KR101164556B1 - 2-Photon Fluorescent Probe for Thiols in Live Cells and Tissues - Google Patents

Abstract

(화학식에서 X = S(황))
본 발명에 따른 티올 감지용 이광자 프로브는 생체 세포 및 생체 조직 내 90 내지 180㎛ 깊이에 존재하는 티올을 높은 선택성으로 감지할 수 있다. 또한, 종래의 프로브들에 비하여 형광 강도가 약 10배 증가되고, pH에 대한 의존성이 낮으므로 티올 감지를 위한 프로브로서 적용 범위가 매우 넓다.The two-photon fluorescent probe of the present invention includes 2-methylamino-6-acetylnaphthalene as a reporter, a disulfide group as a thiol reaction site, and may be represented by the following Chemical Formula 1.
Formula 1

Where X = S (sulfur)
The thiol-sensing two-photon probe according to the present invention can detect a thiol present at a depth of 90 to 180 μm in living cells and tissues with high selectivity. In addition, since the fluorescence intensity is increased by about 10 times compared to the conventional probes, and the dependency on pH is low, the application range is very wide as a probe for thiol detection.

Description

2-Photon Fluorescent Probe for Thiols in Live Cells and Tissues}

The present invention relates to two-photon fluorescent probes for detecting thiols in living cells and tissues.

Thiols are components of many amino acids and peptides, such as Cysteine (Cys) and glutathione (GSH). Glutathione is the most abundant cellular thiol with a concentration of 1 to 15 mM (Ref. 1). Glutathione reduces the disulfide bonds in cytoplasmic proteins to thiols in the course of oxidation to GSSG, and can be reproduced from GSSG by the enzyme glutathione reductase. In healthy cells and tissues, at least 90% of total glutathione is present in reduced form (GSH) and the increased ratio of GSSG to GSH is considered an indication for oxidative stress (Ref. 2). Various fluorescent probes have been developed for detecting thiols in biological systems (Ref. 3). Most of them use fluorophores, rhodamines or green fluorescent proteins as fluorophores and functional groups that react with thiols to produce strong luminescent products. However, the use of such probes with one-photon microscopy (OPM) results in relatively short excitation wavelengths (<525 nm) that limit their use in tissue imaging due to their thin transmission depth (<80 μm). Require.

In order to detect thiols deeply located inside biological tissues, it is essential to use two-photon microscopy (TPM). Two-photon microscopy using two near-infrared photons as excitation sources has the advantage that increased transmission depth (approximately 500 μm), local excitation and extension observations are allowed, thereby allowing tissue imaging (Ref. 4). . Recently, two-photon microscopic images of cells labeled with 8-oxo-acenaphthopyrrole derivatives have been reported. These probes change the λ _max from 430 nm to 580 nm when cysteine is added, and the strength with the mold increases by 75 times when an additional homocysteine (40 equivalents) is added, so that thiol can be detected by visual change. To be. However, the two-photon cross section is so small (about 10 GM) that a high laser power (50 mV) is required to excite the fluorescence source. Moreover, there are no reports related to two-photon probes that can detect thiols located deep within intact tissue. Therefore, the present invention discloses a two-photon probe comprising 2-methylamino-6-acetylnaphthalene as a reporter and a disulfide group as a thiol reaction site. . The two-photon probe according to the present invention can detect thiols in living cells and tissues at a depth of 90 to 180 μm, and thus can be applied to synthesis and biological imaging applications.

The first problem to be solved by the present invention is to provide a two-photon probe that can effectively detect the thiol in living cells and tissues.

The second problem to be solved by the present invention is to provide a method for manufacturing the two-photon probe.

The third object of the present invention is to provide a method for detecting thiols using the two-photon probe.

The present invention provides a two-photon probe for the detection of the thiol represented by the formula (1) to solve the first problem.

Formula 1

Where X = S (sulfur)

The present invention provides a method for producing a two-photon probe of claim 1 prepared by the following scheme 1 to solve the second problem.

