Ratiometric spiropyran-based fluorescent pH probe

Article abstract of DOI:10.1039/c3ra41456f

Spiropyran (SP) is an attractive molecular platform for the design of fluorescent OFF-ON probes for the detection of cations and anions operative on a ring-opening/closing mechanism. By incorporating a benzimidazole moiety at the ortho-position of the oxy

Full text of DOI:10.1039/c3ra41456f

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Ratiometric spiropyran-based fluorescent pH probe3
Qi-Hua You,a Li Fan,b Wing-Hong Chan,*a Albert W. M. Leea and Shaomin Shuangb
Cite this: RSC Advances, 2013, 3, 15762
Spiropyran (SP) is an attractive molecular platform for the design of fluorescent OFF-ON probes for the
detection of cations and anions operative on a ring-opening/closing mechanism. By incorporating a
benzimidazole moiety at the ortho-position of the oxygen atom of the pyran unit, a ratiometric fluorescent
probe in the NIR range for the measurement of pH change in aqueous ACN solution was designed and
investigated. The ring-opening mechanism and cis–trans interconversion of the probe are also
substantiated by 1H NMR titration. The probe showed a ratiometric change in the pH range from 7.0 to
5.5 and the pKa of the probe was calculated to be 5.9. The relative intensity ratio (I690 nm/I517 nm) changed
from 0.08 to 2.11 with a more than 26-fold enhancement in response to the pH variation.
Received 25th March 2013,
Accepted 1st July 2013
DOI: 10.1039/c3ra41456f
www.rsc.org/advances
metric output: intramolecular charge transfer (ICT), fluores-
cence resonance electron transfer (FRET), monomer/excimer
Introduction
In recent years, chemical sensors capable of measuring pH by
optical methods have been widely used in analytical chemistry,
physiology and the biosciences.1–5 Fluorescent probes have
demonstrated outstanding characteristics in the detection of
various analytes such as high selectivity, low detection limits,
real-time detection, and high-throughput. Currently, a number of
fluorophores such as rhodamine,6,7 fluorescein,8 BODIPY,9–11
naphthalenediimide,12 cyanine,13,14 coumarin,15 and others16–18
have been used to design fluorescent pH probes covering the
UV/Vis to NIR operation range. Amongst them, most of the
sensing probes change intensity in single-emission window in
response to the variation of pH. However, fluorescence signals
are easily influenced by environmental factors, such as
temperature, solvent polarity and the concentration of the
analytes. Therefore, these factors make single-emission fluor-
escence detection inappropriate in the quantitation of chemical
species.19
switching and excited state intramolecular proton transfer
(ESIPT). Until now, a number of probes based on these
mechanisms have been reported.20–23 Amongst them, the most
remarkable photophysical property of the ESIPT probes is the
large Stokes shift due to the change from the enol-form to the
keto-form of the probe. For instance, via ESIPT, the Stokes
shift of 2-(2-hydroxyphenyl)benzoxazole (HBO) and 2-(2-hydro-
xyphenyl)benzothiazole (HBT) could be as large as 180 nm.24
Therefore, the ESIPT mechanism is an ideal strategy for the
design of fluorescent probes.
Recently, more and more scientists have become interested
in the design of near-infrared (NIR) fluorophoric probes due to
the high penetration of NIR radiation through organic tissues
and their low fluorescence background.25–29 Spiropyrans (SPs)
are well known photochromic materials. The closed spiro-
pyran form can transform to its ring-opened merocyanine
(MC) form under photochemical irradiation.30 Furthermore,
spiropyran derivatives could also undergo ring-opening in the
presence of acid to form MC-OH+ and then convert back to the
SP form after the addition of base (Scheme 1).31 Inspired by
this transformation, we hypothesized that the incorporation of
the benzimidazole moiety into the ortho-position of the
phenolate oxygen atom in the spiropyran scaffold could
undergo the ESIPT process after spiropyran ring-opening in
acidic conditions. Thus, a ratiometric fluorescent output for
the measurement of pH in the NIR range could be achieved.
