JP2001064279A - New selenium compounds - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a reagent (denaturing agent) capable of forming or cleaving a disulfide bond by a cysteine residue in a protein.
TT (Dithiothreitol) and its derivative 1,2-dithane-4,5-diol
Although DTT and the like are widely known, DTT and the like largely depend on pH, and the reaction proceeds only under alkaline conditions of pH 8.0 or more, so that a reaction near neutrality close to the in vivo environment can be performed. Did not. Kind Code: A1 A compound represented by the following formula (1), a compound represented by the formula (2) obtained by reducing it, and a compound represented by the formula (3), which are highly reactive near neutrality: ), A novel selenium compound such as a compound represented by the formula (4) obtained by oxidizing the compound, and a method for producing them. Embedded image Embedded image Embedded image Embedded image

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel selenium compound which is a reagent capable of forming or cleaving a disulfide bond by a cysteine residue in a protein, and a method for producing the same.

[0002]

2. Description of the Related Art As a reagent (denaturing agent) capable of forming or cleaving a disulfide bond by a cysteine residue in a protein, DTT (Dithiothreitol = 1,4) is known.
-Dimercapto-2,3-butanediol) and its derivative 1,2-dithiane-4,5-diol are widely known. These DTTs are useful reagents,
It is widely used for studying the process of protein three-dimensional structure formation.

[0003]

However, the above-mentioned DTT and the like greatly depend on pH, and the reaction proceeds only under alkaline conditions of pH 8 or more, so that the reaction takes place near neutrality close to the in vivo environment. I couldn't do that. That is, there is a demand for the development of a modifying agent having high reactivity near neutrality.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies on a modifying agent having high reactivity near neutrality, and as a result, obtained by reducing a compound represented by the following formula (1) and a compound represented by the following formula (1). Novel selenium compounds such as a compound represented by the formula (2), a compound represented by the formula (3) and a compound represented by the formula (4) obtained by oxidizing the compound, and methods for producing them Was found.

That is, the present invention has the following constitution. [1] A compound represented by the formula (1).

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[2] A compound represented by the formula (2):

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[3] A compound represented by the formula (3):

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[4] A compound represented by the formula (4):

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[5] Equation (5)

Embedded image Wherein the selenium compound represented by the general formula (6) is caused to act on the compound represented by the general formula (6). M ₂ Se ₂ (6) (where M is Li, Na or K)

[6] The method for producing a compound represented by the formula (1) according to the above item [1], wherein M in the general formula (6) is Na in the item [5].

[7] The above item [2], wherein a compound represented by the general formula (7) is allowed to act on the compound represented by the formula (1) described in the above item [1]. A method for producing a compound represented by the formula (2) described in (1). MBH ₄ (7) (where M is Li, Na or K)

[8] The method for producing a compound represented by the formula (2) according to the above item [2], wherein M in the general formula (7) is Na in the item [7].

[9] The item [3], wherein a selenium compound represented by the general formula (8) is allowed to act on the compound represented by the formula (5) described in the item [5]. The method for producing a compound represented by the formula (3) described in (1). MHSe (8) (where M is Li, Na or K)

[10] The method for producing a compound represented by the formula (3) according to the above item [3], wherein M in the general formula (8) is Na in the above item [9].

[11] The compound represented by the formula (4) according to the above item [4], wherein an organic peroxide is allowed to act on the compound represented by the formula (3) according to the above item [3]. A method for producing a compound represented by the formula:

The novel selenium compound useful as a protein denaturant provided by the present invention is a compound represented by the above formula (1) (hereinafter abbreviated as compound (1)) or a compound represented by the formula (2). (Hereinafter abbreviated as compound (2)) and a compound represented by the formula (4) (hereinafter abbreviated as compound (4)). Further, a compound represented by the formula (3) (hereinafter, abbreviated as compound (3)) has also been newly discovered, and all of them are expected to act as a catalyst.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The compounds of the present invention are each produced by the following methods. The compound (1) is reacted with a compound trans-1,3-butadiene diepoxide represented by the formula (5) and a selenium compound represented by the above general formula (6), for example, Na ₂ Se _2, and It can be obtained by the reaction route shown in Scheme 1.

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The compound (1) is dissolved in methanol, reduced with a reducing agent represented by the general formula (7), for example, sodium borohydride, and then treated with concentrated hydrochloric acid to obtain the compound (2). (Scheme 2).

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In the above example, trans-butadiene diepoxide is used as a starting material, but cis-form compounds (1) and (2) can be obtained by changing this to cis-butadiene diepoxide. .

The compound (3) is reacted with trans-1,3-butadiene diepoxide and a selenium compound represented by the general formula (8), for example, NaHSe, to give the following Schem.
It can be obtained by the reaction route shown in e3.

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The compound (3) is oxidized by using an organic peroxide such as formic acid, peracetic acid, perbenzoic acid, metachloroperbenzoic acid, or t-butyl peroxide.
Can be converted to compound (4) (Scheme
4).

