Method for in-situ monitoring of progress of photochemical cycloaddition reaction by means of fluorescence spectroscopy

Abstract

The present invention relates to the field of the monitoring of photochemical cycloaddition reactions, and relates to a method for in-situ monitoring of the progress of a photochemical cycloaddition reaction by means of fluorescence spectroscopy. The method for in-situ monitoring of the progress of a photochemical cycloaddition reaction by means of fluorescence spectroscopy of the present invention comprises: preparing a fluorescent material that can be subjected to a photochemical cycloaddition reaction; causing the fluorescent material to be subjected to a photochemical cycloaddition reaction under the irradiation of ultraviolet visible light; monitoring a fluorescence change of the material by means of fluorescence spectroscopy; and performing in-situ monitoring on the photochemical cycloaddition reaction to monitor the reaction progress of the occurred photochemical reaction. Compared with conventional in-situ monitoring technology, the method for in-situ monitoring of the progress of a photochemical cycloaddition reaction by means of fluorescence spectroscopy has the advantages of being simple to operate, rapid and efficient, and easy to capture signals, having a wide range of application, and reducing the experimental costs and the operation difficulty.

Description

A method for in situ monitoring of photochemical cycloaddition reaction progress using fluorescence spectroscopy Technical Field

[0001] The present invention relates to the field of in-situ monitoring of photochemical cycloaddition reactions, in particular to a method for in-situ monitoring of the progress of photochemical cycloaddition reactions using fluorescence spectroscopy technology. Background Art

[0002] Real-time monitoring of chemical processes through in situ / online monitoring technology is the key to understanding intermediate processes and reaction mechanisms, and has received widespread attention and application in the fields of catalysis, synthesis, energy, sensors, etc. (Qian, GD, et al. Chem. Rev. 2012, 112(2), 1126-1162). Among them, a series of structural characterization techniques such as X-ray diffraction / scattering / absorption, electron microscopy (EM), optical microscopy (OM), scanning probe microscopy (SPM), X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance spectroscopy, various spectra, mass spectrometry, chromatography, etc. have been applied to monitor reaction intermediates and products on time scales from years to picoseconds and spatial scales from meters to angstroms, greatly expanding and deepening the understanding of chemical reactions and having great benefits for optimizing material properties (Hess, C. Chem. Soc. Rev. 2021, 50(5), 3519-3564).

[0003] Solid-state [2+2] photocycloaddition reactions can be used to synthesize cyclobutane derivatives with stereochemical configurations that are difficult to obtain using solution reaction methods (Barbour, LJ et al. Angew. Chem., Int. Ed. 2018, 57(47), 15563-15566). However, the dynamic processes and kinetics of the photoreaction process remain difficult to understand (Medishetty, R., et al. Angew. Chem., Int. Ed. 2014, 53(23), 5907-5911). This is due to the limitations of experimental conditions and technical levels, as well as the influence of starting materials, intermediate product mixtures, etc. during the chemical reaction process. There are relatively few reports on in situ monitoring of the specific evolution of photocycloaddition reaction products. In particular, when there are intermediates in the photoreaction and the concentration of the photoreaction products is very low in a short period of time, it is difficult for traditional characterization techniques to accurately track and identify them (Wang, Y., et al. Angew. Chem., Int. Ed. 2018, 57(24), 7120-7125). Based on this, using appropriate in situ techniques to visualize and monitor the entire photoreaction process is particularly important for understanding its structural dynamic transformation.

[0004] Common methods used to characterize photocycloaddition reactions include nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (FT-IR), Raman spectroscopy, thermal analysis, and X-ray diffraction (XRD). However, few methods can be used in situ. NMR is often used to monitor the real-time production of products in solution reactions, but it is not easily used to identify products produced in solid-state reactions. Infrared and Raman spectroscopy primarily measure changes in functional group structure during the reaction, but have difficulty identifying the interconversion of isomers. Thermal analysis, mainly including thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), uses temperature changes to study the progress of photochemical reactions. However, excessive heating or high temperatures often trigger thermal ring-opening reactions, making this method a less preferred technique for measuring the progress of photoreactions. XRD techniques mainly include single-crystal X-ray diffraction and powder X-ray diffraction based on Bragg diffraction. Although there have been many reports on SCSC transformations in solid-state [2+2] photocycloaddition reactions, in situ crystallographic studies of SCSC transformations remain limited, and current reports mainly focus on initial and final states. At the same time, when the proportion of cyclobutane products produced under short-term illumination is very small, the low spatial and temporal resolution of XRD technology makes it impossible to detect local sites or components (Li, X., et al. J. Am. Chem. Soc. 2020, 142(21), 9567-9581). Therefore, it is challenging to find a highly sensitive and fast-response in situ characterization technique to monitor the dynamic evolution of solid-state [2+2] photocycloaddition reaction intermediates.

[0005] Due to the extremely high sensitivity and good selectivity of fluorescence detection, fluorescence sensing systems have attracted a lot of attention (Filez, M., et al. Angew. Chem., Int. Ed. 2021, 60 (25), 13803-13806). The olefin ligands used in the solid-state [2+2] photocycloaddition reaction usually exhibit interesting fluorescence properties due to the π conjugated system in their structure. Reactions or changes in intermolecular forces will change the conjugation and structure of the coordination polymer, which will in turn affect the fluorescence properties of the compound structure before and after the reaction (Mizobe, Y., et al. Chem. 2009, 15 (33), 8175-8184). In solid fluorescence tests, if the surface conditions are slightly different, the fluorescence intensity will change greatly. Therefore, monitoring the changes during the entire solid-state [2+2] photocycloaddition reaction by in situ fluorescence spectroscopy is of great significance for qualitative and quantitative analysis of the entire process.

