Design and synthesis of dual probes for detection of metal ions by LALDI MS and fluorescence: Application in Zn(II) imaging in cells

Article abstract of DOI:10.1039/c6ra27302e

A Label-Assisted Laser Desorption/Ionization (LALDI) technique has recently been applied to the detection of metal ions through a time of flight (TOF) mass spectrometric measurement. In this paper, we report the synthesis of two terpyridine based ligands

Full text of DOI:10.1039/c6ra27302e

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Design and synthesis of dual probes for detection
of metal ions by LALDI MS and fluorescence:
application in Zn(II) imaging in cells†
Cite this: RSC Adv., 2017, 7, 7163
a
a
b
a
Arundhoti Mandal, Asim Maity, Swarnendu Bag, Prabuddha Bhattacharya,
c
a
Amit K. Das and Amit Basak*
A Label-Assisted Laser Desorption/Ionization (LALDI) technique has recently been applied to the
detection of metal ions through a time of flight (TOF) mass spectrometric measurement. In this
paper, we report the synthesis of two terpyridine based ligands L1 and L2 containing a pyrene moiety.
The presence of the latter helped the ligand metal complex to desorb from the surface and ionize
2
+
2+
upon laser irradiation and finally show up in the TOF MS. Both ligands were able to detect Zn , Ni
Received 24th November 2016
Accepted 29th December 2016
2+
2+
2+
2+
and Co ions while Cu , Fe and Mg remained LDI silent. Complexation with the ligands also
2
+
2+
caused a red fluorescence only with Zn ions that allowed Zn imaging in cells. Thus the pyrene
moiety acted as a dual probe for metal ion detection exploiting both LALDI MS and fluorescence
techniques.
DOI: 10.1039/c6ra27302e
www.rsc.org/advances
playing critical roles in living systems, even at low concen-
trations, is of utmost importance for assessing health risk and
Metal ions play a crucial role in normal cellular function. In environmental monitoring. The usual detection procedure
Introduction
1
1
0
biological systems, they can function in a number of different includes traditional techniques like UV-VIS spectroscopy,
1
1
ways. They act as cofactors for many critical enzymatic reac- uorescence spectroscopy, Liquid Chromatography-Mass
tions. Some metals operate as structural elements and Spectrometry (LC-MS), Atomic Absorption Spectroscopy
maintain charge and osmotic balance. Transition metal ions (AAS), Inductively Coupled Plasma Atomic Emission Spec-
2
3
12
4
13
1
4
such as zinc(II), function as structural elements in superoxide trometry (ICP/AES),
Inductively Coupled Plasma Mass
5
6
15
16
dismutase and in zinc nger proteins. There are also Spectrometry (ICP/MS), Neutron Activation Analysis (NAA),
1
7
a number of reports on activation of chemical reactivity of X-ray uorescence (XRF), Ion Chromatography (IC) and High
7
18
small molecules upon metal ion chelation. For example, Performance Liquid Chromatography (HPLC),
surface
1
9
acyclic enediynes with terminal ligands undergo Bergman plasmon resonance based optical sensors and microuidics
8
20
cyclization at a lower temperature for the metal complexed based sensors.
However, these methods have their
system as compared to the uncomplexed one. Interestingly, own limitations like interference from other analytes. Even
2
1
presence of metal ions in excess or less in biological systems electrospray mass spectrometry (ESI MS) may be difficult
can have adverse effects. Every essential element follows for mixture of analytes as all the constituents will show up
a dose–response curve. At very low dosages, the organism in the spectrum and make the analysis difficult. A method
struggles to survive, whereas in deciency regions, the which can detect specic metal ions in biological or envi-
organism exists with sub-optimal function. Beyond the ronmental samples will be extremely useful and the LALDI MS
concentration plateau in the optimum dosage region, higher technique coupled with uorescence can offers a viable
dosages cause toxic effects in the organism, eventually solution.
9
22
becoming lethal. Thus detection of metal ions that are
Kozmin et al. used an LALDI MS technique for new
reaction-discovery. Their strategy was based on labeling one of
the reactants with a polyaromatic chemical tag (pyrene), which
Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India. selectively underwent a photoionization/desorption process
a
E-mail: absk@chem.iitkgp.ernet.in
upon laser irradiation, without the assistance of an external
matrix, and enabled rapid mass spectrometric detection of
products originating from such labeled reactants in complex
reaction mixtures without the need of any chromatographic
separation. An example along with the principle behind their
work is shown in Fig. 1.
b
Centre for Healthcare Science and Technology, Indian Institute of Engineering Science
and Technology, Shibpur-711103, India
c
Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302,
India
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c6ra27302e
1
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Fig. 1 Kozmin's work on reaction discovery.
In recent years, our group has demonstrated the use of
Fig.
technique.
3
Schematic diagram of working principle of LALDI-MS
LALDI MS technique involving an oxine carboxylic acid–pyrene
2+
23
conjugate ligand for selective detection of metal ions like Zn .
However, although the detection was easily discernible, the
quality of the spectra was less than satisfactory. This is because
of low sensitivity and low absorption of the ligand at laser
wavelength of 337 nm as well as appearance of extra peaks
originating from the ligand. With the aim of improving the
spectra particularly at the low level of detection and removal of
extraneous peaks, we have designed the terpyridine based
ligands L1 and L2 (Fig. 2) attached with a pyrene moiety through
ester linkage or through conjugation. Terpyridine is a tridentate
ligand that binds metals at three meridional sites and it forms
complexes with transition metal ions. The ligand has been well
exploited for complexation with various metal ions. Recently,
was also expected to serve as the uorophore changing its
emission spectra upon complexation. In this paper, we describe
the synthesis of ligands L1 and L2 and report their ability to
detect metal ions by using a combination of LALDI MS and

