A method for preparing a conjugate

Abstract

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

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201911403882.5 (Filing Date: December 31, 2019) “6-Phosphogluconate Dehydrogenase Mutant and Its Use in Preparing Phenytoin Detection Reagent”. TECHNICAL FIELD

[0002] The present application relates to the field of biological detection, in particular to a multi-site mutant enzyme 6-phosphogluconate dehydrogenase (abbreviated as G6PDH) and its application in a phenytoin detection kit. BACKGROUND

[0003] Hapten, certain small molecule substances (molecular weight less than 4000 Da), which alone cannot induce immune response, i.e. not immunogenic, but when it is cross-linked or combined with a large molecular protein or non-antigenic polylysine carrier, it can obtain immunogenicity and induce immune response. These small molecule substances can be combined with response effect products and have antigenicity, which only has immunoreactivity and not immunogenicity, also known as incomplete antigen.

[0004] Hapten can bind to corresponding antibodies to produce antigen-antibody reaction, and cannot alone stimulate human or animal body to produce antibodies. It only has immunoreactivity and not immunogenicity, also known as incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs belong to haptens. If the haptens are combined with a certain protein molecule (carrier) by chemical methods, new immunogenicity can be obtained, and the corresponding antibodies can be stimulated in animals. Once the haptens are combined with the protein, an antigen cluster of the protein is formed. Some chemical active groups of substances with specific structure (such as penicillin and sulfonamide) with smaller molecular weight than general haptens are called simple haptens.

[0005] Small molecule antigens or haptens lack two or more sites for sandwich method, so they cannot be determined by double antibody sandwich method, and are mostly used in competition mode. The principle is that the antigens in the sample and a certain amount of enzyme-labeled antigens compete for binding to the solid-phase antibody. The more the amount of antigens in the sample, the less the enzyme-labeled antigens bound to the solid phase, and the lighter the color. Small molecule hormones, drugs, etc. are mostly determined by ELISA using this method.

[0006] Phenytoin (PTN) is a specific example of a hapten. Phenytoin is an exogenous small molecule drug for anti-epilepsy. There is no such substance in the serum and plasma of normal people, and the sodium salt form is generally used in clinical.

[0007] Phenytoin sodium, as a traditional anti-epileptic drug, its pharmacological mechanism is mainly to block voltage-dependent Na + channels, reduce Na +Stabilize the cell membrane of the intrinsic flow neuron, reduce its excitability, and thus prevent the spread of local abnormal discharge to normal brain tissue.

[0008] Phenytoin sodium reaches peak concentration 1.5 to 30 hours after oral administration, and an effective blood drug concentration can be reached after 6 to 10 days of continuous administration. It is cheap and effective, and is widely used in clinical practice. However, due to its special pharmacokinetics and narrow therapeutic window, it is easy to cause drug overdose and poisoning, and the clinical manifestations after poisoning are diverse, which is easy to misdiagnose. Therefore, in the process of clinical diagnosis, it is necessary to continuously monitor the drug concentration of phenytoin in the blood.

[0009] Generally, the optimal therapeutic effect can be achieved when the blood drug concentration is 10-20 μg / ml, and no toxic effects on the human body will occur. However, when the blood drug concentration is higher than 20 μg / ml, the liver and kidney of the human body can be toxic, causing coma or even death. Generally, after stopping the use of the drug for 2-4 weeks, the blood drug concentration of phenytoin can be reduced to 0.5 μg / ml or lower through the metabolism of the liver and kidney of the human body.

[0010] Currently known detection methods for phenytoin mainly include enzyme-linked immunosorbent assay, homogeneous enzyme immunoassay, chemiluminescence immunoassay, high-performance liquid chromatography, and fluorescence polarization method. However, these detection methods have many defects, such as radioactive contamination of isotopes in radioimmunoassay, short effective period, and inconvenient operation. Enzyme-linked immunosorbent assay is relatively cumbersome and time-consuming, and is not suitable for clinical use. Although chemiluminescence has good sensitivity, it requires a special device, which is not conducive to popularization due to high use cost. In the process of clinical detection and diagnosis, homogeneous enzyme immunoassay (EMIT) detection is mainly used.

[0011] The principle of homogeneous enzyme immunoassay is as follows: in a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6PDH-phenytoin) and non-labeled antigen (phenytoin) compete to bind to a quantitative antibody (phenytoin antibody). The more the antibody binds to the non-labeled antigen, the more the enzyme-labeled antigen is released, and the more NAD+ is generated by enzyme-catalyzed substrate NADH. The change in absorbance of NADH at 340 nm wavelength can be detected, and the content of phenytoin in the liquid can be calculated. SUMMARY

[0012] In view of the needs in the art, the present application provides a novel 6-phosphogluconate dehydrogenase mutant and its use in preparing a phenytoin detection kit.

