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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.

Histone PTMs play a crucial role in regulating chromatin structure and function, with impact on gene expression. MS is nowadays widely applied to study histone PTMs systematically. Because histones are rich in arginine and lysine, classical shot-gun approaches based on trypsin digestion are typically not employed for histone modifications mapping. Instead, different protocols of chemical derivatization of lysines in combination with trypsin have been implemented to obtain "Arg-C like" digestion products that are more suitable for LC-MS/MS analysis. Although widespread, these strategies have been recently described to cause various side reactions that result in chemical modifications prone to be misinterpreted as native histone marks. These artefacts can also interfere with the quantification process, causing errors in histone PTMs profiling. The work of Paternoster V. et al. is a quantitative assessment of methyl-esterification and other side reactions occurring on histones after chemical derivatization of lysines with propionic anhydride [Proteomics 2016, 16, 2059-2063]. The authors estimate the effect of different solvents, incubation times, and pH on the extent of these side reactions. The results collected indicate that the replacement of methanol with isopropanol or ACN not only blocks methyl-esterification, but also significantly reduces other undesired unspecific reactions. Carefully titrating the pH after propionic anhydride addition is another way to keep methyl-esterification under control. Overall, the authors describe a set of experimental conditions that allow reducing the generation of various artefacts during histone propionylation.

Acid brings the oxygen: a general method for palladium-catalyzed C-H oxygenation has been developed for the facile synthesis of a wide range of functionalized phenols with readily available aryl ketones, benzoates, benzamides, acetanilides, and sulfonamides. A trifluoroacetic acid/trifluoroacetic acid anhydride solvent system serves as the oxygen source and is the critical factor for C-H activation.

The reaction mechanism for the biomimetic synthesis of tryptophan from indole and serine in the presence of Ac(2)O in AcOH was investigated. Although the time-course (1)H-NMR spectra of the reaction of 5-methoxyindole with N-acetylserine were measured in the presence of (CD(3)CO)(2)O in CD(3)CO(2)D, the reactive intermediate could not be detected. This reaction was conducted without 5-methoxyindole in order to elucidate the reactive intermediate, but the intermediate could not be isolated from the reaction mixture. Since the intermediate would be expected to have a very short life time, and therefore be very difficult to detect by conventional analytical methods, the structure of the intermediate was elucidated using a 2D-NMR technique, diffusion-ordered spectroscopy (DOSY). Two intermediates were detected and confirmed to be 2-methyl-4-methyleneoxazol-5(4H)-one and 2-methyl-4-hydroxymethyloxazol-5(4H)-one. The present results demonstrated that DOSY is a powerful tool for the detection of unstable intermediates.

The effects of succinylation and acetylation on some functional properties and the in vitro trypsin digestibility of kidney bean protein isolate (KPI) were investigated. The extent of succinylation or acetylation progressively increased from 0% to 96% to 97%, as the anhydride-to-protein ratio increased from 0 to 1 g/g. Polyacrylamide gel electrophoresis (PAGE) and zeta potential analyses indicated that acylation, especially succinylation, considerably increased the net charge and hydrodynamic radius of the proteins in KPI, especially vicilin. Acylation treatment at various anhydride-to-protein ratios (0.05 to 1 g/g) remarkably improved the protein solubility (PS) and emulsifying activity index (EAI) at neutral pH, but the improvement by succinylation was much better than that by acetylation. Succinylation resulted in a marked decrease in mechanical moduli of heat-induced gels of KPI, while the mechanical moduli were, on the contrary, increased by acetylation. Additionally, in vitro trypsin digestibility was improved by the acylation in an anhydride-type and level-dependent manner. The results suggest that the functional properties of KPI could be modulated by the chemical acylation treatment, using succinic or acetic anhydride at appropriate anhydride-to-protein ratios.

The synthesis of the tripeptide D-Phe-Pro-Arg with the nitrile group instead of the carboxylgroup is described. Initially, the corresponding peptide amide was synthesized by conventional methods in solution using Boc and Fmoc as the protecting group for D-Phe. The dehydration in order to create the nitrile moiety was achieved by treating the peptide amide with phosphorus oxichloride or trifluoroacetic anhydride. Best results were obtained by the use of phosphorus oxichloride in pyridine as the solvent in the presence of imidazole. After deprotection of the N-terminal amino acid the crude product was purified by chromatography on Butyl-Fractogel HW-40 (S). The purity of the final product was checked on a RP18 phase by hplc. The existence of the nitrile group was demonstrated by i.r. and 13C-n.m.r. spectra. The peptide nitrile exhibited a strong inhibition of thrombin compared to the tripeptide amide.

