Targeting SHP2 fluorescent probe, preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a fluorescent probe targeting SHP2, a preparation method and application of the fluorescent probe. A core pharmacophore is determined by replacing an amino group in a central region B of SHP2-099 into a p-methyl group and introducing a sulfur atom as a connecting atom, an SBD fluorophore is integrated in the core pharmacophore, and a series of SHP-PS (1-4) targeting to SHP2 and having connecting arms with different lengths is developed. The series of SHP2-targeting fluorescent probes provided by the invention have high specificity and good target affinity, can selectively mark SHP2 protein in living cells, have an environmentally sensitive switch mechanism, provide a new tool for researching the effect of SHP2 in signal transduction and the significance of SHP2 in disease progression, and have broad application prospects. The research on diseases such as cancer is expected to be accelerated, and a new way is provided for diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a fluorescent probe targeting SHP2, a preparation method and an application thereof. Background Art

[0002] SHP2 is a non-receptor protein tyrosine phosphatase belonging to the PTP family. It plays a vital role in cell signal transduction. It can participate in cell survival, proliferation and migration, and regulate a series of cell signals, including metabolism, growth, differentiation, transcription and cancer transformation, etc. In addition, SHP2 plays a central role in the oncogenic signaling pathways that include but are not limited to the interaction of JAK / STAT, PI3K / AKT, RAS / Raf / MAPK and PD-1 / PD-L1 immune checkpoints. It is essential for mediating the biological responses of growth factors, hormones, cytokines, cell adhesion molecules, and the signal transduction pathways that control the developmental process and hematopoietic function.

[0003] Dysregulation of SHP2, manifested by overexpression or overactivation, has been associated with a range of diseases and is frequently detected in various solid tumors, making it a promising target for cancer therapy.

[0004] Existing SHP2 detection methods, such as western blot (WB), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and substrate-based assays, have greatly enhanced our understanding of SHP2 biology. However, these methods are often limited by low throughput, low sensitivity, and the inability to monitor in real time in living cells.

[0005] In recent years, fluorescence imaging has become increasingly important in the field of target visualization, and small molecule fluorescent probes have emerged as a powerful analytical tool and are now widely used in the labeling, observation, and detection of proteins, ions, nucleic acids, and the physical microenvironment of cells.

[0006] However, to date, no small molecular fluorescent probe specific for SHP2 has been reported. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a fluorescent probe targeting SHP2, a preparation method and application thereof.

[0008] The novel small molecule fluorescent probe provided by the present invention can track SHP2 in real time using imaging technology with high sensitivity and spatiotemporal resolution, has an environmentally sensitive switching mechanism, high specificity, good binding affinity, and can selectively label SHP2 in living cells.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention is to provide a fluorescent probe targeting SHP2, wherein the fluorescent probe has the following structure:

[0011]

[0012] Among them, n=1-10.

[0013] The second aspect of the present invention is to provide a method for preparing the fluorescent probe targeting SHP2, which specifically comprises the following steps:

[0014] (1) Reaction between pharmacophore and fluorophore

[0015] The pharmacophore, fluorophore and N,N-diisopropylacetic acid are dissolved in acetonitrile and reacted at room temperature to obtain a fluorescent probe, wherein the molar ratio of pharmacophore:fluorophore:N,N-diisopropylacetic acid is 1:1-2:1-1.5, and the reaction equation is shown below;

[0016]

[0017] (2) using trifluoroacetic acid to remove the protecting group of the fluorescent probe prepared in (1) to obtain a fluorescent probe targeting SHP2, as shown in the following formula;

[0018]

[0019] In the above formula, R1 is independently selected from H, Cl, Br or I;

[0020] R2 is independently selected from H or tert-butyloxycarbonyl; n=1-10.

[0021] Preferably, n=2, 5, 7, 9.

