JP2014152248A - Water-soluble, luminescent group-labeled polymer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a water-soluble, luminescent group-labeled polymer to which a luminescent functional group is quantitatively bound and which can be used for a biological purpose.SOLUTION: Provided is a water-soluble, luminescent group-labeled polymer having a structure exemplified in Figure 1. By introducing the luminescent functional group to a polymer side chain quantitatively, light emission intensity can be increased compared to the luminescent functional group alone.

Description

  The present invention relates to a water-soluble luminescent label polymer in which a luminescent functional group and a terminal functional group capable of binding to a biomolecule are quantitatively introduced.

  In recent years, there is an increasing need for highly sensitive biotools that can be detected from extremely small amounts of specimens. As part of this increase in sensitivity, attention has been focused on the application of a new luminescent material having high luminescence and high stability as a label for detection. In order to use the illuminant in a biochemical system such as a biotool, in addition to its luminescence performance, it is soluble in water, has excellent biocompatibility, and has a luminescent ability even in an aqueous solution. It is mentioned that it is held as an essential condition (see Non-Patent Document 1).

  Among them, organic fluorescent dyes are often used as detection labels for biomolecules such as nucleic acids and proteins because of their small size and abundant selectivity of luminescent colors (see Patent Document 1). However, most organic fluorescent dyes are highly soluble in organic solvents and have a relatively high quantum yield, but they tend to decrease significantly in aqueous solutions. This is because molecules of organic fluorescent dyes with high hydrophobicity are dispersed in an aqueous solution due to intermolecular association, thereby reducing dispersion stability and suppressing fluorescence. Low-molecular water-soluble substituents such as sulfonic acids and carboxylic acids are used. Even if it is introduced, it is difficult to completely solve it. In addition, in order to use an organic fluorescent dye as a label for detection, modification of a functional group capable of binding to various biomolecules is indispensable, but this is complicated in synthesis and affects the structure of the dye molecule. Various problems such as facilitating quenching have been pointed out (see Non-Patent Document 2).

  As means for solving the above-mentioned problems, a method is known in which a plurality of organic fluorescent dyes are labeled on a water-soluble polymer or nucleic acid to improve the total emission intensity. For example, a method of chemically bonding an organic fluorescent dye at random positions along a water-soluble polymer chain having a reactive functional group (see Patent Document 2), a monomer labeled with an organic fluorescent dye (for example, a vinyl monomer, Synthetic or natural oligonucleotides) are synthesized in advance, and then a method of copolymerizing a hydrophilic comonomer in which a label moiety is not incorporated is known (see Non-Patent Document 3 and Patent Document 3). However, in the former case, it is a polymer obtained by a radical polymerization mechanism, and it is difficult to control the polymer chain length or terminal functional group, so the number of organic fluorescent dyes per polymer or bioactive ligand cannot be quantitatively labeled. There were difficulties. In the latter case, it is often necessary to synthesize a monomer to which an organic fluorescent dye to be labeled is bound each time, and there is a problem that it is difficult to maintain the optical properties of the dye molecule depending on the polymerization conditions.

JP 2000-106876 A JP-T-2001-520893 JP 2005-529606 Gazette

Dubertret, B.M. et. al. , SCIENCE. 2002, 298, 1759-1762. Ow, H .; et. al. , Nano Lett. 2005, 5, 113-117. Nese, A .; et. al. , Macromolecules. 2011, 44, 5905-5910.

  An object of the present invention is to provide a water-soluble light-emitting labeling polymer quantitatively bound with a light-emitting functional group that can be used for biochemical purposes.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a linear-type polyethyleneimine derivative has a hydrophilic functional group, a luminescent functional group, and a terminal functional group capable of binding to a biomolecule. It was found that a water-soluble light-emitting labeled polymer bound quantitatively could be synthesized, and the present invention was completed.

That is, the present invention provides the following [1] to [5].
[1] Repeating unit represented by the following formula (1)

Wherein R ₁ is —COOH, —OH or — (OCH ₂ CH ₂ ) _p R _{1 ′} (where R _{1 ′} is —COOH, —OH, —CH═CH ₂ , —C≡CH or — N ₃ and p are integers of 1 to 30), and l is an integer of 1 to 10).
Repeating unit represented by the following formula (2)

(Wherein R ₂ is a luminescent functional group, —NHR ₃ , —SR ₄ (where R ₃ is a hydrogen atom or a tert-butoxycarbonyl group, a benzyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, A protective group for an amino group selected from 2,2,2-trichloroethoxycarbonyl group, allyloxycarbonyl group, phthaloyl group, p-toluenesulfonyl group and 2-nitrobenzenesulfonyl group, R ₄ is a hydrogen atom or a benzyl group , Methoxybenzyl group, N- (acetyl) aminomethyl group, t-butyl group, methylbenzyl group, 3,4-dimethylbenzyl group, triphenylmethyl group, benzhydryl group, acetamidemethyl group, carbomethoxysulfenyl group, 3- Nitro-2-pyridinesulfenyl group, ethylcarbamoyl group, 9-fluoro It is a protecting group for a thiol group selected from a renylmethyl group and a pyridyl sulfide group.) Or — (OCH ₂ CH ₂ ) _p R _{2 ′} (where R _{2 ′} is a luminescent functional group, —NHR ₃ or —SR). ₄ and p are integers of 1 to 30), and the repeating unit number a includes a luminescent functional group in the repeating unit number y, the repeating unit number b including -NHR ₃ and the repeating unit including -SR ₄ The number of units c is b / a + b = 0 to 0.90 and c / a + c = 0 to 0.95, m is an integer of 1 to 10, and n is an integer of 0 to 10), and the following formula (3 ) Repeating unit

The number of repeating units x in formula (1), the number y of repeating units in formula (2), and the number of repeating units z in formula (3) are x / (x + y + z) = 0.01-0.90, y /(X+y+z)=0.01-0.50 and z / (x + y + z) = 0.01-0.90, which are composed of repeating units of the above formulas (1), (2) and (3) At least one terminal of the polymer main chain is selected from the group consisting of —COOH, —OH, —NH ₂ , —SH, —NCO, —CHO, —CH═CH ₂ , —C≡CH, —N ₃ and a biotinyl group. A water-soluble luminescent labeling polymer comprising at least one functional group selected.
[2] The spacer includes — (OCH ₂ CH ₂ ) _q — (wherein q is an integer of 1 to 120) between the polymer main chain and the terminal functional group. , [1] The water-soluble luminescent labeling polymer.
[3] The luminescent functional group is BODIPY bonded through an amino group, Cy3 bonded through an amino group, Cy5 bonded through an amino group, a europium complex or fluorescein bonded through an amino group. , [1] or [2].
[4] The water-soluble luminescent labeled polymer according to any one of [1] to [3], wherein x, y and z are x + y + z = 10 to 350.
[5] A reagent containing the water-soluble luminescent labeled polymer according to any one of [1] to [4].

  According to the present invention, it is possible to form a water-soluble luminescent labeling polymer having a plurality of luminescent functional groups and a functional group capable of binding to biomolecules. In addition, the water-soluble luminescent labeling polymer of the present invention is stable in water and has the characteristics that the luminescent property can be enhanced by controlling the number of luminescent functional groups, so that it can be used for biochemical applications. it can.

FIG. 1 is a synthesis scheme of a water-soluble luminescent labeled polymer that quantitatively binds a luminescent functional group. FIG. 2 is an explanatory diagram of a ¹ H-NMR spectrum of Boc-NH-PEG-b-PEI (Synthetic Intermediate 1-4). FIG. 3 is an explanatory diagram of a GPC diagram of Boc-NH-PEG-b-PEI (Synthetic intermediate 1-4). FIG. 4 is an explanatory diagram of a GPC diagram of Biotin-PEG-b-PEI (Synthetic intermediate 1-5A). FIG. 5 is an explanatory diagram of the ¹ H-NMR spectrum of Biotin-PEG-b-PEI (Synthetic Intermediate 1-5B). FIG. 6 is an explanatory diagram of a ¹ H-NMR spectrum of Boc-NH-PEG-b-PEI [COOH (18): EI (5)] (Synthetic intermediate 1-6). FIG. 7 shows (A) Biotin-PEG-b-PEI [COOH (9): EI (14)] (Synthetic intermediate 1-7A), (B) Biotin-PEG-b-PEI [COOH (7): EI (16)] (synthesis intermediate 1-7b) and (C) Biotin-PEG-b -PEI [COOH (3): EI (20)] 1 H-NMR spectrum of the (synthetic intermediate 1-7C) It is explanatory drawing. FIG. 8 is an explanatory diagram of a ¹ H-NMR spectrum of Boc-NH-PEG-b-PEI [COOH (1): EI (5): CASSPy (17)] (Synthetic intermediate 1-8). FIG. 9 shows (A) Biotin-PEG-b-PEI [COOH (1): EI (14): NH-Boc (8)] (Synthetic Intermediate 1-10A), (B) Biotin-PEG-b- PEI [COOH (3): EI (16): NH-Boc (4)] (Synthetic Intermediate 1-10B) and (C) Biotin-PEG-b-PEI [COOH (1): EI (20): NH -Boc (2)] (Synthetic intermediate 1-10C) is an explanatory diagram of the ¹ H-NMR spectrum. FIG. 10 shows (A) Boc-NH-PEG-b-PEI [COOH (15): EI (5): fluorescein (3)] (polymer 3-1A) and (B) Boc-NH-PEG-b. -PEI [COOH (17): EI (5): fluorescein (1) is an explanatory view of the ¹ H-NMR spectrum of the (polymer 3-1B). FIG. 11 is an explanatory diagram of the ¹ H-NMR spectrum of Biotin-PEG-b-PEI [COOH (1): EI (20): NH ₂ (1): BODIPY (1)] (polymer 3-2A). is there. FIG. 12A shows Biotin-PEG-b-PEI [COOH (1): EI (20): NH ₂ (1): BODIPY (1)] and Biotin-PEG-b-PEI [COOH (1): _{EI (14): NH 2 (} 4): is a graph showing the fluorescence spectra of BODIPY (4)]. FIG. 12B shows Biotin-PEG-b-PEI [COOH (1): EI (20): Eu (2)] and Biotin-PEG-b-PEI [COOH (1): EI (14): Eu in aqueous solution. It is a graph showing the fluorescence spectrum of (8)]. FIG. 13A shows Biotin-PEG-b-PEI [COOH (1): EI (20): NH ₂ (1): BODIPY (1)] and Biotin-PEG-b-PEI [COOH (1): EI (14 ): NH ₂ (4): BODIPY (4)] is a graph showing a comparison of light emission intensity with a DNA chip. FIG. 13B shows Biotin-PEG-b-PEI [COOH (1): EI (20): Eu (2)] and Biotin-PEG-b-PEI [COOH (1): EI (14): Eu (8). It is a graph showing the comparison of the emitted light intensity in the DNA chip | tip.

