A fluorescent naphthalimide NADH mimic for continuous and reversible sensing of cellular redox state

Hemant Sharma a, Nian Kee Tan b, Natalie Trinh b, Jia Hao Yeo b, Elizabeth J. New b and Frederick M. Pfeffer *a
aSchool of Life and Environmental Sciences, Deakin University, Waurn Ponds, Victoria 3216, Australia. E-mail: fred.pfeffer@deakin.edu.au
bSchool of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia

Received 16th December 2019 , Accepted 22nd January 2020

First published on 22nd January 2020

A fluorescent, naphthalimide-based, NADH mimic has been synthesised as a reversible, biocompatible, “on–off” probe for the detection of changes in intracellular redox environment (both oxidation and reduction). Interconversion was confirmed by means of electrochemistry and also 1H NMR, UV-vis and fluorescence spectroscopy. The reversibility was also successfully detected in A549 cells under simulated redox stress.

Dysregulation of the cellular redox environment can lead to either oxidative stress or hypoxia. Both conditions are associated with serious health concerns including cardiovascular disease,1 stroke2 obesity,3 diabeties4 and arthritis.5 Although the exact mechanistic links between oxidative stress/hypoxia and disease have not been fully elucidated, a number of studies have confirmed that afflicted tissues consistently suffer from an imbalance in the cellular redox environment.6 Thus, for diagnostic purposes, a means to indicate hypoxia and oxidative stress under biological conditions would be of considerable use. Furthermore, for understanding the fundamental mechanistic links, a probe capable of full reversibility, without degradation is essential.

Given the physiological importance of redox homeostasis, a number of techniques have been developed to identify both hypoxia and oxidative stress including positron emission tomography7,8 magnetic resonance imaging,9,10 phosphorescence imaging,11,12 immunostaining13 and fluorescence imaging.14–16 Of these, fluorescence imaging has significant advantages including low cost and a good balance of selectivity and sensitivity.17–19

The development and use of fluorescent probes has received considerable attention in the study of biological systems and various fluorescent probes have been reported for the detection of either oxidative stress or hypoxia.20–24 The development of probes that can detect both oxidative stress and hypoxia in a reversible manner are less common.25,26

Herein we describe the development of a naphthalimide-based NADH mimic 4 (NapNic) as a reversible fluorescent probe to continually monitor and report the cellular redox environment. The reversibility of the probe was confirmed through electrochemical, UV/vis, fluorescence and 1H NMR spectroscopic techniques.

In the design of the probe, the use of nicotinamide was inspired by nature as the moiety confers the redox activity, and most importantly the reversibility, of natural coenzymes NADPH and NADH.27,28 One of the challenges in incorporating nicotinamide into a small molecule probe is ensuring that the coupling does not shift the reduction potential out of the biologically-relevant range. We have previously shown that coupling through the pyridine nitrogen dramatically alters the reduction potential, and in turn reversibility,29 while attachment through the alkyl group retains the biologically-relevant redox properties and reversibility.30 In the present study, we chose to chemically modify the nicotinamide structure through the amide. For probe function, the reversibility of the NAD+/NADH redox system must be converted to a fluorescent response and to achieve this, the nicotinamide amide was directly coupled to a 1,8-naphthalimide. Substituted 1,8-naphthalimides are well known in the field of fluorescent probe development as they possess excellent photophysical properties including photostability, large Stokes shift and high quantum yield.31–33

Probe 4 (NapNic) was synthesised in only three steps (Scheme 1) beginning with microwave mediated condensation of 4-bromo-1,8-naphthalic anhydride with n-propylamine to give the known naphthalimide 2.34,35 The key step involved direct Pd mediated amidation34 of imide 2 with nicotinamide36 using G3-xantphos catalyst to afford the desired 4-amidonaphthalimide 3 in 80% yield after simple aqueous work-up. Finally, N-alkylation of amide 3 was accomplished using 4-fluorobenzylbromide to give probe 4 (NapNic) in 84% yield as the bromide salt.

image file: c9cc09748a-s1.tif
Scheme 1 Synthesis of probe 4 (NapNic) showing structures for both the oxidised and reduced forms.

