Amino(oligo)thiophene-based environmentally sensitive biomembrane chromophores

Article abstract of DOI:10.1021/jo800852h

(Graph Presented) There is a growing need for cellular imaging with fluorescent probes that emit at longer wavelengths to minimize the effects of absorption, autofluorescence, and scattering from biological tissue. In this paper a series of new environmentally sensitive hemicyanine dyes featuring amino(oligo)thiophene donors have been synthesized via aldol condensation between a 4-methylpyridinium salt and various amino(oligo)thiophene carboxaldehydes, which were, in turn, obtained from amination of bromo(oligo)thiophene carboxaldehyde. Side chains on these fluorophores impart a strong affinity for biological membranes. Compared with benzene analogues, these thiophene fluorophores show significant red shift in the absorption and emission spectra, offering compact red and near-infrared emitting fluorophores. More importantly, both the fluorescence quantum yields and the emission peaks are very sensitive to various environmental factors such as solvent polarity or viscosity, membrane potential, and membrane composition. These chromophores also exhibit strong nonlinear optical properties, including two-photon fluorescence and second harmonic generation, which are themselves environmentally sensitive. The combination of long wavelength fluorescence and nonlinear optical properties make these chromophores very suitable for applications that require sensing or imaging deep inside tissues.

Full text of DOI:10.1021/jo800852h

Amino(oligo)thiophene-Based Environmentally Sensitive
Biomembrane Chromophores
Ping Yan, Aifang Xie, Meide Wei, and Leslie M. Loew*
Richard D. Berlin Center for Cell Analysis and Modeling, UniVersity of Connecticut Health Center,
Farmington, Connecticut 06030
les@Volt.uchc.edu
ReceiVed May 6, 2008
There is a growing need for cellular imaging with fluorescent probes that emit at longer wavelengths to
minimize the effects of absorption, autofluorescence, and scattering from biological tissue. In this paper
a series of new environmentally sensitive hemicyanine dyes featuring amino(oligo)thiophene donors have
been synthesized via aldol condensation between a 4-methylpyridinium salt and various amino(oligo)th-
iophene carboxaldehydes, which were, in turn, obtained from amination of bromo(oligo)thiophene
carboxaldehyde. Side chains on these fluorophores impart a strong affinity for biological membranes.
Compared with benzene analogues, these thiophene fluorophores show significant red shift in the absorption
and emission spectra, offering compact red and near-infrared emitting fluorophores. More importantly,
both the fluorescence quantum yields and the emission peaks are very sensitive to various environmental
factors such as solvent polarity or viscosity, membrane potential, and membrane composition. These
chromophores also exhibit strong nonlinear optical properties, including two-photon fluorescence and
second harmonic generation, which are themselves environmentally sensitive. The combination of long
wavelength fluorescence and nonlinear optical properties make these chromophores very suitable for
applications that require sensing or imaging deep inside tissues.
Introduction
power relationship between light scattering and wavelength, long
wavelength probes permit deep tissue detection of optical
signals.
The ultimate goal in the field of biomedical imaging is to
visualize cellular structures and functions with submicrometer
spatial and submillisecond temporal resolutions. Fluorescence
has been playing a leading role in this direction due to its high
sensitivity and the ability to target specific molecules or cellular
2,3
The past decade has witnessed significant progress in long-
4
-7
wavelength bioprobes, from conventional organic dyes
to
8,9
10,11
fluorescent proteins,
quantum dots,
and lanthanide
1
compartments. Imaging cells and biological tissues can often
(
(
(
2) Weissleder, R. Nat. Biotechnol. 2001, 19, 316.
be complicated by the autofluorescence of natural chromophores
such as NADH. Scattering and absorption are two other major
interfering factors, especially in thick tissues. Probes, emitting
in the so-called medical spectral window (650-900 nm), can
potentially minimize these interferences and offer high signal/
background ratios. Furthermore, because of the inverse fourth
3) Frangioni, J. V. Curr. Opin. Chem. Biol. 2003, 7, 626.
4) Wuskell, J. P.; Boudreau, D.; Wei, M. D.; Jin, L.; Engl, R.; Chebolu, R.;
Bullen, A.; Hoffacker, K. D.; Kerimo, J.; Cohen, L. B.; Zochowski, M. R.; Loew,
L. M. J. Neurosci. Methods 2006, 151, 200.
(
5) Mujumdar, S. R.; Mujumdar, R. B.; Grant, C. M.; Waggoner, A. S.
Bioconj. Chem. 1996, 7, 356.
6) Sasaki, E.; Kojima, H.; Nishimatsu, H.; Urano, Y.; Kikuchi, K.; Hirata,
Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 3684.
7) Umezawa, K.; Nakamura, Y.; Makino, H.; Citterio, D.; Suzuki, K. J. Am.
(
(
Chem. Soc. 2008, 130, 1550.
(
1) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science
(8) Matz, M. V.; Lukyanov, K. A.; Lukyanov, S. A. BioEssays 2002, 24,
2
006, 312, 217.
953.
1
0.1021/jo800852h CCC: $40.75  2008 American Chemical Society
J. Org. Chem. 2008, 73, 6587–6594 6587
Published on Web 07/30/2008

Yan et al.
