Protein bioconjugates are in high demand for applications in biomedicine, diagnostics, chemical biology and bionanotechnology. Proteins are large and sensitive molecules containing multiple different functional groups and in particular nucleophilic groups. In bioconjugation reactions it can therefore be challenging to obtain a homogeneous product in high yield. Numerous strategies for protein conjugation have been developed, of which a vast majority target lysine, cysteine and to a lesser extend tyrosine. Likewise, several methods that involve recombinantly engineered protein tags have been reported. In recent years a number of methods have emerged for chemical bioconjugation to other amino acids and in this review, we present the progress in this area.

Oligonucleotide conjugates constitute a versatile tool for research and bioanalytical purposes. Often, such conjugates are prepared by reaction between a thiol on the protein with a maleimide-modified oligonucleotide. Unlike most other chemical handles the maleimide functionality cannot be introduced directly during the solid-phase oligonucleotide synthesis, and therefore the standard method to introduce the maleimide functionality is to react an amino-modified DNA with a heterobifunctional linker containing an activated ester and a maleimide. Here, we present an alternative method for preparation of maleimide and monobromomaleimide-modified oligonucleotides from the corresponding amine using N-methoxycarbonylmaleimide and N-methoxycarbonylbromomaleimide, respectively. In this method, no additional linker is attached to the oligonucleotide, as the maleimide functionality is formed directly on the existing amine. The maleimide can thereby be positioned close to the oligonucleotide, providing a high degree of control over the final construct. The reaction occurs in 30–60 min under alkaline conditions. Maleimide-modified oligonucleotides prepared in this manner were conjugated to bovine serum albumin, and the reaction shows comparable reactivity to the corresponding oligonucleotide modified using the 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) linker.