Chelation of specific metal ions imparts coplanarity and fluorescence in two imidazo[1,2-a]pyridine derivatives: Potential chemosensors for detection of metal ions in aqueous and biosamples

Article abstract of DOI:10.1016/j.saa.2019.117236

Synthesis and chelation induced fluorescence emission from two imidazo[1,2-a]pyridine derivatives are described. The nonfluorescent molecule 1 containing N and O donor atoms, achieves coplanarity upon interactions with trivalent cations Al³⁺, F

Full text of DOI:10.1016/j.saa.2019.117236

Accepted Manuscript
Chelation of specific metal ions imparts coplanarity and
fluorescence in two imidazo[1,2-a]pyridine derivatives: Potential
chemosensors for detection of metal ions in aqueous and
biosamples
Ramanjaneyulu Mala, Koorathota Suman, Manivannan
Nandhagopal, Mathivanan Narayanasamy, Sathiah Thennarasu
PII:
S1386-1425(19)30626-2
DOI:
https://doi.org/10.1016/j.saa.2019.117236
Article
Number:
Reference:
117236
SAA 117236
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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1 June 2019
1 June 2019
Please cite this article as: R. Mala, K. Suman, M. Nandhagopal, et al., Chelation of
specific metal ions imparts coplanarity and fluorescence in two imidazo[1,2-a]pyridine
derivatives: Potential chemosensors for detection of metal ions in aqueous and biosamples,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/
10.1016/j.saa.2019.117236
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ACCEPTED MANUSCRIPT
Chelation of specific metal ions imparts coplanarity and fluorescence in two
imidazo[1,2-a]pyridine derivatives: potential chemosensors for detection of
metal ions in aqueous and biosamples
Ramanjaneyulu Malaa,b, Koorathota Sumana Manivannan Nandhagopalc, Mathivanan
Narayanasamyc and Sathiah Thennarasu*a,b
b
aOrganic and biorgnic Chemistry Laboratory, Academy of Scientific and Innovative Research
(AcSIR), CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India.
c Biocontrol and Microbial Metabolites Lab, Centre for Advanced Studies in Botany, University
of Madras, Guindy Campus, Chennai 600025, India.
*Corresponding Author; E-mail:thennarasu@gmail.com; Tel: +91 44 24437269
Abstract
Synthesis and chelation induced fluorescence emission from two imidazo[1,2-a]pyridine
derivatives are described. The nonfluorescent molecule 1 containing N and O donor atoms,
achieves coplanarity upon interactions with trivalent cations Al3+, Fe3+ and Cr3+, that favours
fluorescence emission. Molecule 2 containing two N donor atoms attains coplanarity upon
interaction with the only Zn2+ and becomes fluorescent. Both molecules 1 and 2 form a 1:1
complex with interacting metal ions. Other trivalent metal ions (including Bi3+ and In3+) and
common divalent metal ions (including Hg2+ and Cd2+) fail to form any complex with 1 or 2, and
they do not interfere in the detection of Zn2+, Al3+, Fe3+ or Cr3+ ions. Noninterference of other
metal ions renders 1 and 2 suitable for the detection of fungal cells contaminated with Zn2+, Al3+,
Fe3+ or Cr3+ ions.
Keywords
Imidazo[1,2-a]pyridine derivatives; Chelation induced fluorescence; Coplanar structure;
Detection of metal ions; Cell imaging.

