KR20210094892A - A novel organic fluorescence compound and a device comprising the same - Google Patents

Abstract

The present invention relates to a novel organic fluorescent compound exhibiting excellent redox potential and a device including the same. The present invention can accurately check blood glucose concentration information in real time.

Description

A NOVEL ORGANIC FLUORESCENCE COMPOUND AND A DEVICE COMPRISING THE SAME

The present invention relates to a novel organic fluorescent compound and a biosensor comprising the same.

In recent years, the incidence of diabetes or other metabolic disorder-related diseases is increasing due to causes such as excessive nutrient intake or lack of exercise.

For diagnosis, treatment, and follow-up management of such diabetes or metabolic disorder-related diseases, it is necessary to monitor the blood glucose concentration. In this regard, in order to accurately monitor the blood glucose concentration in real time, many studies have been conducted to minimize human invasion.

For example, the irreversibility of a complexation reaction between a carbohydrate containing glucose and phenyl boronic acid and a method for separating saccharide compounds using chromatography have been known for a long time.

And U.S. Patent No. 5,503,770 and the like disclose a fluorescent boronic acid-containing compound that emits high intensity fluorescence when bound to a saccharide containing glucose. These fluorescent compounds are sensed by controlling the receptor for interaction with the above-described saccharide, a fluorescence generating group (fluorophore, or reporter, Fluorophore, or Reporter) that causes fluorescence from this binding, and the properties of the entire compound. It is common to have a molecular structure that includes an auxiliary group (linker, or spacer, Linker, or spacer) that can increase efficiency, and includes a nitrogen atom at a position adjacent to the phenyl boronic acid.

The nitrogen atom is located adjacent to the phenyl boronic acid moiety, and when the saccharide forms a bond with the phenyl boronic acid, fluorescence is emitted through intramolecular interaction with the boronic acid moiety and the fluorescence generating group.

In this regard, anthryl-boronic acid-containing compounds, naphthalimide-based compounds, and the like, which are used for detecting blood glucose, are known.

However, compounds currently used are relatively expensive and cumbersome to use, but there is a problem in that it is difficult to accurately check real-time blood glucose concentration information.

An object of the present specification is to provide an organic fluorescent compound having a novel structure and a biosensor including the same.

According to the present invention, there is provided an organic fluorescent compound represented by the following formula (1).

[Formula 1]

In Formula 1,

R1 to R4 are each independently hydrogen, an alkyl group having 1 to 5 carbon atoms, an aryl group having 6 to 20 carbon atoms, an oxyalkyl group having 2 to 20 carbon atoms, or an oxyaryl group having 6 to 20 carbon atoms, The alkyl group, aryl group, oxyalkyl group, or oxyaryl group may each independently be unsubstituted or substituted with a halogen atom, a hydroxy group, an amine group or an amino group,

provided that at least one of R1 to R4 is a group of Formula 1-1,

[Formula 1-1]

In Formula 1-1,

A1, A2 and A3 are each independently a single bond, alkylene having 1 to 5 carbon atoms, phenylene, or phenylalkylene having 7 to 10 carbon atoms;

D is an amine (-NR22-) or an amide (-CO-NR23-), R21 to R23 are each independently hydrogen or an alkyl group having 1 to 5 carbon atoms;

R11 to R15 are each independently hydrogen, an alkyl group having 1 to 5 carbon atoms, a halogen atom, or -B(OH) ₂ , provided that at least one of R11 to R15 is -B(OH) ₂ .

In this case, it may be preferable that any two or more of R1 to R4 is a group represented by Formula 1-1.

And, among R1 to R4, those other than Formula 1-1 are each independently an alkyl group having 1 to 5 carbon atoms substituted with a halogen atom, an aryl group having 6 to 20 carbon atoms substituted with a halogen atom, and 2 to 20 carbon atoms. of an oxyalkyl group, or an oxyaryl group having 6 to 20 carbon atoms.

In addition, A1, A2, and A3 may each independently represent a single bond or alkylene having 1 to 5 carbon atoms.