Scheme 1

, a: 피리딘, 디메틸포름아미드, 4-디메틸아민피리딘, b: 트리플루오로아세틱 에시드, 디클로로메탄)(One:

, a: pyridine, dimethylformamide, 4-dimethylaminepyridine, b: trifluoroacetic acid, dichloromethane)

The present invention provides a method for detecting thiol in living cells or tissue using the two-photon probe to solve the third problem.

According to one embodiment of the invention, the depth of the living cells or tissue tissue is detected thiol is preferably from 90 to 180㎛.

According to another embodiment of the present invention, the living cell may be a hema cell or a rat hippocampal cell.

According to one embodiment of the invention, the biological tissue may be mitochondria or nucleus.

The thiol-sensing two-photon probe according to the present invention can detect a thiol present at a depth of 90 to 180 μm in living cells and tissues with high selectivity. In addition, since the fluorescence intensity is increased by about 10 times compared to the conventional probes, and the dependency on pH is low, the application range is very wide as a probe for thiol detection.

(A), (b), (c) and (d) of FIG. 1 show the normalization of ASS (a, c) and compound 1 (b, d) of 1,4-dioxane, DMF, EtOH and H ₂ O. Normalized absorbption and emission spectra are shown, respectively.
Figure 2 summarizes the photophysical properties of ASS and Compound 1 in various solutions (Solvent).
3 (a), (b) and (c) show two-photon fluorescence over time for the reaction of ASS (5 uM) and 2-aminoethanethiol (10 mM) in a MOPS buffer. Change in intensity is plotted against ln (F _∞ -F) vs. time for the reaction of ASS (5 uM) and 2-aminoethanethiol (10 mM) in MOPS buffer for single- and two-photon processes. Plots of ln (F _∞ -F) versus time for the reaction of ASS (uM) and various 2-aminoethanethiol concentrations (10-100 mM) in buffer, respectively.
4 (a), (b) and (c) show HPLC traces for the reaction product between ASS, Compound 1 and ASS and 2-aminoethanethiol.
5 (a), (b), (c) and (d) show the single-photon fluorescence intensity over time for the reaction of ASS (5 uM) and 2-AET (10 mM) in MOPS buffer. Changes, plots for k _obs versus (2-AET) concentrations, fluorescence reaction of ASS against GSH, Cys, DTT, 2-ME and 2-AET and ASS, ASS + 2-AET and compounds in MOPS buffer The two-photon excitation spectrum of 1 is shown respectively.
FIG. 6 shows the fluorescence response of ASS to GSH and other analytes.
FIG. 7 shows the effect of pH on the single-photon fluorescence intensity of the reaction product (white circle) between ASS and 2aminoethanethiol in ASS (black square) and MOPS buffer.
8A shows TPMs (a to c) and bright regions (d) of HeLa cells incubated with ASS (3 uM), respectively.
8B shows bright regions and TMP images of HeLa cells labeled 3 uM ASS, respectively.
9 shows TPM and OPM images of HeLa cells labeled together with ASS (3 uM), mitotracker (1 uM) and Hoechst (1 uM), respectively.
FIG. 10 shows light regions and TPM images of fresh rat hippocampal slices stained with 20 uM ASS at a depth of about 120 um.
FIG. 11 shows TMP images of fresh rat hippocampal slices stained with 20 uM ASS.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The two-photon fluorescent probe of the present invention includes 2-methylamino-6-acetylnaphthalene as a reporter, a disulfide group as a thiol reaction site, and may be represented by the following Chemical Formula 1.

Formula 1

Where X = S (sulfur)

The two-photon probe (ASS) according to the present invention can be synthesized from Scheme 1 below, and the contrasting probe (ACC) can be synthesized through the process shown in Scheme 2.

Scheme 1

Scheme 2

What is indicated by 1 to 6 in Scheme 1 and Scheme 2 represents compound 1 to compound 6. Compound 1 represents 2-methylamino-6-acetylnaphthalene and ASS or ACC means two-photon probe. A is pyridine, dimethylformamide (DMF) and 4-dimethylaminepyridine (DMAP), and b is trifluoroacetic acid (TFA) and dichloromethane. , CH ₂ Cl ₂₎ .

Compound 1 is represented by the following formula (2).

Formula 2

The products of Scheme 1 and Scheme 2 are represented by the following formula (2).

