Tetrazine probe synthon as well as preparation method and application thereof

Abstract

The invention discloses a tetrazine probe synthon and a preparation method and application thereof.The preparation method comprises the following steps that under the inert atmosphere, diethyl cyanomethylphosphonate, 3-hydroxypropionitrile, 3-mercaptopropionic acid and hydrazine hydrate are mixed and then react at the room temperature, a sodium nitrite solution is added after the reaction is finished, then acid is continuously added till the reaction system reaches acidity, and a tetrazine probe synthon product is obtained; continuously reacting to obtain an intermediate; and dissolving the intermediate in a solvent in an inert atmosphere, adding triethylamine, slowly adding methanesulfonyl chloride, and reacting at room temperature to obtain the tetrazine probe synthon shown in the formula I. The invention further discloses a preparation method of the tetrazine probe synthon. The tetrazine probe synthon provided by the invention can perform series Heck reaction and HWE reaction, so that controllable combination of functional groups on two sides of a tetrazine coupling probe is realized under mild reaction conditions, and the functional groups on two sides are designed according to actual requirements to regulate and control photophysical properties of the probe. A brand new way is provided for developing a high-performance biological orthogonal fluorescent probe.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tetrazine compound preparation, and particularly relates to a tetrazine probe synthon, a preparation method thereof, and application of the tetrazine probe synthon in preparation of a variety of bifunctional tetrazine coupling probes. BACKGROUND

[0002] Biofluorescence imaging can reveal life processes in real time, and has important significance for analyzing the molecular mechanism of life activities, disease diagnosis and drug development, etc. An excellent performance fluorescent probe can realize high-resolution and high-contrast imaging, and one of the core challenges in its synthesis is to reasonably construct an efficient pi conjugated structure to realize efficient energy transfer and signal output. At present, tetrazine compounds based on inverse electron demand Diels-Alder (IEDDA) reaction can realize the conversion of fluorescent signal from "dark" to "bright" due to the rapid and high-selectivity combination between the tetrazine compounds and alkenes, and have become an ideal module for constructing an efficient conjugated system.

[0003] In recent years, researchers have designed various strategies to optimize tetrazine fluorescent probes, thereby significantly improving their optical performance and application potential. In the prior art, the Devaraj team synthesized an alkenyl-linked BODIPY-tetrazine probe through Heck reaction, realized efficient energy transfer and hundreds of times fluorescence enhancement, and the Park team used Suzuki and C-N coupling reaction to modify the BODIPY probe in the middle position, and obtained a probe with a fluorescence opening ratio of more than 2000 times and a quantum yield of 0.32; the Tang team further combined an aggregation-induced emission (AIE) unit to develop a tetrazine-TPE probe with AIE characteristics.

[0004] The inventor's team has previously reported that the tetrazine unit is efficiently grafted on various fluorophore skeletons through palladium-catalyzed Heck reaction, thereby obtaining a series of pi-conjugated alkenyl tetrazine probes. In addition, the inventor's team has also developed a modular synthesis strategy based on Horner-Wadsworth-Emmons (HWE) reaction, which uses a tetrazine precursor containing a phosphate functional group to react with various aldehyde derivatives to directionally construct a Huaxi-Fluor series of fluorescent probes with push-pull electron structure. These probes not only realize thousands of times fluorescence enhancement, but also exhibit excellent performance in wash-free organelle labeling and 500 μm deep tumor imaging.

[0005] However, due to the low electron density of the tetrazine skeleton and the multi-site reactivity, it is extremely challenging to simultaneously introduce different functional groups at both ends of the tetrazine skeleton to achieve precise and orthogonal bifunctionalization. Therefore, the existing tetrazine probe construction strategies are limited to single-sided functionalization, and there is no effective strategy for differential and pi-conjugated bifunctional modification of both sides of the tetrazine skeleton, making it difficult for existing strategies to synthesize flexible and functional asymmetric tetrazine probes. SUMMARY

[0006] One object of the present application is to provide a tetrazine probe synthon having a methanesulfonate group and a diethyl phosphate group connected to the 3 and 6 positions of the 1,2,4,5-tetrazine, respectively, so as to utilize the tandem reaction of Heck reaction and HWE reaction to prepare a series of tetrazine coupling probes with differential functional groups on both sides, thereby facilitating the diversification and functionalization of tetrazine coupling probes.

[0007] The present application is achieved by the following technical solutions:

[0008] A tetrazine probe synthon has the structure shown in Formula I:

[0009] Formula I: .

