A series of fluorene-based two-photon absorbing molecules: Synthesis, linear and nonlinear characterization, and bioimaging

Article abstract of DOI:10.1021/jo1005075

The synthesis, structural, and photophysical characterization of a series of new fluorescent donor-acceptor and acceptor-acceptor molecules, based on the fluorenyl ring system, with two-photon absorbing properties is described. These new compounds exhibited large Stokes shifts, high fluorescence quantum yields, and significantly, high two-photon absorption cross sections, making them well suited for two-photon fluorescence microscopy imaging. Confocal and two-photon fluorescence microscopy imaging of COS-7 and HCT 116 cells incubated with probe I showed endosomal selectivity, demonstrating the potential of this class of fluorescent probes in multiphoton fluorescence microscopy.

Full text of DOI:10.1021/jo1005075

pubs.acs.org/joc
A Series of Fluorene-Based Two-Photon Absorbing Molecules: Synthesis,
Linear and Nonlinear Characterization, and Bioimaging
Carolina D. Andrade,† Ciceron O. Yanez,† Luis Rodriguez,‡ and Kevin D. Belfield*,†,‡
‡
†Department of Chemistry and CREOL, The College of Optics and Photonics,
University of Central Florida, Orlando, Florida 32816
belfield@mail.ucf.edu
Received March 17, 2010
The synthesis, structural, and photophysical characterization of a series of new fluorescent donor-
acceptor and acceptor-acceptor molecules, based on the fluorenyl ring system, with two-photon
absorbing properties is described. These new compounds exhibited large Stokes shifts, high fluore-
scence quantum yields, and significantly, high two-photon absorption cross sections, making them
well suited for two-photon fluorescence microscopy imaging. Confocal and two-photon fluorescence
microscopy imaging of COS-7 and HCT 116 cells incubated with probe I showed endosomal selec-
tivity, demonstrating the potential of this class of fluorescent probes in multiphoton fluorescence
microscopy.
Introduction
The development of multiphoton absorbing materials has
attracted the interest of the scientific community in the past
decade as a result of their potential applications in areas such
as optical data storage systems, microscopy imaging, optical
limiting materials, and photodynamic therapy. In one-photon
absorption processes, the absorbed light is directly propor-
tional to the incident light intensity, whereas in two-photon
absorption (2PA) processes, the probability of absorbing
two photons simultaneously is proportional to the square of
the incident light intensity.1 This nonlinear dependence pro-
vides several advantages, including highly confined excita-
tion, increased 3D resolution (Figure 1), and the potential
increase of penetration depth in tissue, important features in
FIGURE 1. One-photon (left arrow) versus two-photon (right arrow)
excitation, showing the high 3D spatially localized excitation in 2PA in
a solution of dye I.
fields such as optical data storage,2-4 3D microfabrication,5-7
and fluorescence microscopy.1,8
(1) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73–76.
(2) Parthenopoulos, D. A.; Rentzepis, P. M. Science 1989, 245, 843–845.
(3) Belfield, K. D.; Schafer, K. J. Chem. Mater. 2002, 14, 3656–3662.
(4) Yanez, C. O.; Andrade, C. D.; Yao, S.; Luchita, G.; Bondar, M. V.;
Belfield, K. D. ACS Appl. Mater. Interfaces 2009, 1, 2219-2229.
(5) Belfield, K. D.; Schafer, K. J.; Liu, Y. U.; Liu, J.; Ren, X. B.;
Van Stryland, E. W. J. Phys. Org. Chem. 2000, 13, 837–849.
(7) Belfield, K. D.; Ren, X.; Hagan, D. J.; Van Stryland, E. W.;
Dubikovsky, V.; Miesak, E. J. J. Am. Chem. Soc. 2000, 122, 1217–1218.
(8) Schafer-Hales, K. J.; Belfield, K. D.; Yao, S.; Frederiksen, P. K.;
Hales, J. M.; Kolattukudy, P. E. J. Biomed. Opt. 2005, 10, 015402–1-8.
(6) Maruo, S.; Nakamura, O.; Kawata, S. Opt. Lett. 1997, 22, 132–134.
DOI: 10.1021/jo1005075
r
Published on Web 05/19/2010
J. Org. Chem. 2010, 75, 3975–3982 3975
2010 American Chemical Society

Andrade et al.
JOCArticle
FIGURE 2. Structures of new donor-π-acceptor (D-π-A) (I and III) and acceptor-π-acceptor (A-π-A) (II and IV) fluorenyl derivatives.
Benzothiazole moieties were used as acceptor groups while diphenylamino groups were used as donor moieties. Thiophene groups and alkynes
were incorporated to extend the π-conjugation.
We previously reported a series of fluorescent dyes and
photoacid generators that use fluorene as a core structure.9-14
This core exhibited high thermal and photochemical stabi-
lity, making this class of compounds promising in areas such
as lasing applications.15
including thiophene and alkynes were incorporated in order
to increase the length of the π-conjugation and to evaluate
the effect of these functionalities on the linear absorption
maxima, fluorescence quantum yields, and 2PA cross sections.
The chemical structures of the new fluorescent compounds
presented in this paper are illustrated in Figure 2.
For the work described herein, the thiophene ring was chosen
as a π electron bridge because it has a higher degree of aro-
maticity than other five-membered rings, e.g., furan, it is stable
toward oxidation, and its chemical reactivity to allow functio-
nalization is higher than that observed for benzene or furan.
Incorporation of these heterocyclic rings in the molecular
design of chromophores has been reported to enhance the
optical properties, increasing both the maximum of absorption
(λabsmax) and the 2PA cross section values, particulary when the
thiophene ring is close to the fluorene core.17,18
One of our goals was to compare this new series of com-
pounds with our previously prepared and studied probes that
contain benzothiazole groups. Thus, the benzothiazole ring
system was used as an electron-withdrawing group. Previous
studies suggested that compounds containing this moiety are
more robust than analogous compounds containing benzox-
azole groups. Moreover, benzothiazole groups are relatively
strong electron-withdrawing groups, and their ability to
accept charge transferred from a diphenylamino moiety
appears to be higher than that observed for benzoxazolyl-
containing compounds.19,20
Recently, a computational quantum chemical study of nume-
rous fluorene derivatives was reported in order to predict and
rationalize certain 2PA parameters, such as 2PA cross sec-
tions and 2PA wavelength. Though insightful, little compa-
rison and validation of computed results with experimental
data was presented, emphasizing the need to accurately mea-
sure 2PA spectra and cross sections of new compounds.16
In the interest of developing more efficient 2PA fluorophores,
it is essential to improve the key photophysical properties such
as quantum yield, absorption maxima, and 2PA cross sections
in the tuning range of commercial Ti:sapphire lasers (700-
1000 nm). In this work, we present the design, synthesis, and
photophysical (linear and nonlinear) characterization of a new
series of two-photon absorbing molecules based on a fluorenyl
core and demonstration of their use in in vitro confocal and two-
photon fluorescence microscopy (2PFM) bioimaging.
