Synthesis and characterization of BODIPY-DPA-Cu(II)-conjugate HNO fluorescent probes: Effect of linker length on imaging properties

Introduction

Nitroxyl (HNO) has been characterized as protonated species and one-electron-reduced protonated variant of nitric oxide (NO). HNO was a distinguished signaling molecule involved in numerous physiological functions, exhibiting a unique set of biological and chemical characteristics distinct from NO.^{1 –3} HNO can react with thiols to provide either disulfides or sulfinamides, and inhibit the enzyme’s activity.^4,5 Then, HNO has recognized to serve as a potential cardiovascular function regulator that might supply a beneficial tool for cardiac ischemia-reperfusion damage ⁶ and cardiovascular illnesses.^7,8 Currently, there is growing evidence that HNO acts an ideal biomarker for drug-induced liver injury (DILI) diagnosis ⁹ and an potential Parkinson’s biomarker. ¹⁰ Although great progresses regarding the biologically relevant chemistry of HNO of HNO have been achieved, certain questions continue to elude comprehensive exploration. The most captivating queries revolve around whether HNO is indeed synthesized within living organisms, the mechanisms by which it is produced, and if its generation is integral to the biological signaling processes involving redox reactions. Consequently, the creation of methods that are both sensitive and selective for the detection of HNO in biological contexts is of paramount significance.

Regrettably, the exploration of HNO’s multifaceted chemical and physiological attributes is complicated by its inherent tendency to spontaneously dimerized into N₂O in aqueous environments. ¹¹ Various conventional methodologies are available for the identification of NO and HNO, composed of headspace gas chromatography, electron paramagnetic resonance (EPR) analysis, and High-Performance Liquid Chromatography–Mass Spectrometry (HPLC-MS).^{12 –15} These methods often entail prolonged processing times or necessitate the disruption of cellular and tissue integrity, which limited their in vitro and in vivo applications. Fluorescence-probing serves as the most attractive approach to monitor biologically HNO owing to its remarkable sensitivity, exceptional selectivity, and real-time observation capacity.^16,17 To date, a diverse array of fluorescent probes have been judiciously engineered, capitalizing on the distinctive reactivity of HNO with metal complexes^{18 –21} or metalloporphyrins, ²² thiols, ²³ organic phosphines,^{24 –26} and nitroso compounds. ¹⁵ These efforts have significantly propelled the field of HNO fluorescent detection forward. Nonetheless, certain limitations of these probes remain, including slow reaction rates, ²⁶ suboptimal sensitivity, ²⁷ susceptibility to interference by coexisting biological molecules, ²¹ and the necessity for additional surfactants to enhance solubility. ²⁸ Consequently, there remains a critical need to develop new HNO probes that feature rapid response kinetics, superior sensitivity, and exceptional selectivity, to overcome the shortcomings of current options.

Recently, the “covalent-assembly” approach is widely used to construct the fluorescent probes with a strong fluorescence “off–on” response. ²⁹ So far, a kind of BODIPY-DPA-Cu(II)-based fluorescent probes used a dipyrromethene boron difluoride (BODIPY) moiety as fluorescence indicator, a DiPicolylAmine (DPA)-Cu(II) complex as HNO recognition site and a trizole structure as linker. ¹⁹ Those BODIPY-based probes have been widely used for the measurement of HNO levels on the basis of the reduction reaction. ³⁰ However, the impact of varying linker chain lengths on the physicochemical attributes and imaging performance of BODIPY–based fluorescent probes remained uncertain. Therefore, in this study, we constructed four BODIPY-DPA-Cu(II)-conjugate HNO probes such as Cu^II[BD1], Cu^II[BD2], Cu^II[BD3], and Cu^II[BD4] (Chart 1) using ethyl, propyl, benzyl, and biphenyl group as linker (Scheme 1), which have gradually increasing lengths (3.88 Å, 5.02 Å, 6.67 Å, and 10.77 Å). The characterization of these constructed probes involved UV spectra, ¹H NMR, HRMS (High-Resolution Mass Spectrometry), and fluorescence spectra analysis. Those probes show high sensitivities (within 1 min) toward HNO with excellent sensitivity (the lowest detection limit was 0.25 μM). In addition, our findings revealed that the linker length played a crucial role in the imaging characteristics of BODIPY-based fluorescent probes. The fluorescence detection efficiency increases with the growth of the linker chains within 6.67 Å. Moreover, those BODIPY-based probes exhibit high stability toward other species under physiological pH. Finally, the potential in vivo bioimaging application of Cu^II[BD3] was evaluated in the rat blood. Those notable imaging performances displayed by those BODIPY-based probes make them as useful tools to reveal HNO-related dynamic changes in live samples and supply a new methodology for the construction of novel BODIPY-based HNO fluorescent probes.