[0010] In the present technical solution, the tetrazine skeleton of the tetrazine probe synthon is connected to a methanesulfonate group on one side, which can undergo Heck reaction with a variety of halogenated compounds containing electron-rich or electron-deficient substituents and different fluorophores, thereby connecting the desired first functional group on one side of the tetrazine skeleton. At the same time, the other side of the tetrazine skeleton is also connected to a diethyl phosphate group, which can undergo HWE reaction with a variety of aldehyde compounds, thereby connecting the desired second functional group on the other side of the tetrazine skeleton, and finally obtaining a tetrazine coupling probe. In one or more embodiments, the second functional group can be the same as or different from the first functional group. In the present technical solution, the tandem Heck reaction and HWE reaction based on the tetrazine probe synthon can achieve controlled combination of functional groups on both sides of the tetrazine skeleton under mild reaction conditions, and the functional groups on both sides can be designed according to actual needs to regulate the photophysical properties of the tetrazine coupling probe, thereby providing a new approach for developing high-performance bioorthogonal fluorescent probes.

[0011] In the technical solution, the tetrazine probe synthon can be subjected to the Heck reaction first and then the HWE reaction, or subjected to the HWE reaction first and then the Heck reaction when used for preparing the tetrazine conjugate probe. In the process, the methanesulfonate groups and diethyl phosphate groups on both sides of the tetrazine probe synthon are almost not affected. For example, the diethyl phosphate groups are not affected by the Heck reaction conditions and reagents when the methanesulfonate groups are subjected to the Heck reaction to prepare a mono-conjugated tetrazine phosphate; similarly, the methanesulfonate groups are not affected by the HWE reaction conditions and reagents when the diethyl phosphate groups are subjected to the HWE reaction to prepare a mono-conjugated tetrazine methanesulfonate. This significantly improves the flexibility of research and design of the tetrazine conjugate probe, and avoids the failure of probe synthesis due to the instability of the tetrazine itself when different functional groups are connected on both sides of the tetrazine skeleton. Moreover, the tetrazine synthon has excellent stability, and it is found through experiments that the tetrazine synthon can be stored at -4 °C for several months.

[0012] Another object of the present application is to provide a preparation method of the aforementioned tetrazine probe synthon, specifically comprising the following steps:

[0013] A preparation method of a tetrazine probe synthon, comprising the following steps:

[0014] In an inert atmosphere, the cyano methyl diethyl phosphate, 3-hydroxy propionitrile, 3-mercapto propionic acid and hydrazine hydrate are mixed and reacted at room temperature, sodium nitrite solution is added after the reaction is completed, and then acid is continuously added until the reaction system reaches acidity, and the reaction is continued to obtain an intermediate;

[0015] In an inert atmosphere, the intermediate is dissolved in a solvent, triethylamine is added, and then methanesulfonyl chloride is slowly added, and the reaction is carried out at room temperature to obtain the tetrazine probe synthon shown as formula I.

[0016] In some preferred embodiments, the mixture obtained by mixing cyano methyl diethyl phosphate, 3-hydroxy propionitrile, 3-mercapto propionic acid and hydrazine hydrate is stirred at room temperature for 12-18 hours, after the reaction is completed, the reaction solution is cooled with ice water, sodium nitrite solution is added, and then acid such as hydrochloric acid is continuously added under ice bath conditions until the reaction solution is acidic, and the stirring reaction is continued until the reaction is completed to obtain the intermediate.

[0017] In some preferred embodiments, the intermediate is dissolved in a solvent such as anhydrous dichloromethane, triethylamine is added, and then methanesulfonyl chloride is slowly added at low temperature, and the reaction solution is reacted at room temperature for 1-4 hours, and then quenched with water to prepare the tetrazine probe synthon.

[0018] Another object of the present application is to provide the use of the aforementioned tetrazine probe synthon in the preparation of a tetrazine conjugate probe. Specifically, the tetrazine probe synthon is used to prepare a tetrazine conjugate probe shown as formula II:

[0019] Formula II:

[0020] In Formula II, the group R 1 is selected from substituted or unsubstituted C6-C 12 aryl, C4-C 12 heteroaryl, a fluorescent reporter group, or a polyarylene ethylene polymer; the group R 2 is selected from C1-C8 linear alkyl, C2-C4 alkenyl, C4-C 12 heteroaryl, a fluorescent reporter group, a triphenylamine compound, an amino acid derivative, or a conjugated heteroaryl.

[0021] Further, the group R 1 is selected from substituted or unsubstituted C6-C 12 aryl, C4-C 12 heteroaryl, a coumarin compound, a BODIPY compound, a triphenyl ethylene, a tetraphenyl ethylene, or a pentaphenyl ethylene; the group R 2 is selected from substituted or unsubstituted C1-C8 linear alkyl, C2-C4 alkenyl, C4-C 12 heteroaryl, a BODIPY compound, a coumarin compound, or a bis(4-methoxyphenyl)amino, N-tert-butoxycarbonyl-2-aminopropionic acid methyl ester group, a 4-(N,N-dimethylamino)styrylthiophenyl group.

