A Doubly Caged Bis(salicylamide)-Based Anion Transporter for Phototriggered Breast Cancer Therapy

Abstract

Formulation of ion transporters, which hold great potential for anticancer therapy, as prodrugs/protransporters can overcome their nontarget activity, allowing for local activation using specified stimuli. Photocleavable protecting groups offer a robust strategy for caging transporter activity. Their reliance on light as a stimulus ensures ease of application and precise spatiotemporal control. Thus, there is a need to develop protransporters that are activatable in the biologically benign visible region of the electromagnetic spectrum. Herein, we report a series of bis­(salicylamide)-based anion antiporters caged with o-nitrobenzyl groups to create protransporters that can be activated by 405 nm light. Active transporter release via two-step photocleavage and the subsequent photoinduced recovery of ion transport activity was verified. In vitro photoactivation of protransporters using 405 nm light efficiently induced cell death in MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines.

Introduction

Synthetic ion transport systems can induce apoptotic cell death in cancer cells due to their ion homeostasis-disrupting ability , but have limited therapeutic application due to their undesirable toxicity toward healthy nonmalignant cells, − a drawback they share with traditional broad-spectrum drugs. , To overcome this, ion transporters can be formulated as prodrugs or "protransporter" by conjugation to a labile group that inhibits ion transport, which can be regained in the presence of an internal or external stimulus, allowing for localized activation. The high level of spatiotemporal control offered by light and its benign nature make it an ideal external trigger for use in biological systems, − and hence suitable for protransporter activation in cancer tissue. Most existing reports of photoactivated protransporters used UV light, − which is harmful to surrounding healthy tissue and has low penetration. , There is a need to shift to visible light for protransporter activation, given its nontoxicity and better tissue-penetrating properties. , This can be achieved using photocleavable protecting groups (PPGs), which are removable in the visible region of the electromagnetic spectrum. Reports of such systems are few in number and better systems with faster activation times need to be developed. The ortho-nitrobenzyl (ONB) group is a highly versatile PPG whose activation wavelength can be tuned by varying the phenyl ring substitution. It was thus chosen for the preparation of the protransporter.

In a previous report, we demonstrated the salicylamide system as an effective ion transporter, where the OH group played a key role in the transport process. Based on the predicted structure of the ion-transport complex in the salicylamide system, a dipodal, preorganized bis­(salicylamide) system was designed (Figure ). The bis­(salicylamide) core has so far been used only as a ligand for metal coordination − but has potential for anion binding and, in light of our previous observation, can form efficient transmembrane ion transporters. Here, tuning of both −OH pK _a and molecular logP is possible through variation of substitution of the salicyl ring, and caging of ion transport activity can be achieved by attachment of ONB moieties to the OH groups. A 405 nm light source was chosen for photoactivation, as substituted ONB groups are known to absorb in this spectral region. Advances in optical fiber technology, combined with the internal application of light through minimally invasive surgical procedures, , have made the use of the shorter-wavelength regime of the visible spectrum viable for therapeutic applications.

1.

Design of bis­(salicylamide) transporters and protransporters.

Results and Discussion

Synthesis

The bis­(salicylamide) transporters (1a–1e) were synthesized starting from the respective substituted salicylic acid derivatives (4a–4e) (Scheme ). Methylation of salicylic acid OH and COOH groups was conducted using iodomethane, followed by base hydrolysis of the resultant ester. The resulting methylated salicylic acid (6a–6e) was coupled to o-phenylenediamine using EDC·HCl/HOBt to form the amide (7a–7e), followed by demethylation of the OMe groups using BBr₃ to yield the described transporter derivatives (1a–1e). The protransporters 2 and 3 were synthesized by linking the corresponding ONB moieties to transporter 1c by reaction of their benzyl bromide derivatives (8a–8b) in the presence of K₂CO₃ as the base. The photocleavage controls 9 and 10 were synthesized as per a previous report.