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1. Introduction
Development of new chemosensors for collective or selective detection of heavy and
transition metal ions has attracted the attention of a large number of research groups in recent
years, owing to the importance of these metal ions on human health and their impact on the
environment [1-4]. Significant effort has been devoted for the synthesis and evaluation of
chemosensors that bind more than one trivalent cation [5-7] or more than one divalent cation [8-
10]. Chemosensors with binding affinity for both trivalent and divalent metal ions have also been
reported [11-13].
Fluorescent chemosensors are very attractive as they possess the potential for real-time
monitoring of molecular interactions with high selectivity and sensitivity. Fluorescence turn-on
response upon interaction with metal ions offers a non-destructive analytical approach for the
detection of toxic metal ions by using low-cost instrumentation and simple operational
techniques. Mechanisms that involve fluorescence turn-on response include internal charge
transfer (ICT) [14], twisted intramolecular charge transfer (TICT) [15], excited state
intramolecular proton transfer (ESIPT) [16], photoinduced electron transfer (PET) [17],
chelation-enhanced fluorescence (CHEF) [18], Förster resonance energy transfer (FRET) [19],
spirolactam ring-opening in rhodamine based sensors [20] and deprotonation [21] upon
complexation. Among these, CHEF approach is unique in that chelation of metal ion with
organic receptor ligands drastically increases the conjugation, which results in enhanced
fluorescence emission [22]. However, examples of chemosensors attaining coplanar structure
upon interaction with specific metal ions are rare [23].
In continuation of our research on fluorescence turn-on chemosensors [24], herein, we report the
synthesis and photophysical properties of two imidazo[1,2-a]pyridine based probes 1 and 2 with
NO and NN combination of coordinating donor atoms, respectively. Experimental studies
revealed that both probes are nonfluorescent in aqueous solutions, and they emit fluorescence
upon interaction with specific metal ions. Probe 1 displays CHEF effect in response to trivalent
Al3+, Fe3+, and Cr3+ ions, while probe 2 is highly selective towards Zn2+ ions. This study assumes
significance in that the development of fluorescence turn-on chemosensors is challenging due to
the paramagnetic quenching nature of Fe3+ and Cr3+, and the weak coordinating ability of Al3+

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ions [25]. Similarly, the absence of any vacant d orbitals and unpaired electrons in Zn2+ hinder
spectrometric and magnetic detection of Zn2+ ions [26]. Using spectroscopic methods, we have
shown the specific interactions of 1 with Al3+, Fe3+ and Cr3+ ions, and those of 2 with Zn2+ ions
leading to coplanar geometry and metal chelation induced enhancement in fluorescence emission
intensity. The colour change associated with CHEF effect permits naked-eye detection.
Microscopic imaging of fungal cells contaminated with these metal ions is also demonstrated.
Scheme 1 Synthetic route for imidazo[1,2-a]pyridine derivatives: 1, 2 and reference molecule
2. Experimental section
Dry acetonitrile (analytical grade) and double distilled water were used in all experiments. All
the materials for synthesis were purchased from commercial suppliers and used without further
purification. The solutions of metal ions were prepared from the corresponding nitrate and
chloride salts. Absorption spectra were recorded on SPECORD 200 PLUS UV-visible
spectrophotometer. Fluorescence measurements were performed on Agilent Technologies Cary
Eclipse fluorescence spectrophotometer (Excitation wavelength 338 nm; Slit width 5 nm).
Fluorescence quantum yields were calculated by using quinine sulfate (ՓR = 0.577 in 0.1M
H2SO4) as a standard report. NMR spectra were recorded using Bruker Advance 400 MHz
spectrometer. HRMS spectra were obtained on Thermo Exactive PE Sciex API3000 mass
analyzer.
2.1 Synthesis of 2-(3-(tert-butylamino)imidazo[1,2-a]pyridin-2yl) phenol (1)
2-Aminopyridine (0.94g, 10 mmol) and 2-hydroxy benzaldehyde (1.06 mL, 10 mmol) were
solubilized in ethanol (25 mL). To this solution, tert-butylisocyanide (1.13 mL, 10 mmol) and
iodine (0.5 mmol) were added. The reaction mixture was stirred at room temperature overnight.
The white precipitate formed was separated and purified further using silica-gel column

