Specific ca2+ fluorescent sensor: Signaling by conformationally induced pet suppression in a bichromophoric acridinedione

Article abstract of DOI:10.1002/ejoc.200900570

A series of acridinedione-based bichromophoric podand systems 1a-c were synthesized and characterized. Among these, bichromophore 1c shows specific binding of Ca²⁺ in the presence of other biologically important metal ions like Na⁺, K⁺, Mg²⁺, and Zn²⁺, The selective complexation was proved by steady-state emission, time-resolved emission, and ¹H NMR titration. Signaling of the binding event was achieved by Ca²⁺-induced folding of the bichromophore, resulting in PET suppression in the acridinedione chromophore. Involvement of a PET process in the optical signaling was confirmed by comparing bichromophores 1a-c with non-PET compound 2 -and monochromophore model compound 3. Non-PET compound 2 failed to give optical response upon Ca²⁺ binding as a result of the absence of a POT process in the Ca²⁺-bound complex. Monochromophore 3 shows a similar optical response, which is the same as that in 1c. Titration of the metalion-bound complex of 1c with EDTA released the metal ion from the complex, thereby regaining the original photophysical properties of the bichromophore.

Full text of DOI:10.1002/ejoc.200900570

FULL PAPER
DOI: 10.1002/ejoc.200900570
Specific Ca2+ Fluorescent Sensor: Signaling by Conformationally Induced PET
Suppression in a Bichromophoric Acridinedione
Pichandi Ashokkumar,[a] Vayalakkavoor T. Ramakrishnan,[a] and Perumal Ramamurthy*[a]
Keywords: Sensors / Calcium / Fluorescence / Electron transfer / Photophysics / Acyclic polyethers
A series of acridinedione-based bichromophoric podand sys-
tems 1a–c were synthesized and characterized. Among these,
bichromophore 1c shows specific binding of Ca2+ in the pres-
ence of other biologically important metal ions like Na+, K+,
Mg2+, and Zn2+. The selective complexation was proved by
steady-state emission, time-resolved emission, and 1H NMR
titration. Signaling of the binding event was achieved by
Ca2+-induced folding of the bichromophore, resulting in PET
suppression in the acridinedione chromophore. Involvement
of a PET process in the optical signaling was confirmed by
comparing bichromophores 1a–c with non-PET compound 2
and monochromophore model compound 3. Non-PET com-
pound 2 failed to give optical response upon Ca2+ binding as
a result of the absence of a PET process in the Ca2+-bound
complex. Monochromophore 3 shows a similar optical re-
sponse, which is the same as that in 1c. Titration of the metal-
ion-bound complex of 1c with EDTA released the metal ion
from the complex, thereby regaining the original photophysi-
cal properties of the bichromophore.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
must be removed from the system as Mg(OH)2 in high pH
solutions. If this evaluation is able to be achieved in one
step, it will be of great advantage for fast and reliable
analysis.
Introduction
Fluorescent chemosensors capable of selectively recog-
nizing metal ions have potential analytical applications in
Even though a large number of cyclic crown ethers and
different fields including chemistry, biology, and medicine.[1] related macrocyclic chemosensors have been reported, the
The design of chemosensors for the selective detection of corresponding acyclic polyether-based sensors are relatively
biologically important metal ions such as Na+, K+, Mg2+
,
rare.[9,10] Cyclic crown ethers have been found to be power-
Ca2+, and Zn2+ is important,[2–6] because many physiologi- ful extracting agents for alkali metal salts.[11] They form ri-
cal processes are controlled by these ions.[5b] Ca2+ is an in- gid and highly stable complexes with alkali and alkaline
tracellular divalent cation with the largest concentration earth metal ions. The corresponding acyclic polyether
variations, which plays a critical role as a signal transmit- shows only weak binding, but it changes its conformation
ter.[7] Because of the importance of Ca2+ in biological pro- from a linear to a pseudocyclic structure upon complex for-
cesses, particular interest has been paid to the design of mation with metal ions. If this conformational change could
chemosensors for the specific detection of Ca2+ in the pres- be converted into physical signs, such as absorption and
ence of other similar metal ions like Na+, K+, and Mg2+
.
