JPH05320203A - Methylated galactosamine polymer and antibacterial containing the same - Google Patents

Abstract

PURPOSE:To obtain a methylated galactosamine polymer which is improved in solubility and physiological activity especially antibacterial activity by methylating the amino or hydroxyl groups of a specified alpha-1,4-polygalactosamine. CONSTITUTION:A methylated galactosamine polymer of the formula (wherein R1 to R3 are each amino, methylamino, dimethylamino or trimethylamino; R4 to R11 are each H or methyl; and n is 0-20000) is obtained by dispersing alpha-1,4- polygalactosamine produced by a mold paecylomyces sp. I-1, a partially hydrolyzed polygalactosamine prepared by hydrolyzing it with an acid or an enzyme and filtering the product through an ultrafilter membrane to form a fraction of a definite molecular weight, a galactosamine oligosaccharide prepared by separating the hydrolyzate by ion exchange chromatography or a hydrochloride thereof in N-methyl-2-pyrrolidone, leaving the dispersion at rest at room temperature for 6-24hr, adding 1-2 N NaOH to it, and methylating the amino or hydroxyl groups of the galactosamine with methyl iodide and sodium iodide. The amino groups may be selectively methylated.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel methylated galactosamine polymer and its use.

[0002]

2. Description of the Related Art α-1,4 polygalactosamine (polymer of galactosamine) is a filamentous fungus Peethilomyces sp., Which was newly isolated from the soil of Wakayama prefecture by the present inventors. I-1 (Paecilomyces sp. I)
-1) (FERM BP-1180) is a basic polysaccharide produced in large amounts outside the cells, the main body of which is a homopolysaccharide in which galactosamine is α-1,4-linked (Japanese Patent Publication No.
6-12639, Japanese Patent Publication No. 1-41160), and its average molecular weight is 300,000. And this α-1,4 polygalactosamine has an antibacterial activity (monthly food chemical 4
Monthly issue, 27 (1989)) and immunostimulatory action (J. Pha
rmacobio-Dyn, 11 , 58-65 (198).
9)) has been revealed. However, there are problems such as solubility in various solvents, and improvement of physical properties has been desired.

Also, with respect to chitin and chitosan, which are similar polysaccharides, many attempts have been made by chemical modification for the purpose of improving their physical properties and physiological functions, and for example, water solubility by chitin carboxymethylation has been investigated. A number of such achievements have been reported (improvement of metal ion adsorption power, improvement of water solubility by trimethylation of chitosan, application of chitin and chitosan, Gihodo (199).
0)).

The present invention provides a derivative of a novel galactosamine polymer having improved physical properties and physiological activity by chemically modifying a galactosamine polymer to prepare a methylated derivative, and the derivative as an active ingredient. However, such a methylated derivative is a novel compound which has not been known so far, and an antibacterial agent containing it as an active ingredient has not been known so far and is novel.

[0005]

[Means for Solving the Problems] In view of the above-mentioned state of the art, the present inventors have earnestly studied for the purpose of improving physical properties and physiological activity of galactosamine polymers.
The present invention has been completed by finding that the introduction of a methyl group into an amino group or a hydroxyl group significantly improves the solubility and improves the physiological activity, particularly the antibacterial activity.

The galactosamine polymer used in the present invention is
α-1,4 polygalactosamine may be used, or α-
After hydrolyzing 1,4 polygalactosamine with an acid or enzyme, it was fractionated to a certain molecular weight using an ultrafiltration membrane (partially degraded polygalactosamine), or the hydrolyzate was separated by ion exchange chromatography. You may use the galactosamino oligosaccharide obtained by taking it, or those hydrochlorides.

The method for preparing and fractionating partially decomposed polygalactosamine will be described.
N hydrochloric acid and polygalactosamine may be reacted at 60-100 ° C for 6-24 hours. On the other hand, hydrolysis with polygalactosamine degrading enzyme is performed by adding polygalactosamine degrading enzyme to a polygalactosamine solution at 35 to 40 ° C.
You can react with. Examples of the polygalactosamine degrading enzyme include Pseudomonas sp H881 (Japanese Patent Publication No.
-8757, FERM P-8955) -derived enzyme can be used.

The polygalactosamine thus hydrolyzed by an acid or an enzyme can be fractionated by an ultrafiltration membrane or the like to be divided into various fractionated molecular weight units. For example, the partially hydrolyzed product is subjected to molecular weight cut-off of 1,000
The molecular weight cutoff is 100,000 or more, 30,000 to 100,000, 1 or more by ultrafiltration with an ultrafiltration membrane of 00, 30000, 10000, 3000, etc.
0000-30000, 3000-10000, 300
It can be fractionated to 0 or less. These can be lyophilized to obtain partially decomposed polygalactosamine of each molecular weight.

