Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Push−Pull Stilbene: Visible Light Activated Photoremovable Protecting Group for Alcohols and Carboxylic Acids with Fluorescence Reporting Employed for Drug Delivery Amrita Paul,† Angana Biswas,‡ Sreyashi Sinha,† Sk. Sheriff Shah,† Manoranjan Bera,† Mahitosh Mandal,‡ and N. D. Pradeep Singh*,† Department of Chemistry, ‡School of Medical Science and Technology, Indian Institute of Technology Kharagpur, 721302 Kharagpur, West Bengal, India
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†
S Supporting Information *
ABSTRACT: For the first time we have utilized push−pull stilbene as a visible light activated photoremovable group (PRPG) for the uncaging of alcohols and carboxylic acids. The PRPG efficiently release caged molecules photochemical quantum yield. It is capable of monitoring the release in real time owing to its fluorescence phenomenon upon photorelease in polar medium. The efficient photorelease and real time monitoring abilities of stilbene were employed for in vitro drug delivery.
P
electron-donating and -accepting group at the para positions of stilbene, forming a donor−π-acceptor system, popularly known as push−pull stilbenes drive their absorption in the visible region of the electromagnetic spectrum due to the charge transfer process (Figure 1a).22,23 The photocyclization of diarylethenes like stilbenes is among the most extensively studied photochromic reactions. Stilbenes undergo cis−trans isomerization on light irradiation followed by photochemically allowed conrotatory 6π electrocyclization to form highly unstable intermediate trans-dihydrophenanthrene. The intermediate then undergoes oxidative dehydrogenation to yield phenanthrene. But if a leaving group is introduced at the point of cyclization, then stilbene derivatives undergo photocyclization to form phenanthrene through a thermally driven spontaneous nonoxidative elimination process (Figure 1b).24−28 Although stilbene to phenanthrene photocyclizations by both oxidative and nonoxidative paths were quite wellknown, use of stilbene as a PRPG for uncaging active molecules has not been pursued. Therefore, herein we utilized photocyclization of push−pull stilbene for the first time to develop a new visible light activable PRPG for the uncaging of alcohols and carboxylic acids (Figure 1c). The newly designed PRPG was based on trans-4-(N,N-dimethylamino)-4′-nitrostilbene (DANS) with a
hotoremovable protecting groups (PRPGs) are the chemical moieties that cage active molecules through covalent linkage and uncage them on demand upon light irradiation. Light offers precise spatiotemporal control and subepidermal penetration that led to extensive utilization of PRPGs in the field of drug delivery1 and tissue engineering.2 Most widely utilized PRPGs for biological studies are nitrobenzyl,3 phenacyl,4 benzoyl,5 quinolinyl,6 anthracenyl,7 and coumariny8 systems. In the literature fluorogenic PRPGs, such as cinnamates9 and thiochromone S,S-dioxide,10 are explored as fluorescent reporters. With only a few exceptions these PRPGs utilize UV light for the photouncaging of active molecules.11−15 Now researchers are interested in developing PRPGs that can be activated by visible and near-infrared (NIR) light to avoid the complications of cytotoxicity and poor penetration associated with UV light.16 Recently boron− dipyrromethene (BODIPY),17,18 benzoquinone,19,20 and cyanine21 based visible and NIR light activated PRPGs were developed. But these PRPGs do not have real time monitoring ability, which is a very convenient tool for monitoring and quantifying the release of active molecules in biological systems. So there is an urgent need to develop a new visible light activated PRPG with self-contained real time monitoring ability for the release of caged active molecules. The search for a new visible light activated PRPG has directed our attention toward a diarylethene system like stilbene. As it has been observed that the presence of an © XXXX American Chemical Society
protecting with good “turn on” push−pull
Received: January 10, 2019
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DOI: 10.1021/acs.orglett.9b00124 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Figure 2. (a) Absorption and (b) emission spectra of caged stilbene 2c in different solvents.
