Fluorescent probe sensor based on (R)-(?)-4-phenyl-2-oxazolidone for effective detection of divalent cations

Article abstract of DOI:10.1002/bio.3830

Significant progress attained in sensor science in recent years has resulted in the development of highly efficient fluorescence probes for sensing metal ions. Fluorescent molecular probes based on (R)-(?)-4-phenyl-2-oxazolidone are reported here. Fluores

Full text of DOI:10.1002/bio.3830

Received: 4 February 2020
DOI: 10.1002/bio.3830
Revised: 27 April 2020
Accepted: 6 May 2020
R E S E A R C H A R T I C L E
Fluorescent probe sensor based on (R)-(−)-4-phenyl-
2-oxazolidone for effective detection of divalent cations
Shyamal Baruah | Merangmenla Aier | Amrit Puzari
Department of Chemistry, National Institute of
Abstract
Technology Nagaland, Chumukedima,
Dimapur, Nagaland, India
Significant progress attained in sensor science in recent years has resulted in the
development of highly efficient fluorescence probes for sensing metal ions. Fluores-
cent molecular probes based on (R)-(−)-4-phenyl-2-oxazolidone are reported here.
Fluorescence studies indicated that the molecular probe could be used successfully
to sense divalent metal cations such as Cu2+, Co2+, Pb2+, and Zn2+. The addition of
divalent metal cations to the molecular probe produced a specific interaction pattern
under UV–visible and fluorescence spectroscopy. These molecules could detect
metal cations using fluorescence quenching. Stern–Volmer plots were used to deter-
mine quenching rate coefficients, which were calculated to be 2 × 101, 1.06 × 103
and 7.39 × 102 M−1 s−1 for copper, cobalt, and zinc respectively. Calculation of limit
of detection for heavy metal cations revealed that the reported molecular probes
improved the limit of detection compared with available standard data. Limit of quan-
titation values were also well within the permissible range. The frontier energy gap of
highest occupied molecular orbital to the lowest unoccupied molecular orbital was
evaluated using the density functional theory approach and Gaussian 09 W software,
which complemented the coordination of azetidinones with divalent metal ions.
Correspondence
Amrit Puzari, Department of Chemistry,
National Institute of Technology Nagaland,
Chumukedima, Dimapur, Nagaland, India.
Email: amrit09us@yahoo.com
K E Y W O R D S
(R)-(−)-4-phenyl-2- oxazolidone, fluorescent probe, metal cations, optical sensor, quenching
1
|
INTRODUCTION
become an important research objective.[11] In recent years, the selec-
tive recognition of cations by synthetic receptors has accelerated.[12]
Chemosensor is a term used in host–guest chemistry describing a
molecular structure in close association .[1] Sensors are molecules in a
system that on stimulation by any form of energy undergo a charac-
teristic change of their own state resulting in one or more other
changes.[2] Chemosensors are used for ion recognition, and have
increasingly found application in various fields.[3] The roles of ions as
To design artificial receptors with optimal selectivity toward a particu-
lar ion, several strategies need to be followed.[13] Receptors with their
various functionalities can be organized to complement the size and
shape of the analyte.[14] For overall receptor–ion interactions, the
topology of the receptor is important.[15] Receptors can form com-
plexes with many cations ranging from transition metals to alkali[16]
and alkaline earth metals[17] due to the special characteristics of their
flexible donor atoms[18] plus geometrical and interaction complemen-
tarity between the associating partners. This is generally implied by
recognition, i.e. the optimal information content of a receptor con-
cerning associating partners.[19] The ‘lock and key’ concept represents
the double complementarity principle extending over energetic fea-
tures, as well as over geometric ones.[20] The steric fit concept of Emil
cations and anions are more prevalent in industry[4], and in farming[5]
,
as well as in the environment.[6] Ni2+, Cu2+, Co2+ and Zn2+ are the
most relevant cations in different fields[7] and are additionally signifi-
cant for the life cycle of cells.[8] Macromolecular receptors, which are
proteins in nature, generally influence cellular biological process.[9]
Several biochemical transformations are triggered by their cation–
protein complexes.[10] Therefore cationic species recognition has
Luminescence. 2020;1–11.
wileyonlinelibrary.com/journal/bio
© 2020 John Wiley & Sons, Ltd.
1

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BARUAH ET AL.
