JPH08253495A - Mannose structure-containing styrene derivative and polystyrene derivative and their production - Google Patents

Abstract

PURPOSE: To obtain a new styrene derivative having mannose structure, presenting specific affinity to cells, thus applicable to cell culture base materials, hybrid-type artificial internal organs, etc., by binding, via an amide linkage, mannobiose, to a styrene derivative, and to obtain a polystyrene derivative by polymerizing the above styrene derivative. CONSTITUTION: This new styrene derivative containing mannose structure is obtained by binding a mannobiose of formula I, via an amide linkage, to a styrene derivative. This new derivative is capable of giving a new monomer having mannose structure nd a mannose structure-bearing polymer of formula II ((n) is degree of polymerization), contains mannose structure having specific affinity to cells, therefore, having c:luster effect owing to the mannose structure and being applicable to cell culture base materials, hybrid-type artificial internal organs, etc. Specifically, this new derivative can be obtained by lactonizing mannobiose into mannobioselactone which is then reacted with vinylbenzylamine to synthesize vinylbenzyl mannobioselactonamide.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel sugar chain-containing compound,
In particular, the present invention relates to a styrene derivative having a mannose structure, a method for producing the same, a polystyrene derivative having a mannose structure obtained by polymerizing the styrene derivative, and a method for producing the same.

[0002]

2. Description of the Related Art Recent advances in sugar chain engineering have been remarkable. For example, as a biopolymer having a sugar chain,
Proteoglycans of the cell wall of plant cells that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc., and glycoproteins involved in cell-cell interaction and cell recognition, etc. However, the mechanism by which sugar chains of these macromolecules control high-precision biological reactions while acting, assisting, amplifying, regulating, or inhibiting functions of each other is gradually being clarified.

Furthermore, if the relationship between such sugar chains and cell differentiation / proliferation, cell adhesion, immunity, and cell carcinogenesis is clarified, this sugar chain engineering and medical, cell engineering, or organ engineering is performed. It can be expected that new developments will be made by closely linking and.

[0004] As one example, sugar chains on the cell surface and sugar chains
-Research on the role of sugar chains in the occurrence of diseases caused by abnormal interactions between receptors or in the infection of viruses such as AIDS is becoming active. In addition, studies on the involvement of sugar chains in cell-cell interactions and cell-matrix interactions have also become important for understanding biological reactions.

To illustrate the specificity of sugar chain recognition by various cells, hepatic parenchymal cells selectively recognize galactose (Gal) and mannose (Man), whereas hepatic non-parenchymal cells have Man and glucose (Glc). , N-acetylglucosamine (GlcNAc) has a high affinity. Serum tissue was Man, GlcNAc, and N-acetylmannosamine (ManNAc), and lymphoid tissue was Ma.
It specifically recognizes nNAc and GlcNAc.

[0006] Further, sugar chain recognizing proteins such as asialoglycoprotein and lectin-like protein on macrophages existing on the surface of various cells also selectively recognize sugar chains. For example, a protein with a molecular weight of 175K on alveolar macrophages is represented by Man, Fuc, and Gl.
cNAc, 180K molecular weight protein on peritoneal macrophages Man, Fuc, and GlcNAc, 30K molecular weight protein on rat Kupffer cells, 42K molecular weight protein on peritoneal macrophage, and 45-60K molecular weight on activated macrophages. The proteins are Gal and N-acetylgalactosamine (GalN
Ac) is specifically recognized.

[0007] The inventors of the present invention have been intensively researching by focusing on the specific affinity of sugar chains. For example, as a model of a ligand for an asialoglycoprotein receptor, a polymer having galactose as a side chain is used. A poly (Np-vinylbenzyl- [O-β-D-galactopyranosyl- (1 → 4) -D-gluconamide]) (abbreviated as PV-LA) was designed and synthesized.

[0008] For example, in an experiment for culturing hepatocytes on a Petri dish coated with PV-LA, hepatocytes are selectively selected through the specific affinity between PV-LA and the asialoglycoreceptor on the surface of hepatocytes. It was found that the three-dimensional self-assembly was guided by the adhesion and the specific adhesion form. (Yura, Y., Akaike, Toshihiro, "Cell Culture", Vol. 19, 317-322, 1993).

However, no sugar chain-containing polymer containing a mannose structure has been obtained so far. The present inventors have completed the present invention as a result of detailed investigations on the design and synthesis of a monomer containing this mannose structure and the polymerization of the monomer.

