KR100776830B1 - Two-photon dyes for real time imaging of lipid rafts - Google Patents

Abstract

A two-photon dye for real time imaging lipid raft is provided to increase the solubility in water, to remove cytotoxicity and to improve the intensity of fluorescence in a cellular membrane. A two-photon dye is represented by the formula 1, wherein R1 is a C5-C17 alkyl group; R2 is -COOH, -CH2SO3Na, or (CH3)3N^+ -CH2CH2-O-PO3^-CH2C(OCO-)HCH2-O-C(=O)-R3; and R3 is a C5-C17 alkyl group. Preferably the two-photon dye shows an absorption efficiency of 20 GM or more in vivo and has an excitation wavelength of 750-1,000 nm and a fluorescence wavelength of 400-700 nm.

Description

Two-photon dyes for real time imaging of lipid rafts

1 is a result of measuring the change of the single photon emission wavelength according to the polarity of the solvent for the two-photon dyes prepared by Comparative Example 1 and Example 1.

FIG. 2 is a graph showing the spectroscopic properties of two-photon dyes prepared by Example 1 and Comparative Example 1 on DOPC, DOPC / sphingo / chol and DPPC liposomes.

Figure 3 is a result showing the fluorescence emitted by polarization after dyeing the two-photon dye prepared by Example 1 and Comparative Example 1 in a large single membrane vesicle consisting of DOPC, DOPC / Sphingo / Chol, DPPC.

4 is a two-photon fluorescent GP image measured after dyeing two-photon dyes prepared according to Example 1 and Comparative Example 1 in a large single membrane vesicle consisting of DOPC, DOPC / Sphingo / Chol, DPPC.

5 is a two-photon fluorescence GP distribution curve measured after dyeing two-photon dyes prepared according to Example 1 and Comparative Example 1 in a large single membrane vesicle consisting of DOPC, DOPC / Sphingo / Chol, DPPC.

6 is a GP image and a GP distribution curve measured after staining A431 cells with a two-photon dye prepared in Example 1. FIG.

7 is a result of performing MTT assay of the two-photon dye prepared in Example 1.

8 is a time-lapse image taken after staining A431 cells with a two-photon dye prepared in Example 1.

The present invention relates to two photon dyes for real-time imaging of lipid rafts, and more particularly, to two-photon dyes suitable for real-time imaging of lipid rafts because of their high solubility in water, no cytotoxicity, and the high intensity of two-photon fluorescence. It is about.

Lipid rafts have recently been proposed as organelles through which signaling occurs, and research into the mechanisms by which disease occurs is being actively conducted. Repeat raft means a raft that floats in the 'phosphate of phospholipids'. Specifically, a rigid area composed of glycosphingolipids and cholesterol is formed in a sea of glycerophospholipids. As a floating plasma membrane, it means a fractionated plasma membrane in which signaling materials are present. If the signaling materials are located in a fluid membrane, the efficiency and speed of the signaling process itself can be reduced considerably. Therefore, the mediators can be organized by gathering these signaling materials in a certain place to make them efficient. This mediator is Rapid Raft. This rapid raft is not only a receptor for EGF, PDGF, insulin, IGF, VEGF, TNF, etc., but also various signal transduction agents, and thus can be referred to as a center of signal transduction, and mad cow disease, dementia, cancer, and bacterial diseases in the body. It is known to have a close relationship with the mechanism of occurrence.

Therefore, research on lipid rafts is actively conducted to understand the signal transmission, mad cow disease, dementia, cancer, and the like. Since the diameter of the rapid raft is 100 nm or less, it is impossible to observe the reality with an optical microscope. Do. Therefore, a method using AFM has been attempted, but since real cells have proteins and glycoconjugates attached to the cell surface in addition to the lipid component, they have very complex topologies, and thus the existence of lipid rafts in the living cells cannot be confirmed. . In addition, a method using fluorescence microscopy was attempted, which was attempted to attach the Green Fluorescent Protein (GFP) to the lipid raft proteins, and then observed the lipid raft protein in living cells. The pattern should be shown, but so far none of the lipid raft proteins show such spot pattern, and there has been controversy over whether lipid rafts exist in real cells.

As a result, recent attempts have been made to obtain molecular images of lipid rafts using fluorescent markers. The most widely used fluorescent molecules for lipid rafts have been developed by Professor Weber 20 years ago and are now commercialized by Molecular Probe. (Laurdan). This material is widely used in the study of cell membrane models because it can explain the behavior of the liquid ordered and liquid disordered phases of model membranes. A study was attempted to find lipid rafts present in cell membranes (PNAS, 1996, 93, 11443), but there was a problem that the experimental results were not reproducible.

Rodan of Formula 1 was developed as a single photon fluorescent dye and absorbs about 365 nm of photons to reach an excited state. The excited lodan returns to the ground state and emits fluorescence of a specific wavelength. The fluorescence is emitted at different wavelengths depending on the polarity of the solvent in which the lodan is dissolved. (Weber G, et al. , Biochemistry 18 : 3075-3078 (1979)). Recently, rodan has been used as a two-photon fluorescent dye for use in cell imaging.