Fischer[21], electrostatic interactions (ion–ion, ion–dipole, and dipole–
dipole)[22], hydrogen bonding [23], π–π stacking interactions[24], disper-
sion and induction forces (van der Waals forces)[25], and hydrophobic
or solvatophobic effects are also components of recognition.[26] Het-
erocyclic supramolecular entities are an important precursor for appli-
cation studies in various research fields.[27] These types of molecules
have been successfully employed in molecular recognition28 and for
sensing applications.[29] A diverse range of chemosensor probes has
been developed based on various structures ranging from small mole-
2.2 | General procedure for the synthesis of imines
The synthesis of imines was performed following a published proce-
dure.[41] Aromatic aldehyde (2.0 mmol) was dissolved in 1.0 ml methanol
taken in a test tube. An equimolar amount of amine was dissolved sepa-
rately in another test tube containing 1.0 ml of 0.02 M H2SO4 solution.
The solutions were then mixed thoroughly in a 25.0 ml round bottomed
flask. The reaction mixture was then transferred into a microwave oven
and maintained at 40ꢀC. The reaction was allowed to continue for 15 min
under microwave irradiation. Methanol was removed from the reaction
mixture under reduced pressure and the product of the reaction was
extracted with dichloromethane (2 × 10.0 ml). The solvent was removed
under reduced pressure to obtain the product. Products were further
purified by passing through a small column of silica gel using a 10% ethyl
acetate solution in hexane as the eluent.
cules [30], metal complexes[31], macrocycles through to polymers[32]
,
carbon nanotubes[33], quantum dots[34], and nanoparticles.[35] These
systems have been developed for sensing and detection of substrates
of varying sizes and charges, ranging from cations[36] and anions[37] to
small molecules such as explosives[38] and biologically important
motifs.[39]
Oxazolidinone-based compounds have the scope for application
in various fields including molecular recognition and sensory applica-
tions.[40] Therefore, many research projects are still needed to develop
new molecules based on oxazolidinone for application in diverse
fields. (R)-(−)-4-phenyl-2 oxazolidone-based molecular building blocks
have been rarely described and therefore can be considered as poten-
tial molecules for application studies in various research fields. The
study presented here outlines the synthesis of a series of azetidinones
based on (R)-(−)-4-phenyl-2 oxazolidone and describes the investiga-
tion of their application as sensory materials for detection of cations
using UV–vis and fluorescence spectroscopy.
2.2.1 | Analytical data for
2,4-dichlorobenzyliminepropane
Yield: 79.5%. Appearance: colourless liquid. IR (νmax cm−1): 1578
(CH=N), 3089 (C–H), 1461, 1386, 1035, 918, 833, 717, 557. 1H NMR
(CDCl3, 400 MHz) δ = 8.22 (s, 1H, 1CH imine), 7.23, 7.35, 7.44 (m,
3H, 3CH arom.), 3.54 (t, 2H, 1CH2), 1.72 (m, 2H, 1CH2), 0.96 (t, 3H,
1CH3). 13C NMR (CDCl3, 100 MHz) δ = 158.00, 130.56, 134.38,
133.01, 129.54, 127.11, 63.42, 23.95, 11.83. Mass 216 (M),
218 (M + 2). Elemental anal. Calcd. for C10H11NCl2, C-55.56, H-5.09,
N-6.48%. Found C-54.94, H-5.23, N-6.56%.
2
| EXPERIMENTAL
2.1 | Materials and methods
2.2.2 | Analytical data for
2,4-dichlorobenzyliminebutane
All chemicals used for the synthesis were procured from Sigma-
Aldrich or TCI, Japan and were used as received. The reaction pro-
gress was monitored using thin layer chromatography (GF254). Infra-
red (IR) spectra were recorded on a Cary 630 Fourier transform
infrared (FTIR) spectrometer and the values were expressed as νmax
Yield: 78.9%. Appearance: colourless liquid. IR (νmax cm−1): 1585 (CH=N),
3099 (C–H), 1393, 1083, 815, 738, 521. 1H NMR (CDCl3, 400 MHz)
δ = 8.62 (s, 1H, 1CH), 7.76, 7.33, 7.27 (m, 3H, 3CH), 3.68 (t, 2H, 1CH2)
3.62 (t, 2H, 1CH2), 1.65 (m, 2H, 1CH2), 0.95 (t, 3H, 1CH3). 13C (1H) NMR
(CDCl3, 100 MHz) δ = 156.63, 136.82, 132.15, 130.62, 59.11, 33.05,
20.62, 14.03. Mass 232 (M + 2). Elemental anal. Calcd. for C11H13NCl2,
C-57.39, H-5.65, N-6.09%. Found C-56.87, H-5.32, N-6.39%.
cm–1 1H nuclear magnetic resonance (NMR) and 13C (1H) NMR spec-
.
