A Reversible Protection Strategy To Improve Fmoc‐SPPS of Peptide Thioesters by the N‐Acylurea Approach

Abstract

C-terminal peptide thioesters are an essential component of the native chemical ligation approach for the preparation of fully- or semi-synthetic proteins. However, efficient generation of C-terminal thioesters via Fmoc solid phase peptide synthesis remains a challenge. The recent N-acylurea approach to thioester synthesis relies on 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 by 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 may be purified and converted in situ to thioester under ligation conditions. This method is compatible with automated preparation of peptide-Nbz conjugates. We demonstrate that Dbz protection improves synthetic purity of Gly-rich peptide sequences derived from histone H4, as well as a 44-residue peptide from histone H3.

Histone Core Phosphorylation Regulates DNA Accessibility

“…Peptides H3(47-90)A47Thz with or without K56ac and H3(1-46) with or without Tyr(P)-41 were prepared on Fmoc-Dbz(Alloc)-derivatized resin (where Dbz is 3,4-diaminobenzoic acid and Alloc is allyloxycarbonyl) as described in Ref. 42 and cleaved and purified as the C-terminal N-acylurea derivatives. Synthetic histones were analyzed by RP-HPLC and MALDI-TOF MS (Fig.…”

New insights into nucleosome and chromatin structure: an ordered state or a disordered affair?

“…With the advent of even more refined techniques to study nucleosome structure (for example, REFS 65–67), paired with the ability to generate ‘designer nucleosomes’ bearing all kinds of combinations of PTMs, histone variants and DNA sequence 68–72 , we are likely to see a more systematic investigation of biologically relevant modifications on nucleosome structure. It is intuitively obvious that alternative nucleosome structures potentially affect all interactions that they are engaged in, especially the formation of higher-order chromatin structures.…”

New Methods for Chemical Protein Synthesis

“…For example, glycine rich sequences tend to lead to acylation at the second amino group of the Dbz linker, leading to branched Nbz derivatives of varying length. Recently, Ottesen and co-workers have proposed using Alloc as a protecting group for the second amino group of the Dbz resin [130], potentially eliminating almost all problems with branched peptide derivatives and over-acylation. The Alloc group is orthogonal to almost all common Fmoc SPPS protecting groups, stable under SPPS conditions, and can be easily removed with palladium(0) before Dbz activation to form the peptide Nbz derivative.…”