An ESIPT fluorescent dye based on HBI with high quantum yield and large Stokes shift for selective detection of Cys

Article abstract of DOI:10.1039/c4tb00190g

We report 3-(4,5-diphenyl-1H-imidazol-2-yl)naphthalen-2-ol (DPIN) as an interesting luminescent material displaying ESIPT with a large Stokes shift of ～180 nm even in protic/polar solvents. Stable homo-dispersed nanoparticles formed by inter- and intramol

Full text of DOI:10.1039/c4tb00190g

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Materials Chemistry B
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An ESIPT fluorescent dye based on HBI with high
quantum yield and large Stokes shift for selective
detection of Cys†
Cite this: J. Mater. Chem. B, 2014, 2,
4159
b
a
a
b
Ying Zhang,ab Jun-Hao Wang, Wenjie Zheng, Tianfeng Chen, Qing-Xiao Tong
*
b
*
and Dan Li
We report 3-(4,5-diphenyl-1H-imidazol-2-yl)naphthalen-2-ol (DPIN) as an interesting luminescent
material displaying ESIPT with a large Stokes shift of ꢀ180 nm even in protic/polar solvents. Stable
homo-dispersed nanoparticles formed by inter- and intramolecular H-bonds in aqueous media and the
corresponding aggregation induced enhanced emission with a high quantum yield up to 0.45 were
observed. Factors such as pH value and ions (cations and anions) showed a negligible effect on the
Received 5th February 2014
Accepted 20th April 2014
fluorescence performance.
A probe of 3-(4,5-diphenyl-1H-imidazol-2-yl)naphthalen-2-yl-acrylate
DOI: 10.1039/c4tb00190g
(DPIN-A) based on this molecule was designed. The results revealed that it can be used for sensing of
www.rsc.org/MaterialsB
Cys with high selectivity and sensitivity.
much shorter wavelengths); and (ii) very low uorescence
quantum yields of 0.02 (for HBO) and 0.005 (for HBT).19 All
1. Introduction
these problems have severely limited their applications in bio-
sensing and bio-imaging.21 Many efforts have been devoted to
increase uorescence quantum yields of these ESIPT molecules
by introducing bulky dendrimer structures to inhibit the
isomerization efficiency;22 or by introducing moieties with a H-
bond for variation of inter- and intramolecular interactions to
restrict molecular motions.23,26 On the other hand, aggregation
induced enhanced emission (AIEE) uorophores have shown
distinct advantages in many areas of applications such as
chemo/biosensors24 and OLEDs.25 ESIPT materials with AIEE
properties have also been developed23,26 due to high quantum
yields in aggregation states and avoidance of interactions with
solvents. It is very important to generate such homo-dispersed
aggregation of nanoparticles in solution for detection applica-
tions. Kim et al. obtained organic nanoparticles in a poor
solvent with almost exclusively the abnormal emission assigned
to the keto tautomer.11 However, the high critical concentration
of the ESIPT molecules for the keto-emission results in a turbid
solution, which is not favourable for the detection efficiency.
Yang, et al. solved the above problem by dispersing the probes
in the CTAB micelle with smaller aggregates and consequently
clear solution, in which high reactivity towards Fꢁ was
observed.12 The probing medium was further extended to pure
water by dispersing an insoluble organic dye in PVP hydrogel
substrates with enhanced sensing performance.18 Although the
advances are signicant, the preparation of the detection
systems is complicated and time-consuming. Thus, it is still of
signicance to develop new ESIPT dyes which can form homo-
dispersed and stable nanoparticles in aqueous solution with
more simplicity and higher quantum yields.
Molecules with excited-state intra-molecular proton transfer
(ESIPT) have emerged as a class of promising luminescent
materials in many practical applications, such as optical
memories1 and switches,2 UV absorbers,3 laser dyes,4 radiation
detection scintillators,5 white light OLEDs6 and particularly
uorescent probes.7–18 ESIPT molecules can undergo a fast (k z
1012 sꢁ1) photo-tautomerization reaction to yield an excited keto
form (K*), mediated by an intra-molecular hydrogen bonds
(H-bonds) upon photo-irradiation19 (Scheme S1†), which are
famous for an abnormal emission with large Stokes shi
emission (100–200 nm) without self-absorption.
