High-throughput discovery of a dearomative cycloaddition enables dual photochemical-photophysical perturbative probing of protein function
Proteins exist in multiple conformations or states, often associated with different functional capacities. The ability to selectively identify and study these states is crucial for understanding the dynamic nature of proteins and their roles in cellular processes, yet can be currently limited by the artefacts of existing molecular labels and the lack of strategies to exploit them in diverse ways. Whilst i) methods for the photochemical perturbation of proteins and ii) photophysical protein probes and sensors are both known powerful tools, simultaneous methods for site- and context-specific photochemical modification of a protein side chain with concomitant modulation of photophysical properties would allow interrogation of protein function via diverse modes (potentially reporting simultaneously on structure and dynamics of ground- and reactive-state properties). Due to their mechanistic complexity, rational approaches for the design of suitable photochemical strategies can be difficult and non-intuitive. Here, we describe a high-throughput experimentation (HTE) strategy designed to allow the photochemical modification of a genetically-encoded, minimally disruptive ( zero-size), photophysically-active - yet typically unreactive and biologically-inert - unnatural amino acid (uAA) residue in proteins. The discovery of a novel dearomative, formal [4+3] photocycloaddition of imidazopyrimidine reagents which proceeds via photochemically generated C* intermediates with polyaromatic substrates enabled chemoselective application to the normally unreactive Trp-mimetic residue, 2-naphthylalanine (Npa). Npas site-selective installation allows dual photochemical and photophysical dearomative perturbation benignly and efficiently in proteins. Replacement of Trp in the archetypal apoptotic biosensor protein Annexin A5 (AnxV) enabled dual photochemical-photophysical determination of switched, conformationally-determined reactive states induced by Ca2+-binding, without any apparent functional perturbation of its in vitro or cellular recognition of phospholipids, indicating an ordered annexin mechanism. This ready and benign unification of photochemistry and photophysics suggests a general approach for using high-throughput light-mediated experimentation in artefact-free chemical biology to capture different functional states of proteins and to interrogate those states using combined photochemical-photophysical methods. HighlightsO_LIDevelopment of a general, high-throughput experimentation (HTE) approach enabled identification of an unprecedented photochemical reaction with potential for editing amino acid side chains within proteins. C_LIO_LIGenetic incorporation of 2-naphthylalanine, a minimally-disruptive ( zero-size) Trp mimic, enabled photochemical side chain editing with concomitant modulation of its distinctive photophysical properties in proteins. C_LIO_LIA naphthalene-tagged variant of the apoptotic biomarker protein AnxV enabled dual photochemical-photophysical exploration of its dynamic conformational states induced by Ca2+-binding whilst fully maintaining its functional capacity for in vitro or cellular recognition of phospholipids. C_LI