JP6349091B2 - Probe for super-resolution fluorescence imaging - Google Patents
Probe for super-resolution fluorescence imaging Download PDFInfo
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- JP6349091B2 JP6349091B2 JP2014006456A JP2014006456A JP6349091B2 JP 6349091 B2 JP6349091 B2 JP 6349091B2 JP 2014006456 A JP2014006456 A JP 2014006456A JP 2014006456 A JP2014006456 A JP 2014006456A JP 6349091 B2 JP6349091 B2 JP 6349091B2
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- SXZIXHOMFPUIRK-UHFFFAOYSA-N diphenylmethanimine Chemical compound C=1C=CC=CC=1C(=N)C1=CC=CC=C1 SXZIXHOMFPUIRK-UHFFFAOYSA-N 0.000 description 1
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Pyrane Compounds (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
本発明は、スピロ環の環化平衡による蛍光発光特性を利用した新規な超解像蛍光イメージング用プローブ、及び当該プローブを用いた超解像蛍光イメージング方法に関する。 The present invention relates to a novel probe for super-resolution fluorescence imaging using fluorescence emission characteristics due to cyclization equilibrium of a spiro ring and a super-resolution fluorescence imaging method using the probe.
一般に、光の回折により点像は波長の半分程度に広がってしまうため、光学顕微鏡の空間分解能は、Ａｂｂｅの回折限界に制限され、点像の広がりよりも近接に位置する２点を分離してイメージングすることは不可能と考えられてきた。しかしながら、１９９０年代から回折限界を上回る分解能での超解像イメージング手法がいくつか報告されてきており（Ｎａｔ．Ｒｅｖ．Ｍｏｌ．Ｃｅｌｌ．Ｂｉｏｌ．，９,９２９，２００８等）、生細胞に適用することで従来法では観測できなかった生物学的現象を捉える可能性が広がりつつある。 In general, the point image spreads to about half of the wavelength due to light diffraction, so the spatial resolution of the optical microscope is limited to the Abbe diffraction limit, and the two points located closer to the point image spread are separated. It has been considered impossible to image. However, several super-resolution imaging techniques with resolution exceeding the diffraction limit have been reported since the 1990s (Nat. Rev. Mol. Cell. Biol., 9,929, 2008, etc.) and applied to living cells. Therefore, the possibility of capturing biological phenomena that could not be observed by conventional methods is expanding.
超解像イメージング法の１種であるＳＴＯＲＭ（ｓｔｏｃｈａｓｔｉｃｏｐｔｉｃａｌｒｅｃｏｎｓｔｒｕｃｔｉｏｎｍｉｃｒｏｓｃｏｐｙ）やＰＡＬＭ（ｐｈｏｔｏ−ａｃｔｉｖａｔｅｄｌｏｃａｌｉｚａｔｉｏｎｍｉｃｒｏｓｃｏｐｙ）に代表されるＳＬＭ法（ｓｉｎｇｌｅｍｏｌｅｃｕｌｅｌｏｃａｌｉｚａｔｉｏｎｍｉｃｒｏｓｃｏｐｙ）では、試料内にある蛍光分子を十分距離が離れた１分子ずつ確率的に発光させ、その分子の位置を正確に特定し、重ね合わせることで数１０ｎｍの解像度の蛍光画像を構築することができる（例えば、Ｚｈｕａｎｇら、米国特許公開公報ＵＳ２００８/００３２４１４）。 The SLM method (single molecular localization), which is a single molecule of the SLM method (single molecular localization), which is one of the SLM methods (single molecular localization microscopy), which is one of the super-resolution imaging methods, is represented by the SLM method (single molecular localization) in the sample, and in the SLM method (single molecular localization) A fluorescent image with a resolution of several tens of nanometers can be constructed by emitting light, pinpointing the position of the molecule, and superimposing them (for example, Zhuang et al., US Patent Publication No. US2008 / 0032414).
このようなＳＬＭでは、主に、市販の蛍光色素や蛍光蛋白質が用いられている。しかしながら、このような市販の有機蛍光色素を確率的に発光させるためには、一般的に１０〜１００ｍＭ程度のチオール存在下、酸素を除去した条件において、無蛍光状態を達成するための０．８ｋＷ/ｃｍ２程度の高強度のレーザーにより励起し、三重項等の長寿命の無蛍光状態を作り出すことが必須であり、このような条件は生細胞におけるイメージングには適さない。また、分子内の可逆的な光反応に基づき確率的に発光させるという試みもなされているが、その構造最適化は不十分であり、超解像蛍光イメージングへの適用例は殆ど報告されておらず、汎用されるに至っていない。 In such SLM, commercially available fluorescent dyes and fluorescent proteins are mainly used. However, in order to cause such a commercially available organic fluorescent dye to emit light stochastically, it is generally 0.8 kW for achieving a non-fluorescent state in the presence of 10 to 100 mM thiol and in the condition where oxygen is removed. It is essential to excite with a high-intensity laser of about / cm 2 to create a long-lived non-fluorescent state such as a triplet, and such conditions are not suitable for imaging in living cells. In addition, attempts have been made to emit light stochastically based on a reversible photoreaction within the molecule, but the structure optimization has been insufficient, and there have been few reports on applications to super-resolution fluorescence imaging. However, it has not been widely used.
これまでの、超解像蛍光イメージング法の開発は、主に光学系や解析ソフトウェアの観点からの開発がほとんどであり、重要な蛍光プローブの開発が遅れているのが現状である。 The development of super-resolution fluorescence imaging methods so far has mostly been developed from the viewpoint of optical systems and analysis software, and the development of important fluorescent probes has been delayed.
本発明の解決しようする課題は、チオール等の還元剤非存在下かつ酸素存在下において、データ取得前に蛍光性のプローブを無蛍光状態にするために必要であったレーザー照射を省くことができ、かつ低出力のレーザー照射でも機能する超解像蛍光イメージングに適した蛍光プローブ化合物を開発し、生細胞にも適用可能な超解像蛍光イメージング手法を提供することである。 The problem to be solved by the present invention is that in the absence of a reducing agent such as thiol and in the presence of oxygen, it is possible to omit the laser irradiation that was necessary to bring the fluorescent probe into a non-fluorescent state before data acquisition. The aim is to develop a fluorescent probe compound suitable for super-resolution fluorescence imaging that functions even with low-power laser irradiation, and to provide a super-resolution fluorescence imaging technique that can be applied to living cells.
本発明者らは、上記課題を解決するべく鋭意検討を行った結果、ローダミン類の熱的な分子内スピロ環化平衡に着目し、分子内求核基と蛍光団の適切な組み合わせによって自発的な蛍光ＯＮ／ＯＦＦ挙動を制御することで、従来のようなチオールとの反応やレーザー照射に依らずに、上記の生細胞にも適用可能な条件下における超解像蛍光イメージングを可能とする蛍光プローブ分子が得られることを見出した。より具体的には、蛍光プローブ分子内の求核基の求核性と蛍光団の求電子性を変化させて、開環分子の存在比や熱的な閉環速度を最適化することによって、超解像蛍光イメージングに適した確率的な発光が可能となることを見出した。これらの知見に基づき、本発明を完成するに至ったものである。 As a result of intensive studies to solve the above-mentioned problems, the present inventors focused on the thermal intramolecular spirocyclization equilibrium of rhodamines, and spontaneously achieved by an appropriate combination of an intramolecular nucleophilic group and a fluorophore. Fluorescence that enables super-resolution fluorescence imaging under conditions applicable to the above living cells, regardless of conventional reactions with thiols or laser irradiation, by controlling the ON / OFF behavior of the fluorescence It has been found that a probe molecule can be obtained. More specifically, by changing the nucleophilicity of the nucleophilic group in the fluorescent probe molecule and the electrophilicity of the fluorophore, the abundance ratio of the ring-opening molecule and the thermal ring-closing rate can be optimized. We found that stochastic emission suitable for resolving fluorescence imaging is possible. Based on these findings, the present invention has been completed.
すなわち、本発明は、一態様において、以下の式（Ｉ）で表される化合物又はその塩を含む超解像蛍光イメージング用プローブを提供するものである。
式中、Ｘは、酸素原子又はＣ（Ｒａ）（Ｒｂ）又はＳｉ（Ｒａ）（Ｒｂ）を表し（ここで、Ｒａ及びＲｂは、それぞれ独立に水素原子、又はアルキル基を表す）；Ｒ１は、水素原子、アルキル基、カルボキシル基、エステル基、アルコキシ基、アミド基、又はアジド基を表し；Ｙは、Ｃ０-Ｃ３アルキレン基を表し；Ｒ２は、置換されていてもよいアミノ基、置換されていてもよいアミド基、ヒドロキシル基、チオール基、又はカルボキシル基を表し；Ｒ３、Ｒ４、Ｒ７、及びＲ８は、それぞれ独立に水素原子、ヒドロキシル基、アルキル基、スルホ基、カルボキシル基、エステル基、アミド基、アジド基、又はハロゲン原子を表し；Ｒ５及びＲ６は、それぞれ独立に水素原子又はアルキル基を示し（ここで、Ｒ５及びＲ６は、それぞれＲ３及びＲ７と一緒になって、それらが結合する窒素原子を含む環構造を形成してもよい）；及び、Ｒ９及びＲ１０は、それぞれ独立に水素原子もしくはアルキル基を示し、又は、Ｎ（Ｒ９）（Ｒ１０）がアミド基もしくはカルバメート基を形成する（ここで、Ｒ９及びＲ１０がアルキル基である場合、それぞれＲ８及びＲ４と一緒になって、それらが結合する窒素原子を含む環構造を形成してもよい）。 In the formula, X represents an oxygen atom, C (R a ) (R b ), or Si (R a ) (R b ) (where R a and R b are each independently a hydrogen atom or an alkyl group) R 1 represents a hydrogen atom, an alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, or an azide group; Y represents a C 0 -C 3 alkylene group; R 2 represents a substituted group Represents an optionally substituted amino group, an optionally substituted amide group, a hydroxyl group, a thiol group, or a carboxyl group; R 3 , R 4 , R 7 , and R 8 are each independently a hydrogen atom, hydroxyl group, A group, an alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, an azide group, or a halogen atom; R 5 and R 6 each independently represents a hydrogen atom or an alkyl group (where R 5 and R 6 , Respectively together with R 3 and R 7, they may form a ring structure containing a nitrogen atom to which they are attached); and, R 9 and R 10 each independently represent a hydrogen atom or an alkyl group Or N (R 9 ) (R 10 ) forms an amide group or carbamate group (where R 9 and R 10 are alkyl groups together with R 8 and R 4 , respectively) May form a ring structure containing a nitrogen atom to which.
好ましくは、Ｘが、Ｓｉ（Ｒａ）（Ｒｂ）であり、Ｙが、メチレン基でありＲ２が、置換されていてもよいアミノ基又はヒドロキシル基であり、Ｒ５、Ｒ６、Ｒ９及びＲ１０が、いずれもメチル基である。 Preferably, X is Si (R a ) (R b ), Y is a methylene group and R 2 is an optionally substituted amino group or hydroxyl group, and R 5 , R 6 , R 9 and R 10 are both methyl groups.
好ましくは、上記式中、Ｘが、酸素原子であり、Ｙがメチレン基であり、Ｒ２が、置換されていてもよいアミノ基又はヒドロキシル基であり、Ｒ５及びＲ６が、いずれも水素原子であり、Ｎ（Ｒ９）（Ｒ１０）がアミド基である。 Preferably, in the above formula, X is an oxygen atom, Y is a methylene group, R 2 is an optionally substituted amino group or hydroxyl group, and R 5 and R 6 are both hydrogen. An atom, and N (R 9 ) (R 10 ) is an amide group.
好ましくは、上記式中、Ｘが、酸素原子であり、Ｙがメチレン基であり、Ｒ２が、置換されていてもよいアミノ基又はヒドロキシル基であり、Ｒ５、Ｒ６、Ｒ９及びＲ１０が、いずれも水素原子である。 Preferably, in the above formula, X is an oxygen atom, Y is a methylene group, R 2 is an optionally substituted amino group or hydroxyl group, and R 5 , R 6 , R 9 and R 10 are all hydrogen atoms.
好ましい態様では、本発明は、上記式（Ｉ）における
式（Ｉ）における
In formula (I)
より好ましい態様では、式（Ｉ）で表される化合物は、以下よりなる群から選択される。
別の態様において、本発明は、好ましくは、以下の平衡反応
上記平衡反応における平衡定数Ｋｃｙｃｌが、ｐＫｃｙｃｌとして３〜７の範囲内となることが好ましく、４〜６の範囲内がより好ましい。 The equilibrium constant K cycl in the above equilibrium reaction is preferably in the range of 3-7 as pK cycl , and more preferably in the range of 4-6.
上記平衡反応における反応速度定数ｋが、中性条件の水系溶液中において、１〜１．０ｘ１０６Ｓ−１であることが好ましく、１〜１００Ｓ−１であることがより好ましい。 The reaction rate constant k in the equilibrium reaction is preferably 1 to 1.0 × 10 6 S −1 and more preferably 1 to 100 S −1 in an aqueous solution under neutral conditions.
さらに別の態様において、本発明は、上記の超解像蛍光イメージング用プローブを用いる超解像蛍光イメージング方法であって、生体分子にプローブ分子を結合し、これにレーザー光を照射して前記プローブ分子からの蛍光発光を撮影した画像データを取得し、一定の時間間隔でこれを繰り返して得られた複数の前記画像データを解析したうえで重ね合わせることによって、前記生体分子の構造に対する超高解像のイメージ画像を得ることを含む、方法に関する。 In still another aspect, the present invention provides a super-resolution fluorescence imaging method using the above-described super-resolution fluorescence imaging probe, wherein a probe molecule is bound to a biomolecule, and the probe molecule is irradiated with laser light. Ultra high resolution for the structure of the biomolecule is obtained by acquiring image data obtained by photographing fluorescence emission from the molecule, and analyzing and superimposing a plurality of the image data obtained by repeating this at a predetermined time interval. The present invention relates to a method comprising obtaining an image of an image.
本発明の超解像蛍光イメージング方法は、チオールを含む化合物の非存在下で行われることが好ましく、同様に、酸素の存在下で行われることが好ましい。 The super-resolution fluorescence imaging method of the present invention is preferably performed in the absence of a thiol-containing compound, and is preferably performed in the presence of oxygen.
本発明によれば、蛍光プローブ分子内の求核基の求核性と蛍光団の求電子性を変化させて、分子内スピロ環化平衡における開環分子の存在比や熱的な閉環速度を検出器であるＣＣＤカメラ等のフレームレートに最適化することによって、従来のような無蛍光状態を達成するためのチオールとの反応や高強度のレーザー照射に依らずに、超解像蛍光イメージングに適した確率的な発光が達成できる。 According to the present invention, by changing the nucleophilicity of the nucleophilic group in the fluorescent probe molecule and the electrophilicity of the fluorophore, the abundance ratio of the ring-opened molecule and the thermal ring closure rate in the intramolecular spirocyclization equilibrium can be changed. By optimizing the frame rate of a CCD camera, etc., which is a detector, super-resolution fluorescence imaging can be performed without relying on the reaction with thiols and high-intensity laser irradiation to achieve a conventional non-fluorescence state. Suitable stochastic light emission can be achieved.
