JPH0616687A - Heparin unsaturated tetrose and its production - Google Patents

Abstract

PURPOSE:To provide a new heparin tetrose containing glucosamine-3-O-sulfuric acid. CONSTITUTION:The heparin unsaturated tetrose expressed by formula (R1 is SO3<-> or COCH3; R2 is SO3<-> or H) and its salt. The compound of formula can be produced by decomposing heparin with an enzyme at least containing heparitinase II originated from Flavobacterium and separating the compound from the decomposition product.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to glucosamine-3-O.
-A novel heparin unsaturated tetrasaccharide containing sulfuric acid. The unsaturated tetrasaccharide is a sugar chain that constitutes a functional region that is the center of heparin's anticoagulant activity. The present invention also relates to a method for producing the unsaturated tetrasaccharide by an enzymatic method using heparin as a raw material.

[0002]

2. Description of the Related Art Heparin is widely used as a drug because of its strong anticoagulant activity. It is known that its activity is because antithrombin III (ATIII) existing in blood and heparin form a complex to suppress the activity of coagulation factor. The part where ATIII and heparin bind to each other has been studied in detail, and heparin has the following pentasaccharide containing a sugar chain having an O-sulfate ester at the 3-position carbon of glucosamine as a structure necessary for the activity expression (see below ( The structure of 2)) has been determined.

[0003] (In the formula, R represents an acyl group such as an acetyl group. GlcA represents glucuronic acid, GlcN represents glucosamine, (3,6-di-SO ₃ ) represents 3-O and 6-O-sulfate, and IdoA Represents iduronic acid.) Disaccharides that characterize this structure ((3) above), GlcA → GlcNSO
₃ (3,6-di-SO ₃ ) is a minor component in heparin sugar chains. Since this disaccharide (3) is an essential component for binding to ATIII, it is the most suitable marker for heparin having anticoagulant activity.

The inhibitory activity against the coagulation factor expressed by binding of heparin to ATIII is ATII.
It has been known that it changes depending on the molecular weight of the I-binding region, and it is possible to change the degree of suppression of blood coagulation factor Xa (factor-Xa) and thrombin activity by adjusting this size. That is, it is considered that by reducing the molecular weight of heparin, the inhibitory activity on thrombin is reduced to suppress the bleeding action, and the inhibitory activity on Xa activity is preserved to suppress thrombus formation. Based on this idea, a low-molecular-weight heparin, a thrombolytic agent with few side effects of bleeding, has been invented. (For example, US Pat. No. 4,303,651, US Pat. No. 4,396,76
No. 2).

Conventionally, only raw materials having a disaccharide (3) structure among sugar chains obtained from nature have been known, except for heparin / heparan sulfate. However, there is no good method for efficiently extracting an oligosaccharide containing a disaccharide (3) from heparin, and it has been extremely difficult to obtain a large amount of this oligosaccharide by chemical synthesis. That is, when these low-molecular-weight heparins are produced by the limited chemical treatment of heparin, only products having a polydisperse molecular weight and composed of various constituent sugars are obtained, and it is not possible to prepare only oligosaccharides containing an active region. It was difficult.

As a relatively simple method, heparin is thoroughly decomposed using heparinase derived from Flavobacterium heparinum and heparinase I containing heparitinase I, and then heparin is decomposed into disaccharides to octasaccharides. A method of separating an oligosaccharide isomer mixture by high performance liquid chromatography (HPLC) to obtain an unsaturated hexasaccharide containing a disaccharide (3) on the reducing end side (KG Rice & RJ
Linhardt, Carbohydr. Res. 190, 219-233, 1989) is known. The purity of the unsaturated hexasaccharide obtained here is about 80%. However, this method does not produce unsaturated tetrasaccharides containing disaccharide (3).

