α-Amino Acid Derived Benzimidazole-Linked Rhodamines: A Case of Substitution Effect at the Amino Acid Site toward Spiro Ring Opening for Selective Sensing of Al3+ Ions

Article abstract of DOI:10.1021/acs.inorgchem.7b00835

α-Amino acid derived benzimidazole-linked rhodamines have been synthesized, and their metal ion sensing properties have been evaluated. Experimentally, l-valine- and l-phenylglycine-derived benzimidazole-based rhodamines 1 and 2 selectively recognize Al³⁺ ion in aqueous CH₃CN (CH₃CN/H₂O 4/1 v/v, 10 mM tris HCl buffer, pH 7.0) over the other cations by exhibiting color and "turn-on" emission changes. In contrast, glycine-derived benzimidazole 3 remains silent in the recognition event and emphasizes the role of α-substitution of amino acid undertaken in the design. The fact has been addressed on the basis of the single-crystal X-ray structures and theoretical calculations. Moreover, pink 1·Al³⁺ and 2·Al³⁺ ensembles selectively sensed F^- ions over other halides through a discharge of color. Importantly, compounds 1 and 2 are cell permeable and have been used as imaging reagents for the detection of Al³⁺ uptake in human lung carcinoma cell line A549.

Full text of DOI:10.1021/acs.inorgchem.7b00835

Article
pubs.acs.org/IC
α‑Amino Acid Derived Benzimidazole-Linked Rhodamines: A Case of
Substitution Effect at the Amino Acid Site toward Spiro Ring
Opening for Selective Sensing of Al3+ Ions
Anupam Majumdar,† Subhendu Mondal,† Constantin G. Daniliuc,‡ Debashis Sahu,§
Bishwajit Ganguly,*,§ Sourav Ghosh,∥ Utpal Ghosh,∥ and Kumaresh Ghosh*,†
†Department of Chemistry, University of Kalyani, Kalyani 741235, India
‡Organisch-Chemisches Institut, Universitat Munster, Corrensstrasse 40, 48159 Munster, Germany
̈
̈
̈
§Computation and Simulation Unit (Analytical Discipline and Centralized Instrument Facility), CSIR-Central Salt and Marine
Chemicals Research Institute, Bhavnagar, Gujarat 364002, India
∥Department of Biochemistry & Biophysics, University of Kalyani, Kalyani 741235, India
S
* Supporting Information
ABSTRACT: α-Amino acid derived benzimidazole-linked
rhodamines have been synthesized, and their metal ion sensing
properties have been evaluated. Experimentally, L-valine- and
L-phenylglycine-derived benzimidazole-based rhodamines 1
and 2 selectively recognize Al3+ ion in aqueous CH3CN
(CH3CN/H2O 4/1 v/v, 10 mM tris HCl buffer, pH 7.0) over
the other cations by exhibiting color and “turn-on” emission
changes. In contrast, glycine-derived benzimidazole 3 remains
silent in the recognition event and emphasizes the role of α-
substitution of amino acid undertaken in the design. The fact
has been addressed on the basis of the single-crystal X-ray
structures and theoretical calculations. Moreover, pink 1·Al3+ and 2·Al3+ ensembles selectively sensed F− ions over other halides
through a discharge of color. Importantly, compounds 1 and 2 are cell permeable and have been used as imaging reagents for the
detection of Al3+ uptake in human lung carcinoma cell line A549.
only a difference in substituents of the amino acid derived
benzimidazoles which corroborate different sensing behaviors
toward metal ions. While compounds 1 and 2 are fluorometri-
cally and colorimetrically sensitive to Al3+ ions in aqueous
CH3CN, under identical conditions compound 3 has been
observed to be insensitive to all the metal ions examined.
INTRODUCTION
■
The design and synthesis of optical signaling systems based on
organic scaffolds for sensing and recognition of biologically and
environmentally important metal ions has attracted a great deal
of attention.1 Optical cation sensors, where the interaction with
a cation leads to a change in the absorbance (color) or
fluorescence properties of the receptor, are the most widely
suitable class of cation sensors. Fluorometric and colorimetric
methods for sensing of metal ions are important tools due to
the operational simplicity and low detection limits.2 As a
fluorophore and chromophore, the rhodamine fluorochrome
has drawn the attention of chemists to develop sensors due to
its excellent photophysical properties and, in this regard,
various rhodamine-based fluorescent probes for cations, anions,
and other analytes have been reported.3 In spite of significant
reports on rhodamine-based molecular probes for metal ions,
the use of amino acid derived benzimidazole-based rhodamines
in this capacity are unknown.
Our continued interest in ion sensing4 inspired us to develop
α-amino acid derived benzimidazole-linked rhodamine mole-
cules 1−3 for metal ion sensing, both colorimetrically and
fluorimetrically. Structural analysis of the compounds reveals
that all three compounds are structurally the same. There is
In cation recognition, among various metal ions aluminum is
biologically relevant. It is the third most abundant element
Received: April 1, 2017
© XXXX American Chemical Society
A
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a
Scheme 1
a
Legend: (a) (i) o-phenylenediamine, DCC, dry CH2Cl2, 19 h, (ii) AcOH, 80-90 °C, 2 h; (iii) 50% TFA in CH2Cl2, 3 h; (b) (i) POCl3,
ClCH2CH2Cl, reflux, 2 h; (ii) 10/11/12, Et3N, dry CH2Cl2, 6 h.