このように、熱的な分子内スピロ環化平衡を確率的な蛍光発光の原理とするため、本発明による超解像蛍光イメージングでは、高濃度のチオールの添加は不要であり、従来法のようにデータ取得前に蛍光性のプローブを無蛍光状態にするために必要であったレーザー照射を省くことができるため、細胞毒性を低減することができる。さらに、レーザー強度を従来の１／１０程度に低減することができ、酸素存在下においても十分に測定可能である。実際に、かかる条件下において、ｉｎ ｖｉｔｒｏで構築した微小管やプラスミドＳＮＡ上のＲｅｃＡ ｆｉｌａｍｅｎｔｓといった微小構造や固定細胞及又は生細胞中の微小管の超解像蛍光イメージング観察に成功した。従って、生細胞へのダメージを最小限に抑えた測定を行うことができるため、今後の生物学的な現象の理解に貢献できると考えられる。 In this way, since the thermal intramolecular spirocyclization equilibrium is the principle of stochastic fluorescence emission, the super-resolution fluorescence imaging according to the present invention does not require the addition of a high concentration of thiols. In addition, since it is possible to omit the laser irradiation necessary to bring the fluorescent probe into a non-fluorescent state before data acquisition, cytotoxicity can be reduced. Furthermore, the laser intensity can be reduced to about 1/10 of the conventional level, and measurement is possible even in the presence of oxygen. In fact, under such conditions, we succeeded in super-resolution fluorescence imaging observation of microstructures such as microtubules constructed in vitro and RecA filaments on plasmid SNA, microtubules in fixed cells and living cells. Therefore, since it is possible to perform measurement with minimal damage to living cells, it is considered that it can contribute to the understanding of biological phenomena in the future.
さらに、本発明の超解像蛍光イメージング用プローブが、その分子内にタンパク質等への結合部位（カルボキシル基等）を有する場合には、目標タンパク質等へのラベル化にも適しており、固定細胞だけでなく、生細胞系における広範な研究に利用できると期待できる。また、当該プローブに基づく超解像蛍光イメージング法は、市販の顕微鏡を光学系として利用することができるため、汎用性の高い手法であると考えられる。このように、開発したプローブの基礎研究上、産業上の利用価値、経済効果は極めて大きいものといえる。 Furthermore, when the probe for super-resolution fluorescence imaging of the present invention has a binding site (carboxyl group or the like) to a protein or the like in the molecule, it is also suitable for labeling to a target protein or the like, and fixed cells It can be expected that it can be used for a wide range of research in living cell systems. In addition, the super-resolution fluorescence imaging method based on the probe is considered to be a highly versatile technique because a commercially available microscope can be used as an optical system. In this way, it can be said that the basic utility of the developed probe has extremely high industrial utility value and economic effect.
以下、本発明の実施形態について説明する。本発明の範囲はこれらの説明に拘束されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜変更し実施することができる。 Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.
本明細書において、「アルキル基」は直鎖状、分枝鎖状、環状、又はそれらの組み合わせからなるアルキル基のいずれであってもよい。アルキル基の炭素数は特に限定されないが、例えば炭素数１〜６個程度、好ましくは炭素数１〜４個程度である。本明細書において、アルキル基は任意の置換基を1個以上有していてもよい。該置換基としては、例えば、アルコキシ基、ハロゲン原子（フッ素原子、塩素原子、臭素原子、又はヨウ素原子のいずれであってもよい）、アミノ基、モノ若しくはジ置換アミノ基、置換シリル基、アシル基、又はアリール基などを挙げることができるが、これらに限定されることはない。アルキル基が２個以上の置換基を有する場合には、それらは同一でも異なっていてもよい。アルキル部分を含む他の置換基（例えばアルキルオキシ基やアラルキル基など）のアルキル部分についても同様である。 In the present specification, the “alkyl group” may be any of an alkyl group composed of linear, branched, cyclic, or a combination thereof. Although carbon number of an alkyl group is not specifically limited, For example, it is about C1-C6, Preferably it is C1-C4. In the present specification, the alkyl group may have one or more arbitrary substituents. Examples of the substituent include an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group. A group, an aryl group, and the like can be mentioned, but are not limited thereto. When the alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (for example, an alkyloxy group or an aralkyl group).
また、本明細書において、ある官能基について「置換基を有していてもよい」と定義されている場合には、置換基の種類、置換位置、及び置換基の個数は特に限定されず、２個以上の置換基を有する場合には、それらは同一でも異なっていてもよい。置換基としては、例えば、アルキル基、アルコキシ基、水酸基、カルボキシル基、ハロゲン原子、スルホ基、アミノ基、アルコキシカルボニル基、オキソ基などを挙げることができるが、これらに限定されることはない。これらの置換基にはさらに置換基が存在していてもよい。このような例として、例えば、ハロゲン化アルキル基、ジアルキルアミノ基などを挙げることができるが、これらに限定されることはない。 In the present specification, when a functional group is defined as “may have a substituent”, the type of substituent, the substitution position, and the number of substituents are not particularly limited, When it has two or more substituents, they may be the same or different. Examples of the substituent group include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group. These substituents may further have a substituent. Examples of such include, but are not limited to, a halogenated alkyl group, a dialkylamino group, and the like.
（１）超解像蛍光イメージング用プローブ
本発明の超解像蛍光イメージング用プローブは、一態様において、以下の一般式（Ｉ）で表される構造を有する化合物である。
上記一般式（Ｉ）において、Ｘは、酸素原子又はＣ（Ｒａ）（Ｒｂ）又はＳｉ（Ｒａ）（Ｒｂ）を表す。ここで、Ｒａ及びＲｂは、それぞれ独立に水素原子、又はアルキル基を表す。Ｒａ及びＲｂが、アルキル基である場合、それらは１以上の置換基を有することができ、そのような置換基としては、例えば、アルキル基、アルコキシ基、ハロゲン原子、水酸基、カルボキシル基、アミノ基、スルホ基などを１個又は２個以上有していてもよい。Ｒａ及びＲｂは、いずれもメチル基であることが好ましい。また、場合によっては、Ｒａ及びＲｂは互いに結合して環構造を形成していてもよい。例えば、Ｒａ及びＲｂがともにアルキル基である場合に、Ｒａ及びＲｂが互いに結合してスピロ炭素環を形成することができる。形成される環は、例えば５ないし８員環程度であることが好ましい。 In the general formula (I), X represents an oxygen atom, C (R a ) (R b ), or Si (R a ) (R b ). Here, R a and R b each independently represent a hydrogen atom or an alkyl group. When R a and R b are alkyl groups, they can have one or more substituents, and examples of such substituents include alkyl groups, alkoxy groups, halogen atoms, hydroxyl groups, carboxyl groups, You may have 1 or 2 or more amino groups, sulfo groups, etc. R a and R b are preferably both methyl groups. In some cases, R a and R b may be bonded to each other to form a ring structure. For example, when R a and R b are both alkyl groups, R a and R b can be bonded to each other to form a spirocarbocycle. The ring formed is preferably about 5 to 8 membered ring, for example.
Ｒ１は、水素原子、アルキル基、カルボキシル基、エステル基、アルコキシ基、アミド基、又はアジド基を表す。これらの置換基は、タンパク質等の生体分子と共有結合等により結合し得る置換基（ラベル化置換基）を含むことが好ましい。その場合、それら自身がラベル化置換基であってもよいし、又はラベル化置換基によって置換されていてもよい。例えば、カルボキシル基がＮ-ヒドロキシスクシンイミド等の脱離基やＳＮＡＰ−ｔａｇの基質であるベンジルグアニン誘導体やＨａｌｏＴａｇ（登録商標）で用いられるリガンドで置換されていてもよい。そのようなラベル化置換基は、特に限定されず、当該技術分野において公知のものであれば用いることができる。Ｒ１が水素原子以外である場合、そのベンゼン環における位置は特に限定されないが、Ｙを有する置換基に対してメタ位であることが好ましい。また、ベンゼン環上に２個以上の置換基を有する場合には、それらは同一でも異なっていてもよい。 R 1 represents a hydrogen atom, an alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, or an azide group. These substituents preferably include a substituent (labeled substituent) that can be bonded to a biomolecule such as a protein by a covalent bond or the like. In that case, they may themselves be a labeled substituent or may be substituted by a labeled substituent. For example, the carboxyl group may be substituted with a leaving group such as N-hydroxysuccinimide, a benzylguanine derivative that is a substrate of SNAP-tag, or a ligand used in HaloTag (registered trademark). Such a labeled substituent is not particularly limited, and any known substituent in the technical field can be used. When R 1 is other than a hydrogen atom, the position in the benzene ring is not particularly limited, but is preferably a meta position with respect to the substituent having Y. Moreover, when it has two or more substituents on the benzene ring, they may be the same or different.
Ｙは、Ｃ０-Ｃ３アルキレン基を表す。当該アルキレン基は直鎖状アルキレン基又は分枝鎖状アルキレン基のいずれであってもよい。例えば、メチレン基（−ＣＨ２−）、エチレン基（−ＣＨ２−ＣＨ２−）、プロピレン基（−ＣＨ２−ＣＨ２−ＣＨ２−）のほか、分枝鎖状アルキレン基として−ＣＨ（ＣＨ３）−、−ＣＨ２−ＣＨ（ＣＨ３）−、−ＣＨ（ＣＨ２ＣＨ３）−なども使用することができる。これらのうち、メチレン基又はエチレン基が好ましく、閉環速度の観点からメチレン基がさらに好ましい。なお、Ｃ０アルキレン基とは、Ｙが結合であること、すなわち、Ｒ２がベンゼン環に直接結合している場合を表している。 Y represents a C 0 -C 3 alkylene group. The alkylene group may be a linear alkylene group or a branched alkylene group. For example, in addition to a methylene group (—CH 2 —), an ethylene group (—CH 2 —CH 2 —), a propylene group (—CH 2 —CH 2 —CH 2 —), a branched alkylene group such as —CH ( CH 3 ) —, —CH 2 —CH (CH 3 ) —, —CH (CH 2 CH 3 ) — and the like can also be used. Among these, a methylene group or an ethylene group is preferable, and a methylene group is more preferable from the viewpoint of a ring closing speed. The C 0 alkylene group represents that Y is a bond, that is, R 2 is directly bonded to the benzene ring.
Ｒ２は、置換されていてもよいアミノ基、置換されていてもよいアミド基、ヒドロキシル基、チオール基、又はカルボキシル基を表す。好ましくは、置換されていてもよいアミノ基又はヒドロキシル基である。Ｒ２が、置換されていてもよいアミノ基又は置換されていてもよいアミド基である場合、当該置換基を、例えば、アルキル基、アルコキシ基、ハロゲン原子、水酸基、カルボキシル基、アミノ基、スルホ基などが挙げられるが、これらに限定されるわけではない。また、当該置換基を１個又は２個以上有していてもよい。
R 2 represents an optionally substituted amino group, an optionally substituted amide group, a hydroxyl group, a thiol group, or a carboxyl group. Preferably, it is an optionally substituted amino group or hydroxyl group. When R 2 is an optionally substituted amino group or an optionally substituted amide group, the substituent is, for example, an alkyl group, an alkoxy group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a sulfo group. Examples include, but are not limited to, groups. Moreover, you may have the said
Ｒ３、Ｒ４、Ｒ７、及びＲ８は、それぞれ独立に水素原子、ヒドロキシル基、アルキル基、スルホ基、カルボキシル基、エステル基、アミド基、アジド基、又はハロゲン原子を表す。Ｒ３及びＲ４がいずれも水素原子であることが好ましい。また、Ｒ７及びＲ８が、いずれも水素原子であるか、又は、いずれもスルホ基であることが好ましい。場合によっては、Ｒ３、Ｒ４、Ｒ７、又はＲ８のいずれかが、上記のような、タンパク質等の生体分子と共有結合し得るラベル化置換基を含むこともできる。その場合、それら自身がラベル化置換基であってもよいし、又はラベル化置換基によって置換されていてもよい。 R 3 , R 4 , R 7 , and R 8 each independently represent a hydrogen atom, a hydroxyl group, an alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, an azide group, or a halogen atom. R 3 and R 4 are preferably both hydrogen atoms. Moreover, it is preferable that both R 7 and R 8 are hydrogen atoms, or both are sulfo groups. In some cases, any of R 3 , R 4 , R 7 , or R 8 can include a labeled substituent that can be covalently bound to a biomolecule such as a protein, as described above. In that case, they may themselves be a labeled substituent or may be substituted by a labeled substituent.
Ｒ５及びＲ６は、それぞれ独立に水素原子又はアルキル基を示す。Ｒ５及びＲ６がともにアルキル基を示す場合には、それらは同一でも異なっていてもよい。例えば、Ｒ５及びＲ６はそれぞれ独立に、メチル基又はエチル基であることが好ましく、Ｒ５及びＲ６がいずれもメチル基である場合がさらに好ましい。また、Ｒ５及びＲ６がアルキル基の場合に、それぞれＲ３及びＲ７と一緒になって、それらが結合する窒素原子を含む環構造を形成してもよい。その場合、Ｒ５とＲ３、又はＲ６及びＲ７の組み合わせのいずれか一方のみが環構造を形成してもよいし、いずれもが環構造を形成してもよい。 R 5 and R 6 each independently represent a hydrogen atom or an alkyl group. When R 5 and R 6 both represent an alkyl group, they may be the same or different. For example, each of R 5 and R 6 is preferably independently a methyl group or an ethyl group, and more preferably R 5 and R 6 are both methyl groups. When R 5 and R 6 are alkyl groups, they may be combined with R 3 and R 7 to form a ring structure containing a nitrogen atom to which they are bonded. In that case, only one of R 5 and R 3 , or a combination of R 6 and R 7 may form a ring structure, or all may form a ring structure.
Ｒ９及びＲ１０は、それぞれ独立に水素原子、アルキル基を示し、又は、Ｎ（Ｒ９）（Ｒ１０）がアミド基もしくはカルバメート基を形成するものであることができる。Ｒ９及びＲ１０がともにアルキル基を示す場合には、それらは同一でも異なっていてもよい。例えば、Ｒ９及びＲ１０はそれぞれ独立に、メチル基又はエチル基であることが好ましく、Ｒ９及びＲ１０がいずれもメチル基である場合がさらに好ましい。Ｒ９又はＲ１０の一方が水素原子であって、他方がカルボニル基であることも好ましい。この場合、Ｎ（Ｒ９）（Ｒ１０）はアミド結合を形成する。カルボニル基に結合する他の置換基は特に限定されないが、アルキル基が好ましく、メチル基がより好ましい。場合によっては、Ｎ（Ｒ９）（Ｒ１０）が、上記のような、タンパク質等の生体分子と共有結合し得るラベル化置換基を含むこともできる。その場合、それら自身がラベル化置換基であってもよいし、又はラベル化置換基によって置換されていてもよい。 R 9 and R 10 each independently represent a hydrogen atom or an alkyl group, or N (R 9 ) (R 10 ) may form an amide group or a carbamate group. When R 9 and R 10 both represent an alkyl group, they may be the same or different. For example, R 9 and R 10 are each independently preferably a methyl group or an ethyl group, and more preferably R 9 and R 10 are both methyl groups. It is also preferred that one of R 9 or R 10 is a hydrogen atom and the other is a carbonyl group. In this case, N (R 9 ) (R 10 ) forms an amide bond. Although the other substituent couple | bonded with a carbonyl group is not specifically limited, An alkyl group is preferable and a methyl group is more preferable. In some cases, N (R 9 ) (R 10 ) can also contain a labeled substituent that can be covalently bonded to a biomolecule such as a protein, as described above. In that case, they may themselves be a labeled substituent or may be substituted by a labeled substituent.
また、Ｒ９及びＲ１０がアルキル基の場合に、それぞれＲ８及びＲ４と一緒になって、それらが結合する窒素原子を含む環構造を形成してもよい。その場合、Ｒ５とＲ３、又はＲ６及びＲ７の組み合わせのいずれか一方のみが環構造を形成してもよいし、いずれもが環構造を形成してもよい。 When R 9 and R 10 are alkyl groups, they may be combined with R 8 and R 4 to form a ring structure containing a nitrogen atom to which they are bonded. In that case, only one of R 5 and R 3 , or a combination of R 6 and R 7 may form a ring structure, or all may form a ring structure.
