BODIPY based 'click on' fluorogenic dyes: Application in live cell imaging

Article abstract of DOI:10.1016/j.tetlet.2013.11.003

The design, synthesis, and photophysical properties of new BODIPY-based fluorogenic 'click on' dyes are reported. CuAAC reaction of non-fluorescent BODIPY azide with a series of non-fluorescent alkyne molecules resulted in fluorescent triazoles which displayed up to 532-fold enhancement of fluorescence in the red region. Imaging studies confirmed the general trend of cell permeability and a cholesterol linked derivative exhibited selective localization into intracellular membranes.

Full text of DOI:10.1016/j.tetlet.2013.11.003

Accepted Manuscript
BODIPY based “click on” fluorogenic dyes: Application in live cell imaging
Dinesh Pratapsinh Chauhan, Tanmoy Saha, Mayurika Lahiri, Pinaki Talukdar
PII:
S0040-4039(13)01939-4
DOI:
http://dx.doi.org/10.1016/j.tetlet.2013.11.003
TETL 43798
Reference:
Tetrahedron Letters
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Revised Date:
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2 November 2013
5 November 2013
Please cite this article as: Chauhan, D.P., Saha, T., Lahiri, M., Talukdar, P., BODIPY based “click on” fluorogenic
dyes: Application in live cell imaging, Tetrahedron Letters (2013), doi: http://dx.doi.org/10.1016/j.tetlet.
2013.11.003
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Application in live cell imaging
Dinesh Pratapsinh Chauhan,‡a Tanmoy Saha,‡a Mayurika Lahirib and Pinaki Talukdar

Tetrahedron Letters
journal homepage: www.elsevier.com
BODIPY based “click on” fluorogenic dyes: Application in live cell imaging
Dinesh Pratapsinh Chauhan,‡a Tanmoy Saha, ‡a Mayurika Lahirib and Pinaki Talukdara,
a
Department of Chemistry, Mendeleev Block, Indian Institute of Science Education and Research, Pune, 411008, India.
Department of Biology, Indian Institute of Science Education and Research, Pune, 411008, India.
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The design, synthesis and photophysical properties of new BODIPY-based fluorogenic “click
on” dyes are reported. CuAAC reaction of non-fluorescent BODIPY azide with a series of non-
fluorescent alkyne molecules resulted in fluorescent triazoles which displayed upto 532-fold
enhancement of fluorescence in the red region. Imaging studies confirmed the general trend of
cell permeability and a cholesterol linked derivative exhibited selective localization into
intracellular membranes.
Available online
Keywords:
BODIPY
2009 Elsevier Ltd. All rights reserved.
Azide-Alkyne Click Reaction
Fluorescence
Solvatochromic Properties
Cell Imaging
Traditional small-molecule fluorescent probes used for
labelling and sensing of chemical and biological events, exhibit
an “always on” fluorescence which can mask valuable outcome
of the experiments. To overcome this limitation, “latent”
fluorophores are reacted with the molecules of interest, resulting
in “turn on” of fluorescence. This phenomenon, also termed as
fluorogenic reaction, has been used as a powerful analytical tool
in diverse chemical context, e.g. detection of phenols,1
thiophenols2 and nitrosothiols in water3 and acetylenes and
copper in polymers,4 etc. Biological applications of fluorogenic
transformations are associated with labelling, detection and
sensing of biothiols,5 H2S,6 DNA,7 proteins,8 single biocatalytic
events and bioconjugation processes.9 Among various
fluorogenic bioconjugation methods, copper(I)-catalyzed azide-
alkyne cycloaddition (CuAAC) reaction or click reaction,10 and
its non-catalytic, strain-promoted versions with either
cyclooctynes11 or oxanorbornadienes12 have gained burgeoning
attention.
lone pair is part of the aromatic system) and induces fluorescence
activation.13 In 2004, Wang and coworkers reported series of 3-
azidocoumarins e.g. 1 in which the fluorescence is quenched due
to the electron-rich α-nitrogen of the azido group.13a CuAAC
reactions of these alkynes with various non-fluorescent alkynes
resulted in the formation of fluorescent triazoles. In 2006, Wong
and coworkers reported the 4-azido-1,8-naphthalimide 2 in which
represents a non-fluorescent state.13b The CuAAC reaction of 2
with alkyne-linked sugars afforded strongly fluorescent triazolo-
compounds. In 2008, Xie et. al.
The design of CuAAC reaction based fluorogenic probes are
focused on the construction of either an alkyne or azide
connected to a profluorophore to provide the quenched state. The
probe after reacting with a cycloaddition partner provided a
triazole compound which displayed strong fluorescence. Often, a
photoinduced electron transfer (PET) process from the azide
moiety (from nitrogen lone pair) to the profluorophore is
responsible for the quenching of fluorescence (Figure 1A). On
Figure 1. CuAAc based fluorogenic click reaction (A). Structures of
fluorogenic azides 1 - 4 (B).
t—he—oth—er hand, triazole does not permit the PET process (as the
‡ Equal contributions of both authors.
 Corresponding author. Tel.: +91-20-25908098; fax: +91-20-25865315; e-mail: ptalukdar@iiserpune.ac.in

