Unraveling the Hydrolysis of Merocyanine-Based Probes in Biological Assay

Article abstract of DOI:10.1021/acs.analchem.6b02113

Merocyanine dyes, owing to their unique photochemical properties, are widely used to fabricate probes for the detection of biologically active small molecules and bioimaging. In this paper, merocyanine-based probes were proved of undergoing unwanted hydrolysis. To explore the strategies toward avoiding the hydrolysis, the detailed hydrolysis mechanism was first investigated, which was also confirmed by density functional theory (DFT) calculation. Then a series of merocyanine dyes were rationally designed. Influences of molecular structures of the probes, the analytical media such as pH and components of the solution on the hydrolysis were systematically studied. The experimental results suggest that merocyanine based probes with low electron density are more likely to suffer the hydrolysis, which could be exacerbated by the well-accepted strategy for constructing type-II probes. It is worth noting that chemical surroundings could also exert distinctive influence on the hydrolysis. The hydrolysis could be obviously aggravated when fetal calf serum or DMSO was deployed. Our findings will definitely provide an effective and reliable approach for guiding the rational design of highly robust merocyanine-based probes and the optimization of the analytical media, which is helpful in terms of avoiding the hydrolysis of the probes and hydrolysis caused analytical errors.

Full text of DOI:10.1021/acs.analchem.6b02113

Article
pubs.acs.org/ac
Unraveling the Hydrolysis of Merocyanine-Based Probes in Biological
Assay
Liqiang Wang,†,∥ Yuanqiang Hao,†,‡,∥ Jianhan Huang,† Yonghui He,§ Ke Zeng,† Juan Li,†
Johnny Muya Chabu,† Wansong Chen,† Minghui Yang,† Liu Deng,*,† and You-Nian Liu*,†
†College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, P. R. China
‡Henan Key Laboratory of Biomolecular Recognition and Sensing, College of Chemistry and Chemical Engineering, Shangqiu
Normal University, Shangqiu, Henan 476000, P. R. China
§Key Laboratory of Chemistry in Ethnic Medicine Resource, State Ethnic Affairs Commission & Ministry of Education, Yunnan
Minzu University, Kunming, Yunnan 650031, P. R. China
S
* Supporting Information
ABSTRACT: Merocyanine dyes, owing to their unique
photochemical properties, are widely used to fabricate probes
for the detection of biologically active small molecules and
bioimaging. In this paper, merocyanine-based probes were
proved of undergoing unwanted hydrolysis. To explore the
strategies toward avoiding the hydrolysis, the detailed
hydrolysis mechanism was first investigated, which was also
confirmed by density functional theory (DFT) calculation.
Then a series of merocyanine dyes were rationally designed.
Influences of molecular structures of the probes, the analytical
media such as pH and components of the solution on the
hydrolysis were systematically studied. The experimental
results suggest that merocyanine based probes with low electron density are more likely to suffer the hydrolysis, which could
be exacerbated by the well-accepted strategy for constructing type-II probes. It is worth noting that chemical surroundings could
also exert distinctive influence on the hydrolysis. The hydrolysis could be obviously aggravated when fetal calf serum or DMSO
was deployed. Our findings will definitely provide an effective and reliable approach for guiding the rational design of highly
robust merocyanine-based probes and the optimization of the analytical media, which is helpful in terms of avoiding the
hydrolysis of the probes and hydrolysis caused analytical errors.