Among the reports on ESIPT molecules in probe design,
derivatives of 2-(2-hydroxyphenyl)benzoxazole (HBO) and
2-(2-hydroxyphenyl)benzothiazole (HBT) are the most attractive
showing remarkable results with very high selectivity and
sensitivity for detection of cellular transmeꢁmbrane potential,7,8
apoptosis,9 Mg2+,10 protein tyrosine,11 F ,12,18 cysteine and
homocysteine,13 Zn2+ (ref. 14, 16 and 17) and Pd2+
. However,
15
there are still some critical aspects that should be improved: (i)
ESIPT emissions are usually sensitive to protic/polar
solvents11,20 and oen inhibited with only normal emission (at
aDepartment of Chemistry, School of Life Science and Technology, Jinan University,
Guangzhou 510632, China
bDepartment of Chemistry and Research Institute for Biomedical and Advanced
Materials, Shantou University, Guangdong 515063, China. E-mail: qxtong@stu.edu.
cn; dli@stu.edu.cn
† Electronic supplementary information (ESI) available: Schematic representation
of the ESIPT photocycle, synthetic routes of the HBIs and HBOs, and crystal data
for DPIN. CCDC 983882. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c4tb00190g
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It is well known that HBIs (2-(2-hydroxyphenyl)benzimid- response to the analytes.11–18 Based on the excellent uores-
azole derivatives) have much higher quantum yields than HBOs cence performance of DPIN, we designed a new probe denoted
and HBTs.19 Nevertheless, to the best of our knowledge, very few as DPIN-A (Scheme 1) by protecting the OH-moiety of DPIN with
derivatives or analogues of HBI have been photophysically an acryloyl group which can react with biothiol Cys selectively.
studied and rare uorescent probes based on them have been The results show that DPIN-A has a high selectivity and sensi-
reported by taking advantage of the ESIPT originated emis- tivity towards Cys in pure water which is of important signi-
sion.29 There are two drawbacks that hampered the develop- cance in clinical diagnose.
ment of HBIs displaying ESIPT emissions in probe design: (i)
the keto emission of classical HBIs locates mainly in the blue
region (470 nm), which exhibited much smaller Stokes shi
2. Results and discussion
2.1 Design of HBIs and organic probe for Cys
compared with HBOs (500 nm) and HBTs (520 nm); (ii) the
potential interference from other cations or anions towards the
binding-able site (NH, N]) of the imidazole ring would block
intramolecular H-bonds involved in the ESIPT process, which
severely reduces quantum yields of the ESIPT emission. In this
work, we tried to modify the structure of HBIs to improve the
ESIPT emission performance by not only increasing their Stokes
shis but also quantum yields. With a simple one-pot reaction,
we obtained DPIN (3-(4,5-diphenyl-1H-imidazol-2-yl)naph-
thalen-2-ol, Scheme 1). The compound exhibited obvious keto-
form emission even in some polar and protic solvents (DMF,
DCM and ethanol) at low concentration with large Stokes shi
and a high quantum yield up to 0.30 in DMF. Transparent
solution can be obtained by dispersing DPIN in water in which
stable homo-dispersed aggregated nanoparticles of DPIN were
DPIN was designed based on HBIs with following consider-
ations: (i) HBIs have a high uorescent quantum yield of about
one order of magnitude higher than that of HBOs and HBTs; (ii)
the NH of the imidazole core as a H-bonding donor would
enhance the intermolecular interaction which is benecial for
molecular aggregations; (iii) the two phenyls as tails of imid-
azole with a large twisted angle would restrict intermolecular
p–p stacking thus inhibiting aggregation caused uorescent
quenching; (iv) expanding the p-conjugation length by intro-
ducing the 3-OH-2-naphthalene ring as the head of the mole-
cule would result in a keto emission in longer wavelength with
larger Stokes shi, with which the distinct colour difference
compared with normal emission would be discerned by naked
eyes.
formed, showing almost exclusive keto-emission with
a
By protecting the OH of DPIN with an allyl group, we
synthesized the probe DPIN-A (Scheme 1) with enol emission
quenched due to the PET (Photoinduced Electron Transfer)
process between the skeleton and the propylene moiety. Aer
the conjugate addition of the three similar biothiols with acry-
lates, it will generate thioethers which will interrupt the PET
process with enol-like emission. It can further undergo an
intramolecular cyclization to free the OH of DPIN-A which can
undergo the ESIPT process with keto emission (Scheme 1).
DPIN-A was expected to have a high selectivity towards Cys over
HCy and GSH for the different intra-molecular cyclization
rates.13
quantum yield of 0.20. The uorescence stability of DPIN in
water was also demonstrated by interference experiments
involving factors such as pH value and ions (cations and
anions).
