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

giesler, riley; Spaltenstein, Paul; Jacobsen, Michael T.; Xu, Weiliang; Maqueda, Mercedes; Kay, Michael S.

doi:10.1039/d1ob01611c

Cited by 6 publications

(3 citation statements)

References 73 publications

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“…During our efforts put into the total synthesis of cyclic bacteriocin AS-48, a potent broad-spectrum antimicrobial peptide produced by enterococci (such as E. faecalis), 24−26 it was found that the chemical synthesis of related peptide intermediates was very difficult due to their highly hydrophobic segments and compact secondary structures. 27 In this study, we chose fragment AS-48 (33−44) H-TAVGSGGLSL-LA-OH as a model peptide. The results showed that stepwise SPPS could not give any products (Figure S5b); however, incorporation of the ψ 2-hydroxyphenyl pro derivative Fmoc-Gly-Ser(ψ 2-hydroxyphenyl pro)-OH allowed the facile synthesis of the desired peptide in a high yield (71.18%, Figure S5a).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…During our efforts put into the total synthesis of cyclic bacteriocin AS-48, a potent broad-spectrum antimicrobial peptide produced by enterococci (such as E. faecalis), – it was found that the chemical synthesis of related peptide intermediates was very difficult due to their highly hydrophobic segments and compact secondary structures . In this study, we chose fragment AS-48 (33–44) H-TAV GS GGLSLLA-OH as a model peptide.…”

Section: Results and Discussionmentioning

confidence: 99%

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Robust Chemical Synthesis of “Difficult Peptides” via 2-Hydroxyphenol-pseudoproline (ψ^{2-hydroxyphenol}pro) Modifications

Wang,

Jin

2024

J. Org. Chem.

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The challenging preparation of “difficult peptides” has always hindered the development of peptide-active pharmaceutical ingredients. Pseudoproline (ψpro) building blocks have been proven effective and powerful tools for the synthesis of “difficult peptides”. In this paper, we efficiently prepared a set of novel 2-(oxazolidin-2-yl)phenol compounds as proline surrogates (2-hydroxyphenol-pseudoprolines, ψ2‑hydroxyphenolpro) and applied it in the synthesis of many well-known “difficult peptides”, including human thymosin α1, amylin, and β-amyloid (1–42) (Aβ42).

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Robust Chemical Synthesis of “Difficult Peptides” via 2-Hydroxyphenol-pseudoproline (ψ^{2-hydroxyphenol}pro) Modifications

Wang,

Jin

2024

J. Org. Chem.

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“…The hydrophilic tags must be removed when the synthesis is complete and consequently, the linker should be designed to be a cleavable structure that can be removed in a chemoselective fashion. To date, many groups have developed solubilizing tags semi-permanently attached to the C-terminus, 13,14) side chain functionalities, [15][16][17][18][19][20][21][22][23][24][25] or backbone amides. [26][27][28][29] Using hydrazide chemistry (Chart 1B), we recently reported a novel solubilizing tag.…”

Section: Introductionmentioning

confidence: 99%

Hydrazide-Mediated Solubilizing Strategy for Poorly Soluble Peptides Using a Dialkoxybenzaldehyde Linker

et al. 2022

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Proteins modified in a controlled manner with artificial moieties such as fluorophores or affinity tags have been shown to be a powerful tool for functional or structural analysis of proteins. A reliable way to prepare proteins with a well-defined modification is protein synthesis. Although many successful syntheses have been reported, the poor aqueous solubility of synthetic intermediates causes difficulty in the chemical synthesis of proteins. Here we describe a solubilizing strategy for poorly soluble peptides which uses chemoselective incorporation of a hydrophilic tag onto a hydrazide in a peptide. We found that a hydrophilic tag possessing a dialkoxybenzaldehyde moiety can react with peptide hydrazides through reductive N-alkylation. No protecting groups are required for this reaction, and peptides modified in this way show enhanced solubility and consequently good peak separation during HPLC purification. The tag can be removed subsequently by treatment with trifluoroacetic acid to generate a free hydrazide, which can be converted in a one-pot reaction to a thioester for further modification. This method was validated by synthesis of a Lys 63 -linked ubiquitin dimer derivative. This late-stage solubilization can be applied in principal to any peptide and opens the possibility of the synthesis of proteins that have previously been considered inaccessible due to their poor solubility.

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Selective Activation of Peptide‐Thioester Precursors for Templated Native Chemical Ligations

Spaltenstein,

Giesler,

Scherer

et al. 2024

Angewandte Chemie

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Chemical protein synthesis enables access to proteins that would otherwise be difficult or impossible to obtain with traditional means such as recombinant expression. Chemoselective ligations provide the ability to join peptide segments prepared by solid‐phase peptide synthesis. While native chemical ligation (NCL) is widely used, it is limited by the need for C‐terminal thioesters with suitable reaction kinetics, properly placed native Cys or thiolated derivatives, and peptide segment solubility at low mM concentrations. Moreover, repetitive purifications to isolate ligated products are often yield‐sapping, hampering efficiency and progress. In this work, we demonstrate the use of Controlled Activation of Peptides for Templated NCL (CAPTN). This traceless multi‐segment templated NCL approach permits the one‐pot synthesis of proteins by harnessing selective thioester activation and orthogonal conjugation chemistries to favor formation of full‐length ligated product while minimizing side reactions. Importantly, CAPTN provides kinetic enhancements allowing ligations at sterically hindered junctions and low peptide concentrations. Additionally, this one‐pot approach removes the need for intermediate purification. We report the synthesis of two E.coli ribosomal subunits S16 and S17 enabled by the chemical tools described herein. We anticipate that CAPTN will expedite the synthesis of valuable proteins and expand on templated approaches for chemical protein synthesis.

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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...

Cited by 6 publications

References 73 publications

Robust Chemical Synthesis of “Difficult Peptides” via 2-Hydroxyphenol-pseudoproline (ψ^{2-hydroxyphenol}pro) Modifications

Robust Chemical Synthesis of “Difficult Peptides” via 2-Hydroxyphenol-pseudoproline (ψ^{2-hydroxyphenol}pro) Modifications

Hydrazide-Mediated Solubilizing Strategy for Poorly Soluble Peptides Using a Dialkoxybenzaldehyde Linker

Selective Activation of Peptide‐Thioester Precursors for Templated Native Chemical Ligations

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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...

Cited by 6 publications

References 73 publications

Robust Chemical Synthesis of “Difficult Peptides” via 2-Hydroxyphenol-pseudoproline (ψ2-hydroxyphenolpro) Modifications

Robust Chemical Synthesis of “Difficult Peptides” via 2-Hydroxyphenol-pseudoproline (ψ2-hydroxyphenolpro) Modifications

Hydrazide-Mediated Solubilizing Strategy for Poorly Soluble Peptides Using a Dialkoxybenzaldehyde Linker

Selective Activation of Peptide‐Thioester Precursors for Templated Native Chemical Ligations

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Robust Chemical Synthesis of “Difficult Peptides” via 2-Hydroxyphenol-pseudoproline (ψ^{2-hydroxyphenol}pro) Modifications

Robust Chemical Synthesis of “Difficult Peptides” via 2-Hydroxyphenol-pseudoproline (ψ^{2-hydroxyphenol}pro) Modifications