  Below, the suitable form for implementing this invention is demonstrated.

[Synthesis of water-soluble luminescent labeled polymer]
The water-soluble luminescent label polymer is obtained by quantitatively binding a luminescent functional group to a side chain of the water-soluble polymer. By labeling the water-soluble luminescent labeling polymer with a plurality of luminescent functional groups, it has an effect of improving the luminescent properties. Specifically, an optimal number of luminescent organic molecules that do not inhibit the dispersion stability in water are bound to a water-soluble polymer to produce a water-soluble luminescent label polymer, thereby improving the luminescence intensity (see FIG. 1).

The water-soluble luminescent label polymer of the present invention includes a segment having a hydrophilic functional group composed of a repeating unit represented by the formula (1), and a functional group having a luminescent property composed of a repeating unit represented by the formula (2). (Hereinafter referred to as “luminescent functional group”), a segment composed of the repeating unit represented by the formula (3), and a repeating unit of the formulas (1), (2) and (3). at least one end of the polymer backbone to be is a functional group capable of binding to a _{biomolecule, -COOH, -OH, -NH 2,} -SH, -NCO, -CHO, -CH = CH 2, It contains at least one functional group selected from the group consisting of —C≡CH, —N ₃ and biotinyl group.

The segment having a hydrophilic functional group composed of the repeating unit represented by the formula (1) has a hydrophilic functional group (R ₁ ) having a property of improving dispersion stability in water. Specific examples of R ₁ include —COOH, —OH or (OCH ₂ CH ₂ ) _p R _{1 ′} (where R _{1 ′} is —COOH, —OH, —CH═CH ₂ , —C≡CH or N _3, p is an integer of 1-30.) Although the like, preferably -COOH, -OH or _{(OCH 2 CH 2) p R} 1 '( wherein, R _1' is -COOH, -OH, p is an integer of 1 to 5.), more preferably —COOH. Further, l is an integer of 1 to 10, but preferably l is an integer of 2 to 4.

The segment composed of the repeating unit represented by the formula (2) has a portion containing an amino group or a thiol group that can be substituted for the light-emitting functional group, and part or all of the light-emitting polymer emits light. It is substituted with a luminescent functional group capable of imparting properties. In addition, the luminescent functional group may be bonded via a spacer structure containing an amino group or a thiol group. Specific examples of the spacer structure include — (OCH ₂ CH ₂ ) _p — (where p is 1 To 120, preferably an integer of 1 to 30). In addition, the amino group or thiol group may be partially protected by a protective group, thereby adjusting the number of bonds of the luminescent functional group bonded to the segment composed of the repeating unit represented by the formula (2). Can do. Specific examples of protecting groups for amino groups include tert-butoxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, allyloxycarbonyl group, phthaloyl group. , P-toluenesulfonyl group and 2-nitrobenzenesulfonyl group. Specific examples of the protecting group for thiol group include benzyl group, methoxybenzyl group, N- (acetyl) aminomethyl group, t-butyl group, methylbenzyl. A group, 3,4-dimethylbenzyl group, triphenylmethyl group, benzhydryl group, acetamidemethyl group, carbomethoxysulfenyl group, 3-nitro-2-pyridinesulfenyl group, ethylcarbamoyl group, 9-fluorenylmethyl group and Pyridyl sulfide group It is.

  A luminescent functional group is an organic dye molecule that exhibits luminescent properties, specifically fluorescent or phosphorescent, that are particularly useful in various industrial applications. Specific examples include fluorescent dyes and rare earth fluorescent complexes. It is done.

  A fluorescent dye is a highly π-conjugated organic dye that, when excited by absorbing energy such as light, emits light rather than spending energy in vibration or rotational movement within the molecule. It is known to be used for brighteners, fluorescent markers, clinical diagnostic reagents and the like. By labeling a plurality of fluorescent dyes with a water-soluble polymer, the fluorescence intensity of the fluorescent dye alone is amplified, and the present invention makes it possible to apply the fluorescent dye as a fluorescent reagent. In the present invention, a fluorescent dye containing BODIPY, Cy3, Cy5 or fluorescein is preferably used.

  Rare earth fluorescent complexes are preferably used. A rare earth fluorescent complex is an organic dye that absorbs ultraviolet light and emits visible light. Compared with the fluorescent dye, it has a longer fluorescence lifetime (several tens to several hundreds of microseconds compared to several nanoseconds of ordinary fluorescent dyes). It is known that it is used as a labeling agent for time-resolved fluorescence measurement because it has fluorescence characteristics such as a large Stokes shift and a sharp fluorescence peak. When time-resolved fluorescence measurement is used, interference by short-lived background fluorescence derived from excitation light or a biological sample can be avoided, and highly sensitive measurement can be performed. By labeling a plurality of rare earth fluorescent complexes with a water-soluble polymer, the fluorescence intensity of the rare earth fluorescent complex alone is amplified. Therefore, according to the present invention, the rare earth fluorescent complex can be applied as a fluorescent reagent. In the present invention, a europium complex is preferably used.

  In addition, since the luminescent functional group is bonded via an amino group or a thiol group in the segment composed of the repeating unit represented by the formula (2), an amino group or a thiol group is previously added to the luminescent functional group. It is necessary to provide a functional group that can be bonded through the intermediate. Examples of the functional group that can be bonded to the light-emitting functional group through an amino group include a carboxyl group, an active ester group such as an N-hydroxysuccinimide group, a cyanuric chloride, an isocyanate group, and an isothiocyanate group, and a thiol group. Examples of functional groups that can be bonded via a maleimide group, an iodoacetamide group, a halogenated alkyl group, a halogenoacetyl group (for example, an iodoacetyl group, a bromoacetyl group, a chloroacetyl group), and a function capable of forming a disulfide bond Group (for example, thiol group, methanethiosulfonate (MTS)). In the reaction, an additive or a catalyst for accelerating the reaction may be used if necessary. For example, when a carboxyl group is used as a functional group that can be bonded to the luminescent functional group via an amino group, a condensing agent is used. May be.

  In the segment represented by the formula (2), the number of units a including the luminescent functional group, the number of units b and c not including the luminescent functional group are b / a + b = 0 to 0.90 and c / a + c = 0 to 0.95, preferably b / a + b = 0 to 0.88 and c / a + c = 0 to 0.94. M is an integer of 1 to 10, preferably m is an integer of 2 to 4, and n is an integer of 0 to 10, but preferably n is an integer of 0 to 4.

  The segment composed of the repeating unit represented by the formula (3) is a segment composed of polyethyleneimine obtained from a polymerization mechanism effective for controlling the amount of the luminescent functional group introduced into the polymer. Compared to vinyl polymers or nucleic acid polymers having a known luminescent functional group, the water-soluble luminescent labeled polymer of the present invention having a polyethyleneimine skeleton is synthesized from the living cationic polymerization mechanism, so that the chain length control and terminal functionality of the polymer are controlled. Since selective bonding of groups is easy, it is presumed that this method is effective for forming a water-soluble luminescent labeling polymer having a plurality of luminescent organic molecules and biomolecules quantitatively.

Furthermore, at least one terminal of the polymer main chain composed of the repeating units of the formulas (1), (2) and (3) is a functional group having a binding ability to a biomolecule, -COOH, -OH , —NH ₂ , —SH, —NCO, —CHO, —CH═CH ₂ , —C≡CH, —N _3, and a biotinyl group. Examples of biomolecules that can be bound via these functional groups include nucleic acids such as DNA and RNA, proteins, peptides, sugars, lipids, etc., preferably nucleic acids, proteins, or peptides, more preferably nucleic acids. .

