Design and Synthesis of Live-cell Fluorescent Probes

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Andrew Anzalone, Virginia W. Cornish, Tracy Y. Wang, Zhixing Chen, Ruben L. Gonzalez

Columbia University, United States

Engineered fluorescent dyes are increasingly being developed for use as live cell sensors, extending their utility beyond traditional imaging reagents. The improved photophysical and fluorescence properties of oxazine and xanthene dyes make them ideal components for these powerful tools, which are capable of providing dynamic information on signaling networks, molecular/chemical processes, and protein-protein interactions. As such, fluorescent sensors can be harnessed for screening of many cellular activities of interest. In aim to develop novel methodologies that exploit chemical reagents for directed cellular evolution, we report a novel and scalable synthetic approach to the widely used oxazine and xanthene fluorophores through a common diaryl-ether intermediate. Taking advantage of recent advances in transition-metal catalysis, we prepared electronically activated diaryl-ethers to serve as tethered di-nucleophiles. These diaryl-ethers react with a range of electrophiles to undergo cyclization to oxazine and xanthene fluorophores. Using yet another transition metal catalyzed reaction, a late-stage modification of the synthetic route allows for the preparation of fluorogenic (pro-fluorescent) dyes bearing a series of sensitive caging groups. When encountering the appropriate stimulus, the pro-fluorescent compounds are capable of providing inducible readouts in response to specific cellular processes. We believe these cell permeable dyes will enable new methodology for selectively probing desired functions. By teaming synthetic chemistry with synthetic biology, this work expands the toolkit available for engineering living systems.