Furanyl near-infrared fluorescent probes for nad(p)h detection and preparation method and application thereof

Abstract

The application discloses a furan group near-infrared fluorescent probe for detecting total reducing power of NADH and NADPH, and a preparation method and application thereof, and belongs to the field of organic small-molecule fluorescent probes. The probe takes hemicyanine as a signal unit and quinolinium salt as a recognition site. The probe can produce a rapid fluorescent response to NADH and NADPH under room temperature and physiological pH conditions, cannot distinguish NADH and NADPH, can realize rapid response, semi-quantitative detection and dynamic trend monitoring of total reducing power in a system, has a response time of less than or equal to 2 min, a detection limit of nmol / L level, high sensitivity and high selectivity, and can effectively eliminate biological matrix interference; the probe has both mitochondrial targeting characteristics and fluorescence imaging potential, can be used for real-time monitoring of total reducing power of a catalytic fermentation system of P450 monooxygenase, steroid hydroxylase, dehydrogenase, reductase and peroxidase, and the detection result has good consistency with that of an HPLC method. The probe is suitable for dynamic monitoring of reducing power in a biological fermentation process, rapid screening in vitro and cell metabolism related research, solves technical defects of low sensitivity, harsh conditions and difficulty in real-time in-situ monitoring of a traditional NAD(P)H detection method, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically involving a novel type of furan-based near-infrared fluorescent probe, its preparation method, and its application. Background Technology

[0002] Reduced nicotinamide adenine dinucleotide (NADH) and its phosphate ester (NADPH) are important coenzymes for numerous redox reactions in eukaryotic cells. As a core coenzyme for cellular energy metabolism and redox states, the detection of its total reducing power level is crucial for life science research and disease diagnosis. Currently, reported methods for NAD(P)H detection include enzymatic methods, high-performance liquid chromatography (HPLC), electrochemical analysis, and methods based on its own fluorescence (emission at approximately 465 nm). However, these traditional methods generally suffer from limitations such as insufficient sensitivity, poor specificity, and difficulty in applying them to live cell systems. In particular, methods based on NAD(P)H's own fluorescence cannot achieve high spatiotemporal resolution and dynamic real-time monitoring of NAD(P)H in live cells due to their low fluorescence quantum yield and susceptibility to interference from the testing environment.

[0003] To address the aforementioned issues, small molecule fluorescent probe technology has attracted significant attention due to its advantages such as ease of operation, rapid response, and compatibility with live-cell imaging. However, most NAD(P)H fluorescent probes currently disclosed in this field still suffer from the following key technical defects: (1) insufficient response rate, making it difficult to capture the rapid dynamic changes of NAD(P)H during cellular metabolism; (2) demanding detection conditions, often requiring specific temperatures (e.g., 37°C), pH values, or long incubation times, which limits their real-time applications. Therefore, developing a fluorescent probe that can respond rapidly at room temperature, is suitable for real-time imaging of fermentation broth and live cells, and is used for monitoring the total reducing power of NADH and NADPH has become a pressing technical challenge in this field.

[0004] Hemicyanine dyes are a class of excellent fluorophores with large Stokes shifts, near-infrared / deep red emission, high fluorescence quantum yield, and excellent photostability. More importantly, their derived probes often possess unique mitochondrial targeting capabilities, and mitochondria are the key site of NAD(P)H metabolism in cells. Although hemicyanine dyes have wide applications in biosensing, there is still a lack of successful precedents and effective design strategies for rationally designing and constructing fluorescent probes based on them as signaling units that can rapidly respond to the total reducing power of NADH and NADPH and operate under mild conditions. Summary of the Invention

[0005] This invention aims to provide a near-infrared fluorescent probe for detecting the total reducing power of NADH and NADPH and its preparation method, so as to achieve highly selective, real-time and rapid detection of the total reducing power of NADH and NADPH in biological fermentation broth at room temperature.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first objective of this invention is to provide a furanyl NAD(P)H near-infrared fluorescent probe, the molecular structure of which is as follows:

