JP7125107B2 - Block copolymer, phosphate compound detection agent, and method for detecting phosphate compound - Google Patents

Description

TECHNICAL FIELD The present invention relates to a block copolymer containing a fluorene block unit and a five-membered heterocyclic block unit, a phosphate compound detection agent containing the same, and a method for detecting a phosphate compound using them.

Many phosphate compounds such as ATP and DNA are present in living organisms, and detection of phosphate compounds is required not only in the medical field but also in the industrial field. In the industrial field, there is a particular demand for a detection technique for measuring phosphoric acid compounds produced by microorganisms or phosphoric acid compounds serving as nutrients for microorganisms.

So far, techniques for detecting ATP from changes in potential after dephosphorylation of ATP by ATPase and changes in absorption wavelength of specific compounds in the presence of ATP have been reported (References 1 and 2). However, the former has drawbacks in that direct detection is not possible, and the latter has a small change in absorption wavelength.

On the other hand, it has been reported that a thiophene polymer with a cationic phosphonium group, which has the property of adsorbing an anionic phosphate compound through electrostatic interaction, is useful as a detection agent for DNA and the like. Here, since thiophene polymers show strong self-organization, DNA recognition is performed by utilizing changes in their cohesiveness (Patent Document 3).

JP-A-4-131096 Japanese Patent Application Publication No. 2013-533903 JP 2015-158425 A

An object of the present invention is to provide a novel block copolymer useful for detecting phosphoric acid compounds.

In order to achieve the above objects, the present inventors have proposed a block copolymer comprising a fluorene block unit exhibiting excellent light-emitting properties and a 5-membered heterocyclic block unit exhibiting strong self-organization, wherein a fluorene block unit and a 5-membered heterocyclic ring Cationic substituents were introduced into both block units, and the molecular recognition ability of the phosphoric acid compound of the resulting block copolymer was evaluated from the viewpoint of spectral characteristics. The inventors have found that it is possible to detect compounds, and have completed the present invention.

That is, the present invention is as follows.
[1] Formula (I):

(In the formula,
R ¹ to R ⁸ each independently represent -Y-R ¹¹ , hydrogen atom, halogen atom, hydrocarbon group having 1 to 10 carbon atoms, -NR ¹² R ¹³ , -OR ¹² , -SR ¹² , - COR ¹² , —SO ₂ R ¹² , —NR ¹³ COR ¹² , —CONR ¹² R ¹³ , —OCOR ¹² , —COOR ¹² , —NR ¹³ CSR ¹² , —CSNR ¹² R ¹³ , —SCOR ¹² or —COSR ¹² wherein at least one of R ¹ to R ⁸ is —Y—R ¹¹ ;
Y represents a spacer structure having 1 to 30 atoms in the main chain,
R ¹¹ represents a cationic nitrogen-containing group or a cationic phosphorus-containing group,
R ¹² represents a hydrocarbon group having 1 to 10 carbon atoms,
R ¹³ represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms,
n represents 2 to 100,
Each of the n monomer units is the same or different. )
A block unit represented by
Formula (II):

(In the formula,
R ⁹ and R ¹⁰ each independently represent -Y-R ¹¹ , hydrogen atom, halogen atom, hydrocarbon group having 1 to 10 carbon atoms, -NR ¹² R ¹³ , -OR ¹² , -SR ¹² , - COR ¹² , —SO ₂ R ¹² , —NR ¹³ COR ¹² , —CONR ¹² R ¹³ , —OCOR ¹² , —COOR ¹² , —NR ¹³ CSR ¹² , —CSNR ¹² R ¹³ , —SCOR ¹² or —COSR ¹² and at least one of R ⁹ and R ¹⁰ is —Y—R ¹¹ ;
X represents S, O, or NR ¹⁴ ;
Y, R ¹¹ , R ¹² and R ¹³ are the same as in formula (I);
R ¹⁴ represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms,
m represents 2 to 100,
Each of the m monomer units is the same or different. )
A block copolymer containing a block unit represented by
[2] The block copolymer according to [1], wherein n/m is 3 or less.
[3] The block copolymer according to [1] or [2], wherein n is 15 or more.
[4] The block copolymer according to any one of [1] to [3], wherein m is 8 or more.
[5] The block according to any one of [1] to [4], wherein the block unit represented by formula (I) and the block unit represented by formula (II) are directly bonded. copolymer.
[6] The block copolymer according to any one of [1] to [5], wherein R ¹ to R ⁶ are hydrogen atoms.
[7] The block copolymer according to any one of [1] to [6], wherein R ⁷ and R ⁸ are each independently —Y—R ¹¹ .
[8] The block copolymer according to any one of [1] to [7], wherein one of R ⁹ and R ¹⁰ is —Y—R ¹¹ and the other is a hydrogen atom.
[9] A phosphate compound detecting agent comprising the block copolymer according to any one of [1] to [8].
[10] measuring the fluorescence spectrum or ultraviolet-visible light absorption spectrum of the block copolymer according to any one of [1] to [8] in a solution containing a phosphoric acid compound, and the resulting fluorescence intensity or A method for detecting a phosphoric acid compound, comprising detecting the phosphoric acid compound from the waveform or absorbance of an ultraviolet-visible light absorption spectrum.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide novel block copolymers useful for detecting phosphoric acid compounds.

FIG. 1 shows the UV-visible absorption spectra of the block copolymer of Example 1 in TRIZMA buffer solutions with and without various phosphoric acid compounds. FIG. 2 shows the UV-visible absorption spectra of the block copolymer of Example 2 in TRIZMA buffer solutions with and without various phosphoric acid compounds. FIG. 3 shows the UV-visible absorption spectra of the block copolymer of Example 3 in TRIZMA buffer solutions with and without various phosphoric acid compounds. FIG. 4 shows UV-visible light absorption spectra of the block copolymer of Comparative Example 1 in TRIZMA buffer solutions with and without various phosphoric acid compounds. FIG. 5 shows fluorescence spectra of the block copolymer of Comparative Example 2 in TRIZMA buffer solutions with and without various phosphoric acid compounds. FIG. 6 shows fluorescence spectra of the block copolymer of Example 1 in TRIZMA buffer solutions with and without various phosphoric acid compounds. FIG. 7 shows fluorescence spectra of the block copolymer of Example 2 in TRIZMA buffer solutions with and without various phosphoric acid compounds. FIG. 8 shows fluorescence spectra of the block copolymer of Example 3 in TRIZMA buffer solutions with and without various phosphoric acid compounds. FIG. 9 shows fluorescence spectra of the block copolymer of Comparative Example 1 in TRIZMA buffer solutions with and without various phosphoric acid compounds. FIG. 10 shows fluorescence spectra of the block copolymer of Comparative Example 2 in TRIZMA buffer solutions with and without various phosphoric acid compounds. FIG. 11 shows fluorescence spectra of the block copolymer of Example 3 in TRIZMA buffer solution at pH=7.4 with or without various concentrations of ATP. FIG. 12 shows fluorescence spectra of the block copolymer of Example 3 in TRIZMA buffer solution at pH=8.0 with or without various concentrations of ATP. FIG. 13 shows fluorescence spectra of the block copolymer of Example 3 in TRIZMA buffer solution at pH=9.0 with or without various concentrations of ATP.

Hereinafter, embodiments of the present invention will be described in detail.

The present invention provides a compound of formula (I):

and a block unit represented by the formula (II):

A block copolymer represented by offer.

In formula (I), R ¹ to R ⁸ are each independently —Y—R ¹¹ , a hydrogen atom, a halogen atom, a hydrocarbon group having 1 to 10 carbon atoms, —NR ¹² R ¹³ , —OR ¹² , —SR ¹² , —COR ¹² , —SO ₂ R ¹² , —NR ¹³ COR ¹² , —CONR ¹² R ¹³ , —OCOR ¹² , —COOR ¹² , —NR ¹³ CSR ¹² , —CSNR ¹² R ¹³ , —SCOR ¹² , or —COSR ¹² , and at least one of R ¹ to R ⁸ is —Y—R ¹¹ . Y represents a spacer structure having a main chain of 1 to 30 atoms. R ¹¹ represents a cationic nitrogen-containing group or a cationic phosphorus-containing group. R ¹² represents a hydrocarbon group having 1 to 10 carbon atoms. R ¹³ represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms.

As used herein, "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

In the present specification, the "hydrocarbon group having 1 to 10 carbon atoms" includes, for example, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an alkynyl group having 2 to 10 carbon atoms. a cycloalkyl group having 3 to 10 carbon atoms, a cycloalkenyl group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 10 carbon atoms, and the like. A hydrocarbon group having 1 to 10 carbon atoms is preferably an alkyl group having 1 to 10 carbon atoms.

As used herein, the term "alkyl group having 1 to 10 carbon atoms" means a linear or branched monovalent saturated hydrocarbon group having 1 to 10 carbon atoms, such as methyl, ethyl , propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3- Dimethylbutyl, 2-ethylbutyl, heptyl, octyl, nonyl, decyl and the like. The number of carbon atoms in the alkyl group is preferably 1-6, more preferably 1-5, even more preferably 1-4, and particularly preferably 1-3.

As used herein, the term "alkenyl group having 2 to 10 carbon atoms" refers to a linear or branched monovalent unsaturated hydrocarbon containing one or more double bonds having 2 to 10 carbon atoms. means a hydrogen group, such as vinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, 5-hexenyl and the like. The alkenyl group preferably has 2 to 6 carbon atoms, more preferably 2 to 5 carbon atoms, still more preferably 2 to 4 carbon atoms, and particularly preferably 2 or 3 carbon atoms.

