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Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry
Published on: April 9, 2021
6.1K
Copper-Catalyzed Click Reaction on/in Live Cells.
Siheng Li1, Lin Wang2, Fei Yu1
1Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
Chemical Science
|March 29, 2017
Summary
Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions were optimized for live mammalian cells. Biothiols were identified as major inhibitors of cytosolic CuAAC reactions, leading to a 3-fold efficiency improvement.
Area of Science:
- Biochemistry
- Chemical Biology
- Cell Biology
Background:
- Copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a versatile click chemistry reaction.
- Performing CuAAC reactions within live mammalian cells presents challenges due to cellular environments.
- Optimizing CuAAC for intracellular applications requires understanding and overcoming reaction limitations.
Purpose of the Study:
- To develop and optimize an efficient copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction within live mammalian cells.
- To investigate the factors limiting CuAAC reaction efficiency in the cellular cytosol.
- To enhance the utility of *in situ* CuAAC for biological applications like inhibitor screening.
Main Methods:
- Metabolic modification of newly synthesized proteins with homopropargylglycine (HPG) in OVCAR5 cells.
- Utilizing a *tris*(triazolylmethyl)amine Cu(I) ligand tethered to a cell-penetrating peptide to promote the reaction.
- Quantifying reaction efficiency on membrane and cytosolic proteins using mass spectrometry.
- Assessing the impact of biothiol reduction on reaction yields.
Main Results:
- The optimized CuAAC reaction achieved yields over 18% on membrane proteins and 0.8% on cytosolic proteins in live cells, with 75% cell viability maintained.
- A cell-penetrating peptide-tethered *tris*(triazolylmethyl)amine Cu(I) ligand significantly promoted the reaction.
- Reducing biothiols in the cytosol improved cytosolic protein modification yields to ~9-14%, indicating biothiol-mediated catalyst deactivation.
- Overall CuAAC reaction efficiency in live cells was improved by 3-fold.
Conclusions:
- Biothiols are identified as the primary cause of low CuAAC reaction yields in the cellular cytosol.
- The developed *in situ* CuAAC methodology shows promise for screening cell-specific enzyme inhibitors or biomarkers.
- Further optimization of CuAAC catalysts and reaction conditions can enhance intracellular bioorthogonal chemistry applications.