fluorescence, it would be possible to establish more sensitive
As a result of the similar chemical properties of Mg2+ and metal ion sensors. The pseudocyclic structure increases the
Ca2+, selective detection between these two metal ions is a binding force and plays important roles not only in artificial
difficult task. Only a few examples of chemosensors for se- ionophores but also in natural ionophores for the selective
lective detection of Ca2+ over Mg2+ have been reported.[8] binding of metal ions.[12] Among the early reports on acy-
EDTA is a well known strong chelator of Mg2+ and Ca2+
,
clic polyether-based chemosensors, reported by Nakamura
and it is commonly used to determine the concentration et al. and Ajayaghosh et al., the signaling of the binding
of these ions in solution. However, it does not show any event was achieved by excimer formation,[10d] exciton inter-
selectivity for the two metal ions and forms two stable action,[8b,8c,10e] twisted intramolecular charge transfer
complexes. To evaluate the concentration of Ca2+, Mg2+ (TICT)[8a,8e] process, and electron[10a] or energy transfer[10b]
process between two terminal chromophore units. None of
the systems utilize the well-established photoinduced elec-
[a] National Centre for Ultrafast Processes, University of Madras,
tron transfer (PET) process,[1c] which can operate in a single
Taramani Campus, Chennai 600113, India
Fax: +91-44-24546709
chromophore unit. Herein, we describe the concept of PET
suppression resulting from the metal-ion-induced confor-
mational folding of the acridinedione bichromophore.
E-mail: prm60@hotmail.com
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900570.
Eur. J. Org. Chem. 2009, 5941–5947
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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P. Ashokkumar, V. T. Ramakrishnan, P. Ramamurthy
FULL PAPER
Acridinedione (ADD) dyes have been reported as laser
dyes with lasing efficiency comparable to that of coumarin-
102, and these dyes have structural similarities with
NADH.[13] Because both PET and intramolecular charge
transfer (ICT) mechanisms can operate in acridinedione, it
will be good to use it as a signaling unit in sensor molecules.
We have already reported a few acridinedione-based anion
and metal ion sensors, which operate through PET and ICT
mechanisms.[14] In the present chemosensors 1a–c, an oxye-
thylene moiety (ionophore) is linked with an acridinedione
chromophore. In addition, an OCH3 group having electron-
donating ability is incorporated as an additional binding
site as well as a signal transducing group based on PET
process. It has already been reported that an electron-do-
nating OCH3 substituent increases the HOMO level of the
donor, which makes the substituted phenyl ring a better
electron donor in the PET process.[15] Involvement of a PET
process in the optical signaling was confirmed by reference
compound 2, which does not have a PET process, and mon-
ochromophore model compound 3, which has a PET pro-
cess (Scheme 1).
Scheme 2. Synthesis routes to compounds 1, 2, and 3.
the compounds are shown in Table 1. A relatively lower
quantum yield and shorter fluorescence lifetime of 1a–c, 3,
and 5 compared to those of 2 and 7 are attributed to the
intramolecular PET process through space from the elec-
tron-rich OCH3 group to the relatively electron-deficient ex-
cited state of the ADD fluorophore. Non-PET dyes 2 and
7 show a single exponential decay, whereas all other PET-
based dyes show biexponential decay with relatively shorter
fluorescence lifetimes. This kind of PET process through
[16]
[14a]
space from p-OCH3
and p-N(CH3)2
groups to the
ADD moiety has already been reported. In this study, the
PET process from the m-OCH3 group is utilized as a signal-
ing mechanism to monitor metal ion binding.
Table 1. Fluorescence quantum yields and lifetimes of 1a–c, 2, 3,
5, and 7 in CH3CN.
1a
0.122 0.122 0.123 0.314 0.140 0.122 0.282
τf, ns 0.76 0.78 0.78 5.15 0.92 0.76 4.22
(B)[b] (17.3) (18.1) (18.2) (100) (13.9) (16.8) (100)
2.60 2.80 2.83 3.65 2.94
(82.7) (81.9) (81.8) (86.1) (83.2)
1b
1c
2
3
5
7
Scheme 1. Structures of chemosensors 1, 2, and 3.
Φf[a]
–
–
Results and Discussion
[a] Fluorescence quantum yields were determined by exciting the
sample at 366 nm with the use of quinine sulfate as the standard
(Φf = 0.546 in 0.1  H2SO4); Ϯ5%. [b] Relative amplitude corre-
sponding to the lifetime.
Synthesis
The synthesis of bichromophores 1a–c and 2 and mono-
chromophore 3 is outlined in Scheme 2. The acridinediones
were obtained by refluxing methylamine with the respective
tetraketones. Reaction of acridinedione with the corre-
sponding ditosylate 6a–c in the presence of K2CO3 yielded
bis(acridinedione)s 1a–c and 2; the monotosylate yielded 3.
All the above compounds were characterized by spectral
analyses.