Scheme 1. Schematic Illustration for the Hydrolysis of
Merocyanine-Based Probes (X = C, N, O, S)
erocyanine dyes, due to their remarkable photochemical
Mproperties, have been extensively utilized for fabricating
probes for in vivo molecular recognition and bioimaging.1−3 On
one hand, the merocyanine-based probes can be easily obtained
via condensing the aldehyde and corresponding active
methylene, resulting in a D-π-A system. An ICT (intermo-
lecular charge transfer) effect, usually from the donor group to
N+ within the merocyanine skeleton could be tuned via
modifying the merocyanine molecules, based on which
numerous probes were designed.4−6 It is also worthy to note,
the merocyanine molecules were imparted with the ability of
targeting mitochondrial by N+.7
Generally, two main kinds of probes have been reported
according to their sensing mechanism. As depicted in Scheme
1, the first one (type-I) is based on the nucleophilic addition of
the target analytes to the corresponding carbon atom (named
C-2). The π-conjugation system of the fluorescent probes was
disrupted via nucleophilic addition, leading to corresponding
photochemical changes.7 Another one (type-II) is based on the
introduction of a target receptor (trigger) to cage the donor
part in the D-π-A system. The assay can be achieved by spectral
changes before and after cleavage of the trigger in the presence
of analyte targets.8,9
Received: May 30, 2016
Accepted: August 24, 2016
© XXXX American Chemical Society
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However, the probes are likely to suffer the unwanted attack
by OH− in aqueous solution owing to the inherent structural
features, especially the polarization effect of N+.10 In typical
merocyanine-based probes, the adjacent C-2 can be polarized
by N+, rendering C-2 sensitive toward analytes of nucleophil-
icity, OH−, for example. On the basis of such consumptions,
when merocyanine-based probes were utilized to determine
nucleophilic molecules, the side effects arising from the
ubiquitous OH− seem to be inevitable. An important but
often ignored issue is the strategy utilized for constructing type-
II probes. To fabricate a type-II probe, the donor group is often
protected as mentioned above. Such a protective strategy is able
to further decrease the electron density on C-2, thus possibly
aggravating the hydrolysis.
Undoubtedly, the analytical results can be affected negatively
if the hydrolysis takes place. As shown in Scheme 1, for both
types of probes the inherent spectral signals will be inevitably
weakened due to the hydrolysis induced structure damage.
Meanwhile, the reactive sites originally for analytes (for type-I
probe) will be occupied by the OH−, thus interfering the
analytical outcomes. Moreover, dynamic signals which is very
important for biological analysis will be therefore affected by
the unwanted OH− addition. Thus, as a prerequisite toward
reliable assay system based on merocyanine-based probes, it is
crucial to inquire the influences of the structures of
merocyanine-based probes as well as assay situation on the
hydrolysis. However, few attempts have been devoted to such
studies.
probe for the detection of H2O2 was fabricated and their
hydrolysis in the biological system was investigated in detail.
EXPERIMENTAL SECTION
■
Materials and Instruments. Iso-propylmethyl ketone,
phenylhydrazine, ethyl iodide, p-substituted benzaldehyde,
and indocyanine green were purchased from Heowens,
China. Dulbecco’s modified Eagle’s medium (DMEM), fetal
bovine serum, and penicillin were obtained from Dingguo
Changsheng Biotech Co., Ltd. (Beijing, China). Solvents were
purified by standard methods prior to use. Twice-distilled water
was used throughout the whole experiments. Silica gel (mesh
200−300) was utilized for both thin layer chromatography
(TLC) analysis and column chromatography was obtained
from Ocean Chemicals (Qingdao, China). All reagents were of
analytical grade and used without further purification unless
stated.
1H NMR and 13C NMR spectra were recorded on a Bruker
AVANCE III 400 or 500 MHz NMR spectrometer
(tetramethylsilane as an internal standard). High-resolution
mass spectrometry (HRMS) were obtained on a micrOTOF-Q
II mass spectrometer. UV−vis titration experiments were
performed on a Shimadzu UV2450 spectrophotometer
equipped with a quartz cuvette (path length, 0.5 or 1 cm).
The absorption points of merocyanine molecules incubated in a
96 well plate was obtained via Bio-TeK ELx800. The solution
pH measurements were carried out with a pH meter (Leici
PHSj-4a, Shanghai, China).
