Linear and nonlinear photophysics and bioimaging of an integrin-targeting water-soluble fluorenyl probe

Article abstract of DOI:10.1039/b925934a

Linear photophysical characterization and two-photon absorption (2PA) properties of a new water-soluble fluorene derivative, 3-(9-(2-(2-methoxyethoxy) ethyl)-2,7-bis{3-[2-(polyethyleneglycol-550-monomethylether-1-yl)]-4-(benzo[d] thiazol-2-yl)styryl}-9H-f

Full text of DOI:10.1039/b925934a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Linear and nonlinear photophysics and bioimaging of an integrin-targeting
water-soluble fluorenyl probe†
Alma R. Morales,a Gheorghe Luchita,a Ciceron O. Yanez,a Mykhailo V. Bondar,b Olga V. Przhonskab and
Kevin D. Belfield*a,c
Received 9th December 2009, Accepted 23rd March 2010
First published as an Advance Article on the web 8th April 2010
DOI: 10.1039/b925934a
Linear photophysical characterization and two-photon absorption (2PA) properties of a new
water-soluble fluorene derivative, 3-(9-(2-(2-methoxyethoxy)ethyl)-2,7-bis{3-[2-(polyethyleneglycol-550-
monomethylether-1-yl)]-4-(benzo[d]thiazol-2-yl)styryl}-9H-fluoren-9-yl)propanoic acid (1), were
investigated in several organic solvents and water at room temperature. A comprehensive analysis of the
steady-state absorption, emission and excitation anisotropy spectra revealed electronic structures of 1,
including mutual orientation of the transition dipoles, relatively weak solvatochromic effects, high
fluorescence quantum yield (~0.5–1.0), and strong aggregation in water. The 2PA spectra of 1 were
obtained in the 600–900 nm spectral range by two-photon induced fluorescence (2PF) and open
aperture Z-scan methods using femtosecond laser sources. No discrete 2PA bands were apparent and
values of the corresponding 2PA cross sections monotonically increased in the short wavelength range
up to 3000 GM in organic solvents and ~6000 GM in aqueous solution, reflecting relatively high
two-photon absorptivity. The 2PA efficiency of 1 in water increased 2–3 times relative to aprotic
solvents and can be explained by cooperative electronic effects of molecular aggregates of 1 produced in
aqueous media. The carboxylic acid fluorenyl probe 1 was conjugated with the cyclic peptide
RGDfK. Two-photon fluorescence microscopy (2PFM) imaging of U87MG cells (and MCF-7 as
control), incubated with fluorene-RGD peptide conjugate 2, demonstrated high avb3 integrin selectivity,
making this probe particularly attractive for integrin imaging.
plication in two-photon bioimaging has been reported.16,21–24 The
Introduction
development of new water-soluble chromophores with biphenyl
The synthesis and characterization of new molecules with high
and dialkylfluorenyl core and three dodecyloxy end donor groups
fluorescence quantum yield and efficient two-photon absorption
was described in ref. 16. These compounds exhibited 2PA cross
(2PA) has been a subject of intense interest for numerous scien-
tific and practical applications, such as two-photon fluorescence
sections up to 400 GM in the 500–550 nm range and modest
values of d2PA in the near IR (~100 GM at 720 nm). Efficient 2PA
microscopy,1–4 fluorescence-based sensing methodologies,5–7 two-
of [2.2]paracyclophane-based fluorophores with high fluorescence
photon-induced lasing,8,9 and 3D optical data storage.10–12 Multi-
photon fluorescence microscopy is a powerful tool for biological
imaging and for the study of dynamic processes in living cells.13–15
quantum yield (~0.5–0.9) and corresponding cross sections of
~2000 GM in the presence of sodium dodecyl sulfate micelles
was reported in ref. 22. The interaction of 2PA chromophores
act
This application is dependent on the development of highly
with micelles caused a large increase in the d2PA and d2PA
,
fluorescent, water-soluble, photochemically stable, multiphoton
absorbing chromophores.16,17 Most known fluorescent probes
that can be utilized in one-photon fluorescence bioimaging are
not optimized for two-photon excitation, and, as a rule, are
characterized by relatively small 2PA cross sections d2PA ~ 10–100
among the highest reported in aqueous media. Hence, there still
exists tremendous need for water-soluble probes containing high
fluorescence quantum yield and high 2PA with the ability for
conjugation for cell-, organelle-, or protein-specific targeting.
In this paper, the synthesis, linear photophysical charac-
GM18,19 and corresponding action cross sections d2PA = d2PA·h,20
act
terization and 2PA properties of
a new fluorene deriva-
where h is the fluorescence quantum yield. The synthesis and
tive, 3-(9-(2-(2-methoxyethoxy)ethyl)-2,7-bis{3-[2-(polyethylene-
glycol-550-monomethylether-1-yl)]-4-(benzo[d]thiazol-2-yl)-
styryl}-9H-fluoren-9-yl)propanoic acid (1), is described in both
aprotic organic solvents and water. Comprehensive spectral anal-
ysis of the linear and nonlinear optical properties of this new
water-soluble fluorenyl derivative 1, along with its high fluores-
cence quantum yield and photochemical stability, makes this an
intriguing candidate for use as a fluorescence probe. Additionally,
conjugation of carboxylic acid functionality in 1 with the cyclic
peptide RGDfK is presented. The resulting RGDfK conjugate
(2) exhibited high avb3 integrin selectivity in both conventional
characterization of water-soluble 2PA organic compounds for ap-
aDepartment of Chemistry, University of Central Florida, Orlando, FL
32816-2366, USA
bInstitute of Physics, Prospect Nauki, 46, Kiev-28, 03028, Ukraine
cCREOL, The College of Optics and Photonics, University of Central
Florida, Orlando, FL 32816-2366, USA. E-mail: belfield@mail.ucf.edu;
Fax: 4078232252; Tel: 4078231028
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra are included for compounds A, C, D, 1, and 2. See DOI:
10.1039/b925934a
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Table 1 Main photophysical parameters of 1 in solvents with different
(one-photon) and two-photon fluorescence microscopy imaging,
demonstrating the potential of this probe for integrin imaging.