A novel method is described for the cyclization of peptides - or segments of polypeptides - which requires a free A′-terminal α-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal α-amino group with iodoacetic anhydride at pH 6. This acylation reaction has >90% specificity for peptide α-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al, Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications. However, peptides cyclized via Met slowly decompose at 37° with ring cleavage to yield a C-terminal lactone peptide in which the Met methylthio-group is effectively transferred to the JV-terminus. The reactions described here have several applications which take advantage of their unique ability to efficiently cyclize naturally-derived (as well as chemically synthesized) peptides in aqueous solution with little or no protection/deprotection chemistry.

This paper described a novel approach for the determination of bisphenol A by dispersive liquid-phase microextraction with in situ acetylation prior to GC-MS. In this derivatization/extraction method, 500 microL acetone (disperser solvent) containing 30.0 microL chlorobenzene (extraction solvent) and 30.0 microL acetic anhydride (derivatization reagent) was rapidly injected into 5.00 mL aqueous sample containing bisphenol A and K2CO3 (0.5% w/v). Within a few seconds the analyte was derivatized and extracted at the same time. After centrifugation, 1.0 microL of sedimented phase containing enriched analyte was determined by GC-MS. Some important parameters, such as type and volume of extraction and disperser solvent, volume of acetic anhydride, derivatization and extraction time, amount of K2CO3, and salt addition were studied and optimized. Under the optimum conditions, the LOD and the LOQ were 0.01, 0.1 microg/L, respectively. The experimental results indicated that there was linearity over the range 0.1-50 microg/L with coefficient of correlation 0.9997, and good reproducibility with RSD 3.8% (n = 5). The proposed method has been applied for the analysis of drinking water samples, and satisfactory results were achieved.

A method for quantitative proteomic analysis based on the selective isolation of multiply charged peptides (RH peptides) containing arginine and histidine residues is described. Two pools of proteins are digested in tandem with lysyl-endopeptidase and trypsin and the primary amino groups of proteolytic peptides are separately labeled with d3- and d0-acetic anhydride. This reaction has a dual purpose: (i) to allow the relative protein quantification in two different conditions and (ii) to restrict the positive charges of peptides to the presence of arginine and histidine. The N-acylated peptides are separated by cation-exchange chromatography into two groups, neutral and singly charged peptides (R + H ≤ 1) that are neither retained nor analyzed, whereas the multiply charged peptides (R + H>1) are retained into the column and can be eluted in batch or further fractionated using a saline gradient before LC-MS/MS analysis. In silico analysis revealed that the selective isolation of RH peptides considerably simplifies the complex mixture of peptides (three RH peptides/protein) and at the same time they represent 84% of the whole proteomes. The selectivity, and recovery of the method were evaluated with model proteins and with a complex mixture of proteins extracted from Vibrio cholerae.

gamma-Hydroxybutyrate (GHB) is a DEA Schedule I drug of abuse commonly spiked into beverages to incapacitate victims of sexual assault. GHB is a challenging drug for analysis by GC/MS because of its small size, charged nature, low volatility, and intramolecular esterification leading to gamma-butyrolactone (GBL). In this work an extractionless technique has been developed that allows for the use of an aqueous sample for direct derivatization. The technique uses a solution of trifluoroacetic anhydride (TFAA) and 2,2,3,3,4,4,4-heptafluoro-1-butanol (HFB) to derivatize the active hydrogens of GHB. The conversion of GBL into GHB can be forced under alkaline conditions by diluting the sample in 10 mM borate buffer, pH 12. GBL found in beverages intended for human consumption is treated as a Schedule I control substance analogue. Spikes of the two compounds into several beverage matrices gave quantitative recovery of GHB by GC/MS. The derivatization produces higher molecular mass products whose fragmentation pattern provides multiple peaks for confirmation and quantification. The concentration of GBL can also be indirectly determined by the method developed. Therefore, this extractionless technique is rapid, sensitive, and selective for the confirmation of the presence of GHB and GBL in commercial beverages.

Binding constants between the glycopeptides teicoplanin (Teic) and ristocetin (Rist) and their derivatives to D-Ala-D-Ala terminus peptides were determined by on-column receptor synthesis coupled to partial-filling affinity capillary electrophoresis (PFACE) or affinity capillary electrophoresis (ACE). In these techniques, the column is first partially filled with increasing concentrations of D-Ala-D-Ala terminus peptides. This is followed by plugs of buffer, antibiotic and two noninteracting standards, and acetic and/or succinic anhydride (and buffer in the case of ACE). The order of the reagent plugs containing the antibiotic and anhydride varies with the charge of the glycopeptide. Upon electrophoresis, the antibiotic reacts with the anhydride yielding a derivative of Teic or Rist. Continued electrophoresis results in the overlap of the derivatized antibiotic and the plug of D-Ala-D-Ala peptide. Analysis of the change in the relative migration time ratio (RMTR) of the new glycopeptide relative to the standards, as a function of the concentration of the D-Ala-D-Ala ligand yields a value for the binding constant K(b). The techniques described here can be used to assess how the derivatization of drugs alters their affinities for target molecules.