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chromatography to obtain ligand 1 in 80% yield. Mass calculated for C17H19N3O was 281.10;
ESI m/z found was 282.20 (M+H)+. 1H NMR (400 MHz, CDCl3): δ 12.48 (s, 1H), 8.21 (d, J =
6.9 Hz, 1H), 8.13 (dd, J = 7.8, 1.6 Hz, 1H), 7.57 – 7.49 (m, 1H), 7.22 (tdd, J = 8.8, 7.0, 1.5 Hz,
2H), 7.04 (dd, J = 8.2, 1.1 Hz, 1H), 6.91 (td, J = 7.8, 1.3 Hz, 1H), 6.86 (td, J = 6.8, 1.1 Hz, 1H),
3.16 (s, 1H), 1.18 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 156.94, 140.58, 129.34, 128.02,
124.79, 122.94, 121.90, 118.57, 117.64, 116.75, 112.02, 57.06, 30.36.
2.2 Synthesis of N-(tert-butyl)-2-(pyridin-2-yl)imidazo[1,2a]pyridin-3-amine (2)
2-Aminopyridine (0.94g, 10 mmol) and picolinaldehyde (0.95 mL, 10 mmol) were solubilized in
ethanol (40 mL). To this solution, tert-butylisocyanide (1.13 mL, 10 mmol) and iodine (0.5
mmol %) were added. The reaction mixture was stirred at room temperature overnight. The
white solid formed was separated by filtration and purified using silica-gel column
chromatography that afforded the ligand 2 in 90% yield. Calculated mass for C16H18N4 is 266.15
Observed mass (ESI m/z) = 267.33 (M + H) +.1H NMR (400 MHz,CDCl3) δ 8.57 (d, J = 4.3 Hz,
1H),8.28 (d, J = 7.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H),7.76 (td, J = 7.8, 1.7 Hz, 1H), 7.51 (d, J =
9.1 Hz, 1H), 7.27 (s, 1H), 7.07-7.18 (m, J = 19.1, 2H), 6.73 (t, J = 7.2 Hz, 1H), 5.44 (N-H)(s,
1H), 1.15 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 155.13, 148.29, 141.72, 136.45, 134.84,
128.90, 124.25, 123.88, 121.60, 121.27, 117.50, 111.16, 57.08, 30.04.
2.3 Preparation of test solutions
UV-Vis and fluorescence titrations were conducted in low concentration range at room
temperature to avoid dilution-error. Stock solutions of the ligands 1 and 2 were prepared by
dissolving 2.81 mg of 1 or 3.0 mg of 2 in CH3CN and making up to the mark in a 10 mL
volumetric flask. Stock solutions of metal ions (1.0 M) were prepared in de-ionized water. We
used 10 µM solution of 1 and 5.6 μM solution of 2 for metal ion titration experiments. Buffer
solutions were prepared using tablets as per the specifications supplied by SRL Chemicals, India.
2.4 Microscopic observation
The fluorescent microscopic observation of fungal mycelia was carried out under wet
mount technique. Rhizoctonia solani (Obtained from Biocontrol Microbial Metabolites Lab,
Centre for Advanced Studies in Botany, University of Madras, and Guindy) used as the test

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organisms, Fungal growth medium potato dextrose Agar (PDA) (potato 20 g, dextrose 2 g &
agar 2 g per 100 mL) obtained from Sisco Reserch Laboratories (PM033), New Mumbai, was
prepared and sterilized at 121 °C for 20 minutes at 15 lbs. medium poured in to sterile petriplate
and allowed to solidify small portion of fungal mycelium placed in center of PDA agar plates
and incubated for 6 days at 20°C. After incubation small portion of fungal mycelium was taken
using inoculation needle and treated with 5 µL of 10 mM solution of target metal ions (Fe3+,
Al3+, Cr3+ and Zn2+) at room temperature for 5 minutes. After washing the contaminated mycelia
with sterile distilled water, 5 µL of probe 1 (0.0035 mmol) or probe 2 (0.0037 mmol) plus 10 µL
distilled water were added to each microscopic slide separately, mixed using inoculation needle,
covered with cover slip, and observed under bright field and fluorescent field with 40X
magnification. Optika fluorescent microscope (EM535AF45) was used with a cut-on wavelength
510 ± 2 nm and cut-off wavelength 560 ± 5 nm.
3. Results and Discussion
The synthesis and photophysical properties of imidazo[1,2-a]pyridine derivatives have been
reported recently [27]. While exploring the synthesis of imidazo[1,2-a]pyridine and imidazo[1,2-
a]pyrazine derivatives through alternate methods (Scheme 1, Fig. S1-6), we observed that 4-(3-
(tert-butylamino)imidazo[1,2-a]pyridine-2-yl)phenol was fluorescent and 2-(3-(tert-butylamino)
imidazo[1,2-a]pyridine-2-yl)phenol (ligand 1) was non-fluorescent (Fig. S7). A comparison of
MM2 energy minimized structures of both compounds revealed the loss of co-planarity in ligand
1. Consequently, single crystal analysis (Fig. 1, Table S1) confirmed the loss of co-planarity in
ligand 1, supporting the notion that loss of co-planarity led to the loss of fluorescence. We
envisaged that ligand 1 would emit fluorescence if co-planarity is established through chelation
of metal ions. With only two potential chelating atoms, ligand 1 could be expected to behave like
the bipyridyl ligand, as a subtle variation in donor atoms is known to modulate metal ion specific
binding [28, 29]. By the same analogy, ligand 2 (Fig. 1, S8, Table S1) was also envisaged to
display turn-on fluorescence upon chelation with metal ions.
………………………………………Figure 1 …………………………………………………..
3.1 Interaction of metal ions with ligand 1

