Coumarin-based fluorescent sensors for zinc(II) and hypochlorite

Article abstract of DOI:10.1080/10610278.2015.1077958

Two new fluorescent chemosensors have been designed and synthesised through functionalisation of a common 7-aminocoumarin precursor. One sensor features a di(2-picoyl)amine metal ion-binding moiety that allows for selective detection of Zn²⁺ ions over other common cations in aqueous solution, with the sensor exhibiting a 5.4-fold increase in fluorescence upon Zn²⁺ binding (limit of detection = 26 nM). The second sensor incorporates an aminophenyl group designed to react with highly reactive oxygen species (hROS). This sensor reacts with ClO^- in preference to other ROS, exhibiting a 27-fold increase in fluorescence upon the addition of 150 equivalents of ClO^- (limit of detection = 2 M).

Full text of DOI:10.1080/10610278.2015.1077958

Supramolecular Chemistry
ISSN: 1061-0278 (Print) 1029-0478 (Online) Journal homepage: http://www.tandfonline.com/loi/gsch20
Coumarin-based fluorescent sensors for zinc(II)
and hypochlorite
Margaret L. Aulsebrook, Bim Graham, Michael R. Grace & Kellie L. Tuck
To cite this article: Margaret L. Aulsebrook, Bim Graham, Michael R. Grace & Kellie L. Tuck
(2015) Coumarin-based fluorescent sensors for zinc(II) and hypochlorite, Supramolecular
Chemistry, 27:11-12, 798-806, DOI: 10.1080/10610278.2015.1077958
To link to this article: http://dx.doi.org/10.1080/10610278.2015.1077958
View supplementary material
Published online: 26 Nov 2015.
Submit your article to this journal
Article views: 15
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=gsch20
Download by: [University of Sussex Library]
Date: 01 December 2015, At: 07:41

Supramolecular Chemistry, 2015
Vol. 27, Nos. 11–12, 798–806, http://dx.doi.org/10.1080/10610278.2015.1077958
Coumarin-based fluorescent sensors for zinc(II) and hypochlorite
a
Margaret L. Aulsebrook , Bim Graham , Michael R. Grace and Kellie L. Tuck *
b
a
a
a b
School of Chemistry, Monash University, Clayton, VIC, Australia; Monash Institute of Pharmaceutical Sciences, Monash University,
Melbourne, VIC, Australia
(
Received 29 May 2015; accepted 24 July 2015)
Two new fluorescent chemosensors have been designed and synthesised through functionalisation of a common
-aminocoumarin precursor. One sensor features a di(2-picoyl)amine metal ion-binding moiety that allows for selective
7
detection of Zn ions over other common cations in aqueous solution, with the sensor exhibiting a 5.4-fold increase in
2þ
2þ
fluorescence upon Zn binding (limit of detection ¼ 26 nM). The second sensor incorporates an aminophenyl group
2
2
designed to react with highly reactive oxygen species (hROS). This sensor reacts with ClO in preference to other ROS,
exhibiting a 27-fold increase in fluorescence upon the addition of 150 equivalents of ClO (limit of detection ¼ 2 mM).
Keywords: chemosensor; coumarin; zinc; reactive oxygen species; hypochlorite
z2
(27). ROS includes the superoxide anion radical (O ),
2
1
.
Introduction
z
hydrogen peroxide (H O ), alkylperoxyl radicals (ROO),
In recent years, there has been significant interest in the
development of chemosensors for the detection and
quantification of various analytes in environmental and
biological samples. A vast number of fluorescent sensors
have been reported, with many showing good selectivity
and sensitivity for specific analytes of interest (1–15).
Broadly speaking, analyte detection using fluorescent
sensors can be achieved via one of four mechanisms:
protonation/deprotonation, redox reaction, complexation
or bond cleavage/formation (7). Despite significant
advances, the development of sensors that are applicable
to use within aqueous systems is still an important
objective. Fluorescent precursors that can be readily and
diversely functionalised to achieve selectivity for a wide
range of analytes are particularly valuable to the field.
For a number of years, we (14, 16) and others (1–5,
2 2
1
singlet oxygen ( O ) (28–30), and hROS includes the
2
z
hydroxyl radical ( OH), peroxynitrite (ONOO ) and
2
2
hypochlorite (ClO ) (28–30). Although hROS are important
signalling molecules for various pathological processes, their
over-production has been implicated in the pathogenesis of
human diseases due to oxidative stress (31). The detection of
ROS and hROS in environmental samples is also of interest,
as their concentrations can have a direct effect on the health
of an ecosystem (32–34). A range of chemical sensing
probes have been reported within the literature for the
detection of hROS and ROS (see (7, 28–31) and references
within); however, many of these probes suffer from the
inability to distinguish between different hROS.
Herein, we report a facile route to access two new
2þ
fluorescent sensors – one for free Zn ions and one for
hypochlorite (Figure 1) – from a common 7-aminocou-
marin-based precursor. 7-Aminocoumarins exhibit exci-
tation/emission wavelengths ranging from 360 to 550 nm and
represent attractive scaffolds for sensor development because
they possess high fluorescent quantum yields and large
2
þ
17–26) have been interested in the detection of Zn in
1
biological or environmental samples. The d electronic
0
2
þ
configuration of Zn renders it spectroscopically and
magnetically inactive, making its detection problematic.
