A new pyrene based highly sensitive fluorescence probe for copper(ii) and fluoride with living cell application

Article abstract of DOI:10.1039/c4ob00067f

A new pyrene based fluorescence probe has been synthesized for fluorogenic detection of Cu²⁺ in acetonitrile-aqueous media (7:3 CH ₃CN-HEPES buffer, v/v, at pH 7.5) with bioimaging in both prokaryotic (Candida albicans cells) and eukaryotic (Tecoma stans pollen cells) living cells. The anion recognition properties of the sensor have also been studied in acetonitrile by fluorescence methods which show remarkable sensitivity toward fluoride over other anions examined.

Full text of DOI:10.1039/c4ob00067f

Volume 12 Number 19 21 May 2014 Pages 2981–3138
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
ISSN 1477-0520
PAPER
Shyamaprosad Goswami et al.
A new pyrene based highly sensitive fluorescence probe for copper(II) and
fluoride with living cell application

Organic &
Biomolecular Chemistry
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PAPER
A new pyrene based highly sensitive fluorescence
probe for copper(II) and fluoride with living cell
application†
Cite this: Org. Biomol. Chem., 2014,
12, 3037
Shyamaprosad Goswami,*a Shampa Chakraborty,a Sima Paul,a Sandipan Halder,b
Sukanya Panjac and Subhra Kanti Mukhopadhyayc
A new pyrene based fluorescence probe has been synthesized for fluorogenic detection of Cu2+ in aceto-
nitrile–aqueous media (7 : 3 CH3CN–HEPES buffer, v/v, at pH 7.5) with bioimaging in both prokaryotic
(Candida albicans cells) and eukaryotic (Tecoma stans pollen cells) living cells. The anion recognition pro-
perties of the sensor have also been studied in acetonitrile by fluorescence methods which show remark-
able sensitivity toward fluoride over other anions examined.
Received 9th January 2014,
Accepted 20th February 2014
DOI: 10.1039/c4ob00067f
www.rsc.org/obc
catalyze the formation of reactive oxygen species (ROS) that
can damage lipids, nucleic acids, and proteins. Research has
Introduction
Recognition of cations and anions is an expanding area of connected the cellular toxicity of copper ions with serious
research which has attracted growing interest in the field of diseases including Alzheimer’s disease,9 Indian childhood
supramolecular chemistry owing to its significant role in a cirrhosis (ICC),10 prion disease,11 and Menkes and Wilson
wide variety of environmental, clinical, chemical, and biologi- diseases.12,13 Due to its extensive applications, the copper ion
cal applications. Hence, considerable attention has been paid is also a significant metal pollutant. The limit on copper in
to the design of artificial synthetic fluorescent receptors that drinking water set by the US Environmental Protection Agency
can selectively recognize and sense the anionic species.1 (EPA) is 1.3 ppm (∼20 μM). Numerous methods for the detec-
Various structural motifs including amide,2 pyrrole,3 urea4 and tion of copper ions in a sample have been proposed, including
thiourea5 along with the positively charged imidazolium,6 gua- atomic absorption spectrometry,14 inductively coupled plasma
nidinium,7 pyridinium,8 etc. have been well studied for anion mass spectroscopy (ICPMS),15 inductively coupled plasma
recognition. The design and synthesis of sensors capable of atomic emission spectrometry (ICP-AES),16 and voltammetry.17
binding and sensing fluoride selectively have also drawn con- Most of these methods cannot be used for assays because they
siderable attention in supramolecular chemistry,8b because the entail the destruction of the sample. Consequently, more
fluoride anion plays a very important role in clinical treat- attention has been focused on the development of fluorescent
ments for osteoporosis and fluoride toxicity.8c Recently, con- chemosensors for the detection of Cu2+ ions.18–24 In this
siderable effort has been devoted to the development of regard, pyrene based frameworks represent an ideal template
artificial fluoride sensors through visible, optical and electro- for the construction of new fluorescence probes for the
chemical responses.8d–f Also, copper plays an important role in metal ions owing to their excellent photophysical properties,
various biological processes. Many proteins contain copper such as emission wavelengths elongated to the visible
ions as part of a catalytic center. Free copper ions in a live cell region, high fluorescence quantum yield and large absorption
coefficient. Taking all these properties into account, herein we
report a new pyrene based probe (BMPA) which can recognize
aDepartment of Chemistry, Bengal Engineering and Science University, Shibpur,
F− and Cu2+ ions through chelation-enhanced fluorescence
Howrah 711103, West Bengal, India. E-mail: spgoswamical@yahoo.com;
(CHEF).
