JPS63243101A - Cyclodextrin derivative and coloring - Google Patents

Abstract

PURPOSE:To obtain a cyclodextrin derivative useful in the practical use side of test field of clinical chemistry, capable of effectively coloring nitrophenols under neutral or weakly acidic conditions, containing a basic functional group linked by covalent bond. CONSTITUTION:A cyclodextrin derivative containing a basic functional group linked by covalent bond. For example, a group (e.g. diethylaminoethyl group) shown by the formula (R1 and R2 are 1-3C alkyl or H; n is 1-3) is preferable as the basic functional group and is linked to alpha- or beta-cyclodextrin by covalent bond to give the aimed derivative. The derivative is used for measuring activity various enzymes such as phosphatase, phosphodiesterase by coloring nitrophenols liberated from a substrate. The cyclodextrin derivative of this invention includes nitrophenols and drastically promotes coloring in a visible part.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to cyclodextrin modified with a basic functional group, and is applied to measurement methods using a combined r&substrate, and is applicable to fields such as clinical chemistry tests. It is effectively used in Furthermore, the present invention relates to a reagent and method for coloring nitrophenols under neutral to weakly acidic conditions.

[Prior art] 4-nitrophenol (also called para-nitrophenol), a type of nitrophenol, is a substance produced when various synthetic substrates containing a 4-nitrophenol group are decomposed by the action of specific enzymes. For example, in order to measure the activity of phosphatase, the activity of the enzyme can be easily measured by quantifying the increase in this substance over time. Determine 4-ditrophenol. The activity of phosphosiesterase is measured in the same manner using bis(4-nitrophenyl) phosphate as a substrate. β-N
In the activity measurement of -7 cetylhexosaminidase and β-N-7 cetylglucosaminidase, 4-nitrophenyl N-7 cetyl-β-D-glucosaminid is used as one synthetic substrate. In addition, various glycosidases (enzymes that catalyze the hydrolysis of glycosidic bonds), phospholipase C
Synthetic substrates with 4-nitrophenyl groups have been developed for serine proteases such as trypsin and chymotrypsin, and D-glyceraldehyde-3-phosphate dehydrogenase, and have been put to practical use in various fields including clinical testing. has been done.

[Problem to be Solved by the Invention 1] The principle that makes it possible to measure the activity of various enzymes as shown in the examples above is that, for example, in the case of a synthetic substrate with a 4-nitrophenyl group, It is based on the fact that there are differences in the absorption spectra among 4-nitrophenols. In other words, a synthetic substrate with a 4-nitrophenyl group can be used in the ultraviolet region (wavelength 300 to 300 nm) in a wide pH range.
It has a strong absorption peak in the visible region (wavelength 40 nm).
4-nitrophenol is ionized in the alkaline pH region, and 4-nitrophenol shows almost no absorption in
The 4-nitropherulate anion has a strong absorption peak centered around 0 Ons. Therefore, if the activity of the enzyme to be measured can be measured in the alkaline pi region, the color development in the visible region as the enzymatic reaction progresses. can be continuously monitored as an increase in absorbance at 400 nm, allowing accurate rate assays. However, if the optimum pH of the enzyme to be measured is in the neutral or markedly slightly acidic region, the absorption spectrum of 4-nitrophenol will almost overlap with the absorption spectrum of the synthetic substrate with 4-nitrophenyl group. The color is weak and the color is weak.
Continuous monitoring of the reaction over time becomes extremely inconvenient. Therefore, in the current measurement method, after a certain period of time after starting the enzyme reaction, alkali is added to stop the reaction, and at the same time, the produced 4-nitrophenol is colored, and its absorbance at around 400 nm is measured. Many methods have been adopted. That is, with the current measurement method, not only is it difficult to perform an ideal rate assay, which is said to be a more accurate and simple format for enzyme activity measurement, but it also requires the extra operation of adding alkali.

Enzymes that have an optimum pH in the neutral to slightly acidic range include, for example, enzymes that are important in clinical chemistry, such as acid phosphatase and β-N-7 cetylglucosaminidase. If we could obtain a useful means to continuously monitor the reactions of these enzymes over time, we would not only be able to simplify the measurement operation and shorten the measurement time, but also improve measurement accuracy and enable early detection and diagnosis of various diseases. can provide accurate information.