Although many spiropyran-based probes for the detection of
metal ions, anions and thiols have been reported,32–35
spiropyran-based ratiometric NIR pH probes that function in
aqueous solutions, however, have been rarely exploited.36
To alleviate the problems associated with single-emission
measurement, the development of ratiometric measurement
appears to be a valid approach. Ratiometric measurements
record the fluorescence intensity at two wavelengths simulta-
neously and their ratio is calculated for correlating with the
concentration of the analytes. As
a result, ratiometric
measurements can provide better accuracy by the built-in
correction of two emission bands in the detection rather than
that obtained by using a single wavelength. In general, several
types of signal transduction mechanisms can provide ratio-
aDepartment of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong
SAR, China. E-mail: whchan@life.hkbu.edu.hk; Fax: +852-3411-7348;
Tel: +852-3411-7076
bCenter of Environmental Science and Engineering Research, School of Chemistry
and Chemical Engineering, Shanxi University, Taiyuan, 030006, P.R. China
Electronic supplementary information (ESI) available. See DOI: 10.1039/
3
c3ra41456f
15762 | RSC Adv., 2013, 3, 15762–15768
This journal is ß The Royal Society of Chemistry 2013

View Article Online
RSC Advances
Paper
Scheme 1 Structure of HBO and HBT and reversible photochromism of
spiropyrans induced by thermal, photochemical and acid–base stimulation.
Results and discussion
Fig. 1 Absorption spectra of 1 (50 mM) in ACN-phosphate buffer (20 mM, 1 : 1,
v/v) under different pH. Inset: Plot of absorbance at 397, 450 and 545 nm of 1
(50 mM) in ACN-phosphate buffer (20 mM, 1 : 1, v/v) as a function of pH,
respectively.
Synthesis
As shown in Scheme 2, the synthesis of probe 1 was started
from the commercially available material tert-butylphenol. The
known procedure of diformylation of tert-butylphenol with
hexamethylenetetramine (HMTA) in trifluoroacetic acid (TFA)
gave 2 in 52% yield. The reaction of 2 with 1,2-diaminoben-
zene in ethanol afforded imine 3 in 70% yield. Then 3 was
oxidized by DDQ to afford the strongly fluorescent compound
4 in 56% yield. In the presence of morpholine, the target
product 1 was obtained in 30% yield from the reaction of 4
with 1,2,3,3-tetramethyl-3H-indol-1-ium iodide in ethanol. The
relatively low yield of 1 was due to the strong ring-opening
tendency of the spiropyran ring system in column chromato-
graphy. The resulting zwitterionic form of 1 strongly increased
the polarity of the compound, thus making it difficult to be
separated from impurities. All of the new compounds were
well characterized by 1H NMR, 13C NMR, ESI-MS and HRMS
Hz) of the vinyl protons of the pyran moiety) unambiguously
confirmed its structure that existed mainly in the spirocyclic
form.37
Photophysical properties
With the new sensing material in hand, we started to study the
spectroscopic properties of probe 1 (50 mM) in ACN-phosphate
buffer (20 mM, 1 : 1, v/v). As shown in Fig. 1, at a pH above 6.5,
the probe showed almost no absorption in the range from 370
to 700 nm. Upon acidification of 1 from 6.5 to 4.0, the
absorbance at 259, 309 and 338 nm decreased with con-
comitant formation of new peaks at 318, 397, 450 and 555 nm,
which induced a color change of 1 from colorless to brown
(see Fig. S8–S10, ESI ). To facilitate the proton signal assign-
3
(Fig. S17, ESI ). Clear isosbestic points at 342, 331, 311, 306
3
ment in the 1H NMR spectrum of 1, two-dimensional H,H-
COSY and H,H-NOESY experiments were performed and
interacting protons of the molecule were identified (Fig. S11–
and 296 nm were observed, which indicate the acidochromic
interconversion of the spirobenzopyran closed form of 1 to the
extended conjugated merocyanine ring-opened form (vide
infra).
The time course of fluorescence measurement of the probe
revealed that a longer time was required to reach equilibrium
S16, ESI ).
3
The 1H NMR spectrum of the probe 1 (i.e. two signals at 1.30
and 1.34 ppm from the magnetically non-equivalent geminal
methyl groups, and the spin–spin coupling constant (J = 10.4
in more acidic conditions (Fig. S18, ESI ). Thus, to acquire
3
steady readings, the fluorescence spectra were collected after
30 min of incubation. The fluorescence response of probe 1 to
pH was shown in Fig. 2a and 2b. A ratiometric fluorescence
pattern was clearly observed under acidic conditions. Under
neutral conditions, the probe showed a maximum fluores-
cence intensity at 517 nm (I517 nm) due to the fluorescent spiro-
form of 132 and negligible fluorescence in the NIR range was
observed. When pH was decreased, I517 nm decreased with a
concomitant increase of the fluorescence intensity at 690 nm
(I690 nm). A clear isoemission point at 620 nm was observed.
