ESIPT-induced fluorescent: O -hydroxycinnamate: A self-monitoring phototrigger for prompt image-guided uncaging of alcohols

Article abstract of DOI:10.1039/c7ob02280h

O-Hydroxycinnamate derivatives are well-known phototriggers for fast and direct release of alcohols and amines without proceeding through the cleavage of carbonate or carbamate linkages. Despite these unique features, o-hydroxycinnamates lack extensive applications in biological systems mainly because of their non-fluorescent nature. To overcome this limitation, we have attached a 2-(2′-hydroxyphenyl) benzothiazole (HBT) moiety, capable of rapid excited-state intramolecular proton transfer (ESIPT) to the o-hydroxycinnamate group. The ESIPT effect induced two major advantages to the o-hydroxycinnamate group: (i) large Stokes' shifted fluorescence (orange colour) properties and (ii) distinct fluorescence colour change upon photorelease. In vitro studies exhibited an image guided, photoregulated release of bioactive molecules by the o-hydroxycinnamate-benzothiazole-methyl salicylate conjugate and real-time monitoring of the release action.

Full text of DOI:10.1039/c7ob02280h

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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ESIPT-Induced Fluorescent o-Hydroxycinnamate: Self-Monitoring
Phototrigger for Prompt Image-Guided Uncaging of Alcohols
Received 00th January 20xx,
Accepted 00th January 20xx
Amrita Paul,a Rakesh Mengjib, Olive Abraham Chandy,a Surajit Nandi,a Manoranjan Bera,a Avijit
Jana,*b,c,d Anakuthil Anoop,*a and N. D. Pradeep Singh*a
DOI: 10.1039/x0xx00000x
The o-hydroxycinnamate derivatives are well known phototriggers for fast and direct release of alcohols and
amines without proceeding through the cleavage of carbonate or carbamate linkages. Despite these unique
features, the o-hydroxycinnamates lack extensive applications in the biological systems mainly because of its
non-fluorescent nature. To overcome this limitation, we have attached 2-(2’-hydroxyphenyl) benzothiazole (HBT)
moiety, capable of rapid excited-state intramolecular proton transfer (ESIPT) to the o-hydroxycinnamate group.
The ESIPT effect induced two major advantages to the o-hydroxycinnamate group: i) large Stokes’ shifted
fluorescence (orange colour) property, ii) distinct fluorescence colour change upon photorelease. In vitro studies
exhibited image guided, photoregulated release of bioactive molecule by o-hydroxycinnamate-benzothiazole-
methyl salicylate conjugate and real time monitoring of the release action.
www.rsc.org/
Therefore, if we could modify o-hydroxycinnamate as a
Introduction
fluorescent phototrigger, it might open a new dimension in its
In recent years, we have witnessed a sustained development
and upgradation of phototriggers based on their photophysical
and photochemical characteristics in order to ensure better
prospects in the field of drug delivery.[1] The o-
hydroxycinnamate is a well known phototrigger which was
introduced by Porter et al. to cage alcohols and amines.[2-7] The
salient features which attracted us towards this phototrigger
are: (i) clean and rapid uncaging, (ii) structural simplicity, (iii) it
belongs to the category of very few phototriggers which can
directly cage alcohols and amines without carbonate or
carbamate linkage that are prone to be labile under
physiological condition,[8-10] (iv) the resulting photoproduct
coumarin serve as a fluorescent reporter, and (v) sufficient
sensitivity towards both one photon (1PE) and two-photon
excitation (2PE).[11-13] Despite these interesting features, the
major limitation of the cinnamate series is that they are
intrinsically non-fluorescent. This impedes their extensive
usage in the area of drug delivery, where real time tracking of
the drug delivery system by the means of highly sensitive
fluorescence imaging technique is always beneficial to attain
higher spatial and temporal control over the uncaging.
application. Firstly, it can be extensively utilized for image-
guided delivery of active molecules in the biological systems.
Secondly, it will be helpful in self-monitoring the release action
and quantifying the released substrate.
