Heterogeneous catalysis for azide-alkyne bioconjugation in solution via spin column: Attachment of dyes and saccharides to peptides and DNA

Abstract

Copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry is widely used and has demonstrated particular utility for bio-orthogonal conjugation reactions. Here we describe a one-pot, heterogeneous bioconjugation and purification method for selectively activated CuAAC. A Cu(II) precursor, with either the neutral ligand 1,10-phenanthroline-5,6-dione or the anionic ligand 4,7-diphenyl-1,10-phenanthroline-disulfonic acid, is converted to the active Cu(I) species within an ion-exchange matrix using zinc amalgam as the reducing agent. The Cu(I) complexes are then layered on top of a size-exclusion matrix within a commercial microcentrifuge spin column; passing a mixture of an ethynyl-labeled biomolecule and an azide-bearing ligand through the column results in clean and efficient coupling. The methodology is demonstrated by glycosylating a DNA oligonucleotide as well as by labeling a membrane-penetrating peptide (octa-arginine) with a coumarin dye.

Keywords: DNA glycosylation; bioconjugation; cell-penetrating peptide; heterogeneous click conjugation.