JP5561692B2 - Novel fluorescent compound and method for detecting intracellular cholesterol using the same - Google Patents
Novel fluorescent compound and method for detecting intracellular cholesterol using the same Download PDFInfo
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 title claims description 72
- 235000012000 cholesterol Nutrition 0.000 title claims description 36
- 230000003834 intracellular effect Effects 0.000 title claims description 13
- 238000000034 method Methods 0.000 title claims description 9
- 239000007850 fluorescent dye Substances 0.000 title description 5
- 150000001875 compounds Chemical class 0.000 claims description 44
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 10
- 229910052760 oxygen Inorganic materials 0.000 claims description 10
- 239000001301 oxygen Substances 0.000 claims description 10
- 229910052739 hydrogen Inorganic materials 0.000 claims description 9
- 239000001257 hydrogen Substances 0.000 claims description 9
- 150000002431 hydrogen Chemical class 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 8
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 7
- 229910052711 selenium Chemical group 0.000 claims description 7
- 239000011669 selenium Chemical group 0.000 claims description 7
- 229910052717 sulfur Inorganic materials 0.000 claims description 7
- 239000011593 sulfur Chemical group 0.000 claims description 7
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical group [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 6
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 6
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 12
- GZDFHIJNHHMENY-UHFFFAOYSA-N Dimethyl dicarbonate Chemical compound COC(=O)OC(=O)OC GZDFHIJNHHMENY-UHFFFAOYSA-N 0.000 description 10
- 238000010521 absorption reaction Methods 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- ZCVAGTPWBAZXAL-UHFFFAOYSA-N 4-nitro-2,1,3-benzoxadiazole Chemical compound [O-][N+](=O)C1=CC=CC2=NON=C12 ZCVAGTPWBAZXAL-UHFFFAOYSA-N 0.000 description 5
- 238000000862 absorption spectrum Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000002189 fluorescence spectrum Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- HIAJCGFYHIANNA-QIZZZRFXSA-N 3b-Hydroxy-5-cholenoic acid Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@@H](CCC(O)=O)C)[C@@]1(C)CC2 HIAJCGFYHIANNA-QIZZZRFXSA-N 0.000 description 3
- IGHBXJSNZCFXNK-UHFFFAOYSA-N 4-chloro-7-nitrobenzofurazan Chemical compound [O-][N+](=O)C1=CC=C(Cl)C2=NON=C12 IGHBXJSNZCFXNK-UHFFFAOYSA-N 0.000 description 3
- YZWANFXENNWOOR-UHFFFAOYSA-N 7-fluoro-n,n-dimethyl-2,1,3-benzoxadiazole-4-sulfonamide Chemical compound CN(C)S(=O)(=O)C1=CC=C(F)C2=NON=C12 YZWANFXENNWOOR-UHFFFAOYSA-N 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 3
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 2
- BMTZEAOGFDXDAD-UHFFFAOYSA-M 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium;chloride Chemical compound [Cl-].COC1=NC(OC)=NC([N+]2(C)CCOCC2)=N1 BMTZEAOGFDXDAD-UHFFFAOYSA-M 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
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- 150000003904 phospholipids Chemical class 0.000 description 2
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- 239000002356 single layer Substances 0.000 description 2
- AOCSUUGBCMTKJH-UHFFFAOYSA-N tert-butyl n-(2-aminoethyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCN AOCSUUGBCMTKJH-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MHUPZGBELMNFET-HGZXIFPPSA-N (4r)-4-[(8s,9s,10r,13r,14s,17r)-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoic acid Chemical compound C1C=C2CCCC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@@H](CCC(O)=O)C)[C@@]1(C)CC2 MHUPZGBELMNFET-HGZXIFPPSA-N 0.000 description 1
- CITHEXJVPOWHKC-UUWRZZSWSA-N 1,2-di-O-myristoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCC CITHEXJVPOWHKC-UUWRZZSWSA-N 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- QSVJYFLQYMVBDR-UHFFFAOYSA-N Ergosterin Natural products C1C(O)CCC2(C)C3=CCC4(C)C(C(C)C=CC(C)C(C)C)CCC4C3=CC=C21 QSVJYFLQYMVBDR-UHFFFAOYSA-N 0.000 description 1
- 208000001145 Metabolic Syndrome Diseases 0.000 description 1
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 description 1
- 150000007960 acetonitrile Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 150000001841 cholesterols Chemical class 0.000 description 1
- -1 dansyl hydrazone Chemical class 0.000 description 1
- QSVJYFLQYMVBDR-CMNOFMQQSA-N dehydroergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)C3=CC[C@]4(C)[C@@H]([C@H](C)/C=C/[C@H](C)C(C)C)CC[C@H]4C3=CC=C21 QSVJYFLQYMVBDR-CMNOFMQQSA-N 0.000 description 1
- 229960003724 dimyristoylphosphatidylcholine Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- XDWBVLYUTTXKNE-UHFFFAOYSA-N n,n-dimethyl-2,1,3-benzoxadiazole-4-sulfonamide Chemical compound CN(C)S(=O)(=O)C1=CC=CC2=NON=C12 XDWBVLYUTTXKNE-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 125000003748 selenium group Chemical group *[Se]* 0.000 description 1
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- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Steroid Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
本発明は新規蛍光化合物に関する。本発明はまた、当該化合物を用いた細胞内コレステロール検出法および検出キットに関する。 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.