Nowadays, excited-state intramolecular proton transfer
(ESIPT) process have attracted much attention because of their
many advantageous properties, such as a strong fluorescence
with a large Stokes’ shift,[14] a low inner-filter effect, and low
self-quenching ability.[15-23] ESIPT is an ultrafast enol–keto
phototautomerization process exhibited by intramolecularly H
bonded molecules which occur in their excited-states.[24-29]
Recently, our group has reported ESIPT assisted photorelease
of carboxylic acids using p-hydroxyphenacyl phototrigger.[30]
Now, we are interested to design a phototrigger which can
exhibit ESIPT induced fluorescence and image-guided direct
uncaging of hydroxyl groups with the ability of self-monitoring
the course of uncaging in real-time. Since a large number of
bioactive molecules contains hydroxyl as a terminal functional
group.
Scheme 1. Photoinduced uncaging of alcohols from the ESIPT induced fluorescent o-
hydroxycinnamate phototrigger. ROH= ethanol, methyl salicylate.
Herein, we present o-hydroxycinnamate appended with ESIPT
moiety as a new fluorescent phototrigger for the direct
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uncaging of alcohols (Scheme 1). For our investigation, we Interestingly, caged compound
Journal Name
5
exhibits a strong fluorescence
chose ethyl o-hydroxy-5-methylcinnamate-benzothiazole (orange colour) with a large Stokes’ shift due to the presence
conjugate ( ) as a model caged compound. Our designed of HBT moiety which is known to exhibit an ESIPT process upon
5
phototrigger contains 2-(2’-hydroxyphenyl)benzothiazole photoexcitation (Fig. 1b).[33] Its Fluorescence quantum yield
(HBT) as a ESIPT moiety. ESIPT process from the hydroxyl was calculated as 14.6% (in acetonitrile), taking quinine
group of o-hydroxycinnamate to the benzothiazole moiety in sulphate as the standard (quinine sulphate quantum yield:
our newly designed phototrigger provided advantages like (i) 54%) as shown in Table S1 in the ESI†.[34]
induces large Stokes’ shifted fluorescence (orange colour), (ii)
causes distinct fluorescence colour change on photoinduced
uncaging, which in turn leads to excellent self-monitoring the
uncaging process and (ii) helps in quantification of the uncaged
substrate. Finally, image-guided release of
a biologically
relevant molecule methyl salicylate and self-monitoring of the
release by our phototrigger in real time was demonstrated in
vitro. Biocompatibility of our caged compound before and
after photolysis was also displayed by MTT (3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide,
yellow tetrazole) assay.
a
Results and discussion
Synthesis of caged compound 5
The ESIPT based fluorescent o-hydroxycinnamate phototrigger
was synthesized following the sequence of reactions as shown
in Scheme 2. Commercially available p-cresol was converted
into compound
with 2-aminothiophenol in presence of iodine at room
temperature furnished compound 3 [32]
Compound was
converted to by Duff reaction using hexamethylenetetramine
and trifluoroacetic acid as the solvent.
condensed with ethyl triphenylphosphoranylideneacetate in
toluene to provide caged compound with the desired E
configuration. Products obtained in each step were
characterized by 1HNMR, 13CNMR (Fig. S1–S4 in in the ESI†
and HRMS (Fig. S7–S9, ESI†)
2
by Reimer-Tiemann reaction.[31] Treatment of
2
.
3
4
[32]
Compound
4 was
5
)
.
Fig. 1. (a) Absorption and (b) emission spectra of caged compound 5 in different
solvents.
Further, due to ESIPT process, the caged compound
exhibited dual emission, one is normal Stokes’ shifted emission
maximum at 430 nm (enol form ( )) and another is a large
Stokes’ shifted emission maximum at 590 nm (keto form ( ))
due to proton transfer tautomer. The dual emission process of
caged compound is strongly dependent on the solvent
5
8
9
5
system. In non-hydrogen bonding solvents (cyclohexane,
benzene, chloroform), only one emission maximum at 590 nm,
Scheme 2. Synthesis of caged compounds 5. Reagents and conditions: (a) NaOH,
chloroform, reflux, 2 h; (b) 2-aminothiophenol, I2, methanol, room temperature, 2 h; (c)
corresponding to the keto form (9), was observed (Fig. 1b). In
hexamine,
trifluoroacetic
acid,
reflux,
overnight;
(d)
non-hydrogen bonding solvents there is an ultrafast proton
transfer from the hydroxyl group of the o-hydroxycinnamate
to the nitrogen atom of the benzothiazole moiety (ESIPT),
carboethoxymethylidenetriphenylphosphorane, toluene, 60 0C, 4 h.
