Total Chemical Synthesis of RNF4 by Sequential Native Chemical Ligation: C-To-N Versus N-To-C Strategies

RNF4, a RING-type E3 ubiquitin ligase, targets polySUMOylated proteins for ubiquitination and subsequent proteasomal degradation. The ability to chemically synthesize RNF4 will enable future studies of its structure and biological function, particularly its role in degrading the oncoprotein PML-RARα in acute promyelocytic leukemia. To achieve this, we performed a total chemical synthesis of RNF4 using sequential native chemical ligation. The presence of nine cysteine residues enables stepwise ligation of five peptide fragments to assemble the full-length protein. Two synthetic strategies were explored: the first employed a convergent C-to-N ligation, while the second used an N-to-C ligation. In the convergent C-to-N approach, cysteine residues were protected with acetamidomethyl groups to prevent side reactions during ligation, although this required multiple deprotection and purification steps. Conversely, the N-to-C synthesis method proceeded efficiently without cysteine protection, thereby simplifying the workflow and reducing the number of purification steps. This research presents a reliable and accessible method for the complete chemical synthesis of RNF4, addressing significant challenges in synthesizing large proteins and opening up new opportunities for future biological research.