max
max
polarity Df : absorption labs
and fluorescence lfl
maxima, Stokes
shifts, maximum extinction coefficients, emax, fluorescence lifetimes, t, and
quantum yields, U
Results and discussion
N/N
Chloroform
0.149
ACN
Watera
Synthesis
Df
0.305
409
0.32
max
l
abs /nm
fl/nm
412
452
40
1
1
2
1
1
432
509
77
1
1
max
To induce increased solubility of fluorene derivatives in water,
oligo(ethylene glycol) moieties were substituted on the phenyl ring
of the styryl moiety. Poly- and oligo-(ethylene glycols) (PEG and
OEG) have frequently been employed to prepare water-soluble
polymers and therapeutic agents.25 We prepared a new fluorescence
probe of the A-p–p-p-A type, where A represents an electron-
withdrawing moiety while p is a conjugated p-electron system,
bearing long side chains averaging fifteen oxyethylene repeat units
on the styryl phenyl ring (Scheme 1). The key step for the synthesis
of the fluorenyl probe was a Pd-catalyzed Heck coupling reaction
between oligo(2-methoxyethoxy)-4-vinylphenyl)benzo[d]thiazole
l
479
Stokes shift/nm
70
125 10
0.05
0.98 0.04
2
2
emax/10-3 M-1 cm-1
115 10
0.05
0.80 0.04
107 10
U
t/ns
1
1
0.5 0.03
1.21 0.05
0.4 0.1
a Concentration of 1 in water C ª 10-6 M.
(D)
and
3-(9-(2-(2-methoxyethoxy)ethyl)-2,7-dibromo-9H-
fluoren-9-yl)propanoic acid (E) (Scheme 1). The 2-(4-
bromophenyl)benzo[d]thiazole (B) derivative was prepared
by condensation of commercially available 2-aminobenzenethiol
and 4-bromo-2-hydroxybenzoic acid in the presence of
PPSE in o-dichlorobenzene, providing
B
as previously
described.26 Poly(ethylene glycol) monomethyl ether tosylate,
CH3(OCH2CH2)nOTs, was prepared by tosylation of the
poly(ethyleneglycol)monomethylether at the alcohol terminus
with tosyl chloride,27 providing derivative A in 97% yield.
Etherification was performed by refluxing tosylate
A
and 2-(4-bromophenyl)benzo[d]thiazole (B), together with
K2CO3 in DMF at reflux. The oligo(2-methoxyethoxy)-4-
bromophenyl)benzo[d]thiazole derivative was purified by column
chromatography, and obtained in 85% yield (Scheme 1). Pd-
catalyzed Stille coupling was subsequently performed between
derivative C and tributyl(vinyl)tin in refluxing THF in a
high pressure Schlenk tube with Pd(dppf)Cl2·CH2Cl2 and
CuI. After column chromatography, precursor D was obtained
in 70% isolated yield. Pd-catalyzed Heck coupling reaction
between
3-(9-(2-(2-methoxyethoxy)ethyl)-2,7-dibromo-9H-
fluoren-9-yl)propanoic acid (E) and oligo(2-methoxyethoxy)-
4-vinylphenyl)benzo[d]thiazole (D) was conducted in a high
pressure Schlenk tube with Pd(OAc)2, tri-o-tolylphosphine,
and Et3N as base in DMF at 90 ◦C, providing 1 in 70% yield
(Scheme 1). EDC-mediated coupling between carboxylic acid 1
and cyclic RGDfK was carried out in the presence of sulfo-NHS
in water at room temperature, affording the fluorene-RGD
peptide conjugate 2 in 80% yield as shown in Scheme 2.
Fig. 1 Normalized absorption (1, 2), fluorescence (1¢, 2¢) and excitation
anisotropy (3) spectra of 1 in: (a) chloroform (1, 1¢), ACN (2, 2¢) and
poly-THF (pTHF) (3); (b) water with chromophore concentration C ª
10-3 M (1, 1¢), C ª 10-6 M (2, 2¢) and glycerol with C ª 10-6 M (3).
Linear spectral properties
explained by molecular aggregation of 1 in polar protic solvent.
The absorption band of the aggregates was not very sharp nor
intense, as typically observed for large aggregated clusters.28,29 In
order to understand the nature of this aggregation, the depen-
dencies of scattered light intensity, I, on excitation wavelength, l,
were obtained in the spectral range of one-photon transparency,
l ≥ 720 nm, in protic and aprotic media, and presented in Fig. 2.
The steady-state absorption, fluorescence, and excitation
anisotropy spectra, and the primary photophysical parameters
of probe 1 are presented in Fig. 1 and Table 1, respectively. The
shape of absorption spectra and maximum values of extinction
coefficients, emax, were concentration-independent in aprotic sol-
vents up to C ª 2 ¥ 10-3 M. This indicates a negligible amount
of aggregated molecules in aprotic media. In contrast, the shape
of the absorption spectra in water exhibited a strong dependence
on molecular concentration (Fig. 1b, curves 1, 2), which can be
4
As can be deduced from Fig. 2, the dependencies I = f (1/l ) are
close to a linear function for all the investigated solutions, strongly
suggestive of a Rayleigh-type scattering mechanism.30 Taking into
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Scheme 1 Synthesis of water-soluble fluorescent probe 1.
Scheme 2 Synthesis of fluorene-RGDfK peptide conjugate 2.