Figure 1. (a) Fluorescence spectra of 1 (c = 1.5 × 10−5 M) with different metal ions (15 equiv) in CH3CN/H2O (4/1 v/v; 10 mM tris HCl buffer;
pH 7). (b) Fluorescence titration spectra of 1 (c = 1.5 × 10−5 M) in CH3CN/H2O (4/1 v/v; 10 mM tris HCl buffer; pH 7) upon addition of Al3+ (c
= 1.2 × 10−3 M; λexc 510 nm). Inset: emission intensity of 1 at 584 nm as a function of Al3+ concentration and color change of the solution of 1
under illumination of UV light.
(∼8% of total mineral components) in the earth’s crust.5
Though aluminum is a nonessential and toxic element for
biological systems, our daily life is closely allied with aluminum-
based compounds such as in food additives, medicines, cooking
utensils, cars, computers etc. Due to the increased solubility of
aluminum minerals through acid rain, aluminum toxicity
disturbs aquatic life and agricultural production.6 According
to the World Health Organization (WHO), the permissible
limit of daily intake of Al3+ by humans is about 3−10 mg with a
weekly tolerable dietary intake of 7 mg kg−1 body weight.7
Excessive accumulation of Al3+ ions in the human body affects
neurological systems that may lead to Parkinson’s disease,
Alzheimer’s disease, etc.8 This ion also acts as a competitive
inhibitor for various essential elements such as Mg2+, Ca2+, and
Fe3+ and affects the metabolism of iron by influencing the
absorption of iron via the intestine, hindering iron transport in
the serum and displacing iron by binding to transferrin.9
The poor coordination and strong hydration ability of Al3+
ion in water are some important features that have drawn
attention to the development of new classes of compounds for
its sensing.10 A literature survey reveals that rhodamine-coupled
architectures for colorimetric detection of Al3+ ions are fewer in
number in comparison to other cations.11
RESULTS AND DISCUSSION
■
Synthesis. The synthetic pathways of compounds 1−3 are
outlined in Scheme 1. The N-Boc-protected amino acids were
transformed into the corresponding benzimidazole amines
(10−12) after performing a series of reactions as mentioned in
Scheme 1a. The amines were next reacted with the rhodamine
B acid chloride 13 in the presence of Et3N in dry CH2Cl2 to
afford the desired compounds 1−3 in appreciable yields. All of
the compounds were fully characterized by the usual
spectroscopic techniques.
Interaction Study. The photophysical properties of the
rhodamine-based compounds 1−3 were studied by monitoring
emission and absorption changes upon addition of various
metal ions such as Al3+, Cr3+, Fe3+, Hg2+, Co2+, Pb2+, Fe2+, Cu2+,
Ni2+, Mg2+, Zn2+, Cd2+, and Ag+ (taken as their perchlorate
salts, but for Al3+, Al2(SO4)3 was used) in CH3CN/H2O
B
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Figure 2. (a) Fluorescence titration spectra of 2 (c = 1.5 × 10−5 M) in CH3CN/H2O (4/1 v/v; 10 mM tris HCl buffer; pH 7) upon addition of Al3+
(c = 1.2 × 10−3 M). Inset: emission intensity of 2 at 587 nm as a function of Al3+ concentration and color change of the solution of 2 under
illumination of UV light. (b) Fluorescence spectra of 2 (c = 1.5 × 10−5 M) with different metal ions (15 equiv) in CH3CN/H2O (4/1 v/v; 10 mM
tris HCl buffer; pH 7) (λexc 510 nm).
Figure 3. UV−vis titration spectra of (a) 1 (c = 1.5 × 10−5 M) and (b) 2 (c = 1.5 × 10−5 M) with Al3+ (c = 1.2 × 10−3 M) ions in CH3CN/H2O (4/1
v/v; 10 μM tris HCl buffer; pH 7.0). Inset: absorbance of 1 and 2 at ∼560 nm as a function of Al3+ concentration and color changes upon addition of
Al3+ ions.
Figure 4. (a) Suggested binding modes of 1 and 2 with Al3+ ion and 1H NMR (d6-DMSO, 400 MHz). Changes of (b) 1 (c = 1.6 × 10−2 M) and (c)
2 (c = 1.6 × 10−2 M) with 1 equiv of Al2(SO4)3·18H2O.
C
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(CH3CN/H2O 4/1 v/v, 10 mM tris HCl buffer, pH 7). In the
absence of metal ions, all three benzimidazole-coupled probes
were almost nonfluorescent and colorless. However, upon
interaction with only Al3+ ions, the L-valine-derived benzimi-
dazole-coupled rhodamine 1 (Φ = 0.011), on excitation at 510
nm, gave a nonstructured emission at 584 nm (Φ = 0.518) with
significant intensity and the solution became pink. Other metal
ions did not bring any change in emission spectra (Figure 1a
and Figure S1 in the Supporting Information). Figure 1b shows
the emission titration spectra of 1 with Al3+ ions and also the
associated color change of the solution under illumination of
UV light.
ppm upon complexation with an equivalent amount of Al3+.
The signals at 7.23 and 7.13 ppm for the protons of types a and
b (see labeling in Figure 4a) merge together at 7.22 ppm during
complexation (Figure 4b). A similar pattern was also observed
for the probe 2. The signal for benzimidazole NH at 11.48 ppm
also underwent a downfield chemical shift by 0.11 ppm. The
overlapping of the signals at 7.43 and 7.34 ppm for the protons
of types a and b was observed as for 1 (Figure 4c). These
observations in 1H NMR altogether reveal that the
benzimidazole −NHs in 1 and 2 remain intact during
interaction with Al3+ and follow the binding mode shown in
Figure 4a.
