Donor-acceptor-donor fluorene derivatives for two-photon fluorescence lysosomal imaging

Article abstract of DOI:10.1021/jo100554j

As part of a strategy to achieve large two-photon absorptivity in fluorene-based probes, a series of donor-acceptor-donor (D-A-D) type derivatives were synthesized and their two-photon absorption (2PA) properties investigated. The synthesis of D-A-D fluorophores was achieved by efficient preparation of key intermediates for the introduction of central electron acceptor groups. To accomplish the synthesis of two of the new derivatives, a high-yield method for a one-step direct dibromomethylation of phenyl sulfide was developed. The linear and nonlinear optical properties, including UV-vis absorption, fluorescence emission, fluorescence anisotropy, and two-photon absorption (2PA), of the new D-A-D compounds were measured and compared to their D-A or D-D counterparts. Fully conjugated acceptor moieties in the center of the D-A-D fluorophore led to the greatest increase in the 2PA cross section, while weakly conjugated central acceptors exhibited only a modest increase in the 2PA cross section relative to D-A diploar analogs. Encapsulation of the new probes in Pluronic F 108NF micelles, and subsequent incubation in HCT 116 cells, resulted in very high lysosomal colocalization (>0.98 colocalization coefficient) relative to commercial Lysotracker Red, making the micelle-encapsulated dyes particularly attractive as fluorescent probes for two-photon fluorescence microscopy lysosomal imaging.

Full text of DOI:10.1021/jo100554j

pubs.acs.org/joc
Donor-Acceptor-Donor Fluorene Derivatives for Two-Photon
Fluorescence Lysosomal Imaging
Sheng Yao,† Hyo-Yang Ahn,† Xuhua Wang,† Jie Fu,‡ Eric W. Van Stryland,‡
David J. Hagan,‡ and Kevin D. Belfield*,†,‡
†Department of Chemistry and ‡CREOL: The College of Optics and Photonics, University of Central Florida,
P.O. Box 162366, Orlando, Florida 32816-2366
belfield@mail.ucf.edu
Received March 23, 2010
As part of a strategy to achieve large two-photon absorptivity in fluorene-based probes, a series of donor-
acceptor-donor (D-A-D) type derivatives were synthesized and their two-photon absorption (2PA)
properties investigated. The synthesis of D-A-D fluorophores was achieved by efficient preparation of
key intermediates for the introduction of central electron acceptor groups. To accomplish the synthesis of
two of the new derivatives, a high-yield method for a one-step direct dibromomethylation of phenyl sulfide
was developed. The linear and nonlinear optical properties, including UV-vis absorption, fluorescence
emission, fluorescence anisotropy, and two-photon absorption (2PA), of the new D-A-D compounds
were measured and compared to their D-A or D-D counterparts. Fully conjugated acceptor moieties in
the center of the D-A-D fluorophore led to the greatest increase in the 2PA cross section, while weakly
conjugated central acceptors exhibited only a modest increase in the 2PA cross section relative to D-A
diploar analogs. Encapsulation of the new probes in Pluronic F 108NF micelles, and subsequent incuba-
tion in HCT 116 cells, resulted in very high lysosomal colocalization (>0.98 colocalization coefficient)
relative to commercial Lysotracker Red, making the micelle-encapsulated dyes particularly attractive as
fluorescent probes for two-photon fluorescence microscopy lysosomal imaging.
Introduction
by Webb et al. by using a femtosecond pulsed Ti:sapphire laser
scanning microscope.1 It was soon clear that the advantageous
features of two-photon fluorescence bioimaging include the
reduction of photobleaching and photodamage of imaging
probes and cellular structures, the capability of relatively deep
penetration by exciting at longer wavelength, an enhanced
image quality due to less light scattering in turbid media, such
as cells, by eliminating out of focus influences and repressing
background fluorescence, and the ability to bring about precise
3D spatially localized photosensitization, photolysis, abla-
tion, and cutting at the subcellular level.1-3
Two-photon-induced fluorescence involves two photons
being absorbed “simultaneously” by the molecule under inves-
tigation to excite an electron in a molecule to an electronic ex-
cited state. Subsequently, once in the S1 state (through internal
conversion if a higher excited state was populated) the excited
electron relaxes back to the ground state and emits a photon via
identical processes as in single-photon absorption and fluores-
cence processes. This nonlinear process is characterized by the
high spatial localization inherent in the quadratic relationship
between the excitation and fluorescence intensity.1 In the early
1990s, two-photon fluorescence (2PF) imaging was pioneered
(2) Konig, K. J. Microsc. 2000, 200, 83–104.
(3) Piston, D. W. Trends Cell Biol. 1999, 9, 66–69.
(1) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73–76.
DOI: 10.1021/jo100554j
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Published on Web 05/19/2010
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2010 American Chemical Society

Yao et al.
JOCFeatured Article
These advantages have been exploited to develop a number of
amine-reactive fluorene-based fluorophores for 2PFM bioima-
ging (Figure 1).5-8
Among the fluorophores shown in Figure 1, it has been
found that even hydrophobic 2-diphenylamino-7-benzothia-
zole-9, 9-didecylfluorene (A) is useful in staining cells such as
H9c2 rat cardiomyoblasts cells for epi-fluorescence and two-
photon fluorescence microscopy imaging.5 In addition,
rather than the diphenylamino group in A, an isothiocyanato
group was incorporated into two amino reactive analogs B
and C.5,6 Amine-reactive probes B and C can be directly used
for 2PFM imaging or to form bioconjugates, imparting
specificity. Importantly, the cytotoxicity of the fluorophores
is low. However, the conjugation length of fluorene deriva-
tives mentioned above is relatively short, leading to relatively
low 2PA action cross sections, i.e., <100 GM. Recently, two
hydrophilic fluorene derivatives with increased conjugation
length were reported by our group for integrin-targeting, and
2PFM cell imaging was demonstrated.6-8 The probe with the
most extended conjugation system showed a 2PA action
cross section of 680 GM at 740 nm in DMSO. Although this
2PA action cross section is already ca. 5 times higher than
that of rhodamine B, further efforts are warranted to in-
crease the 2PA cross section, the wavelength of 2PF, and the
emission wavelength.
The 2PA efficiency is, in general, related to the conjuga-
tion length and the donor/acceptor strength of the chromo-
phore’s substituents. Additionally, D-A-D-type chromo-
phores provide higher 2PA cross sections compared to those
of D-A- or D-D-type structures with similar conjugation
length and donor/acceptor groups.13,14 With the combina-
tion of a D-A-D architecture and large conjugation sys-
tem, it is reasonable to expect an increase in the 2PA cross
sections of a molecule. Interestingly, very few fluorenyl-
based D-A-D structures have been reported,15,16 and the
reported examples had short conjugation lengths, conse-
quently leading to low 2PA cross sections.
Herein, we report the synthesis of D-A-D type chromo-
phores with increased conjugation length, realizing a signifi-
cant increase in 2PA cross section, and their comprehensive
linear and nonlinear photophysical characterization. The
D-A-D fluorene derivatives that were prepared incorpo-
rated novel bifunctional electron accepting moieties as the
core. Significantly, a high yield method for a one-step direct
dibromomethylation of phenyl sulfide was developed to
prepare two of the new derivatives. The 2PA action cross
sections of these fluorophores was determined and compared
to those of their D-A or D-D type counterparts to evaluate
both the benefit of this design paradigm and a larger con-
jugation system on enhancement of two-photon absorptiv-
ity. Significantly, the new dyes were used in both one- and
two-photon fluorescence microscopy imaging after encapsu-
lation in Pluronic micelles, demonstrating outstanding lyso-
somal selectivity.
FIGURE 1. Structures of fluorene derivatives for bioimaging.
