A new selective turn-on fluorogenic dipodal-cobalt(II) ensemble probe for nitrite ion detection and live cell imaging

Article abstract of DOI:10.1016/j.tetlet.2012.12.083

A new dipodal-triazole-Co²⁺-ensemble with a ligand containing quinoline moiety was designed and synthesized as a highly sensitive fluorescent sensor for nitrite in aqueous media. Based on in situ-formed Co²⁺-DTQ (Co²⁺ chelated dipodal of triazole-quinoline) ensembles having the specific binding affinity for nitrite anions over other anions, this sensory system allows rapid recognition and quantitative detection of nitrite in neutral aqueous media in an 'off-on' fashion. To confirm the suitability of DTQ for biological applications, we also employed it for the fluorescence detection of the changes of intracellular NO2- in three different species of cultured cells in the presence of nitrite ions.

Full text of DOI:10.1016/j.tetlet.2012.12.083

Accepted Manuscript
A new selective turn-on fluorogenic dipodal-cobalt(II) ensemble probe for ni‐
trite ion detection and live cell imaging
Ajit Kumar Mahapatra, Giridhari Hazra, SubhraKantiMukhopadhyay, Anindita
Roy Mukhopadhyay
PII:
S0040-4039(12)02236-8
DOI:
http://dx.doi.org/10.1016/j.tetlet.2012.12.083
TETL 42331
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
7 August 2012
17 December 2012
19 December 2012
Please cite this article as: Mahapatra, A.K., Hazra, G., Mukhopadhyay, S.K., Mukhopadhyay, A.R., A new selective
turn-on fluorogenic dipodal-cobalt(II) ensemble probe for nitrite ion detection and live cell imaging, Tetrahedron
Letters (2012), doi: http://dx.doi.org/10.1016/j.tetlet.2012.12.083
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A new selective turn-on fluorogenic dipodal-cobalt(II) ensemble
probe for nitrite ion detection and live cell imaging
*
a
a
Ajit Kumar Mahapatra , Giridhari Hazra , Subhra Kanti Mukhopadhyay , Anindita Roy
Mukhopadhyay
b
c
a
Department of Chemistry, Bengal Engineering and Science University, Shibpur, Howrah – 711103, India.
b
Department of Microbiology, The University of Burdwan, Burdwan, West Bengal, India.
c
Department of Microbiology, MUC Womens College, West Bengal, India.
*
Corresponding author: Tel.: +91 33 2668 4561; fax: +91 33 26684564;
e-mail: mahapatra574@gmail.com
2
A new dipodal-triazole-Co -ensemble with a ligand containing quinoline moiety was
+
designed and synthesized as a highly sensitive fluorescent sensor for nitrite in aqueous media.
2
+
2+
Based on in situ-formed Co –DTQ (Co chelated dipodal of triazole-quinoline) ensembles
having the specific binding affinity for nitrite anions over other anions, this sensory system
allows rapid recognition and quantitative detection of nitrite in neutral aqueous media in an
‘
off-on’ fashion. To confirm the suitability of DTQ for biological applications, we also
-
employed it for the fluorescence detection of the changes of intracellular NO
different species of cultured cells in the presence of nitrite ions.
2
in three
The nitrite ion is an important intermediate in the biological nitrogen cycle and is widely
1
present in the environment . Nitrites are usually used as a food preservative agent of meat
2
3
and fish and which are also essential nutrients for the growth of plants. On the other hand,
4
nitrite can interact with amines and proteins for producing carcinogenic N-nitrosamines,
which enhances the possibility of gastric cancer and deformities. Water is another major
accessible source through which the safety of human health is threatened by dangerous levels
5
of nitrite (or nitrate). A number of medical issues are believed to be associated with nitrite
intake. One typical example is the infant methemoglobinemia, also known as “blue baby
6
syndrome” due to passage of nitrite into the blood stream, which results in the irreversible
conversion of hemogloblin to metahemogloblin with oxygen uptake and transportation
compromised. Therefore, it is of significant importance to detect nitrite ion in water, food and
agricultural products. The World Health Organization (WHO) recommends a maximum limit
7
of nitrite ion in drinking water to be 65 µM.
1