Metal Ion Binding Studies
Addition of Ca2+ to an acetonitrile solution of 1c shows
a marginal but reproducible (3 nm) redshifted absorption
maximum with an isosbestic point at 381 nm. The corre-
sponding fluorescence spectrum, when excited at its isosbes-
tic point, shows a fluorescence enhancement without any
spectral shift (Figure 1). Evidence for 1:1 complex forma-
Photophysical Studies
The absorption and emission spectra of all the com- tion is provided by the linear relationship obtained in the
pounds show a maximum at 371 and 436 nm, respectively, Benesi–Hildebrand plot[17] of 1/Io – I against [Ca2+ –1
(Sup-
in acetonitrile, which are assigned to the ICT from the ring porting Information, Figure S2). stability constant
]
A
nitrogen atom to the ring carbonyl center within the ADD (logK) of 4.83 was estimated from this plot, which is rea-
moiety. The fluorescence quantum yield and lifetime of all sonably high for an acyclic polyether. Addition of other
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Eur. J. Org. Chem. 2009, 5941–5947

Specific Ca2+ Fluorescent Sensor
metal ions, such as Na+, K+, Mg2+, and Zn2+, did not show
any significant change in the absorption and emission spec-
tra, whereas the addition of Ca2+ to this solution caused a
similar kind of fluorescence enhancement as that shown in
Figure 1. These observations show the unique ability of 1c
to selectively detect Ca2+, which is clear from the plot (Fig-
ure 2) of the variation of fluorescence intensity against the
ratio of the metal ion and 1c.
Figure 3. Fluorescence decay profiles of 1c (11 µ) at different con-
centrations of Ca2+ in acetonitrile; λex = 375 nm and λem = 436 nm:
(a) laser profile, (b) 1c alone, (c) 4.46, (d) 6.70, (e) 8.93, (f) 11.62,
(g) 15.62, (h) 22.32 µ of Ca2+
.
Metal ion binding studies of bichromophores 1a and 1b
showed similar behavior with weak response when com-
pared to those of 1c. However, both the bichromophores
showed maximum response towards Ca2+, which is the
same as in the case of 1c (see Supporting Information). Ad-
dition of Mg2+ showed weak response, and Na+, K+, and
Zn2+ did not show any measurable response. Binding con-
stants of 1a–c with various metal ions were calculated from
the changes in the fluorescence spectra, and these values
are shown in Table 2.
Figure 1. Fluorescence enhancement of 1c (11 µ) upon addition
of Ca2+ (0Ǟ16 µ) in acetonitrile; λex = 381 nm.
Table 2. Binding constants (logK) of 1a–c with various metal ions
in acetonitrile at 22 °C.
Mg2+
Ca2+
Na+
K+
Zn2+
[a]
[a]
[a]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
1a
1b
1c
3.69
3.83
4.83
[a] Changes were too small to calculate the binding constant.
[b] No response.
Figure 2. Plot of I/I0 vs. the ratio between metal ion and 1c in
acetonitrile, which illustrates the selectivity for Ca2+ (᭿) over Na+
(o), K+ (᭡), Mg2+ (᭹), and Zn2+
E
( ).
The highest binding constant was obtained for the bind-
The complex formation between Ca2+ and 1c was also ing of Ca2+ to bichromophore 1c. Compounds 1a and 1b
investigated by time-resolved fluorescence. Bichromophore showed relatively weak binding towards Ca2+. The binding
1c shows biexponential decay with a PET quenched lifetime constants of other metal ions could not be determined be-
of 0.78 ns (18.2%) and 2.83 ns (81.8%). Figure 3 presents cause of the insignificant changes in the corresponding
the fluorescence decay of 1c at different concentrations of fluorescence spectra. The difference in the binding ability
Ca2+ in acetonitrile. During the addition of Ca2+ to 1c, the of 1a–c with Ca2+ can be attributed to the number of oxy-
shorter component disappears gradually, and the longer gen atoms and the size of the pseudo-crown cavity. Binding
lifetime increases (2.83 to 5.68 ns) along with an increase affinity of macrocyclic crown ethers mainly depends on the
in the amplitude. An increase in the lifetime of the longer ionic size of the cation, whereas in pseudocyclic systems,
component in the presence of Ca2+ and the disappearance combined effects of the size of the pseudo-crown ether cav-
of the short component indicates the suppression of PET ity, number of oxygen atoms, the charge density, and the
process during the Ca2+ binding. We observe single ex- coordination number of the cations play a considerable role.
ponential decay with longer lifetime component (5.68 ns) This may be the reason for the specific binding of Ca2+ over
on complete complex formation between Ca2+ and 1c. Ad- other cations. This observation is in analogy to the previous
dition of other metal ions did not show any significant reports on selective binding of Ca2+ to similar podand
change in the decay profile of 1c.
chains.[8b,9b]
Eur. J. Org. Chem. 2009, 5941–5947
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P. Ashokkumar, V. T. Ramakrishnan, P. Ramamurthy
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1H NMR Binding Studies
EDTA are shown in Figure 5. Addition of EDTA shows
gradual quenching of emission intensity without any spec-
tral shift. When 1 equivalent of EDTA was added, the emis-
sion spectrum restored its original intensity of 1c. Time-
resolved studies also support the similar kind of decomplex-
ation. These results clearly indicate that the observed
changes in the photophysical properties of 1c with Ca2+ are
due to reversible complexation.
