Peptide News from Bachem: pNA and AMC Substrates in Milligram to Multi-Gram Quantities

One of the earliest applications of synthetic peptides was for screening purposes to improve our understanding of biochemical processes. To enable quantitative studies of enzyme kinetics, enzyme substrates have been manufactured bearing chromogenic or fluorogenic moieties to allow for visualization using ultraviolet absorption and/or fluorescence spectroscopy.

In both of these applications, the spectroscopic properties of the chromophore or fluorophore are red-shifted upon cleavage of the peptide bond adjacent to this aromatic moiety. Whereas the adjacent amino acid effectively suppresses and blue-shifts the absorbance or fluorescence of the attached aromatic group, cleavage of this bond restores the usual photochemical properties of the attached chromophore or fluorophore.

The most common chromogenic and fluorogenic substrates are based on the p-nitroanilide (pNA) and 7-amino-4-methylcoumarin (AMC) systems.

pNA substrates have been used for decades for the quantitative analysis of enzyme kinetics and substrate specificity profiling. The analysis of these substrates by UV spectroscopy hinges on the absorption of p-nitroaniline at 410nm (ε = 8800 L-mol-1cm-1) which is much lower when N-acylated by the C-terminal amino acid residue of the attached peptide.

Similarly, AMC substrates are analyzed by fluorescence spectroscopy using the fluorescence of 7-amino-4-methylcoumarin (λex = 365nm, λem = 440nm), which is markedly reduced in this wavelength range by N-acylation.

Bachem has a long history of manufacturing both pNA and AMC substrates for the academic, pharmaceutical, and diagnostic communities on scales ranging from milligram to multi-gram quantities.

For small-scale projects, we now offer peptides as C-terminal 7-amino-4-carbamoylmethylcoumarin (ACC) derivatives1-4. The ACC fluorophore exhibits fluorescence with comparable excitation and emission maxima as the AMC fluorophore, but with an approximately three-fold higher fluorescence yield, thus offering improvements in both sensitivity and signal-to-noise ratio.

Quantitative analysis of ACC substrates has verified the near-identical proteolysis rates of ACC and AMC substrates, underscoring the minimal impact of the carbamoyl substituent on the coumarin skeleton. From a practicality perspective, the ACC derivative allows for more cost-effective manufacturing of milligram quantities of coumarin-based substrates and is therefore a useful tool in the arsenal of high throughput screening reagents.

If your research requires chromogenic or fluorogenic substrates for substrate specificity profiling, quantitative kinetic analysis, or high throughput screening applications, contact the experts at Bachem to discuss your project. Whether your project requires modification of a peptide or amino acid as a pNA, AMC, or ACC substrate, we have the facilities and expertise to prepare your molecule expeditiously, using a robust, economical manufacturing process. Quality is also a critical concern in chromogenic and fluorogenic substrate manufacturing, given the importance of reproducible solubility and low background absorbance or fluorescence.

For 40 years, Bachem has set the benchmark for quality in peptide and amino acid manufacturing; you can trust us to provide the highest quality substrates available in mg quantities for research through multi-gram and larger quantities for commercial products.


  1. J.L. Harris et al. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc. Natl. Acad. Sci. U. S. A. 97, 7754-7759 (2000)
  2. K. Paschalidou and C. Tzougraki. A general and direct method for the solid phase synthesis of intramolecularly quenched fluorogenic protease substrates using a bifunctional coumarin derivative. Lett. Pept. Sci. 7, 249-253 (2001)
  3. D.J. Maly et al. Expedient solid-phase synthesis of fluorogenic protease substrates using the 7-amino-4-carbamoylmethylcoumarin (ACC) fluorophore. J. Org. Chem. 67, 910-915 (2002)
  4. Q. Zhu et al. Facile synthesis of 7-amino-4-carbamoylmethylcoumarin (ACC)-containing solid supports and their corresponding fluorogenic protease substrates. Bioorg. Med. Chem. Lett. 13, 1033-1036 (2003)

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