Synthesis and nanoparticle encapsulation of 3,5-difuranylvinyl-boradiaza-s- indacenes for near-infrared fluorescence imaging

Article abstract of DOI:10.1039/b813396d

We report molecular design, synthesis, and photophysical study of a series of near-infrared absorbing and fluorescing dyes, 3,5-difuranylvinyl-boradiaza-s- indacenes, bearing various 5-membered heteroaromatic heads at the 8-position (FBs). The correlation

Full text of DOI:10.1039/b813396d

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PAPER
www.rsc.org/materials | Journal of Materials Chemistry
Synthesis and nanoparticle encapsulation
of 3,5-difuranylvinyl-boradiaza-s-indacenes for near-infrared
fluorescence imaging†
*
Sehoon Kim,‡ Tymish Y. Ohulchanskyy, Alexander Baev and Paras N. Prasad
Received 1st August 2008, Accepted 19th February 2009
First published as an Advance Article on the web 31st March 2009
DOI: 10.1039/b813396d
We report molecular design, synthesis, and photophysical study of a series of near-infrared absorbing
and fluorescing dyes, 3,5-difuranylvinyl-boradiaza-s-indacenes, bearing various 5-membered
heteroaromatic heads at the 8-position (FBs). The correlation between the molecular structure and the
spectral shift has been studied by quantum chemical calculations at various levels (B3LYP/6-31G*, HF/
6-31G* and HF/PM3), which conclude that the planarity determined by the bulkiness of the head unit
controls the optical bandgap of FBs, by energetically affecting the lowest unoccupied molecular orbital
(LUMO) rather than the highest occupied molecular orbital (HOMO). We also show that
incorporation of heavy atoms increased the capability of singlet oxygen generation as a result of
enhanced intersystem crossing, which makes FBs potentially useful for near-infrared photodynamic
therapy. In vitro near-infrared fluorescence imaging of live tumor cells has been demonstrated through
a nanocarrier approach, by encapsulating one of the FB dyes in a stable aqueous formulation of
organically modified silica nanoparticles which retains the fluorescence efficiency of the hydrophobic
dye in water.
have been attempts at wavelength tuning of BODIPYs by
Introduction
structural modification, among which distyryl-substitution at the
3,5-positions has proved to be the most successful way to
produce a spectral red shift.6b,7b,8 Another promising approach is
the utilization of pulse NIR lasers for two-photon excitation of
BODIPYs.4
Near-infrared (NIR) photonics has great potential as a nonin-
vasive method for biological research, owing to the spectral
coincidence with the tissue transparent window (700–1000 nm).1
Photons in this region are less absorbed and scattered by tissues,
which enables deeper light penetration as well as better signal-
to-noise ratio by minimal interference from the background
autofluorescence. Promising applications include visualization of
deep tissues by the near-infrared fluorescence (NIRF) imaging2
and noninvasive treatment of subcutaneous deep tumors by
photodynamic therapy (PDT),3 both of which are effected by
utilizing NIR-active dyes as a fluorescence probe or a singlet
oxygen (1O2)-generating photosensitizer, respectively.
Recently, we aimed to develop a series of BODIPY derivatives
applicable to NIRF imaging. First, in order to achieve efficient
spectral red shift, we designed heteroaromatic versions of dis-
tyryl-BODIPY, i.e., 3,5-difuranylvinyl-BODIPYs (FBs) with an
additional heteroaromatic head unit at the central 8-position
(Chart 1). Furan and other 5-membered heteroaromatic rings
were chosen for their known beneficial properties, such as good
fluorescence capability and lower electron delocalization energy
barrier relative to benzene which generally promotes optical
bandgap narrowing by lengthening the effective p-conjugation.9
As another approach toward NIR application, we have also
developed a different type of BODIPY with enhanced two-
photon absorptivity, which will be reported elsewhere. In this
study, we report the synthesis and optical properties of FBs,
As an effort to develop high-performance fluorescent probes,
various classes of difluoroboradiaza-s-indacenes (boron dipyr-
romethene dyes, BODIPYs) have been investigated. BODIPY
derivatives are well-known versatile dyes that span the visible
spectral region.4 Due to their advantageous optical properties
(high extinction coefficient, high fluorescence quantum yield, and
good photostability, etc.), BODIPYs have been utilized for
various biophotonic applications such as biological fluorescence
probing,5 ion sensing,6 and photosensitizing for photodynamic
therapy,7 etc. To extend their application to the NIR range, there
The Institute for Lasers, Photonics and Biophotonics, Department of
Chemistry, State University of New York, Buffalo, New York,
14260-3000, USA
1
† Electronic supplementary information (ESI) available: The H NMR
spectra of the prepared compounds. See DOI: 10.1039/b813396d
‡ Present address: Biomedical Research Center, Korea Institute of
Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul
136-791, Korea.