As used herein, the term "alkynyl group having 2 to 10 carbon atoms" means a linear or branched monovalent unsaturated hydrocarbon containing one or more triple bonds having 2 to 10 carbon atoms. groups such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1,1-dimethyl Prop-2-yn-1-yl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl and the like. The alkynyl group preferably has 2 to 6 carbon atoms, more preferably 2 to 5 carbon atoms, still more preferably 2 to 4 carbon atoms, and particularly preferably 2 or 3 carbon atoms.

As used herein, "a cycloalkyl group having 3 to 10 carbon atoms" refers to a cyclic monovalent saturated hydrocarbon group having 3 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

As used herein, the term "cycloalkenyl group having 3 to 10 carbon atoms" refers to a cyclic monovalent unsaturated hydrocarbon group containing one or more double bonds having 3 to 10 carbon atoms, For example, cycloalkamonoenyls such as cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl; 1,3-cyclobutadien-1-yl, 1,3-cyclopentadien-1-yl, 1,4-cyclopentadiene-1- yl, 2,4-cyclopentadien-1-yl, 1,3-cyclohexadien-1-yl, 1,4-cyclohexadien-1-yl, 1,5-cyclohexadien-1-yl, 2,4- and cycloalkadienyls such as cyclohexadien-1-yl and 2,5-cyclohexadien-1-yl.

As used herein, the term “aryl group” refers to a cyclic monovalent aromatic hydrocarbon group having 6 to 10 carbon atoms, such as phenyl, 1-naphthyl and 2-naphthyl.

As used herein, the term "aralkyl group" refers to an alkyl group substituted with an aryl group and having 7 to 10 carbon atoms, such as benzyl and phenethyl.

As used herein, the term "spacer structure having A to B atoms in the main chain" (A and B are integers) means A to It means a divalent group having a backbone formed of B atoms.

As used herein, a "cationic nitrogen-containing group" means a group having a +1 valent nitrogen atom. The nitrogen atom includes those that have a valence of +1 in an aqueous solution even if the block copolymer has a valence of 0 when the block copolymer is solid. Examples of the "cationic nitrogen-containing group" include -N ⁺ H ₃ , -N ⁺ H ₂ R ²⁰ , -N ⁺ HR ²⁰ R ²¹ , or -N ⁺ R ²⁰ R ²¹ R ²² (wherein R ²⁰ , R ²¹ and R ²² each independently represent a hydrocarbon group having 1 to 10 carbon atoms.); imidazolium group, oxazolium group, thiazolium group, oxadiazolium group, triazolium group, pyrrolidinium group, pyridinium group, piperidinium group, pyrazolium group, pyrimidinium group, pyrazinium group, triazinium group, indolium group (respectively hydrogen on any C, N, N ⁺ of the ring means a monovalent group excluding any one of the atoms, and the other C, N, N ⁺ may be substituted with a hydrocarbon group having 1 to 10 carbon atoms.), etc. cationic nitrogen-containing heterocyclic group.

As used herein, "cationic phosphorus-containing group" means a group having a +1 valent phosphorus atom. The phosphorus atom includes those that have a valence of +1 in an aqueous solution even if the block copolymer has a valence of 0 when the block copolymer is solid. Examples of the "cationic phosphorus-containing group" include -P ⁺ H ₃ , -P ⁺ H ₂ R ²⁰ , -P ⁺ HR ²⁰ R ²¹ , or -P ⁺ R ²⁰ R ²¹ R ²² (wherein R ²⁰ , R ²¹ and R ²² each independently represent a hydrocarbon group having 1 to 10 carbon atoms.

R ¹ to R ⁸ each independently represent -Y-R ¹¹ , hydrogen atom, halogen atom, hydrocarbon group having 1 to 10 carbon atoms, -NR ¹² R ¹³ , -OR ¹² , -SR ¹² , - COR ¹² , —SO ₂ R ¹² , —NR ¹³ COR ¹² , —CONR ¹² R ¹³ , —OCOR ¹² , —COOR ¹² , —NR ¹³ CSR ¹² , —CSNR ¹² R ¹³ , —SCOR ¹² or —COSR ¹² and at least one of R ¹ to R ⁸ is —Y—R ¹¹ .

R ¹ to R ⁸ are each independently a hydrogen atom or —Y—R ¹¹ , and at least one of R ¹ to R ⁸ is preferably —Y—R ¹¹ .

In one embodiment, R ¹ -R ⁶ are preferably hydrogen atoms. R ⁷ and R ⁸ are preferably each independently —Y—R ¹¹ .

In certain embodiments, R ¹ to R ⁸ preferably 1, 2, 3 or 4 of them are —Y—R ¹¹ , more preferably 1, 2 or 3 of them are — YR ¹¹ , more preferably one or two of which are —YR ¹¹ , particularly preferably two of which are —YR ¹¹ .

Y represents a spacer structure having a main chain of 1 to 30 atoms. Y is preferably a spacer structure having a main chain of 1 to 20 atoms, more preferably a spacer structure having a main chain of 2 to 15 atoms, still more preferably an atom of the main chain. It is a spacer structure with a number of 4-10.

In one embodiment, Y is preferably a group represented by -Y ¹ -Y ² -. Here, Y ¹ is a bond, -NH-, -O-, -S-, -CO-, -SO ₂ -, -NHCO-, -CONH-, -OCO-, -COO-, -NHCS- , -CSNH-, -SCO-, or -COS-. Y ² is represented by the formula (1): -[(CR ¹⁵ ₂ ) _a -Y ³ -] _b -(CR ¹⁵ ₂ ) _c - [wherein each R ¹⁵ is independently a hydrogen atom or a carbon atom represents a hydrocarbon group having a number of 1 to 10, and Y ³ is —NR ¹⁶ —, —O—, or —S— (wherein R ¹⁶ is each independently a hydrogen atom or a 10 hydrocarbon groups), a represents 2 or 3, b and c each independently represents an integer of 0 or more, and b(a+1)+c is 1 or more and 20 or less , preferably 2 or more and 15 or less, more preferably 4 or more and 10 or less. ] or (2) an alkenylene group having 2 to 20 carbon atoms.

As used herein, the term "alkenylene group having 2 to 20 carbon atoms" refers to a linear or branched divalent unsaturated carbonized chain containing one or more double bonds having 2 to 20 carbon atoms. means a hydrogen group, such as -CH=CH-, -CH _{2 -CH=CH-, -CH=CH-CH 2 -} , -CH=CH-CH=CH-, -CH ₂ -CH=CH- CH=CH-, -CH=CH-CH=CH-CH ₂ -, -CH=CH-CH=CH-CH=CH-, -CH=CH-CH=CH-CH=CH-CH=CH-, -CH=CH-CH=CH-CH=CH-CH=CH-CH=CH- and the like. The number of carbon atoms in the alkenylene group is preferably 2 or more and 15 or less, more preferably 4 or more and 10 or less.

Y ¹ is a bond, and Y ² is —(CH ₂ ) _C — [wherein c is an integer of 1 or more and 20 or less, preferably 2 or more and 15 or less, more preferably 4 10 or less. ] is preferably a divalent group represented by

R ¹¹ represents a cationic nitrogen-containing group or a cationic phosphorus-containing group.

The "cationic nitrogen-containing group" and "cationic phosphorus-containing group" represented by R ¹¹ can usually have a counter anion.

As used herein, the term "counter anion" means an organic or inorganic monovalent to pentavalent anion. Examples of the "counter anion" that the "cationic nitrogen-containing group" and "cationic phosphorus-containing group" may have include halide ions (e.g., chloride ion, bromide ion, iodide ion, etc.). .

R ¹¹ is preferably -N ⁺ R ²⁰ R ²¹ R ²² or -P ⁺ R ²⁰ R ²¹ R ²² (wherein R ²⁰ , R ²¹ and R ²² each independently have 1 to 10), more preferably -P ⁺ R ²⁰ R ²¹ R ²² , and particularly preferably -P ⁺ (CH ₃ ) ₃ .

R ¹² represents a hydrocarbon group having 1 to 10 carbon atoms. R ¹³ represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms.

n represents 2-100. n is preferably 3 or more, more preferably 5 or more, even more preferably 10 or more, and particularly preferably 15 or more. On the other hand, n is preferably 80 or less, more preferably 60 or less, even more preferably 40 or less, and particularly preferably 30 or less. n may be the average value of each molecule in the molecular assembly.

Each of the n monomer units is the same or different.

In formula (II), R ⁹ and R ¹⁰ are each independently -Y-R ¹¹ , hydrogen atom, halogen atom, hydrocarbon group having 1 to 10 carbon atoms, -NR ¹² R ¹³ , -OR ¹² , —SR ¹² , —COR ¹² , —SO ₂ R ¹² , —NR ¹³ COR ¹² , —CONR ¹² R ¹³ , —OCOR ¹² , —COOR ¹² , —NR ¹³ CSR ¹² , —CSNR ¹² R ¹³ , —SCOR ¹² , or —COSR ¹² , and at least one of R ⁹ and R ¹⁰ is —Y—R ¹¹ . X is S, O, or NR ¹⁴ (wherein R ¹⁴ represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms). Y, R ¹¹ , R ¹² and R ¹³ are the same as in formula (I).

R ⁹ and R ¹⁰ are each independently a hydrogen atom or —Y—R ¹¹ , and at least one of R ⁹ and R ¹⁰ is preferably —Y—R ¹¹ , and R ⁹ and R More preferably, one of ¹⁰ is —Y—R ¹¹ and the other is a hydrogen atom.

