A p-Hydroxyphenacyl-Benzothiazole-Chlorambucil Conjugate as a Real-Time-Monitoring Drug-Delivery System Assisted by Excited-State Intramolecular Proton Transfer

Article abstract of DOI:10.1002/anie.201508901

Among the well-known phototriggers, the p-hydroxyphenacyl (pHP) group has consistently enabled the very fast, efficient, and high-conversion release of active molecules. Despite this unique behavior, the pHP group has been ignored as a delivery agent, particularly in the area of theranostics, because of two major limitations: Its excitation wavelength is below 400 nm, and it is nonfluorescent. We have overcome these limitations by incorporating a 2-(2′-hydroxyphenyl)benzothiazole (HBT) appendage capable of rapid excited-state intramolecular proton transfer (ESIPT). The ESIPT effect also provided two unique advantages: It assisted the deprotonation of the pHP group for faster release, and it was accompanied by a distinct fluorescence color change upon photorelease. In vitro studies showed that the p-hydroxyphenacyl-benzothiazole-chlorambucil conjugate presents excellent properties, such as real-time monitoring, photoregulated drug delivery, and biocompatibility.

Full text of DOI:10.1002/anie.201508901

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International Edition: DOI: 10.1002/anie.201508901
German Edition: DOI: 10.1002/ange.201508901
Photoregulated Drug Release
A p-Hydroxyphenacyl–Benzothiazole–Chlorambucil Conjugate as
a Real-Time-Monitoring Drug-Delivery System Assisted by Excited-
State Intramolecular Proton Transfer
Shrabani Barman, Sourav K. Mukhopadhyay, Sandipan Biswas, Surajit Nandi,
Moumita Gangopadhyay, Satyahari Dey,* Anakuthil Anoop,* and N. D. Pradeep Singh*
Abstract: Among the well-known phototriggers, the p-
hydroxyphenacyl (pHP) group has consistently enabled the
very fast, efficient, and high-conversion release of active
molecules. Despite this unique behavior, the pHP group has
been ignored as a delivery agent, particularly in the area of
theranostics, because of two major limitations: Its excitation
wavelength is below 400 nm, and it is nonfluorescent. We have
overcome these limitations by incorporating a 2-(2’-hydrox-
yphenyl)benzothiazole (HBT) appendage capable of rapid
excited-state intramolecular proton transfer (ESIPT). The
ESIPT effect also provided two unique advantages: It assisted
the deprotonation of the pHP group for faster release, and it
was accompanied by a distinct fluorescence color change upon
photorelease. In vitro studies showed that the p-hydroxyphe-
nacyl–benzothiazole–chlorambucil conjugate presents excel-
lent properties, such as real-time monitoring, photoregulated
drug delivery, and biocompatibility.
excitation wavelength is below 400 nm, and it is nonfluor-
escent. If we could modify the pHP group to obtain
a fluorescent phototrigger that can be excited at wavelengths
greater than 400 nm, while maintaining its salient features, the
resulting phototrigger might exhibit rapid and clean release of
active molecules in the visible wavelength region with strong
fluorescence. This modified pHP group might be a promising
alternative to the existing o-nitrobenzyl,[7,8] coumaryl-
methyl,[9] and quinoline[10] derivatives for the design of
photoresponsive drug-delivery systems (DDSs).
