CN1306009A

CN1306009A - Se-contained cyclodextrin and its application in preparing medicine to treat cataract

Info

Publication number: CN1306009A
Application number: CN 00133040
Authority: CN
Inventors: 罗贵民; 刘俊秋; 任晓君; 阎岗林; 牟颖; 杨同书; 沈家骢
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2000-11-17
Filing date: 2000-11-17
Publication date: 2001-08-01
Anticipated expiration: 2020-11-17
Also published as: CN1136854C

Abstract

The present invention relates to Se-contained cyclodextrin. A micro-molecular Se-contained cyclodextrin with higher glutathion peroxidase acitivity is prepared from cyclodextrin having hydrophobic cavity as the binding position of enzyme substrate and dual-Se bridge as catalytic radical by chemical synthesis, and can be used to treat cataract. Its advantages include simple preparing process and high output rate, glutathion peroxidase activity and stability.

Description

Selenium-containing cyclodextrin and application thereof in preparation of medicine for treating cataract

The invention belongs to the field of high-activity artificial enzymes, and particularly relates to a method for preparing selenium-containing cyclodextrin with Glutathione Peroxidase (GPX) activity by using cyclodextrin.

The micro-environment of the active site of the mimic enzyme on the molecular level, the factors which play a leading role in catalysis in the absorption enzyme, and the design and synthesis of the non-protein molecule which is much simpler than that of the natural enzyme are one of the leading subjects in the field of natural science at present as the artificial mimic enzyme. Biologists have been using chemical modeling methods to elucidate the catalytic behavior of enzymes in nature. Because the artificial enzyme molecule may have specificity and catalytic function comparable to natural enzyme, and is quite stable, the artificial enzyme molecule has wide application prospect in the fields of chemical industry, enzymology, medicine and pharmacology and the like. For this reason, artificial simulation of enzymes is included in the development programs of future studies in both the U.S. and european countries. In recent years, many small molecule enzyme-like systems have been established, such as cyclodextrins, crown ethers, cyclophanes, cycloarenes and porphyrins.

Glutathione Peroxidase (GPX) is an important selenium-containing enzyme in the body. Because of its excellent oxidation resistance, it has great potential for treating and preventing cataract, keshan disease, cardiovascular disease, inflammation and cancer. However, the enzyme has the defects of unstable source, limited source, large molecular weight, human body immune response and the like, and greatly limits the development and application of the enzyme in the aspect of medicine and pharmacology. The selenium-containing artificial enzyme can overcome the weakness of natural enzyme, so that the artificial simulation of the enzyme is more and more paid attention by scientists. The GPX mimetic Ebselen (PZ51), called "small molecule selenase", is a prominent representation of artificial enzymes, the catalytic mechanism and biological activity of which have been studied in detail. Animal experiments show that PZ51 has broad-spectrum anti-inflammatory activity, has curative effects on diabetes, tumor and cardiovascular diseases (documents 1 and 2), and has entered clinical trials at present abroad. However, as a medicine, the PZ51 has the greatest defects of low activity and poor water solubility, and in order to overcome the defect of the PZ51, solve the problem of poor water solubility and further improve the activity, cyclodextrin is selected to simulate GPX.

The invention aims to: provides the GPX artificial enzyme with simple process, high yield, high stability and high activity.

The principle of the invention is that the hydrophobic cavity of cyclodextrin is used as the substrate binding site of enzyme, the hydroxyl on cyclodextrin is activated by chemical method, and then substituted by NaHSe, the hydroxyl is changed into seleno-hydrogen group, so that the catalytic group of enzyme is introduced on cyclodextrin, and then oxidized to obtain the product containing selenium cyclodextrin with GPX activity (see synthesis scheme).

There are three types of cyclodextrins, α -cyclodextrin, β -cyclodextrin, and γ -cyclodextrin, whichever can be made into selenium-containing cyclodextrins using the above principles (synthetic route), however, the positions of the activated hydroxyl groups on the cyclodextrins are different, and the GPX activity of the finally obtained selenium-containing cyclodextrins is also different.