tra were recorded on a Bruker AvIII HD- 300 MHz FT NMR instru-
ment and Varian Mercury Plus 300 MHz NMR spectrometer using
tetramethylsilane (TMS) as an internal standard. Mass spectral data
were recorded on a Waters UPLC-TQD (ESI-MS) instrument and Tri-
ple Quadrupole (LC–MS/MS) mass spectrometer. Elemental analysis
was performed with aThermo Finnigan Flash EA 1112 CHN/CHNS/O
analyzer and PerkinElmer PR 2400 series II elemental analyzer. A Cary
Eclipse fluorescence spectrometer was used to study fluorescence
spectroscopic properties. Similarly, a Cary 100 UV–vis spectropho-
tometer was used for the UV–vis absorption spectra within the wave-
length range 200–800 nm. For the UV–vis and fluorescence
measurements, stock solutions of azetidinones (0.01 M) and metal
ions (0.01 M) were prepared in H2O/methanol (50/50, v/v). A circular
dichroism (CD) study was performed using a J-815 spectropolarimeter
and a JASCO spectrophotometer at room temperature (25ꢀC).
2.2.3 | Analytical data for
3,4-dichlorobenzyliminepropane
Yield: 81.0%.Appearance: colourless liquid. IR (νmax cm−1): 1578
(CH=N), 3089 (C–H). 1H NMR (CDCl3, 400 MHz) δ = 8.23 (s, 1H, 1CH
imine), 7.27, 7.36, 7.43 (m, 3H, 3CH arom.), 3.53 (t, 2H, 1CH2), 1.72
(m, 2H, 1CH2), 0.96 (t, 3H, 1CH3). 13C (1H) NMR (CDCl3, 100 MHz)
δ = 158.00 (CH=N), 130.56, 127.11, 129.52 (aromatic), 134.38,
133.01 (C–Cl), 63.45 (–CH2–N), 23.94 (–CH2), 11.82 (–CH3). Mass
216 (M), 218 (M + 2). Elemental anal. Calcd. for C10H11NCl2, C-55.56,
H-5.09, N-6.48%. Found C-54.94, H-5.23, N-6.56%.

BARUAH ET AL.
3
2.3 | Synthesis of (2-oxo-4-phenyl-oxazolidin-3yl)
acetic acid
1CH3), 1.73 (s, 1CH2), 0.98 (t, 1CH3). 13C (1H) NMR (CDCl3,
400 MHz): 165.15 (C=O, azetidinone), 162.54 (C=O, oxazolidinone),
141.01, 135.93, 129.22, 128.13, 127.55, 126.83, 74.17 (–CH2), 57.25,
52.64, 49.37, 42.44, 21.62, 11.34. Mass 312.11 (C6H10Cl2), 268.06
(C12H13 ClN2O3). Elemental anal. Calcd. for C21H20N2O3Cl2 C-60.27,
H- 4.78, N-6.70%. Found C-58.91, H-5.13, N-6.90%.
(R)-4- phenyl-2-oxazolidone (2.0 mmol) was dissolved in 3.0 ml of
methanol taken in a 50.0 ml round bottomed flask, followed by the
addition of an equimolar amount of chloroacetic acid (2.0 mmol).
Sodium ethoxide (3.0 mmol) was added to the reaction mixture and
the reaction mixture was kept at 40ꢀC for 24 h. The progress of the
reaction was monitored by thin layer chromatography. Methanol was
removed from the reaction mixture and the product of the reaction
was extracted with diethyl ether (3 × 10 ml) and was dried using anhy-
drous sodium sulfite. Finally, the solvent was removed under reduced
pressure to obtain the product.
2.4.2 | Analytical data for 3[1-butyl-2-(2,4-dichloro-
phenyl)-4-oxo-azetidin-3-yl]-4-phenyl-azetidin-2-one
(C2)
Yield: 73.5%. Appearance: yellow crystal. Melting point (m.p.): 85.5ꢀC.
IR (νmax cm−1): 3372, 3058, 3012, 2919, 2872, 1728, 1687 (C=O),
1596, 1488, 1445, 1213, 1071, 753. 1H NMR (CDCl3, 400 MHz: 7.57,
7.52, 7.47, 7.43, 7.39, 7.37, 7.34, 7.32 (m, 8H, CH aromatic), 5.54,
5.23, 4.92 (d, 2H, 1CH2 aliphatic), 3.23, 1.64 (s, 1CH2), 1.37 (s, 2H,
1CH2), 0.96 (t, 3H, 1CH3).13C (1H) NMR (CDCl3, 400 MHz): 168.34
(C=O, azetidinone), 162.57 (C=O, oxazolidinone), 136.14, 135.76,
135.16, 131.45, 131.15, 130.62, 130.63, 130.32, 130.13, 129.79,
129.54, 128.14, 127.13, 74.06, 57.55, 49.77, 49.58, 49.36, 47.93,
30.64, 20.87, 14.00. Mass 288 (C16H20N2O3), 232.08 (C12H12N2O3).
