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ChemComm
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COMMUNICATION
Journal Name
formylated enzymes 10f and 10i is conserved and highlights be distinguished through a chemical transformation to provide
DOI: 10.1039/C6CC09555K
the mild nature of reaction conditions (ESI Fig. S37 and S38
†).
a platform for site-selective labeling. The protocol renders
access to single site formylation of ε-amine in native proteins,
a recently identified post-translational modification.17 It is
noteworthy that the aldehyde auto-oxidation generates
formylating pre-reagent in physiological conditions. Contrary
to the general belief, the free radicals involved in the process
demonstrate mild nature and do not promote alternative
reaction pathways. The site-selective reversible protection of
Nα-NH2, regulation of formate generation and effective Nɛ-NH2
concentration serves as the key to one-pot site-selective Nε-
NH2 labeling. Even though a complex combination of multiple
parameters is involved, operationally simple front-end
protocol would also excite the non-experts. The
transformation is efficient in the modification of peptides and
structurally diverse proteins with a wide range of Lys residues
(3-19). Interception of formylation of protein by a distinct
electrophile confirms extended application of this chemical
platform. We believe that the demonstration of unique
reactivity and kinetic selectivity of protein side chain residue
delivers a breakthrough that will fuel the ongoing efforts for
site-selective modification of native proteins.
Fig. 5 Site-selective native protein modification enabled by
auto-oxidation of aldehydes.
In the pursuit to examine the generality of Nɛ-NH2 labeling
protocol, we designed an experiment for interception of
formylation by an acylating reagent (Fig. 6). It was exciting to
note that appropriate selection of electrophile 11 leads to site-
selective and homogeneous acylation of RNase A. There is no
indication of formylation or oxidative pathway under the
optimized conditions. The labeled protein K37-Nɛ-COR1-RNase
A (10m) is primed with maleimide and offers a bio-orthogonal
site for late-stage modification with the desired tag of interest.
For example, the reaction of 10m with 7-mercapto-4-
methylcoumarin 12 delivers 13 efficiently. To gain insight to
acylation, we performed the reaction in the absence of
aldehyde 1a, keeping the other parameters constant (ESI Fig.
Notes and references
1
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8
2
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C. D. Spicer and B. G. Davis, Nat. Commun., 2014,
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(a) J. I. MacDonald, H. K. Munch, T. Moore and M. B. Francis,
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(a) R. Singudas, S. R. Adusumalli, P. N. Joshi and V. Rai, Chem.
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Pfisterer, B. Neumann and T. Weil, Bioconjugate Chem.,
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Leung, J.-S. Huang, M.-K. Wong and C.-M. Che, J. Am. Chem.
Soc., 2012, 134, 2589.
6
7
S9†). It results in a mixture of mono-, bis- and tris-labeled
products. We expect the labeling of Nα-NH2 and K37-Nɛ-NH2 in
the acylation. However, the labeling of an additional Nɛ-NH2
suggests that the reduced effective concentration of free Lys
residues, ascribed to imine formation in the presence of
aldehyde 1a, has a role in regulating the site-selectivity.
8
9
J. C. Gildersleeve, O. Oyelaran, J. T. Simpson and B. Allred,
Bioconjugate Chem., 2008, 19, 1485.
K. M. Lum, V. J. Xavier,M. J.-H. Ong, C. W. Johannes and K.-P.
Chan, Chem. Commun., 2013, 49, 11188.
10 P. Agarwal, J. van der Weijden, E. M. Sletten, D. Rabuka and
C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 46.
11 M. Raj, H. Wu, S. L. Blosser, M. A. Vittoria and P. S. Arora, J.
Am. Chem. Soc., 2015, 137, 6932.
12 L. S. Witus, C. Netirojjanakul, K. S. Palla, E. M. Muehl, C.
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13 J. R. McNesby and Jr. C. A. Heller, Chem. Rev., 1954, 54, 325.
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Fig.
6
Interception of formylation renders site-selective
) and offers late-stage modification.
18 P. N. Joshi, P. Landa, N. K. Das, S. Mukherjee and V. Rai, RSC
acylation (ESI Fig. S10
†
Adv., 2016, 6, 208.
In summary, we have successfully demonstrated that a subtle
difference in reactivity of native protein backbone residues can
19 T. Jiang, X. Zhou, K. Taghizadeh, M. Dong and P. C. Dedon,
Proc. Natl. Acad. Sci. U.S.A., 2007, 104, 60.
4 | J. Name., 2012, 00, 1-3
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