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The fluorescent markers based on oxazolopyridine unit for imaging organelles
Journal Pre-proofs The fluorescent markers based on oxazolopyridine unit for imaging organelles Ya-Nan Wang, Bing-Xu, Li-Hua Qiu, Ru Sun, Yu-Jie Xu, Jian-Feng Ge PII: DOI: Reference:
S0960-894X(20)30059-7 https://doi.org/10.1016/j.bmcl.2020.126996 BMCL 126996
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
4 December 2019 1 January 2020 24 January 2020
Please cite this article as: Wang, Y-N., Bing-Xu, Qiu, L-H., Sun, R., Xu, Y-J., Ge, J-F., The fluorescent markers based on oxazolopyridine unit for imaging organelles, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.126996
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.
Graphical Abstract
The fluorescent markers based on oxazolopyridine unit for imaging organelles
Leave this area blank for abstract info.
Ya-Nan Wanga, Bing-Xub, Li-Hua Qiua*, Ru Suna, Yu-Jie Xub and Jian-Feng Gea,c*
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com
The fluorescent markers based on oxazolopyridine unit for imaging organelles Ya-Nan Wang,a Bing-Xu,b Li-Hua Qiu a Ru Sun,a Yu-Jie Xu,b Jian-Feng Gea,c College of Chemistry, Chemical Engineering and Material Science, Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, P. R. China. b State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China c Jiangsu Key Laboratory of Medical Optics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, P. R. China. a
———
ARTICLE INFO
ABSTRACT
Corresponding author: E-mail: [email protected], [email protected] (J.-F. Ge).
Article history: Received Revised Accepted Available online Keywords: Bioactive Oxazolopyridine Fluorescent markers Dual targetable Photogenic precursors
Bioactive oxazolopyridine unit was used in the synthesis of fluorescent markers for specific organelles in this paper. The compounds 1a-c are linked with double bond between oxazolopyridine ring and photogenic precursors (3a-c). Compounds 1a-c showed higher fluorescence yield (0.47-0.86), larger stokes shift (103-224) and good photo stability. In lipid vesicles environment, they also showed good optical properties. In addition, the three compounds are biomarkers with lower cytotoxicity. Among them, compound 1a based on oxazolopyridine and coumarin unit is a dual targetable fluorescent marker for mitochondria and lipid droplets; while the other two compounds 1b-c are only biomarkers for lipid droplets.
Fluorescent markers have been favored by most scientists in recent years because of their high sensitivity, good selectivity and convenient to be used1-3. Most fluorescent markers with good optical properties have a large rigid structure resulting in insufficient biocompatibility. Therefore, it is important to design and synthesize fluorescent markers with good biological activity and optical properties. Oxazolo[4,5-b]pyridine is particularly popular in biomedicine and is often used in the synthesis of antiinflammatory or antibacterial medicines, so it has good biological activity; Although oxazolo[4,5-b] pyridine is involved in medicine, it is rarely involved in dye and organelle targeting4-6. Mitochondria and lipid droplets are two important organelles in cells. Mitochondria are the providers of energy in cells, providing almost all the energy needed for cellular processes, and are closely related to other aspects of cell biology7; lipid droplets are mainly found in eukaryotic cells and are used in cells for storage. The organelle of lipids mainly regulates the storage and hydrolysis of neutral lipids8. Moreover, lipid droplets are dynamic organelle surfaces and enzymes involved in metabolism9, 10. Reported studies on biomarkers for mitochondrial targeting are relatively thorough, while studies on lipid droplet-labeled fluorescent markers are relatively rare and a few specific groups can be used to identify targeted lipid droplets11. Therefore, it is necessary to study fluorescent markers that are capable of specifically labeling lipid droplets. At the same time, there are close relationships between mitochondria and lipid droplets in the cells, which affect the normal functioning of the living body12. For example, when cells need
.