In another report, an unsaturated hexasaccharide having a disaccharide (3) in the middle was obtained by a similar method, and the reducing end was converted to an anhydromannose form by decomposing nitrous acid to this unsaturated tetrasaccharide. It has been reported that sugar can be obtained (J. Choay, J.
C. Lormeau, M. Petitou, P. Snay & J. Fareed, Annals
of the New York Academy of Sciences, 370, 644-64
9, 1981).

As described above, in the previous reports, the minimum oligosaccharide containing disaccharide (3) was obtained from heparin by using an enzyme. However, including these reports, the enzymatic decomposition method, the chemical decomposition method, and the chemical decomposition method were used. To date, there has been no report of preparing an unsaturated tetrasaccharide containing the disaccharide unit of the above formula (3) by any of the synthetic methods. If such a tetrasaccharide is obtained, it can be expected to be utilized as a raw material for synthesizing sugar chains involved in the antithrombin III binding region and as a new blood coagulation factor inhibitor.

[0009]

SUMMARY OF THE INVENTION The first object of the present invention is to provide a novel heparin tetrasaccharide containing glucosamine-3-O-sulfate. Another object is to provide a method for obtaining such a heparin tetrasaccharide efficiently and easily by using heparin as a raw material and an enzymatic means.

[0010]

The present inventors have studied the specificity of heparin / heparan sulphate-degrading enzymes in order to obtain unsaturated tetrasaccharides containing heparin-derived disaccharide (3), and applied them. As a result, they have found that a novel heparin unsaturated tetrasaccharide can be efficiently and easily produced, and completed the present invention.

That is, the present invention provides a heparin unsaturated tetrasaccharide and a salt thereof represented by the following general formula (1).

[0012]

[Chemical 2]

(In the formula, R ₁ represents SO ₃ ^— or COCH ₃ (acetyl group; hereinafter abbreviated as “Ac”), and R ₂ represents SO ₃ ^— or H). In the structural formula of 1), the terminal unsaturated sugar residue is numbered 4 and the other sugar residues are numbered 1 to 3, and the basic unit sugar of each sugar residue is shown. Further, although only the ionized state is described in the structural formula of the general formula (1), it is clear that the present invention is not limited to this, and a structure having a proton added thereto or an alkali metal (sodium,
It includes salts with potassium, lithium, etc.), alkaline earth metals (calcium, barium, etc.), or ammonium ions.

Further, the present invention is characterized in that heparin is decomposed by using at least flavobacterium-derived heparitinase II, and fractionated from this decomposition product. The present invention provides a method for producing a saturated tetrasaccharide and a salt thereof. The present invention will be specifically described below.

[Heparin Unsaturated Tetrasaccharide] The heparin unsaturated tetrasaccharide of the present invention is preferably compounds I to III in which the following substituents are selected in the general formula (1). Compound I: ΔGlcA-GlcNAc (6S) -GlcA-GlcN (NS, 3S) (when R ₁ is Ac and R ₂ is H.) Compound II: ΔGlcA-GlcNAc (6S) -GlcA-GlcN (NS, 3S, 6
S) (R ₁ is Ac, R ₂ is SO ₃ ^- in the case) Compound III:. ΔGlcA-GlcN (NS , 6S) -GlcA-GlcN (NS, 3S, 6
S) (R ₁ is SO ₃ ^-, R ₂ is SO ₃ ^-. That it is a case) (the Δ in the formula unsaturated sugar, GlcA is glucuronic acid, GlcN is glucosamine, Ac is an acetyl group , NS indicates N-sulfate, 3S indicates 3-O-sulfate, and 6S indicates 6-O-sulfate.) These unsaturated tetrasaccharides of the present invention have a reducing end sugar residue of 3-O. -Has a common structure which is sulphated glucosamine. As shown in Examples below, the structure of the unsaturated tetrasaccharide of the present invention has a proton nuclear magnetic resonance spectrum ( ¹ H-NM
It was analyzed by measuring R).

