Rapid synthesis, screening, and identification of xanthone- And xanthene-based fluorophores using click chemistry

Article abstract of DOI:10.1021/ol9010344

A panel of new fluorophores with emission wavelengths from blue to yellow regions using the Cu(l)-catalyzed 1,3-dipolar cycloaddition reaction of alkyne-functionalized xanthones and xanthenes with various azides have been synthesized. Screening of the "cl

Full text of DOI:10.1021/ol9010344

ORGANIC
LETTERS
2009
Vol. 11, No. 14
3008-3011
Rapid Synthesis, Screening, and
Identification of Xanthone- and
Xanthene-Based Fluorophores Using
Click Chemistry
Junqi Li,† Mingyu Hu,† and Shao Q. Yao*,†,‡
Departments of Chemistry and Biological Sciences, NUS MedChem Program of the
Life Sciences Institute, National UniVersity of Singapore, Singapore 117543
chmyaosq@nus.edu.sg
Received May 12, 2009
ABSTRACT
A panel of new fluorophores with emission wavelengths from blue to yellow regions using the Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction
of alkyne-functionalized xanthones and xanthenes with various azides have been synthesized. Screening of the “click” products led to the
identification of “hit” fluorophores which showed a fluorescence increase upon triazole formation. These novel “click” fluorophores could
potentially be used for bioconjugation and bioimaging purposes.
Small organic dyes and fluorescent probes are well-
established labeling agents and sensors in both chemical and
biological systems.1 Despite their wide-ranging applications
and popularity, the underlining photophysical properties of
some of these dyes in relation to their molecular structures
are not yet well understood. Consequently, the rational design
of novel fluorophores possessing highly predictable and
desirable properties has remained elusive. The lack of well-
defined rules to govern fluorophore design has driven
combinatorial efforts in fluorophore discovery in recent
years.2 The most straightforward synthetic approach in the
combinatorial fluorophore synthesis is the modification of
core structures from known fluorophores to furnish analogues
of the parental molecules. This has thus far led to the
discovery of new fluorescent molecules with a range of
spectroscopic properties.3
† Department of Chemistry.
‡ Department of Biological Sciences.
We are particularly interested in the use of the Cu(I)-
(1) (a) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997,
97, 1515–1566. (b) Haugland, R. P.; Spence, M. T. Z.; Johnson, I. D.; Basey,
A. The Handbook: A Guide to Fluorescent Probes and Labeling Technolo-
gies, 10th ed.; Molecular Probes: Eugene, OR, 2005.
catalyzed azide-alkyne cycloaddition,4 the representative
(2) (a) Finney, N. S. Curr. Opin. Chem. Biol. 2006, 10, 238–245. (b)
Ljosa, V.; Carpenter, A. E. Trends Biotechnol. 2008, 26, 527–530.
10.1021/ol9010344 CCC: $40.75
Published on Web 06/12/2009
 2009 American Chemical Society

