Al3+-ion-triggered conformational isomerization of a rhodamine b derivative evidenced by a fluorescence signal - A crystallographic proof

Article abstract of DOI:10.1002/ejic.201403091

A newly designed rhodamine B anisaldehyde hydrazone exhibits Al³⁺-ion-induced cis (L) to trans (L) conformational isomerization with respect to the xanthene moiety through a rotation about a N-N bond; the isomerization is indicated by a detectable naked-eye color change and a turn-on red fluorescence in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (EtOH/Water 1:9 v/v; pH 7.4) at 25 C. In support of this observation, detailed spectroscopic and physicochemical studies along with density function theory (DFT) calculations have been performed. This cis-to-trans conformational isomerization is due to Al³⁺ ion coordination, which induces this visual color change and the turn-on fluorescence response. To strengthen our knowledge of the conformational isomerization, detailed structural characterizations of the cis and trans isomers in the solid state were performed by single-crystal X-ray diffraction. To the best of our knowledge, this is the first structural report of both cis and trans conformational isomers for this family of compounds. Moreover, this noncytotoxic probe could be used to image the accumulation of Al³⁺ ions in HeLa and MCF-7 cell lines. A noncytotoxic rhodamine B anisaldehyde hydrazone exhibits an aluminium-ion-induced cis (L) to trans (L) conformational isomerization with respect to the xanthene moiety through a rotation about a N-N bond; the change is signaled by a red fluorescence response.

Full text of DOI:10.1002/ejic.201403091

FULL PAPER
DOI:10.1002/ejic.201403091
Al3+-Ion-Triggered Conformational Isomerization of a
Rhodamine B Derivative Evidenced by a Fluorescence
Signal – A Crystallographic Proof
Buddhadeb Sen,[a] Siddhartha Pal,[a] Samya Banerjee,[b]
Somenath Lohar,[a] and Pabitra Chattopadhyay*[a]
Keywords: Isomerization / Aluminum / Rhodamine derivatives / Structure elucidation / Fluorescence
A newly designed rhodamine B anisaldehyde hydrazone
exhibits Al3+-ion-induced cis (L) to trans (LЈ) conformational
isomerization with respect to the xanthene moiety through a
rotation about a N–N bond; the isomerization is indicated by
a detectable naked-eye color change and a turn-on red
nation, which induces this visual color change and the turn-
on fluorescence response. To strengthen our knowledge of
the conformational isomerization, detailed structural charac-
terizations of the cis and trans isomers in the solid state were
performed by single-crystal X-ray diffraction. To the best of
our knowledge, this is the first structural report of both cis
and trans conformational isomers for this family of com-
pounds. Moreover, this noncytotoxic probe could be used to
image the accumulation of Al3+ ions in HeLa and MCF-7 cell
lines.
fluorescence in
4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid (HEPES) buffer (EtOH/Water 1:9 v/v; pH 7.4) at
25 °C. In support of this observation, detailed spectroscopic
and physicochemical studies along with density function
theory (DFT) calculations have been performed. This cis-to-
trans conformational isomerization is due to Al3+ ion coordi-
In this manuscript, we report a rhodamine B hydrazone
derivative (L), which exhibits Al3+-ion-induced isomeriza-
tion from the cis conformation (L) to the trans conforma-
tion (LЈ) with respect to the xanthene moiety; the change is
evidenced by a noticeable naked-eye color change and a
turn-on red fluorescence signal. The selective transforma-
tion of the weakly fluorescent cis conformational isomer
(L) to its corresponding trans conformational isomer (LЈ)
within a short time through the addition of Al3+ ions was
confirmed by structural characterization of the trans isomer
(LЈ), which was isolated from the LЈ–Al complex upon
treatment with F– ions. Owing to the coordination of Al3+
ions to L, a rotation about the N–N bond results in the cis–
trans conformational isomerization from L to LЈ.
Introduction
Rhodamines are an advantageous class of photostable
dyes as their long-wavelength absorption with a high ab-
sorption coefficient and emission with excellent quantum
efficiency make them competent fluorescent biomarkers.[1–3]
Conformational isomerization plays a vital role in many
biological systems for the transmission of information
through the binding of an ion or molecule to another bind-
ing site, as structural changes result in dissimilarities in
physiological activities and pharmacological effects.[4,5]
Conformational isomerization is also significantly impor-
tant in the design of ion-controlled switching devices and
the manufacture of fine chemicals.[6–9] There are some re-
ports on thermally induced and photoinduced cis–trans
conformational isomerization,[10,11] but metal-ion-induced
cis–trans conformational isomerization is scarce.[12] Herein,
we describe the first observation of Al3+-ion-induced cis–
trans conformational isomerization, which was observed in
a new rhodamine B derivative through detailed structural
elucidations.