Fax: +91-3326682916
bDepartment of Chemistry, Indian Institute of Technology, Kanpur 208016, India
The fluorescence sensor was synthesised by condensing
acetyl acetone with pyrene aldehyde to afford a new dehydrated
conjugated pyrenyl ene-dione (1). The final receptor BMPA
was then synthesised from the reaction of compound 1 with
2-aminophenol (Scheme 1) which was refluxed with
CuCl2·2H2O in methanol to yield the target complex.
cDepartment of Microbiology, The University of Burdwan, Golapbag, Burdwan-
713104, India
†Electronic supplementary information (ESI) available: Detailed characterization
of the compound sensor BMPA along with the intermediates, additional spectro-
scopic details, spectra and crystallographic analysis. See DOI: 10.1039/
c4ob00067f
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Scheme 1 Synthetic route for preparation of the receptor (BMPA). (a) L-Proline, DMSO, room temperature, overnight. (b) Ethanol, reflux, 2 h. (c)
Ethanol, reflux, 12 h.
Results and discussion
UV-vis and fluorescence study for copper ions
The objective in designing our probe BMPA is to achieve an
efficient emission signal and a change in the photophysical
properties upon interaction with anions/metal ions. The
sensor in the aprotic solvent, only acetonitrile, exhibits weaker
fluorescence than the mixed solvent CH3CN–HEPES buffer
(7 : 3, v/v, at pH 7.5), due to the hydrogen bonding of the
solvent to the N and O lone electron pairs. This weakens the
intramolecular radiationless transition from the nπ* state and
makes the emission maxima (λem) undergo a gradual red shift
with increasing protonation by the solvent. Therefore, the
receptor appears to be a promising candidate for enhancing
Fig. 1 The emission intensity change (λex = 330 nm) after the stepwise
the fluorescence emission upon the binding of suitable metal
ions if their radiationless channel could be well blocked. The
detection of metal ions using fluorescent sensors based on
electron donors/acceptors is usually affected by the presence of
protons. The photophysical properties of BMPA are investi-
gated by monitoring the fluorescence behaviour upon the
addition of several metal ions such as Na+, K+, Ba2+, Ga3+,
Mg2+, Ca2+, Mn2+, Fe2+, Ni2+, Co2+, Zn2+, Al3+, Ag+, Cd2+, Hg2+,
and alternate 1.0 equivalent addition of Cu2+ (c = 2 × 10−4 M) to the
BMPA solution of concentration c = 1 × 10−5 M in HEPES buffer solu-
tion in CH3CN–HEPES-buffer (7 : 3, v/v, at pH 7.5). Visual color change
of BMPA with the addition of 2 equivalents of CuCl2·2H2O under UV
light (inset).
observed changes in the fluorescence emission spectra with
Cu2+ are shown in Fig. 1. It seems that the fluorescence
enhancement is derived from the more widespread formation
of the π-conjugation system which occurs upon metal binding,
and the fluorescence is sensitive to specific metal ions. The
Pb2+, In3+, Cr3+, Fe3+ and Cu2+, and the anions investigated are
−
AcO−, Cl−, Br−, I−, F−, BzO−, SH−, H2PO4−, PO43−, S2−, N3
,
4−
P2O7 and ADP in CH3CN (at pH 7.5) as their salts. The
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Fig. 2 The change in the emission intensity of BMPA after the addition
of 2.0 equivalents of each of the investigated guest cations in CH3CN–
HEPES-buffer (7 : 3, v/v, at pH 7.5) at 414 nm and
fluorescence intensity vs. [Cu2+].
a plot of the
Fig. 3 The emission intensity change (λex = 330 nm) after the stepwise
and alternate 1.0 equivalent addition of F− (c = 2 × 10−4 M) to the BMPA
solution of concentration c = 1 × 10−5 M in HEPES buffer solution at
pH 7.5 in CH3CN. Visual color change of BMPA with the addition of
2 equivalents of tetrabutyl ammonium fluoride under UV light (inset).
selectivity for 2 equivalents of Cu2+ with 10−5 M of BMPA over
other metal ions is plotted as a bar graph in Fig. 2. In addition,
the Job’s plot (Fig. S1, ESI†), which is based on the changes in
the emission at 414 nm, confirmed the formation of a 1 : 1
complex of BMPA with Cu2+. ESI LC-MS spectral analysis also
indicates the formation of a mononuclear complex of BMPA with
Cu2+. The positive ion mass spectrum of BMPA upon the
addition of 2 equivalents of Cu2+ exhibited the most intense peak
at m/z = 557, which corresponds to the ion [BMPA + Cu2+] (ESI†).
