Navegando por Palavras-chave "peptide synthesis"
Agora exibindo 1 - 12 de 12
Resultados por página
Opções de Ordenação
- ItemSomente MetadadadosComparative evaluation of the synthesis and purification of transmembrane peptide fragments - Rat bradykinin receptor fragment 64-97 as model(Munksgaard Int Publ Ltd, 1997-04-01) Oliveira, E.; Miranda, A.; Albericio, F.; Andreu, D.; Paiva, ACM; Nakaie, C. R.; Tominaga, M.; Universidade Federal de São Paulo (UNIFESP); UNIV BARCELONAThe 34-residue peptide CTVAEIYLGNLAGADLILASGLPFWAITIANNFD (TM-34), corresponding to the 64-97 sequence of the rat bradykinin receptor, was selected as a model of hydrophobic transmembrane peptide segment for systematic study of synthesis and purification strategies. Application of conventional Boc/Bzl chemistry resulted in very low yield of the synthesis (around 4%) when DMF was used as the solvent for coupling reactions. As shorter resin-bound fragments of TM-34 showed improved swelling in 80% NMP/DMSO, the synthesis was repeated in this mixed solvent and the yield increased to 12%. A comparative synthesis using optimized Fmoc chemistry and Fmoc-(FmocHmb) derivatives of Ala and Leu to prevent aggregation did not provide any detectable TM-34. Taken together, these results illustrate the synthetic problems associated with hydrophobic sequences, almost regardless of the chemistry used. As expected, the hydrophobicity of TM-34 and of most of its minor fragments made them scarcely soluble in common solvents. Purification could be achieved by loading the crude materials dissolved in 90% AcOH onto a C-4 HPLC column and eluting with a TFA/MeCN linear gradient. CD studies of the TM-34 and of the shorter fragment with the 74-97 sequence (TM-24) showed a higher percentage of cc-helix structure for the latter. This suggests that the shorter sequence may better represent the correct transmembrane region of the second helix of the rat bradykinin receptor. (C) Munksgaard 1997.
- ItemSomente MetadadadosComparative investigation of the cleavage step in the synthesis of model peptide resins: Implications for N-alpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis(Pharmaceutical Soc Japan, 2007-03-01) Jubilut, Guita Nicolaewsky [UNIFESP]; Cilli, Eduardo Maffud [UNIFESP]; Crusca Junior, Edson; Silva, Elias Horacio [UNIFESP]; Okada, Yoshio; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Estadual Paulista; Kobe Gakuin UnivBased on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the N-alpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. the present paper continues along this line of research but examines the N-alpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. the vasoactive peptide angiotensin II (All, DRVYIHPF) and its [Gly(8)]-All analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. the same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. the results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%.) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.
- ItemSomente MetadadadosFluorescent properties of amino acids labeled with ortho-aminobenzoic acid(Wiley-Blackwell, 1998-01-01) Ito, A. S.; Turchiello, RDF; Hirata, I. Y.; Cezari, MHS; Meldal, M.; Juliano, L.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Dept Chemortho-Aminobenzoic acid (Abz) has been used as a convenient fluorescent donor group in internally quenched fluorescent peptides, which are employed as substrates for several proteolytic enzymes. As Abz is usually bound to the N-amino terminal of these peptides, it is of interest to investigate the Abz group fluorescent properties bound to different amino acids. We report in this article the optical absorption and fluorescent properties, in aqueous media, of Abz bound to the alpha-amino group of Ala, Gly, Leu, ne, Val, Pro, Phe, Arg, Glu, Met, Asn, Tyr, and Trp, with monomethyl-amidated alpha-carboxyl group, in order to explore the origin of the drastic reduction of Abz attached to N-alpha amino group of prolyl-peptides, we also examined the fluorescence properties of Abz-NHCH3, Abz-N(CH3)(2), and Abz-pyrrolidine. Molecular dynamics simulation and NMR data indicated a lack of periplanarity of the Abz-dimethylamide, which could be the origin of low fluorescence quantum yield of Abz-prolyl-peptides. (C) 1998 John Wiley & Sons, Inc.
