M. Bodanszky, Y. Klausner, C. Y. Lin
Jul 24, 1974
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0
Influential Citations
58
Citations
Journal
Journal of the American Chemical Society
Abstract
An octacosapeptide with the amino acid sequence proposed for the vasoactive intestinal peptide (VIP) was synthesized in solution through isolated intermediates. The protected heptapeptide derivative, terf-butyloxycarbonyl-~-threonyl-~-aspartyl-~-asparaginyl-~-tyrosyl-~-threonyl-~-arginyl-~-leucine azide, was coupled to the partially deprotected pentadecapeptide ~-arginyl-N‘-benzyloxycarbonyl-~-lysyl-~-glutaminyl-~-methionyl-~-alanyl~-valyl-N‘-benzyloxycarbonyI-~-lysyl-N‘-benzyloxycarbonyI-~-lysyl-~-tyrosyl-~-leucyl-~-asparaginyl-~ seryl L isoleucyl-L-leucyl-L-asparaginamide (hydrochloride). The resulting protected docosapeptide, corresponding to sequence VIPi--28, was partially deprotected and acylated with tert-butyloxycarbonyI-L-histidyl-L-seryl-L-asparty1-Lalanyl-L-valyl-L-phenylalanine azide to afford a protected octacosapeptide encompassing the entire sequence of VIP. The protecting groups were removed in a single operation with trifluoroacetic acid, After purification by countercurrent distribution, the product was compared with natural (porcine) VIP. The synthetic peptide showed the characteristic biological activities of the natural material in systemic vasodilation and reduction of arterial blood pressure in intact dogs and relaxation of several isolated smooth muscle preparations, Comparisons of the fragments formed in side-by-side degradations of samples of natural and synthetic VIP with specific enzymes support the amino acid sequence (Figure 1) proposed for porcine VIP. vasoactive intestinal peptide (VIP) was isolated A from hog intestines by Said and Its effects on peripheral and splanchnic blood flow and on smooth muscle preparations have been described by Said and Mutt3 and by Piper, Said, and Vane.4 The amino acid sequence of the single chain octacosapeptide was determined by Mutt and Said5 and is shown in Figure 1. The synthesis of VIP was carried out with the principal aim of providing independent evidence for the correctness of the sequence elucidated by degradation. Even the scheme of the synthesis was influenced in part by considerations involving proof of structure by synthesis. E.g., a hexapeptide sequence, VIP1-6,6 with phenylalanine at its N-terminus was selected as a fragment because this is one of the points where chymotrypsin cleaves the molecule of VIP and a comparison of a fragment from natural VIP with the corresponding synthetic peptide was an attractive possibility. For similar reasons, the C-terminal cyanogen bromide fragment, VIPls-zs,7 was chosen as a larger building unit. The scheme of the synthesis is shown in Chart I. Strategy. The stepwise approach, building a peptide chain from its C-terminal residue with the addition of a single amino acid at a time, preferentially introduced as an active ester, is often the method of choice, for reasons described earlier.8 In the synthesis of VIP, technical difficulties rendered an attempted stepwise building of the chain impractical. The protected intermediates, from the C-terminal tripeptide on, were extremely insoluble in the solvents commonly used in peptide (1) S. I. Said and V. Mutt, Nature(London1, 225,863 (1970). (2) S. I. Said and V. Mutt, Eur. J. Biochem., 28,199 (1972). (3) S. I. Said and V. Mutt, Science, 169, 1217 (1970). (4) P. J. Piper, S. I. Said, and J. R. Vane, Nature (London), 225, 1144 ( 5 ) V. Mutt and S. I. Said, Eur. J . Biochem., in press. (6) Y. S. Klausner, V. Mutt, and M. Bodanszky, Bioorg. Chem., 2, (7) M. Bodanszky, Y. S. Klausner, and V. Mutt, Bioorg. Chem., 2, (8) M. Bondanszky, Ann. N. Y. Acad. Sci., 88,655 (1960). (1970).