Recognition of Hg2+ ion through restricted imine isomerization: Crystallographic evidence and imaging in live cells

Article abstract of DOI:10.1021/ol3009733

A newly synthesized imine-based receptor (L) showed remarkable specificity toward the Hg²⁺ ion in aqueous media over other metal ions. Coordination of L to Hg²⁺ induces a turn-on fluorescence response. This was explained based on the restricted imine isomerization along with PET on coordination to Hg²⁺. X-ray structural evidence tends to favor a C-C bond rotation rather than C=N isomerization for adopting a favorable conformation in L for coordination to Hg²⁺. This reagent could be used for imaging the accumulation of Hg²⁺ ions in HeLa cells.

Full text of DOI:10.1021/ol3009733

ORGANIC
LETTERS
2
þ
2
012
Vol. 14, No. 12
980–2983
Recognition of Hg Ion through
Restricted Imine Isomerization:
Crystallographic Evidence and
Imaging in Live Cells
2
†
†
†
†
Amal Kumar Mandal, Moorthy Suresh, Priyadip Das, E. Suresh, Mithu Baidya,
‡
,
Sudip K. Ghosh,* and Amitava Das*
‡
,†
Central Salt and Marine Chemicals Research Institute (CSIR), Bhavnagar 364002,
Gujarat, India, and Department of Biotechnology, Indian Institute of Technology,
Kharagpur, West Bengal 721302, India
amitava@csmcri.org; sudip@hijli.iitkgp.ernet.in
Received April 14, 2012
ABSTRACT
2
A newly synthesized imine-based receptor (L) showed remarkable specificity toward the Hg ion in aqueous media over other metal ions.
þ
2
Coordination of L to Hg induces a turn-on fluorescence response. This was explained based on the restricted imine isomerization along with
þ
2þ
PET on coordination to Hg . X-ray structural evidence tends to favor a CꢀC bond rotation rather than CdN isomerization for adopting a favorable
2
conformation in L for coordination to Hg . This reagent could be used for imaging the accumulation of Hg ions in HeLa cells.
þ
2þ
Mercury is one of the most prevalent toxic metals in the
environment and gains access to the body orally or dermally,
causing cell dysfunction that consequently leads to health
problems. The concern over its deleterious effects on human
health has actually led the sensing community to develop
new mercury detection methods that are cost-effective, rapid,
facile, and applicable to the environmental and biological
2
milieus. In this regard, receptor molecules that can provide
2þ
optical feedback on binding to the Hg ion in aqueous or
mixed aqueous environments are crucial for developing
1
3
either colorimetric staining agents or fluorescent imaging
4
reagents for biological applications. Being one of the
2þ
heavier metal ions, Hg generally causes an efficient
quenching of the fluorescence through an efficient spinꢀ
†
Central Salt and Marine Chemicals Research Institute.
5
orbit coupling. However, any optical feedback that trans-
‡
Indian Institute of Technology.
1) (a) Hutchinson, T. C.; Meena, K. M. In Lead, Mercury, Cadmium
(
lates the metal ion-receptor binding phenomena into
a fluorescence turn-on response is preferred over the
and Arsenic in the Environment; JohnWiley: New York, 1987. (b) Harada,
M. Crit. Rev. Toxicol. 1995, 25, 1–24. (c) Tchounwou, P. B.; Ayensu,
W. K.; Ninashvili, N.; Sutton, D. Environ. Toxicol. 2003, 18, 149–175.
(
2) (a) Ros-lis, J. V.; Marcos, M. D.; Martinez-Manez, R.; Rurack,
(4) (a) Lee, M. H.; Lee, S. W.; Kim, S. H.; Kang, C.; Kim, J. S. Org.
Lett. 2009, 11, 2101–2104. (b) Suresh, M.; Mandal, A. K.; Saha, S.;
Suresh, E.; Mandoli, A.; Liddo, R. D.; Parnigotto, P. P.; Das, A. Org.
Lett. 2010, 12, 5406–5409. (c) Dave, N.; Chan, M. Y.; Huang, P. J. J.;
Smith, B. D.; Liu., J. J. Am. Chem. Soc. 2010, 132, 12668–12673. (d)
Kumar, M.; Kumar, N.; Bhalla, V.; Singh, H.; Sharma, P. R.; Kaur, T.
