A fluorogenic 1,3-dipolar cycloaddition reaction of 3-azidocoumarins and acetylenes

Article abstract of DOI:10.1021/ol047955x

(Chemical equation presented) Copper(I)-catalyzed 1,3-dipolar cycloaddition reaction of nonfluorescent 3-azidocoumarins and terminal alkynes afforded intense fluorescent 1,2,3-triazole products. The mild condition of this reaction allowed us to construct a large library of pure fluorescent coumarin dyes. Since both azide and alkyne are quite inert to biological systems, this reaction has potential in bioconjugation and bioimaging applications.

Full text of DOI:10.1021/ol047955x

ORGANIC
LETTERS
2004
Vol. 6, No. 24
4603-4606
A Fluorogenic 1,3-Dipolar Cycloaddition
Reaction of 3-Azidocoumarins and
Acetylenes†
Krishnamoorthy Sivakumar, Fang Xie, Brandon M. Cash, Su Long,
Hannah N. Barnhill, and Qian Wang*
Department of Chemistry & Biochemistry, UniVersity of South Carolina,
631 Sumter Street, Columbia, South Carolina 29208
wang@mail.chem.sc.edu
Received October 5, 2004
ABSTRACT
Copper(I)-catalyzed 1,3-dipolar cycloaddition reaction of nonfluorescent 3-azidocoumarins and terminal alkynes afforded intense fluorescent
1,2,3-triazole products. The mild condition of this reaction allowed us to construct a large library of pure fluorescent coumarin dyes. Since
both azide and alkyne are quite inert to biological systems, this reaction has potential in bioconjugation and bioimaging applications.
Bioconjugation has recently emerged as a fast-growing
technology that affects almost every discipline of life science.
It aims at the ligation of two or more molecules (or
supramolecules) to form a new complex with the combined
properties of its individual components.1 One important
application of bioconjugation is to modify cellular compo-
nents selectively with signaling probes for proteomics,
functional genomics, and cell biology research.2 A multistep
procedure is commonly employed wherein the cellular entity
is first attached with a detectable tag, such as fluorescent
dyes or biotin, followed by purification of the ligated product
and detection. However, excess prelabeled reagents are
generally hard to remove from the intracellular environment
or from tissues of living organisms, which prohibits the
application of a multistep labeling procedure in many
situations. An ideal alternative would be a chemoselective
process that is orthogonal to biological components and the
ligated product will afford strong detectable signal while the
unbound reagent does not contribute any background. Very
few such ligations have been reported.3,4
As a prototype of “click chemistry”,5 the recent advance
of Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of
azides and alkynes affords superior regioselectivity and
almost quantitative transformation under extremely mild
conditions.6 Alkyne and azide groups are very small in size,
are highly energetic, and have a particularly narrow distribu-
tion of reactivity. They can be conveniently introduced to
organic compounds and are quite indifferent to solvent and
pH. Therefore, they have been employed as a pair of
(3) Gaietta, G.; Deerinck, T. J.; Adams, S. R.; Bouwer, J.; Tour, O.;
Laird, D. W.; Sosinsky, G. E.; Tsien, R. Y.; Ellisman, M. H. Science 2002,
296, 503-507.
(4) (a) Lemieux, G. A.; De Graffenried, C. L.; Bertozzi, C. R. J. Am.
Chem. Soc. 2003, 125, 4708-4709. (b) Saxon, E.; Bertozzi, C. R. Science
2000, 287, 2007-2010.
(5) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2001, 40, 2004-2021. (b) Kolb, H. C.; Sharpless, K. B. Drug DiscoV.
Today 2003, 8, 1128-1137. (c) Lewis, W. G.; Green, L. G.; Grynszpan,
F.; Radic, Z.; Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 1053-1057.
(6) (a) Rostovtsev, V. V.; Green, L. G.; Folkin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596-2599. (b) Tornoe, C. W.;
Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057-3062.
† This paper is dedicated to Professor Manfred Schlosser on the occasion
of his 70th birthday.
(1) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San
Diego, 1996.
(2) (a) Choy, G.; Choyke, P.; Libutti, S. K. Mol. Imaging 2003, 2, 303-
312. (b) Johnsson, N.; Johnsson, K. ChemBioChem 2003, 4, 803-810. (c)
Cravatt, B. F.; Sorensen, E. J. Curr. Opin. Chem. Biol. 2000, 4, 663-668.
10.1021/ol047955x CCC: $27.50
© 2004 American Chemical Society
Published on Web 11/04/2004