An elevated level of biothiols (Cys, GSH and HCy) could bring
out different diseases, however, they are difficult to be distin-
guished because of their similarity in structures.28 For sensors
displaying the ESIPT, the signaling transduction is oen ach-
ieved by the turn-on uorescence of keto-form emission upon
2.2 Synthesis and crystal structures
Synthetic routes of DPIN and the target probe are outlined in
Scheme 1. Another four ESIPT dyes of HPO-1, HPO-2, HPI-1 and
HPI-2 were synthesized for comparison (Scheme S2†). DPIN,
HPO-1, and HPO-2 were obtained by reuxing benzyl/phenan-
threne-9,10-dione, araldehyde, NH4Ac in HAc as the solvent.
HPI-1 and HPI-2 were obtained by reuxing phenanthrene-
9,10-dione and araldehyde together in the mixed solvent of
CH2Cl2–ethanol, catalyzed by a small quantity of HAc in one pot
reaction (for details, see Experimental section). The resulting
compounds were characterized by 1H-NMR, elemental analysis,
and MS. Single crystals of DPIN were obtained by layering
hexane on CH2Cl2. X-ray single-crystal diffraction analysis
revealed that DPIN crystallized in a triclinic, p-1 group. As
shown in Fig. 1, three different kinds of weak interactions were
˚
observed: (i) the short distance of 1.7 A between H and N
indicated strong intramolecular O–H/N hydrogen bonds,
Scheme 1 Synthetic routes of DPIN and the target probe DPIN-A and
the reaction process of the probe towards Cys (M: NH4Ac/HAc/reflux). which favours the mobility of the proton from –OH to Lewis
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Fig. 1 Crystal packing structure of DPIN, dot lines represent weak
intermolecular interactions: O–H/N (green), N–H/O (red), C–H/p
(blue).
base ]N sites in the imidazole centre. (ii) The intermolecular
˚
N–H/O hydrogen bond with a distance of 2.0 A and (iii)
intermolecular C–H/p interactions within a distance of 3.08–
˚
3.29 A are benecial for the molecular aggregation in the
solution with a high concentration and an insoluble solvent. As
expected, the intermolecular p–p stacking was suppressed due
to the large twisted dihedral angle between the phenyl and
imidazole rings (ca. 51.87ꢂ or 43.97ꢂ). The crystal stacking is
shown in Fig. S1.† Selected dihydral angles and weak interac-
tions are given in Table S1.† Structural parameters and data are
listed in Tables S2 and S3.†
Fig. 2 Fluorescence of DPIN (10 mM) in different solvents excited at
371 nm (top); pictures of DPIN in different solvents (10 mM) excited with
a hand lamp at 365 nm (down).
2.3 Emission of HBIs
which can act as a hydrogen donor for intermolecular
H-bonding interactions. HPO-1, HPO-2, HPI-1 and HPI-2
showed smaller Stokes shi than DPIN in spite of the involve-
ment of more conjugated 9,10-phenanthrenequinone instead of
benzyl as the tail of these molecules. It has been demonstrated
that the highest occupied molecular orbitals (HOMO) of the
keto form of HPI are mainly located at the imidazole ring and
the head substituents which extend their conjugation length
can reduce the energy gap efficiently.6 The introduction of
3-OH-2-naphthalene as the head of DPIN is essential to the large
Stokes shi of the keto-emission for its longer conjugation
length than 2-OH-naphtanlene and benzene. These results
prove that the –NH in the imidazole ring and 3-OH-2-naph-
thalene are crucial for the stable keto emission with large Stokes
shi. Compared with other ESIPT molecules, the excitation and
emission of DPIN at longer wavelength (ex, ꢀ371 nm; enol
emission, ꢀ415 nm; keto emission, ꢀ550 nm) are much more
benecial for biosensing and bioimiaging.11–18
Strong keto emission with large Stokes shi in polar/protic
solvents are important for ESIPT molecules. As shown in Fig. 2,
DPIN exhibited strong keto emissions (maximized at >540 nm)
with large Stokes shis in normal strong polar and protic
solvents, except that in methanol. Different colours were
observed from blue, green, yellow and red under the radiation
under a UV lamp with 365 nm, due to different ratios between
keto and enol emissions in different solutions. The bath-
ochromic-shi of the keto-form emission in different solvents
(553, 610 nm in DMF and cyclohexane, respectively) is well in
accordance with the n–p* transition character of the keto
tautomer. However, at a low concentration of 10 mM, the enol
emission at ꢀ415 nm is very weak, while the keto emission is
strong with large Stokes shi peaked at ꢀ553 nm even in polar
(DMF) and protic solvents (ethanol) with yellow and green
emissions, respectively. In nonpolar solvents the emissions
show orange yellow at ꢀ600 nm for benzene and DCM and red
(ꢀ610 nm) for cyclohexane. To elucidate the origination of the
excellent uorescence performance of DPIN, emission spectra
of four other molecules with similar structures (HPO-1, HPO-2,
HPI-1 and HPI-2) were also characterized and are shown in
2.4 Emission performance of DPIN in DMF with different
concentrations
Fig. S2.† The emissions of HPO-1 and HPO-2 showed much Many organic dyes suffer aggregation caused quenching (ACQ)