I690 nm reached its highest value at around pH 5.5. In the pH
range from 7.0 to 5.5, the probe showed a ratiometric change
and the ratio (I690 nm/I517 nm) changed from 0.08 to 2.11 with a
more than 26-fold enhancement in the relative ratiometric
intensity. When pH of the solution was decreased from 5.5 to
4.0, I690 nm remained almost constant while I517 nm was still in
the decreasing trend (Fig. 2c). Under basic conditions, I517 nm
Scheme 2 Synthesis of probe 1.
This journal is ß The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 15762–15768 | 15763

View Article Online
Paper
RSC Advances
and a weak band at 455 nm. Upon addition of TFA, the
emission at 455 nm disappeared quickly and the emission at
500 nm decreased with concomitant increase of a new
emission at 700 nm (Fig. 3a). After the addition of 0.5 equiv.
of TFA, I700 nm did not change any more although I500 nm still
retained in a small decreasing trend (Fig. 3b). The ratio of
I700 nm/I500 nm varied from 0.02 to 5.94 after the addition of 0.9
equiv. of TFA, with a more than 290-fold enhancement in the
relative ratiometric intensity. Furthermore, the ratio of I700 nm
/
I500
showed a linear response (R2 = 0.994) to the given
nm
concentration of TFA (Fig. 3b, Inset), indicating that probe 1
could quantitatively detect H+ concentration (0–45 mM) in
ACN. The fluorescence spectral changing pattern of 1 in ACN
solution upon addition of triethylamine (TEA) showed a small
difference from that in aqueous ACN solution. I500
and
nm
I455
remained in a steady increasing trend upon the
nm
addition of TEA.
Fig. 2 Fluorescence spectra of 1 (50 mM) in ACN-phosphate buffer (20 mM,
1 : 1, v/v) in the pH range of (a) 4.0–7.0 and (b) 7.0–10.0. (c) Plot of I517 nm and
Based on above results, the proposed signal transduction
mechanism for the fluorescent probe under acidic and basic
conditions is depicted in Fig. 4. Under neutral conditions, the
probe was considered to form an intramolecular hydrogen
bond between the NH in the benzimidazole moiety and the
oxygen atom in the spiropyran unit. As a consequence,
rotation of the benzimidazole group was restricted and a
bathochromic emission at y517 nm compared with that of
spiropyran group at y475 nm was formed.32 The working
mechanism under acidic conditions was based on the change
in structure between the closed form (SP) and opened form
(MC-OH+) mediated by protons and the ESIPT mechanism.31
With an increase of H+ concentration to pH >5.5 in aqueous
solution or upon addition of 0–0.5 equiv. of TFA in ACN
solution, in the excited state, the ring-opening of probe 1
underwent an ESIPT process from MC-OH+ to MC-NH+. A
proton in the oxygen atom of the phenolic group would
transfer to the proximal nitrogen atom of the imine group,
thus the relative position of resulting amino group and the
I
690 nm of 1 (50 mM) in ACN-phosphate buffer (20 mM, 1 : 1, v/v) as a function of
pH, respectively. (d) Plot of ratio of I690 nm/I517 nm as a function of pH.
also underwent a decreasing trend and a slightly bath-
ochromic shift (27 nm) when the pH was increased from 7.2
to 8.6. A new hypsochromic emission at 460 nm emerged and
increased significantly at a pH larger than 8.6 (Fig. 2b).
Conceivably, the conformation change of the benzimidazole
moiety relative to the SP system could trigger the observed
change. The complete de-coupling of the benzimidazole and
SP p-system could cause the hypochromic shift to 460 nm.32
We also conducted the pH titration experiment of 1 in ACN
with TFA (Fig. 3a). The changing spectral pattern was well
consistent with that in aqueous ACN solution. In the absence
of TFA, probe 1 showed an intense emission band at 500 nm
Fig. 3 (a) Fluorescence spectra of 1 (50 mM) in ACN upon the addition of TFA.
(b) Plot of I500 nm and I700 nm and the ratio of I700 nm/I500 nm (Inset) versus the
ratio of [TFA]/[1], respectively. (c) Fluorescence spectra of 1 (50 mM) in ACN
upon the addition of TEA. (d) Plot of I455 nm and I500 nm versus the ratio of [TEA]/
[1], respectively.
Fig. 4 Proposed ring-opening and ESIPT mechanism under acidic and basic
conditions.
