Journal of the American Chemical Society
Communication
microtubule labeling was confirmed by two-color overlay21 in
which the microtubules were immunostained using an anti-α-
tubulin antibody (Figure S22 (SI)). It is noteworthy that the in
situ formed pyrazoline-docetaxel gave higher efficiency in
labeling microtubules than treating cells with the preformed
pyrazoline-docetaxel at the identical concentration (Figure S23
(SI)), presumably due to poorer permeability of the pyrazoline-
docetaxel, indicating a unique advantage for our two-step in situ
labeling procedure.
(5) (a) Lim, R. K.; Lin, Q. Acc. Chem. Res. 2011, 44, 828−839.
(b) Wang, Y.; Hu, W. J.; Song, W.; Lim, R. K.; Lin, Q. Org. Lett. 2008,
10, 3725−3728. (c) Yu, Z.; Ho, L. Y.; Wang, Z.; Lin, Q. Bioorg. Med.
Chem. Lett. 2011, 21, 5033−5036.
(6) McClure, D. S. J. Chem. Phys. 1954, 22, 1668−1675.
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Biol. 2007, 9, 7−14. (b) Kim, H. M.; Kim, B. R.; Choo, H.-J.; Ko, Y.-
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2830−2838. (c) Kim, H. M.; Cho, B. R. Acc. Chem. Res. 2009, 42,
863−872.
In summary, we have developed a new type of two-photon-
triggered chemistry in which the two-photon generated, highly
reactive intermediates undergo spontaneous and specific 1,3-
dipolar cycloaddition reactions with the suitable dipolarophiles
to generate in situ the fluorescent cycloadducts. This
naphthalene-tetrazole mediated two-photon reaction showed
higher two-photon reaction cross-sections than the two-photon
uncaging action cross-sections for commonly used two-photon
caging groups.22 Moreover, the utilities of this two-photon-
triggered, fluorogenic photoclick chemistry in labeling an
alkene-encoded protein site-selectively in vitro and ligand-
bound microtubules in cultured cells were demonstrated. Since
the genetic methods to encode the unnatural amino acids
containing reactive alkene dipolarophiles have been reported
for both cultured mammalian cells23 and Drosophila mela-
nogaster fruit fly,24 we expect this naphthalene-tetrazole-based
two-photon chemistry to offer a new tool to dissect protein
function in living organisms with a high spatiotemporal
precision.
(8) (a) Zheng, S. L.; Wang, Y.; Yu, Z.; Lin, Q.; Coppens, P. J. Am.
́ ́
Chem. Soc. 2009, 131, 18036−18037. (b) Begue, D.; Qiao, G. G.;
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5438.
(10) See Table S1 (SI) for photophysical characterization including
fluorescence turn-on ratios for all the pyrazolines.
(11) The following equation, δcT = δurCrNP/CTNr, was used, where Cr
and CT are the initial concentrations of the reference compound
(BHC-OAc) and tetrazole, respectively, NP is the number of
pyrazoline molecules formed per unit time, and Nr is the number of
uncaged molecules per unit time. The literature value of the uncaging
action cross-section of BHC-OAc, δur = 0.95 GM, was used in the
calculation. See Figure S9 (SI) for details.
(12) (a) Lee, Y. J.; Wu, B.; Raymond, J. E.; Zeng, Y.; Fang, X.;
Wooley, K. L.; Liu, W. R. ACS Chem. Biol. 2013, 8, 1664−1670. (b) Li,
F.; Zhang, H.; Sun, Y.; Pan, Y.; Zhou, J.; Wang, J. Angew. Chem., Int.
Ed. 2013, 52, 9700−9704.
(13) While tetrazoles 5 and 6 show similar two photon cycloaddition
cross-sections (4.0 GM for 5 and 3.8 GM for 6), we selected tetrazole
6 in our in vivo studies because the pyrazoline cycloadduct derived
from tetrazole 6 was found to be far more resistant to photobleaching
in the fluorescence microscopic studies compared to the pyrazoline
cycloadduct derived from tetrazole 5.
ASSOCIATED CONTENT
■
S
* Supporting Information
(14) Yu, Z.; Ho, L. Y.; Lin, Q. J. Am. Chem. Soc. 2011, 133, 11912−
11915.
Supplemental figures and table, synthetic schemes, experimen-
tal procedures, and characterization of all new compounds. This
material is available free of charge via the Internet at http://
(15) (a) Miller, M. L.; Roller, E. E.; Zhao, R. Y.; Leece, B. A.; Ab, O.;
Baloglu, E.; Goldmacher, V. S.; Chari, R. V. J. J. Med. Chem. 2004, 47,
4802−4805. (b) Matesanz, R.; Rodríguez-Salarichs, J.; Pera, B.;
́
Canales, A.; Andreu, J. M.; Jimenez-Barbero, J.; Bras, W.; Nogales, A.;
Fang, W.-S.; Díaz, J. F. Biophys. J. 2011, 101, 2970−2980.
(16) Zhuang, Y. D.; Chiang, P. Y.; Wang, C. W.; Tan, K. T. Angew.
Chem., Int. Ed. 2013, 52, 8124−8128.
AUTHOR INFORMATION
■
Corresponding Author
(17) Tetrazole 6 and IPFAD showed no cytotoxicity toward CHO
cells at concentrations ≤100 μM; see Figure S19 (SI) for details.
(18) The cytosolic fluorescence outside the rectangle boundary is due
to rapid diffusion of the in situ generated nitrile imine intermediate
within the cytosolic space of the individual irradiated cells.
(19) The decrease in fluorescence intensity after 43 s is due to
photobleaching of the pyrazoline fluorophore at 405 nm excitation; see
Figure S20 (SI) for details.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the National Institutes of Health
(GM 085092) for financial support. We thank Dr. Guang S. He
at ILPB for helpful discussions, Alan Siegel at SUNY Buffalo
Biological Sciences Imaging Facility (supported by the National
Science Foundation Major Research Instrumentation Grant
DBI-0923133) for assistance with microscopy, and Dr. Wenshe
Liu at Texas A&M University for generously providing us the
plasmids pEvol-PylT-PylKRS, pEvol-AcrKRS, and pET-
sfGFPS2TAG.
(20) Song, W.; Wang, Y.; Yu, Z.; Vera, C. I.; Qu, J.; Lin, Q. ACS
Chem. Biol. 2010, 5, 875−885.
(21) The Pearson’s correlation coefficient was determined to be 0.66.
The reaction inside the cells was triggered by a hand-held 365 nm UV
lamp because the subsequent fixation and immunostaining steps make
it difficult to retrack the femtosecond 700 nm laser beam scanned area.
(22) Davis, M. J.; Kragor, C. H.; Reddie, K. G.; Wilson, H. C.; Zhu,
Y.; Dore, T. M. J. Org. Chem. 2009, 74, 1721−1729.
(23) Yu, Z.; Pan, Y.; Wang, Z.; Wang, J.; Lin, Q. Angew. Chem., Int.
Ed. 2012, 51, 10600−10604.
(24) Bianco, A.; Townsley, F. M.; Greiss, S.; Lang, K.; Chin, J. W.
Nat. Chem. Biol. 2012, 8, 748−750.
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