JPH0368552A - Water-soluble dinitrophenyl compound - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I [A is X1 or formula II (X1 to X3 are alkylene); Z<+> is hydrogen ion or alkali metal ion]. EXAMPLE:Sodium N-hydroxysulfosuccinimidyl-6-[(2,4-dinitrophenyl)amino]- hexanoate. USE:A modifier for proteins capable of dinitrophenylation of the proteins in an aqueous solvent. PREPARATION:A compound expressed by formula III is reacted with an alkali metal salt of N-hydroxysulfosuccinimide, in the presence of a condensing agent (e.g. N,N-dicyclohexylcarbodiimide) in a solvent, such as dimethylformamide, at 10-30 deg.C to afford the compound expressed by formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a water-soluble dinitrophenyl compound. The novel compounds according to the invention are particularly effective as 2,4-dinitrophenyl modifiers.

[Conventional technology]

The 2,4-dinitrophenyl group is the most common hubten commonly used in immunological experiments. In the case of a substance modified with a dinitrophenyl group, it is possible to further introduce a label via an anti-dinitrophenyl antibody. A commonly known dinitrophenyl modifier is 2,4-dinitrobenzaldehyde [Shiroya, K.R., Nakane,
P, Ni: Journal Saibu Selby TIII, Vol. 97.

No. 377a, 1983 (Shroyer,
K.R., Nakane, PK.: Jour.
naI of Ce1l Biology,
97. 377 a (1983)] and 2,4-dinitrophenyl-lysine [Handa, S.: Analytical Letters, Vol. 21, No. 7, No. 1141-
No. 1154, 1988 (Hashida, S.
, :^analyticalLetters, 21 (
7) 1141-1154 (1988))), and these aldehyde groups and amino groups react with the amino group of the target substance to be modified with a dinitrophenyl group.

[Problems to be Solved by the Invention] However, 2,4-dinitrobenzaldehyde is poorly soluble in water, and when reacting with many water-soluble substances targeted in immunological experiments, it is difficult to react with organic solvents and water. Because it is carried out in a mixed solvent, the reaction rate is low and often results in denaturation of the target substance. Moreover, since the modified dinitrophenyl group is located near the target substance, there is a problem in that the reaction with anti-dinitrophenyl antibodies is inhibited.

Although t-2,4-dinitrophenyl-L-lysine is water-soluble, the reaction between amino groups complicates the process and tends to reduce yield and reproducibility (Heshida, S.
, report: same as above).

Currently, there are no drugs that are water-soluble and do not inhibit the reaction between the dinitrophenyl group and the anti-dinitrophenyl antibody.
In addition, no dinitrophenyl compound has been found that requires a small number of reaction steps with the target substance and has a high yield, and the appearance of such a compound is expected.

The purpose of the present invention is precisely in this respect, and is to provide a new water-soluble dinitrophenyl compound useful as a dinitrophenyl modifier.

[Means to solve the problem]

The present inventor has conducted various studies with the aim of developing a new water-soluble dinitrophenyl compound, and found that
The inventors succeeded in creating a compound represented by the general formula (1) described below, and completed the present invention through further research.

That is, since the new compound in the present invention is water-soluble,
It does not require the use of organic solvents as is the case with known modifiers, allows uniform reaction to occur in water, and allows easy control of the reaction.

Furthermore, since it is possible to lengthen the crosslink between the dinitrophenyl group and the target substance, it is possible to alleviate the steric hindrance of the target substance itself, which is involved in the reactivity between the dinitrophenyl group and the anti-dinitrophenyl antibody. can.

Furthermore, a one-step reaction with the target substance is possible.

The compound according to the present invention has the general formula (1). Preferred examples of the alkali metal ion include sodium ion and potassium ion.

Among the compounds represented by the general formula (1), when 2' is an alkali metal ion, the general formula (ff)X represents an alkylene group), and Zo represents a hydrogen ion or an alkali metal ion. ] A dinitrophenyl compound represented by It is characterized by having a dinitrophenyl group and a sulfosuccinimide group.

In the compound represented by general formula (I), A1! X,
Also shows X2-C-N11-Xs, Kokote x 1°L,
LIt71 Indicates a alkylene group.

The alkylene group preferably includes a C1-1° straight or branched lower alkylene group.

Z' represents a hydrogen ion or an alkali metal ion.

[In the formula, A is the same as above. ]

It is prepared by reacting a starting compound of the formula with an alkali metal salt of N-hydroxysulfosuccinimide in the presence of a condensing agent. This reaction is carried out in a polar organic solvent at a temperature of 10-30°C.

Examples of suitable solvents include dimethylformamide and dimethylsulfoxide. The reaction components and reaction temperature can be selected as appropriate, and the reaction time will vary accordingly.