Photophysical properties of caged compound 5
leading to the existence of only keto form (9). On the contrary,
Photophysical properties of caged compound
5
(1×10-5 M)
in polar protic solvents (EtOH, MeOH), two emission peaks,
one near 430 nm and another at 590 nm corresponding to the
were studied in different solvent systems. It shows a strong
absorption peak at 370 nm in all solvents
(Fig. 1a).
enol (8) and keto (9) form, respectively was observed. This is
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due to the presence of hydrogen bond between the solvent
ARTICLE
and the hydroxyl group of the caged compound
restricts the ESIPT process.
5 which
Fig. 3. Photolysis of caged compound 5 monitored by 1H NMR in CDCl3.
The NMR studies indicated clean photoconversion of o-
hydroxycinnamate ( ) to the corresponding benzothiazole-
5
Fig. 2. Absorption spectra of the caged compound 5 as the irradiation time is gradually
increased (0–60 min).
coumarin (14) photoproduct along with the uncaging of
ethanol. The photochemical quantum yield for the uncaging
reaction was calculated as 0.10 in acetonitrile/H2O (ACN/H2O)
(1:1 v/v) solution (Table S2, ESI†). We observed that almost
Photolysis of caged compound 5
The photoinduced uncaging of ethanol by our caged
87% of ethanol was released from caged compound
5 after
compound
5 was studied by irradiating the solution of 5
irradiation with light (λ ≥ 365 nm) for 60 min (Fig. 4a).
Furthermore, the precise control over the photoinduced
uncaging was demonstrated by alternative exposure of our
(1×10-4 M) in acetonitrile/H2O (ACN/H2O) (1:1 v/v) using 125 W
medium pressure Hg lamp as the UV light source (λ ≥ 365 nm)
and 1 M CuSO4 solution as the UV cut−off filter. The photolysis
caged compound 5 to light and dark. It was observed that
ethanol was uncaged only upon irradiation of light (Fig. 4b).[35]
of
Fig. 3), emission spectroscopy (Fig. 6), and reversed-phase
5
was monitored by absorption spectroscopy (Fig. 2) 1HNMR
(
1
HPLC (Fig. S12, ESI†). The HNMR spectra of 5 were recorded
at different irradiation time intervals in CDCl3. At 0 min, signals
at 4.29 ppm, 6.74 ppm and 13.16 ppm corresponding to the
methylene protons (Ha) olefinic protons (Hb) and hydroxyl
proton (Hc) of caged compound 5 were noted. With increase in
irradiation time from 0 to 60 min, the intensity of the signals
for Ha, Hb, and Hc decreases indicating the gradual
photodecomposition of caged compound
5. On the other
hand, appearance and increase in the intensity of two new
signals at 6.54 ppm (Hd) and 8.16 ppm (He) corresponding to
two olefinic protons of the benzothiazole-coumarin
(14)
indicates the formation of the photoproduct. The uncaging of
ethanol is indicated by the gradual appearance of quartet
signal at 3.72 ppm corresponding to the methylene protons
(Hf) of ethanol.
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Further to support the ESIPT mechanism computationally, a
relaxed surface scan using density functional theory (See
supporting information for details) was performed from
5 to 9
by decreasing the N—H bond length (from 1.67 to 0.97 Å). The
potential energy surface (PES) of S1 state was constructed from
the vertical excitation energies calculated at each of the points
on the ground-state PES. The PES of the S1 state showed a
monotonous decrease in energy on going from
Moreover, when the ground state geometry of
optimized in the S1 state, the geometry was converged to
5
towards
9.
