Acylation of Amino Acids with Furancarboxylic Acid Chlorides

Acylation of aromatic amino acids with furancarboxylic acid chlorides effectively proceeds in water-acetone medium at pH 8-9. Aliphatic amino acids are acylated at higher pH values, but under these conditions hydrolysis of acid chlorides becomes the main process. Acylation of chlorohydrates of methyl esters of aliphatic amino acids proceeds smoothly in chloroform in the presence of triethylamine. Alkaline hydrolysis of obtained products leads to N-acylated amino acids containing furan heteroring in the acyl radical.     

ClonedBacillus subtilis alkaline protease (aprA) gene showing high level of keratinolytic activity

TheBacillus subtilis alkaline protease(aprA) gene was previously cloned on a pUBHO-derivative plasmid. High levels of expression and gene stability were demonstrated whenB. subtilis cells were grown on the laboratory medium 2XSG.B. subtilis cells harboring the multicopyaprA gene were grown on basal medium, supplemented with 1 % chicken feather as a source of energy, carbon, and nitrogen. Proteolytic and kera-tinolytic activities were monitored throughout the cultivation time. A high level of keratinolytic activity was obtained, and this indicates that alkaline protease is acting as a keratinase. Furthermore, considerable amounts of soluble proteins and free amino acids were obtained as a result of the enzymatic hydrolysis of feather. Biodegradation of feather waste using these cells represents an alternative way to improve the nutritional value of feather, since feather waste is currently utilized on a limited basis as a dietary protein supplement for animal feedstuffs. Moreover, the release of free amino acids from feather and the secreted keratinase enzyme would promote industries based on feather waste.     

Development of a derivatisation method for the analysis of aldehyde modified amino acid residues in proteins by Fourier transform mass spectrometry

A method was developed for the analysis of amino acids within bovine serum albumin (BSA) which had been modified by reaction with different enals. BSA was reacted with the aldehydes and the reaction products were stabilised by reaction with NaBH₄. The protein was then hydrolysed with 6N HCl and the hydrolysis products were analysed by liquid chromatography-mass spectrometry (LC-MS). The modified amino acids were derivatised with propylchloroformate. High resolution mass spectrometry carried out using an LTQ-Orbitrap instrument which was able to characterise a wide range of adducts. In addition double adducts were observed to be formed with 4-hydroxynonenal (HNE) and lysine or lysine + histidine. Qualitatively it was possible to consistently observe a pyridinium adduct formed between lysine and pentenal in human plasma from normal subjects.     

Synthesis of Optically Active,Ring-Substituted N-Benzyloxycarbonylphenylalanines via 2-Benzyloxycarbonylamino-2-arylalkylmalonates

Diethyl or dimethyl benzyloxycarbonylaminomalonate was reacted with ring substituted benzyl halides and the resulting arylalkyl derivatives ( 3 to 6 ) half-saponified to the DL -monoacid-monoesters ( 7 to 10 ). Decarboxylation by refluxing in dioxane afforded the N-benzyl oxycarbonyl-DL -amino acid esters ( 11 to 14 ), which were resolved into their optical antipodes by enzymic hydrolysis of the ester group with Subtilisin, type Carlsberg. Enzymic hydrolysis led to the N-benzyloxycarbonyl-L -amino acids ( 15 to 18 ) and to the corresponding D -amino acid esters. The latter were converted to the N-benzyloxycarbonyl-D -amino acids ( 19 and 20 ) by alkaline hydrolysis of the ester groups. These derivatives could be used directly for further peptide synthesis. The following compounds were prepared: N-benzyloxycarbonyl derivatives of p-methyl-L -phenylalanine ( 15 ), p-methyl-D -phenylalanine ( 19 ), p-fluoro-L -phenylalanine ( 16 ), m-fluoro-L -phenylalanine ( 17 ), m-fluoro-D -phenylalanine ( 20 ) and penta-fluoro-L -phenylalanine ( 18 ). The free amino acids were obtained by removal of the benzyloxycarbonyl group with HBr in acetic acid.     