The photophysical properties of the new probe were investigated in phosphate buffer (10 mM, pH 7.4) and the oxidised form of probe 4 (i.e.4+(NapNic+)) displayed a strong absorption band centred at 360 nm with a broad shoulder at 431 nm (see ESI, Fig. S7). The emission spectrum was typical of 4-amidonaphthalimides,34 with a peak at 474 nm in phosphate buffer (λex = 360 nm, ΦF = 0.03 and εM = 16[thin space (1/6-em)]322 M−1 cm−1 see ESI, Fig. S8).

To investigate how the redox behaviour of NapNic, would influence the photophysical properties, reduction was trialled using a range of mild reducing agents including sodium dithionite, dithiothreitol (DTT) and glutathione (GSH). These agents all readily produced the reduced form of NapNic (i.e.NapNic-H, Scheme 1) with clear changes in both absorption and emission spectra. For example, the incremental addition of sodium dithionite into a solution of probe NapNic (Fig. 1a) was accompanied by stepwise fluorescence quenching.

image file: c9cc09748a-f1.tif
Fig. 1 (A) Change in the emission spectra of probe 4 (NapNic) (10 μM, λex = 360 nm) with the incremental addition of Na2S2O4 (0–500 μM) in phosphate buffer (10 mM, pH 7.4). Inset: Visible fluorescence from cuvettes under UV lamp with (left) no Na2S2O4 and (right) after addition of 500 μM Na2S2O4. (B) Change in fluorescence intensity (at 474 nm) of probe 4 (NapNic) to cycles of oxidation and reduction (reduction performed with Na2S2O4 (500 μM) and oxidation using H2O2 (1 mM)).

Upon reduction, a new peak at 410 nm was observed in the absorption spectrum (Fig. S9, ESI). This reduced form of NapNic could be readily re-oxidised using H2O2 (or slowly in air) and the original photophysical properties were recovered. The reduction–oxidation cycle could be repeated several times without loss in the fluorescence intensity of the oxidised form (Fig. 1b). The kinetics of this reduction–oxidation process were investigated by monitoring the fluorescence intensity at 474 nm over 6 h (see ESI, Fig. S10). The reduction process using Na2S2O4 was considerably faster (1–2 min) than the oxidation process using H2O2 (25–30 min). The effect of pH on the fluorescence profile of NapNic was investigated and under acidic conditions a slight enhancement in intensity was noted (Fig. S11 and S12, ESI). However, significant quenching and a red shift in emission was observed under basic conditions; a response that was attributed to deprotonation of the amide.34

The photophysical properties of NapNic-H/NapNic+ compare favourably with those of NADH/NAD+. NADH has two absorption peaks at 259 nm and 339 nm, but NAD+ has absorption only in ultraviolet region (259 nm).28 As mentioned above, oxidised NapNic has a significantly red-shifted absorption spectra with two absorption peaks (360 nm and 431 nm). A similar red-shift was noted in the absorption spectra of reduced NapNic when compared to that of NADH. The red shifted nature of the absorption is consistent with involvement of the naphthalimide in the electronic transitions. This hypothesis was further supported by comparing the fluorescence spectra. While the reduced form of NAD (NADH) emits at 460 nm (ΦF = 0.019),37 NAD+ does not fluorescence.28 In contrast, for NapNic the fluorescence behaviour is reversed with only the oxidised form being fluorescent. Based on these studies it is clear that the photophysical properties of the naphthalimide dominate the emission behaviour of NapNic.

Additional studies were performed to confirm the redox activity of NapNic and cyclic voltammetry identified clear reversibility with reduction and oxidation potentials at −0.544 and −0.294 V vs. SHE respectively (Fig. 2).

image file: c9cc09748a-f2.tif
Fig. 2 Cyclic voltammogram of probe 4 (NapNic) (1 mM) in degassed acetonitrile, with 0.1 M tetrabutylammonium hexafluorophosphate as the supporting electrolyte and ferrocene/ferrocenium as an internal standard. Scan rate was 100 mV s−1 at 25 °C.

As the redox potential of probe NapNic lies within the biologically relevant reduction potential range,38 the probe was expected to be able to report on any cellular redox events.