4
account, that, for Rayleigh scattering, I ~ I0r6/l (r is the size of
scattering centers), we can estimate the difference in the size r for
aggregated and monomer molecules from the data in Fig. 2, which
were obtained under the same experimental conditions. Analysis
of this data revealed dimers as the most probable aggregates of
1 in water. This is consistent with the relatively broad absorption
spectrum of these aggregates (Fig. 1b, curve 1).
The absorption spectra of 1 in aprotic solvents were nearly
independent of solvent polarity, Df (Fig. 1a, curves 1, 2), and
exhibited a well-defined vibrational structure in low concentration
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In contrast to these aprotic solvents, the value of r0 in glycerol
(Fig. 1b, curve 3) was not constant in the main absorption band,
indicative of the rather complex electronic nature of 1 in polar
protic media. This nature can be attributed to specific solute–
solvent effects such as hydrogen bonding32 and inhomogeneous
broadening of the solvated molecules.33 Fluorescence decay of 1 in
protic polar media corresponded to a double exponential process
with short (~0.4 ns) and long (~1.2 ns) components and decreased
fluorescence quantum yield (see Table 1). The pre-exponential
coefficients were A1 = 0.88 and A2 = 0.12 for the short (t1 =
0.4 ns) and long (t2 = 1.21 ns) lifetime components, respectively,
suggesting predominant aggregate formation in water. This pro-
vides further evidence of specific solvent effects and is consistent
with the excitation anisotropy spectra.
2PA properties of 1
The efficiency of 2PA processes for 1 was investigated in aprotic
solvents and water at room temperature. The corresponding
spectra are presented in Fig. 3. No discrete 2PA peaks were
observed in the main one-photon absorption (1PA) band, l ª 350–
450 nm, and the values of two-photon cross sections d2PA increased
monotonically in the short wavelength spectral range, and were
independent of solvent polarity in aprotic media. The molecular
structure of 1 is unsymmetrical (C1 point group symmetry), but
the p-conjugated system of this chromophore possesses nearly
symmetrical electronic distribution. Alkyl groups in the 9-position
and oxygen-containing substituents in 1 do not strongly affect
the p-electronic distribution. Therefore, the efficiency of the two-
photon transition S0 → S1 is noticeably suppressed by dipole
selection rules34 and no 2PA maximum was detected in this spectral
region. This behavior is typical for symmetrical (C2V) fluorene
derivatives (see ref. 35). Nevertheless, the absolute values of the
2PA cross sections reach d2PA ~ 100–1000 GM at l ª 690–800 nm,
values that are quite suitable for two-photon bioimaging using
commercially available femtosecond lasers. The extreme points at
l ª 300–310 nm in the excitation anisotropy spectra of 1 in aprotic
solvents and water (see Fig. 1, curves 3) indicates weak one-photon
electronic transitions that are slightly resolved in the 1PA spectra
(curves 1, 2). These transitions can be effective in the 2PA processes
4
Fig. 2 Dependences I = f (1/l ) for 1 in: (a) water; (b) ACN (1) and
chloroform (2).
aqueous solution (Fig. 1b, curve 2). The fluorescence spectra of 1
are characterized by a weak solvatochromic effect with increased
Stokes shift up to ~80 nm in water. It is interesting to note
that in high concentrations in aqueous solution, the formation
of molecular dimers of 1 exhibited an increased Stokes shift
(~100 nm) with no apparent vibrational structure (Fig. 1b, curves
1, 1¢). In this case, the fluorescence spectrum is similar to the
charge transfer emission band.31 It should be mentioned that
fluorescence spectra were independent of excitation wavelength
in aprotic solvents and exhibited this dependence only in protic
media (water, glycerol).
The excitation anisotropy spectrum of 1 in viscous pTHF
(Fig. 1a, curve 3) provided information on the nature of the
one-photon absorption band in aprotic media. Nearly constant
values of r0 ª 0.38 in the 400–450 nm spectral range is close to
the theoretical limit (0.4),31 and primarily corresponds to a single
electronic transition, S0–S1. This means that there is nearly parallel
orientation (a ≤ 10◦, see eqn (1) in the Experimental section) of
the absorption S0 → S1 and emission S1 → S0 transition dipoles
of 1, allowing one to estimate the angles between S0 → S1 and
S0 → Sn absorption transitions (n = 2, 3, …). From Fig. 1a
(curve 3), the values of r0 ≥ 0.2 in the spectral range l < 400 nm
corresponded to a ≤ 35◦. The decay of fluorescence emission of 1
in chloroform and ACN (polar aprotic solvents) was characterized
by a single exponential process with typical lifetimes t ª 0.8–
1 ns (see Table 1) and high fluorescence quantum yield U ª 1.0.
Fig. 3 2PA spectra of 1 in chloroform (1), ACN (2) and water (3).
Normalized linear absorption (1PA) of 1 in chloroform (1¢), ACN (2¢)
and water (3¢). Experimental errors of (1–3) 15%.
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and used to determine the monotonic increase in d2PA in the short
wavelength spectral range. On the other hand, the increase in
d2PA is also induced by the change in the detuning energy, E =
max
hc(1/l ab - 1/l), where c and h are the speed of light in vacuum
and Planck’s constant, respectively.36 It should be mentioned that
the differences observed in d2PA for 1 in aprotic solvents and water
cannot be explained by the corresponding changes in the value of E
only. A comprehensive analysis of the linear spectral data (Table 1)
supports a significant role of aggregation processes in increasing
the 2PA cross section of 1 in aqueous medium. According to Fig. 3,
the value of d2PA increased 2–3 times in water relative to aprotic
solvents, reaching 6000 GM at l ª 600 nm. Similar cooperative
effects in 2PA were observed for PIC and porphyrin J-aggregates
in aqueous solutions.37,38
Fig. 4 One-and two-photon fluorescence micrographs of U87MG cells
incubated with probe 2 (50 mM, 30 min). (A) DIC, (B) one-photon
fluorescence, (C) 3D reconstruction from overlaid 2PFM images, 76 MHz,
115 fs pulse width, 700 nm, 60¥ objective (NA = 1.35, Olympus). Scale:
10 mm grid.
