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Chemically Stable Diazopeptides as Cystein Protease Inhibitors Active in Living Cells

Affiliation
Department of Biology, Chemistry and Pharmacy, Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Str.2+4, 14195 Berlin, Germany
Wahl, Juliane;
Affiliation
Department of Biology, Chemistry and Pharmacy, Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Str.2+4, 14195 Berlin, Germany
Ahsanullah;
Affiliation
Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany
Zupan, Hana;
Affiliation
Department of Biology, Chemistry and Pharmacy, Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Str.2+4, 14195 Berlin, Germany
Gottschalk, Franziska;
Affiliation
Charité, Berlin, Germany
Mieth, Maren;
Affiliation
Department of Biology, Chemistry and Pharmacy, Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Str.2+4, 14195 Berlin, Germany
Arkona, Christoph;
Affiliation
Charité, Berlin, Germany
Hocke, Andreas;
Affiliation
Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany
Keller, Bettina;
Affiliation
Department of Biology, Chemistry and Pharmacy, Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Str.2+4, 14195 Berlin, Germany
Rademann, Jörg

Diazo-compounds show selective reactivity and are therefore intensely investigated for versatile applications in chemical biology. The combination of the diazo reactivity and the selectivity of peptide sequences, tuned to a target, evokes a promising new class of enzyme inhibitors. So far, C- and N-terminal diazo peptides have been reported, but their poor stability towards even mild acidic conditions limits their applicability. We set our aim to the synthesis of internal diazo peptides, stabilized by two adjacent electron withdrawing groups, with higher stability towards challenging conditions.
Internal diazo peptides were obtained, as esters or amides, by C-acylation of phosphorane ylides and subsequent cycloaddition-reversion mechanism with electron-deficient azides (a). Starting from polymer supported triphenylphosphine the procedure was compatible with Fmoc-based solid phase peptide synthesis. The formation of both, diazo ester- and amide-peptides, was established in useful yields.
The so acquired diazo peptides are stable between pH 1-14 for amides and 1-8 for esters, as well as against thiol nucleophiles. These findings inspired us to explore their potential as cysteine protease inhibitors. A diazo peptide with the well-established sequence of the human Caspase-3 (hC-3) substrate was synthesized and tested for activity against the enzyme, showing IC50-values in the nanomolar range with a time-dependant covalent inhibition mechanism (b). A fluorescent diazo peptide was used to image apoptosis in living cells by labelling of active hC3-protease (c).

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