Synthesis of bioconjugation reagents for use in covalent cross-linking of proteins by azide-alkyne cycloadditionSuhorepec, Nadja (Avtor) Ciber, Luka (Avtor) Grošelj, Uroš (Avtor) Petek, Nejc (Avtor) Štefane, Bogdan (Avtor) Novinec, Marko (Avtor) Svete, Jurij (Avtor) protein cross-linkingazide-alkyne cycloadditionfluorescein dyesamidationbenzotriazolidesA series of azide- and cyclooctyne-functionalized N-hydroxysuccinimidyl esters (NHS esters) and benzotriazolides were prepared and used as N-acylation reagents to obtain azide-(BSA-1) and cyclooctyne-functionalized bovine serum albumin proteins (BSA-2), fluorescein derivatives 5 and 6, and homobifunctional linkers 3 and 4. Strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) of azide-functionalized fluorescent probe 5 and alkyne-functionalized fluorescent probe 6 with complementary functionalized proteins BSA-2 and BSA-1 yielded fluorescent cycloadducts BSA-2-5 and BSA-1-6. These cycloadducts were used to determine the loading of BSA-1 and BSA-2 with the respective azido and cyclooctyne groups based on their molar absorbances and fluorescence intensities. Dimerization through covalent cross-linking of BSA was then performed by SPAAC between azide-functionalized BSA-1 and cyclooctyne-functionalized BSA-2, and by treating BSA-1 and BSA-2 with 0.5 equiv. of complementary bis-cyclooctyne linker 4 and bis-azide linker 3. Although the formation of covalent dimers BSA-1-2-BSA, BSA-1-6-1-BSA, and BSA-2-5-2-BSA was detected by SDS-PAGE analysis, this was a minor process, and most of the functionalized BSA did not form covalent dimers.20252025-12-03 11:04:48Članek v reviji176538UDK: 547.79:577.112ISSN pri članku: 1420-3049DOI: 10.3390/molecules30234623COBISS_ID: 259740931sl