[0013] According to some embodiments, a 6-phosphogluconate dehydrogenase mutant is provided. The 6-phosphogluconate dehydrogenase mutant of the present application comprises a mutation selected from the group consisting of D306C, G426C, D375C.

[0014] According to some embodiments, a 6-phosphogluconate dehydrogenase mutant is provided, which is selected from the group consisting of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4.

[0015] According to some embodiments, a polynucleotide encoding the 6-phosphogluconate dehydrogenase mutant of the present application is provided.

[0016] According to some embodiments, an expression vector comprising the polynucleotide of the present application is provided.

[0017] According to some embodiments, a host cell comprising the expression vector of the present application is provided. The host cell can be prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast).

[0018] According to some embodiments, a conjugate is provided, which is the 6-phosphogluconate dehydrogenase mutant of the present application coupled with a hapten at a molar ratio of 1 :n.

[0019] In some embodiments, n is 1 to 50, 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.

[0020] In some specific embodiments, the 6-phosphogluconate dehydrogenase mutant of the present application is coupled with a hapten at a molar ratio of 1 : 1.

[0021] In some particular embodiments, the molecular weight of the hapten is between 100 Da and 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, 1000, 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 the present application, the skilled person will understand that "hapten" also encompasses forms of derivatives thereof. In order to facilitate coupling with 6-phosphogluconate dehydrogenase, for those haptens that do not bear a coupling group (e.g. a group reactive with a thiol group) themselves (e.g. phenytoin), the hapten can be engineered to bear a linker in order to covalently bind to a thiol group. Thus, in the present application, a hapten derivative refers to a hapten engineered to bear a thiol reactive group.

[0023] The hapten is selected from the group consisting of: small molecule drugs (e.g. antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, amino acids.

[0024] Haptens include, for example, but are not limited to, phenytoin, vitamin D, 25 hydroxy vitamin D, 1,25 dihydroxy vitamin D, folate, cardiac glycosides, enzyme phenolic acid, rapamycin, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the urinary metabolite of nicotinic acid, cotinine, urinary morphine, urinary monohydroxyphenyl derivatives, neuropeptide tyrosine, plasma cholinesterase, 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, reverse triiodothyronine, free thyroxine, free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone sulfate, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17a hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-keto prostaglandin F1a, prostacyclin, epinephrine, catecholamines, norepinephrine, cholecystokinin, natriuretic, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive intestinal peptide, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0025] In particular embodiments, the hapten is phenytoin or a derivative thereof.

[0026] In particular embodiments, the hapten is a phenytoin derivative that carries a thiol reactive group, such as a maleimide, a bromoacetyl group, a vinyl sulfone, or an aziridine. In particular embodiments, the hapten is a phenytoin derivative, such as shown in Formula I:

[0027]

[0028] According to some embodiments, there is provided a reagent comprising a conjugate of the application.

[0029] According to some embodiments, there is provided a use of a 6-phosphogluconate dehydrogenase mutant of the application in the manufacture of a phenytoin detection reagent.

[0030] According to some embodiments, there is provided a use of a conjugate of the application in the manufacture of a phenytoin detection reagent.

[0031] In particular embodiments, the detection reagent is selected from the group consisting of: an enzyme-linked immunoassay detection reagent, a chemiluminescent immunoassay detection reagent, a homogeneous enzyme immunoassay detection reagent, a latex-enhanced immunoturbidimetric detection reagent.

[0032] In a particular embodiment, the detection reagent is preferably a reagent based on a competition method of detection.

[0033] According to some embodiments, there is provided a phenytoin detection kit comprising:

[0034] - a first reagent comprising a substrate and a phenytoin antibody; the substrate being a substrate for 6-phosphogluconate dehydrogenase;

[0035] - a second reagent comprising a conjugate according to the application;

[0036] - optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 mg / L to 42 mg / L phenytoin; and

[0037] - optionally, a quality control comprising 10 mM to 500 mM buffer, 0 mg / L to 42 mg / L phenytoin.

[0038] According to one embodiment, there is provided a phenytoin detection kit comprising:

[0039] a first reagent comprising:

[0040] 10 mM to 500 mM buffer,

[0041] 5 mM to 25 mM substrate,

[0042] 0.1 mg / L to 5 mg / L of a phenytoin antibody,

[0043] 10 mM to 300 mM NaCl,

[0044] 0.1 g / L to 5 g / L stabilizer,

[0045] 0.1 g / L to 5 g / L surfactant,

[0046] 0.1 g / L to 5 g / L preservative.