5
was
9
which confirmed a barrierless ESIPT process. To understand
the subsequent trans-cis isomerization, we have performed a
rigid surface scan corresponding to one-bond flip (OBF)
mechanism in the ground and photochemically excited
states.[38] The PES (Fig. 5) indicates that the trans (
9) to cis (12)
photoisomerization proceeds through a conical intersection at
Fig. 4. (a) The amount of ethanol released from caged compound 5 on photolysis (≥ 365
nm) at different time intervals. (b) Release of ethanol under bright and dark conditions. a 90° torsion angle of the flipping bond. The vertical excitation
“ON” and “OFF” implies the switching on and off of the light source, respectively.
energy (ΔES1-S0) at the highest point of the S0 surface was 1.13
eV at CASSCF(4,4)/cc-pVDZ[39] level of theory. The barrierless
Mechanism of the photolysis of caged compound 5
ESIPT and a low value of the ΔES1-S0 confirmed that the two
consecutive photochemical processes will be fast.
A possible mechanism for the photoinduced uncaging of
ethanol by caged compound
described in Scheme Upon exposure to light, caged
compound gets excited to the singlet state (supported by
5 in ACN/H2O solution is
3.
5
fluorescence quenching study in the presence of
benzophenone in Fig. S13 in ESI†. The average fluorescent
lifetime (singlet state lifetime) of the caged compound
5 was
1.15 ns, obtained from a time-correlated single photon
counting (TCSPC) experiment (Fig. S14, ESI†).[36] In the excited
state, caged compound
5 undergoes rapid ESIPT from the
hydroxyl group of o-hydroxycinnamate moiety to the
benzothiazole moiety, resulting in deprotonation of the
hydroxyl group to give
immediately gets transformed to the more stable keto form
). The keto form then undergoes rapid trans–cis
a zwitterionic form (11) which
(
9
photoisomerization to give 12, followed by thermally driven
lactonization through a tetrahedral intermediate (13) leading
to quantitative release of ethanol[37] and 8-benzthiazole-6-
methylcoumarin (14) as the photoproduct.
Fig. 5. Reaction profile for the ESIPT and the cis-trans isomerization at the CAM-
B3LYP/cc-pVTZ//CAM-B3LYP/cc-pVTZ level of theory. The crossing point indicates the
conical intersection. The black line indicates the PES in the S0 state and the red line
indicates the PES in the S1 state. The OBF was performed by rotating the double bond.
Here, the structure 15 corresponds to the conical intersection. The compound 12 is the
cis isomer in the ground state.
Real time monitoring of the release
Another interesting feature of our designed system is that it
shows distinct fluorescence colour change from orange to blue
on photoinduced uncaging (Fig. 6). At 0 min the emission
spectrum shows only an orange emission band corresponding
to caged compound
5
(λ
= 590 nm, Fig. 6). As the
max
irradiation time gradually increased (0–60 min) the intensity of
the orange emission band gradually decreased with
concomitant appearance and increase in the intensity of a new
blue emission band at 450 nm corresponding to the
Scheme 3. Possible mechanism of ESIPT and photoinduced uncaging of ethanol from
caged compound 5.
photoproduct benzothiazole-coumarin conjugate
(14) was
noted. The blue shift in the fluorescence spectrum of the
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photoproduct, benzothiazole-coumarin conjugate, is because
of the absence of ESIPT process.
Fig. 7. Absorption and emission spectra of caged compound 7 in acetonitrile/HEPES
buffer (1:9) solution.
Photolysis of caged methyl salicylate. Photolysis of caged
compound
7
(1×10-4 M) was carried out in acetonitrile/HEPES
(1:1) buffer (HEPES was used to resemble biological condition)
using 125 W medium pressure Hg lamp as UV light source (λ ≥
365 nm). The course of the photoinduced uncaging from caged
Fig. 6. Emission spectra of the caged compound 5 as the irradiation time is gradually
increased (0–60 min).
Synthesis, photophysical, photochemical, cellular imaging and
cytotoxic properties of caged methyl salicylate (7)
compound
7
was monitored by fluorescence spectroscopy (Fig.
efficiently
8
). It was noted that the caged compound
7
released methylsalicylate (Scheme 4) in acetonitrile/HEPES
buffer (1:1 v/v) solution with almost similar chemical (87 %)
and quantum yield (0.10) (Table S2, ESI†).