[0010]

Therefore, an object of the present invention is to provide a novel monomer having a mannose structure, a process for producing the same, and a sugar chain-containing polymer obtained by polymerizing the monomer. Especially.

[0011]

In the present invention, mannobiose (formal name: O-β-D-mannopyranosyl- (1 → 4)) is used.
The above-mentioned problems were solved by a styrene derivative having a mannose structure, which is formed by binding -D-mannopyranose) to a styrene derivative through an amide bond. That is, the present invention provides a mannose structure-containing monomer synthesized from a styrene derivative and mannobiose, and a mannose structure-containing polymer obtained by polymerizing the monomer.

The mannose structure-containing styrene derivative of the present invention comprises a styrene derivative and mannobiose lactone linked via an amide bond. The position for introducing mannobiose is preferably the p-position, but is not limited thereto, and may be the o-position, m-position, or a mixture thereof. Further, the mannose structure-containing styrene derivative of the present invention has a hydrogen atom of a benzene ring forming the styrene derivative, other than an alkyl group such as methyl, ethyl and propyl, an alkoxy group, a hydroxy group, a halogen atom, an alicyclic group and the like. It may be substituted by the substituent of.

In the present invention, among them, as shown by the following formula (1), vinyl in which an amide bond is formed by an amino group of p-vinylbenzylamine and a carbonyl group of mannobiose lactone. Benzylmannobiose lactonamide (formal name: Np-vinylbenzyl- [O-
β-D-mannopyranosyl- (1 → 4) -D-mannamide]) is preferred.

[0014]

Embedded image

This vinylbenzylmannobiose lactone amide (hereinafter abbreviated as VB-Man) is obtained by lactonizing the 1-position of mannobiose to mannobiose lactone, and mannobiose lactone and benzylamine of vinylbenzylamide. It is synthesized by forming an amide bond between. The method for producing VB-Man will be described in detail below.

(1) Lactonization of mannobiose Mannobiose is dispersed in distilled water and diluted with methanol. The diluted dispersion is added to a warm iodine methanol solution, and after stirring, a potassium hydroxide / methanol solution is gradually added until the color of iodine disappears.
Subsequently, the reaction solution is cooled with ice, and the deposited precipitate is collected by filtration.
The acid potassium salt is obtained by washing and recrystallizing the precipitate.

The potassium salt thus obtained is passed through an ion exchange resin to give an acid form, and methanol is added to the acid form fraction to concentrate under reduced pressure to obtain crystals. The crystals are dissolved in a small amount of methanol, and ether is further added to cause precipitation, which is repeated several times, and then dried under reduced pressure to obtain mannobiose lactone.

(2) Synthesis of VB-Man The above mannobiose lactone was dissolved in methanol,
A solution of p-vinylbenzylamine in methanol was added with heating under reflux. After heating under reflux for 120 minutes, acetone is added for crystallization. The obtained crystals are purified by column, and VB
-The Man fraction is lyophilized to obtain the desired VB-Man.

The mannose structure-containing styrene derivative of the present invention may be produced by lactonizing mannobiose and forming an amide bond between the mannobiose lactone and the styrene derivative to synthesize it. It is not limited. For example, as the oxidizing agent used when mannobiose is lactonized, bromine, chlorine and the like can be used in addition to iodine. Further, when the styrene derivative has another substituent and thus the solubility is changed, an appropriate solvent is selected accordingly.

Mannobiose used in the production method of the present invention can be prepared by the method of Kusakabe et al. (Kusube Kusaku et al., Agric. Biol.
m., Vol.51, No.10, pp.2825-2826, (1987)),
The β-mannanase obtained from a microorganism belonging to the genus Aspergillus, the genus Bacillus, the genus Trichoderma, and the genus Penicillium can be caused to act on the copra oil residue mannan. Although commercially available p-vinylbenzylamine may be used, it can be synthesized by various methods. For example, it is preferable to synthesize as follows. P-Chloromethylstyrene and phthalimide are used as starting materials, and they are reacted to obtain p-vinylbenzylphthalimide. The obtained p-benzylphthalimide is reduced with hydrazine to obtain p-vinylbenzylamine.

The present invention also provides a polystyrene derivative having a mannose structure. The mannose structure-containing polystyrene derivative has a basic structure obtained by polymerizing the mannose structure-containing styrene derivative.
That is, it is preferable that the mannose structure-containing polystyrene of the present invention has a polystyrene derivative as a main chain and amide bonds with mannobiose as a side chain on the main chain.