[0022] Further, the substituent is a C1-C8 linear alkyl, a halogen, a trifluoromethyl, a methoxy, an ethoxy, a dimethylamino, a hydroxyl, or a cyano.

[0023] Further, the preparation method of the tetrazine conjugated probe based on the tetrazine probe synthon comprises the following steps:

[0024] The tetrazine probe synthon is first subjected to a Heck reaction with a halogenated compound to prepare a first intermediate, and the first intermediate is then subjected to a HWE reaction with an aldehyde compound to prepare the tetrazine conjugated probe; or

[0025] The tetrazine probe synthon is first subjected to a HWE reaction with an aldehyde compound to prepare a second intermediate, and the second intermediate is then subjected to a Heck reaction with a halogenated compound to prepare the tetrazine conjugated probe.

[0026] Still another object of the present application is to provide a tetrazine conjugated probe prepared based on the aforementioned tetrazine probe synthon as a reactant, after a Heck reaction with a halogenated compound and a HWE reaction with an aldehyde compound, the tetrazine conjugated probe has a structure shown in Formula II or Formula III:

[0027] Formula II: ;

[0028] Formula III: ;

[0029] Formula II, Formula III, the group R 1 is selected from substituted or unsubstituted C6-C 12 aryl, C4-C 12 heteroaryl, fluorescent reporter group, or polyarylethene polymer; the group R 2 is selected from C1-C8 linear alkyl, C2-C4 alkenyl, C4-C 12 heteroaryl, fluorescent reporter group, triphenylamine compound, amino acid derivative, or conjugated heteroaryl.

[0030] Further, the group R 1 is selected from substituted or unsubstituted C6-C 12 aryl, C4-C 12 heteroaryl, coumarin compound, BODIPY compound, triphenyl ethylene, tetraphenyl ethylene, or pentaphenyl ethylene; the group R 2 is selected from substituted or unsubstituted C1-C8 linear alkyl, C2-C4 alkenyl, C4-C 12 heteroaryl, BODIPY compound, coumarin compound, or bis(4-methoxyphenyl)amino, N-tert-butoxycarbonyl-2-aminopropionic acid methyl ester, 4-(N,N-dimethylamino)styrylthiophenyl.

[0031] Further, the group R 1 is selected from substituted or unsubstituted phenyl, naphthyl, biphenyl, styryl, thienyl, imidazolyl, indolyl, quinolyl, benzothienyl, benzofuranyl, BODIPY, coumarin, or tetraphenyl ethylene; the group R 2 is selected from substituted or unsubstituted methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, vinyl, propenyl, allyl, thienyl, pyrrolyl, pyridyl, indolyl, indazolyl, BODIPY, coumarin, or bis(4-methoxyphenyl)amino, N-tert-butoxycarbonyl-2-aminopropionic acid methyl ester, 4-(N,N-dimethylamino)styrylthiophenyl.

[0032] wherein the substituents are selected from any one of methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, halogen, trifluoromethyl, methoxy, ethoxy, dimethylamino, hydroxyl, cyano.

[0033] Further, the tetrazine conjugation probe has any one of the following structures:

[0034] , 、 、 、 、 、 、 、 、 、 .

[0035] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0036] 1. The tetrazine probe synthon provided by the present application can perform tandem Heck reaction and HWE reaction, thereby realizing controllable combination of functional groups on both sides of the tetrazine coupling probe under mild reaction conditions, designing the functional groups on both sides according to actual requirements to regulate the photophysical properties of the probe, and providing a brand-new way for developing high-performance biological orthogonal fluorescent probes;

[0037] 2. The tetrazine coupling probe synthesis strategy based on the tetrazine probe synthon of the present application can not only significantly improve the flexibility of research and design of the tetrazine coupling probe, but also effectively avoid the failure of probe synthesis due to instability of the tetrazine itself when different functional groups are connected on both sides of the tetrazine skeleton;

[0038] 3. The tetrazine probe synthon of the present application has excellent stability and can be stored at low temperature for several months;

[0039] 4. In the present application, the various tetrazine coupling probes prepared based on the tetrazine probe synthon have excellent photophysical properties and stability, are compatible with aqueous reaction environment, can expand the excitation wavelength to the near-infrared region while maintaining large stokes shift and high signal-to-noise ratio, and provide a new technical way for developing fluorescent imaging suitable for deep tissue and living body environment. BRIEF DESCRIPTION OF DRAWINGS

[0040] The accompanying drawings, which are included to provide a further understanding of the embodiments of the present application and constitute a part of the application, illustrate embodiments of the present application and do not limit the present application. In the drawings:

[0041] Figure 1 a flowchart of the preparation method of the tetrazine probe synthon in the specific embodiments of the present application;

[0042] Fig. 2 shows the nuclear magnetic resonance spectra of the tetrazine probe synthon prepared in the specific embodiments of the present application, which is newly prepared (A) and stored at-4°C for 2 months (B) after preparation;

[0043] Figure 3The following are shown in a specific embodiment of the present invention: (A) absorption spectrum in an aqueous phase of the precursor TPA-CHO, mono-coupled tetrazine phosphate 12, tetrazine coupling probe 20, and the tetrazine coupling probe 20 after being coupled with BCN; (B) emission spectrum in an aqueous phase.

