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Two rhodamine-azo based fluorescent probes for

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Cite this: Dalton Trans., 2022, 51,

recognition of trivalent metal ions: crystal
15555 structure elucidation and biological applications†
Jayanta Mandal,a Kunal Pal,b Sougata Ghosh Chowdhury,b Parimal Karmakar, b

Anangamohan Panja, c Snehasis Banerjee d and Amrita Saha *a

Two rhodamine and azo based chemosensors (HL1 = (3’,6’-bis(ethylamino)-2-((2-hydroxy-3-methoxy-5-

( phenyldiazenyl)benzylidene)amino)-2’,7’-dimethylspiro[isoindoline-1,9’-xanthen]-3-one) and HL2 =
(3’,6’-bis(ethylamino)-2-(((2-hydroxy-3-methoxy-5-(p-tolyldiazenyl)benzylidene)amino)-2’,7’-dimethyl-
spiro[isoindoline-1,9’-xanthen]-3-one) have been synthesized for colorimetric and fluorometric detection
of three trivalent metal ions, Al3+, Cr3+ and Fe3+. The chemosensors have been thoroughly characterized
by different spectroscopic techniques and X-ray crystallography. They are non-fluorescent due to the
presence of a spirolactam ring. The trivalent metal ions initiate an opening of the spirolactam ring when
excited at 490 nm in Britton–Robinson buffer solution (H2O/MeOH 1 : 9 v/v; pH 7.4). The opening of the
spirolactam ring increases conjugation within the probe, which is supported by an intense fluorescent
pinkish-yellow colouration and an enhancement of the fluorescence intensity of the chemosensors by
∼400 times in the presence of Al3+ and Cr3+ ions and by ∼100 times in the presence of Fe3+ ions. Such a
type of enormous fluorescence enhancement is rarely observed in other chemosensors for the detection
of trivalent metal ions. A 2 : 1 binding stoichiometry of the probes with the respective ions has been
confirmed by Job’s plot analysis. Elucidation of the crystal structures of the Al3+ bound chemosensors (1
and 4) also justifies the 2 : 1 binding stoichiometry and the presence of an open spirolactam ring within
the chemosensor framework. The limit of detection (LOD) values for both the chemosensors towards the
respective metal ions are in the order of ∼10−9 M which supports their application in the biological field.
Received 9th February 2022, The biocompatibility of the ligands has been studied with the help of the MTT assay. The results show that
Accepted 25th August 2022
no significant toxicity was observed up to 100 µM of chemosensor concentration. The capability of our
DOI: 10.1039/d2dt00399f synthesized chemosensors to detect intracellular Al3+, Cr3+ and Fe3+ ions in the cervical cancer cell line
rsc.li/dalton HeLa was evaluated with the aid of fluorescence imaging.

Introduction research.1–3 Various techniques such as atomic absorption

spectroscopy,4 inductively coupled plasma-mass spectroscopy,5
Colorimetric and fluorescent chemosensors which are plasma emission spectrometry,6 neutron activation analysis,7
designed for selective detection of metal ions play a crucial chromatography8 and voltammetry9 are available for detection
role in the development of medicinal and environmental of different metal ions in food, biological systems, and
environmental and industrial samples. Most of these detection
techniques are expensive and users face challenges in sample
a
Department of Chemistry, Jadavpur University, Kolkata-700032, India. preparation, instrument handling and their costly mainten-
E-mail: amritasahachemju@gmail.com, amrita.saha@jadavpuruniversity.in, ance charges. In this regard, the fluorescence study is a highly
asaha@chemistry.jdvu.ac.in; Tel: +91-33-24572146
b
sensitive, user friendly, low cost and real time monitoring
Department of Life Science and Biotechnology, Jadavpur University, Kolkata-
700032, India
process. Among different metal ions, trivalent metal ions, Al3+,
c
Department of Chemistry, Gokhale Memorial Girls’ College, 1/1 Harish Mukherjee Cr3+ and Fe3+ deserve special mention. Extensive application
Road, Kolkata-700020, India of these metals in industry and daily life results in their
d
Department of Higher Education, University Branch, Bikash Bhavan, Salt Lake, diffusion and contamination in living systems and causes a
Sector-3, Kolkata, 700091, India
wide variety of diseases.10 Aluminium is the most abundant
† Electronic supplementary information (ESI) available. CCDC 2051844–2051847
for HL1, HL2, and complexes 1 and 4, respectively. For ESI and crystallographic
metal in the Earth’s crust and used vastly for domestic pur-
data in CIF or other electronic format see DOI: https://doi.org/10.1039/ poses. The excessive concentration of Al3+ in the human body
d2dt00399f causes myopathy, encephalopathy, microcytic hypochromic

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 View Article Online

Paper Dalton Transactions

anemia, Parkinson’s disease and Alzheimer’s disease.11 Iron is yields. Their off/on-type of sensing property is owing to their
an important dietary element since it is present in the active structural properties. The presence of protons or metal ions
site of different metalloenzymes which play crucial roles in initiates the opening of their spirolactam ring, resulting in a col-
different physiological processes, such as oxygen uptake,12 orimetric response and strong fluorescence emission.20 In the
oxygen metabolism,13 electron transfer14 etc. Therefore, iron chemosensors, an azo unit is introduced due to its high photo-
deficiency leads to low blood pressure, anemia, etc,15,16 sensitivity and it may also initiate longer conjugation.21
whereas excess iron storage can generate reactive oxygen Recently, two rhodamine and azo based chemosensors which
species, which can damage lipids, proteins and nucleic selectively detect Al3+ and Cu2+ ions were reported. In the case of
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acids.17,18 Cr3+ is an essential nutritional trace element for the Al3+, rhodamine 2B and nitro substituted azo units are present
human body and its deficiency causes cardiovascular disease in the chemosensor.22 Rhodamine 2B and simple azo units are
and diabetes and affects the glucose and lipid metabolism present in the case of the Cu2+ sensor.23 Interestingly, replace-
resulting in nervous system disorder.19 Again, Cr3+ from indus- ment of the rhodamine 2B unit with a rhodamine 6G unit and a
trial waste causes damage to the environment and living simple azo or methyl substituted azo unit results in chemo-
systems. Generally, chemosensors designed for sensing indi- sensors HL1 and HL2 which selectively detect trivalent metal
vidual metal ions are well known in the literature, but only a ions. Chemosensors HL1 and HL2 exhibit an ∼400, ∼400 and
handful of examples of dual or multi-metal ion sensing ∼100 times enhancement of fluorescence intensity at 555 nm
chemosensors are reported. Such a type of chemosensor wavelength in the presence of Al3+, Cr3+ and Fe3+, respectively.
reduces the synthesis cost and analytical time. The permissible limit of Al3+, Cr3+ and Fe3+ ions in water is
Here, we have demonstrated rhodamine and azo containing 2.9 mg L−1, 0.3 mg L−1, and 0.05 mg L−1, respectively. Therefore,
fluorescent and colorimetric probes HL1 and HL2 [HL1 = 3′,6′- a low detection limit (LOD) of HL1 and HL2 against Al3+, Cr3+
bis(ethylamino)-2-((2-hydroxy-3-methoxy-5-(phenyldiazenyl)ben- and Fe3+ ions (∼10−9 M order) will allow us to use them in real
zylidene)amino)-2′,7′-dimethylspiro[isoindoline-1,9′-xanthen]-3- world applications and cell imaging studies. We have success-
one and [HL2 = 3′,6′-bis(ethylamino)-2-((2-hydroxy-3-methoxy-5- fully elucidated the X-ray crystal structures of both the chemo-
(p-tolyldiazenyl)benzylidene)amino)-2′,7′-dimethylspiro[isoindo- sensors and their Al3+ bound complexes. Their X-ray crystal
line-1,9′-xanthen]-3-one] for monitoring trivalent ions, Al3+, Cr3+ structures confirm the 1 : 2 binding stoichiometry between the
and Fe3+ (Scheme 1). Common fluorophore units present in metal ion and the chemosensors. The crystal structures of Al3+
fluorescent chemosensors are coumarin, pyrene, 1,8-naphthali- bound rhodamine-based chemosensor complexes are scarce in
mide, rhodamine, squaraine, cyanine, boron dipyrromethene the literature. Some recently reported rhodamine-based chemo-
difluoride (BODIPY), nitrobenzofurazan, etc. Among them, rho- sensors are given in Chart S124 (ESI†). A literature survey on pre-
damine-based chemosensors are capable of both naked eye viously reported rhodamine based chemosensors24–26 (both
detection and fluorescence emission. They have excellent single metal ion and multiple metal ion detectors) and other
photophysical properties with greater photostability, visible chemosensors27–30 (both single metal ion and multiple metal
wavelength emission, high extinction coefficients and quantum ion sensors) reveals that our reported chemosensors, which sim-
ultaneously detect three trivalent metal ions, have certain advan-
tages like easy synthetic procedures involving less expensive
chemicals, a high fluorescence enhancement in the presence of
metal ions, low LOD values (10−9 M order), dual sensing charac-
ter (colorimetric and fluorescence), X-ray structures of both free
chemosensors and their metal bound forms and real world and
biological applications. One main drawback of these chemo-
sensors is their partial solubility in an aqueous medium.31,32,24m

