WO2017188355A1 - Establishment of synthesis method for cysteine with isotopically labeled sulfur atom and cysteine derivative - Google Patents

Abstract

The present invention relates to synthesis methods for cysteine with an isotopically labeled sulfur atom and a cysteine derivative. The present invention also relates to a kit for use in the synthesis methods. The present invention makes it possible to easily and efficiently synthesize cysteine with an isotopically labeled sulfur atom and a cysteine derivative.

Description

Establishment of synthetic methods for cysteine and cysteine derivatives isotopically labeled with sulfur atoms

The present invention relates to a method for synthesizing cysteine and cysteine derivatives in which a sulfur atom is labeled with an isotope. The present invention also relates to a kit for use in the synthesis method.

With recent developments in mass spectrometry technology, proteomics and metabolomics have been actively analyzed. For precise quantification by mass spectrometry, it is necessary to use a compound labeled with a stable isotope as an internal standard. However, there are a wide variety of commercially available stable isotopes of carbon atoms and nitrogen atoms, while stable isotopes of sulfur atoms are hardly commercially available except for custom-made products.

The method known so far as a method for synthesizing an amino acid in which a sulfur atom is labeled with an isotope is based on organic chemical synthesis or biosynthesis. In the case of synthesizing an isotope-labeled amino acid by organic chemical synthesis, it is necessary to go through many complicated steps (Non-Patent Document 1), and the yield is as low as about 0.01%. It was. The biosynthetic technique is a technique in which a microorganism such as Escherichia coli is cultured in a medium containing an isotope-labeled sulfur-containing substrate as a sole sulfur source, and cysteine or methionine produced by the microorganism is recovered (non-native). Patent Document 2). The yield of cysteine labeled with an isotope-labeled sulfur atom obtained by a biosynthetic technique is about 10%, which is not a sufficiently high yield.

As described above, a stable isotope-labeled compound is required for precise quantification by mass spectrometry. Therefore, establishment of a technique for obtaining cysteine and cysteine derivatives in which sulfur atoms are isotopically labeled with high yields has been demanded.

The present invention provides a method for efficiently synthesizing cysteine and a cysteine derivative in which a sulfur atom is isotopically labeled and a kit used in the method.

In view of the above, the inventors of the present case have started paying attention to cysteine synthase. As a result of intensive studies, it was found that cysteine and cysteine derivatives in which sulfur atoms are isotopically labeled can be efficiently synthesized by an enzymatic reaction using cysteine synthase. Based on this finding, the present invention has been completed.

That is, in one aspect, the present invention may be as follows.

[1] A method for synthesizing cysteine in which a sulfur atom is isotopically labeled, wherein O-acetyl-L-serine and a sulfur atom are isotopically labeled in the presence of cysteine synthase (EC: 2.5.1.47) Reacting the substrate, wherein the isotopically labeled substrate for the sulfur atom is sodium hydrogen sulfide (NaHS), sodium sulfide (Na ₂ S), or hydrogen sulfide (H ₂ S); and Wherein said sulfur atom isotope label is selected from the group consisting of stable isotope ³² S, stable isotope ³⁴ S, radioactive isotope ³³ S and radioactive isotope ³⁵ S.

[2] O-acetyl-L-serine was isotopically labeled for nitrogen and / or carbon atoms in the presence of the following steps: serine-O-acetyltransferase (EC: 2.3.1.30) The method according to [1] above, further comprising obtaining serine.

[3] The method according to [1] or [2] above, wherein the cysteine synthase is cysteine synthase A (CysK) or cysteine synthase B (CysM).

[4] The method according to [3] above, wherein the cysteine synthase A is derived from a microorganism belonging to the genus Salmonella.

[5] The method according to any one of [1] to [4] above, wherein the cysteine synthase is a recombinant protein containing a histidine tag.

[6] A method for synthesizing cystine in which a sulfur atom is isotopically labeled, comprising synthesizing a cysteine labeled with an isotopically sulfur atom in accordance with the method described in [1] or [2] above, and using the cysteine as an oxidizing agent. Said method comprising the step of obtaining cystine by oxidation in the presence.

[7] The method according to [6] above, wherein the oxidizing agent is iodine (I ₂ ).

[8] A method for synthesizing a cysteine derivative,
(I) the cysteine derivative is a sulfocysteine in which a sulfur atom is isotope-labeled, and comprises reacting O-acetyl-L serine and sodium sulfite (Na ₂ SO ₃ ) in the presence of cysteine synthase, The sulfur atom is selected from the group consisting of a stable isotope ³² S, a stable isotope ³⁴ S, a radioactive isotope ³³ S and a radioactive isotope ³⁵ S;
(Ii) The cysteine derivative is S-sulfocysteine in which a sulfur atom is isotopically labeled, and O-acetyl-L-serine and sodium thiosulfate (Na ₂ S ₂ O ₃ ) are reacted in the presence of cysteine synthase B. Wherein the sulfur atom is selected from the group consisting of a stable isotope ³² S, a stable isotope ³⁴ S, a radioactive isotope ³³ S and a radioactive isotope ³⁵ S;
(Iii) The cysteine derivative is a cysteine persulfide in which a sulfur atom is isotope-labeled, and includes a step of reacting O-acetyl-L-serine and sodium disulfide (Na ₂ S ₂ ) in the presence of cysteine synthase. Where the sulfur atom is selected from the group consisting of stable isotope ³² S, stable isotope ³⁴ S, radioactive isotope ³³ S and radioactive isotope ³⁵ S;
(Iv) The cysteine derivative is a cysteine persulfide in which a sulfur atom is isotopically labeled. (A) In the presence of cysteine synthase, an O-acetyl-L-serine and a substrate that is isotopically labeled with a sulfur atom are reacted. Obtaining a cysteine having an isotope-labeled sulfur atom, wherein the substrate isotope-labeled for the sulfur atom is sodium hydrogen sulfide (NaHS) or sodium sulfide (Na ₂ S), wherein the sulfur atom Isotope label is selected from the group consisting of stable isotope ³² S, stable isotope ³⁴ S, radioactive isotope ³³ S and radioactive isotope ³⁵ S, and (b) the sulfur atom obtained in step (a) Reacting isotope-labeled cysteine with sodium disulfide; or (v) a cysteine derivative is seleno A stain in the presence of a cysteine synthase, comprising the step of reacting O- acetyl -L- serine and sodium selenide _(Na 2 Se);
Said method.