コレステロールは生体膜の重要な構成成分であり、主に肝臓で生合性される内因性コレステロールの分泌・代謝異常は、メタボリックシンドロームやホルモン分泌異常の起因となると考えられている。そのため、コレステロールの細胞内分布・動能を精密に計測する技術が重要視されている。
生体内のコレステロールの分布や動態を解析する上で、蛍光分光法は、高感度な分光法のため有用である。現在では、共焦点レーザー顕微鏡、二光子励起蛍光顕微鏡など測定機器が飛躍的に進歩し、高い時空間分解能で分布や動態を解析することができる。しかしながらコレステロールは蛍光を示さないため、これらの技術を用いるためには、コレステロールに蛍光分子を標識させる必要があり、そのような蛍光性コレステロールが開発されている。ここで、重要な要素として蛍光団を結合させても、コレステロール本来の性質を保持させなければならないことが挙げられる。以下、これまで開発されている蛍光性コレステロールおよび問題点を示す。デヒドロエルゴステロール（A）はコレステロールに分子構造が類似しているため内因性コレステロールに近い性質を有する。しかしながら、蛍光強度が弱いため精度の高い解析が困難である。Molecular Probes社（米国）において、コレステロールの３位のヒドロキシル基をエステル化して蛍光団を組み込んだ化合物（B）が販売されている。しかしながら、細胞内に存在するコレステロールは、３位のヒドロキシル基が遊離して存在しており、細胞内コレステロールの動態解析には不向きである。また、同社は、コレステロールの２２位にニトロベンゾフラザンを置換した化合物（C）を市販している。しかしながら、Cはミトコンドリアの膜に特異的に集積してしまいコレステロールの性質を保持していないことが報告されている[非特許文献1]。コレステロールの７位にダンシルヒドラゾン、24位にダンシルスルホンアミドを置換した化合物（D、E）が開発されている。D、Eは、内因性コレステロールの性質に近いことが報告されており、蛍光性コレステロールアナログとして使用されている[非特許文献2、特許文献１]。しかしながら、吸収が紫外光領域のため、市販の共焦点レーザー顕微鏡を用いることが困難である。
In analyzing the distribution and dynamics of cholesterol in a living body, fluorescence spectroscopy is useful because of high-sensitivity spectroscopy. At present, measuring instruments such as confocal laser microscopes and two-photon excitation fluorescence microscopes have made great progress and can analyze distribution and dynamics with high spatiotemporal resolution. However, since cholesterol does not exhibit fluorescence, in order to use these techniques, it is necessary to label the cholesterol with a fluorescent molecule, and such fluorescent cholesterol has been developed. Here, it is mentioned that even if a fluorophore is bound as an important element, the original properties of cholesterol must be maintained. Hereinafter, fluorescent cholesterol and problems that have been developed so far are shown. Dehydroergosterol ( A ) has properties similar to endogenous cholesterol because of its molecular structure similar to cholesterol. However, since the fluorescence intensity is weak, it is difficult to analyze with high accuracy. Molecular Probes (USA) sells a compound ( B ) in which a hydroxyl group at the 3-position of cholesterol is esterified to incorporate a fluorophore. However, cholesterol present in cells is present in a state where the hydroxyl group at the 3-position is liberated, and is not suitable for analyzing the dynamics of intracellular cholesterol. The company also sells a compound ( C ) in which nitrobenzofurazan is substituted at the 22-position of cholesterol. However, it has been reported that C accumulates specifically in the mitochondrial membrane and does not retain the properties of cholesterol [Non-patent Document 1]. Compounds ( D , E ) in which dansyl hydrazone is substituted at the 7th position of cholesterol and dansylsulfonamide at the 24th position have been developed. D and E have been reported to be close to the properties of endogenous cholesterol, and are used as fluorescent cholesterol analogs [Non-patent Document 2, Patent Document 1]. However, since the absorption is in the ultraviolet region, it is difficult to use a commercially available confocal laser microscope.