The condensing agent used includes condensing agents commonly used in esterification reactions, such as N,N-dicyclohexylcarbodiimide.

Among the starting compounds represented by general formula (II), A is ×1
The compound can be easily synthesized by a known method [Sanf-1F5: Pit Chemical Journal, Vol. 39, p. 507, 1945 (Sange
r, F,:Biochemical Journal
l, 39. 507 (1945))].

Starting compounds in which A is L-C-NH-X3 have the general formula (
A compound represented by 1) (wherein A is X) and 1
It can be easily produced by reacting with -X3-C0OH (in this case, x2 represents the same alkylene group as xI).

The reaction is carried out in water at a temperature of 10-30°C and a pH of 7-10. The reaction components and reaction temperature can be selected as appropriate, and the reaction time will vary accordingly.

In this method, a compound represented by the general formula (1) (A, X,)) and 1. By arbitrarily combining N-X, -COD)I, it is possible to arbitrarily extend the crosslinking portion between the dinitrophenyl group and the sulfosuccinimide group of the compound represented by the general formula (1).

The alkali metal salt of N-hydroxysulfosuccinimide is a known compound N-hydroxymaleimide (Met
ody Poluch, Khim, Reaktiv
av Prep.

1967, No. 17.113-15) with pyrosulfite (Na or K).

Furthermore, among the compounds represented by the general formula (I), compounds in which Zl is a hydrogen ion can be used to adjust the pH of the final product (pH
5 to 7).

When using the compounds according to the invention as dinitrophenyl modifiers, the following method applies.

The compound represented by the general formula (1) reacts with a substance to be modified (a protein, a nucleic acid, etc. that provides at least one amine group for the reaction, hereinafter referred to as B-NL) as follows.

〔Example〕

Example 1.8-hydroxysulfosuccinimidyl-6-
C(2,4-dinitrophenyl)aminoco-hexanoate sodium salt This reaction is normally carried out in water at a temperature of 10-30°C and a pH of 7-10. The reaction components and reaction temperature can be selected as appropriate, and the reaction time will vary accordingly.

The reaction solvent may be hydrophilic depending on the nature of the reaction components85! Solvents can be used. Furthermore, it is relatively easy to adjust the modification rate by changing these reaction conditions.

Next, the present invention will be further explained with reference to Examples.

(Blank below c)] (1) Synthesis of N-hydroxysulfosuccinimide sodium salt 260 mg (2.3 mmol) of N-hydroxymaleimide (Sigma Chemical CD, ST, Louis) was dissolved in 99.5% ethanol 4d. . 244tng
(1.3 mmol) of sodium pyrosulfite was dissolved in 4 d of M water and added dropwise to the ethanol solution of N-hydroxymaleimide over 5 minutes while stirring under a nitrogen stream.

It was then kept at room temperature for 2 hours with stirring, and then kept in air for an additional 15 minutes. The reaction solvent was distilled off under reduced pressure at 40° C. or below, and the residue was dissolved in 10 ml of purified water. The aqueous solution was filtered and the filtrate was lyophilized. Ether 20- was added to the pale yellow solid obtained after freeze-drying and kept at room temperature for 30 minutes with stirring.

A milky white powder was collected by filtration and dried under reduced pressure. 480 mg (2.2 mmol) of N-hydroxysulfosuccinimide sodium salt were thus obtained. The yield was 96%. The product was analyzed by mass spectrometry and ')l-1i
It was analyzed by MR.

Mass spectrometry Molecular weight calculation value (!”) 217 analysis measurement value
218 (M + ) I) 240 (M ” + Na
)' H-111JRδ (DMSO-d,) 10, 4-1
0.8 (1)1. Broad, Ho4>(2) 297 mg (1,0 mmol) of N-hydroxysulfosuccinimidyl-6-((2,4-dinitrophenyl)aminocohexanonite sodium salt) of 6-[2, 4-dinitrophenyl)amino] with bovine sanoic acid and 217a+g (
1°0 mmol) of N-hydroxysulfosuccinimide sodium salt was dissolved in anhydrous dimethylformamide 2.5-. To this solution was added 227 mg (1.1 mmol) of dicyclohexylcarbodiimide (DCC, Wako Pure Chemical Industries, Osaka) with stirring. Thereafter, the mixture was stirred at room temperature for 20 hours, then cooled to 4° C. and maintained for 1 hour. The precipitated dicyclohexylurea was filtered off. The filtrate was heated to 40°C or lower under reduced pressure for 2rnf! It has been condensed below. 50% of ether was added to precipitate N-hydroxysulfosuccinimidyl-6-C(2,4-dinitrophenyl)aminocohexanoate sodium salt.