The preparation of the selenium-containing cyclodextrin using the hydroxyl group at the 2-position of β -cyclodextrin is illustrated below by the synthetic route to bis-selenium-bridged cyclodextrins:

1) β -2-hydroxy of cyclodextrin is activated by p-tolylsulfonyl chloride to prepare β -cyclodextrin-2-hydroxy phenyl sulfonate, 2) selenization of β -cyclodextrin-2-hydroxy phenyl sulfonate, namely substituting phenylsulfonate group by seleno group, 3) oxidation of seleno group in air, wherein seleno group is oxidized in air to generate diseleno-bridged cyclodextrin, 2, the diseleno-bridged cyclodextrin is prepared by the following steps:

the synthetic route is shown as the figure:(1) NaHSe production (see reference 3)

81.1mg (2.15mmole) of NaBH₄Dissolved in a vial containing 1ml of water, and 79mg (1mmole) of selenium powder was slowly added to NaBH₄The solution of (2) is covered with a rubber cover inserted with a needle, so that a large amount of gas generated in water can be seen, and heat is released along with the gas. Since the reaction is too vigorous, it is necessary to control the reaction rate in an ice bath, and the reaction equation is as follows:

due to NaBH₄And H⁺Reaction, with a small amount of H₂Se gas is generated and must be reacted in a fume hood to prevent poisoning. After the reaction is completed, a large amount of salt is precipitated. NaHSe is very easy to oxidize, so N should be introduced₂Storage (2) β -sulfonylation at CD 2-position (see reference 4):

β -CD-2-hydroxy benzene sulfonate, 2-OTs- β -CD for short, 2.0g (1.76mmole) of β -CD is dissolved in 80ml of NaOH (0.15 mole/L) solution, 5ml of acetonitrile solution containing 2.0g (9.66mmole) of P-TsCl is dripped at room temperature, 1 mole/L of NaOH is continuously added during the dripping to ensure that the pH of the solution is more than 12.5, the solution is stirred for 1h after 3h of dripping is finished, 1 mole/L of HCl is added for neutralization to neutrality, 100ml of methanol is added, insoluble substances are filtered out, the methanol and most of water are evaporated under reduced pressure to 20ml, salting-out is carried out after one week in a refrigerator at 5 ℃, after filtration, the first peak is collected by using water as eluent through Bio-gel P-2 column chromatography, freeze-drying is carried out to obtain 1.38g of the product, the yield is 65% (3) of the preparation of the double selenium-bridged cyclodextrin:

dissolving 100mg (0.075mmole) of 2-OTs- β -CD in 50mM phosphate buffer solution, introducing high-purity nitrogen to remove oxygen, adding 100 mu L of 1mol/L NaHSe (0.10mmole), reacting at 60 ℃ for 36h, then fully exposing the reaction system to air for oxidation, centrifuging to remove selenium, separating and purifying the supernatant by a Bio-gel P-2 column (5.0X 50cm), taking distilled water as eluent, collecting a first peak by ultraviolet monitoring at the flow rate of 1ml/min and at 254nm, freeze-drying, washing with acetone three times, and drying in vacuum to obtain 79mg of light yellow powdered diselenide bridged cyclodextrin, wherein the yield is 80%. 3. the structural characterization (1) of the diselenide bridged β -CD shows that the element analysis is carried out

TABLE 1 elemental analysis of bis-selenium-bridged β -CD

Theoretical value (%)		Experimental value (%)
Theoretical value (%)		Experimental value (%)		C	H	C	H
2-SeCD (C₈₄H₁₃₈Se₂·6H₂O)	40.14	6.00	40.32	C	H	C	H	5.96

The results of elemental analysis of the synthesized 2-position bis-selenium-bridged β -CD are shown in Table 1.

As shown in Table 1, the experimental values of the elemental analysis of 2-SeCD are basically consistent with the theoretical values, and the molecular formula of the compound is proved to be: c₈₄H₁₃₈Se₂·6H₂And O. (2) Infrared spectroscopic analysis

The results of infrared spectroscopic analysis (KBr pellet) are shown in Table 2.

