KR101322615B1 - Thiol-targeting chemodosimetric fluorescent probe, method for preparing the same, and method for bioimaging of thiol using the same - Google Patents

Abstract

본 발명에 따른 티올 선택성을 갖는 화학정량 형광 표지자는 티올에 대해서 높은 선택성을 나타내며, 우수한 발광 특성을 보이고, 생체 내에서, 특히 간 티올을 직접적이면서도 실시간으로 영상화하는 것을 가능케 한다.The present invention relates to a chemical quantitative fluorescent marker having a thiol selectivity, a method for preparing the same, and an in vivo thiol imaging method using the same, and more particularly, a chemical quantitative fluorescent marker having a thiol selectivity of the formula (1), a method for preparing the same, and a method of using the same. A method for in vivo thiol imaging:
≪ Formula 1 >

Chemistry quantitative fluorescent markers with thiol selectivity according to the present invention exhibit high selectivity for thiols, show good luminescent properties, and enable direct and real-time imaging of liver thiols, particularly in vivo.

Description

Chemistry quantitative fluorescent markers having thiol selectivity, preparation methods thereof, and in vivo thiol imaging methods using the same {thiol-targeting chemodosimetric fluorescent probe, method for preparing the same, and method for bioimaging of thiol using the same}

The present invention relates to a quantitative fluorescent marker having thiol selectivity, a method for preparing the same, and an in vivo thiol imaging method using the same.

Glutathione (GSH) is the highest thiol in the cell, and its concentration in the cell ranges from 1 to 10 mM and plays a very important role in the homeostasis of biological redox systems. Intracellular glutathione / oxidized glutathione (GSH / GSSG) ratio is an important indicator of intracellular redox status, which exceeds 100: 1 under normal physiological conditions, and this abnormality is often associated with many diseases and cancers. ((a) SC Lu, Curr . Top . Cell . Regul . 2000 , 36 , 95-116; (b) TP Akerboom, M. Bilzer, H. Sies, J. Biol . Chem . 1982 , 257 , 4248-4252 ;.. (c) DM Townsend , KD Tew, H. Tapiero, Biomed Pharmacother 2003, 57, 145-155).

The liver supplies GSH to the blood and other organs, such as the kidneys, lungs, colon and small intestine, so it is a very important organ that contributes to maintaining the redox state of the body. When severe oxidative stress, the GSH / GSSG ratio of hepatocytes is 4 Can be reduced to less than 1 (OW Griffith, Free Radic . Biol . Med . 1999 , 27 , 922-935). In addition, interstitial migration of GSH has a profound effect on cancer metastasis to other tissues. Therefore, it is important to grasp the change in the GSH / GSSG ratio in the liver and the quantitative liver thiol content.

Various chemoquantitative fluorescent markers (chemodosimetric) with distinct advantages over other methods in terms of sensitivity, specificity, and ease of use, for the purpose of detecting specific molecules in vivo, particularly for imaging thiols in vivo and ex vivo. fluorescent probes have been developed. However, many fluorescent markers for thiol detection have been studied ((a) PK Pullela, T. Chiku, MJ Carvan, DS Sem, Anal . Biochem . 2006 , 352 , 265-273; (b) N. Shao, J. Jin, H. Wang, J. Zheng, R. Yang, W. Chan, Z. Abliz, J. Am . Chem . Soc . 2010 , 132 , 725-736; (c) X. Chen, Y Zhou, X. Peng, J. Yoon, Chem . Soc . Rev. 2010 , 39 , 2120-2135; (d) L.-L. Yin, Z.-Z. Chen, L.-L. Tong, K.-H. Xu, B. Tang, Chin . J. Anal . Chem . 2009 , 37 , 1073-1081; (e) HS Jung, KC Ko, GH Kim, AR Lee, YC Na, C. Kang, JY Lee, JS Kim, Org . Lett . 2011 , 13 , 1498-1501; (e) HS Jung, JH Han, Y. Habata, C. Kang, JS Kim, Chem . Commun . 2011 , 47 , 5142-5144), and the actual detection of thiols in certain organs such as the liver has not been reported so far.