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We first investigated the absorbance profile of ligand 1 in the presence of metal ions such as,
Na+, K+, Ag+, Zn2+, Co2+, Ni2+, Ca2+, Ba2+, Pb2+, Cu2+, Mn2+, Hg2+, Cd2+, Fe2+, Mg2+, Fe3+,Cr3+,
Al3+ , In3+ and Bi3+ in acetonitrile medium to explore the chelation potential of 1. In the absence
of any metal ions, the UV-Visible spectrum of 1 (10 µM) showed an absorption maximum at
~338 nm ( = 122000 M-1 cm-1), corresponding to the imidazo[1,2-a]pyridine moiety. When of
10 equiv. of the indicated metal ions were added, only Al3+, Fe3+ and Cr3+ induced significant
changes in the absorption profile of 1 (Fig. 2a). Serial addition of Al3+, Fe3+ or Cr3+ ions resulted
………………………………………Figures 2,3 and 4 ……………………………………..
in a steady blue-shift of the absorption maximum at ~338 nm, and upon addition of 13, 25 and 15
equiv. of Al3+, Fe3+ or Cr3+, the absorption maximum appeared at ~317 nm (Fig. 3). When other
trivalent and divalent metal ions were added to ligand 1 in acetonitrile, the almost negligible
effect was observed (Fig 2a). These observations implied that only Al3+, Fe3+ and Cr3+ had the
ability to form a complex with 1. The observed blue-shift in absorption is attributed to trivalent
cation binding to ligand 1, which results in a more rigid structure of the probe after complex
formation. As the metal ions Al3+, Fe3+ and Cr3+ possess the same charge; the one with the right
ionic radius is expected to induce the greatest blue-shift. It is imperative to note that metal
complex formation with 1 could also be detected by naked-eye when illuminated under a UV
lamp (Fig. 4).
3.2 Chelation of specific metal ions with ligand 1 turns on fluorescence
The observed blue-shift in absorption upon complex formation prompted us to investigate the
fluorescence profiles of 1 (10 µM) in the presence of 100 equiv. of the metal ions in acetonitrile
medium (Fig.2b). Ligand 1 in acetonitrile and in the presence of other metal ions did not show
any detectable fluorescence. However, the addition of 100 equiv. of Al3+, Fe3+ or Cr3+ rendered a
remarkable increase in fluorescence emission at 471 nm. The observed 197, 183 and 134 fold
increments in the emission intensity was ascribed to the selective interactions of Fe3+, Al3+ or
Cr3+, respectively. The fluorescence quantum yields [30] of the putative 1-Al3+, 1-Fe3+ and 1-
Cr3+ complexes were calculated to be 0.445, 0.339 and 0.202, respectively, with respect to
quinine sulphate (ՓR=0.577 in 0.1M H2SO4). Effect of pH on the fluorescence emission from
probe 1, with and without Al3+, Fe3+ or Cr3+ ions, showed that probe 1 is useful only in the
neutral and basic pH range for detection purposes (Fig. S9a).