With the decreasing cost of the two-photon fluorescence
microscopy (2PFM) hardware, 2PFM imaging is increasingly
being adopted by biological and biomedical researchers. How-
ever, the two-photon absorption (2PA) cross sections of most
commercially available fluorescent probes, which were actually
designed for one-photon fluorescence, are low. For 2PFM
imaging, the two-photon excited fluorescence action cross-sec-
tion ηδ, the product of the fluorescence quantum yield η and
2PA cross section δ, should be a more salient feature to
characterize a fluorophore. Since η is always lower than 1, the
action 2PA cross sections of these probes are even lower. For
example, among the commercial fluorophores, rhodamine B has
one of the highest 2PA cross section values, 200 GM (1 GM =
10-50 cm4 s photon-1) at 840 nm in MeOH. Considering the
3 3
fluorescence quantum yield of rhodamine B is 0.7 in MeOH, the
2PA action cross section is only 140 GM.4 Therefore, there is
compelling need to develop efficient 2PA fluorophores with
higher 2PA action cross sections, as a higher 2PA action cross
section translates to less input laser power to observe 2PF,
thereby reducing photobleaching and background noise, e.g.,
autofluorescence.
In an effort to develop 2PF probes with improved 2PA effi-
ciencies, fluorenyl-based fluorescence probes have been prepared
(Figure 1, A-C) and successfully used for fluorescence bioima-
ging both under one-photon and two-photon excitation.5-8
Structurally, fluorenyl derivatives have a number of advantages
for use as bioimaging fluorescence probes. The conjugated
planar ring system provides a well delocalized π-system, which
is essential to achieve high 2PA, and the rigidity of the fused ring
system generally affords high fluorescence quantum yield. Im-
portantly, fluorene derivatives are typically quite photostable
under both one- and two-photon irradiation.9-12 Another
advantage of using fluorene derivatives as biological probes is
that the hydrophilicity/hydrophobicity of the fluorene deriva-
tives may be easily tuned by introducing various substituents at
the 9-position (Figure 1, R0). In addition to modulating solubility,
the substituents at the 9 position also help prevent aggrega-
tion of the dye, thereby reducing fluorescence self-quenching.
(4) Xu, C.; Williams, R. M.; Zipfel, W.; Webb, W. W. Bioimaging 1996
4, 198–207.
(5) Schafer-Hales, K. J.; Belfield, K. D.; Yao, S.; Frederiksen, P. K.;
Hales, J. M.; Kolattukudy, P. E. J. Biomed. Opt. 2005, 10, 051402–1-8.
(6) Morales, A. R.; Schafer-Hales, K. J.; Marcus, A. I.; Belfield, K. D.
Bioconjugate Chem. 2008, 19, 2559–2567.
(7) Morales, A. R.; Yanez, C. O.; Schafer-Hales, K. J.; Marcus, A. I.;
Belfield, K. D. Bioconjugate Chem. 2009, 20, 1992–2000.
(8) Morales, A. R.; Luchita, G.; Yanez, C. O.; Bondar, M. V.; Przhonska,
O. V.; Belfield, K. D. Org. Biomol. Chem. 2010, 8, in press, DOI: 10.1039/
b925934a.
(9) Corredor, C. C.; Belfield, K. D.; Bondar, M. V.; Przhonska, O. V.;
Yao, S. J. Photochem. Photobiol. A Chem. 2006, 184, 105–112.
(10) Belfield, K. D.; Bondar, M. V.; Przhonska, O. V.; Schafer, K. J.
J. Photochem. Photobiol. A Chem. 2004, 162, 489–496.
(11) Belfield, K. D.; Bondar, M. V.; Przhonska, O. V.; Schafer, K. J.
J. Photochem. Photobiol. A Chem. 2004, 162, 569–574.
(12) Belfield, K. D.; Bondar, M. V.; Przhonska, O. V.; Schafer, K. J.
Photochem. Photobiol. Sci. 2004, 3, 138–141.
(13) Albota, M.; Beljonne, D.; Bredas, J.-L.; Ehrlich, J. E.; Fu, J.-Y.;
Heikal, A. A.; Hess, S. E.; Kogej, T.; Levin, M. D.; Marder, S. R.; McCord-
Maughon, D.; Perry, J. W.; Rockel, H.; Rumi, M.; Subramaniam, G.; Webb,
W. W.; Wu, X.-L.; Xu, C. Science 1998, 281, 1653–1656.
(14) Beljonne, D.; Kogej, T.; Marder, S. R.; Perry, J. W.; Bredas, J. L.
MCLC S&T, Sect. B: Nonlinear Opt. 1999, 21, 461–480.
(15) He, G. S.; Lin, T.-C.; Prasad, P. N. Opt. Express 2002, 10, 566–574.
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L.-S. J. Phys. Chem. B 2002, 106, 11081–11084.
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FIGURE 3. Synthesis of compounds 1-4. Key: (a) HNO3/H2SO4,
0 °C, 1.5 h, 41%; (b) DMF, NaH, rt, 20 h, 55- 85%.
FIGURE 2. Structures of probes 1-8.
Lysosomes are membrane-bound vesicles containing various
hydrolytic enzymes necessary for digesting exogenetic macro-
molecules and breaking down old nonfunctioning organelles
that out lived their usefulness.17 Lysosomes are involved in a
number of cell life activities, including intracellular transporta-
tion, metabolism, cell membrane recycling,17 and even apop-
tosis.18 The malfunction of lysosomes has been implicated in
several diseases, such as inflammation, tumors, silicosis, and
various lysosomal storage diseases, e.g., Tay-Sachs disease, or
Pompe’s disease.19,20 In addition to faciliating study of these
conditions, the highly specific lysosome-targeting of the micelle
delivery system described herein may find application as gene
or drug carriers for lysosomal-targeting therapeutics.
Results and Discussion
Synthesis. The structures of the four pairs of 2PA fluor-
ophores synthesized in this work are shown in Figure 2. The
donor-π-donor-type compound 1, where two dibutylami-
no groups as donor and fluorenyl as π bridge, is a reference
compound to the D-A-D archetype 2. Similarly, com-
pounds 4, 6, and 8 will be compared to 3, 5, and 7, respec-
tively. Key to the synthesis of these D-A-D molecules was
the introduction of the central acceptor group.
The preparation of donor-π-acceptor-π-donor (D-
A-D) 2 was achieved by successfully introducing two nitro
groups in the key intermediate 10 by nitration of 9 in a 1:1
mixture of fuming HNO3/concentrated H2SO4 at 0 °C.21 The
reaction was clean and no byproducts, except the mononi-
trated product, were detected. The final product 2 was then
easily prepared by Horner-Emmons reaction as shown in
FIGURE 4. Synthesis of 5 and 6. Key: (a) Pd(AcO)2/PPh3, K2CO3,
Bu4NCl, DMF, reflux, 20 h, 70%; (b) P(OEt)3, reflux, 2 h; (c) 15,
DMF, NaH, rt, 20 h, 37-53%; (d) paraformaldehyde, 33% HBr in
HOAc, 70 °C, 48 h, 77%; (e) H2O2, HOAc, 90 °C, 30 min, 68%.
Figure 3. Compounds 1, 3, and 4 were also synthesized by
Horner-Emmons reactions using similar conditions indi-
cated in Figure 3. The synthesis of compound 3, which has
been reported previously via Heck coupling in 31% yield, was
synthesized in this work by a Horner-Emmons reaction in a
greatly increased yield (81%). Identical NMR spectra were
observed. The yields 1-4 were relatively good (55-85%).
Compounds 5 and 6 were prepared by Horner-Emmons
reactions as shown in Figure 4. Aldehyde 15, used in the
Horner-Emmons reaction, was synthesized by Heck coupling
of bromofluorenyl aldehyde 13 with N,N-diphenylami-
nostryene 14 in the presence of n-Bu4NCl in 71% yield.
(17) deDuve, C. Eur. J. Biochem. 1983, 137, 391–397.
(18) Stoka, V.; Turk, B.; Schendel, S. L.; Kim, T. H.; Cirman, T.; Snipas,
S. J.; Ellerbyi, L. M.; Bredeseni, D.; Freeze, H.; Abrahamson, M.; Bromme,
D.; Krajewski, S.; Reed, J. C.; Yin, X. M.; Turk, V.; Salvesen, G. S. J. Biol.
Chem. 2001, 276, 3149–3157.
(19) Jedeszko, C.; Sloane, B. F. Biol. Chem. 2004, 385, 1017–1027.
(20) Fehrenbacher, N.; Jaattela, M. Cancer Res. 2005, 65, 2993–2995.
(21) Yao, S.; Belfield, K. D. J. Org. Chem. 2005, 70, 5126–5132.
