Design, synthesis and performance test of a hydrogen peroxide fluorescent probe based on selenomorpholine and pyrimidine

ABSTRACT As a vital biological molecule, H2O2(Hydrogen Peroxide) is involved in different physiological and pathological processes. And in addition to this, H2O2 is suitable for medical wound disinfection, environmental disinfection and food disinfection. Accordingly, it is of high significance to conveniently and effectively detect H2O2 in the environment and organisms. This subject synthesised a fluorescent probe Pyrimidine-Se by using selenomorpholine and pyrimidine dye, which detect H2O2 in an aqueous environment. Pyrimidine-Se has a large Stokes shift (Δλ = 155 nm), which can specifically and quickly quantitatively detect H2O2. When the probe Pyrimidine-Se reacted with H2O2, the Se(II) in the selenomorpholine was oxidised to Se(IV), so the electron withdrawing ability of the electron withdrawing group of Pyrimidine-Se was improved, and the fluorescence intensity of the probe were enhanced. Interestingly, Se(IV) can be reduced by GSH (Glutathione) to Se(II) to quench the fluorescence, and this redox cycle can continue several times, which indicated that it can have a potential of real-time monitoring the redox state in vivo. The probe was also satisfactorily used to detect H2O2 in Argentina Bloodfin larvaes and abnormal H2O2 content in some organs was detected.


Introduction
H 2 O 2 is the most stable type of ROSs.High concentrations of H 2 O 2 are cytotoxic to animals, plants and bacteria [1,2].It is generally known that H 2 O 2 in cells was mainly produced by the NADPH oxidase complex, and H 2 O 2 appears as a by-product of various enzymatic reactions.For instance, the conversion of glucose into glucose lactone by the oxidation of glucose oxidase can synthesise H 2 O 2 in the cell [3][4][5][6].H 2 O 2 in the normal concentration range significantly regulates cell proliferation, differentiation, senescence and signal transduction [7].An abnormal cell content of H 2 O 2 may cause cancer, Alzheimer's disease, etc. [8][9][10][11][12][13].The redox state of cells is dynamically adjustted by reactive oxygen species and biological thiols, which can maintain the normal life activities of organisms [14][15][16][17].Thus, a method is required to dynamically monitor the oxidationreduction state of organisms.
On the whole, the main methods that have been developed to detect H 2 O 2 include: iodometric determination [18], spectrophotometry [19], bioluminescence method [20], mass spectrometry [21], chromatography [22], electrochemistry [23], resonance spectroscopy [24], as well as fluorescence analysis [14,[25][26][27][28][29].Compared with other detection methods, fluorescence analysis has been recognised as an essential tool for the biological imaging detection because of its prominent selectivity, high sensitivity, high spatial and temporal resolution, non-destructive detection and low cost of use.Various H 2 O 2 fluorescent probes have been reported previously, most of which are intensity-based turn-on fluorescent probes.However, there are rare H 2 O 2 fluorescent probes that can circulate redox reactions [30,31].
Thus, this subject developed a novel type of H 2 O 2 fluorescent probe Pyrimidine-Se, consisting of a selenomorpholine group [32] and a pyrimidine group connected to the N, N-dimethylaminophenyl group (Scheme 1).H 2 O 2 can oxidise Se(II) in the selenomorpholine group to Se(IV), while GSH is capable of reducing Se(IV) to Se(II), and this redox cycle can last several times.Pyrimidine-Se reacts exclusively and rapidly to H 2 O 2 , and the fluorescence intensity of the probe shows a good linear relationship with the concentration of H 2 O 2 (0-100 μM).Gaussian 09 programme and mass spectrometry were used to explore the mechanism of probe detection of H 2 O 2 .After Pyrimidine-Se responded to H 2 O 2 , the valence state of Se changed from Se(II) to Se(IV), and the electron transfer intensity inside the probe increased, thereby resulting in the increase in the fluorescence intensity of Pyrimidine-Se.Pyrimidine-Se was satisfactorily applied to the detection of H 2 O 2 in Argentine Bloodfin larvae, and it was found that some organs in the body had abnormal H 2 O 2 content.

