SYNTHESIS AND CHARACTERIZATION OF SITE-SPECIFIC BIOTINYLATED PROBES FOR THE MOTILIN RECEPTOR

The solid-phase synthesis of two porcine motilin derivatives, specifically biotinylated on the side chain of Lys(20), was accomplished by preactivation of the protected amino acids N-alpha-(9-fluorenylmethoxycarbonyl)-N(epsilon)biotinyl- L-lysine and N-alpha-(9-fluorenylmethoxycarbonyl)N-epsilon-[N-(biotinyl)-6-aminohexanoyl]-L-lysine with BOP/ HOBt/DIEA (1:1:2.5) followed by coupling to the support-bound peptide substrate. The biotin moiety was stable to TFA cleavage and repetitive cycles of acylation, as evidenced by the high level of purity (> 80%) of the crude peptides. This direct synthetic approach complements existing orthogonal protection strategies for the site-specific biotinylation of peptides. The derivatized peptides were purified by RP-HPLC and characterized by mass spectral and amino acid analysis. In binding studies using a rabbit antral smooth muscle homogenate, both [Leu(13), Lys(20) (N-epsilon-biotinyl)]porcine motilin (3) and [Leu(13), Lys(20) (N-epsilon-[N-(biotinyl)-6-aminohexanoyl])]porcine motilin (4) possessed nearly equal affinities for the motilin receptor (IC50 = 0.89 and 1.2 nM, respectively) as native porcine motilin (1) (IC50 = 0.76 nM). The biotinylated peptides were also highly potent in tissue bath assays employing rabbit duodenal smooth muscle segments. In contrast, commercially available [N-alpha-biotinylPhe(1)]porcine motilin (5) had markedly lower affinity in the binding assay (IC50 = 30 nM). The relative bioactivities of these receptor probes are in accord with previous synthetic studies on motilin which demonstrated the importance of the amino-terminal segment in the high affinity interaction between the peptide and its receptor. Analog 3 retained high affinity for the motilin receptor in the presence of avidin. Therefore, this peptide is expected to be a valuable tool for the isolation and identification of motilin receptors. (C) Munksgaard 1994.