1
To unravel the structure of the complex in detail, a H
NMR spectroscopic study was carried out in [D3]acetoni-
trile at 22 °C. The spectra of 1c before and after addition
of Mg2+ and Ca2+ are shown in Figure 4.
Figure 5. Changes in the emission spectra of 1c-Ca2+ (11 µ:
11 µ) in acetonitrile with the addition of EDTA. λex = 381 nm: (1)
1c alone, (2) 1c + Ca2+, (3–9) 1c + Ca2+ + EDTA (1.8 µ Ǟ11 µ).
To confirm the involvement of a PET process from the
OCH3 group in the optical signaling of bichromophores
1a–c upon Ca2+ binding, reference compound 2 without a
PET group (OCH3) and monochromophore compound 3
with a PET group were synthesized and treated with all
four metal ions. No significant change in the absorption,
emission, or fluorescence lifetime was observed in 2. It is
interesting to see that 2 is optically silent towards Ca2+ de-
Figure 4. Partial 1H NMR spectra of 1c in CD3CN: (i) 1c alone,
(ii) 1c-Mg2+, and (iii) 1c-Ca2+
.
After the addition of Ca2+, the well-resolved oxyethylene
proton peaks (a–d) became broad and shifted to lower mag-
netic field (∆δ = 0.32–0.17 ppm) due to the reduction in the
electron density on the oxygen atom by the coordinated
metal ion. In contrast, the oxyethylene proton peak e (∆δ
= 0.16 ppm) nearest to the benzene ring and aromatic pro-
ton f (∆δ = 0.73 ppm) shifted to higher magnetic field as a
result of shielding by the uncoordinated oxygen atom. A
similar high magnetic field shift of aromatic proton f was
also observed in complex 1b-Ca2+ with a relatively lower ∆δ
value of 0.45 ppm, whereas in complex1a-Ca2+ all the pro-
tons appeared at lower magnetic field. This confirms that
the two oxygen atoms attached to the benzene rings move
away and effectively shield the adjacent protons in 1c and
1b-Ca2+, whereas in 1a all the oxygen atoms are involved in
Ca2+ binding. Addition of Mg2+ showed a smaller change,
whereas the other metal ions did not show any change in
the entire proton spectrum. This clearly reveals that there
is no binding between these ions and bichromophores 1a–
c.
1
spite their binding to 2. This is proved by the H NMR
spectroscopic studies of 2 in CD3CN in the presence of
Ca2+ (Supporting Information, Figure S17). Addition of
Ca2+ results in a considerable downfield shift of the oxye-
thylene proton peaks. Thus, whereas the NMR spec-
troscopy experiment clearly supports the binding of Ca2+
to 2, the optical silence strongly supports the proposed
mechanism where there is no possibility for a PET process.
In contrast, the addition of Ca2+ to monochromophore
model compound 3 shows a change similar to that observed
for 1c (Supporting Information, Figure S13). This clearly
proved that the optical output did not arise from the inter-
action of two terminal chromophore units; instead, it arises
within the single chromophore through a PET suppression
mechanism. Addition of Ca2+ induced folding of the
bichromophore, and the Ca2+ ions become nestled within
the oxyethylene moiety and the OCH3 groups. This binding
suppresses the PET process and also results in the rigidifica-
tion of the host molecular framework. This, in turn, results
Metal Ion Decomplexation Studies
Because of the high stability constant of the EDTA-Ca2+ in the observed fluorescence intensity and lifetime enhance-
complex, it was anticipated that addition of EDTA would ment.
induce decomplexation of Ca2+, thereby restoring the origi-
Even though the simple monochromophoric system 3 is
nal photophysical properties of 1c. The changes in the emis- equally suitable for Ca2+ sensing, the selectivity ratio of
sion spectrum of 1c-Ca2+ upon addition of a solution of Ca2+ over Mg2+ is comparatively lower in the monochromo-
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Specific Ca2+ Fluorescent Sensor
phore. The selectivity plot of monochromophore 3 towards matically and the sensor molecule eventually did not re-
different metal ions is shown in Figure 6. Because of the spond at all in 20% water. Even though the present system
greater flexibility of the oxyethylene chain and steric free- works well in acetonitrile medium, the concept of PET sup-
dom of the monochromophore, Mg2+ also shows a slight pression from the additional binding site incorporated into
enhancement in fluorescence intensity. So, we prefer the the spacer unit is the first of its kind.
metal ion binding studies with a bichromophore instead of
a monochromophore.