[0047] a second reagent comprising:

[0048] 10 mM to 500 mM buffer,

[0049] 0.1 mg / L to 1 mg / L of a conjugate according to the application,

[0050] 0.1 g / L to 5 g / L stabilizer,

[0051] 0.1 g / L to 5 g / L surfactant,

[0052] 0.1 g / L to 5 g / L preservative.

[0053] In some embodiments, the buffer is selected from one or a combination of: Amino-tris buffer, Phosphate buffer, Tris-HCl buffer, Citric acid-sodium citrate buffer, Barbiturate buffer, Glycine buffer, Borate buffer, Tris-hydroxymethyl methane buffer; preferably, phosphate buffer; the concentration of the buffer is 10 mmol / L to 500 mmol / L, preferably 100 mM; the pH of the buffer is 6-8, preferably 7.2 or 7.0.

[0054] In some embodiments, the stabilizer is selected from one or a combination of: Bovine serum albumin, Trehalose, Glycerol, Sucrose, Mannitol, Glycine, Arginine, Polyethylene glycol 6000, Polyethylene glycol 8000; preferably, bovine serum albumin.

[0055] In some embodiments, the surfactant is selected from one or a combination of: Brij 35, Triton X-100, Triton X-405, Tween 20, Tween 30, Tween 80, Coconut oil fatty acid diethanolamide, AEO7, preferably, Tween 20.

[0056] In some embodiments, the preservative is selected from one or a combination of: Azide, MIT, PC-300, Thiomersal; the azide is selected from: Sodium azide, Lithium azide.

[0057] In some embodiments, the substrate comprises: 6-phosphogluconate, β-nicotinamide adenine dinucleotide.

[0058] In some embodiments, a method for preparing a conjugate is provided, comprising steps of:

[0059] 1) providing a phenytoin derivative, preferably in an aprotic solvent;

[0060] 2) providing a 6-phosphogluconate dehydrogenase mutant as defined in claim 2, preferably in a buffer;

[0061] 3) contacting the phenytoin derivative and the 6-phosphogluconate dehydrogenase mutant at 18-28 °C for 1-4 hours, preferably 2-3 hours, so that the phenytoin derivative and the 6-phosphogluconate dehydrogenase mutant are coupled to obtain the conjugate;

[0062] 4) optionally, purifying the conjugate, preferably desalting;

[0063] Steps 1) and 2) can be interchangeable or parallel;

[0064] said buffer is selected from the group consisting of PBS, Tris, TAPS, TAPSO, said buffer having a pH comprised between 6.0 and 8.0;

[0065] said aprotic solvent is selected from the group consisting of acetonitrile, dimethylformamide, dimethylsulfoxide, alone or in combination;

[0066] Preferably, before step 3), said 6-phosphogluconate dehydrogenase mutant comprises a free thiol group; more preferably, said 6-phosphogluconate dehydrogenase mutant has a free thiol group at position 306, 375 or 426. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 Structure of phenytoin.

[0068] Figure 2 Structure of phenytoin derivative.

[0069] Figure 3A G6PDH (wild type) amino acid sequence (SEQ ID No. 1); from Leuconostoc pseudomesenteroides.

[0070] Figure 3B G6PDH (D306C) amino acid sequence (SEQ ID No. 2).

[0071] Figure 3C G6PDH (D375C) amino acid sequence (SEQ ID No. 3).

[0072] Figure 3D G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[0073] EXAMPLE

[0074] Example 1. Synthesis of phenytoin derivative

[0075] 1. Synthesis of compound 2

[0076] Phenytoin (2.00 g, 7.94 mmol) and K2CO3(1.65 g, 11.90 mmol) were dissolved in DMF (10 mL) and compound 1 (1.42 g, 6.35 mmol) was added to the reaction, which was heated to 50°C for 16 h.

[0077] The reaction system was brought to room temperature (20-25°C), water was added to the reaction system and extraction was performed using ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, the organic solvent was removed under reduced pressure and purification was performed on a chromatographic column (EA / PE = 1 :3) to obtain compound 2 as a colorless oil (2.04 g, 81.6%).

[0078] 2. Synthesis of compound 3

[0079] Compound 2 (2.04 g, 5.20 mmol) was dissolved in ethanol (50 mL) and NaOH (2N, 10 mL) was added to the reaction system. Stirring was performed at room temperature (20-25°C) for 2 h. The pH was adjusted to 5 using HC1 (1 N) and the solvent was removed under reduced pressure. Purification was performed on a chromatographic column (MeOH / DCM = 1 :20) to obtain compound 3 as a white solid (1.20 g, 60.0%).