Excited-state intramolecular proton transfer (ESIPT)
processes have generated much attention because of their
desirable properties,[1] such as a fluorescence band with
a large Stokes shift, a low inner-filter effect, and low self-
quenching. Excited-state intramolecular proton transfer is an
ultrafast enol–keto phototautomerization process occurring
on the excited-state surface of many intramolecularly H-
bonded molecules. This process leads from an initial enol
form to its phototautomeric keto form or vice versa
[Scheme 1, Eq. (b)]. As a result, these chromophoric systems
give rise to dual fluorescence emission: fluorescence origi-
nating from the enol form and fluorescence with a large
redshift from its tautomeric keto form. Because of these
interesting fluorescence properties, a variety of applications
have been explored in major areas, such as laser dyes,
photostabilizers,[11] radiation scintillators, luminescent mate-
rials,[12] molecular probes,[13] sensing ions,[14] and photoswitch-
ing of polymorphs.[15] Among the various ESIPT molecules, 2-
(2’-hydroxyphenyl)benzothiazole (HBT) has been a com-
pound of great interest because of its very fast and efficient
ESIPT effect. The HBT moiety also shows dual emission,
which is highly sensitive to solvent polarity and the
pH value.[16]
T
he p-hydroxyphenacyl (pHP) group is an efficient photo-
trigger for the study of very fast biological processes, such as
the release of neurotransmitters and secondary messengers,[2]
enzyme switches, and the release of nucleotides.[3,4] On
excitation, cleavage of the pHP group proceeds efficiently
through four steps:[5] I) formation of the triplet intermediate,
II) deprotonation of the phenolic group, III) bond reorgan-
ization to a putative spirodienedione (Favorskii intermedi-
ate), and IV) hydrolytic ring opening of the spirodiketone to
yield p-hydroxyphenylacetic acid [Scheme 1, Eq. (a)]. The
pHP group has been used extensively to cage biomolecules
owing to its salient features:[6] I) rapid and clean release,
II) high photochemical efficiency, III) synthetic accessibility,
IV) ready installation on most substrates, and V) the forma-
tion of a transparent (no inner-filter effect) biocompatible
photoproduct. Despite these unique properties, its use as
a delivery agent in theranostics has remained unexplored,
mainly because of two major limitations of the pHP group: Its
For these and other reasons, we have designed a novel
photoresponsive DDS, designated as p-hydroxyphenacyl–
benzothiazole–chlorambucil (pHP-Benz-Cbl), with a built-in
ESIPT substituent by incorporating the HBT moiety on the
pHP group. This system has a number of advantages: The
excitation wavelength of the pHP group is extended to greater
than 400 nm, and the combined chromophores can act as an
environment-sensitive fluorophore. This fluorophore can
assist in the deprotonation of the phenol moiety of the pHP
group to effect faster release and is now capable of a distinct
fluorescence color change on photorelease, thus leading to
excellent real-time monitoring during rapid delivery of the
anticancer drug [Scheme 1, Eq. (c)].
[*] S. Barman, S. Biswas, S. Nandi, M. Gangopadhyay, A. Anoop,
N. D. P. Singh
Department of Chemistry, Indian Institute of Technology
Kharagpur 721302, West Bengal (India)
E-mail: ndpradeep@chem.iitkgp.ernet.in
S. K. Mukhopadhyay, S. Dey
Department of Biotechnology, Indian Institute of Technology
Kharagpur 721302, West Bengal (India)
Supporting information for this article can be found under http://dx.
doi.org/10.1002/anie.201508901.
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solvents was assigned to 6,
the keto form of pHP-Benz-
Cbl. However, in polar hydro-
gen-bonding solvents, we
observed a red-shifted absorp-
tion maximum at 380 nm,
which corresponds to 5, the
enol form of pHP-Benz-Cbl
(Figure 1a). The existence of
keto–enol tautomers of pHP-
Benz-Cbl and their sensitivity
to the solvent system are due
to the ESIPT effect.[18]
The influence of the
ESIPT process is further
manifested in the emission
behavior of pHP-Benz-Cbl.
In non-hydrogen-bonding sol-
vents (cyclohexane, benzene,
chloroform), only one emis-
sion maximum at 510 nm, cor-
responding to the keto form of
pHP-Benz-Cbl, was observed
(Figure 1b). The existence of
the keto form of pHP-Benz-
Scheme 1. a) Mechanism of the photorelease of the pHP group. b) ESIPT mechanism of HBT. c) Mecha-
nism of the ESIPT-assisted photorelease of the pHP group. ATP =adenosine 5’-triphosphate, GABA=g-
aminobutyric acid.