Herein, to confirm our speculation that hydrolysis can take
place in merocyanine molecules, some typical merocyanine
probes were designed and synthesized (shown in Scheme 2).
Synthesis and Characterization of Merocyanine Dyes
A1−A6. Merocyanine dyes A1−A6 were synthesized as
illustrated in Scheme 2. Briefly, compound 4 (1 equiv) and
the corresponding aldehyde (1.2 equiv) were placed in a 25 mL
flask, and then ethanol (15 mL) was added. The mixtures were
heated to 85 °C and kept for 12 h under N2 atmosphere. Then
the crude product was recrystallized from ethanol, giving pure
a
Scheme 2. Synthesis of Merocyanines (A1−A6)
1
products. The final products were confirmed by H NMR, 13C
NMR, and HRMS (Figures S17−S21).
General Procedures for Preparation of the Testing
Solution for UV−Visible Titration. Stock solutions (A1−
A6) (5 mM) of the corresponding merocyanine dyes were
prepared in methanol. The testing solution was prepared as
following: 10 μL of stock solution was added into a 3 mL tube
and then required percentages of organic solvents, CTAB, or
serum were added followed by adding buffer solution (20 mM)
to form 2.5 mL of mixture solution containing a certain amount
of specific organic solvents. The corresponding UV−vis data
were obtained at room temperature or 37 °C.
Preparation of the Testing Solution for H2O2
Determination. A stock solution of A6 (5 mM) was prepared
in methanol. The testing solution was prepared by adding 12
μL of A6 stock solution into test tubes, followed by adding
required volume of PBS (pH 7.4, 20 mM) or PBS containing
CTAB (2 mM). Then H2O2 sample solution were added from
3 mL of mixture solutions with different H2O2 concentrations.
The mixture solutions were incubated in 37 °C for 1 h and then
subjected to UV−vis analysis.
General UV−Visible Titration for A1−A6 in Various
Analytical Media. (1) Absorption spectra were obtained by
recording the absorption intensity at different time points at RT
or at 37 °C. (2) Dynamic assay was achieved by recording the
absorption intensity at appointed wavelength according to
different times at RT. It is worthy to note, solutions were
subjected to the analysis of UV−vis immediately once they have
a
Synthetic conditions: (a) 1.0 equiv of 1, 3.0 equiv of 2, CH3COOH,
reflux overnight. (b) 1.0 equiv of 3, CH3CH2I, reflux, overnight. (c)
1.0 equiv of 4, 1.2 equiv of corresponding aldehyde, EtOH, reflux
overnight.
Then influences of their structures and the analytical media,
such as the pH and components of the solutions, on the
hydrolysis of the probes in biological assay were systemically
studied. Density functional theory (DFT) calculation was
subsequently applied for further confirming the hydrolysis
mechanism. Finally, to demonstrate the attention brought on
merocyanine-based probes is well deserved particularly for
type-II probes in which the introduction of the caging group
will aggravate their hydrolysis, a type-II merocyanine-based
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been prepared. (3) Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum and 1%
penicillin was utilized to incubate the as-prepared merocyanine
dyes to test their stability. To mimic the practical condition, the
incubation was taken under 5% CO2 and 95% air at 37 °C for
required time intervals, after which the corresponding
absorbance were collected by a microplate reader.
Calculation Method. Calculation was carried out by
density functional theory (DFT) with 6-31G basis set using a
suite of Gaussian 09 programs. To begin with, the optimized
structures of various merocyanine dyes were obtained. Then at
the optimized geometry, the excitation properties such as
excitation energies and simulated UV−vis absorption spectra
through TD-DFT calculations were obtained. Finally, Gaussian
View 5.0.8 was employed for analyzing the calculation results
such as MAC and the ΔG of the reaction in different solvent
systems.
Figure 2. 1H NMR of A1 (5 mg/mL) before (defined as A1) and after
(defined as A1 + OH−) hydrolysis in the mixture solution of 600 μL of
DMSO and 200 μL of MeOD. A1 + OH− adducts were obtained by
addition of NaOD (20 μL of 30% NaOD solution).