15764 | RSC Adv., 2013, 3, 15762–15768
This journal is ß The Royal Society of Chemistry 2013

View Article Online
RSC Advances
Paper
positively charged nitrogen atom in the keto-form MC-NH+ are
suitable for charge transfer reaction via p electron delocaliza-
tion in the excited state.38 As a result, the keto-form displaying
an emission at 690 nm increased at the expense of the enol-
like form emission and the pKa of the probe was calculated to
chemically equivalent. By the addition of 0–0.6 equiv. of TFA,
no obvious chemical shift change of the N-methyl and the gem-
dimethyl groups was observed, indicating that the spiropyran
ring remained intact. A downfield shift of the broad peak at d
7.2, ascribed to chemically equivalent H-9 and H-12, indicated
that protonation of the imidazole moiety occurred in this
stage. The formation of the imidazolium moiety (SP-H+) also
induced a small downfield shift of protons at d 5.89, 6.67, 7.01
and 8.39 which were ascribed to H-5, H-1, H-6, and H-8
respectively. Upon further addition of 0.8–3.2 equiv. of TFA,
the gem-dimethyl signals at d 1.30 and 1.34 gradually coalesced
to a single downshifted signal at d 1.86, while the N-methyl
signal at d 2.76 disappeared with the concomitant appearance
of two new singlet signals at d 4.06 and 4.17, indicating the
formation of ring-opening products comprising both cis and
trans isomers (cis-MC-OH+ and trans-MC-OH+). Along with
further addition of TFA, the intensity of the N-methyl
resonance at d 4.17 increased at the expense of the signal at
be 5.9 (Fig. S19, ESI ).13 The nitrogen atom of the benzimida-
3
zole moiety can be further protonated (MC-NH22+) when pH
,5.5 or upon addition of >0.5 equiv. of TFA. The protonation
of MC-NH+ will inhibit the charge transfer within the molecule
and reduce its fluorescence. Therefore, the emission intensity
of the enol-like form kept on decreasing while the emissive
peak of the keto-form decreased slowly (Fig. 2c).
Under basic conditions, the intramolecular hydrogen bond
was weakened by the increasing basicity of the solution. As a
result, the fluorescence intensity at 517 nm decreased
gradually along with the increase of pH (SPASP-B1). At pH
>8.6, the hydroxyl ion present was strong enough to break the
intramolecular hydrogen bond, making free rotation of the
benzimidazole moiety possible. Thus, the de-coplanarity of the
benzimidazole group and the spiropyran unit caused a
hypsochromic shift to 460 nm (SP-B1ASP-B2). In ACN
solution, probe 1 existed in an equilibrium of SP and SP-B2
forms. The addition of TEA also caused the de-coplanarity of
the SP form to give the hypsochromic emission at 455 nm.
d
4.06. Presumably, the less stable cis-MC-OH+ finally
isomerized to trans-MC-OH+. At the same time, the vinyl
proton H-5 doublet of the ring-closed compound at d 5.90 (J
10.4 Hz) decreased with the concomitant increase of a new
doublet signal at d 8.59 which was attributed to the trans-
coupled pair of H-5 (J 16.2 Hz). Also, the signals of H-1 and H-6
disappeared gradually and moved downfield to the range of d
7.3–7.8, and the signal of H-8 underwent a small upfield shift
and decrease, with the concomitant increase of a new doublet
signal at d 8.55, suggesting the conversion of the cis-open form
to trans-open form. Interestingly, neutralization of the acid by
the stepwise addition of portions of TEA caused a reverse
change pattern of the spectra compared with that of stepwise
addition of TFA to the probe, suggesting that the probe 1 is a
1H NMR titration
1
The ring-opening mechanism was also supported by H NMR
titration (Fig. 5). To a solution of the probe 1 in deuterochloro-
form (CDCl3) was added sequentially portions of TFA, to a total
of 3.2 equiv, followed by the addition of portions of
1
triethylamine (TEA) to a total of 3.2 equiv., and the H NMR
spectrum was recorded after each addition (Fig. S20–S22, ESI ).
3
Similar to the solution of probe 1 in ACN, 1 in CDCl3 solution
mainly existed in the spirocyclic form with intramolecular
hydrogen bonding. Two far-away doublets at d 6.69 and 7.72
ascribed to H-9 and H-12, respectively, confirmed that these
two protons are in a non-equivalent chemical environment.
However, the addition of small amount of TFA caused the
disappearance of these two doublets and the appearance of a
new broad peak at d 7.2, suggesting the breakage of the
intramolecular hydrogen bond. Thus, the free rotation of the
benzimidazole moiety made the non-equivalent protons
reversible pH sensor (Fig. S22, ESI ).
Finally, we investigated the interference of metal ions on
probe 1 for the pH sensing effect. As shown in Fig. 6 and Fig.