Galactosamino oligosaccharides can be hydrolyzed by hydrolysis with an acid or a degrading enzyme in the same manner as in the case of obtaining the partially degraded polygalactosamine, and then each oligosaccharide can be obtained by cation exchange chromatography (special Kaihei 2-19392). Moreover, a commercially available product (manufactured by Funakoshi Co., Ltd.) can also be used.

Methylation of the thus obtained partially decomposed polygalactosamine or α-1,4 polygalactosamine or galactosamino oligosaccharide of each molecular weight cut off may be carried out by a conventional method, for example, Domard. Law (I
nt. J. Biol. Macromo1. , 8 , 105
(1986)) or the Muzzarelli method (Ca
rbohydr. Polym. , 5 , 297 (198
5)). For example, these are dispersed in N-methyl-2-pyrrolidone and 6-
After standing for 24 hours, 1.0-2.0 M NaOH is added and methyl iodide (CH ₃ I) and sodium iodide (NaI) are added to effect methylation. Then, 4-fold amount of ethanol is added, and the precipitated precipitate fraction is collected by centrifugation. The recovered precipitate fraction is dissolved in water, passed through an ion exchange resin column, and the column-passed fraction is lyophilized to obtain a methylated galactosamine polymer in a high yield.

To selectively methylate only amino groups, for example, α-1,4 polygalactosamine, partially decomposed polygalactosamine or galactosamino oligosaccharide is suspended in water and acetic acid is added to dissolve it. 20%-
Add 40% formaldehyde and react at room temperature for 10-30 hours. Next, sodium borohydride is gradually added, and then the pH is adjusted to 9.0 with NaOH and dispersed in water. The precipitate fraction obtained by centrifugation is washed with acetone, acetone is added to the solution fraction, and the precipitated precipitate is collected by centrifugation. These were dried under reduced pressure, and the obtained powder was dried at 60 ° C to 80 ° C for 10 to 20 hours, dispersed in acetonitrile and CH ₃ I solution, stirred and reacted for 20 to 40 hours, and extracted with diethyl ether using a Soxhlet extractor. The galactosamine polymer N-methylated product can be obtained by performing, concentrating and drying.

In the preparation of the galactosamine polymer methylated product by the above-mentioned Domard's method, the amino group and the hydroxyl group of the galactosamine residue are randomly methylated.
While the galactosamine polymer methylated product can be obtained in a very good yield, the galactosamine polymer N-methylated product prepared by the Muzzarelli method has a slightly inferior yield as compared with the former, but selectively has amino groups. It has a feature that a methyl group can be introduced into, and this method can be advantageously used.

The galactosamine polymer methylated product and the galactosamine polymer N-methylated product thus obtained are novel substances having the structures of the following chemical formulas 3 and 4.

[0014]

[Chemical 3]

[0015]

[Chemical 4]

The various methylated galactosamine polymers thus obtained have improved antibacterial properties, and are useful as an active ingredient of the antibacterial agent of the present invention. Also, when used, each may be used alone,
You may mix and use several kinds of them. The present invention is a novel substance, that is, a methylated α-1,4 polygalactosamine, a methylated partially decomposed polygalactosamine, a methylated galactosaminooligosaccharide, and an antibacterial agent containing them as an active ingredient. In the present invention, the active ingredient can be used as an additive as it is, or can be used as an antibacterial agent by formulating into various liquids or powders, or as a raw material or an intermediate thereof.

Next, production examples and examples of the present invention will be shown.

[0018]

[Production Example 1. Production of α-1,4 polygalactosamine] Glucose 600 g, polypeptone 60 g, CaCl ₂ ·
125 g of 2H ₂ O was dissolved in 17 liters of tap water, and concentrated NaO
The solution was adjusted to pH 7.0 with H solution and transferred to a 30 l jar fermenter.

By injecting steam into this medium solution, pressurization and heat sterilization (121 ° C., 20 minutes) were performed. The cooled medium (final liquid volume 20 l) was shaken in a 500 ml Erlenmeyer flask with 150 ml medium of the same composition (glucose 3%, polypeptone 0.3%, CaCl ₂ 0.5%, pH 7.0) at 26 ° C. for 4 days. Cultured Pesylomyces I-1
(FERM BP-1180) was aseptically inoculated at a volume ratio of about 10%. After inoculation, the cells were cultivated for 5 days under the conditions of 27 ° C., aeration rate of 0.5 VVM, and stirring number of 200 RPM.