上記各置換基の好ましい組み合わせとしては、例えば、Ｘが、Ｓｉ（Ｒａ）（Ｒｂ）であり、Ｙが、メチレン基であり、Ｒ２が、置換されていてもよいアミノ基又はヒドロキシル基であり、Ｒ５、Ｒ６、Ｒ９及びＲ１０が、いずれもメチル基である場合が挙げられる。同様に、Ｘが、酸素原子であり、Ｙがメチレン基であり、ＲＲ２が、置換されていてもよいアミノ基又はヒドロキシル基であり、Ｒ５及びＲ６が、いずれも水素原子であり、Ｎ（Ｒ９）（Ｒ１０）がアミド基である場合も好ましい。さらに、Ｘが、酸素原子であり、Ｙがメチレン基であり、Ｒ２が、置換されていてもよいアミノ基又はヒドロキシル基であり、Ｒ５、Ｒ６、Ｒ９及びＲ１０が、いずれも水素原子である場合も好ましい。 Preferable combinations of the above substituents include, for example, X is Si (R a ) (R b ), Y is a methylene group, and R 2 is an optionally substituted amino group or hydroxyl group. And R 5 , R 6 , R 9 and R 10 are all methyl groups. Similarly, X is an oxygen atom, Y is a methylene group, RR 2 is an optionally substituted amino group or hydroxyl group, R 5 and R 6 are both hydrogen atoms, It is also preferred when N (R 9 ) (R 10 ) is an amide group. Furthermore, X is an oxygen atom, Y is a methylene group, R 2 is an optionally substituted amino group or hydroxyl group, and R 5 , R 6 , R 9 and R 10 are all Also preferred is a hydrogen atom.
このような置換基の組み合わせは、式（Ｉ）における求核基である
式（Ｉ）における求核基の部分構造の具体例としては、
本発明の超解像蛍光イメージング用プローブとして特に適切な式（Ｉ）の化合物の具体例としては、
上記式（Ｉ）で表される化合物は、Ｒ５及びＲ６が連結するＮ原子において１価の正電荷を有するため、通常は塩として存在する。そのような塩としては、塩基付加塩、酸付加塩、アミノ酸塩などを挙げることができる。塩基付加塩としては、例えば、ナトリウム塩、カリウム塩、カルシウム塩、マグネシウム塩などの金属塩、アンモニウム塩、又はトリエチルアミン塩、ピペリジン塩、モルホリン塩などの有機アミン塩を挙げることができ、酸付加塩としては、例えば、塩酸塩、硫酸塩、硝酸塩などの鉱酸塩、メタンスルホン酸塩、パラトルエンスルホン酸塩、クエン酸塩、シュウ酸塩などの有機酸塩を挙げることができる。アミノ酸塩としてはグリシン塩などを例示することができる。もっとも、これらの塩に限定されることはない。 Since the compound represented by the above formula (I) has a monovalent positive charge at the N atom to which R 5 and R 6 are linked, it usually exists as a salt. Examples of such salts include base addition salts, acid addition salts, amino acid salts and the like. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, and acid addition salt. Examples thereof include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, paratoluenesulfonate, citrate, and oxalate. Examples of amino acid salts include glycine salts. However, it is not limited to these salts.
式（Ｉ）で表される化合物は、置換基の種類に応じて１個または２個以上の不斉炭素を有する場合があり、光学異性体又はジアステレオ異性体などの立体異性体が存在する場合がある。純粋な形態の立体異性体、立体異性体の任意の混合物、ラセミ体などはいずれも本発明の範囲に包含される。 The compound represented by the formula (I) may have one or more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as optical isomers or diastereoisomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.
式（Ｉ）で表される化合物又はその塩は、水和物又は溶媒和物として存在する場合もあるが、これらの物質はいずれも本発明の範囲に包含される。溶媒和物を形成する溶媒の種類は特に限定されないが、例えば、エタノール、アセトン、イソプロパノールなどの溶媒を例示することができる。 The compound represented by the formula (I) or a salt thereof may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention. Although the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, can be illustrated.
上記の蛍光プローブは、必要に応じて試薬の調製に通常用いられる添加剤を配合して組成物として用いてもよい。例えば、生理的環境で用いるための添加剤として、溶解補助剤、ｐＨ調節剤、緩衝剤、等張化剤などの添加剤を用いることができ、これらの配合量は当業者に適宜選択可能である。これらの組成物は、粉末形態の混合物、凍結乾燥物、顆粒剤、錠剤、液剤など適宜の形態の組成物として提供され得る。 The above-described fluorescent probe may be used as a composition by blending additives usually used in the preparation of reagents as required. For example, additives such as solubilizers, pH adjusters, buffers, and tonicity agents can be used as additives for use in a physiological environment, and the amount of these can be appropriately selected by those skilled in the art. is there. These compositions can be provided as a composition in an appropriate form such as a powder-form mixture, a lyophilized product, a granule, a tablet, or a liquid.
本明細書の実施例には、式（Ｉ）で表される本発明の化合物に包含される代表的化合物についての製造方法が具体的に示されているので、当業者は本明細書の開示を参照することにより、及び必要に応じて出発原料や試薬、反応条件などを適宜選択することにより、式（ＩＩ）に包含される任意の化合物を容易に製造することができる。 In the examples of the present specification, production methods for representative compounds included in the compounds of the present invention represented by the formula (I) are specifically shown. Any compound included in the formula (II) can be easily produced by referring to, and appropriately selecting starting materials, reagents, reaction conditions and the like as necessary.
（２）本発明のプローブを用いた超解像蛍光イメージング方法
本発明の超解像蛍光イメージング方法は、生体分子に上記で説明した式（Ｉ）のプローブ分子を接触させ、これにレーザー光を照射して前記プローブ分子からの蛍光発光を撮影したＣＣＤカメラ等によって画像データを取得し、一定の時間間隔でこれを繰り返して得られた複数の前記画像データを解析したうえで重ね合わせることによって、前記生体分子の構造に対する超高解像のイメージ画像を得ることを含むものである。観測対象となる生体分子としては、細胞膜（脂質）、タンパク質、ＤＮＡ、ＲＮＡ等が挙げられる。
(2) Super-resolution fluorescence imaging method using the probe of the present invention In the super-resolution fluorescence imaging method of the present invention, the probe molecule of the formula (I) described above is brought into contact with a biomolecule, and laser light is applied thereto. By acquiring image data by a CCD camera or the like that has been irradiated and photographed fluorescence emission from the probe molecules, and by repeating a plurality of the image data obtained by repeating this at a fixed time interval, The method includes obtaining an ultrahigh resolution image for the structure of the biomolecule. Examples of biomolecules to be observed include cell membranes (lipids), proteins, DNA, RNA, and the like.
ここで、ＳＬＭによる超解像蛍光イメージングの原理については、非特許文献２に詳述されている。従って、本発明の超解像蛍光イメージング方法における基本手順は、式（Ｉ）のプローブを用いる点以外は、当該文献に記載された超解像蛍光イメージング手法を参照することができる。
Here, the principle of super-resolution fluorescence imaging by SLM is described in detail in
本発明の超解像蛍光イメージング方法は、式（Ｉ）の化合物が、以下に示される
ＳＬＭによる超解像蛍光イメージングでは、光の回折限界よりも近接した場所において蛍光プローブ分子が発光することは好ましくないため、一度の測定において蛍光を発する状態のプローブ分子は、一定割合以下である必要がある。従って、上記平衡における式（Ｉ）の化合物と式（ＩＩ）の化合物の存在比は、中性条件の水系溶液中において、室温で、１：１００００〜１：１０であることが好ましく、１：１０００〜１：１００がより好ましい。このような存在比となるためには、上記平衡の平衡定数Ｋｃｙｃｌが、ｐＫｃｙｃｌとして３〜７の範囲が好ましく、４〜６の範囲内となることがより好ましい。当該平衡定数は、Ｋｃｙｃｌ＝［平衡状態における（Ｉ）の濃度］／［平衡状態における（ＩＩ）の濃度］[Ｈ＋の濃度]で表され、当該技術分野における周知の手法によって実験的に算出することができる。 In super-resolution fluorescence imaging by SLM, it is not preferable that the fluorescent probe molecule emits light at a location closer than the diffraction limit of light. Therefore, the number of probe molecules that emit fluorescence in one measurement needs to be less than a certain percentage. There is. Therefore, the abundance ratio of the compound of formula (I) to the compound of formula (II) in the above equilibrium is preferably 1: 10000 to 1:10 at room temperature in an aqueous solution under neutral conditions. 1000 to 1: 100 is more preferable. In order to achieve such an abundance ratio, the equilibrium constant K cycl of the above equilibrium is preferably in the range of 3 to 7 as pK cycl and more preferably in the range of 4 to 6. The equilibrium constant is expressed as K cycl == (concentration of (I) in equilibrium state) / [concentration of (II) in equilibrium state] [concentration of H + ], and is experimentally determined by a well-known method in the art. Can be calculated.
また、検出器であるＣＣＤカメラ等のフレームレートに対応させるためには、開環状態の式（Ｉ）の化合物の寿命は、１マイクロ秒〜１秒の範囲であることが好ましく、１０ミリ秒〜１秒の範囲であることがより好ましい。そのような寿命の範囲となるためには、上記平衡における反応速度定数が、中性条件の水系溶液中において、室温で、１〜１．０ｘ１０６Ｓ−１の範囲であることが好ましく、１〜１００Ｓ−１の範囲であることがより好ましい。当該反応速度については、当該技術分野において公知のレーザーフラッシュフォトリシス等のシステムを用いて、ナノ秒レーザーによって蛍光プローブを励起し、ナノ秒〜ミリ秒オーダーの過渡吸収スペクトルや蛍光スペクトルを測定することによって算出することができる。 In order to correspond to the frame rate of a CCD camera or the like as a detector, the lifetime of the compound of formula (I) in the ring-opened state is preferably in the range of 1 microsecond to 1 second, and 10 milliseconds. More preferably, it is in the range of ˜1 second. In order to reach such a life range, the reaction rate constant in the above equilibrium is preferably in the range of 1 to 1.0 × 10 6 S −1 at room temperature in an aqueous solution under neutral conditions. More preferably, it is in the range of -100S- 1 . Regarding the reaction rate, using a system such as laser flash photolysis known in the technical field, a fluorescent probe is excited by a nanosecond laser, and a transient absorption spectrum or a fluorescence spectrum of nanosecond to millisecond order is measured. Can be calculated.
上述のとおり、これら平衡定数及び反応速度定数は、式（Ｉ）の化合物における求核基の求核性と蛍光団骨格の求電子性のバランスに依存するため、これら部分構造の適切な組み合わせを選択することによって、所望の平衡定数及び反応速度を得ることができる。 As described above, since these equilibrium constants and reaction rate constants depend on the balance between the nucleophilicity of the nucleophilic group and the electrophilicity of the fluorophore skeleton in the compound of formula (I), an appropriate combination of these partial structures must be selected. By selecting, a desired equilibrium constant and reaction rate can be obtained.
以下、実施例により本発明をさらに詳細に説明するが、本発明はこれらによって限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these.
以下のスキーム１に従って、本発明の超解像蛍光イメージング用プローブである化合物７（ＨＭＳｉＲＣＯＯＨ）及び化合物１１(ＡＭＳｉＲＣＯＯＨ)を合成した。なお、化合物６の合成については、ＡＣＳＣｈｅｍ.Ｂｉｏｌ.２０１１,６,６００−６０８に開示されており、これを参照することができる。
化合物（2）の合成
化合物（１）5.03g(23.39mmol,1eq)の四塩化炭素（CCl4）懸濁液（100mL）にN-ブロモスクシンイミド(NBS)9.16g(51.5mmol,2.2eq),アゾビスイソブチロニトリル（AIBN）76.8mg（0.47mmol,0.02eq）を加え、18時間加熱還流した。室温に戻し、10%炭酸ナトリウム水溶液を200mL加え、ジクロロメタンで２回洗浄した。濃塩酸30mLを加え酢酸エチルで2回抽出し、飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥し、溶媒を減圧除去した。残渣をジクロロメタン／メタノール＝10／１で洗浄し、目的化合物(2)(5.93g,68%)を得た。
1HNMR(400MHz,MeOD)δ：7.29(s,1H),7.71(d,J=8.3Hz,1H),7.86(dd,J=2.1,8.3Hz, 1H),8.63(d,J=2.1Hz,1H)
13CNMR(400MHz,MeOD)δ：39.9,125.5,132.9,133.0,133.2,134.4,142.5,167.9
Synthesis of compound (2) Compound (1) 5.03 g (23.39 mmol, 1 eq) in carbon tetrachloride (CCl4) suspension (100 mL) N-bromosuccinimide (NBS) 9.16 g (51.5 mmol, 2.2 eq), 76.8 mg (0.47 mmol, 0.02 eq) of azobisisobutyronitrile (AIBN) was added, and the mixture was heated to reflux for 18 hours. The temperature was returned to room temperature, 200 mL of 10% aqueous sodium carbonate solution was added, and the mixture was washed twice with dichloromethane. Concentrated hydrochloric acid (30 mL) was added, and the mixture was extracted twice with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was washed with dichloromethane / methanol = 10/1 to obtain the target compound (2) (5.93 g, 68%).
1 HNMR (400 MHz, MeOD) δ: 7.29 (s, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.86 (dd, J = 2.1, 8.3 Hz, 1H), 8.63 (d, J = 2.1 Hz , 1H)
13 C NMR (400 MHz, MeOD) δ: 39.9, 125.5, 132.9, 133.0, 133.2, 134.4, 142.5, 167.9
化合物（3）の合成
化合物（2)5.65g(15.15mmol,1eq)を10%炭酸ナトリウム水溶液100mLに溶かし、70℃で1時間撹拌した。反応液をろ過し、ろ液に濃塩酸30mLを加え酢酸エチルで2回抽出し、有機相を飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥し、溶媒を減圧除去し、目的化合物(3)(3.29g,95%)を得た。
1HNMR(400MHz,Acetone-d6)δ：7.94(d,J=8.3Hz,1H),8.17(dd,J=2.2,8.3Hz,1H), 8.46(d,J=2.2Hz,1H),10.37(s,1H)
13CNMR(400MHz,Acetone-d6)δ：131.5,131.5,131.7,134.7,135.5,136.5,166.1,191.3
HRMS(ESI-):m/zcalcdfor[M]-,226.93493;found,226.93679(err.-1.9mDa)
Synthesis of compound (3) 5.65 g (15.15 mmol, 1 eq) of compound (2) was dissolved in 100 mL of 10% aqueous sodium carbonate solution and stirred at 70 ° C. for 1 hour. The reaction mixture was filtered, 30 mL of concentrated hydrochloric acid was added to the filtrate, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the target compound (3) (3.29 g, 95%) was obtained.
1 HNMR (400 MHz, Acetone-d 6 ) δ: 7.94 (d, J = 8.3 Hz, 1H), 8.17 (dd, J = 2.2, 8.3 Hz, 1H), 8.46 (d, J = 2.2 Hz, 1H), 10.37 (s, 1H)
13 C NMR (400 MHz, Acetone-d 6 ) δ: 131.5, 131.5, 131.7, 134.7, 135.5, 136.5, 166.1, 191.3
HRMS (ESI -): m / zcalcdfor [M] -, 226.93493; found, 226.93679 (err.-1.9mDa)
化合物（4）の合成
化合物（3)500mg(2.18mmol,1eq)をテトラヒドロフラン10mLに溶かし、0℃に冷やした。水素化ホウ素ナトリウム123.8mg(3.27mmol,1.5eq)を加え、0℃で5時間撹拌した。1N塩酸を加え、酢酸エチルで抽出し、有機相を水、飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥し、溶媒を減圧除去し、目的化合物(4)(492.7mg,98%)を得た。
1HNMR(400MHz,MeOD)δ：4.69(s,2H),7.67(d,J=8.2Hz,1H),7.81(dd,J=2.2,8.2Hz, 1H),8.21(m,1H)
13CNMR(400MHz,MeOD)δ：64.3,128.0,130.3,130.7,131.5,133.7,142.4,169.1
HRMS(ESI+):m/zcalcdfor[M+Na]+,252.94708;found,252.94774(err.-0.7mDa,mSigma:9.2)
Synthesis of compound (4) 500 mg (2.18 mmol, 1 eq) of compound (3) was dissolved in 10 mL of tetrahydrofuran and cooled to 0C. Sodium borohydride (123.8 mg, 3.27 mmol, 1.5 eq) was added, and the mixture was stirred at 0 ° C. for 5 hours. 1N Hydrochloric acid was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the target compound (4) (492.7 mg, 98%). It was.
