Fluorescent probes and their use in detection of idh2 mutant leukemia cells

Abstract

The application discloses a near-infrared hydrogen peroxide fluorescent probe and application thereof in distinguishing IDH2 mutant leukemia cells. The fluorescent probe IRS constructed in the application can be used for selectively and rapidly detecting hydrogen peroxide. The fluorescent probe can be used for distinguishing IDH2 mutant leukemia cells from wild-type leukemia cells.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a near-infrared mitochondrial-targeted fluorescent probe and its application in differentiating IDH2 mutant leukemia cells. Background Technology

[0002] In adult patients with acute myeloid leukemia (AML), approximately 8% to 19% have mutations in the isocitrate dehydrogenase 2 (IDH2) gene. Mutant IDH2 (mutIDH2) utilizes NADPH to reduce α-ketoglutarate to (R)-2-hydroxyglutarate, thereby disrupting the NADPH / NADP⁺ balance in mitochondria, hindering glutathione (GSH) synthesis and the clearance of reactive oxygen species (ROS). Elevated ROS levels induce an oxidative state, which may contribute to AML. Inhibition of mutIDH2 has induced myeloid differentiation in various preclinical models. Therefore, detecting IDH2 mutations is essential for AML subtyping, personalized treatment, and early relapse prediction.

[0003] Researchers have developed various methods for assessing IDH2 status. While Sanger sequencing is widely used to detect IDH mutations, its sensitivity is limited (10–15%). Several highly sensitive and accurate alternative methods have been used to detect mutIDH2, such as intraoperative mass spectrometry, droplet digital PCR, and next-generation sequencing. However, these methods are cumbersome, require expensive reagents, and are time-consuming. Therefore, there is an urgent need to develop economical, rapid, and simple methods to distinguish between IDH2 mutant and wild-type IDH2.

[0004] Hydrogen peroxide, as the most stable reactive oxygen species (ROS), is primarily produced in mitochondria. Visual monitoring of mitochondrial hydrogen peroxide levels could provide a direct and simple method for assessing IDH2 mutation status in AML. Fluorescence imaging analysis based on synthetic fluorescent probes has advanced biological research due to its advantages such as high selectivity, rapid response, ease of operation, non-invasive detection, and real-time imaging. Near-infrared (NIR) fluorescent probes with large Stokes shifts are particularly beneficial for biological research because they possess advantages such as strong penetration, low background fluorescence, and minimal photodamage. To date, fluorescent probes have not been used to distinguish between IDH2 mutant and wild-type leukemia cells.

[0005] Therefore, this application proposes a novel method for detecting IDH2 mutant leukemia cells by designing a large Stokes shift hydrogen peroxide-responsive fluorescent probe. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a new hydrogen peroxide fluorescent probe to achieve efficient and rapid differentiation between IDH2 mutant and wild-type leukemia cells.

[0007] The technical solution of the present invention is: the compound shown in formula (Ⅰ), which mainly uses benzoindole, thiophene and phenol to construct fluorescent groups and uses acetate as recognition groups.

[0008]

[0009] (I).

[0010] A kit containing the compounds described above.

[0011] The use of the above-described compounds in the preparation of reagents for detecting IDH2 mutant leukemia cells.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The fluorescent probe constructed in this invention can sensitively, specifically, and rapidly detect hydrogen peroxide in IDH2 mutant leukemia cells. This fluorescent probe can quickly distinguish between IDH2 mutant and wild-type leukemia cells. Attached Figure Description

[0014] Figure 1 IRS synthesis route;

[0015] Figure 2 A schematic diagram illustrating how the IRS distinguishes between IDH2 mutant and wild-type leukemia cells;

[0016] Figure 3 Spectroscopic performance analysis of the IRS response to hydrogen peroxide;

[0017] Figure 4 Selectivity analysis of hydrogen peroxide by IRS;

[0018] Figure 5 Cytotoxicity analysis for IRS;

[0019] Figure 6 Mitochondrial localization analysis for IRS;

[0020] Figure 7 IRS for intracellular hydrogen peroxide fluorescence imaging in IDH2 mutant leukemia cells;

[0021] Figure 8 Comparison of fluorescence imaging of IDH2 mutant and wild-type leukemia cells by IRS;

[0022] Figure 9 The proton NMR spectrum confirming the structure of the IRS ( 1 H NMR);

[0023] Figure 10 Carbon NMR spectrum confirming the structure of IRS (13 (C NMR).

