Donor–Acceptor
Pyridinium Salts for Photo-Induced
Electron-Transfer-Driven Modification of Tryptophan in Peptides, Proteins,
and Proteomes Using Visible Light
posted on 2022-04-02, 05:20authored byCaleb
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
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 that 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 the probe and short irradiation times
(10–60 min) with violet light enabled efficient reactivity
toward 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
fluorescence 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 labeling
experiments identified 290 Trp modifications, with 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.