Single and two-photon fluorescence cell imaging
To demonstrate the potential of this new dye in selective two-
photon fluorescence bioimaging, a targeting vector was incor-
porated. To achieve target selectivity, the sequence Arg-Gly-Asp
(RGD) was chosen as it is recognized by a number of integrins.39 In-
tegrins are a family of plasma membrane glycoproteins formed by
several a and b subunits. These subunits are long molecular weight
polypeptide chains that form a receptor when a–b heterodimers
are formed. Of the 25 distinct pairs of heterodimers that have been
reported, avb3 integrin has unique properties, in that it is known
to bind to specific extracellular matrix molecules (fibronectin,
fibrogen, and vibronectin). It is also upregulated in invasive tumor
cells, yet not significantly expressed in quiescent endothelial cells
nor in normal tissue,40 making this integrin of particular interest
when targeting angiogenic vasculature and tumor cells alike.41
In order to test the selectivity of fluorene-RGD peptide conju-
gate 2 in vitro, U87MG human gliobastoma cells were selected as
a positive control due to its elevated avb3 integrin expression. Low
avb3 integrin expressing MCF-7 human breast cancer cells served
as a negative control.41 Cells incubated for the same period of time
at the same dye concentration showed significantly different results
for the two different cell lines (positive and negative controls), as
shown in Fig. 4 and 5, respectively. In the positive control images,
specific staining of the cells was observed. Furthermore, enhanced
image resolution for two-photon fluorescence microscopy (2PFM)
vs. conventional or one-photon fluorescence microscopy imaging
was clearly observed, both in terms of spatial resolution and in
contrast (Fig. 4).
The specificity of fluorene-RGD probe 2 enabled the labeling
and visualization of sites where higher concentrations of the
integrin are localized within the cells. Integrins play an important
role in cell adhesion to the extra cellular matrix, ECM, and in
the signaling processes that depend on or regulate adhesion. This
means that integrin is at the center of an array of cell processes
such as cell migration, shape, survival, proliferation, and gene
transcription.40,42 Once integrins bind to their extracellular ligand,
they cluster in focal spots that are rich in actin and actin-associated
proteins. These spots, called focal adhesions (FA’s) or focal contacts,
can clearly be seen in cultured fibroblast, where the cell surface
is separated from the substratum by 10 to 15 mm only at these
localized regions (whereas in the rest of the cell the distance is
approximately 50 mm).43,44
Fig. 5 One-and two-photon fluorescence micrographs of MCF-7 cells
incubated with probe 2 (50 mM, 30 min). (A) DIC, (B) one-photon
fluorescence, (C) 3D reconstruction from overlaid 2PFM images, 76 MHz,
115 fs pulse width, 700 nm, 60¥ objective (NA = 1.35, Olympus). Scale:
10 mm grid.
In the U87MG cells that were incubated with probe 2, it was
observed that a higher concentration of the probe 2 was present
in areas that appear to be FA’s (Fig. 6) in the motile zone (IV)
and culling zone (III) of a polarized cell. In the magnification of
the motile zone (Fig. 6B), these apparent FA’s are visualized quite
clearly by 2PFM. In the persistence zone of the cell (Fig. 6A,
III), more FA’s were observed and higher fluorescence in other
localized spots can be attributed to the endoplasmic reticulum
with newly synthesized or stored integrin. Furthermore, nascent
focal adhesion points can be seen in the formation zone (I) of the
cell, as indicated by the arrow in Fig. 6A. These micrographs show
the selectivity that fluorenyl-RGD peptide 2 has for avb3 integrin,
and the potential that this 2PA fluorescent probe has for studying
integrin-mediated processes in cells or tissue in vitro, and, perhaps,
in vivo.
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Fig. 6 (A) Two-photon fluorescence micrograph of U87MG cells incubated with probe 2 (50 mM, 30 min). 3D reconstruction from overlaid 2PFM
images, 76 MHz, 115 fs pulse width, 700 nm, 60¥ objective (NA= 1.35, Olympus). (B) Magnification of volume indicated by cube in A. Scale: 10 mm grid
in A and 5 mm in B.
CH3+Na]+
=
649.28, requires [M+Na, 649.29]+; found
605.25, requires [M+Na,
Experimental
[C7H8O3S(C2H4O)9CH3+Na]+
=
605.26]+; found [C7H8O3S(C2H4O)8CH3 +Na]+ = 561.23, requires
[M+Na, 561.23]+; found [C7H8O3S(C2H4O)7CH3+Na]+ = 517.20,
requires [M+Na, 517.21]+; found [C7H8O3S(C2H4O)6CH3+Na]+ =
450.16, requires [M+Na, 450.19]+; found [C7H8O3S(C2H4O)5-
Materials and Synthetic Procedures
The synthesis of 2-(benzo[d]thiazol-2-yl)-5-bromophenol (B)
and 3-(9-(2-(2-methoxyethoxy)ethyl)-2,7-dibromo-9H-fluoren-9-
yl)propanoic acid (E) was described previously.26,45 Pd-catalyzed
reactions were performed in high pressure Schlenk tubes under N2.
