RESUMEN
The Ag(I)-promoted coupling of amino acids and peptides with amino ester thioamides generates peptide imides without epimerisation. The peptide imides undergo regioselective hydrolysis under mild conditions to generate native peptides. This method was employed to prepare the pentapeptide thymopentin in the NâC direction, in high yield and purity.
Asunto(s)
Péptidos/síntesis química , Plata/química , Tioamidas/química , Hidrólisis , Imidas/química , Péptidos/química , Estereoisomerismo , Timopentina/síntesis química , Timopentina/químicaRESUMEN
Effective global control of tuberculosis (TB) is increasingly threatened by the convergence of multidrug-resistant TB and the human immunodeficiency virus (HIV) infection. TB/HIV coinfections exert a tremendous burden on the host's immune system, and this has prompted the clinical use of immunomodulators to enhance host defences as an alternative therapeutic strategy. In this study, we modified the clinically used synthetic immunomodulatory pentapeptide, thymopentin (TP-5, RKDVY), with six arginine residues (RR-6, RRRRRR) at the N- and C-termini to obtain the cationic peptides, RR-11 (RKDVYRRRRRR-NH2) and RY-11 (RRRRRRRKDVY-NH2), respectively. The arginine residues conferred anti-mycobacterial activity to TP-5 in the peptides as shown by effective minimum inhibitory concentrations of 125 mg/L and killing efficiencies of >99.99% against both rifampicin-susceptible and -resistant Mycobacterium smegmatis. The immunomodulatory action of the peptides remained unaffected as shown by their ability to stimulate TNF-α production in RAW 264.7 mouse macrophage cells. A distinct change in surface morphology after peptide treatment was observed in scanning electron micrographs, while confocal microscopy and dye leakage studies suggested bacterial membrane disruption by the modified peptides. The modified peptides were non-toxic and did not cause hemolysis of rat red blood cells up to a concentration of 2000 mg/L. Moreover, RY-11 showed synergism with rifampicin and reduced the effective concentration of rifampicin, while preventing the induction of rifampicin resistance. The synthetic peptides may have a potential application in both immunocompetent and immunocompromised TB patients.
Asunto(s)
Antibacterianos/farmacología , Factores Inmunológicos/farmacología , Mycobacterium smegmatis/efectos de los fármacos , Péptidos/síntesis química , Péptidos/farmacología , Timopentina/síntesis química , Timopentina/farmacología , Secuencia de Aminoácidos , Animales , Recuento de Colonia Microbiana , Farmacorresistencia Bacteriana/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Factores Inmunológicos/síntesis química , Factores Inmunológicos/química , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Pruebas de Sensibilidad Microbiana , Microscopía Confocal , Datos de Secuencia Molecular , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/ultraestructura , Péptidos/química , Ratas , Rifampin/farmacología , Timopentina/química , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The ethyl ester derivative of thymopentin was synthesized and characterized. The biological activities of thymopentin were evaluated by mouse spleen lymphocyte proliferation test and superoxide dismutase activity assay. Compared with thymopentin, the synthesized ethyl ester showed more potent immunoregulation activity in the MTT assay and anti-oxidation activity in the immune-suppressed rat model induced by hydrocortisone. The half life of the ester derivate in rat plasma determined by HPLC was slightly longer than that of thymopentin. The ester derivate gained advantages in activity and stability compared to thymopentin.
Asunto(s)
Ésteres/síntesis química , Ésteres/farmacología , Timopentina/síntesis química , Timopentina/farmacología , Animales , Antioxidantes/farmacología , Proliferación Celular/efectos de los fármacos , Colorimetría , Ésteres/farmacocinética , Semivida , Hidrocortisona/farmacología , Inmunosupresores/farmacología , Lípidos/química , Linfocitos/efectos de los fármacos , Masculino , Espectrometría de Masas , Ratas , Ratas Sprague-Dawley , Espectrofotometría Ultravioleta , Superóxido Dismutasa/metabolismo , Sales de Tetrazolio , Tiazoles , Timopentina/farmacocinéticaRESUMEN
The protease-catalyzed, kinetically controlled synthesis of a precursor dipeptide, Z-Asp-Val-NH(2) of thymopentin (TP-5), in organic solvents was studied. Z-Asp-OMe and Val-NH(2) were used as the acyl donor and the nucleophile, respectively. An industrial alkaline protease alcalase was used to catalyze the synthesis of the target dipeptide in water-organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-NH(2). The optimum conditions using alcalase as the catalyst are pH 10.0, 35 degrees C, in acetonitrile/Na(2)CO(3)-NaHCO(3) buffer system (9:1, V/V), reaction time 5 h, with a yield of 63%. The dipeptide product was confirmed by LC- MS.
