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Issue 48, 2019
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Chemical synthesis of Shiga toxin subunit B using a next-generation traceless “helping hand” solubilizing tag

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Abstract

The application of solid-phase peptide synthesis and native chemical ligation in chemical protein synthesis (CPS) has enabled access to synthetic proteins that cannot be produced recombinantly, such as site-specific post-translationally modified or mirror-image proteins (D-proteins). However, CPS is commonly hampered by aggregation and insolubility of peptide segments and assembly intermediates. Installation of a solubilizing tag consisting of basic Lys or Arg amino acids can overcome these issues. Through the introduction of a traceless cleavable linker, the solubilizing tag can be selectively removed to generate native peptide. Here we describe the synthesis of a next-generation amine-reactive linker N-Fmoc-2-(7-amino-1-hydroxyheptylidene)-5,5-dimethylcyclohexane-1,3-dione (Fmoc-Ddap-OH) that can be used to selectively introduce semi-permanent solubilizing tags (“helping hands”) onto Lys side chains of difficult peptides. This linker has improved stability compared to its predecessor, a property that can increase yields for multi-step syntheses with longer handling times. We also introduce a new linker cleavage protocol using hydroxylamine that greatly accelerates removal of the linker. The utility of this linker in CPS was demonstrated by the preparation of the synthetically challenging Shiga toxin subunit B (StxB) protein. This robust and easy-to-use linker is a valuable addition to the CPS toolbox for the production of challenging synthetic proteins.

Graphical abstract: Chemical synthesis of Shiga toxin subunit B using a next-generation traceless “helping hand” solubilizing tag

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Publication details

The article was received on 17 Sep 2019, accepted on 15 Nov 2019 and first published on 20 Nov 2019


Article type: Paper
DOI: 10.1039/C9OB02012H
Org. Biomol. Chem., 2019,17, 10237-10244

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    Chemical synthesis of Shiga toxin subunit B using a next-generation traceless “helping hand” solubilizing tag

    J. M. Fulcher, M. E. Petersen, R. J. Giesler, Z. S. Cruz, D. M. Eckert, J. N. Francis, E. M. Kawamoto, M. T. Jacobsen and M. S. Kay, Org. Biomol. Chem., 2019, 17, 10237
    DOI: 10.1039/C9OB02012H

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