2
þ
Speciation of Zn
context since, in addition to the ‘free’ form (the octahedral
2
cation, Zn(H O)6 ), it may exist in colloidal or particulate
is important in an environmental
2þ
Stokes’ shifts (21, 35). Sensor 1 is a complexation-type Zn
sensor featuring a di-(2-picolyl)amine (DPA) metal ion-
þ
2
2þ
forms, which are considered non-bioavailable and non-
2
toxic. Current methods used for the quantification of Zn
are unable to distinguish between the non-toxic forms of
zinc and the potentially toxic, free cationic form.
binding moiety. In the absence of Zn , it was anticipated
that the fluorescence of this compound would be significantly
quenched because of photo-induced electron transfer (PET)
from the lone pair of the tertiary amine of the DPA to the 7-
þ
2þ
We have also been interested in the development of
fluorogenic sensors for the detection of reactive oxygen
species (ROS) or highly reactive oxygen species (hROS),
chemical entities formed via incomplete reduction of oxygen
aminocoumarin group. Upon Zn binding, however, the
sensor was expected to switch to a more emissive state due to
2þ
the lone pair forming a coordinative bond with the Zn (36)
(it is highly unlikely that the aniline nitrogen of the
*Corresponding author. Email: kellie.tuck@monash.edu
q 2015 Taylor & Francis

Supramolecular Chemistry
799
from commercially available 3-aminophenol 5 and
phenylchloroformate 6, according to the reported pro-
cedure by Higuchi and co-workers (37) (Scheme 2, see
Supplemental data for experimental procedures to
synthesise compound 3). Sensor 1 was then accessed in
good yield through a Finkelstein-like reaction between 3
and di-(2-picolyl)amine (DPA) (Scheme 2). Sensor 2 was
synthesised in two steps from 3, beginning with reaction of
3 with Boc-protected 4-aminophenol 9 (38), followed by
cleavage of the Boc group under acidic conditions
H N
O
O
2
H N
O
O
2
O
N
N
N
NH2
1
2
detection of Zn2+
detection of hROS
(
Scheme 2).
H N 7
O
O
2
2
1
2
.2 Testing of Zn sensor
The absorbance and fluorescence emission spectra of sensor
were recorded at a concentration of 4 mM in aqueous buffer
solution (pH 7.4, 10 mM HEPES). The maximum absorption
4
Cl
3
1
sensor precursor
3
21
21
Figure 1. (Colour online) Structures of the 7-aminocoumarin-
based sensors 1 and 2 and the precursor, 7-amino-4-
chloromethylcoumarin 3.
of the compound 1 (e ¼ 6.0 £ 10 M ·cm ) was centred
at 360 nm (Supplemental data, Figure S1), and the maximum
fluorescence emission at 470 nm (Figure 2).
As anticipated, the fluorescence intensity of 1 increased
2þ
2
þ
aminocoumarin unit binds to Zn due to the delocalisation
of its lone pair into the ring system). Sensor 2 is a cleavage-
based probe, designed for the fluorogenic detection of hROS
in aqueous media. It features an electron-rich aminophenyl
group appended to 7-aminocoumarin, which serves to
initially quench fluorescence through a PET mechanism, as
previously demonstrated by Nagano and co-workers for
fluorescein- and rhodamine-based sensor designs (29, 30).
Upon exposure to hROS, it was expected that cleavage of the
aminophenyl group would occur to produce a highly
emissive 7-aminocoumarin derivative, 4 (Scheme 1). It has
been previously reported that O-dearylation of ipso-
substituted oxyaryl moieties occurs selectively through
reaction with hROS, but not other ROS (29, 30).
linearly with the addition of up to 1.0 equiv of Zn ions. The
intensity continued to increase slightly upon the addition of
2þ
further Zn , with no further increase observed after addition
2þ
of 6 equiv of Zn ions. An overall 5.4-fold increase in
fluorescence intensity was observed. The quantum yield of
2þ
the Zn complex of 1 was determined to be 0.70, compared
to 0.22 for uncomplexed 1. Job plot analysis (Supplemental
data, Figure S2) revealed a 1:1 binding stoichiometry.
2
þ
The effect of addition of a range of cations (Mg
2þ
þ
,
,
þ
2þ
2þ
þ
2þ
2þ
2þ
3þ
Na , Mg , Hg , Co , K , Ni , Pb , Cd , Fe
2
Ca ) on the fluorescent intensity of the sensor was
investigated in order to assess the degree of selectivity for
2
Zn (Figure 3). The measurements were made in buffered
þ
2
solution in the presence and absence of Zn . Only Pb
þ
2þ
2
þ
and Cd resulted in an increase in the basal fluorescence
intensity, while a quenching effect was observed for both
2
.
Results and discussion
2
þ
2þ
the paramagnetic ions, Cu and Ni . All other cations
had little effect on the basal fluorescent intensity of the
2.1 Synthesis of sensors
2
sensor. In the presence of Zn , little deviation from the
þ
The common advanced intermediate compound, 7-amino-
-chloromethylcoumarin 3, was synthesised in three steps
2
expected fluorescence for just the 1-Zn complex was
þ
4
H N
O
O
2
H N
O
O
2
hROS
O
OH
O
NH2
4
strongly fluorescent
NH2
2
almost non-fluorescent
Scheme 1. Anticipated fluorogenic reaction between sensor 2 and hROS.

8
00
M.L. Aulsebrook et al.