In 1932, Maria Gopper-Mayer (Annn , Phys. 9 (1931) 273) predicted spontaneous absorption of two photons, but this two photon excitation phenomenon was long before the development of a strong laser light source. It has not been put to practical use. Two-photon excitation refers to a phenomenon in which two photons are simultaneously absorbed by the same luminophore with a sufficiently high unit volume and photon density per unit time by irradiating a strong light source. The energy absorbed here is the sum of the energy of two photons, and the possibility of two-photon excitation depends on the square of the photon density. Thus, the two photon absorption is a secondary non-linear optical property. On the other hand, the excited state transitions to the ground state and emits energy corresponding to the band gap energy as fluorescence, which is called two photon fluorescence, and the energy of the emitted photons is naturally higher than that of the irradiation light source. Big. Such a photon excited material is usually called a two-photon dye, which can be excited by a light source capable of providing photon energy corresponding to the band gap energy as well as the one-photon excitation. . The spectrum of fluorescence emission by two-photon excitation has the same spectral characteristics as the fluorescence emission spectrum by one-photon excitation.

The first characteristic of this two-photon excitation is that excitation occurs only in the vicinity of the restricted three-dimensional region of the light irradiation point, so that the background fluorescence can be minimized because the fluorescence emission by the excitation is localized in three dimensions. The second characteristic is that the wavelength of the irradiated light and the wavelength of the luminescent fluorescence are different from each other. In particular, the two-photon excitation is suitable for observing a small volume sample because the excitation volume is quite small.

On the other hand, due to the above-mentioned features, two-photon microscopy, which can cause two-photon excitation by irradiating light in the near infrared region, has attracted much attention in the field of bio imaging. The reason is that because of the near-infrared irradiation, it can be applied to living cells with little damage to biomolecules, deep penetration depth of near-infrared light, and minimization of tissue auto-fluorescence. Because.

Two-photon dyes used in these two-photon spectroscopy must: i) have a large two photon cross section (δ _TPA ) in the near infrared region, ii) have a moderate solubility in water, ii) have a high photostability And iv) good binding to the living body.

The rodan has a structurally hydrophobic tail and may be incorporated into the cell membrane, and the rodan present on the cell membrane may be an important indicator for measuring the fluidity of the cell membrane. Currently, the presence of lift rafts can be confirmed by using a method such as fluorescence resonance energy transfer (FRET). The cell membranes of eukaryotic cells have a high fluidity phospholipid region and a low fluidity rapid raft region. The phospholipid region contains unsaturated fatty acids so that water can freely pass, that is, has a relatively hydrophilic environment, whereas the lipid raft region mainly composed of glycolipids and cholesterol has a small fluidity and hardness due to saturated fatty acids and cholesterol. It has a relatively hydrophobic environment that does not pass easily. Rodan has been used to visualize lipid rafts in cell membranes because it emits 470-530 nm fluorescence in phospholipids with hydrophilic environments and 400-460 nm fluorescence in lipid rafts with hydrophobic environments (Gaus et al., PNAS, 100, 15554, 2003; Gaus et al., JCB, 171, 121, 2005). However, Lodan has a very low solubility in water, which precipitates and emits 450 nm of fluorescence, which is confused with the emission wavelength in the rapid raft region, limiting the visibility of the actual rapid raft. There was a problem that the intensity of fluorescence emitted from the intracellular environment was not sufficient. In addition, due to a problem of preferentially binding to a membrane having low fluidity in the cell membrane in which phospholipids and repeat rafts are mixed, it is difficult to properly distinguish images of a membrane having a low fluidity and a membrane having a high fluidity.

Therefore, the technical problem to be achieved by the present invention is to equally control the interaction of the hydrophilic region and the hydrophobic region in the cell membrane by appropriately adjusting the polarity of the two-photon dye, the fluorescence intensity in the environment in the cell membrane is large and in the cell membrane It is possible to clearly distinguish between the part with low fluidity and the part with high fluidity, so that it is possible to provide a two-photon dye suitable for real time imaging and rapid rafting.

The present invention provides a two-photon dye for real-time imaging of the rapid raft of the formula (2) to achieve the above technical problem.

(Wherein R ₁ is an alkyl group having 5 to 17 carbon atoms,

이며,R ₂ is —COOH, —CH ₂ SO ₃ Na, or

Is,

R ₃ is an alkyl group having 5 to 17 carbon atoms)

According to an embodiment of the present invention, the compound of Formula 2 may be a compound of Formula 3.

Wherein R ₁ is an alkyl group having 5 to 17 carbon atoms

According to another embodiment of the present invention, the compound of Formula 2 may be a compound of Formula 4.

Wherein R ₁ is an alkyl group having 5 to 17 carbon atoms

In addition, the compound of Formula 2 may be a compound of Formula 5.

Wherein R ₁ And R ₃ are each an alkyl group having 5 to 17 carbon atoms)

According to another embodiment of the present invention, the compound of Formula 3 may be any one compound selected from the group represented by the following formula (6).

In addition, the compound of Formula 4 may be any one compound selected from the group represented by the following formula (7).

In addition, the compound of Formula 5 may be any one compound selected from the group represented by the following formula (8).

According to a preferred embodiment of the present invention, the two-photon absorption efficiency of the two-photon dye for real-time imaging of the lipid raft is 20 GM (10 ^-50 cm ⁴ s / photon) or more in vivo, the wavelength range of the two-photon excitation light source is 750 ~ 1000 nm, and the wavelength range of the fluorescence may be 400 to 700 nm.

More preferably, the two-photon absorption efficiency of the two-photon dye for real-time imaging of the lipid raft is 150 GM or more, and in a high hydrophobic environment (solvent, liposome, and GUV), fluorescence of 430 to 470 nm is obtained, and the hydrophilic environment (solvent, liposome) And GUV) may emit fluorescence of 470 to 530 nm.

