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

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

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.

• Language: English
• Keywords: protein cross-linking, azide-alkyne cycloaddition, fluorescein dyes, amidation, benzotriazolides
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Publication status: Published
• Publication version: Version of Record
• Year: 2025
• Number of pages: 26 str.
• Numbering: Vol. 30, iss. 23, art. 4623
• PID: 20.500.12556/RUL-176538
• UDC: 547.79:577.112
• ISSN on article: 1420-3049
• DOI: 10.3390/molecules30234623
• COBISS.SI-ID: 259740931
• Publication date in RUL: 03.12.2025

Record is a part of a journal

• Title: Molecules
• Shortened title: Molecules
• Publisher: MDPI
• ISSN: 1420-3049
• COBISS.SI-ID: 18462981

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: povezovanje proteinov, azid-alkin cikloadicija, fluoresceinska barvila, amidiranje, benzotriazolidi