[0008]

[0009] In general formula I, R is Me or Et or

[0010] The second objective of this invention is to provide a method for preparing the above-mentioned rapid detection fluorescent probe for the total reducing power of NADH and NADPH, comprising the following steps:

[0011] (1) Under argon protection, quinoline-3-boronic acid and 5-bromofuran-2-carboxaldehyde were dissolved in a solvent and stirred until homogeneous. Then, a small amount of tetra(triphenylphosphine)palladium and a small amount of cesium carbonate were added, and the mixture was heated under reflux for 6 hours. After cooling to room temperature, the solvent was removed by vacuum distillation. The residue was treated with cold water, then extracted with ethyl acetate. The organic phase was collected and dried over anhydrous magnesium sulfate. The mixture was filtered, and the organic solvent was removed under vacuum. The residue was separated by column chromatography to obtain the product 5-(quinoline-3-yl)furan-2-carboxaldehyde.

[0012] (2) Under argon protection, 1,3,3-trimethyl-3H-indole was dissolved in an organic solvent with iodomethane or iodoethane or 6-bromohexanoic acid or 1,3-propylsulfonate lactone or 1,4-butylsulfonate sodium lactone or 3-bromopropyltrimethylammonium bromide. A small amount of piperidine was added, and after the addition was complete, the solution was heated to reflux. The reaction was monitored by thin-layer chromatography. After the reaction was completed, the temperature was lowered to room temperature, and the solvent was removed under reduced pressure. The crude product was washed with ice-cold diethyl ether to obtain the product 1,2,3,3-tetramethyl-3H-indole-1- Onium or 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium or 1-(4-carboxybutyl)-2,3,3-trimethyl-3H-indole-1-onium or 2,3,3-trimethyl-1-(3-(trimethylammonium)propyl)-3H-indole-1-onium or 3-(2,3,3-trimethyl-3H-indole-1-onium-1-yl)propane-1-sulfonate or 4-(2,3,3-trimethyl-3H-indole-1-onium-1-yl)butane-1-sulfonate.

[0013] (3) The above compounds 1,2,3,3-tetramethyl-3H-indol-1-onium, 1-ethyl-2,3,3-trimethyl-3H-indol-1-onium, 1-(4-carboxybutyl)-2,3,3-trimethyl-3H-indol-1-onium, 2,3,3-trimethyl-1-(3-(trimethylammonium)propyl)-3H-indol-1-onium, and 3-(2,3,3-trimethyl-3H-indol-1-onium-1-yl)propane-1-sulfonate, 4-(2,3,3-trimethyl-3H-indol-1-onthiol-1-yl)butane-1-sulfonate was dissolved in anhydrous ethanol with 5-(quinolin-3-yl)furan-2-carboxaldehyde. Piperidine was added, and the mixture was refluxed at 65-95°C under argon protection. After the reaction was completed, the mixture was cooled to room temperature, the solvent was removed by vacuum distillation, and the product (E)-1,3,3-trimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol- 1-Ondole or (E)-1-ethyl-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indole-1-ondole or (E)-1-(4-carboxybutyl)-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indole-1-ondole or (E)-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl) (E)-1-(3-(trimethylammonium)propyl)-3H-indol-1-onium or (E)-3-(3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium-1-yl)propane-1-sulfonate or (E)-4-(3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium-1-yl)butane-1-sulfonate.

