Preparation of unsymmetrical distyryl BODIPY derivatives and effects of the styryl substituents on their in vitro photodynamic properties

Article abstract of DOI:10.1039/c1cc10727e

A series of unsymmetrical distyryl BODIPYs have been prepared which function as highly potent photosensitisers with in vitro IC₅₀ values as low as 15 nM. Their cellular uptake, subcellular localisation and photocytotoxicity depend greatly on th

Full text of DOI:10.1039/c1cc10727e

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 4748–4750
www.rsc.org/chemcomm
COMMUNICATION
Preparation of unsymmetrical distyryl BODIPY derivatives and effects
of the styryl substituents on their in vitro photodynamic propertiesw
Hui He,a Pui-Chi Lo,*a Sin-Lui Yeung,b Wing-Ping Fongb and Dennis K. P. Ng*a
Received 7th February 2011, Accepted 24th February 2011
DOI: 10.1039/c1cc10727e
A series of unsymmetrical distyryl BODIPYs have been prepared
which function as highly potent photosensitisers with in vitro
IC50 values as low as 15 nM. Their cellular uptake, subcellular
localisation and photocytotoxicity depend greatly on the styryl
substituents.
well as the effects of the styryl substituents on their in vitro
properties are described below.
Compounds 1–4 were prepared by sequential Knoevenagel
condensation of diiodo BODIPY 610 with two different aryl
aldehydes (Scheme 1). Treatment with bis(triethylene glycol)-
substituted benzaldehyde 7 first afforded the mono-styryl
tris(triethylene glycol)-appended BODIPY 8. The triethylene
glycol chains were introduced to enhance the hydrophilicity,
biocompatibility and cellular uptake of the dyes.11 It was then
followed by further condensation with aldehydes 9–12, respec-
tively, to give the corresponding distyryl BODIPYs 1–4.
Compounds 2 and 3 have a naphthyl or aminophenyl group
attached directly to the p system, which can further shift the
absorption to the red. Having a trialkylamino moiety, com-
pound 4 could be N-methylated readily with iodomethane to
give the cationic analogue 5 (Scheme 1). The experimental
details and the characterising data are given in ESI.w
The electronic absorption spectra of compounds 1–5 were
recorded in DMF (Fig. S1, ESIw) and the data are summarised
in Table 1. It can be seen that the Q bands of 2–5 are red-
shifted compared with that of 1, particularly for compound 3,
which exhibits strong intramolecular charge transfer (ICT)
due to the amino group.1b For all these compounds, the
Q-band absorbance strictly followed Lambert–Beer’s law [see
the spectra of 1 (Fig. S2, ESIw) for exemplification], which
suggested that they are essentially non-aggregated in DMF.
Upon excitation at 610 nm, all the compounds (except 3)
Distyryl boron dipyrromethene (BODIPY) derivatives can be
prepared readily by Knoevenagel condensation of 3,5-dimethyl
BODIPYs with aryl aldehydes.1 Having an extended p system,
these compounds absorb strongly at ca. 620–660 nm and emit
in the near-infrared region. These spectral properties can be
tuned readily by chemical modification of the p skeleton.
