Cell-permeable iminocoumarine-based fluorescent dyes for mitochondria

Article abstract of DOI:10.1021/ol200908r

A class of small molecule fluorophores, 2-iminocoumarin-3-carboxamide derivatives, has been developed by a rapid microwave-assisted process. These fluorescent probes are cell membrane permeable with low cytotoxicity and able to selectively stain organelles in living cells.

Full text of DOI:10.1021/ol200908r

ORGANIC
LETTERS
2011
Vol. 13, No. 11
2884–2887
Cell-Permeable Iminocoumarine-Based
Fluorescent Dyes for Mitochondria
Diliang Guo,†,§, Tao Chen,‡, Deju Ye,† Jinyi Xu,§ Hualiang Jiang,† Kaixian Chen,†,§
Hui Wang,*,‡ and Hong Liu*,†,§
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Shanghai, P. R. China, and, Key
Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, 320 YueYang Road,
Shanghai, P. R. China, and Department of Medicinal Chemistry, China Pharmaceutical
University, 24 TongjiaXiang, Nanjing, P. R. China
hliu@mail.shcnc.ac.cn; huiwang@sibs.ac.cn
Received April 7, 2011
ABSTRACT
A class of small molecule fluorophores, 2-iminocoumarin-3-carboxamide derivatives, has been developed by a rapid microwave-assisted
process. These fluorescent probes are cell membrane permeable with low cytotoxicity and able to selectively stain organelles in living cells.
Fluorescent probes with high sensitivity and ease of
handling have been extensively employed in biological
science, clinical diagnosis, and drug discovery.1 The devel-
opment of fluorescent probes, particularly synthetic small
molecular fluorescent probes with various core skeletons,
such as fluorescein, BODIPY, and rhodamine, has re-
ceived special attention.1 The use of small molecule fluo-
rescent dyes in designing imaging probes is generally
advantageous as they offer a greater number of options
for optimizing delivery, and a single dye can be used on
multiple targeting carrier molecules.1e Although signifi-
cant progress has been made in this field, there is still a
great demand for novel fluorescent core skeletons with
flexible synthetic strategies and good photophysical prop-
erties for bioimaging.2
Coumarin-based fluorescent dyes have been used ex-
tensively in biological studies,3 and iminocoumarins
have recently been investigated as a sensitive zinc sensor4
and a selective detector of dual-specific protein tyrosine
~
(2) For selected examples, see: (a) Abet, V.; Nunez, A.; Mendicuti, F.;
Burgos, C.; Alvarez-Builla, J. J. Org. Chem. 2008, 73, 8800. (b) Kim, E.;
Koh, M.; Ryu, J.; Park, S. B. J. Am. Chem. Soc. 2008, 130, 12206. (c) Liu,
Y.; Yan, W.; Chen, Y.; Petersen, J. L.; Shi, X. Org. Lett. 2008, 10, 5389.
(d) Shimizu, M.; Mochida, K.; Hiyama, T. Angew. Chem., Int. Ed. 2008,
47, 9760. (e) Wu, Y.-T.; Kuo, M.-Y.; Chang, Y.-T.; Shin, C.-C.; Wu, T.-
C.; Tai, C.-C.; Cheng, T.-H.; Liu, W.-S. Angew. Chem., Int. Ed. 2008, 47,
9891. (f) Zhao, D.; Wang, W.; Yang, F.; Lan, J.; Yang, L.; Gao, G.; You,
J. Angew. Chem., Int. Ed. 2009, 48, 3296. (g) Chui, C. H.; Wang, Q.;
Chow, W. C.; Yuen, M. C. W.; Wong, K. L.; Kwok, W. M.; Cheng,
G. Y. M.; Wong, R. S. M.; Tong, S. W.; Chan, K. W.; Lau, F. Y.; Lai,
P. B. S.; Lam, K. H.; Fabbri, E.; Tao, X. M.; Gambari, R.; Wong, W. Y.
Chem. Commun. 2010, 46, 3538. (h) Kim, D.; Jun, H.; Lee, H.; Hong, S.-
S.; Hong, S. Org. Lett. 2010, 12, 1212.
† Shanghai Institute of Materia Medica.
§ China Pharmaceutical University.
Authors equally contributed to this work.
‡ Shanghai Institutes for Biological Sciences.
(3) For recent examples, see: (a) Kuo, P.-Y.; Yang, D.-Y. J. Org.
Chem. 2008, 73, 6455. (b) Lin, W.; Yuan, L.; Cao, Z.; Feng, Y.; Long, L.