In addition, it may be preferable that D is an amide (-CO-NR23-), and R23 is, each independently, hydrogen or an alkyl group having 1 to 5 carbon atoms.

According to another aspect of the present invention, a device including the above-described organic fluorescent compound may be provided.

And, the device may be a biosensor, specifically, a biosensor for sugar detection.

In the present invention, terms such as first, second, etc. are used to describe various components, and the terms are used only for the purpose of distinguishing one component from other components.

In addition, the terminology used herein is only used to describe exemplary embodiments, and is not intended to limit the present invention.

The singular expression includes the plural expression unless the context clearly dictates otherwise.

In this specification, terms such as "comprises", "comprises" or "have" are intended to describe embodied features, numbers, steps, components, or combinations thereof, and include one or more other features, numbers, or steps. , components, combinations or additions thereof are not excluded.

Also in this specification, when it is said that each layer or element is formed "on" or "on" each layer or element, it means that each layer or element is formed directly on each layer or element, or other It means that a layer or element may additionally be formed between each layer, on the object, on the substrate.

Since the present invention may have various changes and may have various forms, specific embodiments will be illustrated and described in detail below. However, this is not intended to limit the present invention to the specific disclosed form, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

Hereinafter, the present invention will be described in detail.

According to the present invention, there is provided an organic fluorescent compound represented by the following formula (1).

[Formula 1]

In Formula 1,

R1 to R4 are each independently hydrogen, an alkyl group having 1 to 5 carbon atoms, an aryl group having 6 to 20 carbon atoms, an oxyalkyl group having 2 to 20 carbon atoms, or an oxyaryl group having 6 to 20 carbon atoms, The alkyl group, aryl group, oxyalkyl group, or oxyaryl group may each independently be unsubstituted or substituted with a halogen atom, a hydroxy group, an amine group or an amino group,

provided that at least one of R1 to R4 is a group of Formula 1-1,

[Formula 1-1]

In Formula 1-1,

A1, A2 and A3 are each independently a single bond, alkylene having 1 to 5 carbon atoms, phenylene, or phenylalkylene having 7 to 10 carbon atoms;

D is an amine (-NR22-) or an amide (-CO-NR23-), R21 to R23 are each independently hydrogen or an alkyl group having 1 to 5 carbon atoms;

R11 to R15 are each independently hydrogen, an alkyl group having 1 to 5 carbon atoms, a halogen atom, or -B(OH) ₂ , provided that at least one of R11 to R15 is -B(OH) ₂ .

The inventors of the present invention, when employing diketopyrrolopyrrole rather than an anthryl structure or a naphthalimide structure that can be seen as a reporter part in a compound previously used for sugar detection, such a compound binds to a saccharide compound By emitting strong fluorescence, it can be used as a biosensor for saccharide detection, and it was discovered that using such a biosensor, blood glucose concentration information can be accurately checked in real time while minimizing invasive behavior to the subject. invention was completed.

An organic fluorescent compound used for glucose detection exhibits detectable properties that change in a concentration-dependent manner when exposed to a sample containing glucose. Such properties have already been known through numerous literatures.

These organic fluorescent compounds include a receptor for interaction with saccharides, a fluorescence-generating group that causes fluorescence from the interaction of saccharides and phenyl boronic acid groups (fluorophores, or reporters, Fluorophore, or Reporter), and compounds An auxiliary group may be included to increase the sensing efficiency by adjusting the overall properties.

The detectable characteristic described above is, for example, a characteristic in which fluorescence or quenching occurs depending on the presence or absence of glucose in the sample and thus the intensity of light emission varies, but the wavelength of the emission spectrum due to fluorescence according to the presence or absence of glucose in the sample Spectral shift (Emission Spectrum Shift), which varies in range, and the like.

First, when a mechanism by fluorescence-quenching is used, the fluorescence-generating group may include a fluorescence-generating group and a quencher having a structure in which the fluorescence-generating group is quenched by a quencher in the absence of glucose.

