A dimethyltryptamine artificial hapten, artificial antigen, and preparation method and application thereof

Abstract

The present invention discloses a dimethyltryptamine artificial hapten, an artificial antigen, and a preparation method and application thereof. The molecular structural formula of the dimethyltryptamine artificial hapten is shown in Formula I, and the molecular structural formula of the dimethyltryptamine artificial antigen is shown in Formula II. The present invention also discloses the application of the dimethyltryptamine artificial antigen in the preparation of anti-dimethyltryptamine antibodies. The dimethyltryptamine artificial hapten of the present invention retains the characteristic structure of dimethyltryptamine to the greatest extent, and has an active group that can be coupled with a carrier protein, and can serve as an antigenic determinant. The dimethyltryptamine artificial antigen obtained by further preparation can be immunized to obtain anti-dimethyltryptamine antibodies with high affinity, high sensitivity, and strong specificity. The titer of the immune serum obtained by immunizing New Zealand white rabbits is as high as 1:85,000, and can be used for rapid and accurate immunodetection and immunoanalysis of dimethyltryptamine.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to a dimethyltryptamine artificial hapten, an artificial antigen, and a preparation method and application thereof. Background Art

[0002] Dimethyltryptamine (DMT) is a new psychoactive substance of the tryptamine class.

[0003] According to available data, tryptamine hallucinogens are a class of compounds containing the tryptamine structural framework and possessing hallucinogenic properties. These hallucinogenic effects are typically manifested by physical symptoms such as dizziness, weakness, tremors, nausea, drowsiness, paresthesias, and blurred vision; sensory symptoms such as distorted shapes and colors, difficulty concentrating, a perceived hearing loss, and, in rare cases, sensory disturbances; and psychiatric symptoms such as mood changes, nervousness, a distorted sense of time, an inability to express one's thoughts, depersonalization, dreamlike sensations, and visual hallucinations. Long-term use of these hallucinogens can lead to psychiatric symptoms and even addiction. For regulatory purposes, a rapid method for detecting dimethyltryptamine is needed.

[0004] Currently, the detection of dimethyltryptamine mainly relies on high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), mass spectrometry (MS), etc. However, these methods have disadvantages such as expensive instruments, time-consuming detection, and the need for professional technicians to operate, which cannot meet the modern detection requirements of rapidity and accuracy.

[0005] Immunoassays can overcome all of these shortcomings. Immunoassays are analytical methods that utilize the specific binding reaction between antigens and antibodies to detect various substances (drugs, hormones, proteins, microorganisms, etc.). The key to developing immunoassays for small molecule compounds is the ability to produce antibodies with high affinity and specificity for these compounds. However, since most small molecule compounds (molecular weight less than 1000), including dimethyltryptamine, are not immunogenic, meaning they lack T cell epitopes and cannot directly induce the production of specific antibodies in animals, small molecules are therefore called haptens. Through appropriate chemical modification, a linker with an active group at a specific position in the hapten molecular structure is added. This is then combined with a macromolecular carrier to produce a hapten-carrier conjugate (i.e., an artificial antigen). Artificial antigens can indirectly induce the proliferation and differentiation of B cells through the use of T cell epitopes, leading to the production of specific antibodies. Therefore, the efficient synthesis of artificial antigens is a prerequisite and key to ensuring the effectiveness of immunoassays.

[0006] CN 115521240A discloses a dimethyltryptamine hapten and an artificial antigen, as well as a preparation method and application thereof. The dimethyltryptamine artificial hapten disclosed in this patent is derivatized using the amino group on the indole ring. Although the derivatization group is subsequently removed, in practice, the derivatization group is not completely removed. The NH group, as the main characteristic functional group of dimethyltryptamine, plays a decisive role in the specificity of the antigen. If the removal is not complete, the main characteristic functional group NH is shielded, and the prepared antigen has poor specificity and low potency. Furthermore, this patent directly connects anhydride at the 5-position, resulting in two overly long hydrocarbon chains in the hapten molecule. If the hydrocarbon chains are too long, the hapten molecule itself is easily folded and cannot be fully unfolded, resulting in the characteristic structural portion of the hapten molecule being easily masked by the carrier protein, affecting the specificity and affinity of the antigen. Summary of the Invention

[0007] The present invention provides a dimethyltryptamine artificial hapten, which retains the characteristic structure of dimethyltryptamine to the greatest extent, has an active group that can be coupled with a carrier protein, and can serve as an antigenic determinant.

[0008] To achieve the above technical objectives, the present invention is implemented through the following technical solutions:

[0009] The first object of the present invention is to provide a dimethyltryptamine artificial hapten, the molecular structure of which is shown in I:

[0010]

[0011] A second object of the present invention is to provide a method for preparing the dimethyltryptamine artificial hapten, which comprises the following steps:

[0012] (1) indole and oxalyl chloride undergo a substitution reaction to prepare a compound represented by formula A;

[0013] (2) the compound represented by formula A is subjected to a substitution reaction with methylamine to obtain the compound represented by formula B;

[0014] (3) The compound represented by formula B is subjected to a reduction reaction with lithium aluminum hydride to obtain the compound represented by formula C;

[0015] (4) The compound represented by Formula C is subjected to a substitution reaction with ethyl 4-bromobutyrate to obtain a compound represented by Formula D;

[0016] (5) hydrolyzing the compound represented by Formula D and adjusting the pH to be acidic to obtain the dimethyltryptamine artificial hapten represented by Formula I;

[0017] The reaction formula of the method is shown below:

[0018]

[0019] Furthermore, the method is preferably carried out according to the following steps:

[0020] (1) Indole is dissolved in anhydrous ether, stirred and mixed with oxalyl chloride at 0-5°C, and then sealed and placed at -20--15°C for reaction. After the reaction is completed, the solvent is evaporated from the reaction solution to obtain the compound represented by formula A;

[0021] Furthermore, the indole is preferably dissolved in anhydrous ether, stirred at 0-5°C (generally in an ice bath) for 0.5-1 hour, and then oxalyl chloride is added dropwise. After the addition is complete, stirring is continued at 0-5°C (generally in an ice bath) for 0.5-1 hour, and then the mixture is sealed and placed at -20--15°C for a reaction of 4-6 hours.

