Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.     

Synthesis, characterization, X-ray crystallography, and cytotoxicity of a cymantrene keto carboxylic acid for IR labelling of bioactive peptides on a solid support

Cym-CO-CH₂-CH₂-COOH was prepared in good yield by Friedel-Crafts reaction of cymantrene (Cym, CpMn(CO)₃) with succinic anhydride for the IR labelling of peptides and fully characterized, including an X-ray structure analysis (monoclinic space group P2(1)/n, a = 5.727(3) Å, b = 19.865(9) Å, c = 10.518(5) Å, β = 91.211(9)°). The compound was isolated in pure form without the need for chromatographic work-up and subsequently used for solution-phase synthesis of a bioconjugate with phenylalanine methyl ester to allow a complete spectroscopic characterization of this model system. The cymantrene keto carboxylic acid also turned out to be a very robust marker in automated microwave-assisted solid phase peptide synthesis (SPPS). [Leu⁵]-enkephalin (Tyr-Gly-Gly-Phe-Leu) was prepared on a Wang resin and labelled with the cymantrene derivative on the solid support under microwave irradiation in all steps. The metal-carbonyl marker stayed intact during cleavage from the resin with concentrated trifluoroacetic acid. After simple precipitation and lyophilization, the cymantrene-enkephalin bioconjugate could be obtained in analytically pure form without the need of HPLC purification. As required, the compound is non-cytotoxic against MCF-7 cells at up to 100 μM. This protocol thus allows one to introduce organometallic IR spectroscopic labels to peptides in a very straightforward way.     

5-(pyrrolidin-2-yl)-1H-tetrazole and 5-[(pyrrolidin-2-yl)methyl]-1H-tetrazole: proline surrogates with increased potential in asymmetric catalysis

Catalytic enantioselective methodology has dramatically been enriched by the re-discovery of the simple amino acid proline as a chiral catalyst in the year 2000. Although no catalyst offers such a simple and broad access to quite complex reaction products, as does proline, its synthetic potential is not unrestricted, what is especially connected to its poor solubility in organic media. Exchange of the carboxylic moiety by a tetrazole unit leads to proline surrogates, that by far outperform proline with respect to yield, enantioselectivity, reaction time, substrate and solvent scope, catalyst loading, and stoichiometry of the compounds used in excess. These factors are discussed and critically compared with selected representative proline-catalyzed reactions.     

Characterization of glutathione conjugates of pyrrolylated amino acids and peptides by liquid chromatography-mass spectrometry and tandem mass spectrometry with electrospray ionization

High-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (MS-MS) was used to identify the products formed upon reaction of lysine-containing peptides with the neurotoxicant 2,5-hexanedione (2,5-HD). In addition, secondary autoxidative reaction products of the resultant alkylpyrroles with the biological thiol, glutathione, were characterized. ES mass spectra of the HPLC-separated conjugates showed intense [M+H]⁺ ions as well as several ions formed by amide and C-S bond cleavage. The glutathione conjugates of pyrrolylated amino acids and peptides were analyzed by ES ionization and MS-MS, and product-ion spectra showed fragmentation pathways typical of glutathione conjugates. ES-MS-MS analysis of a synthetic nonapeptide modeling a sequence found in neurofilament proteins showed pyrrole formation after incubation with 2,5-HD, and sequence ions were used to assign the position of the pyrrole adduct. Subsequent reaction of the pyrrolylated peptide with reduced glutathione was evidenced by a shift in m/z of the sequence ions of the reaction products with or without prior methylation. The results demonstrate the utility of ES-MS and ES-MS-MS in the characterization of xenobiotic-modified peptides and confirm that stable pyrrole-thiol conjugates are formed by the reaction of biological thils with pyrrolylated peptides.     