H
H
N
O
N
OH
O
O
O
H N
OH
O
Cl
2
(i)
(ii)
+
O
O
O
7
8
5
6
Cl
(
iii)
O
O
H N
2
H N
O
O
2
(
iv)
N
N
N
Cl
v)
1
3
(
H N
O
O
2
O
NHR
1
0 R = Boc
(
vi)
2
.TFA R = H
Scheme 2. Synthesis of the 7-aminocoumarin-based sensors 1 and 2. Reagents and conditions: (i) EtOAc, D, 1 h, 79%; (ii) Ethyl
-chloroacetoacetate, H SO :H O (3:1), RT, 24 h, 56%; (iii) TBAF, THF, RT, 1 h, 82%; (iv) di-(2-picolyl)amine (DPA), Na CO , NaI,
4
2
4
2
2
3
2 3
DCM, RT, 3 days, 82%; (v) tert-butyl(4-hydroxyphenyl)carbamate 9 (38), K CO , ACN, D, 2 h, 61%, (vi) TFA, DCM, RT, 24 h, 73%.
Figure 2. (Colour online) (a) Changes in the fluorescent emission spectrum (lex ¼ 360 nm) of sensor 1 (4 mM) upon the addition of
2
þ
Zn ions (0 ! 24 mM; spectra measured in 10 mM HEPES buffer (pH 7.4)). (b) Changes in fluorescence intensity detected at 470 nm
2þ
(
average of triplicate experiments). (c) Sensor 1 (4 mM) in the absence and presence of Zn (4 mM), 10 mM HEPES buffer (pH 7.4),
illuminated with a hand-held UV lamp, lex ¼ 365 nm.

Supramolecular Chemistry
801
Figure 3. (Colour online) Fluorescence intensity changes (lex ¼ 360 nm) for sensor 1 (4 mM, 10 mM HEPES buffer pH 7.4) in the
2
presence of various cations. Red bars: 1 alone (4 mM); blue bar 1 (4 mM) and Zn (4 mM). (a) Black bars: 1 (4 mM) and cation (4 mM);
þ
2þ
grey bars: 1 (4 mM), cation (4 mM) and Zn (4 mM). (b) Black bars: 1 (4 mM) and cation (40 mM); grey bars: 1 (4 mM), cation (40 mM)
and Zn (4 mM) (average of triplicate experiments).
2þ
2
þ
observed in the presence of all of the cations. These effects
were the same, but more pronounced, upon the addition of
The limit of detection (LOD) of sensor 1 for Zn ions
was determined to be 26 ^ 1 nM, and thus it is readily able
2
0 equiv of the cations, with the exception of Co , which
þ
2þ
1
produced a quenching effect.
to detect the toxic threshold of Zn ions (7.8 mM), as
specified by the US EPA (Environment Protection
Authority) (39). To demonstrate its potential utility for
2
environmental monitoring of Zn , an environmental
þ
1
water sample was spiked with a known amount of
2
þ
2þ
Zn (3 mM) and subject to Zn analysis using 1 (the
standard addition method was used in order to eliminate
interference from the sample matrix; see Supplemental
N
N
HO
O
O
N
N
2þ
data, Figure S3). The Zn concentration was determined
to be 3.1 ^ 0.1 mM, which closely agrees with the known
H CO
3
N
N
2
concentration of Zn in the spiked water sample.
þ
H CO
O
O
3
2
þ
Two other coumarin-based Zn sensors have been
reported in the literature that are relevant to this work –
compounds 11 (21) and 12 (22) (Figure 4). Compound
11 features two electron-donating dimethoxy groups at the
11
12
2þ
Figure 4. Previously reported coumarin-based Zn fluorescent
sensors 11 (21) and 12 (22).

8
02
M.L. Aulsebrook et al.
Figure 5. (Colour online) Fluorescent ‘turn-on’ response of sensor 2 to various ROS. All ROS were added at a concentration of 200 mM.
Bars represent emission intensities taken at 0 (white), 15 (light grey), 30 (dark grey), 45 (black) and 60 (purple) min after ROS addition to
2
(4 mM). Data acquired at 23 8C in 10 mM HEPES buffer, pH ¼ 7.4, lex ¼ 360 nm, lem ¼ 470 nm (average of triplicate experiments).
2
þ
2þ
2þ
6- and 7-positions of the coumarin group and the Zn
binding group is attached at the 4-position. Compound 12
response for Zn in the presence of Mg (21), while
compound 12 suffers considerable interference from
2
þ
2þ 2þ
has an electron-donating group at position 6 and the Zn
binding group is attached at position 8. In comparison,
sensor 1 has an electron-donating group at the 7-position
Mg , and Cd produces a similar fluorescent response
2
to Zn with this sensor (22). Our fluorescent sensor is not
þ
þ
þ
2þ
2þ
subject to interference by Na , K , Ca and Mg , which
are the most common cations in aquatic systems.
2
þ
and the Zn binding group is attached at position 4. It has
been reported that the most emissive coumarin dyes
feature electron-donating groups at position 7 and
electron-accepting groups at positions 3, 4 and 6 (35).
