KR101011347B1 - Rhodamine derivative, fluorescein derivative and probes theirof - Google Patents

Abstract

The present invention relates to a copper ion (Cu ^{+ 2)} rhodamine derivative, and fluorescein-derivatives, which in vivo method of detecting copper ions and copper ion in vivo detection probe containing them by having a selectivity.

Rhodamine derivatives and fluorescein derivatives according to the present invention have very strong bonds with copper ions, and thus have excellent selectivity to copper ions, and can be usefully used as a method for detecting copper ions in vivo.

Rhodamine derivatives, fluorescein derivatives, copper ions

Description

Rhodamine derivatives and fluorescein derivatives for copper ion detection, and probes using the same {Rhodamine derivative, fluorescein derivative and probes theirof}

The present invention relates to a rhodamine derivative and a fluorescein derivative having copper ion selectivity, a method for preparing the same, a probe including the same, and a method for detecting copper ion in vivo using the same.

Metal-selective fluorescent chemical sensors serve as useful tools for the detection of metal ions in cells, and thus have been extensively developed to detect biologically related metal ions in cells and / or organs.

Among the various transition metal ions, copper ions are the third most abundant heavy metal ions present in the human body and include various metal enzymes, including superoxide dismutase, cytochrome c oxidase and tyrosinase. It plays an important role as a catalytic cofactor for. However, when present in excess, copper can be said to be toxic in that it is involved in the production of reactive oxygen species, leading to neurodegenerative diseases. Due to the toxic nature of copper, cells have tight control over the distribution of copper in the cell. Nevertheless, the uptake and release of intracellular copper proceeds rapidly, so the intracellular copper concentration can be increased 20-fold within an hour. Cytoplasm, mitochondria, endoplasmic reticulum enzymes, and the like require copper as a redox cofactor, but the reaction of oxygen and reactive oxygen species with unregulated copper ions causes oxidation of proteins, nucleic acids and lipids.

As such, the metal ions need to be easily and accurately determined in concentration due to role or environmental aspects in the living body, and as much as research is being conducted, the detection method may be important in terms of environment. In this regard, fluorescent probes for copper ions have been widely developed because of the biological importance of these metal ions.

The design and research of new sensors for key substances and ions in vivo has been actively conducted. Recent understanding and research on supramolecule chemistry has shown great potential in the design of host compounds that can selectively bind ions or other types of guest compounds. It is a great help in the development of a fluorescent chemosensor that can easily observe the selective binding with a guest compound using fluorescence change.

Fluorescence is a photochemical phenomenon that occurs when photons with a specific wavelength (excitation wavelength) collide with an indicator molecule, and electrons are excited at a high energy level as a result of the collision. Among the various analytical methods, fluorescence has the advantage of being able to observe the signal even at 10 ^-9 M concentration due to its very high sensitivity. Recently, studies on fluorescence chemical sensors for cations, anions and neutral organic molecules using these properties have been published (AP de Silva et al., Chem. Rev. 1997, 97, 1515).

Boronic acid is known to have high affinity for materials containing adjacent diol groups and thus has been used as a fluorescent chemical sensor for carbohydrates. Further, the boron steel benil antibody to monitor the acid Hg ^{2 +} - in the line sensor has been utilized as a co-factor. However, there is no precedent for the use of boronic acid based fluorescent chemical sensors to directly detect metal ions.

Accordingly, the present inventors have been studying a fluorescence chemical sensor selective for copper ions (Cu ^{2 +} ) among metal ions, and synthesized rhodamine derivatives and fluorescein derivatives containing boronic acid, the rhodamine Derivatives and fluorescein derivatives have been found to be able to be used for the identification of copper ion binding phenomenon by selectively acting on copper ions in vivo and completed the present invention.

The present invention is directed to providing a rhodamine derivative, and fluorescein derivatives, including the acid with copper ion (Cu ^{+ 2)} selectivity.

In addition, the present invention is to provide a method for producing the rhodamine derivatives and fluorescein derivatives.

