Coumarin-decorated Schiff base hydrolysis as an efficient driving force for the fluorescence detection of water in organic solvents

Article abstract of DOI:10.1039/c6cc04285f

A coumarin based Schiff base was found to be an excellent indicator of moisture, via rapid in situ hydrolysis. A structure-relationship examination of a small library of Schiff bases revealed the critical importance of hydrogen bond acceptors in close proximity to the imine bond, and this observation was further supported by theoretical calculations as well as the solid state structure analysis. The most sensitive compound demonstrated a limit of detection and quantification of 0.18% and 0.54% v/v water in DMSO, respectively.

Full text of DOI:10.1039/c6cc04285f

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Coumarin-decorated Schiff base hydrolysis as an efficient driving
force for the fluorescent detection of water in organic solvents
Received 00th January 20xx,
Accepted 00th January 20xx
Won Young Kim,a, ‡ Hu Shi,b, ‡ Hyo Sung Jung,a, ‡ Daeheum Cho,b Peter Verwilst,a Jin Yong Lee,b,* and
Jong Seung Kima,*
DOI: 10.1039/x0xx00000x
www.rsc.org/
environements,8 as well as their generally low cost and easy of
A coumarin based Schiff base was found to be an excellent
indicator of moisture, via rapid in situ hydrolysis. A structure-
relationship examination of a small library of Schiff bases revealed
the critical importance of hydrogen bond acceptors in close
proximity to the imine bond, and this observation was further
supported by theoretical calculations as well as the solid state
structure analysis. The most sensitive compound demonstrated a
limit of detection and quantification of 0.18% and 0.54% v/v water
in DMSO, respectively.
manufacturing, sensors bearing this type of bond are a prime
candidate in the design of water activatable fluorophores.
Despite this, to date a sole example of a water-triggered
chemodosimeter consisting of a 3,5-dichlorosalicylaldehyde
Schiff base has been reported,9 and no in depth study exploring
the influence of structural modification on the activity of this
class of water sensors has been yet reported. In the current
report, we therefore investigated the structure-activity relation
of a small library of coumarin based fluorophores bearing a
Schiff base, inducing water sensitivity through hydrolysis, and
have rationalised our findings with the aid of theoretical
calculations and crystal structure analysis.
In the current molecular design, coumarin was used as the
fluorophore skeleton and derivatives of aniline and pyridine
were introduced as Schiff bases, as they not only extend the
conjugation inducing a fluorescence shift but also enhance the
fluorescence intensity. It is thus expected that the presence of
water in organic solvents containing probes 3-6 (Scheme 1)
results in the dissociation of the Schiff-base, inducing
observable changes in colour and fluorescence (Scheme 2).
Water is a typical contaminant in a most organic solvents,
which commonly represents an impediment to chemical
reactions and industrial production processes, where dry
operations should be guaranteed. Crucially, under the operation
conditions of chemical production plants, water can induce the
corrosion of metals, quench metal based reagents and cause the
deactivation and deterioration of catalysts, especially so in the
case of the refinery industry.1,2 As such, the detection and
quantification of water in organic media is of vital importance.
Currently, traditional methods for the quantification of
water content in gas streams or liquid phases are widespread,
though are associated with drawbacks such as the cost of
operation and instrumentation and their time-consuming
nature.3 Recently, optical sensors for detecting water contents
have received substantial attention due to the possibility of
remote and in situ monitoring as well as their cost-effective
fabrication.4
Fluorescent indicators, due to their innate high sensitivity,
have received the lion’s share of attention, with a lot of effort
being devoted to the investigation of fluorescent sensors using
sensing mechanisms such as photo-induced electron transfer,
intramolecular charge transfer, proton transfer, water-induced
decomplexation of dyes, water-induced interpolymer π-stacking
aggregation, solvatochromism, etc.4-7
Coumarin probes
synthetic route depicted in Scheme 1. First, coumarin derivative
was synthesized from 4-(diethylamino)salicylaldehyde in a
3-6 were synthesized following the
1
93% yield, followed by the introduction of an aldehyde
functionality using Vilsmeier–Haack conditions, providing a
convenient handle to allow for further decoration, producing
compound
2
A particularly intriguing class of water liable chemicals are
the Schiff bases. With their known instability in aqueous
a. Department of Chemistry, Korea University, Seoul 136-701, Korea. E-mail:
jongskim@korea.ac.kr; Fax: +82-2-3290-3121
b. Department of Chemistry, Sungkyunkwan University, Suwon 16419, Korea. E-mail:
jinylee@skku.edu
Electronic Supplementary Information (ESI) available: Synthesis procedures, NMR,
MS, fluorescence, cell imaging data and crystallographic data. CCDC 1481023 and
CCDC 1481024. See DOI: 10.1039/x0xx00000x
Scheme 1 Synthetic route towards water-sensing probes 3-6.