These characteristics, together with their high stability and
solubility in many solvent systems, render them to be promising
materials for various applications. In particular, these compounds
serve as near-infrared fluorescent probes for various analytes,2
building blocks for artificial photosynthetic models,3 sensitisers
for dye-sensitised solar cells4 and fluorophores for one- and
two-photon cell imaging.5 By incorporating heavy halogen
atoms onto the BODIPY core, these compounds behave as
efficient photosensitisers which can generate singlet oxygen
upon illumination.6 This interesting property suggests that
these compounds are potentially useful as photosensitisers
for photodynamic therapy, which is a promising therapeutic
modality for a variety of pre-malignant and malignant diseases.7
However, this research field has been virtually unexplored. To
our knowledge, only one example has been demonstrated to
exhibit photodynamic activity at the cellular level so far.8 We
report herein a new series of unsymmetrical distyryl BODIPY
derivatives (compounds 1–5). By introducing two different
styryl substituents, we hoped to impart a higher amphiphilicity
to the compounds which may facilitate their cellular uptake9
and control their subcellular localisation. The preparation and
basic photophysical properties of this series of compounds, as
a Department of Chemistry, The Chinese University of Hong Kong,
Shatin, N. T., Hong Kong, China. E-mail: pclo@cuhk.edu.hk,
dkpn@cuhk.edu.hk; Fax: +852 2603 5057
b School of Life Sciences, The Chinese University of Hong Kong,
Shatin, N. T., Hong Kong, China
w Electronic supplementary information (ESI) available: Experimental
details and characterising data, absorption and fluorescence spectra of
1–5 in DMF and DMEM; absorption spectra of 1 at various concen-
trations in DMF, cytotoxic effects of 1, 4 and 5, results of subcellular
localisation studies of 4 and 5, and 1H and 13C{1H} NMR and ESI mass
spectra of 1–5 and 8. See DOI: 10.1039/c1cc10727e
Scheme 1 Synthesis of unsymmetrical distyryl BODIPYs 1–5.
c
4748 Chem. Commun., 2011, 47, 4748–4750
This journal is The Royal Society of Chemistry 2011

View Article Online
Table 1 Electronic absorption and fluorescence emission data for compounds 1–5 in DMF
lem (nm)a
b
FF
Compound
lmax (nm) (log e)
1
2
3
4
5
340 (4.55), 450 (4.12), 653 (4.88)
677
688
—
695
686
0.19
0.15
—
0.18
0.15
340 (4.44), 390 (4.54), 465 (4.19), 659 (4.88)
339 (4.53), 410 (4.53), 517 (4.35), 709 (4.81)
327 (4.43), 382 (4.58), 453 (4.21), 665 (4.91)
327 (4.40), 377 (4.57), 450 (4.19), 661 (4.89)
a
b
Excited at 610 nm. Relative to ZnPc in DMF as the reference [fluorescence quantum yield (FF) = 0.28].
showed a fluorescence emission at 677–695 nm (Fig. S3, ESIw)
with a fluorescence quantum yield (FF) of 0.15–0.19 relative to
unsubstituted zinc(II) phthalocyanine (ZnPc) (FF = 0.28)
(Table 1).12 The Stokes shifts were in the range of 24–30 nm.
Compound 3 was essentially non-fluorescent as a result of ICT
in the excited state.1b
naphthyl group of 2 can induce a bathochromic shift on the
absorption and emission positions, the photocytotoxicity of
this compound is the lowest in this series. Compound 3 is also
a less efficient photosensitiser.
To account for their different photocytotoxicity, their aggre-
gation behavior in the Dulbecco’s modified Eagle’s medium
(DMEM) was first examined by absorption and fluorescence
spectroscopic methods (Fig. S5, ESIw). For all the compounds,
the Q band remained relatively sharp and an intense emission
band was observed (except 3, which was essentially non-
fluorescent due to ICT). These observations suggested that
these compounds remained relatively non-aggregated in the
culture medium.
The efficiency of these compounds in generating singlet
oxygen, as reflected by the rate of decay of the singlet-oxygen
quencher 1,3-diphenylisobenzofuran (DPBF) in DMF was
also compared. As shown in Fig. 1, all these distyryl BODIPYs
can induce photo-degradation of DPBF with a comparable
efficiency, which is also similar to that of ZnPc. The only
exception is compound 3, which is clearly a less efficient
singlet-oxygen generator. Presumably, the strong ICT process
shortens the singlet-state lifetime and disfavors the population
of triplet state to sensitise the formation of singlet oxygen.
