Near-infrared two-region activatable probe as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared two-region activatable probe as well as a preparation method and application thereof. The near-infrared second-region activatable probe comprises a near-infrared second-region fluorescent light-emitting unit and an analyte specific response unit; the near-infrared second-region light-emitting unit comprises a hemicyanine fluorophore structure; when the analyte specific response unit is chemically coupled with the near-infrared second-region light-emitting unit, the intramolecular charge transfer process of the light-emitting unit can be inhibited, and fluorescence quenching is caused; the analyte specific response unit is separated from the light-emitting unit after being subjected to specific reaction with an analyte, so that fluorescence is activated. The near-infrared two-region hemicyanine fluorophore selected by the near-infrared two-region activatable probe has higher fluorescence quantum efficiency, and the near-infrared two-region probe which can be activated by a specific analyte and has a high on-off ratio can be constructed by introducing an analyte specific response unit, so that high-temporal-spatial-resolution fluorescence detection of deep tissues can be realized; the method has a wide application prospect in the field of biomedical detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of near-infrared second-region fluorescent probes, and in particular relates to a class of near-infrared second-region activatable fluorescent probes and a preparation method and application thereof. Background Art

[0002] In the biomedical field, highly sensitive, high signal-to-noise ratio detection of active molecules, enzymes, and ions in physiological and pathological processes is the key to early diagnosis and precise treatment of diseases. Compared with traditional clinical detection methods, fluorescence detection has shown great application potential in the field of clinical detection due to its outstanding advantages such as high specificity, good sensitivity, no ionizing radiation, non-invasiveness, and real-time dynamic monitoring. In particular, near-infrared II (NIR-II, 900-1700nm) fluorescence imaging shows deeper biological tissue penetration (up to centimeter level), lower biological autofluorescence background interference, and higher spatiotemporal resolution, providing a new tool for clinical diagnosis and treatment fields such as in vivo tumor detection and intraoperative fluorescence navigation.

[0003] However, due to the highly heterogeneous physiological microenvironment, the constant-on characteristic of traditional NIR-II region fluorescent probes may cause nonspecific signal interference, thereby generating false positive results, limiting their application in complex biological environments. Therefore, the development of activatable NIR-II region probes to achieve high specificity and high signal-to-noise ratio detection of specific biomarkers (such as enzyme activity, biomolecules, ions, pH, etc.) has become a cutting-edge direction in clinical diagnostic research.

[0004] Semicyanine fluorescent dye is a commonly used molecular probe platform for designing activatable fluorescent probes, which can be used to change the intramolecular charge transfer process of the probe by introducing a specific recognition unit in the probe molecular structure, and realize the regulation and control of the fluorescent probe luminescence wavelength or fluorescence brightness. However, the luminescence wavelength of the current semicyanine fluorescent probe is usually in near-infrared zone I (NIR-I, 650-900nm), and it is urgent to develop the semicyanine molecular probe platform in NIR-II zone, so as to develop activatable fluorescent probes in NIR-II zone. CN119264098A discloses a stereoisomer pair with near-infrared zone II luminescence properties, fluorescent probe and its preparation method and application. The method prepares near-infrared zone II fluorescent probes with oxonium ions as raw materials, but the fluorescent probe prepared by the technology does not have analyte-specific response characteristics. Summary of the Invention

[0005] The main purpose of the present invention is to provide a class of near-infrared second-region activatable fluorescent probes with high specificity, high fluorescence on / off ratio and high sensitivity and a preparation method thereof, so as to overcome the shortcoming of the existing semi-cyanine activatable probes with short wavelength.

[0006] Another object of the present invention is to provide an application of the near-infrared second region activatable fluorescent probe.

[0007] To achieve the aforementioned object of the invention, the technical solutions adopted by the present invention include:

[0008] An embodiment of the present invention provides a class of near-infrared second-region activatable probes, comprising a near-infrared second-region fluorescent light-emitting unit and an analyte-specific response unit connected to each other; the near-infrared second-region fluorescent light-emitting unit comprises a semi-cyanine fluorophore structure; the analyte-specific response unit quenches the fluorescence of the near-infrared second-region fluorescent light-emitting unit when chemically coupled with the near-infrared second-region fluorescent light-emitting unit; and the analyte-specific response unit can activate the fluorescence of the analyte-specific response unit after a specific reaction with the analyte.

[0009] In some embodiments, the structural formula of the near-infrared region II activatable probe is as shown in formula (I):

[0010]

[0011] Among them, X - Including ClO4 - 、BF4 - CF3COO - 、CH3COO - 、F - 、Cl - Br - , I - 、NO3 - Any one of; Z includes any one of O, S, NH; n is 0 or 1;

[0012] R1 includes -H, Any of the following;

[0013] R2 includes any one of -H, -F, -Cl, -Br, and -I;

[0014] R3 is the analyte-specific recognition unit;

[0015] R4 includes Any one of , wherein m is any integer from 0 to 17.

[0016] The present invention provides a method for preparing a near-infrared second-region hemicyanine platform molecular probe, which comprises:

[0017] A first condensation reaction is performed on a first compound containing an indole salt and pentadienaldehyde diphenylamine hydrochloride to obtain an intermediate;

[0018] The intermediate is further subjected to a second condensation reaction with a second compound containing an oxonium ion to prepare a near-infrared second-region hemicyanine platform molecular probe;

[0019] Wherein, the first compound comprising an indole salt has a structure as shown in formula (II):

[0020]

[0021] X - Including ClO4 - 、BF4 - CF3COO - 、CH3COO - 、F - 、Cl - Br - , I - 、NO3 - Any of the following;

[0022] R4 includes Any of the following;

[0023] The second compound containing an oxonium ion has a structure as shown in formula (III):

[0024]

[0025] Z includes any one of O, S, and NH; n is 0 or 1;

[0026] R1 includes -H, Any of the following;

[0027] R2 includes any one of -H, -F, -Cl, -Br, and -I.

