Synthesis and spectroscopic and DNA-binding properties of fluorogenic acridine-containing cyanine dyes

Article abstract of DOI:10.1021/jo901820t

(Chemical Equation Presented) The synthesis of a new subclass of mono- and polymethine cyanine dyes that incorporate an acridinium moiety and that absorb in the orange to near-infrared region of the spectrum is reported. The mono-, tri-, and pentamethine dyes in particular exhibit promising fluorogenic properties. Their ability to aggregate in solution and to interact with B-DNA is also discussed.

Full text of DOI:10.1021/jo901820t

pubs.acs.org/joc
the wide selection of fluorescent dyes available in the litera-
Synthesis and Spectroscopic and DNA-Binding
Properties of Fluorogenic Acridine-Containing
Cyanine Dyes
ture, those belonging to the Cyanine dye (Cy)4 and Acridine5
families have been extensively used as oligonucleotide labels
or probes. Although those dyes can be covalently attached to
a DNA strand for labeling purposes, of particular interest is
their capacity to bind to DNA through noncovalent inter-
actions either by intercalation between base pairs (acridines,
monomethine cyanine dyes)6 or aggregation into the minor
groove of double-stranded DNA (trimethine and penta-
methine cyanine dyes).7 The family of cyanine dyes gener-
ically consists of a conjugated system based on a polymethine
chain linking two nitrogen-containing heterocycles (most
commonly indoles, benzothiazoles, benzoxazoles, or quino-
lines).4 A well-known fluorogenic monomethine cyanine dye
is Thiazole orange (TO), which is nonfluorescent when free
in solution (due to rapid nonradiative deactivation to the
ground state) but becomes highly fluorescent under condi-
tions where motions of the polymethine chain are restricted
(e.g., upon intercalation into DNA or when in viscous
solvents).6b,8 Similar properties are commonly observed with
unsymmetrical polymethine cyanine dyes.9 Here, we report a
new family of fluorogenic and unsymmetrical mono-, tri-,
penta-, and heptamethine cyanine dyes that incorporate one
tricyclic N-methylacridinium heterocycle and cover a large
spectral range from orange to near-infrared. The synthesis
and spectroscopic properties of these dyes are described as
well as their DNA binding properties.
Trimethine, pentamethine, and heptamethine cyanine
dyes were synthesized by reaction of commercially available
2-methylene-1,3,3-trimethylindoline with the appropriate
activated acridine hemicyanine (3b-d). The activated hemi-
cyanines, or acridine “half-dyes”, were obtained by reaction
of 9,10-dimethylacridinium iodide 210 with N,N-diphenyl-
formamidine generated in situ, malonaldehyde bisphenyli-
mine hydrochloride or glutaconaldehydedianil hydro-
chloride in a mixture of acetic anhydride, and either pyridine
or triethylamine and used crude for the subsequent step of
cyanine dye formation (Scheme 1).
Tariq Mahmood, Alexis Paul, and Sylvain Ladame*
ISIS Universiteꢀ de Strasbourg 8, Alleꢀe Gaspard Monge,
BP 70028, 67083 Strasbourg, France
s.ladame@isis.u-strasbg.fr
Received August 24, 2009
The synthesis of a new subclass of mono- and poly-
methine cyanine dyes that incorporate an acridinium
moiety and that absorb in the orange to near-infrared
region of the spectrum is reported. The mono-, tri-, and
pentamethine dyes in particular exhibit promising fluoro-
genic properties. Their ability to aggregate in solution and
to interact with B-DNA is also discussed.
Fluorescent molecules have been widely used as probes in
chemical biology.1 Depending on the nature of the biological
process to be visualized or the molecular species that needs to
be sensed it is possible to choose among a large variety of
fluorescent probes that exhibit specific structural and spec-
troscopic properties. Of particular interest are molecules that
can reversibly switch between a nonemissive state and an
emissive state. Most representative examples are fluorogenic
unsymmetrical cyanine dyes which become fluorescent upon
interaction with specific nucleic acids or proteins,2 and
photochromic compounds which can undergo a reversible
change of their optical properties under illumination.3 Within
For the synthesis of monomethine dyes, a different route
was chosen starting from commercially available 10-methyl
acridone 5. Reaction of 5 with thionyl chloride afforded the
corresponding 9-chloro-10-methylacridinium chloride 6,
which was subsequently reacted with 2-methylene-1,3,3-
trimethylindolenin to produce the desired monomethine
cyanine dye 4a (Scheme 2).