As a prior art, Korean Patent Laid-Open Publication No. 2011-0090417 discloses a fluorescein derivative having thiol selectivity, which relates to a compound that selectively binds to thiol and exhibits a green fluorescence enhancing effect. In addition, Korean Patent Publication No. 2011-0085270 discloses a two-photon fluorescent probe for detecting thiol in living cells and tissues, which relates to a compound comprising 2-methylamino-6-acetylnaphthalene and disulfide groups. However, no quantitative fluorescent markers have been reported that can be directly imaged in vivo in real-time by inducing endocytic processes recognized by receptors present on the cytoplasmic membrane and undergoing quantitative luminescence in vivo. .

Therefore, given that the regulation of hepatic thiols is important for the maintenance of intracellular redox metabolism, quantitative fluorescence markers that allow for the direct and real-time imaging of hepatic thiols in living organs have significant implications. In addition, it will be a powerful means to directly measure the level of thiol in the liver in healthy and pathological conditions.

The first technical problem to be achieved by the present invention is a chemical quantitative fluorescent marker which shows high selectivity to thiols, shows excellent luminescence properties, and in particular has a thiol selectivity capable of imaging thiols directly and in real time in vivo. To provide.

The second technical problem to be achieved by the present invention is to provide a method for preparing the chemical quantitative fluorescent marker.

The third technical problem to be achieved by the present invention is to provide an in vivo thiol imaging method using the chemical quantitative fluorescent markers.

The present invention provides a chemical quantitative fluorescent marker having a thiol selectivity of Formula 1 to achieve the first technical problem:

In order to achieve the second technical object of the present invention,

There is provided a method of preparing a quantitative fluorescent marker having thiol selectivity comprising the step of preparing a compound of Formula 1 by performing a deacetylation reaction on a compound of Formula 2:

According to an embodiment of the present invention, the compound of formula 2 may be prepared by reacting the compound of formula 3 with triphosgene and 2,2'-dithioethanol:

According to another embodiment of the present invention, the compound of formula 3 may be prepared by reacting a compound of formula 4 with a galactose tetraacetate derivative having an -NH ₂ terminal:

The present invention provides an in vivo thiol imaging method comprising the step of injecting the compound of Formula 1 in vivo to monitor the emitted fluorescence in order to achieve the third technical problem.

Chemistry quantitative fluorescent markers with thiol selectivity according to the present invention exhibit high selectivity for thiols, show good luminescent properties, and enable direct and real-time imaging of liver thiols, particularly in vivo.

1 is a schematic diagram of a process for detecting liver thiol by Compound 1 according to the present invention through endocytosis mediated by ASGP-R into liver cells.
2 is a normalized absorption and emission spectrum for Compound 1 measured under physiological conditions.
3A is a diagram comparing the reactivity of compounds having a thiol group with respect to compound 1 and the reactivity of other compounds having no thiol group, and FIG. 3B is a fluorescence (Flu) and absorption (Abs) photograph of a solution in which compound 1 is not present ( Figures A and C) and fluorescence and absorption pictures (B and D in the figure) of the solution in which compound 1 is present.
FIG. 4 is a graph showing the ratio of the fluorescence intensity at 540 nm (F ₅₄₀ / F ₄₇₃ ) to the fluorescence intensity at 473 nm of Compound 1, with and without GSH, as a function of pH. to be.
5A and 5B are graphs showing the change in fluorescence intensity of Compound 1 with time in the presence of GSH.
6 is a graph showing the change in fluorescence intensity of Compound 1 with time in the presence of Cys.
FIG. 7 shows possible reaction mechanisms of Compound 1 with thiol groups.
8a and 8b are graphs showing changes in fluorescence spectra of Compound 1 according to changes in concentration of GSH (0˜1.0 mM).
9 are photographs showing confocal microscopic images of HepG2, C2C12, HaCat and N2a cells treated with Compound 1 (10 μM) according to the present invention and Compound 6 (1.0 μM) according to the comparative example.
FIG. 10 shows confocal microscopy images of HepG2 cells treated with Compound 1 (10 μM) according to the present invention and Compound 6 (1.0 μM) according to a comparative example at various biotin concentration ranges from 0 to 10 ⁻⁸ M. Photos.
FIG. 11 shows confocal microscopy images of HepG2 cells treated with Compound 1 (10 μM) according to the present invention and Compound 6 (1.0 μM) according to a comparative example at various concentration ranges of N -ethylmaleimide (NEM). One picture.
12 are photographs showing fluorescence images of compounds 1 and 6 for liver, lung, kidney, pancreas and heart.