[0022] The molar ratio of indole to oxalyl chloride is 1:3-4.

[0023] The volume of the anhydrous ether used is 200-400 mL / g based on the mass of indole.

[0024] Under the reaction conditions, indole is relatively soluble in anhydrous ether. The solvent is evaporated from the reaction solution to obtain a yellow solid, which is the compound represented by Formula A. The yield and purity are high, and the post-processing procedure is relatively simple.

[0025] Preferably, when adding oxalyl chloride dropwise, the addition rate of oxalyl chloride is controlled by a syringe to reduce the by-products produced by the reaction, thereby achieving the purpose of improving the yield and purity. Preferably, the addition rate is controlled at 1 to 10 drops / minute, more preferably 5 drops / minute.

[0026] (2) The compound represented by formula A and a methylamine aqueous solution are stirred at a temperature of 0 to 5° C. (generally in an ice bath) to carry out a substitution reaction. After the reaction is completed, the reaction solution is post-treated to obtain the compound represented by formula B;

[0027] The mass fraction of methylamine in the methylamine aqueous solution is 30-40%, preferably 40%.

[0028] Preferably, compound A represented by formula A is added to the methylamine aqueous solution in batches, which can control the generation of reaction by-products and improve the yield and purity.

[0029] The reaction time is preferably 2 to 3 hours.

[0030] The molar ratio of the compound represented by formula A to methylamine is 1:10-20, preferably 1:13.

[0031] The post-treatment steps of the reaction solution in step (2) are generally as follows: after the reaction is completed, the reaction solution is filtered, washed with water and anhydrous ether, and the solid precipitate is collected and dried to obtain the compound shown in formula B.

[0032] Under these reaction conditions, the yield of light yellow solid B is high, the post-processing procedure is relatively simple, and purification is relatively easy. Washing the precipitate with purified water and anhydrous ether can remove byproducts or impurities in the reaction system, thereby achieving the purpose of general purification.

[0033] (3) Under nitrogen protection, the compound represented by formula B is heated to 65-70° C. in anhydrous tetrahydrofuran solvent to carry out a reduction reaction. After the reaction, the reaction solution is post-treated to obtain the compound represented by formula C;

[0034] The molar ratio of the compound represented by formula B to lithium aluminum hydride is preferably 1:5-15, more preferably 1:9-10.

[0035] The reaction time is preferably 15 to 25 hours.

[0036] The volume usage of the anhydrous tetrahydrofuran is generally 50 to 150 mL / g based on the mass of the compound represented by formula B.

[0037] In the step (3), the compound represented by formula B is dissolved in anhydrous tetrahydrofuran, and the solution is green and turbid. Lithium aluminum hydride is slowly added, and the solution is heated under nitrogen protection to undergo a reduction reaction, and the solution changes from green turbidity to off-white turbidity.

[0038] The post-treatment step of the reaction solution in step (3) is generally as follows: after the reaction is completed, the reaction solution is cooled to room temperature, placed in an ice bath, a small amount of water is added, the amount ratio of the added water to the lithium aluminum hydride is 4 to 5:1, and then the pH is adjusted to 8 to 9 with a 30 wt % sodium hydroxide aqueous solution, filtered, the residue is washed with a small amount of tetrahydrofuran, the filtrate is combined and the solvent is evaporated to obtain an oily substance, which is dissolved in dichloromethane and then washed with water and saturated brine. The organic phase is collected, dried, filtered, and the solvent is evaporated. The obtained oily substance is then purified by thin layer chromatography to obtain the compound shown in Formula C;

[0039] The chromatographic solution used in the thin layer chromatography method is a mixed solvent of ethyl acetate and ethanol in a volume ratio of 5:1, and the product Rf=0.2.

[0040] (4) Adding the compound represented by Formula C and ethyl 4-bromobutyrate to an isopropanol solvent, heating to 58-63°C (preferably 60°C) in the presence of N,N-diisopropylethylamine (DIPEA) and sodium iodide to carry out a substitution reaction, and after completion of the reaction, post-treating the reaction solution to obtain the compound represented by Formula D;

[0041] The molar ratio of the compound represented by formula C, ethyl 4-bromobutyrate, N,N-diisopropylethylamine and sodium iodide is 1:1-1.5:2-3:1-1.5, preferably 1:1.5:2:1.

[0042] The reaction time is preferably 15 to 25 hours.

[0043] The volume usage of the isopropyl alcohol is generally 20 to 50 mL / g based on the mass of the compound represented by Formula C.

[0044] The post-treatment steps of the reaction solution in step (4) are generally as follows: after the reaction is completed, the reaction solution is evaporated to remove the solvent, the residue is dissolved with dichloromethane, washed with saturated sodium bicarbonate aqueous solution, water, and saturated brine, the organic phase is collected, dried, filtered, and the solvent is evaporated to obtain the compound represented by formula D;

[0045] The residue is washed with a saturated sodium bicarbonate aqueous solution and saturated brine to remove by-products or impurities in the reaction system, thereby achieving the purpose of approximate purification.