3
S23–S25, ESI, , the fluorescence intensity values at 517 nm and
3
690 nm were not influenced by the addition of a large excess
(>3 mM) of biologically relevant metal ions (i.e. Na+, K+, and
Ca2+) and 50 mM of various transition metal ions at pH 4.0 and
7.0, respectively. Only the presence of Cu2+ quenched the
fluorescence completely and produced a red colored solution.
It is well-known that many multifunctional spiropyran-based
probes are responsive to Cu2+, displaying the characteristic red
color due to the formation of the ICT complexes.32 The
paramagnetic properties of Cu2+, which induce intrinsic
fluorescence quenching, turned off the fluorescence of the
probes.39 Those results imply that probe
1 might be
considered as a selective fluorescent probe for pH sensing.
Conclusions
In summary, we have designed a ratiometric dual-emission pH
probe 1 through the coupling of a pH-responsive spiropyran
moiety with a benzimidazole segment suitable for the ESIPT
process. This probe could be used for the ratiometric
measurement of pH values in a range from 7.0 to 5.5, and
the pKa of the probe was calculated to be 5.9. In the pH range
Fig. 5 Proposed structural change of probe 1 caused by the titration with TFA
(0–3.2 equiv.) and TEA (0–3.2 equiv.) in CDCl3.
This journal is ß The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 15762–15768 | 15765

View Article Online
Paper
RSC Advances
combined glass-calomel electrode. Double-distilled water was
used throughout.
All reagents for synthesis were obtained commercially and
were used without further purification. Solvents such as
trifluoroacetic acid (TFA), ethanol (EtOH), acetonitrile (ACN)
and dichloromethane (DCM) were purchased from commer-
cial sources and were the highest grade. Silica gel (200–300
mesh, MACHEREY-NAGEL GmbH & Co. KG) was used for
column chromatography. Analytical thin-layer chromatogra-
phy was performed using TLC silica gel 60 254 (aluminium
sheets, Merck KGaA). In titration experiments, all the cations
in the form of perchlorate or chloride and other substrates
were purchased from Sigma-Aldrich, USA, and stored in a
vacuum desiccator.
Sample preparation
The probe and TFA were dissolved in ACN as a stock solution
(5 mM). TFA was dissolved in ACN as a stock solution (5 mM).
Buffer solution was prepared by dissolving disodium hydrogen
phosphate dodecahydrate in DI water (20 mM). Slight
variations in the pH of the solution were achieved by adding
the minimum volumes of NaOH or HCl.
Absorption and fluorescence analysis
Absorption spectra and fluorescence emission spectra were
obtained with 1.0 cm quartz cells. The pH titration procedures
were as follows: phosphate buffer solution (20 mM) at
different pH values was mixed with ACN in a 1 : 1 ratio (v/v),
then 20 mL of stock solution was added, the mixture was
equilibrated for 30 min before measurement, or 20 mL of stock
solution was added to ACN (2 mL) followed by a stepwise
addition of TFA stock solution (2 mL), and the mixture was
equilibrated for 5 min before measurement. The excitation
wavelength was 397 nm. The excitation and emission slits were
set to 15.0 nm and 15.0 nm, respectively.
Fig. 6 Fluorescence intensity of 517 nm and 690 nm of 1 (50 mM) in ACN-
phosphate buffer (20 mM, 1 : 1, v/v) at pH 4.0 (top) and pH 7.0 (bottom) upon
addition of various metal ions: Na+ (150 mM), K+ (150 mM), Ca2+ (3 mM), Mg2+
(3 mM), Li+ (50 mM), Ag+ (50 mM), Cu2+ (50 mM), Fe2+ (50 mM), Fe3+ (50 mM), Zn2+
(50 mM), Co2+ (50 mM), Ni2+ (50 mM), Cd2+ (50 mM), Hg2+ (50 mM), Pb2+ (50 mM).
from 7.0 to 5.5, the probe showed a ratiometric change and the
ratio (I690 nm/I517 nm) increased from 0.08 to 2.11 with a more
than 26-fold increase in the relative ratiometric intensity. The
probe could quantitatively detect H+ concentration (0–45 mM)
in ACN. Also, the probe suffered no interference from most of
.
metal ions with the exception of Cu2+ 1H NMR titration
confirmed the reversible ring-opening/closing mechanism and
1H NMR titration
Probe 1 (4.6 mg) was dissolved in CDCl3 (0.5 mL) in an NMR
tube. Then portions of 2.0 mL of a solution of TFA in CDCl3 (1.0
M) were introduced successively into the probe solution. After
shaking for 1 min, the acquisition of the NMR spectrum was
carried out. After the addition of 3.2 equiv. of TFA, a stepwise
addition of 2.0 mL of TEA in CDCl3 (1.0 M) and 1H NMR
acquisition followed.
cis–trans conversion of the spiropyran moiety.
