JPH0867695A - Glycate poly-l-lysine obtained from polylysine and methyl glucose - Google Patents

Abstract

PURPOSE: To obtain a new glycate poly-L-lysine having active oxygen forming ability and cleaving action on nucleic acid, by reacting ε-poly-L-lysine or α-poly- L-lysine with 3-O-methyl-D-glucose under physiological conditions. CONSTITUTION: An ε-poly-L-lysine of formula I ((n) is degree of polymerization) or α-poly-L-lysine of formula II is reacted with 3-O-methyl-D-glucose of formula III under physiological conditions to give a new glycate poly-L-lysine having active oxygen forming ability and cleaving action on nucleic acid. The glycate poly-L-lysine is obtained by incubating ε-poly-L-lysine or α-poly-L-lysine produced by Streptomyces albulus subsp. lysinopolymerus No.436-D strain (FERM P-3,834) with 3-O-methyl-D-glucose in a phosphoric acid buffer solution at 37 deg.C for 7 days.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to ε-poly-L-lysine or α
-Poly-L-lysine and glycan poly-L-lysine obtained by reacting 3-O-methyl-D-glucose under physiological conditions.

[0002]

2. Description of the Related Art The glycation reaction of proteins is known as one of the aminocarbonyl reactions of food ingredients and as a typical non-enzymatic reaction of proteins at the molecular level. In the glycation reaction, a reducing sugar reacts with an amino group of a protein under physiological conditions to generate an Amadori rearrangement product (1-substituted amino-2-ketose) via glucosylamine, which further undergoes oxidative decomposition, polymerization, etc. Dyes, fluorescent substances, dimers, trimers, low-molecular peptides and the like are produced through complicated reactions.

[0003]

Polylysine is a polypeptide in which L-lysine is linearly linked. Polylysine is considered as a kind of polyamine, and its reaction and basic research have been conducted as a food preservative and a bioactive amine.
The present inventor has taken polylysine as a model of protein, and discovered this substance as a novel product in the process of reacting polylysine and various reducing sugars under physiological conditions and examining the product. As is clear from the above description, an object of the present invention is to provide a novel polymer compound, glycate poly-L-lysine.

[0004]

The present invention has the following constitution (1). (1) General formula (I)

[Chemical 4] Or general formula (II)

[Chemical 5] A polymer represented by the general formula (III)

[Chemical 6] Glycate poly-L-lysine obtained by reacting the monomer represented by

The structure and effect of the present invention will be described in detail below. The polylysine according to the present invention has a structure in which lysine, which is an amino acid having two amino groups in one molecule, is condensed, and in general, α-polylysine and ε-in which an amino group at the α-position of lysine is condensed with a carboxyl group. There are two types of ε-polylysine in which the amino group at the position and the carboxyl group are condensed.

Ε-Polylysine is an essential amino acid L-
It is a polypeptide in which the amino group at the ε-position of lysine is condensed, and is strictly called ε-poly-L-lysine. The ε-polylysine used as the reaction raw material according to the present invention is Streptomyces albulus (St
oreptomyces arbulus or Streptomyces noorusii
s noursei) is a substance produced, and is a polylysine in which 25 to 30 residues of L-lysine are ε-bonded.

This ε-polylysine is, for example, Japanese Patent Publication No. 59-
As described in Japanese Patent No. 20359, Streptomyces arbus subspecies lisinopolymeris (Streptomyces arbulus).
subspecies lysinopolymer
us) No. Strain 346-D (Ministry of Microbiology Research No. 3834)
Can be obtained by a method of culturing in a medium and separating and purifying from the obtained culture.

Α-Polylysine is an essential amino acid L-
It is a polypeptide in which the amino group at the α-position of lysine is condensed, and is strictly called α-poly-L-lysine. The α-polylysine used in the present invention can be easily obtained by a usual chemical synthesis method, but can also be easily obtained as a commercial product (product of Merck & Co., Inc.).

The 3-O-methyl-D-glucose used in the present invention can be easily obtained by a conventional chemical synthesis, but it can be obtained from Aldrich Chemical Co. It is also possible to use the reagents sold by.

The glycation reaction of polylysine is carried out as follows. (Ε-, α-) polylysine 10 mg and 0.
1M 3-O-methyl-D-glucose at 0.1M, p
It was dissolved in 1.0 ml of H7.2 phosphate buffer. After adding 0.1% gentamicin as a preservative, degassing and nitrogen substitution, incubate at 37 ° C. for 7 days. The water used for preparing the buffer solution and gel filtration was Fent.
In order to prevent generation of hydroxyl radicals due to the on reaction, it is desirable to use ultrapure water that has been degassed and replaced with nitrogen.

The 3-O-methylglycate polylysine obtained by the above method is an optically active substance and has a specific optical rotation. However, it has no unique melting point.

3-O-methyl glycate polylysine has the ability to generate active oxygen. When cytochrome C receives one electron, it changes to a reduced form with strong absorption at 550 nm. This property can be used to investigate the ability of glycated polylysine to generate oxygen radicals.
-Mixing methyl glycate polylysine and cytocloque C increases the absorbance of the mixed solution at 550 nm over time. In addition, this substance has a nucleic acid cleaving action.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 TBA (Thiobarbituric Acid) reacts with 3-O-methyl-D-glycate ε-polylysine to form a yellow-colored substance having absorption at 443 nm. Used as a method for quantifying D-glycate ε-polylysine.