After the completion of the culture, the culture was filtered through a filter cloth to obtain 17 l of culture filtrate. This culture filtrate is heated to 50 ° C to 60 ° C.
By passing through an ultrafiltration membrane (UF membrane tubular module F type manufactured by Mitsubishi Rayon Engineering Co., Ltd.) having a molecular weight cutoff of 160,000 while heating to ℃, the low molecular weight fraction is removed until the liquid volume is approximately 3 liters. did. Furthermore,
By centrifuging at about 14,000 × G, cell residue,
The heat-denatured protein was removed.

After centrifugation, about 75 ml of salt was added to 3 l of the supernatant fraction.
0 g (about 25% concentration) was added and stirred, and after dissolution, concentrated NaO
The pH was adjusted to 7.0-8.5 with H. After allowing to stand overnight to sufficiently deposit the salted-out product, the salted-out product was recovered on a cloth made of Saran (copolymer of vinylidene chloride and vinyl chloride). Furthermore, a large amount of slightly alkaline water (pH
(7.0 or more) was removed to wash away excess salt and neutral sugars and other contaminants simultaneously mixed in the culture solution.

Next, the salted out product after washing with water was dissolved by adding a 0.1 M hydrochloric acid solution in an amount of about 3 times by volume. Concentrated Na in this lysate
Addition of OH solution and pH, which is the isoelectric point of polygalactosamine
It was set to 8.5. After allowing to stand overnight to sufficiently deposit the precipitate, the precipitate was recovered on a Saran cloth as in the above, and washed with a large amount of tap water. This washed product was again dissolved in 0.1 M hydrochloric acid, and then subjected to isoelectric point precipitation and repeated washing with water for purification. The purified precipitate was sterilized at 121 ° C. for 15 minutes and then freeze-dried to obtain 7 g of purified powder of α-1,4 polygalactosamine.

[0023]

[Production Example 2. Preparation of partially decomposed polygalactosamine] 100 g of purified α-1,4 polygalactosamine was added to 4N hydrochloric acid 2
in an Erlenmeyer flask equipped with a cooling tube.
Hydrochloric acid was hydrolyzed at 6 ° C for 6 hours.

After decomposition, this hydrochloric acid hydrolyzed solution was neutralized to pH 7 with 10N NaOH. This solution was first treated with an ultrafiltration membrane having a molecular weight cutoff of 100,000 (manufactured by Grace Japan) to obtain a concentrated fraction (molecular weight cutoff of 100,000 or more).
And a filtration fraction (molecular weight cutoff of 100,000 or less), and then this filtration fraction is similarly ultrafiltered with an ultrafiltration membrane (Grease Japan) having a molecular weight cutoff of 30,000 to obtain a concentrated fraction. Min (molecular weight cut-off 30,000-10000)
0) and the filtered fraction (molecular weight cutoff of 30,000 or less). Further, this filtration fraction was subjected to ultrafiltration in the same manner using an ultrafiltration membrane having a molecular weight cutoff of 10,000 (manufactured by Grace Japan) to obtain a concentrated fraction (molecular weight cutoff of 10,000-3000).
0) and a filtered fraction (molecular weight cutoff of 10,000 or less), and then the filtered fraction was filtered through an ultrafiltration membrane (made by Grace Japan) having a molecular weight cutoff of 3000 to obtain a concentrated fraction (molecular weight cutoff). 3000-10000) and filtered fraction (fraction molecular weight 3
000 or less). Each of these was desalted with Micro Acylyzer G3 (manufactured by Asahi Kasei Co., Ltd.), freeze-dried, and molecular weight cut-off by an ultrafiltration membrane was 100,000.
Above, 30,000-100,000, 10,000-300
A partially decomposed polygalactosamine of 00, 3000-10000, 3000 or less was obtained.

[0025]

Example 1. Methylation of galactosamine polymer]

Preparation of TM2-PG (methylated α-1,4 polygalactosamine) 10 g of α-1,4 polygalactosamine was added to 500 ml of N 2.
Dispersed in -methyl-2-pyrrolidone and left at room temperature for 12 hours. To this, 86 ml of 1.4 M NaOH solution was added, then 128 g of methyl iodide (CH ₃ I) and 18 g of sodium iodide (NaI) were added, and the mixture was shaken at 36 ° C. for 3 hours. Then, 4-fold amount of ethanol was added, and the precipitated fractions were collected by centrifugation, washed with ethanol, and then dissolved in water to dissolve in an anion exchange resin column (DAIAION WA
I went to 10). The fraction passed through the column was freeze-dried to obtain 9 g of TM-PG (α-1,4 polygalactosamine methylated product). The same operation was repeated to further increase the amount of introduced methyl groups, to obtain 8.1 g of TM2-PG.
The elemental analysis value C: H: N = 38.2: 6.78: 4.
58, an average of 3.74 per galactosamine residue
It was presumed that individual methyl groups were introduced into the amino group and the hydroxyl group.

