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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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Characterization of the oxidative coupling reactions usingsmallmolecules. (a) Amine coupling partners were reacted with 4-methylcatecholas a model substrate. The reaction was followed by monitoring theproduct absorbance at 520 nm. Reactions were run under pseudo-firstorder conditions with 100 μM catechol, 1 mM amine, 10 mM ferricyanidein 50 mM phosphate buffer. (b) A peptide containing both an N-terminalproline and a p-aminophenylalanine residue (PAD(pAF)SWAG) was tested for reactivity with 2 equiv of 2-amino-p-cresol at pH 6. An aliquot of the reaction was quenchedand analyzed by LC–MS. The remainder of the reaction was purifiedand then reacted with 2 equiv of the aminophenol at pH 7.5 and analyzedby LC–MS.
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fig2: Characterization of the oxidative coupling reactions usingsmallmolecules. (a) Amine coupling partners were reacted with 4-methylcatecholas a model substrate. The reaction was followed by monitoring theproduct absorbance at 520 nm. Reactions were run under pseudo-firstorder conditions with 100 μM catechol, 1 mM amine, 10 mM ferricyanidein 50 mM phosphate buffer. (b) A peptide containing both an N-terminalproline and a p-aminophenylalanine residue (PAD(pAF)SWAG) was tested for reactivity with 2 equiv of 2-amino-p-cresol at pH 6. An aliquot of the reaction was quenchedand analyzed by LC–MS. The remainder of the reaction was purifiedand then reacted with 2 equiv of the aminophenol at pH 7.5 and analyzedby LC–MS.

Mentions: In the process of characterizing the reaction products, it was observedthat the colored products absorbed light at wavelengths greater than500 nm (with λmax between 505 and 525 nm dependingon the amine coupling partner). As the starting coupling partnersand ferricyanide did not absorb at these wavelengths, this uniqueabsorbance provided a means to monitor the reaction progress. Thedifferent amine coupling partners (p-toluidine, H-Pro-OMe,and H-Phe-OMe) were reacted with 4-methylcatechol in the presenceof 10 mM ferricyanide, and the absorbance of the resulting solutionwas monitored at 520 nm to determine the relative rates of reactivity(Figure 2a; for unnormalized data see Supporting Information Figure S12). The catecholsubstrate was used for these studies to simplify the reaction pathwayby eliminating the imine hydrolysis step. The reactions were run underpseudo-first order conditions with 0.1 mM catechol and 1 mM aminecoupling partner. When the reaction was carried out at pH 6.0, onlythe aniline coupling partner exhibited rapid coupling with the catechol.However, at pH 7.5 all three amines reacted efficiently. The anilinecoupling partner demonstrated the fastest coupling (<30 s). Thereaction with the proline analogue reached completion nearly as rapidly(∼2 min), but the reaction with the primary aliphatic amineof phenylalanine required longer reaction times (∼10 min).This demonstrates how the reaction can have very high selectivityfor aniline residues.


N-terminal modification of proteins with o-aminophenols.

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

Characterization of the oxidative coupling reactions usingsmallmolecules. (a) Amine coupling partners were reacted with 4-methylcatecholas a model substrate. The reaction was followed by monitoring theproduct absorbance at 520 nm. Reactions were run under pseudo-firstorder conditions with 100 μM catechol, 1 mM amine, 10 mM ferricyanidein 50 mM phosphate buffer. (b) A peptide containing both an N-terminalproline and a p-aminophenylalanine residue (PAD(pAF)SWAG) was tested for reactivity with 2 equiv of 2-amino-p-cresol at pH 6. An aliquot of the reaction was quenchedand analyzed by LC–MS. The remainder of the reaction was purifiedand then reacted with 2 equiv of the aminophenol at pH 7.5 and analyzedby LC–MS.
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fig2: Characterization of the oxidative coupling reactions usingsmallmolecules. (a) Amine coupling partners were reacted with 4-methylcatecholas a model substrate. The reaction was followed by monitoring theproduct absorbance at 520 nm. Reactions were run under pseudo-firstorder conditions with 100 μM catechol, 1 mM amine, 10 mM ferricyanidein 50 mM phosphate buffer. (b) A peptide containing both an N-terminalproline and a p-aminophenylalanine residue (PAD(pAF)SWAG) was tested for reactivity with 2 equiv of 2-amino-p-cresol at pH 6. An aliquot of the reaction was quenchedand analyzed by LC–MS. The remainder of the reaction was purifiedand then reacted with 2 equiv of the aminophenol at pH 7.5 and analyzedby LC–MS.
Mentions: In the process of characterizing the reaction products, it was observedthat the colored products absorbed light at wavelengths greater than500 nm (with λmax between 505 and 525 nm dependingon the amine coupling partner). As the starting coupling partnersand ferricyanide did not absorb at these wavelengths, this uniqueabsorbance provided a means to monitor the reaction progress. Thedifferent amine coupling partners (p-toluidine, H-Pro-OMe,and H-Phe-OMe) were reacted with 4-methylcatechol in the presenceof 10 mM ferricyanide, and the absorbance of the resulting solutionwas monitored at 520 nm to determine the relative rates of reactivity(Figure 2a; for unnormalized data see Supporting Information Figure S12). The catecholsubstrate was used for these studies to simplify the reaction pathwayby eliminating the imine hydrolysis step. The reactions were run underpseudo-first order conditions with 0.1 mM catechol and 1 mM aminecoupling partner. When the reaction was carried out at pH 6.0, onlythe aniline coupling partner exhibited rapid coupling with the catechol.However, at pH 7.5 all three amines reacted efficiently. The anilinecoupling partner demonstrated the fastest coupling (<30 s). Thereaction with the proline analogue reached completion nearly as rapidly(∼2 min), but the reaction with the primary aliphatic amineof phenylalanine required longer reaction times (∼10 min).This demonstrates how the reaction can have very high selectivityfor aniline residues.

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