Incorporation of triazole into a quinoline-rhodamine conjugate imparts iron(iii) selective complexation permitting detection at nanomolar levels

Article abstract of DOI:10.1039/c2dt31316b

Two new rhodamine based probes 1 and 2 for the detection of Fe³⁺ were synthesized and their selectivity towards Fe³⁺ ions in the presence of other competitive metal ions tested. The probe 1 formed a coloured complex with Fe³⁺ as well as Cu²⁺ ions and revealed the lack of adequate number of coordination sites for selective complexation with Fe³⁺. Incorporation of a triazole unit to the chelating moiety of 1 resulted in the probe 2, that displayed Fe³⁺ selective complex formation even in the presence of other competitive metal ions like Li ⁺, Na⁺, K⁺, Cu²⁺, Mg²⁺, Ca²⁺, Sr²⁺, Cr³⁺, Mn²⁺, Fe ²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd ²⁺, Hg²⁺ and Pb²⁺. The observed limit of detection of Fe³⁺ ions (5 × 10^-8 M) confirmed the very high sensitivity of 2. The excellent stability of 2 in physiological pH conditions, non-interference of amino acids, blood serum and bovine serum albumin (BSA) in the detection process, and the remarkable selectivity for Fe³⁺ ions permitted the use of 2 in the imaging of live fibroblast cells treated with Fe³⁺ ions.

Full text of DOI:10.1039/c2dt31316b

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PAPER
Incorporation of triazole into a quinoline-rhodamine conjugate imparts
iron(III) selective complexation permitting detection at nanomolar levels†
Narendra Reddy Chereddy,a Sathiah Thennarasu*a and Asit Baran Mandal*b
Received 19th June 2012, Accepted 26th July 2012
DOI: 10.1039/c2dt31316b
Two new rhodamine based probes 1 and 2 for the detection of Fe3+ were synthesized and their selectivity
towards Fe3+ ions in the presence of other competitive metal ions tested. The probe 1 formed a coloured
complex with Fe3+ as well as Cu2+ ions and revealed the lack of adequate number of coordination sites
for selective complexation with Fe3+. Incorporation of a triazole unit to the chelating moiety of 1 resulted
in the probe 2, that displayed Fe3+ selective complex formation even in the presence of other competitive
metal ions like Li+, Na+, K+, Cu2+, Mg2+, Ca2+, Sr2+, Cr3+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+, Hg2+ and
Pb2+. The observed limit of detection of Fe3+ ions (5 × 10−8 M) confirmed the very high sensitivity of 2.
The excellent stability of 2 in physiological pH conditions, non-interference of amino acids, blood serum
and bovine serum albumin (BSA) in the detection process, and the remarkable selectivity for Fe3+ ions
permitted the use of 2 in the imaging of live fibroblast cells treated with Fe3+ ions.
towards competitive metal ions like Cu2+ and Cr3+.11 Hence,
Introduction
a probe that detects Fe3+ selectively, even in the presence of
Iron is an essential trace element for both plants and animals,
and plays an important role in cellular metabolism1 and enzyme
catalysis.2 In humans, Fe3+ acts as the oxygen carrier in haemo-
globin and a cofactor in many enzymatic reactions involved in
the mitochondrial respiratory chain.2a Consequently, Fe3+
deficiency leads to anemia, liver and kidney damages, diabetes,
and heart diseases.3 Therefore, selective detection of Fe3+
has assumed importance in recent years. Both qualitative and
quantitative techniques like atomic absorption spectroscopy,4
colorimetry,5 spectrophotometry,6 and voltammetry7 have
been used for the detection of Fe3+ ions. But these require
sophisticated equipment, tedious sample preparation procedures
and trained analysts. Therefore, probes which allow naked-eye
detection, have advantages over other methods in being easy to
other competitive metal ions would be more attractive. As the
