Kinetically Controlled Ligation for the Convergent Chemical Synthesis of Proteins

Abstract

Converging on the goal: New chemistry enables the key Cys‐peptide‐(αthioester) intermediate to be extended by ligation at either the N or C termini in a controlled fashion. This approach forms the basis for a set of novel tactics for the fully convergent synthesis of proteins by the chemical ligation of multiple peptide segments.

Synthesis of granulocyte–macrophage colony-stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material

“…Merrifield's SPPS method permits stepwise elongation of a growing peptide (18,19). Convergence in the construction of longer polypeptides, including small proteins, may be achieved through the use of the seminal NCL technology of Kent and coworkers, whereby fragments are merged at N-terminal cysteine residues (20,21). The possibility of expanding the scope of NCL to encompass, for instance, alanine ligation was demonstrated in a key paper by Yan and Dawson (22), who accomplished dethiylation…”

Chemical synthesis of the ATAD2 bromodomain

“…Subsequently, metal-free dethiylation (MFD) (34) resulted in a major expansion of NCL by enabling disconnection strategies of alanine, valine, leucine, threonine, and proline, among others. In the convergent retrosynthesis as planned in this work, the first disconnection was between the second and third fragment (F2 and F3), so as to avoid the necessity of tandem kinetic ligations (35) in the assembly of the first half. Thus, to take advantage of the Leu22-Ala23 disconnection site for alanine ligation and obviate the construction an Ala22-Ile78 second fragment, the F2-F3 disconnection site we favored was at Tyr65-Leu66.…”

Total chemical synthesis and biophysical characterization of the minimal isoform of the KChIP2 potassium channel regulatory subunit

“…In order to facilitate the chemical synthesis of the necessary peptide segments, we introduced a cysteine at residue 34, which we then subsequently alkylated to from a ''cGln'' residue (Kochendoerfer et al 2003). The alkylation of only this Cys residue demanded that we use a strategy that involved ligating from the N terminus of the protein toward the C terminus (Bang et al 2006b), rather than the typical C-to N-terminal ligation by native chemical ligation. The kinetically controlled ligation of segments 1-33:COSFCH 2 COOH and Cys34-52:COSR was followed by alkylation with iodoacetamide in quantitative yield.…”

“…The strategy adopted for the synthesis of KChIP2d is shown in Figure 2B. Selective alkylation of Cys34 required assembly of the protein from N terminus to C terminus, rather than the conventional C-terminal to N-terminal strategy used in most three segment syntheses (Bang et al 2006b). This strategy necessitated a kinetically controlled ligation (Bang et al 2006b) between the first two thioestercontaining peptide segments to give a unique peptidethioester product (Bang et al 2006b).…”

“…Selective alkylation of Cys34 required assembly of the protein from N terminus to C terminus, rather than the conventional C-terminal to N-terminal strategy used in most three segment syntheses (Bang et al 2006b). This strategy necessitated a kinetically controlled ligation (Bang et al 2006b) between the first two thioestercontaining peptide segments to give a unique peptidethioester product (Bang et al 2006b). This reaction was accomplished by first exchanging the alkyl thioester of 1-33:COSR with mercaptophenylacetic acid to give a more reactive peptide:COSFCH 2 COOH aryl thioester synthon (Johnson and Kent 2006a), thus favoring formation of the desired 1-52:COSR product (Fig.…”