RESUMO
Coupling reagents have been proposed for peptide synthesis ever since the introduction of the method because of the convenience of the procedure, which consists of peptide bond formation by addition of a specific condensing agent to the mixture of carboxyl and amine components. However, truly efficient and yet innocuous coupling reagents are hard to find. Most coupling reagents give rise to unwanted reactions, which can be traced to certain characteristic structural features in their molecules. Such features are illustrated with the intermediates generated by dicyclohexylcarbodiimide and N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's reagent K). Some side reactions can be avoided if peptide bond formation is carried out in two separate steps: activation of the carboxyl group of the carboxyl component in the form of an active ester, followed by the catalyzed aminolysis of the active ester.
Assuntos
Indicadores e Reagentes , Peptídeos/síntese química , Aminas/química , Dicicloexilcarbodi-Imida/químicaRESUMO
Incorporation of single amino acid residues into peptide chains built on insoluble polymeric supports a priori appeared promising: the use of isolated, well defined (and potentially commercially available) reactive intermediates were expected to reduce the extent of undesired side reactions. In spite of these expectations active esters were only infrequently used in solid-phase peptide synthesis, mainly because the reaction rates achieved with them were insufficient for rapid chain-lengthening that became possible with automated instruments. In recent years, however, a certain revival of the active ester principle can be noted. This is the consequence of two factors: the application of highly reactive esters and the discovery of efficient catalysts of the ester-aminolysis reaction. The mechanism of catalysis and its explantation for further improvements are also discussed.
Assuntos
Peptídeos/síntese química , Acilação , Catálise , Ésteres , MétodosRESUMO
The significance of a well defined molecular architecture in hormone receptor interaction and the methods available for the study of preferred conformations are discussed. The conformational freedom in glucagon is a major obstacle in the determination of its biologically relevant geometry. In the secretin molecule intramolecular forces generate a folded, partially helical conformation. In respect of long range cooperative interactions resulting in a compact molecule with secondary-tertiary structure secretin is similar to globular proteins. In VIP some characteristics of secretin and also of glucagon can be recognized. Further progress in conformation analysis can be expected from the study of rigid, cyclic analogs in which the biological activities of the parent hormones are retained or even enhanced. Such analogs have well defined conformations without external stabilization from membrane mimicking lipids. Therefore, they provide information on the biologically relevant geometry of the hormones and contribute also to our knowledge of receptor sites.
Assuntos
Glucagon , Secretina , Peptídeo Intestinal Vasoativo , Sequência de Aminoácidos , Sítios de Ligação , Glucagon/análogos & derivados , Peptídeos Cíclicos/síntese química , Conformação Proteica , Receptores de Superfície Celular/metabolismo , Secretina/análogos & derivados , Peptídeo Intestinal Vasoativo/análogos & derivadosRESUMO
An attempt was made to discern ideas and trends in the development of peptide synthesis and to recognize general principles of the discipline. Introduction of efficient methods of activation and coupling during the early years of the reviewed period was followed by only moderate further improvements. Major advances were achieved by the discovery of novel methods of protection and by techniques of facilitation. Improvements in the methods of deblocking hold considerable promise and might bring significantly closer the goal of peptide synthesis: the direct preparation of homogeneous products.
Assuntos
Peptídeos/síntese química , Sequência de Aminoácidos , Anidridos , Fenômenos Químicos , Química , Ésteres , Indicadores e Reagentes , Relação Estrutura-AtividadeRESUMO
Self-association of peptides (related to the C-terminal sequence of porcine secretin) in methylene chloride was disrupted by adding dimethylsulfoxide in increasing amounts. This structural transition was monitored by the disappearance of the amide-I C = O stretching band of strongly intermolecularly hydrogen-bonded molecules (1625-1630 cm-1) in the infrared absorption spectra. The effects induced by main-chain length and sequence, type of N alpha-protection, and concentration were assessed. Hexamethylphosphortriamide was compared for its structure-disrupting properties to dimethylsulfoxide. The increasing propensity to aggregate displayed by these peptides is paralleled by a decrease in their solubility. The impact of these results on the planning of peptide syntheses is briefly discussed.
