Stereoselective syntheses of 3-mercaptoproline derivatives protected for solid phase peptide synthesis.

The incorporation of cis- and trans-3-mercaptoproline (3-MPc and 3-MPt) into analogs of biologically active peptides has been shown to be an effective means for reducing the conformational mobility of the peptide backbone. We report herein a novel stereoselective synthetic route to L-1 and L-2, derivatives of 3-MPt and 3-MPc suitably protected for solid phase peptide synthesis. The optically active starting material was the previously reported cis-3-hydroxyprolinol derivative L-3. Oxidation of the C1 alcohol to the carboxylic acid, formation of the methyl ester and deprotection of the C3 alcohol yielded L-6 in an overall yield of 68%. Reaction of the secondary alcohol with thiolacetic acid under Mitsunobu conditions gave the thiolacetate L-7 in 77% yield with clean inversion of configuration. Conversion of L-7 to L-1 was accomplished in a one-pot sequence consisting of three steps: hydrolysis of the thiolacetate, formation of the thioether and hydrolysis of the methyl ester. The overall yield of L-1 from L-3 was 38%. Synthesis of L-2 required an epimerization of L-6, which was accomplished using a standard Mitsunobu inversion to give the trans-3-hydroxyproline derivative L-8. Transformation of L-8 to L-2 followed that described for overall yield of L-2 from L-3 was 18%. The availability of pure enantiomers of 3-MPt and 3-MPc protected for SPPS will greatly facilitate their use as conformational constraints for studying peptide-receptor interactions.

Conformationally readdressed CCK-B/delta-opioid peptide ligands.

The sequence of a cholecystokinin (CCK) related peptide was modified to obtain analogues, which interact selectively either with CCK-B, or with delta-opioid receptors. Two kinds of peptides were designed, namely, the cyclic peptides of the H-Tyr-cyclo (D-Pen-Gly-Trp-L/D-3-transmercaptoproline)-Asp-Phe-NH2 sequence (compounds 1a and 1b, respectively), and the linear peptides of the H-Tyr-D-Val-Gly-Trp-L/D-3-trans-methylmercaptoproline-Asp-Phe- NH2 sequence (compounds 2a and 2b, respectively). The only difference between the chemical structures of the linear analogues compared to the cyclic ones is that one covalent bond has been eliminated and a sulfur atom is replaced by a methyl group. Molecular modeling showed that, among low-energy conformers of cyclic compounds 1, there are three-dimensional structures compatible to the model for delta-receptor-bound conformer, suggested earlier [G. V. Nikiforovich, V.J. Hruby, O. Prakash, and C.A. Gehrig (1991) Biopolymers, vol. 31, pp. 941-955]. Results of binding assays fully supported the rationale for the design of compounds 1 and 2. The cyclic analogue 1a has Ki values of 4.5 and > 5000 nM at delta- and mu-opioid receptors, respectively; and IC50 values of 1.6 and > 10,000 nM for CCK-A and CCK-B receptors, respectively. The results of this study demonstrate a possibility to redirect a peptide sequence that interacts with one type of receptors (CCK-B receptors) toward interaction with another type (delta-opioid receptors) belonging to a different physiological system. This redirection could be performed by changing the conformational properties of the peptide with very minimal changes in its chemical structure.

Ac-[3- and 4-alkylthioproline31]-CCK4 analogs: synthesis and implications for the CCK-B receptor-bound conformation.

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.