Chart 1 3,5-Difuranylvinyl-boradiaza-s-indacenes (FBs)
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together with quantum-chemical calculations for discussions on
the correlation between molecular structure and spectral shift.
Also presented is the encapsulation of a resulting hydrophobic
dye in organically modified silica (ORMOSIL) nanoparticles, to
produce cell-permeable delivery formulations. Through this
nanocarrier approach, we have demonstrated FB-based NIRF
imaging of live tumor cells for potential NIR biophotonic
application. In addition, we report efficient singlet oxygen
generation under laser excitation, using heavy atom substituted
FBs.
Results and discussion
FBs, substituted with phenyl or 5-membered heteroaromatic
subunits at the 8-position, were synthesized in moderate-to-high
yields by acid-catalyzed condensation and subsequent cyclization
with BF3$Et2O between a furanylvinyl-substituted pyrrole (2)
and aromatic aldehydes (Ar-CHO), following a standard
procedure for BODIPY synthesis (Scheme 1).6–8 Furanyl, thio-
phenyl, and selenophenyl groups were chosen for the hetero-
aromatic subunit (Ar in Chart 1), to study the heteroatom effect
on the optical properties. FB-O derivatives bearing one and two
iodines (FB-O-I1 and FB-O-I2, respectively) were prepared to
evaluate the heavy-atom effect10 which is generally expected to
improve the singlet oxygen (1O2) generation capability for PDT
through the enhanced intersystem crossing (ISC) efficiency.7,11
FB-O-I2 was prepared by iodination of FB-O-I1 in the presence
of I2 and HIO3. On comparing the coupling constants and
splitting patterns in the 1H NMR spectra of FB-O-I1 and FB-O-
I2,12 as well as from the mass spectrometric results, it has been
found that the iodination of FB-O-I1 under the given conditions
predominantly gave a product which is mono-iodinated at the
2-position of the central 4-bora-3a,4a-diaza-s-indacene ring.
All the obtained FBs exhibit absorption and fluorescence in
the deep red and NIR ranges (Fig. 1) with moderate extinction
coefficients and fluorescence quantum yields (log3 ¼ 4.79–4.94,
Ff ¼ 0.05–0.29). Table 1 summarizes the optical properties of the
synthesized FBs in CHCl3, as well as a distyryl derivative (3,5-
distyryl-8-phenyl-BODIPY, SB-Ph)6b in Et2O with a solvent
polarity similar to CHCl3.13,14 On comparing the spectral data of
FB-Ph and SB-Ph, it is found that the 3,5-difuranylvinyl
substitution in place of 3,5-distyryl groups causes a bath-
ochromic shift of ꢀ40 nm, unambiguously attributed to the
Fig. 1 Absorption (a) and fluorescence (b) spectra of FBs, measured at
5 and 1 mM in CHCl3, respectively (lex ¼ 610 nm).
bandgap narrowing effect of the furan ring due to enhanced
p-electron delocalization. Additional substitution of 5-membered
heteroaromatic rings (furan, thiophene, and selenophene) in
place of the central phenyl head at the 8-position results in
a further spectral red shift (>10 nm), where the bathochromic
effect is of the order of furan > selenophene $ thiophene.
As a result, the 5-membered heteroaromatic derivatization
produced actually NIR-active BODIPY dyes with fluorescence
peaks at around and above 700 nm.
As listed in Table 1, the fluorescence decay rate constants (kfl)
are quite similar for all FBs. On the other hand, the nonradiative
decay rate constants (knr) exhibit a correlation with the fluores-
cence peak wavelength (lmax.fl), among FB-O, FB-S, and FB-Ph.
An increase in knr (a decrease in Ff) is correlated with the red
shift of lmax.fl (the decrease in fluorescence transition energy),
presumably through the energy-gap law,15 which means that the
rate of internal conversion (IC, one of nonradiative decay
pathways) increases as the energy gap between two electronic
states (S1 and S0 in our case) narrows. In the case of FB-Se,
a notably high knr is observed, indicating that the intersystem
crossing (ISC) exerts a significant influence on nonradiative
decay, due to the presence of a heavy atom (selenium). It is
known that the interaction with heavy atoms gives rise to an
increase in the spin–orbit interaction which leads to an increase
in the rate of spin-forbidden ISC (S1 / Tn), and thereby
a decrease in the efficiency of the competing radiative transition
Scheme 1 Synthetic procedures for FB-O, FB-S, FB-Se, FB-Ph, and
FB-O-I1, where Ar-CHO is 2-furaldehyde, 2-thiophenecarboxaldehyde,
2-selenophenecarboxaldehyde, benzaldehyde, and 5-iodo-2-furaldehyde,
respectively.