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………………………………………Figures 3,4,5 and 6……………………………………..
3.3 Understanding metal ion interactions with ligand 1
To understand metal ion induced fluorescence emission, 1H and 13C NMR spectra of ligand 1
and 1-Fe3+ were recorded. Involvement of the hydroxyl group of 1 was readily observed by
comparing the 1H NMR spectra of 1 and 1-Fe3+ (Fig. 5). Analysis of 13C NMR spectra of 1 and
1-Fe3+ revealed the involvement of N-atom of the imidazo[1,2-a]pyridine moiety as the
resonance positions of these carbons were shifted significantly. To confirm the formation of a
metal complex with ligand 1, we used the proton difference NOE experiment. As would be
expected, 5H of imidazo[1,2-a]pyridine moiety and 3H of phenol moiety in ligand 1 showed
NOE enhancements upon saturation of tert-butyl protons. The ratio of the area of the peaks
corresponding to 5H and 3H was calculated to be 2.0. A similar analysis of the area of the peaks
corresponding to 5H and 3H after adding Al3+ was found to be 1.3 (Fig 6). This observation 
increment in the NOE enhancement of 3H  was attributed to the attainment of co-planarity in
ligand 1 upon complex formation. Co-planarity in 1-Al3+ complex can bestow fluorescence
emission[31] and hence, ligand 1 can be used as a chemosensor for the detection of low levels of
Al3+, Fe3+ and Cr3+ ions.
3.4 Exploring the binding behaviour of ligand 2 with different metal ions
Encouraged by the observations described above, we set out to explore ligand 2 for chelation
induced co-planarity and the associated fluorescence emission from its complex with metal ions.
When the absorption profiles of ligand 2 were studied under the conditions applied to ligand 1,
only Zn2+ (and not the other divalent and trivalent metal ions) showed noticeable changes in the
absorption profile of 2 (Fig. 7a). Serial addition of Zn2+ ions resulted in a steady increase in
absorption, and the maximum absorption was observed upon addition of 10 equiv. of Zn2+ (Fig.
8). Under comparable conditions, ligand 2 in CH3CN-H2O (1:1) mixture did not show any
detectable fluorescence in the presence of common metal ions. However, the addition of 10
equiv. of Zn2+ resulted in a remarkable increase in fluorescence emission at 460 nm (Fig. 7b).
The observed 4 fold increment in the fluorescence intensity was attributed to the Zn2+ selective
interactions of ligand 2. The fluorescence quantum yield of the putative 2-Zn2+ complex was
calculated to be 0.463. The structure of the 2-Zn2+ complex was analyzed using single crystals
obtained from ethanol solution. Single crystal XRD studies of 2 and 2-Zn2+ complex proved the