本発明は、細胞内コレステロールの検出などに有用な、新規な蛍光化合物を提供することを課題とする。 An object of the present invention is to provide a novel fluorescent compound useful for detection of intracellular cholesterol and the like.
本発明者は上記課題を解決すべく鋭意検討を行った。その結果、下記一般式（Ｉ）および（II）で表される蛍光化合物（以下、本発明の化合物とも呼ぶ）を合成することに成功
し、さらに、当該化合物が細胞内でコレステロールと同様の挙動を示すことを見出して本発明を完成するに至った。
The present inventor has intensively studied to solve the above problems. As a result, the inventors succeeded in synthesizing fluorescent compounds represented by the following general formulas (I) and (II) (hereinafter also referred to as the compounds of the present invention), and the compounds behave in the same manner as cholesterol in cells. As a result, the present invention has been completed.
すなわち、本発明は以下の通りである。
（１）下記一般式（I）または（II）で表される化合物。
（２）ｎが２であり、R1およびR2が水素であり、Ｘが酸素であり、ｍが０である、（１）に記載の化合物。
（３）（１）または（２）に記載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）に記載の化合物を含む、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) or (II).
(2) The compound according to (1), wherein n is 2, R 1 and R 2 are hydrogen, X is oxygen, and m is 0.
(3) A method for detecting intracellular cholesterol, comprising adding the compound according to (1) or (2) to a cell and measuring fluorescence.
(4) A cholesterol detection kit comprising the compound according to (1) or (2).
本発明の蛍光化合物は、細胞内に取り込まれ、コレステロールと同様の挙動を示すため、細胞内コレステロールの検出に好適に使用することができる。例えば、細胞内のコレステロール含有小胞の同定など、細胞内コレステロール分布や動態の解析などに使用することができる。本発明の化合物は吸収が可視光領域にあるため、検出が容易である。 Since the fluorescent compound of the present invention is taken into cells and exhibits the same behavior as cholesterol, it can be suitably used for detection of intracellular cholesterol. For example, it can be used for analysis of intracellular cholesterol distribution and dynamics, such as identification of intracellular cholesterol-containing vesicles. Since the compound of the present invention has absorption in the visible light region, it is easy to detect.
以下に本発明を詳しく説明する。
本発明の化合物は、以下の一般式（I）または（II）で表される。
The compound of the present invention is represented by the following general formula (I) or (II).
この中では、ｎが２であり、R1およびR2が水素であり、Ｘが酸素であり、ｍが０である、下記の化合物が特に好ましい。
これらの化合物は後述の合成例１、２に記載の方法によって合成することができる。なお、ｎが２であり、R1およびR2が水素であり、Ｘが酸素であり、ｍが０である化合物以外の化合物も原料を代えることによって同様にして合成することができる。 These compounds can be synthesized by the methods described in Synthesis Examples 1 and 2 described later. A compound other than the compound in which n is 2, R 1 and R 2 are hydrogen, X is oxygen, and m is 0 can be synthesized in the same manner by changing the raw materials.