Materials and equipment
All chemicals used were purchased from suppliers such as Anaiji, Macleans, Aladdin, etc., and no further purification was necessary before use.TLC (Thin Layer Chromatography) was used in the synthesis process, and the product was purified using Qingdao Marine Silica Gel (200-300 meshes). 1 H NMR and 13 C NMR spectra were recorded on Bruker DRX-600 nuclear magnetic resonance spectrometer.HR-ESI-MS was tested by Bruker Solarix XR FTMS of Analytical Instrumentation Center (Peking University).The structure of Pyrimidine-Se single crystal was analysed by Bruker D8 VENTURE single crystal diffractometer.Toshiba F-2700 fluorescence spectrophotometer was used for all fluorescence data detection(Photomultiplier voltage: 700 V, slits width of 5 × 5 nm), and SP-1920 ultraviolet-visible spectrophotometer of Shanghai Spectrometer Co., Ltd. was used for absorption spectrum detection.The pH measurement used METTLER TOLEDO FE28 pH metre.The computer platform used to optimise compounds using density functional theory (DFT) on the Gaussian 09 programme is Dell OptiPlex 3050.

Synthesis
The synthesis of Pyrimidine-Se followed the method in Scheme 1.The preparation of compound 1 was through the use of Knoevenagel Condensation reaction will be 4 -Dimethylaminobenzaldehyde and 2 -Chloro -4, 6 -dimethylpyrimidine together.In an alkaline environment, Selenomorpholine forms a chemical bond with the carbon on compound 1 by nucleophilic substitution to form Pyrimidine-Se. The structures of compound 1 and Pyrimidine-Se were characterised by 1 H NMR, 13 C NMR and HR-ESI-MS.(Fig. S3-S8) Fortunately, we slowly evaporated the Pyrimidine-Se solution(methanol) at room temperature to get its crystal (Fig. S11, Table S1) (CCDC number:2097485).

Synthesis of selenomorpholine
Under Ar atmosphere, the powder of selenium (7.9 g, 0.10 mol) was stirred in absolute ethanol (100 ml) at −10°C.Then sodium borohydride (4.3 g, 0.11 mol) was slowly added to the mixture in portions to react until the solution became colourless.Sodium hydroxide (4.4 g, 0.11 mol) was added to the solution in portions, followed by stirring for 30 minutes.Then, an ethanol solution of bis(2-chloroethyl)ammonium hydrochloride (17.8 g, 0.10 mol) was gradually added and refluxed for 6 h.After the mixture was cooled to room temperature, the reaction solution was filtered and concentrated by rotary evaporation.A colourless oil (boiling point 56°C/1.32kP)was obtained by distillation under reduced pressure, and the yield was 30%.The selenomorpholine obtained by the reaction was stored at low temperature under an argon atmosphere. 1 H NMR (600 MHz, CDCl 3 , TMS) δ(ppm): 3.20-3.14(m, 4 H), 2.60-2.54(m, 4 H), 1.59 (s, 1H).(Fig. S2)

Pyrimidine-Se spectral data
Table S2 UV

Fluorescence reaction of Pyrimidine-Se on redox cycle
Glutathione, cysteine, homocysteine and other biological thiols can reduce the action groups oxidised in the cell.Accordingly, biological thiols can significantly maintain the balance of active oxygen concentration in life.Selenium has been found as an essential trace element in the human body.Due to its unique chemical properties, it can significantly eliminate active oxygen and free radicals.GSH can reduce Pyrimidine-Se oxidised by H 2 O 2 .(Scheme 10) After the addition of H 2 O 2 (50 μM), the fluorescence of the Pyrimidine-Se increased significantly.After the addition of 5 eq of GSH, the fluorescence  of the probe dropped close to the initial state.Subsequently, after the addition of H 2 O 2 , the solution waited for the fluorescence to increase.The redox process could be cycled at least 4 times.Thus, the probe Pyrimidine-Se can achieve a continuous cyclic response between Se(II) and Se(IV), thereby indicating that Pyrimidine-Se has the potential to dynamically monitor the REDOX cycle.

Argentine-bloodfin culture
The Argentine bloodfin larvaes, due to their transparent and non-fluorescent bodies((A), (B) and (C) in Scheme 11), are selected as ideal models for testing the ability of Pyrimidine-Se to detect H 2 O 2 in organisms in real-time.Commercially available  Argentine Bloodfin larvae was employed as the experimental material, incubated in water containing Pyrimidine-Se (10 μM) for 1 h, and subjected to a microscope for imaging detection.According to Scheme 11, Argentine Bloodfin showed blue fluorescence under 365 nm UV lamp.However, compared with tissues (fish fins and muscles), some of its internal organs showed bright yellow-green fluorescence, which indicated that some organs have a higher H 2 O 2 content.NAC(N-Acetyl-L-cysteine) is an effective ROS scavenger that can remove H 2 O 2 or inhibit its production in cells.According to Huang's [34] work, incubation of zebrafish with NAC could remove ROSs in their bodies effectively.An  Argentine Bloodfin larvae was incubated in water containing NAC (1 mM) for 6 h and was treated with the probe Pyrimidine-Se (10 μm) for 1 h.The fluorescence of viscera of Argentine bloodfin larvae decreased ((G), (H) and (I) in Scheme 11).Thus, Pyrimidine-Se exhibits high biocompatibility and it is a very promising fluorescent probe that can monitor H 2 O 2 in animals in real-time.

