Rapid and Quantitative Cyclization of Multiple Peptide Loops onto Synthetic Scaffolds for Structural Mimicry of Protein Surfaces

Structure-based design of synthetic peptide-based molecules that mimic the functional site of natural proteins, plays an important role in drug discovery nowadays. Their application is widespread, ranging from synthetic antiviral, 6] antifertility, 2, 7] or antitumor 7, 8] agents to therapeutic agents that are able to mimic or disrupt 10] protein–protein interactions. A variety of structural mimics exist for a-helices, 12] b-turns or hairpins, 13] and b-sheets. 14] However, more complex topologies, like four-helix bundles, are often needed in order to mimic protein function adequately. The total synthesis of such complex structures is generally demanding; this limits their application and emphasizes the need for high-efficiency synthetic strategies. In this communication, we describe a onestep procedure for the immobilization of (multiple) peptide loops onto a synthetic scaffold (Scheme 1) starting from a linear peptide. The reaction is extremely fast and clean and runs very well with linear peptides that are 2–30 amino acids long (>30 not tested). It is compatible with all possible unprotected side-chain functionalities (except for free cysteine). It therefore avoids the need for complex synthetic strategies and this makes the reaction highly versatile with a very wide scope. As part of our research program on the mapping and reconstruction of the discontinuous epitope of follicle-stimulating hormone (FSH), which is a heterodimeric member of the cysteine-knot protein family, we recently discovered the fast and quantitative cyclization of dicysteine-containing peptides upon their treatment with a,a’-dibromoxylenes (T2). In organic solvents such as ACN, the reaction is rather slow and unselective, but it becomes unusually fast and entirely selective for cysteines when performed in aqueous solutions. For example, treatment of a 0.5 mm solution of the peptide *CRVPGDAHHADSLC# (1 a, where * = acetyl and # = amide) with 1.05 equiv of m-T2 in a 1:7 mixture of ACN/NH4HCO3 (20 mm, pH 7.8) gives the corresponding monocyclic product 2 a with >80 % yield in less than 15 min at RT (see Table 1). The corresponding intramolecular SS-dimer 3 is not formed (<5 %) as oxidative cyclization is not competitive under these conditions. There is no doubt that the reaction takes place exclusively at the free sulfhydryl groups, since corresponding peptides without sulfhydryl groups do not react at all with T2 scaffolds in the solvent system used. The difference in reactivity amongst various dicysteine-containing peptides that we have studied is negligible. The half-lives of peptides 1 a–g in the reaction with m-T2 vary only slightly (t1/2 = 1.4– 3.0 min, see Table 1), despite the fact that their length (14–42) and the number of amino acids that separate the two cysteines (0–22) are very different. In sharp contrast to this, there is a large difference in reactivity amongst different scaffolds. o-T2 (average t1/2 = 1.4 min) is slightly more reactive than mT2 (average t1/2 = 2.2 min), but both react much faster than p-T2 (average t1/2 = 8.6 min). Even with a fivefold excess of m-T2 the reaction predominantly gives the monocyclic product 2, whereas the 1:2 product 3 (Scheme 2) is formed with <10 % yield. Cyclization of dicysteine-containing peptides is a two-steps process: initial formation of linear intermediate 4 (Scheme 2) followed by intramolecular cyclization to give cyclic peptide 2. All intermediates (o-4, m-4, and p-4) are highly reactive and cyclize rapidly at low concentration, but their reactivity does not follow that of the template itself. For example, intermediates o-4 and p-4 are the most reactive by far and can only be observed by HPLC for larger ring sizes (e.g. , peptide 1 f), whereas m-4 is significantly more stable and was observed in significant amounts (= 15 %) for most peptides (see Table 1). Most likely, intermediates o-4 and p-4 are activated by a stabilizing resonance-effect in which the sulfur of the first thioether bond activates the second bromomethyl group for nucleophilic attack. Scheme 1. Schematic representation of the one-step synthesis of single-, double-, and tripleloop peptide constructs by treating di-, tri-, and tetracysteine containing peptides with bis-,