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FIGURE 6. Absorption (1ab, 2ab), fluorescence (1fl, 2fl), fluores-
cence anisotropy (1aniso, 2aniso), and 2PA spectra (12PA, 22PA) of 1
(gray) and 2 (black) in cyclohexane. Fluorescence anisotropy was
measured in polyTHF.
to a one-photon allowed S0-S1 transition (S0 and S1 are the
ground and first excited electronic states, respectively), in
which the fluorescence anisotropy was constant at about 0.35.
As expected from quantum mechanical selection rules, for mole-
cules with relatively high symmetry such as 1 and 2, the S0-S1
transition is a two-photon forbidden transition, a prediction
borne out in the 2PA spectra where very low two-photon ab-
sorptivity was observed in the long-wavelength region. As the
wavelength decreased, a change in the fluorescence anisotropy
values was observed at ca. 360 nm for 1 and 425 nm for 2, indi-
cative of a different electronic transition, e.g., S0-S2. This transi-
tion is obviously a two-photon allowed transition since the
maximum 2PA cross sections of both dyes were obtained at
wavelengths corresponding to this transition. Similar behavior
was observed for symmetrical molecules.23 The 2PA cross
section of dinitrofluorene 2 at 850 nm was nearly 1200 GM,
slightly higher than the 1000 GM 2PA cross section observed for
1 at 720 nm. This moderate increase of the 2PA cross section is
attributed to weak conjugation of the nitro groups with the
terminal amino electron-donating groups. Considering the fluor-
escence quantum yields of 1 and 2 are 1.0 and 0.44, respectively,
the lower fluorescence quantum yield of 2 resulted in the two-
photon action cross section of 2 being less than that for 1.
For the more electron-deficient compound 3, bearing
terminal benzothiazole moieties, the introduction of two
nitro groups in the fluorenyl bridge (4) produced only a slight
bathochromic shift in the absorption maximum (28 nm)
and did not exert much of an effect on the 2PA spectrum,
as shown in Figure 7. This is understandable since, in
general, a large 2PA cross section is considered as a result
of strong intramolecular charge transfer directly related to
the strength of the donor and acceptor groups. The ben-
zothiazole group itself can be considered as a very weak
electron donor (relative to a nitro group), and the intramo-
lecular charge transfer from benzothiazole to the nitro
groups is not strong enough to achieve a high 2PA cross
section. When considering the absorption, 2PA, and fluor-
escence anisotropy data, both 3 and 4 behave like typical
symmetrical molecules. The low energy one-photon allowed
transition is two-photon forbidden, while the 2PA allowed
transition appeared at higher energy (shorter wavelength).
FIGURE 5. Synthesis of 7 and 8: (a) DMF, NaH, rt, 20 h, 43%;
(b) Pd(AcO)2/PPh3, K2CO3, Bu4NCl, DMF, reflux, 20 h, 71%;
(c) NMP, 110 °C, 3 h, 70%; (d) P(OEt)3, o-dichlorobenzene, reflux,
16 h, 93%; (e) 15, DMF, NaH, rt, 20 h, 43%.
Intermediate 16 was prepared according to a literature
method in high yield.22 The development of a one-step direct
dibromomethylation of phenyl sulfide in 77% yield greatly
simplified the synthesis of bisbromomethylphenyl sulfide 18,
a key intermediate in the preparation of 6. Oxidation of sul-
fide 18 to the corresponding sulfone 19 occurred in good
yield. Phosphonylation of both 16 and 19, followed by reac-
tion with 15 in dry DMF using NaH as base, afforded
products 5 and 6 in yields of 53 and 37%, respectively.
Unsymmetrical 7 was prepared in a one-pot reaction by mix-
ing 9 with 11 and 12 in a 1:1:1 mol ratio, resulting in an isolated
yield of 43%. However, the same strategy was unsuccessful for
the synthesis of 8. This compound was prepared following the
same synthetic methodology used in the preparation of 5 and 6,
as shown in Figure 5. Here, aldehyde 21 was again prepared via
Heck coupling of bromofluorenyl aldehyde 13 with N,N-di-n-
butyl-4-vinylaniline (20) in the presence of n-Bu4NCl in good
yield (71%). Phosphonate 25 was prepared from the corre-
sponding chloromethyl derivative 24. We developed a simple
method preparation of 24 by reflux of commercially available 17
with 16 in NMP for a yield of 70%. Dye 8 was then obtained
1
43% yield. All compounds were characterized by H NMR,
13C NMR, and elementary analysis or high-resolution mass
spectroscopy.
Linear and Nonlinear Optical Properties. Shown in Figure
6 are the UV-vis (linear) absorption, fluorescence, fluores-
cence anisotropy, and two-photon absorption (nonlinear)
spectra of aminostyrylfluorenes 1 and 2 in cyclohexane. The
absorption maximum of 491 nm for dinitro-containing 2 had
a bathochromic shift of 84 nm relative to 1 (parent com-
pound). This long-wavelength absorption band corresponds
(23) Hales, J. M.; Hagan, D. J.; Van Stryland, E. W.; Schafer, K. J.;
Morales, A. R.; Belfield, K. D.; Pacher, P.; Kwon, O.; Zojer, E.; Bredas, J. L.
J. Chem. Phys. 2004, 121, 3152–3160.
(22) Yanez, C. O.; Andrade, C. D.; Belfield, K. D. Chem. Commun. 2009,
827–829.
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FIGURE 7. Absorption (3ab, 4ab), fluorescence (3fl, 4fl), fluores-
cence anisotropy (3aniso, 4aniso), and 2PA spectra (32PA, 42PA) of 3
(gray) and 4 (black) in cyclohexane. Fluorescence anisotropy was
measured in polyTHF.
FIGURE 9. Absorption (7ab, 8ab), fluorescence (7fl, 8fl), fluores-
cence anisotropy (7aniso, 8aniso), and two-photon absorption spectra
(72PA, 82PA) of 7 (gray) and 8 (black) in cyclohexane. Fluorescence
anisotropy was measured in polyTHF.
A more profound enhancement of 2PA was observed for
benzobisthiazolylfluorene 8 relative to aminobenzothiazo-
lylfluorene 7, as indicated in Figure 9. Although the absorp-
tion and fluorescence maxima of 8 exhibited only a slight
bathochromic shift (14 and 10 nm, respectively) with respect
to the smaller analogue 7, and the fluorescence anisotropy
profiles were also similar, the 2PA absorption of benzo-
bisthiazolylfluorene 8 was substantially larger than 7 over a
broad spectral range, i.e., from 550 to 930 nm. At its
maximum 2PA wavelength of 790 nm, 8 had an impressive
2PA cross section of 2517 GM, a 10-fold enhancement
relative to 7 at the same excitation wavelength (250 GM).
This is not unexpected since the 2PA cross section is quad-
ratically dependent on the stationary or transition dipole
moments; a small increase in either of these will result in a
significant increase in the 2PA cross section. Taking in
account of the fluorescence quantum yield of 0.75 for 8
and 1.00 for 7 in cyclohexane, the 2PA action cross section
8 (1887 GM) was 7.5 times greater than that of 7. This was
the largest 2PA enhancement observed for this series of dyes.
The high 2PA cross sections over a broad wavelength range
offers the flexibility of 2PF wavelength selection for bioima-
ging, depending on the nature of the biological sample.
Fluorescence Colocalization Studies and Two-Photon
Fluorescence Imaging. Pluronic materials are a series of block
copolymers based on ethylene oxide (forming a hydrophilic
block) and propylene oxide (forming a hydrophobic block)
and widely used as carriers for drug delivery.24,25 In this
work, probes 5-8 were encapsulated in Pluronic F 108NF
upon formation of micelles and their interaction with cells
was investigated by both confocal and two-photon fluores-
cence imaging. Dye-encapsulated micelles were uptaken by
cells without apparent difficulty, as shown in Figure 10 (where
probe 6 was used). To determine whether any oganelle-
selectivity occurred, colocalization studies were performed
with known probes.
FIGURE 8. Absorption (5ab, 6ab), fluorescence (5fl, 6fl), fluores-
cence anisotropy (5aniso, 6aniso), and two-photon absorption spectra
(52PA, 62PA) of 5 (gray) and 6 (black) in cyclohexane. Fluorescence
anisotropy was measured in polyTHF.