availability of metal specific chelating moieties is limited,
chemical modification of the available ligands for the selective
complexation with the metal ion of interest is attaining
importance.11a,12
8-Hydroxyquinoline is one of the widely used chelating moi-
eties for metal ion coordination, and quite a number of chemi-
cally modified 8-hydroxyquinoline derivatives have been used as
metal binding probes.13 But, the major drawbacks of these
probes are their photo physical properties in the far UV region,
low quantum yield, and high solvent dependent fluorescence
characteristics. Rhodamine is one of the most attractive fluoro-
chromes available because of its photo physical properties such
as high quantum yield, photo stability, and absorption and emis-
sion in the visible region.14 Recently, much effort has been
focussed on the development of rhodamine based probes for the
detection of heavy metal ions.15
Recently, Zeng’s group reported a rhodamine–quinoline con-
jugate with ‘O–N–N’ metal chelating moiety as a fluorescent
sensor for Cu2+ ions, without interference from its competitive
metal ions.16 In the present work, we have explored the effect of
additional coordinating moieties to tune the selectivity from
Cu2+ to Fe3+ ion. We synthesized two rhodamine conjugates 1
and 2 containing 8-hydroxyquinoline and 8-((1-benzyl-1H-1,2,3-
triazol-4-yl)methoxy)quinoline, with ‘O–N–N–O’ and ‘O–N–N–
O–N’ combination of chelating moieties, respectively. The sensi-
tivity and selectivity of 1 and 2 for the detection of Fe3+ in the
presence of other competing metal ions were established. The
stability and fluorescence characteristics of 2 in physiologically
relevant conditions were exploited for the detection of live fibro-
blast cells exposed to Fe3+ ions.
operate,
portable,
and
not
requiring
sophisticated
instrumentation.
The metal ion Fe3+ is a fluorescence quencher because of its
paramagnetic nature.8 Therefore, development of probes that
exhibit fluorescence enhancement upon binding with Fe3+ would
be very attractive. Although several probes have been reported in
the literature for Fe3+ detection,9–11 some of the probes devel-
oped for selective detection of Fe3+ suffer from cross-sensitivity
aOrganic Chemistry Division, CSIR-Central Leather Research Institute,
Adyar, Chennai-600 020, India. E-mail: thennarasu@gmail.com
bChemical Laboratory, CSIR-Central Leather Research Institute, Adyar,
Chennai-600 020, India. E-mail: abmandal@hotmail.com
†Electronic supplementary information (ESI) available: 1H/13C NMR,
ESI-MS spectra of C, 1 and 2, Job plot, ESI-MS spectra of 2-Fe3+
complex, selectivity graphs of probe 2 for Fe3+ ions in presence of
various amino acids, BS, BSA, effect of pH on 2-Fe3+ complex, and cell
viability assay. See DOI: 10.1039/c2dt31316b
This journal is © The Royal Society of Chemistry 2012
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Fig. 2 Metal ion (10 μM) induced variations in the absorbance (a),
and fluorescence (b) spectra of 1 (10 μM).
Scheme 1 Synthesis of chemosensors 1 and 2.
Hg2+, and Pb2+ did not show any significant enhancement in the
intensity of pink colour, only the addition of Cu2+ ions showed
pink colour albeit at low intensity (Fig. 1a). The observed prefer-
ential binding of Fe3+ over Cu2+ with probe 1 gave an indication
that the ‘O’ atom present in hydroxy quinoline provided O–N–
N–O combination of chelating moiety and played a vital role in
attaining the selectivity for Fe3+ ions over Cu2+ ions. Since Fe3+
is a stronger hard acid as compared to Cu2+, the incorporation of
a hard base resulting in the O–N–N–O combination of chelating
moiety would impart preferential interaction with Fe3+ over Cu2+
in accordance with the HSAB concept.
