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An oxazetidine amino acid for chemical protein synthesis by rapid, serine-forming ligations

Nature Chemistry volume 7, pages 668–672 (2015)Cite this article

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Abstract

Amide-forming ligation reactions allow the chemical synthesis of proteins by the union of unprotected peptide segments, and enable the preparation of protein derivatives not accessible by expression or bioengineering approaches. The native chemical ligation (NCL) of thioesters and N-terminal cysteines is unquestionably the most successful approach, but is not ideal for all synthetic targets. Here we describe the synthesis of an Fmoc-protected oxazetidine amino acid for use in the α-ketoacid–hydroxylamine (KAHA) amide ligation. When incorporated at the N-terminus of a peptide segment, this four-membered cyclic hydroxylamine can be used for rapid serine-forming ligations with peptide α-ketoacids. This ligation operates at low concentration (100 μM–5 mM) and mild temperatures (20–25 °C). The utility of the reaction was demonstrated by the synthesis of S100A4, a 12 kDa calcium-binding protein not easily accessible by NCL or other amide-forming reactions due to its primary sequence and properties.

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Figure 1: Synthesis of enantioenriched Fmoc-oxazetidine amino acid 1.
Figure 2: A comparison between KAHA ligations with oxazetidine- and oxaproline-containing peptides.
Figure 3: Structure and synthesis of S100A4.
Figure 4: Monitoring of the coupling and folding processes in the synthesis of S100A4.

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Acknowledgements

This work was supported by the Swiss National Science Foundation (200020_150073) and ETH Zürich. F. Thuaud, S. Baldauf and M. Dao are thanked for contributions to the synthesis of compound 1, V. Pattabiraman for discussions, F. Saito for advice on kinetics and C. Wolfrum for biological evaluation.

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Authors and Affiliations

  1. Department of Chemistry and Applied Biosciences, Laboratorium für Organische Chemie, Vladimir Prelog Weg 3, ETH Zürich, Zürich, 8093, Switzerland

    Ivano Pusterla & Jeffrey W. Bode

  2. Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya, 464-8602, Japan

    Jeffrey W. Bode

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  1. Ivano Pusterla
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Contributions

J.W.B. and I.P. contributed equally to the design of the study. J.B., with contributions from I.P., wrote the paper. I.P performed the experiments and wrote the Supplementary Information.

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Correspondence to Jeffrey W. Bode.

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Crystallographic data for compound 9. (CIF 16 kb)

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Pusterla, I., Bode, J. An oxazetidine amino acid for chemical protein synthesis by rapid, serine-forming ligations. Nature Chem 7, 668–672 (2015). https://doi.org/10.1038/nchem.2282

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