In the present invention, there is provided a quantitative fluorescent marker having thiol selectivity of formula (I):

≪ Formula 1 >

The compound of Formula 1 has a disulfide bond showing a redox reaction specific to thiol, and a galactose residue as a liver cell targeting unit, and due to this property, it is possible to image liver thiol in real time, particularly in vivo. The disulfide bond may react with GSH to cause a change in fluorescence, whereas the galactose residue is recognized by the desialalo glycoprotein receptor (ASGP-R) on the plasma membrane of liver cells, indicating that Compound 1 is endocytosis. Make it possible. As shown in FIG. 1, Compound 1 easily penetrates into hepatocytes by endocytosis mediated by ASGP-R, and thus, disulfide bonds are separated by GSH in hepatocytes, indicating a change in fluorescence. In other words, in the present invention, by introducing a hydrophilic galactose group having a polarity into a fluorescent marker which is generally hydrophobic, excellent solubility and selective permeability into liver cells can be simultaneously achieved.

In addition, Compound 1 employs 4-aminonaphthalimide as a skeleton of the fluorescent marker, which has excellent spectroscopic properties such as internal charge transfer (ICT) efficiency, quantum yield, and structural flexibility for various modifications, and disulfide here. By introducing a carbamate group having a bond, the disulfide bond is separated by reaction with thiol, and then subjected to a separation reaction, whereby the emission spectrum (473 nm → 540 nm) and absorption spectrum (367 nm → 428 nm) of Compound 1 are obtained. Primary amine is formed which causes a large change in).

As described in the Examples below, Compound 1 according to the present invention exhibits excellent water solubility, has a ratio equivalent signal change characteristic, and has a higher selectivity to thiol than other non-thiol based amino acids and biologically important metal ions. Its pH range is wide. Confocal microscopy of HepG2, C2C12, HaCat, and N2a cell lines also showed that Compound 1 undergoes a redox fluorescence reaction at 540 nm, following a redox reaction induced by thiol after selective penetration into HepG2 cells. It was confirmed to exhibit characteristics.

The present invention also provides a process for the preparation of a quantitative fluorescent marker having thiol selectivity comprising the step of preparing a compound of Formula 1 by performing a deacetylation reaction on a compound of Formula 2:

(2)

As the deacetylation reaction, a general reaction of dissociating the acetyl group bound to O by hydrolysis to regenerate the OH group may be used, for example, may be performed using a cation exchange resin in the presence of NaOMe / MeOH. .

Preferably, the compound of formula 2 may be prepared by reacting a compound of formula 3 with triphosgene and 2,2'-dithioethanol:

<Formula 3>

The reaction is for introducing a carbamate group having a disulfide bond into a fluorescent marker skeleton on which 4-aminonaphthalimide is based, and the disulfide bond introduced is separated by reaction with a thiol in vivo and consequently, a primary Producing an amine causes a change in the emission spectrum of compound 1.

Preferably, the compound of formula 3 may be prepared by reacting a compound of formula 4 with a galactose tetraacetate derivative having an -NH ₂ terminal:

&Lt; Formula 4 >

The reaction is for introducing galactose residues as liver cell targeting units into the fluorescent marker skeleton upon which 4-aminonaphthalimide is based, wherein the introduced galactose residues are desialalo glycoprotein receptor (ASGP-) on the plasma membrane of liver cells. Recognized by R) enables compound 1 to be endocytosis. In addition, since the galactose residues exhibit hydrophilic properties, Compound 1 also contributes to the expression of excellent solubility in the cellular environment.