[9] O-acetyl-L-serine is reacted with isotope-labelled serine for nitrogen and / or carbon atoms in the presence of serine-O-acetyltransferase (EC: 2.3.3.10) The method according to [8] above, further comprising:

[10] The method according to [8] or [9] above, wherein the cysteine synthase is cysteine synthase A (CysK) or cysteine synthase B (CysM).

[11] The method according to [10] above, wherein the cysteine synthase A is derived from a microorganism belonging to the genus Salmonella.

[12] The method according to any one of [8] to [11] above, wherein the cysteine synthase is a recombinant protein containing a histidine tag.

[13] A kit for synthesizing cysteine or cystine, or a cysteine derivative, wherein a sulfur atom is isotopically labeled,
Cysteine synthase; and sodium hydrogen sulfide (NaHS), sodium sulfide (Na ₂ S), sodium sulfite (Na ₂ SO ₃ ), sodium thiosulfate (Na ₂ S ₂ O ₃ ), and A substrate containing at least one sulfur selected from the group consisting of sodium sulfide (Na ₂ S ₂ ), wherein the isotope label of the sulfur atom is a stable isotope ³² S, a stable isotope ³⁴ S, a radioactive isotope ³³ Selected from the group consisting of S and radioisotope ³⁵ S, or sodium selenide (Na ₂ Se);
The kit.

[14] A method for synthesizing a cysteine derivative,
(I) the cysteine derivative is a cystine polysulfide in which a sulfur atom is isotopically labeled, and (a) a step of synthesizing a cysteine in which a sulfur atom is isotopically labeled according to the method described in [1] or [2] above, b) reacting the cysteine labeled with an isotope-labeled sulfur atom obtained in step (a) with an oxidizing agent, and (c) further reacting by adding sodium sulfide (Na ₂ S); or ( ii) the cysteine derivative is N-acetylcysteine obtained by isotopically labeling a sulfur atom, and (a) a step of synthesizing a cysteine labeled with an isotopically labeled sulfur atom according to the method described in [1] or [2] above, And (b) reacting cysteine labeled with an isotopically labeled sulfur atom obtained in step (a) with an acetylating agent;
Said method.

The present invention is useful in that cysteine and cysteine derivatives labeled with a sulfur atom isotope-labeled can be synthesized with few steps and high yield.

The present invention will be specifically described below, but the present invention is not limited to these. Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

Enzyme (1) Cysteine Synthase Cysteine synthase (EC: 2.5.1.47) is an enzyme involved in cysteine biosynthesis in vivo, and has the following chemical reaction:

It is an enzyme that catalyzes. That is, this enzyme produces L-cysteine and acetic acid using O3-acetyl-L-serine and hydrogen sulfide as substrates. Cysteine transferase belongs to the family of transferases, and there are two types, cysteine synthase A (CysK) and cysteine synthase B (CysM), based on differences in substrate specificity for sulfur sources such as hydrogen sulfide.

The cysteine synthase used in the present invention is not particularly limited as long as it is an enzyme that catalyzes the above reaction. Cysteine synthase A or cysteine synthase B is preferable, and cysteine synthase A is more preferable. The origin of cysteine synthase is not particularly limited, but examples include those derived from Gram-negative bacteria such as Escherichia coli and Salmonella, and Gram-positive bacteria such as Staphylococcus and Streptococcus. Preferably, it is cysteine synthase A derived from Salmonella enterica serover Typhimurium.

The cysteine synthase used in the present invention may be extracted from a microorganism or may be prepared by genetic recombination. When cysteine synthase prepared by gene recombination is used, one modified with a histidine tag (a tag comprising 6 to 11 consecutive histidine residues) or the like may be used. Since the histidine tag has a high affinity for metal ions such as nickel, a recombinant protein modified with a histidine tag can be supported on a resin on which a metal such as nickel is immobilized. By supporting a cysteine synthase containing a histidine tag on a resin in which a metal such as nickel is immobilized, the enzyme can be easily recovered from the reaction system, so that it can be easily separated from the product. It can be reused. In this regard, it is advantageous to use a recombinant cysteine synthase containing a histidine tag.

When cysteine synthase A (CysK) is used, it is known that the enzyme activity of CysK requires pyridoxal phosphate (PLP), which is a coenzyme. When CysK is produced from recombinant E. coli, PLP is purified while bound to CysK. Therefore, in the present invention, when recombinant cysteine synthase A is used, it is not always necessary to add PLP to the reaction system during the enzymatic reaction.

(2) Serine-O-acetyltransferase Serine-O-acetyltransferase (EC: 2.3.3.10) is a reaction that produces O-acetyl-L-serine and CoA using L-serine and acetyl CoA as substrates. An enzyme that catalyzes the following chemical reactions:

It is an enzyme that catalyzes.

The serine-O-acetyltransferase used in the present invention is not particularly limited as long as it is an enzyme that catalyzes the above-mentioned reaction. The thing derived from a microbe is mentioned. Serine-O-acetyltransferase may be extracted from microorganisms or may be prepared by genetic recombination. When serine-O-acetyltransferase prepared by gene recombination is used, it is modified with a histidine tag (a tag comprising 6 to 11 consecutive histidine residues) or the like for the reason described in (1) cysteine synthase above. A thing may be used.

An isotope-labeled isotope refers to a relationship between nuclides having the same atomic number but different numbers of neutrons. As used herein, “isotopically labeled” means a state in which a specific nuclide of a compound is substituted with an isotope other than the isotope having the highest natural abundance ratio. “Isotope labeling” means an isotope other than the isotope having the highest natural abundance ratio for a specific nuclide of a compound. The isotope label may be a stable isotope or a radioisotope. Here, the radioisotope has a radioactivity for emitting radiation by decay, and the stable isotope is a stable isotope that does not cause decay.

Isotope label used for sulfur atoms in the present invention is not particularly limited as long as the isotope of sulfur, preferably, stable isotopes ^{32 S,} stable isotope ^{34 S,} radioisotope ^{33 S} and radioactive isotopes ³⁵ S Selected from the group consisting of More preferably, the isotope label used for sulfur atoms in the present invention are stable isotope ^{34 S.}

The isotope label used for the selenium atom in the present invention is not particularly limited as long as it is an isotope of selenium, but is preferably selected from the group consisting of stable isotope ⁷⁸ Se, stable isotope ⁷⁷ Se and stable isotope ⁷⁶ Se. The More preferably, the isotope label used for the selenium atom is the stable isotope ⁷⁸ Se.

