One-pot native chemical ligation of peptide hydrazides enables total synthesis of modified histones

Abstract

One of the rising demands in the field of protein chemical synthesis is the development of facile strategies that yield the protein in workable quantities and homogeneity, with fewer handling steps. Although the native chemical ligation of peptide hydrazides has recently been shown to be useful for the chemical synthesis of proteins carrying acid-sensitive modification groups, previous hydrazide-based protein synthesis studies have used sequential ligation strategies. Here, we report a practical method for a "one-pot" native chemical ligation of peptide hydrazides that would circumvent the need for the isolation of the intermediate products. This method employed a fast and selective arylboronate oxidation reaction mediated by H2O2, which draws attention to the potential applications of the thus far under-exploited boron-based functionalities in protein chemical synthesis. To demonstrate the practicality and efficiency of the new one-pot method, we report its application to a scalable total synthesis of modified histones (with five analogues of H3 and H4 as examples) on a multi-milligram scale, with good homogeneity.

Hybrid phase ligation for efficient synthesis of histone proteins

“…23, 24 In order to minimize the number of segments, one-pot approaches require long peptide sequences resulting in challenging syntheses. 21 Solid phase ligation (SP-NCL) has been reported to improve efficiency and yield for histone H2B, 22 but we show in this work that for H4 and CpA, overall yields with SP-NCL are unacceptably low despite efficient chemistry at each step. Here, we demonstrate that carrying out initial ligation steps on the solid phase followed by final ligation steps in solution provides optimal efficiency and yield for heavily modified synthetic histone proteins, contrary to previous reports that suggest fully solid phase ligation is inherently superior to mixed phase in other systems.…”

“…Several strategies have been explored for total synthesis of histone proteins, including sequential ligation, 19 convergent ligation, 20 one-pot ligation using peptide hydrazides, 21 and solid phase native chemical ligation. 22 Each approach has both advantages and disadvantages.…”

Chemical and Biological Tools for the Preparation of Modified Histone Proteins

“…Overall, this one pot approach resulted in 20% isolated yield of histone H3-K4me3, a substantial improvement over the Ottesen approach. The Liu group extended the one-pot chemical ligation approach with peptide hydrazides and Dobz protection to the total synthesis of histone H4 [120]. Using the same approaches, they achieved an average 18% overall isolated yield of H4-K16ac.…”

Protein/peptide secondary structural mimics: design, characterization, and modulation of protein–protein interactions

“…18 But to date our ability to produce homogeneous proteins, especially those with posttranslational modications, have been limited. [19][20][21][22] Examples of monoclonal antibodies performing as antitumor drugs do exist, such as Bevacizumab, 23 Nivolumab and Ipilimumab. 24 However, the immune effects and safety prole over the long term are still concerns.…”