Bioconjugation reactions are examples of bioorthogonal reactions in which a covalent bond is formed between two molecules, one of which is a biological molecule or its fragment. Such bioorthogonal reactions are also suitable for chemical cross-linking of proteins without the use of genetic engineering. Various reagents are used for covalent binding to proteins, with N-hydroxysuccinimidyl esters (NHS esters) being among the most common. These molecules preferentially react with free amine groups of lysine residues or thiol groups of cysteine residues. In this way, proteins can be covalently labelled with a desired functional group by conjugation (binding) with a suitably functionalized reagent.
As part of this thesis, we prepared various NHS esters and benzotriazolides that were functionalized with either an azide group or a cyclooctyne group. We developed a protocol for binding these molecules to proteins and an analytical method for quantifying the binding (loading) to proteins. Quantification consisted of a ‘click’ reaction between the azide-labelled fluorescein and the alkyne-labelled protein (and vice versa), followed by measuring the fluorescence intensity from the gel obtained by SDS-PAGE. With known loading of labels on the protein in hand, we were able to perform experiments on induced protein dimerization via azide–cyclooctyne [3+2] cycloaddition reaction, resulting in formation of a covalent 1,2,3-triazole linker. To extend the linker, we also prepared bifunctional reagents that could act as intermediate connectors of the two protein molecules.
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