Ｘが硫黄である化合物は、例えば、後述の合成例１、２において、NBD-Cl、DBD-Fの代わりに、下記の手順で合成された化合物を用い、これを3β-ヒドロキシ-Δ5-コレン酸と反応させることによって合成することができる。
Ｘがセレンである化合物は、例えば、後述の合成例１、２において、NBD-Cl、DBD-Fの代わりに、下記の手順で合成された化合物を用い、これを3β-ヒドロキシ-Δ5-コレン酸と反応させることによって合成することができる。
本発明の化合物は、蛍光を発する。したがって、蛍光標識剤や蛍光プローブとして使用することができる。
特に、本発明の化合物は、細胞内に取り込まれ、コレステロールと同様の挙動を示すため、細胞内コレステロールの検出に好適に使用することができる。
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.
具体的には、本発明の化合物を細胞に添加し、蛍光顕微鏡などで蛍光を測定することによって、細胞内コレステロールの分布などを検出することができる。
検出対象の細胞の種類は特に制限されないが、コレステロールを蓄積する培養細胞が好ましい。
細胞に化合物を添加する場合、本発明の化合物を単独で添加してもよいし、同化合物の溶解を助ける働きをする他の化合物とともに添加してもよい。
本発明の化合物を単独で添加する場合、例えば、同化合物をDMSOなどに溶解させることができる。
本発明の化合物は培養細胞の培地中などに加えることができる。その濃度は細胞の種類によっても異なるが、好ましくは、０．５μＭ〜５０μＭである。
蛍光顕微鏡などの蛍光測定装置を使用することによって本発明の化合物による蛍光を検出することができる。
なお、本発明の化合物を認識する抗体を用いて検出することも可能である。そのような抗体としては、本発明の化合物に含まれるニトロベンゾフラザン（NBD）基や4-(N,N-dimethylaminosulphonyl)-2,1,3-benzoxadiazole（DBD）基に対する抗体が挙げられる。
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.
When the compound is added to the cells, the compound of the present invention may be added alone, or may be added together with other compounds that function to help dissolve the compound.
When the compound of the present invention is added alone, for example, the compound can be dissolved in DMSO or the like.
The compound of the present invention can be added to the culture medium of cultured cells. The 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 antibodies include antibodies against nitrobenzofurazan (NBD) groups and 4- (N, N-dimethylaminosulphonyl) -2,1,3-benzoxadiazole (DBD) groups contained in the compounds of the present invention.
本発明はまた、本発明の化合物を含むコレステロール検出キットに関する。該キットは、本発明の化合物を溶解するための溶媒や、本発明の化合物の溶解を助ける働きをする物質をさらに含むものであってもよい。また、本発明の化合物に対する抗体を含むものであってもよい。 The present invention also relates to a cholesterol detection kit comprising the compound of the present invention. The kit may further contain a solvent for dissolving the compound of the present invention and a substance that functions to help dissolve the compound of the present invention. Moreover, the antibody with respect to the compound of this invention may be included.
以下に実施例を示し、本発明をさらに具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples.
合成例１：24NBDChoの合成
NBD-Cl（600mg、3mmol）、N-BOC-1,2-ジアミノエタン（532mg、3.3mmol）をDMF（8ml）中に溶解させ、DIEA（0.6ml、3.3mmol）を加え、N2ガスを充填し20h攪拌した。反応液にクロロホルムを加え、水で2回洗浄した。有機層をNa2SO4で乾燥させた後、エバポレーターで減圧乾固させ、カラムクロマトグラフィー（充填剤：酸化アルミニウム、展開溶媒：酢酸エチル）を用いて単離した。得られたBOC-NBD（689mg、2.1mmol）を酢酸エチル中に（8.8ml）に溶解させ、2Mの塩化水素水溶液（2M HClaq、2.1ml）を加え、3h攪拌した。反応液を酢酸エチルで洗浄し、乾燥させた。得られたNBD・HCl（495mg、1.9mmol）の286mg（1.1mmol）をメタノール中に（28ml）に溶解させ、3β-ヒドロキシ-Δ5-コレン酸（376mg、1mmol）、DIEA（250μl、1.2mmol）、DMT-MM（559mg、2.2mmol）を加え、15h攪拌し、反応液にクロロホルムを加え、エバポレーターで減圧乾固させ、カラムクロマトグラフィー（充填剤：酸化アルミニウム、展開溶媒：CH2Cl2：MeOH=9：1）を用いて単離した（収量：360mg、収率：40%）。
1H HNR （300 MHz、DMSO、RT）：δ8.47（1H、d）、7.86（1H、m）、6.15（1H、d ）、5.34 （1H、m ）、4.06 （1H、t）、3.83-3.49 （5H、m）、2.32-0.62（35H、m）.