uorescence.
Results and discussion
Both the ligands were synthesized starting from a common
0
intermediate, namely 4 -(4-bromomethylphenyl)terpyridine 2
(Fig. 4). For this, the tolyl terpyridine 1 was rst synthesized
24
Ghosh et al. have reported in an interesting paper the design
following the literature procedure reported by Zecher and
and synthesis of an alkynyl terpyridine which has been shown to
26
Kr ¨o hnke. This synthetic protocol involves condensation reac-
tion between 2-acetyl pyridine and 4-methylbenzaldehyde in
presence of ammonia and NaOH to yield intermediate 1. Allylic
bromination was performed using NBS and AIBN on 1 to obtain
2
+
2+
2+
form octahedral complexes with metal ions like Fe , Zn , Cd ,
2
+
2+
Ru and Pb . Another recent work has been reported by Patra
25
2+
et al. on terpyridine based sensor for detection of Cd .
However, to our knowledge, there is no report of any pyrene
based terpyridine ligand which has been used to serve the dual
purpose of detection of metal ions by both LALDI MS as well as
by uorescence sensing. Pyrene moiety is known for its ability to
act as an excellent LDI tag, due to its chemical inertness, ability
to form radical cations and most importantly, very high molar
extinction coefficient in the range 335–360 nm which allowed it
to absorb laser radiation and subsequent desorption-cum-
ionization. The pyrene–terpyridine conjugate should form
chelate complex with analyte metal ions and the entire complex
upon laser irradiation is expected to undergo desorption-cum-
ionization and should be detectable by LDI-MS. The working
principle is shown in the Fig. 3. In addition, the pyrene moiety
Fig.
2 Target polyaromatic ligands for LALDI based metal ion
detection.
Fig. 4 Synthetic pathway of ligands L1 and L2.
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Fig. 5 LALDI mass spectrum of ligand L1.
4 2
Fig. 7 LALDI mass spectrum of ligand L1 + Zn(ClO ) .
perchlorate salts at 1 mM in acetonitrile. The reaction mixtures
ꢀ
were incubated for 2 h at 40 C. An aliquot of 1 mL from the
reaction mixture were taken from each reaction mixture and
analyzed (TOF-MS) in positive ion mode. The captured
2
+
2+
2+
complexes of Zn , Co , Ni were LDI active with typical
27
2+
2+
isotopic distributions while Fe , Cu remained LDI silent.
The summarized results of the screening process are given
below in Table 1. The spectrum for Zn with L1 and L2 are
2
+
shown in Fig. 7 and 8 respectively.
Fig. 6 LALDI mass spectrum of ligand L2.
2+
Regarding sensitivity of detection for Zn using the present
2
+
protocol, we could determine Zn at an impressive level of
.025 mM (Fig. 9). The isotopic distribution also followed the
0
compound 2. The desired ligand L1 was formed when 2 was
reacted with sodium salt of pyrene butyric acid 3. The ligand L2
was synthesized via Wittig reaction between the Wittig salt 4
and pyrene aldehyde in presence of n-BuLi. The structures of L1
theoretically predicted one (Fig. 10).
The LALDI mass spectrum of ligand L2 and Co(ClO ) was
4 2
compared with the spectra obtained via conventional MALDI
technique (Fig. 11). It clearly showed the advantage of LALDI
over MALDI for small molecule detection.
Moreover our synthesized probe can detect metal ion in
presence of other organic impurities, ubiquitous in biological
1
13
and L2 have also been conrmed by H, C NMR (included in
ESI†).
28
With the ligands in hand, we rst checked whether these are
LDI active. For this, an aliquot of 1 mL of the prepared ligand
solution of 1 mM in chloroform was spotted in the LDI plate and
the mass spectrum (TOF-MS) was recorded. Due to high molar