‡ These authors contributed equally to this work.
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DOI: 10.1039/C6CC04285F
Fig. 2 UV/Vis spectra and fluorescence emission spectra of
various organic solvents (1% DMSO) at rt. λex = 447 nm.
6 (10 μM) in
Scheme 2 Conceptual illustration of the water-sensing probe.
in a 76% yield. After introducing the aldehyde group, probes
possessing moisture sensitive imine bonds were obtained from
fluorescence spectra, which indicates that
6 could potentially be
used as a water sensor in a range of organic solvents.
The time-dependent fluorescence intensity of compounds 3-6
was subsequently monitored (Fig. 3a) and products 4 and 6 were
examined in greater detail to characterise and quantify their
differential time-dependent behaviour. The kinetics of the hydrolysis
of Schiff bases are known to follow a pseudo-first order kinetics
profile. As such, considering the first order kinetics of the hydrolysis
reaction of a reactant (R), generating a product (P), the concentration
of reactant diminishes exponentially, as described in equation (1),
with [R] and [R]0 being the actual and initial concentration of the
reactant:
the reaction of compound
2 with anilines and an amine bearing
derivative of pyridine in anhydrous ethanol. All final
compounds were fully characterized by 1H, 13C NMR, and ESI-
mass spectrometry and the probes were further characterised by
the determination of their molar absorption coefficient (see Fig.
S1-S10 and Table S1 in the ESI†).
Probes
and fluorescence assay in an aqueous DMSO solution (Fig. 1
and S11, ESI†). The maximum of absorbance of probes
exhibited a blue shift upon hydrolysis (green to red line), in
contrast to probe demonstrating a red-shift upon hydrolysis.
After complete cleavage of the imine bond, the released
compound shows strong absorption intensity at 436 nm. As
can be seen from this figure, the reactivity of probe clearly
outperformed other probes. As the fluorescence spectra of the
probes showed the same tendency (Fig. S11, ESI†), probe
was selected for further examination and assessment as a water
sensing probe and the hydrolysis of this probe was further (2)). For this time dependent reaction, the intensity of
3-6 were subjected to a time–dependent colorimetric
4-6
3
푅 = 푅 0푒−푘푡
[ ] [ ]
(1)
(2)
(3)
2
[ ] [ ] [ ]
푅
= 푅 + 푃
6
0
[ ]
퐼휆 = 휖휆,푅 푅 + 휖휆,푃
[ ]
푃
6
The system is further characterised by its mass balance (equation
fluorescence Iλ at a certain wavelength (λ) and time comprises a
contribution from the reactant and the product, depending on
their relative concentrations and emissivities, as described in
equation (3), with ϵ being the wavelength dependent emissivity of
reactant and product.
For a system being characterised at two different wavelengths
(λ1 and λ2), the concentration of reactant remaining at a given
point in time can be written as a function of the intensities and
the emissivities of reactant and product at these two wavelengths
(equation (4)). Combining equations (4) and (1) resulting in
equation (5), a nonlinear regression using the time dependent
fluorescence allows for the determination of the rate constants (k).
supported by ESI-MS, showing the disappearance of a peak at
m/z 322.10 (corresponding to [
new peak at m/z 246.05 (corresponding to [
ESI†).
