KR101171919B1 - Fluorescein derivatives having selectivity for thiol and method for in vivo monitoring thiol using the same - Google Patents

Abstract

The present invention relates to a fluorescent sensor comprising a fluorescein derivative having a thiol selectivity and a thiol detection method using the same. More particularly, a fluorescein derivative which selectively binds to a thiol and exhibits a green fluorescence enhancement effect is detected in an aqueous solution. Alternatively, the thiol-containing material in the body can be quantitatively or qualitatively analyzed by injecting the body into fluorescence intensity.

Description

Fluorescence sensor comprising a fluorescein derivative having thiol selectivity and a thiol detection method using the same {Fluorescein derivatives having selectivity for thiol and method for in vivo monitoring thiol using the same}

The present invention relates to a fluorescent sensor comprising a fluorescein derivative having a thiol selectivity and a thiol detection method using the same. More particularly, a fluorescein derivative which selectively binds to a thiol and exhibits a green fluorescence enhancement effect is detected in an aqueous solution. Or a fluorescent sensor comprising a fluorescein derivative having a thiol selectivity capable of quantitatively or qualitatively analyzing a thiol-containing material in a body in real time by measuring fluorescence intensity by injecting into a body and a thiol detection method using the same.

Intracellular thiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play many important roles in the physiological matrix. For example, Cys and Hcy are essential biomolecules required for cell and tissue growth in a biological system. Deficiency of cysteine causes various health problems, such as growth retardation, hair discoloration, lethargy, liver damage, muscle and fat loss, and skin damage. Increased levels of Hcy in human plasma are risk factors for Alzheimer's disease, cardiovascular disease, neural tube defects, inflammatory bowel disease, and osteoporosis. GSH, the most abundant intracellular non-proteinogenic thiol, plays a pivotal role in maintaining a reducing environment and acting as a redox regulator in cells. Therefore, the detection and identification of substances containing thiols in biological samples is very important. To date, several thiol detection methods have been developed, such as HPLC, capillary electrophoresis and UV-Vis detection / colorimetric analysis. These methods are useful for monitoring thiols in vitro, but due to their limitations for in vivo studies, few methods can be used for intracellular detection. Fluorescent methods for thiol detection are more preferred because they are simple, sensitive and effective. In the past few years, various fluorescent probes for thiols based on different mechanisms have been developed, such as Michael addition, ring formation with aldehydes, separation reactions with thiols, and the like.

It is an object of the present invention to provide novel fluorescein derivative fluorescent probes having high selectivity and sensitivity to thiols in aqueous solution or in vivo and methods for their preparation.

In order to achieve the above object, the present invention provides a compound represented by the following formula (1):

[Formula 1]

Where

R ₁ and R ₄ are each independently hydrogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₂ and R ₃ are each independently hydrogen, halogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₅ is hydrogen, C _1-6 alkyl, C _1-6 alkoxy, hydroxy, carboxyl, CO (R ₆ ), or CON (R ₆ ), wherein R ₆ is C _1-6 alkyl, or 5 to 12 represents aryl of the reduction,

P, Q, Y and Z are each independently an oxygen atom or a sulfur atom,

X is oxygen, sulfur, or NR ₇ , wherein R ₇ represents hydrogen or C _1-6 alkyl,

n represents the integer of 1-4.

The present invention also provides a process for preparing a compound of formula 1 comprising reacting a compound represented by formula 2 with a compound represented by formula 3 under a solvent:

[Formula 2]

(3)

[Formula 1]

Where

R ₁ and R ₄ are each independently hydrogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₂ and R ₃ are each independently hydrogen, halogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₅ is hydrogen, C _1-6 alkyl, C _1-6 alkoxy, hydroxy, carboxyl, CO (R ₆ ), or CON (R ₆ ), wherein R ₆ is C _1-6 alkyl, or 5 to 12 represents aryl of the reduction,

P, Q, Y and Z are each independently an oxygen atom or a sulfur atom,

X is oxygen, sulfur, or NR ₇ , wherein R ₇ represents hydrogen or C _1-6 alkyl,

n is an integer of 1 to 4,

R 'represents 4-7 reduced cycloalkenyl.

The present invention also provides a sensor or composition for detecting a thiol comprising a compound of the formula:

[Formula 1]

Where

R ₁ and R ₄ are each independently hydrogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₂ and R ₃ are each independently hydrogen, halogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₅ is hydrogen, C _1-6 alkyl, C _1-6 alkoxy, hydroxy, carboxyl, CO (R ₆ ), or CON (R ₆ ), wherein R ₆ is C _1-6 alkyl, or 5 to 12 represents aryl of the reduction,

P, Q, Y and Z are each independently an oxygen atom or a sulfur atom,

X is oxygen, sulfur, or NR ₇ , wherein R ₇ represents hydrogen or C _1-6 alkyl,

n represents the integer of 1-4.