To investigate the structural changes that accompany the reduction and oxidation reactions the process was monitored using 1H NMR spectroscopy in DMSO-d6 (Fig. 2). Addition of sodium dithionite to the oxidised form of NapNic resulted in considerable changes to the entire spectrum (Fig. 3b). In particular, the resonances (a–e) assigned to the nicotinamide group (Fig. 3, highlighted in red) migrated upfield. For proton He a significant change in chemical shift accompanied the transformation (from δ = 9.36 → 4.51 ppm, Fig. 3). Similarly, the protons of the benzylic methylene group also experienced a significant change (5.99 → 4.66 ppm, Fig. 3) and these changes are in line with those reported for simple, non-fluorescent, NADH mimics.39 An excess of H2O2 was added to the same NMR tube and after only 15 minutes both the oxidised and reduced forms of NapNic were evident in the 1H NMR spectrum (see ESI, section S5). After 6 hours the spectrum (Fig. 3C) matched perfectly with that of the original.

image file: c9cc09748a-f3.tif
Fig. 3 Partial 1H NMR spectra of probe 4 in DMSO-d6 (A) oxidised probe as synthesised (B) reduced probe following reaction with Na2S2O4 (50 equiv.) and (C) re-oxidised probe using H2O2 (100 equiv.) after 6 h. Full 1H NMR spectra for this process are available (see ESI, section S5).

The combination of NMR, UV-vis, fluorescence and cyclic voltammetry techniques unequivocally demonstrated the redox reversibility of the new probe NapNic. With these promising results, the probe was then applied to biological systems to assess its capability as a cellular redox probe.

Imaging of the probe in A549 lung adenocarcinoma cells was performed to observe whether NapNic could respond to changes in oxidation state within cells. NapNic exhibited localisation in the lipid droplets, confirmed by co-localisation with the lipid droplet stain Nile Red (see ESI, Fig. S15). This is consistent with previously-reported lipophilic naphthalimides that show similar localisation.40 The probe showed an intracellular spectral profile with maximum emission between 450 and 500 nm, consistent with the in vitro emission profile (Fig. 1A and ESI, Fig. S16). Finally, analysis of the confocal images of NapNic in A549 cells revealed a statistically significant cycle of fluorescence increase, then decrease, when cells initially treated with H2O2 were then subject to reducing conditions (Fig. 4). Importantly, both sodium dithionite and N-acetylcysteine were effective reductants, indicating that the sensitivity of NapNic fluorescence is suited to the whole cell environment. The fluorescence of NapNic in cells could be increased upon treatment with a second aliquot of H2O2, highlighting the reversible and reusable nature of the probe.

image file: c9cc09748a-f4.tif
Fig. 4 (A) Confocal microscopy images of A549 cells stained with NapNic (50 μM, 30 min) and Nile Red (50 μM, 30 min): (i) channel 1 (430–470 nm; NapNic emission) (ii) channel 2 (570–670 nm; Nile Red emission) (iii) merged images of channel 1 and 2. Pearson's coefficient of R = 0.762 with Nile Red. Scale bars represent 30 μm (B and C) fluorescence intensities (analysis performed using ImageJ) from confocal imaging of A549 cells treated with (B) NapNic (35 μM, 30 min), followed by sequential addition of H2O2 (50 μM, 30 min), Na2S2O4 (50 μM, 30 min) and H2O2 (50 μM, 30 min). Error bars represent the standard error of the mean fold change (SEM.) across 3 independent experiments; N = 3 independent experiments, *p < 0.05. (B) As for A with N-acetylcysteine (NAC) (50 μM, 30 min) used as the reducing agent.

In conclusion, we have developed a new class of redox probe exemplified by NapNic. While other redox sensitive probes are available,15,26,41NapNic is unique in successfully employing the nicotinamide component of the naturally occurring NAD+/NADH as the reversible reactive component. The new probe NapNic can be reduced by mild reducing agents and re-oxidised by simple oxidants and even air. Complete reversibility was established using a number of techniques including 1H NMR spectroscopy. The reduction/oxidation cycle can be repeated many times without loss of fluorescence intensity. The fluorescence response of NapNic to oxidation and reduction was evident in A549 cells, with NapNic being best suited to reporting on redox changes in lipophilic regions of the cell such as lipid droplets.

The authors would like to acknowledge Deakin University for an Alfred Deakin Fellowship (HS), the Australian Research Council (DP180101353, DP180101897) for funding, and the University of Sydney for a SOAR Fellowship (EJN). We acknowledge the scientific and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Australian Centre for Microscopy and Microanalysis (ACMM).

