An intramolecular crossed-benzoin reaction based KCN fluorescent probe in aqueous and biological environments

Article abstract of DOI:10.1039/c5cc02234g

A turn-on fluorescent probe IND-1 was designed for selective cyanide anion sensing in aqueous and biological environments. The probe underwent an intramolecular crossed-benzoin reaction in the presence of KCN to expel the fluorophore resorufin. This probe was sensitive to KCN concentrations as low as 4 nM in aqueous media.

Full text of DOI:10.1039/c5cc02234g

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An intramolecular crossed-benzoin reaction based
KCN fluorescent probe in aqueous and biological
environments†
Cite this:DOI: 10.1039/c5cc02234g
Received 17th March 2015,
Accepted 28th March 2015
Jae Hong Lee,‡a Joo Hee Jang,‡a Nithya Velusamy,‡b Hyo Sung Jung,a
Sankarprasad Bhuniya*bc and Jong Seung Kim*a
DOI: 10.1039/c5cc02234g
www.rsc.org/chemcomm
A turn-on fluorescent probe IND-1 was designed for selective cyanide or fluorescence changes; a few cases showed both colorimetric
anion sensing in aqueous and biological environments. The probe and fluorescence changes that could be observed by the naked
underwent an intramolecular crossed-benzoin reaction in the presence eye.10c,11a However, most of the reported CNꢀ sensors work with
of KCN to expel the fluorophore resorufin. This probe was sensitive to NH4CN in non-aqueous environments;8,9,11e,12 there are few
KCN concentrations as low as 4 nM in aqueous media.
reports on KCN or NaCN sensing in aqueous environments.11a,c,g
Additionally, KCN and NaCN are more highly toxic than NH4CN;
Fluorogenic probes are widely used to detect biologically active thus, it is crucial to devise a fluorescent probe to detect KCN and
molecules, therapeutics, proteins, and ions because they are easy to NaCN in aqueous environments. Herein we report a strategy-based
modify, cheap, and exhibit rapid responses to the analytes.1 chemodosimeter as a fluorescent probe for KCN sensing in aqueous
A fluorogenic probe for sensing CNꢀ is crucial as this ion is environments (Scheme 1). The probe, IND-1, was synthesized in
extremely poisonous to human health, especially in its strong a single step by esterifying 20-formyl[1,10-biphenyl]-2-carboxylic
ionic form, e.g. KCN and NaCN. CNꢀ ions inhibit the cellular acid with resorufin. The details of the synthetic procedure and
respiratory process by disrupting the function of cytochrome c spectroscopic evidence are provided in the ESI.†
oxidase; this ion forms a stable complex with a heme unit in the
In the presence of KCN, we presumed that a carbanion
active site of cytochrome a3.2 In cellular environments, CNꢀ would be generated at the aldehyde carbonyl of IND-1 via a
ions induce an enhancement of the level of intracellular calcium benzoin-type reaction. The close proximity of the ester carbonyl
ions, causing cascades of enzymatic reactions that increase the would allow intramolecular cross-coupling with the expulsion
cellular reactive oxygen species (ROS), which eventually damage of free resorufin, which shows strong fluorescence.
the human oxidative immune defence system.3 Nevertheless,
To determine the sensing ability of IND-1, we examined the
cyanide is widely used in mining and other industries for gold UV-absorption changes of IND-1 in the presence and absence of
and silver extraction, tanning, electroplating, etc. Moreover, KCN in PBS buffer. The UV-absorption centred at 565 nm
cyanide can be used in chemical warfare for mass-destruction increased 20-fold in the presence of KCN (10 equiv.) with a
in human society.4 Thus, the rapid, unambiguous, and selective visible change from pale orange to deep pink, as seen in Fig. S1a
detection of CNꢀ ions in aqueous environments by employing an (ESI†); a new emission band appeared at 595 nm (Fig. S1b, ESI†)
eye-catching optical method is crucial. Efforts have been made to when excited at 565 nm. To determine the sensitivity of IND-1
develop CNꢀ sensors based on H-bonding motifs,5 oxazines,6 toward CNꢀ ions, we measured the concentration dependent
cationic borane derivatives,7 acridinium salts,8 b-turn motifs,9 fluorescence changes. In Fig. 1a, the fluorescence intensity at
fluorogenic ensembles,10 and reaction-based chemodosimeters.11
lem = 595 nm gradually increases with increasing concentrations
Most of these systems give a response either through colorimetric of CNꢀ (0–3 equiv.). We determined the lower detection limit to
a Department of Chemistry, Korea University, Seoul 136-701, Korea.
