Luminescent marker, luminescent marker intermediate solution and preparation method thereof, luminescent marker compound and application thereof, and kit

Abstract

The invention provides a light-emitting marker and application thereof, a light-emitting marker activated ester reaction solution and a preparation method thereof, and a kit. The luminous marker provided by the invention has a structure as shown in a general formula (I). The luminous marker provided by the invention not only has good luminous intensity, but also can generate a luminous marker intermediate directly coupled with protein under the action of the activator, the marking process is simple, the steric hindrance is lower during marking, and the marking efficiency can be effectively improved while the luminous property is ensured. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of luminescent markers, and in particular to a luminescent marker, a luminescent marker intermediate solution and a preparation method, a luminescent marker complex and an application and a kit. Background Art

[0002] Chemiluminescent markers play a vital role in in vitro diagnostics and are widely used as an efficient labeling tool, especially for the labeling of proteins such as antigens, antibodies and binding proteins. This labeling technology relies on the principle of chemiluminescence and measures the strength of the luminescent signal to achieve accurate quantification of the concentration of the target substance. Chemiluminescent immunoassays show excellent sensitivity and accuracy, and the operation process is simple and fast, which is very suitable for rapid screening and in-depth quantitative analysis of large-scale samples. Therefore, it plays an indispensable and important role in many key areas such as rapid diagnosis of infectious diseases, predictive assessment of genetic disease risks, and target analysis in new drug development.

[0003] Existing luminescent markers have the problem of complex labeling process in protein labeling applications, which affects the labeling efficiency in the actual application process. Therefore, there is an urgent need for a new luminescent marker that can simplify the labeling process and improve the labeling efficiency while ensuring performance, so as to better meet the needs of biomedical research, clinical diagnosis and drug development for efficient, fast and stable labeling technology. Summary of the invention

[0004] The main purpose of the present invention is to provide a luminescent marker, a luminescent marker intermediate solution and a preparation method, a luminescent marker complex and its application and a kit, which can solve the problem of complex labeling process in the prior art and affect the labeling efficiency in the actual application process while ensuring the luminescent performance of the luminescent marker.

[0005] In order to achieve the above object, according to one aspect of the present invention, a luminescent marker is provided, which has a structure shown in general formula (I):

[0006]

[0007] Wherein, Ar is selected from any one of the general formulas (II) to (IV):

[0008]

[0009] m is an integer between 0 and 2, when m=2, each R is the same or different; n is an integer between 0 and 4, when n≥2, each R is the same or different; R is selected from C1-C10 alkyl; X is selected from C1-C10 alkylene, and the C atom or H atom therein can be selectively replaced by O atom, S atom, aryl or heteroaryl; Y is selected from C1-C10 alkyl, C1-C10 alkylsulfonic acid group; and the C atom in the alkyl can be selectively replaced by O atom, S atom, aryl or heteroaryl.

[0010] Furthermore, R is selected from C1-C3 alkyl groups.

[0011] Furthermore, m is 0 or 1.

[0012] Furthermore, n is 0.

[0013] Furthermore, X is selected from C1-C6 alkylene groups, and the C atoms therein may be selectively substituted by O atoms.

[0014] Furthermore, Y is selected from C1-C7 alkyl sulfonic acid groups and C1-C6 alkyl groups, and the C atoms in the alkyl groups may be selectively substituted by O atoms.

[0015] Furthermore, R is selected from a C1-C2 alkyl group.

[0016] Furthermore, m is 0.

[0017] Furthermore, X is selected from C3-C6 alkylene groups, and the C atoms therein may be selectively substituted by O atoms.

[0018] Furthermore, Y is selected from C2-C4 alkyl sulfonic acid groups and C1-C5 alkyl groups, and the C atoms in the alkyl groups may be selectively substituted by O atoms.

[0019] Furthermore, the luminescent marker is selected from at least one of the following compounds:

[0020]

[0021] According to the second aspect of the present invention, the present application provides a luminescent labeling intermediate solution, the components of which include a luminescent labeling intermediate and an organic solvent, wherein the luminescent labeling intermediate is formed by activating the luminescent label provided by the first aspect above through an activator.

[0022] Further, the activator includes an amide condensation agent.

[0023] Further, the amide condensation agent includes at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or dicyclohexylcarbodiimide.

[0024] Furthermore, the activator also includes N-hydroxysuccinimide, and the luminescent labeling intermediate has a structure shown in the following formula (V):

[0025]

[0026] According to the third aspect of the present application, a method for preparing the above-mentioned luminescent marker intermediate solution is also provided, and the preparation method comprises: dispersing the luminescent marker in an organic solvent, adding an activator to carry out an activation reaction, so that the luminescent marker is activated by the activator to form a luminescent marker intermediate, and obtaining a luminescent marker intermediate solution.

[0027] Further, the activating agent includes an amide condensing agent and optionally N-hydroxysuccinimide.

[0028] Furthermore, the molar ratio of N-hydroxysuccinimide to the luminescent marker is ≥1, preferably (1.2-4):1.

[0029] Furthermore, the temperature of the activation reaction is 10-50° C., and the time of the activation reaction is 8-20 hours.

[0030] According to the fourth aspect of the present application, there is also provided the use of the luminescent marker provided in the first aspect or the luminescent marker intermediate solution provided in the second aspect in the preparation of a luminescent marker complex.

[0031] According to the fifth aspect of the present application, a luminescent marker complex is also provided. The luminescent marker complex is formed by coupling the luminescent marker provided in the first aspect with a protein. The luminescent marker complex has a structure shown in the following formula (VI):

[0032]

[0033] According to the sixth aspect of the present application, a method for preparing the above-mentioned luminescent marker complex is also provided, and the preparation method comprises: mixing the luminescent marker intermediate solution provided in the second aspect with a protein to perform a coupling reaction to obtain the luminescent marker complex.

[0034] According to the seventh aspect of the present application, there is also provided the use of the luminescent marker provided in the first aspect, the luminescent marker intermediate solution provided in the second aspect, or the luminescent marker complex provided in the fifth aspect in luminescent immunoassay.

[0035] According to the eighth aspect of the present application, a kit is also provided, which includes the luminescent marker provided in the first aspect, the luminescent marker intermediate solution provided in the second aspect, or the luminescent marker complex provided in the fifth aspect.

[0036] By applying the technical solution provided in the present application, the luminescent marker provided in the present application not only has good luminescent intensity, but also can generate a luminescent marker intermediate directly coupled to the protein under the action of an activator. The labeling process is simple, and the steric hindrance during labeling is lower, which can effectively improve the labeling efficiency. DETAILED DESCRIPTION

[0037] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0038] As analyzed in the background technology of this application, the widely used markers are relatively complicated in the labeling process, which affects the labeling efficiency in the actual application process. In order to solve this problem, this application provides a luminescent marker, a luminescent marker intermediate solution and a preparation method, a luminescent marker complex and its application and a kit.

[0039] In a first typical embodiment of the present application, a luminescent marker is provided, and the luminescent marker has a structure shown in general formula (I):

[0040]

[0041] Wherein, Ar is selected from any one of the general formulas (II) to (IV):

[0042]

[0043] The above Rm refers to m Rs, and Rn refers to n Rs; m is an integer between 0 and 2, such as 0, 1 or 2, and when m=2, each R is the same or different; n is an integer between 0 and 4, and when n≥2, each R is the same or different; R is selected from C1-C10 alkyl; X is selected from C1-C10 alkylene, and the C atom or H atom therein can be selectively replaced by O atom, S atom, aryl and its derivatives, heteroaryl and its derivatives; Y is selected from C1-C10 alkyl, C1-C10 alkylsulfonic acid group; and the C atom in the alkyl can be selectively replaced by O atom, S atom, aryl and its derivatives, heteroaryl and its derivatives.

[0044] In the present application, "aryl" means an aromatic group or an aromatic (sub) group, which refers to a monocyclic or condensed polycyclic group derived from an aromatic hydrocarbon, and includes phenyl, biphenyl, terphenyl, naphthyl, binaphthyl, phenylnaphthyl, naphthylphenyl, fluorenyl, phenylfluorenyl, benzofluorenyl, dibenzofluorenyl, phenanthrenyl, phenylphenanthrenyl, anthracenyl, indenyl, triphenylene, pyrene, condensed tetraphenyl, perylenyl, quinacyl, naphthacene, allenylfluorenyl, and the like.

[0045] "Heteroaryl" refers to a heteroaryl or (sub)heteroaryl group, wherein the heteroaryl group refers to an aromatic group containing at least one heteroatom selected from the group consisting of N, O, and S as the main chain atom of the ring, and can be a monocyclic ring system such as furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, triazinyl, tetrazinyl, triazolyl, tetrazolyl, furazanyl, pyridinyl, Pyrazinyl, pyrimidinyl, pyridazinyl, etc., or a condensed ring condensed with at least one benzene ring, such as benzofuranyl, benzothiophenyl, isobenzofuranyl, dibenzofuranyl, dibenzothiophenyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, carbazolyl, phenanthroxazolyl, phenanthridinyl, benzodiacenaphthenyl, dihydroacridinyl, etc.

[0046] "C1-C10 alkyl" means an alkyl group having 1 to 10, in particular up to 6, carbon atoms, which is straight-chain or branched with one or more branches, for example butyl, such as n-butyl, sec-butyl, isobutyl, tert-butyl; propyl, for example n-propyl or isopropyl; ethyl or methyl; more particularly methyl, isopropyl or tert-butyl.

[0047] The "C1-C10 alkylsulfonic acid group" refers to "C1-C10 alkyl-SO3H", and particularly ethylsulfonic acid, butylsulfonic acid, pentylsulfonic acid, hexylsulfonic acid and the like.

[0048] The luminescent marker provided in the present application is obtained through a series of structural designs. It not only has good luminescent intensity, but also can generate a luminescent marker intermediate directly coupled to the protein under the action of an activator. The labeling process is simple and the steric hindrance is lower during labeling, which can effectively improve the labeling efficiency while ensuring the luminescent performance.

[0049] In some embodiments, R is selected from C1-C6 alkyl.

[0050] In some embodiments, X is selected from C1-C6 alkylene groups, and the C atoms therein may be selectively replaced by O atoms, which is more conducive to improving the labeling efficiency and luminescence intensity.

[0051] In some embodiments, Y is selected from C1-C7 alkyl sulfonic acid groups and C1-C6 alkyl groups, and the C atoms in the alkyl groups can be selectively replaced by O atoms, which is more conducive to improving labeling efficiency and luminescence intensity.

[0052] In some embodiments, X is selected from C3-C6 alkylene, wherein the C atom is optionally substituted by an O atom.

[0053] In some embodiments, Y is selected from C2-C4 alkyl sulfonic acid groups and C1-C5 alkyl groups, and the C atoms in the alkyl groups can be selectively replaced by O atoms, which is more conducive to improving labeling efficiency and luminescence intensity.

[0054] In some specific embodiments, the luminescent marker is selected from at least one of the following compounds:

[0055]

[0056] In the present application, the above-mentioned luminescent marker is prepared according to conventional methods in the art and is not limited here.

[0057] In some embodiments, taking the luminescent marker LM-A as an example, the preparation and activation process of the luminescent marker are as shown below (A).

[0058]

[0059] In some embodiments, the synthesis route of the luminescent marker TM-A is shown below (B).

[0060]

[0061] The above-mentioned "yield" refers to "yield rate".

[0062] Specifically, the luminescent marker TM-A is prepared according to the following steps:

[0063] (1) Synthesis of compound 2a.

