trace amounts of nitrofurans in feedingstuffs due to contamination. It is well known that very low concentrations of these compounds can be the source of residues of nitrofuran metabolites in meat and other edible products obtained from animals consuming the contaminated feed. The present multi-compound method was capable of measuring very low concentrations of nitrofurantoin (NFT), nitrofurazone (NFZ), furazolidone (FZD) and furaltadone (FTD) in animal feed using nifuroxazide (NXZ) as internal standard. Following ethyl acetate extraction at mild alkaline conditions and purification on NH2 column, the nitrofurans are determined using liquid chromatography with photodiode-array detection (LC-DAD). It was observed a CCα ranged from 50 to 100μgkg−1. The liquid chromatography-tandem mass spectrometric (LC-MS/MS) procedure was used to confirm the identity of the suspected presence of any of the nitrofuran compounds.

Enzymatically oxidized lactose and derivatives thereof as potential protein cross-linkers

The enzyme galactose oxidase [EC 1.1.3.9] was applied to convert lactose, lactylamine and lactobionic acid into their corresponding 6′-aldehyde compounds. The potential protein cross-linking ability of these oxidized lactose and derivatives thereof was investigated using n-butylamine as the model compound. First, oxidized lactose gave double Maillard reaction products that were stable under mild alkaline conditions. Second, reductive amination of lactose followed by enzymatic oxidation gave cross-links that were stable under both neutral and alkaline conditions. Third, stable cross-links were obtained through enzymatic oxidation and amidation of lactobionic acid.

Stability of polyphenols in chokeberry ( Aronia melanocarpa) subjected to in vitro gastric and pancreatic digestion

Berries and red fruits are dietary sources of polyphenols with reported health benefits. As part of the diet, polyphenols are ingested as complex mixtures immersed in a food matrix which is digested in the gut. Epithelial cells lining the gut are regularly exposed to these digested mixtures. To understand the effects of dietary polyphenols on human gut health it is essential to determine their stability and fate in the lumen. In this work, we investigated the effects of an in vitro gastric and pancreatic digestion on the stability and composition of the major polyphenols in chokeberry juice. Digestion was carried out with a mixture of pepsin-HCl for 2 h, followed by a 2 h incubation with pancreatin and bile salts at 37 °C with shaking, in the absence of light and under N 2. After digestion, the chokeberry samples were acidified, filtered and HPLC-DAD/HPLC–MS–MS analysed to determine the content of total soluble recovered phenolics. Gastric digestion had no substantial effect on any of the major phenolic compounds in chokeberry, namely anthocyanins, flavan-3-ols, flavonols and caffeic acid derivatives. However, these compounds were significantly altered during the pancreatic digestion and this effect was more marked for anthocyanins (approximately 43% was lost during the 2 h treatment with pancreatin). Flavonols and flavan-3-ols decreased by 26% and 19%, respectively. Neochlorogenic acid decreased by 28% whereas chlorogenic acid was increased by 24%. In vitro digestion of standard phenolic compounds, representing each of the groups of phenolics in chokeberry, confirmed some of the observed changes. Interactions with the digestive enzymes were not responsible for the observed losses which were mostly due to the chemical conditions during pancreatic digestion. Our results, in accordance with previously published results, show that dietary polyphenols are highly sensitive to the mild alkaline conditions in the small intestine and that a good proportion of these compounds can be transformed into other unknown and/or undetected structural forms with different chemical properties and, consequently, different bioaccessibility, bioavailability and biological activity.

Structure-Activity Relationships of Neuromedin U. V. Study on the Stability of Porcine Neuromedin U-8 at the C-Terminal Asparagine Amide under Mild Alkaline and Acidic Conditions