木槿叶中混合氨基酸的提取工艺研究

以浓盐酸和乙醇溶液为提取剂,分别通过加热回流和超声波组织破碎法提取了木槿叶中的混合氨基酸,确定了氨基酸提取的最佳工艺条件。结果表明,用盐酸回流提取,盐酸与木槿干粉的液料比为25∶1,盐酸浓度6 mol/L,100℃下回流提取5 h,提取3次,氨基酸的提取效果最佳,提取率可达16.76%,产品收率可达8.6%。用80%乙醇溶液,室温下超声波提取30 min,提取3次,提取效果最好,提取率可达17.42%,产品收率可达8.93%。     

Determination of amino acids in selenium-enriched yeast by gas chromatography–mass spectrometry after microwave assisted hydrolysis

A simple, rapid microwave digestion procedure for protein hydrolysis preceding the determination of amino acids in yeast using gas chromatography–mass spectrometry (GC–MS) is described. Protein hydrolysis was performed in a focused microwave using 4 M methanesulfonic acid (MAS). Amino acids were derivatized with methyl chlorofomate (MCF) and extracted into chloroform prior to GC–MS analysis. The microwave parameters, including power, temperature and heating time, were optimized. It was found that temperature and heating time were the most influential factors. A total of 17 amino acids were determined in selenium-enriched yeast with use of standard addition calibration. Limits of detection and quantitation (LODs/LOQs) of the amino acids measured were in the sub-nmol range, suitable for monitoring of amino acids in yeast and other food products.     

The effect of hydrolysis time on amino acid analysis

Determining the amino acid content of a protein involves the hydrolysis of that protein, usually in acid, until the protein-bound amino acids are released and made available for detection. Both the variability in the ease of peptide bond cleavage and differences in the acid stability of certain amino acids can significantly affect determination of a protein's amino acid content. By using multiple hydrolysis intervals, a greater degree of accuracy can be obtained in amino acid analysis. Correction factors derived by linear extrapolation of serial hydrolysis data are currently used. Compartmental modeling of the simultaneous hydrolysis (yield) and degradation (decay) of amino acids by nonlinear multiple regression of serial hydrolysis data has also been validated and applied to determine the amino acid composition of various biological samples, including egg-white lysozyme, human milk protein, and hair. Implicit in the routine application of serial hydrolysis in amino acid analysis, however, is an understanding that correction factors, derived either linearly or through the more accurate nonlinear multiple regression approach, need to be determined for individual proteins rather than be applied uniformly across all protein types.     

Determination of amino acids by capillary electrophoresis-electrospray ionization-mass spectrometry: an evaluation of different protein hydrolysis procedures

In this work, a CE equipment, online hyphenated to an IT MS analyzer by a linear sheath liquid interface promoting ESI, was used to develop a method for quantitative determination of amino acids. Under appropriate conditions (BGE composition, 0.8% HCOOH, 20% CH3OH; sheath liquid composition, 0.8% HCOOH, 60% methanol; V ESI, +4.50 kV), analytical curves of all amino acids from 3 to 80 mg/L were recorded presenting acceptable linearity (r >0.99). LODs in the range of 16-172 micromol/L were obtained. BSA, a model protein, was submitted to different hydrolysis procedures (classical acid and basic, and catalyzed by the H+ form of a cation exchanger resin) and its amino acid profiles determined. In general, the resin-mediated hydrolysis yields were overall similar or better than those obtained by classical acid or basic hydrolysis. The resulting experimental-to-theoretical BSA concentration ratios served as correction factors for the quantitation of amino acids in Brazil nut resin generated hydrolysates.     

o-Nitrobenzoyl group as a new amino protecting group for peptide synthesis and the synthesis of some anthranilyl peptides

N (o-nitrobenzoyl)amino acids can be coupled with other amino acids using DCC and the resulting product on hydrogenation gives peptides, containing the anthranilyl group as —NH₂ end group. N (anthranilyl)amino acids or peptides can also be obtained by reaction of isatoic anhydride on amino acids or peptides. The anthranilyl end group is easily cleaved by metal (Cu⁺²) catalysed hydrolysis to give α-amino acid peptides and the insoluble copper(II) anthranilate.     

