news & views BIOCONJUGATION
How to pick a single amine?
Single-step site-specific labeling of native proteins is one of the holy grails in the chemical biology field. 2-Pyridinecarboxyaldehyde derivatives are shown to react selectively at the N terminus of proteins to form stable conjugates, irrespective of the nature of the N-terminal amino acid, enabling the straightforward introduction of useful functional groups into a wide array of proteins.
Floris P J T Rutjes
npg
© 2015 Nature America, Inc. All rights reserved.
R
esearch at the interface of chemistry and biology requires chemical tools for the selective modification of biomolecules to visualize, quantify or selectively isolate them from complex mixtures or even living systems. This raises the question how one can selectively and under mild conditions introduce tailor-made chemical reporter tags in proteins, a prominent class of biomolecules. Many of the existing strategies rely on sitespecific introduction of a bioorthogonal reactive group, followed by conjugation with a reporter tag via the complementary reactive group. One of the most powerful approaches consists of site-specific introduction of an azide-containing amino acid1, followed by azide-alkyne cycloaddition to form the corresponding conjugated triazole-based system2. Other approaches involve chemical modification of the N terminus, for example, oxidative cleavage of an N-terminal serine, followed by selective reaction of the resulting glyoxylic amide derivative. Examples include Pictet-Spengler–type conjugation3 and nitrone formation followed by nitrone-alkyne cycloaddition4. MacDonald et al.5 have now taken the N-terminal modification of peptides a major step further by showing that native proteins can be selectively reacted at the N terminus using 2-pyridinecarboxyaldehyde (2-PCA) derivatives. Even in the presence of multiple lysine residues, 2-PCA residues react with high selectivity at the N-terminal amino acid, facilitating the single-step introduction of desired functional tags in a variety of proteins. Selective functionalization at the N terminus of native proteins, i.e., selective reaction at the N-terminal amino group in the presence of multiple lysine residues, is generally possible owing to the fact that the amino group at the N terminus is slightly less basic (pK value ~8) than the lysine amino groups (pK value ~10). Hence, by proper adjustment of the pH, the free amino group at the N terminus will be more nucleophilic than the protonated lysine side chains and thus may, for example, be selectively acylated in reaction with activated esters.
Chan et al.6 published a recent improvement of this protocol involving the use of ketenes as the electrophile, leading to a higher selectivity than that in the protocol using activated esters. This method, however, may still be sensitive to subtle changes in pH. In this report, MacDonald et al.5 discovered a reaction to selectively functionalize the N terminus, which to some extent is reminiscent of the first steps of the Edman degradation7. In this powerful sequencing reaction (Fig. 1a), the N-terminal amino group reacts with phenylisothiocyanate (the Edman reagent), followed by participation of the adjacent amide group and subsequent cleavage of the N-terminal amino acid. Lysine residues may react with the isothiocyanate but will not be cleaved, owing to the absence of the participating amide bond. As in the Edman degradation sequence, the initial 2-PCA–protein adduct reacts, probably aided by the pyridine substituent, intramolecularly with the adjacent amide group to form a stable cyclic aminal, thereby driving the equilibrium of the first step to completion (Fig. 1b). As ring formation does not generate a reactive tertiary alcohol, as in the Edman degradation, the reaction does not lead to a cleaved residue. Additionally, because this second reaction cannot occur in the lysine a Ph R
C
R
S
Edman reagent
H N
H2N
N
Peptide
Ph
O
Peptide
H N
HN
O
S
H+
R
H N
HN N H
side chains, the reaction is intrinsically selective for the N-terminal amino group. The authors have shown that the conjugation with 2-PCA works for all amino acids, although glycine and proline give somewhat lower yields. It was also shown that by blocking the nucleophilicity of the second amino acid (for example, by incorporation of proline), conjugation was prevented. Furthermore, RNase A was used as a model protein to show the feasibility of conjugation with 2-PCA under many different conditions; 2-PCA conjugation was also demonstrated on a range of different proteins, confirming the wide applicability of the chemistry. Perhaps more importantly, conjugation with RNase A was also successful for modified 2-PCA molecules, allowing the site-selective introduction of functional groups such as a PEG chain or a biotin label; other groups such as fluorescent dyes, MRI labels or affinity tags could also be incorporated through this strategy. The authors demonstrated the potential utility of the new chemistry in applied settings through N-terminal biotinylation of the estrogen receptor α (ERα′), which was then immobilized on a streptavidin-containing resin to detect endocrine-disrupting compounds in drinking water.