[0006] Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the problems in the prior art of slow and complicated in-situ monitoring of photochemical cycloaddition reactions and difficulty in capturing the dynamic evolution of reaction intermediates.

[0008] To address the above-mentioned technical problems, the present invention provides a method for in situ monitoring of photochemical cycloaddition reactions using fluorescence spectroscopy. This method involves designing and synthesizing a fluorescent material capable of undergoing a solid-state photochemical reaction; subjecting the prepared material to a solid-state photochemical cycloaddition reaction under ultraviolet-visible light irradiation; and utilizing fluorescence spectroscopy and confocal laser scanning microscopy to monitor the fluorescence changes of the material during the solid-state photochemical cycloaddition reaction, thereby monitoring the progress of the solid-state photochemical reaction. This overcomes the problems in the prior art of in situ monitoring of photochemical cycloaddition reactions, which are slow and complex, and difficult to capture the dynamic evolution of reaction intermediates. Compared to other monitoring methods, this method has the advantages of being simple to operate, rapid and efficient, and easy to capture signals, thus reducing experimental costs and operational difficulty.

[0009] The first object of the present invention is to provide a method for in situ monitoring of the progress of a photochemical cycloaddition reaction using fluorescence spectroscopy, comprising the following steps:

[0010] Providing a fluorescent material that undergoes a photochemical cycloaddition reaction;

[0011] The fluorescent material undergoes a photochemical cycloaddition reaction under ultraviolet-visible light irradiation, and the fluorescence spectroscopy technology is used to monitor the fluorescence changes of the fluorescent material during the photochemical cycloaddition reaction, thereby realizing in-situ monitoring of the reaction process of the photochemical cycloaddition reaction.

[0012] In one embodiment of the present invention, the photochemical cycloaddition reaction is an intermolecular or intramolecular cycloaddition reaction induced by light.

[0013] In one embodiment of the present invention, the intermolecular or intramolecular cycloaddition reaction is a [2+2], [4+2], [4+4] cycloaddition reaction, a Diels-Alder reaction, or a polymerization reaction of acetylene molecules.

[0014] In one embodiment of the present invention, the fluorescence spectroscopy technique uses a fluorescence spectrometer or a laser confocal scanning microscope to perform spectral measurement.

[0015] In one embodiment of the present invention, the method for in situ monitoring of a photochemical cycloaddition reaction using fluorescence spectroscopy is as follows:

[0016] S1. Evenly dispersing a fluorescent material in an organic solvent to obtain a mixed solution containing the fluorescent material; dropping the mixed solution onto a reaction carrier and drying it;

[0017] S21. The reaction carrier in S1 is subjected to a staged light irradiation reaction to obtain a cycloaddition product; wherein, the stage is divided into N stages, the first stage is: reaction temperature -173°C to 0°C, reaction time is 0.01 to 72h; the second stage is: reaction temperature is 0 to 210°C, reaction time is 1 to 120h; wherein N is an integer greater than 1; further, preferably N is 2, 3, 4, etc.

[0018] S22. Use a fluorescence spectrometer to monitor the fluorescence changes of the fluorescent material in the solid-state photochemical cycloaddition reaction at different stages, and the steps are as follows: collect the fluorescence emission spectra of the fluorescent material in the reaction carrier at different time points after the first stage light irradiation reaction in S21; collect the fluorescence emission spectra of the fluorescent material in the reaction carrier at different time points after the second stage light irradiation reaction; collect the fluorescence emission spectra of the fluorescent material in the reaction carrier at different time points after the Nth stage light irradiation reaction; count the fluorescence intensity values ​​at different time points, and according to the changes in fluorescence intensity, realize qualitative or quantitative monitoring of the reaction progress of the photochemical cycloaddition.

[0019] In one embodiment of the present invention, at least one or more of the following conditions are met:

[0020] The reaction carrier is selected from a quartz plate and a glass plate;

[0021] The organic solvent is a conventional volatile organic solvent in the art, preferably one or more of ethanol, methanol, and acetonitrile;

[0022] The thickness of the fluorescent material on the reaction carrier after drying is 0.2 to 0.5 mm.

[0023] In one embodiment of the present invention, the method for in situ monitoring of a photochemical cycloaddition reaction using fluorescence spectroscopy is as follows:

[0024] (1) Select crystals of fluorescent material after being irradiated with ultraviolet-visible light at different time points for scanning observation; at the same time, set up a control group, which is a crystal of fluorescent material that has not been irradiated with light; wherein during the scanning observation process, the sample is excited by a semiconductor laser at 200-700nm, and the fluorescence emission signal is collected at 250-800nm ​​in the blue channel of a laser confocal scanning microscope;

[0025] (2) Determine the progress of the reaction based on the changes in fluorescence intensity and the collected confocal microscope photos.

[0026] In one embodiment of the present invention, in step (1), at least one or more of the following conditions are met:

[0027] The organic solvent is a conventional volatile organic solvent in the art, preferably one or more of ethanol, methanol, and acetonitrile;

[0028] The transparent carrier is selected from quartz plate and glass plate.