Conclusions
In conclusion, a chemosensor for the selective detection
of Ca2+ in the presence of other similar metal ions such as
Na+, K+, Mg2+, and Zn2+ involving an acridinedione-de-
rived foldamer, which works on the principle of a PET sup-
pression, was described. The optical silence of non-PET ref-
erence compound 2 and a similar response of mono-
chromophore compound 3 with Ca2+ support the suggested
signaling mechanism. The reversible complex formation be-
tween the bichromophore and Ca2+ was proved by restoring
the initial photophysical properties of 1c with the addition
of EDTA to the 1c-Ca2+ complex. However, for practical
Figure 6. Plot of I/I0 vs. the ratio between metal ion and 3 in aceto-
nitrile, which shows the response for Ca2+ (᭿) and Mg2+ (᭹) over
applications, it is necessary that the molecular probe de-
scribed here is able to detect Ca2+ under biological condi-
tions. The challenge here is the synthesis of highly water
soluble acridinedione derivatives that can act as a good
fluorophore under aqueous physiological conditions.
Na+ (o), K+ (᭡), and Zn2+
E
( ).
To clarify the structural change of monochromophore 3
in the presence of Ca2+, a 1H NMR spectroscopy study was
carried out in [D3]acetonitrile (Supporting Information,
Figure S18). During the addition of Ca2+, all the oxyeth-
ylene protons showed a lower magnetic field shift (∆δ =
0.21–0.08 ppm). This confirms that all of the oxygen atoms
are involved in Ca2+ binding, whereas in 1c and 1b, the
two oxygen atoms attached to the benzene rings were not
involved in Ca2+ binding as a result of steric crowdedness
and the flexibility of the oxyethylene moiety.
Experimental Section
General Procedures and Materials: Dimedone was purchased from
Lancaster (India), Ltd. Pentaethylene glycol, vanillin, and all metal
perchlorates were purchased from Sigma Aldrich Chemicals Pvt.
Ltd. and were used as received. Acetonitrile used in this investiga-
tion was of HPLC grade, purchased from Qualigens India, Ltd.
Pentaethylene glycol ditosylate and pentaethylene glycol monotos-
ylate were synthesized according to a reported procedure.[18] Ab-
sorption spectra were recorded with an Agilent 8453 diode array
spectrophotometer. Emission spectra were recorded with a Perkin–
Elmer MPF-44B fluorescence spectrophotometer interfaced with a
PC through Rishcom-100 multimeter and a HORIBA JOBIN
YVON Fluoromax 4P spectrophotometer. Fluorescence decays
were recorded by using an IBH time-correlated single-photon
counting technique as reported elsewhere. NMR spectra were re-
corded with Bruker Avance III 500 MHz and Bruker 300 MHz in-
struments in deuterated solvents as indicated; TMS or the residual
solvent peaks were used as internal standards. NMR peak assign-
ments were made possible by using HSQC and HMBC 2D NMR
spectral studies. Chemical shifts are reported in ppm and coupling
On the basis of 1H NMR and fluorescence spectral
changes of all the compounds, an expected structural
change in 1c in the presence of Ca2+ is depicted in Figure 7.
Before the formation of the complex, 1c shows weak fluo-
rescence due to the PET process from the OCH3 group.
After the addition of Ca2+, the PET process is suppressed
and results in a fluorescence enhancement. Because of the
practical application of the selective detection of Ca2+ in
aqueous media, we attempted the titration of Ca2+ with 1c
in various acetonitrile/water mixtures. As the percentage of
water was increased, the sensitivity for Ca2+ decreased dra-
1
constants (JX–XЈ) are reported in Hz. In the case of HNMR mea-
surements of metal complexes, a 3.1ϫ10–2  concentration of
chromophore were taken in [D3]acetonitrile. Metal ions were added
in excess amount to ensure complete formation of the complex.
MS (ESI) were performed with an ECA LCQ Thermo system with
ion-trap detection in positive and negative mode. Elemental C, H,
and N analyses were performed with a Euro EA Elemental ana-
lyzer.
Synthesis of BisADD-1c
4: To a solution of dimedone (5.0 g, 36 mmol) in aqueous methanol
(25 mL) was added vanillin (2.72 g, 18 mmol), and the mixture was
warmed until the solution became cloudy. (4-Hydroxy-3-methoxy-
benzylidene)bis(dimedone) (4; 6.95 g, 94%) started to separate out.
Figure 7. Schematic representation of Ca2+-induced folding of
bichromophore 1c in acetonitrile.
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5945

P. Ashokkumar, V. T. Ramakrishnan, P. Ramamurthy
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The reaction mixture was diluted with water and allowed to stand
overnight in the refrigerator; the tetraketone was collected by fil-
tration, dried, and recrystallized from methanol.