Synthesis of caged methyl salicylate. To explore the versatility
of our fluorescent o-hydroxycinnamate phototrigger in the
biological system, we caged methyl salicylate as depicted in
Scheme 3. At first compound
NaOH to produce compound
chloride and attached to methyl salicylate in the presence of
trimethylamine to yield caged compound . Products obtained
in each step were characterized by HNMR, 13CNMR (Fig. S5,
S6, ESI† and HRMS (Fig. S10, S11, ESI†) Methyl salicylate was
5
was hydrolysed in presence of
6. 6
was converted into acid
7
1
)
.
selected because of its excellent rubefacient, analgesic and
antiseptic properties.
Scheme 4. Photorelease of caged methyl salicylate from compound 7.
Scheme 3. Synthesis of caged methyl salicylate (7). Reagents and conditions: (a) NaOH,
water, reflux, 6 h; (b) SOCl2, methyl salicylate, triethylamine, dichloromethane, 10 h
Photophysical properties of caged methyl salicylate.
Photophysical properties of caged compound
investigated in acetonitrile/HEPES buffer (1:9) solution and the
results were similar to caged compound Fig. 7). The
fluorescence quantum yield of caged compound in
7
(1×10-5 M) was
5
(
7
acetonitrile/HEPES (1:9 v/v) buffer solution was calculated as
16.3%, taking quinine sulphate as the standard (quinine
sulphate quantum yield: 54%) as shown in Table S1 in the ESI†.
Fig. 8. Emission spectra of caged compound 7 during photolysis at different time
intervals.
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Fig. 10: Percentage of methyl salicylate released from caged methyl salicylate (7) as a
In vitro cell imaging and real time monitoring studies. The
function of fluorescence intensity change.
photoinduced release of methyl salicylate by caged compound
7
inside HeLa cell line was monitored in real time by confocal
Cell cytotoxicity assay of caged methyl salicylate (7). In vitro cell
viability of caged compound 7 was determined by performing MTT
microscopy (Fig. 9). Initially, within the HeLa cells only orange
fluorescence (Fig. 9i) was observed due to the internalization
of caged methyl salicylate. Upon exposure to light of
wavelength 365 nm for 30 min, both orange and blue
fluorescence (Fig. 9ii) was observed which indicated partial
release of methyl salicylate from the caged compound (7).
Finally, after 60 min of irradiation, a complete change in
fluorescence colour from orange to blue (Fig. 9iii) was noted,
thus suggesting a greater extent of photorelease of methyl
salicylate. The percentage of photorelease of methyl salicylate
was calculated as a function of change in the fluorescence
intensity (Fig. 10).[40]
(3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide,
a
yellow tetrazole) assay in normal cells (NIH 3T3). The percentage of
cell viability vs. concentration of caged compound 7 before and
after photolysis is depicted in Fig. 11 respectively. The figure
indicates that caged compound 7 is biocompatible at any given
concentration before and after irradiation.
Fig. 11. Cell viability assay of caged compound 7 in NIH 3T3 cells in before and after
irradiation. Values are presented as mean ± SD.
Conclusion
In conclusion, we have utilized the ESIPT process to upgrade
the photophysical and photochemical properties of the well
known o-hydroxycinnamate phototrigger for the photoinduced
uncaging of alcohols. For the first time o-hydroxycinnamate
Fig. 9. Confocal fluorescence images of the cellular internalization of caged compound
7 (10 µM) in HeLa cell line and image-guided, self-monitored uncaging of methyl
salicylate at different time intervals during photoirradiation: (i) 0 min, (ii) 30 min, and has been converted to a fluorescent phototrigger by simple
(iii) 60 min. (a) Blue emission: emission channel was 440 nm with band width of 40 nm,
attachment of ESIPT moiety. The fluorescence (orange colour)
(b) Orange emission: emission channel was 610 nm with band width of 40 nm. In Fig (i)
property of the newly designed o-hydroxycinnamate
and (ii) HeLa cell nuclei were stained with 4’,2-diamino-2-phenylindoline (DAPI). [Scale
phototrigger was utilized for the in vitro image-guided release
bar = 20 µm].
of bioactive molecule. Quantification of the released active
molecule by our designed phototrigger has been
demonstrated by self-monitoring the release action in real
time, owing to the distinct fluorescence colour change (from
orange to blue) on photorelease. Induction of fluorescence
property in the o-hydroxycinnamate has surely made it a
better phototrigger encouraging a wider scope of applications.
