JP2009091287A - New fluorescent compound and method for detecting intracellular cholesterol using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent compound useful for detection and visualization of cholesterol in immobilized cells and raw cells in higher sensitivity than those of fluorescent staining and antibody staining and to provide a method and a kit for detecting cholesterol using the compound. <P>SOLUTION: The compound is prepared by introducing a dansyl group into amphotericin B, represented by the general formula (I) (wherein n is an integer of 1-5; and R is a 1-4C alkyl), forms a stable complex with cholesterol in a cell lipid membrane and has strong fluorescence intensity. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel fluorescent compound. The present invention also relates to an intracellular cholesterol detection method and detection kit using the compound.

Filipin III, a polyene macrolide antibiotic, has been used as a probe for observing cholesterol distribution in fixed tissues. However, Philippine III, which binds to biological membrane cholesterol, binds to the high cholesterol domain of the biological membrane and punctures it, causing cell damage. In addition to Philippine III, θ-toxin, cholera toxin B, etc. bind to sphingolipids in biological membranes and are used for observation of lipid rafts. However, they are mainly used for staining plasma membrane cholesterol and cholesterol distribution in intracellular organelles. Not suitable for observing the whole. Furthermore, since Philippine III has a fast fluorescence quenching and no antibody that recognizes it, the development of a new probe capable of antibody staining has been desired.
On the other hand, amphotericin B is a polyene macrolide antibiotic (Non-Patent Document 1), but has never been applied for the purpose of detecting cholesterol.
M. Murata et al. Org. Lett. 2002, 4, 2087-2089.

  An object of the present invention is to provide a novel fluorescent compound useful for detection of intracellular cholesterol and the like.

  The present inventors have intensively studied to solve the above problems. As a result, we succeeded in synthesizing a new AmB derivative in which dansyl group was introduced into amphotericin B (AmB). And since this compound forms a stable complex with cholesterol in the lipid membrane, it has been found that by using this compound, intracellular cholesterol can be detected with high sensitivity by fluorescence staining or antibody staining, and the present invention has been completed. It was.

ｎは１〜５の整数、Ｒは炭素数１〜４のアルキルを示す。
（２）ｎが２であり、Ｒがメチルである、（１）の化合物。
（３）（１）または（２）の化合物を細胞に添加し、蛍光を測定することを特徴とする、細胞内コレステロールの検出方法。
（４）（１）または（２）の化合物を含む、コレステロール検出キット。
That is, the present invention is as follows.
(1) A compound represented by the following general formula (I).

n represents an integer of 1 to 5, and R represents an alkyl having 1 to 4 carbon atoms.
(2) The compound of (1), wherein n is 2 and R is methyl.
(3) A method for detecting intracellular cholesterol, comprising adding the compound of (1) or (2) to a cell and measuring fluorescence.
(4) A cholesterol detection kit comprising the compound of (1) or (2).

The novel fluorescent compound of the present invention has higher fluorescence intensity than conventional products and can be immunostained, and therefore can be widely applied to visualization of cholesterol in fixed cells and living cells.

ここで、ｎは１〜５の整数、Ｒは炭素数１〜４のアルキルを示す。なお、Ｒは互いに異なっていてもよい。一般式（Ｉ）の化合物としては、ｎが２であり、Ｒがメチルである、下記の化合物が特に好ましい。

The present invention is described in detail below.
The compound of the present invention is represented by the following general formula (I).

Here, n represents an integer of 1 to 5, and R represents an alkyl having 1 to 4 carbon atoms. Note that R may be different from each other. As the compound of the general formula (I), the following compounds in which n is 2 and R is methyl are particularly preferable.

This compound can be synthesized by reacting amphotericin B with dansylated ethylenediamine by the following synthesis method. In addition, compounds other than the compound in which n is 2 and R is methyl can be synthesized in the same manner by changing raw materials.

The compounds of the present invention fluoresce. Therefore, it can be used as a fluorescent labeling agent or a fluorescent probe.
In particular, since the compound of the present invention is taken up into cells and exhibits the same behavior as cholesterol, it can be suitably used for detection of intracellular cholesterol.

Specifically, the distribution of intracellular cholesterol and the like can be detected by adding the compound of the present invention to cells and measuring the fluorescence with a fluorescence microscope or the like.
The type of cells to be detected is not particularly limited, but cultured cells that accumulate cholesterol are preferred. Fluorescence may be observed directly in a living cell, or fluorescence may be observed after fixing the cell.
When the compound of the present invention is added alone, for example, the compound can be dissolved in DMSO and added.
The compound of the present invention can be added to the culture medium of cultured cells. The addition concentration varies depending on the cell type, but is preferably 0.5 μM to 50 μM.
Fluorescence due to the compound of the present invention can be detected by using a fluorescence measuring device such as a fluorescence microscope.
It is also possible to detect using an antibody that recognizes the compound of the present invention. Examples of such an antibody include an antibody against a dansyl group contained in the compound of the present invention (Molecular Probes: Anti-Dansyl Antibody (Catalog. #: A-6398)).

The present invention also relates to a cholesterol detection kit comprising the compound of the present invention. The kit may also contain an antibody against the compound of the present invention.

  The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples. In the following examples, a compound having n = 2 and R = methyl in the general formula (I) is referred to as dansylated amphotericin B (Amp-en-Ds).