In this approach, a series of dyes were developed with the
general structural design donor-π-acceptor (D-π-A) and
acceptor-π-acceptor (A-π-A) using benzothiazole moieties
as electron-acceptor groups and diphenylamino groups as
electron-donor moieties. To enhance the solubility of the fluo-
rene core in organic solvents, both protons on the 9-position
of fluorene were substituted by alkyl chains. Functionalities
Results and Discussion
Synthesis of New Fluorescent Probes. The model 2PA fluore-
scent probe I was synthesized starting from the transformation
of benzothiazole derivative 1 into tin derivative 2, followed by
Pd-catalyzed Stille coupling with key fluorenyl intermediate 3,
(9) Belfield, K. D.; Bondar, M. V; Morales, A. R.; Yavuz, O.; Przhonska,
O. V. J. Phys. Org. Chem. 2003, 16, 194–201.
(10) Belfield, K. D.; Morales, A. R.; Hales, J. M.; Hagan, D. J.;
Van Stryland, E. W.; Chapela, V. M.; Percino, J. Chem. Mater. 2004, 16,
2267–2273.
(11) 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.
(12) Yao, S.; Belfield, K. D. J. Org. Chem. 2005, 70, 5126–5132.
(13) Belfield, K. D.; Yao, S.; Bondar, M. V. Adv. Polym. Sci. 2008, 213,
97–156.
(14) Yanez, C. O.; Andrade, C. D.; Belfield, K. D. Chem. Commun. 2009,
827–829.
(15) Belfield, K. D.; Bondar, M. V.; Yanez, C. O.; Hernandez, F. E.;
Przhonska, O. V. J. Mater. Chem. 2009, 19, 7498–7502.
(16) Moura, G. L. C.; Simas, A. M. J. Phys. Chem. C 2010, 114, 6106–
6116.
(17) Reinhardt, B. A.; Brott, L. L.; Clarson, S. J.; Dillard, A. G.; Bhatt,
J. C.; Kannan, R.; Yuan, L. X.; He, G. S.; Prasad, P. N. Chem. Mater. 1998,
10, 1863–1874.
(18) Mongin, O.; Porres, L.; Charlot, M.; Katan, C.; Blanchard-Desce,
M. Chem.;Eur. J. 2007, 13, 1481–1498.
(19) Belfield, K. D.; Schafer, K. J.; Mourad, W.; Reinhardt, B. A. J. Org.
Chem. 2000, 65, 4475–4481.
(20) Kannan, R.; He, G. S.; Yuan, L. X.; Xu, F. M.; Prasad, P. N.;
Dombroskie, A. G.; Reinhardt, B. A.; Baur, J. W.; Vaia, R. A.; Tan, L. S.
Chem. Mater. 2001, 13, 1896–1904.
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SCHEME 1
SCHEME 2
as shown in Scheme 1, to yield nitrofluorenyl 4 in excellent
yield.19,21 Reduction of the nitrofluorenyl 4 and subsequent
reaction of resulting amine 5 with iodobenzene under Ullman
conditions generated the D-π-A type fluorescent dye I.19,22
Stille coupling between dibromofluorene derivative 6 and the
tin thiophene benzothiazole intermediate 2 yielded symmetrical
dye II11,19,21 (Scheme 1).
The synthesis of alkyne-containing dyes was achieved by
means of successive Sonogashira coupling.23 To prepare
the asymmetrical compound III, 2-(5-bromothiophen-2-yl)-
benzothiazole 1 was converted into protected alkyne 7, which
after hydrolysis in toluene with KOH generated alkyne 8
(Scheme 2).24,25 Pd-catalyzed Sonogashira coupling between
(23) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975,
4467–4470.
(21) Kosugi, M.; Koshiba, M.; Atoh, A.; Sano, H.; Migita, T. Bull. Chem.
Soc. Jpn. 1986, 59, 677–679.
(24) Umezawa, H.; Okada, S.; Oikawa, H.; Matsuda, H.; Nakanishi, H.
J. Phys. Org. Chem. 2005, 18, 468–472.
(22) Yang, C. P.; Lin, J. H. J. Polym. Sci., Part A: Polym. Chem. 1994, 32,
369–382.
(25) Mongin, O.; Krishna, T. R.; Werts, M. H. V.; Caminade, A. M.;
Majoral, J. P.; Blanchard-Desce, M. Chem. Commun. 2006, 915–917.
J. Org. Chem. Vol. 75, No. 12, 2010 3977

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SCHEME 3
TABLE 1. Photophysical Characterization of New Fluorene Derivatives I, II, III, and IV in Hexane
compound
λabsmax, nm
λemmax, nm
ΔλSt, nm
φa
εmax ꢀ 10-3, M-1 cm-1
53
I
II
III
IV
403 ( 1
405 ( 1
398 ( 1
397 ( 1
452 ( 1
446 ( 1
444 ( 1
433 ( 1
49 ( 2
41 ( 2
46 ( 2
36 ( 2
0.86 ( 0.05
0.98 ( 0.05
0.92 ( 0.05
0.95 ( 0.05
31
50
135
aFluorescence quantum yield measured relative to 9,10-diphenylanthracene in cyclohexane.
FIGURE 3. Normalized absorption (left) and fluorescence (right) spectra for I, II, III, and IV in hexane. Absorption and emission spectra were
collected at room temperature using hexane solutions with optical density of ∼0.1 (approximately 10-6 M) without degassing the solutions.
Emission was collectad by exciting samples at their λabsmax value.
the alkyne 8 and key intermediate 3 yielded nitro derivative 9,
which was reduced with hydrazine to produce amine 10 in
high yield. Amine 10 was treated with iodobenzene under
Ullman conditions to generate the asymmetrical D-π-A III
(Scheme 2).19,22
measuring the two-photon exited fluorescence of the dyes in
the range of 730-860 nm in hexane. The 2PA cross section
measurements were performed with a tunable Ti:sapphire
femtosecond laser system (220 fs pulse width, 76 MHz repe-
tition rate) pumped by a 10 W diode laser (532 nm) as the
excitation source and a spectrofluorimeter with PMT detec-
tors (Figure S-1, Supporting Information).