The mixture was centrifuged at 3000 rpm for 10 minutes to precipitate the sodium salt and separate it from the supernatant. The precipitate was dissolved by adding 10 g of purified water and freeze-dried. There was thus obtained 400 mg (0.8 mmol) of N-hydroxysulfosuccinimidyl-6-C(2,4-dinitrophenyl)aminocohexanoate sodium salt as a yellow powder. Yield is 80%
Met.

The product was analyzed by mass spectrometry and ')I-N! It was analyzed by JR.

Mass spectrometry Calculated molecular weight (M"> 496 Analysis measurement value' H-NMRδ (DMSO-66) cH, -e > 497 (M" + H) 520 (,'J" + Na) 1, 3-1.8 (6) 1. m, N-CH2-(C
L)! - Example 2. N-hydroxysulfosuccinimidyl-6-(6-((2,4-dinitrophenyl)amino)hexanoylaminocohexanoate sodium salt U (1)6-1:6-((2,4-dinitrophenyl)amino) phenyl)
Synthesis of amino)hexanoylaminocohexanoic acid + L
N-C)1. (CL) 3C)I, C00H1”)1
．． C0D)1 238 mg (1.8 mmol) of 6-aminohexanoic acid (Nacalai Tesque, Kyoto) was dissolved in 10 ml of 0.05 M sodium carbonate buffer (p)I 9.2).

150 μm (0.3 mmol) of N-hydroxysulfosuccinimidyl-6-C(2°4-dinitrophenyl) aminocohexanoate sodium salt was dissolved in purified water 5- and a 6-aminohexanoic acid solution was added. The mixture was added dropwise over 15 minutes while stirring.

Then, after being kept at room temperature for 1.5 hours with stirring, l
Gradually add N-HCl to pH 6.
~6.5 and the product precipitated out.

After adding 20 g of purified water, the mixture was extracted with 100 g of dimethyl acetate. Ethyl acetate was distilled off under reduced pressure at 40°C or below, and 104 m
g (0.25 mmol) of a yellow oil product was obtained. The yield was 83%. The product was analyzed by 'H-NMR.

)1-NMRδ(DMSO-d-) 1.2-1.7
(12B, m.

CHz-C[10)1), 2.0-2.3 (4H, m,
-C,H-C-N-C)12-fO)1).

■ (2) N-hydroxysulfosuccinimidyl-6-[6
Synthesis of -((2,4-dinitrophenyl)amino)hexanoylaminocohexanoate sodium salt 174■(0,424 mmol) of 6-[:6-((
2,4-dinitrophenyl)amino)hexanoylamino]hetanoic acid and
The hydroxysulfosuccinimidylurium salt was dissolved in anhydrous dimethylformamide l-. 97 mg in this solution
To (0,466 mm+J mol) of dicyclo was added handylcarbodiimide (Wako Tsumugi Kogyo) with stirring. Thereafter, while stirring, the mixture was kept at room temperature for 20 hours, then cooled to 4°C and kept for 1 hour.

The precipitated dicyclohexylurea was removed by filtration and then condensed to 0.5-a by reduced pressure. Add ethyl acetate 30- and N
-Hydroxysulfosuccinimidyl-6-C6-((2
,4-dinitrophenyl)amino)hexanoylaminocohexanoate sodium salt was precipitated. 300
Centrifuge at 0 rpm for 10 minutes to precipitate the sodium salts,
It was separated from the top.

10 ml of purified water was added to the precipitate to dissolve it, and the mixture was freeze-dried.

Thus 182 mg (0,299 mmol,) of N-hydroxysulfosuccinimidyl, a yellow powder, was obtained.
6-(6-((2,4-dinitrophenyl)amino)hexanoylaminocohexanoate sodium salt was obtained. The yield was 70%.

The product was analyzed by mass spectrometry and 'It-NMR. Mass spectrometry Calculated molecular weight (M”) 609 analysis measurement value
610 (M” + II) 632 (M” + Na
) 'H-N! AR 6(0!JSCl-ds) 1,3-1.8 (12N, m.

〔Effect of the invention〕

The present invention provides a water-soluble dinitrophenyl compound that is new and useful as a protein modifier. This compound has made it possible to dinitrophenylate proteins in aqueous solvents, which was previously impossible. This can be applied not only to the field of immunological measurements but also to biochemical techniques in general, and will contribute widely to the field.

Claims (3)

[Claims] (1) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, A represents X_1 or ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (X_1, X_2, X_3 represent alkylene groups) , Z^+ represents a hydrogen ion or an alkali metal ion. ] A dinitrophenyl compound represented by (2) The dinitrophenyl compound according to claim 1, wherein A is X_1 and X_1 is a lower alkylene group. (3) A is ▲There are mathematical formulas, chemical formulas, tables, etc.▼,
and a claim in which X_2 and X_3 are lower alkylene groups (
1) The dinitrophenyl compound described above.