6
+ H]+) and the emergence of a
2
+ H]+) (Fig. S12,
The UV and fluorescence spectra of
examined in different organic solvents (Fig. 2). The absorption
band of showed only a slight solvent dependency, however,
the fluorescence spectra showed dramatic change in
6 were subsequently
6
a
wavelength and intensity. The probe stability is excellent in
various organic solvents as assessed by UV/Vis and
휖휆1,푃퐼휆2−휖휆2,푃퐼휆1
[ ]
푅 = 휖
(4)
(5)
휆1,푃휖휆2,푅−휖휆1,푅휖휆2,푃
휖휆1,푃퐼휆2−휖휆2,푃퐼휆1
0푒−푘푡
=
휖휆1,푃휖휆2,푅−휖휆1,푅휖휆2,푃
[ ]
푅
Fig. 3 (a) Fluorescence spectra of probes (10 μM) in water solution (2%
DMSO) at rt. λex = 402 nm (3); 456 nm (4); 456 nm (5); 447 nm (6). (b)
ln(ϵ499,2 I544- ϵ544,2 I499) vs. time for the hydrolysis of 4 and 6 in distilled water.
(c) pH dependence of rate constants for the hydrolysis of 4 and 6 in various
buffer solutions.
Fig. 1 UV/Vis absorption spectra of water-sensing probes (10 μM) in an
aqueous solution (2% DMSO) at rt.
2 | J. Name., 2012, 00, 1-3
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Fig. 4 Crystal structures of (a) 4 and (b) 6
Fig. 5 Energy profiles for the hydrolysis of
4 (a) and 6 (b) in acidic,
neutral, and basic solutions. Energies are given in kcal/mol.
Using this approach, the hydrolysis of the probe was monitored
by fluorescence at 499 nm and 544 nm, and the emissivity of the
probes at these wavelengths was determined to be ϵ544,4 = 30.53,
ϵ544,6 = 109.36, ϵ544,2 = 30.06, ϵ499,4 = 10.74, ϵ499,6 = 50.30, and
ϵ499,2 = 97.64. The rate constants were determined, revealing a
nearly 2.6-fold increase in reaction rate in the case of 6 vs. 4 in
distilled water.
The behaviour of the probes was further assessed across a wide
range of pH values, but the reaction rate of 6 under acidic conditions
was too fast in order to make accurate measurements (Fig. S13,
ESI†), limiting the pH range for which rate constants of probe 6
could be determined as opposed to probe 4, where the rate constants
were reported in the pH = 3 to 13 range (Fig. 3c). The hydrolysis
rate in basic solutions was strongly diminished, but greatly increased
under acidic conditions for both 4 (with rates of 1.5 – 2 min-1) and 6
(with an estimated rate of > 4 min-1), following a trend previously
reported for the pH dependent hydrolysis rates of 2-
aminothiophenol Schiff bases.10
strengths (fab) of 1.1848, 1.2420, and 0.8557, respectively. The
calculated emission wavelengths of 4, 6, and product 2 were 504,
476, and 408 nm with oscillator strengths (fem) of 1.1055, 1.4172,
and 0.7805, respectively. The fluorescence intensity should be
proportional to the product (fab×fem) of the oscillator strengths of
absorption and emission. The values of fab×fem for 4, 6, and 2 were
1.3098, 1.7602, and 0.6679 at their emission maximum wavelengths,
respectively. Comparing these values with molar emissivity values,
we can expect that the molecular photophysical property is retained
in solution of 4 and 6, but there is some additional solution
chemistry in the case of 2.