Conflicts of interest

There are no conflicts to declare.

Notes and references

  1. J. F. Garvey, C. T. Taylor and W. T. McNicholas, Eur. Respir. J., 2009, 33, 1195–1205 CrossRef CAS PubMed .
  2. Y. Tang, A. Lu, B. J. Aronow, K. R. Wagner and F. R. Sharp, Eur. J. Neurosci., 2002, 15, 1937–1952 CrossRef PubMed .
  3. S. Furukawa, T. Fujita, M. Shimabukuro, M. Iwaki, Y. Yamada, Y. Nakajima, O. Nakayama, M. Makishima, M. Matsuda and I. Shimomura, J. Clin. Invest., 2017, 114, 1752–1761 CrossRef PubMed .
  4. J. W. Baynes, Diabetes, 1991, 40, 405–412 CrossRef CAS PubMed .
  5. M. M. Maurice, H. Nakamura, E. A. van der Voort, A. I. van Vliet, F. J. Staal, P. P. Tak, F. C. Breedveld and C. L. Verweij, J. Immunol., 1997, 158, 1458–1465 CAS .
  6. W. R. Wilson and M. P. Hay, Nat. Rev. Cancer, 2011, 11, 393–410 CrossRef CAS PubMed .
  7. J. L. Dearling, J. S. Lewis, G. E. Mullen, M. J. Welch and P. J. Blower, JBIC, J. Biol. Inorg. Chem., 2002, 7, 249–259 CrossRef CAS PubMed .
  8. D. J. Yang, S. Wallace, A. Cherif, C. Li, M. B. Gretzer, E. E. Kim and D. A. Podoloff, Radiology, 1995, 194, 795–800 CrossRef CAS PubMed .
  9. J. Pacheco-Torres, P. López-Larrubia, P. Ballesteros and S. Cerdán, NMR Biomed., 2011, 24, 1–16 CrossRef CAS PubMed .
  10. S. Iwaki, K. Hanaoka, W. Piao, T. Komatsu, T. Ueno, T. Terai and T. Nagano, Bioorg. Med. Chem. Lett., 2012, 22, 2798–2802 CrossRef CAS PubMed .
  11. S. Zhang, M. Hosaka, T. Yoshihara, K. Negishi, Y. Iida, S. Tobita and T. Takeuchi, Cancer Res., 2010, 70, 4490–4498 CrossRef CAS PubMed .
  12. S. Sakadžić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov and D. A. Boas, Nat. Methods, 2010, 7, 755 CrossRef PubMed .
  13. J. H. A. M. Kaanders, K. I. E. M. Wijffels, H. A. M. Marres, A. S. E. Ljungkvist, L. A. M. Pop, F. J. A. van den Hoogen, P. C. M. de Wilde, J. Bussink, J. A. Raleigh and A. J. van der Kogel, Cancer Res., 2002, 62, 7066–7074 CAS .
  14. E. W. Miller, S. X. Bian and C. J. Chang, J. Am. Chem. Soc., 2007, 129, 3458–3459 CrossRef CAS PubMed .
  15. A. Kaur, J. L. Kolanowski and E. J. New, Angew. Chem., 2016, 128, 1630–1643 CrossRef .
  16. S. Takahashi, W. Piao, Y. Matsumura, T. Komatsu, T. Ueno, T. Terai, T. Kamachi, M. Kohno, T. Nagano and K. Hanaoka, J. Am. Chem. Soc., 2012, 134, 19588–19591 CrossRef CAS PubMed .
  17. T. D. Ashton, K. A. Jolliffe and F. M. Pfeffer, Chem. Soc. Rev., 2015, 44, 4547–4595 RSC .
  18. T. Kowada, H. Maeda and K. Kikuchi, Chem. Soc. Rev., 2015, 44, 4953–4972 RSC .
  19. J. Yin, Y. Hu and J. Yoon, Chem. Soc. Rev., 2015, 44, 4619–4644 RSC .
  20. S. A. Hilderbrand, M. H. Lim and S. J. Lippard, J. Am. Chem. Soc., 2004, 126, 4972–4978 CrossRef CAS PubMed .
  21. Y. Koide, M. Kawaguchi, Y. Urano, K. Hanaoka, T. Komatsu, M. Abo, T. Terai and T. Nagano, Chem. Commun., 2012, 48, 3091–3093 RSC .
