Structure–Activity Relationships of GHRP-6 Azapeptide Ligands of the CD36 Scavenger Receptor by Solid-Phase Submonomer Azapeptide Synthesis

Abstract

The cluster of differentiation 36 (CD36) class B scavenger receptor binds a variety of biologically endogenous ligands in addition to synthetic peptides (i.e., growth hormone-releasing peptides, GHRPs), which modulate biological function related to anti-angiogenic and anti-atherosclerotic activities. Affinity labeling had previously shown that GHRP-6 analogues such as hexarelin, [2-Me-W(2)]GHRP-6 (1), bind to the lysine-rich domain of the CD36 receptor. Moreover, the azapeptide analogue [aza-F(4)]GHRP-6, 2, exhibited a characteristic β-turn conformation as described by CD and NMR spectroscopy and a slightly higher CD36 binding affinity relative to hexarelin (1.34 and 2.37 μM, respectively), suggesting receptor binding was mediated by the conformation and the aromatic residues of these peptide sequences. Ligand-receptor binding interactions were thus explored using azapeptides to examine influences of side-chain diversity and backbone conformation. In particular, considering that aromatic cation interactions may contribute to binding affinity, we have explored the potential of introducing salt bridges to furnish GHRP-6 azapeptide ligands of the CD36 receptor. Fifteen aza-glutamic acid analogues related to 2 were prepared by submonomer solid-phase synthesis. The azapeptide side chains were installed by novel approaches featuring alkylation of resin-bound semicarbazone with Michael acceptors and activated allylic acetates in the presence of phosphazene base (BTPP). Moreover, certain Michael adducts underwent intramolecular cyclization during semicarbazone deprotection, leading to novel pyrrazoline and aza-pyroglutamate N-terminal residues. Structural studies indicated that contingent on sequence the [aza-Glu]GHRP-6 analogues exhibited CD spectra characteristic of random coil, polyproline type II and β-turn secondary structures in aqueous media. In covalent competition binding studies with the GHRP-6 prototype hexarelin bearing a radiotracer, certain [aza-Glu]GHRP-6 azapeptides retained relatively high (2-27 μM) affinity for the CD36 scavenger receptor.

Diversity-Oriented Synthesis of Azapeptides with Basic Amino Acid Residues: Aza-Lysine, Aza-Ornithine, and Aza-Arginine

“…Employing a similar synthetic strategy [azaLys 3 ]-GHRP-6 analogues 23 were prepared from benzylideneazaGly-Trp(Boc)-D-Phe-Lys(Boc) rink amide resin (Table 1). 31 Finally, [azaLys 6 ]-GHRP-6 analogs 25 were prepared using methyl-, dimethyl-, and trimethylamines (Scheme 4). Benzylidene-azaGly rink amide resin 24 was obtained by removal of the Fmoc protection from rink amide resin, and treatment of the amine resin with 4-nitrophenyl 2-(diphenylmethylidene)-carbazate, which was prepared from the reaction of benzophenone hydrazone with p-nitrophenylchloroformate (Scheme 4).…”

N-Amino-imidazolin-2-one Peptide Mimic Synthesis and Conformational Analysis

“…10,11 The aza-propargylglycine side chain was later reacted in copper-catalyzed 1,3-dipolar cycloadditions to make aza-1,2,3-triazole-3-alaninyl peptide mimics. 12 Aza-propargylglycinamides have now been explored in 5- exo-dig cyclizations to access N -amino-imidazolin-2-one peptidomimetics, as well as in Sonogashira cross-coupling reactions prior to cyclization to provide their 4-arylmethyl analogues, which may mimic phenylalanine and tryptophan residues.…”

A Review on Azapeptides: The Promising Peptidomimetics

“…Ten aza analogs of GHRP-6 were synthesized, among which the [aza-phe 4 ]-GHRP-6, showed stable β-turn and favoured selective binding to cluster of differentiation 36 (CD36) receptor thereby presenting a lead for angiogenic disorders [99].…”