JP7623858B2 - Oxygen isotope labeled compound and method for producing oxygen isotope labeled compound - Google Patents

Description

The present invention relates to oxygen isotope-labeled compounds and methods for producing oxygen isotope-labeled compounds.

Oxygen isotopes in nature are present in the proportions of ^16O at 99.759 atom%, ^17O at 0.037 atom%, and ^18O at 0.204 atom%. Of these oxygen isotopes, isotope-labeled compounds deoxygenated with the heavy isotope ^18O are useful in the fields of medicine, agrochemicals, and biochemistry as internal standard substances for quantitative analysis by mass spectrometry. The proportion (atom%) of a specific oxygen atom or all oxygen atoms constituting a compound that is composed of the oxygen isotope ^18O is sometimes referred to as "oxygen isotope ( ^18O ) enrichment" or simply "oxygen isotope enrichment".

Deuterium is often used as such a stable isotope element, but there are problems such as the difficulty of deuterium substitution in some compounds, and the insufficient number of isotope labels with deuterium substitution alone. In addition, the harmful effects of isotope effects have been pointed out. For this reason, the use of the oxygen isotope ^18O for labeling has been attracting attention in recent years.

Like carbon and nitrogen, oxygen has a larger mass number than hydrogen, so the physicochemical properties of the labeled form are closer to those of the unlabeled form; ¹⁸ O can increase the mass number by two per label; and it is less expensive than the carbon isotope ¹³ C and the nitrogen isotope ¹⁵ N. Despite these great advantages, oxygen is not often used in practice. The reason for this is that there are extremely few types of commercially available oxygen isotope compounds compared to deuterium-labeled compounds, carbon isotope-labeled compounds, and nitrogen isotope-labeled compounds. In addition, when introducing ¹⁸ O, it is essential to set strict reaction conditions that are not affected by ¹⁶ O water.

Since there are very few commercially available oxygen isotope labeling reagents and the types are limited, there is a need for oxygen isotope labeling reagents that can follow commonly used known reaction conditions. The ability to apply known reaction conditions has the great advantage of simplifying preliminary studies.

Among oxygen isotope-labeled reagents, oxygen isotope-labeled amino acids are used in proteome analysis of proteins, and are used as a method for introducing oxygen isotope-labeled amino acids as internal standards into protein or peptide samples and performing mass spectrometry.

As a method for labeling an amino acid having a carboxyl group with an oxygen isotope, Non-Patent Document 1 discloses a method in which an amino acid is reacted under strong acidic conditions in which H ₂ ¹⁸ O is saturated with hydrogen chloride gas, thereby exchanging the oxygen atom in the carboxyl group with an oxygen isotope.

However, there were problems with the exchange reaction of the carboxyl group in the amino acid skeleton under acidic conditions, such as the occurrence of decomposition reactions in amino acids such as tryptophan, cysteine, asparagine, and glutamine.

On the other hand, a glycine ester benzophenone Schiff base compound can be reacted with a specific organic compound in the presence of a catalyst and a basic compound to obtain a desired α-amino acid equivalent. Therefore, glycine ester benzophenone Schiff base compounds are useful compounds that can be used as raw materials for the synthesis of various α-amino acids such as asparagine and glutamine (Non-Patent Document 2).

E. Ponnusany, C. R. Jones, D. Fiat, Journal of Labeled Compounds and Radiopharmaceuticals, 14, 7, (1986) M. J. O’Donnell, Aldrichimica Acta, 34, 1, (2001)

The present invention was made in consideration of the above circumstances, and aims to provide a novel oxygen isotope-labeled compound that is useful as a raw material reagent for oxygen isotope-labeled α-amino acid equivalents, and a method for producing the same.

上記式（１）中、Ｙは^１８Ｏ又は^１７Ｏであり、Ｒ^１は炭素数１～２のアルキル基である。
［２］ 前記Ｙに示す酸素同位体の濃縮度が、天然存在比を超える、［１］に記載の酸素同位体標識化合物。
［３］ 下記式（２）に示す、酸素同位体標識化合物。