[0024] Figure 11 High-resolution mass spectra (HRMS) confirming the structure of IRS. Detailed Implementation

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0026] Example 1 Synthesis of IRS

[0027] The synthesis is achieved through a two-step organic reaction; the synthetic route is as follows: Figure 1 As shown:

[0028] (1) Synthesis of IRS-OH

[0029] Compound 1 (0.68 g, 1.86 mmol) and compound 2 (0.42 g, 2.04 mmol) were dissolved in ultradry ethanol (15 mL), and then 0.25 mL of piperidine was slowly added to the mixture. The mixture was heated to reflux at 80 °C for 16 h, cooled to room temperature, and the solvent was removed by vacuum distillation. The mixture was then purified by silica gel column chromatography (CH2Cl2:CH3OH = 30:1, v / v) to give the purple solid product IRS-OH (0.32 g, 33%). 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 10.17 (s, 1H), 8.76 (d, J = 15.7 Hz,1H), 8.40 (d, J = 8.5 Hz, 1H), 8.26 (d, J = 8.9 Hz, 1H), 8.24 – 8.17 (m, 2H), 8.08 (d, J = 9.0 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.73 – 7.66 (m, 4H), 7.22 (d, J = 15.8 Hz, 1H), 6.91 (d, J = 8.6 Hz, 2H), 4.85 – 4.65 (m, 2H), 2.01 (s,6H), 1.49 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6): δ (ppm) 180.66,159.53, 155.03, 145.22, 140.17, 138.29, 138.09, 137.99, 133.06, 131.16,130.14, 128.51, 128.10, 127.07, 126.96, 125.01, 123.76, 123.11, 116.44,113.02, 108.37, 53.47, 25.81, 13.87. HRMS (ESI): m / z, 424.1726 [M] + , calcdfor C 28 H 26 NOS, 424.1729.

[0030] (2) Synthesis of IRS

[0031] Compound IRS-OH (0.03 g, 0.056 mmol) was dissolved in ultradry CH2Cl2 (3 mL) and placed in an ice bath. Then, acetyl chloride (0.079 mL, 1.12 mmol) and triethylamine (0.03 mL, 0.22 mmol) were added to the mixed solution under continuous N2 purging. The mixture was stirred overnight at room temperature, and the solution changed from purple to purplish-red. After the raw material was almost completely consumed, the solvent was removed by vacuum distillation, and the product was purified by silica gel column chromatography (CH2Cl2 : CH3OH = 30:1, v / v) to give the red solid product IRS (0.019 g, 57%). 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 8.79 (dd, J = 15.9, 3.0 Hz, 1H), 8.42 (d, J = 8.5 Hz, 1H), 8.32 – 8.18 (m, 3H), 8.10 (dd, J = 8.9, 2.7Hz, 1H), 7.94 – 7.63 (m, 5H), 7.38 – 7.26 (m, 3H), 4.84 – 4.72 (m, 2H), 2.31 (s, 3H), 2.02 (s, 6H), 1.51 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz, DMSO-d6): δ(ppm) 181.04, 169.21, 152.28, 151.58, 144.97, 139.63, 139.13, 138.41, 138.24,133.21, 131.24, HRMS (ESI): m / z,466.1831 [M] + calcd for C 30 H 28 NO2S, 466.1835.

[0032] Example 2: Spectroscopic performance analysis of IRS response to hydrogen peroxide

[0033] IRS and hydrogen peroxide were tested in PBS (0.1 M, pH = 7.4) buffer containing 5% DMSO at 37 °C. Figure 3 ).

[0034] Figure 3 a shows the UV absorption spectra of IRS (5 µM) before and after 2 h of reaction with hydrogen peroxide (300 µM). The maximum UV absorption wavelength of IRS is 484 nm. After IRS reacts fully with H2O2, its maximum absorption wavelength redshifts to 516 nm.

[0035] Figure 3 b represents the fluorescence intensity change of IRS (5 µM) reacting with different concentrations of H2O2 for 30 min. The fluorescence of the IRS solution is very weak under 516 nm excitation. After 30 min of reaction between IRS and H2O2, the probe solution emits red fluorescence with a maximum emission wavelength of 656 nm. The fluorescence intensity increases with the concentration of H2O2 (100 μM). −1 The increase in mM) continuously strengthens the effect;

[0036] Figure 3 c represents the kinetic study of the reaction between IRS (5 µM) and H2O2 (500 µM) for 100 min. Real-time monitoring of the reaction between IRS (5 μM) and H2O2 (500 μM) for 90 min revealed that the fluorescence intensity of IRS at 656 nm continuously increased over time, reaching a maximum equilibrium at 60 min, while the IRS solution itself did not show significant fluorescence changes.