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

The two-photon dye for real-time imaging of lipid rafts according to the present invention has a good interaction with lipids, so that the dye is well stained on the cell membrane, and the intensity of fluorescence emitted under the environment in the moisture-rich cell membrane is high and the hydrophobic environment (solvent) is high. , Liposomes and GUV) emit fluorescence of 430 to 470 nm, and hydrophilic environment (solvent, liposome and GUV) to emit fluorescence of 470 to 530 nm. And, when applied to the cell is not toxic, it is characterized in that the real-time image of the lipid raft over a long time can be obtained.

The two-photon dye for real-time imaging of the rapid raft according to the present invention is characterized by having a structure of the following formula (1).

(1)

(One)

(Wherein R ₁ is an alkyl group having 5 to 17 carbon atoms,

이 며,R ₂ is —COOH, —CH ₂ SO ₃ Na, or

This is

R ₃ is an alkyl group having 5 to 17 carbon atoms)

That is, the _two- photon dye for real-time imaging of the rapid raft according to the present invention can improve the solubility in water because the R ₂ is a polar functional group, and because the interaction with lipids in the cell membrane is excellent, staining on the cell membrane is evenly distributed. And a brighter image can be obtained. In addition, since it absorbs near-infrared light and emits light, photobleaching does not occur at all and damages cells. Therefore, it is very advantageous to obtain a real-time image for a long time. The different polar functional groups of carbonyl (TPD1), sulfonyl (TPD2) and phosphatidylcholine (TPD3) may be introduced into R ₂ to have a shape similar to that of actual fatty acids and phospholipids of cells.

R ₁ serves to be incorporated into the cell membrane as a hydrophobic moiety. The R ₁ is an alkyl group having 5 to 17 carbon atoms. When the carbon number is less than 5, the hydrophobicity is weak, and when the carbon number is more than 17, the solubility in water is drastically reduced, resulting in precipitation. It is not suitable as a probe because of a problem.

The two-photon dye for real-time imaging of the lipid raft according to the present invention is preferably a compound of the following formulas 2 to 4.

(2)

(2)

(Wherein R ₁ is an alkyl group having 5 to 17 carbon atoms: hereinafter referred to as TPD1)

(3)

(3)

(Wherein, R ₁ is an alkyl group having 5 to 17 carbon atoms: hereinafter referred to as TPD3)

(4)

(4)

Wherein R ₁ And R ₃ are each an alkyl group having 5 to 17 carbon atoms: hereinafter referred to as TPD2)

In addition, in the two-photon dye for real-time imaging of the rapid raft of the formula 2 or 3 according to the present invention, R ₁ is preferably an alkyl group having 5, 11 or 17 carbon atoms, because of the hydrophobic carbon number of the cell membrane This is because the hydrophilic and hydrophobic positions can be selectively stained, so that images of various cellular activities can be obtained.

In addition, in the two-photon dye for real-time imaging of the rapid raft of the formula 4 according to the present invention, R ₁ is an alkyl group having 5, 9 or 11 carbon atoms, R ₃ is preferably an alkyl group having 11 or 15 carbon atoms It is also possible to selectively stain the hydrophilic and hydrophobic positions of the cell membrane according to the length of the hydrophobic carbon number, because it is possible to obtain images of various cellular actions.

The two-field absorption efficiency of the two-photon dye for real-time imaging of the rapid raft according to the present invention is 20GM or more in a living body, the wavelength range of the excitation light source is characterized in that the wavelength range of fluorescence is 400-700nm, More preferably, the two-photon absorption efficiency is at least 150 GM.

As mentioned above, Rodan, a conventional representative two-photon dye, emits 470-530 nm of fluorescence in a phospholipid having a hydrophilic environment, and 400-460 nm of fluorescence in a lipid raft having a hydrophobic environment. Therefore, although it has been used to visualize lipid rafts in cell membranes, it has a low solubility in water and precipitates, thereby limiting the visualization of lipid rafts because it emits 450 nm fluorescence even in a hydrophilic environment. However, the two-photon dye according to the present invention does not form a precipitate at a concentration suitable for use as a marker because of its high solubility in water, and emits a fluorescence of 430 to 470 nm in a highly hydrophobic environment (solvent, liposome and GUV). In hydrophilic environments (solvents, liposomes and GUV), fluorescence emits 470-530 nm, which can be clearly distinguished by the hydrophobic environment of the cell membrane and the fluorescence that emits hydrophilic environment, and various stresses (thermal shock, hypoxia, hypertonic shock, etc.). It is well suited to confirm the dynamics of lipid rafts as well as changes in the plasma membrane fluidity in cells.

In addition, as described below, the two-photon dye according to the present invention is a lipid raft because the distribution of Generalized Polarization (GP) values for the hydrophilic and hydrophobic environment of the cell membrane is mutually symmetric and is evenly distributed in the hydrophilic region and the hydrophobic region in the cell membrane. There is an advantage that can be observed clearly.

Hereinafter, the present invention will be described in more detail with reference to preferred examples, but the present invention is not limited thereto.

