WO2010027108A1 - Fluorescent sugar derivative compound and sensor using same - Google Patents

Abstract

Disclosed are a method for sensing saccharide-binding protein easily, quickly, and with high sensitivity, and a fluorescent compound necessary for this sensing. Specifically, disclosed is a method for sensing saccharide-binding protein that utilizes the fact that when a saccharide-binding protein acts on a molecule having a conjugate structure whereby a sugar chain is joined directly or via an appropriate linker, the fluorescent intensity is significantly greater than when there is no saccharide-binding protein.

Description

Fluorescent sugar derivative compound and sensor using the same

The present invention relates to a target detection method capable of detecting sugar binding proteins as targets and detecting them with high sensitivity, speed and convenience, and a target detection material suitably used for the target detection.

As a detection method for targeting sugar-binding proteins contained in pathogenic substances, biological substances, etc., a conventional method is to introduce a structure containing a sugar chain into the target detection substance and measure the interaction between the target and the sugar chain Known from. However, sugar chains themselves do not exhibit absorption or fluorescence in the normal UV-Vis region, and do not have properties such as conductivity that are easy to measure, so that detection of sugar chain-protein interactions involves surface plasmon resonance, Precise measurement using an electrochemical measurement method, a quartz crystal microbalance measurement method, an atomic force microscope, a field effect transistor, etc. is necessary, and there is a problem in that a special device is required for that purpose. Yes.
As a method for avoiding the problems related to the necessity of such a special apparatus, a method for introducing a structure exhibiting UV-Vis absorption into a sugar chain and detecting it by a general spectrophotometric method has been studied. . For example, Journal of the American Chemical Society, 2001, Vol. 123, p. 8226; Langmuir, 2003, 19, p. 7141; Biochmica et Biophysica Acta, 2006, 636, p. 1760, a material in which the surface of a gold nanoparticle is modified with a sugar chain, Bioconjugate Chemistry, 2007, Vol. 18, p. 146, CdS-ZnS quantum dots modified with sugar chains, Bioconjugate Chemistry, 2000, 11, p. 777; Science, 1993, 261, p. 585; Journal of the American Chemical Society, 1993, 115, p. 1146; Langmuir, 1997, vol. 13, p. 5049; Langmuir, 1997, 13, p. In 6524, a method for observing protein detection as a change in color (visible region) using a material in which a sugar chain is introduced into a side chain group of a pi-conjugated polymer is reported. However, in these methods, since changes in absorbance and absorption wavelength are actually observed, it is difficult to measure a suspended or colored sample solution as it is, and a relatively high concentration detection target is difficult to measure. In general, molecules are required, and it takes usually several tens of minutes to several hours for detection, and there are problems in detection sensitivity and rapidity.
As a method for avoiding the above-described problems in detection using UV-Vis absorption, studies have been conducted to introduce a structure exhibiting fluorescence into a sugar chain and detect it by a change in fluorescence intensity. For example, Journal of the American Chemical Society, 2004, 126, p. 13343; Chemical Communications, 2005, p. 1273; Bioconjugate Chemistry, 2006, 17, p. 575; Biomacromolecules, 2006, 7, p. 2470; Journal of the American Chemical Society, 2008, 130, p. 6952, using a material in which a sugar chain is introduced into a side chain of a light-emitting conjugated polymer, Chemical Communications, 2003, p. In 1250, a method of detecting an interaction with a protein with a decrease in fluorescence intensity using a metal complex having a sugar chain introduced in the periphery is reported. However, these methods that try to detect by lowering the fluorescence intensity are so-called turn-off detection methods in which the fluorescence switch is turned off by the interaction with the protein, but the switch is activated by the interaction with the protein. It is not completely extinguished, and the degree of decrease in fluorescence intensity is 40 to 60% of the case where there is no interaction. To measure this degree of decrease in fluorescence intensity, precise measurement with a precise fluorometer is required. And has problems with sensitivity and simplicity.
As another method of using fluorescence as a detection means, a method has been proposed in which a so-called fluorescent probe that modifies a protein with a fluorescent substance is used to detect and detect changes in fluorescence intensity due to interaction with sugar chains. . However, in this case as well, in order to measure the change in fluorescence intensity, it is necessary to perform precise measurement with a precise fluorometer as before, and there is a problem in speediness and simplicity (for example, JP 2000-338444).

The present invention does not require pretreatment of a target, can detect a sugar-binding protein rapidly, with high sensitivity and high selectivity, and can be easily determined even with the naked eye, and target detection suitably used for the target detection It is an object to provide materials.
During the study on the absorption / luminescence function of a conjugated molecule containing a phosphorus atom, the present inventors introduced a sugar chain into the conjugated molecule and brought it into contact with a lectin, which is a sugar-binding protein. We found an interesting fact that the fluorescence intensity increases significantly, not the decrease in intensity, that is, the start of a so-called turn-on detection method in which strong fluorescence is switched on by interaction with the sugar-binding protein. Therefore, as a result of further diligent research based on this unexpected result, it was found that various conjugated molecules into which sugar chains were introduced exhibited similar turn-on behavior, and the present invention was completed based on these findings. .
The present invention provides the following [1] to [33].
[1] General formula (1)
^{(S 1 -S 2 -S 3 -S} 4 -S 5 -S 6 -A m) n B (1)
[Wherein S ¹ represents a residue obtained by removing a hydrogen atom from one hydroxyl group of a sugar structure, and S ² , S ³ , S ⁴ , S ⁵ , S ⁶ are independently absent, or Represents a residue in which one hydroxyl group is removed from the sugar structure and a hydrogen atom is removed from the other hydroxyl group, and S ¹ , S ² , S ³ , S ⁴ , S ⁵ , and S ⁶ are linked to each other by a glycosidic bond. A represents a divalent organic group having 2 to 6 carbon atoms that may not exist or may contain an oxygen atom, and B emits fluorescence when irradiated with light. Represents a residue obtained by removing n hydrogen atoms from a fluorescent compound, which residue may be substituted with a substituent having 7 or less carbon atoms, and when A is present, m is a positive integer of 6 or less N represents a positive integer of 12 or less. S ² , S ³ , S ⁴ , S ⁵ and S ⁶ may be the same as or different from each other. By removing a hydrogen atom from one hydroxyl group, the structure of the parent sugar that becomes S ¹ (hereinafter referred to as the structure of the parent sugar of S ¹ or simply the structure of the parent sugar) is one hydroxyl group. And by removing a hydrogen atom from the other hydroxyl group, the structure of the parent sugars to be S ² , S ³ , S ⁴ , S ⁵ , and S ⁶ (hereinafter referred to as S ² to S ⁶ parent sugars). The structure, or simply the structure of the parent sugar). ] The fluorescent sugar derivative compound characterized by the above-mentioned.
[2] The parent sugar structure of S ¹ is glucose, mannose, galactose, fructose, allose, talose, psicose, gulose, idose, sorbose, ribose, lyxose, xylose, apiose, deoxyglucose, deoxymannose, deoxygalactose, The fluorescent sugar derivative compound according to [1], which is deoxyallose, deoxytalose, or deoxyribose.
[3] The fluorescent sugar derivative compound according to [1], wherein S ² , S ³ , S ⁴ , S ⁵ or S ⁶ is not present.
[4] The structure of the S ² , S ³ , S ⁴ , S ⁵ , S ⁶ parent sugar is independently selected from glucose, mannose, galactose, fructose, allose, talose, psicose, gulose, idose, sorbose, ribose. The fluorescent sugar derivative compound according to [1], which is lyxose, xylose, apiose, deoxyglucose, deoxymannose, deoxygalactose, deoxyalose, deoxytalose, or deoxyribose.
[5] The fluorescent sugar derivative compound according to [1], wherein A is absent.
[6] The fluorescent sugar derivative compound according to [1], wherein A is CH ₂ CH ₂ O.
[7] The fluorescent sugar derivative compound according to [1], wherein the base fluorescent compound that becomes B by removing n hydrogen atoms is a fluorescent compound having a conjugated structure.
[8] The parent fluorescent compound that becomes B by removing n hydrogen atoms is represented by the general formula (2).

(Wherein R ¹ represents a saturated hydrocarbon group having 6 or less carbon atoms or an unsaturated hydrocarbon group having 12 or less carbon atoms, and may be substituted with a substituent having 7 or less carbon atoms). [1] The fluorescent sugar derivative compound according to [1].
[9] The parent fluorescent compound that becomes B by removing n hydrogen atoms is represented by the general formula (3).

