The reaction mechanism of p-hydroxystyryl-substituted BODIPY with ABTS•+ and Fe3+ in solutions and in liposomes

Qinhai Xu; Kang Li; Peng Wang

doi:10.1016/j.arabjc.2024.106041

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01 2024

:18;

106041

doi:

10.1016/j.arabjc.2024.106041

The reaction mechanism of p-hydroxystyryl-substituted BODIPY with ABTS^•+ and Fe³⁺ in solutions and in liposomes

Qinhai Xu^{^a,⁎}, Kang Li^{^b}, Peng Wang^{^b}

aCollege of Chemistry, Chemical Engineering and Materials Science, Zaozhuang University, Zaozhuang 277160, Shandong Province, China

bDepartment of Chemistry, Renmin University of China, Beijing 100872, China

⁎Corresponding author. 1249661455@qq.com (Qinhai Xu)

Received: 2024-4-24, Accepted: 2024-11-1,

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Abstract

The reaction mechanism of p-hydroxystyryl-substituted BODIPY (BOH) with two oxidative cations, namely ABTS^•+ and Fe³⁺, was investigated in a water–ethanol mixed solution and in liposome suspensions, respectively, using different spectroscopic methods. In solution, the oxidation of BOH (with orange fluorescence) by the two cations occurred at the ethylene group (C⚌C) locating between the dipyrrole and phenol groups and resulted in conjugation-truncated products exhibiting characteristic green fluorescence emission. In heterogeneous small unilamellar vesicles (SUV), water soluble ABTS^•+ was evidenced to oxidize BOH embedded in the lipid bilayers of SUV, while Fe³⁺ did not. The lack of reaction between Fe³⁺ and BOH was attributed to the complexation between Fe³⁺ and the phenolic hydroxyl group of BOH on the surface of the SUV. The reaction kinetics results indicated that, in homogeneous solution, the oxidation rate of Fe³⁺ was three orders of magnitude slower than that of ABTS^•+ for BOH. The location and orientation of BOH within the SUV were discussed based on the reaction phenomena. BOH could be as a good antioxidant fluorescent prober for ABTS^•+ detection with a detection limit of 1.5 * 10⁻⁷ M and a linear rang of 0–4.93 μM. What’s more, the amphiphilic BOH dispersed in the round GUV (BOH + GUV) could show the bright red fluorescence. This research suggests the significant potential of BOH as an antioxidant fluorescent probe for in situ bioimaging.

Keywords

BODIPY derivatives

Antioxidant fluorescent probe

Reaction mechanism

Small unilamella vesicle

Water-soluble oxidants

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1 Introduction

Redox homeostasis is crucial for maintaining the balance in biological systems. When an imbalance between the production of oxidants and antioxidant defenses occurs, it can result in damage to biological systems, leading to diseases such as Parkinson's disease, Alzheimer's disease, neurodegeneration, and cancer (Razzokovet al., 2017; Stern and McNew, 2021). Therefore, it is important to develop probes with antioxidant capacity to detect oxidative species. Due to the advantages of non-toxicity, sensitivity, easy operation and potential biological value, fluorescent probes could be used as powerful tools for detecting oxidative species and assessing antioxidant activity in both biological and environmental samples (Chen et al., 2016; Itoh et al., 2007; Mondal et al., 2018; Mondal et al., 2019; Roy et al., 2019; Roy et al., 2021; Yan et al., 2019; Zhang et al., 2018 ). P. Varandas et al. presented a fluorescent head-labeled phospholipid-coumarin bioconjugate that aimed to provide antioxidant protection in liposomes (Varandas et al., 2023). A. Akhuli et al. designed a nanoscale fluorescence sensor system using fluorescent copper nanoclusters to detect reactive oxidative species and antioxidants (Akhuli et al., 2022). Y. Huang et al. developed molecular fluorescent probes for imaging and evaluating peroxynitrite fluctuations in living cells and under hypoxic stress conditions (Huang et al., 2022). Consequently, in order to identify and suppress oxidative species, as well as to clarify the reaction mechanism, the use of an antioxidant fluorescent probe is necessary.

BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) derivatives have been identified as potential functional fluorescent probes due to their excellent photostability, high molar extinction coefficient, high fluorescence quantum yield, sharp absorption and emission spectra, and ease of functionalization (Kolemen and Akkaya, 2018; Kowada et al., 2015). Recently, there has been significant interest in p-hydroxystyryl-substituted BODIPY derivatives as antioxidant fluorescent probes for detecting oxidizing species (Jantra et al., 2019; Qu et al., 2019; Xu et al., 2021 ). Suthikorn Jantra et al. synthesized a 'switchable' orange fluorescent probe based on styryl-BODIPY for detecting hypochlorite in water, methanol solution, and cells, through the oxidation of ClO⁻ to the C⚌C bond in the p-hydroxystyryl group (Jantra et al., 2019). X. Qu et al. developed a visible BODIPY probe for detecting intracellular Fe³⁺ by oxidizing Fe³⁺ to the C⚌C bond in the p-hydroxystyryl groups (Qu et al., 2019). Y. Xu et al. reported the regioselective oxidative cleavage of C⚌C bonds from a BODIPY-based photocage by illumination at 630 nm, resulting in a free aldehyde and a thiol fluorescent probe (Xu et al., 2021). These works highlighted the advantages of the p-hydroxystyryl group structure in BODIPY antioxidant fluorescent probes. However, the applications of these BODIPY antioxidant fluorescent probes have mainly been limited in homogeneous solutions in vitro or directly in cells, with no reported applications in heterogeneous model systems, e.g. liposomes. Liposomes, consisting of an amphiphilic phospholipid bilayer and an aqueous compartment, are commonly used as simplified models for biomembranes (Liang et al., 2012; Reddi et al., 1991). Y. Lu et al. developed a ratiometric electrochemiluminescence resonance energy transfer platform based on novel dye-BODIPY derivatives for the sensitive detection of lactoferrin (Lu et al., 2020). Notably, the effects of the reported p-hydroxystyryl-substituted BODIPY derivative as an antioxidative fluorescent probe for oxidizing species in liposome have been rarely investigated. Among various oxides, ferric ion (Fe³⁺) as a biologically essential trace element plays a crucial role in chemical and physiological processes such as enzymatic catalysis, electron transport, nucleic acid synthesis, and cell metabolism (Lin et al., 2017; Song et al., 2020). 2,2-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^•+), a synthesized and water-soluble substance, is generally used as the oxidant to assess the Trolox equivalent antioxidant capacity (TEAC) of the natural and artificial antioxidant from foods, drinks, and other nutrients in aqueous solution (Walker and Everette, 2009).

