JP2883824B2 - Fluorescent compound having a boronic acid group - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel fluorescent compound, and more particularly to a fluorescent compound which can be used for detecting glucose and other saccharides, or a saccharide using such a fluorescent compound. It relates to the detection method.

[0002]

2. Description of the Related Art Sugar is one of the essential organic compounds in living organisms, and plays an important role in information transmission, energy metabolism, structure formation, and the like. For example,
Glucose (D-glucose, glucose) is important as an energy source for various tissue cells constituting the body.
This glucose is converted to glycogen, stored in the liver, released into body fluids as needed, and consumed for energy activities.In the normal human body, the balance between glucose production and consumption is maintained, Is in a certain range. Therefore, diseases such as diabetes and adrenal disease can be diagnosed by measuring glucose in blood and urine.

[0003] A glucose sensor using an enzyme is well known as a means of detecting saccharides currently in practical use. In this method, glucose is decomposed using an enzyme (glucose oxidase), and the concentration of glucose is determined by measuring hydrogen peroxide generated at that time by an appropriate means (for example, using an electrode). Although this method is an established technique, since the enzyme used is derived from a living organism, it is irreversibly deteriorated with time and cannot be reused. Since glucose is also decomposed, the sample cannot be used for another measurement. Such a sugar sensor measures a sample taken out of a living body. If saccharides could be detected in vivo and their behavior could be known in situ, it would provide very useful information for disease treatment and drug development. However, current sensors do not meet such expectations.

[0004]

Means for Solving the Problems The present invention relates to a novel fluorescent compound, and the fluorescent compound of the present invention has many uses, but is particularly suitable for detecting saccharides.

That is, the compound of the present invention has a fluorescent group or a functional group, at least one phenylboronic acid moiety, and at least one amine nitrogen. It is characterized by having a molecular structure such that it is located near a site and bonds intramolecularly to the phenylboronic acid, and can emit fluorescence when bound to a saccharide.

A representative compound belonging to the fluorescent compound according to the technical idea of the present invention is represented by the following general formula [Chemical formula 5]
Can be represented by

[0007]

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In the above formula [5], F represents a fluorescent atomic group or a functional group (fluorophore). As F (fluorescent atomic group or functional group), many atomic groups or functional groups including a π-electron system can be applied. Preferred fluorescent groups include a naphthyl group, anthryl group, a pyrenyl group, and a phenanthryl group, which are represented by the following structural formulas, respectively.
[Formula 7], [Formula 8] and [Formula 9].

[0009]

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[0010]

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[0011]

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[0012]

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The fluorescent group or functional group as described above may have a suitable substituent as long as it does not affect the fluorescent property. Included in the invention. For example, a sulfonic acid group (SO ₃ H)
Is substituted, water solubility is imparted to the compound,
This is preferable when the compound is dissolved in an aqueous sample solution to detect saccharides contained therein. A preferred example of the fluorescent group is an anthryl group that gives good fluorescence.

In the above formula [5], a nitrogen atom (N)
R is an aliphatic or aromatic functional group, and is generally an alkyl group having 1 to 4 carbon atoms (methyl group, ethyl group, propyl group, butyl group) or phenyl group.

Further, m in the formula [Formula 5] is 0, 1 or 2. That is, in the compound of the present invention, the nitrogen atom is located close to the boronic acid site, and specifically, is located at the ortho position of the phenylboronic acid group via a methyl group or an ethyl group or directly. Are combined. Preferably, m is 1, ie the nitrogen atom is connected to the phenyl group via a methylene group.

Further, in the formula [5], n is also 0,1.
Or 2, where n + m is 2 or 3.
That is, the nitrogen atom and the boronic acid are located not far from the fluorophore or functional group. Preferably n is 1.

The phenyl group constituting the phenylboronic acid group may be substituted with a suitable substituent as long as it does not adversely affect the fluorescence characteristics. Examples of such a substituent include a methyl group, an ethyl group, a propyl group, a butyl group, a phenyl group, a methoxy group, an ethoxy group, a butoxy group, and a phenoxy group.