In addition, the present invention is to provide a method for detecting copper ions (Cu ^{2 +} ) in vivo using the rhodamine derivative and fluorescein derivative.

The present invention also provides a probe for detecting copper ions (Cu ^{2 +} ) in vivo including the rhodamine derivative and fluorescein derivative.

The present invention provides a rhodamine derivative represented by the following formula (1).

[Formula 1]

Where R is B (OH) ₂ .

The present invention also provides a fluorescein derivative represented by the following formula (2).

[Formula 2]

이다)Where R is

to be)

Rhodamine derivatives and fluorescein derivatives according to the present invention can be used as boronic acid-based fluorescent and colorimetric chemical sensors for copper ions in combination with copper ions in a unique process of opening a ring included in the structure. The chemical sensor can be practically used for the detection of copper ions in mammalian cells and vertebrates.

The present invention also provides a method for preparing a rhodamine derivative having copper ion selectivity.

Method for producing a rhodamine derivative according to the invention is characterized in that it comprises the step of reacting 2-formylphenylboronic acid and rhodamine hydrazide.

The present invention also provides a method for detecting copper ions in vivo using the rhodamine derivative of the formula (1). The detection principle of copper ions is as follows.

The rhodamine derivative forms a colorless solution in 20 mM HEPES (5% CH ₃ CN) at pH 7.4 and is mostly present in the form of spirolatam. Applying a Cu ^{2 +} in the solution, kind of pink is generated and produced shows a dark orange fluorescence (FIG. 1b). The amount of change in absorption (λ _max = 556㎚) and emission (λ _max = 572㎚) is Cu ^{+ 2} - it is associated with a spiro-lactam ring opening induced Cu ²⁺ to form the rhodamine derivative complex (Fig. 1a). Sickle with an excess of copper ions chelator alkylene (cyclen) the Cu ^{2 +} - a when added to the solution containing the rhodamine derivative complex, disappears, and pink and yellow fluorescent color, and reproducing the rhodamine derivative, which sickle alkylene treatment Cu ² It can be confirmed by HPLC analysis of the ⁺ -rhodamine derivative complex.

Therefore, Cu ^{2 +} - induced opening of spiro lactam ring is a reversible process. Job plots (job plot) results, the combination of the rhodamine derivative and Cu ^{2 +} a 1: It can be seen that it has a stoichiometric ratio of 1. Cu ^{2 +} - the loader coupling constant (association constant) of Min derivative of the result is the size of 10 ⁴ M ^-1 fluorescence titration experiments, and preferably from 4.5 × 10 ⁴ to 5.4 × 10 ⁴ M ^-1.

^{Ag +, Ca 2 +, Co} 2 +, Cd 2 +, Cs +, Hg 2 +, K +, Li +, Mg 2 +, Mn 2 +, Na +, Ni 2 +, Rb +, Sr 2 + , and Other metal ions, such as Zn ²⁺ , do not promote color and fluorescent changes in the rhodamine derivatives (FIG. 1C). Metal ion competition experiments show that the binding of the rhodamine derivative to copper ions is not affected by other metal ions. In other words, the results indicate that the rhodamine derivative reacts highly selective to copper ions. In addition, the rhodamine derivatives did not show any significant fluorescent changes upon addition of adjacent diol-containing carbohydrates such as glucose and fructose (1 mM each).

In addition, as a result of processing the rhodamine derivative substituted with hydrogen instead of acid to Cu ^{2 +} in HEPES (5% CH ₃ CN) at pH 7.4, it can be seen that there is no change in the fluorescence property. That is, the importance of boronic acid moieties for bonding with copper ions in rhodamine derivatives.