Example 1

1- (1) 6- Dodecanoyl -N-methyl-2- naphthylamine Manufacture

MeNH ₂ HCl (11.0 g, 0.13 mol) 2-hydroxy-6-dodecanoylnaphthalene (8.0 g, 25 mmol), Na ₂ S ₂ O ₅ (12 g, 61 mmol), NaOH (5.2 g, 0.13 mol) And water (100 ml) were added to the mixed mixture, which was then stirred at 140 ° C. for 48 hours in a high pressure tube. The resulting solid was filtered off, washed well with water, and then recrystallized with CH ₂ Cl ₂ -EtOH to prepare 6-dodecanoyl-N-methyl-2-naphthylamine. (5.6 g, yield: 67%)

¹ H-NMR (300 MHz, CDCl ₃ ): δ 8.31 (s, 1H), 7.94 (dd, J = 9.0, 3.0 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.64 (d , J = 9.0 Hz, 1H), 6.91 (dd, J = 9.0, 3.0 Hz, 1H), 6.77 (s, 1H), 3.97 (br s, 1H), 3.03 (t, J = 7.5 Hz, 2H), 2.97 (s, 3H), 1.77 (quin, J = 7.5 Hz, 2H), 1.26 (m, 16H), 0.88 (t, J = 7.5 Hz, 3H)

¹³ C-NMR (75 MHz, CDCl ₃ ): δ 200.59, 149.30, 138.23, 130.91, 130.87, 130.08, 126.22, 126.19, 125.05, 118.60, 103.28, 38.64, 32.15, 29.88, 29.86, 29.78, 29.77, 29.76, 29.74 , 29.58, 25.09, 22.92, 14.36;

1- (2) TPD1 of  Produce

6-dodecanoyl-N-methyl-2-naphthylamine (3.0 g, 8.8 mmol) prepared in Example 1- (1), methyl bromoacetate (2.0 g, 13 mmol), A mixture of Na ₂ HPO ₄ (1.9 g, 13 mmol), and NaI (0.5 g, 3.5 mmol) was stirred for 18 h at 80 ° C. under a nitrogen atmosphere. The mixture was extracted with ethyl acetate and washed well with brine. It was possible to recrystallize with ethanol to obtain the product as an intermediate of a yellow solid. The intermediate (2.0 g, 4.9 mmol) and KOH (0.70 g, 12 mmol) was stirred for 5 h in ethanol (50 ml). The solution was diluted with ice water (100 mL) and concentrated hydrochloric acid was added until the pH was 3 below 5 ° C., and the resulting precipitate was filtered out. The precipitate was washed with distilled water sufficiently and then recrystallized with chloroform petroleum ether to obtain a white solid TPD1. (1.5 g, yield: 79%)

¹ H-NMR (300 MHz, DMSO d ₆ ): δ 12.70 (br s, 1 H), 8.43 (s, 1 H), 7.89 (d, J = 9.0 Hz, 1H), 7.80 (d, J = 9.0 Hz , 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.20 (d, J = 9.0 Hz, 1H), 6.92 (s, 1H), 4.26 (s, 2H), 3.09 (s, 3H), 3.03 (t, J = 7.5 Hz, 2H), 1.61 (quin, J = 7.5 Hz, 2H), 1.22 (m, 16H), 0.83 (t, J = 7.5 Hz, 3H);

¹³ C-NMR (75 MHz, DMSO d ₆ ): δ 199.91, 172.44, 149.83, 137.75, 131.30, 130.71, 130.46, 126.66, 125.44, 124.69, 116.70, 105.56, 100.29, 39.75, 38.17, 31.99, 29.72, 29.70, 29.68, 29.65, 29.46, 29.41, 24.92, 22.80, 14.66;

Example 2

TPD2 Manufacture

6-dodecanoyl-N-methyl-2-naphthylamine (3.0 g, 8.8 mmol) prepared in Example 1- (1), BrCH ₂ CH ₂ SO ₃ Na (2.1 g, 10.0 mmol), and Proton-sponge (2.2 g, 10.3 mmol) was stirred in DMF (50 mL) for 12 h at 150 ° C. under a nitrogen atmosphere. The mixture was extracted with water and dichloromethane and washed well with brine. The solvent was then evaporated and the product was purified by silica gel column using a solvent of methanol: ethyl acetate = 1: 5, and recrystallized from chloroform to prepare TPD2. (1.8 g, yield: 44%)

¹ H-NMR (300 MHz, CDCl ₃ ): δ 8.42 (s, 1H), 7.90 (d, J = 9.0 Hz, 1H), 7.89 (dd, J = 9.0, 3.0 Hz, 1H), 7.62 (d , J = 9.0 Hz, 1H), 7.20 (dd, J = 9.0, 3.0 Hz, 1H), 6.90 (s, 1H), 3.71 (t, J = 7.5 Hz, 2H), 3.08 (t, J = 7.5 Hz , 2H), 3.01 (s, 3H), 2.68 (t, J = 7.5 Hz, 2H), 1.61 (quin, J = 7.5 Hz, 2H), 1.26 (m, 16H), 0.83 (t, J = 7.5 Hz , 3H)

Example 3

TPD3 Manufacture

TPD1 (1.6 g, 4.0 mmol) prepared in Example 1- (2), a compound of Formula 9 (1.0 g, 2.0 mmol), DCC (2.4 g, 11.6 mmol), and a mixture of dimethyl amino pyridine (DMAP) (0.24 g, 2.0 mmol) were stirred in chloroform (50 mL) for 96 h under a nitrogen atmosphere.

