A novel $\mathit{\alpha }$-ketoamide reactivity-based two-photon fluorogenic probe for visualizing peroxynitrite in Parkinson’s disease models

1. Introduction

Parkinson’s disease (PD) is a common neurodegenerative disorder with a high incidence in people over 65.^1,2,3 The functional loss of dopaminergic neurons in the substantia nigra is considered as the pathogenesis of PD.^4,5 Despite great efforts in the last few decades, it is difficult to insight the pathway of the loss of dopaminergic neurons.^6,7,8 Recent evidence indicates that abnormal levels of reactive oxygen species (ROS) can cause oxidative stress and induce DA neuronal degeneration during the pathogenesis of PD.^9,10 In addition, ROS overproduction is closely relative to other PD pathogenic factors, including mitochondrial dysfunction, inflammation and metal dyshomeostasis.^11,12 Therefore, exploring the role of elevated ROS level-induced neurotoxicity in PD is beneficial for early diagnosis and treatment.

Peroxynitrite (ONOO${}^{-})$, a common ROS, is produced via the diffusion-limited coupling superoxide radical (O${}_2^{•-})$ and radical nitric oxide (NO${}^{•-})$.¹³ Normal concentrations of ONOO⁻ can maintain the redox balance and act as secondary messengers to regulate several pivotal signal-transduction pathways in cells.¹⁴ On the contrary, excess ONOO⁻ levels can lead to oxidative stress because of the strong oxidizing and nitrification ability in living biological systems.¹⁵ Under such oxidative stress, lipids, nucleic acids, enzymes and proteins were changed in structure and function.¹⁶ Ultimately, the disorders of biomolecular function result in severe PD disease.^17,18,19 From this point of view, ONOO⁻ can be used as a PD biomarker. Thus, developing forceful tools to monitor the levels of ONOO⁻ is beneficial for understanding the relationship between PD and ONOO⁻ and for early diagnosis of PD.

At present, some methods have been exploited to detect ONOO⁻ including electrochemical, electron spin resonance, liquid chromatography-mass spectrometry and fluorescence approaches.²⁰ Compared to the other techniques, fluorogenic probe-based detection are noninvasive and sensitive, which is ideal for monitoring ONOO⁻ levels in biological systems.^{21,22,23,24,25} Consequently, many ONOO⁻ fluorogenic probes have been exploited relying on different recognition moieties, such as aryl boronic, aryl borate ester, N-aminophenol, trifluorocarbonyl, $\alpha$-ketoamide and so on.^18,26,27,28 Among them, $\alpha$-ketoamide has been widely used to detect the ONOO⁻ levels in cardiovascular and hepatotoxicity disease models via combined inhibition of internal charge transfer (ICT) and photo-induced electron transfer (PET).²⁹ However, the $\alpha$-ketoamide-based ONOO⁻ fluorescence probe to track ONOO⁻ fluctuations in the PD model has not been reported. Moreover, the existing $\alpha$-ketoamide-based ONOO⁻ fluorescence probes utilize aryl dicarbonyl which contains an electron-withdrawing substituent (−NO₂ or −CN) to increase the activity of $\alpha$-ketoamide reaction with ONOO⁻.³⁰ As the reactivity increases, other reactive species may interfere with the selective detection of ONOO⁻, including H₂O₂ and ClO⁻, which limits its application.^31,32 Therefore, developing a novel $\alpha$-ketoamide-based recognition moiety with excellent selectivity toward ONOO⁻ in PD model remains a challenge.

In this work, we designed a new $\alpha$-ketoamide recognized group without 4-nitrophenyl aryl dicarbonyl construction unit to detect ONOO⁻. Moreover, given the advantages of two-photon imaging technology in monitoring abnormal ROS-level in vivo models associated with PD models (Table S1), we employed naphthylamine ketone ( Flu) as the two-photon fluorophore owing to its desirable two-photon action cross-section.^{17,18,33,34,35,36,37,38,39} Ultimately, a non4-nitrophenyl aryl dicarbonyl $\alpha$-ketoamide reactivity-based two-photon ONOO⁻ fluorogenic probe ( DFlu) was developed. The systematical investigation of photophysical properties demonstrated that the DFlu possesses excellent selectivity and suitable two-photon fluorescence intensity towards ONOO⁻. In vitro experiments further proved that DFlu is able to sensitively visualize and monitor the dynamic changes of ONOO⁻. Moreover, manipulation of ONOO⁻ levels in adult zebrafish could also be observed by two-photon imaging. These results might guide designing novel two-photon probes for ONOO⁻ in multiple PD models.

