Method for producing oxygen isotope-labeled compounds

Abstract

A method for producing oxygen isotope-labeled compounds with enriched concentrations that can be used as internal standards is provided. [Solution] A method for producing an oxygen isotope-labeled compound, in which an oxygen isotope-labeled compound is obtained by labeling a substrate with a stable oxygen isotope through the action of the enzyme in a reaction solution containing an enzyme, a substrate, and a stable oxygen isotope. The method for producing an oxygen isotope-labeled compound includes the steps of preparing a substrate solution containing the substrate and a solvent, bubbling an inert gas into the substrate solution, adding the enzyme to the substrate solution, and further bubbling a gas containing the stable oxygen isotope to obtain a reaction solution containing the enzyme, the substrate, and the stable oxygen isotope, and synthesizing the oxygen isotope-labeled compound in the reaction solution.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a compound labeled with a stable isotope of oxygen. [Background technology]

[0002] Stable isotope-labeled compounds are useful as internal standards for quantitative analysis using mass spectrometers, and are used in a wide range of research fields, including medical and pharmaceutical research, such as pharmacokinetic studies. Oxygen is the third most abundant element in pharmaceuticals and in vivo metabolites, after carbon and hydrogen. 18 O is an isotope with high utility as an internal standard because the mass increase per label is +2 and it can be multi-labeled with other stable isotopes. 18 The O labeling method is 18 By pH control in water 16 O- 18 However, this method is limited to compounds with carboxyl groups, and is costly. 18 Transfer reactions from O 18 There are synthesis methods using O-labeled metal oxidizers, but none of them require 18 O-labeled materials are difficult to obtain and not widely available.

[0003] Non-Patent Document 1 proposes a method for analyzing drug metabolites by mass spectrometry using oxygen isotope labeling with liver microsomal enzymes. Previous methods had difficulty identifying compounds based on mass spectrometry and chromatographic retention times or fragmentation spectra, but the local anesthetic bupivacaine was used to identify more than 20 unknown metabolites without a standard substance. 18 The concentration of O-labeled metabolites is 20-45%, and their low concentration makes them difficult to use as internal standards.

[0004] Non-patent document 2 describes a method using a dioxygenase called clavaminate synthase (CS). 18A method for producing O-labeled metabolites is disclosed. CS catalyzes three oxidation reactions in the biosynthetic pathway of clavulanic acid: hydroxylation, oxidative cyclization, and desaturation. 18 Under O2 gas atmosphere 18 O labeling was performed, achieving an enrichment of 70%, but this low enrichment makes it difficult to use as an internal standard. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Int J Mol Sci. 2023 Feb 26;24(5):4569. [Non-patent document 2] Tetrahedron Volume 55, Issue 33, 13 August 1999, Pages 10201-10220. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a method for producing enriched oxygen isotope labeled compounds that can be used as internal standards. [Means for solving the problem]

[0007] [1] A method for producing an oxygen isotope-labeled compound, in which an oxygen isotope-labeled compound is obtained by labeling a substrate with a stable oxygen isotope through the action of the enzyme in a reaction solution containing an enzyme, a substrate, and a stable oxygen isotope, the method comprising the steps of: preparing a substrate solution containing the substrate and a solvent; bubbling an inert gas into the substrate solution; adding the enzyme to the substrate solution; and further bubbling a gas containing the stable oxygen isotope to obtain a reaction solution containing the enzyme, the substrate, and the stable oxygen isotope; and synthesizing the oxygen isotope-labeled compound in the reaction solution. [2] A method for producing an oxygen isotope-labeled compound, in which an oxygen isotope-labeled compound is obtained by labeling a substrate with a stable oxygen isotope through the action of the enzyme in a reaction solution containing an enzyme, a substrate, and a stable oxygen isotope, the method comprising the steps of: preparing a substrate solution containing the substrate and a solvent previously bubbled with an inert gas; adding the enzyme to the substrate solution; and further bubbling a gas containing the stable oxygen isotope to obtain a reaction solution containing the enzyme, the substrate, and the stable oxygen isotope; and synthesizing the oxygen isotope-labeled compound in the reaction solution. [3] The method for producing an oxygen-isotope-labeled compound according to [1] or [2], wherein the reaction solution is placed in a container, a gas containing the stable oxygen isotope is injected into the remaining space in the container, and the container is then sealed to synthesize the oxygen-isotope-labeled compound in the reaction solution. [4] The method for producing an oxygen isotope-labeled compound according to any one of [1] to [3], wherein the volume ratio of the volume V1 of the reaction solution to the volume V2 of the remaining space in the container, expressed as V2 / V1, is 3 to 100. [5] The method for producing an oxygen-isotope-labeled compound according to any one of [1] to [4], wherein after the container is sealed, the oxygen-isotope-labeled compound is synthesized in the reaction solution while mixing by inverting the container. [Effects of the Invention]