This Al3+-ion-induced selective isomerization of L has
been ascertained by UV/Vis, 1H NMR, and 13C NMR spec-
troscopy and ESI-MS, and the detailed structural analyses
of the cis and trans conformational isomers were established
by single-crystal X-ray diffraction studies support by theo-
retical calculations. This is an interesting type of Al3+-ion-
induced conformational isomerization that signals through
turn-on red fluorescence.
[a] Department of Chemistry, The University of Burdwan,
Golapbag, Burdwan, 713104, India
Results and Discussion
E-mail: pabitracc@yahoo.com
[b] Department of Inorganic and Physical Chemistry, Indian Insti-
tute of Science,
The cis derivative with respect to the xanthene moiety of
the rhodamine B hydrazone (L) was isolated from the reac-
tion of rhodamine B hydrazide with ortho-anisaldehyde in
Bangalore, 560012, India
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201403091.
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Figure 1. ORTEP views of (A) cis (L) and (B) trans (LЈ) conformers (30% probability) with atom numbering scheme (H atoms are
excluded for clarity).
a 1:1 molar ratio in dry ethanol (Scheme 2). Crystals of L π* charge-transfer (CT) transition. On stepwise addition of
were collected after the slow evaporation of the reaction Al3+ ions (0–20 μm), a new absorption peak at λ ≈ 563 nm
mixture over a few days and characterized by physicochemi- gradually developed owing to the formation of an LЈ–Al
cal and spectroscopic methods (Figures S1–S4, Supporting complex through ring opening, and a visual color change
Information); these crystals were identical to the crystals from colorless to pink was observed (Figure 2). In the pres-
obtained by the slow evaporation of a methanolic solution ence of excess biologically relevant alkali ions (Na+, K+),
of L. The detailed structural analysis of L, which is in the alkaline earth ions (Ca2+, Mg2+), transition metal ions
cis conformation with respect to the xanthene moiety, was (Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+), and
established by collecting the crystallographic data of a other heavy metal ions (Hg2+, Pb2+), no new bands were
single crystal suitable for X-ray diffraction study (Figure 1, produced, as they were unable to induce the ring-opening
A, Table 1 and Supporting Information, Tables S1–S2). The step.
crystal structure of L revealed the spirolactam configura-
tion (spiro atom C-16) with the metal-chelating residue or-
thogonal to the oxotricyclic ring system of the rhodamine
moiety, and this conformation makes the probes weakly
fluorescent. Therefore, both rings are supposed to act as
signal switchers, which show a turn-on response when
bound to Al3+ ions.
Table 1. Crystal data and details of refinements for L and LЈ.
L
LЈ
Empirical formula
Formula weight
Crystal system
Space group
a [Å]
b [Å]
c [Å]
C36 H38N4O3
574.70
C36 H38N4O3
574.70
monoclinic
P21/c
13.0204(5)
11.8790(5)
20.0062(7)
90
95.061(2)
90
triclinic
¯
P1
9.9945(4)
11.6134(5)
15.4804(7)
90.227(2)
102.174(2)
115.296(2)
1.209
1578.96(12)
296(2)
2
α [°]
β [°]
γ [°]
Figure 2. UV/Vis titration spectra of L (10 μm) in HEPES buffer
(1 mm, EtOH/water 1:9 v/v, pH 7.4) at 25 °C upon the incremental
addition of Al3+ ions (0–20 μm). Right: the naked-eye visual color
change from L to LЈ–Al (1).
Density [Mg/m3]
Volume [Å3]
Temperature [K]
Z
1.238
3082.3(2)
296(2)
The cis conformational isomer (L) exhibited weak
fluorescence at λ ≈ 580 nm (λex = 525 nm), which is inde-
pendent of the pH of the medium over the range 6.0–10.0
(Figure S10). On the gradual addition of Al3+ ions to the
solution of L, a fluorescence maximum at λ = 593 nm be-
comes visible with a redshift of 13 nm, and its intensity
increased with Al3+ concentration to 2 equiv. (Figure 3).