Results and discussion
The rhodamine derivatives 1 and 2 containing ‘O–N–N–O’ and
‘O–N–N–O–N’ chelating moieties, respectively, were syn-
thesized in a simple and straight forward organic synthesis pro-
cedure as shown in Scheme 1, and characterized using
spectroscopic methods (Fig. S3–S8, ESI†). Although both 1 and
2 contained the rhodamine moiety, they were colourless and
fluorescence inactive in both aqueous buffer [0.01 M Tris HCl–
CH3CN (pH 7.4)] and other organic solvents, presumably due to
their spirocyclic ring structure. The existence of the ring-closed
spirolactam as the predominant species was confirmed by the
13C resonances at ∼66.1 ppm, ∼66.3 ppm seen in the 13C-NMR
spectrum of 1 and 2, respectively.17
Th metal binding properties of 1 were further explored by
using UV-Visible, and fluorescence spectroscopic techniques.
A 10 μM solution of 1 in aqueous buffer (pH 7.4) showed negli-
gible level of absorbance (Fig. 2a) and fluorescence emission
(Fig. 2b) in the characteristic regions of rhodamine indicating the
stability of spirolactam ring of 1 in the physiological pH con-
dition. However, injection of Fe3+ ions (10 μM) led to the
appearance of a broad band with two absorption maxima at
∼490 and ∼530 nm, arising from the xanthane moiety of
rhodamine-6G (Fig. 2a). Similarly, addition of Fe3+ ions
(10 μM) resulted in the appearance of a new band at ∼552 nm in
the fluorescence emission spectrum of 1 (Fig. 2b). Thus, about
617-fold enhancement in absorbance and 398-fold enhancement
in fluorescence emission intensities were observed (Fig. 2) by
mixing a 10 μM solution of 1 with an equimolar concentration
of Fe3+ ions. Other competitive metal ions like Li+, Na+, K+,
Mg2+, Ca2+, Sr2+, Cr3+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+,
Hg2+, and Pb2+ did not influence the absorption and emission
behaviour of probe 1 as shown in Fig. 2. However, addition of
Cu2+ ions (10 μM) to an equimolar solution of 1 resulted in
about 221-fold increase in absorption and 114-fold increase in
fluorescence emission intensities (Fig. 2). The observed preferen-
tial binding affinity of probe 1 for Fe3+ over Cu2+ ions was
further confirmed from IR studies. The intensity of the band at
∼1694 cm−1 in the IR spectrum of 1 (Fig. S9a, ESI†) corre-
sponds to the spirolactam carbonyl streching.18 The differential
decrement in the magnitude of carbonyl band at ∼1694 cm−1
upon addition of 0.5 equivalents of Cu2+ or Fe3+ ions clearly
suggested that the interaction of probe 1 with Fe3+ was stronger
than that observed with Cu2+. These values were in good agree-
ment with the intensity of pink colour (Fig. 1a). Therefore, the
‘O–N–N–O’ combination in the chelating moiety of 1 appeared
to confer the preferential binding affinity for Fe3+ over Cu2+
ions.
Metal ion selectivity and sensitivity of probes 1 and 2
The probe 1 in which the 8-hydroxyquinoline was conjugated
with rhodamine offered ‘O–N–N–O’ chelating moiety for metal
coordination. The absorbance and fluorescence characteristics of
1 were greatly affected by the addition of Fe3+ ions. The addition
of equimolar concentration of Fe3+ ions to a 10 μM solution of 1
in aqueous buffer induced a bright fluorescence emission with a
clear colour change from colourless to pink as shown in Fig. 1a.
While the addition of other competitive metal ions like Li+, Na+,
K+, Mg2+, Ca2+, Sr2+, Cr3+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+,
Fig. 1 Effect of addition of various metal ions (10 μM) to 10 μM sol-
utions of fluorescent probe 1 (a) and fluorescent probe 2 (b) in 1 : 1 v/v
0.01 M Tris HCl–CH3CN, pH 7.4.
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Fig. 4 Fe3+ concentration dependent variations in the absorbance
(0–20 μM of Fe3+ ions) (a) and fluorescence (0–20 μM of Fe3+ ions) (b)
spectra of 2 (10 μM).
Fig. 3 Metal ion (10 μM) induced variations in the absorbance (a),
and fluorescence (b) spectra of 2 (10 μM).
