Synthesis and in vitro evaluation of a PDT active BODIPY-NLS conjugate

Article abstract of DOI:10.1016/j.bmcl.2013.03.128

Two new photosensitizers based on the BODIPY scaffold have been synthesized, of which one bears an NLS peptide, which is linked to the BODIPY's core using the copper catalysed azide-alkyne click reaction. The phototoxicities of these BODIPY based photosensitizers have been determined, as well as their dark toxicities. Although the conjugation of a single NLS peptide to the BODIPY did not lead to any observable nuclear localization, the photosensitizer did exhibit a superior photoxicity. Cellular co-localization experiments revealed a localization of both dyes in the lysosomes, as well as a partial localization within the ER (for the peptide-bearing BODIPY).

Full text of DOI:10.1016/j.bmcl.2013.03.128

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3204–3207
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro evaluation of a PDT active BODIPY–NLS
conjugate
Peter Verwilst a, Charlotte C. David b, Volker Leen a,b, Johan Hofkens b, Peter A. M. de Witte c,
Wim M. De Borggraeve a,
⇑
a Division of Molecular Design and Synthesis, Department of Chemistry, University of Leuven, Celestijnenlaan 200F, P.O. Box 2404, 3001 Heverlee, Belgium
b Division of Molecular Imaging and Photonics, Department of Chemistry, University of Leuven, Celestijnenlaan 200F, P.O. Box 2404, 3001 Heverlee, Belgium
c Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, University of Leuven, Herestraat 49, P.O. Box 824, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new photosensitizers based on the BODIPY scaffold have been synthesized, of which one bears an
NLS peptide, which is linked to the BODIPY’s core using the copper catalysed azide–alkyne click reaction.
The phototoxicities of these BODIPY based photosensitizers have been determined, as well as their dark
toxicities. Although the conjugation of a single NLS peptide to the BODIPY did not lead to any observable
nuclear localization, the photosensitizer did exhibit a superior photoxicity. Cellular co-localization exper-
iments revealed a localization of both dyes in the lysosomes, as well as a partial localization within the ER
(for the peptide-bearing BODIPY).
Received 20 February 2013
Revised 27 March 2013
Accepted 30 March 2013
Available online 8 April 2013
Keywords:
Nuclear localization sequence
BODIPY
Ó 2013 Elsevier Ltd. All rights reserved.
Photodynamic therapy
Singlet oxygen
Confocal microscopy
Photodynamic therapy (PDT) is a minimally invasive cancer
treatment depending on three elements: a photosensitizer, light
and oxygen.1 Administration of the photosensitizer (either topi-
cally or systemically) is followed by local irradiation with visible
light, resulting in the formation of singlet oxygen (1O) and other
reactive oxygen species (ROS).2–5 The formation of ROS leads to
oxidative damage, resulting in cell death either through apoptosis
or necrosis.3,6,7
substitution patterns lead to a very broad range of absorbance max-
ima of these dyes.19,20
Due to short lifetime of 1O in aqueous media (0.6
l
s), resulting
in a diffusion distance of 0.1
l
m,21 the localization of the photo-
sensitizers within the cell is of vital importance. It is known that
the DNA contained in the nucleus is far more sensitive to the oxi-
dative damage of singlet oxygen than the phospholipids of the
plasma membrane.22,23
Nuclear localization sequences (NLS) are short peptides govern-
ing the active transport of proteins to the nucleus of the cell, of
Due to the body’s therapeutic window (650–900 nm),8–10
photosensitizers that are systemically administered are generally
designed to absorb red light, whereas light with a shorter wave-
length can be used when tissue penetration is not necessary (e.g.
treatment of carcinoma of the bladder wall).11
which the NLS of the simian virus (SV40) large
T antigen
(PKKKRKV) was the first to be discovered.24–27 In order for the
NLS to work, the NLS conjugate must be located in the cyto-
plasm28,29 and since most synthetic NLS conjugates undergo endo-
somal uptake, it is vital for the conjugates to escape from these
vesicles.30,31 Previously a wide range of synthetic molecules, such
as peptide-nucleic acids (PNAs),32–36 gold nanoparticles,37 metal
complexes,38–40 fullerene41 and porphyrins (or tetrapyrrole macro-
cycles)42–48 have been conjugated to the SV40 large T antigen NLS,
with variable success, most likely due to the inability of certain
conjugates to escape the endosomes or lysosomes following endo-
cytosis. A hypothesis that was further confirmed by the successful
nuclear localization of various conjugates inserted by microinjec-
tion26,49,50 and streptolysin O based reversible membrane perme-
abilization,51 thus circumventing the endocytosis pathway. The
successful or unsuccessful nuclear delivery of many of the NLS
Most of the photosensitizers currently used in clinical environ-
ments are tetrapyrrole macrocycles. The drawback of this class of
molecules however, is the cumbersome synthesis and purification,
as well as the relatively low absorbance of these dyes.12 The groups
of O’Shea,12 Nagano,13 Akkaya,14 Burgess15 and Monti16 previously
demonstrated that halogenated (aza) BODIPYs (boron dipyrrometh-
enes), utilizing the internal heavy atom effect,17,18 are viable candi-
dates as photosensitizers, generally exhibiting higher absorbances
and excellent singlet oxygen producing abilities. Furthermore the
synthesis of these dyes is relatively straightforward and different
⇑
Corresponding author. Tel.: +32 16 3 27990; fax: +32 16 32 76 93.
E-mail address: wim.deborggraeve@chem.kuleuven.be (W.M. De Borggraeve).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.128