[0029] In one embodiment of the present invention, in step (2), when the fluorescent material is solid, the reaction observation is performed directly using solid crystals.

[0030] In one embodiment of the present invention, in step (2), the scanning observation is an xyz three-dimensional scan, and the specific process is as follows: the three-dimensional scan starts from the top plane of the crystal, monitoring the fluorescence of multiple sections (for example, a total of 427 sections are tested), wherein the thickness of each section is set to 100 nm, and these sections form a three-dimensional image of the crystal. An external LED light source is placed above the single crystal and irradiated with ultraviolet and visible light for a period of time t, and a three-dimensional scan is performed; the time series scanning process (blank control scanning) is as follows: a fluorescence image of the single crystal is collected every 3 to 10 seconds until the reaction ends, preferably the end time is 1 hour.

[0031] In a specific embodiment, the above method is used to monitor the [2+2] photochemical cycloaddition reaction of CP1 ([Cd2(F-1,3-bpeb)2(3,5-DBB)4], wherein F-1,3-bpeb is (5-fluoro-1,3-)bis[(cis)2-(pyridyl-4-)vinyl]benzene, 3,5-DBB is a unit that loses a hydrogen ion after the reaction of 3,5-dibromobenzoic acid, and the synthesis method of CP1 is based on Lang JP, et al. Nat. Commun. 2022, 13(1), 2847.), as follows:

[0032] Method 1: Fluorescence spectrometer method:

[0033] (1) The synthesized CP1 or prepared CP1-1 ([Cd2(bfpvpcd)(3,5-DBB)4], wherein bfpvpcd = 4,4'-(3,4-bis(3-fluoro-5-(2-pyridine-4-(vinyl)phenyl)cyclobutane-1,2-)bipyridine), 3,5-DBB is a unit that loses hydrogen ions after the reaction of 3,5-dibromobenzoic acid. The synthesis method of CP1-1 refers to Lang JP, et al. Nat. Commun. 2022, 13(1), 2847.) crystals are placed in a mortar and ground into powder. The powder is evenly dispersed in ethanol to obtain a suspension. The suspension is dropped on a quartz plate and dried in air. The sample thickness is about 0.2 to 0.5 mm.

[0034] (2) The [2+2] photochemical cycloaddition reaction is divided into two steps. In the first step of the photocycloaddition reaction, CP1 is irradiated with UV-visible light at –50 °C for 10 min to generate the intermediate product CP1-1. The in situ time-correlated emission spectra under different conditions are collected. In the second step of the photochemical cycloaddition reaction, CP1-1 is irradiated with ultraviolet-visible light at room temperature to generate the final product CP1-1β ([Cd4(tpbfimc)2(3,5-DBB)8], where bfpvpcd = 4,4'-(3,4-bis(3-fluoro-5-(2-pyridine-4-(vinyl)phenyl)cyclobutane-1,2-)bipyridine), 3,5-DBB is a unit that loses a hydrogen ion after the reaction of 3,5-dibromobenzoic acid. The synthesis method of CP1-1β refers to Lang JP, et al. Nat. Commun. 2022, 13(1), 2847.), and the in situ time-correlated emission spectra under different conditions are collected. The fluorescence intensity of the fluorescent material at different time points is counted, and the progress of the reaction is judged according to the fluorescence intensity.

[0035] The illumination process involves the following: In the first step of the reaction (low-temperature illumination), a quartz plate containing the sample is placed in a long glass tube, which is then placed in a low-temperature reaction bath. After stabilization for 30 minutes, the sample is illuminated with an LED light source (250-700nm, 2-100W) for a period of time to form photoproducts. The distance between the sample and the LED light source is fixed at approximately 17cm.

[0036] The second step of the reaction is illumination (room temperature illumination): The sample on the quartz plate is illuminated with an LED light source (250-700nm, 2-100W) for a period of time to form photoproducts. The distance between the sample and the LED light source is fixed at about 10cm.

[0037] Method 2: Laser confocal scanning microscopy:

[0038] CP1 was dispersed in ethanol solution and then sandwiched between a pair of clean glass slides for fluorescence observation. CP1 was excited at 405 nm using a semiconductor laser, and the fluorescence emission signal was collected at 515–600 nm in the blue channel.

[0039] Two CP1 single crystals of different sizes were selected for scanning observations. One crystal was used to test xyz 3D scanning (z-stack scan) and 3D reconstruction, and the other crystal was used for time series scanning.

[0040] The specific process of xyz 3D scanning is as follows: a. A CP1 single crystal was selected from a glass sheet. It was an irregular hexagonal prism with a cross-sectional size of approximately 233.4×38.1μm. The 3D scanning started from the top plane of the crystal. The thickness of each section was set to 100nm. A total of multiple (427) sections were tested, and these sections formed a 3D image of the crystal (crystal 3D model). b. An external LED light source (250-700nm, 2-100W) was placed approximately 10cm above the crystal. After each time the selected crystal was exposed to UV-visible light for a period of time t (t = 1min, 3min, 10min, 20min, 40min and 60min), a 3D scan was performed. During the entire illumination process, the crystal position, light source position and all test conditions remained unchanged.

[0041] The specific process of the time series scanning (blank control scanning) is as follows: a fluorescence image of the CP1 single crystal is collected every 3-10 seconds until the photochemical cycloaddition reaction is complete.