[0064]

[0065] Compound 1a (3 g, 17.03 mmol) and tert-butyl 5-bromovalerate (20.2 g, 85.15 mmol), triethylamine (8.6 g, 85.15 mmol), DMF (45 mL) were added to the Shrek tube, and the mixture was heated at 80 ° C for 20 hours under argon protection. The reaction solution was monitored by LC-MS, and the raw materials were basically converted into the target product. After the reaction was completed, the crude liquid was purified by reverse phase preparation and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain compound 2a solution. The acetonitrile was removed by rotary evaporation, and the aqueous solution containing the product was extracted with ethyl acetate. After rotary drying, the crude compound 2a (5.37 g, 95% yield) was obtained, which can be used directly in the next step. MS [M-(t-BuO - ) ]+ Calculated value: 259.11, measured value: 259.0.

[0066] (2) Synthesis of compound 3a.

[0067]

[0068] Compound 2a (1.0 g, 3.01 mmol), DMF (10 mL), 2-bromoethyl ether (4.6 g, 30.1 mmol), potassium carbonate (1.25 g, 9.03 mmol) were added to the sealed tube, and the mixture was stirred at 130 ° C for 48 hours. Samples were taken for LC-MS detection, and the raw material conversion rate was about 20%. The reaction was stopped, and the crude liquid was purified by reverse phase preparation, and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain compound 3a solution, and acetonitrile was removed by rotary evaporation, and the aqueous phase was extracted with dichloromethane, and then the solvent was dried to obtain the crude product of compound 3a. The crude product was purified by silica gel column to remove some impurities and pigments, and then the pure compound 3a (209.6 mg, 20% yield) was obtained. MS [M+H] + Calculated value: 349.17, measured value: 349.0.

[0069] (3) Synthesis of LM-A.

[0070]

[0071] Compound 3a (200 mg, 0.574 mmol), ethanol (20 mL), hydrazine hydrate (574 mg, 11.5 mmol) were added to a Shrek tube, and the mixture was heated and stirred at 60°C for 6 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation to obtain a crude product, and 3N hydrochloric acid was slowly added to the crude product until it became acidic (pH = 1-2) (stirring was required during the neutralization process, and the pH endpoint was required to remain stable for more than 10 minutes). H2O and acetonitrile were added to dissolve the sample, and the crude sample solution was directly used for reverse phase purification, and reverse phase (CH3CN / H2O, 0.1% FA) C18 column purification was performed to obtain pure compound LM-A (86 mg, 43% yield). 1 H NMR (400MHz, DMSO) δ11.42(s,3H),7.82(d,J=8.9Hz,1H),7.21(dd,J=9.1,2.8Hz,1H),7.06(s,1H),3.62–3. 53(m,4H),3.47–3.41(m,4H),2.29–2.23(m,2H),1.56(dd,J=7.0,4.0Hz,4H),1.09(t,J=7.0Hz,3H).MS[M+H] + Calculated value: 350.16, measured value: 350.0.

[0072] In a second typical embodiment of the present application, a luminescent marking intermediate solution is also provided, the components of which include a luminescent marking intermediate and an organic solvent, wherein the luminescent marking intermediate is formed by activating the luminescent marker provided in the first typical embodiment through an activator.

[0073] In the present application, "luminescent marker intermediate" refers to the activated state of the above-mentioned luminescent marker. After the luminescent marker is activated to the activated state, the coupling efficiency between the luminescent marker and the protein can be further improved. Among them, the activated state can be a luminescent marker activated ester. After the luminescent marker is prepared into the luminescent marker activated ester, the stability of the activated state of the luminescent marker can be improved.

[0074] "Organic solvent" refers to a type of solvent that uses organic matter as a medium. It has the characteristics of small molecular weight, being liquid at room temperature and pressure, being volatile, and being able to dissolve some organic compounds that are insoluble in water. There are many types of commonly used organic solvents. According to their chemical structure, they can be divided into more than ten categories, including aromatic hydrocarbons (such as benzene and toluene), aliphatic hydrocarbons (such as pentane and hexane), halogenated hydrocarbons (such as chlorobenzene and dichloromethane), alcohols (such as methanol and ethanol), ethers (such as ether), esters (such as methyl acetate), ketones (such as acetone), etc.

[0075] The above activating agent is a commonly used activating agent for activating carboxyl groups, and the present application does not impose any limitation on this.

[0076] In some embodiments, the activating agent comprises an amide condensing agent.

[0077] In the present application, "amide condensation agent" refers to a reagent that can activate a luminescent marker to an activated state. They can accelerate and promote the condensation reaction between carboxylic acid and amine to form an amide bond. Common amide condensation agents include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), diisopropylcarbodiimide (DIC) and acetylene amides.

[0078] In some embodiments, when the activator further comprises N-hydroxysuccinimide, the luminescent labeling intermediate has a structure as shown in the following formula (V):

[0079]

[0080] In the above formula (V), Ar, X, and Y each have the same meaning as in the luminescent marker provided in the first typical embodiment, and will not be described in detail here.

[0081] In some embodiments, the organic solvent provides a reaction environment for the luminescent marker and N-hydroxysuccinimide, and is a commonly used organic solvent in the art, including but not limited to DMSO (dimethyl sulfoxide), DMF (dimethylformamide), and the like.

[0082] In some embodiments, the activation of the carboxyl group of LM-A to generate the luminescent labeled intermediate LM-AS is performed according to the following steps:

[0083]

[0084] Compound LM-A (115 mg, 0.328 mmol) was dissolved in DMSO (5 mL), and then EDC (125.6 mg, 0.655 mmol) was added. After stirring at room temperature for 20 minutes under argon protection, NHS (75.4 mg, 0.655 mmol) was added, and the mixture was stirred at 40°C for 12 hours. After the reaction was completed, compound LM-AS was separated by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column, and lyophilized to obtain compound LM-AS (53 mg, 36% yield). MS [M+H] + Calculated value: 447.2, measured value: 447.3.

[0085] In the third typical embodiment of the present application, a method for preparing the above-mentioned luminescent marker intermediate solution is provided, and the preparation method comprises: dispersing the luminescent marker in an organic solvent, adding an activator and mixing to carry out an activation reaction, so that the luminescent marker is activated by the activator to form a luminescent marker intermediate, and obtaining a luminescent marker intermediate solution.

[0086] The meaning of the activator is the same as that described in the second typical embodiment above, and will not be repeated here.

[0087] In some embodiments, the activator includes an amide condensation agent and N-hydroxysuccinimide, and the molar ratio of N-hydroxysuccinimide to the luminescent marker is ≥1, especially when the molar ratio of N-hydroxysuccinimide to the luminescent marker is (1.2-4):1, it is more conducive to improving the labeling efficiency while saving energy.

[0088] Specifically, the molar ratio of N-hydroxysuccinimide to the luminescent marker can be 1.2:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or a range consisting of any two values.

[0089] In order to further improve the reaction efficiency, in some embodiments, the temperature of the activation reaction is 10-50°C, and the time of the activation reaction is 8-20h. Specifically, the temperature of the activation reaction can be 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, or a range consisting of any two values; the time of the activation reaction can be 8h, 10h, 12h, 15h, 18h, 20h, or a range consisting of any two values.

[0090] In a fourth typical embodiment of the present application, there is also provided the use of the luminescent marker provided in the first typical embodiment or the luminescent marker intermediate solution provided in the second typical embodiment in the preparation of a luminescent marker complex.

[0091] In the present application, the luminescent marker complex is formed by coupling a luminescent marker with a protein, and the luminescent marker complex has a structure shown in the following formula (VI):

[0092]

[0093] Herein, "Protein" refers to a protein containing an amino group, which is coupled with a luminescent labeling intermediate through the amino group to form a structure shown in formula (VI).

[0094] In the above formula (VI), Ar, X, and Y each have the same meaning as in the luminescent marker provided in the first typical embodiment, and will not be repeated here.

[0095] In some embodiments, the activator further comprises N-hydroxysuccinimide, and the luminescent labeling intermediate has a structure as shown in the following formula (V):

[0096]

[0097] In a third typical embodiment of the present application, a luminescent labeling intermediate solution is provided, the components of which include the luminescent labeling intermediate provided in the second typical embodiment and an organic solvent.

[0098] In a fourth typical embodiment of the present application, a method for preparing a luminescent marker activated intermediate solution is also provided, the preparation method comprising: dispersing a luminescent marker in an organic solvent, adding an amide condensation agent and N-hydroxysuccinimide, mixing and reacting, and obtaining a luminescent marker intermediate solution. The luminescent marker intermediate in the solution can be stably stored.

[0099] In some embodiments, in the luminescent marker activation intermediate solution provided in the present application, the amide condensation agent is selected from EDC or DCC, and the above amide condensation agent can stably and efficiently react with the luminescent marker, and the reaction conditions are mild.

[0100] In a sixth typical embodiment of the present application, a luminescent marker complex is further provided, which is formed by coupling the above-mentioned luminescent marker with a protein, and the luminescent marker complex has a structure shown in the following formula (VI):

[0101]

[0102] Exemplarily, the reaction principle of coupling the above-mentioned luminescent labeling intermediate with the protein (protein containing an amino group) is shown as follows (C):

[0103]

[0104] In a seventh typical embodiment of the present application, there is provided the use of the above-mentioned luminescent marker, the above-mentioned luminescent marker intermediate solution or the above-mentioned luminescent marker complex in luminescent immunoassay.

[0105] The luminescent marker, luminescent marker intermediate solution or the luminescent marker complex provided in the present application is more conducive to improving the labeling efficiency when used in the field of luminescent immunoassay and has broad application prospects.

[0106] In an eighth typical embodiment of the present application, a kit is also provided, which includes the above-mentioned luminescent marker, luminescent marker active intermediate solution or luminescent marker complex.

[0107] The kit provided in the present application uses a luminescent marker as a main raw material. The kit provided in the present application utilizes the carboxyl group on the luminescent marker to couple with the amino group on the protein to form a luminescent marker complex. The labeling process is simple, the labeling efficiency is effectively improved, and it has a wide range of applications in the field of luminescent immunoassay.

[0108] The beneficial effects of the present application will be further illustrated below with reference to examples and comparative examples.

[0109] Example 1

[0110] This example provides a method for preparing and activating a luminescent marker LM-A, which is carried out according to the following steps:

[0111] The LM-A synthesis route is as follows:

[0112]

[0113] (1) Synthesis of compound 2a.

[0114]

[0115] Compound 1a (3 g, 17.03 mmol) and tert-butyl 5-bromovalerate (20.2 g, 85.15 mmol), triethylamine (8.6 g, 85.15 mmol), DMF (45 mL) were added to the Shrek tube, and the mixture was heated at 80 ° C for 20 hours under argon protection. The reaction solution was monitored by LC-MS, and the raw materials were basically converted into the target product. After the reaction was completed, the crude liquid was purified by reverse phase preparation and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain compound 2a solution. The acetonitrile was removed by rotary evaporation, and the aqueous solution containing the product was extracted with ethyl acetate. After rotary drying, the crude compound 2a (5.37 g, 95% yield) was obtained, which can be used directly in the next step. MS [M-(t-BuO - )] + Calculated value: 259.11, measured value: 259.0.

[0116] (2) Synthesis of compound 3a.

