Trimethylacetic Anhydride-Based Derivatization Facilitates Quantification of Histone Marks at the MS1 Level.

Histone post-translational modifications (hPTMs) are epigenetic marks that strongly affect numerous processes, including cell cycling and protein interactions. They have been studied by both antibody- and MS-based methods for years, but the analyses are still challenging, mainly because of the diversity of histones and their modifications arising from high contents of reactive amine groups in their amino acid sequences. Here, we introduce use of trimethylacetic anhydride (TMA) as a new reagent for efficient histone derivatization, which is a requirement for bottom-up proteomic hPTM analysis. TMA can derivatize unmodified amine groups of lysine residues and amine groups generated at peptide N-termini by trypsin digestion. The derivatization is facilitated by microwave irradiation, which also reduces incubation times to minutes. We demonstrate that histone derivatization with TMA reliably provides high yields of fully derivatized peptides and thus is an effective alternative to conventional methods. TMA afforded more than 98% and 99% labeling efficiencies for histones H4 and H3, respectively, thereby enabling accurate quantification of peptide forms. Trimethylacetylation substantially improves chromatographic separation of peptide forms, which is essential for direct quantification based on signals extracted from MS1 data. For this purpose, software widely applied by the proteomics community can be used without additional computational development. Thorough comparison with widely applied propionylation highlights the advantages of TMA-based histone derivatization for monitoring hPTMs in biological samples.

The Annealing of Acetylated Potato Starch with Various Substitution Degrees.

This study aimed to determine the effect of "annealing" acetylated potato starch with a homogenous granule size and various degrees of substitution on the thermal pasting characteristics (DSC), resistance to amylases, rheology of the prepared pastes, swelling power and dynamics of drug release. A fraction of large granules was separated from native starch with the sedimentation method and acetylated with various doses of acetic anhydride (6.5, 13.0 or 26.0 26 cm3/100 g starch). The starch acetates were then annealed at slightly lower temperatures than their pasting temperatures. The annealing process caused an almost twofold increase in the resistance to amylolysis and a threefold increase in the swelling power of the modified starch preparations. The heat of phase transition decreased almost two times and the range of starch pasting temperatures over two times, but the pasting temperature itself increased by ca. 10 °C. The 40 g/100 g addition of the modified starch preparation decreased the rate of drug release from a hydrogel by ca. one-fourth compared to the control sample.

Surface hydrophobization of pulp fibers in paper sheets via gas phase reactions.

Hydrophobization of cellulosic materials and particularly paper products is a commonly used procedure to render papers more resistant to water and moisture. Here, we explore the hydrophobization of unsized paper sheets via the gas phase. We employed three different compounds, namely palmitoyl chloride (PCl), trifluoroacetic anhydride/acetic anhydride (TFAA/Ac2O)) and hexamethyldisilazane (HMDS) which were vaporized and allowed to react with the paper sheets via the gas phase. All routes yielded hydrophobic papers with static water contact angles far above 90° and indicated the formation of covalent bonds. The PCl and TFAA approach negatively impacted the mechanical and optical properties of the paper leading to a decrease in tensile strength and yellowing of the sheets. The HMDS modified papers did not exhibit any differences regarding relevant paper technological parameters (mechanical properties, optical properties, porosity) compared to the non-modified sheets. XPS studies revealed that the HMDS modified samples have a rather low silicon content, pointing at the formation of submonolayers of trimethylsilyl groups on the fiber surfaces in the paper network. This was further investigated by penetration dynamic analysis using ultrasonication, which revealed that the whole fiber network has been homogeneously modified with the silyl groups and not only the very outer surface as for the PCl and the TFAA modified papers. This procedure yields a possibility to study the influence of hydrophobicity on paper sheets and their network properties without changing structural and mechanical paper parameters.

Fabrication of hydrophobic and high-strength packaging films based on the esterification modification of galactomannan.

There is an increasing interest in substituting current packaging films with biologically-derived films without compromising mechanical properties and hydrophobicity. In this work, the esterified galactomannan (E-GM) films with good hydrophobicity, excellent oxygen barrier performance and high tensile mechanical strength were synthesized using anhydride esterification method prior to film formation. The hydrophobicity, mechanical properties, barrier properties, thermal stability and ultraviolet absorption of the prepared films were determined to fully investigate the features of galactomannan-based films. The results indicated that GM films can be successfully obtained by esterification. Compared to neat GM film, E-GM-1.5 film (acetic anhydride to GM of 1.5:1) achieved the highest degree of esterification (0.05), hydrophobicity (107°) and mechanical strength (92.0 MPa). In addition, the esterified GM films had lower toxicity for macrophages cells. The prepared E-GM films may provide more opportunities for further advancement and applications in the development of food packaging from natural resources.