Synthesis Example 1 Synthesis of mono-N-dansyl-ethylendiamine (Ds-en) 3.34 ml (50 mmol) of ethylenediamine was dissolved in 40 ml of distilled dichloromethane and stirred at room temperature. To this solution, 2.698 g (10 mmol) of Dansyl chloride was added little by little over 10 minutes and reacted at room temperature for 15 hours. 50 ml of water was added to the reaction mixture and transferred to a separatory funnel. The organic phase was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After concentration under reduced pressure, hexane was added to the residue, and the resulting pale yellow solid was collected by filtration to obtain 2.04 g (6.95 mmol) of Ds-en. (Yield 69.5% (calculated based on Dansyl chloride))
mp: 153-154 ° C, ¹ H NMR (300MHz, CDCl ₃ ) δ8.54 (1H, d, J = 8.5Hz), 8.30 (d, 1H, J = 8.6Hz), 8.25 (d, 1H, J = 7.3Hz), 7.56 (dd, 1H, J ₁ = 8.5Hz, J ₂ = 7.6Hz), 7.52 (dd, 1H, J ₁ = 8.5Hz, J ₂ = 7.6Hz), 7.18 (d, 1H, J = 7.4Hz), 2.90 (dd, 2H, J ₁ = 5.3Hz, J ₂ = 6.5Hz), 2.89 (s, 6H), 2.69 (dd, 2H, J ₁ = 5.3Hz, J ₂ = 6.5Hz)

2. Synthesis of dansylated amphotericin B (Amp-en-Ds)
N-Fmoc-amphotericin B (N-Fmoc-Amp) was synthesized by a method known in the literature. (Reference: M. Murata et al. Org. Lett. 2002, 4, 2087-2089.) N-Fmoc-Amp 55 mg (0.044 mmol) was dissolved in 5 ml of DMF, and Ds-en 16 mg (0.05 mmol) was stirred with ice cooling. ), PyBOP (trademark: Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) 27 mg (0.05 mmol), DIEA (diisopropylethylamine) 18 μl (0.106 mmol) were added, and the mixture was stirred for 1 hour under ice cooling and 15 hours at room temperature. After distilling off DMF under reduced pressure, distilled diethyl ether was added to the residue, and the deposited precipitate was collected by filtration. The obtained crude product was crystallized from diethyl ether-methanol to obtain 46 mg (0.032 mmol) of Fmoc-Amp-en-Ds. (Yield 73%) This was dissolved in 5 ml of 2% DBU (diazabicycloundecene) / DMF solution and treated at room temperature for 10 minutes to remove the Fmoc group. After concentration under reduced pressure, distilled ether was added to the resulting residue, and the resulting precipitate was collected by filtration. This precipitate was washed with distilled diethyl ether and dried to obtain 38 mg of Amp-en-Ds. The (quantitative) Amp-en-Ds ¹ H NMR spectrum (300 MHz, DMSO-d ₆ ) is shown in FIG.
mp:> 300 ℃, atmospheric pressure ionization mass spectrum (APCI-MS, positive) m / z = 1199.9 ([M + 3H] ⁺ )

Example 2: Absorption / fluorescence spectrum of Amp-en-Ds
The ultraviolet absorption spectra of Amp-en-Ds and amphotericin B (Amp) were measured with an ultraviolet / visible spectrophotometer (JASCO, Ubest V-550 UV / VIS). The sample was dissolved in MeOH for spectroscopic measurement and measured at room temperature using a square quartz cell with an optical path length of 1 cm. The fluorescence spectrum was measured with a fluorescence spectrophotometer (Hitachi, F-4010). For the measurement, the same sample solution as the ultraviolet absorption spectrum measurement was used, and the measurement was performed at room temperature using a square quartz cell having an optical path length of 1 cm.
The results are shown in FIGS. Amp-en-Ds was confirmed to absorb dansyl groups in addition to the absorption of amphotericin B. In addition, fluorescence was observed at about 510 nm.

Example 3: Fluorescence measurement in cultured cells Macrophage cells (RAW264.7) were taken up with amphotericin B, Amp-en-Ds, Philippine III DMSO solution (concentration 5μM, 75μM) for 1 hour and stained with a fluorescence microscope Was taken. (Figure 4)
The upper two rows are microscopic images when 5 μM and 75 μM of each compound are added to formalin-immobilized cells, and the lower two rows are microscopic images when each compound is added to living cells at 5 μM and 75 μM.
As a result, it was found that Amp-en-Ds was taken up into cells and showed stronger fluorescence than Philippines III, which is a known cholesterol probe. No fluorescence was seen with amphotericin B alone.

¹ H NMR spectrum of Amp-en-Ds. Amp-en-Ds (solid line) and Amp (dashed line) ultraviolet absorption spectra (MeOH). Amp-en-Ds (solid line) and Amp (dashed line) fluorescence spectra (MeOH excitation wavelength 340 nm) The figure (photograph) which shows the fluorescence-staining result of the macrophage cell (RAW264.7) by amphotericin B, Amp-en-Ds, or Philippines III (concentration 5 micromol, 75 micromol). The upper two rows show the results of fluorescent staining when each compound was added to formalin-fixed cells at 5 μM and 75 μM, and the lower two rows show the results of fluorescent staining when each compound was added to live cells at 5 μM and 75 μM.

Claims (4)

The compound represented by the following general formula (I).

n represents an integer of 1 to 5, and R represents an alkyl having 1 to 4 carbon atoms. 2. A compound according to claim 1 wherein n is 2 and R is methyl. A method for detecting intracellular cholesterol, comprising adding the compound according to claim 1 or 2 to a cell and measuring fluorescence. A cholesterol detection kit comprising the compound according to claim 1 or 2.

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