This strong fluorescence emission is due to the ring opening
4
F(000)
θ range [°]
Collected reflections
Independent reflections
Goodness-of-fit
R1 [IϾ 2σ(I)]
wR1 [IϾ 2σ(I)]
612
1224
2.668 to 28.359
7657
3789
1.030
0.0594
0.1560
2.478 to 28.293
7793
5436
1.049
0.0497
0.1244
The UV/Vis spectrum of the cis isomer (L) in 4-(2- of the spirolactam system of rhodamine B (Scheme 3). In
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) addition, the visual and fluorescence color changes owing
buffer (1 mm, EtOH/water 1:9 v/v, pH 7.4) was recorded at to the Al3+ ions were not perturbed by the presence of an
25 °C. The cis Isomer showed an absorption maximum at λ excess of biologically relevant alkali and alkaline earth
= 318–323 nm, which is attributed to the intramolecular π– metal ions (Na+, K+, Ca2+, Mg2+), transition metal ions
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(Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+), or other
A time-correlated single photon counting (TCSPC)
heavy metal ions (Hg2+, Pb2+), and these ions did not offer experiment was also performed to measure the average
this type of visual and fluorescence color change through fluorescence lifetimes (τav) of L and LЈ–Al at λem = 593 nm
ring opening. Therefore, the organic moiety (L) has an (Figure 4). From this study, it was observed that the average
excellent selectivity and specificity towards Al3+ ions over lifetime of the LЈ–Al species (2.54 ns) was greater than that
the other cations (Figures S11–S12).
of L (0.82 ns). From the equations τ–1 = kr + knr and kr =
Φf/τ, in which kr is the radiative rate constant and knr is the
total nonradiative rate constant, the values of kr and knr
for both the organic moiety L and the LЈ–Al species were
calculated and are tabulated in Table S3.[16] The values
clearly indicate the enhancement of the fluorescence owing
to the increase of the kr/knr ratio from 0.03 to 1.4 through
a chelation-enhanced fluorescence (CHEF) process.
Figure 3. Emission spectra of L (10 μm) in HEPES buffer (1 mm,
EtOH/water 1:9 v/v, pH 7.4) at 25 °C upon the incremental
addition of Al3+ ions (0–20 μm); λex = 525 nm. Inset: naked-eye
fluorescence color change from L to LЈ–Al (1).
This selectivity for Al3+ ions over all other ions is due to
selective chelate formation with L to afford an LЈ–Al com-
plex (Scheme 3), which was found to have 1:1 stoichiometry
through a Job plot of the fluorescence titration data (Fig-
ure S13). The probable formulation of the LЈ–Al complex
was also verified by the isolation of the LЈ–Al complex in
the solid state and its characterization by physicochemical
and spectroscopic methods (Figures S5–S8). The selectivity
of L towards Al3+ ions can also be ascribed to the strong
binding constant (K) of 1.7ϫ105 m–1, as determined by the
Benesi–Hilderbrand method (Figure S14).[13] To validate
the sensitivity and affinity of L towards Al3+ ions, the
detection limit was also calculated from the slope of the
calibration curve (S) in the lower region (Figure S15) and
the standard deviation of seven replicate measurements of
the zero level (σzero) through the equation: detection limit
= 3σ/S, and it was found to be 17.3 nM.[14]
Figure 4. Time-resolved fluorescence decay of L (10 μm) and L in
presence of added Al3+ ions in HEPES buffer (1 mm, pH 7.4) at
25 °C with a nano-LED of 550 nm as the light source; λem
=
593 nm.
Interestingly, on treatment with F– or OAc– ions, the
color of the LЈ–Al complex changed from pink to colorless
and the fluorescence was quenched promptly (Figure 5).[17]
On the basis of this observation, crystalline LЈ in the trans
conformation was obtained by treatment of an ethanolic
solution of LЈ–Al with an ethanolic sodium fluoride solu-
tion in an equimolar ratio.