All reagents and solvents were used as received from commercial
suppliers, unless otherwise noted. 1H and 13C NMR spectroscopic
measurements were performed using a Varian 500 NMR spec-
trometer at 500 or 125 MHz, respectively, with tetramethysilane
(TMS) as internal reference; 1H (referenced to TMS at d =
0.0 ppm) and 13C (referenced to CDCl3 at d = 77.0 ppm). Chemical
shifts of 1H and 13C spectra were interpreted with the support of CS
ChemDraw Ultra version 5.0. High-resolution mass spectrometry
(HR-MS) analysis was performed in the Department of Chemistry,
University of Florida, Gainesville, FL.
CH3+Na]+
=
429.13, requires [M+Na, 429.16]+; found
[C7H8O3S(C2H4O)4CH3+H]+ = 363.19, requires [M+Na, 362.14]+;
found [C7H8O3S(C2H4O)3CH3+H]+ = 319.17, requires [M+H,
318.11]+; found [C7H8O3S(C2H4O)2CH3+H]+ = 275.14, requires
[M+H, 274.09]+.
Synthesis of 2-[2-(polyethyleneglycol-550-monomethylether-
1-yl)-4-bromophenyl]benzo[d]thiazole
(C).
A
mixture
of
benzothiazol-2-yl-5-bromophenol (2.45 g, 7.95 mmol) and
CH3(OCH2CH2)nOTs (7.27 g, 10.33 mmol) was dissolved in
25 mL of DMF. To this, K2CO3 (23.85 g) was added and the
solution was stirred at reflux for 4 h. After cooling to room
temperature, the solution was filtered, poured into water, and
extracted thrice with CH2Cl2. The combined organic layers were
washed with water and then with brine, dried over magnesium
sulfate, filtered, and the solvent was removed at reduced pressure.
Chromatography (silica gel, ethyl acetate–MeOH 9 : 1) provided
C (6.04 g, 85%) as colorless oil.1H NMR (500 MHz, CDCl3)
d: 8.42 (d, J = 8 Hz, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.93
(d, J = 7.5 Hz, 1H), 7.51 (t, J = 7 Hz, 1H), 7.39 (t, J =
7 Hz, 1H), 7.27 (t, J = 8.5 Hz, 1H), 4.38 (t, J = 10 Hz, 2H),
4.07 (t, J = 5.5 Hz, 2H), 3.80 (t, J = 5 Hz, 2H), 3.71-3.53
(m, 51H), 3.37 (s, 3H). 13C NMR (125 MHz,d CDCl3) d:
162.15, 156.74, 151.93, 151.91, 136.07, 136.05, 130.71, 130.45,
126.20, 125.87, 125.42, 124,97, 124.78, 124.58, 124.33, 122.84,
122.71, 121.54, 121.25, 116.36, 115.92, 71.89, 70.88, 70.74,
70.67, 70.64, 70.52, 70.41, 70.28, 69.53, 69.37, 69.20, 68.85,
Synthesis of CH3(OCH2CH2)nOTs (A). Sodium hydroxide
(2.18 g, 54.52 mmol) in water (12 mL), and poly(ethylene
glycol) methylether CH3(OCH2CH2)nOH (12 g, 21.81 mmol) in
THF (12 mL) were cooled in an ice bath with stirring, and p-
toluenesulfonyl chloride (5.03 g, 26.39 mmol) in THF (12 mL)
was added dropwise over 1 h. The reaction mixture was stirred for
◦
additional 2 h at 5 C, poured into ice water, and extracted with
CH2Cl2 (200 mL). The combined organic extracts were washed
with dilute HCl followed by brine (50 mL), then dried over
MgSO4, and the solvent removed in vacuo to yield A (12.6 g,
1
82% as colorless oil). H NMR (500 MHz, CDCl3) d: 7.80 (d,
J = 8 Hz, 2H), 7.36 (d, J = 8 Hz, 2H), 4.38 (bs, 6H), 4.16,
(t, J = 5, 2H), 3.73-3.54 (bm, 45H), 3.39 (s, 3H), 2.45 (s, 3H).
13C NMR (125 MHz) d: 144.80, 132.81, 130.03, 129.58, 127.95,
127.83, 127.76, 71.78, 71.22, 70.97, 70.64, 70.58, 70.50, 70.39,
70.28, 70.16, 69.42, 69.25, 69.09, 68.99, 68.79, 68.55, 58.89, 21.67.
HRMS-ESI: found [C7H8O3S(C2H4O)16CH3+Na]+ = 913.43, re-
quires [M+Na, 913.44]+; found [C7H8O3S(C2H4O)15CH3+Na]+ =
869.41, requires [M+Na, 871.42]+; found [C7H8O3S(C2H4O)14
CH3+Na]+ = 825.38, requires [M + Na, 825.39]+; found
59.04. HRMS-ESI: found [C13H7BrNOS(C2H4O)15CH3+Na]+
=
1004.27, requires [M+Na, 1004.35]+; found [C13H7BrNOS-
(C2H4O)14CH3+Na]+ = 960.30, requires [M+Na, 960.32]+; found
[C13H7BrNOS(C2H4O)13CH3+Na]+ = 916.29, requires [M+Na,
916.30]+; found [C13H7BrNOS(C2H4O)12CH3+Na]+ = 872.26,
requires [M+Na, 872.27]+; found [C13H7BrNOS(C2H4O)11-
[C7H8O3S(C2H4O)13CH3+Na]+
=
781.36, requires [M+Na,
781.37]+; found [C7H8O3S(C2H4O)12CH3+Na]+ = 737.37, re-
quires [M+Na, 737.34]+; found [C7H8O3S(C2H4O)11CH3+Na]+ =
693.31, requires [M+Na, 693.31]+; found [C7H8O3S(C2H4O)10-
CH3+Na]+
=
828.23, requires [M+Na, 828.25]+; found
[C13H7BrNOS(C2H4O)10CH3+Na]+ = 784.21, requires [M+Na,
784.22]+; found [C13H7BrNOS(C2H4O)9CH3+Na]+ = 740.19,
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requires [M+Na, 740.19]+; found [C13H7BrNOS(C2H4O)8
67.20, 59.10, 58.59, 58.36, 58.18, 57.86, 57.62, 52.56, 36.68, 31.08.