Cysteine and cysteine derivatives Cysteine is a kind of natural amino acid and is L-cysteine unless otherwise specified.

In the present specification, the cysteine in which a sulfur atom is isotopically labeled means a cysteine in which the sulfur atom of the mercapto group (—SH) of cysteine is isotopically labeled.

As used herein, cysteine derivatives include the following:
(I) a compound that is cystine (Cys-SS-Cys), wherein at least one sulfur atom in the molecule is isotopically labeled;
(Ii) a compound having sulfocysteine (Cys-SO ₃ H) or S-sulfocysteine (Cys-SSO ₃ H), in which a sulfur atom in the molecule is isotopically labeled;
(Iii) Cysteine persulfide (Cys-SSH), a compound in which at least one sulfur atom in the molecule is isotopically labeled;
(Iv) selenocysteine (Cys-SeH), wherein the selenium atom may or may not be isotopically labeled;
(V) a cystine polysulfide (Cys-SS ( _1-3) -S-Cys), wherein at least one sulfur atom in the molecule is isotopically labeled;
(Vi) N-acetylcysteine, which is an isotope-labeled sulfur atom in the molecule.

In one embodiment of the method for synthesizing cysteine, the method for synthesizing cysteine labeled with an isotopically labeled sulfur atom, comprising the step of reacting O-acetyl-L-serine and a substrate labeled with an isotope labeled with a sulfur atom in the presence of cysteine synthase. ,I will provide a.

The isotopically labeled substrate for the sulfur atom is sodium hydrogen sulfide (NaHS), sodium sulfide (Na ₂ S), or hydrogen sulfide (H ₂ S), which may be derived from sodium sulfide. The types of isotope labeling of sulfur atoms are as described above in the section “Isotope labeling”.

The reaction conditions are not particularly limited as long as the enzyme activity of cysteine synthase is maintained. For example, 25 to 37 ° C., pH 7 to 8 and the like can be mentioned.

In one embodiment of the method for synthesizing cystine , the following steps:
(I) reacting isotope-labeled substrates for O-acetyl-L-serine and sulfur atoms in the presence of cysteine synthase;
(Ii) Cystine is obtained by oxidizing the cysteine labeled with the isotope-labeled sulfur atom obtained in step (i) with an oxidizing agent;
A method for synthesizing a cystine labeled with an isotope of a sulfur atom is provided.

Cystine is 3,3′-dithiobis (2-aminopropionic acid) and has a structure in which two molecules of cysteine in the cysteine are linked by forming a disulfide bond. In the present specification, cystine is sometimes referred to as Cys-SS-Cys for convenience.

The step (i) can be performed as described in the above item “Method for synthesizing cysteine”.

The oxidizing agent used in the step (ii) is not particularly limited as long as it is a reagent that can promote the formation of a disulfide bond by oxidizing a mercapto group. For example, iodine (I ₂ ), heavy metal, hydrogen peroxide ( H ₂ O ₂₎ or the like can be preferably used. In a preferred embodiment, the oxidizing agent is iodine.

The reaction conditions in the above step (ii) are not particularly limited as long as they are suitable for the formation of disulfide bonds, and examples include conditions such as 25 ° C. to 37 ° C. and pH 7 to 8.

In one embodiment of the method for synthesizing sulfocysteine, a sulfur atom is reacted with O-acetyl-L-serine and sodium sulfite (Na ₂ SO ₃ ) isotopically labeled for sulfur atom in the presence of cysteine synthase. A method of synthesizing isotopically labeled sulfocysteine is provided. The type of isotope labeling of the sulfur atom is as described above in the section “Isotope labeling”.

The structure of sulfocysteine can be represented by the following formula: H ₂ N—CH (CH ₂ —SO ₃ H) —COOH. In the present specification, sulfocysteine is sometimes referred to as Cys-SO ₃ H for convenience.

The reaction conditions are not particularly limited as long as the enzyme activity of cysteine synthase is maintained. For example, 25 to 37 ° C., pH 7 to 8 and the like can be mentioned.

In one embodiment of the method for synthesizing S-sulfocysteine, in the presence of cysteine synthase B, reacting sodium thiosulfate (Na ₂ S ₂ O ₃ ) isotopically labeled with O-acetyl-L-serine and a sulfur atom And a method for synthesizing S-sulfocysteine, wherein the sulfur atom is isotopically labeled. The type of isotope labeling of the sulfur atom is as described above in the section “Isotope labeling”.

The structure of S-sulfocysteine can be represented by the following formula: H ₂ N—CH (CH ₂ —SSO ₃ H) —COOH. In the present specification, S-sulfocysteine is sometimes referred to as Cys-SSO ₃ H for convenience.

The reaction conditions are not particularly limited as long as the enzyme activity of cysteine synthase B is maintained. For example, 25 to 37 ° C., pH 7 to 8 and the like can be mentioned.

In one embodiment of the method for synthesizing cysteine persulfide, sulfur comprising the step of reacting O-acetyl-L-serine and isotopically labeled sodium disulfide (Na ₂ S ₂ ) with respect to a sulfur atom in the presence of cysteine synthase. Provided is a method for synthesizing cysteine persulfide having an isotope-labeled atom. The type of isotope labeling of the sulfur atom is as described above in the section “Isotope labeling”.

The structure of cysteine persulfide can be represented by the following formula: H ₂ N—CH (CH ₂ —SSH) —COOH. In this specification, cysteine persulfide may be referred to as Cys-SSH for convenience.

The reaction conditions are not particularly limited as long as the enzyme activity of cysteine synthase is maintained. Examples thereof include 30 to 42 ° C., pH 7 to 8 and the like.

In another embodiment, a sulfur atom isotope-labeled cysteine par comprising the step of reacting a sulfur atom isotopically labeled with a sulfur atom obtained by the method described in the above-mentioned section of “Method for synthesizing cysteine” and sodium disulfide. A method for the synthesis of sulfides is provided. In this case, in particular, the sulfur atom bonded to the β carbon of cysteine is isotopically labeled, but the terminal SH group may be an isotope having the highest natural abundance ratio. The conditions for reacting cysteine labeled with an isotope with sulfur atoms and sodium disulfide are not particularly limited as long as they allow persulfide to be formed. An example is 30 to 42 ° C.

