JP3231103B2 - Method for treating pyridyl aminated derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for easily qualifying and quantifying saccharides such as monosaccharides or oligosaccharides, and more particularly to monosaccharides or oligosaccharides utilizing a pyridyl amination reaction. The present invention relates to a method for analyzing saccharides, which enables simple and quick qualitative and quantitative determination of saccharides.

[0002]

2. Description of the Related Art Conventionally, saccharides such as monosaccharides or oligosaccharides or mixtures thereof have been easily and quantitatively determined by directly qualifying and quantifying each component using a chromatograph. After being converted into various suitable sugar derivatives, qualitatively, it is roughly divided into methods for quantification, and the qualitative and quantitative methods are, for example, those using various spectroscopic instruments or the like, or gas chromatographs or liquid chromatographs. The thing is mentioned as a typical thing.

[0003] Among these methods, qualitative and quantitative methods after conversion into sugar derivatives include trimethylsilylation method, dansylation method, phenylthiocarbamyl derivatization method, tritium labeling method, methylated derivative method, and acetyl acetylation method. Derivative method, pyridyl aminated derivative method, etc., depending on the characteristics of each derivative, methods using various spectroscopic instruments such as ultraviolet absorption analysis, fluorescence analysis, and methods using gas chromatography or liquid chromatography are used. .

Among them, the pyridyl aminated derivative method is
In recent years, this method has been researched and developed by Hase et al. [A
analytical biochemistry, 14
5,245-250 (1985)], and has been widely used in the past because of its excellent features such as simplicity of the derivatization reaction and high separation accuracy by reverse-layer chromatography.

In the method using the pyridyl aminated derivative, salt compounds and unreacted excess reagent by-produced in the pyridyl aminated step remain in the reaction mixture and hinder the subsequent qualitative and quantitative steps. It is desirable to carry out a pre-treatment for removing the.

For this purpose, Kondo et al. Have proposed a pretreatment method using a sublimation method [Agric. Bio
l. Chem. , 54.2169-2170 (199).
0)], ion exchange, gel filtration chromatography,
A purification method by a liquid-liquid distribution method or the like has been proposed.
However, all of these methods are complicated in operation, and are not always satisfactory in terms of simplicity and processing of a large amount of samples, and in terms of recovery rate, influence on subsequent analysis accuracy, and the like. There was a problem.

[0007]

In view of such circumstances, the present inventors aimed at overcoming these problems in the conventional method for analyzing saccharides using a pyridylaminated derivative to prepare the pyridylamination reaction mixture. As a result of accumulating various studies on the treatment method, the reaction mixture obtained in the pyridyl amination reaction step of the analyte, qualitative, by performing the treatment by electrodialysis using an ion exchange membrane as a pretreatment of the quantitative step, It can efficiently remove by-product salts and unreacted excess reagents, and has little effect on pyridylaminated derivatives of saccharides. The inventors have found that the present invention can be obtained, and have completed the present invention.

Although the electrodialysis method is conventionally known to be effective for desalting an aqueous solution containing amino acids, nucleic acid components, amino sugars, acidic sugars and the like, the pyridyl amination of saccharides according to the present invention is effective. Derivatives, or in relation to the analysis of saccharides using the pyridyl aminated derivative,
There have been no examples examined so far, and no research reports or the like have been reported.

Accordingly, the present invention provides a simple pretreatment method for a pyridyl amination reaction mixture when analyzing a saccharide such as a monosaccharide or an oligosaccharide or a mixture thereof using a pyridylaminated derivative. Is what you do.

Further, the present invention provides a method for subjecting a monosaccharide or an oligosaccharide or a mixture thereof to a pyridylamination reaction, wherein the corresponding pyridylaminated derivative is subjected to a qualitative or quantitative or fractionation step. It is an object of the present invention to provide a method for easily pretreating a reaction mixture of a corresponding pyridylaminated derivative obtained by subjecting the mixture to a reaction for the reaction.

[0011] The present invention also provides a method for analyzing saccharides, which enables simple and quick qualitative and quantitative determination of saccharides such as monosaccharides or oligosaccharides using the pyridylaminated derivative. It is the purpose.

