Aryl‐Modified Pentamethyl Cyanine Dyes at the C2’ Position: A Tunable Platform for Activatable Photosensitizers

Abstract Pentamethyl cyanine dyes are promising fluorophores for fluorescence sensing and imaging. However, advanced biomedical applications require enhanced control of their excited‐state properties. Herein, a synthetic approach for attaching aryl substituents at the C2’ position of the thio‐pentamethine cyanine (TCy5) dye structure is reported for the first time. C2’‐aryl substitution enables the regulation of both the twisted intramolecular charge transfer (TICT) and photoinduced electron transfer (PET) mechanisms to be regulated in the excited state. Modulation of these mechanisms allows the design of a nitroreductase‐activatable TCy5 fluorophore for hypoxic tumor photodynamic therapy and fluorescence imaging. These C2’‐aryl TCy5 dyes provide a tunable platform for engineering cyanine dyes tailored to sophisticated biological applications, such as photodynamic therapy.


Singlet Oxygen Detection
We used 1,3-diphenylisobenzofuran (DPBF) to measure singlet oxygen produced by cyanine dyes.The absorbance of DPBF at 415 nm was adjusted to about 1.0 and the absorbance of the dye to be tested at 660 nm was adjusted to about 0.35-0.55 in dichloromethane (3 mL).The cuvette was irradiated with 660 nm (5 mW cm -2 ) monochromatic light for various time, and absorption spectra were measured immediately.Φ∆ rel was calculated by the following equation: Where "sam" and "std" represent the "PSs" and "ICG", respectively."k" is the slope of absorbance change curve of DPBF at 415 nm, F=1-10 -O.D. (O.D. is the absorbance of the solution at 660 nm).
At the same time, we used Singlet Oxygen Sensor Green (SOSG) to detect 1 O2 production in aqueous solution.Cyanine dyes and SOSG were prepared as 5 μM and 1 μM in water, respectively.The cuvette was irradiated by 660nm (5 mW cm -2 ) monochromatic light for various time, and the fluorescence spectra were measured immediately.As control, SOSG aqueous solution without photosensitizers was subjected to irradiation (λex = 488 nm).

Superoxide Anion Radical (O2 •− ) Detection
We used dihydrorhodamine 123 (DHR123) to detect O2 •− production, DHR123 can be converted to Rhodamine 123 in the presence of O2 •− .Cyanine dyes and DHR123 were prepared as 5 μM and 10 μM in water, respectively.The cuvette was irradiated by 660nm (5 mW cm -2 ) monochromatic light for various time, and the fluorescence spectra were measured immediately.As control, DHR123 aqueous solution without photosensitizers was subjected to irradiation (λex = 488 nm).
At the same time, we also used Droethidium (DHE) as indicator for detection of O2 •− in solution, When O2 •− is generated in the system, DHE can be oxidized to form ethidium which intercalates into DNA and emits bright fluorescence at ~580 nm. 5 µM of cyanine dyes were dissolved in 3 mL water containing 15 µM of DHE and 100 μg/mL ctDNA.The mixture was then placed in a cuvette and irradiated with 660nm (10 mW cm -2 ) monochromatic light for various time.The fluorescence change of sample was recorded by the fluorescence spectrometer.

Computational Methods
Density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations were employed to elucidate the mechanism of cyanine compounds.The calculations were performed using Gaussian 16A software. 1Geometry optimizations in both the ground and excited singlet states were conducted utilizing the ωB97XD functional with the def2-SVP basis set in dichloromethane. 2 To validate the stability of the structures on the potential energy surfaces (PES), frequency analysis was carried out.The solvent effects were considered using the SMD model, 3 and the electronic energies were calculated using the corrected linear response (cLR) solvent formalism.
For the modeling of the twisted intramolecular charge transfer (TICT) mechanism, 4 two dihedral angles along the rotation bond were systematically varied from approximately 0 (or 180; planar) to 90 degrees (perpendicular), with a step size of about 10 degrees.The remaining parameters were freely optimized, and the energies of these geometries were further corrected using the corrected linear solvation formalism.To account for potential state-crossing between the S1 and S2 states, optimization of both states was performed to construct the S1 potential energy surface (PES).From this PES, the energy barriers of rotation and the driving energy required to enter the TICT state were extracted.
During the modeling of the photon-induced electron transfer (PET) mechanism, 5 the phenyl ring at the C2'-position was fixed at a 90-degree angle to ensure complete charge transfer (or electron transfer).Subsequently, the electronic energy was corrected using the corrected linear solvation formalism.
In most cases, the triplet state energy was calculated using the DFT method based on the corresponding optimized singlet states.In situations involving multiple triplet states, such as both T1 and T2, TD-DFT calculations were performed on the geometries of the optimized singlet excited state, along with the corrected linear solvation formalism, to determine the energy levels of the triplet state.
Spin-orbit coupling values were calculated using ORCA 5.0 software, 6 employing the ωB97XD functional with the def2-SVP basis set in dichloromethane.
The SMD model was utilized to account for solvent effects.