Org. Lett. 2011, 13, 1422–1425.
K.; Soto, J. Angew. Chem., Int. Ed. 2005, 44, 4405–4407. (b) Nolan,
E. M.; Lippard, S. J. Chem. Rev. 2008, 108, 3443–3480.
(
15–418. (b) Li, T.; Li, B.; Wang, E.; Dong, S. Chem. Commun. 2009,
551–3553. (c) Aragay, G.; Pons, J.; Merko c- i, A. Chem. Rev. 2011, 111,
433–3458. (d) Ma, X.; Wang, J.; Shan, Q.; Tan, Z.; Wei, G.; Wei, D.;
3) (a) Brummer, O.; La Clair, J. J.; Janda, K. D. Org. Lett. 1999, 1,
4
3
3
Du, Y. Org. Lett. 2012, 14, 820–823. (e) Saha, S.; Chhatbar, M. U.;
Mahato, P.; Praveen, L.; Siddhanta, A. K.; Das, A. Chem. Commun.
(5) (a) McClure, D. S. J. Chem. Phys. 1952, 20, 682–686. (b) Masuhara,
H.; Shioyama, H.; Saito, T.; Hamada, K.; Yasoshima, S.; Mataga, N. J.
Phys. Chem. 1984, 88, 5868–5873.
2012, 48, 1659–1661.
1
0.1021/ol3009733 r 2012 American Chemical Society
Published on Web 06/07/2012

fluorescence turn-off response for obvious ease in the
detection process. Among different signaling mechanisms,
were also confirmed from single-crystal X-ray structure
analysis.
Spectroscopic properties of L, L , and L were investi-
6
photoinduced electron (PET)/energy transfer (PeT), me-
1
2
7
talꢀligand charge transfer (MLCT), and intramolecular
gated in mixed aqueous organic medium (THF/aqueous
phosphate buffer (6:4, v/v; pH = 7.2). The UVꢀvis spectra
of L exhibited a strong band at 255 nm (ε = 58823 L
8
charge transfer (ICT) are most common. However, to
achieve a reversible switch-on fluorescence response on
2þ
ꢀ1
ꢀ1
ꢀ1
selective binding to Hg , two common methodologies
have been adopted, namely conversion from the nonfluor-
escent lactam to a strongly fluorescent xanthene form or
mol
cm ) with two broad band at 295 nm (ε = 22768
ꢀ
1
ꢀ1
ꢀ1
L mol cm ) and 385 nm (ε = 16096 L mol cm ).
Electronic spectra of L werecomparedwiththoseofL and
1
2
þ
9
through achieving a structural rigidity on Hg ion binding.
Literature reports are there for demonstrating the second
L . The bands at ∼255 and 295 nm could be attributed to
2
N- and Q-based intracomponent charge-transfer (CT)
transitions, respectively, while the broad band at 385 nm
was ascribed to a intercomponent CT band with N as
the donor and Q as the acceptor fragment. Addition of a
perchlorate salt of alkali, alkaline earth, and common
4b
process using azine-based receptors. However, such an
example using analogous imine isomerization as a signaling
mechanism has not been used so far for designing any
2þ
colorimetric or fluorogenic receptor for Hg ion, although
9a
þ
þ
þ
þ
þ
2þ
2þ
it has been documented previously for other metal ions.
Herein, we report a novel imine-based molecule (L),
having naphthalene and quinoline as two photoactive
transition-metal ions (Li , Na , K , Cs , Ag , Ca
,
,
2
þ 2þ 2þ 3þ 2þ 3þ 2þ 2þ
Mg , Sr , Ba , Cr , Fe , Fe , Co , Ni , Cu
2þ
2þ
Zn , Cd ) did not show any change in electronic spectra
of L. However, an appreciable change was observed when
the spectrum was recorded in the presence of Hg
(Supporting Information, SI).
2
þ
units, for the specific recognition of Hg ions through
2þ
2þ
restricted imine isomerization on binding of L to Hg in
mixed aqueousꢀorganic medium. To establish molecular
preorganization that takes place prior to the coordination
2
þ
Spectralresponsesof L, L , and L onbinding toHg in
1
2
2þ
to the Hg center, two reference compounds (L and L )
2
CHCl /CH CN (1:4 v/v) were different, which were pri-
3 3
1
were synthesized (Figure 1), and their optical responses on
marily due to the intrinsic difference in the nature of the
donor and acceptor fragments in these three receptors.