Real-time naked-eye detection of Fe3+ ions using probe 2
As the ‘O–N–N–O’ combination in the chelating moiety of 1
displayed affinity for both Fe3+ and Cu2+ ions (Fig. 1a and 2),
we reasoned that a new chemosensor with ‘O–N–N–O–N’ com-
bination in the chelating moiety would provide the adequate
number of coordination sites for selective complexation with
Fe3+ ions, and be free from interference of other metal ions,
including Cu2+ ions. Also, we reasoned that incorporation of a
triazole unit to the ‘O–N–N–O’ combination in the chelating
moiety of 1 would yield the probe 2 with ‘O–N–N–O–N’ combi-
nation in the chelation moiety as shown in Scheme 1. As pre-
dicted, the probe 2 was colourless in aqueous buffer, turned pink
when Fe3+ ions were added (Fig. 1b) and did not show any inter-
ference from other metal ions including Cu2+. The short response
time of the probe 2 (<30 seconds) could place 2 as a real-time
naked-eye chemosensor for Fe3+ ions.
Fig. 5 Metal-ion selectivity of 2 (10 μM) in 1 : 1 v/v 0.01 M Tris HCl–
CH3CN, pH 7.4. The dark bars represent the fluorescence emission of a
solution of 2 (10 μM) and 5 equiv of the cation of interest. The light
bars show the fluorescence change that occurs upon addition of 1 equiv
of Fe(III) to the solution containing 2 (10 μM) and the cation (50 μM).
Selectivity and sensitivity of probe 2
Negligible variations were observed in the absorbance and fluor-
escence characteristics of 2 (10 μM) in aqueous buffer with and
without various metal ions (Fig. 3). Interestingly, addition of
equimolar quantities of Cu2+ ions did not show any significant
level of absorbance at ∼530 nm (Fig. 3a) or fluorescence emis-
sion at ∼552 nm (Fig. 3b), indicating the stability of the spirolac-
tam ring of 2 in the presence of Cu2+ ions. However, injection of
equimolar concentrations of Fe3+ ions led to the development of
a new 744-fold intense absorption band with a maximum at
∼530 nm (Fig. 3a) and a 427-fold intense fluorescence emission
band with a maximum at ∼552 nm (Fig. 3b) as compared to the
absorbance and fluorescence properties of 2 (10 μM) in aqueous
buffer without Fe3+ ions. Clearly, the incorporation of ‘O–N–N–
O–N’ combination in the chelation moiety of 2 imparts selectiv-
ity for Fe3+ ions over other competitive metal ions including
Cu2+ ions.
association constant for 2-Fe3+ complex was calculated using
Benesi-Hildebrand plot and found to be 4.5 × 104 M−1. The stoi-
chiometry of 2-Fe3+ complex was determined using Job plot
(Fig. S10, ESI†) indicated that the probe 2 formed a fluorescent
1 : 1 complex with Fe3+ ion. The stoichiometry of the 1 : 1
complex was further confirmed from the ESI-MS data (m/z =
880–882) of 2-Fe3+ complex (Fig. S11, ESI†). Further, competi-
tive metal binding studies shown in Fig. 5, clearly established
the non-interference of other metal ions (5 equivalents). Thus,
probe 2 appeared to be highly sensitive and selective displaying
excellent binding affinity for Fe3+ ions.
Furthermore, we investigated the effect of biological entities
such as amino acids, blood serum (BS) and bovine serum
albumin (BSA) on the sensing ability of 2. Probe 2 (10 μM) was
non-fluorescent and colourless in the presence of excess amounts
(50 μM) of various amino acids. Upon addition of Fe3+ ions
(10 μM), this colourless solution turned to pink and displayed
intense fluorescence (Fig. 6), indicating the non-interference of
amino acids in 2-Fe3+ complex formation. Also, the fluorescence
emission of 2-Fe3+ complex was impassive to the addition of
various amino acids (Fig. S12, ESI†), BS (Fig. S13, ESI†) and
BSA (Fig. S14, ESI†). These competitive experiments revealed
that the interaction of Fe3+ with 2 was stronger than that of Fe3+
with the above biological entities and the existence of the latter
did not affect the detection ability of 2.