Femtosecond transient absorption experiments
Femtosecond TA experiments.Femtosecond TA experiments were based on a Pharos laser (1030 nm, 100 kHz, 230 fs pulse-duration; Light Conversion) and Orpheus-HP optical parameter amplifier (OPA; Light Conversion).The 1030-nm output laser was split into two beams with 80/20 ratio.The 80% parts was used to pump the OPA to generate a wavelength tunable pump beam.The 20% parts was further split into two parts with 75/25 ratio.The 75% parts was attenuated with a neutral density filter and focused into a BBO crystal to generate a 515 nm beam, which was further focused into a Sapphire crystal to generate a white light continuum used as the probe beam.The probe beam was focused with an Al parabolic reflector onto the sample.After the sample, the probe beam was collimated and then focused into a fiber-coupled spectrometer with CMOS cameras and detected at a frequency of 10 kHz.The pump pulses were chopped by a synchronized chopper at 5 kHz and the absorbance change was calculated with twenty adjacent probe pulses (ten pumpblocked and ten pump-unblocked).The intensity of the pump pulse used in the experiment was controlled by a variable neutral-density filter wheel.The delay between the pump and probe pulses was controlled by a motorized delay stage.The linear polarization angle difference between the pump and probe light keeps magic angle (54.7°) to record the isotropic response.

Nanosecond Time-resolved Transient Absorption Spectra
The triplet lifetimes of dyes were recorded on a LP980 laser flash photolysis spectrometer (Edinburgh Instruments Ltd.) in combination with a Nd:YAG laser (Surelite I-10, Continuum Electro-Optics, Inc.).Samples (10 μM) in deaerated DCM were excited by a 610 nm laser pulse (1 Hz, 100 mJ per pulse, fwhm ≈ 7 ns) at room temperature.The triplet state decay kinetics was measured at λabs.

Cell and Culture Conditions
HepG2 cells, MCF7 cells and 4T1 cells were purchased from the Institute of Basic Medical Science (IBMS) of the Chinese Academy of Medical Sciences and cultured with Dulbecco's modified Eagle's medium (DMEM, Invitrogen), all of them were supplemented with 1% penicillin streptomycin and 10% fetal bovine serum, and atmosphere of CO2/air = 5%/95% at 37 ºC.

Confocal Fluorescence Imaging of Cells
HepG2 cells were cultured for 24 hours.For cell colocalization assay, first add LysoTracker Green DND 26 (100 nM), MitoTracker Green FM (100 nM) or Hoechst 33342 (100 nM) to the culture medium and incubate for 30 min, then add C2-NO2 (0.5 μM) and then observe the cells with confocal laser microscope.The excitation wavelength for C2-NO2 is 660 nm, while the excitation wavelength for LysoTracker Green DND 26 and MitoTracker Green FM is 488 nm, and the excitation wavelength for Hoechst 33342 is 405 nm.The emission wavelength was collected from 690 to 740 nm for C2-NO2, 500 to 540 nm for Lyso Tracker Green and Mito Tracker Green, and 440 to 480 nm for Hoechst 33342.

In Vitro Photo-Cytotoxicity Assays
To simulate a hypoxic tumor environment, cells were cultured in an incubator at monitored using an oxygen detector (Nuvair, O Qucikstick).
HepG2 cells, MCF7 cells or 4T1 cells were seeded into 96-well plates at 5000 cells per well and incubated at 37 ºC for 24 hours.Cells were cultured for an additional 6 h under normoxia or hypoxia.For normoxic photocytotoxicity assessment, DMEM medium containing various concentrations of photosensitizers from 0 to 4 μM was added to wells culturing normoxic cells.For hypoxic photocytotoxicity assessment, DMEM medium containing different concentrations of photosensitizers 0 to 4 μM was added to the wells of hypoxic cells, respectively.Then, the cells were further cultured under normoxia or hypoxia for 2 hours, respectively.Subsequently, cells were irradiated with 660 nm light (20 mW cm -2 for 15 min).The cells were then incubated for a further 12 hours at 37 ºC.Next, add the formulated MTT-containing DMEM solution (5 mg/mL) to each well.After 4 h of cell culture, carefully remove the solution from each well, add 100 μL of DMSO to each well, and measure the absorbance at 540 nm and 620 nm with a Bio-Rad microplate reader, respectively.The viability was expressed as a percent of the controlled one using the following equation: For dark toxicity measurement of different dyes, light irradiation step was canceled and all other steps were the same.

