After 1 h reaction, label 15a was added to the reaction mixture
along with a catalytic quantity of Cu(I), and the mixture was then
stirred overnight. The mixture was then subjected to size
exclusion chromatography using Sephadex G-25 to remove
unreacted linkers and labels from tagged HSA.
References and notes
1. Lin, Y.; Weissleder, R.; Tung, C. H. Bioconjugate Chem. 2002,
1
3, 605.
2
3
4
.
.
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Cserép, G. B.; Enyedi, K. N.; Demeter, A.; Mező, G.; Kele, P.
Chem. Asian J. 2013, 8, 494.
Nagy, K.; Orbán, E.; Bősze, Sz.; Kele, P. Chem. Asian J. 2010, 5,
The excitation and emission spectra of the free dye and the
fluorescently modified HAS are shown in Figure 2. The spectra
show substantial differences: a) the emission maximum of the
labeled HSA is shortwave-shifted by 40 nm relative to that of the
free dye (from 714 nm to 673 nm); b) the excitation maxima
remained the same (564 nm), however, the shape of the band is
significantly altered. Such changes are in accordance with the
rules of solvatochromism and suggest that the overall
microenvironment of the label on the Tyr is less polar than the
bulk of the buffer.
7
73.
Link, M; Kele, P.; Achatz, D. E.; Wolfbeis, O. S. Bioorg. Med.
Chem. Lett. 2011, 21, 5538.
5. Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13.
6. Debets, M. F.; van Berkel, S. S.; Dommerholt, J.; Dirks, A. T. J.;
Rutjes, F. P. J. T.; van Delft, F. L. Acc. Chem. Res. 2011, 44, 805.
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.
Sletten, E. M.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48,
974.
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Varga, B. R.; Kállay, M.; Hegyi, K.; Béni, Sz.; Kele, P. Chem.
Eur. J. 2012, 18, 822.
9. Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666.
1
0. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
In order to exclude non-selective physical adsorption of the
fluorophore on the surface of the protein, we also performed the
experiments either without the chemical reporter, the Pd(II)
catalyst or the Cu(I) catalyst. In none of these cases did we
observe the formation of fluorescent HSA. This indicates that
covalent binding of the chemical linkers and subsequent click
labeling are mandatory for successful tagging of the Tyr residues
of HSA.
Angew. Chem. Int. Ed. 2002, 41, 2596.
11. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
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1
2. Hermanson, G. T. Bioconjugate Techniques, 1st ed., Academic
Press: San Diego, CA, 1996.
3. Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc. 2006, 128, 1080.
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6. Kele, P.; Mező, G.; Achatz, D.; Wolfbeis, O. S. Angew. Chem. Int.
Ed. 2009, 48, 344.
1
1
In conclusion, we have shown that the use of our new linkers
and appropriately functionalized fluorescent labels with a large
Stokes shift enables specific labeling of proteins directly at Tyr
units. The sequential tagging scheme reported here combines the
selectivity of the Pd(II) catalyzed reaction between an aromatic
hydroxy group and allyloxy acetates with the robustness of the
Cu(I) catalyzed azide-alkyne dipolar cycloaddition reaction. We
beleive that (a) the method is likely to be applicable to various
kinds of azido / alkyne-modified fluorophores, and that (b) the
Pd(II)-catalyzed modification of the tyrosine hydroxy group may
also enable the introduction of other kinds of tags, e.g. biotin or
nonfluorescent labels, provided that they carry a complementary
function.
1
7. Achatz, D. E.; Mező, G.; Kele, P.; Wolfbeis, O. S. ChemBioChem
2
009, 10, 2316.
1
1
8. Jewett, J. C.; Bertozzi, C. R. Chem. Soc. Rev. 2010, 39, 1272.
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1. b) Nadler, A.; Schultz, C. Angew. Chem. Int. Ed. 2013, 452, 2408.
2. Herner, A.; Nikić, I.; Kállay, M.; Lemke, E. A.; Kele, P. Org.
Biomol. Chem. 2013, 11, 3297.
3. At temperatures above 60 °C the allyloxy-acetate moiety
decomposes and mainly forms a diene product (according to NMR
and HRMS), which is in accordance with Tilley’s observations.
4. Lemieux, G. A.; de Graffenried, C. L; Bertozzi C. R. J. Am. Chem.
Soc. 2003, 125, 4708.
2
2
2
2
Supplementary Material
Supplementary data
(experimental
procedures
and
characterization data for all new compounds associated with this
article can be found, in the online version, at http://dx.doi.org/
Figure 2. Excitation and emission spectra of the free dye 15a (grey) and the
labeled HSA protein (black) in water.
Acknowledgments
Financial support of the Hungarian Scientific Research Fund
(
“
(
OTKA, grant numbers K-100134 and NN-110214) and the
Lendület” Program of the Hungarian Academy of Sciences
LP2013-55/2013) is greatly acknowledged.