The caged stilbene molecules exhibited a solvent-dependent fluorescence property. It was observed that, in a nonpolar solvent (hexane), the fluorescence intensity of 2c was comparatively higher with an emission maximum (λf) at 500 nm. Whereas in polar solvents (e.g., tetrahydrofuran, methanol, tetrahydrofuran, etc.), it exhibited comparatively lower fluorescence intensity along with a red-shifted emission maximum (λf) in the range 570−675 nm (Figure 2b). In polar solvents, twisted intramolecular charge transfer (TICT), a nonradiative decay process from the excited state, was more favorable, due to the fact that the fluorescence of caged stilbene (2c) became quenched in polar solvents.22 The absorption maximum (λabs), emission maximum (λf), molar absorptivity (εmax), Stokes shift, and fluorescence quantum yield (Φf) of all the caged stilbenes (2a−e) in different solvents are summarized in Table S1. For the determination of the fluorescence quantum yield of caged stilbene compounds in different solvents, a N2-outgassed solution of coumarin 334 (Φf = 0.69 in methanol)29 was used as a standard, with solvent refractive index correction. To study the photorelease ability of the caged alcohols and carboxylic acids from our newly developed stilbene based PRPG (2a−e), we irradiated the nitrogen gassed solution of caged stilbene 2a−e (1 × 10−4 M) in acetonitrile individually under visible light (≥410 nm) from a medium-pressure mercury lamp (125 W), incident intensity (I0) = 2.886 × 1016 quanta s−1, with a UV cutoff filter (1 M NaNO2 solution). The photolysis was monitored by reversed-phase (RP) HPLC analysis, mass spectrometry (see Figure S12 in SI), 1H NMR spectroscopy (see Figure S13 in SI), and absorption spectroscopy (see Figure S15 in SI). The chemical and quantum yield (Φp) of uncaging of the caged stilbenes (2a−e) in acetonitrile, to release respective alcohols and carboxylic acids, are depicted in Table 1. The photochemical quantum yield (Φp) was calculated by using potassium ferrioxalate as an actinometer30 (see Supporting Information for further details). Photolysis of caged stilbenes 2a−e (1 × 10−4 M) in nitrogen gassed acetonitrile were also performed under a 23 W CFL lamp, and 80−87% uncaging was observed after 6 h of irradiation (see Table S2 in SI). Caged stilbene 2d was used as a representative example to monitor the release of a carboxylic acid by RP-HPLC with acetonitrile/water (9:1) as the mobile phase at a constant flow rate (1 mLmin−1). The RP-HPLC profile (Figure 3) shows a gradual depletion of the peak at retention time (tR) 7.40 min with an increase in irradiation time, indicating the photodegradation of caged stilbene 2d. At the same time, the appearance and gradual increase in the intensity of two new peaks at tR 3.41 min and tR 3.16 min indicated the formation of photoproduct N,N-diethyl-7-nitrophenanthren-3-amine (5) and the release of anisic acid upon photolysis. The release of
Figure 1. (a) Visible light absorbing push−pull stilbene; (b) photocyclization of stilbene to form phenanthrene by a nonoxidative elimination process; (c) visible light activated release of caged molecules from the push−pull stilbene based PRPG.