[0046] (5) The compound represented by Formula D is dissolved in tetrahydrofuran and anhydrous methanol, and a 1N aqueous sodium hydroxide solution is added. The mixture is rapidly stirred at room temperature to carry out a hydrolysis reaction. After the reaction is completed, the pH is adjusted to 4-5. The resulting reaction solution is post-treated to prepare the dimethyltryptamine artificial hapten represented by Formula I;

[0047] The reaction time is preferably 4 to 6 hours.

[0048] The molar ratio of the compound represented by formula D to NaOH in a 1N sodium hydroxide aqueous solution is generally 1:10 to 30;

[0049] The volume of anhydrous methanol is generally 10 to 20 mL / g based on the mass of the compound represented by formula D;

[0050] The volume amount of tetrahydrofuran is generally 10 to 20 mL / g based on the mass of the compound represented by formula D;

[0051] After the reaction is completed, 1N hydrochloric acid is generally used to adjust the pH to 4-5.

[0052] The post-treatment steps of the reaction solution in step (5) are generally as follows: the reaction solution is extracted with dichloromethane, the organic phases are combined, dried, filtered, the filtrate is evaporated to remove the solvent, and the obtained oil is purified by thin layer chromatography to obtain the dimethyltryptamine artificial hapten shown in formula I

[0053] The chromatographic solution used in the thin layer chromatography method is a mixed solvent of 95% ethanol, 1,4-dioxane, dichloromethane, and ammonia water in a volume ratio of 8:1:10:1, and the product Rf is 0.3.

[0054] The mass fraction of ammonia water is 25-28%.

[0055] Preferably, under the reaction conditions, the compound represented by Formula D is easier to hydrolyze, the subsequent treatment procedure is simpler, and it is easier to purify.

[0056] Preferably, extraction with dichloromethane three times can maximize the extraction of the target product.

[0057] The present invention retains the NH characteristic functional group of dimethyltryptamine through the above method, and introduces a linker arm at the N position at the end of the hydrocarbon chain of dimethyltryptamine. The introduction of the linker arm at the modification site can retain the characteristic structure of dimethyltryptamine to the greatest extent, and the modification site is as far away from the characteristic functional group of dimethyltryptamine as possible, so that the characteristic part of the dimethyltryptamine is exposed to the outside to the greatest extent, thereby avoiding interference with specific antigenic determinants and maximizing recognition by the immune body.

[0058] Compared with a circular connecting arm, the connecting arm used in the present invention is chain-shaped and has an appropriate length, which ensures that the dimethyltryptamine hapten small molecule can be fully exposed on the surface of the artificial antigen, thereby reducing the recognition of the connecting arm by T cells during immunization as much as possible. In this way, the antibodies obtained by immunization have stronger specificity and affinity for dimethyltryptamine.

[0059] The third object of the present invention is to provide a dimethyltryptamine artificial antigen, the molecular structure of which is shown in II:

[0060]

[0061] In formula II, KLH is keyhole limpet hemocyanin.

[0062] The fourth object of the present invention is to provide a method for preparing the dimethyltryptamine artificial antigen, which comprises combining the dimethyltryptamine artificial hapten shown in formula I with keyhole limpet hemocyanin by a mixed anhydride method to obtain the dimethyltryptamine artificial antigen shown in formula II.

[0063] Furthermore, when the mixed anhydride method is used to prepare the dimethyltryptamine artificial antigen, the method comprises the following steps:

[0064] (a) A dimethyltryptamine artificial hapten represented by Formula I, isobutyl chloroformate, and triethylamine are stirred in N,N-dimethylformamide (DMF) solvent at 0-5°C (generally in an ice bath) for 2-4 hours. After the reaction, the supernatant is collected by centrifugation;

[0065] The molar ratio of the dimethyltryptamine artificial hapten represented by formula I, isobutyl chloroformate, and triethylamine is 1:1.5-2.5:1-1.5, preferably 1:2:1.

[0066] The volume usage of the DMF solvent is 30-80 mL / g, preferably 50 mL / g, based on the mass of the dimethyltryptamine artificial hapten shown in Formula I.

[0067] (b) The supernatant is added dropwise to the keyhole limpet hemocyanin solution, and the resulting mixture is allowed to stand at 3-5°C overnight (usually for 10-24 hours). The supernatant is dialyzed and centrifuged to obtain the dimethyltryptamine artificial antigen represented by Formula II.

[0068] Unless otherwise specified, the keyhole limpet hemocyanin solution of the present invention is prepared by dissolving keyhole limpet hemocyanin in 0.01 M PBS buffer solution with a pH of 7.2 to 7.4.

[0069] In step (b), the concentration of the keyhole limpet hemocyanin solution is 5 mg / mL, and the volume ratio of the supernatant to the keyhole limpet hemocyanin solution is 1:5-6.

[0070] The present invention uses keyhole limpet hemocyanin (KLH) as a macromolecular carrier, which has the following advantages over bovine serum albumin (BSA): ① KLH has stronger immunogenicity, possesses numerous antigenic determinants, and is likely to induce a stronger immune response; ② Because KLH originates from mollusks, it is phylogenetically distant from mammalian species and is less likely to cross-react with target samples during assays. BSA has the disadvantage that, in many experiments, it is used as a blocking agent. If antisera derived from hapten-BSA conjugates are used in such assays, false positives often occur because these sera contain anti-BSA antibodies; ③ KLH has many lysine residues, making it easy to couple with haptens with high coupling efficiency, and is less likely to generate polyclonal antibodies, making it easier to enhance antibody specificity. Anti-DMT antibodies obtained by immunizing animals with the DMT artificial antigen formed by combining KLH with the DMT artificial hapten have better specificity.

