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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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Effect of N-terminalproline on protein modification. (a) A prolinewas introduced to the N-terminus of GFP. Reactivity with o-aminophenol-PEG was monitored by SDS-PAGE. The proline terminalvariant showed much higher levels of modification than the wild-typeprotein. No modification of N-terminal proline-GFP occurred at pH6. The band doubling is due to a gel artifact, and appears in alllanes. (b) Mutants of TMV were reacted with 5 equiv of 2-amino-p-cresol and 1 mM K3Fe(CN)6 for 30 min and analyzed by LC–MS.
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fig4: Effect of N-terminalproline on protein modification. (a) A prolinewas introduced to the N-terminus of GFP. Reactivity with o-aminophenol-PEG was monitored by SDS-PAGE. The proline terminalvariant showed much higher levels of modification than the wild-typeprotein. No modification of N-terminal proline-GFP occurred at pH6. The band doubling is due to a gel artifact, and appears in alllanes. (b) Mutants of TMV were reacted with 5 equiv of 2-amino-p-cresol and 1 mM K3Fe(CN)6 for 30 min and analyzed by LC–MS.

Mentions: The oxidative coupling reactionwith N-terminal amino groups wasfirst tested on proteins with native N-termini. Several proteins werereacted with o-aminophenol-functionalized 5 kDa PEGunder the optimized reaction conditions (Figure 3). The native proteins showed moderate levels of reactivity, whichcould be attributed to inaccessible N-termini or simply to the lessreactive N-terminal residues. To test if proline terminal proteinswere more reactive, a proline residue was introduced to the N-terminusof GFP and the tobacco mosaic virus (TMV) coat protein. The N-terminusof TMV was also slightly extended from the native sequence (additionof PAG). The proline-GFP was treated with a variety of conditionsto determine the specificity of the reaction (Figure 4a). Only at basic pH in the presence of both the o-aminophenol substrate and the oxidant was modification observed.Additionally, the proline-terminal variant showed significantly improvedreactivity compared to that of the wild-type N-terminus. These highlevels of modification were maintained even when only 1–2 equivof the o-aminophenol PEG was used. The site of modificationwas confirmed to be the N-terminal proline by LC–MS/MS analysisof a tryptic digest of proline-GFP modified with 2-amino-p-cresol (Supporting Information FigureS16).


N-terminal modification of proteins with o-aminophenols.

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

Effect of N-terminalproline on protein modification. (a) A prolinewas introduced to the N-terminus of GFP. Reactivity with o-aminophenol-PEG was monitored by SDS-PAGE. The proline terminalvariant showed much higher levels of modification than the wild-typeprotein. No modification of N-terminal proline-GFP occurred at pH6. The band doubling is due to a gel artifact, and appears in alllanes. (b) Mutants of TMV were reacted with 5 equiv of 2-amino-p-cresol and 1 mM K3Fe(CN)6 for 30 min and analyzed by LC–MS.
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Related In: Results  -  Collection

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fig4: Effect of N-terminalproline on protein modification. (a) A prolinewas introduced to the N-terminus of GFP. Reactivity with o-aminophenol-PEG was monitored by SDS-PAGE. The proline terminalvariant showed much higher levels of modification than the wild-typeprotein. No modification of N-terminal proline-GFP occurred at pH6. The band doubling is due to a gel artifact, and appears in alllanes. (b) Mutants of TMV were reacted with 5 equiv of 2-amino-p-cresol and 1 mM K3Fe(CN)6 for 30 min and analyzed by LC–MS.
Mentions: The oxidative coupling reactionwith N-terminal amino groups wasfirst tested on proteins with native N-termini. Several proteins werereacted with o-aminophenol-functionalized 5 kDa PEGunder the optimized reaction conditions (Figure 3). The native proteins showed moderate levels of reactivity, whichcould be attributed to inaccessible N-termini or simply to the lessreactive N-terminal residues. To test if proline terminal proteinswere more reactive, a proline residue was introduced to the N-terminusof GFP and the tobacco mosaic virus (TMV) coat protein. The N-terminusof TMV was also slightly extended from the native sequence (additionof PAG). The proline-GFP was treated with a variety of conditionsto determine the specificity of the reaction (Figure 4a). Only at basic pH in the presence of both the o-aminophenol substrate and the oxidant was modification observed.Additionally, the proline-terminal variant showed significantly improvedreactivity compared to that of the wild-type N-terminus. These highlevels of modification were maintained even when only 1–2 equivof the o-aminophenol PEG was used. The site of modificationwas confirmed to be the N-terminal proline by LC–MS/MS analysisof a tryptic digest of proline-GFP modified with 2-amino-p-cresol (Supporting Information FigureS16).

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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