E-mail: jongskim@korea.ac.kr
b Amrita Centre for Industrial Research & Innovation, Amrita Vishwa Vidyapeetham,
Ettimadai, Coimbatore 641112, India. E-mail: b_sankarprasad@cb.amrita.edu
c Department of Chemical Engineering & Materials Science,
Amrita Vishwa Vidyapeetham, India 641112
† Electronic supplementary information (ESI) available: The synthesis procedure,
NMR, MS, fluorescence, and cell imaging data. See DOI: 10.1039/c5cc02234g
Scheme 1 Synthesis of probe IND-1.
‡ These authors contributed equally to this work.
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Fig. 2 1H-NMR spectra (400 MHz, DMSO-d6 with 10% D2O) of IND-1 (red),
IND-1 with CNꢀ (blue), resorufin (green), and 9,10-phenanthrenequinone
(black). Inset: photograph of IND-1 (1), IND-1 (10 mM) with 10 equiv. of CNꢀ
(2), and resorufin (3) taken under a UV-lamp at 365 nm.
Fig. 1 (a) Fluorescence spectra of IND-1 (10 mM) with various concentrations
of CNꢀ (0–3 equiv.). (b) Time dependent changes in the fluorescence intensity
of IND-1 (10 mM) with and without 10 equiv. of CNꢀ in PBS buffer (10 mM, pH
7.4) with 1% DMSO (lex = 565 nm, lem = 595 nm, excitation and emission slit
The pH sensitivity of the IND-1 reaction with KCN was
widths: 1.5 nm). (c) Fluorescence intensity of the probe, normalized for the determined by monitoring the changes in the fluorescence
minimum fluorescence intensity in the absence of CNꢀ (shown on the graph as
intensity at 595 nm at various pH values (4–9). There was no
1 nM) and the maximum fluorescence intensity (1 mM CNꢀ). (d) Color changes
change in the fluorescence intensity over this pH range (Fig. S4,
of test strips containing IND-1 (10 mM) treated with various anions (CNꢀ, none,
ESI†), indicating that IND-1 can be used to sense CNꢀ ions over
Fꢀ, Clꢀ, Brꢀ, Iꢀ, CH3CO2ꢀ, HPO42ꢀ, HCO3ꢀ, NO3ꢀ, ClO4ꢀ, SCNꢀ, GSH, Cys,
a wide range of pH values.
and Hcy; 10 equiv.).
Curiosity drove us to determine the mechanism of action of
IND-1 toward CNꢀ ions. Thus, we carried out a 1H-NMR titration
experiment in the presence of KCN at variable time intervals.
Within 5 min, the aldehyde proton peak in IND-1 at 9.78 ppm
be 4 nM (Fig. 1c), which is much lower than the permissible
cyanide concentration (1.9 mM) in drinking water prescribed by the
World Health Organization (WHO).13 To the best of our knowledge,
this is the lowest detection limit reported for the sensing of CNꢀ
ions. Interestingly, IND-1 is capable of detecting CNꢀ ions in
human blood serum using the fluorescence enhancement at
lem = 595 nm at CNꢀ ion concentrations as low as 50 nM, as
shown in Fig. S2 (ESI†). This result implied that IND-1 could be
used to detect CNꢀ ions in living systems.
(
) was observed to disappear completely (Fig. 2) with concomitant
appearance of a new set of peaks at 7.52–8.22 ppm. To identify the
new set of proton peaks, we recoded the 1H-NMR spectra of
resorufin and 9,10-phenanthrenequinone. The proton signals and
integration ratios of resorufin and 9,10-phenanthrenequinone were
1
found to exactly match those observed in the H-NMR spectra of
KCN-treated IND-1 (Fig. 2).
To implement sensing in real systems, the detection time for
CNꢀ ions is crucial. Thus, we carried out time-course experiments
in the presence of KCN (10 equiv.) in PBS buffer (Fig. 1b). Within
10 s resorufin was fully released from IND-1; consequently, the
fluorescence signal recovered and reached saturation. This result
suggested that IND-1 is very sensitive and has a fast response to
CNꢀ ions.