- ItemAcesso aberto (Open Access)Importance of the solvation degree of peptide-resin beads for amine groups determination by the picric acid method(Sociedade Brasileira de Química, 2000-10-01) Cilli, Eduardo Maffud [UNIFESP]; Jubilut, Guita Nicolaewsky [UNIFESP]; Ribeiro, Suely C. F. [UNIFESP]; Oliveira, Eliandre [UNIFESP]; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The classic and important picric acid method used in polymers biochemical and chemical fields of polymers for amine group quantification was chosen in this work as a model for evaluating the influence of the resin bead solvation during an analytical procedure. It was observed that this method, proposed almost three decades ago, failed to quantify amine groups of peptidyl-resin containing aggregating and polar sequence. This was due to inefficient solvation of resin beads when only CH2Cl2 was used for picrate anion binding and subsequent washing steps. It was demonstrated that the use of CH2Cl2/DMF (dimethylformamide) and CH2Cl2/EtOH solutions during these steps allows correct determination of peptidyl-resin amine groups. Besides the importance for the solid phase peptide synthesis methodology itself, these findings also represent the first quantitative demonstration of the relationship between solvation degree and the efficiency of a polymer-supported analytical method.
- ItemSomente MetadadadosInhibition of angiotensin converting enzyme and potentiation of bradykinin by retro-inverso analogues of short peptides and sequences related to angiotensin I and bradykinin(Elsevier B.V., 1996-04-26) Carmona, A. K.; Juliano, L.; Universidade Federal de São Paulo (UNIFESP)There is pharmacological evidence indicating that, in addition to the inhibition of angiotensin converting enzyme (ACE; EC 3.4.15.1), the potentiation of bradykinin (BK) responses may also involve the BK receptor or some binding site in the structures involved in the contractile response to this peptide. Dipeptides such as Val-Trp and some of its analogues as well as tripeptide homologues, including total and partial retroinverso peptides, were synthesized and assayed for their ability to inhibit purified guinea pig plasma ACE and to potentiate the action of BK on the isolated ileum of the same species. the peptides containing the P-2-P-1, P-1-P-1', and P-1'-P-2' inverted amide bonds inhibited ACE, were resistant to hydrolysis, and, depending on the amino acid composition, some of them potentiated the contractile response to BK while others did not. Des-[Arg(1)]-BK, which has an intrinsic activity at concentrations higher than 10(-5) M, and the very dissimilar angiotensin I (AI) analogue [Cys(5)-Cys(10)]-angiotensin-I-(5-10)-amide, which has no detectable contractile activity, were able to inhibit ACE and potentiate BK. in contrast to these peptides, BPP5a and BPP9a from Bothrops jararaca venom, and Potentiators B and C from Agkistrodon halys blomhoffii venom were more effective as BK potentiators than as ACE inhibitors. in conclusion, we have synthesized and assayed compounds that preferentially inhibit ACE, e.g. retro-inverso tripeptides, or potentiate the response of smooth muscle to BK, e.g. snake venom peptides.
- ItemSomente MetadadadosMixtures of trifluoroethanol or hexafluoroisopropanol and dimethylformamide are not of general applicability for peptide condensations catalyzed by trypsin(Munksgaard Int Publ Ltd, 1998-01-01) Bemquerer, Marcelo P.; Liria, Cleber W.; Kitagawa, Kouki; Miranda, M Teresa M; Tominaga, Mineko; Universidade de São Paulo (USP); Niigata Coll Pharm; Universidade Federal de São Paulo (UNIFESP)Mixtures of a good hydrogen bond donor, 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluroisopropanol, and an acceptor, dimethylformamide (DMF) (1:1,v/v), containing 4% buffer have been described as adequate solvent systems for trypsin-catalyzed peptide fragment condensations [Mihara et al. (1993) Int. J. Pept. Protein Res. 41, 405]. Thus, we decided to study the behaviour of trypsin in such solvent systems. We investigated whether this protease would efficiently catalyze condensations between fragments derived from an analogue of the gp-41 capsid protein of HIV virus or from cholecystokinin-22. None of the reactions carried out yielded the desired condensation products. However, when Fmoc-NLQNLDPSHR-OH and cholecystokinin-12 (H-ISDRDYMGWMDF-NH2) were used as substrates, the last had its R-D peptide bond hydrolyzed producing cholecystokinin-8. The proteolytic activity of this enzyme measured against a fluorogenic peptide derivative was 50 times lower in DMF/TFE containing 5% of aqueous phase than in buffer. Steady-state fluorescence studies in DMF/TFE buffer were performed to examine the structure of this protease in these media. Steady-state spectra obtained with increasing proportions of these two organic solvents in buffer showed that the emission intensities built up. Quenching studies with iodide revealed that the I-o/I ratio (where I,and I are the fluorescence emission intensities in the absence and presence of quencher, respectively) changed from 1.2 in aqueous media to 2.2 in DMF/TFE (1:1, v/v) containing 11% 0.2 M Tris-HCl buffer, pH 8.0, for 0.5 M iodide. The complete data indicated a higher exposure of tryptophan residues to the quencher in organic media, probably because of the partial unfolding of the enzyme. (C) Munksgaard 1997.
