Methods of preparing conjugates

Abstract

The present application relates to a preparation method of a conjugate. Specifically, the 6-phosphogluconate dehydrogenase mutant of the present application comprises one mutation or a combination thereof selected from D306C, D375C, G426C compared with wild-type 6-phosphogluconate dehydrogenase. The detection kit prepared by using the 6-phosphogluconate dehydrogenase mutant of the present application has high specificity, high sensitivity, convenient operation, short detection time, accurate quantification, and is suitable for high-throughput detection.

Description

[0001] This application is a divisional application of Chinese patent application filed on January 8, 2020, entitled “6-phosphate dehydrogenase mutant and its use in the preparation of rapamycin detection reagent” (application number 2020100165353). Technical Field

[0002] This application relates to the field of biological detection, and in particular to a mutant enzyme, glucose-6-phosphate dehydrogenase (G6PDH), and its application in a rapamycin detection kit. Background Technology

[0003] Haptens are certain small molecules (molecular weight less than 4000 Da) that, when alone, cannot induce an immune response (i.e., they lack immunogenicity). However, when they cross-link or bind with large protein molecules or non-antigenic carriers such as polylysine, they acquire immunogenicity and induce an immune response. These small molecules can bind to response effect products and thus possess antigenicity. They only exhibit immunoreactivity but lack immunogenicity; they are also known as incomplete antigens.

[0004] A hapten is an antigen that can bind to a corresponding antibody to produce an antigen-antibody reaction, but cannot independently stimulate the production of antibodies in humans or animals. It only has immunoreactivity, not immunogenicity, and is also called an incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If a hapten is chemically bound to a protein molecule (carrier), it will acquire new immunogenicity and stimulate the animal to produce corresponding antibodies.

[0005] Small molecule antigens or haptens lack two or more sites suitable for sandwich assays, therefore they cannot be detected using the double-antibody sandwich method; instead, a competitive mode is often used. The principle is that the antigen in the sample and a certain amount of enzyme-labeled antigen compete for binding to the solid-phase antibody. The higher the antigen content in the sample, the less enzyme-labeled antigen binds to the solid phase, resulting in a lighter color development. This method is commonly used for the ELISA assay of small molecule hormones and drugs.

[0006] The structural formula of rapamune is shown below:

[0007]

[0008] Rapamycin (also known as sirolimus) is a hydrophobic macrocyclic triene lactone synthesized by actinomycetes and belongs to the lipophilic molecule family. It carries a lactone ring, with hydroxyl, methyl, or ethane substitutions at the 12-, 14-, or 16-positions. When used in combination with cyclosporine or glucocorticoids, rapamycin can reduce the incidence of acute rejection in kidney transplant recipients. This is because rapamycin binds to tacrolimus-binding protein-12, inhibiting the proliferation of T lymphocytes.

[0009] Rapamycin is distributed in human red blood cells, metabolized by the liver, and eliminated through feces and bile. Side effects of rapamycin include headache, nausea, dizziness, nosebleeds, and joint pain. Laboratory tests may reveal abnormalities in the following indicators: thrombocytopenia, leukopenia, hypertriglyceridemia, and hypercholesterolemia. These side effects are dose-dependent and reversible.

[0010] For the reasons mentioned above, timely monitoring of rapamycin blood concentration is necessary during treatment. This is an effective way to assist clinical treatment, improve treatment efficacy, and reduce toxicity risks.

[0011] Currently known methods for detecting rapamycin include high-performance liquid chromatography (HPLC), luminescent immunoassay, and enzyme-linked immunosorbent assay (ELISA). HPLC can separate the drug from its metabolites and endogenous substances, exhibiting high specificity and serving as the gold standard for detecting MTX plasma concentrations. However, this method requires complex pretreatment and a long assay time, making it unsuitable for rapid detection of large samples. In clinical diagnostic testing, homogeneous enzyme immunoassay (EMIT) and latex-enhanced immunoturbidimetric assay are the primary methods of detection.