[0028] The embodiment of the present invention also provides a near-infrared second-region hemicyanine platform molecular probe prepared by the above preparation method.

[0029] The present invention provides a method for preparing a near-infrared second-region activatable probe, which comprises:

[0030] According to the above preparation method, a near-infrared second-region semi-cyanine platform molecular probe was prepared;

[0031] The near-infrared zone II hemicyanine platform molecular probe is chemically coupled with a substance having an analyte-specific recognition unit to prepare a near-infrared zone II activatable probe.

[0032] The embodiment of the present invention also provides a near-infrared second-region activatable probe prepared by the above-mentioned preparation method.

[0033] Correspondingly, the embodiments of the present invention also provide applications of the aforementioned near-infrared region II activatable probe in fields such as biological imaging detection or drug screening.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1) The present invention provides a class of near-infrared region II activatable fluorescent probes that can be obtained by reacting near-infrared region II hemicyanine platform molecules with analyte-specific recognition units. This method has the characteristics of modular preparation and can be used to efficiently design and synthesize the desired types of activatable near-infrared region II fluorescent probes, which is easy to produce on a large scale.

[0036] 2) The near-infrared second-region activatable fluorescent probe provided by the present invention has high fluorescence brightness and response on-off ratio, and can be used for specific high signal-to-noise ratio detection of analytes;

[0037] 3) The near-infrared second region activatable fluorescent probe provided by the present invention can be used in the fields of living disease detection, biological imaging, medical diagnosis or drug screening research. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0039] Figure 1 Schematic diagram of the response mechanism of a near-infrared second region activatable fluorescent probe in a typical embodiment of the present invention;

[0040] Figure 2 This is the H-NMR spectrum of the CR11-OH platform molecule prepared in Example 1 of the present invention;

[0041] Figure 3 This is the C NMR spectrum of the CR11-OH platform molecule prepared in Example 1 of the present invention;

[0042] Figure 4 This is the H-NMR spectrum of the CR12-OH platform molecule prepared in Example 2 of the present invention;

[0043] Figure 5 This is the C NMR spectrum of the CR12-OH platform molecule prepared in Example 2 of the present invention;

[0044] Figure 6 This is the H-NMR spectrum of the CR13-OH platform molecule prepared in Example 3 of the present invention;

[0045] Figure 7 This is the C NMR spectrum of the CR13-OH platform molecule prepared in Example 3 of the present invention;

[0046] Figure 8This is the H NMR spectrum of the CR11-Cys activatable probe prepared in Example 9 of the present invention;

[0047] Figure 9 This is the NMR carbon spectrum of the CR11-Cys activatable probe prepared in Example 9 of the present invention;

[0048] Figure 10 This is the H NMR spectrum of the CR12-Cys activatable probe prepared in Example 10 of the present invention;

[0049] Figure 11 This is the NMR carbon spectrum of the CR12-Cys activatable probe prepared in Example 10 of the present invention;

[0050] Figure 12 This is the H NMR spectrum of the CR13-Cys activatable probe prepared in Example 11 of the present invention;

[0051] Figure 13 This is the NMR carbon spectrum of the CR13-Cys activatable probe prepared in Example 11 of the present invention;

[0052] Figure 14 This is the H NMR spectrum of the CR13-APN activatable probe prepared in Example 15 of the present invention;

[0053] Figure 15 This is the NMR carbon spectrum of the CR13-APN activatable probe prepared in Example 15 of the present invention;

[0054] Figure 16 Schematic diagram of the reaction process in Example 16 of the present invention;

[0055] Figure 17a-Figure 17i is a schematic diagram of the responsiveness of CR11-OH, CR12-OH and CR13-OH to different pH values ​​in Example 17 of the present invention;

[0056] Figure 18a and Figure 18b 17 is a comparison diagram of the fluorescence images of CR11-OH, CR12-OH and CR13-OH at different pH values ​​and the corresponding fluorescence intensities;

[0057] Figure 19a-Figure 19i 1 is a schematic diagram of the test results of the response sensitivity of CR11-Cys, CR12-Cys and CR13-Cys to Cys in Example 18 of the present invention;

[0058] Figure 20a-20f 1 is a schematic diagram of the test results of the response speed of CR11-Cys, CR12-Cys and CR13-Cys to Cys in Example 19 of the present invention;

[0059] Figure 21a-Figure 21f20 is a schematic diagram of the test results of the response specificity of CR11-Cys, CR12-Cys and CR13-Cys to different analytes in Example 20 of the present invention;

[0060] Figure 22 This is a schematic diagram of the fluorescence detection results of Cys by CR13-Cys at the cellular level in Example 21 of the present invention. DETAILED DESCRIPTION

[0061] By extending the conjugated chain or modifying the end group, the electronic orbital energy level and intramolecular charge transfer process of the probe can be regulated, which is expected to develop a semi-cyanine molecular probe platform in the NIR-II region. In addition, on the basis of extending the conjugated chain of the semi-cyanine probe, the system can be further functionalized, such as introducing a benzoic acid structure (to reduce the quenching effect of the probe in the solvent and increase the fluorescence intensity) and a halogen atom (to increase the charge separation effect of the probe ground state / excited state), which will help to improve the fluorescence brightness and response switching ratio of the semi-cyanine molecular platform in the NIR-II region, and optimize the high-performance NIR-II region activatable semi-cyanine probe platform.

[0062] The analyte-specific response unit is directly bound to the phenolic hydroxyl group or aniline on the semi-cyanine molecular structure in the NIR-II region through a covalent bond or by using a self-eliminable bridging structure, and blocks or inhibits the intramolecular charge transfer process of the semi-cyanine probe, causing the fluorescence wavelength of the semi-cyanine probe to blue-shift or the fluorescence intensity to weaken. The analyte-induced "deprotection" process can restore the intramolecular charge transfer of the probe, thereby activating the fluorescence of the probe. For specific biomarkers, there are currently many analyte-specific response units to choose from, such as nitroreductase, cysteine, reactive oxygen species (ROS), phosphatase, reactive nitrogen (RNS), etc., and corresponding response units can be selected to design NIR-II region-specific activation probes. In addition, the introduction of functional groups such as amino, carboxyl, and alkynyl groups into the probe structure for targeted or nano-modification can help improve the response properties and application scenarios of the activated probe.