Probing Substrate/Catalyst Effects Using QSPR Analysis on Friedel-Crafts Acylation Reactions over Hierarchical BEA Zeolites.

Attempts to optimize heterogeneous catalysis often lack quantitative comparative analysis. The use of kinetic modelling leads to rate (k) and relative sorption equilibrium constants (K), which can be further rationalized using Quantitative Structure-Property Relationships (QSPR) based on Multiple Linear Regressions (MLR). Friedel-Crafts acylation using commercial and hierarchical BEA zeolites as heterogeneous catalysts, acetic anhydride as the acylating agent, and a set of seven substrates with different sizes and chemical functionalities were herein studied. Catalytic results were correlated with the physicochemical properties of substrates and catalysts. From this analysis, a robust set of equations was obtained allowing inferences about the dominant factors governing the processes. Not entirely surprising, the rate and sorption equilibrium constants were found to be explained in part by common factors but of opposite signs: higher and stronger adsorption forces increase reaction rates, but they also make the zeolite active sites less accessible to new reactant molecules. The most relevant parameters are related to the substrates' molecular size, which can be associated with different reaction steps, namely accessibility to micropores, diffusion capacity, and polarizability of molecules. The relatively large set of substrates used here reinforces previous findings and brings further insights into the factors that hamper/speed up Friedel-Crafts reactions in heterogeneous media.

Efficient Synthesis of α-Branched Purine-Based Acyclic Nucleosides: Scopes and Limitations of the Method.

An efficient route to acylated acyclic nucleosides containing a branched hemiaminal ether moiety is reported via three-component alkylation of N-heterocycle (purine nucleobase) with acetal (cyclic or acyclic, variously branched) and anhydride (preferentially acetic anhydride). The procedure employs cheap and easily available acetals, acetic anhydride, and trimethylsilyl trifluoromethanesulfonate (TMSOTf). The multi-component reaction is carried out in acetonitrile at room temperature for 15 min and provides moderate to high yields (up to 88%) of diverse acyclonucleosides branched at the aliphatic side chain. The procedure exhibits a broad substrate scope of N-heterocycles and acetals, and, in the case of purine derivatives, also excellent regioselectivity, giving almost exclusively N-9 isomers.

Study and optimization of parameters affecting the acetylation process of lignin sulfonate biopolymer.

Here, firstly lignin sulfonate was produced from sulfite liquor provided by Mazandaran wood & paper industries (Chookam). Then, the role of effective parameters including reaction temperature, duration time, and amounts of pyridine and acetic anhydride respectively as the catalyst and the esterification agent on the acetylation rate of lignin sulfonate were studied and the process parameters were optimized through multiple experiments. In this investigation, using 1 g lignin sulfonate, the effect of various levels of temperature (ranged from room temperature to 140 °C), reaction time (12-72 h), pyridine volume (0-30 mL), and acetic anhydride volume (5-30 mL) were evaluated. Based on the results of several esterification processes, the optimal values of temperature and reaction time were obtained to be 100 °C and 48 h, respectively, and the optimal volumes of acetic anhydride and pyridine were 20 mL (with equal amounts). Besides, the characterization tests of lignin sulfonate and acetylated lignin sulfonate were performed using FT-IR and NMR techniques. Also in this paper, the morphology and crystallinity/amorphicity of lignin sulfonate and acetylated lignin sulfonate were examined using SEM images and XRD patterns.

Chitin degradation enzyme-responsive system for controlled release of fibroblast growth factor-2.

Chitin is widely found in fungal cell walls and arthropod exoskeletons, and is used as a biomedical material. However, chitin is not water-soluble, restricting its use for controlled release materials. We found that water-soluble chitosan can be acetylated to produce a chitin equivalent, or chitin gel. Chitin gel, produced by mixing chitosan solution with acetic anhydride, can be degraded by lysozyme and fetal bovine serum, so could provide an ideal means for controlled release in biological systems. We tested a combination of chitin gel with a chitin binding domain (CBD) fusion protein as a controlled release system regulated by chitin degradation. A fusion protein consisting of fibroblast growth factor 2 (FGF-2) fused to CBD bound the chitin gel, and was released time-dependently rather than as an initial burst during lysozyme degradation, suggesting that this system could provide a means for controlled drug release in biological systems. Contrastingly, the trinitrophenyl residue (TNP-X) covalently bound to chitin gel, and was released by lysozyme degradation with an initial burst. If release of CBD-FGF-2 were simply dependent on lysozyme degradation of the chitin gel, the release behavior of CBD-FGF-2 would be similar to that of TNP-X, with an initial burst. Therefore, we propose that CBD-FGF-2 repeats the cycle of binding, release, and re-binding to the chitin gel during degradation. This system allows for a time-dependent, controlled release of CBD-FGF-2 without an initial burst.