[0044] Figure 4 The fluorescence of MeTz-BODI-BCN,23-BCN under ultraviolet light excitation is shown in a specific embodiment of the present invention. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0046] All raw materials used in this invention are not particularly limited in their source; they can be purchased commercially or prepared using conventional methods well-known to those skilled in the art. The purity of all raw materials used in this invention is not particularly limited; however, analytical grade or the purity requirements conventional in the field of fluorescent probes are preferred. All raw materials used in this invention have brand names and abbreviations that are conventional in the art, and each brand name and abbreviation is clearly defined within its relevant application. Those skilled in the art can obtain them commercially available or prepare them using conventional methods based on the brand name, abbreviation, and corresponding application.

[0047] The present invention does not impose any particular restrictions on the expression of the substituents, and all expressions are well known to those skilled in the art. Based on common sense, those skilled in the art can correctly understand their meaning according to their expression.

[0048] The terms "first," "second," etc., used in this document (e.g., first intermediate, second intermediate; first functional group, second functional group, etc.) are used merely for clarity of description to distinguish the corresponding components and are not intended to restrict any order or emphasize importance. Furthermore, the term "linked" as used herein, unless otherwise specified, can refer to a direct link or an indirect link via other groups.

[0049] I. Preparation of tetrazine probe synthon

[0050]

Example 1

[0051] In this embodiment, a tetrazine probe synthon was synthesized, and its synthetic route is as follows:

[0052]

[0053]

[0054] Under argon protection, diethyl cyanomethyl phosphate A (1 eq.), 3-hydroxypropionitrile B (2.5 eq.), 3-mercaptopropionic acid (1 eq.), and hydrazine hydrate (15 eq.) were added sequentially to a round-bottom flask to obtain a mixture. The mixture was stirred at room temperature for 15 hours. After the reaction was complete, the reaction solution was cooled with ice water, and sodium nitrite (150 mmol) dissolved in ice water was slowly added. Then, 1 M hydrochloric acid solution was slowly added dropwise under ice-water bath conditions, during which the solution turned bright red and gas was released. The addition of 1 M hydrochloric acid continued until the gas emission stopped and the pH reached acidity. The resulting mixture was stirred and extracted three times with dichloromethane. The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain intermediate C.

[0055] Under argon protection, intermediate C (1 eq.) was dissolved in 20 mL of anhydrous dichloromethane, followed by the addition of triethylamine (1 eq.), and then methanesulfonyl chloride (1 eq.) was slowly added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 2 hours, quenched with water, and extracted six times with dichloromethane. The resulting mixture was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give tetrazine probe synthon 1 in 45% yield.

[0056] The proton NMR spectrum of the tetrazine probe synthon 1 is as follows:

[0057] 1 H NMR (400 MHz, CDCl3) δ 4.88(t, J = 6.2 Hz, 2H), 4.26 – 4.15 (m,4H), 3.95 (d, J = 22.2 Hz, 2H), 3.80 (td, J = 6.2, 1.6 Hz, 2H), 3.02 (s, 3H),1.34 (t, J = 7.0Hz, 6H).

[0058] By comparing the NMR spectra of the newly prepared sample and the sample stored for two months, it was found, as shown in Figure 2, that the tetrazine probe synthon 1 has good stability and can be stored at -4℃ for 2 months.

[0059] II. Preparation of tetrazine conjugated probes

[0060] Examples 2 through 15

[0061] In this embodiment, the tetrazine probe synthon from Example 1 is first reacted with a halogenated compound via a Heck reaction to obtain the corresponding mono-coupled tetrazine phosphate. The synthetic route is as follows:

[0062]

[0063] Specifically, the test method is as follows:

[0064] To a 10 mL microwave reaction tube equipped with a stir bar, add catalyst [Pd2(dba)3] (3 mol%), ligand 1,2,3,4,5-pentanylphenyl-1′-(di-tert-butylphospho)ferrocene (QPhos) (12 mol%), tetrazine probe synthon 1 (1 eq., 7.0 mg, 0.02 mmol), bromide (1.5 eq., 0.03 mmol), and dimethyldicyclohexylamine Cy2NMe (3.0 eq.), and dissolve in 1.5 mL of anhydrous N,N-dimethylformamide (DMF).

[0065] In one or more embodiments, it may also be based on group R 1 Depending on the specific bromine derivative, the corresponding iodide derivative is used instead of the bromine derivative.