[0063] In view of the shortcoming of the existing semi-cyanine activatable probes with a short wavelength, the inventors of this case proposed this technical solution after long-term research and a large number of experiments. It is mainly based on indole salt structure compounds and oxonium ion structure compounds containing -OH, -SH, and -NH2 active groups. Through conjugated chain extension or end group modification strategies, the electronic orbital energy level and intramolecular charge transfer process of the probe are regulated to provide a class of near-infrared region II (NIR-II region) semi-cyanine molecular probe platforms with high specificity, high fluorescence switching ratio, and high sensitivity. Analyte-specific activation fluorescent probes are developed based on the near-infrared region II semi-cyanine molecular platform, and their applications in biomedical diagnosis and drug screening are provided.

[0064] The technical solution, its implementation process and principles are further explained below.

[0065] As one aspect of the technical solution of the application, a class of near-infrared zone II activatable probes involved therein includes a near-infrared zone II fluorescent luminescent unit and an analyte-specific response unit; the near-infrared zone II fluorescent luminescent unit contains a semi-cyanine fluorophore structure; when the analyte-specific response unit is chemically coupled with the near-infrared zone II fluorescent luminescent unit, it will inhibit the intramolecular charge transfer process of the luminescent unit, thereby causing fluorescence quenching of the luminescent unit; after the analyte-specific response unit undergoes a specific reaction with the analyte, it will detach from the fluorescent luminescent unit, thereby activating the fluorescence of the analyte-specific response unit.

[0066] In some embodiments, the structural formula of the near-infrared region II activatable probe is as shown in formula (I):

[0067]

[0068] Among them, X - Including ClO4 - 、BF4 - CF3COO - 、CH3COO - 、F - 、Cl - Br - , I - 、NO3 - etc.; Z includes any one of O, S, NH;

[0069] n is 0 or 1;

[0070] R1 includes -H, Any of the above;

[0071] R2 includes any one of -H, -F, -Cl, -Br, -I, etc.;

[0072] R3 is the analyte-specific recognition unit;

[0073] R4 includes Any one of the above, wherein m is any integer from 0 to 17.

[0074] In some preferred embodiments, R3 comprises Any one of the above, but not limited to these.

[0075] After the near-infrared second-region activatable probe of the present invention specifically reacts with the analyte, it triggers the analyte-specific recognition unit to be removed from the fluorescent probe structure, so that the intramolecular charge transfer process of the probe is restored, thereby realizing the turning on of fluorescence.

[0076] Furthermore, the near-infrared second-region activatable fluorescent probe provided by the present invention has high fluorescence brightness and response on-off ratio, and can be used for specific high signal-to-noise ratio detection of analytes.

[0077] As another aspect of the technical solution of the present invention, a method for preparing a type of near-infrared second-zone semi-cyanine platform molecular probe involved therein comprises:

[0078] A first condensation reaction is performed on a first compound containing an indole salt and pentadienaldehyde diphenylamine hydrochloride to obtain an intermediate;

[0079] The intermediate is further subjected to a second condensation reaction with a second compound containing an oxonium ion to prepare a near-infrared second-zone hemicyanine platform molecular probe.

[0080] In some embodiments, the first compound comprising an indole salt (abbreviated as C) used in the present invention has a structure as shown in formula (II):

[0081]

[0082] Among them, X - Including ClO4 - 、BF4 - CF3COO - 、CH3COO - 、F - 、Cl - Br - , I - 、NO3 - Any of the above;

[0083] R4 includes Any one of the above.

[0084] In some embodiments, the second compound containing an oxonium ion (abbreviated as R) used in the present invention has a structure as shown in formula (III):

[0085]

[0086] Wherein, Z includes any one of O, S, NH, etc.;

[0087] n is 0 or 1;

[0088] R1 includes -H, Any of the above;

[0089] R2 includes any one of -H, -F, -Cl, -Br, -I, etc.

[0090] In some embodiments, the preparation method specifically includes: mixing a first compound containing an indole salt (abbreviated as C), pentadienaldehyde diphenylamine hydrochloride and a first reaction solvent, and performing a first condensation reaction in a protective atmosphere to obtain an intermediate (abbreviated as M).

[0091] In some embodiments, the preparation method specifically includes: mixing the intermediate M, a second compound containing an oxonium ion (which can be simply referred to as R) and a second reaction solvent, and continuing a second condensation reaction in a protective atmosphere to obtain a near-infrared second-region semi-cyanine platform molecular probe with near-infrared second-region luminescence properties.

[0092] The reaction equation of the preparation method of a type of near-infrared second-zone hemicyanine platform molecular probe of the present invention is as follows:

[0093]

[0094] The preparation mechanism of a class of near-infrared second-range semi-cyanine platform molecular probes of the present invention is: first, an indole salt is used to undergo a condensation reaction with pentadienaldehyde diphenylamine hydrochloride to obtain an intermediate M, and then the intermediate M is further subjected to a condensation reaction with an oxonium ion to obtain a near-infrared second-range semi-cyanine platform molecular probe.

[0095] In some more preferred embodiments, the method for preparing a class of near-infrared second-region hemicyanine probe platform molecules of the present invention comprises the following steps:

[0096] (1) Synthesis of intermediate M: dissolving a first compound C containing an indole salt and pentadienaldehyde diphenylamine hydrochloride in a first reaction solvent, followed by heating in an inert protective gas atmosphere. After the reaction, heating is stopped, and a large amount of water is added for precipitation to obtain intermediate M;

[0097] (2) Synthesis of near-infrared zone II semi-cyanine probe platform molecules: Add a second compound R containing an oxonium ion, a base, and a second reaction solvent to the intermediate M solution obtained in step (1), and continue the reaction under an inert protective gas. After the reaction is completed, add an anti-solvent for precipitation and purification to obtain a near-infrared zone II semi-cyanine platform molecular probe.

