Poster Presentation 10th Modern Solid Phase Peptide Synthesis & Its Applications Symposium 2025

Peptide ligation mediated by N-aminoacyl-N-hydroxy amino acid (128961)

Junya Hayashi 1 , Ayane Masui 1 , Masaya Denda 1 , Akira Otaka 1
  1. Tokushima University, Tokushima, TOKUSHIMA, Japan

Many attempts have been made to achieve protein chemical synthesis by utilizing Native Chemical Ligation (NCL)1, featuring chemoselective fragment coupling between a thioester and N-terminal cysteine residue. Combination of the NCL with desulfurization of thiol-bearing residue at the ligation junction has allowed the coupling residues to reach beyond the Xaa-Cys2. However, this NCL-desulfurization sequence does not allow for fragment coupling at the Xaa-Gly junction because there is no thiol-containing side chain at the Gly site. Incorporation of a thiol-embedded auxiliary at the amine of Gly residue have improved such situation that Xaa-Gly ligation is not achievable; nonetheless, there is still substantial opportunity for advancing the ligation method to facilitate Gly site ligation.

In this context, being encouraged by the Asx (Asp or Asn) site ligation which features the O-selective acylation followed by O-N acyl transfer at the N-terminal alanyl hydroxamic acid (AlaHA) and subsequent conversion of the AlaHA to Asx3, we attempted the glycyl hydroxamic acid (GlyHA) site ligation with thioester with aiming at the Gly-site ligation. The expected O-acylation at the GlyHA site unambiguously proceeded; however, no subsequent O-N acyl transfer occurred, and the GlyHA was converted to glyoxylyl unit through Lossen rearrangement and subsequent hydrolysis4. The mechanistic insight into the unexpected reaction outcome prompted us to hypothesize that a hydroxamic acid moiety in the peptide main chain should allow for the O-N acyl transfer, achieving amide formation without accompanying Lossen rearrangemet. As the hydroxamic acid moiety, we incorporated the N-amino acyl-N-hydroxy-amino acid (NAHA) into the N-terminal at acyl acceptor peptides. Attempted ligation of a NAHA-incorporated peptide with peptide thioester proceeded efficiently through the O-selective acylation followed by O-N acyl transfer via 6-membered transition state to give NAHA-containing ligation product. Finally, the N-O bond in the NAHA residue was reduced through zinc-mediated single electron transfer (SET) process to give the desired ligation product. Of note is that the developed ligation allows any amino acid, including Gly, to serve as the N-terminal residue in the acyl acceptor fragment.

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  2. Agouridas, V.; Mahdi, O. E.; Diemer, C.; Cargoët, M.; Monbaliu, J.-C. M.; Melnyk, O. Chem. Rev. 2019, 119, 7328.
  3. Dunkelmann, D. L.; Hirata, Y.; Totaro, K. A.; Cohen, D. T.; Zhang, C.; Gates, Z. P.; Pentelute, B. L., Proc. Natl. Acad. Sci. U.S.A. 2018, 115, 3752-3757.
  4. Hayashi, J.; Kobayashi, D.; Namikawa, C.; Denda, M.; Otaka, A., Org. Lett. 2024, 4246-4250.