Asunto(s)
Dipéptidos/síntesis química , Compuestos Orgánicos/química , Péptido Hidrolasas/química , Solventes/química , Timopentina/síntesis química , Catálisis , Concentración de Iones de Hidrógeno , Temperatura , AguaRESUMEN
Three conjugates of thymopentin (TP5), an oligopeptide derived from the thymic hormone thymopoietin, with lipoamino acid (LAAs) have been obtained by solid-phase peptide synthesis. Both linear and dendrimer structures have been prepared to achieve enhanced lipophilicity. After incubation in foetal calf serum the lipophilic conjugates showed a higher stability to hydrolysis with respect to the parent drug. In a preliminary in vitro biological assay, LAA conjugates showed the ability to retain or improve the growth inhibitory activity of the parent peptide against a human lymphoblastoid cell line. The interaction of the prepared conjugates with 1,2-L-alpha-dimiristoylphosphatidylcholine multilamellar liposomes, chosen as a biological membrane model, was studied. The higher lipophilicity of TP5 conjugates was reflected in a better penetration through phospholipid bilayers, whose thermal behaviour was altered in a concentration-dependent way. Such enhanced affinity of TP5-LAA conjugates for this membrane model could anticipate a better interaction with cell membranes and, ultimately, an improved biological activity of compounds compared with the parent pentapeptide.
Asunto(s)
Aminoácidos/química , Aminoácidos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Lípidos/química , Lípidos/farmacología , Timopentina/química , Timopentina/farmacología , Anisotropía , Antineoplásicos/síntesis química , Rastreo Diferencial de Calorimetría , Línea Celular , Línea Celular Tumoral , Fenómenos Químicos , Química Física , Cromatografía Líquida de Alta Presión , Difusión , Estabilidad de Medicamentos , Humanos , Hidrólisis , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Leucemia-Linfoma de Células T del Adulto/patología , Liposomas , Timopentina/síntesis químicaRESUMEN
Trypsin-catalyzed, kinetically controlled synthesis of a precursor, dipeptide of thymopentin (TP-5), Bz-Arg-Lys-OH (or-OEt) in organic solvents was studied. Bz-Arg-OEt was used as the acyl donor and Lys-OH and Lys-OEt were used as the nucleophiles. Ethanol was selected as the organic solvent from ethanol, methanol, acetonitrile, and ethyl acetate tested under the experimental conditions. As expected, Lys-OEt is not a suitable nucleophile in trypsin-catalyzed reaction, due to its competition with the protective Arg-OEt as acyl donor for the active site of trypsin, while Lys-OH does not have this problem. The optimal reaction condition for the synthesis of Bz-Arg-Lys-OH was set up as 20% Tris-HCl buffer, pH 8.0, 35 degrees C for 6 h with the yield of 52.5%, or for 18-24 h with the yield of about 60%.