If the addition processing of a different control, pyrene boronic simple boronic acid derivatives, there are no other metal recognition site like a mountain by Cu ^{2 +} under the same conditions, it exhibited a unique excimer peak at 455 ㎚ the Cu ^{2 +} addition of acetonitrile, This represents the interaction between boronic acid groups and Cu ²⁺ . Job plots (job plot) results, pyrene boronic acid and a combination of Cu ^{2 +} 2: It can be seen that it has a stoichiometric ratio of 1. Accordingly, excimer tends to be formed are laminated between pyrene ring through the interaction between the acid moiety and Cu ^{+ 2.} As well as the control experiment on the basis of the fluorescence and coloring property change of the rhodamine derivative, a combination of the rhodamine derivative according to the invention of Cu ^{2 +} is a ring of the spiro-lactam as shown in Fig. 1a - may be described in the opening process.

The present invention also provides a method for preparing a fluorescein derivative of formula (2) having copper ion selectivity.

The method for preparing a fluorescein derivative according to the present invention comprises the step of condensing 2,7-dichlorofluorescein and 2- (N-methylaminomethyl) phenylboronic acid through a Mannich reaction. It is done.

The fluorescein derivatives exhibit a completely different type of fluorescent response when reacted with copper ions than the rhodamine derivatives.

That is, in the absence of copper ions, the solution of the fluorescein derivative in 20 mM HEPES (5% CH ₃ CN) at pH 7.4 is pale yellow and exhibits strong yellow fluorescence. The addition of Cu ^{+ 2} in the solution is a color change and at the same time of the fluorescent pink significant reduction in pale yellow, and shows a weak blue color transition (~ 9㎚) (Fig. 2). Job plots (plot job) Results The combination of the program Luo fluorescein derivative and Cu ^{2 +} a 1: It can be seen that it has a stoichiometric ratio of 2. A coupling constant of the fluorescein derivative for Cu ^{+ 2} are the results of fluorescence titration experiments 10 ³ M ^- it is the size of ^2, preferably 7.5 × 10 ³ to 9.5 × 10 ³ M ^-2. Finally, the fluorescein derivative exhibits a high selectivity for Cu ^{2 +} compared to other metal ions (Figure 2c).

Here, the boron-nitrogen interaction is a fact known to those skilled in the art and can block the photo-induced electron transfer (PET) mechanism derived from benzyl nitrogen. In other words, the fluorescein derivative of Formula 2 exhibits relatively strong fluorescence. On addition of Cu ^{+ 2,} the ions are OH Chemistry and complexes of boronic acids and phenols as shown in Figure 2a.

The process liberates the tertiary amine moiety, which can regenerate the PET process and induce fluorescence disappearance. Color changes may also assist in the Cu ^{2 +} combines with oxygen of the phenol, which may form a phenolate.

Effect of acid for copper combined with the fluorescein derivative may be seen by incubation of the compound and the Cu ^{2 +} a substituted boronic acid group in HEPES (5% DMSO) at pH 7.4 with hydrogen. In other words, the hydrogen substituted fluorescein derivatives induced by copper ions do not exhibit fluorescence change, indicating the importance of the boronic acid moiety for bonding with copper ions.

Rhodamine derivative, and fluorescein derivatives, including the acid according to the present invention, a copper ion (Cu ^{2 +)} and excellent so by a strong binding selectivity for copper ion (Cu ^{2 +),} In addition, in vivo copper ions can be effectively used as a (Cu ^{+ 2)} detection method.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited thereto.

Example 1 Preparation of Rhodamine Derivatives of Formula 1

Rhodamine hydrazide (0.46 g, 1 mmol) was dissolved in 20 mL anhydrous ethanol. Excess 2-formylphenylboronic acid (4 mmol) was added and refluxed for 6 hours in an air bath. Ethanol was then completely evaporated and the crude product was purified on a column (neutral alumina; MC + MeOH; 98 + 2) to yield 0.280 g (47%) of the compound of formula 1 as a pink solid.