[0042] In one embodiment of the present invention, the wavelength of the ultraviolet-visible light is 250-700 nm.

[0043] The second object of the present invention is to provide an application of the method in monitoring the kinetic process of a photochemical cycloaddition reaction.

[0044] The above technical solution of the present invention has the following advantages over the prior art:

[0045] (1) The fluorescence spectroscopy technique described in the present invention was used to monitor the two-step solid-phase [2+2] photocycloaddition process of CP1 under UV-visible light irradiation at room temperature, and its reaction kinetics were studied. The photocycloaddition reaction of CP1 is a two-step solid-phase [2+2] photocycloaddition reaction synergistically acted upon by temperature and light. Under UV-visible light irradiation at 25°C, a two-step reaction occurs: first, CP1 rapidly generates a monocyclobutane compound, CP1-1, which then reacts to generate a bicyclobutane compound, CP1-1β, after further UV-visible light irradiation (Figure 2).

[0046] (2) Due to the different fluorescence properties of the products during the photoreaction process, the [2+2] photocycloaddition reaction of CP1 single crystal under UV-visible light irradiation is accompanied by a clear trend of fluorescence intensity from weak to strong and then weak again, and a blue shift of the maximum emission wavelength. This provides a basis for the in situ monitoring of photochemical cycloaddition reactions by fluorescence spectroscopy.

[0047] (3) The fluorescence emission intensity of the in situ photochemical cycloaddition reaction monitoring method of the present invention can still change significantly even in a very short time of ultraviolet-visible light irradiation, when the photoreaction progress is very weak and the amount of cyclobutane product is very small. By monitoring the fluorescence emission intensity change trend in these two steps of the reaction by in situ fluorescence spectroscopy, the changes in the products and the kinetic process during the photoreaction process can be qualitatively and quantitatively analyzed.

[0048] (4) The fluorescence spectroscopy monitoring in situ photochemical cycloaddition reaction method described in the present invention directly observed the asynchronous fluorescence changes of the surface and inner layer of a single CP1 crystal during the solid-phase [2+2] photocycloaddition process using a laser confocal microscope (LSCM), confirming the heterogeneity of the photocycloaddition reaction process. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0050] FIG1 is a schematic diagram of an apparatus for the in-situ fluorescence spectroscopy monitoring method of Test Example 1 of the present invention.

[0051] FIG2 shows the two-step solid-phase [2+2] photocycloaddition process of CP1 under UV-visible light irradiation at room temperature in Test Example 1 of the present invention.

[0052] FIG3 shows the structure of CP1 in Test Example 1 of the present invention.

[0053] FIG4 is a diagram of the light irradiation reaction in Test Example 1 of the present invention. 19 F NMR spectra (DMSO-d6), where (a) is CP1; (b) is CP1 irradiated with UV-visible light (λ = 365 nm, 2 W) at –50 °C for 10 min; (c) is CP1 irradiated with UV-visible light (λ = 365 nm, 2 W) at 25 °C for 1 h or CP1-1 irradiated with UV-visible light (λ = 365 nm, 2 W) at 25 °C for 35 min. 19 F NMR spectrum (DMSO-d6).

[0054] Figure 5 shows the irradiation time of CP1 under ultraviolet visible light (λ = 365nm, 2W) at 25°C in Test Example 1 of the present invention. 19 F NMR spectrum (DMSO-d6).

[0055] Figure 6 is the powder X-ray diffraction patterns of (a) CP1, (b) CP1-1 and (c) CP1-1β in Test Example 1 of the present invention (the black one is the simulated curve of single crystal data, the red one is the measured curve, and the blue one is the test curve after CP1 is kept in ethanol for 24 hours after grinding).

[0056] FIG7 is a diagram of solid-state fluorescence excitation and emission spectra of compounds F-1,3-bpeb, CP1, CP1-1, and CP1-1β in Test Example 2 of the present invention.

[0057] FIG8 is an in situ solid fluorescence emission spectrum (λ) of CP1 in Test Example 2 of the present invention (a) under ultraviolet-visible light (λ = 365 nm, 2 W) at 25° C. ex =365nm). (b) Changes in the solid-state fluorescence intensity of CP1 at 437nm as a function of irradiation time with UV-visible light (λ = 365nm, 2W) at 25°C. Inset: A partial view showing the increase in the solid-state fluorescence intensity of CP1 at 437nm as a function of irradiation time with UV-visible light (λ = 365nm, 2W) at 25°C. (1s) λ represents the fluorescence emission intensity of CP1 at 437 nm after 1 second of UV-visible light (λ = 365 nm) irradiation at 25°C. F represents the fluorescence emission intensity of CP1 at 437 nm after irradiation with UV-visible light (λ = 365 nm) at 25°C for different durations. The fluorescence emission intensity of CP1 increased 2.7-fold after only 1 second of UV-visible light irradiation.

[0058] FIG9 is a fluorescence photograph of the CP1 powder in Test Example 2 of the present invention under irradiation with ultraviolet-visible light (λ=365 nm) at room temperature.