1 HNMR (400 MHz, MeOD) δ: 4.69 (s, 2H), 7.67 (d, J = 8.2 Hz, 1H), 7.81 (dd, J = 2.2, 8.2 Hz, 1H), 8.21 (m, 1H)
13 CNMR (400 MHz, MeOD) δ: 64.3, 128.0, 130.3, 130.7, 131.5, 133.7, 142.4, 169.1
HRMS (ESI + ): m / zcalcdfor [M + Na] + , 252.94708; found, 252.94774 (err.-0.7mDa, mSigma: 9.2)
化合物（5）の合成
硫酸マグネシウム83.4mg(0.69mmol,8eq)の脱水ジクロロメタン懸濁液（2mL）に濃硫酸4.6mL（0.087mmol,1eq）を加え室温で15分間撹拌した。化合物（4)20mg(0.087mmol,1eq)、tert-ブチルアルコール64.2mg(0.87mmol,10eq)を加え、密栓して20時間撹拌した。飽和炭酸水素ナトリウム水溶液を加え、酢酸エチルで抽出し、有機相を水、飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥し、溶媒を減圧除去し、シリカゲルクロマトグラフィーで粗精製し（ヘキサン／酢酸エチル=98/2-87/13(6min)）、目的化合物(5)(10.5mg,35%)を得た。
1HNMR(400MHz,CDCl3)δ：1.31(s,9H),1.58(s,9H),4.50(s,2H),7.54 (d,J=8.3Hz,1H),7.71(dd,J=2.2,8.3Hz,1H),8.14(d,J=8.3Hz,1H)
13CNMR(400MHz,MeOD)δ：27.7(CH3),28.3(CH3),63.5(CH),74.1(C),81.3(C), 127.2(C),129.3(CH),130.1(CH),131.4(C),132.3(CH),139.4(C),165.3(C)
HRMS(ESI+):m/zcalcdfor[M+Na]+,365.07228;found,365.07232(err.-0.0mDa,mSigma:9.8)
Synthesis of compound (5) 4.6 mL (0.087 mmol, 1 eq) of concentrated sulfuric acid was added to 83.4 mg (0.69 mmol, 8 eq) of magnesium sulfate in a dehydrated dichloromethane suspension (2 mL), and the mixture was stirred at room temperature for 15 minutes. 20 mg (0.087 mmol, 1 eq) of compound (4) and 64.2 mg (0.87 mmol, 10 eq) of tert-butyl alcohol were added, sealed and stirred for 20 hours. A saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was roughly purified by silica gel chromatography (hexane / acetic acid). Ethyl = 98 / 2-87 / 13 (6 min)) and the target compound (5) (10.5 mg, 35%) was obtained.
1 HNMR (400 MHz, CDCl 3 ) δ: 1.31 (s, 9H), 1.58 (s, 9H), 4.50 (s, 2H), 7.54 (d, J = 8.3 Hz, 1H), 7.71 (dd, J = 2.2 , 8.3Hz, 1H), 8.14 (d, J = 8.3Hz, 1H)
13 C NMR (400 MHz, MeOD) δ: 27.7 (CH 3 ), 28.3 (CH 3 ), 63.5 (CH), 74.1 (C), 81.3 (C), 127.2 (C), 129.3 (CH), 130.1 (CH) , 131.4 (C), 132.3 (CH), 139.4 (C), 165.3 (C)
HRMS (ESI + ): m / zcalcdfor [M + Na] + , 365.07228; found, 365.07232 (err.-0.0mDa, mSigma: 9.8)
化合物(7)（ＨＭＳｉＲＣＯＯＨ）の合成
化合物(5)103.3mg(0.301mmol,5eq)を脱水テトラヒドロフラン（THF）5mLに溶かし、アルゴン雰囲気下、-78℃で10分間撹拌した。1Msec-ブチルリチウムシクロヘキサン、n-ヘキサン溶液301μL(0.301mmol,5eq)を８分間かけて加え、25分間撹拌した。化合物(1)33.4mg(0.060mmol,1eq)のTHF(2mL)溶液を加え、-78℃で10分間、室温で1時間撹拌した。1N塩酸を0.5mL加え、室温で10分間撹拌した。飽和炭酸水素ナトリウム水溶液を加え、ジクロロメタンで抽出し、有機相を水、飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥し、溶媒を減圧除去した。トリフルオロ酢酸3mLを加え、室温で42時間撹拌した後、減圧除去し、HPLCで精製し(eluentA(H2O,1%MeCN,0.1%TFA)及びeluentB(MeCN,1%H2O) (A/B=90/10 to 0/100 40min))、目的化合物(7)(2.4mg,24%)を得た。
1HNMR(400MHz,MeOD)δ：0.61(s,3H),0.62(s,3H),3.35(s,12H),4.36(s,2H), 6.77(dd,J=2.7,9.6Hz,2H),7.03(d,J=9.6Hz,2H),7.28(d,J=7.9Hz,1H),7.37(d,J=2.7Hz,2H),8.10(d,J=7.0Hz,1H),8.41(s,1H)
13CNMR(400MHz,MeOD)δ：-1.33,-1.09,40.93,62.01,115.32,122.34,128. 14,129.22,129.45,130.65,132.77,141.51,142.11,142.97,149.39,155.82,167.92,169.14
HRMS(ESI+):m/zcalcdfor[M]+,459.20985;found,459.21126(err.-1.4mDa)
Synthesis of compound (7) (HMSiRCOOH) Compound (5) (103.3 mg, 0.301 mmol, 5 eq) was dissolved in dehydrated tetrahydrofuran (THF) (5 mL) and stirred at -78 ° C for 10 minutes in an argon atmosphere. 301 μL (0.301 mmol, 5 eq) of 1 Msec-butyllithium cyclohexane and n-hexane solution was added over 8 minutes and stirred for 25 minutes. A solution of compound (1) 33.4 mg (0.060 mmol, 1 eq) in THF (2 mL) was added, and the mixture was stirred at −78 ° C. for 10 minutes and at room temperature for 1 hour. 0.5 mL of 1N hydrochloric acid was added and stirred at room temperature for 10 minutes. A saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with dichloromethane. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After adding 3 mL of trifluoroacetic acid and stirring at room temperature for 42 hours, it was removed under reduced pressure and purified by HPLC (eluentA (H 2 O, 1% MeCN, 0.1% TFA) and eluentB (MeCN, 1% H2O) (A / B = 90/10 to 0/100 40 min)), and the target compound (7) (2.4 mg, 24%) was obtained.
1 HNMR (400 MHz, MeOD) δ: 0.61 (s, 3H), 0.62 (s, 3H), 3.35 (s, 12H), 4.36 (s, 2H), 6.77 (dd, J = 2.7, 9.6 Hz, 2H) , 7.03 (d, J = 9.6Hz, 2H), 7.28 (d, J = 7.9Hz, 1H), 7.37 (d, J = 2.7Hz, 2H), 8.10 (d, J = 7.0Hz, 1H), 8.41 (s, 1H)
13 CNMR (400 MHz, MeOD) δ: -1.33, -1.09,40.93,62.01,115.32,122.34,128. 14,129.22,129.45,130.65,132.77,141.51,142.11,142.97,149.39,155.82,167.92,169.14
HRMS (ESI + ): m / zcalcdfor [M] + , 459.20985; found, 459.21126 (err.-1.4mDa)
化合物(8)の合成
化合物(7)5.9mg(0.013mmol,1eq)をジクロロメタン1.5mLとメタノール1.5mLに溶かし、0℃に冷やした。2Mトリメチルシリルジアゾメタンジエチルエーテル溶液192μLをゆっくり加え、10分間撹拌した後、溶媒を減圧除去し、目的化合物(8)(4.7mg,77%)を得た。
1HNMR(400MHz,CDCl3)δ：0.54(s,3H),0.62(s,3H),2.95(s,12H),3.93(s,3H), 5.29(s,2H),6.61(dd,J=2.9,8.9Hz,2H),6.94(d,J=8.8Hz,2H),6.96(d,J=2.8Hz,2H),7.09(d,J=8.0Hz,1H),7.93(d,J=8.0Hz,1H),8.00(s,1H)
13CNMR(400MHz,CDCl3)δ：-0.96,0.65,40.60,52.30,72.24,92.60,113.95,116.76, 122.86,124.57,128.69,129.26,129.50,135.48,137.70,140.11,148.94,151.83,167.16
HRMS(ESI+):m/zcalcdfor[M+Na]+,495.20504;found,495.20372(err.1.3mDa)
Synthesis of compound (8) 5.9 mg (0.013 mmol, 1 eq) of compound (7) was dissolved in 1.5 mL of dichloromethane and 1.5 mL of methanol and cooled to 0 ° C. After slowly adding 192 μL of 2M trimethylsilyldiazomethane diethyl ether solution and stirring for 10 minutes, the solvent was removed under reduced pressure to obtain the target compound (8) (4.7 mg, 77%).
1 HNMR (400 MHz, CDCl 3 ) δ: 0.54 (s, 3H), 0.62 (s, 3H), 2.95 (s, 12H), 3.93 (s, 3H), 5.29 (s, 2H), 6.61 (dd, J = 2.9,8.9Hz, 2H), 6.94 (d, J = 8.8Hz, 2H), 6.96 (d, J = 2.8Hz, 2H), 7.09 (d, J = 8.0Hz, 1H), 7.93 (d, J = 8.0Hz, 1H), 8.00 (s, 1H)
13 C NMR (400 MHz, CDCl 3 ) δ: −0.96, 0.65, 40.60, 52.30, 72.24, 92.60, 113.95, 116.76, 122.86, 124.57, 128.69, 129.26, 129.50, 135.48, 137.70, 140.11, 148.94, 151.83, 167.16
HRMS (ESI + ): m / zcalcdfor [M + Na] + , 495.20504; found, 495.20372 (err.1.3mDa)
化合物(11)(ＡＭＳｉＲＣＯＯＨ)の合成
化合物(8)25mg(0.053mmol,1eq)をTHF5mLに溶かし、パラジウム炭素26mgを加え、水素雰囲気下室温で30分間激しく撹拌した。セライト上でろ過し、ろ液を減圧除去した。残渣をトルエン5mLに溶かし、ジアザビシクロウンデセン(DBU)15.8μL(0.106mmol,2eq)、ジフェニルリン酸アジド(DPPA)23.7μL(0.106mmol,2eq)を加え、アルゴン雰囲気下、80℃で3時間撹拌した。飽和炭酸水素ナトリウム水溶液を加え、酢酸エチルで抽出し、水、飽和食塩水で洗浄後、無水硫酸ナトリウムで乾燥し、溶媒を減圧除去した。THF5mL、水0.5mLに溶かし、トリフェニルホスフィン23mg(0.088mmol,2eq)を加え、アルゴン雰囲気下で12時間加熱還流した。溶媒を減圧除去し、シリカゲルクロマトグラフィーで粗精製した（ジクロロメタン／メタノール=92/8-85/15(6min)）。0.4M水酸化リチウム水-メタノール（1/3）溶液を1mL加え、室温で6時間撹拌した。1N塩酸を加え、ジクロロメタンで抽出し、飽和食塩水で洗浄、無水硫酸ナトリウムで乾燥した後、溶媒を減圧除去した。ジクロロメタン5mLに溶かし、クロラニル22mgを加え、室温で12時間撹拌した。溶媒を減圧除去し、HPLCで精製し(eluentA(H2O,1%MeCN,0.1%TFA)及びeluentB(MeCN,1%H2O)(A/B=90/10 to 0/100 40min))、目的化合物(11)(AMSiRCOOH)(2.1mg,9%)を得た。
1HNMR(400MHz,MeOD)δ：0.51(s,3H),0.64(s,3H),2.97(s,12H),4.45(s,2H), 6.63,6.70(m,4H),6.96(d,J=8.0Hz,1H),7.07(d,J=2.5Hz,2H),8.01(d,J=8.0Hz,1H),8.05(s,1H)
13CNMR(400MHz,MeOD)δ：-2.04,0.73,40.45,79.95,115.17,118.01,124.52, 126.24,131.17,131.30,134.13,137.91,138.59,140.33,147.96,151.11,174.25
HRMS(ESI+):m/zcalcdfor[M]+,458.22583;found,458.22584(err.-0.0mDa)
Synthesis of compound (11) (AMSiRCOOH) 25 mg (0.053 mmol, 1 eq) of compound (8) was dissolved in 5 mL of THF, 26 mg of palladium carbon was added, and the mixture was vigorously stirred at room temperature for 30 minutes in a hydrogen atmosphere. Filtration over Celite and the filtrate was removed under reduced pressure. Dissolve the residue in 5 mL of toluene, add diazabicycloundecene (DBU) 15.8 μL (0.106 mmol, 2 eq), diphenyl phosphate azide (DPPA) 23.7 μL (0.106 mmol, 2 eq), and add 3 3 at 80 ° C. under an argon atmosphere. Stir for hours. A saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with ethyl acetate, washed with water and saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. It was dissolved in 5 mL of THF and 0.5 mL of water, 23 mg (0.088 mmol, 2 eq) of triphenylphosphine was added, and the mixture was heated to reflux for 12 hours under an argon atmosphere. The solvent was removed under reduced pressure, and the residue was roughly purified by silica gel chromatography (dichloromethane / methanol = 92 / 8-85 / 15 (6 min)). 1 mL of 0.4M lithium hydroxide aqueous-methanol (1/3) solution was added and stirred at room temperature for 6 hours. 1N Hydrochloric acid was added, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Dissolved in 5 mL of dichloromethane, added 22 mg of chloranil, and stirred at room temperature for 12 hours. Solvent was removed under reduced pressure and purified by HPLC (eluentA (H 2 O, 1% MeCN, 0.1% TFA) and eluentB (MeCN, 1% H2O) (A / B = 90/10 to 0/100 40 min)) The target compound (11) (AMSiRCOOH) (2.1 mg, 9%) was obtained.
1 HNMR (400 MHz, MeOD) δ: 0.51 (s, 3H), 0.64 (s, 3H), 2.97 (s, 12H), 4.45 (s, 2H), 6.63, 6.70 (m, 4H), 6.96 (d, J = 8.0Hz, 1H), 7.07 (d, J = 2.5Hz, 2H), 8.01 (d, J = 8.0Hz, 1H), 8.05 (s, 1H)
13 CNMR (400 MHz, MeOD) δ: −2.04, 0.73,40.45, 79.95, 115.17, 118.01, 124.52, 126.24, 131.17, 131.30, 134.13, 137.91, 138.59, 140.33, 147.96, 151.11, 174.25
HRMS (ESI + ): m / zcalcdfor [M] + , 458.22583; found, 458.22584 (err.-0.0mDa)
化合物（12）の合成
化合物(7)8.4mg（0.018mmol,1eq）を脱水N,N-ジメチルホルムアミド2mLに溶かし、N-ヒドロキシコハク酸イミド10.5mg(0.091mmol,5eq),1-エチル-3-（3-ジメチルアミノプロピル）カルボジイミド塩酸塩（WSCD）17.5mg(0.091mmol,5eq)を加え、24時間撹拌した。溶媒を減圧除去し、HPLCで精製し(eluentA(H2O,1%MeCN,0.1%TFA)及びeluentB(MeCN,1%H2O) (A/B=90/10 to 0/100 30min))、目的化合物(12)(8.1mg,79%)を得た。
1HNMR(400MHz,MeOD)δ:0.62(s,3H),0.62(s,3H),2.95(s,4H),3.36(s,12H),4.38(s,2H),6.80(dd,J=2.8,9.6Hz,2H),7.03(d,J=9.6Hz,2H),7.38(d,J=2.8Hz,2H),7.42(d,J=7.9Hz,1H),8.23(d,J=7.9Hz,1H),8.52(s,1H)
13CNMR(400MHz,MeOD)δ:-1.3,-1.1,26.6,41.0,61.7,115.5,122.5,127.3,127.8, 129.7,131.4,141.9,142.6,145.1,149.4,155.9,163.1,166.5,171.8
HRMS(ESI+):m/zcalcdfor[M+H]+,556.22622;found,556.22596(err.0.3mDa)
Synthesis of compound (12) 8.4 mg (0.018 mmol, 1 eq) of compound (7) was dissolved in 2 mL of dehydrated N, N-dimethylformamide, and 10.5 mg (0.091 mmol, 5 eq), 1 of N-hydroxysuccinimide was obtained. -Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSCD) 17.5 mg (0.091 mmol, 5 eq) was added and stirred for 24 hours. Solvent was removed under reduced pressure and purified by HPLC (eluentA (H2O, 1% MeCN, 0.1% TFA) and eluentB (MeCN, 1% H2O) (A / B = 90/10 to 0/100 30min)) (12) (8.1 mg, 79%) was obtained.
