JP2009127044A - Inclusion supramolecular complex - Google Patents

Abstract

Provided is an inclusion supramolecular complex that has a low possibility of invading a living tissue and a possibility of leaking from a gap between living tissues, and can increase the microscopic density of the oxygen complex.
The inclusion supramolecular complex of the present invention comprises a cyclodextrin containing a porphyrin metal complex portion of a linear polymer compound having a porphyrin metal complex in at least one side chain of a linear polymer having a hydrophilic side chain. A dimer is included. In addition, if the side chain of the linear polymer having a hydrophilic side chain and the porphyrin metal complex are bonded via a spacer, the stability of the oxygen complex in which oxygen is adsorbed to the inclusion supramolecular complex is further improved. .
[Selection figure] None

Description

  The present invention relates to an inclusion supramolecular complex that can be used as a material for oxygen infusion that stably transports oxygen in the human body. In particular, the present invention relates to the possibility of invading living tissues and the possibility of leaking from gaps in living tissues. Is an inclusion supramolecular complex that has a low microscopic density and can increase the microscopic density of the oxygen complex.

  One of the biomolecules that play the most important role in life activity is metalloprotein. These metal proteins are composed of a metal complex (heme) constituting the active center or a metal ion and a protein surrounding it three-dimensionally.

  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. Since this oxygen binding site is composed of hydrophobic amino acid residues, 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. Can absorb and desorb oxygen.

  Hemoglobin, which carries oxygen, is composed of four subunits that have a structure very similar to that of globin proteins of heme and myoglobin. Without being oxidized, oxygen can be adsorbed and desorbed so as to be reversible and to have a constant oxygen partial pressure. 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. Therefore, many researchers are trying to imitate the structure of the active center of metalloproteins with artificially synthesized molecules. 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, and so on. Modeling is being attempted.

  For example, Groves et al. Synthesized a clathrate complex in which a porphyrin-based compound is clathrated with a compound in which the inner hydroxyl group of cyclodextrin is substituted with pyridine and the outer hydroxyl group is substituted with a hydrophobic group. It has been reported that the inclusion complex adsorbs oxygen (see Non-Patent Document 1).

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

Therefore, the inventors of the present invention have included an inclusion complex that can be used as a constituent material of a heme protein model that can reversibly absorb and desorb molecular oxygen in water, a synthesis method thereof, and a constituent material of the inclusion complex. A certain cyclodextrin dimer has already been developed (see Patent Document 1).
JP 2006-2077 A “Biophysical Chemistry” (USA), 2003, volume 105, p. 639-648

  However, the inclusion complex has the following problems because of its low molecular weight. First, there is a possibility of entering a living tissue and a possibility of entering a gap between living tissues and leaking from a tissue such as a blood vessel.

  Further, the microscopic density of the oxygen complex, for example, the density of the oxygen complex on the cell surface cannot be increased when the cell surface is used as a microscopic environment. Therefore, in order to raise the oxygen partial pressure in the living tissue to a practical level, a large amount of oxygen complex has to be sent to the capillaries.

  Therefore, it is an object to provide an inclusion supramolecular complex that has a low possibility of entering a living tissue and a possibility of leaking from a gap between living tissues and can increase the microscopic density of the oxygen complex.

  As a result of intensive studies on a complex containing a porphyrin metal complex and a cyclodextrin dimer, the inventors have found that an inclusion complex capable of forming a stable oxygen complex having a large molecular weight by polymerizing the porphyrin metal complex. It was found that a supramolecular complex can be obtained.

  That is, the inclusion supramolecular complex of the present invention is obtained by converting a porphyrin metal complex portion of a linear polymer compound having a porphyrin metal complex to at least one side chain of a linear polymer having a hydrophilic side chain into a cyclodextrin dimer. The body is an inclusion.

  Since the inclusion supramolecular complex of the present invention has a large molecular weight, it is unlikely to enter a living tissue or leak from a gap between living tissues, and the microscopic density of the oxygen complex, for example, on the cell surface. The oxygen complex density can be increased. Therefore, this inclusion supramolecular complex can be 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.

  The inclusion supramolecular complex of the present invention is (1) a linear polymer compound and (2) a cyclodextrin dimer is included. The details will be described below.

(1) Linear polymer compound A linear polymer compound has a porphyrin metal complex in at least one side chain of a linear polymer having a hydrophilic side chain.

(I) Polymer having a hydrophilic side chain The polymer having a hydrophilic side chain is a hydrophilic functional group capable of strongly interacting with water such as a carboxyl group, a hydroxyl group and an amino group. Specific examples thereof include polyacrylic acid, polyvinyl alcohol, and starch. Among these, polyacrylic acid is preferable because the porphyrin metal complex is easily substituted. The molecular weight of the polymer having a hydrophilic side chain can be arbitrarily selected depending on the application.