Photophysical data for fluorenylsulfones 5 and 6 are illu-
strated in Figure 8. The linear absorption and emission of 6
were nearly identical to 5. The profile of the 2PA absorption
spectra of 5 is consistent with a typical 2PA spectra of D-π-A
dipolar chromophores. Here the one-photon-allowed S0 to S1
transition, accompanied by constant fluorescence anisotropy,
is accessible by 2PA due to the low molecular symmetry.
Although the 2PA absorption spectrum of 6 is similar to that
of 5, the situation of6 is more complex since no clear transitions
can be identified from fluorescence anisotropy data. For such a
large molecule, it is not unusual that a number of electronic
transitions are closely spaced, resulting in constantly changing
anisotropy values. However, the 2PA cross section of 6 (1520
GM at 720 nm) was significantly larger than the smaller mole-
cule 5 (362 GM at 720 nm). The fluorescence quantum yields of
both 5 and 6 were similar (0.94 and 0.95), providing a 4-fold
enhancement of the 2PA action cross section for the larger
fluorenylsulfone 6 compared to 5. Moreover, at two-photon
excitation wavelengths from 690 to 840 nm, sulfone 6 showed
2PA action cross sections >750 GM. As expected, the 2PA
cross section of 6 was somewhat lower in THF relative to
cyclohexane (ca. 4000 vs 1300 GM at 700 nm, respectively).
With high 2PA action cross sections over such a broad spectral
range, this derivative is particularly attractive as a probe for
2PFM imaging.
In fluorescence microscopy, colocalization refers to an
analysis method to characterize the degree of overlap be-
tween two different fluorescent labels, each having a separate
emission wavelength, to determine if two different cellular
(24) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled
Release 2002, 82, 189–212.
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Rapoport, N. Y. J. Controlled Release 2002, 84, 39–47.
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FIGURE 10. Colocalization images of HCT 116 cells coincubated with probe 6 in Pluronic micelles and LysoTracker Red. Images were taken
using a 60ꢀ, oil immersion objective: (A) DIC, 500 ms; (B) one-photon confocal fluorescence image using a custom-made filter cube (Ex 377/50,
DM 409, Em 525/40), 4 ms; (C) one-photon confocal LysoTracker fluorescence image using a Texas Red filter cube (Ex 562/40, DM 593, Em
624/40) 350 ms; (D) overlapped image of A, B, and C (colocalization coefficient 0.99).
FIGURE 11. Images of HCT 116 cells incubated with fluorescence probe 8 in Pluronic micelles (30 μM, 3 h) taken with 60ꢀ, oil immersion
objective: (a) DIC, 400 ms; (b) one-photon fluorescence image (filter cube Ex 377/50, DM 409, Em 525/40); (c) 3D reconstruction from overlaid
2PFM images (Ex 700 nm, Em long-pass filter 690 nm).
“targets” are located in the same area. Here commercially
available LysoTracker was used for colocalization studies.
Commonly used lysosomal markers include DAMP, neutral
red, acridine orange, dextran, and bovine serum albumin
(BSA) labeled with a fluorophore and LysoTracker probes.
However, DAMP is not fluorescent, neutral red and acridine
orange lack staining specificity, and fluorescent dextran and
BSA have low-photostability and short-circulating life in
living cells.26 The LysoTracker probes are fluorescent acid-
otropic probes for labeling and tracking acidic organelles in
live cells, primarily lysosomes with high selectivity and
effective labeling of living cells at nanomolar concentrations,
although their two-photon action cross sections (ηδ) are very
low, e.g., a value of 10 GM was reported for LysoTracker
Red.27 It is important to note that cross-talk or “bleed-
through” may occur if the emission spectra of the two
fluorophores are similar. Accurate colocalization determi-
nation can only occur if emission spectra are sufficiently
separated between fluorophores and the correct filter sets
are used during the acquisition step. For this reason, Lyso-
Tracker Red was chosen since the emission wavelength of
LysoTracker Red can be detected at 624 nm, well separated
from the 525 nm emission maximum of probe 6 (Figure 10).
The fluorescence images, collected from two different wave-
lengths, i.e., 525 nm for probe 6 and 624 nm for Lysotracker
Red, are shown in Figure 10B and C, respectively, as well as the
differential interference contrast (DIC) image (Figure 10 A),
along with the overlap image of A, B, and C (Figure 10 D). Simi-
lar images were also collected for probes5-7 and are included in
the Supporting Information. It is obvious that the fluorescent
images of probe 6and LysoTracker are almost identical. Quanti-
tatively, the colocalization coefficient indicates the relative de-
gree of overlap between signals. While there are several methods
to calculate the colocalization coefficient, the one presented here
is the Pearson’s correlation coefficient, calculated within Slide-
book 4.1, imaging processing software. The correlation coeffi-
cients of dye 5-8relative to LysoTracker Red are all higher than
0.97, supporting lysosomal colocalization. An additional experi-
ment was performed for long-term tracking of lysosomes. A high
colocalization coefficient (0.80) was observed after 2 h of
incubation with both dye 6 and LysoTracker Red, followed by
an additional 9 h postincubation. The colocalization images are
included in the Supporting Information.
Two-photon fluorescence microscopy (2PFM) images
of the cells incubated with Pluronic micelle-encapsulated
probes 5-8 were obtained using a modified Olympus
Fluoview 3000 microscope system coupled to a femtosecond
Ti:sapphire femtosecond laser as excitation source. As an
example, a 2PFM image of HCT 116 cells incubated with
micelles containing probe 8 is shown in Figure 11 C. For
comparison, DIC and one-photon fluorescence images are
(26) Shi, H.; He, X.; Yuan, Y.; Wang, K.; Liu, D. Anal. Chem. 2010, 82,
2213–2220.
(27) Kim, H. M.; An, M. J.; Hong, J. H.; Jeong, B. H.; Kwon, O.; Hyon,
J.-Y.; Hong, S.-C.; Lee, K. J.; Cho, B. R. Angew. Chem. 2008, 120,
2263–2266.
3970 J. Org. Chem. Vol. 75, No. 12, 2010

Yao et al.
JOCFeatured Article
also presented (Figure 11A and B). The 2PFM 3D volume
view image provides higher resolution than conventional
confocal imaging, supporting further investigation of these
probes for bioimaging applications. Additional 2PFM
images of cells incubated with probes 5-7 can be found in
the Supporting Information.
triphenylamine (14),33 1-(bromomethyl)-4-(phenylsulfonyl)ben-
zene (16),22 and N,N-di-n-butyl-4-vinylaniline (20)34 were prepared
according to literature methods. A new route was used for the
synthesis of 3, which was different from that previously reported,35
1
and the H NMR spectrum was consistent with the previously
reported spectrum. 2, 5-Diaminobenzene-1,4-dithiol (22) was a gift
from the Air Force Research Laboratory/Polymer Branch at
Wright-Patterson Air Force Base. 4-(Chloromethyl)benzoyl chlor-
ide (17) was purchased from Acros Organics. Pluronic F 108NF
was obtained from BASF. LysoTracker Red was purchased from
Invitrogen. All other reagents and solvents were used as received
Conclusion
Synthetic methodology was developed to prepare a series
of donor-acceptor-donor (D-A-D) type fluorene deriva-
tives. The efficient synthesis of the precursor of the central
acceptor 10, 19, and 25 was key for the synthesis of 2, 4, 6,
and 8. Direct nitration of 2, 7-diformylfluorene introduced
two nitro groups into the fluorenyl ring at the 4 and 5
positions (10). The development of one-step direct dibromo-
methylation of phenyl sulfide, followed by oxidation, facili-
tated the synthesis of sulfone 19. An efficient method was
developed in this work for preparation of bisphosphonate 25
from commercially available starting materials. In general,
D-A-D compounds exhibited high fluorescence quantum
yields and improved 2PA cross sections relative to their
D-A or D-D counterparts with similar donor/acceptor
groups and conjugation length. The conjugated acceptor
core of fluorophore 8 led to the greatest 2PA cross section
enhancement, while the poorly conjugated central acceptor
core in 2 resulted in only a modest increase in the 2PA cross
section. The strength of the terminal donor moiety also
played a role in the D-A-D molecules; with the weak
electron donor in 4 having little effect on 2PA efficiency.