In this work, the reaction mechanisms between p-hydroxystyryl-substituted BODIPY derivative (BOH) and two water-soluble oxidizing species, i.e. Fe³⁺ and ABTS^•+ cations, in a homogeneous water–ethanol solutions and in the small unilamellar vesicle (SUV) systems, respectively, were investigated by various spectroscopic methods. The structures of oxidation products were characterized by mass spectra and the kinetics were determined by absorption spectra. The location and orientation of BOH in lipid bilayers were discussed based on the reaction phenomena. BOH as a good antioxidant fluorescent prober was used for ABTS^•+ detection. Additionally, the amphiphilic BOH dispersed in the round GUV (BOH + GUV) was prepared to directly show the fluorescence. This research proved that as an antioxidant fluorescent probe, BOH have significant potential for in situ detection of oxidized species.

2 Experimental

2.1

2.1 Materials and methods

2,4-dimethylpyrrole, 4-carboxybenzaldehyde, Trifluoroacetic acid (TFA), 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), Triethylamine, Boron trifluoride diethyl etherate, p-hydroxybenzaldehyde, Acetic acid, Piperidine, Toluene, DMSO‑d₆, FeCl₃ and ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid ammonium salt)] were purchased from Aladdin Industrial Corporation (Shanghai, China). Soybean L-α-phosphatidylcholine (Soybean PC, 23 %; Product No. P5638) was purchased from Sigma-Aldrich (St. Louis, MO, US).

¹H NMR and ¹³C NMR spectra were analyzed using a Bruker 600 MHz, the chemical shifts were recorded on a delta scale in ppm relative to DMSO‑d₆ (δ = 2.50 ppm) for ¹H NMR and DMSO‑d₆ (δ = 39.51 ppm) for ¹³C NMR. The absorption spectra were recorded using a Cary 60 UV–Vis spectrophotometer (Agilent Technologies, USA). The fluorescence spectra were measured with a fluorescence spectrophotometer (HITACHI F-4600, Japan). QTOF-MS spectra were obtained using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS) with positive mode electron spray ionization (ESI). (Water, Xevo G2 Qtof, USA). Ultrapure water was obtained from a Millipore Mingche™-D 24^UV water purification system. Fluorescence lifetime was measured using an Edinburgh FLS980 all-function steady-state/transient fluorescence spectrometer (Edinburgh Instruments, UK). The absolute fluorescence quantum yield was measured using an FLS 980 spectrometer with an integrating sphere detector. Small unilamellar vesicles with BOH were sonicated with an ultrasonic homogenizer (JY-IIN, Ningbo Scientz Biotechnology CO., LTD). Dynamic light scattering analysis (DLS) was performed using a Nano ZS90 Zetasizer (Malvern Instruments Corp, UK).

2.2

2.2 Synthesis of BOH

BOD was synthesized using Safacan Kolemen’s method (Fig. 1) (Kolemen et al., 2011). The synthesis of BOH was briefly described as followings. In a 500 mL round bottom flask, BOD (2.5 mmol, 0.92 g) and p-hydroxybenzaldehyde (7.5 mmol, 0.92 g) were added to 200 mL of toluene, along with piperidine (0.5 mL) and acetic acid (0.5 mL) (Lu et al., 2020). The reaction was refluxed using a Dean-Stark trap; and monitored by thin layer chromatography with CHCl₃/MeOH (v/v, 97/3). After the starting materials were consumed, the solution was cooled to room temperature and washed three times by water. The organic phase was dried with anhydrous Na₂SO₄ and evaporated using a rotary evaporator. The residue was then purified using silica gel column chromatography with CHCl₃/MeOH (v/v, 97/3) as the eluent. The obtained product is purple-black solid (0.27 g, 23 %). BOH: ¹H NMR (600 MHz, DMSO‑d₆) δ_H 8.12 (d, J = 8.1 Hz, 2H), 7.54 (d, J = 7.8 Hz, 2H), 7.50 (s, 1H), 7.47 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 16.3 Hz, 1H), 6.94 (s, 1H), 6.90 (d, J = 4.3 Hz, 2H), 6.18 (s, 1H), 2.49 (s, 3H), 1.39 (s, 3H), 1.34 (s, 3H). ¹³C NMR (600 MHz, DMSO‑d₆): δ_C 167.35, 159.67, 154.30, 154.03, 142.87, 141.56, 39.06, 138.98, 138.54, 138.48, 132.26, 30.75, 130.52, 129.52, 129.07, 127.54, 121.59, 118.75, 116.54, 115.11, 55.25, 14.72, 14.39 ppm. QTOF-MS (m/z): [M + H]⁺ calc'd for C₂₇H₂₄BF₂N₂O₃: 473.1843, Found: 473.1842, Δ = 0.21 ppm.

Optimized molecular structures of BOD (a) and BOH (b) obtained from the DFT-based theoretical calculations at the B3LYP (6-31G (d, p)) level using the Gaussian09 package. The molecular structure of ABTS•+ (c).