X represents S, O, or NR ¹⁴ (R ¹⁴ represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms). X is preferably S.

m represents 2 to 100; m is preferably 3 or more, more preferably 4 or more, still more preferably 5 or more, and particularly preferably 8 or more. On the other hand, m is preferably 80 or less, more preferably 60 or less, even more preferably 40 or less, and particularly preferably 30 or less. m may be the average value of each molecule in the molecular assembly.

n/m is preferably 20 or less, more preferably 10 or less, even more preferably 5 or less, and particularly preferably 3 or less. On the other hand, n/m is preferably 0.1 or more, more preferably 0.5 or more, still more preferably 1 or more, and particularly preferably 1.5 or more.

Each of the m monomer units is the same or different.

In certain embodiments, when one of R ⁹ and R ¹⁰ is —Y—R ¹¹ and the other is a hydrogen atom, R ⁹ is —Y for m−1 bonds of m monomer units. The ratio of bonds between monomer units with -R ¹¹ or bonds between monomer units with R ¹⁰ being -YR ¹¹ (so-called head to tail bonds (HT bonds) to all bonds) is 90% or more. is preferred, and 95% or more is more preferred. When the ratio is high, the connection of the π-π conjugated system in the block unit represented by formula (II) becomes long, and the twist of the bond between the rings does not exist so much, so that the rings are coplanar. It is thought that it becomes a structure that exists side by side for a long time. The ratio may be the average value of each molecule in the molecular assembly.

The block unit represented by formula (I) and the block unit represented by formula (II) may be directly bonded, or another structural unit may be bonded between them. Further, another structural unit may be bound to the terminal of the block unit represented by formula (I) and the terminal of the block unit represented by formula (II). Here, the "other structural unit" is not particularly limited as long as it does not inhibit the effects of the present invention. For example, the block unit represented by formula (I) and the block unit represented by formula (II) are Combinations of polymer units and monomer units having different pyrrole rings, furan rings, thiophene rings or benzene rings as skeletons, known low-molecular weight compounds, and the like can be mentioned. "Other structural units" are preferably less than 10% by mass, more preferably less than 5% by mass, still more preferably less than 2% by mass, and particularly preferably It is less than 1% by mass. The block unit represented by formula (I) and the block unit represented by formula (II) are preferably directly bonded.

In the block copolymer of the present invention, a known solid phase carrier may be bound to the terminal of the block unit represented by formula (I) or the terminal of the block unit represented by formula (II). Examples of solid phase carriers include carriers such as silica (SiO ₂ ).

The block copolymer of the present invention preferably has a number average molecular weight (M _n ) of 1,000 or more, more preferably 2,000 or more. On the other hand, the number average molecular weight (M _n ) is preferably 100,000 or less, more preferably 50,000 or less.

The molecular weight dispersity (M _w /M _n ) of the block copolymer of the present invention is preferably 5.0 or less, more preferably 3.0 or less, still more preferably 2.0 or less.

The block copolymer of the present invention has high solubility in polar solvents such as water, methanol and dimethylsulfoxide (DMSO). Therefore, it is useful as a detection agent for highly polar phosphate compounds.

The block copolymer of the present invention can change the phosphoric acid compound to be detected by controlling the copolymerization ratio and degree of polymerization. It is also expected to be applied to mapping of phosphate compounds in cell culture.

The block copolymer of the present invention can be synthesized using known methods. A method for producing the following polymer (III) among the block copolymers of the present invention will be described below. Polymer (III) can be produced, for example, by a method using the Kumada catalyst transfer condensation polymerization (KCTP) method, as shown in the synthesis scheme below. The production method shown in the synthesis scheme below is a representative production method, and the production method of polymer (III) is not limited thereto.

Synthetic scheme

[In the formula, R ^1′ to R ^10′ have the same definition as R ¹ to R ¹⁰ except that “-Y-R ¹¹ ” is “-Y-Br”, and L ₂ is represents a ligand of a nickel catalyst, other symbols are the same as defined above, X ¹ represents a halogen atom, preferably a bromine atom or an iodine atom, and X ² is a Grignard reagent-derived Indicates a halogen atom. ]

(Steps 1 and 5)
Compounds (V) and (IX) can be obtained, for example, by magnesiating compounds (IV) and (VIII), respectively, by a halogen metal exchange method using secondary or tertiary alkyl Grignard reagents. can be done.

Compounds (IV) and (VIII) may be commercially available or may be prepared based on known methods.

Secondary or tertiary alkyl Grignard reagents include, for example, isopropylmagnesium chloride, isopropylmagnesium bromide, tert-butylmagnesium chloride, sec-butylmagnesium chloride, isopropylmagnesium chloride/lithium chloride, sec-butylmagnesium chloride/ Lithium chloride etc. are mentioned. The amount of secondary or tertiary alkyl Grignard reagents to be used is generally 1 to 10 molar equivalents, preferably 1 to 5 molar equivalents, relative to compound (IV) or (VIII).

Also, 1,4-bis(hexyloxy)benzene (DHB) or the like may be added as necessary.

Examples of the reaction solvent include chain ethers such as diethyl ether and diisopropyl ether; and ether solvents such as cyclic ethers such as 1,4-dioxane and tetrahydrofuran. The reaction temperature is usually -80 to 50°C. The reaction time is usually 0.1 to 200 hours.

(Steps 2 and 6)
Polymers (VI) and (X) can be obtained by adding a nickel catalyst reagent (NiL ₂ Cl ₂ ) to compounds (V) and (IX), respectively, and subjecting them to chain condensation polymerization.

Nickel catalytic reagents include, for example, [1,3-bis(diphenylposphino)propane]nickel(II) dichloride (Ni(dppp)Cl ₂ ) and the like. The degree of polymerization of the copolymer can be controlled by the amount of nickel catalyst. The amount of the nickel catalyst reagent to be used is generally 0.005 to 50 molar equivalents, preferably 0.01 to 50 molar equivalents, relative to compound (V) or (IX).

The reaction solvent is the same as in Steps 1 and 5. The reaction temperature is usually -80 to 50°C. The reaction time is usually 0.1 to 200 hours.

(Steps 3 and 7)
Polymer (VII) can be obtained by adding compound (IX) to polymer (VI) or adding compound (V) to polymer (X). The amount of compound (IX) or (V) added is generally 0.01 to 100 molar equivalents, preferably 0.05 to 50 molar equivalents, relative to polymer (VI) or (X).

The reaction solvent is the same as in Steps 1 and 5. The reaction temperature is usually -80 to 50°C. The reaction time is usually 0.1 to 200 hours.

(Step 4)
Polymer (III) can be obtained from polymer (VI) by a known method for converting the "-Y-Br" structural moiety into the "-Y-R ¹¹ " structural moiety.

The intermediate compound in each of the above steps can be used as it is in the next step, or can be isolated and purified after completion of the reaction. For example, steps 1-3 and steps 5-7 can be performed in one pot without isolation and purification. When isolating and purifying, for example, common isolation procedures such as filtration, concentration, extraction, etc. are performed to separate the crude reaction product, which is then subjected to column chromatography, recrystallization, etc. It can be isolated and purified from the reaction mixture by subjecting it to common purification procedures.

The present invention also provides a phosphate compound detection agent containing the block copolymer of the present invention.

As used herein, the term "phosphate compound" means a compound to which phosphoric acid having anionic properties is bound, and is preferably a biological substance, such as adenosine monophosphate (AMP), adenosine diphosphate ( ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP) , uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP) and other ribonucleotides; deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate acid (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), Deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP) and other deoxynucleotides; Molecular phosphate compounds are included. Here, the nucleic acid is not limited to a double-stranded nucleic acid, and may be a single-stranded nucleic acid. Alternatively, it may be a nucleic acid having a higher-order structure such as a supercoil structure, a histone complex, or a triple-strand structure.

Since the phosphate compound detection agent of the present invention contains the block copolymer of the present invention, it can be obtained by measuring the fluorescence spectrum or the ultraviolet-visible light absorption spectrum in a solution containing the phosphate compound (1) fluorescence intensity , (2) the waveform of the UV-visible light absorption spectrum, or (3) the absorbance, the phosphate compound can be detected.

The present invention measures the fluorescence spectrum or ultraviolet-visible light absorption spectrum of the block copolymer of the present invention in a solution containing a phosphoric acid compound, thereby obtaining (1) fluorescence intensity, (2) ultraviolet-visible Provided is a method for detecting a phosphate compound, which includes detecting the phosphate compound by the waveform of the light absorption spectrum or (3) the difference in absorbance.

Phosphate compound detection is usually obtained from the spectrum of the block copolymer in the solution containing the phosphate compound and the spectrum of the block copolymer in the solution not containing the phosphate compound (1) fluorescence intensity, ( 2) by comparing the waveform of the UV-visible light absorption spectrum, or (3) the absorbance.

In the method for detecting a phosphate compound of the present invention, for example, a phosphate compound can be detected simply by dissolving the phosphate compound-detecting agent of the present invention or the block copolymer of the present invention in a solution to be detected. be.

Detectable temperatures are from 0°C to 100°C. The pH range of the detection target solution is, for example, pH 5.5 to 9.5.

The concentration of the phosphate compound in the solution to be detected is preferably 0.01 μM or higher, more preferably 0.1 μM or higher, even more preferably 1 μM or higher, and particularly preferably 10 μM or higher. .

The concentration of the block copolymer of the present invention required in the solution to be detected is preferably 0.01 mg/mL or higher, more preferably 0.1 mg/mL or higher.

The solvent of the solution to be detected is preferably a polar solvent in which the block copolymer of the present invention can be dissolved, such as water, methanol, dimethylsulfoxide (DMSO).