2. Materials and Methods

2.1. Materials and instrumentation

All chemical reaction and cell imaging reagents were obtained from internet medicine suppliers without further purification unless otherwise noted. The progress in the chemical reaction and column chromatography was surveilled with thin-layer chromatography (TLC) on precoated silica plates (250$$$\mu$m thickness), and the spots were observed with UV light. The nuclear magnetic resonance was recorded in Bruker ADVANCE NEO 500 spectrometer using CDCl₃ or $d_6$-DMSO at room temperature. U-3900H and F-7100, respectively, collected absorption and fluorescence data. High-performance liquid chromatography (HPLC) was performed on UltiMate 3000. A high-resolution mass spectrum was obtained by Micromass GCT-MS (ESI source).

2.2. Preparation and Characterization

1-(6-hydroxynaphthalen-2-yl)ethan-1-one ( A1) and 1-(6-(methylamino)naphthalen-2-yl)ethan-1-one ( Flu) were detailed described in detail in the experimental section of Supplementary Material. The date of ¹H NMR and ${}^{13}$C NMR were shown in the Supporting Information.

2.2.1. Preparation of 1-methyl-2,3-dioxo-2,3-dihydro-1H-benzo[f]indole-6-carbonitrile (SFlu)

Oxalyl chloride (85.6$$$\mu$L, 1 mmol) was added into 25 mL three-neck flask containing dry tetrahydrofuran (10$$mL) in a nitrogen atmosphere. Subsequently, Flu (0.20$$g, 1.00$$mmol) was added dropwise and kept stirring for 4$$h at 37^∘C. The product was collected by filtration to obtain red powder SFlu (0.21$$g, 85%). ¹H NMR (500$$MHz, $d_6$-DMSO) $\delta$ (ppm): 8.71 (s, 1H), 8.56 (d, J = 8.7$$Hz, 1H), 8.48 (d, J = 8.7$$Hz, 1H), 8.16 (d, J = 8.8$$Hz, 1H), 7.62 (d, J = 8.7$$Hz, 1H), 3.24 (s, 3H), 2.69 (s, 3H). ${}^{13}$C NMR (126$$MHz, $d_6$-DMSO) $\delta$ (ppm): 196.4, 182.3, 160.1, 157.6, 140.4, 133.4, 132.2, 130.9, 128.4, 121.9, 111.7, 108.3, 27.0, 26.7. [M+H]${}^{+}$: calculated for 254.07, found 254.26.

2.2.2. Preparation of N,N-bis(6-acetylnaphthalen-2-yl)-N1,N2-dimethyloxalamide (DFlu)

Flu (0.20$$g, 1.00$$mmol) and K₂CO₃(0.70$$g, 5.00$$mmol) were added into 25$$mL three-neck flask containing dry tetrahydrofuran (10$$mL) at ice bath conditions. After stirring for 10$$min, oxalyl chloride (42.8$$$\mu$L, 0.50$$mmol) was added dropwise at 0^∘C. Then, the white suspension was observed and stirred at room temperature for 4$$h. Using filtration, the product was obtained as a white powder (0.20$$g, 90%). ¹H NMR (500$$MHz, CDCl₃) $\delta$ (ppm): 8.51 (s, 2H), 8.11 (dd, J = 8.6, 1.7$$Hz, 2H), 7.87 (d, J = 8.7$$Hz, 2H), 7.70 (d, J = 8.5$$Hz, 2H), 7.17 (d, J = 2.2$$Hz, 2H), 6.90 (dd, J = 8.7, 2.2$$Hz, 2H), 3.10 (s, 6H), 2.79 (s, 6H). ${}^{13}$C NMR (126$$MHz, CDCl₃) $\delta$(ppm): 197.8, 164.8, 140.2,135.3, 131.2, 130.5, 129.6, 128.4, 125.3, 124.5, 123.7, 35.5, 26.8. [M+H]${}^{+}$: calculated for 453.1736, found 453.1819.

