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N-terminal modification of proteins with o-aminophenols.

Obermeyer AC, Jarman JB, Francis MB - J. Am. Chem. Soc. (2014)

Bottom Line: Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive.Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete.This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California, Berkeley , Berkeley, California 94720, United States.

ABSTRACT
The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.

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N-terminal oxidative coupling for proteinswith free cysteines.(a) PAG S123C TMV was reacted with small molecule substrates and analyzedby LC–MS. Cysteine residues were protected as a disulfide usingEllman’s reagent (DTNB) before oxidative coupling. Subsequentreduction of the disulfide resulted in selective modification of theN-terminus. (b) PAG S123C TMV was labeled with two fluorophores. Thecysteine was first alkylated with an Alexa Fluor maleimide. The N-terminalproline was then modified with a rhodamine-functionalized o-aminophenol.
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fig5: N-terminal oxidative coupling for proteinswith free cysteines.(a) PAG S123C TMV was reacted with small molecule substrates and analyzedby LC–MS. Cysteine residues were protected as a disulfide usingEllman’s reagent (DTNB) before oxidative coupling. Subsequentreduction of the disulfide resulted in selective modification of theN-terminus. (b) PAG S123C TMV was labeled with two fluorophores. Thecysteine was first alkylated with an Alexa Fluor maleimide. The N-terminalproline was then modified with a rhodamine-functionalized o-aminophenol.

Mentions: The compatibility of the reaction withcysteine residues was alsotested using TMV. A single cysteine residue (S123C) was introducedinto the TMV coat protein with a proline N-terminus (PAG S123C TMV).This mutant was reacted with 2-amino-p-cresol andanalyzed by LC–MS (Supporting Information Figure S20). The cysteine residue also reacted with the o-aminophenol, resulting in two modifications.61,62 However, it was found that the N-terminal proline could be modifiedselectively if the cysteine was first capped (Figure 5a,b, see Supporting Information Figures S21–22 for wider mass range and ion series). To dothis, the cysteine residue was protected as a disulfide bond by reactionwith 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’sreagent). After the oxidative coupling step the disulfide bond wasreadily reduced by TCEP, leaving the free cysteine and the modifiedN-terminus. Alternatively, the cysteine residue was modified witha maleimide, followed by modification at the N-terminus with an o-aminophenol reagent.


N-terminal modification of proteins with o-aminophenols.

Obermeyer AC, Jarman JB, Francis MB - J. Am. Chem. Soc. (2014)

N-terminal oxidative coupling for proteinswith free cysteines.(a) PAG S123C TMV was reacted with small molecule substrates and analyzedby LC–MS. Cysteine residues were protected as a disulfide usingEllman’s reagent (DTNB) before oxidative coupling. Subsequentreduction of the disulfide resulted in selective modification of theN-terminus. (b) PAG S123C TMV was labeled with two fluorophores. Thecysteine was first alkylated with an Alexa Fluor maleimide. The N-terminalproline was then modified with a rhodamine-functionalized o-aminophenol.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4353012&req=5

fig5: N-terminal oxidative coupling for proteinswith free cysteines.(a) PAG S123C TMV was reacted with small molecule substrates and analyzedby LC–MS. Cysteine residues were protected as a disulfide usingEllman’s reagent (DTNB) before oxidative coupling. Subsequentreduction of the disulfide resulted in selective modification of theN-terminus. (b) PAG S123C TMV was labeled with two fluorophores. Thecysteine was first alkylated with an Alexa Fluor maleimide. The N-terminalproline was then modified with a rhodamine-functionalized o-aminophenol.
Mentions: The compatibility of the reaction withcysteine residues was alsotested using TMV. A single cysteine residue (S123C) was introducedinto the TMV coat protein with a proline N-terminus (PAG S123C TMV).This mutant was reacted with 2-amino-p-cresol andanalyzed by LC–MS (Supporting Information Figure S20). The cysteine residue also reacted with the o-aminophenol, resulting in two modifications.61,62 However, it was found that the N-terminal proline could be modifiedselectively if the cysteine was first capped (Figure 5a,b, see Supporting Information Figures S21–22 for wider mass range and ion series). To dothis, the cysteine residue was protected as a disulfide bond by reactionwith 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’sreagent). After the oxidative coupling step the disulfide bond wasreadily reduced by TCEP, leaving the free cysteine and the modifiedN-terminus. Alternatively, the cysteine residue was modified witha maleimide, followed by modification at the N-terminus with an o-aminophenol reagent.

Bottom Line: Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive.Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete.This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California, Berkeley , Berkeley, California 94720, United States.

ABSTRACT
The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.

Show MeSH
Related in: MedlinePlus