  When the biomolecule is a nucleic acid, it is possible to bind to the nucleic acid by introducing a reactive group into a part of the nucleic acid and reacting the reactive group with the terminal functional group of the water-soluble luminescent labeling polymer. For example, a double-stranded nucleic acid having a terminal amino group can be prepared by performing a polymerase chain reaction (PCR) reaction using an oligonucleotide having a terminal amino group as a primer. By condensing the terminal amino group of the nucleic acid and the carboxyl group introduced at the end of the water-soluble luminescent label polymer, the water-soluble luminescent label polymer can be bound to the nucleic acid.

  When the biomolecule is a protein or peptide, the water-soluble luminescent label polymer is bound to the protein or peptide by binding the amino group of the lysine residue in the protein or peptide to the carboxyl group at the end of the water-soluble luminescent label polymer. It becomes possible. When a water-soluble luminescent labeled polymer having a biotinyl group is used, it can be immobilized on an avidin molecule by an avidin-biotin interaction. In addition, since avidin has four binding sites per molecule, for example, avidin is first bound to a nucleic acid into which a biotinyl group has been introduced, and a water-soluble luminescent labeled polymer having a biotinyl group at the end is attached to the terminal binding site of avidin. By acting on the nucleic acid, the nucleic acid can be labeled with a water-soluble luminescent labeling polymer. On the other hand, when a water-soluble luminescent labeling polymer having a primary amino group is used, since the avidin molecule can be directly labeled with the water-soluble luminescent labeling polymer without using a biotinyl group, the biotin-avidin-biotin sandwich structure staining method is used. It becomes possible to suppress a decrease in the binding force.

  Furthermore, when the biomolecule is a sugar or a lipid, it becomes possible to bind to the sugar or lipid by activating a part of the hydroxyl group and reacting the activated reactive group with the terminal functional group of the water-soluble luminescent labeling polymer. . For example, after converting a sugar chain hydroxyl group to an active ester group with an activating reagent, a water-soluble light-emitting label polymer having a terminal amino group is subjected to a condensation reaction, whereby the water-soluble light-emitting label polymer can be bound to the sugar chain. .

The composition ratio of each segment is such that the number of repeating units represented by the formula (1) is x, the number of repeating units represented by the formula (2) is y, and the number of repeating units represented by the formula (3). Is z (x, y and z are integers), x / (x + y + z) = 0.01-0.90, y / (x + y + z) = 0.01-0.50 and z / (x + y + z) = 0 0.01 to 0.90, preferably x / (x + y + z) = 0.04 to 0.80, y / (x + y + z) = 0.04 to 0.40 and z / (x + y + z) = 0 20-0.90, more preferably x / (x + y + z) = 0.04-0.74, y / (x + y + z) = 0.04-0.35 and z / (x + y + z) = 0.22-0. 87. If x, y, and z are out of this numerical range, it is not preferable because the water-soluble luminescent labeling polymer becomes poorly dispersed in water. In addition, x / (x + y + z), y / (x + y + z) and z / (x + y + z) can be calculated based on the ¹ H-NMR spectrum and UV absorbance measurement of the water-soluble luminescent labeled polymer (see Examples). .

The total value of x, y and z representing the size of the water-soluble luminescent labeled polymer of the present invention, that is, the degree of polymerization (DP) of the polymer is not particularly limited, but the water-soluble luminescent labeled polymer described below is dispersed in water. And from the viewpoint that the light emission characteristics are controlled by the total value of x, y and z, it is preferably in the range of 10 to 350, more preferably in the range of 20 to 310. The degree of polymerization (DP) of the water-soluble luminescent labeling polymer can be measured by ¹ H-NMR spectrum (see Examples).

Furthermore, the water-soluble luminescent labeling polymer of the present invention has-(OCH as a spacer) between a polymer main chain composed of the repeating units of the formulas (1), (2) and (3) and the terminal functional group. ₂ CH ₂ ) _q — (q is an integer of 1 to 120) is preferably included. This spacer is presumed to have a function of improving the dispersion stability in water of the water-soluble luminescent labeling polymer as a hydrophilic group and the ability of the terminal functional group to bind to a biomolecule.

The water-soluble luminescent labeling polymer of the present invention comprises a step of polymerizing a polyoxazoline having a polydispersity (PDI: weight average molecular weight (Mw) / number average molecular weight (Mn)) of 1.3 or less, at least of the polyoxazoline main chain. A step of introducing a functional group or a spacer having a functional group at one end, a step of converting the polyoxazoline side chain into a linear polyethyleneimine by deprotection, the linear polyethyleneimine and a hydrophilic reactive group; A step of reacting a fatty acid anhydride or a fatty acid halide containing to introduce a segment of a hydrophilic functional group (R ₁ ); and reacting the linear polyethyleneimine with a fatty acid halide containing a luminescent functional group to produce a luminescent functional group It can be obtained by introducing (R ₂ ). The polydispersity (PDI) of linear polyethyleneimine can be measured by gel permeation chromatography (GPC) diagrams.

  Polymerization of the polyoxazoline and introduction of a spacer having the above-mentioned functional group or functional group at at least one end of the polyoxazoline main chain is carried out using the following formula using an oxazoline derivative, a cationic polymerization initiator and a nucleophilic terminator as monomers. It is achieved by polymerizing the polyoxazoline represented by (4) and introducing the above-mentioned functional group or a spacer having a functional group into at least one terminal of the polyoxazoline main chain.

(In the formula, In represents a residue derived from a cationic polymerization initiator, NP represents a residue derived from a nucleophilic terminator, and R ₅ represents a linear or branched alkyl group having 1 to 20 carbon atoms. (Preferably methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, hexyl, octyl, dodecyl, octadecyl, eicosyl, and 18-methylnonadecanyl), and r is x, y, The total value of z (preferably an integer of 10 to 350).

  In, which is a residue derived from a cationic polymerization initiator, has the aforementioned functional group or functional group at the start terminal of the polymer main chain composed of the repeating units of the above formulas (1), (2) and (3). There is no particular limitation as long as it is a residue of a cationic polymerization initiator capable of introducing a spacer. Similarly, NP, which is a residue derived from a nucleophilic terminator, provides a functional group and a spacer at the terminating end of the polymer main chain composed of the repeating units of the above formulas (1), (2) and (3). There is no particular limitation as long as it is a residue of a nucleophilic terminator that can be used.

  In addition, by using a cationic polymerization initiator having a functional group or a spacer to which a functional group has been added in advance, the polyoxazoline polymerization step represented by the formula (4) and at least one terminal of the polyoxazoline main chain are described above. The step of introducing a functional group or a spacer having a functional group may be performed at once. Similarly, by using a nucleophilic terminator having a functional group or a spacer to which a functional group has been added in advance, the polyoxazoline polymerization step represented by the formula (4) and at least one terminal of the polyoxazoline main chain You may implement the process of introduce | transducing the functional group or the spacer to which the functional group was added at once.

Examples of the cationic polymerization initiator include a wide variety of tosylates represented by the general formula: TsOR ₆ , and the residue corresponding to the R group is not limited. For example, — (CH ₂ ) _1-10 R _{6 ′} (where R _{6 ′} represents —COOH, —OH, —NH ₂ , —SH, —NCO, —CHO, —CH═CH ₂ , —C≡CH, —N ₃ and a biotinyl group) Or — (OCH ₂ CH ₂ ) _s R _{6 ′} (where R _{6 ′} is —COOH, —OH, —NH ₂ , —SH, —NCO, —CHO, —CH═CH ₂ , — C≡CH, —N ₃ and a biotinyl group, s is an integer from 1 to 120). A part of R _{6 ′} may be protected during the polymerization reaction so as not to adversely affect the cationic ring-opening polymerization of oxazoline. For example, —COOH is methyl ethyl ester, benzyl ester, tert-butyl ester, protected with any of tert-butyldimethylsilyl group, -OH is methyl, benzyl, p-methoxybenzyl, tert-butyl, methoxymethyl (MOM), 2-tetrahydropyranyl (THP), Ethoxyethyl group (EE), acetyl group, pivaloyl group, benzoyl group, trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldimethylsilyl (TBS or TBDMS), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS) -SH is benzyl group, methoxybenzyl group, N- (acetyl) aminomethyl group, t-butyl group, methylbenzyl group, 3,4-dimethylbenzyl group, triphenylmethyl group, benzhydryl group, acetamidemethyl group, carbomethoxysulfur phenyl, 3-nitro-2-pyridine sulfenyl group, ethylcarbamoyl group, are protected with either 9-fluorenylmethyl group or pyridyl sulfide group, -NH ₂ is tert- butoxycarbonyl group, benzyloxycarbonyl group , 9-fluorenylmethyloxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, allyloxycarbonyl group, phthaloyl group, p-toluenesulfonyl group or 2-nitrobenzenesulfonyl group, Is dimethylacetal, cyclic acetal Lumpur, can be protected by any of the dithio acetal.

Examples of the nucleophilic terminator include an anionoid compound that generates an anion active terminal corresponding to the NP residue, and examples thereof include, but are not limited to, H ₂ O, NaOH, KOH, NaSH, KSH, NH ₃ , NHR ₇ R ₈ (wherein R ₇ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ₈ is — (CH ₂ ) _1-10 R _{8 ′} (where R _{8 ′} is —COOH, —OH, —NH ₂ , —SH, —NCO, —CHO, —CH═CH ₂ , —C≡CH, —N ₃ and a biotinyl group.) Or — (OCH ₂ CH ₂ ) _t R _{8 ′} ( Here, R _{8 ′} is the same as defined above, and t is an integer of 1 to 120.) and cyclic secondary amine compound derivatives represented by the following formula (5) are given as specific examples. A part of R _{8 ′} is protected during the reaction as described above. May be.