Therefore, the development of reagents that effectively promote color development in the neutral to weakly acidic pH range of 4-nitrophenol and other nitrophenols has become an important practical issue in the field of clinical chemistry testing. ing.

[Means for Solving the Problems] The present invention is a cyclodextrin derivative to which a basic functional group is covalently bonded.

Furthermore, the present invention is a coloring method characterized by including a nitrophenol derivative using a cyclodextrin derivative to which a basic functional group is covalently bonded.

The present invention will be explained in detail below.

Aromatic compounds including 4-nitrophenol are
It is known that phenyl groups with hydrophobic properties are incorporated into cyclodextrins to form clathrates.

If this causes a subtle change in the ionic state of the aromatic ring, it is expected that ionization will be accelerated. Therefore, when α-cyclodextrin or β-cyclodextrin is added to 4-nitrophenol etc., ionization progresses and an increase in absorbance around 400n- is observed, but the effect is still satisfactory in the neutral to weakly acidic region. There are many problems in putting it into practical use, including the fact that it is not possible to make it, and its solubility is low.

As a result of intensive studies, the present inventors have determined whether or not covalently bonding a basic functional group to cyclodextrino would promote the ionization of phenolic hydroxyl groups, and whether this would promote color development of 4-nitrophenols. We synthesized a cyclodextrin derivative with a basic group fused to it to determine whether 4-nitrophenol and other nitrophenols would occur in the neutral to slightly acidic pH range.
When the effect of the compounds on the absorption spectrum was investigated, it was found that they had a remarkable coloring effect, and the present invention was achieved.

To describe the present invention in more detail, for the purpose of the present invention, a diethylaminoethyl group was first selected as a basic functional group, and a cyclodextrin conjugate thereof was synthesized according to the following principle.

A diethylaminoethyl conjugate of α-cyclodextrin is obtained in the same manner. In addition to diethylaminoethyl groups, conjugates of dimethylaminoethyl, dimethylaminopropyl, aminoethyl groups, etc. can also be obtained. Cyclodextrins encapsulated with basic functional groups, such as diethylaminoethylated α-cyclodextrin (hereinafter abbreviated as DEα) and diethylaminoethylated β-cyclodextrin (hereinafter abbreviated as DEβ), surprisingly have 1 Compared to ordinary cyclodextrin, it was much more soluble in water and easier to put into practical use. β-cyclodextrin, which is currently particularly inexpensive and commonly used, is relatively sparingly soluble in water, and this property determines its practical range in clinical chemistry tests and the like.

That is, when a poorly soluble target substance is dissolved by inclusion of β-cyclodextrin, its solubility is limited by the solubility of the cyclodextrin itself.

Specifically, this is the case when dissolving synthetic substrates that are often used to measure the activity of specific enzymes. In the case of hydrolytic enzymes, a poorly soluble nitrophenol derivative is often bound as a coloring agent to monitor the progress of enzyme reactions. , 4-nitrophenyl (al or 2-chloro-4-nitrophenyl)-N-acetyl-β-D-glucosaminide, attempts are made to dissolve the necessary amount with the help of β-cyclodextrin, but the cyclodextrin itself In comparison, DEα and DEβ according to the present invention cannot fully achieve their purpose due to poor solubility.
etc. can be easily dissolved in water, and their entrapping ability allows them to dissolve the necessary amount of the above-mentioned substrates, etc., so this aspect also adds practical value to the present invention.

Furthermore, the coloring effect of nitrophenol derivatives under neutral or weakly acidic conditions, which is the essence of the present invention, was investigated using various derivatives. 4-ditrophenol, 2-chloro-4-ditrophenol, 5-nitrosalicylic acid, 5-
0M for methyl nitrosalcylate, 2,4-dinitrophenol, etc. (Of these, 0M should be the most neutral or especially weakly ionized under weak acidity, i.e. p
In the vicinity of H5,0, even 4-nitrophenol, which does not show an ionizing visible color peak near 400 nm, has a large new ionization peak near 400 nm in the presence of a normal concentration of DEα or DEβ of 1% or less. It was shown that the action and effect of DEα and DEβ are remarkable.No ordinary cyclodextrin has such an effect. Also DEα, DE
Since β has good solubility in water, it can be effectively enhanced by increasing the amount used. Regarding other nitrophenols, 1
% or less, the visible region coloring effect was several times greater.