energy, the fatty acids that are absorbed by the cells and stored in the lipid droplets are hydrolyzed, and then ATP is produced by the action of mitochondria, to provide the energy needed by the cells. The injury in this process can lead to metabolic disorders, disease and even death13, 14. Therefore, it is important to design fluorescent marker that can label two organelles simultaneously. However, some articles on organelle markers have basically labeled single organelles in recent years, while labeling two organelles with few fluorescent markers. This may be because small molecule fluorescent markers will accumulate in a specific organelle or subcellular region after entering the cell, so it is relatively difficult to enter two organelles simultaneously15. Therefore，up to now, there are few articles have used a single molecule to mark two organelles in living cells at the same time and fewer biomarkers target lipid droplets and mitochondria simultaneously. So it is of great significance to study the biomarkers of lipid droplets and mitochondria simultaneously. The structure of a nitrogen-containing heterocyclic is often observed in anticancer, antitumor, antiviral and the like, but is rarely involved in fluorescent labels16, 17. The oxazolo[4,5b]pyridine (2) used in this paper has good biological activity and is widely used in the synthesis of anti-inflammatory and antibacterial medicines. Compound 2 and photogenic precursors 3a-c (coumarin (3a), triphenylamine (3b), phenothiazine (3c)) were reacted by Knoevenagel reaction to obtain fluorescent markers 1a-c. The specific design and synthesis route of the three fluorescent markers are as follows:
Commented [user1]: According to the comment of reviewer 2, we supplement the novelty of this research in this manuscript.
Scheme 1. Synthesis of compounds 1a-c. As shown in Scheme 1, compounds 1a-c were synthesized by compound 2 and compounds 3a-c at 130 oC in toluene. The yields were 41–53.1 % and all structures were confirmed by 1H NMR, 13C NMR and HRMS in detail (Fig. S7-15). The photo stability is also one of the criteria for measuring the performance of a compound. Therefore, the photo stability of the three compounds in acetonitrile was tested (Fig. S1), using Nile red with better photo stability as control. After irradiation with 500 W Philips iodine tungsten lamp for six hours, the maximum relative residual absorption of Nile red was 97%, and the maximum relative residual absorption of the three compounds was 94% (1a), 93% (1b) and 92% (1c), respectively. These data show that the compounds 1a-c have good photo stability. Fluorescent markers can exhibit different optical properties in different solvents, so the optical properties of fluorescent markers 1a-c in different solvents were tested18. The UV-vis absorption and fluorescence emission spectra of fluorescent markers 1a-c in water (H2O), dimethyl sulfoxide (DMSO), methanol (CH3OH), chloroform (CHCl3), tetrahydrofuran (THF), toluene (TOL) were tested (Fig. 1 and Figs. S2-S3). Further detailed data on the fluorescent markers 1a-c in different solvents were recorded in Table S1. It can be clearly seen from the Fig. 1 that the compound 1a shows different optical properties in different solvents. Generally, the fluorescence intensity of compound 1a increased with the decrease of solvent polarity, and the fluorescence peak showed a significant blue shift. This is, from water to toluene, the maximum absorption peaks of compound 1a blue shifted from 490 nm to 445 nm, and the maximum emission peaks had a hypsochromic shift from 550 nm to 480 nm. In the Fig. 1(b), the fluorescence intensity of compound 1a is the strongest in THF with the weaker polar, and the fluorescence is the weakest in water with the stronger polar. Compounds 1b and 1c also have the similar variation trend as 1a. These phenomena may be due to the fact that the charge separation of the compounds are more serious in a solvent with a higher polarity and the energy of the relaxed states are lower, resulting in a rapid dissipation of energy in the case of twisted intramolecular charge transfer (TICT), which cause a weaker fluorescence intensity and the large red shift19-21. The general physiological environment is in a neutral or weak alkaline one, so we tested the pH influenced optical properties of three compounds (Fig. S4). Fig. S4 shows that the three compounds are relatively stable in the neutral and alkaline environment, which is consistent with the pH environment of cell growth, making them suit for biological application22.
Fig. 1. Optical responses of compound 1a (10 μM) toward different solvents. (a) Absorption spectra; (b) Emission spectra (λex = 464 nm, slit width: 3 nm/1.5 nm); (c) photographs of compound 1a in different solvents; (d) photographs of compound 1a in different solvents under a lamp at 365 nm in dark room.
