Caging and Photo-triggered Release of Singlet Oxygen by Excited-State Engineering of Molecular Sensors Bound to an Electron Donor-Acceptor

General

All chemicals used in this research were analytical grade and used as received, unless otherwise specified. Potassium carbonate (K2CO3), potassium iodide (KI), hydrochloric acid (HCl) and sodium azide (NaN3) were obtained from FUJIFILM Wako Pure Chemical Corporation, Japan. 7-Amino-4-methyl coumarin, 7-ethylamino-4-methyl coumarin, 9-chloromethyl anthracene, 9-methylanthracene, tetrakis(4-carboxyphenyl)porphyrin (TCPP) and Rose Bengal (RB) from Tokyo Chemical Industry (TCI) , Japan. We obtained 2,2,6,6-tetramethylpiperidine (TEMP) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) from Sigma Aldrich, USA. The SOSG was obtained from the Sigma sensor and SiDMA from DOJINDO, Japan. All solvents were reagent grade and sourced from FUJIFILM Wako Pure Chemical Corporation, Japan.

Absorption spectra were recorded using an Evolution 220 UV-visible spectrophotometer (ThermoFisher Scientific) and fluorescence (FL) spectra were recorded using a Hitachi F-4500 FL spectrofluorimeter. The NMR measurements were carried out using an Agilent Unity INOVA 500 or JEOL ECX-400 spectrometer. Continuous wave EPR measurements were performed using a Bruker EMXplus spectrometer. For the photoirradiation of the samples, we used a 532 nm DPSS green laser (Shanghai Dream Laser Technology), a Xenon/Mercury lamp (Hamamatsu Photonics KK, Japan) or an 800 nm femtosecond laser (Coherent Mira 900, the pulse width is 140 fs). The 404 nm laser (Thorlabs, 600 mW) with neutral density filters was used to vary the power.

Synthesis and characterization

1 was prepared and characterized according to the reported procedure with a slight modification20. 7-Amino-4-methylcoumarin (0.175 g, 1.00 mmol) and 9-chloromethylanthracene (0.227 g, 1.00 mmol) were dissolved in 20 mL of acetonitrile. Then DBU (304 mg, 2.00 mmol) was added to the solution and the reaction mixture was stirred at 82°C for 6 h. The reaction mixture was cooled to room temperature and excess water was added giving a yellow residue. The pH of the solution was adjusted to ~6–7 using aq. HCI. The residue was filtered and dried. The yellow powder was redissolved in hot THF then reprecipitated by adding excess toluene, and the residue was filtered and washed with toluene then acetone to give a pale yellow powder (0.332 g, 92%). λmaximum (DMF): 354, 370, 389nm. 1: 1H NMR (400 MHz, CDCl3) δ= 8.50 (1H), 8.21 (2H), 8.05 (2H), 7.57–7.40 (m, 5H), 6.74 (1H), 6.58 (1H), 6 .02 (sec, 1H), 5.21 (2H), 4.30 (1H), 2.40 (3H).

2 was prepared according to the following method.

7-(ethylamino)-4-methylcoumarin (0.10 g, 0.49 mmol) and 9-chloromethylanthracene (0.16 g, 0.73 mmol) were dissolved in 10 mL of DMF. Then K2CO3 (47 mg, 2.9 mmol) and potassium iodide (5 mg, 0.03 mmol) were added to the solution, and the reaction mixture was stirred at 85°C for 5 h. The reaction mixture was cooled to room temperature and excess water was added giving a yellow residue. The pH of the solution was adjusted to ~6–7 using aq. HCI. The residue was filtered and dried. The yellow powder was redissolved in hot THF then reprecipitated by adding excess toluene, and the residue was filtered and washed with toluene then acetone to give a pale yellow powder (0.10 g, 51% ). λmaximum (DMF): 354, 370, 389nm. 1H NMR (500 MHz, CDCl3) δ = 8.52 (s, 1H; Ar–H), 8.14–8.16 (d, 2H; Ar–H), 8.05–8.07 (d, 2H; Ar–H), 7.55–7.48 (m, 5H; Ar–H), 6.95–6.98 (dd, 1H; Ar–H), 6.91–6.92 (d, 1H; Ar–H) , 6.05 (s, 1H; allylic), 5.37 (s, 2H; N-CH2), 3.06–3.10 (q, 2H; N-CH2), 2.42 (s, 3H; CH3), 0.77–0.80 (t, 3H; CH3).

Steady State and Absorption FL Spectroscopic Studies of 1O2 detection

A sample solution of a sensor molecule (1 Where 2; 10.0 μM) and photosensitizer (5.00 μM) in DMF was photosensitized under selective excitation of the photosensitizer. The sample solution containing RB was irradiated with a 532 nm continuous wave laser (DPSS, 50 mW). The one containing TCPP was illuminated by a xenon lamp equipped with a 410-430 nm bandpass filter or a 404 nm continuous wave laser (Thorlabs, 70 mW). The sample solution was irradiated with a UV LED lamp (Asahi-spectra. Co. CL) (365 nm, 10 nm band path, 1.0 mW cm-2). FL and absorption spectra were recorded before and after irradiation.