The quencher is a portion that serves to quench fluorescence by interaction with a fluorescence generating group. When the quencher fulfills its role (quenching), quenching occurs, fluorescence is not emitted to the outside, and quenching If I do not do my job (quenching), fluorescence is emitted to the outside and has detectable properties.

In the case of fluorescence-quenching, it can work by two kinds of quenching mechanism:

First, in the organic fluorescent compound according to an embodiment of the present invention, the quenching mechanism by the amine group adjacent to the fluorescence generating group. In this case, photoinduced electron transfer (PET) occurs between the nitrogen atom of the amine group and the excited electrons of the fluorescence generating group and is quenched, so that fluorescence is not emitted to the outside.

Specifically, in the absence of glucose in the test sample, i) the boronic acid (B-OH) form is maintained, ii) the fluorescence generating group and the quencher move sufficiently close to cause quenching, and iii) thus, fluorescence is emitted to the outside. doesn't happen

Conversely, when glucose is present in the sample to be tested, i) boronic acid and glucose form a boron-glucose ester, ii) an intramolecular structural change occurs accordingly, so that the quencher is separated from the fluorescence group by a sufficient distance, iii ), so fluorescence is emitted as is from the fluorescence generating group.

The quenching mechanism by such light-induced electron transfer is described in Acc. Chem. Res. 1994, 27, 10, 302-308, (Chemical Communication in Water Using Fluorescent Chemosensors; Anthony W. Czarnik) and the like.

Another is, for the boronic acid group in the molecule, quenching by vibration of the B-OH bond (vibrational quenching). In this case, since the presence or absence of the B-OH bond directly affects the quenching, the presence or absence of glucose can be confirmed more immediately and with higher sensitivity than the PET mechanism. For the mechanism of vibrational quenching of B-OH bonds, see J. Am. Chem. Soc. 2018, 140, 6, 2348-2354 (Arresting “Loose Bolt” Internal Conversion from B(OH)2 Groups is the Mechanism for Emission Turn-On in ortho-Aminomethylphenylboronic Acid-Based Saccharide Sensors; Xiaolong Sun) or Nature Chemistry volume 11, pages 768-778 (2019) (The mechanisms of boronate ester formation and fluorescent turn-on in ortho-aminomethylphenylboronic acids; Xiaolong Sun) et al.

In this case, there is an advantage that the molecular structure can be designed more freely because it is not limited by the distance between the fluorescence-generating group and the nitrogen atom and each group such as boronic acid. Needs to be. In the case of poor water solubility, the quenching mechanism by vibration of the B-OH bond does not work efficiently, and the difference between fluorescence and fluorescence emitted to the outside during quenching is not large, making it difficult to distinguish the presence and absence of glucose. can occur

In the case of the organic fluorescent compound according to an aspect of the present invention, as described above, including the dendrimer having the structure of Formula 1-1, it may have excellent water solubility, and thus the above-described fluorescence-quenching mechanism may work efficiently. and the presence and absence of glucose can be more easily distinguished.

And, in the case of using the mechanism by the spectral shift, the wavelength region of the emission spectrum generated in the fluorescence group is changed according to the change in the chemical structure and electron density of the fluorescence group and its neighboring groups depending on the presence or absence of glucose.

In an organic fluorescent compound comprising a fluorescence-generating group (reporter), a linker, and a receptor, the fluorescence-generating group can be viewed as a kind of electron donor group, and the receptor can be viewed as a kind of electron-donating group (acceptor group).

When the structural change of this fluorescence-generating group, that is, the energy-donating group, occurs and the electron density is reduced, the length of the total conjugated bond is shortened, and the electron-donating ability is also decreased, so that the wavelength length of the absorbed light in the absorption spectrum is short. When it is lost, a Blue Shift occurs.

Conversely, when a substance to be detected is bound to a receptor, that is, an electron-donating group, the property of attracting electrons from the receptor is further amplified by the Enhanced Conjugative Effect, and generally absorbed light and A red-shift phenomenon in which the wavelength of light emission becomes longer occurs.