[0008] According to the present invention, it is possible to produce oxygen isotope-labeled compounds with such a high degree of enrichment that they can be used as internal standards. DETAILED DESCRIPTION OF THE INVENTION

[0009] Although the embodiments of the present invention will be described in detail below, the present invention is not limited to the embodiments described below, and various modifications are possible without departing from the gist of the present invention.

[0010] The meanings and definitions of terms used in this specification are as follows. A numerical range expressed by "to" means that the numerical values ​​before and after "to" are the lower and upper limits of the numerical range. "Oxygen isotope labeled compound" refers to a compound containing an oxygen isotope 16 O is one of the stable isotopes of oxygen. 18 O or 17 O-substituted compounds. "Oxygen isotopes ( 18 O) enrichment is the degree to which a given oxygen atom in a compound is an oxygen isotope. 18 This means the ratio (atom%) of O. "Oxygen isotopes ( 17 O) enrichment is the degree to which a given oxygen atom in a compound is an oxygen isotope. 17 This means the ratio (atom%) of O.

[0011] <Method for producing oxygen isotope-labeled compound> A first aspect of the present invention is a method for producing an oxygen-isotope-labeled compound, comprising: a reaction solution containing an enzyme, a substrate, and a stable oxygen isotope; and obtaining an oxygen-isotope-labeled compound by labeling the substrate with the stable oxygen isotope through the action of the enzyme. This embodiment preferably includes the following bubbling step and synthesis step: It may also include a preparation step of preparing a substrate solution to be used in the bubbling step.

[0012] [Preparation process] This step is a step of preparing a substrate solution containing a substrate and a solvent. The substrate is preferably dissolved in a solvent such as water in advance and used as a substrate solution in the subsequent bubbling step. A preferred embodiment of this aspect involves bubbling an inert gas through a solvent such as water or a pH buffer solution in which the substrate is dissolved. The water or pH buffer solution often accounts for 50% by mass of the volume of the reaction solution. In this case, the dissolved oxygen in the solvent is expelled in advance by bubbling, thereby allowing the target oxygen-isotope-labeled compound to be released. 18 O enrichment can be further increased. Furthermore, it is preferable to degas the solvent, such as water or a pH buffer solution, in which the substrate is dissolved, or the substrate solution before bubbling with an inert gas. Examples of degassing treatment include known methods such as ultrasonic treatment and vacuum treatment. By performing degassing treatment in advance, the concentration of the target oxygen isotope-labeled compound can be reduced. 18 O enrichment may be even greater. The substrate is not particularly limited as long as it is an organic compound that can introduce dissolved oxygen in the substrate solution into the chemical structure of the substrate through an enzymatic reaction, and any known organic compound can be used as the substrate. The concentration of the substrate contained in the substrate solution is not particularly limited and is set appropriately taking into consideration the efficiency of the enzyme reaction and the yield of the product, and is, for example, approximately 0.1 mM to 20 mM.