[0071] The present invention also provides the use of the dimethyltryptamine artificial antigen in preparing anti-dimethyltryptamine antibodies.

[0072] The fifth object of the present invention is to provide the use of the dimethyltryptamine artificial hapten or dimethyltryptamine artificial antigen in the preparation of anti-dimethyltryptamine antibodies

[0073] The sixth object of the present invention is to provide an anti-dimethyltryptamine antibody, which is a globulin obtained by immunizing animals with the dimethyltryptamine artificial antigen and can produce a specific immune reaction with dimethyltryptamine.

[0074] The seventh object of the present invention is to provide the use of the anti-dimethyltryptamine antibody in detecting dimethyltryptamine.

[0075] The eighth object of the present invention is to provide a reagent for detecting dimethyltryptamine, wherein the reagent comprises the anti-dimethyltryptamine antibody.

[0076] A ninth object of the present invention is to provide a kit for detecting dimethyltryptamine, wherein the kit comprises the anti-dimethyltryptamine antibody.

[0077] Experiments have shown that the titer of the immune serum obtained by immunizing New Zealand white rabbits with the artificial dimethyltryptamine antigen was 1:85,000. This indicates that the artificial dimethyltryptamine antigen of the present invention can be used to immunize New Zealand white rabbits to obtain anti-dimethyltryptamine antibodies with high affinity, high sensitivity, and strong specificity. The anti-dimethyltryptamine antibodies can be used for immunodetection and analysis of dimethyltryptamine.

[0078] Compared with the prior art, the present invention has the following beneficial effects:

[0079] The artificial dimethyltryptamine hapten of the present invention retains the characteristic structure of dimethyltryptamine to the greatest extent, and has an active group that can be coupled with a carrier protein, and can serve as an antigenic determinant. The artificial dimethyltryptamine antigen further prepared can be used for immunization to obtain anti-dimethyltryptamine antibodies with high affinity, high sensitivity and strong specificity. The titer of the immune serum obtained by immunizing New Zealand white rabbits is as high as 1:85,000, and the hapten can be used for rapid and accurate immunodetection and immunoanalysis of dimethyltryptamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is the reaction formula for preparing the dimethyltryptamine artificial antigen II in Example 1 of the present invention.

[0081] Wherein, DEE represents anhydrous diethyl ether, DIPEA represents N,N-diisopropylethylamine, DMF represents N,N-dimethylformamide, THF represents tetrahydrofuran, MeOH represents anhydrous methanol, KLH represents keyhole limpet hemocyanin, and Et3N represents triethylamine, the same below;

[0082] Figure 2 This is a liquid chromatogram of the dimethyltryptamine artificial hapten I prepared in Example 1 of the present invention.

[0083] The horizontal axis of the spectrum is time, unit is min; the vertical axis is the response value, unit is mAU.

[0084] Figure 3 This is the mass spectrum of the dimethyltryptamine artificial hapten I prepared in Example 1 of the present invention.

[0085] Wherein, RelativeAbundance represents relative abundance; m / z represents charge-to-mass ratio.

[0086] Figure 4 These are UV scans of keyhole limpet hemocyanin, dimethyltryptamine artificial hapten I, and dimethyltryptamine artificial antigen II.

[0087] Wherein, Abs represents the UV-visible absorption spectrum, and WL (nm) represents the wavelength (nm).

[0088] Figure 5 This is the reaction formula for preparing dimethyltryptamine artificial antigen IV in comparative example 1.

[0089] Wherein, Pyridine represents pyridine, BSA represents bovine serum albumin, and DCC represents N,N'-dicyclohexylcarbodiimide, and the same applies hereinafter.

[0090] Figure 6 This is the reaction formula for preparing dimethyltryptamine artificial antigen V in comparative example 2.

[0091] Figure 7 This is the reaction formula for the preparation of dimethyltryptamine artificial antigen VI in comparative example 3.

[0092] Figure 8 This is the reaction formula for the preparation of dimethyltryptamine artificial antigen VII in comparative example 4.

[0093] Figure 9 This is the reaction formula for the preparation of dimethyltryptamine artificial antigen VIII in comparative example 5.

[0094] Figure 10 This is the reaction formula for the preparation of dimethyltryptamine artificial antigen IX in comparative example 6.

[0095] Figure 11 This is the reaction formula for preparing dimethyltryptamine artificial antigen X in comparative example 7. DETAILED DESCRIPTION

[0096] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings, specific embodiments and comparative examples, but the protection scope of the present invention is not limited thereto.

[0097] Example 1

[0098] Preparation method of dimethyltryptamine artificial antigen II (reaction formula as shown in Figure 1 ), comprising the following steps:

[0099] (1) Preparation of artificial hapten I:

[0100] ① Dissolve 637 mg (5.444 mmol) of indole in 150 ml of anhydrous ether and place in a 250 ml three-necked round-bottom flask. Stir and react in an ice bath for 0.5 hour. Slowly add 1.4 ml (16.332 mmol) of oxalyl chloride dropwise with a syringe (drop rate 5 drops / minute). The solution gradually turns yellow. Continue stirring and react in an ice bath for 0.5 hour until a yellow precipitate is produced. Seal the reaction flask with sealing film and place in a -20°C refrigerator for 4 hours. After the reaction is complete, directly dry to obtain 1.138 g of yellow solid A.