Compounds 11 and 12 show similar sensing properties
to 1; however, neither of these compounds has been used to
test an aqueous environmental sample and 11 has only
been used in methanol. Compound 11 shows a diminished
2
.3 Testing of hROS sensor
The absorbance and fluorescence emission spectra of
sensor 2 were recorded at a concentration of 4 mM in
aqueous buffer solution (pH 7.4, 10 mM HEPES). The
2
Figure 6. (Colour online) (a) Changes in fluorescent emission spectrum (lex ¼ 350 nm) of sensor 2 (4 mM) upon the addition of ClO
ions (0 ! 800 mM; spectra measured in 10 mM HEPES buffer (pH 7.4)). (b) Changes in fluorescence intensity detected at 470 nm
2
(
average of triplicate experiments). Each measurement was recorded after 30 min. (c) Sensor 2 (4 mM) in the absence and presence of
ClO (200 mM), 10 mM HEPES buffer (pH 7.4), illuminated with a hand-held UV lamp, lex ¼ 365 nm.

Supramolecular Chemistry
803
interference from other common aquatic cations. Sensor 2
NH2
2
showed a high degree of selectivity for ClO over other
ROS tested. It exhibited a linear increase in fluorescence
2
upon the addition of up to 150 equiv of ClO , and was able
to detect this species down to a concentration of ca. 2 mM.
O
O
O
NH2
CO2–
N
O
N
4.
Experimental
4.1 General remarks
13
14
Preparative high-performance liquid chromatography
HPLC) was performed on an 1260 Infinity Quaternary LC
(
Figure 7. Fluorescent sensors developed by Nagano and co-
workers for the detection of ROS (29, 30).
System with a 1290 Infinity Diode-Array Detector using a
Phenomenex Luna C8 column (150 £ 21.5 mm, 5 micron)
with a 10 mL per minute flow rate. The elution method used
for HPLC purification was: 100% buffer A for 4 min, then
gradient from 100% buffer A to 100% buffer B over 30 min
3
21
21
maximum absorption of 2 (e ¼ 3.7 £ 10 M ·cm ) was
centred at 350 nm (Supplemental data, Figure S4), and the
maximum fluorescence emission at 470 nm. The fluor-
escent quantum yield of 2 was determined to be 0.01,
compared to 0.89 for the product expected upon reaction
with hROS (Scheme 1).
(
buffer A ¼ 0.1% TFA in MilliQ water, buffer B ¼ 0.1%
TFA in 80% ACN/20% MilliQ water). Analytical HPLC was
conducted using a Phenomenex Luna C8 column (150
£ 4.6 mm, 5 micron). Proton nuclear magnetic resonance
·
· t
When treated with the ROS H O , TBHP, OH, O Bu
2
2
1
1
and O , no change in fluorescent intensity was observed
( H-NMR) spectra were recorded on either a Bruker AV400
2
or Bruker AV600 spectrometer operating at 400 and
600 MHz respectively, as solutions in deuterated solvents
as specified. Each resonance was assigned according to the
following convention: chemical shift; multiplicity; number of
protons; observed coupling constants (J in Hertz) and proton
assignment. Chemical shifts (d), measured in parts per
million (ppm), are referenced to residual solvent signal.
(
measurements made every 15 min for 1 h; Figure 5).
2
However, when treated with ClO , the fluorescence
increased 12-fold over the 1 h period. LC–MS analysis
2
confirmed ClO had cleaved the aminophenyl moiety as
predicted, to produce compound 4 in Scheme 1. Nitric
oxide (NO) also produced an increase in fluorescence,
2
although not as dramatic as that observed for ClO . These
results suggest that 2 can be used as a selective fluorescent
13
Carbon-13 nuclear magnetic resonance ( C-NMR) spectra
were recorded on either a Bruker AV400 or Bruker AV600
spectrometer operating at 100 and 150 MHz, respectively, as
solutions in deuterated solvents as specified. Assignments
were determined from J-Modulated Spin Echo experiments
showing quaternary and methylene signals in the opposite
phase to those of methine and methyl resonances. Correlation
spectroscopy (COSY) was used to correlate chemical shifts
of protons coupled to one another. Heteronuclear multiple
quantum correlation (HMQC) spectroscopy was used to
2
sensor for ClO in the presence of other ROS.
2
Addition of increasing amounts of ClO to sensor 2
with 30 min intervals between titres) produced a linear
(
increase in fluorescence, with the intensity reaching 27
times the initial value after the addition of 150 equiv of
2
ClO (Figure 6). Plateauing was observed upon adding
2
further ClO . The LOD was determined to be
2
.0 ^ 0.8 mM. The hROS-sensing capabilities of 2 are
similar to those of the fluorescein- and rhodamine-based
compounds, 13 and 14, reported by Nagano and co-
workers (Figure 7) (29, 30).
13
1
correlate directly bonded C– H nuclei. Melting point
analyses were performed on a Stuart Scientific SMP3 melting
point apparatus. High-resolution mass spectrometry (HRMS)
was performed on a Waters LCT TOF LC/MS Mass
Spectrometer coupled to a 2795 Alliance Separations
module. All data were acquired and mass corrected via a
dual-spray Leucine Enkephaline reference sample. Mass
spectra were created by averaging the scans across each peak
and background subtracted of the total ion count (TIC).
Acquisition and analysis were performed using the Masslynx
software version 4.1. Low-resolution mass spectrometry
(LRMS) was conducted using a Micromass Platform II QMS
(ESI). Infrared spectra (IR) were recorded on an Agilent
Technologies Cary 630 FTIR as thin films of compressed
powders. UV-visible absorption spectra were recorded at
room temperature using a Varian Cary 1E UV-visible
3
.