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Chart 1.

Molecular structures of Cu^II[BD1], Cu^II[BD2], Cu^II[BD3], and Cu^II[BD4].

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Scheme 1.

Synthesis of Cu^II[BD1], Cu^II[BD2], Cu^II[BD3], and Cu^II[BD4].

Results and discussion

Reaction of probes with HNO

To ascertain whether these probes could serve effectively as HNO detectors, their responsiveness to HNO was thoroughly examined. Moreover, to explore the impact of varying linker chain lengths on the physical, chemical attributes, and the detection proficiency of BODIPY-derived HNO probes, a kind of BODIPY-based fluorescent probes, which used BODIPY moiety as fluorescence indicator and DPA-Cu(II) complex as HNO recognition site. Cu^II[BD1] (3.88 Å), Cu^II[BD2] (5.02 Å), Cu^II[BD3] (6.67 Å), and Cu^II[BD4] (10.77 Å) contain gradually increasing lengths of linker chain. The fluorescence intensity of Cu^II[BD1] (3.88 Å), Cu^II[BD2] (5.02 Å), and Cu^II[BD3] (6.67 Å) increased steadily with increasing Angeli’s salt (Na₂N₂O₃, a HNO donor) concentration (see Figure 1) until they reached a steady state at 20 μM Na₂N₂O₃, which aligned with 7.17-, 8.20-, 9.3-fold fluorescent enhancement individually compared to the blank [HNO] within 1 min. Cu^II [BD3] using benzyl group as linker has the biggest fluorescent enhancement (9.3-fold). However, Cu^II[BD4] (10.77 Å) using biphenyl group as linker with the longest length of linker chain has the lowest fluorescent enhancement (1.5-fold), which can be attributed to the weak photo-induced electron transfer (PET) effect of Cu(II) toward BODIPY fluorophore with the longest length of linker chain (10.77 Å).

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Figure 1.

Fluorescence titration of (a) Cu^II[BD1] (1 μM); (b) Cu^II[BD2] (1 μM); (c) Cu^II[BD3] (1 μM); (d) Cu^II[BD4] (1 μM) in PIPES buffer (50 mM, 0.1 M KCl, pH =7.4) in the presence of different amounts of Angeli’s salt. The fluorescence intensity was measured over an elongated duration of 30 min subsequent to adding Na₂N₂O₃, λ_ex: 500 nm, λ_em: 510 nm).

In addition, the fluorescence response of Cu^II[BD1] (3.88 Å), Cu^II[BD2] (5.02 Å), Cu^II [BD3] (6.67 Å), and Cu^II[BD4] (10.77 Å) to HNO displayed a linear relationship during 0–20 μM HNO (Figure S3) and their detection limits were measured as 0.31, 0.3, 0.25, and 0.9 μM (3s/k) respectively (Figure 1). Probe Cu^II [BD3] (6.67 Å) using benzyl group as linker has the highest sensitivity (LOD = 0.25 μM). The fluorescence detection efficiency increases with the growth of the linker chain within 6.67 Å. Therefore, the length of linker chain has an important influence on the detection efficiency. Apart from the increasing chain length of benzyl group, the rigidity of the benzene ring also plays an important role in enhancing the fluorescence detection efficiency.

