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Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

preprint
submitted on 10.07.2020 and posted on 13.07.2020 by Patrick Erickson, James Fulcher, Michael Kay

Chemoselective ligation reactions, such as native chemical ligation (NCL), enable the assembly of synthetic peptides into proteins. However, the scope of proteins accessible to total chemical synthesis is limited by ligation efficiency. Sterically hindered thioesters and poorly soluble peptides can undergo incomplete ligations, leading to challenging purifications with low yields. This work describes a new method, ClickAssisted NCL (CAN), which overcomes these barriers. In CAN, peptides are modified with traceless “helping hand” lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles for strain-promoted alkyne-azide cycloaddition (SPAAC) reactions. This cycloaddition templates the peptides to increase their effective concentration and greatly accelerate ligation kinetics. After ligation, mild hydroxylamine treatment tracelessly removes the linkers to afford the native ligated peptide. Although DBCO is incompatible with standard Fmoc solid-phase peptide synthesis (SPPS) due to an acid-mediated rearrangement that occurs during peptide cleavage, we demonstrate that copper(I) protects DBCO from this side reaction, enabling direct production of DBCO-containing synthetic peptides. Excitingly, low concentrations of triazole-linked model peptides reacted ~1,200-fold faster than predicted for non-templated control ligations, which also accumulated many side products due to the long reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that the SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in a one-pot fashion. CAN will be useful for overcoming ligation challenges to expand the reach of chemical protein synthesis.

Funding

Center for the Structural Biology of Cellular Host Elements in Egress, Trafficking, and Assembly of HIV (CHEETAH Center)

National Institute of Allergy and Infectious Diseases

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D-peptide Inhibitors of HIV-1 Entry

National Institute of Allergy and Infectious Diseases

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University of Utah Graduate Research Fellowship

Marjorie Riches Gunn Award for Graduate Student Excellence

History

Email Address of Submitting Author

kay@biochem.utah.edu

Institution

University of Utah

Country

USA

ORCID For Submitting Author

0000-0003-3186-9684

Declaration of Conflict of Interest

None

Licence

Exports