If the solution to be detected contains a negatively charged molecule such as phosphoric acid, it will interact electrostatically with the block copolymer and hinder the detection of the phosphoric acid compound, so it can be a target for detection. It is preferred that no negatively charged molecules are included other than the phosphate compound. The detection target solution may contain an inert salt. The required amount of the solution to be detected is not particularly limited as long as it allows the measurement of the ultraviolet-visible light absorption spectrum and fluorescence spectrum.

In one embodiment, (A) the fluorescence intensity emitted from the block copolymer when the block copolymer of the present invention is irradiated with excitation light in the presence of a phosphate compound, and (B) the absence of the phosphate compound. It has a characteristic that the fluorescence intensity emitted from the block copolymer is different from that emitted from the block copolymer when irradiated with light of the same wavelength. Therefore, various phosphate compounds can be detected based on the difference.

For example, when the phosphate compound is a nucleic acid such as DNA or RNA, the fluorescence intensity of (A) is much lower than that of (B). In a specific block copolymer, when the phosphate compound is ADP, the fluorescence intensity of (A) greatly exceeds the fluorescence intensity of (B). In a specific block copolymer, the fluorescence intensity of (A) greatly exceeds the fluorescence intensity of (B) even when the phosphate compound is AMP in addition to ADP, and when the phosphate compound is ATP, ( The fluorescence intensity of A) is much lower than that of (B).

In this embodiment, it can be detected by a known method of measuring the fluorescence spectrum at the time of detection, but it can also be detected using a device that can irradiate excitation light in a dark space (such as an illuminator). It is possible. As a specific operation, the above (A) and (B) are prepared as separate samples, and the fluorescence intensity of each is compared to detect the presence of the phosphate compound. At this time, if the amount of the phosphate compound is above a certain level, it can be easily detected even when visually compared with the naked eye.

Further, in the embodiment, the phosphate compound is electrophoresed using a gel, and the gel after electrophoresis is stained with an aqueous solution containing a block copolymer to remove the band (the portion where the phosphate compound is present) from other It is also possible to detect it as a signal that emerges from the gel portion of (phosphate compound-free region).

As the “excitation light” for the block copolymer during fluorescence spectrum measurement, light of any wavelength can be employed as long as it is light of a wavelength at which fluorescence emission occurs. For example, light with a wavelength of 450-750 nm can be employed.

In one embodiment, (A) the waveform of the UV-visible absorption spectrum of the block copolymer of the present invention in the presence of a phosphoric acid compound, and (B) the block copolymer of the present invention in the absence of a phosphoric acid compound. It has characteristics different from the waveform of the ultraviolet-visible light absorption spectrum. Therefore, various phosphate compounds can be detected based on the difference.

For example, when the phosphate compound is a nucleic acid such as DNA or RNA, the absorption peak derived from the thiophene block unit appearing at 450 to 550 nm (especially around 500 nm) is longer in the case of (A) than in (B). Shift to the wavelength side. A specific block copolymer shifts to the longer wavelength side even when the phosphate compound is ATP.

In this embodiment, a known technique for measuring ultraviolet-visible light absorption spectrum can be used.

In one embodiment, (A) the absorbance of the block copolymer of the present invention in the presence of a phosphoric acid compound and (B) the absorbance of the block copolymer of the present invention in the absence of a phosphoric acid compound are different. have. Therefore, various phosphate compounds can be detected based on the difference.

For example, when the phosphate compound is a nucleic acid such as DNA or RNA, the absorbance derived from the fluorene block unit appearing at 350 to 450 nm (especially around 400 nm) is significantly higher in case (A) than in case (B). Decrease. In a specific block copolymer, even when the phosphate compound is ATP, it is greatly reduced.

In this embodiment, a known technique for measuring ultraviolet-visible light absorption spectrum can be used.

The present invention will be further described in detail by the following examples, comparative examples and test examples, but these are not intended to limit the present invention and may be changed without departing from the scope of the present invention.

"Room temperature" in the following examples and comparative examples usually means about 10°C to about 35°C. % indicates % by mass unless otherwise specified. FAB-MS (fast atom bombardment ionization mass spectrometry) was measured using JMS-SX102A (JEOL Ltd.). Nitrobenzyl alcohol (NBA) was used as the matrix. IR was measured using Nicolet 6700 (Thermofisher Scientific). ¹ H NMR (proton nuclear magnetic resonance spectrum) was measured by Fourier transform NMR (JNM-ECZR (JEOL)) at room temperature. Elemental analyzes were determined using a Perkin Elmer PE 2400-II.

Examples 1-3
Poly{9,9-bis[6-(trimethylphosphonium)hexyl]fluorene}-b-poly{3-[6-(trimethylphosphonium)hexyl]thiophene} (PTMPHF-b-PTMPHT) (Example 1 = m: n=7:14; Example 2=m:n=4:16; Example 3=m:n=9:18)

(Step 1) Synthesis of 2-iodo-7-bromo-9,9-bis(6-bromohexyl)fluorene (BIBHF)

A Dimroth condenser and a nitrogen inlet tube were connected to a three-necked round-bottomed flask, and 2-iodo-7-bromofluorene (4.86 g, 1.30×10 ⁻² mol) and tetrabutylammonium bromide (TBAB) (5.02 g, 1.20×10 ⁻⁴ mol), 1,6-dibromohexane (30 g, 0.12 mol) and 50% NaOH aqueous solution (15 mL) were added. The mixture was stirred at 75° C. for 12 hours under nitrogen atmosphere. The resulting reaction solution was poured into dichloromethane (100 mL). This solution was extracted with saturated saline (100 mL) and purified water (100 mL), and the organic layer was distilled off with an evaporator. Next, in order to remove 1,6-dibromohexane, vacuum distillation was carried out at 100° C. and 0.5 kPa. Purification was performed by column chromatography (Wakogel C-300) using a mixed solvent of hexane/chloroform (v/v=9:1) as a developing solvent, and a crude product was recovered. Thereafter, recrystallization was performed using chloroform as a good solvent and methanol as a poor solvent. Suction filtration was performed using a membrane filter (hydrophilicity 0.1 μm) to obtain white crystals. After drying under reduced pressure at 40° C., BIBHF (5.34 g, yield 58%) was recovered.

FAB-MS: m/z = 695 (M ⁺ )
IR (KRS cm ⁻¹ ): 2960 (w, νC−H), 2810 (w, νC−H), 1610 (s, νC=C), 1430 (w, δC−H), 1254 (m, δC− H), 910 (s, δC=C), 558 (m, νC-Br)
¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 0.578 (4H, m, CH ₂ ), 1.08 (4H, m, CH ₂ ), 1.20 (4H, m, CH ₂ ), 1.64 (4H, quin, _CH2 ), 1.92 (4H, m, _CH2 ), 3.29 (4H, t, _CH2 ), 7.43 (2H, d, _JHH = 1.8 Hz, CH) , 7.46 (2H, dd, J _HH =8.0 Hz, J _HH =1.8 Hz, CH), 7.53 (2H, d, J _HH =8 Hz, CH).
Anal. Calcd. for (C25H31Br3I): C, _43.07 %; H, 4.34%; Br _{, 34.39} %. Found: C, 43.09%; H, 4.25%; S, 34.18%.

(Step 2) Synthesis of 3-(6-bromohexyl)thiophene (BHT)

3-bromothiophene (5.22 g, 3.20×10 ⁻² mol) and hexane (40 mL) were measured into a 500 mL three-necked flask, and 1,6-dibromohexane (8.78 g, 3.56× 10 ⁻² mol) and hexane (10 mL) were added. A 300 mL three-necked round-bottomed flask was equipped with a drain tube, a dropping funnel, a septum rubber, and a nitrogen inlet tube. As for the two nitrogen introduction tubes, silicon tubes were attached to both the IN and OUT sides of the nitrogen introduction, and were closed with pinch cocks. After removing from the glove and stirring at −78° C. for 10 minutes under an argon atmosphere, n-BuLi (20 mL) was added dropwise using a syringe, and the mixture was stirred for 10 minutes after dropping. After that, 5 mL of tetrahydrofuran (THF) was added dropwise and stirred for 1 hour. After the temperature was raised from -78°C to -10°C, 1,6-dibromohexane was added dropwise from the dropping funnel, and the mixture was stirred at room temperature for 2 hours. The reaction solution was quenched by adding methanol and dilute hydrochloric acid. After that, hexane was added, and the quenched solution was transferred to a 500 mL separating funnel and washed with purified water (100 mL×3 times). The obtained organic layer was dried with anhydrous magnesium sulfate. The solvent was distilled off with an evaporator to recover the crude product. Purification was performed using silica gel (Wakogel C-300) with hexanes. BHT (5.06 g, yield 64%) was then separated using silica gel (Wakogel FC-40).

FAB-MS: m/z = 246 (M ⁺ )
IR (KRS cm ⁻¹ ): 3090 (w, νC−H), 2938 (w, νC−H), 2857 (w, νC−H), 2360 (s, νC=C), 1425 (w, δC− H), 772 (m, νC-Br)
¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 1.40 (2H, q, CH ₂ ), 1.48 (2H, q, CH ₂ ), 1.74 (2H, t, CH ₂ ), 1.86 (2H, t, _CH2 ), 2.77 (2H, t, _CH2 ), 3.31 (2H, d, _CH2 ), 6, 92 (2H, d, _JHH = 1.8 Hz, CH) , 7.32 (H, dd, J _HH =8.0 Hz, J _HH =1.8 Hz, CH).
Anal. Calcd. for ( _C10H15SBr ): C, 59.24% _; H, 7.46%; Br, 17.49%. Found: C, 59.20%; H, 7.47%, Br, 17.46%.

