Chemical and enzymatic synthesis of fluorinated-dehydroalanine-containing peptides.

Michael acceptors have long been recognized as reactive functionalities that may link a biologically active molecule to its cellular target. 1,2-Dehydro amino acids are potential Michael acceptors present in a large number of natural products, but their reactivity is modulated by the deactivating nature of the alpha-amino group engaged in an amide bond. We describe here the preparation of 3-fluoro-1,2-dehydroalanine moieties within peptides that significantly enhance the reactivity of the Michael acceptor. Two different routes were designed to access these compounds, one relying on chemical means to introduce the desired functionality and the second taking advantage of a peptide epimerase. In the chemical approach, the fluoro-Pummerer reaction of cysteine derivatives afforded 3-fluorocysteine residues that were oxidized to the corresponding sulfoxides, followed by thermolytic elimination to provide the desired 3-fluorodehydroalanines. The mechanism of the fluoro-Pummerer reaction was investigated and several possible pathways were ruled out. The enzymatic approach utilized the dipeptide epimerase YcjG from Escherichia coli. Difluorinated alanine was incorporated at the C terminus of a dipeptide by chemical means. The resulting peptide proved to be a substrate for YcjG, which catalyzed fluoride elimination to provide the 3-fluorodehydroalanine-containing peptide. Mechanistic investigations showed that fluoride elimination occurred faster than epimerization and at a rate close to that of epimerization of Ala-Ala.

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