(Ii) Porphyrin metal complex A porphyrin metal complex is a water-soluble porphyrin complex with a metal ion coordinated at the center, which can be covalently bonded to the hydrophilic side chain of the polymer by a chemical reaction and bonded to the polymer. Any cyclodextrin dimer can be used without particular limitation.

  The porphyrin metal complex and the polymer can be bonded by a known amide bond, ester bond, ether bond or the like. Note that the number of porphyrin metal complexes bonded to one polymer (hereinafter, abbreviated as the number of bonds) may be freely adjusted depending on the application.

  The polymer having a hydrophilic side chain and the porphyrin metal complex may be bonded via a spacer. By using the spacer, the distance between the main chain of the polymer and the porphyrin metal complex is increased, and the cyclodextrin dimer can more stably include the porphyrin metal complex.

The spacer can be used without particular limitation as long as the distance between the polymer and the porphyrin metal complex is increased, and examples thereof include a lower alkylene group having about 1 to 8 carbon atoms, such as an ethylene group. In addition, such a spacer is formed by, for example, a polymer having a hydrophilic side chain and a porphyrin metal complex by a normal chemical reaction using an achiral amino acid having a structure of NH _2- (CH ₂ ) _n -COOH and a protecting group. And can be interposed.

（式中、R1は、カルボキシル基、スルホニル基、水酸基、リン酸基の何れかを表し、MはFe²⁺、Mn²⁺、Co²⁺、Zn²⁺の何れかを表す。）で示される構成単位を含むもの、及び下記の化学式（II）

（式中、R1は、カルボキシル基、スルホニル基、水酸基、リン酸基の何れかを表し、MはFe²⁺、Mn²⁺、Co²⁺、Zn²⁺の何れかを表す。）で示される構成単位を含むもの、
が挙げられる。 (Iii) Specific Examples As specific examples of the linear polymer compound, the following chemical formula (I)

(Wherein R1 represents any of a carboxyl group, a sulfonyl group, a hydroxyl group, and a phosphate group, and M represents any of Fe ²⁺ , Mn ²⁺ , Co ²⁺ , and Zn ²⁺ ). And the following chemical formula (II)

(Wherein R1 represents any of a carboxyl group, a sulfonyl group, a hydroxyl group, and a phosphate group, and M represents any of Fe ²⁺ , Mn ²⁺ , Co ²⁺ , and Zn ²⁺ ). Including structural units
Is mentioned.

(2) Cyclodextrin dimer A cyclodextrin dimer is a dimerized by covalently bonding cyclodextrin and can include a porphyrin metal complex part of a linear polymer compound. It can be used without any particular limitation.

  Cyclodextrins include α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, and β-cyclodextrin is preferable because it easily includes a porphyrin metal complex.

（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。）で示されるものが挙げられる。なかでも、シクロデキストリンがβ−シクロデキストリンであるｎ＝２、かつ、酸素錯体を形成し易いｍ＝１であることが好ましい。 Specific examples of the cyclodextrin dimer include the following chemical formulas (III), (IV), (V)

(Wherein m represents any number of 1 or 2, and n represents any number of 1, 2 or 3). Especially, it is preferable that it is n = 1 which cyclodextrin is (beta) -cyclodextrin, and m = 1 which is easy to form an oxygen complex.

(3) Inclusion supramolecular complex An inclusion supramolecular complex is obtained by dissolving a linear polymer compound and a cyclodextrin dimer in an appropriate solvent such as a phosphate buffer, and then mixing these solutions. Can be prepared. The concentration of the linear polymer compound in the solution, the concentration of the cyclodextrin dimer, and the amount ratio in the case of mixing may be appropriately adjusted according to the concentration of the inclusion supramolecular complex to be prepared.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, the claim of this invention is not restrict | limited in any meaning by a following example.

1. Synthesis of Linear Polymer Compound and Cyclodextrin Dimer A linear polymer compound and a cyclodextrin dimer constituting an inclusion supramolecular complex were synthesized along the synthesis route shown in FIGS. In addition, in order to clarify the relationship between FIG. 1 to FIG. 3 and the following description, the same number is assigned to the same compound.

(1) Synthesis of linear polymer compound P-1 (i) Synthesis of 4-nitrobenzaldehyde ethylene acetal
In a 500 mL eggplant-shaped flask, 500 mL of benzene, 4-nitrobenzaldehyde (hereinafter abbreviated as Compound 1, 9.0 g, 0.06 mol), ethylene glycol (5.0 mL, 0.090 mol) and p-toluenesulfonic acid monohydrate (1.0 g, 5.2 mmol) was added and the Dean-Stark apparatus was assembled, and then heated to reflux for 3 hours. After completion of the reaction, the reaction mixture was cooled to room temperature and the solvent was distilled off under reduced pressure.