PDC-109, the major heparin-binding protein of bull seminal plasma, binds to sperm choline lipids at ejaculation and modulates capacitation mediated by heparin. Affinity chromatography on heparin-Sepharose showed that polydisperse, but not monomeric, PDC-109 displayed heparin-binding capability. We sought to characterise the surface topology of the quaternary structure-dependent heparin-binding region of PDC-109 by comparing the arginine- and lysine-selective chemical modification patterns of the free and the heparin-bound protein. A combination of reversed-phase peptide mapping of endoproteinase Lys-C-digested PDC-109 derivatives and mass spectrometry was employed to identify modified and heparin-protected residues. PDC-109 contains two tandemly arranged fibronectin type II domains (a, Cys24-Cys61; b, Cys69-Cys109). The results show that six basic residues (Lys34, Arg57, Lys59, Arg64, Lys68, and Arg104) were shielded from reaction with acetic anhydride and 1,2-cyclohexanedione in heparin-bound PDC-109 oligomers. In the 1H-NMR solution structures of single fibronectin type II domains, residues topologically equivalent to PDC-109 Arg57 (Arg104) and Lys59 lay around β-strand D on the same face of the domain. In full-length PDC-109, Arg64 and Lys68 are both located in the intervening polypeptide between domains a and b. Our data suggest possible quaternary structure arrangements of PDC-109 molecules to form a heparin-binding oligomer.

To investigate the function of tRNAs or any other aminoacylable RNAs in vivo, it is important to be able to estimate the amounts and species of aminoacylated RNAs in living cells. We have developed a method of analyzing amino acids attached to particular tRNAs obtained from cells. After the ester bond between the amino acid and the 3′-adenosine moiety of a specific aminoacyl-tRNA is stabilized by acetylation of the amino acid with [14C]acetic anhydride, the aminoacyl-tRNA can be fished out with a solid-phase-attached DNA probe. The 14C-labeled acetylamino acid is then released from the thus purified acetyl-aminoacyl-tRNAs by alkaline treatment and detected by TLC analysis.

Modification (acetylation) of 2'-OH groups of mRNA ribose residues does not result in a loss of their template activity in a cell-free translation system from wheat embryos and in some cases activates inactive mRNAs. Modification of 70–75% of 2'-OH groups makes mRNA stable to the effect of pancreatic ribonuclease.

This paper reports the nature of abnormally expressed Forssman (F) antigen in the lymph node cells of MRL/MpJ-lpr/lpr, autoimmune mice, and also reports its autoantibody in sera. By acetylation study of the F antigen with [14C]acetic anhydride, we concluded that the F antigen was not a glycolipid but a glycoprotein. Several bands of F-active glycoproteins were identified on a nitrocellulose sheet after purification by an anti-F antibody affinity column. Hemolysis of SRBC by some sera from MRL/MpJ/lpr/lpr was inhibited by purified F glycoprotein and also by F glycolipid. The antibody in the serum, however, seemed to be more specific for F glycoproteins than F glycolipid, but the opposite was the case for rabbit anti-F glycolipid antibody. No significant difference of the SRBC hemolysis levels was observed between the sera from MRL/MpJ-lpr/lpr and its congenic MRL/MpJ-+/+ mice.

Re(2)O(7), which is known primarily as a strong oxidant, was found to be a highly selective Lewis acid catalyst that affects the heteroacylative dimerization of THF at room temperature. This multicomponent reaction, which involves THF, trifluoroacetic anhydride (TFAA), and a carboxylic acid, produces a nonsymmetrical diester, RCO(2)(CH(2))(4)O(CH(2))(4)OCOCF(3), in high yields. The reaction is quite general with respect to the carboxylic acid but is highly selective for unsubstituted THF in preference to other cyclic ethers. It is also highly selective for TFAA in preference to other anhydrides. Isotope labeling experiments indicate that two of the five oxygen atoms in the product originate from THF; one comes from rhenium oxide, and the two carbonyl oxygens originate from the carboxylic acid and from TFAA. The catalytic cycle, which is proposed on the basis of these experiments, involves a multistep sequence of nucleophilic attacks, starting with an attack of a rhenium oxo ligand on a coordinated THF, then attack of the resultant alkoxide ligand on a second coordinated THF, nucleophilic addition of the resultant alkoxide ligand to the coordinated carboxylic acid (an intramolecular metal-oxygen bond metathesis), and, finally, electrophilic cleavage of the other coordinated alkoxide by TFAA to produce the nonsymmetrical diester. This synthetically useful reaction highlights the unique, frequently avoided Lewis acidity of transition-metal oxides.