[0013] In addition to the substrate and the solvent, the substrate solution may contain various additives to ensure that the enzyme reaction proceeds appropriately. Examples of additives include pH buffers, coenzymes, surfactants, enzyme supports, etc. The concentrations of additives can be those applied to known enzyme reactions.

[0014] [Bubbling process] This step involves bubbling an inert gas into a substrate solution, adding an enzyme to the substrate solution, and then bubbling a gas containing the stable oxygen isotope to obtain a reaction solution containing the enzyme, the substrate, and the stable oxygen isotope. However, if the solvent is previously bubbled with an inert gas in the preparation step as in the preferred embodiment described above, the operation of "after bubbling the inert gas into the substrate solution" in this step may be performed or may be omitted.

[0015] As the inert gas, argon gas and nitrogen gas are preferable. They do not affect the enzyme reaction and do not dissolve oxygen gas ( 16Other gases may be used as the inert gas as long as they are capable of expelling O2. In this process, by expelling the oxygen gas originally dissolved in the reaction solution by bubbling an inert gas, the concentration of the stable oxygen isotope dissolved in the reaction solution can be increased by subsequent bubbling of a gas containing the oxygen isotope, and as a result, the desired oxygen isotope-labeled compound can be easily obtained with a high concentration.

[0016] Gases containing stable oxygen isotopes include: 17 O2 or 18 A gas containing either O or O is preferred. The concentration of the stable oxygen isotope in this gas is preferably 90 atom% or more, more preferably 95 atom% or more, and even more preferably 99 atom% or more, relative to the total oxygen gas (100 atom%). The higher the concentration of the stable oxygen isotope in the bubbling gas containing the stable oxygen isotope, the higher the concentration of the stable oxygen isotope dissolved in the reaction solution, and as a result, the desired oxygen isotope-labeled compound can be easily obtained with a high degree of enrichment.

[0017] The method for bubbling a gas into a solution is not particularly limited, and known methods can be used. For example, a method of inserting a gas supply pipe into the solution and blowing the gas into the solution can be used. In this case, it is preferable to blow the gas so as to generate fine bubbles in the solution. The amount and time of gas injection are adjusted appropriately depending on the volume of the solution. According to the experience of the present inventors, bubbling for about 1 minute with a gas amount that does not cause the main body of the solution to splash is sufficient for 1 mL of solution.

[0018] [Synthesis process] This step is a step of synthesizing the oxygen isotope-labeled compound in the reaction solution obtained in the previous step. The enzyme is not particularly limited as long as it is an enzyme that can introduce dissolved oxygen in a substrate solution into the chemical structure of the substrate through an enzymatic reaction, and known enzymes can be used. For example, monooxygenase and dioxygenase are preferred enzymes.

[0019] The enzyme concentration in the reaction solution is not particularly limited and is set appropriately taking into consideration the substrate concentration, the efficiency of the enzyme reaction, and the yield of the product. As a guideline, for example, it may be about 0.1 μM to 20 μM.

[0020] The pH of the reaction solution is preferably adjusted to an optimum pH suitable for the enzymatic reaction. The temperature of the reaction solution is preferably adjusted to an optimum temperature suitable for the enzyme reaction. The time for the enzymatic reaction is adjusted appropriately depending on the reaction conditions such as the substrate concentration, the enzyme concentration, the reaction temperature, etc. According to the experience of the present inventors, a reaction time of 1 to 3 hours is sufficient under the conditions of optimum pH and optimum temperature at a substrate concentration:enzyme concentration ratio of 1000:1.