2.3

2.3 Preparation of small unilamellar vesicles with BOD (or with BOH)

To investigate the spectral changes and chemical kinetics of BOH reaction with oxidized species in a heterogeneous system, the small unilamellar vesicle (SUV) was chosen as a model. Small unilamellar vesicles with BOD (referred to as BOD in SUV) and small unilamellar vesicles with BOH (referred to as BOH in SUV) were prepared using the solid dispersion method (de Freitas et al., 2019; Haller et al., 2018). Specifically, 40 mL of BOD or BOH (10 μM) solutions in ethanol was added to two separate copies of 40 mg of soybean PC dissolved in 3 mL of CHCl₃. The mixed solutions were evaporated by a rotary evaporator and dried in a vacuum oven for 2 h, yielding dried thin films. These films were then hydrated with 40 mL of ultrapure water and subjected to ultrasonic treatment for 10 min. Subsequently, the solutions were processed for an additional 15 min using an ultrasonic homogenizer. The resulting small unilamellar vesicles with BOD (BOD in SUV) and small unilamellar vesicles with BOH (BOH in SUV), respectively, were collected and stored at 4 °C.

3 Results and discussion

3.1

3.1 Absorption and emission spectra of BOD or BOH in ethanol solution

Optimized molecular structure of BOD and BOH obtained from the DFT-based theoretical calculations at the B3LYP (6-31G (d, p)) level using the Gaussian09 package were shown in Fig. 1. The total energy scans were calculated for the model compounds BOD and BOH as a function of the dihedral angles (Fig. S4). The most stable structure of BOD or BOH was observed at dihedral angles of 90 and 270 degrees. It was clearly seen that in both BOD and BOH, the dipyrrolic plane was perpendicular to the meso-substituted phenyl group, while in BOH the p-hydroxystyryl group and the dipyrrolic group were coplanar, consequentially resulting in the significant extension of conjugation systems in BOH than in BOD.

The physicochemical parameters of BOD and BOH measured in pure ethanol solution were summarized in Table 1. The absorption maxima of BOD and BOH were observed at 499 nm and 572 nm, respectively (Fig. 2). Correspondingly, the fluorescence emission maxima of BOD and BOH were found at 509 nm and 589 nm, respectively (Fig. 2). It was noted that both the absorption maximum and fluorescence emission maximum of BOH were red-shifted by approximately 70 nm compared to those of BOD. These shifts were attributed to the introduction of a conjugated p-hydroxystyryl group, which resulted in extended conjugation system in BOH compared to that in BOD. The spectral results were supported by theoretical calculations (as seen in Fig. 1). The absolute fluorescence quantum yield (Φ) of BOD and BOH, measured using an integrating sphere detector, were determined as 26.1 % and 79.90 %, respectively. The greatly enhanced fluorescence quantum yield of BOH was attributed to the presence of a hydroxyl group, which acted as a strong electron donating group at the end of BOH (Su et al., 2014). This indicated that BOH was a superior choice as a fluorescence probe for fluorescence sensing and bioimaging. The fluorescence lifetimes (τ_FL) of BOD and BOH measured in ethanol were 3.37 and 4.08 ns, respectively, using the time-correlated single photon counting (TCSPC) method. These results confirmed the fluorescent characteristics for the emissions of both BOD and BOH.

Table 1 Physicochemical parameters of BOD or BOH in ethanol solution.

	ε/L mol⁻¹ cm⁻¹	$λ_{max}^{Abs}$ /nm	$λ_{max}^{Em}$ /nm	Φ_FL/%	τ_FL/ns	TEAC	Redox potential (V vs. RHE)	pKa	Log P_Cal
BOD	68,038	499	509	26.10	3.37	0	0.55	4.07	−0.86
BOH	107,758	572	589	79.90	4.08	1.31	0.17	4.19, 9.33	1.32

Normalized absorption and fluorescence emission (FL) spectra of BOD or BOH measured in ethanol solution.

The antioxidant capacity of BOD and BOH in aqueous solutions was measured by Trolox equivalent antioxidant capacity (TEAC) test. This test measured the ability of an antioxidant to scavenge ABTS^•+ relative to the standard antioxidant of Trolox (Arts et al., 2004; Jibril et al., 2017; Re et al., 1999 ). The results indicated that BOH was a good antioxidant in comparison with Trolox while BOD wasn’t, since TEAC values of BOH and BOD were 1.31 and 0, respectively (Fig. S6a–b and Table 1). In addition, the redox potentials of BOD and BOH were determined as 0.55 and 0.17 V versus RHE, respectively, by cyclic voltammetry (Fig. S6c and Table 1). The lower redox potential of BOH was beneficial for the reaction with ABTS^•+ (0.68 V versus RHE).

There were one pH-sensitive functional group in BOD (4′-carboxyl group on meso-phenyl ring) and two in BOH (4′-carboxyl group on meso-phenyl ring and phenoxy group at the end of hydroxystyryl group). Since the meso-phenyl group plane is perpendicular to the dipyrrole skeleton based on the optimized molecular conformation by DFT (Fig. 1), the electron-withdrawing ability of carboxyl group has negligible effect on the absorption and emission properties of BOD and BOH. The pKa values of BOD and BOH were measured as 4.07, and 4.19 and 9.33, respectively. These results indicated that the carboxyl groups (−COOH) in both BOD and BOH existed in deprotonation form (i.e., —COO⁻) under neutral conditions.

Additionally, the lipid-water partition coefficients (logP_Cal) for BOD and BOH were theoretically calculated as −0.86 and 1.32, respectively. The results revealed that BOH is more lipophilic than BOD, and consequently had the potential to be utilized as a fluorescent probe in lipid phase.

The different metal cations (Ca²⁺, Cd²⁺, CO²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺ and Zn²⁺, 10⁻³ M) were individually added to BOD, the normalized absorption spectra and fluorescence spectra showed no change (Fig. S10a–b). It indicated that the metal cations had no influence on BOD. Similarly, when the different metal cations were added to BOH, the absorption spectra and fluorescence emission spectra also remained unchanged (Fig. S10c–d), suggesting that these metal cations had no effect on BOH. However, when Fe³⁺ was added to BOH, the absorption spectrum and fluorescence emission spectrum of the mixed solution exhibited a blue shift. The absorption peak shifted to 504 nm (Fig. S10c), and the fluorescence emission peak shifted to 520 nm (Fig. S10d), indicating the formation of new species. This shift was attributed to the redox reaction between BOH and Fe³⁺. It also showed that BOH had good selectivity for Fe³⁺, which could be used as a fluorescence sensor.