The concentration dependent variation in Fe3+ induced absor-
bance characteristics of 2 (Fig. 4a) indicated that the addition of
as low as 0.1 μM concentration of Fe3+ ions to 2 (10 μM) in
aqueous buffer (pH 7.4) was enough to induce a clear absorption
band ∼530 nm. A similar titration to monitor the fluorescence
characteristics of 2 upon addition of Fe3+ ions (Fig. 4b) covering
a linear range from 3.0 × 10−8 to 9.0 × 10−6 M (Fig. 4b inset)
revealed the limit of detection to be 5 × 10−8 M (552I5×10−8 552I0
/
= 3.77).12 It is imperative to note that the limit of detection (5 ×
10−8 M) observed in the case of 2 was lower than those values
reported for most of the rhodamine based Fe3+ sensors.10 The
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Fig. 7 Microscopic images of (a) untreated fibroblast cells, (b) cells
incubated with 2 (1 μM), (c) cells incubated with 2 (1 μM) and Fe3+
(2 μM), and, fluorescence microscopic images of (d) untreated cells, (e)
cells incubated with 2 (1 μM), and (f) cells incubated with 2 (1 μM) and
Fe3+ (2 μM).
Fig. 6 Fe3+ ion selectivity of 2 (10 μM) in 1 : 1 v/v 0.01 M Tris HCl–
CH3CN, pH 7.4 in the presence of different amino acids. The dark bars
represent the fluorescence emission of a solution of 2 (10 μM) and 5
equiv of the amino acid of interest. The light bars show the fluorescence
change that occurs upon addition of Fe(III) (10 μM) to the solution con-
taining 2 (10 μM) and the amino acid (50 μM).
conditions was studied. The probe 2 was colourless in all
aqueous solutions in the pH range 6–10, indicating the stability
of the spirolactam ring. However, a pink colour developed in the
solutions in which the pH value was below 5.0 (Fig. S15, ESI†)
indicating the susceptibility of the spirolactam ring. As the pH of
most biofluids is about 7.4, the observed stability of the spirolac-
tam ring in the pH range 6–10 would make 2 suitable for
bioassays.
Detection, imaging and viability of live cells
The selective binding with Fe3+ ions, high sensitivity, stability at
physiological pH conditions and non-interference of amino
acids, blood serum and BSA in the detection process, prompted
us to use the probe 2 for the detection of contaminant Fe3+ ions
on live mouse fibroblast cells. Mouse fibroblast cell line NIH
3T3 incubated with Fe3+ ions (2 μM) in Dulbecco’s Modified
Eagle Medium (DMEM) for 2 min at 37 °C, was washed with
PBS buffer (pH 7.4) to remove excess metal ions. The cells were
then treated with 2 (1 μM) in the DMEM culture medium for
30 min at 37 °C, and washed with PBS buffer (pH 7.4) to
remove unbound 2. Fibroblast cells treated with both 2 and Fe3+
displayed intense red fluorescence as shown in Fig. 7f. A visual
comparison of normal and fluorescence microscopic images
clearly indicated that 2 could be used to detect live cells exposed
to micro molar concentrations of Fe3+ ions. Fibroblast cells not
contaminated with Fe3+ ions did not show any fluorescence.
Further, the cytotoxicity of probe 2 assessed by performing MTT
assay indicated that the cell viability was significant
(∼80–100%) up to ∼6 μM concentration of probe 2 (Fig. 8).
Scheme 2 Perspective binding interactions of 2 with Fe3+ ions.
