Amide-to-Ester Substitution Allows Fine-Tuning of the Cyclopeptide Conformational Ensemble

Abstract

Natural or designed peptide ligands rarely bind to cognate receptors in their most stable conformation when in solution. The receptor, through reciprocal induced fitting, applies pressure on the conformational ensemble of the peptide to select its complementary conformation. Given the flexible nature of the peptides’ modular architecture, a bioactive sequence must often be primed for recognition of its target to achieve high binding affinity or specificity. For rational peptide design, cyclization or structure-inducing residues have been successfully used to accomplish ligand preorganization. However, deconvolution of the averaged NMR spectra of strained peptides or other macrocycles had shown that the bound-state conformations are poorly populated in the free uncomplexed state. In small cyclic peptides, the stereochemistry of the backbone, rather than interactions with or among side chains, determines the conformational ensemble by establishing a defined pattern of local torsional preferences. Intramolecular H bonds act on the equilibrium distribution of the conformational ensemble and favor specific conformations. Thus, we envisioned amide-to-ester substitution or “Ester Scan” as an interesting modification for the peptide backbone, which influences the conformational ensemble as well as its equilibrium distribution; this influence is achieved through modulation of the backbone torsional preferences and H-bonding pattern, respectively. As a model we choose cilengitide (CIL), which is an ArgGly-Asp (RGD) peptide of sequence cyclo[RGDfNMeV] (see Scheme 1), with well-characterized biological and conformational properties. CIL displays nanomolar inhibition of vitronectin binding to the isolated avb3 and avb5 integrin receptors, and it blocks integrin-dependent adhesion of tumor and endothelial cells to immobilized extracellular matrix (ECM) proteins and reduces angiogenesis and tumor growth in vivo. Modulation of the internal H-bond pattern of nonmethylated CIL precursors—achieved by changing the flexibility or the chirality of the backbone—was found to influence the antagonist activity on the vitronectin (VN) and fibrinogen (FB) receptors, and has been proposed to control laminin P1 vs. vitronectin receptor specificity. We synthesized all five depsi-analogues of the depsipeptide CIL (D1–D5) by stepwise assembling of the linear precursors on 2-CTC resin (Fmoc/tBu strategy) and cyclization in solution. For the introduction of the a-hydroxy acid residues onto the growing peptide chain, their HFA-activated/protected derivatives were used (Scheme 1). For the acylation of the free hydroxy group, DIPDCI/DMAP-activation was used. For conventional peptide cyclization, the optimal site for macrocycle formation is between the Gly (acting as the C terminus) and Asp (acting as the N terminus) residues because Gly cannot epimerize. This strategy was used for the synthesis of parent CIL, D1, and D2 depsipeptides with PyBOP/HOAt. The macrolactonization required for the preparation of D3 was successful with MSNT activation and NMI as the base. For the depsipeptides D4 and D5, certain particularities of the ester bond had to be taken into account. Our attempts to synthesize the linear precursor of D4 (OGly analogue) starting from OGly as the C terminus, resulted in low yields, and was likely because of cleavage of the ester bond that was mediated by base during repeated treatment with piperidine. Therefore, NMeVal was chosen as the C terminus. For the preparation of D5, the cyclization was performed at the (apparently) less attractive position between (d)Phe and NMeVal. Macrolactamization at less hindered sites was impeded by intramolecular nucleophilic attack on the ester bond, which occurred during peptide chain elongation, and eliminated the (D)Phe–NMeVal couple as dioxopiperazine. Given the increased steric demand of the N-terminal NMeVal, we chose the more reactive PyAOP as the coupling reagent and HOAt as the additive. After cleavage from the solid support, all linear peptide and depsipeptide precursors were obtained in over 85% yield. Head-to-tail cyclization was performed in solution and gave good yields in all cases and, finally, the protecting groups on the side chains were removed using [Pd(PPh3)4]/phenylsilane [*] T. Cupido, Dr. J. Spengler, Dr. J. Ruiz-Rodriguez, Prof. F. Albericio Institute for Research in Biomedicine (IRB), Barcelona Science Park (PCB) and CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine PCB Baldiri Reixac 10, 08028 Barcelona (Spain) Fax: (+34)93-403-7126 E-mail: tommaso.cupido@irbbarcelona.org albericio@irbbarcelona.org