Chiral separation and determination of the optical purity of thiamphenicol-related chiral compound by capillary zone electrophoresis with cyclodextrins as chiral selectors

Summary One classical method for quantitation of amino acids in proteins is hydrolysis of the proteins and determination of the free amino acids. Although the drastic experimental conditions necessary for complete hydrolysis always cause degradation of some of the amino acids, if mild hydrolysis conditions are used, a mixture of amino acids and oligopeptides is obtained. If these conditions are adequately tuned, the oligopeptides are almost exclusively dipeptides. For this reason we have initiated a study to find a derivatizing agent suitable for the analysis of amino acids and dipeptides by an absolute method of quantitation already tested for amino acids. FMOC-Cl was found to be a suitable derivatizing agent for this purpose.     

Determination of the configuration of histrelin,and LHRH analog,by chiral capillary gas chromatography

Summary Summary The assigned chirality at each center of the synthetic nonapeptide histrelin (L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-N^im-benzyl-histidyl-L-leucyl-L-arginyl-L-proline-ethylamide) was verified using chiral gas chromatography. The procedure involved acid hydrolysis of histrelin to the constituent amino acids, derivatization as the N-pentafluoropropionyl/isopropyl esters and the analysis of the mixture using a commercially available 25m chiral capillary column (Chirasil-L-Val). There was no significant difference in the retention time of the amino acids obtained from the hydrolysate mixture when compared to the appropriate standards. Additionally, the hydrolysate was spiked with the D and L amino acids to prove the identity of closely eluting peaks. Luteinizing hormone-releasing hormone     

Complete separation of ephedrines by graphite-layer open-tubular (GLOT) column

Summary A reversed phase high performance liquid chromatographic (RP-HPLC) method for the analysis of amino acids in kelp, using precolumn derivatization, is described. Phenylisothiocyanate (PITC) was used as the reagent for derivatization. The kelp samples were prepared by microwave hydrolysis in only 5 min; seventeen PTC amino acids were separated after hydrolysis and derivatization within 12 min. The coefficients of variation were >1.94 and the correlation coefficients for concentration versus response were >0.999 for all derivatives. The ratio of branched amino acid (BAA) to aromatic amino acid (AAA) was also studied. The method has the advantage of shorter hydrolysis and analysis times with optimum separation. In addition, it gives high repeatability of retention times and peak areas for all the amino acids present.     

Electrochemical sensor for amino acids and albumin based on composites containing carbon nanotubes and copper microparticles

This work reports on the analytical performance of composites obtained by dispersing copper microparticles and multi-wall carbon nanotubes within a mineral oil binder (CNTPE-Cu) for the determination of amino acids and albumin. The strong complexing activity of amino acids towards copper makes possible an important improvement in the sensitivity for the determination of amino acids and albumin. This new electrode permits the highly sensitive amperometric detection of amino acids, even the non-electroactive ones, at very low potentials (0.000 V) and physiological pH (phosphate buffer solution pH 7.40). The response of the electrode is highly dependent on the amount of copper, demonstrating the crucial role of the metal in the analytical performance of the sensor. The best analytical performance is obtained for the electrode containing 6.0% (w/w) copper. The resulting sensor shows a fast response (7 s) and a sensitivity that depends on the nature of the amino acid. The electrode surface demonstrates an excellent resistance to surface fouling, with R.S.D. of 4% for the sensitivities of 10 successive calibration plots. Albumin is determined with CNTPE-Cu using a protocol based on the accumulation of the protein for 10 min at −0.100 V, followed by the square-wave voltammetric analysis. The quantification of albumin concentration in lyophilized control serum gives excellent agreement with the classical spectrophotometric methodology and with the value informed for the supplier.     