The solvent was then evaporated and the product was purified by silica gel column using solvent with chloroform: methanol: water = 18: 9: 1 to give TPD3. (2.2 g, yield: 63%)

¹ H-NMR (600 MHz, CDCl ₃ ): δ 8.27 (s, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 9.0 Hz, 1H), 7.08 (d, J = 9.0 Hz, 1H), 6.85 (s, 1H), 5.23 (br m, 1H), 4.39 (d, J = 9.5 Hz, 1H), 4.26 (d, J = 18 Hz, 1H), 4.18 (d, J = 18 Hz, 1H), 4.16 (s, 2H), 4.06 (dd, J = 12 Hz, 6.6 Hz, 1H), 3.94 (br s, 2H), 3.57 (br s, 2H), 3.14 (br s, 12H), 2.99 (t, J = 7.2 Hz, 2H), 2.08 (t, J = 7.8 Hz, 2H), 1.72 (m, 2H), 1.43 (m , 2H), 1.38-1.14 (m, 40H), 0.85 (t, J = 7.0 Hz, 6H)

Comparative Example 1

1- (1) 2- Methoxy -6- dodecanoylnaphthalene Manufacture

Aluminum chloride (32.9 g, 0.25 mol) was dissolved in anhydrous nitrobenzene and 2-methoxynaphthalene (30.1 g, 0.19 mol) was added under a nitrogen atmosphere. To this mixture, lauryl chloride (41.6 g, 0.19 mol) was slowly added dropwise between 10 and 13 DEG C over 15 minutes, stirred at 5 DEG C for 2 hours, and then stirred at room temperature for 12 hours. The mixture was poured into 300 mL of 3M HCl at 0 ° C., extracted with chloroform, and washed well with brine. The solvent was evaporated under reduced pressure, dissolved with chloroform and washed with 0.1 M NaOH. Finally, chloroform was evaporated and recrystallized from ethanol to prepare 2-methoxy-6-dodecanoylnaphthalene. (41.5 g, yield: 64.2%)

¹ H-NMR (300 MHz, CDCl ₃ ): δ 8.40 (s, 1H), 8.01 (dd, J = 9.0, 3.0 Hz, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.77 (d , J = 9.0 Hz, 1H), 7.20 (dd, J = 9.0, 3.0 Hz, 1H), 7.16 (s, 1H), 3.95 (s, 3H), 3.06 (t, J = 7.5 Hz, 2H), 1.78 (quin, J = 7.5 Hz, 2H), 1.26 (m, 16H), 0.87 (t, J = 7.5 Hz, 3H).

1- (2) Rodan  Produce

Li (0.5 g, 70 mmol) was added to HMPA (20 mL) and benzene (30 mL), and dimethylamine (3.5 g, 78 mmol) was added dropwise and stirred vigorously for 1 hour. 2-methoxy-6-dodecanoylnaphthalene prepared in Comparative Example 1- (1) was added to this mixture. (7.3 g, 22 mmol) was added at once and stirred at room temperature for 5 hours. The mixture was poured into ice water, extracted with ether, the solvent was evaporated and recrystallized from ethanol to give Laurdan. (5.1 g, yield: 66%)

¹ H-NMR (300 MHz, CDCl ₃ ): 8.31 (s, 1H), 7.94 (dd, J = 9.0, 3.0 Hz, 1H), 7.81 (d, J = 9.0 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.17 (dd, J = 9.0, 3.0 Hz, 1H), 6.87 (s, 1H), 3.11 (s, 6H), 3.04 (t, J = 7.5 Hz, 2H), 1.78 (t , J = 7.5 Hz, 2H), 1.37-1.27 (m, 16H), 0.89 (t, J = 7.5 Hz, 3H)

Test Example 1

Reflectivity test of solvent

In order to measure the change in the wavelength of single photon fluorescence according to the polarity of the solvent for the two-photon dyes prepared by Comparative Example 1 and Example 1, time-resolved fluorescence (TRF) was measured and the results are shown. 1 is shown. In this test example, hexane, DMF, ethanol, and water were used as the solvent, and the polarity was increased in the order of hexane, DMF, ethanol, and water. Referring to FIG. 1, in the case of the two-photon dye according to Comparative Example 1 (hereinafter referred to as rodan), the maximum fluorescence wavelength is increased as the polarity of the solvent increases, but the maximum fluorescence wavelength is decreased in water. It is shifted toward the blue wavelength (blue-shifted, blue shifted), and it can be seen that the wavelength is getting wider, which means that the rodan forms an aggregate because it is almost insoluble in water. Because Rodan has a long hydrocarbon tail, the dye surrounded by the tails of neighboring molecules in the aggregate is placed in a hydrophobic environment, showing a blue wavelength shift. That is, it is confirmed that rodan present in water does not reflect a hydrophilic environment, but emits light having a fluorescent wavelength of 400 to 460 nm, which is emitted from a hydrophobic environment, and thus is not suitable for a real time image of a rapid raft. However, in the case of the two-photon dye according to Example 1 of the present invention (hereinafter referred to as TPD1. FIG. 1 (b)), as the polarity of the solvent increases, the emission wavelength also increases and reflects the change of the polarity of the solvent well. Therefore, it can be seen that the light emitting regions are clearly distinguished according to the difference in polarity of the cell membrane.

Test Example 2

Measurement of Spectroscopic Properties

Single photon maximum absorption wavelength (λ _max ) and single photon maximum emission wavelength (λ ^fl ) when the two-photon dyes prepared in Example 1 and Comparative Example 1 were dissolved in four solvents (hexane, DMF, ethanol and water). _max ), ^Two- photon maximum absorption wavelength (λ ⁽²⁾ _max ), Quantum yield (Φ), two-photon absorption efficiency (δ _max ) was measured and the results are shown in Table 1 below. Absorption spectra were measured using a Hewlett-Packard 8453 diode array spectrophotometer, and fluorescence spectra were measured using an Amico Bowman series 2 luminescence spectrometer. The two-photon absorption efficiency means the maximum value of the two-photon cross section (δ _TPA ), and the two-photon cross section can be obtained by Equation 1 below.