Synthesis of the bradykinin B1 antagonist [desArg10]HOE 140 on 2-chlorotrityl resin

Summary This paper describes the synthesis of the bradykinin B₁ antagonist [desArg¹⁰]HOE 140 (d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Tic-Oic-OH) by the solid-phase method. This synthesis is predicted to be a difficult one because the C-terminal sequence d-Tic-Oic, when linked to the resin, could easily undergo an intramolecular aminolysis, releasing the corresponding diketopiperazine. This reaction is greatly favored by the imino acidic structure of these two residues and by the d-configuration of the second one. When using the Fmoc strategy, the reaction takes place during the Fmoc removal with piperidine. In this case, it has been suggested previously that it is possible to prevent the side reaction by reducing the time of the base treatment. In our hands, this expedient worked correctly for the synthesis of a test tetrapeptide (H-Gly-Pro₃-OH), but under the same conditions the synthesis of the target peptide failed completely. In contrast, the use of the very hindered 2-chlorotrityl resin reduced diketopiperazine formation to undetectable levels.     

Stereodesign of chiral para-substituted calix[4]arenes

According to the hypothesis that the chirality of molecule hosts is a cause of their enantioselectivity, the chirality of para-substituted calix[4]arenes was analyzed quantitatively. The relationship between types of para-substituents and the dissymmetry of the 2D (two-dimensional) entrance into the cavity and the whole 3D (three-dimensional) cavity of calix[4]arenes was studied by means of the enantiomer dissimilarity factor (EDF) method for quantitative evaluation of molecular chirality. The design of the most chiral, and probably enantioselective, para-substituted calix[4]arenes was planned such that all four substituents should be different and the two largest should be near each other (adjacent). It was, on the other hand, shown that the 2D chiral entrance determines chirality of the whole 3D structures of these molecules. This phenomenon is interpreted as an example of the chirality transition from 2D into 3D space.Electronic Supplementary Material available.     

Comparative solution and solid-phase glycosylations toward a disaccharide library

A comparative study on solution-phase and solid-phase oligosaccharide synthesis was performed. A 16-member library containing all regioisomers of Glc–Glc, Glc–Gal, Gal–Glc, and Gal–Gal disaccharides was synthesized both in solution and on solid phase. The various reaction conditions for different approaches and corresponding yields are analyzed and discussed.     

Synthesis of bis-(methyl 3,4,6-tri-O-acetyl-D-glucopyranosid-2-yl)-oxamides

The synthesis of a new bis-(D-glucopyranosid-2-yl)oxamides via the key intermediate, N-acetyl N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl) oxamic acid chloride (2alpha) is described. Treatment of compound 2alpha with methyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-beta-D-glucopyranoside afforded N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl)-N'-(methyl 3,4,6-tri-O-acetyl-beta-D-glucopyranosid-2-yl)-oxamide. Reaction of 2alpha with 1,2-diaminoethane afforded 1,2-bis-[N,N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)]ethyloxamide as a main product, while 2-N-[N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)oxamide]-ethyl acetamide was formed as a side product. Reaction of 2alpha with 1,3-diamino-2-hydroxypropane gave only 1,3-bis-N,N-[N'-(methyl 3',4',6'-tri-O-acetyl-2'-deoxy-alpha-D-glucopyranosid-2'-yl)-oxamido]-2-propanol.     

Designing nanoporous crystalline materials by self-assembly: 2D hydrogen-bonded networks from upper rim calix[4]arene diamide derivatives

The self-assembly properties in the solid state of tectons derived from the reaction of calix[4]arene aminomethyl derivatives with a carboxylic acid or a protected amino acid are described, along with their conformational properties in solution. The X-ray crystal structures show that these compounds self-assemble in the lattice through a network of hydrogen-bonds between the amide groups, which overall results in infinite nanotubes formed by the calixarene aromatic cavities. Small changes in the upper rim substituents do not disturb the basic self-assembly motif which appears general and quite useful for controlling crystal growth by design and obtaining novel nanoporous crystalline materials.     