[0014] (4) Under argon protection, the above compounds (E)-1,3,3-trimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium, (E)-1-ethyl-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium, (E)-1-(4-carboxybutyl)-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium, (E)-1-1-(4-carboxybutyl)-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium, (E)-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium, (E)-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium, and (E)-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium, (E)-3-(3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onyl)propane-1-sulfonate, (E)-4-(3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onyl)butane-1-sulfonate were dissolved in organic solvents respectively, and methyl trifluoromethanesulfonate was added to each system until the reaction was complete. Subsequently, the corresponding products (E)-1,3,3-trimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium, (E)-3-(5-(2-(1-ethyl-3,3-dimethyl-3H-indol-1-onium-2-yl)vinyl)furan-2-yl)-1-methylquinolin-1-onium, (E)-3-(5-(2-(1-(4-carboxybutyl)-3,3-dimethyl-3H-indol-1-onium-2-yl)vinyl)furan-2-yl)-1-methylquinolin-1-onium, (E)-3-(5-(2-(1-(4-carboxybutyl)-3,3-dimethyl-3H-indol-1-onium-2-yl)vinyl)furan-2-yl)-1-methylquinolin-1-onium, (E)-3-(5-(2-(3-) ,3-Dimethyl-1-(3-(methylammonium)propyl)-3H-indol-1-onth-2-yl)vinyl)furan-2-yl)-1-methylquinoline-1-onthium,(E)-3-(3,3-dimethyl-2-(2-(5-(1-methylquinoline-1-onth-3-yl)furan-2-yl)vinyl)-3H-indol-1-onth-1-yl)propane-1-sulfonate,(E)-4-(3,3-dimethyl-2-(2-(5-(1-methylquinoline-1-onth-3-yl)furan-2-yl)vinyl)-3H-indol-1-onth-1-yl)butane-1-sulfonate.

[0015] The synthetic route of the furanyl near-infrared fluorescent probe of this invention is as follows:

[0016]

[0017] In a further embodiment, the solvent used in step (1) is water and toluene, with a volume ratio of 1:1-9.

[0018] In a further embodiment, the molar ratio of tetra(triphenylphosphine)palladium and cesium carbonate used in step (1) is 1:1.1-1.5.

[0019] In a further embodiment, the molar ratio of tetra(triphenylphosphine)palladium and quinoline-3-boronic acid used in step (1) is 1:10-15.

[0020] In a further embodiment, the molar ratio of 5-bromofuran-2-carboxaldehyde and quinoline-3-boronic acid in step (1) is 1:1.1-1.5.

[0021] In a further embodiment, the molar ratio of 1,3,3-trimethyl-3H-indole to 6-bromohexanoic acid or 1,3-propylsulfonate lactone or 1,4-butylsulfonate sodium lactone or 3-bromopropyltrimethylammonium bromide in step (2) is 1:1.3-1.7.

[0022] In a further embodiment, the solvent used in step (3) is anhydrous ethanol.

[0023] In a further embodiment, the solvent used in step (4) is anhydrous dichloromethane or anhydrous acetonitrile.

[0024] In a further embodiment, the (E)-1,3,3-trimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indole-1-onium or (E)-1-ethyl-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indole-1-onium or (E)-1-(4-carboxybutyl)-3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indole-1-onium or (E)-3,3-dimethyl-2-( The molar ratio of 2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-1-(3-(trimethylammonium)propyl)-3H-indol-1-onium or (E)-3-(3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium-1-yl)propane-1-sulfonate or (E)-4-(3,3-dimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onium-1-yl)butane-1-sulfonate to methyl trifluoromethanesulfonate is 1:1-5.

[0025] In a further embodiment, the methylation reaction time in step (4) is 6-16 hours, and the temperature is 30-70℃.

[0026] A third objective of this invention is to provide a method for applying the fluorescent probe, comprising the following steps:

[0027] 1) Spectroscopic determination of the interaction between the probe and NAD(P)H

[0028] The above fluorescent probe stock solution (1.0 × 10⁻⁶) was prepared in N,N-dimethyl sulfoxide (DMSO). -3 (mol / L) to be used;

[0029] 40 μL of probe stock solution was added to a 4 mL test system. Different volumes of NAD(P)H solution were added to the bulk solution to keep the probe concentration in the sample constant at 1.0 μmol / L and increase the NAD(P)H concentration gradient. The UV-Vis absorption and fluorescence spectra of the solution were measured at room temperature.