larger dependence than HPI-1, HPI-2 and DPIN on solvents with in concentrated solution or solid states for the p–p stacking. To
just moderate keto-form emission in non-polar solvents. Aer avoid ACQ, we introduced benzyl with large twisted angles into
careful examination of their structures, we deduce that the less DPIN. As shown in Fig. 3, the uorescent spectra at a low
dependence on solvents of the keto-emission of DPIN and HPI-1 concentration of 2.5 mM in DMF mainly at ꢀ480 nm which
and HPI-2 might be mainly attributed to the molecular aggre- originated from the species resulting from the interaction of the
gation due to the presence of –NH within the imidazole ring solvent with DPIN. Two shoulders at 415 nm and 565 nm can be
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Fig. 3 PL of DPIN in DMF with different concentrations ranging from
2.5 mM to 100 mM under 371 nm excitation. Inset picture: emission
colour of DPIN in DMF with different concentrations, excited under a
365 nm hand lamp.
Fig. 4 (a) Fluorescence of DPIN in DMF and H2O mixed solution (10
mM), with a proportion of H2O increasing from 0 to 100% water v/v in
DMF. Excited wavelength: 371 nm. Inset picture: fluorescence of DPIN
solution with different contents of H2O, excited under a 365 nm hand
lamp. (b) Ratio of intensity of keto emission toward enol emission. (c)
Size distribution of DPIN in pure water by DLS.
assigned to enol emission and keto emission, respectively,
according to previous reports. Upon increasing the concentra-
tion from 2.5 mM to 100 mM, the intensity of keto emission
mainly at 565 nm appears and increases rapidly. This was
further demonstrated by the colour change discerned by naked
eyes from blue (2.5 mM), yellow (10 mM) to bright yellow (50 mM)
under 365 nm radiation (Fig. 3, inset). This enhanced keto
tautomer emission can be attributed to the increased planarity
and rigidity between imidazole and naphthalene rings as well as
the restriction of the molecular motion by the J-aggregation
with a head-to-tail arrangement which is more facile for the
tautomerization of DPIN from the enol-form to the keto-form
upon photo excitation.18,23
which may be the critical concentration for the aggregation of
DPIN to nanoparticles (Fig. 4b). Only keto-form emission can be
observed in 100% water, which can be attributed to the
formation of nanoparticles. Consequently, the emission colour
can be varied from yellowish green (0%), yellow (60%) to green
(100%) with different water contents, which are in well accor-
dance with the spectral change (Fig. 4a, inset). DLS analysis
(dynamic light scattering) (Fig. 4c) showed that the size of the
nanoparticles is about 205 nm in pure water. The formed
solution is stable which is still clear aer standing for 12 h with
a negligible change in the emission spectra (Fig. S4†).
2.5 Effect of H2O fraction on emission performance
We also investigated addition of H2O on the uorescence
performance of DPIN in DMF solution (10 mM). The uores-
cence spectra showed an obvious change upon increasing the
ratio of H2O from 0 to 100% (Fig. 4a). A similar uorescent
performance of DPIN in THF with different H2O proportions is
shown in Fig. S3.† In pure DMF, besides the strong keto-form
emission at ca. 565 nm and weak enol-form emission at ca. 415
nm, the emission spectra of DPIN displayed a third weak peak at
ca. 480 nm which might be due to the formation of species that
are intermolecularly hydrogen-bonded to DMF molecules. Upon
The absolute quantum yields of keto-emission of DPIN in
DMF, pure water and DMF–H2O mixed solution (10 mM, v/v,
2 : 3) were measured by using an integrating sphere. It shows
high quantum yields of 0.30 (keto emission quantum yield:
0.28), 0.45 (keto emission quantum yield: 0.39), and 0.20 in
DMF, DMF–H2O (2 : 3) and pure water, respectively. These are
much higher than that of the reported AIEE ESIPT molecules.23
2.6 pH effects
addition of water, this peak disappeared and the intensity of As known, HBIs have amphoteric properties due to the presence
enol-form emission increases upon the concentration of H2O up of both Lewis acidic –NH– and basic ]N– sites in the imidazole
to 70% while the keto-form emission showed an irregular ring. It is very important to examine the pH effect on the uo-
change. The decreasing intensity ratio between the keto- and rescence performance of DPIN. The experiments were con-
enol-form emissions below addition of water up to 80% may be ducted in PBS buffer (20 mM). The results showed that the
due to the formation of the hydrogen bond between DPIN and performance of the uorescence of DPIN hardly changed under
water, which is an obstacle for the tautomerization of DPIN different pH values from 6.0 to 8.8 (Fig. 5a). The experiment
from the enol form to the keto form. The keto-form emission results indicate that DPIN is a good candidate dye displaying
becomes more prominent up to the concentration of 90%, ESIPT for applications in the physiology environment.