[0098] In some preferred embodiments, in step (1) of the above-mentioned preparation method, the first reaction solvent may include at least any one of acetic anhydride, toluene / n-butanol, benzene / n-butanol, ethanol, methanol, etc., preferably acetic anhydride.

[0099] In some preferred embodiments, in step (1) of the above-mentioned preparation method, the molar ratio of the first compound comprising an indole salt to pentadienaldehyde diphenylamine hydrochloride can be 1:(0.1-2).

[0100] In some preferred embodiments, in step (1) of the preparation method, the temperature of the first condensation reaction is 50-150°C, preferably 90-110°C.

[0101] In some preferred embodiments, in step (1) of the above-mentioned preparation method, the time of the first condensation reaction is 0.5 to 12 hours, preferably 2 to 3 hours.

[0102] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the base may include at least one of sodium acetate, potassium acetate, potassium carbonate, sodium carbonate, triethylamine, etc., preferably sodium acetate or potassium acetate. In the toluene and n-butanol reaction system, the addition of a base may not be required.

[0103] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the second reaction solvent may include at least any one of acetic anhydride, toluene / n-butanol, benzene / n-butanol, etc., preferably acetic anhydride.

[0104] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the molar ratio of the intermediate to the second compound containing an oxonium ion is 1:(0.5-10).

[0105] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the molar ratio of the intermediate to the base is 1:(1-30).

[0106] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the temperature of the second condensation reaction is 0 to 150°C, preferably 20 to 60°C.

[0107] In some preferred embodiments, in step (2) of the above-mentioned preparation method, the time of the second condensation reaction is 0.5 to 24 hours, preferably 6 to 12 hours.

[0108] As another aspect of the technical solution of the present invention, a near-infrared second-zone hemicyanine platform molecular probe prepared by the aforementioned preparation method is also provided.

[0109] As another aspect of the technical solution of the present invention, a method for preparing a type of near-infrared second-region activatable probe involved therein includes:

[0110] Prepare the near-infrared second-region hemicyanine platform molecular probe according to the aforementioned preparation method;

[0111] The near-infrared second-zone hemicyanine platform molecular probe is chemically coupled with a substance having an analyte-specific recognition unit to prepare an analyte-specifically activated near-infrared second-zone activatable probe.

[0112] In some embodiments, the substance having an analyte-specific recognition unit may include Any one of the above, but not limited to these.

[0113] In some embodiments, the molar ratio of the near-infrared second-region hemicyanine platform molecular probe to the substance having an analyte-specific recognition unit is 1:1-5.

[0114] In some embodiments, the preparation method further comprises chemically coupling the near-infrared region II activatable probe with functional units such as amino, carboxyl, alkyne, azide, and maleimide to improve the probe's targeting, water solubility, and other properties.

[0115] In summary, the present invention provides a method for preparing a class of near-infrared second-region activatable fluorescent probes, which has a simple synthesis method, a clear molecular design principle, high scalability, and is easy to customize and mass produce.

[0116] As another aspect of the technical solution of the present invention, a near-infrared second-region activatable probe prepared by the above-mentioned preparation method is also provided.

[0117] See also Figure 1 As shown, the near-infrared second-region activatable probe provided by the present invention can specifically respond to the analyte and restore the probe fluorescence from the "off" state to the "activated" state, thereby realizing the activation detection of the specific analyte.

[0118] In some embodiments, the near-infrared region II activatable probe exhibits specific activation properties for cysteine ​​(Cys). More specifically, the fluorescence brightness of the probe is significantly enhanced upon the addition of cysteine, while the fluorescence brightness of the probe is not significantly enhanced upon the addition of other analytes.

[0119] The near-infrared second-region activatable fluorescent probe in the present invention has strong designability and high scalability, and has broad application prospects in the fields of biological imaging detection, drug screening and development, and disease diagnosis and treatment.

[0120] Using the technical solution of the present invention, the probe uses a near-infrared region II hemicyanine fluorophore with high fluorescence quantum efficiency. This fluorophore can serve as a platform molecule, enabling the construction of near-infrared region II probes with high on / off ratios that can be activated by specific analytes by introducing different analyte-specific response units. Furthermore, within the near-infrared region II optical window, light absorption and scattering in biological tissues are reduced, significantly reducing biological autofluorescence and significantly improving tissue penetration depth. This enables fluorescence detection in deep tissues with high temporal and spatial resolution, promising broad applications in biomedical testing.

[0121] The following will be combined with the examples and drawings to describe the specific embodiments of the present invention in more detail, but the examples do not constitute a limitation of the present invention. All variations associated with or derived from the contents disclosed in the present invention are considered to be within the scope of protection of the present invention.

[0122] Example 1

[0123] Indole salt C1 (0.1mmol) and pentadienaldehyde diphenylamine hydrochloride (0.1mmol) are added in 10mL diacetic anhydride and ultrasonically dissolved. The air in the reaction system is removed by double-row pipe, and then reacted in 110°C oil bath for 3 hours. After the reaction is completed, the reaction solution is poured into 1000mL of ice water and precipitated. After standing for 2 hours, the supernatant is removed by centrifugation to obtain intermediate M1. Above-mentioned intermediate M1 (0.06mmol), oxonium ion R1 (0.1mmol) and sodium acetate (0.3mmol) are dissolved in 15mL diacetic anhydride, and air is removed by double-row pipe, and reacted in 45°C water bath for 3 hours. Then, the petroleum ether of 500mL is added into the reaction system and precipitated. The precipitation is purified using a silica gel column, and the eluent is dichloromethane and methanol.

[0124]

[0125] The NIR II semi-cyanine platform molecule CR11-OH obtained in this example has a hydrogen NMR spectrum as shown in FIG. Figure 2 As shown, the NMR carbon spectrum of CR11-OH is as follows Figure 3 shown.