In one embodiment of the method for synthesizing selenocysteine, a method for synthesizing selenocysteine is provided, which comprises the step of reacting O-acetyl-L-serine and sodium selenide (Na ₂ Se) in the presence of cysteine synthase. Sodium selenide may or may not be isotopically labeled. When sodium selenide is isotopically labeled, selenocysteine in which the selenium atom is isotopically labeled is obtained. The type of isotope labeling of the selenium atom is as described above in the section “Isotope labeling”.

The structure of selenocysteine can be represented by the following formula: H ₂ N—CH (CH ₂ —SeH) —COOH. That is, selenocysteine has a structure in which the sulfur atom of cysteine is substituted with selenium. In the present specification, selenocysteine is sometimes expressed as Cys-SeH for convenience.

The reaction conditions are not particularly limited as long as the enzyme activity of cysteine synthase is maintained. For example, 25 to 37 ° C., pH 7 to 8 and the like can be mentioned.

In one embodiment of the method for synthesizing cystine polysulfide ,
(I) reacting isotope-labeled substrates for O-acetyl-L-serine and sulfur atoms in the presence of cysteine synthase;
(Ii) reacting the cysteine labeled with an isotope of the sulfur atom obtained in step (i) with an oxidizing agent sodium nitrite (NaNO ₂ );
(Iii) A method for synthesizing a cystine polysulfide in which a sulfur atom is isotopically labeled is provided, which further comprises adding sodium sulfide (Na ₂ S) to react to obtain a cystine polysulfide.

The structure of cystine polysulfide can be represented by the following formula: HN ₂ —CH (COOH) —CH ₂ —SS _(1-3) —S—CH ₂ —CH (COOH) —NH ₂ . In the present specification, the cystine polysulfide is sometimes referred to as Cys-SS _(1-3) -S-Cys for convenience.

The step (i) can be carried out as described in the item “Method for synthesizing cysteine” above.

The oxidizing agent used in the above step (ii) is not particularly limited as long as it is a reagent that can promote the reaction of extracting a hydrogen atom from a mercapto group. For example, sodium nitrite (NaNO ₂ ), iodine (I ₂ ), metal Salts (eg, iron (II) sulfide (FeSO ₄ ), iron (II) chloride (FeCl ₂ ), manganese (II) chloride (MnCl ₂ ), manganese (III) acetate (Mn (OCOCH ₃ ) ₃ , copper sulfide ( II) (CuSO ₄ ), zinc chloride (ZnCl ₂ )), metal-containing enzymes (eg, hemin, superoxide dismutase (SOD), catalase, horseradish peroxidase (HRP)), etc. can be suitably used. The oxidizing agent is sodium nitrite (NaNO ₂ ).

The sodium sulfide (Na ₂ S) used in the above step (iii) may be an isotope-labeled sulfur atom or an unisotopically labeled sulfur atom.

The reaction conditions (ii) and (iii) are not particularly limited as long as they are suitable for forming a polysulfide bond, and examples thereof include 25 to 37 ° C.

In one embodiment of the method for synthesizing N-acetylcysteine , the following steps:
(I) reacting isotope-labeled substrates for O-acetyl-L-serine and sulfur atoms in the presence of cysteine synthase;
(Ii) N-acetylcysteine is obtained by reacting the cysteine labeled with an isotopically labeled sulfur atom obtained in step (i) with an acetylating agent;
A method for synthesizing N-acetylcysteine labeled with an isotope of a sulfur atom is provided.

The structure of N-acetylcysteine can be represented by the following formula: CH ₃ —CONH—C (CH ₂ —SH) —COOH.

The step (i) can be carried out as described in the item “Method for synthesizing cysteine” above.

The acetylating agent used in the above step (ii) is not particularly limited as long as it is a reagent capable of acetylating the amino group of cysteine. For example, acetyl CoA, acetic anhydride and the like can be preferably used. In a preferred embodiment, the acetylating agent is acetyl CoA.

The reaction conditions in the above step (ii) are not particularly limited as long as they are suitable for the acetylation reaction, and examples include conditions such as 25 ° C. to 37 ° C. and pH 7 to 8.

Provision of O-acetyl-L-serine in which nitrogen and / or carbon atoms are isotopically labeled The above cysteine, cystine, sulfocysteine, S-sulfocysteine, cysteine persulfide, cystine polysulfide, N-acetylcysteine or selenocysteine In this synthesis method, nitrogen and / or carbon and sulfur are isotopically labeled by using O-acetyl-L-serine in which nitrogen and / or carbon atoms are isotopically labeled for the reaction with cysteine synthase ( Ie, nitrogen and sulfur isotope labeled, carbon and sulfur isotope labeled, nitrogen, carbon and sulfur isotope labeled) cysteine, cystine, sulfocysteine, S-sulfocysteine, cysteine persulfide, Cystine polysulf De, can be obtained N- acetyl cysteine or selenocysteine.

To this end, in another embodiment, in the above method, O-acetyl-L-serine is isotopically labeled for nitrogen and / or carbon atoms in the presence of the following steps: serine-O-acetyltransferase. A method further comprising: Isotope labels for nitrogen and / or carbon atoms may be stable isotopes ¹⁵ N and / or ¹³ C, respectively.

Recovery and purification of cysteine and cysteine derivatives Cysteine or cysteine derivatives labeled with a sulfur atom, or cysteine labeled with nitrogen and / or carbon and sulfur, obtained by the above synthesis method or the above reaction Alternatively, the cysteine derivative can be recovered and purified by techniques known to those skilled in the art such as chromatography.

In one embodiment of the kit, a kit for synthesizing cysteine or cystine or a cysteine derivative isotopically labeled with a sulfur atom is provided. The kit includes a cysteine synthase; and a substrate that is isotopically labeled for a sulfur atom, or sodium selenide (Na ₂ Se), wherein the selenium atom of sodium selenide may or may not be isotopically labeled. May be).

The cysteine synthase included in the kit is as described in the item “Cysteine synthase” above.