CHART 1. Structures of Indocyanine Green (ICG), Di-4-ANEPPS, and Amino(oligo)thiophene-Based Hemicyanines (1-4)
1
2,13
SCHEME 1. A General Route to
Amino(oligo)thiophene-Based Hemicyanines
complexes.
Cyanine dyes have been dominating near-IR
imaging applications and some of them even have been
approved by the FDA (e.g., indocyanine green, Chart 1). The
absorption peak of a symmetric cyanine red-shifts ∼100 nm
for every vinylene extension and can be calculated by using a
1
4
classical free electron gas model.
Hemicyanine chromophores (e.g., di-4-ANEPPS, Chart 1)
have one quaternary nitrogen within a heterocycle and one
acyclic tertiary nitrogen end group; they generally absorb at
much shorter wavelengths than the corresponding cyanines,
which typically have a pair of symmetrically arrayed hetero-
cyclic end groups. Unlike symmetric cyanines, hemicyanines
have distinct donor and acceptor groups so that there is
significant electron shift from donor to acceptor ends of the
chromophore upon photoexcitation; thus, they encompass an
important class of push-pull chromophores. Our laboratory has
been exploring the chemistry and photophysics of these hemi-
4,20
from problems of product stability and synthetic difficulty.
An alternative strategy is to use thienylene, which represents a
good balance between stability and wavelength-shifting ability
1
5-20
cyanines (also called amino-styryl dyes) for many years.
Their optical properties lead to many useful traits such as
24,25
in comparison with phenylene and vinylene.
Indeed, thie-
nylene has proven effective in red-shifting voltage-sensitive dyes
1
6,21
environmentally sensitive spectra,
signals,
good nonlinear optical
4,26
(
VSD) while preserving voltage sensitivities.
In this paper,
22,23
16,20
and voltage-sensitive fluorescence.
These prop-
we describe a new group of hemicyanines featuring the
amino(oligo)thiophene-pyridinium structure (Chart 1), includ-
ing detailed syntheses, systematic structure-property relation-
ships, environment sensitivities, and general applicability as long
wavelength fluorescent sensors of cell membrane physiology.
erties, largely absent in symmetrical cyanines, make the
hemicyanine chromophores ideally suited for incorporation into
biosensor probes. The development of longer wavelength
hemicyanines with compact structures can therefore have
significant impact in the field of biophotonics and live cell
imaging.
Results and Discussion
Conventional vinylene extension met with only limited
success in making long-wavelength hemicyanines and suffered
Synthesis. Unlike aniline, amino(oligo)thiophenes are ex-
tremely sensitive to air and mostly known in disubstituted forms.
More often than not, there are electron-withdrawing groups
attached to help stabilize them. In our series of dyes, the
N-alkylpyridinium moiety serves this purpose and concomitantly
supplies a π-electron acceptor to complete the push-pull
chromophore. Shown in Scheme 1 is a general route for the
synthesis of amino(oligo)thiophene-based hemicyanines from
commercially available bromo(oligo)thiophene carboxaldehydes.
The first step is an aromatic nucleophilic substitution (SNAr)
reaction with a secondary amine, bypassing unstable primary
amino(oligo)thiophenes. For n ) 1 (see Scheme 1), direct
(
9) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. ReV. Mol.
Cell Biol. 2002, 3, 906.
10) Lim, Y. T.; Kim, S.; Nakayama, A.; Stott, N. E.; Bawendi, M. G.;
Frangioni, J. V. Mol. Imaging 2003, 2, 50.
11) Raschke, G.; Brogl, S.; Susha, A. S.; Rogach, A. L.; Klar, T. A.;
(
(
Feldmann, J.; Fieres, B.; Petkov, N.; Bein, T.; Nichtl, A.; Kurzinger, K. Nano
Lett. 2004, 4, 1853.
(
12) Halim, M.; Tremblay, M. S.; Jockusch, S.; Turro, N. J.; Sames, D. J. Am.
Chem. Soc. 2007, 129, 7704.
13) Zhang, J.; Badger, P. D.; Geib, S. J.; Petoud, S. Angew. Chem., Int. Ed.
(
Engl. 2005, 44, 2508.
(
(
(
14) Kuhn, H. J. Chem. Phys. 1949, 17, 1198.
15) Loew, L. M.; Bonneville, G. W.; Surow, J. Biochemistry 1978, 17, 4065.
16) Loew, L. M.; Scully, S.; Simpson, L.; Waggoner, A. S. Nature 1979,
2
7
2
81, 497.
17) Loew, L. M.; Simpson, L.; Hassner, A.; Alexanian, A. J. Am. Chem.
amination in water proceeds well and is preferred for its
(
28,29
simplicity; for higher homologues, CuI-catalyzed amination
Soc. 1979, 101, 5439.
(
(
(
(
18) Loew, L. M.; Simpson, L. L. Biophys. J. 1981, 34, 353.
19) Hassner, A.; Birnbaum, D.; Loew, L. M. J. Org. Chem. 1984, 49, 2546.
20) Fluhler, E.; Burnham, V. G.; Loew, L. M. Biochemistry 1985, 24, 5749.