Mechanism of Fe3+ ion sensing
The mechanistic aspects of Fe3+ ion sensing by 2 were deci-
phered (Scheme 2) using EDTA assay. As would be expected,
binding of the hard acid Fe3+ to 2 resulted in enhanced absor-
bance and fluorescence intensities presumably due to the
opening of the spirolactam ring. The Job plot and ESI-MS data
(Fig. S10 and S11, ESI†) clearly confirmed the formation of a
1 : 1 complex containing two chloride ligands.19 Further, the
pink coloured solution formed by the addition of Fe3+ ions
turned colourless upon addition of EDTA. This colourless solu-
tion regained its pink colour upon addition of an excess amount
of Fe3+ ions. ESI-MS analysis of both colourless and pink
colour solutions confirmed the interconversion between ring-
closed spirolactam form and Fe3+ bound ring-open form of 2
(Scheme 2). Thus, the EDTA titration experiment clearly estab-
lished the underlying mechanism and the fact that the colour
development was due to the formation of 2-Fe3+ complex and
not because of any catalytic action of Fe3+ ions.
Effect of pH
Since the probe 2 showed Fe3+ induced fluorescence response in
aqueous buffer (pH 7.4), the stability of 2 at various pH
Fig. 8 The cell viability percentage of NIH 3T3 cells after treatment
with different concentrations of probe 2.
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commercial suppliers and used without further purification. The
solutions of metal ions were prepared from the corresponding
chloride salts. Absorption spectra were recorded on a Cary 50
Bio UV-visible spectrophotometer. Fluorescence measurements
were performed on a Cary Eclipse fluorescence spectropho-
tometer. All pH measurements were made with a Systronics μpH
System Model 361. NMR spectra were recorded using a Bruker
Avance 400 MHz spectrometer operated at 400 MHz and a
Bruker 300 MHz spectrometer operated at 300 MHz. ESI MS
spectra were obtained on a PE Sciex API3000 Mass Spec-
trometer. Fluorescence imaging experiments were performed
using a Leica DM IRB microscope equipped with EBQ-100 UV-
lamp. All measurements were carried out at room temperature.
Stock solutions of rhodamine derivatives (chemosensors) were
prepared by dissolving 1.0 mmol of chemosensors (5.83 mg, and
7.54 mg of 1 and 2, respectively) in 1 : 1 v/v 0.01 M Tris HCl–
CH3CN (pH 7.4) and making up to the mark in a 10 mL volu-
metric flask. Further dilutions were made to prepare 100 μM
solutions for the experiments. Stock solutions of all amino acids
(1 M) and metal ions (1 M) were prepared in de-ionized water.
The blood serum was obtained via centrifugation of the human
blood and stored at −20 °C. The serum stock solution was pre-
pared by dissolving 50 μL of serum in 1.5 mL of PBS. The
stock solution of BSA protein was prepared by dissolving
1.0 mg of BSA in 1.0 mL of PBS. Absorption and fluorescence
measurements were made using a 3.0 mL cuvette. Poly(methyl
methacrylate) PMMA (500 mg) and probe 2 (100 mg) were dis-
solved in DCM (0.5 mL), poured onto clean microscopic glass
plates and air dried. The PMMA films formed were removed
from the glass plates and used for Fe3+ sensing.
Fig. 9 Images of poly(methyl methacrylate) polymer sheets: (A)
Doped with probe 2; (B) Doped with probe 2 and sprayed with FeCl3
(50 μM) solution.
Hence, at 1 μM of 2, the concentration used for fibroblast cell
imaging experiments, the probe is unlikely to be cytotoxic.
These results augment the suitability of 2 for the detection and/
or imaging of live cells.
Polymer thin films for Fe3+ sensing
Thin films of poly(methyl methacrylate) doped with 2 were pre-
pared by dissolving the polymer (500 mg) and probe 2 (100 mg)
in DCM (0.5 mL) and pouring the solution onto clean micro-
scopic glass plates. The films formed upon air drying were
removed from the glass plates and used for Fe3+ sensing. The
films were non-fluorescent and colourless, but upon spraying a
solution of FeCl3 (50 μM) in acetonitrile, the colourless films
turned to pink (Fig. 9). This observation opens up the possibility
of designing polymer fabrics for sensing Fe3+ ions.
