A reversible protection strategy to improve Fmoc-SPPS of peptide thioesters by the N-Acylurea approach.

C-terminal peptide thioesters are an essential component of the native chemical ligation approach for the preparation of fully or semisynthetic proteins. However, the efficient generation of C-terminal thioesters by Fmoc solid-phase peptide synthesis remains a challenge. The recent N-acylurea approach to thioester synthesis relies on the deactivation of one amine of 3,4-diaminobenzoic acid (Dbz) during Fmoc SPPS. Here, we demonstrate that this approach results in the formation of side products through the over-acylation of Dbz, particularly when applied to Gly-rich sequences. We find that orthogonal allyloxycarbonyl (Alloc) protection of a single Dbz amine eliminates these side products. We introduce a protected Fmoc-Dbz(Alloc) base resin that may be directly used for synthesis with most C-terminal amino acids. Following synthesis, quantitative removal of the Alloc group allows conversion to the active N-acyl-benzimidazolinone (Nbz) species, which can be purified and converted in situ to thioester under ligation conditions. This method is compatible with the automated preparation of peptide-Nbz conjugates. We demonstrate that Dbz protection improves the synthetic purity of Gly-rich peptide sequences derived from histone H4, as well as a 44-residue peptide from histone H3.