1a (1.06 g, 62%) as a yellow powder. M.p. 94–96 °C. FTIR (KBr):
˜
1
ν = 1631 [vs. (conj. CO)], 1369 [s (–C=C–) cm–1] cm–1. H NMR
(500 MHz, CDCl3, TMS): δ = 1.00 and 1.03 (2 s, 24 H, gem-di-
methyl), 2.22 (s, 8 H, C2 and C7 CH2), 2.34 and 2.61 (2 d, J = 16.5,
17 Hz, 8 H, C4 and C5 CH2), 3.25 (s, 6 H, NCH3), 3.67 (s, 4 H,
OCH2), 3.79 (t, 10 H, OCH2 and OCH3), 4.07 (t, 4 H, OCH2), 5.21
(s, 2 H, C9-H), 6.55–6.57 (dd, J = 2, 8.5 Hz, 2 H, ArH), 6.69 (d, J
= 8.5 Hz, 2 H, ArH), 6.94 (d, J = 2 Hz, 2 H, ArH) ppm. 13C NMR
(300 MHz CDCl3, TMS): δ = 28.7, 31.0, 32.7, 33.4, 40.6, 50.0, 55.9,
68.5, 69.7, 70.7, 112.5, 113.8, 115.0, 118.7, 139.3, 146.4, 149.2,
151.1, 195.5 ppm. MS (ESI): m/z = 934.26 [M + 1]+. C56H72N2O10
(933.18): calcd. C 72.08, H 7.78, N 3.00; found C 72.16, H 7.75, N
2.98.
5: A mixture of tetraketone 4 (2 g, 4.83 mmol) and methylamine
(40% solution, 0.42 mL, 4.83 mmol) was kept under reflux in acetic
acid (20 mL) for 6 h. After completion of the reaction as indicated
by TLC, the reaction mixture was cooled and poured into crushed
ice. The solid obtained was purified by recrystallization from
CHCl3/MeOH (8:2) to isolate acridinedione 5 (1.52 g, 77%) as a
brown solid. M.p. 223–225 °C. FTIR (KBr): ν = 3170 [br. (OH)],
˜
1640 [vs. (conj. CO)], 1367 [s (–C=C–)] cm–1. 1H NMR (300 MHz,
CDCl3, [D6]DMSO, TMS): δ = 1.00 and 1.03 (2 s, 12 H, gem-
dimethyl), 2.14 (s, 4 H, C2,C7 CH2), 2.41 and 2.75 (2 d, J = 17.4 Hz,
4 H, C4,C5 CH2), 3.27 (s, 3 H, NCH3), 3.68 (s, 3 H, OCH3), 4.96
(s, 1 H, C9-H), 6.42–6.45 (dd, J = 1.8, 8.1 Hz, 1 H, ArH), 6.54 (d,
J = 8.1 Hz, 1 H, ArH), 6.67 (d, J = 1.8 Hz, 1 H, ArH), 8.49 (s, 1
H, OH) ppm. 13C NMR (300 MHz, CDCl3, [D6]DMSO, TMS): δ
= 27.8, 28.4, 29.8, 32.1, 33.1, 40.4, 49.6, 55.3, 111.5, 113.3, 114.8,
118.9, 137.0, 144.3, 146.8, 152.0, 194.7 ppm. MS (ESI): m/z =
410.37 [M + 1]+. C25H31N1O4 (409.52): calcd. C 73.32, H 7.63, N
3.42; found C 73.53, H 7.68, N 3.39.
Synthesis of BisADD-2
6: Treatment of 4-hydroxybenzaldehyde (2.20 g, 18 mmol) with di-
medone (5.0 g, 36 mmol) afforded (4-hydroxybenzylidene)bis(di-
medone) (6; 5.62 g, 82%).
7: Refluxing a mixture of tetraketone 6 (2.0 g, 5.2 mmol) with meth-
ylamine (0.45 mL, 5.2 mmol) yielded acridinedione 7. The crude
compound was purified by recrystallization from CHCl3/MeOH
(6:4) to isolate 7 (1.48 g, 75%) as a bright yellow crystalline solid.
1c: To a solution of 5 (1.5 g, 3.66 mmol) in acetonitrile (20 mL)
was added anhydrous K2CO3 (1.52 g, 10.98 mmol). The reaction
mixture was stirred for 2 h at room temperature under an atmo-
sphere of nitrogen, to which pentaethylene glycol ditosylate
(0.80 mL, 1.83 mmol) in acetonitrile (10 mL) was added dropwise
with stirring. The whole mixture was then kept under reflux for
16 h. The hot reaction mixture was filtered, and the resultant solu-
tion was evaporated under reduced pressure. The residue was dis-
solved in dichloromethane, washed with distilled water, and dried
with MgSO4, and the solvents were evaporated. The product was
purified by column chromatography over silica gel (CHCl3/MeOH,
98:2) to isolate pure 1c (1.06 g, 57%) as a yellow powder. M.p. 80–
M.p. Ͼ260 °C. FTIR (KBr): ν = 3268 [br. (OH)], 1629 [vs. (conj.