[0021] In this step, it is preferable to place the reaction solution in a container, inject the gas containing the stable oxygen isotope into the remaining space in the container, and then seal the container to synthesize the oxygen isotope-labeled compound in the reaction solution. By sealing the container and allowing the enzyme reaction to occur, the outside air 16 This prevents O2 from dissolving in the reaction solution, making it easy to obtain the desired oxygen isotope-labeled compound with a high concentration. In addition, by injecting a gas containing stable oxygen isotopes into the remaining space in the vessel after removing the reaction solution, the oxygen originally contained in the remaining space can be reduced. 16 O2 can be expelled, and as a result, the desired oxygen isotope-labeled compound can be easily obtained with high enrichment. The phrase "injecting a gas containing a stable oxygen isotope into the remaining space in the container excluding the reaction solution" may be any method as long as it can fill the remaining space in the container with the gas containing the stable oxygen isotope. For example, the reaction solution may be sufficiently bubbled with the gas containing the stable oxygen isotope to fill the gas phase of the container, for example. 16 There are several methods, such as expelling O2, or injecting a gas containing a stable oxygen isotope into the gas phase of the container from outside the container, and then sealing the container.

[0022] When the enzymatic reaction is carried out in a sealed container as described above, the volume ratio of the volume V1 of the reaction solution in the container to the volume V2 of the remaining space in the container, expressed as V2 / V1, is preferably 3 to 100, more preferably 10 to 60, and even more preferably 20 to 40. Here, the remaining space refers to the space obtained by subtracting the volume of the reaction solution in the container from the volume of the sealed container. When the concentration is equal to or greater than the lower limit of the above range, the stable oxygen isotope filled in the remaining space is easily dissolved in the reaction solution, and the concentration of the stable oxygen isotope dissolved in the reaction solution is always maintained at a high level, making it possible to easily obtain the desired oxygen isotope-labeled compound with a high concentration. When the content is equal to or less than the upper limit of the above range, it is possible to prevent the gas containing oxygen isotopes from filling unnecessary spaces, which is economical.

[0023] When the enzymatic reaction is carried out in a sealed container as described above, it is preferable to synthesize an oxygen isotope-labeled compound in the reaction solution while mixing the container by inverting it after sealing it. Here, mixing by inverting the container means an operation of inverting the positional relationship between the ceiling and bottom of the container, moving the reaction solution at the bottom of the container to the ceiling side, and then moving it back to the bottom. By performing the enzyme reaction while mixing by inversion, the stable oxygen isotopes that fill the remaining space are more easily dissolved into the reaction solution, and the concentration of the stable oxygen isotopes dissolved in the reaction solution is maintained at a constantly high level, making it possible to easily obtain the desired oxygen isotope-labeled compound with a high concentration.

[0024] When the enzyme to be added to the reaction solution is in the form of an enzyme solution in which it has been dissolved in a solvent in advance, the volume ratio of the volume V3 of this enzyme solution to the volume V1 of the reaction solution, expressed as V1 / V3, is preferably 20 to 200, more preferably 40 to 150, and even more preferably 60 to 150. Degassing or bubbling the enzyme solution can deactivate the enzyme, so it is difficult to degas or bubbling the enzyme solution. 16 Since O2 is dissolved in the reaction mixture, when the enzyme solution is added, 16This will bring in O2. To minimize this amount of carryover, it is preferable that the volume ratio of V1 / V3 is as high as possible (for example, 20 or more). On the other hand, reducing the volume V3 of the enzyme solution increases the enzyme concentration of the enzyme solution. If the enzyme concentration becomes extremely high, the viscosity of the enzyme solution increases, making it difficult to handle. To prevent this, it is preferable that the upper limit of the volume ratio of V1 / V3 is, for example, about 100. By adjusting the volume ratio of V1 / V3 above, 16 The amount of O2 can be reduced, and the concentration of stable oxygen isotopes dissolved in the reaction solution is maintained at a high level at all times, making it possible to easily obtain the desired oxygen isotope-labeled compound with a high concentration. [Example]

[0025] [Example 1] P450BM3 RT2APAI mutant 18 Synthesis of O-labeled indoxyl (1) Degassing 1 M potassium phosphate buffer (pH 7.4), DMSO, and ultrapure water were each placed in a glass screw-cap bottle, which was then immersed in an ultrasonic water bath and degassed for 5 minutes. A 500 mM indole solution was prepared using degassed DMSO as a solvent. A 100 mM NADPH solution was prepared using degassed ultrapure water as a solvent.

