PH Induced dual "oFF-ON-OFF" switch: Influence of a suitably placed carboxylic acid

Article abstract of DOI:10.1039/c2ob26630j

The design and synthesis of molecular probes competent for pH signaling within or beyond a certain range is a complicated matter. Herein a new mechanism for ''OFF-ON-OFF'' absorbance and fluorescence intensities vs. pH behaviour is described. The probe design is based on the connection of carboxylic acid derivatized benzoxazole and 7-hydroxycoumarin/iminocoumarin parts. The protonation/deprotonation of the carboxylic acid (-COOH), N atom of benzoxazole ring and hydroxy part of the coumarin ring have been used for this mechanistic study. We have designed the molecule in such a fashion that deprotonation of the hydroxy part takes place at a lower pK_a compared to deprotonation of the -COOH. The dual ''OFF-ON-OFF'' properties of our probes depend on the C-C bond between the two different heterocyclic parts. Quantum mechanical calculations showed that the particular 'C-C' bond has an additional π-character. The twisting around this bond in different forms is responsible for such an ''OFF-ON-OFF'' property. This mechanism is new in fluorescence alteration processes. The delocalization of charge from one heterocyclic part to the other heterocyclic part in the mono- and dianionic forms controls the ''OFF-ON-OFF'' properties. The role of the carboxylic acid group was examined using an acetyl substituted derivative. One of our probes was successfully applied in live cell imaging studies in media at different pH.

Full text of DOI:10.1039/c2ob26630j

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PAPER
pH Induced dual “OFF–ON–OFF” switch: influence of a suitably placed
carboxylic acid†
Kalyan K. Sadhu,a Shin Mizukami,a,b Akimasa Yoshimuraa and Kazuya Kikuchi*a,b
Received 7th June 2012, Accepted 21st September 2012
DOI: 10.1039/c2ob26630j
The design and synthesis of molecular probes competent for pH signaling within or beyond a certain
range is a complicated matter. Herein a new mechanism for ‘‘OFF–ON–OFF’’ absorbance and
fluorescence intensities vs. pH behaviour is described. The probe design is based on the connection of
carboxylic acid derivatized benzoxazole and 7-hydroxycoumarin/iminocoumarin parts. The protonation/
deprotonation of the carboxylic acid (–COOH), N atom of benzoxazole ring and hydroxy part of the
coumarin ring have been used for this mechanistic study. We have designed the molecule in such a
fashion that deprotonation of the hydroxy part takes place at a lower pKa compared to deprotonation of
the –COOH. The dual ‘‘OFF–ON–OFF’’ properties of our probes depend on the C–C bond between the
two different heterocyclic parts. Quantum mechanical calculations showed that the particular ‘C–C’ bond
has an additional π-character. The twisting around this bond in different forms is responsible for such an
‘‘OFF–ON–OFF’’ property. This mechanism is new in fluorescence alteration processes. The
delocalization of charge from one heterocyclic part to the other heterocyclic part in the mono- and
dianionic forms controls the ‘‘OFF–ON–OFF’’ properties. The role of the carboxylic acid group was
examined using an acetyl substituted derivative. One of our probes was successfully applied in live cell
imaging studies in media at different pH.
revealed strong fluorescence for iminocoumarin in acidic and
Introduction
neutral solutions, but little fluorescence for coumarin over a wide
pH region, except under highly acidic conditions.7
Several biological processes have an effect on the intracellular or
The pH dependent fluorescence properties of umbelliferone8
extracellular pH. For cellular diagnosis, numerous fluorogenic
pH probes have been developed to estimate intracellular pH.1
Recent research also pursues activatable probes for acidic and
neutral pH.2
and 3-(2-benzoxazolyl)umbelliferone9 (U and BU respectively,
Chart 1) revealed the fluorescence “ON” state in basic medium
due to loss of a proton from the phenolic hydroxy group.