Under identical conditions, compound 2 (Φ = 0.01) was
titrated with the same metal ions and, in the study, a
fluorescence enhancement (∼600-fold) at 587 nm (Φ =
0.642) was observed upon interaction with Al3+ ions (Figure
2a) and the solution became pink, like that of 1. Emission at
587 nm was not observed in the presence of other metal ions
examined (Figure 2b and Figure S2 in the Supporting
Information). The plots in Figure 1a and Figure 2b, in this
regard, clearly show that the probes 1 and 2 are selective to Al3+
over other metal ions by exhibiting “turn-on” emission changes.
In a UV−vis study, the change in absorption of 1 upon
titration with Al3+ was followed. Upon addition of Al3+, a new
absorption band centered at 559 nm (ε = 2.02 × 104 M−1
cm−1) appeared with increasing intensity and there was a color
change from colorless to pink (Figure 3a). The absorption band
at 313 nm was increased to a small extent. Other metal ions in
the study were silent and did not induce any change in either
color or absorption spectra (Figure S3 in the Supporting
Information). A similar change in the absorption profile was
noticed for 2 on titration with Al3+ ions (Figure 3b). The
absorption at 275 nm decreased with the appearance of a new
absorption at 561 nm (ε = 4.8 × 104 M−1 cm−1) upon gradual
addition of Al3+. This resulted in an isosbestic point at 313 nm.
As for 1, other metal ions used in the study did not produce
such changes in the absorption spectra of 2 (Figure S4 in the
Supporting Information).
Interestingly, when we move to compound 3 (Φ = 0.016)
with an aim to understand the interaction with the same metal
ions under similar conditions, no measurable change in either
absorption or emission spectra (Figure S7 in the Supporting
Information) was observed except for Al3+. In fluorescence, on
excitation at 510 nm, a small fluorescence enhancement (∼7-
fold) at ∼580 nm (Φ = 0.066) was observed after addition of
greater amounts of Al3+ ions (50 equiv) and the colorless
solution of the probe remained colorless (Figure S8a in the
Supporting Information).
In the UV−vis spectrum, a very low intense absorption band
at ∼560 nm (ε = 3.26 × 103 M−1 cm−1) was found in the
presence of greater amounts of Al3+ ion, but the probe solution
was colorless. Figure S8b in the Supporting Information
represents the UV−vis titration spectrum of 3 with Al3+ ions.
Thus, the probe 3 failed to exhibit any appreciable “turn-on”
response to Al3+ ions, which clearly indicated that the spiro ring
remained intact during the interaction.
The differential sensing behaviors of the three compounds
are understandable from the plots of fluorescence ratios in
Figure 5.
It should be pointed out that the appearance of a new
emission peak at ∼585 nm and an absorption peak at ∼560 nm
for 1 and 2 in the presence of Al3+ ions is attributed to the
opening of spirolactam rings and generation of the delocalized
xanthene moieties, as shown in Figure 4a. In relation to this, the
excitation spectra of the aluminum complexes of 1 and 2 (λem
580 nm) gave good correspondence with their absorption
spectra (Figure S5 in the Supporting Information).
To support the proposed binding, we recorded the FTIR and
1H NMR of both the probes 1 and 2 in the presence and
absence of Al salt. The amide carbonyl stretching appearing at
1669 cm−1 in 1 was reduced to 1645 cm−1 upon complexation
of Al3+ (Figure S6 in the Supporting Information). Similarly,
the carbonyl stretching appearing at 1685 cm−1 in 2 moved to
1654 cm−1 in the presence of Al3+ (Figure S6). Such significant
reduction in carbonyl stretching in both cases substantiated the
lactam ring opening in the interaction process. In addition, the
CN stretching for benzimidazoles in 1 and 2 which appeared
at 1616 and 1615 cm−1, respectively, underwent a blue shift and
merged with the amide carbonyl stretching frequencies. The
blue shift essentially reflects the strengthening of the CN
bond in the presence of Al3+, and it is ascribed to the inhibition
of electronic delocalization around the benzimidazolic unit.12 In
1H NMR of 1 in d6-DMSO, we further note that the signal at
11.07 ppm for benzimidazole NH moves downfield by 0.15
Figure 5. Fluorescence ratios ((I − I0)/I0) of compounds 1−3 (c = 1.5
× 10−5 M) in CH3CN/water (4/1 v/v; 10 mM tris HCl buffer, pH 7)
at 584 nm.
The inertness of 3 toward metal ion sensing in contrast to
structures 1 and 2 is interesting. In our opinion, this is
associated with the substitutions at the α-positions of the
benzimidazoles that presumably influence the orientation of the
spirolactam rings with the benzimidazoles and xanthene
nucleus and regulate the ring opening differently toward
binding of metal ion. To understand this, we tried to grow
single crystals of the three compounds. Only single crystals of 2
and 3 were obtained from CH3OH (Figure 6). For compound
2, we observe in the solid state the formation of an
intramolecular hydrogen bond between the CO group and
the N−H unit from the benzimidazole derivate (N2−H1A···O2
2.205 Å, angle N2−H1A−O2 134.9°; see Figure 6a). The
D
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Figure 6. Crystal structures of compounds (a) 2 and (b) 3. Thermal ellipsoids are shown at 30% probability.
packing diagram of 2 presents the formation of linear chains
along the a axis involving weak C−H···O and C−H···N
intermolecular interactions (see Figure S9a in the Supporting
Information; C41−H41···O2B 2.649 Å, angle C41−H41−O2B
157.2°; C35−H35A···N3A 2.692 Å, angle C35−H35A-N3A
128.6°). The π−π distance between the phenyl rings is about
3.6 Å, which corroborates a weak stacking interaction.