(Wherein R _{8 ′} is the same as defined above).

  Conversion of the polyoxazoline to linear polyethyleneimine is achieved by a hydrolysis reaction using a strongly acidic or strongly basic reagent. Examples of the strongly acidic reagent include hydrochloric acid, sulfuric acid, and nitric acid, and hydrochloric acid is preferably used. In addition, examples of the strongly basic reagent include calcium hydroxide, barium hydroxide, sodium hydroxide, and potassium hydroxide, and sodium hydroxide or potassium hydroxide is preferably used.

In the step of introducing the hydrophilic functional group (R ₁ ), when a fatty acid anhydride containing a hydrophilic functional group is used, it is not particularly limited as long as a desired structure can be introduced into the linear polyethyleneimine. Is, for example, a linear fatty acid anhydride represented by the following formula (6).

(Wherein R ₁ and l are as defined above).

A part of R ₁ may be protected during the reaction as described above.

In the case of introducing -COOH as R _1, a cyclic anhydride of a divalent carboxylic acid represented by the following formula (7), preferably to use succinic anhydride, glutaric anhydride or adipic anhydride Can do.

(Wherein l is as defined above).

In the step of introducing the hydrophilic functional group (R ₁ ), when a fatty acid halide containing a hydrophilic reactive group is used, it is not particularly limited as long as a desired structure can be introduced into the linear polyethyleneimine. A compound represented by the following formula (8) is preferable.

(Wherein R ₁ and l are as defined above).

A part of R ₁ may be protected during the reaction as described above.

  In the step of introducing an amino group or thiol group for introducing the luminescent functional group, when a fatty acid halide containing an amino group or a thiol group is used, a desired structure can be introduced into the linear polyethyleneimine If it is, it will not specifically limit, However, The compound represented by the following Formula (9) can be used preferably.

(Wherein R ₉ is an amino group or thiol group, and m and n are as defined above).

R ₉ may be partially protected by the aforementioned amino group or thiol protecting group during the reaction.

In addition, when -COOH is introduced as R ₁ of the hydrophilic reactive group, an amino group for introducing the luminescent functional group in a step of reacting the -COOH with an amine compound containing the amino group or thiol group or Thiol groups can be introduced. Specifically, an amine compound represented by the following formula (10), preferably ethylenediamine, propylenediamine, butylenediamine, 2-mercaptoethylamine, 3-mercaptopropylamine, or 4-mercaptobutylamine is condensed with —COOH. As a result, part of the introduced hydrophilic reactive group can be converted to an amino group or a thiol group.

(Wherein R ₉ and n are as defined above).

R ₉ may be partially protected during the reaction as described above.

The segment consisting of the repeating unit represented by the formula (2) has an amino group or thiol group of the segment consisting of the repeating unit represented by the formula (9) or (10) in advance via an amino group or thiol group. Without reacting the luminescent functional group to which the functional group capable of binding is reacted or the amino group or thiol group of the segment composed of the repeating unit represented by the formula (9) or (10) Specifically, in the case where the hydrophilic functional group introduced as R ₁ is —COOH, the above-mentioned light emission to which a functional group that can be bonded to —COOH via —COOH in advance, for example, an amino group is added. It can also be obtained by a method of reacting a functional group.

  The composition ratio of each segment in the water-soluble luminescent label polymer of the present invention, that is, x (number of repeating units represented by the formula (1)): y (number of repeating units represented by the formula (2)): z ( The number of repeating units represented by the formula (3) is adjusted to a desired ratio of a reactive group containing a hydrophilic reactive group and a light-emitting functional group with respect to the secondary amine of the linear polyethyleneimine. It can be adjusted by reacting sequentially. Specifically, for example, after introducing x by reacting a linear fatty acid anhydride containing a hydrophilic functional group represented by the chemical formula (6) with a secondary amine of a linear polyethyleneimine, After reacting the fatty acid halide containing the amino group or thiol group represented by the chemical formula (9), the light-emitting functional group to which a functional group capable of being bonded via the amino group or thiol group is previously reacted is reacted. Or a method of adjusting the composition ratio of x: y: z by introducing y, or after introducing x by reacting a fatty acid halide containing a hydrophilic functional group represented by the chemical formula (8), After reacting the fatty acid halide containing an amino group or thiol group represented by 9), the luminescent functional group to which a functional group that can be bonded via an amino group or thiol group has been reacted is reacted. Introduce The method of adjusting the composition ratio of x: y: z by the above, after reacting the cyclic anhydride of the divalent carboxylic acid represented by the chemical formula (7) and introducing x, it is represented by the chemical formula (10). After reacting an amine compound containing an amino group or a thiol group, x is introduced by reacting the luminescent functional group to which a functional group that can be bonded in advance through an amino group or a thiol group is reacted to introduce y. : The method of adjusting the composition ratio of y: z, or after reacting the cyclic anhydride of the divalent carboxylic acid represented by the chemical formula (7) and introducing x, it can be bonded in advance via —COOH. Examples include a method of adjusting the composition ratio of x: y: z by reacting the luminescent functional group to which a functional group has been added and introducing y.

  The constituent ratio of a, b and c in the segment consisting of the repeating unit represented by the formula (2) of the water-soluble luminescent labeling polymer of the present invention is such that a part of the amino group or thiol group is protected with a protecting group, It can be adjusted by a method of reacting the remaining amino group or thiol group with a luminescent functional group, or a method of reacting the luminescent functional group of the number of molecules to be introduced after deprotecting all of the amino group or thiol group. it can.

  The number of light-emitting functional groups bonded to the water-soluble light-emitting label polymer of the present invention is, for example, from the viewpoint of improving the characteristics of the light-emitting organic molecules and maintaining the dispersion stability in water, for example, the polymer chain length ( For DP) = 23, a range of 2 to 17 is preferable, and a range of 2 to 8 is more preferable.

The chemical composition and emission characteristics of the water-soluble luminescent labeling polymer of the present invention can be confirmed by ¹ H-NMR spectrum, UV absorption or fluorescence spectroscopy.

[reagent]
The water-soluble luminescent labeled polymer of the present invention can be applied as a detection reagent in various techniques for detecting an interaction between substances (hereinafter referred to as “sensing technique”), and is present in the body or produced in the body. Therefore, it can be used in a wide range of fields such as diagnosis / prediction of human health, drug discovery, and other fields. Examples of biomolecules to be detected by the sensing technique include nucleic acids such as genomic DNA and RNA, proteins, peptides, sugars, and lipids as described above.

  Specifically, when detecting nucleic acid in a living body, a DNA chip having a large number of probe nucleic acids immobilized on a substrate is known as a tool for detecting the nucleic acid. A nucleic acid labeled with a luminescent labeling polymer or a replica thereof is hybridized with a probe nucleic acid on the substrate, and the signal on the substrate derived from the water-soluble luminescent labeled polymer is detected, thereby determining the presence or absence of nucleic acid in the living body. Can be detected. Alternatively, the water-soluble luminescent label is obtained by hybridizing the nucleic acid in vivo or a replica thereof with the probe nucleic acid on the substrate without labeling, and then binding the water-soluble luminescent label polymer to the nucleic acid by a chemical reaction or the like. Signals on the polymer-derived substrate can be detected.

  In addition, when detecting a protein in a living body, by utilizing the interaction between the protein and a protein or nucleic acid immobilized on a substrate in the same manner as the method for detecting a nucleic acid, The presence or absence can be detected.

  Hereinafter, the features of the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples. Moreover, about the various compounds used in the Examples, those sold as reagents were used.

Example 1 Synthesis of water-soluble polymer (Synthetic intermediate 1-1A: OH-PEI)
Under a dry argon atmosphere, initiating methyl paratosylate (8 mmol, 1.2 mL) and monomer 2-ethyl-2-oxazoline (EtOx) (176.8 mmol, 17.84 mL) in a solvent of acetonitrile (50 mL). In addition, cationic ring-opening polymerization was performed. After reacting for 7 days in a thermostatic bath at 50 ° C., the mixture was cooled to room temperature and subjected to termination reaction with 1M sodium hydroxide / methanol mixed solvent (introduction of hydroxyl group (—OH) to the termination end). After purification by water dialysis, about 7 g of polymer was recovered by drying under reduced pressure. The degree of polymerization and polydispersity of the obtained poly (2-ethyl-2-oxazoline) (PEtOx) were confirmed by ¹ H-NMR spectrum and GPC diagram, respectively (polymerization degree (DP) = 23, polydispersity ( PDI: Mw / Mn) = 1.1, hydroxyl group (—OH) introduction rate = 100%).

In order to obtain linear polyethyleneimine (PEI) by side chain decomposition reaction, PEtOx (3 g) was dissolved in a mixed solvent of 37% hydrochloric acid (30 mL) and water (24 mL) and refluxed at about 110 ° C. for 24 hours. While adjusting the pH to 9-10 while adding sodium hydroxide pellets until the reaction solution became completely transparent, 1.2 g of PEI was recovered by water dialysis and lyophilization. As a result of ¹ H-NMR measurement of the obtained polymer, the PEtOx skeleton and side chain hydrogen-derived peaks were all disappeared, and the PEI skeleton ethyleneimine (-NHCH ₂ CH _2- ) derived from methylene hydrogen Since the peak exists in the vicinity of 3.2 to 3.4 ppm, it was confirmed that the side chain decomposition reaction was quantitatively performed.