Depending on the type of compound to be synthesized, a substrate that is isotopically labeled with a sulfur atom may be sodium hydrogen sulfide (NaHS), sodium sulfide (Na ₂ S), or sodium sulfite (Na ₂ SO) that is isotopically labeled with a sulfur atom. ₃ ), at least one substrate selected from the group consisting of sodium thiosulfate (Na ₂ S ₂ O ₃ ) and sodium disulfide (Na ₂ S ₂ ). Here, the types of isotope labeling of sulfur atoms are as described above in the section of “isotope labeling”.

The types of isotope labeling of selenium atoms are as described in the “Isotope labeling” section above.

Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto.

Example ^{1: 34} S-labeled cysteine ^(Cys- 34 SH) in the synthesis 100mM phosphate buffer (pH 7.6) in, so that 20 mM O-acetyl -L- serine, 20 mM sodium hydrogen sulfide _(Na ^{2 34} S) Dissolved. To this solution, 0.05 mg / ml of cysteine synthase (CysK) prepared by the method of Reference Example 1 was added and reacted at 37 ° C. for 1 hour.

For the purpose of facilitating confirmation of cysteine production, methanol and an excess amount of monobromobiman (MBB) were added to the reaction solution after the above reaction and incubated at 37 ° C. for 15 minutes.

The reaction product was analyzed by high performance liquid chromatography (column: YMC Triat C18 plus (4.6 mm × 150 mm) (YMC Co., Ltd.); column temperature: 35 ° C; injection volume: 10 μl; moving bed: solution A / 0.1 % Formic acid and B solution / acetonitrile; Gradient: 5% B solution (0 min)-80% B solution (22 min)-80% B solution (23 min)-5% B solution (24 min)-5% B Liquid (30 minutes); flow rate 0.8 ml / min). The peak of the target product (cysteine-biman) was confirmed at an elution time of 9 to 10 minutes. From the peak area, it was confirmed that a product of 15 mM was generated. Since 20 mM O-acetylserine and sodium hydrogen sulfide were used as starting materials, this means a reaction yield of 75%.

Further, when mass analysis was performed on this product, a peak at m / z: 312.1 was detected in a sample not labeled with cysteine-biman, and in the product, m / z: 314 A peak of .1 was detected. This product, indicates that it is isotopically labeled compounds with a stable isotope ^{34 S.}

Example ^{2: 34} S-labeled cystine synthesis 100mM phosphate buffer (pH 7.6) in ^{(Cys- 34 S- 34 S-Cys} ), 20mM O- acetyl -L- serine, 20 mM sodium hydrogen sulfide _(Na ^{2 34} S ) Was dissolved. To this solution, 0.05 mg / ml of cysteine synthase (CysK) prepared by the method of Reference Example 1 was added and reacted at 37 ° C. for 1 hour.

After the above reaction, iodine (I ₂ ) was added to the reaction solution to 40 mM and incubated at 37 ° C. for 10 minutes.

The reaction product was analyzed by high performance liquid chromatography (column: Intrada Amino Acid (100 × 3 mm) (Intact Corporation); column temperature: 35 ° C .; injection volume: 10 μl; moving bed: solution A / acetonitrile and solution B / 20 mM ammonium formate; Gradient: 14% solution B (0 minutes)-14% solution B (3 minutes)-100% solution B (20 minutes)-14% solution B (20.5 minutes)-14% solution B ( 30 minutes); flow rate 0.6 ml / min). The peak of the target product (cystine) was confirmed at an elution time of about 18 minutes. The reaction yield was almost 100%.

In addition, when mass analysis was performed on this product, a peak at m / z: 240.9 was detected for a sample not labeled with cystine, whereas the product had an m / z: 244.9. The peak was detected. This product indicates that the compound is isotopically labeled with stable isotopes ^{34 S.}

Example 3: Synthesis of ¹⁵ N-labeled / ³⁴ S-labeled cysteine ([ ¹⁵ N] Cys- ³⁴ SH) 1 mM ¹⁵ N-labeled L-serine, 1 mM acetyl CoA, 1 mM hydrogen sulfide in 100 mM phosphate buffer (pH 7.6) It was dissolved at a sodium _(Na ^{2 34} S). To this solution, 0.05 mg / ml of cysteine synthase (CysK) prepared by the method of Reference Example 1 and serine-O-acetyltransferase (CysE) prepared by the method of Reference Example 2 were added and reacted at 37 ° C. for 5 minutes. I let you. In order to facilitate confirmation of cysteine production, methanol and an excess amount of monobromobiman (MBB) were added to the reaction solution after the above reaction, and the mixture was incubated at 37 ° C. for 15 minutes.

The reaction product was analyzed by high performance liquid chromatography (column: YMC Triat C18 plus (4.6 mm × 150 mm) (YMC Co., Ltd.); column temperature: 35 ° C .; injection volume: 10 μl; moving bed: solution A / 0.1 % Formic acid and B solution / acetonitrile; Gradient: 5% B solution (0 min)-80% B solution (22 min)-80% B solution (23 min)-5% B solution (24 min)-5% B Liquid (30 minutes); flow rate 0.8 ml / min). The peak of the target product (cysteine-biman) was confirmed at an elution time of 9 to 10 minutes. Further, when mass analysis was performed on this product, a peak at m / z: 312.1 was detected for a sample not labeled with a cysteine-biman isotope, whereas the product had an m / z: 315 A peak of .1 was detected. This indicates that the product is a compound isotopically labeled with stable isotopes ³⁴ S and ¹⁵ N.