[0126] Example 2

[0127] Indole salt C1 (0.1mmol) and pentadienaldehyde diphenylamine hydrochloride (0.15mmol) are added to 10mL acetic anhydride and ultrasonically dissolved. The air in the reaction system is removed by double-row pipe, and then reacted in 90°C oil bath for 5 hours. After the reaction is completed, the reaction solution is poured into 1000mL of ice water for precipitation. After standing for 2 hours, the supernatant is removed by centrifugation to obtain intermediate M1. The above-mentioned intermediate M1 (0.06mmol), oxonium ion R2 (0.1mmol) and sodium acetate (0.5mmol) are dissolved in 15mL acetic anhydride, and the air is removed by double-row pipe, and reacted in 60°C water bath for 1 hour. Afterwards, 500mL of cyclohexane is added to the reaction system for precipitation. The precipitation is purified using a silica gel column, and the eluent is ethyl acetate and methanol.

[0128]

[0129] The NIR II semi-cyanine platform molecule CR12-OH obtained in this example has a hydrogen NMR spectrum as shown in FIG. Figure 4 As shown, the NMR carbon spectrum of CR12-OH is as follows Figure 5 shown.

[0130] Example 3

[0131] Indole salt C1 (0.1mmol) and pentadienaldehyde diphenylamine hydrochloride (0.1mmol) are added in 10mL methanol and ultrasonically dissolved. Utilize double-row pipe to remove the air in the reaction system, then react in 110 ℃ of oil baths for 3 hours. After reaction completes, reaction solution is poured into 1000mL ice water and precipitated, after standing for 2 hours, centrifuge and remove supernatant to obtain intermediate M1. Above-mentioned intermediate M1 (0.06mmol) and oxonium ion R3 (0.2mmol) are dissolved in 15mL toluene / n-butanol mixed solvent, and remove air with double-row pipe, at room temperature react 12 hours, then in reaction system, add the petroleum ether of 500mL and precipitate. Described precipitation uses silica gel column to be purified, and eluent is methylene chloride and methanol.

[0132]

[0133] The NMR hydrogen spectrum of the near-infrared second-zone semi-cyanine platform molecule CR13-OH obtained in this example is as follows Figure 6 As shown, the NMR carbon spectrum of CR13-OH is as follows Figure 7 shown.

[0134] Example 4

[0135] Indole salt C2 (0.1mmol) and pentadienaldehyde diphenylamine hydrochloride (0.1mmol) are added in 10mL diacetic anhydride and ultrasonically dissolved. Utilize double-row pipe to remove the air in the reaction system, then react in 110 ℃ of oil baths for 0.5 hour. After reaction completes, reaction solution is poured into 1000mL ice water and precipitated, after standing for 0.5 hour, centrifuge and remove supernatant to obtain intermediate M2. Above-mentioned intermediate M2 (0.08mmol), oxonium ion R4 (0.1mmol) and potassium carbonate (0.3mmol) are dissolved in 50mL diacetic anhydride, and remove air with double-row pipe, react at 60 ℃ for 1 hour, then in reaction system, add the ether of 1000mL and precipitate. Described precipitation uses silica gel column to be purified, and eluent is methylene chloride and methanol.

[0136]

[0137] Example 5

[0138] Indole salt C3 (0.1mmol) and pentadienaldehyde diphenylamine hydrochloride (0.1mmol) are added to 10mL acetic anhydride and ultrasonically dissolved. The air in the reaction system is removed by double-row pipe, and then reacted in 110°C oil bath for 1 hour. After the reaction is completed, the reaction solution is poured into 1000mL of ice water and precipitated. After standing for 2 hours, the supernatant is removed by centrifugation to obtain intermediate M3. The above-mentioned intermediate M3 (0.09mmol) and oxonium ion R5 (0.1mmol) are dissolved in 50mL of benzene / n-butanol mixed solvent, and air is removed by double-row pipe, and reacted under 150°C water bath for 0.5 hour. The petroleum ether of 500mL is added to the reaction system and precipitated. The precipitation is purified using a silica gel column, and the eluent is dichloromethane and methanol.

[0139]

[0140] Example 6

[0141] Indole salt C4 (0.1mmol) and pentadienaldehyde diphenylamine hydrochloride (0.01mmol) are added in 10mL diacetic anhydride and ultrasonically dissolved. The air in the reaction system is removed by double-row pipe, and then reacted in 110 DEG C of oil baths for 3 hours. After the reaction is completed, the reaction solution is poured into 1000mL of ice water and precipitated. After standing for 2 hours, the supernatant is removed by centrifugation to obtain intermediate M4. Above-mentioned intermediate M4 (0.01mmol), oxonium ion R6 (0.1mmol) and sodium carbonate (0.3mmol) are dissolved in 15mL diacetic anhydride, and air is removed by double-row pipe, and reacted under 0 DEG C of water baths for 24 hours. The petroleum ether of 500mL is added to the reaction system and precipitated. The precipitation is purified using a silica gel column, and eluents are dichloromethane and methanol.

[0142]

[0143] Example 7

[0144] Indole salt C5 (0.1mmol) and pentadienaldehyde diphenylamine hydrochloride (0.1mmol) are added in 10mL diacetic anhydride and ultrasonically dissolved. The air in the reaction system is removed by double-row pipe, and then reacted in 50 ℃ of oil baths for 12 hours. After the reaction is completed, the reaction solution is poured into 1000mL of ice water and precipitated. After standing for 2 hours, the supernatant is removed by centrifugation to obtain intermediate M5. Above-mentioned intermediate M5 (0.01mmol), oxonium ion R7 (0.005mmol) and sodium acetate (0.3mmol) are dissolved in 15mL diacetic anhydride, and air is removed by double-row pipe, and reacted under 45 ℃ of water baths for 3 hours. The petroleum ether of 500mL is added in the reaction system and precipitated. The precipitation is purified using a silica gel column, and the eluent is dichloromethane and methanol.