Elemental anal. Calcd. for C22H22Cl2N2O3, C-61.09, H-5.09, N-6.48%.
Found, C-59.60, H-5.72, N-6.54%.
2.3.1 | Analytical data for (2-oxo-4-phenyl-
oxazolidin-3yl)acetic acid
Yield: 69.5%.Appearance: white crystal. Melting point (m.p.): 85ꢀC. IR
(νmax cm−1): 3450 (–OH), 2989, 2879, 1678 (C=O). 1H NMR (CDCl3,
400 MHz): 11.00 (carboxyl –OH), 7.42, 7.41, 7.40, 7.39, 7.38, 7.36
(aromatic), 4.72, 4.13 (methylene). 13C (1H) NMR (CDCl3, 100 MHz)
δ = 171.32 (carboxylic), 49.76 (–CH2), 162.00 (amide), 74.03 (–CH2
aliphatic), 141.82, 130.44, 130.17, 129.58, 127.12 (aromatic –CH),
Mass 221 (M), Elemental anal. Calcd. for C10H11NCl2, C-59.72, H-
4.90, N-6.30%. Found C-58.90, H-5.21, N-7.20%.
2.4.3 | Analytical data for 4-(3,4-dichloro-phenyl)-
3-(2-oxo-4-phenyl-tetrahydro-furan-3-yl)-1-propyl-
azetidin-2-one (C3)
2.4 | Synthesis of (R)-(−)-4-phenyl-2-oxazolidone-
based azetidinones
Yield: 74.0%. Appearance: creamy yellow crystal. Melting point (m.p.):
(2-Oxo-4-phenyl-oxazolidin-3yl)acetic acid (2.0 mmol) was dissolved
in 3.0 ml of methanol taken in a 250.0 ml round bottomed flask
followed by the addition of an equimolar amount of thionyl chloride
(2.0 mmol). The reaction mixture was kept at 40ꢀC for 1 h. This was
followed by the addition of an equimolar amount of imine and
triethylamine as a base. The reaction mixture was further stirred for
4 h at 40ꢀC. The progress of the reaction was monitored by thin layer
chromatography. Methanol was removed from the reaction mixture
under reduced pressure and the product was extracted with diethyl
ether (3 × 10 ml). The product was dried using anhydrous sodium sul-
fite. Finally, the solvent was removed to obtain the product.[42]
82.3ꢀC. IR (νmax cm−1): 3377, 3061, 3015, 2929, 2873, 1728, 1682
(C=O), 1596, 1491, 1448, 1217, 1073, 758 cm−1 1H NMR (CDCl3,
.
400 MHz: 7.58, 7.55, 7.43, 7.42, 7.32, 7.33, 7.32, 7.31, 7.30 (m, 8H,
CH aromatic), 5.38, 5.23, 5.12, 4.90 (d, 2H, 1CH2 aliphatic), 4.12, 3.82,
3.23 (t, 2H, 1CH2), 2.86, 1.76 (s, 2H, 1CH2), 1.33, 0.93 (t, 3H, 1CH3).
13C (1H) NMR (CDCl3, 400 MHz): 165.17 (C=O, azetidinone), 162.58
(C=O, oxazolidinone), 141.01, 140.00, 135.79, 129.13, 128.04,
127.57, 126.72, 74.10, 57.12, 52.56, 49.22, 42.37, 21.57, 11.29. Mass
387 (C21H21ClN2O3), 345 (C15H15N2O3Cl2). Elemental anal. Calcd. for
C21H20N2O3Cl2, C-60.27, H-4.78, N-6.70%. Found C-59.70, H-6.30,
N-6.66%.
2.4.1 | Analytical data for 4-(2,4-dichloro-phenyl)-
3-(2-oxo-4-phenyl-tetrahydro-furan-3-yl)-1-propyl-
azetidin-2-one (C1)
3 | RESULTS AND DISCUSSION
3.1 | Synthesis of (R)- (−)-4-phenyl-2- oxazolidone-
based azetidinones
Yield: 72.0%. Appearance: Creamy yellow crystal. Melting point (m.p.):
82ꢀC. IR (νmax cm−1): 3375, 3059, 3011, 2920, 2863, 1725, 1650
A series of (R)-(−)-4-phenyl-2- oxazolidone-based azetidinones was
prepared using the synthesis procedure outlined in Scheme 1. The
reaction involved in situ conversion of (2-oxo-4-phenyl-oxazolidin-
3yl)-acetic acid to its corresponding acid chloride by reacting with
(C=O), 1590, 1480, 1438, 1217, 1073, 767 cm−1 1H NMR (CDCl3,
.