The limitation of our phototrigger is that it operates in UV
region and the photoproduct (coumarin) is
a strong
chromophore which acts as optical filter. In future, we are
interested to develop a system based on o-hydroxycinnamte
overcoming the above mentioned limitations, to utilize it as a
drug delivery vehicle.
Experimental Section
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NMR (400 MHz, CDCl3) δ 12.31 (s, 1H), 7.98 (d, J = 8.2 Hz, 1H),
7.90 (d, J = 8.0 Hz, 1H), 7.54–7.45 (m, 2H), 7.40 (t, J = 7.2 Hz,
1H), 7.19 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 2.36 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 169.3, 155.7, 151.9, 133.7,
132.5, 128.6, 128.3, 126.5, 125.3, 122.0, 121.4, 117.6, 116.3,
20.4. HR-MS calc for C14H12NOS [MH+]: 242.0595, found:
242.0608.
Chemicals and starting materials
All reagents were purchased from Sigma Aldrich and were
used without further purification. Dimethyl sulfoxide and
dichloromethane were distilled from CaH2 before use. All
anhydrous reactions were performed under a dry nitrogen
atmosphere.
Methods and techniques
Synthesis
of
3-(benzo[d]thiazol-2-yl)-2-hydroxy-5-
(1.5 g, 6 mmol) was
1H NMR spectra were recorded on a BRUKER-AC 400-MHz methylbenzaldehyde (4). Compound
3
spectrophotometer. Chemical shifts are reported in ppm from
tetramethylsilane with the solvent resonance as the internal
standard (deuterochloroform: 7.26 ppm, DMSO-d6: 3.313 ppm
and 2.484 ppm). Data are reported as follows: chemical shifts,
multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet),
coupling constant (Hz). 13C NMR (100 MHz) spectra were
dissolved in 10 mL of trifluoroacetic acid and then
hexamethylenetetramine (1.05 g, 7 mmol) was added. The
mixture was refluxed over night until all the starting material
was consumed. The reaction mixture was then cooled to room
temperature and poured into 6 M HCl (30 mL) and extracted
with CH2Cl2. The combined organic extracts were washed with
saturated brine. Next purification was done by column
chromatography (ethyl acetate: petroleum ether = 1:5) to
afford the pure product as a yellow solid (1.16 g, yield 87%). 1H
NMR (400 MHz, CDCl3) δ 13.01 (s, 1H), 10.49 (s, 1H), 8.02 (d, J
= 8.1 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.89 (s, 1H), 7.71 (s, 1H),
7.54 (t, J = 7.7 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 2.41 (s, 3H, CH3).
13C NMR (100 MHz, CDCl3) δ 190.7, 158.4, 151.4, 135.0, 133.1,
recorded on
a BRUKER–AC 400-MHz spectrometer with
complete proton decoupling. Chemical shifts are reported in
ppm from tetramethylsilane with the solvent resonance as the
internal standard (deuterochloroform: 77.0 ppm, DMSO-d6:
39.96 ppm). UV/Vis absorption spectra were recorded on a
Shimadzu UV–2450 UV/ Vis spectrophotometer; fluorescence
emission spectra were recorded on
a Hitachi F–7000
fluorescence spectrophotometer; HRMS spectra were
recorded on a JEOL–AccuTOF JMS–T100L mass spectrometer.
Photolysis of the caged compounds was carried out using 125-
132.4, 128.8, 126.8, 125.7, 123.7, 122.3, 121.5, 118.6, 115.8
,
20.3. HR-MS calc. for C15H12NO2 S [MH+]: 270.0544, found
270.0552.
W
medium-pressure Hg lamp supplied by SAIC (India).
