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1.
J Pept Res ; 52(2): 155-64, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9727872

RESUMO

A novel analog of vasoactive intestinal peptide (VIP) has been reported which exhibits high potency and enhanced duration of in vivo biological activity. This VIP analog, cyclo-(Lys21-Asp25)Ac[Glu8 Lys12 Nle17 Ala19, Asp25 Leu26,Lys27,28,Gly29,30,Thr31]-VIP, which also has a lactam bridge, has been reported to have relaxant effects that are significantly more potent than other beta-agonists such as salbutamol and salmeterol. Because it has potential use for the treatment of bronchial asthma in humans, various convergent syntheses were evaluated to enable the economic preparation of large quantities of this medium-sized hentriacontapeptide. From these studies we developed a combined solid-phase/solution synthesis which uses four protected fragments (each prepared by solid-phase synthesis with highly acid-labile resins) possessing Nalpha-9-fluorenylmethyloxycarbonyl and side-chain tert-butyl protection. Only equivalent amounts of each fragment were required to achieve near-quantitative coupling reactions using N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmeth anaminium hexafluorophosphate N-oxide/N-hydroxybenzotriazole. All reagents and side products were removed at each stage by simple extraction procedures. Final deprotection was carried out with 90% trifluoroacetic acid. Under these conditions only low levels of epimerization were observed (<2%). These diastereoisomers and other trace impurities were removed from the product in a single purification by preparative high-performance liquid chromatography. The procedure has been scaled up (10-g scale) and the final product obtained in an overall (nonoptimized) yield of 24%. This procedure for the repetitive coupling of fragments, without isolation of intermediates, may be generally applicable for the economic synthesis of other medium-sized and longer peptides.


Assuntos
Bioquímica/métodos , Fragmentos de Peptídeos/síntese química , Peptídeo Intestinal Vasoativo/análogos & derivados , Peptídeo Intestinal Vasoativo/síntese química , Sequência de Aminoácidos , Dados de Sequência Molecular
2.
Int J Pept Protein Res ; 46(3-4): 279-89, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-8537181

RESUMO

From previous work, the primary functional groups, i.e. side chains, of the vasoactive intestinal peptide which are responsible for interaction with the VIP receptor have been identified. One of these sites, the side chain of tyrosine22 is essential for high receptor affinity. The present work aims to examine this site in greater detail. Several Boc-substituted-phenylalanine derivatives were prepared and incorporated into VIP analogs as replacement for tyrosine22. These analogs, of the form Ac-[Lys12,Nle17,X22,Val26,Thr28]-VIP, were assayed as smooth muscle relaxants and found to be full agonists of native VIP. Most of the analogs, however, proved to be less potent than the parent analog by up to 300-fold. A few analogs, all possessing electron-donating substituents, retained nearly full potency. Two compounds, 3-F,4-OH-Phe, 42 and 3-OCH3,4-OH-Phe, 43, were found to be 1.5- and 3.4-fold more potent than the parent compound, which equates to being 8.9- and 20-fold more potent than native VIP. Compound 43 was also found to be active as a bronchodilator in vivo in guinea pigs, with slightly over 2-fold enhanced potency and a significantly longer duration of action (> 20 min) when compared to the parent compound (5 min). The physical characteristics of the various substituents and their effect on biological activity are discussed with a brief analysis by QSAR techniques.


Assuntos
Tirosina/química , Peptídeo Intestinal Vasoativo/análogos & derivados , Peptídeo Intestinal Vasoativo/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Broncodilatadores/química , Broncodilatadores/farmacologia , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Cobaias , Dados de Sequência Molecular , Estrutura Molecular , Relaxamento Muscular/efeitos dos fármacos , Parassimpatolíticos/química , Parassimpatolíticos/farmacologia , Fenilalanina/análogos & derivados , Fenilalanina/química , Relação Estrutura-Atividade , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo
3.
Biopolymers ; 37(2): 57-66, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-7893947

RESUMO

Analogs of vasoactive intestinal peptide (VIP) were synthesized and screened as bronchodilators with the ultimate goal of enhancing the potency and extending the duration of action of the native peptide. Several design approaches were applied to the problem. First, the amino acid residues required for receptor binding and activation were identified. A model of the active pharmacophore was developed. With knowledge of the secondary structure (NMR) of the peptide, various analogs were synthesized to stabilize alpha-helical conformations. Having achieved a level of enhanced bronchodilator potency, our approach then concentrated on identification of the sites of proteolytic degradation and synthesis of metabolically-stable analogs. Two primary cleavage sites on the VIP molecule were identified as the amide bonds between Ser25-Ile26 and Thr7-Asp8. This information was used to synthesize cyclic peptides which incorporated disulfide and lactam ring structures. Analog work combined the best multiple-substitution sites with potent cyclic compounds which resulted in identification of a cyclic lead peptide. This compound, Ro 25-1553, exhibited exceptionally high potency, metabolic stability, and a long duration of action and may be an effective therapeutic for the treatment of bronchospastic diseases.