[0012]

The structure of the present invention for achieving the above object comprises the following technical means (1) and (2). (1) Subjecting a monosaccharide or oligosaccharide or a mixture thereof to a pyridyl amination reaction and subjecting the obtained corresponding pyridylaminated derivative to a qualitative or quantitative or preparative step, to the pyridylamination reaction A method for treating a pyridyl aminated derivative, comprising subjecting a reaction mixture of the corresponding pyridyl aminated derivative obtained by the above to a pretreatment by an electrodialysis method.

(2) When the monosaccharide or oligosaccharide or a mixture thereof is subjected to a pyridylamination reaction, and the obtained corresponding pyridylaminated derivative is subjected to a qualitative or quantitative or fractionation step, the pyridylamination reaction is carried out. A method for analyzing saccharides, comprising subjecting a reaction mixture of a corresponding pyridylaminated derivative obtained by applying the same to a pretreatment by an electrodialysis method.

Next, the method of the present invention will be described in more detail.

The treatment method according to the present invention can be applied without limitation to the analysis of saccharides from which the reducing end has been released, to which the pyridyl amination method was conventionally applicable. As the oligosaccharide or a mixture thereof, specifically, for example, glucose, pyranose such as galactose, furanose such as fructose, glucosamine, amino sugar such as galactosamine, and the like,
Lactose, disaccharides such as maltose, various oligosaccharides,
Furthermore, sugar chains of glycolipids, sugars of glycoproteins, polysaccharides on bacterial cell walls, and the like can be mentioned.

In the treatment method according to the present invention, first, a monosaccharide or an oligosaccharide to be analyzed or a mixture thereof is subjected to a pyridyl amination reaction. The step of the pyridyl amination reaction is performed by a conventionally known method, and a method performed under aqueous solution conditions and a method performed under anhydrous conditions are generally used, and any of these methods can be used. When the reaction is performed in an aqueous solution, for example, a hydrochloric acid solution of 2-aminopyridine is added to a trace amount of sample, heated, and then reduced by adding sodium borohydride.

When the reaction is carried out under anhydrous conditions, for example, if the saccharide mixture to be analyzed is in a powder or solid form, it is used as it is. After concentrating to dryness, an acetic acid solution of 2-aminopyridine was added as a pyridyl amination reagent,
A pyridylaminated derivative can be quantitatively obtained by heating and reacting and then performing a reduction reaction.

The reaction mixture of the pyridylaminated derivative of oligosaccharide thus obtained is used together with the pyridylaminated derivative of a saccharide for analysis or the like, together with salts produced as a result of the reaction, unreacted saccharides, and unreacted excess reagent. Is contained in a large amount, so that qualitative and quantitative determinations are impossible or extremely inaccurate.

Therefore, in order to remove the by-produced salts and unreacted excess reagent, the pyridyl amination reaction mixture is used in the present invention as electrodialysis as a pretreatment for qualitative and quantitative determination of saccharides. Apply to processing step.

The electrodialysis method used in the present invention is:
Basically, using an apparatus comprising three tanks of a positive electrode tank, a processing solution passage tank partitioned via an anion exchange membrane, and a negative electrode tank further partitioned via a cation exchange membrane. Do.

That is, according to the present invention, in the electrodialysis treatment step, the positive electrode tank and the negative electrode tank are filled with purified water or an appropriate electrolytic solution, and an appropriate direct current is applied between the two electrodes. The aqueous solution of the reaction mixture to be treated is passed through.

At this time, by appropriately selecting the passage pore size of the anion exchange membrane and the cation exchange membrane, only the low molecular weight electrolyte in the treatment solution is removed through the exchange membrane, and the pyridylaminated derivative of the saccharide is removed. It remains in the treatment liquid as it is, and as a result, desalting is achieved. The ion exchange membrane suitable in the present invention is a cation exchange membrane having a molecular weight cut off of about 100 and an anion exchange membrane having a molecular weight cut off of about 300.

As for the passage of the treatment solution, an appropriate infusion system may be combined, but by using a recyclable system, the degree of desalination can be easily controlled. Also, by combining an appropriate sensor for measuring the salt concentration in the treatment solution, it is possible to easily know the degree of desalination.