[0101] The yellow solid A was subjected to TLC detection, the chromatographic solvent was ethyl acetate, and the product Rf = 0.2.

[0102] ② Place 7 ml of 40 wt% methylamine aqueous solution in a 50 ml round-bottom flask and stir under ice bath conditions. Add 1.138 g of yellow solid A to the reaction flask in three batches. During the process, white mist is generated and the solution becomes turbid. Continue stirring under ice bath for 2 hours. Filter the reaction solution and wash the precipitate with 30 ml of water and 30 ml of anhydrous ether. Collect the solid and place it in a 50 ml flask and dry it to obtain 575 mg of light yellow solid B.

[0103] The light yellow solid product B was subjected to TLC detection. The chromatographic solvent was ethyl acetate:ethanol in a volume ratio of 5:1. The product Rf was 0.8.

[0104] ③ 575 mg (2.847 mmol) of light yellow solid B was dissolved in 50 ml of anhydrous tetrahydrofuran. The solution was green and turbid. 1082 mg (28.47 mmol) of lithium aluminum hydride was slowly added. Under nitrogen protection, the reaction was refluxed at 68 ° C for 17 hours. The solution changed from green turbidity to off-white turbidity. After the reaction was completed, it was cooled to room temperature and placed in an ice bath. 2.05 ml (113.88 mmol) of purified water was added and the pH was adjusted to 8-9 with 30 wt% aqueous sodium hydroxide solution. The mixture was filtered and the residue was washed with 5 ml of tetrahydrofuran. The filtrate was dried to obtain a light yellow oil. The oil was dissolved in 30 ml of dichloromethane and washed with 20 ml × 3 purified water and 20 ml of saturated brine in sequence. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and dried to obtain 398 mg of a yellow oil. 127 mg of a yellow oil C was obtained by thin layer chromatography. The solvent and eluent were anhydrous ethanol.

[0105] The yellow oily product C was subjected to TLC detection. The chromatographic solvent was ethyl acetate:ethanol volume ratio = 5:1, Rf = 0.2;

[0106] The chromatographic solvent used in the thin layer chromatography method was ethyl acetate:ethanol = 5:1, and the product Rf = 0.2.

[0107] ④ 127 mg (0.730 mmol) of the obtained yellow oily product C was dissolved in 3.7 ml of isopropanol, and 157 ul (1.095 mmol) of ethyl 4-bromobutyrate, 254 ul (1.46 mmol) of N,N-diisopropylethylamine, and 110 mg (0.730 mmol) of sodium iodide were added in sequence (molar ratio of reactants: yellow oily product C / ethyl 4-bromobutyrate / N,N-diisopropylethylamine / sodium iodide = 1 / 1.5 / 2 / 1). The reaction was refluxed at 60°C for 17 hours. After the reaction was completed, the mixture was directly dried. The residue was dissolved in 20 ml of dichloromethane and washed in sequence with 20 ml of saturated sodium bicarbonate aqueous solution, 20 ml of purified water, and 20 ml of saturated brine. The organic phase was collected, dried, filtered, and dried to obtain 204 mg of brown oily product D.

[0108] The brown oil D was subjected to TLC detection. The chromatographic solvent was ethyl acetate:ethanol in a volume ratio of 5:1. The product Rf was 0.7.

[0109] ⑤ Dissolve 204 mg of brown oil D in 2.28 ml of tetrahydrofuran and 2.77 ml of anhydrous methanol, add 12.24 ml of 1N aqueous sodium hydroxide solution, react with rapid stirring at room temperature for 4 hours, adjust the pH to 4-5 with 1N hydrochloric acid, extract with 30 ml x 3 of dichloromethane, combine the organic phases, dry, filter, and transfer to dryer to obtain 170 mg of brown-red oil, which is purified by thin-layer chromatography to obtain 67 mg of dimethyltryptamine artificial hapten I, with anhydrous ethanol as solvent and eluent;

[0110] The dimethyltryptamine artificial hapten I was subjected to TLC detection, the chromatographic solution was 95 vol% ethanol:1,4-dioxane:dichloromethane:25 wt% ammonia water in a volume ratio of 8:1:10:1, and the product Rf was 0.3;

[0111] The chromatographic solution used in the thin layer chromatography method was 95% ethanol:1,4-dioxane:dichloromethane:ammonia water=8:1:10:1, and the product Rf=0.3.

[0112] The liquid chromatogram of dimethyltryptamine artificial hapten Ⅰ is shown in Figure 2 (UV detector, wavelength 290nm), the mass spectrum of dimethyltryptamine artificial hapten I is shown in Figure 3 .

[0113] from Figure 2 It can be seen that the purity of the purified dimethyltryptamine artificial hapten reaches more than 99.9%. Figure 3It can be seen that the mass-to-charge ratio (m / z) of the M+H peak of the dimethyltryptamine artificial hapten obtained in this example is 261.15, which is consistent with its theoretical relative molecular weight of 260. The mass-to-charge ratios (m / z) of its other three major fragment ion peaks are 260.15, 246.15, and 216.16, respectively, which are consistent with the theoretical molecular weights of its three major fragments of 259, 245, and 215. Based on the above data, it can be preliminarily determined that the final compound obtained in step ⑤ is the dimethyltryptamine artificial hapten I designed by the present invention.