[0059] FIG10 is an in situ solid-state fluorescence emission spectrum (λ) of CP1 in Test Example 2 of the present invention (a) under ultraviolet-visible light (λ = 365 nm, 2 W) irradiation at -50°C. ex =365nm). Inset: Photographs of solid powder samples of CP1 and CP1-1 under UV-visible light (b) In-situ solid fluorescence emission spectrum of CP1-1 irradiated with UV-visible light (λ = 365nm, 2W) at 25°C (λ ex =365nm). Inset: Photographs of solid powder samples of CP1-1 and CP1-1β under UV-visible light. (c) During the conversion of CP1 to CP1-1, c0-c t The relationship between the photocycloaddition reaction from CP1 to CP1-1 is a zero-order reaction. t represent the conversion rates calculated based on the fluorescence intensity of CP1 during UV-visible light irradiation at –50°C. (d) During the conversion of CP1-1 to CP1-1β, Ln(c0 / c t ) versus illumination time, indicating that the photocycloaddition reaction from CP1-1 to CP1-1β is a first-order reaction. t represent the conversion rates calculated based on the fluorescence intensity of CP1-1 during UV-visible light irradiation at 25 °C.

[0060] FIG11 shows the fluorescence lifetimes of CP1, CP1-1, and CP1-1β in Test Example 3 of the present invention.

[0061] FIG12 is a graph showing the conversion rate curves of the photochemical products of CP1 to CP1-1 (red) and CP1-1 to CP1-1β (blue) at different time intervals in Test 4 of the present invention. The conversion rate is calculated based on the fluorescence intensity.

[0062] Figure 13 shows the irradiation time of CP1 under ultraviolet visible light (λ = 365nm, 2W) at -50°C in Test 4 of the present invention. 19 F NMR (DMSO-d6).

[0063] Figure 14 shows the irradiation time of CP1-1 under UV-visible light (λ=365nm, 2W) at 25°C in Test 4 of the present invention. 19 F NMR (DMSO-d6).

[0064] FIG15 is a graph showing the conversion rate of photochemical products from CP1 to CP1-1 (red) and CP1-1 to CP1-1β (blue) at different time intervals in Test 4 of the present invention. The conversion rate is based on 19 Calculated by F NMR.

[0065] FIG. 16 shows the process of converting CP1 to CP1-1 in Test 4 of the present invention, c0-c t The relationship between the photocycloaddition reaction from CP1 to CP1-1 is a zero-order reaction. t Represent the CP1 at -50℃ UV-visible light irradiation process 19 Conversion rate calculated by F NMR. (b) During the conversion of CP1-1 to CP1-1β, Ln(c0 / c t ) versus illumination time, indicating that the photocycloaddition reaction from CP1-1 to CP1-1β is a first-order reaction. t Respectively represent the UV-visible light irradiation process of CP1-1 at 25℃ 19 Conversion calculated by F NMR.

[0066] FIG. 17 is a diagram of the present invention in test 4 according to 19 Linear relationship between the conversion rate calculated by F NMR and the conversion rate calculated by fluorescence intensity: (a) The conversion of CP1 to CP1-1. (b) The conversion of CP1-1 to CP1-1β.

[0067] Figure 18 shows (a) a time series scan of a CP1 crystal under 405 nm laser light for 1 hour in Test 5 of the present invention, with a scale bar of 50 μm. (b) quantitative luminescence intensity of a CP1 crystal after continuous scanning at different times under 405 nm laser light.

[0068] Figure 19 shows (a) a fluorescence image of a CP1 crystal in a 3D scan, scale bar 50 μm, in Test 5 of the present invention. (b) a 3D image of CP1 derived from the 3D scan, scale bar 50 μm.

[0069] Figure 20 shows (a) fluorescence images of CP1 at different layers after UV-visible light irradiation for different times (t = 1 min, 3 min, 10 min, 20 min, 40 min, 60 min) in Test 5 of the present invention. Scale bar, 50 μm. Z represents the layer number. (b) Quantitative fluorescence intensity of (c) ROIs at different layers after UV-visible light irradiation for different times of CP1 as a function of irradiation time. (d) Schematic diagram of the fluorescence intensity changes of a single CP1 crystal under UV-visible light irradiation at 25°C. DETAILED DESCRIPTION

[0070] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0071] Example 1 Preparation of fluorescence test samples

[0072] The sample is sandwiched between two quartz plates for testing. Take the preparation of sample CP1 as an example:

[0073] The synthesized CP1 (synthesis method, see Lang JP, et al. Nat. Commun. 2022, 13(1), 2847) crystals were ground into powder in a mortar. Next, 10 mg of the powder was evenly dispersed in 1 mL of ethanol. The suspension (100 μL) was evenly dropped onto a quartz glass plate (12 × 20 mm), dried in air, and then covered with another quartz glass plate to prepare a sample. The sample thickness was approximately 0.3 mm.

[0074] Example 2 UV-visible light experiment

[0075] Grind the synthesized CP1 or prepared CP1-1 crystals into a powder in a mortar. Next, evenly disperse 10 mg of the powder in 1 mL of ethanol. Drop 200 μL of the suspension onto a quartz plate (25 × 25 mm) and air-dry. The sample thickness is approximately 0.3 mm.

[0076] Room temperature illumination: The sample on the quartz plate was illuminated with an LED light source (365 nm, 2 W) for a period of time to form photoproducts. The distance between the sample and the LED light source was fixed at approximately 10 cm.

[0077] Low-temperature illumination: Place the quartz plate containing the sample in a long glass tube, which is then placed in a low-temperature reaction bath. After stabilization for 30 minutes, illuminate the sample with an LED light source (365nm, 2W) for a period of time to form photoproducts. The distance between the sample and the LED light source is fixed at approximately 17cm.