Formula 2

In Formula 2, when X = S (sulfur), it becomes a two-photon probe (ASS) according to the present invention, and when X = C (carbon), it becomes a two-photon probe and the control two-photon probe (ACC). The two-photon probe according to the present invention is characterized by having a disulfide group in which X = S.

The application process of the two-photon probes ASS and ACC will be described.

The mechanisms presented below show the structure of ASS, ACC, Compound 1 and Compound 2 and the mechanism of thiol-induced oxidation of ASS.

mechanism

The mechanism presented below is described in detail.

1 (a), (b), (c) and (d) show ASS (a, c) in 1,4-dioxane (1,4-dioxane), dimethylformamide (DMF), ethanol and water And Normalized absorbption and emission spectra of Compound 1 (b, d), respectively. FIG. 2 summarizes the photophysical properties of ASS and Compound 1 in various solvents. The emission spectrum is expressed in normalized fluorescence intensity. Absorption spectra were recorded on a Hewlett-Packard 8453 diode array spectrophotometer, and fluorescence spectra were obtained with an Amico-Bowman series 2 luminescence spectrometer using a 1-cm standard quartz cell. Fluorescence quantum yield was determined by Demas, JN; Crosby, GA J. Phys . Chem . Determined using coumarin 307 and rhodamine B according to the methods disclosed in 1971, 75 , 991. In Figure 2 [a] the numbers in parentheses are Reichardt, C. Chem. Rev. Normal experimental parameters of solution polarity are shown as disclosed in 1994, 94, 2319-2358, the single-photon absorption and emission spectra of [b] are in nm, and [c] is the fluorescent photon product. The margin of error is 15%.

The solubility of water in the ASS determined by the fluorescence method is from 4.0 to 8.0 μM, preferably from 5.0 to 7.0 uM and most preferably from 6.0 uM, which is sufficient to stain the cells. Absorption and emission spectra of ASS were not detected in solution polarity whereas absorption and emission spectra for Compound 1 showed large red transitions in order of 1,4-dioxane <ethanol <water (Figs. 1 b and c and Fig. 2). Reference). The effect was that the emission spectrum (76 nm) was larger than the absorption spectrum (12 nm), which showed the utility of compound 1 by polarity detection.

(A), (b) and (c) of FIG. 3 show two-photon fluorescence intensities over time for the reaction of ASS (5 uM) and 2-aminoethanethiol (10 mM) in MOPS buffer. Change in photon fluorescence intensity is plotted against ln (F _∞ -F) vs. time for the reaction of ASS (5 uM) and 2-aminoethanethiol (10 mM) in MOPS buffer for single and two-photon processes and MOPS buffer Plots for ln (F _∞ -F) vs. time for the reaction of ASS (uM) with various 2-aminoethanethiol concentrations (10-100 mM).

4 (a), (b) and (c) show HPLC traces for the reaction product between ASS, Compound 1 and ASS and 2-aminoethanethiol. HPLC conditions are detected at 1.0 mL / min flow rate at 5% A to 90% A and 307 nm over 20 minutes. Solution A is acetonitrile containing 0.1% TFA and solution B is water containing 0.1% TFA.

5 (a), (b), (c) and (d) show the change in single-photon fluorescence intensity over time for the reaction of ASS (5 uM) and 2-AET (10 mM) in a MOPS buffer. , plot the concentrations of k _obs vs. (2-AET), fluorescence response of ASS for GSH, Cys, DTT, 2-ME and 2-AET and ASS, ASS + 2-AET and Compound 1 in MOPS buffer. Each of the two-photon excitation spectra is shown. In (c), the white and black bars represent the fluorescence intensity of ASS before the addition of thiol and at 1 hour after the addition of thiol, respectively.

6 shows fluorescence response of ASS to GSH and other analytes in MOPS buffer. Other analytes were 30 equivalents, 1, Ca ²⁺ 2, K ⁺ 3, Na ⁺ 4, Fe ³⁺ 5, Zn ²⁺ 6, Mg ²⁺ 7, Glu 8, Ser 9, Val 10, Met 11, Ala 12, Ile 13, H ₂ O ₂ 14, GSH and white and black bars show the fluorescence intensity of ASS (5 uM) in the presence of various analytes before and 3 hours after addition of GSH.

