Synthesis, S Atom Promoted Photodynamic Therapy and Two- Photon Fluorescence Imaging of Phenothiazine Fluorescent Protein Chromophore Analogue

doi:10.6023/cjoc202104040

Abstract

The green fluorescent protein (GFP) chromophore has attracted much attention due to its good biocompatibility, low dark cytotoxicity and high photostability. However, there are rare reports about GFP chromophore analogues in photodynamic therapy (PDT). In this paper, a new photosensiter phenothiazine fluorescent protein (Ptz-FP) chromophore was synthesized by [2+3] cycloaddition reaction using N-butylphenothiazine-3-carbaldehyde, tert-butyl glycine hydrochloride and imine ester as raw materials. Ptz-FP was a heavy-atom free photosensitizer that promoted by S atom and showed excellent singlet oxygen generation which can be used in PDT. In dimethyl sulfoxide (DMSO) solution, the absorption wavelength of Ptz-FP photosensitizer was at 436 nm, the molar extinction coefficient was 1.4×10⁴ L•mol^–1•cm^–1, the emission wavelength was at 626 nm, stokes shift was 190 nm, and the fluorescence quantum yield was 1.5%. The singlet oxygen yield of Ptz-FP was 33.1% using Ru(bpy)₃Cl₂ as a reference in methanol solution. The dark cytotoxicity and phototoxicity of the photosensitizer Ptz-FP were tested by methyl thiazolyl tetrazolium (MTT) assay. The results showed that it had negligible dark toxicity (≥89.9%) and excellent phototoxicity. Besides that, DCFH-DA (ROS indicator) successfully detected the production of reactive oxygen in A-549 cells within 30 min, and the apoptosis process of A-549 cells was monitored by the AO/EB stain assay. Finally, the photosensitizer Ptz-FP was successfully used in two-photon fluorescence imaging. Therefore, the photosensitizer Ptz-FP is expected to lay a foundation for FP chromophore based photosensitizers on fluorescence imaging guided PDT in the future.

Key words: fluorescent protein chromophore, containing S atom photosensitizer, photodynamic therapy, two-photon fluorescence imaging

Cite this article

Wenhui Xiang, Lei Zhang, Xu Zhi, Ying Qian. Synthesis, S Atom Promoted Photodynamic Therapy and Two- Photon Fluorescence Imaging of Phenothiazine Fluorescent Protein Chromophore Analogue[J]. Chinese Journal of Organic Chemistry, 2021, 41(9): 3578-3584.

Export EndNote|Reference Manager|ProCite|BibTeX|RefWorks

share this article

Fig. & Tab. 9

Figure 1. (a) Recent reported GFP chromophore analogues; (b) the photosensitizer Ptz-FP synthesized in this work and its photodynamic therapy in A-549 cells

Figure 2. (a) Energy level diagram of singlet oxygen generation in Ptz-FP photosensitizer containing S atom; (b) Synthetic route of Ptz-FP

Figure 3. Absorbance spectrum and fluorescence spectrum of Ptz-FP (20 μmol•L–1) in DMSO

Compd.	λmax abs/nm^a	ε^max/(10⁴ L•mol^–1•cm^–1)^b	λmax em/nm^c	Stokes shift/nm	φ_F^d	IC₅₀^e/(μmol•L^–1)
Ptz-FP in DMSO	436	1.4	626	190	1.5%	3
Ptz-FP in CH₂Cl₂	436	1.8	626	190	28%	—

Figure 4. One-photon fluorescence imaging of Ptz-FP in A-549 cells (Group A, 2 μmol•L–1, Ex: 488 nm, Em: 577~677 nm), and two-photon fluorescence imaing of Ptz-FP in A-549 cells (Group B, 2 μmol•L–1, Ex: 800 nm) Scale bar: 100 μm

Figure 5. (a) Absorbance decrease at 414 nm of DPBF in the presence of presence of Ptz-FP in CH3OH and blue light (460 nm, 4 mW/cm2) irradiation; (b) Linear fitting of the absorption at 414 nm of DPBF in the presence of Ptz-FP; (c) Absorbance decrease at 414 nm of DPBF in the presence of Ru(bpy)3Cl2 in CH3OH and blue light (4 mW/cm2) irradiation; (d) Linear fitting of the absorption at 414 nm of DPBF in the presence of Ru(bpy)3Cl2