orthogonal linkers for chemoselective ligations through a Cu-
(I)-mediated cycloaddition reaction by many research groups.7
Here, we report a “clicking-and-probing” ligation protocol,
a fluorogenic version of 1,3-dipolar cycloaddition of azides
and alkynes, which has potential in covalently linking any
two biomolecules or supramolecular complexes. The reaction
design is schematically illustrated in Figure 1. The fluorescent
cycloaddition products A1 at room temperature quantita-
tively. A1 fluoresces at 478 nm due to the elimination of
the quenching through the formation of the triazole ring. The
quantum yield of A1 is 0.30 in comparison to 0.0 of the
starting materials A. This result encouraged us to further
explore the impact of the structural features of azidocou-
marins and alkynes to the fluorescent properties of the
products.
As shown in Figure 2, eight 3-azidocoumarins and one
4-azidocoumarin were synthesized in addition to twenty-four
Figure 1. Schematic illustration of the “clicking-and-probing
ligation”. Profluorophores (or “prelinkers”) containing azide or
alkyne moieties are fluorescent inactive, which upon cycloaddition
would lead to the formation of triazolyl units with enhanced
fluorescent emissions.
signals will be triggered by the formation of triazole rings,
which have the potential to be used as probes for imaging
or as reporters to monitor the ligation efficiency.
Coumarin was chosen as the profluorophore since it is
small in size, biocompatible, and easy to manipulate syn-
thetically. Substitutions at the 3- and 7-positions of coumarin
dyes are known to have a strong impact on their fluorescence
properties.8 Compound A was first synthesized with an azido
group attached to the 3-position (Scheme 1). As we expected,
Scheme 1
A showed no fluorescence due to the quenching effect from
the electron-rich R-nitrogen of the azido group. With catalytic
Cu(I), the azide reacted smoothly with phenylacetylene (1)
in a mixed solution of ethanol and water (1:1) to afford
(7) (a) Wang, Q.; Chan, T.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.;
Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193. (b) Speers, A. E.;
Adam, G. C.; Cravatt, B. F. J. Am. Chem. Soc. 2003, 125, 4686-4687. (c)
Seo, T. S.; Li, Z.; Ruparel, H.; Ju, J. J. Org. Chem. 2003, 68, 609-612. (d)
Lee, L. V.; Mitchell, M. L.; Huang, S. J.; Fokin, V. V.; Sharpless, K. B.;
Wong, C. H. J. Am. Chem. Soc. 2003, 125, 9588-9589. (e) Link, A. J.;
Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 11164-11165. (f) Deiters, A.;
Cropp, T. A.; Mukherji, M.; Chin, J. W.; Anderson, J. C.; Schultz, P. G. J.
Am. Chem. Soc. 2003, 125, 11782-11783. (g) For a review, see:
Breinbauer, R.; Kohn, M. ChemBioChem 2003, 4, 1147-1149.
(8) (a) Schiedel, M. S.; Briehn, C. A.; Bauerle, P. Angew. Chem., Int.
Ed. 2001, 40, 4677-4680. (b) Yee, D. J.; Balsanek, V.; Sames, D. J. Am.
Chem. Soc. 2004, 126, 2282-2283.
Figure 2. Building blocks for triazolylcoumarin dyes.
alkynes (1-24) which were purchased or synthesized for
our study. The synthesis of 3-azidocoumarins was quite
straightforward starting from the respecting substituted
salicylaldehyde and N-acetylglycine or ethyl nitroacetate
4604
Org. Lett., Vol. 6, No. 24, 2004