(In the formula, R ² and R ³ represent a saturated hydrocarbon group having 6 or less carbon atoms or an unsaturated hydrocarbon group having 12 or less carbon atoms, and may be the same or different from each other. The fluorescent sugar derivative compound according to [1], which may be substituted with the following substituent.
[10] General formula (1)
^{(S 1 -S 2 -S 3 -S} 4 -S 5 -S 6 -A m) n B (1)
(Wherein, S ¹ , S ² , S ³ , S ⁴ , S ⁵ , S ⁶ , A, B, m, and n have the same meanings as described above) are used as detection materials. A detection target detection method.
[11] The detection method according to [10], wherein the detection target is a substance containing a sugar binding protein.
[12] The detection method according to [10] or [11], wherein a change in fluorescence is used as the detection means.
[13] A detection material capable of interacting with a detection target, and when it does not interact with the detection target, it does not substantially emit fluorescence even when it is electronically excited by light irradiation, or interacts with the detection target. Even if it is not acting, it emits fluorescence when excited by light itself, and emits fluorescence when interacting with the detection target, and emits fluorescence when interacting with the detection target. A method for detecting a detection target, comprising using a detection material whose intensity is greater than the intensity of fluorescence when not interacting with the detection target.
[14] General formula (1) as a detection material
^{(S 1 -S 2 -S 3 -S} 4 -S 5 -S 6 -A m) n B (1)
(Wherein S ¹ , S ² , S ³ , S ⁴ , S ⁵ , S ⁶ , A, B, m and n represent the same meaning as described above), and a compound represented by [13] is used. Detection method.
[15] The detection method according to [13] or [14], wherein the detection target is a substance containing a sugar-binding protein.
[16] A method for detecting a substance containing a sugar-binding protein utilizing the interaction between a substance containing a sugar-binding protein and a fluorescent detection material, wherein the fluorescent detection material substituted with a sugar chain is converted into a fluorescent detection material A method for detecting a substance containing a sugar-binding protein, characterized by being used as:
[17] General formula (1) as a detection material
^{(S 1 -S 2 -S 3 -S} 4 -S 5 -S 6 -A m) n B (1)
(Wherein S ¹ , S ² , S ³ , S ⁴ , S ⁵ , S ⁶ , A, B, m and n represent the same meaning as described above), and a compound represented by [16] is used. Detection method.
[18] The detection method according to any one of [10] to [17], which is performed in an aqueous solution.
[19] The detection method according to [18], wherein the aqueous solution is an aqueous solution containing a buffer.
[20] The detection method according to [19], wherein the buffer is a phosphate buffer, Tris buffer, or Tris hydrochloride buffer.
[21] The detection method according to any one of [11] to [20], which is performed in an aqueous solution in the presence of an agent that activates a sugar-binding protein.
[22] The detection method according to [21], wherein the aqueous solution is an aqueous solution containing a buffer.
[23] The detection method according to [22], wherein the buffer is a phosphate buffer, Tris buffer, or Tris hydrochloride buffer.
[24] An aggregate composed of a sugar-binding protein and the compound according to any one of [1] to [9].
[25] The aggregate according to [24], which is prepared by reacting a sugar-binding protein and the compound according to any one of [1] to [9] in an aqueous solution.
[26] The aggregate according to [25], wherein the aqueous solution is an aqueous solution containing a buffer.
[27] The aggregate according to [26], wherein the buffer is a phosphate buffer, Tris buffer, or Tris hydrochloride buffer.
[28] The aggregate according to any one of [25] to [27], which is prepared by reacting in an aqueous solution in the presence of a drug that activates a sugar-binding protein.
[29] A method for detecting a sugar-binding protein comprising observing fluorescence from the aggregate according to claim 24.
[30] The detection method according to [29], which utilizes the fact that the fluorescence from the aggregate increases in intensity as compared to the fluorescence from the compound itself according to any one of [1] to [9].
[31] The detection method according to [29] or [30], wherein the fluorescence is observed in an aqueous solution.
[32] The detection method according to [31], wherein the aqueous solution is an aqueous solution containing a buffer.
[33] The detection method according to [32], wherein the buffer is a phosphate buffer, Tris buffer, or Tris hydrochloride buffer.
EFFECT OF THE INVENTION The fluorescent sugar derivative compound represented by the general formula (1) of the present invention can be easily synthesized by applying a known reaction technique, and separation and purification can also be performed by chromatography or recrystallization. . In addition, since the fluorescent sugar derivative compound itself represented by the general formula (1) exhibits a significantly stronger fluorescence due to selective interaction with the sugar-binding protein and aggregate formation, the sugar-binding protein is It is useful as a material for detecting a contained substance quickly and with high sensitivity, and can also be easily detected on site, so that it is extremely useful as a practical technique.

FIG. 1 shows changes in fluorescence intensity when ConA is added to the compound of formula (17) (excitation wavelength = 370 nm).
FIG. 2 shows the effect of ConA concentration on the increase in fluorescence intensity at 500 nm when ConA is added to the compound of formula (17) or (21) (excitation wavelength = 370 nm, I ₀ = initial intensity before adding ConA, I = strength when ConA is added). ○ indicates the change of the fluorescence intensity with respect to the initial intensity when ConA is added to the compound of the formula (17), and x indicates the case where ConA is added to the compound of the formula (21).
FIG. 3 shows the change over time in the fluorescence intensity change (excitation wavelength: 370 nm) when ConA is added to the compound of formula (17). An experiment conducted at a concentration of the compound of formula (17) of 17.8 μM and a ConA concentration of 8.0 μM is shown.
FIG. 4 shows the change in fluorescence intensity when PNA is added to the compound of formula (21) (excitation wavelength = 370 nm).
FIG. 5 shows the effect of PNA concentration on the increase in fluorescence intensity at 500 nm when PNA is added to the compound of formula (17) or (21) (excitation wavelength = 370 nm, I ₀ = initial intensity before adding PNA, I = intensity when PNA is added). ○ indicates a change in fluorescence intensity with respect to the initial intensity when PNA is added to compound 21, and x indicates a case where PNA is added to the compound of formula (17).
FIG. 6 shows changes in fluorescence intensity when ConA is added to the compound of formula (25) (excitation wavelength = 320 nm).
FIG. 7 shows the effect of ConA concentration on the increase in fluorescence intensity at 460 nm when ConA is added to the compound of formula (25) (excitation wavelength = 320 nm, I ₀ = initial intensity before adding ConA, I = ConA Strength when added).
FIG. 8 shows changes in fluorescence intensity when ConA is added to the compound of formula (29) (excitation wavelength = 320 nm).
FIG. 9 shows the effect of ConA concentration on the increase in fluorescence intensity at 470 nm when ConA is added to the compound of formula (29) (excitation wavelength = 320 nm, I ₀ = initial intensity before adding ConA, I = ConA Strength when added).
FIG. 10 shows changes in fluorescence intensity (excitation wavelength = 300 nm) when ConA is added to the compound of formula (36) (concentration = 7.3 μM).
FIG. 11 shows the effect of ConA concentration on the increase in fluorescence intensity at 450 nm when ConA is added to the compound of formula (36) (excitation wavelength = 300 nm, I ₀ = initial intensity before adding ConA, I = ConA Strength when added).
FIG. 12 shows the change in fluorescence intensity (excitation wavelength = 312 nm) when ConA is added to the compound of formula (40).
FIG. 13 shows the detection of ConA by a handy UV lamp with the compound of formula (17). The left is a test solution in which only the compound of formula (17) is dissolved in the salt-containing buffer prepared in Example 6 [the concentration of the compound of formula (17) is 19 μM], and the right is the salt prepared in Example 6. ConA was added to a solution in which the compound of formula (17) was dissolved in the containing buffer and stirred for 10 minutes. The concentration of the compound of formula (17) was 19 μM and the concentration of ConA was 8 μM. When it is used and irradiated with 365 nm ultraviolet light, the right test solution emits extremely strong fluorescence.
FIG. 14 shows the change in fluorescence intensity when RCA120 is added to the compound of formula (43) (concentration = 6.7 μM) (excitation wavelength = 320 nm).
FIG. 15 shows the emission intensity ratio (I / I ₀ = emission intensity after addition of RCA120 / initial emission intensity) at 400 nm when RCA120 is added to the compound of formula (43).

Each group shown in this specification is specifically as follows.
Examples of the saturated hydrocarbon group having 6 or less carbon atoms include n-hexyl group, cyclohexyl group, n-pentyl group, isopentyl group, n-butyl group, sec-butyl group, isobutyl group, n-propyl group, isopropyl group, and ethyl. Examples thereof include linear, branched or cyclic aliphatic saturated hydrocarbon groups such as a group and a methyl group.
Examples of the unsaturated hydrocarbon group having 12 or less carbon atoms include vinyl group, isopropenyl group, 1-propenyl group, allyl group, 1-buten-1-yl group, 2-buten-1-yl group, and 3-butene- 1-yl group, 3-buten-2-yl group, 2,2-dimethylvinyl group, 1-penten-1-yl group, 2-penten-1-yl group, 3-penten-1-yl group, 3 -Penten-2-yl group, 4-penten-1-yl group, 4-penten-2-yl group, 4-penten-3-yl group, 4-penten-4-yl group, 1,2-dimethyl- 1-buten-1-yl group, 1-octen-1-yl group, 1-decene-1-yl group, 1-dodecene-1-yl group, 1-cyclohexen-1-yl group, 1-cyclohexene-3 -Yl group, 1-cycloocten-1-yl group, 1-methyl-1-cyclohe Sen-2-yl group, phenyl group, alpha naphthyl group, beta naphthyl group, beta anthryl group, orthobiphenyl group, metabiphenyl group, parabiphenyl group, benzyl group, 1- or 2-phenylethyl group, etc. Examples thereof include a chain, branched or cyclic aliphatic unsaturated hydrocarbon group, and an aromatic hydrocarbon group.
Examples of the substituent having 7 or less carbon atoms include those having 7 or less carbon atoms in the saturated hydrocarbon group having 6 or less carbon atoms, n-heptyl group, and unsaturated hydrocarbon groups having 12 or less carbon atoms, benzyl group , Methoxy group, ethoxy group, butoxy group, phenoxy group, benzyloxy group, methylthio group, phenylthio group, methoxymethyl group, methoxyethyl group, dimethylamino group, carbomethoxy group, carboethoxy group, cyano group, acetyl group, benzoyl group , Trimethylsilyl group, fluorine atom, chlorine atom, bromine atom and the like.
General formula (1) of the present invention
^{(S 1 -S 2 -S 3 -S} 4 -S 5 -S 6 -A m) n B (1)
In the fluorescent sugar derivative compound represented by the formula: S ¹ , S ² , S ³ , S ⁴ , S ⁵ , and S ⁶ constituting the compound are those in which the structure of the parent sugar is selected from monosaccharides, S ¹ -S ² -S ³ -S ⁴ -S ⁵ -S ⁶ form a sugar chain structure in which these monosaccharides are linked to each other by glycosidic bonds. Any monosaccharide can be used depending on the detection target, and preferable examples include glucose, mannose, galactose, fructose, allose, talose, psicose, gulose, idose, sorbose and other 6-carbon sugars and ribose. And pentoses such as lyxose, xylose and apiose. The monosaccharide may further be a so-called deoxy sugar in which the hydroxyl group of the hexose or pentose is substituted with hydrogen. Examples of these deoxy sugars include deoxyglucose, deoxymannose, deoxygalactose, deoxyallose, Examples include deoxytalose and deoxyribose.
In one embodiment of the present invention, S ¹ , S ² , S ³ , S ⁴ , S ⁵ , and S ⁶ specified by the general formula (1) are limited to a linear structure connected by a glycosidic bond. Alternatively, a structure in which the non-reducing terminal side is branched may be used. As a branched structure, for example,