[0012] The principle of homogeneous enzyme immunoassay: In a homogeneous liquid reaction system, enzyme-labeled antigen (such as G6PDH-rapamycin) and unlabeled antigen (rapamycin) compete for binding with a quantitative amount of antibody (rapamycin antibody). The more the antibody binds to the unlabeled antigen, the more activity of the enzyme-labeled antigen is released, and the more NADH is generated from the substrate NAD+ by the enzyme. By detecting the change in absorbance of NADH at a wavelength of 340 nm, the content of rapamycin in the liquid can be calculated.

[0013] Existing homogeneous enzyme immunoassays rely on activating the reactive groups of small molecule drugs themselves before reacting with enzymes. This strategy makes it difficult to guarantee a directional 1:1 reaction between small molecule drugs and enzymes, resulting in large batch-to-batch variations. Summary of the Invention

[0014] In view of the needs of the art, this application provides a novel glucose-6-phosphate dehydrogenase mutant and its use in the preparation of a rapamycin detection kit.

[0015] According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. Unlike the published glucose-6-phosphate dehydrogenase mutant in patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the glucose-6-phosphate dehydrogenase mutant of this application contains mutations selected from the following: D306C, D375C, and G426C.

[0016] According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided, said glucose-6-phosphate dehydrogenase mutant being selected from the sequences shown below: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4.

[0017] According to some embodiments, a polynucleotide is provided that encodes the glucose-6-phosphate dehydrogenase mutant of this application.

[0018] According to some implementation schemes, an expression vector is provided that contains the polynucleotides of this application.

[0019] According to some embodiments, a host cell is provided that contains the expression vector of this application. The host cell can be prokaryotic (such as bacteria) or eukaryotic (such as yeast).

[0020] According to some embodiments, a conjugate is provided, which is formed by conjugating the glucose-6-phosphate dehydrogenase mutant of this application with a hapten in a molar ratio of 1:1.

[0021] In some specific implementations, the molecular weight of the hapten is from 100 Da to 4000 Da, for example: 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 520, 550, 570, 600, 620, 650, 700, 750, 800, 850, 900, 950, 1 000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000.

[0022] According to this application, those skilled in the art will understand that "hapten" also includes its derivative forms. To facilitate coupling with glucose-6-phosphate dehydrogenase, haptens that do not inherently possess a coupling group (e.g., a group that reacts with a thiol group) (e.g., rapamycin) can be modified to have a linker for covalent binding with a thiol group. Therefore, in this application, a hapten derivative refers to a hapten modified to possess a thiol-reactive group.

[0023] Haptens are selected from: small molecule drugs (such as antibiotics and psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0024] Haptens include, but are not limited to: vancomycin, theophylline, phenytoin, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin and digitoxin), phenolic acids, rapamycin, cyclosporine A, methotrexate, amiodarone, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, urinary nicotinic acid metabolite cotinine, urinary morphine, urinary monohydroxyphenol derivatives, neuropeptide tyrosine, plasma glycoproline, polyamines, histamine, thyroid-stimulating hormone, prolactin, placental lactogen, growth hormone, follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, transtriiodothyronine, free thyroxine. Hormones, free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and its sulfate, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17α-hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-ketoprostaglandin F1α, prostacyclin, adrenaline, catecholamines, norepinephrine, cholecystokinin, natriuretic peptide, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptide, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0025] In a specific implementation plan, the hapten is rapamycin or a derivative thereof.

[0026] In a specific implementation, the hapten is a rapamycin derivative with a thiol reactive group, such as lemiimide, bromoacetyl, vinyl sulfone, or aziridine.

[0027] In a specific implementation scheme, the hapten is a rapamycin derivative, as shown in Formula I:

[0028]

[0029] in,

[0030] SIRO represents

[0031] In some implementations, m is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, 5.

