JP2008209361A - Lipid membrane localized fluorescent probe - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent probe for accurately obtaining a behavior of a hydroxyl radical in a lipid membrane. <P>SOLUTION: A structure includes: a functional fluorescent molecular unit for reacting to the hydroxyl radical, and changing an intensity or a wavelength; and a hydrophobic molecular unit including a phospholipid structure for applying a lipid membrane localization to the fluorescent probe. The fluorescent probe is a compound expressed by formula (A) for example. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a fluorescent probe for detecting a physiologically active species to be measured in a lipid membrane and in or near the lipid membrane.

  The concentration of physiologically active species present in the living body is constantly changing, which leads to maintaining the homeostasis of the living body. Therefore, if it is desired to understand the biological system in detail, it is indispensable to obtain dynamic information on the biologically active species in a state where the living organism is alive.

  There is an analysis method using a fluorescent probe as a method for obtaining dynamic information on a biologically active species in a living state.

  The non-destructive analysis method using a fluorescent probe can obtain temporal and spatial information on bioactive species that could not be obtained by the conventional destructive analysis method of measuring activity after disrupting cells. In recent years, it has attracted a great deal of attention as an effective tool for elucidating the dynamics of physiologically active species in cells (see Non-Patent Document 1).

Various fluorescent probes have been proposed, but the beginning of the fluorescent probe is a fluorescent probe for Ca ²⁺ measurement (see Non-Patent Document 2-4), and thereafter, various fluorescent probes for measuring metal ions are mainly used. Fluorescent probes have been developed and their usefulness has been proven.

  However, in the development of conventional fluorescent probes, the main focus is on capturing the behavior of physiologically active species in the cytoplasm, and attention is focused on capturing the behavior of physiologically active species in areas other than the cytoplasm, that is, lipid membranes (cell membranes). It wasn't.

  In recent years, there has been an increasing demand for a comprehensive understanding of intracellular processes, and accordingly, the importance of elucidating the behavior of physiologically active species other than the cytoplasm, that is, lipid membranes (cell membranes) and the like is also increasing.

  For example, lipid peroxidation, which is thought to be deeply involved in various diseases and aging, is a reaction that proceeds in the lipid membrane. If the behavior of physiologically active species involved in this lipid peroxidation can be clarified, It helps to know the mechanism of diseases and aging.

This lipid peroxidation reaction is said to be mainly induced by hydroxyl radicals. However, terephthalic acid known as an existing fluorescent probe for measuring hydroxyl radical (see Non-Patent Documents 5 and 6), fluorescent group-labeled nitroxide (see Non-Patent Documents 7-10), HPF and AFP (see Non-Patent Document 11), Fluorescent group-labeled DNA (see Non-Patent Document 12), CCA and SECCA (see Non-Patent Documents 13-15) are both developed for the purpose of measuring hydroxyl radicals in aqueous solutions. It was difficult to use it for the purpose of accurately capturing the behavior.
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  The problem to be solved is that it has been difficult to use conventional fluorescent probes for the purpose of accurately capturing the behavior of physiologically active species such as hydroxyl radicals in lipid membranes.

  The fluorescent probe according to the present invention has a functional fluorescent molecular unit responsible for changing fluorescence by capturing or reacting with a biologically active species, and a role of imparting lipid membrane localization to the fluorescent probe. The main feature is to have in its structure a hydrophobic (lipid-soluble) molecular unit responsible for. The functional fluorescent molecular unit and the hydrophobic (lipid-soluble) molecular unit may be directly bonded or via a linker. A linker will not be specifically limited if both are couple | bonded. For example, the C1-C6 alkyl group which may have a substituent can be mentioned.

  Here, examples of the functional fluorescent molecular unit include those that react with reactive oxygen species, more specifically, hydroxyl radicals, and change in fluorescence intensity or wavelength. Examples of the functional fluorescent molecular unit that changes its fluorescence intensity or wavelength upon reaction with a hydroxyl radical include those having a coumarin skeleton or a phenoxazone skeleton.

  Examples of the hydrophobic molecular unit include those having a lipid structure in the molecular structure. Examples of lipids constituting the lipid structure include simple lipids, complex lipids, and isoprenoids. Complex lipids are preferred as lipids constituting this lipid structure, and examples of complex lipids include phospholipids, glycolipids, and sphingolipids. Examples of phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), and the like. When the various lipids described above are chemically bonded to the functional fluorescent molecular unit, the structure of the binding site is partly changed from the structure of the original lipid molecule. Therefore, the lipid mentioned here includes a lipid derivative formed with this chemical bond.