From the fluorescence titration data it was revealed that
a minimum of 1.21 µM of copper can be detected by using
10 µM of the receptor using the equation DL = K × Sb1/S,
where K = 3, Sb1 is the standard deviation of the blank solu-
tion and S is the slope of the calibration curve (ESI†).
UV-vis and fluorescence study for fluoride ions
With the addition of F−, in the emission spectra, there is an
enhancement in fluorescence with an emission band shift from
419 nm to 448 nm (29 nm red shift, excitation at 330 nm). With
the exception of F−, there is a negligible change in fluorescence
upon the addition of other anions. The observed changes in the
fluorescence emission spectra for BMPA with the addition of F−
are shown in Fig. 3 and the changes caused by the addition of
other ions are shown as a bar graph in Fig. 4.
Fig. 4 The change in the emission intensity of BMPA after the addition
of 2.0 equivalents of each of the investigated guest anions in CH3CN
(pH = 7.5) at 448 nm and a plot of the fluorescence intensity vs. F−.
anions other than F− do not interfere in the sensing properties
(Fig. 5). Furthermore, to examine the selectivity of the sensor
in a complex background of potentially competing species, the
fluorescence enhancement of BMPA with Cu2+ was investigated
in the presence of other metal ions and the fluorescence
enhancement of BMPA with F− was investigated in the pres-
ence of other common anions. The experiment is performed
by adding the species under investigation, i.e. Cu2+ or F−
(2.0 equivalents), to the sensor in the presence of commonly
employed interfering species, i.e. metal ions or anions, respect-
ively (8.0 equivalents). With the exception of Cd2+ and Co2+ no
other competing metal ions interfered in the detection of Cu2+
by BMPA in CH3CN–H2O (7 : 3, v/v, at pH 7.5) (Fig. 6). The fluo-
rescence enhancement by Cu(II) as well as fluoride binding
Study for complexation and binding mode for copper and
fluoride ions
In addition, the Job’s plot (Fig. S2†), which is based on the
changes in the emission at 448 nm, confirmed the formation
of a 1 : 1 complex of F− with BMPA, and the detection limit of
2.91 μM (ESI†) suggests that the receptor is very sensitive
towards F−. However, when titrations of other anions such as
H2PO4−, PO43−, ADP, ATP, AcO−, Cl−, Br−, I−, F−, BzO−, SH−, S2−
−
and N3 were performed under similar experimental con-
ditions, no significant changes were observed in the emission
spectra. The selectivity for 2 equivalents of F− with 10−5 M of
BMPA was plotted as a bar graph in Fig. 4. Interestingly,
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and protons of the benzene ring of the amino phenol group
are not much affected; the
δ value is shifted to just
∼0.017 ppm upfield with the addition of 1 equivalent of metal
ions. In contrast, the amine protons are most affected in the
complexation process of BMPA with metal ions. From the pres-
ence of this type of change in the NMR spectra, we can con-
clude that a structural change must have occurred during the
complexation of BMPA with Cu2+, and probably because of
this, disappearance of the phenolic protons of amino phenol
rings occurs due to the influence of the metal ions (Fig. 7).
The protons of the aromatic ring are shifted to a lower mag-
netic field due to the reduction of electron density upon
coordination to the metal ion and are broadened upon com-
plexation. Thus the NMR data also demonstrate the adduct
formation between Cu2+ and BMPA which results in these
chemical shift changes. The spectra of BMPA before and after
treatment with 1 equivalent of F− are shown in Fig. 8.
The −OH protons of the amino phenol moiety at δ 9.763
and δ 9.16 disappear on addition of 1 equivalent of F− which
1
is the same as what happens in the case of Cu2+. The H NMR
Fig. 5 The anion sensitivity profile for BMPA: the change in the emis-
sion intensity of BMPA + 8.0 equivalents of the investigated interfering
anions + 2.0 equivalents of F−.
is much cleaner after addition of 1 equivalent of F−, which
means the complexation process has happened (ESI†). And an
abrupt upfield shift occurs (0.89 ppm) to the phenolic protons
(Ha) of the amino phenol ring upon the association of BMPA
with F− and an upfield shift occurs to the pyrene ring protons
(0.26 ppm) with the addition of 1 equivalent of F−. In the case
of complexation with Cu2+ the same upfield shift is less when
compared to the complexation of F− with BMPA.
