High Affinity Neutral Bodipy Fluorophores for Mitochondrial Tracking

Article abstract of DOI:10.1021/acsmedchemlett.8b00022

We report the first high affinity neutral Bodipy fluorophores for selective imaging of mitochondria with notable sensitivity (～100 nM) and insignificant cytotoxicity even at very high concentration (～100 μM), when tested against HeLa cells. Further, these fluorophores are chemically robust and require no special conditions for storage.

Full text of DOI:10.1021/acsmedchemlett.8b00022

Subscriber access provided by Stony Brook University | University Libraries
Letter
High Affinity Neutral Bodipy Fluorophores for Mitochondrial Tracking
Thumuganti Gayathri, Santosh Karnewar, Srigiridhar Kotamraju, and Surya Prakash Singh
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00022 • Publication Date (Web): 20 Jun 2018
Downloaded from http://pubs.acs.org on June 20, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 5
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
High Affinity Neutral Bodipy Fluorophores for Mitochondrial
Tracking
†
,‡
§,‡
§,‡
†,‡
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Thumuganti Gayathri, Santosh Karnewar, Srigiridhar Kotamraju,* and Surya Prakash Singh*
†
Polymers and Functional Materials Division, CSIRꢀIndian Institute of Chemical Technology (IICT), Uppal Road, Tarnaka,
Hyderabad, 500007, India
‡
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India
§
Department of Applied Biology, CSIRꢀ Indian Institute of Chemical Technology, Hyderabadꢀ500007
KEYWORDS: Bodipy, fluorescent probes, mitochondrial staining, cytotoxicity
ABSTRACT: We report the first high affinity neutral Bodipy fluorophores for selective imaging of mitochondria with notable
sensitivity (~100 nM) and insignificant cytotoxicity even at very high concentration (~100 ꢁM), when tested against HeLa cells.
Further, these fluorophores are chemically robust and
require no
special conditions for storage.
1
0ꢀ13
Mitochondria are unusual intracellular organelles involved
in many cellular processes and play a key role in cell survival
and death. Their main function, however, is to provide cellular
energy in the form of ATP for various cellular needs. An imꢀ
balance in mitochondrial bioenergetics leads to formation of
excessive reactive oxygen species (ROS), that in turn cause
oxidative stressꢀmediated cellular responses. A wide range of
human diseases, such as metabolic disorders, cancers, cardioꢀ
vascular and neurological diseases are associated with abnorꢀ
ternate fluorescent mitochondrial probes.
Neto et al. have
succinctly discussed the relevance of neutral fluorophores by
incorporating strong electron acceptor groups in the molecular
skeleton. The low electron density (+δ) results in specific reꢀ
gime of a neutral structure, enabling the fluorophore to localꢀ
14
ize in the mitochondria. Borondipyrromethene (Bodipy)
derivatives show bright fluorescence, fair chemical stability
and structural features with magnificient photophysical
properties . They are often used as biological probes,
1
15
mal functions in mitochondria. Thus numerous therapeutic
fluorescent stains, and protein labelling reagents. Also,
applications targeting the mitochondria have been developed
and have been playing vital role in diagnosis and biomedical
research.
various cationicꢀBodipy dyes have been reported for staining
16ꢀ19
20
21
mitochondria tagged with TPP,
cis-platin, phosphonium,
2
22,23
and quarternized pyridine moieties.
The morphological changes in mitochondria in living cells
are typically discerned by fluorescence microscopy using miꢀ
tochondriaꢀselective dyes. A simple, yet efficient, approach to
distinguish mitochondria from other organelles is to take the
advantage of substantial negative electrochemical potential
The present communication reports the design and synthesis
of three neutral Bodipy derivatives tethered with
phenoxymethylpyridine unit namely BIP, CFꢀMONO, and CFꢀ
BI (Figure 1), which provide highly efficient molecules for
targeting mitochondria. To the best of our knowledge, this is
the first report on highly efficient neutral Bodipy based specifꢀ
ic mitochondrial staining.