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Table 1 Photophysical parameters for FBs in CHCL3
a
b
c
c
lmax.absa(nm)
3max (Mꢁ1cmꢁ1
)
lmax.flb (nm)
Ffl
Stokes’ shift (cmꢁ1
)
tflb (ns)
kfl (10sꢁ1
)
knr (108sꢁ1
)
FB-O
FB-S
FB-Se
FB-O-I1
FB-O-I2
FB-Ph
SB-Phd
695
683
685
702
713
671
628
62 000
70 100
86 500
67 500
79 100
80 500
—
718
694
697
730
746
681
641
0.20
0.25
0.19
0.09
0.05
0.29
0.81
461
232
251
546
620
219
323
2.3
2.7
2.2
1.2
0.6
3.5
4.8
0.87
0.93
0.86
0.75
0.83
0.83
1.7
3.48
2.78
3.68
7.58
15.8
2.03
0.4
a
Measured at 5 mM. b Measured at 1 mM. c kfl ¼ Ffl/tfl, knr ¼ (1 ꢁ Ffl)/tfl. d Data for 3,5-distyryl-8-phenyl-BODIPY (SB-Ph) in Et2O are from ref. 6b.
(S1 / S0).10 This heavy-atom effect is seen even more remark-
ably by the substitution of a heavier atom (iodine). Proportional
to the number of iodine (FB-O-I1 and FB-O-I2 in Table 1),
iodine incorporation into FB-O leads to significant decreases in
Ff, along with moderate bathochromic shifts (transition energy
decrease), indicating that the rates of ISC and IC increase at the
same time by spin–orbit coupling and energy-gap narrowing,
respectively, to produce a dramatic increase in knr.
The enhanced ISC efficiency is further evidenced by moni-
toring the capability of singlet oxygen generation. Singlet oxygen
(1O2) is produced through the reaction between an excited triplet
state of a dye and the surrounding molecular oxygen in the triplet
ground state (3O2). Therefore, the efficiency of generation of 1O2
is determined to a great extent by the population of triplet states,
i.e., the efficiency of ISC. Highly reactive 1O2 is a key therapeutic
species in PDT, killing malignant cells, and the dye capable of
producing 1O2-generating triplet states is called a photosensitizer
(PS). Fig. 2 shows the phosphorescence spectra of 1O2 at
1270 nm, generated by photoexcited FBs in CHCl3. Notable 1O2
phosphorescence was observed only for the heavy atom-bearing
FBs, where the generation efficiency is in the order of FB-O-I2 >
FB-O-I1 > FB-Se, as determined by the phosphorescence
intensity. This tendency is well correlated with that of knr,
strongly supporting that an increase in knr by heavy atoms results
dominantly from the enhanced ISC rate. The observed capability
of singlet oxygen generation suggests a possible application of
FBs as NIR-active drugs for PDT.
To gain an insight into quantum-chemical origins determining
spectral shifts of FBs, the electronic properties of four model
compounds (mFBs in Fig. 3a) were computed by the ab initio
Fig. 2 Phosphorescence spectra of singlet oxygen generated by FBs in
CHCl3. The excitation wavelength is 532 nm, at which the absorbances of
all samples were matched to ꢀ0.076.
Fig. 3 (a) Model structures (mFBs) used for molecular orbital calculations. (b) HOMO/LUMO diagrams (HF/PM3) for mFB-Ph (top) and mFB-O
(bottom). (c) Traces of HOMO–LUMO levels and calculated absorption wavelength (inset) for mFB-Ph, obtained by HF/PM3 as functions of torsion
angle (4 in (a)).
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Table 2 Calculated geometry parameters, HOMO/LUMO levels (eV), and transition wavelengths, lcalcd (nm)
B3LYP/6-31G*
HF/6-31G*b
HF/PM3b
qa
4a
HOMO/LUMO
lcalcd
HOMO/LUMO
lcalcd
HOMO/LUMO
lcalcd
mFB-O
mFB-S
mFB-Se
mFB-Ph
108.9ꢂ
110.1ꢂ
110.1ꢂ
118.7ꢂ
34.9ꢂ
47.9ꢂ
49.6ꢂ
55.0ꢂ
ꢁ5.02/ꢁ3.04
ꢁ5.06/ꢁ3.05
ꢁ5.07/ꢁ3.07
ꢁ5.03/ꢁ2.97
626
618
621
603
ꢁ6.59/ꢁ0.47
ꢁ6.62/ꢁ0.44
ꢁ6.62/ꢁ0.45
ꢁ6.59/ꢁ0.31
536
531
532
521
ꢁ8.34/ꢁ2.70
ꢁ8.35/ꢁ2.69
ꢁ8.35/ꢁ2.74
ꢁ8.35/ꢁ2.57
559
554
558
546
a
Defined in Fig. 3a. b Calculated with B3LYP/6-31G* geometries.