[0080] 3. Synthesis of phenytoin derivative

[0081] Compound 3 (117 mg, 0.32 mmol) and compound 4 (56 mg, 0.32 mmol) were dissolved in DCM (5 mL) and triethylamine (98 mg, 0.96 mmol) was added dropwise. HATU (147 mg, 0.39 mmol) was added and stirring was performed at room temperature (20-25°C) for 5 h. Water (30 mL) was added to the reaction system and extraction was performed using DCM. The organic phase was washed with saturated brine, dried over anhydrous Na2S04, the solvent was removed under reduced pressure and purification was performed on a preparative column to obtain the phenytoin derivative (100 mg, 67%).

[0082]

[0083] The role of this example is to provide phenytoin with a group that can bind to the enzyme.

[0084] Example 2. Coupling of phenytoin derivative to G6PDH molecule

[0085] The G6PDH-phenytoin conjugate according to the present application is coupled in the following manner: the thiol-reactive group on the phenytoin derivative molecule (such as, but not limited to, a maleimide group) covalently binds to the thiol group on the G6PDH molecule.

[0086] 1. Solution preparation:

[0087] Phenytoin derivative solution: 10 mg / ml of the phenytoin derivative prepared in Example 1 was dissolved in DMF;

[0088] G6PDH solution: G6PDH (mutant according to the present application or prior art mutant), PB 100 mmol, NaCl 100 mmol, pH = 8.0;

[0089] Coupling solution: 100 mM PB / K, 100 mM EDTA, 150 mM NaCl, pH = 7.2;

[0090] Desalting solution: 100 mM PB / K, 100 mM EDTA, 150 mM NaCl, pH = 7.2.

[0091] 2. Coupling operation: 4 ml G6PDH solution, 15.2 ml coupling solution and 0.8 ml phenytoin derivative solution, reaction at room temperature (20 to 25°C) for 4 h.

[0092] 3. After the above reaction system is shaken at room temperature for 4 h, desalting column elution is performed using the above desalting solution, and the protein peak is collected. The obtained product is G6PDH-phenytoin conjugate.

[0093] Example 3. Preparation of a kit

[0094] The following kit for detecting phenytoin is prepared, which comprises:

[0095] Reagent R1, comprising:

[0096] 100 mM PB buffer, pH 7.2

[0097] 15 mM 6-phosphogluconate

[0098] 15 mM β-nicotinamide adenine dinucleotide

[0099] 2.5 mg / L phenytoin antibody (commercially available antibody, no special restriction)

[0100] 150 mM NaCl

[0101] 1 g / L bovine serum albumin

[0102] 1 g / L Tween 20

[0103] 1 g / L sodium azide;

[0104] Reagent R2, comprising:

[0105] 100 mM PB buffer, pH 7.2

[0106] 0.1 mg / L G6PDH-phenytoin conjugate

[0107] 1 g / L bovine serum albumin

[0108] 1 g / L Tween 20

[0109] 1 g / L sodium azide;

[0110] Calibrator: 100 mM PB buffer, pH 7.2, and 0, 2.5, 5.0, 10, 20, 42 mg / L Phenytoin (or add as needed);

[0111] Quality control: 100 mM PB buffer, pH 7.2, and 5.0, 15, 25 mg / L Phenytoin (or add as needed).

[0112] Detection Example

[0113] Reaction time: 10 min, in which the incubation time is 4.7 min, after adding reagent R2, incubate for 1 min, then measure the absorbance A1, and after another 1 min incubation, measure the absorbance A2, calculate ΔA = (A2-A1) / min. Calculate the content of phenytoin in the sample by the calibration curve: Phenytoin = sample tube absorbance * calibration sample concentration / calibration sample absorbance.

[0114] The performance of the phenytoin detection kit prepared in Example 3 was detected, and the main detection performances were total imprecision, repeatability, recovery, linearity, and 37°C accelerated stability.

[0115] Table 1. Automatic biochemical instrument parameters

[0116]

[0117]

[0118] Detection Example 1. Phenytoin detection kit calibration absorbance

[0119] Table 2. Phenytoin detection kit calibration absorbance

[0120]

[0121] Note: the mutant with code A45C in the prior art, the mutation site corresponds to Figure 3A the 46th position.

