Fatty acids are key components and abundant constituents of all biological systems and are subject of many clinical, nutritional, and metabolic studies as they play a crucial role for normal functioning at all levels of an organism. Protein lipidation/fatty acylation, a common co- and post-translational modification of proteins plays important roles in human physiology. As previously reported in our newsletter, palmitoylation represents one of the major post translational modifications responsible for protein activity control.

Dysfunction in fatty acid metabolism is related to various pathological states, e.g. cancer and neurodegenerative diseases. Thus, the study and tracing of fatty acids during metabolism has become an important focus of biomedical research. Furthermore, this topic gained interest as the modification of peptides with fatty acids may lead to increased serum half-life and improved in vivo performance via binding to albumin. The most prominent example in this context is the marketed blockbuster drug liraglutide with sales of US$ 4,142 million in 2019.

In former times, the major technology for the analysis of fatty acid metabolism was radioisotope tracing being regulated by special laboratory requirements as well as high costs for purchase of substances as well as disposal of radioactive waste. As radioactivity-free alternative Click Chemistry-based labeling methods are gaining ground.

There is an increasing number of reports on the incorporation of alkynyl and azido fatty acid analogs for the detection and tracing of fatty acylated proteins. Click Chemistry allows for the sensitive and specific detection of those modified building blocks containing azido groups or alkynes, which can be integrated into fatty acids without major disturbance of the structure of the hydrophobic hydrocarbon chains. The term Click Chemistry includes a set of chemical reactions but most commonly refers to the Cu(I)-catalyzed azido-alkyne [3+2] cycloaddition reaction of an alkyl group and an azido group under formation of a triazole.

As an example, S-palmitoylation or N-myristylation can be tracked by incorporating bio-orthogonal alkynyl-palmitate or alkynyl-myristate into cells thus providing labeled proteins. For the analysis of protein acylation, after cell lysis and immunoprecipitation of the protein of interest, the covalent linkage of the alkyne moiety to an azide of an affinity probe via Click Chemistry is performed. Alternatively, Click Chemistry can also be performed on the total cell lysate and the fatty acylated proteins can be affinity purified for identification by mass spectrometry or labeled with a fluorescent probe to examine protein localization.

Interested to find out about the effect of fatty acylation on your peptide’s localization, function, and stability?

→ Iris Biotech offers various (protected) azide- and alkyne-modified fatty acid derivatives suitable for Click Chemistry.

→ You could not find your desired building block? Get in contact for a custom synthesis!

→ Do you need more information on Click Chemistry and its applications? Check-out the recording of our workshop!

References:

Clickable Lipids: Azido and Alkynyl Fatty Acids and Triacylglycerols; J. A. Zerkowski, A. Nuñez, G. D. Strahan, D. K. Y. Solaiman; J Am Oil Chem Soc 2009; 86: 1115.  https://doi.org/10.1007/s11746-009-1442-z.

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry; L. M. Q. Liao, R. A. V. Gray, D. D. O. Martin; J. Vis. Exp. 2021; 170: e62107. https://doi.org/10.3791/62107.

Tracing Fatty Acid Metabolism by Click Chemistry; C. Thiele, C. Papan, D. Hoelper, K. Kusserow, A. Gaebler, M. Schoene, K. Piotrowitz, D. Lohmann, J. Spandl, A. Stevanovic, A. Shevchenko, L. Kuerschner; ACS Chem. Biol. 2012; 7(12): 2004-2011. https://doi.org/10.1021/cb300414v.

Rapid and selective detection of fatty acylated proteins using omega-alkynyl-fatty acids and click chemistry; M. C. Yap, M. A. Kostiuk, D. D. O. Martin, M. A. Perinpanayagam, P. G. Hak, A. Siddam, J. R. Majjigapu, G. Rajaiah, B. O. Keller, J. A. Prescher, P. Wu, C. R. Bertozzi, J. R. Falck, L. G. Berthiaume; J. Lipid. Res. 2010; 51(6): 1566-1580. https://doi.org/10.1194/jlr.D002790.

A Decade of Click Chemistry in Protein Palmitoylation: Impact on Discovery and New Biology; X. Gao, R. N. Hannoush; Cell Chem. Biol. 2018; 25(3): 236-246. https://doi.org/10.1016/j.chembiol.2017.12.002.

Synthetic peptide API manufacturing: A mini review of current perspectives for peptide manufacturing; J. H. Rasmussen; Bioorg. Med. Chem. 2018; 26(10): 2914-2918. https://doi.org/10.1016/j.bmc.2018.01.018.

The Discovery and Development of Liraglutide and Semaglutide; L. B. Knudsen; J. Lau; Front Endocrinol (Lausanne) 2019; 10: 155. https://doi.org/10.3389/fendo.2019.00155.