UAA! Discover Aminocoumarinyl Lysine

In contrast to the simple transfection of an active protein like it is done in overexpression studies by expressing photo-caged proteins, deliberate amounts of active protein can be produced at exactly defined time points and intracellular locations, allowing for precise pulse-chase-style experiments. Placed in the active site of an enzyme to block its activity, incorporation of a photo-protected amino acid may be used to activate enzymes and to control protein interaction.

Structure of HAA9645 (H-L-Lys(AMC)-OH*TFA): the 7-aminocoumarinyl moiety is connected to the ε-amino group of the lysine via a methyl-oxocarbonyl linker. Upon irradiation, the coumarin moiety is photoactivated, the linker disintegrates, and the lysine is deprotected. 

This spatiotemporal deprotection by light is possible with the help of lasers and microscopes. The technique may be used to study the effects of enzyme or protein activity, e.g., in cell and developmental biology. Photocaged lysine is of special scientific value, as many nucleotide-binding proteins make use of this amino acid in their active site, because the positively charged side chain interacts with nucleotide phosphates. The caging moiety here is not only occupying space; it also neutralizes the positive charge of the protonated lysine side chain. Besides, this feature also allows you to deliberately manage the accessibility for posttranslational modifications. 7-Aminocoumarinyl-lysine (ACK) has been used, e.g., to control the activity of a designed Cre recombinase, of small G-proteins and the levels of cellular ATP. 

ACK has a λ_max of 348 nm and a wide absorption range. It emits in the 450 nm range: When illuminated at 405 nm, the compound readily decages within a pH range of 5 to 8. A release rate of more than 50% could be achieved with an irradiation time of just 10 s, and after 120 s, more than 90% deprotection was observed. Wavelengths of 365 nm and 388 nm have been used successfully, too. Thus, many common light sources can be used, mercury and xenon lamps, as well as LEDs. Two photon absorption photolysis at 760 nm is another option, allowing for irradiation with a wavelength which penetrates deeper into tissue and is of low risk to damage living cells due to lower photon energy levels.

For observing coumarin fluorescence, the same filters may be used as for the DNA dyes DAPI or H33258. 

Illustration of the deprotection of an AMC-protected lysine in a peptide chain: The carbamate link is cleaved, and CO₂ and hydroxymethyl-AMC are released while liberating the lysine side-chain. 

The genetically encoded incorporation of non-canonical amino acids into proteins is achieved by engineered pyrrolysyl-tRNA systems which make use of the rare codon UAA, normally serving as a stop codon. Three plasmid encoded genes are required for this method: one for an evolved aminoacyl-tRNA synthetase accepting the special amino acid (in this case: ACK), the engineered tRNA for ACK and the target gene with the protein to be modified, where one or more UAA codon(s) represent the aminocoumarin modified lysine(s). 

→ Looking for photocaged amino acids? Interested in other AMC derivatives? Download our brochure “Photochemistry” and discover our portfolio! 

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References: 

Genetically Encoded Optochemical Probes for Simultaneous Fluorescence Reporting and Light Activation of Protein Function with Two-Photon Excitation; J. Luo, R. Uprety, Y. Naro, C. Chou, D. P. Nguyen, J. W. Chin, A. Deiters; J. Am. Chem. Soc. 2014; 136(44): 15551-15558. https://doi.org/10.1021/ja5055862 

Genetically Encoded Photocaged Proteinogenic and Non-Proteinogenic Amino Acids; X. Yang, X. C. Su, W. Xuan; ChemBioChem. 2024; 25(17): e202400393. https://doi.org/10.1002/cbic.202400393 

Genetically Encoded Aminocoumarin Lysine for Optical Control of Protein–Nucleotide Interactions in Zebrafish Embryos; W. Brown, J. Wesalo, S. Samanta, J. Luo, S. E. Caldwell, M. Tsang, A. Deiters; ACS Chem. Biol. 2023; 18(6): 1305-1314. https://doi.org/10.1021/acschembio.3c00028 

Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis; T. Furuta, S. S. H. Wang, J. L. Dantzker, T. M. Dore, W. J. Bybee, E. M. Callaway, W. Denk, R. Y. Tsien; Proc. Natl. Acad. Sci. 1999; 96(4): 1193-1200. https://doi.org/10.1073/pnas.96.4.1193 

Synthesis, Photophysical, Photochemical and Biological Properties of Caged GABA, 4-[[(2H-1 -Benzopyran-2-one-7-amino-4-methoxy) carbonyl] amino] Butanoic Acid; B. Curten, P. H. M. Kullmann, M. E. Bie, K. Kandler, B. F. Schmidt; Photochem. Photobiol. 2005; 81(3): 641-648. https://doi.org/10.1111/j.1751-1097.2005.tb00238.x 

Optogenetics with Atomic Precision─A Comprehensive Review of Optical Control of Protein Function through Genetic Code Expansion; M. Charette, C. Rosenblum, O. Shade, A. Deiters; Chem. Rev. 2025; 125(4): 1663-1717. https://doi.org/10.1021/acs.chemrev.4c00224 

Optical Control of Cellular ATP Levels with a Photocaged Adenylate Kinase; W. Zhou, C. P. Hankinson, A. Deiters; ChemBioChem. 2020; 21(13): 1832-1836. https://doi.org/10.1002/cbic.201900757