Meanwhile, the compound of formula 4, which is a naphthalimide derivative (prepared by reducing the compound of formula 5), the compound of formula 5 and galactose tetraacetate derivatives for the introduction of galactose residues may be prepared by a conventionally known method. ((A) LGF Patrick, A. Whiting,Dys Pigments 2002,55, 123-132; (b) D. Yuan, R. G. Brown, J. D. Hepworth, M. S. Alexiou, J. H. P. Tyman,J. Heterocyclic Chem . 2008,45, 397-404; (c) H. Sato, E. Hayashi, N. Yamada, M. Yatagai, Y. Takahara,Bioconjugate Chem . 2001, 12, 701-710).

For reference, the following scheme shows an exemplary schematic reaction scheme for preparing the compound of Formula 1 from the compound of Formula 5.

The present invention also provides a method for in vivo thiol imaging comprising injecting the compound of Formula 1 in vivo and then monitoring the emitted fluorescence.

The compound 1 according to the present invention can be easily injected into the body by intravenous injection or the like, and the injected compound 1 is selectively absorbed by the tissue in vivo, in particular the liver tissue. Subsequently, Compound 1 absorbed into the liver tissue exhibits red shifted fluorescence due to a redox reaction induced by thiol, and the in vivo thiol imaging method according to the present invention provides a thiol in a living sample by quantitatively monitoring such emission fluorescence. Numerical values can be imaged directly and in real time. Thus, Compound 1 according to the present invention can be used for selective in vivo delivery of useful molecules, in addition to use for such imaging.

Hereinafter, the present invention will be described in more detail by way of examples, but the following examples are not intended to limit the scope of the present invention but are described for the purpose of helping understanding of the present invention.

Production example. Preparation of Fluorescent Markers According to the Invention

According to the following preparation example was prepared a compound of formula 1, a fluorescent marker according to the present invention.

&Lt; Formula 1 >

Preparation Example 1.1. Synthesis of Compound (5)

The compound of Formula 4, the compound of Formula 5 and acetylated galactosamine (AcGal-NH ₂ ) was prepared by a conventionally known method ((a) LGF Patrick, A. Whiting, Dyes Pigments 2002 , 55 , 123-132; (b) D. Yuan, RG Brown, JD Hepworth, MS Alexiou, JHP Tyman, J. Heterocyclic Chem . 2008 , 45 , 397-404; (c) H. Sato, E. Hayashi, N. Yamada, M. Yatagai, Y. Takahara, Bioconjugate Chem . 2001 , 12 , 701-710).

Preparation Example 1.2 Synthesis of Compound of Chemical Formula 3

A mixture of compound 4 (0.9 g, 3.1 mmol), EDCI (0.7 g, 3.1 mmol) and DMAP (0.8 g, 6.2 mmol) dissolved in anhydrous DMF was stirred for 10 minutes at room temperature in a nitrogen atmosphere. Then, to the mixture AcGal-NH ₂ (2.0 g, 3.1 mmol) was added. The resulting reaction mixture was stirred overnight, the solvent was evaporated, CH ₂ Cl ₂ (100 mL) and water (100 mL) were added, and the organic layer formed was separated. The CH ₂ Cl ₂ layer was dried over anhydrous MgSO ₄ , the solvent was removed, and the crude product obtained was purified on silica gel using CH ₂ Cl ₂ / MeOH (v / v, 97: 3) as eluent to give a yellow oil. Compound 3 was obtained (1.7 g, 72%).

IR (Precipitate of DCM solution was analyzed on NaCl plate, cm ⁻¹ ): 3370, 3250, 2920, 2870, 1750, 1640, 1580. ESI-MS m / z (M ⁺ ) calculated 745.7, measured 768.7 (M + Na ⁺ ). ¹ H NMR (CDCl ₃ , 400 MHz): δ 8.45 (d, 1H, J = 7.2 Hz); 8.25-8.18 (m, 2H); 7.49 (t, 1H, J = 8.0 Hz); 6.76 (d, 1 H, J = 8.2 Hz); 6.66 (t, 1H, J = 4.40 Hz); 5.80 (s, 2H); 5.40 (d, 1H, J = 3.3 Hz); 5.31-5.19 (m, 1H); 5.05-5.01 (m, 1H); 5.56 (d, 1 H, J = 4.6 Hz); 4.47 (d, 2H, J = 7.2 Hz); 4.19-3.71 (m, 3H); 3.77-3.47 (m, 12H); 2.70 (t, 2H, J = 7.2 Hz); 2.13 (s, 3H); 2.07 (s, 3H); 2.04 (s, 3H). 2.00 (s, 3H). ¹³ C NMR (CDCl ₃ , 100 MHz): 171.3, 170.6, 169.8, 164.7, 164.0, 151.2, 134.2, 131.5, 129.9, 128.3, 122.4, 119.9, 110.1, 109.2, 101.5, 71.0, 70.8, 70.3, 69.8, 69.0 , 67.3, 61.5, 39.5, 36.9, 35.3, 20.9 ppm.

