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

Abstract

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.

Photocatalytic Late-Stage C–H Functionalization

“…Taylor and co-workers aimed at leveraging tryptophan tendency to engage in photoinduced electron transfer with N -amido-pyridinium species ( 59.1 ), as opposed to its usual nucleophilicity (cf. Schemes and ) . Upon photon absorption from EDA-complex 59.2 , a transient charge separation can trigger N–N bond fragmentation to generate a new C–N linkage and release pyridinium salt 59.5 .…”

Chemical modification of proteins – challenges and trends at the start of the 2020s

“…167 Using a related donor–acceptor biaryl N -carbamoylpyridinium salt, the group was able to extend the method to Trp profiling in cell lysates and live HEK293T cells. 168…”

Carbon-Centered Radicals in Protein Manipulation

“…Trp radical cations have also been invoked as putative intermediates formed from in situ photoexcited electron donor•acceptor pairs (Trp donor•Katritzsky-type pyridnium acceptors) that allow elegant methodology for Trp reactivity under a range of wavelengths. 104,105 Met-Derived Radicals. Met is also of relatively low abundance within the protein structures.…”