Experimental section
Materials and physical measurement description
All reagent or analytical grade chemicals and solvents were col-
lected from commercial sources and used without further puri-
fication. Elemental analysis was carried out using a Perkin-
Elmer 240C elemental analyzer. Infrared spectra
(400–4000 cm−1) were recorded using KBr pellets on a Nicolet
Magna IR 750 series-II FTIR spectrophotometer. Absorption
spectral data were collected using a Cary 60 spectrophotometer
(Agilent) with a 1 cm-path-length quartz cell. Electron spray
Scheme 1 Route to preparation of the azo-aldehyde and chemo- ionization mass (ESI-MS positive) spectra were recorded using
sensors (HL1 and HL2). a MICROMASS Q-TOF mass spectrometer. A Fluromax-4 spec-

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Dalton Transactions Paper

trofluorimeter was used to collect emission spectral data at Yield: 1.094 g (80%). Anal. Calc. for C42H40N6O4Cl6: C
room temperature (298 K) in Britton–Robinson buffer at pH = 55.71%; H 4.45%; N 9.28%; Found: C 55.69%; H 4.40%; N
7.4 solution under degassed conditions. A time-resolved spec- 9.26%. IR (cm−1, KBr): ν(CvN) 1624s; ν(O–H) 3433s; ν(CvO)
trofluorometer from IBH, UK was used to collect fluorescence 1690s (Fig. S1†). ESI-MS ( positive) in MeOH: the base peak
lifetime data. 1H and 13C NMR spectral data were collected appeared at m/z = 689.27, corresponding to [HL1 + Na]+
using Bruker 300 and Bruker 400 spectrometers in CD3OD and (Fig. S2†). UV-Vis, λmax (nm), (ε (dm3 mol−1 cm−1)) in Britton–
DMSO-d6 solvents. Cyclic voltammetric experiments were per- Robinson buffer at pH = 7.4: 307 (39 020).
formed using a PC-controlled PAR model 273A electrochemical 1
H NMR (300 MHz, d6-DMSO) δ ppm: 1.20 (–CH3) (t, 6H, J =
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system under a nitrogen atmosphere using an Ag/AgCl refer- 7.2 Hz), 1.86 (Ar–CH3) (s, 6H), 3.34–3.099 (–CH2) (m, 4H), 3.84
ence electrode, with a Pt disk working electrode and a Pt wire (–OCH3) (s, 3H), 5.09 (NH) (t, 2H, J1 = 4.8 Hz, J2 = 5.1 Hz), 6.23
auxiliary electrode in acetonitrile containing a supporting elec- (Ar–CH) (s, 2H), 6.34 (Ar–CH) (s, 2H), 7.06 (Ar–CH) (d, 1H, J =
trolyte, 0.1 M Bu4NClO4. 6.2 Hz), 7.44 (Ar–CH) (s, 1H), 7.49–7.67 (Ar–CH) (m, 4H),
7.82–7.96 (Ar–CH) (m, 3H), 8.32 (Ar–CH3) (s, 1H), 9.05
X-ray crystallography (–CHvN) (s, 1H), 11.55 (–OH) (s, 1H) (Fig. S3a†).
Single crystal X-ray data of the chemosensors (HL1 and HL2) 1
H NMR (400 MHz, d4-CD3OD) δ ppm: 1.31 (–CH3) (t, 6H),
were collected on a Bruker SMART APEX-II CCD diffractometer 1.92 (Ar–CH3) (s, 6H), 3.21–3.27 (–CH2) (m, 4H), 3.91 (–OCH3)
using graphite mono-chromated Mo Kα radiation (λ = (s, 3H), 6.29 (Ar–CH) (s, 2H), 6.49 (Ar–CH) (s, 2H), 7.53–8.29
0.71073 Å) at room temperature. Data processing, structure (Ar–CH) (m, 11H), 8.84 (–CHvN) (s, 1H), 11.54 (–OH) (s, 1H)
solution, and refinement were examined using the Bruker (Fig. S3b†).
Apex-II suite program. All available reflection data in the 2θmax 13
C NMR (d6-DMSO, 75 MHz) δ ppm: 14.62, 17.47, 37.94,
range were collected and corrected for Lorentz and polariz- 56.36, 66.08, 96.29, 104.06, 104.70, 118.92, 119.28, 119.77,
ation factors with Bruker SAINT plus.33 Reflections were then 122.74, 123.63, 124.31, 127.27, 128.71, 128.96, 129.32, 129.85,
corrected for absorption, inter-frame scaling, and different sys- 131.33, 134.59, 145.06, 146.67, 147.40, 148.39, 149.26, 150.56,
tematic errors with SADABS.34 The structures were solved by 151.47, 151.99, 152.35, 165.25 (Fig. S4†).
the direct methods and refined with the help of the full matrix
least-square technique based on F2 with the SHELX-2017/1
software package.35 All the non-hydrogen atoms were refined Synthesis of chemosensor HL2
with anisotropic thermal parameters. C–H hydrogen atoms A mixture of N-(rhodamine-6G)lactam-hydrazine (2.0 mmol,
were attached to the geometrical positions with Uiso = 1/2Ueq 0.8564 g) and 2-hydroxy-3-methoxy-5-( p-tolyldiazenyl)benz-
for those atoms they were attached to. Some restraints were aldehyde (2.0 mmol, 0.540 g) was heated under refluxing con-
applied when refining disordered DMF molecules and nitrate ditions for ca. 2 h in methanol–chloroform (9 : 1, v/v). Light
ions to make it reasonable. One of the dichloromethane mole- yellow crystals were formed from slow evaporation of the
cules present as the solvent of crystallization in HL1 was methanol–chloroform solvent mixture.
highly disordered, which was very difficult to model, and thus Yield: 1.201 g (86%). Anal. Calc. for C42H41N6O4Cl3: C
their final contribution to the R value was excluded through 63.04%; H 5.16%; N 10.50%. Found: C 62.98%; H 5.10%; N
the SQUEEZE procedure. Similarly in complex 1, two water and 10.46%. IR (cm−1, KBr): ν(CvN) 1621s; ν(O–H) 3421s; ν(CvO)
two DMF molecules present as solvents of crystallization are 1696s (Fig. S1†). ESI-MS ( positive) in MeOH: the molecular ion
highly disordered and it is very difficult to produce a good peak appeared at m/z = 681.27, corresponding to [HL2 + 1]+
model to resolve the issue and thereby their contributions (Fig. S5†). UV-Vis, λmax (nm), (ε (dm3 mol−1 cm−1)) in Britton–
were removed by a squeeze procedure from the final R values. Robinson buffer (10 mM) at pH = 7.4: 310 (38 440).
Crystal data and details of data collection and refinement for 1
H NMR (300 MHz, d6-DMSO) δ ppm: 1.21 (–CH3) (t, 6H),
the chemosensors (HL1 and HL2) and complexes (1 and 4) are 1.86 (Ar–CH3) (s, 6H), 2.39 (Ar–CH3) (s, 3H), 3.18–3.09 (–CH2)
collected in Table S2.† (m, 4H), 3.85 (–OCH3) (s, 3H), 5.09 (NH) (t, 2H, J1 = 4.5 Hz, J2 =
5.4 Hz), 6.22 (Ar–CH) (s, 2H), 6.34 (Ar–CH) (s, 2H), 7.06 (Ar–
Synthesis of N-(rhodamine-6G)lactam-hydrazine and azo- CH) (d, 1H, J = 6.6 Hz), 7.36 (Ar–CH) (d, 2H, J = 8.4 Hz), 7.42
aldehydes (Ar–CH) (s, 1H), 7.65–7.56 (Ar–CH) (m, 2H), 7.75 (Ar–CH) (d,
N-(Rhodamine-6G)lactam-hydrazine and azo-aldehydes were 2H, J = 8.1 Hz), 7.94 (Ar–CH) (d, 2H, J = 6.6 Hz), 8.32 (Ar–CH)
prepared by following literature procedures.24h,21 (s, 1H), 9.03 (–CHvN) (s, 1H), 11.51 (–OH) (s, 1H) (Fig. S6a†).
1
H NMR (400 MHz, d4-CD3OD) δ ppm: 1.29 (–CH3) (t, 6H),
Preparation of chemosensor HL1 1.92 (Ar–CH3) (s, 6H), 2.42 (Ar–CH3) (s, 3H), 3.26–3.21 (–CH2)
A mixture of N-(rhodamine-6G)lactam-hydrazine (2.0 mmol, (m, 4H), 3.89 (–OCH3) (s, 3H), 6.26 (Ar–CH) (s, 2H), 6.44 (Ar–
0.8564 g) and 2-hydroxy-5-( phenyldiazenyl)benzaldehyde CH) (s, 2H), 8.02–7.30 (Ar–CH) (m, 9H), 8.30 (Ar–CH) (s, 1H),
(2.0 mmol, 0.512 g) was heated under refluxing conditions for 8.82 (–CHvN) (s, 1H), 11.50 (–OH) (s, 1H) (Fig. S6b†).
ca. 2 h in methanol–chloroform (9 : 1, v/v). Light yellow crystals 13
C NMR (d6-DMSO, 75 MHz) δ ppm: 14.62, 17.47, 21.92,
were formed from slow evaporation of the methanol–chloro- 37.94, 56.36, 66.08, 96.29, 104.06, 104.70, 118.92, 119.28,
form solvent mixture. 119.77, 122.74, 123.63, 124.31, 127.27, 128.71, 128.96, 129.32,