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Fig. 5 The fluorescent spectra of DPIN (10 mM) in PBS buffer (20 mM):
(a) with a pH from 6.0 to 8.8; (b) with common cations and ions (100
eq.) added, (pH, 7.2).
2.7 Interference of common ions
Imidazole behaves as not only an excellent hydrogen bond
donor to an anion receptor but also an electron donor that can
bind metal ions by coordination bonds. In other words, the
uorescence of dyes involving imidazole rings may be affected
profoundly by the presence of ions.27 On this account, it is
necessary to investigate the effect of ions on the uorescence
performance of DPIN. The experiments were carried out in PBS
buffer (20 mM, PH, 7.2) with common cations and anions. As
shown in Fig. 5b, all the ions examined showed a negligible
effect on the intensity of the emissions. It is concluded that the
uorescence performance of DPIN would not be disturbed by
common ions.
Fig. 6 (a) The fluorescent spectra of DPIN-A (10 mM) in PBS buffer (20
mM, PH, 7.2) in the presence of 10 eq. Cys from 0 to 150 min. (b) The
fluorescent emission intensity for keto emission (l ¼ 553 nm) and enol
emission (l ¼ 425 nm) as time increases. (c) The fluorescent spectra of
keto emission in the presence of different amounts of Cys after 30 min.
(d) The fluorescent intensity at 553 nm toward different amounts of
Cys from 0–30 mM.
of the Cys plot shows a linear correlation to the amounts of Cys
from 0 to 30 mM (Fig. 6d). The detection limit of Cys was
calculated to be 0.21 mM which is below the requisite detection
limits for Cys assays,30 indicating that DPIN-A is suitable for
sensing Cys with good sensibility in pure water.
2.8 Optical responses of probe DPIN-A towards Cys
In view of the excellent uorescence performance of DPIN, we
designed a new probe DPIN-A for Cys by protecting the –OH
group with an allyl group. The formed acrylate quenched both
2.9 Selective detection of biothiols
the enol- and keto-form emissions due to the PET process and To evaluate the selectivity of DPIN-A towards Cys, we studied the
block the –OH group. Condensation of acrylates with Cys which uorescence performance of DPIN-A corresponding to biothiols
could release the –OH group in two step reactions restored the (10 eq.) and other amino acids (100 eq.) in pure water (PBS
DPIN emission. This uorescent turn-on process had been buffer, 20 mM, pH, 7.2). The emission spectra changed slightly
reported to detect Cys in the CTAB micelle with an obvious color compared with the blank sample in the presence of other 19
change discerned by eyes.12 Here, we investigated the sensing kinds of amino acids, indicating that the other amino acids did
performance for Cys of DPIN-A in a more simple protocol. The not react with DPIN-A. Biothiols including HCy, Cys and GSH
experiments were carried out using a 10 mM solution of DPIN-A have similar structures which just differ by a single methylene
in pure water (PBS buffer, 20 mM, pH, 7.2). Fig. 6a shows the unit in their side chains.28 It can be seen that (Fig. 6b and 7a) all
uorescent spectra of the assay system in the presence of Cys of them can give rise to enol emission (l ¼ 425 nm) indicating
(10 eq.) for 150 min. Aer the addition of Cys, the intensity of their conjugate addition reactions with DPIN-A. Interestingly,
keto-form emission increases gradually, while that of enol-form only the addition of Cys can signicantly increase the intensity
emission increased quickly at rst (ꢀ10 min) then decreased ratio between the keto- and enol-form emissions. It displays
gradually. The emission intensity is relatively stable in about 20 strong emission at ꢀ553 nm while the enol emission peaked at
minutes for both enol- and keto-emission (Fig. 6b). The enol ꢀ425 nm is very weak. The emission intensity change of DPIN-A
emission increases initially due to the conjugate addition, towards HCy and GSH with time are shown in Fig. S5.†
which removes the alkene-induced PET quenching and the Compared with Cys, the emission at 425 nm for HCy and GSH
increase of the keto emission is due to the formation of lactam was much stronger than that of Cys, indicating that they cannot
releasing DPIN exhibiting the ESIPT process.