[Production of Heparin Unsaturated Tetrasaccharide] In order to excise the heparin unsaturated tetrasaccharide from heparin, means for utilizing an enzyme that acts on the sugar chain structure around the disaccharide (3) in the active region was examined. That is, as enzymes that may be used in such applications, 6 produced by two types of Flavobacterium microorganisms having different substrate specificities are used.
Heparin-degrading enzymes of various types (heparinase, heparitinase I, I ₀ , II, IV and V) were selected, and various combinations thereof were used sequentially or simultaneously to decompose heparin, and the composition of the product was investigated. Specifically, with respect to heparin degradation products by each enzyme, the degradation pattern of the oligosaccharides produced was examined by gel filtration, and the difference in the constituent 2 sugar composition was examined by anion exchange HPLC.

As a result, the main structure of heparin, Ido (2S
Regarding the cluster part of O ₃ ) -GlcNSO ₃ (6SO ₃ ), heparinase is likely to decompose more rapidly to form various oligosaccharides of unsaturated disaccharide to octasaccharide, and heparinina is more likely to react with this oligosaccharide. -When the enzymes I and II are allowed to act simultaneously, the three unsaturated tetrasaccharides of the compounds I to III of the present invention and one unsaturated oligosaccharide (ΔGlcA-GlcNAc (6S) -Gl at the sugar-protein binding site are combined.
It was found that only four kinds of unsaturated oligosaccharides (cA-Gal-Gal-Xyl-Ser) remained, and all other oligosaccharides became unsaturated disaccharides. Among them, one kind of unsaturated oligosaccharide occurs only when the heparin used as a raw material has a structure in which a peptide is bound to the reducing end. This oligosaccharide was decomposed by heparitinase V into a disaccharide and a tetrasaccharide-Ser, but the three unsaturated tetrasaccharides of the present invention were affected by any of the above six heparin degrading enzymes (eliminase). It was found that the above was not decomposed and unsaturated disaccharide was not produced. In addition, these 6
For the properties of general enzymes, general substrate specificity, etc., see “Course of New Chemistry 3, Sugar II, Proteoglycans and Glycosaminoglycans”, pp. 244-249 (1991,
(Published by Tokyo Kagaku Doujinshi), AT
It was unclear how it acts on the structure around the disaccharide unit (3) involved in binding to III.

From the above experimental results, regarding the main sugar chain of heparin (excluding the bridge structure to the protein), only the portion containing glucosamine-3-O-sulfate (including the disaccharide structure of the above (3)) It was found that only the unsaturated tetrasaccharide of the present invention can be easily concentrated and recovered because it is left as an unsaturated tetrasaccharide by enzymatic decomposition. The above is a basic experiment leading to the present invention, and the method for producing the heparin unsaturated tetrasaccharide of the present invention was completed based on this result.

The production method of the present invention is based on heparin, heparitin-
It is an essential requirement to let Ze II act. Various heparin degrading enzymes can be used in combination with heparitinase II in the present invention. That is, heparitinase II
Heparinase,
Known heparin-degrading enzymes such as heparitinase I, I ₀ , IV and heparitinase V can be used in combination. The heparin-degrading enzyme used in the present invention includes a microorganism of the genus Flavobacterium (for example, Flavobacterium heparinum (ATCC 13125; US Patent Application No. 180,78).
No. 0), Flavobacterium sp. Hp206 (Ministry of Microbiological Research, No. 10207; JP-A-2-57183); Xth
International Symposium on Glycoconjugates (Proceed
ings), No228, p.330-331 (1989)), but enzymes of other origins (for example, Bacillus microorganisms; JP-A-2-1424) as long as they are compatible with the object of the present invention.
70, Japanese Patent Application No. 3-63707).