(Step 3) Synthesis of 2,5-dibromo-3-(6-bromohexyl)thiophene (DBrBHT)

Under a nitrogen atmosphere, BHT (8.29 g, 3.36×10 ⁻² mol) and N,N-dimethylformamide (DMF) (10 mL) were added to a 300 mL two-necked round bottom flask, and DMF (40 mL) was added to the dropping funnel. N-bromosuccinimide (NBS) (11.96 g, 6.72×10 ⁻² mol) dissolved in rt was added and a reflux tube was attached. It was immersed in a low-temperature constant temperature bath set at 0° C., and NBS was added over 2 hours. After that, the reaction was continued for another 2 hours. After completion of the reaction, the mixture was neutralized with a saturated NaHCO ₃ aqueous solution, added with diethyl ether, and extracted with purified water and saturated brine. After concentrating with an evaporator, purification was performed using silica gel (Wakogel C-300) with hexane as a single solvent. Separation of DBrBHT (6.12 g, 45% yield) was then performed using silica gel (Wakogel FC-40).

FAB-MS: m/z = 404 (M ⁺ )
IR (KRS cm ⁻¹ ): 3085 (w, νC−H), 2942 (w, νC−H), 2862 (w, νC−H), 1550 (s, νC=C), 1440 (w, δC− H), 1360 (m, δC-H), 794 (m, νC-Br)
¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 1.48 (2H, q, CH ₂ ), 1.51 (2H, q, CH ₂ ), 1.66 (2H, t, CH ₂ ), 1.79 (2H, t, _CH2 ), 2.53 (2H, t, _CH2 ), 3.31 (2H, d, _CH2 ), 6, 92 (H, d, _JHH = 1.8 Hz, CH) .
Anal. Calcd. for ( _C10H13SBr3 ): C, _29.66 % _; H, 3.24%; Br, 59.19%. Found: C, 29.34%; H, 3.12%, Br, 59.70%.

(Step 4) Synthesis of poly[9,9-bis(6-bromohexyl)fluorene]-b-poly[3-(6-bromohexyl)thiophene] (PBHF-b-PBHT)

DBrBHT was dissolved in THF (4 mL) in a 10 mL eggplant flask in a glove box, 1 ^PrMgCl.LiCl was added, and the mixture was reacted in an ice bath at 0° C. for 2 hours. BIBHF and 1,4-bis(hexyloxy)benzene (DHB) were placed in a 50 mL eggplant flask, dissolved in THF (10 mL), and ⁱ PrMgCl·LiCl was added. Removed from the glove box, reacted for 1 hour with stirring in a low-temperature constant temperature bath set at −20° C., added 2 mL of the prepared THF (5 mL) solution of Ni(acac) ₂ /dppp using a syringe, and added ice bath 0. C. for 30 minutes. After the reaction, all the Grignard solution of DBrBHT was added into the polymerization solution of BIBHF and reacted at room temperature for 30 minutes. After completion of the reaction, the product was quenched by adding it to a mixed solution of 10 mL of hydrochloric acid and 300 mL of methanol, recovered by suction filtration, and then reprecipitated with a good solvent of chloroform and a poor solvent of hexane for purification. It was recovered by suction filtration to recover yellow powdery PBHF-b-PBHT.

Usage amounts of BIBHF, 1,4-bis(hexyloxy)benzene (DHB), ⁱ PrMgCl·LiCl, Ni(acac) ₂ /dppp, DBrBHT in the production of intermediate PBHF-b-PBHT in Examples 1-3 , and the yield of PBHF-b-PBHT are as follows.

The degree of polymerization (m:n), number average molecular weight (M _n ), and molecular weight dispersity (M _w /M _n ) of each PBHF-b-PBHT were determined from GPC and ¹ H NMR measurements. The results are as follows.

The measurement method of GPC is as follows.

(GPC measurement method)
PBHF-b-PBHT (1 mg) was dissolved in high performance liquid chromatography (HPLC) grade THF containing 3,5-di(tert-butyl)-4-hydroxytoluene (BHT) (0.03 wt%), A solution was prepared. The solution was filtered using a syringe fitted with a sample preparation (MILLIPORE MillexFG 0.45 μm) and the sample was prepared and measured. Number average molecular weight (M _n ) and molecular weight dispersity (M _w /M _n ) were calculated based on a calibration curve prepared using polystyrene as a standard sample. HLC-8320GPC (TOSOH) was used as a measuring device.

(Example 1=m:n=7:14)
IR (KRS cm ⁻¹ ): 3064 (w, νC−H), 2963 (w, νC−H), 2888 (w, νC−H), 1483 (s, νC=C), 1241 (m, δC− H), 680 (m, νC-Br), 604 (s, δC=C).
¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 0.832 (4H, m, CH ₂ ), 1.18 (4H, m, CH ₂ ), 1.21 (4H, m, CH ₂ ), 1.68 (4H, quin, _CH2 ), 1.92 (4H, m, _CH2 ), 2.16 (4H, m, _CH2 ), 2.90 (4H, m, _CH2 ), 3.29 (4H , t, CH ₂ ), 3.34 (2H, t, CH ₂ ), 7.02 (2H, d, J _HH =1.8 Hz, CH), 7.88 (2H, dd, J _HH =8. 0 Hz, J _HH =1.8 Hz, CH), 7.94 (2H, d, J _HH =8 Hz, CH).
Anal. Calcd. for _{( C315H434Br28S14} ): C, _57.00 %; H, 6.15%; Br, _32.50 %. Found: C, 57.42%; H, 6.32. %; Br, 31.89%.

(Step 5) Synthesis of poly{9,9-bis[6-(trimethylphosphonium)hexyl]fluorene}-b-poly{3-[6-(trimethylphosphonium)hexyl]thiophene} (PTMPHF-b-PTMPHT)

PBHF-b-PBHT (4.66×10 ⁻¹ g, 1.58×10 ⁻⁴ mol) was dissolved in a mixed solvent of THF:DMF=10 mL:10 mL in a 50 mL eggplant flask, and trimethylphosphine (6 .32 mL, 6.32×10 ⁻³ mol) was added and stirred at 80° C. for 24 hours under an argon atmosphere. Thereafter, DMSO (10 mL) was added to partially promote phosphoniumation, dissolve the precipitated polymer, and react for an additional 24 hours. After completion of the reaction, the solution was cooled to room temperature and reprecipitated in diethyl ether. This was filtered using a membrane filter (hydrophobicity 0.1 μm) to recover PTMPHF-b-PTMPHT (4.99×10 ⁻¹ g, yield 83%) as a red-orange solid.

(Example 1=m:n=7:14)
IR (KRS cm ⁻¹ ): 2992 (w, νC-H), 2862 (w, νC-H), 2520 (s, νP-H), 1484 (w, δC-H), 1032 (m, δP- C), 764 (m, νPC).
¹ H NMR (500 MHz, CDCl ₃ ) δppm: 0.835 (4H, m, CH ₂ ), 1.28 (8H, m, CH ₂ ), 1.31 (4H, m, CH ₂ ), 1.66 (6H, _CH2 , _CH2 ), 1.72 (2H, q, _CH2 ), 1.87 (18H, m, _CH2 ), 1.92 (9H, m, _CH2 ), 2.06 ( 4H, q, _CH2 ), 2.12 (6H, _CH2 , _CH2 ), 2.89 (2H, t, _CH2 ), 7.10 (2H, d, _JHH = 1.8 Hz, CH) , 7.91 (4H, dd, J _HH =8.0 Hz, J _HH =1.8 Hz, CH), 7.95 (2H, d, J _HH =8 Hz, CH).
Anal. Calcd. for _{( C409H686P28Br28S14} ): C, _54.72 %; H, _7.51 %; Br, _24.82 %. Found: C, 54.12%; H, 7.22%; Br, 24.98%.

Comparative example 1
Poly(9,9-dihexylfluorene)-b-poly{3-[6-(trimethylphosphonium)hexyl]thiophene} (PHF-b-PTMPHT) (m:n=6:25)

(Step 1) Synthesis of 2-iodo-7-bromo-9,9-dihexylfluorene (BIHF)

A Dimroth condenser and a nitrogen inlet tube were connected to a three-necked round bottom flask, and 2-iodo-7-bromofluorene (12.5 g, 3.37×10 ⁻² mol) and TBAB (3.62 g, 1.13×10 ⁻² ) were added. ² mol), 1-bromohexane (11.7 g, 7.08×10 ⁻² mol) and 50% NaOH aqueous solution (75 mL) were added. The mixture was stirred at 75° C. for 12 hours under nitrogen atmosphere. The reaction solution was poured into dichloromethane (100 mL). This solution was extracted with saturated saline (100 mL) and purified water (100 mL), and the organic layer was distilled off with an evaporator. Next, in order to remove 1-bromohexane (b.p. 247°C), vacuum distillation was carried out at 100°C and 0.5 kPa. Purification was performed by column chromatography (Wakogel C-300) using a hexane/chloroform (v/v=9:1) mixed solvent as a developing solvent, and a component with an Rf value of 0.81 was recovered. Thereafter, recrystallization was performed using chloroform as a good solvent and methanol as a poor solvent. Suction filtration was performed using a membrane filter (hydrophilicity 0.1 μm) to obtain white crystals. After drying under reduced pressure at 40° C., BIHF (15.1 g, yield 83%) was recovered.

