Benzimidazolinone-Free Peptide o-Aminoanilides for Chemical Protein Synthesis

Abstract

The thioester surrogate 3,4-diaminobenzoic acid (Dbz) facilitates the efficient synthesis of peptide thioesters by Fmoc chemistry solid phase peptide synthesis and the optional attachment of a solubility tag at the C-terminus. The protection of the partially deactivated ortho-amine of Dbz is necessary to obtain contamination-free peptide synthesis. The reported carbamate protecting groups promote a serious side reaction, benzimidazolinone formation. Herein we introduce the Boc-protected Dbz that prevents the benzimidazolinone formation, leading to clean peptide o-aminoanilides suitable for the total chemical synthesis of proteins.

Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli

“…To validate the refolded Pf AMA1 protein's functional activity, we needed to synthesize the peptide ligand (RON2ed) that is known to bind with Pf AMA1 protein. We chemically synthesized the 39 mer RON2ed polypeptide by Fmoc-chemistry SPPS using an automated peptide synthesizer [ 17 ]. The chemically synthesized peptide was then allowed to undergo oxidative folding under air oxidation conditions in buffer H (2 M Gu.HCl and 100 mM Tris, pH 8.4) for 18 h. After the folding with a disulfide bond formation was complete, as monitored by LCMS ( Fig.…”

“…The peptide was synthesized on 2-chlorotrityl chloride (CTC) resin. Disulfide bond formation of the peptide was achieved by air oxidation in Tris buffer at pH 8 [ 17 ]. The folded peptide was purified by reverse-phase HPLC and lyophilized.…”

“…Plasmodium falciparum 3D7 cell line culture was synchronized tightly before growth inhibition assay. Intraerythrocytic late trophozoite or early schizont stage with parasitemia 0.3% were subjected to treatment with varying concentrations (1 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM and 50,000 nM) of chemically synthesized Pf RON2ed in nutrient-rich complete media and allowed to incubate for 72 h as described previously [ 17 , 18 ]. Upon completing the incubation period, the standard Giemsa counting assay was performed by preparing a thin smear by taking a 4 μL palette culture after centrifugation.…”

Efficient Chemical Protein Synthesis using Fmoc‐Masked N‐Terminal Cysteine in Peptide Thioester Segments

“…The same strategy was used for the synthesis of several other moderately sized protein molecules, where peptide thioester syntheses were performed using Boc‐chemistry SPPS [4b,c] . Liu and co‐workers introduced peptide hydriazides [7a] and Dawson's group introduced peptide o ‐aminoanilides [7b–d] as surrogates for peptide thioester segments used in chemical protein synthesis. Because peptide hydrazides or o ‐aminoanilides are readily prepared by Fmoc‐chemistry SPPS, they are widely used.…”

Efficient Chemical Protein Synthesis using Fmoc‐Masked N‐Terminal Cysteine in Peptide Thioester Segments

“…The same strategy was used for the synthesis of several other moderately sized protein molecules, where peptide thioester syntheses were performed using Boc‐chemistry SPPS . Liu and co‐workers introduced peptide hydriazides and Dawson's group introduced peptide o ‐aminoanilides as surrogates for peptide thioester segments used in chemical protein synthesis. Because peptide hydrazides or o ‐aminoanilides are readily prepared by Fmoc‐chemistry SPPS, they are widely used.…”