[0117]

[0118] Compound 2a (1.0 g, 3.01 mmol), DMF (10 mL), 2-bromoethyl ether (4.6 g, 30.1 mmol), potassium carbonate (1.25 g, 9.03 mmol) were added to the sealed tube, and the mixture was stirred at 130 ° C for 48 hours. Samples were taken for LC-MS detection, and the raw material conversion rate was about 20%. The reaction was stopped, and the crude liquid was purified by reverse phase preparation, and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain compound 3a solution, and acetonitrile was removed by rotary evaporation, and the aqueous phase was extracted with dichloromethane, and then the solvent was dried to obtain the crude product of compound 3a. The crude product was purified by silica gel column to remove some impurities and pigments, and then the pure compound 3a (209.6 mg, 20% yield) was obtained. MS [M+H] + Calculated value: 349.17, measured value: 349.0.

[0119] (3) Synthesis of LM-A.

[0120]

[0121] Compound 3a (200 mg, 0.574 mmol), ethanol (20 mL), hydrazine hydrate (574 mg, 11.5 mmol) were added to a Shrek tube, and the mixture was heated and stirred at 60°C for 6 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation to obtain a crude product, and 3N hydrochloric acid was slowly added to the crude product until it became acidic (pH = 1-2) (stirring was required during the neutralization process, and the pH endpoint was required to remain stable for more than 10 minutes). H2O and acetonitrile were added to dissolve the sample, and the crude sample solution was directly used for reverse phase purification, and reverse phase (CH3CN / H2O, 0.1% FA) C18 column purification was performed to obtain pure compound LM-A (86 mg, 43% yield). 1 H NMR (400MHz, DMSO) δ11.42(s,3H),7.82(d,J=8.9Hz,1H),7.21(dd,J=9.1,2.8Hz,1H),7.06(s,1H),3.62–3. 53(m,4H),3.47–3.41(m,4H),2.29–2.23(m,2H),1.56(dd,J=7.0,4.0Hz,4H),1.09(t,J=7.0Hz,3H).MS[M+H] + Calculated value: 350.16, measured value: 350.0.

[0122] LM-A carboxyl activation.

[0123]

[0124] Compound LM-A (115 mg, 0.328 mmol) was dissolved in DMSO (5 mL), and then EDC (125.6 mg, 0.655 mmol) was added. After stirring at room temperature for 20 minutes under argon protection, N-hydroxysuccinimide (NHS) (75.4 mg, 0.655 mmol) was added, and the mixture was stirred at 40°C for 12 hours. After the reaction was completed, compound LM-AS was separated by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column, and lyophilized to obtain compound LM-AS (53 mg, 36% yield). MS [M+H] + Calculated value: 447.2, measured value: 447.3.

[0125] Example 2

[0126] This example provides a method for preparing a luminescent marker LM-B, which is carried out according to the following steps:

[0127] The synthetic route of LM-B is as follows:

[0128]

[0129] (1) Synthesis of compound 2b.

[0130]

[0131] Compound 1b (300 mg, 1.7 mmol) and tert-butyl 2-(2-bromoethoxy)acetate (1.02 g, 4.25 mmol), potassium carbonate (704.8 mg, 5.1 mmol), and DMF (10 mL) were added to a Shrek tube, and the mixture was heated at 120°C for 16 hours under argon protection. The reaction solution was monitored by TLC (PE:EA 2:1, RF:0.6). After the reaction was completed, the product was purified by silica gel column chromatography, and the product eluate was collected and rotary evaporated to obtain pure compound 2b (113.5 mg, 20% yield). MS [M-(t-BuO - )] + Calculated value: 261.09, measured value: 262.1.

[0132] (2) Synthesis of compound 3b.

[0133]

[0134] Compound 2b (200 mg, 0.598 mmol), potassium carbonate (248 mg, 1.79 mmol), iodoethane (1.87 g, 11.96 mmol), and DMF (4 mL) were added to a sealed tube, and the mixture was stirred at 80°C for 72 hours. Samples were taken for LC-MS detection, and the raw material conversion rate was about 50%. After the reaction was completed, the crude solution was purified by reverse phase preparation, and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain compound 3b solution, and acetonitrile was removed by rotary evaporation, and then lyophilized to obtain pure compound 3b (91.5 mg, 50% yield). MS [M+H] + Calculated value: 307.12, measured value: 307.0.

[0135] (3) Synthesis of LM-B.

[0136]

[0137] Compound 3b (90 mg, 0.294 mmol), ethanol (2 mL), hydrazine hydrate (294 mg, 5.88 mmol) were added to a Shrek tube, and the mixture was heated and stirred at 60°C for 6 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation to obtain a crude product, and 3N hydrochloric acid was slowly added to the crude product until it became acidic (pH = 1-2) (stirring was required during the neutralization process, and the pH endpoint was required to remain stable for more than 10 minutes). H2O and acetonitrile were added to dissolve the sample, and the crude sample solution was directly used for reverse phase purification, and reverse phase (CH3CN / H2O, 0.1% FA) C18 column purification was performed to obtain pure compound LM-b (31.6 mg, 35% yield). 1 H NMR (400MHz, DMSO) δ7.83(d,J=8.9Hz,1H),7.22(dd,J=9.1,2.7Hz,1H),7.06(d,J=2.7Hz,1H),4.04(s, 2H),3.66(d,J=4.8Hz,2H),3.62(d,J=4.9Hz,2H),3.51(q,J=7.0Hz,2H),1.14(t,J=7.0Hz,3H).MS[M+H] + Calculated value: 308.12, measured value: 308.0.

[0138] The activation steps are the same as LM-A.

[0139] Example 3

[0140] This example provides a method for preparing and activating a luminescent marker LM-C, which is carried out according to the following steps:

[0141] The synthetic route of LM-C is as follows:

[0142]

[0143] (1) Synthesis of compound 2c.

[0144]

[0145] Compound 1c (500 mg, 2.8 mmol), 3-bromopropylpyridine hydrobromide (1.5 g, 5.3 mmol), and DMF (30 mL) were added to a three-necked flask, and the mixture was heated at 100°C for 16 hours under argon protection. LCMS monitoring showed that the raw materials were basically converted. After completion, the mixture was cooled to room temperature and purified by reverse phase purification (CH3CN / H2O+0.1% FA) to obtain pure compound 2c (300 mg, 38% yield). MS [M+H] + Calculated value: 296.13, measured value: 296.4.

[0146] (2) Synthesis of compound 3c.

[0147]

[0148] Compound 2c (1.0 g, 3.6 mmol), 1,3-propane sultone (0.82 g, 6.0 mmol), and DMF (40 mL) were added to a Shrek tube, and the mixture was heated at 110°C for 6 hours under argon protection. LCMS monitoring showed that the raw materials were basically converted. After completion, the mixture was cooled to room temperature and purified by reverse phase purification (CH3CN / H2O+0.1% FA) to obtain pure compound 3c (797 mg, 53% yield). MS [M+H] + Calculated value: 418.14, measured value: 418.1.

[0149] (3) Synthesis of compound 4c.

[0150]

[0151] Compound 3c (500 mg, 1.20 mmol), pentabromovaleronitrile (1.94 g, 12.0 mmol), and DMF (5 mL) were added to a sealed tube and heated at 130°C for 26 hours. LCMS monitoring showed that the raw materials were basically converted. After completion, the mixture was cooled to room temperature and purified by reverse phase purification (CH3CN / H2O+0.1% FA) to obtain pure compound 4c (479 mg, 80% yield). MS [M+H] + Calculated value: 499.19, measured value: 499.2.

[0152] (4) Synthesis of compound 5c.

[0153]

[0154] Add H2O (0.9 mL), glacial acetic acid (0.9 mL), and concentrated sulfuric acid (1.8 mL) in a sealed glass tube, mix well, and then add compound 4 (900 mg, 1.8 mmol). The reaction solution was heated and stirred at 70 ° C for 48 hours. LC-MS monitoring reaction was stopped when the conversion rate of the target product was higher than 85%. After the reaction was completed, cool to room temperature, add H2O (15 mL) to dilute the reaction solution, and the clear solution was directly used for reverse phase purification. Purification by reverse phase (CH3CN / H2O, 0.1% FA) C18 column gave pure compound 5 (606 mg, 65% yield). MS [M+H] + Calculated value: 518.19, measured value: 518.2.

[0155] (5) Synthesis of compound LM-C.

[0156]

[0157] In a Shrek tube, compound 5c (500 mg, 0.97 mmol), ethanol (5 mL), hydrazine hydrate (966.0 mg, 19.3 mmol) were added, and the mixture was heated and stirred at 50°C for 6 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation to obtain a crude product, and 3N hydrochloric acid was slowly added to the crude product until it became acidic (pH = 1-2) (stirring was required during the neutralization process, and the pH endpoint was required to remain stable for more than 10 minutes). H2O (10 mL) and acetonitrile (10 mL) were added to dissolve the sample, and the crude sample solution was directly used for reverse phase purification, and reverse phase (CH3CN / H2O, 0.1% FA) C18 column purification was performed to obtain pure compound LM-C (415 mg, 80% yield). 1 HNMR(400MHz,DMSO)δ9.05(s,1H),8.94(d,J=6.0Hz,1H),8.50(d,J=7.9Hz,1H),8.05(dd,J=8 .0,6.0Hz,1H),7.82(d,J=9.0Hz,1H),7.19(dd,J=9.1,2.8Hz,1H),6.96(d,J=2.7Hz,1H),4.6 9(t,J=7.0Hz,2H),3.48(t,J=7.6Hz,2H),3.42(t,J=6.9Hz,2H),2.89(t,J=7.7Hz,2H),2.45( t,J=7.1Hz,2H),2.25(q,J=7.1Hz,4H),1.96(p,J=7.9Hz,2H),1.56(p,J=3.4Hz,4H).MS[M+H] + Calculated value: 519.18, measured value: 519.2.

[0158] The carboxyl group of LM-C was activated to generate the luminescent labeling activated ester LM-CS.

[0159]

[0160] LM-C (100 mg, 0.192 mmol) and NHS (88 mg, 0.768 mmol) were added to a Shrek tube, and DMSO (5 mL) was added. After argon replacement, the mixture was stirred at 40°C for 5 minutes. Then, the entire batch of EDC (110 mg, 0.576 mmol) was quickly added to the reaction solution and stirred at 40°C for 6 hours. The reaction was stopped by LC-MS monitoring until the conversion rate of the target product was higher than 70%. After the reaction was completed, a luminescent labeled activated ester (LM-CS) reaction solution was obtained, which can be directly used to label proteins. MS [M+H] + Calculated value: 616.20, measured value: 616.5.

[0161] Example 4

[0162] This example provides a method for preparing and activating a luminescent marker TM-D, which is carried out according to the following steps:

[0163] The synthetic route of TM-D is as follows:

[0164]

[0165] (1) Synthesis of compound 2d.

[0166]

[0167] Compound 1d (2 g, 11.35 mmol) and sodium 2-bromoethylsulfonate (24 g, 113.7 mmol), potassium carbonate (7.9 g, 57.2 mmol), DMF (40 mL) were added to the Shrek tube, and the mixture was heated at 110 ° C for 72 hours under argon protection. The reaction solution was monitored by LC-MS, and the reaction was stopped when the conversion rate was above 70%. The reaction solution was cooled to room temperature, H2O was added to the system, and ultrasonic vibration was performed until it was clear. The crude solution was purified by reverse phase preparation, and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain compound 2 solution, and the solvent was removed by rotary evaporation to obtain pure compound 2d (1.6 g, 50% yield). MS [M+H] + Calculated value: 285.1, measured value: 285.1.

[0168] (2) Synthesis of compound 3d.

