Reversible synthesis and characterization of dynamic imino analogues of trimethine and pentamethine cyanine dyes

Article abstract of DOI:10.1021/ol802913b

A new family of unsymmetrical imine-based trimethine and pentamethine cyanine dye analogues is reported that can form under reversible and thermodynamically controlled conditions from non-or weakly emissive amine and aldehyde building blocks. These dynami

Full text of DOI:10.1021/ol802913b

ORGANIC
LETTERS
2009
Vol. 11, No. 5
1123-1126
Reversible Synthesis and
Characterization of Dynamic Imino
Analogues of Trimethine and
Pentamethine Cyanine Dyes
Kamel Meguellati, Martin Spichty, and Sylvain Ladame*
ISIS UniVersite´ Louis Pasteur 8, Alle´e Gaspard Monge,
BP 70028, 67083 Strasbourg, France
s.ladame@isis.u-strasbg.fr
Received December 19, 2008
ABSTRACT
A new family of unsymmetrical imine-based trimethine and pentamethine cyanine dye analogues is reported that can form under reversible
and thermodynamically controlled conditions from non- or weakly emissive amine and aldehyde building blocks. These dynamic fluorophores
show spectroscopic properties comparable to those of their parent cyanine dyes and are responsive to external effectors.
Cyanine dyes generically consist of a conjugated system
based on a polymethine chain linking two nitrogen-containing
heterocycles (e.g., indoles, benzothiazoles).1 They are gener-
ally named based on the number of carbon atoms in the
polymethine chain. Trimethine and pentamethine dyes exhibit
absorption maxima at 550 and 650 nm, respectively, and
emission maxima around 570 and 670 nm, respectively, in
the green or red part of the spectrum. Due to their relative
chemical stability, high molar absorption coefficient, and high
fluorescence quantum yield, cyanine dyes have been exten-
sively used in photography,2 optical data storage,3 and more
recently, proteomics4 and biomolecular labeling.5 Of par-
ticular interest are molecules that can reversibly switch
between a nonemissive state and an emissive state. Most
representative examples are fluorogenic unsymmetrical cya-
nine dyes which become fluorescent upon interaction with
(2) James, T. H. AdV. Photochem. 1986, 13, 329.
(3) (a) Heilemann, M.; Margeat, E.; Kasper, R.; Sauer, M.; Tinnefeld,
P. M. J. Am. Chem. Soc. 2005, 127, 3801. (b) Spitler, T.; Ehret, A.;
Kietzmann, R.; Willig, F. J. Phys. Chem. B 1997, 101, 2552.
(4) (a) Anaya, C.; Church, N.; Lewis, J. P. Proteomics 2007, 7, 215.
(b) Karp, N. A.; Lilley, K. S. Proteomics 2005, 5, 3105.
(5) (a) Fegan, A.; Shirude, P. S.; Balasubramanian, S. Chem. Commun.
2008, 17, 2004. (b) Toutchkine, A.; Nguyen, D. V.; Hahn, K. M.
Bioconjugate Chem. 2007, 18, 1344.
(1) Hamer, F. M. The Cyanine Dyes and Related Compounds; Weiss-
berger, A., Ed.; Interscience: London, U.K., 1964.
10.1021/ol802913b CCC: $40.75
Published on Web 02/04/2009
2009 American Chemical Society