Porcine neuromedin U-8 (X-Asn-NH(2), X=H-Tyr-Phe-Leu-Phe-Arg-Pro-Arg) is occasionally unstable in the biological fluids used for bioassay as well as in the acidic solutions used for purification of synthetic peptides. In this study, HPLC examination of an incubate solution of X-Asn-NH(2) revealed that the main decomposition products in Tyrode's solution (pH 7.4) were either alpha- or beta-monocarboxylic acid analogs (X-Asn-OH or X-Asp-NH(2)), and that no dicarboxylic acid analog (X-Asp-OH) was produced. Further investigation, employing a model peptide (Y-Asn-NH(2), Y=Benzoyl-Pro-Arg) incubated in a 0.1 M sodium bicarbonate solution at 60 degrees C, revealed that the decomposition of C-terminal Asn-NH(2) occurred through the formation of an aminosuccinimide intermediate (Y-Asu), at a rate faster than that of Y-Asn-Ser peptide but slower than that of Y-Asn-Gly peptide. Mild acid hydrolysis of X-Asn-NH(2) examined in a 1 M HCl solution at 60 degrees C yielded X-Asn-OH and X-Asp-NH(2), which further decomposed to yield X-Asp-OH. The C-terminal degradation of X-Asn-NH(2) resulted in reduced biological and immunochemical binding activities.

Forward and backward extractions of cytochrome c using reverse micellar system of sucrose fatty acid ester

The effects of solvent, pH, and ionic strength on the reverse micellar extraction of cytochrome c have been examined, when sucrose fatty acid esters were employed as surfactants of reverse micelles. The transparent and stable reverse micellar organic phase was formed, when the mixture of isooctane and n-butanol (7:3 v/v) was used as the bulk organic phase. The high forward extraction ratio was obtained under mild alkaline and low ionic strength conditions, while the high backward extraction ratio was obtained for acidic pH values or at high ionic strength. The activity of cytochrome c recovered from the reverse micellar phase was sufficiently retained.

Derivatization chemistries for determination of serotonin, norepinephrine and dopamine in brain microdialysis samples by liquid chromatography with fluorescence detection

The present paper provides an overview on currently developed derivatization chemistries and techniques for determination of monoamine neurotransmitters serotonin (5-HT), norepinephrine (NE) and dopamine (DA) in microdialysis samples by microbore liquid chromatography with fluorescence detection. In mild alkaline conditions, 5-hydroxyindoles and catecholamines react with benzylamine (BA), forming highly fluorescent 2-phenyl-4,5-pyrrolobenzoxazoles and 2-phenyl(4,5-dihydropyrrolo) [2,3-f]benzoxazoles, respectively. However, for derivatization of DA a higher fluorescence intensity was achieved for reaction with 1,2-diphenylethylenediamine (DPE) rather than with BA, therefore for simultaneous determination of 5-HT, NE and DA in brain microdialysates, a two-step derivatization with BA followed by DPE was developed. The detection limits for 5-HT, NE and DA were 0.2, 0.08 and 0.13 fmol, respectively, in an injection volume of 20 microL, which corresponds to concentrations of 30, 12 and 19.5 pm, respectively in standard solution prior to derivatization. The experimental data presented demonstrate the ability of the technique to simultaneously monitor neuronally releasable pools of monoamine neurotransmitters in the rat and mouse brains at basal conditions and following pharmacological treatments or physiological stimuli. These techniques play an important role in drug discovery and clinical investigation of psychiatric and neurological diseases such as depression, schizophrenia and Parkinson's disease.

Scission of the Lactyl Ether Bond of N-Acetylmuramic Acid by Escherichia coli “Etherase”

The ubiquitous bacterial cell wall sugar N-acetylmuramic acid (MurNAc) carries a unique D-lactyl ether substituent at the C3 position. Recently, we proposed an etherase capable of cleaving this lactyl ether to be part of the novel bacterial MurNAc dissimilation pathway (Dahl, U., Jaeger, T., Nguyen, B. T., Sattler, J. M., Mayer, C. (2004) J. Bacteriol. 186, 2385-2392). Here, we report the identification of the first known MurNAc etherase. The encoding gene murQ is located at 55 min on the Escherichia coli chromosome adjacent to murP, the MurNAc-specific phosphotransferase system. A murQ deletion mutant could not grow on MurNAc as the sole source of carbon and energy but could be complemented by expressing murQ from a plasmid. The mutant had no obvious phenotype when grown on different carbon sources but accumulated MurNAc 6-phosphate at millimolar concentrations from externally supplied MurNAc. Purified MurQ-His6 fusion protein and extracts of cells expressing murQ both catalyze the cleavage of MurNAc 6-phosphate, with GlcNAc 6-phosphate and D-lactate being the primary products. The 18O label from enriched water is incorporated into the sugar molecule, showing that the C3-O bond is cleaved and reformed by the enzyme. Moreover, an intermediate was detected and identified as an unsaturated sugar molecule. Based on this observation, we suggested a lyase-type mechanism (beta-elimination/hydration) for the cleavage of the lactyl ether bond of MurNAc 6-phosphate. Close homologs of murQ were found on the chromosome of several bacteria, and amino acid sequence similarity with the N-terminal domain of human glucokinase-regulatory protein (GckR or GKRP) was recognized.