The residue was dissolved in 100 mL of ether, transferred to a separatory funnel, and washed with saturated aqueous sodium hydrogen carbonate and brine. The organic layer was separated, and the organic layer was dehydrated with anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure to obtain 4-nitrobenzaldehyde ethylene acetal (hereinafter abbreviated as Compound 2) as a brown solid ( Yield 11.7 g, yield 90.1%). The product was identified by ¹ H NMR spectrum.

(Ii) Synthesis of 4-aminobenzaldehyde ethylene acetal
Compound 2 (5.4 g, 25.1 mmol), ethanol 300 mL, and platinum (IV) oxide (300 mg, 1.32 mmol) were added to a 300 mL eggplant-shaped flask, and the mixture was stirred at room temperature for 15 hours in a hydrogen atmosphere. The reaction solution was filtered through Celite, and the solvent of the filtrate was distilled off under reduced pressure to obtain 4-aminobenzaldehyde ethylene acetal (hereinafter abbreviated as Compound 3) as a yellow oily liquid. Since compound 3 is unstable in air, it was directly used in the next reaction.

(Iii) Synthesis of 4-azidobenzaldehyde First, compound 3 (3.7 g, 25.1 mmol), concentrated hydrochloric acid 45 mL and acetic acid 135 mL were added to a 500 mL reaction vessel equipped with a dropping funnel, and cooled to 0 ° C. using an ice bath. did. Next, an aqueous sodium nitrite solution (5.72 g in 24 mL) was slowly added dropwise to the reaction vessel, followed by stirring at 0 ° C. for 30 minutes. Further, a sodium azide aqueous solution (8.09 g in 30 mL) was slowly added dropwise to the reaction solution, followed by stirring at 0 ° C. for 30 minutes.

100 mL of water was added to the reaction solution and extracted with diethyl ether. The extract was transferred to a separatory funnel and washed with saturated aqueous sodium hydrogen carbonate and saturated brine, and the organic layer was separated. The solvent was distilled off from the organic layer under reduced pressure to obtain 4-azidobenzaldehyde (hereinafter abbreviated as compound 4) as a brown liquid (yield 2.74 g, yield 69.4%). The product was identified by ¹ H NMR spectrum.

(Iv) Synthesis of 5,10,15-tris (4′-methoxycarbonylphenyl) -20- (4′-azidophenyl) porphyrin First, compound 4 (0.74 g, 5.00 mmol), terephthalaldehyde in a 1 L reaction vessel Methyl acid (2.46 g, 15.0 mmol), pyrrole (1.32 mL, 20.0 mmol) and 1 L of chloroform were added, and argon gas was blown for 30 minutes while stirring at room temperature. Next, boron trifluoride diethyl ether complex (0.34 mL, 2.69 mmol) was added to the reaction vessel, and the mixture was stirred at room temperature for 1.5 hours under light shielding. Furthermore, dichlorodicyanobenzoquinone (DDQ, 3.2 g, 14.1 mmol) was added to the reaction vessel, and the mixture was stirred at room temperature for 3 hours.

The reaction solution was transferred to a separatory funnel and washed with saturated aqueous sodium hydrogen carbonate and brine, and then the organic layer was dehydrated with anhydrous sodium sulfate. After separating the organic layer, the solvent was distilled off from the organic layer under reduced pressure. The residue was purified by silica gel chromatography (developing solvent: chloroform), and 5,10,15-tris (4′-methoxycarbonylphenyl) -20- (4′-azidophenyl) porphyrin (hereinafter referred to as compound 5) which was a purple solid. (The yield was 0.343 g, the yield was 10.2%). The product was identified by FAB-MS spectrum and ¹ H NMR spectrum.

(V) Synthesis of 5,10,15-tris (4′-methoxycarbonylphenyl) -20- (4′-azidophenyl) porfinatoiron (III) In a 50 mL eggplant-shaped flask equipped with a reflux tube, 100 mg of compound 5 was added. Then, 5 mL of chloroform and 5 mL of a saturated methanol solution of iron (II) chloride n hydrate were added, and the mixture was heated to reflux for 24 hours under an argon atmosphere.

  After cooling the reaction solution to room temperature, the reaction solution was transferred to a separatory funnel and washed with distilled water. The organic layer of the separatory funnel was dehydrated with anhydrous sodium, and after separating the organic layer, the solvent was distilled off under reduced pressure. The residue is dissolved in a minimum amount of chloroform, re-precipitated by adding hexane, the precipitate is collected by filtration, and 5,10,15-tris (4'-methoxycarbonylphenyl) -20- ( 4'-azidophenyl) porfinato iron (III) (hereinafter abbreviated as compound 6) was obtained (yield 0.090 g, yield 85.0%). The product was identified by FAB-MS spectrum.

