Issue 36, 2025

Expanding the fluorescence toolkit: molecular design, synthesis and biological application of unnatural amino acids

Abstract

Fluorescence imaging has become an indispensable tool in modern biology, enabling the visualisation of dynamic molecular processes with spatial and temporal precision. Traditional strategies rely heavily on the conjugation of large, extrinsic fluorophores, such as green fluorscent protein or organic dyes, through linkers to proteins or peptides of interest. While sometimes effective, these bulky labels can interfere with native protein structure, function, and interactions, limiting their utility in studying sensitive or compact biological systems. In contrast, fluorescent unnatural amino acids that can be site-specifically incorporated into proteins with minimal perturbation, provide high-resolution insights into molecular behaviour while preserving biological integrity. This perspective focuses on recent advances in the synthesis and application of fluorescent unnatural amino acids, particularly through the structural modification of natural aromatic-containing residues such as phenylalanine, tyrosine, and tryptophan. Additionally, the incorporation of small-molecule fluorophores as amino acid side chains to create versatile probes is also discussed. The utility of these fluorescent amino acids in biological chemistry is highlighted through their application in imaging protein dynamics, measuring enzyme activity and studying biomolecular interactions. Together, these advances establish fluorescent amino acids as a valuable and evolving toolkit for minimally invasive biological imaging.

Graphical abstract: Expanding the fluorescence toolkit: molecular design, synthesis and biological application of unnatural amino acids

Article information

Article type
Perspective
Submitted
30 Jul 2025
Accepted
25 Aug 2025
First published
25 Aug 2025
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2025,16, 16414-16432

Expanding the fluorescence toolkit: molecular design, synthesis and biological application of unnatural amino acids

O. Marshall and A. Sutherland, Chem. Sci., 2025, 16, 16414 DOI: 10.1039/D5SC05745K

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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