Hartree–Fock self-consistent field method (HF/6-31G*) and the
hybrid density functional theory treatment (B3LYP/6-31G*), as
well as a semiempirical approach (HF/PM3). The ground-state
geometry optimization at the B3LYP/6-31G* level reveals that
the structures of common main body (3,5-difuranylvinyl-BOD-
IPY) are planar and almost identical for all mFBs. The aromatic
head fragments at the 8-position of BODIPY are twisted out of
the main body plane with the torsion angle (4) depending on the
magnitude of steric hindrance between spatially adjacent
hydrogen atoms in BODIPY and in head fragments. As listed in
Table 2, mFB-Ph shows the largest torsion angle (4 ¼ 55ꢂ)
among mFBs, since the hexagonal phenyl ring has a larger inner
angle (q ¼ 118.7ꢂ) than the heteroaromatic rings, which makes
phenyl hydrogens closer to the BODIPY hydrogens at the 1,7-
positions, to cause more steric hindrance. In the case of FBs
bearing a 5-membered heteroaromatic head unit, the resulting
torsion angle (4) is correlated with the size of the heteroatom, in
the order of mFB-O < mFB-S < mFB-Se, indicating that the
bigger heteroatom causes an increase of steric hindrance.
head unit, are notably stabilized with 5-membered hetero-
aromatic heads over the bulky phenyl unit, due to increased
planarity by less bulkiness and other possible electronic effects
from the 5-membered ring structure and heteroatoms. As
a result, the calculated absorption wavelengths (lcalcd) exhibit the
same trends as the experimental ones, which can be correlated
with the head planarity in a way that the most planar mFB-O has
the longest lcalcd. These calculation results suggest that the
derivatization of BODIPY at the 8-position with less bulky
p-conjugated segments can be another potential strategy for
spectral tuning toward the NIR.
Photostability is another important requirement for dyes to be
used for the NIRF imaging applications. In general, NIR fluo-
rescent dyes are labile to photobleaching, since they are mostly
composed of photochemically unstable, elongated olefin struc-
tures to achieve a narrow optical bandgap.16 The structures of
FBs, however, are rather aromatic and do not carry such long
unsaturated hydrocarbon chains, providing an opportunity
for better photostability. Among FBs, FB-O seems to be most
promising as NIRF imaging probes, with suitable spectral
position and quantum yield of fluorescence. Its photostability
was examined by comparing with that of a typical NIRF probe,
As representatively shown by orbital diagrams for mFB-Ph
and mFB-O in Fig. 3b, all mFBs have the same patterns of
electronic distribution for the highest occupied and the lowest
unoccupied molecular orbitals (HOMO and LUMO), which are
involved in a main electronic transition (lcalcd) corresponding
to the absorption maximum wavelength. In the HOMO, the
p-electron distribution is confined within the main body (3,5-
difuranylvinyl-BODIPY), whereas in the LUMO it is extended
up to the distorted head fragment, suggesting that the energy
level of the LUMO might be governed more remarkably by the
planarity of the head fragment. The effect of the head planarity
on the electronic properties was calculated with mFB-Ph at
the HF/PM3 level, by varying its torsion angle (Fig. 3c). With
decreasing torsion angle (4), the HOMO–LUMO gap narrows
and the transition wavelength (the inset of Fig. 3c) increases, in
a way that the LUMO level is notably stabilized while keeping
the HOMO level less affected. This indicates that only the
effective p-conjugation of the LUMO is extended by planariza-
tion due to the electronic nature of the HOMO and the LUMO,
as discussed with Fig. 3b. On comparing mFB-Ph and the
other mFBs with a 5-membered heteroaromatic subunit, similar
tendencies depending on the head planarity are observed in the
calculation results obtained at the levels of B3LYP/6-31G*, HF/
6-31G* and HF/PM3 (Table 2). Regardless of the head structure,
the HOMO levels are close to each other for all mFBs, suggesting
that the head units have a minimal effect on the HOMO due to
the absence of electron distribution on them. In contrast, the
levels of the LUMO, which has p-electron distribution on the
Fig. 4 Comparison of photostability of FB-O and thiadicarbocyanine
dye (C5), shown by the change of the absorption spectra in EtOH upon
pulse laser illumination (532 nm, 20 Hz, 18 mJ/pulse). The initial
absorbance (A0) at the excitation wavelength was matched to 0.021 for
both dye solutions. The inset shows the relative absorbance change (A/
A0) at each lmax, as a function of irradiation time.
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thiadicarbocyanine dye (C5).17 Fig. 4 shows the temporal
evolutions of absorption spectra of FB-O and C5 upon repetitive
laser illumination. The traces of absorbances at each lmax (the
inset of Fig. 4) clearly indicate that FB-O is much more resistant
to photobleaching than C5, even with a narrower optical
bandgap.
denoted as OSNP), in the nonpolar core of Tween-80/1-butanol/
water micelles. As shown in the transmission electron micro-
scopic (TEM) image of OSNP (Fig. 5a), the obtained nano-
particles are spherical in shape, with a narrow size distribution of
15.2 ꢃ 2.2 nm. Fig. 5b shows the absorption and fluorescence
spectra of an aqueous suspension of OSNP. The band shapes
and the peak positions (lmax.abs ¼ 695 nm and lmax.fl ¼ 719 nm)
are almost identical to those of the CHCl3 solution, with no sign
for any self-aggregation of FB-O. Note that in water, hydro-
phobic FBs are not soluble and form aggregates with changed
absorption and quenched fluorescence, as observed for common
organic dyes.20 Consequently, the efficient NIR fluorescence
from an aqueous suspension of OSNP indicates that the FB-O
molecules have successfully been embedded in the hydrophobic
part of the ORMOSIL matrix, in the molecularly dispersed form.