reaction in “click” chemistry,4b for the rapid assembly of
fluorophores. The Wang and Fahrni groups independently
introduced the concept of fluorogenic “click” reactions in
which a weakly fluorescent azido- or alkynylcoumarin is
converted into a fluorescent molecule by triazole formation
using “click” chemistry.3f,5 This unique feature, coupled with
the bioothorgonal nature of the cycloaddition reaction, has
found useful applications in the fluorescence labeling and
visualization of glycans,6 newly synthesized proteins,7 and
lipids.8 In addition, the combinatorial discovery of new
fluorescent dyes is facilitated by the modular and highly
efficient characteristics of the “click” reaction. At present,
only the coumarins,3f,5 carbostyrils,3g anthracenes,9 naphtha-
limides,6a and pyridyloxazole mimics3h comprise the family
of “click” fluorophores in which “click” chemistry has been
used as a fluorogenic reaction and/or for diversification to
generate analogues of the parental fluorophore. One major
drawback of the current “click” fluorophores is that all of
them are UV-excited dyes, making them undesirable choices
for bioimaging applications where cells or tissues are used.
The key aim in the current work is to extend the “click”
chemistry-mediated discovery of fluorescent dyes to previ-
ously unexplored fluorophore scaffolds, especially those with
excitation wavelengths in the visible range.
Figure 1. Design of xanthone- and xanthene-based fluorophores
from known fluorophores.
Recently, our group introduced a new fluorophore, Sin-
gapore Green,10 a structural hybrid of Tokyo Green (a
fluorescein analogue),11 and Rhodamine 110 with emission
and excitation properties similar to both (Figure 1). We
reasoned that replacement of the oxygen electron donor at
the 6′ position with an alkyne in both Singapore Green and
Tokyo Green will significantly decrease the fluorescence
output of their xanthene core. We further extended this design
to Rhodamine B by similarly substituting the diethylamino
group at the 6′-position with an alkyne, as well as replacing
the carboxylic acid moiety in Rhodamine B with a methyl
group at the 2-position to lock the xanthene core in the
conjugated quinol-iminium form. We anticipate that the
formation of a triazole ring at this position using “click”
chemistry will result in a fluorescence change in these
xanthene-alkynes through an extended π-conjugated system
and that this change can be tuned by the use of azides with
different electronic properties. In the interest of extending
the emission range of our “click” fluorophores from the blue
to the yellow region, we also used the blue-light emitting
xanthones which are synthetic precursors of our xanthenes
(Figure 1). Similar to the design of our xanthene-alkynes,
we replaced the heteroatom at the 6-position with an alkyne
to yield the xanthone-alkyne for “click” modification. We
noted that while there are several reports on the synthesis
and spectroscopic characterizations of rosamine dyes from
3,6-disubstituted xanthones,3b,12 to the best of our knowledge
there are no detailed studies on xanthone-based fluorophores
and their fluorescence properties. In this paper, we describe
the synthesis of 6 xanthone- and xanthene-alkynes, the
rapid microplate-based assembly of the “click” fluorophores,
and the subsequent characterization and identification of “hit”
fluorophores with varying fluorescence characteristics.
As shown in Scheme 1, the general synthetic strategy for
the alkynes A, B, D, and E involves the desymmetrization
of the common starting material 3,6-dihydroxyxanthone 1
to give the appropriate substituent at the 6-position, leaving
the other phenolic group for conversion into a triflate which
serves as the substrate for Sonogashira coupling with
trimethylsilylacetylene. Deprotection of the TMS group
affords alkynes A and B, while Grignard addition to the
xanthone followed by removal of the protecting groups gave
alkynes D and E (see the Supporting Information for full
synthetic details). The synthesis of alkynes C and F followed
the same strategy. Starting from 3-nitro-6-hydroxyxanthone
(3) For selected examples, see: (a) Wang, S.; Chang, Y.-T. J. Am. Chem.
Soc. 2006, 128, 10380–10381. (b) Ahn, Y.-H.; Lee, J.-S.; Chang, Y.-T.
J. Am. Chem. Soc. 2007, 129, 4510–4511. (c) Schiedel, M.-S.; Briehn, C. A.;
Ba¨uerle, P. Angew. Chem., Int. Ed. 2001, 40, 4677–4680. (d) Hirano, T.;
Hiromoto, K.; Kagechika, H. Org. Lett. 2007, 9, 1315–1318. (e) Zhu, Q.;
Yoon, H.-S.; Parikh, P. B.; Chang, Y.-T.; Yao, S. Q. Tetrahedron Lett.
2002, 43, 5083–5086. (f) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.;
Barnhill, H. N.; Wang, Q. Org. Lett. 2004, 6, 4603–4606. (g) Glasnov,
T. N.; Kappe, C. O. QSAR Comb. Sci. 2007, 11, 1261–1265. (h) Shi, J.;
Liu, L.; He, J.; Meng, X.; Guo, Q. Chem. Lett. 2007, 36, 1142–1143
.
(4) For reviews, see: (a) Meldal, M.; Tornoe, C. W. Chem. ReV. 2008,
108, 2952–3015. (b) Kolb, H. C.; Sharpless, K. B. Drug Disc. Today 2003,
8, 1128–1137
.
(5) Zhou, Z.; Fahrni, C. J. J. Am. Chem. Soc. 2004, 126, 8862–8863
.
(6) (a) Sawa, M.; Hsu, T.-L.; Itoh, T.; Sugiyama, M.; Hanson, S. R.;
Vogt, P. K.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 12371–
12376. (b) Hsu, T.-L.; Hanson, S. R.; Kishikawa, K.; Wang, S.-K.; Sawa,
M.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2614–2619.
(7) (a) Beatty, K. E.; Xie, F.; Wang, Q.; Tirrell, D. A. J. Am. Chem.
Soc. 2005, 127, 14150–14151. (b) Beatty, K. E.; Liu, J. C.; Xie, F.; Dieterich,
D. C.; Schuman, E. M.; Wang, Q.; Tirrell, D. A. Angew. Chem., Int. Ed.
2006, 45, 7364–7367.
(8) Neef, A. B.; Schultz, C. Angew. Chem., Int. Ed. 2009, 48, 1498–
1500.
(9) Xie, F.; Sivakumar, K.; Zeng, Q.; Bruckman, M. A.; Hodges, B.;
Wang, Q. Tetrahedron 2008, 64, 2906–2914.
(10) Li, J.; Yao, S. Q. Org. Lett. 2009, 11, 405–408.
(11) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano,
T. J. Am. Chem. Soc. 2005, 127, 4888–4894.
(12) (a) Wu, L.; Burgess, K. Org. Lett. 2008, 10, 1779–1782. (b) Wu,
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.
Org. Lett., Vol. 11, No. 14, 2009
3009