[0026] (2) Bubbling with argon gas Argon gas was bubbled into the degassed 1M potassium phosphate buffer (pH 7.4) and ultrapure water for approximately 30 seconds, which expelled the dissolved oxygen and replaced it with argon. The 500 mM indole solution was kept at room temperature because the solvent, DMSO, solidified when cooled on ice. All other reagents were cooled on ice.

[0027] (3) Preparation of reaction mixture Reaction solutions were prepared on ice by adding all reagents except the enzyme according to the composition table in Table 1. The reaction solutions were prepared in four different volume containers: 0.6 mL, 2 mL, 5 mL, and 15 mL.

[0028] [Table 1]

[0029] The reaction solution (substrate solution) to which no enzyme was added was bubbled with argon gas and then allowed to stand on ice. Add the enzyme to the reaction mixture placed on ice and immediately 18 After bubbling the reaction solution with O2 gas, the lid of the reaction vessel containing the reaction solution was closed, wrapped with parafilm to seal, and placed on a rotator in a gas-phase incubator at 37°C. The labeling reaction was carried out while gently stirring the reaction vessel (Samples 1 to 4).

[0030] Degassing and argon gas bubbling were performed to determine the concentration of the reaction product. 18 The following test was conducted to investigate the effect on the concentration of O. First, a reagent solution with the same composition as above was prepared without degassing or argon gas bubbling for the reagent solution and ultrapure water used to prepare the reaction solution. Next, a reaction solution with the same composition as above but without enzyme added was prepared in a 2 mL container, and immediately after adding the enzyme, the reaction solution was heated without argon gas bubbling. 18 The labeling reaction was carried out in the same manner as above with bubbling of O2 gas (Sample 5). In addition, a reaction solution with the same composition as above but without enzyme was prepared in a 2 mL container, and the reaction solution was bubbled with argon gas. 18 O2 gas was bubbled through the solution, the enzyme was added, and the labeling reaction was carried out in the same manner as above (Sample 6). In addition, 18 When bubbling the reaction solution with O2 gas was performed without sealing the lid of the reaction vessel and leaving a gap, the remaining space in the reaction vessel was 18 The chamber was filled with O2 gas.

[0031] [Table 2]

[0032] (4)Analysis After the labeling reaction (90 min) for each sample was completed, the reaction mixture was cooled on ice for 3 min. Then, 100 μL of the reaction mixture was added to 900 μL of ice-cold methanol and mixed thoroughly. The mixture was centrifuged at 20,000 x g for 15 min at 4°C to precipitate the denatured enzyme. 950 μL of the supernatant containing the reaction product was transferred to a separate tube. The mixture was then dried for approximately 2 hours using a centrifugal evaporator. The dried sample was reconstituted in 100 μL of 40% acetonitrile and analyzed by LCMS.

[0033] The LCMS instruments used were an ACQUITY H class (Waters) and a XEVO-G2X SQTOF (Waters). Mobile phase A was ultrapure water containing 0.1% (v / v) formic acid, and mobile phase B was acetonitrile containing 0.1% (v / v) formic acid. The flow rate was 0.4 mL / min. The separation column was a COSMOSIL 3PBr packed column, 2.0 mm LD × 100 mm (Nacalai). The ionization mode was positive. The capillary voltage was 1.5 kV, the cone voltage was 40, the source temperature was 150 °C, the desolvation gas temperature was 500 °C, the desolvation gas flow rate was 1000 L / h, and the cone gas flow rate was 50 L / h.