21) Jin, L.; Millard, A. C.; Wuskell, J. P.; Dong, X. M.; Wu, D. Q.; Clark,
(24) Roncali, J. Chem. ReV. 1997, 97, 173.
(25) Effenberger, F.; W u¨ rthner, F.; Steybe, F. J. Org. Chem. 1995, 60, 2082.
(26) Salama, G.; Choi, B. R.; Azour, G.; Lavasani, M.; Tumbev, V.; Salzberg,
B. M.; Patrick, M. J.; Ernst, L. A.; Waggoner, A. S. J. Membr. Biol. 2005, 208,
125.
H. A.; Loew, L. M. Biophys. J. 2006, 90, 2563.
(
22) Huang, Y. A.; Lewis, A.; Loew, L. M. Biophys. J. 1988, 53, 665.
(23) Marder, S. R.; Cheng, L. T.; Tiemann, B. G.; Friedli, A. C.; Blanchard-
(27) Prim, D.; Kirsch, G.; Nicoud, J. F. Synlett 1998, 383.
(28) Lu, Z.; Twieg, R. J. Tetrahedron 2005, 61, 903.
desce, M.; Perry, J. W.; Skindhoj, J. Science 1994, 263, 511.
6
588 J. Org. Chem. Vol. 73, No. 17, 2008

Amino(oligo)thiophene Optical Biomembrane Probes
SCHEME 2. Synthesis of a Bridged Hemicyanine
is necessary to obtain the desired products. Here the aldehyde
group not only facilitates the amination reaction but also
stabilizes the product, which is the required intermediate for
the subsequent aldol condensation reactions with appropriate
salts, i.e., 4-methylpyridinium, 4-methylquinolinium, or 10-
methylacridinium salts. The two-step yield critically depends
effective solvation in the vertically excited states due to the
migration of positive charge to the amino end of the chro-
17
mophore. The fluorescence quantum yield of 1d is 0.10 when
bound to lipid vesicles, but it is less than 0.001 in ethanol and
PBS buffer. Such dramatic enhancement of fluorescence upon
binding has long been known for traditional hemicyanine dyes
and may be attributed to the shielding of excited states from
1
on the bulkiness of substituent R , ranging from ∼70% for
2
0
methyl to ∼10% for n-butyl.
polar-molecule-induced nonradiative pathways and slowing
4
0
It should be noted that (oligo)thiophene derivatives like 6
have been of considerable interest as donor moieties in organic
down of torsional relaxation pathways. This unique feature
of hemicyanine dyes offers the advantage of low background
signals even in situations where the removal of excess dyes is
too challenging, e.g., intracellular membrane studies. Further-
more, for most of the dyes, there is a large Stokes shift (∆λ ≈
140 nm for 1d) that allows convenient filtering of the excitation
and emission in fluorescence microscopic studies.
3
0
31
nonlinear optical materials and photorefractive materials.
However, there are only a few reports on the synthesis of higher
2
5,32-35
analogues (n ) 2, 3)
and all of them require 4-6 steps
from commercially available starting materials. Our one-step
route to these critical intermediates will greatly accelerate the
discovery of new applications of amino(oligo)thiophene deriva-
tives. Here the yields of Cu-catalyzed amination reaction, except
for a dimethylamine case, are still low due to a competing
debromination process. Development of more selective amina-
tion catalysts is highly desirable.
B. Effect of Conjugation Length. We assessed how the
absorption and emission spectra changed as a function of the
number of thienylene units. For comparison, the absorption and
emission spectra, measured in vesicle suspensions, of dyes 1a
(n ) 1), 1d (n ) 2), and 1i (n ) 3) are shown together in Figure
2. Although it is known that vinylene extension does not induce
A bridged version, 4, has also been prepared for comparison
2
0
(
3
see Scheme 2). Bridged bithiophene 8 was prepared from
large bathchromic shift in absorption of hemicyanines, it is
still surprising to find that thienylene extension actually produces
small hypsochromic shift when the dyes are bound to lipid
membranes (see Figure 2). However, when measured in ethanol
the absorption peaks of 1d (λmax ) 614 nm) and 1i (λmax )
588 nm) are indeed red-shifted relative to 1a (λmax ) 569 nm)
(Table 1). This trend can be understood by the larger charge
separation in the vertical excited state of the more conjugated
members of this family, causing them to display greater
solvatochromism. On the other hand, when their fluorescence
properties are compared, the longer the conjugation length, the
longer the emission wavelength even for the lipid vesicle-bound
3
6-39
-bromothiophene according to literature methods.
Sub-
sequent methylation, formylation, and bromination afforded the
properly functionalized 11 that can be used following the general
route in Scheme 1.
Absorption and Fluorescence Properties. A. General
Properties and Effect of Solvent Environment. Hemicyanines
20
containing aniline donor moieties, instead of aminothiophene,
have absorbance maxima in a range of 485-510 nm. The new
amino(oligo)thiophene-based hemicyanines have absorbance
maxima in ethanol in the 600 nm region and their solutions in
ethanol are blue or green. As a typical example, absorption and
emission spectra of 1d are shown in Figure 1. The absorption
spectra in lipid vesicles and PBS buffer are blue-shifted by about
7
0 nm from that in ethanol; this can be explained by solvent
stabilization of the positive charge in the ground state, but less
(
(
29) Zhang, H.; Cai, Q.; Ma, D. W. J. Org. Chem. 2005, 70, 5164.