- ItemSomente MetadadadosNovel Copoly(Styrene-Divinylbenzene)-Resins with Different Phenylmethylamine Groups for Use in Peptide Synthesis Method(Bentham Science Publ Ltd, 2015-01-01) Souza, Sinval Estevam Gregorio de [UNIFESP]; Malavolta, Luciana [UNIFESP]; Cilli, Eduardo Maffud [UNIFESP]; Schreier, Shirley [UNIFESP]; Jubilut, Guita Nicolaewsky [UNIFESP]; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Santa Casa Sao Paulo Sch Med Sci; Unesp; Universidade de São Paulo (USP)Differently than the 4-methylbenzhydrylamine-resin (MBHAR) which contains a methyl group coupled to the phenylmethylamine-functionalized copoly(styrene-divinilbenzene) structure, alternative resins containing the electron-donating 4-tert-butyl-(BUBHAR) or the electron-withdrawing 2-chloro-(ClBHAR) and 2,4-chloro-(diClBHAR) groups were developed as potential supports for carboxamide peptide synthesis. Initially, a time-course investigation of HF cleavage reaction (0 degrees C) with these resins bearing the vasoconstrictor angiotensin II (AngII, DRVYIHPF) or its Gly(8)-AngII analogue revealed that the peptide-BUBHAR linkage is much more labile than those with ClBHAR or diClBHAR. HF cleavage times of near 2 h or longer than 24 h were needed for complete removal of peptide chains from these two classes of resin, respectively. By including MBHAR and benzhydrylamine-resins (BHAR) in this comparative study, the decreasing order of acid stability of the peptidyl-resin linkage was diClBHAR > ClBHAR > BHAR > MBHAR similar to BUBHAR. The same stability order was observed for the HCl/ propionic acid hydrolysis reaction (130 degrees C) with the Phe-or Gly-resins. These findings thus suggest that ClBHAR and diClBHAR are not appropriate for use in peptide synthesis. Nevertheless, these supports could still be tested as stationary phases for affinity chromatography. When placed into more apolar solvents, the beads of all of these resins exhibited a greater swelling (as measured by a microscope) or higher mobility of the polymer matrix (as measured with EPR experiments using spin-labeled beads). Moreover, under the latter approach, BUBHAR displayed a comparatively higher solvation degree than did MBHAR (in DCM, DMF and NMP), with slightly higher peptide synthesis yields as well.