  The synthesis conditions and composition of OH-PEI (Synthetic Intermediates 1-1B, 1-1C, 1-1D) having different chain lengths obtained by the same synthetic operation as that of Synthetic Intermediate 1-1A were as shown below. It was.

(Synthetic intermediate 1-1B: OH-PEI)
Charge amount: acetonitrile (30 mL), methyl paratosylate (2 mmol, 0.3 mL), EtOx (88.4 mmol, 8.92 mL), yield: 8 g, degree of polymerization (DP) = 43, polydispersity (PDI: Mw) /Mn)=1.1, hydroxyl group (—OH) introduction rate = 100%).

(Synthetic intermediate 1-1C: OH-PEI)
Charge amount: acetonitrile (30 mL), methyl paratosylate (1 mmol, 0.15 mL), EtOx (88.4 mmol, 8.92 mL), yield: 8 g, degree of polymerization (DP) = 93, polydispersity (PDI: Mw) /Mn)=1.1, hydroxyl group (—OH) introduction rate = 100%).

(Synthetic intermediate 1-1D: OH-PEI)
Charge amount: acetonitrile (36 mL), methyl paratosylate (0.3 mmol, 0.045 mL), EtOx (96 mmol, 9.7 mL), yield: 7 g, degree of polymerization (DP) = 302, polydispersity (PDI: Mw) /Mn)=1.1, hydroxyl group (—OH) introduction rate = 100%).

(Synthetic intermediate 1-2: NH ₂ -PEI)
Under a dry argon atmosphere, initiating methyl paratosylate (8 mmol, 1.2 mL) and monomer 2-ethyl-2-oxazoline (EtOx) (176.8 mmol, 17.84 mL) in a solvent of acetonitrile (50 mL). In addition, cationic ring-opening polymerization was performed. After reacting for 7 days in a thermostatic bath at 50 ° C., 4- (N-Boc-amino) piperidine (16 mmol, 3.2 g) was added as a terminator, and then the reaction was stopped in a thermostatic bath at 50 ° C. for 1 day. After purification by water dialysis, 7 g of polymer was recovered by drying under reduced pressure. The polymerization degree of the obtained polymer (PEtOx) and the introduction rate of N-Boc-amino group at the terminal end were confirmed by ¹ H-NMR spectrum (polymerization degree (DP) = 23, polydispersity (PDI : Mw / Mn) = 1.1, introduction rate = 100%).

In order to obtain a linear PEI by side chain decomposition reaction while introducing a primary amino group (—NH ₂ ) by deprotection reaction of the terminal end Boc, PEtOx (7 g) was added with 37% hydrochloric acid (70 mL) and water (56 mL). ) And refluxed at about 110 ° C. for 24 hours. While adjusting the pH to 9-10 while adding sodium hydroxide pellets until the reaction solution became completely transparent, 2.5 g of polymer was recovered by water dialysis and freeze-drying. When ¹ H-NMR measurement of the obtained polymer was performed, the PEtOx skeleton and the side chain ethylhydrogen-derived peaks were all gone, and the methylene hydrogen of the PEI skeleton ethyleneimine (—NHCH ₂ CH ₂ —). Since the peak derived from 3.2 to 3.4 ppm is present and the peak derived from the methyl hydrogen of the N-Boc protecting group at the end of PEtOx disappeared completely, the side chain decomposition reaction was quantitatively performed. I confirmed it.

(Synthetic intermediate 1-3: Biotin-PEI)
In a mixed solvent of PEI (Synthetic intermediate 1-2) (0.18 mmol, 180 mg) having a primary amino group introduced at the terminal end in 0.1 M NaHCO ₃ (15 mL) / DMSO (25 mL), Biotin-NHS (0. 89 mmol, 305 mg) was added and reacted at room temperature with stirring for 24 hours, followed by ethanol dialysis and water dialysis sequentially. Biotin-PEI (yield: 146 mg) having a biotinyl group introduced at the end was recovered by lyophilization after dialysis. The introduction rate of the terminal biotinyl group of the obtained polymer was calculated from the methylene hydrogen of the ethyleneimine skeleton (—NHCH ₂ CH ₂ —) of the ¹ H-NMR spectrum and the methylene hydrogen of the amide bond site (the introduction rate of the biotinyl group). Is 100%).

(Synthetic intermediate 1-4: Boc-NH-PEG-b-PEI)
PEI (Synthetic intermediate 1-2) (0.45 mmol, 513.7 mg) in which a primary amino group was introduced at the terminal end was dissolved in ultrapure water (7 mL). Further, polyethylene glycol (Boc-NH-PEG ₁₀₃ -NHS) having a Boc-protected primary amino group and an active ester group at both ends (manufactured by NOF Corporation, BO-050TS, MW = 4522) (0.45 mmol) 2.12 g) was dissolved in ultrapure water (8 mL). Subsequently, both aqueous polymer solutions were mixed and reacted at room temperature with stirring for 48 hours, followed by sequential water dialysis and ion exchange chromatography purification. Thereafter, Boc-NH-PEG-PEI (yield: 1 g) having a protected primary amino group introduced via a PEG spacer at the terminal end was lyophilized. When ¹ H-NMR measurement of the polymer was performed, the peak (b) derived from methylene hydrogen of the ethylene oxide (—OCH ₂ CH ₂ —) of the PEG skeleton was present in the vicinity of 3.5 to 3.7 ppm, the PEI skeleton The peak (a) derived from methylene hydrogen of ethyleneimine (—NHCH ₂ CH ₂ —) in the vicinity of 2.6 to 2.7 ppm and the peak derived from methyl hydrogen of the Boc protecting group at the PEG termination end (c ) In the vicinity of 1.4 ppm, it was confirmed that Boc-NH-PEG (103) -b-PEI (23) having a Boc-protected primary amino group via a PEG spacer at the terminal end was obtained ( Figure 2). Further, the polymer purity (96%) after purification was confirmed by measurement with aqueous GPC (high-speed GPC system “HLC-8320GPC EcoSEC” manufactured by Tosoh Corporation, TSKgel G3000PWXL-CP, TSK guard column) (FIG. 3). .

(Synthetic intermediate 1-5A: Biotin-PEG-b-PEI)
PEI (Synthetic intermediate 1-2) (0.03 mmol, 120 mg) in which a primary amino group was introduced at the terminal end was dissolved in ultrapure water (1 mL). In addition, polyethylene glycol (Biotin-PEG ₁₀₃ -NHS) having a biotinyl group and an active ester group at both ends (manufactured by NOF Corporation, BI-050TS, MW = 5003) (0.02 mmol, 100 mg) is ultrapure water. (1 mL) was dissolved. Subsequently, both aqueous polymer solutions were mixed and reacted at room temperature with stirring for 48 hours, followed by sequential water dialysis and ion exchange chromatography purification. Then, Biotin-PEG-b-PEI (yield: 200 mg) into which a biotinyl group via a PEG spacer was introduced at one end was recovered by lyophilization. As a result of ¹ H-NMR measurement of the polymer, a peak derived from methylene hydrogen of ethylene oxide (—OCH ₂ CH ₂ —) of the PEG skeleton was present in the vicinity of 3.5 to 3.7 ppm, ethylene imine of the PEI skeleton. Since the peak derived from methylene hydrogen of (—NHCH ₂ CH ₂ —) is present in the vicinity of 2.6 to 2.7 ppm and the peak derived from hydrogen of the biotinyl group is present at 4.6 ppm, PEG is present at the terminal end. It was confirmed that Biotin-PEG (103) -b-PEI (23) into which a biotinyl group via a spacer was introduced was obtained. In addition, the purity of the polymer after purification or the introduction rate of biotinyl group (100%) is confirmed by measurement with aqueous GPC (high-speed GPC system “HLC-8320GPC EcoSEC” manufactured by Tosoh Corporation, TSKgel G3000PWXL-CP, TSK guard column). (FIG. 4).

(Synthetic intermediate 1-5B: Biotin-PEG-b-PEI)
Boc-NH-PEG (103) -b-PEI (23) (Synthetic Intermediate 1-4) (0.078 mmol, 300 mg) having a Boc-protected primary amino group via a PEG spacer at the terminating end was added to ultrapure water (12 mL). ), 35% hydrochloric acid (3 mL) was added, and Boc deprotection reaction was performed for 1 hour. ^Since the peak derived from methyl hydrogen of the Boc protecting group via the PEG spacer disappeared completely by ¹ H-NMR measurement, NH ₂ -PEG-b-PEI into which 100% of the free primary amino group was introduced was obtained. I confirmed. Next, in a 0.1 M NaHCO ₃ (1 mL) aqueous solution of NH ₂ -PEG-b-PEI (300 mg), polyethylene glycol having both a biotinyl group and an active ester group at both ends (Biotin-PEO ₄ -NHS) (MW = 588.67) (0.27 mmol, 157.7 mg) was added and allowed to react for 24 hours. Thereafter, Biotin-PEG-b-PEI (yield: 351 mg) into which a biotinyl group via a PEG spacer was introduced at the stop end was recovered by water dialysis and freeze-drying. When ¹ H-NMR measurement of the polymer was performed, it was found that the peak (b) derived from methylene hydrogen of the ethylene oxide (—OCH ₂ CH ₂ —) of the PEG skeleton was present in the vicinity of 3.6 to 3.8 ppm, the PEI skeleton The peak (a) derived from methylene hydrogen of ethyleneimine (—NHCH ₂ CH ₂ —) in 2.9 to 3.1 ppm is present, and the peak (c) derived from methylene hydrogen of an amide bond is 2.3 ppm. And biotin-PEG (103) -b-PEI (23 having a biotinyl group introduced through a PEG spacer at the terminal end, since a peak (d) derived from hydrogen of the biotinyl group is present at 4.6 ppm. ) Was obtained (FIG. 5). The introduction rate of the biotinyl group based on the peaks (c) and (d) was 97%.