Mechanism of Pyrimidine-Se detection to H 2 O 2
Scheme 12 predicts the detection mechanism of Pyrimidine-Se on H 2 O 2 .The oxidation reaction of Pyrimidine-Se with H 2 O 2 was verified by MS spectroscopy.After the addition of H 2 O 2 , the valence state of Se in Pyrimidine-Se varied from Se (II) to Se (IV), thereby forming Pyrimidine-SeO.In the MS spectrum (Fig. S1), [M+] = 386.8and [M+] = 402.7,thereby indicating that Pyrimidine-SeO was synthesised.The charge attraction ability of selenomorpholine to pyrimidinyl groups facilitated the ICT process and improved the fluorescence intensity of the probe molecules.
1 H NMR and 13 C NMR titration experiments were conducted in order to better explain the mechanism of H 2 O 2 reaction with Pyrimidine-Se.As shown in Fig. S9, the 1 H NMR and 13 C NMR data showed obviously change in the presence of 1 equiv.H 2 O 2 using CDCl 3 as the solvent.After the probe Pyrimidine-Se reacted with H 2 O 2 , the chemical shift has significance changed with respect to probe Pyrimidine-Se.This was because of the oxidation of selenium (II), resulting in the charge of selenomorpholine was affected.Density functional theory (DFT) was used to optimise the structure of Pyrimidine-Se, Pyrimidine-SeO, Pyrimidine-SeH + and Pyrimidine-SeOH + at the b3lyp/6-31* opt freq scrf(pcm,solvent = water) level.Correspondingly, the structure, electron density and molecular electrostatic potential were analysed.According to Fig. S10, the selenomorpholine groups in Pyrimidine-Se, Pyrimidine-SeO, Pyrimidine-SeH + and Pyrimidine-SeOH + exhibit stable boat-like structure.The ground state (HOMO) electrons of Pyrimidine-Se and Pyrimidine-SeO mostly concentrate on N, N-dimethylaminophenyl.In the excited state (LUMO) of the two, the charge is attracted to the pyrimidine group that has showed a strong ability to attract the charge.When Pyrimidine-Se was oxidised by H 2 O 2 , Se(II) was oxidised to Se(IV), which reduced the ability of selenomorpholine to supply charge to the pyrimidine group, thereby improving the charge transfer (ICT) in the molecule strength.Subsequently, the fluorescence of the probe increased.Due to the basicity of selenomorpholine, the fluorescence intensities of Pyrimidine-Se and Pyrimidine-SeO are greatly affected in strongly acidic environments.In acidic environment, the selenomorpholines of Pyrimidine-Se and Pyrimidine-SeO rotate nearly 90° (Fig. S10).DFT was used to optimise the structure of the two molecules.The charge of Pyrimidine-SeH + is mainly concentrated near the pyrimidine group, and the charge of Pyrimidine-SeOH + is mainly concentrated near the pyrimidine group and the oxidised selenomorpholine.The intramolecular charge transfer is severely restricted, so the fluorescence of Pyrimidine-Se and Pyrimidine-SeO is quenched under acidic conditions.

Conclusion
In brife, this project synthesised a new type of H 2 O 2 fluorescent probe (Pyrimidine-Se) that can detect H 2 O 2 in water.It was synthesised by a simple reaction between selenomorpholine and pyrimidine fluorophore.After the probe was oxidised with H 2 O 2 , the Se(II) in the selenomorpholine became Se(IV), which affected the electron absorption capacity of the pyrimidine in the pyrimidine fluorophore, thereby achieved the change in the fluorescence spectrum.The fluorescence intensity of the probe shows a real good linear relationship with the concentration of H 2 O 2 (0-100 μM).Interestingly, H 2 O 2 can oxidise the probe and increase its fluorescence intensity, while GSH can reduce it.This redox cycle can continue several times.Thus, it allows for the real-time imaging of redox processes.Furthermore, the probe was also successfully implemented to detect H 2 O 2 in the organs of Argentine Bloodfin larvaes.It is noteworthy that the H 2 O 2 content of some organs far exceeded that of other tissues.Therefore, Pyrimidine-Se has potential biological application value.