[0169]

[0170] Compound 2d (1.0 g, 3.52 mmol), DMF (10 mL), pentabromovaleronitrile (5.7 g, 35.2 mmol) were added to a sealed tube, and the mixture was stirred at 130°C for 16 hours. Samples were taken for LC-MS detection, and the raw materials were basically converted into the target product. After the reaction was completed, the crude liquid was purified by reverse phase preparation and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain compound 3d solution, and the solvent was removed by rotary evaporation to obtain pure compound 3d (1.0 g, 78% yield). MS [M+H] + Calculated value: 366.1, measured value: 366.1.

[0171] (3) Synthesis of compound 4d.

[0172]

[0173] Add H2O (1mL), glacial acetic acid (1mL), and concentrated sulfuric acid (2mL) to the sealed tube in sequence, mix well, and then add compound 3d (500mg, 1.37mmol). The mixture is heated and stirred at 70°C for 48 hours. After the reaction is completed, cool to room temperature, add H2O (15mL) to dilute the reaction solution, and then purify with reverse phase. Purify with reverse phase (CH3CN / H2O, 0.1% TFA) C18 column to obtain pure compound 4d (400mg, 76% yield). MS [M+H] + Calculated value: 385.1, measured value: 385.1.

[0174] (4) Synthesis of LM-D.

[0175]

[0176] Compound 4d (200 mg, 0.52 mmol), ethanol (2 mL), 85% hydrazine hydrate (520 mg, 8.83 mmol) were added to a Shrek tube, and the mixture was heated and stirred at 50°C for 6 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation to obtain a crude product, and 3N hydrochloric acid was slowly added to the crude product until it became acidic (pH = 1-2) (stirring was required during the neutralization process, and the pH endpoint was required to remain stable for more than 10 minutes). H2O and acetonitrile were added to dissolve the sample, and the crude sample solution was directly used for reverse phase purification, and reverse phase (CH3CN / H2O, 0.1% FA) C18 column purification was performed to obtain pure compound LM-D (150 mg, 75% yield). 1 H NMR (400MHz, DMSO) δ7.91(s,1H),7.83(d,J=9.0Hz,1H),7.15(dd,J=9.1,2.8Hz,1H),7.05(d,J=2.8Hz,1H),3.71–3.62 (m,2H),3.41(t,J=6.9Hz,2H),2.73(t,J=7.9Hz,2H),2.18(t,J=6.8Hz,2H),1.55(dq,J=9.8,6.1,5.2Hz,4H).MS[M+H] + Calculated value: 386.1, measured value: 386.2.

[0177] LM-D carboxyl activation.

[0178]

[0179] LM-D (100 mg, 0.26 mmol) and NHS (119 mg, 1.03 mmol) were added to the Shrek tube, followed by DMSO (5 mL). After argon replacement, the mixture was stirred at 40°C for 5 minutes. The whole batch of EDC (149 mg, 0.78 mmol) was then quickly added to the reaction solution and stirred at 40°C for 6 hours. The reaction was stopped by LC-MS monitoring until the conversion rate of the target product was higher than 70%. After the reaction was completed, the luminescent labeled activated ester LM-DS reaction solution was obtained, which can be directly used to label proteins. MS [M+H] + Calculated value: 483.1, measured value: 483.1.

[0180] Example 5

[0181] This example provides a method for preparing and activating a luminescent marker LM-E, which is carried out according to the following steps:

[0182] The synthetic route of LM-E is as follows:

[0183]

[0184] (1) Synthesis of compound 2e.

[0185]

[0186] Add compound 1e (3 g, 17.03 mmol) and 1,3-propane sultone (20.8 g, 170.3 mmol), dioxane (55 mL) to a Shrek tube, replace argon, and heat the mixture at 100 ° C for 20 hours. Monitor the reaction solution with LC-MS, and stop the reaction when the conversion rate is above 70%. Filter the reaction solution while hot (filter immediately after taking out the oil bath pot), rinse the pulped filter cake repeatedly with 80 ° C dioxane solution three times, and discard the filtrate. Collect the filter cake solid, remove the solvent by rotary evaporation to obtain compound 2e (3.2 g, 70% yield), which can be directly used in the next step reaction. MS [M + H] + Calculated value: 269.09, measured value: 299.0.

[0187] (2) Synthesis of compound 3e.

[0188]

[0189] Compound 2 (800 mg, 2.98 mmol), DMF (8 mL), pentabromovaleronitrile (4.35 g, 26.85 mmol) were added to the sealed tube, and the mixture was stirred at 130°C for 16 hours. Samples were taken for LC-MS detection, and the raw materials were basically converted into the target product. After the reaction was completed, the crude liquid was purified by reverse phase preparation and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain compound 3 solution, which was rotary evaporated and freeze-dried to obtain pure compound 3 (904 mg, 80% yield). MS [M+H] + Calculated value: 380.12, measured value: 380.1.

[0190] (3) Synthesis of compound 4e.

[0191]

[0192] H2O (4 mL), glacial acetic acid (4 mL), and concentrated sulfuric acid (8 mL) were added to the sealed tube in sequence, and after mixing evenly, compound 3e (800 mg, 2.1 mmol) was added, and the mixture was heated and stirred at 70 °C for 48 hours. After the reaction was completed, it was cooled to room temperature, acetonitrile was added to dilute the reaction solution, and then purified by reverse phase. Pure compound 4e (635 mg, 76% yield) was obtained by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column purification. MS [M+H] + Calculated value: 399.11, measured value: 399.1.

[0193] (4) Synthesis of LM-E.

[0194]

[0195] Compound 4e (700 mg, 1.76 mmol), ethanol (7 mL), and hydrazine hydrate (1.76 g, 35.2 mmol) were added to a Shrek tube, and the mixture was heated and stirred at 50°C for 6 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by rotary evaporation to obtain a crude product, and 3N hydrochloric acid was slowly added to the crude product until it became acidic (pH = 1-2) (stirring was required during the neutralization process, and the pH endpoint was required to remain stable for more than 10 minutes). H2O and acetonitrile were added to dissolve the sample, and the crude sample solution was directly used for reverse phase purification, and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain pure compound LM-E (527 mg, 75% yield). 1H NMR (400MHz, DMSO) δ7.95(s,1H),7.81(d,J=9.5Hz,1H),7.22(dd,J=9.1,2.8Hz,1H),7.01(s,1H),3.50(t,J=7.6Hz,2H) ,3.42(t,J=6.7Hz,2H),2.47(t,J=7.3Hz,2H),2.31–2.22(m,2H),1.84(p,J=7.5Hz,2H),1.56(p,J=3.6Hz,4H).MS[M+H] + Calculated value: 400.11, measured value: 400.0.

[0196] The activation steps are the same as LM-D.

[0197] Example 6

[0198] This example provides a method for preparing and activating a luminescent marker LM-F, which is carried out according to the following steps:

[0199] The synthetic route of LM-F is as follows:

[0200]

[0201] (1) Synthesis of compound 2f.

[0202]

[0203] Compound 1f (3 g, 17.03 mmol) and 1,4-butane sultone (23.2 g, 170.3 mmol), triethylamine (5.16 g, 51.09 mmol), dioxane (60 mL) were added to a Shrek tube, and the mixture was heated at 100°C for 48 hours under argon protection. The reaction solution was monitored by LC-MS, and the conversion rate was about 10%. After the reaction, the crude solution was purified by reverse phase preparation, and purified by reverse phase (CH3CN / H2O, 0.1% FA [formic acid]) C18 column to obtain compound 2f solution, which was rotary evaporated and freeze-dried to obtain pure compound 2f (531 mg, 10% yield). MS [M+H] + Calculated value: 313.08, measured value: 313.0.

[0204] (2) Synthesis of compound 3f.

[0205]

[0206] Compound 2f (460 mg, 1.47 mmol), DMF (4 mL), pentabromovaleronitrile (2.38 g, 14.7 mmol) were added to the sealed tube, and the mixture was stirred at 130°C for 16 hours. Samples were taken for LC-MS detection, and the raw materials were basically converted into the target product. After the reaction was completed, the crude liquid was purified by reverse phase preparation and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain compound 3f solution, which was rotary evaporated and freeze-dried to obtain pure compound 3f (173 mg, 30% yield). MS [M+H] + Calculated value: 394.14, measured value: 394.0.

[0207] (3) Synthesis of compound 4.

[0208]

[0209] Add H2O (1.5mL), glacial acetic acid (1.5mL), and concentrated sulfuric acid (3mL) in the sealed tube, mix well, add compound 3f (160mg, 0.4mmol), and heat the mixture at 70℃ with stirring for 20 hours. After the reaction is completed, cool to room temperature, add acetonitrile (15mL) to dilute the reaction solution, and then purify with reverse phase. Purify with reverse phase (CH3CN / H2O, 0.1% TFA) C18 column to obtain pure compound 4f (138mg, 84% yield). MS [M+H] + Calculated value: 413.13, measured value: 413.1.

[0210] (4) Synthesis of LM-F.

[0211]

[0212] Compound 4f (140 mg, 0.34 mmol), ethanol (7 mL), hydrazine hydrate (340 mg, 6.8 mmol) were added to a Shrek tube, and the mixture was heated and stirred at 50°C for 6 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation to obtain a crude product, and 3N hydrochloric acid was slowly added to the crude product until it became acidic (pH = 1-2) (stirring was required during the neutralization process, and the pH endpoint was required to remain stable for more than 10 minutes). H2O and acetonitrile were added to dissolve the sample, and the crude sample solution was directly used for reverse phase purification, and reverse phase (CH3CN / H2O, 0.1% FA) C18 column purification was performed to obtain pure compound LM-F (42 mg, 30% yield). 1H NMR (400MHz, DMSO) δ7.82(d,J=9.0Hz,1H),7.19(dd,J=9.0,2.7Hz,1H),7.02(d,J=2.6Hz,1H),3.40(q,J =6.3Hz,4H),2.56(t,J=7.3Hz,2H),2.28–2.24(m,2H),1.69–1.62(m,4H),1.56(p,J=3.8Hz,4H).MS[M+H] + Calculated value: 414.13, measured value: 414.0.

[0213] The activation steps are the same as LM-D.

[0214] Example 7

[0215] This example provides a method for preparing and activating a luminescent marker LM-G, which is carried out according to the following steps:

[0216] LM-G synthesis route:

[0217]

[0218] (1) Synthesis of compound 2g.

[0219]

[0220] Compound 1g (5.0g, 28.4mmol) and diethyl sulfate (50mL, 379mmol) were added to a three-necked flask, and the mixture was heated at 110°C for 24 hours under argon protection. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 10:1) until the raw material conversion was complete. After completion, the mixture was cooled to room temperature, H2O (50mL) was added to the system, and after stirring for 5 minutes, it was extracted with dichloromethane (200mL). The organic phase was dried and the solvent was removed to obtain a crude compound 2g, which was further purified by column chromatography to obtain a pure compound 2g (2.32g, 40% yield). MS [M+H] + Calculated value: MS205.1, measured value: MS205.5.

[0221] (2) Synthesis of compound 3g.

[0222]

[0223] Compound 2g (2.32g, 11.4mmol) was dissolved in DMF (50mL), and then pentabromovaleronitrile (3.7g, 22.8mmol) was added. The mixture was heated at 120°C for 24 hours under argon protection. After the reaction was completed, it was extracted with ethyl acetate, the organic phase was dried, and the solvent was removed under reduced pressure to obtain a crude compound 3g. Column chromatography was further purified to obtain a pure compound 3a (2.3g, 71% yield). MS [M+H] + Calculated value: MS286.2, found value: MS286.4.