The in vitro photodynamic activities of 1–5 in Tween
80 emulsions were then investigated against HT29 human
colorectal carcinoma cells. Fig. 2 shows their dose-dependent
survival curves. It can be seen that all of them are essentially
non-cytotoxic in the absence of light. However, upon illumina-
tion with red light (l 4 610 nm), they exhibit different degrees
of cytotoxicity. The IC50 and IC90 values, defined as the dye
concentrations required to kill 50% and 90% of the cells,
respectively, are summarised in Table 2. The photocytotoxicity
follows the order 4 E 5 4 1 4 3 4 2. Compound 4 and its
N-methylated derivative 5 are particularly potent with IC50
values of 15–17 nM, which are much lower than those of the
classical photosensitiser porfimer sodium (IC50 = 4.6 mg mLÀ1
under the same experimental conditions vs. 22 ng mLÀ1
for 4 and 5) and pheophorbide a (IC50 = 0.5 mM),13 and are
comparable with those of the most potent phthalocyanine-
based photosensitisers reported by us so far.14 Although the
The cellular uptake of these compounds was then examined
by fluorescence microscopy. HT29 cells were incubated respec-
tively with all these compounds (2 mM) for 2 h. Upon excitation
at 633 nm, the bright field and fluorescence (650–720 nm)
images of the cells were then captured (Fig. 3a), and the intra-
cellular fluorescence intensities were determined (Fig. 3b).
Compounds 4 and 5 showed much stronger intracellular
fluorescence throughout the cytoplasm. The intensity, which is
an indicator of the cellular uptake, followed the order: 4 E 5 4
1 4 2 E 3. Generally, it is in good agreement with the trend
observed for the photocytotoxicity. It is expected that 3 has a
very different fluorescence emission efficiency compared with
the other compounds. Hence its intracellular fluorescence
intensity may not directly reflect the cellular uptake. However,
in light of its low photocytotoxicity, we did not pursue further
the exact uptake of this compound, and put our focus on the
highly photocytotoxic 4 and 5.
For 4 and 5, their subcellular localisation was also investi-
gated. The cells were first incubated with either 4 or 5 in the
medium for 2 h, then stained with LysoTracker DND 26,
MitoTracker Green FM or ER-Tracker Green (for 10–30 min),
which are specific fluorescent dyes for lysosomes, mitochondria
and endoplasmic reticulum, respectively. As shown in Fig. 4,
both compounds show substantial intracellular fluorescence,
but compound 4 is distributed throughout the cytoplasm,
while compound 5 tends to localise in the cell membrane.
To take a closer examination, the fluorescence caused by 4
(excited at 633 nm, monitored at 675–720 nm) could superimpose
with the fluorescence caused by the LysoTracker (excited at
488 nm, monitored at 510–560 nm) and partially overlap with
the fluorescence caused by the MitoTracker (excited at 488 nm,
monitored at 510–560 nm), but not with that caused by
the ER-Tracker (Fig. S6, ESIw). The results suggested that
compound 4 has high affinity to the lysosomes and can also
bind to the mitochondria. For the cationic N-methylated
derivative 5, it is not exclusively localised in any organelles,
Fig.
1
Comparison of the rates of decay of DPBF (initial
concentration = 75 mM) in DMF, as monitored spectroscopically at
415 nm, using 1–5 as the photosensitisers (4 mM) and ZnPc (4 mM) as
the reference.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 4748–4750 4749

View Article Online
Fig. 4 Bright field (left column) and fluorescence (right column)
images of HT29 after incubation with 4 or 5 in the medium for 2 h.
Fig. 2 Comparison of the cytotoxic effects of 1 (squares), 2 (circles),
3 (triangles), 4 (rhombuses) and 5 (stars) on HT29 cells in the absence
(closed symbols) and presence (open symbols) of light (l 4 610 nm,
40 mW cmÀ2, 48 J cmÀ2). Fig. S4 (ESIw) shows the enlarged region
for 1, 4 and 5. Data are expressed as mean value Æ standard error
of the mean value (SEM) of three independent experiments, each
performed in quadruplicate.
In conclusion, we have prepared a new series of unsymmetrical
distyryl BODIPYs and evaluated their in vitro photodynamic
activities. It has been found that these properties depend
greatly on the styryl substituents. The amino derivative 4
and its N-methylated analogue are highly photocytotoxic with
IC50 values down to 15 nM. The high potency could be
attributed to their low aggregation tendency, high efficiency
in generating singlet oxygen and high cellular uptake. These
compounds, however, show a remarkably different subcellular
localisation property as a result of minor modification of the
substituent.