P. Verwilst et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3204–3207
3205
conjugates above was established by covalent attachment of
fluorescein38–41 or rhodamine36 to the conjugates. Remarkably,
conjugates simply consisting of fluorescein and an NLS did not
exhibit nuclear localization (and did not escape the endosomes
or lysosomes),38 whereas the structurally related rhodamine dye
of a rhodamine–NLS conjugate did exhibit nuclear localization,36
even in the case where the NLS peptide was replaced by a hybrid
PNA–peptide mimetic. Also noteworthy is that, although the
NLS-conjugated porphyrins did not manage to enter the nucleus,
the presence of an NLS peptide did result in increased
phototoxicities.47,48
Here, we report the synthesis of a dihalogenated BODIPY, conju-
gated with an NLS peptide utilizing the chemical orthogonality of
the copper catalysed azide–alkyne cycloaddition,52,53 as well as a
reference dye, also containing a triazole. We study the phototoxic-
ity and subcellular localization behavior of these BODIPY dyes after
endocytosis to assess the ability of these BODIPY dyes to undergo
nuclear localization.
The synthesis of the NLS containing BODIPY (3) and the refer-
ence molecule (2) was initiated by the deprotection of a TMS (tri-
methylsilyl) protected acetylene containing diiodoBODIPY (1)54 by
tetra-n-butyl ammonium fluoride in THF (tetrahydrofuran) at
À78 °C. The deprotected BODIPY subsequently underwent a copper
(I) catalyzed azide–alkyne cycloaddition (also known as the click
reaction),52,53 using tetrakis(acetonitrile)copper(I) hexafluoro-
phosphate as a copper source. The reaction was carried out under
an inert atmosphere and protected from the light, to eliminate the
risk of decomposition due to singlet oxygen formation. The reac-
tion proceeded in an excellent yield in the case of 2. By contrast,
the reaction yielding 3 only resulted in a 19% yield, which may
be explained by
p
–
p
stacking of the unreacted BODIPY (or its
decomposition products) with the target molecule, preventing full
recovery of this molecule after the filtration of the reaction mixture
(Scheme 1).
The absorption and emission spectra of the halogenated BOD-
IPY 2 were determined in methanol (Fig. S1, see supplementary
material). The absorption spectrum reveals
a maximum at
531 nm and the emission spectrum (upon excitation at 531 nm)
presents a maximum at 549 nm, resulting in a Stokes shift of
18 nm. The quantum yield of fluorescence of 2 in methanol was
determined relative to rhodamine 6G in ethanol and was found
to be 2%, which is in good accordance with the reported quantum
yield of fluorescence of a previously reported diiodinated BODI-
PY.13 A virtually identical absorbance and emission profile was
found for 3 (Fig. S2, see supplementary material). In order to as-
sess 1O production efficiency of dyes 2 and 3, the 1O lumines-
cence emission of both dyes was determined relative to Rose
Bengal (RB) in dimethylformamide (DMF) (Table 1). Judging from
these corrected relative emission intensities, both BODIPY dyes
show a similar 1O generation capacity. The relative emission
intensities are in good accordance with the proportion of the 1O
luminescence emission of the diiodinated BODIPY core versus
RB in MeOH.13
The in vitro phototoxic activity of 2 and 3 against T24, a human
urinary bladder carcinoma cell line,55 was determined after an irra-
diation with 4 J/cm2 of a broad spectrum light using an MTT (meth-
ylthiazolyl-diphenyltetrazolium bromide) assay.15,56
A second
assay in which the cells were not irradiated was also carried out
to determine the dark toxicity. The IC50 (the concentration of sen-
sitizer that inhibits the proliferation rate by 50% compared to cells
Scheme 1. Synthesis of BODIPYs 2 and 3.

