Selective Lysine Ubiquitination Using Activated Phenol Esters

ChemBioChem, vol. 26, pp. e202500266

Abstract

Ubiquitination of target proteins is an essential post-translational modification influencing a wide variety of cellular processes. Herein, the use of a novel water-soluble acylation reagent based on the 2,4-dichloro-6-sulfonic acid phenol ester of ubiquitin is described for efficient and selective ubiquitin modification of peptides. Under alkaline conditions, this reagent is swift and regioselective toward lysine acylation, while at neutral pH it shows loss of regioselectivity and is able to acylate both lysine and N-terminal modification at reduced speeds. As proof of concept, a model peptide is utilized to demonstrate this strategy, proving to be successful. Then the ubiquitination of a synthetic protein called Fau gene encoded Ubiquitin-like protein (FUBI) is performed under alkaline conditions followed by tandem MS analysis, proving that the selective lysine ubiquitination works to prepare protein-protein conjugates.

Authors 10

1. Halana C. Vlaming Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Dept. Cell and Chemical Biology Leiden University Medical Centre 2333 ZC Leiden The Netherlands

2. Yara Huppelschoten Aachen

   Leiden University · Novo Nordisk (Denmark) · Leiden University Medical Center

   Affiliation as printed

   Dept. Cell and Chemical Biology Leiden University Medical Centre 2333 ZC Leiden The Netherlands

   Global Research Technologies Novo Nordisk A/S Novo Nordisk Research Park DK‐2760 Måløv Denmark

3. Rayman T.N. Tjokrodirijo Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Centre for Proteomics and Metabolomics Leiden University Medical Centre 2300 RC Leiden The Netherlands

4. Peter A. van Veelen Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Centre for Proteomics and Metabolomics Leiden University Medical Centre 2300 RC Leiden The Netherlands

5. Francesca D’Amico Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Dept. Cell and Chemical Biology Leiden University Medical Centre 2333 ZC Leiden The Netherlands

6. Kim B. Jensen

   Novo Nordisk (Denmark)

   Affiliation as printed

   Global Research Technologies Novo Nordisk A/S Novo Nordisk Research Park DK‐2760 Måløv Denmark

7. Jens Buchardt

   Novo Nordisk (Denmark)

   Affiliation as printed

   CMC API Development Novo Nordisk A/S Novo Nordisk Park DK‐2880 Bagsvaerd Denmark

   Global Research Technologies Novo Nordisk A/S Novo Nordisk Research Park DK‐2760 Måløv Denmark

8. Thomas E. Nielsen

   Novo Nordisk (Denmark)

   Affiliation as printed

   Global Research Technologies Novo Nordisk A/S Novo Nordisk Research Park DK‐2760 Måløv Denmark

9. Bhavesh Premdjee

   Novo Nordisk (Denmark)

   Affiliation as printed

   Global Research Technologies Novo Nordisk A/S Novo Nordisk Research Park DK‐2760 Måløv Denmark

10. Gerbrand J. van der Heden van Noort corresponding Aachen

    Leiden University · Leiden University Medical Center

    Affiliation as printed

    Dept. Cell and Chemical Biology Leiden University Medical Centre 2333 ZC Leiden The Netherlands