(Synthetic intermediate 1-6: Boc-NH-PEG-b-PEI [COOH: EI])
Anhydrous Boc-NH-PEG (103) -b-PEI (23) (Synthetic Intermediate 1-4) (500 mg) in aqueous solution (6.2 mL) having a Boc protected primary amino group via a PEG spacer at the terminating end A solution of succinic acid (626 mg) in acetonitrile (4.7 mL) and pyridine (0.337 mL) were added and reacted at room temperature with stirring for 24 hours, followed by ethanol dialysis and water dialysis sequentially. After dialysis, Boc-NH-PEG-b-PEI [COOH: EI] (624 mg) in which a carboxyl group was introduced into the side chain was obtained by lyophilization. Each component of the obtained polymer is methylene hydrogen (b, a) derived from an ethyleneimine skeleton (—NHCH ₂ CH ₂ —) and an ethylene glycol skeleton (—OCH ₂ CH ₂ —) of ¹ H-NMR spectrum, Methylene hydrogen (d) derived from the carboxyl group of the ethyleneimine side chain and methyl hydrogen (c) derived from the Boc protecting group could be assigned respectively (FIG. 6, introduction rate of side chain carboxyl group from ¹ H-NMR spectrum. 78%, Boc-NH-PEG-b-PEI [COOH (18): EI (5)]).

(Synthetic intermediate 1-7A: Biotin-PEG-b-PEI [COOH: EI])
Biotin-PEG (103) -b-PEI (23) (Synthetic intermediate 1-5B) (100 mg) having biotinyl group via a PEG spacer at the terminal end in water (150 μL) / acetonitrile (1 mL) in anhydrous solvent Succinic acid (18.4 mg) / acetonitrile (25 μL) and pyridine (59.4 μL) were added and reacted at room temperature with stirring for 24 hours, followed by ethanol dialysis and water dialysis sequentially. Biotin-PEG-b-PEI [COOH: EI]) (94 mg) in which a carboxyl group was introduced into the side chain was obtained by lyophilization after dialysis. Each component of the obtained polymer is methylene hydrogen (b, a) derived from an ethyleneimine skeleton (—NHCH ₂ CH ₂ —) and an ethylene glycol skeleton (—OCH ₂ CH ₂ —) of ¹ H-NMR spectrum, Methylene hydrogen (e) in the side chain carboxyl group, methylene hydrogen (c) in the amide bond and hydrogen (d) in the biotinyl group could be assigned respectively (FIG. 7A, introduction of side chain carboxyl group from ¹ H-NMR spectrum) The rate is 73%, corresponding to 9 out of 23 secondary amine units in the PEI skeleton) (Biotin-PEG-b-PEI [COOH (9): EI (14)]).

(Synthetic intermediate 1-7B: Biotin-PEG-b-PEI [COOH: EI])
Biotin-PEG (103) -b-PEI (23) (Synthetic intermediate 1-5B) (100 mg) having biotinyl group via a PEG spacer at the terminal end is anhydrous in a mixed solvent of water (20 μL) / acetonitrile (1 mL) Succinic acid (12.3 mg) / acetonitrile (10 μL) and pyridine (59.4 μL) were added and reacted at room temperature with stirring for 24 hours, followed by ethanol dialysis and water dialysis sequentially. Biotin-PEG-b-PEI [COOH: EI]) (46 mg) in which a carboxyl group was introduced into the side chain was obtained by lyophilization after dialysis. Each component of the obtained polymer is methylene hydrogen (b, a) derived from an ethyleneimine skeleton (—NHCH ₂ CH ₂ —) and an ethylene glycol skeleton (—OCH ₂ CH ₂ —) of ¹ H-NMR spectrum, Methylene hydrogen (e) of the side chain carboxyl group, methylene hydrogen (c) of the amide bond and hydrogen (d) of the biotinyl group could be assigned respectively (FIG. 7B, introduction of side chain carboxyl group from ¹ H-NMR spectrum. The rate is 88%, corresponding to 7 out of 23 secondary amine units of the PEI skeleton) (Biotin-PEG-b-PEI [COOH (7): EI (16)]).

(Synthetic intermediate 1-7C: Biotin-PEG-b-PEI [COOH: EI])
Biotin-PEG (103) -b-PEI (23) (Synthetic intermediate 1-5B) (100 mg) having biotinyl group via a PEG spacer at the terminal end is anhydrous in a mixed solvent of water (20 μL) / acetonitrile (1 mL) Succinic acid (4.1 mg) / acetonitrile (3 μL) and pyridine (59.4 μL) were added and reacted at room temperature for 24 hours, followed by ethanol dialysis and water dialysis sequentially. Biotin-PEG-b-PEI [COOH: EI]) (54 mg) in which a carboxyl group was introduced into the side chain was obtained by lyophilization after dialysis. Each component of the obtained polymer is methylene hydrogen (b, a) derived from an ethyleneimine skeleton (—NHCH ₂ CH ₂ —) and an ethylene glycol skeleton (—OCH ₂ CH ₂ —) of ¹ H-NMR spectrum, Methylene hydrogen (e) of the side chain carboxyl group, methylene hydrogen (c) of the amide bond and hydrogen (d) of the biotinyl group could be assigned respectively (FIG. 7C, introduction of side chain carboxyl group from ¹ H-NMR spectrum) The rate is 85%, corresponding to 2 out of 23 secondary amine units of the PEI skeleton) (Biotin-PEG-b-PEI [COOH (3): EI (20)]).

(Synthetic intermediate 1-8: Boc-NH-PEG-b-PEI [COOH: EI: SH])
Boc-NH-PEG-b-PEI [COOH (18): EI (5)] (Synthetic Intermediate 1-6)
(50 mg) in aqueous solution (0.5 mL) was added to the protected thiol derivative 2- (2-pyridyldithio) ethylamine hydrochloride (CASSPy) (0.165 mmol, 12.7 mg), 1-ethyl-3- (3-dimethyl). Aminopropyl) carbodiimide (EDAC) (0.165 mmol, 31.6 mg) was added and reacted at room temperature for 12 hours, and then 55 mg of polymer was recovered by water dialysis and lyophilization. The composition ratio of each segment of the water-soluble polymer calculated by the integral value ratio of the ¹ H-NMR spectrum is Boc-NH-PEG-b-PEI [COOH (1): EI (5): CASSPy (17)]. This was confirmed (FIG. 8). Then, as a result of performing deprotection reaction of the polymer side chain CASSPy, the peak (be, 7.0 to 8.5 ppm) derived from hydrogen of the 2-pyridyl group of the CASSPy protecting group completely disappeared. It was confirmed that Boc-NH-PEG-b-PEI [COOH (1): EI (5): SH (17)] was obtained.

(Synthesis Intermediate 1-9: Boc-NH-PEG- b-PEI [COOH: EI: NH 2])
Boc-NH-PEG-b-PEI [COOH (18): EI (5)] (Synthetic Intermediate 1-6)
(50 mg) in aqueous solution (0.5 mL) was added ethylenediamine (0.03 mmol, 1.8 mg), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) (0.15 mmol, 28.8 mg). The mixture was allowed to react at room temperature for 24 hours, and 50 mg of polymer was recovered by water dialysis and freeze-drying. The composition ratio of each segment of the water-soluble polymer calculated by the integral value ratio of the ¹ H-NMR spectrum is Boc-NH-PEG-b-PEI [COOH (16): EI (5): NH ₂ (2)]. I confirmed that there was.

(Synthesis intermediate 1-10A: Biotin-PEG-b- PEI [COOH: EI: NH 2])
Biotin-PEG-b-PEI [COOH (9): EI (14)] (Synthetic intermediate 1-7A) (94 mg) in aqueous solution (5 mL) was added N- (tert-butoxycarbonyl) -1,2-diamino. Ethane (NH-Boc) (1.04 mmol, 166.6 mg) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) (1.04 mmol, 199.4 mg) were added, and the mixture was stirred at room temperature for 24 hours. After the reaction, 90 mg of the polymer was recovered by water dialysis and lyophilization. Each component of the obtained polymer is methylene hydrogen (b, a) derived from an ethyleneimine skeleton (—NHCH ₂ CH ₂ —) and an ethylene glycol skeleton (—OCH ₂ CH ₂ —) of ¹ H-NMR spectrum, It could be attributed from methylene hydrogen (e) of the side chain carboxyl group, methylene hydrogen (c) of the amide bond and hydrogen (f) of the Boc group, respectively. The composition ratio of each segment of the water-soluble polymer calculated by the integral value ratio of (c) and (f) is Biotin-PEG-b-PEI [COOH (1): EI (14): NH-Boc (8 ]] (FIG. 9A). Thereafter, as a result of deprotection reaction of the polymer side chain Boc, the peak (f, 1.4 ppm) derived from methyl hydrogen of the Boc protecting group disappeared completely, and thus Biotin-PEG-b-PEI [COOH (COOH ( 1): EI (14): NH ₂ (8)] was confirmed to be obtained.