(1) For recent reviews see: (a) Terai, T.; Nagano, T. Curr. Opin. Chem.
Biol. 2008, 12, 515–521. (b) Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008,
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(2) (a) Yang, P.; De Cian, A.; Teulade-Fichou, M. P.; Mergny, J. L.;
Monchaud, D. Angew. Chem., Int. Ed. 2009, 48, 2188–2191. (b) Ozhalici-
Unal, H.; Pow, C. L.; Marks, S. A.; Jesper, L. D.; Silva, G. L.; Shank, N. I.;
Jones, E. W.; Burnette, J. M. 3rd; Berget, P. B.; Armitage, B. A. J. Am. Chem.
Soc. 2008, 130, 12620–12621. (c) Constantin, T. P.; Silva, G. L.; Robertson,
K. L.; Hamilton, T. P.; Fague, K.; Waggoner, A. S.; Armitage, B. A. Org.
Lett. 2008, 10, 1561–1564. (d) Karlsson, H. J.; Eriksson, M.; Perzon, E.;
Akerman, B.; Lincoln, P.; Westman, G. Nucleic Acids Res. 2003, 31, 6227–
6234.
(4) Hamer, F. M. The Cyanine Dyes and Related Compounds; Weissberger,
A., Ed.; Interscience: London, U.K., 1964.
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Chem. 2007, 7, 139–169.
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(b) Nygren, J.; Svanvik, N.; Kubista, M. Biopolymers 1998, 46, 39–51.
(7) Karlsson, H. J.; Bergqvist, M. H.; Lincoln, P.; Westman, G. Bioorg.
Med. Chem. 2004, 12, 2369–2384.
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€
(8) Jarikote, D. V.; Krebs, N.; Tannert, S.; Roder, B.; Seitz, O. Chem.;
Eur. J. 2006, 13, 300–310.
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(b) Tomasulo, M.; Kaanumal, S. L.; Sortino, S.; Raymo, F. M. J. Org. Chem.
2007, 72, 595–605.
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204 J. Org. Chem. 2010, 75, 204–207
Published on Web 12/02/2009
DOI: 10.1021/jo901820t
r
2009 American Chemical Society

Mahmood et al.
JOCNote
SCHEME 1. Synthetic Route to Trimethine (4b), Pentamethine
(4c), and Heptamethine (4d) Acridine Cyanine Dyes
TABLE 1. UV Absorption Properties of Dyes 4a-d (in their monomeric
state)
methanol
ε (M-1 cm-1
buffera
compd
λabs (nm)
)
λabs (nm)
ε (M-1 cm-1
)
3
3
4a
4b
4c
595
677
43690
62650
83300
592
672
26720
25630
-
c
774
(700b)
872
771
(696b)
872
c
c
-
4d
-
(718b)
(668b)
b
aTRIS HCl 50 mM þ 100 mM KCl. Maximum wavelength of H-
3
aggregates. cNot following Beer’s law.
FIGURE 1. UV absorption spectra of 4a (red), 4b (black), 4c
(green), and 4d (blue) taken in MeOH.