Summarily, based on its favorable physical and chemical properties, BOH, a p-hydroxystyryl-substituted BODIPY derivative, might be employed as a liposoluble fluorescent probe responding to oxidizing species.

3.2

3.2 The reactions with ABTS^•+ or Fe³⁺ in water–ethanol solutions

The redox reactions of BOH with ABTS^•+ or Fe³⁺ in water–ethanol solution were investigated using the absorption and fluorescence emission spectra, and the results were shown as Fig. 3. The absorption band peaked at 572 nm and the fluorescence emission band peaked at 589 nm (with orange color) are characteristics of BOH, which gradually decreased and eventually disappeared during reaction with ABTS^•+ or Fe³⁺, respectively. The oxidized product is simultaneously generated with absorption peaked at ∼503 nm and fluorescent emission at ∼518 nm (with green color). BOH was oxidized by ABTS^•+ to generate spectral blue shifted products. The characteristic absorption peak of ASTS^•+ at 734 nm decreased and disappeared, indicating that blue-green ABTS^•+ was reduced to colorless ABTS. The prominent emission color change of BOH upon oxidation by ABTS^•+ or Fe³⁺ suggested that BOH could function as a color-changing fluorescent probe. The absorption and fluorescence emission of oxidized products exhibited similar spectral characteristics with those of BOD, indicating the great similarities in their conjugation systems. In another word, the oxidized product of BOH has truncated conjugation systems which is similar with that of BOD. Then the deduction of the oxidation occurring at the C⚌C bond in the p-hydroxystyryl functional group is easily to be drawn.

(a) The normalized absorption spectra of BOD, BOH without ABTS•+, BOH with ABTS•+ in 5 min. (b) The normalized fluorescence emission (FL) spectra of BOD, BOH without ABTS•+, BOH with ABTS•+ in 5 min, λex = 365 nm. (c) The normalized absorption spectra of BOD, BOH without Fe3+, BOH with Fe3+ in 5 min. (d) The normalized fluorescence emission (FL) spectra of BOD, BOH without Fe3+, BOH with Fe3+ in 5 min, λex = 365 nm.

The oxidized products of BOH upon reaction with ABTS^•+ or Fe³⁺ were characterized using LC-QTOF-MS. The possible structures for the products were proposed based on the exact molecular mass as shown in Scheme 1. Two of them were proposed as 1a and 2a with molecular mass of 503.1971 and 517.2100 (Fig. S11), respectively, for the reaction with ABTS^•+. The extra carbonyl group in 1a and 2a versus BOD might cause the corresponding red-shifts of absorption and fluorescence spectra (Fig. 3a and b). Similar mechanism was proposed in refs. (Jantra et al., 2019; Jantra et al., 2021). Furthermore, two oxidized products with the exact molecular mass 383.1375 and 397.1542, respectively, were proposed as 1f and 2f (Fig. S12) for reaction with Fe³⁺. In this case, the p-hydroxystyryl group were oxidized as aldehyde or carboxyl group (Qu et al., 2019; Xu et al., 2021). The proposed product’s structural formulas had very similar conjugation system with BOD but obviously shorter than BOH, which were in consistent with their absorption and fluorescence spectra results as seen in Fig. 3. In summary, the proposed structural formulas reasonably explained the spectral characteristics observed upon the reaction of BOH with ABTS^•+ or Fe³⁺, respectively. Such results further confirmed that the C⚌C bond in p-hydroxystyryl group of BOH was the reaction site for oxidation by ABTS^•+ or Fe³⁺ in homogeneous solutions.

The proposed products and intermediates from reactions between BOH in methanol solution and ABTS•+ or Fe3+, characterized by LC-QTOF-MS. The color represents the corresponding fluorescence emission region of molecules.

3.3

3.3 The reactions with ABTS^•+ or Fe³⁺ in small unilamellar vesicle (SUV) system

The redox reaction between BOH and ABTS^•+ or Fe³⁺ was further investigated in a heterogeneous system, i.e. small unilamellar vesicles. Since BOH was a lipophilic compound based on the calculated logP results (Table 1.), BOH was embedded in lipid phase during sample preparation and then reacted with the oxidants staying in water phase. The reactions were monitored by absorption and fluorescence spectra as well, and the results were shown as Fig. 4a–d.

(a) Normalized absorption spectra of BOD in SUV, BOH in SUV without ABTS•+, BOH in SUV with ABTS•+ in 5 min, (I) Photo of BOH in SUV, (Ⅱ) Photo of BOH in SUV with ABTS•+. (b) Normalized fluorescence emission (FL) spectra of BOD in SUV, BOH in SUV without ABTS•+, BOH in SUV with ABTS•+ in 5 min, λEx = 365 nm, (I) Fluorescence photo of BOH in SUV, (Ⅱ) Fluorescence photo of BOH in SUV with ABTS•+, λEx = 365 nm. (c) Normalized absorption spectra of BOD in SUV, BOH in SUV without Fe3+, BOH in SUV with Fe3+ in 5 min, (I) Photo of BOH in SUV, (Ⅱ) Photo of BOH in SUV with Fe3+. (d) The fluorescence emission (FL) spectra of BOD in SUV, BOH in SUV without Fe3+, BOH in SUV with Fe3+ in 5 min, (I) Fluorescence photo of BOH in SUV, (Ⅱ) Fluorescence photo of BOH in SUV with Fe3+, λEx = 365 nm.

Fig. 4a and b showed the results of BOH reacting with ABTS^•+ in SUV suspension. In consistent with the results measured in solution, the oxidized product observed in SUV owned absorption band peaked at ∼500 nm and fluorescence emission at ∼520 nm, which were similar to those of BOD in SUV. The oxidation occurring at C⚌C bond of p-hydroxystyryl group resulting in the conjugation system truncated product could be easily deduced based on the spectral changes. Additionally, upon oxidation by ABTS^•+, the oxidized product of BOH in lipid bilayers emitted green fluorescence. This suggested that BOH could also serve as a color-changing fluorescent probe in heterogeneous SUV systems responding to the oxidative species in water phase.