1H NMR (400 MHz, MeOD) δ: 0.62 (s, 3H), 0.62 (s, 3H), 2.95 (s, 4H), 3.36 (s, 12H), 4.38 (s, 2H), 6.80 (dd, J = 2.8 , 9.6Hz, 2H), 7.03 (d, J = 9.6Hz, 2H), 7.38 (d, J = 2.8Hz, 2H), 7.42 (d, J = 7.9Hz, 1H), 8.23 (d, J = 7.9 Hz, 1H), 8.52 (s, 1H)
13C NMR (400MHz, MeOD) δ: -1.3, -1.1, 26.6, 41.0, 61.7, 115.5, 122.5, 127.3, 127.8, 129.7, 131.4, 141.9, 142.6, 145.1, 149.4, 155.9, 163.1, 166.5, 171.8
HRMS (ESI +): m / zcalcdfor [M + H] +, 556.22622; found, 556.22596 (err.0.3mDa)
また、以下のスキーム２に従って、本発明の超解像蛍光イメージング用プローブである化合物１４(ＨＭＡｃＲＧ)、１６(ＡＭＡｃＲＧ)、及び１７(ＡＭＲＧ)を合成した。なお、化合物１３及び１４の合成については、Ｊ.Ａｍ.Ｃｈｅｍ.Ｓｏｃ.,２０１３,１３５,４０９−４１４に開示されており、これを参照することができる。
化合物(１４)(ＨＭＡｃＲＧ)の合成
化合物(13)30mg(0.095mmol)をピリジン1mLに溶かし、0度に冷やした。無水酢酸9.0μL（0.095mmol,1eq）のピリジン（1mL）溶液を加え、アルゴン雰囲気下、室温で23時間撹拌した。溶媒を減圧除去し、HPLCで精製し(eluentA(H2O,1%MeCN,0.1%TFA)及びeluentB(MeCN,1%H2O)(A/B=90/10 to 0/100 60min))、目的化合物(14)(HMAcRG)(23.6mg,69%)を得た。
1HNMR(300MHz,MeOD)δ：7.64(d,1H,J=7.7Hz),7.56(t,1H,J=7.6Hz),7.44(t,1H,J=7.5Hz),7.17(d,1H,J=7.5Hz),7.03-7.00(m,2H),6.71-6.74(m,4H),4.23(s,2H).13CNMR(75MHz,MeOD)δ：161.5,159.9,159.6,141.0,133.4,132.2,131.3,130.3,129.5,128.8,118.0,115.0,98.4,62.8.
HRMS(ESI+):m/zcalcdfor[M]+,317.12900;found,317.12862(err.-0.38mDa)
Synthesis of compound (14) (HMAcRG) 30 mg (0.095 mmol) of compound (13) was dissolved in 1 mL of pyridine and cooled to 0 ° C. A solution of acetic anhydride (9.0 μL, 0.095 mmol, 1 eq) in pyridine (1 mL) was added, and the mixture was stirred at room temperature for 23 hours under an argon atmosphere. Solvent was removed under reduced pressure and purified by HPLC (eluentA (H 2 O, 1% MeCN, 0.1% TFA) and eluentB (MeCN, 1% H2O) (A / B = 90/10 to 0/100 60 min)) The target compound (14) (HMAcRG) (23.6 mg, 69%) was obtained.
1 HNMR (300 MHz, MeOD) δ: 7.64 (d, 1H, J = 7.7 Hz), 7.56 (t, 1H, J = 7.6 Hz), 7.44 (t, 1H, J = 7.5 Hz), 7.17 (d, 1H , J = 7.5 Hz), 7.03-7.00 (m, 2H), 6.71-6.74 (m, 4H), 4.23 (s, 2H). 13 C NMR (75 MHz, MeOD) δ: 161.5, 159.9, 159.6, 141.0, 133.4 , 132.2, 131.3, 130.3, 129.5, 128.8, 118.0, 115.0, 98.4, 62.8.
HRMS (ESI + ): m / zcalcdfor [M] + , 317.12900; found, 317.12862 (err.-0.38mDa)
化合物(１６)(ＡＭＡｃＲＧ)の合成
化合物(１４)7.3mg(0.020mmol,1eq)をTHF2mLに溶かし、パラジウム炭素34mgを加え、水素雰囲気下室温で40分間激しく撹拌した。セライト上でろ過し、ろ液を減圧除去した。残渣をTHF2mL,トルエン2mLに溶かし、ジアザビシクロウンデセン(DBU)6.1μL(0.041mmol,2eq)、ジフェニルリン酸アジド(DPPA)9.1μL(0.041mmol,2eq)を加え、アルゴン雰囲気下、80℃で18時間撹拌した。飽和炭酸水素ナトリウム水溶液を加え、酢酸エチルで抽出し、水、飽和食塩水で洗浄後、無水硫酸ナトリウムで乾燥し、溶媒を減圧除去し、シリカゲルクロマトグラフィーで粗精製した（ジクロロメタン／メタノール=95/5-88/12(6min)）。THF3mL、水0.3mLに溶かし、トリフェニルホスフィン7.8mg(0.030mmol,2eq)を加え、アルゴン雰囲気下で6時間加熱還流した。溶媒を減圧除去し、ジクロロメタン2mLに溶かし、クロラニル7.3mgを加え、室温で24時間撹拌した。溶媒を減圧除去し、HPLCで精製し(eluentA(H2O,1%MeCN,0.1%TFA)及びeluentB(MeCN,1%H2O)(A/B=90/10 to 0/100 40min))、目的化合物(１６)(AMAcRG)(2.0mg,27%)を得た。
1HNMR(400MHz,MeOD)δ2.12(s,3H),4.35(d,J=5.0Hz,2H),6.41(dd,J=2.2,8.4Hz,1H),6.52(d,J=2.3Hz,1H),6.59(d,J=8.4Hz,1H),6.74(d,J=8.6Hz,1H),6.90(d,J=7.6Hz,1H),7.07(dd,J=2.1,8.5Hz,1H),7.30(t,J=7.4Hz,1H),7.39(dt,J=1.0,7.4Hz,1H),7.45(d,J=7.5Hz,1H),7.59(d,J=2.1Hz,1H)
HRMS(ESI+):m/zcalcdfor[M]+,358.15500;found,358.15672(err.-1.7mDa)
Synthesis of Compound (16) (AMAcRG) 7.3 mg (0.020 mmol, 1 eq) of Compound (14) was dissolved in 2 mL of THF, 34 mg of palladium carbon was added, and the mixture was vigorously stirred at room temperature for 40 minutes under a hydrogen atmosphere. Filtration over Celite and the filtrate was removed under reduced pressure. Dissolve the residue in 2 mL of THF and 2 mL of toluene, add diazabicycloundecene (DBU) 6.1 μL (0.041 mmol, 2 eq), diphenylphosphate azide (DPPA) 9.1 μL (0.041 mmol, 2 eq), and add 80 ° C. under argon atmosphere. For 18 hours. Saturated aqueous sodium hydrogen carbonate solution was added, extracted with ethyl acetate, washed with water and saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was roughly purified by silica gel chromatography (dichloromethane / methanol = 95 / 5-88 / 12 (6min)). It was dissolved in 3 mL of THF and 0.3 mL of water, 7.8 mg (0.030 mmol, 2 eq) of triphenylphosphine was added, and the mixture was heated to reflux for 6 hours under an argon atmosphere. The solvent was removed under reduced pressure, dissolved in 2 mL of dichloromethane, 7.3 mg of chloranil was added, and the mixture was stirred at room temperature for 24 hours. Solvent was removed under reduced pressure and purified by HPLC (eluentA (H 2 O, 1% MeCN, 0.1% TFA) and eluentB (MeCN, 1% H2O) (A / B = 90/10 to 0/100 40 min)) The target compound (16) (AMAcRG) (2.0 mg, 27%) was obtained.
1 HNMR (400MHz, MeOD) δ2.12 (s, 3H), 4.35 (d, J = 5.0Hz, 2H), 6.41 (dd, J = 2.2,8.4Hz, 1H), 6.52 (d, J = 2.3Hz , 1H), 6.59 (d, J = 8.4Hz, 1H), 6.74 (d, J = 8.6Hz, 1H), 6.90 (d, J = 7.6Hz, 1H), 7.07 (dd, J = 2.1,8.5Hz , 1H), 7.30 (t, J = 7.4Hz, 1H), 7.39 (dt, J = 1.0,7.4Hz, 1H), 7.45 (d, J = 7.5Hz, 1H), 7.59 (d, J = 2.1Hz , 1H)
HRMS (ESI + ): m / zcalcdfor [M] + , 358.15500; found, 358.15672 (err.-1.7mDa)
化合物(1７)(ＡＭＲＧ)の合成
化合物(1５)6.7mg(0.017mmol,1eq)をTHF5mL、水2mLに溶かし、トリフェニルホスフィン9.1mg(0.035mmol,2eq)を加え、アルゴン雰囲気下で18時間、80℃で撹拌した。溶媒を減圧除去し、1N塩酸5mLを加えアルゴン雰囲気下で1時間、加熱還流した。飽和炭酸水素ナトリウム水溶液を加え、ジクロロメタンで抽出し、情話食塩水で洗浄、無水硫酸ナトリウムで乾燥した。溶媒を減圧除去し、HPLCで精製し(eluentA(H2O,1%MeCN,0.1%TFA)及びeluentB(MeCN,1%H2O) (A/B=90/10 to 0/100 60min))、目的化合物(12)(AMRG)(3.1mg,56%)を得た。
1HNMR(400MHz,MeOD)δ3.88(s,2H),6.86,6.89(m,4H),7.09(d,J=9.4Hz,2H), 7.42(d,J=7.4Hz,1H),7.68(d,J=2.2,10.9Hz,1H),7.76-7.81(m,2H)
HRMS(ESI+):m/zcalcdfor[M]+,316.14444;found,316.14521(err.-0.8mDa)
Synthesis of compound (17) (AMRG) 6.7 mg (0.017 mmol, 1 eq) of compound (15) was dissolved in 5 mL of THF and 2 mL of water, 9.1 mg (0.035 mmol, 2 eq) of triphenylphosphine was added, and the atmosphere was argon. And stirred at 80 ° C. for 18 hours. The solvent was removed under reduced pressure, 5 mL of 1N hydrochloric acid was added, and the mixture was heated to reflux for 1 hour under an argon atmosphere. A saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with dichloromethane, washed with narrative saline, and dried over anhydrous sodium sulfate. Solvent was removed under reduced pressure and purified by HPLC (eluentA (H 2 O, 1% MeCN, 0.1% TFA) and eluentB (MeCN, 1% H2O) (A / B = 90/10 to 0/100 60 min)) The target compound (12) (AMRG) (3.1 mg, 56%) was obtained.
1 HNMR (400MHz, MeOD) δ3.88 (s, 2H), 6.86,6.89 (m, 4H), 7.09 (d, J = 9.4Hz, 2H), 7.42 (d, J = 7.4Hz, 1H), 7.68 (d, J = 2.2,10.9Hz, 1H), 7.76-7.81 (m, 2H)
HRMS (ESI + ): m / zcalcdfor [M] + , 316.14444; found, 316.14521 (err.-0.8mDa)
また、以下のスキームに従って、本発明の超解像蛍光イメージング用プローブである化合物２１(ＨＭＡｃＲＧＣＯＯＨ)を合成した。なお、化合物１８の合成については、Ｊ．Ｏｒｇ．Ｃｈｅｍ., ２００８, ７３, ８７１１−８７１８に開示されており、これを参照することができる。
化合物（１９）の合成
化合物（１８）２ｇ（４．０６２ ｍｍｏｌ, １ ｅｑ）をトルエン７０ｍLに溶かし、トリス(ジベンジリデンアセトン)二パラジウム(０)(クロロホルム付加物) ８４０ｍｇ（０．８１２５ｍｍｏｌ, ０．２ ｅｑ）、キサントホス１．１７６ｇ（２．０３１ｍｍｏｌ, ０．５ｅｑ）、炭酸セシウム４ｇ（１２．１９ｍｍｏｌ, ３ ｅｑ）、ベンゾフェノンイミン４．０９ｍL（２４．３７ｍｍｏｌ, ６ｅｑ ） を加え、１００℃で１３時間撹拌した。セライト上で濾過し、飽和炭酸水素ナトリウム水溶液を加え、酢酸エチルで抽出し、水、飽和食塩水で洗浄後、無水硫酸ナトリウムで乾燥し、溶媒を減圧除去した。シリカゲルクロマトグラフィーで粗精製し（ヘキサン／酢酸エチル＝８３／１７―６２／３８（１５ｍｉｎ））、酢酸エチルで再結晶することで目的化合物（１９）（１．３８０５ｇ、６１％）を得た。
1H NMR (400 MHz, CDCl3): δ 8.05 (d, J = 8.5 Hz, 2 H), 7.77 (d, J = 7.0 Hz, 4 H), 7.52-7.49 (m, 2 H), 7.45-7.44 (m, 4 H), 7.29-7.27 (m, 6 H), 7.16 (m, 4 H), 6.75 (d, J = 1.9 Hz, 2 H) , 6.66 (dd, J = 8.5, 1.9 Hz, 2 H); 13C NMR (101 MHz, CDCl3): δ176.1 (C), 169.3 (C), 157.6 (C), 157.0 (C), 138.9 (C), 135.5 (C), 131.5 (CH), 129.7 (CH), 129.4 (CH), 128.5 (CH), 128.3 (CH), 127.3 (CH), 117.7 (CH), 117.6 (C), 108.4 (CH); HRMS (m/z): [M+Na]+ calcd. for C39H26N2NaO2, 577.18865; found, 577.18802.(err. 0.6 mDa)
Synthesis of compound (19) 2 g (4.062 mmol, 1 eq) of compound (18) was dissolved in 70 mL of toluene, and 840 mg (0. 0 of chloroform adduct) of tris (dibenzylideneacetone) dipalladium (0). 8125 mmol, 0.2 eq), 1.176 g (2.031 mmol, 0.5 eq) xanthophos, 4 g (12.19 mmol, 3 eq) cesium carbonate, 4.09 mL (24.37 mmol, 6 eq) benzophenone imine, and 100 Stir at 13 ° C. for 13 hours. The mixture was filtered over celite, saturated aqueous sodium hydrogen carbonate solution was added, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was roughly purified by silica gel chromatography (hexane / ethyl acetate = 83 / 17-62 / 38 (15 min)) and recrystallized from ethyl acetate to obtain the target compound (19) (1.3805 g, 61%).
