"Triazole bridge": disulfide-bond replacement by ruthenium-catalyzed formation of 1,5-disubstituted 1,2,3-triazoles.

About one fourth of the peptidic macromolecular structures deposited in the protein data base (PDB) contain at least one disulfide bridge. In nature, disulfide bonds are formed in a milieu where oxidizing conditions prevail, for example, on the cell surface or in the extracellular matrix. Many proteins benefit from disulfide contributions to their conformational stability. In particular, the defined tertiary folding of oligopeptides smaller than 30 residues essentially relies on macrocyclization through the cystine motif because of the restricted number of noncovalent intramolecular interactions available. Moreover, formation of the disulfide pattern results in structural rigidity of the peptidic framework, as for example, in the family of cystine knot miniproteins, leading to conformationally constrained scaffolds with extraordinary thermal stability and resistance against proteolytic degradation. Hence, the discovery and development of disulfidebridged peptides suitable for diagnostic and therapeutic applications remains a field of intense research. The in-vitro generation of disulfide bonds in peptides is usually achieved post-synthetically and mediated by DMSO, air oxygen, or other oxidizing agents. Although this reaction step can be achieved under relatively mild conditions in solution, it remains one of the most demanding obstacles towards high-yield peptide synthesis, especially for disulfiderich species in which the controlled regiospecific formation of several disulfide bonds is not trivial to control. In addition, to suppress unwanted intermolecular reactions of the thiol groups of individual peptides, oxidative folding usually has to be conducted in highly diluted solutions. In spite of the use of gluthathione-based redox buffers, polymer-supported oxidation systems, macrocyclization on the solid support and/or orthogonal protecting groups, control over the topology of the disulfide bridges formed is still a challenge. 5] In view of these difficulties and to improve the redox stability of bridged peptides, several routes towards synthetic disulfide surrogates have been developed. Straightforward approaches usually employ thioether, olefin, or alkane-based isosters. However, cystathione bridges require multiple synthetic steps and careful choice of orthogonal protection, and dicarba bridges give cis/trans isomers during ring-closing metathesis (RCM). Only an additional purification step or the subsequent palladium-catalyzed hydrogenation of the unsaturated species to the corresponding alkane leads to a construct with defined configuration. In 2004, Meldal et al. described the utility of copper(I)catalyzed azide–alkyne cycloaddition (CuAAC) for a triazole-based disulfide replacement. Owing to the compelling characteristics of this prototypic “click” reaction, it has been extensively applied in peptide chemistry exploiting the almost perfect orthogonality to side-chain reactivities. The introduction of 1,4-disubstituted 1,2,3-triazoles into peptides has also been used to mimic and rigidify conformations of the amide backbone. Moreover, a variety of examples of CuAAC-based macrocyclizations of peptides in solution and on solid supports has been reported. Using the same azideand alkyne-functionalized buidling blocks, 1,5-disubstituted 1,2,3-triazoles can be generated in the ruthenium(II)-catalyzed variant (RuAAC) of the CuAAC. This reaction expands the range of peptidomimetic structures selectively accessible from the same precursor and having different biological activities governed by the architecture of the incorporated triazole. To our knowledge, 1,5-disubstitiuted 1,2,3-triazoles have not been taken into consideration as disulfide mimics to date. Herein, we report the facile introduction of 1,4and 1,5disubstituted 1,2,3-triazoles into a monocyclic variant of the sunflower trypsin inhibitor-I (SFTI-1[1,14], 1; Figure 1) and show that the macrocyclic peptidomimeticum 2 with the “1,5” substitution pattern retains nearly full biological activity in contrast to the “1,4” variants 3 and 4. The choice of 1 as the model peptide for the investigation of triazole-based disulfide replacements had several reasons. SFTI-1 is a small, though very potent, inhibitor of trypsin. Therefore, the influence of different modes of macrocyclization on the bioactivity of the corresponding synthetic variant can be routinely examined by serine protease inhibition assays. [*] M. Empting, Dr. O. Avrutina, Dr. R. Meusinger, S. Fabritz, M. Reinwarth, Prof. Dr. H. Kolmar Clemens-Sch pf-Institut f r Organische Chemie und Biochemie Technische Universit t Darmstadt Petersenstrasse 22, 64287 Darmstadt (Germany) Fax: (+49)6151-16-5399 E-mail: kolmar@biochemie-tud.de Homepage: http://www.chemie. tu-darmstadt.de/kolmar