30) Breitung, E. M.; Shu, C.-F.; McMahon, R. J. J. Am. Chem. Soc. 2000,
1
22, 1154.
(
31) W u¨ rthner, F.; Yao, S.; Schilling, J.; Wortmann, R.; Redi-Abshiro, M.;
Mecher, E.; Callego-Gomez, F.; Meerholz, K. J. Am. Chem. Soc. 2001, 123,
2
810.
(
32) Raposo, M. M. M.; Fonseca, A. M. C.; Kirsch, G. Tetrahedron 2004,
6
0, 4071.
(
(
33) Raposo, M. M. M.; Kirsch, G. Tetrahedron 2003, 59, 4891.
34) Ikemoto, N.; Estevez, I.; Nakanishi, K.; Berova, N. Heterocycles 1997,
4
6
2
6, 489.
(
35) Bedworth, P. V.; Cai, Y.; Jen, A.; Marder, S. R. J. Org. Chem. 1996,
1, 2242.
(
36) Kraak, A.; Wiersema, A. K.; Jordens, P.; Wynberg, H. Tetrahedron 1968,
4, 3381.
(
37) Beyer, R.; Kalaji, M.; Kingscote-Burton, G.; Murphy, P. J.; Pereira,
V.; Taylor, D. M.; Williams, G. O. Synth. Met. 1998, 92, 25.
38) Nenajdenko, V. G.; Baraznenok, I. L.; Balenkova, E. S. J. Org. Chem.
998, 63, 6132.
39) Amer, A.; Burkhardt, A.; Nkansah, A.; Shabana, R.; Galal, A.; Mark,
H. B.; Zimmer, H. Phosphorus, Sulfur, Silicon 1989, 42, 63.
FIGURE 1. Absorption and emission spectra of 1d in different solvent
environments. The emission curves in ethanol and PBS buffer have
been scaled 10 and 100 times, respectively. Here lipid vesicles prepared
from soybean phosphatidylcholine are used to represent the environment
of cell membranes. Excitation wavelength: 480 nm.
(
1
(
J. Org. Chem. Vol. 73, No. 17, 2008 6589

Yan et al.
2
observed for the R series (1d, 1g, and 1h, see Table 1) since
these alkyl chains are quite remote from the chromophore.
Although the effects on spectra are not substantial, side chains
do play critical roles in optimizing solubility, membrane binding
kinetics and avidity, and even voltage sensitivities (vide infra).
Applicability Assessment A: Sensitivity to Transmem-
brane Potential. Historically hemicyanines are best known for
their voltage sensitivities. A typical hemicyanine dye has a
hydrophobic tail and a hydrophilic head that promote binding
and alignment within cell membranes. Since optical excitation
of a hemicyanine dye involves charge redistribution, the
transmembrane potential has a measurable effect on the excita-
tion energy, and indeed voltage sensitivities can be conveniently
1
8
measured by using a hemispherical lipid bilayer apparatus.
The voltage sensitivities of these dyes, expressed in fluorescence
change per 100 mV, are included in Table 1. A few points are
clear from a glance at the table: first, the average sensitivity of
monothiophene series (1a,b), 1.5%, is much lower than that of
the bithiophene series (1c-h), 10%; second, the side chains
have significant effects on the sensitivity, i.e., 1d and 1e differ
only in side chains but have very different sensitivities.
Extensive optimization of side chains results in 1e with a
sensitivity of 18%, which is among the highest reported for fast
FIGURE 2. Normalized absorption and emission spectra of a series
of dyes with different numbers of thienylene units, measured in vesicle
suspensions containing 1 mg/mL soybean phosphatidylcholine in PBS
buffer.
dyes (see Figure 2 and Table 1). The fluorescence quantum
yields of 1a and 1d are around 10%, but that of 1i is only 2%,
discouraging further work on higher homologues. This low
fluorescence quantum yield is most likely caused by torsional
26,42
40
voltage sensitive dyes.
On the other hand, no attempts have
relaxation by rotation around intervening single bonds, which
are most abundant in 1i.
been made to optimize other series (1i, 2, 3, and 4) due to their
relatively low fluorescence quantum yields (e2%).
C. Effect of Acceptor. Using a larger heterocyclic acceptor
has proven effective in red-shifting the optical spectra of a
Applicability Assessment B: Sensitivity to Lipid Composi-
tions. Even in the absence of proteins, the cell membrane is a
highly heterogeneous 2-dimensional liquid phase. “Rafts”,
domains enriched in cholesterol and saturated lipids, floating
in a fluidic lipid bilayer have been proposed as a feature of cell
4
hemicyanine dye. Shown in Figure 3 are the absorption and
emission spectra of a series of dyes with different acceptors:
pyridinium for 1d, quinolinium for 2, and acridinium for 3. For
each additional fused benzo ring, there is an ∼75 nm red-shift
in both absorption and emission spectra, but there is also a
successive drop in fluorescence quantum yield, with acridinium
salt 3 being not fluorescent at all. The mechanism might be
due to steric hindrance induced by the fused benzo rings, which
effectively prevents the chromophore from attaining a fluores-
cent planar conformation.