Example 4: Synthesis of ¹⁵ N-labeled sulfocysteine ([ ¹⁵ N] Cys-SO ₃ H) In 100 mM phosphate buffer (pH 7.6), 1 mM ¹⁵ N-labeled L-serine, 1 mM acetyl CoA, 1 mM sodium sulfite ( Na ₂ SO ₃ ) was dissolved. To this solution, 0.05 mg / ml of cysteine synthase (CysK) prepared by the method of Reference Example 1 and serine-O-acetyltransferase (CysE) prepared by the method of Reference Example 2 were added and reacted at 37 ° C. for 30 minutes. I let you. The reaction product was analyzed by a mass spectrometer (column: Imtakt Scherzo SS-C18 (2.0 mm × 150 mm) (Intact Corporation); column temperature: 45 ° C .; injection volume: 3 μl; moving bed: solution A / 0.2 % Formic acid, 0.2% acetic acid, and B solution / 100 mM ammonium acetate, 50% methanol; Gradient: 0% solution B (0 minutes)-0% solution B (1 minute)-2% solution B (4 minutes)- 45% solution B (15 minutes)-0% solution B (15.1 minutes)-0% solution B (20 minutes); flow rate 0.3 ml / min); mass analysis conditions: precursor ion 168, product ion 81, flag Mentor voltage 90V, collision energy 21, polarity negative. A peak of the target product (sulfocysteine) was confirmed at an elution time of 3 to 4 minutes. In addition, the product detected a peak at m / z: 169, while a peak at m / z: 168 was detected for the product of this product which was not isotopically labeled with sulfocysteine. This indicates that the product is a compound isotopically labeled with ¹⁵ N.

Example 5: Synthesis of ¹⁵ N-labeled S-sulfocysteine ([ ¹⁵ N] Cys-SSO ₃ H) In 100 mM phosphate buffer (pH 7.6), 1 mM ¹⁵ N-labeled L-serine, 1 mM acetyl CoA, 1 mM thio It was dissolved at a sodium sulphate _{(Na 2 S 2 O 3)} . To this solution, 0.05 mg / ml of cysteine synthase (CysM) prepared by the method of Reference Example 1 and serine-O-acetyltransferase (CysE) prepared by the method of Reference Example 2 were added and reacted at 37 ° C. for 30 minutes. I let you. The reaction product was analyzed by a mass spectrometer (column: Imtakt Scherzo SS-C18 (2.0 mm × 150 mm) (Intact Corporation); column temperature: 45 ° C .; injection volume: 3 μl; moving bed: solution A / 0.2 % Formic acid, 0.2% acetic acid, and B solution / 100 mM ammonium acetate, 50% methanol; Gradient: 0% solution B (0 minutes)-0% solution B (1 minute)-2% solution B (4 minutes)- 45% solution B (15 minutes)-0% solution B (15.1 minutes)-0% solution B (20 minutes); flow rate 0.3 ml / min); mass analysis conditions: precursor ion 201, product ion 136, flag Mentor voltage 90V, collision energy 9, polarity negative. A peak of the target product (S-sulfocysteine) was confirmed at an elution time of 4 to 5 minutes. Further, for this product, a peak at m / z: 200 was detected for the sample not labeled with S-sulfocysteine, while the product detected a peak at m / z: 201. This indicates that the product is a compound isotopically labeled with ¹⁵ N.

Example 6: Synthesis of ¹⁵ N-labeled selenocysteine ([ ¹⁵ N] Cys-SeH) In 100 mM phosphate buffer (pH 7.6), 1 mM ¹⁵ N-labeled L-serine, 1 mM acetyl CoA, 1 mM sodium selenide (Na ₂ Se). To this solution, 0.05 mg / ml of cysteine synthase (CysK) prepared by the method of Reference Example 1 and serine-O-acetyltransferase (CysE) prepared by the method of Reference Example 2 were added and reacted at 37 ° C. for 30 minutes. I let you. The reaction product was analyzed by a mass spectrometer (column: Imtakt Scherzo SS-C18 (2.0 mm × 150 mm) (Intact Corporation); column temperature: 45 ° C .; injection volume: 3 μl; moving bed: solution A / 0.2 % Formic acid, 0.2% acetic acid, and B solution / 100 mM ammonium acetate, 50% methanol; Gradient: 0% solution B (0 minutes)-0% solution B (1 minute)-2% solution B (4 minutes)- 45% solution B (15 minutes) −0% solution B (15.1 minutes) −0% solution B (20 minutes); flow rate 0.3 ml / min); mass analysis conditions: precursor ion 336.9, product ion 247 .8, fragmentor voltage 90V, collision energy 9, polarity positive. Selenocysteine was detected because almost all selenocysteine was oxidized in the reaction solution to form selenocystine (Cys-Se-Se-Cys) in which the molecules were linked by a disulfide bond. A peak of selenocystine, an oxidant of the target product, was confirmed at an elution time of 4 to 5 minutes. Further, for this product, a peak at m / z: 336.9 was detected for the sample not labeled with selenocystine, whereas the product detected a peak at m / z: 338.9. This indicates that the product is a compound isotopically labeled with ¹⁵ N.

Example 7: Synthesis of ³⁴ S-labeled cysteine persulfide (Cys- ³⁴ SSH) 0.1 mM ³⁴ S-labeled cysteine and 0.1 mM disulfide prepared by the procedure of Example 1 in 100 mM phosphate buffer (pH 7.6) Sodium (Na ₂ S ₂ ) was mixed and reacted at 37 ° C. for 30 minutes.

The reaction product was analyzed by a mass spectrometer (column: YMC Triat C18 plus (2.1 mm × 150 mm) (YMC Co., Ltd.); column temperature: 45 ° C .; injection volume: 1 μl; moving bed: solution A / 0.1% Formic acid and B solution / acetonitrile; 0.2% B solution isocratic; flow rate 0.2 ml / min); mass analysis conditions: precursor ion 153.9, product ion 73.1, fragmentor voltage 50 V, collision energy 10 Positive polarity. A peak of the target product (cysteine persulfide) was confirmed at an elution time of 3 to 4 minutes. In addition, the product detected a peak at m / z: 155.9, whereas the product detected a peak at m / z: 153.9 for an unlabeled sample of cysteine persulfide. . This indicates that the product is a compound isotopically labeled with ³⁴ S.

Example ^{8: 34} S labeled-sulfocysteine during the synthesis 100mM phosphate buffer (pH 7.6) in _{^{(Cys- 34 SO 3 H),}} 1mM O- acetyl -L- serine, ^{1 mM 34} S labeled sodium sulfite (Na ₂ ³⁴ SO ₃ ). To this solution, 0.05 mg / ml of cysteine synthase (CysK) prepared by the method of Reference Example 1 was added and reacted at 37 ° C. for 30 minutes. Under the same conditions as described in Example 4, the reactants were analyzed with a mass spectrometer. A peak of the target product (sulfocysteine) was confirmed at an elution time of 3 to 4 minutes. The product detected a peak at m / z: 170, whereas a peak at m / z: 168 was detected for the product of the product, which was not isotopically labeled with sulfocysteine. This indicates that the product is a compound isotopically labeled with ³⁴ S.