The compound of the present invention represented by the above formula [Formula 5] does not emit fluorescence while having a fluorescent group. This is because, in this compound, the fluorescence derived from the fluorophore is quenched by the effect of the lone pair of the nitrogen atom-the electron of the nitrogen atom moves to the ground state level of the fluorophore. It is understood that the original fluorescence does not occur. In contrast, when sugar molecules are present, the compounds of the invention fluoresce very strongly. This phenomenon is understood as follows: when a sugar molecule is present and the compound of the present invention forms a stable complex with the sugar molecule via its boronic acid moiety, a nitrogen atom (N) and a boron atom ( B)
(The boron atom has an electron deficiency and combines with the electron-rich nitrogen atom) and is no longer quenched. That is, the lone pair of electrons of the nitrogen atom is used for bonding with boron, so that the electron transfer does not contribute to the quenching of the fluorescence, and the original fluorescence is restored.

Specific examples of preferred compounds according to the formula [Chemical Formula 5] of the present invention are those wherein F (fluorescent atom) is an anthryl group, R
Is a methyl group and each of n and m is 1.

[0020]

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The characteristics of this compound include D-glucose and D-glucose.
The presence of a monosaccharide such as fructose results in a very high fluorescence intensity. Thus, this compound can be used to detect monosaccharides, in which case it can be used not only to generally detect the presence of monosaccharides, but also to detect specific monosaccharides from a sample containing multiple monosaccharides. It can also be used to detect saccharides. In the latter case, usually, the sample is subjected to a pretreatment (for example, chromatography) for separating each monosaccharide, followed by detection using the fluorescent compound of the present invention.

Thus, in order to detect a saccharide using the compound of the present invention, the compound of the present invention is added to a saccharide-containing sample, and the generation of fluorescence or an increase in fluorescence intensity due to the binding of the compound to the saccharide is performed. It may be measured spectroscopically. As another embodiment, it is possible to detect sugar by a chromatographic method using the fluorescent compound of the present invention. In this case, the compound of the present invention is supported on a suitable carrier,
When a sample containing a saccharide passes through the sample, generation of fluorescence or an increase in fluorescence intensity derived from a complex formed between the compound and the saccharide may be detected by an appropriate detection means.

Further, according to the present invention, it is also possible to obtain a compound which selectively binds to a specific saccharide and emits extremely strong fluorescence. Thus, according to the present invention, there is provided a fluorescent compound represented by the following general formula [Formula 11].

[0024]

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In the formula, n and m are both integers, and n + m is 2 or 3. n and m are each an integer from 0 to 2 (that is, 0, 1 or 2), and one of n and m may be zero. n + m is preferably 2, and particularly preferably, both n and m are 1. The compound of the present invention has a methylene group (CH ₂ ) having such a length to provide a molecular structure suitable for binding to glucose via two boronic acid groups.

In the formula, F represents a fluorescent atomic group or a functional group (fluorophore). As F, many atomic groups or functional groups including a π-electron system can be applied. Preferred examples of the fluorescent atomic group include a naphthyl group, anthryl group, a pyrenyl group and a phenanthryl group, which are respectively represented by the aforementioned structural formulas [Chemical Formula 6], [Chemical Formula 7], [Chemical Formula 8], and [Chemical Formula 8]. 9].
A particularly preferred example of the fluorescent group is an anthryl group.

In the compound of the present invention represented by the formula [11], a phenyl ring (benzene ring) to which a fluorescent atom group (functional group) and / or boronic acid B (OH) OH is bonded. ) May have a suitable substituent as long as it does not affect the fluorescence, and such a substituted product (derivative) is also included in the compound of the present invention. In particular, when the compound of the present invention is dissolved in an aqueous sample solution to detect a saccharide contained therein, a group that improves the solubility of the compound, for example, a sulfonic acid group (SO ₃ H)
And the like are preferred.