linium or a benz[e]indolium, respectively.11 Similar effects were
observed within the trimethine family with red shifts of 80 and
110 nm, respectively.11
Typically, the visible absorption spectrum of a cyanine dye
shows two close maxima: the band of longest wavelength is
the most intense while the intensity and resolution of the
vibronic shoulder at shorter wavelength differ from dye to
dye. In solution, cyanine dyes can spontaneously assemble to
form dimers or also polymer-type aggregates which can be
composed of up to thousands of molecules. These aggregates
can be of H- or J-type depending on whether the dyes
assemble in a face-to-face or end-to-end manner.12
The absorption spectra of mono- and trimethine dyes 4a
and 4b (Figure 1) were typical of nonaggregated cyanine dyes
and similar in shape when taken in either methanol or buffer,
thus suggesting the existence of the dyes in solution mainly
under their monomeric form. This was no longer the case for
the pentamethine dye 4c for which a broadband at shorter
wavelength (compared to that of the monomeric dye) and
corresponding to the formation of H-aggregates12 became
apparent in methanol and predominant in buffer. This
aggregation phenomenom was even more pronounced for
the heptamethine dye 4d, which was mainly present in
solution as an H-aggregate (characterized by a very broad
absorption band around 600-800 nm) in both methanol and
buffer. In the conditions of our experiments, however, i.e.,
in aqueous or organic solvent at dye concentrations up to
200 μM, no J-aggregate was ever observed by absorption
SCHEME 2. Synthetic Route to Monomethine Acridine Dye 4a
The spectroscopic properties of the four acridine cyanine
dyes 4a-d were then determined with absorption and fluor-
escence spectroscopy. Absorption coefficients and maxi-
mum absorption wavelengths were measured in methanol
and in an aqueous buffer (50 mM TRIS HCl pH 7.4 contain-
3
ing 100 mM KCl). The data are summarized in Table 1.
As commonly observed for cyanine dyes, the elongation of
the polymethine bridge resulted in a significant red shift of the
maximum absorption wavelength at a rate of ∼100 nm per two
methine groups added. Of high interest for future applications
is the large spectral range, from orange to near-infrared,
covered by this family of acridine-containing cyanine dyes.
The contribution of the acridine moiety on the maximum
absorption wavelength of the dye can be assessed by comparing
the UV spectra of our dyes with those of similar unsymmetrical
cyanine dyes lacking the acridine. For instance, the acridinium
moiety induces a red shift of the λabs of 135 and 120 nm when
compared with similar unsymmetrical pentamethine dyes in
which the acridinium moiety has been substituted by a quino-
(12) (a) Biver, T.; Boggioni, A.; Secco, F.; Turriani, E.; Venturini, M.;
Yarmoluk, S. Arch. Biochem. Biophys. 2007, 465, 90–100. (b) Armitage, B. A.
Top. Curr. Chem. 2005, 253, 55–76. (c) Slavnova, T. D.; Chibisov, A. K.;
€
Gorner, H. J. Phys. Chem. A 2005, 109, 4758–4765. (d) West, W.; Pearce, S.
J. Phys. Chem. 1965, 69, 1894–1903.
(11) Mason, S. J.;Hake, J. L.;Nairne, J.; Cummins, W. J.; Balasubramanian,
S. J. Org. Chem. 2005, 70, 2939–2949.
J. Org. Chem. Vol. 75, No. 1, 2010 205

Mahmood et al.
JOCNote
FIGURE 3. Fluorescence emission spectra (λexc = 670 nm) of 4b
(5 μM, left) and 4a (5 μM, right) in solution in buffer (blue),
methanol (red), and 85% glycerol (black).
upon binding to CT DNA, i.e., partial disappearance of the
H-aggregate at the expense of the monomeric dye.
Finally, the fluorescence properties of dyes 4a-d were
examined in methanol, buffer, and 85% glycerol on a Jobin
Yvon Fluorolog 3.22 instrument. In buffer, monomethine
cyanine dye 4a showed a maximum emission band around
552 nm with a shoulder of weaker intensity around 594 nm.
As commonly observed for monomethine cyanine dyes,
compound 4a exhibits fluorogenic behavior. Its fluorescence
is very low in aqueous buffer and methanol (Φf = 0.20 and
0.23%, respectively) while it increases significantly (ca. 25-
fold, Φf = 5.6%) when measured in 85% glycerol (Figure 3).
This is due at least in part to a restriction in conformational
freedom of the monomethine bridge. A similar fluorogenic
property was observed for dyes 4b,c. While almost no
fluorescence was observed when exciting trimethine dye 4b
around its maximum absorption wavelength (λexc = 670 nm)
in buffer, a moderate emission was detected in methanol
(Φf = 0.10%), which significantly increased (ca. 15-fold)
when measured in 85% glycerol (Figure 3). An analogous effect,
although of weaker amplitude, was observed with the penta-
methine dye 4c for which a moderate emission of fluorescence
around 810 nm (λexc = 770 nm) was detectable in glycerol
solution only. Unfortunately, no fluorescence was ever obtained
when exciting heptamethine dye 4d around its maximum absorp-
tion wavelength (λexc = 870 nm), even in glycerol solution.
