JP2003073397A - Synthetic mucin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an artificially synthesized mucin having a structural characteristic property as a mucin and stably producible, and further to provide an artificially synthesized mucin useful as an antiviral agent having infection inhibiting activities against a wide range of influenza virus isolates. SOLUTION: An artificial sugar chain polymer substituted with a sialyl oligosugar chain. Poly[para-aminophenyl(N-acetylneuraminyl-(2,3)-N-acetyl-β- lactosaminide)-L-glutamine-co-glutamic acid]. Poly[para-aminophenyl(N- acetylneuraminyl-(2,6)-N-acetyl-β-lactosaminide)-L-glutamine-co-glutamic acid].

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a sialyl oligosaccharide chain-substituted artificial sugar chain polymer having a structure similar to mucin and capable of being stably supplied as an artificially synthesized mucin.

[0002]

BACKGROUND OF THE INVENTION Mucin is a huge glycoprotein having a high molecular weight of 200,000 or more, which is a main component of mucus produced and secreted mainly by mucosal epithelial cells. Most of the molecular weight (50 to 90%) is occupied by sugar chains composed of D-galactose, sialic acid, L-fucose, N-acetyl-D-galactosamine, etc., and a straight chain with a characteristic repeating structure. It is thought to have a large number of O-linked brush-like bindings to the core-like protein part, and to have important significance in the expression of mucin function. Mucin plays an important role as a sugar chain-presenting molecule so that it is considered to be a molecule designed in the process of evolution to display sugar chains. For example, it has a function as a ligand for cell adhesion molecules with a lectin-like domain,
It has been suggested that it influences the mutual recognition of lymphocytes and leukocytes with vascular endothelium and plays a role in homing of lymphocytes and accumulation of leukocytes at sites of inflammation. In addition, it strongly influences intercellular recognition in cancer cell invasion, metastasis, angiogenesis, etc., and it has been suggested to be associated with enhancement of malignancy associated with cancer progression in colorectal cancer and renal cancer. On the contrary, due to the structural feature that the extracellular domain is large, anti-adhesiveness that does not allow cells to adhere to each other and suppressive action on immune cells that suppress T cell proliferation have attracted attention. In this way, mucin expresses various biological functions on the cell membrane surface via its mucin-type sugar chains, and is involved in the development, morphogenesis, maintenance of ecological homeostasis, pathogenesis, etc. of multicellular organisms. There is no doubt. Therefore,
Mucin is expected as a bioregulatory functional material that acts as a functional element for molecular recognition. In addition, it has been reported that influenza virus specifically binds to the receptor of host cells and at this time recognizes the sugar chain structure containing sialic acid as a receptor {E. Nobusawa et al. [Birology ( Virology), Vol. 82, pages 475-485 (1991)]}.
Therefore, mucin containing sialic acid in the non-reducing end sugar chain part is expected to have an anti-influenza action that inhibits binding to the virus receptor, which is the first step of influenza virus infection. , There are various subtypes, and in order to overcome drug resistance as a more effective anti-influenza agent, an anti-influenza action that widely inhibits receptor binding is desired regardless of the virus subtype. It is also known that mucin-like glycoprotein is expressed in healthy ocular surface epithelium, and that this positively contributes to the function of the tear film and its retention. Keratoconjunctivitis sicca (dry eye), various types of keratoconjunctivitis, allergies, infection with microorganisms (viruses, bacteria, fungi, etc.),
In addition, it is said that keratoconjunctival epithelial damage occurs due to the cytotoxicity of eye drops, chemical corrosion due to acid and alkali, trauma, etc., but at this time, the tear film breaks down, causing a vicious circle that worsens epithelial damage. It is believed that. Therefore, if mucin can be used to stabilize the tear film, it is expected to act as a therapeutic agent for keratoconjunctival disorders. If the mucin can be stably supplied as described above, the above-mentioned useful actions are expected.