Cyanamide mediated syntheses of peptides containing histidine and hydrophobic amino acids

Summary Using the model of a primitive earth evaporation pond, the synthesis of three histidyl peptides in yields of up to 11% was demonstrated when aqueous solutions of histidine, leucine, ATP, cyanamide, and MgCl₂ were evaporated and heated for 24 h at 80°C. In addition, peptides were formed in yields of up to 56%, 35%, and 21%, respectively for phenylalanine, leucine, and alanine when aqueous solutions of the appropriate amino acid were evaporated and heated with cyanamide and one or more of the following components: ATP, AMP, 4-amino-5-imidazole carboxamide, or MgCl₂. The greatest peptide yield occurred at pH 3. But peptide formation was demonstrated for a system of Leu, cyanamide, and MgCl₂ adjusted to pH 7 with NH₄OH.Peptide synthesis was also studied in the presence of CaCl₂, ZnCl₂, different adenosine nucleotides, and UTP to compare their effects on peptide synthesis. The optimum conditions for cyanamide mediated peptide synthesis were also studied in terms of pH, reaction time, reaction temperature, and cyanamide concentration. The major side product in nearly all reactions studied appears to be an amino acid-cyanamide adduct. Peptides were analyzed and identified by thin layer chromatography, acid hydrolysis, and enzymatic degradation.     

Thermal stability of dehydrophenylalanine-containing model peptides as probed by infrared spectroscopy: a case study of an alpha-helical and a 3(10)-helical peptide

The temperature-dependent secondary-structural changes in the two known helical model peptides Boc-Val-deltaPhe-Ala-Leu-Gly-OMe (1; alpha-helical) and Boc-Leu-Phe-Ala-deltaPhe-Leu-OMe (2; 3(10)-helical), which both comprise a single dehydrophenylalanine (deltaPhe) residue, were investigated by means of FT-IR spectroscopy (peptide film on KBr). Both the first-order and the better-resolved second-order derivative IR spectra of 1 and 2 were analyzed. The nu(NH) (3240-3340 cm(-1)), the Amide-I (1600-1700 cm(-1)), and the Amide-II (1510-1580 cm(-1)) regions of 1 and 2 showed significant differences in thermal-denaturation experiments (22 degrees --> 144 degrees), with the 3(10)-helical peptide (2) being considerably more stable. This observation was rationalized by different patterns and strengths of intramolecular H-bonds, and was qualitatively related to the different geometries of the peptides. Also, a fair degree of residual secondary-structural elements were found even in the 'denatured' states above 104 degrees (1) or 134 degrees (2).     

The use of gel electrophoresis to study the reactions of activated amino acids with oligonucleotides

We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonucleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5-phosphates and ethylenediamine. We find that arginine and amino acids that can interact with oligonucleotides through stacking interactions react most efficiently. D-and L-amino acids give indistinguishable families of products.Correspondence to: L.E. Orgel 1444     

Synthesis and antimicrobial activity of novel benzo[f]coumarin compounds

The acetyl benzo[f]coumarin condensed with phenyl hydrazine to afford the corresponding phenyl hydrazone which cyclized into the pyrazolyl benzocoumarin under Vilsmeier reaction conditions. The pyrazolylaldehyde was used as starting material for synthesis of other heterocyclic compounds containing pyrazolylbenzocoumarin moiety. The ethyl benzo[f]coumarin carboxylate were subjected to react with other reagents to synthesize thiazolidinyl and oxadiazolyl derivatives attached to benzocoumarin system. Some of novel synthesized compounds showed highly antibacterial and antifungal activities.     

Structure-lipophilicity relationships of peptides and peptidomimetics

Summary Understanding the physicochemical and structural properties of peptides are important prerequisites for the rational design of bioactive peptides and peptidomimetics. The present contribution reviews methods used for the assessment and prediction of lipophilicity (or hydrophobicity) and their correlation with structural elements of peptides and closely related peptidomimetics.     

Synthesis of (<Emphasis Type="Italic">2S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4-phenylamino-5-oxoproline derivatives

Summary. The paper describes the synthesis of (2S,4S)-4-(N-Ts)- and (2S,4S)-4-(N-Boc)-phenylamino-5-oxoprolines (pyroglutamic acid). These derivatives have been shown to be useful for synthesis of their amides and peptides in spite of steric hindrances caused by bulky groups adjacent to the reaction centre. Under the conditions applied no lactam ring opening and no loss of stereochemical integrity of any of the chiral centres were observed, which has been confirmed by NMR techniques. Received December 29, 2000 Accepted June 26, 2001     

