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.

Synthesis Concepts for Peptides and Proteins

“…The solid-phase chemical ligation (SPCL) [146,147] of unprotected peptide segments in aqueous solution can be performed in both N ! C and C !…”

Highly efficient solid phase synthesis of large polypeptides by iterative ligations of bis(2-sulfanylethyl)amido (SEA) peptide segments

“…[24][25][26][27] Elongation in the C-to-N direction can facilitate the synthesis of large N-terminal cysteinyl peptides, and elongation in the N-to-C direction potentially gives access to large C-terminal peptide thioester surrogates (Fig. [24][25][26][27] Elongation in the C-to-N direction can facilitate the synthesis of large N-terminal cysteinyl peptides, and elongation in the N-to-C direction potentially gives access to large C-terminal peptide thioester surrogates (Fig.…”

Exploratory synthesis of peptide–α‐thioester segments spanning the polypeptide sequence of the δ‐opioid receptor, a G protein‐coupled receptor

“…Because protein polypeptide chains must be synthesized by the ligation of smaller peptide building blocks of between 30 and 60 amino acids in length, and because recovered yields depend on the number of manipulations and intermediate purifications that must be performed during the synthesis, typical research laboratory‐scale syntheses have so far been limited to <200 amino acids in the polypeptide chain. Recently, our laboratory has reported two advances that promise to significantly extend this practical size limit: a convergent ligation scheme9 and solid phase chemical ligation 10. Syntheses that rely on the convergent ligation of peptides are inherently more efficient than sequential ligation, and covalent attachment of the growing polypeptide chain to a cross‐linked polymer support eliminates the need for intermediate purifications that can greatly reduce synthetic yields.…”

“…The second challenge is the hydrophobic nature of a large fraction of the polypeptide sequence of GPCRs, which complicates peptide ligations performed in aqueous solutions. Covalent attachment of the growing polypeptide chain to a water soluble cross‐linked polymer support,10 in addition to simplifying manipulations during protein synthesis, is also expected to maintain the polypeptide in solution as it does for the fully protected peptide chains in solid phase peptide synthesis 11…”