reported azido-anthracene 3 to act as fluorogenic probes via the
CuAAC reaction.13c Recently, Bertozzi and several other groups
applied the concept of copper-free click reaction which has been
extensively used for bioconjugation and fluorogenic probe
development.14
7i were re-crystallized from CH2Cl2/hexane system and single
crystal X-ray diffraction data also confirmed assigned structures
of triazoles 7a (Figure S1), 7b (Figure S2), 7c (Figure 2A), 7f
(Figure 2B) and 7i (Figure S5).
Herein, we report the design and synthesis of a new boron-
dipyrromethene (BODIPY) based fluorogenic probe 4 with an
azide connected at the 2-position of the BODIPY ring.
Advantages due to the intense absorption in visible light,
relatively high molar extinction coefficient (ε), biocompatibility,
chemical and photochemical stability encouraged us to explore
the “click on” chemistry with BODIPY fluorophore.15 Synthesis
of the azide 4 was carried out from the 3-bromo-BODIPY 5 by
reacting with NaN3 in acetone/H2O (1:1) with 84% yield
(Scheme 1).6b The click reactions were carried out on azide 4
with various alkyne analogues 6a - 6k. Selection of these
substituents were based on choice of aliphatic (6h and 6i) and
aromatic ones (6a - 6g) with additional possibility of further
derivatization (6b, 6c and 6i). Examples also covered a
representative protected sugar analogue (6j). Considering the
importance of BODIPY-cholesterol in sterol trafficking in living
cells and organisms,16 a substituent consisting of cholesterol side
chain (6k) was also selected. Reactions were carried out in
presence of CuSO4 and sodium ascorbate in CH2Cl2/H2O (2:1) at
room temperature and stirred for 8 - 12 h to furnish triazoles 7a -
7k with yields ranging from 30 to 90%. Relatively lower yields
in few cases were possibly due to the reduction of BODIPY-
azide 4 to corresponding BODIPY-amine in presence of sodium
ascorbate also acting as a reducing agent.17 This possibility can
be rationalized by the electron withdrawing nature of the
BODIPY ring which contributes to the reduction of the azide 4
during triazole formation reaction.18
Figure 2. Crystal structures of (A) 7c and (B) 7f.
All BODIPY derivatives displayed appreciable solubility in
water and ranges of organic solvents. Photophysical properties of
the azide 4 (10 μM) and triazoles 7a - 7k (10 μM each) were
recorded in HEPES buffer (10 mM, pH = 7.4). In solution, all the
compounds showed a strong S0S1 (ππ*) transition between
502 - 540 nm (Figure S6 - S17), unambiguously assigned to the
boradiazaindacene chromophore.19 At higher energy, a weaker
band around 370 - 390 nm, can be attributed to the S0S2
(ππ*) transition of the BODIPY (Figure S6-S17). Although,
high molar absorption coefficient, ε = 23190 M-1 cm-1 was
calculated for the azide 4, it did not exhibit any significant
2
fluorescence when excited at its λmax value. The off-fluorescence
can be corroborated to the PET mechanism from the azide to the
BODIPY core.13a On the other hand, trizoles 7i and 7j exhibited
2
λem = 530 and 528 nm, respectively when excited at respective λmax
values. Other triazoles displayed relatively higher λem in the range
of 574 - 650 nm (Table 1). All triazoles when excited at
1
respective λmax values, exhibited emission spectra identical to
2
those observed during λmax excitations (Figure S6 - S17). The
turn-on fluorescence of 7a - 7k confirms the deactivation of the
PET pathway due to conjugation of nitrogen (N1) lone pair in
triazole ring (Figure 1A). In HEPES (10 mM, pH = 7.4) buffer,
azide 4 displayed quantum yield, Φ =  0 (standard: rhodamine G
in water, Φ = 0.76). On the other hand, appreciable high Φ =
0.013, 0.025 and 0.021 were determined for 7c, 7i, and 7j,
respectively (in HEPES buffer, 10 mM, pH = 7.4). Other triazole
derivatives provided Φ values ranging from 0.02 - 0.005 (Table
1).
Table 1. Photophysical properties of azide 4 and triazoles 7a-7k in
HEPES buffer (10 mM, pH = 7.4).
2
2 b
BODIPY
λmax1 (nm)
λmax
ε a
(M-1 cm-1)
23190
34500
30560
27200
22700
15880
9410
21290
24830
12250
13830
λem
Φ
(nm)
515
538
534
538
538
540
535
540
530
502
502
535
(nm)
0
4
370
390
390
390
390
380
390
377
380
383
380
380
0
7a
7b
7c
7d
7e
7f
7g
7h
7i
580
575
580
580
594
582
650
574
530
528
578
0.012
0.005
0.013
0.013
0.008
0.018
0.002
0.019
0.025
0.021
0.002
7j
7k
7500
a
2
b
Calculated at corresponding λmax value.
Fluorescence spectra of
compounds were recorded at 10 M concentrations in HEPES buffer ( with
0.5% DMSO) at pH = 7.4.
Scheme 1. Synthesis of the probe BODIPY-triazole derivatives 7a -
7k.
The quantitative fluorescence intensity enhancement for
triazoles 7a - 7k (10 μM each) were determined relative to the
azide 4 (10 μM) at 580 nm in HEPES buffer (10 mM, pH = 7.4).
This data indicated a 532-fold fluorescence enhancement for
All the synthesized triazole derivatives were characterized by
NMR and Mass spectrometric analysis. Triazoles 7a - 7c, 7f and