˜
CO)], 1369 [s (–C=C–)] cm–1. 1H NMR (500 MHz, [D6]DMSO,
TMS): δ = 0.95 and 1.00 (2 s, 12 H, gem-dimethyl), 2.11 (d, J =
6.5 Hz, 4 H, C2 and C7 CH2), 2.41 and 2.76 (2 d, J = 17 Hz, 4 H,
C4 and C5 CH2), 3.26 (s, 3 H, NCH3), 4.92 (s, 1 H, C9-H), 6.52 (d,
J = 8.5 Hz, 2 H, ArH), 6.87 (d, J = 8.5 Hz, 2 H, ArH), 9.00 (s, 1
H, -OH) ppm. 13C NMR (500 MHz, [D6]DMSO, TMS): δ = 28.2,
28.8, 30.2, 32.6, 33.7, 40.5, 50.0, 113.7, 115.0, 128.4, 137.1, 152.7,
155.6, 195.3 ppm. MS (ESI): m/z = 380.43 [M + 1]+. C24H29N1O3
(379.49): calcd. C 75.96, H 7.70, N 3.69; found C 75.85, H 7.66, N
3.64.
82 °C. FTIR (KBr): ν = 1631 [vs. (conj. CO)], 1369 [s (–C=C–)]
˜
2: The reaction of 7 (1.5 g, 3.96 mmol) with pentaethylene glycol
ditosylate (0.86 mL, 1.98 mmol) in the presence of K2CO3 (1.52 g,
10.98 mmol) afforded bis(acridinedione) 2, which was purified by
column chromatography over silica gel (CHCl3/MeOH, 98:2) to
1
cm–1. H NMR (300 MHz, CDCl3, TMS): δ = 1.04 and 1.08 (2 s,
24 H, gem-dimethyl), 2.23 (s, 8 H, C2 and C7 CH2), 2.36 and 2.60
(2 d, J = 16.8 Hz, 8 H, C4 and C5 CH2), 3.26 (s, 6 H, NCH3), 3.63
(s, 4 H, OCH2), 3.64–3.68 (m, 8 H, OCH2), 3.78–3.81 (m, 10 H,
OCH2 and OCH3), 4.07 (t, 4 H, OCH2), 5.22 (s, 2 H, C9-H), 6.54–
6.57 (dd, J = 1.8, 8.2 Hz, 2 H, ArH), 6.68 (d, J = 8.4 Hz, 2 H,
ArH), 6.95 (d, J = 1.8 Hz, 2 H, ArH) ppm. 13C NMR (300 MHz,
CDCl3, TMS): δ = 28.7, 31.0, 32.7, 33.4, 40.6, 50.0, 55.9, 68.5, 69.6,
70.6, 70.7, 112.6, 113.7, 115.0, 118.7, 139.3, 146.4, 149.2, 151.0,
195.5 ppm. MS (ESI): m/z = 1022.23 [M + 1]+. C60H80N2O12
(1021.28): calcd. C 70.56, H 7.90, N 2.74; found C 70.43, H 7.92,
N 2.72.
isolate 2 (0.83 g, 44%) as a pale yellow powder. M.p. 140–142 °C.
1
FTIR (KBr): ν = 1631 [vs. (conj. CO)], 1368 [s (–C=C–)] cm–1. H
˜
NMR (500 MHz, CDCl3): δ = 0.98, 1.02 and 1.07, 1.09 (4 s, 24 H,
gem-dimethyl), 2.16 and 2.22 (2 d, J = 16.5, 16 Hz, 8 H C2 and C7
CH2), 2.35 and 2.58 (2 d, J = 17 Hz, 8 H, C4 and C5 CH2), 3.26
(s, 6 H, NCH3), 3.63 (s, 4 H, OCH2), 3.65–3.70 (m, 8 H, OCH2),
3.76–3.80 (m, 4 H, OCH2), 4.02 (t, 4 H, OCH2), 4.69 (s, 1 H, C9-
H); 5.19 (s, 1 H, C9Ј-H), 6.70 (d, J = 8.5 Hz, 2 H, ArH), 6.75 (d,
J = 8.5 Hz, 2 H, ArH), 7.11 (d, J = 8.5 Hz, 2 H, ArH); 7.18 (d, J
= 8.5 Hz, 2 H, ArH) ppm. 13C NMR (500 MHz, CDCl3, TMS): δ
= 27.3, 28.6, 28.7, 29.3, 30.9, 31.0, 32.2, 32.7, 33.4, 40.6, 40.9, 50.0,
50.8, 67.2, 69.7, 69.8, 70.6, 70.8, 114.2, 115.2, 115.8, 128.2, 128.5,
129.0, 129.3, 136.7, 138.4, 150.8, 156.9, 157.2, 162.1, 195.4,
196.4 ppm. MS (ESI): m/z = 962.17 [M + 1]+. C58H76N2O10
(961.23): calcd. C 72.47, H 7.97, N 2.91; found C 72.29, H 7.93, N
2.88.