[0022] In the above method for preparing the fluorescent probe targeting SHP2, the method for preparing the pharmacophore is:

[0023] (I) using 2-chloro-3-fluoroaniline and tert-butyl mercaptan as raw materials, reacting under alkaline conditions to obtain compound 2;

[0024] (II) heating the compound 2 obtained in (I) in concentrated hydrochloric acid to remove the tert-butyl group to obtain compound 3;

[0025] (III) cross-linking 2-bromo-5-chloro-3-methylpyrazine with the compound 3 obtained in (II) to obtain compound 4;

[0026] (IV) taking tert-butyl carbamate (4-methylpiperidin-4-yl) and reacting it with the compound 4 obtained in (III) to obtain compound 5;

[0027] (V) Compound 6, i.e., the pharmacophore, is obtained by reacting compound 5 obtained in (IV) with bromoacetyl chloride.

[0028] Preferably, in the method for preparing the pharmacophore, the reaction conditions of each step are as follows:

[0029] (I) the molar ratio of 2-chloro-3-fluoroaniline to tert-butyl mercaptan is 1:2-4, and the reaction is carried out at 100-150° C. in the presence of Cs 2 CO 3 and with N,N-dimethylformamide as solvent;

[0030] The heating temperature in (II) is 60 to 100°C;

[0031] (III) the molar ratio of 2-bromo-5-chloro-3-methylpyrazine to compound 3 is 1:1-2, and a cross-linking reaction is carried out in the presence of CuI, 1,10-phenanthroline, K3PO4, and dioxane, and the cross-linking reaction temperature is 70-150° C.;

[0032] (IV) taking tert-butyl (4-methylpiperidin-4-yl)carbamate, N,N-diisopropylethylamine and the compound 4 obtained in (III), and reacting them at 80-120° C. in the presence of dimethyl sulfoxide, wherein the molar ratio of compound 4: tert-butyl (4-methylpiperidin-4-yl)carbamate: N,N-diisopropylethylamine is 1.8-3:1:2-4;

[0033] (V) The molar ratio of compound 5 to bromoacetyl chloride is 1:5-7.

[0034] Preferably, the preparation method of the fluorophore is as follows:

[0035] Using 4-chlorobenzofuran as the raw material, compound 8 and compound 9 are obtained in sequence through a two-step substitution reaction of chlorosulfonic acid and dimethylamine, and then the obtained compound 9 is connected with a fatty diamine to obtain compound 10, i.e., the fluorophore.

[0036] Further preferably, the preparation conditions of the fluorophore are as follows:

[0037] The reaction temperature of 4-chlorobenzofuran and chlorosulfonic acid is 100-150°C;

[0038] The molar ratio of compound 9 to fatty diamine is 1:15-20.

[0039] The third aspect of the present invention provides a series of fluorescent probes targeting SHP2 for use in SHP2 detection.

[0040] The beneficial effects of the present invention are:

[0041] (1) A series of small molecule fluorescent probes SHP-PS (1-4) targeting SHP2 protein are provided. These fluorescent probes exhibit excellent optical properties and biocompatibility, high signal-to-noise ratio, and stable fluorescent signals, which can meet the requirements of imaging. Moreover, these fluorescent probes maintain good affinity for SHP2 and can specifically label SHP2 in living cells, showing excellent protein selectivity, and homologous proteins such as SHP1 and other proteins have minimal interference with imaging.

[0042] In addition, these fluorescent probes have a selective switching function, are easy to use, and do not require washing before imaging. Cell imaging shows that they can clearly locate the SHP2 protein;

[0043] (2) A method for preparing such probes has been developed, which can achieve large-scale synthesis of such probes, which will help enhance researchers' understanding of the role of SHP2 in signal transduction and its significance in disease progression, thereby accelerating research and potentially revealing new diagnostic and therapeutic pathways for conditions such as cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a design diagram of a fluorescent probe targeting SHP2 in Example 1 of the present invention;

[0045] Figure 2 A diagram showing the synthesis method of a series of fluorescent probes SHP-PS (1-4) targeting SHP2 in Example 2 of the present invention;

[0046] Figure 3 The emission spectra (A1, A2, A3, A4) of the series of fluorescent probes SHP-PS (1-4) targeting SHP2 in Example 3 of the present invention in different polar solvents (water, methanol, ethanol, propanol, butanol) and the emission spectra (B1, B2, B3, B4) in different viscosity systems;

