JPH09302033A - Acrydan-group-containing polymer and method for chemiluminence - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a production process and a method for chemiluminescence by giving an acrydan-group-containing polymer comprising specified repeating units. SOLUTION: An acrydan-group-containing monomer represented by formula I [wherein R<1> and R<2> are each H or methyl; X<1> is O or S; Y is an eliminable group represented by formula II (wherein X<2> is O or S; R<5> is a 1-3C alkyl; and n<5> is 0-5) or formula III (wherein R<6> is H, a 1-10C alkyl or an aryl; and R<7> is a 1-10C alkyl or an aryl); and n<2> is 1-10] and a monomer represented by formula IV (wherein R<3> and R<4> are each H or methyl; n<4> is 2-100) are radical- polymerized or anionically polymerized to obtain an acrydan-group-containing polymer comprising repeating units each represented by formula V [wherein R<1> to R<7> are as defined in formulas I, II, III and IV; X<1> and X<2> are as defined in formulas I and II; Y is as defined in formula I; n<1> is 1-100,000; n<2> is 1-10; n<3> is 0-100,000; n<4> is 2-100; and the repeating units are bonded with each other so as to form a random or block copolymer]. This polymer is treated with an acid and reacted with hydrogen peroxide water having a concentration of 0.001-30wt.% and a base to allow it to emit chemiluminescence.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel acridan group-containing polymer, a method for producing the same, and a chemiluminescence method. The polymer of the present invention is used as a detecting agent for various diagnostic agents, various luminescent probes, an emergency light source, and the like.

[0002]

2. Description of the Related Art Chemiluminescence refers to a phenomenon in which an atom or molecule is excited by energy generated by a chemical reaction to emit light. To date, chemiluminescent compounds having various skeletons have been developed, and luminol, acridinium ester, oxalate ester, etc. are known (“Bioluminescence and Chemiluminescence” edited by Kazuhiro Imai, Hirokawa). Bookstore Publishing, pages 75-150, 1989). These compounds are used as a detecting agent for diagnostic agents or as a light source for emergencies. In particular, acridinium ester has an emission quantum yield of about 5% among existing chemiluminescent substances
And high values [EH White et al., J. Am. Chem.
Soc., 109 , 5189-5196 (1987), K. -D. Gundermann,
F. McCapra, “Chemiluminescence in Organic Chemist
ry "Springer-Verlag (1987)]. Furthermore, acridan has been reported as a protected form of acridinium ester, and is intended to improve the stability of the acridinium ester (Japanese Patent Laid-Open No. Hei 10-1999)
3-501772). However, for existing compounds, 1
The amount of chemiluminescence per molecule is not sufficient, and there is a demand for a more sensitive detection agent. Moreover, there has been no example in which a polyacrylate or polymethacrylate derivative has been used as a chemiluminescent substance.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel acridan group-containing polymer and a method for producing the same. A further object of the present invention is to provide a method for chemiluminescence of the novel acridan group-containing polymer of the present invention.

[0004]

According to the present invention, there is provided a compound represented by the general formula (1):

[0005]

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[Wherein R ¹ , R ² , R ³ and R ⁴ represent a hydrogen atom or a methyl group, X ¹ represents an oxygen atom or a sulfur atom, and Y represents the following general formula (2):

[0007]

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(In the formula, X ² represents an oxygen atom or a sulfur atom, R ⁵ represents an alkyl group having 1 to 3 carbon atoms, and n ⁵ represents 0.
Represents an integer of from 5 to 5. ) Or the following general formula (3)

[0009]

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(In the formula, R ⁶ is a hydrogen atom or a carbon number of 1 to
10 represents an alkyl group or an unsubstituted or substituted aryl group. R ⁷ represents an alkyl group having 1 to 10 carbon atoms, an unsubstituted or substituted aryl group. )

It is a leaving group represented by: n ¹ is 1 to 1
00000, n ² is 1 to 10, n ³ is 0 to 10000
0, n ⁴ is an integer of 2 to 100. Further, the repeating unit may be random or block. ]

It is an acridan group-containing polymer having a repeating unit represented by:

The present invention also has the general formula (4)

[0014]

[Chemical 12]

[Wherein R ¹ and R ² represent a hydrogen atom or a methyl group, X ¹ represents an oxygen atom or a sulfur atom, and Y 1
Is the following general formula (2)

[0016]