Specifically, in the absence of glucose in the test sample, i) the form of boronic acid (B-OH) is maintained, and ii) an emission spectrum due to fluorescence unique to the fluorescence generating group is emitted.

Conversely, when glucose is present in the sample to be tested, i) boronic acid and glucose form a boron-glucose ester, and ii) an emission spectrum due to fluorescence inherent to the original fluorescence group due to the Enhanced Conjugative Effect described above. Rather, the wavelength length of the actually observed emission spectrum becomes longer.

According to an embodiment of the present invention, it may be preferable that any two or more of R1 to R4 are a group represented by Formula 1-1. As described above among the compounds, when any two or more of R1 to R4 is a group of Formula 1-1, the water solubility of the entire molecule may be greatly increased by the amine or amide group used as a linking group, The presence of phenyl boronic acid may increase selectivity for sugar molecules such as glucose.

And, among R1 to R4, those other than Formula 1-1 are each independently an alkyl group having 1 to 5 carbon atoms substituted with a halogen atom, an aryl group having 6 to 20 carbon atoms substituted with a halogen atom, and 2 to 20 carbon atoms. of an oxyalkyl group, or an oxyaryl group having 6 to 20 carbon atoms.

On the other hand, the fluorescent indicator compound used for detecting sugar molecules must selectively bind to sugar molecules in the blood. In particular, in the case of a recently used fluorescent sensor, after being inserted into the human body, a separate invasive treatment is not required. In many cases, it is manufactured so that it is possible to check blood sugar in real time. In this case, the fluorescent indicator compound should have sufficient water solubility, and at the same time, it needs to be fixed on the insoluble surface of a polymer material such as plastic, which is harmless to the human body, or inside or outside a matrix, etc., and for this purpose, it is preferable to further include a functional group do.

The organic fluorescent compound according to an aspect of the present invention contains diketopyrrolopyrrole as a reporter and a central structure as described above, and at least one of the substituents of R1 to R4 connected thereto serves as a receptor and a linker. contains the structure of 1-1, and among R1 to R4, the rest other than the group of Formula 1-1 are each independently hydrogen, an alkyl group having 1 to 5 carbon atoms, an aryl group having 6 to 20 carbon atoms, and carbon number An oxyalkyl group having 2 to 20 carbon atoms, or an oxyaryl group having 6 to 20 carbon atoms, wherein the alkyl group, aryl group, oxyalkyl group, or oxyaryl group is each independently a halogen atom, a hydroxy group, an amine group or an amino group It may be substituted or unsubstituted.

The alkyl group having 1 to 5 carbon atoms is, specifically, a methyl, ethyl, propyl, butyl, or pentyl group, and when it has 3 or more carbon atoms, it may be in a straight or branched chain form.

Preferably, among R1 to R4, those other than Formula 1-1 are each independently an alkyl group having 1 to 5 carbon atoms substituted with a halogen atom, an aryl group having 6 to 20 carbon atoms substituted with a halogen atom, and 2 carbon atoms. to 20 oxyalkyl group, or an oxyaryl group having 6 to 20 carbon atoms.

Among them, an alkyl group substituted with a halogen atom or an aryl group substituted with a halogen atom is another functional group capable of modifying the physical and chemical properties of the compound, for example, an ethylenically or acrylate unsaturated group, a silane group, It may act as a precursor for the introduction of an amine group, etc.

When these other functional groups are introduced, they may be used for immobilization of insoluble surfaces or matrices such as glass or other polymers, and other organic fluorescent compounds are copolymerized with molecules, other indicator compounds, or other hydrophilic monomers to form hydrophilic macromolecules. It can also be used for the introduction of functional groups to control water solubility, pKa, and the like.

In addition, an oxyalkyl group having 2 to 20 carbon atoms or an oxyaryl group having 6 to 20 carbon atoms may serve to improve the water solubility of the entire molecule. Such an oxyalkyl group or oxyaryl group is polyoxyalkylene, or polyoxyarylene, in which an alkylene oxide repeating unit (-[O-Alkylene]-) or an arylene oxide repeating unit-[O-Arylene]- is repeated. It may be in the form of, specifically, may be in the form of polyethylene glycol or the like.