[0047] Figure 4 The in vitro inhibition curves of different concentrations of the fluorescent probes SHP-PS (1-4) and SHP-099 on SHP2 in Example 4 of the present invention;

[0048] Figure 5 The bright field imaging (L) and fluorescence imaging (R) of SHP-PS1 (A), SHP-PS2 (B), SHP-PS3 (C) and SHP-PS4 (D) in Example 5 of the present invention, wherein A1-D1 represent the fluorescent probe (2 μmol / L), and A2-D2 represent the fluorescent probe (2 μmol / L) + SHP-099 (20 μmol / L); the scale bar is 50 μm;

[0049] Figure 6The flow cytometry method for detecting the binding of the fluorescent probe to SHP2 in living cells in Example 6 of the present invention;

[0050] Figure 7 The fluorescence turn-on characteristics of the fluorescent probe SHP-PS2 after incubation of SHP2 proteins or living cells at different concentrations at 37° C. for 30 minutes in Example 7 of the present invention;

[0051] Figure 8 This is a result diagram of the fluorescence intensity (1 μmol / L) of different proteins and fluorescent probes after incubation for 30 minutes at 37° C. in Example 8 of the present invention;

[0052] Fig. 9 The fluorescence polarization values ​​of the fluorescent probe and SHP2 protein of different concentrations (B) detected by the fluorescence polarization method in Example 9 of the present invention. DETAILED DESCRIPTION

[0053] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0054] It should be noted that the terms used in the embodiments of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. In the following specific embodiments, if the experimental methods of specific conditions are not indicated, they are generally in accordance with conventional methods and conditions in the art.

[0055] In a typical embodiment of the present invention, the design of the fluorescent probe includes a structural optimization process of SHP-099, by replacing the amino group in the central region B of SHP-099 with a p-methyl group (RWC-4550) and introducing a sulfur atom as a connecting atom (TNO-155) to enhance the activity, and finally determining structure 1 as the pharmacophore, and introducing the SBD fluorophore therein, and designing and synthesizing a series of fluorescent probes SHP-PS with different lengths of connecting arms.

[0056] In another typical embodiment of the present invention, a series of fluorescent probes are chemically synthesized, which involves synthesizing the pharmacophore and the fluorophore through a series of substitution and cross-linking reactions of the starting materials, connecting the two through a nucleophilic reaction, and finally deprotecting to obtain the target fluorescent probe.

[0057] In addition, the present invention also measured the optical properties of a series of fluorescent probes, such as UV absorption, fluorescence excitation and emission wavelengths. In addition, the environmental sensitivity of the fluorescent probes was also evaluated.

[0058] Furthermore, the present invention also evaluated the inhibitory ability of the fluorescent probe on the enzyme activities of SHP1 and SHP2. By adding the substrate DiFMUP after pre-incubation with the enzyme and measuring the fluorescence signal, the results showed that the fluorescent probe had a strong inhibitory effect on SHP2 but had no effect on SHP1, showing the selective labeling ability for SHP2. Although the introduction of the fluorophore may affect the inhibitory activity, the fluorescent probe still exhibited strong binding ability and satisfactory selectivity for SHP2.

[0059] In another specific embodiment of the present invention, a fluorescent probe for specific labeling of SHP2 in living cells is described. Through direct imaging after incubation, it is found that all fluorescent probes can specifically bind to SHP2, among which SHP-PS2 performs best in cell imaging, has good SHP2 inhibitory activity and environmental sensitivity, and SHP-PS1 has the best inhibitory activity, but its imaging result is the worst, which is presumably because its short connecting arm hinders the fluorophore from entering the hydrophobic environment of the binding site. Furthermore, the present invention verifies the specific labeling ability of the fluorescent probe for SHP2 in living cells through flow cytometry experiments. The results show that the fluorescence intensity of the cells increases significantly after the probe is incubated, and the fluorescence intensity decreases under the competition of SHP-099, confirming the specific binding of the fluorescent probe to SHP2.