4
3
membrane structure. Rafts play important roles in cellular
functions such as signal transduction and membrane traffick-
44
ing. Fluorescence imaging of domains in model biomembranes
can be realized by using two dyes having complementary
4
5
staining preferences or one dye with domain-sensitive fluo-
rescence characteristics (polarization, wavelength, lifetime,
2
1,46-49
etc.).
The large Stoke shifts and solvatochromic effects
D. Effect of Rigidification. Incorporating methine groups
in a ring has been very successful in red-shifting the absorption
of these new hemicyanines prompted us to assess the sensitivity
to membrane composition. Model membranes for liquid disor-
dered phase and liquid ordered phase were prepared from 1,2-
dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 7:3 1,2-
dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol,
respectively. Figure 4 shows the emission spectra of two
representative hemicyanines, 1b and 1d, in two types of vesicle
suspensions. 1d shows increased and blue-shifted (30 nm)
fluorescence in 7:3 DPPC/cholesterol vesicles relative to DOPC
vesicles; in contrast, 1b shows decreased and red-shifted (20
nm) fluorescence in 7:3 DPPC/cholesterol vesicles. All mono-
thiophene and bithiophene derivatives tested show similarly
contrasting lipid composition dependent emission spectra.
41
peaks and improving the photostabilities of polymethine dyes.
To test the effect of rigidification in our amino(oligo)thiophene-
based dyes we synthesized a CMe2-bridged dye, 4 (see Scheme
2
). Compared with the nonbridged 1d, 4 shows a large red-
shift (∆λ ) 65 nm) in the absorption spectrum and a small red-
shift (∆λ ) 18 nm) in the fluorescence spectrum, both of which
were measured in vesicle suspensions.
E. Effect of Side Chains. Two series of dyes with varying
1
2
alkyl substitutes, R on the hydrophobic side and R on the
1
hydrophilic side, have been synthesized. The R series (1c-f,
see Table 1) show progressive red-shifts in both absorption and
emission spectra when the alkyl chain increases from methyl
to n-butyl. This trend may be understood in terms of the
HOMO-LUMO gap: for hemicyanines the HOMO resides
mostly on the amine side and the LUMO on the pyridinium
(42) Kuhn, B.; Fromherz, P. J. Phys. Chem. 2003, 107, 7903.
(
43) Simons, K.; Ikonen, E. Nature 1997, 387, 569.
(
44) Simons, K.; Toomre, D. Nat. ReV. Mol. Cell Biol. 2000, 1, 31.
15
side. The larger the size of the alkyl chain the more the amino
nitrogen is shielded from hydrogen-bonded solvation, destabiliz-
ing the ground state relative to the excited state, thus red-shifting
the absorption and emission maxima. It is not surprising that
only minimal variations in absorption and emission spectra are
(45) Baumgart, T.; Hess, S. T.; Webb, W. W. Nature 2003, 425, 821.
(
46) Parasassi, T.; Gratton, E.; Yu, W. M.; Wilson, P.; Levi, M. Biophys. J.
997, 72, 2413.
47) Owen, D. M.; Lanigan, P. M. P.; Dunsby, C.; Munro, I.; Grant, D.;
Neil, M. A. A.; French, P. M. W.; Magee, A. I. Biophys. J. 2006, 90, L80.
48) Margineanu, A.; Hotta, J. I.; Van der Auweraer, M.; Ameloot, M.; Stefan,
1
(
(
A.; Beljonne, D.; Engelborghs, Y.; Herrmann, A.; Muellen, K.; De Schryver,
F. C.; Hofkens, J. Biophys. J. 2007, 93, 2877.
(49) Kim, H. M.; Jeong, B. H.; Hyon, J.-Y.; An, M. J.; Seo, M. S.; Hong,
J. H.; Lee, K. J.; Kim, C. H.; Joo, T.; Hong, S.-C.; Cho, B. R. J. Am. Chem.
Soc. 2008, 130, 4246.
(
40) Silva, G. L.; Ediz, V.; Yaron, D.; Armitage, B. A. J. Am. Chem. Soc.
007, 129, 5710.
41) Reynolds, G. A.; Drexhage, K. H. J. Org. Chem. 1976, 42, 885.
2
(
6
590 J. Org. Chem. Vol. 73, No. 17, 2008

Amino(oligo)thiophene Optical Biomembrane Probes
TABLE 1. Spectroscopic Properties of Amino(oligo)thiophene-Based Hemicyanines
a
b
c
d
e
Ethanol. PBS buffer. Lipid vesicles. FQY < 0.001. Fluorescence changes for a 100 mV step in membrane potential were measured with
f
excitation through a monochromator (20 nm bandwidth) and emission through a long pass filter at the indicated wavelengths. Sensitivity cannot be
measured due to low fluorescence.
Although we cannot provide a detailed explanation for the
sensitivity of these dyes to membrane environment, it is
important to note that only the emission spectra are sensitive,
not the absorption spectra. Therefore, any explanation would
have to involve the detailed interactions of the relaxed excited
state structure with its lipid environment. These lipid composi-
tions do produce very different intramembrane electric potential;
in particular, cholesterol imparts a very strong dipole electric
5
0,51
field at the membrane surface.
