Elsevier

Dyes and Pigments

Volume 145, October 2017, Pages 461-468
Dyes and Pigments

Development of fluorescent mitochondria probe based on 1,2-dihydropyrrolo[3,4-b]indolizine-3-one

https://doi.org/10.1016/j.dyepig.2017.06.014Get rights and content

Abstract

Mitochondria are one of the most important organelles in our body. Recent studies showed morphology of polymorphous organelle mitochondria can be used as a relevant biological marker for certain diseases. In this study, we developed new fluorescent mitochondrial probe, 5, for monitoring mitochondrial morphology via rational probe design using 1,2-dihydropyrrolo[3,4-b]indolizine-3-one. To overcome the drawback of triphenylphosphonium incorporation, pyridinium group was introduced as a mitochondria targeting moiety. Additional incorporation of olefin moiety between pyridinium and indolizine core skeleton in 5 induced a bathochromic shift of both absorption and emission wavelength along with increased hydrophobicity. Finally, we found 5 selectively stains the mitochondria under the live-cell condition. This work indicated that a simple change of chemical structure in a certain organic fluorophore can cause a significant transition in staining pattern of bioprobes.

Introduction

Mitochondria are one of the most important cellular organelle and play pivotal roles in cellular processes such as ATP synthesis [1], signaling [2], apoptosis [3], and redox homeostasis [4]. Therefore, mitochondrial dysfunction may cause various human diseases including cancer, metabolic diseases, aging and degenerative diseases [5] [6] [7]. Interestingly, recent studies revealed that cancer cells have different patterns in mitochondrial morphology [8] and, therefore, mitochondrial phenotyping can be used as a biological marker for assessing cancer phenotype and drug response [8] [9] [10]. In other word, specific monitoring of mitochondrial morphology can help to study the cellular function and disease states of the cells. Owing to the advantageous features, such as high sensitivity, real-time monitoring, and easy accessibility, fluorescent imaging technique became an indispensable scientific tool for modern biomedical and life sciences [11] [12]. In this context, there are high demands for the development of fluorescent mitochondrial bioprobes to monitor mitochondrial phenotypes as molecular diagnostic tools for the related diseases. One of the most straightforward approaches for the development of fluorescent mitochondrial probes is the conjugation of triphenylphosphonium (TPP) moiety to the fluorochromes [13] [14] [15]. Because of strong negative potential at the mitochondrial membrane, lipophilic cationic molecules tend to pass through the inner membrane, and be accumulated inside of mitochondria. Therefore, simple conjugation of a TPP to fluorochrome can guide the molecule to the mitochondria. However, the major drawbacks of TPP-containing fluorochromes are their poor solubility in the aqueous condition, disruptive effect on mitochondrial function via proton leaking [16], [17] and inhibition of mitochondrial respiration [17] [18]. In this regards, alternative molecular design strategies or elaborated molecular system are highly desirable. After the discovery of new fluorescent molecular framework, 1,2-dihydropyrrolo[3,4-b]indolizin-3-one (Seoul-Fluor), the underlying principles of structure-photophysical property relationship of Seoul-Fluor have been unveiled [19] [20] [21] [22]. Through a series of systematic studies, we demonstrated Seoul-Fluor system can serve as a versatile molecular platform for the development of fluorescent bioprobes in an efficient manner [20] [23] [24] [25] [26]. Among several advantageous features of Seoul-Fluor system, it is notable that various functional groups can be easily introduced to the system in a combinational manner and the photophysical properties of Seoul-Fluor, including emission wavelength, extinction coefficient, and quantum yield, could be controlled by changing the functional groups with an excellent predictability [19] [20] [22]. Therefore, we envisioned the development of new fluorescent mitochondria probes using Seoul-Fluor system without TPP moiety through rational molecular design.

Section snippets

Materials and instruments

All reactions were carried out under an atmosphere of nitrogen or argon in air-dried glassware with magnetic stirring. Air- and/or moisture-sensitive liquids were transferred with syringe. Organic solutions were concentrated by rotary evaporation at 25–60 °C at 15–30 torr. All solvents and common materials were purchased from suppliers and used without further purification. Column chromatography was carried out as “Flash Chromatography” using Biotage MPLC machine. 1H NMR and 13C NMR data were

Rational probe design and efficient synthesis of mitochondria targeting probe

Because of negative mitochondrial membrane potential, lipophilic cationic molecules are known to equilibrate across the membranes in a Nernstian fashion [27] and accumulated into the mitochondrial membrane matrix. Therefore, we first designed the probe 4 having (+1) net charge through the introduction of pyridinium moiety at the R1 position (Fig. 1) [28], [29], [30]. To induce the bathochromic shift and increase hydrophobicity, we also designed another probe 5 having olefin moiety between

Conclusion

In summary, we rationally designed novel fluorescent mitochondrial probes using indolizine-based Seoul-Fluor system. Instead of the conventional TPP moiety, pyridinium moiety was incorporated to Seoul-Fluor system for targeting negatively charged mitochondrial matrix. Notably, we discovered that the insertion of olefin moiety between the indolizine fluorophore and pyridinium moiety caused drastic differences in terms of absorption, emission wavelength and mitochondrial staining ability of

Acknowledgements

Following are results of a study on the “Leaders INdustry-university Cooperation” Project, supported by the Ministry of Education. This research was also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2009-0093826) and by National Research Foundation of Korea grant (NRF-2016R1C1B2014699, NRF-2016M3A9C4939668, and NRF-2012M3A9C4048780).

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