[0030] 2) Probe biological applications

[0031] HepG2 cells were seeded in 96-well culture dishes and then cultured in an incubator at 37°C. After cell adhesion, a probe was added to some of the HepG2 cells, and after co-culturing for 20 min, some cells were washed with PBS buffer and fluorescence imaging analysis was performed. A portion of the HepG2 cells incubated with the probe were then co-cultured with NADH, and after 20 min, the cells were washed with PBS buffer and fluorescence imaging observation was performed. (The last sentence is a repetition of the previous one and can be omitted.)

[0032] 3) Application of probes in fermentation systems

[0033] Take an appropriate amount of fermentation broth, centrifuge, and set aside. Take 40 μL of probe stock solution into a 4 mL test system, add an appropriate amount of filtered fermentation broth, DMSO, and deionized water, fix the probe concentration at 1.0 μmol / L, measure the fluorescence intensity of the system, and calculate the total reducing power levels of NADH and NADPH based on the changes in fluorescence intensity.

[0034] The fluorescence spectrum testing conditions for the system after the near-infrared fluorescent probe reacts with NAD(P)H are: excitation wavelength 589 nm and emission wavelength 775 nm.

[0035] Compared with existing NADH detection technologies, the present invention has the following significant advantages:

[0036] (1) Excellent detection performance: The furanyl NAD(P)H near-infrared fluorescent probe provided by this invention can work stably at room temperature and physiological pH conditions without harsh reaction conditions. It responds to both NADH and NADPH, but cannot distinguish between the two. It can realize rapid monitoring of total reducing power, effectively eliminate interference from biological matrix such as amino acids, has high sensitivity (detection limit can reach nmol / L level) and rapid response characteristics (response time ≤2min), and can accurately capture the dynamic changes of NADH and NADPH during cell metabolism and fermentation.

[0037] (2) It can be used for rapid detection of the total reducing power of NADH and NADPH in fermentation broth catalyzed by P450 monooxygenase, steroid hydroxylase, dehydrogenase, reductase, peroxidase and other enzymes. It can also achieve in situ fluorescence imaging of the total reduced state of NADH and NADPH in living cells. It has mitochondrial targeting capability, providing a tool for studying the coenzyme metabolism mechanism in mitochondria. Its application scope covers multiple fields such as bio-fermentation quality control, life science research and disease diagnosis assistance. Attached Figure Description

[0038] Figure 1 This is an example of the ultraviolet-visible absorption spectrum of the interaction between the NADH near-infrared fluorescent probe HC-1 and NADH in one embodiment of the present invention.

[0039] Figure 2 The fluorescence spectrum of the interaction between the NADH near-infrared fluorescent probe HC-2 and NADH is shown in one embodiment of the present invention.

[0040] Figure 3 This is a schematic diagram of the experimental results of the near-infrared fluorescent probe HC-3 on the selectivity of NADH in one embodiment of the present invention.

[0041] Figure 4 This is a schematic diagram showing the change in fluorescence intensity over time when the NADH near-infrared fluorescent probe HC-4 interacts with NADH in one embodiment of the present invention. Detailed Implementation

[0042] The present invention elaborates on its objectives, technical solutions, and advantages through the following specific embodiments. The embodiments described below are for illustrative purposes only and should not be construed as limiting the scope of the invention. It should be noted that all modifications and improvements to NAD(P)H based on the principles of this invention fall within the protection scope of this invention.

[0043] Example 1: Preparation of a high-efficiency NAD(P)H near-infrared fluorescent probe

[0044]

[0045] Under a nitrogen atmosphere, 2.08 g of quinoline-3-boric acid and 1.75 g of 5-bromofuran-2-carboxaldehyde were weighed and added to a 250 mL round-bottom flask, dissolved in an organic solvent of toluene / water = 9 / 1. Then, 0.60 g of tetrakis(triphenylphosphine)palladium and 2-3 g of cesium carbonate were added. After the addition was complete, the resulting solution was heated under reflux at 90 °C for 12 h. The reaction mixture was cooled, and the solvent was removed under reduced pressure. The residue was treated with cold water and extracted with ethyl acetate (3 extractions, 20 mL each). The bound organic layer was dried over anhydrous magnesium sulfate and evaporated under reduced pressure to give a pale yellow solid. The product 5-(quinoline-3-yl)furan-2-carboxaldehyde was purified by column chromatography in 62% yield.

