Leveraging the Knorr Pyrazole Synthesis for the Facile Generation of Thioester Surrogates for use in Native Chemical Ligation

Abstract

Facile synthesis of C‐terminal thioesters is integral to native chemical ligation (NCL) strategies for chemical protein synthesis. We introduce a new method of mild peptide activation, which leverages solid‐phase peptide synthesis (SPPS) on an established resin linker and classical heterocyclic chemistry to convert C‐terminal peptide hydrazides into their corresponding thioesters via an acyl pyrazole intermediate. Peptide hydrazides, synthesized on established trityl chloride resins, can be activated in solution with stoichiometric acetyl acetone (acac), readily proceed to the peptide acyl pyrazoles. Acyl pyrazoles are mild acylating agents and are efficiently exchanged with an aryl thiol, which can then be directly utilized in NCL. The mild, chemoselective, and stoichiometric activating conditions allow this method to be utilized through multiple sequential ligations without intermediate purification steps.

Chemoenzymatic Semisynthesis of Proteins

“…The use of N-acyl hydrazides, one of the first methods of acyl activation, has been reimagined as a thioester precursor for NCL reactions. 63,64 Chemoselective conversion of this comparatively stable moiety to an α-thioester can be carried out in situ prior to ligation reactions, a method that has been widely adopted for both protein total synthesis and semisynthesis. 50 Importantly, resins prefunctionalized with thioester precursors are now commercially available (including the Dbz, bis(2-sulfanylethyl)amino (SEA), 59 and N-acyl hydrazide linkers), significantly reducing the barrier to adoption of these powerful technologies.…”

Relative configuration of Cys‐Pro ester peptides in thioester formation

“…The thiolactone intermediate 6, formed from the D-Cys-L-Pro ester or L-Cys-D-Pro ester, has a turn-like structure, which might facilitate intramolecular cyclization. Therefore, the reactions of these two peptides, D-Cys-Pro-Cys-Arg-Phe-Ala-Cys-Pro-OCH 2 CONH 2 (9) and Cys-Arg-Phe-Ala-D-Cys-Pro-OCH 2 CONH 2 (10), were compared, because they would be predicted to be transformed into the same cyclic peptide, cyclo(-Cys-Arg-Phe-Ala-D-Cys-Pro-) (13), through the DKP thioester 11 and the thiolactone 12, respectively (Figure 4). When peptide 9 was reacted at a concentration of 1 mM, a complex F G U R E 4 Formation of the cyclic Cys-Pro-Cys (CPC) peptide 13 from CPE peptides 9 and 10.…”

“…In such cases, 9‐fluorenylmethoxycarbonyl (Fmoc) SPPS is more suitable for use with such modified peptides, but the thioester is decomposed by piperidine, which is used for the deprotection of the Fmoc group 5 . Therefore, indirect methods were proposed such as safety catch, 6,7 Nbz, 8 hydrazide, 9 pyrazole, 10 ynamide 11 strategies, and so on. An N‐to‐S acyl shift reaction is also used as an indirect method for the preparation of the peptide thioester, in which the peptide chain is elongated on the amide structure and then transformed to the thioester at the final stage 12,13 .…”

Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

“…S1 and S2). The Hir(1-38)-COSR N-terminal peptides were similarly prepared by Fmoc-SPPS, bearing a C-terminal thioester surrogate 40,41 and Sec at different specific positions (6, 14, 16, 22 and/or 28) as needed (for details see SI, and Fig. S3-S7).…”