[0032] In a specific implementation scheme, the rapamycin derivative is shown in Formula II:

[0033]

[0034] According to some embodiments, a reagent is provided that comprises the conjugate of this application.

[0035] According to some implementation schemes, the use of the glucose-6-phosphate dehydrogenase mutant of this application in the preparation of rapamycin detection reagents is provided.

[0036] According to some implementation schemes, the use of the conjugates of this application in the preparation of rapamycin detection reagents is provided.

[0037] In the specific implementation plan, the detection reagents are selected from: enzyme-linked immunosorbent assay (ELISA) reagents, chemiluminescent immunoassay (CIA) reagents, homogeneous enzyme immunoassay (HIA) reagents, and latex-enhanced immunoturbidimetric assay (LTIA) reagents.

[0038] In a specific implementation plan, the detection reagent is preferably a reagent based on a competitive detection method.

[0039] According to some implementation schemes, the use of the conjugate of this application in the preparation of a rapamycin detection device is provided.

[0040] In a specific implementation, the detection device can be prepared in the form of a well plate (e.g., a 96-well plate), for example, the plate is coated with the reagent according to this application.

[0041] In a specific implementation, the detection device can be prepared in the form of particles (e.g., latex, magnetic beads), such as particles coated with the reagent according to this application.

[0042] According to some implementation schemes, a rapamycin detection kit is provided, comprising:

[0043] - First reagent, comprising a substrate, a buffer solution, and a rapamycin antibody; said substrate is a substrate of glucose-6-phosphate dehydrogenase;

[0044] - A second reagent, comprising the conjugate and buffer solution of this application;

[0045] - Optionally, a calibrator comprising a 10 mM to 500 mM buffer solution, a known concentration of rapamycin; and

[0046] -Optionally, a quality control sample comprising 10 mM to 500 mM buffer solution and a known concentration of rapamycin.

[0047] According to one embodiment, a rapamycin detection kit is provided, comprising:

[0048] The first reagent comprises:

[0049] 10mM to 500mM buffer solution

[0050] 5mM to 50mM substrate,

[0051] 0.01 μg / ml to 10 μg / ml rapamycin antibody, 0.1 g / L to 5 g / L stabilizer,

[0052] 0.1 g / L to 5 g / L surfactant,

[0053] 0.1 g / L to 5 g / L of preservatives;

[0054] The second reagent comprises:

[0055] 10mM to 500mM buffer solution

[0056] 0.01 μg / ml to 10 μg / ml of the conjugate according to this application,

[0057] 0.1 g / L to 5 g / L stabilizer

[0058] 0.1 g / L to 5 g / L surfactant,

[0059] 0.1 g / L to 5 g / L of preservatives;

[0060] In some embodiments, the buffer is selected from one or a combination of the following: TAPS, glycerol buffer, phosphate buffer, Tris-HCl buffer, citrate-sodium citrate buffer, barbiturate buffer, glycine buffer, borate buffer, trimethylolpropane buffer; preferably, phosphate buffer; the concentration of the buffer is from 10 mmol / L to 500 mmol / L, preferably from 50 to 300 mM; the pH of the buffer is from 7 to 8.4.

[0061] In some embodiments, the stabilizer is selected from one or a combination of the following: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, and polyethylene glycol 8000; preferably bovine serum albumin.

[0062] In some embodiments, the surfactant is selected from one or a combination of the following: Brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween20.

[0063] In some embodiments, the preservative is selected from one or a combination of the following: azides, MIT, biological preservatives PC (such as PC-300), and thimerosal; the azides are selected from: sodium azide and lithium azide.

[0064] In some embodiments, the substrate comprises: 6-phosphoglucose and β-nicotinamide adenine dinucleotide.

[0065] In some specific implementations, the rapamycin antibody is derived from: rabbits, mice, rats, goats, sheep, cats, guinea pigs, dogs, primates, cattle, horses, camels, birds, and humans.