[0078] In the single crystal structure of CP1 and its solid-phase [2+2] photocycloaddition reaction, CP1-1 can be considered an intermediate in the process of forming CP1-1β. In the present invention, a powder sample of CP1 was irradiated with UV-visible light using a 2W UV-visible lamp (λ = 365nm). The powder sample of CP1 was directly irradiated with UV-visible light at 25°C to quickly form CP1-1, and CP1-1β was generated after 1 hour. In the intermediate process, a mixture of CP1-1 and CP1-1β existed. After irradiation at a low temperature of -50°C for 10 minutes, it was completely converted into CP1-1, and then irradiated at room temperature for 35 minutes to produce CP1-1β.

[0079] Example 3 Study on the Fluorescence Properties of CP1 in Solid-Phase [2+2] Photocycloaddition Reaction

[0080] During the photoreaction of CP1, when the illumination time is less than 10 seconds, the concentration of photoproducts is low, and neither nuclear magnetic resonance spectroscopy nor single-crystal X-ray diffraction can detect their presence. However, when CP1 is exposed to UV-visible light, the sample rapidly brightens. This phenomenon inspired in situ fluorescence spectroscopy monitoring of the reaction process, as shown in Figure 1. Compounds CP1, CP1-1, and CP1-1β are stable in air, and their crystal structures remain intact even after immersion in ethanol for 24 hours (Figure 2).

[0081] Example 4 In situ monitoring of the two-step [2+2] photocycloaddition reaction process by fluorescence spectroscopy

[0082] Fluorescence spectroscopy was used to monitor the two-step [2+2] photocycloaddition reaction in situ. In the first step, CP1 was irradiated with UV-visible light at –50°C to generate CP1-1, and the in situ time-correlated emission spectrum was collected under these conditions.

[0083] Example 5: Laser confocal scanning microscope observation of crystal fluorescence changes

[0084] Selected high-quality CP1 was dispersed in ethanol and sandwiched between a pair of clean glass slides for fluorescence observation. CP1 was excited at 405 nm using a semiconductor laser, and the fluorescence emission signal was collected in the blue channel at 515–600 nm. Two CP1 single crystals of different sizes were selected for scanning observation. One crystal was used to test xyz 3D scanning (z-stack scanning) and 3D reconstruction, while the other was used for time-lapse scanning.

[0085] XYZ 3D Scanning: A CP1 single crystal selected from a glass slide was an irregular hexagonal prism with a cross-sectional dimension of approximately 233.4 × 38.1 μm. The 3D scan began at the top plane of the crystal. Each slice was 100 nm thick, and a total of 427 slices were tested, from which a 3D image of the crystal was generated. An external LED light source (365 nm, 2 W) was positioned approximately 10 cm above the crystal. After each period of UV-visible light exposure (t = 1 min, 3 min, 10 min, 20 min, 40 min, and 60 min), the selected crystal was subjected to a 3D scan. Throughout the entire illumination process, the crystal position, light source position, and all test conditions remained unchanged.

[0086] Time series scanning: a fluorescence image of the CP1 single crystal was acquired every 5 s for up to 1 h.

[0087] Test Example 1 Characterization of the [2+2] Photocycloaddition Reaction Process of CP1 under UV-Vis Illumination

[0088] The two-step photoreaction process (Figure 3) was confirmed by SCXRD (Figure 4) and 19 The results were characterized by F NMR (Figures 5 and 6).

[0089] Test Example 2 Fluorescence Test of CP1 in Solid-Phase [2+2] Photocycloaddition Reaction

[0090] At room temperature, CP1 exhibits a weak emission band at 451 nm under 365 nm excitation with a quantum yield (QY) of 0.8%. ex =350nm,λ em =451nm) and F-1,3-bpeb(λ ex =346nm, λ em =441 nm) have approximately the same emission wavelength, so the fluorescence mechanism of CP1 may be attributed to the charge transfer between and within the ligands (Figure 7).

[0091] In situ fluorescence emission spectra of CP1 were collected at room temperature under UV-visible light (λ = 365 nm, 2 W) to investigate fluorescence changes during the photoreaction. An ethanol suspension of CP1 was spread as evenly as possible on a glass slide and dried at room temperature for fluorescence measurement. As shown in Figure 8, the [2+2] photocycloaddition reaction of CP1 is accompanied by a clear trend of fluorescence intensity shifting from weak to strong and then weak again, and a blue shift in the maximum emission wavelength. Within 3 minutes of UV-visible light irradiation, the fluorescence intensity of the CP1 powder sample increased dramatically, reaching a 45-fold increase after 3 minutes, and the maximum emission wavelength shifted to 437 nm. With continued UV-visible light irradiation, the fluorescence intensity gradually decreased to a minimum. Notably, the fluorescence intensity of the CP1 powder sample increased 2.7-fold after only 1 second of UV-visible light irradiation, demonstrating the sample's high sensitivity to UV-visible light (Figure 3-8b). Microscopic images of a large number of powder samples during this photocycloaddition reaction were also recorded as a function of irradiation time. As shown in Figure 9, the CP1 powder can be easily observed with the naked eye, with obvious fluorescence enhancement and reduction, which is consistent with the fluorescence spectroscopy data. This phenomenon also inspired the systematic study of the [2+2] photocycloaddition reaction by in situ fluorescence spectroscopy.