2þ
binding to Hg were compared with that of L. Also,
naphthalene (N) and quinolone (Q) form a FRET pair,
and thus, the choice of these two chromophores provides
the opportunity to achieve a larger Stokes shift on the
sensing event. More importantly, the possibility of using
Systematic titration in CHCl /CH CN (1:4 v/v) medium
3 3
for L revealed the formation of a broader band at longer
2þ
wavelength (∼480 nm). Coordination to Hg was ex-
pected to favor the intercomponent CT transition and
thus the shift to longer wavelength. However, spectral
responses for L in THFꢀaqueous phosphate buffer
(6:4, v/v; pH = 7.2) were quiet different (Figure 2a and
inset) and were attributed to a weaker coordination of L to
2þ
this reagent for imaging application and detection of Hg
accumulated in cervical cancer cells was explored to check
the viability of the cells under the experimental conditions.
2
þ
2þ
hydrated Hg ion, as compared to the nonaqueous Hg
ion in organic medium. Binding affinity of the reagent L
2
þ
toward Hg in mixed THFꢀaqueous buffer medium was
evaluated by monitoring changes in absorbance at 385 nm
(
Figure 2b) with varying [Hg þ] (0ꢀ27 molar equiv).
2
Figure 1. Structure of the imine-based receptor molecules.
Reagent L and the two reference compounds (L and L )
1
2
were synthesized following a typical one-step reaction (SI).
Various analytical and spectroscopic data agree well with
the structure proposed for these reagents and the desired
purity. Further, trans conformations for three receptors
(
6) (a) de Silva, A. P.; Fox, D. B.; Huxley, A. J. M.; Moody, T. Coord.
Chem. Rev. 2000, 205, 41–57. (b) Gunnlaugsson, T.; Davis, A. P.;
O’Brien, J. E.; Glynn, M. Org. Lett. 2002, 4, 2449–2451.
Figure 2. Changes in UVꢀvis spectra recorded in 25 °C (a)
ꢀ5
CHCl /CH CN (1:4 v/v) medium for L (1.8 ꢁ 10 M) with
(7) Beer, P. D. Acc. Chem. Res. 1998, 31, 71.
3
3
2
þ
ꢀ4
(8) (a) Xu, Z.; Xiao, Y.; Qian, X.; Cui, J.; Cui, D. Org. Lett. 2005, 7,
varying [Hg ] (0 ꢀ 1.1 ꢁ 10 M). Spectra in red indicate L.
ꢀ 2þ
5
8
89. (b) Wang, J. B.; Qian, X. F.; Cui, J. N. J. Org. Chem. 2006, 71, 4308.
9) (a) Wu, J.; Liu, W.; Ge, J.; Zhang, H.; Wang, P. Chem. Soc. Rev.
011, 40, 3483 and the references cited therein. (b) Saha, S.; Mahato, P.;
Inset: titration profile for L (2.3 ꢁ 10 M) with varying [Hg
]
ꢀ
4
(
(0 ꢀ 6.2 ꢁ 10 M) in THF/aqueous phosphate buffer (6:4, v/v;
pH = 7.2) and its corresponding; (b) BꢀH plot for evaluation of
2
Reddy, U. G.; Suresh, E.; Chakrabarty, A.; Baidya, M.; Ghosh, S. K.;
Das, A. Inorg. Chem. 2012, 51, 336. (c) Mahato, P.; Saha, S.; Suresh, E.;
Liddo, R. D.; Parnigotto, P. P.; Conconi, M. T.; Kesharwani, M. K.;
Ganguly, B.; Das, A. Inorg. Chem. 2012, 51, 1769.
2þ
the binding constant for L with Hg . The good fit of the linear
plot (R = 0.99) confirmed the 2:1 binding stoichiometry.
2
Org. Lett., Vol. 14, No. 12, 2012
2981

Weaker binding in aqueous buffer medium was also
N-moiety-based luminescence in L, as observed for L2.