leaving group (caged molecule) at the point of cyclization.27,28 DANS exhibited a solvent-dependent fluorescence property.22 It was almost nonfluorescent in the polar medium due to twisted intramolecular charge transfer (TICT), but after the formation of phenanthrene upon photorelease of caged molecules, it showed blue fluorescence. This fluorescence “turn-on” phenomenon upon photorelease displayed by our developed PRPG was exploited for monitoring the release of caged molecules and anticancer drug chlorambucil (cbl) in real time. The desired stilbene based PRPGs with corresponding caged molecules viz. alcohols (2a, 2b), carboxylic acids (2c, 2d), and drug (cbl) (2e) were synthesized according to the Scheme 1. Scheme 1. Synthesis of Caged Stilbene PRPGs (2a−e)
Wittig reaction between O-protected 4-(diethylamino)-2hydroxybenzaldehyde (1a−e) and (4-nitrobenzyl)triphenylphosphonium bromide in the presence of t-BuOK in THF in reflux conditions yielded our desired caged stilbene PRPGs (2a−e). Products obtained were characterized by 1H NMR, 13 C NMR, and HRMS (Figures S1−S10 in the Supporting Information (SI)). The photophysical properties of the caged stilbene 2c were investigated. The UV/vis absorption spectrum of a degassed 10−5 M solution of 2c was recorded in different solvents (Figure 2a). It was observed that, in nonpolar solvents like hexane caged stilbene, 2c exhibited an absorption maximum (λabs) at 410 nm and, in polar solvents like acetonitrile, tetrahydrofuran, methanol, etc., it becomes red-shifted to 430 nm. The absorption of caged stilbene 2c in the visible region was attributed to the charge transfer process due to the presence of an electron donor N,N diethyl and acceptor −NO2 group at the para positions of the stilbene molecule (2c). B
DOI: 10.1021/acs.orglett.9b00124 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
period of 20 days. The caged stilbenes were studied using 1H NMR. We observed only 3−6% decomposition of the caged stilbenes (Table S4 in SI). The aqueous solubility of the 2e is found to be 12 mg/100 mL at 25 °C. Interestingly our designed stilbene based PRPG showed a distinct fluorescence “turn-on” phenomenon on photorelease of active molecules. At 0 min of irradiation, 2c in acetonitrile is nonfluorescent, but with the increase in irradiation time, a steady increase in the emission intensity at 450 nm was observed. After 60 min of irradiation, it was observed that the solution of 2c in acetonitrile changed from almost nonfluorescent to blue fluorescent (Figure 4). This phenomenon was observed due to the formation of the phenanthrene derivative as a photoproduct.
Table 1. Photochemical Data of Caged Stilbene Compounds (2a−e)a
a
% of uncaging from caged stilbenes was determined by RP-HPLC; photochemical quantum yield of decomposition of starting material, Φp, calculated using potassium ferrioxalate as an actinometer. Error limit: ±5%.
Figure 4. Time-dependent fluorescence spectra for photorelease of 2c.
From the literature study,27,28,31 we proposed a mechanism of photorelease of caged carboxylic acids and alcohols from our designed PRPG taking 2c as a representative example (Scheme 2). According to our proposed mechanism, the photorelease of Scheme 2. Possible Mechanism of Photorelease of Acetic Acid from Caged Stilbene 2c
Figure 3. RP-HPLC chromatograms of 2c at regular time intervals on irradiation with visible light (≥410 nm). AU = arbitrary units.
anisic acid was confirmed by coinjection of standard anisic acid, and the peak at tR 3.41 min was determined as phenanthrene derivative 5 from 1H NMR (see Figure S14 in SI) and HRMS analysis (see Figure S12 in SI) after isolating it (see Figure S11 in SI). The alcohol release ability of our PRPG was studied by 1H NMR spectroscopy using caged stilbene 2a as a representative example in acetonitrile d3 (see Figure S13 in SI). The quantum yield for the formation of phenanthrene photoproduct (5) was calculated (see Table S3 in the SI). To examine whether the photorelease from our stilbene based PRPG was dependent on the solvent system, we studied the photolysis of caged stilbene 2c (1 × 10−5 M) in three different types of solvents viz. hexane (nonpolar), acetonitrile (polar aprotic), and water containing 0.001% DMSO (polar protic) under visible light (≥410 nm). The photolysis was monitored by absorption spectroscopy (see Figure S16 in SI), and photodecomposition of 2c was observed in all three solvent systems after 1 h of irradiation. The hydrolytic stability of the caged stilbenes (2a−e) were examined individually in acetonitrile/water (1:1 v/v) solvent (of pH = 7) by keeping them under dark conditions for a