1. Synthesis of Bis­(salicylamide) Transporters (1a–1e), Protransporters (2–3), and Photocleavage Controls (9–10).

Transporter Activity Screening

The ion transport abilities of the transporters 1a–1e were tested in egg yolk phosphatidylcholine (EYPC) large unilamellar vesicles (LUVs) entrapping the pH-sensitive dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), i.e., EYPC-LUVs⊃HPTS (Figure S1), across whose membrane a pH gradient was applied (pH_in = 7.0, pH_out = 7.8). Data from dose-dependent studies of 1a–1e (Figure S2–S6) were fitted to the Hill equation to yield the EC ₅₀ values (Table ), which showed the order of transport activity to be 1e > 1c > 1d > 1b ≫ 1a. The electron-withdrawing CF₃ and halogen (Br, Cl) substituted bis­(salicylamides) (1e, 1c, 1d) were observed to exhibit higher ion transport activities, where the activity increased with an increase in logP values of the transporters (5.31, 5.09, and 4.76, respectively). The derivative 1b, though having the highest logP value (5.53) due to the presence of the naphthyl group, exhibited significantly lower ion transport activity.

1. Summary of LogP calculations, Dose-dependent Studies, and Cancer Cell (MCF-7) Cytotoxicity.

compound	logP	EC 50 ,	n	cell viability
1a	3.55	15.86	1.30	63%
1b	5.53	6.84	1.40	50%
1c	5.09	2.00	1.31	35%
1d	4.76	2.08	1.14	39%
1e	5.31	1.87	1.01	59%

^aValues calculated using MarvinSketch logP plugin.

^b EC ₅₀ and Hill coefficient (n) determined across EYPC-LUVs⊃HPTS.

^cUnit: μM.

^dCell viability (%) in MCF-7 cells following 24 h treatment with 50 μM transporter.

Hill coefficients (n) of transporters were found to be ∼ 1, indicating the formation of an ion transport complex by one molecule of the transporter and one ion. The cytotoxicity of transporters 1a–1e was assessed in the MCF-7 breast cancer cell line (Figure S25A), where treatment at 50 μM concentration yielded activity of the order 1c > 1d > 1b > 1e > 1a (Table ). The Br-substituted transporter 1c was observed to exhibit the highest cytotoxicity despite being only the second most active ion transporter as assessed in EYPC liposomes. This was likely due to differences in lipid membrane composition between the cancer cell and the liposome. Further studies were conducted using transporter 1c since it was most suitable for anticancer applications. The IC ₅₀ of 1c in MCF-7 was determined to be 28.7 μM (Figure S26A).

Mechanism of Ion Transport

To elucidate the ion transport mechanism of the bis­(salicylamide) system, the transport activity of 1c (3 μM) was assessed across EYPC-LUVs⊃HPTS while varying the anions (Cl^–, Br^–, I^–, NO₃ ^–, ClO₄ ^–, AcO^–) in the extravesicular buffer (Figure S7). A significant difference in ion transport ability was observed with this variation (Figure A), indicating that the transport process is anion-dependent. Hence, the transporter was tested across EYPC-LUVs entrapping lucigenin (a Cl^– sensing dye), i.e., EYPC-LUVs⊃lucigenin (Figure S8), where a Cl^– gradient was generated across the lipid membrane. The dissipation of this gradient by 1a–1e was observed with the order of activity 1e > 1c > 1d > 1b > 1a (Figure S9), which was in agreement with the observations across EYPC-LUVs⊃HPTS. Dose-dependent studies of 1c yielded n ≈ 1 (n = 1.34, EC ₅₀ = 3.66 μM) (Figure S10), consistent with data from the HPTS assay. This was cross-verified using 1e, which yielded n = 1.13 and EC ₅₀ = 2.15 μM (Figure S11). Variation of extravesicular cations (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺) in EYPC-LUVs⊃lucigenin showed no change in ion transport activity of 1c (5 μM) (Figure B), confirming a cation-independent mechanism of ion transport. The chloride transport ability of 1c was significantly enhanced in the presence of valinomycin (K⁺ transporter), indicating an anion antiport mechanism of transport (Figure C). The carrier mechanism of transport by the bis­(salicylamide) was verified through the classical U-tube experiment (Figure D). , Transport of chloride from the source arm (0.5 M NaCl) to the receiver arm (0.5 M NaNO₃) through bulk CHCl₃ in the presence of transporter 1c (1 mM) was observed, indicating a carrier mode of anion transport. A dye leakage assay was performed across EYPC-LUVs⊃carboxyfluorescein (CF) to probe for the possibility of lipid membrane disruption by the transporters (Figure S14). Negligible CF leakage was observed on treatment with 1c and 1e up to 20 μM concentration, indicating that liposomal membrane integrity is preserved during the transport process (Figure S15). The chloride ion transport ability of the transporter 1c was also verified in MCF-7 cells using the fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), which undergoes fluorescence quenching with increasing chloride concentration. , A time-dependent decrease in fluorescence intensity of the dye was observed in cells treated with transporter 1c at 10 and 25 μM concentrations, indicating transporter-induced chloride influx (Figure S27).

