Poly-Ubiquitin Chains

Employing the strategy of hydrazide-based native chemical ligation and microwave-assisted solid phase peptide synthesis (SPPS), we achieved to synthesis the eight forms of di-ubiquitin (linear, K6/K11/K27/K29/K33/K48/K63-linked di-ubiquitin). We also successfully synthesized the tri-ubiquitin and tetra-ubiquitin. 

Taking K27-linked di-ubiquitin as an example, a trifluoroacetic acid (TFA)-labile 1-(2,4-dimethoxyphenyl)-2-mercaptoethyl auxiliary (Aux) was employed to enable the construction of the isopeptide bond. The K27-linked diUb containing 152 amino acids was divided in to four peptide segments, namely Ub[Cys(Acm)46-Gly75]-NHNH2 (1), Ub[Met1-Lys(Aux-Gly)27-Phe45]-NHNH2 (2), Ub[Met1-Phe45]-NHNH2 (5) and Ub[Cys46-Gly76] (7). Ala46 of each Ub unit was temporarily mutated to Cys as the ligation site. Gly76 of the distal Ub was incorporated to 2 through the building block glycyl auxiliary. An acetamidomethyl (Acm) group was used to protect the thiol group of N-terminal Cys of segment 1 to avoid oligomerization or self-cyclization. The synthesis started with ligation of 1 and 2 to produce 3 with the specific Lys27-linked isopeptide bond. The Acm and Aux groups were then removed to afford 4. After two more steps of ligation reactions, desulfurization was conducted to convert all the Cys residues back to the native Ala residues. The full-length peptide 8 was thus obtained on a 50 mg scale with an overall yield of 4.5% (Figure 1).

Figure 1. Synthetic route of K27-linked di-ubiquitin



1.Pan, M. et al. Quasi-Racemic X-ray Structures of K27-Linked Ubiquitin Chains Prepared by Total Chemical Synthesis. Journal of the American Chemical Society138, 7429-7435, doi:10.1021/jacs.6b04031 (2016).

2.  Gao, S. et al. Monomer/Oligomer Quasi-Racemic Protein Crystallography. Journal of the American Chemical Society138, 14497-14502, doi:10.1021/jacs.6b09545 (2016).

3. Liang, J. et al. Chemical Synthesis of Diubiquitin-Based Photoaffinity Probes for Selectively Profiling Ubiquitin-Binding Proteins. Angewandte Chemie56, 2744-2748, doi:10.1002/anie.201611659 (2017).

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