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(Wherein X ² represents an oxygen atom or a sulfur atom, R ⁵ represents an alkyl group having 1 to 3 carbon atoms, n ⁵ represents an integer of 0 to ⁵ ) or the following general formula (3 )

[0018]

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(Wherein R ⁶ is a hydrogen atom or a carbon number of 1 to
10 represents an alkyl group or an unsubstituted or substituted aryl group. R ⁷ represents an alkyl group having 1 to 10 carbon atoms, an unsubstituted or substituted aryl group. )

It is a leaving group represented by: n ² is 1 to 1
Indicates an integer of 0. ]

An acridan group-containing monomer represented by:
General formula (5)

[0022]

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(Wherein R ³ and R ⁴ represent a hydrogen atom or a methyl group, and n ⁴ represents an integer of 2 to 100), and are polymerized. Formula (1)

[0024]

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(Wherein R ¹ , R ² , R ³ , R ⁴ , X ¹ ,
Y, n ² and n ⁴ have the same meaning as previously defined,
n ¹ is an integer of ¹ to 100,000, and n ³ is 0 to 10.
Indicates an integer of 0000. )

A method for producing an acridan group-containing polymer having a repeating unit represented by:

In the present invention, the acid and hydrogen peroxide are added to the acridan group-containing polymer having the repeating unit represented by the general formula (1) in any order or simultaneously, and then the base is mixed to react. The chemiluminescence method is characterized in that chemiluminescence is caused by the reaction.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION The acridan group-containing polymer of the present invention (hereinafter referred to as an acridan polymer) is represented by the general formula (1). In the formula, hydrogen atom and methyl group can be mentioned as specific examples of R ¹ , R ² , R ³ and R ⁴ , and they can be selected independently. Further, Y in the formula is a leaving group represented by the general formulas (2) and (3). Specific examples of R ⁵ in the general formula (2) include a methyl group, an ethyl group, a propyl group and an isopropyl group. Specific examples of R ^{6 in} the general formula (3) are hydrogen atom, methyl group, ethyl group, propyl group, butyl group, pentyl group, phenyl group, tolyl group, xylyl group, mesityl group, benzyl group, phenethyl group. However, the present invention is not limited to these. R ⁷
Specific examples thereof include, but are not limited to, methyl group, ethyl group, propyl group, butyl group, pentyl group, phenyl group, tolyl group, xylyl group, mesityl group, benzyl group, phenethyl group and the like. .

The acridan polymer of the present invention is obtained by copolymerizing the acrydan group-containing monomer represented by the general formula (4) and the monomer represented by the general formula (5), that is, polyethylene glycol (meth) acrylate. To be As the polymerization method used at this time, radical polymerization or anionic polymerization can be selected. When radical polymerization is performed, both bulk polymerization and solution polymerization can be used. Examples of the solvent used for solution polymerization include benzene, toluene, xylene, tetrahydrofuran, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl isobutyl ketone, methanol, ethanol, dimethylformamide, water and the like. The polymerization monomer concentration is used in the range of 1 to 50% by weight. As the polymerization initiator used at this time, an azo initiator represented by azobisisobutyronitrile or a peroxide represented by benzoyl peroxide and a redox initiator formed by combining a peroxide and a metal catalyst are selected. It can be carried out at a polymerization temperature of 30 to 100 ° C.

If desired, various functional groups may be introduced at the ends of the main chain of the acridan polymer of the present invention by using a polymerization initiator and a chain transfer agent. Specifically, an amino group, a carboxyl group, a hydroxyl group or the like can be easily introduced. 2,2′-azobis [N- (4-aminophenyl) -2-methylpropionamidine] tetrahydrochloride as a polymerization initiator for introducing the functional group,
4,4′-azobis (4-cyanovaleric acid), 2,2′-
Azobis [N- (2-hydroxyethyl) -2-methylpropionamidine] dihydrochloride, 2,2′-azobis [2-methyl-N- (2-hydroxyethyl) propionamide], 2,2′-azobis [2- (Hydroxymethyl) propionitrile] can be preferably mentioned. Further, as a chain transfer agent for introducing the above functional group, 2-
Aminoethanethiol, 3-mercaptopropionic acid,
2-Hydroxyethanethiol can be preferably mentioned.