Synthesis of calix[n]arene-based silica polymers for lipase immobilization

The objective of this study was to prepare new calix[n]arene-based silica polymers for immobilization of Candida rugosa lipase. The amino functionalized calix[4]arene (C4P), calix[6]arene (C6P) and calix[8]arene (C8P)-based silica polymers were used for the covalent attachment of C. rugosa lipase using glutaraldehyde as a coupling agent. The characterization of synthesized CnP polymers and immobilized lipases were made by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. The hydrolytic activities of immobilized lipases (CnP-L) were evaluated and compared with the free enzyme. The activity recovery of immobilized CRL (C. rugosa lipase) based on the carrier C4P, C6P and C8P reaches 74.6%, 68.5% and 51.4%, respectively. The optimal pH and temperature region of the immobilized lipases for the hydrolysis of p-NPP were 7.0 and 50 °C. Nevertheless, the immobilized lipase has good stability, adaptability and reusability in comparison with the free enzyme.     

In vivo synthesis of cholecystokinin in the rat cerebral cortex: identification of COOH-terminal peptides with labeled amino acids

The synthesis of the COOH-terminal octa- and tetrapeptides of cholecystokinin (CCK) has been studied in rat cerebral cortex after intraventricular administration of radioactive amino acids characteristic of the porcine COOH-terminal octapeptide of CCK, CCK-8. After immunosorption with a COOH-terminal directed antibody, cortical CCK was fractionated on Sephadex G-50 columns. The experiments demonstrated newly synthesized CCK forms which coeluted with porcine CCK-8 and CCK-4. Except for threonine the amino acids employed, methionine, tryptophan, aspartic acid, glycine and phenylalanine were incorporated. The sequence-specific radioimmunoassay, the incorporation of the employed labeled amino acids, and the elution pattern by gel filtration, suggest an almost identical structure of porcine and rat cortical CCK-8, and a concomitant synthesis of CCK-8 and CCK-4 in rat cerebral cortex.     

Synthesis and characterization of electronically varied XCX palladacycles with functional arene groups

X-ray crystallographic studies show that varying the nature of the S-aryl ligands in SCS-Pd(II) pincer complexes and the electronic nature of the aryl substituent para to the Pd(II) group in PCP-Pd(II) pincer complexes do not lead to structural changes in these palladacycles that can be correlated with the changing nature of the ligands. While the original C2 symmetry for the S-aryl groups in SCS-Pd(II) pincer complexes seen in the case of the 2,5-bis(thiophenylmethyl)phenylpalladium chloride pincer complex is also seen in other SCS-Pd(II) pincer complexes, the relative stereochemistry of the S-aryl rings is not consistently maintained in 2,5-bis((4-dimethylaminothiophenyl)methyl)-phenylpalladium chloride.     

Synthesis,Structure, and Biological Applications of α‐Fluorinated β‐Amino Acids and Derivatives

This review gives a broad overview of the state of play with respect to the synthesis, conformational properties, and biological activity of α‐fluorinated β‐amino acids and derivatives. General methods are described for the preparation of monosubstituted α‐fluoro‐β‐amino acids (Scheme 1). Nucleophilic methods for the introduction of fluorine predominantly involve the reaction of DAST with alcohols derived from α‐amino acids, whereas electrophilic sources of fluorine such as NFSI have been used in conjunction with Arndt? Eistert homologation, conjugate addition or organocatalyzed Mannich reactions. α,α‐Difluoro‐β‐amino acids have also been prepared using DAST; however, this area of synthesis is largely dominated by the use of difluorinated Reformatsky reagents to introduce the difluoro ester functionality (Scheme 9). α‐Fluoro‐β‐amino acids and derivatives analyzed by X‐ray crystal and NMR solution techniques are found to adopt preferred conformations which are thought to result from stereoelectronic effects associated with F located close to amines, amides, and esters (Figs. 2–6). α‐Fluoro amide and β‐fluoro ethylamide/amine effects can influence the secondary structure of α‐fluoro‐β‐amino acid‐containing derivatives including peptides and peptidomimetics (Figs. 7–9). α‐Fluoro‐β‐amino acids are also components of a diverse range of bioactive anticancer (e.g., 5‐fluorouracil), antifungal, and antiinsomnia agents as well as protease inhibitors where such fluorinated analogs have shown increased potency and spectrum of activity.