(Synthesis intermediate 1-10B: Biotin-PEG-b- PEI [COOH: EI: NH 2])
Biotin-PEG-b-PEI [COOH (7): EI (16)] (Synthetic intermediate 1-7B) (30 mg) in aqueous solution (2 mL) was added N- (tert-butoxycarbonyl) -1,2-diamino. Ethane (NH-Boc) (0.165 mmol, 26.4 mg) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) (0.165 mmol, 31.6 mg) were added, and the mixture was stirred at room temperature for 24 hours. After the reaction, 44.6 mg of polymer was recovered by water dialysis and lyophilization. As with the synthetic intermediate 1-10A, the composition ratio of each segment of the water-soluble polymer calculated by the integral value ratio of the ¹ H-NMR spectrum is Biotin-PEG-b-PEI [COOH (4): EI (16) : NH-Boc (3)] (FIG. 9B). Thereafter, as a result of deprotection reaction of the polymer side chain Boc, the peak (f, 1.4 ppm) derived from methyl hydrogen of the Boc protecting group disappeared completely, and thus Biotin-PEG-b-PEI [COOH (COOH ( 3): EI (16): NH ₂ (4)] was confirmed to be obtained.

(Synthesis intermediate 1-10C: Biotin-PEG-b- PEI [COOH: EI: NH 2])
Biotin-PEG-b-PEI [COOH (2): EI (21)] (Synthetic intermediate 1-7C) (55 mg) in aqueous solution (3 mL) was added N- (tert-butoxycarbonyl) -1,2-diamino. Ethane (NH-Boc) (0.187 mmol, 30 mg) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) (0.187 mmol, 35.85 mg) were added and reacted at room temperature for 24 hours. Thereafter, 45.1 mg of polymer was recovered by water dialysis and freeze-drying. As with the synthetic intermediate 1-10A, the composition ratio of each segment of the water-soluble polymer calculated by the integral value ratio of the ¹ H-NMR spectrum is Biotin-PEG-b-PEI [COOH (1): EI (21) : NH-Boc (1)] (FIG. 9C). Thereafter, as a result of deprotection reaction of the polymer side chain Boc, the peak (f, 1.4 ppm) derived from methyl hydrogen of the Boc protecting group disappeared completely, and thus Biotin-PEG-b-PEI [COOH (COOH ( 1): EI (20): NH ₂ (2)] was confirmed to be obtained.

Example 2 Synthesis of Europium Complex (DTBTA-Eu) 12 mg of ATBTA-Eu complex (manufactured by Tokyo Chemical Industry) was dissolved in 360 μL of an acetate buffer solution (0.1 M, pH 4.9). To this solution, 2.58 mg of cyanuric chloride dissolved in 150 μL of acetone was added and stirred for 30 minutes. A precipitate is precipitated by adding acetone to the reaction solution, and the precipitate is collected by centrifugation, washed twice with acetone, and then dried by drying under reduced pressure to obtain a europium complex having a cyanuric acid chloride group (DTBTA-Eu). Obtained (yield: 12.9 mg).

Example 3 Synthesis of Water-Soluble Luminescent Labeled Polymer (Polymer 3-1A. Boc-NH-PEG-b-PEI [COOH: EI: fluorescein])
Boc-NH-PEG-b-PEI [COOH (18): EI (5)] (Synthetic Intermediate 1-6)
To a solution of (50 mg) / 10 mM NaHCO ₃ (pH 8.4) (0.4 mL), an amine compound of fluorescein (0.063 mmol, 21.9 mg) / DMSO (0.1 mL) and EDAC (0.126 mmol, 24.2 mg). ) / 10 mM NaHCO ₃ (pH 8.4) (0.1 mL), reacted at room temperature for 24 hours, water dialysis, ultrafiltration (Amicon Ultra-4, centrifugal filter devices, NMWL = 3,000) Recovered 55 mg of polymer. Composition of each segment of water-soluble luminescent labeled polymer calculated by integration ratio of ¹ H-NMR spectrum (comparing hydrogen (b) derived from fluorescein and hydrogen (a) of methyl group derived from polymer terminal Boc protecting group)) The ratio was confirmed to be Boc-NH-PEG-b-PEI [COOH (15): EI (5): fluorescein (3)] (FIG. 10A).

(Polymer 3-1B. Boc-NH-PEG-b-PEI [COOH: EI: fluorescein])
Boc-NH-PEG-b-PEI [COOH (18): EI (5)] (Synthetic Intermediate 1-6)
To a solution of (50 mg) / 10 mM NaHCO ₃ (pH 8.4) (0.4 mL), an amine compound of fluorescein (0.021 mmol, 7.3 mg) / DMSO (0.1 mL) and EDAC (0.126 mmol, 24.2 mg). ) / 10 mM NaHCO ₃ (pH 8.4) (0.1 mL), reacted at room temperature for 24 hours, water dialysis, ultrafiltration (Amicon Ultra-4, centrifugal filter devices, NMWL = 3,000) Recovered 50 mg of polymer. Composition of each segment of water-soluble luminescent labeled polymer calculated by integration ratio of ¹ H-NMR spectrum (comparing hydrogen (b) derived from fluorescein and hydrogen (a) of methyl group derived from polymer terminal Boc protecting group)) The ratio was confirmed to be Boc-NH-PEG-b-PEI [COOH (17): EI (5): fluorescein (1)] (FIG. 10B).

(Polymer 3-2A. Biotin-PEG-b-PEI [COOH: EI: BODIPY])
Biotin-PEG-b-PEI [COOH (1): EI (20): NH ₂ (2)] (Synthetic Intermediate 1-10C) (10 mg) / DMF (0.1 mL) in a BODIPY succinimide compound ( BODIPY630 / 650-NHS) (0.0017 mmol, 1.15 mg) / DMF (0.15 mL) and DIEA (0.013 mmol, 1.7 mg) were added and reacted at room temperature for 24 hours. 8 mg of the polymer was recovered by external filtration (Amicon Ultra-4, centrifugal filter devices, NMWL = 3,000). ¹ H-NMR spectrum of each segment of the water-soluble luminescent labeled polymer calculated by the integral value ratio (compare hydrogen (b) derived from BODIPY and hydrogen (a) of methylene group derived from amide bond of polymer main chain)) The composition ratio was confirmed to be Biotin-PEG-b-PEI [COOH (1): EI (20): NH ₂ (1): BODIPY (1)] (FIG. 11).

(Polymer 3-2B. Biotin-PEG-b-PEI [COOH: EI: BODIPY])
Biotin-PEG-b-PEI [COOH (1): EI (14): NH ₂ (8)] (Synthetic Intermediate 1-10A) (5 mg) / DMF (0.1 mL) in a BODIPY succinimide compound ( BODIPY630 / 650-NHS) (0.015 mmol, 10.48 mg) / DMF (0.15 mL) and N, N-diisopropylethylamine (DIEA) (0.013 mmol, 1.7 mg) were added and reacted at room temperature for 24 hours. Then, 6 mg of the polymer was recovered by water dialysis and ultrafiltration (Amicon Ultra-4, centrifugal filter devices, NMWL = 3,000).

The amount of BODIPY introduced was determined by measuring the absorbance at a UV maximum absorption wavelength of 630 nm based on the Lambert-Beer law. Hereinafter, the calculation method is simply shown. Biotin-PEG-b-PEI [COOH (1): EI (20): BODIPY (2)] (Polymer 3-2A) aqueous solution (1 μM) having a known composition has an absorbance at 630 nm of 0.018. The absorbance at 630 nm of the water-soluble luminescent labeled polymer aqueous solution (1 μM) was 0.004. When comparing the absorbance per mole of polymer, it was about 4 times higher than that of polymer 3-2A, so the number of BODIPY introduced per polymer was calculated to be 8. Therefore, the composition ratio of each segment of the water-soluble luminescent labeled polymer is, Biotin-PEG-b-PEI [COOH (1): EI (14): NH 2 (4): BODIPY (4)] which was the substance to be produced.

(Polymer 3-3A. Biotin-PEG-b-PEI [COOH: EI: Eu])
Biotin-PEG-b-PEI [COOH (1): EI (14): NH ₂ (8)] (Synthetic intermediate 1-10A) (5 mg) / 10 mM NaHCO ₃ (pH 8.4) (0.5 mL) solution Was charged with a cyanuric chloride compound of a europium complex (DTBTA-Eu) (8.8 mg) / 10 mM NaHCO ₃ (pH 8.4) (0.1 mL) and reacted at room temperature for 24 hours. 500 μL of the aqueous polymer solution was recovered by external filtration (Amicon Ultra-4, centrifugal filter devices, NMWL = 3,000).

  Since the number of Eu complexes per polymer was calculated as 8 from the amount of Eu complex (6.7 mg) determined by absorbance measurement at a UV maximum absorption wavelength of 345 nm, the composition ratio of each segment of the water-soluble luminescent labeled polymer was Biotin-PEG-b-PEI [COOH (1): EI (14): Eu (8)].