400 MHz: 7.55, 7.50, 7.49, 7.48, 7.45, 7.36 (m, 8H, CH aromatic)
7.35, 7.27, 5.02 5.01 4.93 (d, 2H, 1CH2 aliphatic) 4.81, 4.73, 3.27 (t,

4
BARUAH ET AL.
molecules. The secondary structure or conformation of sup-
ramolecules can be analyzed using this technique. The technique
reveals the structural, kinetic, and thermodynamic information of
macromolecules. CD signals are observed only when there is
absorption of radiation, and therefore the spectral bands are easily
assigned to distinct structural features of the molecule. An advan-
tage of the CD technique in the study of proteins is that comple-
mentary structural information can be obtained from several
spectral regions. Circular dichroism spectra recorded for the com-
pounds (R)-(−)-4-phenyl-2 oxazolidone and 4-(2,4-dichloro-phenyl)-
3-(2-oxo-4-phenyl-tetrahydrofuran-3-yl)-1-propyl-azetidin-2-one are
represented in Figure 2.
SCHEME 1 Synthesis of (R)- (−)-4-phenyl-2-oxazolidone-based
azetidinones
thionyl chloride and then treating the product with an aromatic imine
in the presence of triethylamine as base, leading to the formation of
the compounds (C1–C3) in 76%, 77.5%, and 76.5% yields, respec-
tively (Figure 1).
The absorption region at 240 nm and below in Figure 2 indicated
the cis steric orientation of the aromatic chromophore. This directly
reflects the difference in folding of the overall structure. From
Figure 2(a), which represents the CD spectra of the parent (R)-
(−)-4-phenyl-2 oxazolidone, it was observed that the cis isomer has
strong peaks in the region 230–240 nm. Whereas, for the compound
synthesized from (R)-(−)-4-phenyl-2 oxazolidone, i.e. for the
azetidinone as represented in Figure 2(b), strong absorption peaks
were observed below 230 nm. Therefore the study shows that incor-
poration of azetidinone into the structure influences the positive sign
of the band relative to the parent compound regardless of the relative
orientation of the ring substituents.
The molecules were characterized using 1H NMR, 13C (1H) NMR,
FTIR, and LC–MS spectroscopy. Observation of two distinct bands of
the two (C=O) groups in the FTIR spectra of each compound strongly
supported the formation of the product. 1H and 13C (1H) NMR spectra
of the compounds also supported the formation of azetidinone rings
in the molecules, as represented in Figure 1. The chemical shift posi-
tions of the azetidinone protons in the 1H NMR spectra were
observed in the range 4.9–5.2 ppm. Chemical shift position for the
azetidinone carbons in 13C (1H) NMR spectra was observed in the
range 155–160 ppm.
3.2 | Circular dichroism study of the molecules
3.3 | The change of electronic absorption by
divalent metal cations
Circular dichroism spectroscopy is an extensively used technique in
the study of chiral molecules of all types and sizes. The technique
has gained far more importance in the study of large biological
It has been observed that the azetidinones reported in this study, on
interaction with metal cations exhibit significant changes in their
FIGURE 1 Chemical structures of (R)-(−)-4-phenyl-
2-oxazolidone-based molecules (a) C1 (b) C2 and (c) C3
FIGURE 2 Circular dichroism
spectra of (a) (R)-(−)-4-phenyl-2
oxazolidone (b) 4-(2,4-dichloro-
phenyl)-3-(2-oxo-4-phenyl-
tetrahydrofuran-3-yl)-1-propyl-
azetidin-2-one (for the
experiment methanol was used as
solvent)

BARUAH ET AL.
5
electronic absorption spectra. Such changes in electronic absorption
spectra can be attributed to the specific interaction of the metal cat-
ion with the azetidinone moiety as represented in Scheme 2.
Specific interaction patterns of these azetidinones observed with the
metal cations can be exploited for sensing such metal cations and the
same was established experimentally. Additionally, the molecules exhibited
important photoluminescence characteristics, reflecting the scope for
using these molecules as optical sensors and fluorescence probes.
3.4 | Cation sensing
For biological metabolism Fe, Zn, Cu, Co, Cr, Mn, and Ni, are essential
in trace amounts; however higher doses of these ions in living organ-
isms may cause toxic effects. Conversely, metals like Pb, Hg, Cd, and
SCHEME 2 Schematic diagrams showing the interaction of
azetidinones with metal cations
FIGURE 3 UV–vis absorption pattern from interactions of (a) C1 with Cu2+ (b) C2 with Co2+ (c) C3 with Zn2+ (d) C1 with Pb2+ and (e) C3 with
Cu2+

6
BARUAH ET AL.
As are positively toxic and are not suitable for biological functions.