caged acetic acid from 2c occurred through the following steps: (i) upon irradiation, the caged stilbene 2c becomes excited to its singlet excited state (2c*); (ii) from the excited state the trans-stilbene isomer undergoes cis−trans photoisomerization to form a cis-stilbene isomer (3c); (iii) then 3c becomes re-excited to the singlet state (3c*); (iv) 3c* via a conrotatory, 6π electrocyclic ring closure affords the trans isomer of a dihydrophenanthrene derivative (4c); (v) the tricyclic polyene (4c) is unstable (non aromatic), and therefore it spontaneously eliminates the leaving group (acetic acid), producing stable phenanthrene, 5. To validate the cyclization of cis caged stilbenes proceeds only under light, we carried out the cyclization of 3d under both light and dark conditions and C
DOI: 10.1021/acs.orglett.9b00124 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters observed that the cyclization leading to uncaging occurred only in the presence of light (see Figure S17 in SI). The rate constant of the dark step was 7.1 × 103 s−1 (Figure S18a and b). The newly developed stilbene based PRPG was further employed as a photoinduced drug delivery system (DDS) for the on-demand release of anticancer chlorambucil (cbl). The photorelease of cbl from the DDS (2e) was investigated by irradiating the solution of 2e (1 × 10−4 M) in acetonitrile/ HEPES (1:19) buffer under visible light (≥410 nm). The photolysis was monitored by RP-HPLC (Figure S19 in SI) with acetonitrile/water (9:1) as the mobile phase at a constant flow rate (1 mLmin−1). It was observed about 75% of the drug was released after 1 h of irradiation with a quamtum yield of 0.02. Finally, we demonstrated the real-time monitoring of the drug release by our DDS (2e) inside the MCF-7 breast cancer cell line by confocal microscopy (Figure 5). Initially, the cells
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
N. D. Pradeep Singh: 0000-0001-6806-9774 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank DST SERB (Grant No. EMR/2016/005885) for financial support and DST-FIST for 600 and 400 MHz NMR. A.P. is thankful to IIT Kharagpur for a fellowship.
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REFERENCES
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Figure 5. Confocal images of real time monitoring of the drug release from 2e at different times during photoirradiation: (a) 0 min, (b) 15 min, and (c) 30 min.
were nonfluorescent, and after exposure to visible light (λ ≥ 410 nm) for 30 min, we observed a gradual increase in blue fluorescence after the release of the caged drug (cbl) (Figure 5). So this “turn on” fluorescence after the release of drug helped in monitoring the drug release in real time. We also examined the cytotoxicity of the caged stilbene (2a) and DDS (2e) in the MCF-7 breast cancer cell line by using the MTT assay32 [MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, a yellow tetrazole] before and after light irradiation. It was observed that, before photolysis, the cell viability of 2a and 2e remained more than 75% at 100 μM concentration (Figure S20a in SI). After 30 min of irradiation, 2a exhibited above 60% viability (IC50 at 230 μM) and DDS (2e) 15% viability (IC50 at 230 μM) individually (Figure S20b in SI). Thus, the efficient anticancer activity of our DDS 2e was validated by the MTT assay. In conclusion, we have developed a new visible light activable PRPG based on push−pull stilbene for the release of caged alcohols and carboxylic acids. The stilbene based PRPG showed efficient photorelease ability in both polar and nonpolar solvents with good chemical and quantum yields. Our developed PRPG exhibited a fluorescence “turn on” phenomenon upon photorelease in polar medium which was utilized for real time monitoring of the release. The efficient release and real time monitoring ability of our PRPG was further employed for anticancer drug delivery in vitro.
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Experimental procedures and copies of NMR, HRMS, RP-HPLC, and UV−vis spectra (PDF)
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00124. D
DOI: 10.1021/acs.orglett.9b00124 Org. Lett. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.orglett.9b00124 Org. Lett. XXXX, XXX, XXX−XXX