The present invention also provides a method for detecting a thiol comprising the step of reacting the compound represented by Formula 1 with a thiol.

The present invention has the effect of providing a novel fluorescein derivative fluorescent probe with high selectivity and sensitivity to thiols.

The fluorescent probe can effectively detect thiol in vivo as well as in aqueous solution.

Figure 1 shows the fluorescence spectrum (a) and UV / Vis absorption spectrum (b) of the fluorescein derivative of the present invention for a variety of analytes in HEPES buffer.
Figure 2 shows the fluorescence (a) and absorption (b) spectrum of the fluorescein derivative of the present invention according to the concentration of GSH in HEPES buffer solution.
The present invention resulting from the FIG. 3 is added to concentrations GSH in the fluorescence titration results (a) and CH ₃ CN-HEPES buffer solution of fluorescein derivatives of the present invention resulting from the addition of GSH at a low concentration in CH ₃ CN-HEPES buffer Fluorescence intensity change at 520 nm of (b) is shown.
Figure 4 shows the fluorescence intensity at 520nm of the fluorescein derivative of the present invention with or without GSH by pH.
FIG. 5 shows a Job's plot of the reaction between the fluorescein derivative and GSH of the present invention in a CH ₃ CN / HEPES solution.
Figure 6 shows the FAB mass spectrum of the product obtained by mixing the fluorescein derivative and 2-mercaptoethanol of the present invention.
FIG. 7 is a ¹ H NMR spectrum of a fluorescein derivative of the present invention according to the presence (lower) or no (top) of 2-mercaptoethanol in DMSO- d ₆ .
FIG. 8 shows phase contrast microscopy images and fluorescence images of mammalian cells and organs. (A) shows the results of treatment of the fluorescein derivative of the present invention on mouse P19 fetal cancer cells, and (b) shows P19 fetal Pretreatment of cancer cells with NMM for 20 minutes followed by treatment with the fluorescein derivative of the present invention (left: phase contrast microscopy; middle is fluorescent image; right is merged image), and (c) shows three-day-old zebrafish. Incubated with the fluorescein derivative of the present invention, (d) is a result of treating the fluorescein derivative of the present invention after pretreatment of the three-day-old zebrafish with NMM (top: phase contrast microscope image; bottom) : Fluorescent image).
Figure 9 shows an image of the organs of zebrafish treated with the fluorescein derivative of the present invention (top: phase contrast microscope image, bottom: fluorescence image).
FIG. 10 shows the fluorescein derivatives of the present invention having high selectivity and sensitivity to thiols and biopsies in zebrafish using the same.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention relates to a compound represented by the following general formula (1):

[Formula 1]

Where

R ₁ and R ₄ are each independently hydrogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₂ and R ₃ are each independently hydrogen, halogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₅ is hydrogen, C _1-6 alkyl, C _1-6 alkoxy, hydroxy, carboxyl, CO (R ₆ ), or CON (R ₆ ), wherein R ₆ is C _1-6 alkyl, or 5 to 12 represents aryl of the reduction,

P, Q, Y and Z are each independently an oxygen atom or a sulfur atom,

X is oxygen, sulfur, or NR ₇ , wherein R ₇ represents hydrogen or C _1-6 alkyl,

n represents the integer of 1-4.

The terms used in the substituent definitions of the compounds of the present invention are as follows.

"Halo" is -F, -Cl, -Br or -I.

"Alkyl" refers to a straight or branched chain or cyclic saturated hydrocarbon having 1 to 30 carbon atoms unless otherwise stated. Examples of C _1-30 alkyl groups are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, isopropyl, isobutyl, sec-butyl and tert-butyl, isopentyl, neopentyl, isohexyl , Isoheptyl, isooctyl, isononyl and isodedecyl, but is not limited thereto. The alkyl also includes "cycloalkyl". The cycloalkyl includes a monocyclic and fused ring as a non-aromatic, saturated hydrocarbon ring having 3 to 12 carbon atoms, unless otherwise specified. Representative examples of C _3-12 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

"Alkoxy" means an alkyl group having 1 to 6 carbon atoms, such as 1 to 4 carbon atoms, bonded to an oxygen atom unless otherwise specified. Examples of C _1-4 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, and butoxy.