Conclusions
This study demonstrates the potential to rapidly access
fluorescent sensors for a diverse range of analytes from a
common 7-aminocoumarin scaffold. Two new fluorescent
sensors, 1 and 2, were synthesised through simple
nucleophilic substitution reactions of compound 3 with
2
þ
groups designed to enable selective sensing of free Zn
ions and hROS, respectively. Both sensors were shown to
function in aqueous solution, at a biologically/environ-
mentally relevant pH (7.4). Sensor 1 was able to detect
2
þ
Zn ions at a concentration as low as 26 nM, with little

8
04
M.L. Aulsebrook et al.
spectrophotometer with a 10 mm path length cell. Fluor-
escent emission spectra were recorded at room temperature
using an Agilent Technologies Cary Eclipse fluorescence
spectrophotometer. A quartz cell with a path length of 10 mm
and a volume of 400 mL was used. The instrument excitation
and emission slit widths were both set at 5 nm.
(d, J ¼ 1.1 Hz, 1H, H1), 6.68 (dd, J ¼ 4.3, 2.2 Hz, 1H,
1
3
H5), 7.05 (m, 2H, H12), 7.51 (m, 2H, H11/H4); C-NMR
(d -Acetone, 100 MHz) d: 28.6 (C16), 79.7 (C15), 100.4
6
(C1), 107.3 (C7), 108.3 (C8), 112.2 (C5), 115.9 (C12),
120.6 (C4), 126.2 (C11), 134.5 (C6), 152.3 (C3), 153.6
(C14), 153.9 (C10), 154.5 (C13), 157.1 (C2), 161.5
(C ¼ O); IR (ATR): 3451, 3358, 3328, 2981, 1691, 1601,
All starting materials and solvents were of reagent or
analytical grade and were used as purchased.
2
1
þ
1515 cm ; LRMS (ESI þ ): m/z [M þ Na] 405.1
þ
þ
(
100%); HRMS (ESI ): calcd. for [M þ H] : 405.1421,
found: 405.1427.
4
.2 Syntheses
4
2
.2.1 4-[[bis(2-Pyridylmethyl)amino]methyl]-7-amino-
H-1-benzopyran-2-one (1)
4.2.3 7-Amino-4-((4-aminophenoxy)methyl)-2H-
chromen-2-one TFA salt (2.TFA)
7
-Amino-4-(chloromethyl)-2H-1-benzopyran-2-one
(20 mg, 97 mmol) was added to a stirring solution of
3
tert-Butyl (4-((7-amino-2-oxo-2H-chromen-4-yl)meth-
oxy)phenyl)carbamate 10 (11 mg, 0.02 mmol) was dis-
solved in a 1:1 DCM:TFA mixture (2 mL). The reaction
was allowed to stir at RT overnight. Following this, excess
DCM (4 mL) containing di-(2-picolyl)amine (36 mg,
81 mmol) and Na CO (65 mg, 612 mmol). The reaction
1
2
3
was refluxed for 3 days in the presence of NaI (26 mg,
71 mmol). The mixture was then filtered and dried under
vacuum, producing a yellow solid residue. The residue was
1
TFA and DCM were removed by a stream of N . The
2
residual was then dissolved in a mixture of H O and ACN
2
dissolved in a H O–ACN mixture and purified by
2
and purified by preparative HPLC. The fractions contain-
ing the pure product were concentrated in vacuo to yield
preparative reverse phase HPLC. Fractions containing
the pure product were combined and concentrated under
reduced pressure to yield the desired product as a viscous
the final product as the TFA salt (5.5 mg, 0.02 mmol);
1
H-NMR (D O, 400 MHz) d: 4.77 (s, 2H, partially
2
1
yellow oil (31 mg, 82%): H-NMR (D O, 400 MHz) d:
2
4
obscured by HOD peak), 6.11 (s, 1H), 6.73 (d, J ¼ 1.8 Hz,
.01 (s, 2H, H9), 4.40 (s, 4H, H10), 6.42 (s, 1H, H7), 6.85
1
H), 6.82 (d, J ¼ 8.8 Hz, 2H), 6.86 (dd, J ¼ 8.8, 1.8 Hz,
(
m, 1H, H1), 7.04 (m, 1H, H5), 7.55 (d, J ¼ 8.6 Hz, 1H,
1H), 7.16 (d, J ¼ 8.8 Hz, 2H), 7.29 (d, J ¼ 8.8 Hz, 1H);
H4), 7.90 (m, 2H, H14), 8.06 (d, J ¼ 8.0 Hz, 2H, H12),
1
H-NMR (DMSO-d , 600 MHz) d: 5.25 (s, 2H, H9), 6.08
6
8
1
.46 (td, J ¼ 8.0, 1.5 Hz, 2H, H13), 8.61 (m, 2H, H15);
(
s, 1H, H7), 6.45 (d, J ¼ 1.7 Hz, 1H, H1), 6.58 (dd,
3
C-NMR (D O, 100 MHz) d: 56.4 (C10), 57.6 (C9), 107.0
2
J ¼ 1.7, 8.8 Hz, 1H, H5), 7.05 (m, 4H, H11/H12), 7.47 (d,
(
C1), 113.6 (C5), 114.1 (C3), 117.0 (C7), 127.1 (C4),
27.2 (C14), 128.2 (C12), 141.9 (C15), 147.7 (C13), 152.6
C6), 153.0 (C2), 154.7 (C8), 163.3 (C11), 163.7 (C ¼ O);
IR (ATR): 3225, 3065, 2853, 1676, 1597, 1544, 1467,
13
J ¼ 8.8 Hz, 1H, H4); C-NMR (DMSO-d , 150 MHz) d:
6
1
6
1
1
5.8 (C9), 98.7 (C1), 105.0 (C7), 106.0 (C8), 111.3 (C5),
15.9 (C11), 117.1 (q, J ¼ 31.2 Hz, TFA), 120.0 (C12),
25.7 (C4), 133.6 (C3), 152.0 (C6), 153.3 (C13), 153.4
(
2
1
1
401, 1177, 766 cm ; LRMS (ESI þ ): m/z 373.3
(
C10), 155.7 (C2), 158.1 (q, J ¼ 298.1 Hz, TFA), 160.8
þ þ
[
M þ H] (100%), 395.3 [M þ Na] (12%); HRMS
(
C ¼ O); IR (ATR): 3362, 2910, 1690, 1608, 1511,
þ
(ESI þ ): calcd. for C H N O [M þ H] : 373.1659,
found: 373.1650.