However, it is not particularly limited. In the branched structure, when one or more sugar residues among S ² , S ³ , S ⁴ , S ⁵ , and S ⁶ are non-reducing terminals, the definition of such sugar residues is , S ¹ shall be followed.
In the fluorescent sugar derivative compound represented by the general formula (1), A is defined as a divalent organic group having 2 to 6 carbon atoms which may not exist or may contain an oxygen atom. . The presence or absence of A is presumed to affect the interaction with the detection target and the difficulty of aggregate formation. In the case where A is present, as A, dimethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, 1,2-propylene group and other alkylene groups, ethyleneoxy group, 1,3-propyleneoxy group , 1,2-propyleneoxy group, alkylenemethylene group containing an oxygen atom in the chain, such as a tetramethyleneoxy group, etc. are included, but an alkyleneoxy group having a flexible structure that can be adapted to the interaction with the detection target In view of solubility and availability, an ethyleneoxy group is particularly preferable.
In the fluorescent sugar derivative compound represented by the general formula (1), B represents a residue obtained by removing n hydrogen atoms from a fluorescent compound that emits fluorescence when irradiated with light. The parent fluorescent compound excluding n hydrogen atoms may be any fluorescent compound that has been reported in the past in photochemical research. From the viewpoint of ease of detection, the visible fluorescent region can be used. Those exhibiting fluorescence are preferable, and various conjugated molecules including structures that have been researched and developed as electroluminescent materials can be used.

(Wherein R ¹ represents a saturated hydrocarbon group having 6 or less carbon atoms or an unsaturated hydrocarbon group having 12 or less carbon atoms, and may be substituted with a substituent having 7 or less carbon atoms). Compound, general formula (3)

(In the formula, R ² and R ³ represent a saturated hydrocarbon group having 6 or less carbon atoms or an unsaturated hydrocarbon group having 12 or less carbon atoms, and may be the same or different from each other. A compound represented by formula (4), which may be substituted with the following substituents:

A compound represented by formula (5):

A compound represented by formula (6):

A compound represented by formula (7):

A compound represented by formula (8):

A compound represented by the general formula (9)

(In the formula, R ⁴ and R ⁵ represent a saturated hydrocarbon group having 6 or less carbon atoms or an unsaturated hydrocarbon group having 12 or less carbon atoms, and may be the same or different from each other. A compound represented by the following general formula (10):

(In the formula, R ⁶ and R ⁷ represent a saturated hydrocarbon group having 6 or less carbon atoms or an unsaturated hydrocarbon group having 12 or less carbon atoms, and may be the same or different from each other. A compound represented by formula (11), which may be substituted with the following substituents:

The compound etc. which are represented by these can be illustrated.
General formula (1) of the present invention
^{(S 1 -S 2 -S 3 -S} 4 -S 5 -S 6 -A m) n B (1)
In the fluorescent sugar derivative compound represented by the formula, a sugar chain or a substance containing a sugar binding protein is changed because sugar chains arranged in the periphery change the fluorescence behavior by interacting with or forming an aggregate with the sugar binding protein. It can be used as a detection material for detection. In the highly sensitive detection of a sugar-binding protein or a substance containing a sugar-binding protein according to the present invention, when a detection target is contained in a sample, the sample is added to the compound solution of the general formula (1) and irradiated with light. It can be easily performed by observing that the intensity of the emitted fluorescence changes, and preferably the intensity increases. There is no particular limitation on the sugar-binding protein or substance containing a sugar-binding protein that is a detection target as long as it recognizes sugar and strongly interacts, binds or forms an aggregate. Any substance having a protein capable of recognizing a sugar may be used, for example, various biological substances, cells, microorganisms, viruses and the like.
As a medium used for detection, a mixed solvent in which a water-soluble solvent such as methanol, ethanol, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, and acetone is mixed with water can be used, but water is preferably used. Interaction and aggregation between the sugar-binding protein and the detection material of the present invention often depend on pH, and fluorescence used as a detection means is also easily affected by pH. On the other hand, the pH of the solution at the time of detection may change depending on external factors such as atmospheric carbon dioxide, protein structures, and internal factors such as the microorganism's own metabolites. Considering these, in order to perform reliable detection, it is preferable to use a buffer solution to keep the pH of the solution constant. The preferred pH generally depends on the combination of the specimen and the detection material, but can be used without any problem as long as it can maintain a pH of around 7 in relation to handling biologically relevant proteins. For example, Tris buffer Tris-HCl buffer, phosphate buffer, Hepes buffer, and the like can be suitably used.
In order to promote the interaction and aggregation between the sugar-binding protein or the substance containing the sugar-binding protein and the detection material of the present invention, the detection operation may be performed in the presence of an agent that activates the sugar-binding protein. This is an advantageous embodiment. As such agents, various inorganic salts are used, and preferred examples include sodium salts, potassium salts, calcium salts, magnesium salts, manganese salts and the like.
The light source used for light irradiation may be any light source that emits light having a wavelength near the absorption wavelength of the fluorescent sugar derivative compound. Depending on the structure of the fluorescent sugar derivative compound, a xenon lamp, a low-pressure mercury lamp may be used. A medium pressure mercury lamp, a high pressure mercury lamp, etc. can be used, but if necessary, the emitted light from the xenon lamp may be spectrally used. For convenience, an ultraviolet lamp used for detection in the chromatographic operation is used. You can also In general, detection can be easily performed by visual observation with the naked eye, but quantitative data can also be obtained by using a fluorometer.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
Example 1
The following reaction formula (12)

According to formula (17)

This compound was synthesized.
Add a compound of formula (16) (0.03 g, 0.017 mmol), sodium methoxide (0.003 g, 0.054 mmol), and methanol (2 ml) to a 10 ml flask equipped with a magnetic stir bar, and stir at room temperature for 30 minutes. did. Thereafter, a cation exchange resin (DOWEX 50WX8-100) was added until the reaction solution became acidic, filtered, and the solvent was distilled off to obtain a compound of the formula (17) (0.013 g, 0.012 mmol, yield 72). %).
This compound is a new compound not yet published in the literature, and its properties, spectroscopic data, and analytical data were as follows. Yellow solid; mp 267.3-268.9 ° C .;¹H NMR (300 MHz, CD₃OD) δ 1.75-1.84 (m, 4H), 2.75-2.88 (m, 4H), 3.51-3.58 (m, 4H), 3.69-3.75 (m) , 12H), 3.82-3.86 (m, 4H), 3.95 (s, 4H), 5.40 (s, 4H), 6.82 (s, 2H), 6.92 (s, 4H), 7.46-7.53 (m, 3H), 7.68-7.75 (m, 2H);¹³C NMR (75 MHz, CD₃OD) δ 23.4, 28.8, 62.6, 68.2, 71.8, 72.3, 75.5, 100.3, 106.4, 112.0, 130.3 (d,³J (P, C) 12.4 Hz), 130.5 (d,¹J (P, C) 98.5 Hz), 130.6 (d,¹J (P, C) 98.3 Hz), 131.9 (d,²J (P, C) 11.0 Hz), 133.7 (d,⁴J (P, C) 2.9 Hz), 135.6 (d,²J (P, C) 11.0 Hz), 151.9 (d,²J (P, C) 26.1 Hz), 158.9;³¹P {¹H} NMR (162 MHz, CD₃OD) δ 46.4; HRMS (FAB, glycerin matrix) C₅₀H₆₃O₂₅PLi ([M + Li]⁺Calculated value as 1101.3556, measured value; 1101.3533.
Reference example 1
Formula (14) which is a raw material of the compound represented by Formula (16) used in Example 1 above