ESI-MS m/z ： Calcd for C32H45N5NaO5 + （［M+Na］+） 602.3、m/z ： Found for 602.4
NBD-Cl (600 mg, 3 mmol), N-BOC-1,2-diaminoethane (532 mg, 3.3 mmol) are dissolved in DMF (8 ml), DIEA (0.6 ml, 3.3 mmol) is added, and N 2 gas is added. Filled and stirred for 20 h. Chloroform was added to the reaction solution and washed twice with water. The organic layer was dried over Na 2 SO 4 , dried under reduced pressure with an evaporator, and isolated using column chromatography (filler: aluminum oxide, developing solvent: ethyl acetate). The obtained BOC-NBD (689 mg, 2.1 mmol) was dissolved in ethyl acetate (8.8 ml), 2M aqueous hydrogen chloride solution (2M HClaq, 2.1 ml) was added, and the mixture was stirred for 3 h. The reaction solution was washed with ethyl acetate and dried. 286 mg (1.1 mmol) of the obtained NBD · HCl (495 mg, 1.9 mmol) was dissolved in methanol (28 ml), and 3β-hydroxy-Δ 5 -cholenic acid (376 mg, 1 mmol), DIEA (250 μl, 1.2 mmol) ), DMT-MM (559 mg, 2.2 mmol) was added, and the mixture was stirred for 15 h. Chloroform was added to the reaction solution, and the mixture was evaporated to dryness with an evaporator. Column chromatography (packing agent: aluminum oxide, developing solvent: CH 2 Cl 2 : MeOH = 9: 1) (yield: 360 mg, yield: 40%).
1 H HNR (300 MHz, DMSO, RT): δ 8.47 (1H, d), 7.86 (1H, m), 6.15 (1H, d), 5.34 (1H, m), 4.06 (1H, t), 3.83 -3.49 (5H, m), 2.32-0.62 (35H, m).
ESI-MS m / z: Calcd for C 32 H 45 N 5 NaO 5 + ([M + Na] + ) 602.3, m / z: Found for 602.4
合成例２：24DBDChoの合成
DBD-F（100mg、0.41mmol）、N-BOC-1,2-ジアミノエタン（72mg、0.44mmol）を脱水アセトニトリル（2.3ml）中に溶解させ、ジイソプロピルエチルアミン（DIEA、80μl、0.44mmol）を加え、N2ガスを充填し15h攪拌した。反応液に酢酸エチルを加え、水で2回洗浄した。有機層をNa2SO4で乾燥させた後、エバポレーターで減圧乾固させた。得られたBOC-DBDを4Mの塩酸/ジオキサン（4M HCl/dioxane）に溶解させ、1h攪拌した。反応液にTHFを加え、エバポレーターで減圧乾固させた。得られたDBD・HCl（120mg、0.38mmol）の70mg（0.22mmol）をメタノール中に（10ml）に溶解させ、3β-ヒドロキシ-Δ5-コレン酸（76mg、0.2mmol）、DIEA（43μl、0.24mmol）、クロリドn水和物（DMT - MM、55mg、0.4mmol）を加え、15h攪拌し、エバポレーターで減圧乾固させ、カラムクロマトグラフィー（充填剤：酸化アルミニウム、展開溶媒：CH2Cl2：MeOH = 95：5）を用いて単離した（収量：30mg、収率：21%）。
1H HNR （300 MHz、DMSO、RT） ： δ8.05 （1H、s）、7.80 （1H、d ）、6.35 （1H、d）、5.23（1H、m）、 4.6 （1H、d）、 3.55-3.13 （5H、m）、 2.73（6H、m）、 2.60-0.53（35H、m）.