[0003]

However, in order to supply the above-mentioned useful mucins, it is considered to extract and purify them from the mucin-containing living tissue. In the process, the mucins are easily cleaved. -There is a problem that it is difficult to obtain a stable substance while being decomposed and maintaining physiological activity. Although it is possible to artificially synthesize mucin, there is a problem that mucin is a macromolecule and it is impossible to artificially synthesize mucin itself. At present, the characteristics of what is called a mucin-type sugar chain are that the sugar chain part is linked to the amino acid of the protein part in an O-linked form, and many of them contain sialic acid. It has been found to have important significance in the. In addition, as another structural feature of mucin, the protein part has a repeating structure of peptide chains (tandem repeat),
A large number of mucin-type sugar chains may be uniformly added to the protein part. An object of the present invention is to provide an artificially synthesized mucin which has the above-mentioned structural characteristics as mucin and can be stably supplied. Another object is to provide an artificially synthesized mucin useful as an antiviral agent having an infection inhibitory activity against a wide range of influenza isolates.

[0004]

Means for Solving the Problems As a result of earnest research from the above viewpoints, the present inventor has introduced an oligosaccharide containing a mucin-type sugar chain into a side chain of a polypeptide-based polymer,
Furthermore, they have found that an artificially synthesized mucin can be provided by synthesizing a sialyl oligosaccharide chain-substituted artificial sugar chain polymer in which sialic acid is added to the non-reducing end portion of the introduced oligosaccharide chain, and arrived at the present invention. That is, the present invention is obtained by introducing a sialyl oligosaccharide into polyglutamic acid,
A structural feature is that it is a sialyl oligosaccharide chain-substituted artificial sugar chain polymer having a molecular weight of 2,000 to 1,000,000 and having repeating units represented by the following general formulas (1) and (2),
In addition, the polyglutamic acid has a degree of polymerization of 10 to 1,000, and the introduction rate of the sialyloligosaccharide chain to the glutamic acid residue is 10% to 100%. The gist is the characteristic synthetic synthetic mucin.

[0005]

[Chemical 3]

[0006]

[Chemical 4]

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION A sialyl oligosaccharide chain-substituted artificial sugar chain polymer of the present invention is described by T. Usui et al. [Carbohydrate Research, Vol. 244, Vol.
5 to 323 (1993)], the paranitrophenyl glycoside (para-nitrophenyltolu-N-acetyl-β-lactosaminide) synthesized by the transglycosylation reaction of β-galactosidase was used as X.Zen (X. Zeng) et al. [Carbohydr Res, Vol. 312, 209-217 (199)
8)] was introduced into polyglutamic acid, and then X.
X.Zeng et al. [Archbus Biochemistry
Biophysics (Arch. Biochem. Biophys.), No. 383
Vol. 28-37 (2000)], α2, 3- (N)-and α2,
It can be prepared by sialylating the introduced oligosaccharide with 6- (N) -sialyltransferase. The sialyl oligosaccharide chain-substituted artificial sugar chain polymer of the present invention thus obtained is useful as an antiviral agent having an infection inhibitory activity against a wide range of influenza isolates. Next, the present invention will be specifically described with reference to examples.

[0008]

EXAMPLES Example 1: Synthetic intermediate of the present invention, namely
Para-nitrophenyl tol N-acetyl-β-lactosaminide [Galβ1-4GlcNAcβ-pNP] was prepared by the following method. 20 mM sodium phosphate buffer containing 2.4 g lactose and 2.3 g para-nitrophenyl tolu N-acetylglucopyranoside (Sigma) in 20% acetonitrile.
Dissolve in (pH 7.0) (12 mL) and add 20 units of Bacillus circulans-derived β-galactosidase (Daiwa Kasei Co., Ltd.) to 40
The reaction was carried out at ℃ for 6 hours. Then, the reaction solution was heated at 95 ° C. for 10 minutes to stop the reaction and then centrifuged to collect the supernatant. The supernatant is applied to a Toyopearl HW-40S column (5x100 cm, Tosoh Corporation), the eluate is fractionated (20 mL / tube), and a portion of it is measured for absorbance at 300 nm to remove para-nitrophenyl residues. Hydrocarbons were quantified by measuring the absorbance at 485 nm by the phenol-sulfuric acid method. Fraction containing para-nitrophenyl tol N-acetyl-β-lactosaminide (120 mL)
Were collected and collected, and after concentration, methanol was gradually added. The deposited precipitate was collected by filtration, dried under reduced pressure and dried to obtain 292 mg of para.
Crystals of -nitrophenyltolu-N-acetyl-β-lactosaminide were obtained.

