Protein Chemical Synthesis DOI: 10.1002/ange.201107222 Synthesis of a Biologically Active Triazole-Containing Analogue of Cystatin A Through Successive Peptidomimetic Alkyne–Azide Ligations** Ibai E. Valverde, Fabien Lecaille, Gilles Lalmanach, Vincent Aucagne,* and Agns F. Delmas* Amide surrogates are common in naturally occurring pep- tides and in synthetic peptides used in therapy. Whereas backbone-engineered proteins are, to date, extremely labori- ous to produce by genetic means, [1] the advent of chemo- selective peptide chemical ligation reactions [2, 3] paved the way to such complex molecular architectures of considerable potential for protein therapeutics. To date, the most popular strategy to introduce amide bond surrogates in proteins relies on an elaborate combination of 1) solution- phase synthesis to provide a suitably pro- tected pseudo-dipeptide, 2) solid-phase pep- tide synthesis (SPPS) to incorporate the modification in a peptide fragment, and 3) native chemical ligation [2b] (NCL) to yield a full-length backbone-engineered pro- tein. [4] A valuable alternative for the introduc- tion of amide-bond mimics in proteins would be a peptidomimetic ligation strategy com- bining in a single step the formation of the amide surrogate, its incorporation in a pep- tide backbone, and ligation of fragments. Besides the pioneering study on thioester backbone-engineered proteins, [2a] only few examples have been reported, [5] including a recent study concerning a ligation of thio- acid- and aziridine-terminated model pep- tides, giving a reduced form (Y[CH 2 NH 2 ]) of an amide bond. [6] To enlarge the palette of the synthetic protein chemist, we envisioned developing a new peptidomimetic ligation prototype that leads to bioactive backbone-modified proteins. Herein, we report for the first time the use of the Cu I -mediated cycloaddition of azides and terminal alkynes (CuAAC) [7] for the assembly of unprotected peptide fragments into a bioactive triazole-containing pro- tein. The rapid and regioselective method for the synthesis of 1,4-disubstituted 1,2,3-triazoles (Tz) through CuAAC, inde- pendently discovered by the Meldal [7a] and Sharpless [7b] groups, has incredibly accelerated the studies involving Tz- containing novel compounds. This heterocycle is well recog- nized as an amide bioisostere: it closely mimics the geometric, steric, and electronic features of a trans-amide bond and can likewise participate in hydrogen bonding and dipole–dipole interactions [8] (Scheme 1). The triazole moiety is considered as an universal peptidomimetic group that can accommodate any peptide secondary structure. [9] The introduction of triazole amide surrogates (Y[Tz]) by a peptidomimetic ligation strategy requires peptides modified at their C and N termini with a-amino alkynes and a-azido acids, respec- tively. The latter building blocks can be easily prepared [11] and introduced into peptide fragments using standard peptide chemistry, and the synthesis of chiral a-amino alkynes can be Scheme 1. Protein synthesis through fragment condensation using NCL and PTL. [10] [*] Dr. I.E. Valverde, Dr. V. Aucagne, Dr. A.F. Delmas Centre de Biophysique MolØculaire, CNRS UPR 4301 Rue Charles Sadron, 45071 OrlØans cedex 2 (France) E-mail: aucagne@cnrs-orleans.fr delmas@cnrs-orleans.fr Dr. F. Lecaille, Prof. Dr. G. Lalmanach ProtØases et Vectorisation Pulmonaires, INSERM U618 UniversitØ FranÅois Rabelais 10, boulevard TonnellØ, 37032 Tours cedex (France) [**] The RØgion Centre council is gratefully acknowledged for financial support (FibroCat project and PhD fellowship for I.E.V.). We thank the CBM spectrometric platforms, and in particular Guillaume Gabant, for recording most of the MALDI-TOF spectra, HervØ Meudal for the NMR spectra, and Dr. Sandrine Villette for her help in the circular dichroism experiments. We thank Dr. StØphane Bourg (molecular modeling lab, University of Orleans-CNRS FR 2708 research federation) for DFT calculations. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201107222. . Angewandte Zuschriften 742  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2012, 124, 742 –746