The 2PA spectra for this series of compounds are shown
in Figure 4. Compound I exhibited a maximum 2PA cross
section of ∼248 GM at 730 nm, which can be attributed
to the tail of the band corresponding to the two-photon
allowed S0 f S2 transition; a second maximum of ∼217 GM
at 800 nm, assigned to S0 f S1 2PA forbidden transition,
was also observed. Compound II exhibits a maximum 2PA
cross section of ∼335 GM at 730 nm. Comparing these
results with our previously reported data for similar
molecules that did not contain the thiophene moiety, a
significant increase in the 2PA cross section values was
observed when the thiophenyl moiety is introduced in the
fluorophore.9-14
The symmetrical intermediate 11 was prepared using
Sonogashira coupling between 9,9-didecyl-2,7-dibromofluorene
6 and 2-methylbut-3-yn-2-ol.24,25 After hydrolysis, the dia-
lkyne 12 was coupled through a second Sonogashira reaction
with 1, yielding symmetrical A-π-A fluorenyl dye IV (Scheme 3).
Linear Photophysical Properties. Table 1 summarizes the
photophysical properties of the four new fluorescent dyes
(I-IV). As can be seen in Figure 3, all molecules exhibited
an intense absorption between 397 and 405 nm, fluorescence
maximum in the range of 433-452 nm, and significant Stokes
shift values in hexane (see Table 1). The new compounds also
exhibited high fluorescence quantum yields (0.86-0.98),
while a slight blue shift in the absorption maximum was
observed when alkynyl moieties were incorporated in both
symmetrical and asymmetrical derivatives.
Nonlinear Photophysical Properties. Two-photon absorp-
tion (2PA) values were determined by the fluorescence method,
To observe the effect of the presence of alkynyl triple
bonds in the new derivatives, comparisons of compound I to
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FIGURE 5. Confocal fluorescence images of COS-7 cells incubated
with probe I (20 μM, 1 h 50 min) and Lysotracker Red (100 nM,
1 h 50 min). (A) DIC, one-photon fluorescence image showing
(B) Lysotracker Red (Texas Red filter cube (Ex 562/40; DM 593;
Em 624/40)), (C) probe I (custom-made filter cube (Ex 377/50;
DM 409; Em 525/40)), and (D) colocalization (overlay of B and C).
Scale bar = 10 μm.
FIGURE 4. 2PA spectra obtained for compounds I-IV in hexane.
Incorporation of thiophene and alkynyl moieties resulted in an
increase in the 2PA cross section values.
compound III (D-π-A) and compound II to compound IV
(A-π-A) were conducted. Compound III exhibited a maxi-
mum 2PA cross section of ∼563 GM at 730 nm, and a second
maximum of ∼356 GM at 800 nm was observed. Comparing
these values to those obtained for compound I, it can be
inferred that incorporating alkynyl triple bonds in these
molecules results in an enhancement in the 2PA cross section
for this unsymmetrical compound.
The 2PA cross section value obtained for compound II
(∼335 GM at 730 nm) can be compared with the large value
obtained for compound IV at the same wavelength (∼1093 GM).
A significant increase in the 2PA cross section was observed
when the number of alkynyl triple bonds was doubled.
Since the number of π electrons in these molecules is
different, it is also useful to compare the ratios of 2PA cross
section (δ) over the number of π-electrons (Ne) in the
chromophores (normalized cross section).18,26 When com-
paring I to III, it was observed that the incorporation of a
triple bond in the π system resulted in a 2-fold increase in the
normalized cross section value (7 to 14 GM per π electron).
Similarly, a 3-fold increase of the normalized cross section
was observed when comparing II and IV (9 and 27 GM per
π electron, respectively).
In general, a considerable increase in the value of 2PA
cross section was obtained when thiophene moieties were
introduced in both fluorophore archetypes (D-π-A and A-π-A)
studied in this series. In addition, extending the π-conjugation
with alkynyl triple bond functionalities also significantly
increased the 2PA for this class of molecules.
Fluorescence Imaging. In order to evaluate the efficiency of
these archetypes as fluorophores in 2PFM cell imaging,
fluorene I was incubated in COS-7 and HCT 116 cell lines
at different concentrations and incubation times. Probe I was
chosen as model compound because of its relatively constant
two-photon absorptivity across a useful NIR wavelength
range (750-860 nm for the Ti:sapphire laser, Figure 4) and
because it exhibited better solubility in DMSO than the more
symmetrical compounds II and IV (important for cell
incubation). Fluorescence micrographs indicated the distinct
localization of the probe in well-confined areas within the
cytosol in both COS-7 and HCT 116 cell lines (Figures 5 and
6). Probe I was incubated with a commercially available
lysosomal selective dye (Lysotracker Red) and incubated
FIGURE 6. Two-photon fluorescence micrograph of HCT 116 cells
incubated with probe I (20 μM, 1 h 50 min). (A) DIC and (B) 3D
reconstruction from overlaid 2PFM images obtained from a modi-
fied laser scanning confocal microscopy system equipped with a
broadband, tunable Ti:sapphire laser (220 fs pulse width, 76 MHz
repetition rate), pumped by a 10 W frequency doubled Nd:YAG
laser. (60X objective, NA = 1.35). Scale: 5 μm grid.
for 10 min, 30 min, 50 min, 1 h 10 min, 1 h 30 min, and 1 h
50 min, and after incubation the cells were fixed, mounted,
and imaged. Early colocalization with Lysotracker dye sug-
gests a similar uptake mechanism for both the Lysotracker
dye and probe I. However, longer incubation times (50 min
and 1 h 50 min) indicate a difference in the progression of the
probe from the endosomal vesicles to the lysosomes, where
the Lysotracker dye reached the lysosomes more quickly
than probe I (see Figure 5). The slower progression of probe
I may prove useful for the study of late endosomal processes.
The high contrast and good resolution observed in 2PFM
images of HCT 116 cells, incubated with probe I, support use
of the fluorenyl core as an integral part of the fluorescent
probe’s molecular architecture. The excellent linear and non-
linear photophysical properties point toward their potential
as efficient 2PA fluorophores. The 2PFM image, shown in
Figure 6, where the vesicles (presumably late endosomes) are
clearly visualized in both 2D and 3D (see also Supporting
Information), is a testament to the efficacy of the 2PA
fluorophore. The potential of these dyes exceeds that of
imaging the endosomes, as further functionalization of the
dye can be performed to modulate the solubility properties
and, more importantly, impart selectivity to other organelles
or biomarkers of interest, aspects of current investigation.
The new fluorenyl derivatives provide the basis for the
development of fluorescent probes for 2PFM.
Conclusions
Efficient synthetic routes were developed to prepare four
new fluorescent compounds containing functionalities such
(26) Pawlicki, M.; Collins, H. A.; Denning, R. G.; Anderson, H. L.
Angew. Chem., Int. Ed. 2009, 48, 3244–3266.