Since the water and lipid vesicles environment are important for biological applications23, it is necessary to study the optical properties of fluorescent markers 1a-c in water and lipid vesicles environment. It can be seen from the UV-vis absorption and fluorescence spectra of the three compounds (Fig. 2) that they have fluorescence signal in both water and lipid vesicles environment, and the fluorescence signal is stronger in lipid vesicles environment. This made compounds 1a-c more suitable for biological application.
Commented [user2]: According to the fifth comment of editor, we have placed the experimental part of this article in Supporting Information.
Commented [user4]: According to the third comment of reviewer 1, this sentence has been corrected.
Fig. 2. Optical responses of compounds 1a-c (10 μM) toward water and lipid vesicles (PC:PE=80:20). (a) Absorption spectrum; (b) Emission spectrum (λex = 440 nm, slit width: 3 nm/1.5 nm).
A CCK-8 was used to verify the cytotoxicity of fluorescent markers 1a-c. Fig. S5 shows that the three compounds have high cell survival rate in HeLa and L929 cells. At the concentration of 100-300 nmoL-1, when compounds 1a-c were cultured in HeLa and L929 cells for 6 hours, the survival rate of compounds 1a-c was over 90%. The survival rate of normal cells was more than 100% when compounds 1a and 1c were cultured in cells for six hours. Therefore, probe 1a and 1c could promote the growth of normal cells. These results indicate that 1a-b has low cytotoxicity, which is one of the key criteria for living cell imaging. From the pH optical response test of fluorescent markers 1a-c, they have good optical properties in a physiological environment. Therefore, L929 mouse fibroblastic normal cells were selected for cell experiments. Laser confocal imaging (Fig. 3) was
Commented [user3]: According to the second comment of reviewer 1, this sentence has been corrected.
Commented [user5]: According to the fourth comment of reviewer 1, this sentence has been corrected.
performed on the double target compound 1a. It can be seen compound 1a is roughly positioned in the lipid droplets (Fig. 3(b)), the same as commercial lipid droplet markers (Fig. 3(c)). Meanwhile, from the two channels being well overlapped (Fig. 3(d)), it can be determined that the compound 1a is specifically labeled with lipid droplets. Similarly, the cells were incubated with compound 1a and the commercial mitochondrial marker MitoTracker Red FM (50 nM) respectively, and strong fluorescence was observed in the green channel (Fig. 3(g)) and the red channel (Fig. 3(h)). It can be seen from Fig. 3(i) that compound 1a also has a good superposition with mitochondria, and the ROI curves further show that compound 1a has a similar trend with commercial mitochondrial markers (Fig. 3(j)). Thus, the results indicate that the compound 1a can well localize to lipid droplets and mitochondria in cells. The laser confocal experiment of compound 1b shows strong fluorescence in green channel (Fig. 4(b)) and red channel (Fig. 4(c)). Moreover, the two channels are well overlapped (Fig. 4(d)). From the ROI curve, the fluorescence trends of the two are similar. Similarly, the laser confocal experiments of compounds 1b-c have been done with L929 cells that are incubated with compounds 1b-c and the commercial lipid droplets marker Nile red or commercial mitochondrial marker MitoTracker Red FM (Fig. 4 and Fig. S6). Unfortunately, the results present compounds 1b-c can only locate lipid droplets in L929 cells well.
Commented [user6]: According to the fifth comment of reviewer 1, Mito Tracker has been corrected to MitoTracker.
Fig. 4. Fluorescence confocal images of L929 cells. (a) Bright–field images; (b) confocal images (green channel) of cells with compound 1b (5 μM); (c) confocal images (red channel) of cells with Nile red (50 nM); (d) merged images of green and red channels; (e) fluorescence intensity of the regions of interest (ROIs) across the cells. Green channel emission was collected in 500–571 nm upon excitation at 458 nm, and red channel emission was collected in 571–750 nm upon excitation at 561 nm.