The potential of FB-O as a NIR fluorescent probe was eval-
uated in vitro by fluorescence imaging of live tumor cells, in the
nanoassembled form of OSNP. Fig. 6 shows the microscopic
images of Cos-1 cells treated with an aqueous formulation of
OSNP, where for the confocal fluorescence image only the NIR
signal above 750 nm was taken using a longpass 750LP filter. An
intense NIR fluorescence signal is observed from the cells
(Fig. 6b), indicating an active uptake of OSNP by tumor cells. As
shown in the merged transmission and fluorescence images
(Fig. 6c), the intracellular uptake pattern of OSNP is quite
similar to those of other ORMOSIL nanoparticles,18,19 with
significant accumulation in the cytoplasm. This in vitro demon-
stration of NIRF imaging suggests the potential utility of 3,5-
difuranylvinyl-BODIPYs for NIR biophotonics.
For practical applications of the hydrophobic FBs toward
NIRF bioimaging, we attempted encapsulation in a cell-perme-
able carrier, by utilizing a stable aqueous formulation of
organically modified silica (ORMOSIL) nanoparticles.18,19 Trie-
thoxyvinylsilane (VTES) was used as a silicate precursor, to
prepare ORMOSIL nanoparticles encapsulating FB-O (hereafter
Conclusion
Novel NIR-absorbing and fluorescing boradiaza-s-indacene
(BODIPY) dyes, with and without heavy atoms, have been
designed, synthesized, and photophysically investigated. Spectral
red shift of the BODIPY chromophore has successfully been
achieved up to the NIR region at around and above 700 nm,
through the heteroaromatic derivatization by incorporating
difuranylvinyl groups at the 3,5-positions as well as another
5-membered heteroaromatic head at the 8-position. It has
been concluded from quantum chemical calculations that the
planarity determined by the bulkiness of the head unit plays an
important role in achieving further spectral red shift of FBs.
A cell-permeable NIRF imaging nanoprobe has successfully
been prepared, by encapsulating one FB dye in ORMOSIL
Fig. 5 TEM image (a) and absorption/fluorescence spectra (b) of
ORMOSIL nanoparticles encapsulating FB-O (OSNP).
Fig. 6 Transmission (a), fluorescence (b) and merged (c) images of Cos-1 cells stained with OSNP. In the merged image, transmission and fluorescence
signals are shown in blue and red, respectively.
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nanoparticles while retaining its fluorescence efficiency. It has
been demonstrated by heavy atom-effected singlet oxygen
generation and in vitro cellular NIRF imaging that our hetero-
aromatized BODIPY dyes hold considerable potential for NIR
biophotonic applications.
recrystallized from n-hexane, to give a trans isomer of compound
1
2. Yield 0.8 g (39%). H NMR (CDCl3): d 7.16 (d, 1H, J ¼ 3.6
Hz), 7.13 (d, 1H, J ¼ 16.4 Hz), 6.86 (m, 1H), 6.48 (d, 1H, J ¼ 16.4
Hz), 6.44 (m, 1H), 6.33 (d, 1H, J ¼ 3.6 Hz), 6.27 (m, 1H), 4.37 (q,
2H, J ¼ 7.2 Hz), 1.39 (t, 3H, J ¼ 7.2 Hz).
Experimental
General procedure for difluoroboradiaza-s-indacene (BODIPY)
formation
Chemical structures were identified by 1H NMR (Varian
INOVA-400, 400 MHz) and electrospray ionization (ESI) mass
spectra (Themo Finnigan LCQ Advantage mass spectrometer).
Cosurfactant 1-butanol and NH4OH (28.0ꢀ30.0%) are products
of J. T. Baker. All other chemicals used were purchased from
Aldrich. UV-vis absorption and fluorescence spectra were
recorded using a Shimadzu UV-3600 spectrophotometer and
a Jobin-Yvon Fluorog FL-311 spectrofluorometer, respectively.
Relative fluorescence quantum yields (Ff) were estimated using
3,30-diethylthiadicarbocyanine iodide (C5) in ethanol (Ff ¼ 0.35)
as a reference.17 Fluorescence decays were obtained by using an
EasyLife fluorescence lifetime system (Photon Technology
International, Birmingham, NJ). Singlet oxygen luminescence
spectra peaking at 1270 nm were recorded by using a SPEX
270M spectrometer (Jobin Yvon) equipped with an InGaAs
photodetector (Electro-Optical Systems Inc.), under the excita-
tion at 532 nm from the diode-pumped solid-state laser (Millenia,
Spectra-Physics).