An SDS-PAGE based proteomic approach for N-terminome profiling.

Initial sample quantity, solubilization, separation, and visualization of proteins or their proteolytically altered products are some of the challenges of the currently available solution-based N-termini enrichment methods. We therefore took advantage of the conventional SDS-PAGE system and attempted to address these challenges by proposing a simple yet reproducible, negative selection N-termini enrichment strategy coupled with mass spectrometry based protein identification. It includes in-gel protein level labeling of primary amines using d6-acetic anhydride and post-digestion negative selection of labeled N-terminal peptide(s) using N-hydroxysuccinimide activated agarose beads. We demonstrated the superiority of our method by successfully enriching protein N-termini from as low as 10 ng of bovine serum albumin. The method was validated for its applicability to a complex mixture of proteins by selectively enriching neo-N-termini generated by a site specific protease Glu-C. Its effectiveness for deep N-terminome profiling was also shown using human cell lysate. In addition, a system-wide label-free quantitative proteomic analysis of N-termini in MMP2-perturbed HCT8 cell secretome revealed substrates of several extra- and intra-cellular proteases, which are part of cell growth and proliferation and degradation pathways. In brief, the proposed method demonstrates an effective strategy not only to detect N-termini from a single protein but also for the deep and quantitative analysis of N-terminome from a limited sample amount.

Selective acetolysis of primary benzyl groups in carbohydrate derivatives under the mild reaction condition.

Selective acetolysis of the primary benzyloxy groups in a wide variety of carbohydrate derivatives was achieved in excellent yield using acetic anhydride and perchloric acid supported over silica (HClO4-SiO2) as a solid acid catalyst in a fast reaction condition without using any organic solvent. The reaction condition is significantly rapid and can be scaled up for its use in the multi-step oligosaccharide synthesis.

Amphipathic chitosans improve the physicochemical properties of siRNA-chitosan nanoparticles at physiological conditions.

Chitosan has received a lot of attention as a carrier for small interfering RNA (siRNA), due to its capacity for complexation and intracellular release of these molecules. However, one of its limitations is its insolubility at neutral pH and the tendency towards aggregation of its nanoparticles in isotonic ionic strength. In this study, a series of amphipathic chitosans were synthesized by varying the degree of acetylation (DA) from ˜2 to ˜30 mol% and the degree of substitution (DS) from 5 to 25%. by tertiary amino groups (DEAE) The results showed that the adjustment of these parameters decreases the interparticle interactions mediated by hydrogen bonding to obtain nanoparticles with improved colloidal stability. siRNA-containing nanoparticles of 100 to 150 nm with low polydispersities (0.15-0.2) and slightly positive zeta potentials (˜+ 5 mV) were resistant to aggregation at pH 7.4 and ionic strength of 150 mM. This resistance to aggregation is provided by changes on the nanoparticle surface and highlights the importance of more organized self-assembly in providing colloidal stability at physiological conditions. Additionally, the PEGylation of the most promising vectors conferred favorable physicochemical properties to nanoparticles. The chitosans and their nanoparticles exhibited low toxicity and an efficient cell uptake, as probed by confocal microscopy of rhodamine labeled vectors. The results provide a new approach to overcome the limited stability of chitosan nanoparticles at physiological conditions and show the potential of these amphipathic chitosans as siRNA carriers.

Simultaneous derivatization and lighter-than-water air-assisted liquid-liquid microextraction using a homemade device for the extraction and preconcentration of some parabens in different samples.

Simultaneous derivatization and air-assisted liquid-liquid microextraction using an organic that is solvent lighter than water has been developed for the extraction of some parabens in different samples with the aid of a newly designed device for collecting the extractant. For this purpose, the sample solution is transferred into a glass test tube and a few microliters of acetic anhydride (as a derivatization agent) and p-xylene (as an extraction solvent) are added to the solution. After performing the procedure, the homemade device consists of an inverse funnel with a capillary tube placed into the tube. In this step, the collected extraction solvent and a part of the aqueous solution are transferred into the device and the organic phase indwells in the capillary tube of the device. Under the optimal conditions, limits of detection and quantification for the analytes were obtained in the ranges of 0.90-2.7 and 3.0-6.1 ng/mL, respectively. The enrichment and enhancement factors were in the ranges of 370-430 and 489-660, respectively. The method precision, expressed as the relative standard deviation, was within the range of 4-6% (n = 6) and 4-9% (n = 4) for intra- and interday precisions, respectively. The proposed method was successfully used for the determination of methyl-, ethyl-, and propyl parabens in cosmetic, hygiene and food samples, and personal care products.

