JP2006002077A - Cyclodextrin dimer, inclusion complex and method for producing the same - Google Patents

Abstract

【選択図】なしPROBLEM TO BE SOLVED: To provide an inclusion complex which can be used as a constituent material of a heme protein model capable of reversibly adsorbing and desorbing molecular oxygen in water, a method for producing the same, and cyclodextrin which is a constituent material of the inclusion complex Provide a mer.
A cyclodextrin dimer represented by the following chemical formula (1) includes a water-soluble metalloporphyrin, for example, a 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrin (II) iron complex. It is an inclusion complex. This inclusion complex is produced by mixing a cyclodextrin dimer represented by the chemical formula (1) and a water-soluble metalloporphyrin with water.
[Chemical 1]

[Selection figure] None

Description

  The present invention relates to an inclusion complex that can be used as a material for oxygen infusion that stably transports oxygen in the human body, a method for producing the same, and a cyclodextrin dimer that is part of the raw material of the inclusion complex.

  One of the biomolecules that play the most important role in life activity is metal protein, which is a metal complex (heme) or metal ion that constitutes the active center and a protein that surrounds it sterically. Consists of.

  For example, myoglobin, which is a metal protein responsible for oxygen storage, contains a porphyrin iron complex called heme and a globin protein embedded in an oxygen binding site. This oxygen binding site is composed of hydrophobic amino acid residues, so it is a hydrophobic environment. Heme iron (II) is not oxidized even if oxygen is attached to or removed from heme, and heme is reversible. Thus, oxygen can be absorbed and desorbed.

  Hemoglobin, which carries oxygen, is composed of four subunits that have a structure very similar to that of globin proteins of heme and myoglobin. As with myoglobin, heme iron (II) remains even if oxygen is attached to or removed from the heme. Without being oxidized, oxygen can be adsorbed and desorbed so that it is reversible and the oxygen partial pressure is constant. The oxygen partial pressure is kept constant because the local structural change around heme caused by the binding of oxygen to heme iron is transmitted to the whole protein, and the binding force with oxygen changes, so-called Due to the positive allosteric effect.

  Many metal proteins play various functions in the living body, but most of them are driven by the formation of coordination bonds to the active center of the substrate. The reaction is going on. For this reason, many researchers have previously focused on the structure of the active center of a metalloprotein and attempted to imitate it using an artificially synthesized molecule. In particular, modeling of hemoproteins that adsorb and desorb molecular oxygen is important for the development of artificial myoglobin, artificial hemoglobin, oxygen storage materials, and oxygen selective membranes. Modeling is being attempted.

For example, Groves et al. Produced a clathrate complex in which a porphyrin compound was clathrated with a compound in which the inner hydroxyl group of cyclodextrin was substituted with pyridine and the outer hydroxyl group was substituted with a hydrophobic group. It has been reported that the inclusion complex adsorbs oxygen (see Non-Patent Document 1).
“Biophysical Chemistry” (USA), 2003, volume 105, p. 639-648

  However, most of the heme protein models described in the above prior art documents can reversibly absorb and desorb molecular oxygen only in organic solvents, and in water, which is the same environment as the blood vessels of humans and animals, Oxygen could not be absorbed and desorbed reversibly. For this reason, oxygen infusions using these models as artificial myoglobin or artificial hemoglobin could not be made.

  Therefore, the present invention is an inclusion complex that can be used as a constituent material of a heme protein model capable of reversibly adsorbing and desorbing molecular oxygen in water, a production method thereof, and a constituent material of the inclusion complex. An object is to provide a cyclodextrin dimer.

（式中、ｍは１又は２の何れかの数字を表し、ｎは１、２又は３の何れかの数字を表す） In order to solve the above problems, an inclusion complex according to the present invention is characterized in that a cyclodextrin dimer represented by the following chemical formula (1) includes a water-soluble metalloporphyrin.