RESULTS AND DISCUSSION
■
Influence of Molecular Structure of the Probe on the
Hydrolysis. For merocyanine molecules, electron distribution
can be affected by the para-substituted group, which is closely
linked to the electron density of C-2. Thus, merocyanine
molecules with various electron density of C-2 were designed
by introducing different para-substituted groups to investigate
the hydrolysis mechanism of the probes. Bromine, a well-
accepted electro-withdrawing group, is supposed to reduce the
electron density on C-2. Br was introduced at the para-position
(A1) and it was found that A1 could undergo an obvious
alteration in the spectral profile over 12 h in NaHCO3−
Na2CO3 buffer solution at pH 9.4, a typical pH used for the
detection of nucleophiles such as CN− (see Figure 1a). Similar
reported, further confirming that OH− in aqueous solution is
able to attack C-2 and converts N+C into N−C−OH.11
Like spiropyran and its derivatives,12 A1 was found to
undergo pH-dependent structural transition, i.e., the hydro-
lyzed structure was able to turn back to its original structure
under acidic environment and vice versa (see Figure S3).
Because of that, when α-cyano-4-hydroxycinnamic acid
(HCCA) was utilized as a matrix for HRMS analysis, the A1-
OH peaks were not observed. However, after adding
triethylamine (TEA), a small peak with m/z 371.0877
appeared, which can be assigned to [A1-OH]+ (see Figure 3).
The hydrolysis tendency of merocyanine molecules could be
aggravated by the introduction of electron-withdrawing groups
like -Br, which can reduce the electron density on C-2.
Figure 1. Hydrolysis induced spectral changes (a) A1 (20 μM) and
(b) A2 (20 μM) after incubation in buffer solution (pH 9.4, 20 mM
NaHCO3−Na2CO3) for 12 h. The inset in part a shows the digital
photos of A1 solution after incubating for 0 h (pale yellow) and 12 h
(colorless).
Figure 3. HRMS characterization of of A1 after incubating with NaOH
solution for 10 min by using (A) HCCA as matrix with addition of
TEA and (B) HCCA as matrix without addition of TEA.
On the other hand, when electron-withdrawing group (-Br)
in A1 was replaced by a strong electron-donating group
-N(CH3)2 (A2), the nucleophilic addition seems to be
completely restrained at the pH of 7.4 (see Figure S4). Even
the pH was elevated to 9.4, as depicted in Figure 1b, only
negligible spectral changes were observed. Compared with A1,
A2 was empowered with strong electron-donating group
-N(CH3)2 which affords C-2 higher electron density thus
disfavoring the nucleophilic addition. In fact, in merocyanine
molecules, their active groups such as -NH2 are often protected
via acylation. Such a process is believed to decrease the electron
donating ability and may further lead to a different hydrolysis
behavior. Once -N(CH3)2 group in A2 was substituted by
-NHCOCH3 (A3), the absorbance of A3 greatly decreased in
the buffer solution of pH 9.4, even in the initial 30 min (see
result could be even observed in aqueous solution (see Figure
S1). Since the electron-withdrawing ability of indolenium in
merocyanine molecules could be decreased by the nucleophilic
addition, the chemical shift of neighboring atom will be
therefore affected. On the basis of such consideration, 1H
NMR, 13C NMR were utilized to test whether OH− is able to
induce such an addition on C-2. As illustrated in Figure 2,
almost all protons underwent an obvious upfield shift upon the
addition of OD−, meanwhile the dimethyl protons of C(c) were
driven nonequivalent, causing the original singlet signals to split
into multiple signals. Moreover, the structural transition was
also proved by 13C NMR, where the signal of C-2 located at
77.3 ppm instead of 182.0 ppm, demonstrating the conversion
of N+C into N−C (see Figure S2). Similar results were
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Figure S5). These results indicate that the structure-induced
electron distribution on C-2 is able to exert great impact on the
stability of the merocyanine molecules. To sum up, approaches
that could effectively evaluate electron density on C-2 is
beneficial in terms of avoiding the hydrolysis. Usually,
merocyanine-based probes with longer conjugation system
will increase the electron density on C-2 and that could help us
understand why some merocyanine-based probes with longer
conjugation systems are relatively stable.
