Push-pull amino succinimidyl ester thiophene-based fluorescent dyes: Synthesis and optical characterization

Article abstract of DOI:10.1002/chem.201100142

The design and synthesis of new fluorescent dyes with emission range at 490-650 nm are described. Their structural and electronic properties have been characterized by both experimental techniques and quantum-chemical calculations. The chromophores are do

Full text of DOI:10.1002/chem.201100142

FULL PAPER
DOI: 10.1002/chem.201100142
Push–Pull Amino Succinimidyl Ester Thiophene-Based Fluorescent Dyes:
Synthesis and Optical Characterization
Giovanna Sotgiu,*[a] Matteo Galeotti,[b] Cristian Samorꢀ,[a] Alessandro Bongini,[c] and
Andrea Mazzanti[d]
Abstract: The design and synthesis of
new fluorescent dyes with emission
range at 490–650 nm are described.
Their structural and electronic proper-
ties have been characterized by both
experimental techniques and quantum-
chemical calculations. The chromo-
phores are donor–p-bridge–acceptor
push–pull compounds with a p bridge
of phenyl and thiophene rings and
their combination. Compared with pre-
vious thiophene fluorophores, these
dyes show significant redshift in the ab-
sorption and emission spectra and offer
compact, red-emitting fluorophores.
The dyes have amino succinimidyl
active ester and can be readily conju-
gated to proteins, polymers and other
amino-group-containing materials.
Keywords: dyes/pigments · fluores-
cent probes · oligothiophenes · sin-
glet oxygen · sulfine
Introduction
thiophenes as fluorescent dyes encouraged us to further in-
vestigate their suitability as fluorophores.[4]
The analysis of biomolecules and their interaction by means
of fluorescent labels is a common technique in todayꢀs bio-
logical and biochemical research because of its noninvasive
nature and its high intrinsic sensitivity.[1] Fluorescence
allows qualitative and quantitative determinations and can
be performed easily with rapid and inexpensive methodolo-
gy.
Many fluorescent tags are organic dyes that are also used
to trace the presence of biomolecules in cells and other bio-
logical systems.[2] Among the reactive dyes, amine-reactive
dyes are most often used to prepare various bioconjugates
for biological applications since amino groups are either
abundant or easily introduced into biomolecules.[2,3] Current
organic dyes suffer from many limitations including narrow
excitation spectra, broad emission spectra, and photobleach-
ing. On the other hand, our previous papers regarding oligo-
Oligothiophenes are an important class of p-conjugated
organic materials that have been investigated as active ma-
terials for optoelectronic devices; moreover, they have been
employed in photovoltaic solar cells, in light-emitting diodes
(LEDs), in photochromic switches, and as field-effect tran-
sistors (FETs).[5]
Oligothiophene compounds are chemically stable mole-
cules with a common fluorescent skeleton that can be op-
portunely functionalized.[6] They display intense absorption
bands, the positions of which can be tailored to fall any-
where from the UV to deep-red spectrum by carefully
choosing the number of aromatic rings and by introducing
suitable substituents at the a positions.[7]
We have recently reported the synthesis of fluorescent
thiophene derivatives constituted by an electron-donor (D)
and an electron-acceptor (A) group, which interact each
other through a p-conjugated spacer. These compounds are
characterized by an efficient emission in the blue-orange in-
terval and can be conveniently used to label monoclonal an-
tibodies, toxins, and other amino-group-containing materials.[8]
Donor–p-acceptor fluorescent dyes are characterized by
three key components (donor, acceptor, and connecting p
system). We report herein on the design, synthesis, and opti-
cal characteristics of new fluorescent oligothiophenes with a
modified donor group and p bridge.