(In the formula, m represents a number of 1 or 2, and n represents a number of 1, 2, or 3)

（式中、R₁及びR₂は、それぞれカルボキシル基、スルホニル基、水酸基の何れかを表し、MはFe²⁺、Mn²⁺、Co²⁺、Zn²⁺の何れかを表す） In the present invention, the cyclodextrin used as a raw material for the cyclodextrin dimer is either α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, and among them, it is easy to include water-soluble metalloporphyrin. -Preferably cyclodextrin is used as raw material. The water-soluble metalloporphyrin in the present invention is a porphyrin-based compound that coordinates with a metal ion at the center and dissolves in water, and is a cyclodextrin dimer represented by the chemical formula (1) (hereinafter referred to as cyclodextrin). Although it is not particularly limited as long as it can be included by dextrin dimer), the following chemical formula (2) or (3) may be mentioned from the viewpoint that molecular oxygen can be adsorbed and desorbed with certainty. 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrin (II) iron complex (hereinafter abbreviated as “Fe (II) TPPS”) or 5,15-bis (3,5- And dicarboxylatophenyl) -10,20-diphenylporphyrin (II) iron complex (hereinafter abbreviated as “Fe (II) -trans-2DC”).

(In the formula, R ₁ and R ₂ each represent a carboxyl group, a sulfonyl group, or a hydroxyl group, and M represents any one of Fe ²⁺ , Mn ²⁺ , Co ²⁺ , and Zn ²⁺ )

  In addition, the method for producing an inclusion complex according to the present invention is characterized in that a cyclodextrin dimer represented by the chemical formula (1) and a water-soluble metalloporphyrin are mixed in water such as a phosphate buffer.

  In the inclusion complex according to the present invention, the water-soluble metalloporphyrin is included in the cyclodextrin dimer, so that it is possible to adsorb and desorb molecular oxygen repeatedly and stably in a wider pH range than in vivo. And this inclusion complex can be easily produced from the cyclodextrin dimer according to the present invention and the water-soluble metalloporphyrin by the production method according to the present invention.

  In the cyclodextrin dimer according to the present invention, as shown by the chemical formula (1), two cyclodextrin molecules in which all hydroxyl groups are methylated are bonded via 3,5-dimercaptomethylpyridine which is a linker molecule. Is. The cyclodextrin dimer is produced, for example, by tosylating and epoxidizing cyclodextrin, methylating the hydroxyl group of cyclodextrin, and combining the methylated cyclodextrin with a linker molecule. The reason why the hydroxyl group of cyclodextrin was methylated in advance is to prevent the internal pores of cyclodextrin from becoming hard due to hydrogen bonds generated by the hydroxyl groups, and preventing the water-soluble metalloporphyrin from being included in the internal pores.

  The inclusion complex according to the present invention is an inclusion complex of the above-mentioned cyclodextrin dimer with a water-soluble metalloporphyrin. Water-soluble metalloporphyrins can be used without any particular limitation, but those shown in chemical formulas (2) and (3), more specifically Fe (II) TPPS, Fe (II) -trans-2DC, etc. are used. can do. This inclusion complex is produced by mixing cyclodextrin dimer and water-soluble metalloporphyrin in water.

  In order to clarify the features of the present invention more specifically, β-cyclodextrin (hereinafter, abbreviated as “β-CD”) as an example to a cyclodextrin dimer represented by the formula (1) (hereinafter referred to as “β-CD”) , Abbreviated as “CD2”), and an inclusion complex of this CD2 and FeTPPS was produced, and its characteristics were examined. The synthesis route is shown in FIG. Further, the present invention is not limited by this embodiment.

[Manufacture of CD2]
(1) Synthesis of 2-monotosyl-β-cyclodextrin (hereinafter abbreviated as “2-monotosyl-β-CD”) 200 mL of anhydrous DMF and dry β-CD in a 300 mL three-necked flask under an argon atmosphere (17.0 g, 15.0 mmol) was added, sodium hydride (60-70% in oil, 513 mg, 15.0 mmol) was added thereto, and the mixture was stirred at room temperature for 15 hours. Tosyl chloride (2.9 g, 15.2 mmol) was added thereto and stirred at room temperature. After 3 hours, the reaction solution was poured into 3 L of acetone, and the resulting white precipitate was separated by filtration and dried under reduced pressure. The dried solid was dissolved in distilled water and injected into a column packed with Diaion HP20 (manufactured by Mitsubishi Chemical Corporation). Only distilled water was added to elute unreacted β-CD, and after elution of β-CD was completed, the developing solvent was changed to a 40% methanol aqueous solution to elute only 2-monotosyl-β-CD. The elution was confirmed by TLC (developing solvent n-BuOH: EtOH: H ₂ O = 5: 4: 3, anisaldehyde color development, Rf = 0.45). The solvent of the eluted part was distilled off under reduced pressure to obtain 2-monotosyl-β-CD (6.3 g, yield 33%) as a colorless solid.