[0066] The reaction system was protected under nitrogen gas and then heated by microwave irradiation (50℃, 45 min). After the reaction solution cooled to room temperature, 20 mL of ethyl acetate (EtOAc) was added, followed by washing three times with 20 mL of water. The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by thin-layer chromatography (Agela Technologies, silica gel 200×200 mm, pH=5, fluorescence wavelength 254 nm, glass backing plate; developing solvent: dichloromethane (DCM): ethyl acetate = 4:1) to obtain mono-coupling tetrazine phosphate.

[0067] In some embodiments, depending on the halogenated compound, the group R 1 C6-C, whether substituted or not 12 Aryl, C4-C 12 Heteroaryl groups, fluorescent reporter groups, or polyaryl vinyl polymers.

[0068] In some preferred embodiments, group R 1 It can be a substituted or unsubstituted aryl group, which can be a monocyclic aryl group or a fused-ring aryl group. The preferred number of carbon atoms in the aryl group is C6-C. 12 In one or more embodiments, the aryl group may be phenyl, naphthyl, biphenyl, styryl, etc.

[0069] In some preferred embodiments, group R 1 It can be a substituted or unsubstituted heteroaryl group, preferably with C4-C carbon atoms. 12 In one or more embodiments, the heteroaryl group may be thienyl, imidazolyl, indolyl, quinolinyl, benzothienyl, benzofuranyl, etc.

[0070] In some embodiments, the aryl or heteroaryl substitution can be monosubstituted or polysubstituted. In one or more embodiments, the substituent can be a chain alkyl group, preferably with C1-C8 carbon atoms, more preferably C1-C4. In one or more embodiments, the chain alkyl group is methyl, ethyl, propyl, isopropyl, n-butyl, or tert-butyl. In some preferred embodiments, the substituent can also be selected from trifluoromethyl, methoxy, ethoxy, dimethylamino, hydroxy, or cyano.

[0071] In some preferred embodiments, group R 1 It can be a fluorescent reporter group, which is the core unit in the probe responsible for generating a fluorescent signal. In one or more embodiments, the fluorescent reporter group can be a coumarin compound, a fluorescein compound, a rhodamine compound, a BODIPY compound, or anthocyanin compound.

[0072] In some preferred embodiments, group R 1 It can also be a polyaryl vinyl polymer, which is a molecular segment that can undergo significant enhancement after aggregation. In one or more embodiments, the polyaryl vinyl polymer can be triphenylethylene, tetraphenylethylene (TPE), pentaphenylethylene, etc.

[0073] In some embodiments, the amount of [Pd2(dba)3] is preferably 3-12 mol%; the amount of QPhos is preferably 12-48 mol%, wherein the chemical formula of QPhos is […]. The preferred amount of Cy2NMe is 3.0 to 4.0 equivalents; the preferred reaction temperature is 50 to 60°C.

[0074] Table 1 shows the structural formulas, characterization, and yields of the mono-coupled tetrazine phosphates prepared in Examples 2 through 15.

[0075] Table 1:

[0076]

[0077] Examples 16 to 26

[0078] Next, based on the mono-coupled tetrazine phosphate, HWE reactions were carried out with various aldehyde compounds to prepare the corresponding tetrazine-coupled probes. The synthetic route is as follows:

[0079]

[0080] Specifically, the tetrazine-coupled probe is prepared by mixing mono-coupled tetrazine phosphate, an aldehyde compound, a base, and a solvent, and then performing an HWE reaction at low temperature.

[0081] In some embodiments, depending on the selected aldehyde compound, the group R 2 It can be C1-C8 chain alkyl, C2-C4 alkenyl, C4-C 12 Heteroaryl groups, fluorescent reporter groups, triphenylamine compounds, amino acid derivatives, or conjugated heteroaryl groups.

[0082] In some embodiments, the aldehyde compound may be 2,5-thiophenedicarboxaldehyde, thereby forming a tetrazine coupling probe of formula III with respect to thiophene symmetry.

[0083] In this embodiment, group R 2 It can be a C1-C8 chain alkyl group, preferably a C1-C4 chain alkyl group, such as methyl, ethyl, propyl, isopropyl, n-butyl, or tert-butyl. Group R 2 It can be C2-C4 alkenyl, for example, vinyl, propenyl, allyl. The group R... 2 It can be C4-C 12 Heteroaryl groups, such as thiophene, pyrrolyl, pyridyl, indole, and indazole. Group R 2 It can be a fluorescent reporter group, such as in BODIPY compounds or coumarin compounds. Group R 2 It can be a triphenylamine compound, such as bis(4-methoxyphenyl)amino. The R group... 2 It can be an amino acid derivative, such as N-tert-butoxycarbonyl-2-aminopropionic acid methyl ester. The group R... 2 It can also be a conjugated heteroaryl group, such as 4-(N,N-dimethylamino)styrylthiophene.