Compound 2 (0.2 g, 0.86 mmol) and aromatic aldehyde
(Ar-CHO, 0.43 mmol) in CH2Cl2 (15 mL) were stirred in the
presence of 1 drop of trifluoroacetic acid (TFA) at room
temperature for 5 hrs. p-Chloranil (tetrachloro-1,4-benzoqui-
none, 0.212 g, 0.86 mmol) in CH2Cl2 (10 mL) was added, and the
mixture was stirred for 20 min. Then, triethylamine (1 mL) and
BF3$OEt2 (1 mL) were added slowly in sequence. After stirring
for another 5 hrs, the diluted mixture with excess CH2Cl2 was
washed with water, and dried over MgSO4. The crude product
obtained after solvent evaporation was purified by column
chromatography on a silica gel (ethyl acetate/CH2Cl2 ¼ 1/6), and
then reprecipitated from CH2Cl2 into n-hexane, to give pure FBs.
3,5-Bis(2-{5-[ethoxycarbonyl]-furan-2-yl}vinyl)-8-(furan-2-yl)-
BODIPY (FB-O)
1
0.15 g isolated. Yield 59%. H NMR (CDCl3): d 7.77 (m, 1H),
2-Selenophenecarboxaldehyde
7.74 (d, 2H, J ¼ 16.4 Hz), 7.36 (d, 2H, J ¼ 4.0 Hz), 7.25 (d, 2H,
J ¼ 3.6 Hz), 7.20 (d, 2H, J ¼ 16.4 Hz), 7.01 (m, 1H), 6.94 (d, 2H,
J ¼ 4.0 Hz), 6.85 (d, 2H, J ¼ 3.6 Hz), 6.68 (m, 1H), 4.41 (q, 4H, J
¼ 7.2 Hz), 1.42 (t, 6H, J ¼ 7.2 Hz). MS (EI): calcd for
C31H25BF2N2O7, m/z ¼ 586.17; found, m/z ¼ 586.4. Anal. calcd
for C31H25BF2N2O7: C, 63.50; H, 4.30; N, 4.78. Found: C, 63.7;
H, 4.3; N, 4.7%.
To a solution of selenophene (3 g, 22.9 mmol) in DMF (14 mL),
phosphorus oxychloride (5.6 mL, 60 mmol) was added and
heated for 2 hrs at 85 ꢂC. After cooling, the resulting mixture was
poured onto excess ice-cold water, neutralized with sodium
acetate and heated to boil. The cooled mixture was extracted
with ethyl acetate. The organic extracts were washed with brine
and dried over MgSO4. The crude product obtained after solvent
evaporation was purified by column chromatography on silica
3,5-Bis(2-{5-[ethoxycarbonyl]-furan-2-yl}vinyl)-8-(thiophen-2-
yl)-BODIPY (FB-S)
1
gel (ethyl acetate/n-hexane ¼ 1/3). Yield 2 g (55%). H NMR
(CDCl3): d 9.82 (s, 1H), 8.50 (d, 1H, J ¼ 5.2 Hz), 8.03 (d, 1H,
J ¼ 4.0 Hz), 7.48 (m, 1H).
0.187 g isolated. Yield 72%. 1H NMR (CDCl3): d 7.72 (d, 2H, J ¼
16.4 Hz), 7.62 (d, 1H, J ¼ 4.8 Hz), 7.43 (d, 1H, J ¼ 3.2 Hz), 7.24–
7.22 (m, 3H), 7.19 (d, 2H, J ¼ 16.4 Hz), 7.15 (d, 2H, J ¼ 4.4 Hz),
6.91 (d, 2H, J ¼ 4.4 Hz), 6.84 (d, 2H, J ¼ 3.6 Hz), 4.41 (q, 4H, J ¼
7.2 Hz), 1.42 (t, 6H, J ¼ 7.2 Hz). MS (EI): calcd for
C31H25BF2N2O6S, m/z ¼ 602.15; found, m/z ¼ 602.1. Anal. calcd
for C31H25BF2N2O6S: C, 61.81; H, 4.18; N, 4.65. Found: C, 61.5;
H, 4.1; N, 4.7%.