[0224] (3) Synthesis of compound 4g.

[0225]

[0226] Add H2O (2mL), glacial acetic acid (2mL), and concentrated sulfuric acid (2mL) to the reaction flask in sequence, mix well, add compound 3g (2.2g, 7.71mmol), and heat the mixture at 70°C with stirring for 16 hours. After the reaction is completed, cool to room temperature, add H2O (10mL) to dilute the reaction solution, and then slowly add solid sodium bicarbonate until no bubbles are generated. Add DMF (10mL) to clarify the solution, and the clear solution is directly used for reverse phase purification. Purification by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column gave pure compound 4g (1.64g, 70% yield). MS [M+H] + Calculated value: MS 305.1, found: MS 305.5.

[0227] (5) Synthesis of compound LM-G.

[0228]

[0229] Compound 4g (5.5g, 18.1mmol) was dissolved in ethanol (55mL), hydrazine hydrate (18.12g, 362mmol) was added, and the mixture was heated and stirred at 50°C for 24 hours. After the reaction was completed, 1N hydrochloric acid was slowly added dropwise until the solution was weakly acidic, and the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation. DMF (100mL) was added to dissolve the sample to obtain a clear solution, which was directly used for reverse phase purification. Pure compound LM-G (4.1g, 75% yield) was obtained by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column purification. 1HNMR(400MHz,DMSO)δ11.53(s,3H),7.82(d,J=8.9Hz,1H),7.17(dd,J=9.0,2.7Hz,1H),7.02(s,1H),3.51–3 .44(m,2H),3.40(t,J=5.1Hz,2H),2.31–2.23(m,2H),1.57(p,J=3.6Hz,4H),1.13(t,J=7.0Hz,3H).MS[M+H] + Calculated value: MS 306.1, found: MS 306.5.

[0230] LM-G carboxyl activation.

[0231]

[0232] Compound LM-G (100 mg, 0.328 mmol) was dissolved in DMSO (5 mL), and then EDC (125.6 mg, 0.655 mmol) was added. After stirring at room temperature for 20 minutes under argon protection, NHS (75.4 mg, 0.655 mmol) was added, and the mixture was stirred at 40°C for 12 hours. After the reaction was completed, the compound LM-GS was separated by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column, and lyophilized to obtain compound LM-GS (58 mg, 44% yield). MS [M+H] + Calculated value: 403.2, measured value: 403.2.

[0233] Example 8

[0234] This example provides a method for preparing and activating a luminescent marker LM-H, which is carried out according to the following steps:

[0235] LM-H synthesis route:

[0236]

[0237] (1) Synthesis of compound 2h.

[0238]

[0239] Compound 1h (5.0 g, 28.4 mmol) and dimethyl sulfate (30 mL, 317 mmol) were added to a three-necked flask, and the mixture was heated at 110 ° C for 24 hours under argon protection. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 10:1) until the raw material was completely converted. After the reaction was completed, it was cooled to room temperature, H2O (50 mL) was added to the system, and it was stirred for 5 minutes and then extracted with dichloromethane. The organic phase was dried and the solvent was removed to obtain the crude compound 2h, which was further purified by column chromatography to obtain the pure compound 2h (1.94 g, 36% yield). MS [M+H] + Calculated value: 191.07, measured value: 191.1.

[0240] (2) Synthesis of compound 3h.

[0241]

[0242] Compound 2h (280 mg, 1.472 mmol) was dissolved in DMF (5 mL), and then pentabromovaleronitrile (1.2 g, 7.41 mmol) was added. The mixture was heated at 120 ° C for 24 hours under argon protection. LC-MS detected the reaction, and the raw material was basically converted into the target product. After completion, the reaction crude liquid was directly purified by silica gel column chromatography to obtain pure compound 3h (278 mg, 69.2% yield). MS [M+H] + Calculated value: 272.13, measured value: 273.0.

[0243] This step was repeated twice.

[0244] (3) Synthesis of compound 4h.

[0245]

[0246] H2O (2mL), glacial acetic acid (2mL), and concentrated sulfuric acid (4mL) were added to the reaction flask in sequence, and after mixing evenly, compound 3h (500mg, 1.843mmol) was added, and the mixture was heated and stirred at 70°C for 26 hours. After the reaction was completed, it was cooled to room temperature, and H2O (10mL) was added to dilute the reaction solution, and the solution was directly used for reverse phase purification. Purification by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column gave pure compound 4h (261mg, 48.6% yield). MS [M+H] + Calculated value: 291.13, measured value: 291.0.

[0247] (4) Synthesis of compound LM-H.

[0248]

[0249] Compound 4h (200 mg, 0.689 mmol) was dissolved in ethanol (5 mL), and then hydrazine hydrate (689 mg) was added, and the mixture was heated and stirred at 60 ° C for 8 hours. Sampling LC-MS detection showed that the raw material was basically converted into the target product. After the reaction was completed, 1N hydrochloric acid was slowly added dropwise until the solution was weakly acidic, and the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation. DMF (10 mL) was added to dissolve the sample to obtain a clear solution, which was directly used for reverse phase purification. Pure compound LM-H (131 mg, 64.8% yield) was obtained by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column purification. 1 H NMR (400MHz, DMSO) δ11.41(s,3H),7.84(d,J=8.9Hz,1H),7.22(dd,J=9.0,2.7Hz,1H),7.02(d ,J=2.6Hz,1H),3.51–3.44(m,2H),3.02(s,3H),2.28–2.23(m,2H),1.61–1.49(m,4H).MS[M+H] + Calculated value: 292.12, measured value: 292.0.

[0250] LM-H carboxyl activation.

[0251]

[0252] Compound LM-H (100 mg, 0.3435 mmol) was dissolved in DMSO (5 mL), and NHS (158 mg, 1.375 mmol) was added. The mixture was stirred at room temperature for 5 minutes under argon protection, and then EDC (200 mg, 1.05 mmol) was added. The mixture was stirred at 37°C for 12 hours. After the reaction, the mixture was purified using a reverse phase (CH3CN / H2O, 0.1% TFA) C18 column to obtain a product solution, which was further lyophilized to obtain a pure compound LM-HS (68 mg, 51% yield). MS [M+H] + Calculated value: 389.14, measured value: 389.0.

[0253] Example 9

[0254] This embodiment provides a preparation method and an activation method of a luminescent marker LM-I, which is carried out according to the following steps:

[0255] The synthetic route of LM-I is as follows:

[0256]

[0257] (1) Synthesis of compound 2i.

[0258]

[0259] Compound 1i (3 g, 17.03 mmol) and tert-butyl 6-bromohexanoate (21.4 g, 85.15 mmol), DMF (45 mL), triethylamine (8.6 g, 85.15 mmol) were added to the Shrek tube, and the mixture was heated at 80°C for 16 hours under argon protection. The reaction solution was monitored by LC-MS, and the raw materials were basically converted into products. After the reaction was completed, the crude solution was purified by reverse phase preparation and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain a solution of compound 2i. The acetonitrile was removed by rotary evaporation and freeze-dried to obtain a crude compound 2i (5.1 g, 86.5% yield), which can be directly used in the next step reaction. MS [M-(t-BuO - )] + Calculated value: 273.12, measured value: 272.0.

[0260] (2) Synthesis of compound 3i.

[0261]

[0262] Compound 2i (1g, 2.89mmol), potassium carbonate (1.2g, 8.67mmol), DMF (10mL), 2-bromoethyl ether (4.42g, 28.9mmol) were added to the sealed tube, and the mixture was stirred at 130°C for 48 hours. Samples were taken for LC-MS detection, and the raw material conversion rate was about 30%. After the reaction was completed, the crude liquid was purified by reverse phase preparation, and the compound 3i solution was obtained by reverse phase (CH3CN / H2O, 0.1% FA) C18 column purification. The acetonitrile was removed by rotary evaporation, and the aqueous phase was extracted with dichloromethane. The solvent was dried to obtain the crude compound 3i. Afterwards, a silica gel chromatography column was used to remove some impurities and pigments, and the product eluate was collected and the solvent was dried to obtain the pure compound 3 (230.3mg, 22% yield). MS [M+H] + Calculated value: 363.18, measured value: 363.0.

[0263] (3) Synthesis of LM-I.

[0264]

[0265] Compound 3 (230 mg, 0.635 mmol), ethanol (10 mL), and hydrazine hydrate (635 mg, 12.7 mmol) were added to a Shrek tube, and the mixture was heated and stirred at 60°C for 6 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation to obtain a crude product, and 3N hydrochloric acid was slowly added to the crude product until it became acidic (pH = 1-2) (stirring was required during the neutralization process, and the pH endpoint was required to remain stable for more than 10 minutes). H2O and acetonitrile were added to dissolve the sample, and the crude sample solution was directly used for reverse phase purification, and purified by reverse phase (CH3CN / H2O, 0.1% FA [formic acid]) C18 column to obtain pure compound LM-I (99.2 mg, 43% yield). 1 H NMR (400MHz, DMSO) δ11.40(s,3H),7.82(d,J=9.0Hz,1H),7.19(dd,J=9.0,2.7Hz,1H),7.05(s,1H),3.63–3.52(m,4H),3.4 4(q,J=7.0Hz,4H),2.22(t,J=7.3Hz,2H),1.55(dq,J=15.3,7.7Hz,4H),1.37–1.28(m,2H),1.09(t,J=7.0Hz,3H).MS[M+H] + Calculated value: 364.18, measured value: 364.0.

[0266] LM-I carboxyl activation

[0267]

[0268] LM-I (100 mg, 0.275 mmol) and NHS (126.7 mg, 1.10 mmol) were added to a Shrek tube, and DMSO (4 mL) was added. After argon replacement, the mixture was stirred at 40°C for 5 minutes. Then, the whole batch of EDC (158.2 mg, 0.825 mmol) was quickly added to the reaction solution and stirred at 40°C for 6 hours. The reaction was monitored by LC-MS, and the raw materials were basically converted into the target product. After the reaction was completed, the crude sample solution was directly used for reverse phase purification and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain an eluent containing the target product, which was lyophilized to obtain the target product LM-IS pure lyophilized powder (60.7 mg, 48% yield). MS [M+H] + Calculated value: 461.20, measured value: 461.2.

[0269] Example 10

[0270] This example provides a method for preparing and activating the luminescent marker LM-J, which is carried out according to the following steps:

[0271] The synthetic route of LM-J is as follows:

[0272]

[0273] (1) Synthesis of compound 2j.

[0274]

[0275] Compound 1j (3 g, 17.03 mmol) and tert-butyl 4-bromobutyrate (19 g, 85.15 mmol), DMF (45 mL), triethylamine (8.6 g, 85.15 mmol) were added to a Shrek tube, and the mixture was heated at 80°C for 16 hours under argon protection. The reaction solution was monitored by LC-MS, and the conversion rate was about 25%. After the reaction was completed, the crude solution was purified by reverse phase preparation and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain a solution of compound 2j, which was rotary evaporated and freeze-dried to obtain pure compound 2j (1084 mg, 20% yield). MS [M-(t-BuO - )] + Calculated value: 245.09, measured value: 245.0.

[0276] (2) Synthesis of compound 3j.