uids like glucose, amino acid, a-ketoglutaric acid and tartaric
acid, which cannot be accomplished by any other LDI tech-
niques (Fig. 12). Even it can detect the presence of all three
extinction coefficient of pyrene, ligand L1 showed a strong
2+
2+
2+
metal ions like Co , Zn and Ni in a mixture (Fig. 13).
We have also recorded the uorescence and UV-spectral
changes upon complexation (Fig. 14 and 15). The UV and uo-
+
[
MH] peak at m/z 610 (Fig. 5) while ligand L2 showed the peak
at m/z 536 (Fig. 6). Expectedly, because of greater conjugation,
the ligand L2 absorbs the laser to a greater extent and hence the
corresponding LALDI spectrum of ligand L2 appeared to be
clearer than ligand L1.
For screening the metal ion complexation, the concentration
of probe (L1 or L2) was maintained at 0.1 mM and that of metal
rescence experiments were carried out in acetonitrile medium at
ꢁ5
10
M concentration of ligand solution upon addition of
incremental amount of metal perchlorate (in order of 200 mM).
The UV-VIS spectrum of the ligand L1 exhibits maxima at 242 nm
2+
2+
2+
and 275 nm. For Zn , Ni and Co , upon gradual increase of
Table 1 Metal ion screening results with ligand L1 and L2
Metal
ions
Obtained peak of metal complexes
of ligand L1 (m/z)
Obtained peak of metal complexes
ligand L2 (m/z)
2
+
Zn
Ni
708, 772
667, 684, 702, 766
668, 685, 703
No signicant peak
No signicant peak
No signicant peak
634
2+
593, 610, 628, 692
594, 611
No signicant peak
No signicant peak
No signicant peak
2
+
Co
2+
Fe
2
+
Cu
2
+
Mg
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Fig. 10 Isotopic distribution of zinc–ligand L2 complex in LALDI mass
spectra (a) experimentally (b) calculated obtained.
4 2
Fig. 8 LALDI mass spectrum of ligand L2 + Zn(ClO ) .
2
+
metal ion concentration, a new peak (284 nm for Co and
2
+
2+
2
85 nm for Zn and Ni ) was observed. No change was observed
2
+
2+
2+
for Fe , Cu , Mg . Ligand L2 exhibited maxima at 384 nm. For
Zn , Ni and Co shi of ligand peak was observed i.e., 384 nm
to 407 nm for Zn , to 403 nm for Co and to 405 nm for Ni . No
change was observed for Fe , Cu , Mg . This also indicates the
2
+
2+
2+
2
+
2+
2+
2
+
2+
2+
2
+
2+
greater chelating ability of terpyridine moiety towards Zn , Ni
2
+
and Co .
Fluorescence data for ligand L2 were recorded at excitation
wavelength of 360 nm. The free ligand showed emission
maxima at 487 nm. Upon gradual increase of metal ion
concentration, quenching of uorescence was observed for all
metal ions. Many transition metals are known to be uores-
cence quenchers, and they can quench uorescence through
Fig. 11 MALDI mass spectrum of ligand L2 and Co(ClO
of a-CHCA matrix.
4 2
) in presence
29
energy transfer or electron transfer mechanisms.
phenomenon of quenching in case of Zn , Ni and Co was
The
L1 had very low uorescent power, thus the experiment was
carried out using a greater slit width of the uorimeter. The
2
+
2+
2+
2
+
2+
2+
very high compared to Fe , Mg , Cu . The greater extent of