Figure 6. Cell viability of A-549 cells treated with various concentrations of Ptz-FP in the dark and exposed to light (460 nm) for 10 and 30 min

Figure 7. ROS generation in Ptz-FP (3 μmol•L–1, Ex: 488 nm, Em: 577~677 nm) treated A-549 cells

Figure 8. AO/EB staining assay of Ptz-FP to A-549 cells Group a: No light+Ptz-FP (3 μmol•L–1)+AO/EB; Group b: Blue light (irradiation time:15 min, 460 nm, 4 mW/cm2)+AO/EB; Group c: Ptz-FP (3 μmol•L–1)+blue light (irradiation time: 5 min, 460 nm, 4 mW/cm2)+AO/EB; Group d: Ptz-FP (3 μmol•L–1)+blue light (irradiation time:10 min, 460 nm, 4 mW/cm2)+AO/EB; Group e: Ptz-FP (3 μmol•L–1)+blue light (irradiation time: 15 min, 460 nm, 4 mW/cm2)+AO/EB. Scale bar: 100 μm

References 31

[1]	Zimmer, M. Chem. Rev. 2002, 102, 759. pmid: 11890756
[2]	Tsien, R. Y. Angew. Chem., nt. Ed. 2009, 48, 5612.
[3]	Walker, C.; Lukyanov, K.; Yampolsky, I.; Mishin, A.; Bommarius, A.; Duraj-Thatte, A.; Azizi, B.; Tolbert, L.; Solntsev, K. Curr. Opin. Chem. Biol. 2015, 27, 64. doi: 10.1016/j.cbpa.2015.06.002
[4]	Deng, H.; Zhu, X. Mater. Chem. Front. 2017, 1, 619. doi: 10.1039/C6QM00148C
[5]	Baldridge, A.; Samanta, S. R.; Jayaraj, N.; Ramamurthy, V.; Tolbert, L. M. J. Am. Chem. Soc. 2011, 133, 712. doi: 10.1021/ja1094606 pmid: 21174415
[6]	Liu, Y.; Wolstenholme, C. H.; Carter, G. C.; Liu, H.; Hu, H.; Grainger, L. S.; Miao, K.; Fares, M.; Hoelzel, C. A.; Yennawar, H. P.; Ning, G.; Du, M.; Bai, L.; Li, X.; Zhang, X. J. Am. Chem. Soc. 2018, 140, 7381. doi: 10.1021/jacs.8b02176 pmid: 29883112
[7]	Mirajkar, A. L.; Mittapelli, L. L.; Nawale, G. N.; Gorea, K. R. Sens. Actuator, B 2018, 265, 257. doi: 10.1016/j.snb.2018.03.068
[8]	Sun, H.; Leng, H.; Liu, J.; Roy, G.; Yan, J.; Zhang, L. Sens. Actuators, B 2020, 305, 127509. doi: 10.1016/j.snb.2019.127509
[9]	Shen, B.; Zhu, W.; Zhi, X.; Qian, Y. Talanta 2020, 208, 120461. doi: 10.1016/j.talanta.2019.120461
[10]	Zhi, X.; Shen, B.; Qian, Y. New. J. Chem. 2020, 44, 8823 doi: 10.1039/D0NJ01477J
[11]	Zhi, X.; Qian, Y. Talanta 2021, 222, 121503. doi: 10.1016/j.talanta.2020.121503 pmid: 33167216
[12]	Li, M. L.; Peng, X. J. Acta Chim. Sinica 2016, 74, 959. (in Chinese). doi: 10.6023/A16100553
	( 李明乐, 彭孝军, 化学学报, 2016, 74, 959.)
[13]	Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D.; Korbelik, M.; Moan, J.; Mroz, P.; Nowis, D.; Piette, J.; Wilson, B. C.; Golab, J. Ca-Cancer J. Clin. 2011, 61, 250.