It is noteworthy that dyes 4b-d also share two common
fluorescence emission maxima at shorter wavelengths
(i.e., 552 and 654 nm upon excitation at 520 and 620 nm,
respectively). These could be assigned to the formation after
excitation of locally excited (LE) states that could also result
in excited state intramolecular charge transfer (ICT).13
Fluorescence titration experiments were carried out by
adding increasing concentrations of CT DNA into a solution
of acridine dye 4a or 4b (5 μM in buffer). For monomethine
dye 4a, the addition of CT DNA resulted in a significant
decrease of the fluorescence intensity, which is consistent
with a nonintercalative binding mode (i.e., no restriction of
conformational freedom upon binding to DNA), likely due
to steric hindrances preventing the dye from adopting a
planar conformation (see the Supporting Information).9
Interestingly, a modest (ca. 3-fold, see the Supporting
Information) fluorescence enhancement was observed for
trimethine dye 4b upon addition of CT DNA, which was
accompanied by a 23 nm red shift of the emission maximum.
Although the fluorescence intensity remains very moderate
FIGURE 2. UV-vis spectra recorded during titration of CT-
dsDNA into an aqueous solution of 4a (top left), 4b (top right), 4c
(bottom left), and 4d (bottom right). CT DNA concentration varies
from 0 to 300 μM (CT DNA concentration is given in base pairs and
is estimated assuming an extinction coefficient at 260 nm of 12 824
M-1 cm-1).
3
spectroscopy. All together, these observations are consistent
with the data accumulated in the literature, although mainly
on symmetrical cyanine dyes, which demonstrated that in
aqueous solution, (i) the tendency of cyanine dyes to aggre-
gate increases with the length of the polymethine chain
(i.e., as hydrophobicity and polarizability increase) and
(ii) unsubstituted dyes favor H-aggregation rather than
J-aggregation.12b
Next, the ground state interaction between cyanine dyes
4a-d and double-stranded DNA was investigated. Titration
experiments were carried out by adding increasing amounts
of Calf-Thymus (CT) DNA to a 20 μM solution of acridine
dye (in TRIS HCl buffer 50 mM also containing 100 mM
3
KCl). Interestingly, different behaviors were obtained with
each dye that can be interpreted in terms of different DNA
binding mode. For monomethine cyanine dye 4a, a decrease
in intensity of the UV band was observed when increasing the
DNA concentration, which was accompanied by a very small
(4 nm) red shift (Figure 2). For trimethine dye 4b, a similar
decrease in intensity was observed along with a significant
red shift of 24 nm. Although the bathochromic shifts
observed for compounds 4a and 4b upon binding to DNA
were suggesting intercalation of the dyes between base pairs,
viscosity experiments showed no DNA lengthening upon
addition of either dye, suggestive of a nonintercalative
or heterogeneous mode of binding (see the Supporting
Information).
While pentamethine cyanine dyes typically aggregate into
the minor groove of double-stranded DNA, results obtained
with compound 4c indicate a different binding mode. While
4c existed predominantly as an H-aggregate in aqueous
buffer, addition of increasing amounts of CT DNA led to
the appearance of a new band, red-shifted (29 nm) and of
increasing intensity with respect to the initial band for the
monomeric dye in solution. This corresponds to a complete
disassembly of the H-aggregate upon binding to DNA.
Comparable effect was observed with heptamethine dye 4d
(13) Peng, X.; Song, F.; Lu, E.; Wang, Y.; Zhou, W.; Fan, J.; Gao, Y.
J. Am. Chem. Soc. 2005, 127, 4170–4171.
206 J. Org. Chem. Vol. 75, No. 1, 2010

Mahmood et al.
JOCNote
compared to that obtained in glycerol, it demonstrates the
fluorogenic properties of 4b upon binding to B-DNA.