uorescence spectrum (Fig. 15a) of L1 was recorded at an
2
+
2+
2+
quenching in case of Zn , Ni and Co suggested formation of
complexes causing easier energy transfer. This is also supported
by LALDI-MS and UV-VIS studies. In case of Zn , apart from
excitation wavelength of 280 nm and it showed emission
maxima at 378 nm. Upon gradual addition of Zn concentra-
2+
2
+
tion, the nature of the curve completely changed indicating
strong complexation. However, unlike L2, no new band in the
visible region was observed. Small changes were also noticed for
quenching of uorescence, a new peak was observed at 620 nm
2
+
with increasing Zn concentration (Fig. 15b and c), it also
showed a vivid colour change from yellow to red under UV lamp
2+
2+
2+
2+
2+
Ni and Co , but no change was observed for Fe , Mg , Cu .
(
Fig. 16). The new peak may be due to ligand to metal ion
30
intramolecular charge transfer (ICT). The synthesized ligand
Fig. 12 LALDI mass spectrum of ligand L2 and Co(ClO
Fig. 9 Sensitivity detection of zinc ion by LALDI-MS using ligand L2, of other organic impurities like amino acids, a-ketoglutaric acid, tar-
blown up spectra for 0.025 mM concentration is shown in the inset. taric acid and glucose.
4 2
) in presence
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4 2 4 2
Fig. 13 LALDI mass spectrum of ligand L2 + Zn(ClO ) + Co(ClO ) +
Ni(ClO ) .
4 2
Encouraged by the outcome of uorescence study we pro-
ceeded to the in vitro cell imagine for the staining of the zinc ion
by the ligand L2. In this experiment we targeted HaCaT cell line
for imaging studies. HaCaT is a spontaneously transformed
aneuploid immortal keratinocyte cell line from adult human
31
skin, widely used in scientic research.
Considering the merits required for cell-imaging study, our
object was to locate zinc ion in living cell by employing the
higher uorescent property of the ligand L2 and its greater
affinity for zinc ion at biologically relevant environment.
As predicted beforehand, the ligand L2 can selectively sense
zinc ions in cell. The control cells and the cells only treated with
Zn(ClO ) showed no red (Fig. 17A and B) but blue uorescence
4
2
was observed due to staining of the nucleus by DAPI. The cells
treated with only ligand L2 showing the red uorescence having
less intensity (Fig. 17C) but the cells treated with both zinc salt
Fig. 15 Comparative fluorescence titration curve for (a) ligand L1
4 2
when titrated with Zn(ClO ) and (b) ligand L2 when titrated with
Zn(ClO (c) expanded portion of (b).
4 2
)
and ligand showed more intense red uorescence in high
intensity (Fig. 17D).
When HaCaT cell were incubated with only ligand L2 very
faint uorescence expression was observed due to the zinc
Fig. 16 Color change under UV radiation for different metal ions
2+
Fig. 14 Comparative UV-VIS titration curve: (a) ligand L1 and (b) ligand incubated with ligand L2: (a) ligand L2 (b) ligand L2 + Co (c) ligand L2
2+
2+
L2, each with increasing concentration with Zn(ClO
4
2
) .
+ Zn and (d) ligand L2 + Ni .
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2
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4 2 4 2
Zn(ClO ) (C) cell treated with ligand L2 (D) cell treated with Zn(ClO )
+
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arrow indicates the staining by the red fluorescence. Blue color indi-
0
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8
9
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1
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2
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1
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Conclusion
In conclusion, we have successfully synthesized pyrene–terpyr-
idine ligands for detection of their metal ion complexation
property. One of the ligand also acted as a new uorescent
probe highly selective for zinc ion in presence of other metal
ions in the biologically relevant range. Zinc ion imaging was
demonstrated using ligand L2 in HaCaT cell by observing
1
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2
+
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+

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1
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1
1
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Acknowledgements
We thank Professor Sanjib K. Patra, Department of Chemistry,
IIT Kharagpur, for helpful discussion and suggestion. DST is
acknowledged for an SERC grant (SB/S1/OC-94/2013) and for the
JC Bose Fellowship to AB which supported this research. AM is
grateful to IIT Kharagpur for a senior research fellowship and
PB is grateful to CSIR, Government of India, for a research
fellowship.
1
1
1
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2
2
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2
2
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where the two ligands H
However the absence of isotopic peaks due to Cl and Cl
made us to believe that it is indeed OH and H O acting as
2
O and OH are replaced by Cl.
3
5
37
2
the ligands. We have not seen the formation of dimers as 30 A. Vogler and H. Kunkely Coord, Chem. Rev., 2000, 208, 321.
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