triazole 7i (Figure 3). Similarly, 334- and 332-fold enhancements
in fluorescence were observed for triazoles 7a and 7c,
respectively. For other triazoles, the increase in fluorescence
intensity varied from 15- to 300-fold.
between the excited state of 7c and H2O may be responsible for
the stabilization of the state compared to that in other solvents.
As a result, bathochromic shifts in both λmax and λem were
observed in the solvent leading to a large Δν = 1390 cm-1.
Table 2. Solvatochromic properties of 7a (10 μM) in various
solvents.
Solvent
P’ a
λex
(nm)
531
532
524
528
518
516
517
535
νex
λem
(nm)
545
548
540
545
537
538
542
578
νem
Δλ
Δν
(cm-1)
18832
18796
19083
18939
19305
19379
19342
18691
(cm-1)
18348
18248
18518
18348
18621
18587
18450
17301
(nm) (cm-1)
Cyclohexane
CCl4
0.2
1.6
3.9
4.1
5.1
5.8
6.4
10.2
14
16
16
17
19
22
25
43
483
548
565
590
683
792
892
1390
2-Propanol
Chloroform
Methanol
Acetonitrile
DMF
Water
a Solvent polarity index.
Figure 3. Comparison of fluorescence intensities (at λ = 580 nm with
excitation at respective λmax values) of BODIPY triazoles 7a - 7k (10
μM each) relative to the azide 4 (10 μM).
Naked eye detection of fluorogenic click reactions was
studied in the next stage. Briefly, first 12-wells of a 24-well plate
were used in which each well represents either azide 4 or a single
triazole product (50 µM) in HEPES (10 mM, pH = 7.4). All
BODIPY compounds 4 and 7a - 7k displayed pink color under
ambient light (Figure 4A). When placed under the hand-held UV
lamp (λex = 365 nm), the well containing the azide 4 remained
dark (Figure 4B). Significantly, low fluorescence illuminations
were observed for 7e and 7g as expected from the Figure 3.
Triazoles 7i and 7j exhibited green fluorescence which
corroborates to their observed λem values. Other triazoles (7a-7d,
7f, 7h and 7k) exhibited fluorescence in the red region.
Figure 5. Major contributing resonance structures of BODIPY-
triazole.
Red shifted excitation wavelength and low cytotoxicity of
BODIPY inspired us to evaluate applicability of synthesized
analogs as fluorescent markers of specific cellular organism.
Initial screening of cell permeability was conducted with 7a
(Figure S19A). Based on the higher fluorescence intensity and Φ
values BODIPY-triazoles 7c, 7f and 7i were selected for live cell
imaging studies. On the other hand, 7k bearing a cholesterol unit
with BODIPY-triazole core was selected to evaluate its
membrane accumulation. When each of triazoles 7c, 7f and 7i
(500 nM in 1:1000 DMSO/DMEM v/v, pH = 7.4) was separately
Figure 4. Naked eye detection of change in color (A) and
fluorescence (B) of 7a - 7k (50 μM each) compared to 4 (50 μM).
Photographs were taken either under ambient light (A) or under a