[0277]

[0278] Compound 2j (450 mg, 1.41 mmol), potassium carbonate (586 mg, 4.24 mmol), DMF (5 mL), 2-bromoethyl ether (2.16 g, 14.1 mmol) were added to the sealed tube, and the mixture was stirred at 130°C for 48 hours. Samples were taken for LC-MS detection, and the raw material conversion rate was about 20%. After the reaction was completed, the crude liquid was purified by reverse phase preparation, and purified by reverse phase (CH3CN / H2O, 0.1% FA [formic acid]) C18 column to obtain compound 3j solution, and acetonitrile was removed by rotary evaporation, and the remaining aqueous phase was extracted with DCM. The solvent was dried to obtain the crude compound 3j, and then a silica gel chromatography column was used to remove some impurities and pigments, and the product eluate was collected and the solvent was dried to obtain the pure compound 3j (84.8 mg, 18% yield). MS [M+H] + Calculated value: 335.15, measured value: 335.0.

[0279] (3) Synthesis of LM-J.

[0280]

[0281] Compound 3j (85 mg, 0.254 mmol), ethanol (4 mL), hydrazine hydrate (101.6 mg, 5.08 mmol) were added to a Shrek tube, and the mixture was heated and stirred at 60°C for 6 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation to obtain a crude product, and 3N hydrochloric acid was slowly added to the crude product until it became acidic (pH = 1-2) (stirring was required during the neutralization process, and the pH endpoint was required to remain stable for more than 10 minutes). H2O and acetonitrile were added to dissolve the sample, and the crude sample solution was directly used for reverse phase purification, and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain pure compound LM-J (34 mg, 40% yield). 1 H NMR (400MHz, DMSO) δ8.42(s,1H),7.82(d,J=8.9Hz,1H),7.27(d,J=9.0Hz,1H),7.11(s,1H),3.57(dd,J=12.6, 4.7Hz,4H),3.44(q,J=7.0Hz,4H),2.21(t,J=7.1Hz,2H),1.75(q,J=7.5Hz,2H),1.09(t,J=7.0Hz,3H).MS[M+H] + Calculated value: 336.15, measured value: 336.0.

[0282] The activation steps are the same as TM-I.

[0283] Embodiment 11

[0284] This example provides a method for preparing the luminescent marker LM-K, which is carried out according to the following steps:

[0285] The synthetic route of LM-K is as follows:

[0286]

[0287] (1) Synthesis of compound 2k.

[0288]

[0289] Compound 1k (2.51 g, 8.69 mmol) was added to a pressure tube and methanol (100 mL) was added to dissolve until it was clear, and then concentrated sulfuric acid (100 mL) was added under an ice-water bath and the pressure tube was sealed. The reaction was heated at 90°C for 48 h and monitored by TLC (PE:EA=8:1) until the conversion of the raw materials was complete. After the reaction was completed, the reaction solution was quenched with ice water (1 L), and then ethyl acetate (1 L) was added for extraction, and the organic phase was separated by a separatory funnel. The organic phase was dried and spin-dried to obtain a crude compound 2k (2.64 g, 95.8% yield).

[0290] (2) Synthesis of compound 3k.

[0291]

[0292] Compound 2k (2.64 g, 8.33 mmol) was added to the reaction flask and dissolved in methanol (150 mL). Ammonium chloride (2.2 g, 41.6 mmol) was dissolved in water (50 mL) and then added to the above solution. Finally, iron powder (4.66 g, 83.3 mmol) was added. The reaction was heated at 65 ° C for 2 hours under N2 protection and monitored by TLC (PE: EA = 2: 1) until the conversion of the raw materials was complete. The reaction solution was filtered with diatomaceous earth, and the filtrate was collected and dried to obtain a crude compound 3k. The crude product was purified by silica gel column to obtain pure compound 3k (1.16 g, 48.5% yield).

[0293] (3) Synthesis of compound 4k.

[0294]

[0295] Compound 3k (1.1 g, 3.83 mmol), tert-butyl 4-bromobutyrate (8.5 g, 38.3 mmol) and triethylamine (3.88 g, 38.3 mmol) were added to the pressure tube in sequence and the pressure tube was sealed. The reaction was heated at 80°C for 24 hours and monitored by TLC (PE:EA=2:1) ​​until most of the raw materials were converted. After completion, the reaction solution was extracted with EA, and the organic phase was dried and then spin-dried to obtain a crude compound 4k. The crude product was purified by silica gel column to obtain pure compound 4k (0.56 g, 47.8% yield).

[0296] (4) Synthesis of compound 5k.

[0297]

[0298] Compound 4k (0.56 g, 1.3 mmol) was added to a pressure tube and dissolved in DMF (7 mL), followed by iodoethane (6.08 g, 39 mmol) and potassium carbonate (0.54 g, 3.9 mmol), and the pressure tube was sealed. The reaction was heated at 70 °C for 14 hours, and TLC (PE: EA = 4: 1) was used to monitor the conversion of most of the raw materials. After the reaction was completed, EA was added to the reaction solution for extraction, and the organic phase was dried and then spin-dried to obtain a crude compound 5k. The crude product was purified by silica gel column to obtain a pure compound 5k (0.29 g, 48.8% yield). MS [M+H] + Calculated value: MS 458.12, found value: MS 457.9.

[0299] (5) Synthesis of compound 6k.

[0300]

[0301] Compound 5k (0.15 g, 0.33 mmol), tetrakis(triphenylphosphine)palladium (38 mg, 0.033 mmol), sodium carbonate (277.7 mg, 2.62 mmol), dioxane (5 mL) and water (1 mL) were added to the Shrek tube. The system was replaced with nitrogen. Trimethylcyclotriboroxane (383 μL, 1.31 mmol) was added under nitrogen protection. The reaction was heated at 90 °C for 6 hours under nitrogen protection, and LC-MS was monitored until the conversion of the raw materials was complete. After completion, the reaction solution was filtered through diatomaceous earth, and the filtrate was dried to obtain the crude compound 6k. The crude product was passed through a silica gel column, eluted with PE:EA=4:1, and the corresponding eluent was collected and dried to obtain the pure compound 6k (106 mg, 82% yield). MS [M+H] + Calculated value: MS 394.22, found value: MS 394.2.

[0302] (6) Synthesis of compound 7k.

[0303]

[0304] Compound 6k (106 mg, 0.27 mmol) was added to a Shrek tube and dissolved in ethanol (5 mL), followed by 85% hydrazine hydrate (0.6 mL, 10 mmol). The reaction was heated at 50 °C for 24 h, and LC-MS monitored the conversion of most of the raw materials. The reaction solution was dried by spin drying, water (50 mL) was added and the pH was adjusted to 2, EA (50 mL) was added, and extraction was performed. The organic phase was collected and dried to obtain a crude compound 7k (90 mg, 92.2% yield). MS [M+H] + Calculated value: MS 362.21, found: MS 362.4.

[0305] (7) Synthesis of compound LM-K.

[0306]

[0307] Take all the crude compound 7k obtained in the previous step, add 4M HCl dioxane solution (3mL), stir at room temperature for 20min, and monitor by LC-MS until most of the raw materials are converted. The reaction solution is spin-dried and purified by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column to obtain pure compound LM-K (45mg, 54.7% yield). 1 H NMR (400MHz, DMSO) δ6.97 (s, 1H), 6.91 (s, 1H), 2.71 (s, 3H), 2.31 (t, J = 7.1Hz ,2H),1.76(q,J=6.4,5.3Hz,4H),1.35(s,2H),1.12(t,J=6.9Hz,3H).MS[M+H]+ Calculated value: MS 306.14, found: MS 306.0.

[0308] The activation steps are the same as LM-B.

[0309] Example 12

[0310] This example provides a method for preparing a luminescent marker LM-L, which is carried out according to the following steps:

[0311] The synthetic route of LM-L is as follows:

[0312]

[0313] (1) Synthesis of compound 21.

[0314]

[0315] Compound 1l (3 g, 10.4 mmol) and ammonium chloride (2.8 g, 52.3 mmol) were added to a round-bottom flask and dissolved with methanol (90 mL) and water (30 mL). Then, reduced iron powder (5.8 g, 103.6 mmol) was weighed and added to the above mixture. The reaction was carried out at 65 ° C for 12 hours under nitrogen protection. TLC was used for monitoring and the reaction was stopped when the raw materials reacted completely. After the reaction was completed, the filtrate was collected by diatomaceous earth filtration. After the filtrate was evaporated to remove most of the methanol, water (20 mL) was added, and then the crude liquid was extracted with dichloromethane, the organic phase was collected, and the solvent was removed by vortexing to obtain the crude compound 2l (2.5 g, 9.6 mmol, 92% yield). MS [M+H] + Calculated value: 260.08, measured value: 228.4.

[0316] (2) Synthesis of compound 31.

[0317]

[0318] Compound 21 (1 g, 3.9 mmol), DMF (15 mL), tert-butyl 4-bromobutyrate (3.6 mL, 20.3 mmol) and triethylamine (2.8 mL, 20.3 mmol) were added to a sealed pressure tube in sequence, and the tube was sealed and reacted at 80°C for 12 hours. After the reaction, the reaction solution was cooled to room temperature, the crude solution was directly liquid injected, and purified by reverse phase (CH3CN / H2O, 0.1% TEA) C18 column to obtain pure compound 31 (1.0 g, 2.5 mmol, 63% yield).

[0319] (3) Synthesis of compound 41.

[0320]

[0321] Compound 3l (1.0 g, 2.5 mmol), DMF (15 mL), potassium carbonate (1 g, 7.2 mmol) and iodoethane (4 mL, 50 mmol) were added to a sealed pressure tube in sequence, and the tube was sealed and reacted at 50°C for 12 hours. After the reaction, the reaction solution was cooled to room temperature, the crude solution was directly injected, and purified by reverse phase (CH3CN / H2O, 0.1% TEA) C18 column to obtain pure compound 4l (816.1 mg, 1.9 mmol, 76% yield). MS [M+H] + Calculated value: 430.22, measured value: 431.0.

[0322] (4) Synthesis of compound 51.

[0323]

[0324] Compound 41 (816.1 mg, 1.9 mmol) was dissolved in ethanol (8 mL), hydrazine hydrate (2.9 mL, 57 mmol) was added, and the mixture was reacted at 50 °C for 10 hours. The reaction was monitored by LCMS until most of the raw materials were converted. After the reaction, ethanol was removed by rotary evaporation, and the crude product was redissolved in water (20 mL) and acetonitrile (20 mL), and concentrated hydrochloric acid was added dropwise to adjust the pH to 1-2 to obtain a crude solution of compound 51. MS [M+H] + Calculated value: 398.20, measured value: 398.0.

[0325] (5) Synthesis of compound LM-L

[0326]

[0327] Hydrochloric acid (4M in dioxane, 48mL, 192.0mmol) was added dropwise to the crude solution of compound 51 obtained in the previous step. After the addition was complete, the reaction was allowed to react at room temperature for 1 hour. The reaction was monitored by LCMS and stopped when most of compound 51 was converted. After removing most of the organic solvent by vacuum rotary evaporation with a water pump, a small amount of DMF was added to the crude solution until the solution was clarified. The crude solution was directly injected and purified by reverse phase (CH3CN / H2O, 0.1% FA) C18 column to obtain pure compound LM-L (530mg, 1.6mmol, 84% yield). 1HNMR (400 MHz, DMSO) δ 11.73 (s, 3H), 9.33 (s, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.90 (d, J = 9.1 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H), 7.39 (dd, J = 9.1, 2.7 Hz, 1H), 3.52 (q, J = 7.1 Hz, 2H), 3.45 (t, J = 7.6 Hz, 2H), 2.35 (t, J = 7.3 Hz, 2H), 1.85 (p, J = 7.4 Hz, 2H), 1.19 (t, J = 6.9 Hz, 3H). MS [M+H]+ calculated value: 342.14, found value: 342.0.