[0066] In some specific implementation schemes, the rapamycin antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen-binding fragments.

[0067] According to some implementation schemes, a method for preparing a coupling compound is provided, including the following steps:

[0068] 1) Provide rapamycin derivatives according to the present application, especially in aprotic solvents (e.g., but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0069] 2) Provide a glucose-6-phosphate dehydrogenase mutant, preferably provided in a buffer (which provides a reaction environment, such as, but not limited to, PBS, Tris, TAPS, TAPSO, said buffer pH 6.0 to 8.0);

[0070] 3) At 18°C ​​to 28°C, the rapamycin derivative and the glucose-6-phosphate dehydrogenase mutant are contacted at a molar ratio of n:1 for 1 hour to 4 hours (preferably 2 hours to 3 hours) to couple the rapamycin derivative and the glucose-6-phosphate dehydrogenase mutant, thereby obtaining the conjugate.

[0071] 4) The conjugate may be purified as needed, for example, by desalting.

[0072] In some embodiments, the contact molar ratio of enzyme to hapten in the reaction system is 1:n, where n is from 1 to 500, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 and any of the above values; preferably n is from 20 to 60, for example, 50.

[0073] In some specific implementations, steps 1) and 2) can be interchanged or run in parallel.

[0074] In some specific implementations, prior to coupling, the glucose-6-phosphate dehydrogenase contains one or more free sulfhydryl groups, thereby allowing a directed reaction with rapamycin.

[0075] Wild-type glucose-6-phosphate dehydrogenase does not contain a free sulfhydryl group. Therefore, in some specific implementations, glucose-6-phosphate dehydrogenase is genetically engineered to mutate the amino acid at a specific site (positions 306, 375, or 426) to cysteine, thereby giving it a free sulfhydryl group. Attached Figure Description

[0076] Figure 1 .G6PDH (wild-type) amino acid sequence (SEQ ID No.1); derived from Leuconostoc pseudomesenteroides.

[0077] Figure 2 .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0078] Figure 3 The amino acid sequence of G6PDH(D375C) (SEQ ID No. 3).

[0079] Figure 4 The amino acid sequence of G6PDH (G426C) (SEQ ID No. 4). Detailed Implementation

[0080] Example

[0081] Example 1. Synthesis of rapamycin derivatives

[0082]

[0083] Add 10 mL of dry DCM to a round-bottom flask, then add compound 1 (200 mg, 1.18 mmol) and DIPEA (183 mg, 1.42 mmol). Cool to 0 °C, and slowly add compound 2 (244 mg, 1.18 mmol) under nitrogen protection. Raise the reaction temperature to room temperature (18-28 °C, preferably 20-25 °C), continue stirring, and monitor with TLC. The reaction is complete in about 4 hours. The solution is used directly in the next step without further processing.

[0084] Rapamycin (1.08 g, 1.18 mmol) was added to the above reaction system, followed by DMAP (432 mg, 3.54 mmol). The mixture was stirred at room temperature and detected by TLC. After the reaction was completed, the solvent was removed under reduced pressure, and the rapamycin derivative (480 mg) was purified by column chromatography with a yield of 38%.

[0085] The product structure was confirmed using conventional methods. In this example, rapamycin contains a group that can bind to an enzyme.

[0086] Example 2. Coupling of rapamycin derivatives with G6PDH molecules

[0087] I. Coupling Method of This Application

[0088] According to the G6PDH-rapamycin conjugate of this application, the coupling is carried out in the following manner: the thiol reactive group (such as, but not limited to, maleimide group) on the rapamycin derivative molecule is covalently bonded to the thiol group on the G6PDH molecule.