Experimental
General
Synthesis
1H NMR and 13C NMR spectra were recorded on a Bruker
Advance-III 400 MHz Spectrometer (at 400 and 100 MHz,
respectively) using trimethylsilane (TMS) as an internal
standard. 2-D COSY and NOESY experiments were performed
using Bruker microprograms. Low-resolution mass spectra
were recorded on a Finnigan MAT SSQ-710 mass spectrometer
while high-resolution mass spectra were performed using a
2,6-Diformyl-4-tert-butylphenol (2). To a solution of 4-tert-
butylphenol (2.0 g, 13.3 mmol) in TFA (25 mL) was added
hexamethylenetetramine (3.9 g, 27.8 mmol) and then the
mixture was heated to reflux under N2 atmosphere for 24 h.
After cooling, the reaction mixture was poured into 6 M HCl
(80 mL) and stirred for 20 min. The aqueous phase was
extracted with DCM (2 6 60 mL) and the combined organic
phases were washed with 4 MHCl (2 6 80 mL), water (80 mL)
and brine (80 mL) successively, then dried over Na2SO4. After
removal of the solvent, the residue was purified by column
chromatography on silica gel (PE : EA = 40 : 1) to give a light
yellow solid 2 (1.44 g, yield: 52%); m.p. 103–104 uC.40
Bruker
Autoflex
mass
spectrometer
(MALDI-TOF).
Fluorescence spectra and UV-Vis spectra were collected on a
PE LS50B and a Cary UV-300 spectrometer, respectively. The
melting point was determined with MEL-TEMPII melting point
apparatus (uncorrected). The pH measurements were per-
formed on an Orion 420A pH mV temperature meter with a
15766 | RSC Adv., 2013, 3, 15762–15768
This journal is ß The Royal Society of Chemistry 2013

View Article Online
RSC Advances
Paper
1H-NMR (400 MHz, CDCl3) d 11.48 (1H, s), 10.25 (2H, s), 7.99
(2H, s), 1.36 (9H, s).
Acknowledgements
13C-NMR (100 MHz, CDCl3) d 192.44, 161.70, 143.14, 134.67,
122.67, 34.32, 31.10.
The work was supported by a grant from the Research
Committee of Hong Kong Baptist University (FRG2/11-12/121).
(E)-3-(((2-Aminophenyl)imino)methyl)-5-(tert-butyl)-2-hydro-
xybenzaldehyde (3). The mixture 2 (0.31 g, 1.5 mmol) and 1,2-
diaminobenzene (0.17 g, 1.6 mmol) in absolute EtOH (20 mL)
was heated to 50 uC for 6 h. After cooling, the solid was filtered
and washed with small portions of EtOH to give a yellow solid
3 (0.31 g, yield: 70%); m.p.: 264–265 uC.
Notes and references
1 J. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd edn,
Kluwer Academic/Plenum Publishers, New York, 1999, pp.
531–572.
1H-NMR (400 MHz, CDCl3) d 13.56 (1H, s), 8.63 (1H, s), 7.42
(1H, d, J = 2.3 Hz), 7.34 (1H, d, J = 2.3 Hz), 7.2827.24 (m, 1H),
7.06 (1H, dd, J = 7.7 Hz, J = 1.4 Hz), 6.97 (1H, d, J = 8.1 Hz), 6.79
(1H, dt, J = 7.6 Hz, J = 1.1 Hz), 6.32 (1H, t, J = 5.6 Hz), 4.46 (2H,
d, J = 5.8 Hz), 1.33 (9H, s).
2 J.-P. Desvergne and A. W. Czarnik, Fluorescent
Chemosensors for Ion and Molecule Recognition, Kluwer
Academic Publishers, Dordrecht, The Netherlands, 1997.
3 R. P. Haugland, Molecular Probes Handbook, A Guide to
Fluorescent Probes and Labeling Technologies, 10th edn,
Molecular Probes Inc., Eugene, OR, 2005, pp. 935–947.
4 B. Valeur, Molecular Fluorescence: Principles and
Applications, Wiley-VCH, Weinheim, Germany, 2002.
5 R. P. Haugland and M. T. Z. Spence, Handbook of
Fluorescent Probes and Research Products, Molecular
Probes Inc., 9th edn, Eugene, OR, 2002, pp. 827–848.
6 Q. A. Best, R. Xu, M. E. McCarroll, L. Wang and D. J. Dyer,
Org. Lett., 2010, 12, 3219–3221.