However, similarities in the luminescence responses of L
and L on binding to Hg revealed that the Q-moiety is
1
primarily involved in the photoinduced geometrical iso-
merization either through rotation about the CdN or by
A
evident from the much lower affinity constant (K
7
2þ
Hg ] L
2
[
ꢀ2
2
2þ
=
1.48 ꢁ 10 M L ) than the one that was evaluated in
A
9
CHCl /CH CN (1:4 v/v) medium (K 2þ
[Hg ] L
2
= 2.79 ꢁ 10
3
3
ꢀ
2
2
M
L ) under otherwise identical condition. Good linear
fit of the BenesiꢀHildebrand plot (BꢀH plot) confirmed
C[Q]ꢀN
bond rotation.
CdN
2þ
2þ
The binding affinity of Hg toward L, L , and L was
1 2
the 2:1 binding stoichiometry and formation of [Hg ] L
2
complex. This binding stoichiometry in solution was also
confirmed from the ESIꢀmass spectral data (SI). Spectral
evaluated from a BenesiꢀHildebrand (BꢀH) plot using
the results of the systematic titrations either by probing the
change in fluorescence or absorption band intensity with
2þ
2þ
2
data alsoconfirmed formation of[Hg ] L and [Hg ] L
2
2
1
2þ
in solution. Further, the single-crystal structure for
2þ
varying [Hg ]. Analysis of the BꢀH plots for L and L
2
2þ
[
dence for the stepwise formation of 1:1 and then 2:1
Hg ]2L and [Hg ] L also corroborated this. No evi-
confirmed the 2:1 binding stoichiometry (SI). No spectral
evidence for stepwise complex formation was apparent.
However, for L , two distinct changes in fluorescence
2
2
complexcould beobtainedfor L and L , which was evident
2
1
from the electronic spectral titration profile for L (SI).
1
spectra were observed when spectra were recorded with
2þ
Thus, for reagent L, the observed binding constant could
be better assigned as the composite binding constant for
varying [Hg ] and allowed us toevaluate stepwise binding
2
þ
constants for formation of [Hg ]L and [Hg ] L (SI).
1 1
2þ
2
2þ
the formation of {L(Hg ) }(ClO ) .
2
The respective binding constants evaluated from different
methodologies are summarized in Table 1. The reversible
4 4
Emission quantum yields for L (Φ = 0.0011) and one
L
2
þ
of the two model receptors (ΦL1 = 0.0118) were poor.
However, relative fluorescence quantum yield for L2
binding of Hg
to L was checked by addition of
an aqueous solution of KI (SI). This was also demon-
1
strated by H NMR spectroscopic studies. Because of
1
limited solubility, the H NMR spectra were recorded in a
(
ΦL2 = 0.1090) was much higher than that of L . Because
1
of the flexible nature of the imino compound, the thermal
barrier between the two geometrical isomers is low, so the
relaxation through photoinduced geometrical change is
2þ
CDCl ꢀDMSO-d (4:1, v/v) medium. On addition of Hg ,
3
6
the H
proton showed a drastic downfield shift along
HCdN
1
0
extremely rapid and efficient. The photoinduced electron
transfer (PET) process involving the lone pairs of electrons
of NCdN and the excited Q-based fluorophore could also
contribute to the observed weaker emission for L and L1.
with a decrease of signal intensity for the rest of the protons
(SI). On addition of excess of thiourea to this solution, the
initial spectrum for L was restored with a subsequent
increase in signal intensity (SI).
Detailed H NMR spectroscopic studies were carried
out with an attempt to address the critical issue of bond
1
Interestingly, the fluorescence enhancement for L was
2
2
þ
[Hg2
þ
]
2
L
2
only 2-fold on binding to Hg (Φ
= 0.2245), while
= 0.2390) and L1
[
Hg2
þ
] L
2
for similar experiment with L (Φ
rotation about the CdN or by C ꢀN
bond in L, L1,
and L on binding to Hg . The 1D-NOE spectra of L on
[Q]
CdN
2þ
[
Hg
] L
2 1
2þ
( = 0.2053) a switch-on fluorescence response was
observed (Figure 3). This observed enhancement on lumi-
Φ
2
selective irradiation of the HHCdN proton at 8.95 ppm
showed a correlation at 7.35 ppm, which signified a trans
orientation of the two N-atoms of Q-ring and NHCdN with
respect to each other in solution. No such 1D-NOE
nescence response for L, L and L2 on binding to
1
2þ
Hg could be ascribed due to the loss of molecular flex-
ibility of these receptors because of restricted imine iso-
merization. In addition, an interrupted PET process on
2þ
correlation was observed in presence of Hg (SI). Similar
observations were also witnessed for L and L (SI).
