Alkyne-phenol-II

Alkyne tyramide-II (Alkyne-phenol-II, Alk-Ph-II, Ph_Amide_Yne) is a clickable alkyne-phenol substrate engineered to overcome the yeast cell wall permeability barrier that renders standard biotin-phenol (BP) probes ineffective. By replacing the bulky biotin with a small, highly soluble alkyne handle, Alk-Ph-II enables efficient labeling in intact cells, circumventing the need for artifact-inducing pretreatments. Upon H₂O₂ activation, genetically targeted APEX2 catalyzes the one-electron oxidation of Alk-Ph-II, generating a short-lived phenoxyl radical. This intermediate irreversibly tags proximal proteins and RNA within a ~10 nm radius in a rapid, 1-minute reaction, which is then captured via CuAAC click chemistry. This strategy delivers high-specificity (94%) proteomic maps, identifying 165 mitochondrial matrix proteins, and uniquely facilitates the first successful proximity-dependent transcriptome profiling (APEX-seq) in yeast.