[a] Dr. G. Sotgiu, Dr. C. Samorꢁ
Consiglio Nazionale Ricerche CNR-ISOF
Via Gobetti 101, 40129 Bologna (Italy)
Fax : (+39)051-6398349
E-mail: sotgiu@isof.cnr.it
[b] Dr. M. Galeotti
Mediteknology srl
Via Gobetti 101, 40129 Bologna (Italy)
[c] Prof. A. Bongini
Department of Chemistry ’G.Ciamician’
University of Bologna
Via F. Selmi 2, 40122 Bologna (Italy)
Results and Discussion
[d] Dr. A. Mazzanti
Design and synthesis of oligothiophenes: There is a growing
need for cellular imaging fluorescent probes that are able to
emit at longer wavelengths and minimize the effects of ab-
sorption, autofluorescence, and scattering from biological
tissue.[2,3]
Department of Organic Chemistry ’A.Mangini’
University of Bologna
Viale Risorgimento 4, 40136 Bologna (Italy)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201100142.
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It is commonly known that a
shift of the absorption band to
longer wavelength in a thio-
phene-based dye might be ob-
tained by increasing the elec-
tron-withdrawing power of the
chromophore, by increasing the
electron-releasing power of the
auxochromes, and by extending
the length of conjugation.[9] Fur-
thermore, the thiophene ring
not only increases the length of
conjugation between donor and
acceptor, but also acts as an
auxiliary electron donor in
push–pull structures.[10] The
photochemistry of the thio-
phene unit has been investigat-
ed in a number of thiophene-
linked donor–p-bridge–acceptor
push–pull chromophores for
their optoelectronic proper-
ties.[11–16]
Scheme 1. Synthesis of the electron-donor side of chromophores.
For this reason, to obtain flu- Scheme 2. Synthesis of the electron-acceptor side of chromophores.
orescent dyes with longer ab-
sorption and emission wavelengths, we have chosen 4-meth-
ylpiperidine as the donor group, phenyl and/or thienyl rings
have been selected for the design of the p-conjugated
spacer, and N-hydroxysuccinimide (NHS) was chosen to be
the acceptor group. Fluorescent dyes rapidly react with pri-
mary amines in slightly alkaline conditions (pH 7.2–8.5) to
yield stable amide bonds.[17] The labeling reaction complete-
ly releases N-hydroxysuccinimide within 30 min.
2-(tributylstannyl)thiophene and subsequently brominated
with NBS under dark conditions in a mixture of acetic acid
and dichloromethane, thereby affording 8 in 79% yield.
The donor and acceptor precursors were coupled using
the Stille palladium-catalyzed cross-coupling to provide the
desired chromophores in good yield (Scheme 3). All reac-
Fluorescent push–pull dyes were assembled in a conver-
gent manner as outlined below. Scheme 1 shows the routes
followed to prepare the donor sides of the chromophore, re-
spectively, N-piperidinyl phenyl 1 and 2-(N-piperidinyl)thio-
phene 4. They were synthesized from bromobenzene and 2-
iodothiophene by means of a copper-catalyzed amination of
the corresponding phenyl or thienyl halogen derivates and
by using N,N-dimethylaminoethanol (deanol) as solvent.[18]
The amination reaction conditions are different for bro-
mide and iodide substrates. For example, for bromobenzene,
a mixture of copper metal and CuI is used as the catalyst.
Instead, for 2-iodothiophene, only copper metal is em-
ployed. Compound 2 was obtained by regio-bromination re-
action of 4-methyl-1-phenylpiperidine in aqueous suspension
using cetyltrimethylammonium tribromide (CTAB3) as
transfer in 98% yield.[19] Stannanes 3 and 5 were obtained in
good yields and as pure compound by metalation with
nBuLi followed by transmetalation with tributyltin chloride.
The acceptor sides of the chromophores were prepared
starting from commercial 5-bromo-2-thiophenecarboxylic
acid and 4-bromobenzoic acid, respectively (Scheme 2).
Carboxyl groups were converted to N-succinimidyl ester
by reaction with NHS and dicyclohexylcarbodiimide (DCC)
in THF. Compound 6 was further treated with commercial
Scheme 3. Synthesis of chromophores 10–15.
