Single-Step Azide Introduction in Proteins via an Aqueous Diazo Transfer

Abstract

The controlled introduction of azides in proteins provides targetable handles for selective protein manipulation. We present here an efficient diazo transfer protocol that can be applied in an aqueous solution, leading to the facile introduction of azides in the side chains of lysine residues and at the N-terminus of enzymes, e.g. horseradish peroxidase (HRP) and the red fluorescent protein DsRed. The effective introduction of azides was verified by mass spectrometry, after which the azido-proteins were used in Cu(I)-catalyzed [3 + 2] cycloaddition reactions. Azido-HRP retained its catalytic activity after conjugation of a small molecule. This modified protein could also be successfully immobilized on the surface of an acetylene-covered polymersome. Azido-DsRed was coupled to an acetylene-bearing protein allowing it to act as a fluorescent label, demonstrating the wide applicability of the diazo transfer procedure.

Staudinger Ligation as a Method for Bioconjugation

“…The azide is preferred as the chemical handle because of the high degree of oxidation of the phosphine moiety in aqueous solutions. The azide can be introduced into a protein chemically, that is, by a diazotransfer reaction [39] or biochemically, either by using genetic engineering for the incorporation of azide-containing amino acids into proteins [40] or by post-translational modifications. The latter concept was used in a combined effort by the research groups of Tirrell and Bertozzi.…”

Functionalization of Block Copolymer Vesicle Surfaces

“…Glucose oxidase (GOx) was encapsulated in the interior of these polymersomes and Candida antarctica lipase B (CalB) was incorporated into the vesicle bilayer membranes. Horseradish peroxidase (HRP) was reacted with imidazole-1-sulfonyl azide hydrochloride to introduce azide functionalities to this enzyme [57,58]. The HRP was then conjugated to the outer surface of the polymersomes via click-chemistry.…”

A DNA-based strategy for dynamic positional enzyme immobilization inside fused silica microchannels

“…Candida antarctica lipase B and its azidefunctionalized counterpart (AHA-CalB) as well as the azidefunctionalized HRP (azido-HRP) and the substrate glucose mono-acetate (1-O-acetyl-D-gluco-pyranose, further denoted as Gluc-Ac), were obtained as described elsewhere. 23,24 Horseradish peroxidase (E.C. 1.11.1.7) type VI and glucose oxidase (E.C.…”

“…GOx was left in solution as it is the ''slowest'' enzyme in sequence. HRP was prepared following literature procedures 24 and coupled with CuAAC to hexynyl-5 0 -AGT ATT GAC CTA AGT ATT GAC-3 0 , under similar conditions as applied for DNA-CalB to obtain DNA-HRP. As previous findings showed that unreacted CalB did not bind to the modified capillaries, the DNA-HRP was only filter dialyzed to remove the excess of unreacted DNA.…”