Where s and r are subscripts and reference molecules, δ is a two-photon cross-section to obtain, s is the intensity of the signal collected by the CCD detector, S is the fluorescence quantum efficiency is expressed as Φ , and φ is The total fluorescence collection efficiency of the test equipment. The number density of molecules in the solution is indicated by c. δ _r means the two-photon cross-sectional area of the reference molecule.

 menstruum λ _max λ ^fl _max λ ⁽²⁾ _max Φ δ _max (GM) Example 1 Comparative Example 1 Example 1 Comparative Example 1 Example 1 Comparative Example 1 Example 1 Comparative Example 1 Example 1 Comparative Example 1 Hexane 345 344 404 407 - - 0.10 0.11 - - DMF 363 356 446 454 780 780 0.36 1.00 167 50 ethanol 383 363 494 487 780 780 0.43 1.00 153 57 water 384 354 522 431 820 - 0.11 0.01 323 -

Referring to Table 1, it can be seen that the two-photon dyes prepared by Example 1 and Comparative Example 1 are both significantly high in quantum yield in DMF and ethanol. However, when the solvent is changed from ethanol to water, it can be seen that in Comparative Example 1, the quantum yield is reduced to 1/100, whereas in the case of the two-photon dye according to the present invention, the quantum yield is reduced to only 1/4. The reason why the quantum yield decreases rapidly when Lodan is present in the water in Comparative Example 1 is due to the quenching phenomenon due to precipitation. When the quantum yield drops sharply, the intensity of fluorescence also decreases. On the other hand, in the case of the dye according to Example 1, the two-photon absorption efficiency is at least 153GM and the maximum 323GM, it can be seen that it is a two-photon dye more efficient than Lodan because it is about 3 to 6 times larger than the dye according to Comparative Example 1.

Test Example 3

Liposome  Reflectivity test

Three types of liposomes having different fluidity (polarity) were prepared to investigate the degree to which the two-photon dyes prepared in Example 1 and Comparative Example 1 reflect the polarity of liposomes. The first DOPC liposome is made from 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC) containing unsaturated fatty acids and has relatively high fluidity. To reflect the fluidity of the phospholipids of the plasma membrane, the second DOPC / Sphingo / Chol liposomes were mixed with DOPC, sphingomyelin and cholesterol (cholesterol) in the same ratio, to reflect the phospholipids and lipid rafts at the same time, The third DPPC liposome consists of 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, DPPC) with saturated fatty acids. The film with the least flowability was intended to reflect the rapid raft. Next, rodan and TPD1 were stained on the three types of liposomes, and their spectroscopic characteristics were examined, and the results are shown in FIG. 2. Referring to Figure 2 (a) (lodan) and (b) (TPD1) it can be seen that the single-photon absorption wavelength of Lodan and TPD1 shows a maximum at 360nm and 395nm regardless of the type of liposomes. However, the intensity of fluorescence increased with greater hydrophobicity of liposomes (DOPC <DOPC / Sphingo / Chol <DPPC). In all cases, it can be seen that TPD1 emits fluorescence 2-5 times stronger than Lodan. This means that the single photon fluorescence efficiency of TPD1 is greater than Lodan. Since the di-photon tolerance of the dipole molecule is very similar to the Frank-Condon state, the above results are in good agreement with the fact that the TPD1 absorption efficiency of TPD1 at 780 nm is greater. On the other hand, referring to Figure 2 (c) which is a normalized fluorescence spectra of Lodan according to Comparative Example 1, the maximum fluorescence wavelength of Lodan in DPPC is 440nm, the spectrum in DOPC is more widespread and at 439nm and 484nm It shows two peaks, which means that the lodan is well dissolved in DOPC liposomes, and the aggregates exist at the same time. On the other hand, in the DOPC / Sphingo / Chol and DPPC, it can be seen that the fluorescence wavelength of the lodan overlaps considerably. This means that it binds preferentially to the gel membrane, which is less fluid than the membrane, and makes it difficult to distinguish between them in an image. On the other hand, referring to Figure 2 (d) TPD1 according to the first embodiment of the present invention emits 442nm fluorescence in DPPC, 486nm fluorescence in DOPC, the fluorescence spectrum of TPD1 in DOPC / Sphingo / Chol liposome It is widely spread and it can be confirmed that it is almost similar to the sum of the fluorescence spectra of DPPC and DOPC. The two-photon dye according to the present invention is uniform in liposomal and raft-mixed liposomes, which is uniform in the low-flowing and high-flowing membranes. To show that it is distributed. Therefore, the two-photon dye according to the present invention is uniformly distributed regardless of the polarity (fluidity) in the cell membrane and emits fluorescence in different wavelength bands according to the polarity (fluidity) difference, thereby clearly indicating the polarity (fluidity) of the cell membrane. It can be distinguished and is suitable for real time image of rapid raft.