（式中、Rは水素原子又はＣ1-18アルキル基を示し、かつn = ８〜２０）で表される化合物又はその塩が提供される。 That is, as the fluorescent probe according to the present invention, for example, the following general formula (A):

(Wherein R represents a hydrogen atom or a C1-18 alkyl group, and n = 8 to 20) or a salt thereof is provided.

  As a more preferred embodiment of the above invention, there is provided a compound or a salt thereof, wherein, in the general formula (A), R is a hydrogen atom and n = 14.

  The fluorescent probe according to the present invention is based on the hydrophobic interaction between the hydrophobic molecular unit and the lipid membrane, and is localized in the lipid membrane, and further based on the action of the functional fluorescent molecule, Since the fluorescence intensity or wavelength changes by reacting with hydroxyl radical, there is an advantage that a physiologically active substance existing in or near the lipid membrane can be effectively detected.

  Fluorescence responsiveness of functional fluorescent molecular units with a simple configuration that binds hydrophobic molecular units to functional fluorescent molecular units that respond to biologically active species in order to accurately capture the behavior of biologically active species in lipid membranes Realized without compromising. As an example, a fluorescent probe in which a phosphatidylethanolamine (PE) derivative is bound to a functional fluorescent molecular unit having a coumarin skeleton that responds to hydroxyl radicals has been developed, and the above object has been achieved. EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.

(Experiment 1) Synthesis of fluorescent probe for hydroxyl radical measurement (1,2-dipalmitoylglycerophosphorylethanolamine labeled with coumarin: DPPEC) [Experimental operation]
Coumarin-3-carboxylic acid (CCA), 1-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride (EDC), 1-hydroxy-7-azabenzo-triazole (HOAt) as a mixed solvent of CHCl ₃ and DMF Dissolved and stirred at room temperature for 1 hour. Thereafter, 1,2-dipalmitoylglycerophosphorylethanolamine (DPPE) and triethylamine (TEA) were added to the reaction solution and stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure using an evaporator, followed by extraction and purification by silica gel column chromatography to obtain a white solid.

〔Experimental result〕
¹ H-NMR measurement and mass spectrometry (MALDI-TOF / MS) measurement confirmed that the intended DPPEC was obtained.

(Experiment 2) Evaluation of DPPEC's ability to detect hydroxyl radicals (generates various amounts of hydroxyl radicals)
In order to evaluate DPPEC's ability to detect hydroxyl radicals, the hydroxyl radicals generated by the redox decomposition reaction of copper-mediated H ₂ O ₂ to DPPEC introduced into the liposomes of 1,2-dipalmitoyl-sn-glycerophosphorylcholine (DPPC) Fluorescence spectrum measurement was performed.

[Experimental operation]
DPPC 8.5 mg was dissolved in CHCl ₃ and 50 mg of 10 mg / mL DPPEC solution was added. Then, the solvent was removed while flowing N ₂ in a 55 ° C. water bath. A 0.1 M phosphate buffer (pH 7.4) was added, vortexed, and then irradiated with ultrasound to form DPPEC-introduced liposomes (100 μM DPPEC / 2 mM DPPC).

To 250 μL of 100 μM DPPEC-introduced liposome solution, 0.1 M phosphate buffer (pH 7.4) was added, and excitation and fluorescence spectra were measured (final concentration: 50 μM DPPEC). Then, various concentrations of CuSO ₄ aqueous solution and H ₂ O ₂ aqueous solution were added, and time course measurement was started (final concentration: 50 μM DPPEC, 0, 0.1, 1, 5, 10, 50 μM CuSO ₄ , 1 mM H ₂ O ₂ ). 5 minutes after the start of measurement, sodium ascorbate aqueous solution was added (final concentration: 50 μM DPPEC, 0, 0.1, 1, 5, 10, 50 μM CuSO ₄ , 1 mM H ₂ O ₂ , 100 μM ascorbic acid. ). After an additional 60 minutes, the fluorescence spectrum was measured. The measurement conditions were 37 ° C., excitation wavelength 400 nm, and fluorescence wavelength 444 nm.

〔Experimental result〕
FIG. 1 shows changes over time in the fluorescence intensity ratio when various concentrations of hydroxyl radicals are allowed to act on DPPEC-introduced liposomes, and FIG. 2 shows the results of fluorescence spectrum measurement. As shown in FIG. 1, it became clear that DPPEC immediately increased the fluorescence intensity ratio at the maximum fluorescence wavelength with the addition of ascorbic acid. Furthermore, it was confirmed that this increase in the fluorescence intensity ratio increases with increasing Cu ²⁺ concentration. In addition, as shown in FIG. 2, it was confirmed that the fluorescence intensity of the fluorescence spectrum increases as the Cu ²⁺ concentration increases. Therefore, it is considered that the coumarin part of DPPEC formed a 7-hydroxy form, which is a strong fluorescent substance, by reaction with the hydroxyl radical. From the above results, it was shown that DPPEC has hydroxyl radical detection ability.