2.

(A) Anion selectivity assay of 1c (3 μM) across EYPC-LUVs⊃HPTS. (B) Cation selectivity assay of 1c (5 μM) and (C) valinomycin (Val, 0.5 μM) coupled assay of 1c (3 μM) across EYPC-LUVs⊃lucigenin. (D) Chloride transport across CHCl₃ layer in U-tube experiment in the presence (2 mM) and absence of transporter 1c.

Prediction of Ion Transport Complex Structure

The transporter 1c was titrated with TBACl (Cl^– source) and monitored through ¹H NMR to assess its anion binding ability (Solvent: 13.5% DMSO-d ₆ in CD₃CN). Both N–H (H_a) and O–H (H_b) protons were observed to show downfield shifts along with aromatic C–H protons H_c and H_d (Figure S16). The binding constant was calculated from the titration data using the BindFit program. A fit to the 1:1 transporter-ion binding model was obtained with K _a(1:1) = 205.7 ± 5.3 M^–1. The binding stoichiometry was the same as that observed for the ion transporter complex, where n ≈ 1. The locations of protons involved in the anion binding indicate the presence of more than one ion binding mode, as it is not possible for all four protons to interact with the anion in a single conformation. This was likely due to the salicylamide arms adopting "open-ring" and "closed-ring" conformations (Figure S17). The contributions of both conformations to chloride binding in different binding modes were observed and analyzed in our work on salicylamides. Theoretical calculations were conducted to shed light on the structure of the [1c+Cl^–] complex based on evidence from ion transport and ion titration (NMR) experiments.

Predictions for the most probable geometry of [1c+Cl^–] were carried out with the CONFLEX 8 conformation search software package. , The output yielded a single major conformation (CBr1), with salicylamide arms in the "open-ring" conformation (Figure S19A). The conformer with "closed-ring" salicylamide arms was built using Chem3D, and the chloride ion was positioned in the likely binding site (CBr2) (Figure S19B). The density functional theory (DFT) geometry optimization of these conformers was conducted on Gaussian 09 using B3LYP exchange-correlation functional , and 6–31G­(d,p) basis set. , The structures of the two possible interchangeable conformers of [1c+Cl^–] were obtained. In the "open-ring" CBr1 conformation, the O–H_b and C–H_c protons are involved in anion binding (Figure , S19C), while in the "closed-ring" CBr2 conformation, the N–H_a and C–H_d protons bind to the anion (Figure , S19D). Both structures have similar HF and binding energies, indicating both are equally likely as ion transport complexes and might exist in equilibrium with each other.

3.

Predicted structures of the [1c+Cl^–] ion transport complex and calculated binding energies (BE).