Synthesis of (E)-ethyl 3-(3-(benzo[d]thiazol-2-yl)-2-hydroxy-5-
Chromatographic purification was done with 60–120-mesh
silica gel (Merck). For reaction monitoring, precoated silica gel
methylphenyl)acrylate (5). A mixture of
4 (500 mg, 2 mmol)
60 F254 TLC sheets (Merck) were used. RP–HPLC was recorded and carboethoxymethylidenetriphenylphosphorane (970 mg, 3
using acetonitrile in mobile phase, at a flow rate of 1 mL/min.
mmol) in toluene (10 mL for 1 mmol of aldehyde) was heated
at 60 °C under argon upon protecting from light. The course of
the reaction was followed by TLC. After 4 h, the reaction was
completed. After cooling to room temperature, toluene was
removed in a vacuum. The crude residue was purified by flash
chromatography on silica gel (ethyl acetate: petroleum ether =
1:9) to yield the desired cinnamate in high yield (90%; about
10% of the coumarin resulting from thermal trans-cis
isomerization followed by lactonization was formed during the
reaction). 1H NMR (400 MHz, CDCl3) δ 13.16 (s, 1H), 8.05 (d, J =
16.2 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H),
7.55 – 7.49 (m, 2H), 7.45 – 7.40 (m, 2H), 6.71 (d, J = 16.1 Hz,
1H), 4.28 (q, J = 7.1 Hz, 2H), 2.37 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H).
13C NMR (100 MHz, CDCl3) δ 169.1, 167.5, 155.2, 151.6, 139.5,
132.8, 132.6, 130.3, 128.4, 126.8, 125.7, 123.4, 122.2, 121.5,
119.6, 117.0, 60.4, 20.5, 14.4. HR-MS calc. for C19H18NO3S
[MH+] : 340.0963, found: 340.0965.
General Procedure for the preparation of caged compounds
Synthesis of 2-hydroxy-5-methylbenzaldehyde (2). A solution of
the p cresol (5.15 g, 47.6 mmol) in 120 mL of 10 N NaOH (3
mol) was heated to 65 °C. Then 40 mL of CHCl3 was added in
three portions over 15 min. The mixture was heated at reflux
in chloroform for 2 h. After cooling, the mixture was acidified
to pH 1 with 12 N HCl, the organic layer collected and the
aqueous layer extracted with chloroform. The combined
chloroform solution was dried and evaporated to give a crude
product which was distilled or recrystallized from an
appropriate solvent to yield 2-hydroxy-5-methylbenzaldehyde
1
as white solid, yield 95 %. H NMR (CDCl3, 400 MHz), δ = 2.37
(s, 3H), 6.90 (d, 1H, J = 8.60 Hz, 1H), 7.25-7.50 (m, 2H), 9.80 (s,
1H), 10.75 (s, 1H,). 13C NMR (100 MHz, CDCl3) δ 196.6, 159.3,
138.2, 133.2, 129.9, 128.9, 117.4, 20.1.
Synthesis of 2-(benzo[d]thiazol-2-yl)-4-methylphenol (3). To a
solution of 2-aminobenzenethiol (1.09 g, 8.7 mmol) and 2-
hydroxy-5-methylbenzaldehyde (2 g, 14 mmol) in anhydrous
CH3OH (10 mL) was added I2 (1.89 g, 7 mmol). The mixture
was stirred at room temperature. After about 5 min, yellow-
green precipitate generated gradually. The reaction mixture
was further stirred for another 2 h. The solid was collected on
a filter and washed with cold CH3OH. Further dried in a
Synthesis
of
(E)-3-(3-(benzo[d]thiazol-2-yl)-2-hydroxy-5-
(500 mg,
methylphenyl)acrylic acid (6). To the solution of
5
1.5 mmol) in 10 ml ethanol 5 M sodium hydroxide (1 ml) and
water (10 ml) was added and refluxed for 6 h. The dark
solution obtained was cooled to room temperature and
acidified with 12 N HCl to get white precipitate. The solid was
filtered, washed with dichloromethane to yield 260 mg (73%)
of the pure acid. 1H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H),
8.20 (d, J = 7.9 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 16.1
vacuum afforded
3 H
as a pale-yellow solid (1.5, yield 50.1%). 1
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Hz, 1H), 7.73 (d, J = 6.1 Hz, 2H), 7.59 (t, J = 7.6 Hz, 1H), 7.51 (t, J
= 7.4 Hz, 1H), 6.65 (d, J = 16.1 Hz, 1H), 2.34 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 168.9, 168.2, 154.2, 151.3, 138.4,
132.8, 130.8, 129.4, 127.5, 126.4, 123.1, 122.8, 122.4, 120.5,
117.2, 20.3. HR-MS calc. for C17H14NO3S [MH+]: 312.0650,
found: 312.0651.