"Cycloalkenyl" means a non-aromatic mono or multicyclic ring system having 3 to 10 carbon atoms containing one or more carbon-carbon double bonds. Preferred cycloalkenyl rings contain 4 to 7 ring atoms. Cycloalkenyl may be the same or different and may be optionally substituted with one or more "ring system substituents" as defined above. Non-limiting examples of suitable monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl, and the like. Non-limiting examples of suitable multicyclic cycloalkenyls are norbornylenyl.

"Aryl" refers to 5 to 12-reduced aromatic cyclic compounds unless otherwise stated. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl and anthracenyl.

The compound of Formula 1 is more specifically

R ₁ and R ₄ are each independently hydrogen, C _1-4 alkyl, C _1-4 alkoxy, or hydroxy,

R ₂ and R ₃ are each independently hydrogen, halogen, C _1-4 alkyl, C _1-4 alkoxy, or hydroxy,

R ₅ is hydrogen, C _1-4 alkyl, C _1-4 alkoxy, hydroxy, carboxyl, CO (R ₆ ), or CON (R ₆ ), wherein R ₆ is C _1-4 alkyl, or 5 to 12 represents aryl of the reduction,

P, Q, Y and Z are oxygen atoms,

X is oxygen, sulfur, or NR ₇ , wherein R ₇ represents hydrogen or C _1-4 alkyl,

n can represent the integer of 1-2.

Compound of Formula 1 is more specifically

R ₁ to R ₅ each independently represent hydrogen or hydroxy,

P, Q, Y and Z are oxygen atoms,

X is oxygen, or NR ₇ , wherein R ₇ represents hydrogen, or C _1-2 alkyl,

n can represent the integer of 1-2.

The compound of formula 1 is most specifically

R ₁ , R ₂ , R ₃ and R ₅ are hydrogen,

R ₄ is hydroxy,

P, Q, X, Y and Z are oxygen atoms,

n may represent 2.

The present invention also relates to a method for preparing a compound represented by the following Chemical Formula 1, comprising reacting a compound represented by the following Chemical Formula 2 and a compound represented by the following Chemical Formula 3 under a solvent:

[Formula 2]

(3)

[Formula 1]

Where

R ₁ and R ₄ are each independently hydrogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₂ and R ₃ are each independently hydrogen, halogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₅ is hydrogen, C _1-6 alkyl, C _1-6 alkoxy, hydroxy, carboxyl, CO (R ₆ ), or CON (R ₆ ), wherein R ₆ is C _1-6 alkyl, or 5 to 12 represents aryl of the reduction,

P, Q, Y and Z are each independently an oxygen atom or a sulfur atom,

X is oxygen, sulfur, or NR ₇ , wherein R ₇ represents hydrogen or C _1-6 alkyl,

n is an integer of 1 to 4,

R 'represents 4-7 reduced cycloalkenyl.

Method for preparing a compound of Formula 1

Preparing a compound of Formula 2 wherein an aldehyde group is substituted from fluorescein; And

It is preferred to include the step of preparing the compound of formula 1 by the Baileys Hillman and Michael addition reaction of the compound of formula 2 and the compound of formula 3 in a solvent.

The compound of Formula 2 may be fluorescein monoaldehyde, but is not particularly limited thereto.

The compound of Formula 2 is J. Am. Chem. Soc . It may be prepared according to the method disclosed in 2005, 127, 15949-15958, but is not particularly limited thereto.

According to one embodiment, the method for preparing fluorescein monoaldehyde with the compound of Formula 2 will be described in more detail.

Fluorescein, methanol and 15-crown-5 were mixed and reacted by the addition of NaOH solution, followed by acidification of the mixture after cooling, and the precipitate was purified by silica gel column chromatography to give the pale yellow solid of Chemical Formula 2 Compounds can be obtained.

In the method for preparing the compound of Formula 1 according to the present invention, the compound of Formula 1 may be prepared by the Baileys Hillman and Michael addition reaction of the compound of Formula 2 and the compound of Formula 3 obtained in the above step under a solvent.

2-cyclopenten-l-one may be used as the compound of Formula 3, but is not particularly limited thereto.

The Baileys Hillman and Michael addition reactions are carried out by further addition of imidazole to the mixture.

The solvent may be used alone or two or more of tetrahydrofuran, dichloromethane, acetonitrile and the like.

Hereinafter, the steps for preparing the compound of Formula 1 from the compound of Formula 2 will be described in more detail.