Example 2: The synthetic intermediate of the present invention, para-aminophenyltolu N-acetyl-β-lactosaminide [Galβ1-4GlcNAcβ-pAP], was prepared by the following method. Para-nitrophenyl tol N-obtained in Example 1
100 mg of acetyl-β-lactosaminide was dissolved in 20 mL of methanol, 300 mg of ammonium formate and 20 mg of 10% palladium / activated carbon powder were added to the solution, and the reaction was carried out at 40 ° C. The reaction was followed by high performance liquid chromatography over time. After 40 minutes, after confirming that the peak of para-nitrophenyltolu-N-acetyl-β-lactosaminide disappeared, the reaction solution was returned to room temperature to stop the reaction. The reaction solution was sequentially filtered through Celite and filter paper, and the filtrate was concentrated and then preliminarily equilibrated with 12% methanol Chromatolex-ODS DM1.
It was subjected to 020T column chromatography. The eluate was fractionated (30 mL / tube), and the peak fraction expected to be an amino-reduced disaccharide derivative with consistent absorption at 210 nm and 300 nm was concentrated,
Lyophilization gave 70.7 mg of crystals of para-aminophenyltolu N-acetyl-β-lactosaminide.

Examples 3-4: The synthetic intermediate of the present invention, namely poly (para-aminophenyltolu N-acetyl-β-lactosaminide-L-glutamine-co-glutamic acid) [Poly (Ga
lβ1-4GlcNAcβ-pAP / Gln-co-Glu)] was prepared by the following method. Dissolve 20 mg of sodium poly-L-glutamate (Sigma) in 0.4 mL of dimethyl sulfoxide,
Benzotriazol-1-yloxytris hexafluorophosphate pre-dissolved in 0.2 mL dimethylsulfoxide.
160 mg of (dimethylamino) phosphonium and 18 mg of 1-hydroxybenzotriazole monohydrate were added, and the mixture was stirred at room temperature for 20 minutes. Further, 60 mg of para-aminophenyl-N-acetyl-β-lactosaminide obtained in Example 2 was dissolved in 0.4 mL of dimethyl sulfoxide and added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was applied to Sephadex G-25 column.
(2.0x26 cm, Amersham Pharmacia Biotech) and eluted with 0.02 M sodium phosphate buffer (pH 7.4) containing 0.1 M sodium chloride (flow rate 1.0 mL / min). Fraction collection of the eluate (2.0 mL / tube)
The absorbance at 485 nm was measured by the sulfuric acid method, and the fractions containing hydrocarbon were collected and collected (13 mL). The solution was concentrated with an ultrafilter (Amicon) equipped with a YM-3 membrane (2 kg /
cm ² ), and further freeze-dried to obtain 46 mg of the standard product (Example 3). The results are shown in Table 1. The ¹ H-NMR spectrum chart (D ₂ O solution) and ¹³ C-NMR spectrum chart (D ₂ O solution) of this compound are shown in FIG. 1. Synthesis was performed in the same manner as described above, except that poly-L-glutamic acid having a different degree of polymerization was used as a raw material and the amount of each raw material compound charged was changed as shown in Table 1 (Example 4). . The results are shown in Table 1.