Systems competent for fluorescence “OFF–ON–OFF” by vari-
ation of pH are particularly important for the direct visualization
of pH windows with implications in the life sciences. However,
there have been only a few reports with such pH windows based
on molecular probes.6,10 Most of these previously reported pH
sensitive molecules involved protonated pyridines (acting as
electron acceptors in the PET process) and unprotonated tertiary
The most common technique for fluorescence enhancement at
acidic pH involves the blocking of the photoinduced electron
transfer (PET) process of dialkylamine based probes.3 In another
example, a phenyl ring plays the role of fluorescence quencher in
basic medium.4 Fluorescence turn-on with protonation around
physiological pH was achieved by a pentamethine cyanine dye
with pKa 7.5.5 Recently, the direct correlation between the emis-
sion of a fluorescein based probe and its proton equilibria over a
wide pH range was established.6 The difference between the fluo-
rescence properties of iminocoumarin and coumarin probes
aDivision of Advanced Science and Biotechnology, Graduate School of
Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871,
Japan. E-mail: kkikuchi@mls.eng.osaka-u.ac.jp,
http://www-molpro.mls.eng.osaka-u.ac.jp/; Fax: (+81) 6-6879-7875
bImmunology Frontier Research Center, Osaka University,
3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
†Electronic supplementary information (ESI) available: Experimental
and theoretical details, UV–visible absorption spectra, and fluorescence
spectra of the probes. See DOI: 10.1039/c2ob26630j
Chart 1 Chemical structures of the probes U, BU, ONO, ONNH, and
AcNO.
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amines (acting as electron donors in the PET process). The pres-
ence of both functional groups within a multicomponent system
generates several equilibria in the medium. The pKa values of
such protonated functional groups cover different pH regions.
Some probes contain coordinative interactions to gain such fluor-
escence properties. The deprotonation of the phenolic hydroxy
group was mostly explored for the basic pH region. However,
deprotonation of the carboxylic acid group in fluorescence-based
pH modulation remains unexplored. Herein, our target was to
develop a new pH sensor on the basis of deprotonation of the
carboxylic acid group and also the influence of its position in the
probe. In our probe design, we combined the carboxylic acid and
phenolic hydroxy group together for pH monitoring. We syn-
thesized 4-carboxylic acid-substituted coumarin and imino-
coumarin derivatives (ONO and ONNH, Chart 1) for
monitoring the effect of a wide range of pH.
Fig. 1 Absorption spectra of 10 μM ONO were measured in 100 mM
sodium phosphate buffer containing 1% DMSO at various pH values (a)
pH 4.8–7.8 and (b) 8.9–12.2 at 25 °C.
Results and discussion
Design and synthesis
Since the development of BU, there have been some reports of
5-substituted derivatives including a carboxy group.11 However,
there has been only one report of a 4-substituted BU derivative.12
In our design a 4-substituted acid derivative was chosen to study
the influence of the nitrogen of the benzoxazolyl part in the
vicinity during the deprotonation process.
In these probes, three protonation/deprotonation processes
occur, one from the hydroxy group of the 7-hydroxycoumarin
part and the others from the carboxylic acid and N atom of the
substituted benzoxazolyl part. These two heterocyclic rings are
connected via a ‘C–C’ bond. In order to compare the role of the
–COOH in the dual spectroscopic modulation, our aim was to
synthesize another 4-substituted derivative having a similar elec-
tronic effect without any protonation/deprotonation properties.
The acetyl substituted aniline derivative showed a similar elec-
tron withdrawing effect compared to the acid analogue.13–15 For
our purpose, a new acetyl substituted AcNO (Chart 1) probe was
also synthesized, where acetyl substitution played a significant
contribution to the fluorescence properties compared to the
carboxy group.
Fig. 2 Relative fluorescence intensities of ONO at 470 nm (λex
=
415 nm) and ONNH at 480 nm (λex = 435 nm) in 100 mM sodium
phosphate buffer containing 0.1% DMSO at various pH values
(2.6–12.2) at 25 °C.
absorption peak was shifted to 435 nm after the first deproto-
nation step with an isosbestic point at 399 nm. After the second
deprotonation, the peak was blue shifted to 400 nm with low
extinction coefficient (Fig. S4b†). The pH dependent absorption
intensities at 415 nm and 435 nm for ONO and ONNH respecti-
vely (Fig. S5†) revealed absorption “OFF–ON–OFF” properties
for both the probes.