- ItemSomente MetadadadosReduction of ortho-aminobenzoyl-proline fluorescence and formation of pyrrolobenzodiazepine-5,11-dione(Kluwer Academic Publ, 1998-01-01) Hirata, Izaura Yoshico [UNIFESP]; Cezari, Maria Helena Sedenho [UNIFESP]; Boschcov, Paulo [UNIFESP]; Garratt, Richard Charles; Oliva, Glaucius; Ito, Amando Siuiti; Spisni, Alberto; Franzoni, Lorella; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Univ ParmaThe ortho-aminobenzoic acid (Abz) group is widely employed as a fluorescent marker for peptides used as substrates for the study of proteolytic enzyme activity. in fact, a direct correlation has been observed between fluorescence intensity and enzyme activity. An unusual behavior of the fluorescence properties of this group, which would lead to erroneous evaluation of the enzyme activity, was observed when it is bound directly to proline. Here we report a systematic NMR, fluorescence and X-ray diffraction study of the compounds obtained from Boc-Abz-Pro-NH2, Boc-Abz-Pro-OH, as well as from various other Boc-Abz-Pro-X derivatives, after treatment with HCl or TFA under anhydrous conditions. We verified that, as recently reported, even under these synthetic conditions, deprotection of Boc-Abz-Pro-NH2 or Boc-Abz-Pro-OH leads to the formation of the same product: pyrrolobenzodiazepine-5,11-dione. However, the formation of this compound was not detected with Abz-Pro-N(CH3)(2), Abz-Pro-Leu-Gly-NH2 or Abz-pyrrolidine. for all these compounds we observed an unusual behavior for the fluorescence quantum yield of Abz that can be explained as the consequence of a non-radiative deactivation process produced, specifically, by the amidation of the Abz carboxyl group with proline or a similar secondary amine such as pyrrolidine. in conclusion, these results indicate that Abz cannot be used as an internal fluorescence marker for proteolytic enzyme activity when bound directly to proline.
- ItemSomente MetadadadosResin selection based on the lability of peptidyl-resin linkage towards HF and TFA steps: Dependence on the C-terminal amino acid and peptide length(Pharmaceutical Soc Japan, 1999-11-01) Jubilut, Guita Nicolaewsky [UNIFESP]; Miranda, Maria Teresa; Tominaga, Mineko; Okada, Yoshio; Miranda, Antonio de [UNIFESP]; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Kobe Gakuin UnivIdeally, the solid support used for teut-butyloxycarbonyl (Boc)-peptide synthesis method must allow sufficient stability of the peptide linkage towards TFA-alpha-amino deprotection but adequate lability to final HF cleavage. Due to these conflicting characteristics, the choice of the correct resin for peptide synthesis is complex and dependent upon many factors. Aiming to clarify this issue, a time-course study of the trifluoroacetic acid (TFA) and HF steps using model peptidyl-resins was developed. The peptidyl-resin bond stability was strongly dependent upon the resin and the carboxy-terminus residue, The decreasing order of acid stability for resins was: benzhydrylamine-resin (BHAR)>p-methylbenzhydrylamine-resin (MBHAR)congruent to 4-(oxymethyl)-phenylacetamidomethyl-resin (PAMR)>chloromethyl-resin (CMR) and Phe>Gly congruent to His congruent to Asp for C-terminal amino acids. HF-cleavage times of near 6 h (BHAR) and 2-3 h (MBHAR and PAMR) were necessary for quantitative cleavage of hydrophobic Phe residue-containing sequence at its C-terminal portion. When premature chain loss in TFA and incomplete cleavage in HF values were both quantitatively considered, a significant decrease in the overall yield (up to 35%) was observed in some resins, Moreover, MBHAR was more suitable than BHAR only when the peptide C-terminal residue is hydrophohic. The data also allow the prediction that due to more significant chain loss in TFA when MBHAR is used, BHAR will be the resin of choice for much longer than 40-mer peptide sequences containing C-terminal hydrophilic residues. Otherwise PAMR is the best resin for the synthesis of free carboxyl peptides but significantly low HF cleavage was observed when the C-terminal amino acid is of the hydrophobic-type.
- ItemSomente MetadadadosSynthesis and pharmacological properties of TOAC-labeled angiotensin and bradykinin analogs(Elsevier B.V., 2002-01-01) Nakaie, Clovis Ryuichi [UNIFESP]; Silva, Eneida de Gusmão [UNIFESP]; Cilli, Eduardo Maffud [UNIFESP]; Marchetto, Reinaldo [UNIFESP]; Schreier, Shirley [UNIFESP]; Paiva, Therezinha Bandiera [UNIFESP]; Paiva, Antonio Cechelli de Mattos [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); UNESP; Universidade de São Paulo (USP)Angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label were synthesized by solid phase methodology. Ammonium hydroxide (pH 10, 50degreesC, 1 h) was the best means for reverting nitroxide protonation occurring during peptide cleavage. EPR spectra yielded rotational correlation times for internally labeled analogs that were nearly twice as large as those of N-terminally labeled analogs. Except for TOAC(1)-AngII and TOAC(0)-BK, which showed high intrinsic activities, other derivatives were inactive in smooth muscle preparations. These active paramagnetic analogs may be useful for conformational studies in solution and in the presence of model and biological membranes. (C) 2002 Elsevier Science Inc. All rights reserved.