1 H NMR (400 MHz, CDCl 3 ): δ 8.05 (d, J = 8.5 Hz, 2 H), 7.77 (d, J = 7.0 Hz, 4 H), 7.52-7.49 (m, 2 H), 7.45- 7.44 (m, 4 H), 7.29-7.27 (m, 6 H), 7.16 (m, 4 H), 6.75 (d, J = 1.9 Hz, 2 H), 6.66 (dd, J = 8.5, 1.9 Hz, 2 H); 13 C NMR (101 MHz, CDCl 3 ): δ176.1 (C), 169.3 (C), 157.6 (C), 157.0 (C), 138.9 (C), 135.5 (C), 131.5 (CH ), 129.7 (CH), 129.4 (CH), 128.5 (CH), 128.3 (CH), 127.3 (CH), 117.7 (CH), 117.6 (C), 108.4 (CH); HRMS (m / z): [ M + Na] + calcd.for C 39 H 26 N 2 NaO 2 , 577.18865; found, 577.18802. (Err. 0.6 mDa)
化合物（２０）（ＨＭＲＧＣＯＯＨ）の合成
化合物(５)３７１．３ｍｇ(１．０８１８ｍｍｏｌ, ３ ｅｑ)を脱水テトラヒドロフラン（ＴＨＦ）１５ ｍＬに溶かし、アルゴン雰囲気下、-７８℃で１０分間撹拌した。1M sec-ブチルリチウム シクロヘキサン、n-ヘキサン溶液１．０８ ｍL(１．０８ｍｍｏｌ, ３ ｅｑ)を５分間かけて加え、３０分間撹拌した。化合物(１９) ２００ ｍｇ (０．３６０６ ｍｍｏｌ, １ ｅｑ)のTHF (８ ｍＬ)溶液を加え、-７８℃で１０分間、室温で1時間半撹拌した。飽和炭酸水素ナトリウム水溶液を５ mL加え、酢酸エチルで抽出し、有機相を水、飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥し、溶媒を減圧除去した。シリカゲルクロマトグラフィーで粗精製し（ヘキサン／酢酸エチル＝６７／３３―４６／５４（９ｍｉｎ））、トリフルオロ酢酸 ５ｍＬを加え、室温で１８時間撹拌した後、減圧除去し、HPLCで精製し(eluent A (H2O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H2O) (A/B = 90/10 to 0/100 40min))、目的化合物(２０) (６４．９ mg, ３８％)を得た。
1H NMR (400 MHz, MeOD): δ8.41 (d, J = 1.0 Hz, 1 H), 8.17 (dd, J = 7.9, 1.5 Hz, 1 H), 7.39 (d, J = 7.9 Hz, 1 H), 7.05 (d, J = 9.2 Hz, 2 H), 6.83 (dd, J = 9.2, 2.1 Hz, 2 H), 6.75 (d, J = 2.1 Hz, 2 H), 4.34 (s, 2 H); 13C NMR (101 MHz, MeOD): δ168.9 (C), 161.5 (C), 159.6 (C), 157.9 (C), 141.7 (C), 136.5 (C), 133.8 (C), 133.0 (CH), 130.7 (CH), 130.4 (CH), 129.8 (CH), 118.2 (CH), 114.5 (C), 98.6 (CH), 62.4 (CH2); HRMS (m/z): [M]+ calcd. for C21H17N2O4, 361.11828; found, 361.11837.(err. -0.1 mDa)
Synthesis of compound (20) (HMRGCOOH) Compound (5) 371.3 mg (1.0818 mmol, 3 eq) was dissolved in 15 mL of dehydrated tetrahydrofuran (THF) and stirred at -78 ° C for 10 minutes under an argon atmosphere. did. 1M sec-butyllithium cyclohexane / 1.08 mL (1.08 mmol, 3 eq) of n-hexane solution was added over 5 minutes and stirred for 30 minutes. A solution of compound (19) 200 mg (0.3606 mmol, 1 eq) in THF (8 mL) was added, and the mixture was stirred at −78 ° C. for 10 minutes and at room temperature for 1.5 hours. 5 mL of a saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Crudely purified by silica gel chromatography (hexane / ethyl acetate = 67 / 33-46 / 54 (9 min)), 5 mL of trifluoroacetic acid was added, stirred at room temperature for 18 hours, then removed under reduced pressure, and purified by HPLC (eluent A (H 2 O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H 2 O) (A / B = 90/10 to 0/100 40 min)), target compound (20) (64. 9 mg, 38%).
1 H NMR (400 MHz, MeOD): δ8.41 (d, J = 1.0 Hz, 1 H), 8.17 (dd, J = 7.9, 1.5 Hz, 1 H), 7.39 (d, J = 7.9 Hz, 1 H), 7.05 (d, J = 9.2 Hz, 2 H), 6.83 (dd, J = 9.2, 2.1 Hz, 2 H), 6.75 (d, J = 2.1 Hz, 2 H), 4.34 (s, 2 H ); 13 C NMR (101 MHz, MeOD): δ168.9 (C), 161.5 (C), 159.6 (C), 157.9 (C), 141.7 (C), 136.5 (C), 133.8 (C), 133.0 (CH), 130.7 (CH), 130.4 (CH), 129.8 (CH), 118.2 (CH), 114.5 (C), 98.6 (CH), 62.4 (CH 2 ); HRMS (m / z): [M] + calcd.for C 21 H 17 N 2 O 4 , 361.11828; found, 361.11837. (err.-0.1 mDa)
化合物（２１）（ＨＭＡｃＲＧＣＯＯＨ）の合成
化合物(２０)４５．８ｍｇ(０．１２７１ｍｍｏｌ, １ ｅｑ)を脱水ピリジン５ ｍＬと脱水Ｎ，Ｎ−ジメチルホルムアミド５ ｍＬに溶かし、無水酢酸１８ mＬ（０．１９０６ ｍｍｏｌ, １．５ ｅｑ）を加え、アルゴン雰囲気下、室温で１２時間撹拌した。溶媒を減圧除去し、HPLCで粗精製し(eluent A (H2O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H2O) (A/B = 90/10 to 0/100 40min))、シリカゲルクロマトグラフィーで精製し（ヘキサン／酢酸エチル＝８７／１３―７９／２１（６ ｍｉｎ））、目的化合物(２１) (１４．６ mg, ２９％)を得た。
1H NMR (400 MHz, MeOD): δ8.08 (s, 1 H), 7.93 (d, J = 8.0 Hz, 1 H), 7.61 (d, J = 2.0 Hz, 1 H), 7.07 (dd, J = 8.6, 2.0 Hz, 1 H), 6.87 (d, J = 8.0 Hz, 1 H), 6.84 (d, J = 8.6 Hz, 1 H), 6.65 (d, J = 8.5 Hz, 1 H), 6.50 (d, J = 2.2 Hz, 1 H), 6.41 (dd, J = 8.5, 2.2 Hz, 1 H), 5.29 (s, 2 H), 2.12 (s, 3 H); HRMS (m/z): [M-H]- calcd. for C23H17N2O5, 401.11430; found, 401.11696.(err. -2.7 mDa)
Synthesis of compound (21) (HMAcRGCOOH) Compound (20) 45.8 mg (0.1271 mmol, 1 eq) was dissolved in
1 H NMR (400 MHz, MeOD): δ8.08 (s, 1 H), 7.93 (d, J = 8.0 Hz, 1 H), 7.61 (d, J = 2.0 Hz, 1 H), 7.07 (dd, J = 8.6, 2.0 Hz, 1 H), 6.87 (d, J = 8.0 Hz, 1 H), 6.84 (d, J = 8.6 Hz, 1 H), 6.65 (d, J = 8.5 Hz, 1 H), 6.50 (d, J = 2.2 Hz, 1 H), 6.41 (dd, J = 8.5, 2.2 Hz, 1 H), 5.29 (s, 2 H), 2.12 (s, 3 H); HRMS (m / z) :.. [MH] - calcd for C 23 H 17 N 2
さらに、以下のスキームに従って、本発明の超解像蛍光イメージング用プローブである化合物２１(ＨＭＭｏＲＣＯＯＨ)を合成した。なお、化合物２２の合成については、Ｊ．Ｏｒｇ．Ｃｈｅｍ., ２００８, ７３, ８７１１−８７１８に開示されており、これを参照することができる。
化合物（２３）（ＨＭＲＧＣＯＯＨ）の合成
化合物(５)５８．５ｍｇ(０．１７０３ ｍｍｏｌ, ２ ｅｑ)を脱水テトラヒドロフラン（ＴＨＦ）７ ｍＬに溶かし、アルゴン雰囲気下、-７８℃で１０分間撹拌した。１Ｍ sec-ブチルリチウム シクロヘキサン、n-ヘキサン溶液０．１７ ｍL(０．１７ ｍｍｏｌ, ２ ｅｑ)を２分間かけて加え、３０分間撹拌した。化合物(２２) ３１．２ ｍｇ (０．０８５２ ｍｍｏｌ, １ ｅｑ)のＴＨＦ (５ ｍＬ)溶液を加え、-７８℃で３０分間、室温で２３時間半撹拌した。飽和炭酸水素ナトリウム水溶液を３ mL加え、酢酸エチルで抽出し、有機相を水、飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥し、溶媒を減圧除去した。トリフルオロ酢酸 ３ ｍＬを加え、室温で２３時間撹拌した後、減圧除去し、HPLCで精製し(eluent A (H2O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H2O) (A/B = 90/10 to 0/100 40min))、目的化合物(２０) (３９．０ mg, ７５％)を得た。
1H NMR (400 MHz, MeOD) dδ: 3.77 (t, J = 4.7, 8 H), 3.85 (t, J = 4.7, 8 H), 4.38 (s, 2 H), 7.21-7.28 (m, 6 H), 7.43 (d, J = 7.9 Hz, 1 H), 8.18 (dd, J = 7.9, 1.6 Hz, 1 H), 8.41 (d, J = 1.0 Hz, 1 H); 13C NMR (400 MHz, MeOD) δ: 48.5 (CH2), 62.6 (CH2), 67.4 (CH2), 98.6 (CH), 115.6, 116.2 (CH), 129.9 (CH), 130.6 (CH), 130.8 (CH), 132.7 (CH), 134.0 (C), 136.3 (C), 141.9 (C), 158.2 (C), 159.0 (C), 159.8 (C), 168.8 (C); HRMS (ESI +): m/z calcd for [M] +, 501.20201; found, 501.20237 (err. -0.4 mDa)
Synthesis of compound (23) (HMRGCOOH) Compound (5) 58.5 mg (0.1703 mmol, 2 eq) was dissolved in dehydrated tetrahydrofuran (THF) 7 mL, and at -78C for 10 minutes under argon atmosphere. Stir. 1 M sec-butyllithium cyclohexane and n-hexane solution 0.17 mL (0.17 mmol, 2 eq) was added over 2 minutes, and the mixture was stirred for 30 minutes. A solution of compound (22) 31.2 mg (0.0852 mmol, 1 eq) in THF (5 mL) was added, and the mixture was stirred at −78 ° C. for 30 minutes and at room temperature for 23 and a half hours. 3 mL of a saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 3 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 23 hours, then removed under reduced pressure, and purified by HPLC (eluent A (H 2 O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H 2 O) (A / B = 90/10 to 0/100 40 min)), the target compound (20) (39.0 mg, 75%) was obtained.
1 H NMR (400 MHz, MeOD) dδ: 3.77 (t, J = 4.7, 8 H), 3.85 (t, J = 4.7, 8 H), 4.38 (s, 2 H), 7.21-7.28 (m, 6 H), 7.43 (d, J = 7.9 Hz, 1 H), 8.18 (dd, J = 7.9, 1.6 Hz, 1 H), 8.41 (d, J = 1.0 Hz, 1 H); 13 C NMR (400 MHz , MeOD) δ: 48.5 (CH 2 ), 62.6 (CH 2 ), 67.4 (CH 2 ), 98.6 (CH), 115.6, 116.2 (CH), 129.9 (CH), 130.6 (CH), 130.8 (CH), 132.7 (CH), 134.0 (C), 136.3 (C), 141.9 (C), 158.2 (C), 159.0 (C), 159.8 (C), 168.8 (C); HRMS (ESI + ): m / z calcd for [M] + , 501.20201; found, 501.20237 (err. -0.4 mDa)
また、以下のスキームに従って、本発明の超解像蛍光イメージング用プローブである化合物を合成した。なお、化合物２４の合成については、Ｎａｔ．Ｂｉｏｔｅｃｈ．，２００３， ２１，８６−８９に開示されており、化合物２５の合成については、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．２０１３，１３５，６１８４−６１９１に開示されており、これらを参照することができる。
化合物（２６）（ＨＭＳｉＲ−ＢＧ）の合成
化合物（７）９．２ ｍｇ（０．０２０１ ｍｍｏｌ, １ ｅｑ）を脱水Ｎ，Ｎ−ジメチルホルムアミド１ ｍＬに溶かし、化合物（２４）５．４ ｍｇ（０．０２０１ ｍｍｏｌ, １ ｅｑ）、１-ヒドロキシベンゾトリアゾール２．７ ｍｇ（０．０２０１ ｍｍｏｌ, １ ｅｑ）、1-エチル-3-（3-ジメチルアミノプロピル）カルボジイミド塩酸塩３．８ ｍｇ（０．０２０１ ｍｍｏｌ, １ ｅｑ）、トリエチルアミン５．６ mL（０．０４０２ ｍｍｏｌ, ２ ｅｑ）を加え、アルゴン雰囲気下、室温で１７時間撹拌した。溶媒を減圧除去し、HPLCで精製し(eluent A (H2O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H2O) (A/B = 90/10 to 0/100 50min))、目的化合物(２６) （ＨＭＳｉＲ−ＢＧ）(９．９ mg, ６０％)を得た。
1H NMR (400 MHz, MeOD): δ 8.33 (s, 1 H), 8.25 (s, 1 H), 7.95 (d, J = 7.1 Hz, 1 H), 7.56 (d, J = 8.0 Hz, 2 H), 7.46 (d, J = 8.0, 2 H), 7.37 (d, J = 2.6 Hz, 2 H), 7.27 (d, J = 7.9 Hz, 1 H), 7.04 (d, J = 9.6 Hz, 2 H), 6.75 (dd, J = 9.6, 2.6 Hz, 2 H), 5.66 (s, 2 H), 4.66 (s, 2 H), 4.36 (s, 2 H), 3.35 (s, 12 H), 0.61 (s, 3 H), 0.60 (s, 3 H); 13C NMR (101 MHz, MeOD): δ 169.4, 168.0, 161.2,158.3, 155.8, 153.7, 149.4, 143.5, 142.2, 141.8, 141.6, 141.0, 136.2, 135.5, 130.7, 130.3, 128.8, 128.2, 127.3, 126.9, 122.3, 115.2, 108.4, 70.8, 62.1, 44.3, 40.9, -1.1, -1.3; HRMS (m/z): [M]+ calcd. for C40H43N8O3Si, 711.32219; found, 711.32339. (err. 1.2 mDa)
Synthesis of compound (26) (HMSiR-BG) Compound (7) 9.2 mg (0.0201 mmol, 1 eq) was dissolved in dehydrated N, N-
1 H NMR (400 MHz, MeOD): δ 8.33 (s, 1 H), 8.25 (s, 1 H), 7.95 (d, J = 7.1 Hz, 1 H), 7.56 (d, J = 8.0 Hz, 2 H), 7.46 (d, J = 8.0, 2 H), 7.37 (d, J = 2.6 Hz, 2 H), 7.27 (d, J = 7.9 Hz, 1 H), 7.04 (d, J = 9.6 Hz, 2 H), 6.75 (dd, J = 9.6, 2.6 Hz, 2 H), 5.66 (s, 2 H), 4.66 (s, 2 H), 4.36 (s, 2 H), 3.35 (s, 12 H) , 0.61 (s, 3 H), 0.60 (s, 3 H); 13 C NMR (101 MHz, MeOD): δ 169.4, 168.0, 161.2,158.3, 155.8, 153.7, 149.4, 143.5, 142.2, 141.8, 141.6, 141.0, 136.2, 135.5, 130.7, 130.3, 128.8, 128.2, 127.3, 126.9, 122.3, 115.2, 108.4, 70.8, 62.1, 44.3, 40.9, -1.1, -1.3; HRMS (m / z): [M] + calcd for C 40 H 43 N 8 O 3 Si, 711.32219; found, 711.32339. (err. 1.2 mDa)
化合物（２７）（ＨＭＳｉＲ−Ｈａｌｏ）の合成
化合物（７）１０．６ ｍｇ（０．０１８５ ｍｍｏｌ, １ ｅｑ）を脱水Ｎ，Ｎ−ジメチルホルムアミド２ ｍＬに溶かし、化合物（２５）７．２ ｍｇ（０．０２７８ ｍｍｏｌ, １．５ ｅｑ）、１-ヒドロキシベンゾトリアゾール３．８ ｍｇ（０．０２７８ ｍｍｏｌ, １．５ ｅｑ）、1-エチル-3-（3-ジメチルアミノプロピル）カルボジイミド塩酸塩５．３ ｍｇ（０．０２７８ ｍｍｏｌ, １．５ ｅｑ）、トリエチルアミン１２．９ mL（０．０９２６ ｍｍｏｌ, ５ ｅｑ）を加え、アルゴン雰囲気下、室温で１３時間撹拌した。溶媒を減圧除去し、HPLCで精製し(eluent A (H2O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H2O) (A/B = 90/10 to 0/100 40min))、目的化合物(２７) （ＨＭＳｉＲ−Ｈａｌｏ）(５．２ mg, ３６％)を得た。
