A flow platform for degradation-free CuAAC bioconjugation

Abstract

The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a cornerstone method for the ligation of biomolecules. However, undesired Cu-mediated oxidation and Cu-contamination in bioconjugates limits biomedical utility. Here, we report a generic CuAAC flow platform for the rapid, robust, and broad-spectrum formation of discrete triazole bioconjugates. This process leverages an engineering problem to chemical advantage: solvent-mediated Cu pipe erosion generates ppm levels of Cu in situ under laminar flow conditions. This is sufficient to catalyze the CuAAC reaction of small molecule alkynes and azides, fluorophores, marketed drug molecules, peptides, DNA, and therapeutic oligonucleotides. This flow approach, not replicated in batch, operates at ambient temperature and pressure, requires short residence times, avoids oxidation of sensitive functional groups, and produces products with very low ppm Cu contamination.

An Overview of Antibody Conjugated Polymeric Nanoparticles for Breast Cancer Therapy

“…In the same way, optimum polyethylenglycol (PEG) coverage over trastuzumab conjugated ACNPs was also prepared via CuAAC conjugation to improve the pharmacokinetics [111]. Of note, developments in copper-free variants of AAC were achieved by introducing a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to overcome the CuAAC limitations [112][113][114].…”

Site-selective lysine conjugation methods and applications towards antibody–drug conjugates

“…33 More recently, its benefits have been utilised for the synthesis of bioconjugates. 34,35 Bernardes et al investigated the use of sulfonyl acrylate reagents for selective antibody conjugation. 36 Computer-assisted reagent design helped the group select an appropriately structured sulfonyl acrylate (Scheme 2c), with identification of an intermolecular hydrogen bond between this acrylate and the lysine side-chain in a chair-like conformation found to be key for desirable reactivity.…”

Miniaturized and Automated Synthesis of Biomolecules—Overview and Perspectives

“…As one example, the use of Cu‐pipes as the catalytic medium for a Cu‐catalyzed alkyne–azide cycloaddition (CuAAC) reaction yielding triazoles was shown as an interesting example for flow chemistry recently . The authors could show that the use of Cu‐pipes yielded the target compounds with very low content of Cu, which allowed use in biological systems that are sensitive to the amount of copper impurities usually obtained in CuAAC reactions.…”