Total Chemical Synthesis, Folding, and Assay of a Small Protein on a Water‐Compatible Solid Support

Abstract

Chemical ligation of unprotected peptides has enabled the successful synthesis of a large number of diverse proteins. [1] The native chemical ligation (NCL) reaction, [2] which proceeds in water at neutral pH to yield a native amide bond at the ligation site, has proven to be the most effective chemistry for the ligation of unprotected peptides. A major limitation of protein synthesis by NCL, and indeed all peptide chemicalligation methods, is the steady decrease in yield because of losses from intermediate handling and purification. Although recent one-pot schemes [3] for chemical protein synthesis have helped eliminate some intermediate purification steps and, therefore, increased yields, one-pot reactions can, over time and multiple ligations, lead to a large accumulation of byproducts that are difficult to purify from the target polypeptide. For certain classes of proteins, such as integral membrane proteins, an additional major problem is the poor solubility of many intermediate polypeptide products in the solvents used for ligation. There is currently no effective and general strategy for solubilizing poorly soluble intermediate polypeptides in ligation buffers without introducing modifications to the peptide building blocks. We aimed to perform protein synthesis by NCL on a cross-linked polymer support to address these limitations.Polymer-supported synthetic chemistry, originally introduced by Merrifield for peptide synthesis, [4] is known to have a number of advantages over traditional solution-phase chemistry. These advantages include simple purification through[*] Prof.

Towards the Simplification of Protein Synthesis: Iterative Solid‐Supported Ligations with Concomitant Purifications

“…[11] A simple filtration work-up separates the truncated peptides, and the subsequent cleavage of the tag results in a traceless release of the purified peptide into solution.A few of these approaches [10c,d] have been utilized for solid-phase NCLs, [12] but the corresponding studies focus only on minimizing the chromatographic and handling steps. [13] All the examples of sequential solid-phase ligations reported so far were limited to polypeptides smaller than 100 amino acids.We thus decided to re-examine the potential of applying this strategy to develop a self-purifying method by using contemporary ligation techniques, as we believe that new reagents or methods that allow for facile and rapid purification of the starting materials and intermediates are critical to the field of protein chemical synthesis. We disclose herein a new N-terminal linker [14] and its application to the nonchromatographic purification of peptides that are difficult to synthesize by existing methods, as well as to self-purifying Nto-C ligations.…”

“…A few of these approaches [10c,d] have been utilized for solid-phase NCLs, [12] but the corresponding studies focus only on minimizing the chromatographic and handling steps. [13] All the examples of sequential solid-phase ligations reported so far were limited to polypeptides smaller than 100 amino acids.…”

Ultrafiltration, a useful method for isolation of intermediates in native chemical ligation exemplified with the total synthesis of Sortase AΔN59

“…My hypothesis was that at each stage of the synthesis, carried out in homogenous solution, reactants would be separated from intermediate products by ultrafiltration. In contrast to solid phase chemical protein synthesis, homogenous reaction system may avoid diffusion problems in resins [9].…”

Solid‐phase peptide synthesis using acetonitrile as a solvent in combination with PEG‐based resins

“…Excess of reagents and soluble side products are removed by simple washings with solvents. Examples of peptides synthesized via the solid‐phase method involve small bicyclic peptides6, 7, large peptides or small proteins8, 9, and multi‐kilogram‐scale production of APIs10, 11. Since the first scheme proposed by Merrifield was introduced, there has been a great evolution in the majority of parameters involved in the synthetic process.…”