2.3. Absorption and fluorescence measurements

The absorption and fluorescence were tested in phosphate buffer ($1\times$ PBS). SFlu and DFlu (1$$mM) were dissolved in DMSO. The ion and amino acid (10$$mM) was dissolved in deionized water. The fluorescence data were collected at 350$$nm excitation wavelength with a 5$$nm slit. A cytation 5 microplate reader recorded the stable response of the probe at different temperatures and pH.

2.4. Cell culture and imaging

SH-SY5Y cells were cultured in Dulbecco’s modified medium (DMEM) containing 100 units/mL penicillin, 10% fetal bovine serum and 100$$$\mu$g/mL streptomycin. When the cell density reaches 60–70% and maintains exponential growth, it is needed in several confocal dishes, and the cell density is observed to be around 60%. The cells are treated for imaging according to the requirements of different groups.

3-morpholinosydnonimine hydrochloride (SIN-1, 500$$$\mu$M) was used to treat cells for 1$$h at 37^∘C with or without the 4$$h pretreatment of uric acid (UA, ONOO⁻ scavenger, 200$$$\mu$M) to image exogenous ONOO⁻. To image endogenous ONOO⁻ in PD models, 1-Methyl-4-Phenylpyridinium ion (500$$$\mu$M) was applied to the cells for 4$$h at 37^∘C with or without 4$$h pretreatment of 200$$$\mu$M UA. Subsequently, the cells were incubated with 10$$$\mu$M DFlu for 1$$h at 37^∘C, rinsed with PBS and imaged with Nikon confocal microscope.

2.5. Zebrafish culture and imaging

For exogenous ONOO⁻ imaging, the five-day-old zebrafish were treated with 1$$mM SIN-1 for 1$$h with or without 4$$h pretreatment of 500$$$\mu$M UA. For endogenous ONOO⁻ imaging in PD models, the zebrafish were treated with 3$$mM MPP${}^{+}$ for 2$$h with or without 4$$h pretreatment of 500$$$\mu$M UA. Subsequently, DFlu were incubated for 3$$h with zebrafish, rinsed with PBS and anesthetized with 0.01–0.02% tricaine prior to imaging. Zeiss LSM880 NLO Confocal Microscope was used for the imaging collection.

2.6. Statistical analysis

GraphPad Prism (GraphPad Software, San Diego, CA) was used to analyze all the experimental data. Normally distributed datasets were compared using a one-way ANOVA followed by Dunnet’s post-hoc test where appropriate. A $P-$value of ≤ 0.05 was considered statistically significant. Data were expressed as mean ± SD.

3. Results and Discussion

3.1. Design and synthesis

To achieve high ONOO⁻ selectivity, avoid autofluorescence interference and diminish light damage in living PD models during imaging, we chose the dicarbonyl group instead of aryldicarbonyl with a strong electron-withdrawing. Moreover, we utilize Flu as a two-photon-based fluorophore. DFlu was synthesized via the Stollé synthesis reaction between aldehyde oxalyl chloride and Flu. In the synthesis process, we found an interesting phenomenon: Oxalyl chloride is added in different orders to get different products ( DFlu or SFlu), but both products contain $\alpha$-ketoamide reactivity-based ONOO⁻ recognition groups. To our best knowledge, the amide bond of the $\alpha$-ketoamide group could be broken after reacting with nucleophilic ONOO⁻ and then resulting in the change in fluorescence signals of SFlu and DFlu. Therefore, with the help of the fluorescence imaging technique can detect the dynamic changes of ONOO⁻ in PD models. The detailed synthetic route is shown in Fig. S1. These compounds were well-validated through ¹H NMR, ${}^{13}$C NMR and mass spectrometry.