After reacting the compound of Formula 2, the compound of Formula 3 and imidazole by mixing in deionized water, and then evaporating under reduced pressure, the final mixture was solvent extracted, and the organic layer was concentrated under reduced pressure and purified by silica gel column chromatography to obtain yellow crystals. A compound of formula 1 in form can be obtained.

According to one embodiment, the compound of formula 1 may be prepared according to the following scheme 1:

[Reaction Scheme 1]

The present invention also relates to a sensor for detecting thiol, which comprises a compound of the formula:

[Formula 1]

Where

R ₁ and R ₄ are each independently hydrogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₂ and R ₃ are each independently hydrogen, halogen, C _1-6 alkyl, C _1-6 alkoxy, or hydroxy,

R ₅ is hydrogen, C _1-6 alkyl, C _1-6 alkoxy, hydroxy, carboxyl, CO (R ₆ ), or CON (R ₆ ), wherein R ₆ is C _1-6 alkyl, or 5 to 12 represents aryl of the reduction,

P, Q, Y and Z are each independently an oxygen atom or a sulfur atom,

X is oxygen, sulfur, or NR ₇ , wherein R ₇ represents hydrogen or C _1-6 alkyl,

n represents the integer of 1-4.

The expected mechanism for thiol selectivity of the compounds of Formula 1 of the present invention can be represented by Scheme 2 below. In other words, in the aqueous solution, the compound of Formula 1 reacts with the thiol compound and thiol is added at positions 1 and 4 of the α, β-unsaturated ketone in the compound of Formula 1 to generate fluorescence by spiro ring opeining. Since the compound-thiol adduct of Formula 1 is formed to increase fluorescence and change in UV-Vis spectrum, the compound of Formula 1 can be used as a fluorescent probe having high selectivity and sensitivity to thiol.

Scheme 2

The compound of formula 1 of the present invention shows a selective fluorescence increase effect at an excitation wavelength of 520 nm depending on the addition of thiol in 100% aqueous solution, and thus can be used as a fluorescent probe of "OFF-ON" type to detect thiol. It is.

According to one embodiment, no change in fluorescence occurs for amino acids such as Gly, Phe, Ser, Glu, Lys, Arg, His, Ala, Gln, Met, Tyr, and cystine, but cysteine, an amino acid containing thiols ( Cys), homocysteine (Hcy), or glutathione (GSH) show large fluorescence changes in a concentration-dependent manner.

According to one embodiment, the concentration of thiol-containing material, such as glutathione, detectable with the compound of formula 1 is 50 to 350 nM.

The present invention also relates to a thiol detection composition comprising the compound of formula (1).

The thiol detecting composition or the thiol detecting sensor may include a buffer solution in addition to the compound represented by Chemical Formula 1. The type and concentration of the buffer solution are not particularly limited, but may be appropriately changed depending on the application of the thiol detection sensor. For example, when the sensor for detecting thiol of the present invention is to be used for detecting thiol in vivo, the buffer solution may preferably have a buffer range corresponding to pH in vivo.

The present invention also relates to a method for detecting a thiol comprising the step of reacting a thiol with a compound represented by the formula (1).

The compound of formula 1 of the present invention is a "OFF-ON" type sensor that can detect a thiol compound in an aqueous solution or in vivo, and the fluorescence intensity increases when the thiol is detected in a low fluorescence intensity (OFF). Is a sensor.

When the thiol compound is detected in the aqueous solution, the pH of the aqueous solution may be 5 to 12 in consideration of the detectable fluorescence intensity.

The thiol compound is not particularly limited as long as it contains a thiol group. For example, the thiol compound may be a thiol-containing amino acid such as cysteine (Cys), homocysteine (Hcy), or glutathione (GSH).

In addition, when detecting a thiol compound in vivo, the compound of Formula 1 may be mixed culture with living cells, or administered in vivo to detect the thiol by measuring the fluorescence intensity of the cells or in vivo.

The living body may be a fish such as zebrafish, a mouse, a mouse, a guinea pig, a rabbit, a cat, a dog, a sheep, a pig, a cow, a monkey, a baboon, or a chimpanzee.

The thiol detection can be carried out by measuring an increase in fluorescence intensity using a conventional measuring device, for example, a fluorescence microscope.

The method for detecting thiols according to the invention is to detect an increase in fluorescence intensity.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention, but the scope of the present invention is not limited by the following Examples.

Example 1 Synthesis of Fluorescein Derivatives and Investigation of Thiol Selectivity

Compounds used for the synthesis of fluorescein derivatives and the thiol selectivity of the derivatives were purchased and used without further purification unless otherwise noted.