By encapsulation of the dyes in Pluronic F 128 NF micelles,
probes 5-8 were uptaken by cells, presumably through endo-
cytosis, resulting in high lysosomal selectivity as demonstrated
through colocalization (>0.98 coefficients) experiments with
Lysotracker Red. Preliminary results using these dyes as fluor-
escence probes for 2PFM cell imaging indicating they afford
high resolution and lysosomal selectivity when encapsulated in
micelles. The high specificity and 2PA cross section of our
probes suggest a strategy to overcome limitations for currently
used lysosomal trackers for 2PFM applications. Recently,
hydrophobic 2PF probes encapsulated in Pluronic micelles were
also used to image mice brain vasculature in vivo by 2PFM,28 a
further testament to the potential of this class of probes and
micelle-encapsulation strategy.
1
from commercial suppliers. Melting points are uncorrected. H
NMR and 13C NMR spectra were recorded at either 300 or 500
MHz and at 75 or 125 MHz, respectively. Absorption spectra were
measured with an UV-vis spectrophotometer. Steady-state fluor-
escence spectra were obtained at room temperature with a spectro-
fluorimeter using 10 mm quartz cuvettes. Fluorescence anisotropy
spectra were measured using three polarizers in the L-format
method, with correction for background signals, in the high
viscosity solvent polytetrahydrofuran (pTHF) at room tempera-
ture. Experimental details of anisotropy measurements were pre-
viously reported.36,37 Fluorescence quantum yields were measured
for all compounds by a standard method,38 relative to rhodamine
6G in ethanol at room temperature.
Preparation of 9,9-Didecyl-4,5-dinitro-9H-fluorene-2,7-dicar-
baldehyde (10). A mixture of fuming HNO3 (8 mL, d = 1.42 g/
cm3) and 98% H2SO4 was cooled to 0 °C. 9,9-Didecyl-9H-
fluorene-2,7-dicarbaldehyde (9, 2.0 g, 3.4 mmol) was then added
slowly, and the resulting mixture was stirred at 0 °C for 1.5 h.
The reaction was terminated by addition of 20 g of ice. The
organic phase was separated, and the aqueous phase was
extracted with hexane. The combined organic phase was then
washed with water and dried over MgSO4. Solvent was removed
under reduced pressure, and the crude product was purified via
column chromatography (4:3 hexanes/CH2Cl2), affording 0.96
g of off-white solid (41% yield): mp 57-58 °C; 1H NMR (300
MHz, CDCl3) δ 10.09 (s, 2H), 8.36 (s, 2H), 8.09 (s, 2H), 2.10 (m,
4H), 1.16-0.95 (m, 28H), 0.76 (t, J = 6.8 Hz, 6H), 0.46 (m, 4H);
13C NMR (75 MHz, DMSO-d6) δ 187.7, 155.3, 145.9, 135.9,
133.0, 124.5, 124.4, 55.7, 38.7, 30.7, 28.5, 28.3, 28.1, 28.0, 22.7,
21.5, 13.0. Anal. Calcd for C35H48N2O6 (592.77): C, 70.92; H,
8.16; N, 4.73. Found: C, 71.20; H, 8.12; N, 4.83.
General Procedure for Preparation of Dyes 1-5. 9,9-Didecyl-
9H-fluorene-2,7-dicarbaldehyde (9) or 9,9-didecyl-4,5-dinitro-
9H-fluorene-2,7-dicarbaldehyde (10, 0.5 mmol), 4-(N,N-
dibutylaminobenzyl)triphenylphosphonium iodide (11), and/or
diethyl 4-(benzo[d]thiazol-2-yl)benzylphosphonate (12, 1.0 mmol)
in 10 mL of dry DMF were degassed with Ar for 30 min. NaH
(0.24 g, 10.0 mmol) was then added, and the mixture was stirred
at room temperature under Ar for 20-24 h. The crude pro-
duct precipitated upon slow addition of water and was collected
by filtration, followed by column chromatographic purifica-
tion to afford pure compounds 1-4 and 7 (characterization
details below).
Experimental Section
General Methods. 9,9-Didecyl-9H-fluorene-2,7-dicarbalde-
hyde (9),29 (4-(dibutylamino)benzyl)triphenylphosphonium iodide
(11),30 diethyl 4-(benzo[d]thiazol-2-yl)benzylphosphonate (12),31
7-bromo-9,9-didecyl-9H-fluorene-2-carbaldehyde (13),32 4-vinyl-
4,40-(1E,10E)-2,20-(9,9-Didecyl-9H-fluorene-2,7-diyl)bis(ethane-
2,1-diyl)bis(N,N-dibutylaniline) (1): 3:1 hexanes/CH2Cl2 as elu-
ent (62% yield); mp 95-96 °C; 1H NMR (300 MHz, CDCl3) δ
7.56 (d, J = 8.1 Hz, 2H), 7.41-7.36 (m, 8H), 7.06 (d, J = 16.2
Hz, 2H), 6.94 (d, J = 15.9 Hz, 2H), 6.61 (d, J = 9.0 Hz, 4H), 3.27
(t, J = 7.5 Hz, 8H), 1.97 (m, 4H), 1.58 (m, 8H), 1.35 (m, 8H),
(28) Maurin, M.; Vurth, L.; Vial, J.-C.; Baldeck, P.; Marder Seth, R.; Van
der Sanden, B.; Stephan, O. Nanotechnology 2009, 20, 235102.
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Van Stryland, E. W.; Chapela, V. M.; Percino, J. Polym. Adv. Technol. 2005,
16, 150–155.
(30) Bredereck, H.; Simchen, G.; Griebenow, W. Chem. Ber. 1973, 106,
3732–42.
(31) Yoshino, K.; Kohno, T.; Uno, T.; Morita, T.; Tsukamoto, G.
J. Med. Chem. 1986, 29, 820–825.
(32) Kannan, R.; Tan, L.-S.; Reinhardt, B. A.; Vaia, R. A. US Patent
6730793, 2004.
(33) Xia, H.; He, J.; Xu, B.; Wen, S.; Li, Y.; Tian, W. Tetrahedron 2008,
64, 5736–5742.
(34) Cho, M. J.; Lim, J. H.; Hong, C. S.; Kim, J. H.; Lee, H. S.; Choi,
D. H. Dyes Pigm. 2008, 79, 193–199.
(35) Belfield, K. D.; Morales, A. R.; Kang, B.-S.; Hales, J. M.; Hagan,
D. J.; Van Stryland, E. W.; Chapela, V. M.; Percino, J. Chem. Mater. 2004,
16, 4634–4641.
(36) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer
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(37) Belfield, K. D.; Bondar, M. V.; Przhonska, O. V.; Schafer, K. J.
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J. Org. Chem. Vol. 75, No. 12, 2010 3971

Yao et al.
JOCFeatured Article
1.25-1.05 (m, 28H), 0.95 (t, J = 7.2 Hz, 12H), 0.82 (t, J = 6.6
Hz, 6H), 0.69 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 151.3,
147.6, 139.8, 137.0, 128.0, 127.7, 125.0, 124.8, 124.4, 120.1,
119.6, 111.7, 55.1, 51.0, 40.9, 32.2, 30.4, 30.0, 29.9, 29.8, 29.6,
24.1, 23.0, 20.7, 14.5, 14.4. Anal. Calcd for C65H96N2 (905.47):
C, 86.22; H, 10.69; N, 3.09. Found: C, 86.18; H, 10.83; N, 3.16.
4,40-(1E,10E)-2,20-(9,9-Didecyl-4,5-dinitro-9H-fluorene-2,7-diyl)-
bis(ethene-2,1-diyl)bis(N,N-dibutylaniline) (2): 2:1 hexanes/
CH2Cl2 as eluent (55% yield); mp 68-69 °C; 1H NMR (300
MHz, CDCl3) δ 7.98 (s, 2H), 7.56 (s, 2H), 7.42 (d, 4H, J = 8.7
Hz), 7.18 (d, J = 15.9 Hz, 2H), 6.94 (d, J = 16.2 Hz, 2H), 6.65
(d, J = 9.0 Hz, 4H), 3.32 (t, J = 7.2 Hz, 8H), 2.07 (m, 4H),
1.61 (m, 8H), 1.36 (m, 8H), 1.25-1.00 (m, 28H), 0.97 (t, J =
7.4 Hz, 12H), 0.82 (t, J = 6.8 Hz, 6H), 0.63 (m, 4H); 13C
NMR (75 MHz, CDCl3) δ 154.8, 148.6, 146.5, 139.8, 132.5,
129.6, 128.5, 128.5, 123.4, 123.2, 120.9, 111.7, 56.1, 51.2,
40.9, 32.3, 32.1, 30.2, 30.0, 29.9, 29.7, 29.6, 24.0, 23.1, 20.8,
14.6, 14.5. Anal. Calcd for C65H94N4O4 (995.47): C, 78.43;
H, 9.52; N, 5.63. Found: C, 78.06; H, 9.57; N, 5.44.
