1,3a,6a-Triazapentalene derivatives as photo-induced cytotoxic small fluorescent dyes

1,3a,6a-Triazapentalene (TAP) is a compact fluorescent chromophore whose fluorescence properties vary greatly depending on the substituents on the TAP ring. This study investigated the photo-induced cytotoxicities of various TAP derivatives. Among the derivatives, 2-p-nitrophenyl-TAP showed significant cytotoxicity to HeLa cells under UV irradiation but no cytotoxicity without UV. In addition, the photo-induced cytotoxicity of 2-p-nitirophenyl-TAP was found to be cancer cell selective and effective against HeLa cells and HCT 116 cells. Under UV irradiation, 2-p-nitrophenyl-TAP generated reactive oxygen species (ROS) that induced an apoptosis and ferroptosis in cancer cells. Therefore, it was revealed that 2-p-nitrophenyl-TAP is the most compact dye that can generate ROS by photoirradiation.


• Supplementary Tables
Supplementary Table 1 | Fluorescence properties of TAP derivatives.
Florescence properties of TAP derivatives used in Fig. 2 were summarized. The samples used in this study were the same quality as the references. Synthetic procedure, spectral data and charts of these

Supplementary Table 2 | Colocalization analysis
The Pearson correlation (Rcoloc) value for organelle markers and 1d in the colocalization studies were calculated using the colocalization analysis of the ImageJ software.

Supplementary Figure 1 | UV irradiation
TAP derivatives treated cells were irradiated with UV (Wave length: 365nm, 6 W) light from a distance of 5 cm for 1h in CO2 incubator.

Supplementary Figure 2 | Cell viability of UV irradiation without compound
HeLa cells were cultured without compound (Untreated) and cell viability upon UV irradiation was measured by the WST8 assay. N=4 biologically independent samples. The error bars represent the standard deviation of the mean.

Supplementary Figure 3 | Cytotoxicity of UV or Blue LED irradiation with 1d.
Blue LED irradiation also showed 1d concentration-dependent photo-induced cytotoxicity, although weaker than UV irradiation.

Supplementary Figure 4 | Dose dependency of photo-induced cytotoxicity under different conditions of 1d treatment
HeLa cells were cultured with several conditions of 1d and the photo-induced cytotoxic effect of UV irradiation was determined by WST8 assay. A: Cells were treated with 1d for 1h, and then UV irradiation 1h as is (total 1d treatment time: 2h), B: Cells were treated with 1d for 1h, and then the medium was replaced before UV irradiation 1h as is (total 1d treatment time: 1h), C: Cells were irradiated UV 1h as soon as 1d added (total 1d treatment time: 1h). Scale bar =20 μm.

Supplementary Figure 6 | Observation of 1d or Alexa555 in HeLa cells.
HeLa cells were cultured with 100 µM of 1d or 2µg/mL of Alexa555 conjugated anti-mouse IgG (Cell Signaling Technology) for 1 h, and then the cells were monitored by fluorescent microscopy (BIOREVO BZ-9000; KEYENCE) in fluorescence images with TRITC filter. Scale bar =50 μm.

Supplementary Figure 7 | Localization of 1d in HeLa cells.
HeLa cells were cultured with 100 µM of 1d for 1 h, and then the cells were fixed with 4% paraformaldehyde (PFA) after incubation at 4 °C for 24 h. Fixed cells were treated with primary antibodies of organelle markers at 4 °C for 24 h. The cells were washed with phosphate buffered saline (PBS), and then the cells were incubated with Alexa488 conjugated secondary antibodies and Hoechst33258 for 2h at room temperature. The cells were washed with PBS, and observed using confocal fluorescent microscopy (LSM700, Carl Zeiss). Scale bar =20 μm.

Supplementary Figure 8 | Magnified cell imaging of Fig. 5a
Magnified cell imaging of Fig. 5a in the main text. Scale bar =50 μm.

Supplementary Figure 9 | Production of reactive oxygen species (ROS) in 1d, Rose bengal and Talaporfin after photo-irradiation.
HeLa cells were cultured with 50 µM of compounds for 1 h, and then the cells were iraddiated 365nm (1d) or 520nm (Rose bengal) or 660nm (Talaprfin) for 1h, and then ROS in the cells was measured by a DHE probe (ROS Detection Cell-Based Assay Kit ; Cayman). N=5 biologically independent samples.
The error bars represent the standard deviation of the mean. Kanto Chem. Co. Silica Gel 60N (particle size 0.040-0.050 mm) was used for column chromatography.
High-resolution images of cells were obtained using a ZEISS LSM700 (Carl Zeiss) confocal microscope.