Test Example 4

Giant Monolayer In the vesicle (GUV) Photoselectivity  Test

Irradiation of two-photon fluorescence according to the angle of polarization reveals information about the relative arrangement of two-photon markers present in the lipid bilayer. After producing giant unilamellar vesicels (GUVs) consisting of DOPC, DOPC / Sphingo / Chol, and DPPC, dyeing RPD according to Comparative Example 1 and TPD1 according to Example 1 on the GUVs and then polarizing them After examining the distribution of fluorescence emitted, the results are shown in FIG. 3. 3 (a), (b) and (c) are fluorescence distribution photographs in the case of dyeing DOdan GUV, DOPC / Sphingo / Chol GUV and DPPC GUV, respectively, according to Comparative Example 1 in sequence, and FIG. ), (f) and (g) are fluorescence distribution photographs when the TPD1 according to Example 1 was sequentially stained with DOPC GUV, DOPC / Sphingo / Chol GUV, and DPPC GUV, respectively. Referring to FIG. 3, in the case of a GUV composed of DOPC / Sphingo / Chol and DPPC, fluorescence intensity is very weak in a region perpendicular to polarization when dyed with Lodan or TPD1. However, referring to FIG. 3 (a), the DOPC GUV stained with Lodan shows uniform fluorescence in a uniform state in all regions, indicating that Lodan is arranged randomly in DOPC GUV. On the other hand, in the DOPC GUV stained with TPD1 (FIG. 3 (e)), the fluorescence is very weak in the region perpendicular to the polarization, while the intensity of the fluorescence is high at other sites. It is shown that it is arranged parallel to the tail and the two-photon dye according to the present invention has a photoselection in the DOPC GUV and it can be seen that the dye is uniformly well. On the other hand, Figure 3 (d) and (h) is a schematic diagram showing the position of the two-photon markers present in the lipid bilayer, both Rodan and TPD1 is arranged in parallel with the lipid molecule in the gel state (small flow state), but the fluidity is large In the state, rodan is randomly arranged or aggregated, but TPD1 is thought to be arranged parallel to lipid molecules due to hydrophilic interaction between water and carboxylate near the head of lipid.

Test Example 5

GP  Giant using image Monolayer Follicular  Reflectivity test of hydrophilicity and hydrophobicity

In order to obtain GP images using fluorescent dyes, first, dye cells or model membranes, and calculate the intensity of fluorescence at 400 to 460 nm and 470 to 530 nm. To get the final image.

Where I _(400-460) is the fluorescence intensity at the wavelength of 400-460 nm, I _(470-530) is the fluorescence intensity at the wavelength of 470-530 nm, and G is the sensitivity in the two wavelength ranges. It is a sensitive correction factor.

 The closer the GP value is to -1, the more hydrophilic the material is. The color is violet and blue. In the case of hydrophobic materials, the GP value is close to 1 and is represented by red and orange. After staining Lodan according to Comparative Example 1 and TPD1 according to Example 1 of the present invention to each of the three types of GUV, the GP value was calculated from the cross section of the GUV, and the result was converted to an image, and the result is shown in FIG. 4. . 4 (a), 4 (b) and 4 (c) are fluorescence GP images when rhodan was stained with DOPC GUV, DOPC / Sphingo / Chol GUV and DPPC GUV, respectively, in order. ), (e) and (f) are fluorescence GP images when TPD1 according to Example 1 was sequentially stained with DOPC GUV, DOPC / Sphingo / Chol GUV, and DPPC GUV, respectively. On the other hand, Figure 5 shows the GP distribution curves for Lodan and TPD1, respectively (blue: DOPC, red: DPPC, black: DOPC / Sphingo / Chol). Referring to FIG. 5, the GP distribution in the DPPC GUV was very narrow and the GP value was 0.54 for Lodan and 0.41 for TPD1. In the DOPC GUV, the GP value was 0.044 for Lodan and -0.35 for TPD1. Because DOPC GUV has high hydrophilicity, the reason why the GP value of Rodan is positive, although the GP value should be negative, may be because Lodan forms an aggregate in the flowable membrane. However, since TPD1 does not form aggregates in flowable membranes, it can be clearly identified as a hydrophilic environment with negative GP values in DOPC GUV. This is a result showing that TPD1 can distinguish the large fluid film and the small film more clearly than Lodan. On the other hand, similar results can be seen in the GP distribution of the GUV composed of DOPC / sphingo / chol, showing that the GP value is somewhat higher in the GUV stained with Rodan, and that the GP distribution is more symmetric in the GUV stained with TPD1. When each distribution was converted to a bimodal distribution by Gaussian curve fitting, Lodan had a positive GP value of 0.09 and 0.36, but TPD1 showed GP values of -0.14 and 0.18. The two rods are symmetrical with each other and the GP value is almost symmetrical. On the other hand, here, too, the GP value of Lodan is considerably larger than that of TPD1, and the distribution of low GP value is considerably wider than that of high GP value, which is exposed to various environments with different polarities and To show that it is present as a precipitate. However, the width and shape of the low GP and high GP distributions in the GUV stained with TPD1 are very similar and mutually symmetrical, indicating that TPD1 in GUV has a better environment for low fluidity (hydrophobic) and large membranes (hydrophilic). It can be reflected, and the two can be distinguished more clearly.

In the present invention, single photon and two photon fluorescence images were measured using confocal multiphoton microscopes (manufactured by Leica, TCS SP2). The single photon fluorescent microscope used an argon laser (488 nm) as the light source, and the two photon fluorescent microscope used a mode-locked titanium / sapphire laser (780 nm). On the other hand, the GP value calculation was performed using a visual C ++ based program developed in-house.