The electrodialysis device for implementing the above basic configuration is not particularly limited as long as it meets the purpose of the present application, and as the device, for example, a commercially available micro-acylizer (G -1 type, manufactured by Asahi Kasei Kogyo Co., Ltd.). The cartridge for the electrodialysis part of the apparatus is AC-120-10.
Or, one having the same performance as this is preferable.

By subjecting the reaction mixture of the pyridyl amination reaction to a pretreatment step by using an appropriate electrodialysis apparatus for carrying out the above basic constitution, it becomes difficult to perform subsequent analysis operations. By-products and excess reagents can be reliably removed, and a relatively large amount of a pyridylaminated derivative can be easily prepared and fractionated, which is sufficient to enable various instrumental analysis thereafter. It is possible to supply a sample volume.

Conventionally, when a reaction mixture of a pyridylaminated derivative is subjected to desalting treatment by gel filtration and then purified, it takes about two days for fractionation by gel filtration and concentration, desalting, drying and the like of the fractionated solution. Although required, the preparation of the pyridylaminated derivative can be performed in about 4 hours by performing the pretreatment by the electrodialysis method of the present invention. On the other hand, the pretreatment step hardly affects the pyridylaminated derivative of the saccharide to be analyzed, and as a result, highly accurate analysis can be performed.

[0026]

The present invention will be described more specifically below with reference to examples.

A pyridyl amination reagent (PA) was added to the following sample 1 and sample 2 saccharide samples (in the case of an aqueous solution, after concentrating to dryness ) for the purpose of analyzing the saccharide sample for the pyridyl amination reaction (general method). 2.2 g of 2-aminopyridine dissolved in 1.3 ml of acetic acid)
Is added (2- to 1000-fold molar amount), and the mixture is heated and reacted at 90 ° C. for 60 minutes. Then, an acetic acid solution of a reducing agent (390 mg of borane-dimethylamine complex in 2 ml of acetic acid) prepared at the time of use was added in a 25- to 500-fold molar amount,
The reaction was carried out at 80 ° C. for 50 minutes to prepare a reaction mixture of a pyridylaminated derivative. Sample 1 (standard); Molar mixture of glucose, maltose, maltotriose, maltotetraose, maltopentaose, etc. Sample 2: Galactooligosaccharide mixture (Oligomate 50, manufactured by Yakult Honsha)

Pretreatment by electrodialysis (method of the present invention) For pretreatment by electrodialysis, a micro-acyler (G-1 type, manufactured by Asahi Kasei Kogyo) was used as an apparatus. The ion exchange membrane used was a cationic dialysis membrane (AC-120-10) having a molecular weight cut off of 100. Also, the anode tank,
A 0.5% Na nitrate solution or distilled water was circulated as an electrode solution in the cathode tank.

First, the above-mentioned pyridyl amination reaction was carried out on a scale of 0.1 to 100 μmol, and after the reaction, distilled water was added to dilute the mixture to a precise 6 ml, followed by electrodialysis.
Using the change in current flowing between the electrodes as an index,
The sample solution was circulated until it reached 2A or 0.00A. Furthermore, when performing the thorough treatment, a pre-treatment operation was further performed for 10 minutes or 20 minutes after the pressure reached 0.00A.

A desalination treatment by gel chromatography (conventional method) A reaction mixture of the pyridylaminated derivative reacted in the same manner was dissolved in 2 ml of distilled water, and the mixture was dissolved in biogel P2.
A desalting operation was performed using a column (16 × 1000 mm, manufactured by Bio-Rad). Comparison with the sample subjected to the pretreatment operation by the electrodialysis method described above showed no loss of the sample and no problem in the quantitative property.

Next, the obtained sample was analyzed and quantified using the following reversed-phase high-performance liquid chromatography and a solvent. Apparatus: High-performance liquid chromatography (600E, manufactured by Waters) Column: ODS STR-H300 (manufactured by Shimadzu Techno Research) Solvent: 0.2 M citrate buffer (pH 4.5)

The analysis results for the standard sample and each sample are shown below. 1. Examination of pretreatment conditions by electrodialysis method (1) Sample: maltopentaose When the above sample was used and the current between the electrodes was set to 0.02 A and 0.00 A, and thorough pretreatment was performed for 10 minutes.
And for 20 minutes, the pyridylaminated derivative was quantified. Table 1 shows the results.