Nitrite is known to be chemically unstable in the environment. Owing to this reactivity, a
rapid detection procedure is preferable, especially for on-site analysis. Many analytical
8
methods based on different principles, including UV-vis absorbance. Electrochemistry
,9
1
0,11
12
fluorescence, and chemiluminescence
(
(
such as conductimetry and amperometry),
1
3-15
CL),
have been developed. UV-vis absorbance determination is usually based on
diazotization of aromatic amine using acidified nitrite. A highly colored azo chromophore is
formed using a subsequent coupling reaction. The process requires close control of coupling
1
conditions. Among all of the techniques, spectrofluorimetric protocol is widely used
6
because of its simplicity, sensitivity and low cost. A number of different fluorescent reagents
1
have been developed, such as 5-aminofluorescein, 4-hydroxycoumarin, acetaminophen,
7
18
19
2
0
21
22
23
safranine, rhodamine 110, 2,3-diaminonaphthalene (DAN), carbon dot and so on.
There are a significant number of fluorescent sensors for metal ions and small biomolecules
in aqueous media. However, bioimaging studies of fluorescent chemosensors toward anions
have been rarely reported, probably because of low solubility in aqueous media, low
2
4
selectivity, and low sensitivity. The development of fluorescent sensors for anions in
aqueous media is a challenging task owing to the strong hydration nature of anions and the
competition of water for the hydrogen bonding sites, which weakens the interactions of the
2
sensors with the target anions. One way to tackle this hurdle is by employing the
5
2
6
displacement method, in which the sensor-ligand-metal ion “ensemble” is nonfluorescent
due to metal-ion-induced fluorescence quenching. However, the addition of anions may
release the sensor ligand into the solution with revival of fluorescence.
27
Recently, a few fluorescent chemosensors for nitrite anions have been constructed, but all
of them have metal reduction without fluorophore displacement or sensing through the
nitrous acid specific diazotization of aromatic amine and subsequent coupling reaction with
suitable aromatic reagent to yield a highly colored azo dye, the intensity of which is related to
28
the original nitrite concentration. The classic Griess reaction also used for the colorimetric
29
detection of nitrite with Au NPs probe. Despite the significant development in this domain,
its ensemble version and bioimaging studies has never been explored yet for nitrite detection
due to its structural simplicity. New methods have been of keen interest in molecular
recognition research. Furthermore, the development of metal-based probes to fluorescent
2

imaging of nitrite in live cells were not satisfactory. Therefore, more nitrite ion sensors with
high sensitivity and good biocompatibility are still under investigation.
By incorporating a quinoline moiety as the fluorescence active unit, we synthesized a new
2
Co -complex fluorescence probe Co –DTQ for detection of nitrite in organo-aqueous
+
2+
2
media (Scheme 1). Such detection may be achievable when Co chelated dipodal of triazole-
+
2
+
quinoline (Co –DTQ) moiety is generated followed by demetalization to generate
fluorescence ‘turn on’. Based on these results, we designed a probe for nitrite including a
quinoline fluorophore and a dipodal phenyl platform receptor, which was attached via a
2
+
triazole bridge. The receptor provides a rigid Co binding site spacer between the quinoline
fluorophore and chelating ligand. Quinoline and its derivatives are used extensively as
fluorescence labeling reagents for their excellent photophysical properties of high
3
fluorescence quantum yield and efficient membrane permeability. The probe also provides
0
2
O bridges to minimize the distance between the Co binding site and quinoline fluorophore,
+
2
which will ensure strong fluorescence quenching in the off state of Co quinoline. At the
+
2
beginning, the fluorescence of DTQ is quenched by coordinating to Co ion, attributing to
+
2
+
2+
the paramagnetic of Co . The Co –DTQ -based nitrite probe is formed in situ by treating
2
+
DTQ with one equiv of cobalt chloride. After addition of nitrite solution, nonemissive Co –
DTQ -“ensemble” formed of a metal-nitrite adduct, releasing a fluorophore that was initially
quenched by coordination to a paramagnetic transition-metal center by electron or energy
3
transfer. Fluorophores can bind the metal center as axial ligands. Introduction of nitrite
1
-
causes their displacement with concomitant fluorescence turn on. Changes in [NO
2
] under
physiological conditions will exhibit “turn-on” type fluorogenic behavior, which can be
detected by measuring the ratio of fluorescence intensity with good sensitivity and selectivity.
It was also expected to exhibit high sensitivity for the biological imaging of nitrite ions in
living cells.
The target compound DTQ was readily synthesized in main two steps as shown in Scheme
3
2
1
. The starting triazide-compound 2 was prepared according to the literature. Reaction of 8-
hydroxyquinoline with propargylbromide in dry acetone and K CO under a nitrogen
atmosphere gave compound 3. The key intermediate, compound 2, was then treated with
2
3
3
compound 3 in the presence of a copper (I)-mediated click cycloaddition reaction in 1:1
3
THF : H
2
O solvent mixture to afford product DTQ (1) in 65% yield. All the new compounds
3