specific nucleic acids6 or proteins7 and photochromic com-
pounds8 which can undergo a reversible change of their
optical properties under illumination. Such dynamic systems
can find valuable applications as fluorescent supramolecular
devices, in particular for the design of smart materials.9
Herein, we introduce a new family of dynamic fluorophore
analogues of the well-known cyanine dyes (from the trime-
thine and pentamethine family) that can be obtained via a
reversible and covalent reaction between non- and/or weakly
fluorescent benzothiazolium amine and indolenin aldehyde
building blocks (Scheme 1). These cyanine dye analogues
zole with methyl iodide. Aldehydes 2 and 310 were synthe-
sized by reaction of 1,2,3,3-tetramethylindolenin with (chlo-
romethylene)dimethyl ammonium chloride and malonaldehyde
bisphenylimine hydrochloride, respectively, followed by
alkaline hydrolysis of the activated hemicyanine intermediate.
In order to monitor the reaction between amine 1 and Fisher’s
base aldehyde 2, a 10 mM stoichiometric mixture of both
1
building blocks was heated (50 °C) in DMSO-d6, and H
NMR spectra were recorded every 24 h for 3 days. A new
set of signals slowly appeared at the expense of the signals
of monomers 1 and 2 along with a pale orange coloration of
the solution. Particularly striking was the appearance of two
doublets at 8.52 and 6.50 ppm that could be assigned to the
imino proton and the adjacent methine proton, respectively,
of the predicted C3 analogue 4 (Figure 1a). The intensity of
Scheme 1. General Structures of Unsymmetrical Trimethine and
Pentamethine Cyanine Dyes (C3 and C5). Proposed Synthetic
Strategies for Their Imine Analogues 4 and 5 and Synthetic
Route for Aldehydes 2 and 3
Figure 1.
1H NMR spectra of (a) a stoichiometric mixture of amine
1 and aldehyde 2 after reacting for 72 h at 50 °C in DMSO-d6 and
(b) isolated C3 imine analogue 4. The all-trans structure of 4 is
represented in the inset (arrows indicate the observed NOESY
correlations).
these signals slowly increased for 3 days until saturation was
reached.
differ from the original dyes solely by the introduction of
an imine bond into the polymethine chain, thus making their
formation reversible and adaptive to the pressure of external
conditions.
In order to unambiguously characterize this new species,
a mixture of amine 1 and aldehyde 2 in a mixture of toluene
and DMF was heated at 95 °C in a Dean-Stark apparatus
for 3 days. The imine derivative 4 was obtained pure as a
bright orange solid and was subsequently fully characterized.
The 1H NMR of the isolated imine (Figure 1b) was consistent
with the new set of signals appearing when mixing 1 and 2.
This allowed us to estimate around 15% the maximal
conversion of aldehyde and amine into the corresponding
imine at 50 °C. It is noteworthy that despite the possible
cis/trans isomerization of the imine bond formed, only one
As a model system, the reactions between an N-methyl-
2-amino benzothiazole and two functionalized indolenin
aldehydes were investigated. Amine 1 was obtained by
quarternization of commercially available 2-aminobenzothia-
(6) (a) Constantin, T. P.; Silva, G. L.; Robertson, K. L.; Hamilton, T. P.;
Fague, K.; Waggoner, A. S.; Armitage, B. A. Org. Lett. 2008, 10, 1561.
(b) Monchaud, D.; Allain, C.; Bertrand, H.; Smargiasso, N.; Rosu, F.;
Gabelica, V.; De Cian, A.; Mergny, J.-L.; Teulade-Fichou, M.-P. Biochimie
2008, 90, 1207. (c) Socher, E.; Jarikote, D. V.; Knoll, A.; Ro¨glin, L.;
Burmeister, J.; Seitz, O. Anal. Biochem. 2008, 375, 318.
1
isomer was always observed by H and 13C NMR. The all-
trans conformation of the polymethine/imine chain was
assigned by NOESY, thus confirming that this imino dye
adopts the same thermodynamically stable conformation as
a trimethine cyanine dye.
¨
¨
(7) Ozhalici-Unal, H.; Lee Pow, C.; Marks, S. A.; Jesper, L. D.; Silva,
G. L.; Shank, N. I.; Jones, E. W.; Burnette, J. M., III; Berget, P. B.;
Armitage, B. A. J. Am. Chem. Soc. 2008, 130, 12620.
(8) (a) Yumoto, K.; Irie, M.; Matsuda, K. Org. Lett. 2008, 10, 2051.
(b) Tomasulo, M.; Kaanumal, S. L.; Sortino, S.; Raymo, F. M. J. Org.
Chem. 2007, 72, 595. (c) Raymo, F. M.; Tomasulo, M. J. Phys. Chem. A
2005, 109, 7343.
(10) The formation of aldehyde 3 as a C5 byproduct resulting from the
hydrolysis of immobilized hemicyanines was recently proposed based on
M.S. analysis in: Mason, S. J.; Hake, J. L.; Nairne, J.; Cummins, W. J.;
Balasubramanian, S. J. Org. Chem. 2005, 70, 2939.
(9) (a) Mendes, P. M. Chem. Soc. ReV. 2008, 37, 2512. (b) Yagai, S.;
Kitamura, A. Chem. Soc. ReV. 2008, 37, 1520.
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Org. Lett., Vol. 11, No. 5, 2009

