Selective detection and bio-imaging of Pd2+ with novel 'C-CN' bond cleavage of cyano-rhodamine, cyanation with diaminomaleonitrile

Article abstract of DOI:10.1039/c3dt51591e

A new rhodamine based chemosensor, cyano-rhodamine, has been designed and synthesized with a green approach which shows a specific 'C-CN' bond breaking with the action of the Pd²⁺ ion to produce the specific color and fluorescence of rhodamine

Full text of DOI:10.1039/c3dt51591e

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Selective detection and bio-imaging of Pd2+ with novel
‘C–CN’ bond cleavage of cyano-rhodamine, cyanation
with diaminomaleonitrile†
Shyamaprosad Goswami,*a Abhishek Manna,a Anup Kumar Maity,b Sima Paul,a
Avijit Kumar Das,a Manas Kumar Das,a Partha Saha,b Ching Kheng Quahc and
Hoong-Kun Func,d
Cite this: Dalton Trans., 2013, 42, 12844
Received 15th June 2013,
Accepted 11th July 2013
DOI: 10.1039/c3dt51591e
www.rsc.org/dalton
A new rhodamine based chemosensor, cyano-rhodamine, has
Being a paramagnetic species, most of the reported fluo-
been designed and synthesized with a green approach which rescence sensors of Pd2+ readily undergo fluorescence quench-
shows a specific ‘C–CN’ bond breaking with the action of the Pd2+ ing.9 Despite advances in the development of highly selective
ion to produce the specific color and fluorescence of rhodamine and sensitive fluorescent indicators for palladium,10 there are
6G itself in solution and in HeLa cells.
still some limitations on the quantitative detection and bio-
imaging including complicated synthetic procedures, poor
water solubility and selectivity over metal ions including Pd(0).
Furthermore, so far, few reports on the imaging of palladium
in living systems have been published.10e,h Thus, novel cell-
permeable fluorescence indicators for palladium species became
our target.
Herein, we report a new cyano-rhodamine (Rh-CN) based
colorimetric ‘off–on’ fluorescence probe that can selectively
detect Pd2+ over other examined metal ions studied including
Pd(0) in aqueous solution. In general, the rhodamine architec-
ture provides an ideal mode for the construction of ‘off–on’
type fluorescence-enhanced probes based on spirolactam (col-
orless, nonfluorescence) to ring-open amide (pink colored,
fluorescence) equilibrium,11 but in this paper we report a
novel selective ‘C–CN’ bond breaking approach on the rhoda-
mine moiety to gain its color and fluorescence with the
addition of Pd2+ in solutions and in cells.
Recently, a great deal of attention has been paid to the deter-
mination of palladium (Pd) species among the metal ions
because of its extensive use in numerous materials such as
alloys, jewellery, dental crowns, fuel cells and catalysts.1 Palla-
dium(II) salts are predominantly used as oxidizing reagents
and pre-catalysts for many cross-coupling reactions.2 However,
the largest use of palladium today is in catalytic converters of
automobiles that frequently release a large quantity of palla-
dium to the environment3 which may cause a serious health
problem4 owing to the formation of complexes between Pd
and some biomacromolecules such as proteins, DNA and
RNA.5 Thus, government restrictions on the levels of residual
heavy metals in end-products are very strict, and its threshold
for palladium is 5–10 ppm.6
Hence, considerable attention has been paid to the develop-
ment of methods for selective sensing of palladium. Colori-
metric and fluorimetric methods for the quick detection of
Pd2+ have been very popular in recent years because they are
inexpensive and nondestructive in nature7 compared with high
cost instrumental analysis.8
The cyano-rhodamine moiety (Ortep view: Fig. 4a) is pre-
pared from rhodamine 6G with the help of diaminomaleoni-
trile, water and triethyl amine in ethanol (Scheme 1). For the
preparation of nitrile compounds, conventional methods are
most often used: the Sandmeyer12 and Rosenmund-von
Braun13 procedures employ stoichiometric amounts of CuCN
as the cyanating reagent. While the recent development of the
aDepartment of Chemistry, Bengal Engineering and Science University, Shibpur,
West Bengal, India. E-mail: spgoswamical@yahoo.com
bCrystallography and Molecular Biology Division, Saha Institute of Nuclear Physics,
Kolkata, West Bengal, India
cX-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia,
11800 USM Penang, Malaysia
dDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud
University, Riyadh 11451, Kingdom of Saudi Arabia
†Electronic supplementary information (ESI) available: Details of synthetic pro-
cedure, X-ray crystal structure and spectral data available. CCDC 934861. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c3dt51591e
Scheme 1
12844 | Dalton Trans., 2013, 42, 12844–12848
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Scheme 2
transition-metal-catalyzed cyanation14 allows the use of certain
metal- or metalloid bound cyanide sources,15 but the sources
of cyanation are also toxic in nature. In response to these
issues, the feasibility of using nonmetallic cyanation species
has been actively investigated in recent years.16
In fact, a range of organic precursors bearing a “CN” moiety
have been examined: acetone cyanohydrin and its analogues,17
alkyl nitriles,18 malononitrile,19 phenyl cyanates,20 benzyl thiocya-
nates,21 N-cyanobenzimidazole,22 TMSCN,23 nitromethane,24 and
triselenium dicyanide (TSD).25 Herein, we report the diaminoma-
leonitrile as a source of cyanide in the presence of water.
