Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes

Abstract

Described herein are the synthesis and photophysical characterization of a library of aryl-substituted oxazole- and thiazole-based dipeptidomimetic analogues, and their incorporation into position 66 of green fluorescent protein (GFP) in lieu of the natural fluorophore. These fluorescent analogues resemble the fluorophore formed naturally by GFP. As anticipated, the photophysical properties of the analogues varied as a function of the substituents at the para position of the phenyl ring. The fluorescence emission wavelength maxima of compounds in the library varied from ~365 nm (near-UV region) to ~490 nm (visible region). The compounds also exhibited a large range of quantum yields (0.01–0.92). The analogues were used to activate a suppressor tRNACUA and were incorporated into position 66 of GFP using an in vitro protein biosynthesizing system that employed engineered ribosomes selected for their ability to incorporate dipeptides. Four analogues with interesting photophysical properties and reasonable suppression yields were chosen, and the fluorescent proteins (FPs) containing these fluorophores were prepared on a larger scale for more detailed study. When the FPs were compared with the respective aminoacyl-tRNAs and the actual dipeptide analogues, the FPs exhibited significantly enhanced fluorescence intensities at the same concentrations. Part of this was shown to be due to the presence of the fluorophores as an intrinsic element of the protein backbone. There were also characteristic shifts in the emission maxima, indicating the environmental sensitivity of these probes. Acridon-2-ylalanine and oxazole 1a were incorporated into positions 39 and 66 of GFP, respectively, and were shown to form an efficient Förster resonance energy transfer (FRET) pair, demonstrating that the analogues can be used as FRET probes.

In vitro genetic code reprogramming and expansion to study protein function and discover macrocyclic peptide ligands

“…For example, Hecht introduced 6-cyano tryptophan as a FRET pair to study the binding of several proteins to DNA labeled with fluorescent nucleobases [26]. Using the dipeptidyl-tRNA tolerant ribosomes, he incorporated aryl oxazole and thiazole dipeptide mimics into the GFP structure, leading to the formation of modified fluorophores and fluorescent proteins [27]. Introduction of ncAAs has also made its mark in the synthesis of radiolabeled proteins for PET imaging.…”

Continuous multistep synthesis of 2-(azidomethyl)oxazoles

“…In addition, ongoing studies show the potential of amino and amido-oxazoles to act as fluorescent dipeptidomimetics ( Fig. 1 ) [ 9 ]. Due to their diene character, oxazoles find also use as intermediates in the synthesis of other organic scaffolds such as furans and pyridines, via cycloaddition/retro-cycloaddition tandem processes ( Fig.…”

Posttranslational chemical installation of azoles into translated peptides

“…To the best of our knowledge, the only adequate approach reported in the literature is the use of a mutated ribosome that incorporates azolecontaining dipeptide units into the nascent peptide chain [30][31][32][33][34] . Unfortunately, the incorporation of such azole-containing dipeptide units into the nascent peptide chain was very modest where only 1-7% efficiency was observed for the expression of green fluorescent protein (GFP) mutants compared to the wildtype GFP expression even using the dedicated mutant ribosome (i.e., the conventional wildtype ribosome could not do the job at all) 31,32 . This implies that direct incorporation of azole moiety via such a peptidyl-transfer reaction is yet an arduous task.…”