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129.85, 131.33, 134.59, 145.06, 146.67, 147.40, 148.39, 149.26, Yield: 1.302 g (75%). Anal. Calc. for C80H74FeN13O11: C
150.56, 151.47, 151.99, 152.35, 165.27 (Fig. S7†). 66.74%; H 5.15%; N 12.56%; Found: C 66.55%; H 5.01%; N
12.41%. IR (cm−1, KBr): ν(CvN) 1603s; ν(NO3−) 1300s and
Synthesis of [Al(L1)2](NO3)·3DMF·2H2O (1) 809s; ν(CvO) 1641s (Fig. S8†). The molecular ion peak
Firstly, 2 mL of a methanol solution of aluminum nitrate non- appeared at m/z = 1386.51 and 693.83, corresponding to [Fe
ahydrate (1.0 mmol, 0.375 g) was added carefully to 20 mL of a (L1)2]+ and [Fe(L1)2 + H]2+, respectively (Fig. S13†). UV-Vis, λmax
methanol–chloroform (9 : 1, v/v) solution of HL1 (2.0 mmol, (nm), (ε (dm3 mol−1 cm−1)) in Britton–Robinson buffer at pH =
1.332 g). Then, the reaction mixture was stirred for ca. 3 h. Red 7.4: 525 (80 470).
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crystals were collected in high yield after slow evaporation of

the methanol–DMF solvent mixture. Synthesis of [Al(HL2)(L2)](NO3)2·4H2O (4)
Yield: 1.348 g (79%). Anal. Calc. for C89H99AlN16O16: C Firstly, 2 mL of a methanol solution of aluminum nitrate non-
63.79%; H 5.95%; N 13.37%; Found: C 63.68%; H 5.86%; N ahydrate (1.0 mmol, 0.375 g) was added carefully to 20 mL of a
13.29%. IR (cm−1, KBr): ν(CvN) 1601s; ν(NO3−) 1300s and methanol–chloroform (9 : 1, v/v) solution of HL2 (2.0 mmol,
809s; ν(CvO) 1646s (Fig. S8†). The molecular ion peak 1.362 g). Then, the reaction mixture was stirred for ca. 3 h. Red
appeared at m/z = 1357.37 and 679.26, corresponding to [Al crystals were collected in high yield after slow evaporation of
(L1)2]+ and [Al(L1)2 + H]2+, respectively (Fig. S9†). UV-Vis, λmax the solvent.
(nm), (ε (dm3 mol−1 cm−1)) in Britton–Robinson buffer at pH = Yield: 0.802 g (76%). Anal. Calc. for C82H87AlN14O18: C
7.4: 525 (59 558). 62.19%; H 5.54%; N 12.38%; Found: C 62.01%; H 5.29%; N
1
H NMR (400 MHz, d6-DMSO) δ ppm: 1.20 (–CH3) (t, 6H, J = 12.07%; IR (cm−1, KBr): ν(CvN) 1608s; ν(NO3−) 1300s and
7.2 Hz), 1.87 (Ar–CH3) (s, 6H), 3.12–3.19 (–CH2) (m, 4H), 5.93 822s; ν(CvO) 1640s (Fig. S14†). The molecular ion peak
(NH) (s, 1H), 6.29 (Ar–CH) (d, 2H, J = 6 Hz), 6.40 (Ar–CH) (d, appeared at m/z = 1385.58 and 693.29, corresponding to [Al
2H, J = 6.3 Hz), 7.47–7.61 (Ar–CH) (m, 6H), 7.63–7.88 (Ar–CH) (L2)2]+ and [Al(L2)2 + H]2+, respectively (Fig. S15†). UV-Vis, λmax
(m, 4H), 8.41 (NH) (d, 2H), 9.07 (–CHvN) (s, 1H) (Fig. S10a†). (nm), (ε (dm3 mol−1 cm−1)) in Britton–Robinson buffer at pH =
1
H NMR (400 MHz, d4-CD3OD) δ ppm: 1.32 (–CH3) (t, 6H), 7.4: 525 (59 847).
2.09 (Ar–CH3) (s, 6H), 3.12–3.19 (–CH2) (m, 4H), 6.53 (NH) (s, 1
H NMR (400 MHz, d6-DMSO) δ ppm: 1.20 (–CH3) (t, 6H, J =
1H), 6.89 (Ar–CH) (d, 4H), 7.42–8.45 (Ar–CH) (m, 10H), 8.90 7.2 Hz), 1.87 (Ar–CH3) (s, 6H), 2.39 (Ar–CH3) (s, 3H), 3.12–3.19
(–CHvN) (s, 1H) (Fig. S10b†). (–CH2) (m, 4H), 5.93 (NH) (s, 1H), 6.29 (Ar–CH) (d, 2H, J = 6
13
C NMR (d6-DMSO, 75 MHz) δ ppm: 14.55, 17.92, 38.10, Hz), 6.40 (Ar–CH) (d, 2H, J = 6.3 Hz), 7.47–7.61 (Ar–CH) (m,
56.35, 96.62, 104.16, 105.21, 118.29, 119.09, 119.67, 122.41, 4H), 7.63–7.88 (Ar–CH) (m, 4H), 8.41 (Ar–CH) (d, 1H), 9.07
122.74, 123.63, 124.30, 125.12, 127.34, 128.74, 129.35, 130.33, (–CHvN) (s, 1H) (Fig. S16a†).
134.60, 141.42, 145.08, 148.17, 149.22, 150.23, 150.40, 151.47, 1
H NMR (400 MHz, d4-CD3OD) δ ppm: 1.34 (–CH3) (t, 6H),
151.91, 156.01, 157.22, 164.47, 168.07 (Fig. S11†). 2.09 (Ar–CH3) (s, 6H), 2.68 (Ar–CH3) (s, 3H), 3.04–2.90 (–CH2)
(m, 4H), 6.24 (Ar–CH) (s, 2H), 6.46 (Ar–CH) (s, 2H), 7.43–8.18
Synthesis of [Cr(L1)2](NO3) (2) (Ar–CH) (m, 10H), 8.45 (Ar–CH) (d, 1H), 8.99 (–CHvN) (s, 1H)
Firstly, 2 mL of a methanol solution of chromium nitrate non- (Fig. S16b†).
ahydrate (1.0 mmol, 0.400 g) was added carefully to 20 mL of a 13
C NMR (d6-DMSO, 75 MHz) δ ppm: 14.55, 17.91, 21.91,
methanol–chloroform (9 : 1, v/v) solution of HL1 (2.0 mmol, 38.10, 56.34, 96.60, 104.16, 105.01, 118.99, 119.09, 119.67,
1.332 g). Then, the reaction mixture was stirred for ca. 3 h. Red 122.41, 122.74, 123.63, 124.30, 125.12, 127.34, 128.74, 129.35,
microcrystals were collected in high yield after slow evapor- 130.33, 134.60, 141.46, 145.08, 148.17, 149.23, 150.23, 150.40,
ation of the solvent. 151.47, 151.91, 156.01, 157.25, 164.27, 168.27 (Fig. S17†).
Yield: 1.385 g (80%). Anal. Calc. for C80H74CrN13O11: C
66.74%; H 5.16%; N 12.60%; Found: C 66.65%; H 5.01%; N Synthesis of [Cr(L2)2](NO3) (5)
12.50%. IR (cm−1, KBr): ν(CvN) 1601s; ν(NO3−) 1300s and Firstly, 2 mL of a methanol solution of chromium nitrate non-
810s; ν(CvO) 1655 s (Fig. S8†). The molecular ion peak ahydrate (1.0 mmol, 0.400 g) was added carefully to 20 mL of a
appeared at m/z = 1382.51 and 691.76, corresponding to [Cr methanol–chloroform (9 : 1, v/v) solution of HL2 (2.0 mmol,
(L1)2]+ and [Cr(L1)2 + H]2+, respectively (Fig. S12†). UV-Vis, λmax 1.362 g). Then, the reaction mixture was stirred for ca. 3 h. Red
(nm), (ε (dm3 mol−1 cm−1)) in Britton–Robinson buffer at pH = coloured microcrystals were collected in high yield after slow
7.4: 525 (80 235). evaporation of the solvent.
Yield: 1.448 g (82%). Anal. Calc. for C82H78CrN13O11: C
Synthesis of [Fe(L1)2](NO3) (3) 66.84%; H 5.34%; N, 12.36%; Found: C 66.75%; H 5.28%; N
Firstly, 2 mL of a methanol solution of ferric nitrate nonahy- 12.31%; IR (cm−1, KBr): ν(CvN) 1600s; ν(NO3−) 1312s and
drate (1.0 mmol, 0.404 g) was added carefully to 20 mL of a 811s; ν(CvO) 1641s (Fig. S14†). The molecular ion peak
methanol–chloroform (9 : 1, v/v) solution of HL1 (2.0 mmol, appeared at m/z = 1411.55 and 706.28, corresponding to [Cr
1.332 g). Then, the reaction mixture was stirred for ca. 3 h. A (L2)2]+ and [Cr(L2)2 + H]2+, respectively (Fig. S18†). UV-Vis, λmax
red powder was collected in high yield after slow evaporation (nm), (ε (dm3 mol−1 cm−1)) in Britton–Robinson buffer at pH =
of the solvent. 7.4: 525 (80 235).