In case of the reaction between BOH in SUV and Fe³⁺ in water phase, the absorption spectra had hardly changed (Fig. 4c), while the fluorescence emission of BOH was drastically quenched (Fig. 4d). All these results could be explained by the complexation of Fe³⁺ by the phenolic hydroxyl group occurring on the surface of SUV and no oxidation happened in this system, which was supported by the absorption spectrum of the complexation of Fe³⁺ and phenol (Fig. S13), since the similar purple color observed in the suspension of Fe³⁺ mixing with BOH in SUV (inset II in Fig. 4c) (Qi et al., 2019). Based on the different reaction mechanisms of BOH towards ABTS^•+ or Fe³⁺ in SUV, it had certain selectivity towards two water-soluble oxide species.

3.4

3.4 Chemical kinetics and reaction mechanism

Chemical kinetics (the analysis of the rates of chemical reactions) was further used to study the reaction mechanism of the effects of ABTS^•+ or Fe³⁺ on BOH, which was widely applied in the physical and chemical sciences (Bar et al., 2021; Bar et al., 2024). The oxidation reaction kinetics of BOH with ABTS^•+ or Fe³⁺ in solutions and in SUV were studied by monitoring the absorbance change at representative wavelengths. The results were shown in Fig. S16. For BOH reaction with ABTS^•+, the kinetics were measured by UV–Vis spectrophotometer using the stop-flow technique at 734 nm (Fig. S16a and c), the characteristic absorption band of ABTS^•+, since this reaction was very fast. For BOH reaction with Fe³⁺, the kinetics were examined by absorption at 504 nm and 570 nm (Fig. S16b), the representative wavelengths of oxidized product and starting material, respectively. The reaction rates were obtained by mono-exponential fitting the kinetic curves, and the results were summarized as rate constants in s⁻¹ as shown in Table 2. For reaction of BOH with ABTS^•+, either in solution or in SUV, the rate constant was much larger than that for reaction of BOH with Fe³⁺. Due to the lower redox potential of ABTS^•+ (0.68 V versus RHE) compared to Fe³⁺ (0.771 V versus RHE), the larger rate constant might originate from the differences in molecular structure. ABTS^•+ owns larger and organic π skeleton, which has more priority to closely attach to BOH (due to π–π stacking) and then to promote the oxidation reaction rate. This deduction was strongly supported by the experimental results. In solution, the oxidation rate of BOH by Fe³⁺ was three orders of magnitude slower than that of by ABTS^•+. While in SUV suspension, even the oxidation rate by ABTS^•+ became slower than that in solutions, its still much larger than the oxidation rate by Fe³⁺ in solution. For the case of BOH interaction with Fe³⁺ in SUV, no oxidation reaction actually happened, and only complexation of Fe³⁺ by phenolic group occurred.

Table 2 The chemical reaction rate constants of reactions between BOH and ABTS^•+ or Fe³⁺.

	Reaction with ABTS^•+ (734 nm, s⁻¹)	Reaction with Fe³⁺ (570 nm, s⁻¹)
In water–ethanol solutions In small unilamellar vesicle	5.88 1.04	0.0043

ABTS^•+ in the aqueous phase demonstrated the capability to oxidize BOH in the lipid phase. This capability could be attributed to its organic skeleton and extensive conjugated system, which enabled it to penetrate the lipid membrane to a certain extent. Ferric ions mainly existed as hydrated ions and were impeded from further accessing the oxidation sites in the lipid phase due to their complexation with phenolic hydroxyl groups.

Based on the experimental results of BOH in the SUV with Fe³⁺ or ABTS^•+, the position and orientation of BOH in SUV was discussed. Additionally, BOH exhibited amphiphilic properties and could serve as a fluorescent probe on the surface of the liposome, which was supported by the calculated lipid-water partition coefficient of BOH (logPcal) being 1.32. BOH could stay in the lipid phase. The phenol group and carboxyl group were hydrophilic in comparison with the main conjugation plane. Both functional groups could be located on the surface of the lipid-bilayers. Based on these two points, the orientation of BOH was assigned. Considering the water-soluble ABTS^•+ still reacted with BOH in lipid phase efficiently, the oxidation site (C⚌C in the p-hydroxystyryl group) must stay close to the surface of lipid-bilayers. The proposed alignment of BOH was shown in Scheme 2. Due to its position and orientation at the lipid/water interface, BOH could serve as a valuable antioxidant fluorescent probe for detection the oxidation of oxidized species on biomembranes in situ.

Schematic representation of the reactions of the antioxidant fluorescent probe BOH with ABTS•+ or Fe3+ in small unilamellar vesicles containing BOH (BOH in SUV), a heterogeneous system.

3.5

3.5 Sensitivity of BOH as an antioxidant fluorescent sensor for ABTS^•+ detection

Chemical kinetics studies showed that the oxidation rate of BOH by Fe³⁺ was three orders of magnitude slower than that of by ABTS^•+ in solution. Therefore, the sensitivity of BOH as an antioxidant fluorescent prober for ABTS^•+ detection was investigated. The fluorescence intensity at = 590 nm of BOH decreased gradually in the presence of different concentrations of ABTS^•+ in 5 min in Fig. 5a–b. From Fig. 5c, the linear regression equation with a correlation coefficient of 0.9998 showed a good linearity in the range of 0–4.93 μM. The limit of detection (LOD) was calculated to be 1.5 * 10⁻⁷ M based on three times the standard deviation rule (LOD = 3 S_k/k, S_k as the standard deviation and k as the slope). BOH could be as a good antioxidant fluorescent prober for ABTS^•+ detection.

(a) Fluorescence emission specta of BOH in the presence of different concentrations of ABTS•+ in 1 min, the concentration of ABTS•+ from top to bottom: 0, 1.64, 3.29, 4.93, 6.75, 8.21 and 9.86 μM), λex = 400 nm. (b) The dependence of FL intensity of BOH at 590 nm on the concentration of ABTS•+ in the range of 0 to 9.86 μM. (c) The linear relationship between FL intensity of BOH at 590 nm and the concentration of ABTS•+ in the range of 0 to 4.93 μM.

