Exogenous phenol probes, especially biotin-phenol, can be transformed into reactive phenoxyl radicals by genetic fusions of engineered ascorbic acid peroxidase (e.g. APEX2) enzymes to label the subcellular proteome in organelles as well as classify protein complex members under various conditions on a minute time scale. Even with the broad application of biotin-phenol as APEX2 substrate, there may be concerns over the cellular responses to biotin-phenol. The labeling radius of the phenoxyl radical and thus the labeling specificity of the proximity labeling was predicted to be modulated by changing the aromatic ring with chemical substituents. Herein, we introduce a new APEX2 substrate for spectroscopy and microscopy. A panel of aromatic containing alkyne compounds was synthesized and screened for APEX activity. 4-((4-Ethynylphenyl)buta-1,3-diyn-1-yl)phenol (4-EPBDP) was identified as a novel probe with significantly greater reactivity towards proteins in various subcellular compartments including nucleus, the endoplasmic reticulum, and mitochondria. 4-EPBDP possesses a large, conjugated structure connecting two internal alkynes, two phenyl groups, and one terminal alkyne for strong Raman scattering signal while the terminal alkyne serves as a clickable tag for fluorescence microscopy or mass spectrometry for proteomic profiling. From confocal fluorescence microscopy, we found that 4-EPBDP enables rapid labeling with remarkably high specificity and low cell toxicity in comparison to biotin-phenol which suffers from non-specific labeling of mitochondria and apoptotic response. Moreover, 4-EPBDP probe demonstrated vibrational solvatochromism, making it a promising molecule for sensing the local environment of a specific subcellular compartment by measuring the frequency shift of the alkyne peak at 2200 cm<sup>-1</sup> in the cell-free window. With a standard Raman spectrometer, we measured statistically significant Raman frequency shifts in various organelles. Therefore, we opened a new application of proximity labeling for probing variety of subcellular environments by Raman spectroscopy.

Diyne based Raman and clickable probes for proximity-dependent proteomic labeling

ACS Spring Meeting 2021

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