Assuntos
Peptídeos/análise , Secretina/análise , Animais , Fenômenos Químicos , Química , Conformação Proteica , Solubilidade , Espectrofotometria Infravermelho , SuínosRESUMO
The decrease in the solubility of peptides when their methionine residues are replaced by methionine sulfoxides prompted the exploration of an alternative approach to the protection of the thioether in methionine side chains. Alkylation of tert.-butyloxycarbonyl methionine p-nitrophenyl ester with methyl p-toluenesulfonate yielded the crystalline derivative of methionine S-methyl p-toluenesulfonate which could be incorporated into peptide chains. Alternatively, methionine S-methyl p-toluenesulfonate (Mmt) residues could be generated by the action of methyl p-toulensulfonate on methionine containing peptides. The protecting group remained intact under the conditions of aminolysis and ammonolysis commonly used in peptide synthesis, and it was unchanged after the removal of other blocking groups with trifluoroacetic acid or diethylamine. On treatment with hydrobromic acid in acetic acid the toluenesulfonate anion was replaced by bromide ion, while hydrogenation resulted in the decomposition of the modified methionine side chain. In the process of deprotection Mmt residues could be smoothly converted to methionine residues by thiolysis. Thus, the protecting group functioned well in several respects but an increase in solubility (in dimethylformamide) on alkylation was observed only in a part of the peptide derivatives tested. Therefore, the value of the new approach for the protection of the methionine side chain in peptide synthesis remains to be established.
Assuntos
Metionina , Peptídeos/síntese química , Sulfetos , Alquilação , Estabilidade de Medicamentos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Rotação OcularRESUMO
The 9-fluorenylmethyl (Fm) group was examined with respect to its potential for blocking the sulfhydryl function. The S-Fm group is resistant to acids and to catalytic hydrogenation but is cleaved by ammonia in methanol or by organic bases, such as a 20% solution of piperidine in dimethylformamide. Synthesis of N-tert.-butyloxycarbonyl-S-9-fluorenylmethyl-L-cysteine p-nitrophenyl ester and of cysteinyl peptides protected with the S-Fm group are described.
Assuntos
Cisteína/análogos & derivados , Fluorenos/síntese química , Peptídeos/síntese química , Cisteína/síntese química , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Rotação OcularRESUMO
In order to avoid base catalyzed side reactions during coupling, attempts were made to render superfluous the addition of tertiary amines to the reaction mixture. Weak acids were applied for the removal of acid labile protecting groups. Acetic acid and other carboxylic acids were considered unsuitable for this purpose coupling step. Pentachlorophenol and 2,4-dinitrophenol cleaved the Bpoc, Nps and Trt groups but more practical rates were reached with solutions of 1-hydroxybenzotriazole (HOBt) in trifluoroethanol, in acetic acid, or in a mixture of phenol and p-cresol. In addition to acidolysis, HOBt salts of amino components could also be obtained through hydrogenolysis of the Z group or thiolysis of the Nps group in the presence of HOBt, or by the displacement of acetic acid from acetate salts with HOBt. Acylation of HOBt salts of amino components with symmetrical or mixed anhydrides or with active esters did not require the addition of tertiary amine.
Assuntos
Aminas , Peptídeos/síntese química , Acilação , Fenômenos Químicos , Química , Concentração de Íons de Hidrogênio , TriazóisRESUMO
To explore the importance of the charge of the amino acid in position 15 in influencing the apparent affinities of VIP and secretin for their receptors on pancreatic acinar cells, we tested the synthetic C-terminal 23-peptide fragment of secretin (S5-27) and two analogues containing substitutions in position 15 for their abilities to interact with secretin-preferring and VIP-preferring receptors on pancreatic acinar cells. In inhibiting the increase in cyclic AMP caused by VIP acting through the VIP-preferring receptors, 15-Lys-S5-27 was equipotent with 15-Asn-S5-27, and these analogues were significantly more potent than S5-27. In inhibiting the increase in cyclic AMP caused by secretin acting through the secretin-preferring receptors, S5-27 was equipotent with 15-Asn-S5-27, and these peptides were significantly more potent than 15-Lys-S5-27. These findings indicate that the affinities of these 23-peptides for the VIP-preferring receptors and for the secretin-preferring receptors were influenced primarily by the absence of a particular charge in position 15 but not by the presence of the opposite charge.