上記式（２）中、Ｙは^１８Ｏ又は^１７Ｏであり、Ｒ^１は炭素数１～２のアルキル基であり、Ｒ^２は、同一又は互いに異なる、水素、重水素、炭素数１～２のアルキル基、炭素数１～２のアルコキシ基、及びハロゲン原子のいずれかであり、ｎはそれぞれ１～５のいずれかである。
［４］ 前記Ｙに示す酸素同位体の濃縮度が、天然存在比を超える、［３］に記載の酸素同位体標識化合物。
［５］ ［１］又は［２］に記載の酸素同位体標識化合物の製造方法であって、
下記式（３）に示す化合物と、フタルイミドカリウムとを反応させる、酸素同位体標識化合物の製造方法。

上記式（３）中、Ｙは^１８Ｏ又は^１７Ｏであり、Ｒ^１は炭素数１～２のアルキル基であり、Ｘはハロゲン原子である。
［６］ ［３］又は［４］に記載の酸素同位体標識化合物の製造方法であって、
下記式（３）に示す化合物と、フタルイミドカリウムとを反応させて、下記式（１）に示す化合物を得、
得られた下記式（１）に示す化合物と下記式（４）に示す化合物とを反応させた後、塩化水素・１，４－ジオキサン溶液と反応させ、次いでベンゾフェノンイミンと反応させる、酸素同位体標識化合物の製造方法。

上記式（１）及び式（３）中、Ｙは^１８Ｏ又は^１７Ｏであり、Ｒ^１は炭素数１～２のアルキル基であり、Ｘはハロゲン原子である。
また、上記式（４）中、Ｚは水素、又はメチル基である。 In order to solve the above problems, the present invention has the following configuration.
[1] An oxygen isotope-labeled compound represented by the following formula (1):

In the above formula (1), Y is ¹⁸ O or ¹⁷ O, and R ¹ is an alkyl group having 1 to 2 carbon atoms.
[2] The oxygen isotope-labeled compound according to [1], wherein the enrichment of the oxygen isotope represented by Y exceeds its natural abundance.
[3] An oxygen isotope-labeled compound represented by the following formula (2):

In the above formula (2), Y is ¹⁸ O or ¹⁷ O, R ¹ is an alkyl group having 1 to 2 carbon atoms, R ² is the same or different and is any one of hydrogen, deuterium, an alkyl group having 1 to 2 carbon atoms, an alkoxy group having 1 to 2 carbon atoms, and a halogen atom, and n is any one of 1 to 5.
[4] The oxygen isotope-labeled compound according to [3], wherein the enrichment of the oxygen isotope represented by Y exceeds its natural abundance.
[5] A method for producing an oxygen isotope-labeled compound according to [1] or [2], comprising the steps of:
A method for producing an oxygen isotope-labeled compound, comprising reacting a compound represented by the following formula (3) with potassium phthalimide:

In the above formula (3), Y is ¹⁸ O or ¹⁷ O, R ¹ is an alkyl group having 1 to 2 carbon atoms, and X is a halogen atom.
[6] A method for producing an oxygen isotope-labeled compound according to [3] or [4], comprising the steps of:
A compound represented by the following formula (3) is reacted with potassium phthalimide to obtain a compound represented by the following formula (1):
The resulting compound represented by the following formula (1) is reacted with a compound represented by the following formula (4), and then the resulting mixture is reacted with a hydrogen chloride/1,4-dioxane solution and then with benzophenone imine, thereby producing an oxygen isotope-labeled compound.

In the above formulas (1) and (3), Y is ¹⁸ O or ¹⁷ O, R ¹ is an alkyl group having 1 to 2 carbon atoms, and X is a halogen atom.
In addition, in the above formula (4), Z is hydrogen or a methyl group.

The oxygen isotope-labeled compounds of the present invention are novel compounds (oxygen isotope-labeled glycine ester benzophenone Schiff base compounds and oxygen isotope-labeled phthalimidoacetic ester compounds serving as the raw materials) that are useful as raw material reagents for oxygen isotope-labeled α-amino acid equivalents.
The method for producing an oxygen isotope-labeled compound of the present invention can produce the novel compound described above.

<Oxygen isotope labeled compounds>
The oxygen isotope-labeled compound of the present invention is an oxygen isotope-labeled phthalimidoacetic acid ester compound represented by the following formula (1), and an oxygen isotope-labeled glycine ester benzophenone Schiff base compound represented by the following formula (2).

In the above formula (1) and formula (2), Y is ¹⁸ O or ¹⁷ O, R ¹ is an alkyl group having 1 to 2 carbon atoms, R ² is the same or different and is any one of hydrogen, deuterium, an alkyl group having 1 to 2 carbon atoms, an alkoxy group having 1 to 2 carbon atoms, and a halogen atom, and n is any one of 1 to 5.