When various heparin-degrading enzymes are used in combination, these enzymes can be used in an arbitrary combination and mixed, or they can be used individually or in a mixture of two or more kinds to act separately in any order. In this case, heparitinase II and (a: heparinase and heparitinase are preferred.
One or more enzymes selected from the group consisting of IV) (group a enzyme) and one or more enzymes selected from (b: a group consisting of heparitinase I, heparitinase I ₀ and heparitinase V) (group b enzyme) It is possible to use together with. It is also possible to use heparitinase II in combination with one of the group a enzyme and the group b enzyme.

Particularly preferably, heparitinase II and
A combination of heparitinase I and heparinase or heparitinase IV can be mentioned. Examples of the combined use include, for example, a method of mixing heparinase, heparitinase I and II to act on heparin, a method of mixing heparitinase I, II and IV to act on heparin,
After heparin was digested with heparinase or heparitinase IV, the unsaturated disaccharide was removed and heparinase I was added to it.
A method of adding and II and thoroughly digesting again is exemplified. However, in any case, heparitina
The use of ZE II is mandatory.

The heparin used as a raw material is not particularly limited as long as it is a commonly available heparin. For example, heparin derived from a mammalian organ (eg, small intestine, lung) such as pig, cow, sheep, whale can be used. Usually, since commercially available heparin is treated with an alkali, it often loses the peptide or bridging structure at the reducing end, and even if the enzyme treatment according to the present invention is carried out, the unsaturated sugar at the binding site between sugar and protein is Often does not occur.

The specific method and reaction conditions for carrying out the above-mentioned enzymatic reaction are not particularly limited as long as they are the usual methods and conditions adopted for each heparin-degrading enzyme. That is, an aqueous solution containing heparin (aqueous solution, a solution containing an arbitrary buffer (for example, a phosphate buffer, an acetate buffer, a borate buffer, etc.)) is added by mixing all of the various heparin degrading enzymes, Incubation can be performed, or heparin-degrading enzymes, which are used alone or as a mixture of two or more, can be sequentially added in any order and incubated. Further, a single method or two or more kinds of enzymes can be used by a conventional method (US Patent Application No. 196,72).
It is also possible to react using an immobilized enzyme immobilized with No. 0) (for example, immobilized enzyme immobilized on Sepharose (Pharmacia Fine Chemical Co.)).

When two or more kinds of enzymes are mixed and used, it is necessary to set the reaction conditions such that each of the enzymes can act, but when two or more kinds of enzymes are acted separately, each enzyme can act. The reaction conditions can be selected by selecting the optimum reaction conditions. The reaction time depends on the production of the unsaturated tetrasaccharide of the present invention.
Those skilled in the art can easily determine by analyzing by chromatography such as PLC. The optimum reaction conditions (pH, temperature) for each heparin-degrading enzyme are as follows.

Heparitinase II: optimum pH of about 7.5, optimum temperature of about 40 ° C. Heparinase: optimum pH of about 7.0, optimum temperature of about 30 ° C. Heparitinase I: optimum pH of about 6.5 to 7.5. Optimum temperature about 45 ° C Heparitinase I ₀ : optimum pH about 7.5, optimum temperature about 45
℃ Heparitinase IV: optimum pH about 7.5, optimum temperature about 40
℃ Heparitinase V: optimum pH about 7.0, optimum temperature about 40
As the reaction conditions common to each enzyme, for example, a substrate solution having a heparin concentration of 1.0 to 100 g / L is used, and the pH is 5 to 5.
Examples of conditions include reaction at 8, 10 to 40 ° C. for 0.1 to 100 hours.

The amount of enzyme used can be variously selected depending on the kind of enzyme used in combination. For example, when heparitinase II, heparitinase I, and heparinase or heparitinase IV are used in combination as the enzymes, the amount of each enzyme is 10 to 1 g of heparinase II for 1 g of heparin.
00 units, 5-50 units of heparitinase I,
20 to 200 units of heparinase or heparitinase IV can be exemplified. Here, the unit means a titer capable of producing 1 μmol of unsaturated sugar per minute. However, in the titer measurement, heparinase I, I ₀ , II, V is used as a substrate, heparan sulfate derived from bovine kidney is used, and for heparinase and heparitinase IV, heparin derived from porcine small intestine is used.