In the formula, R bonded to the nitrogen atom (N) is an aliphatic or aromatic functional group, and is generally an alkyl group having 1 to 4 carbon atoms (methyl group). , An ethyl group, a propyl group, a butyl group) or a phenyl group. Particularly preferred examples of R are a methyl group and an ethyl group.

Thus, as a preferred example of the compound according to the present invention represented by the following formula [11], the following formula [12],
Compounds represented by [Chemical formula 13], [Chemical formula 14], and [Chemical formula 15] are exemplified, and among them, the compound of [Chemical formula 12] is particularly preferable.

[0030]

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[0031]

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[0032]

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[0033]

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Surprisingly, the compound of the present invention as represented by the formula [Formula 11] is present in an aqueous solution having a pH value near neutrality and has a concentration corresponding to the measured concentration in human biological fluid. Concentration (50-250 mg / dl, approximately 0.0005
M-0.001M) in the presence of glucose (D-glucose).
The presence of other sugars (eg, galactose or fructose) does not change.

The compound of the present invention as represented by the formula [11] emits a particularly strong fluorescence in the presence of glucose, because two boronic acid moieties are present in the structure represented by the formula [11]. And the arrangement is 1,2,2 of glucose
It is most suitable for covalently bonding with four hydroxyl groups (OH groups) at the 4,6 positions, so that a stable 1: 1 complex is formed between the compound and glucose to form a nitrogen atom It is understood that the quenching function is surely eliminated.
On the other hand, other sugars (eg, fructose) are considered to have weak fluorescence because they are bonded only to one boronic acid site. In fact, at concentrations where fructose and galactose are present in biological fluids, little fluorescence is observed.

As described above, the compound of the present invention has a structure in which a fluorescent atom group, two phenylboronic acids and a nitrogen atom are specifically arranged as shown in the above-mentioned formula [Chemical Formula 11] to glucose. It emits strong fluorescence specifically and is used for detecting glucose. In the case of detecting glucose in a solution, if the pH is changed using an appropriate acid after the measurement, the bond between glucose and the fluorescent compound is easily released, and glucose returns to the original state. .

The compound of the present invention is generally prepared by combining a phenylboronic acid having an alkyl halogen at the ortho-position with a reaction reagent having an alkylaminomethyl group bound to a fluorescent atom (F) using a base. It is obtained by mixing and reacting in an appropriate solvent.

As is apparent from the above description, according to the present invention, since sugar is detected using a stable synthetic compound, the problem caused by enzyme instability unlike the conventional enzyme method is solved. You. In addition, in the detection of sugar using the synthetic compound of the present invention, the sample is left as it is (inta
ct) Since the measurement is performed and the sample is not decomposed unlike the enzymatic method, the sample can be used for another measurement.
Furthermore, the detection of sugar according to the present invention is advantageous in that it can be performed in a wide pH range as well as near neutral.

As a future prospect, the present invention measures sugars without decomposing them, so that sugars at specific sites in the body (eg, specific cell surfaces) can be measured without destroying the tissues. It is also possible to expand to various technologies. At this time, if it is combined with a means such as an optical fiber (for example, the tip of the fiber is coated with the compound of the present invention), information on sugars in the body is continuously monitored to be clinically useful. You could also get data. In addition, it can be said that the method for detecting a saccharide of the present invention using a synthetic compound has opened the way to design change so as to be applicable to various saccharides by appropriately changing the compound.

Hereinafter, the present invention will be described with reference to Examples to further clarify the features of the present invention.

In the structural formulas shown in connection with the description in this specification, carbon atoms and hydrogen atoms of a methyl group (CH ₃ ) and a methylene group (CH ₂ ) are commonly used in the art. May be omitted.

[0042]

【Example】

 [Example 1: Preparation of fluorescent compound]

The compound of the above formula [Formula 10] was prepared as a fluorescent compound according to the present invention.