For the synthesis of pHP-Benz-Cbl, salicylaldehyde (1)
was first converted into compound 2 by Friedel–Crafts
acylation with bromoacetyl bromide according to a previously
reported procedure (Scheme 2).[17] Next, 2 was treated with
the anticancer drug chlorambucil (Cbl) in the presence of
potassium carbonate (K2CO3) in dry dimethylformamide
(DMF) at room temperature for 4 h to afford 3. Finally, the
treatment of 3 with 2-aminothiophenol at reflux for 1 h in
dimethyl sulfoxide (DMSO) yielded our DDS, pHP-Benz-Cbl
(4), in moderate yield (70%). The product obtained in each
step was characterized by 1H NMR, 13C NMR, and FTIR
spectroscopy as well as HRMS (see Figures S1–S8 in the
Supporting Information).
Cbl in non-hydrogen-bonding solvents can be attributed to
ultrafast proton transfer at intrinsically rapid rates from the
hydroxy group of pHP to the nitrogen atom of the benzo-
thiazole moiety (ESIPT). In contrast, in polar aprotic solvents
(ACN, THF) and polar protic solvents (EtOH, MeOH), we
observed two emission maxima, one near 450 nm (enol form)
and another at 515 nm (keto form). The new emission
maximum at 450 nm exhibited by pHP-Benz-Cbl in hydro-
gen-bonding solvents corresponds to the enol form that arises
as a result of the restricted ESIPT process. Furthermore, as we
increased the hydrogen-bond-forming ability of the solvent,
higher fluorescence intensity was observed from the enol
form as compared to the keto form (see Figure S11).
The photophysical properties of pHP-Benz-Cbl are highly
sensitive to the solvent system (Figure 1). An absorption
maximum observed at 350 nm in non-hydrogen-bonding
We examined photorelease from our DDS in the visible
wavelength region by exposing a solution of pHP-Benz-Cbl
(1 ” 10À4 m) in acetonitrile/HEPES (1:19) buffer (20 mL)
to visible light (ꢀ 410 nm) from
a
medium-pressure mercury lamp
(125 W, incident intensity (I0) =
2.886 ” 1016 quantasÀ1) with
a
UV
cutoff filter (1m NaNO2 solution). The
photorelease of Cbl was analyzed by
reversed-phase HPLC with acetonitrile
as the mobile phase at a constant flow
rate (1 mLminÀ1). The HPLC trace
(Figure 2) showed the gradual disap-
pearance of a peak at tR = 9.15 min
corresponding to pHP-Benz-Cbl, thus
indicating gradual photodecomposi-
tion. On the other hand, the appear-
ance and gradual increase in intensity
of two new peaks at tR = 7.20 and
8.20 min indicated the formation of
photoproducts pHP-Benz-COOH (see
Scheme 2. Synthesis of the DDS p-hydroxyphenacyl–benzothiazole–chlorambucil (pHP-Benz-Cbl).
Reagents and conditions: a) bromoacetyl bromide, AlCl3, CH2Cl2, 358C, 15 h; b) chlorambucil,
K2CO3, DMF, room temperature, 4 h; c) 2-aminothiophenol, DMSO, reflux, 1108C, 1 h.
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was entirely responsible for drug release (see Figure S12b).[19]
Furthermore, we found that almost 90% of the drug had been
released from pHP-Benz-Cbl after irradiation for 15 min with
visible light (see Figure S12a).