TABLE 2 Infrared Spectroscopy of bis-selenium-bridged β -CD

	Wave number(cm^-1) (wave number)
	Wave number(cm^-1) (wave number)	β-CD	3385v_as-OH；2927vC-H(CH,CH₂)；1634δ-OH；1153, 1076,1028v(-O-)；947,756,707,580,530.
2-OTs-β-CD 2-SeCD	3423v_as-OH；2930vC-H(CH,CH₂)；1632δ-OH；1598,1496 vC=C(benzyl)；1364v_as(-SO3R)；1177v_s(-SO3R)；1155, 1083,1030v(-O-)；815γC-H(benzyl)；946,845,755, 706,580,530. 3392v_as-OH；2928vC-H(CH,CH₂)；1632δ-OH；1152, 1078,1029v(-O-)；946,756,708,581,532.	β-CD

From the analysis in Table 2, the IR spectrum showed 1177,1364cm characteristic absorption peak of the sulfonic acid ester^-1And characteristic absorption peaks 1598,1496,845 and 815cm of the benzene ring^-1Whereas the characteristic absorptions of the sulfonate and the benzene ring in SeCD disappeared, indicating that the sulfonate had been replaced by seleno (-SeH), while the structural characteristics of CD were unchanged.(3) NMR analysis Table 3 of bis-selenium-bridged Cyclodextrins¹HNMR、¹³CNMR spectra

δ(ppm)
δ(ppm)	¹H NMR 2-SeCD 4.94(H₁)；3.9-3.6(H₃,H₆,H₅)；3.6-3.4(H₂,H₄) ¹³CNMR 2-SeCD 103.3(C₁)；101.3(C₁’)；80.4(C₄)；79.0(C₄’)； 78.40(C₂’)；72.0(C₂)；71.6(C₃)；71.5(C₅)；71.0 (C₅’)；60.9(C₆)

By using D₂And O is a solvent for determining the double selenium-bridged cyclodextrin.¹H NMR and¹³c NMR, the results of which are shown in table 3, and the data obtained show structural features substituted with the hydroxyl at the 2-position, indicating that the seleno-hydrogen group is introduced into the 2-position of the cyclodextrin. (4) Mass spectrometric analysis

The molecular weight of the diselenide-bridged cyclodextrin measured by laser mass spectrometry was 2393.7 (theoretical value: C)₈₄H₁₃₈Se₂2393) which closely matches the calculated values, further demonstrating that the synthesized compound is present in a selenium-bridged form. (5) Selenium valence state and content analysis

Using X-ray photoelectron spectroscopy with magnesium (Mg)_Kα) As a target, the valence state of selenium in the mimic was determined to be-1. The electron energy spectrum of the standard selenocysteine (Se-Cyss) has Se (3d,5/2) electron binding energy of 55.1eV, and the electron binding energy of the mimic is 54.9eV, indicating that the valence state of selenium is very close to that of the standard. Further confirms that the selenium binding form of the mimic is consistent with selenocysteine and is a selenium bridge connection form.

By combining the above structural analysis, the structure of the diselenide-bridged cyclodextrin is determined as follows:

4. enzymatic Activity and stability of bis-selenium-bridged Cyclodextrin (1) GPX Activity of selenium-bridged Cyclodextrin [5]]

Measuring the living line to 700. mu.L, containing 50mmol/L potassium phosphate buffer, pH7.0,1mmol/L sodium azide, 1mmol/L glutathione, 0.25mmol NADPH,1U glutathione reductase, adding 0.5mmol/L H₂O₂The reaction was initiated and NADPH was monitored by oxygen at 340nmChange in absorbance of the reaction. When measuring enzyme activity, steamingDistilled water instead of artificial enzyme was blank control. The mimic enzyme activity unit is defined as one activity unit of 1 micromole NADPH consumed per micromole of artificial enzyme per minute.