1H NMR (400 MHz, MeOD): δ 8.22 (s, 1 H), 7.92 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 2.6 Hz, 2H), 7.26 (d, J = 7.8 Hz, 1 H), 7.05 (d, J = 9.6 Hz, 2H), 6.76 (dd, J = 9.6, 2.6 Hz, 2H), 4.37 (s, 2 H), 3.71-3.61 (m, 8 H), 3.53-3.49 (m, 4 H), 3.35 (s, 12 H), 1.77-1.70 (m, 2H), 1.63-1.56 (m, 2 H), 1.48-1.37 (m, 4 H), 0.62 (s, 3 H), 0.61 (s, 3 H); 13C NMR (101 MHz, MeOD): δ169.5 (C), 168.1 (C), 155.8 (C), 149.5 (C), 142.2 (CH), 141.6 (C), 141.5 (C), 136.4 (C), 130.6 (CH), 128.3 (C), 127.3 (CH), 126.8 (CH), 122.3 (CH), 115.2 (CH), 72.2 (CH2), 71.3 (CH2), 71.2 (CH2), 70.6 (CH2), 62.2 (CH2), 45.7 (CH2), 41.1 (CH2), 40.9 (CH3), 33.7 (CH2), 30.5 (CH2), 27.7 (CH2), 26.5 (CH2), -1.1 (CH3), -1.3 (CH3); HRMS (m/z): [M]+ calcd. for C37H51ClN3O4Si, 664.33319; found, 664.33434.(err. -1.2 mDa)
Synthesis of compound (27) (HMSiR-Halo) Compound (7) 10.6 mg (0.0185 mmol, 1 eq) was dissolved in dehydrated N, N-
1 H NMR (400 MHz, MeOD): δ 8.22 (s, 1 H), 7.92 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 2.6 Hz, 2H), 7.26 (d, J = 7.8 Hz, 1 H), 7.05 (d, J = 9.6 Hz, 2H), 6.76 (dd, J = 9.6, 2.6 Hz, 2H), 4.37 (s, 2 H), 3.71-3.61 (m, 8 H), 3.53-3.49 (m, 4 H), 3.35 (s, 12 H), 1.77-1.70 (m, 2H), 1.63-1.56 (m, 2 H), 1.48-1.37 (m, 4 H), 0.62 (s , 3 H), 0.61 (s, 3 H); 13 C NMR (101 MHz, MeOD): δ169.5 (C), 168.1 (C), 155.8 (C), 149.5 (C), 142.2 (CH), 141.6 (C), 141.5 (C), 136.4 (C), 130.6 (CH), 128.3 (C), 127.3 (CH), 126.8 (CH), 122.3 (CH), 115.2 (CH), 72.2 (CH 2 ) , 71.3 (CH 2 ), 71.2 (CH 2 ), 70.6 (CH 2 ), 62.2 (CH 2 ), 45.7 (CH 2 ), 41.1 (CH 2 ), 40.9 (CH 3 ), 33.7 (CH 2 ), 30.5 (CH 2 ), 27.7 (CH 2 ), 26.5 (CH 2 ), -1.1 (CH 3 ), -1.3 (CH 3 ); HRMS (m / z): [M] + calcd. For C 37 H 51 ClN 3 O 4 Si, 664.33319; found, 664.33434. (Err.-1.2 mDa)
化合物（２８）（ＨＭＡｃＲＧ−ＢＧ）の合成
化合物（２１）２．４ ｍｇ（０．００６０ ｍｍｏｌ, １ ｅｑ）を脱水Ｎ，Ｎ−ジメチルホルムアミド１ ｍＬに溶かし、化合物（２４）４．８ ｍｇ（０．０１８０ ｍｍｏｌ, ３ ｅｑ）、１-ヒドロキシベンゾトリアゾール２．４ ｍｇ（０．０１８０ ｍｍｏｌ, ３ ｅｑ）、1-エチル-3-（3-ジメチルアミノプロピル）カルボジイミド塩酸塩３．４ ｍｇ（０．０１８０ ｍｍｏｌ, ３ ｅｑ）、トリエチルアミン８．３ mL（０．０５９６ ｍｍｏｌ, １０ ｅｑ）を加え、アルゴン雰囲気下、室温で２０時間撹拌した。溶媒を減圧除去し、HPLCで精製し(eluent A (H2O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H2O) (A/B = 90/10 to 0/100 40min))、目的化合物(２８) （ＨＭＡｃＲＧ−ＢＧ） (２．１ mg, ４６％)を得た。
1H NMR (400 MHz, MeOD): δ8.48 (s, 1 H), 8.27 (s, 1 H), 8.23 (s, 1 H), 8.04 (d, J = 7.9 Hz, 1 H), 7.56 (d, J = 8.0 Hz, 2 H), 7.48-7.44 (m, 4 H), 7.34-7.30 (m, 2 H), 7.04 (d, J = 9.1 Hz, 1 H), 6.98 (s, 1 H), 5.65 (s, 2 H), 4.67 (s, 2 H), 4.39 (s, 2 H), 2.23 (s, 3 H); HRMS (m/z): [M+Na]+ (closed form + Na) calcd. for C36H30N8NaO5, 677.22314; found, 677.22302.(err. 0.1 mDa)
Synthesis of compound (28) (HMAcRG-BG) Compound (21) 2.4 mg (0.0060 mmol, 1 eq) was dissolved in dehydrated N, N-
1 H NMR (400 MHz, MeOD): δ8.48 (s, 1 H), 8.27 (s, 1 H), 8.23 (s, 1 H), 8.04 (d, J = 7.9 Hz, 1 H), 7.56 (d, J = 8.0 Hz, 2 H), 7.48-7.44 (m, 4 H), 7.34-7.30 (m, 2 H), 7.04 (d, J = 9.1 Hz, 1 H), 6.98 (s, 1 H), 5.65 (s, 2 H), 4.67 (s, 2 H), 4.39 (s, 2 H), 2.23 (s, 3 H); HRMS (m / z): [M + Na] + (closed form + Na) calcd.for C 36 H 30 N 8 NaO 5 , 677.22314; found, 677.22302. (err. 0.1 mDa)
化合物（２９）（ＨＭＡｃＲＧ−Ｈａｌｏ）の合成
化合物（２１）４．２ ｍｇ（０．０１０４ ｍｍｏｌ, １ ｅｑ）を脱水Ｎ，Ｎ−ジメチルホルムアミド２ ｍＬに溶かし、化合物（２５）７．０ ｍｇ（０．０３１２ ｍｍｏｌ, ３ ｅｑ）、１-ヒドロキシベンゾトリアゾール４．２ ｍｇ（０．０３１２ ｍｍｏｌ, ３ ｅｑ）、1-エチル-3-（3-ジメチルアミノプロピル）カルボジイミド塩酸塩６．０ ｍｇ（０．０３１２ ｍｍｏｌ, ３ ｅｑ）、トリエチルアミン１４．６ mL（０．１０４４ ｍｍｏｌ, １０ ｅｑ）を加え、アルゴン雰囲気下、室温で２１時間撹拌した。溶媒を減圧除去し、HPLCで精製し(eluent A (H2O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H2O) (A/B = 90/10 to 0/100 40min))、目的化合物(２９)（ＨＭＡｃＲＧ−Ｈａｌｏ）(２．０ mg, ２１％)を得た。
1H NMR (400 MHz, MeOD): δ 8.50 (s, 1 H), 8.21 (s, 1 H), 8.02 (d, J = 7.4 Hz, 1 H), 7.47-7.44 (m, 2 H), 7.34-7.30 (m, 2 H), 7.05 (d, J = 9.1 Hz, 1 H), 6.97 (d, J = 2.0 Hz, 1 H), 4.40 (s, 2 H), 3.73-3.62 (m, 8 H), 3.54-3.49 (m, 4 H), 2.23 (s, 3 H), 1.75 (quin, J = 6.9 Hz, 2 H), 1.59 (quin, J = 6.9 Hz, 2 H), 1.48-1.37 (m, 4 H); HRMS (m/z): [M+Na]+ (closed form + Na) calcd. for C33H38ClN3NaO6, 630.23413; found, 630.23342.(err. -0.7 mDa)
Synthesis of compound (29) (HMAcRG-Halo) Compound (21) 4.2 mg (0.0104 mmol, 1 eq) was dissolved in dehydrated N, N-
1 H NMR (400 MHz, MeOD): δ 8.50 (s, 1 H), 8.21 (s, 1 H), 8.02 (d, J = 7.4 Hz, 1 H), 7.47-7.44 (m, 2 H), 7.34-7.30 (m, 2 H), 7.05 (d, J = 9.1 Hz, 1 H), 6.97 (d, J = 2.0 Hz, 1 H), 4.40 (s, 2 H), 3.73-3.62 (m, 8 H), 3.54-3.49 (m, 4 H), 2.23 (s, 3 H), 1.75 (quin, J = 6.9 Hz, 2 H), 1.59 (quin, J = 6.9 Hz, 2 H), 1.48- 1.37 (m, 4 H); HRMS (m / z): [M + Na] + (closed form + Na) calcd.for C 33 H 38 ClN 3 NaO 6 , 630.23413; found, 630.23342. (Err.-0.7 mDa)
化合物（３０）（ＨＭＭｏＲ−ＢＧ）の合成
化合物（２３）１０．３ ｍｇ（０．０２０６ ｍｍｏｌ, １ ｅｑ）を脱水Ｎ，Ｎ−ジメチルホルムアミド５ ｍＬに溶かし、化合物（２４）１６．７ ｍｇ（０．０６１８ ｍｍｏｌ, ３ ｅｑ）、１-ヒドロキシベンゾトリアゾール８．３ ｍｇ（０．０６１８ ｍｍｏｌ, ３ ｅｑ）、1-エチル-3-（3-ジメチルアミノプロピル）カルボジイミド塩酸塩１１．８ ｍｇ（０．０６１８ ｍｍｏｌ, ３ ｅｑ）、トリエチルアミン２８．７ mL（０．２０６０ ｍｍｏｌ, １０ ｅｑ）を加え、アルゴン雰囲気下、室温で１５時間撹拌した。溶媒を減圧除去し、HPLCで精製し(eluent A (H2O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H2O) (A/B = 90/10 to 0/100 40min))、目的化合物(３０)（ＨＭＭｏＲ−ＢＧ）(１２．６ mg, ７１％)を得た。
1H NMR (400 MHz, MeOD): δ 8.38 (s, 1 H), 8.25 (d, J = 1.4 Hz, 1 H), 8.03 (dd, J = 7.9, 1.4 Hz, 1 H), 7.57 (d, J = 8.1 Hz, 2 H), 7.47 (d, J = 8.1 Hz, 2 H), 7.43 (d, J = 7.9 Hz, 1 H), 7.28-7.22 (m, 6 H), 5.68 (s, 2 H), 4.67 (s, 2 H), 4.38 (s, 2 H), 3.85 (dd, J = 4.9, 4.2 Hz, 8 H), 3.77 (dd, J = 4.9, 4.2 Hz, 8 H); 13C NMR (101 MHz, MeOD): δ169.1,161.1, 159.8, 159.1, 158.3, 158.0, 153.4, 143.9, 142.0, 141.1, 137.5, 135.4, 135.1, 132.7, 130.9, 130.3, 128.9, 128.4, 127.6, 116.2, 115.7, 107.8, 98.5, 71.0, 67.4, 62.7, 48.5, 44.4; HRMS (m/z): [M]+ calcd. for C42H41N8O6, 753.31436; found, 753.31450.(err. -0.1 mDa)
Synthesis of compound (30) (HMMoR-BG) Compound (23) 10.3 mg (0.0206 mmol, 1 eq) was dissolved in dehydrated N, N-
1 H NMR (400 MHz, MeOD): δ 8.38 (s, 1 H), 8.25 (d, J = 1.4 Hz, 1 H), 8.03 (dd, J = 7.9, 1.4 Hz, 1 H), 7.57 (d , J = 8.1 Hz, 2 H), 7.47 (d, J = 8.1 Hz, 2 H), 7.43 (d, J = 7.9 Hz, 1 H), 7.28-7.22 (m, 6 H), 5.68 (s, 2 H), 4.67 (s, 2 H), 4.38 (s, 2 H), 3.85 (dd, J = 4.9, 4.2 Hz, 8 H), 3.77 (dd, J = 4.9, 4.2 Hz, 8 H); 13 C NMR (101 MHz, MeOD): δ169.1,161.1, 159.8, 159.1, 158.3, 158.0, 153.4, 143.9, 142.0, 141.1, 137.5, 135.4, 135.1, 132.7, 130.9, 130.3, 128.9, 128.4, 127.6, 116.2, 115.7, 107.8, 98.5, 71.0, 67.4, 62.7, 48.5, 44.4; HRMS (m / z): [M] + calcd.for C 42 H 41 N 8 O 6 , 753.31436; found, 753.31450. (Err.-0.1 mDa)
化合物（３１）（ＨＭＭｏＲ−Ｈａｌｏ）の合成
化合物（２３）９．８ ｍｇ（０．０１９６ ｍｍｏｌ, １ ｅｑ）を脱水Ｎ，Ｎ−ジメチルホルムアミド２ ｍＬに溶かし、化合物（２４）５．９ ｍｇ（０．０７１１ ｍｍｏｌ, ３．６ ｅｑ）、１-ヒドロキシベンゾトリアゾール７．９ ｍｇ（０．０５８７ ｍｍｏｌ, ３ ｅｑ）、1-エチル-3-（3-ジメチルアミノプロピル）カルボジイミド塩酸塩１１．３ ｍｇ（０．０５８７ ｍｍｏｌ, ３ ｅｑ）、トリエチルアミン２７．３ mL（０．１９６０ ｍｍｏｌ, １０ ｅｑ）を加え、アルゴン雰囲気下、室温で１３時間撹拌した。溶媒を減圧除去し、HPLCで精製し(eluent A (H2O, 1% MeCN, 0.1% TFA) and eluent B (MeCN, 1% H2O) (A/B = 90/10 to 0/100 40min))、目的化合物(３１) （ＨＭＭｏＲ−Ｈａｌｏ）(１０．０ mg, ６２％)を得た。
1H NMR (400 MHz, MeOD): δ 8.23 (d, J = 1.2 Hz, 1 H), 8.01 (dd, J = 7.9, 1.7 Hz, 1 H), 7.42 (d, J = 7.9 Hz, 1 H), 7.28-7.23 (m, 6 H), 4.39 (s, 2 H), 3.86 (dd, J = 5.0, 4.2 Hz, 8 H), 3.77 (dd, J = 5.0, 4.2 Hz, 8 H), 3.73-3.56 (m, 8 H), 3.54-3.49 (m, 4 H), 1.79-1.70 (m, 2 H), 1.63-1.56 (m, 2 H), 1.49-1.35 (m, 4 H); 13C NMR (101 MHz, MeOD): δ169.2 (C), 159.8 (C), 159.1 (C), 158.4 (C), 142.0 (C), 137.6 (C), 135.0 (C), 132.7 (CH), 130.8 (CH), 128.5 (CH), 127.5 (CH), 116.2 (CH), 115.8 (C), 98.5 (CH), 72.2 (CH2), 71.3 (CH2), 71.2 (CH2), 70.5 (CH2), 67.4 (CH2), 62.8 (CH2), 48.9 (CH2), 45.7 (CH2), 41.1 (CH2), 33.7 (CH2), 30.5 (CH2), 27.7 (CH2), 26.5 (CH2); HRMS (m/z): [M]+ calcd. for C39H49ClN3O7, 706.32536; found, 706.32555.(err. -0.2 mDa)
Synthesis of compound (31) (HMMoR-Halo) Compound (23) 9.8 mg (0.0196 mmol, 1 eq) was dissolved in dehydrated N, N-
1 H NMR (400 MHz, MeOD): δ 8.23 (d, J = 1.2 Hz, 1 H), 8.01 (dd, J = 7.9, 1.7 Hz, 1 H), 7.42 (d, J = 7.9 Hz, 1 H ), 7.28-7.23 (m, 6 H), 4.39 (s, 2 H), 3.86 (dd, J = 5.0, 4.2 Hz, 8 H), 3.77 (dd, J = 5.0, 4.2 Hz, 8 H), 3.73-3.56 (m, 8 H), 3.54-3.49 (m, 4 H), 1.79-1.70 (m, 2 H), 1.63-1.56 (m, 2 H), 1.49-1.35 (m, 4 H); 13 C NMR (101 MHz, MeOD): δ169.2 (C), 159.8 (C), 159.1 (C), 158.4 (C), 142.0 (C), 137.6 (C), 135.0 (C), 132.7 (CH ), 130.8 (CH), 128.5 (CH), 127.5 (CH), 116.2 (CH), 115.8 (C), 98.5 (CH), 72.2 (CH 2 ), 71.3 (CH 2 ), 71.2 (CH 2 ), 70.5 (CH 2 ), 67.4 (CH 2 ), 62.8 (CH 2 ), 48.9 (CH 2 ), 45.7 (CH 2 ), 41.1 (CH 2 ), 33.7 (CH 2 ), 30.5 (CH 2 ), 27.7 ( CH 2 ), 26.5 (CH 2 ); HRMS (m / z): [M] + calcd. For C 39 H 49 ClN 3 O 7 , 706.32536; found, 706.32555. (Err.-0.2 mDa)
平衡定数（ｐＫ ｃｙｃｌ ）の算出
本発明のプローブ化合物について、溶液中での吸収スペクトル変化及び蛍光スペクトル変化を測定し、そのｐＨ依存性から平衡定数を算出した。化合物７（ＨＭＳｉＲＣＯＯＨ）、化合物１１（ＡＭＳｉＲＣＯＯＨ）、化合物１４（ＨＭＡｃＲＧ）、化合物１６（ＡＭＡｃＲＧ）、化合物１７（ＡＭＲＧ）、及び化合物２１（ＨＭＡｃＲＧＣＯＯＨ）の各ｐＨにおける吸収スペクトル変化及び蛍光スペクトル変化（励起波長６２０ｎｍ）を図１中の（ａ）及び（ｂ）にそれぞれ示す。測定は、化合物３が０．５μＭの１％ＤＭＳＯ水溶液で行った。その結果、化合物７ではｐＫｃｙｃｌ＝５．８が得られた。その他も同様にして、ｐＫｃｙｃｌを測定し、得られた結果を以下の表１に示す。
表１によれば、これら化合物のｐＫｃｙｃｌは、いずれも３．６〜６．7の範囲であり、これら化合物が中性条件下では大多数が閉環状態であり、開環状態のものの存在比率がＳＬＭによる超解像蛍光イメージングに適した範囲内であることを示している。 According to Table 1, the pK cycl of these compounds is in the range of 3.6 to 6.7, and the majority of these compounds are in a ring-closed state under neutral conditions, and the abundance ratio of those in the ring-opened state Is within the range suitable for super-resolution fluorescence imaging by SLM.