The outline of the synthesis route is as shown in FIG. First, phenylboronic acid (A in the figure) was prepared as follows.

Orthobromotoluene was reacted with magnesium (1.1 equivalents) at 25 ° C. in diethylene. The Grignard reagent was added dropwise at −78 ° C. to a solution of trimethylboronic acid (10 equivalents) in diethyl ether. The resulting mixture was stirred for 2 hours, then warmed to room temperature and stirred for another 2 hours. The diethyl ether was removed under reduced pressure and recrystallized in water to give a solid product.
The product (boronic acid) was dried in vacuo overnight to give boronic anhydride (A).

N-bromosuccinimide (1.1 equivalent) and azobisisobutyronitrile (catalyst) were mixed with the boronic anhydride in a carbon tetrachloride solvent. The obtained mixture was refluxed while being irradiated with a 200 W lamp for 2 hours. The obtained solution was filtered to remove the solvent, thereby obtaining bromomethylboronic anhydride (B).

The bromomethylboronic anhydride was reacted with 9-methylaminomethylanthracene (2.1 equivalents) in chloroform. The mixture was filtered and the solvent was removed.
The solid product was washed with diethyl ether and recrystallized in ethyl acetate to give the desired compound [Chemical Formula 10].

Product analysis: Proton NMR (CDCl
1.300 MHz): Chemical shift (ppm)
2 (3H, S), 3.9 (2H, S), 4.5 (2H,
S), 7.4 (4H, m), 8.0 (4H, m), 8.
4 (1H, S). Mass spectrometry (SIMS negative):
M + (glycerol-2H2O) proton 410.

[Example 2: Fluorescence test]

The compound [Chemical Formula 10] obtained in Example 1 was subjected to a fluorescence experiment to examine its applicability to saccharide detection.

1.2 / 10 ⁵ M (0.00001) of the compound containing sodium chloride (0.05 M)
2M) An aqueous solution was prepared. Sugar (D-glucose or D-fructose) was added thereto to a concentration of 0.05 M, and the total volume was adjusted to 100 ml. The measurement was performed while gradually decreasing the pH from around 12 by adding a small amount of HCl. That is, when the pH was stabilized, a part of the solution was taken out as a sample, and the fluorescence spectrum was measured. After the measurement, the sample was returned to the original solution, the pH was further adjusted by adding HCl, and the same measurement was repeated. The measurement of the fluorescence spectrum was performed using a fluorescence spectrophotometer F-4500 manufactured by Hitachi. Excitation for fluorescence was performed by UV (ultraviolet light). The result is shown in FIG.

As shown in this figure, when no sugar is present (blank), the fluorescence intensity of the compound of the formula [Chem. 10] is low from around pH 3 to high pH, and the fluorescence derived from anthracene is quenched. Understood. However, the presence of sugars significantly increases the fluorescence intensity, and its effects are particularly wide-ranging at pH 4-10. Thus, it is understood that the compound [Chemical Formula 10] can be used for detecting sugar by fluorescence.

Example 3 Preparation of Fluorescent Compound

The compound of the above formula [Formula 12] was prepared as a fluorescent compound according to the present invention.

The outline of the synthesis route is as shown in FIG.

First, using AIBN (azobisisobutyronitrile) as an initiator, orthomethylboronic acid was brominated with NBS (N-bromosuccinimide) dissolved in carbon tetrachloride for 3 hours under reflux. Orthobromomethylboronic acid was obtained in a yield of 60% (reaction a in the figure).

The obtained ortho-bromomethylboronic acid was reacted with 2,2-dimethyl-1.3-propanediol (in toluene) all day and night while azeotropically removing water (reaction (b) in the figure).