Influence of Strategy Utilized for Preparing Type-II
Probes. As mentioned above, caging strategy has been
extensively utilized for constructing type-II probes. Typically,
the donor group within the D-π-A system of the merocyanine-
based probes is caged with a trigger which can be selectively
cleaved by the analyte.13−17 However, when electron donor
groups, such as -OH and -NH2, were once caged, the electron
donating ability will be weakened along with an obvious blue
shift of the absorption spectra due to the attenuation of the
ICT effect. Meanwhile, hydrolysis may be therefore aggravated,
due to the decrease of C-2 electron density as a consequent of
the decrease in electron donating ability. As displayed in Figure
S6, once caged by -CH3, the resulting A5 shows high hydrolysis
preference even at low pH because A4 tends to be transformed
into its keto structure, bringing in more electron intensity on C-
2, the process of which could be restricted once modified with
-CH3 (see Scheme S1).16 Those could lead to the hydrolysis of
A5 when incubated in aqueous solution.
Influence of Analytical Media on the Hydrolysis.
Because of the limited aqueous solubility of merocyanine
molecules, organic solvents, surfactants, and even serum are
often utilized to help to enhance their solubility. However,
those chemicals can also exert great influences on their
hydrolysis, as both the nucleophilic interactions and the
molecule structures are highly dependent on the surround-
ings.18,19 Thus, the hydrolysis of merocyanine molecules was
tested in different surroundings. Interestingly, although A2 is
resistant to the nucleophilic attack of OH− even in relatively
higher pH buffer solution, the spectrum of A2 changed upon
the addition of dimethyl sulfoxide (DMSO). As DMSO content
was within the ranges of 0 to 25% (v/v), the absorbance of A2
showed a slight increase with the increase amounts of DMSO.
In contrast, as the DMSO content got to 50% and finally to
75%, the absorbance at 542 nm dropped remarkably along with
an increase at ∼300 nm ascribed to the hydrolyzed products
(see Figure 4 and Figure S7). Absorbance of A2 in pure DMSO
declines to some extent compared with that in pure water,
which is owing to the difference in molar extinction coefficient
between water and DMSO. The fluctuation in absorbance of
A2 in solution with DMSO content ranging from 50% to 100%
imply that the hydrolysis is solvent-reversible, i.e., the
absorbance decrease induced by addition of DMSO can be
restored by decreasing the relative amount of DMSO. Although
the absorption intensity of A2 in solution containing 75%
DMSO decreased greatly, the absorption intensity could be
restored to a certain level by adding some amount of water to
the mixture solution (see Figure S8).
Figure 4. Absorption changes of A2 (20 μM) in buffer solution (pH
9.4, 20 mM NaHCO3−Na2CO3) with an increase amount of DMSO
(vol %) from 0% to 100%.
donating ability (α) and hydrogen bond accepting ability (β),
play important role during the hydrolysis process.20 The binary
DMSO/H2O with high β and low α value could accelerate the
nucleophilic addition especially considering the preferential
solvation effects. In the mixture solution of DMSO/H2O, the
merocyanine dye tends to exhibit its properties that are similar
to that in pure DMSO. Accordingly, other polar aprotic
solvents, for example, DMF, of similar α and β values to that of
DMSO, can also accelerate the OH− nucleophilic attack (Figure
S9). Comparatively, other solvents of high α and low β value,
such as MeCN (α 0.19, β 0.31) and MeOH (α 0.93, β 0.62),
showed limited ability to enhance such a nucleophilic addition.