7 W. Zhang, B. Tang, X. Liu, Y. Liu, K. Xu, J. Ma, L. Tong and
G. Yang, Analyst, 2009, 134, 367–371.
8 X. Chen, Z. Li, Y. Xiang and A. Tong, Tetrahedron Lett.,
2008, 49, 4697–4700.
9 S. Madhu, M. R. Rao, M. S. Shaikh and M. Ravikanth, Inorg.
Chem., 2011, 50, 4392–4400.
10 Y. Chen, H. Wang, L. Wan, Y. Bian and J. Jiang, J. Org.
Chem., 2011, 76, 3774–3781.
13C-NMR (100 MHz, CDCl3) d 162.17, 157.25, 143.47, 141.83,
136.18, 130.90, 128.23, 128.06, 124.98, 119.16, 118.03, 117.46,
111.77, 47.28, 34.07, 31.44.
3-(1H-Benzo[d]imidazol-2-yl)-5-(tert-butyl)-2-hydroxybenzal-
dehyde (4). To a suspension of 3 (0.16 g, 0.5 mmol) in DCM (20
mL) was added DDQ (0.24 g, 1.0 mmol) and the reaction
mixture was stirred at room temperature for 2 h. After removal
of the solvent, the residue was purified by column chromato-
graphy on silica gel (PE : EA = 20 : 1) to afford 4 as a yellow
solid (90 mg, 56% yield); m.p.: 249–251 uC.
1H-NMR (400 MHz, MeOD) d 10.42 (1H, s), 8.41 (1H, d, J =
2.5 Hz), 7.90 (1H, d, J = 2.5 Hz), 7.65–7.63 (2H, m), 7.31–7.28
(2H, m), 1.42 (9H, s).
13C-NMR (100 MHz, MeOD) d 193.54, 160.38, 151.79, 143.61,
138.78, 131.79, 129.16, 124.28, 116.22, 115.87, 35.41, 31.70.
ESI-MS: m/z calcd for C18H18N2O2 [M + H+] 295.14, found,
295.3.
11 H. He and D. K. P. Ng, Org. Biomol. Chem., 2011, 9,
2610–2613.
8-(1H-Benzo[d]imidazol-2-yl)-6-(tert-butyl)-19,39,39-trimethyl-
spiro[chromene-2,29-indoline] (1). A mixture of 4 (40 mg, 0.14
mmol) and 1,2,3,3-tetramethyl-3H-indol-1-ium iodide (42 mg,
0.14 mmol) in absolute EtOH (10 mL) was heated to reflux
under N2 atmosphere. A solution of morpholine (24 mg, 0.28
mmol) in absolute EtOH (10 mL) was added in a dropwise
manner. After addition, the reaction mixture was refluxed for 2
h. Then the solvent was evaporated under reduced pressure
and the residue was purified by column chromatography on
silica gel (PE : EA = 50 : 1) to afford 1 as a pink solid (19 mg,
30% yield).
12 C. Zhou, Y. Li, Y. Zhao, J. Zhang, W. Yang and Y. Li, Org.
Lett., 2010, 13, 292–295.
13 B. Tang, F. Yu, P. Li, L. Tong, X. Duan, T. Xie and X. Wang,
J. Am. Chem. Soc., 2009, 131, 3016–3023.
14 B. Tang, X. Liu, K. Xu, H. Huang, G. Yang and L. An, Chem.
Commun., 2007, 3726–3728.
15 X. Zhou, F. Su, H. Lu, P. Senechal-Willis, Y. Tian, R.
H. Johnson and D. R. Meldrum, Biomaterials, 2012, 33,
171–180.
16 C. N. Carroll, B. A. Coombs, S. P. McClintock, C. A. Johnson
II, O. B. Berryman, D. W. Johnson and M. M. Haley, Chem.
Commun., 2011, 47, 5539–5541.
17 E. Kim, S. Lee and S. B. Park, Chem. Commun., 2011, 47,
7734–7736.
18 P. Song, X. Chen, Y. Xiang, L. Huang, Z. Zhou, R. Wei and
A. Tong, J. Mater. Chem., 2011, 21, 13470–13475.
19 A. Yayon, Z. I. Cabantchik and H. Ginsburg, EMBO J., 1984,
3, 2695–2700.
20 X. Lv, J. Liu, Y. Liu, Y. Zhao, Y.-Q. Sun, P. Wang and
W. Guo, Chem. Commun., 2011, 47, 12843–12845.
21 L. Yuan, W. Lin, Y. Xie, B. Chen and J. Song, Chem.
Commun., 2011, 47, 9372–9374.