The plausible mechanism for cyanation of rhodamine 6G is
presented in Scheme 2. At first the diaminomaleonitrile is
hydrolysed to give HCN. HCN is then neutralised by the organic
base triethyl amine to give the unmask cyanide ion which
readily attacks to give the product, cyano rhodamine (Rh-CN).
The photophysical properties of the receptor was investi-
gated by monitoring the absorption and fluorescence behav-
iour upon the addition of several metal ions such as K+, Mg2+,
Ni2+, Co2+, Zn2+, Cu2+, Cd2+, Hg2+, Fe2+, Fe3+, Pd0, Pd2+, Pt2+ in
MeOH–H2O (2 : 8, v/v) at pH 7.4 by using 20 mM HEPES
buffer. When we evaluated changes in the absorbance of a
receptor upon treatment with Pd2+ ions up to 4 equiv., the
Fig. 1 (a) UV-vis absorption titration spectra of the receptor (c = 1 × 10−5 M) in
the presence of Pd2+ (c = 2 × 10−4 M) in MeOH–H2O (2 : 8, v/v, at pH = 7.4,
20 mM HEPES buffer) with the interval of 5 minutes (naked eye color change in
the inset). (b) Absorption intensity of Rh-CN after the addition of 6.0 equiv. of
each of the guest cations (except Pd2+: 4 equiv.).
intensity of the absorption bands of the receptor at 525 nm the bond breaking mechanism, an almost 21 fold enhancement
increases rapidly and gives a sigmoidal type curve (Fig. 3a). of the emission intensity at 555 nm (Fig. 2a) observed upon
Thus the strong coordination by the cyanide group present in gradual addition of Pd2+ (0–4.0 equiv.) with an 88 fold enhance-
the receptor to the Pd2+ ion breaks the ‘C–CN’ bond to re- ment of quantum yield (ESI†). It is noteworthy to mention that
produce the characteristics color of rhodamine 6G itself which for the rest of the cations except Pd2+, neither pink coloration
is absent with the addition of other metal ions (Fig. 1a). In nor a large fluorescence intensity is observed demonstrating
addition, the same group element Pt2+ also shows an increased that the sensor is insensitive towards the other metal ions.
absorption band at 525 nm but the intensity is less than for Fig. 2b displays a comparative view of the emission intensity of
Pd2+. This indicates that the receptor is highly selective and the receptor, Rh-CN fluorophore, after the addition of 6.0 equiv.
sensitive for Pd2+ which is shown in bar graph representation of each of the guest cations (except Pd2+: 4 equiv.).
(Fig. 1b). The receptor is inert towards all other cations except
The mass spectrum (ESI MS) of the Rh-CN with the treat-
Pd2+ and Pt2+, i.e. all other cations are unable to break the ment of Pd2+ shows peaks at m/z 443.22 possibly for the rhoda-
‘C–CN’ bond of the Rh-CN and hence show no such effect in mine moiety, which also proves the cleavage of the ‘C–CN’
absorption spectroscopy (ESI†). As the absorption band at bond of the Rh-CN moiety, m/z 470.23, with the action of Pd2+
525 nm appears with a high absorption value, the “naked eye” (ESI†). From 13C-NMR data (ESI†) we also conclude the elimin-
detection of the metal ion is possible (Fig. 1b). Thus without ation of the cyanide group from Rh-CN (Fig. 4b).
using any instrument we can sense Pd2+ using aqueous solu-
In fluorescence spectroscopy, there is also a small enhance-
tion of the receptor, Rh-CN. From the linear dynamic graph, it ment occurring during the gradual addition of the Pt2+ ion.
is clearly indicated that the receptor can easily detect 0.83 µM The reason is the same as for the absorption phenomenon.