OH
S Ph
H+
H2N
Peptide
R
N T R
H N
H2N O
N S
O
Ph
O H+
2-PCA reagent Peptide
O
HN
N
H
b
Peptide +
R
H N
N N
O
R Peptide
N
Peptide
HN N
T T
Figure 1 | Basis of the selectivity for the N terminus of proteins by participation of the adjacent amide substituent. (a) N-terminal conjugation with phenylisothiocyanate in Edman degradation. (b) N-terminal conjugation with 2-PCA.
nature chemical biology | Advance online publication | www.nature.com/naturechemicalbiology
1
news & views to mixtures of proteins. Notwithstanding, the contribution of MacDonald et al.5 represents a robust method for single-step N-terminal functionalization of many proteins and is flexible in the functionality that is to be coupled to the protein of interest and therefore may well turn into a widely used conjugation approach in the near future. ■ Floris P. J. T. Rutjes is at Radboud University, Institute for Molecules and Materials, Nijmegen, the Netherlands. e-mail: [email protected]
Published online 30 March 2015 doi:10.1038/nchembio.1791 References
1. Kiick, K.L. et al. Proc. Natl. Acad. Sci. USA 99, 19–24 (2002). 2. Debets, M.F. et al. Org. Biomol. Chem. 11, 6439–6455 (2013). 3. Agarwal, P. et al. Proc. Natl. Acad. Sci. USA 110, 46–51 (2013). 4. Ning, X. et al. Angew. Chem. Int. Ed. Engl. 49, 3065–3068 (2010). 5. MacDonald, J.I. et al. Nat. Chem. Biol. doi:10.1038/nchembio.1792 (30 March 2015). 6. Chan, A.O.-Y. et al. J. Am. Chem. Soc. 134, 2589–2598 (2012). 7. Edman, P. Acta Chem. Scand. 4, 283–293 (1950).
Competing financial interests The author declares no competing financial interests.
npg
© 2015 Nature America, Inc. All rights reserved.
It is only recently that chemists have more systematically started to develop molecular tools to unravel complex mechanisms in biological systems. Over the years, a toolbox has been developed that allows the sitespecific introduction of functional groups in biomolecules. However, numerous challenges still remain. Despite the promises that the 2-PCA molecules may hold, they certainly also have their limitations. This conjugation strategy will most likely only work outside the cell and not be applicable inside cellular systems. The method works well on a single protein but will not be selective when applied
2
nature chemical biology | Advance online publication | www.nature.com/naturechemicalbiology
How to pick a single amine?
Single-step site-specific labeling of native proteins is one of the holy grails in the chemical biology field. 2-Pyridinecarboxyaldehyde derivatives are shown to react selectively at the N terminus of proteins to form stable conjugates, irrespective of the nature of the N-terminal amino acid, enabling the straightforward introduction of useful functional groups into a wide array of proteins.
Floris P J T Rutjes
npg
© 2015 Nature America, Inc. All rights reserved.
R
esearch at the interface of chemistry and biology requires chemical tools for the selective modification of biomolecules to visualize, quantify or selectively isolate them from complex mixtures or even living systems. This raises the question how one can selectively and under mild conditions introduce tailor-made chemical reporter tags in proteins, a prominent class of biomolecules. Many of the existing strategies rely on sitespecific introduction of a bioorthogonal reactive group, followed by conjugation with a reporter tag via the complementary reactive group. One of the most powerful approaches consists of site-specific introduction of an azide-containing amino acid1, followed by azide-alkyne cycloaddition to form the corresponding conjugated triazole-based system2. Other approaches involve chemical modification of the N terminus, for example, oxidative cleavage of an N-terminal serine, followed by selective reaction of the resulting glyoxylic amide derivative. Examples include Pictet-Spengler–type conjugation3 and nitrone formation followed by nitrone-alkyne cycloaddition4. MacDonald et al.5 have now taken the N-terminal modification of peptides a major step further by showing that native proteins can be selectively reacted at the N terminus using 2-pyridinecarboxyaldehyde (2-PCA) derivatives. Even in the presence of multiple lysine residues, 2-PCA residues react with high selectivity at the N-terminal amino acid, facilitating the single-step introduction of desired functional tags in a variety of proteins. Selective functionalization at the N terminus of native proteins, i.e., selective reaction at the N-terminal amino group in the presence of multiple lysine residues, is generally possible owing to the fact that the amino group at the N terminus is slightly less basic (pK value ~8) than the lysine amino groups (pK value ~10). Hence, by proper adjustment of the pH, the free amino group at the N terminus will be more nucleophilic than the protonated lysine side chains and thus may, for example, be selectively acylated in reaction with activated esters.