Only negligible changes could be obtained when A2 was
incubated in mixed solution of MeCN and buffer solution. The
nucleophilic addition of OH− on dyes did not occur in MeOH
solution (see Figure S10). Likely, in practical biological assay,
serum can enhance the solubility of organic molecules through
hydrophobic effects. However, as depicted in Figure 5, when
Figure 5. (a) Time-dependent spectral changes of A5 (20 μM) in
bovine serum incubating at 37 °C; (b) dynamic absorption changes of
A5 at room temperature in bovine serum (λabs. = 430 nm, time
interval = 1 s).
incubated in fetal calf serum, A5 was hydrolyzed quickly within
60 min and the hydrolysis features zero order reaction. As
mentioned above, enlarging the conjugation system of the
merocyanine molecules is supposed to enhance their ability to
resist the hydrolysis. As expected, when indocyanine green, a
commercial available cyanine derivates with longer conjuga-
tions, was incubated with both bovine serum and assigned
DMSO/water mixture, the hydrolysis could be inhibited to
some extent (see Figure S11).
In general, the absorbance of merocyanine molecules is
negatively correlated with the index of refraction (n) of the
solvents. The absorbance of A2 showed some slight bath-
ochromic shift with increasing the percentages of DMSO
(nDMSO > nwater), which further demonstrates that solvent could
pose distinctive effects on the electron distribution of
merocyanine.3 Two solvent parameters, i.e., hydrogen bond
Merocyanine is well-known for its aggregation proper-
ties.18,21−23 Although the reason why serum is able to hasten
its hydrolysis is still undertaken, we speculate that once the dye
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was dissolved into serum, the serum is able to force them to
aggregate through hydrophobic interaction, then exist in a state
which is more sensitive toward nucleophilic reagents. To
confirm that, CTAB (cetyltrimethylammonium bromide)
which possesses similar solubilization effects as serum was
also employed to test the hydrolysis of A6. As expected, the
addition of CTAB is able to enhance the hydrolysis rate to
some extent (see Figure S12).
DFT Calculation for the Hydrolysis. Theoretical
calculation can help us understand how the structures and
media affect the hydrolysis process by comparing the optimized
molecule structures and related thermodynamics data of the
hydrolysis process. First, the optimized structure of A1 and A1-
OH depicted in Figure 6 showed how hydrolysis could affect
To further confirm the important role played by C-2 in the
hydrolysis, Mulliken atomic charges (MAC) were gathered. As
listed in Table 1, it is found that more positive charges on C-2
support the hydrolysis. In particular, compared with A1, A3
with C-2 imparting less MAC is unlikely to suffer the attack of
OH−. This is also the case for A3 whose C-2 is entitled to less
MAC compared with A1, which can be deduced from the
comparison of the hydrolysis slope (see Figure S14). Finally,
the thermodynamic parameters for the hydrolysis reaction in
different solvents were obtained. Gibbs free energy (ΔG) of the
hydrolysis of A2 are −45.468 and −44.906 J/mol in DMSO
and water, respectively, which suggested that the addition of
DMSO is more likely to support the hydrolysis.
Type-II Merocyanine-Based Probe in Biological Assay.
Caging strategy is widely utilized for fabricating type-II probes,
but such a strategy is supposed to aggravate the hydrolysis as
mentioned above. To confirm our allegations, a merocyanine
dye based probe for the detection of H2O2 was fabricated. As
illustrated in Scheme 3, the merocyanine molecule was
Scheme 3. Illustration of the Sensing Mechanism of
Merocyanine for Detection of H2O2
Figure 6. Optimized structures of A1 and A1-OH. (The left and right
represent the top view and front view, respectively).