Furthermore, cholesterol is
(50) Starke-Peterkovic, T.; Turner, N.; Vitha, M. F.; Waller, M. P.; Hibbs,
D. E.; Clarke, R. J. Biophys. J. 2006, 90, 4060.
J. Org. Chem. Vol. 73, No. 17, 2008 6591

Yan et al.
(
(
532 nm) as the most common product utilizing DPPS laser
1064 nm) through an SHG process. To our delight, most of
the dyes (1a-i) have a significant absorbance at 532 nm, making
them perfect for 2PF and SHG excited with mode locked 1064
nm lasers. Although the SHG process does not involve absorp-
tion of photons, it is resonance enhanced when the frequency-
doubled light is close to the absorption peak. The bithiophene
series (1c-h) shows strong nonlinear optical properties and
high-quality 2PF and SHG images are readily obtained. Panels
A and B of Figure 5 show SHG and 2PF images of the same
neuroblastoma cell before differentiation. Filopodia, filamentous
structures emanating from the round cell, can be seen clearly
in the 2PF image, but are absent in the SHG image. This is
because SHG requires a noncentrosymmetric distribution of
harmonophores on the scale of the optical coherence length,
and thus is unable to image highly symmetric or tiny structures
like filopodia. The images of differentiated neuroblastoma cells,
Figure 5C,D, illustrate another interesting phenomenon: internal
membranes such as the endoplasmic reticulum are not imaged
in SHG (Figure 5C) even when the dye has apparently been
internalized to an extent that the 2PF contrast pattern is
dominated by interior fluorescence (Figure 5D). This is par-
ticularly apparent in the color overlay image of Figure 5E, where
many of the neurites display red interiors from the 2PF channel
and green peripheries from the SHG channel. This is because
the highly convoluted fine structure of the endoplasmic reticulum
has the effect of randomly orienting the dye molecules on the
spatial scale of the optical coherence length. The use of chiral
dyes has been shown to relax the requirement of structural
FIGURE 3. Normalized absorption and emission spectra of a series
of dyes with different acceptors, measured in vesicle suspensions
containing 1 mg/mL soybean phosphatidylcholine in PBS buffer.
5
7
asymmetry.
On the other hand, the monothiophene dyes (1a,b) generally
show signals that are more than an order of magnitude lower.
An example of weak 2PF and SHG images of neuroblastoma
cells stained with 1b is provided in Figure S1 in the Supporting
Information; these images were obtained with the same settings
on our nonlinear microscopy apparatus as the images in Figure
5. The inferior nonlinear optical properties of the monothiophene
series may be attributed to their different donor-acceptor
distances.
FIGURE 4. Emission spectra of 1b and 1d in vesicle suspensions
prepared from DOPC or 7:3 DPPC/cholesterol in PBS buffer. Lipid
concentration ) 0.017 mg/mL.
known to produce a liquid ordered phase that is more rigid than
4
3
the liquid disordered phase associated with DOPC. The
relatively large shifts of fluorescence peak, together with long
emission wavelengths, make them very promising probes for
the detection of lipid variation in cell membranes.
Applicability Assessment C: Nonlinear Optical Imaging.
5
2-55
Two-photon fluorescence (2PF)
and second harmonic
Conclusions
56,57
generation (SHG)
are two useful nonlinear optical phenom-
A series of hemicyanines based on amino(oligo)thiophene
donors have been developed. Owing to thiophene’s balanced
stability and wavelength-shifting ability, these hemicyanines
show fluorescence in the red and near-infrared region but are
still compact and readily synthesized. Every element of a
hemicyanine structuresdonor, acceptor, bridge, and even side
chainsshas been varied to study the structure-property rela-
tionship and to identify the most promising probes. Some of
these dyes have shown superior sensitivities to trans-membrane
potentials and membrane lipid compositions. In addition they
have demonstrated strong nonlinear optical properties and, due
to their absorption peaks around 532 nm, an inexpensive and
compact diode-pumped solid-state fiber laser (1064 nm) can be
employed in two photon fluorescence and second harmonic
generation imaging. The relatively compact structure, facile
synthesis, high environment sensitivity, long wavelength fluo-
rescence, and strong nonlinear optical properties make them
promising probes for a wide range of biological applications.
ena for biomedical imaging. Since such phenomena occur only
at the diffraction-limited focal point, nonlinear optical micros-
copy has intrinsic 3D sectioning capability and minimal out-
of-focus photobleaching. Furthermore, in nonlinear microscopy,
near-IR lasers can be used even for fluorophores that are
traditionally used with UV/vis sources. Recent advances in
diode-pumped solid state (DPSS) lasers have made near-IR
lasers extremely compact and affordable, with green laser pointer
(
(
(
51) Latorre, R.; Hall, J. E. Nature 1976, 264, 361.
52) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73.
53) Kim, H. M.; Jung, C.; Kim, B. R.; Jung, S.-Y.; Hong, J. H.; Ko, Y.-G.;
Lee, K. J.; Cho, B. R. Angew. Chem., Int. Ed. Engl. 2007, 46, 3460.