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as thiophene and alkynyl moieties on a fluorene core,
affording systematic variation of molecular symmetry and
extent of conjugation. Comprehensive characterization of
the new compounds revealed excellent linear and nonlinear
photophysical properties, such as high fluorescence quan-
tum yields (0.86-0.98) and high two-photon absorptivity
(220-1060 GM). Incorporation of thiophene and alkynyl
moieties resulted in an increase in the 2PA cross section
values. Probe I proved to have low cytotoxocity when incu-
bated with COS-7 and HCT 116 cells, exhibiting endosomal
selectivity, as evidenced by one-photon fluorescence imaging
and colocalization studies. The advantageous properties of
probe I facilitated 2PFM endosomal imaging, with the
2PFM images providing much better resolution, allowing
the visualization of individual endosomes. Due to their linear
and nonlinear photophysical properties, these new fluorenyl
derivatives should find use in a number of emerging photo-
nics applications such as targeted two-photon fluorescence
imaging and two-photon-based optical data storage.
8.05 (d, J=8.0 Hz, 1H, Ph-H), 7.88 (d, J=7.8 Hz, 1H, Ph-H),
7.79-7.84 (m, 2H, Ph-H), 7.72-7.76 (m, 1H, Ph-H), 7.69 (d, J=
1.4 Hz, 1H, Ph-H), 7.66 (d, J=3.7 Hz, 1H, Thy-H), 7.48-7.53
(m, 1H, Ph-H), 7.46 (d, J = 3.7 Hz, 1H, Thy-H), 7.37-7.42
(m, 1H, Ph-H), 2.01-2.09 (m, 4H, CH2), 0.99-1.24 (m, 28H,
CH2), 0.82 (t, J = 7.0 Hz, 6H, CH3), 0.54-0.69 (m, 4H, CH2).
13C NMR (125 MHz, CDCl3) δ 161.0, 153.7, 153.4, 152.2, 147.9,
147.2, 146.8, 139.0, 136.6, 134.7, 134.4, 129.5, 126.6, 125.3,
124.40, 124.36, 123.5, 122.0, 121.9, 121.5, 120.4, 120.0, 118.3,
55.9, 40.1, 31.8, 29.8, 29.49, 29.47, 29.24, 29.19, 23.8, 22.6, 14.1.
Anal. Calcd for C44H54N2O2S2: C, 74.74; H, 7.70; N, 3.96.
Found: C, 74.97; H, 7.85; N, 3.97.
Synthesis of 7-(5-(Benzothiazol-2-yl)thiophen-2-yl)-9,9-didecyl-
9H-fluoren-2-amine (5). 2-(5-(9,9-Didecyl-7-nitro-9H-fluoren-
2-yl)thiophen-2-yl)benzothiazole 4 (160 mg, 0.23 mmol) and
10% Pd/C (16 mg) were dissolved in a mixture 1:1 of THF/
EtOH (8 mL). NH2NH2 2H2O (136 mg, 2.7 mmol) was added
3
to the mixture slowly at room temperature and then heated to 70 °C
for 20 h. The mixture was filtered though a silica plug with CH2Cl2,
and after removing the solvent under reduced pressure, the mate-
rial was purified by column chromatography using as a solvent a
mixture of hexanes/EtOAc (9:1), to yield 130 mg (84%) of dark
yellow oil that was used directly since the primary amine is prone
to oxidation. 1H NMR (500 MHz, CDCl3) δ 8.04 (d, J=8.0 Hz,
1H, Ph-H), 7.85 (d, J = 8.0 Hz, 1H, Ph-H), 7.59-7.64 (m, 2H,
Ph-H, Thy-H), 7.54-7.58 (m, 2H, Ph-H), 7.45-7.51(m,2H,Ph-H),
7.34-7.39 (m, 2H, Ph-H, Thy-H), 6.65-6.69 (m, 2H, Ph-H),
3.80 (s, 2H, NH2), 1.83-2.00 (m, 4H, CH2), 0.99-1.28 (m, 28H,
CH2), 0.83 (t, J = 7.0 Hz, 6H, CH3), 0.62-0.73 (m, 4H, CH2).
13C NMR (125 MHz, CDCl3) δ 161.4, 153.8, 153.1, 150.7, 149.6,
146.4, 142.4, 135.0, 134.6, 131.6, 130.5, 129.5, 126.4, 125.1, 124.7,
123.0, 122.8, 121.4, 120.8, 120.1, 118.8, 114.1, 109.7, 54.9, 40.6,
31.9, 30.1, 29.6, 29.5, 29.29, 29.28, 23.8, 22.7, 14.1.
Experimental Section
Materials and Methods. 2-(5-Bromothiophen-2-yl)benzothia-
zole 1, 9,9-didecyl-2-iodo-7-nitro-9H-fluorene 3, and 2,7-dibromo-
9,9-didecyl-9H-fluorene 6 were prepared as described in the
literature.19,27,28 All reactions were carried out under N2. All
other reagents and solvents were used as received from com-
1
mercial suppliers. H and 13C NMR spectra were recorded in
CDCl3 on a NMR spectrometer at 500 and 125 MHz, respec-
tively. MALDI MS analyses were performed at the University of
Florida.
Synthesis of 2-(5-(Tributylstannyl)thiophen-2-yl)benzothiazole
(2). At -78 °C 2-(5-bromothiophen-2-yl)benzothiazole 1 (200 mg,
0.67 mmol) was dissolved in dry THF (5 mL). A solution of
n-BuLi in hexanes (0.27 mL, 2.5 M, 0.68 mmol) was added
dropwise into the reaction mixture. After stirring for 1 h, Sn-
(Bu)3Cl (360 mg, 1.10 mmol) was added, and the mixture was
allowed to reach room temperature, and stirred overnight. The
mixture was added to water, extracted with Et2O twice, dried
over Mg2SO4, and purified by column chromatography using a
mixture of hexanes/EtOAc (9:1) as an eluent to yield 240 mg
(70%) of viscous oil. 1H NMR (500 MHz, CDCl3) δ 8.02(d, J=
8.2 Hz, 1H, Ph-H), 7.82 (d, J=8.0 Hz, 1H, Ph-H), 7.74 (d, J=
3.5 Hz, 1H, Thy-H), 7.43-7.47 (m, 1H, Ph-H), 7.31-7.35 (m,
1H, Ph-H), 7.18 (d, J=3.5 Hz, 1H, Thy-H), 1.55-1.63 (m, 6H,
CH2), 1.31-1.39 (m, 6H, CH2), 1.12-1.19 (m, 6H, CH2), 0.90
(t, J=7.34 Hz, 9H, CH3). 13C NMR (125 MHz, CDCl3) δ 161.4,
153.9, 143.5, 142.4, 136.2, 134.7, 129.6, 126.2, 125.1, 122.9,
121.4, 28.9, 27.3, 13.7, 11.0. Anal. Calcd for C23H33NS2Sn: C,
54.56; H, 6.57; N, 2.77. Found: C, 54.85; H, 6.67; N, 2.86.