FIG. 7 shows the effect of pH on the single-photon fluorescence intensity of the reaction product (white circle) between ASS and 2-aminoethanethiol in ASS (black square) and MOPS buffer. The reaction of ASS (5 uM) and 2-aminoethanol thiol (10 mM) proceeds for 2 hours in a buffer solution (2 mL) at room temperature and the excitation wavelength is 365 nm.

2-aminoethanethiol (2-AET, 10 mM) is added to a buffer solution of 3- (N-morpholino) propane sulfonic acid (MOPS) buffer solution (30 mM, 100 mM KCl, 10 mM EGTA, pH 7.2) , The single and two-photon fluorescence intensities gradually increased over time (see FIG. 5 (a) and FIG. 3 (a)). The reaction made compound 1 as the only product as revealed by HPLC analysis (see FIG. 4). Moreover, the plot of time versus ln (F _∞ -F) is a straight line, where F _∞ and F represent infinity (> 10 half-life) and fluorescence intensity measured at a given time, respectively (FIG. 5 (b), FIG. See (b) and (c) of 3). The slope of the plot is the observed pseudo-first-order rate constant (k _obs ). The plot for k _obs versus (2-AET) concentration is a straight line through the origin. These results indicate that the response is second order, first order for ASS, and first order for 2-AET, with k ₂ = 2.2 ㅧ 10 ^-5 M ^-1 s ^-1 ( (B) of FIG. 5). The observed results can therefore be most reasonably attributed to the rate-limiting attack of thiols in disulfide bonds followed by cleavage of CN to give compound 1 as outlined in the reaction scheme. .

ASS exhibits strong response to GSH, Cys, dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and 2-AET, and to thiol groups (glu, ser, val, met, ala, ile), metal ions (Ca ²⁺ , K ⁺ , Na ⁺ , Fe ³⁺ , Zn ²⁺ , Mg ²⁺ ) and H ₂ O ₂ showed a negligible reaction (Fig. 5 (c) And pH 6) and pH independent of biologically relevant pH (see FIG. 7). Thus, such probes can detect intracellular thiols without interference from other biologically relevant analytes and pH.

The TP operating spectrum of the ASS and the reaction product between ASS and 2-AET in the MOPS buffer solution exhibited two-photon working cross section (Φ δ) values of 11 and 113 GM at 780 nm. This represents a 10-fold increase in two-photon excitation fluorescence (TPEF) intensity when ASS is reduced to compound 1. As expected, the two-photon operating spectrum of the reaction product is almost the same as the two-photon operating spectrum of compound 1. In addition, the two-photon working cross-sectional value of Compound 1 is 11 times larger than the two-photon working cross-sectional value of Compound 2, and it is expected that the TPM for ASS-labeled cells should be significantly brighter than the TPM image stained with Compound 2.

The ability of ASS to detect thiols in living cells is described below.

8A shows TPMs (a to c) and bright regions (d) of HeLa cells cultured with ASS (3 uM), respectively. In FIG. 8A, (a) shows an image for 30 minutes, (b) and (c) with lipoic acid (500 uM) for 1 day and NEM (30 minutes) before being labeled with ASS. Pretreated with 100 uM). Two-photon excitation is provided at 780 nm using femto second pulses and scale bars represent representative images from replication experiments (n = 5).

FIG. 8B shows bright regions and TMP images of HeLa cells labeled 3uM ASS, respectively.

In FIG. 8B, (a) shows bright regions and (b) and (c) show that TPEP was collected at 360-460 nm and 500-620 nm, respectively. And (d) of Figure 9 shows the TPM image of HeLa cells labeled with 3uM ACC. Two-photon excitation is provided at 780 nm using femto-second pulses and the cells shown represent a representative image obtained from a copy experiment (n = 5) and the scale bar is 30 um.

9 shows TPM and OPM images of HeLa cells labeled together with ASS (3 uM), mitotracker (1 uM) and Hoechst (1 uM), respectively. The wavelengths for single and two photon excitation are 514 and 780 nm and the scale bar is 30 um and the cells shown represent representative images from replication experiments (n = 5).

