Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N‐terminus, one compound, Ac‐[D‐C∼S6,D‐Cys11,Lys12,Nle17,Val26,Thr28]‐VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N‐terminal region were found to be inactive. A second analog, Ac‐[Lys12,Cys17,Val26,Cys28]‐VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (>40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor–active conformation of VIP.

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,Va126,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 00 300‐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 (>20min) 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.

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Pig tracheal epithelium, a site of extensive mucin biosynthesis, contained polypeptide N-acetylgalactosaminyltransferase activity directed towards L-threonine residues. The enzyme preparation was broadly similar in properties to preparations from other tissues, e.g. pig and bovine submaxillary glands, bovine colostrum, BW5147 mouse lymphoma and baby-hamster kidney cells. Enzyme was membrane-bound and was released from microsomal preparations by extraction with Triton X-100. Extracted enzyme had a pH optimum of 7.5, had a requirement for Mn2+ (10 mM) and was inhibited by Na2EDTA. The Km for UDP-N-acetylgalactosamine was 110 microM and that for an octapeptide acceptor (VTPRTPPP) was 3.0 mM at 37 degrees C. Using a range of synthetic peptides of known structure related to TPPP it was established that L-threonine residues were specifically O-glycosylated probably in the alpha-configuration. Synthetic peptides containing the TPPP sequence required a peptide length of five or more for significant acceptor activity. In VTPRTPPP the two threonine residues were similarly glycosylated, as revealed by tryptic cleavage of the glycosylated product and separation of the 3H-labelled fragments. The enzyme preparation also specifically catalysed the transfer of N-acetylgalactosaminyl residues from UDP-N-acetyl[1-3H]galactosamine to bovine submaxillary mucin core protein and to myelin basic protein.

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