Chromatography was performed on silica gel 60 (230-400 mesh ASTM; Merck). Thin layer chromatography (TLC) was used Merck 60 F ₂₅₄ plate (thickness: 0.25 mm). Preparative TLC used a Merck 60 F ₂₅₄ plate (thickness: 1 mm). ¹ H NMR and ¹³ C NMR spectra were recorded using Bruker 250. Mass spectra were acquired on a Jeol JMS 700 high resolution mass spectrometer. UV absorption spectra were obtained on a UVIKON 933 Double Beam UV / VIS Spectrometer. Fluorescence emission spectra were obtained on RF-5301 / PC Spectrofluorophotometer (Shimadzu).

Preparation Example 1 Synthesis of Fluorescein Derivatives

[Reaction Scheme 1]

Fluorescein monoaldehyde (2) in the above formula was synthesized according to the previously reported method ( J. Am. Chem. Soc . 2005, 127, 15949-15958). Fluorescein (4 g, 12 mmol), 10 mL CHCl ₃ , 6 mL MeOH and 0.06 g 15-crown-5 were placed in a 100 mL-flask. 20 g of 50% NaOH solution (50%) were carefully added while the reaction temperature was maintained at 55 ° C. The mixture was stirred at this temperature for 5 hours. After cooling, the mixture was acidified by treatment with 10M H ₂ SO ₄ . The precipitate was collected and dried in vacuo. Silicagel chromatography (15:85 EtOAc: DCM) and crystallization in CH ₃ OH gave 1.13 g of a pale yellow solid product (yield: 26%).

¹ H NMR (250 MHz, with small amount of CD ₃ OD) δ (ppm): 10.59 (1H, s), 7.95 (1H, d, J = 7.5 Hz), 7.69-7.56 (2H, m), 7.12 ( 1H, d, J = 7.5 Hz), 6.82 (1H, d, J = 9.0 Hz), 6.71 (1H, s), 6.55 (1H, d, J = 9.0 Hz), 6.54 (2H, s).

Next, to prepare compound 1 of the above formula, fluorescein monoaldehyde (360 mg, 1 mmol) and 2-cyclopenten-l-one (164 mg, 2) dissolved in tetrahydrofuran (THF, 10 mL) mmol) and imidazole (68 mg, 1 mmol) solution and deionized water (10 mL) were mixed. The mixture was stirred at rt for 72 h. After evaporation under reduced pressure, the final mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was concentrated under reduced pressure. Silica gel chromatography of crude extract using ethyl acetate and hexane (60:40) as eluent and crystallization using CH ₃ OH / H ₂ O (50:50, v / v) yielded a yellow chemical formula. 90 mg (yield 21%) of compound 1 were obtained.

_{^{1 H NMR (DMSO- d 6,}} 250 MHz) δ (ppm): 10.31 (1H, s), 8.75 (1H, d, J = 7.5 Hz), 7.88-7.78 (2H, m), 7.69 (1H, s ), 7.37 (1H, t, J = 7.5 Hz), 6.79 (1H, d, J = 8.25 Hz), 6.80-6.76 (2H, m), 6.67-6.65 (2H, m), 5.46 (1H, t, J = 8 Hz), 2.79-2.65 (2H, m), 2.20-2.00 (2H, m).

¹³ C NMR (DMSO- d ₆ , 62.5 MHz) δ (ppm): 200.7, 168.4, 159.6, 156.2, 151.7, 151.4, 151.1, 149.1, 135.7, 131.8, 131.7, 131.4, 130.3, 129.0, 126.2, 124.7, 124.1 , 120.0, 113.2, 112.6, 112.5, 110.4, 109.3, 109.0, 102.7, 82.3, 81.9, 75.6, 36.7, 27.4.

FAB MS m / z = 425.1026 [M + H] ⁺ , calc. for C ₂₆ H ₁₇ O ₆ = 425.1025.

As above, the fluorescent probe compound of Formula 1 was synthesized as shown in Scheme 1. Fluorescein-monoaldehyde reacted with 2-cyclopentenone in the presence of imidazole (Baylis-Hillman and intramolecular Michael addition reactions) dissolved in THF to synthesize the fluorescent probe 1 in 21% yield. .