54) Kim, H. M.; Kim, B. R.; Hong, J. H.; Park, J.-S.; Lee, K. J.; Cho, B. R.
Angew. Chem., Int. Ed. Engl. 2007, 46, 7445.
55) 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., Int. Ed. Engl. 2008,
(
(
4
7, 2231.
(56) Campagnola, P. J.; Loew, L. M. Nat. Biotechnol. 2003, 21, 1356.
57) Yan, P.; Millard, A. C.; Wei, M.; Loew, L. M. J. Am. Chem. Soc. 2006,
(
1
28, 11030.
6
592 J. Org. Chem. Vol. 73, No. 17, 2008

Amino(oligo)thiophene Optical Biomembrane Probes
FIGURE 5. (A) SHG and (B) 2PF images of the same undifferentiated neuroblastoma cell; (C) SHG, (D) 2PF, and (E) merged images of differentiated
neuroblastoma cells. The samples were stained with 5 µM 1d and excited at 1064 nm. A bandpass filter (615-665 nm) was used in 2PF imaging.
SHG was collected in the transmitted light path at 532 nm.
Experimental Procedures
8 H), 4.73 (t, J ) 7.8 Hz, 2 H), 7.99 (d, J ) 6.4 Hz, 2 H), 8.98 (d,
13
J ) 6.4 Hz, 2 H); C NMR (100 MHz, CD
4.4, 54.5, 58.3, 130.1, 145.2, 161.9; HRMS (FAB+) m/z 315.1428
: 315.1430).
3
OD) δ 8.0, 22.1, 25.2,
General. 4H-Cyclopenta[2,1-b:3,4-b′]dithiophene (8) was syn-
5
[
3
6-39
thesized according to the literature methods.
-Diethylaminothiophene-2-carboxaldehyde (6a). 5-Bro-
mothiophene-2-carboxaldehyde (380 mg, 2.0 mmol), diethylamine
440 mg, 6.0 mmol), and 10 mL of H O were stirred at 100 °C in
a pressure vessel for 41 h. After cooling, the organic compounds
were separated by extraction with CH Cl and purified by column
chromatography (SiO , solvent gradient: CH Cl to 1:1 CH Cl
EtOAc) to furnish 192 mg of product (52%). R
M - Br]+ (calcd for C15
H28BrN
2
5
Hemicyanine 1a. 5-Diethylaminothiophene-2-carboxaldehyde
(
18 mg, 0.10 mmol), 1-(3-triethylammoniopropyl)-4-methylpyri-
(
2
dinium dibromide (40 mg, 0.1 mmol), 0.1 mL of pyrrolidine, and
5
1
mL of ethanol were stirred at 100 °C in a pressure vessel for
6 h. The solution turned red during the reaction. After cooling,
2
2
2
2
2
2
2
/
the solvent was evaporated under vacuum and the residue was
purified by column chromatography (SiO -amino, 2:98 MeOH/
CH Cl to elute impurities; 1:4 MeOH/CH Cl to elute a purple
product) to give 1a as a purple solid (47.0 mg, 84%). R (silica gel,
O/AcOH) 0.30; H NMR (400
OD) δ 1.28 (t, J ) 7.2 Hz, 6 H), 1.33 (t, J ) 7.2 Hz, 9
H), 2.40 (m, 2 H), 3.34-3.43 (m, 8 H), 3.52 (q, J ) 7.2 Hz, 4 H),
.44 (t, 2 H), 6.11 (d, J ) 4.4 Hz, 1 H), 6.36 (d, J ) 15.0 Hz, 1
H), 7.33 (d, J ) 4.4 Hz, 1 H), 7.70 (d, J ) 7.2 Hz, 2 H), 7.98 (d,
f
(silica gel, 1:1
) δ 1.26 (t, J )
2
1
CH
2
Cl
2
/EtOAc) 0.63; H NMR (400 MHz, CDCl
3
2
2
2
2
7
7
.2 Hz, 6 H), 3.42 (q, J ) 7.2 Hz, 4 H), 5.92 (d, J ) 4.4 Hz, 1 H),
f
13
.46 (d, J ) 4.4 Hz, 1 H), 9.48 (s, 1 H); C NMR (100 MHz,
) δ 12.1, 47.6, 102.7, 125.3, 140.8, 166.4, 179.6; MS (EI)
1
3 2
24:4:16:6:6 CHCl /i-PrOH/MeOH/H
CDCl
m/z 183 [M] .
3
MHz, CD
3
+
5′-Dimethylamino-2,2′-bithiophene-5-carboxaldehyde (6c). 5′-
4
Bromo-2,2′-bithiophene-5-carboxaldehyde (100 mg, 0.37 mmol),
dimethylamine (40% solution in H
mg, 0.073 mmol), Cu (4.7 mg, 0.074 mmol), K
mg, 0.73 mmol), and 1 mL of N,N-dimethylethanolamine were
stirred at 80 °C in a pressure vessel for 87 h. The solution turned
red during the reaction. After cooling, the reaction mixture was
filtered through a short column of silica gel and eluted with more
EtOAc. The eluent was concentrated under vacuum and purified
by column chromatography (SiO
red solid (86%). The product shows strong green fluorescence when
2
O, 1 g, 8.9 mmol), CuI (13.9
PO ·H O (155.4
13
J ) 15.0 Hz, 1 H), 8.43 (br, 2 H); C NMR (100 MHz, CD
δ 7.9, 12.6, 24.9, 54.4, 54.5, 56.5, 105.5, 112.6, 121.8, 124.9, 138.4,
40.7, 143.3, 156.1, 165.6; HRMS (FAB+) m/z 480.2054 [M -
3
OD)
3
4
2
1
+
3
Br] (calcd for C24H39BrN S: 480.2048).