Preparation Example 1.3 Synthesis of Compound of Chemical Formula 2

DIPEA (0.3 mL, 1.4 mmol) was added dropwise to a mixture of compound 3 (0.3 g, 0.4 mmol) and triphosgene (0.3 g, 0.8 mmol) dissolved in 20 mL of anhydrous toluene. The resulting solution was refluxed for 3 hours. After cooling to room temperature, the reaction mixture was flushed with nitrogen gas. After removal of the unreacted tripphosgene gas, 2,2'-dithiodiethanol (0.3 g, 2.0 mmol) dissolved in distilled CH ₂ Cl ₂ (5 mL) was added to the mixture. After stirring the obtained reaction mixture overnight, the solvent was evaporated, CH ₂ Cl ₂ (100 mL) and water (100 mL) were added, and the organic layer was separated. The CH ₂ Cl ₂ layer was dried using anhydrous MgSO ₄ . After removal of the solvent, the resulting crude product was purified on silica gel using ethyl acetate / MeOH (v / v, 98: 2) as eluent to afford compound 2 in yellow oil (150.0 mg, 60%). IR (Precipitate of DCM solution was analyzed on NaCl plate, cm ⁻¹ ): 3342, 3250, 2919, 1650, 1538. ESI-MS m / z (M ⁺ ) calculated 925.3, measured 924.4 (MH ⁺ ). ¹ H NMR (CDCl ₃ , 400 MHz): δ 8.55 (s, 1H); 8.39-8.28 (m, 3H); 8.11 (d, 1H, J = 8.2 Hz); 7.55 (t, 1H, J = 8.0 Hz); 6.79 (t, 1H, J = 4.40 Hz); 5.38 (d, 1 H, J = 3.1 Hz); 5.22-5.17 (m, 1H); 5.03-4.99 (m, 1H); 4.56-4.50 (m, 3H); 4.41 (t, 2H, J = 7.1 Hz); 4.14-3.67 (m, 3H); 3.66-3.46 (m, 12H); 3.08 (t, 2H, J = 6.4 Hz); 2.96 (t, 2H, J = 6.0 Hz); 2.69 (t, 2H, J = 7.1 Hz); 2.14 (s, 3H); 2.06 (s, 3H); 2.03 (s, 3H); 1.97 (s, 3H). ¹³ C NMR (CDCl ₃ , 100 MHz): 171.2, 170.6, 169.8, 164.1, 163.6, 153.6, 139.9, 132.4, 131.3, 128.7, 127.5, 126.4, 123.2, 122.7, 117.2, 101.5, 71.0, 70.8, 70.3, 69.8 , 69.0, 67.2, 64.0, 63.7, 61.5, 60.6, 41.8, 39.5, 37.7, 37.1, 34.8, 39.9, 20.8 ppm.

Preparation Example 1.4 Synthesis of Compound of Chemical Formula 1

To compound 2 (60.0 mg, 0.06 mmol) dissolved in MeOH (2 mL) was added sodium methoxide (0.5 M solution in MeOH, 129 μl). The reaction mixture obtained was stirred at room temperature until the starting material disappeared. Cation exchange resin (H ⁺ ) was added to the resulting solution, neutralized, filtered and washed with MeOH, and the solvent was removed in vacuo. The obtained solid was recrystallized in DCM to give compound 1 as a yellow solid (30.0 mg, 61%).

