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 re-mains 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 enabling 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 450 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.

Triazolinedione protein modification: from an overlooked off-target effect to a tryptophan-based bioconjugation strategy

“…20 This approach was further refined and now allows for the use of visible light in presence of ambient air. 21 In 2010, Barbas and co-workers reported a click like reaction for the more abundant tyrosine (Tyr, 3.3% abundance 7 ) using triazolinedione chemistry, 22 after which many applications and refinements for protein conjugation followed. [23][24][25][26][27][28][29] Interestingly, when exploring this powerful Tyr click reaction on Trp containing peptides, we observed a high degree of off-target labelling on Trp residues, even in aqueous buffers.…”

Photocatalytic Late-Stage C–H Functionalization

“…Schemes 58 and 59). 250 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. In order to use visible-light irradiation, 251 and avoid photodegradation of labile proteins, the highly conjugated chromophore system of 59.1 features a donor−acceptor system to enforce bathochromic shift.…”

“…Their often delicate structure renders visible-light photocatalysis a valuable mean toward their mild functionalization. 130,133,136,137,139,250,251 The deuteration/tritiation of the α-position of amino acids can offer crucial insights both in in vitro and in in vivo studies. Furthermore, the kinetic isotope effect can reduce their epimerization.…”

A Toolkit for Engineering Proteins in Living Cells: Peptide with a Tryptophan-Selective Ru-TAP Complex to Regioselectively Photolabel Specific Proteins

“…Histidine-, 8−10 tyrosine-, 11−15 methionine-, 16−18 and tryptophan-reactive 19−21 reagents have been developed for in vitro labeling of peptides and native proteins. One example of chemoselectivity for tryptophan (Trp) involves a photoinduced electron transfer (PeT) from a Trp-containing peptide, photoexcited in the near UV, toward differently substituted pyridinium salts in solution, 22,23 allowing the transfer of various chemical groups from pyridinium to the peptide Trp.…”