In summary, we have developed the first family of acri-
dine-containing fluorogenic cyanine dyes that cover a broad
spectral range from orange to near-infrared. The introduc-
tion of an acridine moiety accounts for a red shift of the
maximum absorption wavelength of ca. 100-120 nm when
compared to the corresponding quinoline dyes. Of particular
interest are (i) trimethine dye 4b, which emits maximally
at 697 nm under conditions of restricted mobility only, and
(ii) pentamethine 4c, which absorbs in the near-infrared
region of the spectrum (770 nm). Cyanine dyes from this
family could find valuable application as fluorogenic sensors
(e.g., viscosity sensors) and their versatile synthesis should
allow the development of optimized dyes with increased
fluorogenic properties.
3 h. Et2O (100 mL) was added and the dark blue solid was
collected by filtration and washed with Et2O. The crude material
was purified by silica gel chromatography (CH2Cl2, then
CH2Cl2/MeOH 95:5) to give 4c as a blue/green solid (235 mg,
72%). 1H NMR (CDCl3, 400 MHz) δ 1.75 (s, 6H), 3.89 (s, 3H),
4.03 (s, 3H), 7.12 (d, 2H, J = 8 Hz), 7.27-7.33 (m, 3H), 7.38
(t, 1H, J = 8 Hz), 7.44-7.50 (m, 4H), 7.67 (t, 2H, J = 8 Hz),
7.76 (t, 2H, J = 12 Hz), 7.95 (t, 1H, J = 12 Hz), 8.04 (d, 2H, J =
8 Hz). 13C NMR (CDCl3, 100 MHz) δ 27.8, 27.8, 34.5, 35.6,
50.4, 109.9, 112.4, 114.5, 121.7, 122.5, 123.2, 127.2, 132.2,
132.2, 140.8, 141.9, 142.5, 146.6, 153.0, 154.0, 176.4, 207.1.
HRMS (ESI) m/z calcd for C30H29N2 [Mþ] 417.233, found
417.239.
Preparation of Compound 4d. Compound 4d was synthesized
following the same protocol as for compound 4c but starting
from 9,10-dimethylacridinium iodide and glutaconaldehydedia-
nil hydrochloride. 1H NMR (CDCl3, 400 MHz) δ 1.75 (s, 6H),
3.73 (s, 3H), 4.30 (s, 3H), 6.78 (t, 1H, J = 12 Hz), 6.81 (d, 1H,
J = 8 Hz), 7.07 (t, 1H, J = 12 Hz), 7.13 (d, 1H, J = 12 Hz), 7.24
(t, 2H, J = 8 Hz), 7.30 (d, 2H, J = 8 Hz), 7.40-7.56 (m, 8H),
7.82 (d, 2H, J = 8 Hz), 7.97 (t, 1H, J = 12 Hz). 13C NMR
(CDCl3, 100 MHz) δ 27.5, 34.6, 35.0, 50.7, 110.7, 112.7, 113.9,
121.9, 122.3, 122.4, 122.4, 123.5, 126.7, 127.5, 129.2, 130.3,
131.0, 131.9, 140.8, 142.0, 142.1, 142.1, 146.9, 153.3, 153.6,
177.1. HRMS (ESI) m/z calcd for C32H31N2 [Mþ] 443.248,
found 443.250.
Preparation of Compound 4a. A solution of 10-methylacri-
done (100 mg, 0.48 mmol) in thionyl chloride (2 mL) was stirred
at room temperature for 1 h. Excess SOCl2 was then removed
under reduced pressure to afford the desired 9-chloro-10-methyl-
acridinium chloride intermediate 6 as an orange solid. To a
solution of 6 in anhydrous DMF (1 mL) was added 2-methylene-
1,3,3-trimethylindoline (124 mg, 0.72 mmol). The reaction
mixture was then stirred at room temperature for 3 h and the
product was extracted with CH2Cl2 and washed with water. The
organic layer was dried (MgSO4) and the solvent was evapo-
rated off under reduced pressure. The crude material was finally
purified by silica gel chromatography (CH2Cl2, then CH2Cl2/
MeOH 97:3) to give 4a as a dark blue solid (125 mg, 65%). 1H
NMR (CDCl3, 400 MHz) δ 1.82 (s, 6H), 2.66 (s, 3H), 4.76
(s, 3H), 6.58 (s, 1H), 7.03 (d, 1H, J = 8 Hz), 7.28 (t, 1H, J = 8
Hz), 7.41 (t, 1H, J = 8 Hz), 7.45 (d, 1H, J = 8 Hz), 7.61 (t, 2H,
J = 8 Hz), 8.14 (t, 2H, J = 8 Hz), 8.19 (d, 2H, J = 8 Hz), 8.46
(d, 2H, J = 8 Hz). 13C NMR (CDCl3, 100 MHz) δ 28.6, 36.5,
38.3, 50.0, 87.9, 110.6, 118.6, 122.5, 123.7, 124.7, 125.6, 136.6,
138.5, 140.4, 144.2, 157.4, 174.8. HRMS (ESI) m/z calcd for
C26H25N2 [Mþ] 365.201, found 365.203.