Assuntos
Hormônios Gastrointestinais/metabolismo , Pâncreas/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores dos Hormônios Gastrointestinais , Secretina/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Cobaias , Receptores Acoplados a Proteínas G , Receptores de Peptídeo Intestinal Vasoativo , Relação Estrutura-AtividadeAssuntos
Hormônio Adrenocorticotrópico/metabolismo , Hormônio Liberador da Corticotropina/farmacologia , Hormônios Hipotalâmicos/farmacologia , Oligopeptídeos/farmacologia , Adeno-Hipófise/metabolismo , Animais , Bovinos , Hormônio Liberador da Corticotropina/isolamento & purificação , Distribuição Contracorrente , Hormônios Hipotalâmicos/isolamento & purificação , Técnicas In Vitro , Oligopeptídeos/isolamento & purificação , Adeno-Hipófise/efeitos dos fármacosRESUMO
The C-terminal tricosapeptide of secretin (S5-27) and two analogues, one with asparagine replacing aspartic acid in position 15 (15-Asn-S5--27) and one with lysine replacing aspartic acid in position 15 (15-Lys-S5-27) were tested for their abilities to interact with hormone receptors on pancreatic acinar cells. In interacting with the receptors which prefer vasoactive intestinal peptide (vasoactive intestinal peptide-preferring receptors), the apparent affinity of 15-Asn S5-27 was equal to that of 15-Lys-S5-27 and was greater than that of S5-27. In interacting with secretin-preferring receptors, the apparent affinity of 15-Asn-S5--27 was equal to that of S5-27 and was greater than that of 15-Lys-S5-27. In interacting with the secretin-preferring receptors each of the secretin fragments was approximately 2% as effective as secretin in causing an increase in cellular cyclic AMP. None of these fragments was able to cause a detectable increase in cyclic AMP mediated by the vasoactive intestinal peptide-preferring receptors. The dose vs. response curves for the action of secretin and vasoactive intestinal peptide on cellular cyclic AMP and on amylase secretion as well as the pattern of effects of secretin fragments on these actions indicated that the increase in amylase secretion caused by vasoactive intestinal peptide and secretin is mediated exclusively by the vasoactive intestinal peptide-preferring receptors. Furthermore, occupation of approximately 50% of the vasoactive intestinal peptide-preferring receptors is sufficient to cause maximal stimulation of amylase secretion.
Assuntos
Hormônios Gastrointestinais/metabolismo , Pâncreas/metabolismo , Receptores de Superfície Celular/metabolismo , Secretina/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Amilases/metabolismo , Animais , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Cobaias , Masculino , Pâncreas/citologia , Fragmentos de Peptídeos/metabolismo , Peptídeos/farmacologia , Secretina/análogos & derivados , Secretina/farmacologia , Relação Estrutura-AtividadeRESUMO
Acylation of the hydroxyl groups in the side chains of serine, threonine or tyrosine occurs in coupling with active esters. This side reaction, which is quite pronounced in histidine-containing peptides, can be prevented with additives. From a series of compounds tested, 2,4-dinitrophenol and pentachlorophenol were the most effective.
Assuntos
Peptídeos/síntese química , Acilação , Sequência de Aminoácidos , Dinitrofenóis , Ésteres , Pentaclorofenol , Serina , Treonina , TirosinaRESUMO
The base-catalyzed ring closure of beta-benzylaspartyl peptides was efficiently suppressed by the addition of phenols (with electron-withdrawing substituents) to the reaction mixtures. From a series of compounds tested, 2,4-dinitrophenol and pentachlorophenol were the most effective. No direct relationship was found between the acidity of the additives and their ability to suppress the formation of aminosuccinyl peptides. The applicability of 2,4-dinitrophenol and pentachlorophenol in practical syntheses was also examined.
Assuntos
Peptídeos/síntese química , Fenóis , Acilação , Catálise , Fenômenos Químicos , Química , Dinitrofenóis , PentaclorofenolRESUMO
The solution conformation of a 7-peptide with the C-terminal sequence of cholecystokinin was investigated by evaluation of intramolecular resonance energy transfer between tyrosine (donor) in position 1 and tryptophan (acceptor) in position 4. From the relative enhancement of acceptor fluorescence a transfer efficiency of 0.70 +/- 0.04 was determined. The use of this parameter in Förster's equation permitted the calculation of the average intramolecular tyrosine-tryptophan separation, whereby the assumption of random donor-acceptor orientation was made. The resulting average distance of 10.0 +/- 0.3 A suggests some type of a folded conformation and excludes the existence of a fully extended chain in the N-terminal part of the peptide. A comparison with tyrosine-tryptophan distances observed in other biologically active polypeptides is made.
Assuntos
Colecistocinina/análogos & derivados , Peptídeos/análise , Triptofano/análise , Tirosina/análise , Sequência de Aminoácidos , Transferência de Energia , Conformação MolecularRESUMO
The acid catalyzed O leads to C migration of the benzyl group in the side chain of tyrosine could be reduced by applying HBr in a mixture of phenol and p-cresol instead of BHr in trifluoroacetic acid for acidolytic deprotection. This side reaction occurs also during the removal of Boc groups. The loss of O-benzyl protection and the formation of 3-benzyltyrosine residues could be suppressed by the application of a 7:3 mixture of trifluoroacetic acid and acetic acid. The acid- and base-catalyzed ring closure of beta-benzylaspartyl residues to aminosuccinyl derivatives was also studied. In this case HBr in trifluoroacetic acid was found to be relatively harmless. Deprotection with HBr in a mixture of trifluoroacetic acid and p-cresol can be applied for peptides that contain both beta-benzylaspartyl and O-benzyltyrosyl residues. An attempt to reduce the rate of the base-catalyzed side reaction by application of hindered tertiary amines was abandoned because the tertiary amines which were effective in this respect let to significant reduction of the rate of the desired reaction, the aminolysis of active esters, as well. A satisfactory solution for the problem was found in the selective catalysis of the active ester reaction with 1-hydroxybenzotriazole or 4-dimethyl-aminopyridine. These catalysts do not enhance the rate of ring closure and in their presence essentially pure beta-benzylaspartyl peptides can be produced in good yield.