A Zn-salen-modified cavitand templates the catalytic formation of acetylcholine from choline and acetic anhydride.

Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine-reactive agents tested, acetic anhydride, N-acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3–9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.

Solvent-free per-O-acetylation of hexoses with a stoichiometric amount of acetic anhydride employing 0.03 mol % Cu(OTf)2 proceeded in high yields (90-99%) at room temperature to give exclusively pyranosyl products as an anomeric mixture, the alpha/beta ratio of which was dependent on the temperature and amount of catalyst used. Sequential anomeric substitution with p-thiocresol in the presence of BF3.etherate gave the thioglycosides, isolated exclusively or predominantly as one anomer in 66-75% yields.

A nucleophile-catalyzed asymmetric intermolecular C-acylation of silyl ketene acetals by anhydrides has been developed, furnishing quaternary stereocenters with high enantioselectivity. Mechanistic studies support the hypothesis that the reaction involves activation both of the silyl ketene acetal (generation of an enolate) and of the anhydride (formation of an acylpyridinium ion).

A procedure for the determination of the proportions of diacyl, alkenylacyl and alkylacyl subclasses of glycerophospholipids was developed. The procedure involves: (1) acid methanolysis of the phospholipid followed by Bligh/Dyer extraction of fatty acid methyl esters (FAME) derived from acyl chain types, dimethylacetals (DMA) derived from alkenyl ether chain types, and lysoalkyl phosphatidic acids (lysoalkyl-PA) derived from alkyl ether chain types; and (2) subsequent acetolysis to convert the lysoalkyl-PA to monoalkyl glycerol diacetates (MAGD). GLC analysis and quantitation (using internal standard, 21:0 FAME) of FAME, DMA and MAGD allowed calculation of the proportions of the three molecular subclasses. The methanolysis/acetolysis procedure gave an overall mean phospholipid recovery of 95 +/- 3%. Analysis of the major phospholipids in four separate preparations of fresh resting human platelets by this procedure showed the following range of molecular subclasses: phosphatidylcholine (PC), 86-92 mol % diacyl, 6-10 mol % alkylacyl and 2-3 mol % alkenylacyl; and phosphatidylethanoline (PE), 39-60 mol % diacyl, 5-8 mol % alkylacyl and 34-55 mol % alkenylacyl. The results of these subclass analyses were in general agreement with those reported in the literature.

Isolated glycosaminoglycans (GAGs) were quantified biochemically in the cerebella of 6-day-old rats. 14C-Labeled hyaluronic acid (HA) and chondroitin-4-sulfate (C-4-S), added prior to isolation of GAGs from tissue, served as internal standards to allow correction for unknown losses during the purification procedure and exact quantification of GAGs in the intact tissue. Three main constituents--HA, chondroitin sulfate (CS), and heparan sulfate (HS)--were found at concentrations of 1.82, 1.52, and 0.76 micrograms/mg protein amounting to 44%, 37%, and 19% of the total GAG fraction, respectively. Incorporation of [3H]glucosamine precursor into GAGs was higher for HS (56% of incorporated precursor) and lower for HA (29%) and CS (15%). The specific activities of individual GAGs were 64.7 nCi/micrograms for HS, 14.2 for HA, and 8.3 for CS.

The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenyglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation. Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imimpramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N-acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non-active-site tyrosine residues.

The applicability of the Finnigan MAT Ion Trap Detector (ITD) mass spectrometer for structure determination in some selected fatty acids and their derivatives has been investigated. Isopropylidene derivatives of novel glyceryl ethers isolated from cod flesh and of phenolic acetates are included to indicate the potential for diverse structures and to clarify the protonation of ions. The ITD is a simple and unsophisticated gas liquid chromatograph-mass spectrometer, but the spectra obtained are in most respects comparable to those from more conventional electron impact mass spectrometers. However, due to the comparatively high background pressure (approximately 10(-3) torr) in the ionization chamber of the ITD, there is a tendency for both neutral and ionized molecules to acquire protons from other molecules or fragments through collision. In many cases, the molecular ion was observed as the protonated molecular ion (M + 1), as in chemical ionization mass spectrometry. These interactions can be minimized if the sample load is decreased. Phenolic acetates exhibit not only protonation of the molecular ion, but also protonation of stable fragmented neutral molecules or ions.