Mp 109-110 ° C. IR (Precipitate of MeOH solution was analyzed on NaCl plate, cm ⁻¹ ): 3340, 2920, 1650, 1540. ESI-MS m / z (M ⁺ ) calculated 757.2, measured 756.3 (MH ⁺ ). ¹ H NMR (CD ₃ OD, 400 MHz): δ 8.56-8.49 (m, 3H); 8.22 (d, 1 H, J = 8.2 Hz); 7.81-7.78 (m, 1H); 4.51 (t, 2H, J = 6.4 Hz); 4.41 (t, 2H, J = 7.0 Hz); 4.18 (d, 1 H, J = 7.5 Hz); 3.95-3.30 (m, 20H); 3.08 (t, 2H, J = 6.5 Hz); 2.88 (t, 2H, J = 6.7 Hz); 2.60 (t, 2H, J = 7.0 Hz). ¹³ C NMR (CDCl ₃ , 100 MHz): 173.8, 165.6, 165.0, 155.8, 142.2, 133.1, 132.4, 130.0, 129.9, 127.6, 125.7, 124.0, 119.5, 118.9, 105.1, 76.7, 74.8, 72.5, 71.3, 71.0 , 70.5, 70.2, 69.4, 64.7, 62.5, 61.2, 42.2, 40.4, 38.3, 38.1, 35.7 ppm.

Comparative Example

As a comparative example, a compound of Formula 6, which is a structural analogue in which galactose is absent, was prepared.

A compound of formula 7 (0.2 g, 0.7 mmol) dissolved in 20 mL of toluene (LGF Patrick, A. Whiting, Dyes Pigments 2002 , 55 , 123-132 and D. Yuan, RG Brown, JD Hepworth, MS Alexiou, JHP Tyman, J. Heterocyclic Chem . DIPEA (0.5 mL, 2.5 mmol) was added dropwise to a mixture of Triphosphene (0.4 g, 1.4 mmol) and triphosphene (produced by the method described in 2008 , 45 , 397-404). The resulting solution was refluxed for 3 hours, cooled to room temperature and the reaction mixture was flushed with nitrogen gas. After removal of the unreacted triphosgene gas, 2,2'-dithiodiethanol (0.6 g, 3.5 mmol) dissolved in CH ₂ Cl ₂ (5 mL) was added to the mixture. The resulting reaction mixture was stirred overnight, then the solvent was evaporated, CH ₂ Cl ₂ (100 mL) and water (100 mL) were added and the organic layer was separated. The CH ₂ Cl ₂ layer was dried using anhydrous MgSO ₄ . After removing the solvent, the obtained crude product was purified on silica gel using ethyl acetate / hexane (v / v, 3: 2) as eluent to give compound 6 as a yellow solid (200.0 mg, 60%).

Mp 123-124 ° C. IR (Precipitate of DCM solution was analyzed on NaCl plate, cm ⁻¹ ): 3430, 2910, 1640, 1360, 1260. ESI-MS m / z (M ⁺ ) calculated 448.1, measured 447.1 (MH ⁺ ). ¹ H NMR (CDCl ₃ , 400 MHz): δ 8.62-8.56 (m, 2H); 8.34 (d, 1 H, J = 8.2 Hz); 8.25 (d, 1 H, J = 8.5 Hz); 7.82 (br, 1 H); 7.75 (t, 1H, J = 8.0 Hz); 4.56 (t, 2H, J = 6.3 Hz); 4.16 (d, 2H, J = 7.5 Hz); 3.96 (t, 2H, J = 5.6 Hz); 3.06 (t, 2H, J = 6.3 Hz); 2.96 (t, 2H, J = 5.8 Hz); 2.42 (br, 1 H); 1.74-1.67 (m, 2H); 1.47-1.39 (m, 2H); 0.97 (t, 3H, J = 7.3 Hz). ¹³ C NMR (CDCl ₃ , 100 MHz): 164.1, 163.6, 152.9, 138.8, 132.4, 131.2, 128.8, 126.6, 126.1, 123.3, 123.0, 117.9, 116.9, 63.8, 60.5, 41.5, 40.2, 37.4, 30.2, 20.4 , 13.8 ppm.