In the chemiluminescence method of the present invention, the acridan group-containing polymer represented by the general formula (1) is treated with an acid and then reacted with hydrogen peroxide and a base to cause chemiluminescence. At this time, hydrogen peroxide may be added before the acid treatment, simultaneously with the acid treatment, or after the acid treatment. As the acid,
Inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and nitric acid, and organic acids such as acetic acid and trifluoroacetic acid can be used. The base is, for example, sodium methoxide, potassium-t-
Butoxide, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium hydrogen carbonate, tetrabutylammonium hydroxide and the like can be preferably mentioned. The concentration of hydrogen peroxide used in the chemiluminescence method of the present invention is preferably 0.001 to 30% by weight, and particularly preferably 0.1 to 30% by weight.

[0032]

The acridan group-containing polymer of the present invention is novel and has a high luminescence amount per molecule and is a chemiluminescent compound excellent in stability, and is used as a detecting agent for various diagnostic agents,
It can be used as various light emitting probes, emergency light sources, and the like. In addition, the production method of the present invention makes it possible to easily produce an acridan group-containing polymer in a short time, at a high yield. Furthermore, the chemiluminescence method of the present invention can be applied to the above various uses.

[0033]

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

Example 1 Structural formula (6)

[0035]

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2- {10-methyl-9-represented by
(N-Butyl-N-toluenesulfonylcarboxamide) acridyl} oxyethyl methacrylate 29 mg
(0.050 mmol) and polyethylene glycol methacrylate (n = 22) 500 mg (0.47 mmol)
And azobisisobutyronitrile 5.5 mg (0.033 m
was dissolved in 1.5 ml of benzene, and then the tube was degassed and sealed and reacted at 50 ° C. for 20 hours. The reaction solution was distilled off and gel filtration chromatography (Sephadex LH2
0, manufactured by Pharmacia Biotech Co., Ltd. to remove the monomer component, and 275 mg (yield 52%) (0.0052 m)
(mol) polymer was obtained. The result of the absorbance measurement, 2-
It was found that about 6 pieces of {10-methyl-9- (N-butyl-N-toluenesulfonylcarboxamido) acridyl} oxyethyl methacrylate were introduced in one molecule of the polymer.

¹ H NMR (C ₆ D ₆ ) δ3.64-3.42 (brs, 88H), 3.4
0, 3.18 (brs, 3H), 2.61-1.85 (br, 2H), 1.70-0.90 (br,
3H) IR (cm ^-1 ); 2874, 1728, 1645, 1456, 1351, 1252, 110
8, 952, 848 UV visible (Abs. In DMF; Conc .: 0.35mg / ml) 0.342 (312n
m) Mn = 5.29 × 10 ⁴ , Mw / Mn = 4.2

Example 2 Structural formula (7)

[0039]

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2- {10-methyl-9-represented by
(N-butyl-N-toluenesulfonylcarboxamide) acridyl} oxypropyl methacrylate 9 mg
(0.015 mmol) and polyethylene glycol methacrylate (n = 22) 200 mg (0.19 mmol)
And azobisisobutyronitrile 3 mg (0.018 mm
ol) was dissolved in 0.6 ml of benzene, a degassed tube was sealed, and the mixture was reacted at 50 ° C. for 20 hours. The reaction solution was distilled off,
Gel filtration chromatography (Sephadex LH2
0, manufactured by Pharmacia Biotech Co., Ltd. to remove the monomer component, and 100 mg (yield 48%) (0.0015 m)
(mol) polymer was obtained. The result of the absorbance measurement, 2-
It was found that about 5 {10-methyl-9- (N-butyl-N-toluenesulfonylcarboxamide) acridyl} oxyethyl methacrylate were introduced in one molecule of the polymer.

¹ H NMR (C ₆ D ₆ ) δ 3.84-3.44 (brs. 88H), 3.5
3. 3.19 (brs, 3H), 2.50-1.90 (br, 2H), 1.34, 0.90 (br,
3H) IR (cm ^-1 ); 2871, 1727, 1643, 1455, 1350, 1252, 110
5, 952, 849 UV visible (Abs. In DMF; Conc .: 0.43 mg / ml) 0.259 (312n
m) Mn = 6.85 × 10 ⁴ ,, Mw / Mn = 4.38

Example 3 The acridan group-containing polymer obtained in Example 1 was dissolved in dimethylformamide. Add 30 μl of this solution to 0.
300μ of 0.1N nitric acid aqueous solution containing 5 wt% hydrogen peroxide
After adding 1 l, it was set in a luminescence reader (chemiluminescence measuring instrument Aloka BLR201). 0.2 from the outside
A 5N NaOH aqueous solution was added, and luminescence was measured for 1 minute.
Based on this measurement result, the amount of luminescence per molecule was determined, and Table 1
It was shown to.