[0060] In addition, the present invention also verified the switching characteristics of the fluorescent probe, which was activated and released through binding to SHP2 protein and living cells. It was found that the performance of the fluorescent probe was affected by the length of the connecting arm, among which SHP-PS1 performed poorly, while SHP-PS2, SHP-PS3 and SHP-PS4 showed good switching characteristics.

[0061] In addition, the selectivity of the fluorescent probe for SHP2 protein was further verified through experiments. The results showed that compared with other proteins, the binding of the fluorescent probe to SHP2 resulted in a significant increase in fluorescence intensity, and the fluorescence intensity decreased under competition with SHP-099, confirming the high selectivity and specific binding of the obtained fluorescent probe to SHP2.

[0062] Furthermore, the present invention utilizes fluorescence polarization technology to accurately reflect the interaction between molecules by measuring the change in fluorescence polarization value after the fluorescent probe binds to the SHP2 protein, and is used for affinity determination and the establishment of competitive fluorescence screening methods. The results show that the prepared fluorescent probe has a strong affinity for SHP2, which is consistent with the results of SHP2 enzyme activity inhibition.

[0063] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0064] Example 1 Design of fluorescent probe

[0065] The design route of the fluorescent probe provided by the present invention is as shown in the attached Figure 1 As shown, attached Figure 1 In the formula, Structure 1 is structure 1, Fharmacophore is pharmacophore, Fluorophore is fluorophore, SHP-PS is a series of fluorescent probes, Formula 1 is the two-dimensional structure of SHP-099, in Formula 1, A is hydrophobic region, B is central region, C is polar region, and L is connecting atom.

[0066] From the perspective of two-dimensional structure, SHP-099 is mainly divided into hydrophobic region A, central region B and polar region C. If the synthesized fluorescent probe uses a similar structure to SHP-099 as the pharmacophore, the binding pocket where the pharmacophore binds to the protein is flanked by large-surface solvent-soluble regions, providing potential binding sites for the optimization of the inhibitor molecular structure.

[0067] RWC-4550 is a compound obtained by replacing the 2-amino group of the pyrazine ring in the middle B region of SHP099 with a p-methyl group, which improves the activity.

[0068] TNO-155 introduces sulfur as a linker atom, which increases the flexibility and activity of the molecule.

[0069] Taking into account the pharmacophore activity and synthetic feasibility, the present invention determined that structure 1 was the pharmacophore, introduced the SBD fluorophore into its hydrophobic region, and designed and synthesized a series of fluorescent probes SHP-PS with different lengths of linkers.

[0070] Example 2 Chemical synthesis of probe

[0071] A typical fluorescent probe consists of a fluorophore, a pharmacophore, and a linker between the two. Figure 2 The specific steps are as follows:

[0072] (1) Reaction between pharmacophore and fluorophore

[0073] Chemical Synthesis of S1 Pharmacophore

[0074] (I) 68.7 mmol of 2-chloro-3-fluoroaniline, 206 mmol of 2-methyl-2-propanethiol and 206 mmol of cesium carbonate were added to 100 mL of N,N-dimethylformamide (DMF), and then the mixture was stirred at 120° C. for 24 h to obtain compound 2 as a yellow oil (yield 85.7%);

[0075] (II) Compound 2 (55.6 mmol) obtained in (I) was added to hydrochloric acid (100 mL), and stirred at 80° C. for 6 h to obtain compound 3 as a white solid (yield 92.3%);

[0076] (III) 2.50 g of compound 3, 2.60 g of 2-bromo-5-chloro-3-methylpyrazine, 597 mg of CuI, 1.13 g of o-phenanthroline, and 9.97 g of K3PO4 were added to 100 mL of 1,4-dioxane, and then stirred at 90 °C for 10 h under nitrogen protection to obtain compound 4 (yield 45.3%);

[0077] (IV) 1.50 g of tert-butyl (4-methylpiperidin-4-yl) carbamate and 1.87 mL of diisopropylethylamine (DIPEA) were added to a 15 mL solution of 1.00 g of compound 4 in dimethyl sulfoxide (DMSO), and the mixture was stirred at 100 °C for 3 h to obtain compound 5 (yield 77.2%);

[0078] (V) A solution of 1.00 g of compound 5 and 2.14 mL of DIPEA in 20 mL of dichloromethane (DCM) was cooled in an ice bath, and then 1.08 mL of bromoacetyl chloride was added dropwise. The mixture was stirred at room temperature for 3 h, and then n-hexane was added. The precipitate was filtered out to obtain pharmacophore compound 6 as a yellow solid (yield 88.9%).