[0089] 1. Dissolve the rapamycin derivative in N,N-dimethylformamide (10 mg / ml);

[0090] 2. Add 200 μl of G6PDH (the mutant of this application or the prior art mutant) solution (6.4 mg / ml, 0.2 M phosphate buffer, pH 8.0) to 750 μl of buffer solution (0.05 M Na2HPO4, 150 mM NaCl, 10 mM EDTA, 0.1% NaN3, pH 7.2);

[0091] 3. Then add 50 μl of an N,N-dimethylformamide solution of rapamycin derivative;

[0092] 4. The above mixed solution should be thoroughly shaken at room temperature (18 to 28°C) for 2-3 hours;

[0093] 5. Molecular sieve chromatography was used to obtain G6PDH-rapamycin conjugates (concentrations of 0.1 mg / mL to 2.0 mg / mL).

[0094] II. Non-directional control conjugation method (relying on the activation of the group inherent in rapamycin)

[0095] 1. Weigh out G6PDH and dissolve it in PBS buffer at room temperature;

[0096] 2. Dissolve a certain amount of rapamycin, 1-ethyl-3-carbodiimide, and N-hydroxythiosuccinimide in Mes solution and activate by stirring at room temperature for 15-60 minutes.

[0097] 3. Add the activated rapamycin solution dropwise to the dissolved G6PDH and stir to dissolve;

[0098] 4. Stir and dissolve overnight at 2-8℃;

[0099] 5. After purification, the glucose dehydrogenase-rapamycin conjugate was obtained and stored at 2-8℃.

[0100] Example 3. Preparation of the reagent kit

[0101] Prepare the following kit for detecting rapamycin, comprising:

[0102] 1. Preparation of the first reagent:

[0103]

[0104]

[0105] 2. Preparation of the second reagent:

[0106]

[0107] 3. Quality control products and calibrators:

[0108] The quality control samples were obtained by diluting pure rapamycin with buffer solution, and their concentrations were 4-6 ng / ml, 8-12 ng / ml, and 22-28 ng / ml, respectively.

[0109] The calibrators were pure rapamycin diluted with buffer solution, with concentrations of 0 ng / ml, 3 ng / ml, 6 ng / ml, 12 ng / ml, 24 ng / ml, and 36 ng / ml, respectively.

[0110] 4. Reagent kit assembly:

[0111] Assemble the above reagents (optionally including quality control and calibrators) into a homogeneous enzyme immunoassay kit for rapamycin.

[0112] Detection example

[0113] In a homogeneous reaction system, rapamycin and the G6PDH-rapamycin conjugate in the sample simultaneously compete for binding sites to anti-rapamycin antibodies. Since enzyme activity decreases after antibody binding to the conjugate, the more free rapamycin in the sample, the more antibody sites compete for binding, and the less antibody binds to the enzyme conjugate. The unbound enzyme conjugate catalyzes the oxidation of β-nicotinamide adenine dinucleotide (NAD). + Rapamycin is converted into β-nicotinamide adenine dinucleotide (NADH). The concentration of rapamycin in the sample is directly proportional to the amount of NADH generated. The concentration of rapamycin in the sample can be obtained by observing the change in absorbance.

[0114] Table 1. Parameters of Fully Automated Biochemical Analyzer

[0115]

[0116]

[0117] Example 1. Performance of the reagent kit of this application

[0118] 1. Calibration absorbance

[0119] Table 2. Calibration Absorbance

[0120]

[0121] 2. Precision Experiment

[0122] Using the calibration curves established above, high, medium, and low quality control products are measured.

[0123] Table 3. Precision (D306C mutant)

[0124]

[0125]

[0126] 3. Repeatability

[0127] Table 4. Repeatability

[0128]

[0129]

[0130] 4. Recycling

[0131] Table 5. Recycling

[0132]

[0133] 5. Linear

[0134] Table 6. Linear

[0135]

[0136] 6. Stability

[0137] Table 7. Accelerated stability at 37℃

[0138]

[0139]

[0140] Example 2. Antibody inhibition rate in conjugates

[0141] 1. Detection principle of antibody inhibition rate

[0142] When the antibody binds to the G6PDH-rapamycin conjugate, the steric hindrance affects the activity of the G6PDH enzyme, thereby reducing its efficiency in catalyzing the conversion of NAD to NADH. By detecting the change in the amount of NADH, the difference between the experimental groups with and without the antibody can be compared. This difference reflects the inhibitory ability of the antibody on G6PDH.

