Pseudopeptidic Cages as Receptors for N‑Protected Dipeptides Enrico Faggi, † Alejandra Moure, † Michael Bolte, ‡ Cristian Vicent, § Santiago V. Luis,* ,∥ and Ignacio Alfonso* ,† † Departamento de Química Bioló gica y Modelizació n Molecular, Instituto de Química Avanzada de Cataluñ a (IQAC-CSIC), Jordi Girona 18-26, E-08034 Barcelona, Spain ‡ Institut fü r Anorganische Chemie, J.-W.-Goethe-Universitä t, Max-von-Laue-Strasse 7, D-60438 Frankfurt/Main, Germany § Serveis Centrals d’Instrumentació Científica and ∥ Departamento de Química Inorga ́ nica y Orga ́ nica, Universitat Jaume I, Avda. Sos Baynat, s/n, E-12071 Castelló n, Spain * S Supporting Information ABSTRACT: The molecular recognition of short peptides is a challenge in supramolecular chemistry, and the use of peptide- like cage receptors represents a promising approach. Here we report the synthesis and characterization of a diverse family of pseudopeptidic macrobicycles, as well as their binding abilities toward N-protected dipeptides using a combination of different techniques (NMR, ESI-MS, and fluorescence spec- troscopy). The cage hosts were assayed for dipeptide binding using competition ESI-MS experiments as high-throughput screening to obtain general trends for the recognition phenomena. Selected hosts were additionally studied by NMR spectroscopy ( 1 H NMR titration and diffusion-ordered spectroscopy experiments) in different solvents. The results unambiguously demonstrated the formation of the [cage·dipeptide] supramolecular complexes and rendered quantitative information about the strength of the interaction (K ass ). The structural variables within the pseudopeptidic cage framework that produced a stronger and more selective recognition were thus identified. The cages showed a remarkable selectivity for N-protected dipeptides with an aromatic amino acid at the carboxylic terminus, which prompted us to propose a mode of binding based on polar and nonpolar noncovalent interactions. Accordingly, we faced the molecular recognition of a target dipeptide (Ac-EY-OH) mimicking a biologically relevant sequence by NMR and fluorescence spectroscopy in highly competitive media. ■ INTRODUCTION Macrocyclic structures derived from amino acids are attractive molecules with a formidable potential in the molecular recognition field. 1 The different functionalities of the side chains could implement a large molecular diversity and, concomitantly, many potential noncovalent binding sites and different chemical properties (such as charge or polarity). Moreover, the peptide bond also represents a structural and interactional functional group. 2 This combination has allowed the use of pseudopeptidic macrocycles for the recognition of different species. 3 Regarding that, the addition of another dimension to the binding motif could improve the recognition abilities, both by increasing the strength of the interaction and by improving the selectivity of the process. 4 In this context, molecular cages have recently emerged as interesting entities for the binding of specific guests. 5 Accordingly, the macro- bicyclic compounds containing amino acid moieties can be considered as pseudopeptidic cages with an inner cavity surrounded by functional groups defining a binding pocket for potential substrates. 6 On the other hand, although some important biological processes are closely related to the noncovalent interaction with specific peptide sequences, 7 the molecular recognition of short peptides is still a challenge in supramolecular chemistry. 8 The external intervention on these interactions could serve for the design of therapeutic or diagnostic tools. 9 The use of synthetic receptors for dipeptides is a fundamental approach to the problem, since the relative simplicity of the systems could allow the full understanding of the rules controlling the process. 10 During the past decade, we have designed and prepared different pseudopeptidic macrocycles with interesting supra- molecular properties. 11 They were shown to be efficient receptors for different species with positive or negative charge. 12 More interestingly, they are hosts for N-protected amino acids and dipeptides. 13 However, the strength and selectivity of the binding are moderate, and in most cases, the process has been studied in noncompetitive solvents. More recently, we have increased the complexity of the pseudopepti- dic receptors by adding a third arm defining a cage structure. 14 This second generation of pseudopeptidic receptors is able to partially or completely include the guest even in more Received: March 18, 2014 Published: April 21, 2014 Article pubs.acs.org/joc © 2014 American Chemical Society 4590 dx.doi.org/10.1021/jo500629d | J. Org. Chem. 2014, 79, 4590−4601