Chem.;Eur. J. 2009, 15, 5096. (c) Do, J. H.; Kim, H. N.; Yoon, J.; Kim,
J. S.; Kim, H.-J. Org. Lett. 2010, 12, 932. (d) Yuan, L.; Lin, W.; Song, J.
Chem. Commun. 2010, 46, 7930.
(4) Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem.
Soc. 2007, 129, 13447.
(1) For reviews, see: (a) Giepmans, B. N. G.; Stephen, R. A.;
Ellisman, M. H.; Tsien, R. Y. Science 2006, 312, 217. (b) Rao, J.;
Dragulescu-Andrasi, A.; Yao, H. Curr. Opin. Biotechnol. 2007, 18, 17.
(c) Gonc-alves, M. S. T. Chem. Rev. 2009, 109, 190. (d) Renatus, W. S.;
Nicholas, J. G.; Yitzhak, T. Chem. Rev. 2010, 110, 2579. (e) Kobayashi,
H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y. Chem. Rev. 2010,
110, 2620. (f) Qian, X.; Xiao, Y.; Xu, Y.; Guo, X.; Qian, J.; Zhu, W.
Chem. Commun. 2010, 46, 6418. (g) Chen, X.; Zhou, Y.; Peng, X.; Yoon,
J. Chem. Soc. Rev. 2010, 39, 2120.
(5) Kim, T.-I.; Jeong, M. S.; Chung, S. J.; Kim, Y. Chem.;Eur.
J. 2010, 16, 5297.
r
10.1021/ol200908r
Published on Web 05/06/2011
2011 American Chemical Society

phosphatases in vitro.5 Iminocoumarins can be excited at
longer wavelengths than coumarin analogues, hence in-
creasing their suitability for biological applications.5 How-
ever, there have been very few investigations of their
3-carboxamide analogues as a scaffold for fluorescence
probes. Inourongoing effortstodevelop novel approaches
to the synthesis of biologically active heterocyclic com-
pounds,6 we have designed and synthesized a 2-iminocou-
marin-3-carboxamide7 fluorescent coreskeletonwithgood
photophysical properties, which can be easily derivatized
and potentially used for fluorescent intracellular imaging.8
intermediates 1aꢀ1g in 30 min. Then, convenient conden-
sation of the corresponding cyanoacetamides and com-
mercially available substituted salicylaldehydes via a
Knoevenagel reaction at 60 °C under microwave irradiation
produces the desired 2-iminocoumarin-3-carboxamide
2aꢀ2p derivatives after an additional 30 min. It is important
to emphasize that in most cases analytically pure products
are obtained in moderate to good yields (ranging from 55%
to 97%, Table 1) from the reaction mixture via simple
filtration the exception being compound 2o.
In an endeavor to further examine the fluorescent dyes
with good photophysical properties and potential bio-
logical applications, we attempted to further modify the
2-iminocoumarin-3-carboxamide probes with direct de-
rivation (Scheme 1). For example, by direct reaction of
2a and p-anisidine in acetic acid at 60 °C for 3 h, product
Table 1. Synthesis of Compounds 2aꢀ2p
Scheme 1. Synthesis of 3a and NPE-caged 2o
entry
1
R
R0
2
yielda (%)
1
1a Bn
1b Ph
7-Diethylamino 2a
7-Diethylamino 2b
7-Diethylamino 2c
7-Diethylamino 2d
7-Diethylamino 2e
76
95
67
78
55
73
80
64
57
97
85
78
95
73
70b
72
2
3
1c
CH2CH2Ph
4
1d 4-OMeBn
5
1e
1f
3,4-OMeBn
6
3,4,5-OMeBn 7-Diethylamino 2f
7-Diethylamino 2g
7
1g 4-CF3Bn
1a Bn
1a Bn
1a Bn
1a Bn
1a Bn
1a Bn
1a Bn
1a Bn
1a Bn
8
ꢀ
2h
2i
9
5-Methoxy
6-Methoxy
7-Methoxy
8-Methoxy
6-Bromo
6-Hydroxy
7-Hydroxy
8-Hydroxy
3a possessing a 4-methoxyphenylimino substituent at
position 2 was obtained in 81% yield. Now that the
photocaged fluorescent probes10 have wide applications
in tracking the spatiotemporal dynamics of molecular
movements in biological systems, NPE-caged 2o was
prepared in moderate yield by incorporating a 1-(2-
nitrophenyl)ethyl (NPE)10a group onto the 7-hydroxyl
group of 2o. NPE-caged 2o is nonfluorescent before
10
11
12
13
14
15
16
2j
2k
2l
2m
2n
2o
2p
a Isolated yields via filtration. b Purification by chromatography.