Photoactivation Studies of Protransporters

The photoactivated release of the active transporter 1c from 2 and 3 (DMSO-d ₆ solutions) on photoirradiation was monitored using ¹H NMR (Figure A, S21, S23). The appearance of a new set of peaks, which then disappeared with an increase in photoirradiation time, was observed, which corresponded to the formation of a mono-ONB protected intermediate (2′ , 3′) having one free OH. These results indicate a two-step photorelease mechanism involving the initial cleavage of one ONB group to form intermediate (2′, 3′), which subsequently undergoes a second photoactivation step (Figure B, S22, S24). Overall, with increasing time of photoirradiation, the disappearance of the benzyl CH₂ (H_d) peak was observed along with the appearance of the free OH (H_b) peak. Formation of the CHO (H_c) peak from the o-nitrosobenzaldehyde byproduct (N2, N3) was transiently observed owing to the unstable nature of the molecule.

4.

(A) Change in ¹H NMR spectrum of 3 on irradiation with 405 nm light. Solvent: DMSO-d ₆. (B) Mechanism of two-step photoactivation of 3 on irradiation with 405 nm light to release active transporter 1c and photocleavage byproduct N3. (C) Comparison of ion transport activities of transporter 1c, and protransporters 2 and 3 (5 μM) across EYPC-LUVs⊃HPTS. (D) Recovery of ion transport activity (across EYPC-LUVs⊃HPTS) of 2 and 3 (5 μM) on irradiation under 405 nm light for different time intervals. Activity recovery normalized with respect to the activity of 5 μM 1c at 290 s.

The ion transport activities of protransporters 2 and 3 (5 μM) were tested across EYPC-LUVs⊃HPTS. Both compounds showed negligible ion transport activity relative to the free transporter 1c (Figure C), confirming that the ONB groups successfully caged the activity of the transporter. Absorbance spectra of 2 and 3 were recorded in DMSO, where protransporter 3 was found to have a significant absorbance at 405 nm, while 2 showed only a very mild absorption at that wavelength (Figure S20). Hence, 3 was expected to undergo faster photoactivation under 405 nm light than 2. Following verification of photoactivated transporter (1c) release, activation of ion transport activity upon irradiation with violet light was confirmed. Stock solutions of protransporters 2 and 3 (0.5 mM) in DMSO were irradiated under 405 nm and then tested for ion transport activity across EYPC-LUVs⊃HPTS at a final working concentration of 5 μM. Recovery of ion transport activity was observed with increasing photoirradiation time (Figure D). Transport activity in 3 recovered within 3 min of photoirradiation. This quick release can be attributed to the stronger absorbance of the substituted ONB group at 405 nm. In contrast, 2 took 10 min for complete activation due to the weakly absorbing unsubstituted ONB group.

Study of Photoinduced Anticancer Activity

The protransporters (2 and 3) were tested in MCF-7 cells, where they were found to be inactive under normal conditions; however, upon irradiation with 405 nm light, both compounds exhibited concentration-dependent cytotoxicity (Figure A). Compound 3 showed excellent cytotoxicity within 10 min of photoirradiation, owing to its faster activation time. Compound 2 exhibited only moderate toxicity at 10 min of irradiation but showed much higher activity at 20 min. To assess the effect of the photocleavage byproduct on cells, the same photoactivation experiments were performed using control compounds 9 and 10, which release the o-nitrosobenzaldehyde byproducts (N2, N3) corresponding to those of 2 and 3, respectively, along with the nontoxic methoxyphenol (MP). Compounds 9, 10, and MP exhibited negligible cytotoxicity even at 50 μM following 20 min of irradiation at 405 nm (Figure S28). Photoactivated transporter release-induced cytotoxicity by protransporter 3 was further verified using a flow cytometry-based apoptosis assay with propidium iodide (PI). MCF-7 cells incubated with 3 (25 μM) with and without photoactivation (405 nm, 5 min) were treated with PI, and the percentage of dead cells was assessed through flow cytometry (Figure B, S30–S34). Significant levels of cell death (>80%) were observed in the photoactivated sample, comparable to cells treated with the active transporter 1c (25 μM). The nonirradiated sample resembled the DMSO-treated negative control with little cell death. The PI assay was also performed on DMSO-treated MCF-7 cells irradiated at 405 nm (5 min) to assess direct phototoxicity, and no change was observed compared with the negative control.

