Steric-Free Bioorthogonal Labeling of Acetylation Substrates Based on a Fluorine–Thiol Displacement Reaction

Abstract

We have developed a novel bioorthogonal reaction that can selectively displace fluorine substitutions alpha to amide bonds. This fluorine–thiol displacement reaction (FTDR) allows for fluorinated cofactors or precursors to be utilized as chemical reporters, hijacking acetyltransferase-mediated acetylation both in vitro and in live cells, which cannot be achieved with azide- or alkyne-based chemical reporters. The fluoroacetamide labels can be further converted to biotin or fluorophore tags using FTDR, enabling the general detection and imaging of acetyl substrates. This strategy may lead to a steric-free labeling platform for substrate proteins, expanding our chemical toolbox for functional annotation of post-translational modifications in a systematic manner.

New opportunities for immunomodulation of the tumour microenvironment using chemical tools

“…196 These sialic acids showed substantially higher affinity for different Siglec family members, leading to the activation of a downstream immunoreceptor tyrosine-based inhibitory motif or immunoreceptor tyrosine-based activation motif, thus enhancing immunomodulatory effects. In addition to reactions between azide and alkyne groups, other biorthogonal reactions may also have wide applications, such as the cycloaddition reaction between tetrazine and cyclopropene or isonitrile, 193 the substitution reaction between fluorine and thiol on fluoroacetamide, 197 and the phospha-Michael addition reaction. 198 These reactions have potential applications in labelling as well as in corresponding modifications; however, their in vivo usages require further validation.…”

Molecular probes for cellular imaging of post-translational proteoforms

“…[92][93][94] Beyond effects on PTM transfer processes, functional effects on the receiving end-the proteoform-must be carefully characterized. Continual developments to create minimally perturbative tools to label PTMs (for instance, the development of isosteric fluorinated cofactors taggable by the fluorine-thiol displacement reaction 95 ) are crucial to the characterization of native proteoform function.…”

Steric-free bioorthogonal profiling of cellular acetylation and glycosylation via a fluorine-selenol displacement reaction (FSeDR)

“…We attempted the reaction kinetics studies (Figure 3E) at concentrations of 5 mM and 10 mM, respectively, and the resulting 2 nd order rate constant is ~ 4.25 ± 0.18 x 10 -3 M -1 s -1 , which is at least 4-5 times faster than the previously reported FTDR reaction. 21 Labeling and pull down of the model protein using FSeDR gradually reaching saturation after 6h of reaction. The minimal signal observed from the control group (unmodified BSA) indicated the relative specificity of Biotin−SeH/SH (Figure 4B).…”

“…29 Followed with copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, one can expend these tags with functional groups such as fluorophores for detection or biotin for pull-down and chemical proteomics studies (Figure 1). Yet, the alkyne or azide tags were recently revealed to be bulky in size, 21 and thereby cannot be incorporated by most acetyltransferases 30 or even accommodated well by glycosyltransferases. 31 While mutations in protein pockets can be pursued, 30,[32][33] the tedious process may compromise the correct folding of PTM 'writers' or even further perturb the intrinsic acylome complex.…”

“…20 Detecting these PTMs and understanding the substrate proteins would be of urgent importance for diagnostic and therapeutic purposes but were largely limited by the currently available tools. 21 Classical methods to detect and analyze PTMs rely on antibodies and antibody-based LC-MS/MS proteomics analysis. 22 However, the small-sized PTMs makes their recognition and enrichment challenging and the results varied significantly per antibody.…”