3.2. Spectroscopic response of probe to ONOO⁻

The absorption spectra were first employed to determine the optimal reaction conditions between ONOO⁻ and the probe to confirm the molecular design concept. The absorption spectra of SFlu and DFlu in different solvents were shown in Figs. S2 and S3, respectively. The absorption wavelength of DFlu was obviously changed only in DMSO because DMSO as an aprotic solvent may inhibit the generation of hydrogen bonds and improve the solubility of DFlu (Fig. S3).⁴⁰ Furthermore, the absorption spectra of SFlu and DFlu in the PBS/DMSO mixture solvent were tested (Figs. S4 and S5). When the ratios of DMSO and PBS are 8:2, the presence of 40$$$\mu$M ONOO⁻ modifies the absorption spectra of SFlu and DFlu dramatically. We next investigated the alteration in the absorption spectrum of SFlu and DFlu with various concentrations of ONOO⁻ in the PBS/DMSO mixture solvent. As depicted in Fig. 1(a), the absorption spectra of DFlu gradually occurred as a new peak at 360$$nm accompanied by the peak decline at 300$$nm with a clear isosbestic point of 313$$nm. In contrast, the absorption spectra of SFlu exhibited a noticeable hypochromic effect at 488$$nm and a blue shift ranging from 480$$nm to 412$$nm [Fig. 1(b)]. The fluorescence titration experiments were shown in Fig. 1(b), and it demonstrated that DFlu displayed an apparent fluorescence turn-on changes at 490$$nm. On the other hand, SFlu showed a fluorescence turn-off in the existance of ONOO⁻ [Fig. 1(d)]. In order to reduce the background interference of the biological environment, the turn-on of fluorescence intensity of DFlu was selected for subsequent experiments.

We next investigated the sensitivity of DFlu for ONOO⁻. In Fig. 1(e), the peak intensity at 490$$nm increased gradually with increasing content of ONOO⁻ and the detection limit of DFlu was calculated to be 9.89$$nM. The rate constant of the reaction between the DFlu and ONOO⁻ was further evaluated (k = 0.0282$$min${}^{-1}$ at 37^∘C, Fig. S6 and Table S2), which is slower than that of $\alpha$-ketoamide with strong electron absorption.²⁹ However, the change of fluorescence signal of DFlu is obvious after reacting with ONOO⁻ [Fig. 1(f)]. In addition, the DFlu and ONOO⁻ reaction also had a yield of 64% (Fig. S7). These results indicated that DFlu is considered a powerful imaging tool to monitor ONOO⁻ in the living system at low concentrations.

3.3. Selective and stable response of DFlu towards ONOO⁻

To test the specificity against ONOO⁻, DFlu was tested with different anions (HCO${}_3^{-}$; F⁻; I⁻; Br⁻; NO${}_3^{-}$; CO${}_3^{2-}$; SO${}_4^{2-})$, cations (Mg${}^{2+}$; Al${}^{3+}$; Cu${}^{2+}$, Fe${}^{3+}$; Mn${}^{2+}$; Zn${}^{2+}$; Ni${}^{2+})$, amino acids (Glycine; Alanine; Valine; Methionine; Proline; Serine; Tyrosine; Histidine) and ROS (ONOO⁻; ROO⁻; ¹O₂; ⋅OH; ClO⁻; H₂O${}_2)$. As shown in Fig. 2(a), ONOO⁻ was the only substance that enhanced the fluorescence of DFlu. In addition, the competitive experiments manifested that the fluorescence response of DFlu to ONOO⁻ was not affected by other phycological substances [Fig. 2(b)]. Considering that ONOO⁻ probes frequently cross-reacted with H₂O₂ due to their similar reactivity and peroxide moiety, high level of H₂O₂ was added to DFlu solution [Figs. 2(c) and 2(d)]. Inspiringly, no fluorescence increase was seen in the presence of 500$$$\mu$M H₂O₂. Moreover, in the presence of biologically relevant reducing agents (GSH or CO${}_2)$, ONOO⁻-dependent reactions are often altered.⁴¹ ONOO⁻ can be rapidly reduced by reductive reagents such as GSH and CO₂ which results in its short lifetime. However, DFlu is still able to detect ONOO⁻ in a relatively short time [Figs. S8(a) and 8(b)], and as shown in Figs. S8(c) and 8(d), DFlu exhibited high sensitivity in the presence of high concentrations of GSH (100$$$\mu$M) and CO₂ (12$$mM), respectively. On the basis of these results, DFlu could selectively identify ONOO⁻.^42,43