[0011]

[Table 1] ¹⁾ : benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate ²⁾ : 1-hydroxybenzotriazole monohydrate ³⁾ : para-aminophenyltolu N-acetyl-β-lactosaminide ⁴⁾ : The rate of introduction of sugar chains to glutamic acid residues, ¹ H-
Calculated from NMR (at 60 ° C)

Examples 5-6: Poly [para-aminophenyl (N-acetylneuraminyl- (2-3) -N-acetyl- of the present invention.
β-lactosaminide) -L-glutamine-co-glutamic acid] [P
oly (Neu5Acα2-3Galβ1-4GlcNAcβ-pAP / Gln-co-Glu)] was prepared by the following method. The poly (para-paraffin obtained in Example 3
Aminophenyltolu-N-acetyl-β-lactosaminide-L-
Glutamine-co-glutamic acid) [Poly (Galβ1-4GlcNAcβ-
pAP / Gln-co-Glu)] 10 mg and cytidine 5'-monophospho-N-
Sodium acetylneuraminate 15 mg 250 mM Manganese chloride 10 μL, 10% bovine serum albumin 10 μL and alkaline phosphatase 2 μL were dissolved in 50 mM cacodylate buffer (pH 6.0) 950 μL, and 30 mm units of α2,3- (N) -
Sialyltransferase (rat recombinant,
Spodoptera frugiperda-derived, Calbiochem) was added and the reaction was carried out at 37 ° C for 48 hours. This reaction solution was applied to a Sephadex G-25 column (2.0x26 cm, Amersham Pharmacia Biotech) to obtain 10.9 mg of a standard sample (Example 5). The results are shown in Table 2. Further, the ¹ H-NMR spectrum chart (D ₂ O solution) and the ¹³ C-NMR spectrum chart (D ₂ O solution) of this compound are shown in FIG. Poly with different sugar chain introduction rates
(Para-aminophenyltolu N-acetyl-β-lactosaminide-L-glutamine-co-glutamic acid) [Poly (Galβ1-4Glc
NAcβ-pAP / Gln-co-Glu)] was used as the raw material, and the charged amount and the solvent amount of each raw material compound and the reaction time were changed as shown in Table 2, except that the same method as described above was used. Synthesis was performed (Example 6). The results are shown in Table 2. In addition, ¹ H-NMR spectrum chart of this compound (D ₂ O solution)
Is shown in FIG.

[0013]

[Table 2] : Those obtained in Example 3: those obtained in Example 4

Examples 7-8: Poly [para-aminophenyl (N-acetylneuramiminyl- (2-6) -N-acetyl- of the present invention.
β-lactosaminide) -L-glutamine-co-glutamic acid] [P
oly (Neu5Acα2-6Galβ1-4GlcNAcβ-pAP / Gln-co-Glu)] was prepared by the following method. The poly (para-paraffin obtained in Example 3
Aminophenyltolu N-acetyl-β-lactosaminide-L-
Glutamine-co-glutamic acid) [Poly (Galβ1-4GlcNAcβ-
pAP / Gln-co-Glu)] 5 mg and cytidine 5'-monophospho-N-
Sodium acetylneuraminate 7.5 mg Further 250 mM manganese chloride 5 μL, 10% bovine serum albumin 5 μL and alkaline phosphatase 1 μL were dissolved in 50 mM cacodylate buffer (pH 6.0) 474 μL, and 15 milliunits of α2,6- (N) -Sialyltransferase (derived from rat liver, Calbiochem) was added and the reaction was carried out at 37 ° C for 48 hours. This reaction solution was applied to a Sephadex G-25 column (2.0x26 cm, Amersham Pharmacia Biotech) to obtain 6.3 mg of the standard product (Example 7). The results are shown in Table 3. The ¹ H-NMR spectrum chart (D ₂ O solution) and ¹³ C-NMR spectrum chart (D ₂ O solution) of this compound are shown in FIG. Poly (para-aminophenyltolu N-acetyl-β- with different sugar chain introduction rates
Lactosaminide-L-glutamine-co-glutamic acid) [Poly
(Galβ1-4GlcNAcβ-pAP / Gln-co-Glu)] was used as a raw material, and the charged amount, solvent amount and reaction time of each raw material compound were changed as shown in Table 3, except that Synthesis was performed in the same manner (Example 8). The results are shown in Table 3.
Shown in. In addition, the ¹ H-NMR spectrum chart of this compound
(D ₂ O solution) is shown in FIG.