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co-planar structure of 2-Zn2+ complex (Fig. 9, Table S2) and the lack of co-planarity in the
structure of 2. Thus, metal chelation stabilized the coplanar structure of 2-Zn2+ complex, and
fluorescence emission due to the attainment of coplanar structure were confirmed. It is
imperative to note that complex formation between 2 and Zn2+ could also be detected by naked-
eye when illuminated under a UV lamp (Fig. 4b).
………………………………………Figures 7,8,9 and 10 ……………….…………………..
3.5 Exploiting chelation induced fluorescence for detection of specific metal ions
As ligand 1 showed an affinity for trivalent ions (Fe3+, Al3+ and Cr3+) and ligand 2 had an
affinity for only Zn2+, we exploited their potential for chemosensor applications. As shown in
Fig. 10 and Fig. 11, the plot of emission intensity versus the concentration of trivalent and
divalent cations showed a linear relationship. The detection limits for Al3+, Fe3+, Cr3+ and Zn2+
ions were calculated to be 1.8 × 10-9 M, 1.9 × 10-9 M, 3.2 × 10-9 M, and 1.64×10-10 M,
respectively (detection limit, DL = 3σ/S, where σ is the standard deviation of the blank solution,
and S is the slope of the calibration curve). Curiously, Job plot measurements clearly showed the
formation of complexes 1-Al3+, 1-Fe3+, 1-Cr3+ and 2-Zn2+ in 1:1 stoichiometry (Fig. 12). The
ESI-MS spectra of these 1-Al3+, 1-Fe3+, 1-Cr3+ and 2-Zn2+ also confirmed the 1:1 stoichiometry
of these complexes (Fig.S10-S13). The calculated association constants based on Benesi-
Hildebrand method were 5.5 × 106 M-1, 2.77 × 106 M-1, 5.0 × 106 M-1 and 1.69 ×10-9 M-1 for 1-
Al3+, 1-Fe3+, 1-Cr3+ and 2-Zn2+ complexes, respectively (Fig. S14). Effect of pH on the
fluorescence emission from probe 2, with and without Zn2+ ions, showed that probe 2 is useful
only in the neutral and basic pH range for detection purposes (Fig. S9b).
………………………………………Figures 11,12 and 13 …………………………………..
Further, the fluorescence emission of 1-Al3+, 1-Fe3+, 1-Cr3+ and 2-Zn2+ complexes (10 M)
at 471 nm, in the presence of 50 M quantity of other metal ions (Na+, K+, Ag+, Zn2+, Co2+,
Ni2+, Ca2+, Ba2+, Pb2+, Cu2+, Mn2+, Hg2+, Cd2+, Fe2+, Mg2+, Fe3+,Cr3+, Al3+ ,and In3+ and Bi3+)
indicated little or no interference in the detection process (Fig.S15). The non-interference of
competing for metal ions clearly suggest that ligands 1and 2 can be used as sensors for the
detection of specific metal ions. The major distinction between the two ligands is the difference
between the two donor-atoms present in them. Ligand 1 that senses trivalent metal ions contains

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N and O combination of donor-atoms, separated by four intervening bonds. Ligand 2 that detects
divalent Zn2+ ions has N and N combination of donor-atoms, separated by three bonds. Common
divalent metal ions (including Hg2+ and Cd2+), and trivalent cations (including Bi3+ and In3+) do
not interference in the formation of 1-Al3+, 1-Fe3+, 1-Cr3+ and 2-Zn2+ complexes. These
observations clearly demonstrate the role of donor atoms vis-à-vis ionic radius in the selective
recognition of Zn2+ by ligand 2, and Fe3+, Al3+ and Cr3+ by ligand 1.
………………………………………Figure 13 ………………………………………………..
To assess the potential of the probes 1and 2 for possible applications in biological samples,
we explored their suitability as a turn-on sensors for the detection of Fe3+, Al3+, Cr3+ and Zn2+
ions in live Rhizoctonia solani cells. The mycelia of R. solani were exposed to these four metal
ions as described in the experimental section, and then washed with sterile distilled water to
remove excess metal ions. When the mycelia were viewed through the fluorescence microscope,
bright blue fluorescence was observed (Fig. 13) only from the cells treated with both the metal
ion and the probe 1 or 2. R. solani mycelia treated only with the probe or only with the metal
ions, did not show any fluorescence. The fact that ~3.5 μM concentration of probes was enough
to exhibit the desired level of fluorescence for imaging cells clearly demonstrated the sensitivity
of 1 and 2 (Fig. S16).
4. Conclusions
In this report, we provide evidence for chelation of specific metal ions with two non-
luminescent imidazo[1,2-a]pyridine derivatives that leads to chelation induced fluorescence turn-
on behaviour. The ability of ligand 1 to form a 1:1 complex with Fe3+, Al3+ or Cr3+ has been
proved using Job plot. The selectivity displayed by ligand 2 for Zn2+ is also confirmed by the 1:1
geometry of the complex, as deduced from Job plot. Both ligands 1 and 2 attain coplanar
structure upon complex formation as revealed by difference NOE experiments and XRD
analysis. Complex formation with Zn2+, Fe3+, Al3+ or Cr3+ by ligands 1 and 2 is not disrupted by
common monovalent, divalent and trivalent metal ions. In the case of ligand 1, the direct
involvement of donor atoms O and N in complex formation has been demonstrated using NMR
methods. In the case of probe 2, the involvement of pyridyl and imidazolyl N-atoms in complex

