Signalling probes appended with two rhodamine derivatives: Inter-component preferences, Fe(III)-ion selective fluorescence responses and bio-imaging in plant species

Article abstract of DOI:10.1039/c7nj03364h

The photophysical investigation of two amino-alkyl receptor-based probes, 1 and 2, appended with two different rhodamine derivatives but differing in their receptor constituents revealed their selective absorption and fluorescence signalling responses to Fe(iii) among all metal ions investigated. Although the output channels in the complexation-induced signalling patterns of these probes are in proximity to each other, the spectroscopic signatures of Fe(iii)-complexation spiro-ring opening of the individual rhodamine derivatives and associated kinetic measurements revealed a preferential coordination affinity of the rhodamine 6G (Rho-6G) component over the rhodamine-B (Rho-B) segment. The Fe(iii)-selective signalling response was observed to be higher in flexible DETA(bis(2-aminoethyl)amine) receptor-incorporated probe 1 than in 2, which incorporates a rigid ethylenediamine receptor. The ratio of the fluorescence of the two wavelength output channels arising from the two different signalling subunits in the architectures of these probes enhances their sensitivity levels in Fe(iii) ion detection. The utility of the Fe(iii)-complexation-induced orange fluorescence output of 1 was demonstrated in the detection and localization of bio-accumulated Fe(iii) ions in root tissues of Grevillea pteridifolia plant species.

Full text of DOI:10.1039/c7nj03364h

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Signalling probes appended with two rhodamine derivatives: Inter-component
preferences, Fe(III)-ion selective fluorescence responses and bio-imaging in plant
species
Debajani Mallicka,b, Biswonath Biswala, M. Thirunavoukkarasuc, Roshnara Mohantyc,
Bamaprasad Baga,b,*
b
aColloids and Materials Chemistry Department, Academy of Scientific and Innovative
c
Research, Environment and Sustainability Department, CSIR-Institute of Minerals and
Materials Technology, P.O.: R.R.L., Bhubaneswar-751 013, Odisha, India. Email:
bpbag@immt.res.in
Abstract:
The photophysical investigation of two amino-alkyl receptor based probes 1 and 2 appended
with two different rhodamine derivatives but differing in their receptor constituents revealed
to their Fe(III) selective absorption and fluorescence signalling responses among all metal
ions investigated. Although the output channels of these probes in their complexation induced
signalling pattern are in proximity to each other, the spectroscopic signatures of Fe(III)-
complexation spiro-ring opening of individual rhodamine derivatives and associated kinetic
measurements revealed to a preferential coordination affinity of Rhodamine 6G (Rho-6G)
component over their Rhodamine-B (Rho-B) segment. The Fe(III) selective signalling
responses was observed to be higher in flexible DETA(bis(2-aminoethyl)amine) receptor
incorporated probe 1 than that in 2 which incorporates a rigid ethylenediamine receptor. The
ratio of two wavelength output channel in their fluorescence due to the presence of two
different signalling subunits in their architecture enhances the sensitivity level of these probes
in their Fe(III) ion detection. The utility of Fe(III)-complexation induced orange fluorescence
output of 1 was demonstrated in detection and localization of bio-accumulated Fe(III) ions in
root tissues of Grevillea pteridifolia plant species.
Introduction
1

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Iron in its ionic form is one of the essential physiologically relevant metal ion that performs
vital and versatile functional activities in living organisms, human-being in particular1-3, as it
present in active sites of many enzymes and proteins. Its potency of facile redox reactions
and higher oxygen affinity facilitate2-6 its involvement in oxygen uptake and transport,
oxygen metabolism and electron transfer mechanisms. Being such an essential constituent,
the average requirement of ionic iron by a human has been approximated to be 1-10mg
depending upon age, gender and physiological conditions and rate of iron loss through
various excretion activities. Despite its importance, requirement and utility, a deviation from
its requisite concentration threshold (hyperferremia and hypoferremia) impart a detrimental
toxic effect on living organisms that lead to various health hazards7. The consequence of bio-
accumulation of iron through contaminated water, diet and other sources result in
physiological disorder and multi-organ failure. Primitively labile iron potentially catalyzes
the generation of highly reactive oxygen species such as hydroxyl radicals via the Fenton
reaction which interacts with most bio-molecules including sugars, lipids, proteins, and
nucleic acids and consequently, results in peroxidative cellular dysfunction and DNA
damage8. On the contrary, deficiency of iron concentration in human being also has a vicious
impact in form of anaemia and other health implications9. Therefore, there exists an utmost
need for detection of ionic iron species and their subsequent quantification of concentration
for biological, clinical, environmental impact assessment. Many analytical techniques are
commonly used for selective detection of ionic iron10; however these methods involve much
complicated sampling procedures and high-cost sophisticated instrumentation. In this regard,
fluorescent signalling methodologies with molecular probes have proven to be advantageous
due to high sensitive and selective detection, rapid and ‘easy to handle’ operational
principles, low-cost and non-destructive sampling methodologies. Many chemosensors for
selective detection of Fe(III) ions were developed11 exploring designing principles of
2

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covalent receptors for effective coordination and subsequent perturbation of operational
photophysical processes which manifested into a quantifiable signalling of the recognition
event. Concerning signalling pattern and sensitivity of detection, probes which strategically
exhibit fluorescence enhancement rather than quenching as a consequence of probe-metal ion
interaction is advantageous. This is because ionic iron is a well-known quencher of
florescence signal by virtue, as rationalized through various mechanisms, therefore a
fluorescence turn-off strategy may fail to detect metal ion in real samples when present in
trace amount. In context of preferential molecular entities as chemosensors, rhodamine
derivatives which render chromogenic and fluorogenic dual mode signalling relying on metal
ion coordination induced spiro-ring opening protocol have proven to be potential12, as
xanthene-based dyes have excellent photophysical properties such as long wavelength
absorption and emission, high fluorescence quantum yield, large extinction coefficient and
high photo-bleaching threshold, contrast structure-function correlation and have the ability to
execute signalling action in aqueous media. With synthetic and operational advantages, few
rhodamine derivatives were already known to selectively detect Fe(III) ions13. Despite
advantages, such probes quite often fail to respond at a very low concentration level of metal
ions, where a low signal to noise ratio in spectral amplification becomes detrimental
parameter and restricts limit of detection. On contrary, bi-fluorophoric ratiometric probes
strategically overcome such limitation in determining sensitivity level of detection which
allows (a) measurement of fluorescence intensities at two different wavelengths along with
increased dynamic range, (b) render a built-in correction for environmental effects in respect
of smaller concentration variation and (c) lowers dependency of signal to noise ratio to
achieve a higher sensitivity since sensitivity and dynamic range of such probes are controlled
by ratio of emission intensities11a,14. Considering the advantages of rhodamine-based probes,
herein we report two amino-alkyl receptor based probes (1 and 2, Scheme 1) incorporating
3

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two different xanthene dye derivatives [rhodamine-6G(Rho-6G) and rhodamine-B(Rho-B)]
that selectively detect Fe(III) ion in terms of enhancements in chromogenic and fluorogenic
signals of corresponding dyes and a visual perception of colour change. Both probes differ in
their receptor constituents; 1 incorporates a flexible DETA (bis(2-aminoethyl)amine) while 2
consists of a rigid ethylenediamine as receptor component, thus offers possibilities of
adaptation of different coordination environment for metal ion complexation by these iso-
componential probes. An optimal spectral overlap between these two different yet
complementary rhodamine derivatives as fluorophores is anticipated by virtue of their
proximities in absorption and fluorescence properties, thus, enabling those to act as an inter-
componential intra-molecular donor-acceptor cascade with Rho-6G as excitation energy
donor and Rho-B as an energy acceptor. In that context, it is imperative to investigate
whether complexation leads to initiation of fluorescence resonance energy transfer(FRET)
process in these probes. Examples of Fe(III)-complexation induced FRET modulation are
already available in probes with these xanthene based dyes (Rho-6G or Rho-B) as either
excitation energy donor15 or as acceptor14c,15,16. However to best of our knowledge, probes
containing both Rho-6G and Rho-B are not explicitly known. Further, this kind of energy
donor-acceptor embedded molecular probes raise an obvious question that need to be
convincingly addressed; whether both the xanthene based fluorophoric groups individually
experience their direct excitation to exhibit complexation mediated fluorescence signalling
through two different output channels or undergoes any FRET mediated ratiometric
fluorescence signalling as observed in other donor-acceptor based systems. Therefore, the
photophysical studies of 1 and 2 were carried out to understand the stereo-electronic
contribution in their Fe(III) selective signaling, metal-probe kinetics, role of spacer and
solvent medium and extent of energy transfer if operative between these rhodamine moieties.
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Further, substituted rhodamine derivatives have already been proven11a,14c,17 as imaging
constituent in not only in-situ detection and intracellular monitoring of various chemical
analytes and bio-analytes, but also the estimation of their concentration level, distribution and
dynamics. However, the utility of rhodamine probes for such detection and quantification in
complex bio-systems has been focused on animal cells, animal models and
microorganisms11a,13d,14c,18; not many attempts were made towards bio-imaging for selective
detection of metal ions in plant species. Identification and assessment of bio-accumulation of
essential and non-essential metal ions in plant species, which they uptake along with other
nutrients from the medium where they are being nurtured, is highly desirable and
indispensable in context of their utility and usage. In our earlier effort on detection and
concentration level monitoring of bio-accumulated toxic metal ions in living cells or tissues
of plant species, the potentiality of rhodamine derivatives was demonstrated in the detection
of lead19 and cobalt20 ions in root/shoot tissues of Aerva lanata and Hybanthus
enneaspermus plant species respectively. With probe 1, herein we also report detection and
subsequent localization of Fe(III) ion in roots of Grevillea pteridifolia Knight, a Fern-leaved
plant of Proteaceae family which suitably grow21 on wasteland soils with a texture varying
from pulverized lateritic soil to sandy loam. The fluorescence imaging investigations
therefore establish the probes’ potential capabilities and utility as a marker agent in detection
and localization of bio-accumulated metal ions in complex plant systems.
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O
O
Cl
i
O
N
O
N
N
O
N
I
O
O
O
N
NH2
O
N
NH2
N
H
ii
iii
N
H
O
N
H
N
H
O
N
H
N
H
O
1a
N
H
2a
iv
I
I
iv
H
N
H
N
H
N
H
N
O
O
O
N
N
N
O
N
HN
O
O
N
O
N
N
O
N
2
1
Scheme 1: Synthetic route to 1 and 2. Reaction conditions: (i) DCM, POCl3, reflux, 4h., (ii)
diethylenetriamine, EtOH, Reflux, 12h, (iii)ethylenediamine, EtOH, Reflux, 12h, (iv) MeCN,
Et3N, 0 °C, 1h
Result and discussion
The probes 1 and 2 were synthesized by reaction of rhodamine-B acyl chloride (I) with either
diethylenetriamine(DETA) coupled rhodamine-6G (1a) or its ethylenediamine analogue(2a)
respectively in presence of triethylamine (Scheme 1). The compounds were characterized by
1H-NMR, 13C-NMR, FT-IR, ESI-MS, UV-Vis and fluorescence spectroscopy. The peaks in
their 13C-NMR spectra in CDCl3 corresponding to characteristics of quaternary carbon(C-
9) of spiro-rings were observed at 60.87ppm(Rho-6G) and 65.65ppm(Rho-B) for 1 and at
6

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60.48ppm(Rho-6G) and 64.83ppm(Rho-B) for 2 respectively. This inferred to a predominant
existence of both rhodamine derivatives in their spirolactam form, which is well supported by
their FT-IR spectrum where the stretching frequency at ~1690 cm-1 assignable to that of
spiro-lactam carbonyl groups. The UV–VIS absorption spectra of 1 and 2 in an aqueous-
organic binary mixture medium(EtOH-H2O, 1:1v/v, 0.1M PBS, pH7.2) revealed to
appearance of absorption peaks at 275nm and 350nm which were attributed to higher energy
ligand localized π→π* and n→π* transitions. The absence of any peak in 500–600nm
region of their absorption spectra and corresponding weak fluorescence on excitation at
500nm also ascertained the spirocyclic form of each xanthene derivatives in both probes.
In order to investigate metal ion induced signalling responses in these bifluorophoric
probes appended with two different xanthene dyes, perchlorate salts of various metal ions
such as Na(I), K(I), Ca(II), Mn(II), Fe(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Ag(I), Cd(II),
Hg(II) and Pb(II) were added to their colourless solution in EtOH-H2O (1:1 v/v, 0.1M PBS,
pH 7.2) binary mixture medium. Among all the metal ions under investigation, addition of
Fe(III) ions to 1 resulted(Fig. 1a) in appearance of absorption peaks at 525nm and 557nm
along with a change in colour of the solution to pink[(ε
)
1+Fe(III) 525=11293, (ε1+Fe(III))557=8277
dm3mol−1cm−1)]. A component distribution analysis through deconvolution of absorption
spectra revealed that the Fe(III)-complexation induced absorption peaks at 525 nm(A525) and
557nm (A557) corresponds to be those of spiro-ring opened form of rhodamine 6G (Rho-6G)
and rhodamine B(Rho-B) components respectively. The dual output channel also inferred to
the opening of spiro-ring of both xanthene dyes upon complexation of 1 with Fe(III) ion, in
turn, the involvement of both xanthene derivatives in complexation process. Such enhanced
absorption
transitions (A557 and A525) in 1 were also observed in presence of
1+Fe(II) 557=4367 dm3mol−1cm−1] and Hg(II)[(ε
1+Hg(II) 557=3454 dm3mol−1cm−1] ions. However, the extents of absorption spectral
Fe(II)[(ε
)
1+Fe(II) 525=5078, (ε
)
1+Hg(II))525=2849,
(ε
)
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enhancements in 1 with these metal ions(factors of 15 and 8 folds respectively with Fe(II)
and Hg(II) ions] were much lower in comparison to that with Fe(III) ions(EF525=33 fold).
Other metal ions did not show any appreciable absorption spectral enhancement. The
absorption spectra of 2 on addition of various metal ions exhibited(Fig. 1b) similar pattern as
those of 1. The appearance absorption spectral peaks predominantly at 530nm with a shoulder
peak 557nm in 2 and corresponding enhancements were found to be optimal in the presence
of Fe(III) ion[(ε
)
2+Fe(III) 530=41951, (ε )
2+Fe(III) 557=14500 dm3mol−1cm−1)]. Absorption peaks
corresponding to complexation mediated spiro-ring opening in 2 were also observed in
presence of few other metal ions, but to a lesser extent in comparison to that of Fe(III)(log
ε530= 4.62, 4.45, 4.20, 3.73 with Fe(III), Fe(II), Cr(III) and Cu(II)/Ni(II) respectively). It is
worth mentioning here that the comparatively rigid ethylenediamine receptor containing
probe 2 was observed to exhibit higher metal ion induced absorption spectral enhancement in
comparison to its flexible diethylenetriamine receptor containing analogue 1. On the contrary,
the contribution of A525 and A557 components which correspond to metal ion induced ring
opened Rho-6G and Rho-B moieties respectively in the complete absorption spectral profile
is more pronounced in 1 than 2. The component ratio of corresponding metal ion induced
absorption spectral enhancements(ARho-6G/ARho-B) with Fe(III) and Fe(II) ions were found to
be 1.36 and 1.16 respectively in 1, while those were observed to be 2.89 and 2.96 in 2. On
contrary and although to a lesser extent, such component ratio (ARho-6G/ARho-B) with Hg(II)
were observed to follow an opposite trend as 0.82 in 1 and 0.71 in 2 (Fig. 1c). The
complexation induced individual absorption components (ARho-6G and ARho-B) of 1 and 2
varied with the nature of the metal ions. A correlation therefore was established that metal
ions other than Hg(II) ions have preferential affinity for rho-6G fraction over its rho-B
component in their spiro-ring opening to result in observable spectral enhancements(Fig. 1d).
On contrary, complexation of these probes with Hg(II) ion involves in spiro-ring opening
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preferentially of rho-B over its rho-6G component. The observation is in alignment with our
earlier reports where preferences of rhodamine-B derivatized probes22 in metal ion induced
spectral enhancements could selectively be tuned towards Hg(II) ion as a function of
presence of aqueous component in organic solvent medium. However, overall enhanced
spectral responses here predominantly contributed by optimal effective metal ion-probe
interaction which is effected through spatial arrangements of donor atoms in their receptor
unit.
(a)
(b)
0.15
0.6
A525
0.10
Fe(III)
0.4
A
557
Fe(II)
Blank and other
metal ions
Fe(III)
Blank and other
metal ions
Cr(III)
Fe(II)
Hg(II)
0.05
0.00
0.2
0.0
Cu(II), Ni(II)
Hg(II)
600
2
300
400
500
700
400
500
Wavelength(nm)
600
Wavelength(nm)
35
0.20
0.15
0.10
0.05
0.00
(c)
(d)
1.0
30
25
20
15
10
5
2 + Hg(II)
2 + Fe(III)
λ
λ
525
557
0.8
0.6
0.4
0.2
0.0
0.0
0.2
0.4
0.6
0.8
1.0
Mole fraction of Fe(III) added
450
500
550
600
Wavelength (nm)
Added metal ions
Fig. 1. (a) Absorption spectra of (a) 1 and (b) 2 in presence of various metal ions in EtOH–
H2O (1:1v/v, 0.1M PBS, pH 7.2) medium. (c) Extent of metal ion induced absorption
enhancement factor in 1 monitored through two absorption spectral positions (λobs = 525 and
557nm), (Inset c): Jobs plot for determination of stoichiometry of complexation of 1 with
Fe(III). (d) Normalized absorption spectra of 2 with Fe(III) and Hg(II) showing componential
preferences of spiro-ring opening. Absorption: [1 or 2] = 10µM, [M(I/II/III)] = 50µM.
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From absorption spectral data, the complexation stoichiometry of 1 and 2 with Fe(III) was
determined to be in 1:1 (1:Fe(III)) ratio using continuous variation method (Job’s plot, Fig.
1d). A molecular ion peak at 1179 (m/z)+ in ESI-MS spectral data of the [1.Fe(III)] complex
has also supported to its 1:1 (1:Fe(III)) stoichiometry. The FT-IR spectrum of the [1.Fe(III)]
complex ascertained complexation of 1 with Fe(III) as its carbonyl (C=O) stretching
frequencies were lowered to be observed at 1680cm-1. Similarly, a 10cm-1 shift in carbonyl
(C=O) stretching frequencies in 2(1698 cm-1) inferred to its complexation with Fe(III) ion.
The complexation induced fluorescence spectral pattern also followed their absorption
spectral behaviour(Fig. 2a). Upon excitation at 500nm in EtOH-H2O (1:1v/v, 0.1M PBS, pH
7.2) binary mixture medium, the weakly fluorescing 1 exhibited complexation induced
fluorescence recovery(φF=0.51) in presence of Fe(III) ion with signature spectral features of
spiro-ring opened rhodamine species. The extent of fluorescence enhancement in 1 was found
to be maximum in presence of Fe(III) ion(IF/I0 = 91 fold), while other metal ions rendered
none or negligible change in fluorescence of the probe except Fe(II) and Hg(II) ions where
such enhancement was observed to be up to a quantifiable extent[(IF/I0) = 8 and 3 folds
respectively for Fe(II) and Hg(II)]. Therefore, 1 preferentially exhibited optimal fluorescence
enhancement in presence of Fe(III) ion among all metal ions under investigation. The
component distribution diagram(Inset, Fig. 1c) of the fluorescence spectra of 1 with Fe(III)
ion revealed to emissive contributions of metal complexation induced spiro-ring opened Rho-
6G and Rho-B components with emission maxima at 557nm and 583nm respectively. 2 also
exhibited similar fluorescence(Fig. 2b) trend, it exhibited optimal fluorescence enhancement
in presence of Fe(III) ion (φF=0.63) whereas metal ions such as Fe(II) and Cr(III) also
rendered appreciable enhancements[φF(2+Fe(II))=0.31, φF(2+Cr(III))=0.24]. Other metal ions
did not result in appreciable fluorescence enhancements in 2, rather shown quenching trends,
indicating their failure in complex formation.
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1.2
1.0
0.8
0.6
0.4
0.2
0.0
1 + Fe(III)
(a)
Fe(III)
F
583
F553
1.0
0.5
0.0
550
600
650
700
Wavelength (nm)
Blank and
other metal ions
Fe(II)
Hg(II)
550
600
Wavelength(nm)
650
700
1.0
0.8
0.6
0.4
0.2
0.0
2.5
(b)
Fe(III)
2+Fe(III)
2+Hg(II)
2.0
1.5
1.0
0.5
0.0
Fe(II)
Cr(III)
500
550
600
650
700
Wavelength (nm)
Blank and other
metal ions
Hg(II)
500
550
600
650
700
Wavelength(nm)
Fig. 2. Fluorescence spectra of (a) 1 and (b) 2 in presence of various metal ions in EtOH–
H2O (1:1v/v, 0.1M PBS, pH 7.2) medium. Insets: (a) component distribution pattern of
fluorescence spectra of 1 in presence of Fe(III) and (b) Normalized fluorescence spectra of 2
with Fe(III) and Hg(II) showing preferences of spiro-ring opening of Rho-6G and Rho-B
components. [1] = [2] = 1 µM, [M(I/II/III)] =5 µM, λex = 500nm, RT, ex./em. b. p. = 5nm.
The excited state life-time of 1 and its Fe(III) complex through time correlated single photon
counting(TCSPC) measurements was carried to determine contribution of their individual
component distribution pattern to the total emission process. The average life-time, estimated
based on the average of obtained tri-exponential decay constants of the decay profile on
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excitation of the solution at 470nm in EtOH-H2O (1:1 v/v, 0.1M PBS) medium, were found
to be 2.92ns and 3.01ns respectively for 1 and its Fe(III) complexes. With estimated
fluorescence quantum yield of 1 and its Fe(III) complex as 0.0001 and 0.51 respectively, the
rate of non-radiative decay(knr) were calculated to be 0.342 for 1 which decreased as
anticipated to 0.162 upon its complexation with Fe(III). The quenched non-radiative
processes operative in 1-Fe(III) complex on comparison to that of 1 complement well to their
steady-state fluorescence spectral data. The fluorescence multi-exponential decay
corresponds to contributions from various conformational orientations of both fluorophoric
segments in excited state. The decay profile of 1 could approximately be fitted (χ2 = 1.195) to
be sum of three exponential components; 0.1ns(τ1, 26.88%), 2.64ns(τ2, 40.92%) and
5.66ns(τ3, 32.2%), where τ2(2.64ns) with maximum relative appearance and τ3(5.66ns)
components correspond to spirocyclic Rho-6G and Rho-B segments of 1 and the lower decay
(τ1, 0.1ns) component probably corresponds to inter-fluorophoric interaction. Similarly, the
tri-exponential fitting to the decay profile of 1-Fe(III) complex(χ2 = 1.107) revealed
components of excited-state life time to be 0.07ns(τ1, 20.91%), 3.18ns(τ2, 61.09%) and
5.62ns(τ3, 18.00%) respectively where τ2 and τ3 components corresponds to Rho-6G and
Rho-B in their ring-opened form.
In a control experiment, the absorption and fluorescence spectral behaviour of 1 in presence
of various metal ion were carried out in different solvent (dry EtOH as well as MeCN-
H2O(9:1 v/v, PBS, pH 7.2) medium in order to verify if the solvent contribute or alter the
probe’s optimal photophysical responses along with its colourization in presence of Fe(III)
ion. The choice of an aqueous-acetonitrile binary mixture medium is based on our earlier
observations22 that aqueous component in solvent medium promotes fluorescence and
absorption spectral enhancements in “derivatized amino-alkyl-rhodamine” based probes
selectively in presence of Hg(II) ion. The absorption(A525) and fluorescence(F550
)
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enhancements in 1 were observed to be maximum((IF/I0)1+Fe(III) = 16 fold, aq. MeCN) in
presence of Fe(III) ion irrespective of the solvent medium, which inferred to a higher probe-
metal ion interaction in 1 with Fe(III) among other metal ions investigated.
With two xanthene dyes coupled to the receptor unit in the architecture of both 1and 2, it is
imperative to understand and determine contributions of both Rho-6G and Rho-B in its over-
all Fe(III) ion induced spectroscopic signal amplifications. Therefore, absorption and
fluorescence titrations of 1 and 2 were carried out as a function of added Fe(III) ion. The
absorption titration of 1(6.6 ꢀM) with Fe(III) ion in EtOH-H2O (1:1v/v, 0.1M PBS, pH
7.2) binary mixture rendered better comprehension of metal-probe interaction in its ground
state complexation pattern(Fig. 3a). On gradual addition of Fe(III), both A525 and A557 were
observed to increase simultaneously up to addition of 1eq. and subsequent addition up to 4.5
eq. led enhancement of A525 component more than that of A557. Similar component
enhancement profile (F550 and F580) were also observed in its fluorescence titration profile,
where predominant F550 fluorescence component prevails over that of F580 at saturation of
complexation. The absorption and fluorescence titration profile of 2(Fig. 3b) upon addition of
Fe(III) also revealed to spectral enhancements and attained maximum(>10 eq.); where the
individual spectral components of Rho-6G predominantly enhanced over that of Rho-B
component. The spectroscopic observations of both probes as a function of added Fe(III) ion
therefore inferred that Rho-G component facilitates higher affinity in complexation with
Fe(III) than its Rho-B component. The association constant(Ka) of complexation were
calculated23 for a 1:1 (1:Fe(III)) complexation stoichiometry through a non-linear fit to
change in absorption(λobs=525nm) and fluorescence(λobs=550nm) data as a function of
concentration of Fe(III) added(ln[Fe(III)]). The Ka were estimated to be 1.58×104 M-1 and
2.17×104 M-1 from the respective fluorescence and absorption titration data of 1 with Fe(III)
ion in EtOH-H2O(1:1v/v, 0.1M PBS, pH 7.2) binary solvent medium. Similarly, estimation of
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Ka for Fe(III) complexation with 2 was also found to be similar on determination from
respective fluorescence(Ka=1.21×105 M-1) and absorption(Ka=3.63×104 M-1) titration data.
The correlation factor of association constants estimated from both fluorescence and
absorption spectral data, being close to unity ([logKa(fluo.) / logKa(abs)= 0.97(for 1) and
1.11(for 2)], validated the adapted methodology for Ka determination.
0.4
(a)
A
525
0.3
0.6
0.2
A
557
0.1
0.4 0.0
0
1
2
Eq. of Fe(III) added
3
4
5
10.0 eq
0.0 eq
0.2
0.0
400
450
500
550
600
Wavelength(nm)
(b)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
2+Fe(III)
2
2
12.0 eq.
[Fe(III)]
0.0 eq.
500
550
600
650
700
Wavelength(nm)
Fig. 3: (a) Absorption titration spectra of 1 and (b) fluorescence titration spectral profile of 2
as a function of added Fe(III) ion. The spectra were recorded immediately after addition of
solutions of metal ions to that of probes. Abs: [1] = 6.6µM, Fluo: [2] = 1µM, λex = 500nm,
RT, ex. and em. b. p. = 5nm.
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It is worth mentioning here that in total spectral pattern of absorption and fluorescence
titration, the individual contributions of ring-opened Rho-B component (A557, F583) were
observed to be prominent in comparison to that of ring-opened Rho-6G component (A525
,
F550) when recorded after 1h of mixing solutions of Fe(III) ion with 1. This inferred that
metal-probe kinetics plays a significant role in the probe’s complexation preferences
expressed through their respective signatory spectral pattern. In order to investigate
coordination affinity of both Rho-G and Rho-B segments of 1 towards Fe(III) ions,
complexation induced change in absorption in 1 were assessed as a function of time with
stoichiometric addition of Fe(III) ions to its solution in EtOH-H2O(1:1 v/v, 0.1M PBS, pH
7.2) binary mixture medium and monitored at both 525nm and 557nm corresponding to
spiro-ring opening of Rho-6G and Rho-B fractions respectively. As the interaction between
probe and added metal ions being determining factor for rates of their spiro-ring opening
profile24 and considering complexation mediated spiro-ring opening of both xanthene dyes to
follow a first-order kinetics, the rate constant was determined through a non-linear regression
fit (A = A0 e-kt) of first-order kinetics at the desired time. The Fe(III) complexation induced
rate constant(k) of spiro-ring opening in 1 were found to be 0.00254s−1 and 0.00025s−1
respectively at 525nm and 557nm(Fig. 4a). A one order higher rate constant at 525nm over
that of at 557nm ascertained that coordination affinity of Rho-6G segment towards Fe(III) ion
is higher than its Rho-B fraction. Further, the rate constant(k) of spiro-ring opening in 1
monitoring at 525nm was estimated to be 0.00139s−1 from the time driven Fe(III)-
complexation induced spectral data of 1 in dry EtOH. The faster kinetics rate of 1-Fe(III)
complexation in EtOH-H2O (1:1 v/v, 0.1M PBS) medium in comparison to that in EtOH
suggest to a crucial contribution of solvent composition in the kinetic rate of complexation, in
turn, probe-metal ion interaction. This observation is well complemented by the fact that
intensities of Fe(III) complexation induced absorption spectral enhancements(A525) in 1 was
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observed to be lower in dry EtOH than that in its EtOH-H2O (1:1 v/v, 0.1M PBS) binary
mixture solution. The Fe(III) complexation induced rate constant(k) of spiro-ring opening in
2 was found to be 0.00028s−1 at 530nm (Fig. 4b). The lower rate constant of spiro-ring
opening with Fe(III) in 2 in comparison to that in 1 attributed to the stereo-electronic
environment of the probes. The rigid ethylenediamine receptor segment in 2 tends to induce
higher rigidity for its effective interaction with metal ion while the flexible diethlenetriamine
receptor in 1 facilitates faster metal ion-probe interaction. The kinetic studies of probe-metal
ion interaction in these probes also inferred that metal ion induced signaling perturbations is
not as fast here as observed for other rhodamine based probes22, however, rendered a
comprehension of preferential complexation affinity between its two distinct coordinating
sites and provided the basis for recording the spectra at a time scale after addition of solutions
of metal ion to the probe.
(b)
0.08
0.06
0.04
0.02
0.00
0.30
0.25
0.20
0.15
0.10
0.05
0.00
A
(k =0.00254s-1)
(aq. EtOH)
(a)
525
A
[A = A e^ (-3475.13)t]
0
557
A
(k = 0.00025s-1)
557 (aq. EtOH)
A
(k =0.00139 s-1)
(EtOH)
525
2+Fe(III)
0
2000
4000
6000
8000 10000 12000 14000
0
300
600
900
1200
1500
1800
Time (s)
Time(s)
Fig. 4: Plot of absorption of (a) 1 and (b) 2 as a function of time on addition of equivalent
Fe(III) ions. [1] = [Fe(III)] = 5µM, [2] = [Fe(III)] = 5µM.
As the probe exhibited fluorescence output at two different wavelengths(F550 and F580), a plot
of fluorescence intensity ratio (I580/I550) as a function of added Fe(III) ion concentration at
lower concentration level (<0.15µM) also potentially indicated the coordination of its both
ring-opened xanthene derivatives to Fe(III) ion. From the fluorescence spectral responses of 1
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with Fe(III) at very low concentrations, its limit of Fe(III) detection was calculated25 to be
11nM in EtOH-H2O (1:1v/v, 0.1M PBS) solution(λex = 500nm) where as the visual detection
limit were found to be 3µM as a function of colour change observable through naked eye.
Similarly, the limit of Fe(III) detection with 2 was found to be 0.16µM. The detection limits
for 1 and 2 towards Fe(III) thus obtained are found to be lower in comparison to previously
reported probes13e,f which appends either two Rho-B units in their architecture or other
rhodamine derivatized entities26. Further, these probes accomplish the sensitivity criteria for
Fe(III) detection in human serum which normally contains iron in 10-30mM concentration
range.
A higher spectral overlap between fluorescence of Rho-6G(~F550) and absorption spectra of
rho-B(A557) segments of both 1 and 2 on complexation with Fe(III) suggests towards a
possible fluorescence resonance energy transfer between excited donor (Rho-6G*) and
ground state acceptor (Rho-B) components. However, the ground and excited state spectral
features in these probes did not support for the occurrence of any detectable FRET process
between these derivatives in their complexation mediated ring-opened form. The absorption
and fluorescence titration profile of 1 and 2 with Fe(III) ion indicated that spectral features
corresponding to their both Rho-6G and Rho-B components increased simultaneously as a
function of added Fe(III) ion which in-turn, indicated that fluorescence resulted through
metal ion induced spiro-ring opening process of both xanthene components are independent
of each other. If significant FRET process had been operative between these anticipated
energy donor-acceptor pair, the fluorescence corresponding to Rho-6G component would
have decreased and that of Rho-B component would have increased simultaneously in a
ratiometric manner, which is unlikely in these cases. Evaluation of component distribution
diagram of each fluorescence spectral pattern of 1 and 2 on titration with Fe(III) ion also
inferred to direct excitation of both fluorophore derivatives. Complementing to steady-state
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fluorescence spectral observation, the longer(τ3=5.62ns) life-time of Rho-6G component in
comparison to that (τ2=3.18ns) of Rho-B component in excited state lifetime decay profile of
1 in presence of Fe(III) also did not render the possibilities of excitation energy transfer
process between the DRho-6G−ARho-B pair. Despite of spectroscopic evidences overruling
existence of operative and perturbation of FRET process between these xanthene derivative
pair, the steady-state fluorescence spectral pattern and multi-component exponential decay
profiles of these probes and their Fe(III) complex obtained are complicated to be
comprehensively resolved in terms of energy transfer process and is a matter of separate
investigation before putting any conclusion in this regard. The spectroscopic evidence here
inferred to preferences of ring-opened Rho-6G and Rho-B components for their direct
excitation rather than an energy transfer, although spectral overlap of both fluorophores is
much close to each other. Nevertheless, the spectral responses of these probes established
their potentiality in selective and sensitive Fe(III) ion detection irrespective of the output
signalling window. With all the spectroscopic evidence, a possible mechanism of
complexation of 1 with Fe(III) is depicted in Scheme 2.
H
N
NH
H
N
O
O
O
N
O
H
N
N
O
Fe(III)
N
H
N
N
O
N
NH
N
O
N
O
N
Scheme 2: Proposed complexation mechanism of 1 with Fe(III).
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The reversibility in signal responses resulted through metal ion induced spiro-ring opening in
these probes was determined with subsequent addition of ammonium salts of various counter
anions in a EtOH–H2O(1:1v/v) medium(Fig. 5a). The Fe(III) induced enhancement in
absorption (A525) and (A557) of 1 decreased almost to that of its metal-free state and the pink
coloured solution turned colourless within 1min. after addition of the AcO- anion. Addition of
other counter anions exhibited a time-dependent decrease in spectral responses and
corresponding decolourization of its solution. Apart from anions, its spectral responses
revealed that initial spirocyclic state was regenerated upon the subsequent addition of
chelating agents such as EDTA and ethylenediamine to the 1–Fe(III) complex in solution.
Subsequent addition of Fe(III) ions to the decolourized solution of anion(AcO-) mediated
Fe(III) decomplexed 1 resulted in reappearance of colour of 1-Fe(III) complex along with re-
appearance of enhanced A525 and A557 spectral transitions almost to the same extent as those
upon initial Fe(III) addition to 1. Exploiting higher affinity of counter anion/chelating agents
towards metal ions than metal ion-probe interaction, similar lowering in Fe(III) complexation
mediated enhanced absorption transition were also observed in 2. Such subsequent metal ion-
counter anion induced ‘off-on-off’ cycles of absorption spectra of these probes therefore
establish their reusability as chemosensory probes for selective Fe(III) ion detection.
0.3
(a)
Blank
AcO-
Cl-
CO3-
0.2
0.1
0.0
I-
PO 3-
4
SCN-
SO42-
[1+Fe(III)]
[1+Fe(III)]+AcO-
EDTA
300
400
500
600
700
Wavelength(nm)
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8
7
6
5
4
3
2
1
0
(b)
1 + M(I/II/III)
1 + M(I/II/III) +Fe(III)
Added Metal ions
Fig. 5: (a) Absorption spectra of in-situ 1-Fe(III) complex on addition of various
anions/chelating reagents in EtOH–H2O(1:1v/v) and corresponding colour mapping
photograph (Inset a). (b) Comparative representation of fluorescence intensities of solution of
1 in presence of various metal ions and followed by subsequent addition of Fe(III) depicting
non-interference of other metal ions in its Fe(III) complexation. Spectra were recorded
immediately after mixing. [1] = 0.67 µM, [M(I/II/III)] =2.5 µM, Fe(III) = 2.5 µM, λex
500nm, RT, ex. and em. b. p. = 5nm.
=
In another control experiment to investigate cross-sensitivity of these probes towards other
metal ions in their selective Fe(III) ion detection, absorption and emission spectra of 1 in
EtOH–H2O(1:1v/v, 0.1 M PBS) were recorded after addition of either (a) other competitive
metal ions followed by Fe(III) or (b)Fe(III) followed by other individual metal ions to its
solution(Fig. 5b). The addition of Fe(III) to the solutions containing 1 and each of other metal
ions resulted in optimal absorption spectral enhancements to a comparable extent of that of 1
on addition of only Fe(III). On contrary, the Fe(III) induced absorption spectral responses of
1 remained unperturbed on addition of other metal ions. The observations hence supported its
ability to render Fe(III) selective signalling amplification amidst presence of other
competitive metal ions.
The absorption and fluorescence data of 1 and 2 in EtOH–H2O(1:1v/v, PBS) under various
pH conditions of the solvent medium revealed that metal ion induced signal amplification
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were highly affected at lower pH due to proton induced spiro-ring opening(Fig. 6). However,
when measured under approximately physiological conditions (pH = 6-7) or under basic
conditions, no appreciable enhancement in absorption (A525 and A557) or fluorescence (F550
and F581) spectra were observed in 1 and 2. Therefore, the absorption and fluorescence
spectra of these probes resulted in a stable Fe(III) induced enhanced signals in 5-10 pH range,
without any interference by protons. Their fluorogenic and chromogenic signaling responses
alone as well as in the presence of Fe(III) ions in varying pH and corresponding signal
stability within 5–10 pH range promotes for their potential utility in various practical
applications, such as detection of bio-accumulated metal ions in plant tissues.
0.3
0.6
(a)
0.5
0.2
0.4
0.3
A
(1+Fe(III))
525
0.1
0.0
0.2
0.1
0.0
A
(1)
525
A
(2)
A
(2+Fe(III))
530
530
(b)
4
6
8
10
2
4
6
8
10
pH
pH
Fig. 6: Absorption spectral responses of (a) 1 and (b) 2 alone and in presence of Fe(III) ion as
a function of varying pH in EtOH:H2O (1:1 v/v, PBS) medium. [1] = [2] =10µM. Inset b:
photograph depicting colour of the solution of 2 in varying pH.
To demonstrate the probes’ utility in detection of accumulated metal ions in compatible plant
species, the bio-imaging experiments were carried out with Grevillea pteridifolia species
exploiting Fe(III) selective dual mode signaling of 1. One-month old Grevillea pteridifolia
seedlings with the well-developed root system, which were hydroponically grown in nutrient
solution27(1/4 Murashige Skoog salts), were taken as control and further treatment in a
solution of either metal ion or the probe. To assess the phytotoxic effect of 1 on these plants,
few healthy seedlings were co-cultivated with a solution(2% DMSO in H2O) of 1 (50 ppm) in
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the clean test tubes and their morphological and anatomical changes in root and shoot cross
sections were observed, even for a week. No observable morphological differences were
found among control plant and those treated with 1 (50 ppm) till a couple of weeks. However,
those plants were started to wilt beyond that time frame. Therefore, the time of anatomical
section for imaging was chosen to be 14 days after treatment in each case.
Fig. 7: (a) Grevillea pteridifolia seedlings being treated with aqueous solution of Fe(III) of
variable concentrations (0, 5, 10, 15, 20, 30, 40, 50, 60 and 75 ppm), Note: appearance of
plant leaves showing brownish colour on uptake of Fe(III) (Conc. ≥ 50 ppm). (b) Cross
section of G. pteridifolia root stained with safranin (0.5 % w/v) solution (10x objective)
showing cellular details; P, phloem; PF, phloem fibre; Xy, xylem; XyR, xylem ray cells.
On contrary, the plants which were also grown hydroponically with aqueous solution of ferric
nitrate of variable concentrations (0, 5, 10, 15, 20, 30, 40, 50, 60 and 75 ppm) allowing them
to absorb and accumulate metal ion for two weeks growth period, a visible toxic effect was
observed on plants treated with more than 50ppm of Fe(III) showed visible colour change on
the leaves (green to brown) during second week of treatment period(Fig. 7). No such
browning of green leaves and wilting effects were observed in control plants as well as those
treated with 25 ppm of Fe(III) solution or lesser concentration. This ascertained that the plant
species uptake the Fe(III) ion through its roots. As mentioned in the experimental section, the
root segments from each plant treated with Fe(III) were further were dipped in an aqueous
ethanolic solution of probe 1 of respective concentration for 15 min followed by washing
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with deionized water and were subjected to hand-section for fluorescence image analysis(Fig.
8). The bio-imaging studies revealed that Fe(III) ions were localized in the roots of treated G.
pteridifolia, as was confirmed by the appearance of strong orange color fluorescence(Fig. 8b)
in comparison to the non-fluorescing condition of those plants treated with 1(Fig. 8a). The
ferric ion deposition was mostly observed in the xylem ray cells, the main functions of the
ray cells are storage and radial transport (Fig 8b).
Fig. 8: Fluorescence images of (a) cross section of root segment of G. pteridifolia plant
grown on nutrient solution treated with 1 (50 ppm); (b) cross section of G. pteridifolia root
treated with solution of Fe(III) (50 ppm) followed by 1 (50 ppm) at 15 min duration. The
orange fluorescence appeared due to spiro-ring opening of 1 on complexation with Fe(III)
accumulation in the xylem ray cells.
Similar fluorescence microscopic images of the cross-sections of stem segments also
indicated detection of bio-accumulated Fe(III) ions with 1. In context of interference of
chelation mediated fluorescence signals of these probes with bio-accumulated Fe(III) ions in
plant tissues, various phyto-hormones such as Indole-3-acetic acid (IAA) and salicylic acid
derivatives present in the plant species are potential in forming complexes28 with Fe(III) ions.
Considering the UV-VIS absorption of these auxins which occurs in 200-400 nm region on
their Fe(III)-complexation whereas these rhodamine derivatives(1 and 2) exhibits absorption
and emission at a longer wavelength upon complexation, the optical signalling interference
by complexes of phyto-hormones present in the plant segments may be overruled on the
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fluorescence signals of these probes. The quantitive analysis of bio-accumulated Fe(III) is
anticipated to get affected on their complexation with phyto-hormones and needs a thorough
investigation before putting a conclusion. Nevertheless, the probe 1 demonstrated its
potentiality in detection and localization of Fe(III) ion accumulated in the roots of G.
pteridifolia plant species.
Experimental Section
Materials and Methods
All reagent grade chemicals were used without purification unless otherwise specified.
Rhodamine B base, rhodamine-6G, diethylenetriamine(DETA), ethylenediamine and metal-
perchlorate salts were obtained from Sigma–Aldrich (India) and used as received. Anhydrous
sodium sulphate, triethyl amine, silica gel for column chromatography, acids and the solvents
were received from Spectrochem Pvt. Ltd. (India). All solvents were freshly distilled before
use for absorption and fluorescence measurements.
Analysis and measurements
1
The compounds were characterized by elemental analyses, H NMR, 13C NMR and Mass
1
(ESI) spectroscopy. H-NMR and 13C-NMR spectra were recorded on a JEOL JNM-AL400
FT V4.0 AL 400 (400 MHz and 100 MHz respectively) instrument in CDCl3 with Me4Si as
the internal standard. Electrospray mass spectral data were recorded on a MICROMASS
QUATTRO II triple quadruple mass spectrometer following standardized operational
protocols. Melting points were determined with a melting point apparatus by Stuart SMP20,
India and were uncorrected. Elemental analyses were done in an ElementarVario EL III Carlo
Erba 1108 elemental analyzer. UV–visible spectra were recorded on a Perkin Elmer Lambda
650 UV/VIS spectrophotometer at 298 K in 10−5M concentration. Steady-state fluorescence
spectra were recorded in a Fluoromax 4P spectrofluorometer at 298 K. Fluorescence quantum
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yields were determined29 in each case by comparing the corrected spectrum with that of
Rhodamine 6G (φF = 0.95) in EtOH30 by taking the area under total emission. The time-
resolved fluorescence measurements were evaluated in an Edinburgh FLS980
photoluminescence spectrometer through time-correlated single photon counting (TCSPC)
technique. The excitation wavelength was 500nm and the decays were monitored at emission
wavelengths at 550nm and 580nm respectively corresponding to Rho-6G and Rho-B
fluorescing units.
The association constant (Ka) of the complexes were determined from changes in absorbance
or fluorescence intensities resulted from titration of dilute solutions (~10−5-10−7 M) of these
probes against metal ion solutions following equation23 reported earlier. For a 1:1(probe:
metal ion) stoichiometry of complex formation, the non-linear curve fitting to the plot of
change in intensities of absorption or fluorescence spectra against the logarithm of
concentrations of metal ions added determined Ka. From the plot of change in fluorescence
(I/I0) as a function of concentration of added metal ion in a linear range determined the limit
of detection (S/N=5) as 3σ/s where σ is the standard deviation of y-intercept and s is the slope
of linear regression of graph25.
Plant material and treatment procedure
G. pteridifolia seeds were immersed in 0.5 % Bavistin(BASF, India) solution for five minutes
to avoid fungal contamination, followed by thorough washing with deionized water and
subsequent germination on a pre-soaked filter paper. Germinated seedlings were transferred
on to plastic pots filled with vermiculite and the seedlings were maintained inside incubation
room with mean temperature of 25 2 ºC with 50-70% relative humidity. One month old
seedlings were used for bio-imaging experiments. The young plants were cultured in test
tubes containing 30 mL of aqueous solution of one-fourth strength Murashige Skoog (MS)
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salts27. One group of plants were continued to be nurtured as control group, while another
group of plants with well-developed root system were hydroponically grown in test tubes
containing 0, 25, 50, and 75ppm concentrations of ferric nitrate in nutrient solution and
maintained in an incubation where pH was adjusted to 7.00 using 1 M KOH or 1 N HCl. In
each concentration 5 uniform plants were cultured and allowed for 14 days growth period.
Procedure for histological studies
Plants from control groups and plants that have been exposed to the different concentrations
of ferric nitrate were taken out at the end of the exposure period (14 days) and rinsed
thoroughly in three changes of deionized water. After rinsing, individual plants of treated and
few of control origin were dipped in an aqueous ethanolic solution of probe 1 of respective
concentration for 15 min followed by washing with deionized water. The root was severed
from the plant for anatomic sections, making thinner cross sections for fluorescence
microscopy (Nikon i80) and microanalysis. In order to prepare thinner (<20 micron) sections
we employed a simple technique that involved the hand-sectioning of the samples using sharp
razor. Observations on images were recorded with the aid of Nikon DS-Fi1 camera connected
to Nikon Digital Sight DS-U3 interface.
Synthesis of intermediate rhodamine B acyl chloride (I)
Rhodamine B acyl chloride was prepared, as described, following a procedure reported31 in
literature. To a solution of rhodamine B base (1.0 g, 2.3 mmol) in dichloromethane (15 mL),
phosphorusoxychloride (0.75 mL, 8.2 mmol) was added in a drop-wise manner over 5 min
under continuous stirring and the reaction mixture was heated to reflux for 4 h. After cooling
the reaction mixture down to room temperature, the solvent was removed under reduced
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pressure to obtain rhodamine B acyl chloride which was directly used in subsequent steps
without purification.
Synthesis of 1a and 2a
The compounds 1a32 and 2a33 were synthesized following a procedure reported earlier by
reacting rhodamine-6G with diethylenetriamine (for 1a) and ethylenediamine(for 2a)
respectively.
Generalized Synthesis of 1and 2
To a stirring solution of either 1a (1.0g, 2 mmol) or 2a (0.97g, 2 mmol) in acetonitrile(30
mL) added with and triethylamine (0.2g, 2mmol), rhodamine-B acylchloride I (1.0g,
2.16mmol) was added drop-wise at 0°C over a period of 1h after which the reaction mixture
was allowed to react at room temperature for 1h, followed by heated to reflux to 12h until the
solution turned dark-orange in colour. The solution mixture was then filtered and the solvent
was evaporated under reduced pressure. Water (10 mL) was added to the brownish solid and
extracted with CHCl3 (3×20 mL). The combined organic layer were dried over anhydrous
Na2SO4 and evaporated to dryness under reduced pressure to obtain a brown residual mass,
which was further purified by passing through a column (100–200 mesh silica gel) with
chloroform and methanol (99 : 1 v/v) as eluent to yield 1 or 2 as the desired product.
Compound 1: Yield: 1.078g (58 %); mixed m. p.= 120-122 °C; ESI-MS (C58H65N7O4) m/z+
1
(%): 924.91 [1 +H]+ (100); H-NMR (400MHz, CDCl3, 25°C, TMS, δ): 7.992 (d, J = 7.19Hz,
2H), 7.610 (td, J1 = 1.19Hz, J2 = 7.19Hz, 4H), 7.202(d, J = 7.19Hz, 2H), 6.584(br s, 2H),
6.561(br s, 2H), 6.442(d, J = 2.79Hz, 2H), 6.323(dd, J1 =2.39Hz, J2 = 8.39Hz, 4H), 3.331(q,
J =7.19 Hz, 12H), 3.086(d, J = 2.79Hz, 2H), 3.045(d, J = 2.39Hz, 2H), 2.693(m, 4H),
2.308(s, 6H), 1.152(t, J = 6.79 Hz, 18H); 13C-NMR(100MHz, CDCl3, 25 °C, TMS, δ):153.94,
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152.89, 151.84, 149.04, 148.88, 147.82, 132.66, 131.06, 128.99, 126.55, 123.96, 118.49,
110.75, 108.40, 106.23, 105.80, 98.04, 96.86, 76.80, 68.35, 65.53, 47.52, 44.53, 40.29, 38.94,
37.29, 32.95, 31.44, 30.57, 29.88, 27.28, 23.95, 22.87, 19.92, 16.85, 14.80; Anal. calcd. for
C58H65N7O4 (Mw = 923.5098), %: C, 75.38; H, 7.09; N, 10.61; found: C, 76.21; H, 7.62; N,
9.47.
Compound 2: Yield: 0.76g (43%); mixed m. p. = 161-163 °C; ESI-MS (C56H60N6O4) m/z+
1
(%): 881.47 [2 +H]+(100), 903.45[2 +Na]+ (57) ; H-NMR (400MHz, CDCl3, 25°C, TMS, δ):
7.782(dq, J1 = 8.99Hz, J2 = 1.99Hz, 2H), 7.306(d, J = 2.79Hz, 2H), 6.860(t, J = 5.59Hz, 2H),
6.324(d, J = 2.4Hz, 4H), 6.266(br s, 2H), 6.111(d, J = 2.39Hz, 2H), 6.111(s, 4H), 4.092(q, J
=6.79Hz, 4H), 3.362(dd, J1 = 6.79Hz, J2 = 5.19Hz, 8H), 3.332(t, J = 7.19Hz, 4H), 3.036(s,
6H), 2.043 (brs, 2H, NH), 1.369(t, J = 7.19Hz, 3H), 1.256(t, J = 7.19Hz, 3H), 1.201(t, J =
6.79Hz, 12H); 13C-NMR (100MHz, CDCl3, 25°C, TMS, δ): 168.325, 168.162, 154.286,
153.098, 151.584, 148.565, 147.137, 128.422, 128.269, 127.713, 123.545, 123.506, 122.855,
117.661, 107.992, 106.056, 105.683, 98.304, 97.029, 64.908, 64.831, 60.481, 44.382, 38.556,
38.479, 38.383, 16.544, 14.972, 14.282, 12.845; Anal. calcd. for C56H60N6O4 (Mw
880.4676), %: C, 76.34; H, 6.86; N, 9.54; found: C, 76.11; H, 7.03; N, 9.29.
=
Conclusion
Two rhodamine derivative(Rho-6G and Rho-B) coupled alkylamino-receptor based probes 1
and 2 were shown to exhibit absorption and fluorescence signalling responses selectively in
presence of Fe(III) ion in a EtOH-H2O(1:1v/v, pH 7.2, 0.1M PBS) binary mixture medium
through their complexation induced spiro-ring opening. The dual wavelength output channel
in their both absorption and fluorescence spectra revealed to the simultaneous occurrence of
ring-opening of both spiro-centres upon complexation to a Fe(III) ion with a 1:1 (probe:
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New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ03364H
metal) stoichiometry. The kinetic experiments for spiro-ring opening along with absorption
and fluorescence spectral features revealed that the Rho-G segment has higher and faster
affinity towards Fe(III) ion than the Rho-B segments in both the probes. The excited-state life
time measurement experiments complemented well to the steady-state fluorescence
behaviour of the metal ion induced ring-opened probes. With various control experiments for
determination of sensitivity and reversibility, these probe establish their potency as
chemosensory probes for detection of Fe(III) ion, where control of their receptor unit in
preferential complexation pattern is driven by the affinity of the individual spiro-ring opened
rhodamine derivatives. The probe 1 was conveniently demonstrated as an optical imaging
module in detection and subsequent localization of Fe(III) ions accumulated in root tissues of
G. pteridifolia plant species. Despite complex photophysical behaviour, such bi-fluorophoric
probes with dual mode, dual wavelength output spectral responses upon complexation to a
selective metal ion not only promotes viable methodologies in designing probes for selective
detection of metal ion, Fe(III) ion in particular, but also render comprehensive insight
towards multi-functional complex logic circuitry.
Conflict of interest
There are no conflicts to declare.
Acknowledgements
BPB wishes to thank DST, New Delhi for financial support (SB/EMEQ-226/2013); the
Director, CSIR-IMMT for support through CSIR-IMMT-ESC-401; UGC-New Delhi for a
senior research fellowship (RGNF-SRF) to DM. The authors are thankful to Dr. Y. S.
Chaudhary, CSIR-IMMT for TCSPC measurements (CSIR-IMMT-MLP-020).
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