In order to investigate the practical application of the
sensor (BMPA), we performed a biological study to test the
ability of the receptor to image copper in living cells. As shown
in Fig. 9, we used here two types of cells for the bioimaging
application of BMPA. Candida albicans cells and Tecoma stans
pollen grains were treated with the receptor (5 mM) for
45 minutes at ambient temperature which showed a weak
green color emission from the intracellular area and then the
cells were washed and treated with 5 mM BMPA solution and
the green fluorescence changed to blue in both cases. These
results suggest that the probe BMPA is cell membrane per-
meable and could be used as a sensor to detect Cu2+ in both
prokaryotic and eukaryotic type living cells and it is also con-
cluded that BMPA would provide detection of intracellular
copper present in a biological system (Fig. 9) incubated with
copper perchlorate salt (1 mg mL−1) for 45 minutes.
Fig. 6 The metal ion sensitivity profile for BMPA: the change in the
emission intensity of BMPA + 8.0 equivalents of the investigated inter-
fering metals + 2.0 equivalents of Cu2+
.
This type of selective displacement behavior of the receptor
BMPA mimics the OR logic gate by combining two different
may be due to several reasons. One probable reason is that the
energy gap between the ground state and the excited state of wavelengths, i.e. at λmax = 448 nm or λmax = 414 nm. The truth
the metal bound species gets reduced by possible metal– table and the circuit for the OR logic gate are shown in Fig. 10.
ligand charge transfer (ICT) and chelation and another reason
Initially, when no metal ions are added, i.e. input 1 and input
may be other forms of the energy decay process which is not 2 are zero, the output is nothing. When we add Cu2+, i.e. input
prominent.
The binding mode of BMPA with Cu2+ and F− was examined
1 is ‘1’ and input 2 is ‘0’, the fluorescence is on at λmax = 414,
i.e. the output is ‘1’. When we add F−, i.e. input 1 is ‘0’ and
by 1H NMR in DMSO-d6. Spectra of BMPA before and after input 2 is ‘1’, the fluorescence is on at λmax = 448, i.e.
treatment with 0.5 and 1 equivalent of Cu2+ are shown in the output is ‘1’. When we add both Cu2+ and F−, i.e. input 1
Fig. 7. On complexation of BMPA with Cu2+, the −OH protons
is ‘1’ and input 2 is ‘1’, the fluorescence is on at λmax = 448,
of the amino phenol moiety at δ 9.763 and δ 9.16 disappear i.e. output is ‘1’.
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Fig. 7 Partial 1H NMR spectra (400 MHz) of BMPA in DMSO-d6 at 25 °C and the corresponding changes after the gradual addition of different
equivalents of copper chloride from (a) BMPA, (b) BMPA + 0.5 equiv. Cu2+ and (c) BMPA + 1.0 equiv. Cu2+
.
completion of the reaction. It was worked up with water and
ethyl acetate. Column chromatography was done to afford
the pure yellow colored compound. Yield 67%. Color yellow.
Experimental section
General
The chemicals and solvents were purchased from Sigma- Mp >280 °C.
Aldrich Chemicals Private Limited and were used without
1H NMR (DMSO-d6, 400 MHz). δ (ppm): 8.70 (s, 1H), 8.31
further purification. 1H-NMR and 13C-NMR spectra were (m, 5H), 8.8.21 (m, 3H), 8.12 (d, 2H, J = 8.24), 2.56 (s, 3H), 2.07
recorded on Bruker 400 MHz instruments respectively. For (s, 3H).
NMR spectra, DMSO-d6 was used as a solvent with TMS as an
HRMS: M+ calculated for C22H16O2 is 312.12; found: 313.12
internal standard. Chemical shifts are expressed in δ units and (M + H)+.
1H–1H coupling constants in Hz. UV-vis titration experiments
Synthesis of ligand (BMPA). To the stirred hot solution of
were performed on a JASCO UV-V530 spectrophotometer and this yellow colored compound 1 (50 mg, 0.151 mmol) in
fluorescence experiments were done using a PTI fluorescence methanol, 2-amino phenol (33 mg, 0.302 mmol) was added
spectrophotometer with a fluorescence cell of 10 mm path and stirred for one hour. TLC showed the completion of the
length. IR spectra were recorded on a JASCO FT/IR-460 plus reaction and the product was filtered through a sintered
spectrometer using KBr discs.
funnel to afford a brick red colored compound followed by
purification through column chromatography to afford a brick
red colored compound (C34H26N2O2 from HRMS): yield 25%,
mp >280 °C.