21
þ
2
2 21 4 2
1
070 cm ; LRMS (ESI þ ): m/z [M þ H] 283.2 (100%);
þ
HRMS (ESI þ ): calcd. for [M þ H] : 283.1077, found:
2
83.1077.
4
.2.2 tert-Butyl (4-((7-amino-2-oxo-2H-chromen-4-yl)
methoxy)phenyl)carbamate (10)
tert-Butyl(4-hydroxyphenyl)carbamate 9 (38) (52 mg,
4.3 General analytical procedures
4
.3.1 UV-visible absorption measurements
0
2
0
.2 mmol), 7-amino-4-(chloromethyl)-2H-1-benzopyran-
-one 3 (52 mg, 0.2 mmol) and K CO (90 mg,
.7 mmol) were dissolved in ACN (4 mL) and refluxed
2
3
The absorption spectra of sensors 1 (50 mM) and 2 (100 mM)
were separately measured in 10 mM HEPES aqueous buffer
at pH 7.4.
for 2 h. The reaction mixture was then allowed to cool and
solvent was removed in vacuo. The crude was re-dissolved
in EtOAc (15 mL) and washed with water and brine. The
crude compound was purified by basic alumina chroma-
tography (1% MeOH in DCM) to yield the product as a
4.3.2 Quantum yield measurements
1
yellow solid (58 mg, 61%): mp: 212.2–214.8 8C; H-NMR
Fluorescence quantum yields were determined by
comparing the spectrum with that of anthracene
(F ¼ 0.27) in ethanol by taking the area under the
(
d6-Acetone, 400 MHz) d: 1.46 (s, 9H, H16)), 5.23
d, J ¼ 1.3 Hz, 2H, H9), 6.21 (t, J ¼ 1.2 Hz, 1H, H7), 6.56
(

Supramolecular Chemistry
805
emission spectra following the equation:
the concentrations range of 0–2 mM, with the fluorescence
spectrum recorded after each addition (lex ¼ 360 nm and
lem ¼ 470 nm).
ꢀ
ꢁ
ꢀ
ꢁ
2
Gradx
hX
FX ¼ FST
£
GradST
hST
where subscripts X and ST denote sample and standard,
respectively, F is the quantum yield, Grad is the gradient
of the plotted integrated fluorescence intensity vs.
absorbance and h is the refractive index of solvents. The
fluorescence measurements in solutions were carried out at
4
.4.5 Job plot analysis
The fluorescence spectra of solutions containing varying
2
þ
2þ
concentrations of sensor 1 and Zn (1 þ [Zn ] ¼ 4 mM)
were measured in 10 mM HEPES buffer at pH 7.4
(lex ¼ 360 nm and l ¼ 470 nm). Corresponding blanks
em
2
6
1
0
M.
were prepared which contained 1 only.
2
.4 Testing of Zn sensor
1
4
4
.4.1 Fluorescence measurements
4
.5 Testing of hROS sensor
The fluorescence spectrum of sensor 1 (4 mM) was
measured in 10 mM HEPES buffer at pH 7.4,
4.5.1 Fluorescence measurements
2
þ
The fluorescence spectrum of sensor 2 (4 mM) was
measured in 10 mM HEPES buffer at pH 7.4,
(
lex ¼ 360 nm), with addition of varying Zn amounts
(
0.00, 0.05, 0.10, 0.20, 0.25, 0.38, 0.50, 0.62, 0.75, 0.88,
2
2
.00, 2.00, 3.00, 4.00, 5.00, 6.00 equiv of Zn ).
þ
(
(
lex ¼ 350 nm), with addition of varying ClO amounts
2
1
0.00, 6.25, 12.50, 18.75, 25.00, 31.25, 37.50, 43.75,
5
0.00, 62.50, 75.00, 75.00, 100.00, 150.00 equiv of ClO ).
4
.4.2 Metal ion selectivity measurements
The fluorescent emission from sensor 1 (4 mM) was
measured in 10 mM HEPES buffer (pH 7.4, lex ¼ 360 nm,
lem ¼ 470 nm), with addition of various amounts of metal
ions (1.0 and 10.0 equiv of metals). Metals were added as
NaI, HgCl , CoCl , CaCl , KCl, CuCl , MgCl , NiCl , Pb
2
4
.5.2 Limit of detection for ClO
Solutions of 2 (4 mM) in 10 mM HEPES buffer at pH 7.4
were incrementally spiked with a 5.0 mM standard solution
2
of ClO over the concentration range of 0–600 mM, with the
2
2
2
2
2
2
(
utions were prepared also with the addition of Zn (1.0
OAc) z3H O, FeCl zH O and Cd(NO ) . Identical sol-
2
2
2
3
2
3 2
þ
fluorescence signal recorded after each addition
(l ¼ 350 nm and l ¼ 470 nm). From the measured
equiv of Zn(NO ) z6H O).