(8) For selected examples of intracellular imaging, see: (a) Zhang, L.;
Murphy, C. S.; Kuang, G.-C.; Hazelwood, K. L.; Constantino, M. H.;
Davidson, M. W.; Zhu, L. Chem. Commun. 2009, 7408. (b) Huang, Y.-
Y.; Mroz, P.; Zhiyentayev, T.; Sharma, S. K.; Balasubramanian, T.;
Target compounds containing the core skeleton were
successfully synthesized under microwave irradiation via a
two-step process (Table 1). First, efficient amidation9 of
methyl cyanoacetate with aryl amines under microwave
irradiation at 135 °C gives rise to the key cyanoacetamide
ꢀ
Ruzie, C.; Krayer, M.; Fan, D.; Borbas, K. E.; Yang, E.; Kee, H. L.;
Kirmaier, C.; Diers, J. R.; Bocian, D. F.; Holten, D.; Lindsey, J. S.;
Hamblin, M. R. J. Med. Chem. 2010, 53, 4018. (c) Jose, J.; Loudet, A.;
Ueno, Y.; Barhoumi, R.; Burghardt, R. C.; Burgess, K. Org. Biomol.
Chem. 2010, 8, 2052. (d) Liu, Y.; Lok, C.-N.; Ko, B. C.-B.; Shum, T. Y.-
T.; Wong, M.-K.; Che, C.-M. Org. Lett. 2010, 12, 1420. (e) Signore, G.;
Nifosı, R.; Albertazzi, L.; Storti, B.; Bizzarri, R. J. Am. Chem. Soc. 2010,
132, 1276.
(9) For selected examples, see: (a) Varma, R. S.; Naicker, K. P.
Tetrahedron Lett. 1999, 40, 6177. (b) Luque, R.; Budarin, V.; Clark,
J. H.; Macquarrie, D. J. Green Chem. 2009, 11, 459. (c) Dhake, K. P.;
Qureshi, Z. S.; Singhal, R. S.; Bhanage, B. M. Tetrahedron Lett. 2009, 50,
2811.
(10) For recent examples of caged fluorescent probes, see: (a) Zhao,
Y.; Zheng, Q.; Dakin, K.; Xu, K.; Martinez, M. L.; Li, W.-H. J. Am.
Chem. Soc. 2004, 126, 4653. (b) Zheng, G.; Guo, Y.-M.; Li, W.-H. J. Am.
Chem. Soc. 2007, 129, 10616. (c) Belov, V. N.; Wurm, C. A.; Boyarskiy,
V. P.; Jakobs, S. S.; Hell, W. Angew. Chem., Int. Ed. 2010, 49, 3520. (d)
Lin, W.; Long, L.; Tan, W.; Chen, B.; Yuan, L. Chem.;Eur. J. 2010, 16,
3914.
(6) For recent examples, see: (a) Li, Z.; Sun, H.; Jiang, H.; Liu, H.
Org. Lett. 2008, 10, 3263. (b) Ye, D.; Zhang, X.; Zhou, Y.; Zhang, D.;
Zhang, L.; Wang, H.; Jiang, H.; Liu, H. Adv. Synth. Catal. 2009, 351,
2770. (c) Zhang, X.; Ye, D.; Sun, H.; Guo, D.; D. Wang, D.; Huang, H.;
Zhang, X.; Jiang, H.; Liu, H. Green Chem. 2009, 11, 1881. (d) Zhou, Y.;
Zhai, Y.; Ji, X.; Liu, G.; Feng, E.; Ye, D.; Zhao, L.; Jiang, H.; Liu, H.
Adv. Synth. Catal. 2010, 352, 373.
(7) For selected examples of synthesis of 2-iminocoumarin-3-carbox-
amides, see: (a) Rajagopal, R.; Seshadri, S. Dyes Pigm. 1990, 13, 29. (b)
Burke, T. R., Jr.; Lim, B.; Marquez, V. E.; Li, Z.-H.; Bolen, J. B.;
Stefanova, I.; Horaks, I. D. J. Med. Chem. 1993, 36, 425. (c) Gorobets,
N. Y.; Yousefi, B. H.; Belaj, F.; Kappe, C. O. Tetrahedron 2004, 60,
8633. (d) Borisov, A. V.; Dzhavakhishvili, S. G.; Zhuravel, I. O.;
Kovalenko, S. M.; Nikitchenko, V. M. J. Comb. Chem. 2007, 9, 5. (e)
Proenc-a, F.; Costa, M. Green Chem. 2008, 10, 995.