Mp: 195-197 ° C .;

¹ H NMR (250 MHz, CDCl ₃ ) δ 8.13 (s, 1H), 8.11 (m, 1H), 7.94 (m, 1H), 7.85 (2, 2H, B- (O H ) ₂ ), 7.39 (m , 2H), 7.24 (m, 2H), 7.01 (m, 2H), 3.22 (q, 8H, J = 7.1 Hz), 1.06 (t, 12H, J = 7.1 Hz);

¹³ C NMR (62.5 MHz, CDCl ₃ ) δ 165.2, 152.9,152.7, 149.2, 138.4, 138.2, 133.8, 133.3, 130.1, 129.6, 129.2, 128.4, 128.2, 127.6, 123.7, 123.6, 108.4, 104.5, 97.7, 65.1 , 44.3, 12.6;

FAB Mass m / z = 571.3 (M−H ₂ O + H) ⁺ , calc. for C ₃₅ H ₃₆ BN ₄ O ₃ = 571.3.

Example 2 Preparation of Fluorescein Derivatives of Formula 2

2,7-dichlorofluorosane (0.8 g, 2.00 mmol) and paraformaldehyde (0.47 g, 16.00 mmol) were dissolved in 15 mL of CH ₃ CN and stirred. 2-methylaminomethylboronic acid (1.3 g, 8.00 mmol) contained in 20 mL of CH ₃ CN was added, followed by 15 mL of H ₂ O. The reaction mixture was refluxed for 24 hours. The solvent was then removed in vacuo and the residue was removed by column chromatography using acetone (100%). The resulting product was purified by column chromatography using ethyl acetate-methanol (9: 1, v / v) to obtain a pure product having a yield of 24%.

Mp: 124-126 ° C .;

¹ H NMR (250 MHz, CD ₃ OD) δ 8.16 (m, 1H), 7.91 (s, 1H), 7.65-7.70 (m, 2H), 7.39-7.49 (m, 8H), 7.22 (m, 2H) , 4.42 (m, 4H), 4.34 (s, 4H), 2.73 (s, 6H);

¹³ C NMR (62.5 MHz, CD ₃ OD) δ 174.3, 172.9, 157.7, 155.8, 140.2, 134.6, 131.9, 131.8, 131.2, 131.1, 131.0, 130.9, 130.5, 130.3, 128.2, 112.1, 108.7, 105.9, 79.5, 60.8, 52.4, 50.0, 49.7, 49.3, 49.0, 48.7, 48.3, 48.0, 40.4;

FAB Mass m / z = 755.19 (M + H) ⁺ , calc. for C ₃₈ H ₃₅ B ₂ Cl ₂ N ₂ O ₉ = 754.19.

Example 3 Preparation of Fluorometric Metal Ion Titration Solution

1 mM metal perchlorate stock solution was prepared using double distilled demineralized water. In addition, a stock solution of a compound represented by Formula 1 and Formula 2 in acetonitrile was prepared. The stock solutions were used on the day of preparation.

The test solution was prepared by adding 40 μl of probe stock solution to the test tube, adding an appropriate amount (0 μl) of each metal stock, and then diluting the solution to 4 mL with 20 mM HEPES buffer (pH 7.4).

In all measurements, the excitation wavelength was 505 nm or 504 nm. The slit width of the excitation and emission was 1.5 or 3 nm.

Experimental Example 1 Imaging of Mammalian Cells Incubated with Compound of Formula 1 and Copper Ions

Murine P19 cells were seeded in 6-well plates at a density of 10 ⁴ cells per well in culture medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS)). After 24 hours, P19 cells were incubated with 0.5 mM Cu (ClO ₄ ) ₂ for 30 minutes in culture medium. After washing with PBS to remove residual copper ions, the cells were incubated for 30 minutes at 37 ℃ in 50 μM compound of formula (1).

Rat (Murine 3T3-L1) fibroblasts were seeded in 6-well plates at a density of 10 ⁴ cells per well in culture medium. After 24 hours, 3T3-L1 cells were incubated with 0.5 mM Cu (ClO ₄ ) ₂ for 30 minutes in culture medium. After washing with PBS to remove residual copper ions, the cells were incubated for 30 minutes at 37 ℃ in 50 μM compound of formula (1).

