Donor–Acceptor Pyridinium Salts for Photo-Induced Electron-Transfer-Driven Modification of Tryptophan in Peptides, Proteins, and Proteomes Using Visible Light

Autor: Caleb R. Hoopes, Francisco J. Garcia, Akash M. Sarkar, Nicholas J. Kuehl, David T. Barkan, Nicole L. Collins, Glenna E. Meister, Taylor R. Bramhall, Chien-Hsiang Hsu, Michael D. Jones, Markus Schirle, Michael T. Taylor
Rok vydání: 2022
Předmět:
Zdroj: J Am Chem Soc
ISSN: 1520-5126
0002-7863
Popis: Tryptophan (Trp) plays a variety of critical functional roles in protein biochemistry however, owing to its low natural frequency and poor nucleophilicity, the design of effective methods for both single protein bioconjugation at Trp as well as for in situ chemoproteomic profiling remains a challenge. Here, we report a method for covalent Trp modification that is suitable for both scenarios by invoking photo-induced electron transfer (PET) as a means of driving efficient reactivity. We have engineered biaryl N-carbamoyl pyridinium salts that possess a donor-acceptor relationship which enables optical triggering with visible light whilst simultaneously attenuating the probe’s photo-oxidation potential in order to prevent photodegradation. This probe was assayed against a small bank of eight peptides and proteins, where it was found that micromolar concentrations of probe and short irradiation times (10–60 min) with violet light enabled efficient reactivity towards surface exposed Trp residues. The carbamate transferring group can be used to transfer useful functional groups to proteins including affinity tags and click handles. DFT calculations and other mechanistic analyses reveal correlations between excited state lifetimes, relative fluorescent quantum yields, and chemical reactivity. Biotinylated and azide-functionalized pyridinium salts were used for Trp profiling in HEK293T lysates and in situ in HEK293T cells using 440 nm LED irradiation. Peptide level enrichment from live cell labelling experiments identified 290 Trp modifications, with an 82% selectivity for Trp modification over other π-amino acids; demonstrating the ability of this method to identify and quantify reactive Trp residues from live cells.
Databáze: OpenAIRE
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