Novel fluorescent substrates for detection of trypsin activity and inhibitor screening by self-quenching

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• 作者：Daisuke Sato ; Tamaki Kato ; ^{tmkato@life.kyutech.ac.jp" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Fluorescent peptide ; Self-quenching substrate ; Enzyme reaction ; Trypsin detection ; Inhibitor evaluation
• 刊名：Bioorganic & Medicinal Chemistry Letters
• 出版年：2016
• 出版时间：1 December 2016
• 年：2016
• 卷：26
• 期：23
• 页码：5736-5740
• 全文大小：838 K

文摘

A group of self-quenching-based substrates with two fluorescent peptides for detection of trypsin activity was designed and synthesized. The substrates could be easily synthesized using simple solid-phase peptide synthesis techniques. Two fluorescent peptide substrates for trypsin were conjugated to the amino groups of lysine as a branched unit. The fluorescence of these substrates was self-quenched owing to the highly assembled fluorophores on the substrates. The release of these concentrated fluorophores by proteases allows for fluorescence recovery. Self-quenching reduced the fluorescence of the substrates by 64.1%, and the fluorescence intensity was recovered by the release of the fluorophores from the substrate peptides via tryptic cleavage. The kinetic assay revealed that the k_cat/K_m values of the substrates were almost comparable to those of the standard fluorescent probe, peptide-MCA. The detection limit for trypsin was 111 pM, and the calculation of IC₅₀ and K_i values for the Bowman–Birk inhibitor was achieved using these substrates. These easily synthesizable self-quenching-based substrates have the potential to be useful for the detection of other disease-related protease activities.