Therefore, scientific research focused on cation sensing in the biologi-
cal system has become an important research objective. Here we
investigated the cation sensing ability of (R)-(−)-4-phenyl-2
oxazolidone-based azetidinones for the metal cations Cu2+, Co2+, Zn2
+, and Pb2+. The study was performed by introducing a dilute aqueous
solution of the metal salt (0.01 M) to a solution of C1, C2, or C3 in
methanol (0.01 M) under UV–vis spectroscopy. The UV–vis spectra
obtained for these four metal cations are represented in Figure 3.
A specific absorption pattern was observed for interaction of the
azetidinone molecules with metal cations. For each metal cation, the
absorption band observed at 360 nm for the azetidinone unit dis-
appeared on interaction with the cation. Absorption peaks observed
at 310 nm for the azetidinone molecule exhibited a blue shift from
310 nm to 300 nm. The intensity of the absorption band gradually
decreased with time (Figure 3a). A similar observation was noted for
the Co2+ cation (Figure 3b). In this case, the intensity of the absorp-
tion band also gradually decreased with time, as is the case for the
absorption spectra of the Zn2+cation and Pb2+cation, Figure 3(c) and
Figure 3(d), respectively. The observed blue shift in the UV–vis
absorption peak at 310 nm and the subsequent decrease in the
absorption intensity of the peak at 300 nm suggested the ability of
the azetidinones to sense metal cations. Interaction of the metal cat-
ion with the organic molecular entity (C1, C2, and C3) led to the for-
mation of a ligand to metal charge transfer complex, in which the
metal cation acts as acceptor and ligand molecule as a donor. The
absorption peaks observed at 220, 275, 300 and 360 nm in Figure 3
(a) are due to n–π* and π–π* transitions. After interaction with Cu2+
ion solution, the absorption peak at 300 nm was degraded up to 50%
and the peak at 360 nm was completely degraded in 45 min. Similarly,
for the Co2+ cation, the absorption peak at 300 nm was degraded up
to 90% and the one at 360 nm was fully degraded after 60 min. We
also observed that for the Zn2+ ion, the interaction led to almost com-
plete degradation of both the peaks at 300 nm and 360 nm. For other
heavy metals like the lead (Pb) cation, for interaction with azetidinone
C1, there was intercrossing and an isosbestic point was observed
around 380 nm. The appearance of the isosbestic point indicates that
the interaction took place through a two-step process involving an
equilibrium. However, nonappearance of isosbestic point in other
cases does not mean that the interaction of the two species has not
proceeded through equilibrium. Similarly, for copper cations, C3
showed an exclusive ligand to metal charge transfer (LMCT) interac-
tion (Figure 3e). As metal ions have an empty d orbital, therefore the
d–d transition can also contribute to the decrease in the absorption
peaks. Therefore, it can be stated that the forbidden d–d transition
has resulted in a decrease in absorption intensity.
FIGURE 4 Job's plot for the stoichiometry of binding between C3
and Cu2+
of the ligand and the metal [C3–Cu2+ (10 ml)] was considered
along with a continuous variable mole fraction of the guest. From
the figure, it was observed that the maximum absorption intensity
was attained with a mole fraction of 0.5 for C3. This fact con-
firmed that there was a 1:1 stoichiometry for the C3–Cu2+ com-
plex. Similar 1:1 stoichiometry was also observed for the metal
cations Ni, Co, and Zn.
3.6 | Fluorescence study
Fluorescence response is generally considered more sensitive than
UV–vis absorption and therefore sensing and binding properties of
the molecules were examined using fluorescence spectroscopy.
Reported azetidinones are expected to have fluorescence properties
as they contain the electron-rich conjugated aromatic system. As
expected, they showed a significant change in their absorption spectra
in the presence of certain divalent transition metal cations. The
changes in the emission spectra of the ligands in the presence of
those metal ions were also measured. Fluorescence spectra of C1 and
C3 along with metal cations like Cu2+, Co2+, and Zn2+ are represented
in Figure 5.
Fluorescence titration of 0.01 M solution of C1 and C3 in
methanol was carried out with metal cations Cu2+, Co2+, and Zn2+
.
Upon titration of molecules C1 and C3 with various metal cations,
significant changes in the spectral intensity were observed. Fluores-
cence titration spectra for the three metal cations are shown in
Figure 6. Taking the example of Cu2+, a decrease in emission
intensity was observed for ligand C3 upon addition of Cu2+ ion
solution. This dramatic quenching of fluorescence was monitored
for the ligand C3 in the presence of Cu2+ at varying
concentrations. Here, on increasing the metal ion concentration, a
sharp quenching of the fluorescence was observed. This sharp
quenching of fluorescence was attributed to the formation of a
3.5 | Binding mode study
To find out the stoichiometry of binding between the receptor and
the metals, the Job's method of continuous variation was utilized
as shown in Figure 4. For the study, a constant total concentration

BARUAH ET AL.
