Synthesis of bioconjugation reagents for use in covalent cross-linking of proteins by azide-alkyne cycloaddition

Suhorepec, Nadja (avtor), Ciber, Luka (avtor), Grošelj, Uroš (avtor), Petek, Nejc (avtor), Štefane, Bogdan (avtor), Novinec, Marko (avtor), Svete, Jurij (avtor)

A 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.

• Jezik: Angleški jezik
• Ključne besede: protein cross-linking, azide-alkyne cycloaddition, fluorescein dyes, amidation, benzotriazolides
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2025
• Št. strani: 26 str.
• Številčenje: Vol. 30, iss. 23, art. 4623
• PID: 20.500.12556/RUL-176538
• UDK: 547.79:577.112
• ISSN pri članku: 1420-3049
• DOI: 10.3390/molecules30234623
• COBISS.SI-ID: 259740931
• Datum objave v RUL: 03.12.2025

Gradivo je del revije

• Naslov: Molecules
• Skrajšan naslov: Molecules
• Založnik: MDPI
• ISSN: 1420-3049
• COBISS.SI-ID: 18462981

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: povezovanje proteinov, azid-alkin cikloadicija, fluoresceinska barvila, amidiranje, benzotriazolidi