  22. Q. Cai, T. Yu, W. Zhu, Y. Xu and X. Qian, Chem. Commun., 2015, 51, 14739–14741 RSC .
  23. R. Kumari, D. Sunil and R. S. Ningthoujam, Bioorg. Chem., 2019, 88, 102979 CrossRef CAS PubMed .
  24. X. Lv, X. Yuan, Y. Wang and W. Guo, New J. Chem., 2018, 42, 15105–15110 RSC .
  25. Z. Lou, P. Li and K. Han, Acc. Chem. Res., 2015, 48, 1358–1368 CrossRef CAS PubMed .
  26. A. Kaur and E. J. New, Acc. Chem. Res., 2019, 52, 623–632 CrossRef CAS PubMed .
  27. T. S. Blacker and M. R. Duchen, Free Radicals Biol. Med., 2016, 100, 53–65 CrossRef CAS PubMed .
  28. A. Mayevsky and G. G. Rogatsky, Am. J. Physiol.: Cell Physiol., 2007, 292, C615–C640 CrossRef CAS PubMed .
  29. K. Leslie, J. Kolanowski, N. Trinh, S. Carrara, M. Anscomb, K. Yang, C. Hogan, K. Jolliffe and E. New, Aust. J. Chem. DOI:10.1071/CH19398 .
  30. M. Harris, J. L. Kolanowski, E. S. O’Neill, C. Henoumont, S. Laurent, T. N. Parac-Vogt and E. J. New, Chem. Commun., 2018, 54, 12986–12989 RSC .
  31. R. M. Duke, E. B. Veale, F. M. Pfeffer, P. E. Kruger and T. Gunnlaugsson, Chem. Soc. Rev., 2010, 39, 3936–3953 RSC .
  32. H. Sharma, J. M. White, J. Lin, E. J. New and F. M. Pfeffer, Sens. Actuators, B, 2019, 126825 CrossRef .
  33. S.-Y. Xu, X. Sun, H. Ge, R. L. Arrowsmith, J. S. Fossey, S. I. Pascu, Y.-B. Jiang and T. D. James, Org. Biomol. Chem., 2015, 13, 4143–4148 RSC .
  34. K. N. Hearn, T. D. Nalder, R. P. Cox, H. D. Maynard, T. D. M. Bell, F. M. Pfeffer and T. D. Ashton, Chem. Commun., 2017, 53, 12298–12301 RSC .
  35. T. N. Konstantinova, P. Meallier and I. Grabchev, Dyes Pigm., 1993, 22, 191–198 CrossRef CAS .
  36. T. Ezawa, Y. Kawashima, T. Noguchi, S. Jung and N. Imai, Tetrahedron: Asymmetry, 2017, 28, 1690–1699 CrossRef CAS .
  37. T. G. Scott, R. D. Spencer, N. J. Leonard and G. Weber, J. Am. Chem. Soc., 1970, 92, 687–695 CrossRef CAS .
  38. F. Q. Schafer and G. R. Buettner, in Signal Transduction by Reactive Oxygen and Nitrogen Species: Pathways and Chemical Principles, ed. H. J. Forman, J. Fukuto and M. Torres, Springer, Netherlands, Dordrecht, 2003, pp. 1–14 Search PubMed .
  39. C. E. Paul, S. Gargiulo, D. J. Opperman, I. Lavandera, V. Gotor-Fernández, V. Gotor, A. Taglieber, I. W. C. E. Arends and F. Hollmann, Org. Lett., 2013, 15, 180–183 CrossRef CAS PubMed .
  40. K. G. Leslie, D. Jacquemin, E. J. New and K. A. Jolliffe, Chem. – Eur. J., 2018, 24, 5569–5573 CrossRef CAS PubMed .
  41. X. Chen, F. Wang, J. Y. Hyun, T. Wei, J. Qiang, X. Ren, I. Shin and J. Yoon, Chem. Soc. Rev., 2016, 45, 2976–3016 RSC .


Electronic supplementary information (ESI) available: Full details of the synthesis of NapNic as well as spectroscopic characterisation and biological imaging studies. See DOI: 10.1039/c9cc09748a

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