In summary, three fluorescent markers with specific organelle targeting ability were constructed in this paper. They were synthesized from bioactive oxazolopyridine unit and photogenic precursors (coumarin, triphenylamine and phenothiazine). The three compounds have good photo stability, high fluorescence quantum yield and large stokes shifts. In addition, all of three compounds have lower cytotoxicity and high security for imaging living cells. Compound 1a based on oxazolopyridine and coumarin unit is reported as a dual targetable fluorescent marker for lipid droplets and mitochondria in L929 cells, while compounds 1b-c are single target fluorescent markers for lipid droplets. Therefore, the biocative oxazolopyridine unit has potential significance in improving the biological activity of fluorescent markers.
Acknowledgments We sincerely thank the National Natural Science Foundation of China (21977078), the Natural Science Found of Jiangsu Province (BK20181429), the Project of Scientific and Technological Infrastructure of Suzhou (SZS201708) and the Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher Education Institutions.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version.
References and notes
Fig. 3. Fluorescence confocal images of L929 cells. (a, f) Bright–field images; (b, g) confocal images (green channel) of cells with compound 1a (5 μM); (c) confocal image (red channel) of cells with Nile red (50 nM); (h) confocal image (red channel) of cells with MitoTracker Red FM (50 nM); (d, i) merged images of green and red channels; (e, j) fluorescence intensity of the regions of interest (ROIs) across the cells. Green channel emission was collected in 510–580 nm upon excitation at 488 nm, and red channel emission was collected in 575–750 nm upon excitation at 561 nm.
1. Liu H-W, Chen L, Xu C, et al. Recent progresses in smallmolecule enzymatic fluorescent probes for cancer imaging. Chem Soc Rev. 2018;4718:7140. 2. Ren M, Deng B, Wang J-Y, et al. A fast responsive two-photon fluorescent probe for imaging H2O2 in lysosomes with a large turnon fluorescence signal. Biosens Bioelectron. 2016;79:237. 3. Qi F, Zhang Y, Wang B, et al. A fluorescent probe for the dicriminatory detecion of Cys/Hcy, GSH and H2S in living cells and zebrafish. Sens Actuators, B. 2019;296:126533. 4. Tantray MA, Khan I, Hamid H, et al. Synthesis of Novel Oxazolo[4,5-b]pyridine-2-one based 1,2,3-triazoles as Glycogen Synthase Kinase-3β Inhibitors with Anti-inflammatory Potential. Chem Bio Drug Des. 2016;876:918. 5. Savelon L, Bizot-Espiard JG, Caignard DH, et al. 6Aminoalkyloxazolo[4,5-b]pyridin-2(3H)-ones: Synthesis and Evaluation of Antinoceptive Activity. Bioorg Med Chem. 1998;611:1963.
Commented [user7]: According to the sixth comment of reviewer 1, the fluorescence confocal imaging of compound 1a has been changed.
6. Marie-Claude V, Patricia J, Marie-Laure B, Laurence S, Gérald G. Acylation of oxazolo[4,5-b]pyridin-2(3H)-ones, 2phenyloxazolo[4,5-b]pyridines and pyrrolo[2,3-b]pyridin-2(2H)-ones. Tetrahedron. 1997;5314:5159. 7. Chakrabarty S, Kabekkodu SP, Singh RP, Thangaraj K, Singh KK, Satyamoorthy K. Mitochondria in health and disease. Mitochondrion. 2018;43:25. 8. Chen Y, Wei X-R, Sun R, Xu Y-J, Ge J-F. The fluorescent biomarkers for lipid droplets with quinolone-coumarin unit. Org Biomol Chem. 2018;1641:7619. 9. Beller M, Thiel K, Thul PJ, Jäckle H. Lipid droplets: A dynamic organelle moves into focus. FEBS Lett. 2010;58411:2176. 10. Zhang C, Liu P. The New Face of the Lipid Droplet: Lipid Droplet Proteins. Proteomics. 2019;1910:170 0223. 11. Wu W-L, Ma H-L, Xi LL, et al. A novel lipid dropletstargeting ratiometric fluorescence probe for hypochlorous acid in living cells. Talanta. 2019;194:308. 