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Synthesis of [Fe(L2)2](NO3) (6) were cultured in an enriched cell culture medium, DMEM
Firstly, 2 mL of a methanol solution of ferric nitrate nonahy- (Dulbecco’s modified Eagle medium), supplemented with 10%
drate (1.0 mmol, 0.404 g) was added carefully to 20 mL of a FBS (fetal bovine serum) and a mixture of appropriate anti-
methanol–chloroform (9 : 1, v/v) solution of HL2 (2.0 mmol, biotics (streptomycin and penicillin) at a dose of 100 units per
1.362 g). Then, the reaction mixture was stirred for ca. 3 h. A ml. The cells were incubated at a temperature of 37 °C and in
red coloured powder was collected in high yield after slow the prevalence of 5% CO2.
evaporation of the solvent.
Cell internalisation studies
Yield: 1.377 g (78%). Anal. Calc. for C82H78FeN13O11: C
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66.66%; H 5.32%; N, 12.32%; Found: C 66.55%; H 5.25%; N The cervical cancer cell line HeLa was cultured on coverslips
12.30%; IR (cm−1, KBr): ν(CvN) 1605s; ν(NO3−) 1300s and for a period of 24 h. Then these cells were either left untreated
812s; ν(CvO) 1642s (Fig. S14†). The molecular ion peak or were exposed to a dose of ligands (10 µM) and Al3+ salt
appeared at m/z = 1414.54 and 707.77, corresponding to [Fe (5 µM), Cr3+ salt (5 µM), and Fe3+ salts (5 µM). These treated
(L2)2]+ and [Fe(L2)2 + H]2+, respectively (Fig. S19†). UV-Vis, λmax cells were then incubated for 24 h at a temperature of 37 °C.
(nm), (ε (dm3 mol−1 cm−1)) in Britton–Robinson buffer at pH = Afterwards the cells were thoroughly washed with the help of
7.4: 525 (82 471). 1× PBS. Finally the cells were mounted on a glass slide and
visualized with the aid of a fluorescent microscope (Leica).
UV-visible and fluorescence spectroscopic experiment
Computational method
Stock solutions of different ions (1 × 10−3 M) were prepared in
deionized water medium. A stock solution of the chemo- All computations were performed using the GAUSSIAN 09
sensors (HL1 and HL2) (1 × 10−3 M) was prepared in methanol (G09)37 software package. For optimization, we used the
medium. The chemosensor (HL1 and HL2) solution was then density functional theory method at the CAM-B3LYP level38,39
diluted to 1 × 10−5 M as per requirements. Competitive assays and the standard 6-31+G(d) basis set for C, H, N and O
of various cations and anions and other spectroscopic experi- atoms.40,41 TDDFT calculations were performed with the opti-
ments were performed in aqueous-methanol Britton–Robinson mized geometry to ensure only positive eigen values. Time-
buffer (10 mM) medium at pH 7.4. In competitive assay experi- dependent density functional theory (TDDFT)42–44 was per-
ments, the test samples were prepared by mixing appropriate formed using the conductor-like polarizable continuum model
amounts of the cation stock in 3 mL of the chemosensor (HL1 (CPCM)45–47 and the same CAM-B3LYP level and basis sets in a
and HL2) solution (1 × 10−5 M). methanol solvent system. GAUSSSUM48 was used to calculate
the fractional contributions of various groups to each mole-
Binding stoichiometry (Job’s plot) studies cular orbital.
Binding stoichiometry of the chemosensors with Al3+, Cr3+ and
Fe3+ ions was determined by Job’s continuous variation
method using absorption spectroscopy. At 25 °C temperature, Results and discussion
the absorbance was recorded for the solutions where the con- Synthesis and characterization
centrations of both chemosensors and Al3+, Cr3+ and Fe3+ ions N-(Rhodamine-6G)lactam-hydrazine and azo-aldehyde were pre-
were varied but the sum of their concentrations was kept con- pared following the published procedures.24h,21 The Schiff base
stant at 1 × 10−5 M. The relative change in absorbance (ΔA/A0) condensation reaction between rhodamine-6G based amine and
was plotted against mole fraction. The break point in the 2-hydroxy-5-(phenyldiazenyl)benzaldehyde or 2-hydroxy3-
resulting plot represents the mole fraction of the chemosensor methoxy-5-(p-tolyldiazenyl)benzaldehyde finally generated the
in the Al3+, Cr3+ and Fe3+ complexes. From the break point the chemosensors, HL1 and HL2, respectively (Scheme 1). Different
stoichiometry was determined. The final results were the spectroscopic techniques (UV-Vis, FT-IR and NMR), X-ray crys-
average of at least three experiments. tallography, ESI-mass and elemental analysis were utilized for
their complete characterization (Fig. S1–S21; ESI†).
Protocol of real sample analysis
The reaction between M(NO3)3·9H2O (where, M3+ = Al3+,
A stock solution of the chemosensors (HL1 and HL2) (1 × 10−2 Cr and Fe3+) and HL1/HL2 in a 1 : 2 ratio produced com-
3+