100 mg of ε-polylysine and 0.1 M of 3
-O-methyl-D-glucose 0.1M, pH 7.2
It was dissolved in 10.0 ml of the phosphate buffer solution. 0.1% gentamicin was added as a preservative, the mixture was degassed, replaced with nitrogen, and then incubated at 37 ° C for 7 days. As a control, 3-O-methyl-D-only with ε-polylysine
Those without glucose were incubated under similar conditions. The water used for preparing the buffer solution, gel filtration, etc. was degassed from ultrapure water and replaced with nitrogen in order to prevent generation of hydroxyl radicals by the Fenton reaction. 3-O-methyl-during incubation
When the production state of D-glycate ε-polylysine was measured by the TBA method, the results shown in FIG. 1 were obtained. As can be seen from FIG. 1, after 4 days, an increase in absorbance was not observed so much that glycation of ε-polylysine was 4
Peak on day one. As a control, TBA was prepared by incubating only ε-polylysine without adding 3-O-methyl-D-glucose.
No absorption was observed by law.

By this reaction, 3-O-methyl-D-
120 mg of glycate ε-polylysine was obtained.

The 3-O-methyl-D-glycate ε-polylysine obtained by this reaction was analyzed by JASCO DIP36.
The optical rotation measured with 0 (digital polarimeter)

[Chemical 7] Met.

As a result of measurement by Seiko Denshi SSC-5020 (differential scanning calorimeter), there was no melting point as shown in FIG.

For infrared analysis, JASCO FT / IR
As a result of measurement by −300 (KBr method), a chart as shown in FIG. 3 was obtained.

Example 2 Cytocloak C changes to a reduced form having strong absorption at 550 nm when receiving one electron. Although the rate of reduction reaction of cytochrome C by active oxygen is not fast, it is widely used as a method for detecting active oxygen because it is easy to measure and there is no chain reaction. Utilizing this property, the ability to generate glycated polylysine oxygen radicals was investigated. (Preparation of sample) 0.1 reaction solution obtained in Example 1
The ml was subjected to gel filtration to separate the target glycated polylysine and unreacted low molecular weight compounds such as sugars. That is, 0.1 ml of the reaction solution was collected, and PD-10 for desalting Se was used.
Gel filtration was performed using a phadex G-25M prepacked column. In this gel filtration, the solution is 0.5m
Glycated polylysine was not detected in the first 2.0 ml of the eluate, but it was discarded and the subsequent 1.5 ml was collected and used for the measurement of oxygen radical generation ability. (Measurement of reduction reaction rate of cytochrome C) 1.5 ml of the sample prepared by the above method was used for measurement of reduction reaction rate of cytochrome C. To 1.5 ml of the sample solution, 1.0 ml of a cytochrome C solution prepared with a phosphate buffer was added, and the increase in absorbance at 550 nm was measured over time. The result is shown in FIG. The reducing power, which shows the ability to generate radicals, is
It was expressed by the rate of increase in absorbance (Δ550 nm) per unit time. It was shown that the glycated polylysine produced oxygen radicals.

Example 3 100 mg of α-polylysine and 0.1 M of 3-O-methyl-D-glucose were dissolved in 10.0 ml of 0.1 M phosphate buffer of pH 7.2. Add 0.1% gentamicin as a preservative, degas, replace with nitrogen, then 37 ℃
And incubated for 7 days. As a control
-Polylysine alone without the addition of 3-O-methyl-D-glucose was incubated under similar conditions. The water used for preparing the buffer solution and gel filtration was Fent.
In order to prevent hydroxyl radicals from being generated by the on reaction, ultrapure water was deaerated and replaced with nitrogen. When the production state of 3-O-methyl-D-glycate α-polylysine during the incubation was measured by the TBA method, the results shown in FIG. 5 were obtained. As can be seen from FIG. 5, the increase in absorbance was not observed much after 4 days, so that the glycation of ε-polylysine reaches a peak on the 4th day. As a control, it was not desired that absorption was observed by the TBA method in the case of incubation with ε-polylysine alone without the addition of 3-O-methyl-D-glucose.

By this reaction, 3-O-methyl-D-
118 mg of glycated α-polylysine was obtained.

The 3-O-methyl-D-glycate α-polylysine obtained by this reaction was analyzed by JASCO DIP36.
The optical rotation measured with 0 (digital polarimeter)

Embedded image Met.

As a result of measurement by Seiko Denshi SSC-5020 (differential scanning calorimeter), no melting point was recognized as shown in FIG.

For infrared analysis, JASCO FT / IR
As a result of measurement by −300 (KBr method), a chart as shown in FIG. 7 was obtained.

Example 4 3-O-methyl-D-glycate ε-polylysine was added to a double-stranded φ × 175 DNA in a phosphate buffer (pH 7.2).
When 200 ng, sugar 1 nM, and Cu ²⁺ 10 μM were treated at 37 ° C. for 3 hours, single-strand single cleavage (FormII) and generation of linear DNA (FormII) were examined.
%; 84.3.

[Brief description of drawings]

FIG. 1 shows a glycation reaction of 3-O-methyl glucose and ε-polylysine.

FIG. 2 shows the melting point of 3-O-methylglycate-ε-polylysine.

FIG. 3 shows an infrared absorption chart of 3-O-methylglycate-ε-polylysine.

FIG. 4 shows changes in active oxygen-producing ability of 3-O-methylglycate-ε-polylysine over time.

FIG. 5 shows a glycation reaction of 3-O-methyl glucose and α-polylysine.

FIG. 6 shows the melting point of 3-O-methylglycate-α-polylysine.

FIG. 7 shows an infrared absorption chart of 3-O-methylglycate-α-polylysine.

Claims (1)

[Claims] 1. A compound of the general formula (I) Alternatively, the compound represented by the general formula (II): And a polymer represented by the general formula (III): Glycate poly-L-lysine obtained by reacting the monomer represented by