(Vi) Synthesis of 5,10,15-tris (4′-methoxycarbonylphenyl) -20- (4′-aminophenyl) porphinatoiron (III) Compound 6 () was added to a 500 mL eggplant-shaped flask equipped with a reflux condenser. 50 mg, 0.058 mmol), chloroform (100 mL), methanol (100 mL) and sodium sulfide nonahydrate (48 mg, 0.20 mmol) were added, and the mixture was heated to reflux for 6 hours. The reaction solution was cooled to room temperature, and methanol was removed by distillation under reduced pressure.

  Chloroform 200 mL was added to the residue, transferred to a separatory funnel, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate and brine. After separating the organic layer, 5,10,15-tris (4'-methoxycarbonylphenyl) -20- (4'-aminophenyl) porfinatoiron (III), which is a purple solid, is distilled off under reduced pressure. (Hereinafter abbreviated as Compound 7) was obtained (yield 0.043 g, yield 89.2%). The product was identified by FAB-MS spectrum.

(Vii) Synthesis of linear polymer compound P-1
In a 100 mL eggplant-shaped flask, compound 7 (50 mg, 60 μmol), polyacrylic acid (average molecular weight 5000, 24 mg, 5.0 μmol), O- (7-azabenzotriazol-1-yl) -1,1,3,3- Tetramethyluronium hexafluorophosphate (hereinafter abbreviated as HATU, 180 mg, 480 μmol), N, N-diisopropylethylamine (hereinafter abbreviated as DIPEA, 170 mL, 950 μmol), N, N-dimethylformamide (hereinafter, (Abbreviated as DMF, 10 mL) was added, and the mixture was stirred at room temperature for 2 days. The solvent was distilled off from the reaction solution under reduced pressure, and 50 mL of THF and 30 mL of an aqueous sodium hydroxide solution (0.64 mol / L) were added to the residue, followed by stirring at room temperature for 2 hours.

The reaction solution was neutralized with HCl, the solvent was distilled off under reduced pressure, and the residue was dissolved in a small amount of aqueous sodium hydroxide solution. This aqueous solution was dialyzed in water for 3 days using a dialysis membrane having a molecular weight cut off of 5000. The dialysate was acidified with HCl to precipitate the product and filtered through a membrane filter to obtain a purple polymer linear polymer compound P-1 (hereinafter abbreviated as compound 8) (yield). 42 mg, yield 36.0%). The product was identified by ¹ H NMR spectrum.

(2) Synthesis of linear polymer compound P-2 (i) Synthesis of 5,10,15-tris (4'-methoxycarbonylphenyl) -20- (4'-aminophenyl) porphyrin A reflux condenser was attached. Compound 5 (500 mg, 0.58 mmol), chloroform 100 mL, methanol 100 mL, sodium sulfide nonahydrate (480 mg, 2.00 mmol) was added to a 200 mL eggplant-shaped flask, and the mixture was heated to reflux for 5 hours. The reaction solution was cooled to room temperature, and methanol was removed by distillation under reduced pressure.

Chloroform 200 mL was added to the residue, transferred to a separatory funnel, the organic layer was washed with saturated aqueous sodium hydrogen carbonate and brine, and then the organic layer was separated. The organic layer was dehydrated with anhydrous sodium sulfate, and after separating the organic layer, the solvent was distilled off from the organic layer under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent: chloroform: acetone = 20: 1) and 5,10,15-tris (4'-methoxycarbonylphenyl) -20- (4'-aminophenyl), a purple solid ) Porphyrin (hereinafter abbreviated as compound 9) was obtained (yield 0.433 g, yield 89.2%). The product was identified by MALDI-TOF MS spectrum and ¹ H NMR spectrum.

(Ii) Synthesis of 5,10,15-tris (4'-methoxycarbonylphenyl) -20- (4'-Fmoc-β-alaninephenyl) porphyrin
Compound 9 (178.0 mg, 0.224 mmol), Fmoc-β-alanine (760 mg, 2.240 mmol), HATU (423 mg, 1,120 mmol), DIPEA (0.980 μL, 2.240 μmol) and DMF 150 mL are added to a 100 mL eggplant-shaped flask. Stir at room temperature for 13 hours. The reaction solution was cooled to room temperature, and DMF was distilled off under reduced pressure.

To the residue was added 100 mL of chloroform, transferred to a separatory funnel, washed with saturated aqueous sodium hydrogen carbonate and brine, and the organic layer was separated. After dehydrating the organic layer with anhydrous sodium sulfate, the organic layer was separated again, and the solvent was distilled off from the organic layer under reduced pressure. The residue was purified by silica gel chromatography (developing solvent: chloroform: acetone = 40: 1) and 5,10,15-tris (4′-methoxycarbonylphenyl) -20- (4′-Fmoc-β) was a purple solid. -Alaninephenyl) porphyrin (hereinafter abbreviated as compound 10) was obtained (yield 0.105 g, yield 43.0%). The product was identified by MALDI-TOF MS spectrum and ¹ H NMR spectrum.