[0033]

[Table 1]

As is clear from the results in Table 1, the recovery of the pyridylaminated derivative was found to be good.

[0035] 2. Examination of pretreatment conditions by electrodialysis (2) Sample: Equivalent mixture of glucose, maltose, maltotriose, maltotetraose, and maltopentaose. Using the above sample, the amount of the pyridylaminated derivative was determined when the interelectrode current was set to 0.02 A and 0.00 A and when the thorough pretreatment was performed for 20 minutes. Table 2 shows the results.

[0036]

[Table 2]

As is evident from the results in Table 2, when the monosaccharide (glucose) was thoroughly pretreated for 20 minutes, a decrease in the recovery rate of the sample was observed, but in other cases, there was no problem. .

3. Confirmation of pretreatment effect by electrodialysis method Sample: Galactooligosaccharide mixture (oligomate 5
0, manufactured by Yakult Honsha) Using the above sample, the pyridylaminated derivative was quantified when the interelectrode current was set to 0.02 A and 0.00A, and when thorough pretreatment was performed for 30 minutes. On the other hand, as a control, pyridylaminated derivatives were quantified for those desalted by gel filtration. The result is shown in FIG.

In FIG. 1, a) shows the case of the pretreatment in which the interelectrode current by the electrodialysis method is set to 0.02 A, and b) shows the interelectrode current by the electrodialysis method of 0.00 A.
, C) shows the case of thorough pretreatment by electrodialysis for 30 minutes, and d) shows the case of a desalted sample by gel filtration. As is clear from FIG. 1, when thorough pretreatment was performed by the electrodialysis method, 2
It was found that the peak of AP (2-aminopyridine) was efficiently removed by the electrodialysis method.

[0040] 4. Relationship between the molar ratio of the pyridyl amination reagent and the quantification result Sample; Galactooligosaccharide mixture (oligomate 5
0, manufactured by Yakult Honsha Co., Ltd.)
Table 3 shows the analysis results when the pyridyl amination reaction was carried out using 50 times and 500 times, and the reaction mixture of the pyridyl aminated derivative was pretreated by the electrodialysis method.

[0041]

[Table 3]

As is evident from Table 3, when the pretreatment was performed by the electrodialysis method, the molar ratio of the pyridyl amination reagent hardly affected the quantitative results.

[0043]

As described in detail above, the pretreatment method by the electrodialysis method of the present invention has a specific action and effect not found in the conventional desalting treatment by the gel filtration method and the like.
According to the method of the present invention, the pretreatment of the pyridylaminated derivative, which was conventionally required for at least about 2 days, can be performed in about 4 hours, and the time required for qualitative and quantitative analysis of saccharides utilizing the pyridylamination reaction Can be greatly reduced. Further, since the method of the present invention can be applied to a relatively large amount of sample, a sample of unknown structure is also subjected to a sample separation step,
It is possible to supply a sufficient amount of sample to enable subsequent instrumental analysis.

[Brief description of the drawings]

FIG. 1 shows the results of analysis and quantification of pyridylaminated derivatives by reversed-layer high-performance liquid chromatography.

──────────────────────────────────────────────────続 き Continued on the front page (56) References Agric. Biol. Chem. , Vol. 54 (1990) p. 2169-2170 (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 30/00-30/96 CA (STN)

Claims (2)

(57) [Claims] 1. A monosaccharide or oligosaccharide or a mixture thereof is subjected to a pyridylamination reaction, and the corresponding pyridylaminated derivative is subjected to a qualitative or quantitative or preparative step. A method for treating a pyridyl aminated derivative, comprising subjecting a reaction mixture of the corresponding pyridyl aminated derivative obtained by the above process to a pretreatment by an electrodialysis method. 2. When the monosaccharide or oligosaccharide or a mixture thereof is subjected to a pyridyl amination reaction and the corresponding pyridyl aminated derivative obtained is subjected to a qualitative or quantitative or preparative step, the pyridyl amination reaction is carried out. A method for analyzing saccharides, comprising subjecting a reaction mixture of a corresponding pyridylaminated derivative obtained as described above to a pretreatment by an electrodialysis method.