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Notes and references
1 (a) J. M. McFarland and M. B. Francis, J. Am. Chem. Soc., 2005,
127, 13490; (b) J. M. McFarland, N. S. Joshi and M. B. Francis, J. Am.
Chem. Soc., 2008, 130, 7639; (c) M. J. Han, D. C. Xiong and X. S. Ye,
Chem. Commun., 2012, 48, 11079; (d) P. Agarwal, R. Kudirka,
A. E. Albers, R. M. Barfield, G. W. de Hart, P. M. Drake, L. C. Jones
and D. Rabuka, Bioconjugate Chem., 2013, 24, 846; (e) 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; ( f ) L. S. Witus, C. Netirojjanakul,
K. S. Palla, E. M. Muehl, C. H. Weng, A. T. Iavarone and M. B. Francis,
J. Am. Chem. Soc., 2013, 135, 17223.
2 (a) I. I. Geschwind and C. Hao Li, Biochim. Biophys. Acta, 1954, 15, 442;
(b) I. S. Carrico, B. L. Carlson and C. R. Bertozzi, Nat. Chem. Biol., 2007,
3, 321; (c) J. S. Rush and C. R. Bertozzi, J. Am. Chem. Soc., 2008, 130, 12240;
(d) P. Wu, W. Shui, B. L. Carlson, N. Hu, D. Rabuka, J. Lee and C. R.
Bertozzi, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 3000; (e) J. E.
Hudak, H. H. Yu and C. R. Bertozzi, J. Am. Chem. Soc., 2011, 133, 16127;
( f ) O. El-Mahdi and O. Melnyk, Bioconjugate Chem., 2013, 24, 735.
3 (a) L. Wang, Z. Zhang, A. Brock and P. G. Schultz, Proc. Natl. Acad. Sci.
U. S. A., 2003, 100, 56; (b) Z. Zhang, B. A. Smith, L. Wang, A. Brock,
C. Cho and P. G. Schultz, Biochemistry, 2003, 42, 6735; (c) Y. Huang,
W. Wan, W. K. Russell, P. J. Pai, Z. Wang, D. H. Russell and W. Liu,
Bioorg. Med. Chem. Lett., 2010, 20, 878; (d) B. Wu, Z. Wang, Y. Huang
and W. R. Liu, ChemBioChem, 2012, 13, 1405; (e) H. Zeng, J. Xie and
P. G. Schultz, Bioorg. Med. Chem. Lett., 2006, 16, 5356.
Fig. 4 Selective labelling of E. coli cells expressing OmpX-1 and OmpX-2.
The labelling was carried out in the presence of 100 mM aniline and at pH 7
for 1 h. The top panel shows bright field imaging of E. coli cells, the middle
panel shows green fluorescent imaging of same cells, and the bottom
panel shows their composite images (scale bars are 10 mm).
4 (a) A. Dirksen, T. M. Hackeng and P. E. Dawson, Angew. Chem., Int.
Ed., 2006, 45, 7581; (b) A. Dirksen and P. E. Dawson, Bioconjugate
Chem., 2008, 19, 2543; (c) Y. Zeng, T. N. Ramya, A. Dirksen,
P. E. Dawson and J. C. Paulson, Nat. Methods, 2009, 6, 207;
(d) M. Wendeler, L. Grinberg, X. Wang, P. E. Dawson and M. Baca,
Bioconjugate Chem., 2014, 25, 93.
5 (a) Y. S. Wang, W. K. Russell, Z. Wang, W. Wan, L. E. Dodd, P. J. Pai,
D. H. Russell and W. R. Liu, Mol. Biosyst., 2011, 7, 714; (b) Y. S. Wang,
X. Fang, A. L. Wallace, B. Wu and W. R. Liu, J. Am. Chem. Soc., 2012,
134, 2950; (c) Y. S. Wang, X. Fang, H. Y. Chen, B. Wu, Z. U. Wang,
C. Hilty and W. R. Liu, ACS Chem. Biol., 2013, 8, 405; (d) J. M. Tharp,
Y. S. Wang, Y. J. Lee, Y. Yang and W. R. Liu, ACS Chem. Biol., 2014,
9, 884; (e) A. Tuley, Y. S. Wang, X. Fang, Y. Kurra, Y. H. Rezenom and
W. R. Liu, Chem. Commun., 2014, 50, 2673.
and labelled with 4 in the presence of 100 mM aniline and at
pH 7 for 1 h. After labelling, cells were washed with PBS buffer
six times to remove the residual dye and then imaged by
fluorescent microscopy. Cells grown in the absence of 1 or 2
were used as a control. As shown in Fig. 4, 4 specifically labelled
cells expressing OmpX-1 and OmpX-2 were observed, but not
for cells in the control experiment. Although cells expressing
OmpX-2 were fluorescently labelled, their intensities are weaker
than those of cells expressing OmpX-1, indicating a slower
labelling kinetics of OmpX-2. However, we have found that this
concentration of aniline is toxic to E. coli, which underscores
the need for developing less toxic catalysts for this reaction.