Org. Lett., Vol. 13, No. 11, 2011
2885

activation; however, the photolabile protecting group
can be removed by ultraviolet (UV) illumination (365
nm) to progressively generate the fluorescent probe 2o
(Figure S2 shows HPLC chromatograms of NPE-caged
2o and its photolysis products).
and fluorescence emission brightness going from or-
ganic to water media largely decreased (Figure S1 and
Table S1).
Table 2. Photophysical Data of 2aꢀ2p and 3aa
λabs
(nm)b
λem
(nm)c
Stokes
log εmax
compd
shift [nm]
(Mꢀ1 cmꢀ1 d
)
Φfe
2a
2b
2c
2d
2e
2f
397
414
402
396
401
403
402
352
357
381
365
382
363
382
370
350
418
475
480
470
470
473
475
473
407
446
449
442
449
445
466
483
453
504
78
66
68
74
72
72
71
55
89
68
77
67
82
84
113
103
86
4.61
4.54
4.28
4.56
4.41
4.52
4.50
4.03
4.04
3.95
4.25
3.19
3.91
3.93
4.31
3.81
4.18
0.59
0.20
0.39
0.51
Figure 1. (a) Fluorescence images of HeLa cells initially incubated
with 1 μM 2a for 30 min in DMEM medium (with 10% FBS
and 1% penicillin/streptomycin) and then fixed with 4% parafor-
maldehyde for 10 min (λex = 390 nm); (b) 4ꢁ amplification
image of (a).
0.42
0.50
2g
2h
2i
0.61
<0.05
<0.05
<0.05
0.09
2j
2k
2l
In order to examine the potential use of these 2-imino-
coumarin-3-carboxamide dyes for biological imaging, we
initially incubated HeLa cells with a representative com-
pound 2a (1 μM) at 37 °C for 30 min. As shown in Figure 1,
strong blue fluorescence was detected inside the cells
(except for the nucleus) with excitation at 390 nm, which
indicates that compound 2a has good cell membrane
permeability. HeLa cells were extensively stained at either
1 or 5 μM (Figure S4) incubation concentration (Figure 1).
On the basis of cell morphology, compound 2a appears to
have low cell toxicity. We further evaluated the toxicity of
the compounds 2a and 2k to HeLa and 7702 (Normal
Human Liver cell line) cells; the IC50 values are over 100
μM (Figure S3). Moreover, 2a can be used in either HBSS
buffer (Figure S4a and S4b) or DMEM medium (Figure 1a
and 1b), which indicates its stability when used for live cell
imaging.
To further investigate the subcellular localization of com-
pound 2a, organelle-specific fluorescent dyes were employed
for colocalization studies. As shown in Figure 2, we found
that compound 2a can colocalize strongly with MitoTracker
Red CMXRos in mitochondria, with weighted colocaliza-
tion coefficient values to 0.992 (Figure 2c and 2f, analyzed
using LSM 510 software). The extensive overlapping with
MitoTracker Red CMXRos indicated that mitochondria
are probably the main site of compound 2a accumulation. In
cell biology, mitochondria are membrane-enclosed energy
producing organelles found in most eukaryotic cells, they
participate in a range of cellular processes, such as signaling,
cellular differentiation, and cell death. Mitochondrial dys-
functions or defects involved in a variety of diseases
ranging from aging and metabolic diseasestodegenerative,
hyper-proliferative diseases and cancers.11 The imaging of
<0.05
<0.05
<0.05
0.16
2m
2n
2o
2p
3a
<0.05
0.15
a Determined in chloroform. b Absorption maxima. c Emission max-
ima. d Maximum molar extinction coefficient. e Absolute quantum yield
determined by a calibrated integrating sphere system.
With the fluorescent compound library in hand, we
investigated the optical properties of the compounds,
and the results are summarized in Table 2. In general,
the iminocoumarin derivatives containing an electron-
donating group R0 at position 7 and an electron-with-
drawing group CONHR at position 3 afforded a strong
fluorescence emission. The introduction of a benzyl
group at position R with a fixed R0 substituent such as
a diethylamino group leads to larger Stokes shifts and
higher quantum yields than with a phenyl or phenethyl
group (2a vs 2bꢀ2c). However, no significant change in
the fluorescence properties is observed with alternation
of the substituents on the benzyl group (2dꢀ2g). Fur-
thermore, the fluorescence emissions, in agreement with
the electron-donating potentials of group R0, are increa-
sed in the following order: bromo (2m) < methoxy (2iꢀ
2k)<hydroxyl (2nꢀ2p)<diethylamino (2aꢀ2g). On the
other hand, the substituted position of the electron-
donating group R0 is crucial for the photonic property.