[0145]

[0146] Example 8

[0147] Indole salt C6 (0.1mmol) and pentadienaldehyde diphenylamine hydrochloride (0.2mmol) are added in 10mL diacetic anhydride, ultrasonically dissolved. Utilize double-row pipe to remove the air in the reaction system, afterwards in 150 ℃ of oil baths, react 3 hours. After reaction terminates, reaction solution is poured into the frozen water of 1000mL and precipitated, after standing for 2 hours, centrifuge and remove supernatant, obtain intermediate M6. Above-mentioned intermediate M6 (0.09mmol), oxonium ion R8 (0.1mmol) and sodium acetate (0.09mmol) are dissolved in 15mL diacetic anhydride, and remove air with double-row pipe, under 45 ℃ of water baths, react 3 hours, in reaction system, add the petroleum ether of 500mL and precipitate. Described precipitation uses silica gel column to be purified, and eluent is methylene chloride and methanol.

[0148]

[0149] Example 9

[0150] The near-infrared second zone semi-cyanine platform molecule CR11-OH (0.1mmol) and triethylamine (0.2mmol, providing an alkaline environment to neutralize the hydrochloric acid generated during the reaction of the acyl chloride) obtained in Example 1 were added to 10mL of dichloromethane, and then acryloyl chloride (0.15mmol) diluted with dichloromethane was slowly added dropwise under an ice bath, and the reaction was continued under an ice bath for 1 hour. Afterwards, it was returned to room temperature and the reaction was continued for 4 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, and the crude product was separated and purified by preparative high performance liquid chromatography (HPLC) to obtain a near-infrared second zone activatable probe CR11-Cys.

[0151]

[0152] The NMR hydrogen spectrum of the near-infrared second region activatable probe CR11-Cys obtained in this example is as follows Figure 8 As shown, the NMR carbon spectrum of CR11-Cys is as follows Figure 9 shown.

[0153] Example 10

[0154] The near-infrared second-zone semi-cyanine platform molecule CR12-OH (0.1 mmol) and triethylamine (0.2 mmol) obtained in Example 2 were added to 10 mL of dichloromethane, and then acryloyl chloride (0.15 mmol) diluted with dichloromethane was slowly added dropwise under an ice bath, and the reaction was continued under an ice bath for 1 hour. After returning to room temperature, the reaction was continued for 4 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, and the crude product was separated and purified by preparative high performance liquid chromatography (HPLC) to obtain a near-infrared second-zone activatable probe CR12-Cys.

[0155]

[0156] The NMR hydrogen spectrum of the near-infrared second region activatable probe CR12-Cys obtained in this example is as follows Figure 10 As shown, the NMR carbon spectrum of CR12-Cys is as follows Figure 11 shown.

[0157] Example 11

[0158] The near-infrared second-zone semi-cyanine platform molecule CR13-OH (0.1 mmol) and triethylamine (0.2 mmol) obtained in Example 3 were added to 10 mL of dichloromethane, and then acryloyl chloride (0.15 mmol) diluted with dichloromethane was slowly added dropwise under an ice bath, and the reaction was continued under an ice bath for 1 hour. After returning to room temperature, the reaction was continued for 4 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, and the crude product was separated and purified by preparative high performance liquid chromatography (HPLC) to obtain a near-infrared second-zone activatable probe CR13-Cys.

[0159]

[0160] The NMR hydrogen spectrum of the near-infrared second region activatable probe CR13-Cys obtained in this example is as follows Figure 12 As shown, the NMR carbon spectrum of CR13-Cys is as follows Figure 13 shown.

[0161] Example 12

[0162] The near-infrared second-zone hemicyanine platform molecule CR46-SH (0.1 mmol) prepared in Example 6 was added to 10 mL of anhydrous acetonitrile, and then phosphorus oxychloride (0.15 mmol) diluted in anhydrous acetonitrile was slowly added dropwise under an ice bath, and the reaction was continued under an ice bath for 30 minutes. The reaction was then returned to room temperature and the reaction progress was monitored by a plate. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the crude product was separated and purified by preparative high-performance liquid chromatography (HPLC) to obtain the near-infrared second-zone activatable probe CR46-ALP.

[0163]

[0164] Example 13

[0165] The near-infrared second zone semi-cyanine platform molecule CR35-NH2 (0.1mmol) obtained in Example 5 was added to 10mL of anhydrous acetonitrile, and 0.2mmol of trifluoromethanesulfonic anhydride was added. The reaction system was then transferred to 45°C to continue the reaction. The reaction progress was monitored using TLC. After the raw materials were basically gone, 50mL of water was added to the reaction system to quench the reaction. The product was extracted with a large amount of dichloromethane. After the solvent was removed by distillation under reduced pressure, the crude product was separated and purified by preparative high performance liquid chromatography (HPLC) to obtain the near-infrared second zone activatable probe CR35-O2.

[0166]

[0167] Example 14

[0168] The near-infrared region II hemicyanine platform molecule CR24-NH2 (0.1 mmol) prepared in Example 4 was added to 10 mL of dry dichloromethane, followed by 0.15 mmol of β-gal-NHS. The mixture was stirred at room temperature for 12 hours. After the reaction, the solvent was removed by distillation under reduced pressure, and the crude product was separated and purified by preparative high-performance liquid chromatography (HPLC) to obtain the near-infrared region II activatable probe CR24-β-gal.

[0169]

[0170] Example 15

[0171] CR13-Cys (0.1 mmol) prepared in Example 11 was added to 10 mL of DMF, and 0.2 mmol of an APN recognition group was added. The reaction system was then allowed to react overnight at room temperature in the dark. TLC was used for monitoring. After the starting material had substantially disappeared, 500 mL of n-hexane was added to the reaction system for precipitation. The crude product was separated and purified by preparative high-performance liquid chromatography (HPLC) to obtain the near-infrared region II activatable probe CR13-APN.

