FULL PAPER DOI: 10.1002/ejoc.200901045 A Versatile, Modular Platform for Multivalent Peptide Ligands Based on a Dendritic Wedge Edith H. M. Lempens, [a][‡] Brett A. Helms, [a][‡] Andrea R. Bayles, [a] Maarten Merkx, [a] and E. W. Meijer* [a] Keywords: Dendrimers / Peptides / Multivalency / Modular approach / Native chemical ligation / Orthogonal functionaliza- tion A general methodology for the synthesis of multifunctional AB 2 , AB 3 , AB 4 , and AB 5 dendritic wedges is described based on an orthogonal protection strategy. Asymmetric polyamide dendrons that possess N-terminal cysteine residues at the pe- riphery were quantitatively functionalized with C-terminal thioester peptides using native chemical ligation. Conjuga- tion of biologically relevant groups at the focal point resulted Introduction Over the past two decades, multiple synthetic strategies have been developed to arrive at functional dendrimers. The ability of this type of macromolecule to present multiple groups at the surface, while maintaining a well-defined structure with low polydispersity makes them ideal vehicles for use in biomedical applications. [1] Inherent to their struc- ture are opportunities to employ multivalent interactions to yield ligands with high affinity and specificity for biological targets. Beyond that, the preparation of well-defined multi- functional dendrimers – for example, by conjugation of therapeutic agents, targeting groups and radio- or fluores- cent labels onto a single platform – is limited by the fact that most dendritic molecules possess uniformly protected functional groups in a spherical (or cylindrical) topology. This symmetry-related synthesis constraint is inherently contrary to generating molecular complexity within a sin- gle, well-defined molecular species. [2] Methodologies for the preparation of dendrimers with asymmetric functionaliza- tion have appeared recently: for example, by conjugation of functional units (e.g. biotin or chromophores) at the focal point of dendritic wedges; [3] by merging two separate wedges using click chemistry, native chemical ligation or Diels–Alder reactions; [4] or by a combination of convergent [a] Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, P. O. Box 513, 5600 MB Eindhoven, The Netherlands Fax: +31-40-245-1036 E-mail: e.w.meijer@tue.nl [‡] Edith H. M. Lempens and Dr. Brett A. Helms contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejoc.200901045. Eur. J. Org. Chem. 2010, 111–119 © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 111 in a series of related structures with a highly controlled val- ency (2–5) that can directly be used in a systematic study on the strength of multivalent interactions. Using our modular approach various ligands, functional groups and spacers can readily be combined in order to generate a toolbox for the development of smart biomaterials used in molecular medi- cine and imaging. and divergent growth strategies. [5] While these desymme- trized dendrimers possess a highly controlled surface func- tionalization, they are limited by synthetic procedures based on either 1  2 branching or 1  3 branching. As a result the number of end groups increases exponentially with each generation. [6] For many biomedical applications, however, an arithmetic branching scheme is more attractive for eluci- dating and quantifying the fundamental aspects of multi- valent ligand interactions for targeting. We present here a new type of bifunctional building block that enables the modular synthesis of multivalent peptide dendritic wedges with arithmetic control over the degree of branching (Fig- ure 1). The design of our biocompatible platform allows an opti- mal presentation of multiple functional groups and is in- spired by Newkome’s polyamide dendrimers. [7] In contrast to previously reported strategies, the number of ligands in- creases arithmetically, allowing a systematic study on the nature and strength of multivalent interactions. Two, three, four or five ligands can be presented at the periphery of a polyamide dendritic wedge, while another functional group is site-specifically introduced at the focal point. Poly(ethyl- ene glycol) (PEG) units are used to append reactive cysteine residues to the dendritic core. Besides the fact that the length of this spacer can be easily tuned to inter-receptor distances, PEG-dendrimer hybrids are clinically approved and have shown to decrease toxicity and increase circu- lation times in vivo. [8] N-Terminal cysteine residues at the PEGylated wedge periphery were used for the introduction of multiple peptide-based ligands via their C-terminus using native chemical ligation, a highly efficient and specific cou- pling reaction. [9] Minor modifications will even allow the use of other ligation reactions such as oxime or “click”