1 13
were well characterized by H NMR, C NMR, and mass spectroscopy (See supplementary
2
data Figs. S1-S5).The chelated ensemble Co –DTQ was easily prepared by addition of
+
CoCl
2
to DTQ (See supplementary data Fig. S6a).
N3
N
3
N3
O
N
N
N
N
N
N
N
N
N
N
N
(
i)
N
(ii)
N
N
2+
Co
O
O
N3
O
O
N
N
N
N3
2
3
1
2+__
DTQ
Co
DTQ
4 2 2 2
Reagent and conditions: (i) Na-ascorbate, CuSO .5H O, THF, H O, rt. 16h. (ii) CoCl
2
+
Scheme1. Synthesis of Dipodal-Triazole-Quinoline (DTQ) and ensamble of Co –DTQ.
With compound DTQ in hand, we examined its optical properties in the absence or
presence of various heavy and transition metal species. The free compound DTQ exhibited
an absorption band at around 261 nm in pH 7.4 HEPES buffer (30 mM, pH 7.4, containing
7
3
0% CH CN).
-
Figure 1. Absorption titration spectra of DTQ (c = 1.0×10 M) upon addition of Co ions
5
2+
(
0-2 equiv) in pH 7.4 HEPES buffer (30 mM, pH 7.4, containing 70% CH CN).
3
Gradual addition of cobalt perchlorate to a solution of DTQ in HEPES buffer, induces a 19
nm red shift of the absorption from 261 to 280 nm along with a shoulder peak at 295 nm
(
Fig.1). A clear isosbestic point was observed at 269 nm, which is consistent with the
4

2+
presence of only two species, free ligand, and Co –ligand ensemble. No marked changes in
3
the absorption profiles were noted upon the addition of Al , Ba , Ca , Mg , Cd , Zn ,
+
2+
2+
2+
2+
2+
2
+
2+
2+
3+
2+
2+
Pb , Hg , Mn , Fe , Ni , and Cu , respectively. These results clearly suggested that the
metal complexation of DTQ show a great preference for cobalt ion over other cations. The
5
binding constant was determined to be (1.25±0.25)×10 M using linear regression Benesi–
-1
3
Hildebrand equation analysis (See supplementary data Fig. S6b).
4
The corresponding fluorescence spectra have been measured for the DTQ’s solution (1.0
3
µM, pH 7.4, containing 70% CH CN) in the absence and presence of various metal ions. The
3
5
free ligand DTQ is fluorescent with an emission peak at around 395 nm (Φfl=0.71). From
the Figure 2a and its inset, it can be seen that its intense emission peak at 395 nm is nearly
2
quenched (9.5 fold) upon addition of 1.2 equiv of Co , concomitant with a new weak band at
+
around 325 nm and a distinct isoemissive point at 344 nm was observed. This could be
3
attributed to the photoinduced electron transfer (PET) from the quinoline fluorophore to the
6
2
chelated Co and probably due to the paramagnetic nature of the Co ion.
+
2+
37
(
a)
(b)
-6
Figure 2. (a) Fluorescence titration of DTQ (c = 1.0×10 M) in HEPES buffer (50mM, pH
.4) in the presence of different amounts of cobalt perchlorate (λex=285 nm). The inset shows
the fluorescence intensity changes at 395 nm of compound DTQ (10 µM) with the amount of
7
2
Co ions. (b) Fluorescence emission changes in DTQ (10 µM), upon addition of different
+
metal ions (1.0 equiv) in 70% CH CN-water at pH 7.4 (λex=285 nm).
3
However, addition of heavy and transition metal ions caused fluorescence quenching to a
different extent (See supplementary data Fig. S7), which is shown in Figure 2b. In particular,
5