(2) Synthesis of 2,3-epoxy-β-cyclodextrin (hereinafter abbreviated as “2,3-Epo-β-CD”)
2-monotosyl-β-CD (6.0 g, 4.7 mmol) and 300 mL of 0.2 M aqueous sodium hydroxide were added to a 500 mL eggplant-shaped flask, and the mixture was stirred at room temperature for 40 hours. The reaction solution was immersed in an ice bath and the solution was neutralized with dilute hydrochloric acid. The solvent was distilled off to about 100 mL with an evaporator, and the solution was poured into 1 L of acetone. The resulting white precipitate was dissolved in a minimum amount of DMF, and insoluble salts were removed by filtration. The filtrate was poured into 1 L of acetone, and the resulting white precipitate was separated by filtration and dried under reduced pressure. The dried solid was dissolved in distilled water and injected into a column packed with Diaion HP20. The elution of 2,3-Epo-β-CD was confirmed by TLC (developing solvent n-BuOH: EtOH: H ₂ O = 5: 4: 3, anisaldehyde color development, Rf = 0.25), and the part eluted only with water Was taken out. The solvent in the extracted part was distilled off under reduced pressure to obtain 2,3-Epo-β-CD (3.5 g, yield 67%) as a colorless solid.

(3) Synthesis of 2,3-epoxy-permethyl-β-cyclodextrin (hereinafter abbreviated as “2,3-Epo-perMe-β-CD”) In a 200 mL three-necked flask under an argon atmosphere, 3-Epo-β-CD (2.00 g, 1.45 mmol), 80 mL anhydrous DMF and 30 mL anhydrous THF were added and the vessel was immersed in an ice bath. Sodium hydride (washed with hexane and dried in vacuo (1.39 g, 58.00 mmol) was added thereto, and immersed in an ice bath and stirred for 1 hour. Methyl iodide (3.60 mL, 58.00 mmol) was added dropwise thereto. Then, 4 mL of methanol was added to the reaction solution, and after foaming stopped, chloroform was added and transferred to a separatory funnel, washed with distilled water and aqueous sodium thiosulfate solution, organic The layer was dehydrated with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (developing solvent chloroform only → chloroform / acetone = 5/2) to give a colorless solid 2, 3-Epo-perMe-β-CD (1.50 g, 61% yield) was obtained.

(4) Synthesis of 3,5-dimercaptomethylpyridine In a 50 mL eggplant-shaped flask equipped with a reflux tube, 3,5-dichloromethylpyridine (200 mg, 0.94 mmol), thiourea (180 mg, 2.4 mmol) and 6 mL of ethanol was added and heated to reflux. After 2 hours, 3 mL of 5 M aqueous sodium hydroxide solution was added, and the mixture was further heated under reflux for 3 hours. After cooling to room temperature, the reaction solution was neutralized with hydrochloric acid, transferred to a separatory funnel, and extracted with methylene chloride. The organic layer was dehydrated with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give 3,5-dimercaptomethylpyridine (100 mg, yield 49%) as a brown oil.

(5) Synthesis of CD2 2,3-Epo-perMe-β-CD (1.0 g, 0.72 mmol) and 60 mL of 0.1 M NaHCO ₃ aqueous solution were added to a 100 mL eggplant-shaped flask equipped with a reflux tube. 3,5-dimercaptomethylpyridine (50 mg, 0.29 mmol) dissolved in 2 mL of methanol was added thereto, and the mixture was heated to reflux for 24 hours. After cooling to room temperature, the solution was transferred to a separatory funnel and extracted three times with chloroform. The organic phase was dehydrated with anhydrous sodium sulfate and the solvent was distilled off under reduced pressure. The residue was purified by gel filtration chromatography (Shemadex G-25, manufactured by Amersham Biosciences), and fractions containing the target product were collected and the solvent was distilled off. The obtained residue was purified by silica gel column chromatography (developing solvent chloroform / acetone = 5/2 → chloroform only → chloroform / methanol = 10/1), and colorless solid CD2 (0.21 g, 20%, melting point 126-127 ^o C) was obtained.