7
FIGURE 5 Fluorescence spectra of (a) C1 and (b) C3 (0.01 M) with copper, cobalt, and zinc cation (0.01 M) in MeOH–H2O (1/1, v/v) solutions
FIGURE 6 Fluorescence titration (10 and 20 μl titration) spectra of C3 (0.01 M) with (a) copper, (b) cobalt, and (c) zinc cations (0.01 M) in
MeOH–H2O (1/1, v/v) (excitation wavelength was fixed at 360 nm)
complex between the ligand and metal ion. But the pattern of
changes in fluorescence spectra of ligand C3 was different for
cobalt (Co) and zinc (Zn) cations. The metal ions under study pro-
duced a varying degree of luminescence quenching effects in which
the quenching effect was more prominent for the Cu2+ ion, indicat-
ing more effective binding of Cu2+ ion with the azetidinone fluo-
rophore than the Co2+ and Zn2+ ions. However, Co2+ and Zn2+
also produced significant quenching effects. This fact implied that
the azetidinone molecule can successfully be used for detection of
metal cations.
Here the fluorophore receptor conjugate, with a benzene ring as
the fluorophore unit, serves as a chemosensor for detection of ana-
lyte. Strong and selective interaction between the metal ion analyte
and the fluorophore receptor conjugate assumes unique coordination
geometries which allows tuning of the photophysical properties. How-
ever, depending on the occupancies of the d-orbitals, the interac-
tion will vary from one metal ion to another and this fact explains
the selective nature of metal ion detection by the azetidinones. On
the mechanistic front it can be ascertained that binding of the
receptor unit with the metal ion through lone pairs of electrons
attached to the two carbonyl groups, led to lowering of the energy
for orbitals containing them. This might induce effectively one elec-
tron transfer from the receptor HOMO to the fluorophore HOMO,
leading to fluorescence quenching. Therefore the process follows a
static quenching mechanism. The resulting complexes formed had
their own unique properties that influenced the observed
quenching process.
Fluorescence titration revealed that the azetidinones are capable
of detecting specific changes in fluorescence properties even at very
low concentrations of metal ions. Therefore, these ligands can be effi-
ciently used as fluorescent probes for detection of certain specific
transition metal ions in the presence of other metal ions, implying the
potential applicability of these species as fluorescence-enhanced
detectors for heavy metals.

8
BARUAH ET AL.
3.7 | Stern–Volmer plot for C3 with copper from
fluorescence titration
The quenching rate coefficient was determined using the Stern–
Volmer relationship. It provides the kinetics of a photophysical inter-
molecular deactivation process. The Stern–Volmer equation is given
below:
F0=F = 1 + KQ:½Qꢁ
where F0 = intensity or rate of fluorescence without a quencher, F is
the intensity or rate of fluorescence with the quencher, KQ is the
quencher rate coefficient and Q is the concentration of the quencher.
The Stern–Volmer plot obtained for the fluorescence titration of C3
with copper (20 μl titration) is shown in Figure 7.
Quenching rate coefficients calculated for the metal cations were
2 × 103 M−1 s−1, 1.06 × 103 and 7.39 × 102 M−1 s−1, respectively, for
copper, cobalt and zinc, indicating the presence of a static quenching
process.
As a representative example, the interaction of C3 with different
metal cations was compared and plotted in the form of a histogram,
as shown in Figure 8. It is evident from the histogram that, compara-
tively, the interaction of the C3 molecule with Cu2+ cations produced
more prominent changes in intensities for both wavelengths, followed
by Zn2+ cations.
FIGURE 8 Histogram for comparison of photoluminescence
titration spectra of C3 with copper, cobalt, and zinc cation
TABLE 1
Limit of detection and limit of quantitation calculation
for metal ions
Standard
Slope
(b)
Limit of
Limit of
Metal
deviation (Sa)
detection
quantitation
Copper
(Cu)
16.64
0.365
0.129
0.130
0.085 mg/L
0.086 mg/L
0.096 mg/L
0.289 mg/L
0.463 mg/L
0.314 mg/L
Cobalt
(Co)
6.37
6.46
FIGURE 7 Stern–Volmer plot for C3 with (a) copper, (b) cobalt,
and (c) zinc (10 and 20 μl titrations)
Zinc
(Zn)

BARUAH ET AL.
9
FIGURE 9 Frontier HOMO–LUMO energy gap of (a) C1, C2, C3 and (b) C3 complex with Cu2+

10
BARUAH ET AL.
Intensities were plotted in the histogram particularly at the
performed. The HOMO–LUMO frontier orbital compositions for C1,
C2, C3, and C3 complex (with Cu) are depicted in Figure 9(a, b).