A possible mechanism for the photochemical release of
Cbl from pHP-Benz-Cbl in aqueous solution is summarized in
Scheme 3. Except for the initial step involving a rapid ESIPT
process and deprotonation, our mechanism is similar to that
suggested by Givens and co-workers.[20] Upon irradiation,
pHP-Benz-Cbl is excited to the singlet state, where it
undergoes rapid ESIPT from the pHP group to the benzo-
thiazole moiety via 7, thus resulting in deprotonation of the
pHP group to give intermediate 8. The zwitterionic form then
passes through efficient intersystem crossing (ISC) to its
triplet excited state, which undergoes photo-Favorskii rear-
rangement to give a putative spirodiketone 10 with the
concomitant release of the drug. The spirodiketone is then
subject to hydrolytic ring opening to yield pHP-Benz-COOH
(11).
When the photolysis of pHP-Benz-Cbl was carried out in
the presence of the triplet quencher potassium sorbate
(50 mm), drug release by pHP-Benz-Cbl was completely
arrested, thus clearly indicating that the photorelease occurs
from the triplet excited state (see Figure S13). The higher
photochemical quantum yield (0.46) is only possible if ESIPT
assists the deprotonation process, which further triggers the
formation of the zwitterionic form 9 in the triplet excited state
on exposure to light.
To support the singlet-state ESIPT mechanism, we also
studied the process on the basis of a model system, a p-
hydroxyphenacyl–benzothiazole–anisic acid conjugate (pHP-
Benz-AA; see Figure S14), by using time-dependent DFT
(CAM-B3LYP/6-31 + G (d,p)//CAM-B3LYP/6-31G(d)) with
the Gaussian09 software. We located the transition state on
the S1 surface. The computed activation energy for ESIPT is
9.92 kcalmolÀ1. Proton transfer in the S1 state is exothermic
by À2.50 kcalmolÀ1, and the barrier for the reverse process is
12.42 kcalmolÀ1. On the other hand, proton transfer in the
ground state is unlikely, as it is endothermic by 9.96 kcal
molÀ1; moreover, the reverse process is nearly barrierless. The
subsequent ISC is expected to be more favorable from the S1
state of pHP-Benz-AA K than
Figure 1. a) Absorption and b) emission spectra of the DDS pHP-Benz-
Cbl in different solvents. ACN=acetonitrile, DCM=dichloromethane.
Figures S9 and S10) and Cbl, respectively. The quantum yield
for the photorelease of the drug was calculated as 0.46 (see
Table S1 in the Supporting Information).
To demonstrate the precise control over the photolytic
release of the anticancer drug Cbl, we exposed pHP-Benz-Cbl
to light and dark conditions separately, and found that light
from pHP-Benz-AA, because
the S–T gap (DES1–T2) gap is
smaller (2.40 kcalmolÀ1) after
proton transfer than before
(DES1–T3 = 6.94 kcalmolÀ1). The
mechanism of the next step after
the ISC, the photo-Favorskii rear-
rangement in the triplet state, is
well-known[21] and was not calcu-
lated. Our computations support
singlet-state ESIPT followed by
ISC and triplet-state photo-Favor-
skii rearrangement.
Interestingly, we noted a dis-
tinctive fluorescence color change
from green to blue during the
photolysis of pHP-Benz-Cbl
Figure 2. Overlay of HPLC chromatograms of the DDS pHP-Benz-Cbl at regular time intervals of
irradiation with visible light (ꢀ410 nm). The y axes are offset by 10 mAU and the x axes are offset by
15 s to facilitate visualization. AU=arbitrary units.
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time increased. We also observed
a decrease in steady-state fluo-
rescence intensity[23] (see Fig-
ure S16) with an increase in irra-
diation time.
Real-time monitoring of drug
release by our DDS (pHP-Benz-
Cbl) inside malignant neoplastic
disease cells (MDA-MB 231) was
analyzed by confocal microscopy
(Figure 4). Initially, the cells
showed green fluorescence due
to cellular uptake of pHP-Benz-
Cbl (see Figure S17). After expo-
sure to visible light (ꢀ 410 nm)
for 10 min, we observed both
Scheme 3. Possible photorelease mechanism of the DDS pHP-Benz-Cbl.
