Tools for the Identification of Receptor Dimmers: Synthesis and Biological Evaluation of On-Resin Dimerized, Photosensitive Analogues of Angiotensin II

Abstract

Molecular tools for the identification of homodimers of G protein coupled receptors are not available yet. We intended to investigate the possibility of functional dimerization of the known angiotensin II (AngII, DRVYIHPF) receptors AT1 and AT2; either as homo-dimers of AT1 or AT2 or as heterodimers of AT1–AT2. For this purpose, we synthesized AngII dimer ligands with a photosensitive residue (p-benzoylphenylalanine, B) in the C-terminal position. The dimers were held together with linkers anchored either to the Nα-terminus (I), or to Ne of Lys in position 1 (II), or to Ne of Lys in position 3 (III), or to p-NH2-Phe replacing Y in position 4 (VI). The linker was of the general structure (CO(CH2)nNH)2 X, where n = 10 and X is either CO or CO(CH2) m CO with m = 1–10. Fmoc peptide synthesis was carried out on Tentagel resin (0.06 meq/g) or Wang resin (0.7 meq/g). Orthogonality for linker introduction was achieved with Nα-Boc-Ne-Fmoc-Lys for group II, with Nα-Fmoc-Ne-Dde-Lys for III, and Nα-Fmoc-Nar-Dde-p-NH2-Phe for VI. Couplings were performed with TPTU. On-resin dimerization was achieved after Fmoc deprotection of the first linker moiety, by coupling for 2 h with either carbonyl-diimidazole or the respective diacid disuccinimide in half-equivalent amount, followed by a five-fold excess of dimerization reagent until a negative ninhydrine test. Peptide resins were cleaved with 95% TFA & 2% triisopropylsilane; crude peptides were purified by prep RP-HPLC, lyophilized and analyzed by electrospray MS.