12. Chen B, Li C, Zhang J, et al. Sensing and imaging of mitochondrial viscosity in living cells using a red fluorescent probe with a long lifetime. Chem Commun. 2019;5551:7410. 13. Benador IY, Veliova M, Liesa M, Shirihai OS. Mitochondria Bound to Lipid Droplets: Where Mitochondrial Dynamics Regulate Lipid Storage and Utilization. Cell Metab. 2019;294:827. 14. He L, Cao J-J, Zhang D-Y, et al. Lipophilic phosphorescent iridium(III) complexes as one- and two-photon selective bioprobes for lipid droplets imaging in living cells. Sens Actuators, B. 2018;262:313. 15. Zheng X, Zhu W, Ni F, et al. Simultaneous dual-colour tracking lipid droplets and lysosomes dynamics using a fluorescent probe. Chem Sci. 2019;108:2342. 16. El-Wakil MH, Ashour HM, Saudi MN, Hassan AM, Labouta IM. Design, synthesis and molecular modeling studies of new series of antitumor 1,2,4-triazines with potential c-Met kinase inhibitory activity. Bioorg Chem. 2018;76:154. 17. Sha X-L, Niu J-Y, Sun R, Xu Y-J, Ge J-F. Synthesis and optical properties of cyanine dyes with an aromatic azonia skeleton. Org Chem Front. 2018;54:555. 18. Wu Q, Yin C, Wen Y, Zhang Y, Huo F. An ICT lighten ratiometric and NIR fluorogenic probe to visualize endogenous/exogenous hydrogen sulphide and imaging in mice. Sens Actuators, B. 2019;288:507. 19. Xie Z, Kong X, Feng L, et al. A novel highly selective probe with both aggregation-induced emission enhancement and intramolecular charge transfer characteristics for CN− detection. Sens Actuators, B. 2018;257:154. 20. Yin J, Peng M, Ma Y, Guo R, Lin W. Rational design of a lipiddroplet-polarity based fluorescent probe for potential cancer diagnosis. Chem Commun. 2018;5485:12093. 21. Jiang M, Gu X, Lam JWY, et al. Two-photon AIE bio-probe with large Stokes shift for specific imaging of lipid droplets. Chem Sci. 2017;88:5440.
22. Branagan D, Breslin CB. Electrochemical detection of glucose at physiological pH using gold nanoparticles deposited on carbon nanotubes. Sens Actuators, B. 2019;282:490. 23. Liu Z, Li G, Wang Y, et al. Quinoline-based ratiometric fluorescent probe for detection of physiological pH changes in aqueous solution and living cells. Talanta. 2019;192:6.
Declaration of interests √ The authors declare that they have no known
competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Graphical abstract: Bioactive heterocycle was used for the design of biomarkers, and one of the final products is a dual targetable fluorescent marker for organelles.
S0960-894X(20)30059-7 https://doi.org/10.1016/j.bmcl.2020.126996 BMCL 126996
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
4 December 2019 1 January 2020 24 January 2020
Please cite this article as: Wang, Y-N., Bing-Xu, Qiu, L-H., Sun, R., Xu, Y-J., Ge, J-F., The fluorescent markers based on oxazolopyridine unit for imaging organelles, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.126996
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.
Graphical Abstract
The fluorescent markers based on oxazolopyridine unit for imaging organelles
Leave this area blank for abstract info.
Ya-Nan Wanga, Bing-Xub, Li-Hua Qiua*, Ru Suna, Yu-Jie Xub and Jian-Feng Gea,c*
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com
The fluorescent markers based on oxazolopyridine unit for imaging organelles Ya-Nan Wang,a Bing-Xu,b Li-Hua Qiu a Ru Sun,a Yu-Jie Xu,b Jian-Feng Gea,c College of Chemistry, Chemical Engineering and Material Science, Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, P. R. China. b State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China c Jiangsu Key Laboratory of Medical Optics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, P. R. China. a
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ARTICLE INFO
ABSTRACT
Corresponding author: E-mail: [email protected], [email protected] (J.-F. Ge).