Synthesis of 2-(5-(9,9-Didecyl-7-nitro-9H-fluoren-2-yl)thiophen-
2-yl)benzothiazole (4). 9,9-Didecyl-2-iodo-7-nitro-9H-fluorene
3 (200 mg, 0.32 mmol), 2-(5-(tributylstannyl)thiophen-2-yl)benzo-
thiazole 2 (191 mg, 0.38 mmol), and Pd(PPh3)2Cl2 (6 mg, 0.008
mmol) were dissolved in toluene (4 mL). The mixture was heated
under reflux for 5 h. The solvent was removed under reduced
pressure, and the crude was purified by column chromatogra-
phy using a mixture of hexanes/EtOAc (9.5:0.5) as an eluent to
yield 222 mg (98%) of yellow oil that solidified upon standing
(mp 73.2-74.9 °C). 1H NMR (500 MHz, CDCl3) δ 8.28 (dd, J=
8.3 Hz, J=2.0 Hz, 1H, Ph-H), 8.22 (d, J=2.0 Hz, 1H, Ph-H),
Synthesis of 7-(5-(Benzothiazol-2-yl)thiophen-2-yl)-9,9-didecyl-
N,N-diphenyl-9H-fluoren-2-amine (I). 7-(5-(Benzothiazol-2-yl)-
thiophen-2-yl)-9,9-didecyl-9H-fluoren-2-amine 5 (130 mg, 0.19
mmol), iodobenzene (157 mg, 0.77 mmol), Cu-bronze (61 mg,
0.96 mmol), 18-crown-6 (15 mg, 0.058 mmol), and K2CO3 (212 mg,
1.54 mmol) were combined with 1,2-dichlorobenzene (3 mL).
The mixture was heated to 180 °C for 48 h. The crude product
was passed through a silica plug with CH2Cl2. The solvent was
removed under reduced pressure, and the crude was purified by
column chromatography on silica gel using as a solvent a
mixture of hexanes/EtOAc (9:1) to yield 150 mg (94%) of yellow
solid (mp 107.5-109.5 °C). 1H NMR (500 MHz, CDCl3) δ 8.04
(d, J = 8.0 Hz, 1H, Ph-H), 7.84 (d, J = 8.0 Hz, 1H, Ph-H),
7.58-7.65 (m, 4H, Ph-H, Thy-H), 7.56 (d, J=8.0 Hz, 1H, Ph-H),
7.45-7.50 (m, 1H, Ph-H), 7.33-7.39 (m, 2H, Ph-H, Thy-H),
7.22-7.30 (m, 5H, Ph-H), 7.11-7.16 (m, 5H, Ph-H), 7.00-7.05
(m, 2H, Ph-H), 1.82-1.95 (m, 4H, CH2), 1.01-1.23 (m, 28H,
CH2), 0.85 (t, J = 7.0 Hz, 6H, CH3), 0.66-0.75 (m, 4H, CH2).
13C NMR (125 MHz, CDCl3) δ 161.3, 153.8, 152.5, 151.6, 149.2,
147.9, 147.5, 141.6, 135.4, 134.6, 131.4, 129.6, 129.5, 129.24, 129.16,
125.2, 125.0, 124.0, 123.9, 123.4, 123.3, 122.8, 122.6, 121.5,
121.4, 120.1, 105.0, 55.2, 40.2, 31.9, 30.0, 29.6, 29.6, 29.3, 23.9,
22.7, 14.1. Anal. Calcd for C56H64N2S2: C, 81.11; H, 7.78; N,
3.38. Found: C, 80.81; H, 7.74; N, 3.30.
Synthesis of 2,20-(5,50-(9,9-Didecyl-9H-fluorene-2,7-diyl)bis-
(thiophene-5,2-diyl))dibenzothiazole (II). 2-(5-(Tributylstannyl)-
thiophen-2-yl)benzothiazole 2 (460 mg, 0.90 mmol), 2,7-dibromo-
9,9-didecyl-9H-fluorene 6 (250 mg, 0.41 mmol), and Pd(PPh3)2Cl2
(7.2 mg, 0.01 mmol) were dissolved in toluene. The mixture was
heated under reflux for 24 h. The solvent was removed under
reduced pressure, and the crude was purified by column chro-
matography using a mixture of hexanes/EtOAc (9.9:0.1) to yield
126 mg (35%) of yellow oil that solidified upon standing (mp
(27) Zeng, D. X.; Chen, Y. J. Photochem. Photobiol. A 2007, 186, 121–
124.
(28) Belfield, K. D.; Yao, S.; Morales, A. R.; Hales, J. M.; Hagan, D. J.;
Van Stryland, E. W.; Chapela, V. M.; Percino, J. Polym. Adv. Technol. 2005,
16, 150–155.
1
125.2-126.5 °C). H NMR (500 MHz, CDCl3) δ 8.05 (d, J =
7.76 Hz, 2H, Ph-H), 7.87 (d, J=7.82 Hz, 2H, Ph-H), 7.74 (d, J=
3980 J. Org. Chem. Vol. 75, No. 12, 2010

Andrade et al.
JOCArticle
7.63 Hz, 2H, Ph-H), 7.68-7.71 (m, 2H, Ph-H), 7.64-7.67 (m,
4H, Ph-H, Thy-H), 7.47-7.52 (m, 2H, Ph-H), 7.43 (d, J=4.10
Hz, 2H, Thy-H), 7.36-7.40 (m, 2H, Ph-H), 2.00-2.06 (m, 4H,
CH2), 1.04-1.23 (m, 28H, CH2), 0.80 (t, J=7.04 Hz, 6H, CH3),
0.67-0.74 (m, 4H, CH2). 13C NMR (125 MHz, CDCl3) δ 161.2,
153.7, 152.1, 148.8, 140.9, 135.8, 134.7, 132.6, 129.5, 126.5,
125.2, 125.0, 123.7, 122.9, 121.5, 120.5, 120.3, 55.4, 40.4, 31.9,
30.0, 29.6, 29.5, 29.3, 29.2, 23.8, 22.6, 14.1. HRMS (MALDI)
m/z calcd 877.3712 [M þH]þ, m/z found 877.3702 [M þ H]þ.