M) was prepared in methanol : H2O (9 : 1) medium. 3 mL of plexes 1–6, respectively. They were characterized by 1H NMR,
the real sample (saloon waste water or laboratory tap water) 13
C NMR, FT-IR spectroscopy, X ray-crystallography, and C, H
was taken in a vial and 3 µL stock solution of the chemo- and N analyses. In ESI-MS analysis, both the chemosensors
sensors (HL1 or HL2) was added to it. An instantaneous colour exhibit the base peak at m/z 689.27 and 681.27, corresponding
change was observed. After that, we recorded the images of the to [HL1 + Na]+ and [HL2 + 1]+, respectively. All six complexes
vials under a UV-lamp and visible light. 1–6 exhibit ESI-MS peaks at 1357.37, 1382.60, 1386.51,
1385.51, 1411.49, and 1414.64 corresponding to the [M(L1/
Cell culture L2)2]+ (where M = Al, Cr, Fe) species. Interestingly, all the
The cervical cancer cell line HeLa was procured from the experimental data are well matched with their simulated
National Center for Cell Science (NCCS) Pune, India. The cells patterns.

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Crystal structure descriptions of the chemosensors (HL1 and

HL2)
X ray-crystallographic analysis reveals that HL1 is crystallized
in the monoclinic system with the P21/c space group, while
HL2 in the triclinic system with the P1 ˉ space group
(Table S2†). The crystal structure of chemosensor HL1 is pre-
sented in Fig. 1, while that of HL2 is depicted in Fig. S20.†
Both chemosensors are non planar and their crystal structures
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confirm imine bond formation, and the presence of a spirolac-

tam ring, xanthene unit and azo chromophore within the
molecules. The C–O and C–N bond distances of HL1 vary
within the ranges 1.220–1.425 Å and 1.280–1.494 Å,
respectively.

Crystal structure descriptions of complexes 1 and 4

The crystals of complex 1 were developed from a methanol–
DMF solvent mixture whereas, the crystals of complex 4 were
collected from a chloroform–methanol solvent mixture.
Complex 4 crystallized in the triclinic system with space group Fig. 2 Crystal structure of one of the complex cations of 4. Atoms are
ˉ where the asymmetric unit consists of two complex cations shown as 30% thermal ellipsoids. H atoms are omitted for clarity.
P1
and four non-coordinating nitrate anions together with four
water molecules as the solvent of crystallization. The crystal
structure of one of the complex cations is shown in Fig. 2. chemosensor in a meridional fashion. The Al3+–Ophenoxide,
Al3+–Nimine bond distances vary within the ranges 1.807–1.929
Crystallographic data are collected in Table S2† and important
(3) Å, 1.811–1.863(2) Å and 1.957–2.000(3) Å, respectively. In
bond lengths and angles are given in Table S3.† The crystal
structure of complex 4 confirms the presence of an open spiro- complex 4, spirolactam amide is present in amide form in one
chemosensor and in iminolate form in the other one. The
lactam ring within the chemosensor and 2 : 1 binding stoichio-
presence of both amide and iminolate forms of spirolactam
metry between the chemosensor and the Al3+ ion. The struc-
tures of the complex cations in the asymmetric unit are similar amide is further supported by shorter C–O (C67–O6, 1.247(5)
Å) and longer C–N (C67–N9, 1.348(6) Å) bond distances in one
and the structure of only one unit is briefly described here. In
chemosensor unit and relatively long C–O (C26–O2, 1.297(5) Å)
the complex cations, the metal centre is distorted octahedral
being coordinated with two imine N-atoms (N4 and N10), two and short C–N (C26–N3, 1.323(5) Å) bond distances in another
chemosensor unit. The azo bonds (N11–N12 and N5–N6)
phenoxide O-atoms atoms (O3 and O7) of both ligands and
exhibit double bond character with distances of 1.265(6) Å and
two O-atoms of open spirolactam amide (O2 and O6) of the
1.277(6) Å, repetitively. In the complex the azo bound phenyl
ring is slightly twisted with respect to the phenolate ring. The
dihedral angles between these two phenyl rings are 21.91° and
36.92° for complex 4 (Fig. 2).
Complex 1 crystallizes in the same crystal system as
complex 4 does but the asymmetric unit of complex 1 consists
of a complex cation, comprising an Al3+ ion and two chemo-
sensors, and one non-coordinating nitrate anion. Three DMF
and two water molecules are also present as solvents of crystal-
lization. In complex 1, spirolactamide is present exclusively in
iminolate form. The presence of the iminolate form is con-
firmed by longer C–O (C10–O1 = 1.290 Å, C11–O2 = 1.291 Å)
and shorter C–N (C10–N10 = 1.312 Å, C11–N2 = 1.307 Å) bond
distances within the complex cation (Fig. S21 and Table S3;
ESI†).

NMR studies
All 1H NMR spectra are recorded in DMSO-d6 solvent. The 1H
Fig. 1 Crystal structure of chemosensor HL1. Atoms are shown as 30%
NMR spectrum of HL2 is discussed as a representative
thermal ellipsoids. H atoms and the solvent molecule are omitted for example. Here, both phenolic –OH and imine proton appear
clarity. as sharp singlets at 11.51 and 9.04 ppm, respectively. Aromatic