Conformational Aspects of the O-acetylation of C-tetra(phenyl)calixpyrogallol[4]arene.

Reaction between pyrogallol and benzaldehyde results in a conformational mixture of C-tetra(phenyl)pyrogallol[4]arene (crown and chair). The conformer mixture was separated using crystallization procedures and the structures were determined using FTIR, ¹H-NMR, and 13C-NMR. O-acetylation of C-tetra(phenyl)pyrogallol[4]arene (chair) with acetic anhydride, in pyridine results in the formation of dodecaacetyl-tetra(phenyl)pyrogallol[4]arene. The structure was determined using ¹H-NMR and 13C-NMR finding that the product maintains the conformation of the starting conformer. On the other hand, the O-acetylation reaction of C-tetra(phenyl)pirogallol[4]arene (crown) under same conditions proceeded efficiently, and its structure was determined using ¹H-NMR and 13C-NMR. Dynamic ¹H-NMR of acetylated pyrogallolarene was studied by means of variable temperature in DMSO-d6 solution, and it revealed that two conformers are formed in the solution. Boat conformations for acetylated pyrogallolarene showed a slow interconversion at room temperature.

Synthesis of New Glycosylated Flavonoids with Inhibitory Activity on Cell Growth.

Natural flavonoids and xanthone glycosides display several biological activities, with the glycoside moiety playing an important role in the mechanism of action of these metabolites. Herein, to give further insights into the inhibitory activity on cell growth of these classes of compounds, the synthesis of four flavonoids (5, 6, 9, and 10) and one xanthone (7) containing one or more acetoglycoside moieties was carried out. Acetyl groups were introduced using acetic anhydride and microwave irradiation. The introduction of one or two acetoglycoside moieties in the framework of 3,7-dihydroxyflavone (4) was performed using two synthetic methods: the Michael reaction and the Koenigs-Knorr reaction. The in vitro cell growth inhibitory activity of compounds 5, 6, 7, 9, and 10 was investigated in six human tumor cell lines: A375-C5 (malignant melanoma IL-1 insensitive), MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), U251 (glioblastoma astrocytoma), U373 (glioblastoma astrocytoma), and U87MG (glioblastoma astrocytoma). The new flavonoid 3-hydroxy-7-(2,3,4,6-tetra-O-acetyl-ß-glucopyranosyl) flavone (10) was the most potent compound in all tumor cell lines tested, with GI50 values < 8 µM and a notable degree of selectivity for cancer cells.

Pushing the limits: Quantification of chromophores in real-world paper samples by GC-ECD and EI-GC-MS.

Widening the methodology of chromophore analysis in pulp and paper science, a sensitive gas-chromatographic approach with electron-capture detection is presented and applied to model samples and real-world historic paper material. Trifluoroacetic anhydride was used for derivatization of the chromophore target compounds. The derivative formation was confirmed by NMR and accurate mass analysis. The method successfully detects and quantifies hydroxyquinones which are key chromophores in cellulosic matrices. The analytical figures of merit appeared to be in an acceptable range with an LOD down to approx. 60ng/g for each key chromophore, which allows for their successful detection in historic sample material.

Influence of amylose content and oxidation level of potato starch on acetylation, granule structure and radicals' formation.

This study was aimed at determining the effect of the amylose content of starch and oxidation level of potato starch on the structure of starch granules, and susceptibility to chemical modification (acetylation) and subsequent generation of radicals. Potato starch and waxy potato starch were oxidised with sodium hypochlorite applied in doses corresponding to 10, 20, and 30gCl/kg starch, and then acetylated with acetic acid anhydride. The carboxyl, carbonyl, acetyl groups were determined in modified starches. Structural properties of starch granules were evaluated based on gelatinisation, crystallinity, specific surface, intrinsic viscosity, and microphotographs by SEM microscope. The electron paramagnetic resonance (EPR) measurements were carried out to establish starch susceptibility to radical creation upon chemical modification and UV radiation. The amount of formed radicals was treated as a measure of the starch structure stability. The higher amount of amylose and the highest level of oxidation led to strong starch structure destruction and consequently facilitated radical generation. Study results showed also that amylose content as well as the degree of starch oxidation modified consecutive acetylation process. The different effectiveness of the acetylation processes influenced the morphology and structure of starch granules.