Test Example 6

In the cell GP  Visual test

Samples treated with A431 cells and methyl-β-cyclodextrin (MβCD), which remove cholesterol from the plasma membrane of the cells, were stained with two-photon dyes according to Example 1 and Comparative Example 1, followed by two-photon fluorescence microscopy. The result of converting this to a GP image and a GP distribution are shown in FIG. 6. Treatment with MβCD is known to destroy lipid rafts of cell membranes as cholesterol is removed. Therefore, when the cell membrane is treated with MβCD, the low GP distribution peak showing hydrophilicity should be unchanged and the high GP distribution peak showing hydrophobicity should disappear. Referring to the GP distribution diagram of FIG. 6, when the cells stained with Lodan were treated with MβCD, a high GP distribution peak (center GP value = 0.49) did not disappear and the median GP value was shifted slightly to 0.51. These results lead to the incorrect conclusion that lipid rafts still exist in cholesterol-free cells. In contrast, the cells stained with TPD1 showed brighter and more uniform images than in Comparative Example 1, and the size of the GP distribution peak of the MβCD-treated cells was significantly reduced, although not completely disappeared. have. Meanwhile, referring to the GP image of FIG. 6, in the case of Lodan, the hydrophobic region (the rapid raft) and the hydrophilic region (phospholipid) cannot be clearly distinguished, and even if the image is treated with MβCD, the disappeared region is irregular and random. You can't tell which part of the image was processed as a rapid raft. However, in the case of the two-photon dye according to the present invention, the hydrophobic region and the hydrophilic region of the cell membrane can be clearly distinguished and imaged, and the portion represented by the red fluorescence is the hydrophobic region (the rapid raft) and the portion represented by the green is the hydrophilic region. It is clearly classified as (phospholipid), and the image after the treatment with MβCD shows that all the portions (the repeat raft regions) that appeared as red fluorescence before MβCD treatment have disappeared. Thus, it can be seen that the two-photon dye according to the present invention can properly image the actual repeat raft region.

Test Example 7

Cytotoxicity test

In order to determine the cytotoxicity of the two-photon dye according to the invention was carried out MTT assay for the two-photon dye prepared in Example 1. That is, TPD1 1 × 10 ⁻⁵ M was stained on A431 cells and DMSO 1% was used as a control solvent, and the results are shown in FIG. 7. Referring to FIG. 7, the y-axis is cell viability. Since the cytotoxicity of TPD1 was found to be little different from the control solvent until 6 hours of incubation, the two-photon dye according to the present invention can be confirmed to be a dye suitable for imaging of living cells.

Test Example 8

Fluorescence for Living Cells GP  Video shooting

After staining A431 cells with dye 1 × 10 ⁻⁵ M according to Example 1 of the present invention, 22 minutes of the image was taken, and the fluorescent GP images thereof are shown in FIG. 8. Referring to Figure 8, it can be seen that the two-photon dye according to the present invention can be very useful for the visualization of the lipid raft over a long time, the lipid raft (red area) of the cell does not always stay in a certain place You can see that it is moving dynamically.

As described above, the two-photon dye for real-time imaging of the rapid raft according to the present invention has a large intensity of fluorescence emitted under the environment in the cell membrane, and can clearly distinguish between the hydrophobic region and the hydrophilic region of the cell membrane. It can be imaged, and since it is not toxic when applied to cells, it can be usefully used in the field of biological imaging such as real time image of lipid raft over a long time.

Claims (9)

Two-photon dye for real-time imaging of the lipid raft of the following formula 1.

(One) (Wherein R ₁ is an alkyl group having 5 to 17 carbon atoms, R ₂ is —COOH, —CH ₂ SO ₃ Na, or

Is, R ₃ is an alkyl group having 5 to 17 carbon atoms) The two-photon dye for a real-time image of a lipid raft, according to claim 1, wherein the compound of Formula 1 is a compound of Formula 2.

(2) Wherein R ₁ is an alkyl group having 5 to 17 carbon atoms The two-photon dye for a real-time image of a lipid raft, according to claim 1, wherein the compound of Formula 1 is a compound of Formula 3.

(3) Wherein R ₁ is an alkyl group having 5 to 17 carbon atoms The two-photon dye for a real-time image of a lipid raft, according to claim 1, wherein the compound of Formula 1 is a compound of Formula 4.

(4) Wherein R ₁ And R ₃ are each an alkyl group having 5 to 17 carbon atoms) The two-photon dye for real-time imaging of a lipid raft, according to claim 2, wherein the compound of Formula 2 is any compound selected from the group represented by the following Formula 5.

(5) The two-photon dye for real-time imaging of a lipid raft, according to claim 3, wherein the compound of Formula 3 is any compound selected from the group represented by the following Formula 6.

(6) The two-photon dye for a real-time image of a lipid raft, according to claim 4, wherein the compound of Formula 4 is any compound selected from the group represented by the following Formula 7.

(7) The two-photon absorption efficiency of the two-photon dye for real-time imaging of the lipid raft is at least 20 GM in a living body, and the wavelength range of the two-photon excitation light source is 750 to 1000 nm, and the wavelength of fluorescence according to any one of claims 1 to 7. A two-photon dye for a real time image of a rapid raft, wherein the range is 400 to 700 nm. The two-photon absorption efficiency of the two-photon dye for real-time imaging of the lipid raft is at least 150 GM, and fluorescence of 430 to 470 nm is generated in a highly hydrophobic environment (solvent, liposome and GUV). , Two-photon dye for real-time imaging of lipid raft, characterized in that the fluorescence emission of 470 ~ 530nm in the hydrophilic environment (solvent, liposomes and GUV).

Images