This compound was synthesized as follows.
To a 50 ml flask equipped with a magnetic stir bar, a compound of formula (13) (0.31 g, 0.73 mmol), 30% aqueous hydrogen peroxide (0.09 g, 0.80 mmol) and THF (30 ml) were added, For 20 minutes. Thereafter, the solvent was distilled off and the residue was isolated by silica gel column chromatography (ethyl acetate) to obtain a compound of the formula (14) (0.22 g, 0.50 mmol, yield 69%).
Yellow solid; mp 215.1-219.3 ° C .;¹1 H NMR (300 MHz, acetone-d₆) 1.71 (m, 4H), 2.61-2.89 (m, 4H), 6.24 (s, 2H), 6.49 (s, 4H), 7.37-7.49 (m) , 3H), 7.73-7.79 (m, 2H), 8.59 (m, 4H);¹³C NMR (75 MHz, acetone-d₆) Δ 22.6, 28.0 (d,³J (P, C) 14.7 Hz), 103.0, 107.7 (d,³J (P, C) 5.6 Hz), 129.3, (d,³J (P, C) 11.9 Hz), 130.2 (d,¹J (P, C) 93.6 Hz), 130.5 (d,¹J (P, C) 97.3 Hz), 131.2 (d,²J (P, C) 10.3 Hz), 132.3 (d,⁴J (P, C) 2.6 Hz), 135.0 (d,²J (P, C) 11.3 Hz), 149.5 (d,²J (P, C) 26.6 Hz), 159.0;³¹P {¹H} NMR (162 MHz, acetone-d₆) 44.5; HRMS (FAB) C₂₆H₂₄O₅P ([M + H]⁺Calculated value as 447.1361, measured value: 447.1371.
Reference example 2
Formula (16) used as a raw material in Example 1 above

This compound was synthesized as follows.
A 25 ml three-necked flask equipped with a magnetic stirring bar was prepared and purged with nitrogen. Under a nitrogen atmosphere, methylene chloride (5 ml), the compound of formula (14) (0.076 g, 0.017 mmol), the compound of compound (15) (0.42 g, 0.85 mmol), and molecular sieves 4A were added, Boron trifluoride diethyl ether complex (0.20 g, 2.03 mmol) was added dropwise and stirred at room temperature for 20 hours. Thereafter, the reaction solution is poured into a saturated aqueous solution of sodium bicarbonate, the organic layer is washed with a saturated aqueous solution of sodium bicarbonate, water and saturated saline, the organic layer is dried over magnesium sulfate, the solvent is distilled off, and the silica gel column chromatography is performed. (Methylene chloride → hexane / ethyl acetate = 1/1 → methylene chloride / acetone = 10/1) to obtain a compound of the formula (16) (0.094 g, 0.053 mmol, yield 32%) ).
Yellow solid; melting point 123.1-125.9 ° C .;¹H NMR (300 MHz, CDCl₃) Δ 1.75 (m, 4H), 1.99-2.01 (m, 36H), 2.16 (s, 12H), 2.72-2.78 (m, 4H), 3.95-3 .98 (m, 8H), 4.18-4.24 (m, 4H), 5.27-5.46 (m, 16H), 6.69 (s, 2H), 6.76 (s, 2H) ), 6.85 (s, 2H), 7.36-7.45 (m, 3H), 7.61-7.67 (m, 2H);¹³C NMR (75 MHz, CDCl₃) Δ 20.6, 20.8, 22.1, 27.4, 60.3, 61.8, 65.5, 68.7, 69.0, 95.5, 104.4, 111.2, 128 .6 (d,¹J (P, C) 93.7 Hz), 129.0 (d,³J (P, C) 11.9 Hz), 130.3 (d,¹J (P, C) 97.3 Hz), 130.5 (d,²J (P, C) 10.4 Hz), 132.1, 135.1 (d,²J (P, C) 11.5 Hz), 149.3 (d,²J (P, C) 26.2 Hz), 156.3;³¹P {¹H} NMR (162 MHz, CDCl₃) Δ 42.8; Elemental analysis C₈₂H₉₅O₄₁Calculated as P: C, 55.72; H, 5.42. Found: C, 55.63; H, 5.75.
Example 2
The following reaction formula (18)

According to formula (21)

This compound was synthesized as follows.
Add a compound of formula (20) (0.011 g, 0.006 mmol), sodium methoxide (0.001 g, 0.019 mmol), and methanol (2 ml) to a 10 ml flask equipped with a magnetic stir bar, and stir at room temperature for 40 minutes. did. Thereafter, a cation exchange resin (DOWEX 50WX8-100) was added until the reaction solution became acidic, filtered, and the solvent was distilled off to obtain a compound of formula (21) (0.0006 g, 0.005 mmol, yield 88). %).
This compound is a novel compound not yet published in the literature, and its properties, spectroscopic data, and analytical data were as follows. Yellow solid; mp 230.0-233.5 ° C;¹H NMR (300 MHz, CD₃OD) δ 1.75-1.83 (m, 4H), 2.89-3.07 (m, 4H), 3.53-3.56 (m, 4H), 3.69-3.77 (m) , 16H), 3.87-3.88 (m, 4H), 4.80-4.81 (m, 4H), 6.79-6.82 (m, 4H), 6.87 (s, 2H) ), 7.43-7.54 (m, 3H), 7.66-7.72 (m, 2H);¹³C NMR (75 MHz, CD₃OD) δ 23.5, 28.9, 62.5, 70.3, 72.1, 74.8, 77.1, {102.2, 102.4}, 105.6, {111.5, 111 .9}, 130.3 (d,³J (P, C) 11.7 Hz), 130.3 (d,¹J (P, C) 98.8 Hz), 131.3 (d,¹J (P, C) 98.8 Hz), 131.9 (d,²J (P, C) 10.3 Hz), 133.6, 135.3 (d,²J (P, C) 11.6 Hz), 151.4, 160.0;³¹P {¹H} NMR (162 MHz, CD₃OD) δ 47.2; HRMS (FAB, matrix; glycerol) C₅₀H₆₄O₂₅P ([M + H]⁺Calculated value as 109.53474, measured value; 10953468.
Reference example 3
Formula (20) used as a raw material in Example 2 above

This compound was synthesized as follows.
A 30 ml two-necked flask equipped with a magnetic stirring bar was prepared and purged with nitrogen. Under a nitrogen atmosphere, methylene chloride (5 ml), the compound of formula (14) (0.15 g, 0.34 mmol), the compound of formula (19) (0.52 g, 1.34 mmol) were added, and SnCl 2 was added.₄(0.71 g, 2.72 mmol) was added dropwise and stirred at room temperature for 5 days. Thereafter, the reaction solution was poured into a saturated aqueous sodium hydrogen carbonate solution and extracted with methylene chloride. The organic layer was washed with saturated aqueous sodium hydrogen carbonate solution, water and saturated brine, dried over sodium sulfate, suction filtered through celite, evaporated, and silica gel column chromatography (hexane / ethyl acetate = 1/3 → acetone). / Methylene chloride = 1/5) to give a compound of formula (20) (0.065 g, 0.037 mmol, 11% yield).
Yellow solid; melting point 146.8-149.7 ° C .;¹H NMR (300 MHz, CDCl₃) Δ 1.84 (m, 4H), 2.00-2.01 (m, 18H), 2.06 (m, 18H), 2.15-2.16 (m, 12H), 2.77 (m) , 4H), 3.91-3.93 (m, 2H), 4.01-4.16 (m, 10H), 4.91-4.94 (m, 2H), 5.02-5.13. (M, 6H), 5.36-5.44 (m, 8H), 6.51 (s, 2H), 6.63 (s, 2H), 6.75 (s. 2H), 7.44- 7.49 (m, 3H), 7.68-7.75 (m, 2H);¹³C NMR (75 MHz, CDCl₃) Δ 20.5, 20.6, 20.72, 20.74, 22.3, 27.5 (d,³J (P, C) 13.9 Hz), 60.8, 66.6, 68.4, 70.7, 70.8, 98.5, 105.2, 110.9, 129.15, 129.2. (D,¹J (P, C) 93.0 Hz), 130.2 (d,¹J (P, C) 96.6 Hz), 130.7 (d,²J (P, C) 10.4 Hz), 132.2, 134.7 (d,²J (P, C) 11.4 Hz), 149.5 (d,²J (P, C) 23.5 Hz), 157.4, 169.3, 170.0, 170.1, 170.4;³¹P {¹H} NMR (162 MHz, CDCl₃) Δ 42.8; Elemental analysis C₈₂H₉₇O₄₂PH₂Calculated as O: C, 55.16; H, 5.48. Found: C, 54.92; H, 5.43.
Example 3
The following reaction formula (22)

According to formula (25)

This compound was synthesized as follows.
To a 10 ml flask equipped with a magnetic stir bar was added the compound of formula (24) (0.025 g, 0.015 mmol), sodium methoxide (0.003 g, 0.05 mmol), methanol (2 ml), THF (2 ml), Stir at room temperature for 60 minutes. Thereafter, a cation exchange resin (DOWEX 50WX8-100) was added until the reaction solution became acidic, filtered, and the solvent was distilled off to obtain a compound of the formula (25) (0.013 g, 0.012 mmol, yield 87). %).
This compound is a novel compound not yet published in the literature, and its properties, spectroscopic data, and analytical data were as follows. Pale yellow solid; mp 255.2-258.9 ° C .;¹H NMR (300 MHz, D₂O) δ 3.52-3.58 (m, 8H), 3.67-3.70 (m, 4H), 3.77-3.83 (m, 4H), 3.93-4.00 (m , 8H), 5.34 (s, 4H), 6.61-6.67 (m, 8H), 6.71-6.75 (m, 8H). ;¹³C NMR (75 MHz, D₂O) δ 61.3, 67.1, 71.1, 71.6, 74.2, 99.2, 117.0, 133.5, 139.0, 139.7, 155.3; HRMS (FAB, Glycerin matrix) C₅₀H₆₁O₂₄([M + H]⁺Equivalent value); 1045.3553, measured value; 1045.3533.
Reference example 4
Formula (24) used as raw material in Example 3 above