The selectivity of probes toward HNO

Given their exceptional responsiveness to HNO, a critical next step was to assess their selectivities with different bioactive substances including reactive nitrogen species (RNS), reactive oxygen species (ROS), cations, and anions over an elongated duration of 30 min (see Figure 2). When exposed to 30-fold of NaNO₂, those probes exhibited minimal fluctuation in their fluorescence emission profiles. This observation suggested that the activation observed in the presence of Angeli’s salt is attributed to the generation of HNO, rather than being influenced by the NO²⁻ byproduct. Those probes showed a pronounced specificity toward HNO, as evidenced by their emission behaviors, which were significantly more responsive to HNO than to any other reactive species that are common constituents of the biological milieu. The biologically active compounds, including NO³⁻, ClO⁻, H₂O₂, HO^•, O₂^•−, ONOO⁻, ROO^•, and EDTA, did not result in a notable increase in the emission intensity of the Cu^II[BD] complex (see Figures 2 and 3). The slight increase in emission seen on exposing these probes to NO is particularly striking, highlighting the system’s potential utility in elucidating the hypothesized divergent functions of NO and HNO within biological systems. The above results indicate those copper-based probes were highly selective to HNO over other biorelated species. However, Hcy, Cys, GSH, and H₂S could also restore the fluorescence signal intensity of Cu^II[BD3] (7.44-, 7.77-, 7.90- and 13.95-fold, see Figure S3 in the Supporting Information) because of the reduction of chelated Cu(II)-DPA. To improve the stability and selectivity of BODIPY-based HNO probes, Cu(II)-DPA group would be replaced with Cu(II)-azamacrocyclic in the following work. ³¹

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Figure 2.

Normalized integrated fluorescence intensity of probes (1 μM) in aqueous buffer (50 mM PIPES, 100 mM KCl, pH = 7) over an elongated duration of 30 min subsequent to adding. λ_ex: 500 nm, λ_em: 510 nm.

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Figure 3.

The photo of Cu^II[BD3] under ultraviolet lamp (365 nm) after the addition of different ROS and RNS.

Considering the common pH range of 4.5–8.0 encountered within live cellular environments, we explored the impact of pH on the emission behavior of Cu^II[BD3] in the presence of HNO. Acid-base fluorescence titrations revealed that the fluorescence intensity of Cu^II[BD3] remained constant within the pH spectrum from 6 to 9. The result suggested that this probe is well suited for functioning optimally under physiological pH condition (Figure 4).

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Figure 4.

Fluorescence responses of Cu^II[BD3] (10 µM) at pH 2 ~ 12. λ_ex: 500 nm, λ_em: 510 nm.

To further explore in vivo application potential of those BODIPY-DPA-Cu(II)-Conjugated HNO probes, the application of Cu^II[BD3] in whole blood from rat was also investigated. As shown in Figure 5, Cu^II[BD3] exhibited minimal fluctuation in the fluorescence intensity after 6-h incubation in the blood; when exposed to 30-fold of HNO, a 8.6-fold increase in fluorescent intensity was observed. This significant enhancement demonstrated the potential in vivo application of Cu^II[BD3].

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Figure 5.

Fluorescence responses of Cu^II[BD3] (1 µM) at PBS solution, blood, and HNO.

Summary and conclusion

We have constructed a new series of BODIPY-DPA-Cu(II)-conjugate HNO probes such as Cu^II[BD1] (3.88 Å), Cu^II[BD2] (5.02 Å), Cu^II[BD3] (6.67 Å), and Cu^II[BD4] (10.77 Å) using ethyl, propyl, benzyl, and biphenyl group as linker, which contained increasing lengths of linker chains. Those probes show exceptional selectivity sensitivity toward HNO, especially probe Cu^II[BD3] (6.67 Å) using benzyl group as linker has the highest sensitivity (LOD = 0.25 μM). The fluorescence detection efficiency increases with the growth of the linker chain within 6.67 Å. The length of linker chain has an important influence on the detection efficiency. Apart from the increasing chain length of benzyl group, the rigidity of the benzene ring also plays an important role in enhancing the fluorescence detection efficiency. Furthermore, these BODIPY-DPA-Cu(II)-conjugate probes exhibited superior specificity for HNO, distinguishing it effectively from other biologically relevant ROS and RNS under physiological pH, which make them hold great potential for practical applications in living system. Finally, the potential in vivo bioimaging application of BODIPY-DPA-Cu(II)-conjugate HNO probe was evaluated in rat blood. Therefore, our present study provides a novel strategy for the construction of new BODIPY-based HNO fluorescent probes with enhanced characteristics which holds the potential to significantly broaden the scope of in vivo biomedical imaging practices.

Supplemental Material