Conclusions
Synthesis of rhodamine 6G hydrazide A
We have presented the synthesis of two new rhodamine 6G
derivatives, 1 and 2, containing ‘O–N–NO’ and ‘O–N–N–O–N’
combination, respectively, in the chelation moieties. The number
and spatial disposition of coordinating atoms have been
exploited for the selective detection of metal ions. The require-
ment of ‘O–N–N–O–N’ combination in the chelation moiety for
Fe3+ ion selective complex formation has been demonstrated.
The absence of any interference from competitive metal ions on
the absorbance and fluorescence characteristics of the probe 2 in
aqueous buffer has been presented as evidence for the selective
detection of Fe3+ ions. The probe 2 is highly sensitive and forms
a 1 : 1 complex with Fe3+ ions. The limit of detection of Fe3+
ions (5 × 10−8 M) observed using 2 is very much less than those
values reported for rhodamine based Fe3+ sensors. The probe 2
is stable in the physiological pH range and non-cytotoxic up to
∼6 μM. Non-interference of amino acids, human blood serum
and BSA protein with the probe 2 could augment the detection
and/or imaging of live cells exposed to Fe3+ ions. Further, the
probe 2 could be formulated into a polymeric thin film sensor
for Fe3+ detection.
Rhodamine 6G hydrazide was synthesized according to the
reported procedure.20
Synthesis of triazole appended quinoline aldehyde C
The triazole appended quinoline aldehyde C was synthesized in
a three-step procedure. To a solution of SeO2 (1.66 g, 15 mmol)
in 1,4-dixane (20 mL) under N2 atmosphere, 2-methyl 8-quinoli-
nol (1.59 g, 10 mmol) in 1,4-dixane (20 mL) was added drop-
wise, the resulted mixture was allowed to stir for 8 h under N2
atmosphere at 70 °C and progress of the reaction was monitored
by TLC. After the completion of reaction, the reaction mixture
was allowed to cool to room temperature, filtered and subjected
silica gel 100–200 mesh column chromatography using
95 : 5 hexane–ethylacetate as eluent to get 0.87 g (50%) of
8-hydroxyquinoline 2-aldehyde (B) in pure form as yellow colour
solid. To a solution of 8-hydroxyquinoline 2-aldehyde (B)
(0.87 g, 5 mmol) in DMF (20 mL) was added potassium carbon-
ate (1.04 g, 7.5 mmol) and the solution was stirred at room temp-
erature. Propargyl bromide (0.7 mL, 7.5 mmol) was added drop
wise and the resulting mixture was allowed to stir overnight.
After completion of reaction, the reaction mixture was parti-
tioned between DCM and water, and the DCM layer was col-
lected. The aqueous layer was extracted three times with DCM,
and the combined organic extracts was dried over anhydrous
Experimental
Materials and instrumentation
Dry acetonitrile and double distilled water were used in all
experiments. All the materials for synthesis were purchased from
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Na2SO4, and concentrated under vacuum to obtain the desired
propargylated aldehyde that was later on converted to a triazole
derivative using click chemistry. Propargylated aldehyde (0.63 g,
3 mmol) was dissolved in 1 : 1 THF/H2O mixture and triethyl
amine (0.7 mL, 5 mmol) was added. Sodium azide (0.26 g,
4 mmol), benzyl bromide (0.48 mL, 4 mmol) and cuprous
iodide (ca.) were added to this solution. The resulting mixture
was allowed to stir overnight at room temperature. Upon com-
pletion of the reaction, the mixture was filtered, extracted with
ethyl acetate, concentrated under vacuum and then subjected to
column chromatography to obtain the desired product. The
overall yield was 0.84 g, (40%).1H NMR (CDCl3, 300 MHz),
δ (ppm): 5.53 (2H, s, NCH2), 5.59 (2H, s, OCH2), 7.27 (2H, m,
Ar–H), 7.37 (3H, m, Ar–H), 7.42 (1H, d, J = 9.1 Hz, Ar–H),
7.49 (1H, d, J = 7.6 Hz, Ar–H), 7.60 (1H, m, Ar–H), 7.71 (1H,
s, Ar–H), 8.04 (1H, d, J = 8.3 Hz, Ar–H), 8.27 (1H, d, J =
8.3 Hz, Ar–H), 10.24 (1H, s, aldehyde-H); ESI MS: Calcd m/z
344.1; found 345.1 (M + 1+).