By reacting the thus obtained orthobromomethylboronic acid with a protecting group with anthryldiamine and potassium carbonate (in THF) overnight under reflux conditions, the boronic acid moiety is protected as an ester. The desired diboronic acid compound obtained was obtained (yield 5%, reaction (c) in the figure). PH using 33.3% methanol / water solution
7.77, the protecting group was removed under room temperature conditions (reaction 2 in the figure).

Identification of the desired diboronic acid compound (having a protecting group) can be performed by mass spectrometry (MS) and nuclear magnetic resonance (N
MR). MS: SIMS (NPOE)
m + 668. Proton NMR (CDCl3) Chemical shift (ppm): 0.9 (s, CH3 (protecting group), 12
H), 2.2 (s, CH3N, 6H), 3.6 (s, C
H2 (protecting group), 8H), 3.9 (s, CH2 (benzyl), 4H), 4.4 (s, CH2 (anthryl), 4
H), 7.2-8.4 (m, aromatic ring, 16H).

Example 4 Fluorescence Test

A fluorescence experiment was performed on the diboronic acid compound [Chemical Formula 12] obtained in Example 3 to examine its adaptability to glucose detection.

The diboronic acid compound is buffered (pH 7.7).
7, 0.01M KCl, 0.00262M KH ₂ P
O3,0.002642M Na ₂ HPO3) was dissolved in aqueous methanol (33% methanol in water). Glucose and other sugars were added to 100 ml of this solution, and the fluorescence spectrum (423 nm) was measured. The measurement of the fluorescence spectrum was carried out using a fluorescence spectrophotometer F-4500 manufactured by Hitachi (Hewlett-Packard VETRA 286/12 computer). The wavelength of the excitation spectrum for fluorescence emission was 370 nm.

The results are shown in the graph of FIG. The horizontal axis is
Log (molar concentration of sugar (M)), and the vertical axis indicates relative fluorescence intensity. As shown in the figure, glucose emits strong fluorescence from a relatively low concentration, and particularly at a concentration of 0.0005 M to 0.001 M corresponding to a measured concentration in a human biological fluid, the fluorescence is linearly increased as the concentration increases. It has increased. This tendency does not change not only when glucose is present alone, but also when other sugars such as galactose and fructose coexist.

On the other hand, the ethylene glycol used as a control showed little fluorescence, and sugars such as galactose and fructose showed almost no fluorescence at the maximum concentration of about 0.0001M found in human biological fluids. Does not emit
Fluorescence is observed only when the concentration is increased by one or two orders of magnitude.

From these results, it is understood that the compound [Chemical Formula 12] according to the present invention emits particularly strong fluorescence in the presence of glucose and is useful for detecting glucose.

[Brief description of the drawings]

FIG. 1 outlines a synthetic route of a specific example of a fluorescent compound according to the present invention.

FIG. 2 shows the fluorescence intensity of a fluorescent compound according to the present invention in the presence of a monosaccharide.

FIG. 3 shows a synthetic route of another embodiment of the fluorescent compound according to the present invention.

FIG. 4 shows changes in the fluorescence intensity of the fluorescent compound according to the present invention in the presence of glucose and other sugars.

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁶ , DB name) C07F 5/02 CA (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] 1. A fluorescent compound represented by the following general formula [1]. Embedded image In the formula, F is a fluorescent group or a functional group selected from a naphthyl group, anthryl group, pyrenyl group or phenanthryl group, R is an alkyl group having 1 to 4 carbon atoms or a phenyl group, and n and m are Each is 0, 1 or 2, and n + m is 2 or 3. 2. The fluorescent compound according to claim 1, represented by the following formula: Embedded image 3. A fluorescent compound represented by the following general formula [3]. Embedded image In the formula, F is a fluorescent group or a functional group selected from a naphthyl group, anthryl group, pyrenyl group or phenanthryl group, R is an alkyl group having 1 to 4 carbon atoms or a phenyl group, and n and m are Each is 0, 1 or 2, and n + m is 2 or 3. 4. The fluorescent compound according to claim 3, represented by the following formula: Embedded image