[0122] Detection Example 2. Total imprecision of the phenytoin detection kit

[0123] Table 3. Total imprecision

[0124]

[0125] Detection Example 3. Repeatability of the phenytoin detection kit

[0126] Table 4. Repeatability

[0127]

[0128] Detection Example 4. Recovery of the phenytoin detection kit

[0129] Table 5. Recovery

[0130]

[0131]

[0132] Detection Example 5. Linearity of phenytoin detection kit

[0133] Table 6. Linearity

[0134]

[0135] Detection Example 6. 37°C accelerated stability

[0136] Table 7. 37°C accelerated stability

[0137]

[0138] The calibration absorbance of the reagent of the present application decreased by about 15% after 37°C acceleration for 7 days, and the calibration absorbance of the control reagent decreased by about 95% after 37°C acceleration for 7 days.

[0139] Detection Example 7. Antibody inhibition rate

[0140] 1. Detection principle of antibody inhibition rate

[0141] When the antibody binds to the G6PDH-phenytoin conjugate, the G6PDH enzyme activity is affected due to steric hindrance, thereby reducing the efficiency of its catalysis of NAD to NADH. By detecting the change in the amount of NADH, the difference between the experimental group with and without the addition of the antibody is compared, which reflects the inhibition ability of the antibody to G6PDH.

[0142] 2. Reaction system

[0143] Table 8. Preparation of detection reagent for antibody inhibition rate

[0144]

[0145] Table 9. Parameters for detection of antibody inhibition rate

[0146] Detection mode Abbott C16 0000 Analysis / time / point Rate / 10 min / 28-32 points R1 / S 120:20 Wavelength (secondary / primary) 405 / 340 Reaction type Increasing

[0147] 3. Results

[0148] By comparing the detection of G6PDH-phenytoin conjugate absorbance with and without the addition of the antibody, the inhibition of the antibody to G6PDH can be obtained.

[0149] The antibody inhibition rate = [1 - (absorbance change value of G6PDH-phenytoin with antibody / absorbance change value of G6PDH-phenytoin without antibody)] x 100%.

[0150] Compared with the published mutation sites, the mutant of the application has a significant increase in antibody inhibition rate, which can reach more than 30%, and the highest can reach 55%. The inhibition rate of the commonly used mutation sites (such as A45C, K55C) is only about 40% or even lower.

[0151] Table 10. Antibody inhibition rate of different G6PDH mutants

[0152]

[0153] Although not limited to a specific theory, it can be partially explained as follows: compared with the G6PDH mutants in the prior art, the mutation site (i.e. the site where the free thiol group is introduced) in the enzyme mutant (D306C, D375C, G426C) of the application is the position where the hapten (such as hormones, small molecule drugs, etc.) is coupled. When the hapten is combined with the hapten-specific antibody at this position, the steric hindrance formed has the greatest impact on the activity of G6PDH enzyme, and at the same time, after introducing the mutation, it cannot substantially affect the spatial folding of the molecule. Therefore, the position of this mutation site is very important, and it needs to take into account the activity of G6PDH enzyme, the spatial folding of the coupled molecule, and the sufficient exposure of the hapten epitope.

[0154] Since the enzyme mutant has a significant increase in antibody inhibition rate, after the enzyme mutant is coupled with phenytoin to form a conjugate, the reagent kit prepared from the conjugate has a significant performance improvement in repeatability, total imprecision, linearity, stability and other performance.

Claims

1. A method for preparing a coupling agent, comprising the steps of: Provide glucose-6-phosphate dehydrogenase mutant; Provide phenytoin derivatives; The glucose-6-phosphate dehydrogenase mutant is coupled with the phenytoin derivative; The phenytoin derivative is shown in Formula I: Compared to wild-type glucose-6-phosphate dehydrogenase, the glucose-6-phosphate dehydrogenase mutant contains the mutant D306C; 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 phenytoin derivative; 2) Provide the glucose-6-phosphate dehydrogenase mutant; 3) The phenytoin derivative and the glucose-6-phosphate dehydrogenase mutant were contacted at 18°C ​​to 28°C for 1 hour to 4 hours to couple the phenytoin 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 phenytoin derivative is provided in an aprotic solvent; 2) Provide the glucose-6-phosphate dehydrogenase mutant in a buffer solution; 3) The phenytoin derivative and the glucose-6-phosphate dehydrogenase mutant were contacted at 18°C ​​to 28°C for 2 to 3 hours to couple the phenytoin derivative and the glucose-6-phosphate dehydrogenase mutant to obtain the conjugate. 4) Purify the conjugate; Steps 1) and 2) can be interchanged or performed in parallel; The buffer solution is selected from: PBS, Tris, TAPS, and TAPSO; The pH of the buffer solution is 6.0 to 8.0; The aprotic solvent is selected from: acetonitrile, dimethylformamide, and dimethyl sulfoxide.

4. The method for preparing the coupling compound according to claim 3, in step 4): The conjugate was purified by desalting.

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