To establish the formation of the LЈ–Al species, a 1H
NMR titration of L in [D6]dimethyl sulfoxide ([D6]DMSO)
was performed. The addition of Al3+ ions caused downfield
shifts of some characteristic peaks (from δ = 3.699 to
3.684 ppm for the methoxy group) and a reduction of the
singlet peak for the imine hydrogen atom (at δ = 9.00 ppm
in L) with an upfield shift (δ = 8.98 ppm in LЈ–Al complex),
but the peaks corresponding to the other hydrogen atoms
of the phenyl and xanthene moieties did not show any sig-
nificant changes (Figure S16). The formation of the LЈ–Al
species through ring opening has also been ascertained
from the 13C NMR spectra of L in absence and presence of
Al3+ ions, from which it was observed that the signal at δ
= 66.225 ppm, attributable to the tertiary carbon atom (sp3-
hybridized) of the spirolactam ring in L (C-4), was absent in
Figure 5. Anion selectivity of LЈ–Al in the presence of different
anions in HEPES buffer (1 mm, EtOH/water 1:9 v/v, pH 7.4) at
the spectrum of the LЈ–Al complex (Figures S3 and S7).[15] 25 °C.
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The single crystals of LЈ (trans conformation) collected the band at λ = 341.68 nm is dominated by the HOMO–
on slow evaporation of this reaction mixture were studied 2ǞLUMO excitation, the band at λ = 320.44 nm is domi-
by single-crystal X-ray diffraction. The structural analysis nated by the HOMOǞLUMO+1 excitation, the band at λ
of LЈ showed that the methoxy group of the phenyl ring is = 287.78 nm is mainly due to HOMOǞLUMO+3 transi-
in a trans orientation with respect to the xanthene moiety tions, and the band at λ = 274.21 nm is mainly due to
(Figure 1, B). Compound LЈ crystallized in the monoclinic HOMO–2ǞLUMO+1 transitions (Figure S18). The de-
system in space group P21/c, whereas L (cis conformation) tails of the vertical excitation energies, oscillator strengths,
¯
crystallized in the triclinic system in space group P1. Al- and salient transitions are shown in Table S7. For LЈ–Al,
though electron delocalization is present in the N1/N2/C8 the band at λ = 528.54 nm is dominated by the HOMO–
fragment and there is a small amount of double-bond char- 1ǞLUMO and HOMO–1ǞLUMO+1 transitions, the
acter in the N–N bond (the single N–N bond length is band at λ = 351.02 nm is mainly due to HOMO–
1.47 Å, and an average value of 1.38 Å was measured here), 5ǞLUMO,
HOMO–2ǞLUMO,
and
HOMO–
the two molecules can be seen as different conformations 2ǞLUMO+1 transitions (Figure S19), as tabulated in
that result from a rotation of ca. 180° about the N1–N2 Table S8. The detailed theoretical studies, including the
bond. The C16–N1–N2–C8 torsion angles are 173.9° in LЈ TDDFT calculations (Table S5–S8), are in good agreement
and 13.7° in L. The molecules are closely comparable with with the experimental observations.
a slight difference in the bending of the two phenyl groups
of the rhodamine moiety, which form dihedral angles of
13.06 and 22.12° in LЈ and L, respectively. Under the same
reaction conditions, this isolated LЈ gave a similar LЈ–Al
species with Al3+ ions to that obtained from L. Further-
more, LЈ was also recovered from this isolated LЈ–Al com-
plex upon treatment with F–/OAc– ions by following the
above-mentioned procedure (Scheme 1).
Scheme 1. Detailed mechanistic pathway of metal-ion-induced con-
formational isomerization.
The UV/Vis spectrum of LЈ in HEPES buffer is identical
to that of L, but the fluorescence intensity of LЈ is some-
Figure 6. Molecular orbitals (MOs) of L, LЈ, and their correspond-
what greater than that of L by a factor of ca. 2.5 (Fig-
ing Al complexes (H atoms are omitted for clarity).
ure S17). Upon the addition of 2.0 equiv. of Al3+ ions to
the solution of L, an approximately 34-fold enhancement of
Here, the coordination of Al3+ ions with the O atom of
the fluorescence intensity was observed with a increase in the C–O group of the rhodamine moiety and the imine N
quantum yield from 0.03 to 0.59, and the same amount of atom assists the cis-OMe group (with respect to the
added Al3+ ions to the solution of LЈ caused an increase of xanthene moiety) in L to overcome the steric hindrance be-
fluorescence of ca. 11 times, and the quantum yield tween the cis-OMe group and the xanthene moiety and
changed from 0.2 to 0.59 (Table S4).
