Utilization of alkyne bioconjugations to modulate protein function

Abstract

The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein.

Playing with the Molecules of Life

“…193, 194 In addition ncAAs have been incorporated at residue 66 in GFP to alter the fluorescence properties of the protein. 37, 190, 195–198 , and into the coelenterazine binding site of aequorin to dramatically red-shift the bioluminescence of the interaction to afford in vivo bioluminescence reporters in mice. 197 …”

Development of optimized conditions for Glaser-Hay bioconjugations

“…1C) [32–34]. With incorporation of p PrF into green fluorescent protein (GFP), we generated a protein-fluorophore bioconjugate at 4 °C after 4 h. Despite having successfully employed the Glaser-Hay coupling in a biological context, we observed noticeable protein degradation after about 6 h. We proposed that this degradation was potentially due to hydroxyl radicals generated from the copper (II)-hydroxyl intermediate in the catalytic cycle of the Glaser-Hay reaction, which is known to be deleterious to living systems [19,35].…”

Genetic Encoding of a Bioconjugation Handle for [2+2+2] Cycloaddition Reactions

“…One common bioorthogonal reaction employed to prepare protein bioconjugates is the copper‐catalyzed azide/alkyne cycloaddition (CuAAC), known as the “copper click” reaction, in which an UAA with either an alkyne or azide group is reacted with an azide‐containing or alkyne‐containing reaction partner, respectively . More recently, we reported the development of a Glaser–Hay bioconjugation reaction involving the coupling of an alkynyl UAA with a terminal alkyne reaction partner to afford a covalent diyne linkage . However, there are a limited number of bioconjugation reactions available, as they necessitate physiological conditions and must lack cross‐reactivity with endogenous biological systems .…”