[0015]

[Table 3] : Those obtained in Example 3: those obtained in Example 4

Two kinds of artificial oligosaccharide chain substitution artificial oligosaccharide chain polymers which are synthetic intermediates of the present invention synthesized in Examples 3 to 4 and sialyloligosaccharide chain substitution of the present invention synthesized in Examples 5 to 8 Table 4 shows the physical properties and the like of the four types of artificial sugar chain polymers.

[0017]

[Table 4] ¹⁾ : Degree of polymerization of poly-L-glutamic acid used as a synthetic raw material ²⁾ : ¹ G-NMR of sugar chain introduction ratio to glutamic acid residue
(At 60 ° C) ³⁾ : Value calculated from the structural formula

Example 9: Sialyloligosaccharide chain-substituted artificial sugar chain polymers (2 types) of the present invention, artificial sugar chain polymers (2 types) in which a sialyl oligosaccharide chain was added to a polyacrylamide carrier, and para of sialyl oligosaccharide chains -Nitrophenyl glycosides (2 types), a total of 6 types of compounds were subjected to influenza virus infection inhibition test by the following method. 96
100 μL of canine kidney-derived cell line (MDCK cells) suspended in MEM medium containing 10% fetal calf serum was placed in a well plastic plate, and allowed to adhere and grow. After cells became monolayer confluent, sugar chains or sugar chain polymers as test compounds were added at various concentrations, and then influenza virus was added and cultured. At this time, the virus is A / PR / 8/34, A / M
Four strains of emphis / 1/71, B / Lee / 40 and B / Gifu / 2/73 were used.
After 5 hours of culture, the cells were washed to remove a virus that did not infect the cells and sugar chains or sugar chain polymers that did not adsorb. Then, the medium was replaced with a fresh medium and cultivated. After 20 hours of culture, a reagent for measuring lactate dehydrogenase (LDH) (LDH CII Test Wako, Wako Pure Chemical Industries, Ltd.) was added, and after incubation, the absorbance at 630 nm was used as a control, and the absorbance at 550 nm was measured. The amount of LDH leaked from the cells was determined, and the virus infection rate was calculated from this. The group to which the test compound was not added was used as a positive control, and the absorbance value was defined as 100% virus infection, and a dose-correlation curve based on the virus infection rate of each test compound concentration was prepared, and the concentration of the test compound that inhibits virus infection by 50% (IC ₅₀ values) were calculated. The results are shown in Table 5.

[0019]

[Table 5] ¹⁾ : Polyglutamic acid ²⁾ : Polyacrylamide ³⁾ : Para-nitrophenyl group ND: Not tested

As shown in Table 5, the sialyl oligosaccharide chain-substituted artificial sugar chain polymer of the present invention prepared in Example 6 had a strong virus infection inhibitory activity against three types of influenza virus strains. However, the polyacrylamide-type polymer with the same sugar chain added showed only antiviral infection inhibitory activity against only one virus strain. On the other hand, the para-nitroglycoside having the same sialyl oligosaccharide did not show any virus infection inhibitory activity. Furthermore, the sialyl oligosaccharide chain-substituted artificial sugar chain polymer of the present invention prepared in Example 8 had a strong virus infection inhibitory activity against three influenza virus strains. However, the polyacrylamide type polymer to which the same sugar chain was added showed antiviral infection inhibitory activity against two virus strains, and the activity is 1/2 to 1/3 that of the polyglutamic acid type of the present invention.
Met. On the other hand, the para-nitroglycoside having the same sialyl oligosaccharide did not show any virus infection inhibitory activity.