This compound was synthesized as follows.
A 50 ml two-necked flask equipped with a magnetic stirring bar was prepared and purged with nitrogen. To this were distilled methylene chloride (15 ml), a compound of formula (23) (0.20 g, 0.50 mmol), a compound of formula (15) (1.21 g, 2.46 mmol), and molecular sieves 4A in a nitrogen atmosphere. In addition, boron trifluoride diethyl ether complex (0.76 ml, 6.0 mmol) was added dropwise, and the mixture was stirred at room temperature for 16 hours. Thereafter, the reaction solution is poured into a saturated aqueous solution of sodium bicarbonate, the organic layer is washed with a saturated aqueous solution of sodium bicarbonate, water and saturated saline, the organic layer is dried over sodium sulfate, the solvent is distilled off, and the silica gel column chromatography is performed. Isolation by chromatography (methylene chloride / acetone = 20/1) and preparative HPLC gave the compound of formula (24) (0.028 g, 0.016 mmol, 3% yield).
White solid; melting point 114.1-118.4 ° C .;¹H NMR (300 MHz, CDCl₃) 2.02 (m, 24H), 2.04 (s, 12H), 2.17 (s, 12H), 4.04-4.12 (m, 8H), 4.25-4.31 (m) 4H), 5.31-5.54 (m, 16H), 6.82 (d, 8H, J = 8.7 Hz), 6.91 (d, 8H, J = 8.7 Hz);¹³C NMR (75 MHz, CDCl₃) Δ20.6, 20.8, 62.0, 65.9, 68.8, 69.1, 69.4, 95.9, 115.9, 132.5, 138.5, 138.8, 154 2, 169.7, 169.86, 169.93, 170.4; RMS (FAB, 2-nitrobenzyl alcohol matrix) C₈₂H₉₂O₄₀([M]⁺); Calculated value as 1716.5165, actually measured value; 1716.5171.
Example 4
The following reaction formula (26)

According to formula (29)

This compound was synthesized as follows.
Add a compound of formula (28) (0.024 g, 0.011 mmol), sodium methoxide (0.002 g, 0.04 mmol), and methanol (2 ml) to a 10 ml flask equipped with a magnetic stir bar, and stir at room temperature for 80 minutes. did. Thereafter, a cation exchange resin (DOWEX 50WX8-100) was added until the reaction solution became acidic, filtered, and the solvent was distilled off to obtain a compound of the formula (29) (0.014 g, 0.009 mmol, yield 88). %).
This compound is a novel compound not yet published in the literature, and its properties, spectroscopic data, and analytical data were as follows. Pale yellow solid; mp 86.6-89.3 ° C .;¹H NMR (300 MHz, CD₃OD) [delta] 3.59-3.73 (m, 28H), 3.81-3.87 (m, 20H), 4.02-4.05 (m, 8H), 4.81 (s, 4H), 6.68 (d, 8H, J = 8.4 Hz), 6.89 (d, 8H, J = 8.4 Hz);¹³C NMR (75 MHz, CD₃OD) δ 62.9, 67.8, 68.5, 68.6, 70.8, 71.5, 72.1, 72.5, 74.6, 101.8, 114.8, 133.7, 138.3, 140.0, 158.7; HRMS (FAB, glycerin matrix) C₆₆H₉₂O₃₂([M]⁺) Calculated value as); 1396.5572, actually measured value: 1396.5605.
Reference Example 5
Formula (27) used as a raw material for the compound of Formula (28) used in Example 4 above

This compound was synthesized as follows.
A 300 ml three-necked flask equipped with a magnetic stirrer was prepared and purged with nitrogen. Under nitrogen atmosphere, DMF (100 ml), compound of formula (23) (0.40 g, 1.00 mmol), 2-chloroethoxyethanol (1.25 g, 10.0 mmol), potassium carbonate (4.16 g, 30 0.1 mmol) and potassium iodide (0.21 g, 1.27 mmol) were added, and the mixture was stirred at 100 ° C. for 8 hours. Thereafter, the solvent was distilled off, ethyl acetate (100 ml) was added, and the mixture was washed with a saturated aqueous ammonium chloride solution and saturated brine. The organic layer was dried over sodium sulfate, the solvent was distilled off, and silica gel column chromatography (methylene chloride / Acetone = 1/3) to give a compound of formula (27) (0.35 g, 0.47 mmol, 47% yield).
White solid; melting point 138.6-140.0 ° C .;¹H NMR (300 MHz, CDCl₃) Δ 2.5-2.29 (m, 4H), 3.63-3.66 (m, 8H), 3.72-3.75 (m, 8H), 3.81-3.84 (m, 8H), 4.06-4.87 (m, 8H), 6.65 (d, 8H, J = 8.4 Hz), 6.91 (d, 8H, J = 8.4 Hz);¹³C NMR (75 MHz, CDCl₃) 61.8, 67.2, 69.7, 72.6, 113.7, 132.5, 137.1, 138.5, 156.9; Elemental analysis C₄₂H₅₂O₁₂Calculated as: C, 67.36; H, 7.00. Found: C, 67.30; H, 6.97.
Reference Example 6
Formula (28) used as a raw material in Example 4 above

This compound was synthesized as follows.
A 50 ml two-necked flask equipped with a magnetic stirring bar was prepared and purged with nitrogen. To this were distilled methylene chloride (15 ml), a compound of formula (27) (0.23 g, 0.30 mmol), a compound of formula (15) (0.73 g, 1.48 mmol), and molecular sieves 4A in a nitrogen atmosphere. In addition, boron trifluoride diethyl ether complex (0.45 ml, 3.6 mmol) was added dropwise and stirred at room temperature for 20 hours. Thereafter, the reaction solution is poured into a saturated aqueous solution of sodium bicarbonate, the organic layer is washed with a saturated aqueous solution of sodium bicarbonate, water and saturated saline, the organic layer is dried over sodium sulfate, the solvent is distilled off, and the silica gel column chromatography is performed. Isolated by chromatography (methylene chloride / acetone = 10/1) to give the compound of formula (28) (0.31 g, 0.15 mmol, yield 50%).
Pale yellow solid; melting point 57.3-62.0 ° C .;¹H NMR (300 MHz, CDCl₃) 1.9.95-1.96 (m, 24H), 2.05 (s, 12H), 2.10 (s, 12H), 3.68-3.78 (m, 24H), 4.02-4 .07 (m, 16H), 4.21-4.25 (m, 4H), 4.85 (s, 4H), 5.24-5.31 (m, 12H), 6.60 (d, 8H) , J = 7.2 Hz), 6.85 (d, 8H, J = 7.2 Hz);¹³C NMR (75 MHz, CDCl₃) Δ 20.5, 20.6, 20.7, 62.3, 66.0, 67.0, 67.2, 68.3, 68.9, 69.4, 69.7, 70.0, 97 6, 113.5, 132.4, 136.9, 138.2, 156.8, 169.6, 169.7, 169.8, 170.4; Elemental analysis C₉₈H₁₂₄O₄₈Calculated as: C, 56.86; H, 6.04. Found: C, 57.15; H, 5.96.
Example 5
The following reaction formula (30)

According to formula (36)

This compound was synthesized as follows.
Add a compound of formula (35) (0.041 g, 0.011 mmol), sodium methoxide (0.004 g, 0.07 mmol), and methanol (3 ml) to a 10 ml flask equipped with a magnetic stir bar, and stir at room temperature for 2 hours. did. Thereafter, a cation exchange resin (DOWEX 50WX8-100) was added until the reaction solution became acidic, filtered, and the solvent was distilled off to obtain a compound of formula (36) (0.024 g, 0.0092 mmol, yield 83). %).
This compound is a novel compound not yet published in the literature, and its properties, spectroscopic data, and analytical data were as follows. Pale yellow solid; mp 83.7-87.6 ° C;¹H NMR (300 MHz, CD₃OD) δ 3.59-3.72 (m, 72H), 3.81-3.93 (m, 40H), 4.82 (s, 8H), 6.28 (s, 8H), 6.37 ( s, 4H);¹³C NMR (75 MHz, CD₃OD) δ 62.9, 67.8, 68.6, 68.7, 70.6, 71.5, 72.1, 72.5, 74.6, 101.8, 102.1, 111.0, 142.3, 146.5, 160.8.
Reference Example 7
Formula (35) used as raw material in Example 5 above