[0328] LM-L carboxyl activation

[0329]

[0330] LM-L (200 mg, 0.59 mmol) and NHS (276 mg, 2.36 mmol) were added to a Shrek tube, and DMSO (10 mL) was added. After argon replacement, the mixture was stirred at 40°C for 5 minutes. Then, the entire batch of EDC (344 mg, 1.77 mmol) was quickly added to the reaction solution and stirred at 40°C for 6 hours. The reaction was stopped when the conversion rate of the target product was higher than 80% by LC-MS monitoring. After the reaction was completed, an activated solution was obtained, which can be directly used for labeling. MS [M+H] + Calculated value: 439.15, measured value: 438.9.

[0331] Example 13

[0332] This example provides a method for preparing a luminescent marker LM-M, which is carried out according to the following steps:

[0333] The synthetic route of LM-M is as follows:

[0334]

[0335] (1) Synthesis of compound 2m.

[0336]

[0337] Compound 1m (3 g, 10.4 mmol) was dissolved in methanol (90 mL), and then reduced iron powder (5.8 g, 104 mmol) was added and stirred to obtain a mixed solution. Ammonium chloride (2.8 g, 52 mmol) was dissolved in H2O (30 mL) and added to the mixed solution. The reaction solution was heated to reflux at 65°C for 12 hours, and TLC (DCM, RF = 0.3) was used to monitor the reaction until it was complete. After the reaction was completed, the solution was filtered through diatomaceous earth, and the filtrate was collected and the methanol was removed by rotary evaporation under reduced pressure. Then, the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and then rotary evaporation was performed to remove dichloromethane to obtain a crude compound 2m (2.3 g, 8.9 mmol, 86% yield). MS [M+H] + Calculated value: 259.08, measured value: 228.4.

[0338] (2) Synthesis of compound 3m.

[0339]

[0340] Compound 2m (1 g, 3.9 mmol) was dissolved in DMF (15 mL), and then tert-butyl 6-bromohexanoate (6.4 mL, 31.2 mmol) and triethylamine (4.3 mL, 31.2 mmol) were added, and the mixture was heated at 80 ° C for 12 hours. The reaction was monitored by TLC (DCM, RF = 0.7) until the raw material was basically converted. DMF (15 mL) was added to clarify the solution, and the clear solution was directly used for reverse phase purification. Purification by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column gave pure compound 3m (1.1 g, 2.5 mmol, 66% yield).

[0341] Add materials to two pots in parallel.

[0342] (3) Synthesis of compound 4m.

[0343]

[0344] Compound 3m (1.87 g, 4.4 mmol), DMF (30 mL), 1-bromo-ethoxyethane (5.2 mL, 44 mmol) and potassium carbonate (1.82 g, 13.2 mmol) were added to a sealed tube in sequence, and the reaction was heated at 130°C for 12 hours. TLC (DCM: MeOH = 10: 1, RF = 0.5) was used to monitor until the conversion of the raw materials was complete. After completion, the mixture was cooled to room temperature, and the filtrate was collected by filtration and directly used for purification on a reverse phase (CH3CN / H2O, 0.1% TFA) C18 column to obtain crude compound 4m, which was further purified by silica gel (DCM / MeOH) to obtain pure compound 4m (180 mg, 0.4 mmol, 9% yield). MS [M+H] +Calculated value: 446.21, measured value: 446.0.

[0345] (4) Synthesis of compound LM-M.

[0346]

[0347] Compound 4m (180 mg, 0.4 mmol) was dissolved in ethanol (8 mL), hydrazine hydrate (405 uL, 8 mmol) was added, and the mixture was heated and stirred at 50 ° C for 10 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation, 1N hydrochloric acid was slowly added dropwise until the solution was weakly acidic, and DMF (5 mL) was added to dissolve the sample to obtain a clear solution, which was directly used for reverse phase purification. The pure compound LM-M (120 mg, 0.29 mmol, 73% yield) was obtained by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column purification. 1 H NMR (400MHz, DMSO) δ11.73(s,2H),9.35(s,1H),8.11(d,J=8.6Hz,1H),7.89(d,J=9.2Hz,1H),7.59(s,1H),7.36(dd,J=9.2,2.7Hz,1H),3.62(dt, J=9.5,5.0Hz,4H),3.48(q,J=6.9Hz,4H),2.23(t,J=7.3Hz,2H),1.60(dq,J=30.7,7.4Hz,4H),1.43–1.34(m,2H),1.11(t,J=7.0Hz,3H).MS[M+H] + Calculated value: 414.20, measured value: 414.0.

[0348] The carboxyl activation procedure was the same as that of LM-L.

[0349] Embodiment 14

[0350] This example provides a method for preparing a luminescent marker LM-N, which is carried out according to the following steps:

[0351] The LM-N synthesis route is as follows:

[0352]

[0353] (1) Synthesis of compound 2n.

[0354]

[0355] Compound 1n (3 g, 10.4 mmol) was dissolved in methanol (90 mL), and then reduced iron powder (5.8 g, 104 mmol) was added and stirred to obtain a mixed solution. Ammonium chloride (2.8 g, 52 mmol) was dissolved in H2O (30 mL) and added to the mixed solution. The reaction solution was heated to reflux at 65°C for 12 hours, and TLC (DCM, RF = 0.3) was used to monitor the reaction until it was complete. After the reaction was completed, the solution was filtered through diatomaceous earth, and the filtrate was collected and the methanol was removed by rotary evaporation under reduced pressure. Then, the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and then rotary evaporation was performed to remove dichloromethane to obtain a crude compound 2n (2.4 g, 9.3 mmol, 89% yield). MS [M+H] + Calculated value: 259.08, measured value: 228.4.

[0356] (2) Synthesis of compound 3n.

[0357]

[0358] Compound 2n (1 g, 3.9 mmol) was dissolved in dioxane (20 mL), and propane sultone (2.4 g, 19.3 mmol) was then added and heated at 100 °C for 12 hours under argon protection. The reaction was monitored by LC-MS until the raw material was substantially converted. After completion, the mixture was cooled to room temperature, and DCM (40 mL) was added to precipitate the product, and the filter cake was collected by filtration. The filter cake was dissolved in water (20 mL), and the clear solution was directly used for reverse phase purification. Purification by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column gave pure compound 3n (780 mg, 2.1 mmol, 53% yield). MS [M+H] + Calculated value: 382.09, measured value: 382.0.

[0359] (3) Synthesis of compound 4n.

[0360]

[0361] Compound 3n (500 mg, 1.3 mmol), DMF (15 mL), tert-butyl 6-bromohexanoate (5.5 mL, 26.2 mmol) and potassium carbonate (542 mg, 3.9 mmol) were added to a sealed tube in sequence, and the reaction was heated at 130°C for 6 hours. LC-MS monitoring was performed until most of the raw materials were converted. After completion, the mixture was cooled to room temperature, and the filtrate was collected by filtration and directly used for purification on a reverse phase (CH3CN / H2O, 0.1% TFA) C18 column to obtain crude compound 4n, which was further purified by a silica gel column to obtain pure compound 4n (200 mg, 0.4 mmol, 30% yield). MS [MH] - Calculated value: 494.16, measured value: 494.0.

[0362] (4) Synthesis of compound LM-N.

[0363]

[0364] Compound 4n (200 mg, 0.4 mmol) was dissolved in ethanol (8 mL), hydrazine hydrate (405 uL, 8 mmol) was added, and the mixture was heated and stirred at 55 ° C for 10 hours. After the reaction was completed, the solvent and excess hydrazine hydrate were removed by vacuum rotary evaporation, 1N hydrochloric acid was slowly added dropwise until the solution was weakly acidic, and water (5 mL) was added to dilute the sample to obtain a clear solution, which was directly used for reverse phase purification. The pure compound LM-N (140 mg, 0.3 mmol, 75% yield) was obtained by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column purification. 1 HNMR (400MHz, DMSO) δ9.68(s,1H),8.25(d,J=8.6Hz,1H),8.09(s,1H),7.80(s,1H),7.68(s,1H),3.65(t,J=8.1Hz,2H),3.53(t, J=8.1Hz,2H),2.56(t,J=7.3Hz,2H),2.18(t,J=7.3Hz,2H),1.91-1.81(m,2H),1.57-1.47(m,4H),1.34(q,J=7.4Hz,2H).MS[MH] - Calculated value: 462.14, measured value: 461.9.

[0365] The carboxyl activation procedure was the same as that of LM-L.

[0366] Embodiment 15

[0367] This example provides a method for preparing and activating a luminescent marker LM-O, which is carried out according to the following steps:

[0368] The synthetic route of LM-O is as follows:

[0369]

[0370] (1) Synthesis of compound 2o.

[0371]

[0372] Compound 1o (3.2 g, 19.7 mmol) and NH4SCN (3 g, 39.5 mmol) were added to methanol (150 mL), and the mixture was stirred in an ice bath and cooled to 0°C. Liquid bromine (1.7 mL) was taken, diluted with methanol (50 mL), and slowly added to the mixture in an ice bath using a constant pressure dropping funnel. After the addition, the mixture was reacted in an ice bath for 1 h, and then at 25°C for 48 h. The reaction was monitored by LCMS until most of the raw materials reacted completely. Pure compound 2o (777 mg, 18% yield) was purified by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column. MS [M+H] + Calculated value: 220.0, measured value: 220.4.

[0373] Repeat this step several times.

[0374] (2) Synthesis of compound 3o.

[0375]

[0376] Compound 2o (1.5 g, 6.85 mmol) and hydrazine hydrate (20 g, 400.0 mmol) were added to ethanol (20 mL), and the mixture was heated under reflux for 24 hours and monitored by LCMS until the reaction was complete. After the reaction was completed, ethanol and hydrazine hydrate were removed by rotary evaporation under reduced pressure, and then the pH was adjusted to 2-3 with 1N HCl to obtain crude compound 3o. Pure compound 3o (1.24 g, 87% yield) was obtained by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column purification. MS [M+H] + Calculated value: 210.0, measured value: 210.1.

[0377] (3) Synthesis of compound 5o.

[0378]

[0379] Compound 4o (1.0 g, 6.71 mmol), cesium carbonate (4.3 g, 13.2 mmol) and pentabromovaleronitrile (22 g, 135.8 mmol) were added to 1,4-dioxane (2 mL), argon was replaced, and the mixture was heated under reflux for 16 hours and monitored by TLC until the reaction was complete. After the reaction was completed, the solvent was removed by rotation to obtain a crude mixture. Further purification by silica gel chromatography (hexane / EA=10:1) gave pure compound 5o (927 mg, 60% yield). MS [M+H] + Calculated value: 231.1, measured value: 231.5.

[0380] Repeat this step several times.

[0381] (4) Synthesis of compound 6o.

[0382]

[0383] Compound 5o (1.0 g, 4.34 mmol) was added to a mixture of concentrated sulfuric acid (1 mL), purified water (1 mL), and glacial acetic acid (1 mL), and the mixture was heated to 70°C for 16 hours and monitored by LCMS until the reaction was complete. After the reaction was completed, a small amount of water was added to dilute, and then sodium bicarbonate was slowly added in batches to adjust the pH to 2. Compound 6o (789 mg, 73% yield) was purified by C18 reverse phase column (CH3CN / H2O, 0.1% FA). MS [M+H] + Calculated value: 250.1, measured value: 250.5.