Live and Dead Cell Staining
HepG2 cells were cultured on cell culture plates for 24 h.Cells were cultured under normoxic or hypoxic conditions for an additional 10 hours and then exposed to different following treatments: 1) normoxic cells; 2) normoxic cells were irradiated with 660 nm light (20 mW cm -2 , 15 min); 3) normoxic cells were incubated with 0.5 μM C2-NO2 for 1 h and irradiated with 660 nm light (20 mW cm -2 , 15 min); 4) hypoxic cells; 5) hypoxic cells were irradiated with 660 nm light (20 mW cm -2 , 15 min); 6) hypoxic cells were incubated with 0.5 μM C2-NO2 for 1 h and irradiated with 660 nm light (20 mW cm -2 , 15 min); After different treatments, calcein AM and PI costaining was performed.The excitation wavelength is 488 nm, the emission wavelength is 505 to 545 nm for the green channel, and 600 to 700 nm for the red channel.

In Vivo Solid Tumor Phototherapy Evaluation
To evaluate the effect of photodynamic treatment with C2-NO2 in vivo, mice were divided into four groups of five tumor-bearing mice each: 1) saline injection and irradiation; 2) ICG (50 μmol, 100 μL) injection and irradiation; 3) C2-NO2 injection; and 4) C2-NO2 (50 μmol, 100 μL) injection and irradiation.After 3 h of injection, the tumor areas were irradiated with 671 nm laser (100 mW cm -2 , 15 min).After different treatments, the volume (V) of the tumor was calculated according to the following formula: V = a× b 2 / 2, where a represented the maximum diameter (mm) among solid tumor diameters, and b represented the diameter (mm) perpendicular to a.The tumor growth curve was calculated by comparing the tumor volume (V) with the initial tumor volume (V0).Mice were executed after 14 d of treatment, and tumor tissues were removed and stained with hematoxylin-eosin (H&E) for histological analysis.

In Vivo Biosafety Assay
The in vivo biosafety assay was performed by measuring the body weight of mices and histological analysis of H&E sections.Mice were executed after 14 d of treatment, and the major organs, such as heart, liver, spleen, lung, and kidney, were removed and stained with hematoxylin-eosin (H&E) for histological analysis.

Synthesis of 1:
Benzyl bromide (4.6 g, 26.8 mmol) was added to a stirred solution of 2methylbenzothiazole (2.0 g, 13.4 mmol) in dry acetonitrile (10 mL).The reaction was heated to 60 °C under N2 atmosphere and stirred for 24 h.Subsequently, the reaction was allowed to cool to room temperature.The precipitate was filtered and washed with ethyl acetate and dried under vacuum to afford compound 1 as a light green solid in 88.5% yield (3.8 g, 11.9 mmol). 2)

Scheme S2. Preparation of compounds 2-6
Synthesis of 2 POCl3 (3 mL, 32.1 mmol) was added dropwise to dry DMF (15 mL) at 0 °C.The mixture was stirred at room temperature for 3 h.Subsequently, acetophenone (1 g, 8.32 mmol) was added to the reaction solution.The red solution formed was heated at 75°C for 12 h.After cooling to room temperature, the solution was poured into 100 mL of ice water and then 50 ml of saturated NaClO4 solution was added.The solid obtained was filtered and washed twice with cold aqueous water and finally dried under vacuum.The product 2 was obtained as a yellow solid (1.3 g, 51.6% yield).

Synthesis of 3-6
Compound 3-5 were synthesized according to the synthetic procedure of compound 2. The compound 3 was gotten as brown white solid (0.7 g, 33.2 % yield).he synthesis of compound 7 follows the same procedure as that of compounds 3-6.However, due to the reaction system being acidic, the amino group reacts with DMF to form a Schiff base.As a result, the para-substituent in compound 7 is not an amino group, but rather a Schiff base.The compound 7 was gotten as yellow solid     Table S1: Calculated data of relative singlet oxygen yield.

Supplementary Content
All animal operations were in accordance with institutional animal use and care9 regulations approved by the Model Animal Research Center of Dalian Medical University (MARC).Female BALB/c mice, 5 weeks old, were purchased from Liaoning Changsheng Biotechnology co.Ltd. mouse solid tumor model was then established by subcutaneous injection of 5 × 10 6 4T1 cells at selected axillary locations.Tumors were allowed to grow to a volume of approximately 100 mm 3 .The mouse model construction and imaging experiments involved in this work were carried out under Guide for the Care and Use of Laboratory Animals (8th edition) published by the US National Institutes of Health in 2011, all manipulations were followed by USA National Research Council regulation.The animal protocol was approved by the local research ethics review board of the Animal Ethics Committee of Dalian University of Technology (2019-016).For in vivo tumor imaging, 4T1 tumor-bearing BALB/c mice were divided two groups: 1) injecting C2-NO2 (50 μmol, 100 μL) into the tumor; 2) preinjecting dicumarin (100 μmol, 100 μL) into the tumor for 1h and then C2-NO2 (50 μmol, 100 μL) was injected.The fluorescence signals were monitored at different post injection time (30 min, 60 min, 120 min, 180 min, 240 min and 300 min).