This compound was synthesized as follows.
A 50 ml two-necked flask equipped with a magnetic stirring bar was prepared and purged with nitrogen. Thereto were distilled methylene chloride (15 ml), a compound of formula (34) (0.33 g, 0.28 mmol), a compound of formula (15) (1.43 g, 2.90 mmol), and molecular sieves 4A under a nitrogen atmosphere. In addition, boron trifluoride diethyl ether complex (0.85 ml, 6.7 mmol) was added dropwise, and the mixture was stirred at room temperature for 41 hours. Thereafter, the reaction solution is poured into a saturated aqueous solution of sodium bicarbonate, the organic layer is washed with a saturated aqueous solution of sodium bicarbonate, water and saturated saline, the organic layer is dried over sodium sulfate, the solvent is distilled off, and the silica gel column chromatography is performed. Isolation by chromatography (methylene chloride / acetone = 5/1) gave the compound of formula (35) (0.16 g, 0.042 mmol, 15% yield).
Pale yellow solid; melting point 59.2-62.9 ° C .;¹H NMR (300 MHz, CDCl₃) Δ 1.96 (s, 24H), 2.01 (s, 24H), 2.06 (s, 24H), 2.12 (s, 24H), 3.64-3.73 (m, 40H), 3.78-3.85 (m, 24H), 4.05-4.08 (m, 16H), 4.24-4.29 (m, 8H), 4.85 (s, 8H), 5. 25-5.33 (m, 24H), 6.17 (s, 8H), 6.24 (s, 4H);¹³C NMR (75 MHz, CDCl₃) Δ 20.61, 20.64, 20.7, 20.8, 62.3, 66.0, 67.2, 67.2, 68.3, 69.0, 69.4, 69.6, 70 0.0, 97.7, 100.6, 109.8, 140.6, 144.8, 159.0, 169.7, 169.8, 169.9, 170.6; Elemental analysis C₁₇₀H₂₂₈O₉₆Calculated as: C, 53.63; H, 6.04. Found: C, 53.84; H, 5.92.
Reference Example 8
Formula (34) used as a raw material for the compound of Formula (35)

This compound was synthesized as follows.
A 200 ml three-necked flask equipped with a magnetic stirrer was prepared and purged with nitrogen. Under nitrogen atmosphere, DMF (100 ml), compound of formula (33) (0.38 g, 0.82 mmol), 2-chloroethoxyethanol (2.04 g, 16.4 mmol), potassium carbonate (6.83 g, 49 0.4 mmol) and potassium iodide (0.33 g, 2.02 mmol) were added, and the mixture was stirred at 100 ° C. for 13 hours. Thereafter, the solvent was distilled off, methylene chloride (200 ml) was added, washed with a saturated aqueous ammonium chloride solution and saturated brine, the organic layer was dried over sodium sulfate, the solvent was distilled off, and silica gel column chromatography (chloroform / methanol). = 5/1) to give the compound of formula (34) (0.42 g, 0.36 mmol, 44% yield).
Pale yellow oil;¹H NMR (300 MHz, CDCl₃) 3.17-3.20 (m, 8H), 3.54-3.55 (m, 16H), 3.62-3.71 (m, 16H), 3.84-3.91 (m, 16H), 6.21 (s, 8H), 6.26 (s, 4H);¹³C NMR (75 MHz, CDCl₃) 61.5, 67.2, 69.2, 72.6, 100.9, 110.0, 140.7, 144.8, 159.0; HRMS (FAB, 2-nitrobenzyl alcohol matrix) C₅₈H₈₅O₂₄([M + H]⁺Calculated value as 1165.5431, measured value; 1165.5413.
Reference Example 9
Formula (33) used as a raw material for the compound of Formula (34)

This compound was synthesized as follows.
A 200 ml three-necked flask equipped with a magnetic stirrer was prepared and purged with nitrogen. Dichloromethane (50 ml) and a compound of the formula (32) (1.25 g, 2.18 mmol) were added thereto under a nitrogen atmosphere, and 1M boron tribromide dichloromethane solution (34.9 ml, 34.34) was added at -50 ° C. 9 mmol) was added dropwise and stirred at room temperature for 18 hours. The reaction solution is then poured into ice water (200 ml) and extracted with ethyl acetate (300 ml). The organic layer is washed with a saturated aqueous ammonium chloride solution, dried over magnesium sulfate, the solvent is distilled off, and silica gel column chromatography (methylene chloride) is used. / Acetone = 1/1) to give a compound of the formula (33) (0.792 g, 1.72 mmol, yield 79%).
White solid; melting point <300 ° C;¹1 H NMR (300 MHz, acetone-d₆) Δ 6.07 (s 8H), 6.12 (s, 4H), 8.04 (s, 8H);¹³C NMR (75 MHz, acetone-d₆) Δ101.7, 110.0, 140.5, 146.6, 158.3. HRMS (FAB, 2-nitrobenzyl alcohol matrix) C₂₆H₂₁O₈(M + H]⁺461.1236, measured value; 461.1230.
Reference Example 10
Formula (32) used as a raw material for the compound of Formula (33)

This compound was synthesized as follows.
A 300 ml three-necked flask equipped with a magnetic stirring bar was prepared and purged with nitrogen. Under a nitrogen atmosphere, zinc powder (4.47 g, 68.3 mmol) and dehydrated THF (100 ml) were added, and titanium chloride (IV) (6.48 g, 34.2 mmol) was added dropwise at -10 ° C. Then, the mixture was stirred at 70 ° C. for 2 hours. Thereafter, the compound of the formula (31) (3.57 g, 11.8 mmol) dissolved in dehydrated THF (50 ml) was added at 0 ° C., and the mixture was stirred at 70 ° C. for 5 hours. Then return to room temperature, 10% K₂CO₃Aqueous solution (100 ml) was added and extracted with methylene chloride (200 ml). The organic layer was washed with saturated brine, dried over magnesium sulfate, evaporated, and silica gel column chromatography (methylene chloride / hexane = 2 / Isolation in 1) gave the compound of formula (32) (1.45 g, 2.53 mmol, 43% yield).
White solid; melting point 210.0-210.8 ° C .;¹H NMR (300 MHz, CDCl₃) Δ 3.65 (s, 24H), 6.21-6.25 (m, 12H);¹³C NMR (75 MHz, CDCl₃) Δ 55.3, 99.4, 109.0, 140.9, 145.0, 160.0; HRMS (FAB, 2-nitrobenzyl alcohol matrix) C₃₄H₃₆O₈([M]⁺); 572.2410, measured value; 572.2407; elemental analysis C₃₄H₃₆O₈Calculated as: C, 71.31; H, 6.34. Found: C, 71.20; H, 6.04.
Example 6
The sugar-binding protein was detected using the compound of the formula (17) modified with mannose synthesized in Example 1 as a detection agent. CaCl3 to a concentration of 1 mg / 1 ml in 10 mM Tris-HCl buffer (pH 7.6)₂In addition, MnCl to a concentration of 1 mg / 1 ml₂A stock solution is prepared in which a solution is added in advance (hereinafter, this solution is referred to as a salt-containing buffer), and this solution is added to the compound of formula (17) (1.05 mg) to a volume of 10 ml. did. To 1 ml of this stock solution, 0 mg, 0.1 mg, 0.3 mg, 0.5 mg, 1.0 mg, 2.1 mg, 4.2 mg, 8.3 mg of Concanavalin A (hereinafter abbreviated as “Con A”) is added and the salt is added. A test solution was prepared by adding the contained buffer to a total volume of 5 ml. After preparing these test solutions for 10 minutes, fluorescence spectra were measured respectively. The concentration of compound (17) in the test solution is 19 μM, and the concentration of ConA is 0 μM, 0.2 μM, 0.6 μM, 1.0 μM, 2.0 μM, 4.0 μM, 8.0 μM, 16.0 μM, respectively. It becomes. The measurement was carried out using a fluorescence spectrophotometer manufactured by Hitachi, Ltd., excited with light having a wavelength of 370 nm using a xenon lamp, and the fluorescence spectrum was measured at room temperature. As shown by the circled data in FIG. 1 and FIG. 2, when the fluorescent sugar derivative compound is the compound of the formula (17), it was found that the fluorescence near 500 nm became remarkably strong with the increase in ConA concentration. .
Example 7
The same operation as in Example 6 was performed except that the compound of formula (21) modified with galactose instead of the compound of formula (17) was used as a detection agent and the concentration in the test solution was changed to 22 μM instead of 19 μM, Experiments for detecting sugar-binding proteins were performed. As indicated by the crosses in FIG. 2, no fluorescence was observed in the vicinity of 500 nm regardless of the ConA concentration. Considering together with the result of Example 6, ConA is detected in the compound of formula (17) but not in the compound of formula (21), and it is found that the selectivity varies depending on the structure of the fluorescent sugar derivative compound. It was.
Example 8
Using the compound of the formula (17) as a detection agent, the same operation as in Example 6 was performed to prepare a detection agent solution. However, the concentration of the compound of formula (17) in the test solution after addition of ConA was adjusted to 17.8 μM instead of 19 μM. This solution was attached to a fluorometer, ConA was added so that the ConA concentration after addition was 8 μM, and fluorescence was measured immediately. As the data in FIG. 3 show, the intensity of the 500 nm fluorescence after the addition increased rapidly and reached a saturation value within seconds. Considering the time lag required for operation, the increase in intensity is also considered instantaneous, indicating that ConA can be detected very quickly.
Example 9
Sugar-binding protein was detected using the compound of formula (21) modified with galactose as a detection agent. Compound (21) was added to 10 mM phosphate buffer (pH 7.4) to prepare a detection agent solution. A peanut agglutinin (hereinafter abbreviated as PNA) was added thereto at room temperature to prepare a test solution. At that time, the concentration of the compound (21) in the test solution was adjusted to 20 μM. Furthermore, test solutions having different PNA concentrations were prepared by changing the amount of PNA to be added. These were also prepared so that the concentration of the compound of formula (21) in the test solution was 20 μM. About each test liquid, after adding PNA solution, after stirring for 10 minutes, the fluorescence spectrum was measured at room temperature similarly to Example 6. FIG. As shown by the circled data in FIG. 4 and FIG. 5, it was found that when the fluorescent sugar derivative compound is the compound of formula (21), the fluorescence near 500 nm becomes stronger with increasing PNA concentration.
Example 10
Using the compound of formula (17) as a detection agent instead of the compound of formula (21), a test solution was prepared in the same manner as in Example 6, and fluorescence was measured. As shown by the data with crosses in FIG. 5, the intensity of fluorescence near 500 nm hardly increased even when the concentration of PNA was increased. When taken together with the results of Example 9, it was found that PNA was detected in the compound (21) but not substantially detected in the compound of the formula (17).
Example 11
ConA detection experiment was conducted in the same manner as in Example 6 using the compound of formula (25) as a detection agent. However, unlike the case of Example 6, the concentration of the detection agent in the test solution was adjusted to 7.2 μM, and the excitation wavelength was 320 nm. As shown in FIG. 6 and FIG. 7, it was found that even when the detection agent was the compound of the formula (25), the fluorescence near 460 nm was remarkably increased with the increase in ConA concentration.
Example 12
ConA detection experiment was conducted in the same manner as in Example 6 using the compound of formula (29) as a detection agent instead of the compound of formula (25). However, unlike the case of Example 11, the concentration of the detection agent in the test solution was adjusted to 7.1 μM. As shown in FIGS. 8 and 9, it was found that also in the case where the detection agent was a compound (29) containing 2 units of an ethyleneoxy group as a linker, the fluorescence around 470 nm was remarkably increased as the ConA concentration was increased.
Example 13
ConA detection experiment was conducted in the same manner as in Example 12 using the compound of formula (36) as a detection agent instead of the compound of formula (29). However, unlike the case of Example 11, the concentration of the detection agent in the test solution was adjusted to 7.3 μM, and the excitation wavelength was 300 nm. As shown in FIGS. 10 and 11, even in the case of the compound of the formula (36) containing 8 sugar residues through a 2 unit ethyleneoxy group as a linker, the fluorescence around 450 nm is markedly increased with the increase in ConA concentration. I found it stronger. When considered together with the results of Example 12, it was found that the increase in fluorescence intensity with respect to the increase in ConA concentration was steeper and more sensitive than in the case of the compound of formula (29).
Example 14
The following reaction formula (37)

