Peptide a-thioesters

C-terminal peptide a-thioester, mUdly activated peptide ester acting as a valuable key intermediate for the synthesis/semi-synthesis of polypeptides and proteins by both chemical ligation and the Aimoto thioester approach. The synthesis of the peptide a-thioester (—r thioester) can be performed by standard SPPS using Boc- or Fmoc-based chemistry or, for larger target polypeptides, by application of intein-based bacterial expression systems. Peptide a-thioester synthesis can also be carried out based on an N-S acyl shift reaction mediated by a thiol ligation auxiliary [F. B. Perler, E. Adams, Curr. Opin. Biotechnol. 2000, 377 D. Swinnen, D. Hilvert, Org. Lett. 2000, 2, 2439 R. Quaderer, D. Hilvert, Org. Lett. 2001, 3, 3181 T. W. Muir, Annu. Rev. Biochem. 2003, 72, 249 T. Kawakami et al.. Tetrahedron Lett. 2005, 46, 8805 J. A. Camarero, A. R. Mitchell, Prot. Pept. Lett. 2005, 12, 723]. 

Mende F, Seitz O (2011) 9-Fluorenylmethoxycarbonyl-based solid-phase synthesis of peptide a-thioesters. Angew Chem Int Ed 50 1232-1240... 

Previous AIP syntheses involved a combined solid-phase solution route. According to this new method, a linear unprotected peptide, a-thioester XI, prepared by f rf-butoxycarbonyl (tert-Boc)-SPPS undergoes cycUzation to XII through trani-thioesterification, and elimination of the resin in aqueous buffer to yield the thiolactone peptide XIII (Scheme 12.5) [36]. 

Camarero J, Hackel BJ, de Yoreo JJ, Mitchell AR (2004) Fmoc-based synthesis of peptide a-thioesters using an aryl hydrazine support. J Org Chem 69 4145-4151... 

Sieber P (1987) A new acid-labile anchor group for the solid-phase synthesis of C-terminal peptide amides by the Fmoc method. Tetrahedron Lett 28 2107-2110 Mende F, Seitz O (2011) 9-Fluorenylmethoxycarbonyl-based solid-phase synthesis of peptide a-thioesters. Angew Chem Int Ed 50 1232-1240 Giiillier F, Grain D, Bradley M (2000) Linkers and cleavage strategies in solid-phase organic synthesis and combinatorial chemistry. Chem Rev 100 2091-2158... 

Native chemical ligation has been used successfully to couple two unprotected peptides together during solid phase synthesis, wherein one of the peptides is attached to the resin using a thioester linkage and the other peptide is introduced containing a cysteine at its N-terminal... 

Figure 17.25 The native chemical ligation reaction can be used to form larger peptides from smaller peptides, if one contains a cysteine residue at its N-terminal and the other one contains a thioester on its C-terminal. Reaction of the peptide derivatives gives a native peptide (amide) bond.

FIGURE 7.17 Synthesis of a peptide by construction of a peptide alkyl thioester on a solid support, detachment of the assembled molecule that includes the spacer and transesterfication of the ester into an activated ester which is aminolyzed as it is formed. [Aimoto et al., 1991]. Dhbt = 4-oxo-3,4-dihydrobenzotriazin-3-yl. 

Earlier in this chapter, it was mentioned that many of the nonprotein amino acids are components of nonribosomal peptides. During such a biosynthesis, the peptide is attached to a carrier protein through a thioester bond, until chain termination occurs and the final product is released. The carrier protein is posttranslationally modified by the attachment of a phosphopantetheinyl group from coenzyme A. This step gives rise to the active carrier protein with a phosphopantetheine arm upon which amino acids are added to during NRPS. As an example, loading of isoleucine onto the carrier protein is depicted below (Scheme 5). Further details about nonribosomal peptide syntheses and enzymatic reactions can be found in Chapter 5.19. 

Once assembly of a mature peptide has been completed by the NRPS, the product remains covalently linked to the PCP domain of the last module as a thioester. Release into solution from the assembly line is accomplished by a variety of enzyme-catalyzed reactions as described below (Figure 8). 

Among the electrophilic handles proposed for head-to-tail and side-chain-to-tail cyclization of peptides on solid support by intrachain aminolysis with concurrent detachment of the product from the resin in the protected form (see Section 6.8.3.1.3), generally the oxime resin (also called Kaiser resin)1364 365 and a thioester resin[363l are recommended (see Scheme 14). In addition to the classical head-to-tail cyclization,[3431 the oxime resin is used for side-chain cyclizations as well as for the synthesis of multicyclic peptides vide infra). Due to its dual functions, the oxime resin can be employed only with Boc/Bzl chemistry it is not compatible with Fmoc/tBu chemistry where the basic N -deprotection leads to free amino groups and thus to premature cyclization reactions. To avoid this premature cleavage of the... 

Fig. 2.15A. Pattern of ubiquitinylation of proteins and degradation in the proteosome. Ubiquitin (Ub)is initially activated by an enzyme El, whereby the C-terminal carboxyl group of ubiquitin becomes attached to a SH group of El via a thioester bond. Ubiquitin is then transferred from El-Ub to E2, from which it is transferred with the help of E3 to the target protein. Several ubiquitin molecules can attach to the target protein in a hnear or in a crosshnked fashion. The mono- or polyubiquitinylated protein is degraded to peptides in the 26S proteosome. In the above diagram the filled circles represent the ubiquitin residues attached to the target protein. K lysine residues of the target protein.

Replacement of the peptide bond by a thioester group has been extensively applied in synthetic peptides used as enzyme substrates. Several classes of enzymes such as serine peptidases and metallo-endopeptidases are able to cleave efficiently thioester-modified amino acids and peptides. 61... 

Other very important applications of C-terminal thioester-functionalized peptides include their usage in the condensation of large unprotected peptide fragments (ligation see Vol. E22a, Section 4.1.5). 4,5,74 In this process the thioester-modified unprotected peptide reacts with the N-terminal cysteine of a second unprotected peptide giving a thioester intermediate. This step is followed by a rapid intramolecular S—>N shift with formation of the thermodynamically favored amide bond at the ligation site. 

In this method, the cysteine-thioester cyclization generates a cyclic peptide 86a (see Scheme 23) with a Xaa-Cys bond whose thiol moiety is then used for tethering to the core through an S-alkylation reaction.191 The requirement for the cyclization reaction is a linear precursor 84 containing both an N-terminal Cys and a C-terminal thioester. Such a peptide precursor can be conveniently synthesized by a stepwise solid-phase synthesis on a thioester resin 81 using Boc chemistry (Scheme 22). Cleavage by HF after assembly of the peptide sequence will produce the desired precursor with an N-terminal Cys and a C-terminal thioester 84. The crude peptide is then purified by RP-HPLC and the purified unprotected peptide is then circularized in aqueous conditions buffered at pH > 7.0. 

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