Absorption studies of the probes
The absorbance spectra of ONO probe were monitored in
100 mM sodium phosphate buffers of different pH. An absorp-
tion maximum at 360 nm was observed for pH 4.8 (Fig. 1a).
With increasing pH, another new absorption maxima appeared at
415 nm (pH 6.2, Fig. 1a). The absorption intensity increased at
415 nm with further increase of pH. A clear isosbestic point was
obtained at 387 nm. This process involved the first deprotonation
step of the neutral ONO probe. The absorption peak position
remained in the same position within a certain pH range
(7.8–8.9). With further increase in pH, a second deprotonation
took place and this resulted in a slight blue shift of the absorp-
tion peak to 405 nm with low extinction coefficient (Fig. 1b).
A similar type of absorption profile was obtained for the
ONNH probe. The absorption maximum of this iminocoumarin
probe was obtained at 385 nm for pH 4.8 (Fig. S4a†). The
The ground state pK1 and pK2 values (6.6 and 11.3 for ONO,
6.6 and 11.0 for ONNH) were obtained from the absorption
spectra. These pKa values are the result of electronic charge dis-
tributions during the deprotonation process of the acidic form
and its conjugated base in the ground state.
Steady state emission studies of the probes
The fluorescence spectra of the ONO probe showed maximum
emission at 470 nm (λex = 415 nm) in acidic pH. The fluor-
escence intensity at 470 nm was monitored at different pH
ranging from acidic to basic (Fig. 2). The fluorescence intensity
increased from pH 2.6 to 6.0, but with further increase in pH the
fluorescence intensity decreased in a continuous manner. In the
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case of the ONNH probe, a similar type of fluorescence assay
was obtained by monitoring the emission at 480 nm (λex
=
435 nm). The maximum fluorescence intensity was obtained at
pH 7.4 for ONNH. At extremely basic pH, the emission maxima
for both the probes were blue shifted and obtained at 450 nm
(Fig. S6†). The fluorescence properties of both the probes
showed an “OFF–ON–OFF” trend within a wide pH range. The
stability of the probes was examined by analytical HPLC at
different pH to overrule the immediate degradation of the probes
(Fig. S8†).
The charge distributions of the acidic form and its conjugated
base pair in the excited states are expected to be different from
the ground states. These differences are reflected in the variation
*
*
of acidity (pKa ) in the excited states. The pKa values were cal-
culated by Förster cycle16 and were found to be 4.7 and 7.7 for
ONO, and 6.5 and 8.4 for ONNH. In the presence of biologi-
cally important metal ions, the fluorescence properties of these
probes remained unaltered at different pKa* values.
Fig. 3 Radiative and nonradiative decay rate constants of ONO and
ONNH at different pH around two different pKa.
*
The differences in the second pKa and pKa values for both
regions of ONO probe. On the other hand, knr values for ONNH
were almost constant (Fig. 3) around different pKa* regions. This
indicates that there is no significant change in geometry around
pKa* for ONNH probe.
the probes were reflected in the fluorescence and absorption
spectral changes at higher pH range. The position of the emis-
sion bands were blue shifted for both the probes around pH
10.8. On the other hand, similar kinds of shift for the absorption
spectra were observed around pH 11.7 for both ONO and
ONNH. These changes in the spectra account for the second
deprotonation step.
The trend of radiative decay rate constants (kr) for both the
*
probes (Fig. 3) around different pKa values controlled the fluo-
rescence “OFF–ON–OFF” modulation. The delocalization of the
negative charge between the two heterocyclic parts in the mono-
anionic form was responsible for the “OFF–ON” state. The
second deprotonation from the benzoxazolyl part restricted the
negative charge delocalization from the hydroxycoumarin part.
This resulted in a further fluorescence “OFF” state.
The contribution due to different prototropic zwitterionic
forms17 around the heterocyclic part in the solution also plays an
*
important contribution in the pKa determination. Similar types
of observation have been reported in the literature.18 The contri-
bution from these prototropic forms is also reflected in the mis-
match of the absorbance and excitation spectra.