5.

(A) MTT assay-based cell viability of MCF-7 cells treated with 2 and 3 followed by 405 nm light irradiation for 0, 10, and 20 min. (B) Assessment of cell death, through flow cytometry-based propidium iodide (PI) assay, in protransporter 3 (25 μM) treated MCF-7 cells with (violet bar) and without (black bar) photoactivation under 405 nm light (5 min) in comparison with transporter 1c (25 μM). Cell death following photoactivation was observed to be statistically significant when compared to the DMSO-treated control and was similar to active transporter 1c. DMSO-treated control cells exhibited negligible differences before and after photoactivation (405 nm, 5 min). (C) Comparison of cell viabilities (via MTT assay) of MCF-7 and MDA-MB-231 cells treated with 2 and 3 (10–50 μM) irradiated under 405 nm light for 10 min. For MTT assays, cells treated with DMSO were set at 100% viability, and data for all compound-treated cells was scaled accordingly. **** represents p < 0.0001, ** represents p < 0.01, and ns represents p nonsignificant when compared to DMSO-treated, unirradiated control.

The bis­(salicylamide) system was also tested in the MDA-MB-231, a triple-negative breast cancer cell line that is known to be resistant to many traditional anticancer drugs. The transporters exhibited good cytotoxicity (Figure S25B), with 1c exhibiting an IC ₅₀ of 27.5 μM (Figure S26B). In vitro photoactivation experiments were performed with the protransporters 2 and 3, and both exhibited significant cytotoxicity within 10 min of photoirradiation at 405 nm (Figure S29A). Protransporter 3 was observed to induce complete cell death even at 10 μM concentration following photoactivation (10 min). Photoinduced cytotoxicity of 2 and 3 toward MCF-7 and MDA-MB-231 cells following 10 min of photoirradiation was compared at 10, 25, and 50 μM of the compounds (Figure C). The bis­(salicylamide) protranporters were found to be more efficient in their phototriggered activity toward the triple negative MDA-MB-231 compared to the MCF-7 cells. The effect of photocleavage byproducts on MDA-MB-231 cells was assessed using controls 9 and 10. Compounds 9, 10, and MP did not exhibit significant cytotoxicity even at 50 μM after 10 min of photoirradiation (Figure S29B).

Conclusions

The bis­(salicylamides) were demonstrated to be efficient transmembrane anion antiporters capable of inducing death in MCF-7 and drug-resistant triple-negative MDA-MB-231 breast cancer cell lines. Protransporters were prepared by caging the OH groups of the transporter with o-nitrobenzyl photocleavable groups. Photoactivation of protransporters was conducted using 405 nm light, following which the release of active transporters and recovery of ion transport activity were verified through ¹H NMR and liposomal assays, respectively. Irradiation of protransporter-treated MCF-7 and MDA-MB-231 breast cancer cells with 405 nm light resulted in extensive cell death (with a superior effect in MDA-MB-231), indicating successful activation in cells. Protransporter (3) activation-induced cell death in MCF-7 cells was also verified by determining the percentage of live-to-dead cells through a flow cytometry-based propidium iodide staining assay. The availability of two fuctionalizable OH groups in the bis­(salicylamides) also allows for the formulation of novel protransporter systems with two different labile caging groups. These can be activated by dual stimuli, which can be used in various combinations suitable for the application of choice.