In the oxygen isotope-labeled compound of the present invention, the enrichment (abundance ratio) of the oxygen isotope represented by Y in the above formula (1) and formula (2) preferably exceeds the natural abundance ratio of 0.204 atom%, and more preferably is 85 atom% or more. In other words, since the oxygen isotope-labeled compound of the present invention is used as a raw material reagent for oxygen isotope-labeled α-amino acid equivalents, the closer the enrichment is to 100 atom%, the more preferable it is.

The isotopic enrichment of the oxygen isotope target compound of the present invention can be calculated using the peak area value in EI-MS (electron ionization mass spectrometry) by the same calculation method as in Example 2 described in JP 2006-008666 A.

<Method for producing oxygen isotope-labeled compound>
[Method for producing oxygen isotope-labeled phthalimidoacetic acid ester compound]
The method for producing the oxygen isotope-labeled phthalimide acetic acid ester compound represented by the above formula (1) comprises reacting a compound represented by the following formula (3) with potassium phthalimide.

In the above formula (3), Y is ¹⁸ O or ¹⁷ O, R ¹ is an alkyl group having 1 to 2 carbon atoms, and X is a halogen atom (F, Cl, Br, I).

Specifically, the method for producing an oxygen isotope-labeled compound of the present invention involves first reacting the compound represented by the above formula (3) with potassium phthalimide in an organic solvent. After the reaction is completed, distilled water is added to the reaction solution, and the precipitated powder represented by the above formula (1) is separated from the solvent by filtration. In this way, the oxygen isotope-labeled phthalimide acetic acid ester compound represented by the above formula (1) is obtained (isolated).

In the method for producing an oxygen isotope-labeled compound of the present invention, the enrichment (abundance ratio) of the oxygen isotope ( ^18O or ^17O ) represented by Y in the above formula (3) preferably exceeds the natural abundance ratio, more preferably is 90 atom% or more, and even more preferably is 95 atom% or more.

The organic solvent used in the reaction of the compound represented by formula (3) with potassium phthalimide is not particularly limited. Examples of such organic solvents include alcohols such as methanol and ethanol.

The amount of the compound shown in the above formula (3) used relative to potassium phthalimide is preferably a stoichiometric amount or more. There is no particular upper limit to the amount of the compound shown in the above formula (3) used, but in order to avoid increased costs due to excessive use, it is preferably 1.03 equivalents or less, and more preferably 1.01 equivalents or less.

The reaction temperature between the compound shown in formula (3) and potassium phthalimide is in the range of 0 to 100°C, preferably in the range of 40 to 60°C. A reaction temperature below 0°C is not preferred because the reaction does not proceed, and a reaction temperature above 100°C is not preferred because there is a risk of decomposition of the raw materials and products.

The reaction time between the compound shown in formula (3) and potassium phthalimide is 30 minutes or more. If the reaction time is less than 30 minutes, the reaction will not be completed, which is not preferable.

[Method for producing oxygen isotope-labeled glycine ester benzophenone Schiff base compound]
The method for producing the oxygen isotope-labeled glycine ester benzophenone Schiff base compound represented by the above formula (2) comprises reacting the oxygen isotope-labeled phthalimidoacetic ester compound represented by the above formula (1) with a compound represented by the following formula (4), followed by reacting with a hydrogen chloride-1,4-dioxane solution, and then reacting with benzophenone imine.

In the above formula (4), Z is hydrogen or a methyl group.

Specifically, in the method for producing an oxygen isotope-labeled compound of the present invention, first, the compound of the above formula (1) (oxygen isotope-labeled phthalimidoacetic ester compound) is reacted with the compound shown in the above formula (4) in an organic solvent to produce an oxygen isotope-labeled glycine ester in the system.
The organic solvent is then distilled off under reduced pressure, and a solution of hydrogen chloride in 1,4-dioxane is added to give oxygen isotope-labeled glycine ester hydrochloride, after which the solvent is distilled off under reduced pressure.
Next, benzophenone imine and an organic solvent are added to this reaction system, and the reaction is allowed to proceed with oxygen isotope-labeled glycine ester hydrochloride, after which the product is isolated and purified using column chromatography.
As a result, the oxygen isotope-labeled glycine ester benzophenone Schiff base represented by the above formula (2) is obtained.