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Fluorescent Dyes
FULL PAPER
tions were performed in toluene under nitrogen atmosphere
fects that different combinations of thienyl and phenyl units
have on the effective conjugation of our push–pull chromo-
phores, density functional theory (DFT) calculations were
performed on the model compounds a–d (see Table 2).
and catalyzed by [PdACTHNUTRGNEUNG
(AsPh3)4] generated in situ.[20] The
chemical structures of all the dyes synthesized for our pur-
pose are reported in Scheme 3.
Optical properties: The normal-
ized absorption and emission
spectra of the dyes 10–15 have
been recorded in CH2Cl2 and
are shown in Figure 1. These
spectra span the UV and visible
regions with absorption maxima
ranging from 359 to 460 nm,
and the spectral shifts are the
result of well-known variation
in the charge-transfer character
of the absorption determined
by the nature and the length of
the conjugated network.
Excitation of dichlorome-
thane solutions of dyes with a
standard laboratory UV lamp
(lex =365 nm; Table 1), allowed
us to visualize pale green (10),
deep-green (11), green (12),
yellow (13), orange (14), and
deep-red (15) emissions. The
bluest absorbing variants of the
dyes have a phenyl ring (10, 11,
and 14) attached to the electron
donor N-piperidine. By replac-
ing the phenyl ring with a thio-
phene (12, 13, and 15) the spec-
Figure 1. Normalized absorption and photoluminescence spectra of compounds 10–15 in CH2Cl2. A color ver-
sion of this figure is available in the Supporting Information.
Table 1. Maximum absorption and photoluminescence wavelengths of compounds 10–15 in CH2Cl2 and their
emission color shown under excitation with a 365 nm UV lamp.
Compound
lmax [nm]
lPL [nm]
Stokes shift [cmÀ1
]
e [mÀ1 cmÀ1
]
Emission color
10
359
493
7571
16066
tra are progressively shifted 11
toward red. Because thiophene
393
412
439
422
460
509
515
543
601
650
5799
4854
4363
7058
6497
18690
17194
17964
32628
16685
has
a
lower delocalization
energy than benzene, it produ-
ces better effective conjugation
in donor–acceptor compounds
relative to benzenoid moiet-
ies.[14,21]
12
13
14
15
The Stokes shift is an impor-
tant parameter for the fluores-
cent compounds. Since it pro-
vides information on the prop-
erties of fluorophores and their
structure either at the ground
and the first excited state. All
new compounds exhibit very
large Stokes shifts (difference
between the spectral positions
of the band maxima of the ab-
sorption and emission), from
4363 (13) to 7571 cmÀ1 (10).
Theoretical calculations: To
gain further insight into the ef-
Chem. Eur. J. 2011, 17, 7947 – 7952
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7949

G. Sotgiu et al.
Table 2. Selected geometrical parameters and physical properties obtained by the theoretical calculations.[a]
corresponds to the addition of
molecular oxygen (HRMS: m/
z: 518.0649; see the Supporting
Information). A sample of 15a
Model compound
(CH2)2N-Th-Th-CHO (a)
(CH2)2N-Th-Ph-CHO (b)
(CH2)2N-Ph-Th-CHO (c)
(CH2)2N-Ph-Ph-CHO (d)
w [8]
d [ꢃ]
DEH–L [eV]
m [Debye]
lmax [nm][b]
G
175
15
23
1.438
1.456
1.461
1.478
3.18
3.37
3.45
3.63
7.76
7.24
7.24
6.89
412
360
360
304
ACHTUNGTRENNUNG
E
was generated directly in
a
G
33
NMR spectroscopy tube by ir-
radiating the parent molecule
15 for 2 h with a laboratory UV
lamp (lex =365 nm) and was an-
[a] Inter-ring torsion w [8], inter-ring distance d [ꢃ], HOMO–LUMO energy gap DEH–L [eV], dipolar moment
m [Debye], absorption wavelength lmax [nm]. [b] ZINDO/S calculations on optimized DFT geometries.