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protons appear as singlets at 8.32 and 7.42 ppm, representing We have also performed 1H NMR titration in DMSO-d6
the azo-aromatic part, and doublets at 7.74 and 7.36 ppm, solvent. Gradual addition of Al3+ (0–2 equivalent) to the
representing the O-vanillin part. Aromatic protons adjacent to chemosensor (HL1/HL2) solution shows gradual dis-
the spirolactam ring appear as doublets at 7.94 and 7.06 ppm appearance of the phenolic-OH proton and aliphatic-NH
and multiplets within 7.65–7.56 ppm. Aromatic protons proton confirming the opening of the spirolactam ring fol-
present in the xanthene part appear as sharp singlets with lowed by complexation through phenoxide oxygen, amide
double intensity at 6.34 and 6.22 ppm. Aliphatic amine (–NH) oxygen and imine nitrogen atoms. Broadening of both aro-
protons appear as a triplet at 5.09 ppm. Aromatic –OCH3 matic and aliphatic protons is also observed during this titra-
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protons appear as a singlet at 3.84 ppm. Aliphatic CH2 protons tion process (Fig. S22 and S23; ESI†). Since all the sensing
appear as a multiplet within 3.18–3.09 ppm. Aromatic CH3 experiments are performed in methanol : water solution, we
protons appear as singlets at 2.39 and 1.85 ppm, whereas ali- have performed 1H NMR of the free chemosensors and com-
phatic CH3 protons appear as a triplet at 1.21 ppm (Fig. S6†). plexes 1 and 4 in CD3OD. Well resolved NMR spectra are pre-
The 1H NMR spectrum data of HL1 are collected in the sented in the Experimental section and ESI (Fig. S10b and
Experimental section (Fig. S3; ESI†). S16b†).
Both Al3+-probe complexes (1 and 4) give clean and similar
types of 1H NMR spectra in DMSO-d6 solvent. In complex 4, Absorption spectral analysis
the coordination of metal with the chemosensors results in The UV-Vis spectra of the chemosensors (HL1 and HL2) are
the disappearance of the phenolic –OH proton, whereas the first examined in 10 mM Britton–Robinson buffer solution at
position of the imine proton (singlet, 9.08 ppm) remains pH 7.4 (1 : 9, water : methanol, v/v). Chemosensors HL1 and
almost unchanged. We also observe a downfield shift of aro- HL2 exhibit well-resolved bands at ∼307 and ∼360 nm, respon-
matic protons and broadening of the peaks. Aromatic protons sible for π → π* and n → π* types of transitions. Interestingly
adjacent to the open spirolactam ring, azo-aromatic moiety upon successive addition of Al3+, Fe3+ and Cr3+ ions (0–6 µM,
and o-vanillin part appear as multiplets within the range 10 mM Britton–Robinson buffer solution; pH 7.4; 1 : 9, water :
7.96–7.47 ppm. Protons present in the xanthene part appear as methanol, v/v) to the chemosensor (10 µM), a new peak
doublets at 6.40 and 6.29 ppm, respectively. Aliphatic CH2 appears at ∼525 nm with significant changes in the spectra of
protons appear as a multiplet within 3.19–3.12 ppm and aro- all chemosensors (HL1 and HL2). In the case of Fe3+, an
matic OCH3 protons (merged with water molecules) appear as additional peak appears at ∼395 nm (Fig. 3 and S24; ESI†).
a multiplet within the 3.90–3.69 ppm range. During complexa- The presence of Al3+, Cr3+ and Fe3+ ions initiates spirolactam
tion one aliphatic –NH proton disappears, and the other ali- ring opening followed by the coordination with the chemo-
phatic amine gets protonated (–NH2+) and appears at sensors, resulting in its colour change from faint yellow to
5.93 ppm. In complex 4, the amide group is present in imino- intense fluorescent yellowish pink in visible light. Spirolactam
late form in one chemosensor and amide form in another ring opening increases the delocalization of π electrons within
chemosensor for which the proton appears at 8.42 ppm. the ligand framework followed by charge transfer transitions.
Interestingly, the positions of aromatic and aliphatic CH3 Saturation has been observed in the presence of 0.5 equiva-
protons are almost unchanged upon complexation (2.39, 1.87 lents of trivalent metal ions to the chemosensors. The 2 : 1
and 1.20 ppm, respectively) (Fig. S12; ESI†). The 1H NMR spec- binding stoichiometry of the chemosensors with Al3+, Cr3+ and
tral data of complex 1 are collected in the Experimental Fe3+ ions has been confirmed by Job’s plot analysis at λ =
section (Fig. S9; ESI†). 525 nm (Fig. 4 and S25, ESI†). Addition of different cations
13
C NMR spectra are also recorded in DMSO-d6 solvent (Cd2+, Hg2+, Pb2+, Zn2+, Ag+, Mn2+, Fe2+, Co2+, Ni2+, Na+, K+,
and collected in the Experimental section. In HL2, spirolac- Mg2+, Cu2+, Ca2+, As3+, Ga3+, In3+, Tl3+ and citric acid) did not
tam amide carbon appears at 165.25 and phenolic carbon change the absorption spectrum of the chemosensors appreci-
appears at 152.35 ppm. Imine carbon appears at ably under similar experimental conditions (Fig. S26 and S27,
151.99 ppm. In complex 4, metal coordination results in a ESI†). We have also performed UV-Vis spectroscopic studies of
downfield shift of the spirolactam amide carbon, phenolic the chemosensors in a series of different solvents such as
carbon and imine carbon positions at 168.27, 164.27 and EtOH, DMSO, THF, MeCN and EtOH–H2O (9 : 1, v/v), DMSO–
157.25 ppm respectively. In the free chemosensor, the H2O (9 : 1, v/v), HEPES buffer in MeOH–H2O (9 : 1) but no sol-
carbon atom connecting the xanthene part and spirolactam vatochromic behavior was observed in the case of both chemo-
ring is sp3 hybridized and appears at 66.08 ppm. Al3+ coordi- sensors (Fig. S28, ESI†).
nation results in spirolactam ring opening, therefore, the
carbon atom connecting the xanthene part and spirolactam Fluorescence property analysis
ring shows changed hybridization from sp3 to sp2 and Both chemosensors (10 µM) are non-fluorescent when excited
appears at 141.46 ppm. Interestingly, the positions of the at 360 nm in 10 mM Britton–Robinson buffer (1 : 9, water :
OCH3 carbon, aliphatic CH2 and CH3 carbon atoms remain methanol, v/v; pH = 7.4) medium. The exposure of trivalent
almost unchanged (appear at 56.34, 17.91 and 14.54 ppm, metal ions (Al3+, Cr3+ and Fe3+ ions; 0–6 µM) to the probe
respectively) both in the free and Al3+ bound chemosensor results in great fluorescence enhancement at 555 nm (Fig. 5
(Fig. S11 and S17; ESI†). and S29; ESI†). Fluorescence increases steadily and reaches a

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Fig. 3 Absorption titration study of HL1 (10 µM) with gradual addition of metal ions (Al3+, Cr3+ and Fe3+) (A–C) 0–6 µM in 10 mM Britton–Robinson
buffer at pH 7.4.

Fig. 4 2 : 1 (ligand : metal) binding stoichiometry is shown by Job’s plot of complexes 1–3 (a–c) (at λ = 525 nm). Symbols and solid lines represent
the experimental and simulated profiles, respectively.

maximum at 0.5 equivalents of every trivalent metal ion. Free Hildebrand equation (eqn (1)) involving the fluorescence
chemosensors are non-fluorescent due to the presence of the titration curve.36
closed spirolactam ring. Opening of the spirolactam ring and
metal coordination with imine N-atoms, phenoxide O-atoms and 1=ðF  F 0 Þ ¼ 1=ðF max  F 0 Þ þ ð1=K½CÞf1=ðF max  F 0 Þg ð1Þ
O-atoms of open spirolactam amide initiate fluorescence enhance-
ment. In the case of Al3+ and Cr3+ the emission enhancement is Here, Fmax, F0 and Fx represent fluorescence intensities of
∼400 fold, whereas for Fe3+ the enhancement is ∼100 fold. each chemosensor (HL1/HL2) in the presence of metal ions at
Binding ability of the chemosensors towards Al3+, Cr3+ saturation, free chemosensors (HL1/HL2) and any intermedi-
and Fe3+ ions has been calculated using the Benesi– ate metal ion concentration, respectively. K is denoted as the

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Fig. 5 Fluorescence titration of HL1 (10 µM) in 10 mM Britton–Robinson buffer at pH = 7.4 by successive addition of metal ions (Al3+, Cr3+ and
Fe3+) (A–C) (0–6 µM) with λem = 555 nm.