Asunto(s)
Aminoácidos/química , Dipéptidos/síntesis química , Timopentina/síntesis química , Tripsina/química , Catálisis , Cromatografía Líquida de Alta Presión , Precursores de Proteínas/síntesis química , SolventesRESUMEN
The most commonly used photoaffinity labeling probes are compared, which are aryl azides, aryl diazirines, alpha-diazocarbonyls and benzophenone-derivatives. The compounds were used under identical conditions and crosslinking efficiency, influence of water, irradiation requirements, and by-products were investigated. Using the pentapeptide thymopentin (TP5) as a model system, we synthesized four analogues by solid-phase peptide synthesis and partially N-terminal modification to obtain [p-(3-trifluoromethyl)diazirinophenylalanine5]TP5, [p-benzoylphenylalanine5]TP5, 4-azidobenzoyl-TP5 and 2-diazo-3,3,3-trifluoropropionyl-TP5. The peptides were characterized by HPLC and ion-spray mass spectroscopy. Irradiation of the peptides with two different ultraviolet sources was carried out in water, n-propanol and water/n-propanol to imitate both hydrophobic and hydrophilic peptide/protein-interactions as well as the influence of the aqueous environment. Analysis of the products with HPLC, ion-spray MS, HPLC-MS and HPLC-CID-MS revealed that (Tmd)Phe is a highly potent carbene-precursor, which can be transformed easily into uniform crosslinking products by smooth photolysis. However, the electrophilic nature of the intermediate causes a high tendency to react with water molecules. The 4-azidobenzoyl group showed comparable crosslinking efficiency, but the probability to create non-uniform irradiation products (e.g. through rearrangement) is higher, whereas the reaction with water is less dominant. In contrast, Bpa was found to have an extremely low affinity to react with water, whereas prolonged UV irradiation is needed to get complete rearrangement into a variety of products. As the absorption band of alpha-diazocarbonyls at around 350 nm possesses a low extinction coefficient, 2-diazo-3,3,3-trifluoropropionyl-TP5 could not be activated at all with the optimized irradiation conditions that we have chosen for our comparative studies.
Asunto(s)
Marcadores de Afinidad/química , Timopentina/análogos & derivados , Timopentina/química , Adyuvantes Inmunológicos , Marcadores de Afinidad/efectos de la radiación , Cromatografía Líquida de Alta Presión , Fotoquímica , Espectrometría de Masa de Ion Secundario , Timopentina/síntesis químicaRESUMEN
A discontinuous thymopoietin-like motif, composed of the fragments 97-111 and 277-307 of the molecule, as well as of residues Arg178 and Asn163 of G-actin was found. It was established that G-actin has an immunosuppressive activity regarding the humoral immune response. This activity is probably connected to the thymopentin-like sequence RKDLY, which is present in the 277-307 fragment of G-actin. The immunomodulatory activity of a series of peptide-partial sequences of G-actin was tested using plaque-forming cells (PFC) and delayed type hypersensitivity (DTH) tests. The investigated series consisted of five peptides: RKDLY (I), RKDLYANT (II), DVDIRKDLY (III), DVDIR (NO2)KDLY (IV), DVDIRKDLYANT (V). The peptides have the immunosuppressive activity regarding the humoral and cellular immune response.
Asunto(s)
Actinas/inmunología , Adyuvantes Inmunológicos/fisiología , Fragmentos de Péptidos/inmunología , Timopentina/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/síntesis química , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos/efectos de los fármacos , Células Cultivadas , Eritrocitos/inmunología , Hydra/química , Hipersensibilidad Tardía/etiología , Inmunidad Celular/efectos de los fármacos , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Músculo Esquelético/química , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/síntesis química , Conejos , Ovinos/inmunología , Timopentina/administración & dosificación , Timopentina/síntesis químicaRESUMEN
Four cyclic analogs of thymopentin were synthesized and evaluated for biological activity on the human T cell line CEM. Three of these conformationally restricted analogs were biologically active. The one analog which most closely mimicked the conformation predicted from NMR and theoretical energy minimization calculations proved to be inactive. These studies establish that the biologically active conformations of thymopentin differ from the most probable conformation predicted from solution NMR and theoretical energy minimization studies.
Asunto(s)
Timopentina/síntesis química , Secuencia de Aminoácidos , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Péptidos Cíclicos/química , Conformación Proteica , Relación Estructura-Actividad , Termodinámica , Timopentina/análogos & derivadosRESUMEN
The final catalytic deprotection in the large scale synthesis of thymopentin (Arg-Lys-Asp-Val-Tyr) produced an impurity which had not previously been observed. Isolation by preparative HPLC and spectroscopic characterization led to a postulated structure of the impurity as the 1:1 thymopentin-palladium complex. A complex corresponding to the proposed structure was independently synthesized and shown to have identical chromatographic and spectroscopic properties with the isolated material. Proton and carbon (13) NMR were used to determine the coordination sites of the peptide with palladium. The susceptibility of the complex to hydrogenation indicated a possible source for its formation.