The mass spectrum data of KCN-treated IND-1 (Fig. S10, ESI†)
also revealed that 9,10-phenanthrenequinone was formed. More-
over the TLC profile (Fig. 2, inset) of KCN treated IND-1 exactly
matched with those of resorufin and 9,10-phenanthrenequinone
respectively. These experimental findings strongly supported our
In practice, the selectivity of IND-1 toward a particular analyte
in the presence of other interfering anions is very important. We
recorded the fluorescence changes of IND-1 in the presence of
the potassium salts of various anions, such as CNꢀ, Fꢀ, Clꢀ, Brꢀ,
Iꢀ, CH3CO2ꢀ, HPO42ꢀ, HCO3ꢀ, NO3ꢀ, ClO4ꢀ, and SCNꢀ, and
some neutral species, such as Cys, Hcy, and GSH (Fig. S3, ESI†).
IND-1 was observed to be highly selective for CNꢀ ions; there was
no significant change in the fluorescence signal in the presence
of other anions. Fig. 1d depicts the color changes of test strips
containing IND-1 in the presence of various analytes. Interestingly,
only CNꢀ ions caused a color change to pink, whereas the color was
unaltered with the other anions. This result suggests that IND-1 can
be used for high throughput screening of KCN in immobilized
systems, even in the presence of other ions.
Scheme 2 KCN catalyzed intramolecular crossed-benzoin reaction of
IND-1.
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speculation on KCN catalyzed intramolecular crossed-benzoin
Finally, we evaluated whether IND-1 can show the KCN catalyzed
reaction (Scheme 2) that produces the discrete new molecule, intramolcular crossed-benzoin reaction to give a cell image based on
9,10-phenanthrenequinone.
cellular CNꢀ ions. Fig. 4a indicates that fluorescence intensity
We then evaluated the biocompatibility of IND-1 by the MTT gradually increases with increasing concentration of KCN. The cell
assay. Data in Fig. 3 revealed that until 20 mM IND-1, the cells images in Fig. 4a and Fig. S5 (ESI†) implied that KCN in cells can be
remain healthy without a change in cell viability. This indicates detected using this IND-1 irrespective of various cell lines (HeLa and
that IND-1 can be used in the cellular level to assess the presence A549). In addition, we carried out the co-localization experiment to
of CNꢀ ions.
know the distribution of KCN in cells and justify whether IND-1
is capable of noting the KCN throughout the cells. The Fig. 4b
concluded that fluorescence images of IND-1 co-localized with
Mito-tracker and ER-tracker as well.
Furthermore, Mito-tracker, ER-tracker, and IND-1 were sub-
jected to fluorescence profile studies through the transverse
section of the HeLa cells. The fluorescence profile of the HeLa
cells treated with IND-1 exactly matched the profiles of the
HeLa cells labelled with Mito-tracker and ER-tracker (Fig. S6,
ESI†). These results led us to conclude that IND-1 is an efficient
probe to track KCN throughout the cells, including sub-cellular
organelles.
In conclusion, we developed for the first time a molecular
probe, IND-1, for sensing KCN in aqueous environments based
on an intramolecular crossed-benzoin reaction. The synthesis
of this turn-on fluorescent chemodosimeter probe was very
simple, and IND-1 was shown to be an efficient probe with a
fast response in two modes i.e. as a chromogenic and fluoro-
genic sensor for KCN in aqueous and biological environments.
IND-1 displayed sensitivity toward concentrations of KCN as
low as 4 nM, which is remarkably lower than any previously
reported system. Furthermore, IND-1 is capable of detecting
KCN at a concentration of 50 nM in blood serum by amplifying
the emission signal at 595 nm. Finally, cellular imaging and
co-localization experiments strongly suggested that IND-1 could
be used to track KCN in cellular environments.
Fig. 3 Cell viability assay of IND-1 on HeLa cell lines. IND-1 was incubated
with the cells for 24 h, and the cell viability observed via the MTT assay.
This work was supported by CRI project (no. 2009-0081566,
J.S.K.) and by AVV research grant (S.B.).
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