In contrast, the solid state of compound 3 presents no
relevant intramolecular interaction between the CO and N−
H units. The distance is longer by about 0.5 Å (N2−H1A···O2
2.734 Å, angle N2−H1A-O2 130.7°). This difference can be
explained by the presence of one water molecule found in the
crystal structure. Intermolecular interactions between the water
molecule and the benzimidazole derivate were found in the
solid state (N2−H1A···O3 2.110 Å, angle N2−H1A-O3 150.0°;
O3−H2A···N3 2.060 Å, angle O3−H2A-N3 170.0°). These
interactions are building spiral chains along the b axis (see
Figure S9b in the Supporting Information). No other major
structural differences were found in the solid state of these two
compounds, which can explain the different opening processes
of the spirolactam ring. The angles between the planes of
benzimidazole and xanthene groups are 14.2° in 3 and 19.8° for
compound 2 with a bulky phenyl group. For more information,
a theoretical study (see below) was performed.
similar conditions and thereby ruled out the possibility of any
role of the anionic part of the aluminum salts in the sensing
process (Figure S11 in the Supporting Information). Moreover,
the time course of the fluorescence responses of 1 and 2 upon
addition of Al3+ was tested. The initial weak fluorescence at 580
nm of sensors 1 and 2 in the metal-free state was stable with
time. However, the fluorescence responses of 1 and 2 as tested
in the presence of Al3+ ions indicate that the reaction of the
chemosensors with Al3+ ions was rapid and complete within 4
min, after which the fluorescence intensity hardly changed
(Figure S12 in the Supporting Information). Thus, the
chemosensors are useful in real-time detection of Al3+ ions.
In the interactions, the stoichiometries15 of complexation of
the probes 1 and 2 with Al3+ ions were evaluated to be 1:1
(Figure S13 in the Supporting Information). The binding
constant values of 1 and 2 with Al3+ ions as determined by
nonlinear16 fitting of the fluorescence titration data were found
to be (7.71
0.15) × 103 and (6.99
0.14) × 103 M−1,
respectively (Figure S14 in the Supporting Information). Due
to a poor change in emission titrations, we were unable to
determine the binding constant values for other metal ions.
Analysis of the fluorescence titration data of 1 and 2 with Al3+
ions gave detection limits17 of 6.73 × 10−7 and 8.57 × 10−7 M,
respectively (Figure S15 in the Supporting Information).
The effects of pH on emission and as well as on absorption
of probes 1−3 with and without Al3+ ions were also
investigated to evaluate the practical applicability of the probes
(Figure S16 in the Supporting Information). The results show
that probes 1 and 2 were nonfluorescent in the pH range 5−12
but below this range both were highly fluorescent and pink,
demonstrating that the spirolactam rings of these two probes
were not stable in acidic pH. After addition of Al3+ ions to
solutions of 1 and 2 under different pH conditions, dramatic
enhancement in emission as well as absorption was found from
pH 2 to 8 and the colorless solutions became pink (Figure
S17A,B in the Supporting Information). In comparison, the
probe 3 was almost nonfluorescent and colorless in the wide
range of pHs 3−12 (Figure S16). Only at pH 2 slight changes
in absorption as well as in emission were observed and
indicated that the spiro ring of 3 remained intact at acidic pH.
Upon addition of Al3+ to the different pH solutions of 3, no
change in absorption and emission as that for 1 and 2 was
found and the colorless solution of 3 remained colorless
(Figure S17C). This, in addition, pointed out that the
spirocyclic ring opening in 3 did not take place even in the
presence of Al3+ ion.
The selectivity in sensing of Al3+ ion by the structure 1 was
established by recording the emission of 1 upon adding 15
equiv amounts of Al3+ in the presence and absence of 15 equiv
amounts of other metal ions. The selectivity plot indicates that
the background metal ions have no interference in the binding
of Al3+ with 1 (Figure S10a in the Supporting Information).
Similarly, for structure 2, the pronounced “off−on” type of Al3+
selectivity was also observed even in the presence of other
metal ions (Figure S10b). The preferential binding of a
particular metal ion into the binding site of a receptor structure
depends on various factors such as the nature of coordinating
atoms, size of the metal ion, differential solvations, softness/
hardness, polarity of solvents, etc.13 Thus, while the
benzimidazole motif in some reported receptor structures12b,14
is prone to bind metal ions other than aluminum, the selective
sensing of Al3+ ion over other metal ions in the present study is
likely to be due to the change in coordination environment, the
polarity of the working solvent that modifies the basicity of the
donor atoms involved in binding.
It is notable that chemosensors 1 and 2 both gave the same
results in absorption and emission titrations with Al(NO3)3,
Al(ClO4)3·9H2O, AlCl3, and K2SO4·Al2(SO4)3·24H2O under
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Figure 7. M06-2X/6-31G(d) calculated optimized structures of compounds 1−3.
Figure 8. M06-2X/6-31G(d) calculated relative electronic energies (Erel) of compounds 1−3 interacting with Al3+ in acetonitrile medium when their
spirolactam rings are closed. All distances are given in angstroms. Color scheme of atoms: yellow, C; white, H; blue, N; red, O; green, Al.
The reversibility in complexation was realized from recording
of emission and absorption spectra of the ensembles of 1 and 2
with Al3+ in aqueous CH3CN by adding different halides.
Addition of Cl−, Br−, and I− ions to the ensembles of 1-Al3+ and
2-Al3+ did not bring the reverse changes in both emission and
absorption spectra (Figure S18 and S19 in the Supporting
Information). Only in the presence of F− was the pink color of
Al complexes was discharged and produced reverse changes in
absorption and emission profiles (Figures S18 and S19). The
complete decomplexation of Al3+ from 1-Al3+ and 2-Al3+
ensembles is ascribed to the high affinity of F− ions toward
Al3+ for which in the absence of Al3+ the spiro rings are
regenerated.