transform to its corresponding trans orientation through a
The energy values obtained from ground-state theoretical rotation about the N–N bond. Thus, the LЈ–Al complex,
DFT calculations clearly indicate that both the highest formulated as [Al(LЈ)(NO3)2(H2O)2][NO3], is formed, and
occupied molecular orbital (HOMO) and the lowest un- L (cis conformation) transforms into LЈ (trans conforma-
occupied molecular orbital (LUMO) of LЈ (trans conforma- tion) in situ (Scheme 3). As a result of this type of complex
tion) are more stabilized than those of L (cis conformation; formation, a chelation-enhanced fluorescence (CHEF) pro-
Figure 6). In L, the electron density mainly resides on half cess was observed. Again, fluoride or acetate ions added
of the xanthene moiety, and there is some electron density to the solution of the LЈ–Al complex substituted the aqua
on the C=O moiety. However, in LЈ, the electron density is molecules of the coordination sphere of the Al3+ ions to
mainly on the full xanthene moiety with some on the C=O afford LЈ (trans conformation) from LЈ–Al (Scheme 3).
moiety. Energy optimization calculations revealed that the
Additionally, the stability of the LЈ–Al species has also
LЈ–Al complex is much more stable than the L–Al complex, been examined by employing L in biological systems to
as the energy gap between the HOMO and LUMO in the acquire fluorescence images. Compound L was applied to
LЈ–Al complex (1.231 eV) is lower than that of the L–Al human cervical cancer (HeLa) cells and breast cancer cells
complex (2.369 eV). The UV/Vis spectra in water computed (MCF-7). A very faint intracellular fluorescence was cap-
from time-dependent DFT (TDDFT) calculations show tured after incubation with L (10 μm) for 30 min. However,
two important bands in the range λ = 300–550 nm. For L, the cells exhibited strong fluorescence when exogenous Al3+
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First, the rhodamine B hydrazide 1 was prepared by a literature
method.[18] In brief, 85% hydrazine hydrate (4 mL) was added to a
solution of rhodamine B (1 g, 2.09 mmol) in ethanol (40 mL). The
solution was heated to reflux for 6 h. Then, the reaction mixture
was evaporated under reduced pressure to give an orange oil, which
was then recrystallized from methanol/water to afford rhodamine B
hydrazide as light orange crystals (77%).
ions were applied into the cells (Figures 7, S20, and S22).
In addition, an in vitro study showed no cytotoxic effects
of L on the cells for up to 6 h, and the calculated IC50 value
is greater than 50 μm (Figures S21 and S23).
2-Methoxybenzaldehyde (136 mg, 1.0 mmol) was dissolved in
ethanol, and this solution was added to an ethanolic solution of
rhodamine B hydrazide (456 mg, 1.0 mmol) with stirring. Then, the
resulting solution was heated to reflux for 6 h. The solution was
evaporated to a small volume and cooled. The white crystalline
product was collected by filtration and then recrystallized from
pure methanol. Single crystals were obtained from this solution,
one of which was selected for the crystallographic study, yield 78%,
m.p. (178Ϯ2) °C. C36H38N4O3 (574.70): calcd. C 75.24, H 6.66, N
Figure 7. Fluorescence image of HeLa and MCF-7 cells after
incubation with L (10 μm) for 30 min (1, 1Ј) followed by treatment
with Al3+ ions (10 μm) at 37 °C (2, 2Ј).
9.75; found C 75.49, H 6.54, N 9.93. IR: ν = 3433 [νOH(H O)], 3076
˜
2
(νNH), 2968 [νC=C(aromatic)], 1726 (νC=O), 1614 (νCH=N) cm–1. 1H
NMR (300 MHz, [D6]DMSO): δ = 9.00 (s, 1 H, CH=N), 7.87 (d,
1 H), 7.58–7.55 (m, 2 H), 7.54–7.47 (d, 1 H), 7.30 (t, 1 H), 7.05 (d,
1 H), 6.95 (d, 1 H), 6.87 (t, 1 H), 6.28–6.41 (m, 3 H), 3.69 (s, 3 H,
OMe), 3.27 (q, 8 H, 4CH2), 1.04 (t, 12 H, 4CH3) ppm. 13C NMR
([D6]DMSO): δ = 164.530, 158.675, 153.589, 152.242, 149.356,
143.968, 134.676, 132.559, 129.755, 129.618, 128.518, 125.576,
Conclusions
This study represents the first manifestation of Al3+-ion-
induced conformational isomerization of a rhodamine de- 124.752, 123.845, 123.652, 121.563, 112.931, 108.945, 106.251,
98.141, 66.225, 56.575, 44.590, 13.305 ppm. HRMS (ESI, ethanol):
rivative through a N–N bond rotation. The conformational
change signals through a red fluorescence response and was
studied through thorough structural characterization. Inter-
estingly, this rhodamine derivative may be useful in the field
of ion-controlled switching devices. Although there are a
huge number of reports on rhodamine B derivatives as se-
lective sensors for several ions through chelation-enhanced
fluorescence (CHEF) or Förster resonance energy transfer
(FRET) processes, it is notable that none of the resulting
complexes or ensembles have been structurally established.