129.1, 129.5, 133.9, 134.7, 135.9, 144.7, 147.5, 147.6, 151.5,
153.8, 165.4; ESI MS: Calcd m/z 731.4; found 732.5 (M + 1+).
Acknowledgements
One of the authors, Ch. N. R, thanks the Council of Scientific
and Industrial Research, New Delhi, India, for a research
fellowship.
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Synthesis of rhodamine probe 1
To a solution of rhodamine 6G hydrazide A (0.43 g, 1 mmol)
dissolved in 20 mL methanol, 8-hydroxyquinoline 2-aldehyde
(B) (0.19 g, 1.1 mmol) was added. The red colour mixture thus
obtained was refluxed in an oil bath for 3 h. After that, the solu-
tion was cooled to room temperature. The white colour precipi-
tate formed was filtered and dried in vacuum to yield 0.47 g
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1
(80%) of 1 as colourless solid. H NMR (CDCl3, 300 MHz), δ
(ppm): 1.28 (t, J = 7.2 Hz, 6H, NCH2CH3), 1.84 (s, 6H,
xanthane CH3), 3.19 (q, J = 6.9 Hz, 4H, NCH2CH3), 3.51 (s,
2H, xanthane NH), 6.39 (s, 2H, xanthene-H), 6.46 (s, 2H,
xanthene-H) 7.06 (m, 2H, Ar–H), 7.16 (d, J = 7.5 Hz, 1H,
Ar–H), 7.31 (m, 1H, Ar–H), 7.47 (m, 2H, Ar–H), 7.95 (d, J =
8.7 Hz, 1H, Ar–H) 8.08 (m, 2H, Ar–H) 8.47 (s, 1H, Imine–H).
13C NMR (CDCl3, 75 MHz), δ (ppm): 14.7, 16.7, 38.4, 66.1,
97.0, 105.8, 110.2, 117.7, 118.2, 118.8, 123.7, 123.9, 127.5,
127.7, 128.0, 128.1, 128.4, 128.4, 134.0, 135.9, 137.5, 145.4,
147.7, 151.3, 152.2, 152.3, 152.6, 165.6; ESI MS: Calcd m/z
583.3; found 584.4 (M + 1+).
Synthesis of rhodamine probe 2
To a solution of rhodamine 6G hydrazide A (0.43 g, 1 mmol)
dissolved in 20 mL methanol, triazole appended quinoline alde-
hyde C (0.38 g, 1.1 mmol) was added. The red colour mixture
thus obtained was refluxed in an oil bath for 3 h. After that, the
solution was cooled to room temperature. The white colour pre-
cipitate formed was filtered and dried in vacuum to yield 0.54 g
1
(71%) of 2 as colourless solid. H NMR (CDCl3, 400 MHz),
δ (ppm): 1.26 (t, J = 7.0 Hz, 6H, NCH2CH3), 1.83 (s, 6H,
xanthane CH3), 3.13 (q, J = 7.1 Hz, 4H, NCH2CH3), 3.49 (s,
2H, xanthane NH), 5.46 (s, 2H, CH2), 5.52 (s, 2H, CH2), 6.37
(d, J = 8.8 Hz, 4H, Xanthene-H), 7.07 (m, 1H, Ar–H), 7.18–7.34
(m, 8H, Ar–H), 7.47 (t, J = 3.6 Hz, 1H, Ar–H), 7.74 (s, 1H, tri-
azole-H), 7.94 (d, J = 8.8 Hz, 1H, Ar–H) 8.03 (t, J = 4.1 Hz, 2H,
Ar–H) 8.85 (s, 1H, Imine–H). 13C NMR (CDCl3, 100 MHz),
δ (ppm): 14.8, 16.8, 38.4, 54.2, 63.7, 66.3, 97.0, 106.2, 111.4,
118.1, 118.9, 120.5, 123.5, 127.1, 127.5, 128.0, 128.4, 128.7,
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