This study was further extended with DFT calculations to
examine the stability of compounds 1−3 in an opened
spirolactam ring with the presumption that such ring opening
would occur with Al3+. The relative complexation energies of
Al3+ with opened spirolactam-1 and -2 are −13.1 and −3.7
kcal/mol, respectively, which are energetically preferred over
the opened spirolactam-3 with Al3+ complex (Figure 9). These
results corroborate the experimental finding of the ring opening
of 1 and 2 easily in the presence of Al3+.
Biological Studies of 1 and 2 in the Presence of Al3+.
Due to favorable binding properties of 1 and 2 with Al3+ and
intense emission in the visible region, it was conceived that
both compounds could be exploited for fluorescence imaging of
human lung carcinoma cell line A549, particularly for sensitive
detection of intracellular Al3+ (Figure 10). Hence, to assess the
effectiveness of compounds 1 and 2 as probes for intracellular
detection of Al3+ by fluorescence microscopy, stocks of 2.088
and 1.768 M were prepared in acetonitrile for probes 1 and 2,
respectively. A subsequent substock solution of 1 mM was
prepared for both probes in 1X PBS, from which the
compounds were added in cell suspensions. Cells were
trypsinized and then centrifuged at 300g for 15 min. Cell
pellets were collected and then washed with 1X PBS. Then cells
were incubated with 20 μM of compounds with and without 40
μM Al(ClO4)3 at 37 °C for 1 h. A drop of the cell suspension
Computational Study. We have carried out DFT (M06-
2X) calculations of compounds 1−3 in CH3CN medium at the
molecular level (Figure 7) to understand the nature of
complexation with Al3+.
First, we computed the complexation energies of Al3+ with
compounds 1−3 having a closed spirolactam ring. The M06-
2X/6-31G(d) calculated results show that Al3+ forms a
relatively more stable complex with compound 3 in comparison
to compounds 1 and 2 (Figure 8). The stability of 3 with Al3+
ion in comparison to 1 and 2 suggests that the spirolactam ring
opening would be less preferred for the former compound.
F
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Figure 9. M06-2X/6-31G(d) calculated relative electronic energies (Erel) of compounds 1−3 interacting with Al3+ in acetonitrile medium when their
spirolactam rings are opened. All distances are given in angstroms. Color scheme of atoms: yellow, C; white, H; blue, N; red, O; green, Al.
Figure 10. Fluorescence and bright field images of lung cancer cells (A549 cells): (a) bright field image of normal cells; (b) fluorescence image of
normal cells; (c) bright field image of cells treated with probe 1 (20 μM) for 1 h at 37 °C; (d) fluorescence image of cells treated with probe 1 (20
μM) for 1 h at 37 °C; (e) fluorescence image of cells upon treatment with probe 1 (20 μM) and then with Al(ClO4)3 (40 μM) for 1 h at 37 °C; (f)
bright field image of cells treated with probe 2 (20 μM) for 1 h at 37 °C; (g) fluorescence image of cells treated with probe 2 (20 μM) for 1 h at 37
°C; (h) fluorescence image of cells upon treatment with probe 2 (20 μM) and then with Al(ClO4)3 (40 μM) for 1 h at 37 °C.
was mounted over a microscopic slide and observed under the
40× objective of an Axio Scope A1 microscope (Carl Zeiss) for
both bright field and fluorescence images. Filter Set 43 with
excitation range ∼530−560 nm was used. It is evident from the
figure that untreated normal cells do not have any fluorescence,
as shown in Figure 10b. Treatment of cells with probes 1 and 2
separately did not show fluorescence from cells (Figure 10d,g).
However, when the probe-treated cells in each case were
incubated with Al(ClO4)3, they fluoresced red when filter set 43
filter was used (Figures 10e,h). This fluorescence microscopic
analysis strongly suggested that compounds 1 and 2 could
readily cross the membrane barrier, permeate into cells, and
rapidly sense intracellular Al3+. Further, to verify the cell
viability in the presence of compounds 1 and 2, a conventional
MTT assay was performed. In the study, A549 cells were
seeded in 96-well plates in triplicate manner (Figure S20 in the
Supporting Information). Cells were incubated with 20, 25, and
30 μM compounds for 4 h. It is evident from the result that
compounds 1 and 2 both have very little effect on cell viability.
CONCLUSION
■
In summary, we have designed and synthesized some α-amino
acid derived benzimdazole-linked rhodamine compounds 1−3
with different substituents on the amino acid part. Among the
structures, only compounds 1 and 2 bind and sense Al3+ ion
selectively over the other ions studied by exhibiting “turn-on”
G
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(brs, 1H), 6.09 (s, 1H), 1.42 (s, 9H); FT-IR ν cm−1 (KBr) 3329, 2935,
1673, 1528, 1454, 1442.
response in electronic and fluorescence spectra in the visible
region. The selectivity, binding constant values, and detection
limits are praiseworthy and are comparable with the reported
structures of various architectures (Table S2 in the Supporting
Information). However, the response of the compounds
depends on the nature of the α-substitutions of the amino
acid motifs. Experimental results reveal that the rhodamine−
benzimidazole conjugate without α-substitution at the amino
acid motif (compound 3 in the present case) is evaluated to be
stable to metal ions and acid as well. The substitutions at the α-
positions of the benzimidazoles presumably influence the
orientation of the spirolactam rings with the benzimidazoles
and xanthene nucleus and regulate the ring opening differently
toward binding of metal ion. The crystal structure analysis and
theoretical insight explain the reactivity profiles. As compounds
1 and 2 work under physiological conditions, both compounds
are preferred for use either as colorimetric staining agents or as
reagents for imaging studies with biological and environmental
samples. In practice, both 1 and 2, when applied in human lung
carcinoma cell line A549, could detect successfully the cellular
uptake of Al3+ ion.
tert-Butyl (1H-Benzo[d]imidazol-2-yl)methylcarbamate19 (9).