Imaging System. For full details of our nonlinear imaging
microscope, we refer the reader to previously published descrip-
58
tions. Our microscope combines a Fluoview (Olympus) scan-
2 2 2
, CH Cl ) to furnish 74.8 mg of
head and an Axiovert (Zeiss) inverted microscope. For excitation
we use a Femtopower (Fianium) fiber laser operating at 1064 nm
and an IR-Achroplan (Zeiss) 40×, 0.8 NA water-immersion
objective. 2PF is collected back through the objective, passed
through a 50-nm wide band-pass filter centered at 640 nm (Chroma),
and detected by a Hamamatsu R3896 photomultiplier tube with an
original Fluoview I-to-V converter. SHG is collected in the forward
direction by a 0.55 NA (Zeiss) condenser, filtered through a 20-
nm wide band-pass filter centered at 532 nm (Newport), and
detected by a Hamamatsu H7421-40 photon-counting head.
Lipid Vesicle Preparations. 1,2-Dioleoyl-sn-glycero-3-phos-
phocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
1
dissolved in CH
2 2 f 2 2
Cl . R (silica gel, CH Cl ) 0.36; H NMR (400
MHz, CDCl ) δ 3.00 (s, 6 H), 5.81 (d, J ) 4.0 Hz, 1 H), 6.95 (d,
3
J ) 4.0 Hz, 1 H), 7.13 (d, J ) 4.0 Hz, 1 H), 7.57 (d, J ) 4.0 Hz,
1
3
1
1
H), 9.75 (s, 1 H); C NMR (100 MHz, CDCl
3
) δ 42.6, 102.9,
19.5, 120.7, 127.8, 138.2, 138.6, 149.8, 161.3, 182.0; MS (EI)
m/z 237 [M] .
-(3-Triethylammoniopropyl)-4-methylpyridinium Dibromide
+
1
(
7b). 4-Methylpyridine (307 mg, 3.3 mmol), (3-bromopropyl)tri-
ethylammonium bromide (1.0 g, 3.3 mmol), and 4 mL of DMF
were stirred at 100 °C in a pressure vessel for 41 h. Precipitates
formed during the reaction. After cooling, the precipitates were
(
DPPC), and cholesterol were obtained from Avanti Polar Lipids;
filtered out and washed with CH
2 2
Cl to give 7b as a light pink
soybean phosphatidylcholine was obtained from Sigma Chemical
solid (750 mg, 57%), which was used in the next aldol condensation
1
without further purification. H NMR (400 MHz, CD
3
OD) δ 1.34
(58) Millard, A. C.; Jin, L.; Wei, M.; Wuskell, J. P.; Lewis, A.; Loew, L. M.
(
t, J ) 7.0 Hz, 9 H), 2.50 (m, 2 H), 2.70 (s, 3 H), 3.35-3.47 (m,
Biophys. J. 2004, 86, 1169.
J. Org. Chem. Vol. 73, No. 17, 2008 6593

Yan et al.
Company. At first lipids were dissolved in chloroform, and then
the solvent was removed with an argon stream to yield a lipid film.
Phosphate buffered saline (PBS) was added to hydrate the film and
the mixture was sonicated to clarity with a Branson W-185 probe-
type sonicator.
Cell Preparations and Staining Protocol. N1E-115 mouse
neuroblastoma cells were cultured by using Dulbecco’s modified
Eagle’s medium with 10% fetal bovine serum and 1% antibiotic-
Acknowledgment. This study was supported by the National
Institutes of Health via grant Nos. EB001963 and U54RR022232.
Supporting Information Available: Synthesis and charac-
terization of 6b, 6d, 6e, 6f, 6g, 7a, 7c, 7d, 7e, 9, 10, 11, 12, 1b,
1
c, 1d, 1e, 1f, 1g, 1h, 1i, 2, 3, and 4; SHG and 2PF images of
1
neuroblastoma cells with 1b as the staining reagents; H NMR
13
spectra of all new compounds; C NMR spectra of representa-
tive intermediates and products (6a, 6c, 7b, 1a, 1c, and 1d).
This material is available free of charge via the Internet at
http://pubs.acs.org.
2
antimycotic and maintained at 37 °C with 5% CO . Dye solutions
(
1-10 µM) were prepared by diluting stock solutions in ethanol
with Earle’s balanced salt solution, followed by vigorous sonication.
After removing the culture medium from a dish of cells, 3 mL of
dye solution was added and the cells were imaged at an excitation
wavelength of 1064 nm.
JO800852H
6
594 J. Org. Chem. Vol. 73, No. 17, 2008