3206
P. Verwilst et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3204–3207
Table 1
a clear co-localization with the lysosomes (Fig. S4, see supplemen-
tary material), as well as a weak overlap with the ER (endoplasmic
reticulum). As was the case with 2, 3 showed a localization with
the lysosomes. In contrast with 2, 3 also showed a better partial
overlap with the ER (Fig. 1).
The relative 1O emission intensities vs RB in DMF upon excitation at 532 nm; and the
in vitro phototoxicity of 1 and 2, at a dose of 4 J/cm2, as well as the dark toxicity
BODIPY Relative 1O luminescence emission vs. RB
IC50
0 J/cm2
(l
M)a
4 J/cm2
In view of the similar 1O generating proprieties of 2 and 3, the
difference in cellular localization seems to be the major factor
determining the phototoxicity of these BODIPY photosensitizers.
The presence of 3 in the ER points to the successful liberation of
(a fraction of) this peptide-bearing dye into the cytosol, in contrast
to 2. However, this did not lead to any significant nuclear uptake. A
concentration dependent increase in nuclear uptake could be
imagined, as was previously demonstrated for a ruthenium com-
plex bearing cationic peptide, showing nuclear uptake in concen-
2
3
1.76
1.61
>10
>10
0.042 0.004
0.016 0.003
a
The values represent the mean the standard deviation of 3 measurements.
that were not exposed to the sensitizer) of cells treated with 3 was
2.6 times lower than the IC50 of cells exposed to the reference mol-
ecule without the peptide (2) (Table 1 and Fig. S3, see supplemen-
tary material). Both compounds showed no signs of dark toxicity at
trations higher than 10 l
M.40 Yet, in the present case these
a concentration of 10 lM. These data show an increase in toxicity
of at least 240–625 times upon irradiation with 4 J/cm2 (for 2 and
3, respectively).
experiments would not show any representative cellular distribu-
tion, in view of the IC50 being lower by at least three orders of mag-
nitude. It has been previously observed that targeting
photosensitizers to lysosomes leads to the permeabilization of
these organelles, releasing the photosensitizer as well as hydrolytic
enzymes into the cytosol (which in turn might lead to the photo-
sensitization of tubulin)3,57; photosensitization of the more sensi-
tive ER has been reported to directly lead to apoptosis via
activation of the caspase cascade.58–60 Therefore, it would not be
unlikely that the latter pathway is the cause of the observed
In order to get a better understanding of the underlying cause of
the observed toxicity difference between the BODIPY substituted
with the NLS peptide and the one without, confocal laser scanning
microscopy was performed on T24 cells incubated with either 2 or
3. Co-staining images of the dyes and ER Tracker Blue-White DPX
or LysoTracker Blue DND-22 revealed that neither 2 or 3 showed
any signs of nuclear localization (judged from the absence of fluo-
rescence originating from the cell’s nucleus). The BODIPY 2 showed
Fig. 1. Subcellular localization of 3 in T24 cells at 100 nM for 30 min. Confocal fluorescence images (c and e) and fluorescence topographic profiles (d and f) of T24 cells
costained with 100 nM 3 and respective organelle tracers. (a) Transmission image of T24 cells using DIC. (b) Subcellular localization of 3. (c and d) Endoplasmic reticulum was
labelled with 100 nM ERTracker. (e and f) Lysosomes were labelled with 100 nM LysoTracker. White lines indicate the longitudinal transcellular axis analyzed to generate the
fluorescence topographic profiles. A scalebar of 30 lm is indicated in lightgrey.

P. Verwilst et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3204–3207
3207
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