Analytical study of proteinaceous binding media in works of art by gas chromatography using alkyl chloroformates as derivatising agents

In this work, we present the results obtained in an analytical study of the different types of proteinaceous binding media most commonly used in paintings, using GC-FID as the technique of analysis and GC-MS as a confirmatory technique. The application of this methodology requires prior hydrolysis of the proteins in the binding media to obtain free amino acids and then volatile derivatives, in this case by reaction with chloroformates due to advantages of speed, safety and the aqueous medium in which the reaction occurs. The method proposed for the proteinaceous binding media study is to calculate the proportions of the different amino acids with respect to alanine. This method provided good characterisation of different binding media, such as pork gelatine, beef gelatine, albumin, egg white and casein. The proposed method is used for the identification of binding media (including mixtures of binders) present in real samples from paintings in the city of Valencia, Spain.     

Determination of Free and Total Underivatized Amino Acids Including L-Canavanine in Bitter Vetch Seeds Using Hydrophilic Interaction Liquid Chromatography

A new hydrophilic interaction liquid chromatography method coupled with diode-array detector was developed for the determination of 17 underivatized amino acids including L-canavanine in bitter vetch [Vicia ervilia (L.) Willd.] seeds. Amino acids were extracted as free as well as total extracts after acid hydrolysis, followed by chromatographic separation on a Zorbax Rx-SIL column with a mobile phase of acetonitrile/potassium phosphate buffer (12.5?mM; pH 3.0) using gradient elution and detection at 190?nm. The method is characterized by a wide linear range (0.01–200?µg/mL, r?>?0.9987), sufficient accuracy (relative error 86.3–109.1%), and suitable precision for the results (relative standard deviation <4.9% in the case of intra-day and <9.8% in the case of inter-day precision). The limits of detection and quantification for free amino acids ranged from 0.01 to 0.24?mg/g and 0.03 to 0.72?mg/g, respectively, whereas the total amino acids ranged from 0.02 to 0.47?mg/g and 0.07 to 1.43?mg/g, respectively. The mean recoveries of free and total amino acids in spiked samples exceeded 70.3% for most amino acids. The mean total content of free and total amino acids in bitter vetch seeds was 1.71 and 14.88?g/100?g seed, whereas the corresponding values for canavanine were 0.07 and 0.19?g/100?g seed, respectively.     

4:3-β-naphthapyrone-4-acetic acidN-hydroxysuccinimidyl ester as a fluorescent labeling reagent for amino acids and oligopeptides in high-performance liquid chromatography

Summary 4:3-β-Naphthapyrone-4-acetic acidN-hydroxysuccinimidyl ester (NPA-OSu) is a highly sensitive and moderately reactive derivatizing reagent with a naphthapyrone moiety as fluorophore and anN-hydroxysuccinimidyl active ester as reactive group toward amino compounds. It is readily prepared in two steps. The fluorescence properties of NPA-OSu and its hydrolysis product have been studied in detail, and the conditions for derivatization and separation of the NPA-OSu derivatives of some amino acids and oligopeptides have been investigated. Atλ _ex = 352 nm andλ _em = 422 nm the detection limits (signal-to-noise ratio=3) for amino acids and oligopeptides reached fmol levels, for injection of 20 μL; this sensitivity was comparable with that obtained by use of 7-(diethylamino)coumarin-3-carboxylic acid succinimidyl ester as derivatizing reagent in the analysis of amino acids by capillary electrophoresis with laser-induced-fluorescence detection.     