Experimental Section

Materials and Methods

Reagents and solvents for synthesis were purchased from commercial sources and used without further purification. Lipids and mini-extruder set for vesicle preparation were purchased from Avanti Polar Lipids (Merck). High-power LEDs were procured from Mouser Electronics India, and the associated components were sourced locally. All NMR spectra were recorded on a Bruker Avance III HD 400 MHz NMR spectrometer. The NMR spectra were calibrated with respect to residual solvent peaks. High-resolution mass spectra (HRMS) were recorded on a Waters SYNAPT G2 mass spectrometer equipped with a Waters Z-Spray electrospray ionization source. UV–vis absorption spectra were recorded on a Shimadzu UV-2600 UV–vis spectrophotometer. Fluorescence studies were conducted on a HORIBA Jobin Yvon Fluoromax+ spectrofluorometer with an injector port and a temperature -controlled cuvette holder. Chloride ion measurements were conducted using a Thermo Scientific Orion Chloride ionplus Sure Flow Combination ISE. Absorbance measurements on a 96-well microplate were recorded on a Thermo Scientific Varioskan Flash multimode microplate reader.

Synthesis

Substituted Methyl 2-Methoxybenzoates (5a–5e)

To a stirred suspension of the substituted salicylic acid (1 equiv) and K₂CO₃ (2.5 equiv) in DMF (1 mL per 100 mg substituted salicylic acid) was added MeI (2.42 equiv) and stirred for 18 h at 90 °C. It was then diluted with EtOAc and washed with water. The aqueous layer was further extracted with EtOAc (3x), the organic layers combined, washed with brine, dried over anhydrous Na₂SO₄, and the solvent removed in vacuo.

Substituted 2-Methoxybenzoic Acids (6a–6e)

To a solution of the ester in MeOH (2.7 mL per 100 mg ester) was added 6 N aqueous KOH (26.5 equiv) and stirred for 3 h at rt. MeOH was then removed in vacuo. The mixture was quenched with water (10 mL), followed by conc. HCl until pH ∼ 2. It was then diluted with EtOAc and then washed with water. The aqueous layer was extracted exhaustively with EtOAc, the organic layers combined, washed with brine, dried over anhydrous Na₂SO₄, and the solvent removed in vacuo.

o-Methylated Bis­(salicylamides) (7a–7d)

To a solution of the substituted 2-methoxybenzoic acid (2.2 equiv) in DMF (6 mL per 100 mg o-phenylenediamine) was added o-phenylenediamine (1 equiv), followed by Et₃N (3 equiv) and cooled to 0 °C in an ice bath. Then HOBt (2.6 equiv) was added, and the mixture was stirred at 0 °C for 10 min. Then EDC·HCl (2.6 equiv) was added, and the mixture was stirred at 0 °C for 10 min. The mixture was then allowed to reach rt and stirred for 24 h. It was then diluted with EtOAc and washed with water. The aqueous layer was further extracted with EtOAc (3x), the organic layers combined, washed with brine, dried over anhydrous Na₂SO₄, and the solvent removed in vacuo. The residue was purified by flash column chromatography on silica gel. A variation of this method was used for the synthesis of 7e. Details have been provided in the electronic Supporting Information.

Bis­(salicylamides) (1a–1e)

To a cooled solution (at 0 °C) of substituted methylated bis­(salicylamide) (100 mg, 1 equiv) in DCM (8 mL) was added BBr₃ (1 M in DCM, 5.8 equiv) dropwise. After the addition of BBr₃, the mixture was stirred at rt for 12 h. The progress of the reaction was monitored using TLC. Once the reaction was done, DCM in the reaction mixture was removed, and the crude reaction was cooled in an ice bath, followed by the addition of methanol to quench the excess BBr₃. The formation of a precipitate was observed upon the addition of methanol. The solvent was removed in vacuo, and distilled water was added to the crude reaction mixture. The precipitate was filtered and collected using a Buchner funnel, washed with water, and dried under vacuum in a desiccator for 18 h.

Bis­(salicylamide) Protransporters (2, 3)

To a solution of N,N′-(1,2-phenylene)­bis­(4-bromo-2-methoxybenzamide) in DMF kept in a dark environment was added K₂CO₃ (2.6 equiv) and the substituted benzyl bromide (2.5 equiv), and stirred at 50 °C for 2 h. The progress of the reaction was monitored using TLC. Once the reaction was complete, it was diluted with EtOAc and washed with water. The aqueous layer was further extracted with EtOAc (x3), the organic layers combined, washed with brine, dried over anhydrous Na₂SO₄, and the solvent removed in vacuo. The residue was purified by flash column chromatography on silica gel.