be removed quickly aer conjugation with DPIN-A. The high
We also investigated the uorescent response of DPIN-A selectivity of Cys over HCy and GSH was further proved by the
towards different amounts of Cys (0–100 mM) aer reaction for ratio between emission intensity at 553 nm and 425 nm as time
30 min. The results (Fig. 6c) showed that the increasing inten- increases (Fig. 7b). For Cys, it reached the stable ratio about 12
sity of the major peak at ca. 553 nm correlates with the aer 20 min with yellowish-green emission, whereas HCy and
increasing concentration of Cys. The intensity vs. concentration GSH show a much lower ratio (below 2.0) with blue emission
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change aer being cultured with excessive other amino acids
(100 eq.) for about one hour (red bars). Upon addition of 10 eq.
Cys in the buffer solution of DPIN-A together with other amino
acids (100 eq.) for about an hour, the intensity ratio increased
signicantly (green bars), indicating that DPIN-A can selectively
detect Cys without interference from other amino acids.
3. Experimental section
3.1 Materials and general methods
1H-NMR spectra were recorded using a Varian Gemin-400 MHz
spectrometer. Fluorescence spectra and absolute quantum
yields (measured with an integrating sphere) were measured
using an optical spectrometer (PTI: QM-TM, USA). MS analyses
were recorded using a Perkin Elmer Flexar SQ 3000 mass
spectrometer. Elemental analysis (C, H and N) was performed
using a Vario EL III CHNS elemental analyzer. The single crystal
structure of DPIN was obtained by X-ray diffraction with a
Gemini A Ultra X-ray single crystal diffractometer using graphite
Fig. 7 (a) Fluorescent spectra of DPIN-A (10 mM) towards Cys (10 eq.),
HCy, (10 eq.) GHS (10 eq.). The spectra were recorded after 30 min.
Other amino acids (100 eq. obtained after 1 hour.) (b) The ratio of
fluorescent intensity centred at 553 nm and 425 nm towards Cys, HCy
and GSH with a function of time. (c) Intensity ratio (of keto emission
and enol emission) of the probe (10 mM) towards other amino acids
(100 eq., data were obtained after 1 hour) and biothiols of HCy, GSH
and Cys (10 eq., data were obtained after 30 min).
˚
monochromatized Cu-Ka radiation (Cu-Ka, l¼ 1.54184 A). All
pH measurements were made with a Sartorius (PB-10) pH
meter. Dynamic Light Scattering (DLS) measurement was per-
formed using a nano Zetasizer (Malvern). Flash chromatog-
raphy was performed with silica gel (200–300 mesh).
Spectroscopic grade DMF and Milli-Q water (18.2 MU cm, 25 ꢂC)
were used throughout the detection experiments.
(Fig. 7b inset). In Fig. 7c we can directly see that the emission
intensity ratio of keto vs. enol is high up to ꢀ14 towards Cys of
DPIN-A aer reaction for 30 min. It has a higher selectivity over
other amino acids and biothiols, which exhibits a ratio below
ꢀ2.0 even aer a longer reaction time of 1 h.
3.2 Synthesis of 3-hydroxy-2-naphthaldehyde
3-Hydroxy-2-naphthaldehyde was synthesized according to the
previous reported literature with little modication.31 In a dry
100 mL round bottom ask was typically charged with 6 mmol
of the naphthalen-2-ol, dissolved in 3 mL of anhydrous THP
under N2 protection. Then 5.6 mmol of 1.6 M t-BuLi in pentane
was added dropwise via a syringe in approximately 2 min to the
stirred solution at room temperature. Immediately a rapid
evolution of heat and gases occurred, thus raising the temper-
ature of the reaction mixture to ca. 50 ꢂC. Aer the gas evolution
subsided (15 min), the reaction mixture was le to stir for
another 4 h. A solution of the DMF (3 mL) in THP (3 mL) was
2.10 Interference experiment
To further investigate the selectivity of Cys over other compet-
itive species, interference experiments of other amino acids
towards Cys were carried out. The intensity ratio between
553 nm and 425 nm of the probe (Fig. 8) displays no distinct
ꢂ
then added to the above mixture at the 0 C and, subsequently
le to stir for 24 h at room temperature. Aer the reaction
nished, water was added carefully to the mixture and dropped
1 M HCl until pH was 5–6. The mixture was extracted with ethyl
acetate, dried over anhydrous Na2SO4 and evaporated to
dryness. The resulting residue was chromatographed on silica
gel (petroleum ether–CH2Cl2, 3 : 1), and a bright yellow powder
was then obtained with 60% yield. 1H NMR (400 MHz, DMSO) d
7.30 (s, 1H), 7.36 (ddd, 1H), 7.55 (ddd, 1H), 7.77 (d, 1H), 7.99 (d,
1H), 8.36 (s, 1H), 10.42 (s, 1H), 10.59 (s, 1H).