According to formula (40)

This compound was synthesized as follows.
Add a compound of formula (39) (0.033 g, 0.027 mmol), sodium methoxide (0.002 g, 0.044 mmol) and methanol (2 ml) to a 10 ml flask equipped with a magnetic stir bar and stir at room temperature for 40 minutes. did. Thereafter, a cation exchange resin (DOWEX 50WX8-100) was added until the reaction solution became acidic, filtered, and the solvent was distilled off to obtain a compound of the formula (40) (0.019 g, 0.022 mmol, yield 83). %).
This compound is a novel compound not yet published in the literature, and its properties, spectroscopic data, and analytical data were as follows. White solid; mp 111.2-115.9 ° C .;¹H NMR (300 MHz, CD₃OD) δ 3.60-3.81 (m, 24H), 3.96-4.06 (m, 4H), 4.77-4.83 (m, 2H), 6.64-6.71 (m , 4H), 6.86-7.08 (m, 14H);¹³C NMR (75 MHz, CD₃OD) δ 62.9, 67.8, 68.4, 68.5, 70.8, 71.5, 72.1, 72.5, 74.6, 101.8, 114.8, {127.3 , 127.4}, {128.6, 128.7}, 132.4, 133.6, {137.8, 137.9}, 141.2, {145.5, 145.6}, 158. 7; HRMS (FAB, glycerin matrix) C₄₆H₅₆O₁₆Na ([M + Na]⁺Equivalent value); 887.3466, measured value; 887.3470.
Reference Example 11
Formula (39) used as a raw material in Example 14 above

This compound was synthesized as follows.
A 50 ml two-necked flask equipped with a magnetic stirrer was prepared and purged with nitrogen. Under nitrogen atmosphere, DMF (10 ml), compound of formula (38) (0.19 g, 0.51 mmol), compound of formula (38) (0.56 g, 1.12 mmol), potassium carbonate (0.18 g, 1.31 mmol) and 18-crown-6 (0.007 g, 0.025 mmol) were added, and the mixture was stirred at 80 ° C. for 17 hours. Thereafter, methylene chloride (100 ml) was added, washed with water and saturated brine, the organic layer was dried over sodium sulfate, the solvent was distilled off, silica gel column chromatography (methylene chloride / acetone = 20/1) and fractionation. Isolated by preparative HPLC to give the compound of formula (39) (0.050 g, 0.042 mmol, 8% yield).
White solid; melting point 64.2-68.7 ° C .;¹H NMR (300 MHz, CDCl₃) 1.9.96-2.00 (m, 12H), 2.06-2.09 (m, 6H), 2.11-2.14 (m, 6H), 3.68-3.74 (m, 6H), 3.77-3.83 (m, 6H), 4.00-4.10 (m, 8H), 4.23-4.30 (m, 2H), 4.87-4.90 ( m, 2H), 5.24-5.38 (m, 6H), 6.61-6.67 (m, 4H), 6.87-6.93 (m, 4H), 6.97-7. 10 (m, 10H);¹³C NMR (75 MHz, CDCl₃) Δ20.6, 20.6, 20.7, 20.8, 62.3, 66.0, 67.1, 67.3, 68.3, 69.0, 69.5, 69.8, 70 , 97.7, {113.5, 113.6}, 126.1, {127.5, 127.6}, 131.3, 132.4, 136.5, 139.5, 144.1 , 157.0, 169.7, 169.8, 169.9, 170.5; HRMS (FAB, 2-nitrobenzyl alcohol matrix) C₆₂H₇₂O₂₄([M]⁺); Calculated value as 1200.4414, measured value; 1200.4443.
Example 15
The compound of formula (40) has low water solubility due to the small number of introduced sugars. Therefore, a stock solution was prepared by adding methanol (1 ml) to the compound of formula (40) (0.444 mg) and dissolving it, and adding the salt-containing buffer prepared in Example 6 to a total volume of 5 ml. Take 1 ml of the stock solution and add a salt-containing buffer to make a total of 5 ml [(concentration of compound of formula (40) = 20.6 μM]), 1 ml of stock solution containing ConA (3.94 mg) and salt The test solution [concentration of the compound of formula (40) = 20.6 μM, ConA concentration = 7.9 μM] prepared by adding a buffer solution to make a total of 5 ml and stirring for 10 minutes was measured for each fluorescence spectrum, as shown in FIG. As shown, even for a compound of formula (40) where the amount of sugar introduced is low, the test solution to which ConA is added is about 2.8 times more fluorescent than the test solution containing only the compound of formula (40). It showed an increase in strength.
Example 16
A test solution prepared by dissolving only the compound of formula (17) in the salt-containing buffer prepared in Example 6 was prepared so that the concentration of the compound of formula (17) was 19 μM. Separately, the compound of formula (17) is dissolved in the salt-containing buffer prepared in Example 6, and ConA is further added so that the concentration of the compound of formula (17) is 19 μM and the concentration of ConA is 8 μM. A test solution stirred for 10 minutes was prepared. In a dark room, each sample was placed on a handy UV lamp (manufactured by ASONE Co., Ltd., 9.0 W, 15 A, FL: 8 W × 1) and irradiated with ultraviolet light of 365 nm. A photograph taken in this state is shown in FIG. The left cell of FIG. 13 contains a test solution containing only the compound of formula (17), and the right cell contains a test solution containing both the compound of formula (17) and ConA. Although the fluorescence from the test solution in the left cell not containing ConA, which is a control experiment, is weak and not substantially observed, the test solution in the right cell to which ConA is added exhibits strong fluorescence that can be clearly confirmed with the naked eye. It was found that ConA can be detected very easily and with high sensitivity.
Example 17
The following synthetic route

According to formula (43)

This compound was synthesized as follows.
To a 10 mL flask equipped with a magnetic stir bar was added the compound of formula (42) (14 mg, 4.3 μmol), sodium methoxide (1.4 mg, 26 μmol), methanol (2 mL), tetrahydrofuran (1 mL), and 2 hours at room temperature. Stir. Thereafter, a cation exchange resin (DOWEX 50WX8-100) was added until the reaction solution became acidic, filtered, and the solvent was distilled off to obtain a compound of formula (43) (7.1 mg, 3.5 μmol, yield 81). %).
Compound of formula (43): pale yellow solid; melting point 170.1-173.5 ° C .;¹H NMR (300 MHz, D₂O) δ 3.26-3.36 (m, 8H), 3.49-3.86 (m, 76H), 4.36-4.39 (m, 4H), 6.43-6.51 (m) , 8H), 6.72-6.81 (m, 8H);¹³C NMR (75 MHz, D₂O) δ 61.5, 62.0, 67.9, 69.6, 70.0, 70.8, 72.0, 72.9, 73.6, 73.9, 75.4, 75.8, 76.4, 79.5, 103.3, 104.0, 114.9, 133.4, 138.0, 139.6, 157.6; MALDI-TOFMS (matrix: 2-nitrobenzyl alcohol matrix) C₉₀H₁₃₂O₅₂Na ([M + Na]⁺Calculated value as 207.77; measured value; 207.71.
Reference Example 12
Formula (42) used as a raw material in Example 17 above