(130ꢀ170 mV, negative side) across the inner mitochondrial
membrane. Based on this phenomenon, cationic lipophilic
3
dyes tagged with triphenylphosphonium (TPP) , 5,5’,6,6’ꢀ
tetrachloro 1,1’,3,3’tetraethylꢀbenzimidazol carbocyanine ioꢀ
4
5
dide (JCꢀ1), tetramethylrhodamine methyl ester (TMRM) and
several others were developed for studying mitochondrial
physiology. Majority of the commercial dyes used for mitoꢀ
6
7
chondrial imaging include rhodamine, and cyanine dyes sufꢀ
fer from various tradeꢀoffs such as low specificity, inhibiting
8
,9
mitochondrial respiration and high toxicity. However, most
of the commercially available cationic probes are chemically
o
unstable and thus require storage below ꢀ20 C. It is a wellꢀ
Figure 1. Molecular structures of Bodipy fluorophores
known fact that cationic molecules induce cytotoxicity by
imposing mitochondrial membrane depolarization leading to
unwarranted biological responses, thereby drastically limiting
their applications. On the other hand, extensive studies on
nonꢀcationic mitochondrial probes have shown that they have
a poor fluorescence image profile pertinent to explore for alꢀ
The
probes
CFꢀMONO
and
CFꢀBI
contain
hydroꢀ
oligoethyleneglycol moieties to maintain
a
philic−lipophilic balance and improved cell permeability. The
electron accepting group ꢀCF (trifluoromethyl group) has
3
been incorporated in the molecular structures in order to result
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 5
BIP
CFꢀMONO
CFꢀBI
+
δ character over a specific region, facilitating the fluorophore
BIP
CFꢀMONO
CFꢀBI
1
.0
.8
1.0
0.8
0.6
0.4
0.2
0.0
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
localization in mitochondria.
0
The choice of aldehydes 1 and 3 (Scheme 1) is the key for
designing the present series of Bodipy based probes. The
synthetic routes of compounds BIP, CFꢀMONO, and CFꢀBI
were presented in Scheme 1. The compound BIP was
synthesized by following classic one pot synthesis; by the acid
catalyzed condensation of aldehyde 1 with 2,4 dimethyl
pyrrole followed by the oxidation with DDQ to form dipyrrin,
which in turn complexed with BF .Et O in presence of
0.6
0
.4
.2
0
0.0
3
00
400
500
600
700
500
550
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
3
2
Figure 2. Normalized absorbance and emission spectra of
Bodipy fluorophores in acetonitrile
24
triethylamine (see details in supporting information (SI). The
compounds CFꢀMONO and CFꢀBI were synthesized by
Knoevenegal condensation of C /C methyl groups of CFꢀBDP
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. The photophysical properties of Bodipy fluorophores
3
5
(
for synthetic protocol see SI) with aldehyde 3 in presence of
a
b
c
d
Dye
Λ
max Abs
Λ
max Ems
ԑ
Φ
catalytic amount of piperidine and pꢀTsOH in toluene under
reflux condition. The monoꢀ and biꢀ condensations were
controlled by adjusting mole ratio of the reactants and
monitoring the reaction time, resulting CFꢀMONO and CFꢀBI
derivatives in 22 and 25% yields respectively (Scheme 1)
DCM ACN DMF DCM ACN DMF DCM
ACN
DMF
ACN
0.51
0.42
0.21
BIP
CFꢀMONO
CFꢀBI
502
576
652
498
570
645
501
576
654
514
595
672
510
589
666
515
598
675
1,37,671
1,14,438
1,94,100
89,562
1,22,947
1,26,865
81,707
1,45,454
1,16,333
a. Absorption maximum; b. Fluorescence emission maximum
(obtained by exciting at the absorption maximum of the dye); c.
Extinction coefficient at absorption maximum; d. Fluorescence
quantum yield calculated by a standard relative method using
BodipyꢀCO H, (ϕ = 0.57 in CH Cl ), Rhodamine B (ϕ = 0.70 in
(details are in SI). All the compounds were characterized by
1 13
NMR ( H, C) and HRMS (See SI).
2
2
2
CH OH), Zinc tertꢀbutyl phthalocyanine (ϕ = 0.37 in benzene).
3
Figure 3. Mitochondrial staining by Bodipy fluorophores in HeLa
cells: The cells were incubated with Bodipy fluorophores (100
nM) at 37 C for 30 min. A) Green fluorescence indicates mitoꢀ
0
chondrial staining of BIP compound by confocal microscopy
using FITC filter. Red fluorescence indicates mitochondrial stainꢀ
ing of Mitotracker Red CMXRos (Thermofisher) by confocal
microscopy by using rhodamine filter. Blue fluorescence indicates
nuclear staining by DAPI. B) Same as A, except that red fluoresꢀ
cence indicates mitochondrial staining of CFꢀMONO compound
by confocal microscopy by using rhodamine filter. C) Same as A,
except that red fluorescence indicates mitochondrial staining of
CFꢀBI compound by confocal microscopy by using rhodamine
filter.