[0046] Under argon protection, 0.335 g of 5-(quinoline-3-yl)furan-2-carboxaldehyde and 0.54 g of 1,3,3-trimethyl-3H-indole were dissolved in 30 mL of organic solvent, and 0.5 mL of piperidine was added. After the addition was complete, the solution was reacted at 90 °C for 2 h, and the reaction was monitored by thin-layer chromatography. Then, the solvent was removed under reduced pressure, the residue was treated with 20 mL of cold water, and then extracted with dichloromethane (10 mL × 3 times). The combined organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure to give a crude product (E)-1,3,3-trimethyl-2-(2-(5-(quinoline-3-yl)furan-2-yl)vinyl)-3H-indole-1-onium, which was orange-brown in color, in a yield of 35%.

[0047] 0.38 g of compound (E)-1,3,3-trimethyl-2-(2-(5-(quinolin-3-yl)furan-2-yl)vinyl)-3H-indol-1-onthium was dissolved in 10 mL of organic solvent, and 1 mL of methyl trifluoromethanesulfonate was added. The reaction mixture was stirred at room temperature for 6 h under a nitrogen atmosphere, and monitored by thin-layer chromatography. The solvent was then removed under reduced pressure, the residue was treated with 20 mL of cold water, and then extracted with dichloromethane (10 mL × 3 times). The combined organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure to give a brown crude product (E)-1-methyl-3-(5-(2-(1,3,3-trimethyl-3H-indol-1-onthium-2-yl)vinyl)furan-2-yl)quinolin-1-onthium, in 48% yield. ESI mass: [M-HCO2] - ] + Calculated value: 439.2016; Measured value: 439.2022. ¹H NMR (400MHz, DMSO-d6) δ (ppm): 10.20 (s, ¹H), 9.79 (s, ¹H), 8.55 (dd, 2H), 8.42 (d, ¹H), 8.33 (t, ¹H), 8.14 (t, ¹H), 7.93 (ddd, 2H), 7.81–7.72 (m, 2H), 7.72–7.54 (m, 3H), 4.74 (s, 3H), 4.21 (s, 3H), 1.81 (s, 6H); ¹³C NMR (101MHz, DMSO-d6) δ181.19, 153.36, 153.13, 148.36, 144.06, 142.43, 140.44, 138.03, 136.97, 136.37, 131.3 1, 131.22, 129.97, 129.59, 125.90, 123.42, 119.92, 119.53, 115.72, 114.91, 111.23, 52.49, 46.28, 34.96, 25.74.

[0048] Example 2: NADH response spectroscopy experiment of near-infrared fluorescent probe HC-1

[0049] 1. Prepare DMSO stock solution for HC-1 probe: Accurately weigh 0.0067g of the probe and dissolve it in 10mL of DMSO. Mix well to obtain a 1mM stock solution and store it in a refrigerator at 4℃ for later use.

[0050] 2. Prepare NADH aqueous solution: Accurately weigh 0.0033g of NADH and dissolve it in 5mL of deionized water. Mix well to obtain a 1mM NADH stock solution, which should be prepared immediately before use.

[0051] 3. Sample Preparation: In the 4mL sample cells numbered 1-15, add 40μL of probe HC-3 stock solution, different volumes of NADH stock solution (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, 2.2mL), 1560μL of DMSO, and 200μL of PBS (0.2M, pH=7.4) buffer. Finally, bring the volume to 4.00mL with deionized water. Mix well and let stand for 2 minutes before testing.