The complete characterization spectra for all compounds are provided in the electronic Supporting Information.

Ion Transport Studies

All ion transport studies, HPTS, lucigenin and CF leakage assays, were performed as per standard reported protocols. Details are provided in the electronic Supporting Information.

U-Tube Experiment

A 2 mM solution of transporter 1c in 1:25 MeOH/CHCl₃ was placed into an upright U-tube along with a stir bar. The whole setup was secured to a magnetic stirrer. Solutions of NaCl (0.5 M) and NaNO₃ (0.5 M) in Milli-Q water were carefully filled into the left and right arms of the U-tube, respectively. The Cl^– concentration in the right arm was measured using a Cl^–-ISE to get the zero-point reading. Then, the stirring was turned on, and the Cl^– concentration in the right arm was measured at determined time points for up to 96 h. The data was plotted as Cl^– concentration (U-tube right arm) vs time. A control experiment was set up without a transporter in the organic layer and monitored in parallel.

NMR Titration Experiment

The ¹H NMR titration study was conducted at ambient temperature on a Bruker Avance III HD 400 MHz spectrometer. The titration with Cl^– ion was conducted by the addition of aliquots of tetrabutylammonium chloride (TBACl) solution (0.5 M, Solvent: 13.5% DMSO-d ₆ in CD₃CN) to a solution of receptor 1c (0.005 M, Solvent: 13.5% DMSO-d ₆ in CD₃CN). The spectra were calibrated using the residual signal from CD₃CN (δ_H = 1.94 ppm) as an internal reference. TBACl was dried under vacuum in a desiccator prior to use. The data was fitted using BindFit software.

Theoretical Calculations

Density functional theory (DFT) geometry optimization and energy calculations were conducted using the Gaussian 09 program suite. B3LYP exchange-correlation functional and 6–31G­(d,p) basis set were used. The input structures for optimization were generated using CONFLEX 8 conformation search software package , using MMFF94S (2010–12–04HG) force field or manually using Chem3D.

Photoactivation Studies

Photocleavage studies of the protransporters were conducted by placing a solution of the sample in a cuvette/NMR tube approximately 1 cm from the 405 nm LED (∼3.8 W radiant flux) and irradiating for the specified time.

NMR Analysis of Photocleavage

Solutions of the protransporters 2 and 3 (0.5 mL, ∼ 2 mg/mL) in DMSO-d ₆ were prepared in borosilicate glass NMR tubes (Norell), and the ¹H NMR spectra (32 scans) were recorded. The tubes were then irradiated with 405 nm light for specified time periods, and the ¹H NMR spectra were recorded. ¹H NMR spectrum of compound 1c was recorded under similar conditions for comparison.

Photoactivated Ion Transport

Stock solutions of the protransporters 2 and 3 (0.5 mM) in DMSO were taken in 1 mL quartz cuvettes and irradiated with 405 nm light for specified time periods. The transport activities of the samples were then assessed across EYPC-LUVs⊃HPTS by the addition of 20 μL of the irradiated stocks at the 100 s time point of the assay. Transporter 1c (5 μM working concentration) was used as the positive control and DMSO as the blank.

Cell Lines and Cell Culture

The human breast cancer cell lines MCF-7 and MDA-MB-231 were used for studies. All cell lines were cultured in the presence of high-glucose Dulbecco’s Modified Eagle media (DMEM; Lonza) containing 2 mM l-glutamine. The media was supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; ThermoFisher Scientific), 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in tissue-culture treated 60 mm dishes at 37 °C in a humidified 5% CO₂ incubator (Thermo Scientific).

Assessment of Cellular Viability

Cellular viability for MCF-7 and MDA-MB-231 cell lines were assessed in the presence of the required concentrations of the compounds using MTT assay. Cellular viability was expressed as a percentage of viable cells for each treatment when compared to the negative control (set as 100%).