[0021]

EFFECTS OF THE INVENTION The sialyl oligosaccharide chain-substituted artificial sugar chain polymer of the present invention has the structural characteristics of mucin and can be stably supplied. Therefore, it can be expected to be used as an artificially-synthesized mucin, and is useful as an antiviral agent having an infection inhibitory activity against a wide range of influenza isolates.

[Brief description of drawings]

1 is a synthetic intermediate of the present invention obtained in Example 3, poly (para-aminophenyltolu N-acetyl-β-lactosaminide-L-glutamine-co-glutamic acid) [Poly (Galβ1-4
GlcNAcβ-pAP / Gln-co-Glu)]] ¹ H-NMR spectrum chart and ¹³ C-NMR spectrum chart.

FIG. 2 Poly [para-aminophenyl (N-acetylneuramiminyl- (2-3) -N-acetyl-of the present invention obtained in Example 5
β-lactosaminide) -L-glutamine-co-glutamic acid] [P
oly (Neu5Acα2-3Galβ1-4GlcNAcβ-pAP / Gln-co-Glu)]
^{It is a 1} H-NMR spectrum chart figure and a ¹³ C-NMR spectrum chart figure.

FIG. 3 The poly [para-aminophenyl (N-acetylneuramiminyl- (2-6) -N-acetyl- of the present invention obtained in Example 6
β-lactosaminide) -L-glutamine-co-glutamic acid] [P
oly (Neu5Acα2-6Galβ1-4GlcNAcβ-pAP / Gln-co-Glu)]
^{It is a 1} H-NMR spectrum chart.

FIG. 4 The poly [para-aminophenyl (N-acetylneuramiminyl- (2-3) -N-acetyl- of the present invention obtained in Example 7
β-lactosaminide) -L-glutamine-co-glutamic acid] [P
oly (Neu5Acα2-3Galβ1-4GlcNAcβ-pAP / Gln-co-Glu)]
^{It is a 1} H-NMR spectrum chart figure and a ¹³ C-NMR spectrum chart figure.

FIG. 5: Poly [para-aminophenyl (N-acetylneuramiminyl- (2-6) -N-acetyl- of the present invention obtained in Example 8
β-lactosaminide) -L-glutamine-co-glutamic acid] [P
oly (Neu5Acα2-6Galβ1-4GlcNAcβ-pAP / Gln-co-Glu)]
^{It is a 1} H-NMR spectrum chart.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Kazuhide Toya             913-3 Oga, Shizuoka City, Shizuoka Prefecture F-term (reference) 4C084 AA02 AA06 BA01 BA08 BA22                       BA23 BA34 MA01 NA14 ZA332                       ZB332                 4H045 AA10 AA30 BA53 EA29                 4J001 DA01 DC12 EA36 GA11 JA20                       JB01

Claims (7)

[Claims] 1. A sialyl oligosaccharide chain-substituted artificial sugar chain polymer having a molecular weight of 2,000 to 1,000,000 and having a repeating unit represented by the following general formula (1). [Chemical 1] 2. A degree of polymerization of glutamic acid units is 10 to 100.
The sialyl oligosaccharide chain-substituted artificial sugar chain polymer according to claim 1, which is 0. 3. The sialyl oligosaccharide chain-substituted artificial sugar chain polymer according to claim 1, wherein the introduction ratio of the sialyl oligosaccharide chain to the glutamic acid residue is 10% to 100%. 4. A sialyl oligosaccharide chain-substituted artificial sugar chain polymer having a molecular weight of 2,000 to 1,000,000 and having a repeating unit represented by the following general formula (2). [Chemical 2] 5. A glutamic acid unit having a degree of polymerization of 10 to 100
The sialyl oligosaccharide chain-substituted artificial sugar chain polymer according to claim 4, which is 0. 6. The sialyl oligosaccharide chain-substituted artificial sugar chain polymer according to claim 4, wherein the introduction rate of the sialyl oligosaccharide chain to the glutamic acid residue is 10% to 100%. 7. An anti-influenza agent containing one or more compounds selected from claims 1 to 6 as active ingredients.