In particular, 2-chloro-4-ditrophenol is relatively more likely to develop color than 4-ditrophenol under neutral to weakly acidic conditions, and synthetic substrates for specific enzymes to which it is bound have recently been provided. However, for example, for the above-mentioned N-acetyl-β-D-glucosaminidase, etc., which performs an enzymatic reaction at a pH of 5.0 or less, as a result of the enzymatic reaction, the pH
400 of 2-chloro-4-ditrophenol produced in
The ionization color development in the vicinity of the n-layer is still completely insufficient, and does not have the sensitivity and precision to continuously monitor the amount of produced 2-chloro- Since the absorption spectrum of 4-ditrophenol appears in a nearly completely ionized form and is colored, continuous monitoring and measurement of the enzyme can be performed under ideal conditions.

As mentioned above, DEα, DEβ or DEβ is easily soluble in water, and together with the fact that the required amount of the synthetic acid substrate can be dissolved in the reaction solution, it provides a significant practical effect.

The above effects are similar to that of 4-ditrophenylmaltopentaoside or 2-chloro-4-nitrophenylmaltopentaoside, which is a synthetic substrate for α-amylase that reacts near neutrality. For α-amylase measurement, which continuously monitors nitrophenyl derivatives produced by using nitrophenyl derivatives at around 400 nm, or for acid phosphatase measurement, where the enzymatic reaction is carried out in a lower pH region. Even when synthetic substrates are used, they are used much more effectively than ordinary cyclodextrins.

The effects of the present invention have been described above for DEα and DEβ, but the present invention also applies when using cyclodextrins modified with other basic functional groups and other synthetic substrates bound with nitrophenol derivatives for other enzymatic reactions. The essence is the same.

[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 (Synthesis method of coloring reagent) 1.2-(N,N-dialkylaminoethyl)-α-cyclodextrin (hereinafter abbreviated as DEα): 2.4 g of α-cyclodextrin was added to 4 tan of distilled water. 4 containing 1.5 g of sodium hydroxide while suspending and stirring.
When the tan aqueous solution is added dropwise, a clear liquid is obtained. While continuing to stir, 2 ml of an aqueous solution containing 1.4 g of diethylaminoethyl chloride hydrochloride was added dropwise.
The reaction is completely terminated by continuing stirring at 30-40°C for several hours. From this reaction mixture, low molecular weight substances are removed by Sephadex G-25 column chromatography to obtain DEα of i and e g. Obtained DBα
It was found by neutralization titration and proton nuclear magnetic resonance that 1 to 2 diethylaminoethyl groups were covalently bonded.

2, '2-(N,N-dialkylaminoethyl)
-β-cyclodextrin (hereinafter abbreviated as DEβ) β-cyclodextrin instead of α-cyclodextrin in the above combination tl
- By using cyclodextrin, DEβ can be synthesized with a low recovery rate.

Example 2 Visible coloring effect of DEα on 4-nitrophenol and its application to enzymatic reactions.

FIG. 1 is a comparison of the absorption spectrum of 4-nitrophenol at p) 15.0 and the absorption spectrum when DEα was added to a concentration of 1% as a coloring reagent.

As is clear from this figure, 4-nitrophenol is 40
0n It has almost no absorbance near the intestine, but DE
A remarkable color development promotion effect was shown in a solution containing α, and 400n
The absorbance at m reaches 30% or more of the ultraviolet absorbance of 4-nitrophenol, making it fully measurable. On the other hand, the absorption spectrum of 4-nitrophenol N-7 cetyl-β-D-glucosaminide, which is one of the 4-nitrophenyl synthetic substrates, is slightly shifted to a lower wavelength than the spectrum of 4-nitrophenyl at pH 5.0. Even if DEα is added to this at a concentration of 1%, only a slight shift in the longer wavelength of the absorption peak is observed, and the absorbance near 400 nm is not affected at all. In this reaction solution containing 4-
4-Nitrophenol is produced by the action of N-7 cetyl-β-D-glucosaminidase using nitrophenyl N-acetyl-β-D-glucosaminid as a substrate.
The enzyme activity can be measured by continuously monitoring the amount of change in absorbance at 0 nm. Note that the wavelength that gives the maximum absorbance in the visible region in the coexistence of DEα is 410 nm. In addition, if the spectrophotometer used has the ability to measure the absorbance at 36 on I11, it is possible to measure the absorbance at pH 5,0 by monitoring the absorbance change at the same wavelength.
It is also possible to increase the measurement sensitivity by approximately 25%. The visible absorbance increasing effect of DEa on 4-ditrophenol was also observed to some extent in p)14,0, and the effect was maintained in a wide pH range from neutral to slightly alkaline.