[0052] 4. Test conditions: The excitation wavelength for fluorescence spectroscopy was 689 nm, the width of both the excitation and emission slits was 2 nm, the emission wavelength scanning range was 690-850 nm, the fluorescence intensity was read at 775 nm, and the test temperature was 25℃.

[0053] Example 3: Near-infrared fluorescent probe HC-3 used for the detection of total reducing power in fermentation broth of steroid hydroxylase / monooxygenase.

[0054] 1. Preparation of HC-3 probe DMSO stock solution: Accurately weigh 0.0069g of the probe and dissolve it in 10mL of DMSO. Mix well to obtain a 1mM stock solution and store it in a refrigerator at 4℃ for later use.

[0055] 2. Take 5 mL of fermentation broth from the mycobacterial fermentation system that converts cholesterol to ursene dione, centrifuge, collect 0.1 mL of the supernatant, dilute to 10 mL, and set aside.

[0056] 3. Take 40 μL of the HC-3 probe stock solution into a cuvette, add 1.00 mL of diluted fermentation broth, 1560 μL LDMSO, 200 μL PBS (pH = 7.4) buffer, and 2100 μL double-distilled water. Measure the fluorescence intensity using fluorescence spectroscopy and compare it with the linear relationship between fluorescence intensity and NADH concentration obtained by NADH titration. The NADH concentration in the test system was determined to be 3 μM, and the NADH concentration in the fermentation system was determined to be 1.2 mM. This result is consistent with the HPLC method for detecting NADH in this system, indicating that the established fluorescence analysis method can achieve rapid and semi-quantitative detection of the total reducing power of NADH and NADPH in steroidal transformation fermentation systems.

[0057] Example 4: Detection of total reducing power of near-infrared fluorescent probe HC-1 in dehydrogenase fermentation broth

[0058] 1. Prepare DMSO stock solution for HC-1 probe: Accurately weigh 0.0067g of the probe and dissolve it in 10mL of DMSO. Mix well to obtain a 1mM stock solution and store it in a refrigerator at 4℃ for later use.

[0059] 2. In the Rhodococcus fermentation system that converts androstenedione to 9α-hydroxyandrostenedione, take 5 mL of fermentation broth, centrifuge, take 0.1 mL of the supernatant, dilute to 10 mL, and set aside.

[0060] 3. Take 40 μL of probe HC-1 stock solution into a cuvette, add 1.00 mL of diluted fermentation broth, 1560 μL LDMSO, 200 μL PBS (pH = 7.4) buffer, and 2100 μL double-distilled water. Measure the fluorescence intensity using fluorescence spectroscopy and compare it with the linear relationship between fluorescence intensity and NADH concentration obtained by NADH titration. The NADH concentration in the test system was determined to be 2.5 μM, and the NADH concentration in the fermentation system was determined to be 1.0 mM. This result is consistent with the HPLC method for detecting NADH in this system, indicating that the established fluorescence analysis method can achieve rapid and semi-quantitative detection of the total reducing power of NADH and NADPH in steroidal transformation fermentation systems.

[0061] Example 5: Detection of total reducing power of near-infrared fluorescent probe HC-4 in P450 monooxygenase / steroid hydroxylase fermentation broth.

[0062] 1. Prepare HC-4 probe DMSO stock solution: Accurately weigh 0.0063g of the probe and dissolve it in 10mL of DMSO. Mix well to obtain a 1mM stock solution and store it in a refrigerator at 4℃ for later use.

[0063] 2. In the Saccharomyces cerevisiae fermentation system that converts progesterone to 11α-hydroxyprogesterone, take 5 mL of fermentation broth, centrifuge, take 0.1 mL of the supernatant, dilute to 10 mL, and set aside.