Murine C2C12 cells were seeded in 6-well plates at a density of 10 ⁴ cells per well in culture medium. After 24 hours, C2C12 cells were incubated with 0.5 mM Cu (ClO ₄ ) ₂ for 30 minutes in culture medium. After washing with PBS to remove residual copper ions, the cells were incubated for 30 minutes at 37 ℃ in 50 μM compound of formula (1).

Human HCT-15 colon cancer cells were seeded in 6-well plates at a density of 10 ⁴ cells per well in culture medium. After 24 hours, C2C12 cells were incubated with 0.5 mM Cu (ClO ₄ ) ₂ for 30 minutes in culture medium. After washing with PBS to remove residual copper ions, the cells were incubated for 30 minutes at 37 ℃ in 50 μM compound of formula (1). Cells incubated with copper ions and compound of formula 1 were imaged by fluorescence microscopy (Stemi 2000-C, ZAISS, Germany).

Experimental Example 2 Imaging of Human Muscle Cells Incubated with Compound of Formula 1 and Mercury Ions

Human muscle biopsy was provided in abundance by Dr. Hyun-Woo Kim (Yonsei University College of Medicine). People muscle fibers Bonavaud, S. et al. (In Vitro Cell Dev . Biol. Anim . 2002, 38, 66-72) was isolated and cultured according to the procedure developed by. Muscle samples were digested at 37 ° C. for 90 minutes in Petri dishes containing 0.2% collagenase (Sigma) in Ham's F10 medium (Gibco) and washed several times with PBS. Most human muscle fibers were isolated from tissues after digestion with enzymes. Single muscle fibers were isolated by repeatedly powdering muscle fragments with a Pasteur pipette with a very large opening. The isolated muscle fibers were finally included in a drop of medium, Ham's F10 medium, plated into a 6-well plate (BioCoat) coated with Matrigel (Collative Biomedical Products, 1 mg / mL), and plated medium (Ham's). It was allowed to adhere for at least 4 hours before adding 10% FBS, 1% chick embryo extract (including CEE, Sera Laboratories International) in F10 medium. The number of cells was determined by trypsinization and incubated in DAEM supplemented with 10% FBS, 1% CEE, 50 units / mL penicillin and 50 g / mL streptomycin.

Human muscle cells are seeded in 6-well plates at a density of 10 ³ cells per well in culture medium (DAEM supplemented with 10% FBS, 50 units / mL penicillin and 50 g / mL streptomycin) and 30 in culture medium. Incubate with 1 mM Cu (ClO ₄ ) ₂ for minutes. After washing with PBS to remove residual copper ions, the cells were incubated in a medium with 50 M of compound of formula 1 for 30 minutes at 37 ° C. Cells incubated with copper ions and the compound of formula 1 were imaged by fluorescence microscopy.

Experimental Example 3 Imaging of Zebrafish Incubated with Compound of Formula 1 and Mercury Ions

Zebrafish were stored at 28 ° C. and maintained in optimal breeding conditions. For mating, male and female zebrafish were preserved in a tank with 12 hours light / 12 hours dark cycles at 28 ° C., and egg laying occurred in the morning with light stimulation. Almost all eggs were fertilized immediately. 5-day-old zebrafish was cultured 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 5-day-old zebrafish was incubated with 1 mM Cu (ClO ₄ ) ₂ for 30 minutes at 28 ° C. in E3 medium. After washing with PBS to remove residual copper ions, zebrafish was further incubated with 50 M of compound of formula 1 for 30 minutes at 28 ° C. Zebrafish were imaged with a fluorescence microscope.

