Fluorogenic Building Blocks for Peptide Guided Wash-Free Imaging

Published on 20.02.2024

Live-imaging of dynamic structures and catching events in living cells is a challenge, as most fluorescent probes have a high background signal. For alternatives, please read on!

Fluorogenic Building Blocks for Peptide Guided Wash-Free Imaging

To visualize dynamic and changing non-polar structures like cell membranes in living systems, fluorescent probes are ideal when they only light up after reaching their designated environment. With this property, a probe does not require washing steps, e.g., after adding it to cultured cells, and thus makes it ideal for tracking live events with minimal disturbance.

For peptide guided wash-free imaging, Iris Biotech is offering two novel building blocks, Fmoc-L-Dap(NBSD)-OH (FAA7995) and Fmoc-L-Dap(NBTD)-OH (FAA8020), which may be used for the synthesis of fluorescent peptides by Fmoc-SPPS. The main characteristics of these small non-canonical amino acids are the nitrobenzoselenadiazole and nitrobenzothiadiazole side chains, which confer the desired fluorogenic properties while retaining the binding capabilities of the bioactive peptide due to their small size.

Left: FAA7995, the Fmoc-protected nitrobenzoselenadiazolyl building block; λ_abs,max= 488 nm, λ_em,max = 601 nm. FAA8020, the Fmoc-protected nitrobenzothiadiazolyl building block; λ_abs,max= 460 nm, λ_em,max = 546 nm.

With significant Stokes-shifts of around 90 nm and 120 nm, the absorption maxima of both compounds (measured in their unprotected state) are in the blue range (460 nm/488 nm), whereas their maximal emission is in the orange (546 nm) and red (600 nm) range, respectively. Interestingly, compared to the S-bridged molecule, the Se-bridged amino acid has an emission maximum at a 60nm higher wavelength. Furthermore, both amino acids come with good fluorescence quantum yields of around 20%.

Normalized absorption and emission spectra of FAA7995 (left) and FAA8020 (right).

References:

Small Fluorogenic Amino Acids for Peptide-Guided Background-Free Imaging. F. de Moliner, Z. Konieczna, L. Mendive-Tapia, R. S. Saleeb, K. Morris, J. A. Gonzalez-Vera, T. Kaizuka, S. G. N. Grant, M. H. Horrocks, M. Vendrell; Angew. Chem. Int. Ed. 2023; 62: e202216231.
https://doi.org/10.1002/anie.202216231.

Fluorescent amino acids as versatile building blocks for chemical biology; Z. Cheng, E. Kuru, A. Sachdeva, M. Vendrell; Nat. Rev. Chem. 2020; 4: 275-290.
https://doi.org/10.1038/s41570-020-0186-z