Yakir S. Klausner

A preferred conformation in the vasoactive intestinal peptide (VIP). Molecular architecture of gastrointestinal hormones

Abstract The ORD spectrum of the vasoactive intestinal peptide (VIP) in water indicates a preferred conformation with low helix content. Addition of organic solvents, especially of trifluoroethanol, results, even at low solvent concentration, in spectra with pronounced helical character. The readiness of shorter chains, with C-terminal sequences of VIP, to take up helical conformation under the effect of organic solvents parallels their biological activity. This suggests that an “active architecture” may be required for the interaction between hormone and receptor.

Synthesis of the vasoactive intestinal peptide (VIP) I. The C-terminal cyanogen bromide fragment

Abstract The C-terminal cyanogen bromide fragment of VIP, Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH 2 (all l ), was synthesized to provide evidence for the correctness of the sequence proposed by Mutt and Said ( 1 ). The synthesis of this hendecapeptide (VIP 18–28 ) was carried out by coupling Z-Ala-Val-(Z)Lys to (Z)Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH 2 . After removal of the protecting groups and purification, the synthetic material was indistinguishable from the natural fragment on paper chromatograms and electropherograms. Their identity was further confirmed by comparison of the products formed on enzymatic hydrolysis.

Synthesis of the vasoactive intestinal peptide (VIP): III. The sequence 7–13

Abstract The protected heptapeptide derivative t -butyloxycarbonyl- l -threonyl-β-benzyl- l -aspartyl- l -asparaginyl- O -benzyl- l -tyrosyl- l -threonyl-nitro- l -arginyl- l - leucine methyl ester was prepared by stepwise chain lengthening. The protecting groups on the side chains of arginine, tyrosine, and aspartic acid residues were removed by hydrogenolysis and the partially deprotected heptapeptide ester converted to the hydrazide, an intermediate in the synthesis of the (porcine) vasoactive intestinal peptide (VIP). After the removal of the tert -butyloxycarbonyl group, the heptapeptide ester was exposed to the action of trypsin which split off its C-terminal residue, l -leucine methyl ester. The hexapeptide was then exposed to chymotrypsin, which cleaved it into an acidic, and a basic fragment. The former was, under the conditions used, indistinguishable on paper chromatography and paper electrophoresis from the tetrapeptide threonyl-aspartyl-asparaginyl-tyrosine which had previously been isolated from natural VIP, of which it comprises the sequence 7–10. Similarly, the basic fragment was indistinguishable from threonyl-arginine, the sequence 11–12 of VIP. This intestinal peptide increases visceral blood flow and reduces blood pressure in the dog, and also causes relaxation of different smooth muscle preparations, e.g., the trachea of guinea pigs. The principal aim of the present synthesis is to provide independent evidence for the sequence of (porcine) VIP.

Synthesis of the vasoactive intestinal peptide (VIP): II. The N-terminal hexapeptide

Abstract The protected hexapeptide derivative t -butyloxycarbonyl- l -histidyl- O -benzyl- l -seryl-β-benzyl- l -aspartyl- l -alanyl- l -valyl- l -phenylalanine methyl ester was prepared from l -phenylalanine methyl ester by stepwise chain lengthening. Hydrogenation of the protected hexapeptide ester followed by hydrazinolysis afforded a derivative that is expected to be useful in the synthesis of VIP. Complete removal of the protecting groups yielded a free hexapeptide, indistinguishable from the N -terminal chymotryptic fragment of the natural hormone. Identical products were obtained from side-by-side treatment of the natural and synthetic hexapeptides with thermolysin and with dipeptidyl aminopeptidase I.

Active Esters and the Strategy of Peptide Synthesis

After the epoch-making discovery of the still unsurpassed carbobenzoxy groups by Bergmann and Zervas (1932), the first major breakthrough in peptide synthesis came in the early 1950s with the introduction of mixed anhydrides by Wieland and Bernhard (1951), Boissonnas (1951), and Vaughan and Osato (1952). The impetus provided by the availability of an easily removable aminoprotecting group and efficient carboxyl activation culminated in the synthesis of oxytocin by du Vigneaud and his coworkers (du Vigneaud et al., 1953, 1954; Katsoyannis and du Vigneaud, 1954; Ressler and du Vigneaud, 1954; Swan and du Vigneaud, 1954). For even more ambitious endeavors, however, carboxyl activation in the form of mixed anhydrides seemed to be not entirely satisfactory. Unsymmetrical anhydrides yield—at least in principle—two acylation products, the desired peptide (A) and an acyl derivative (B) of the amino component (Fig. 1). With the proper choice of the “activating” acid in the mixed anhydride, the formation of the undesired byproduct B can be kept at a minimum, but it is unlikely that the relative electrophilicities of the two carbonyl carbons could be so different that the main product, peptide A, would be completely uncontaminated by some small amount of the byproduct B. [A combination of electron release and of steric hindrance, such as in pivaloyl mixed anhydrides (Zaoral, 1962), might approach the ideal of a single acylation product.]