HRMS-ESI: High resolution MS shows a series of ions centered
around m/z 2246 [C51H40N2O6S2(C2H4O)32C2H6 - CH3O]+ which
are fragments related to the various ethoxylate oligomers that
differ by Dm/z 44, an ethoxy unit.
CH3+Na]+ = 696.16, requires [M+Na, 696.17]+.
Synthesis of 2-[2-(polyethyleneglycol-550-monomethylether-1-
yl)-4- vinylphenyl)benzo[d]thiazole (D). Compound C (3 g,
3.94 mmol), Pd(dppf)Cl2·CH2Cl2 (0.32 g, 0.39 mmol), and CuI
(0.15 g, 0.78 mmol) were transferred to a high pressure Schlenk
tube, followed by addition of THF (50 mL) and tributyl(vinyl)tin
(1.62 g, 5.12 mmol). The Schlenk tube was sealed and stirred at
Synthesis of fluorene-RGD conjugate (2). Fluorene 1 (38 mg,
0.017 mmol) was dissolved in 2 mL H2O. After adjust-
ing the pH to 5.0–5.5 with 0.1 N NaOH (aq), sodium
N-hydroxysulfonosuccinimide (5 mg, 0.023 mmol) and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6 mg,
0.026 mmol) were added. After stirring at 4 ◦C for 30 min, the
pH of the reaction mixture was adjusted to 9 with 0.1 N NaOH
(aq) and c(RGDfK) (10 mg, 0.016 mmol), dissolved in 0.5 mL of
0.1 M Na2HPO4 solution (pH = 9), was added. The reaction was
stirred at room temperature for 24 h. Water was removed under
reduced pressure, providing yellow oil, which was redissolved in
CH2Cl2, dried over NaSO4, filtered, and concentrated. The oil was
washed several times with ether, then dried to afford 2 (43 mg,
◦
90 C. After 96 h, silica gel TLC indicated disappearance of the
aryl bromide. Hence, the dark brown reaction mixture was cooled
to room temperature, diluted CH2Cl2, and filtered through a plug
of silica gel. The crude product was concentrated and purified
by column chromatography on silica gel using ethyl acetate–
MeOH (4 : 1) providing D (2.1 g, 71%) as colorless oil. 1H NMR
(500 MHz, CDCl3) d: 8.48 (d, J = 8.5 Hz, 1H), 8.06 (d, J =
6 Hz, 1H), 7.91 (d, J = 8 Hz, 1H), 7.48 (t, J = 15 Hz, 1H),
7.36 (t, J = 14.5 Hz,1H), 7.17 (d, J = 8 Hz, 1H), 7.07 (s, 1H),
6.74 (q, J = 17.5 Hz, 1H), 5.86 (d, J = 17.5 Hz, 1H), 5.36 (d,
J = 10.5 Hz, 1H), 4.40 (t, J = 10 Hz, 2H), 4.07 (t, J = 10 Hz,
2H), 3.79 (t, J = 9 Hz, 2H), 3.69-3.51 (m, 59H), 3.35 (s, 3H).13C
NMR (125 MHz, CDCl3) d: 162.87, 156.62, 152.01, 141.04,
136.19, 136.10, 136.12, 129.71, 129.55, 126.0, 125.74, 124.66,
124.43, 122.66, 121.22, 119.44, 119.22, 110.38, 110.18, 71.88,
70.85, 70.65, 70.60, 70.48, 70.34, 69.69, 69.53, 69.35, 68.44, 59.01,
1
90%) as a yellow oil. H NMR (500 MHz, d-DMSO) d: 8.55 (d,
J = 8 Hz, 2H), 8.09 (d, J = 8 Hz, 2H), 7.94 (d, J = 8 Hz, 2H),
7.67 (d, J = 8 Hz, 2H), 7.60 (s, 2H), 7.52-7.17 (m, 5H), 7.50 (t,
J = 8 Hz, 3H), 7.38-7.09 (m, 15H), 4.47 (t, J = 10 Hz, 5H),
4.12 (t, J = 9.5 Hz, 5H), 3.84 -3.52 (m, 118 H), 3.36 (s, 6H),
3.31-3.10 (m, 10H), 2.83 (t, 15.5 Hz, 2H), 2.74 (bt, 1H), 2.53-2.42
(m,6H), 1.74 (s, 1H), 1.63 (t, 16 Hz, 3H), 1.24 (s, 3H).13C NMR
(125 MHz, CDCl3) d: 175.48, 171.27, 168.48, 162.88, 159.99,
159.81, 156.71, 152.10, 152.12, 149.33, 140.97, 140.55, 136.41,
136.12, 136.10, 129.89, 129.64, 126.14, 125.80, 122.68, 122.53,
121.58, 121.26, 120.81, 119.32, 110.13, 71.83, 71.67, 71.25, 70.84,
70.67, 70.55, 70.43, 70.18, 69.94, 69.54, 69.39, 69.03, 68.48, 58.98,
56.42, 55.81, 55.56, 55.30, 54.99, 54.73, 52.18, 45.95, 44.25, 43.59,
43.19, 42.77, 42.38, 36.96, 36.87, 36.37, 36.29, 36.20, 35.81, 35.63,
35.23, 34.96, 34.70, 34.40, 34.12, 30.50, 29.67, 28.55, 25.81, 25.67,
25.54, 16.49, 15.74, 15.59, 15.44, 15.29, 14.54. HRMS-ESI: High
resolution MS shows a series of ions centered around m/z 2246
[C78H79N11O12S2(C2H4O)30C2H6+Na]+ which are fragments related
to the various ethoxylate oligomers that differ by Dm/z 44, an
ethoxy unit.