3.3 Synthesis of 3-(4,5-diphenyl-1H-imidazol-2-yl)
naphthalen-2-ol (DPIN) (1)
Fig. 8 Bars represent the final fluorescence intensity at 528 nm in PBS
buffer (20 mM, pH, 7.2). Red bars: emission responses of DPIN-A
towards amino acids (100 eq.). Green bars: emission responses of
DPIN-A (10 mM) towards amino acids (100 eq.) in the presence of Cys
(10 eq.) All the data were obtained after 1 h.
The product was prepared by reuxing benzyl (0.21 g, 1 mmol),
3-hydroxy-2-naphthaldehyde (0.32 g, 1.2 mmol) and ammonium
acetate (0.75 g, 10 mmol) in glacial acetic acid (50 mL) for 24 h
under an argon atmosphere. Aer cooling to room temperature,
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a pale yellow mixture was obtained and poured into a methanol
solution under stirring. The separated solid was ltered off,
washed with methanol, and dried to give a light yellow solid.
The solid was puried by column chromatography (petroleum
ether–CH2Cl2, 1 : 1) on silica gel. 1H NMR (400 MHz, DMSO-d6,
TMS) d 5.32 (d, J ¼ 4.7 Hz, 1H), 7.19–7.67 (m, 12H), 7.78 (dd, J ¼
18.1, 8.0 Hz, 2H), 8.66 (s, 1H), 12.93 (s, 1H), 13.32 (s, 1H). MS
(ESI): m/z calculated 362.46, found 362.76. Anal. found: C, 82.36;
H, 5.08; N, 7.45. Calc. for C25H18N2O: C, 82.85; H, 5.01; N,
7.73%.
3.8 Synthesis of 3-(4,5-diphenyl-1H-imidazol-2-yl)
naphthalen-2-yl acrylate (DPIN-A)
DPIN-A was obtained according to the reported literature.15 To a
solution of DPIN (182 mg, 0.5 mmol) and Et3N (2 eq.) in 10 mL
of anhydrous CH2Cl2, acryloyl chloride (1.25 eq., mixed with 4
mL of CH2Cl2) was added dropwise at 0 ꢂC. Aer stirring at this
temperature for 90 min, the mixture was warmed to room
temperature and stirred overnight. The solvent was removed
under vacuum to furnish a crude mixture which afforded DPIN-
A upon ash chromatography as a light orange solid (110 mg,
51% yield) 1H NMR (400 MHz, DMSO) d 5.32 (s, 1H), 5.88 (s, 1H),
5.98–6.31 (m, 1H), 6.41–6.77 (m, 2H), 6.92–7.81 (m, 10H), 7.75–
8.15 (m, 3H), 8.59 (s, 1H), 12.76 (s, 1H). MS (ESI): m/z calculated
416.47, found 416.81. Anal. found: C, 80.30; H, 4.90; N, 6.35.
Calc. for C28H20N2O2: C, 80.75; H, 4.84; N, 6.73%.
3.4 2-(Phenanthro[9,10-d]oxazol-2-yl)phenol (HPO-1)
1
HPO-1 was synthesized using a similar approach for DPIN. H
NMR (400 MHz, DMSO) d, 7.17 (ddd, J ¼ 15.1, 8.3, 4.3 Hz, 2H),
7.41–7.66 (m, 1H), 7.73–7.99 (m, 4H), 8.25 (dd, J ¼ 7.8, 1.5 Hz,
1H), 8.44 (dd, J ¼ 7.9, 1.1 Hz, 1H), 8.58 (dd, J ¼ 7.8, 1.2 Hz, 1H),
9.01 (t, J ¼ 8.6 Hz, 2H), 11.17 (s, 1H). MS (ESI): m/z calculated
311.33, found 311.46. Anal. found: C, 80.94; H, 4.43; N, 4.31.
Calc. for C21H13NO2: C, 81.01; H, 4.21; N, 4.50%.
