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A glutamic acid-based traceless linker to address challenging chemical protein syntheses.

ABSTRACT: Native chemical ligation (NCL) enables the total chemical synthesis of proteins. However, poor peptide segment solubility remains a frequently encountered challenge. Here we introduce a traceless linker that can be temporarily attached to Glu side chains to overcome this problem. This strategy employs a new tool, Fmoc-Glu(AlHx)-OH, which can be directly installed using standard Fmoc-based solid-phase peptide synthesis. The incorporated residue, Glu(AlHx), is stable to a wide range of chemical protein synthesis conditions and is removed through palladium-catalyzed transfer under aqueous conditions. General handling characteristics, such as efficient incorporation, stability and rapid removal were demonstrated through a model peptide modified with Glu(AlHx) and a Lys₆ solubilizing tag. Glu(AlHx) was incorporated into a highly insoluble peptide segment during the total synthesis of the bacteriocin AS-48. This challenging peptide was successfully synthesized and folded, and it has comparable antimicrobial activity to the native AS-48. We anticipate widespread use of this easy-to-use, robust linker for the preparation of challenging synthetic peptides and proteins.

SUBMITTER: Giesler RJ

PROVIDER: S-EPMC8604549 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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A glutamic acid-based traceless linker to address challenging chemical protein syntheses.

Giesler Riley J RJ Spaltenstein Paul P Jacobsen Michael T MT Xu Weiliang W Maqueda Mercedes M Kay Michael S MS

Organic & biomolecular chemistry 20211020 40

Native chemical ligation (NCL) enables the total chemical synthesis of proteins. However, poor peptide segment solubility remains a frequently encountered challenge. Here we introduce a traceless linker that can be temporarily attached to Glu side chains to overcome this problem. This strategy employs a new tool, Fmoc-Glu(AlHx)-OH, which can be directly installed using standard Fmoc-based solid-phase peptide synthesis. The incorporated residue, Glu(AlHx), is stable to a wide range of chemical pr ...[more]

PMID: 34585207

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Project description:Glutamic acid is a commonly used linker to form dimeric peptides with enhanced binding affinity than their corresponding monomeric counterparts. We have previously labeled NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 (NOTA-PRGD2) [1] with [(18)F]AlF and (68)Ga for imaging tumor angiogenesis. The p-SCN-Bn-NOTA was attached to E[c(RGDyK)]2 [2] through a mini-PEG with a thiourea linkage, and the product [1] was stable at radiolabeling condition of 100 °C and pH 4.0 acetate buffer. However, when the same p-SCN-Bn-NOTA was directly attached to the α-amine of E[c(RGDfK)]2 [3], the product NOTA-Bn-NCS-E[c(RGDfK)]2 [4] became unstable under similar conditions and the release of monomeric c(RGDfK) [5] was observed. The purpose of this work was to use HPLC and LC-MS to monitor the decomposition of glutamic acid linked dimeric peptides and their NOTA derivatives. A c(RGDyK) [6] and bombesin (BBN) [7] heterodimer c(RGDyK)-E-BBN [8], and a dimeric bombesin E(BBN)2 [9], both with a glutamic acid as the linker, along with a model compound PhSCN-E[c(RGDfK)] [10] were also studied. All the compounds were dissolved in 0.5 M pH 4.0 acetate buffer at the concentration of 1 mg/mL, and 0.1 mL of each sample was heated at 100 °C for 10 min and the more stable compounds were heated for another 30 min. The samples at both time points were analyzed with analytical HPLC to monitor the decomposition of the heated samples. The samples with decomposition were further analyzed by LC-MS to determine the mass of products from the decomposition for possible structure elucidation. After 10 min heating, the obvious release of c(RGDfK) [5] was observed for NOTA-Bn-NCS-E[c(RGDfK)]2 [4] and Ph-SCN-E[c(RGDfK)] [10]. Little or no release of monomers was observed for the remaining samples at this time point. After further heating, the release of monomers was clearly observed for E[c(RGDyK)]2 [2], E[c(RGDfK)]2 [3], c(RGDyK)-E-BBN [8], and E(BBN)2 [9]. No decomposition or little decomposition was observed for NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 [1], PEG3-E[c(RGDyK)]2 [11], NOTA-E[c(RGDyK)]2 [12], and NOTA-PEG3-E[c(RGDyK)]2 [13]. The glutamic acid linked dimeric peptides with a free α-amine are labile due to the neighboring amine participation in the hydrolysis. The stability of peptides could be increased by converting the free amine into amide. The instability of thiourea derivatives formed from α-amine was caused by participation of thiol group derived from thiourea.

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A glutamic acid-based traceless linker to address challenging chemical protein syntheses.

Publications

A glutamic acid-based traceless linker to address challenging chemical protein syntheses.

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