58.98. HRMS-ESI: found [C15H10NOS(C2H4O)15CH3+Na]+
=
950.32, requires [M+Na, 950.41]+; found [C15H10NOS(C2H4O)14-
CH3+Na]+ = 906.38, requires [M+Na, 906.43]+; found [C15H10-
NOS(C2H4O)13CH3+Na]+ = 862.36, requires [M+Na, 862.4]+;
found [C15H10NOS(C2H4O)12CH3+Na]+
=
818.37, [M+Na,
818.38]+; found [C15H10NOS(C2H4O)11CH3+Na]+ = 774.34, re-
quires [M+Na, 774.35]+; [C15H10NOS(C2H4O)10CH3+Na]+
=
730.32, requires [M+Na, 730.32]+; found [C15H10NOS(C2H4O)9-
CH3+Na]+ = 686.29, requires [M+Na, 686.30]+.
Synthesis of 3-(9-(2-(2-methoxyethoxy)ethyl)-2,7-bis{3-[2-
(polyethyleneglycol-550-monomethylether-1-yl)]-4-(benzo[d]thi-
azol-2-yl)styryl}-9H-fluoren-9-yl)propanoic acid (1). 3-(9-(2-(2-
Methoxyethoxy)ethyl)-2,7-dibromo-9H -fluoren-9-l)propanoic
acid (E) (0.17 g, 0.34 mmol), benzothiazole derivative D (0.55 g,
0.78 mmol), Pd(OAc)2 (0.03 g, 0.13 mmol), and P(o-tolyl)3
(0.068 g, 0.22 mmol) were transferred to a high pressure Schlenk
tube. To this 8 mL of DMF–Et3N (5 : 1 v/v) ◦was added. The
Schlenk tube was then sealed and stirred at 90 C for 72 h. The
mixture was cooled to room temperature and filtered. The solvent
was removed under reduced pressure. The crude product was
purified by column chromatography on silica gel first using ethyl
acetate, then ethyl acetate–MeOH (4 : 1) and MeOH as eluent
to afford 1 (0.4 g, 82%) as yellow-brownish solid. 1H NMR
(500 MHz, d-DMSO) d: 8.46 (d, J = 8 Hz, 2H), 8.12 (d, J =
8 Hz, 2H), 8.06 (d, J = 8 Hz, 2H), 7.86 (t, J = 15.5 Hz, 4H),
7.65-7.42 (m, 14H), 4.52 (s, 4H), 4.03 (bt, J = 8.5 Hz, 4H), 3.73
(t, J = 5.5 Hz 4H), 3.62 -3.10 (m, 125H), 2.71 (bt, 2H), 2.43 (bt,
4H), 2.24 (bt, 2H). 13C NMR (125 MHz, d-DMSO) d: 175.99,
162.58, 156.95, 152.11, 150.64, 141.88, 140.51, 136.62, 136.06,
131.61, 129.47, 129.26, 127.91, 127.34, 127.03, 126.73, 126.29,
125.04, 124.81, 122.83, 122.67, 122.19, 122.01, 121.64, 120.77,
120.11, 119.77, 111.53, 111.24, 110.86, 79.71, 79.45, 79.18, 71.89,
71.78, 71.57, 70.97, 70.78, 70.69, 70.46, 70.39, 70.28, 70.21, 70.02,
69.97, 69.77, 69.64, 69.52, 69.47, 69.41, 69.30, 68.89, 68.53, 68.29,
Cell culture and incubation. U87MG cells were cultured in
DMEM, supplemented with 10% FBS, 1% penicillin, and 1%
streptomycin, at 37 ◦C under a 5% CO2 environment. MCF 7
cells were cultured in MEM, also supplemented with 10% FBS,
1% penicillin, and 1% streptomycin, at 37 ◦C under a 5% CO2
environment. No. 1 round 12 mm coverslips were treated with poly-
D-lysine to improve cell adhesion and washed (3¥) with PBS buffer
solution. The treated coverslips were placed in 24-well plates, and
60 000 cells/well were seeded and incubated at the same conditions
as indicated above until 75–85% confluency was reached on the
coverslips. From a 3.04 ¥ 10-4 M stock solution of probe 2 in PBS
(7.4), a series of 1, 10, and 50 mM solutions in culture media were
used to incubate both cell lines for 1 and 1.5 h. The dye solutions
were removed and the coverslipped cells were washed abundantly
with PBS (4¥).
Cell fixing and mounting. Cells were fixed with 3.7%
paraformaldehyde solution in pH = 7.4 PBS buffer for 10 min.
The fixing agent was removed and washed (2¥) with PBS. To
reduce autofluorescence, a fresh solution of NaBH4 (1 mg mL-1)
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in pH = 8 PBS buffer was used for treating the fixed cells (2¥).
The coverslipped cells were then washed with buffer PBS (2¥) and
mounted on microscope slides using Prolong Gold (Invitrogen) as
mounting media.
MIRA 900 (Coherent) was also used for these measurements. The
dependencies of I on excitation wavelength l and I0 facilitated
estimation of the nature of scattering mechanism and size of
scattering centers.
Confocal one-photon fluorescence imaging. Conventional (one-
photon) fluorescence microsocopy images were recorded on an
Olympus DSU IX-81 confocal microscope equipped with a
Hamamatsu EM-CCD C9100 digital camera. Fluorescent 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 2 and Lysotracker Red,
respectively.