Cellular internalisation and in vitro real-time monitoring of the
release of active molecule by our phototrigger was studied in
HeLa cell line.
Cellular Internalization Study of compound 7
The HeLa cells were grown in its log phase. Cells were seeded
in 96-well plates in Dulbecco's modified Eagle's (DMEM)
medium containing 10 % fetal bovine serum (FBS) for 8 h.
Synthesis of (E)-methyl 2-((3-(3-(benzo[d]thiazol-2-yl)-2-hydroxy-
5-methylphenyl)acryloyl)oxy)benzoate (7). Methyl salicylate (132
mg, 0.87 mmol) was stirred with triethyamine (80 mg, 0.79
mmol) in 5 ml DCM at room temperature for 15 min. To this
stirred solution, acid chloride of 6 was added and further
stirred at room temperature for 10 h. Upon completion of
reaction, monitored by TLC, the reaction mixture was poured
into 1 N HCl an extracted with DCM. The combined organic
extracts were washed with saturated brine. Purification was
done by column chromatography (ethyl acetate: petroleum
ether = 1:9) to afford the pure product as a yellow solid (60
mg, yield 50 %). 1H NMR (400 MHz, CDCl3) δ 13.28 (s, 1H), 8.27
(d, J = 16.1 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 8.1 Hz,
1H), 7.93 (d, J = 7.9 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.56 – 7.50
(m, 3H), 7.44 (t, J = 7.5 Hz, 1H), 7.34 (t, J = 7.7 Hz, 1H), 7.22 (d, J
= 8.0 Hz, 1H), 6.98 (d, J = 16.1 Hz, 1H), 3.86 (s, 3H), 2.40 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 168.9, 165.9, 165.3, 155.5,151.6,
150.7, 141.8, 133.7, 133.2, 132.6, 131.8, 130.8, 128.5,
126.9,125.9, 125.8, 124.0, 123.7, 123.1, 122.2, 121.5, 118.3,
117.2, 52.3, 20.5. HR-MS calc. for C25H20NO5S [MH+]: 446.1017,
found: 446.1008.
Different concentrations of compound
7 in HEPES buffer
containing 10 % ethanol were added and incubated at 37 °C in
5 % CO2 for 4 h. Imaging was done using confocal laser
scanning microscopy (CLSM) with suitable bandpass filters.
In-vitro self-monitoring of the release
Self-monitoring of the release of methyl salicylate from our
phototrigger
studied by fluorescence microscopy. HeLa cells were treated
with 10 μM of compound and put aside for 4 h at 37 °C.
7 after UV light (≥ 365 nm) irradiation was also
7
Then, compound-treated cells were irradiated with UV light (≥
365 nm) for 0–60 min. The live imaging was done to observe
image-guided release of methyl salicylate and self-monitoring
of uncaging reactions inside the cells was visually observed by
CLSM with suitable bandpass filters. For orange emission:
emission channel was 610 nm with band width of 40 nm and
for blue emission: emission channel was 440 nm with band
width of 40 nm.
Acknowledgements
We thank DST (SERB) for financial support and DST-FIST for
400 MHz NMR. A. Paul and M. Bera are thankful to IIT KGP for
their fellowship. A. Jana and R. Mengji are thankful to the
Department of Science & Technology (DST), India, for DST-
INSPIRE Faculty Research project grant (GAP 0546) at CSIR-
IICT, Hyderabad.
Photolysis of caged compounds 5 and 7
A solution of 10−4 M of the caged compound
5 was prepared in
acetonitrile/H2O (1 : 1 v/v). Half of the solution was kept in
dark and to the remaining half nitrogen was passed and
irradiated under UV light (≥365 nm), using a 125 W medium
pressure Hg lamp filtered by suitable filters with continuous
stirring. At regular interval of time, 20 μl of the aliquots was
taken and analyzed by absorption spectroscopy, fluorescence
spectroscopy and RP-HPLC using mobile phase acetonitrile , at
a flow rate of 1 ml min−1 (detection: UV 254 nm). Peak areas
were determined by RP-HPLC with the average of three runs,
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