Over the years, the Genetic Code Expansion (GCE) strategy has allowed the precise incorporation of desired unnatural amino acids (uAAs) into various proteins, thus offering a powerful stage for introducing novel functionalities beyond those specified by the standard 20 natural amino acids [1a,b]. By utilising orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs [1c], and by recoding the TAG stop codon (amber), this technique has been efficiently applied to expand the chemical repertoire of proteins [2a,b], thereby further broadening the scope of protein engineering.

Among the various genetically encoded systems, the superfolder green fluorescent protein (sfGFP) has been used as a highly robust scaffold due to its enhanced folding kinetics, heightened thermal stability, and high fluorescence quantum yield [3a]. In fact, several successful efforts have resulted in the generation of expanded GFP containing unnatural amino acids at 66 positions within the β-barrel structure [3b,c]. All these attributes have made sfGFP an attractive model for studying the site-specific incorporation of uAAs, especially those that are capable of imparting new photophysical or sensing properties to the protein's backbone.

Of the many different classes of unnatural amino acids [4a,b,5a–d], fluorescent triazolyl unnatural amino acids (FTUAAs), synthesised via Cu(I) -catalysed azide alkyne cycloaddition (CuAAC) chemistry, represent a highly promising class of microenvironment-sensitive fluorescent probes [6a]. Some of the most important features of these amino acids include conjugated aromatic systems, the ability to form hydrogen bonds, and excellent resistance to degradation by proteases. These properties make them ideal candidates for biorthogonal labelling purposes.

For a long time, our group has focused on the design of such microenvironment-sensitive fluorescent probes including amino acids [6a] and nucleosides [6b], utilising the CuAAC reaction as a key protocol. We have primarily aimed to utilise the click-derived triazole moiety as an integral part of such biomolecular building blocks. We have exploited the triazole's aromatic stacking, biomolecular associability via hydrogen bonding, and especially its electron-donating ability to install or modulate the fluorescence photophysics of the attached chromophores for the generation of polarity-sensitive fluorescent uAAs. Eventually, 1,4-disubstituted-1,2,3-triazolyl alanine unnatural amino acids (FTUAAs) with chromophores at the 4th position have been reported by us to showcase attractive microenvironment-sensitive photophysical properties. These amino acids have been exploited in various ways, such as: (a) in sensing proteins [6c]/DNA [6d], (b) to generate fluorescent peptidomimetic probes showcasing fundamentally new and novel photophysics, such as: (i) relay FRET event in a designed trichromophoric pentapeptide [6e], (ii) distance-dependent FRET event in a designed fluorescent pentapeptide, (iii) FRET event in a β-turn tripeptide [6f], (iv) fluorescent pentapeptide in OFETs sensor for biomedical/defence application [6g], (v) dual door entry system to excimer emission in a trichromophoric β-sheet pentapeptide [6h]. Many such fluorescent peptides have shown excellent switch-on sensing abilities for BSA proteins while maintaining their secondary conformation.

Therefore, we believe that such fluorescent triazolyl amino acids [7] would provide a great opportunity to further protein engineering research in chemical biology. However, despite their several advantages, these amino acids have not been previously reported to be incorporated into protein sequences, using the standard GCE approach (Fig. 1). This may be primarily due to the lack of development of o-aaRS/tRNA pairs capable of incorporating such classes of bulky uAAs.

Keeping in mind their various benefits, herein we report for the first time, the successful incorporation of four distinct FTUAAs in the 150th residue of sfGFP by utilising the orthogonal Tet 3.0 synthetase/tRNA pair [8a,b] derived from an archaea Methanosarcina mazei. Our results indicate that the variants of the synthetic proteins formed retained the beta-barrel structure of the sfGFP, while exhibiting unique photophysical properties, drawing consistency with triazole-mediated electronic interactions. Our findings thus highlight the immense potential of FTUAAs to be encoded in protein sequences to serve as genetically encodable fluorescent probes. Our report also paves a way towards engineering proteins with environment-sensitive photophysical properties towards applications in the fields of biosensing, imaging and study of biomolecular interactions.