2-(2-{5-[Ethoxycarbonyl]-furan-2-yl}vinyl)-1H-pyrrole (2)
A mixture of ethyl 5-(chloromethyl)-2-furancarboxylate (4 g,
21.2 mmol) and triphenylphosphine (8.34 g, 31.8 mmol) in
toluene (30 mL) was refluxed for 5 hrs in the presence of 0.3 mL
of DMF. After cooling, the precipitate was filtered off and
washed with diethyl ether, to give 7.5 g of pure ({5-[ethox-
ycarbonyl]furan-2-yl}methyl)triphenylphosphonium chloride (1,
78%). To a solution of potassium t-butoxide (1.1 g, 9.8 mmol) in
DMSO (8 mL) was added the triphenylphosphonium salt 1 (4 g,
8.9 mmol) at room temperature. The mixture was stirred for
20 min or until complete dissolution of the salt. Then, pyrrole-2-
carboxaldehyde (0.93 g, 9.8 mmol) dissolved in DMSO (8 mL)
was added dropwise to the ylide solution. After overnight stirring
at room temperature, the mixture was poured into water and
extracted with ethyl acetate. The organic extracts were washed
with brine, and dried over MgSO4. The crude product obtained
after solvent evaporation was purified by column chromatog-
raphy on silica gel (ethyl acetate/n-hexane ¼ 1/4), and then
3,5-Bis(2-{5-[ethoxycarbonyl]-furan-2-yl}vinyl)-8-(selenophen-2-
yl)-BODIPY (FB-Se)
0.189 g isolated. Yield 67%. 1H NMR (CDCl3): d 8.33 (d, 1H, J ¼
5.6 Hz), 7.72 (d, 2H, J ¼ 16.8 Hz), 7.59 (d, 1H, J ¼ 3.2 Hz), 7.46
(m, 1H), 7.23 (d, 2H, J ¼ 3.6 Hz), 7.19 (d, 2H, J ¼ 16.8 Hz), 7.16
(d, 2H, J ¼ 4.4 Hz), 6.90 (d, 2H, J ¼ 4.4 Hz), 6.84 (d, 2H, J ¼ 3.6
Hz), 4.41 (q, 4H, J ¼ 7.2 Hz), 1.42 (t, 6H, J ¼ 7.2 Hz). MS (EI):
calcd for C31H25BF2N2O6Se, m/z ¼ 650.09; found, m/z ¼ 650.1.
Anal. calcd for C31H25BF2N2O6Se: C, 57.34; H, 3.88; N, 4.31.
Found: C, 57.1; H, 3.8; N, 4.4%.
3186 | J. Mater. Chem., 2009, 19, 3181–3188
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3,5-Bis(2-{5-[ethoxycarbonyl]-furan-2-yl}vinyl)-8-phenyl-
BODOPY (FB-Ph)
129 configurations (HF/PM3), by changing the torsion angle (4)
of the B3LYP/6-31G*-optimized geometry.
0.067 g isolated. Yield 26%. 1H NMR (CDCl3): d 7.73 (d, 2H, J ¼
16.8 Hz), 7.54-7.49 (m, 5H), 7.24 (d, 2H, J ¼ 3.6 Hz), 7.19 (d, 2H,
J ¼ 16.8 Hz), 6.88 (d, 2H, J ¼ 4.4 Hz), 6.84 (d, 2H, J ¼ 3.6 Hz),
6.82 (d, 2H, J ¼ 4.4 Hz), 4.41 (q, 4H, J ¼ 7.2 Hz), 1.42 (t, 6H, J ¼
7.2 Hz). MS (EI): calcd for C33H27BF2N2O6, m/z ¼ 596.19;
found, m/z ¼ 596.4. Anal. calcd for C33H27BF2N2O6: C, 66.46;
H, 4.56; N, 4.70. Found: C, 66.3; H, 4.5; N, 4.7%.
Encapsulation of FB-O in ORMOSIL nanoparticles
The aqueous micelle was prepared by dissolving 0.2 g of Tween-
80 and 0.3 mL of 1-butanol in 10 mL of deionized water by
vigorous magnetic stirring. Then, 60 mL of FB-O solution in
DMSO (5 mM) and 200 mL of neat VTES were added to the
micellar solution under magnetic stirring, and the resulting
mixture was sonicated for about 5 min, or until they became
homogeneous. After that, 40 mL of NH4OH was added and
the mixture was magnetically stirred for about 20 hrs at room
temperature, to ensure completion of sol-gel condensation. The
residual DMSO, catalyst and surfactants were removed by dia-
lyzing the nanoparticle dispersion against deionized water in
a 12–14 kDa cutoff cellulose membrane (Spectrum Laboratories,
Inc.) for 48 h. To further remove the residual Tween-80, the
dialyzed dispersions were spin-filtered in a microfuge membrane
filter (NANOSEP 100K OMEGA, Pall Corporation) by centri-
fuging at 11 000 rpm for 40 min. Nanoparticles collected on the
membrane were redispersed in water of the same volume as the
amount before spin filtration.