Experimental Section
Preparation of Compound 4b. A mixture of 9,10-dimethyl-
acridinium iodide10 (200 mg, 0.59 mmol), 4-methoxyaniline (150 mg,
1.18 mmol), and triethyl orthoformate (200 μL, 1.18 mmol) in
ethanol (3 mL) was refluxed at 75 °C for 3 h. The reaction
mixture was cooled to room temperature and then poured into
Et2O (50 mL). A solid precipitated, which was collected by
filtration and washed with Et2O. Crude hemicyanine 3b was
obtained as a dark solid and was used directly for the next step
without further purification. The crude product 3b obtained
above was taken in a mixture of acetic anhydride (250 μL) and
pyridine (5 mL). 2-Methylene-1,3,3-trimethylindoline (120 mg,
0.70 mmol) was added and the resulting mixture was stirred at
room temperature for 2 h. Et2O (50 mL) was added, thus leading
to the formation of a dark blue solid that was filtered off
solution and washed with Et2O. Purification by silica gel
chromatography (CH2Cl2, then CH2Cl2/MeOH 98:2) afforded
the trimethine cyanine dye 4b as dark blue solid (192 mg, 63%).
1H NMR (CD3OD, 400 MHz) δ 1.69 (s, 6H), 3.84 (s, 3H), 4.10
(s, 3H), 6.90 (d, 1H, J = 12 Hz), 7.40 (t, 1H, J = 8 Hz),
7.48-7.54 (m, 4H), 7.59 (d, 1H, J = 8 Hz), 7.86-7.94 (m, 3H),
8.27 (d, 1H, J = 8 Hz), 8.36 (t, 1H, J = 12 Hz). 13C NMR
(CD3OD, 100 MHz) δ 26.6, 31.0, 34.8, 49.8, 108.1, 111.7, 115.3,
118.4, 122.1, 122.2, 122.9, 126.3, 127.0, 128.7, 133.4, 140.6,
141.5, 142.6, 151.7, 154.3, 176.6. HRMS (ESI) m/z calcd for
C28H27N2 [Mþ] 391.217, found 391.217.
Preparation of Compound 4c. To a suspension of 9,10-di-
methylacridinium iodide (200 mg, 0.60 mmol) in acetic anhy-
dride (15 mL) were successively added malonaldehyde
bis(phenylimine) hydrochloride (155 mg, 0.60 mmol) and pyri-
dine (1 mL). After the mixture was stirred at room temperature
for 8 h, Et2O (50 mL) was added and the resulting brown solid
was filtered off and washed extensively with Et2O. The activated
hemicyanine 3c was then collected by extraction in CH2Cl2
(100 mL) and obtained as an orange solid after evaporation of
the solvent under vacuum. To a solution of crude 3c (130 mg) in
CH2Cl2 (25 mL) was added 2-methylene-1,3,3-trimethylindo-
line (103 mg, 0.60 mmol) and triethylamine (1 mL). The reaction
mixture was then stirred at room temperature and in the dark for
Acknowledgment. The authors would like to thank the
ꢀ
Universite de Strasbourg (UdS) and the CNRS for funding.
This work was also supported by the International Center for
Frontier Research in Chemistry (FRC).
Supporting Information Available: Experimental details,
characterization of compounds 4a-d, and spectroscopic
(Ultraviolet, fluorescence) data. This material is available free
of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 1, 2010 207