Communications
porting Information, Figure S5). The staining pattern of
molecule 2 remained the same in the presence of CCCP,
indicating that the staining properties of molecule 2 are
independent of the membrane potential. We also performed
staining experiments with additional cell types: human
embryonic kidney HEK 293, prostate carcinoma DU 145,
hepatocyto carcinoma HepG2, breast adenocarcinoma SK-
BR-3, and mouse myoblast C2C12. The results showed that
staining patterns of molecule 2 in these cell lines are similar to
each other (Supporting Information, Figure S6). These results
suggest that molecule 2 is recognized by mitochondrion-
associated cellular components common in a range of cell
types.
Figure 5. a) The chemical structure of molecule 3, the metabolite of
molecule 2. b) HeLa cells that were double-stained with molecule 3
(20 mgmLÀ1) and MitoTracker Red and then observed by a confocal
microscope.
In conclusion, we have developed a novel fluorescent
probe specific for mitochondrial surfaces. To our knowledge,
this molecule is the first such probe. Mitochondria play an
important role not only in energy metabolism but also in
apoptosis.[12] Furthermore, recent studies revealed that mito-
chondria interacts with endoplasmic reticulum and supports
direct transfer of lipids and Ca2+ ions.[13] Molecule 2 or its
analogues may allow the visualization of cross-talk between
mitochondria and other organelles and for mitochondrial
surface dynamics in response to various stimuli. Cell-based
image screening of larger chemical libraries enriched in
aromatic or fluorescent structures will almost certainly
discover additional cell-permeable probes with various prop-
erties,[14] and some may undergo bioconversion within cells to
achieve their selectivities.
chemically synthesized molecule 3 and compared its physical
and chemical properties to those of the metabolite. The
synthesized compound showed exactly the same HPLC
profile and fluorescence spectrum as the metabolite, and the
1
signatures in its absorbance and H NMR spectra matched
those of the metabolite (Supporting Information, Figure S4
and NMR chart). Both molecule 3 and the metabolite could
be monoacetylated to yield a substance with a molecular mass
of 525.80, which is consistent with the presence of a free
amino group.
Incubation of HeLa cells with fluorescent molecule 3
resulted in similar staining of mitochondrial surfaces as did
incubation with molecule 2 (Figure 5b), suggesting that
molecule 3, not molecule 2, is the molecule that generates
fluorescent images. Nevertheless, the staining pattern of
molecule 3 was more complex and had higher background
than that of molecule 2 at the same concentration. Molecule 2
is likely to penetrate cell membrane and gradually become
fluorescent through bioconversion within the cells, lowering
background levels. Another possible explanation would be in
situ bioconversion of molecule 2 in the mitochondrial matrix.
The pH of the mitochondrial matrix in cells has been
estimated to be 8.05 Æ 0.11.[10] Model reactions of molecule
2 at the pH range of 6.0–8.0 in 150 mm sodium phosphate
buffers showed faster conversion of molecule 2 in alkaline
conditions (see the Supporting Information for details). At
pH 8.0, 65.8% of molecule 2 was converted into molecule 3 in
4 h, while a conversion of only 8.4% and 1.5% of molecule 2
into molecule 3 at pH 7.0 and pH 6.0, respectively, was
observed. Perhaps an acidic a proton of molecule 2 is
gradually released at pH 8.0 to give an enolate, which
undergoes cyclization. Hydrolysis of an ester within cells
has been used for controlling the properties of fluorescent
small molecule probes.[11] This example suggests that a similar
control of a fluorescent probe may be possible through
cyclization of non-fluorescent molecules within cells.
Experimental Section
The chemical library of 12000 molecules consists of relatively large
molecules enriched in aromatic groups that were purchased from
several chemical library companies. HeLa cells were cultured in
DMEM supplemented with 10% fetal bovine serum. Each compound
was screened at 20 mgmLÀ1 by incubation of cells for 2 h, and the
signals were checked in situ by fluorescence microscopy. The details
of the cell-based screening are provided in the Supporting Informa-
tion.
The synthesis of molecules 2 and 3 and other analogues is
described in the Supporting Information. To purify the metabolite,
80 L of cultured HeLa S3 cells were treated with 5 mgmLÀ1 of
molecule 2 for 2 h. The cells were extracted twice with acetonitrile
and then lyophilized. The resulting powder was extracted with
chloroform and lyophilized again. The extracts were fractionated by
sequential column chromatography with Inertsil ODS-3 (GL Scien-
ces, Japan) and COSMOSIL Chlester (Nacalai Tesque, Japan), using a
Shimadzu LC2010C HPLC system.
Fluorescence spectra for each compound were obtained with a
Hitachi F-7000 fluorescence spectrometer at 20 mgmLÀ1 1H NMR
.
spectra were recorded in deuterated solvents at 600 MHz (JOEL
JNM-ECA 600). High-resolution mass spectra were obtained on a
JEOL JMS-700 spectrometer.
Direct molecular targets of molecule 3 remain unknown.
Selectivity of molecule 3 may be achieved by direct physical
association with some of the many proteins in the mitochon-
drial surface. Alternatively, molecule 3 may be recognized by
non-proteins or influenced by the environmental status of the
mitochondrial membrane. We examined the staining of
molecule 2 in the presence of CCCP, an uncoupling reagent
that disrupts the mitochondrial membrane potential (Sup-
Received: February 7, 2011
Published online: May 3, 2011
Keywords: bioconversion · cyclization · fluorescent probes ·
.
live-cell imaging · mitochondria
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 5478 –5481