(Iii) Synthesis of 5,10,15-tris (4′-methoxycarbonylphenyl) -20- (4′-Fmoc-β-alaninephenyl) porfinatoiron (III) 50 mL of a reflux tube attached under argon atmosphere To the eggplant-shaped flask, 10.0 mg of compound 10, 5 mL of chloroform, and 5 mL of a saturated methanol solution of iron (II) chloride n hydrate were added and heated under reflux for 16 hours, and then the reaction solution was cooled to room temperature.

  The reaction solution was transferred to a separatory funnel and washed with distilled water, and the organic layer was separated. The organic layer was dehydrated with anhydrous sodium, the organic layer was separated again, and the solvent was distilled off under reduced pressure. Dissolve the residue in a minimum amount of chloroform, add hexane to reprecipitate, filter the precipitate, and collect 5,10,15-tris (4'-methoxycarbonylphenyl)- 20- (4′-Fmoc-β-alaninephenyl) porphinate iron (III) (hereinafter referred to as Compound 11) was obtained (yield 9.2 mg, yield 85.0%). The product was identified by FAB-MS spectrum.

(Iv) Synthesis of 5,10,15-tris (4'-methoxycarbonylphenyl) -20- (4'-β-alaninephenyl) porfinatoiron (III)
Compound 11 (20.0 mg, 0.224 mmol) and 5 mL of 20 wt% piperidine / DMF solution were added to a 100 mL eggplant-shaped flask and stirred at room temperature for 10 minutes, and then the piperidine / DMF solution was removed by distillation under reduced pressure. The residue was dissolved in 100 mL of chloroform, transferred to a separatory funnel, washed with saturated aqueous sodium hydrogen carbonate and brine, and the organic layer was separated. After dehydrating the organic layer with anhydrous sodium sulfate, the organic layer is separated again, and the solvent is distilled off from the organic layer under reduced pressure to give a purple solid 5,10,15-tris (4′-methoxycarbonylphenyl) -20. -(4'-β-alaninephenyl) porphinatoiron (III) (hereinafter abbreviated as compound 12) was obtained (yield 10 mg, yield 64.5%). The product was identified by FAB-MS spectrum.

(V) Synthesis of linear polymer compound P-2
Compound 12 (80 mg, 56 μmol), polyacrylic acid (average molecular weight 5000, 50 mg, 4.7 μmol), HATU (280 mg, 480 μmol), DIPEA (250 mL, 950 μmol), DMF 10 mL are added to a 100 mL eggplant-shaped flask, and 2 at room temperature. Stir for days. The solvent was distilled off from the reaction solution under reduced pressure, and 50 mL of THF and 30 mL of aqueous sodium hydroxide (0.64 mol / l) were added to the residue, followed by stirring at room temperature for 2 hours.

The reaction solution was neutralized with HCl, the solvent was distilled off under reduced pressure, and the residue was dissolved in a small amount of aqueous sodium hydroxide solution. This aqueous solution was dialyzed in water for 2 days using a dialysis membrane having a molecular weight cut off of 5000. The dialysate was acidified with HCl to precipitate the product, and the precipitate was collected by a membrane filter to obtain a purple solid linear polymer compound P-2 (hereinafter abbreviated as compound 13) (yield 70 mg). Yield 25.0%). The product was identified by ¹ H NMR spectrum.

(2) Synthesis of cyclodextrin dimer (i) Synthesis of 2-monotosyl-β cyclodextrin 200 mL of anhydrous DMF and dry β-cyclodextrin (hereinafter abbreviated as compound 21) in a 300 mL three-necked flask under an argon atmosphere. 17.0 g, 15.0 mmol) and sodium hydride (60-70% in oil, 513 mg, 15.0 mmol) were added, and the mixture was stirred at room temperature for 15 hours. After the reaction was completed, tosyl chloride (2.9 g, 15.2 mmol) was added to the three-necked flask and stirred at room temperature for 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 obtained dry solid was dissolved in distilled water and injected into a column filled with Diaion HP20 (manufactured by Mitsubishi Chemical Corporation). Add only distilled water to elute unreacted β-cyclodextrin, and after elution of unreacted β-cyclodextrin, change the developing solvent to 40% methanol aqueous solution, then only 2-monotosyl-β-cyclodextrin Was eluted. The eluate 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 colorless solid 2-monotosyl-β-cyclodextrin (hereinafter abbreviated as compound 22) (yield 6.3 g, yield 33%).