2þ
coordination to the Hg was also expected to contribute
1
2
1
1
to this observed switched-on emission response. It is
worth mentioning here that the presence of lower lying
CT states (vide supra) could account for the absence of any
A single-crystal X-ray structure for L and L revealed
2
that quinolyl and imine nitrogen atoms oriented trans to
each other (Figure 4a,c), minimizing the protonꢀproton
interactions. Such an orientation is typical for pyridy-
1
2
limine ligands. Crystal structures for L and [(Hg) L]I
2
4
(
(
Figures 4a,b) also revealed change in the twist angle
1
3
θ
)
of 14.6° (42.2° for L to 56.8° for [(Hg) L]I )
Q‑N
2
4
between the terminal Q and central N ring. This change
in θ
for L and [(Hg) L ]I was 14.1° (42.2 for L to
QꢀN
6.3 for [(Hg) L ]I ) (Figures 4c,d). Thus, structural evi-
2
2
2
4
2
5
2 2 4
dence suggest a difference of ∼14° in θQ‑N tomakeeffective
(
10) King, N. R.; Whale, E. A.; Davis, F. J.; Gilbert, A.; Mitchell,
G. R. J. Mater. Chem. 1997, 7, 625–630.
11) (a) Naik, L. R.; Suresh, H. M.; Inamdar, S. R.; Math, N. N.
(
Spectrosc. Lett. 2005, 38, 645. (b) Koner, A. L.; Mishra, P. P.; Jha, S.;
Dutta, A. J. Photochem. Photobiol. A. 2005, 170, 21–26.
(12) (a) Kesslen, E. C.; Euler, W. B. Chem. Mater. 1999, 11, 336–340.
(b) Tuna, F.; Hamblin, J.; Clarkson, G.; Errington, W.; Alcock, N. W.;
Figure 3. Fluorescence spectral changes in THFꢀaqueous phos-
phate buffer medium (6:4, v/v; pH = 7.2) at 25 °C: (a) L (2.0 ꢁ
ꢀ
5
2þ
ꢀ4
1
0
M) with varying [Hg ] (0ꢀ5.2 ꢁ 10 M), λexc = 295 nm;
ꢀ
5
Hannon., M. J. Chem.;Eur. J. 2002, 8, 4957–4964.
(
b) fluorescence scanning of L (2.0 ꢁ 10 M) in the presence of
ꢀ
exc
(
13) θQꢀN is the twist angle between the Q and N rings, while
4
various metal ions (6.0 ꢁ 10 M) (λ = 295 nm).
θ[
CdNꢀCNaph]
the imine functionality and the naphthalene (N) ring.
represents the dihedral angle between planes involving
2
982
Org. Lett., Vol. 14, No. 12, 2012

Table 1. Association Constants for Complex Formation with
Hg with Different Receptors in Different Solvent Media
2
þ
a
L
5
K
2
(10 )
ꢀ
2
2
L
1
1
1
L d
L
2
2
5
ꢀ2
2
K
(M L )
K
and K
2
K
(10 ) (M L )
b
c
ꢀ1
L
abs
abs
148
27900
186
8700 and 1100 M
5.84
364
9
ꢀ2 2e
1.29 ꢁ 10 M
L
Figure 5. Bright field transmission of living HeLa cells incubated
b
ꢀ1
2þ
fluo
10000 and 1500 M
L
7.83
with (a) L (20 μM) and (b) L (20 μM) with Hg (10 μM); (c)
fluorescence image of HeLa cells incubated with L (20 μM) with
Hg (10 μM); (d) merged image of (b) and (c).
[Hg2 ]L
þ
2þ
a
L
[Hg2þ] L
L
1
1
;
L
2
1
[Hg ] L
2
1
L
2
K
2
= K
.