Example 1. Spectral Analysis

GSH was added under physiological conditions (PBS buffer, pH 7.4, 37 ° C.) to investigate the spectroscopic properties of Compound 1 by observing changes in UV / vis and fluorescence spectra. In the absence of GSH, Compound 1 showed broad absorption and emission spectra near 367 nm and 473 nm, respectively. This large Stokes shift (Δλ = 106 nm) shows that the aminonaphthalimide group of Compound 1 has an electron donor and acceptor that exhibits strong ICT (intramolecular charge transfer) characteristics (FIG. 2). ).

FIG. 3A is a comparison of the reactivity of compounds having a thiol group, for example glutathione (GSH), with respect to 1.0 μM Compound 1 and other compounds having no thiol group, for example, valine. In addition, Figure 3b is a fluorescence (Flu) and absorption (Abs) picture (A and C in the figure) of the solution without the compound 1, and a fluorescence and absorption picture (B and D in the figure) of the solution in which the compound 1 is present Figure is a diagram. As can be seen in Figure 3a, 5.0 mM GSH was added to the solution of Compound 1 to increase the fluorescence intensity, and the maximum wavelength of the absorption and fluorescence spectra was 428 nm (λ _abs = 61 nm) and 540 nm (λ _{em, respectively).} = 67 nm) with significant red shift. Also referring to FIG. 3A, compounds having various thiols, but not GSH, such as cysteine (Cys), homocysteine (Hcy), 2-aminoethanethiol (AET), 2-mercaptoethanol (ME) and dty Similar results were obtained with the addition of otreitol (DTT) and the like. However, this change was not observed for non-thiol based amino acids or biologically essential metal ions other than valine.

In addition, the effect of pH on the separation of disulfide bonds by GSH was investigated. 4 shows the ratio of fluorescence intensity at 540 nm (F ₅₄₀ / F ₄₇₃ ) to fluorescence intensity at 473 nm of Compound 1 (1.0 μM) with and without GSH (5.0 mM). , graph as a function of pH (each point was measured after 3 hours at λ _ex = 428 nm at 37 ° C). Referring to FIG. 4, Compound 1 was stable in the pH 2-10 range, but easily reacted with GSH in the biological pH range (5-10). Thus, it can be seen that Compound 1 can detect intracellular thiols, especially the highest content of GSH, without affecting other biological analytes and pH.

5A and 5B show changes in fluorescence intensity of Compound 1 with time in the presence of 5.0 mM GSH. When GSH was added to the solution of Compound 1, the emission intensity at 473 nm temporarily increased and a new emission band (green arrow) appeared at 540 nm at 16 minutes. Afterwards, the emission intensity at 473 nm gradually decreased, the emission intensity at 540 nm was enhanced (red arrow), and a clear iso-absorption point appeared at 492 nm. After 100 minutes, the fluorescence intensity did not change anymore. Similar results were also observed when Cys was added to the solution of Compound 1 (see FIG. 6). This two fluorescence intensities indicate that the intermediate is present before the final compound is obtained. That is, FIG. 7 shows a possible reaction mechanism between Compound 1 and a thiol group. Referring to FIG. 7, Intermediate A is formed by separation reaction of disulfide bond of Compound 1, followed by slow intramolecular cyclization and adjacent carbamate. It can be seen that the red shifted aminonaphthalimide (B in the figure) is formed by separation of the bond.

8a and 8b show the change in fluorescence spectrum of compound 1 according to the change in concentration of GSH (0 ~ 1.0 mM). The fluorescence intensity ratio (F ₅₄₀ / F ₄₇₃ ) increased from 0.7 to 6.7 (10-fold), showing an excellent linear relationship ( R = 0.99) with respect to the concentration of GSH (0-0.05 mM) (see Figure 8b). From these results, it can be seen that the compound 1 can be used to detect the concentration of GSH that maintains a high micromolar level under physiological conditions, which can be quantitatively measured by changing the ratio of fluorescence intensity. In particular, it is meaningful in that the measurement range includes the physiological concentration level of GSH.