[0384] (5) Synthesis of compound LM-O.

[0385]

[0386] Compound 6o (500 mg, 2.0 mmol), compound 3o (836 mg, 4.0 mmol), sodium sulfite (160 mg, 1.27 mmol) and Na2HPO3 (493 mg, 3.47 mmol) were added to DMF (40 mL), and then 1.5 M sulfuric acid (40 mL) was added. The mixture was heated at 80°C for 1.5 hours and monitored by LCMS until the reaction was complete. After the reaction was completed, a small amount of ice water was added, and the mixture was frozen in an ice bath. The solid was precipitated and filtered to obtain the crude compound LM-O, which was then recrystallized from acetonitrile to obtain the pure compound 7 (263 mg, 30% yield). 1 H NMR (400MHz, DMSO) δ11.70(s,3H),8.76(s,1H),8.40(s,1H),7.94(d,J=9.1Hz,2H),6.82(d,J=9.0Hz,2H),3 .46(q,J=7.0Hz,2H),2.33–2.24(m,2H),1.58(t,J=3.4Hz,4H),1.23(s,2H),1.14(t,J=6.9Hz,3H).MS[M+H] + Calculated value: 439.1, measured value: 439.2.

[0387] LM-O carboxyl activation.

[0388]

[0389] Compound LM-O (210 mg, 0.479 mmol) was dissolved in DMSO (10 mL), and then EDC (184 mg, 0.958 mmol) was added. After stirring at room temperature for 20 minutes under argon protection, NHS (110 mg, 0.958 mmol) was added, and the mixture was stirred at 37°C for 12 hours. After the reaction was completed, compound LM-OS was separated by reverse phase (CH3CN / H2O, 0.1% TFA) C18 column and lyophilized to obtain pure compound LM-OS (33 mg, 13% yield). MS [M+H] + Calculated value: 536.2, measured value: 536.1.

[0390] Comparative Example 1

[0391] This comparative example provides luminol, which is commercially available.

[0392] Comparative Example 2

[0393] This comparative example provides ABEI (N-(4-aminobutyl)-N-ethylisoluminol), which is commercially available. ABEI-A is an activated intermediate of ABEI, and its structural formula is ABEI-A can be prepared according to the method described in the patent specification of WO2012066003A1 and can be directly used for protein labeling.

[0394] Test example

[0395] (I) Pure product light intensity verification

[0396] 1) Preparation of luminescent marker solution: Dissolve the luminescent marker in DMF to make 1×10 -3 M DMF solution, and then diluted to 1×10 -8 M for standby use.

[0397] 2) Light intensity test: Take 1×10 -8 10 μL of the test substance solution was tested on the immunoassay analyzer MAGLUMI X3 platform produced by Xinye Biotechnology, and the chemiluminescence intensity information of 0.1-3.1s was collected. The results are shown in Table 1 below.

[0398] Table 1

[0399]

[0400] The results showed that, except for a few markers, the light intensity of most markers was higher than that of luminol, among which the light intensities of LM-A, LM-E, LM-G, LM-H, LM-H, LM-I, and LM-K exceeded that of ABEI-A.

[0401] (II) Verification of light intensity of luminescent labeling complex

[0402] To verify the luminescence intensity of the luminescent labeling complex formed by the aforementioned luminescent labeling substance after protein labeling, the detection steps are as follows:

[0403] 1) Activation of carboxyl group: All luminescent markers must activate the carboxyl group before labeling with BSA (bovine serum albumin) and convert the carboxyl group into NHS activated ester. The luminescent marker (0.05mmol) was dissolved in DMSO (1mL), and then EDC (0.2mmol) was added. After stirring at room temperature for 20 minutes under argon protection, NHS (0.2mmol) was added and the mixture was stirred at 40°C for 12 hours. After the reaction was completed, the reaction conversion rate was monitored by LC-MS, and the molar concentration of the luminescent marker activated ester was calculated accordingly. The luminescent marker activated ester reaction solution was directly used for labeling.

[0404] 2) BSA labeling: Weigh BSA (1 mg) into a vial and dissolve it in 0.1 M NaHCO3 (0.1 mL) solution to a final concentration of 10 mg / mL, corresponding to a BSA molar concentration of approximately 1.5 × 10 -4 M; according to the labeling molar ratio of 10:1, the above NHS activated ester solution and ABEI-A solution were added to the BSA solution (slowly added dropwise under stirring), stirred at room temperature for 1 hour, and then desalted to 0.01M PBS solution (0.1mL). The final concentration of the BSA marker was about 1.5×10 -4 M.

[0405] 3) Light intensity test: dilute the BSA marker with 0.1M PBS to 1.5×10 -8 M, set aside. Take 1.5×10 -8 10 μL of the analyte solution was tested on the MAGLUMI X3 platform of Xinye Biotechnology, and the chemiluminescence intensity information of 0.1-3.1s was collected. The results are shown in Table 2.

[0406] Table 2

[0407]

[0408]

[0409] The results showed that the detection light intensity after BSA labeling with LM-H, LM-L, LM-M, and LM-N was doubled compared with ABEI. The detection light intensity after labeling with LM-A, LM-D, LM-F, LM-G, LM-J, LM-K, and LM-O was improved to varying degrees compared with ABEI. LM-B and LM-E showed light intensities comparable to ABEI. Although the light intensity of LM-I was weaker than that of ABEI, it could still meet the light intensity performance required for detection.

[0410] (III) Label coupling ratio

[0411] The activated state of ABEI (ABEI-A) was used as a control to verify the coupling ratio of the activated state of LM-H and the labeled protein. This experiment continued to use protein BSA to verify the marker coupling ratio. Mass spectrometry was used to determine how many luminescent marker molecules a BSA protein could label. That is, if a BSA protein labeled 2 marker molecules, the coupling ratio was expressed as 2:1. The test results are shown in Table 3.

[0412] Table 3

[0413]

[0414] The results showed that after LM-H was activated, the coupling efficiency of ABEI was significantly increased compared with that of BSA. The reason was that the activation step of ABEI required an intermediate reaction to convert the amino group into a carboxyl group. The chain of the activated intermediate formed was longer. When labeling the protein, the swing of the long chain would produce steric hindrance. The activated structure of LM-H has a shorter chain, which can overcome the above steric hindrance and increase the coupling ratio, which is beneficial to increase the light intensity after labeling.

[0415] (IV) Stability verification

[0416] To verify the stability of the marker, the prepared marker-BSA was placed in a 37°C environment (normal storage temperature is 2-8°C) and monitored continuously for 7 days. The changes in its luminescence intensity were recorded and the relative deviation of the light intensity change was calculated. The calculation method was (initial light intensity-light intensity after 7 days) / initial light intensity×100%. The test concentration of BSA marker was 1.5×10 - 8 M, on the first, third, fifth and seventh days, 10 μL of the test solution was taken and tested on the New Industry Bio MAGLUMI X3 platform to collect 0.1-3.1s chemiluminescence intensity information. The results are shown in Table 4 below:

[0417] Table 4

[0418]

[0419] It can be seen from the above table that the 7-day deviation of most markers in this scheme does not exceed 40%, which can meet the actual needs. Among them, the deviations of LM-H and LM-J are only 19.6% and 16.9%.

[0420] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0421] The luminescent marker provided in the present application not only has good luminescent intensity, but also can generate a luminescent marker intermediate directly coupled to the protein under the action of an activator. The labeling process is simple, and the steric hindrance is lower during labeling, which can effectively improve the labeling efficiency while ensuring the luminescent performance.

[0422] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A luminescent marker, characterized in that: The luminescent marker has a structure shown in general formula (I): Wherein, Ar is selected from any one of the general formulas (II) to (IV): m is an integer between 0 and 2. When m=2, each R is the same or different; n is an integer between 0 and 4, and when n≥2, each R is the same or different; R is selected from C1-C10 alkyl; X is selected from C1-C10 alkylene, wherein the C atom or H atom is optionally substituted by an O atom, a S atom, an aryl group or a heteroaryl group; Y is selected from a C1-C10 alkyl group and a C1-C10 alkyl sulfonic acid group; and the C atom in the alkyl group may be selectively substituted by an O atom, a S atom, an aryl group or a heteroaryl group.

2. The luminescent marker according to claim 1, characterized in that R is selected from C1-C3 alkyl; and / or, m is 0 or 1; and / or, n is 0; and / or, X is selected from C1-C6 alkylene groups, wherein the C atoms are selectively substituted by O atoms; And / or, Y is selected from C1-C7 alkyl sulfonic acid group, C1-C6 alkyl group, and the C atom in the alkyl group may be selectively substituted by an O atom.

3. The luminescent marker according to claim 1, characterized in that R is selected from C1-C2 alkyl; and / or, m is 0; and / or, X is selected from C3-C6 alkylene, wherein the C atom is optionally substituted by an O atom; And / or, Y is selected from C2-C4 alkyl sulfonic acid group, C1-C5 alkyl group, and the C atom in the alkyl group can be selectively substituted by an O atom.

4. The luminescent marker according to claim 1, characterized in that The luminescent marker is selected from at least one of the following compounds:

5. A luminescent labeling intermediate solution, characterized in that: The components of the luminescent labeling intermediate solution include a luminescent labeling intermediate and an organic solvent, wherein the luminescent labeling intermediate is formed by activating the luminescent labeling substance according to any one of claims 1 to 4 by an activator.

6. The luminescent labeling intermediate solution according to claim 5, characterized in that: The activator comprises an amide condensation agent; Preferably, the amide condensation agent includes at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or dicyclohexylcarbodiimide.

7. The luminescent labeling intermediate solution according to claim 6, characterized in that: The activator further comprises N-hydroxysuccinimide, and the luminescent labeling intermediate has a structure as shown in the following formula (V):

8. The method for preparing the luminescent labeling intermediate solution according to any one of claims 5 to 7, characterized in that: The preparation method comprises: Dispersing the luminescent marker in the organic solvent, adding the activator and mixing to perform an activation reaction, so that the luminescent marker is activated by the activator to form a luminescent marker intermediate, thereby obtaining the luminescent marker intermediate solution; Preferably, the activator comprises an amide condensing agent and optionally N-hydroxysuccinimide; Preferably, the molar ratio of the N-hydroxysuccinimide to the luminescent marker is ≥1, preferably (1.2-4):1; Preferably, the temperature of the activation reaction is 10-50° C., and the time of the activation reaction is 8-20 h.

9. Use of the luminescent marker according to any one of claims 1 to 4 or the luminescent marker intermediate solution according to any one of claims 5 to 7 in preparing a luminescent marker complex.

10. A luminescent labeling complex, characterized in that: The luminescent marker complex is formed by coupling the luminescent marker according to any one of claims 1 to 4 with a protein, and the luminescent marker complex has a structure shown in the following formula (VI):

11. A method for preparing the luminescent labeling complex according to claim 10, characterized in that: The method for preparing the luminescent marker complex comprises: mixing the luminescent marker intermediate solution according to any one of claims 5 to 7 with a protein to perform a coupling reaction to obtain the luminescent marker complex.

12. Use of the luminescent marker according to any one of claims 1 to 4, the luminescent marker intermediate solution according to any one of claims 5 to 7, or the luminescent marker complex according to claim 10 in luminescent immunoassay.

13. A kit, characterized in that: The kit comprises the luminescent marker according to any one of claims 1 to 4, the luminescent marker intermediate solution according to any one of claims 5 to 7, or the luminescent marker complex according to claim 10.

Images