2
R. Choudhury et al. / Tetrahedron Letters xxx (xxxx) xxx
-
strong phenolate (PhO ) donor and tricyanofuran (TCF) acceptor
makes the process conspicuous to the naked eyes.
from pale yellow to light purple was also observed within minutes
(inset, Fig. 1). Formation of an ICT fluorophore was confirmed by
comparing the absorption spectra of the mixture after 12 min with
an absorption spectrum of 2. The phenolic form of 2 was previously
prepared by our group to investigate the relationship between
solution pH and the ICT properties of a donor-acceptor fluorophore
[30]. Therefore, with that compound in hand, we readily prepared a
standard graph with different amounts of 2 (Fig. S4). Inserting the
absorbance value of 1 (at 570 nm) into the standard graph shows
In the past, several TCF based donor-acceptor fluorophores have
been developed for selective detection of chemical and biological
analytes and for single molecule imaging of live cells [26–29].
À
Recently, we have established that phenolate (PhO ) and its
derivatives when conjugated with a strong electron acceptor such
as TCF or benz[e]indolium produce intense colors in the red/NIR
regions of the spectrum [30]. This class of compounds is moder-
ately emissive in water. However, fluorescence can be dramatically
enhanced with a viscous liquid or by complexation with a macro-
molecule. Herein, we have described the development of a simple
non-enzyme based colorimetric probe for quantitative determina-
tion of hydrogen peroxide in aqueous solution.
2 2
~90% yield for the H O induced conversion of 1 into 2.
Rate constant and instantaneous rate of reaction were then
measured. Under pseudo first order condition ([1] = 10 mM and
15 mM; [H
2 2
O ] = 2940 mM) plot of natural logarithm of [1] versus
time showed linear relationships (Fig. 2). The observed rate con-
stant for H
2
O
2
induced demasking of boronic acid to the corre-
À3 À1
sponding phenol was kobs = 1.8 Â 10
s . We measured the
Results and discussion
instantaneous rate of the reaction for both concentrations, and
the first four minutes of the reactions were monitored (Fig. S5).
As expected, under similar experimental conditions, the instanta-
In this study (4-formylphenyl)boronic acid was conjugated with
a strong electron acceptor tricyanofuran (TCF) via a simple conden-
sation reaction (Scheme 1a). The goal of this design principle was
to construct a small molecule probe which would undergo hydro-
gen peroxide induced electrophilic displacement reaction at the
À6
À1
À1
neous rate of reaction for 15 mM of 1 (r = 1.82 Â 10 M min
)
À6
was 1.5 times faster than 10 mM (r = 0.82 Â 10 M min ). There-
fore, all these kinetics studies indicate that a rapid colorimetric
assay for hydrogen peroxide can be developed with as low as
10 mM of 1.
boron center to produce a phenol-p-TCF donor–acceptor fluo-
rophore in aqueous solution (Scheme 1b).
Hydrogen peroxide induced conversion of arylboronic acids to
phenols is faster at alkaline pH [33,34]. Therefore, the effect of
pH on the rate of the reaction (1->2) was examined at pH 7.4. As
shown in Fig. 3, absorbance at 410 nm slowly decreased with time;
after ~38 min a saturation reached. Comparison of the absorbance
at 570 nm with the standard graph shows ~46% conversion yield
after 38 min. Moreover, an isobestic point was recorded at
435 nm, which is distinctly different from that of the pH 9.0 solu-
tion. In pH 7.4 solution, the isobestic point was at shorter wave-
length, indicating an equilibrium transformation of 1 to the
phenolic form of 2. We have previously established that the max-
imum absorbance at 463 nm originates from the protonated (phe-
nolic) form of 2, and a high percentage of this form exists in
Knoevenagel condensation between (4-formylphenyl)boronic
acid and 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-ylidene)malonon-
itrile in ethanol resulted 1 in moderate yield (27%) [28]. Details of
the synthesis, purification, and characterization are provided in the
experimental section (electronic Supplementary materials,
1
Figs. S1–S3). In the H NMR spectrum, appearance of a broad signal
at 8.25 ppm in DMSO d
group. Moreover, in the IR spectrum, broad signal at ~3290 cm
6
indicates presence of –OH functional
À1
corroborates to the –OH of phenyl boronic acid [31]. As expected,
À1
a sharp signal appears at ~2225 cm , confirming nitrile groups
on 1. The selectivity to all trans isomer of the Knoevenagel conden-
sation was very high, which was confirmed from the high coupling
constant (J = 16.2 Hz) of the vinylic hydrogens [32].
equilibrium at pH 7.4 [30]. The rate constant under pseudo first
À4 À1
We first studied the reaction kinetics of 1 with hydrogen perox-
order condition was kobs = 2.2 Â 10
s , which is one order of
ide ([H
2
2
O ] = 2.94 mM) by UV–vis spectroscopy. The reaction was
magnitude lower than that of the pH 9.0 solution (Fig. S6). The
À6
monitored by recording absorption spectra of the mixtures in a
buffer (pH = 9.0; 0.1 M) solution. As shown in Fig. 1a—upon addi-
tion of hydrogen peroxide—amount of 1 ([1] = 10 mM) rapidly
decreased. Two absorption peaks were observed before addition
instantaneous rate was also relatively slower (r = 0.28 Â 10
-
À1
M min ), as obtained from the first eight minutes of the reaction
(Fig. S6).
From the pH dependent kinetics study it was established that 1
responds rapidly at alkaline pH. Next, to gain an insight into sensi-
tivity of 1, limit of detection (LOD) was calculated by titrating dif-
ferent amounts of hydrogen peroxide with 1 at pH 9.0 [35]. As
shown in Fig. 4a, a good linear correlation (between 0 and
of H
signal emerged at 570 nm, which leveled-off after ~12 min
Fig. 1b). A clear isobestic point was observed at 465 nm, indicating
transformation of 1 into another species. A visible color change
2 2 2 2
O ; after addition of H O both signals decreased and a new
(
Scheme 1. (a) Synthesis of probe 1. (b) Demasking of 1 by hydrogen peroxide to a new Intramolecular Charge Transfer (ICT) dye.
Choudhury, Rajib