Preparation Example 2 Preparation of Amino Acid Solution for Fluorescence Experiment

Amino acids and other analytes, ie Cys, Hcy, GSH, Gly, Phe, Ser, Glu, Lys, Arg, His, Ala, Gln, Met, Tyr and cystine are dissolved in distilled water and stock solution (10 mM) ) Was prepared. Stock solution of Compound 1 (1 mM) was prepared by dissolving in acetonitrile. In the following experiments, test solutions were prepared by placing 30 μl of probe stock solution in a test tube, diluted to 3 mL by adding 0.01 M HEPES (pH 7.4), and adding the appropriate buffer of each analyte stock solution. In general, the excitation wavelength was 485 nm. The width of the excitation and emission slits is 1.5 nm / 1.5 nm. Fluorescence spectra were measured after addition of the analyte for 5 minutes.

For low concentration titration of GSH, fluorescence spectra were measured after addition of GSH for 5 minutes, and the excitation and emission slit widths were 3 nm and 1.5 nm, respectively.

Experimental Example 1 Detection of Thiol in Aqueous Solution of Fluorescein Derivative (Compound 1) of the Present Invention

1 shows various analytes (100 mM) in HEPS buffer (20 mM, pH 7.4, 1% CH ₃ CN), ie Cys, Hcy, GSH, Gly, Phe, Ser, Glu, Lys, Arg His, Shows fluorescence spectra (λ _ex = 485 nm, slit: 1.5 nm / 1.5 nm) (a) and UV / Vis absorption spectra (b) of compound of formula 1 (10 μM) for Ala, Gln, Met, Tyr and cystine will be.

As shown in FIG. 1, the compound of formula 1 exhibited an increase in fluorescence (λ _max = 520 nm), and UV-Vis spectrum in the presence of thiol containing analytes (Cys, Hcy and GSH) in HEPES buffer. However, thiol-free analytes (Gly, Phe, Ser, Glu, Lys, Arg, His, Ala, Gln, Met, Tyr and cystine) showed little change in fluorescence intensity and UV-Vis spectra under the same conditions.

In addition, the fluorescence increase observed after addition of 10 equivalents of Cys, Hcy and GSH to the compound of Formula 1 was 95.46 and 61 times greater, respectively.

Next, GSH, which is a typical biological thiol, was used to further test the fluorescence reaction of the compound of Formula 1. Figure 2 is a compound of formula 1 (10 μM in GSH (0, 2, 4, 6, 8, 10, 20, 40 and 100 μM) by concentration in HEPES buffer (20 mM, pH 7.4, 1% CH ₃ CN) Fluorescence (a) and absorption (b) spectra of () _ex = 485 nm, slit: 1.5 nm / 1.5 nm), each spectrum was recorded 5 minutes after the addition of GSH to the compound of formula (1).

The compound of Formula 1 did not fluoresce at all in the absence of GSH. However, a dramatic change in the fluorescence spectrum occurred with the addition of GSH. A new strong emission peak appeared at 520 nm and an increase in fluorescence intensity was observed up to 61 times. Incidentally, there was a gradual decrease of the absorption peak at 454 nm and a new absorption peak was observed around 500 nm upon addition of GSH (FIG. 2B). At 467 nm, distinct iso-absorbance points were observed. This means that a new product was formed upon treatment with the compound of Formula 1 and GSH ( vide infra ).

To investigate the detection limit of the compound of formula 1 for GSH, formula 1 with the addition of low concentrations of GSH in CH ₃ CN-HEPES buffer (0.02 M, pH 7.4) (1:99, v / v) (A) (excitation wavelength: 485 nm, slit: 3 nm / 1.5 nm) with CH ₃ CN-HEPES buffer solution (0.02 M, pH 7.4) (1:99, The change in fluorescence intensity (b) at 520 nm of the compound of Formula 1 (1 μM) according to the concentration-specific GSH (50 to 350 nM) at v / v) is shown in FIG. 3.

The fluorescence intensity of the compound of Formula 1 was linearly proportional to the GSH concentration (0-350 nM), and was detected using the compound of Formula 1 having a signal-to-noise ratio of 3 at a low concentration of GSH of 53 nM.

The hourly fluorescence response of the compound of formula 1 to thiol was monitored at 520 nm under pseudo-first-order kinetic conditions (10 μM compound of formula 1 and 500 μM thiol).

Under these conditions, the rate constants ( k _obs ) observed at pH 7.4 were found to be 36.5, 8.0 and 11.5 min ⁻¹ for Cys, Hcy and GSH, respectively.

In order to investigate the effect of pH on the fluorescence reaction of the compound of formula 1 on thiol, the change in fluorescence intensity of the compound of formula 1 induced by thiol was measured by pH. Figure 4 shows the fluorescence intensity at 520 nm of the compound of formula 1 with or without GSH at different pH (10 μM compound of formula 1 in a CH ₃ CN-H ₂ O (1:99, v / v) system; λ _ex = 485 nm; Slit: 1.5 nm / 1.5 nm).