Next, 1H NMR experiments were carried out from amine
1 and aldehyde 3 under similar conditions as with aldehyde
2. As described above for imine 4, the slow formation of
6-31G(d) level of theory using an implicit treatment of the
solvent (DMSO). The scaling scheme of Champagne et al.13
was used to correct for the slight, intrinsic overestimation
of excitation energies by the TD-DFT method. The calculated
energies for vertical excitation of 1.89, 2.06, 2.23, and 2.42
eV for dyes C5, 5, C3, and 4 are in good agreement with
the experimental values of 1.91, 2.14, 2.26, and 2.64 eV.
The experimental shift to larger excitation energies, i.e.,
shorter wavelengths upon introduction of an imino group
into the polymethine chain, is also seen in the calculations:
44 nm for 4 versus C3 and 54 nm for 5 versus C5. While
the energy-minimized structures of C3 and C5 and of their
respective imino analogues 4 and 5 all proved perfectly
planar, the shift in wavelength can be understood in terms
of the larger HOMO-LUMO splitting in the ground state
for imino compounds 4 and 5 in comparison to the parent
C3 and C5 molecules, respectively (Figure 2; note that the
1
imine 5 was evidenced by H NMR. Compound 5 was
isolated as a dark purple solid and was also present in
solution as a unique all-trans isomer (Supporting Informa-
tion). It is noteworthy that both imino dyes 4 and 5 proved
highly stable in their solid state while slowly regenerating
the amine and aldehyde precursors when stored in solution
for more than 1 h. Fresh solutions of analytically pure
samples of compounds 4 and 5 and of their aldehyde
precursors 2 and 3 were prepared in DMSO, and their
spectroscopic properties were investigated using UV-vis and
fluorescence (Table 1).
Table 1. Spectral Properties of the Imino Dyes 4 and 5 and of
Their Aldehyde Precursors 2 and 3a
compd
λabs (nm)
ε (M-1 cm-1
)
λem (nm)
QYb
2
3
4
5
338
410
469
579
547
648
5.41 × 104
5.47 × 104
7.42 × 104
4.83 × 104
5.70 × 104
8.82 × 104
410
460
522
608
572
674
0.00017
0.006
0.010
0.033
0.016
0.038
C311
C511
a All measurements were taken in DMSO, and reported values are the
average of at least two independent measurements. b Fluorescence quantum
yields were determined by comparative method using quinine sulfate,
fluorescein, and sulforhodamine 101 as reference standards.
In comparison to their parent C3 and C5 cyanine dyes
(i.e., lacking the imine bond), imines 4 and 5 have their
maximum emission of fluorescence at slightly shorter
wavelengths (-50 and -68 nm, respectively). However, the
introduction of an imine bond into the conjugated polyme-
thine chain has only a very limited effect on either the Stokes’
shift or the molar absorption coefficient of the dye. Although
notably low (0.01-0.033), fluorescence quantum yields of
both imines proved significantly higher than those of their
corresponding aldehyde precursors and only slightly lower
than those of commonly used trimethine and pentamethine
dyes. It is also noteworthy that the absorption spectra of
aldehydes 2 and 3 do not overlap with those of their
corresponding imines. Therefore, when forming imine 4 (or
5) from amine 1 and aldehyde 2 (or 3), excitation of the
reaction mixture at 470 nm (or 580 nm) enables the specific
detection of the fluorescence emitted by the imino dye only.
Of particular interest is the case of bright orange and
fluorescent C3 imino analogue 4 which results from the
reaction between a dark and colorless amine 1 and a colorless
and minimally fluorescent aldehyde 2 (fluorescence quantum
yield 60-fold lower than that of imine 4).
Figure 2. Highest occupied molecular orbital (HOMO) and lowest
unoccupied orbital molecular orbital (LUMO) of C3 and 4. The
isodensity surfaces of the orbitals were drawn with the program
MOLEKEL 5.314 from GAUSSIAN 0315 cube-files with a contour
value of (0.06.
HOMO-LUMO gap in the ground state does not correspond
to the reported values of the excitation energy as determined
from TD-DFT). Due to the positive charge of the system,
both HOMO and LUMO are largely negative in energy. It
is not surprising that the orbitals of the imino dye 4 are
shifted to lower energy with respect to the all-methine dye,
the electrons being more strongly bound in 4 than in C3 due
To elucidate the changes in electronic structure upon
introduction of an imino group into the polymethine chain
of cyanine dyes we performed a time-dependent density
functional theory (TD-DFT)12 study. Calculations were
carried out for compounds 4 and 5 as well as for reference
compounds C3 and C5 on the B3LYP/6-311G(d)//B3LYP/
(11) For direct comparison, analytically pure samples of the C3 and C5
direct analogues of imines 4 and 5 were prepared according to the literature
procedure, and their spectroscopic properties in DMSO were determined
under identical conditions. See the Supporting Information.
(12) Casida, M. E.; Jamorski, C.; Casida, K. C.; Salahub, D. R. J. Chem.
Phys. 1998, 108, 4439.
(13) Champagne, B.; Guillaume, M.; Zutterman, F. Chem. Phys. Lett.
2006, 425, 105.
Org. Lett., Vol. 11, No. 5, 2009
1125