Table 2 IC50 and IC90 values of compounds 1–5 against HT29 cells
Compound
IC50 (nM)
IC90 (nM)
1
2
3
4
5
42
108
2600
1270
53
1000
470
17
This work was supported by a Direct Grant for Research
(2010/2011) of The Chinese University of Hong Kong.
15
34
Notes and references
1 (a) Z. Dost, S. Atilgan and E. U. Akkaya, Tetrahedron, 2006, 62,
8484; (b) A. Loudet and K. Burgess, Chem. Rev., 2007, 107, 4891.
2 (a) R. Guliyev, A. Coskun and E. U. Akkaya, J. Am. Chem. Soc.,
2009, 131, 9007; (b) O. A. Bozdemir, R. Guliyev, O. Buyukcakir,
S. Selcuk, S. Kolemen, G. Gulseren, T. Nalbantoglu, H. Boyaci
and E. U. Akkaya, J. Am. Chem. Soc., 2010, 132, 8029.
3 (a) X. Zhang, Y. Xiao and X. Qian, Org. Lett., 2008, 10, 29;
(b) R. Ziessel and A. Harriman, Chem. Commun., 2011, 47, 611.
4 (a) S. Erten-Ela, M. D. Yilmaz, B. Icli, Y. Dede, S. Icli and
E. U. Akkaya, Org. Lett., 2008, 10, 3299; (b) S. Kolemen,
Y. Cakmak, S. Erten-Ela, Y. Altay, J. Brendel, M. Thelakkat
and E. U. Akkaya, Org. Lett., 2010, 12, 3812.
5 (a) Q. Zheng, G. Xu and P. N. Prasad, Chem.–Eur. J., 2008, 14,
5812; (b) P. Didier, G. Ulrich, Y. Mely and R. Ziessel, Org. Biomol.
´
Chem., 2009, 7, 3639.
6 (a) S. Ozlem and E. U. Akkaya, J. Am. Chem. Soc., 2009, 131, 48;
(b) S. Erbas, A. Gorgulu, M. Kocakusakogullari and
E. U. Akkaya, Chem. Commun., 2009, 4956.
7 (a) D. E. J. G. J. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev.
Cancer, 2003, 3, 380; (b) S. B. Brown, E. A. Brown and I. Walker,
Lancet Oncol., 2004, 5, 497.
8 S. Atilgan, Z. Ekmekci, A. L. Dogan, D. Guc and E. U. Akkaya,
Chem. Commun., 2006, 4398.
9 I. J. MacDonald and T. J. Dougherty, J. Porphyrins Phthalocyanines,
2001, 5, 105.
10 H. He and D. K. P. Ng, Org. Biomol. Chem., 2011, DOI: 10.1039/
C0OB01252A.
11 J.-Y. Liu, X.-J. Jiang, W.-P. Fong and D. K. P. Ng, Org. Biomol.
Chem., 2008, 6, 4560.
12 I. Scalise and E. N. Durantini, Bioorg. Med. Chem., 2005, 13, 3037.
13 A. Hajri, S. Wack, C. Meyer, M. K. Smith, C. Leberquier,
M. Kedinger and M. Aprahamian, Photochem. Photobiol., 2002,
75, 140.
Fig. 3 (a) Bright field (lower row) and intracellular fluorescence
(upper row) images of HT29 cells after incubation with (i) 1, (ii) 2,
(iii) 3, (iv) 4 and (v) 5 (all at 2 mM) for 2 h. (b) Comparison of the
intracellular fluorescence intensities of compounds 1–5. Data are
expressed as mean value Æ standard deviation (number of cells = 50).
but shows bright fluorescence in the cell membrane (Fig. S7, ESIw).
It is likely that due to the cationic nature of this compound, it
is preferentially retained in the membrane’s lipid bilayer.
14 X.-J. Jiang, S.-L. Yeung, P.-C. Lo, W.-P. Fong and D. K. P. Ng,
J. Med. Chem., 2011, 54, 320 and references cited therein.
c
4750 Chem. Commun., 2011, 47, 4748–4750
This journal is The Royal Society of Chemistry 2011