In light of DFlu excellent anti-interference performance against ONOO⁻, we further explore whether other biological environments, like temperature and pH, would affect the response signals. In Fig. 2(e), in the presence of 40$$$\mu$M ONOO⁻, the DFlu fluorescence intensity did not change significantly between 25^∘C and 45^∘C, indicating that DFlu monitoring ONOO⁻ was not affected by temperature. Additionally, as shown in Fig. 2(f), DFlu exhibited weak fluorescence in the pH ranges from 4 to 10 without ONOO⁻, and strong fluorescence intensity with ONOO⁻ in pH of 6–8. The above results indicated that DFlu is suitable for detecting ONOO⁻ in physiologically relevant conditions.

3.4. Sensing mechanism of DFlu towards ONOO⁻

Next, the mechanism of fluorescence signal activation of DFu was studied from both experimental and theoretical aspects. Firstly, the reaction between DFlu and ONOO⁻ was confirmed by HPLC. The results displayed that the Flu (Rt = 3.34$$min) was the main product after DFlu reacted with ONOO⁻ [Fig. 3(a)]. In Fig. S6, high-resolution mass spectrometry (HR-MS) analysis further confirmed that the reaction produced Flu ($m/z=200.1076$) is consistent with Scheme S1.

In order to further clarify the DFlu sensing mechanism, theoretical studies were conducted utilizing time-dependent DFT (TD-DFT) calculations at the B3LYP/6-31G ($d)$ level using Gaussian 09. The results indicated that DFlu and Flu exhibited characteristic absorption peaks at 317$$nm and 360$$nm, respectively, which corresponded to experimental peaks at 345$$nm and 354$$nm [Figs. 3(b) and 3(c)]. In addition, the oscillator strengths ($f)$ of Flu ($f=0.4349$) is higher than DFlu ($f=0.1955$), which corresponded to the high molar absorption coefficients of Flu compared to DFlu in Table S3. Moreover, the frontier molecular orbitals play crucial roles in the electronic transition. As shown in Fig. 3(d), the electrons of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) were independently distributed at the ends of DFlu, implying that the amide bonds have suppressed the push–pull electron effect of DFlu. Conversely, the electrons on HOMO and LUMO were mainly distributed throughout the entire molecule, which indicated the amide bond was cleaved and produced Flu with a strong fluorescence signal due to the recovery of ICT. Furthermore, as shown in Table S3 and Fig. S9, the high quantum yield (0.918) and emission wavelength redshift with increasing polarity of Flu also shows that the strong fluorescence originates from the nature of ICT. Moreover, the HOMO–LUMO energy gap was 3.6462–3.4944$$eV, which is consistent with the apparent redshift in the absorption spectrum after reactions between DFlu reacting with ONOO⁻.

3.5. The imaging of ONOO⁻ in living cells

Based on the excellent fluorescent response of DFlu to ONOO⁻, we attempted to utilize DFlu to visualize the ONOO⁻ in living cells. Before cell imaging, we evaluated the cytotoxicity of different DFlu concentrations. After being treated with 20$$$\mu$M DFlu for 24$$h, the viability of SH-SY5Y cells and RAW 264.7 cells remained above 80% (Fig. S10). Next, DFlu was employed to monitor the exogenous and endogenous ONOO⁻. For the exogenous ONOO⁻ imaging, we stimulated SH-SY5Y cells with SIN-1 (ONOO⁻ donor). As shown in Figs. 4(a) and 4(b), no fluorescence signals were observed in DFlu-treated cells. However, the cells exhibited significant fluorescence increase SIN-1 stimulation. After being pretreated with UA (ONOO⁻ scavenger), the fluorescence signal intensity induced by SIN-1 was distinctly diminished. These results reveal that DFlu possesses the capability to monitor exogenous ONOO⁻.