The dissociation constant in the excited state includes the
favorable excited state intramolecular proton transfer (ESIPT)19
from the proton of the carboxy group to the benzoxazolyl nitro-
gen atom. ESIPT could also be a possibility for modulation of
fluorescence properties due to the presence of H-chelate ring19c
as a reaction center.
In order to study the participation from the carboxylic acid
proton in the spectral “OFF–ON–OFF” properties, we studied
the spectral properties of the AcNO probe containing a 4-acetyl
substituted derivative. The absorbance and fluorescence proper-
ties of the AcNO probe showed similar types of “OFF–ON”
spectral patterns (Fig. S10†) to those of BU.9 These differences
bring forward the vital role of the acid groups in the “OFF–ON–
OFF” spectral properties of the ONO and ONNH probes. No
spectral change for AcNO at higher pH suggested the absence of
a second deprotonation of this probe.
Mechanism of the fluorescence “OFF–ON–OFF”: theoretical
support
It is a well-established phenomenon that the deprotonation
of carboxy group occurs at lower pH compared to phenolic
proton.20 To rationalize the pH profile of the newly synthesized
probes in the same fashion, deprotonation of the carboxy group
was considered during the first deprotonation step (ONO1′,
Scheme 1). However, the changes in the spectral properties
could not be correlated with this deprotonation. Possibly hydro-
gen bonding between the carboxylic acid and the N atom of the
benzoxazolyl part restricted the deprotonation of the carboxylic
acid. The protonated zwitterionic form (benzoxazolium ion
ONO-z, Scheme 1) could also play an important role in the spec-
tral properties.
In order to check out these possibilities, theoretical calcu-
lations for optimized geometries were carried out at the B3LYP/
6-31G++ level of theory in Hartree–Fock method using the
Gaussian 09W programme package.21 The energies of the opti-
mized geometries in the zwitterionic form (ONO-z) were found
to be comparable with that of ONO (Scheme 1). Electron
density calculations ruled out the possibility of deprotonation of
the carboxylic acid during the first deprotonation (Fig. 4a). This
calculation suggested that the first deprotonation was possible
from the 7-hydroxy group of the coumarin instead of the
carboxylic acid. The favorable interaction of the carboxylic acid
Fluorescence lifetime studies of the probes
In order to check the origin of the “OFF–ON–OFF” spectral
properties of the probes, fluorescence decay profiles of both the
probes were monitored around different pKa* values (Fig. S12†).
In the case of ONO, the trend in nonradiative decay rate constant
values (knr, Fig. 3) around two different pKa confirmed the
maximum rigid geometry in the monoanionic form. The knr
*
*
values were reached to the maximum within the two pKa
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Fig. 4 (a) Calculated electron density contour surface (blue: positive
electrostatic potential, and red: negative electrostatic potential) of ONO
and (b) contour π-MO surface of planar ground state form of ONO
showing the origin of the “CIC” bond.
Scheme 1 Probable protonation equilibria of ONO.
proton with the nitrogen atom of the heterocyclic ring was
involved with the poor acidity of the carboxylic acid proton.
The absence of a pH dependent fluorescence “OFF–ON”
switch in the case of 7-methoxy-substituted coumarin22 also
indirectly confirmed the first switching mechanism to be from
the 7-hydroxy coumarin part of the newly synthesized probes.
Optimized geometry depicted the nature of the bond order
between the two heterocyclic parts as 1.5, with a calculated
‘C–C’ bond distance of 1.45 Å. Contour π-MO of the neutral
planar form of ONO (Fig. 4b) depicted a “CIC” bond instead
of ‘C–C’ between the benzoxazolyl part and the 7-hydroxy-
coumarin part. The deprotonation of the 7-hydroxycoumarin part
could lead to formation of a stable quinoid geometry by reso-
nance (ONO1-r, Scheme 1). The first “OFF–ON” absorption
profile of ONO during the first deprotonation (Fig. 1a) was
similar to the absorption profile of BU.9 However, unlike BU,
the second “OFF” absorption state of ONO was observed with
further increase in pH due to the loss of proton from the car-
boxylic acid (ONO1) or benzoxazolium ion (ONO1-z) to form
ONO2 as shown in Scheme 1.
