A Selenamorpholine and Pyrimidine-based Redox-responsive Fluorescent Probe and Its Response Mechanism

A new type of hydrogen peroxide (H 2 O 2 ) uorescent probe Pyrimidine-Se was synthesized from selenomorpholine and pyrimidinyl and the large Stokes shift (Δλ>140 nm) was exhibited. The uorescence intensity of Pyrimidine-Se is very sensitive to pH, and its pK a value is 9.06. While the probe is reacted with H 2 O 2 , the selenomorpholine changes from Se (II) to Se (IV), which enhances the electron-withdrawing ability of the Pyrimidine-Se electron-withdrawing group. Based on this, the probe Pyrimidine-Se was used to detect H 2 O 2 by the uorescence spectrum. The detection limit of the probe Pyrimidine-Se was 1.3 µM. At the same time, we also found that Pyrimidine-Se displayed the reversibility back and forth between H 2 O 2 and GSH. The reaction mechanism with H 2 O 2 was veried by mass spectrometry and simulation on the Gaussian 09 program.


Introduction
Hydrogen peroxide is a light blue liquid compound combined with a covalent bond. It can be miscible with water in any proportion and easily penetrate the cell membrane. It is generally considered that high concentration of H 2 O 2 has cytotoxicity to a variety of animals, plants, and bacteria [1][2], and it is also an important product of cell life activities. It is known that the H 2 O 2 in the cell is mainly produced by the NADPH oxidase complex, and H 2 O 2 appears as a by-product in a variety of enzymatic reactions. For example, the conversion of glucose to glucose lactone by the oxidation of glucose oxidase can produce H 2 O 2 in the cell [3][4][5][6]. The normal concentration of peroxide concentration plays an important role in regulating cell proliferation, differentiation, aging, and signal transduction [7]. The abnormal cell content of H 2 O 2 may cause cancer, Alzheimer's disease, etc [8][9][10][11][12][13][14]. The redox state of cells is dynamically regulated by reactive oxygen species and biological thiols, which can maintain the normal life activities of cells [15][16][17][18]. Therefore, a means to dynamically monitor the oxidation-reduction state is needed.
At present, the main methods that have been developed to detect hydrogen peroxide are: titration [19], spectrophotometry [20], bioluminescence [21], mass spectrometry [22], chromatography [23], electrochemistry [24], resonance spectroscopy [25][26], and uorescence analysis [27][28][29][30][31][32]. Compared with other detection methods, uorescence analysis has become an important tool for biological imaging detection due to its excellent selectivity, high sensitivity, high spatial and temporal resolution, nondestructive detection and low cost of use. A variety of H 2 O 2 uorescent probes have been reported before, and most of these probes are intensity-based turn-on uorescent sensors. Changes in probe position, probe concentration, probe environment, and excitation intensity may affect the measurement of uorescence intensity as well as emission collection e ciency and excitation intensity, which may further affect the accuracy and reliability of uorescence intensity measurement. Moreover, there are still very few H 2 O 2 uorescent probes capable of cycling redox reactions [33][34]. For this reason, we designed and synthesized a new type of H 2 O 2 uorescent probe Pyrimidine-Se, which is composed of selenomorpholine group [35][36] and pyrimidine group connected with N, N-dimethylaminophenyl group (scheme 1). The selenomorpholine group is the recognition group of H 2 O 2 , and GSH can reduce and oxidize the selenomorpholine group.
Pyrimidine-Se responds quickly to H 2 O 2 , and the reaction with excess H 2 O 2 is almost instantaneous. The uorescence intensity of Pyrimidine-Se has a good linear relationship with 0-20 times the concentration of H 2 O 2 , and the linear correlation coe cient (R 2 ) is 0.996. Pyrimidine-Se has good speci city and some ions can detect a variety of reactive oxygen species. The concentration of reactive oxygen species (ROS), amino acids, ions, etc. (100 µm) is much greater than H 2 O 2 (20 µm), and the uorescence intensity of other detected substances is not signi cant. The change. At the same time, we also tested the probe's responsiveness to H 2 O 2 and GSH, and the results showed that the cyclic response to both can last 4 times. We simulated the reduced, oxidized, and acidi ed probes on the Gaussian 09 software through the B3LYP/6-31* OPT FREQ level. We also used nuclear magnetic titration and mass spectrometry to explain the mechanism of the probe detecting H 2 O 2 : Pyrimidine-Se After responding to H 2 O 2 , the valence state of Se changes from Se(II) to Se(IV), and the electron transfer intensity inside the probe increases, which leads to an increase in the uorescence intensity of Pyrimidine-Se.