Using the strategy followed for compound 7, compound 9 was
1
prepared in 88% yield: mp 174 °C; H NMR (400 MHz, CDCl3) δ
10.30 (s, 1H), 7.70 (br s, 1H), 7.52 (br s, 1H), 7.25−7.23 (m, 2H),
5.55 (br t, 1H), 4.51 (d, 2H, J = 8 Hz), 1.30 (s, 9H); FT-IR ν cm−1
(KBr) 3348, 2978, 2931, 2751, 1688, 1529.
Compound 1. To synthesize compound 1, a solution of
rhodamine B (0.1 g, 0.2 mmol) in 1,2-dichloroethane (10 mL) was
stirred and phosphorus oxychloride (250 μL) was added dropwise at
room temperature. Then the resulting solution was refluxed for 2 h.
The reaction mixture was cooled and evaporated in vacuo to give
rhodamine B acid chloride 13, which was used in the next step directly.
The acid chloride was dissolved in dry dichloromethane (10 mL) and
was added dropwise over 10 min to a solution of amine 10 (0.06 g,
0.317 mmol; obtained from deprotection of Boc-group in 7 using
TFA) and Et3N (70 μL) in dichloromethane (10 mL) at room
temperature and stirred for 8 h. After completion of the reaction as
monitored by TLC, the solvent was removed under pressure and the
residue was dissolved in chloroform, extracted with water, and dried
over anhydrous Na2SO4. The crude mass was purified by silica gel
column chromatography using petroleum ether/ethyl acetate (7/3 v/
v) as eluent, giving a white powder of compound 1 (0.09 g, yield
70%): mp 96 °C; [α]D25 = −112.13 (c = 0.305 g/100 mL in CHCl3);
1H NMR (400 MHz, CDCl3) δ 10.44 (s, 1H), 7.94 (m, 1H), 7.49 (t,
2H, J = 8 Hz), 7.33 (brm, 2H), 7.14 (t, 1H, J = 4 Hz), 7.07 (brm, 2H),
6.55 (d, 1H, J = 8 Hz), 6.43 (d, 1H, J = 4 Hz), 6.38 (d, 1H, J = 8 Hz),
6.33 (d, 1H, J = 8 Hz), 5.64 (d, 1H, J = 8 Hz), 5.25 (d, 1H, J = 8 Hz),
3.82 (d, 1H, J = 10.8 Hz), 3.39−3.34 (m, 4H), 3.20−3.09 (m, 2H),
3.01−2.94 (m, 2H), 1.72 (brs, 1H), 1.18 (t, 6H, J = 7.20 Hz), 0.95 (t,
6H, J = 7.20 Hz), 0.62 (t, 6H, J = 7.20 Hz); 13C NMR (100 MHz,
CDCl3) δ 168.2, 153.6, 153.2, 152.4, 151.5, 148.1, 147.5, 131.8, 131.1,
129.4, 127.2, 125.9, 123.2, 121.5, 120.5, 106.6, 106.4, 103.3, 103.0,
97.02, 96.6, 66.4, 59.4, 43.4, 43.0, 28.4, 19.8, 19.0, 11.56, 11.53; FT-IR
ν cm−1 (KBr) 3333, 2965, 2928, 1669, 1634, 1616, 1515; HRMS
(TOF MS ES+) calcd for (M + H)+ 614.3495, found 614.3495.
Compound 2. According to the procedure as followed for the
synthesis of 1, compound 2 was prepared (0.092 g, yield 68%); mp
216 °C; [α]D25 = −0.8 (c = 0.6 g/100 mL in CH3OH); 1H NMR (400
MHz, d6-DMSO) δ 11.48 (s, 1H), 7.82 (d, 1H, J = 8 Hz), 7.57−7.55
(m, 2H), 7.43 (d, 1H, J = 8 Hz), 7.34 (d, 1H, J = 8 Hz), 7.11−7.02 (m,
6H), 6.88 (d, 2H, J = 4 Hz), 6.45 (d, 1H, J = 8 Hz), 6.38 (s, 1H),
6.28−6.19 (m, 3H), 5.85 (d, 1H, J = 8 Hz), 5.34 (s, 1H), 3.50−3.30
(m, 4H, buried under signal of solvent), 3.16- 3.11 (m, 4H), 1.09 (t,
6H, J = 8 Hz), 0.91 (t, 6H, J = 8 Hz); 13C NMR (100 MHz, d6-
DMSO) δ 166.9, 153.4, 153.3, 153.0, 152.5, 148.8, 148.6, 142.9, 138.4,
134.8, 133.5, 131.7, 129.8, 129.5, 128.8, 128.6, 128.0, 127.3, 124.3,
122.9, 122.1, 121.2, 118.7, 112.1, 108.5, 107.9, 104.8, 104.1, 97.4, 66.4,
56.2, 44.2, 44.0, 12.8, 12.7; FT-IR ν cm−1 (KBr) 3293, 2966, 1685,
1635, 1615, 1545, 1514; HRMS (TOF MS ES+): C calcd for (M + H)+
648.3339, found 648.3364.