As a result, it is not known whether the fluorescence
calcd. for C36H39N4O3 [M + H]+ 575.2944; found 575.3147.
LЈ–Al Complex {[Al(LЈ)(NO3)2(H2O)2][NO3]}: To an ethanolic
solution (10 mL) of L (0.1 mmol, 58 mg), a solution of aluminium
nitrate (0.1 mmol, 38 mg) was added dropwise, and the mixture was
stirred for 4 h. The solvent was reduced to a small volume with a
rotary evaporator, and the solution was kept in a beaker to afford
a crystalline blood red solid on slow evaporation (Scheme 3). The
pure solid was collected by filtration, washed with methanol, and
then dried in vacuo. C36H42AlN6O11 (761.73): calcd. C 56.76, H
5.56, N 11.03; found C 56.57, H 5.50, N 11.17. IR: ν = 3452 (νNH),
˜
1614 (νC=N), 1383 [νN–O(NO ,sym)] cm–1. 1H NMR (300 MHz,
3
changes occurred along with this type of structural change [D6]DMSO): δ = 8.98 (s, 1 H, CH=N), 7.87 (d, 1 H), 7.88–7.56 (m,
in the probes or not.[11c] Thus, this work will help us to
2 H), 7.53–7.46 (d, 1 H), 7.29 (t, 1 H), 7.05 (d, 1 H), 6.94 (d, 1 H),
6.87 (t, 1 H), 6.29–6.42 (m, 3 H), 3.68 (s, 3 H, OMe), 3.27 (q, 8 H,
explain this type of work in a proper way.
4CH2), 1.04 (t, 12 H, 4CH3) ppm. 13C NMR ([D6]DMSO): δ =
165.060, 158.722, 153.486, 152.292, 151.006, 149.445, 143.566,
134.024, 132.728, 129.8805, 129.237, 128.502, 125.747, 124.644,
124.001, 123.359, 121.797, 112.979, 109.121, 105.998, 98.374,
Experimental Section
Probe L: The probe L was synthesized by a two-step reaction
(Scheme 2).
56.581, 44.732, 13.227 ppm. HRMS (ESI, methanol): calcd. for
[M]+ 761.2727; found 761.3310 (ca. 10% abundance).
Scheme 2. Synthesis of the probe (L).
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Scheme 3. Synthetic pathway for LЈ–Al and LЈ from L.
Compound LЈ: To reclaim the probe from the LЈ–Al complex, we
wanted to examine the quenching of fluorescence by adding dif-
tical electronic excitations based on B3LYP-optimized geometries
were computed by the time-dependent density functional theory
–
–
–
ferent anions (CN–, OAc–, F–, Cl–, Br–, I–, N3 , H2PO4 , NO3 , (TD-DFT)[22] formalism with water as the solvent, the conductor-
ClO4 , SO42–, and H2AsO4 ) to ethanolic solutions of LЈ–Al com-
plex. Interestingly, in this experiment, we noticed that the fluores-
cence of the LЈ–Al complex was dramatically quenched by the ad-
dition of only AcO– or F– anions, and no noticeable changes were
observed with the other anions, as shown in Figure 5. Additionally,
it is worth mentioning that this LЈ–Al complex can also be used as
an AcO–/F– anion selective dual sensor.
like polarizable continuum model (CPCM)[23] was used to calculate
the fractional contributions of various groups to each molecular
orbital. The lowest 20 singlet states along the vertical excitation
energies are computed here.