[0034] (5) Results The reaction product of each sample is 18 The enrichment of O-labeled indoxyl is shown in Table 3. In the results of Table 3, comparing Samples 2, 5, and 6, argon gas bubbling was used. <1> "Perform this on the stock solution before preparing the reaction mixture" and <2> It is clear that it is important to perform at least one of the following in order to increase the concentration: "Performing this on a reaction solution to which no enzyme has been added before the reaction" or "Performing this on a reaction solution to which no enzyme has been added before the reaction." In other words, by expelling dissolved oxygen before the reaction by argon gas bubbling, indoxyl- 18 O enrichment is 85 atom% 18 O to 91 atom% 18 It improved to O. In addition, the above <1> In this case, argon gas bubbling of the ultrapure water stock solution, which accounted for more than 50% of the volume of the reaction solution, is thought to have played an important role in removing dissolved oxygen from the reaction solution.

[0035] Samples 1 to 4 and 6 are examples, and Sample 5 is a comparative example. The enrichment of the oxygen isotope-labeled compounds obtained in each example was 87 atom % or more, and they can be said to be effective as internal standard substances for quantification using a mass spectrometer.

[0036] [Table 3]

[0037] <Preparation of monooxygenase> The P450BM3 RT2APAI mutant used in Example 1 was prepared by the following procedure. The gene encoding the P450BM3 RT2APAI mutant was cloned into the pET101 vector to create the expression vector pET101-P450BM3 RT2APAI. The amino acid sequence of the P450BM3 RT2APAI mutant (SEQ ID NO: 1) was obtained from Reference 1 below and converted to a nucleotide sequence consistent with E. coli codon usage. pET101-P450BM3 RT2APAI was transformed into an E. coli strain for protein expression, and the resulting transformant was cultured in TB / Amp medium at 37°C and 180 rpm. Expression was induced when the OD600 reached 0.6-0.8, and the culture was continued overnight at 23°C. The E. coli cells were harvested by centrifugation, disrupted by sonication, and then centrifuged at 30,000 x g for 20 minutes to collect the supernatant. The supernatant was loaded onto an anion exchange column, and the P450BM3 RT2APAI bound to the column was eluted using a potassium chloride gradient. P450BM3 RT2APAI was concentrated by ultrafiltration and then purified using a gel filtration column. The concentration of P450BM3 RT2APAI was quantified by measuring the absorbance at 280 nm.

[0038] <Reference 1> Ren X, Yorke JA, Taylor E, Zhang T, Zhou W, Wong LL. Drug Oxidation by Cytochrome P450BM3 : Metabolite Synthesis and Discovering New P450 Reaction Types. Chemistry. 2015 Oct 12;21(42):15039-47. doi: 10.1002 / chem.201502020.

[0039] <Synthesis Reaction of Example 1> By the above enzymatic reaction, indoxyl was synthesized from indole as shown in the following formula.

[0040] [ka]

[0041] [Example 2] Indoleamine 2,3-dioxygenase 18 Synthesis of O-labeled N-formylkynurenine (1) Degassing 1 M potassium phosphate buffer (pH 7.0), 500 mM ascorbic acid aqueous solution, 1000 μM methylene blue aqueous solution, 10 mM tryptophan aqueous solution, and ultrapure water were each placed in a glass screw-cap bottle, immersed in an ultrasonic water bath, and degassed for 5 minutes.

[0042] (2) Bubbling with argon gas Argon gas was bubbled into each of the stock solutions (reagent solutions) in the degassed screw-cap bottles for about 30 seconds to expel dissolved oxygen and replace it with argon.

[0043] (3) Preparation of reaction mixture A reaction solution was prepared by mixing the stock solutions other than the enzyme on ice according to the composition table in Table 4. The reaction solution was prepared in a 5 mL container.

[0044] [Table 4]

[0045] Add the enzyme to the reaction mixture without adding the enzyme, which is left on ice. 18 After bubbling the reaction solution with O2 gas, the lid of the reaction vessel containing the reaction solution was closed, wrapped with parafilm to seal, and placed on a rotator in a gas-phase incubator at 37°C. The labeling reaction was carried out at 37°C for 60 minutes while gently stirring the reaction vessel (Sample 21).