また、プローブ化合物の蛍光団の部分構造を固定して、種々の求核基を変化させた場合におけるｐＫｃｙｃｌを算出した結果を図２に示す。逆に、プローブ化合物の求核基を固定して、蛍光団の部分構造を変化させた場合におけるｐＫｃｙｃｌを算出した結果を図３に示す。これらの結果から、求核基と蛍光団の組み合わせを変えることによって、ｐＫｃｙｃｌを制御することが可能であることが分かった。 Further, FIG. 2 shows the result of calculating pK cycl when the partial structure of the fluorophore of the probe compound is fixed and various nucleophilic groups are changed. Conversely, FIG. 3 shows the result of calculating pK cycl when the nucleophilic group of the probe compound was fixed and the partial structure of the fluorophore was changed. From these results, it was found that pK cycl could be controlled by changing the combination of the nucleophilic group and the fluorophore.
閉環速度の算出
レーザーフラッシュフォトリシスにより本発明のプローブ化合物の過渡吸収スペクトルの経時変化を測定することによって、開環構造と閉環構造の平衡における反応速度定数ｋ、閉環速度時定数τ、開環量子収率Φoを算出した。溶液条件は、３％ＤＭＳＯ水溶液、ｐＨ７．４（１０ｍＭＮａＰｉバッファー）である。測定条件は２９５Ｋで、光源は１０ｍＪ/ｐｕｌｓｅのＸｅＣｌエキシマーレーザー（３０８ｎｍ）である。各化合物について得られた結果を表２に示す。
微小管の超解像蛍光イメージング
化合物７（ＨＭＳｉＲＣＯＯＨ）のカルボキシル基をエステル化した化合物１２でラベル化したチューブリンをｉｎ ｖｉｔｒｏで構築した微小管を、チオールを添加せず、酸素存在下で、データ取得前に無蛍光状態にするためのレーザーを照射することなく、従来法より極めて低い０．０２ ｋＷ／ｃｍ２の励起強度によりＳＴＯＲＭ顕微鏡で観察し、解析することで超解像画像が取得できた。解析前のｐｒｏｊｅｃｔｉｏｎ画像に比べ、解像度が高い（線が細くなる）ことが確認できた（図４）。
Super-resolution fluorescence imaging of microtubules Microtubules constructed in vitro with tubulin labeled with
ＲｅｃＡｆｉｌａｍｅｎｔｏｎｐｌａｓｍｉｄＤＮＡの超解像蛍光イメージング
直径約５００ｎｍの環状構造を持つプラスミドＤＮＡ上に構築したＲｅｃＡｆｉｌａｍｅｎｔをＳＴＯＲＭ顕微鏡で観察した。蛍光プローブとして、化合物７（ＨＭＳｉＲＣＯＯＨ）のカルボキシル基をエステル化した化合物１２を用いた。解析をしない状態（Ｐｒｏｊｅｃｔｉｏｎ画像）では、環状構造が潰れてしまうのに対して、ＳＴＯＲＭイメージングでは明確に環状構造が確認できた（図５）。
RecAfilamentonplasmid DNA super-resolution fluorescence imaging RecAfilament constructed on plasmid DNA having a circular structure with a diameter of about 500 nm was observed with a STORM microscope. As a fluorescent probe,
実施例４及び５の結果は、本発明のプローブを用いて、チオールを添加せず、酸素存在下で、データ取得前に無蛍光状態にするためのレーザーを照射することなく、超解像蛍光イメージングが可能であることを実証するものである。 The results of Examples 4 and 5 show that, using the probe of the present invention, super-resolution fluorescence without adding a thiol, in the presence of oxygen, and without irradiating a laser for making it non-fluorescent before data acquisition. This demonstrates that imaging is possible.
固定細胞における微小管の超解像蛍光イメージング
化合物７（ＨＭＳｉＲＣＯＯＨ）のカルボキシル基をＳＮＡＰ−ｔａｇの基質であるベンジルグアニン基と結合した化合物２６（ＨＭＳｉＲ−ＢＧ）でＨｅＬａ生細胞のβ−チューブリンをラベル化し、メタノールで固定後、ＰＢＳ中で観察した。細胞膜を通過し、β−チューブリンを選択的にラベル化できていることが確認できた。また、チオールを添加せず、酸素存在下で、データ取得前に無蛍光状態にするためのレーザーを照射することなく超解像画像を取得できた。解析前のｐｒｏｊｅｃｔｉｏｎ画像に比べ、解像度が高い（線が細くなる）だけでなく、１本に見えていた微小管を２本に分離することが出来た（図６）。
Super-resolution fluorescence imaging of microtubules in fixed cells Compound 26 (HMSiRCOOH) conjugated with benzylguanine which is a substrate of SNAP-tag Compound 26 (HMSiR-BG) β -Tubulin was labeled, fixed with methanol, and observed in PBS. It was confirmed that β-tubulin was selectively labeled through the cell membrane. Moreover, a super-resolution image could be acquired without adding thiol and in the presence of oxygen, without irradiating a laser for making it non-fluorescent before data acquisition. Compared to the projection image before the analysis, not only the resolution was high (the line became thinner), but the microtubules that appeared to be one could be separated into two (FIG. 6).
生細胞における微小管の超解像蛍光イメージング
化合物７（ＨＭＳｉＲＣＯＯＨ）のカルボキシル基をＳＮＡＰ−ｔａｇの基質であるベンジルグアニン基と結合した化合物２６（ＨＭＳｉＲ−ＢＧ）でＨｅＬａ生細胞のβ−チューブリンをラベル化し、培地で洗浄後、培地中で観察した。細胞膜を通過し、β−チューブリンを選択的にラベル化できることが確認できた。細胞質内においても、チオールを添加せず、酸素存在下で、データ取得前に無蛍光状態にするためのレーザーを照射することなく、超解像画像を取得できた。解析前のｐｒｏｊｅｃｔｉｏｎ画像に比べ、解像度が高い（線が細くなる）だけでなく、１本に見えていた微小管を２本に分離することが出来た（図７）。
Super-resolution fluorescence imaging of microtubules in living cells Compound 26 (HMSiRCOOH) is bound to benzylguanine group, which is a substrate of SNAP-tag, with compound 26 (HMSiR-BG) β -Tubulin was labeled and washed in the medium and then observed in the medium. It was confirmed that β-tubulin can be selectively labeled through the cell membrane. Even in the cytoplasm, a super-resolution image could be acquired without adding thiol and in the presence of oxygen without irradiating a laser for making it non-fluorescent before data acquisition. Compared to the projection image before the analysis, not only the resolution was high (the line became thinner), but the microtubules that appeared to be one could be separated into two (FIG. 7).
実施例６及び７の結果は、本発明のプローブをＳＮＡＰ−ｔａｇ等の基質特異的にラベル化されるタンパク質を用いることで、任意のタンパク質の超解像蛍光イメージングが固定細胞及び生細胞において可能であることを実証するものである。 The results of Examples 6 and 7 show that by using a protein specifically labeled with a substrate such as SNAP-tag as the probe of the present invention, super-resolution fluorescence imaging of any protein can be performed in fixed cells and living cells. It is proved that.
Claims (13)
Ｘは、酸素原子又はＣ（Ｒａ）（Ｒｂ）又はＳｉ（Ｒａ）（Ｒｂ）を表し（ここで、Ｒａ及びＲｂは、それぞれ独立に水素原子、又はアルキル基を表す）；
Ｒ１は、水素原子、アルキル基、カルボキシル基、エステル基、アルコキシ基、アミド基、又はアジド基を表し；
Ｙは、Ｃ １ -Ｃ３アルキレン基を表し；
Ｒ２は、置換されていてもよいアミノ基、置換されていてもよいアミド基、ヒドロキシル基、チオール基、又はカルボキシル基を表し；
Ｒ３、Ｒ４、Ｒ７、及びＲ８は、それぞれ独立に水素原子、ヒドロキシル基、アルキル基、スルホ基、カルボキシル基、エステル基、アミド基、アジド基、又はハロゲン原子を表し；
Ｒ５及びＲ６は、それぞれ独立に水素原子又はアルキル基を示し（ここで、Ｒ５及びＲ６は、それぞれＲ３及びＲ７と一緒になって、それらが結合する窒素原子を含む環構造を形成してもよい）；及び、
Ｒ９及びＲ１０は、それぞれ独立に水素原子もしくはアルキル基を示し、又は、Ｎ（Ｒ９）（Ｒ１０）がアミド基もしくはカルバメート基を形成する（ここで、Ｒ９及びＲ１０がアルキル基である場合、それぞれＲ８及びＲ４と一緒になって、それらが結合する窒素原子を含む環構造を形成してもよい。）〕 A probe for super-resolution fluorescence imaging containing a compound represented by the following formula (I) or a salt thereof:
X represents an oxygen atom or C (R a ) (R b ) or Si (R a ) (R b ) (where R a and R b independently represent a hydrogen atom or an alkyl group) ;
R 1 represents a hydrogen atom, an alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, or an azide group;
Y represents a C 1 -C 3 alkylene group;
R 2 represents an optionally substituted amino group, an optionally substituted amide group, a hydroxyl group, a thiol group, or a carboxyl group;
R 3 , R 4 , R 7 , and R 8 each independently represent a hydrogen atom, a hydroxyl group, an alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, an azide group, or a halogen atom;
R 5 and R 6 each independently represent a hydrogen atom or an alkyl group (wherein R 5 and R 6 together with R 3 and R 7 respectively contain a nitrogen atom to which they are bonded) And); and
R 9 and R 10 each independently represent a hydrogen atom or an alkyl group, or N (R 9 ) (R 10 ) forms an amide group or a carbamate group (where R 9 and R 10 are alkyl groups) If it is, respectively together with R 8 and R 4, may form a ring structure containing a nitrogen atom to which they are attached.)]
Ｙがメチレン基であり、
Ｒ２が、置換されていてもよいアミノ基又はヒドロキシル基であり、
Ｒ５、Ｒ６、Ｒ９及びＲ１０が、いずれもメチル基である、
請求項１に記載の超解像蛍光イメージング用プローブ。 X is Si (R a ) (R b );
Y is a methylene group;
R 2 is an optionally substituted amino group or hydroxyl group,
R 5 , R 6 , R 9 and R 10 are all methyl groups,
The probe for super-resolution fluorescence imaging according to claim 1.
Ｙがメチレン基であり、
Ｒ２が、置換されていてもよいアミノ基又はヒドロキシル基であり、
Ｒ５及びＲ６が、いずれも水素原子であり、
Ｎ（Ｒ９）（Ｒ１０）がアミド基である
請求項１に記載の超解像蛍光イメージング用プローブ。 X is an oxygen atom,
Y is a methylene group;
R 2 is an optionally substituted amino group or hydroxyl group,
R 5 and R 6 are both hydrogen atoms,
The probe for super-resolution fluorescence imaging according to claim 1, wherein N (R 9 ) (R 10 ) is an amide group.
Ｙがメチレン基であり、
Ｒ２が、置換されていてもよいアミノ基又はヒドロキシル基であり、
Ｒ５、Ｒ６、Ｒ９及びＲ１０が、いずれも水素原子である
請求項１に記載の超解像蛍光イメージング用プローブ。 X is an oxygen atom,
Y is a methylene group;
R 2 is an optionally substituted amino group or hydroxyl group,
R 5, R 6, R 9 and R 10, the super-resolution fluorescence imaging probe according to <br/> claim 1 Both Ru Oh hydrogen atom.
ＲR ９9 及び／又はＲAnd / or R １０10 は、ハロゲン原子で置換されていてもよいアルキル基であるIs an alkyl group optionally substituted with a halogen atom
請求項１に記載の超解像蛍光イメージング用プローブ。The probe for super-resolution fluorescence imaging according to claim 1.
式（Ｉ）における
In formula (I)
生体分子にプローブ分子を結合させ、これにレーザー光を照射して前記プローブ分子からの蛍光発光を撮影した画像データを取得し、一定の時間間隔でこれを繰り返して得られた複数の前記画像データを解析したうえで重ね合わせることによって、前記生体分子の構造に対する超高解像のイメージ画像を得ることを含む、方法。 A super-resolution fluorescence imaging method using the super-resolution fluorescence imaging probe according to any one of claims 1 to 10 ,
A plurality of image data obtained by binding a probe molecule to a biomolecule, irradiating it with a laser beam, and capturing image data obtained by photographing fluorescence emission from the probe molecule, and repeating this at regular time intervals And obtaining a super high resolution image of the structure of the biomolecule by superimposing and analyzing.
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