[Production and various tests of inclusion complex of Fe (II) TPPS and CD2]
(6) Adsorption of oxygen complex at pH 4.5 and its reversibility test
Make a mixed aqueous solution of [Fe (III) TPPS] = 5 x 10 ^-6 M and [CD2] = 6 x 10 ^-6 M (pH 4.5, 5 mM succinate buffer containing 0.1 M NaClO ₄ ) After completely removing oxygen from the experimental system containing this mixed aqueous solution by freezing, degassing and introducing argon, sodium dithionite [Na ₂ S ₂ O ₄ ] = 1 x 10 ^-5 M was added, and Fe in the aqueous solution was added. (III) TPPS-CD2 was reduced to Fe (II) TPPS-CD2 (the absorption spectrum at this time is shown in FIG. 2 (a)). Next, oxygen was blown into this Fe (II) TPPS-CD2 aqueous solution at room temperature, and oxygen was coordinated to Fe (II) TPPS-CD2 (hereinafter referred to as “Fe (II) TPPS (O ₂ ) -CD2”. (Abbreviated as “)” (the absorption spectrum at this time is shown in FIG. 2B). Furthermore, when carbon monoxide was blown into this aqueous solution of Fe (II) TPPS (O ₂ ) -CD2 at room temperature, the inclusion complex (hereinafter referred to as “Fe (II)” in which the ligand was replaced from molecular oxygen to carbon monoxide. (Abbreviated as “TPPS (CO) -CD2”) (the absorption spectrum at this time is shown in FIG. 2 (c)). Thus, since the absorption spectrum of the inclusion complex was changed by the gas to be blown, that is, by the ligand, the inclusion complex of this example can adsorb oxygen even in a weakly acidic aqueous solution at pH 4.5. Substitution with carbon monoxide was confirmed.

(7) Adsorption of oxygen complex at pH 6.0 and its reversibility test
Fe (III) TPPS] = 5 x 10 ^-6 M and [CD2] = 6 x 10 ^-6 M mixed aqueous solution (pH 6.0, 5 mM phosphate buffer containing 5 mM NaClO ₄ ) After completely excluding oxygen from the experimental system containing freezing, degassing, and introducing argon, sodium dithionite [Na ₂ S ₂ O ₄ ] = 1 x 10 ^-5 M was added, and Fe (III) in the aqueous solution was added. TPPS-CD2 was reduced to Fe (II) TPPS-CD2 (the absorption spectrum at this time is shown in FIG. 3 (a)). Next, oxygen was blown into the Fe (II) TPPS-CD2 aqueous solution at room temperature to obtain an aqueous solution of Fe (II) TPPS (O ₂ ) -CD2 (the absorption spectrum at this time is shown in FIG. 3 (b)). Show.) Furthermore, when carbon monoxide was blown into this Fe (II) TPPS (O ₂ ) -CD2 aqueous solution at room temperature, an aqueous solution of Fe (II) TPPS (CO) -CD2 was obtained (the absorption spectrum at this time is shown in ( c)). Thus, since the absorption spectrum of the inclusion complex was changed by the gas to be blown, that is, by the ligand, the inclusion complex of this example can adsorb oxygen even in a weakly acidic aqueous solution at pH 6.0. Substitution with carbon monoxide was confirmed.

(8) Adsorption and reversibility test of oxygen complex at pH 7.5 Experiments similar to (6) and (7) were conducted in phosphate buffer at pH 7.5. It was confirmed that the inclusion complex could adsorb oxygen and replace the adsorbed oxygen with carbon monoxide.

(9) Repeat test
Make a mixed aqueous solution of [Fe (III) TPPS] = 5 x 10 ^-6 M and [CD2] = 6 x 10 ^-6 M (pH 4.5, 5 mM succinate buffer containing 0.1 M NaClO ₄ ) After completely removing oxygen from the experimental system containing this mixed aqueous solution by introducing freeze-degas-argon, sodium dithionite [Na ₂ S ₂ O ₄ ] = 1 x 10 ^-5 M was added, Fe (III) TPPS-CD2 was reduced to Fe (II) TPPS-CD2. Oxygen was blown into this solution, and an absorption spectrum was measured ((1) in FIG. 4). Then, argon gas was blown into this aqueous solution (first time), oxygen was replaced with argon, and then the absorption spectrum was measured ((1 ′) in FIG. 4). After that, oxygen and argon were alternately blown and the absorption spectrum was measured twice.

  The absorption spectrum when oxygen was blown for the second time was blown into (2) of FIG. 4, and the absorption spectrum when oxygen was blown for the second time was blown into (2 ′) of FIG. (3) in FIG. 4 and the absorption spectrum when argon is blown for the third time are shown in (3 ′) of FIG. Gas blowing and absorption spectrum measurements were performed at room temperature.