TABLE 4 enzymatic Activity of bis-selenium-bridged Cyclodextrins

Enzyme and mimetic enzyme	Substrate	Specific activity (U/mu mol)
Enzyme and mimetic enzyme	Substrate	Specific activity (U/mu mol)	PZ51	H₂O₂	0.99
2-SeCD	H₂O₂ t-BuOOH CuOOH	7.40±0.10 4.3±0.10 10.5±0.5	PZ51	H₂O₂	0.99
2-SeCD	H₂O₂ t-BuOOH CuOOH	7.40±0.10 4.3±0.10 10.5±0.5	Natural rabbit liver GPX	H₂O₂	5780

(2) Stability of bis-selenium-bridged cyclodextrins

The double selenium-bridged cyclodextrin is preserved in PBS (pH7.0, 50 mmol/L) solution with half-year activity of 92 percent, and is dry powder

The enzyme activity is kept unchanged. 5. Effect of double selenium bridging cyclodextrin on treating cataract

32 rats with congenital cataract were divided into two groups: treatment and control groups of 16 animals each. In the treatment group, 0.33U (GPX active unit) of double selenium-bridged cyclodextrin is dropped into eyes every day, after 15 days, the cataract of 14 mouse eyes disappears, and the cataract of the other two mouse eyes is also relieved to a great extent. The control group was eye-dropped with the same amount of non-selenized cyclodextrin every day, and after 15 days, the degree of cataract was not reduced. After 15 days of test, the rat has sensitive response and good health condition. Therefore, the double-selenium bridged cyclodextrin can effectively treat cataract.

The invention has the following characteristics:

1. the preparation method is simple, the synthesis steps are few, the yield is high, and the production can be expanded.

2. The artificial enzyme model is simple, the cyclodextrin cavity is taken as a substrate binding site, and the diselenide bridge is taken as a catalytic group.

3. The selenium-containing cyclodextrin GPX prepared by the invention has high activity which is 7 times of that of the best micromolecule GPX simulant PZ51 in the world.

4. The diselenide bridged cyclodextrin as medicine can overcome the defects of unstable natural GPX, limited source, large molecular weight, human body immunoreaction, difficult membrane crossing and the like.

5. The diselenide bridged cyclodextrin is stable.

6. It has excellent antioxidant performance and great application potential in preventing and treating cataract, cardiac vascular disease, inflammation, cancer, etc.

In conclusion, the selenium-containing cyclodextrin disclosed by the invention is simple in preparation process, high in yield, capable of realizing mass production, high in GPX activity and good in stability, and can effectively treat cataract. Meanwhile, the defects of high immunogenicity, difficult membrane passing, instability and the like caused by using natural enzyme as a medicament can be overcome. Therefore, the compound has great application potential in medicine.

Reference documents:

1)Parnharn MJ,Leyck S,Graf E,Dowling EJ.Blake DR,Agents andActions,1991,32:4-9.

2)Sies H,Masumoto H,Advancesin Pharmacology,1997,38:229-246.

3)Klaymen DL,Girffin TS,J.Am.Chem.Soc.1973,95:197-199.

4) haeauiyou, Tonglinhui, Zhang Wei, Jindason, organic chemistry, 1991,11: 265-.

5)Wilson SR,Zucker PA,Spcetor AR,J.Am.Chem.Soc.,1989,111:5936

Claims

1. A kind of cyclodextrin containing selenium prepared from synthetic route (1), wherein the cyclodextrin can be any one of α -cyclodextrin, β -cyclodextrin, gamma-cyclodextrin, activate the hydroxyl group on the cyclodextrin by chemical method, then substitute the hydroxyl group with NaHSe to become seleno-hydrogen group, introduce the catalytic group of enzyme on the cyclodextrin, get cyclodextrin containing selenium with GPX activity by oxidation;

(1)

2. the selenium-containing cyclodextrin as claimed in claim 1, wherein the structural formula of the selenium-containing cyclodextrin obtained by activating the hydroxyl group at position 2 of β -cyclodextrin is shown as (2);(2)

3. the selenium-containing cyclodextrin of claim 1 or 2, wherein: has higher glutathione peroxidase activity;

4. use of a selenium-containing cyclodextrin according to claim 1, 2 or 3 in the manufacture of a medicament for the treatment of cataract.