Scheme 1. Synthetic route of Bodipy fluorophores
The photophysical properties of the Bodipy fluorophores
were recorded in three solvents of varying polarity, including
dicholoromethane (Figure S1, SI), acetonitrile (ACN, Figure
2
) and dimethylformamide (Figure S1, SI) at room
temperature.
Fluorescence microscopy experiments were conducted in
human cervical carcinoma (HeLa) cells to investigate The
ability of Bodipy fluorophores to stain mitochondria was
investigated in in human cervical carcinoma (HeLa) cells. The
intracellular distribution was observed through colocalization
imaging experiments. HeLa cells were incubated with these
Bodipy fluorophores (100 nM) at 37 C for 30 min. Figure 3
displays the fluorescence microscopic images of cells, and
after coincubation with both BIP and Mitotracker
CMXRos, a commercially available mitochondria selective
dye, from Thermofisher.
The normalized absorbance and emission spectra of these
fluorophores in acetonitrile are shown in Figure 2. All these
dyes possess small Stokes shifts (<25 nm) and maintain simiꢀ
lar photophysical properties in different solvents. Fluorescence
quantum yields were calculated using fluorescence emission
spectra, obtained from low concentration solutions (0.045ꢀ0.05
OD), by a standard relative method. BodipyꢀCO H, Rhodaꢀ
mine B and Zinc tertꢀbutyl phthalocyanine were used as referꢀ
ence for BIP, CFꢀMONO and CFꢀBI respectively.
photophysical data are presented in Table1. Superior pH inꢀ
sensitivity and photostability was exhibited by the Bodipy
fluorophores compared to the commercial Mitotracker Red
CMXRos (Figures S2 and S3 in SI).
0
2
TM
25ꢀ27
Red
The
The results of confocal microscopy show that all the newly
synthesized Bodipy fluorophores substantially stained the miꢀ
ACS Paragon Plus Environment

Page 3 of 5
ACS Medicinal Chemistry Letters
tochondria with marginal retention in the cytoplasmic comꢀ
BIP
FCCP+BIP
CFꢀMONO
FCCP+CFꢀMONO
CFꢀBI
FCCP+CFꢀBI
1
2
3
4
5
6
7
8
9
partment (Figure 3). However, no fluorescence was noticed in
the nuclear compartment. In contrast, slight fluorescence was
observed in the nuclear compartment of the cells with the
commercially available Mitotracker Red CMXRos (Figure 3),
implying that these Bodipy fluorophores are very specific in
staining mitochondria in living cells.
Figure 5. HeLa cells were preꢀtreated with FCCP (20 ꢁM) for 8h
before incubating with Bodipy fluorophores for 30 min (100 nM).
Images were taken with fluorescence microscope equipped with
Rhodamine and FITC filters.
The accumulation of these Bodipy fluorophores in the mitoꢀ
chondrial fraction was further confirmed by high resolution
mass spectrometry (HRMS). For HRMS studies, HeLa cells
were treated with 10 ꢁM Bodipy fluorophores for 24 h and the
cells were fractionated into cytosolic and mitochondrial fracꢀ
tions (see SI). From Table 2, it is evident that almost all ( >
99%) the Bodipy fluorophores are localized in the mitochonꢀ
drial fraction. HRMS spectra of mitochondrial and cytosolic
fractions are included in SI.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Cytosolic and mitochondrial uptake of Bodipy fluoroꢀ
phores (from HRMS)
Figure 6. HeLa cells were preꢀtreated with FCCP for 8h before
adding Bodipy fluorophores. Images were quantified with image
proplus software.
Dye
Cytosolic Fraction (N=3)
(nmole/ꢁg protein)
Mitochondrial Fraction (N=3)
(nmole/ꢁg protein)
BIP
1.84 ± 0.91
48.35 ± 9.2
0.0045 ± 0.81
6140.75 ± 300.94
6706.81 ± 163.09
4558.64 ± 229.0
In summary, highly specific neutral Bodipyꢀbased fluoroꢀ
phores with exclusive affinity towards mitochondria were
developed. The chemical robustness of these fluorophores
requires no special conditions for storage. These Bodipy
fluorophores present excellent mitochondrial targeting traits
with exceptionally low cytotoxicity compared to commercially
available mitochondrial probes. We believe, exploring and
commercialization of such fluorophores is highly needed for
better understanding of mitochondrial diseases.