Synthesis of 4-(5-(Benzothiazol-2-yl)thiophen-2-yl)-2-methylbut-
3-yn-2-ol (7). 2-(5-Bromothiophen-2-yl)benzothiazole 1 (200 mg,
0.67 mmol), 2-methyl-3-butyn-2-ol (170 mg, 2.02 mmol), Pd(PPh3)2-
Cl2 (19 mg, 0.027 mmol), and CuI (5 mg, 0.027 mmol) were
dissolved in a 1:4 mixture of Et3N/toluene (5 mL). The mixture
was heated under reflux for 12 h. After cooling to room tempera-
ture, the mixture was filtered through a Celite plug, and purified by
column chromatography using a mixture of hexanes/EtOAc (1:1)
to yield 192 mg (96%) of pale yellow solid (mp 163.9-164.6 °C).
1H NMR (500 MHz, CDCl3) δ 8.02 (d, J=8.04 Hz, 1H, Ph-H),
7.85 (d, J=7.87 Hz, 1H, Ph-H), 7.50 (d, J=3.95 Hz, 1H, Thy-H),
7.46-7.49 (m, 1H, Ph-H), 7.35-7.40 (m, 1H, Ph-H), 7.18 (d, J=
3.95 Hz, 1H, Thy-H), 2.11 (s, 1H, -OH), 1.64 (s, 6H, CH3). 13C
NMR (125 MHz, CDCl3) δ160.4, 153.6, 137.9, 134.7, 132.9, 128.3,
126.4, 125.4, 123.1, 121.5, 121.4, 100.2, 75.2, 65.8, 31.3. Anal. Calcd
for C16H13NOS2: C, 64.18; H, 4.38; N, 4.68. Found: C, 64.10; H,
4.33; N, 4.64.
heating to110 °C for 48 h. The mixture was filtered though a silica
plug with CH2Cl2, and after removing the solvent under reduced
pressure, the material was purified by column chromatography
using a mixture of hexanes/EtOAc (9:1) as eluent to yield 150 mg
(87%) of dark yellow oil that was used immediately due to the
1
oxidative lability of the primary amine. H NMR (500 MHz,
CDCl3) δ 8.03 (d, J=7.45 Hz, 1H, Ph-H), 7.86 (d, J=7.67 Hz,
1H, Ph-H), 7.56(d, J=3.99 Hz, 1H, Thy-H), 7.52(d, J=7.75 Hz,
1H, Ph-H), 7.46-7.50 (m, 3H, Ph-H), 7.43-7.44 (m, 1H, Ph-H),
7.36-7.40 (m, 1H, Ph-H), 7.27 (d, J = 3.99 Hz, 1H, Thy-H),
6.65-6.68 (m, 2H, Ph-H), 3.83 (s, 2H, NH2), 1.84-1.97 (m, 4H,
CH2), 1.03-1.27 (m, 28H, CH2), 0.85 (t, J=7.16 Hz, 6H, CH3),
0.56-0.68 (m, 4H, CH2). 13C NMR (125 MHz, CDCl3) δ 161.0,
160.6, 153.8, 153.1, 150.2, 149.9, 146.6, 146.0, 144.4, 142.7, 141.6,
136.1, 134.6, 133.7, 132.7, 132.2, 131.5, 128.4, 122.9, 121.6, 118.5,
114.0, 109.8, 97.7, 81.9, 54.9, 40.5, 31.9, 30.2, 29.7, 29.6, 29.4, 29.3,
23.9, 22.7, 14.2.
Synthesis of 7-((5-(Benzothiazol-2-yl)thiophen-2-yl)ethynyl)-
9,9-didecyl-N,N-diphenyl-9H-fluoren-2-amine (III). 7-((5-(Benzo-
thiazol-2-yl)thiophen-2-yl)ethynyl)-9,9-didecyl-9H-fluoren-2-amine
10, (0.135mg, 0.19 mmol), iodobenzene(157 mg, 0.77 mmol), Cu-
bronze (61 mg, 0.96 mmol), 18-crown-6 (15 mg, 0.058 mmol), and
K2CO3 (212 mg, 1.54 mmol) were combined with 1,2-dichloro-
benzene (3 mL). The mixture was heated at 180 °C for 48 h. The
crude product was passed through a silica plug with CH2Cl2. The
solvent was removed under reduced pressure, and the crude
product was purified by column chromatography on silica gel
using a mixture of hexanes/EtOAc (9.5:0.5) as eluent to yield
81 mg (50%) of yellow viscous oil. 1H NMR (500 MHz, CDCl3)
δ 8.04 (d, J=8.11 Hz, 1H, Ph-H), 7.85 (d, J=8.11 Hz, 1H, Ph-H),
7.59 (d, J=7.90 Hz, 1H, Ph-H), 7.56 (d,, J=3.72 Hz, 1H, Thy-H),
7.45-7.52 (m, 4H, Ph-H), 7.35-7.39 (m, 1H, Ph-H), 7.24-7.30
(m, 5H, Ph-H), 7.10-7.15 (m, 5H, Phe-H), 7.00-7.05 (m, 3H,
Phe-H), 1.80-1.92 (m, 4H, CH2), 1.03-1.28 (m, 28H, CH2), 0.85
(t, J = 7.14 Hz, 6H, CH3), 0.62-0.69 (m, 4H, CH2). 13C NMR
(125 MHz, CDCl3) δ 160.5, 153.7, 152.6, 150.8, 147.8, 147.8,
141.9, 137.7, 135.2, 134.7, 132.4, 130.8, 129.2, 128.4, 127.4, 126.6,
125.7, 125.4, 124.0, 123.3, 123.01 122.7, 121.5, 120.9, 119.6, 119.1,
118.9, 97.4, 82.2, 55.2, 40.2, 31.9, 30.0, 29.6, 29.6, 29.4, 29.3, 23.9,
22.7, 14.1. Anal. Calcd for C58H64N2S2:C, 81.64; H, 7.56; N, 3.28.
Found: C, 81.63; H, 7.62; N, 3.05.
Synthesis of 4,40-(9,9-Didecyl-9H-fluorene-2,7-diyl)bis(2-methyl-
but-3-yn-2-ol) (11). 2,7-Dibromo-9,9-didecyl-9H-fluorene 6 (1.0 g,
1.65 mmol), 2-methyl-3-butyn-2-ol (0.83 g, 9.92 mmol), Pd(PPh3)2-
Cl2 (95 mg, 0.135 mmol), and CuI (25 mg, 0.13 mmol) were dis-
solved in a 1:4 mixture of Et3N/toluene (15 mL). The mixture was
heated under reflux for 12 h. After it was cooled down, the mixture
was filtered through a Celite plug and purified by column chro-
matography using hexanes as a eluent, followed by a mixture of
hexanes/EtOAc (10:1) to yield 0.64 g (63%) of white solid (mp
89.0-89.9 °C). 1H NMR (500 MHz, CDCl3) δ 7.58-7.61 (m, 2H,
Ph-H), 7.35-7.41 (m, 4H, Ph-H), 2.05 (s, 2H, -OH), 1.89-1.96
(m, 4H, CH2), 1.63-1.68 (s, 12H, CH3), 0.98-1.27 (m, 28H, CH2),
0.85 (t, J=6.49 Hz, 6H, CH3), 0.50-0.58 (m, 4H, CH2). 13C NMR
(125 MHz, CDCl3) δ 150.9, 140.6, 130.7, 126.0, 121.4, 119.8, 93.9,
83.0, 65.7, 55.2, 40.4, 31.9, 31.6, 30.0, 29.6, 29.5, 29.3, 29.2, 23.7,
22.6, 14.1. Anal. Calcd for C43H62O2: C, 84.53; H, 10.23. Found: C,
84.46; H, 10.25.