In summary, we reported the genetic incorporation of a
readily synthesized aldehyde-containing NCAA and demon-
strated its fast labelling kinetics with hydroxylamine dyes in
the presence of the aniline catalyst. This rapid labelling
approach was also successfully applied to label a membrane
protein on the E. coli extracellular surface. Although genetically
encoded ketone-containing NCAAs were reported previously,
labelling of proteins with these NCAAs typically suffers low
labelling efficiencies, attributing to slow labelling kinetics of
ketones. This work resolves this obstacle. Given that a large
variety of hydroxylamine and hydrazine dyes are commercially
available, as well as the existence of numerous aldehyde-based
bioconjugation strategies,9 we anticipate that this approach will
be quickly adopted by others for studies such as protein
folding/dynamics, protein–ligand interactions, high through-
put drug discovery, etc.
6 S. C. Reddington, E. M. Tippmann and D. D. Jones, Chem. Commun.,
2012, 48, 8419.
7 J. D. Pedelacq, S. Cabantous, T. Tran, T. C. Terwilliger and
G. S. Waldo, Nat. Biotechnol., 2006, 24, 79.
8 (a) X. S. Wang, Y. J. Lee and W. R. Liu, Chem. Commun., 2014,
50, 3176; (b) Y. J. Lee, B. Wu, J. E. Raymond, Y. Zeng, X. Fang,
K. L. Wooley and W. R. Liu, ACS Chem. Biol., 2013, 8, 1664; (c) K. Lang,
L. Davis, S. Wallace, M. Mahesh, D. J. Cox, M. L. Blackman, J. M. Fox
and J. W. Chin, J. Am. Chem. Soc., 2012, 134, 10317; (d) A. Borrmann,
S. Milles, T. Plass, J. Dommerholt, J. M. Verkade, M. Wiessler,
C. Schultz, J. C. van Hest, F. L. van Delft and E. A. Lemke, ChemBio-
Chem, 2012, 13, 2094; (e) T. Plass, S. Milles, C. Koehler, J. Szymanski,
R. Mueller, M. Wiessler, C. Schultz and E. A. Lemke, Angew. Chem.,
Int. Ed., 2012, 51, 4166; ( f ) M. L. Blackman, M. Royzen and J. M. Fox,
J. Am. Chem. Soc., 2008, 130, 13518.
9 (a) G. R. Lorello, M. C. Legault, B. Rakic, K. Bisgaard and J. P. Pezacki,
Bioorg. Chem., 2008, 36, 105; (b) J. Y. Byeon, F. T. Limpoco and
R. C. Bailey, Langmuir, 2010, 26, 15430; (c) D. A. Ossipov, X. Yang,
O. Varghese, S. Kootala and J. Hilborn, Chem. Commun., 2010, 46, 8368;
(d) V. Raindlova, R. Pohl, M. Sanda and M. Hocek, Angew. Chem., Int.
Ed., 2010, 49, 1064; (e) B. H. Rotstein, V. Rai, R. Hili and A. K. Yudin, Nat.
Protoc., 2010, 5, 1813; ( f ) G. Iyer, F. Pinaud, J. Xu, Y. Ebenstein, J. Li,
J. Chang, M. Dahan and S. Weiss, Bioconjugate Chem., 2011, 22, 1006;
(g) J. D. Cohen, P. Zou and A. Y. Ting, ChemBioChem, 2012, 13, 888;
(h) P. Crisalli, A. R. Hernandez and E. T. Kool, Bioconjugate Chem., 2012,
23, 1969; (i) V. Raindlova, R. Pohl and M. Hocek, Chem. – Eur. J., 2012,
18, 4080; ( j) P. K. Dhal, S. C. Polomoscanik, D. A. Gianolio,
P. G. Starremans, M. Busch, K. Alving, B. Chen and R. J. Miller,
Bioconjugate Chem., 2013, 24, 865; (k) D. Ossipov, S. Kootala, Z. Yi,
X. Yang and J. Hilborn, Macromolecules, 2013, 46, 4105; (l) K. Tanaka,
Y. Nakamoto, E. R. Siwu, A. R. Pradipta, K. Morimoto, T. Fujiwara,
S. Yoshida, T. Hosoya, Y. Tamura, G. Hirai, M. Sodeoka and K. Fukase,
Org. Biomol. Chem., 2013, 11, 7326; (m) S. Wang, O. P. Oommen, H. Yan
and O. P. Varghese, Biomacromolecules, 2013, 14, 2427.
We are thankful for the financial support from the National
Institute of Health (grant 1R01CA161158), the National Science
Foundation (grant CHE-1148684), and the Welch Foundation
(grant A-1715). We also thank Dr Yohannes H. Rezenom from
the Laboratory for Biological Mass Spectrometry at Texas A&M
University for characterizing our proteins with electrospray
ionization mass spectrometry.
7426 | Chem. Commun., 2014, 50, 7424--7426
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