ESI-MS m/z ：Calcd for C34H52N5O5S+ （［M+H］+） 642.9、 m/z ： Found for 642.6
DBD-F (100 mg, 0.41 mmol), N-BOC-1,2-diaminoethane (72 mg, 0.44 mmol) are dissolved in dehydrated acetonitrile (2.3 ml), and diisopropylethylamine (DIEA, 80 μl, 0.44 mmol) is added. N 2 gas was charged and stirred for 15 h. Ethyl acetate was added to the reaction solution and washed twice with water. The organic layer was dried over Na 2 SO 4 and then evaporated to dryness with an evaporator. The obtained BOC-DBD was dissolved in 4M hydrochloric acid / dioxane (4M HCl / dioxane) and stirred for 1 h. THF was added to the reaction solution, and the mixture was dried under reduced pressure using an evaporator. 70 mg (0.22 mmol) of the obtained DBD · HCl (120 mg, 0.38 mmol) was dissolved in methanol (10 ml), and 3β-hydroxy-Δ 5 -cholenoic acid (76 mg, 0.2 mmol), DIEA (43 μl, 0.24 mmol), chloride n hydrate (DMT-MM, 55 mg, 0.4 mmol), stirred for 15 h, evaporated to dryness in an evaporator, column chromatography (filler: aluminum oxide, developing solvent: CH 2 Cl 2 : MeOH = 95: 5) (yield: 30 mg, yield: 21%).
1 H HNR (300 MHz, DMSO, RT): δ8.05 (1H, s), 7.80 (1H, d), 6.35 (1H, d), 5.23 (1H, m), 4.6 (1H, d), 3.55 -3.13 (5H, m), 2.73 (6H, m), 2.60-0.53 (35H, m).
ESI-MS m / z: Calcd for C 34 H 52 N 5 O 5 S + ([M + H] + ) 642.9, m / z: Found for 642.6
実施例１：24NBDChoおよび24DBDChoの吸収・蛍光スペクトル
＜DMPC膜における24NBDChoおよび24DBDChoの吸収・蛍光スペクトルの測定＞
1-1 DMPC膜の調製
細胞膜のモデルとしてリン脂質DMPC（dimyristoyl-phosphatidylcholine）でできた単層ベシクル (ULV; unilamellar vesicle)を用いた。DMPC単層膜はエタノールインジェクション法を用いて作成した（DMPC濃度 1.0 x 10-3M）。
Example 1: Absorption / fluorescence spectrum of 24NBDCho and 24DBDCho <Measurement of absorption / fluorescence spectrum of 24NBDCho and 24DBDCho in DMPC film>
1-1 Preparation of DMPC membrane A monolayer vesicle (ULV; unilamellar vesicle) made of phospholipid DMPC (dimyristoyl-phosphatidylcholine) was used as a cell membrane model. The DMPC monolayer film was prepared using the ethanol injection method (DMPC concentration 1.0 × 10 −3 M).
1-2 24NBDChoおよび24DBDChoのDMPC膜中への取り込み
22NBDCho（前出の化合物 C）、24NBDChoおよび24DBDChoをDMSOに溶かして、これを上記で調製されたDMPC膜と混合し、常温でDMPC膜に取り込ませた。なお、各化合物の最終濃度は1.0 x 10-5Mとした。
図１に22NBDCho、24NBDChoおよび24DBDChoのDMPC膜中における吸収および蛍光スペクトルを示す。また、吸収極大波長、蛍光極大波長を表１に示す。24NBDChoは吸収および蛍光波長領域は22NBDChoとほぼ同様であった。一方、24DBDChoは、24NBDChoおよび22NBDChoと比較し、吸収は短波長化したが、蛍光は長波長化した。このため、24DBDChoは吸収と蛍光波長のピーク間隔が広くなり、蛍光顕微鏡下での観測において、観測波長で励起光をカットすることが容易となると考えられた。
1-2 Uptake of 24NBDCho and 24DBDCho into DMPC membrane
22NBDCho (compound C ), 24NBDCho and 24DBDCho were dissolved in DMSO, mixed with the DMPC membrane prepared above, and incorporated into the DMPC membrane at room temperature. The final concentration of each compound was 1.0 × 10 −5 M.