(Polymer 3-3B. Biotin-PEG-b-PEI [COOH: EI: Eu])
Biotin-PEG-b-PEI [COOH (1): EI (20): NH ₂ (2)] (Synthetic Intermediate 1-10C) (5 mg) / 10 mM NaHCO ₃ (pH 8.4) (0.5 mL) solution Was charged with a cyanuric chloride compound of a europium complex (DTBTA-Eu) (3.3 mg) / 10 mM NaHCO ₃ (pH 8.4) (0.1 mL) and reacted at room temperature for 24 hours. 500 μL of the aqueous polymer solution was recovered by external filtration (Amicon Ultra-4, centrifugal filter devices, NMWL = 3,000).

  Since the number of Eu complexes per polymer was calculated as 2 from the amount of Eu complex (1.6 mg) determined by absorbance measurement at the UV maximum absorption wavelength of 345 nm, the composition ratio of each segment of the water-soluble luminescent labeled polymer is Biotin-PEG-b-PEI [COOH (1): EI (20): Eu (2)].

(Polymer 3-4. Boc-NH-PEG-b-PEI [COOH: EI: Cy3]
Boc-NH-PEG-b- PEI [COOH (16): EI (5): NH 2 (2)] ( Synthesis Intermediate 1-9) (1mg) / DMSO ( 0.1mL) was added succinimide of Cy3 Compound (Cy3-NHS) (0.00045 mmol) was added and reacted at room temperature for 24 hours, followed by water dialysis and an aqueous polymer solution by ultrafiltration (Amicon Ultra-4, centrifugal filter devices, NMWL = 3,000). 500 μL was recovered.

Example 4 Fluorescence measurement of water-soluble luminescent labeled polymer The fluorescence spectrum of the water-soluble luminescent labeled polymer synthesized in Example 3 was measured. The measurement was performed using a fluorescence spectrometer (“FluoroMax-3”, manufactured by HORIBA JOBIN YVON). Fluorescence measurement was performed by irradiating light with an excitation wavelength of 630 nm (BODIPY) or 345 nm (Eu) at room temperature. As a result, Biotin-PEG-b-PEI [COOH: EI: NH ₂ : BODIPY] was compared with the fluorescence intensity normalized by the polymer concentration, and Biotin-PEG-b-PEI [COOH (1): EI ( 20): NH ₂ (1): BODIPY (1)] (polymer 3-2A), Biotin-PEG-b-PEI [COOH (1): EI (14): NH ₂ (4): BODIPY ( 4)] (Polymer 3-2B) was confirmed to be about 13.5 times higher (based on 650 nm) (FIG. 12A). Similarly, in Biotin-PEG-b-PEI [COOH: EI: Eu], when the fluorescence intensity normalized by the polymer concentration is compared, Biotin-PEG-b-PEI [COOH (1): EI (20): Compared with Eu (2)] (polymer 3-3B), Biotin-PEG-b-PEI [COOH (1): EI (14): Eu (8)] (polymer 3-2A) is about 3.7. Double (617 nm reference) was confirmed (FIG. 12B).

Example 5 Fluorescence Detection of Water-Soluble Luminescent Labeled Polymer with DNA Chip For the water-soluble luminescent labeled polymer (polymer 3-2, polymer 3-3) synthesized in Example 3, a DNA chip ( Fluorescence was detected on a DNA chip using “3D-Gene” (registered trademark) manufactured by Toray Industries, Inc. (FIGS. 13A and 13B). A synthetic oligo DNA added with biotin at the 3 ′ end was hybridized with shaking at 10 pmol / chip at 37 ° C. overnight, and then washed by a method recommended by “3D-Gene” (registered trademark). Next, neutravidin (10 μg / mL, 1M MES, 1M NaCl, 0.05% Tween 20) was allowed to react at 20 ° C. for 1 hour. The washing method was washing at 25 ° C. for 5 minutes in a PBST solution (1 × PBS, 0.05% Tween 20).

Finally, a water-soluble luminescent labeled polymer having a biotinyl group on the chip was reacted at 20 ° C. for 1 hour in the same buffer as described above. The washing buffer and temperature used were the recommended method of “3D-Gene” (registered trademark), and the washing time was shortened to 2 minutes each. After washing, the biotin-PEG-b-PEI [COOH: EI: NH ₂ : BODIPY] (polymer 3-2) was dissolved in water with a DNA chip scanner (“ScanArrayExpress”, PerkinElmer Japan) (ex630 nm, em650 nm). An excitation fluorescence image of the luminescent labeled polymer was obtained, and the fluorescence intensity was digitized by GenePix Pro 6.0 (Molecular Device). For Biotin-PEG-b-PEI [COOH: EI: Eu] (polymer 3-3), Olympus fluorescence microscope (Ex = 345 ± 10 nm, Em = 650 ± 75 nm, dichroic mirror = 409 nm) An excitation fluorescence image of the water-soluble luminescent labeled polymer was acquired, and the fluorescence intensity was digitized. The fluorescence intensity at each probe concentration was an average value of 4 spots, and the synthetic oligo 0 μM was calculated as background (BG).

As a result, in the sandwich structure staining method “(DNA) biotin-avidin-biotin (fluorescent label)”, Biotin-PEG-b-PEI [COOH (1): EI (14): NH ₂ (4): In the case of labeling with BODIPY (4)], it is about 18 times as compared with the case of labeling with Biotin-PEG-b-PEI [COOH (1): EI (20): NH ₂ (1): BODIPY (1)]. It was confirmed that the emission intensity was high as described above (difference in emission intensity value between the spot 100 and BLANK). In addition, when labeled with Biotin-PEG-b-PEI [COOH (1): EI (14): Eu (8)], Biotin-PEG-b-PEI [COOH (1): EI (20): Eu Compared with the case of labeling with (2)], it was confirmed that the emission intensity was about twice or more (difference in emission intensity value between the spot 100 and BLANK). The reason why the correlation between the number of luminescent functional groups introduced into the polymer and the luminescence intensity on the DNA chip differs depending on the system is that the reactivity or binding force of the water-soluble fluorescently labeled polymer at the time of biotin-avidin labeling depends on the luminescent functional group. This was presumed to depend on the type of group and the amount introduced.

  The water-soluble luminescent labeled polymer having a luminescent functional group of the present invention has the characteristics that the luminescent functional group is quantitatively bonded to the polymer side chain, and the dispersion stability in water and the fluorescence intensity are increased. As a sensing reagent, for example, it can be applied to biodiagnosis and biotherapy.

Claims (5)

Repeating unit represented by the following formula (1)

Wherein R ₁ is —COOH, —OH or — (OCH ₂ CH ₂ ) _p R _{1 ′} (where R _{1 ′} is —COOH, —OH, —CH═CH ₂ , —C≡CH or — N ₃ and p are integers of 1 to 30), and l is an integer of 1 to 10).
Repeating unit represented by the following formula (2)

(Wherein R ₂ is a luminescent functional group, —NHR ₃ , —SR ₄ (where R ₃ is a hydrogen atom or a tert-butoxycarbonyl group, a benzyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, A protective group for an amino group selected from 2,2,2-trichloroethoxycarbonyl group, allyloxycarbonyl group, phthaloyl group, p-toluenesulfonyl group and 2-nitrobenzenesulfonyl group, R ₄ is a hydrogen atom or a benzyl group , Methoxybenzyl group, N- (acetyl) aminomethyl group, t-butyl group, methylbenzyl group, 3,4-dimethylbenzyl group, triphenylmethyl group, benzhydryl group, acetamidemethyl group, carbomethoxysulfenyl group, 3- Nitro-2-pyridinesulfenyl group, ethylcarbamoyl group, 9-fluoro It is a protecting group for a thiol group selected from a renylmethyl group and a pyridyl sulfide group.) Or — (OCH ₂ CH ₂ ) _p R _{2 ′} (where R _{2 ′} is a luminescent functional group, —NHR ₃ or —SR). ₄ and p are integers of 1 to 30), and the repeating unit number a includes a luminescent functional group in the repeating unit number y, the repeating unit number b including -NHR ₃ and the repeating unit including -SR ₄ The number of units c is b / a + b = 0 to 0.90 and c / a + c = 0 to 0.95, m is an integer of 1 to 10, and n is an integer of 0 to 10), and the following formula (3 ) Repeating unit

The number of repeating units x in formula (1), the number y of repeating units in formula (2), and the number of repeating units z in formula (3) are x / (x + y + z) = 0.01-0.90, y /(X+y+z)=0.01-0.50 and z / (x + y + z) = 0.01-0.90, which are composed of repeating units of the above formulas (1), (2) and (3) At least one terminal of the polymer main chain is selected from the group consisting of —COOH, —OH, —NH ₂ , —SH, —NCO, —CHO, —CH═CH ₂ , —C≡CH, —N ₃ and a biotinyl group. A water-soluble luminescent labeling polymer comprising at least one functional group selected. Between the terminal functional groups and the polymer main chain, as a spacer _{- (OCH 2 CH 2) q} - (. Wherein, q is an integer of 1 to 120), characterized in that it comprises a claim 2. The water-soluble luminescent label polymer according to 1.   The luminescent functional group is BODIPY bonded through an amino group, Cy3 bonded through an amino group, Cy5 bonded through an amino group, a europium complex or fluorescein bonded through an amino group. 3. The water-soluble luminescent label polymer according to 1 or 2.   The water-soluble luminescent labeling polymer according to claim 1, wherein x, y, and z are x + y + z = 10 to 350.   A reagent comprising the water-soluble luminescent labeling polymer according to claim 1.

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