[0114] (2) Preparation of dimethyltryptamine artificial antigen II:

[0115] ⑥ Place 67 mg (0.258 mmol) of dimethyltryptamine artificial hapten I in a 50 ml round-bottom flask, add 3.35 ml of N,N-dimethylformamide (DMF), then add 35.7 ul (0.258 mmol) of triethylamine, place in an ice bath and stir for 30 minutes, then add 66.7 ul (0.516 mmol) of isobutyl chloroformate, stir in an ice bath for 2 hours, centrifuge after the reaction is completed, take the supernatant and set aside.

[0116] ⑦ Weigh 14.5 g (0.0405 mol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) of sodium chloride, and 1.495 g (0.00958 mol) of sodium dihydrogen phosphate dihydrate and dissolve them in double-distilled water to a volume of 5.0 L to obtain 0.01 M PBS buffer solution with a pH of 7.4.

[0117] ⑧ Weigh 0.2 g of keyhole limpet hemocyanin and dissolve it in 40 ml of the PBS buffer prepared in step ⑦ to obtain a keyhole limpet hemocyanin solution with a concentration of 5 mg / ml.

[0118] ⑨ Under rapid stirring, the supernatant from step ⑥ was slowly added dropwise to the keyhole limpet hemocyanin solution. The volume ratio of the supernatant to the keyhole limpet hemocyanin solution was 1:5. The resulting mixture was stored at 4°C overnight to obtain an artificial antigen mixture.

[0119] ⑩ Transfer the artificial antigen mixture into a dialysis bag and dialyze 9 times with the PBS buffer in step ⑦. After the dialysis is completed, centrifuge and collect the supernatant to obtain artificial antigen II: dimethyltryptamine-keyhole limpet hemocyanin conjugate. The UV scanning images of dimethyltryptamine artificial antigen II before and after preparation are shown in Figure 4 .

[0120] Figure 4In the figure, curve a is the UV scan of dimethyltryptamine artificial hapten I, curve b is the UV scan of dimethyltryptamine artificial antigen II, and curve c is the UV scan of keyhole limpet hemocyanin. The maximum absorption wavelength of dimethyltryptamine artificial hapten I is 290 nm, the maximum absorption wavelength of keyhole limpet hemocyanin is 283 nm, and the maximum absorption wavelength of dimethyltryptamine artificial antigen II is 275 nm. Compared with dimethyltryptamine hapten I and keyhole limpet hemocyanin, the maximum absorption wavelength of dimethyltryptamine artificial antigen II shows a significant shift, indicating that dimethyltryptamine hapten I and keyhole limpet hemocyanin are successfully coupled.

[0121] Comparative Example 1

[0122] Preparation method of dimethyltryptamine artificial antigen IV (reaction formula as shown Figure 5 ), including the following steps:

[0123] (1) Preparation of dimethyltryptamine artificial hapten III:

[0124] ①②③ are the same as in Example 1;

[0125] ④ 127 mg (0.730 mmol) of the obtained yellow oily product C was dissolved in 12.7 ml of pyridine and placed in a 50 ml round-bottom flask. 109.5 mg (1.095 mmol) of succinic anhydride was added and refluxed at 100°C for 20 hours. After the reaction was completed, the mixture was directly dried to obtain 296 mg of a light yellow oil. 108 mg of dimethyltryptamine artificial hapten III was purified by thin layer chromatography using anhydrous ethanol as the solvent and eluent.

[0126] TLC was performed on the artificial hapten III of dimethyltryptamine. The chromatographic solvent was ethyl acetate:ethanol volume ratio = 5:1. The product Rf = 0.5.

[0127] The chromatographic solvent used in the thin layer chromatography method was ethyl acetate:ethanol = 5:1, and the product Rf = 0.5.

[0128] (2) Preparation of dimethyltryptamine artificial antigen IV:

[0129] ⑤ Place 108 mg (0.394 mmol) of dimethyltryptamine artificial hapten III in a 50 ml round-bottom flask, add 5.4 ml of N,N-dimethylformamide (DMF), then add 68 mg (0.591 mmol) of N-hydroxysuccinimide (NHS) and 122 mg (0.591 mmol) of cyclohexylcarbodiimide (DCC), stir at room temperature and react overnight. After the reaction is complete, centrifuge and collect the supernatant for later use.

[0130] ⑥ Weigh 14.5 g (0.0405 mol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) of sodium chloride, and 1.495 g (0.00958 mol) of sodium dihydrogen phosphate dihydrate and dissolve them in double distilled water to 5.0 L to obtain 0.01 M PBS buffer solution with a pH of 7.4.

[0131] ⑦ Weigh 0.135 g of bovine serum albumin and dissolve it in 27 ml of the PBS buffer prepared in step ⑥ to obtain a bovine serum albumin solution with a concentration of 5 mg / ml.

[0132] ⑧ Under rapid stirring, the supernatant of step ⑤ was slowly added dropwise to the bovine serum albumin solution, the volume ratio of the supernatant to the bovine serum albumin solution was 1:5, and the resulting mixture was stored at 4°C overnight to obtain an artificial antigen mixture.

[0133] ⑨ Transfer the artificial antigen mixture into a dialysis bag and dialyze it 9 times with the PBS buffer in step ⑥. After the dialysis is completed, centrifuge and collect the supernatant to obtain artificial antigen IV: dimethyltryptamine-bovine serum albumin conjugate.

[0134] Comparative Example 2

[0135] Preparation method of dimethyltryptamine artificial antigen V (reaction formula as shown in Figure 6 ), including the following steps:

[0136] (1) Preparation of dimethyltryptamine artificial hapten III:

[0137] ①-④ are the same as Comparative Example 1.