Scheme 1 .
Scheme 1. Synthesis and fluorescence action mechanism of the fluorescence probe Pyrimidine-Se.
-Vis spectrophotometer measured the excitation wavelength of the probe Pyrimidine-Se(10 μM)  in different solvent systems (Scheme 1).Using the maximum absorption wavelength of Pyrimidine-Se in different solvents as the excitation wavelength, the fluorescence spectrum of Pyrimidine-Se was detected (Scheme 2).The fluorescence intensity (Scheme 3) of Pyrimidine-Se and Pyrimidine-SeO was found to be sensitive to changes in pH values.The fluorescence intensity of the probe Pyrimidine-Se increased significantly from pH 2 to 7. When the pH value is from 7 to 12, the fluorescence decreased slightly.The fluorescence intensity of Pyrimidine-SeO is also greatly affected by pH.When the pH ranges from 3 to 5, the fluorescence intensity of Pyrimidine-Se at 533 nm varies significantly, almost an increase of 18 times.The Henderson-Hasselbalch equation was used to analyse the data, the pK a of Pyrimidine-Se was estimated as 3.94.The fluorescence intensity of Pyrimidine-Se varies considerably in different solvent systems.Pyrimidine-Se solution(DMSO) exhibited the highest fluorescence brightness, and PBS buffer solution exhibites the weakest fluorescence.When the Pyrimidine-Se reacts with H 2 O 2 , its excitation wavelength wavelength is blue shifted from 390 nm to 378 nm (Scheme 4).(Scheme 5) Pyrimidine-Se and excess H 2 O 2 can quickly complete the reaction.After Pyrimidine-Se reacted with 5eq of H 2 O 2 , its fluorescence at 533 nm increased rapidly, and it remained unchanged after reaching the maximum within 15 min.When the probe did not react to H 2 O 2 , its fluorescence intensity did not vary over time.According to Scheme 6 and Scheme7, the fluorescence intensity of the probe Pyrimidine-Se at 533 nm increased with the increase in H 2 O 2 concentration, while the fluorescence at 460 nm first increased and then decreased.When the concentration of H 2 O 2 was 500 μM, all Pyrimidine-Se was oxidised by H 2 O 2 , and the fluorescence intensity of Pyrimidine-Se reached the peak.When the concentration of H 2 O 2 was between 0-100 μM, the fluorescence intensity of Pyrimidine-Se showed a very good linear relationship with the concentration of H 2 O 2 , which aroused our attention.The fitting equation is written as Y = 36.83X+ 757.28, the linear correlation coefficient was 0.994, and the detection limit of the probe is 1.3 μM.Under 365 nm UV light, the fluorescence of the probe significantly varied from light blue to green in response to H 2 O 2 .(Scheme8 and Scheme 9) The response of Pyrimidine-Se to ROSs was studied in PBS buffer (10 mM, pH = 7.4).The red bars in Scheme 9 represent the fluorescence intensity of Pyrimidine-Se in response to ROSs.Black bars represent the fluorescence intensity of Pyrimidine-Se in the presence of both H 2 O 2 (50 μM) and other ROSs (0.5 mM).Though some studies reported that selenomorpholine as a hypochlorite receptor[33], Pyrimidine-Se exhibits selectivity to H 2 O 2 in ROSs, and the fluorescence spectrum changes significantly.Other analytes (0.5 mM) expressed slight and negligible changes in fluorescence.Pyrimidine-Se can still effectively detect H 2 O 2 when other analytes are present with H 2 O 2 .

Fig 10 .
Fig 10.The fluorescence response of Pyrimidine-Se (10 μM) to the redox cycle.Pyrimidine-Se was first oxidised with 120 μM H 2 O 2 .After 0.5 h, 5 eq of GSH were added to the solution.After the fluorescence intensity returned to the original level, the solution was treated with other H 2 O 2 and This redox cycle was performed 4 times.The detection was performed in PBS Buffer (10 mM, pH = 7.4, λ ex = 388 nm and, λ em = 528 nm).

Fig 11 .
Fig 11.Microscopic images of Argentine Bloodfin larvaes.Imaging detection of commercially available Argentine bloodfin larvae: (A) Argentine Bloodfin larvae under visible light; (B) Argentine Bloodfin larvae under UV light; (C) The Argentine Bloodfin larvae are under UV light and visible light.Fluorescent image of an Argentine Bloodfin larvae incubated with the probe (10 μg/mL) for 60 min: (D)Argentine Bloodfin larvae under visible light;(E) Argentine Bloodfin larvae are under UV light;(F) The Argentine Bloodfin larvae are under UV light and visible light.The larvae were first incubated with NAC (1 mM) for 6 h and then with Pyrimidine-Se (10 μM) for 1 h:(G) Argentine Bloodfin larvae under visible light;(H) Argentine Bloodfin larvae are under UV light;(I) The Argentine Bloodfin larvae are under UV light and visible light.