- ItemSomente MetadadadosTruncation of amidated fragment 33-61 of bovine alpha-hemoglobin: Effects on the structure and anticandidal activity(Wiley-Blackwell, 2007-01-01) Machado, Alessandra; Sforca, Mauricio L.; Miranda, Antonio; Daffre, Sirlei; Pertinhez, Thelma A.; Spisni, Alberto; Miranda, M. Teresa M.; Universidade de São Paulo (USP); Ctr Struct Mol Biol; Universidade Federal de São Paulo (UNIFESP); Univ ParmaPeptides derived from endogenous hemoglobin play important biological roles in a variety of living systems. in previous works we showed that the fragment 33-61 of bovine alpha=hemoglobin (Hb33-61) and its C-terminus amidated analogue (Hb33-61a) exhibit antimicrobial activity and we determined the 3D structure of HB33-61a bound to sodium dodecyl sulfate micelles. Here we report that Hb33-61a is lethal to Candida albicans at 625 mu M probably through disruption of its plasma membrane. in addition, we show that, even when used at 50 mu M, Hb33-61a produces low hemolysis (16% +/- 3.0%). Recognizing that one of the key steps to study new compounds with potential pharmaceutical application is to identify the structural elements essential to express biological activity, we also investigated the anticandidal activity of HB33-61a fragments. the results indicated that Hb40-61a exhibits the same minimal inhibitory concentration as Hb33-61a, whereas HB33-52a and Hb48-61a are significantly less astive. Noteworthy, for all the peptides tested, we observed that C-terminus amidation produces a potentiation of their anticandidal activity and we associate that increased biological activity and we associate that increased biological activity to a preferred structural and spatial organization of the C-terminal region favored by amidation. Finally, the data show that the most active peptides (Hb33-61a and Hb40-61a) are characterized by a central hinge joining the C-terminal region (containing a beta-turn followed by a helical element) to the N-terminal region (that presents only a beta-turn). We hypothesize that these two structured regions, by fluctuating independently in the lipid environment, may act in a coordinated fashion disrupting the yeast plasma membrane. (c) 2007 Wiley Periodicals, Inc.
- ItemAcesso aberto (Open Access)Use of the same polymer for synthesis and purification of peptides(Sociedade Brasileira de Química, 2005-04-01) Silva, Elias Horacio da [UNIFESP]; Etchegaray, Augusto [UNIFESP]; Carvalho, Regina S. H. [UNIFESP]; Jubilut, Guita Nicolaewsky [UNIFESP]; Miranda, Antonio [UNIFESP]; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)This work reveals an uncommon but valuable biotechnological approach regarding the use of a same polymer (benzhydrylamine-resin, BHAR) for synthesis and anion exchange purification of peptides. Initially, the octapeptide DRVYIHPF-NH2 was synthesized in 1% and 3% cross-linked BHAR, attaching 2.5 mmol g-1 ammonium groups. Due certainly to its less rigid polymeric backbone, higher synthesis yield (about 80%) was achieved with the former resin. Next, the negatively charged peptides DEVYEHPF-NH2 and DEVYEDPF-NH2 (-1 and -3 in neutral pH, respectively), both synthesized in 1% BHAR were submitted to chromatographic separation test in this same type of resin (1% and 3%). Following comparative results of peptide synthesis and swelling data of resin beads obtained by microscopy, an improved separation of both peptides occurred with 1% BHAR batch. These findings demonstrated that BHAR applied so far for peptide synthesis, when containing high amount of positively charged ammonium groups, can be also used alternatively as a solid support for chromatographic purification of this type of biological molecule.