Synthesis of 9,9-Didecyl-2,7-diethynyl-9H-fluorene (12).
4,40-(9,9-Didecyl-9H-fluorene-2,7-diyl)bis(2-methylbut-3-yn-2-
ol) 11 (450 mg, 0.736 mmol) and KOH (206 mg, 3.68 mmol) were
heated under reflux in toluene for 2 h. The mixture was filtered,
then purified by column chromatography using hexanes to yield
350 mg (97%) of light yellow oil. 1H NMR (500 MHz, CDCl3)
δ 7.63 (dd, J =7.73 Hz, J =0.65 Hz, 2H, Ph-H), 7.48 (dd, J =
7.73 Hz, J=1.42 Hz, 2H, Ph-H), 7.45-7.46 (m, 2H, Ph-H), 3.15
(s, 2H, CtC-H), 1.90-1.96 (m, 4H, CH2), 0.99-1.25 (m, 28H,
CH2), 0.85 (t, J = 7.0 Hz, 6H, CH3), 0.52-0.58 (m, 4H, CH2).
Synthesis of 2-(5-Ethynylthiophen-2-yl)benzothiazole (8).
4-(5-(Benzothiazol-2-yl)thiophen-2-yl)-2-methylbut-3-yn-2-ol 7
(300 mg, 1.0 mmol) and KOH (300 mg, 5.3 mmol) were heated
under reflux in toluene for 1 h. The mixture was filtered and then
purified by column chromatography eluting with hexanes to
1
yield 180 mg (75%) of a white solid (mp 117.3-118.3 °C). H
NMR (500 MHz, CDCl3) δ 8.03 (d, J=8.21 Hz, 1H, Ph-H), 7.86
(d, J = 8.21 Hz, 1H, Ph-H), 7.51 (d, J = 3.94 Hz, 1H, Thy-H),
7.47-7.50 (m, 1H, Ph-H), 7.37-7.42 (m, 1H, Ph-H), 7.28 (d, J=
3.94 Hz, 1H, Thy-H), 3.50 (s, 1H, CtC-H). 13C NMR (125
MHz, CDCl3) δ 160.2, 153.6, 138.5, 134.8, 133.9, 133.7, 127.8,
126.5, 125.4, 123.2, 121.6, 84.0, 76.5. Anal. Calcd for C13H7NS2:
C, 64.70; H, 2.92; N, 5.80. Found: C, 64.64; H, 2.91; N, 5.75.
Synthesis of 2-(5-((9,9-Didecyl-7-nitro-9H-fluoren-2-yl)ethynyl)-
thiophen-2-yl)benzothiazole (9). 2-(5-Ethynylthiophen-2-yl)benzo-
thiazole 8 (180 mg, 0.75 mmol), 9,9-didecyl-2-iodo-7-nitro-9-
H-fluorene 3(460 mg, 0.75 mmol), Pd(PPh3)2Cl2 (20 mg, 0.03 mmol),
and CuI (5.6 mg, 0.03 mmol) were dissolved in a 1:4 mixture of
Et3N/toluene (5 mL). The mixture was heated under reflux for
12 h. After cooling to room temperature, the mixture was filtered
through a Celite plug and purified by column chromatography
eluting with a mixture of hexanes/EtOAc (9:1) and then recrystal-
lized from hexane to yield 300 mg (55%) of yellow solid (mp
1
142.3-143.6 °C). H NMR (500 MHz, CDCl3) δ 8.28 (dd, J =
8.40 Hz, J=2.18 Hz, 1H, Ph-H), 8.21 (d, J=2.05 Hz, 1H, Ph-H),
8.05 (d, J=7.74 Hz, 1H, Ph-H), 7.88 (d, J=7.74 Hz, 1H, Ph-H),
7.77-7.82 (m, 2H, Ph-H), 7.55-7.61 (m, 3H, Ph-H, Thy-H),
7.48-7.52 (m, 1H, Ph-H), 7.39-7.42 (m, 1H, Ph-H), 7.33 (d,
J=4.08 Hz, 1H, Thy-H), 2.01-2.08 (m, 4H, CH2), 0.99-1.29 (m,
28H, CH2), 0.84 (t, J = 6.93 Hz, 6H, CH3), 0.50-0.64 (m, 4H,
CH2). 13C NMR (125 MHz, CDCl3) δ 160.3, 153.7, 152.5, 152.3,
147.4, 146.6, 139.3, 138.4, 134.8, 132.8, 131.2, 128.5, 128.3, 126.7,
126.2, 126.0, 123.2, 123.1, 121.6, 121.5, 120.2, 118.3, 105.0, 96.3,
83.8, 55.9, 40.0, 31.9, 29.9, 29.51, 29.48, 29.2, 23.8, 22.6, 14.14,
14.06. Anal. Calcd for C46H54N2O2S2: C, 75.57; H, 7.45; N, 3.83.
Found: C, 75.69; H, 7.47; N, 3.85.
Synthesis of 7-((5-(Benzothiazol-2-yl)thiophen-2-yl)ethynyl)-
9,9-didecyl-9H-fluoren-2-amine (10). 2-(5-((9,9-Didecyl-7-nitro-
9H-fluoren-2-yl)ethynyl)thiophen-2-yl)benzothiazole 9 (180 mg,
0.24 mmol) and10% Pd/C(18mg) weredissolvedina 1:1mixture
of THF/EtOH (4 mL). NH2NH2 2H2O (146 mg, 2.8 mmol) was
3
added to the mixture dropwise at room temperature, followed by
J. Org. Chem. Vol. 75, No. 12, 2010 3981

Andrade et al.
JOCArticle
13C NMR (125 MHz, CDCl3) δ 151.0, 141.0, 131.2, 126.5, 120.8,
119.95, 84.5, 77.3, 55.2, 40.2, 31.9, 29.9, 29.5, 29.5, 29.2, 23.7,
22.7, 14.1. Anal. Calcd for C37H50: C, 89.81; H, 10.19. Found: C,
89.79; H, 10.24.
lin, and 1% streptomycin at 37 °C, under 5% CO2 environment.