Scheme 1. Synthesis of Alkynes A-F
4, it was converted to triflate 5 which underwent Sonogashira
coupling to give the TMS-protected nitroxanthone 6. The
nitro group was reduced under mild conditions to aniline 7
with zinc in MeOH/THF buffered at pH 5. Aniline 7 was
protected with a trityl protecting group, followed by Grignard
reaction to give the trityl-protected xanthene 9. Subsequent
deprotection of the trityl and TMS groups furnished the final
alkyne F. Alkyne C was obtained after deprotection of the
TMS group in 7.
and azide in each well. The reaction was initiated by adding
a solution of CuSO4 (2 equiv) and sodium ascorbate (5
equiv). The “click” reaction for the xanthene-alkynes D-F
proved to be highly efficient, with the alkynes consumed
within 12 h to give the products in high purity as monitored
by LC-MS. The xanthone-alkynes A-C were considerably
less reactive with some incomplete reaction after 12 h (see
the Supporting Information). The desired product was
obtained for all compounds with the exception of products
with azides z13 and z14, which did not give the desired
products for all the alkynes and were thus omitted from
subsequent studies. For preliminary evaluation of the fluo-
rescence properties of the library, the crude reaction mixture
was diluted to 400 µM in DMSO, which was further diluted
to 20 µM for fluorescence screening. Since the quantum yield
of fluorophores can be significantly influenced by solvent
effects, we chose four different solventssH2O (aqueous, with
DMSO as cosolvent to prevent precipitation), DMSO (polar
aprotic), EtOH (polar protic), and DCE (apolar aprotic)sto
record the excitation and emission spectra of all the fluoro-
phores in the library (see the Supporting Information for
details).
With the six alkynes in hand, we selected a total of 17
aromatic and aliphatic azides with different electronic
properties for the “click” chemistry reaction (Scheme 2; see
Scheme 2. “Click” Assembly of Fluorophores
The results are summarized in the form of a heat map
displaying the fluorescence intensities of each fluorophore
relative to its alkyne precursor (that is, the xanthone or
xanthene core prior to click chemistry) (Figure 2). Several
observations can be made from the heat map. As anticipated,
the majority of the “click” products registered an increase
in fluorescence intensity (red squares) over the parent alkyne,
but a considerable number also led to a fluorescence decrease
the Supporting Information for the azides used). A 102-
membered fluorophore library was assembled in a 384-deep
well block by mixing each of the alkynes with a different
azide in DMSO/t-BuOH/H2O to give a unique pair of alkyne
3010
Org. Lett., Vol. 11, No. 14, 2009