FAB-MS: m/z = 540 (M ⁺ ), IR (KRS cm ^-1 ): 2954 (w, νC-H), 2851 (w, νC-H), 1598 (s, νC = C), 1428 ( w, δC-H), 1250 (m, δC-H), 902 (s, δC=C).
¹ H NMR (500 MHz, CDCl ₃ ) δppm: 7.54 (d, J = 8.9 Hz, 2H), 7.50-7.45 (m, 4H), 1.99-1.91 (m, 4H ), 1.21-1.02 (m, 12H), 0.83 (t, J=7.0Hz, 6H), 0.68-0.58 (m, 4H).
Anal. Calcd. for ( _C25H33BrI ): C, _77.65 %; H, 10.86%. Found: C, 77.93%; H, 9.71%.

(Step 2) Synthesis of 3-(6-chlorohexyl)thiophene (CHT)

In a glove box THF (40 mL), Mg (1.28 g, 5.25×10 ⁻² mol) are weighed into a 300 mL round bottom flask, 3-bromothiophene (7.47 g, 4.58×10 ⁻² mol). , Ni(dppp)Cl ₂ (0.905 g, 1.67×10 ⁻³ mol), THF (20 mL) were weighed into a 50 mL sample bottle, 1-bromo-6-chlorohexane (9.98 g, 5.00× 10 ⁻² mol), THF (40 mL) was weighed into the addition funnel using a foot length funnel. A dropping funnel was attached to the left of the three-necked round-bottom flask, a Dimroth fitted with two nitrogen inlet tubes was attached to the center, and a septum rubber was attached to the right. A silicon tube was attached to both IN and OUT of nitrogen introduction and closed with a pinch cock. After removing from the glove, the 1-bromo-6-chlorohexane solution was added dropwise from the dropping funnel in a nitrogen atmosphere over about 2 hours, and the mixture was further stirred at 0° C. for 2 hours. Furthermore, a previously prepared THF solution of 3-bromothiophene and Ni catalyst was slowly dropped into the three-necked round-bottomed flask that had been returned to room temperature using a syringe, and the mixture was heated under reflux for 12 hours. After completion of the reaction, quenching was performed with methanol and dilute hydrochloric acid, hexane was added, and the mixture was washed and extracted with NaHCO ₃ saturated aqueous solution (100 mL×3 times), NaCl saturated aqueous solution (100 mL×3 times), and purified water (100 mL×3 times) in this order. . The obtained organic layer was dried with magnesium sulfate, gravity filtered, the solvent was distilled off with an evaporator, and the crude product was recovered. After that, purification was performed using silica gel (Wakogel C-300) with hexane as a single solvent. CHT (6.69 g, yield 72%) was then separated using silica gel (Wakogel FC-40).

FAB-MS: m/z = 202 (M ⁺ )
IR (KRS cm ⁻¹ ): 3118 (w, νC−H), 2955 (w, νC−H), 2870 (w, νC−H), 1510 (s, νC=C), 1475 (w, δC− H), 1340 (m, δC-H), 782 (m, νC-Br)
¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 1.32 (4H, q, CH ₂ ), 1.74 (2H, t, CH ₂ ), 1.86 (2H, t, CH ₂ ), 2.82 (2H, m, _CH2 ), 3.64 (2H, d, _CH2 ), 6, 92 (2H, d, _JHH = 1.8 Hz, CH), 7.32 (H, dd, _JHH = 8.0 Hz, J _HH =1.8 Hz, CH).
Anal. Calcd. for ( _C10H15SCl ): C, 59.24% _; H, 7.46%; Cl, 17.49%. Found: C, 59.17%; H, 7.40%, Cl, 17.32%.

(Step 3) Synthesis of 2,5-dibromo-3-(6-chlorohexyl)thiophene (DBrCHT)

CHT (6.31 g, 2.76×10 ⁻² mol) and DMF (90 mL) were added to a 300 mL round-bottomed flask under a nitrogen atmosphere and fitted with a reflux tube. It was immersed in a low-temperature constant temperature bath set at 0° C., and a DMF solution of NBS (10.8 g, 6.06×10 ⁻² mol) was added dropwise from a dropping funnel over 2 hours. After dropping, the reaction was allowed to proceed for 2 hours. After completion of the reaction, the mixture was neutralized with a saturated _NaHCO3 aqueous solution, extracted with diethyl ether, and washed with purified water and a saturated NaCl aqueous solution. After concentrating the organic layer with an evaporator, column chromatography was performed using silica gel (Wakogel C-300) using hexane as a developing solvent. Furthermore, separation of DBrCHT (6.95 g, yield 65%) was performed using silica gel (Wakogel FC-40).

FAB-MS: m/z = 360 (M ⁺ )
IR (KRS cm ⁻¹ ): 3082 (w, νC−H), 2935 (w, νC−H), 2882 (w, νC−H), 1486 (s, νC=C), 1425 (w, δC− H), 1375 (m, δC-H), 852 (m, νC-Br)
¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 1.34 (2H, q, CH ₂ ), 1.48 (2H, q, CH ₂ ), 1.59 (2H, q, CH ₂ ), 1.77 (2H, q, _CH2 ), 2.54 (2H, t, _CH2 ), 3.54 (2H, t, _CH2 ), 6, 92 (H, d, _JHH = 1.8 Hz, CH) .
Anal. Calcd. for ( _C10H13SBr2Cl ): C, _43.07 % _; H, 4.34%; Cl, 34.39%. Found: C, 43.09%; H, 4.25%, Cl, 34.18%.

(Step 4) Synthesis of poly(9,9-dihexylfluorene)-b-poly[3-(6-chlorohexyl)thiophene] (PHF-b-PCHT)

BIHF (4.31×10 ⁻¹ g, 8.02×10 ⁻⁴ mol), 1,4-bis(hexyloxy)benzene (DHB) (8.90×10 ^{− 2} g, 3.20×10 ⁻⁴ mol) was added and dissolved in THF (10 mL). It was taken out of the glove box and reacted for 1 hour with stirring in a low-temperature constant temperature bath set at -20°C. Then, 1 mL of the prepared THF solution of Ni(acac) ₂ /dppp (4.0 mL, 8.00×10 ⁻⁵ mol) was added to the Grignard solution of BIHF using a syringe, and stirred at 0° C. in an ice bath. React for 30 minutes. Separately, DBrCHT (8.65×10 ⁻¹ g, 2.40×10 ⁻³ mol), 1,4-bis(hexyloxy)benzene (DHB) (2.51×10 ^{− 1} g, 9.02×10 ⁻⁴ mol) and THF (4 mL) were added, and after dissolution, ⁱ PrMgCl·LiCl was added and reacted at 0° C. ice bath for 1 hour. After the reaction, it was added to the fluorene Grignard solution using a syringe and allowed to react at room temperature for 30 minutes. After completion of the reaction, the product was quenched by adding it to a mixed solution of hydrochloric acid (10 mL) and methanol (300 mL), collected by suction filtration, and then reprecipitated with a good solvent, chloroform, and a poor solvent, hexane, for purification. It was recovered by suction filtration to recover yellow powdery PHF-b-PCHT.

The degree of polymerization (m:n), number average molecular weight (M _n ), and molecular weight dispersity (M _w /M _n ) of PHF-b-PCHT were determined from GPC and ¹ H NMR measurements. The results are as follows.

The GPC measurement method is the same as described above.

IR (KRS cm ⁻¹ ): 3076 (w, νC−H), 2953 (w, νC−H), 2868 (w, νC−H), 1542 (s, νC=C), 1481 (s, νC= C), 1241 (m, δC-H), 832 (m, νC-Cl), 689 (s, δC=C).
¹ H NMR (500 MHz, CDCl ₃ ) δppm: 0.890 (6H, CH _{2 ,} CH ₂ ), 1.08 (4H, m, CH ₂ ), 1.34 (6H, m, CH ₂ ), 1. 58 (2H, quin, _CH2 ), 1.84 (8H, q, _CH2 ), 2.14 (4H, t, _CH2 ), 2.80 (2H, t, _CH2 ), 3.34 ( 2H, t, _CH2 ), 7.02 (H, d, _JHH = 1.8Hz, CH), 7.80 (4H, dd, _JHH = 8.0Hz, _JHH = 1.8Hz, CH) , 7.94 (2H, d, J _HH =8 Hz, CH). Anal. Calcd. for ( _{C400H579Cl25S25} ): C, _78.54 %; H, _8.85 %; Cl, _6.62 %. Found: C, 78.40%; H, 8.60. %; Cl, 6.33%.

(Step 5) Synthesis of poly(9,9-dihexylfluorene)-b-poly{3-[6-(trimethylphosphonium)hexyl]thiophene} (PHF-b-PTMPHT)

PHF-b-PCHT (2.00×10 ⁻¹ g, 2.20×10 ⁻⁵ mol) was dissolved in a mixed solvent of THF:DMF=10 mL:10 mL in a 50 mL eggplant flask, and trimethylphosphine (0 .96 mL, 0.96×10 ⁻³ mol) was added and stirred at 80° C. for 24 hours under a nitrogen atmosphere. After 24 hours, DMSO (10 mL) was added to partially phosphonate, dissolve the precipitated polymer, and react for an additional 24 hours. After completion of the reaction, the solution was cooled to room temperature and reprecipitated in diethyl ether. This was filtered using a membrane filter (hydrophobicity 0.1 μm) to recover PHF-b-PTMPHT (3.30×10 ⁻¹ g, yield 89%) as a red-orange solid.

IR (KRS cm ^-1 ): 3003 (w, νC-H), 2930 (w, νC-H), 2513 (w, νP-H), 1492 (s, δC-H), 1035 (m, δP- C), 760 (m, νPC)
¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 0.812 (14H, CH _{2 ,} CH ₂ ), 1.26 (10H, CH _{2 ,} CH ₂ ), 1.71 (2H, m, CH ₂ ), 1 .64 (4H, quin, _CH2 ), 1.92 (4H, m, _CH2 ), 7.04 (H, d, J _HH = 1.8 Hz, CH), 7.98 (2H, dd, J _HH = 8.0 Hz, J _HH = 1.8 Hz, CH), 8.06 (2H, d, J _HH = 8 Hz, CH).
Anal. Calcd. for ( _{C475H804P25I25S25} ): _C , _64.94 %; H, _8.03 %; Br, _18.06 %. Found: C, 64.80%; H, 7.80%; Br, 18.72%.