Materials And Equipment
The chemicals used are all purchased from suppliers such as Anaiji, Macleans, Aladdin, and Daimo, and no further puri cation is required before use. In the synthesis process, the high-e ciency thin-layer plate was used for TLC analysis (thin-layer chromatography (TLC) analysis), and the product was puri ed by silica gel column chromatography using Qingdao Ocean Silica Gel (200-300 mesh). 1 H NMR and 13 C NMR spectra were recorded on Bruker DRX-600. The HR-ESI-MS was detected by Bruker Solarix XR FTMS from the Analysis and Testing Center of Peking University. All uorescence data detection uses Toshiba F-2700 uorescence spectrophotometer, and absorption spectrum detection uses SP-1920 UV-Vis spectrophotometer of Shanghai Spectrometer Co. Ltd. The pH measurement used METTLER TOLEDO's FE28 type pH meter. According to Xu's [35] work, selenomorpholine was synthesized. Reactive oxygen solution (ROS) was prepared according to Jiao Shan's [37] work. The water used in the con guration solution is ultrapure water (micropores, ≥18MΩ).

Synthesis of Selenomorpholine
Under the protection of Ar, selenium powder (7.9 g, 0.10 mmol) was stirred in anhydrous ethanol (100 mL) in a 250 mL three-necked ask at -10 °C. Sodium borohydride (4.3 g, 0.11 mmol) was gradually added to the solution in portions until colorless. Then sodium hydroxide (4.4 g, 0.11 mmol) was added to the solution in portions and stirred for 30 minutes. An ethanol solution of bis(2-chloroethyl)amine hydrochloride (17.8 g, 0.10 mmol) was gradually added and re uxed for 6 h. The mixture was cooled to room temperature, ltered to remove the insoluble matter, and then the solvent was removed by rotary evaporation. Distillation under reduced pressure (56 °C/1.32 kP) gave a colorless oil with a yield of 30%.