The organic solvent used in the reaction between the compound shown in formula (1) and the compound shown in formula (4) is not particularly limited. Examples of such organic solvents include alcohols such as methanol and ethanol.

The amount of the compound shown in formula (4) used relative to the compound shown in formula (1) may be a small stoichiometric excess, preferably 1.1 to 1.3 equivalents. By using a small stoichiometric excess, decomposition of the compound can be suppressed.

The reaction time between the compound shown in formula (1) and the compound shown in formula (4) is preferably 1 to 3 hours. If the reaction time is less than 1 hour, the reaction does not proceed, which is not preferred. On the other hand, if the reaction time exceeds 3 hours, the oxygen isotope-labeled glycine ester produced will decompose, which is not preferred.

The reaction between the compound represented by formula (1) and the compound represented by formula (4) is preferably carried out under ice cooling. If the reaction temperature is higher than room temperature, it is not preferable because it leads to decomposition of the oxygen isotope-labeled glycine ester.

The amount of hydrogen chloride/1,4-dioxane solution used is preferably such that the amount of hydrogen chloride is in stoichiometric excess over the compound shown in formula (1) above.

When adding the hydrogen chloride/1,4-dioxane solution to the reaction system, it is preferable to carry out the reaction under ice cooling. If the reaction temperature is higher than room temperature, it will lead to the decomposition of the oxygen isotope-labeled glycine ester, which is not recommended.

The solvent used in the reaction with benzophenone imine is not particularly limited. Examples of such solvents include dichloromethane and chloroform, and among these, it is preferable to use dichloromethane.

The amount of benzophenone imine used is preferably an amount that is stoichiometrically equivalent to the compound shown in formula (1) above.

In the reaction with benzophenone imine, the reaction temperature is not particularly limited, but is preferably 0 to 50°C. The reaction time is not particularly limited, but is preferably 12 to 24 hours.

As explained above, the oxygen isotope-labeled compound of the present invention is a novel compound.
The oxygen isotope-labeled glycine ester benzophenone Schiff base compound of the present invention is useful as a raw material reagent for oxygen isotope-labeled α-amino acid equivalents.
The oxygen isotope-labeled phthalimidoacetic ester compound of the present invention is a raw material for the above-mentioned oxygen isotope-labeled glycine ester benzophenone Schiff base compound.
The method for producing an oxygen isotope-labeled compound of the present invention can produce the novel compound described above.

The present invention is not limited to such specific embodiments. Furthermore, the present invention may be modified in various ways, including additions, omissions, substitutions, and other changes, within the scope of the gist of the present invention as described in the claims.

The effects of the present invention will be explained in detail below using examples. Note that the present invention is not limited to the contents of the following examples.

Example 1
Methyl phthalimidoacetate- ¹⁸ O was synthesized by the reaction shown in formula (5) below.

Specifically, 2.58 g (16.4 mmol) of methyl bromoacetate- ¹⁸ O was placed in a 10 mL recovery flask, 10 mL of N,N-dimethylformamide was added and stirred, and 3.06 g (16.5 mmol) of potassium phthalimide was added and reacted overnight at 50° C. After the reaction, the liquid was separated and the solvent was distilled off under reduced pressure to obtain methyl phthalimidoacetate- ¹⁸ O (3.30 g, yield 91%, concentration 98.0 atom% ¹⁸ O).

Example 2
Glycine methyl ester benzophenone Schiff base- ¹⁸ O was synthesized by the reaction shown in formula (6) below.

Specifically, 0.44 g (2 mmol) of methyl phthalimidoacetate- ¹⁸ O and 20 mL of ethanol were added to a 50 mL eggplant flask and stirred under ice cooling, then 4.4 mL (4.4 mmol) of 1 M methylhydrazine-ethanol solution was added and reacted under ice cooling for 2 hours, and the solvent was distilled off under reduced pressure. 20 mL of ethanol and 2 mL of 4 M hydrogen chloride-1,4-dioxane solution were added to this reaction system under ice cooling and stirred, and the solvent was distilled off again under reduced pressure. 10 mL of dichloromethane and 0.34 mL (2 mmol) of benzophenone imine were added to the obtained powder and stirred for 24 hours. After the reaction, the reaction solution was filtered, the solvent was distilled off under reduced pressure, and the mixture was purified by column chromatography to obtain glycine methyl ester benzophenone Schiff base- ¹⁸ O (0.09 g, yield 18%, concentration 89.8 atom% ¹⁸ O).