Substitution of Hydroxybiaryls via Directed ortho-Lithiation of N-Silylated O-Aryl N-Isopropylcarbamates

[reaction: see text] Herein we report regioselective substitution reactions of a series of 2- and 3-hydroxybiaryls including BINOL via a new directed ortho-metalation procedure. O-Aryl N-isopropylcarbamates, conveniently prepared from phenols and isopropyl isocyanate, are temporarily and in situ N-protected by means of silyl triflates to form stable intermediates for low temperature lithiation reactions using butyllithium/TMEDA in diethyl ether. The resulting aryllithiums are efficiently substituted by a wide range of electrophilic reagents to afford functionalized biaryls in high yields. N-Desilylation already occurs during aqueous workup. The following deprotection of the urethanes to the corresponding phenols proceeds rapidly and in quantitative yields. Even sensitive substituents (e.g., CO2Me, CHO, SiMe3, I) in the products are preserved under mild alkaline conditions which have been established for carbamate ester cleavage. Furthermore, applications of ortho-substituted products in common cross-coupling reactions for further C-C bond formations are demonstrated.

Preparation and Characterization of Chitosan-Based Nanoparticles

The present investigation describes the synthesis and characterization of novel biodegradable nanoparticles based on chitosan for biomedical applications. Natural di- and tricarboxylic acids were used for intramolecular cross-linking of the chitosan linear chains. The condensation reaction of carboxylic groups and pendant amino groups of chitosan was performed by using water-soluble carbodiimide. This method allows the formation of polycations, polyanions, and polyampholyte nanoparticles. The prepared nanosystems were stable in aqueous media at low pH, neutral, and mild alkaline conditions. The structure of products was determined by NMR spectroscopy, and the particle size was identified by laser light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that particle size depends on the pH, but at a given pH, it was independent of the ratio of cross-linking and the cross-linking agent. Particle size measured by TEM varied in the range 60-280 nm. In the swollen state, the average size of the particles measured by DLS was in the range 270-370 nm depending on the pH. The biodegradable cross-linked chitosan nanoparticles, as solutions or dispersions in aqueous media, might be useful for various biomedical applications.

Structural elucidation and analysis of thermal degradation products of theFusarium mycotoxin nivalenol

The major class of mycotoxins produced by Fusarium moulds are trichothecenes, a large group of sesquiterpenes sharing the same basic chemical structure, a 12,13-epoxytrichothec-9-ene ring system. Their toxic effects range from causing diarrhoea, vomiting and gastro-intestinal inflammation to noncompetitive inhibition of the biosynthesis of proteins in eukaryotic cells. Trichothecenes in general are relatively stable compounds, their degradation is observed only at high temperatures and prolonged heating time. In order to investigate the stability of the trichothecene nivalenol (NIV) under food processing conditions such as cooking or baking, we performed model heating experiments and screened the residue for degradation products using gas chromatography-mass spectrometry (GC-MS). Heating of nivalenol, especially under mild alkaline conditions, gave a mixture of four compounds (norNIV A, norNIV B, norNIV C, and NIV lactone), which where isolated and identified by nuclear magnetic resonance (NMR) and MS experiments. Although their formation was also demonstrated in heating experiments with spiked flour samples, only norNIV B was detectable in a screening of several commercially available samples, possibly due to the very low contamination with nivalenol. Furthermore, cell culture experiments using immortalized human kidney epithelial (IHKE) cells showed that the four compounds are less cytotoxic (formazan dye cytotoxicity assay) compared to nivalenol. Whereas nivalenol revealed an EC50 at 0.9 micromol, all other compounds did not show any significant effect up to 100 micromol.