Furthermore, we used MPP${}^{+}$, an acute clinical parkinsonism inductive agent, to reveal the variation of ONOO⁻ in PD disease at the cellular level.⁴⁴ After treating with MPP${}^{+}$ for 8$$h, a noticeable boost in fluorescence intensity was observed [Fig. 4(c)]. Meanwhile, when a combination of MPP${}^{+}$ and UA was used to treat cells, no remarkable increased fluorescence was observed [Fig. 4(d)]. In addition, we also used DFlu to monitor ONOO⁻ levels in rotenone-induced PD models and obtained the same fluorescence imaging results (Fig. S11). Moreover, we observed that the ONOO⁻ levels were slightly lower in the rotenone-induced PD model than in the MPP${}^{+}$-induced PD model. Meanwhile, DFlu successfully detected excessive ONOO⁻ levels in inflammatory models (Fig. S12). These results further proved that DFlu could detect the exogenous and endogenous ONOO⁻ levels in vitro and serve as a promising molecular imaging tool to visualize abnormal ONOO⁻ levels under pathological conditions.

3.6. The imaging of ONOO⁻ in zebrafish

Encouraged by the capability of the probe to visualize exogenous/endogenous ONOO⁻ and the excellent two-photon properties of the product ( Flu) after the reaction between DFlu and ONOO⁻ (Fig. S13), we further investigated whether DFlu could be applied for visualizing ONOO⁻ levels in vivo by two-photon confocal microscope. As shown in Figs. 5(a) and 5(b), the SIN-1 treated zebrafish using exogenous ONOO⁻ models exhibited strong TP fluorescence compared to the control and SIN-1+UA groups. Moreover, the MPP${}^{+}$-treated zebrafish were used as PD disease models. As shown in Figs. 5(c) and 5(d), strong red two-photon fluorescence was observed when the MPP${}^{+}$-treated zebrafish was incubated with DFlu for 3$$h. Furthermore, the recorded fluorescence signal decreased significantly in the presence of UA. In addition, the same results were obtained in the zebrafish model of PD induced by rotenone (Fig. S14). These results demonstrated that DFlu could accurately discriminate the area with high levels of ONOO⁻ in the zebrafish PD model.

4. Conclusion

In conclusion, we developed novel $\alpha$-ketoamide recognition moieties and used it to construct a two-photon peroxynitrite probe ( DFlu). The conspicuous fluorescence of DFlu at 490 nm was observed to be stimulated by ONOO⁻ and the theoretical calculation showed strong fluorescence from the recovery of ICT capacity. DFlu exhibited good stability and excellent selectivity in the presence of other reactive substances. In addition, DFlu could detect exogenous and endogenous ONOO⁻ in living cells with negligible cytotoxicity. More importantly, DFlu could detect endogenous/exogenous ONOO⁻ in PD zebrafish models by two-photon imaging. These results could guide the design of the probe’s red-shifting absorption and emission based on push and pull electrons and could promote a new way to construct $\alpha$-ketoamide-based two-photon ONOO⁻ probes for the in-depth study of ONOO⁻ stress-related diseases.

Conflict of Interest

The authors declare that there are no conficts of interest relevant to this article.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (22077101), the Open Project Program of Wuhan National Laboratory for Optoelectronics (No. 2020WNLOKF023), Natural Science Foundation of Shaanxi Province (2022JM-130), the Joint Research Funds of Department of Science & Technology of Shaanxi Province and Northwestern Polytechnical University (2020GXLH-Z-008, 2020GXLH-Z-021, 2020GXLH-Z-023), the China Postdoctoral Science Foundation (2022M711595, 2022M722595), Postdoctoral Research Funding Schemes of Jiangsu Province (2021K036A), The Natural Science Foundation of Ningbo (202003N4049, 202003N4065). Tao Shao and Xianning Xu contributed equally to this work.