3.6

3.6 BOH + GUV

To further assess the application of BOH as the fluorescent probe in application of bio-imaging, BOH + GUV was selected and prepared by electroforming method. As shown in Fig. 6, the amphiphilic BOH was well dispersed in the round GUV, showing the bright red fluorescence by laser scanning confocal fluorescence microscope under the excitation of 405 nm. BOH could be used as an antioxidant fluorescent probe with bioimaging capability.

The fluorescent image of BOH + GUV by laser scanning confocal fluorescence microscope, λex = 405 nm.

4 Conclusion

In this work, a p-hydroxystyryl-substituted BODIPY derivative (BOH) was synthesized as an antioxidant fluorescent probe with enhanced quantum yield. No matter in homogenous solution or in heterogenous SUV system, the oxidation occurred at the C⚌C bond in the p-hydroxystyryl group based on spectral changes. There was a complexation between Fe³⁺ and the phenolic hydroxyl group of BOH in SUV system differently. Kinetics results indicated that oxidation rate by ABTS^•+ was much faster than by Fe³⁺, due to the molecular structure of oxidants. The orientation and position of BOH stayed in lipid bilayers were discussed and proposed that the phenol group and carboxyl group were located on the surface of the SUV and the oxidation site (C⚌C in the p-hydroxystyryl group) was close to the surface of lipid-bilayers. BOH could be as a good antioxidant fluorescent prober for ABTS^•+ detection with a detection limit of 1.5 * 10⁻⁷ M and a good linearity in the range of 0–4.93 μM. In addition, the amphiphilic BOH dispersed in the round GUV (BOH + GUV) could show the bright red fluorescence. It is expected that the modifiable BOH could be utilized to recognize oxidized species from both aqueous and lipid phases in situ in the fields of biomembrane and biochemistry.

CRediT authorship contribution statement

Qinhai Xu: Writing – review & editing, Writing – original draft, Investigation, Data curation. Kang Li: Writing – original draft, Software, Data curation. Peng Wang: Writing – review & editing, Data curation.

Acknowledgments

This work has been financially supported by the New Faculty Start-Up Fund of Zaozhuang University (No. 1020752), the National Natural Science Foundation of China (Nos. 21673289, 21273282). We express our grateful thanks to them for their financial support.

Declaration of competing interest

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.

References

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de Freitas C., Calori I., Tessaro A., Caetano W., Hioka N., . Rapid formation of small unilamellar vesicles (SUV) through low-frequency sonication: an innovative approach. Colloid Surface B. 2019;181:837-844.
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[Google Scholar]
Kolemen S., Akkaya E., . Reaction-based BODIPY probes for selective bio-imaging. Coordin. Chem. Rev.. 2018;354:121-134.
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[CrossRef] [Google Scholar]
Lin S., Geng S., Li N., Liu S., Li N., Luo H., . L-histidine-protected copper nanoparticles as a fluorescent probe for sensing ferric ions. Sensor Actuat. B-Chem.. 2017;252:912-918.
[CrossRef] [Google Scholar]
Lu Y., Ke H., Wang Y., Zhang Y., Li H., Huang C., Jia N., . A ratiometric electrochemiluminescence resonance energy transfer platform based on novel dye BODIPY derivatives for sensitive detection of lactoferrin. Biosens. Bioelectron.. 2020;170:112664
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Qi H., Teng M., Liu M., Liu S., Li J., Yu H., Teng C., Huang Z., Liu H., Shao Q., Umar A., Ding T., Gao Q., Guo Z., . Biomass-derived nitrogen-doped carbon quantum dots: highly selective fluorescent probe for detecting Fe³⁺ ions and tetracyclines. J. Colloid Interf. Sci.. 2019;539:332-341.
[CrossRef] [Google Scholar]
Qu X., Bian Y., Yang B., Zhen S., . A visible BODIPY probe for 'Naked-Eye' detection of pH value and intracellular Fe³⁺. Chin. J. Inorg. Chem.. 2019;35:649-657.
[CrossRef] [Google Scholar]
Razzokov J., Yusupov M., Vanuytsel S., Neyts E., Bogaerts A., . Phosphatidylserine flip-flop induced by oxidation of the plasma membrane: a better insight by atomic scale modeling. Plasma. Process Polym.. 2017;14:e1700013.1-e1700013.6.
[CrossRef] [Google Scholar]
Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C., . Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med.. 1999;26:1231-1237.
[CrossRef] [Google Scholar]
Reddi E., Valduga G., Rodgers M., Jori G., . Studies on the mechanism of the hematoporphyrin-sensitized photooxidation of 1,3-diphenylisobenzofuran in ethanol and unilamellar liposomes. Photochem. Photobiol.. 1991;54:633-637.
[CrossRef] [Google Scholar]
Roy D., Fouzder C., Mukhuty A., Pal S., Mondal M., Kundu R., Chowdhury P., . Designed synthesis of dual emitting silicon quantum dot for cell imaging: direct labeling of alpha 2-HS-glycoprotein. Bioconjchem. 2019;30:1575-1583.
[CrossRef] [Google Scholar]
Roy D., Mukhuty A., Fouzder C., Bar N., Chowdhury S., Kundu R., Chowdhury P., . Multi-emissive biocompatible silicon quantum dots: synthesis, characterization, intracellular imaging and improvement of two fold drug efficacy. Dyes Pigm.. 2021;186:109004
[CrossRef] [Google Scholar]
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[CrossRef] [Google Scholar]
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[CrossRef] [Google Scholar]
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[CrossRef] [Google Scholar]
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[CrossRef] [Google Scholar]
Walker R., Everette J., . Comparative reaction rates of various antioxidants with ABTS radical cation. J. Agric. Food Chem.. 2009;57:1156-1161.
[CrossRef] [Google Scholar]
Xu Y., Lin S., He R., Zhang Y., Gao Q., Ng D., Geng J., . C=C bond oxidative cleavage of BODIPY photocages by visible light. Chem. Eur. J.. 2021;27:1-6.
[CrossRef] [Google Scholar]
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Appendix A

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2024.106041.