The result obtained from the fluorescence lifetime experiment
has been further supported by the different energy minimized
structures of twisted and planar geometries of ONO in different
ionic and neutral forms (Fig. 5 and Fig. S13–S15†). The twisted
geometry in the neutral state became planar after the first depro-
tonation. Further deprotonation generated the twisted dianionic
form. These geometries were responsible for the knr values of
ONO. On the other hand, the planar geometries of different
ionic and neutral species of ONNH (Fig. 5) were responsible for
the constant knr values in the different forms. The planar geo-
metries in the case of ONNH originated from the hydrogen
bond between the imine group and the heteroatom of the benz-
oxazolyl part.
Fig. 5 The energy-minimized ground state geometries of ONO and
ONNH in neutral, monoanion, and dianionic forms along with the dihe-
dral angle (D.A.) between the benzoxazolyl part and the coumarin part.
In order to correlate the experimental absorption spectra of the
probes at different pH, the transition energies of the optimized
geometries were calculated by time-dependent density functional
theory (TD-DFT) using the same level of basis set. TD-DFT cal-
culations for different species (Fig. S15†) also supported the
experimentally observed red and blue shifts of the probes.
Live cell imaging studies with ONNH probe
We have applied the ONNH probe to evaluating its applicability
for live cell imaging studies by confocal microscopy. Fluore-
scence images of the living HEK293T cells were taken after
probe treatment in different extracellular pH buffers (Fig. S16†).
The highest fluorescence intensity was observed at pH 7.4. The
fluorescence images at pH 6.5 and 8.0 confirmed the fluore-
scence “OFF–ON–OFF” of ONNH probe in living cell imaging
studies.
The calculated electronic charge distribution on the heteroa-
toms (N and O) of the benzoxazolyl part in the neutral and ionic
states (Table S1†) supported the variation in the kr values for
both the probes. The charge densities on these heteroatoms in
the monoanionic forms are first increased from the neutral states
due to the favorable charge delocalization process. However, the
values are then decreased in the dianionic forms due to the
restriction in the delocalization process across the “CIC” bond.
This is reflected in the maximum kr values in the monoanionic
forms for both ONO and ONNH probes.
Conclusions
In conclusion, we have developed a new mechanism for a rever-
sible dual “OFF–ON–OFF” pH sensor with the influence from a
suitably located carboxylic acid group. The deprotonation of a
carboxylic acid group or benzoxazolium ion was used for the
first time in the alteration of absorption and fluorescence proper-
ties simultaneously. Theoretical study supported the trend of the
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Scheme 3 Synthetic route to AcNO.
Scheme 2 Synthetic route to ONO and ONNH.
Synthesis of AcNO. Compound 2 (9 mg, 0.05 mmol) was
added to an ethanol solution (15 mL) of 3′-hydroxy-2′-amino-
acetophenone (8 mg, 0.05 mmol). Acetic acid (1 mL) was added
to this mixture. The mixture was then set for reflux at 90 °C for
24 h. The desired product AcNO was isolated using preparative
kinetic parameters obtained from experimental studies. In
addition, the iminocoumarin probe ONNH showed the maximum
fluorescence at physiological pH conditions and has been
successfully applied in live cell imaging studies.
1
HPLC (5 mg, y. 30%). H NMR (DMSO-d6, 400 MHz) δ 2.68
(s, 3H), 6.33 (d, 1H), 6.80 (s, 1H), 6.90 (d, 1H), 7.39 (d, 1H),
7.48 (dd, 1H), 7.78 (d, 1H), 7.83 (d, 1H), 8.81 (s, 1H); 13C
NMR (DMSO-d6, 100 MHz) δ 28.2, 110.6, 112.3, 118.3, 122.3,
123.6, 126.9, 135.8, 138.5, 139.2, 143.6, 145.3, 149.9, 152.2,
156.8, 157.1, 162.3, 202.1; HRMS (C18H11NO5, FAB+); Found
321.0642; Calc. 321.0637.