[0138] (2) Preparation of dimethyltryptamine artificial antigen V:

[0139] Keyhole limpet hemocyanin was used as a carrier to couple with dimethyltryptamine artificial hapten III. The coupling steps ⑤ to ⑨ were the same as those in Comparative Example 1 to obtain dimethyltryptamine artificial antigen V.

[0140] Comparative Example 3

[0141] Preparation method of dimethyltryptamine artificial antigen VI (reaction formula as shown in Figure 7 ), including the following steps:

[0142] (1) Preparation of dimethyltryptamine artificial hapten III:

[0143] ①-④ are the same as Comparative Example 1.

[0144] (2) Preparation of dimethyltryptamine artificial antigen VI:

[0145] ⑤ Weigh 108 mg (0.394 mmol) of dimethyltryptamine artificial hapten III and place it in a 50 ml round-bottom flask. Add 5.4 ml of N,N-dimethylformamide (DMF) and then 54.5 μl (0.394 mmol) of triethylamine. Stir on ice for 30 min. Then add 101.9 μl (0.788 mmol) of isobutyl chloroformate. Continue stirring on ice for 2 h. Centrifuge after the reaction is complete and take the supernatant for later use.

[0146] ⑥-⑨ were the same as in Comparative Example 1; dimethyltryptamine artificial antigen VI was obtained.

[0147] Comparative Example 4

[0148] Preparation method of dimethyltryptamine artificial antigen VII (reaction formula as shown in Figure 8 ), including the following steps:

[0149] (1) Preparation of dimethyltryptamine artificial hapten III:

[0150] ①-④ are the same as Comparative Example 1.

[0151] (2) Preparation of dimethyltryptamine artificial antigen:

[0152] Keyhole limpet hemocyanin was used as a carrier to couple with dimethyltryptamine artificial hapten III. The coupling steps ⑤ to ⑨ were the same as those in Comparative Example 3 to obtain dimethyltryptamine artificial antigen VII.

[0153] Comparative Example 5

[0154] Preparation method of dimethyltryptamine artificial antigen VIII (reaction formula as shown in Figure 9 ), including the following steps:

[0155] (1) Preparation of dimethyltryptamine artificial hapten I:

[0156] ①-⑤ are the same as in Example 1.

[0157] (2) Preparation of dimethyltryptamine artificial antigen VIII:

[0158] ⑥ Weigh 67 mg (0.258 mmol) of dimethyltryptamine artificial hapten I into a 50 ml round-bottom flask, add 3.35 ml of N,N-dimethylformamide (DMF), then add 44.5 mg (0.387 mmol) of N-hydroxysuccinimide (NHS) and 79.7 mg (0.387 mmol) of cyclohexylcarbodiimide (DCC), stir at room temperature for 18 hours, centrifuge after the reaction, and collect the supernatant for later use.

[0159] ⑦ Weigh 14.5 g of disodium hydrogen phosphate dodecahydrate, 43.875 g of sodium chloride, and 1.495 g of sodium dihydrogen phosphate dihydrate and dissolve them in double-distilled water to a volume of 5.0 L to obtain 0.01 M PBS buffer solution with a pH of 7.4.

[0160] ⑧ Weigh 0.085g of bovine serum albumin and dissolve it in 17ml of PBS buffer to obtain a 5mg / ml bovine serum albumin solution.

[0161] ⑨ Under rapid stirring, slowly add the supernatant dropwise to the bovine serum albumin solution, the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, and the resulting mixture is stored at 4°C overnight to obtain the artificial antigen mixture.

[0162] ⑩ Transfer the artificial antigen mixture into a dialysis bag and dialyze it 9 times with the above-mentioned PBS buffer. Centrifuge after dialysis and take the supernatant to obtain dimethyltryptamine artificial antigen VIII.

[0163] Comparative Example 6

[0164] Preparation method of dimethyltryptamine artificial antigen IX (reaction formula as shown in Figure 10 ), including the following steps:

[0165] (1) Preparation of dimethyltryptamine artificial hapten I:

[0166] ①-⑤ are the same as in Example 1.

[0167] (2) Preparation of dimethyltryptamine artificial antigen IX:

[0168] Keyhole limpet hemocyanin was used as a carrier and coupled with dimethyltryptamine artificial hapten I. The coupling steps ⑥-⑩ were the same as those in Comparative Example 5 to obtain dimethyltryptamine artificial antigen IX.

[0169] Comparative Example 7

[0170] Preparation method of dimethyltryptamine artificial antigen X (reaction formula as shown in Figure 11 ), including the following steps:

[0171] (1) Preparation of dimethyltryptamine artificial hapten I:

[0172] ①-⑤ are the same as in Example 1.

[0173] (2) Preparation of dimethyltryptamine artificial antigen X:

[0174] Bovine serum albumin was used as a carrier and coupled with dimethyltryptamine artificial hapten I. The coupling steps ⑥ to ⑩ were the same as those in Example 1 to obtain dimethyltryptamine artificial antigen X.

[0175] Test Example: Performance Determination of Dimethyltryptamine Artificial Antigen

[0176] (1) Identification of dimethyltryptamine artificial antigen:

[0177] Molar Absorption Coefficient ε: Prepare dimethyltryptamine hapten solutions at concentrations of 0 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, and 40 μg / ml in PBS buffer. UV scanning indicates that the maximum absorption wavelength of dimethyltryptamine hapten is 290 nm. Measure absorbance at 290 nm, with replicate samples prepared for each concentration. The molar absorption coefficient (i.e., molar absorption coefficient) is calculated as: ε = absorbance / molar concentration.