2,20-(4,40-(1E,10E)-2,20-(9,9-Didecyl-9H-fluorene-2,7-diyl)bis-
(ethene-2,1-diyl)bis(4,1-phenylene))dibenzo[d]thiazole35 (3): 1:2
hexanes/CH2Cl2 as eluent (81% yield); mp 154-155 °C (lit.35
mp 143-144 °C); 1H NMR (500 MHz, CDCl3) δ 8.12 (d, J = 8.0
Hz, 4H), 8.09 (d, J = 8.0 Hz, 2H), 7.93 (d, J = 8.0 Hz, 2H), 7.69
(d þ d, 6H), 7.56 (d, J = 8.0 Hz, 2H), 7.52 (m, 4H), 7.41 (t, J =
8.3 Hz, 2H), 7.35 (d, J = 16.0 Hz, 2H), 7.23 (d, J = 16.0 Hz, 2H),
2.05 (t, J = 7.5 Hz, 4H), 1.22-1.08 (m, 28H), 0.82 (t, J = 7.0 Hz,
6H), 0.68 (m, 4H).
2,20-(4,40-(1E,10E)-2,20-(9, 9-Didecyl-4,5-dinitro-9H-fluorene-
2,7-diyl)bis(ethene-2,1-diyl)bis(4,1-phenylene))dibenzo[d]thiazole
(4): 1:3 hexanes/CH2Cl2 as eluent (82% yield); mp 171-172 °C;
1H NMR (300 MHz, CDCl3) 8.15-8.06 (m, 8H), 7.91 (d, 2H,
J = 8.4 Hz), 7.69 (m, 6H), 7.50 (d, J = 7.5 Hz, 2H), 7.39 (d, J =
7.5 Hz, 2H), 7.32 (s, 4H), 2.10 (m, 4H), 1.15-1.07 (m, 28H), 0.81
(t, J = 6.6 Hz, 6H), 0.62 (m, 4H); 13C NMR (75 MHz, CDCl3) δ
167.3, 155.2, 154.2, 146.8, 138.9, 138.6, 135.2, 133.7, 131.4,
129.8, 128.2, 127.8, 127.6, 126.6, 125.5, 124.3, 123.4, 121.8,
56.4, 40.8, 32.3, 30.2, 29.9, 29.7, 29.6, 24.0, 23.1, 14.6. Anal.
Calcd for C63H66N4O4S2 (1007.35): C, 75.11; H, 6.60; N, 5.56.
Found: C, 75.24; H, 6.78; N, 5.42.
[4-[2-[7-[2-(4-Benzothiazol-2-ylphenyl)vinyl]-9,9-didecyl-9H-
fluoren-2-yl]vinyl]phenyl]dibutylamine (7): 1:3 hexanes/CH2Cl2
as eluent (43% yield); mp 95-96 °C; 1H NMR (300 MHz,
CDCl3) δ 8.07 (m, 3H), 7.89 (d, J = 7.8 Hz, 1H), 7.63 (m, 4H),
7.51-7.38 (m, 8H), 7.31 (d, J = 16.8 Hz, 2H), 7.17 (d, J = 16.2
Hz, 2H), 7.08 (d, J = 16.2 Hz, 2H), 6.95 (d, J = 16.2 Hz, 2H),
6.62 (d, J = 8.1 Hz, 2H), 3.29 (m, 4H), 2.02 (m, 4H), 1.58 (m, 4H),
1.37 (m, 4H), 1.15-1.06 (m, 28H), 0.97 (t, J = 7.1 Hz, 6H), 0.82 (t,
J = 5.9 Hz, 6H), 0.68 (m, 4H). 13C NMR (75 MHz, CDCl3) δ
154.3, 151.6, 147.9, 141.6, 140.5, 139.5, 137.7, 135.5, 135.1, 132.4,
131.3, 128.5, 128.1, 127.9, 127.0, 126.7, 126.5, 126.1, 125.3, 124.8,
124.4, 123.3, 121.8, 121.1, 120.3, 120.1, 119.9, 111.8, 55.3, 51.2,
41.0, 32.3, 32.1, 30.5, 30.1, 30.0, 30.0, 29.7, 24.3, 23.1, 20.9, 14.6,
14.5. Anal. Calcd for C64H82N2S (911.42): C, 84.34; H, 9.07; N,
3.07. Found: C, 84.34; H, 9.28; N, 3.08.
7.42 (d, J = 9.0 Hz, 2H), 7.25-7.29 (m, 4H), 7.11-7.18 (m, 6H),
7.03-7.08 (m, 4H), 2.03 (m, 4H), 1.03-1.25 (m, 28H), 0.83 (t,
J = 7.0 Hz, 6H), 0.62 (m, 4H); 13C NMR (125 MHz, CDCl3) δ
14.1, 22.7, 23.8, 29.3, 29.5, 29.6, 30.0, 31.9, 40.3, 55.2, 119.8,
120.7, 121.2, 123.0, 123.1, 123.4, 124.6, 125.7, 127.1, 127.4,
128.6, 129.3, 130.7, 131.3, 135.1, 138.4, 138.9, 147.4, 147.5,
147.5, 151.7, 152.8, 192.4; HRMS-(APPI) for C54H65NO theo-
retical m/z [M þ H]þ=744.5139, found [M þ H]þ=744.5157.
Preparation of Bis(4-(bromomethyl)phenyl)sulfane 18. Phenyl
sulfide 17 (1.86 g, 0.01 mmol) and paraformaldehyde (1.2 g, 0.04
mmol) in 33% HBr/HOAc (10 mL) were heated at 70 °C for
48 h. Water and 2:1 hexanes/EtOAc were added. White solid
precipitated and was collected by filtration to afford 2.86 g of 18
(77% yield): mp 129-130.5 °C (lit.39 135.3-136.3 °C); 1H NMR
(500 MHz, CDCl3) δ 7.29-7.34 (m, 8H), 4.47 (s, 4H); 13C NMR
(125 MHz, CDCl3) δ 32.9, 129.9, 131.3, 135.8, 136.8; HRMS-
(DIP-CI) for C14H12Br2S theoretical m/z [M]þ = 369.9026,
found [M]þ=369.9008.
Preparation of 4,40-Sulfonylbis((bromomethyl)benzene) 19.
Bis(4-(bromomethyl)phenyl)sulfane 18 (0.5 g, 1.34 mmol) was
dissolved in HOAc (4 mL) at 70 °C. H2O2 (30%, 1 mL) was
slowly added, and the mixture was heated at 90 °C for 30 min.
The product precipitated after cooling and was collected by
filtration, washed with water, and dried, providing 0.37 g of 19
(68% yield): mp 145-146 °C (lit.40 134-135 °C); 1H NMR (500
MHz, CDCl3) δ 7.92 (d, J = 5.0 Hz, 4H), 7.53 (d, J = 5.0 Hz,
4H), 4.46 (s, 4H); 13C NMR (125 MHz, CDCl3) δ 31.3, 128.3,
130.0, 141.1, 143.3; HRMS(DIP-CI) for C14H12Br2O2S theore-
tical m/z [M þ H]þ=402.9003, found [M þ H]þ=402.9010.
Preparation of Dye 5. 1-(Bromomethyl)-4-(phenylsulfonyl)-
benzene (16, 36.2 mg, 0.116 mmol) was refluxed with triethyl
phosphite (1 mL) for 2 h. Excess triethyl phosphite was evapo-
rated, and the residue was dried in vacuo. (E)-9,9-Didecyl-
7-(4-(diphenylamino)styryl)-9H-fluorene-2-carbaldehyde (15,
86.5 mg, 0.116 mmol) was then mixed with the above product in
dry DMF (2 mL) under Ar. NaH (27.8 mg, 1.16 mmol) was
added, and the reaction was stirred at room temperature for 20 h.
