Synthesis of Bioconjugation Reagents for Use in Covalent Cross-Linking of Proteins by Azide-Alkyne Cycloaddition

A series of azide- and cyclooctyne-functionalized <i>N</i>-hydroxysuccinimidyl esters (NHS esters) and benzotriazolides were prepared and used as N-acylation reagents to obtain azide-(<b>BSA-1</b>) and cyclooctyne-functionalized bovine serum albumin proteins (<b>BSA-2</b>), fluorescein derivatives <b>5</b> and <b>6</b>, and homobifunctional linkers <b>3</b> and <b>4</b>. Strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) of azide-functionalized fluorescent probe <b>5</b> and alkyne-functionalized fluorescent probe <b>6</b> with complementary functionalized proteins <b>BSA-2</b> and <b>BSA-1</b> yielded fluorescent cycloadducts <b>BSA-2-5</b> and <b>BSA-1-6</b>. These cycloadducts were used to determine the loading of <b>BSA-1</b> and <b>BSA-2</b> with the respective azido and cyclooctyne groups based on their molar absorbances and fluorescence intensities. Dimerization through covalent cross-linking of <b>BSA</b> was then performed by SPAAC between azide-functionalized <b>BSA-1</b> and cyclooctyne-functionalized <b>BSA-2</b>, and by treating <b>BSA-1</b> and <b>BSA-2</b> with 0.5 equiv. of complementary bis-cyclooctyne linker <b>4</b> and bis-azide linker <b>3</b>. Although the formation of covalent dimers <b>BSA-1-2-BSA</b>, <b>BSA-1-6-1-BSA</b>, and <b>BSA-2-5-2-BSA</b> was detected by SDS-PAGE analysis, this was a minor process, and most of the functionalized <b>BSA</b> did not form covalent dimers.