After completion of the heparin decomposition reaction by the enzyme, the decomposition-produced oligosaccharide can be purified by a usual separation and purification method, for example, various chromatographies, to separate and purify the target heparin unsaturated tetrasaccharide. For example, usually, the decomposition product is obtained by first roughly fractionating oligosaccharides in the vicinity of tetrasaccharide by gel filtration, and then separating compounds I to III by HPLC (for example, HPLC using a polyamine-bonded silica gel column). Fractionation can be mentioned. In this case, the fraction to be separated is not necessarily a single compound as long as it is composed of the compounds satisfying the general formula (1) of the present invention. It may be a mixture of two or more kinds selected. Further, when each of the compounds 1 to III is purified, it may be carried out by refining the above mixture or the like.

As the gel filtration method, a molecular weight cutoff range of 50
The thing of 0-20,000 is preferable. Also, HPLC
Preferably employs anion exchange chromatography. [Use] The unsaturated tetrasaccharide of the present invention is a compound 1 to III or its constituent oligosaccharide, or a monosaccharide, particularly 3-O of residue 1.
-Sulfuric acid-substituted sugar can be provided as various valuable reagents. In addition, heparin unsaturated tetrasaccharide can be expected to be used as a synthetic raw material (material) for sugar chains involved in the ATIII binding region and a new inhibitor of blood coagulation factors. In addition, the reducing or non-reducing end of the unsaturated tetrasaccharide is replaced with other saccharides, ligands, medical materials,
The modified product bound to other synthetic polymer can be expected to be used as a functional material for various chromatography and artificial blood vessel surface as an ATIII-binding affinity carrier. In addition, these modified products can be used as weak anticoagulant active substances.

[0029]

EXAMPLES Hereinafter, specific examples of the present invention will be described.
The present invention is not limited to this. In addition, the name of each heparin-degrading enzyme is the above-mentioned "Shinsei Chemistry Experiment Course 3,
Carbohydrate II, proteoglycans and glycosaminoglycans ”. Example 1: 20 mg of heparin 500 mg derived from porcine small intestine
L (milliliter) of 20 mM acetate buffer (pH 7.
0) (containing 2 mM calcium acetate). To this solution, 30 units of heparinase derived from Flavobacterium heparinum, 12 units of heparinase II and 6 units of heparitinase I were added and digested at 37 ° C for 5 hours. After completion of the reaction, the enzyme was denatured by heating in a boiling bath for 3 minutes, and the resulting insoluble matter was removed by centrifugation at 3000 rpm for 10 minutes.

Salt was added to this supernatant liquid to make a 0.2 molar concentration, which was loaded on a Cellulofine GCL-90m (Seikagaku Corporation) column (4.5 × 105 cm),
Gel filtration was performed with a 0.2 M sodium chloride solution. The tetrasaccharide-equivalent fraction was collected and concentrated, then dissolved in 2 mL of distilled water, desalted using Cellulofine GCL-25 (Seikagaku Corporation) column (2 × 45 cm), and freeze-dried. Lyophilized product was added to 1 mL of 20 mM acetate buffer (pH 7.0).
It was dissolved in (containing 2 mM calcium acetate), 2 units of heparitinase II was added, and digested at 37 ° C. for 3 hours. Thereafter, the enzyme was denatured in the same manner as above, and the resulting insoluble matter was removed by 30
Centrifugation was performed at 00 rpm for 10 minutes to remove, and the tetrasaccharide was recovered with a Cellulofine GCL-90m column (3 x 100 cm), and the cellulofine GCL-25 column (2 x 45c).
m) was used for desalting and lyophilization to obtain a mixture of heparin unsaturated tetrasaccharides containing compounds I, II and III.