[0079] Preparation of S2 fluorophore

[0080] 3.00 g of 4-chlorobenzofuran was slowly added in batches to 30 mL of chlorosulfonic acid under an ice bath, and then the mixture was transferred to 120° C. and reacted for 6 h to obtain compound 8 as an off-white solid (yield 93.5%);

[0081] 4.00 g of compound 8 was dissolved in 40 mL of acetonitrile, and then a mixture of dimethylamine hydrochloride, triethylamine and acetonitrile was added. After that, the reaction mixture was stirred in an ice bath for 30 min and continued to be stirred at room temperature for 3 h to obtain compound 9 as a white solid (yield 73.7%);

[0082] 1.00 g of compound 9 was dissolved in 50 mL of acetonitrile with fatty diamines of different lengths (1,3-propylenediamine, 1,6-hexanediamine, 1,8-octanediamine, 1,10-decanediamine), and stirred at 60° C. for 6 h to obtain fluorophore compounds 10 with linkers of different lengths.

[0083] 200 mg of compound 6, 153 mg of compound 10 and 133 mg of DIPEA were dissolved in 50 mL of acetonitrile and stirred at room temperature for 20 h to obtain fluorescent probe compound 11.

[0084] (2) Using trifluoroacetic acid to remove the protecting group of the fluorescent probe compound 11 obtained in (1) to obtain a fluorescent probe targeting SHP2, the specific operation is as follows:

[0085] Trifluoroacetic acid (TFA) was added dropwise to the DCM solution of compound 11 and stirred at room temperature for 12 h. The solvent was removed under reduced pressure, and the residue was dissolved in DCM and methanol, then washed with saturated sodium bicarbonate solution, and the crude product was purified by silica gel column chromatography to obtain a series of fluorescent probes SHP-PS (1-4) targeting SHP2. The length of the linker increased successively, and the yields of SHP-PS (1-4) were 73.9%, 84.5%, 54.2%, and 66.4%, respectively.

[0086] Example 3 Determination of optical properties of probe

[0087] The optical properties of the fluorescent probe, such as ultraviolet absorption, fluorescence excitation and emission wavelength, were measured, as shown in Table 1. In Table 1, Φ is the relative quantum yield calculated with sodium fluorescein as a reference, λ max is the maximum UV absorption wavelength, λ ex is the maximum fluorescence excitation wavelength, λ em is the wavelength of maximum fluorescence emission.

[0088] Table 1 Optical properties of fluorescent probes

[0089]

[0090] As shown in Table 1, various fluorescent probes show two different UV absorption bands at 355 nm and 445 nm. Among them, the band at 355 nm corresponds to the conjugated system of the pharmacophore, which is known for its minimal fluorescence emission and is not the focus of the present invention.

[0091] Additionally, all fluorescent probes exhibited maximum excitation at 440 nm and 445 nm and peaked at 570 nm, resulting in a Stokes shift of approximately 130 nm, which is advantageous because it can reduce background interference, minimize phototoxicity to biological samples, and improve detection sensitivity.

[0092] In addition, the present invention also evaluates the environmental sensitivity of various fluorescent probes. Figure 3 Emission spectra (A1, A2, A3, A4) of each fluorescent probe SHP-PS (1-4) in solvents of different polarity (water, methanol, ethanol, propanol, butanol) and in systems of different viscosities (B1, B2, B3, B4).

[0093] Figure 3In the graph, the ordinate is the emission / absorption intensity, and the abscissa Wavelength is the wavelength; the viscosity system is realized by a mixture of glycerol and water, where the percentage represents the proportion of the glycerol volume to the total volume.