In Figure 9 (a), (b) and (c) is a TPEF collected at 500 ~ 620nm (ASS), single photon fluorescence collected at 600 ~ 660nm (mitotracker) and 450 ~ 550nm (Hoechst) Represented are collected at and (d) and (e) represent colocalized images, respectively.

The ability of ASS to detect thiols in living cells was tested. TPM images of ASS-labeled HeLa cells were assumed to be bright due to the easy loading and convenient ratio of thiol induced oxidation and the prominent two-photon working cross-sectional values of the reaction product (see FIG. 8A (a)). When cells were pre-cultured with α-lipoic acid for 1 day to increase GSH production (see Figure 8a, (b)), TPEF strength increased significantly and the well-known thiol-blocking agent N-ethylmaleimide (NEM) The treatment decreased sharply (see FIG. 8A (c)). In contrast, little TPEF was detected from cells labeled with ACC (see FIG. 8B). Furthermore, bright area images confirm that cells continue to survive independently through image studies (see (d) of FIG. 8A). Thus, ASS can clearly detect thiols in living cells. In addition, TPM images of HeLa cells stained with ASS, Mito Tracker and well-known mitochondria and Hoechst 33342 (photo9), photon fluorescence detection for the nucleus, merged well with OPM images of mitochondria (Fig. (E) of 9). These results indicate that thiols are superior in mitochondria.

The two-photon probe according to the invention can be applied for tissue imaging.

FIG. 10 shows light regions and TPM images of fresh rat hippocampal slices stained with 20 uM ASS at a depth of about 120 um.

In FIG. 10, (a) is a bright region and (b) is a TPM image 10 Hz magnification with a scale bar of 300 um, and (c) 100 Hz magnification with a scale bar of 30 um. (D) shows the TPM images of (b) pretreated with NEM (200 uM) before being labeled with 20 uM ASS. TPEF was excited at 780 nm using femto second pulses and collected at 500-620 nm.

Figure 11 shows a TMP image of fresh rat hippocampal fragments stained with 20 uM ASS.

Images were obtained at depths of 90-180 um at 10 Hz magnification. TPEF was excited at 780 nm using a femto second pulse, collected at 500-620 nm, and the scale bar was 300 um.

The usefulness of these probes in tissue imaging has been further investigated. The bright area image shows the dentate gyrus (DG) as well as CA1 and CA3 (see FIG. 10 (a)). TPM images of a portion of fresh rat hippocampal slices incubated with 1 μM ASS show abundance of CA1 and DG regions at 120 μm depth (see FIG. 10 (d)). At larger magnifications the image shows thiol distribution in the CA1 region at 100 μm depth (see FIG. 10 (c)). Furthermore, TPM images at 90, 120, 150, and 180 μm show thiol distributions in the xy plane and along the z direction (see FIG. 11). In addition, TPEF strength was drastically reduced when tissue pieces were pretreated with NEM (see FIG. 10 (d)). This fact shows that ASS can detect thiols at 90-180 μm deep in living tissue when using TPM.

The probe according to an embodiment of the present invention shows a 10-fold improvement in TPEF in response to thiol, and is free of pH-effects at biologically relevant pH and releases superior stiff TPEF 11-fold compared to known probes. And the probe according to the invention has the advantage that thiols in living cells and tissues can be detected at a depth of 90 to 180 μm without interference from other biologically related species.

Hereinafter, the present invention will be described in more detail with reference to preferred examples. However, these examples are intended to illustrate the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited thereby.

Processes for the preparation of compound 1, compound 3 and compound 5 are known in the art and are described, for example, in Kim, HM; Jung, C .; Kim, BR; Jung, SY; Hong JH; Ko, YG; Lee, KJ; Cho, BR Angew. Chem. Int. Ed. 2007, 46, 346 b) Pires, MM Chmielewski, J. Org. Lett. 2008, 10, 837, and the like. This document is incorporated herein by reference.