Article history: Received Revised Accepted Available online Keywords: Bioactive Oxazolopyridine Fluorescent markers Dual targetable Photogenic precursors
Bioactive oxazolopyridine unit was used in the synthesis of fluorescent markers for specific organelles in this paper. The compounds 1a-c are linked with double bond between oxazolopyridine ring and photogenic precursors (3a-c). Compounds 1a-c showed higher fluorescence yield (0.47-0.86), larger stokes shift (103-224) and good photo stability. In lipid vesicles environment, they also showed good optical properties. In addition, the three compounds are biomarkers with lower cytotoxicity. Among them, compound 1a based on oxazolopyridine and coumarin unit is a dual targetable fluorescent marker for mitochondria and lipid droplets; while the other two compounds 1b-c are only biomarkers for lipid droplets.
Fluorescent markers have been favored by most scientists in recent years because of their high sensitivity, good selectivity and convenient to be used1-3. Most fluorescent markers with good optical properties have a large rigid structure resulting in insufficient biocompatibility. Therefore, it is important to design and synthesize fluorescent markers with good biological activity and optical properties. Oxazolo[4,5-b]pyridine is particularly popular in biomedicine and is often used in the synthesis of antiinflammatory or antibacterial medicines, so it has good biological activity; Although oxazolo[4,5-b] pyridine is involved in medicine, it is rarely involved in dye and organelle targeting4-6. Mitochondria and lipid droplets are two important organelles in cells. Mitochondria are the providers of energy in cells, providing almost all the energy needed for cellular processes, and are closely related to other aspects of cell biology7; lipid droplets are mainly found in eukaryotic cells and are used in cells for storage. The organelle of lipids mainly regulates the storage and hydrolysis of neutral lipids8. Moreover, lipid droplets are dynamic organelle surfaces and enzymes involved in metabolism9, 10. Reported studies on biomarkers for mitochondrial targeting are relatively thorough, while studies on lipid droplet-labeled fluorescent markers are relatively rare and a few specific groups can be used to identify targeted lipid droplets11. Therefore, it is necessary to study fluorescent markers that are capable of specifically labeling lipid droplets. At the same time, there are close relationships between mitochondria and lipid droplets in the cells, which affect the normal functioning of the living body12. For example, when cells need
.
energy, the fatty acids that are absorbed by the cells and stored in the lipid droplets are hydrolyzed, and then ATP is produced by the action of mitochondria, to provide the energy needed by the cells. The injury in this process can lead to metabolic disorders, disease and even death13, 14. Therefore, it is important to design fluorescent marker that can label two organelles simultaneously. However, some articles on organelle markers have basically labeled single organelles in recent years, while labeling two organelles with few fluorescent markers. This may be because small molecule fluorescent markers will accumulate in a specific organelle or subcellular region after entering the cell, so it is relatively difficult to enter two organelles simultaneously15. Therefore，up to now, there are few articles have used a single molecule to mark two organelles in living cells at the same time and fewer biomarkers target lipid droplets and mitochondria simultaneously. So it is of great significance to study the biomarkers of lipid droplets and mitochondria simultaneously. The structure of a nitrogen-containing heterocyclic is often observed in anticancer, antitumor, antiviral and the like, but is rarely involved in fluorescent labels16, 17. The oxazolo[4,5b]pyridine (2) used in this paper has good biological activity and is widely used in the synthesis of anti-inflammatory and antibacterial medicines. Compound 2 and photogenic precursors 3a-c (coumarin (3a), triphenylamine (3b), phenothiazine (3c)) were reacted by Knoevenagel reaction to obtain fluorescent markers 1a-c. The specific design and synthesis route of the three fluorescent markers are as follows:
Commented [user1]: According to the comment of reviewer 2, we supplement the novelty of this research in this manuscript.
