SARS-CoV / SARS-CoV-2 Main Protease Fluorogenic Substrate 2020/06/23

Peptides Institute Inc. is pleased to announce three types of substrates for the SARS-CoV main protease (SARS-CoV Mpro or 3CLpro) and SARS-CoV-2 main protease (SARS-CoV-2 Mpro). Protease activity can be measured by observing the increase in fluorescence by the main protease cleaving the substrate, useful in the research of inhibitors.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative virus of a new coronavirus infection (COVID-19), which has been spreading worldwide since the end of 2019. When a virus infects a host cell, the RNA from the virus translates a large precursor protein. The precursor protein is cleaved by various proteases, and becomes a functional protein required for virus growth such as RNA-dependent RNA polymerase. SARS-CoV-2 Mpro, which is also one of the cleavage products, plays a major role in cleavage of precursor proteins and is an essential protease for viral growth. Therefore, this “main protease” inhibitor may be an antiviral drug and is being developed all over the world.


Fluorogenic substrate using FRET of SARS-CoV / SARS-CoV-2 Mpro1),2),3)
The precursor protein sequence, Thr-Ser-Ala-Val-Leu-Gln ↓ Ser-Gly-Phe-Arg-Lys-Met [↓ is a cleavage site] and a fluorescent group (Edans) and a quenching group (Dabcyl) are introduced. SARS-CoV Mpro cleaves the Gln-Ser sequence between the fluorescent and quencher groups, resulting in increased fluorescence. By observing this fluorescence, the protease activity can be measured [KM=17μM, kcat=1.9s-1 (kinetic parameter for SARS-CoV Mpro)]1) [KM=28μM (SARS-CoV-2 Kinetic parameters for Mpro)].3)
SARS Mpro substrate

Figure 1. Method of measuring protease activity of SARS-CoV/SARS-CoV-2 Mpro quenching fluorogenic substrate using FRET.

Code Product Name Quant Price
3249-v Dabcyl-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu(Edans)-NH2 1 mg 50,000



Fluorogenic substrate for SARS-CoV / SARS-CoV-2 Mpro 4)
4-methylcoumarin-7-amide (MCA) type fluorogenic substrate in which aminomethylcoumarin (AMC) is introduced into two sequences with high affinity for SARS-CoV-2 Mpro, as reported by Drag et al. When the non-fluorescent MCA-bound substrate is cleaved by SARS-CoV-2 Mpro, AMC with strong fluorescence is released. By observing this fluorescence, protease activity can be measured. (Ex: 380nm / Em: 460nm)

Code Product Name Quant Price
3250-v Ac-Abu-Tle-Leu-Gln-MCA 1 mg 10,000
3251-v Ac-Thz-Tle-Leu-Gln-MCA 1 mg 10,000


Other CoV related products

Code Product Name Quant Price
3252-v Z-Arg-Leu-Arg-Gly-Gly-MCA (Substrate for PLPro) 1 mg 10,000
3206-v Ubiquitin-MCA (Substrate for PLPro) 50 μg 20,000
3155-v Boc-Arg-Val-Arg-Arg-MCA (Substrate for Furin) 5 mg 6,300
3159-v Pyr-Arg-Thr-Lys-Arg-MCA (Substrate for Furin) 5 mg 6,500
3099-v AMC (Reference Compound) 5 mg 2,000
3253-v Ac-Abu-D-Tyr-Leu-Gln-VS (Inhibitor for Mpro) 1 mg 25,000
4041 Leupeptin (Inhibitor for Serine Protease) 25 mg 5,700
4321-v E-64-d (Inhibitor for Thiol Protease) 5 mg 10,000


For questions and inquiries regarding this article

Author: Yukie Nohara


  1. C.-J. Kuo, Y.-H. Chi, J.T.-A. Hsu, and P.-H. Liang, Biochem. Biophys. Res. Commun., 318, 862 (2004).
  2. L. Zhang, D. Lin, X. Sun, U. Curth, C. Drosten, L. Sauerhering, S. Becker, K. Rox, and R. Hilgenfeld, Science, 368, 409 (2020).
  3. M. D. Sacco, C. Ma, P. Lagarias, A. Gao, J. A. Townsend, X. Meng, P. Dube, X. Zhang, Y. Hu, N. Kitamura, B. Hurst, B. Tarbet, M. T. Marty, A. Kolocouris, Y. Xiang, Y. Chen, and J. Wang, Sci Adv., 6, eabe0751 (2020).
  4. W. Rut, K. Groborz, L. Zhang, X. Sun, M. Zmudzinski, B. Pawlik, X. Wang, D. Jochmans, J. Neyts, W. Młynarski, R. Hilgenfeld, and M. Drag, Nat. Chem. Biol., 17, 222 (2021).

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