[14]	Liu, Z. Y.; Zou, H.; Zhao, Z.; Zhang, P. F.; Shan, G. G.; Kwok, R. T. K.; Lam, J. W. Y.; Zheng, L.; Tang, B. Z. ACS Nano 2019, 13, 11283. doi: 10.1021/acsnano.9b04430
[15]	Bai, J.; Qian, Y. Dyes Pigm. 2020, 181, 108615. doi: 10.1016/j.dyepig.2020.108615
[16]	Wang, L.; Bai, J.; Qian, Y. New J Chem 2019, 43, 16829. doi: 10.1039/C9NJ04166D
[17]	Wang, C.; Qian, Y. Org. Biomol. Chem. 2019, 17, 8001-8007. doi: 10.1039/C9OB01242G
[18]	Xu, F.; Li, H.; Yao, Q.; Ge, H.; Fan, J.; Sun, W.; Wang, J.; Peng, X. Chem. Sci. 2019, 10, 10586. doi: 10.1039/C9SC03355F
[19]	Zheng, J.; Liu, Y.; Song, F.; Jiao, L.; Wu, Y.; Peng, X. Chem. Commun. 2020, 56, 5819. doi: 10.1039/D0CC02019B
[20]	Yang, X.; Bai, J.; Qian, Y. Spectrochim. Acta, Part A 2020, 228, 117702. doi: 10.1016/j.saa.2019.117702
[21]	Ning, Y.; Liu, Y.; Yang, Z.; Yao, Y.; Kang, L.; Sessler, J. L.; Zhang, J. J. Am. Chem. Soc. 2020, 142, 6761. doi: 10.1021/jacs.0c01155
[22]	Nguyen, V.; Baek, G.; Qi, S.; Heo, S.; Yim, Y.; Yoon, J. Dyes Pigm. 2020, 177, 108265. doi: 10.1016/j.dyepig.2020.108265
[23]	Tian, R.; Sun, W.; Li, M.; Long, S.; Li, M.; Fan, J.; Guo, L.; Peng, X. Chem. Sci. 2019, 10, 10106. doi: 10.1039/C9SC04034J
[24]	Tang, J.; Wang, L.; Loredo, A.; Cole, C.; Xiao, H. Chem. Sci. 2020, 11, 6701. doi: 10.1039/D0SC02286A
[25]	Nguyen, V.; Qi, S.; Kim, S.; Kwon, N.; Kim, G.; Yim, Y.; Park, S.; Yoon, J. J. Am. Chem. Soc. 2019, 141, 16243. doi: 10.1021/jacs.9b09220
[26]	Nguyen, V.; Park, S. J.; Qi, S.; Ha, J.; Heo, S.; Yim, Y.; Baek, G.; Lim, C. S.; Lee, D. J.; Kim, H. M.; Yoon, J. Chem. Commun. 2020, 56, 11489. doi: 10.1039/D0CC04644B
[27]	Bai, Jin.; Qian, Ying. Spectrochim. Acta, Part A 2021, 252, 119512. doi: 10.1016/j.saa.2021.119512
[28]	Xie, Z. D.; Fu, M. L; Yin, B.; Zhu, Q. Chin. J. Org. Chem. 2018, 38, 1364. (in Chinese). doi: 10.6023/cjoc201712031
	( 谢振达, 付曼琳, 尹彪, 朱勍, 有机化学, 2018, 38, 1364.)
[29]	Tanielian, C.; Wolff, C.; Esch, M. J. Phys. Chem. 1996, 100, 6555. doi: 10.1021/jp952107s
[30]	Jiao, L.; Song, F. L.; C, J.; Peng, X. J. Chem. Commun. 2018, 54, 9198. doi: 10.1039/C8CC04582H
[31]	Kim, S. H.; Sakong, C.; Chang, J. B.; Kim, B.; Ko, M. J.; Kim, D. H.; Hong, K. S.; Kim, J. P. Dyes Pigm. 2013, 97, 262. doi: 10.1016/j.dyepig.2012.12.007

吩噻嗪-荧光蛋白生色团类似物的合成、S原子促进的光动力治疗及双光子荧光成像