Noncovalent interactive fluorescent probe detects flavin analogue in live cells Shayeri Biswas a , Sourav Sarkar b , Kyo Han Ahn b , Sankarprasad Bhuniya* a a JIS University, India, b POSTECH, Pohang, Republic of Korea. E-Mail: spbhuniya@jisiasr.org The isoalloxazine moiety of flavin can easily accept and donate electrons to favor it to participate in various redox reactions in live cells. 1 Flavin adenine dinucleotide (FAD) is one of the analogues that resides in the mitochondrial electron transport chain in the mitochondria inner membrane region and actively participates in oxidative phosphorylation to produce ATP. A few number of fluorescent probes for FAD have been reported. 2-4 The growing interest in the search for cancer markers to unfold various biological events in cancer cells motivates us to develop markers for flavin in cancer cells. The probe was synthesized in three successive steps as shown in the Scheme 1. 5 We observed that the probe FL FAD has UV absorption at 370 nm and corresponding emission at 450 nm.
The fluorescence at 450 nm of the probe ( FL FAD ) has quenched immediately upon addition of flavin analogue only. After addition of various flavin analogues with the variable appended hydrophobic unit (Fig. 1a), a FRET transfer to flavin with emission at 545 nm (Fig. 1b) has been observed. Obviously, this FRET process is because of the overlap of emission spectra of probe FL FAD with absorption spectra of flavin (Fig. 1c). The fluorescence intensity at 545 nm increased with increasing hydrophobicity of the flavin
analogues. Moreover, protons of both quinoline (10.09 ppm, 9.79 ppm, 8.91 ppm) and coumarin (8.26 ppm, 8.14–8.11 ppm) shifted toward the upfield upon interaction with p-electron rich isoalloxazine ring of riboflavin (Fig. 1d). Additionally, we observed in a molecular docking study using Autodoc 4.2 software that the average distance between the isoalloxazine ring of FMN and the aryl ring of probe ( FL FAD ) is only 3.9 A o (Fig. 1e). This study further advocated that noncovalent association between flavin and probe ( FL FAD ) favors FRET transfer process.
Moreover, increment of fluorescence lifetime of the probe ( FL FAD ) from 1.73 ns to 2.58 ns in the presence of FAD (100 µM) approved a stable FRET transfer process. Finally, we noticed that FAD expression in cancer cells (HeLa and MCF7 cells) is higher than the normal cells (C2C12, PNT2 cells) (Fig. 2). In conclusion, the fluorescent probe FL FAD can discriminate cancer cells from normal cells based on the expression of flavin analogues through FRET-based fluorescence imaging. References 1. R. B. Silverman, Acc. Chem. Res. , 1995, 28 , 335. 2. L. Zhang, S. Li, Y. Sun, K. Xiao, G. Song, P. Lu, S. Yin, K. Huang and Z. Yao, Polym. Chem. , 2020, 11 , 3762. 3. S. Hussain, A. H. Malika and P. K. Iyer, J. Mater. Chem. B , 2016, 4 , 4439. 4. H.-W. Rhee, H.-Y. Choi, K. Han and J.-I. Hong, J. Am. Chem. Soc. , 2007, 129 , 4524. 5. S. Biswas, S. Sarkar, A. Shil, T. S. Demina, K. H. Ahn and S. Bhuniya, Anal. Chem. , 2022, 94 , 3494.
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