Comparative example 2
Poly{9,9-bis[6-(trimethylphosphonium)hexyl]fluorene}-b-poly(3-hexylthiophene) (PTMPHF-b-PHT) (m:n=16:15)

(Step 1) Synthesis of 2,5-dibromo-3-hexylthiophene (DBrHT)

Under a nitrogen atmosphere, 3-hexylthiophene (6.31 g, 3.75×10 ⁻² mol) and DMF (30 mL) were added to a 300 mL round bottom flask, NBS dissolved in DMF was added to the dropping funnel, and the reflux tube was added. attached. It was immersed in an ice bath and NBS (13.3 g, 7.50×10 ⁻² mol) was added over 2 hours. The reaction is carried out at ambient temperature for an additional 16 hours. After completion of the reaction, neutralization was performed with a saturated multi-layer solution, diethyl ether was added, and extraction was performed with purified water and saturated brine. After concentrating with an evaporator, purification was performed by column chromatography using silica gel (Wakogel C-300) using hexane as a developing solvent. Furthermore, silica gel (Wakogel FC-40) was used to purify DBrHT (7.46 g, yield 61%).

FAB-MS: m/z = 326 (M ⁺ )
IR (KRS cm ⁻¹ ): 3088 (w, νC−H), 2949 (w, νC−H), 2880 (w, νC−H), 1557 (s, νC=C), 1435 (w, δC− H), 1365 (m, δCH), 1196 (m, νCOC), 1087.
¹ H NMR (500 MHz, CDCl ₃ ) δppm: 0.920 (3H, m, CH ₃ ), 1.32 (6H, m, CH ₂ ), 1.59 (2H, m, CH ₂ ), 7.28 (2H, d, J _HH =1.8 Hz, CH).
Anal. Calcd. for ( _C10H14SBr2 ): C, _77.65 % _; H, 10.86%. Found: C, 77.93%; H, 9.71%.

(Step 2) Synthesis of poly[9,9-bis(6-bromohexyl)fluorene]-b-poly(3-hexylthiophene) (PBHF-b-PHT)

In a glove box, DBrHT (1.64×10 ⁻¹ g, 5.02×10 ⁻⁴ mol) was dissolved in THF (4 mL) in a 10 mL eggplant flask, and ⁱ PrMgCl·LiCl (0.770 mL, 1. 00×10 ⁻³ mol) was added and reacted at room temperature for 1 hour. BIBHF (6.97×10 ⁻¹ g, 1.02×10 ⁻³ mol) and 1,4-bis(hexyloxy)benzene (DHB) (1.11×10 ⁻¹ g, 4.9×10 −1 g, 1.02×10 −3 mol) are placed in a 50 mL eggplant flask. 08×10 ⁻⁴ mol) and THF (10 mL) were added, and after dissolution, ⁱ PrMgCl·LiCl (0.770 mL, 1.00×10 ⁻³ mol) was added. It was taken out of the glove box and reacted for 1 hour with stirring in a low-temperature constant temperature bath set at -20°C. Thereafter, the prepared THF solution of Ni(acac) ₂ /dppp (2.0 mL, 4.00×10 ⁻⁵ mol) was added using a syringe, and the mixture was stirred in an ice bath at 0° C. and reacted for 30 minutes. Then all the Grignard solution of DBrHT was added and allowed to react for 30 minutes at room temperature. After completion of the reaction, the product was quenched by adding it to a mixed solution of hydrochloric acid (10 mL) and methanol (300 mL), collected by suction filtration, and then reprecipitated with a good solvent, chloroform, and a poor solvent, hexane, for purification. It was recovered by suction filtration to recover yellow powdery PBHF-b-PHT (0.23 g, yield 33%).

The degree of polymerization (m:n), number average molecular weight (M _n ), and molecular weight dispersity (M _w /M _n ) of PBHF-b-PHT were determined from GPC and ¹ H NMR measurements. The results are as follows.

The GPC measurement method is the same as described above.

IR (KRS cm ⁻¹ ): 3082 (w, νC−H), 2932 (w, νC−H), 2845 (w, νC−H), 1493 (s, νC=C), 1241 (w, δC− H), 770 (m, νC-Br)
¹ H NMR (500 MHz, CDCl ₃ ) δ ppm: 0.892 (7H, CH _{2 ,} CH ₃ ), 1.27 (6H, t, CH ₂ ), 1.55 (2H, m, CH ₂ ), 1. 86 (8H, _CH2 ), 2.24 (4H, q, _CH2 ), 2.78 (2H, t, _CH2 ), 3.31 (4H, t, _CH2 ), 6.98 (H, d, _JHH = 1.8Hz, CH), 7.64 (4H, dd, _JHH = 8.0Hz, _JHH = 1.8Hz, CH), 7.72 (2H, d, _JHH = 8Hz, CH).
Anal. Calcd. for _{( C550H722Br32S15} ): C, _63.83 %; H, 7.04%; Br, _24.26 %. Found: C, 63.96%; H, 7.18%; Br, 24.12%.

(Step 3) Synthesis of poly{9,9-bis[6-(trimethylphosphonium)hexyl]fluorene}-b-poly(3-hexylthiophene) (PTMPHF-b-PHT)

PBHF-b-PHT (2.00 × 10 ^-1 g, 2.20 × 10 ^-5 mol) was dissolved in a mixed solvent of THF: DMF = 10 mL: 10 mL in a 50 mL eggplant flask, and trimethylphosphine (0.96 g , 0.96×10 ⁻³ mol) was added and stirred at 80° C. for 24 hours under an argon atmosphere. After 24 hours had passed, DMSO (10 mL) was added to dissolve the polymer precipitated by partial phosphoniumation, and the reaction was allowed to proceed for an additional 24 hours. After completion of the reaction, the solvent was distilled off using a vacuum evaporator, and the residue was dissolved in a small amount of methanol and reprecipitated in diethyl ether. This was filtered using a membrane filter (hydrophobicity 0.1 μm) to recover PTMPHF-b-PHT (3.30×10 ⁻¹ g, yield 89%) as a red-orange solid.

IR (KRS cm ⁻¹ ): 2992 (w, νC−H), 2870 (w, νC−H), 2506 (w, νP−H), 1551 (s, νC=C), 1497 (w, δC− H), 1375 (m, δC-H), 1040 (m, δP-C), 702 (m, νP-C)
¹ H NMR (500 MHz, CDCl ₃ ) δppm: 0.820 (11H, CH _{2 ,} CH ₃ ), 1.26 (8H, CH ₂ ), 1.41 (4H, m, CH ₂ ), 1.92 ( 22H, CH2 _{, CH3} ), 2.12 (8H, m, _CH2 ), 6.97 (H, d, _JHH = 1.8 Hz, CH), 7.94 (4H, dd, _JHH = 8.0 Hz, J _HH =1.8 Hz, CH), 8.06 (2H, d, J _HH =8 Hz, CH).
Anal. Calcd. for _{( C646H1010P32Br32S15} ): C, _60.74 %; H, _7.96 %; Br, _19.71 %. Found: C, 60.90%; H, 8.11%; Br, 19.57%.

(Test Example 1: Characterization of various phosphoric acid compounds by ultraviolet-visible (UV-vis) light absorption spectrum measurement)
Evaluation by UV-vis light absorption spectra of the block copolymers of Examples 1 to 3 and Comparative Examples 1 and 2 in TRIZMA buffer solutions containing or not containing phosphoric acid compounds (ATP, ADP, AMP, DNA or RNA) did

A master solution of each block copolymer and a master solution of ATP, ADP, AMP, DNA, or RNA were mixed in the TRIZMA buffer solution so as to have the following predetermined concentrations, and stirred at room temperature for 3 minutes to obtain a sample. A solution was prepared and then placed in a quartz cell to measure the UV-vis light absorption spectrum under the following measurement conditions.

(sample solution)
The pH of the TRIZMA buffer solution was adjusted to pH=7.4 for the block copolymers of Examples 1 to 3, and to pH=8.0 for the block copolymers of Comparative Examples 1 and 2. The block copolymer solution of Comparative Example 2 contained 1% by volume of DMSO. The block copolymer concentration was P ⁺ =1 μM for the block copolymers of Examples 1 to 3, and 3.86×10 ⁻⁷ M for the block copolymers of Comparative Examples 1 and 2. The concentration of ATP, ADP, AMP, DNA or RNA was P ⁻ =1 μM for the block copolymers of Examples 1-3 and 5.00× for the block copolymers of Comparative Examples 1 and 2. 10 ⁻² mg/mL.

(Measurement condition)
Optical absorption spectrometer: SHIMADZU UV-PC3100
Measurement mode: Absorbance Scan speed: Low Slit width: 2.0

The results are shown in Figures 1-5. "Blank" means a solution containing no phosphate compound.

In the obtained light absorption spectrum, the block copolymer of Example 2 (m:n=4:16) has a maximum absorption peak derived from the fluorene block unit at 398 nm, and the block copolymer of Example 1 (m: n = 7:14) at 410 nm, and the block copolymer of Example 3 (m:n = 9:18) at 412 nm. 1-3). As the ratio of thiophene to fluorene increases, the absorption peak derived from the thiophene block unit increases, suggesting that the structure in aqueous solution changes.