2PA measurements. The 2PA spectra of 1 were determined
by a standard two-photon induced fluorescence (2PF) method,
relative to Rhodamine B in methanol.18 A femtosecond laser
Clark-MXR CPA-2010, with pulse duration ª140 fs (FWHM),
pumped an optical parametric generator/amplifier (TOPAS, Light
Conversion) with a tuning range of 580–940 nm, pulse energies
≤0.15 mJ, and repetition rate 1 kHz, were used for excitation. The
intensity of the 2PF was measured with a PTI QuantaMaster spec-
trofluorimeter in 10 mm quartz cuvettes with dye concentrations
~(1–2) ¥ 10-5 M. The quadratic dependence of 2PF on average
excitation power was verified for each excitation wavelength. The
values of 2PA cross sections, d2PA , in the 720–900 nm spectral
range were determined independently by the open aperture Z-
scan method46 using 76 MHz femtosecond excitation (MIRA
900, Coherent). The main characteristics of the MHz Z-scan
methodology were described in ref. 47–49. A space filter with
a 100 mm pinhole, electronic shutter VMM-T1 (Uniblitz) with
5 ms exposure time (less than 0.1 ms time to open), and neutral
density filters were used in the experimental setup in order to
avoid thermooptical distortion and accumulative effects of triplet
population.
Two-photon fluorescence imaging. Two-photon fluorescence
microscopy (2PFM) imaging was performed on a modified
Olympus Fluoview FV300 laser scanning confocal microscopy
system equipped with a tunable Coherent Mira Ti:sapphire laser
(115 fs pulse width, 76 MHz repetition rate), pumped by a 10
W Coherent Verdi frequency doubled Nd:YAG laser. The laser
was tuned to 700 nm, modelocked, and used as the two-photon
excitation source. Two-photon induced fluorescence was collected
by a 60¥ microscope objective (UPLANSAPO 60¥, N.A. = 1.35
Olympus). A high transmittance (>95%) short-pass filter (cutoff
685 nm, Semrock) was placed in front of the PMT detector within
the FV300 scanhead in order to filter out background radiation
from the laser source (700 nm).
Linear photophysical characterization. The absorption, flu-
orescence, and excitation anisotropy spectra of 1 were inves-
tigated in spectroscopic grade chloroform, acetonitrile (ACN),
polytetrahydrofuran (pTHF), glycerol, and distilled water at
room temperature. The steady-state absorption spectra of 1 were
measured with an Agilent 8453 UV-visible spectrophotometer
in 0.1, 1, and 10 mm path length quartz cuvettes with dye
concentrations, C, 10-5 M ≤ C ≤ 2 ¥ 10-3 M. The steady-state
fluorescence and excitation anisotropy spectra were obtained with
a PTI QuantaMaster spectrofluorimeter in 10 mm spectrofluoro-
metric quartz cuvettes with C ~10-6 M. The fluorescence spectra
were corrected for the spectral responsivity of the PTI emission
monochromator and PMT. Excitation anisotropy measurements
were performed in viscous pTHF and glycerol at room temperature
and an “L-format” configuration was used. In viscous solvents, the
influence of rotational depolarization processes are negligible dur-
ing nanosecond fluorescence lifetimes, and excitation anisotropy
reaches its fundamental value:29
Conclusions
Water-soluble fluorene derivative 1 was synthesized in a straight-
forward manner. The steady-state absorption spectra were inde-
pendent of solvent polarity while pronounced aggregation of 1 was
observed in an aqueous medium, indicative of dimer formation, as
suggested by light scattering measurements. Excitation anisotropy
spectra of 1 in aprotic solvents provided valuable information on
the spectral position and mutual orientation of ground to excited
states transition dipoles, and disclosed excited electronic states
participating in two-photon transitions. The fluorescence spectra
of 1 were independent of excitation wavelength in aprotic solvents
and exhibited weak solvatochromic effects. A high fluorescence
quantum yield ~1.0 and a single exponential decay of the fluores-
cence emission was observed. In contrast, the fluorescence spectra
of 1 in water exhibited an excitation wavelength dependence and
double exponential decay in emission, indicative of specific solute–
solvent effects.
The values of the 2PA cross sections increased monotonically
in the short wavelength range (two-photon allowed transition) up
to 3000–6000 GM. In the main 1PA band (formally two-photon
forbidden), the efficiency of 2PA remained sufficiently high for
practical applications (d2PA ~ 100–1000 GM) with commercially
available femtosecond lasers. The increased values of d2PA in water,
relative to the corresponding values in aprotic solvents, can be
explained by possible cooperative electronic effects in molecular
aggregates of 1.
r0 = (3 cos2 a - 1)/5
(1)
where a is the angle between absorption S0 → S1 and emission
S1 → S0 transition dipoles (S0 and S1 are the ground and first
excited electronic state, respectively). Fluorescence lifetimes of 1,
t, were obtained with a single photon counting system PicoHarp
300 under 76 MHz femtosecond excitation (MIRA 900, Coherent)
with time resolution of ª 80 ps. The excitation laser beam was
linearly polarized and oriented by the magic angle. Fluorescence
quantum yields, U, of 1 were measured by a standard method
relative to 9,10-diphenylanthracene in cyclohexane.29 The intensity
of linear light scattering I was measured in a 10 mm spectroflu-
orometric cuvette in the transverse direction to the excitation
laser beam with corresponding intensity I0. The femtosecond laser
The corresponding RGDfK conjugate 2 clearly demonstrated
the integrin-targeting ability of this new probe through 2PFM
imaging of U87MG cells, providing a valuable new tool for the
study of integrin-mediated processes in cells.
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Acknowledgements
We wish to acknowledge the National Institutes of Health
(1 R15 EB008858-01), the U.S. Civilian Research and Develop-
ment Foundation (UKB2-2923-KV-07), the Ministry of Educa-
tion and Science of Ukraine (grant M/49-2008), and the National
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