binding constant of the complexes and the concentration of the The on-field applicability of these colorimetric sensors has
respective metal ion is represented by C. The value of the also been tested. We have performed paper strip experiments
binding constant (K) has been determined using the relation, K to support colorimetric and fluorescence sensing abilities of
= 1/slope. The binding constant values are 5.14 × 105 M−2, 4.91 both the chemosensors. We have used saloon waste water and
× 105 M−2, and 3.37 × 104 M−2 and 5.03 × 105 M−2, 4.86 × 105 our laboratory tap water for real sample analysis. The presence
M−2, and 3.95 × 104 M−2, respectively for the chemosensors HL1 of Al3+ ions in saloon waste water and laboratory tap water is
and HL2 towards Al3+, Cr3+ and Fe3+ ions (Fig. 6 and S30; ESI†). successfully detected by our chemosensors with the naked eye
High selectivity of the chemosensors toward respective and under a UV-lamp (Fig. S43 and S44, ESI†). Although the
metal ions (Al3+, Cr3+ and Fe3+) is again established by the chemosensors have low solubility in water, the use of aqueous-
competition assay experiment. Here in the presence of the methanol solutions of HL1/HL2 in the system and low LOD
chemosensors and respective metal (Al3+, Cr3+ and Fe3+) ions, values of the chemosensors help this type of detection. Both
(0.5 equiv.) different metal ions (Cd2+, Hg2+, Pb2+, Zn2+, Ag+, the chemosensors are highly photostable. In the photostability
Mn2+, Fe2+, Co2+, Ni2+, Na+, K+, Mg2+, Cu2+, Ca2+, As3+, Ga3+, experiments, 10−3 M aqueous solutions of HL1 and HL2 were
In3+ and Tl3+) (Fig. 7 and S31–S35; ESI†) and common anions irradiated with tungsten lamp light (emission in the
(S2O32−, S2−, SO32−, HSO4−, SO42−, SCN−, N3−, OCN−, AsO43−, 400–700 nm range, power 60 W/220 V) over a period of one
H2PO4−, HPO42−, PO43−, ClO4−, AcO−, NO3−, F−, Cl−, PF6−, hour. Interestingly, the fluorescence intensity of the chemo-
P2O74− and ROS such as NaOCl, KO2, H2O2) are added in sensors remains unchanged over a period of time (Fig. S45
excess (10.0 equiv.) in 10 mM Britton–Robinson buffer solu- and S46, ESI†).
tion at pH 7.4. Competition assay experiments clearly express Limit of detection (LOD) values of the chemosensors
high fluorescence recognition of the chemosensors (HL1 and towards Al3+, Cr3+ and Fe3+ ions are estimated using the 3σ
HL2) for Al3+, Cr3+ and Fe3+ ions over most of the metal ions method.49 The detection limit values of the chemosensors
and all common anions (Fig. S36–S41; ESI†). (HL1 and HL2) for Al3+, Cr3+ and Fe3 ions are 2.86 × 10−8 M,
Interestingly both the chemosensors, HL1 and HL2, also 2.67 × 10−8 M, 5.62 × 10−6 M, 2.78 × 10−8 M, 2.61 × 10−8 M and
act as colorimetric probes for selective detection of Al3+, Cr3+ 6.14 × 10−6 M, respectively.
and Fe3+ ions. The Al3+, Cr3+ and Fe3+ ions exhibit fluorescent The effect of pH on chemosensors (HL1 and HL2) both
pinkish yellow colouration in the presence of both probes. under free conditions and in the presence of Al3+, Cr3+ and
Some common cations show light yellow colouration in the Fe3+ ions is studied fluorometrically. It is well known that
presence of the chemosensors. The intensity of the chemo- under acidic conditions, the spirolactam ring of the chemo-
sensors increases in the order Al3+ ≈ Cr3+ > Fe3+. Thus, the sensor opens. A similar observation is also made in the pres-
chemosensors will be a good choice for colorimetric detection ence of Al3+, Cr3+ and Fe3+ ions. Therefore, both free chemo-
of Al3+, Cr3+ and Fe3+ ions both in environmental and biologi- sensor and chemosensor-Al3+, Cr3+, and Fe3+ adducts exhibit
cal fields (Fig. 8 and S42; ESI†). high fluorescence intensity at pH 2–4. At pH 5, a sharp

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Fig. 6 Benesi–Hildebrand plot for complexes 1–3 (a–c). The plot is obtained after adding 5 μM Al3+ Cr3+ and Fe3+ solution to the HL1 solution
(10 μM) (in 10 mM HEPES buffer medium, pH 7.4).

Fig. 7 Relative fluorescence intensity diagram of the [HL1-Al3+] system in the presence of different cations in 10 mM Britton–Robinson buffer at pH
7.4.

decrease in the fluorescence intensity of the free chemosensor fluorescence intensity is observed. This is probably due to the
is observed. Above pH 5 to pH 11 its fluorescence intensity is generation of metal hydroxide and the free chemosensor at
very weak and remains unchanged. This observation suggests higher pH (Fig. 9 and S47; ESI†). Interestingly, the effect of
the reconstruction of the spirolactam ring under neutral and pH is more pronounced in the case of the Fe3+-chemosensor
basic conditions. In the presence of Al3+, Cr3+ and Fe3+ ions, adduct. The pH experiment shows that these
the fluorescence intensity of the chemosensor decreases a chemosensors can act as a selective fluorescent probe for the
little above pH 4 and then it maintains a constant value up to detection of Al3+, Cr3+ and Fe3+ ions in the presence of other
pH 8. At pH 9 a sharp decrease in the fluorescence intensity of metal ions in biological systems under physiological
the chemosensor is observed. Above pH 9 to pH 11, very weak conditions.

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Fig. 8 Visual colour changes of chemosensor HL1 (10 μM) in the presence of common metal ions (0.5 equivalent) in 10 mM Britton–Robinson
buffer ( pH 7.4). The images in the above row and below row were recorded under visible light and UV light respectively.

Fig. 9 Fluorescence intensity of HL1 (10 µM) in the absence and pres-
Fig. 10 Time-resolved fluorescence decay curves (logarithm of nor-
ence of metal ions (Al3+, Cr3+ and Fe3+) (5 µM) at different pH values in
malized intensity vs. time in ns) of HL1.
10 mM Britton–Robinson buffer.

The fluorescence quantum yield (Φ) has been calculated as

follows:
Life time and quantum yield study
Lifetime measurements for the chemosensors (HL1 and HL2) n
and complexes 1–6 are performed at 25 °C in 10 mM Britton– Φsample ¼ ðODstandard  Asample  ηsample 2 Þ=

Robinson buffer ( pH = 7.4) medium. The average values of ðODsample  Astandard  ηstandard 2 Þ  Φstandard
fluorescence decay life time of the chemosensors and com-
plexes 1–6 have been measured using the given formula (τf =
a1τ1 + a2τ2, where a1 and a2 are the relative amplitude of the In the above equation, A is the area under the emission
decay process). The average values of fluorescence lifetime of spectral curve, OD is the optical density of the compound at
the chemosensors (HL1 and HL2) and complexes 1–6 are 2.26 the excitation wavelength and η is the refractive index of the
and 2.21 ns and 4.56, 3.77, 2.14, 4.24, 3.59 and 2.12 ns, solvent. The Φstandard value is taken as 0.52 (for quinine
respectively (Fig. 10, S48 and Table S1; ESI†). sulfate).

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The values of Φ for HL1 and HL2 and complexes 1–6 are From the results, no significant toxicity was observed even at
estimated to be 0.005 and 0.006 and 0.26, 0.24, 0.02, 0.25, enhanced concentrations of 100 µM (as seen in Fig. S49; ESI†).
0.23, and 0.02 respectively (Table S1†). Hence the results clearly depict the biocompatibility of the
ligands and also suggest that these ligands have potential to
Mechanism of fluorescence intensity enhancement in the emerge as promising tools for application in biomedical fields.
chemosensors in the presence of trivalent metal ions
Free chemosensors are non-fluorescent due to the presence of Cell imaging
the spirolactam ring. The fluorescence intensity of the chemo-
The cellular internalization of the chemosensors (HL1 and
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sensor increases ∼400 times in the presence of Al3+ and Cr3+