[0084] Therefore, the method of synthesizing tetrazine-coupled probes based on tetrazine probe synthon can undergo HWE reaction with aliphatic aldehydes that are incompatible with the Heck reaction to obtain the group R. 2 As a chain-like alkyl tetrazine coupling probe, it effectively broadens the selection range of functional groups on both sides of the tetrazine coupling probe, allowing researchers to explore more structures of tetrazine coupling probes.

[0085] In some embodiments, the amount of mono-coupled tetrazine phosphate is 1 equivalent, and the amount of aldehyde compound is 1.1 to 1.4 equivalents, preferably 1.2 equivalents. In one or more embodiments, the base may be lithium hexafluoroisopropoxide (LiHFI) or lithium diisopropylamino (LDA). In one or more embodiments, the reaction temperature is -15 to -25°C; in one or more embodiments, the reaction time is 3 to 12 hours. In one or more embodiments, the solvent is preferably tetrahydrofuran (THF).

[0086] In this embodiment, based on the mono-coupled tetrazine phosphates prepared in Examples 2, 6, 9, 11-15, tetrazine-coupled probes were prepared by reacting them with aldehyde compounds to obtain the tetrazine-coupled probes of [Example 16] to [Example 26]. Table 2 shows the structural formula, characterization and yield of each tetrazine-coupled probe.

[0087] Table 2:

[0088]

[0089] As shown in Table 2, tetrazine-coupled probes constructed based on tetrazine probe synthon can achieve precise functionalization on both sides of the tetrazine structure, thus having broad application value in various application scenarios. For example, tetrazine-coupled probe 21 is expected to become a novel bioorthogonal probe; tetrazine-coupled probe 22 containing two asymmetric fluorescent reporter groups may become a novel FRET probe; tetrazine-coupled probe 23, which simultaneously introduces a fluorescent reporter group and a photodynamic group, may serve as a novel therapeutic probe; and tetrazine-coupled probe 25, which combines the Huaxi fluorescent framework previously developed by the inventors' team with the classic AIE unit TPE, is likely to exhibit new optical properties. Furthermore, by using dialdehydethiophene, a previously difficult-to-obtain symmetrical AIE bis-tetrazine molecule, namely tetrazine-coupled probe 26, can be synthesized efficiently with a yield of 58%. It possesses an electron donor-acceptor unit and may exhibit new properties in the field of tetrazine optoelectronic materials, such as enrichment and refinement.

[0090]

Example 27

[0091] In the method for preparing tetrazine-coupled probes based on tetrazine probe synthon, one can either, as in Examples 2 to 26, first perform a Heck reaction with a halogenated compound to prepare a mono-coupled tetrazine phosphate, and then perform an HWE reaction of the mono-coupled tetrazine phosphate with an aldehyde compound; or one can first perform an HWE reaction with an aldehyde compound, and then perform a Heck reaction with a halogenated compound to prepare a tetrazine-coupled probe.

[0092] In this embodiment, the preparation method is basically the same as in Example 16, except that the tetrazine probe synthon first undergoes an HWE reaction with an aliphatic aldehyde, and then a Heck reaction with a halobenzene. Ultimately, the tetrazine coupling probe 16 can still be obtained in 73% yield, indicating that the order of introduction of the functional groups on both sides of the tetrazine coupling probe is reversible. Those skilled in the art will obviously be able to use a Heck-HWE tandem reaction or an HWE-Heck tandem reaction according to actual needs. Such a method of preparing tetrazine coupling probes by changing the order of the Heck and HWE reactions should also be included within the scope of protection of this invention.

[0093] III. Performance testing of tetrazine conjugated probes

[0094]

Example 28

[0095] In this embodiment, the photophysical properties of the reaction product of tetrazine-coupled probe 20 and bicyclic [6,1,0]nonyne (BCN) were evaluated.

[0096] The experimental method was as follows: a tetrazine-coupled probe (0.2 mM) was reacted with BCN (4 mM), and the reaction was monitored by LC-MS. The product was stored in DMSO as a stock solution. For UV-Vis spectrophotometric measurements, the probe solution was prepared by directly diluting the stock solution to a final concentration of 10 μM with double-distilled water or ethanol. For fluorescence measurements, the stock solution was diluted to a final concentration of 10 μM with water or ethanol. The background fluorescence signal of the solvent was analyzed under the same conditions and subtracted from the fluorescence signal. All optical samples were at a concentration of 10 μM, the solvent was ethanol, and the samples were excited at 361 nm.