1H-NMR (400 MHz, CDCl3) d 9.79 (1H, s), 8.41 (1H, d, J = 2.4
Hz), 7.74 (1H, d, J = 8.1 Hz), 7.39 (1H, dt, J = 7.7 Hz, J = 1.3 Hz),
7.25 (1H, dd, J = 7.3 Hz, J = 0.8 Hz), 7.21–7.16 (2H, m), 7.12 (dt,
J = 7.4 Hz, J = 0.9 Hz), 7.01 (1H, d, J = 10.4 Hz), 6.69 (2H, m),
5.90 (1H, d, J = 10.4 Hz), 2.77 (3H, s), 1.39 (9H, s), 1.34 (3H, s),
1.30 (3H, s).
13C-NMR (100 MHz, CDCl3) d 149.62, 149.51, 148.18, 143.96,
142.58, 136.79, 133.46, 131.09, 128.27, 126.49, 125.97, 122.45,
122.18, 122.03, 120.32, 119.08, 118.97, 117.93, 114.82, 110.94,
108.28, 107.40, 51.90, 34.42, 31.48, 29.55, 24.64, 19.69.
HRMS (MALDI-TOF): m/z calcd for C39H39N5O4 [M + H+]
450.2540, found, 450.2561.
22 B. Ma, F. Zeng, X. Li and S. Wu, Chem. Commun., 2012, 48,
6007–6009.
23 X. Yang, Y. Guo and R. M. Strongin, Angew. Chem., Int. Ed.,
2011, 50, 10690–10693.
This journal is ß The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 15762–15768 | 15767

View Article Online
Paper
RSC Advances
24 J. Zhao, S. Ji, Y. Chen, H. Guo and P. Yang, Phys. Chem.
Chem. Phys., 2012, 14, 8803–8817.
25 C. Bremer, C.-H. Tung, A. J. Bogdanov and R. Weissleder,
Radiology, 2002, 222, 814–818.
33 N. Shao, J. Y. Jin, S. M. Cheung, R. H. Yang, W. H. Chan
and T. Mo, Angew. Chem., Int. Ed., 2006, 45, 4944–4948.
34 Y. Shiraishi, S. Sumiya and T. Hirai, Chem. Commun., 2011,
47, 4953–4955.
26 K. Kiyose, H. Kojima and T. Nagano, Chem.–Asian J., 2008,
3, 506–515.
35 J.-F. Zhu, H. Yuan, W.-H. Chan and A. W. M. Lee, Org.
Biomol. Chem., 2010, 8, 3957–3964.
27 K. Licha and C. Olbrich, Adv. Drug Delivery Rev., 2005, 57,
1087–1108.
28 V. Ntziachristos, C. Bremer and R. Weissleder, Eur. J.
Radiol., 2003, 13, 195–208.
29 D. Oushiki, H. Kojima, T. Terai, M. Arita, K. Hanaoka,
Y. Urano and T. Nagano, J. Am. Chem. Soc., 2010, 132,
2795–2801.
30 S. V. Paramonov, V. Lokshin and O. A. Fedorova, J.
Photochem. Photobiol., C, 2011, 12, 209–236.
31 J. T. C. Wojtyk, A. Wasey, N.-N. Xiao, P. M. Kazmaier,
S. Hoz, C. Yu, R. P. Lemieux and E. Buncel, J. Phys. Chem. A,
2007, 111, 2511–2516.
36 Y. P. Chan, L. Fan, Q. You, W. H. Chan and A. W. M. Lee,
Tetrahedron, 2013, 69, 5874–5879.
37 C. J. Roxburgh and P. G. Sammes, Dyes Pigm., 1995, 27,
63–69.
38 A. P. Demchenko and P. R. Callis, Advanced Fluorescence
Reporters in Chemistry and Biology I: Fundamentals and
molecular design, Springer Heidelberg Dordrecht London
New York, 2010, pp. 250–259.
39 Y. Zheng, J. Orbulescu, X. Ji, F. M. Andreopoulos, S.
M. Pham and R. M. Leblanc, J. Am. Chem. Soc., 2003, 125,
2680–2686.
40 L. F. Lindoy, G. V. Meehan and N. Svenstrup, Synthesis,
1998, 1998, 1029–1032.
32 N. Shao, J. Y. Jin, H. Wang, Y. Zhang, R. H. Yang and W.
H. Chan, Anal. Chem., 2008, 80, 3466–3475.
15768 | RSC Adv., 2013, 3, 15762–15768
This journal is ß The Royal Society of Chemistry 2013