–
–
Preparation of Cell and in Vitro Cellular Imaging with L: Human
cervical cancer cells (HeLa) and breast cancer cells (MCF-7) were
purchased from the National Center for Cell Science (NCCS),
Pune, India and used throughout the study. The cells were cultured
in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL)
supplemented with 10% fetal bovine serum (FBS, Gibco BRL) and
a 1% antibiotic mixture containing penicillin, streptomycin, and
neomycin (PSN, Gibco BRL) at 37 °C in a humidified incubator
with 5% CO2. For the experimental study, the cells were grown to
80–90% confluence, harvested with 0.025% trypsin (Gibco BRL)
and 0.52 mm ethylenediaminetetraacetic acid (EDTA; Gibco BRL)
in phosphate-buffered saline (PBS, Sigma Diagnostics), plated at
the desired cell concentration, and allowed to re-equilibrate for 24 h
before any treatment. The cells were rinsed with PBS and incubated
with DMEM containing L (10 μm, 1% DMSO) for 30 min at 37 °C.
All experiments were conducted in DMEM containing 10% FBS
and 1% PSN antibiotic. The imaging system was composed of a
fluorescence microscope (ZEISS Axioskop 2 plus) with an objective
lens [10ϫ].
On the basis of the above observation, an ethanolic solution of
sodium fluoride (80 mg) was added to a solution of LЈ–Al (300 mg,
0.4 mmol) in ethanol (25 mL) with stirring. The mixture was stirred
for 30 min at room temperature, and then the solution was kept
aside. The crystalline LЈ was obtained from this solution on slow
evaporation within a few days; crystals suitable for single-crystal
X-ray diffraction were also collected, yield 85%, m.p. (150Ϯ2) °C.
C36H38N4O3 (574.70): calcd. C 75.24, H 6.66, N 9.75; found C
75.52, H 6.58, N 9.96. IR: ν = 3460 [ν
], 3076 (νNH), 2968
˜
OH(H2O)
[νC=C(aromatic)], 1762 (νC=O), 1614 (νCH=N) cm–1. HRMS (ESI, eth-
anol): calcd. for C36H39N4O3 [M + H]+ 575.2944; found 575.3021
(100%).
X-ray Crystallography: The X-ray data of suitable crystals of L and
LЈ were collected with a Bruker Apex-II CCD diffractometer with
Mo-Kα radiation (λ = 0.71073). The data were corrected for Lo-
rentz and polarization effects, and empirical absorption corrections
were applied with SADABS from Bruker. The structures were
solved by direct methods with SIR-92 and refined by full-matrix
least-squares refinement methods based on F2 with using SHELX-
97.[19] All calculations were performed with the WinGX package.[20]
Important crystallographic parameters are given in Table 1, and the
corresponding bond lengths and angles are tabulated in Tables S1
and S2. CCDC-993884 (for L) and -1008295 (for LЈ) contain the
supplementary crystallographic data for this paper. These data can
be obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Cell Cytotoxicity Assay: To test the cytotoxicity of L, a 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
was performed by the procedure described previously.[24] After
treatments of the probe (5, 10, 20, 50, and 100 μm), MTT solution
(10 μL, 10 mg/mL PBS) was added to each well of a 96-well culture
plate and incubated continuously at 37 °C for 6 h. All media were
removed from the wells and replaced with acidic 2-propanol
(100 μL). The intracellular formazan crystals (blue-violet) that
formed were dissolved with 0.04 n acidic 2-propanol, and the ab-
sorbance of the solution was measured at 593 nm wavelength with
a microplate reader. The values are means Ϯ standard deviations
(SDs) of three independent experiments. The cell cytotoxicity was
calculated on the basis of a cell viability of 100%.
Theoretical Calculations: To clarify the understanding of the
ground-state configurations of L, LЈ, and their corresponding com-
plexes, DFT calculations were performed with the Gaussian-09
software over a Red Hat Linux IBM cluster. Molecular-level inter-
actions have also been studied by density functional theory (DFT)
with the B3LYP/6-31G (d,p) functional model and basis set.[21] Ver-
Supporting Information (see footnote on the first page of this arti-
cle): Procedures, characterization data, and spectroscopic data.
Eur. J. Inorg. Chem. 2015, 1383–1389
1388
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjic.org
FULL PAPER
Yang, J. X. Zhang, B. W. K. Chu, N. Y. Zhu, Organometallics
2001, 20, 4911.
Acknowledgments
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B. S. is thankful to the University Grants Commission (UGC),
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the single-crystal X-ray diffractometer facility under the PURSE
program and Prof. Ennio Zangrando, Dipartimento di Scienze Chi-
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Received: November 15, 2014
Published Online: February 9, 2015
Eur. J. Inorg. Chem. 2015, 1383–1389
1389
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim