Naphthalene dianhydride based selective detection targetable fluorescent probe for monitoring exogenous Iron in living cells

Article abstract of DOI:10.1016/j.tetlet.2018.09.028

A green emissive PET operating fluorescent turn-on cell permeable novel probe R1 has been successfully developed and utilized for the detection of Fe⁺³ in the pure aqueous system at sub-nanomolar level. Moreover, probe R1 demonstrate highly sensitive and selective towards Fe⁺³ over the other divalent and trivalent metal ions and was established by using fluorescence spectroscopy. The efficiency and aid of R1 was demonstrated by the fluorescence imaging of captured Fe⁺³ within Pollen grains by using fluorescence microscopy. These results indicate that, this is the first fluorescent turn-on PET probe to detect sub-nanomolar Fe⁺³ in the pure aqueous system and in cellular level.

Full text of DOI:10.1016/j.tetlet.2018.09.028

Accepted Manuscript
Naphthalene dianhydride based selective detection targetable fluorescent probe
for monitoring exogenous Iron in living cells
Gandhaveeti Rohini, Konakanchi Ramaiah, Anandaram Sreekanth
PII:
S0040-4039(18)31109-2
https://doi.org/10.1016/j.tetlet.2018.09.028
TETL 50267
DOI:
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
4 July 2018
7 September 2018
11 September 2018
Please cite this article as: Rohini, G., Ramaiah, K., Sreekanth, A., Naphthalene dianhydride based selective detection
targetable fluorescent probe for monitoring exogenous Iron in living cells, Tetrahedron Letters (2018), doi: https://
doi.org/10.1016/j.tetlet.2018.09.028
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Naphthalene dianhydride based selective detection targetable fluorescent
probe for monitoring exogenous Iron in living cells
a
b
a, *
Gandhaveeti Rohini , Konakanchi Ramaiah , Anandaram Sreekanth
a
b
Department of Chemistry, National Institute of Technology, Tiruchirappalli-620015, India.
Department of Chemistry, National Institute of Technology, Warangal -506004, India.
*
Corresponding Author E-mail address: sreekanth@nitt.edu; Tel: +91 431 2503642
ABSTRACT
A green emissive PET operating fluorescent turn-on cell permeable novel probe R1
+
3
has been successfully developed and utilized for the detection of Fe in the pure aqueous
system at sub-nanomolar level. Moreover, probe R1 demonstrate highly sensitive and
+
3
selective towards Fe over the other divalent and trivalent metal ions and was established by
using fluorescence spectroscopy. The efficiency and aid of R1 was demonstrated by the
+
3
fluorescence imaging of captured Fe within Pollen grains by using fluorescence microscopy.
These results indicate that, this is the first fluorescent turn-on PET probe to detect sub-
+
3
nanomolar Fe in the pure aqueous system and in cellular level.
+
3
Keywords: Fe ion sensors; Live cell imaging; Naphthalene dianhydride based fluorescent
probe; PET operating probe.
Introduction
Development of fluorescent sensors for sensitive and selective detection of
1
-4
biologically important ions increased more attention nowadays.
Iron and copper are
5
-8
essential nutrients, excesses or deficiencies of which cause impaired cellular functions. In
case of Iron, it is the most abundant transition metal and found fourth most common element
9
,10
in the earth’s crust. Iron is an essential trace element in animals and plants
and plays an
1
1-13
+3
important key role in the human body.
Fe acts as an oxygen carrier in haemoglobin and

1
4,15
+3
involved in the several electron transfer reactions.
Additionally, Fe involve in many
enzymatic, catalytic reactions which include oxygen exchange and Fenton reaction in cellular
level. The mechanistic reports of these reactions have been well investigated and reported in
the literature. Moreover, iron involves in the generation of highly active oxygen-derived
+
3
hydroxyl radicals. However, the Fe concentrations in the living system have been found to
be important for the smooth functioning of many biological processes. Iron dyshomeostasis
(
either deficiency or overload) may lead dysfunction of various organs and several types of
1
6
diseases such as hemochromatosis, neurodegenerative diseases (Parkinson’s disease and
1
7
18
19,20
21
ALS), cancer, hepatitis
and liver cirrhosis.
From the past few decades, several selective and collective probes have been reported
3
+
for the detection of Fe ion in environmental samples. Moreover, fluorescent turn-on probes
3
+
for the detection and imaging of Fe in biological systems are much less in number. The main
3
+
reasons for this scarcity are the strong paramagnetic quenching nature of Fe ion, probes cell
impermeability, lack of solubility and lack of sensitivity in aqueous solution. In addition, the
3
+
development of fluorescence turn-on probes for Fe has always been a challenging task.
Therefore, there is a need for the development of water-soluble and cell permeable
3
+
fluorescent turn-on probes for the detection of Fe having the potential applications in bio-
3
+
imaging. The importance of Fe ion gave us to motivation to prepare highly selective,
3
+
sensitive and cell permeable fluorescent turn-on probe for the detection of Fe ion in the pure
2
2-27
aqueous system. Recently, naphthalene-based chemosensors,
ratiometric fluorescent
2
8
29,30
chemosensors and polymeric chemo sensors
have been developed for the detection of
heavy metal ions due to its well-known photophysical properties.
Results and Discussion
The PET probe R1 was synthesized by following a three-step condensation reaction
(
Scheme 1). The first step involved the condensation of ethylenediamine and iodobenzene to
form metal ion chelating ethylenediamine derivative R. In the next step, the condensation
reaction between propylamine and naphthalene dianhydride to form an intermediate Rb.
Further, the final step involves the condensation reaction between the compounds Rb with R,
to form the desired probe R1. The final structure of the probe was confirmed by NMR and
HRMS spectroscopy (Supporting Information Figs. S1-S5).

Scheme 1. Synthesis of probe R1
Initially, the photophysical properties of probe R1 were studied by using UV-Vis and
fluorescence spectrometry in H O/DMSO (99.0:1.0, v/v) buffered with HEPES, pH 7.2. The
2
-1
-1
probe R1 displayed the absorption maxima at ~340 nm (ε=8200 M cm ) (Fig.1a) due to the
strong π−π* and n−π* transitions of aromatic moieties and amine functional groups. The
emission spectra showed weak fluorescence at 536 nm, due to polar nature of amine. After
initial photophysical studies, next we examined the absorption and emission properties of
+
+
+
2+
2+
2+
2+
2+
2+
probe R1 towards toxic metal ions such as, Na , K , Ag , Zn , Co , Ni , Ca , Ba , Cu ,
2
+
2+
2+
2+
2+
2+
2+
3+
3+
3+
3+
3+
Fe , Mg , Mn , Hg , Cd , Pb , Fe , Eu , Cr , Al , Nb and Gd in aqueous medium
H
2
O/DMSO (99.0:1.0, v/v) buffered with HEPES, pH 7.2. However, the probe R1 displayed
3
+
slight blue shifts in absorption maxima due to its strong binding affinity towards Fe ions
(
Fig. 1a). Upon the addition of relevant di and trivalent cations, there was no significant
3
+
changes were noticed in the absorption profile of R1. The serial addition of Fe ions resulted
in a steady blue-shift in the absorption maximum at ~340 nm, and upon addition of 10
3
+
equivalents of Fe , the absorption maximum appeared at ~320 nm (Fig. 2a).

Fig. 1. Metal ion-dependent absorption (a) and fluorescence (b) profiles of probe R1 in
H
2
O/DMSO (99.0:1.0, v/v) buffered with HEPES, pH 7.2 (λex was 340 nm).
In order, we investigated the emission properties probe R1 in presence of toxic metal
ions upon excited at 340 nm in the similar solvent system. However, a remarkable
3
+
fluorescence enhancement was observed upon the addition of Fe at 536 nm with a quantum
yield (Φ=0.02(R1) and no significant changes were observed in presence of relative divalent
and trivalent cations (Fig.1b). As expected, the fluorescence intensity of probe R1 was
3
+
3+
remarkably enhanced in presence of Fe was due to the unpaired electron in Fe ions, which
favoured for photoinduced electron transfer (PET) effect. Moreover, the Job plots and HRMS
3
+
results revealed the formation of a 2:1 (R1-Fe ) complex (Fig. S6 and S7). Similarly, in the
mass spectra, the peak arises at m/z 889.3440 (calculated 889.3254), indicates the formation
3
+
of the R2-Fe complex. These results indicate that probe R1 was highly selective towards
+
3
Fe .

3
+
Fig. 2. (a) The absorbance spectrum of probe R1 (10 µM) upon addition of Fe metal ions
(
0−10 μL) (b) The fluorescence profile of probe R1 (10 µM) upon addition of various
3
+
concentrations (0−15 μL) of Fe metal ion: probe R1 in H O/DMSO (99.0:1.0, v/v) buffered
2
with HEPES, pH 7.2 (λex was 340 nm).
3
+
Consequently, the suitability of probe R1 for fluorometric detection of Fe was
3
+
evaluated. While adding various concentrations of Fe to the probe R1, the enhancement of
emission intensity was observed as shown in Fig. 2b. The plot of emission intensity versus
3
+
the concentration of Fe cation showed a linear relationship as shown in Fig. S8. The
3
+
-5
detection limit of Fe ion was calculated to be 5.386 × 10 M, (detection limit, DL = 3σ/S,
where σ is the standard deviation of the blank solution, and S is the slope of the calibration
curve). Curiously, the calculated association constant based on Benesi-Hildebrand method
-8
-1
3+
were 6.516 × 10 M for R1-Fe complex (Fig. S9). Few groups only reported the detection
3
+
of transition metal cations (Fe and other di or trivalent metal ions) by using a naphthalimide
fluorophore. Chereddy et al., synthesized a naphthaline substituted rhodamine derivative for
3
+
3+
3+
detection of more then one trivalent cations (Al ,Fe and Cr ) through FRET “off-on“
with low detection limit and they provided live cell imaging application. Based on the
detection limit and binding constant, the efficiency of probe R1 was compared with the other
reported values as depicted in Table 1.

S.No
Probe
Solvent
system
Detection
limit
Binding
constant
Ion Ref.
-
8
5
-1
-1
3+
1
Naphthalimide
probe
Tris HCl-
3.0 10 M
1.04 10 M
Fe
31
5
CH CN
3
-8
4
3+
2
Rhodamine-based
sensors
DMSO/H
(2:8)
2
O
6.6 10 M
6.74 10 M
Fe
buffered
−
7
4
-1
3+
3+
3
4
Naphthalimide
based probe
H
2
O:CH
9:1, v/v
3
CN 3.5 10 M
3.8 10 M
Fe
32
33
-6
Polyphenyl
derivative
CH
3
OH:H
2
O
4.0 10 M
------
Fe
10:1, v/v
4
-1
3+
5
Imidazole-based
chemosensor
DMSO /bis-
tris buffer
solution
0.27 μM
2.8 10 M
Fe
34
Fe
3
O
4
------
3
+
−
1
6
7
Nanoprobe
Fe
35
36
nanoparticl-
es
3 nmol L
-7
8
-2
3+
Cationic
poly
Water
9.2 10 M
1.32 10 M
Fe
(
perylene diimide)
derivative
−
7
3+
3+
8
9
1,5-
Aqueous
solutions
7.3 10 M
431.22
Fe
37
dihydroxyanthraqui
none
4
-1
4-hydrazine-1,8
naphthalimide
THF:H
2
O
2 μM
1.36 10 M
Fe
38
39
3
+
1
0
0.38 nM
-----
Fe
Chemodosimetric
probe
THF-H
(8:2)
2
O
-
8
3+
3+
1
1
1
2
Spirobenzopyran-
quinoline based
CH
CH
3
3
CN-H
2
O
3.2 10 M
-------
Fe
40
41
4
-1
Rhodamine based
OH/H
2
O
0.29 μM
6.70 10 M
Fe

2
-1
-1
3+
3+
1
1
3
4
Rhodamine–
naphthalic
anhydride based
CH
3
OH/H
2
O
2.90 μM
1.20 10 M
Fe
4
2
-
5
-8
This paper work
H
2
O/DMSO
9:1 v/v
5.39 10
M
6.52 10 M
Fe
-
9
3
+
Table. 1. Comparison of the potentials of probe R1 with other reported Fe
specific/selective molecular probes
Meanwhile, we examined the pH titrations to determine the fluorescence behaviour of
+
3
probe R1 upon the addition of Fe ions. Initially, the probe R1 found weakly fluorescent
+
3
nature within the wide range of pH (pH 3.0-10.0) (Fig. S10). Eventually, the addition of Fe
ions gave a remarkable fluorescence enhancement in the profile of R1 within a wide range
+
3
pH (pH 3.0-10.0) medium. However, the fluorescence enhancement while adding Fe ions
was mainly due to the formation of the deprotonated product as well as favoured for
photoinduced electron transfer (PET) from the donor (amine moiety) to the acceptor
(
naphthalic fluorophore) unit. These results strongly supported and recommended that the
3
+
developed probe R1 was highly selective and sensitive towards Fe under physiological pH.
In order, the selectivity experiment was examined in presence of excess amount of
+
+
+
2+
2+
2+
2+
2+
2+
2+
2+
relevant competitive metal ions (Na , K , Ag , Zn , Co , Ni , Ca , Ba , Cu , Fe , Mg ,
2
+
2+
2+
2+
2+
3+
3+
3+
3+
3+
Mn , Hg , Cd , Pb , Fe , Eu , Cr , Al , Nb and Gd ). Initially, upon the addition of
an excess amount of relevant competitive metal ions, a weak fluorescence enhancement was
+
3
observed in the profile of R1. However, upon addition of Fe ions, a remarkable
fluorescence enhancement was observed (Fig. S11). These results indicate that the probe R1
+
3
was highly selective towards Fe in presence other relevant competitive metal ions.
3
+
Fluorescence Imaging of Fe ion in Pollen grains
The changes were observed in the afore-displayed favourable properties of the probe
3
+
(
R1) encouraged us to examine its potential utility for the detection of Fe in a biological
environment. Therefore, with the great interest, we investigated whether if the present probe
3
+
was capable for fluorescence imaging of Fe in pollen grains. Initially, pollen grains were
incubated with a probe solution (10µM), as a control in Dulbecco’s modified Eagle medium

(
(
pH 7.2) for 30 min at 37 °C. The cells exhibited almost no fluorescence in the green channel
Fig. 3a). These results indicate that the probe system was not interacting with biological
3
+
species. However, upon treating with Fe (10µM), an intense green fluorescence was
observed in the green channel after 30 min incubation (Fig. 3b). These results indicate that
the probe (R1) were highly cell-membrane permeable and more feasible to be used for the
+
3
fluorescence imaging of Fe in a living cell.
3
+
Fig. 3. Fluorescence microscopic imaging of exogenous Fe in living Pollen grains cells
using a probe (R1). a) Pollen grains cells were incubated with a probe (R1) (10 µM) for 30
min. b) Pollen grains cells were incubated with a probe (R1) (10 µM) for 30 min and then
3
+
treated with Fe (300 mM) for 30 min.
Conclusions
Here in, the synthesis of fluorescent turn-on green emissive probe (R1) and its
3
+
photophysical properties for toxic Fe ion detection at a nanomolar level in the pure aqueous
3
+
system and pollen grains were reported. Upon the addition of Fe ion to the probe system in
the pure aqueous buffer, a remarkable fluorescence enhancement was noticed. Moreover,
3
+
probe (R1) shows high selectivity towards Fe ion over other relevant divalent and trivalent
+
3
cations. The reason behind the selectivity of probe R1 towards Fe ion was examined by
using HRMS spectroscopy. The probe R1 was highly cell permeable and utilized to image
3
+
Fe ion in pollen grains.

Acknowledgements
G. R. thank the Council of Scientific and Industrial Research (CSIR), Government of
India, for Senior Research Fellowship [(09/895(0006)/2014-EMR-1]. RK thanks to the
MHRD, Govt. of India for providing Senior Research Fellowship.
Supplementary material
General experimental methods, characterization data, graphs and formulae related to
this manuscript were included.
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Highlights:
+
3




Naphthalene dianhydride based novel probe R1 useful for the detection of Fe
The performance of probe R1 over the other di/trivalent ions
The PET mechanism on fluorescent turn-on cell permeable probe
The efficacy of R1 towards live-cells