This compound was synthesized as follows.
A 50 mL two-necked flask equipped with a magnetic stirring bar was prepared and purged with nitrogen. Under a nitrogen atmosphere, dry methylene chloride (15 mL), the compound of formula (27) synthesized in Reference Example 5 (55 mg, 74 μmol), the compound of formula (41) (0.46 g, 0.59 mmol), and molecular were added. Sieves (4A) was added, boron trifluoride diethyl ether complex (0.47 mL, 3.7 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 4 days. Thereafter, the reaction solution was poured into a saturated aqueous solution of sodium bicarbonate, the organic layer was washed with a saturated aqueous solution of sodium bicarbonate, water and saturated brine, the organic layer was dried over sodium sulfate, and the solvent was distilled off. Then, it was isolated by silica gel column chromatography (methylene chloride / acetone = 5/1) and preparative HPLC to obtain a compound of formula (42) (14 mg, 4.3 μmol, yield 6%).
Compound of formula (42): pale yellow solid; melting point 96.5-99.7 ° C .;¹H NMR (300 MHz, CDCl₃) Δ 1.96 (s, 12H), 2.00 (s, 12H), 2.04 (s, 24H), 2.11 (s, 12H), 2.14 (s, 12H), 3.58- 4.16 (m, 56H), 4.46-4.56 (m, 12H), 4.87-4.97 (m, 8H), 5.07-5.22 (m, 8H), 5. 34-5.36 (m, 4H), 6.62 (d, 8H, J = 8.2 Hz), 6.89 (d, 8H, J = 8.2 Hz);¹³C NMR (75 MHz, CDCl₃) Δ 20.5, 20.6, 20.7, 20.8, 20.9, 60.8, 62.0, 66.6, 67.1, 69.1, 69.2, 69.9, 70 3, 70.6, 71.0, 71.6, 72.6, 72.8, 76.2, 100.6, 101.1, 113.6, 132.5, 137.0, 138.4 , 156.9, 169.1, 169.7, 169.8, 170.0, 170.1, 170.34, 170.35; MALDI-TOFMS (matrix: 2-nitropentyl alcohol matrix) C₁₄₆H₁₈₉O₈₀([M + H]⁺Calculated value as 3); 32.22.07; measured value; 3222.43.
Example 18
Using the compound of formula (43), a detection experiment of RCA120 was performed as a lectin.
In a buffer (10 mM phosphate buffer, pH 7.4), 6.7 μM of the compound of formula (43) is added to 0.0 μM, 0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM and 5.0 μM. RCA120 was added, and the sample was prepared by stirring for 10 minutes. As shown in FIG. 14, in the fluorescence spectrum, the emission intensity in the vicinity of 400 nm of the compound of the formula (43) increased as the concentration of RCA120 increased. As shown in FIG. 15, when the concentration of RCA120 is 1.0 μM, 2.0 μM, and 3.0 μM, the emission intensity increases 1.5 times, 1.8, and 2.7 times, respectively. Was possible.

The present invention does not require pretreatment of a target, can detect a sugar-binding protein rapidly, with high sensitivity and high selectivity, and can be easily determined even with the naked eye, and target detection suitably used for the target detection Material can be provided.
All publications and patent documents cited herein are hereby incorporated by reference in their entirety. While specific embodiments of the invention have been described herein for purposes of illustration, it will be apparent to those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.

Claims (23)

1. General formula (1)
   ^{(S 1 -S 2 -S 3 -S} 4 -S 5 -S 6 -A m) n B (1)
   [Wherein S ¹ represents a residue obtained by removing a hydrogen atom from one hydroxyl group of a sugar structure, and S ² , S ³ , S ⁴ , S ⁵ , S ⁶ are independently absent, or Represents a residue in which one hydroxyl group is removed from the sugar structure and a hydrogen atom is removed from the other hydroxyl group, and S ¹ , S ² , S ³ , S ⁴ , S ⁵ , and S ⁶ are linked to each other by a glycosidic bond. A represents a divalent organic group having 2 to 6 carbon atoms that may not exist or may contain an oxygen atom, and B emits fluorescence when irradiated with light. Represents a residue obtained by removing n hydrogen atoms from a fluorescent compound, which residue may be substituted with a substituent having 7 or less carbon atoms, and when A is present, m is a positive integer of 6 or less N represents a positive integer of 12 or less. S ² , S ³ , S ⁴ , S ⁵ and S ⁶ may be the same as or different from each other. By removing a hydrogen atom from one hydroxyl group, the structure of the parent sugar that becomes S ¹ (hereinafter referred to as the structure of the parent sugar of S ¹ or simply the structure of the parent sugar) is one hydroxyl group. And by removing a hydrogen atom from the other hydroxyl group, the structure of the parent sugars to be S ² , S ³ , S ⁴ , S ⁵ , and S ⁶ (hereinafter referred to as S ² to S ⁶ parent sugars). The structure, or simply the structure of the parent sugar). ] The fluorescent sugar derivative compound characterized by the above-mentioned.
2. Structure of the mother of sugar S ¹ is selected from the group consisting of glucose, mannose, galactose, fructose, allose, talose, psicose, gulose, idose, sorbose, ribose, lyxose, xylose, apiose, deoxy-glucose, deoxy-mannose, deoxy-galactose, deoxy Arrow vinegar, The fluorescent sugar derivative compound according to claim 1, which is deoxytalose or deoxyribose.
3. The fluorescent sugar derivative compound according to claim 1, wherein S ² , S ³ , S ⁴ , S ⁵ or S ⁶ is absent.
4. The structure of the S ² , S ³ , S ⁴ , S ⁵ , S ⁶ parent sugar is independently selected from glucose, mannose, galactose, fructose, allose, talose, psicose, gulose, idose, sorbose, ribose, lyxose, The fluorescent sugar derivative compound according to claim 1, which is xylose, apiose, deoxyglucose, deoxymannose, deoxygalactose, deoxyallose, deoxytalose, deoxyribose.
5. The fluorescent sugar derivative compound according to claim 1, wherein A is absent.
6. The fluorescent sugar derivative compound according to claim 1, wherein A is CH ₂ CH ₂ O.
7. The fluorescent sugar derivative compound according to claim 1, wherein the parent fluorescent compound that becomes B by removing n hydrogen atoms is a fluorescent compound having a conjugated structure.
8. The parent fluorescent compound that becomes B by removing n hydrogen atoms is represented by the general formula (2).
   

   

   Wherein R ¹ represents a saturated hydrocarbon group having 6 or less carbon atoms or an unsaturated hydrocarbon group having 12 or less carbon atoms, and may be substituted with a substituent having 7 or less carbon atoms. Item 6. A fluorescent sugar derivative compound according to Item 1.
9. The parent fluorescent compound that becomes B by removing n hydrogen atoms is represented by the general formula (3).
   

   

   (In the formula, R ² and R ³ represent a saturated hydrocarbon group having 6 or less carbon atoms or an unsaturated hydrocarbon group having 12 or less carbon atoms, and may be the same or different from each other. The fluorescent sugar derivative compound according to claim 1, which may be substituted with the following substituents:
10. General formula (1)
    ^{(S 1 -S 2 -S 3 -S} 4 -S 5 -S 6 -A m) n B (1)
    (Wherein, S ¹ , S ² , S ³ , S ⁴ , S ⁵ , S ⁶ , A, B, m, and n have the same meanings as described above) are used as detection materials. A detection target detection method.
11. The detection method according to claim 10, wherein the detection target is a substance containing a sugar-binding protein.
12. The detection method according to claim 10 or 11, wherein a change in fluorescence is used as the detection means.
13. A detection material that can interact with the detection target, and when it does not interact with the detection target, it does not substantially emit fluorescence even when it is electronically excited by light irradiation, or interacts with the detection target. Even if it is not, it is excited by light itself to emit fluorescence and interacts with the detection target. When it interacts with the detection target, it is excited by light irradiation and emits fluorescence. A method for detecting a detection target, comprising using a detection material that is greater than the intensity of fluorescence when not interacting with the detection target.
14. General formula (1) as detection material
    ^{(S 1 -S 2 -S 3 -S} 4 -S 5 -S 6 -A m) n B (1)
    (Wherein S ¹ , S ² , S ³ , S ⁴ , S ⁵ , S ⁶ , A, B, m, and n have the same meanings as described above). .
15. The detection method according to claim 13 or 14, wherein the detection target is a substance containing a sugar-binding protein.
16. A method for detecting a substance containing a sugar-binding protein utilizing the interaction between a substance containing a sugar-binding protein and a fluorescent detection material, wherein the fluorescent detection material substituted with a sugar chain is used as a fluorescent detection material A method for detecting a substance containing a sugar-binding protein characterized by the following.
17. General formula (1) as detection material
    ^{(S 1 -S 2 -S 3 -S} 4 -S 5 -S 6 -A m) n B (1)
    ^{(Wherein, S 1, S 2, S} 3, S 4, S 5, S 6, A, B, m and n are. As defined above) using the compound represented by the detection of claim 16 Method.
18. The detection method according to claims 11 to 17, which is carried out in an aqueous solution in the presence of a drug that activates a sugar-binding protein.
19. An aggregate composed of a sugar-binding protein and the compound according to any one of claims 1 to 9.
20. The aggregate according to claim 19, which is prepared by reacting a sugar-binding protein and the compound according to any one of claims 1 to 9 in an aqueous solution.
21. 21. The aggregate according to claim 20, which is prepared by reacting in an aqueous solution in the presence of an agent that activates a sugar-binding protein.
22. A method for detecting a sugar-binding protein, comprising observing fluorescence from the aggregate according to claim 19.
23. The detection method according to claim 22, which utilizes the fact that the fluorescence from the aggregate is stronger than the fluorescence from the compound itself according to any one of claims 1 to 9.

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