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 Lys20, 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 [Leu13, Lys20 (N epsilon-biotinyl)]porcine motilin (3) and [Leu13, Lys20 (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-biotinylPhe1]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.
Macielag
M J
MJ
Ohmeda PPD, New Providence, New Jersey, USA.
Peeters
T
T
Depoortere
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin; D004386:Duodenum; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000494:pharmacology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects; D011817:Rabbits; D011964:Receptors, Gastrointestinal Hormone / Q000032:analysis*; D018013:Receptors, Neuropeptide / Q000032:analysis*; D013329:Structure-Activity Relationship; D013552:Swine
D015335:Molecular Probes; D011964:Receptors, Gastrointestinal Hormone; D018013:Receptors, Neuropeptide; C050802:motilin receptor; D001710:Biotin