According to another aspect of the present invention, a device including the above-described organic fluorescent compound may be provided.

When such an organic fluorescent compound is used, various detection techniques for glucose detection are known in the art to which the present invention pertains. For example, the compound of the present invention may be used in a fluorescence sensing device or may be used in a form bound to a polymer material such as test paper for visual inspection, and may be used as a simple reagent in a standard analytical device such as a fluorescence spectrometer or a clinical analyzer. may be used as

In the case of a fluorescence sensing device, it may be in a form including a matrix high-pass filter including stacked organic fluorescent compound molecules and a photodetector. In this device, a light source, eg, a light emitting diode (“LED”) of a specific wavelength, can be placed within the organic fluorescent compound, or in a waveguide over which a matrix is placed, to induce fluorescence in the organic fluorescent compound molecule. The high-pass filter may serve to filter out scattered incident light from the light source while allowing the emitted light to reach the photodetector.

Such a device may be used, for example, as an implantable device for continuously monitoring blood glucose levels in vivo.

The compounds of the present invention can be readily prepared by those skilled in the art without much trial and error using known reaction mechanisms and reagents, including, for example, reaction mechanisms consistent with the general procedures described below.

The organic fluorescent compound according to the present invention can be used in a biosensor for detecting sugars, and by using such a biosensor, blood glucose concentration information can be accurately checked in real time while minimizing an invasive action on a subject.

1 is an image showing a change in the emission spectrum according to the presence or absence of glucose in the organic fluorescent compound according to an embodiment of the present invention.

Hereinafter, through specific examples of the invention, the operation and effect of the invention will be described in more detail. However, these embodiments are merely presented as an example of the invention, and the scope of the invention is not defined thereby.

[Example]

의 합성 Example 1:

synthesis of

3,6-bis(4-chlorophenyl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (hereinafter, DPP) in dimethylformamide (DMF) at a concentration of about 0.3 to about 0.5 M After dissolving, about 3 equivalents of NaH compared to DPP were slowly added under low temperature conditions in an ice bath, and the mixture was stirred for 30 minutes.

About 3 equivalents of methyl bromo acetate compared to DPP was slowly added to the mixed solution, and the mixture was stirred for 12 hours.

After quenching with NaH while checking the progress of the reaction, dimethylformamide (DMF) was removed, and the compound of Intermediate 1 below was obtained by chromatography.

After the Intermediate 1 was dissolved in MeOH to a concentration of about 0.3 to about 0.5 M, N-methyldiamine was added in about 3 equivalents relative to DPP, and the mixture was stirred for about 1 hour. Then, the solvent was removed and the compound of Intermediate 2 was obtained through recrystallization.

Dissolving the intermediate 2 in THF, and adding about 2.5 equivalents of 2-(2-bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolein compared to DPP, Stirred for about 12 hours.

Then, it was purified through recrystallization to finally obtain a compound of the following formula.

의 합성 Example 2:

synthesis of

3,6-bis(4-bromophenyl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (hereinafter, DPP) in dimethylformamide (DMF) at a concentration of about 0.3 to about 0.5 M After dissolving, about 3 equivalents of NaH compared to DPP were slowly added under low temperature conditions in an ice bath, and the mixture was stirred for about 30 minutes.

About 3 equivalents of diethylene glycol 2-bromoethyl methyl ether was slowly added to the mixed solution compared to DPP, and the mixture was stirred for about 12 hours.

After quenching with NaH while checking the progress of the reaction, dimethylformamide (DMF) was removed, and the compound of the following intermediate 3 was obtained by chromatography.

After dissolving the compound of Intermediate 3 in dimethylformamide (DMF) at a concentration of about 0.3 to about 0.5 M, triethylamine is added as a base, and about 3 equivalents of N-methyldiamine compared to DPP are added, and then about 1 hour stirred for a while.