CFꢀMONO
CFꢀBI
We next studied the cytotoxicity of all these compounds in
HeLa cells, treated at 100 ꢁM for 24 h. None of the syntheꢀ
sized compounds caused any noticeable cell death. On the
other hand, incubation of cells with 5 ꢁM CMXRos resulted in
significant cytotoxicity (Figure 4).
ASSOCIATED CONTENT
Supporting Information
1
20
100
The Supporting Information is available free of charge on the
ACS Publications website.
80
60
40
20
0
Experimental procedures, characterization spectra (PDF).
AUTHOR INFORMATION
Corresponding Author
Control
BIP
CFꢀMONO
CFꢀBI
Mitotracker Red
CMX Ros 5uM
*
Tel: +91ꢀ40ꢀ27191384, Eꢀmail: spsingh@iict.res.in
Figure 4. Effect of Bodipy fluorophores on cytotoxicity: HeLa
cells were treated with Bodipy fluorophores (100 ꢁM) and Therꢀ
mofisher’s Mitotracker Red CMXRos (5 ꢁM) for 24h and cell
viability was measured by SRB assay. Cell viability data above is
an average of three independent experiments.
* Tel: +91ꢀ40ꢀ27191867, Eꢀmail: giridhar@iict.res.in
ACKNOWLEDGMENT
TG and SaKa, thank UGC and ICMR respectively for providing
the senior research fellowship. We thank Dr. B. Surendar Reddy
for useful discussions. We sincerely thank Dr. A.C. Kunwar for
his helpful suggestions.
To examine whether the uptake of the dyes was dependent on
2
8
the mitochondrial membrane potential, cells were treated with pꢀ
trifluoromethoxyphenylhydrazone (FCCP) prior to the staining
procedure. FCCP is a mitochondrial uncoupler that causes rapid
acidification of the mitochondria and dysfunction of ATP synꢀ
thase and reduces the mitochondrial membrane potential (MMP or
ABBREVIATIONS
ACN, acetonitrile; ATP, adenosine triphosphate; Bodipy, boꢀ
rondipyrromethene; BF .Et O, boron trifluoride diethyl
3
2
etherate; DAPI, 4',6ꢀdiamidinoꢀ2ꢀphenylindole; DCM,
dichloromethane; DDQ, 2,3ꢀdichloroꢀ5,6ꢀ
Ψ ). All the probes showed considerable reduction in the mean
m
fluorescence intensity upon FCCPꢀinduced MMP depolarization
(Figure 5). The reduction in fluorescence for BIP, CFꢀMONO and
CFꢀBI was 48%, 38 % and 38% respectively (Figure 6). This
result indicates that these fluorophores definitely localize in mitoꢀ
chondria and its cellular uptake is sensitive to the mitochondrial
membrane potential.
dicyanobenzoquinone; DMF, dimethylformamide; FCCP, pꢀ
trifluoromethoxy phenylhydrazone; FITC, fluorescein isothioꢀ
cyanate; HeLa, human cervical carcinoma; HRMS, high
resolution mass spectrometry; JCꢀ1, 5,5’,6,6’ꢀtetrachloroꢀ
1,1’,3,3’tetraethylꢀbenzimidazolcarbocyanine iodide; MMP,
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 5
mitochondrial membrane potential; NMR, nuclear magnetic
resonance; OD, optical density; ROS, reactive oxygen speꢀ
cies; SRB, sulforhodamine B; TFA, trifluoroacetic acid; pꢀ
TsOH, pꢀtoluenesulfonic acid; TMRM, tetramethylrhodamin
emethylester; TPP, triphenyl phosphonium.
excitable BODIPY fluorophores readily modifiable for molecuꢀ
lar probes. J. Org. Chem., 78, 9153−9160.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
1
7. Cheng, G., Fan, J., Sun, W., Sui, K., Jin, X., Wang, J., and Peng,
X. (2013) A highly specific Bodipyꢀbased probe localized in miꢀ
tochondria for HClO imaging. Analyst, 138, 6091ꢀ6096.
8. Sui, B., Tang, S., Woodward, A. W., Kim, B., Belfield, K. D.
(2016) A Bodipyꢀbased waterꢀsoluble fluorescent probe for miꢀ
tochondria targeting. Eur. J. Org. Chem., 2851–2857.
