Extent of the Oxidative Side Reactions to Peptides and Proteins During the CuAAC Reaction

Abstract

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a powerful tool for bioconjugation of biomolecules, particularly proteins and peptides. The major drawback limiting the use of the CuAAC reaction in biological systems is the copper-mediated formation of reactive oxygen species (ROS), leading to the oxidative degradation of proteins or peptides. From the studies on a limited number of proteins and peptides, it is known that, in general, the copper mediated oxidative damage is associated with the copper coordination environment and solvent accessibility. However, there is a lack of data to help estimate the extent of copper-mediated oxidation on a wide range of proteins and peptides. To begin to address this need, we quantitatively measured the degree of copper-mediated oxidation on libraries of 1200 tetrapeptides and a model protein (bovine serum albumin, BSA) using liquid chromatography mass spectrometry (LC-MS). The collected data will be useful to researchers planning to use the CuAAC reaction for bioconjugaton on peptides or proteins.

Tripodal amine ligands for accelerating Cu-catalyzed azide–alkyne cycloaddition: efficiency and stability against oxidation and dissociation

“…Thus, the peptide oxidation is correlated to the amount of reactive oxygen species (ROS) generated by Cu(I)/Cu(II)/ascorbate/O 2 redox cycle. 26–28, 32 …”

“…1 & 2), 23 are the most efficient ligands that enable the reaction to be completed within minutes at tens of micromolar level of copper. 24, 25 However, in the complex biological systems, the cytotoxicity of Cu(I) 26–28 and the presence of strong-coordinating biological chelators that deactivate the copper catalyst 24, 29 greatly hampered the application of CuAAC reaction in living systems. 30 …”

“…2, 31 Nevertheless, the Cu(I)/Cu(II)/ascorbate/O 2 redox cycle rapidly generates reactive oxygen species (ROS) 32 that can degrade the biomolecules and the Cu(I) ligands, 26–28 which is one of the major drawbacks of CuAAC reaction compared to the copper(I)-free bioconjugation methods. 33 The oxidation mechanism of Cu(I) complexes has been extensive studied.…”

Copper-catalyzed click reaction on/in live cells

“…6,15–17 However, its use in living systems has been limited by the cytotoxicity of excess Cu I that mediates the generation of reactive oxygen species (ROS). 18–20 …”

[18F]ODIBO: a prosthetic group for bioorthogonal radiolabeling of macromolecules via strain-promoted alkyne–azide cycloaddition

“…Several recently discovered biocompatible conjugations have found applications in PET radiochemistry in the last two decades 2 . Copper (I) catalyzed acetylene-azide cycloaddition (CuAAC) 3 , considered a prototypical “click” reaction, found somewhat limited utility in biomedical applications, particularly in syntheses of radiopharmaceuticals for clinical nuclear medicine due to the presence of Cu(I) ions 4 which cause oxidative degradation of DNA 5 or coordinate peptides and proteins 6 . Strict current Good Manufacturing Practices (cGMP) regulations require extensive quality control protocols to analyze each batch of radiotracer for the absence of a cytotoxic copper contamination.…”