4. Conclusion
In summary, by introducing benzyl as the tail, imidazole as the
core and 3-OH-2-naphthalene as the head, we successfully
designed and obtained an interesting ESIPT chromophore
named DPIN which showed prominent ESIPT emission in
protic and polar solvents with large Stokes shi and high
quantum yields. The benzyl tail with a large twisted angle
avoided aggregation induced quenching by restricting the
intermolecular p–p stacking. The imidazole core supplies
intermolecular H-bond sites which are in favour of J-aggrega-
tion in solution and ensures the prominent keto-form emission
in protic and polar solvents. 3-OH-2-naphthalene as the head of
DPIN can extend the conjugation length effectively which
results in a keto-emission with large Stokes shi.
DPIN can be dispersed in water forming stable transparent
solution with almost exclusive keto-form emission and a high
uorescence quantum yield up to 0.20. The uorescence
performance is very stable in the physiology pH range of 6.2–9.2
and cannot be interfered by other common ions.
The new turn-on sensor denoted as DPIN-A (Scheme 1) by
protecting the OH-moiety of DPIN with an acryloyl group
showed high selectivity and sensitivity towards Cys and can
discriminate Cys from other amino acids including GSH and
HCy in pure water. Further studies are underway to optimize the
sensing method.
3.5 2-(1H-Phenanthro[9,10-d]imidazol-2-yl)phenol (HPI-1)
9,10-Phenanthrenequinone (2.12 g, 10 mmol) and ammonium
acetate (0.75 g, 10 mmol) were added to the 30 mL mixed solvent
of ethanol and dichloromethane (1 : 1, v/v). Aer reuxing for
10 min, salicylaldehyde (0.32 g, 1.2 mmol) and a catalytic
amount of glacial acetic acid were added. The reaction mixture
was held at reux for another 3 h. Aer cooling to room
temperature, the mixture was ltered. The solid was puried by
column chromatography (petroleum ether–CH2Cl2, 1 : 1) on
silica gel. A white powder was nally obtained aer it was stirred
in reuxing ethanol, subsequently ltered, and dried under
vacuum. 1H NMR (400 MHz, DMSO) d 5.32 (s, 1H), 7.09 (ddd, J ¼
4.5, 3.5, 1.8 Hz, 2H), 7.47–7.28 (m, 1H), 7.90–7.57 (m, 3H), 8.26
(dd, J ¼ 8.1, 1.6 Hz, 1H), 8.56 (dd, J ¼ 40.9, 7.7 Hz, 2H), 13.70 (s,
1H), 8.91 (dd, J ¼ 12.7, 8.3 Hz, 2H), 13.14 (s, 1H). MS (ESI): m/z
calculated 310.35, found 310.70. Anal. found: C, 81.16; H, 4.76;
N, 8.94. Calc. for C21H14N2O: C, 81.27; H, 4.55; N, 9.03%.
3.6 1-(Phenanthro[9,10-d]oxazol-2-yl)naphthalen-2-ol (HPO-2)
1
HPO-2 was synthesized using a similar approach for DPIN. H
NMR (400 MHz, DMSO) d 7.42 (d, J ¼ 9.0 Hz, 1H), 7.48 (t, J ¼ 7.4
Hz, 1H), 7.63–7.75 (m, 1H), 7.77–7.93 (m, 3H), 8.00 (d, J ¼ 7.4 Hz,
1H), 8.12 (d, J ¼ 9.0 Hz, 1H), 8.44 (d, J ¼ 7.7 Hz, 1H), 8.56 (dd, J ¼
11.4, 8.4 Hz, 2H), 9.04 (t, J ¼ 8.6 Hz, 3H), 11.91 (s, 1H). MS (ESI):
m/z calculated 361.39, found 361.75. Anal. found: C, 82.95; H,
4.65; N, 3.98. Calc. for C25H15NO2: C, 83.09; H, 4.18; N, 3.88%.
Acknowledgements
This work was nancially supported by the National Basic
Research Program of China (973 Program, no. 2013CB834803),
the National Science Foundation for Distinguished Young
Scholars of China (no. 20825102) and the National Natural
Science Foundation of China (no. 91222202, 21171114).
3.7 1-(1H-Phenanthro[9,10-d]imidazol-2-yl)naphthalen-2-ol
(HPI-2)
1
HPI-2 was synthesized using a similar approach for HPI-1. H
NMR (400 MHz, DMSO) d 5.32 (d, J ¼ 4.3 Hz, 1H), 7.26–7.44 (m,
7H), 7.46–7.52 (m, 1H), 7.52–7.62 (m, 4H), 7.88 (t, J ¼ 8.5 Hz,
2H), 8.19 (d, J ¼ 8.6 Hz, 1H). MS (ESI): m/z calculated 360.41,
found 360.42. Anal. found: C, 83.60; H, 4.53; N, 7.85. Calc. for
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