Example 1 (Preparation of Compound 4)

To prepare compound 4, N, N-dimethylaminopyridine in a solution of compound 3 (500 mg, 1.6 mmol), pyridine (0.64 mL, 7.9 mmol) and compound 1 (630 mg, 3.2 mmol) in DMF (5 mL) was prepared. (DMAP) is added in a catalytic amount and stirred for 14 hours at room temperature. The solvent is removed in vacuo and the resulting brown residue is dissolved in CH ₂ Cl ₂ and washed twice in 1% HCl (aq). The product containing impurities is purified by silica-gel column chromatography using hexane / ethyl acetate (4: 1) as eluent. The final product is 310 mg (41%). Prepared compound 4 is used for the preparation of ASS or ACC corresponding to two-photon probe.

Example  1-2 ( ASS Manufacture)

Compound 4 (300 mg, 6.8 mmol) is dissolved in CF ₃ CO ₂ H / CH ₂ Cl ₂ (1/1, 10 mL) at 0 ° C. under dark conditions. The solution is stirred at 0 ° C. for 1 hour, heated to room temperature, stirred for 2 hours and the solvent is removed in vacuo. The residue is dissolved in MeOH and filtered. Using pre-HPLC, ASS was purified using a mixture of 10% eluent A (HPLC-grade MeCN with 0.1% TFA) and 90% eluent B (HPLC-grade water with 0.1% TFA). The reaction process proceeds with a linear gradient of 80% eluent A and 20% eluent B at a flow rate of 10 mL / min over a period of 120 minutes. Pure ASS is obtained as brown oil. The final product is 140 mg (46%).

Example 1-3 (Preparation of Compound 6)

Compound 6 is prepared by the same process as the synthesis of Compound 4, except that Compound 5 (140 mg, 0.50 mmol) is used instead of Compound 3. Purification by silica-gel column chromatography using hexane / ethyl acetate (4: 1) as eluent is applied to compound 5. The final yield is 90 mg (41%).

Comparative Example (Manufacture of ACC)

ACC is prepared through the same process as the synthesis process of Example, except that compound 6 (50 mg, 0.11 mmol) is used instead of compound 4. ACC is purified by pre-HPLC using the same procedure as described above. The final product is 32 mg (62%).

Evaluation example  1-1 (Analysis of compound 4)

IR (KBr), ¹ H NMR (CDCl ₃ , 300 MHz) and ¹³ C NMR (CDCl ₃ , 100 MHz) analyzes for Compound 4 prepared according to Example 1-1 were aquatic. The IR peaks were 3361, 1714. , 1681 cm ^-1 , and ¹ H NMR was δ 8.45 (d, J = 1.9 Hz, 1H) 8.06 (dd, J = 1.9, 8.8 Hz, 1H), 7.96 (d, J = 8.8 Hz, 1H) , 7.86 (d, J = 8.8 Hz, 1H), 7.72 (d, J = 1.9 Hz, 1H), 7.55 (d, J = 1.9 Hz, 8.8 Hz, 1H), 4.41 (t, J = 6.6 Hz, 2H ), 3.44 (s, 3H), 3.41 (t, J = 6.6 Hz, 2H), 2.92 (t, J = 6.6 Hz, 2H), 2.75 (t, J = 6.6 Hz, 2H), 2.73 (s, 3H ), Peaks were observed at 1.43 (s, 9H), and ¹³ C NMR was δ 198.2, 155.3, 136.8, 136.0, 134.7, 130.7, 130.4, 130.0, 128.4, 125.7, 124.8, 122.9, 120.8, 63.9, 39.3, 38.8 Peaks were observed at 38.0, 37.4, 28.6, 26.9, and 14.4.