[0097] The test results are as follows Figure 3 As shown, compared to the precursor TPA-CHO and mono-coupled tetrazine phosphate 12, the absorption peak of tetrazine-coupled probe 20 in the aqueous phase exhibits a significant red shift, with its absorption peak located at 475 nm. After undergoing the reverse electron-demanding Diels-Alder reaction with BCN, the fluorescence of probe 20-BCN is turned on by 291 times, with a Stokes shift of 141 nm (excitation wavelength 421 nm, emission wavelength 562 nm). This demonstrates that conjugating TPA and auxochrome to both ends of the tetrazine via a double bond using a tetrazine probe synthon can effectively improve the photophysical properties of the probe.

[0098] Furthermore, the fluorescence properties of the reaction products of tetrazine-coupled probes 21 and 25 with BCN were tested using the same method. The results showed that probe 21-BCN exhibited excellent fluorescence properties in aqueous solution, with an excitation wavelength of 418 nm, a Stokes shift of 148 nm, and a fluorescence onset of 430 times. When probe 25-BCN was tested in ethanol, it showed a Stokes shift of 226 nm, an emission wavelength of 677 nm, and a fluorescence onset of 480 times.

[0099]

Example 29

[0100] In this embodiment, the bioorthogonal reaction kinetics of tetrazine-coupled probes 20 and 22 were tested to examine their potential applicability in biomedical applications.

[0101] Specifically, tetrazine-coupled probes 20 and 22 (10 μM) reacted with trans-cyclooctene (TCO, 200 μM) in PBS / DMSO (1:4, pH 7.2) at 37 °C under pseudo-first-order conditions. The second-order kinetic rate was determined to be 751 M. -1 s -1 and 319M -1 s -1 Under the same conditions, the second-order kinetic rate of the tetrazine-coupled probe 22 with BCN was measured to be 24 M. -1 s -1 This indicates that these tetrazine-coupled probes, using double bonds as a bridge, do not significantly affect the orthogonal reactivity of tetrazines after the introduction of relatively bulky fluorescent reporter groups, making them potentially suitable for live-cell imaging.

[0102]

Example 30

[0103] In this embodiment, the stability of tetrazine-coupled probes 20 and 25 was tested. Specifically, the two tetrazine-coupled probes were stored in PBS / DMSO (1:4, pH 7.2) at 37°C for 24 hours. It was observed that 90% of tetrazine-coupled probe 20 and 70% of tetrazine-coupled probe 25 remained, demonstrating the stability of the tetrazine-coupled probes.

[0104]

Example 31

[0105] In this embodiment, the photodynamic therapeutic effects of the reaction products 23-BCN and 24-BCN of tetrazine-coupled probes 23 and 24 with BCN were tested and compared with those of the single-coupled tetrazine probe MeTz-BODI-BCN.

[0106] The experimental method is as follows: the probe and addition product before the reaction are diluted to the same concentration (e.g., 10 μM) using the same solvent to ensure consistency; then, under controlled conditions (excitation wavelength 361 nm), the sample is irradiated and the singlet oxygen emission intensity in the near-infrared region (1270 nm) is measured, while the absorbance of the sample at the excitation wavelength is recorded; finally, by comparing the emission intensity and absorbance of the sample to be tested with a standard (e.g., methylene blue) with a known ΦΔ value, the quantum yield is calculated using the formula ΦΔ_sample = ΦΔ_std × (I_sample / I_std) × (A_std / A_sample).

[0107] The test results are as follows Figure 4 As shown, compared to MeTz-BODI-BCN, 23-BCN exhibits significant green fluorescence under UV excitation. Furthermore, the singlet oxygen quantum yields (ΦΔ) of tetrazine-coupled probes 23 and 24, and MeTz-BODI before and after reaction with BCN were also analyzed. Table 3 shows that the ΦΔ of 23-BCN is 0.649, indicating that introducing iodolated BODIPY as a photodynamic and fluorescent group at both ends of the tetrazine-coupled probe can construct new molecules with excellent photodynamic therapeutic properties. This may be because the different functional groups at both ends of the tetrazine molecule have complementary effects. For example, the BODIPY group on one side of the tetrazine-coupled probe 23 continues to contribute to efficient fluorescence emission, while the iodolated BODIPY introduced on the other side contributes to photodynamic activity.

[0108] Table 3:

[0109]

[0110] Therefore, the strategy of preparing tetrazine-coupled probes based on the tetrazine probe synthon of this application provides a novel approach for designing multifunctional diagnostic and therapeutic probes that integrate targeted activation, fluorescence imaging, and photodynamic therapy.

[0111] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A tetrazine probe synthon, characterized in that, It has the structure shown in Equation I: Formula I: .

2. A method for preparing a tetrazine probe synthon, characterized in that, Includes the following steps: Under an inert atmosphere, diethyl cyanomethyl phosphate, 3-hydroxypropionitrile, 3-mercaptopropionic acid and hydrazine hydrate were mixed and reacted at room temperature. After the reaction was completed, sodium nitrite solution was added, and then acid was continuously added until the reaction system became acidic. The reaction was continued to obtain the intermediate. Under an inert atmosphere, the intermediate was dissolved in a solvent and triethylamine was added, followed by the slow addition of methanesulfonyl chloride. The reaction was carried out at room temperature to obtain the tetrazine probe synthon as shown in Formula I.