2
the fluorescence of DTQ is almost completely quenched by Co ions. Thus, we decided to
+
2
further evaluate the Co -responsive nature of compound DTQ.
+
3
In good agreement with this finding, the Job plot also shows the formation of a 1:1
8
2
bonding mode between DTQ and Co ions (Fig. 3). The association constant of DTQ with
+
2
+
5
Co was determined to be (1.49±0.51)×10 M on the basis of the fluorescence titration
-1
2
experiments (See supplementary data Fig. S8). The 1:1 binding model of Co and DTQ can
+
2
be further confirmed by mass spectra. The ESI mass spectrum of complex DTQ–Co has a
+
2
major peak with m/z of 670.018 [Co –DTQ], which corresponds to 1:1 complex (See
+
supplementary data Fig. S9).
2
Figure 3. Fluorescence titration Job plot of the receptor DTQ with Co , where [G] = [H] =
+
-
6
1
3 2
.0×10 M in HEPES buffer (CH CN : H O = 7:3, v/v, 50mM, pH 7.4).
2
Based on the above finding results of titration experiments and mass spectrum of Co –
+
2
+
DTQ complex, the chelated complex Co –DTQ was easily prepared by addition of cobalt
chloride to DTQ in the solution of HEPES buffer (50 mM, pH 7.4, containing 70% CH CN).
We found that Co –DTQ ensemble is initially nonfluorescent (Φfl=0.05), the fluorescence
intensity increased steadily with increasing NaNO concentration (Fig. 4a) until it reached a
plateau at 1.0 equiv NaNO , which corresponded to a 9.5-fold increase in fluoresence
instensity compared to the blank [NO
3
2
+
2
2
–
2+
2
]. This indicates that complete displacement of Co
2
+
from Co –DTQ occurred with one equiv NaNO
2
addition. The fluorescence intensities at
-
3
4
95 nm have an excellent linear relationship with the concentrations of [NO
2
] anions (Fig.
2
a, inset). To test this idea, the absorption spectra of Co –DTQ ensemble were recorded by
+
2
+
gradual addition of nitrite to a solution of Co –DTQ in HEPES buffer. As shown in Figure
4
b the absorption spectra, which is essentially identical with the maximal wavelength of the
6