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formation has been confirmed by XRD analysis, and the suitability of 2 for selective detection of
Zn2+ is demonstrated. The suitability of probes 1 and 2 for bioimaging is also demonstrated.
Acknowledgements
One of the authors, MR, thanks to the UGC, New Delhi, India, for Senior Research
Fellowship. Financial support from CSIR 12th plan project CSC0201 is acknowledged.
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Figure Legends
Probe 1
Probe 2
Fig. 1 ORTEP diagrams of ligand 1 and 2 obtained from single crystal XRD analysis
(a)
(b)
Fig. 2 Absorption (a) and fluorescence profiles of probe 1 (10µM) in response to the addition of
indicated metal ions. Only Al3+, Cr3+ and Fe3+ cause an effect.

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(a)
(b)
(c)
Figure 3. Changes in the absorption profile of 1 (10 µM) in CH3CN upon addition of various amounts of
trivalent metal ions: (a) Al3+ (0-13 equiv.) (b) Fe3+ (0-25equiv.) and (c) Cr3+ (0-15 equiv.).

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Cr3+
2+ In3+
Hg2+ Zn2+ Mg
Fe2+
Bi3+
Al3+
Mn2+
Ni2+
Cu2+ Sm3+
Co2+
Ca2+
Fe3
Ba2+
Dye 1
Cd2+
+
(a)
Mn2+
Fe3+
Zn2+
Fe2+
Cu2+
Bi3+ Sm3+
Co2+
Ba2+
Hg2+
Ni2+
Al3+
Cd2+
Mg2+ In3+
Ca2+
Cr3+
Dye 2
(b)
Figure 4. Naked-eye detection of (a) trivalent Al3+/Cr3+/Fe3+ in CH3CN and (b) divalent Zn2+ in water-
CH3CN (1:1, v/v) mixture under UV-light; the concentration of probe 1 was 10 µM, probe 2 was 6.6 µM
and the metal ions was 15µM.

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Figure 5. 1H NMR (a) and 13C NMR (b) spectra of 1 in CDCl3:D2O (8:2) mixture with indicated amounts
of Fe3+ ions.

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Calculation of enhancement in NOE
푃푒푎푘 퐴
0.0585
0.0287
NOE of Probe 1
=
= 2.0383
푃푒푎푘 퐵
푃푒푎푘 퐴
푃푒푎푘 퐵
0.1509
NOE of Probe 1+Al3+ complex
=
= 1.388
0.1087
Figure 6. A comparison of difference NOE spectra of probe 1 and 1-Al3+ complex shows more NOE in
1-Al3+ complex suggesting a coplanar structure for the complex.

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Fig. 7 Absorption (a) and fluorescence profiles of probe 2 (5.6 μM) in response to the addition of
indicated metal ions. Only Zn2+ causes an effect.
1000
[Zn2+
]
800
600
400
200
0
(a)
(b)
400
450
500
550
600
650
Wavelength(nm)
Figure 8. Changes in the absorption (a) and fluorescence (b) profiles of probe 2 (5.6 μM) in water-CH3CN (1:1,
v/v) mixture upon addition of various concentrations of Zn2+ions (0-60 μM).

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Fig. 9. ORTEP diagram of the 2-ZnCl2 complex obtained from single crystal XRD analysis
Fig. 10. Linear fit obtained from the plot of fluorescence intensity versus concentration of metal
ion: (a) probe 1 (10 µM) titrated against serial concentrations of trivalent (Al3+, Cr3+ and Fe3+)
metal ions (em 471 nm); (b) probe 2 (5.6 μM) titrated against indicated concentrations of Zn2+
ions (em 460 nm).

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800
(a)
600
400
200
0
400
450
500
550
600
Wavelength(nm)
800
600
400
200
0
(b)
400
450
500
550
600
Wavelength (nm)

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(c)
Figure 11. Changes in the fluorescence profile of 1 (10 µM) in CH3CN upon addition of various
concentrations of trivalent metal ions: (a) Al3+ (0-15 equiv.), (b) Fe3+ (0-15 equiv.) and (c) Cr3+ (0-15
equiv.)