Ac-[3- and 4-Alkylthioproline31]-CCK4 Analogs: Synthesis and Implications for the CCK-B Receptor-Bound Conformation

Abstract

It has been reported that substitution of the Met31 residue in Boc-CCK4 (Boc-Trp30-Met31-Asp32-Phe33-NH2, CCK33 numbering) by trans-3-propyl-L-proline yields a highly potent and selective CCK-B agonist. To further explore the structural requirements of the Met31 side chain in the receptor-bound conformation of CCK4, we have synthesized several Ac-CCK4 analogs containing substitution of Met31 by 3- and 4-(alkylthio)-substituted proline derivatives. To this end we have developed novel synthetic routes to enantiomerically pure N-Boc-4-cis- and -trans-(methylthio)prolines and racemic N-Boc-3-cis and -trans-[(4-methylbenzyl)thio]prolines. The protected mercaptoprolines were incorporated into Ac-CCK4 analogs using SPPS and were alkylated using various electrophiles following cleavage from the solid support. Binding assays reveal that 3-(alkylthio)prolines analogs have higher affinities at the CCK-B receptor than the corresponding 4-(alkylthio)proline analogs, and that trans-3-(alkylthio)proline analogs had higher affinities than corresponding cis-3-(alkylthio)proline analogs. Within both the cis- and trans-3-(alkylthio)proline series, the order of potency was found to be Me < Et < n-Pr. The trans-3-(n-propylthio)-L-proline analog demonstrates a higher affinity than that reported for Boc-CCK4[trans-3-propyl-L-Pro31]. Comparison of the low-energy structures calculated for several high-affinity Ac-CCK4 analogs reveal a common geometry which we propose to be the CCK-B receptor-bound conformation. This model shows grouping of the hydrophobic side chains of Trp, Met, and Phe at one side of the molecule and the hydrophilic side chain of Asp and the C-terminal carboxamide at the other side.

Towards nonpeptide agonists: Design of ?true? peptidomimetics

“…The synthesis of mercaptoproline derivatives has been described earlier [8] Binding studies for opioid receptors were identical to those described elsewhere [9,10]. They were performed at Washington University by Dr. Bruce Nock.…”

Biological activity of cholecystokinin (30–33) tetrapeptide analogues

“…It was shown that conformational changes in practically all the peptide bonds of CCK-4 affect both affinity and selectivity of analogues for CCRs [26][27][28]. The PROSKURYAKOVA et al . structural modifications have been revealed that resulted in the analogues with pronounced selectivity toward CCR-1 [29].…”

Conformationally readdressed CCK‐B/δ‐opioid pepitide ligands

“…However, we have previously found 24 that the absolute configuration of 3-MP residues in peptides can be assigned by selective reduction of the thiol to the corresponding Dor L-Pro containing analogue using Raney Ni. Comparison of 'H-nmr spectra and reverse phase high performance liquid chromatography ( RP-HPLC) retention times of the reduction products with known compounds allowed the stereochemical assignment to be made.…”

“…, which was prepared as previously described, 24 and BOG( NMe)Nle, which was graciously provided by Dr. Lakmal Boteju (The University of Arizona). Dimethylformamide (DMF) and CH2C12 were HPLC grade and stored over 4 A molecular sieves prior to use.…”