absorption peak of the free DTQ, indicating that addition of nitrite anions to the ensemble
resulted in the release of the free DTQ. Consistent with this observation, treatment of nitrite
caused a significant fluorescence turn-on response at 395 nm (Fig. 4a), and up to a 9.5-fold
fluorescence enhancement (Φfl=0.75) was observed.
(
a)
(b)
2
+
Figure 4. (a) Fluorescence spectra of the Co –DTQ ensemble (1 µM) in pH 7.4 HEPES
–
buffer (25 mM, pH 7.4, containing CH
3
CN : H
2
O = 7:3, v/v) in the presence of NO
2
anions
(
(
0-1 equiv). The inset shows the fluorescence intensity changes at 395 nm of the ensemble
10 µM) in the presence of increasing nitrite concentrations (0-1 equiv). (b) The absorption
2
+
spectra of the Co –DTQ ensemble (10µM) in pH 7.4 HEPES buffer (30 mM, pH 7.4,
containing CH CN : H O = 7:3, v/v) in the presence of nitrite anions.
3
2
2
+
–
To identify whether the Co –DTQ ensemble was highly sensitive to NO
wide array of possible competitive reactive oxygen (ROS) and nitrogen species (RNS) and
other analytes in organo-aqueous media (25 mM, pH 7.4, containing 70% CH CN) at up to
2
, we examined a
3
–
–
–
00-fold excess. As shown in Figure 5a, 100 equiv of F , Cl , Br , I , NO
–
–
–
–
1
3
, N
3
, SCN ,
2
–
–
, SH , PPi and 10 equiv of CN could not induce any marked fluorescence
–
HPO
4
enhancement. By sharp contrast, one equiv of nitrite elicited a large fluorescence
enhancement in the region of 395 nm. Competitive experiments were conducted with one
equiv NaNO
2
and 100 equiv various ROS and RNS. The fluorescence intensity did not vary
–
2+
+Co –DTQ (Fig. 5b). These results indicate
much in comparison with that produced by NO
2
2
+
that Co –DTQ displays high selectivity toward NO
–
2
over other ROS and RNS species.
7

(
a)
(b)
Figure 5. (a) Bars represent the fluorescence responses to various ROS and RNS species at
2
95 nm. The left hand bars represent only DTQ, middle bars Co –DTQ ensemble and right
+
3
2
+
hand bars Co –DTQ ensemble + representative anions respectively. Spectra were acquired
in HEPES buffer (25 mM, pH 7.4, containing CH CN : H O = 7:3, v/v). (b) Normalized
3
2
2
fluorescence spectra of DTQ (green), DTQ +Co (red), and Co –DTQ ensemble+NO
+
2+
–
2
(
black).
To gain insight into the sensing mechanism, we decided to study the sensing process by
1
2+
HNMR spectra in presence of nitrite ions. The product of Co –DTQ + nitrite anions was
1
isolated by a silica gel column and was then subjected to H NMR analysis. The H NMR and
1
mass spectra of the resulting product are essentially identical to that of free sensor DTQ.
NO -
2
n+
M (L)(NO )
-
__
n+
+
FL
FL M (L)
OFF"
2
"
"ON"
N
3
N3
NO -
2
N
N
N
Co2+
N
N
N
N
N
N
N
N
N
2+
Co
O
Co(NO2)2
O
O
O
N
N
N
N
2
+__
Co
DTQ
DTQ
–
2
Scheme 2. Proposed displacement mechanism for sensing of NO anions.
8

Thus, the studies of NMR, mass spectrometry, absorption spectrometry, and fluorescence
spectrometry indicate that the sensor functions by the displacement mechanism (Scheme 2).
2
+
Acid-base fluorescence titrations revealed that the fluorescence intensity of Co –DTQ
ensemble was unaffected by pH values between 6.08 and 9.41 (excitation at 285 nm). This
suggests that the probe would work well at physiological pH, although the fluorescence
2
intensity of Co –DTQ ensemble was quenched slightly at pH <6.0 (See supplementary data
+
Figs. S10).
To further demonstrate the practical biological application of the sensor DTQ, fluorescence
imaging experiments were carried out in living cells. Here we used three different sets of
living cells for imaging purpose. Candida albicans (Set 1), Bacillus subtilis (Gram-positive,
rod shaped bacteria) (Set 2) and Pollen (Set 3) cells were first incubated with 20 µM of sensor
DTQ (in the MEM (modified Eagle’s medium) culture medium containing 1:1 v/v water-
ethanol) for 30 min at 37°C in three different cover glass bottom dish, and then washed with
PBS (containing 2.0% methanol) (0.1M, pH = 7.4) three times to remove excess of DTQ.
(a)
(b)
(c)
Set 1 (Candida Cell)
Set 2 (Bacillus Cell)
(
a)
a)
(b)
(b)
(c)
(c)
(
Set 3 (Pollen Cell)
Figure 6. Comparison of three sets of cells by fluorescence microscopic experiment (a)
images of Candida albicans, Bacillus subtilis and Pollen cells + DTQ, (b) images of Candida
2
albicans, Bacillus subtilis and Pollen cells + DTQ + Co (c) images of Candida albicans,
+
2
+
–
Bacillus subtilis and Pollen cells + DTQ + Co + NO (5 µM).
2
9