[0094] Figure 3 The positive correlation between the fluorescence intensity of each fluorescent probe and the decrease in solvent polarity and the increase in viscosity is shown in Table 1. In addition, the quantum yield of the probe is significantly higher in a low-polarity environment, as shown in Table 1. It is worth noting that the length of the linker between the pharmacophore and the fluorophore significantly affects the environmental sensitivity of the fluorescent probe. For example, the probes SHP-PS1 and SHP-PS2 showed excellent sensitivity, which decreased with the increase in the linker length. It can be seen that the optimization of the linker length in the probe design is extremely important.

[0095] Example 4 Enzyme Activity Inhibition Test of Fluorescent Probe

[0096] The ability of fluorescent probes to inhibit SHP1 and SHP2 enzymatic activity is a key aspect of their activity assessment because it provides insight into the binding affinity of the fluorescent probes to the target proteins and their selectivity for cognate proteins.

[0097] The commercial enzyme was pre-incubated with the probe for 30 min, and the phosphatase substrate DiFMUP was added to measure the fluorescence signal of the product. The in vitro inhibition curves of different concentration series of fluorescent probes SHP-PS (1-4) and SHP-099 on SHP2 are shown in Figure 4 .

[0098] Figure 4 The vertical axis is Inhibition rate, and the horizontal axis is Concentration.

[0099] The results showed that each fluorescent probe had strong inhibitory activity against SHP2, but had no inhibitory effect on SHP1 (Table 2), suggesting that it can selectively label SHP2 among homologous proteins.

[0100] Although the inhibitory activity of each fluorescent probe was reduced compared with SHP-099, SHP-PS1 showed the most obvious activity, which gradually decreased with the extension of the linker and reached about 15 times that of SHP-PS4.

[0101] Table 2 IC values ​​of fluorescent probes for inhibition of SHP1 and SHP2 enzymes 50 value

[0102]

[0103]

[0104] In summary, the introduction of fluorophores may inevitably affect the inhibitory activity, but the fluorescent probe provided by the present invention still shows good binding to the target SHP2 and exhibits satisfactory selectivity.

[0105] Example 5 Labeling of SHP2 in living cells

[0106] The present invention conducted cell imaging experiments to determine whether each fluorescent probe can specifically label SHP2 on living cells.

[0107] After the cells were incubated with each fluorescent probe for 20 min, images were taken using an inverted fluorescence microscope without washing.

[0108] Figure 5 Bright field imaging (L) and fluorescence imaging (R) of different fluorescent probes SHP-PS1 (A), SHP-PS2 (B), SHP-PS3 (C) and SHP-PS4 (D) are shown, where 1 represents the fluorescent probe (2 μmol / L), and 2 represents the fluorescent probe (2 μmol / L) + SHP-099 (20 μmol / L); the scale bar is 50 μm.

[0109] The results showed that all fluorescent probes could label SHP2 in living cells. Under the competition of SHP-099, the decrease in the fluorescence intensity of each fluorescent probe further proved the specific binding of each fluorescent probe to SHP2, rather than the fluorescence being turned on due to nonspecific binding with other proteins.

[0110] Moreover, under the same conditions, the fluorescence properties of the four probes were also different, and SHP-PS2 maintained good SHP2 inhibitory activity (IC 50 =2.88±0.17μmol / L) and environmental sensitivity, and its performance in cell imaging was the most outstanding, showing the prospect of further research.

[0111] In addition, although SHP-PS1 maintained the best SHP2 inhibitory activity (IC 50 =2.10±0.07μmol / L) and is most sensitive to environmental changes, but Figure 5 The results showed that it had the worst imaging effect in living cells compared with the other three fluorescent probes, which was speculated to be due to the short linker of SHP-PS1, which prevented the fluorophore from fully entering the hydrophobic environment of the binding site. In addition, the imaging effects of SHP-PS3 and SHP-PS4 were also satisfactory.

[0112] As described above, various fluorescent probes provided by the present invention can specifically label SHP2 to achieve the location and tracking of SHP2 in biological systems.

[0113] Example 6 Flow cytometry experiment to determine the binding of fluorescent probes to SHP2 in living cells

[0114] Flow cytometry was used to study the labeling effect of the probe on SHP2 in living cells.