[0092] Test Example 3: In-situ fluorescence spectroscopy monitoring of the two-step [2+2] photocycloaddition reaction process

[0093] As shown in Figure 10a, the fluorescence emission intensity gradually increased with increasing UV-visible light irradiation time at –50°C, accompanied by a blue shift in the maximum emission wavelength from 451 nm to 437 nm. When CP1 was irradiated with UV-visible light at –50°C for 10 min, the fluorescence emission intensity reached its highest level, approximately 71 times that of the initial sample CP1, and the quantum yield increased to 58.5%. In the second step, the photocycloaddition reaction of CP1-1 to CP1-1β at 25°C, the fluorescence emission intensity gradually decreased with increasing UV-visible light irradiation time, with no significant blue or red shift in the maximum emission wavelength. When CP1-1 was completely converted to CP1-1β, the fluorescence emission intensity reached its lowest level, approximately 1 / 20 of that of the initial sample CP1-1, and the quantum yield dropped to 0.2% (Figure 10b).

[0094] Test Example 4: In-situ fluorescence spectroscopy monitoring of fluorescence lifetime during a two-step [2+2] photocycloaddition reaction

[0095] The emission lifetimes of these three compounds were measured separately, and the calculated results showed that the fluorescence lifetimes of CP1, CP1-1, and CP1-1β were 6.8 ns, 13.9 ns, and 5.4 ns, respectively, indicating that they all have fluorescence characteristics ( FIG. 11 ).

[0096] Test Example 5 Fluorescence Intensity Kinetic Analysis

[0097] According to the analysis of the fluorescence intensity changes in the above two-step reaction, it was shown that the emission peak intensity showed a significant increase / decrease with the change of CP1-1 yield, and the formation of CP1-1 can be tracked by monitoring the changes in the in situ fluorescence emission intensity (Figure 12). Therefore, the dynamics of each step can be studied from the fluorescence intensity data set (Figures 10c and 10d). At the same time, by 19 F NMR was used to test the two-step photocycloaddition reaction process and study the kinetics of each step (Figures 13–16). The conversion rate calculated from the fluorescence intensity in these two steps was fitted with the illumination time. The results showed that the kinetic types of the two reaction processes were different. Specifically, the first step [2+2] photocycloaddition reaction from CP1 to CP1-1 is a zero-order reaction, while the second step from CP1-1 to CP1-1β is a first-order reaction (Figures 10c and 10d). The obtained kinetic type is consistent with the reaction rate constants k and 19 The results of F NMR are basically consistent. In order to better illustrate that the in situ fluorescence intensity can quantitatively describe the solid phase [2+2] photocycloaddition reaction process, according to 19 The conversion rates calculated by F NMR and fluorescence intensity fitted a linear relationship (Figure 17). The results from the two testing methods were largely consistent, demonstrating that in situ fluorescence spectroscopy is a very effective method for monitoring [2+2] photocycloaddition reactions.

[0098] Test Example 6 Laser Confocal Scanning Microscope (LSCM) Technology Test

[0099] There are many factors that affect the reaction rate of the photoreaction, such as the thermal effect during UV-visible light irradiation, the light intensity of UV-visible light, the size of the crystal, the crystal surface exposed to UV-visible light, the temperature during the reaction, etc. All of these factors will affect the kinetics of the photoreaction. The influence of thermal effect, light intensity and temperature has been excluded as much as possible. Therefore, the fluorescence intensity is calculated and 19 The discrepancies between the conversions calculated by F NMR may result from the inhomogeneous illumination of the complex crystals, which was also confirmed by the subsequent laser confocal microscopy test results.

[0100] In order to visually observe the fluorescence changes of a single crystal during the [2+2] photocycloaddition reaction, laser confocal microscopy (LSCM) data of CP1 under UV-visible light irradiation at room temperature were collected and analyzed.

[0101] Test Example 6.1 LSCM Test of CP1 without UV-Visible Light Irradiation

[0102] First, LSCM data were collected for CP1 every 5 seconds for up to 1 hour without UV-visible light irradiation to verify the reliability of the technique. As shown in Figure 18, the fluorescence of the crystal was clearly observed and the fluorescence intensity remained constant during the 1-hour laser scanning of CP1, indicating that the 405 nm laser in the instrument had no effect on the fluorescence of the CP1 sample during the test.

[0103] Test Example 6.2: Fluorescence changes of each layer of a single crystal obtained by LSCM testing under different UV-visible light exposure times

[0104] Based on the fluorescence emission spectra of CP1 exposed to UV-visible light (λ = 365 nm) at 25°C for various durations, layer scans of a CP1 single crystal were acquired at various UV-visible light exposure times (t = 1 min, 3 min, 10 min, 20 min, 40 min, and 60 min). The scans were taken from the top plane of the crystal, with each layer having a thickness of 100 nm. Due to the high resolution of LCSM, detailed fluorescence changes in each layer of the crystal after UV-visible light exposure were observed (Figure 19).