Appendix A

Supplementary material

The following are the Supplementary data to this article:

Supplementary Data 1

Download

Show Sections

[1] Akhuli A., Preeyanka N., Chakraborty D., Sarkar M., . A Turn-off detection of reactive oxidative species and turn-on detection of antioxidants using fluorescent copper nanoclusters. ACS Appl. Nano Mater.. 2022;5:5826-5837.
[CrossRef] [Google Scholar]

[2] Arts M., Dallinga J., Voss H., Haenen G., Bast A., . A new approach to assess the total antioxidant capacity using the TEAC assay. Food Chem.. 2004;88:567-570.
[CrossRef] [Google Scholar]

[3] Bar N., Chowdhury P., Roy D., Adhikari S., Mondal S., Kanti Das G., Kumar Chandra S., . Photochromism of dye containing Schiff base-metal complex: a revisit through spectro-kinetic, thermodynamic and theoretical analyses for the design of a molecular logic gate. J. Photochem. Photobiol. A. 2021;420:113505-113516.
[CrossRef] [Google Scholar]

[4] Bar N., Chowdhury P., Kanti Das G., . The photodynamic approach to the molecular-level origin of metal-guided photochromism and ultrafast absorption spectroscopy. Spectrochim. Acta A. 2024;312:124031
[CrossRef] [Google Scholar]

[5] Chen X.Q., Wang F., Hyun J.Y., Wei T.W., Qiang J., Ren X.T., Shin I., Yoon J., . Recent progress in the development of fluorescent, luminescent and colorimetric probes for detection of reactive oxygen and nitrogen species. Chem. Soc. Rev.. 2016;45:2976-3016.
[CrossRef] [Google Scholar]

[6] de Freitas C., Calori I., Tessaro A., Caetano W., Hioka N., . Rapid formation of small unilamellar vesicles (SUV) through low-frequency sonication: an innovative approach. Colloid Surface B. 2019;181:837-844.
[CrossRef] [Google Scholar]

[7] Haller B., Göpfrich K., Schröter M., Janiesch J., Platzman I., Spatz J., . Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems. Lab Chip. 2018;18:2665-2674.
[CrossRef] [Google Scholar]

[8] Huang Y., Yu L., Fu L., Hou J., Wang L., Sun M., Wang X., Chen L., . Molecular fluorescent probes for imaging and evaluation of peroxynitrite fluctuations in living cells and in vivo under hypoxic stress. Sensor Actuat. B-Chem.. 2022;370:132410
[CrossRef] [Google Scholar]

[9] Itoh N., Cao J., Chen Z., Yoshida Y., Niki E., . Advantages and limitation of BODIPY as a probe for the evaluation of lipid peroxidation and its inhibition by antioxidants in plasma. Bioorg. Med. Chem. Lett.. 2007;17:2059-2063.
[CrossRef] [Google Scholar]

[10] Jantra S., Butta P., Jithavech P., Rojsitthisak P., Palaga T., Rashatasakhon P., Sukwattanasinitt M., Wacharasindhu S., . “Turn on” orange fluorescent probe based on styryl-BODIPY for detection of hypochlorite and its application in live cell imaging. Dyes Pigm.. 2019;162:189-195.
[CrossRef] [Google Scholar]

[11] Jantra S., Palaga T., Rashatasakhon P., Sukwattanasinitt M., Wacharasindhu S., . A “turn on” fluorometric and colorimetric probe based on vinylphenol-BODIPY for selective detection of Au(III) ion in solution and in living cells. Dyes Pigm.. 2021;191:109341
[CrossRef] [Google Scholar]

[12] Jibril S., Sirat H., Basar N., . Bioassay-guided isolation of antioxidants and α-glucosidase inhibitors from the root of Cassia sieberiana DC (Fabaceae) Rec. Nat. Prod.. 2017;11:406-410.
[Google Scholar]

[13] Kolemen S., Akkaya E., . Reaction-based BODIPY probes for selective bio-imaging. Coordin. Chem. Rev.. 2018;354:121-134.
[CrossRef] [Google Scholar]

[14] Kolemen S., Bozdemir O., Cakmak Y., Barin G., Erten-Ela S., Marszalek M., Yum J., Zakeeruddin S., Nazeeruddin M., Graetzel M., Akkaya E., . Optimization of distyryl-Bodipy chromophores for efficient panchromatic sensitization in dye sensitized solar cells. Chem. Sci.. 2011;2:949-954.
[CrossRef] [Google Scholar]

[15] Kowada T., Maeda H., Kikuchi K., . BODIPY-based probes for the fluorescence imaging of biomolecules in living cells. Chem. Soc. Rev.. 2015;44:4953-4972.
[CrossRef] [Google Scholar]

[16] Liang R., Liu Y., Fu L., Ai X., Zhang J., Skibsted L., . Antioxidants and physical integrity of lipid bilayers under oxidative stress. J. Agric. Food Chem.. 2012;60:10331.
[CrossRef] [Google Scholar]

[17] Lin S., Geng S., Li N., Liu S., Li N., Luo H., . L-histidine-protected copper nanoparticles as a fluorescent probe for sensing ferric ions. Sensor Actuat. B-Chem.. 2017;252:912-918.
[CrossRef] [Google Scholar]

[18] Lu Y., Ke H., Wang Y., Zhang Y., Li H., Huang C., Jia N., . A ratiometric electrochemiluminescence resonance energy transfer platform based on novel dye BODIPY derivatives for sensitive detection of lactoferrin. Biosens. Bioelectron.. 2020;170:112664
[CrossRef] [Google Scholar]

[19] Mondal M., Roy D., Chowdhury P., . Designed functionaliztion of Reduced Graphene Oxide for sorption of Cr(VI) over a wide pH range: a Theoretical and Experimental perspective. New J. Chem. 2018
[CrossRef] [Google Scholar]

[20] Mondal M., Mukherjee S., Joardar N., Roy D., Chowdhury P., Sinha Babu S.P., . Synthesis of smart graphene quantum dots: a benign biomaterial for prominent intracellular imaging and improvement of drug efficacy. Appl. Surf. Sci.. 2019;495:143562
[CrossRef] [Google Scholar]