3. The application of a tetrazine probe synthon, characterized in that, Using a tetrazine probe synthon as described in claim 1, the tetrazine probe synthon is used to prepare the tetrazine-coupled probe of formula II: Formula II: In Formula II, group R 1 Selected from substituted or unsubstituted C6-C 12 Aryl, C4-C 12 Heteroaryl, fluorescent reporter group, or polyaryl vinyl polymers; group R 2 Selected from C1-C8 chain alkyl, C2-C4 alkenyl, C4-C 12 Heteroaryl groups, fluorescent reporter groups, triphenylamine compounds, amino acid derivatives, or conjugated heteroaryl groups.

4. The application of the tetrazine probe synthon according to claim 3, characterized in that, Group R 1 Selected from substituted or unsubstituted C6-C 12 Aryl, C4-C 12 Heteroaryl compounds, coumarins, BODIPY compounds, triphenylethylene, tetraphenylethylene, or pentaphenylethylene; group R 2 Selected from substituted or unsubstituted C1-C8 chain alkyl, C2-C4 alkenyl, C4-C 12 Heteroaryl, BODIPY compounds, coumarin compounds, or bis(4-methoxyphenyl)amino, N-tert-butoxycarbonyl-2-aminopropionic acid methyl ester, 4-(N,N-dimethylamino)styrylthiophene.

5. The application of a tetrazine probe synthon according to claim 3 or 4, characterized in that, The substituent is a C1-C8 chain alkyl, halogen, trifluoromethyl, methoxy, ethoxy, dimethylamino, hydroxy, or cyano group.

6. The application of a tetrazine probe synthon according to claim 3 or 4, characterized in that, The preparation method of tetrazine-coupled probes based on tetrazine probe synthon includes the following steps: The tetrazine probe synthon is first reacted with a halogenated compound via a Heck reaction to prepare a first intermediate, which is then reacted with an aldehyde compound via a HWE reaction to prepare the tetrazine-coupled probe; or The tetrazine probe synthon is first reacted with an aldehyde compound via a HWE reaction to prepare a second intermediate, which is then reacted with a halogenated compound via a Heck reaction to prepare the tetrazine-coupled probe.

7. A tetrazine-coupled probe, characterized in that, The tetrazine-coupled probe is prepared by reacting the tetrazine probe synthon of claim 1 with a halogenated compound via a Heck reaction and with an aldehyde via an HWE reaction, wherein the tetrazine-coupled probe has the structure shown in Formula II or Formula III: Formula II: ; Formula III: ; In Formulas II and III, group R 1 Selected from substituted or unsubstituted C6-C 12 Aryl, C4-C 12 Heteroaryl, fluorescent reporter group, or polyaryl vinyl polymers; group R 2 Selected from C1-C8 chain alkyl, C2-C4 alkenyl, C4-C 12 Heteroaryl groups, fluorescent reporter groups, triphenylamine compounds, amino acid derivatives, or conjugated heteroaryl groups.

8. The tetrazine-coupled probe according to claim 7, characterized in that, Group R 1 Selected from substituted or unsubstituted C6-C 12 Aryl, C4-C 12 Heteroaryl compounds, coumarins, BODIPY compounds, triphenylethylene, tetraphenylethylene, or pentaphenylethylene; group R 2 Selected from substituted or unsubstituted C1-C8 chain alkyl, C2-C4 alkenyl, C4-C 12 Heteroaryl, BODIPY compounds, coumarin compounds, or bis(4-methoxyphenyl)amino, N-tert-butoxycarbonyl-2-aminopropionic acid methyl ester, 4-(N,N-dimethylamino)styrylthiophene.

9. A tetrazine-coupled probe according to claim 7, characterized in that, Group R 1 Selected from substituted or unsubstituted phenyl, naphthyl, biphenyl, styryl, thiophene, imidazolyl, indolyl, quinolinyl, benzothiophene, benzofuranyl, BODIPY, coumarin, or tetraphenylethylene; group R 2 Selected from substituted or unsubstituted methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, vinyl, propenyl, allyl, thiophene, pyrrole, pyridyl, indolyl, indazole, BODIPY, coumarin, or bis(4-methoxyphenyl)amino, N-tert-butoxycarbonyl-2-aminopropionic acid methyl ester, 4-(N,N-dimethylamino)styrylthiophene; The substituent is selected from any one of methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, halogen, trifluoromethyl, methoxy, ethoxy, dimethylamino, hydroxy, and cyano.

10. A tetrazine-coupled probe according to claim 7, characterized in that, It has any of the following structures: 、 、 、 、 、 、 、 、 、 、 。

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