Chan et al.6 published a recent improvement of this protocol involving the use of ketenes as the electrophile, leading to a higher selectivity than that in the protocol using activated esters. This method, however, may still be sensitive to subtle changes in pH. In this report, MacDonald et al.5 discovered a reaction to selectively functionalize the N terminus, which to some extent is reminiscent of the first steps of the Edman degradation7. In this powerful sequencing reaction (Fig. 1a), the N-terminal amino group reacts with phenylisothiocyanate (the Edman reagent), followed by participation of the adjacent amide group and subsequent cleavage of the N-terminal amino acid. Lysine residues may react with the isothiocyanate but will not be cleaved, owing to the absence of the participating amide bond. As in the Edman degradation sequence, the initial 2-PCA–protein adduct reacts, probably aided by the pyridine substituent, intramolecularly with the adjacent amide group to form a stable cyclic aminal, thereby driving the equilibrium of the first step to completion (Fig. 1b). As ring formation does not generate a reactive tertiary alcohol, as in the Edman degradation, the reaction does not lead to a cleaved residue. Additionally, because this second reaction cannot occur in the lysine a Ph R
C
R
S
Edman reagent
H N
H2N
N
Peptide
Ph
O
Peptide
H N
HN
O
S
H+
R
H N
HN N H
side chains, the reaction is intrinsically selective for the N-terminal amino group. The authors have shown that the conjugation with 2-PCA works for all amino acids, although glycine and proline give somewhat lower yields. It was also shown that by blocking the nucleophilicity of the second amino acid (for example, by incorporation of proline), conjugation was prevented. Furthermore, RNase A was used as a model protein to show the feasibility of conjugation with 2-PCA under many different conditions; 2-PCA conjugation was also demonstrated on a range of different proteins, confirming the wide applicability of the chemistry. Perhaps more importantly, conjugation with RNase A was also successful for modified 2-PCA molecules, allowing the site-selective introduction of functional groups such as a PEG chain or a biotin label; other groups such as fluorescent dyes, MRI labels or affinity tags could also be incorporated through this strategy. The authors demonstrated the potential utility of the new chemistry in applied settings through N-terminal biotinylation of the estrogen receptor α (ERα′), which was then immobilized on a streptavidin-containing resin to detect endocrine-disrupting compounds in drinking water.
OH
S Ph
H+
H2N
Peptide
R
N T R
H N
H2N O
N S
O
Ph
O H+
2-PCA reagent Peptide
O
HN
N
H
b
Peptide +
R
H N
N N
O
R Peptide
N
Peptide
HN N
T T
Figure 1 | Basis of the selectivity for the N terminus of proteins by participation of the adjacent amide substituent. (a) N-terminal conjugation with phenylisothiocyanate in Edman degradation. (b) N-terminal conjugation with 2-PCA.
nature chemical biology | Advance online publication | www.nature.com/naturechemicalbiology
1
news & views to mixtures of proteins. Notwithstanding, the contribution of MacDonald et al.5 represents a robust method for single-step N-terminal functionalization of many proteins and is flexible in the functionality that is to be coupled to the protein of interest and therefore may well turn into a widely used conjugation approach in the near future. ■ Floris P. J. T. Rutjes is at Radboud University, Institute for Molecules and Materials, Nijmegen, the Netherlands. e-mail: [email protected]
Published online 30 March 2015 doi:10.1038/nchembio.1791 References
1. Kiick, K.L. et al. Proc. Natl. Acad. Sci. USA 99, 19–24 (2002). 2. Debets, M.F. et al. Org. Biomol. Chem. 11, 6439–6455 (2013). 3. Agarwal, P. et al. Proc. Natl. Acad. Sci. USA 110, 46–51 (2013). 4. Ning, X. et al. Angew. Chem. Int. Ed. Engl. 49, 3065–3068 (2010). 5. MacDonald, J.I. et al. Nat. Chem. Biol. doi:10.1038/nchembio.1792 (30 March 2015). 6. Chan, A.O.-Y. et al. J. Am. Chem. Soc. 134, 2589–2598 (2012). 7. Edman, P. Acta Chem. Scand. 4, 283–293 (1950).
Competing financial interests The author declares no competing financial interests.
npg
© 2015 Nature America, Inc. All rights reserved.
It is only recently that chemists have more systematically started to develop molecular tools to unravel complex mechanisms in biological systems. Over the years, a toolbox has been developed that allows the sitespecific introduction of functional groups in biomolecules. However, numerous challenges still remain. Despite the promises that the 2-PCA molecules may hold, they certainly also have their limitations. This conjugation strategy will most likely only work outside the cell and not be applicable inside cellular systems. The method works well on a single protein but will not be selective when applied
2
nature chemical biology | Advance online publication | www.nature.com/naturechemicalbiology
