<div><div><div><p>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.</p></div></div></div>
Chemical synthesis of proteins using hydrazide intermediates