[0046] [Table 5]

[0047] (4)Analysis After the completion of the labeling reaction (60 minutes), the reaction product was analyzed by LCMS in the same manner as in Example 1. The LC / MS system used was an ACQUITY H class (Waters) and a XEVO-G2X SQTOF (Waters). Mobile phase A was ultrapure water containing 0.1% (v / v) formic acid, and mobile phase B was acetonitrile containing 0.1% (v / v) formic acid. The flow rate was 0.2 mL / min. The separation column was an ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm x 100 mm (Waters). The ionization mode was positive. The capillary voltage was 1.5 kV, the cone voltage was 40, the source temperature was 150 °C, the desolvation gas temperature was 500 °C, the desolvation gas flow rate was 1000 L / h, and the cone gas flow rate was 50 L / h.

[0048] (5) Results is the reaction product 18 The enrichment of O-labeled N-formylkynurenine is shown in Table 6. Sample 21 is an example. The enrichment of the oxygen isotope-labeled compound obtained in this example was 87 atom % or more, and it can be said to be effective as an internal standard substance for quantification using a mass spectrometer.

[0049] [Table 6]

[0050] <Preparation of dioxygenase> The indoleamine 2,3-dioxygenase (abbreviation: IDO1) used in Example 2 was prepared by the following procedure. The human IDO1 gene was cloned into the pET101 vector to create an expression vector. The amino acid sequence of IDO1 (SEQ ID NO: 2) was determined using Uniprot ID: P14902. During cloning, the base sequence was altered to conform to the codon usage of E. coli. IDO1 was expressed and purified according to standard methods. The concentration of IDO1 was quantified by measuring the absorbance at 280 nm.

[0051] <Synthesis Reaction of Example 2> By the above enzymatic reaction, N-formylkynurenine was synthesized from L-tryptophan as shown in the following formula.

[0052] [ka]

Claims

1. A method for producing an oxygen isotope-labeled compound, comprising the steps of: in a reaction solution containing an enzyme, a substrate, and a stable oxygen isotope, obtaining an oxygen isotope-labeled compound by labeling the substrate with the stable oxygen isotope through the action of the enzyme; preparing a substrate solution containing the substrate and a solvent; After bubbling an inert gas into the substrate solution, the enzyme is added to the substrate solution, and the gas containing the stable oxygen isotope is further bubbled therein, obtaining a reaction solution containing the enzyme, the substrate, and the stable oxygen isotope; synthesizing the oxygen isotope-labeled compound in the reaction solution; A method for producing oxygen isotope-labeled compounds.

2. A method for producing an oxygen isotope-labeled compound, comprising the steps of: in a reaction solution containing an enzyme, a substrate, and a stable oxygen isotope, obtaining an oxygen isotope-labeled compound by labeling the substrate with the stable oxygen isotope through the action of the enzyme; preparing a substrate solution containing the substrate and a solvent previously bubbled with an inert gas; The enzyme is added to the substrate solution, and the gas containing the stable oxygen isotope is further bubbled through the substrate solution, obtaining a reaction solution containing the enzyme, the substrate, and the stable oxygen isotope; synthesizing the oxygen isotope-labeled compound in the reaction solution; A method for producing oxygen isotope-labeled compounds.

3. 3. The method for producing an oxygen-isotope-labeled compound according to claim 1, wherein the reaction solution is placed in a container, a gas containing the stable oxygen isotope is injected into a remaining space in the container, and the container is then sealed, and the oxygen-isotope-labeled compound is synthesized in the reaction solution.

4. 4. The method for producing an oxygen isotope-labeled compound according to claim 3, wherein a volume ratio expressed as V2 / V1 of a volume V1 of the reaction solution to a volume V2 of the remaining space in the container is 3 to 100.

5. 4. The method for producing an oxygen-isotope-labeled compound according to claim 3, wherein after the container is sealed, the oxygen-isotope-labeled compound is synthesized in the reaction solution while mixing by inverting the container.

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