EXPERIMENTAL SECTION
■
(S)-tert-Butyl 1-(2-Aminophenylamino)-3-methyl-1-oxobu-
tan-2-ylcarbamate18 (4). To a stirred solution of N-Boc-protected
L-valine acid (1 g, 4.6 mmol) in dry CH2Cl2 (15 mL) was added DCC
(1 g, 4.84 mmol) at 0 °C. The solution was stirred for 30 min, and a
solution of o-phenylenediamine (2.5 g, 23.11 mmol) in dry CH2Cl2
(35 mL) was added to it under a nitrogen atmosphere. After it was
stirred for 19 h, the reaction mixture was filtered off to remove
insoluble DCU. The filtrate was removed in vacuo, and the crude
product was purified by silica gel column chromatography using 1%
CH31OH in CHCl3 to afford compound 4 (0.92 g, yield 65%): mp 132
°C; H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.21 (d, 1H, J = 8
Hz), 7.05 (t, 1H, J = 8 Hz), 6.79−6.75 (m, 2H), 5.12 (br s, 1H), 3.99
(t, 1H, J = 8 Hz), 3.87 (s, 2H), 2.29−2.25 (m, 1H), 1.65 (s, 9H),
1.12−1.01 (m, 6H); FT-IR ν cm−1 (KBr) 3420, 3339, 3257, 3044,
2980, 1703, 1678, 1538.
(S)-tert-Butyl 2-(2-Aminophenylamino)-2-oxo-1-phenyle-
thylcarbamate (5). Using the same strategy as for compound 4,
we synthesized compound 5 (yield 68%); mp 138 °C; 1H NMR (400
MHz, CDCl3) δ 7.78 (s, 1H), 7.44 (d, 2H, J = 8 Hz), 7.38−7.32 (m,
3H), 7.11 (d, 1H, J = 8 Hz), 7.01 (t, 1H, J = 8 Hz), 6.74−6.69 (m,
2H), 5.87 (d, 1H, J = 4 Hz), 5.36 (s, 1H), 3.65 (s, 2H), 1.42 (s, 9H);
FT-IR ν cm−1 (KBr) 3347, 3257, 2977, 1708, 1661, 1523, 1456.
(S)-tert-butyl 1-(1H-benzo[d]imidazol-2-yl)-2-methylpropyl-
carbamate18 (7). Compound 4 (0.5 g, 1.63 mmol) was next heated
in acetic acid (0.5 mL) at 80 °C for 2 h. Then the reaction mixture was
quenched by addition of an aqueous solution of NaHCO3 (15 mL).
The reaction mixture was next extracted with a CHCl3/MeOH solvent
mixture (CHCl3/MeOH 3/1 v/v; 20 mL). The organic layer was
washed with brine, dried over Na2SO4, and evaporated under reduced
pressure. The reaction mixture was purified by silica gel column
chromatography using 40% petroleum ether in ethyl acetate to afford
Compound 3. According to the procedure followed for the
synthesis of 1, compound 3 was prepared (0.12 g, yield 67%): mp 160
°C; 1H NMR (400 MHz, CDCl3) δ 9.90 (s, 1H), 7.96−7.94 (m, 1H),
7.48−7.44 (m, 3H), 7.26 (s, 1H), 7.14−7.07 (m, 3H), 6.37 (d, 2H, J =
2.4 Hz), 6.22 (s, 1H), 6.20 (s, 1H), 5.94 (dd, 2H, J1 = 8 Hz, J2 = 4 Hz),
4.56 (s, 2H), 3.27−3.17 (m, 8H), 1.08 (t, 12H, J = 8 Hz); 13C NMR
(100 MHz, CDCl3) δ 169.2, 153.6, 153.4, 150.8, 148.8, 133.0, 130.3,
128.2, 128.0, 124.0, 122.8, 121.9, 108.4, 107.8, 103.9, 97.8, 65.8, 44.2,
38.2, 12.5; FT-IR ν cm−1 (KBr) 3512, 2970, 1693, 1680, 1634, 1615,
1547, 1515; HRMS (TOF MS ES+) calcd for (M + H)+ 572.3026,
found 572.3018.
Quantum Yield Determination. Quantum yields (Φ) of the
compounds 1−3 by themselves and in the presence of Al3+ were
determined in CH3CN by a relative comparison procedure using
rhodamine B as standard (Φrh 6G = 0.95 in ethanol).20a The general
equation used for determination of relative quantum yields is20b
1
the compound 7 (0.415 g, yield 88%): mp 174 °C; H NMR (400
MHz, CDCl3 containing one drop of d6-DMSO) δ 10.70 (s, 1H), 7.71
(d, 1H, J = 8 Hz), 7.35 (d, 1H, J = 8 Hz), 7.21−7.19 (m, 2H), 5.67 (d,
1H, J = 8 Hz), 4.61 (t, 1H, J = 8 Hz), 2.47−2.45 (m, 1H), 1.43 (s,
9H), 1.06 (d, 3H, J = 4 Hz), 0.93 (d, 3H, J = 4 Hz); 13C NMR (100
MHz, CDCl3 containing one drop of d6-DMSO) δ 155.6, 154.9, 121.7
(four carbons unresolved), 79.2, 55.1, 33.2, 28.2, 19.2, 18.3; FT-IR ν
cm−1 (KBr) 3333, 2974, 2930, 1675, 1628, 1536.
(S)-tert-Butyl (1H-Benzo[d]imidazol-2-yl)(phenyl)-
methylcarbamate (8). Using the strategy followed for compound
7, compound 8 was prepared in 85% yield: mp 190 °C; 1H NMR (400
MHz, CDCl3) δ 10.15 (s, 1H), 7.71 (d, 1H, J = 8 Hz), 7.36 (m, 2H),
7.35−7.29 (m, 2H), 7.27−7.25 (m, 2H), 7.24−7.19 (m, 2H), 6.24
Φ = (ΦF Asλexsη2 )/(FAuλexuη2 )
u
s
u
s
u
s
H
DOI: 10.1021/acs.inorgchem.7b00835
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
where Φ is the quantum yield, F is the integrated area under the
corrected emission spectrum, A is the absorbance at the excitation
wavelength, λex is the excitation wavelength, η is the refractive index of
the solution, and the subscripts u and s refer to the unknown and the
standard, respectively.