Example 3 Visible coloring effect of DEa on 4-ditrophenol By using DEa in place of DEa in Example 1, a coloring effect of about 60% of that of DEa can be obtained.

Example 4 Visible coloring effect of DEa, DEa on 2-chloro-4-ditrophenol and its application to enzyme reaction 2-chloro-4-ditrophenol is ionized by more than 10% at pH 5.0 Therefore, it also exhibits absorption near 400 nm. 0. I IL-0 each in acetate buffer
．． When DEa is added at a concentration of 8% or more, the absorption spectrum of 2-chloro-4-nitrophenol shows a shape almost similar to safe ionization, and the absorbance at 400 nm (7) is 3.8 times that of DEa. It was 3.4 times higher in DEa.

On the other hand, 2-chloro-4-nitrophenyl-N-acetyl-β-D-glucosaminide, which is one of the synthetic substrates, shows almost no absorption around 400r++s at pH 5.0, and a subtle long wavelength shift in DEa or DEa. is observed, and there is almost no effect on the treatment at around 400 nm.Therefore, by treating this substrate solution with N-acetyl-β-D-glucosaminidase, the produced 2-chloro-4-ditrophenol can be detected at 410 nm. It was possible to continuously monitor the amount of change in absorbance.

Example 5 Visible coloring effect of DEa and DEa on other nitrophens This shows the results of an investigation to see if it lasted. Note that DEa used and the concentration of DEa were 0.8%.

(i) 2.4-dinitrophenol grows approximately 90% at pH 5.0, so even if it is as it is, 400n
3 shows strong light absorption. However, this absorption merely forms a shoulder of strong absorption in the ultraviolet region; when DEa is added thereto, an independent absorption peak is formed at 40 On, and its absorbance increases by 35%.

(ii) 5-nitrosalicylic acid has a pH of 5.040
The absorbance at 0 nm is only 2.5% of the ultraviolet absorption maximum, and when DEa or DEa is added to this, the absorbance can be increased 3 to 4 times.

(iii) pH of methyl 5-nitrosalicylate 5.0
The absorbance at 400 nm is 3.
It is 6%. When DEa is added to this, a new absorption peak appears around 400 nm, and the absorbance increases 8.4 times. DEa is weaker than DEa, but has a similar effect.

(ii) Also for ethyl 5-nitrosalicylate, DE
a and DEa show similar effects as on methyl esters, but the effect is more pronounced in the case of methyl esters.

[Effects of the Invention] As is clear from the above, according to the present invention, the cyclodextrin bonded with a basic functional group has the effect of significantly promoting color development in the visible region of the nitrophenol derivative,
Furthermore, since it is more soluble in water than ordinary cyclodextrin, it has unprecedented practical utility when applied to various enzyme activity measurement methods using synthetic substrates bound with nitrophenol derivatives. .

[Brief explanation of drawings]

Figure 1 shows the DE of the absorption spectrum of 4-nitrophenol.
It is a graph diagram showing the influence of α. A...pH 5,020m p-acetate buffer medium...
pH 5,020 mg acetate buffer containing 1% DEα Nakasaka+ (nm) →

Claims (4)

[Claims] (1) Cyclodextrin derivative to which a basic functional group is covalently bonded. (2) The compound according to claim 1, wherein the basic functional group is represented by the following formula. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R_1 and R_2 each represent a lower alkyl group having 1 to 3 carbon atoms or hydrogen, and n represents an integer of 1 to 3.) (3) A coloring method characterized by including a nitrophenol derivative using a cyclodextrin derivative to which a basic functional group is covalently bonded. (4) The method according to claim 3, wherein the reaction system for producing the nitrophenol derivative is made to share the derivative to develop color.