As far as geometrical properties are concerned, it is
known that the planarity of the system and the length of the
bonds are a good indicator of the overall conjugation. In
Table 2, we note an increase of the inter-ring torsion (w)
and of the inter-ring distance (d) going from a to d, which
shows a decrease of the effective conjugation in the same di-
rection. It is interesting to compare these values with the
corresponding values for 2,2’-bithiophene (1608, 1.451 ꢃ), 2-
phenylthiophene (288, 1.469 ꢃ), and biphenyl (388, 1.486 ꢃ)
obtained by DFT calculations at the same level. Experimen-
tal inter-ring torsions in the gas phase for 2,2’-bithiophene
and biphenyl are 148 and 448, respectively.[22,23] It is evident
that the presence of the push–pull groups increases the con-
jugation in each compound, but this effect is more than
marked when more thienyl groups are present. The ob-
served increase of the HOMO–LUMO energy gap going
from a to d is in perfect agreement with a corresponding de-
crease of the conjugation. In fact, the delocalization energy
of thiophene is lower than that of benzene. This behavior is
suitably reflected by the values of the physical properties.
The color chemistry studies evidenced that the replacement
of benzene ring by a less aromatic heterocycle in the p
linker in a typical donor–acceptor chromogen determines a
significant bathochromic shift of the visible absorption
band.[24–26]
ZINDO/S-C.I. calculations show that in all the molecules
the UV absorption band is essentially due to a HOMO–
LUMO p transition with a charge-transfer (CT) character as
shown by the extension of the electronic densities of the
frontier orbitals over the entire molecule (Figure 2). It is
noteworthy that, in contrast to the geometrical properties,
the topology of the alternation of a thienyl with a phenyl
group does not seem to strongly affect the physical proper-
ties of the system. This signifies that is possible to substitute
a thienyl ring with a phenyl ring if necessary without a
strong loss of performance.
alyzed using NMR spectroscopic experiments.
Figure 2. a) ZINDO/S-calculated HOMO and LUMO frontier orbitals of
compound a. b) DFT-calculated HOMO and LUMO frontier orbitals of
compound a.
In addition to the signal of two thiophene rings, the
1H NMR spectrum shows two doublets at d=6.65 and
7.92 ppm with an unexpected constant coupling J=10.2 Hz
(see the Supporting Information), and a new quaternary
carbon at d=181.6 ppm is present in the 13C spectrum. To
better understand the structure of this byproduct, 2D NMR
spectroscopic experiments were acquired. The 1H–13C
HSQC spectrum (see the Supporting Information) showed
that the two hydrogen atoms at d=6.65 and 7.92 ppm are
bonded to carbon atoms at d=115.8 and 133.6 ppm, respec-
tively. These carbon shifts are typical of an ethylenic system,
which would be a cis-ethylene, because of the 10.5 Hz cou-
pling. The HSQC spectrum also exhibits four signals that
correspond to the CH2 carbons that belong to the methyl pi-
peridine ring, which are therefore diastereotopic. The
1H–13C heteronuclear multiple bond coherence (HMBC)
spectrum shows that the hydrogen at d=7.92 ppm is long
range coupled with the carbon at d=181.6 ppm, whereas
the hydrogen at d=6.65 ppm is coupled with the quaternary
carbon at d=164.5 ppm (see the Supporting Information).
1
Finally, an H–15N HMBC spectrum (see the Supporting In-
formation) showed that the chemical shift of the nitrogen of
piperidine is about d=126 ppm, typical of an amidic nitro-
gen.
All the above data can be rationalized if the sulfine de-
picted in Scheme 4 is considered, and several data support
Detailed discussion of the photochemical reaction of com-
pound 15: During optical measures we observed the decol-
oration of freshly prepared solution of 15 in dichlorome-
thane under constant irradiation with a UV lamp (lex =
365 nm) and the formation of a
byproduct (15a).
Structural identification of pho-
toproduct 15a: High-resolution
mass spectrometry indicated
that the mass of byproduct 15a Scheme 4. Photooxidation of compound 15.