[0105] Test Example 6.3: Relationship between fluorescence intensity and time obtained by LSCM at different depths of a single crystal

[0106] As shown in Figure 3-20, the fluorescence intensity within a layer first increases and then decreases with increasing irradiation time, which is consistent with the fluorescence emission spectrum results showing that the sample fluorescence changes from weak to strong and then weak again. The fluorescence intensity changes uniformly within each layer, indicating that [2+2] photocycloaddition occurs in each layer. However, different fluorescence intensity variations were observed at different depths within a single crystal. To further understand the fluorescence intensity variations at different depths within a single crystal, a series of fluorescence images recorded by LSCM layer scanning were intensity quantified, and the relationship between fluorescence intensity at different depths and irradiation time (t) was studied (Figure 20). The results show that the fluorescence intensity is highest in layers 1–260 after 1 minute of CP1 irradiation, while the fluorescence intensity is highest after 3 minutes of UV-visible light irradiation from layer 270 (approximately 27 μm deep in the crystal) to the bottom. This result indicates that the photoreaction rate in the lower layers of the crystal is slower than that in the upper layers. When a single crystal of CP1 is exposed to UV-visible light at 25°C, it undergoes a [2+2] photocycloaddition reaction. CP1 in the structure first generates CP1-1, and the fluorescence intensity at the top of the single crystal increases, while the fluorescence intensity of the unreacted portion below remains unchanged. With increasing illumination time, the lower portion of the crystal gradually reacts to generate CP1-1, while the upper portion of the crystal transforms from CP1-1 to CP1-1β. The fluorescence is manifested by brightening of the lower portion of the single crystal and dimming of the upper portion (Figure 20d). This phenomenon clearly demonstrates that the [2+2] photocycloaddition reaction begins at the top of the crystal, where light contacts it, and gradually reaches the lower layer. This is the first time that the sequence of the [2+2] photocycloaddition reaction has been clearly observed through fluorescence changes. The results further demonstrate that fluorescence is an effective means of monitoring the progress of photocycloaddition reactions.

[0107] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for in situ monitoring of the progress of a photochemical cycloaddition reaction using fluorescence spectroscopy, characterized in that: The following steps are involved: Provide a fluorescent material that undergoes a photochemical cycloaddition reaction; The fluorescent material is subjected to a photochemical cycloaddition reaction under ultraviolet visible light irradiation, and fluorescence spectroscopy is used to monitor changes in the fluorescence intensity of the fluorescent material during the photochemical cycloaddition reaction. The reaction progress of the photochemical cycloaddition reaction can be monitored in situ through the changes in the fluorescence intensity.

2. The method according to claim 1, characterized in that The photochemical cycloaddition reaction is a photoinduced excited cycloaddition reaction occurring between molecules or within molecules.

3. The method according to claim 2, characterized in that The intermolecular or intramolecular cycloaddition reaction is a [2+2], [4+2], [4+4] cycloaddition reaction, a Diels-Alder reaction, or a polymerization reaction of acetylene molecules.

4. The method according to claim 1, characterized in that: The fluorescence spectroscopy technique uses a fluorescence spectrometer or a laser confocal scanning microscope to perform spectral measurement.

5. The method according to claim 1, characterized in that The method for in situ monitoring of the progress of photochemical cycloaddition reaction using fluorescence spectroscopy is as follows: S1, uniformly dispersing the fluorescent material in an organic solvent to obtain a mixed solution containing the fluorescent material; dropping the mixed solution on a reaction carrier and drying; S21, subjecting the reaction carrier in S1 to a staged light irradiation reaction to obtain a cycloaddition product; wherein the reaction stage is divided into N stages, wherein N is an integer greater than 1; S22. Using a fluorescence spectrometer to monitor the changes in fluorescence intensity of the fluorescent material in different stages of the photochemical cycloaddition reaction, the steps are as follows: collecting the fluorescence emission spectrum of the fluorescent material in the reaction carrier at different time points after the first stage light irradiation reaction in S21; collecting the fluorescence emission spectrum of the fluorescent material in the reaction carrier at different time points after the second stage light irradiation reaction; collecting the fluorescence emission spectrum of the fluorescent material in the reaction carrier at different time points after the Nth stage light irradiation reaction; counting the fluorescence intensity values ​​at different time points, and according to the changes in fluorescence intensity, qualitatively or quantitatively monitoring the reaction progress of the photochemical cycloaddition is achieved.

6. The method according to claim 5, characterized in that At least one or more of the following conditions must be met; The reaction carrier is selected from a quartz plate or a glass plate; The organic solvent is selected from one or more of ethanol, methanol and acetonitrile; The thickness of the fluorescent material on the reaction carrier after drying is 0.2 to 0.5 mm.

7. The method according to claim 1, characterized in that The method for in situ monitoring of the progress of photochemical cycloaddition reaction using fluorescence spectroscopy is as follows: (1) Selecting crystals of fluorescent materials after being irradiated with ultraviolet and visible light at different time points for scanning observation; and setting a control group, which is a crystal of fluorescent material that has not been irradiated with light; wherein during the scanning observation process, the sample is excited by a semiconductor laser, and the fluorescence emission signal is collected at 250-800nm ​​in the blue channel of a laser confocal scanning microscope; (2) Determine the progress of the reaction based on changes in fluorescence intensity and collected confocal microscopy photos.

8. The method according to claim 7, characterized in that In step (1), the excitation wavelength of the semiconductor laser is 250-700 nm.

9. The method according to claim 7, characterized in that: In step (2), the scanning is a three-dimensional scanning, and the specific process is as follows: taking a crystal of fluorescent material to irradiate with an ultraviolet visible light source, and performing three-dimensional scanning at different time points.

10. Use of the method according to any one of claims 1 to 8 in monitoring the reaction kinetics in a photochemical cycloaddition reaction.

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