[0115] like Figure 6 As shown in the figure, red: control, blue: fluorescent probe SHP-PS (1-4) only, yellow: fluorescent probe SHP-PS (1-4) + SHP-099 containing competitive inhibitor (SHP-099).

[0116] Figure 6 As can be seen in the figure, after the fluorescent probe was incubated with living cells, the detected fluorescence intensity increased significantly. In addition, under the competition with SHP-099 at a concentration of 10 times, the histogram showed an obvious left shift, indicating that the cell fluorescence intensity decreased, indicating that SHP-099 inhibited the binding of the fluorescent probe to SHP2.

[0117] Flow cytometry experiments further confirmed that the fluorescent probe prepared by the present invention specifically labels SHP2 in living cells.

[0118] Example 7 Verification of Selective Opening of Probes

[0119] In the present invention, an environment-sensitive switch is designed for the fluorescent probe, which is activated when bound to a target to release strong fluorescence, and the present invention verifies the switching properties of the fluorescent probe from the perspective of proteins and living cells.

[0120] Figure 7 The fluorescence turn-on characteristics of SHP-PS2 after incubation with different concentrations of SHP2 protein or living cells at 37°C for 30 minutes, including: (A) fluorescence emission spectrum of the fluorescent probe containing SHP2 protein; (B) fluorescence intensity of the fluorescent probe containing SHP2 protein at the maximum emission wavelength; (C, D) fluorescence intensity of the fluorescent probe containing living cells at the maximum emission wavelength; Wavelength-wavelength, Concentration-concentration, Cell number-cell number.

[0121] Figure 7 As shown in the figure, after the fluorescent probe SHP-PS2 was incubated with SHP2 protein, the fluorescence intensity increased significantly. As the protein concentration gradually increased, the fluorescence intensity reached saturation ( Figure 7 A, 7B). Consistent with previous imaging results, probes SHP-PS2, SHP-PS3, and SHP-PS4 showed good switching performance, while SHP-PS1 performed poorly with weak fluorescence release.

[0122] The present invention verifies the switch characteristics by incubating with living cells ( Figure 7C, 7D), and reached a consistent conclusion.

[0123] In general, the environmentally sensitive fluorescent switch designed by the present invention is well implemented, and its performance is greatly affected by the length of the connector.

[0124] Example 8 Selective determination of fluorescent probe for SHP2

[0125] The present invention selects bovine serum albumin (BSA), papain and trypsin as representatives to compare with SHP2 protein to test the selectivity of the fluorescent probe, thereby verifying whether it may non-specifically bind to other cellular components, and other proteins are present at twice the concentration of SHP2 protein.

[0126] Figure 8 The graph shows the fluorescence intensity of different proteins and fluorescent probes (1 μmol / L) after incubation for 30 min at 37°C. Blank represents the fluorescent probe solution without protein.

[0127] like Figure 8 As shown in the figure, the fluorescence produced by the three fluorescent probes after incubation with bovine serum albumin (BSA), papain (Papain) and trypsin (Trypsin) was significantly weaker than that produced after incubation with SHP2 (P<0.001). In the presence of SHP-099, the competition for binding sites prevented the fluorescent probes from entering the hydrophobic environment to activate the fluorescent switch, resulting in a significant decrease in the fluorescence intensity of the system (P<0.01). Therefore, each fluorescent probe has good selectivity for SHP2 and has minimal nonspecific binding.

[0128] Example 9 Fluorescence polarization experiment to determine the affinity of the fluorescent probe to the SHP2 protein

[0129] When the fluorescent probe is in a free state, the molecule rotates or flips freely, resulting in a low fluorescence polarization value. After binding to the target protein, its free movement is restricted, resulting in a slower rotation or flip rate, thus producing a strong fluorescence polarization. Fluorescence polarization can not only characterize the affinity of fluorescent molecules for their targets, but can also be further used to establish competitive fluorescence screening methods.

[0130] After incubating SHP2 protein with probes of different concentrations, the fluorescence polarization value increased with the increase of probe concentration and gradually reached a plateau. d Values ​​were calculated using Graphpad Prism 7.00.