(Ii) Synthesis of 2,3-epoxy-β-cyclodextrin
Compound 22 (6.0 g, 4.7 mmol) and 300 mL of 0.2 M aqueous sodium hydroxide solution were added to a 500 mL eggplant-shaped flask, and the mixture was stirred at room temperature for 40 hours. With the reaction solution immersed in an ice bath, the reaction solution was neutralized with dilute hydrochloric acid.

The solvent was distilled off from the reaction solution to about 100 mL with an evaporator, and the remaining 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, poured into a column filled with Diaion HP20, and distilled water alone was added to elute 2,3-epoxy-β-cyclodextrin. 2,3-epoxy-β-cyclodextrin was confirmed by TLC (developing solvent n-BuOH: EtOH: H ₂ O = 5: 4: 3, anisaldehyde coloration, Rf = 0.25). The solvent of the eluate was distilled off under reduced pressure to obtain colorless solid 2,3-epoxy-β-cyclodextrin (hereinafter abbreviated as compound 23) (yield 3.5 g, yield 67%).

(Iii) Synthesis of 2,3-epoxy-permethyl-β-cyclodextrin Under an argon atmosphere, add Compound 23 (2.00 g, 1.45 mmol), 80 mL of anhydrous DMF and 30 mL of anhydrous THF to a 200 mL three-necked flask in an ice bath. Soaked in. Sodium hydride (washed with hexane and dried in vacuo (1.39 g, 58.00 mmol)) was added to the reaction solution, and the mixture was stirred in an ice bath for 1 hour.Methyl iodide (3.60 mL, 58.00) was added to the reaction solution. mmol) was added dropwise and stirred at room temperature overnight.

  4 mL of methanol was added to the reaction solution, foam was generated, chloroform was added and transferred to a separatory funnel, and then the extract was washed with distilled water and aqueous sodium thiosulfate solution to separate the organic layer. The organic layer was dehydrated with anhydrous sodium sulfate, and after separating the organic layer, the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (developing solvent: chloroform only → chloroform: acetone = 5: 2), and 2,3-epoxy-permethyl-β-cyclodextrin (hereinafter referred to as Compound 24) as a colorless solid. (Omitted) was obtained (yield 1.50 g, yield 61%).

(Iv) Synthesis of 3,5-dimercaptomethylpyridine 3,5-dichloromethylpyridine (200 mg, 0.94 mmol), thiourea (180 mg, 2.4 mmol) and 6 mL ethanol in a 50 mL eggplant-shaped flask equipped with a reflux tube And heated to reflux for 2 hours. To the reaction solution was added 3 mL of 5M aqueous sodium hydroxide solution, and the mixture was heated to reflux for 3 hours. The reaction solution was cooled to room temperature, neutralized with hydrochloric acid, transferred to a separatory funnel, and extracted with methylene chloride. After dehydrating the organic layer with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure to obtain 3,5-dimercaptomethylpyridine (hereinafter abbreviated as Compound 25) as a brown oil (yield 100 mg, yield 49%). ).

(V) Synthesis of cyclodextrin dimer Compound 25 (50 mg, 50 mg, dissolved in Compound 24 (1.0 g, 0.72 mmol), 60 mL 0.1 M NaHCO ₃ aqueous solution, 2 mL methanol in a 100 mL eggplant-shaped flask equipped with a reflux tube 0.29 mmol) was added, and the mixture was refluxed for 24 hours. After cooling the reaction solution to room temperature, the reaction solution was transferred to a separatory funnel and extracted three times with chloroform. After dehydrating the organic layer with anhydrous sodium sulfate, 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 desired product were collected and the solvent was distilled off.

  The resulting residue was purified by silica gel column chromatography (developing solvent chloroform / acetone = 5/2 → chloroform only → chloroform: methanol = 10: 1) to give a cyclodextrin dimer (hereinafter referred to as Compound 26) as a colorless solid. (Omitted) (yield 0.21 g, yield 20%, melting point 126-127 ° C.).

2. Production of Inclusion Supramolecular Complex and Various Tests (1) Measurement of Bond Number and Molecular Weight of Linear Polymer Compound The bond number and molecular weight of porphyrin metal complexes of linear polymer compounds P-1 and P-2 were examined. Specifically, DMSO solutions (1.0 × 10 ⁻⁶ M) of linear polymer compounds P-1 and P-2 were prepared, their ¹ H NMR spectra were measured, and from the integration ratio of the measured signals The quantity ratio between the polyacrylic acid moiety and the porphyrin moiety was determined. The results are shown in FIGS. In the figure, a is a signal derived from polyacrylic acid, and b is a signal derived from a porphyrin metal complex.

  From FIG. 4, it was found that the linear polymer compound P-1 is a substance having an average molecular weight of about 9,000, with about 5 equivalents of a porphyrin metal complex bonded to one molecule of polyacrylic acid. Further, FIG. 5 shows that the linear polymer compound P-2 is a substance having an average molecular weight of about 12,000, with about 8 equivalents of a porphyrin metal complex bonded to one molecule of polyacrylic acid.

