Koshikamide B, a cytotoxic peptide lactone from a marine sponge Theonella sp

Koshikamide B (1) has been isolated from two separate collections of the marine sponge Theonella sp. as the major cytotoxic constituent. Koshikamide B is a 17-residue peptide lactone composed of six proteinogenic amino acids, two D-isomers of proteinogenic amino acids, seven N-methylated amino acids, and two unusual amino acid residues. The unusual amino acids are N(delta)-carbamoylasparagine and 2-(3-amino-2-hydroxy-5-oxopyrrolidin-2-yl)propionic acid (AHPP); the former is first found as the constituent of peptides, whereas the latter is a new amino acid residue. The N-terminus of koshikamide B is blocked by a methoxyacetyl group. The structure of koshikamide B (1) has been determined by interpretation of spectral data and analysis of chemical degradation products. Koshikamide B (1) exhibits cytotoxicity against P388 murine leukemia cells and the human colon tumor (HCT-116) cell line with an IC50 value of 0.45 and 7.5 microg/mL, respectively.     

A simple microscale method for determining the relative stereochemistry of statine units

A simple method to determine the relative stereochemistry of statine amino acids (gamma-amino-beta-hydroxyacids) by using (1)H NMR spectroscopy is described. Configurational assignment of statine units within complex natural products is possible without degradation or derivatization as the syn and anti diastereomers can be distinguished by using a combination of chemical shift and coupling constant information derived from the alpha-methylene ABX system. Seventy-three examples are provided, demonstrating the scope and limitations of the methodology. These examples range in complexity from simple statine units to cyclic depsipeptides, such as tamandarin B.     

Kulokekahilide-1, a cytotoxic depsipeptide from the cephalaspidean mollusk Philinopsis speciosa

The cytotoxic depsipeptide kulokekahilide-1, which contains two unusual amino acids, 4-phenylvaline and 3-amino-2-methylhexanoic acid, was isolated from the cephalaspidean mollusk Philinopsis speciosa. Structure elucidation of kulokekahilide-1 was carried out by spectroscopic analysis and chemical degradation. The absolute stereochemistry was determined by Marfey analysis for amino acids and chiral HPLC analysis for hydroxy acids. All four stereoisomers of 4-phenylvaline and 3-amino-2-methylhexanoic acid, which were necessary for Marfey analysis, were synthesized by use of the Heck reaction and Evans's method, respectively. Kulokekahilide-1 showed cytotoxicity against P388 murine leukemia cells with an IC(50) value of 2.1 microg/mL.     

Review: derivatization in mass spectrometry-6. Formation of mixed derivatives of polyfunctional compounds

The review describes chemical transformations of multifunctional compounds (amino acids and peptides, amino alcohols, amino thiols, hydroxy acids, oxo acids, oxo alcohols, compounds containing simultaneously three or more different groups etc.) by using step-wise or one-step modification or protection of functional groups. Some chemical aspects of mixed derivatization performed for improving the physical-chemical properties and mass spectral characteristics are discussed. Application of mixed derivatization to qualitative and quantitative analysis of various multifunctional compounds mainly in biological fluids and other matrices by gas chromatography/mass spectrometry in electron ionization, chemical ionization, negative-ion chemical ionization and selected ion monitoring modes is considered.     

Homocyclic o-dicarboxaldehydes: Derivatization reagents for sensitive analysis of amino acids and related compounds by capillary and microchip electrophoresis

Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on-capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o-dicarboxaldehyde compounds, namely o-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet-visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed.     

Gas-liquid chromatographic analysis of protein amino acids as N-isobutyloxycarbonylamino acid methyl esters.

A practical method for the quantitative determination of protein amino acids by gas-liquid chromatography (GLC) is described. All of the common protein amino acids except arginine can be readily converted into their N-isobutyloxycarbonyl (N-isoBOC) methyl ester derivatives by a simple procedure involving isobutyloxycarbonylation with isobutyl chloroformate in aqueous medium, followed by methylation with diazomethane. Arginine was converted into N-isoBOC ornithine methyl ester by treatment with arginase, followed by the above derivatization procedure. The resulting N-isoBOC methyl esters of the amino acids have good GLC properties. Complete resolution of the derivatives of 20 protein amino acids was achieved by using a dual-column system consisting of a 0.65% Poly-A-101A column and a 0.70% FFAP-Poly-A-101A (1:1, w/w) column. The reproducibility of response was found to be good for derivatives carried through the entire chemical and chromatographic procedure. The calibration graphs were linear and showed no statistical bias. The results of recovery experiments with synthetic mixtures containing known amounts of the amino acids were satisfactory, the recoveries ranging from 94.3 to 106.2%.     