Methods for the preparation of the receptor
Synthesis of the aldol dehydration product (1). To a stirred
solution of acetyl acetone (260 mg, 5.21 mmol) in 3 mL of
DMSO, L-proline (75 mg, 1.3 mmol) was added and stirred
for one hour. Pyrene aldehyde (500 mg, 4.34 mmol) was added
to the reaction mixture. The next day, TLC showed the
1H NMR (DMSO-d6, 400 MHz). δ (ppm): 9.76 (s, 1H), 9.43
(d, 1H, J = 9.36), 9.42 (d, 1H, J = 9.36), 8.91 (d, 3H, J = 15.5),
8.31 (d, 1H, J = 6.64), 8.26 (m, 13H), 8.18 (t, 6H, J = 10.24), 8.03
(d, 2H, J = 4.48), 7.50 (t, 1H, J = 10.53), 7.48 (t, 1H, J = 11.52),
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Fig. 8 Partial 1H NMR spectra (400 MHz) of BMPA in DMSO-d6 at 25 °C and the corresponding changes after the gradual addition of different
equivalents of tetrabutyl ammonium fluoride from (a) BMPA, (b) BMPA + 0.5 equiv. F− and (c) BMPA + 1.0 equiv. F−.
Fig. 9 Fluorescence microscopic photographs of (left) Candida albicans cells and (right) Tecoma stans pollen grains, (a) cells without any treatment,
(b) cells treated with copper perchlorate, (c) cells treated with 5 mM BMPA, (d) cells treated with 5 mM copper perchlorate, washed and then treated
with 5 mM BMPA. The emission intensity change for copper at 414 nm (λex = 330 nm).
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receptor solution is 2 mL. Solutions of the sensor of various
concentrations and increasing concentrations of cations and
anions were prepared separately. The spectra of these solutions
were recorded by means of UV-vis and fluorescence methods.
Conclusions
Thus, in conclusion, we have designed and synthesized a
pyrene based fluorophore that easily recognizes Cu2+ and F−
over other interfering cations and anions in CH3CN–HEPES-
buffer (7 : 3, v/v, pH = 7.5) and in acetonitrile solution respecti-
vely by the fluorescence change which makes it an efficient
sensor for possible analytical and biological use in the detec-
tion and determination of Cu2+ and F−.
Fig. 10 (a) The truth table of the OR gate and (b) the logic scheme of
the following gate, with the change in the emission spectra and the
output intensities (bar diagram) of BMPA upon reversing the chemical
inputs of Cu2+ and F−.
6.98 (d, 1H, J = 7.60), 6.97(t, 2H, J = 7.68), 1.28 (s, 3H), 1.25
(s, 3H). HRMS: M+ calculated for C34H26N2O2 is 494.2; found:
518.21 (M + 23 + H)+.
Acknowledgements
Authors thank the DST (SR/S1/0C-58/2010) (Govt. of India) and
UGC (for a fellowship to S. P.) for financial support and
SC thanks TCG Life Sciences Ltd (Chembiotek), Kolkata for
spectral help and Mr M. Adak for assistance.
13C NMR (DMSO-d6, 500 MHz). δ (ppm): 193.32, 155.75,
152.85, 135.93, 134.09, 131.69, 130.85, 129.61, 129.30, 128.50,
127.83, 127.57, 127.19, 126.93, 126.56, 125.11, 124.92, 123.42,
122.74, 122.26, 120.61, 116.35, 115.48. Anal calculated:
C 82.57; H 5.30; N 5.66.
Notes and references
Syntheses of the complexes (BMPA-Cu complex). To a hot
1.0 mL methanolic solution containing 20 mg (0.40 mmol) of
the ligand, 1.0 mL of a methanolic solution containing 70 mg
(0.40 mmol) of CuCl2·2H2O was added. A deep blue turbidity
appears immediately. After stirring for 1.0 h at 50 °C the blue
complexes were filtered, collected and then washed several
times with cold methanol. The complex was dried in a desicca-
tor over anhydrous CaCl2 under vacuum. The dried ligand
and complexes were subjected to spectroscopic analyses.
The complexes are air-stable, non-hygroscopic, and soluble
in H2O ethanol, methanol, DMSO, and DMF. CuBMPA
(C34H26N2O2Cu): yield 80%. Colour blue. Mp >280 °C.
MS (FD). M+ calculated for C34H26N2O2Cu is 557.74; found:
558.10 (BMPA + Cu2+ + H+).
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7.5) for cation titration. The solution of the guest cation was
prepared (2 × 10−4 mL−1) in CH3CN–H2O (7 : 3, v/v, at pH 7.5)
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