Substrate for dengue virus NS2B-NS3 and yellow fever virus NS3 protease. The kcat/Km value of Bz-NleKRR-AMC for the dengue virus type 4 (DEN4) enzyme is > 800 fold higher than that of Boc-Gly-Arg-Arg-AMC, a widely used NS3 substrate (kcat = 2.9 s⁻¹, Km = 8.6 μM for DEN4). For yellow fever virus NS3 protease the kcat value was 0.111 s⁻¹, the Km value 14.6 μM.
CAT No: R1920
CAS No:863975-32-0
Synonyms/Alias:Bz-Nle-Lys-Arg-Arg-AMC;863975-32-0;Bz-Nle-KRR-AMC (hydrochloride);Bz-Nle-Lys-Arg-Arg-AMC.HCl;N-[(2S)-1-[[(2S)-6-amino-1-[[(2S)-5-(diaminomethylideneamino)-1-[[(2S)-5-(diaminomethylideneamino)-1-[(4-methyl-2-oxochromen-7-yl)amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]benzamide;hydrochloride;
Bz-Nle-Lys-Arg-Arg-AMC is a synthetic peptide substrate widely recognized in biochemical research for its utility in the study of protease activity, particularly those enzymes with trypsin-like specificity. Featuring a benzoyl (Bz) group at the N-terminus and an AMC (7-amino-4-methylcoumarin) fluorogenic reporter at the C-terminus, this peptide is engineered to enable sensitive and quantitative detection of enzymatic cleavage events. Its defined amino acid sequence—norleucine, lysine, and two arginine residues—provides a substrate motif that closely mimics physiological targets of several serine proteases. The incorporation of AMC allows for real-time monitoring of proteolytic activity via fluorescence, making this compound an indispensable tool in enzymology, inhibitor screening, and assay development.
Enzyme Activity Assays: Bz-Nle-Lys-Arg-Arg-AMC is extensively applied in the quantitative assessment of serine protease activity, especially for enzymes exhibiting trypsin-like specificity such as thrombin, kallikreins, or related proteolytic enzymes. Upon enzymatic cleavage at the peptide bond adjacent to the AMC moiety, the fluorophore is released, resulting in a measurable increase in fluorescence intensity. This property enables researchers to monitor enzyme kinetics in real time, optimize assay conditions, and compare relative activities across enzyme variants or sample types. The high sensitivity and specificity of the substrate make it ideal for both endpoint and continuous kinetic assays in biochemical research.
Inhibitor Screening: The peptide substrate serves as a robust platform for the evaluation of protease inhibitors, facilitating the discovery and characterization of compounds that modulate enzymatic activity. By monitoring the rate of AMC release in the presence of candidate inhibitors, researchers can derive precise IC50 values and assess inhibitor selectivity. This application is particularly valuable in early-stage drug discovery and academic research focused on elucidating protease function or identifying novel regulatory molecules.
Protease Characterization: Bz-Nle-Lys-Arg-Arg-AMC enables detailed studies of substrate specificity, catalytic efficiency, and mechanistic properties of target proteases. By providing a well-defined peptide sequence, it allows for the systematic investigation of enzyme-substrate interactions, cleavage site preferences, and the influence of sequence modifications on proteolytic processing. Such studies are essential for understanding the physiological roles of proteases, mapping their substrate repertoires, and guiding the rational design of more selective substrates or inhibitors.
High-Throughput Screening: The fluorogenic nature of the AMC reporter makes this substrate highly amenable to automation and high-throughput screening formats. Laboratories can utilize microplate readers or automated liquid handling systems to perform large-scale assays for enzyme activity or inhibitor profiling. The rapid, reproducible, and quantitative readout provided by the released fluorophore streamlines the identification of active compounds or enzyme variants in large libraries, accelerating research in both academic and industrial settings.
Biochemical Pathway Analysis: The substrate's defined sequence and sensitive detection capabilities also support its use in dissecting complex proteolytic cascades and signaling pathways. By incorporating it into cell-free extracts or reconstituted systems, scientists can monitor protease activation, probe the dynamics of proteolytic networks, and identify upstream or downstream regulatory events. These applications provide critical insights into physiological and pathological processes involving protease activity, contributing to a deeper understanding of cellular regulation and protein turnover.
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