Figure 3. Emission spectra of (a) F-z12 and (b) F-z17 in different
solvents (bottom graphs). Top graphs: emission spectra of F-z12
(in DCE; green line) and F-z17 (in EtOH; red line) were compared
against the alkyne precursor F (in each corresponding solvent; black
line). Black arrows indicate the increase in fluorescence after “click”
reaction. See the Supporting Information for full spectra.
Figure 2. Heat map showing relative fluorescence intensities and
structures of the four “hit” fluorophores selected for further studies.
See the Supporting Information for details on the generation of the
heat map.
(blue squares). In particular, a morpholino substituent in the
azide (z4, column 4) led to a consistent diminution of
fluorescence throughout the alkyne series, possibly due to
photoinduced-electron-transfer (PET) quenching from the
nitrogen atom, and a strongly electron-withdrawing nitro
substituent (z8, column 8) also effectively quenched the
fluorescence in alkynes series A-D. Notably, the “click”
reaction between aliphatic azides (column 15-17) and the
xanthene-alkynes was fluorogenic, implying that triazole
formation is itself sufficient for fluorescence activation. This
feature is particularly important when searching for green
light-emitting “click” fluorophores for use in labeling azide-
modified biomolecules for bioimaging purposes.5-7
To examine in detail the fluorescence properties of our “click”
fluorophores, we picked a few “hits” from each of the six alkyne
series for scale-up, purification, and characterization. These
“hits” were selected on the basis of the following points: (i)
they showed significantly higher fluorescence intensity than their
alkyne precursors; (ii) the “hits” give a good representation of
aromatic and aliphatic azides of different properties; and (iii)
their fluorescence intensities showed some solvent sensitivity
(F-z12 and F-z17). The spectroscopic properties of four of the
brightest “hits” (structures shown in Figure 2) and their
corresponding alkyne precursors were further evaluated (see the
Supporting Information for full details). It was found that
triazole formation led to an increase in both molar absorptivity
and quantum yield, leading to an overall increase in brightness
(εΦf) of 2-3-fold for B-z15 and C-z17 in DMSO, and ∼10-
fold for F-z12 in DCE and F-z17 in EtOH (Figure 3). F-z12
and F-z17 also displayed different solvent sensitivity despite
being derivatives of the same alkyne, with F-z12 fluorescing
most brightly in DCE, while in other solvents it is considerably
less bright. F-z17 is the brightest in EtOH and has varying
intensities in other solvents. There is, however, no direct trend
between the fluorescence intensities and solvent dielectric
constants, suggesting that the observed solvent effects are
specific to the molecular structure of the fluorophore. Because
these effects shown in both our microplate screening and
detailed analysis are not easily predicted, the combinatorial
approach to various analogues is an advantage in searching for
the desired fluorophore properties as demonstrated in this report.
In conclusion, we have successfully designed and synthesized
two new classes of “click” fluorophores based on the xanthone
and xanthene scaffolds. The rapid assembly of these fluoro-
phores enabled by “click” chemistry gave easy access to
xanthene analogues, which are traditionally difficult to synthe-
size. We have also identified two fluorophores, F-z12 and
F-z17, in which triazole formation resulted in a significant
fluorescence increase. These fluorophores could potentially be
used as green light-emitting substitutes for the existing “click”
fluorophores in bioconjugation and bioimaging applications.5-7
Their utilities in live cell imaging are in progress.
Acknowledgment. Funding support was provided by the
Ministry of Education (R143-000-390-315), the Agency for
Science, Technology and Research (A*Star) of Singapore
(R154-000-274-305), and the National Research Foundation
(Competitive Research Programme R143-000-218-281).
Supporting Information Available: Experimental pro-
cedures, characterization of new compounds, and fluores-
cence spectra. This material is available free of charge via
the Internet at http://pubs.acs.org.
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