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Fluorescent Dyes
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this hypothesis: a) the quaternary carbon of sulfine is
known[25] to resonate in the range 180–190 ppm; b) the
former piperidine nitrogen is now part of an amidic system,
and its chemical shift has moved to d=126 ppm; c) the
carbon atoms of the piperidine systems are diastereotopic
Labeling of dye 14 to polystyrene microspheres: We used for
our study nominally 6.31 mm amino-modified polystyrene
microspheres, with about 134 meq gÀ1 amino groups attached
to the surface of the particles by alkyl chains. The fluores-
cent dye 14 was covalently bound to microspheres in dry di-
methyl sulfoxide (DMSO). The tagged microspheres were
separated from unconjugated dye using microfiltration
through a 0.45 mm syringe filter and washed with DMSO
and Millipore water several times.
The particles were redispersed in PBS buffer and sonicat-
ed before examination with fluorescent microscope. As
shown in Figure 4, the labeled microspheres–14 have good
photostability under constant illumination with a fluorescent
microscope excitation source for 60 s, whereas under the
same conditions nearly complete degradation occurred for
fluorescein-labeled spheres after only 30 s of exposure.[8]
À
because of the hindered rotation about the N CO bond;
d) the resulting cis-ethylene system shows the typical J cou-
pling of about 10 Hz; e) the conjugation of the oligothio-
phene is lost and the absorption band has been shifted; and
f) the high-resolution mass corresponds to the proposed
structure (i.e., to a neat addition of molecular oxygen).
The photosensitizing properties of bithiophene and ter-
thiophene derivatives are known.[28] Moreover, oligothio-
phenes react with singlet oxygen to give sulfine derivatives
with extremely clean reactions.[29]
Labeling: Dyes 14 and 15 were
tested for protein and micro-
sphere labeling to investigate
their behavior after conjuga-
tion.
Conjugation of terthiophene 15
to saporin: The plant toxin sap-
orin was conjugated to dye 15
with the same procedure de-
scribed in our previous paper.[4c]
HeLa cells, derived from
a
human cervical carcinoma,
were cultured in Roswell Park
Memorial Institute (RPMI)
1640 medium supplemented
with 10% fetal calf serum
Figure 4. Fluorescence microscopy imaging of FITC and dye 14 conjugated with amino-modified polystyrene
(FCS) and antibiotics. HeLa microspheres under continuous light irradiation.
cells were seeded on four-cham-
bered culture slides (Falcon
BD) (5000 cells per well) in complete medium. After 24 h,
cells were incubated with 10 mm saporin–15 for 1 h at 378C.
Conclusion
After treatment, cells were washed twice with phosphate
buffer solution (PBS) and then fixed with cold methanol at
À208C for 15 min. Photoluminescence measurements were
performed by exciting the solutions at optimum wavelengths
for absorbances 0.1–0.2.
After endocytosis of saporin–15 by HeLa cells, no photo-
bleaching of dye was observed after continuous excitation
for 180 s (Figure 3).
We have reported the rational design and synthesis of novel
series of thiophene-based fluorescent push–pull dyes charac-
terized by absorption maxima in the range 359–460 nm and
photoluminescence maxima in the range 493–650 nm with
large Stokes shifts (4300–7500 cmÀ1), which suggests the pos-
sibility of using these labels in bioimaging applications. The
modification of p-conjugating units that link the electron-
donating and -accepting groups of chromophores is a tool
for the development of fluorescent dyes with longer emis-
sion wavelengths and a large Stokes shift, which are re-
quired for fluorophore candidates for biological applications.
The photosensitizing properties of compound 15, which
reacts with singlet oxygen to give sulfine, suggests the possi-
bility of using push–pull terthiophene derivatives in photo-
dynamic therapy.
Figure 3. Binding and endocytosis of 15-labeled saporin by HeLa cells.
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G. Sotgiu et al.
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Synthesis: The synthesis of all compounds 1–15, their characterizations,
and the materials for their preparation are described in detail in the Sup-
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The authors wish to thank L. Zuppiroli for the acquisition of the HRMS
spectrum of compound 15a. We wish to thank Mediteknology S.r.l, Area
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compounds, and Dr. Letizia Polito for experiments with HeLa cells and
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Received: January 14, 2011
Revised: March 30, 2011
Published online: May 26, 2011
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