(2) Structure of inclusion supramolecular complex An inclusion supramolecular complex was prepared and its structure was investigated. Specifically, it was performed as follows. First, a phosphate buffer solution (0.1 M, pH 7.0, 25 ° C.) containing 1.0 × 10 ⁻⁶ mol / L of the linear polymer compound P-1 or P-2, respectively, and a cyclodextrin dimer of 1.0 A phosphate buffer solution (0.1 M, pH 7.0, 25 ° C.) containing × 10 ⁻⁶ mol / L was prepared. Next, an ultraviolet-visible light spectrum was measured while titrating a phosphate buffer containing a cyclodextrin dimer into a buffer containing the linear polymer compound P-1 or P-2.

  From the obtained absorption spectrum, calculate the relationship between the change in the absorption spectrum and the amount of cyclodextrin dimer added and draw a titration curve. From the inflection point of this titration curve, P-1 in the inclusion supramolecular complex Alternatively, the amount ratio of P-2 and cyclodextrin dimer was estimated. The results are shown in FIGS.

  FIG. 6 shows the change in the absorption spectrum when titrated to P-1, and the upper right inset shows the change in absorbance at 388 nm, which is the absorption wavelength of P-1, and P-1 and cyclodextrin dimer. 3 shows the relationship (titration curve) between the change in absorbance at 424 nm, which is the absorption wavelength of the inclusion supramolecular complex consisting of, and the amount of cyclodextrin dimer (Py3CD) dropped. FIG. 7 shows the change in the absorption spectrum when titrated to P-2, and the upper right inset shows the absorption wavelength of the inclusion supramolecular complex composed of P-2 and cyclodextrin dimer at 422 nm and The relationship (titration curve) between the change in absorbance at 489 nm and the amount of cyclodextrin dimer (Py3CD) added dropwise is shown.

  Although there is no clear inflection point in these titration curves, the quantitative ratio of linear polymer compound to cyclodextrin dimer in the inclusion supramolecular complex derived from P-1 is linear polymer compound: cyclo Dextrin dimer = 1: 5, and the ratio of linear polymer compound to cyclodextrin dimer in the inclusion supramolecular complex containing P-2 is linear polymer compound: cyclodextrin dimer = It can be estimated to be 1: 8.

(3) Oxygen binding behavior The oxygen binding behavior of the inclusion supramolecular complex was investigated. Specifically, it was performed as follows. First, a phosphate buffer solution (0.1 M, pH 7.0, 25 ° C.) containing 1.0 × 10 ⁻⁶ mol / L of an inclusion supramolecular complex containing P-1 or P-2 was prepared. For comparison, a phosphate buffer solution (0.1 M, pH 7.0, 25 ° C.) containing 1.0 × 10 ⁻⁶ mol / L of the inclusion complex described in Patent Document 1 was prepared.

Next, the inclusion supramolecular complex and inclusion complex are reduced by adding an excess amount of sodium dithionite [Na ₂ S ₂ O ₄ ] to the inclusion supramolecular complex and the phosphate buffer of the inclusion complex. In this state (hereinafter abbreviated as “P _red” ), an ultraviolet-visible light spectrum was measured. Was measured visible light spectrum - After the measurement, by oxidizing the inclusion supramolecular complexes and inclusion complex by blowing oxygen gas into the phosphate buffer solution, oxidation state ultraviolet rays (hereinafter abbreviated as P _O2.). Was measured visible light spectrum - After the measurement, the oxygen is blown carbon monoxide gas in a phosphate buffer solution was replaced with carbon monoxide, substituted state (hereinafter, abbreviated as P _CO.) Ultraviolet. The measurement results are shown in FIGS.

8 shows the measurement result of the inclusion supramolecular complex containing P-1, FIG. 9 shows the measurement result of the inclusion supramolecular complex containing P-2, and FIG. 10 shows the measurement of the inclusion complex described in Patent Document 1. Each result is shown. Further, deoxy, oxy, and CO in the figure indicate spectra measured in the states of P _red , P _O2 , and P _CO , respectively.

  First, from the spectra of FIGS. 8, 9 and 10, it was confirmed that in any complex, the absorption spectrum was changed by blowing oxygen, and the absorption spectrum was changed by blowing carbon monoxide. This indicates that these complexes can adsorb oxygen and substitute the adsorbed oxygen with carbon monoxide. That is, it was found that the inclusion supramolecular complex containing P-1 or P-2 and the inclusion complex described in Patent Document 1 have oxygen detachment ability in an aqueous solution and can be used as a model compound of hemoglobin.