Determination of the enantiomeric purity of synthetic peptides by gas chromatography-mass spectrometry.

Hydrolysis using 2H-labelled HCl and H2O, derivatization of free amino acids as N.O.S-trifluoroacetyl isobutyl esters, separation by gas chromatography on a chiral stationary phase and detection by mass spectrometry in selected-ion monitoring mode have been used in order to determine the enantiomeric purity of several synthetic peptides. Chromatographic separation has been optimized for proline, whose two enantiomers are difficult to resolve under standard conditions. Electron impact and methane chemical ionization mass spectra and chromatographic resolution of unnatural amino acids, such as 3-(1-naphthyl)-alanine and p-chlorophenylalanine, are reported. For both natural and unnatural amino acids selected-ion monitoring of the different fragmentation peaks has been carried out. The results are interpreted from the point of view of whether or not the fragments contain a hydrogen atom on the alpha-carbon, and a comparison between electron impact and methane chemical ionization has been carried out. The main advantage of the latter method is that a quasimolecular ion can be observed for all the amino acids studied.     

Studies of Australian Ascidians. 5. Virenamides A-C, New Cytotoxic Linear Peptides from the Colonial Didemnid Ascidian Diplosoma virens

Three new linear cytotoxic tripeptides, virenamides A-C have been isolated from extracts of the Didemnid ascidian Diplosoma virens collected on the Great Barrier Reef, Australia. Their structures were deduced from 1D and 2D NMR spectral data and confirmed by HPLC analysis of the constituent amino acids after hydrolysis of the peptides and derivatization with 1-fluoro-2,4-dinitrophen-5-yl-L-alanine amide using Marfey's procedure.     

Simultaneous analysis of carbohydrates,polyols and amines in urine samples using chemical ionization gas chromatography with tandem mass spectrometry

A simple method for the simultaneous derivatization of carbohydrates, polyols, amines and amino acids using hexamethyldisilazane and N,O‐bis(trimethylsilyl)trifluoroacetamide was developed. This method allows the direct derivatization of urine samples without sample pretreatment before derivatization. The method was successfully used for analysis of the selected metabolites in urine samples of healthy individuals and neonates suffering from galactosemia. The limits of detection by positive chemical ionization gas chromatography with tandem mass spectrometry analysis were in the range of 1.0 mgL^‐1 for mannitol to 4.7 mg/L for glucose.     

Leucamide A: a new cytotoxic heptapeptide from the Australian sponge Leucetta microraphis

Leucamide A (1), a bioactive cyclic heptapeptide containing a unique mixed 4,2-bisheterocycle tandem pair consisting of a methyloxazole and thiazole subunit was isolated together with the known compound BRS1 (2), from the dichloromethane extract of the Australian marine sponge Leucetta microraphis. The planar structure of leucamide A (1) was elucidated by employing spectroscopic techniques (NMR, MS, UV, and IR). Its absolute stereochemistry was established by chemical degradation, derivatization, and chiral GC[bond]MS analysis. A conformational analysis of 1 was made using MMFF. Leucamide A (1) was found to be moderately cytotoxic toward several tumor cell lines.     

New triazine spectroscopic reagent for the separation of DL-amino acids by micellar electrokinetic chromatography

An approach to the chiral separation of racemic mixtures of amino acids by means of micellar electrokinetic chromatography after derivatization with a new triazine spectroscopic reagent, 3-(4,6-dichloro-1,3,5-triazinylamino)-7-dimethylamino-2-methylphenazine (DTDP), has been evaluated. It was found that the derivatives of the aliphatic amino acids such as serine, valine and arginine, could produce a strong UV absorption at 282 nm, whose apparent molar absorptivities are of 10(-4) M(-1) cm(-1), and thus the concentration of the amino acids down to 3 x 10(-7) M can still give a detectable signal (S/N = 3). Beta-Cyclodextrin (beta-CD) added to the buffer system was used as a chiral selector, and separation conditions were optimized. The presence of an organic modifier (2-propanol) was also a prerequisite for the chiral separation. The best results for the chiral separation of DTDP-amino acids were achieved in a mixed sodium dodecylsulfate-beta-CD-borate-2-propanol medium at pH 9.0. Compared to some of the commonly used derivatization methods, the present one offers a relatively stable derivative and strong UV absorption for the spectroscopically inert amino acids, thus enabling amino acids to be separated and detected by CE even with a simpler UV detector.     

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.     