Example 3
Ethyl phthalimidoacetate- ¹⁸ O was synthesized by the reaction shown in the following formula (7).

Specifically, 3.00 g (17.8 mmol) of ethyl bromoacetate- ¹⁸ O was placed in a 10 mL recovery flask, 9 mL of N,N-dimethylformamide was added and stirred, and then 3.32 g (17.9 mmol) of potassium phthalimide was added and reacted overnight at 50° C. After the reaction, the liquid was separated and the solvent was distilled off under reduced pressure to obtain ethyl phthalimidoacetate- ¹⁸ O (3.87 g, yield 93%, concentration 97.3 atom% ¹⁸ O).

Example 4
Glycine ethyl ester benzophenone Schiff base- ¹⁸ O was synthesized by the reaction shown in formula (8) below.

Specifically, 0.47 g (2 mmol) of ethyl phthalimide acetate- ¹⁸ O and 20 mL of ethanol were added to a 50 mL eggplant flask and stirred under ice cooling, then 4.4 mL (4.4 mmol) of 1 M methylhydrazine-ethanol solution was added and reacted under ice cooling for 2 hours, and the solvent was distilled off under reduced pressure. 20 mL of ethanol and 2 mL of 4 M hydrogen chloride-1,4-dioxane solution were added to this reaction system under ice cooling and stirred, and the solvent was distilled off again under reduced pressure. 10 mL of dichloromethane and 0.34 mL (2 mmol) of benzophenone imine were added to the obtained powder and stirred at room temperature for 24 hours. After the reaction, the reaction solution was filtered, the solvent was distilled off under reduced pressure, and the mixture was purified by column chromatography to obtain glycine ethyl ester benzophenone Schiff base- ¹⁸ O (0.11 g, yield 20%, concentration 91.2 atom% ¹⁸ O).

The oxygen isotope-labeled compound and the method for producing the oxygen isotope-labeled compound of the present invention have industrial applicability as an oxygen isotope-labeled compound for synthesizing an oxygen isotope-labeled α-amino acid equivalent and as an oxygen isotope-labeled compound that serves as a synthetic intermediate thereof.

Claims (6)

An oxygen isotope-labeled compound represented by the following formula (1).

In the above formula (1), Y is ¹⁸ O or ¹⁷ O, and R ¹ is an alkyl group having 1 to 2 carbon atoms. The oxygen isotope-labeled compound according to claim 1, wherein the enrichment of the oxygen isotope represented by Y exceeds its natural abundance. An oxygen isotope-labeled compound represented by the following formula (2).

In the above formula (2), Y is ¹⁸ O or ¹⁷ O, R ¹ is an alkyl group having 1 to 2 carbon atoms, R ² is the same or different and is any one of hydrogen, deuterium, an alkyl group having 1 to 2 carbon atoms, an alkoxy group having 1 to 2 carbon atoms, and a halogen atom, and n is any one of 1 to 5. The oxygen isotope-labeled compound according to claim 3, wherein the enrichment of the oxygen isotope represented by Y exceeds its natural abundance. A method for producing the oxygen isotope-labeled compound according to claim 1 or 2, comprising the steps of:
A method for producing an oxygen isotope-labeled compound, comprising reacting a compound represented by the following formula (3) with potassium phthalimide:

In the above formula (3), Y is ¹⁸ O or ¹⁷ O, R ¹ is an alkyl group having 1 to 2 carbon atoms, and X is a halogen atom. A method for producing an oxygen isotope-labeled compound according to claim 3 or 4, comprising the steps of:
A compound represented by the following formula (3) is reacted with potassium phthalimide to obtain a compound represented by the following formula (1):
The resulting compound represented by the following formula (1) is reacted with a compound represented by the following formula (4), and then the resulting mixture is reacted with a hydrogen chloride/1,4-dioxane solution and then with benzophenone imine, thereby producing an oxygen isotope-labeled compound.

In the above formulas (1) and (3), Y is ¹⁸ O or ¹⁷ O, R ¹ is an alkyl group having 1 to 2 carbon atoms, and X is a halogen atom.
In addition, in the above formula (4), Z is hydrogen or a methyl group.