Number 1 round 12 mm coverslips were treated with poly-
D-lysine to improve cell adhesion and washed (3ꢀ) with PBS
buffer solution. The treated coverslips were placed in 24-well
plates, seeded with 80,000 cells/well, and incubated at the same
conditions as indicated above until 75-85% confluency was
reached on the coverslips. From a 4.10 ꢀ 10-4 M stock solution
of dye I a series of 0.1, 1, 10, 20, and 50 μM solutions in DMSO
were prepared with all solutions also containing 75 nM of
Lysotracker Red. These solutions were used to incubate the
cells for 10 min to 3 h. The dye solutions were extracted and the
coverslipped cells were washed abundantly with PBS (4ꢀ).
Cell Fixing and Mounting. Cells were fixed with 3.7% solution
of paraformaldehyde in pH = 7.4 PBS buffer for 10 min. The
fixing agent was extracted and washed (2ꢀ) with PBS. To reduce
autofluorescence, a fresh solution of NaBH4 (1 mg/mL) in pH=
8 PBS buffer was used to treat the fixed cells (2ꢀ). The cover-
slipped cells were then washed with buffer PBS (2ꢀ) and moun-
ted on microscope slides using an antifade mounting media.
Confocal One-Photon Fluorescence Imaging. One-photon
(conventional) fluorescence microscopy images were recorded
on an inverted confocal microscope equipped with a EM-CCD
digital camera. One-photon confocal fluorescence images of the
fixed cells were taken using a custom-made filter cube (Ex 377/50;
DM 409; Em 525/40) and a Texas Red filter cube (Ex 562/40; DM
593; Em 624/40) for probe I and Lysotracker Red, respectively.
Two-Photon Upconverted Fluorescence Imaging. Two-photon
fluorescence microscopy (2PFM) imaging was performed on a
modified laser scanning confocal microscopy system equipped
with a broadband, tunable Ti:sapphire laser (220 fs pulse width,
76 MHz repetition rate), pumped by a 10 W frequency doubled
Nd:YAG laser. The Ti:sapphire laser, tuned 700 nm and mode-
locked, was used as the two-photon excitation source. Two-
photon induced fluorescence was collected by a 60X microscope
objective (UPLANSAPO 60X, N.A.=1.35). A high transmit-
tance (>95%) short-pass filter (cutoff 685 nm) was placed in
front of the PMT detector within the scanhead in order to filter
off background radiation from the laser source (700 nm).
Synthesis of 2,20-(5,50-(9,9-Didecyl-9H-fluorene-2,7-diyl)bis-
(ethyne-2,1-diyl)bis(thiophene-5,2-diyl))dibenzothiazole (IV).
9,9-Didecyl-2,7-diethynyl-9H-fluorene 12 (300 mg, 0.61 mmol),
2-(5-bromothiophen-2-yl)benzothiazole 1 (377 mg, 1.27 mmol),
Pd(PPh3)2Cl2 (34 mg, 0.05 mmol), and CuI (10 mg, 0.05 mmol)
were dissolved in a 1:4 mixture of Et3N/toluene (5 mL). The
mixture was heated under reflux for 12 h. After it was cooled
down, the mixture was filtered through a Celite plug, and
purified by column chromatography using a mixture of hex-
anes/EtOAc (10:1) as eluent to yield 310 mg (55%) of yellow
1
solid (mp 74.0-75.5 °C). H NMR (500 MHz, CDCl3) δ 8.04
(dd, J=8.09 Hz, J=0.54 Hz, 2H, Ph-H), 7.87 (dd, J=8.04 Hz,
J=0.53 Hz, 2H, Ph-H), 7.70 (d, J=7.83 Hz, 2H, Ph-H), 7.57 (d,
J=3.89 Hz, 2H, Thy-H), 7.54-7.56 (m, 2H, Ph-H), 7.52-7.53
(m, 2H, Ph-H),7.48-7.51 (m, 2H, Ph-H), 7.37-7.41 (m, 2H, Ph-H),
7.31 (d, J=3.89 Hz, 2H, Thy-H), 1.97-2.04 (m, 4H, CH2), 1.04-
1.23 (m, 28H, CH2), 0.84 (t, J= 6.96 Hz, 6H, CH3), 0.58-0.65
(m, 4H, CH2). 13C NMR (125 MHz, CDCl3) δ 160.5, 153.7,
151.3, 141.1, 138.0, 134.7, 132.6, 130.8, 128.4, 127.1, 126.6,
125.9, 125.5, 123.1, 121.5, 121.3, 120.2, 97.0, 82.9, 55.4, 40.3,
31.9, 30.0, 29.6, 29.5, 29.3, 29.3, 23.8, 22.7, 14.1. Anal. Calcd for
C59H60N2S4: C, 76.58; H, 6.54; N, 3.03. Found: C, 76.79; H,
6.75; N, 2.79.
Measurements. Absorption spectra were recorded with an
UV-vis spectrophotometer. Steady-state fluorescence spectra
were measured with a spectrofluorimeter in the photon counting
regime of the PMT using an L-format configuration. The
fluorescence spectra were corrected for the spectral dependence
of the PMT. All measurements were performed in hexane at
room temperature in 1 cm quartz cuvettes, with dye concentra-
tions on the order of 10-6 M. Fluorescence quantum yields were
determined relative to 9,10-diphenylanthracene in cyclohexane
as a standard.29
Two-photon absorption (2PA) cross sections of the final
compounds were determined by the two-photon induced fluore-
scence method.30 In this method, the sample is excited simulta-
neously by two photons of low energy, resulting in fluorescence
emission. The number of photons emitted can be measured by a
PMT, and the latter related to the numeric value of the 2PA
cross section relative to a reference.
Acknowledgment. The authors wish to acknowledge the
National Science Foundation (CHE-0832622 and CHE-
0840431) and the National Institutes of Health (1 R15
EB008858-01) for support of this work.
Cell Culture and Incubation. COS-7 and HCT 116 cell were
cultured in DMEM, supplemented with 10% FBS, 1% penicil-
Supporting Information Available: Additional experimental
descriptions, NMR spectra, and 3D cell images from layer-by-
layer reconstruction of two-photon fluorescence micrographs
are included. This material is available free of charge via the
Internet at http://pubs.acs.org.
(29) Belfield, K. D.; Bondar, M. V.; Przhonska, O. V.; Schafer, K. J.
J. Fluoresc. 2002, 12, 445–450.
(30) Belfield, K. D.; Bondar, M. V.; Yanez, C. O.; Hernandez, F. E.;
Przhonska, O. V. J. Phys. Chem. B 2009, 113, 7101–7106.
3982 J. Org. Chem. Vol. 75, No. 12, 2010