Gas chromatographic separation of protein amino acids in four minutes

A method is presented for analysis of protein amino acids in five minutes, including derivatization. The chemical treatment involves reaction with ethyl chloroformate: if the composition of the reaction medium is optimum this reagent esterifies carboxylic groups simultaneously with derivatization of other reactive groups and the reaction is complete in seconds. The moderately polar silicone OV-1701 was found to be a convenient liquid phase for separation of the derivatives: a five meter capillary column of internal diameter suitable for routine use was able to separate the amino acids in four minutes. Derivatization of arginine was incomplete and it failed to elute from the column.     

A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.     

Method of intracellular naphthalene-2,3-dicarboxaldehyde derivatization for analysis of amino acids in a single erythrocyte by capillary zone electrophoresis with electrochemical detection

A novel method of intracellular derivatization was developed. In this method, the derivatization reagents [naphthalene-2,3-dicarboxaldehyde (NDA) and CN-] were introduced into living cells by electroporation for the derivatization reaction. After completion of derivatization reaction in cells, a single cell was drawn into the capillary tip by electroosmotic flow. Then the lysing solution was introduced into the capillary by diffusion. Once the individual cell was lysed, the derivatized amino acids in the individual cell were separated by capillary zone electrophoresis and detected by end-column amperometric detection at the outlet of the capillary. This method of intracellular NDA derivatization confined the analytes and the derivatization reagents to the volume of a single cell expanded. For an 8-microm erythrocyte, the contents were diluted by a factor of only ca. 1.6. The method was used to determination of amino acids in single erythrocytes. Six amino acids were identified and quantified.     

Amino acid quantitation in aqueous matrices via trap and release membrane introduction mass spectrometry: homocysteine in human plasma

Trap and release membrane introduction mass spectrometry (T&R-MIMS) using a removable direct insertion membrane probe (DIMP) is employed to determine the total homocysteine concentration (tHcy) directly from human plasma after derivatization with ethyl chloroformate. The method uses no chromatographic separation, is linear, reproducible, and displays limit of quantitation (2 pM) sufficiently below the threshold concentration of tHcy in plasma. It also combines chemical, membrane, and mass spectrometric discrimination, and can be used to determine selected amino acids in human plasma simultaneously. After derivatization with ethyl chloroformate, many amino acids in aqueous solution are observed to be efficiently detected; hence T&R-MIMS is promising as a simple and sensitive technique for simultaneous quantitation of selected amino acids in plasma and urine, and in other aqueous matrices.     

Electrophoresis PDMS/glass chips with continuous on-chip derivatization and analysis of amino acids using naphthalene-2,3-dicarboxaldehyde as fluorogenic agent

In this work, we developed a PDMS electrophoresis device able to carry out on-chip derivatization and quantification of amino acids (AAs) using naphthalene-2,3-dicarboxaldehyde (NDA) as a fluorogenic agent. A chemical modification of the PDMS surface was found compulsory to achieve the derivatization of AAs with NDA and a limit of detection (LOD) of 40 nM was reached for glycine. Finally, we suggested the applicability of this microdevice for the analysis of real biological samples such as a rat hippocampus microdialysate.     

Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser-induced fluorescence detection

A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 microm erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser-induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8-32 amol/single cell.     

CE methods for the determination of non-protein amino acids in foods

In addition to the 20 amino acids universally distributed as protein constituents in living organisms, there are other amino acids of non-protein origin that can be found in foods. The determination of these non-protein amino acids is interesting since they can be indicative of the quality and safety of foods. This work presents for the first time an updated and comprehensive review devoted to show the possibilities of capillary electrophoresis for the determination of non-protein amino acids in food samples. The results reported have been classified according to the chemical structure of the non-protein amino acid studied. Separation conditions as well as detection systems used have been detailed since most of these amino acidic compounds do not possess chromophore groups detectable by conventional UV-Vis detection, being in this case necessary a previous derivatization step. Finally, the application of microchip electrophoresis to the determination of non-protein amino acids in foodstuffs is also included in this review.     

Simultaneous analysis of biologically active aminoalkanephosphonic acids

A new approach for simultaneous analysis of biologically active aminoalkanephosphonic acids, namely glyphosate, phosphonoglycine, phosphonosarcosine, phosphonoalanine, phosphono-beta-alanine, phosphonohomoalanine, phosphono-gamma-homoalanine and glufosinate, is presented. This includes a preliminary 31p NMR analysis of these amino acids, their further derivatization to volatile phosphonates (phosphinates) by means of trifluoroacetic acid-trifluoroacetic anhydride-trimethyl orthoacetate reagent and subsequent analysis of derivatization products using MS and/or GC-MS (chemical ionization and/or electron impact ionization).