Amino acid composition analysis of minute amounts of cysteine-containing proteins using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-fluoro-7-nitro-2,1,3-benzoxadiazole in combination with HPLC

The simultaneous determination of amino acid composition including cysteine of egg albumin, a model protein containing a/s cysteine residue, is reported. All the thiol groups of the cysteine residue(s) of egg albumin were labelled with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, a fluorogenic reagent for thiol groups. The labeled egg albumin was hydrolyzed in 6N HCl at 110 degrees C for 24 h. The hydrolysate was lyophilized, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, a fluorogenic reagent for amines, and subjected to HPLC. 18 derivatized amino acids including double labelled cysteine were separated within 90 min on a Nucleosil ODS column (150 mm X 4.6 mm i.d.; 5 microns), and detected at 530 nm (ex. 470 nm) in a range from 90 fmol (aspartic acid) to 1.3 pmol (cysteine) (S/N = 3). Composition ratios of amino acids of egg albumin were similar to theoretical values except for methionine, which would be destroyed under the present acid hydrolysis condition. Analytical methods for cysteine residues are reviewed, and the availability of fluorogenic reagents having the benzofurazan structure is also discussed.     

Stereocontrolled Synthesis of syn‐β‐Hydroxy‐α‐Amino Acids by Direct Aldolization of Pseudoephenamine Glycinamide

β‐Hydroxy‐α‐amino acids figure prominently as chiral building blocks in chemical synthesis and serve as precursors to numerous important medicines. Reported herein is a method for the synthesis of β‐hydroxy‐α‐amino acid derivatives by aldolization of pseudoephenamine glycinamide, which can be prepared from pseudoephenamine in a one‐flask protocol. Enolization of (R,R)‐ or (S,S)‐pseudoephenamine glycinamide with lithium hexamethyldisilazide in the presence of LiCl followed by addition of an aldehyde or ketone substrate affords aldol addition products that are stereochemically homologous with L ‐ or D ‐threonine, respectively. These products, which are typically solids, can be obtained in stereoisomerically pure form in yields of 55–98 %, and are readily transformed into β‐hydroxy‐α‐amino acids by mild hydrolysis or into 2‐amino‐1,3‐diols by reduction with sodium borohydride. This new chemistry greatly facilitates the construction of novel antibiotics of several different classes.     

Synthesis of enantiomers of 6-nitro- and 6-amino-2-methyl-1,2,3,4-tetrahydroquinolines

Enantiomers of 2-methyl-6-nitro-1,2,3,4-tetrahydroquinoline have been obtained by kinetic resolution of racemic 2-methyl-1,2,3,4-tetrahydroquinoline in acylation with acyl chlorides of N-protected amino acids followed by regioselective nitration of the diastereoisomeric amides and acidic hydrolysis. The introduction of a trifluoroacetyl protecting group into the position 1 of the enantio pure nitro compound followed by the reduction led to (S)-6-amino-2-methyl-1-trifluoroacetyl-1,2,3,4-tetra-hydroquinoline in a high yield.     

Quantification of glycated N‐terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry

A novel method of amino acid analysis using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups) was applied to measure glycated amino acids in order to quantify glycated peptides and evaluate the degree of glycation of peptide. Amino and carboxyl groups of amino acids were derivatized with 1‐bromobutane so that the hydrophobicities and basicities of the amino acids, including glycated amino acids, were improved. These derivatized amino acids could be detected with high sensitivity using LC‐MS/MS. In this study, 1‐deoxyfructosyl‐VHLTPE and VHLTPE, which are N‐terminal peptides of the β‐chains of hemoglobin, were selected as target compounds. After reducing the peptide sample solution with sodium borohydride, the obtained peptides were hydrolyzed with hydrochloric acid. The released amino acids were then derivatized with 1‐bromobutane and analyzed with LC‐MS/MS. The derivatized amino acids, including glycated amino acids, could be separated using an octadecyl silylated silica column and good sharp peaks were detected. We show a confirmatory experiment that the proposed method can be applied to evaluate the degree of glycation of peptides, using mixtures of glycated and non‐glycated peptide. Copyright © 2015 John Wiley & Sons, Ltd.