Chemical Synthesis of Ubiquitin Chains

Abstract

Chemical synthesis of complex biomolecules such as proteins is a challenging adventure, yet rewarding in driving various biochemical and biophysical research activities. Over the years, the refinement of peptide synthesis and invention of ligation methodologies have led to the successful synthesis of several complex protein targets. Ubiquitin bioconjugates, which are being studied intensively by many groups due to their involvement in numerous biological processes, represent a fine example where chemistry is greatly aiding these studies. In this article, we describe the synthetic routes and strategies to prepare different ubiquitin analogs with desired modifications, as well as di‐ubiquitin chains. © 2015 by John Wiley & Sons, Inc.

The Journey for the Total Chemical Synthesis of a 53 kDa Protein

“…42−44 For example, our group found that the use of HCTU/DIEA and three dipeptide building blocks (Ile13-Thr14, Asp52-Gly53, and Leu56-Ser57) were sufficient to achieve a very good synthesis of the various Ub analogues for the synthesis of more complex conjugates. 45 To synthesize the Ub-thioester, one approach is to assemble the Ub sequence with N-methylated Cys at the C-terminus. The N-methylcysteine is utilized as an N → S acyl transfer device for the thioesterification step carried out at pH 1 in the presence of an excess of an alkylthiol such as mercaptopropionic acid (MPA).…”

“…The distal Flag-Ub-thioester was prepared using N-methylcysteine approach, while the other two Ubs having the same functionalities were prepared by using the MeNbz chemistry, followed by switching the C-terminus to hydrazide and deprotecting the δ-mercaptolysine. 45 Having the Ub building blocks in hand, we assembled the Tri-Ub-thioester chain by taking advantage of the stability and tunability of the hydrazide moiety. First, we performed the ICL between Flag-Ubthioester and Ub hydrazide bearing free δ-mercaptolysine.…”

“…Although the approach applying the ligation of two segments to synthesize Ub building blocks was achieved and successfully used to assemble Ub chains, ,,, a direct synthesis of Ub analogues was found to reduce the number of synthetic steps and allow higher efficiency in the synthesis of large Ub conjugates. We and other groups have applied Fmoc-SPPS using different coupling reagents and dipeptide building blocks (pseudoproline dipeptides and dimethoxybenzyl dipeptides) for the efficient synthesis of Ub monomer analogues. − For example, our group found that the use of HCTU/DIEA and three dipeptide building blocks (Ile13-Thr14, Asp52-Gly53, and Leu56-Ser57) were sufficient to achieve a very good synthesis of the various Ub analogues for the synthesis of more complex conjugates …”

“…We then turned our attention to the assembly of the intermediate Flag-triUb-thioester by using two different Ub building blocks. The distal Flag-Ub-thioester was prepared using N -methylcysteine approach, while the other two Ubs having the same functionalities were prepared by using the MeNbz chemistry, followed by switching the C-terminus to hydrazide and deprotecting the δ-mercaptolysine . Having the Ub building blocks in hand, we assembled the Tri-Ub-thioester chain by taking advantage of the stability and tunability of the hydrazide moiety.…”