Water was added to terminate the reaction, and the product
was extracted with EtOAc. Solvent was removed, and the crude
product was purified by column chromatography (1:1 hexanes/
CH2Cl2), producing 58.6 mg of 5 (53% yield) as a soft glassy
material, which turned into viscous liquid upon heating. No
defined melting point was observed: 1H NMR (500 MHz,
CDCl3) δ 7.88 (m, 4H), 7.57 (m, 4H), 7.52-7.33 (m, 8H),
7.23-7.17 (m, 6H), 7.07-6.95 (m, 11H), 1.93 (m, 4H), 1.18-
0.97 (m, 28H), 0.73 (t, J = 5.0 Hz, 6H), 0.58 (m, 4H); 13C NMR
(125 MHz, CDCl3) δ 151.6, 147.5, 147.3, 141.8, 140.0, 139.5,
133.2, 133.1, 131.6, 129.3, 127.6, 127.5, 127.3, 126.9, 126.2, 125.6,
125.5, 124.5, 123.6, 123.0, 121.1, 120.6, 120.1, 119.9, 55.0, 40.5,
31.9, 29.6, 29.6, 29.3, 29.3, 23.8, 22.7, 14.1; HRMS (MALDI-TOF)
for C67H75NO2S theoretical m/z [M þ H]þ = 958.5591, found
[M þ H]þ=958.5587.
Preparation of Dye 6. 4,40-Sulfonylbis((bromomethyl)ben-
zene) (19, 0.1 g, 0.247 mmol) was refluxed with triethyl phos-
phite for 2 h. Excess triethyl phosphite was evaporated, and the
residue was dried in vacuo. (E)-9,9-Didecyl-7-(4-(diphenyl-
amino)styryl)-9H-fluorene-2-carbaldehyde (15, 0.37 g, 0.5 mmol)
was then mixed with the above product in dry DMF (5 mL) under
Ar. NaH was added, and the reaction was stirred at room
temperature for 20 h. Water was added, and the product was
extracted with EtOAc. Solvent was removed, and the crude
product was purified by column chromatography (1:1 hexanes/
CH2Cl2), providing 0.16 g of 6 (37% yield) as a glassy material,
which turned into a viscous liquid upon heating. No defined
Preparation of (E)-9,9-Didecyl-7-(4-(diphenylamino)styryl)-
9H-fluorene-2-carbaldehyde (15). 7-Bromo-9,9-didecyl-9H-flu-
orene-2-carbaldehyde (13) (1.384 g, 2.5 mmol), 4-vinyltripheny-
lamine (14, 0.678 g, 2.5 mmol), PPh3 (65.56 mg, 0.25 mmol), and
K2CO3 (6.25 mmol, 0.864 g) in DMF were degassed with Ar
for 30 min, and then Pd(OAc)2 (84.20 mg, 0.125 mmol) and
n-Bu4NCl (0.695 g, 2.5 mmol) were added, followed by refluxing
the mixture under Ar for 20 h. Water and hexanes were added,
and the organic phase was concentrated. The crude product was
purified by column chromatography (20:1 hexanes/EtOAc),
affording 1.31 g of 15 as yellow sticky oil (70% yield): 1H
NMR (500 MHz, CDCl3) δ 10.05 (s, 1H), 7.86 (m, 2H), 7.80
(d, J = 7.5 Hz, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.48-7.53 (m, 2H),
(39) Oi, F.; Yanase, N.; Yamamoto, M. JP Patent 2005154379, 2005.
(40) Jung, H. K.; Lee, J. K.; Kang, M. S.; Kim, S. W.; Kim, J. J.; Park,
S. Y. Polym. Bull. 1999, 43, 13–20.
3972 J. Org. Chem. Vol. 75, No. 12, 2010

Yao et al.
JOCFeatured Article
melting point was observed: 1H NMR (500 MHz, CDCl3) δ 7.95
(d, J = 10.0 Hz, 4H), 7.65 (m, 8H), 7.41-7.51 (m, 12H), 7.29 (m,
12 H), 7.02-7.13 (m, 20H), 2.01 (m, 8H), 1.04-1.26 (m, 56H),
0.79 (t, J = 5.0 Hz, 12H), 0.65 (m, 8H); 13C NMR (125 MHz,
CDCl3) δ 151.7, 147.6, 147.3, 141.8, 140.0, 139.8, 137.0, 133.1,
131.6, 129.3, 128.1, 127.7, 127.6, 127.3, 126.9, 126.2, 125.7, 125.5,
124.5, 123.6, 123.1, 121.1, 120.6, 120.1, 120.0, 55.0, 40.5, 31.9,
29.6, 29.6, 29.3, 29.3, 23.8, 22.7, 14.1; HRMS (MALDI-TOF) for
7.33-7.27 (m, 4H), 7.10 (d, J = 16.0 Hz, 2H), 7.09 (d, J = 16.0
Hz, 2H), 7.00 (d, J=16.0 Hz, 2H), 6.65 (d, J = 16.0 Hz, 4H),
3.32 (t, J=7.5 Hz, 8H), 2.03 (m, 8H), 1.62 (m, 8H), 1.39 (m,
8H), 1.23-0.98 (m, 68H), 0.83 (m, t, J=7.2 Hz), 0.73 (m, 8H);
13C NMR (125 MHz, CDCl3) δ 168.5, 152.3, 151.6, 151.5, 147.8,
141.5, 140.5, 139.4, 137.6, 135.3, 134.5, 132.1, 128.0, 127.9,
127.7, 127.1, 126.9, 124.7, 121.3, 121.0, 120.6, 120.3, 120.0,
119.9, 115.4, 115.2, 111.6, 55.0, 50.8, 40.6, 31.9, 30.1, 29.5,
29.3, 23.9, 22.7, 20.6, 20.4, 20.3, 14.3, 14.1, 14.0; HRMS (ESI-
TOF) for C122H158N4S2 theoretical m/z [M]2þ=871.5958, found
[M]2þ = 871.5959.
Two-Photon Absorption Measurements. 2PA spectra of com-
pounds 1-8 were determined over a broad spectral region by the
typical two-photon induced fluorescence (2PF) method,41 relative
to rhodamine B in methanol and fluorescein in water (pH 11) as
standards, using a spectrofluorimeter and femtosecond laser-
pumped optical parametric generator/amplifier, with pulse dura-
tion, 140 fs (fwhm), tuning range 580-940 nm, pulse energies
0.15 mJ, and 1 kHz repetition rate. Two-photon fluorescence
measurements were performed in 10 mm fluorometric quartz
cuvettes with dye concentrations ∼3 ꢀ 10-5 M in cyclohexane.
The experimental fluorescence excitation and detection conditions
were conducted with the negligible reabsorption processes to
ensure no effect on 2PA measurements. The quadratic dependence
of two-photon induced fluorescence intensity on the excitation
power was verified for each excitation wavelength by system-
atically varying the excitation power.
General Procedure for Preparation of Dye-Encapsulated Mi-
celles. A solution of dye in CH2Cl2 (5 mL) was mixed with
Pluronic F 108NF (30 mg) in water (5 mL). The resulting
mixture was stirred at room temperature for 48 h to slowly
evaporate the CH2Cl2. The dye concentration was approxi-
mately 0.5 mM in water, as estimated by absorption spectra.
General Procedure for HCT 116 Cells’ Incubation with Fluor-
escent Probes. An epithelial colorectal carcinoma cell line HCT
116, purchased from ATCC (America Type Culture Collection,
Manassas, VA), was used. All cells were incubated in Dulbecco’s
modified Eagle’s Medium (DMEM), supplemented with 10%
fetal bovine serum (FBS), 100 units/mL penicillin-streptomycin,
and incubated at 37 °C in a 95% humidified atmosphere
containing 5% CO2.
