Rapid Biomolecular Trifluoromethylation Using Cationic Aromatic Sulfonate Esters as Visible-Light-Triggered Radical Photocages.

Described here is a photodecaging approach to radical trifluoromethylation of biomolecules. This was accomplished by designing a quinolinium sulfonate ester that, upon absorption of visible light, achieves decaging via photolysis of the sulfonate ester to ultimately liberate free trifluoromethyl radicals that are trapped by π-nucleophiles in biomolecules. This photodecaging process enables protein and protein-interaction mapping experiments using trifluoromethyl radicals that require only 1 s reaction times and low photocage concentrations. In these experiments, aromatic side chains are labeled in an environmentally dependent fashion, with selectivity observed for tryptophan (Trp), followed by histidine (His) and tyrosine (Tyr). Scalable peptide trifluoromethylation through photodecaging is also demonstrated, where bespoke peptides harboring trifluoromethyl groups at tryptophan residues can be synthesized with 5-7 min reaction times and good yields.

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

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.

Selective Modification of Tryptophan Residues in Peptides and Proteins Using a Biomimetic Electron Transfer Process.

We report here a photochemical process for the selective modification of tryptophan (Trp) residues in peptides and small proteins using electron-responsive N-carbamoylpyridinium salts and UV-B light. Preliminary mechanistic experiments suggest that the photoconjugation process proceeds through photoinduced electron transfer (PET) between Trp and the pyridinium salt, followed by fragmentation of the pyridinium N-N bond and concomitant transfer of this group to Trp. The reaction displays excellent site selectivity for Trp and is tolerant to other, redox-active amino-acid residues. Moreover, the reaction proceeds in pure aqueous conditions without the requirement of organic cosolvents or photocatalysts, is enhanced by glutathione, and operates efficiently over a wide range of peptide concentrations (10-700 µM). The scope of the process was explored through the labeling of 6-Trp-containing peptides and proteins ranging from 1 to 14 kDa. We demonstrate the versatility of the N-carbamoylpyridinium salt both by tuning the electrochemical and photochemical properties of the pyridinium scaffold to enable challenging photoconjugation reactions and by using the carbamoyl moiety to tether a plethora of productive functional groups, including reactive handles, purification tags, and removable protecting groups.