The mannobiose is preferably bonded to the p-position of the main chain, but is not limited thereto, and may be the o-position, m-position, or a mixture thereof. Further, in the mannose structure-containing polystyrene derivative of the present invention, the hydrogen atom of the benzene ring forming the polystyrene derivative of the main chain is an alkyl group such as methyl, ethyl or propyl, an alkoxy group, a hydroxy group, a halogen atom,
It may be substituted with another substituent such as an alicyclic group.

Among them, the mannose structure-containing polystyrene of the present invention is, for example, poly (p-vinylbenzylmannobiose lactone amide) represented by the following formula (2) (formal name: poly (Np-vinylbenzyl)). -[O-β-
D-mannopyranosyl- (1 → 4) -D-mannanamide]), hereinafter abbreviated as PV-Man) is preferred.

[0024]

[Chemical 4]

The mannose structure-containing polystyrene derivative of the present invention can be synthesized by polymerizing the mannose structure-containing styrene derivative. The polymerization conditions may be the same as those used for the polymerization of general styrene derivatives, and are not particularly limited. For example, a mannose-containing styrene derivative can be prepared by adding water, dimethyl sulfoxide (DMSO), dimethylformamide (DM
F) dissolved in a suitable solvent, and added with a radical polymerization initiator such as an azo compound such as azobisisobutyronitrile (AIBN), a persulfate such as potassium persulfate, or a peroxide such as benzoyl peroxide. And heat-polymerize. In addition to such solution polymerization, methods such as bulk polymerization, emulsion polymerization and suspension polymerization may be used.

The mannose structure-containing polystyrene derivative of the present application is preferably a homopolymer obtained by homopolymerizing the mannose structure-containing styrene derivative, but may be copolymerized with another monomer. For example, styrene, ethylene,
When copolymerized with a hydrophobic monomer such as propylene, acrylic acid, methacrylic acid, or methyl methacrylate, the obtained mannose structure-containing polystyrene derivative has strong hydrophobicity, and can be treated with a hydrophilic monomer such as vinyl acetate. When copolymerized, the obtained mannose structure-containing polystyrene derivative becomes strongly hydrophilic.

The mannose-containing polystyrene derivative of the present application can also be synthesized by polymerizing a monomer having a functional group capable of forming an amide bond with mannobiose lactone and then binding mannobiose lactone. However, it is preferable to polymerize the mannose-containing styrene derivative because the number of mannose introduced into one molecule can be controlled.

The molecular weight of the mannose structure-containing polystyrene derivative is appropriately set depending on its use. For example, when the mannose structure-containing polystyrene derivative is used in the form of an aqueous solution, the molecular weight of the polystyrene derivative is such that when it is dissolved in the aqueous solution, it forms micelles. In the case of a homopolymer of a styrene derivative containing a mannose structure, a homopolymer having a mannose structure of 30,000 to 50,000 is usually used.
In this case, for example, by using a copolymer with a monomer having a hydrophilic group, a hydrophilic group having a molecular weight outside the above range and maintaining water solubility is also preferably used. It

[0029]

【Example】

(Example 1) (1) Lactonization of mannobiose 5 g of mannobiose was dispersed in 17.5 ml of distilled water,
The dispersion is diluted with 26.3 ml of methanol. The diluted dispersion was added to a methanol solution of iodine (15 g / 175 ml) heated at 40 ° C. and stirred for 30 minutes, and then 4% potassium hydroxide / methanol solution was gradually added until the color of iodine disappeared. Was added to. The color of iodine disappeared upon addition of 350 ml of potassium hydroxide / methanol solution. Subsequently, the reaction solution was ice-cooled, and the deposited precipitate was collected by filtration. The precipitate was washed with cold ethanol and cold ether in this order, and recrystallized from ethanol / water [9: 1 (weight / volume)] to obtain a potassium salt.

The obtained potassium salt was converted to the acid form by passing it through an ion exchange resin (for example, Amberlite IR-12B (H ⁺ type)). Methanol was added to the acid fraction and concentrated under reduced pressure to give crystals. After repeating the procedure of adding a small amount of methanol to the crystals to dissolve the crystals and further adding ethanol to dehydrate and concentrate, the mixture was dried under reduced pressure to give 3.6 g (yield 72%) of the desired mannobiose. The lactone was obtained.

