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

Abstract

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.

Figure 1.

Pyridinium salt reagents for Trp-selective protein modification by mimicking biological PET.

Figure 2.

(A) Transferring group scope. (B) Synthesis of alkyne-labeled 2 via derivatizable pyridinium salt 1c. (C) LC/MS TIC traces of crude reaction mixtures. ^a 120 min reaction time. [GSH] = 5 mM, [1a] = 20 mM. ^b % conversion estimated by TIC, average of 2–3 experiments. Ratio of mono/di label >20:1 unless stated otherwise. ^c Isolated after HPLC purification, average of three experiments.

Figure 3.

Scope of the Trp photobioconjugation process. ^a % conversion was measured by TIC, average of 2 runs. ^b [1a] = 2 mM. ^c 150 min reaction, [GSH] = 5 mM, [1a] = 10 mM. ^d Isolated after HPLC purification, average of two experiments. ^e 20 mM 1a. ^f Tris(2-carboxyethyl)phosphine (TCEP) (1 mM) used as an additive.

Figure 4.

Mechanistic studies. (A) Temporal control experiments with 2 and 1a. (B) Additive and light-perturbation experiments. (C) Absorption spectra of 2, 1a, and 1d. (D) Mechanistic and NMR experiments performed on Trp analogs. ^a % conjugate observed by TIC. ^b Performed in a foiled reaction vessel that was irradiated. ^c UVP 365 nm lamp (8 W).

Figure 5.

(A) Photochemical labeling of 7 and mechanistic reversal of the labeling process using 1d. Trp residues in 7 are highlighted in cyan. (B) Unmodified 7. (C) 7 + 1a. (D) 7 + 1d, 15 min irradiation time at 302 nm. (E) 7 + 1d, 45 min irradiation time with 320 nmlong pass filter. (F) Confirmation of site selectivity by MS-MS. ^a Conversion estimated by TIC, average of 2 runs. ^b 7 mM 1a, 1 mM GSH, 20 mM pH 6.9 NH₄OAc buffer. ^c 3 mM 1d, 6 mM GSH, 20 mM pH 6.9 NH₄OAc buffer. ^d Ratio of +1 to +2 modifications. ^e 3 mM 1d, 3 mM GSH, 20 mM pH 6.9 NH₄OAc buffer, 320 nm long-pass filter used.