[0172]

[0173] The NIR II semicyanine platform molecule CR13-APN obtained in this example has a hydrogen NMR spectrum as shown in FIG. Figure 14 As shown, the NMR carbon spectrum of CR13-Cys is as follows Figure 15 shown.

[0174] In this embodiment, another response structure is introduced on the basis of the CR13-Cys response probe to construct a corresponding activatable probe. This strategy is mainly used when some response groups cannot directly react with the hydroxyl or amino groups on the platform molecule and it is necessary to eliminate the structural transition.

[0175] Example 16

[0176] In order to increase the water solubility and functionality of the prepared probe, CR13-APN (0.1 mmol), azidated β-cyclodextrin (CD-N3, 0.1 mmol), copper sulfate (0.1 mmol) and anti-hematoxylin (0.3 mmol) prepared in Example 15 were added to 10 mL of DMF / water (1:1), and the air in the reaction system was removed by a double-row tube and replaced with high-purity nitrogen. After the reaction was sealed and protected from light at room temperature overnight, it was dialyzed using a dialysis bag with a molecular weight cutoff of 1500 Da. The crude product after dialysis was separated and purified by preparative high performance liquid chromatography (HPLC) to obtain the kidney-clearable near-infrared second-zone activatable probe CR13-APN-CD. The reaction process is as follows: Figure 16 shown.

[0177] In this embodiment, the hydrophilicity of the probe is increased by utilizing the alkyne structure of the CR13-APN molecule and introducing cyclodextrin, thereby giving the probe multifunctionality.

[0178] Example 17

[0179] The inventors of this case also conducted a spectral characterization of the pH responsiveness of the near-infrared second-zone semi-cyanine probe platform obtained in Examples 1-3 above: the near-infrared second-zone semi-cyanine probes CR11-OH, CR12-OH and CR13-OH were dissolved in PBS buffer containing 50% acetonitrile, and the pH of the buffer was adjusted using dilute hydrochloric acid and dilute sodium hydroxide solution. The final probe concentration was 10 μM. After incubation in the corresponding pH buffer at 37°C for 2 hours, the absorption and fluorescence spectra of the samples were tested, as well as the fluorescence imaging results of the probes at different pH values, to evaluate the pH responsiveness of the near-infrared second-zone semi-cyanine platform molecules. The results showed that Figure 17a-Figure 17c The results of absorption, fluorescence spectrum and fluorescence intensity change with pH value of the semi-cyanine platform molecule CR11-OH at different pH values ​​are shown respectively; Figure 17d-17f The results of absorption, fluorescence spectrum and fluorescence intensity change with pH value of the semi-cyanine platform molecule CR12-OH at different pH values ​​are shown respectively; Figure 17g-Figure 17i The results of absorption, fluorescence spectrum and fluorescence intensity change with pH value of the semi-cyanine platform molecule CR13-OH at different pH values ​​are shown respectively; Figure 18a and Figure 18b The fluorescence imaging results of three platform probe molecules at different pH values ​​are shown.

[0180] Example 18

[0181] The inventors of this case also tested the responsiveness and sensitivity of the activatable fluorescent probes obtained in Examples 9-11 above: the three activatable probes CR11-Cys, CR12-Cys, and CR13-Cys were dissolved in PBS buffer containing 50% acetonitrile, with the final probe concentration being 10 μM. Then, different concentrations of Cys were added to the solutions and incubated for 30 minutes. The absorption, fluorescence spectrum, and fluorescence imaging data of the probes were tested respectively. The results showed that Figure 19a-Figure 19c The absorption spectrum, fluorescence spectrum and fluorescence imaging of CR11-Cys are shown as they change with Cys concentration; Figure 19d-19f The absorption spectrum, fluorescence spectrum and fluorescence imaging of CR12-Cys are shown as they change with Cys concentration; Figure 19g-Figure 19i The absorption spectrum, fluorescence spectrum and fluorescence imaging of CR13-Cys are shown as they change with the Cys concentration.

[0182] Example 19

[0183] The inventors of this case also tested the response speed of the activatable fluorescent probes obtained in Examples 9-11 above to analytes: the three activatable probes CR11-Cys, CR12-Cys, and CR13-Cys were dissolved in PBS buffer containing 50% acetonitrile, with the final probe concentration being 10 μM. The absorption and fluorescence spectra of the probes were then tested over time after the addition of 100 μM Cys, as well as fluorescence imaging of the samples before and after the response. The results showed that Figure 20a and Figure 20b The absorption spectrum and fluorescence spectrum of CR11-Cys change with time, respectively. The inset shows the fluorescence imaging results of CR11-Cys before and after the response. Figure 20c and Figure 20d The absorption spectrum and fluorescence spectrum of CR12-Cys change with time, respectively. The inset shows the fluorescence imaging results of CR12-Cys before and after the response. Figure 20e and Figure 20f The absorption spectrum and fluorescence spectrum of CR13-Cys change with time, respectively. The inset shows the fluorescence imaging results of CR13-Cys before and after response.

[0184] Example 20

[0185] The inventors of this case also tested the specific response of the activatable fluorescent probes obtained in Examples 9-11 above to analytes: the three activatable probes CR11-Cys, CR12-Cys, and CR13-Cys were dissolved in PBS buffer containing 50% acetonitrile, with a final probe concentration of 10 μM. Different analytes were then added and incubated at 37°C for 30 minutes. The absorption and fluorescence spectra of the probes were then measured. The results showed that Figure 21a and Figure 21b The absorption and fluorescence spectra of CR11-Cys under the action of different analytes are shown respectively; the results show that Figure 21c and Figure 21d The absorption and fluorescence spectra of CR11-Cys under the action of different analytes are shown respectively; the results show that Figure 21e and Figure 21f The absorption and fluorescence spectra of CR11-Cys under the action of different analytes are shown respectively.