Example 4 The amount of emitted light was determined in the same manner as in Example 3 except that the acridan group-containing polymer obtained in Example 2 was used.

Comparative Example 1 Instead of the polymer containing an acridan group, 10-methyl-
The measurement was performed in the same manner as in Example 3 except that 9-phenoxycarbonylacridinium methosulfate was used.
The results are shown in Table 1.

[0045]

[Table 1] Table 1 ──────────────────────────── Examples or Comparative Examples Luminescence amount per molecule (counts / mol) Example 3 2.28 × 10 ¹⁸ Example 4 1.41 × 10 ¹⁸ Comparative Example 1 1.19 × 10 ¹⁷ ─────────────────────────────

As a result, in Example 3, the amount of emitted light was about 19 times that in Comparative Example 1, and in Example 4, the amount of emitted light was about 12 times, indicating that the acridan group-containing polymer of the present invention is excellent. You can see that

Example 5 The acridan group-containing polymer obtained in Example 1 was adjusted to pH.
After dissolving in 7.0 mM 10 mM phosphate buffer, 4
Stored at ° C. Example 2 with sampling over time
The amount of luminescence was measured in the same manner as in. The results are shown in Table 2.

Comparative Example 2 The same measurement as in Example 5 was carried out except that 10-methylacridinium-9-carboxylic acid phenyl ester methosulfate was used instead of the acridan group-containing polymer. The results are shown in Table 2.

[0049]

[Table 2] Table 2 ──────────────────────────────── Example Residual rate of luminescence (%) 2 days later 7 After 12 days After ──────────────────────────────── Example 5 100 100 100 100 Comparative Example 2 97 90 90 82 ─── ─────────────────────────────

From the above results, it can be seen that the stability of Example 5 is higher than that of Comparative Example 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C08F 220: 28)

Claims (3)

[Claims] 1. A compound of the general formula (1) [Wherein R ¹ , R ² , R ³ and R ⁴ represent a hydrogen atom or a methyl group, X ¹ represents an oxygen atom or a sulfur atom, Y
Is the following general formula (2) (In the formula, X ² represents an oxygen atom or a sulfur atom, R ⁵ represents an alkyl group having 1 to 3 carbon atoms, n ⁵ represents an integer of 0 to ⁵ ) or the following general formula (3): 3] (Wherein R ⁶ represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, an unsubstituted or substituted aryl group. The R ⁷
Represents an alkyl group having 1 to 10 carbon atoms, an unsubstituted or substituted aryl group. ) Is a leaving group. n ¹ is ¹ to 100,000, n ² is 1 to 10, and n ³ is 0 to 100.
000, n ⁴ is an integer of 2 to 100. Further, the repeating unit may be random or block. ] The acridan group containing polymer which has a repeating unit represented by these. 2. A compound represented by the general formula (4): [Wherein R ¹ and R ² represent a hydrogen atom or a methyl group,
X ¹ represents an oxygen atom or a sulfur atom, and Y represents the following general formula (2): (In the formula, X ² represents an oxygen atom or a sulfur atom, R ⁵ represents an alkyl group having 1 to 3 carbon atoms, n ⁵ represents an integer of 0 to ⁵ ) or the following general formula (3): 6] (Wherein R ⁶ represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, an unsubstituted or substituted aryl group. The R ⁷
Represents an alkyl group having 1 to 10 carbon atoms, an unsubstituted or substituted aryl group. ) Is a leaving group. n ² is an integer of 1 to 10. And an acridan group-containing polymer represented by the following general formula (5): (In the formula, R ³ and R ⁴ represent a hydrogen atom or a methyl group, and n ⁴ represents an integer of 2 to 100.) A monomer represented by the general formula (1) is polymerized. ) [Chemical 8] (In the formula, R ¹ , R ² , R ³ , R ⁴ , X ¹ , Y, n ² and n
⁴ has the same meaning as defined above, n ¹ is 1 to 10
0000 integer and n ^3, is an integer of from 0 to 100,000. ) A method for producing an acridan group-containing polymer having a repeating unit represented by: 3. An acid and hydrogen peroxide are added to the acridan group-containing polymer having the repeating unit represented by the general formula (1) of claim 1 in any order or simultaneously, and then a base is mixed to react. A chemiluminescence method, characterized in that chemiluminescence is caused by the reaction.