[0064] 3. Take 40 μL of the HC-4 probe stock solution into a cuvette, add 1.00 mL of diluted fermentation broth, 1560 μL LDMSO, 200 μL PBS (pH = 7.4) buffer, and 2100 μL double-distilled water. Measure the fluorescence intensity using fluorescence spectroscopy and compare it with the linear relationship between fluorescence intensity and NADPH concentration obtained by NADPH titration. The concentration of NADH in the test system was determined to be 2.6 μM, and the concentration of NADPH in the fermentation system was determined to be 1.04 mM. This result is consistent with the HPLC method for detecting NADPH in this system, indicating that the established fluorescence analysis method can achieve rapid and semi-quantitative detection of the total reducing power of NADH and NADPH in steroidal transformation fermentation systems.

Claims

1. A type of fluorescent probe for detecting NAD(P)H, characterized in that, The general formula for this fluorescent probe is as follows: In general formula I, R is Me or Et or 2. The method for preparing a near-infrared fluorescent probe for NAD(P)H detection as described in claim 1, characterized in that... Includes the following steps: Step (1): Under argon protection, 2,3,3-trimethylindole and iodomethane or iodoethane or 6-bromohexanoic acid or 1,3-propylsulfonate lactone or 1,4-butylsulfonate sodium lactone or 3-bromopropyltrimethylammonium bromide are dissolved in an organic solvent to react and obtain the corresponding indoleonium salt. Step (2): Under argon protection, quinoline-3-boronic acid and 5-bromofuran-2-carboxaldehyde are dissolved in a solvent. After the Suzuki reaction is carried out under alkaline conditions with the addition of a catalyst, the product 5-(quinoline-3-yl)furan-2-carboxaldehyde is reacted with the indoleon salt described in step (1) to obtain a vinyl conjugated intermediate. Step (3): Under argon protection, the intermediate obtained in step (2) is dissolved in anhydrous dichloromethane or acetonitrile, and then methyl trifluoromethanesulfonate is added to carry out a methylation reaction to obtain the compound shown in general formula I.

3. The method according to claim 2, characterized in that: In step (1), the molar ratio of 2,3,3-trimethylindole to iodomethane or iodoethane or 6-bromohexanoic acid or 1,3-propylsulfonate lactone or 1,4-butylsulfonate sodium lactone or 3-bromopropyltrimethylammonium bromide is 1:1.2-1.6, and the solvent used in the reaction is acetonitrile or toluene or dichloromethane.

4. The method according to claim 2, characterized in that: In step (2), the molar ratio of 5-bromofuran-2-carboxaldehyde and quinoline-3-boronic acid is 1:1.1-1.5, the solvent used is a water / toluene mixture with a volume ratio of 1:1-9, and the molar ratio of tetra(triphenylphosphine)palladium to cesium carbonate is 1:1.1-1.

5.

5. The method according to claim 2, characterized in that: In step (2), the molar ratio of 5-(quinolin-3-yl)furan-2-carboxaldehyde to indoline salt is 1:1-1.

7.

6. The method according to claim 2, characterized in that: In step (3), the molar ratio of the vinyl conjugated intermediate to methyl trifluoromethanesulfonate is 1:1-5; the methylation reaction temperature is 20-60℃, and the reaction time is 3-16h.

7. The application of the furanyl near-infrared fluorescent probe of claim 1 in the detection of the total reducing power of NADH and NADPH; the probe can respond to NADH and NADPH simultaneously, but cannot distinguish between the two.

8. The application according to claim 7, characterized in that, The detection was performed in a PBS buffer system at room temperature and pH 7.4, with an excitation wavelength of 589 nm and an emission wavelength of 775 nm.

9. The application according to claim 7, characterized in that, The applications include rapid response, semi-quantitative detection, or dynamic trend monitoring of the total reducing power of endogenous NADH and NADPH in fermentation broths of P450 monooxygenase systems, steroid hydroxylase systems, dehydrogenase systems, reductase systems, and peroxidase systems.

10. The application according to claim 7, characterized in that, The application also includes fluorescence imaging observation and in-situ rapid analysis of the total reduced state of mitochondrial NADH / NADPH in HepG2 live cells.

Images