Adequate Reducing Conditions Enable Conjugation of Oxidized Peptides to Polymers by One-Pot Thiol Click Chemistry

Abstract

Thiol­(-click) chemistry has been extensively investigated to conjugate (bio)­molecules to polymers. Handling of cysteine-containing molecules may however be cumbersome, especially in the case of fast-oxidizing coiled-coil-forming peptides. In the present study, we investigated the practicality of a one-pot process to concomitantly reduce and conjugate an oxidized peptide to a polymer. Three thiol-based conjugation chemistries (vinyl sulfone (VS), maleimide, and pyridyldithiol) were assayed along with three reducing agents (tris­(2-carboxyethyl)­phosphine (TCEP), dithiothreitol, and β-mercaptoethanol). Seven out of the nine possible combinations significantly enhanced the conjugation yield, provided that an adequate concentration of reductant was used. Among them, the coincubation of an oxidized peptide with TCEP and a VS-modified polymer displayed the highest level of conjugation. Our results also provide insights into two topics that currently lack consensus: TCEP is stable in 10 mM phosphate buffered saline and it reacts with thiol-alkylating agents at submillimolar concentrations, and thus should be carefully used in order to avoid interference with thiol-based conjugation reactions.

Grafting Strategies of Oxidation-Prone Coiled-Coil Peptides for Protein Capture in Bioassays: Impact of Orientation and the Oxidation State

“…Reducing the disulfide bond could theoretically reactivate the Cys-Kcoil, but we observed that reducing agents deactivated the functional groups of the linkers typically used to perform the thiol chemistry . Hence, reversing the oxidation to subsequently perform the grafting is not practically feasible.…”

“…33 Reducing the disulfide bond could theoretically reactivate the Cys-Kcoil, but we observed that reducing agents deactivated the functional groups of the linkers typically used to perform the thiol chemistry. 34 Hence, reversing the oxidation to subsequently perform the grafting is not practically feasible. Therefore, producing a Kcoil that is devoid of any cysteine residue (Cys-free Kcoil) would be preferable as there would be no need to monitor/control the oxidation level, rendering large-scale production in a bioreactor possible.…”

“…This rapid oxidation phenomenon complicates peptide grafting and introduces variability in the yield of the grafting process. Moreover, increasing the grafting yield using reducing agents is not practically feasible as we have shown that such reagents deactivate the functional groups of the linkers used for thiol chemistry . Since the Cys-Kcoil peptide is difficult and costly to produce by chemical synthesis due to its length, and difficulty to recover from bacterial production, we here evaluate alternative coupling chemistry that would negate the inclusion of a cysteine altogether, and avoid the issues associated with the terminal thiol functional group.…”

Versatile and High-Throughput Strategy for the Quantification of Proteins Bound to Nanoparticles

“…CEE and 10% PLA- b -PEG-maleimide NPs were incubated (1000:1 molar ratio) overnight in 100 mM saline phosphate buffer (pH 7.4) in the presence of the reducing agent tris­(2-carboxyethyl)­phosphine (TCEP, 100:1 TCEP/CEE molar ratio) for the reduction of disulfide bridges that might form between CEE molecules. Previous work in our research group had shown that these reducing conditions prove adequate to promote thiol–maleimide coupling, even when a significant fraction of the cysteine-terminated peptide is oxidized …”

“…Previous work in our research group had shown that these reducing conditions prove adequate to promote thiol−maleimide coupling, even when a significant fraction of the cysteine-terminated peptide is oxidized. 58 The samples then underwent three cycles of purification through an Amicon centrifugal filter unit (cutoff 50 kDa, Millipore Sigma #UFC9050). Supernatants were kept for further quantification of the unreacted CEE.…”