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In the above example, the starting material was changed from trans-butadiene diepoxide to cis-butadiene diepoxide to obtain cis-form compounds (3) and (4).
Can be obtained.

The selenium compound represented by the general formula (6) or (8) can be obtained by suspending metal selenium in water and reacting the metal selenium with a reducing agent represented by the general formula (7). it can. At this time, if the molar ratio of the metal selenium to the reducing agent represented by the general formula (7) is set to 1: 1, the selenium compound represented by the general formula (6) is obtained, and the reaction is performed at a ratio of 1: 2. For example, a selenium compound represented by the general formula (8) is obtained.

[0024]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention. Example 1 Synthesis of compound (1) (1,2-diselenane-4,5-diol) 3.0 g (38 mmol) of selenium at room temperature under a nitrogen stream.
Was suspended in 25 mL of water, and a solution of 3.0 g (76 mmol) of sodium borohydride dissolved in 25 mL of water was slowly added dropwise thereto. 10 minutes later, 3.0 g of selenium
(38 mmol) was added and the mixture was further stirred for 15 minutes. The mixture was heated at 100 ° C. for about 1 hour until selenium was dissolved, then cooled to room temperature, and 1.46 mL (19 mmol) of 1,3-butadiene diepoxide was added. After heating and refluxing for 1 hour, the mixture was stirred overnight in air to oxidize excess reduced selenium (Na ₂ Se ₂ ). After separating the precipitated selenium by filtration, the aqueous layer was subjected to continuous ether extraction using a Soxhlet extractor to obtain 2.54 g of the target compound. The crude yield is 54
%Met. 5 mL of the obtained compound (1) is methanol
Further recrystallization gave yellow columnar crystals.

¹ H-NMR (in DMF-d7): d 3.16 (m, 2H), 3.43 (dd, 2H), 3.5
3 (m, 2H), 5.20 (d, 2H) ¹³ C-NMR (in DMF-d7): d 31.8, 75.4 ⁷⁷ Se-NMR (in DMF-d7): d 268 (broad) IR (liquid paraffin method) : 3450, 3319, 1448, 1412, 12
82, 1248, 1120, 997, 968, 843, 760 cm- ¹ Elemental analysis: C 19.59%, H 3.14% (calc. C 19.53%, H 3.
(28%)

Example 2 Synthesis of compound (3) (selenolan-3,4-diol) 1.5 g (19 mmol) of selenium at room temperature under a nitrogen stream.
l) was suspended in 25 mL of water, and a solution of 1.5 g (38 mmol) of sodium borohydride dissolved in 25 mL of water was slowly added dropwise. After stirring for 15 minutes,
-Butadiene diepoxide 1.2 mL (16 mmol)
Was added. After stirring at room temperature for 10 minutes, the nitrogen line was removed and the mixture was stirred in air overnight. After filtering off the precipitated selenium,
The aqueous layer was subjected to continuous ether extraction using Soxhlet to obtain 1.44 g of selenolane-3,4-diol. The crude yield was 55%. Further, recrystallization was performed with 5 mL of chloroform to obtain white columnar crystals.

[0027] mp ^{79~80 ℃ 1 H-NMR (in} CDCl3): d 2.10 (d, 2H), 2.87 (dd, 2H), 3.16
(dd, 2H), 4.27 (m, 2H) ¹³ C-NMR (in CDCl 3): d 26.8, 78.9 ⁷⁷ Se-NMR (in CDCl 3): d 65.6 IR (liquid paraffin method): 3305, 1460, 1377, 1275 , 11
80, 1134, 1012, 941, 781 cm ^-1 Elemental analysis: C 28.55%, H 4.70% (calc. C 28.76%, H 4.
(83%)

Example 3 Synthesis of compound (4) (3,4-dihydroxy-1-selenolane oxide) Selenolane-3,4-diol 5 obtained in Example 2
4.7 mg (0.33 mmol) of methylene chloride 10m
L, and m-chloroperbenzoic acid 56.3 was added to this solution.
A solution of mg (0.33 mmol) in 10 mL of methylene chloride was added and stirred. The resulting white precipitate was collected by filtration to give 41.1 mg of the desired compound. The crude yield is 69
%Met. Recrystallization from methanol gave white columnar crystals.

Melting point 123.8-125.2 ° C. ¹ H-NMR (in CDCl 3): d 2.87 (d, 1H), 3.08 (dd, 1H), 3.58
(dd, 1H), 3.78 (dd, 1H), 4.63 (m, 1H), 4.72 (dd, 1H), 4.8
0 (s, 2H) ¹³ C-NMR (in CDCl3): d 55.0, 58.0, 79.1, 79.5 ⁷⁷ Se-NMR (in CDCl3): d 941.8 Elemental analysis: C 26.16%, H 4.17% (calc. C 26.24% , H 4.
(405%)

Example 4 Synthesis of compound (2) (2,3-dihydroxy-1,4-butanediselenol) 68.6 mg of 1,2-diselenane-4,5-diol synthesized in Example 1 (0.4%). 279 mmol) was dissolved in 0.5 mL of methanol, and sodium borohydride was added to the solution until the color of the solution disappeared. 6 mL of 6M hydrochloric acid and 80 mL of ether were added to the reaction solution, and ether extraction was performed with vigorous stirring for 1 hour. The extraction was performed twice, and the ether layer was collected with a syringe and dried over magnesium sulfate.
The solvent was distilled off to obtain a white crystalline target compound.

