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.

Keywords: chemoselective ligation; native chemical ligation; peptide hydrazides; protein synthesis; pyrazoles.

Figure 1:

Some previously reported routes to C-terminal thioesters and active esters via A^[17] MeDBZ cyclization and B ^[18-22] acyl hydrazide oxidation. C This work.

Figure 2:

A Peptide thioester formation scheme. B Conversion of 1 (1 mM) to corresponding thioester upon addition of 2.5 equiv acac in 6 M GdmCl at varying pHs

Figure 3:

Proposed mechanism of acid catalyzed acyl-pyrazole formation in aqueous solution

Figure 4:

A Conversion of 1 to corresponding thioester with varying thiol sources, 200 mM MPAA, 2% v/v thiophenol or 2% v/v ethyl-3-mercaptopropionate (1 mM peptide 1, 2.5 mM acac in unbuffered 6 M GdmCl at pH 3). B Conversion of 1 to corresponding thioester with varying acac concentrations (1mM peptide 1, 200 mM MPAA in 6M GdmCl at pH 3)

Figure 5:

A Model peptide variants. B Conversion of 1, 2, 3 and 4 to corresponding thioester over time (1mM peptide, 2.5 mM acac 200mM MPAA in 6M GdmCl at pH 3)

Figure 6:

A Sequential ligations of 1 and 5 to create peptides 6 and 7. B HPLC-MS chromatograms of sequential ligations outlined above. Thioester formed with 1.0 eq. acac and 200 mM MPAA in 6 M GdmCl at pH 3. Subsequent ligation performed with 1 eq. Cys fragment at pH 7.

Figure 7:

A reaction scheme for the chemical synthesis of SpA domain. Thioester formed with 2.5 eq acac in 6 M GdmCl containing 200 mM MPAA. Ligation performed with 1.2 eq cys fragment 10 in 6 M GdmCl at pH 7. B HPLC-MS chromatograms of sequential ligations outlined above. For deconvoluted mass spectra see Supporting Information.

Figure 8:

A reaction scheme for the chemical synthesis of SDF-1a^[45,46] domain. Thioester formed with 2.5 eq acac in 6 M GdmCl containing 200 mM MPAA. Ligation performed with 0.8 eq cys fragment 14 in 6 M GdmCl at pH 7. B Crude deconvoluted mass spectra of the reaction mixtures outlined above.