Coupling in the absence of tertiary amines. V. Removal of the 2-nitrobenzenesulfenyl (NPS) group with 1-hydroxybenzotriazole in the presence of aniline or N-methylaniline.

In a search for conditions of acidolytic removal of amine protecting groups leading to salts of the deblocked amine that can be acylated without addition of a tertiary amine, cleavage of the 2-nitrobenzenesulfenyl (Nps) group with hydroxybenzotriazole (HOBt) in 2,2,2-trifluoroethanol was attempted. The Nps group was smoothly removed, but the resulting salt of the amine component could not be acylated unless deprotonated with a tertiary base. A rationale is now proposed for this unsatisfactory outcome of the cleavage reaction and for the concomitant surprising reduction of HOBt to benzotriazole.

Reactions of C-terminal omega-amino acid residues in liquid hydrogen fluoride.

The benzyl-ester bond linking the C-terminal delta-aminovaleric acid residue of a peptide to a polymeric support was cleaved with liquid hydrogen fluoride in the presence of anisole, added as scavenger. Instead of the expected peptide with a free carboxyl group at the C-terminus, a peptide terminating in a ketone derivative was obtained. The unusual extent of this known side-reaction was attributed to the effect of the distance between the amino group and the carboxyl group in the C-terminal residue.

Contractile activity of vasotocin, oxytocin, and vasopressin on mammalian prostate.

The neurohypophyseal peptides arginine vasotocin, oxytocin and arginine vasopressin contracted guinea pig, rat, canine and human prostates with potencies and efficacies that were comparable to those of noradrenaline and methacholine. All three neuropeptides raised prostatic tone and elicited contractions at 10(-9) or 10(-8) M, with an order of efficacy: arginine vasotocin greater than oxytocin greater than arginine vasopressin. The findings suggest a physiological role for these peptides in prostatic smooth muscle contraction and possibly also in other aspects of male reproductive function.

The myth of coupling reagents.

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.

Active esters in solid-phase peptide synthesis.

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.

Conformation of peptides of the secretin-VIP-glucagon family in solution.

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 search of new methods in peptide synthesis. A review of the last three decades.

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.

Self-association and solubility of peptides. Solvent-titration study of protected C-terminal segments of porcine secretin.

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.

Cyclization studies with a model pentapeptide.

A pentapeptide with the sequence Gly-Ala-D-Val-Leu-Ile was designed for a study of cyclization. Isoleucine was selected as the C-terminal residue in order to determine, from the amount of alloisoleucine in the cyclic product, the extent of racemization during activation and ring closure. The insolubility of cyclo (glycyl-alanyl-D-valyl-leucyl-isoleucyl) in the commonly used solvents facilitated its isolation and thus the evaluation of comparative experiments.

9-Fluorenylmethyl esters.

N alpha-Protected amino acid 9-fluorenylmethyl esters (Fm esters) were prepared by imidazole-catalyzed transesterification of active esters with 9-fluorenylmethanol (9-hydroxymethylfluorene). The new carboxyl protection is unaffected by acids, but is efficiently removed by beta-elimination under the influence of secondary and tertiary amines. Primary amines and ammonia can cause slight amide formation.

Derivatives of S-9-fluorenylmethyl-L-cysteine.

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.

Experiments on the protection of the thioether in methionine.

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.

Coupling in the absence of tertiary amines.

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.

Selective modification of the ability of cholecystokinin to cause residual stimulation of pancreatic enzyme secretion.

In the C-terminal heptapeptide of cholecystokinin (-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2), replacing the aspartic acid residue by beta-aspartic acid did not alter the ability of the peptide to cause stimulation, desensitization, or residual stimulation of enzyme secretion from dispersed pancreatic acini. Replacing the tyrosine sulfate residue by hydroxynorleucine sulfate did not alter the ability of the heptapeptide to cause stimulation or desensitization, but caused a 50-fold decrease in the potency with which the peptide caused residual stimulation of enzyme secretion. These findings suggest that a modification of the N-terminal region of cholecystokinin heptapeptide, which does not alter the ability of the peptide to bind to its receptor on pancreatic acini and by so doing cause stimulation and desensitization of enzyme secretion, can increase the rate at which the bound peptide dissociates when the acini are washed and reincubated.

Allomalformin.

In an attempt to find explanation for the initial erroneous sequence assignment for malformin, a sequence-isomer of the natural product, 3-isoleucine-5-valine malformin or briefly "allomalformin" that on partial acid hydrolysis could have given rise to misleading fragments, was synthesized and compared with both natural and synthetic preparations of malformin. Allomalformin is identical to the parent microbial peptide (malformin A, or briefly malformin) with respect to biological activity and conformation (ORD and CD spectra) and is indistinguishable from it by high pressure liquid chromatography. Yet, the two isomers have slightly different Rf values on thin-layer chromatograms and by this method no allomalformin could be detected in samples of the natural product.

Synthesis and some pharmacological properties of Z-Tyr(SO3H)-Met-Gly-Trp-Met-Asp(Phe-NH2)-OH, a 32-beta-aspartyl analogue of cholecystokinin (pancreozymin) 27-33.

The heptapeptide carbobenzoxy-L-tyrosyl(O-sulfate)-L-methionylglycyl-L-tryptophyl-L-methionyl-L- aspartyl-beta-L-phenylalanine amine (Z-32-beta-Asp-CCK-27-33) was synthesized and tested for its ability to stimulate secretion from dispersed pancreatic acini in vitro, to increase protein secretion from cat pancreas in vivo, and to cause contraction of guinea pig gallbladder in situ. In increasing amylase secretion in vitro, the Z-32-beta-Asp-CCK-27-33 was equal in efficacy with but approximatively one-third as potent as the Boc-CCK-27-33, and when tested in vivo its activity is approximately 10 Ivy dog units (Idu)/microgram. In stimulation of the contraction of the gallbladder, it showed an activity lower than 1 Idu/microgram.

The importance of the amino acid in position 32 of cholecystokinin in determining its interaction with cholecystokinin receptors on pancreatic acini.

In the C-terminal heptapeptide of cholecystokinin, replacement of the penultimate residue, aspartic acid, by beta-alanine caused a 300-fold decrease in the potency with which the peptide stimulated enzyme secretions, whereas replacement by glutamic acid caused a 1000-fold decrease in potency. The beta-alanine-substituted peptide was approximately ten times more potent when the n terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl, and the glutamic acid-substituted peptide was approximately twice as potent when the N terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl. Changes in the ability of the peptide to stimulate amylase secretion were accompanied by corresponding changes in the ability of the peptide to inhibit binding of 125I-labeled cholecystokinin.

Synthesis of 8-L-tryptophan-oxytocin and determination of one of its conformational parameters.

The hormone analog 8-L-tryptophan-oxytocin was synthesized in solution by stepwise chain lengthening from the C-terminal residue. Active esters of 9-fluorenylmethyloxycarbonyl (Fmoc)-amino acids were used for the incorporation of individual residues and thereby exposure to the tryptophan-containing intermediates both to acid conditions and to alkylation could be avoided. In a parallel experiment the parent compound, oxytocin, was prepared similarly.

Importance of the amino acid in position 15 of VIP and secretin in influencing their interactions with membrane receptors on pancreatic acinar cells.

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.