HCT 116 cells were placed onto poly-D-lysine coated coverslips
placedinto24-wellglassplates (40000 cells per well) andincubated
for 36 h before incubating with the fluorescent probes. A 0.5 mM
stock solution of the fluorescent probe in Pluronic F 108NF
micelles was dissolved in water. Diluted solutions of probe (30 μM)
by complete growth medium, Dulbecco’s modification of Eagle’s
medium (DMEM), were then freshly prepared and placed over
the cells for a 3 h period. For colocalization experiments, a
diluted mixture of dye (30 μM) and LysoTracker Red (75 nM)
in the growth medium was used. After the incubation, cells were
washed with PBS (pH = 7.2) three to five times, fixed using 3.7%
formaldehyde solution at 37 °C for 15 min, and incubated with
0.5 mL/well NaBH4 (1 mg/mL) solution in PBS (pH = 8.0, pre-
pared by adding few drops of 6N NaOH solution into PBS) at
37 °C for 15 min. The last step was then repeated. The plates were
washed twice with PBS and once with water. Finally, the glass
coverslips were mounted using Prolong Gold mounting media for
microscopy imaging.
C
1697.0663.
122H140N2O2S theoretical m/z [M]þ = 1697.0636, found [M]þ =
Preparation of (E)-9,9-Didecyl-7-(4-(dibutylamino)styryl)-
9H-fluorene-2-carbaldehyde (21). 7-Bromo-9,9-didecyl-9H-
fluorene-2-carbaldehyde 13 (0.554 g, 1.0 mmol), N,N-di-n-bu-
tyl-4-vinylaniline (20, 0.231 g, 1.0 mmol), PPh3 (26.2 mg, 0.10
mmol), and K2CO3 (0.346 g, 2.5 mmol) in DMF were degassed
for 30 min, and then Pd(OAc)2 (33.7 mg, 0.05 mmol) and
n-Bu4NCl (0.278 g, 1.0 mmol) were added. The mixture was
then refluxed for 20 h under Ar. Water and hexane were added,
the organic phase was concentrated, and the crude product
was purified by column chromatography using 1.5:1 hexanes/
CH2Cl2 as eluent, affording 0.50 g of 21 as yellow oil (71%
1
yield): H NMR (300 MHz, CDCl3) δ 9.95 (s, 1H), 7.77-7.68
(m, 3H), 7.62 (d, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.35-7.32 (m,
3H), 7.05 (d, J = 16.2 Hz, 1H), 6.88 (d, J = 16.2 Hz, 1H), 6.57
(d, J = 8.7 Hz, 2H), 3.23 (t, J = 7.5 Hz, 4H), 1.95 (t, J = 8.0 Hz,
4H), 1.51 (m, 4H), 1.29 (m, 4H), 1.20-0.88 (m, 28H), 0.83-0.74
(m, 12H), 0.54 (m, 4H); 13C NMR (74.5 MHz, CDCl3) δ 13.0,
13.1, 19.3, 21.6, 22.7, 28.2, 28.4, 28.4, 28.5, 28.9, 30.5, 30.8, 39.2,
49.8, 54.0, 107.2, 110.5, 118.4, 119.0, 119.9, 121.7, 122.6, 124.0,
126.6, 128.1, 129.4, 133.6, 136.8, 137.9, 146.3, 146.5, 150.3,
151.4, 191.0; HRMS (ESI) for C50H73NO theoretical m/z
[M þ H]þ=704.5765, found [M þ H]þ=704.5751.
Preparation of 2,6-Bis[4-(chloromethyl)phenyl]benzo[1,2-
d:4,5-d0]bisthiazole (24). 2,5-Diaminobenzene-1,4-dithiol (22,
0.39 g, 2.26 mmol) and 4-(chloromethyl)benzoyl chloride (23,
0.86 g, 4.55 mmol) in 5 mL of anhydrous NMP was heated at
110 °C for 3 h. Upon cooling, water was added, and the pre-
cipitate was collected by filtration. Recrystallization from
toluene/o-dichlorobenzene afforded 0.71 g of 24 (70% yield):
1
mp>500 °C dec; H NMR (300 MHz, DMSO-d6) δ 8.87 (s,
2H), 8.15 (d, J = 7.8 Hz, 4H), 7.66 (d, J = 7.8 Hz, 4H), 4.88 (s,
4H); 13C NMR could not be collected due to the limited
solubility; HRMS (ESI) for C22H14Cl2N2S2 theoretical m/z
[M þ H]þ=441.0052, found [M þ H]þ=441.0061. Anal. Calcd
for (C22H14Cl2N2S2): C, 59.86; H, 3.20; N, 6.35; S, 14.53.
Found: C, 60.01; H, 3.26; N, 6.43; S, 14.59.
Preparation of Tetraethyl 4,40-(Benzo[1,2-d:4,5-d0]bisthiazole-
2,6-diyl)bisbenzylphosphonate (25). A solution of 24 (0.78 g, 1.77
mmol) and triethyl phosphite (1.17 g, 7.08 mmol) in 5 mL of
o-dichlorobenzene was heated at reflux for 16 h. The product
was then precipitated from hexane, collected by filtration, and
purified by column chromatography using 20:1 CH2Cl2/MeOH
as eluent, providing 1.06 g of 24 (93% yield): mp 289.5-290 °C;
1H NMR (300 MHz, CDCl3) δ 8.52 (s, 2H), 8.06 (d, J = 8.1 Hz,
4H), 7.45 (d, J = 8.1 Hz, 4H), 4.05 (m, 8H), 3.23 (d, J =22.2 Hz,
4H), 1.27(t, J=7.1 Hz, 12H); 13C NMR (75 MHz, CDCl3) δ
16.8, 33.4, 35.2, 62.7, 115.6, 128.0, 130.7, 132.3, 134.6, 135.5,
152.3, 168.7. Anal. Calcd for (C30H34N2O6P2S2): C, 55.89; H,
5.32; N, 4.35; S, 9.95. Found: C, 56.11; H, 5.36; N, 4.41; S, 10.04.
Preparation of Dye 8. A solution of 21 (58 mg, 0.082 mmol)
and 25 (26 mg, 0.041 mmol) in 1 mL of DMF was degassed by Ar
for 30 min. NaH (14 mg, 0.82 mmol) was then added, and the
mixture was stirred at room temperature under Ar for 20 h. The
product was precipitated by addition of water and collected by
filtration. The crude product was purified by column chroma-
tography using 1:2 CH2Cl2/hexanes as eluent, affording 31 mg
of 6 (43% yield): mp 196-197 °C; 1H NMR (300 MHz, CDCl3)
δ 8.37 (s, 2H), 7.99 (d, J=8.0 Hz, 4H), 7.54-7.40 (m, 18H),
One-Photon Fluorescence Imaging. An inverted microscope
equipped with a cooled CCD was used for conventional fluor-
escence imaging, where the output of a filtered 100 W mercury
lamp was used as the excitation source. For probes 5-8, a
customized filter cube (Ex 377/50, DM 409, Em 460/50) was
used for fluorescence imaging, while for LysoTracker Red,
(41) Xu, C.; Webb, W. W. J. Opt. Soc. Am. B: Opt. Phys. 1996, 13,
481–491.
J. Org. Chem. Vol. 75, No. 12, 2010 3973

Yao et al.
JOCFeatured Article
a Texas Red filter cube (Ex 562/40, DM 593, Em 624/40)
was used.
Acknowledgment. We acknowledge the National Science
Foundation (CHE-0832622, ECS-0524533, and ECCS-
0621715) and the National Institutes of Health (1 R15
EB008858-01) for support of this work.
Two-Photon Fluorescence Microscopy (2PFM) Imaging.
2PFM images were collected on a modified microscope system
coupled to a tunable Ti:sapphire, 76 MHz, mode-locked, femto-
second laser tuned to 700 nm. An emission long-pass filter (cutoff
690 nm) was placed in the microscope scanhead to avoid back-
ground irradiance from the excitation source. Consecutive layers,
separated by approximately 0.15 μm, were recorded to create a
3D reconstruction from overlaid 2PFM images. The two-photon
induced fluorescence was collected with a 60ꢀ microscope objec-
tive (UPLANSAPO 60ꢀ, NA=1.35).
Supporting Information Available: Copies of 1H and 13C
NMR spectra, fluorescence images of the colocalization of
probes 5, 7, and 8 with Lysotracker Red in HCT 116 cells, and
2PFM images of HCT 116 cells incubated with probes 5-7. This
material is available free of charge via the Internet at http://
pubs.acs.org.
3974 J. Org. Chem. Vol. 75, No. 12, 2010