Experimental section
See ESI† for full experimental details and other supporting
materials.
The synthetic route for the ONO and ONNH is illustrated in
Scheme 2.
Synthesis of 1. Compound 1 was synthesized according to the
Acknowledgements
procedure available in the literature.23
This research is supported by MEXT of Japan (Grants 20675004
to K. K.), by CREST from JST, by Asahi Glass Foundation, by
the Grant-in-Aid from the Ministry of Health, Labour and
Welfare (MHLW) of Japan, and by the Japan Society for the
Promotion of Science (JSPS) through its “Funding Program for
World-Leading Innovative R&D on Science and Technology
(FIRST Program).” We thank Dr Koushik Dhara of Osaka
University for his helpful discussion and Prof. Hirohiko Houjou
at the University of Tokyo for his suggestion regarding the
quantum calculations. KKS and AY acknowledge GCOE Fellow-
ship of Osaka University.
Synthesis of 2. Compound 2 was synthesized according to the
procedure available in the literature.24
Synthesis of 3. Compound 2 (186 mg, 1 mmol) was added to
an ethanol solution (15 mL) of 1 (182 mg, 1 mmol). Acetic acid
(1 mL) was added to this mixture. The mixture was then set for
reflux at 90 °C. After 24 h the desired product was isolated by
flash column chromatography using 9% methanol in dichloro-
methane. Compound 3 was isolated as a yellow solid after com-
1
plete removal of solvent (263 mg, y. 75%). H NMR (CD3OD,
400 MHz) δ 1.38 (t, 3H), 4.33 (q, 2H), 6.43 (d, 1H), 6.61
(s, 1H), 7.51 (m, 1H), 7.60 (d, 1H), 7.92 (d, 1H), 7.99 (d, 1H),
8.35 (s, 1H); 13C NMR (CD3OD, 100 MHz) δ 14.4, 63.0, 60.3,
103.7, 107.8, 108.4, 115.9, 118.0, 123.5, 126.8, 129.6, 130.3,
140.9, 142.3, 148.1, 151.6, 155.7, 157.4, 163.9, 168.2; HRMS
(C19H15N2O5, FAB+); Found 351.0992; Calc. 351.0981.
Synthesis of ONO and ONNH. Compound 3 (20 mg,
0.06 mmol) was added to an aqueous medium (10 mL) contain-
ing 6N hydrochloric acid. The mixture was then set for reflux at
100 °C. The reflux was continued for 2 h. The desired products
ONO (8 mg, y. 45%) and ONNH (5 mg, y. 28%) were isolated
using preparative HPLC.
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ONO characterization: 1H NMR (DMSO-d6, 400 MHz)
δ 6.83 (s, 1H), 6.89 (d, 1H), 7.09 (d, 1H), 7.15 (dd, 1H), 7.22
(d, 1H), 7.84 (d, 1H), 8.90 (s, 1H), 9.90 (br, 1H), 10.95 (br, 1H);
13C NMR (DMSO-d6, 100 MHz) δ 100.4, 102.1, 111.4, 113.3,
114.8, 119.1, 119.3, 120.7, 126.2, 132.6, 148.2, 149.4, 150.8,
156.8, 160.4, 161.4, 164.2; HRMS (C17H10NO6, FAB+); Found
324.0502; Calc. 324.0508.
ONNH characterization: 1H NMR (DMSO-d6, 400 MHz)
δ 6.87 (s, 1H), 6.97 (d, 1H), 7.24 (d, 1H), 7.35 (dd, 1H), 7.55
(d, 1H), 7.73 (d, 1H), 9.53 (s, 1H), 9.63 (s, 1H), 11.21 (s, 1H),
11.84 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 99.9, 107.5,
116.7, 124.8, 125.8, 128.2, 133.6, 134.8, 138.6, 141.1, 146.7,
148.4, 153.1, 161.7, 165.5, 169.8, 173.3; HRMS (C17H11N2O5,
FAB+); Found 323.0680; Calc. 323.0668.
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The synthetic route for AcNO is illustrated in Scheme 3.
This journal is © The Royal Society of Chemistry 2012
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