(Figure 3a). The emission spectrum (Figure 3b) showed only
a green-emission band corresponding to pHP-Benz-Cbl
(lmax = 517 nm) at 0 min. As the irradiation time gradually
increased (0–15 min), the intense green-emission band grad-
ually decreased, whereas a new blue-emission band at 450 nm
corresponding to the photoproduct pHP-Benz-COOH grad-
ually increased in intensity. The blueshift in the fluorescence
spectrum of the photoproduct, pHP-Benz-COOH, is due to
the disruption of conjugation (from a phenolic hydroxy group
to a carbonyl group). A time-resolved fluorescence-decay
curve[22] (see Figure S15) showed that the fluorescence life-
time (see Table S2) gradually decreased as the irradiation
Figure 4. Confocal images of the cellular internalization of pHP-Benz-
Cbl: 1(a) bright-field image, 1(b) fluorescence image, and 1(c) overlay
of bright-field and fluorescence images; and real-time monitoring
(fluorescence images at different times during photoirradiation) of the
release of the anticancer drug from pHP-Benz-Cbl on visible-light
irradiation (ꢀ410 nm): 2(a) 0 min, 2(b) 10 min, and 2(c) 15 min.
green and blue fluorescence, thus indicating the partial
release of Cbl by our DDS (pHP-Benz-Cbl). Finally, after
irradiation for 15 min, we noted a complete fluorescence
color change from green to blue (Figure 4, 2 (c)), thus
suggesting a greater extent of photorelease of Cbl. We also
evaluated in vitro drug release by dialysis. We calculated the
percentage of drug release[24] as a function of the change in
fluorescence intensity (see Figure S18).
We examined the cytotoxicity of pHP-Benz-Cbl, Cbl, and
Phe-Benz-COOH in vitro by using the MTT[25] assay [MTT=
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bro-
mide, a yellow tetrazole] in the MDA-MB-231 cell line. Cell
viability remained above 90% at different concentrations of
pHP-Benz-Cbl and pHP-Benz-COOH, whereas increasing
cytotoxicity was observed on the addition of increasing
amounts of Cbl (see Figure S19a). For the light-exposure
experiment, in cells incubated with different concentrations
of pHP-Benz-Cbl, Cbl was released on irradiation for 15 min
with visible light (ꢀ 410 nm), thereby causing toxicity in
Figure 3. a) Jablonski diagram for the DDS pHP-Benz-Cbl and photo-
product pHP-Benz-COOH. b) Emission spectra of the DDS as the
irradiation time is gradually increased (0–15 min).
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On the other hand, no significant cell death was observed
when cells were irradiated in the presence of pHP-Benz-
COOH, thus indicating that the cytotoxicity was probably
caused by the released drug. In comparison, with the same
concentration of chlorambucil to that of pHP-Benz-Cbl, pHP-
Benz-Cbl showed much lower cytotoxicity than chlorambucil.
However, upon irradiation, pHP-Benz-Cbl showed enhanced
cytotoxicity towards cancer cells in comparison to Cbl
because of the efficient photorelease of Cbl inside the cells.
Furthermore, the highest level of toxicity of pHP-Benz-Cbl
towards MDA-MB-231 cells was observed for an irradiation
time of 15 min (see Figure S19c).
In conclusion, ESIPT-assisted photorelease by the pHP
group has been demonstrated. We have utilized the photo-
removable pHP protecting group for the first time as
a delivery vehicle for a chemotherapeutic agent. The pHP
protecting group was designed as an environment-sensitive
fluorophore, and the excitation wavelength was extended to
the visible wavelength region (ꢀ 410 nm). The dramatic
fluorescence color change (green to blue) upon the efficient
photorelease of the drug demonstrate the excellent photo-
responsive DDS ability of the molecule pHP-Benz-Cbl and its
outstanding properties for real-time monitoring.
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real-time monitoring · theranostics
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Received: September 25, 2015
Revised: January 16, 2016
Published online: February 25, 2016
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