In the absence of GSH, the compound of Formula 1 showed little change in fluorescence intensity in the range of Ph 2.5-11.3 (FIG. 4). When GSH was added to the probe, strong fluorescence was detected above pH 5, indicating that the compound of formula 1 reacts to the analyte under physiological conditions (pH 7.4).

In the detection mechanism, the spiro ring opening of fluorescein after 1,4-addition of thiol to the α, β-unsaturated ketone in the probe seems to affect the increase in fluorescence and UV-Vis spectral change (Scheme 2). To test the validity of this mechanism, the stoichiometry of the binding event between thiol and the compound of formula 1 was first measured. FIG. 5 shows a Job's plot for the reaction between a compound of Formula 1 and GSH in a CH ₃ CN / HEPES (20 mM) (1:99, v / v) solution (pH 7.4). The total concentration was kept constant at 10 μM.

As shown in FIG. 5, according to the results obtained from the Job's plot, the stoichiometry between 1 and GSH was 1: 1.

In addition, mass spectrometry analysis of the product obtained from the reaction between the compound of formula 1 and 2-mercaptoethanol in CH ₂ Cl ₂ may be supported by the formation of the compound-thiol adduct of formula 1.

FIG. 6 shows the FAB mass spectrum of the product obtained by mixing the compound of Formula 1 with 4 equivalents of 2-mercaptoethanol, that is, the peak at 503.12 corresponding to [1-mercaptoethanol + H ⁺ ] ⁺ Clearly observed.

In addition, NMR spectroscopic analysis also provides evidence for 1,4-thiol addition of α, β-unsaturated ketones of the compound of formula (1). When 4 equivalents of 2-mercaptoethanol was added to the compound of Formula 1 in DMSO- d ₆ , the phenol group of fluorescein etherified by 2-cyclopentenone was released due to nucleophilic attack by thiol (Scheme 2, FIG. 7). The final fluorescein fluorophore showed strong fluorescence in the recipient media.

Experimental Example 2 Detection of Thiol in Mammalian Cells of Compound of Formula 1

Murine P19 carcinoma embryonic cells were cultured in an incubator at 37 ° C. in culture medium (DMEM with 10% FBS, 50 unit / mL penicillin, and 50 g / mL streptomycin), The culture medium was replaced with fresh medium every day. The cells were seeded in 3-well plates at a density of 10 ⁴ cells per mL of culture medium. After 24 hours, the cells were treated with 50 μM NMM in the culture medium or else at 37 ° C. for 20 minutes. After washing with PBS (phosphate buffered saline) to remove the remaining NMM, 20μM compound of Formula 1 was added to the culture medium and further incubated for 30 minutes at 37 ℃. The cells were imaged under fluorescence microscopy (Nicon Eclipse TE2000; excitation filter; 450-490 nm, emission filter; 520 nm).

Experimental Example 3 Detection of Thiols in Zebrafish of the Compound of Formula 1

The zebrafish was maintained at 28 ° C. and maintained at optimal breeding conditions. For mating, male and female zebrafish were kept in one tank, maintained at 28 ° C., 12/12 hour light cycle, and spawning was induced by providing light stimulation in the morning (Yang et al. Nat. Protocols 2007 , 2 , 1740-1745). Nearly all eggs were fertilized immediately. Three-day-old zebrafish were harvested in E3 embryo medium (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO ₄ , 1 mM CaCl ₂ , 0.15 mM KH ₂ PO ₄ , 0.05 mM Na ₂ HPO ₄ , 0.7 mM NaHCO ₃ , 10 ^-5 % Methylene blue; pH 7.5). The zebrafish was incubated at 28 ° C. for 30 minutes in E3 medium with or without 40 μM NMM. After washing off with E3 medium to remove the remaining NMM, the zebrafish was further incubated for 30 minutes at 28 ° C. with 20 μM compound of formula 1 in E3 medium. After washing with E3 medium, the zebrafish was imaged under fluorescence microscopy.

Experimental Example 4 Detection of Thiols in the Zebrafish Organ of the Compound of Formula 1

Adult zebrafish (3 month old with identifiable organs) were exposed to 5 μM compound of formula 1 in E3 medium at 28 ° C. for 5 minutes and 60 minutes. After removing the remaining compound of formula 1 by washing with E3 medium, the zebrafish was dissected to separate tissues and organs and imaged under fluorescence and dissecting microscopes (Stemi 2000-C, ZAISS, Germany).