Scheme 1. Synthesis of compounds 1a-c. As shown in Scheme 1, compounds 1a-c were synthesized by compound 2 and compounds 3a-c at 130 oC in toluene. The yields were 41–53.1 % and all structures were confirmed by 1H NMR, 13C NMR and HRMS in detail (Fig. S7-15). The photo stability is also one of the criteria for measuring the performance of a compound. Therefore, the photo stability of the three compounds in acetonitrile was tested (Fig. S1), using Nile red with better photo stability as control. After irradiation with 500 W Philips iodine tungsten lamp for six hours, the maximum relative residual absorption of Nile red was 97%, and the maximum relative residual absorption of the three compounds was 94% (1a), 93% (1b) and 92% (1c), respectively. These data show that the compounds 1a-c have good photo stability. Fluorescent markers can exhibit different optical properties in different solvents, so the optical properties of fluorescent markers 1a-c in different solvents were tested18. The UV-vis absorption and fluorescence emission spectra of fluorescent markers 1a-c in water (H2O), dimethyl sulfoxide (DMSO), methanol (CH3OH), chloroform (CHCl3), tetrahydrofuran (THF), toluene (TOL) were tested (Fig. 1 and Figs. S2-S3). Further detailed data on the fluorescent markers 1a-c in different solvents were recorded in Table S1. It can be clearly seen from the Fig. 1 that the compound 1a shows different optical properties in different solvents. Generally, the fluorescence intensity of compound 1a increased with the decrease of solvent polarity, and the fluorescence peak showed a significant blue shift. This is, from water to toluene, the maximum absorption peaks of compound 1a blue shifted from 490 nm to 445 nm, and the maximum emission peaks had a hypsochromic shift from 550 nm to 480 nm. In the Fig. 1(b), the fluorescence intensity of compound 1a is the strongest in THF with the weaker polar, and the fluorescence is the weakest in water with the stronger polar. Compounds 1b and 1c also have the similar variation trend as 1a. These phenomena may be due to the fact that the charge separation of the compounds are more serious in a solvent with a higher polarity and the energy of the relaxed states are lower, resulting in a rapid dissipation of energy in the case of twisted intramolecular charge transfer (TICT), which cause a weaker fluorescence intensity and the large red shift19-21. The general physiological environment is in a neutral or weak alkaline one, so we tested the pH influenced optical properties of three compounds (Fig. S4). Fig. S4 shows that the three compounds are relatively stable in the neutral and alkaline environment, which is consistent with the pH environment of cell growth, making them suit for biological application22.
Fig. 1. Optical responses of compound 1a (10 μM) toward different solvents. (a) Absorption spectra; (b) Emission spectra (λex = 464 nm, slit width: 3 nm/1.5 nm); (c) photographs of compound 1a in different solvents; (d) photographs of compound 1a in different solvents under a lamp at 365 nm in dark room.
Since the water and lipid vesicles environment are important for biological applications23, it is necessary to study the optical properties of fluorescent markers 1a-c in water and lipid vesicles environment. It can be seen from the UV-vis absorption and fluorescence spectra of the three compounds (Fig. 2) that they have fluorescence signal in both water and lipid vesicles environment, and the fluorescence signal is stronger in lipid vesicles environment. This made compounds 1a-c more suitable for biological application.
Commented [user2]: According to the fifth comment of editor, we have placed the experimental part of this article in Supporting Information.
Commented [user4]: According to the third comment of reviewer 1, this sentence has been corrected.
Fig. 2. Optical responses of compounds 1a-c (10 μM) toward water and lipid vesicles (PC:PE=80:20). (a) Absorption spectrum; (b) Emission spectrum (λex = 440 nm, slit width: 3 nm/1.5 nm).
A CCK-8 was used to verify the cytotoxicity of fluorescent markers 1a-c. Fig. S5 shows that the three compounds have high cell survival rate in HeLa and L929 cells. At the concentration of 100-300 nmoL-1, when compounds 1a-c were cultured in HeLa and L929 cells for 6 hours, the survival rate of compounds 1a-c was over 90%. The survival rate of normal cells was more than 100% when compounds 1a and 1c were cultured in cells for six hours. Therefore, probe 1a and 1c could promote the growth of normal cells. These results indicate that 1a-b has low cytotoxicity, which is one of the key criteria for living cell imaging. From the pH optical response test of fluorescent markers 1a-c, they have good optical properties in a physiological environment. Therefore, L929 mouse fibroblastic normal cells were selected for cell experiments. Laser confocal imaging (Fig. 3) was
Commented [user3]: According to the second comment of reviewer 1, this sentence has been corrected.