FIG. 1 shows absorption and fluorescence spectra of 22NBDCho, 24NBDCho and 24DBDCho in a DMPC film. Further, Table 1 shows the absorption maximum wavelength and the fluorescence maximum wavelength. 24NBDCho had almost the same absorption and fluorescence wavelength region as 22NBDCho. On the other hand, compared with 24NBDCho and 22NBDCho, 24DBDCho has a shorter absorption wavelength but a longer fluorescence wavelength. For this reason, 24DBDCho has a broad peak interval between absorption and fluorescence wavelength, and it is considered that excitation light can be easily cut at the observation wavelength in observation under a fluorescence microscope.
＜各溶媒中における24NBDChoおよび24DBDChoの吸収・蛍光スペクトルの測定＞
24NBDChoおよび24DBDChoの各溶媒中での蛍光量子収率（Φf）を表２に示す。
<Measurement of absorption and fluorescence spectra of 24NBDCho and 24DBDCho in each solvent>
Table 2 shows the fluorescence quantum yield (Φ f ) in each solvent of 24NBDCho and 24DBDCho.
24NBDChoは、22NBDChoと同様にニトロベンゾフラザン部位を有しており、吸収および蛍光波長領域はこれまでとほぼ同様である（図１、表１）。
24DBDChoは、22NBDCho や24NBDChoと比較して、吸収は短波長化するが、蛍光は長波長化する（図１、表１）。このためストークスシフトが大きくなり、蛍光顕微鏡下での観測において、観測波長で励起光をカットすることが容易となる。
24NBDCho has a nitrobenzofurazane moiety similarly to 22NBDCho, and absorption and fluorescence wavelength regions are almost the same as before (FIG. 1, Table 1).
24DBDCho has a shorter absorption wavelength than 22NBDCho and 24NBDCho, but a longer wavelength of fluorescence (FIG. 1, Table 1). For this reason, the Stokes shift becomes large, and it becomes easy to cut the excitation light at the observation wavelength in the observation under the fluorescence microscope.
実施例２：培養細胞での蛍光測定
溶媒DMSOに溶かした22NBDCho、24NBDCho、24DBDChoを、マウス膵β細胞由来MIN6細胞に、最終濃度20μMで添加して2時間培養し、蛍光顕微鏡で各プローブの自家蛍光を観察した。
励起波長：400-440nm、検出波長：> 475nm
Example 2: Measurement of fluorescence in cultured cells 22NBDCho, 24NBDCho, and 24DBDCho dissolved in a solvent DMSO were added to mouse pancreatic β cell-derived MIN6 cells at a final concentration of 20 μM and cultured for 2 hours. Fluorescence was observed.
Excitation wavelength: 400-440nm, detection wavelength:> 475nm
その結果、図２に示されるように、22NBDChoはミトコンドリアに集積したのに対し、24NBDChoと24DBDChoは細胞内コレステロールと同様の分布を示し、細胞内コレステロールの挙動を模倣できることがわかった。 As a result, as shown in FIG. 2, 22NBDCho accumulated in mitochondria, whereas 24NBDCho and 24DBDCho showed the same distribution as intracellular cholesterol, and it was found that the behavior of intracellular cholesterol can be mimicked.
本発明は、細胞生物学、分子生物学、バイオイメージング分野、医学などの分野で有用である。 The present invention is useful in fields such as cell biology, molecular biology, bioimaging, and medicine.
Claims (4)
ここで、ｎは２〜６の整数、R1およびR2はそれぞれ独立して水素、メチルおよびエチルから選択される基であり、Xは酸素、硫黄、またはセレンである。
ここで、ｎは２〜６の整数、ｍは０、R1およびR2はそれぞれ独立して水素、メチルおよびエチルから選択される基であり、Ｘは酸素、硫黄、またはセレンである。 A compound represented by the following general formula (I) or (II).
Here, n is an integer of 2 to 6, R 1 and R 2 are each independently a group selected from hydrogen, methyl and ethyl, and X is oxygen, sulfur or selenium.
Here, n is an integer of 2 to 6, m is 0 , R 1 and R 2 are each independently a group selected from hydrogen, methyl and ethyl, and X is oxygen, sulfur or selenium.
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