Figure 6a showed that when all three types of cells were treated with sensor DTQ only,
bright fluorescence were detected. Then, the treated cells were incubated with 30 µM CoCl
2
and the fluorescence signal was monitored again, which produced very faint intracellular
fluorescence (Fig. 6b). The fluorescence intensities were further observed inside of the cells,
-
6
2
after the NaNO (c = 1.2× 10 M) treatment. The bright field transmission images of these
cells in Figure 6c is exactly the same as the fluorescence image in Figure 6a, confirming that
the imaged fluorescence is intracellular, instead of extracellular. Appearance of the blue
2
fluorescence confirmed the displacement of Co from Co –DTQ ensemble and free DTQ
+
2+
was adsorbed within the bacterial cells. The resluts suggest that sensor DTQ is cell
3
9
–
2
membrane permeable and can also be used for intracellular imaging of NO in living cells
and potentially in vivo.
2
In this contribution, we have synthesized a cobalt (II ) ensemble Co –DTQ with a triazole
+
–
N-donor ligands having a pendant quinoline moiety as a fluorophore probe for NO
2
sensing
2
and bioimaging. DTQ showed excellent selectivity for Co over competing metal ions even
+
2
+
–
2
at low concentrations. This ensemble probe Co –DTQ was NO specific and provided high
2
selectivity over other ROS and RNS species. Fluorescence imaging shows that Co –DTQ
+
–
could be used for detection of changes in NO
2
levels in living cells with low cytotoxicity.
Furthermore, the confocal fluorescence images of Candida albicans, Bacillus subtilis and
2
Pollen cells suggested that Co –DTQ was successfully applied for monitoring intracellular
+
–
NO
2
.
Acknowledgment
We thank CSIR [Project No. 01(2460)/11/EMR-II] for financial support. GH thanks CSIR,
New Delhi for fellowship. We also acknowledge DST, AICTE, UGC-SAP and MHRD for
funding instrumental facilities in our department.
Supporting Information Available:
Experimental details, spectral data, NMR spectra, mass spectra, binding studies.
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3
1
2

39. MTT {3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide} assay on
2
+
experimental cells were done in presence and absence of DTQ and Co -salt and the
result indicates that mitochondrial succinate dehydrogenase reduces yellow coloured
MTT into an insoluble dark purple coloured formazan. Since reduction of MTT can
only occur in metabolically active cells (due to presence of active dehydrogenase) the
level of activity is a measure of the viability of the cells.
1
3

Graphical Abstract
A new selective turn-on fluorogenic dipodal-cobalt(II) ensemble
probe for nitrite ion detection and live cell imaging
Ajit Kumar Mahapatra, Giridhari Hazra, Subhra Kanti Mukhopadhyay, Anindita Roy
Mukhopadhyay
N
3
N3
NO2-
N
N
N
Co2+
N
N
N
N
N
N
N
2+
Co
N
N
O
Co(NO2)
2
O
O
N
O
N
N
N
Co2+__DTQ
DTQ
1
4

Graphical Abstract
A new selective turn-on fluorogenic dipodal-cobalt(II) ensemble
probe for nitrite ion detection and live cell imaging
Ajit Kumar Mahapatra, Giridhari Hazra, Subhra Kanti Mukhopadhyay, Anindita Roy
Mukhopadhyay
N
3
N3
NO2-
N
N
N
Co2+
N
N
N
N
N
N
N
2+
N
Co
N
O
Co(NO2)
2
O
O
N
O
N
N
N
Co2+__DTQ
DTQ
1
5