Pd2+ using the K*S/Sb1 equation12 (ESI†). The fluorogenic Interestingly Pd(0) which is the most interfering species10 for
response of the sensor, Rh-CN, is also in well agreement with the detection of Pd(II) is inert towards Rh-CN under the
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Fig. 3 (a) Absorbance vs. [Pd2+] plot adding up to 4 equivalents of Pd2+
towards Rh-CN (c = 1 × 10−5 M) and (b) fluorescence intensity vs. [Pd2+] plot
between the addition of 3 to 16 µM of Pd2+
.
Fig. 2 (a) Fluorescence titration spectra of the receptor (c = 1 × 10−5 M) in the
presence of Pd2+ (c = 2 × 10−4 M) in MeOH–H2O (2 : 8, v/v, at pH = 7.4, 20 mM
HEPES buffer) with the interval of 5 minutes (λex = 505 nm) (naked eye color
change of Rh-CN with the addition of Pd2+ under UV-light in the inset). (b) Emis-
sion intensity of Rh-CN fluorophore after the addition of 6.0 equiv. each of the
guest cations (except Pd2+: 4 equiv.).
mentioned condition. Further, competition experiment also
reveals that Pd2+-induced fluorescence enhancement remains
unperturbed and does not get any interference by the co-exist-
ing metal ions (Fig. 6). The calculated detection limit from the
fluorescence intensity vs. conc. of the Pd2+ curve is found to be
0.57 µM less than the detection limit observed by using
absorption data calculation with the same equation.26 Under
this condition, the changes of the intensity produced an excel-
lent linear function with the concentration of Pd2+ between
3 and 16 µM (Fig. 3b).
Fig. 4 (a) Ortep view of the receptor, cyano rhodamine (Rh-CN), showing 50%
probability displacement ellipsoids for non-H atoms and the atom-numbering
scheme. (b) Plausible mechanism of ‘C–CN’ bond cleavage of Rh-CN in the pres-
ence of Pd2+
.
In order to determine the membrane permeability of the
receptor (Rh-CN) and its specific binding ability to the Pd2+ in
living cells, HeLa cells were first incubated with the PdCl2 fol-
lowed by the addition of the receptor. A controlled incubation
of the cells with Rh-CN only was also carried out. As shown in
Fig. 5, the cells showed intense red fluorescence, specifically
in the cytoplasm in TRITC channel when they were treated
with Pd2+ followed by the receptor. Nuclei of the cells stained
with DAPI are shown in deep blue colour. In the absence of
PdCl2 no fluorescence was observed in cells. The result clearly
established that the receptor (Rh-CN) could permeate the
Fig. 5 Fluorescence images of HeLa cells incubated with 50 µM of the receptor
(Rh-CN) in the absence (a: blue channel; and b: red channel) and in the pres-
ence (e: blue channel; and f: red channel) of 50 µM of PdCl2. Corresponding
differential interference contrast (DIC) images (c and g) and merged images
(d and h) of the cells are shown.
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in the cytoplasm only in the presence of Pd(II).
In conclusion, we have reported the first synthesis of a
hitherto unknown cyano-rhodamine (Rh-CN) fluorophore with
a green approach; it easily undergoes selective Pd2+ induced
‘C–CN’ bond breaking to produce the characteristics pink
coloration along with fluorescence of rhodamine 6G itself. The
recognition events have been successfully applied to the bio-
imaging of Pd2+ on HeLa cells.
Acknowledgements
We thank DST and CSIR (Govt. of India) for financial support.
A.M., A.K.D., K.A. and M.K.D. acknowledge CSIR and S.P.
acknowledges UGC for providing fellowships. The authors
extend their appreciation to The Deanship of Scientific
Research at King Saud University for funding the work through
research group project no. RGP-VPP-207.
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