The ability of probes comprising boronic acid-bound Rhodamine derivatives of Formula 1 to detect copper ions in mammalian cells and in vivo has been discussed. The results of fluorescence microscopic analysis of the murine P19 fetal carcinoma cells of Experimental Example 1 show that the rhodamine derivative of Formula 1 passes through the cell membrane and showed a strong fluorescence response to copper ions in the cells (FIG. 3A). . On the other hand, cells treated with only the rhodamine derivative of formula 1 without external copper ions show very weak fluorescence (FIG. 3b).

In addition, in the case of 5 day old zebrafish as in Experimental Example 3, the fluorescence microscopic image shows that copper ions in zebrafish were fluorescently detected by the probe (FIG. 3C). However, zebrafish treated only with probes without external copper ions showed a very weak fluorescence signal in the egg yolk region (FIG. 3D).

The probe responds selectively to copper ions. The chemical sensor of the present invention exhibits the opposite fluorescence change upon complexation with Cu ²⁺ . That is, the mono acid-fluorescence of bound rhodamine derivatives, but significant increase in the presence of Cu ^{2 +,} bis-boronic acid-fluorescence of the bound fluorescein derivative is selectively destroyed by Cu ^{2 +.} The chemical sensors all enables the visual detection of the Cu ^{+ 2.}

Figure 1 shows the selectivity for (a) copper ion bonds, (b) fluorescence and color change, and (c) copper ions of the rhodamine derivative of the present invention.

Figure 2 shows the selectivity to (a) copper ion bonds, (b) fluorescence and color change, and (c) copper ions of the fluorescein derivative of the present invention.

Figure 3 shows the results of detection of living and intracellular copper ions using a probe containing a rhodamine derivative according to an embodiment of the present invention.

Figure 4 shows that the chemical sensor (3μM) of formula (I) to Cu ^{2 +} at pH7.4 at various concentrations in _{20 mM HEPES (5% CH 3} CN) of the appropriate fluorescent pH7.4.

5 is of the formula (1) in _{20 mM HEPES (5% CH 3} CN) of pH7.4, in the presence of excess metal ions (250 eq) in pH7.4 according to the Cu ^{+ 2} (50 equivalent) of chemical sensors (3μM) The change in fluorescence is shown.

Figure 6 is (a) CH ₃ fluorescence change of formula 5 upon addition of Cu ^{2 +} in the CN (at 342 nm herein) and (b) CH ₃ in the formula 5 and the job plot (342 nm of Cu ^{2 +} in the CN where Emission at 416 nm).

FIG. 7 shows the fluorescence spectra of the compound of Formula 2 (2 μM) at 20 mM HEPES (5% DMSO) at pH 7.4 with the addition of various metal ions (100 μM).

Figure 8 illustrates the incubation of cells or organisms with the compound, and Cu ^{2 +} of the formula 1 (a) is a c mice (C2C12) near subspecies cells (myoblast), b) the human muscle cells) is a colon cancer cells (HCT -15), d) rat (3T3-L1) fibroblasts).

9 shows ¹ H NMR (250 MHz) of the compound of Formula 1 in CDCl ₃ .

10 shows ¹³ C NMR (62.5 MHz) of the compound of Formula 1 in CDCl ₃ .

FIG. 11 shows ¹ H NMR (250 MHz) of the compound of Formula 2 in CD ₃ OD. FIG.

FIG. 12 shows ¹³ C NMR (62.5 MHz) of the compound of Formula 2 in CD ₃ OD. FIG.

Claims (6)

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

Where R is B (OH) ₂ . Fluorescein derivatives represented by the following formula (2):  [Formula 2]

Where R is

to be. A method for preparing the rhodamine derivative of claim 1 comprising reacting 2-formylphenylboronic acid and rhodamine hydrazide. A method for preparing the fluorescein derivative of claim 2 comprising condensing 2,7-dichlorofluorescein and 2- (N-methylaminomethyl) phenylboronic acid through a Mannich reaction. . A method for detecting copper ions (Cu ²⁺ ) of a sample separated from the human body using the compound of claim 1 or 2. Claim 1 or in vivo copper ions comprising a compound of claim 2 (Cu ^{+ 2)} detecting probe.

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