Assuntos
Asma/tratamento farmacológico , Peptídeo Intestinal Vasoativo/análogos & derivados , Peptídeo Intestinal Vasoativo/síntese química , Sequência de Aminoácidos , Animais , Desenho de Fármacos , Humanos , Dados de Sequência Molecular , Conformação Proteica , Relação Estrutura-Atividade , Peptídeo Intestinal Vasoativo/química , Peptídeo Intestinal Vasoativo/uso terapêutico
4.
J Med Chem ; 35(21): 3774-83, 1992 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-1433191

RESUMO

A series of analogs of Ac-CCK-7 [Ac-Tyr(SO3H)-Met28-Gly29-Trp-Met-Asp- Phe-NH2, (1)] were prepared in which the Met28-Gly29 dipeptide was replaced by omega-aminoalkanoic acids. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors, respectively, and for anorectic activity after intraperitoneal administration to rats. The analog incorporating 4-aminobutanoic acid (5) was only 8 times less potent than 1 in the pancreatic binding assay, was more potent in the striatal binding assay, and was more potent than 1 in reducing food intake in rats. Using a bioactive cyclic analog of Ac-CCK-7 as a template, several rigid spacers were designed and tested as substitutes for the Met28-Gly29 dipeptide. The analogs incorporating 3-aminobenzoic acid (20) and (1S)-trans-2-aminocyclopentanecarboxylic acid (26) proved highly effective in the binding assays and as anorectic agents. We hypothesize that for stimulation of CCK-A receptors, the main function of the N-terminal tripeptide of Ac-CCK-7 is to orient the tyrosine sulfate with respect to Trp30 and that the bioactive arrangement of these elements lies among those which are readily available to both 20 and 26. NOESY and distance-constrained molecular dynamics experiments carried out on 20 and 26 identified conformations in which the relative orientation of the tyrosine hydroxide and the alpha-carbon atom of tryptophan were similar, providing the basis for further drug design efforts.


Assuntos
Colecistocinina/análogos & derivados , Sequência de Aminoácidos , Animais , Bovinos , Colecistocinina/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dipeptídeos/química , Comportamento Alimentar/efeitos dos fármacos , Glicina/química , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Masculino , Metionina/química , Dados de Sequência Molecular , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Conformação Proteica , Ratos , Ratos Sprague-Dawley , Receptores da Colecistocinina/efeitos dos fármacos , Receptores da Colecistocinina/metabolismo
6.
Int J Pept Protein Res ; 39(4): 337-47, 1992 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1428523

RESUMO

Cholecystokinin represents a family of gut hormones which among other activities, have been proposed to participate in satiety signaling. Ac-CCK-7[Ac-Tyr(SO3H)-Met-Gly-Trp30-Met-Asp-Phe-NH2 (2)] possesses the full spectrum of activity and potency of the intact hormone; thus analogs of 2 may be useful as anorectic agents. A series of derivatives has been prepared in which the tryptophan indole moiety of 2 has been modified. The new compounds were assayed in CCK binding assays using homogenated rat pancreatic membranes and bovine striatum as a source of CCK-A and CCK-B receptors respectively and in vivo in rats for anorectic activity. Although previous studies have concluded that the indole ring of Trp30 is a critical pharmacophore for the interaction of CCK with both its A and B type receptors, we find 2-Nal30-Ac-CCK-7 (20) to be nearly equipotent to 2 in both CCK binding and as an anorectic agent sensitive to blockade by the Merck CCK-A receptor antagonist MK-329. The extreme structural sensitivity of this anorectic activity is illustrated by the 1-naphthylalanine30 (19) and (benzo[b]thien-2-yl)alanine30 (21) analogs which are 30 and 100 times less potent than 2 respectively. Other mono- and bicyclic Trp30 replacements, including substituted phenylalanines, 3-quinolinylalanine, and 2-(5,6,7,8-tetrahydro)naphthylalanine, gave inactive compounds.