HL2) (10 µM) and Cr3+ salt (5 µM), Fe3+ salt (5 µM) and Al3+
ions and ∼100 times in the presence of Fe3+ ions due to the
salt (5 µM) has been investigated in detail with the aid of fluo-
opening of its spirolactam ring followed by complexation with
rescence microscopy studies. The images obtained from fluo-
the respective metal ions. 1H, 13C NMR and FT-IR spectroscopy
rescent microscopy shows a distinct red fluorescent signal
and X-ray crystallographic techniques are used to explain the
(Fig. 11 and S50; ESI†). Hence the results suggest that the
mechanistic pathway. The crystal structures of complexes 1
ligands and the Cr3+ salt (5 µM), Fe3+ salt (5 µM), and Al3+
and 4 prove the presence of an open spirolactam ring in the
salts (5 µM) are promptly internalized by the cells which in
Al3+ bound chemosensors. The presence of phenoxide oxygen,
turn is responsible for the emergence of a red fluorescent
amide oxygen and imine nitrogen in the coordination environ-
signal.
ment is also observed in the crystal structures. In the 1H NMR
spectra of the free chemosensors, a phenolic –OH peak, and
DFT and TDDFT study
imine and aliphatic –NH protons appear around 11.5 and 9.0
and 5.0 ppm. Whereas, in the presence of Al3+ ions the dis- We have performed DFT and TDDFT studies to support the
appearance of the phenolic –OH peak and aliphatic –NH structure of the chemosensors. The TDDFT studies also help
proton, and the downfield shift of imine protons and aromatic to understand the nature, origin and contribution of the FMOs
protons, establish the opening the of spirolactam ring and which are involved in the electronic transitions, and quantity
coordination of phenoxide oxygen and imine nitrogen atoms of energy associated with each transition. Here, the optimiz-
with the metal center. In the 13C NMR spectra of the free ation of HL1 and HL2 was performed using DFT with the
chemosensor, the sp3 hybridized carbon atom connecting the Coulomb attenuating method CAM-B3LYP (Fig. S51†). The
xanthene part and spirolactam ring appears at 66.08 ppm. optimized energies (eV) of some selected FMOs are presented
Interestingly, the spirolactam ring opening followed by metal in Table S5.† The contour plots of some selected molecular
coordination results in a change of sp3 hybridization into sp2 orbitals of the chemosensors are presented in Fig. S52 (ESI†).
hybridization and a new peak appears at 141.46 ppm (Fig. S11 Both the ligands HL1 and HL2 can exist in keto or enol form
and S17; ESI†). In the FT-IR spectrum, the free chemosensors in solution. The results show that in the keto and enol forms
exhibit stretching frequency of the amide ‘CvO’ bond and of HL1 and only the keto form of HL2, the electron density in
imine bond at ∼1696 and ∼1621 cm−1, respectively. These
values are shifted significantly to lower values and appear at
∼1640 and ∼1600 cm−1, respectively for all metal bound com-
plexes (complexes 1–6). Apart from that a sharp –OH peak
appears around 3500 cm−1 in the free chemosensor and it also
disappears after complexation. Such types of changes in the
FT-IR spectral pattern again confirm the opening of the spiro-
lactam ring and coordination of phenoxide oxygen, imine
nitrogen and amide oxygen atoms with the metal centers.
Therefore, using the above spectroscopic and X-ray crystallo-
graphic results, we can easily establish the coordination of the
metal centers (Al3+/Cr3+/Fe3+) with the chemosensor followed
by charge transfer within the ligand framework resulting in
strong colorimetric changes and great fluorescence
enhancement.

Biocompatibility study of the ligands

The cellular toxicity of the ligands (HL1 and HL2) was envi-
saged to determine the compatibility against the normal
human lung fibroblast cells, WI-38. The cells were exposed
with various concentrations (20–100 µM ml−1) of the ligands. Fig. 11 Bright field, fluorescence and merged microscopic images of
Then the cells were incubated for 24 h and then the cellular untreated HeLa (control) cells in the presence of the chemosensor (HL1)
survivability was determined with the help of the MTT assay. (10 μM) + M3+ (Al3+, Cr3+ and Fe3+) (5 μM).

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the HOMO and LUMO is distributed over the azo aromatic basis set for all atoms. Time dependent DFT (TD-DFT)
part. In the case of the enol form of HL2, the electron density approaches over 60 states to compute the vertical excitations
in the HOMO and LUMO is mainly distributed on the rhoda- also using the CAM-B3LYP functional to minimize deviations
mine part. in charge-transfer excitation energies.50 To avoid the compu-
In TDDFT, the CAM-B3LYP/CPCM method was used with tational time, we have truncated the structure slightly of the
the same basis sets in methanol. The calculated electronic Al-complex as shown in the cartesian coordinates in the ESI.†
transitions are presented in Table S6.† The theoretical calcu- The solvation effects were applied via a conductor-like screen-
lations showed that the keto and enol form of both chemo- ing model (CPCM) using methanol as the solvent.
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sensors exhibit intense absorption bands at around 310 and The results show that the two strong low-energy transitions
365 nm for the ligand based π → π* and n → π* transitions, are associated with H−2 to L+2 (58%) at 525 nm ( f = 0.48 a.u.)
respectively, which are well matched with experimental obser- and H−2 to L+3 (63%) at 519 nm ( f = 0.45 a.u.). This is in
vations. The major transitions for HL1 are H−2 → L (89%) and good agreement with the experimental results at 555 nm.
H−2 → L+1 (56%) (for enol form) and H−2 → L (83%), H → Further analyses (Table 1 and Fig. 13) of these FMOs show that
L+1 (80%), and H−9 → L+1 (27%) (for keto form), whereas for H−2 is localized (93%) on the azo aromatic part of the chemo-
HL2, the key transitions are H−2 → L (89%) and H−2 → L+1 sensor (1st part, marked with red colour) (see Fig. 14), L+2 is
(53%) (for enol form) and H → L (86%), H → L+1 (84%), and localized (96%) on the open spirolactam ring of the rhodamine
H−9 → L+1 (32%) (for keto form) (Fig. 12). unit (2nd part, marked with sky colour), and L+3 is delocalized
To investigate the mechanism of emission behavior of the over both the open spirolactam ring of the rhodamine part (1st
Al-complex, the structures of the S0 and S1 states were opti- part, marked with green colour) (72%) and the azo aromatic
mized at the DFT level using the Coulomb attenuating method part of the chemosensor (2nd part, marked with blue colour)
CAM-B3LYP hybrid functional and the split-valence 6-31+G(d) (22%). Thus, the peak at 525 nm originated from an electron

Fig. 12 Pictorial representation of key transitions of chemosensors HL1 and HL2.

Table 1 Composition (%) of the FMOs in terms of the central metal and the fragments of the attached ligands for the Al-complex

1st part 2nd part

FMOs Energy (eV) Al Azo (red) Rhodamine (green) Azo (blue) Rhodamine (sky)

L+3 −1.75 0 1 72 22 5
L+2 −1.91 0 0 0 3 96
L+1 −2.62 0 1 1 27 71
LUMO −3.83 0 0 30 20 49
HOMO −4.13 0 0 29 21 50
H−1 −4.28 0 0 0 100 0
H−2 −4.8 0 93 5 0 1
H−3 −5.17 1 77 19 1 3

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Fig. 13 Pictorial representation of the lowest energy vertical excitation of the Al-bound complex.

transition from the azo aromatic part (marked with red colour)
to the rhodamine part (marked with sky colour) of chemo-
sensors. Another peak at 519 nm can be assigned to a transition
from the azo part of the ligand (marked with red colour) to both
the fragments: the rhodamine (marked with green colour) and
azo aromatic part of chemosensors (marked with blue colour).

Natural transition orbital (NTO) study

Additionally, analysis on the electronic structure of the excited
states employing NTO representation showed that the S1 state
Fig. 14 Naming of fragments of the Al-bound complex as used in can be mainly characterized by intra-ligand charge-transfer
Table 1. (ILCT) transitions, by populating the highest-occupied (HO)
NTO and the lowest-unoccupied (LU)NTO which describe the
hole and the excited electron state, respectively. The charge

Fig. 15 The HONTO and LUNTO of the strongest low-energy emission.

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transfer index (Δr) values between the HONTO and LUNTO, Conflicts of interest
and hole–electron overlapping indices (σS) were calculated to
identify the charge transition in the excited-states. The Δr There are no conflicts of interest to declare.
value is a quantitative measure of charge-transfer (CT) length
of electron excitation; higher Δr indices and smaller σS indices
imply a longer CT distance, whereas, smaller Δr values and Acknowledgements
larger σS values are indicators of local-excitation. Here, the
computed values of Δr and σS are 2.47 Å and 0.51 which indi- A. S. gratefully acknowledges the financial support of this work
Published on 12 September 2022. Downloaded by N-List College Programme on 4/2/2025 6:14:21 PM.

cate that the fluorophore originated from the charge transfer by the DST, India (sanction no. SB/FT/CS-102/2014, dated –
within the Schiff base. As shown in Fig. 15, both HONTOs and 18.07.2015) and RUSA 2.0, Government of India (sanction no.
LUNTOs are mainly localized on the ligand Schiff base. R-11/262/19, dated – 08.03. 2019). J. M. heartily acknowledges
Therefore, theoretical studies clearly reveal that in metal the UGC, Government of India for his RGNF fellowship
bound chemosensors, the presence of the metal ion (Al3+) (Registration ID: RGNF-2017-18-SC-WES-33748).
initiates opening of the spirolactam ring of the chemosensor
followed by charge transfer within the ligand framework result-
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