BisADD-1b: By using the above procedure, addition of tetraethyl-
ene glycol ditosylate (0.50 mL, 1.23 mmol) to 5 (1.0 g, 2.44 mmol)
yielded 1b (0.66 g, 55%) as a yellow powder. M.p. 87–89 °C. FTIR
1
(KBr): ν = 1631 [vs. (conj. CO)], 1370 [s (–C=C–)] cm–1. H NMR
˜
(300 MHz, CDCl3, TMS): δ = 1.04 and 1.08 (2 s, 24 H, gem-di-
methyl), 2.23 (s, 8 H, C2 and C7 CH2), 2.36 and 2.59 (2 d, J =
16.8 Hz, 8 H, C4 and C5 CH2), 3.26 (s, 6 H, NCH3), 3.65 (t, 4 H,
OCH2), 3.79–3.82 (m, 11 H, OCH2 and OCH3), 4.06 (t, 4 H,
OCH2), 5.21 (s, 2 H, C9-H), 6.55–6.58 (dd, J = 1.8, 8.2 Hz, 2 H,
ArH), 6.68 (d, J = 8.4 Hz, 2 H, ArH), 6.95 (d, J = 1.8 Hz, 2 H,
ArH) ppm. 13C NMR (300 MHz, CDCl3, TMS): δ = 28.7, 31.0,
32.7, 33.4, 40.6, 50.0, 55.9, 68.5, 70.2, 70.7, 112.5, 113.7, 115.0,
118.7, 139.3, 146.4, 149.2, 151.0, 195.5 ppm. MS (ESI): m/z =
978.18 [M + 1]+. C58H76N2O11 (977.23): calcd. C 71.29, H 7.84, N
2.87; found C 71.18, H 7.87, N 2.84.
Monochromophore 3: The reaction of 5 (0.75 g, 1.83 mmol) with
pentaethylene glycol monotosylate (0.60 mL, 1.82 mmol) in the
presence of K2CO3 (0.76 g, 5.50 mmol) afforded monochromo-
phore 3, which was purified by column chromatography over silica
gel (CHCl3/MeOH, 97:3) to isolate 3 (0.60 g, 52%) as a pale yellow
semisolid. FTIR (KBr): ν = 3300 [br. (OH)], 1634 [vs. (conj. CO)],
˜
1367 [s (–C=C–)] cm–1. 1H NMR (500 MHz, CD3CN): δ = 1.03
and 1.07 (2 s, 12 H, gem-dimethyl), 2.18 (s, 4 H, C2 and C7 CH2),
2.45 and 2.72 (2 d, J = 17.5, 17 Hz, 4 H, C4 and C5 CH2), 2.93 (t,
1 H, -OH), 3.26 (s, 3 H, NCH3), 3.49 (t, 2 H, OCH2), 3.57–3.63
BisADD-1a: By using the above procedure, addition of triethylene
glycol ditosylate (0.84 g, 1.83 mmol) to 5 (1.5 g, 3.66 mmol) yielded
5946
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 5941–5947

Specific Ca2+ Fluorescent Sensor
[7]
[8]
(m, 14 H, OCH2), 3.73–3.74 (m, 5 H, OCH2 and OCH3), 4.02 (t,
2 H, OCH2), 5.05 (s, 1 H, C9-H), 6.67–6.69 (dd, J = 2, 8.5 Hz, 1
H, ArH), 6.74 (d, J = 8.0 Hz, 1 H, ArH), 6.79 (d, J = 2.0 Hz, 1 H,
ArH) ppm. 13C NMR (500 MHz, CD3CN): δ = 27.5, 27.7, 30.7,
32.2, 33.1, 39.7, 49.7, 55.2, 61.0, 68.2, 69.4, 70.1, 70.2, 70.3, 72.3,
111.6, 112.9, 113.6, 119.3, 139.5, 146.5, 149.0, 152.7, 195.5 ppm.
MS (ESI): m/z = 630.70 [M + 1]+. C35H51N1O9 (629.78): calcd. C
66.75, H 8.16, N 2.22; found C 66.68, H 8.19, N 2.18.
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.
Acknowledgments
The authors thank the Department of Science and Technology
(DST), Government of India for financial support through SERC
scheme project number DST/SR/S1/PC-31/2005. Financial support
by DST-IRHPA is also gratefully acknowledged. NMR facilities
provided at Sophisticated Analytical Instruments Facility (SAIF),
IIT Madras by DST is thankfully acknowledged.
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Received: May 23, 2009
Published Online: October 14, 2009
Eur. J. Org. Chem. 2009, 5941–5947
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5947