Fluorescent amino acids as versatile building blocks for chemical biology

Abstract

Fluorophores have transformed the way we study biological systems, enabling noninvasive studies in cells and intact organisms, which increase our understanding of complex processes at the molecular level. Fluorescent amino acids (FlAAs) have become an essential chemical tool because they can be used to construct fluorescent macromolecules, such as peptides and proteins, without disrupting their native biomolecular properties. Fluorescent and fluorogenic amino acids with unique photophysical properties have been designed for tracking protein-protein interactions in situ or imaging nanoscopic events in real-time with high spatial resolution. In this Review, we discuss advances in the design and synthesis of FlAAs and how they have contributed to the field of chemical biology in the past 10 years. Important areas of research that we review include novel methodologies to synthesize building blocks with tunable spectral properties, their integration into peptide and protein scaffolds using sitespecific genetic encoding and bio-orthogonal approaches, and their application to design novel artificial proteins as well as to investigate biological processes in cells by means of optical imaging.

To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models

“…Thus, they are a good example of a molecule of therapeutic and biological interest that must be conjugated to a fluorophore. Recently, unnatural fluorescent amino acids have been tested as an alternative to the use of fluorophores (Mendive-Tapia et al, 2016;Cheng et al, 2020;Subiros-Funosas et al, 2020), presenting several advantages over fluorophores. Nevertheless, the use of fluorophores is still broadly used to investigate peptide biological activity.…”

A rationally designed orthogonal synthetase for genetically encoded fluorescent amino acids

“…In this context, numerous analogs of the canonical amino acids tyrosine and tryptophan have been used as ncAAs because of their spectroscopic properties [ 26 , 27 ]. However, their utility is restricted to auxotroph-based systems and are not yet available for orthogonal translation methods implemented in vitro or in living mammalian cells.…”

“…Among the f-ncAAs that have been genetically encoded in eukaryotes, the most notable so far are dansylalanine and L-Anap [ 27 ]. A useful feature of these ncAAs is that their spectroscopic properties varies depending on the polarity of the medium.…”

A Novel Electroporation System for Living Cell Staining and Membrane Dynamics Interrogation

“…Another important application of electroporation is small particle delivery for drug loading [8], living cell staining or transfection [9], which is crucial for elucidating the biological roles/duties of different cellular components and organelles. Conventional methods of living cell staining rely on the proper characteristics (like reactivity and hydrophilicity) of staining molecules that allow them to pass through the membranes of living cells spontaneously [10]. However, it usually takes hours for small dyes to enter the cell, making it challenging to achieve immediate and real-time staining and monitoring.…”