Scheme 2. Typical Synthetic Procedures of 3-Azidocoumarins
(Scheme 2). All of the azidocoumarin compounds (A-I) show
no or very weak fluorescence.
reactions were generally completed in 12 h at room tem-
perature monitored by TLC or mass spectroscopy. The
formation of the fluorescent or nonfluorescent triazole
compounds could be easily ascertained upon irradiation at
365 nm with a hand-held UV lamp (Figure 3). The reaction
mixtures were further diluted to 1 or 10 µM for quantitative
The mild reaction conditions and high fidelity of the
Cu(I)-catalyzed process allowed us to screen the fluorogenic
properties of the cycloaddition reactions combinatorially. The
synthesis of the coumarins and the screening procedures are
described in detail in the Supporting Information. Briefly, a
96-well microtiter plate was used in which each well-
represented a unique combination of azidocoumarin and
alkyne. The total volume for each reaction was 200 µL
fluorescent detection. The absorbance and fluorescence
Ab
properties of the products were fairly widespread (λmax
)
Em
298-445 nm; λmax ) 388-521 nm). The raw data are
displayed in Figure 4. Electron-donating groups at the
7-position of 3-azidocoumarins (such as B, E, and F) showed
strong enhancement of the fluorescence and dominated the
emission color. The quantum yields of the corresponding
triazolylcoumarin products are around 0.6-0.7 (data not
shown). The structure-property relationships are still under
investigation.
Based on the preliminary screening, selected coumarin
products were then synthesized in large quantity. In a
cosolvent of ethanol and water (1:1), the triazole dyes were
formed quantitatively monitored with 1H NMR. The products
can be collect by simple filtration after evaporation of
ethanol. In most situations, no further purifications were
necessary and the yields were around 80% (Table 1). Due
to the high reactivity of aromatic azides used in the synthesis,
the cycloaddition went to completion even at 0 °C. It will
benefit the real application of ligation between biomolecules,
for which elevated temperature is usually destructive.
Figure 3. Combinatorial synthesis and screening of a triazolyl-
coumarin library in microtiter plates. Azides A-H and alkynes
1-24 were used in the reactions. To take the pictures, UV filters
have been used. Therefore, the colors shown here do not represent
the true fluorescent wavelengths.
(DMSO/H2O ) 1:1) containing 1 mM azide and alkyne
catalyzed with 16 mM CuSO4 and sodium ascorbate. The
The inertness of azides and acetylenes allows further
derivatization and attachment to other entities such as
Figure 4. Intensities and emission wavelengths of the final triazolocoumarin products. The rows and columns refer to different building
blocks as indicated in Figure 2. The relative fluorescent intensities are labeled with different letters, in which w ) weak; m ) medium; and
s ) strong. The color of each cell represents the emission wavelength. The detailed data are listed in Table S-1 of the Supporting Information.
Org. Lett., Vol. 6, No. 24, 2004
4605

Table 1. Synthesis of Triazolylcoumarinsa
a The yield is the isolated yield by direct filtration. For more data, see the Supporting Information.
biomolecules, polymers, nanoparticles, and surfaces. We have
successfully used this “click and probing” ligation protocol
in functionalizing bionanoparticles. The bioconjugation ef-
ficiencies can be reported by the fluorescent emission assays
of the triazolylcoumarin products.9 Therefore, this reaction
should have broad application in the emerging field of cell
biology and functional proteomics due to the high reaction
efficiency at mild reaction conditions and the distinguished
fluorescence properties of the products. In addition, this work
is the first to report using azide-alkyne ligation to synthesize
fluorescent compounds combinatorially;10 the mild reaction
conditions and the easy workup can be easily adopted to
construct a large library of pure fluorescent coumarin dyes
with a parallel synthesizer. Furthermore, the concept of our
design will lead to the convenient synthesis of other
fluorescent compounds.
Acknowledgment. We are grateful to the University of
South Carolina, USC Research and Productive Scholarship,
and ARO-MURI award for support of this work. We thank
Miss Xin Wang for the assistance in the synthesis and
fluorescent measurements.
(9) The results will be published elsewhere.
(10) During preparation of this manuscript, another fluorogenic probe
was reported with Cu(I)-catalyzed azide-alkyne reaction: Zhou, Z.; Fahrni,
C. J. J. Am. Chem. Soc. 2004, 126, 8862-8863. For recent combinatorial
synthesis for fluorescent compounds, see ref 8 as well as: (a) Rosania, G.
R.; Lee, J. W.; Ding, L.; Yoon, H.-S.; Chang, Y.-T. J. Am. Chem. Soc.
2003, 125, 1130-1131. (b) Lavastre, O.; Illitchev, I.; Jegou, G.; Dixneuf,
P. H. J. Am. Chem. Soc. 2002, 124, 5278-5279.
Supporting Information Available: All experimental
procedures and fluorescence data. This material is available
free of charge via the Internet at http://pubs.acs.org/.
OL047955X
4606
Org. Lett., Vol. 6, No. 24, 2004