2,5-Diketopiperazine Formation Facilitated by γ-Substituted Prolines for Direct Synthesis of Tag-Attached Peptide Thioester — ASN Events
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  3. 2,5-Diketopiperazine Formation Facilitated by γ-Substituted Prolines for Direct Synthesis of Tag-Attached Peptide Thioester

2,5-Diketopiperazine Formation Facilitated by γ-Substituted Prolines for Direct Synthesis of Tag-Attached Peptide Thioester (#319)

Koki Nakatsu 1 , Gosuke Hayashi 1 , Hiroshi Murakami 1
  1. Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Japan

     Native chemical ligation (NCL) is a prevalent strategy to ligate side-chain unprotected peptide segments between an N-terminal cysteine peptide and a C-terminal peptide thioester in neutral aqueous conditions.1 To introduce a base-liable thioester moiety through Fmoc solid-phase peptide synthesis (SPPS), many thioester surrogates have been designed and reported. Among them, the Cysteinyl-Prolyl-(Leaving Group) (Cys-Pro-LG) is easily introduced to peptides via Fmoc-SPPS, and it converts into thioester through the sequential reactions consisting of N,S acyl transfer at cysteine and 2,5-diketopiperazine (DKP) formation prompted by proline. The effects of leaving group on the reaction rate in DKP formation have been reported intensively by previous studies.2–4 However, few studies have reported the effect of substitutions at prolyl moiety on DKP formation. Among modified prolines, γ-substituted prolines are relatively readily available and have been intensively investigated mainly in the context of collagen mimetic peptides.5 These substitutions significantly affect cis-trans isomerization of prolyl amide and puckering of the pyrrolidine ring of proline.6 Therefore, we hypothesized that γ-modified proline is an effective tool for tuning the DKP formation.

     Herein, we will discuss the effect of prolyl γ-substitutions of Cys-Pro-LG on DKP-thioester formation using Cys-Pro-pyrazole7 model peptides. By comparing various substitutions, we revealed that prolyl γ-modification greatly affects the rate of DKP-thioester formation. Notably, some modifications showed a significant improvement (up to ~6-fold) in the rate of DKP-thioester formation. Furthermore, by employing DFT calculations, the γ-substitution-mediated acceleration mechanism was rationalized by the preference of the pyrrolidine ring puckering, the rotational barrier of cis-trans isomerization of prolyl amide, and/or the intramolecular hydrogen bonding. Finally, we will report chemical protein synthesis using peptide DKP-thioester with a solubilizing tag connected via an optimized linker at the prolyl γ-position to facilitate the handling of peptides during ligation reaction and purification process.

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  1. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science 1994, 266, 776–779.
  2. Kawakami, T.; Kamauchi, A.; Harada, E.; Aimoto, S. Tetrahedron Lett. 2014, 55, 79–81.
  3. Yanase, M.; Nakatsu, K.; Cardos, C. J.; Konda, Y.; Hayashi, G.; Okamoto, A. Chem. Sci. 2019, 10, 5967–5975.
  4. Nakatsu, K.; Yanase, M.; Hayashi, G.; Okamoto, A. Org. Lett. 2020, 22, 4670–4674.
  5. Shoulders, M. D.; Raines, R. T. Annu. Rev. Biochem. 2009, 78, 929–958.
  6. Newberry, R. W.; Raines, R. T. Top. Heterocycl. Chem. 2017, 48, 1–25.
  7. Flood, D. T.; Hintzen, J. C. J.; Bird, M. J.; Cistrone, P. A.; Chen, J. S.; Dawson, P. E. Angew. Chem. Int. Ed. 2018, 57, 11634–11639.
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