Further, FIGS. 8, 9, were compared the absorption spectra of P _CO of FIG. 10, a carbon monoxide complex of inclusion supramolecular complexes containing P-2 from a comparison of FIGS. 8 and 9 includes a P-1 Inclusion supramolecular complex has an absorption spectrum larger than that of carbon monoxide complex. From the comparison between FIG. 8 and FIG. 10, the inclusion complex of carbon monoxide described in Patent Document 1 is an inclusion supramolecular complex containing P-2. The absorption spectrum was larger than that of the carbon monoxide complex.

  That is, the absorption spectrum of the carbon monoxide complex was large in the order of the inclusion complex described in Patent Document 1, the inclusion supramolecular complex containing P-2, and the inclusion supramolecular complex containing P-1. From this, it was found that the stability of the oxygen complex was higher in the order of the inclusion complex described in Patent Document 1, the inclusion supramolecular complex containing P-2, and the inclusion supramolecular complex containing P-1.

The reason why we compared the absorption spectrum of _PCO rather than the change of the absorption spectrum of _{P02 to} compare the stability of the oxygen complex is that the central iron of the porphyrin metal complex part is trivalent and the ultraviolet-visible light spectrum of the oxygen complex This is because they are similar and difficult to distinguish. In other words, even if oxygen is blown into the complex and the absorption spectrum is changed, it cannot be determined whether this is due to the formation of an oxygen complex or whether the central iron is autoxidized from divalent to trivalent. is there.

On the other hand, when carbon monoxide is blown into P ₀₂ , if the central metal of the complex is trivalent iron, _PCO is not formed and the absorption spectrum does not change, but the central metal of the complex is divalent iron. In some cases, oxygen is replaced by carbon monoxide to form _PCO and change the absorption spectrum. Thus, by measuring the absorption spectrum of the P _CO to replace the oxygen complex of P ₀₂ to carbon monoxide, it is possible to determine the stability of an oxygen complex.

FIG. 2 is a diagram showing an outline of a synthesis route for a linear polymer compound P-1. It is a figure which shows the outline of the synthetic pathway of the linear high molecular compound P-2. It is a figure which shows the outline of the synthetic pathway of a cyclodextrin dimer. 1 is a ¹ H NMR chart of a linear polymer compound P-1. ^{1 is} a ¹ H NMR chart of a linear polymer compound P-2. It is an ultraviolet-visible light absorption spectrum which shows the result of having titrated the phosphate buffer containing the linear polymer compound P-1 with the phosphate buffer containing the cyclodextrin dimer. It is an ultraviolet-visible light absorption spectrum which shows the result of having titrated the phosphate buffer containing the linear high molecular compound P-2 with the phosphate buffer containing the cyclodextrin dimer. It is an ultraviolet-visible light absorption spectrum showing the result of oxygen adsorption on the inclusion supramolecular complex containing P-1 and substitution with carbon monoxide. It is an ultraviolet-visible light absorption spectrum showing the result of oxygen adsorption on the inclusion supramolecular complex containing P-2 and substitution with carbon monoxide. It is the ultraviolet-visible light absorption spectrum which shows the result of having made oxygen adsorb | suck to the conventional inclusion complex, and having substituted with carbon monoxide.

Claims (8)

  An inclusion supramolecular complex formed by inclusion of a cyclodextrin dimer in a porphyrin metal complex part of a linear polymer compound having a porphyrin metal complex in at least one side chain of a linear polymer having a hydrophilic side chain .   The inclusion supramolecular complex according to claim 1, wherein the linear polymer having a hydrophilic side chain is polyacrylic acid.   The inclusion supramolecular complex according to claim 1 or 2, wherein the side chain of the linear polymer having a hydrophilic side chain and the porphyrin metal complex are bonded via a spacer.   The inclusion supramolecular complex according to claim 3, wherein the spacer is a lower alkylene group. The linear polymer compound has the following chemical formula (I)

(In the formula, R1 represents any of a carboxyl group, a sulfonyl group, a hydroxyl group, and a phosphate group, and M represents any of Fe ²⁺ , Mn ²⁺ , Co ²⁺ , and Zn ²⁺ )
The inclusion supramolecular complex according to any one of claims 1 to 4, which comprises a structural unit represented by: The linear polymer compound has the following chemical formula (II)

(In the formula, R1 represents any of a carboxyl group, a sulfonyl group, a hydroxyl group, and a phosphate group, and M represents any of Fe ²⁺ , Mn ²⁺ , Co ²⁺ , and Zn ²⁺ )
The inclusion supramolecular complex according to any one of claims 1 to 4, which comprises a structural unit represented by: The cyclodextrin dimer has the following chemical formula (III)

(In the formula, m represents a number of 1 or 2, and n represents a number of 1, 2, or 3.)
The inclusion supramolecular complex as described in any one of Claims 1-6 shown by these.   The inclusion supramolecular complex according to claim 7, wherein m = 1 and n = 2.

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