The energy profiles for the hydrolysis of
4 and 6 in acidic,
neutral, and basic solutions were shown in Fig. 5. All the
reactant complex (RC), transition state (TS), and product
complex (PC) were confirmed from the frequency calculations
and IRC calculations. In all cases, the hydrolysis proceeds in
two steps excluding equilibrium (protonation and
deprotonation) processes. The first step is the addition of water
to the imine bond (C=N), and the second step is dissociation of
the hemiaminal intermediate into an aldehyde and amine. In
acidic solutions, the second dissociation step is barrierless,
while in basic solutions, the first water addition step is
As shown in their crystal structures, compound
perpendicular aromatic ring with the coumarin moiety,
however, the pyridine ring of is parallel with the imine bond
of coumarin fluorophore (Fig. 4, Table S2, ESI†). This
indicates that the nitrogen atom on pyridine ring of can form
4 exhibits a
6
6
hydrogen bonds (C(6)-H(11) with N(1) in Fig. 4b and Table S3,
ESI†) and affect the imine bond hydrolysis by anchimeric
assistance, an effect well known to accelerate the reaction rate
of 1-hydroxy-7-azabenzotriazole based peptide coupling
reagents.11
In order to gain a better mechanistic insight into the pH
dependent reaction rate as well as to quantify the effect of the
potential anchimeric assistance, DFT and TDDFT calculations for
the reactions of 6 and 4 with water in acidic, neutral, and basic
solution conditions were performed. Initial structures were chosen
from the crystal structures. All the calculations were performed with
B3LYP exchange functional with 6-31G* basis sets using a suite of
Gaussian 09 programs.12 In the optimized structures, the dihedral
angle between coumarin and the phenyl/pyridine ring moiety was
barrierless. In neutral solutions, for both
addition is the rate determining step. It can clearly be seen that
the activation barriers for the hydrolysis of are lower than for
, which is consistent with the kinetic data obtained from the
time-dependent fluorescence changes. It can be therefore
concluded that the hydrolysis can be much enhanced in acidic
solutions as noted from the greatly reduced activation barrier as
compared to neutral solutions, while non-catalytic reactions
were observed in basic solutions. The experimentally observed
non-catalytic feature in basic solution was also supported from
the activation barriers of the dissociation (17.7 and 20.0
4 and 6, the water
6
4
kcal/mol for
those in neutral solution (17.3 and 19.9 kcal/mol for
respectively). The most striking feature is that the hydrolysis
can be much enhanced in acidic solution, and is much faster
than . The calculated energy profile is in excellent agreement
with experimentally observed pH dependent kinetics. Thus, the
probe would most effectively work as a water sensor in acidic
solutions.
4
and
6
, respectively) being virtually identical to
4
and
6,
6
calculated to be 12.2° (6) and 37.2° (4), which is consistent with the
4
solid state structure, though more strongly pronounced in the solid
state due to the packing effect in the crystal. The calculated
maximum absorption wavelengths of 4, 6, and product 2 were 427,
431, and 394 nm, which are in excellent agreement with the
experimental values of 450, 450, and 430 nm with oscillator
6
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In conclusion, we synthesized coumarin-baseVdiewflAuroticrleesOcnelinnet
moisture sensors . Their UV/Vis and fluorescence spectra
3-6
DOI: 10.1039/C6CC04285F
confirmed that these probes are stable in various organic
solvents, yet sensitive to moisture content in organic solvents
and aqueous solutions. We monitored the time-dependent
fluorescence changes for the most efficient probes
4 and 6, and
found that they follow pseudo-first order kinetics as evidenced
from the single exponential decay. The kinetics study in buffer
solutions at various pH levels revealed that the hydrolysis of
and was remarkably enhanced in acidic solutions, in contrast
to basic solutions, not exhibiting catalytic effects. Thus, the
probe is expected to be an efficient potential fluorescent
moisture sensor at neutral or acidic pH. The experimentally
observed kinetics were rationalised by the analysis of the solid
state conformation in the crystal structure as well as pH
dependent theoretical calculations. The probe was further
determined to exhibit a limit of detection (LOD) and limit of
quantitation (LOQ) of 0.18% and 0.54% v/v water in DMSO.
Taken together these results indicate the strong potential of
Schiff base bearing fluorophores as highly sensitive water
sensors in organic solvents via a hydrolysis mechanism.
4
Fig. 6 (a) Fluorescence intensity at 536 nm and of
6 in DMSO with
6
increasing water content in the range of 0-10% (v/v) (b) linear fit of the
range (below 1.0% v/v). λex = 459 nm, data recorded after 800s of
equilibration.