[0143] 2. Reaction system

[0144] Table 8. Preparation of reagents for detecting antibody inhibition rate

[0145]

[0146] 3. Results

[0147] By comparing the absorbance values ​​of the G6PDH-rapamycin conjugate with and without the addition of antibody, the inhibitory effect of the antibody on G6PDH can be obtained.

[0148] Compared to conjugates prepared from previously published mutant sites (A45C, K55C), the enzyme mutant of this application exhibits a significantly improved antibody inhibition rate, reaching over 50% (G426C: 50%; D375C: 51%), with a maximum of 60.4% (D306C). Previously published mutant sites (e.g., A45C, K55C) showed inhibition rates of 38.6% to 45%.

[0149] While not limited to specific theories, this can be partially explained as follows: Compared to existing G6PDH mutants (A45C, K55C), the mutation site (i.e., the site introducing a free thiol group) in the enzyme mutant of this application is the location where it couples with haptens (such as hormones, small molecule drugs, etc.). When a hapten binds to a hapten-specific antibody at this site, the resulting steric hindrance has the greatest impact on the activity of the G6PDH enzyme, while the introduction of the mutation does not substantially affect the spatial folding of the molecule. Therefore, the location of this mutation site is crucial, requiring consideration of G6PDH enzyme activity, the spatial folding of the coupled molecule, and the sufficient exposure of the hapten epitope.

[0150] Because the enzyme mutant exhibits a significant increase in antibody inhibition rate, the reagent kit formulated by conjugating the enzyme mutant with rapamycin shows a marked improvement in performance in terms of batch-to-batch coefficient of variation, linearity, and specificity.

Claims

1. A method for preparing a coupling agent, comprising the steps of: 1) Provide rapamycin derivatives; 2) Provide a glucose-6-phosphate dehydrogenase mutant; 3) The glucose-6-phosphate dehydrogenase mutant is coupled with the rapamycin derivative; The rapamycin derivative is shown in Formula II: in, SIRO represents Compared to wild-type glucose-6-phosphate dehydrogenase, the glucose-6-phosphate dehydrogenase mutant contains the D306C mutation: the glucose-6-phosphate dehydrogenase mutant is shown in SEQ ID No.

2.

2. The method for preparing the coupling compound according to claim 1, comprising the steps of: 1) Provide the rapamycin derivative; 2) Provide the glucose-6-phosphate dehydrogenase mutant; 3) The glucose-6-phosphate dehydrogenase mutant and the rapamycin derivative are contacted at 18°C ​​to 28°C for 1 hour to 4 hours to couple the rapamycin derivative and the glucose-6-phosphate dehydrogenase mutant to obtain the conjugate. Steps 1) and 2) can be interchanged or performed in parallel; Prior to step 3), the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306.

3. The method for preparing the coupling compound according to claim 2, comprising the steps of: 1) The rapamycin derivative is provided in an aprotic solvent; 2) Provide the glucose-6-phosphate dehydrogenase mutant in a buffer solution; 3) At 20°C to 25°C, the glucose-6-phosphate dehydrogenase mutant and the rapamycin derivative are contacted for 2 to 3 hours to couple the rapamycin derivative and the glucose-6-phosphate dehydrogenase mutant to obtain the conjugate. 4) Purify the conjugate; The buffer solution is selected from: phosphate buffer, Tris buffer, Hepes buffer, PBS buffer, and TAPSO buffer; The pH of the buffer solution is 6.0 to 8.0; The aprotic solvent is selected from: acetonitrile, dimethylformamide, and dimethyl sulfoxide.

4. In the method for preparing the coupling compound according to claim 3, in step 4): The conjugate was then desalted and purified.

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