3,5-Bis(2-{5-[ethoxycarbonyl]-furan-2-yl}vinyl)-8-(5-iodofuran-
2-yl)-BODIPY (FB-O-I1)
0.146 g isolated. Yield 47%. 1H NMR (CDCl3): 7.72 (d, 2H, J ¼
16.4 Hz), 7.30 (d, 2H, J ¼ 4.4 Hz), 7.23 (d, 2H, J ¼ 3.6 Hz), 7.19
(d, 2H, J ¼ 16.4 Hz), 6.94 (d, 2H, J ¼ 4.4 Hz), 6.87 (d, 1H, J ¼ 3.6
Hz), 6.84 (d, 2H, J ¼ 3.6 Hz), 6.81 (d, 1H, J ¼ 3.6 Hz), 4.41 (q,
4H, J ¼ 7.2 Hz), 1.42 (t, 6H, J ¼ 7.2 Hz). MS (EI): calcd for
C31H24BF2IN2O7, m/z ¼ 712.07; found, m/z ¼ 712.1. Anal. calcd
for C31H24BF2IN2O7: C, 52.28; H, 3.40; N, 3.93. Found: C, 52.0;
H, 3.3; N, 4.0%.
2-Iodo-3,5-bis(2-{5-[ethoxycarbonyl]-furan-2-yl}vinyl)-8-(5-
iodofuran-2-yl)-BODIPY (FB-O-I2)
In-vitro studies with tumor cells: nanoparticle uptake and NIRF
imaging
To a mixture of FB-I1 (0.1 g, 0.14 mmol) and iodine (0.178 g,
0.7 mmol) dissolved in DMF (4 mL) was added iodic acid (0.1 g,
0.57 mmol) solution in water (0.3 mL) with stirring over 5 min.
After overnight stirring at room temperature, the diluted mixture
with excess CH2Cl2 was washed with saturated sodium thiosul-
fate solution and brine to remove excess iodine and DMF, and
then dried over MgSO4. The crude product obtained after
solvent evaporation was recrystallized from ethyl acetate, to give
For studying nanoparticles uptake and imaging, Cos-1 cells were
used, maintained in DMEM medium with 10% fetal bovine
serum (FBS) and appropriate antibiotic. The cells at a confluency
of 70–75% were treated overnight with the nanoparticles. Next
day, the treated cells were washed thoroughly with PBS and then
directly imaged using a confocal laser scanning microscope
(MRC-1024, Bio-Rad, Richmond, CA). A water immersion
objective lens (Nikon, Fluor-60X, NA ¼ 1.0) was used for cell
imaging. A Ti:sapphire laser (Tsunami from Spectra-Physics)
pumped by a diode-pumped solid state laser (Millenia, Spectra-
Physics) was used as a source of excitation. The Ti:sapphire
output, tuned to 725 nm, was coupled into a single mode fiber
for delivery into the confocal scan head. Long-pass filters, 585 LP
(585 nm) and 750 LP (750 nm), were used as emission filters for
NIR fluorescence imaging.
1
FB-I2. Yield 0.03 g (51%). H NMR (CDCl3): 7.93 (d, 1H, J ¼
17.2 Hz), 7.69 (d, 1H, J ¼ 18.0 Hz), 7.65 (d, 1H, J ¼ 17.2 Hz),
7.44 (s, 1H), 7.37 (d, 1H, J ¼ 4.4 Hz), 7.23 (d, 2H, J ¼ 3.6 Hz),
7.21 (d, 1H, J ¼ 18.0 Hz), 6.98 (d, 1H, J ¼ 4.4 Hz), 6.90 (d, 1H,
J ¼ 3.6 Hz), 6.86 (d, 1H, J ¼ 3.6 Hz), 6.83 (d, 2H, J ¼ 3.6 Hz),
4.41 (q, 4H, J ¼ 7.2 Hz), 1.42 (t, 6H, J ¼ 7.2 Hz). MS (EI): calcd
for C31H23BF2I2N2O7, m/z ¼ 837.97; found, m/z ¼ 838.0. Anal.
calcd for C31H23BF2I2N2O7: C, 44.42; H, 2.77; N, 3.34. Found:
C, 44.1; H, 2.7; N, 3.4%.
Acknowledgements
This work was supported by grant from the National Institute
of Health (R01CA119358) and the John R. Oishei Foundation.
Calculation details
The ground-state geometries of the model structures were opti-
mized by means of the Gaussian electronic structure program,21
making use of the density functional theory (DFT) method with
a hybrid functional B3LYP and a split-valence basis set 6-31G*.
The calculations of electronic properties at various levels were
carried out with the B3LYP/6-31G*-optimized geometries, using
the DALTON program22 (B3LYP/6-31G* and HF/6-31G*) and
the HyperChem 7.5 program (HF/PM3). The linear response
theory applied to either single-determinant self-consistent
field reference state (HF) or Kohn–Sham reference state (DFT)
was applied to calculate absorption wavelengths. For the head
planarity-dependent electronic properties of mFB-Ph (Fig. 3c),
the configuration interaction calculations were performed with
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This journal is ª The Royal Society of Chemistry 2009