Chemoselective and site-selective peptide and native protein modification enabled by aldehyde auto-oxidation

The generation of protein biotherapeutics with improved features compared to the synthetic drugs has received emerging interest. The conjugation of various synthetic functionalities to proteins provides access to new classes of protein conjugates, where the advantages from both the synthetic world and Nature can be combined in a synergistic fashion. Here, we reported that 2-chloromethyl acryl scaffold can serve as a simple yet versatile platform for synthesizing acrylamide or acrylate derivatives by coupling with different end-group functionalities (amino group or hydroxyl group) via a one-pot reaction. The chemical properties of the amide or ester linkage influence their inherent reactivity as bioconjugation reagents, which in turn allows synthetic customization of their features to achieve selective protein modification at cysteine or disulfide sites on demand. 2-Chloromethyl acrylamide reagents with amide linkage favors selective modification at cysteine site with high efficiency and the resultant bioconjugates exhibit superior stability compared to commonly employed maleimide-thiol conjugates. In contrast, 2-chloromethyl acrylate reagents bearing ester linkage can undergo two successive Michael reaction, allowing the selective modification of disulfides with high labelling efficiency and conjugate stability. These reagents could outperform widely applied maleimide reagents in terms of stability of the resultant bioconjugates without compromising on the ease of reagent preparation, reactivity and reaction speed. <br>

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Chloromethyl Acryl Reagents for Simple and Site-Selective Cysteine and Disulfide Modification

10.26434/chemrxiv.14769873 ◽

2021 ◽

Author(s):

Lujuan Xu ◽

Seah Ling Kuan ◽

Tanja Weil

Keyword(s):

Protein Modification ◽

High Efficiency ◽

Chemical Properties ◽

Hydroxyl Group ◽

One Pot ◽

Synthetic Drugs ◽

Protein Conjugates ◽

Ester Linkage ◽

Protein Biotherapeutics ◽

Site Selective

The generation of protein biotherapeutics with improved features compared to the synthetic drugs has received emerging interest. The conjugation of various synthetic functionalities to proteins provides access to new classes of protein conjugates, where the advantages from both the synthetic world and Nature can be combined in a synergistic fashion. Here, we reported that 2-chloromethyl acryl scaffold can serve as a simple yet versatile platform for synthesizing acrylamide or acrylate derivatives by coupling with different end-group functionalities (amino group or hydroxyl group) via a one-pot reaction. The chemical properties of the amide or ester linkage influence their inherent reactivity as bioconjugation reagents, which in turn allows synthetic customization of their features to achieve selective protein modification at cysteine or disulfide sites on demand. 2-Chloromethyl acrylamide reagents with amide linkage favors selective modification at cysteine site with high efficiency and the resultant bioconjugates exhibit superior stability compared to commonly employed maleimide-thiol conjugates. In contrast, 2-chloromethyl acrylate reagents bearing ester linkage can undergo two successive Michael reaction, allowing the selective modification of disulfides with high labelling efficiency and conjugate stability. These reagents could outperform widely applied maleimide reagents in terms of stability of the resultant bioconjugates without compromising on the ease of reagent preparation, reactivity and reaction speed. <br>

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Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

10.26434/chemrxiv.7150097 ◽

2018 ◽

Author(s):

Daniel D. Brauer ◽

Emily C. Hartman ◽

Daniel L.V. Bader ◽

Zoe N. Merz ◽

Danielle Tullman-Ercek ◽

...

Keyword(s):

Small Molecules ◽

Protein Modification ◽

Positive Charge ◽

Specific Protein ◽

High Yield ◽

Site Specific ◽

Protein Cage ◽

Protein Carriers ◽

Site Selective ◽

Targeted Library

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

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Incorporation of 3-Azidotyrosine into Proteins Through Engineering Yeast Tyrosyl-tRNA Synthetase and Its Application to Site-Selective Protein Modification

Methods in Molecular Biology - Cell-Free Protein Production ◽

10.1007/978-1-60327-331-2_19 ◽

2009 ◽

pp. 227-242 ◽

Cited By ~ 5

Author(s):

Takashi Yokogawa ◽

Satoshi Ohno ◽

Kazuya Nishikawa

Keyword(s):

Protein Modification ◽

Trna Synthetase ◽

Site Selective

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Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation

10.26434/chemrxiv.7250201 ◽

2018 ◽

Author(s):

Shang Jia ◽

Christopher Chang

Keyword(s):

Protein Function ◽

Living Cells ◽

Covalent Modification ◽

Native Protein ◽

Selective Labeling ◽

Protein Residues ◽

Mild Conditions ◽

Starting Point ◽

Histidine Phosphorylation ◽

Site Selective

Site-selective bioconjugation to native protein residues is a powerful tool for protein functionalization, with cysteine and lysine side chains being the most common points for attachment owing to their high nucleophilicity. We now report a strategy for histidine modification using thiophosphorodichloridate reagents that mimic post-translational histidine phosphorylation, enabling fast and selective labeling of protein histidines under mild conditions where various payloads can be introduced via copper-assisted alkyne-azide cycloaddition (CuAAC) chemistry. We establish that these reagents are particularly effective at covalent modification of His-tags, which are common motifs to facilitate protein purification, as illustrated by selective attachment of polyarginine cargoes to enhance the uptake of proteins into living cells. This work provides a starting point for probing and enhancing protein function using histidine-directed chemistry.

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