¹ H-NMR (in CDCl 3): d −0.51 (t, 2H), 2.82
(m, 4H), 4.27 ( m, 2H) 13 C-NMR (in CDCl3): d 22.6,80.7 77 Se-NMR (in CDCl3): d -78.3 Elemental analysis: C 26.16%, H 4.17% (calc. C 26.24%, H 4.
(405%)

Comparative Example 1 Oxidation Reactivity of Compound (4) Compound (4) and DTT (1,4-dimercapto-2,3)
-Butanediol) was dissolved at a concentration of 0.1 M in a buffer solution having a pH of 1: 1 at a ratio of 1: 1 and rapidly changed to the compound (3) and 1,2-dithiane-4,5-diol. did. That is, it was found that the compound (4) had a strong oxidizing ability.

Comparative Example 2 The same operation as in Comparative Example 1 was carried out except that the pH of the buffer was changed to 4.0.
Dithiane-4,5-diol was obtained.

Comparative Example 3 The same operation as in Comparative Example 1 was carried out except that the pH of the buffer solution was changed to 8.0.
Dithiane-4,5-diol was obtained.

Comparative Example 4 The same operation as in Comparative Example 1 was carried out except that DTT was changed to glutathione, whereby a compound (3) was obtained. Glutathione oxide could not be identified.

Comparative Example 5 Reactivity to diphenyl disulfide (PhSSPh) Compound (2) and diphenyl disulfide were placed in an NMR sample tube using heavy methanol as a solvent, and NMR analysis was carried out. 1) and the formation of PhSH were confirmed by ¹ H, ¹³ C, ⁷⁷ Se-NMR. On the other hand, when DTT was used, although the production of 1,2-dithiane-4,5-diol, which was considered to be due to air oxidation, was slightly observed, the production of PhSH was not clearly confirmed. From the above results, it was found that the compound (2) had a higher reduction reactivity than DTT.

Comparative Example 6 Literature (J. Am. Chem. Soc., 1994, 11)
6,2557), 10 mol% of compound (1) was added to aqueous hydrogen peroxide and 2 equivalents of thiophenol in methanol. 30 by PhSSPh
From the change in UV absorption intensity at 5 nm, Ph at each time
The SH concentration was determined, and the initial reaction rate was determined from a 1 / v vs. 1 / [PhSH] plot (Lineweaver-Burk Plot). As initial conditions, [H ₂ O ₂ ] =
30 mM, [PhSH] = 1 mM, [catalys
t] = 0.01 mM. (Setting of the initial concentration of H ₂ O ₂ is different from Table 1 in the literature). The obtained initial reaction rate (v0) is 5.2 microMole / m when no catalyst is used.
Whereas, when compound (1) was added,
The value was 7.9 microMole / min.

Comparative Example 7 The same operation as in Comparative Example 6 was carried out except that Compound (1) was replaced with Compound (3), and the reaction rate was 8.4 microMo.
le / min.

[0039]

Industrial Applicability According to the present invention, the conventional protein denaturant DTT and its derivative 1,2-dithiane-
Novel selenium compounds having higher reactivity near neutrality than 4,5-diol have been found. An effect as a biological reaction catalyst is also expected.

Claims (11)

[Claims] 1. A compound represented by the formula (1). Embedded image 2. A compound represented by the formula (2). Embedded image 3. A compound represented by the formula (3). Embedded image 4. A compound represented by the formula (4). Embedded image 5. The formula (5) The method for producing a compound represented by the formula (1) according to claim 1, wherein a selenium compound represented by the general formula (6) is caused to act on the compound represented by the formula (6). M ₂ Se ₂ (6) (where M is Li, Na or K) 6. The method for producing a compound represented by the formula (1) according to claim 1, wherein M in the general formula (6) is Na. 7. The compound according to claim 2, wherein the compound represented by the formula (1) according to claim 1 is reacted with a reducing agent represented by the formula (7). A method for producing the compound represented by 2). MBH ₄ (7) (where M is Li, Na or K) 8. The method for producing a compound represented by the formula (2) according to claim 2, wherein M in the general formula (7) is Na. 9. The compound according to claim 3, wherein the selenium compound represented by the general formula (8) acts on the compound represented by the formula (5) according to claim 5. A method for producing the compound represented by 3). MHSe (8) (where M is Li, Na or K) 10. The method according to claim 9, wherein M
Is a method for producing a compound represented by the formula (3) according to claim 3. 11. The compound represented by the formula (4) according to claim 4, wherein an organic peroxide is allowed to act on the compound represented by the formula (3) according to claim 3. Manufacturing method.