Commented [user5]: According to the fourth comment of reviewer 1, this sentence has been corrected.
performed on the double target compound 1a. It can be seen compound 1a is roughly positioned in the lipid droplets (Fig. 3(b)), the same as commercial lipid droplet markers (Fig. 3(c)). Meanwhile, from the two channels being well overlapped (Fig. 3(d)), it can be determined that the compound 1a is specifically labeled with lipid droplets. Similarly, the cells were incubated with compound 1a and the commercial mitochondrial marker MitoTracker Red FM (50 nM) respectively, and strong fluorescence was observed in the green channel (Fig. 3(g)) and the red channel (Fig. 3(h)). It can be seen from Fig. 3(i) that compound 1a also has a good superposition with mitochondria, and the ROI curves further show that compound 1a has a similar trend with commercial mitochondrial markers (Fig. 3(j)). Thus, the results indicate that the compound 1a can well localize to lipid droplets and mitochondria in cells. The laser confocal experiment of compound 1b shows strong fluorescence in green channel (Fig. 4(b)) and red channel (Fig. 4(c)). Moreover, the two channels are well overlapped (Fig. 4(d)). From the ROI curve, the fluorescence trends of the two are similar. Similarly, the laser confocal experiments of compounds 1b-c have been done with L929 cells that are incubated with compounds 1b-c and the commercial lipid droplets marker Nile red or commercial mitochondrial marker MitoTracker Red FM (Fig. 4 and Fig. S6). Unfortunately, the results present compounds 1b-c can only locate lipid droplets in L929 cells well.
Commented [user6]: According to the fifth comment of reviewer 1, Mito Tracker has been corrected to MitoTracker.
Fig. 4. Fluorescence confocal images of L929 cells. (a) Bright–field images; (b) confocal images (green channel) of cells with compound 1b (5 μM); (c) confocal images (red channel) of cells with Nile red (50 nM); (d) merged images of green and red channels; (e) fluorescence intensity of the regions of interest (ROIs) across the cells. Green channel emission was collected in 500–571 nm upon excitation at 458 nm, and red channel emission was collected in 571–750 nm upon excitation at 561 nm.
In summary, three fluorescent markers with specific organelle targeting ability were constructed in this paper. They were synthesized from bioactive oxazolopyridine unit and photogenic precursors (coumarin, triphenylamine and phenothiazine). The three compounds have good photo stability, high fluorescence quantum yield and large stokes shifts. In addition, all of three compounds have lower cytotoxicity and high security for imaging living cells. Compound 1a based on oxazolopyridine and coumarin unit is reported as a dual targetable fluorescent marker for lipid droplets and mitochondria in L929 cells, while compounds 1b-c are single target fluorescent markers for lipid droplets. Therefore, the biocative oxazolopyridine unit has potential significance in improving the biological activity of fluorescent markers.
Acknowledgments We sincerely thank the National Natural Science Foundation of China (21977078), the Natural Science Found of Jiangsu Province (BK20181429), the Project of Scientific and Technological Infrastructure of Suzhou (SZS201708) and the Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher Education Institutions.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version.
References and notes
Fig. 3. Fluorescence confocal images of L929 cells. (a, f) Bright–field images; (b, g) confocal images (green channel) of cells with compound 1a (5 μM); (c) confocal image (red channel) of cells with Nile red (50 nM); (h) confocal image (red channel) of cells with MitoTracker Red FM (50 nM); (d, i) merged images of green and red channels; (e, j) fluorescence intensity of the regions of interest (ROIs) across the cells. Green channel emission was collected in 510–580 nm upon excitation at 488 nm, and red channel emission was collected in 575–750 nm upon excitation at 561 nm.
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Declaration of interests √ The authors declare that they have no known
competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Graphical abstract: Bioactive heterocycle was used for the design of biomarkers, and one of the final products is a dual targetable fluorescent marker for organelles.