6
In the first water addition step, the reactant complex (RC) of
4
shows two water molecules involved in an hydrogen bonding
network with the imine bonds. The RC of is similar to , but
6
4
shows an additional water molecule interacting with the
nitrogen atom of the pyridine ring through hydrogen bonding as
shown in the SI (Fig. S14, ESI†). The additional water
molecule in
6 plays a role as a blocking group, without which
the nitrogen atom of pyridine destroys the hydrogen bonding
network. Subsequently, the hemiaminal intermediate was
formed through a transition state (TS1) containing a 6-
membered ring as shown in Fig. S14, ESI†. Finally, the
intermediate dissociates into the final products, aldehyde and
amine, through the transition state (TS2) also containing a 6-
membered ring. The natural bond orbital (NBO) population
(charge) on the imine bond can be used as be a simple estimate
for the reactivity of the water addition. The atomic charges of C
and N of the imine bond were calculated to be 0.106e and -
This work was funded by CRI project (No. 2009-0081566,
J.S.K.)
and
the
Basic
Science
Research
Program
(2015R1A6A3A04058789, H.S.J.) from the National Research
Foundation of Korea (NRF) funded by the Ministry of Education as
well as a Korea University Grant (P.V.). The work at SKKU was
supported by Ministry of Science, ICT and Future Planning,
subjected project to the project EDISON (Education-research
Integration through Simulation On the Net, Grant No.
2012M3C1A6035359).
0.481e for
the water addition to the more positive carbon of
easier compared with . In acidic solutions, protonation at the
4
, and 0.123e and -0.519e for
6
, respectively. Thus,
6
would be
Notes and references
4
nitrogen atom of the imine bond can induce some charge
transfer from the carbon atom of the imine bond, making the
carbon more electron deficient, hence prone to water addition.
The atomic charges of C and N of the imine bond under these
1. M. Araujo, L. M. Santos, M. Fortuny, R. L. F. V. Melo, R. C. C.
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B-Chem., 2001, 77, 620.
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conditions were calculated to be 0.176e and -0.495e for
4, and
12191.
0.184e and -0.519e for , respectively. The protonation at N of
6
4. (a) H. S. Jung, P. Verwilst, W. Y. Kim and J. S. Kim, Chem. Soc.
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the imine bond can also reduce the barrier by stabilizing the
accumulated negative charge at the nitrogen atom after water
addition. This charge redistribution is consistent with the
calculated activation barriers.
Finally, the performance of probe
6 in the determination of
the water content in DMSO solutions was assessed. Probe
6
was subjected to increased amounts of water in DMSO. Upon
excitation at 459 nm, the fluorescence at 536 nm was monitored
in the 0–10% v/v water in DMSO range (Fig. 6a). The probe
showed a good linear response in the 0–1% (v/v) range (Fig.
6b), allowing for the determination of the limit of detection
(LOD) and limit of quantitation (LOQ) as defined in equations
6 and 7, with
σ being the standard deviation of the blank and m
8. (a) S. Wang, B. Wu, F. Liu, Y. Gao and W. Zhang, Polym. Chem.,
the slope of the calibration curve (Fig. 6b):
2015, 6, 1127; (b) Z. Yang, M. She, J. Zhang, X. Chen, Y. Huang, H.
Zhu, P. Liu, J. Li and Z. Shi, Sens. Actuator B-Chem., 2013, 176, 482.
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Jiang, Analyst, 2013, 138, 2847.
10. H. B. Hassib, N. S. Abdel-Kader and Y. M. Issa, J. Solution Chem.
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LOD = 3.3휎
(6)
(7)
푚
LOQ = 10휎
푚
11. L. A. Carpino, J. Am. Chem. Soc., 1993, 115, 4397.
12. Gaussian 09 (Revision D.01). M. J. Frisch, et al. Gaussian, Inc.,
Wallingford CT, 2009.
Thus the LOD and the LOQ of
6 for water in DMSO were
determined to be 0.18% and 0.54% v/v demonstrating the
highly sensitive nature of this type of Schiff base bearing
fluorophores for the determination of moisture in organic
solvents.
4 | J. Name., 2012, 00, 1-3
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