Assuntos
Fragmentos de Peptídeos/química , Sincalida/química , Triptofano/química , Sequência de Aminoácidos , Animais , Depressores do Apetite/química , Proteínas Sanguíneas/química , Bovinos , Corpo Estriado/química , Dados de Sequência Molecular , Pâncreas/química , Fragmentos de Peptídeos/análise , Ratos , Sincalida/análise , Relação Estrutura-Atividade
7.
J Med Chem ; 34(3): 1125-36, 1991 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2002454

RESUMO

A series of analogues of the satiety-inducing peptide cholecystokinin (CCK-8) was prepared in which the sulfated tyrosine required for activation of peripheral receptors was replaced with a carboxy(alkyl)- or tetrazolyl(alkyl)-phenylalanine to investigate whether an organic acid could serve the role of the sulfate group at the receptor. The necessary intermediates were prepared by previously reported procedures or by alkylation of carboxy(alkyl)- or tetrazolyl(alkyl)phenylmethyl bromides with a glycine-derived anion followed by protecting-group manipulations, and these were incorporated into derivatives of acetyl-CCK-7 using solid-phase synthesis. Peptide analogues were evaluated in a CCK-binding assay for affinity for either peripheral (CCK-A) receptors using homogenated rat pancreatic membranes as the receptor source or for central (CCK-B) receptors using bovine striatum as the receptor source. They were further evaluated for effects on food intake in rats after intraperitoneal (ip) injection. A number of the compounds reported are active in the CCK-A receptor binding assay although less potent than acetyl-CCK-7 and decrease food intake with comparable potency to acetyl-CCK-7. In a meal feeding model designed to assess appetite suppressant activity, acetyl-CCK-7 has an ED50 of 7 nmol/kg ip, while the ED50s of Ac-Phe(4-CH2CO2H)-Met-Gly-Trp-Met-Asp-Phe-NH2 (28) and Ac-Phe[4-(tetrazol-5-yl)]-Met-Gly-Trp-Met-Asp-Phe-NH2 (34) were 9 and 11 nmol/kg ip, respectively. An analogue of 28 lacking the N-terminal acetamido group, 3-[4-(carboxymethyl)-phenyl]propanoyl-Met-Gly-Trp-Met-Asp-Phe-NH2 (50), was also active in the meal feeding assay with an ED50 of 3 nmol/kg ip. Its anorexic effect was blocked by simultaneous administration of the CCK-A receptor antagonist MK 329, indicating that the observed anorexic activity is mediated by CCK-A receptors. We conclude from this work that the requirement for a negative charge at the CCK-A receptor provided in the natural substrate by a sulfate group can be satisfied by organic acids.


Assuntos
Ácidos Carboxílicos/química , Sincalida/análogos & derivados , Sulfatos/química , Tetrazóis/química , Animais , Bovinos , Membrana Celular/metabolismo , Fenômenos Químicos , Química , Corpo Estriado/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Privação de Alimentos , Masculino , Estrutura Molecular , Pâncreas/metabolismo , Ratos , Ratos Endogâmicos , Receptores da Colecistocinina/metabolismo , Sincalida/química , Sincalida/metabolismo , Sincalida/farmacologia , Relação Estrutura-Atividade
8.
Pept Res ; 4(2): 59-65, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1815779

RESUMO

Analogs of cholecystokinin (CCK-7) in which the N-terminal Tyr(SO3H) was acetylated, Asp6 replaced with Thr(SO3H) and Phe7 replaced with N-methyl-Phe were prepared by solid-phase peptide synthesis and evaluated for their receptor-binding activity and ability to suppress appetite. The receptor binding activities of these synthetic analogs of CCK-7 and their selectivity for the CCK-A and CCK-B receptor subtypes were determined using solubilized membrane preparations from rat pancreatic tissue and bovine striatum. The synthetic peptide Ac-Tyr(SO3H)-Met-Gly-Trp-Met-Thr(SO3H)-N-methyl-Phe-NH2 (referred to as Ro 23-7014) demonstrated superior satiating potency (ED50 = 0.3 micrograms/kg, i.p.), increased selectivity for CCK-A receptors (400-fold), increased resistance to peptidergic degradation and a longer duration of action (4 to 5 hours). This analog also effectively suppressed food intake following intranasal administration (ED50 = 100 micrograms/kg). These studies demonstrate the feasibility of designing analogs of CCK-8 with greater selectivity, potency and duration of action, which may be useful as nonsystemically administered appetite suppressants.


Assuntos
Colecistocinina/análogos & derivados , Receptores da Colecistocinina/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Colecistocinina/síntese química , Colecistocinina/metabolismo , Colecistocinina/farmacologia , Preparações de Ação Retardada , Cobaias , Masculino , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Ensaio Radioligante , Ratos , Ratos Endogâmicos , Sulfatos
9.
Int J Pept Protein Res ; 21(1): 93-9, 1983 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-6826286

RESUMO

The primary structure of the 28-peptide thymosin alpha 1 as determined by Goldstein et al. (1) has been confirmed by independent procedures. Limited dilute acid digestion generated a 26-peptide and a 22-peptide both extending to the C-terminal and lacking the N-terminal blocking group. A combination of Edman microsequencing, carboxypeptidase Y and thermolysin digestion, and fast atom bombardment mass spectrometry was used.