For example, compounds with a substituent at position
7 show relatively higher quantum yields and extinction
coefficients (2iꢀ2l and 2nꢀ2p). It is interesting that
introduction of a 4-methoxyl-phenyl group at position
2 of compound 2a generates a large red shift in the
excitation wavelength, owing to both the extension of
the electronic conjugation and the electron-withdraw-
ing effect of the added aromatic ring (3a). Next, the
solvatochromic behavior of compound 2a was exam-
ined; it can be observed that the extinction coefficients
(11) (a) Wallace, D. C. Science 1999, 283, 1482. (b) Murphy, M. P.;
Smith, R. A. Adv. Drug Delivery Rev. 2000, 41, 235. (c) Henze, K.;
Martin, W. Nature 2003, 426, 127. (d) McBride, H. M.; Neuspiel, M.;
Wasiak, S. Curr. Biol. 2006, 16, 551. (e) Yousif, L. F.; Stewart, K. M.;
Kelley, S. O. ChemBioChem 2009, 10, 1939. (f) Frantz, M. C.; Wipf, P.
Environ. Mol. Mutagen 2010, 51, 462.
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Org. Lett., Vol. 13, No. 11, 2011

lipophilic fluorophores with delocalized positive charges
or with depolarizing effects on mitochondria.8b,c Bearing
this in mind, the lipohilic property of compound 2a, and its
potential depolarizing effects, may contribute to its accu-
mulation in mitochondria.
We further employed ER tracker Red dye (used to label
the endoplasmatic reticulum) and Golgin-97 (an antibody
usedtolabel the Golgi complex) toexamine whether2acan
be localized within other subcellular domains. There is a
relatively low colocalization of compound 2a with ER
tracker Red dye (Figure S5) and within the Golgi complex
(Figure S6), withweighted colocalization coefficient values
only to 0.569 (Figure S5g and S5h) and 0.392 (Figure S6g
and S6h) respectively.
In summary, we have developed a small molecule fluo-
rescent core skeleton, 2-iminocoumarin-3-carboxamide,
with convenient synthesis and easy derivation. The library
of fluorophores can undergo a dramatic change in fluore-
scence properties simply by changing the substituents at the
two variation points of the fluorophore. Furthermore, the
dyes can permeate the living cell membranes with
low cytotoxicity and accumulate preferentially in the mito-
chondria, thus possessing potential utility in intracellular
imaging.
Figure 2. Confocal images of 2a and mitochondrial specific dye
in HeLa cells. (a) Fluorescence image of HeLa cells stained with
compound 2a; (b) Fluorescence image of HeLa cells stained with
mitochondrial specific dye (MitoTracker Red CMXRos); (c)
Merged image of (a) and (b); (d) 4ꢁ amplification image of single
cell stained with compound 2a; (e) 4ꢁ amplification image of
single cell stained with MitoTracker Red CMXRos; (f) Merged
image of (d) and (e); (g) and (h) colocalization analysis of com-
pound 2a and MitoTracker Red CMXRos in image (f).
Acknowledgment. We gratefully acknowledge financial
support from the National Natural Science Foundation of
China (Grants 20721003, 20872153, 81025017, 30870513, and
31070680), National S&T Major Projects (2009ZX09301-
001, 2009ZX09501-001, 2009ZX09501-010, 2009ZX09102-
114, and 2009ZX09301-011). We thank Prof. Dr. Hequan
Yao (China Pharmaceutical University) for helpful discus-
sions. We also thank Prof. Dr. Yingchun Zhu and Zhiyong
Mao (Shanghai Institute of Ceramics, Chinese Academy
Sciences) for absolute quantum yield measurements.
mitochondria with novel synthetic mitochondrial specific
probes will undoubtedly provide a more detailed under-
standing about the mitochondrial biology and signal
propagation.11e Additionally, mitochondria possess a net
negative chargeon the matrix side of the membrane; hence,
common markers for this organelle tend to be moderately
Supporting Information Available. Full experimental
details and characterization data. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
Org. Lett., Vol. 13, No. 11, 2011
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