Example 9: Synthesis of ³⁴ S-labeled S-sulfocysteine (Cys-S ³⁴ SO ₃ H) 1 mM O-acetyl-L-serine, 1 mM ³⁴ S-labeled thiosulfate in 100 mM phosphate buffer (pH 7.6) was dissolved at a sodium _{^{(Na 2 S 34 sO 3)}} . To this solution, 0.05 mg / ml of cysteine synthase (CysM) prepared by the method of Reference Example 1 was added and reacted at 37 ° C. for 30 minutes. Under the same conditions as described in Example 5, the reactants were analyzed with a mass spectrometer. A peak of the target product (S-sulfocysteine) was confirmed at an elution time of 4 to 5 minutes. In addition, for this product, a peak at m / z: 200 was detected for the sample not labeled with an isotope of S-sulfocysteine, whereas the product detected a peak at m / z: 202. This indicates that the product is a compound isotopically labeled with ³⁴ S label.

Example ^{10: 34} S-labeled-cystine polysulfide _{^{(Cys 34 S- 34 S (1-3}} ) - 34 SCys) during synthesis 5% formic acid, 5 mM ³⁴ S-labeled L- cysteine, and 25mM sodium nitrite (NaNO ₂₎ And dissolved at 37 ° C. for 15 minutes. To this solution, 6 mM Na ₂ ³⁴ S was added and further reacted at 37 ° C. for 15 minutes. The reaction product was analyzed by a mass spectrometer (column: YMC Triat C18 plus (2.1 mm × 50 mm) (YMC Co., Ltd.); column temperature: 45 ° C .; injection volume: 3 μl; moving bed: solution A / 0.1% Formic acid, and B solution / acetonitrile; isocratic: 0.2% B solution; flow rate 0.2 ml / min; mass analysis conditions: MS2 scan (m / z: 200-400), fragmentor voltage 90 V, polarity positive. elution time 2-3 minutes, 4-5 minutes, each product of interest 12-13 minutes. (cystine _{^{polysulfide: Cys 34 S- 34 S (1-3}} ) - 34 SCys) peak of was confirmed also For this product, peaks of m / z: 273.0, 305.0, 337.0 were detected for the cystine polysulfide unisotopically labeled preparation, The m / z:. Detects respective peaks of 278.9,312.9,346.8 This ^product, it indicates that it is isotopically labeled compounds ³⁴ S.

Example 11: Synthesis of ³⁴ S-labeled N-acetylcysteine It was dissolved in 100 mM phosphate buffer (pH 7.6) to give 5 mM ³⁴ S-labeled L-cysteine, 5 mM acetyl CoA, and reacted at 37 ° C for 30 minutes. It was. The reaction product was analyzed by a mass spectrometer (column: YMC Triat C18 plus (2.1 mm × 50 mm) (YMC Co., Ltd.); column temperature: 45 ° C .; injection volume: 3 μl; moving bed: solution A / 0.1% Formic acid and solution B / acetonitrile; Gradient: 0.2% solution B (0 minutes) −10% solution B (10 minutes) −0.2% solution B (10.1 minutes) −0.2% solution B ( Flow rate: 0.2 ml / min Mass spectrometry conditions: Precursor ion 166.2, Product ion 99.5, Fragmenter voltage 50V, Collision Energy 21, Polarity positive, target product at elution time 2-3 minutes A peak of (N-acetylcysteine) was confirmed, and a peak at m / z: 164 was detected for the product with no N-acetylcysteine isotopically labeled, whereas the product was A peak at m / z: 166 was detected, indicating that the product is a compound isotopically labeled with ³⁴ S.

Reference Example 1: Preparation of cysteine synthase (CysK, CysM) Escherichia coli expressing histidine tag-fused CysK and CysM derived from Salmonella typhimurium was cultured with shaking in LB medium at 37C. After the turbidity of E. coli reached OD.600 = 0.5, 0.5 mM IPTG was added, and the mixture was further cultured with shaking at 30 ° C. for 2 hours. E. coli was recovered by centrifugation and resuspended in lysis buffer (50 mM phosphate buffer, 300 mM sodium chloride, 10 mM imidazole 1 mg / ml lysozyme, 0.1 μg / ml DNase I pH 7.8). After standing on ice for 30 minutes, Escherichia coli was crushed with an ultrasonic crusher, centrifuged, and the supernatant was collected. After passing the supernatant through a nickel-immobilized agarose-packed column, the column was washed twice with a washing buffer (50 mM phosphate buffer, 300 mM sodium chloride, 20 mM imidazole pH 7.8), and then an elution buffer (50 mM phosphate buffer). The histidine tag-fused CysK was eluted with 300 mM sodium chloride, 300 mM imidazole pH 7.8). The eluted histidine tag-fused CysK was passed through a desalting column equilibrated with 50 mM phosphate buffer pH 7.8, and the resulting solution was used as cysteine synthase (CysK, CysM).

Reference Example 2: Preparation of serine-O-acetyltransferase (CysE) Escherichia coli expressing histidine-tagged CysE derived from Salmonella typhimurium was cultured at 37 ° C with shaking in LB medium. After the turbidity of E. coli reached OD.600 = 0.5, 0.5 mM IPTG was added, and the mixture was further cultured with shaking at 30 ° C. for 2 hours. E. coli was recovered by centrifugation and resuspended in lysis buffer (50 mM phosphate buffer, 300 mM sodium chloride, 10 mM imidazole 1 mg / ml lysozyme, 0.1 μg / ml DNase I pH 7.8). After standing on ice for 30 minutes, Escherichia coli was crushed with an ultrasonic crusher, centrifuged, and the supernatant was collected. After passing the supernatant through a nickel-immobilized agarose-packed column, the column was washed twice with a washing buffer (50 mM phosphate buffer, 300 mM sodium chloride, 20 mM imidazole pH 7.8), and then an elution buffer (50 mM phosphate buffer). The histidine tag-fused CysE was eluted with 300 mM sodium chloride, 300 mM imidazole pH 7.8). The eluted histidine tag-fused CysE was passed through a desalting column equilibrated with 50 mM phosphate buffer pH 7.8, and the resulting solution was used as serine-O-acetyltransferase (CysE).