[0178] Determination of conjugate protein concentration: Prepare 1 ml of each keyhole limpet hemocyanin solution with PBS buffer at concentrations of 0 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, and 120 μg / ml. Add 3 ml of Coomassie Brilliant Blue staining solution, mix immediately, and warm in a 30°C water bath for 5 minutes. Prepare replicates for each concentration, measure the absorbance at 655 nm, and plot a curve showing the relationship between protein concentration and absorbance. Dilute the dimethyltryptamine artificial antigen solution (prepared with PBS buffer) in a specific ratio, measure the absorbance of the dimethyltryptamine artificial antigen at 655 nm, and read the corresponding protein concentration of the dimethyltryptamine artificial antigen solution from the curve.

[0179] Coupling ratio determination: prepare 100 μg / ml keyhole limpet hemocyanin PBS solution, dilute the conjugate (i.e., dimethyltryptamine artificial antigen) to 100 μg / ml with PBS, measure the absorbance value A1 at 275 nm, and measure the absorbance value A2 with PBS as blank. The coupling ratio γ is: γ=[(A1-A2) / ε] / (100×10 -3 / 400000).

[0180] Where ε is the molar absorption coefficient (L / mol), 400,000 is the molecular weight of keyhole limpet hemocyanin, and 100×10 -3 is the keyhole limpet hemocyanin concentration (g / L).

[0181] When bovine serum albumin is used as a carrier, the coupling ratio is calculated as follows: γ = [(A1-A2) / ε] / (100×10 -3 / 65000); wherein 65000 is the molecular weight of bovine serum albumin.

[0182] Table 1 Coupling ratio and molar absorption coefficient of each dimethyltryptamine artificial antigen

[0183] serial number Artificial antigens Coupling ratio Conjugate protein concentration Molar absorption coefficient Example 1 Ⅱ 28 3.816mg / ml 6111.88 Comparative Example 1 Ⅳ 12 3.256mg / ml 6218.38 Comparative Example 2 Ⅴ 5 1.852mg / ml 6218.38 Comparative Example 3 Ⅵ 14 2.845mg / ml 6218.38 Comparative Example 4 Ⅶ 18 0.252mg / ml 6218.38 Comparative Example 5 Ⅷ 3 3.214mg / ml 6111.88 Comparative Example 6 Ⅸ 24 3.568mg / ml 6111.88 Comparative Example 7 Ⅹ 7 1.088mg / ml 6111.88

[0184] As can be seen from Table 1, the structure of the artificial hapten, the activation method of the artificial hapten and the structure of the carrier protein all have an impact on the binding ratio when the artificial hapten is cross-linked with the carrier protein.

[0185] (2) Animal immunization

[0186] Each of the prepared dimethyltryptamine artificial antigens was used to immunize New Zealand white rabbits, and the titer of the obtained immune serum was tested by ELISA. The test results are shown in Table 2.

[0187] Table 2 Titer test results of each immune serum

[0188]

[0189]

[0190] As shown in Table 2, compared with Example 1, the titers of the immune sera obtained by immunizing animals with the dimethyltryptamine artificial antigens in each comparative example were all low, making them unusable in immunoassays. The dimethyltryptamine artificial antigen VII obtained in Comparative Example 4 immediately showed a large amount of precipitation during dialysis, while the dimethyltryptamine artificial antigen V obtained in Comparative Example 2 was relatively turbid and showed a large amount of precipitation after cryopreservation, indicating poor stability and thus unsuitable for use as an immune antigen. However, the immune serum obtained by immunizing animals with the dimethyltryptamine artificial antigen II showed a titer of 1:85,000, making it fully usable in immunoassays and providing a more convenient, rapid, and accurate method for the detection of dimethyltryptamine.

Claims

1. A dimethyltryptamine artificial antigen, the molecular structure of which is shown in II: ， In formula II, KLH is keyhole limpet hemocyanin.

2. The method for preparing the dimethyltryptamine artificial antigen according to claim 1, wherein The method comprises the following steps: combining the dimethyltryptamine artificial hapten represented by formula I with keyhole limpet hemocyanin by a mixed acid anhydride method to prepare the dimethyltryptamine artificial antigen represented by formula II; 。 3. The method for preparing the dimethyltryptamine artificial antigen according to claim 2, wherein The method comprises the following steps: (a) reacting the dimethyltryptamine artificial hapten represented by formula I, isobutyl chloroformate, and triethylamine in N,N-dimethylformamide solvent at 0-5°C with stirring for 2-4 hours, and centrifuging to obtain the supernatant; ， The molar ratio of the dimethyltryptamine artificial hapten represented by formula I, isobutyl chloroformate, and triethylamine is 1:1.5-2.5:1-1.5; (b) adding the supernatant dropwise to the keyhole limpet hemocyanin solution, and allowing the resulting mixture to stand at 3-5°C overnight. The supernatant was collected by dialysis and centrifugation to obtain the dimethyltryptamine artificial antigen represented by Formula II.

4. The method for preparing the dimethyltryptamine artificial antigen according to claim 3, wherein In the step (b), the concentration of the keyhole limpet hemocyanin solution is 5 mg / mL, and the volume ratio of the supernatant to the keyhole limpet hemocyanin solution is 1:5-6.

5. Use of the dimethyltryptamine artificial antigen according to claim 1 in the preparation of anti-dimethyltryptamine antibodies.

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