10.1021/ol302412a r 2012 American Chemical Society Published on Web 09/28/2012 ORGANIC LETTERS 2012 Vol. 14, No. 20 5254–5257 Incorporation of cis- and trans-4,5-Difluoromethanoprolines into Polypeptides Vladimir S. Kubyshkin, † Pavel K. Mykhailiuk,* ,§, ) Sergii Afonin,* ,‡ Anne S. Ulrich, †,‡ and Igor V. Komarov* ,§, ) Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry and CFN, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany, KIT, Institute of Biological Interfaces (IBG-2), POB 3640, 76021 Karlsruhe, Germany, Kyiv National Taras Shevchenko University, Vul.Volodymyrska 64, 01601 Kyiv, Ukraine, and Enamine Ltd., Vul. Oleksandra Matrosova 23, 01103 Kyiv, Ukraine Pavel.Mykhailiuk@gmail.com; Sergiy.Afonin@kit.edu; Igor.Komarov@enamine.net Received August 30, 2012 ABSTRACT Substituted prolines exert diverse effects on the backbone conformation of proteins. Novel difluoro-analogues were obtained by adding difluorocarbene to N-Boc-4,5-dehydroproline methyl ester, which gave the trans-adduct as the sole product with 71% yield. Upon cleavage of the N-protection group the free amino acid decomposed rapidly. Its incorporation into the proline-rich cell-penetrating “sweet arrow peptide” was thus accomplished using a dipeptide strategy. Two building blocks, containing either cis- or trans-4,5-difluoromethanoproline, were obtained by difluorocyclopropanation of the aminoacyl derivatives of 4,5-dehydroproline. The resulting dipeptides were stable under standard conditions of Fmoc solid phase peptide synthesis and, thus, suitable to study conformational effects. Conformational and electronic effects in the side chain of proline and its substituted analogues can have a significant influence on the three-dimentional structure of proteins. They are known to modify the stability of certain conformations by stucturing proline-containing turns, loops, and/or secondary structure boundaries. 1 Even intermolecular protein assemblies can be influenced this way. Numerous studies have suggested that stereo- electronic effects may result, for example, in a modulation of the triple-helical oligomeric structure of collagen-like peptides. A comprehensive understanding of the inter- play between steric and electronic contributions opens the way to rationally control protein structure and there- by function. During a structural study of the proline-rich cell- penetrating “sweet arrow peptide” 2 (SAP) [(VRLPPP) 3 ], † KIT, Institute of Organic Chemistry and CFN. ‡ KIT, Institute of Biological Interfaces (IBG-2). § Kyiv National Taras Shevchenko University. ) Enamine Ltd. (1) (a) Eberhardt, E. S.; Panasik, N., Jr.; Raines, R. T. J. Am. Chem. Soc. 1996, 118, 12261. (b) Holmgren, S. T.; Taylor, K. M.; Bretscher, L. E.; Raines, R. T. Nature 1998, 392, 666. (c) Persikov, A. V.; Ramshaw, J. A. M.; Kirkpatrick, A.; Brodsky, B. J. Am. Chem. Soc. 2003, 125, 11500. (d) Doi, M.; Nishi, Y.; Uchiyama, S.; Nishiuchi, Y.; Nakazawa, T.; Ohkubo, T.; Kobayashi, Y. J. Am. Chem. Soc. 2003, 125, 9922. (e) Barth, D.; Milbradt, A. G.; Renner, C.; Moroder, L. ChemBioChem 2004, 5, 79. (f) Hodges, J. A.; Raines, R. T. Org. Lett. 2006, 8, 4695. (g) Horng, J.-C.; Raines, R. T. Protein Sci. 2006, 15, 74. (h) Shoulders, M. D.; Satyshur, K. A.; Forest, K. T.; Raines, R. T. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 559. (i) Kim, W.; McMillan, R. A.; Snyder, J. P.; Conticello, V. P. J. Am. Chem. Soc. 2005, 127, 18121. (j) Naduthambi, D.; Zondlo, N. J. J. Am. Chem. Soc. 2006, 128, 12430. (k) Ruzza, P.; Siligardi, G.; Donella-Deana, A.; Calderan, A.; Hussain, R.; Rubini, C.; Cesaro, L.; Osler, A.; Guiotto, A.; Pinna, L. A.; Borin, G. J. Pept. Sci. 2006, 12, 462. (l) Steiner, T.; Hess, P.; Bae, J. H.; Wiltschi, B.; Moroder, L.; Budisa, N. PloS ONE 2008, 3, e1680. (m) Beausoleil, E.; Lubell, W. D. Biopolymers 2000, 53, 249. (n) Zhang, R.; Madalengoitia, J. S. J. Org. Chem. 1999, 64, 330. (o) Delaney, N. G.; Madison, V. Int. J. Peptide Protein Res. 1982, 19, 543. (p) Kuemin, M.; Nagel, Y. A.; Schweizer, S.; Monnard, F. W.; Ochsenfeld, C.; Wennemers, H. Angew. Chem., Int. Ed. 2010, 49, 6324.