Assuntos
Ácido Aspártico , Peptídeos/síntese química , Tirosina , Compostos de Benzil , Fenômenos Químicos , Química , MétodosRESUMO
The base catalyzed ring closure in t-Boc-Asp-X beta-napthylamides was examined in a series of 2-peptide derivatives in which position X was occupied by the neutral and acidic amino acid residues that occur in proteins. Bulkiness and functional groups in the side chain of X have a major effect on the rate of cyclization, e.g. acidic groups slow down the formation of aminosuccinyl derivatives. Rate-enhancing effect can be observed in serine and threonine, while the side reaction is unexpectedly slow when X is methionine.
Assuntos
Peptídeos/síntese química , Aminoácidos , Ácido Aspártico , Compostos de Benzil , Fenômenos Químicos , Química , Métodos , Relação Estrutura-Atividade , SuccinatosRESUMO
A new analogue of oxytocin was constructed from L-tyrosyl-L-isoleucyl-L-glutaminyl-L-asparaginyl-L-lysyl-L-prolyl-L-leucyglycinamide. Reaction of this 8-peptide amide with di-p-nitrophenyl carbonate yielded a cyclic compound, in which the -CH2SSCH2-bridging portion of oxytocin formed by the oxidative linking of the two cysteine side chains was replaced by the -CH2CH2CH2CH2-group of lysine, while the epsilon-NH2 group of the same residue took the place of the alpha-CH of cysteine-1. The N-terminal amino group of oxytocin, which is not necessary for its hormonal activities, was omitted. The new analogue, referred to as [1,6-Nepsilon-carbonyl-L-lysine]oxytocin, possessed a rat uterotonic activity in vitro of 3.9 +/- 0.3 units/mg, less than 0.5 unit/mg of rat antidiuretic activity, and caused a marked tachyphylaxis in the rat pressor assay. Moreover, the analogue was a strong competitive inhibitor, with a pA2 value of 7.27 +/- 0.13 of the oxytocin induced vasodepressor response in chickens.
Assuntos
Ocitocina/análogos & derivados , Animais , Galinhas , Feminino , Masculino , Ocitocina/síntese química , Ocitocina/farmacologia , Ratos , Útero/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , Vasopressinas/metabolismoRESUMO
The effects of synetic porcine vasoactive intestinal peptide (VIP) on pancreatic and biliary secretion and on glucose, insulin and calcium blood levels were examined in the dog and compared with the effects of natural porcine VIP. Synthetic VIP was a secretin-like partial agonist of pancreatic and biliary secretion with efficacies relative to secretin of 0.27 and 0.41, respectively. Consistent with its weak secretin-like action, synthetic VIP augmented the pancreatic response to CCK-OP and the submaximal but not the maximal response to secretin. Synthetic VIP also produced a dose-dependent increase in blood glucose levels and a synchronous and proportionate increase in blood insulin levels. A slight increase in total calcium levels was equivocal. The effects of antural VIP on pancreatic and biliary secretion and on glucose and insulin blood levels were identical to those of synthetic VIP. The identity of effects supports the validity of the postulated structure of porcine VIP. Although elicited at high doses, the effects of VIP on exocrine and endocrine secretion may be relevant physiologically in the context of a neurocrine or paracrine role for VIP.
Assuntos
Bile/metabolismo , Glicemia , Hormônios Gastrointestinais/farmacologia , Insulina/sangue , Fígado/metabolismo , Pâncreas/metabolismo , Suco Pancreático/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Bicarbonatos/metabolismo , Glicemia/análise , Pressão Sanguínea/efeitos dos fármacos , Cálcio/sangue , Cães , Relação Dose-Resposta a Droga , Feminino , Frequência Cardíaca/efeitos dos fármacos , Fígado/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/administração & dosagemRESUMO
Acylation of amino acid beta-naphthylamides with protected (Boc) amino acidisobutylcarbonic acid mixed anhydrides resulted in each case in the formation of some undesired by-product: an isobutyloxycarbonylamino acid beta-naphthylamide. The amount of this second acylation product was particularly high, with the hindered amino acids valine and isoleucine as carboxyl-components. The nature of the amino component had no major influence on the extent of this side reaction.