According to the present invention, cysteine and a cysteine derivative in which a sulfur atom is isotope-labeled can be efficiently synthesized, and this is useful in that the cysteine and the cysteine derivative can be stably supplied.

Claims (14)

1. A method for synthesizing cysteine labeled with an isotope of a sulfur atom, wherein the substrate is isotopically labeled for O-acetyl-L-serine and a sulfur atom in the presence of cysteine synthase (EC: 2.5.1.47). Wherein the substrate that is isotopically labeled for the sulfur atom is sodium hydrogen sulfide (NaHS), sodium sulfide (Na ₂ S), or hydrogen sulfide (H ₂ S), and wherein The method, wherein the isotope labeling of the sulfur atom is selected from the group consisting of stable isotope ³² S, stable isotope ³⁴ S, radioactive isotope ³³ S and radioactive isotope ³⁵ S.
2. O-acetyl-L-serine is reacted with isotopically labeled serine for nitrogen and / or carbon atoms in the presence of the following steps: serine-O-acetyltransferase (EC: 2.3.3.10) The method of claim 1, further comprising: obtaining.
3. The method according to claim 1 or 2, wherein the cysteine synthase is cysteine synthase A (CysK) or cysteine synthase B (CysM).
4. The method according to claim 3, wherein the cysteine synthase A is derived from a microorganism belonging to the genus Salmonella.
5. The method according to any one of claims 1 to 4, wherein the cysteine synthase is a recombinant protein containing a histidine tag.
6. A method for synthesizing a cystine labeled with an isotope of a sulfur atom, comprising synthesizing a cysteine labeled with an isotope labeled with a sulfur atom according to the method according to claim 1 or 2, and oxidizing the cysteine in the presence of an oxidizing agent. Said method comprising the step of obtaining cystine by
7. The method of claim 6, wherein the oxidizing agent is iodine (I ₂ ).
8. A method for synthesizing a cysteine derivative,
   (I) the cysteine derivative is a sulfocysteine in which a sulfur atom is isotope-labeled, and comprises reacting O-acetyl-L serine and sodium sulfite (Na ₂ SO ₃ ) in the presence of cysteine synthase, The sulfur atom is selected from the group consisting of a stable isotope ³² S, a stable isotope ³⁴ S, a radioactive isotope ³³ S and a radioactive isotope ³⁵ S;
   (Ii) The cysteine derivative is S-sulfocysteine in which a sulfur atom is isotopically labeled, and O-acetyl-L-serine and sodium thiosulfate (Na ₂ S ₂ O ₃ ) are reacted in the presence of cysteine synthase B. Wherein the sulfur atom is selected from the group consisting of a stable isotope ³² S, a stable isotope ³⁴ S, a radioactive isotope ³³ S and a radioactive isotope ³⁵ S;
   (Iii) The cysteine derivative is a cysteine persulfide in which a sulfur atom is isotope-labeled, and includes a step of reacting O-acetyl-L-serine and sodium disulfide (Na ₂ S ₂ ) in the presence of cysteine synthase. Where the sulfur atom is selected from the group consisting of stable isotope ³² S, stable isotope ³⁴ S, radioactive isotope ³³ S and radioactive isotope ³⁵ S;
   (Iv) The cysteine derivative is a cysteine persulfide in which a sulfur atom is isotopically labeled. (A) In the presence of cysteine synthase, an O-acetyl-L-serine and a substrate that is isotopically labeled with a sulfur atom are reacted. Obtaining a cysteine having an isotope-labeled sulfur atom, wherein the substrate isotope-labeled for the sulfur atom is sodium hydrogen sulfide (NaHS) or sodium sulfide (Na ₂ S), wherein the sulfur atom Isotope label is selected from the group consisting of stable isotope ³² S, stable isotope ³⁴ S, radioactive isotope ³³ S and radioactive isotope ³⁵ S, and (b) the sulfur atom obtained in step (a) Reacting isotopically labeled cysteine with sodium disulfide;
   Or (v) the cysteine derivative is selenocysteine, comprising reacting O-acetyl-L-serine and sodium selenide (Na ₂ Se) in the presence of cysteine synthase;
   Said method.
9. O-acetyl-L-serine is reacted with serine labeled isotopically for nitrogen and / or carbon atoms in the presence of serine-O-acetyltransferase (EC: 2.3.3.10). 9. The method of claim 8, further comprising:
10. The method according to claim 8 or 9, wherein the cysteine synthase is cysteine synthase A (CysK) or cysteine synthase B (CysM).
11. The method according to claim 10, wherein the cysteine synthase A is derived from a microorganism belonging to the genus Salmonella.
12. The method according to any one of claims 8 to 11, wherein the cysteine synthase is a recombinant protein containing a histidine tag.
13. A kit for synthesizing cysteine or cystine, or a cysteine derivative, isotopically labeled with a sulfur atom,
    Cysteine synthase; and sodium hydrogen sulfide (NaHS), sodium sulfide (Na ₂ S), sodium sulfite (Na ₂ SO ₃ ), sodium thiosulfate (Na ₂ S ₂ O ₃ ), and substrate comprising at least one sulfur selected from the group consisting of sodium sulfide (Na 2 _{S 2),} wherein isotopic labeling of the sulfur atoms, a stable isotope ^{32 S,} stable isotope ^{34 S,} radioisotope ³³ Selected from the group consisting of S and radioisotope ³⁵ S, or sodium selenide (Na ₂ Se);
    The kit.
14. A method for synthesizing a cysteine derivative,
    (I) the cysteine derivative is a cystine polysulfide in which a sulfur atom is isotopically labeled, and (a) a step of synthesizing a cysteine labeled with an isotopically sulfur atom in accordance with the method according to claim 1 or 2, step (b) (Ii) comprising reacting cysteine labeled with an isotope-labeled sulfur atom obtained in (a) with an oxidizing agent, and (c) further reacting by adding sodium sulfide (Na ₂ S); or (ii) cysteine The derivative is N-acetylcysteine having an isotopically labeled sulfur atom, and (a) a step of synthesizing a cysteine labeled with an isotopically sulfur atom according to the method of claim 1 or 2, and (b) step ( reacting cysteine labeled with an isotopically labeled sulfur atom obtained in a) with an acetylating agent;
    Said method.