X-ray Diffraction. Data sets for compound 2 were collected with a
Nonius Kappa CCD diffractometer. Programs used: data collection,
COLLECT;21 data reduction, DENZO-SMN;22 absorption correction,
DENZO.23 For compound 3 data sets were collected with a D8
Venture Dual Source 100 CMOS diffractometer. Programs used: data
collection, APEX3;24 cell refinement and data reduction, SAINT;24
absorption correction, SADABS;24 structure solution and structure
refinement, SHELXL25 and SHELXTL;26 and graphics, XP.27 R1
values are given for observed reflections, and wR2 values are given for
all reflections.
Exceptions and Special Features. For compound 2 the hydrogen
atom positions at the N2 atom were refined freely. Two isopropyl
groups were found to be disordered over two positions. Several
restraints (SADI, SAME, ISOR, and SIMU) were used in order to
improve refinement stability. Compound 3 crystallizes by chance in
the Sohncke space group P21 and was refined as a two-component
inversion twin (with BASF 0.20). The hydrogen atom positions at N2
and O3 atoms were refined freely.
X-ray Crystal Structure Analysis of 2. Crystal data are as follows:
formula C42H41N5O2, Mr = 647.80, colorless crystal, 0.22 × 0.18 ×
0.02 mm, a = 16.7855(3) Å, b = 9.6854(2) Å, c = 22.4428(5) Å, β =
111.048(1)°, V = 3405.2(1) Å3, ρcalc = 1.264 g cm−3, μ = 0.079 mm−1,
empirical absorption correction (0.982 ≤ T ≤ 0.998), Z = 4,
monoclinic, space group P21/c (No. 14), λ = 0.71073 Å, T = 223(2) K,
confirm that each stationary point is a local minimum (with zero
imaginary frequency). Single-point calculations were executed at the
same level of theory to account the solvation effects with the
polarizable continuum model (PCM) in acetonitrile (ε = 35.688). All
calculations have been performed with the Gaussian 09 suite of
programs.30
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.7b00835.
Fluorescence and UV−vis titrations of compounds 1−3
with various metal ions, Job plots, binding constant
curves, detection limit plots, and other spectral data
(PDF)
Accession Codes
CCDC 1496178−1496179 contain the supplementary crystal-
lographic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by email-
ing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Authors
*B.G.: e-mail, ganguly@csmcri.org.
■
ω and φ scans, 25844 reflections collected ( h,
k,
l), 8349
independent (Rint = 0.067) and 5459 observed reflections (I > 2σ(I)),
486 refined parameters, R1 = 0.084, wR2 = 0.185, maximum
(minimum) residual electron density 0.26 (−0.31) e Å−3, hydrogen
atom at N2 refined freely; other atoms calculated and refined as riding
atoms.
*K.G.: e-mail, ghosh_k2003@yahoo.co.in; fax,
+913325828282; tel, +913325828750.
ORCID
Bishwajit Ganguly: 0000-0002-9858-3165
Kumaresh Ghosh: 0000-0003-1236-8139
Notes
X-ray Crystal Structure Analysis of 3. A pale yellow prismlike
specimen of C36H37N5O2·H2O, approximate dimensions 0.084 mm ×
0.117 mm × 0.177 mm, was used for the X-ray crystallographic
analysis. The X-ray intensity data were measured. A total of 1054
frames were collected. The total exposure time was 19.17 h. The
frames were integrated with the Bruker SAINT software package using
a wide-frame algorithm. The integration of the data using a monoclinic
unit cell yielded a total of 19806 reflections to a maximum θ angle of
68.30° (0.83 Å resolution), of which 5650 were independent (average
redundancy 3.505, completeness 99.8%, Rint = 3.81%, Rsig = 3.54%)
and 5242 (92.78%) were greater than 2σ(F2). The final cell constants
of a = 11.8491(3) Å, b = 11.1111(3) Å, c = 11.8732(3) Å, β =
100.1190(10)°, and V = 1538.87(7) Å3 are based upon the refinement
of the XYZ centroids of 9937 reflections above 20σ(I) with 7.563° <
2θ < 136.3°. Data were corrected for absorption effects using the
multiscan method (SADABS). The ratio of minimum to maximum
apparent transmission was 0.931. The calculated minimum and
maximum transmission coefficients (based on crystal size) are 0.8930
and 0.9470, respectively. The structure was solved and refined using
the Bruker SHELXTL Software Package, using the space group P21,
with Z = 2 for the formula unit, C36H37N5O2·H2O. The final
anisotropic full-matrix least-squares refinement on F2 with 414
variables converged at R1 = 2.94% for the observed data and wR2 =
7.24% for all data. The goodness of fit was 1.053. The largest peak in
the final difference electron density synthesis was 0.131 e Å−3, and the
largest hole was −0.134 e Å−3 with an RMS deviation of 0.029 e Å−3.
On the basis of the final model, the calculated density was 1.273 g/cm3
and F(000) was 628 e.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
A.M. thanks the CSIR and S.M. thanks the UGC, New Delhi,
India, for fellowships. K.G. thanks the DST, West Bengal, India,
for financial support (project sanc. No. 755(Sanc.)/ST/P/
S&T/4G-3/2014 dated 27.11.2014).
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