MQAE Assay ,

MCF-7 cells were cultured in L-15 medium in black 96-well plates and treated with MQAE dye for 4.5 h. The media was then discarded and fresh L-15 containing transporter 1c (10 and 25 μM concentrations) was added. The dye fluorescence was recorded (λ_ex = 350 nm, λ_em = 460 nm) at different time points (0, 15, 30, and 45 min) on a Revvity EnSight multimode plate reader. Data was plotted as the drop in fluorescence counts over time, relative to the 0 min measurement.

Photoactivation of Compounds in Cells

Cells cultured in L-15 medium in a 96-well plate were treated with the compounds, and the plate was irradiated for the specified time at 405 nm with a high-power LED. The cell viability was assessed following 24 h incubation (37 °C, 5% CO₂) via MTT assay.

Apoptosis Assay (Flow Cytometry)

MCF-7 cells were dispersed in 6-well tissue culture-treated plates (VWR) in complete DMEM at a density of 105 cells/well (per 1.5 mL) and incubated at 37 °C in a 5% CO₂ incubator for 36 h. The DMEM was aspirated, and complete L-15 media (1.5 mL) was added to each well, followed by the respective compound. Treatments: DMSO (2 sets), 25 μM 1c (1 set), 25 μM 3 (2 sets). One set of each cell, treated with DMSO or compound 3, was irradiated with 405 nm light for 5 min. The plates were then incubated at 37 °C in a 5% CO₂ incubator for 13 h. The cells were harvested by trypsinization (300 μL Trypsin, 2 min at 37 °C), transferred to a 2 mL microcentrifuge tube, and pelletized (1000 rpm, 25 °C, 2 min). The cells were washed with PBS (x2) and pelletized. The cells were resuspended in ice-cold PBS, and 200 μL PI was added from the stock solution. The samples were run on a BD Accuri C6 Flow Cytometer, where data were collected using Forward Scattering (FS), Side Scattering (SS), and PI fluorescence (FL2) channels.

Glossary

Abbreviations

ONB

o-nitrobenzyl

EYPC-LUVs

egg yolk phosphatidylcholine-large unilamellar vesicles

HPTS

8-hydroxypyrene-1,3,6-trisulfonate

TBACl

tetrabutylammonium chloride

MP

methoxyphenol

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c01202.

• Characterization spectra, detailed experimental protocols, and details of geometry-optimized structures (PDF)

University of Warsaw, Faculty of Chemistry, Biological and Chemical Research Centre, Żwirki i Wigury 101, Warsaw, Poland (R.N.)

Department of Chemical Biology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, Braunschweig 38124, Germany (A.J.).

Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, U.K. (M.A.).

‡.

B.A. and A.J. contributed equally to the work. N.J.R. and P.T. designed the project. N.J.R., P.T. and M.L. established research plans. P.L.P. performed pilot reactions. N.J.R., R.N., and A.J. synthesized and characterized transporter and protransporters, and conducted ion transport and chemical experiments. M.A. synthesized control compounds. U.S. performed NMR experiments. N.J.R., R.N., and B.A. performed biological experiments. N.J.R. wrote the manuscript. P.T. and M.L. revised the manuscript. All authors have approved the final manuscript version.

†.

M.A. and U.S. contributed equally to the work.

P.T. and M.L. acknowledge the support of the Anusandhan National Research Foundation (ANRF), Government of India (Grant No. CRG/2022/001640), and the Department of Biotechnology, Government of India (Grant No. BT/PR42717/BRB/10/1994/2021), respectively, for funding the research. P.T. and M.L. also acknowledge IISER Pune for funding the research. N.J.R. and B.A. thank CSIR (Council for Scientific and Industrial Research), India, for research fellowships. M.A. thanks UGC (University Grants Commission) India for the research fellowship. U.S. thanks the National Fellowship for Other Backward Classes (UGC-NFOBC), India, for the research fellowship.

The authors declare no competing financial interest.