the molecular structures as well as the spectra. Obviously, sp2-
hybridized C-2 renders the indolenium group and styrene
conjugated and coplanar. An ICT prefers to take place from Br
atom to the positively charged indolenium group through the
conjugated system. While in A1-OH, because of the addition of
OH−, C-2 adopts sp3 hybridized orbits, getting the indole group
nearly perpendicular to styrene. The conjugation system is
therefore broken consequently, resulting in the sharp decline of
the ICT effects. Generally, larger conjugation system
contributes to the red shift of the absorption.2 Hence, once
the conjugated system is interrupted by nucleophilic addition, a
blue-shift absorption occurs theoretically. This was further
confirmed by simulated absorption spectrum carried out by
time-dependent DFT (TDDFT) (see Figure S13). In
particular, the calculated excitation energy of A1 and A1-OH
is 2.8694 and 4.4550 eV, respectively, both of which are
consistent with the experimental results, whose λmax shifted to
264 nm from 394 nm upon addition of OH− (see Table 1).
The DFT calculation as well as experimental results
demonstrated that the nucleophilic attack of OH− on C-2
can decrease the ICT effects, consequently leading to the
spectral changes.
modified with the aryl borate specifically responding toward
H2O2.17,24−26 Before protected with aryl borate, A4 is likely to
transfer into the keto structure, which could be inhibited once
caging A4 with aryl borate. When H2O2 was added, the -OH
could be restored, through which H2O2 could be detected. In
fact, a good linear relationship could be obtained between
spectral changes and H2O2 concentration in physiological pH
conditions (pH 7.4 20 mM PBS) (see Figure 7) with a
Figure 7. Absorption changes of A6 (20 μM) in the presence of
different concentrations of H2O2 (0−30 μM). Inset: plot of the
absorption intensity ratios (A515/A415) of A6 (20 μM) upon
addition of H2O2 (0−30 μM).
Table 1. Excitation Energies and Corresponding Absorption
Wavelength of A1−A5 and the Corresponding Mulliken
Atomic Charges of C-2
theoretical calculation
detection limit of 1 μM. However, A6 was hydrolyzed when
exposed to biological medium, serum for instance as shown in
Figure 8. This is because A6 is unlikely to transform into
corresponding keto isomers that are more stable against the
nucleophilic attack, which can be deduced from the comparison
between optimized structures between A4 and A4′. As
displayed in Figure S15, A4 is readily to exist in the form of
exptl λabs, nm
cmpd
ΔE/eV
MAC of C-2
λabs/nm
A1
A2
A3
A4
A5
3.17
2.30
3.14
2.91
2.86
0.428
0.421
0.426
0.426
0.426
391.0
394
542
418
426
426
539.20
394.51
425.99
433.84
E
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.6b02113.
1
Chemical structures and corresponding H, 13C NMR
and HRMS spectra; additional UV−vis spectra data;
DFT calculation results (PDF)
Figure 8. Time-dependent absorption changes of A6 (20 μM)
incubating in (a) FBS and (b) HS at 37 °C.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: dengliu@csu.edu.cn (L. Deng). Fax: +86 731 8887
9616.
■
keto-structures, however A4′ resembling to A6 prefers to exist
in the form of enol-structure which is more likely to be attacked
by nucleophiles as discussed above. Consequently, the intensity
of A6 dropped remarkably during the analytical periods (30
min).
*E-mail: liuyounian@csu.edu.cn(Y.-N. Liu).
Author Contributions
∥L. Wang and Y. Hao contributed equally.
Also, sensor A6 was dissolved into cell culture medium and
incubated in CO2 incubator at 37 °C for different time, which is
an essential procedure for biological assay. As depicted in
Figure 9, an obvious decrease in absorption peaked at 405, 450,
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (Grant Nos. 21476266, 21276285, and
U1404215)
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Figure 9. (a) Absorption changes of probe A6 (50 μM) in cell culture
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aggravated by biological surrounding such as serum. It is worth
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accepted strategy for the construction of type-II probes, that is
modifying the donor group to tune the ICT effects. Our
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