(Experiment 3) Evaluation of hydroxyl radical detectability of DPPEC (addition of hydroxyl radical scavenger)
DMSO is known as a representative scavenger for hydroxyl radicals. Therefore, in order to confirm that DPPEC reacts with hydroxyl radicals to cause fluorescence changes, the effect of DMSO on hydroxyl radical detection using DPPEC was evaluated.

[Experimental operation]
100 μM DPPEC-introduced liposome solution to 250 μL, 0.1 M phosphate buffer (pH 7.4) containing various concentrations of DMSO, CuSO ₄ aqueous solution, and H ₂ O ₂ aqueous solution were added, and time course measurement was performed. (Final concentrations: 50 μM DPPEC, 10 μM CuSO ₄ , 1 mM H ₂ O ₂ , 0, 0.1, 1, 10 mM DMSO). 5 minutes after the start of measurement, sodium ascorbate aqueous solution was added (final concentration: 50 μM DPPEC, 10 μM CuSO ₄ , 1 mM H ₂ O ₂ , 100 μM ascorbic acid, 0, 0.1, 1, 10 mM DMSO ). The measurement conditions were 37 ° C., excitation wavelength 400 nm, and fluorescence wavelength 444 nm.

On the other hand, 0.1 M phosphate buffer (pH 7.4), CuSO ₄ aqueous solution and H ₂ O ₂ aqueous solution were added to 250 μL of 100 μM DPPEC-introduced liposome solution, and time course measurement was started (final) Concentration: 50 μM DPPEC, 10 μM CuSO ₄ , 1 mM H ₂ O ₂ ). Five minutes after the start of measurement, an aqueous sodium ascorbate solution was added (final concentrations: 50 μM DPPEC, 10 μM CuSO ₄ , 1 mM H ₂ O ₂ , 100 μM ascorbic acid). Furthermore, 7 minutes after the start of measurement, an aqueous DMSO solution was added (final concentrations: 50 μM DPPEC, 10 μM CuSO ₄ , 1 mM H ₂ O ₂ , 100 μM ascorbic acid, 10 mM DMSO). The measurement conditions were 37 ° C., excitation wavelength 400 nm, and fluorescence wavelength 444 nm.

〔Experimental result〕
The results when DMSO is added to the measurement sample in advance are shown in FIG. As can be seen from this, it was confirmed that the increase in the fluorescence intensity ratio accompanying the generation of hydroxyl radicals was reduced by the influence of DMSO. On the other hand, when DMSO was added while the fluorescence intensity was increasing due to the generation of hydroxyl radicals, the increase in the fluorescence intensity ratio was suppressed immediately after the addition, as shown in FIG. These results may be attributed to the fact that the generated hydroxyl radical was eliminated by DMSO, preventing the formation of the 7-hydroxy form of DPPEC. From the above results, it was confirmed that the fluorescence change of DPPEC was based on the reaction with hydroxyl radical.

(Experiment 4) Hydroxyl radical selectivity evaluation of DPPEC
In order to evaluate the selectivity of DPPEC for hydroxyl radical, peroxynitrite (ONOO ⁻ ), hypochlorite ion (OCl ⁻ ), superoxide anion (· O ₂ ⁻ ), Fluorescence measurement was performed by reacting hydrogen oxide (H ₂ O ₂ ), nitric oxide (NO), and peroxy radical (ROO.).

[Experimental operation]
To 250 μL of liposome solution introduced with 100 μM DPPEC, 0.1 M phosphate buffer (pH 7.4) was added, and time course measurement was started (final concentration: 50 μM DPPEC). Five minutes after the start of measurement, various concentrations of reactive oxygen species generator were added (final concentrations: 50 μM TDFT, {50 μM ONOO ⁻ , 50 μM NaOCl (50 μM OCl ⁻ ), 50 μM KO ₂ (50 μM • ^{_{O 2 -), 50 μM H}} 2 O 2, 25 μM NOC 7 (50 μM NO), 25 μM 2, 2'-azobis (2-amidinopropane) dihydrochloride (50 μM ROO ·)}). Similarly, a hydroxyl radical was generated by adding a sodium ascorbate solution 5 minutes after the addition of CuSO ₄ aqueous solution and H ₂ O ₂ aqueous solution (final concentration: 50 μM DPPEC, {[CuSO ₄ ] / [H ₂ O ₂ ] / [ascorbic acid]: 50 μM / 500 μM / 25 μM, 50 μM / 500 μM / 50 μM (50, 100 μM OH)}). The measurement conditions were 37 ° C., excitation wavelength 400 nm, and fluorescence wavelength 444 nm.