[0131] K of SHP-PS1 and SHP-PS2 d The values ​​were between 1 and 2 μmol / L. The K dThe values ​​were 5.7 μmol / L and 11.2 μmol / L ( Fig. 9 ), and their affinity strength was well correlated with the inhibition of SHP2 enzyme activity in vitro. Fluorescence polarization experiments also further demonstrated that each fluorescent probe bound well to the SHP2 protein.

[0132] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A fluorescent probe targeting SHP2, characterized in that: The fluorescent probe has the following structure: Among them, n=1-10.

2. A method for preparing a fluorescent probe targeting SHP2 according to claim 1, characterized in that: The steps include: (1) Reaction between pharmacophore and fluorophore The pharmacophore, fluorophore and N,N-diisopropylacetic acid are dissolved in acetonitrile and reacted at room temperature to obtain a fluorescent probe, wherein the molar ratio of pharmacophore:fluorophore:N,N-diisopropylacetic acid is 1:1-2:1-1.5, and the reaction equation is shown below; (2) using trifluoroacetic acid to remove the protecting group of the fluorescent probe prepared in (1) to obtain a fluorescent probe targeting SHP2, as shown in the following equation; In the above formula, R1 is independently selected from H, Cl, Br or I; R2 is independently selected from H or tert-butyloxycarbonyl; n=1-10.

3. The preparation method according to claim 2, characterized in that: The preparation method of the pharmacophore is: (I) using 2-chloro-3-fluoroaniline and tert-butyl mercaptan as raw materials, reacting under alkaline conditions to obtain compound 2; (II) heating the compound 2 obtained in (I) in concentrated hydrochloric acid to remove the tert-butyl group to obtain compound 3; (III) cross-linking 2-bromo-5-chloro-3-methylpyrazine with the compound 3 obtained in (II) to obtain compound 4; (IV) taking tert-butyl carbamate (4-methylpiperidin-4-yl) and reacting it with the compound 4 obtained in (III) to obtain compound 5; (V) Compound 6, i.e., the pharmacophore, is obtained by reacting compound 5 obtained in (IV) with bromoacetyl chloride.

4. The preparation method according to claim 3, characterized in that: The reaction conditions of each step are as follows: (I) the molar ratio of 2-chloro-3-fluoroaniline to tert-butyl mercaptan is 1:2-4, and the reaction is carried out at 100-150° C. in the presence of Cs 2 CO 3 and with N,N-dimethylformamide as solvent; The heating temperature in (II) is 60 to 100°C; (III) the molar ratio of 2-bromo-5-chloro-3-methylpyrazine to compound 3 is 1:1-2, and a cross-linking reaction is carried out in the presence of CuI, 1,10-phenanthroline, K3PO4, and dioxane, and the cross-linking reaction temperature is 70-150° C.; (IV) taking tert-butyl (4-methylpiperidin-4-yl)carbamate, N,N-diisopropylethylamine and the compound 4 obtained in (III), and reacting them at 80-120° C. in the presence of dimethyl sulfoxide, wherein the molar ratio of compound 4: tert-butyl (4-methylpiperidin-4-yl)carbamate: N,N-diisopropylethylamine is 1.8-3:1:2-4; (V) The molar ratio of compound 5 to bromoacetyl chloride is 1:5-7.

5. The preparation method according to claim 2, characterized in that: The preparation method of the fluorophore is as follows: Using 4-chlorobenzofuran as the raw material, compound 8 and compound 9 are obtained in sequence through a two-step substitution reaction of chlorosulfonic acid and dimethylamine, and then the obtained compound 9 is connected with a fatty diamine to obtain compound 10, i.e., the fluorophore.

6. The preparation method according to claim 5, characterized in that: The preparation conditions of the fluorophore are as follows: The reaction temperature of 4-chlorobenzofuran and chlorosulfonic acid is 100-150°C; The molar ratio of compound 9 to fatty diamine is 1:15-20.

7. Use of the fluorescent probe targeting SHP2 as claimed in claim 1 in the detection of SHP2.

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