9. Dodani, S. C., Leary, S. C., Cobine, P. A., Winge, D. R., and
Chang, C. J. (2011) A targetable fluorescent sensor reveals that
copperꢀdeficient SCO1 and SCO2 patient cells prioritize mitoꢀ
chondrial copper homeostasis. J. Am. Chem. Soc., 133, 8606–
8616.
20. Sun, T., Guan, X., Zheng, M., Jing X., and Xie, Z. (2015) Mitoꢀ
chondriaꢀlocalized fluorescent Bodipyꢀplatinum conjugate. ACS
Med. Chem. Lett., 6, 430−433.
21. Nigam, S., Burke, B. P., Davies, L. H., Domarkas, J., Wallis, J.
F., Waddell, P. G., Waby, J. S., Benoit, D. M., Seymour, A. M.,
Cawthorne, C., Higham, J. L., and Archibald, S. J., (2016) Strucꢀ
turally optimised Bodipy derivatives for imaging of mitochonꢀ
drial dysfunction in cancer and heart cells. Chem. Commun., 52,
7114ꢀ7117.
REFERENCES
1
.
Zielonka, J., Joseph. J., Sikora, A., Hardy, M., Ouari, O., Vivar,
J. V., Cheng, G., Lopez, M., and Kalyanaraman, B. (2017) Mitoꢀ
chondriaꢀtargeted triphenylphosphoniumꢀbased compounds: synꢀ
theses, mechanisms of action, and therapeutic and diagnostic
Applications. Chem. Rev., 117, 10043ꢀ10120.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
.
.
Nunnari, J., and Suomalainen, A. (2012) Mitochondria: in sickꢀ
ness and in health. Cell, 148, 1145–1159.
Murphy, M. P. (2008) Targeting lipophilic cations to mitochonꢀ
dria. Biochimica et BiophysicaActa, 1028–1031.
4. Cossarizza, A., Contri, M. B., Kalashnikov, G., and Franceschi,
C. (1993). A new method for the cytofluorimetric analysis of miꢀ
tochondrial membrane potential using the Jꢀaggregate forming
lipophilic
cation
5,5',6,6'ꢀtetrachloroꢀ1,1',3,3'ꢀ
tetraethylbenzimidazol carbocyanine iodide (JCꢀ1). Biochem Bi-
ophys Res Commun., 1, 40ꢀ45.
5. Floryk D., and Houstek J. (1999) Tetramethyl rhodamine methyl
ester (TMRM) is suitable for cytofluorometric measurements of
mitochondrial membrane potential in cells treated with digitonin.
Biosci Rep.,1, 27ꢀ34.
22. Zhang, S., Wu, T., Fan, J., Li, Z., Jiang, N. J. Wang, B. Dou,
Sun, S., Song, F., and Peng, X. (2013)
A Bodipyꢀ
based fluorescent dye for mitochondria in living cells, with low
cytotoxicity and high photostability. Org. Biomol. Chem., 11,
555ꢀ558.
23. Jiang, N., Fan, J., Liu, T., Cao, J., Qiao, B., Wang, J., Gao, P.,
and Peng, X. (2013) A nearꢀinfrared dye based on BODIPY for
tracking morphology changes in mitochondria Chem. Commun.,
49, 10620ꢀ10622.
24. Wang, M., Zhang, Y., Wang, T., Wang, C., Xue, D., and Xiao, J.
(2016) Story of an ageꢀold reagent: an electrophilic chlorination
of arenes and heterocycles by 1ꢀchloroꢀ1,2ꢀbenziodoxolꢀ3ꢀone.
Org. Lett., 18, 1976–1979.
6
.
Norman, J. P., Perry, S. W., Barbieri, J., Brown, E. B., and Gelꢀ
bard, H. A. (2011) Mitochondrial membrane potential probes
and the proton gradient: a practical usage guide. BioTechniques,
50, 98–115.
Martin P., and Fei, M. Cyanine dyes that stain cells and mitoꢀ
chondria. US 6291203 B1.
Nicholls, D. G., Ward, M. W. (2000) Mitochondrial membrane
potential and neuronal glutamate excitotoxicity: mortality and
millivolts. Trends Neurosci., 23, 166–174.
7
8
.
.