〔Experimental result〕
FIG. 5 shows changes with time in the fluorescence intensity ratio of DPPEC when various reactive oxygen species are allowed to act. As is clear from this, it was revealed that DPPEC does not show a significant fluorescence change for reactive oxygen species other than the hydroxyl radical. This confirmed that DPPEC has excellent selectivity for hydroxyl radicals.

(Experiment 5) Evaluation of lipid membrane localization
DPPEC has a phospholipid site that is hydrophobic to confer lipid membrane localization. Therefore, we evaluated the plasma membrane localization of the fluorescent probe using DPPEHC and 7-OH-CCA, which are phosphors formed by the reaction of hydroxyl radicals with the new fluorescent probe DPPEC and the existing probe CCA. .

[Experimental operation]
DPPEHC or CCA was added to the cultured RAW264 cells and incubated at 37 ° C. Thereafter, washing was performed by centrifugation, a buffer solution was added, and fluorescence was measured with a plate reader. A cell suspension solution to which no fluorescent probe was added was used as a blank.

〔Experimental result〕
The result of fluorescence measurement with a plate reader is shown in FIG. As can be seen from FIG. 6, 7-OH-CCA having no hydrophobic site showed fluorescence intensity equivalent to that of the blank, and it was confirmed that it did not have cell membrane localization. On the other hand, DPPEHC showed stronger fluorescence intensity and was found to be localized in the cell membrane. From the above, it is considered that DPPEC has excellent cell membrane localization.

It is a graph showing the time change of the fluorescence intensity ratio when various concentrations of hydroxyl radicals are allowed to act on DPPEC-introduced liposomes (the generated reagent is a mixed solution of copper (II) sulfate, hydrogen peroxide and ascorbic acid, added The concentration of copper sulfate (II) to be changed was changed). It is a graph showing the relationship between the fluorescence intensity and the fluorescence wavelength (nm) when hydroxyl radicals of various concentrations are allowed to act on DPPEC-introduced liposomes (the generated reagents are copper sulfate (II), hydrogen peroxide, ascorbic acid) The concentration of copper sulfate (II) to be added was changed in the mixed solution). It is a graph which shows the time change (when DMSO is added beforehand) when a hydroxyl radical is made to act on a DPPEC introduction | transduction liposome (A generation reagent is copper sulfate (II), hydrogen peroxide, ascorbine). Finally, ascorbic acid was added in the acid mixture solution). It is a graph which shows the time change (when DMSO is added on the way) when the hydroxyl radical of various concentrations is made to act on DPPEC introduction liposome (the generation reagent is copper (II) sulfate, hydrogen peroxide) Ascorbic acid was finally added in a mixed solution of ascorbic acid, and DMSO was added in the middle of the fluorescence increase observed thereby). It is a graph which shows the time change of the fluorescence intensity ratio at the time of making various reactive oxygen species act with respect to a DPPEC introduction | transduction liposome. It is the graph which measured the fluorescence intensity of the cell suspension solution when DPPEHC or 7-OH-CCA was added to RAW264 cells and then washed, using a plate reader (the result when no fluorescent probe is added is also shown as a reference).

Claims (11)

  A fluorescent probe comprising a functional fluorescent molecular unit that changes fluorescence intensity or wavelength by reacting with a biologically active species, and a hydrophobic molecular unit.   The fluorescent probe according to claim 1, wherein the functional fluorescent molecular unit reacts with reactive oxygen species to change fluorescence intensity or wavelength.   The fluorescent probe according to claim 1 or 2, wherein the functional fluorescent molecular unit reacts with a hydroxyl radical to change fluorescence intensity or wavelength.   The fluorescent probe according to claim 1, wherein the functional fluorescent molecular unit has a coumarin skeleton.   The fluorescent probe according to claim 1, wherein the functional fluorescent molecular unit has a phenoxazone skeleton.   The fluorescent probe according to claim 1, wherein the hydrophobic molecular unit includes a lipid structure.   The fluorescent probe according to claim 1, wherein the hydrophobic molecular unit includes a complex lipid structure.   The fluorescent probe according to claim 1, wherein the hydrophobic molecular unit includes a phospholipid structure.   The fluorescent probe according to claim 1, wherein the hydrophobic molecular unit includes a phosphatidylethanolamine derivative structure. The following general formula (A):

(Wherein R represents a hydrogen atom or a C1-18 alkyl group, and n = 8 to 20) or a salt thereof.   The compound or a salt thereof according to claim 10, wherein R is a hydrogen atom and n = 14.

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