Amino-Methylcoumarins as Fluorescent Labels

Amino-Methylcoumarins as Fluorescent Labels

Published on 11/01/2021

Enzymatically-activatable probes based on 7-amino-4-methylcoumarin (AMC) represent versatile tools for the detection and localization of proteolytic enzyme activities. Read on to find out more.

Fluorescence imaging developed as a unique visualization technique for the non-invasive study of the distribution and kinetics of labelled molecules, e.g. drug candidates, as well as for the expression, localization, and activity of drug targets, e.g. specific enzymes. As further benefit, fluorometric assays avoid the typical problems of radiolabeling and are easy to handle.

Besides fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PeT), one strategy to develop enzymatically activatable probes relies on the alteration of the absorption spectra after reaching the target enzyme. Typically, the fluorophore is covalently bound to a distinct enzyme substrate through an amide or ester bond. Upon enzymatic cleavage, the free amino or hydroxyl compound is liberated as active fluorophore, which leads to an increase in fluorescence and a red-shift of the absorbance maximum.

Despite all benefits, fluorescent conjugates often suffer from small Stokes shifts and photobleaching under the intense illumination required for fluorescence microscopy.

One fluorophore combining a high photostability and fluorescence yield with a large Stokes shift is 7-amino-4-methylcoumarin (AMC), which can be used for the preparation of fluorogenic 7-amido-4-methylcoumarin based substrates for the detection of proteolytic enzyme activity. When bound to a peptide, the AMC-amide fluoresces very weakly and excitation/emission wavelengths are shorter (ca. 330/390 nm). When the free AMC-amine is released by proteolytic cleavage, the fluorescence increases by a factor of approx. 700 and excitation and emission wavelengths are red-shifted. Thus, the low fluorescence of the AMC-amide substrate does not interfere with the fluorometric assay.

 

Principle of AMC-based peptides. Drastic increase in fluorescence upon enzymatic cleavage.

Its acetic acid derivative 7-amino-4-methyl coumarin-3-acetic acid (AMCA) emits in the blue region 440-460 nm upon activation with UV light of 350 nm. Iris Biotech provides its N-hydroxysuccinimide ester RL-1005, which can easily be attached to amino acids or other target molecules under formation of a photostable amide bond.

 

Chemical structures of AMC, AMCA and AMCA-OSu. Point of conjugation to amino acids highlighted in green.

Besides, Iris Biotech offers several AMC and AMCA amino acid amides, e.g. of Leucine (HAA3200), Glycine (HAA7971), Threonine (HAA7760) or of dipeptide recognition motifs such as Phe-Arg (ZAA1480) or Arg-Arg (ZAA1470). The derivatives can easily be incorporated into peptides by Fmoc based solid-phase synthesis techniques.

You could not find your desired AMC/AMCA derivative within the section of related products? Please get in contact for your custom synthesis inquiry!

 

References:

  • Synthesis of a New Fluorogenic Substrate for Cystine Aminopeptidase; Y. Kanaoka, T. Takahashi, H. Nakayama, T. Ueno, T. Sekine; Chem. Pharm. Bull. 1982; 30(40): 1485-1487. https://doi.org/10.1248/cpb.30.1485.
  • Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates; M. Zimmerman, B. Ashe, E. C. Yurewicz, G. Patel; Anal. Biochem. 1977; 78(1): 47-51. https://doi.org/10.1016/0003-2697(77)90006-9.
  • Aminomethyl coumarin acetic acid: a new fluorescent labelling agent for proteins; H. Khalfan, R. Abuknesha, M. Rand-Weaver, R. G. Price, D. Robinson; Histochem J. 1986; 18(9): 497-499. https://doi.org/10.1007/BF01675617.
  • Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries; J. L. Harris, B. J. Backes, F. Leonetti, S. Mahrus, J. A. Ellman, C. S Craik; Proc. Natl. Acad. Sci. USA. 2000; 97(14): 7754-7759. https://doi.org/10.1073/pnas.140132697.
  • A Simple Fluorescent Labeling Method for Studies of Protein Oxidation, Protein Modification, and Proteolysis; A. M. Pickering, K. J. A. Davies; Free Radic Biol Med. 2012; 52(2): 239-246. https://doi.org/10.1016/j.freeradbiomed.2011.08.018.
  • Fluorescence imaging of drug target proteins using chemical probes; H. Zhu, I. Hamachi; J. Pharm. Anal. 2020; 10: 426-433. https://doi.org/10.1016/j.jpha.2020.05.013.
  • A New Fluorogenic Substrate for Chymotrypsin; M. Zimmerman, E. Yurewicz, G. Patel; Anal. Biochem. 1976; 70: 258-262. https://doi.org/10.1016/S0003-2697(76)80066-8.
  • Determination of Caspase Specificities Using a Peptide Combinatorial Library; N. A. Thornberry, K. T. Chapman, D. W. Nicholson; Method Enzymol 2000; 322: 100-110. https://doi.org/10.1016/S0076-6879(00)22011-9.
Related Products
    1. Structure image for AMCA-OSu
      AMCA-OSu

      Product code: RL-1005

      Starting at $162.00

    2. Structure image for H-L-Ala-AMC*TFA
      H-L-Ala-AMC*TFA

      Product code: HAA1174

    3. Structure image for H-L-Arg-AMC*2HCl
      H-L-Arg-AMC*2HCl

      Product code: HAA7630

      Starting at $101.25

    4. Structure image for H-L-Pyr-AMC
      H-L-Pyr-AMC

      Product code: HAA7740

      Starting at $101.25

    5. Structure image for Fmoc-ACA-OH
      Fmoc-ACA-OH

      Product code: RL-1170

      Starting at $270.00

    6. Structure image for H-L-Asp(AMC)-OH
      H-L-Asp(AMC)-OH

      Product code: HAA2890

      Starting at $291.60

    7. Structure image for H-Gly-AMC
      H-Gly-AMC

      Product code: HAA7971

      Starting at $81.00

    8. Structure image for H-L-Leu-AMC
      H-L-Leu-AMC

      Product code: HAA3200

      Starting at $81.00

    9. Structure image for Glt-Gly-Arg-AMC.HCl
      Glt-Gly-Arg-AMC.HCl

      Product code: HAA7645

      Starting at $290.25

    10. Structure image for Boc-L-Lys-AMC*AcOH
      Boc-L-Lys-AMC*AcOH

      Product code: BAA3650

      Starting at $195.75

    11. Structure image for Z-L-Arg-L-Arg-AMC*2HCl
      Z-L-Arg-L-Arg-AMC*2HCl

      Product code: ZAA1470

      Starting at $290.25

    12. Structure image for Z-L-Phe-L-Arg-AMC*HCl
      Z-L-Phe-L-Arg-AMC*HCl

      Product code: ZAA1480

      Starting at $324.00

    13. Structure image for H-L-Ala-AMC
      H-L-Ala-AMC

      Product code: HAA1200

      Starting at $81.00