Multimerization of cRGD Peptides by Click Chemistry: Synthetic Strategies, Chemical Limitations, and Influence on Biological Properties

Integrin alpha(nu)beta(3) is overexpressed on endothelial cells of growing vessels as well as on several tumor types, and so integrin-binding radiolabeled cyclic RGD pentapeptides have attracted increasing interest for in vivo imaging of alpha(nu)beta(3) integrin expression by positron emission tomography (PET). Of the cRGD derivatives available for imaging applications, systems comprising multiple cRGD moieties have recently been shown to exhibit highly favorable properties in relation to monomers. To assess the synthetic limits of the cRGD-multimerization approach and thus the maximum multimer size achievable by using different efficient conjugation reactions, we prepared a variety of multimers that were further investigated in vitro with regard to their avidities to integrin alpha(nu)beta(3). The synthesized peptide multimers containing increasing numbers of cRGD moieties on PAMAM dendrimer scaffolds were prepared by different click chemistry coupling strategies. A cRGD hexadecimer was the largest construct that could be synthesized under optimized reaction conditions, thus identifying the current synthetic limitations for cRGD multimerization. The obtained multimeric systems were conjugated to a new DOTA-based chelator developed for the derivatization of sterically demanding structures and successfully labeled with Ga-68 for a potential in vivo application. The evaluated multimers showed very high avidities-increasing with the number of cRGD moieties-in in vitro studies on immobilized alpha(nu)beta(3) integrin and U87MG cells, of up to 131- and 124-fold, respectively, relative to the underivatized monomer.