[0037] Figure 3 d represents the linear correlation analysis of the fluorescence intensity of IRS at 656 nm with the concentration of hydrogen peroxide. IRS (5 μM) and H2O2 showed a linear relationship in the concentration range of 5–80 μM, with a detection limit of 3.57 μM (3 σ / s, n = 11).

[0038] Example 3: Selectivity analysis of hydrogen peroxide by IRS

[0039] The selectivity of IRS was analyzed by comparing its response to H2O2 and other related intracellular bioactive molecules. The IRS's response to other bioactive molecules besides H2O2 (O2...) was also examined. ·− , 1 O2、HO·、·NO、ONOO − 、·O t Bu and TBHP showed no obvious fluorescence response ( Figure 4 This indicates that IRS can specifically detect H2O2.

[0040] Example 4 Cytotoxicity of IRS

[0041] The effects of IRS on TF-1 and IDH2 were evaluated using the CCK-8 kit. R140Q The toxic effects of TF-1 on two cell lines. Cell viability of the two cell lines after incubation with different concentrations of IRS for 12 h. Although cell viability gradually decreased with increasing IRS concentration, cell viability remained above 80% at an IRS concentration of 20 μM. Figure 5 This indicates that the probe IRS has good biocompatibility.

[0042] Example 5: Fluorescence Imaging Detection of Hydrogen Peroxide in Cellular Mitochondria by IRS

[0043] IDH2 was incubated with 5 μM IRS R140Q TF-1 cells were incubated for 30 min, then 0.2 μM MitoTracker Green reagent was added and the cells were incubated for another 10 min. The supernatant was removed by centrifugation, the cells were resuspended in PBS, and transferred to a confocal dish for fluorescence imaging. Figure 6 As shown, the red and green fluorescence showed good overlap, with a Pearson correlation coefficient of 0.91, indicating that the IRS has good mitochondrial targeting function. (6a) Bright field; (6b) IRS; (6c) Mitochondrial targeting reagent; (6d) Overlap signal diagram; (6e) Fluorescence intensity diagram of the overlap signal between IRS and mitochondrial targeting reagent.

[0044] Example 6 IRS for IDH2 R140QFluorescence imaging detection of hydrogen peroxide in TF-1 cells

[0045] With IDH2 R140Q Using TF-1 cells as a model, four groups of experiments were conducted, and the fluorescence images are shown below. Figure 7 As shown in figure a, the fluorescence intensity is as follows: Figure 7 As shown in b, the first group is a blank control without any treatment; no fluorescence was observed, indicating that IDH2... R140Q TF-1 cells did not produce autofluorescence upon excitation at 561 nm. The second group of experiments involved incubating IDH2 cells with IRS. R140Q After 30 min, TF-1 cells showed obvious intracellular red fluorescence. To verify that this fluorescence was initiated by H2O2, we conducted the third and fourth sets of experiments, first pretreating IDH2 cells with TEMPO, an H2O2 scavenger with mitochondrial targeting function. R140Q TF-1 cells were incubated for 30 min, then incubated with IRS for another 30 min. The intracellular red fluorescence was weakened. Upon further addition of 100 μM H2O2, the intracellular red fluorescence significantly increased again. This study indicates that IRS can inhibit IDH2... R140Q Fluorescence detection and imaging of H2O2 in TF-1 cells.

[0046] Example 7 IRS for TF-1 and IDH2 R140Q Fluorescence imaging analysis of TF-1 leukemia cells

[0047] Using IRS to analyze TF-1 and IDH2 respectively R140Q TF-1 cells were used for fluorescence imaging. IDH2 cells were treated with IRS (5 μM) separately. R140Q Fluorescence imaging images of TF-1 and TF-1 cells at 30 min, IDH2 R140Q TF-1 cells showed obvious red fluorescence ( Figure 8 a) Statistical analysis of fluorescence intensity for the two cell types, IDH2 R140Q The fluorescence intensity of TF-1 cells was 12.1 times that of TF-1 cells. Figure 8 b), indicating that IDH2 R140Q The H2O2 level in the mitochondria of TF-1 cells was significantly higher than that in wild-type leukemia cells. Therefore, the probe IRS constructed in this study can be used to distinguish between IDH2 mutant leukemia cells and wild-type leukemia cells.

Claims

1. The compound represented by formula (Ⅰ): （Ⅰ）。 2. A kit containing the compound of claim 1.

3. Use of the compound of claim 1 in the preparation of hydrogen peroxide detection reagent.

4. Use of the compound of claim 1 in the preparation of a reagent for detecting IDH2 mutant leukemia cells.

Images