[21] Qi H., Teng M., Liu M., Liu S., Li J., Yu H., Teng C., Huang Z., Liu H., Shao Q., Umar A., Ding T., Gao Q., Guo Z., . Biomass-derived nitrogen-doped carbon quantum dots: highly selective fluorescent probe for detecting Fe³⁺ ions and tetracyclines. J. Colloid Interf. Sci.. 2019;539:332-341.
[CrossRef] [Google Scholar]

[22] Qu X., Bian Y., Yang B., Zhen S., . A visible BODIPY probe for 'Naked-Eye' detection of pH value and intracellular Fe³⁺. Chin. J. Inorg. Chem.. 2019;35:649-657.
[CrossRef] [Google Scholar]

[23] Razzokov J., Yusupov M., Vanuytsel S., Neyts E., Bogaerts A., . Phosphatidylserine flip-flop induced by oxidation of the plasma membrane: a better insight by atomic scale modeling. Plasma. Process Polym.. 2017;14:e1700013.1-e1700013.6.
[CrossRef] [Google Scholar]

[24] Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C., . Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med.. 1999;26:1231-1237.
[CrossRef] [Google Scholar]

[25] Reddi E., Valduga G., Rodgers M., Jori G., . Studies on the mechanism of the hematoporphyrin-sensitized photooxidation of 1,3-diphenylisobenzofuran in ethanol and unilamellar liposomes. Photochem. Photobiol.. 1991;54:633-637.
[CrossRef] [Google Scholar]

[26] Roy D., Fouzder C., Mukhuty A., Pal S., Mondal M., Kundu R., Chowdhury P., . Designed synthesis of dual emitting silicon quantum dot for cell imaging: direct labeling of alpha 2-HS-glycoprotein. Bioconjchem. 2019;30:1575-1583.
[CrossRef] [Google Scholar]

[27] Roy D., Mukhuty A., Fouzder C., Bar N., Chowdhury S., Kundu R., Chowdhury P., . Multi-emissive biocompatible silicon quantum dots: synthesis, characterization, intracellular imaging and improvement of two fold drug efficacy. Dyes Pigm.. 2021;186:109004
[CrossRef] [Google Scholar]

[28] Song M., Cheng Y., Tian Y., Chu C., Zhang C., Lu Z., Chen X., Pang X., Liu G., . Sonoactivated chemodynamic therapy: a robust ROS generation nanotheranostic eradicates multidrug-resistant bacterial infection. Adv. Funct. Mater.. 2020;30:2003587
[CrossRef] [Google Scholar]

[29] Stern M., McNew J.A., . A transition to degeneration triggered by oxidative stress in degenerative disorders. Mol. Psychiatr.. 2021;26:736-746.
[CrossRef] [Google Scholar]

[30] Su D., Teoh C., Sahu S., Das R., Chang Y., . Live cells imaging using a turn-on FRET-based BODIPY probe for biothiols. Biomaterials. 2014;35:6078-6085.
[CrossRef] [Google Scholar]

[31] Varandas P., Belinha R., Marques S., Cobb A., Serra V., Segundo M., Silva E., . Fluorescent head-labelled phospholipid coumarin bioconjugate as a chemical reporter for antioxidant protection in liposomes. Dyes Pigments. 2023;218:111440
[CrossRef] [Google Scholar]

[32] Walker R., Everette J., . Comparative reaction rates of various antioxidants with ABTS radical cation. J. Agric. Food Chem.. 2009;57:1156-1161.
[CrossRef] [Google Scholar]

[33] Xu Y., Lin S., He R., Zhang Y., Gao Q., Ng D., Geng J., . C=C bond oxidative cleavage of BODIPY photocages by visible light. Chem. Eur. J.. 2021;27:1-6.
[CrossRef] [Google Scholar]

[34] Yan K., Sedgwick A., Zang Y., Chen G., He X., Li J., Yoon J., James T., . Sensors, imaging agents, and theranostics to help understand and treat reactive oxygen species related diseases. Small Methods. 2019;3:1900013
[CrossRef] [Google Scholar]

[35] Zhang M., Song C., Su S., Du F., Li Z., . ROS-activated ratiometric fluorescent polymeric nanoparticles for self-reporting drug delivery. ACS Appl. Mater. Interf.. 2018;10:7798-7810.
[CrossRef] [Google Scholar]

The reaction mechanism of p-hydroxystyryl-substituted BODIPY with ABTS•+ and Fe3+ in solutions and in liposomes

Abstract

Keywords

BODIPY derivatives

Antioxidant fluorescent probe

Reaction mechanism

Small unilamella vesicle

Water-soluble oxidants

1 Introduction

2 Experimental

2.1 Materials and methods

2.2 Synthesis of BOH

2.3 Preparation of small unilamellar vesicles with BOD (or with BOH)

3 Results and discussion

3.1 Absorption and emission spectra of BOD or BOH in ethanol solution

3.2 The reactions with ABTS•+ or Fe3+ in water–ethanol solutions

3.3 The reactions with ABTS•+ or Fe3+ in small unilamellar vesicle (SUV) system

3.4 Chemical kinetics and reaction mechanism

3.5 Sensitivity of BOH as an antioxidant fluorescent sensor for ABTS•+ detection

3.6 BOH + GUV

4 Conclusion

CRediT authorship contribution statement

Acknowledgments

Declaration of competing interest

References

Appendix A

Supplementary material

Appendix A

Supplementary material

Supplementary Data 1

Suggested read for related articles:

The reaction mechanism of p-hydroxystyryl-substituted BODIPY with ABTS^•+ and Fe³⁺ in solutions and in liposomes

3.2 The reactions with ABTS^•+ or Fe³⁺ in water–ethanol solutions

3.3 The reactions with ABTS^•+ or Fe³⁺ in small unilamellar vesicle (SUV) system

3.5 Sensitivity of BOH as an antioxidant fluorescent sensor for ABTS^•+ detection