[0186] Example 21

[0187] The inventors of this case also tested the responsiveness of the activatable fluorescent probe CR13-Cys obtained in Example 11 above at the cellular level: MCF-7 cells were subjected to blank treatment, MCF-7 cells + CR13-Cys incubation for 30 minutes, MCF-7 cells + NEM thiol blocker pretreatment for 30 minutes + CR13-Cys incubation for 30 minutes, MCF-7 cells + Cys pretreatment for 30 minutes + CR13-Cys incubation for 30 minutes, MCF-7 cells + NEM thiol blocker pretreatment for 30 minutes + Cys pretreatment for 30 minutes + CR13-Cys incubation for 30 minutes, and then fluorescence imaging of each group of cells was performed. The results showed that Figure 22 Shows the fluorescence imaging results of cells after different treatments.

[0188] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0189] Although the present application has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions, and / or additions may be made and that substantial equivalents may be substituted for elements of the described embodiments without departing from the spirit and scope of the present application. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present application without departing from the scope of the present application. Therefore, it is not intended that the present application be limited to the particular embodiments disclosed for carrying out the present application, but rather that the present application will encompass all embodiments falling within the scope of the appended claims.

Claims

1. A type of near-infrared region II activatable probe, characterized in that: The invention comprises a near-infrared second-zone fluorescent light-emitting unit and an analyte-specific response unit connected to each other; the near-infrared second-zone fluorescent light-emitting unit comprises a semi-cyanine fluorophore structure; the analyte-specific response unit quenches the fluorescence of the near-infrared second-zone fluorescent light-emitting unit when chemically coupled with the near-infrared second-zone fluorescent light-emitting unit; and the analyte-specific response unit can activate the fluorescence of the analyte-specific response unit after a specific reaction with the analyte.

2. The near-infrared second region activatable probe according to claim 1, characterized in that: Its general structural formula is shown in formula (I): Wherein, X- includes any one of ClO4, BF4, CF3COO, CH3COO, F, Cl, Br, I, and NO3; Z includes any one of O, S, and NH; and n is 0 or 1; R1 includes -H, Any of the following; R2 includes any one of -H, -F, -Cl, -Br, and -I; R3 is the analyte-specific recognition unit; R4 includes Any one of, wherein m is any integer from 0 to 17; Preferably, R3 includes Any of the following; And / or, the fluorescence brightness of the near-infrared second region activatable probe is 100 to 8000M -1 cm -1 , the switching ratio is greater than 10.

3. A method for preparing a near-infrared second-region hemicyanine platform molecular probe, characterized in that: include: A first condensation reaction is performed on a first compound containing an indole salt and pentadienaldehyde diphenylamine hydrochloride to obtain an intermediate; The intermediate is further subjected to a second condensation reaction with a second compound containing an oxonium ion to prepare a near-infrared second-region hemicyanine platform molecular probe; Wherein, the first compound comprising an indole salt has a structure as shown in formula (II): X - Including ClO4 - 、BF4 - CF3COO - 、CH3COO - 、F - 、Cl - Br - , I - 、NO3 - Any of the following; R4 includes Any of the following; The second compound containing an oxonium ion has a structure as shown in formula (III): Z includes any one of O, S, and NH; n is 0 or 1; R1 includes -H, Any of the following; R2 includes any one of -H, -F, -Cl, -Br, and -I.

4. The preparation method according to claim 3, characterized in that include: Mixing a first compound containing an indole salt, pentadienaldehyde diphenylamine hydrochloride, and a first reaction solvent, and performing a first condensation reaction under a protective atmosphere to obtain an intermediate; Preferably, the first reaction solvent comprises at least any one of acetic anhydride, toluene / n-butanol, benzene / n-butanol, ethanol, and methanol, preferably acetic anhydride; and / or, the molar ratio of the first compound comprising an indole salt to pentadienaldehyde diphenylamine hydrochloride is 1:(0.1-2); and / or, the temperature of the first condensation reaction is 50 to 150° C., preferably 90 to 110° C.; And / or, the first condensation reaction time is 0.5 to 12 hours, preferably 2 to 3 hours.

5. The preparation method according to claim 3, characterized in that include: The intermediate, a second compound containing an oxonium ion, a base, and a second reaction solvent are mixed, and a second condensation reaction is carried out in a protective atmosphere to prepare a near-infrared second-region hemicyanine platform molecular probe; Preferably, the base includes at least any one of sodium acetate, potassium acetate, potassium carbonate, sodium carbonate, and triethylamine, preferably sodium acetate or potassium acetate; Preferably, the second reaction solvent comprises at least any one of acetic anhydride, toluene / n-butanol, and benzene / n-butanol, preferably acetic anhydride; and / or, the molar ratio of the intermediate to the second compound comprising an oxonium ion is 1:(0.5-10); and / or, the molar ratio of the intermediate to the base is 1:(1-30); and / or, the temperature of the second condensation reaction is 0 to 150° C., preferably 20 to 60° C.; And / or, the second condensation reaction time is 0.5 to 24 hours, preferably 6 to 12 hours.

6. A near-infrared second-region hemicyanine platform molecular probe prepared by the preparation method according to any one of claims 3 to 5.

7. A method for preparing a near-infrared second region activatable probe, characterized in that: include: Prepare the near-infrared second-region hemicyanine platform molecular probe according to the preparation method according to any one of claims 3 to 5; The near-infrared zone II hemicyanine platform molecular probe is chemically coupled with a substance having an analyte-specific recognition unit to prepare a near-infrared zone II activatable probe.

8. The preparation method according to claim 7, characterized in that: The substance having the analyte-specific recognition unit includes Any of the following; and / or, the molar ratio of the near-infrared second-region hemicyanine platform molecular probe to the substance having the analyte-specific recognition unit is 1:1 to 5; And / or, the preparation method further comprises: chemically coupling the near-infrared region II activatable probe with any one functional unit selected from the group consisting of amino, carboxyl, alkynyl, azide, and maleimide.

9. A near-infrared second region activatable probe prepared by the preparation method according to claim 7 or 8.

10. Use of the near-infrared second region activatable probe according to any one of claims 1 to 2 and 9 in the field of biological imaging detection or drug screening.

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