Assuntos
Timosina , Hormônios do Timo , Sequência de Aminoácidos , Fragmentos de Peptídeos , Conformação Proteica
10.
Int J Pept Protein Res ; 15(4): 377-98, 1980 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-6252107

RESUMO

A solution synthesis of human beta-endorphin (beta-EP) was carried out by condensation of protected peptide segments bearing N alpha-tert.-butyloxycarbonyl groups and benzyl-derived groups for the protection of functionalities in amino acid side chains. Five intermediate segments were assembled in a stepwise manner starting at the carboxyl terminus. Thus, the segment of sequence region (27-31) was coupled to segment (22-26) by the azide method. Segment (19-21) was incorporated into the growing chain by azide coupling, and segment (10-18) by dicyclohexylcarbodiimide coupling in the presence of 1-hydroxybenzotriazole (DDC-HOBt). Solubility problems in condensing the ensuing 22-peptide with segment (1-9) by DDC-HOBt were overcome by using a dimethylformamidephenol mixture as a solvent. Protecting group cleavage by Na in liquid NH3 was much superior to liquid HF which gave rise to many decomposition products. Homogeneous betah-EP indistinguishable from authentic material in physiochemical and biological properties, was obtained in a single preparative reversed phase liquid chromatographic step after protecting group cleavage.


Assuntos
Compostos de Benzil , Endorfinas/síntese química , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Dicroísmo Circular , Endorfinas/isolamento & purificação , Endorfinas/farmacologia , Cobaias , beta-Endorfina
11.
Int J Pept Protein Res ; 9(2): 129-36, 1977.
Artigo em Inglês | MEDLINE | ID: mdl-190180

RESUMO

A simple preparative system is described for rapid and efficient purification of protected synthetic peptides on a gram scale by high performance liquid chromatography on prepacked silica gel 60 columns. A variety of protected peptides up to tetradecapeptides have been chromatographed at pressures of 50 to 150 psi and obtained in analytically pure from within 2 to 4 h. With such commonly used protecting groups as N-benzyloxycarbonyl (Z), N-2-(p-biphenylyl)-2-propyloxycarbonyl (Bpoc), N-t-butyloxycarbonyl (Boc), O- and S-t-butyl (But), and S-acetamidomethyl (Acm), compounds were sufficiently soluble in chloroform, alcohols, acetic acid, or mixtures of these solvents for column loading. Dimethylformamide was also used as a solvent for loading. Solvent systems for column elution in isocratic, stepwise, or gradient modes were composed of chloroform, isopropanol, ethanol, or methanol and acetic acid in ratios that differed for each protected peptide depending on Rf values on t.l.c. plates. A simple chromatography is described which was self-assembled using standard instruments commonly in use in most laboratories. A shut-off valve was designed to prevent loss of material between fractions.


Assuntos
Cromatografia Líquida/métodos , Peptídeos/isolamento & purificação , Cromatografia em Camada Fina , Géis , Técnicas In Vitro , Peptídeos/síntese química , Dióxido de Silício , Solventes , Fatores de Tempo
12.
J Chromatogr ; 129: 287-93, 1976 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-187608

RESUMO

A variety of protected synthetic peptides were purified by high-performance liquid chromatography on pre-packed silica gel columns. The compounds varied in protecting groups, amino acid composition and molecular weight. Flow-rates of two to four column volumes per hour were employed, with resultant back pressure of less than 150 p.s.i. A typical column load of 0.2-5 g was purified in 2-5 h.


Assuntos
Cromatografia Líquida de Alta Pressão , Peptídeos/síntese química , Sequência de Aminoácidos , Géis , Peso Molecular , Dióxido de Silício , Fatores de Tempo
13.
Nucleic Acids Res ; 2(1): 43-60, 1975 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1093143

RESUMO

A synthetic DNA fragment of 19 residues was enlarged by the enzymatic addition of deoxyadenylate residues to its 3'-end with calf thymus terminal deoxynucleotidyl transferase. The 3'-terminus of this elongated DNA strand was blocked with 2', 3'-dideoxyadenylate to prevent hydrolysis by the 3'-exonuclease function of E. coli DNA polymerase I. This elongated and 3'-blocked fragment was annealed to an oligomeric primer and used as a template for the synthesis of a complementary copy of the synthetic 19-mer. The product of such a repair synthesis was separated by gel filtration and analyzed by nearest neighbor techniques. All template strands were copied with complete repair in over 90% of the chains. Facile recovery of the elongated template by virtue of its size permitted repetition of the copy process, thus allowing accumulation of the desired strand.


Assuntos
DNA Nucleotidiltransferases/metabolismo , Replicação do DNA , DNA Bacteriano/biossíntese , Animais , Bovinos , DNA/biossíntese , Escherichia coli/enzimologia , Métodos , Hibridização de Ácido Nucleico , Oligonucleotídeos/biossíntese , Polinucleotídeo 5'-Hidroxiquinase , Moldes Genéticos , Timo/enzimologia
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