Large Scale Stepwise Synthesis of Ubiquitin

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Ubiquitin is a central member within a group of structurally conserved proteins found throughout (ubiquitous) eukaryotic organisms. The ubiquitin family of proteins regulate multiple processes through covalent binding to a substrate protein in a process called ubiquitination [1].  Covalent attachment can occur through binding to a lysine residue via an isopeptide bond, a cysteine through a thioester bond, serine/threonine residues through an ester bond, or the N-terminus amino group.  Ubiquitination can trigger different cellular processes for the substrate protein such as degradation, activity changes, effects on protein interactions, or altering cellular location. Synthesis of ubiquitin is known to be challenging due to its length (76 aa). The use of pseudoprolines
and/or native chemical ligation have been previously employed to aid in the synthesis [2,3]. Here, we report a stepwise linear synthesis of ubiquitin without pseudoprolines or native chemical ligation by using a new microwave based SPPS process that we recently introduced [4].  Additionally, the synthesis of ubiquitin was scaled from 0.1 mmol – 0.5 mmol to demonstrate the scalability of this approach even for very long peptides.


[1] C.M. Pickart, M.J. Eddins, BBA Mol. Cell. Res. 55, 1695 (2004).
[2] Y.-C. Huang, C.-C. Chen, S. Gao, Y.H. Wang, H. Xiao, F. Wang, C.-L. Tian, Y.-M. Li, Eur. Chem. 22, 7623 (2016).
[3] F.E. Oualid, R. Merkx, R. Ekkebus, D.S. Hameed. J.J. Smit, A.d. Jong, H. Hilkmann, T.K. Sixma, H. Ovaa, Angewandte Chemie 49, 10149 (2010).
[4] J. M. Collins, K. A. Porter, S. K. Singh, G. S. Vanier; High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS), Org. Lett. 16, 940 (2014).