Example 2. Detection of Thiols in Cultured Cells

To identify galactose groups that induce Compound 1 into hepatocytes during receptor mediated endocytosis, Compound 1 and Compound 6 were incubated with various cells, each containing HepG2. It has been reported that the degree of desialalo glycoprotein receptor expression in HepG2 cells is low and can be induced by biotin treatment ((a) AM Pujol, M. Cuillel, O. Renaudet, C. Lebrun, P. Charbonnier, D. Cassio, C. Gateau, P. Dumy, E. Mintz, P. Delangle, J. Am . Chem . Soc . 2011 , 133 , 286-296; (b) JC Collins, E. Paiettall, R. Green, AG Morell, RJ Stockert, J. Biol. Chem . 1988 , 263 , 11280-11283).

9 shows confocal microscopic images of HepG2, C2C12, HaCat and N2a cells treated with Compound 1 (10 μM) and Compound 6 (1.0 μM). Cell images were obtained using a luminescence filter with an excitation wavelength of 458 nm and a band-path of 530-600 nm, with cells at 37 ° C. for 2 days in medium containing 10 ⁻⁸ M biotin. Incubated, washed and incubated with PBS solution containing Compound 1 or 6 for 20 minutes. Bottom pictures of the second row and the fourth row are pictures superimposed fluorescence images with the non-confocal control.

As can be seen in Figure 9, the fluorescence intensity of Compound 1 in HepG2 cells depends on the presence of biotin in the medium, similar changes were not observed in other cells. Unlike compound 1, compound 6 shows strong fluorescence intensity regardless of cell type or presence of biotin in the medium. This relationship of compounds 1 and 6 was also confirmed in various biotin concentration ranges from 0 to 10 ⁻⁸ M (see FIG. 10).

In order to confirm whether the change in fluorescence intensity of compound 1 or 6 is according to the reaction shown in FIG. 7, the cells are treated with N -ethylmaleimide (NEM) which reacts well with thiol groups, Fluorescence intensity was measured. As can be seen in FIG. 11, the fluorescence of compounds 1 and 6 depends on the intracellular thiol concentration. Taken together, it can be seen that Compound 1 is able to specifically penetrate into HepG2 cells, which is thought to be due to endocytosis by desialalo glycoprotein receptors, thereby separating SS binding and It can be inferred that the fluorescence intensity at 540 nm increases due to the formation of primary amines.

On the other hand, in order to confirm whether imaging of liver thiol by Compound 1 is possible in the body of the animal, Compound 1 was injected into the tail vein of the rat. 1.5 hours after injection, rats were anesthetized, sacrificed and sacrificed cervically. Each organ was then removed quickly and washed with cold PBS buffer. In order to confirm the intratracheal distribution of compound 1, confocal microscopy was used to obtain fluorescence images of each tissue section (thickness 10 μm). In addition, mice were treated with Compound 6 without galactose residues for comparison of hepatocyte targeting ability, and normal mice not injected with Compound 1 or Compound 6 were used as controls.

12 shows fluorescence images of compounds 1 and 6 for liver, lung, kidney, pancreas and heart. Referring to FIG. 12, Compound 1 selectively accumulates in the liver (panel b) to allow intramolecular thiol imaging by chemoquantitative reaction, while other organs (panels e, h, k and n) have almost no fluorescence emission. It can be seen that there is no. On the other hand, it was confirmed that Compound 6 accumulates in a significant amount in the liver (panel c) and kidney (panel i). These results confirm that Compound 1 is selectively absorbed into liver tissue by endocytosis mediated by ASGP-R, resulting in red shifted fluorescence due to redox reactions by thiols. Thus, it can be seen that Compound 1 is very useful for detecting liver thiol in vivo.

Claims (5)

Chemistry quantitative fluorescent markers with thiol selectivity of Formula 1:
&Lt; Formula 1 >

A method of preparing a quantitative fluorescent marker having thiol selectivity comprising the step of preparing a compound of Formula 1 by performing a deacetylation reaction on a compound of Formula 2:
(2)

&Lt; Formula 1 >

The method of claim 2, wherein the compound of Chemical Formula 2 is prepared by reacting a compound of Chemical Formula 3 with triphosgene and 2,2'-dithioethanol.
(3)

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