(2) Synthesis of VB-Man 1.3 g of the above mannobiose lactone was added to methanol 3
It was dissolved in 0 ml, and a solution of p-vinylbenzylamine in methanol (0.53 g / 0.5 ml) was added to the solution while heating under reflux. After heating under reflux for 120 minutes, 200 ml of acetone was added for crystallization. The obtained crystals are
Purified by passing through a column (HW40H Toyopearl) with distilled water as a developing solvent, and UV measurement (250 nm, 280 n
m) was confirmed by freeze-drying the VB-Man fraction,
1.0 g (yield 77%) of the desired VB-Man was obtained.

(3) Structure confirmation of VB-Man The structure of the obtained VB-Man was confirmed by ¹ H-NMR and ¹³ C-NMR. The resulting NMR spectrum, ¹
1 H-NMR is shown in FIG. 1 and ¹³ C-NMR is shown in FIG. In FIG. 1, a peak derived from mannobiose at 3.2 to 4.0 ppm, a peak derived from a double bond of a vinyl group at 5.2 to 6.8 ppm, and a peak derived from benzene of a styrene skeleton at around 7.4 ppm. A peak was seen. In FIG. 2, the NH—CH ₂ peak at 43.439 ppm, the mannose peak at 62.277 to 78.198 ppm, and β-bonded at 101.018 ppm 1
Carbon peaks were seen. Furthermore, 115.268
Peak of double bond of vinyl group at ppm: 127.415
The peak of benzene in the styrene skeleton around 138.502 ppm, and C = O of the amide bond at 175.500 ppm.
The peak of was seen. From these results, the structure of VB-Man was clearly confirmed.

Example 2 Polymerization of VB-Man (Synthesis of PV-Man) VB-Man (1 g) synthesized in Example 1 was added to DMSO.
Dissolve in (1 ml) and use AIBN (1 m
g) was added. The solution was heated to 60 ° C. under nitrogen and polymerized for 24 hours to obtain PV-Man. The number average molecular weight was about 40,000.

[0034]

EFFECTS OF THE INVENTION According to the present invention, there are provided a monomer having a mannose structure, which has never existed before, and a polymer having a mannose structure in its side chain. In the mannose structure-containing polystyrene of the present invention, since mannobiose is bonded as a side chain to the hydrophobic main chain, it is easy to adsorb it onto a hydrophobic substrate such as a polystyrene dish, for example, a mannose-modified dish is easy. Can be made into

Further, by polymerizing the polymer, a cluster effect due to the mannose structure existing at high density can be obtained. Since the mannose structure-containing styrene derivative and polystyrene derivative of the present invention have a mannose structure having a specific affinity with cells, they can be applied to, for example, cell culture substrates and hybrid artificial organs.

[Brief description of drawings]

FIG. 1 ¹ H-NM of VB-Man synthesized in Example 1
It is a figure which shows R spectrum.

FIG. 2 shows ¹³ C-NM of VB-Man synthesized in Example 1.
It is a figure which shows R spectrum.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Toshihiro Akaike 4-15-23 Shimohoya, Hoya-shi, Tokyo (72) Inventor Issei Kobayashi 4-7-107-403 Shiratori, Togo-cho, Aichi-gun, Aichi Prefecture

Claims (6)

[Claims] 1. A styrene derivative having a mannose structure, characterized in that mannobiose is bound to a styrene derivative through an amide bond. 2. The styrene derivative having a mannose structure according to claim 1, which is represented by the following formula (1). Embedded image 3. A method for producing a styrene derivative having a mannose structure, which comprises lactonizing mannobiose to give mannobiose lactone and reacting mannobiose lactone with vinylbenzylamine to synthesize vinylbenzyl mannobiose lactone amide. . 4. A mannose structure-containing polystyrene derivative, characterized in that mannobiose is bound to the polystyrene derivative through an amide bond. 5. The polystyrene derivative containing a mannose structure according to claim 4, which is represented by the following formula (2). Embedded image 6. Mannobiose is lactonized into mannobiose lactone, and this mannobiose lactone is reacted with vinylbenzylamine to synthesize vinylbenzyl mannobiose lactone amide, and the obtained vinylbenzyl mannobiose lactone is obtained. A method for producing a polystyrene derivative containing a mannose structure, which comprises polymerizing an amide.