This mixture was dissolved in 0.5 mL of a 16 mM sodium dihydrogen phosphate solution, and an HPLC column (amino silica gel column, PA03 4.6 × 250 mm,
(Manufactured by YMC Co., Ltd.), the concentration of sodium dihydrogen phosphate was linearly increased by HPLC from 200 mM to 800 mM for elution, and the three components I, II, and III were separated by tracing at 232 nm ultraviolet absorption. (See Figure 1).

All the components were concentrated to dryness, dissolved in a small amount of distilled water, desalted with a Cellrefine GCL-25 column, and freeze-dried to obtain heparin unsaturated tetrasaccharides I, II and III. Each dried product was dissolved in distilled water, and one part thereof was diluted with a 0.01 N hydrochloric acid solution to determine the ultraviolet absorption at 232 nm. This value was divided by the molar molecular extinction coefficient of unsaturated disaccharide to estimate the molar concentration.
Yield from 00 mg is 3.8 mg for I and 1 for II.
6.8 mg and III were 2.8 mg. The structure was confirmed from the 500 MHz ¹ H-NMR spectrum and proton assignment of the obtained heparin unsaturated tetrasaccharide.

Table 1 shows the δ values of the ¹ H-NMR spectrum of each structure. In addition, the attribution of each signal is two-dimensional HOH
AHA (Homonuclear Hartmann-Hahn Spectroscopy),
COSY (Corelation Spectroscopy; homonuclear nuclear shift correlation two-dimensional NMR) was used.

[0034]

[Table 1]

Example 2: Heparin 500 from porcine small intestine
mg was dissolved in 20 mL (milliliter) of 20 mM acetate buffer (pH 7.0) (containing 2 mM calcium acetate). Flavobacterium sp. Hp206
Heparitinase IV (25 units), flavobacterium heparinum-derived heparitinase II (15 units) and heparitinase I (5 units)
Digested at ℃ for 5 hours. After that, the same treatment as in Example 1 was performed. The yield from 500 mg heparin is I is 4.2 m.
g and II were 17.5 mg, and III was 3.1 mg.

[0036]

The present invention provides a heparin unsaturated tetrasaccharide represented by the general formula (1), wherein the reducing end is glucosamine-3-O-sulfate and the non-reducing end is unsaturated glucuron. A novel structure containing an acid, which is a very useful structure containing sites involved in the regulation of heparin's anticoagulant activity. This heparin-unsaturated tetrasaccharide can be very easily produced in a large amount as a compound having a single structure by degrading heparin by the enzyme of the present invention and fractionating the resulting mixture. Therefore, the present invention can reduce the cost of an extremely expensive reagent in this field and can inexpensively provide useful raw materials for various medical materials or pharmaceuticals based on the compound of the present invention.

[Brief description of drawings]

FIG. 1 is a chromatogram showing the results of HPLC in Example 1.

Claims (4)

[Claims] 1. A heparin unsaturated tetrasaccharide and a salt thereof represented by the following general formula (1). [Chemical 1] (In the formula, R ₁ represents SO ₃ ⁻ or COCH ₃ , and R ₂ represents SO ₃ ^−.
Or represents H. ) 2. The production of heparin unsaturated tetrasaccharide and a salt thereof according to claim 1, wherein heparin is decomposed by using at least Heparitinase II derived from Flavobacterium and fractionated from the decomposition product. Law. 3. Heparin unsaturation according to claim 2, wherein heparitinase II is used in combination with one or more enzymes selected from the following group a and one or more enzymes selected from group b. Method for producing tetrasaccharide. a: Heparinase and Heparitinase IV b: Heparitinase I, Heparitinase I ₀ and Heparitinase V 4. Heparitinase II and heparitinase I
And heparinase or heparitinase IV in combination with heparin unsaturation 4 according to claim 2 or 3.
Method of manufacturing sugar.