Results And Discussion
The UV-Vis spectrophotometer was used to determine the maximum absorption wavelength of the probe Pyrimidine-Se (10 μM) in different solvent systems (Fig. 2), and the probe Pyrimidine-Se was measured in the range of 400 nm-500 nm with a uorescence spectrophotometer for six different The solvent is screened. The maximum absorption wavelength (λ ex ) of the probe Pyrimidine-Se in the six solvents is 382 nm. The uorescence intensity of Pyrimidine-Se varies greatly in different solvent systems (Fig. 3). The highest brightness is in DMSO, the highest The weak one is in ethanol (the photomultiplier tube voltage is 400V).
We evaluated the optical response of the probe Pyrimidine-Se to pH ( Fig. 4 and Fig. 5). When the pH value is from 3 to 5, the uorescence intensity of Pyrimidine-Se at 525 nm changes greatly, almost an increase of 18 times. Using the Henderson-Hasselbalch equation to analyze the data, the pKa of Pyrimidine-Se was calculated to be 9.06. The absorbance of the probe Pyrimidine-Se at 395 nm decreased rapidly, and the absorbance at 455 nm increased rapidly (Fig. 6). Due to the basicity of the pyrimidine on Pyrimidine-Se and the in uence of the nitrogen atom on the selenomorpholine group, the uorescent probe is greatly affected in a highly acidic environment. By calculating the B3LYP/6-31* OPT FREQ level on the Gaussian 09 program (Fig. 16), the selenomorpholine of Pyrimidine-SeH + is in phase with the selenomorpholine groups of Pyrimidine-Se and Pyrimidine-SeO. The de ection of nearly 90 ° occurs, and the energy difference between the excited state (LUMO) and the ground state (HOMO) becomes smaller. The charge of Pyrimidine-SeH + is mainly concentrated near the benzene ring, which is very different from the uorescence quenching in an acidic environment. Big relationship.
After the probe Pyrimidine-Se reacts with H 2 O 2 , its maximum absorption wavelength is red-shifted from 382 nm to 388 nm (Fig. 7), and the uorescence emission wavelength is also red-shifted (Fig. 8).
The response time of Pyrimidine-Se (10 μm) to H 2 O 2 (10 μm, 20 μM, 100 μM) was tested, and the results showed that the probe has a high sensitivity to H 2 O 2 (Fig. 9). Pyrimidine-Se and excess H 2 O 2 can quickly complete the reaction. In a solution of (pH=7.4, 10 mmol, 30% DMSO), the wavelength of the excitation wave is 388 nm, and the probe shows weak uorescence at 528 nm. After being oxidized by H 2 O 2 , the uorescence of the probe at 528nm increases.
As shown in Fig. 11, the uorescence intensity of the probe Pyrimidine-Se increases with the increase of The reaction of Pyrimidine-Se (10 μM) to reactive oxygen species and other analytes was studied in PBS Buffer (30% DMSO, 10 mM, pH=7.4). According to reports, selenomorpholinyl has competitive adaptability to the detection of H 2 O 2 and hypochlorite. As shown in the Fig. 15, Pyrimidine-Se has high speci city for H 2 O 2 detection, and the uorescence of H 2 O 2 detection (λ em =525 nm) is much stronger than other he uorescence intensity of other analytes increased slightly, but it was negligible. As shown in the gure, when other analytes and H 2 O 2 are both present, the detection of H 2 O 2 by Pyrimidine-Se will not be interfered.
Fluorescence reaction of Pyrimidine-Se on redox cycle Biological thiols such as glutathione, cysteine, and homocysteine can reduce the active groups oxidized in cells. Therefore, biological thiols play an important role in maintaining the balance of intracellular reactive oxygen species concentration. Selenium is an important trace element in the human body. Its unique chemical properties have excellent performance in eliminating reactive oxygen species and free radicals. Pyrimidine-Se responds to the redox cycle of H 2 O 2 and glutathione. It can be seen from Fig. 16 that the uorescence of Pyrimidine-Se probe increased signi cantly after adding 5 eq H 2 O 2 . After adding 3 eq of GSH for two hours, it can be seen that the uorescence of the probe decreases close to the initial state, and then add 5 eq of H 2 O 2 to the solution to wait for uorescence enhancement. The redox process can be cycled at least 4 times. It is proved that the probe Pyrimidine-Se can realize the continuous cyclic response between H 2 O 2 and GSH, indicating that Pyrimidine-Se has the potential for real-time imaging of redox cycle.
Pyrimidine-SeH + Using density functional theory (DFT) to optimize the structure of Pyrimidine-Se, Pyrimidine-SeO and Pyrimidine-SeH + at the B3LYP/6-31* OPT FREQ level on the Gaussian 09 program. The structure, electron density, and molecular electrostatic potential are analyzed accordingly. As shown in the Fig. 17, the selenomorpholines of Pyrimidine-Se, Pyrimidine-SeO, and Pyrimidine-SeH + present boat-like con gurations. The electrons in the ground state (HOMO) of Pyrimidine-Se and Pyrimidine-SeO are mainly concentrated in the N, N-dimethylaminophenyl group. In the excited state (LUMO) state of the two, the charges are attracted by the strongly attracting pyrimidine groups and then migrate. When Se(II) is oxidized to Se(IV), the charge is enriched in Se=O, which enhances the intensity of charge transfer (ICT) in the molecule, and thus the uorescence increases (Fig. 18). The selenomorpholine of Pyrimidine-SeH + undergoes a nearly 90 ° twist after accepting hydrogen ions. From its ESP diagram, it can be seen that its charge is mainly concentrated in the pyrimidine group and mainly exhibits an excited state (LUMO). The ground state (HOMO) and excited state (LUMO) energies of Pyrimidine-SeH + are much smaller than those of Pyrimidine-Se and Pyrimidine-SeO, so the uorescence of Pyrimidine-SeH + is quenched in an acidic environment.

Conclusion
A novel hydrogen peroxide uorescent probe Pyrimidine-Se with large Stokes shift was synthesized. It was synthesized by a simple combination of selenium morpholine and pyrimidine uorescent groups.
Through the change of Se ( ) to Se ( ) after reaction with H 2 O 2 , the probe affects the electron absorption ability of pyrimidine in pyrimidine uorescent group, so as to realize the change of uorescence intensity. The probe can quickly and speci cally recognize H 2 O 2 , which is less affected by other reactive oxygen species (ROS) and ions. The uorescence intensity of the probe MNG has a good linear relationship in the range of H 2 O 2 concentration 0-200 µM In addition, the Pyrimidine-Se redox cycle can last at least four times. Therefore, it has the potential of real-time imaging of the redox process. Declarations