Preparation of TM3-PG (methylated α-1,4 polygalactosamine) 10 g of TM2-PG obtained in Example 1-was methylated again by the same method as in Example 1-
9 g of TM3-PG was obtained. Elemental analysis value C: H: N = 4
Galactosamine residue 1 from 1.3: 6.95: 4.3
An average of 5.2 (3 for the amino group at the 2-position, 3, 6
It was presumed that one methyl group was introduced into each hydroxyl group at each position).

TM2-3000 (fraction molecular weight 3000
-10,000 Polygalactosamine partial degradation product methylated product) The fractionated molecular weight 3000-10000 polygalactosamine partial degradation product 10 g obtained in Production Example 2 was methylated by the same method as in Example 1-7.2 g of TM2-3000. Obtained.
From the elemental analysis values C: H: N = 37.9: 6.85: 4.5, it was estimated that an average of 3.9 methyl groups was introduced into the amino group and the hydroxyl group per galactosamine residue. ..

TM3-3000 (cut off molecular weight 3000
-10,000 Polygalactosamine partial decomposition product methylated product) TM2-3000 (10 g) obtained in Example 1-was methylated again by the same method as in Example 1-to give 7.2 g of TM3-3000 was obtained. From elemental analysis values C: H: N = 41.4: 7.59: 4.33, an average of 5.2 per galactosamine residue (3 in the amino group at the 2-position, hydroxyl groups at the 3 and 6-positions) It was presumed that one methyl group was introduced.

[0030]

Example 2. N-methylation of galactosamine polymer]

Preparation of N-M3-PG (α-1,4 Polygalactosamine N-Trimethylated Compound) 10 g of α-1,4 polygalactosamine was suspended in 400 ml of water and 10 ml of acetic acid was added to dissolve it. %
100 ml of formaldehyde of the above is added and reacted at room temperature for 20 hours. Next, 2.6 g of sodium borohydride was gradually added, and then the pH was adjusted to 9.0 with NaOH, and water 1
Disperse in 500 ml. This was centrifuged, the precipitate fraction was washed with acetone, acetone was added to the solution fraction, and the precipitate precipitated by centrifugation was recovered. These were dried under reduced pressure, pulverized in a mortar and then dried at 80 ° C. for 17 hours. After drying, it is dispersed in 100 ml of acetonitrile and 2.5 ml of CH _{3 I} , reacted with stirring at 36 ° C. for 30 hours, extracted with diethyl ether using a Soxhlet extractor, concentrated and dried to give 4.85 g of N-M3-PG. Obtained. The elemental analysis value C: H: N = 53.3: 9.52: 6.74
From the above, it was presumed that an average of 3.2 methyl groups per galactosamine residue was an N-trimethyl compound introduced into an amino group.

N-M3-3000 (molecular weight cutoff 300
Preparation of 0-10000 polygalactosamine partially decomposed product N-trimethylated product) 10 g of the fractionated molecular weight 3000-10000 polygalactosamine partially decomposed product obtained in Production Example 2 was N-trimethylated in the same manner as in Example 2 to 4.27 g. N-M3-30
I got 00. The elemental analysis value C: H: N = 52.9:
From 9.47: 6.72, N- in which an average of 3.2 methyl groups per galactosamine residue was introduced into an amino group.
It was presumed to be a trimethylated product.

[0033]

Example 3. Antibacterial Action of Various Methylated Galactosamine Polymers TM2-PG, TM3-PG obtained in Example 2
TM2-3000, TM3-3000, N-M3-PG
The antibacterial effect of N-M3-3000 is
eller Hinton Broth (pH 7.0)
After culturing for 1 day at 30 ° C. (manufactured by Difco), each galactosamine polymer methylated product was brought to a final concentration of 10
Mueller Hinton Br added to 0, 50, 25, 12.5, 6.25, 3 μg / ml
Oth (pH 7.0) was inoculated, and the growth of the bacteria after culturing at 30 ° C. for 1 day was measured at OD660 nm to determine the minimum inhibitory concentration (MIC). The results are shown in Table 1 below.

[0034]

[Table 1]

[0035]

The present invention provides a galactosamine polymer (N-) methyl derivative. This derivative is a novel compound which has not been heretofore known, has improved physical properties, and has significantly increased antibacterial activity. Therefore, it can be used as an excellent antibacterial agent.

Claims (3)

[Claims] 1. A methylated galactosamine polymer having a chemical formula shown in the following chemical formula 1. [Chemical 1] 2. A galactosamine polymer N-methylated product having the chemical formula shown below. [Chemical 2] 3. An antibacterial agent comprising the methylated galactosamine polymer according to claim 1 and / or 2 as an active ingredient.