Emulsifying properties of gelatin conjugated to pectin under alkaline conditions

AbstractGelatin–pectin mixed solution incubated under mild alkaline conditions for a period of 4 h exhibited an improvement of emulsion stability in terms of both droplet coalescence and serum separation. Application of SDS–PAGE provided evidence for gelatin–pectin hybrid formation possibly due to amide bonds between the lysine group residues of protein and the esterified carboxyl groups of the polysaccharide. The superior stabilizing properties of the heat‐treated protein–polysaccharide conjugate is attributed to the enhancement of the repulsive steric forces operating between emulsion oil droplets, as a result of conjugate adsorption through their protein moiety. Copyright © 2004 Society of Chemical Industry

Alkalitolerance: a biological function for a multidrug transporter in pH homeostasis.

MdfA is an Escherichia coli multidrug-resistance transporter. Cells expressing MdfA from a multicopy plasmid exhibit multidrug resistance against a diverse group of toxic compounds. In this article, we show that, in addition to its role in multidrug resistance, MdfA confers extreme alkaline pH resistance and allows the growth of transformed cells under conditions that are close to those used normally by alkaliphiles (up to pH 10) by maintaining a physiological internal pH. MdfA-deleted E. coli cells are sensitive even to mild alkaline conditions, and the wild-type phenotype is restored fully by MdfA expressed from a plasmid. This activity of MdfA requires Na(+) or K(+). Fluorescence studies with inverted membrane vesicles demonstrate that MdfA catalyzes Na(+)- or K(+)-dependent proton transport, and experiments with reconstituted proteoliposomes confirm that MdfA is solely responsible for this phenomenon. Studies with multidrug resistance-defective MdfA mutants and competitive transport assays suggest that these activities of MdfA are related. Together, the results demonstrate that a single protein has an unprecedented capacity to turn E. coli from an obligatory neutrophile into an alkalitolerant bacterium, and they suggest a previously uncharacterized physiological role for MdfA in pH homeostasis.

Magnetic properties of α-Fe2O3 nanoparticle synthesized by a new hydrothermal method

Nanoparticles of α-Fe2O3 have been prepared using a hydrothermal synthesis method in aqueous-organic microemulsion under mild alkaline condition. The condensation reaction was optimized in the presence of a cationic surfactant cetyltrimethylammonium bromide (CTAB). It was found that the size and nature of the α-Fe2O3 nanoparticle strongly depends on the pH, oxalic acid and CTAB as well as tetramethylammonium hydroxide (TMAOH, alkali source) concentrations. The uniformity of the particle size was checked by the transmission electron microscopy while the single phase of the nanocrystalline α-Fe2O3 was characterized using powder X-ray diffraction. The Mössbauer study exhibited a sextet pattern with internal field smaller than that of the bulk counterpart. The temperature variation of magnetization showed a broad maximum at around 125K while the field-cooled effect of the magnetization showed the branching between the field cooled and zero field cooled magnetization up to 340K. A large anisotropy has been observed from the analysis of magnetization curve as well as from the large blocking temperature. The estimation of the particle size from the magnetization curve was found to be in close agreement with the TEM results.

Alkali-hydrolysis kinetics of alkali-clearable azo disperse dyes containing a fluorosulphonyl group and their fastness properties on PET/cotton blends

The alkali-hydrolysis kinetics of a series of 4-amino-4′-fluorosulphonylazobenzene disperse dyes were investigated using HPLC and their fastness properties on PET/cotton blends were examined. Most of the dyes showed reasonable level of hydrolysis (%) under relatively mild alkaline condition and the dyes having more electron-accepting group in diazo component exhibited lower rate of hydrolysis. The application of fluorosulphonyl containing disperse dyes on PET/cotton blends showed excellent dyeing fastness properties and offers the option of one-bath dyeing process, replacing time- and energy-consuming conventional two-bath dyeing process.