In the block copolymer of Comparative Example 1, an absorption peak derived from the fluorene block unit was observed near 380 nm, and a peak derived from the thiophene block unit was observed near 470 nm (Fig. 4). In the block copolymer of Comparative Example 2, an absorption peak derived from the fluorene block unit was observed at about 400 nm, and an absorption peak derived from the thiophene block unit was observed at around 500 nm (Fig. 5).

In addition, in the block copolymers of Examples 1 to 3, after the addition of DNA and RNA, a significant decrease in absorption derived from the fluorene block unit and an increase in the wavelength of the maximum absorption derived from the thiophene block unit were observed (Fig. 1-3). It is believed that the electrostatic interaction with biomolecules eased aggregation of the block copolymer and extended the effective conjugation length.

In the block copolymer of Example 3 (m:n=9:18), even when ATP was added, the absorption derived from the fluorene block unit was similarly greatly reduced, and the maximum absorption wavelength derived from the thiophene block unit was lengthened. was seen (Fig. 3). From this result, it was found that the absorption wavelength of the absorption spectrum for ATP changes depending on the copolymerization ratio of the block copolymer.

In the block copolymer of Comparative Example 1, similarly, after the addition of DNA, a significant decrease in absorption derived from the fluorene block unit and a lengthening of the maximum absorption derived from the thiophene block unit were observed. After the addition of RNA, the absorption maxima from the thiophene block units were slightly lengthened (Fig. 4).

On the other hand, in the block copolymer of Comparative Example 2, after the addition of DNA and RNA, the absorbance derived from the fluorene block units increased in contrast to the block copolymers of Examples 1 to 3, and ATP, ADP, and AMP increased. A decrease in absorbance was observed after the addition. Further, in the block copolymer of Comparative Example 2, after the addition of DNA and RNA, no change in the absorption position was observed as observed in the block copolymers of Examples 1 to 3 (Fig. 5).

From the above results, in the block copolymers of Examples 1 to 3, when DNA and RNA were added, a significant decrease in absorption derived from the fluorene block unit and a lengthening of the maximum absorption wavelength derived from the thiophene block unit were observed. Therefore, it is possible to detect DNA and RNA using this phenomenon. In the block copolymer of Example 3, a similar phenomenon is observed even when ATP is added, so ATP can be detected using this phenomenon.

(Test Example 2: Characterization of various phosphoric acid compounds by fluorescence spectrum measurement)
The fluorescence spectra of the block copolymers of Examples 1 to 3 and Comparative Examples 1 and 2 in TRIZMA buffer solutions containing or not containing phosphate compounds (ATP, ADP, AMP, DNA or RNA) were evaluated.

Using the sample solution prepared in Test Example 1, fluorescence spectra were measured under the following measurement conditions.

(Measurement condition)
Fluorescence spectrometer: HITACHI F-4500
Excitation wavelength: 411 nm
Fluorescence start wavelength: excitation wavelength + 5 nm
Fluorescence end wavelength: 800 nm
Scanning speed: 100 nm/min
Response: 0.5sec
Data capture interval: 5.0 nm
Scanning speed: 240 nm/min
Photomultiplier voltage: 950V

The results are shown in Figures 6-10. "Blank" means a solution containing no phosphate compound.

All the block copolymers of Examples 1-3 exhibited turn-off behavior with respect to DNA and RNA (FIGS. 6-8). The block copolymer of Example 2 (m: n = 4: 16) showed a slight turn-on behavior with respect to ATP, ADP, and AMP, and the block copolymer of Example 1 (m: n = 7:14) showed Turn-on behavior with respect to ATP and ADP, and the block copolymer of Example 3 (m:n=9:18) showed Turn-off behavior with respect to ATP. Turn-on behavior was exhibited for AMP and ADP (Figs. 6-8).

In the block copolymer of Comparative Example 1, there was no change in behavior with respect to AMP, and turn-on behavior was observed with respect to DNA, RNA, ATP and ADP (Fig. 9). The block copolymer of Comparative Example 2 showed turn-on behavior only for DNA, and turned-off behavior for others (FIG. 10).

These results show that all the block copolymers of Examples 1-3 behave differently than the block copolymers of Comparative Examples 1 and 2. In addition, it was found that the recognition ability can be changed by controlling the degree of polymerization of the block copolymer. In particular, the block copolymer (m:n=9:18) of Example 3 exhibits different behaviors with ATP and AMP or ADP, so that low-molecular-weight phosphate compounds such as ATP, ADP, and AMP It can be expected to be used as a selective detection agent according to the number of acid groups.

(Test Example 3: pH dependence evaluation by fluorescence spectrum measurement)
The block copolymer of Example 3 (m :n=9:18) were evaluated. A sample solution was prepared in the same manner as in Test Example 1 except that the concentration of the block copolymer was 5 μM, and the fluorescence spectrum was measured under the same measurement conditions as in Test Example 2. The results are shown in Figures 11-13. "Blank" means a solution containing no ATP.

A decrease in fluorescence intensity was confirmed when ATP was added under all pH conditions, and the fluorescence decrease rate increased as the pH value increased. The decrease rate of fluorescence intensity was largest at pH=9.0, and I ₀ /I=2.4. In a previous report, a thiophene block unit having a side chain having a methyl group at the 4-position and a triphenylphosphine group at the end of the 3-position has an I ₀ / It has been reported to quench to I = 4 or less (Huang, B.; Geng, Z.; Yan, S.; Li, Z.; Cai, J.; Wang, Z. Anal. Chem. 2017, 89 , 8816.). However, in ATP sensing using the block copolymer of Example 3, only quenching of about I ₀ /I=2 was confirmed. The reason for this is thought to be that fluorescence energy transition transfer occurs in the fluorene block units of the block copolymer of Example 3, and there is a limit to the decrease in fluorescence intensity due to concentration quenching.

(Test Example 4: solubility evaluation)
Each of the block copolymers of Examples 1 to 3 and Comparative Examples 1 and 2 is dissolved at a concentration of 1 mg/mL in methanol, DMSO, water, acetone, chloroform, DMF, and THF at 40°C. Thus, the solubility in various solvents was evaluated.

Results are shown in the table below. Complete dissolution is expressed as "dissolved", and complete indissolution is expressed as "insoluble".

From the above results, it was found that the block copolymer of the present invention exhibits high solubility in polar solvents such as water, methanol, and dimethylsulfoxide (DMSO). Therefore, the block copolymer of the present invention is generally useful as a detection agent for highly polar biological substances.

The block copolymer of the present invention is useful for detecting phosphate compounds such as ATP, ADP, AMP, DNA or RNA.

Claims (9)

Formula (I):

(In the formula,
R ¹ to R ⁶ each independently represent -Y-R ¹¹ , hydrogen atom, halogen atom, hydrocarbon group having 1 to 10 carbon atoms, -NR ¹² R ¹³ , -OR ¹² , -SR ¹² , - COR ¹² , —SO ₂ R ¹² , —NR ¹³ COR ¹² , —CONR ¹² R ¹³ , —OCOR ¹² , —COOR ¹² , —NR ¹³ CSR ¹² , —CSNR ¹² R ¹³ , —SCOR ¹² or —COSR ¹² wherein R ⁷ and R ⁸ are each independently —Y—R ¹¹ ;
Y represents a spacer structure having 1 to 30 atoms in the main chain,
R ¹¹ represents a cationic nitrogen-containing group or a cationic phosphorus-containing group,
R ¹² represents a hydrocarbon group having 1 to 10 carbon atoms,
R ¹³ represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms,
n represents 2 to 100,
Each of the n monomer units is the same or different. )
A block unit represented by
Formula (II):

(In the formula,
R ⁹ and R ¹⁰ each independently represent -Y-R ¹¹ , hydrogen atom, halogen atom, hydrocarbon group having 1 to 10 carbon atoms, -NR ¹² R ¹³ , -OR ¹² , -SR ¹² , - COR ¹² , —SO ₂ R ¹² , —NR ¹³ COR ¹² , —CONR ¹² R ¹³ , —OCOR ¹² , —COOR ¹² , —NR ¹³ CSR ¹² , —CSNR ¹² R ¹³ , —SCOR ¹² or —COSR ¹² and at least one of R ⁹ and R ¹⁰ is —Y—R ¹¹ ;
X represents S, O, or NR ¹⁴ ;
Y, R ¹¹ , R ¹² and R ¹³ are the same as in formula (I);
R ¹⁴ represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms,
m represents 2 to 100,
Each of the m monomer units is the same or different. )
A block copolymer containing a block unit represented by 2. The block copolymer according to claim 1, wherein n/m is 3 or less. 3. The block copolymer according to claim 1, wherein n is 15 or more. 4. The block copolymer according to any one of claims 1 to 3, wherein m is 8 or more. The block copolymer according to any one of claims 1 to 4, wherein the block unit represented by formula (I) and the block unit represented by formula (II) are directly bonded. . The block copolymer according to any one of claims 1 to 5, wherein R ¹ to R ⁶ are hydrogen atoms. 7. The block copolymer according to any one of claims 1 to 6 , wherein one of R ⁹ and R ¹⁰ is -Y-R ¹¹ and the other is a hydrogen atom. A phosphate compound detection agent comprising the block copolymer according to any one of claims 1 to 7 . Measuring the fluorescence spectrum or ultraviolet-visible light absorption spectrum of the block copolymer according to any one of claims 1 to 7 in a solution containing a phosphoric acid compound, and the resulting fluorescence intensity or ultraviolet-visible A method for detecting a phosphoric acid compound, comprising detecting the phosphoric acid compound from the waveform of the light absorption spectrum or the absorbance.

Images