2
; K
1
= K
K
= K
; K
2
=
2þ
Hg2 ] L
þ
[
2
b
K
medium (6:4, v/v; pH = 7.2). Signifies absorption titration in CHCl /
2
Absorption titration in THFꢀaqueous phosphate buffer
c
3
d
CH CN (1:4 v/v) medium. Stepwise (1:1 and then 2:1 with respect to
3
2þ
Hg and L
THFꢀaqueous buffer medium. Composite binding constant was ob-
tained in CHCl :CH CN (1:4 v/v) medium.
1
) complex formation was observed for reagent L
1
only in
fluorescence was observed for Hela cells pretreated with
2þ
e
Hg and then with L/L , while no such fluorescence was
1
3
3
observed eitehr for untreated HeLa Cells or cells treated
with only Hg(ClO ) (Figure 5).
So, these two probe reagents (L and L ) were cell
4
2
1
2þ
membrane permeable and could detect Hg uptake in
2þ
living cells exposed to [Hg ] as low as 10 μM. The
reversibility of the binding to L/L and the detection of
1
2þ
Hg in HeLa cells was established by treating those
stained cells with KI (20 μM) solution (SI).
In summary, we synthesized an imine-based chemo-
sensor that selectively binds Hg ion in aqueous media.
We exploited two signal transaction mechanisms, namely,
restriction of molecular flexibility and/or PET to achieve
fluorescence on response on binding to Hg ions. Crytallo-
graphic evidence tends to favor the CꢀC bond rotation,
rather than the geometrical isomerization at CdN as one of
the primary reason for the restriction of the molecular
2þ
2þ
Figure 4. Single crystal structure: (a) L, (b) [(Hg)
[
2 4 2
L]I , (c) L ; (d)
(Hg) L ]I (solvent molecule was omitted for clarity).
2 2 4
2þ
flexibility on binding to Hg . Perhaps for a binucleating
receptors like these, cisꢀtrans isomerization is energetically
more demanding than the CꢀC bond rotation. Results of the
2
þ
binding to Hg either by L or L , while θ involving the
2
1
3
N-ring and imine plane (θ[
2.4° for L and L , respectively (SI). Interestingly, a much
smaller change in θ[
center was observed (θ[
and 45.1° for [(Hg) L ]I ), which indicated a nominal
perturbation in θ
)
was 41.8° and
CdNꢀCNaph]
4
fluorescence microscopic studies revealed that L and L are
2
1
on binding to the Hg(II)
of 46.01° for [(Hg) L]I
cell permeable and could be used as nontoxic imaging
CdNꢀCNaph]
2þ
reagents for detection of the uptake of Hg in the HeLa cells.
CdNꢀCNaph]
2
4
2
2 4
upon coordination to the
Acknowledgment. A.D. thanks DST and CSIR (India)
for financial support. A.K.M., M.S., and P.D. acknowledge
CSIR for Senior Research Fellowships. M.B. acknowledges
UGC (New Delhi) for Jr. Research Fellowship. S.K.G.
acknowledges DST for partial grant for the Confocal
facility.
[
CdNꢀCNaph]
2þ
Hg center. Thus, structural evidence tends to indicate
bond rotation was more plausible than
geometrical isomerization around the CdN.
that C[Q]ꢀN
CdN
We explored the possibility of using L and L for the
1
recognition of the Hg ion in living cervical cancer cells
HeLa cells) using fluorescence imaging experiments.
2þ
(
MTT assay studies revealed that >80% Hela cells re-
mained viable even after 12 h of incubation with these
probe molecules having concentration comparable to that
used for imaging studies (SI). For fluorescence imaging
experiments, HeLa cells preexposed to 10 μM Hg were
washed with an appropriate buffer solution and subs-
Supporting Information Available. Synthetic details,
characterization data for the compounds, and selected
spectroscopic data. Three crystal structures have been
deposited with the Cambridge Crystallographic Data
Centre and allocated deposition nos. CCDC 874976ꢀ
2þ
8
74978. This material is available free of charge via the
quently incubeted either with L or with L for 30 min.
1
Internet at http://pubs.acs.org.
On viewing through a fluorescence microscope using a
3
30ꢀ380 nm UV filter asanexcitation source, a bright blue
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 12, 2012
2983