Evaluation example  1-2 ( ASS Analysis of

IR (KBr), ¹ H NMR (CD ₃ OD, 300 MHz) and ¹³ C NMR (CD ₃ OD, 100 MHz) analyzes for ASS prepared according to Examples 1-2 were aquatic. The IR peak was 3442, Observed at 1712, 1680 cm ⁻¹ , ¹ H NMR was δ 8.60 (d, J = 2.0 Hz, 1H), 8.06 (d, J = 8.9 Hz, 1H), 8.04 (dd, J = 2.0 Hz, 8.9 Hz , 1H), 7.93 (d, J = 8.9 Hz, 1H), 7.84 (d, J = 2.0 Hz, 1H), 7.60 (dd, J = 2.0 Hz, 8.9 Hz 1H), 4.42 (t, J = 6.5 Hz , 2H) 3.43 (s, 3H), 3.22 (t, J = 6.5 Hz, 2H), 3.00 (t, J = 6.5 Hz, 2H), 2.90 (t, J = 6.5 Hz, 2H), 2.72 (s, 3H), peaks were observed, and ¹³ C NMR was δ 199.0, 155.7, 147.8, 143.2, 136.1, 134.6, 130.9, 130.2, 130.1, 128.1, 125.6, 124.1, 123.0, 63.7, 37.8, 36.9, 36.6, 34.2, 29.9 The peak was observed at, 25.5.

Evaluation example  1-3 (analysis of compound 6)

IR (KBr), ¹ H NMR (CDCl ₃ , 300 MHz) and ¹³ C NMR (CDCl ₃ , 100 MHz) analyzes for ASS prepared according to Examples 1-2 were aquatic. The IR peaks were 3365, 1710, Observed at 1680 cm ^-1 , ¹ H NMR was δ 8.43 (d, J = 1.6 Hz, 1H), 8.03 (dd, J = 1.6 Hz, 8.7 Hz 1H), 7.93 (d, J = 8.7 Hz, 1H) , 7.83 (d, J = 8.7 Hz, 1H), 7.68 (d, J = 1.6 Hz, 1H), 7.53 (dd, J = 1.6 Hz, 8.7 Hz, 1H), 4.13 (t, J = 6.6 Hz, 2H ) Peaks were observed at 3.41 (s, 3H), 3.06 (t, J = 6.6 Hz 2H), 2.71 (s, 3H), 1.61 (m 4H), 1.42 (s, 9H), 1.30 (m, 4H). , ¹³ C NMR is δ 198.2, 155.8, 143.5, 136.0, 134.5, 130.5, 130.2, 130.1, 128.3, 125.8, 125.1, 124.7, 122.5, 66.2, 40.7, 37.8, 30.2, 29.0, 28.6, 28.3, 27.0, 26.6, A peak was observed at 25.8.

Evaluation example  1-4 ( ACC Analysis of

IR (KBr), ¹ H NMR (CD ₃ OD, 300 MHz) and ¹³ C NMR (CD ₃ OD, 100 MHz) analyzes for ASS prepared according to Examples 1-2 were aquatic. 3423, 1685, 1677 cm ⁻¹ , ¹ H NMR was measured at δ 8.60 (d, J = 1.9 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H), 8.05 (dd, J = 1.9 Hz , 8.8 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.82 (d, J = 1.9 Hz, 1H), 7.58 (dd, J = 1.9 Hz, 8.8 Hz, 1H), 4.15 (t, Peaks observed at J = 6.9 Hz, 2H), 3.41 (s, 3H), 2.86 (t, J = 6.9 Hz, 2H), 2.72 (s, 3H) 1.63 (m, 4H), 1.37 (m, 4H) ¹³ C NMR are δ 199.0, 156.2, 143.4, 136.2, 134, 5, 130.8, 130.1, 128.8, 128.0, 127.5, 125.7, 124.8, 124.1, 65.9, 39.4, 36.8, 28.5, 27.3, 25.8, 25.5, 25.3 , Peaks were observed at 25.1.

Claims (6)

Two-photon probe for the detection of thiols represented by the following formula (1).
Formula 1

Where X = S (sulfur) A method for preparing the two-photon probe of claim 1 according to Scheme 1 below:
Scheme 1

Where 1 is

, a: pyridine, dimethylformamide, 4-dimethylaminepyridine, b: trifluoroacetic acid, dichloromethane) A method for detecting thiol in living cells or living tissue using the two-photon probe of claim 1. The method of claim 3,
A method for detecting thiol in a living cell or living tissue, wherein the depth of the living cell or living tissue in which the thiol is detected is 90 to 180 μm. The method of claim 3,
The living cell is a hematopoietic cell or a rat hippocampal cell, characterized in that the method for detecting thiol in a living cell or living tissue. The method of claim 3,
A method for detecting thiol in a living cell or living tissue, wherein the living tissue is mitochondria or nucleus.

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