3
2
2
ex
em
data, the LOD for each of the complexes was calculated as
3sB/sensitivity, where sB corresponds to the standard
deviation of the blank and the sensitivity is the slope of the
2
þ
4.4.3 Limit of detection for Zn
2
least-squares linear fitted fluorescence signal-versus-ClO
2
calibration curve (r ¼ 0.9884) (40, 41).
Solutions of sensor 1 (4 mM) in 10 mM HEPES buffer at
pH 7.4 were incrementally spiked with a 1.0 mM standard
solution of Zn(NO ) ·6H O over the concentration range
3
2
2
of 0–4 mM, with the fluorescence signal recorded after
each addition (lex ¼ 360 nm and l ¼ 470 nm). From
em
2
4.5.3 Selectivity for ClO over other ROS
the measured data, the LOD for each of the complexes was
calculated as 3sB/sensitivity, where sB corresponds to the
standard deviation of the blank and the sensitivity is the
slope of the least-squares linear fitted fluorescence signal-
Sensor 2 was treated with various ROS as follows.
Hydrogen peroxide (H O ), tert-butyl hydroperoxide
2
2
2
TBHP), hypochlorite (ClO ) and nitrogen dioxide
(
(
2
þ
2
versus-[Zn ] calibration curve (r ¼ 0.9908) (40, 41).
NO ) were delivered from 5 mM aqueous solutions to
2
give a final concentration of 200 mM. Hydroxyl radicals
·
·
OH) and tert-butoxyl radicals ( Ot Bu) were generated by
(
reaction of 5 mM Fe with 200 mM H O and 200 mM
4
.4.4 Analysis of water sample
2þ
2
2
1
TBHP, respectively. Singlet oxygen ( O ) was generated
The water sample was subjected to a modified standard
addition method (40, 41). An aliquot of a spiked water
sample (20 mL) was diluted to 1.0 mL with a solution
of 10 mM HEPES buffer (pH 7.4) containing sensor
2
using the Na MoO –H O system in 0.05 M carbonate
2
4
2 2
buffer of pH 10.5. NO was added using NOC-5 (half-life
of 93 min) at a final concentration of 200 mM. Peroxyl
radicals (ROO) were generated by the addition of 2,2’-
azobis(2-amidinopropane)dihydrochloride (200 mM).
1
(4 mM). This solution was then incrementally spiked
with a 1.0 mM standard solution of Zn(NO ) z6H O over
3
2
2

8
06
M.L. Aulsebrook et al.
(16) Aulsebrook, M.L.; Graham, B.; Grace, M.R.; Tuck, K.L.
Tetrahedron 2014, 70, 4367–4372.
Supplemental data
Experimental procedures for compounds 3, 7, 8, 9, UV-vis
absorbance spectra for sensors 1 and 2, Job plot analysis
(
17) Lin, J.-R.; Chu, C.-J.; Venkatesan, P.; Wu, S.-P. Sensors
Actuat. B-Chem 2015, 207, 563–570.
2
for binding of Zn by sensor 1, and fluorescence of sensor
þ
(18) Khairnar, N.; Tayade, K.; Sahoo, S.K.; Bondhopadhyay, B.;
Basu, A.; Singh, J.; Singh, N.; Gite, V.; Kuwar, A. Dalton
Trans. 2015, 44, 2097–2102.
2
þ
1
upon standard addition of Zn
ions to a spiked
environmental sample. Supplemental data for this article
can be accessed here: http://dx.doi.org/10.1080/10610278.
(
19) Aich, K.; Goswami, S.; Das, S.; Mukhopadhyay, C.D. RSC
Advances 2015, 5, 31189–31194.
2
015.1077958.
(
20) Bhosale, J.; Fegade, U.; Bondhopadhyay, B.; Kaur, S.;
Singh, N.; Basu, A.; Dabur, R.; Bendre, R.; Kuwar,
A. J. Mol. Recognit. 2015, 28, 369–375.
(
21) Lim, N.C.; Schuster, J.V.; Porto, M.C.; Tanudra, M.A.;
Yao, L.; Freake, H.C.; Br u¨ ckner, C. Inorg. Chem. 2005, 44,
Disclosure statement
No potential conflict of interest was reported by the authors.
2
018–2030.
(
(
(
22) Mizukami, S.; Okada, S.; Kimura, S.; Kikuchi, K. Inorg.
Chem. 2009, 48, 7630–7638.
23) Sreejith, S.; Divya, K.P.; Ajayaghosh, A. Chem. Commun.
2008, 25, 2903–2905.
24) Divya, K.P.; Sreejith, S.; Balakrishna, B.; Jayamurthy, P.;
Funding
The authors acknowledge the support of the School of Chemistry
and Monash Institute of Pharmaceutical Sciences, Monash
University. Financial support from the Australian Research
Council is gratefully acknowledged [grant numbers
DP150100383 and FT130100838].
Anees, P.; Ajayaghosh, A. Chem. Commun. 2010, 46,
6
069–6071.