25. Brouwer, A. M. (2011) Standards for photoluminescence quanꢀ
tum yield measurements in solution. Pure Appl. Chem., 83,
2213–2228
9. Rimpelova, S., Briza, T., Kralova, J., Zaruba, K., Kejik, Z., Cisaꢀ
rova, I., Martasek, P., Ruml, T., and Kral, (2013) V. Rational deꢀ
sign of chemical ligands for selective mitochondrial targeting.
Bioconjugate Chem., 24, 1445–1454.
0. Lim, S. H., Thivierge, C., Sliwinska, P. N., Han, J., Bergh, H.
V., Wagnieres, G., Burgess, K., and Lee, H. B. (2010) In Vitro
and In vivo photocytotoxicity of boron dipyrromethene derivaꢀ
tives for photodynamic therapy. J. Med. Chem., 53, 2865–2874.
1. Jose, J., Loudet, A., Ueno, Y., Barhoumi, R., Burghardtb R. C.,
and Burgess, K. (2010) Intracellular imaging of organelles with
new waterꢀsoluble benzophenoxazine dyes. Org. Biomol. Chem.,
8, 2052–2059.
12. Neto, B. A. D., Correa, J. R., Carvalho, P. H. P. R., Santos, D. C.
B. D., Guido, B. C., Gatto, C. C., De Oliveira, H. C. B., Fasciotꢀ
ti, M., Eberlinb, M. N., and da Silva Jr. E. N., (2012) Selective
and efficient mitochondrial staining with designed 2,1,3ꢀ
benzothiadiazole derivatives as live cell fluorescence imaging
probes. J. Braz. Chem. Soc., 23, 770ꢀ781.
13. Denora, N., Laquintana, V., Trapani, A., Suzuki, H., Sawada,
M., and Trapani, G. (2011) New fluorescent probes targeting the
mitochondriallocated translocator protein 18 kda (TSPO) as acꢀ
tivated microglia imaging agents. Pharm Res., 28, 2820–2832.
26. Hart, A. S., Bikram, K. C., Subbaiyan, N. K., Karr, P. A., and
D’Souza, F., Hart, A. S., Bikram, K. C., Subbaiyan, N. K., Karr,
P. A., and D’Souza, F. (2012) Phenothiazineꢀsensitized organic
solar cells: effect of dye anchor group positioning on the cell
performance. ACS Appl. Mater. Interfaces, 4, 5813ꢀ5820.
27. Lawrence D. S., and Whitten, D. G. (1996) Photochemistry and
photophysical properties of novel, unsymmetrically substituted
metallophthalocyanines. Photochem. Photobiol., 64, 923ꢀ935.
28. Yuan, H., Cho, H., Chen, H. H., Panagia, M., Sosnovik, D. E.,
and Josephson, L. (2013) Fluorescent and radiolabeled triꢀ
phenylphosphonium probes for imaging mitochondria. Chem.
Commun., 49, 10361−10363.
29. To, M. S., Aromataris, E. C., Castro, J., Roberts, M. L., Barritt,
G. J., and Rychkov, G. Y. (2010) Mitochondrial uncoupler
FCCP activates proton conductance but does not block storeꢀ
operated Ca(2+) current in liver cells. Arch. Biochem. Biophys.,
152ꢀ158.
1
1
1
4. Neto, B. A. D., Correaa, J. R., and Silva, R. G. (2013) Selective
mitochondrial staining with small fluorescent probes: imꢀ
portance, design, synthesis, challenges and trends for new markꢀ
ers. RSC Adv., 3, 5291–5301.
5. Kowada, T., Maeda, H., and Kikuchi, K. (2015) Bodipyꢀbased
probes for the fluorescence imaging of biomolecules in living
cells. Chem. Soc. Rev., 44, 4953ꢀ4972.
1
16. Zhang, X., Xiao, Y., Qi, J., Qu, J., Kim, B., Yue, X., and Belꢀ
field, K. D. (2013) Longꢀwavelength, photostable, twoꢀphoton
ACS Paragon Plus Environment

Page 5 of 5
ACS Medicinal Chemistry Letters
Table of Contents art work
1
2
3
4
5
6
7
8
9
High Affinity Neutral Bodipy Fluorophores for Mitochondrial Tracking
†
, ‡
§, ‡
§, ‡
, †, ‡
Thumuganti Gayathri , Santosh Karnewar , Srigiridhar Kotamraju* , Surya Prakash Singh*
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
ACS Paragon Plus Environment