to the larger electronegativity of the nitrogen. The HOMO
is also more strongly delocalized over the entire molecule
in 4 than in C3, i.e., the atomic orbital coefficients of the
heterocycles are larger in 4 than in C3. The replacement of
a methine group by an imino group leads to a better overlap
between the polymethine chain and the nitrogen-containing
heterocycles, and thus, the HOMO is more stabilized. On
the other hand, the LUMO is slightly less delocalized in 4
than in C3. The orbital coefficients of the sulfur atom are
smaller in 4 than in C3, whereas for the imino nitrogen the
opposite is true. As a result of the larger delocalization
(stabilization) of the HOMO with respect to the LUMO in 4
than in C3 the gap between these orbitals is larger for the
former. This explains qualitatively the larger excitation
energy and the shorter absorbance wavelength for the imino
dyes 4 and 5 in comparison to C3 and C5, respectively.
Finally, we investigated whether the reaction of formation
of cyanine dye imino analogues from amine and aldehyde
building blocks was truly reversible. The dynamic system
could then undergo constitutional reorganization in response
to an external stimulus.16 A stoichiometric mixture of amine
1 and aldehyde 2 (25 mM each) in DMSO was stirred at
room temperature until thermodynamic equilibrium was
reached (48 h). The perturbation of the system was monitored
by HPLC, UV-vis, and fluorescence spectroscopy. At
equilibrium, only a very small amount of C3 imine 4 was
formed. Despite this low level of conversion, imine 4 was
easily detected by fluorescence spectroscopy and could also
be visualized by UV-vis (yellow color). The mixture was
then heated to 60 °C and was again allowed to reach
equilibrium. Equilibrium was reached after 24 h which was
different from that observed at room temperature. The
absolute amount of C3 analogue 4 present at equilibrium
was increased by about 5-fold (as measured by both HPLC
and fluorescence) (Figure 3). When the reaction mixture was
being cooled to room temperature, the system reorganized
again until it reached an equilibrium position (after 72 h)
that was comparable to that obtained initially before heating,
thus proving true reversibility of the system. Similar results
were obtained when reacting amine 1 (50 mM) with both
aldehydes 2 and 3 (25 mM each) simultaneously.
It is also noteworthy that reorganization of the equilibrating
mixture can be monitored in real time by fluorescence
spectroscopy. While this dynamic system takes 48 h to
equilibrate at room temperature, less than 24 h is required
to reach equilibrium when the reaction is carried out at 60
°C (see the Supporting Information for kinetics of imino dye
formation).
Synthesis of cyanine dyes is generally accomplished by
the irreversible stepwise reaction between nucleophilic
heterocycles (e.g., 1,2,3,3-tetramethylindolenin) and a poly-
ene-chain precursor such as an amidine and proceeds via
the formation of a hemicyanine intermediate. Herein, we
reported the first family of cyanine dye (trimethine and
pentamethine) imine analogues that are easily accessible via
a reversible and thermodynamically controlled reaction from
readily available amine and aldehyde building blocks. We
provided proof-of-concept that this dynamic system could
reorganize in response to an external stimulus, thus leading
to a measurable perturbation of the global UV-vis and
fluorescence spectra of the equilibrating mixture. The
development of smart materials based on this concept is
currently underway in our laboratory.
Acknowledgment. We thank Martin Karplus (ISIS, Stras-
bourg) for fruitful discussions, and we are grateful for the
computational resources provided by the high-performance
cluster of the Universite´ Louis Pasteur. K.M. thanks the
French Ministry of Research and Technology for a doctoral
Fellowship. S.L. thanks the CNRS for funding. M.S. is
grateful to ISIS for hosting him as a guest assistant professor.
Supporting Information Available: General experimental
procedures and spectroscopic data for new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL802913B
(14) MOLEKEL 5.3: Flu¨kiger, P.; Lu¨thi, H. P.; Portmann, S.; Weber,
J. Swiss National Supercomputing Centre CSCS, Manno, Switzerland, 2008.
(15) GAUSSIAN 03, Revision: Frisch, M. J.; Trucks, G. W.; Schlegel,
H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A.,
Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.;
Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.;
Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda,
R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai,
H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken,
V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.;
Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski,
V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick,
D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui,
Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith,
T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.;
Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople,
J. A. Gaussian, Inc, Wallingford, CT, 2004.
Figure 3. HPLC traces and fluorescence spectra showing the
formation of imine 4 from a mixture of amine 1 and aldehyde 2
(25 mM each). Starting position (black) and positions of equilibrium
after 48 h at rt (blue), then after heating 24 h at 60 °C (red), and
after cooling back for 72 h at rt (green) are represented. HPLC
traces show the absolute ratios of aldehyde 2 (retention time 14.5
min) and imine 4 (retention time 18.5 min). Fluorescence emission
spectra were recorded when exciting at 480 nm.
(16) (a) Giuseppone, N.; Lehn, J.-M. Chem.sEur. J. 2006, 12, 1715.
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