Then, the solvent was removed and the compound of Intermediate 4 was obtained by chromatography.

After dissolving the compound of Intermediate 4 in THF, about 2.5 equivalents of 2-(2-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolein compared to DPP After the addition, the mixture was stirred for about 12 hours.

Then, it was purified through recrystallization to finally obtain a compound of the following formula.

Fluorescence evaluation

Using the material obtained in Example 1, a glucose sensing evaluation was performed to determine whether fluorescence for glucose concentration in an aqueous state was properly implemented.

A PBS buffer aqueous solution (pH=7.4) was prepared, and the material obtained in Example 1 was added to the buffer solution to a concentration of 40 μM, respectively.

By mixing 1 ml of glucose solution with 1 ml of each of the buffer solutions, samples were prepared so that final glucose concentrations in each sample were 0 mM and 1.5 mM, respectively.

For each sample, UV-Visible light was irradiated so that fluorescence appeared and this was observed.

1 is an image showing a change in the emission spectrum according to the presence or absence of glucose in the organic fluorescent compound according to an embodiment of the present invention.

Referring to FIG. 1 , it can be seen that the organic fluorescent compound according to an embodiment of the present invention emits yellow-green light close to yellow in the absence of glucose, and emits light close to orange in the presence of glucose.

As described above in the present specification, among the reporter and receptor portions in the organic fluorescent compound according to an embodiment of the present invention, the detection target material is bound to a receptor, that is, an electron-donating group, thereby enhancing the conjugate effect (Enhanced Conjugative). Effect), the property of attracting electrons from the receptor is further amplified, and thus the wavelength of light emission becomes longer.

Accordingly, it is considered that the organic fluorescent compound according to an embodiment of the present invention can be usefully used as a sensor for detecting glucose.

Claims (7)

An organic fluorescent compound of Formula 1 below:
[Formula 1]

In Formula 1,
R1 to R4 are each independently hydrogen, an alkyl group having 1 to 5 carbon atoms, an aryl group having 6 to 20 carbon atoms, an oxyalkyl group having 2 to 20 carbon atoms, or an oxyaryl group having 6 to 20 carbon atoms, The alkyl group, aryl group, oxyalkyl group, or oxyaryl group may each independently be unsubstituted or substituted with a halogen atom, a hydroxy group, an amine group or an amino group,
provided that at least one of R1 to R4 is a group of Formula 1-1,
[Formula 1-1]

In Formula 1-1,
A1, A2 and A3 are each independently a single bond, alkylene having 1 to 5 carbon atoms, phenylene, or phenylalkylene having 7 to 10 carbon atoms;
D is an amine (-NR22-) or an amide (-CO-NR23-), R21 to R23 are each independently hydrogen or an alkyl group having 1 to 5 carbon atoms;
R11 to R15 are each independently hydrogen, an alkyl group having 1 to 5 carbon atoms, a halogen atom, or -B(OH) ₂ , provided that at least one of R11 to R15 is -B(OH) ₂ .
According to claim 1,
The organic fluorescent compound, wherein any two or more of R1 to R4 is a group of Formula 1-1.
According to claim 1,
Among R1 to R4, those other than Formula 1-1 are each independently an alkyl group having 1 to 5 carbon atoms substituted with a halogen atom, an aryl group having 6 to 20 carbon atoms substituted with a halogen atom, and an oxy group having 2 to 20 carbon atoms. An organic fluorescent compound, which is an alkyl group or an oxyaryl group having 6 to 20 carbon atoms.
According to claim 1,
Wherein A1, A2 and A3 are each independently a single bond or alkylene having 1 to 5 carbon atoms.
According to claim 1,
wherein D is an amide (-CO-NR23-), and R23 is, each independently, hydrogen or an alkyl group having 1 to 5 carbon atoms, an organic fluorescent compound.
A device comprising the organic fluorescent compound according to claim 1 .
7. The device of claim 6, which is a biosensor.

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