All the structures showed conjugation and delocalization of
charge over the entire framework. Zero charge density delocalization
was observed on the azetidinone ring for all the structures (C1, C2,
and C3). After formation of the complex, the charge density moved
towards the azetidinone ring. This fact confirmed the coordination of
360 nm wavelength (at which the increase in the excitation intensity
was observed) and at 462 nm (at which the decrease in the emission
intensity was observed). The histogram illustrates that the responses
of copper and zinc cations produce more prominent responses for
photoluminescence sensing in the fluorescence spectra. In addition, a
significantly higher intensity variation was observed in the emission at
462 nm for the Cu2+ cation. The preference of Cu2+ suggests that the
the azetidinone ring with Cu2+
.
large ionic radius of Cu2+ has
a
complementary size to the
HOMO–LUMO energy gaps of C1, C2, and C3 were also calcu-
lated and were found to be 5.34, 5.34 and 5.46 eV, respectively. Con-
versely, the energy gap for the C3 complex with Cu2+ was found at
4.81 eV. In many studies, it has been reported that the HOMO-LUMO
energy gap plays an important role as a stability descriptor. A larger
energy gap is associated with a more stable compound. But in our
case, the energy gap was less compared with the energy gap of the
isolated ligand. This fact revealed that metal chelation reduced the
stability of the independent medium. Another fact established from
the observation was that charge transfer led to a sharp increase in the
electronegativity of the metal that has undergone reduction, leading
to a decrease in the energy gap.
pseudocavity formed by the ligand for encapsulation in the binding
sites. Therefore it can be put forward as an example of better binding
of Cu2+ with the receptor according to its size rather than its acidity.
3.8 | Estimation of limit of detection (LOD) and limit
of quantitation (LOQ) from photoluminescence
titration
For a linear calibration curve, LOD and LOQ are expressed as:
LOD = 3 Sa/b
and
LOQ = 10 Sa/b.
where Sa is the standard deviation of the response and b is the
slope of the calibration curve. The standard deviation of the response
can be estimated by the standard deviation of either y-residuals or y-
intercepts, of regression lines. LOD and LOQ data for heavy metals
copper (Cu), cobalt (Co) and zinc (Zn) are given inTable 1.
For this calculation, fluorescence titrations of 10 μl and 20 μl
were employed; 0.01 M metal solutions of Cu, Co, and Zn were con-
tinuously added to the ligand solution at a fixed concentration
i.e. 0.01 M. Emission intensities were plotted against the molar con-
centration of the ligand. Linear fitting was performed with the plot to
determine the slope.
4 | CONCLUSION
We developed inexpensive and single optical chemical sensors and/or
metal ion receptors based on (R)-(−)-4-phenyl-2-oxazolidone for
detection of toxic metal ions. These compounds produced characteris-
tic absorption patterns in the UV–vis region upon interaction with Pb2
+, Zn2+, Co2+, or Cu2+ cations, which facilitated easy detection of
these cations. Job’s plot analysis revealed a 1:1 stoichiometry of the
C3–M2+ complex that was further supported using DFT studies.
Stern–Volmer studies showed that the binding process between C3
and metal ions exhibited a static quenching mechanism. Another
important observation was that these compounds could detect metal
ions when simultaneously present in a water sample at the ppm level.
Therefore their low detection limit at the ppm level makes the com-
pound a promising sensor for metal ions in wastewater and in nutri-
tional samples.
LOD was found to be 0.085, 0.086 and 0.096 mg/L for copper,
cobalt, and zinc, respectively. According to a standard published
report, the detection limits of copper, cobalt, and zinc are 1 mg/L,
0.05 mg/L and 3.0 mg/L, respectively.[43] Therefore comparison with
the standard data suggests that azetidinones improved the LOD for
these metal cations. Similarly, the LOQ data were also found to be
well within the permissible range.
ACKNOWLEDGEMENTS
The authors would like to acknowledge Ramesh Chandra Deka, Pro-
fessor of Chemical Sciences, Tezpur University for providing the com-
putational facility.
3.9 | DFT study
All calculations were performed using the DFT method with the
6-311G (d, p) basis set using the Gaussian 09w software package.[44]
HOMO–LUMO energies were studied at time-dependent TD-SCF
based on the optimized structure. Molecular orbitals generally provide
an excellent overview of the reactivity of the molecules. They also
give an idea about the active sites by demonstrating the distribution
of the frontier orbitals. Therefore for directing a wide range of chemi-
cal interactions, HOMO–LUMO frontier energy gap analysis was
ORCID
Amrit Puzari
https://orcid.org/0000-0001-9485-6237
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How to cite this article: Baruah S, Aier M, Puzari A.
Fluorescent probe sensor based on (R)-(−)-4-phenyl-2-
oxazolidone for effective detection of divalent cations.
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