In Experimental Examples 2 to 4, due to chemical and spectroscopic characteristics, the compound of formula 1 is suitable for identifying thiols in living cells and organs. To test this, mice were incubated with P19 fetal cancer cells and three-day-old zebrafish with a compound of Formula 1 (20 μM). FIG. 8 shows phase contrast microscopy images and fluorescence images of mammalian cells and organs. (A) shows the results of treatment with 20 μM of the compound of Formula 1 for 30 minutes in mouse P19 fetal cancer cells, and (b) shows P19. Fetal cancer cells were pretreated at 50 μM NMM for 20 minutes and then treated with 20 μM of compound of Formula 1 for 30 minutes (left: phase contrast microscopy image; middle is fluorescent image; right is merged image), (c) is 3 The aged zebrafish was incubated with 20 μM of the compound of Formula 1, and (d) is the result of pretreatment of the 3 day old zebrafish with 40 μM NMM for 30 minutes and then 20 μM of the compound of Formula 1 for 1 hour. (Top: phase contrast microscopy image; bottom: fluorescence image).

Strong fluorescence was seen in the cells and zebrafish (FIGS. 8A and 8C). However, when the cells and zebrafish were pretreated with N-methylmaleimide (NMM) (40-50 μM), a trapping agent of thiol species, a significant decrease in fluorescence intensity was observed, which was specific detection of thiol by the compound of formula (1). It means (Fig. 8b and 8d).

In addition, thiol was analyzed in zebrafish using the compound of Formula 1. Zebrafish were exposed to 5 μM compound of formula 1 for 1 hour, dissected to separate tissues and organs and tested using fluorescence microscopy. Thiol species were strongly detected in the eyes, gallbladder, eggs and fins by the compound of formula 1 and weakly identified in the brain (FIG. 9). These in vivo experiments show that the compound of formula 1 can penetrate cell membranes and fish and react with thiols to form fluorescent products.

From the above results, the present invention provides a novel fluorescein derivative fluorescence probe with high selectivity and sensitivity for detecting thiol-containing substances, wherein the fluorescence increase and UV-Vis spectral change are dependent on the α, β-unsaturated ketone in the fluorescent probe. It is considered that thioether was formed by adding thiol at positions 1 and 4.

Claims (14)

Compound represented by the following formula (1):
[Formula 1]

In this formula,
R ₁ , R ₂ , R ₃ and R ₅ are hydrogen,
R ₄ is hydroxy,
P, Q, X, Y and Z are oxygen atoms,
n represents 2.
delete delete delete Method for preparing a compound of formula 1 comprising the step of reacting a compound represented by the formula (2) and a compound represented by the formula (3) under a solvent:
(2)

(3)

[Formula 1]

In this formula,
R ₁ , R ₂ , R ₃ and R ₅ are hydrogen,
R ₄ is hydroxy,
P, Q, X, Y and Z are oxygen atoms,
n is 2,
R 'represents a 5-membered cycloalkenyl.
The method of claim 5,
The solvent is at least one selected from the group consisting of tetrahydrofuran, dichloromethane and acetonitrile.
Sensor for detecting thiol comprising a compound of Formula 1
[Formula 1]

In this formula,
R ₁ , R ₂ , R ₃ and R ₅ are hydrogen,
R ₄ is hydroxy,
P, Q, X, Y and Z are oxygen atoms,
n represents 2.
A thiol detecting composition comprising a compound of Formula 1
[Formula 1]

In this formula,
R ₁ , R ₂ , R ₃ and R ₅ are hydrogen,
R ₄ is hydroxy,
P, Q, X, Y and Z are oxygen atoms,
n represents 2.
A method for detecting a thiol comprising the step of reacting a compound represented by Formula 1 with a thiol:
[Formula 1]

In this formula,
R ₁ , R ₂ , R ₃ and R ₅ are hydrogen,
R ₄ is hydroxy,
P, Q, X, Y and Z are oxygen atoms,
n represents 2.
10. The method of claim 9,
A method for detecting thiols by reacting a compound of Formula 1 with a thiol compound in an aqueous solution.
The method of claim 10,
A method for detecting thiols having an aqueous pH of 5 to 12.
10. The method of claim 9,
A method of detecting a thiol which measures the fluorescence intensity in vivo by administering the compound of Formula 1 to an animal body other than a human.
The method of claim 12,
The living body detects a thiol which is a fish, mouse, mouse, guinea pig, rabbit, cat, dog, sheep, pig, cow, monkey, baboon, or chimpanzee.
10. The method of claim 9,
Thiol detection is a method of detecting thiols which measures the increase in fluorescence intensity.

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