Skeletal Diversity in Combinatorial Fashion: A New Format for the Castagnoli-Cushman Reaction.

A new format for the Castagnoli-Cushman reaction of structurally diverse dicarboxylic acids, amines, and aldehydes in the presence of acetic anhydride as dehydrating agent is described. The reaction is distinctly amenable to parallel format: the combinatorial array of 180 reactions delivered 157 products of >85% purity without chromatographic purification (of this number, 143 compounds had >94% purity). The new method offers a convenient preparation of the skeletally and peripherally diverse, lead- and druglike γ- and δ-lactam carboxylic acids with high diastereoselectivity in combinatorial fashion.

Quantification of piperazine in chicken and pig tissues by gas chromatography-electron ionization tandem mass spectrometry employing pre-column derivatization with acetic anhydride.

This paper describes a novel method that combines acetic anhydride derivatization with gas chromatography-electron ionization tandem mass spectrometry (GC-EI/MS/MS) for the sensitive and selective determination of piperazine in chicken and pig tissues. Samples were extracted using an accelerated solvent extraction (ASE) apparatus, purified by solid-phase extraction (SPE) and derivatized with acetic anhydride. This optimized method was validated according to the requirements defined by the European Union and the Food and Drug Administration. At the limit of quantification (LOQ) spiked levels of 50.0, 100.0, 500.0, 1000.0 and 2000.0µg/kg, the average recoveries of piperazine in chicken and pig tissues were 77.46-96.26%, with relative standard deviations (RSDs) of 1.55-6.64%. The intra-day RSDs were 1.39-5.92%, and the inter-day RSDs were 2.24-8.39%. The limits of detection (LODs) and the LOQs were 1.4-1.6µg/kg and 4.8-5.2µg/kg, respectively. The decision limits (CCα) were 102.02-105.17µg/kg, and the detection capabilities (CCß) were 104.03-109.09µg/kg. Finally, the new approach was verified for the quantitative determination of piperazine in 30 commercial chicken and pig tissues from local supermarkets.

Folate-Targeted Dendrimers Selectively Accumulate at Sites of Inflammation in Mouse Models of Ulcerative Colitis and Atherosclerosis.

Folate-receptor-positive activated macrophages are critical for the development and maintenance of many chronic inflammatory and autoimmune diseases. Previously, small-molecule folate-targeted conjugates were found to specifically bind to these activated macrophages in vitro and selectively accumulate at sites of inflammation in vivo. While these small-molecule conjugates have shown promise, the use of a folate-targeted, higher cargo capacity nanovehicle may prove superior in delivering imaging or therapeutic agents in vivo. This nanoparticle strategy has been demonstrated in oncology, where targeted dendrimers have shown superior delivery capabilities; however, little research has been pursued in the area of folate-targeted dendrimers for inflammation and autoimmune diseases. Therefore, we endeavored to create a folate-decorated dendrimer to explore its uptake in mouse models of ulcerative colitis and atherosclerosis. We demonstrate that our final poly(ethylene glycol)-coated, acetic-anhydride-capped, folate-targeted poly(amidoamine) dendrimer exhibits no discernible cytotoxicity in vitro, specifically binds to a folate-receptor-expressing macrophage cell line in vitro, and selectively accumulates in areas of inflammation in vivo.

A mild acetolysis procedure for the regioselective removal of isopropylidene in di-O-isopropylidene-protected pyranoside systems.

A mild acetolysis method for the regioselective removal of isopropylidene group from di-O-isopropylidene-protected hexopyranosides is reported. O-Isopropylidene-protected intermediates play an important role in carbohydrate chemistry, as they are often found in commercially available furanosyl and pyranosyl materials, and some of them contain more than one O-isopropylidene groups. Methods that permit regioselective removal of O-isopropylidene groups are extremely valuable, as the number of steps in the total synthesis of complex oligosaccharides could be significantly decreased. Here we report that trifluoroacetic acid (TFA)/acetic anhydride (Ac2O) can be used to regioselectively convert one of the two O-isopropylidene groups to vicinal di-O-acetates in the di-O-isopropylidene-protected galacto- and fructo-pyranosyl systems, and the reagent is compatible with some common functionalities such as sulfonate esters, bromide, azide etc.