Isolation and characterization of the reaction product of 1 and 1O2

2.0mm 1 and 1.0 mM RB were mixed in 800 μL DMF (HPLC grade) and illuminated with a green diode laser (532 nm, 50 mW, 10 min). The reaction mixture was subjected to an HPLC system (Agilent 1220) equipped with a C18-MS-II column (Nacalai; 4.6 mm ID x 250 mm) using DMF as eluent. The fraction with the maximum retention time of 2.8 min was collected and removed from the solvent under vacuum in the dark. DMSO-d6 was added and measured by an NMR spectrometer. Yield 86% (estimated from HPLC profile). λmaximum (DMF): 354nm, 1H-NMR (400 MHz, DMSO-D6) δ= 7.53–7.56 (m, 4H), 7.49 (s, 1H), 7.31–7.33 (m, 4H), 6.98 (d, J= 9.1Hz, 1H), 6.92 (s, 1H), 6.86 (t, J= 4.1Hz, 1H), 6.47 (s, 1H), 5.98 (s, 1H), 4.50 (d, J= 4.1Hz, 2H), 2.35 (s, 3H).

The reaction mixture without the HPLC separation was also measured for comparison. Then, the sample solutions were irradiated with a UV LED lamp (Asahi-spectra. Co. CL) (365 nm, 10 nm band path, 1.0 mW cm-2, 10 min) and again measured by the NMR spectrometer. The results have been presented in Figures S6 and S7.

Estimation of singlet oxygen photo-release quantum yield from EPR studies)

The singlet oxygen photo-release quantum yield is estimated to be 1.6% based on the number of photons absorbed (320 nmol) and detection 1O2 (10.0 nmoles × 50% = 5.00 nmoles). The number of absorbed photons was calculated based on the induced light and the absorbance of the sample solution after RB photosensitization. The detected 1O2 in moles was calculated on the basis of the 1 (10 μM, 0.50 mL) and the ratio of signal change from TEMP to TEMPO (50%).

Steady State Absorption and FL Spectroscopic Studies for 1O2 release

An example of a solution to 1 (10.0 μM) and photosensitizer (5.00 μM) in DMF were photosensitized under selective excitation of the photosensitizer. The sample solution containing RB was irradiated with a 532 nm (50 mW) continuous wave laser. Then, SOSG (10 μM) was added and the sample solution was irradiated with a UV LED lamp (Asahi-spectra. Co. CL) (365 nm, 10 nm band path, 1 mW cm-2). Prior to adding SOSG, the solution was purged with argon (50 mL/min, 20 min) for the oxygen-free condition. FL and absorption spectra were recorded before and after irradiation.

Steady-state absorption and FL spectroscopic studies under NIR activation of the intermediate complex

A sample solution containing 1 (10.0 μM) and rose bengal (10.0 μM) in DMF was irradiated under a 532 nm (50 mW) green laser for the photosensitized generation of 1O2. FL and absorption spectra of the sample (250 μL in a 5 mm path length cuvette) were recorded before and after 30 min of photosensitization. Then, the sample solution was irradiated with an 800 nm fs laser (Coherent Mira 900) for 40 min, and the FL and absorption spectra were recorded at every 5 min interval. The FL quantum yield of the samples was estimated by relative estimation of the FL quantum yield using coumarin 120 as a reference. A control experiment was conducted to compare the enhancement factor by recording the FL spectra of the equivalent sample solution after photosensitization, which was kept in the dark.

Steady State and Absorption FL Spectroscopic Studies of 1O2 detection

A sample solution of a sensor molecule (1 Where 2; 10.0 μM) and photosensitizer (5.00 μM) in DMF was photosensitized under selective excitation of the photosensitizer. The sample solution containing RB was irradiated with a 532 nm continuous wave laser (DPSS, 50 mW). The one containing TCPP was illuminated by a xenon lamp equipped with a 410-430 nm bandpass filter or a 404 nm continuous wave laser (Thorlabs, 70 mW). The sample solution was irradiated with a UV LED lamp (Asahi-spectra. Co. CL) (365 nm, 10 nm band path, 1.0 mW cm-2). FL and absorption spectra were recorded before and after irradiation.

Density Functional Theory (DFT) Calculations

Molecular structures and electronic energies were optimized and obtained by the Gaussian package1622 using ub3lyp/6-311++ theory level G**23.24. Molecular orbital analyzes were performed for natural transition orbitals25 using “Pop=(NTO,SaveNTO)” and “Density=(Check,Transition=not)” after performing the TD-DFT calculations.

Electron paramagnetic resonance (EPR) studies

The generation of 1O2 was monitored indirectly using a TEMP spin probe which undergoes oxidation by 1O2 to form EPR-active TEMPO. The measurement conditions were optimized by evaluating the photosensitization of RB in the presence of SPECT. For this purpose, 5 mM of TEMP was added to 5.00 μM of RB in DMF. The solution was irradiated with a xenon lamp equipped with a high-pass filter > 480 nm for 30 min (50 mW at 532 nm). After photosensitization, EPR spectra of the sample solution were recorded using the X-band frequency of microwaves (9.79 GHz) at 1 mW cm−2 Powerful. To check the possibility of generation 1O2 under UV illumination, 1 or RB was illuminated with a UV lamp with an emission maximum at 365 nm, at 2.0 mW cm−2 for 10 min in the presence of 5.00 mM TEMP.

To examine the UV-activated release of 1O2a sample of solution containing 1 (10 μM) and RB (5 μM) were irradiated with a xenon lamp equipped with a long-pass filter >480 nm for 30 min (50 mW at 532 nm). After photosensitization and generation of the intermediate complex, 5 mM of TEMP was added to the sample solution, and EPR spectra were recorded before and after 10 min of UV illumination (365 nm, 10 nm bandpath , 2 mW cm−2). A control experiment was conducted by illuminating a sample solution containing 1 (10 μM) and RB (5 μM) and 5 mM TEMP with UV light (UV, 2 mW cm−2 at 365 nm).

The enhancement factor of EPR signals was determined assuming the formation of TEMPO in the presence of TEMP and RB without 1 be 100% (Fig. S7).

Jack C. Nugent