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.
Highlights
The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a cornerstone method for the ligation of biomolecules
A significant limitation of the CuAAC reaction conducted under batch conditions is the need for a Cu catalyst; this can be problematic in a number of applications[5,6]
These issues have inspired the development of a series of alternative Cufree click approaches such as strain-promoted azide-alkyne cycloadditions (SPAAC)[12] and inverse electron demand DielsAlder (IEDDA) approaches using tetrazines[13]
Summary
Flow-based technologies offer distinct advantages over batch, such as enhanced mass transfer, which is advantageous for large molecular weight biomolecules where accessibility of functional groups is significantly compromised in the batch regime[25,26,27,28,29] Despite these advantages, application of flow-based CuAAC bioconjugation has not been reported due to the need for (i) excess Cu catalyst, which promotes biomolecule degradation, (ii) ionic scavengers, which can result in residual Cu trapped in bioconjugates, (iii) elevated temperatures, which promotes biomolecule degradation, and (iv) organic solvents, which typically limits biocompatibility. Diyne 18, containing aliphatic alkyne and aromatic ynamine sites, underwent sequential CuAAC ligation, firstly with the coumarin azide at the ynamine site followed by ligation with the nucleobase azide at the aliphatic alkyne site; complete chemoselectivity was observed throughout This demonstrates that established reactivity profiles[43] are replicated in the flow format and that our system enhances overall reaction kinetics but does so at very low [Cu]. Residues with known oxidative susceptibility (27a–e) under conventional CuAAC batch conditions were installed on the N-terminus to report any potential degradation
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