hand-held UV lamp (B) with λex = 365 nm.
o
incubated with the HeLa cells at 37 C for 30 min followed by
washing with PBS, good permeability of these compounds into
the cell and significant fluorescence was observed (Figure 5 and
Figure S19).
Further we studied the solvatochromism properties of
BODIPY triazole 7c (10 μM). This derivative was selected
because of its relatively high ε and Φ values. Absorption and
fluorescence spectra of 7c were recorded in various solvents
including polar, nonpolar, protic and aprotic ones (Table 2,
Figure S18). For the triazole, a decreasing order of λmax was
observed with the increase in solvent polarity (i.e. λmax = 531 nm
in cyclohexane and λmax = 517 nm in DMF). However, in H2O a
significantly high λmax = 535 nm was observed. BODIPY 7c
displayed comparable λem values (537 - 545 nm) in all solvent,
except in H2O (λem = 578 nm). For a symmetrical BODIPY
derivative, two identical zwitterionic structures I and II (ignoring
the triazole ring) are possible (Figure 5). However, introduction
of triazole ring contributes an additional canonical form III with
relatively higher dipole moment. The observed trend in λmax value
(from cyclohexane to DMF) infers a better stabilization of the
ground state in polar solvent compared to the excited state
leading to decrease in λmax. Therefore, the observed increasing
trend Stoke shift, Δν (i.e. Δν = 483 cm-1 in cyclohexane and Δν =
892 cm-1 in DMF) can also be corroborated to the stabilization of
the ground state.20 However, contribution from hydrogen bonding
Figure 6. Images of HeLa cells: brightfield (A), fluorescence (B),
and overlay (C), incubated with probe 7c (500 nM) for 30 min. (D-
F) are the respective brightfield, fluorescence and overlay image of
HeLa cells incubated with probe 7f (500 nM) for 30 min.
When permeability of the cholesterol linked BODIPY-triazole
7k (500 nM) was tested in HeLa cells after 30 minutes of
incubation, its predominant accumulation at the nuclear
membrane was observed (Figure 7A, B). This was further

confirmed (Figure 7D) by marking the location of the nucleus
with Hoechst stain (Figure 6C, blue emission) and overlaying
with cell image of probe 7k (Figure 7D, red emission). However,
localization of the BODIPY at other intracellular membranes
cannot be ruled out. No significant accumulation of 7k in the
plasma membrane was detected. This inference was achieved by
comparing the DIC and fluorescence image for a particular cell.
This is probably due to preferential adherence of cholesterol into
the double lipid bilayer of nuclear membrane over single lipid
bilayer of the cellular membrane.
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