Figure S1 .
Figure S1.Crystal structures of C2-H.All H atoms are omitted for clarity atomic

Figure S2 .
Figure S2.Chemical structural of different dyes

Figure S7 .
Figure S7.Fluorescence spectra for 1 O2 using SOSG as fluorescence probe under 660 nm light irradiation for 3 min in water with Cyanine dyes.

Figure S10 .
Figure S10.electron spin-resonance spectroscopy (ESR) signals of the mixture containing C2-NH2 and TEMP or DMPO under 660 nm light irradiation and the reference signal.

Figure S11 .
Figure S11.(a) Schematic illustration of the TICT mechanism and the excitation/deexcitation energy along with oscillator strength (f) of the key states during this process.(b) Optimized molecular structures of TCy5-H in the ground and excited states, as well as the corresponding electron and hole distributions in dichloromethane.VES and AES denote a vertically excited state and an adiabatic excited state, respectively.

Figure S12 .
Figure S12.(a) Schematic illustration of the TICT and PET mechanisms and the excitation/de-excitation energy along with oscillator strength (f) of the key states during this process.(b) Optimized molecular structures of C2-NO2 in the ground and excited states, as well as the corresponding electron and hole distributions in dichloromethane.VES and AES denote a vertically excited state and an adiabatic excited state, respectively.

Figure S13 .
Figure S13.(a) Schematic illustration of the TICT and PET mechanisms and the excitation/de-excitation energy along with oscillator strength (f) of the key states during this process.(b) Optimized molecular structures of C2-NH2 in the ground and excited states, as well as the corresponding electron and hole distributions in dichloromethane.VES and AES denote a vertically excited state and an adiabatic excited state, respectively.

Figure S14 .
Figure S14.(a) Schematic illustration of the TICT and PET mechanisms and the excitation/de-excitation energy along with oscillator strength (f) of the key states during this process.(b) Optimized molecular structures of C2-H in the ground and excited states, as well as the corresponding electron and hole distributions in dichloromethane.VES and AES denote a vertically excited state and an adiabatic excited state, respectively.

Figure S15 .
Figure S15.(a) Schematic illustration of the TICT and PET mechanisms and the excitation/de-excitation energy along with oscillator strength (f) of the key states during this process.(b) Optimized molecular structures of C2-NMe2 in the ground and excited states, as well as the corresponding electron and hole distributions in dichloromethane.VES and AES denote a vertically excited state and an adiabatic excited state, respectively.

Figure S16 .
Figure S16.(a) Schematic illustration of the TICT and PET mechanisms and the excitation/de-excitation energy along with oscillator strength (f) of the key states during this process.(b) Optimized molecular structures of meso-NO2 in the ground and excited states, as well as the corresponding electron and hole distributions in dichloromethane.VES and AES denote a vertically excited state and an adiabatic excited state, respectively.

Figure S17 .
Figure S17.(a) Schematic illustration of the TICT and PET mechanisms and the excitation/de-excitation energy along with oscillator strength (f) of the key states during this process.(b) Optimized molecular structures of meso-OMe in the ground and excited states, as well as the corresponding electron and hole distributions in dichloromethane.VES and AES denote a vertically excited state and an adiabatic excited state, respectively.

Figure S18 .
Figure S18.(a) Schematic illustration of the TICT and PET mechanisms and the excitation/de-excitation energy along with oscillator strength (f) of the key states during this process.(b) Optimized molecular structures of meso-NH2 in the ground and excited states, as well as the corresponding electron and hole distributions in dichloromethane.VES and AES denote a vertically excited state and an adiabatic excited state, respectively.

Figure S19 .
Figure S19.(a) Schematic illustration of the TICT and PET mechanisms and the excitation/de-excitation energy along with oscillator strength (f) of the key states during this process.(b) Optimized molecular structures of meso-NMe2 in the ground and excited states, as well as the corresponding electron and hole distributions in dichloromethane.VES and AES denote a vertically excited state and an adiabatic excited state, respectively.

Figure S32 .
Figure S32.Cell viability of (a)4T1 cells, (b) A549 cells and (c) MCF7 cells under hypoxia subjected to a range of C2-NO2 concentrations under 20 mW cm -2 light irradiation for 15 min.