Since the absorption spectrum of the complex was changed by the gas to be blown, that is, by the ligand, it was confirmed that the inclusion complex could not only adsorb oxygen but also repeatedly adsorb and desorb oxygen. In addition, the absorbance peak (423 nm) of the Fe (II) TPPS (O ₂ ) -CD2 aqueous solution and the absorbance peak (434 nm) of the Fe (II) TPPS-CD2 aqueous solution decrease each time the oxygen adsorption / desorption cycle is repeated. On the other hand, the absorbance peak (398 nm) of the Fe (III) TPPS-CD2 aqueous solution increased, and as this oxygen adsorption / desorption cycle was repeated several times, Fe (II) TPPS-CD2 was a little It was also confirmed that each was oxidized to Fe (III) TPPS-CD2.

(10) Reaction rate test and pH stability test Using a buffer solution (5 mM succinic acid or phosphate buffer solution containing 0.1 M NaClO ₄ ) from which oxygen was completely removed by freezing-degassing-argon introduction, [ Fe (III) TPPS] = 6 x 10 ^-4 M and [CD2] = 7.2 x 10 ^-4 M and sodium dithionite [Na ₂ S ₂ O ₄ ] = 1.2 x 10 ^-3 M mixed aqueous solutions with different pH It was created. 25 mL was taken from this mixed aqueous solution, and an air-saturated buffer (5 mM succinic acid or phosphate buffer containing 0.1 M NaClO ₄ ) was added thereto to dilute to 3 mL, and the absorption spectrum was measured. Thereafter, the absorption spectrum was measured every two hours, and the logarithm of the absorbance change at 423 nm was plotted against time. The obtained plot was linearly approximated according to the first-order reaction rate, and the decomposition rate kd (h ⁻¹ ) was determined from the slope. The _half -life t (h) was determined from t _1/2 = ln2 / kd.

By the above operations, Fe (II) TPPS (O 2) a-CD2 disappearance rate of tracks spectroscopically, was quantitatively measured the stability of _{Fe (II) TPPS (O 2} ) -CD2. The results are shown in Table 1. As shown in Table 1, the oxygen complex was remarkably stable, and its stability was not significantly affected by pH.

  In general, Fe (III) TPPS becomes a μ-oxo dimer in an aqueous solution having a pH of 8 or higher. However, inclusion of CD2 as in the example suppressed the production of μ-oxo dimer. In addition, Fe (III) TPPS is generally easily decomposed because the oxygen complex is protonated in an acidic aqueous solution, but it is decomposed by inclusion with CD2 as in the examples. It was possible to prevent.

  As described above, it was confirmed that the clathrate complex according to the present application was able to repeatedly and reversibly attach and detach molecular oxygen in aqueous solutions having different pHs, and the stability of an oxygen complex to which oxygen was bonded was also confirmed. Therefore, the inclusion complex according to the present invention is expected to be used as a component of artificial blood for the purpose of holding oxygen, such as artificial myoglobin and artificial hemoglobin, and as an oxygen separator.

It is a figure which shows the outline of the synthetic pathway of the inclusion complex concerning this invention. It is a figure which shows the result of the adsorptivity in pH4.5, and its reversibility test. It is a figure which shows the adsorptivity in pH6.0 and the result of the reversibility test. It is a figure which shows the result of the oxygen adsorption-desorption repetition test with respect to an inclusion complex.

Claims (6)

A cyclodextrin dimer represented by the following chemical formula (1).

(In the formula, m represents a number of 1 or 2, and n represents a number of 1, 2, or 3) The cyclodextrin dimer according to claim 1, wherein m = 1 and n = 2.   An inclusion complex comprising the cyclodextrin dimer according to any one of claims 1 to 2 and a water-soluble metalloporphyrin. The inclusion complex according to claim 3, wherein the water-soluble metalloporphyrin is represented by the following chemical formula (2) or (3).

(In the formula, R ₁ and R ₂ each represent a carboxyl group, a sulfonyl group, or a hydroxyl group, and M represents any one of Fe ²⁺ , Mn ²⁺ , Co ²⁺ , and Zn ²⁺ )   The inclusion complex according to claim 4, wherein the water-soluble metalloporphyrin is 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrin (II) iron complex.   A method for producing an inclusion complex comprising mixing the cyclodextrin dimer according to claim 1 or 2 and the water-soluble metalloporphyrin according to claim 4 or 5 in water.

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