RESUMEN
Branched peptides E(RLAR)2, E[E(RLAR)2]2, E(KLAR)2, and E[E(KLAR)2]2 were synthesized on the basis of tetrapeptides RLAR and KLAR and glutamic acid bis(pentafluorophenyl) ester. Their minimal antimicrobial concentrations were shown to decrease along with increase in branching, achieving 12 microM for Escherichia coli cells, which is comparable to antimicrobial activities of temporin, magainin, and dermaseptin. The branched peptides were found not to act on human erythrocytes.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Escherichia coli/efectos de los fármacos , Oligopéptidos/química , Péptidos Catiónicos Antimicrobianos/síntesis química , Eritrocitos/efectos de los fármacos , Glutamatos/química , HumanosRESUMEN
An epimerization-free system for coupling N-protected peptides with free amino acids was developed. A number of inorganic substances were tested as epimerization suppressant additives during the coupling by various methods (carbodiimide plus additives, uronium salts, Woodward's reagent-K, isobutyl-chloroformate, etc.). Some of them (ZnCl2, RbClO4, LiCl, SnCl4, AlCl3, etc.) in combination with some coupling methods can guarantee coupling with minimal epimerization (D-epimer < 1%). But only a simultaneous use of 1-hydroxybenzotriazole and Cu2+ ions as additives in carbodiimide-mediated peptide couplings appeared to give a standard result (D-epimer < 0.1%). There was no epimerization even in the case when N-methyl amino acid (sarcosine) was used as an amino component, while in the absence of Cu2+ ions an unacceptable level of epimerization was observed (D-epimer, 22% for carbodiimide with the 1-hydroxybenzotriazole method). So far it has been considered that Cu2+ ions prevent obtaining peptides in high yields (< 90%) by various coupling methods. We have found that the use of 1-hydroxybenzotriazole, CuCl2 and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of dicyclohexylcarbodiimide provides a possible method for obtaining the desired peptides in 90-99% yields without epimerization. All these results were shown by employing several model peptide couplings with free amino acids as amino components dissolved in an effective solvent system which readily dissolved them.
Asunto(s)
Técnicas de Química Analítica/métodos , Cobre/química , Biosíntesis de Péptidos , Sales (Química)/química , Aminoácidos/química , Cromatografía Líquida de Alta Presión , Racemasas y Epimerasas , Temperatura , Factores de TiempoRESUMEN
Highly reactive hydrophilic (i.e., water-soluble) p-sulfotetrafluorophenyl esters (Tfs esters) are proposed for peptide synthesis in aqueous and aqueous-organic media, as well as for protein and peptide partial synthesis in an aqueous medium. These esters can serve as a basis for creating a series of protein modifying reagents. As they are analogs of the widely used pentafluorophenyl esters, the Tfs esters possess a high reactivity coupled with good stability during storage. The expression for the reaction rate (for substrates AA1 and AA2) is shown to be v = k[Boc-AA1-OTfs][H-AA2-NH2]0.5 for both water and DMF, i.e., the reaction is not a simple second-order reaction. The reaction rate in water is only slightly lower than that in DMF.
Asunto(s)
Aminoácidos/química , Péptidos/síntesis química , Ésteres del Ácido Sulfúrico/química , Flúor/química , Indicadores y Reactivos , Cinética , Péptidos/químicaRESUMEN
The proteins, AlgR3 and AlgP, are involved in the regulation of alginate synthesis in Pseudomonas. They contain multiple repeats of Ala*Ala*Lys*Pro as do several other proteins that resemble histones. The interactions of synthesis oligopeptides composed of repeated Ala*Ala*Lys*Pro or Lys*Lys*Ser*Pro units with DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl) group attached to the N-termini of the peptides. DNA quenching of the Fmoc fluorescence of the peptides was used to estimate the apparent association constants for the interaction of Fmoc(AAKP)nOH (n = 2, 4, 8, 18, 32) and of Fmoc (KKSP)nOH (n = 2, 4, 8, 16, 20, 32) with DNA. The Fmoc(AAKP)nOH peptides bind to DNA only at low ionic strength; the Fmoc(KKSP)n OH peptides interact with DNA at both low (0.05 M KCl) and high (0.2 M KCl) salt. At low ionic strength an increase in the number of the repeat units causes an increase in the apparent association constant up to approximately 2 x 10(6) M-1 for both types of peptides at N congruent to 24. The insertion of an AAKTA unit into the middle of the Fmoc(AAKP)8OH peptide increases its affinity to DNA. We propose a model of (AAKP)n and of its interaction with DNA. The repeat unit consists of a single turn of alpha-helix followed by a bend necessitated by Pro. The resultant coiled-coil forms a right-handed superhelix with 10 AAKPs per repeat distance of approximately 33 A.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Espectrometría de FluorescenciaRESUMEN
The kinetics of the reaction of Boc-alanine-trifluorophenyl, Boc-alanine-tetrafluorophenyl, Boc-alanine-pentafluorophenyl, and Boc-alanine-p-chlorotetrafluorophenyl esters (BocAlaOTrf, BocAlaOTfp, BocAlaOPfp, and BocAlaTfc, respectively) with leucine amide and with valine methyl ester have been measured using changes in fluorophenyl chromophore emission at 375 nm. The kinetic data cannot be well fit with a simple second-order reaction scheme. Measurements of the reaction kinetics at different concentrations of the reagents showed that the expression for the reaction rate is V = kC(N)0.5C(AE)1.5, in which k is the reaction rate constant, CN is the concentration of either LeuNH2 or ValOCH3, and CAE is the concentration of the fluorophenyl ester. This reaction equation indicates a complex, probably chain-like, reaction mechanism. The order of reactivity for these active esters with ValOCH3 is BocAlaOTfc > BocAlaOPfp > BocAlaOTfp > BocAlaTrf. The apparent rate constant, k, for the reaction with LeuNH2 is higher than that for the reaction with ValOCH3.
Asunto(s)
Alanina/análogos & derivados , Fluorobencenos/análisis , Hidrocarburos Fluorados/análisis , Péptidos/síntesis química , Alanina/análisis , Ésteres/análisis , Fluorescencia , CinéticaRESUMEN
Dipentafluorophenylcarbonate, belonging to transesterifiying reagents, has been prepared and used for the synthesis of pentafluorophenyl esters of amino acids. In contrast to many other reagents of the kind, its preparation is simple, it is highly reactive and at the same time stable upon storage.