(
(
25) Sreejith, S.; Divya, K.P.; Jayamurthy, P.; Mathew, J.;
Anupama, V.N.; Philips, D.S.; Anees, P.; Ajayaghosh,
A. Photochem. Photobiol. Sci. 2012, 11, 1715–1723.
26) Divya, K.P.; Sreejith, S.; Ashokkumar, P.; Yuzhan, K.;
Peng, Q.; Maji, S.K.; Tong, Y.; Yu, H.; Zhao, Y.;
Ramamurthy, P.; Ajayaghosh, A. Chem. Sci. 2014, 5,
Note
1.
This sample was analysed for ICP-MS and the total
concentration Zn in solution was below 5 nM.
3
469–3474.
27) D’Autr e´ aux, B.; Toledano, M.B. Nat. Rev. Mol. Cell Biol.
007, 8, 813–824.
28) Chen, X.; Tian, X.; Shin, I.; Yoon, J. Chem. Soc. Rev. 2011,
40, 4783–4804.
(
(
(
(
(
(
(
(
2
References
29) Koide, Y.; Urano, Y.; Kenmoku, S.; Kojima, H.; Nagano, T.J.
Am. Chem. Soc. 2007, 129, 10324–10325.
(1) Kimura, E.; Aoki, S. BioMetals 2001, 14, 191–204.
(2) Jiang, P.; Guo, Z. Coord. Chem. Rev. 2004, 248, 205–229.
(3) Carol, P.; Sreejith, S.; Ajayaghosh, A. Chem. Asian J. 2007,
30) Setsukinai, K.-I.; Urano, Y.; Kakinuma, K.; Majima, H.J.;
Nagano, T.J. Biol. Chem. 2003, 278, 3170–3175.
31) Xiao, Y.; Ye, Z.; Wang, G.; Yuan, J. Inorg. Chem. 2012, 51,
2, 338–348.
(
(
(
(
(
(
4) Li, L.; Dang, Y.-Q.; Li, H.-W.; Wang, B.; Wu, Y. Tetrahedron
Lett. 2010, 51, 618–621.
5) Xu, Z.; Yoon, J.; Spring, D.R. Chem. Soc. Rev. 2010, 39,
2
940–2946.
32) Rose, A.L.; Waite, T.D. Environ. Sci. Technol. 2005, 39,
645–2650.
2
1
996–2006.
6) Que, E.L.; Domaille, D.W.; Chang, C.J. Chem. Rev. 2008,
08, 1517–1549.
7) Li, X.; Gao, X.; Shi, W.; Ma, H. Chem. Rev. 2014, 114,
90–659.
8) Lavis, L.D.; Raines, R.T. ACS Chem. Biol. 2014, 9,
55–866.
9) Nadler, A.; Schultz, C. Angew. Chem. Int. Ed. 2013, 52,
408–2410.
10) Kitchen, J.A.; Parkesh, R.; Veale, E.B.; Gunnlaugsson,
T. Luminescent Sensing. In Supramolecular
33) Melton, E.D.; Swanner, E.D.; Behrens, S.; Schmidt, C.;
Kappler, A. Nat. Rev. Micro. 2014, 12, 797–808.
34) Murphy, S.A.; Solomon, B.M.; Meng, S.; Copeland, J.M.;
Shaw, T.J.; Ferry, J.L. Environ. Sci. Technol. 2014, 48,
1
5
3
815–3821.
(
(
(
(
35) Kaholek, M.; Hrdlovi cˇ , P.J. Photochem. Photobiol., A-
Chem. 1997, 108, 283–288.
36) de Silva, A.P.; Moody, T.S.; Wright, G.D. Analyst 2009,
34, 2385–2393.
37) Akita, S.; Umezawa, N.; Higuchi, T. Org. Lett. 2005, 7,
565–5568.
38) Burgy, G.; Tahtouh, T.; Durieu, E.; Foll-Josselin, B.;
Limanton, E.; Meijer, L.; Carreaux, F.; Bazureau, J.-P.; Eur,
J. Med. Chem. 2013, 62, 728–737.
8
2
1
(
5
Chemistry: From Molecules to Nanomaterials; Steed, J.
W., Gale, P.A., Eds.; John Wiley: Chichester, 2012;
pp. 2611–2642.
(
(
(
(
(
11) Gale, P.A.; Caltagirone, C. Chem. Soc. Rev. 2015, 44,
4412–4227.
(39) Water: Current Water Quality Criteria, 2015, http://water.
epa.gov/scitech/swguidance/standards/criteria/current/
index.cfm (accessed May 25, 2015).
12) Lee, S.; Yuen, K.K.Y.; Jolliffe, K.A.; Yoon, J. Chem. Soc.
Rev. 2015, 44, 1749–1762.
13) Du, J.; Hu, M.; Fan, J.; Peng, X. Chem. Soc. Rev. 2012, 41,
(40) Miller, J.C.; Miller, J.N. Statistics for Analytical Chemistry;
2nd ed., Chichester, England: E. Horwood; New York:
Halsted Press, 1988.
(41) Skoog, D.A.; Holler, F.; Nieman, T. Principles of
Instrumental Analysis; 5th ed., Philadelphia: Saunders
College Pub., 1992.
4511–4535.
14) Lee, A.E.; Grace, M.R.; Meyer, A.G.; Tuck, K.L.
Tetrahedron Lett. 2010, 51, 1161–1165.
15) Ashton, T.D.; Jolliffe, K.A.; Pfeffer, F.M. Chem. Soc. Rev.
2015, 44, 4547–4595.