JP2018054434A - Method and kit for detecting compound containing formyl-dehydro-piperidine structure - Google Patents
Method and kit for detecting compound containing formyl-dehydro-piperidine structure Download PDFInfo
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Abstract
Description
本発明は、例えば酸化ストレスマーカーとして機能するアクロレイン-アミン付加体等のホルミルデヒドロピペリジン構造を含む化合物の検出方法及び検出キットに関する。 The present invention relates to a detection method and a detection kit for a compound containing a formyl dehydropiperidine structure such as an acrolein-amine adduct that functions as an oxidative stress marker, for example.
有機物(油脂、木炭、材木、樹脂製品等)やタバコが燃えた際に発生する不飽和アルデヒドのアクロレイン(図１Ａ)は、非常に高い毒性を有している(非特許文献１)。このアクロレインは、生体内における酸化ストレス条件(活性酸素種の発生等)下でポリアミン、脂質、アミノ酸等の生体分子からも産生する(非特許文献１)。アクロレインは、非常に高い化学反応性を有しており、様々な基質と反応して、生体にダメージを与える。特に、核酸やタンパク質のアミノ基、チオール基、及び水酸基等と反応した際には、生体機能が損なわれ、種々の免疫・炎症システムの異常を引き起こすと考えられている。それゆえ、アクロレインと様々な酸化ストレス疾患との関係性を分子レベルで理解することは重要であり、またアクロレインの検出法の向上が要求されている。 Unsaturated aldehyde acrolein (FIG. 1A) generated when organic matter (oil, fat, charcoal, timber, resin product, etc.) or tobacco burns has extremely high toxicity (Non-patent Document 1). This acrolein is also produced from biomolecules such as polyamines, lipids and amino acids under oxidative stress conditions (generation of reactive oxygen species, etc.) in the living body (Non-patent Document 1). Acrolein has a very high chemical reactivity, and reacts with various substrates to damage the living body. In particular, when it reacts with amino groups, thiol groups, hydroxyl groups and the like of nucleic acids and proteins, it is considered that biological functions are impaired and various immune / inflammatory system abnormalities are caused. Therefore, it is important to understand the relationship between acrolein and various oxidative stress diseases at the molecular level, and there is a demand for an improved acrolein detection method.
これまでアクロレインの検出は、求核付加による3-アミノフェノールへの変換や蛍光ヒドラジン修飾等の誘導化により行われてきたが、厳しい反応条件や他のアルデヒド共存下での選択性(化学直交性)の低さ等が課題であった。また高速液体クロマトグラフィーを用いずに、高精度且つ高効率な方法として、固相担体を用いる手法も開発されてきた。また、本発明者は独自に見出したアクロレインとアジドのクリック反応を用いて酸化ストレス条件の癌細胞上のアクロレインのイメージングに成功している(非特許文献２)。これは、酸化ストレス条件下の細胞から内因的若しくは外因的に発生するアクロレインに対して、直接的に蛍光アジドプローブを作用させることで可視化することができた。現在は、アクロレイン関連疾患の診断法として、アクロレイン-アミン付加体(非特許文献３)である3-ホルミル-3,4-デヒドロピペリジンを検出する手法が広く知られている。バイオマーカーとしてのホルミルデヒドロピペリジン（FDP）検出はアクロレインの生体濃度を間接的に指し示している。 Until now, detection of acrolein has been carried out by conversion to 3-aminophenol by nucleophilic addition and derivatization of fluorescent hydrazine modification, but selectivity under harsh reaction conditions and other aldehydes (chemical orthogonality) ) Was a problem. In addition, a method using a solid phase carrier has been developed as a highly accurate and highly efficient method without using high performance liquid chromatography. In addition, the present inventor has succeeded in imaging acrolein on cancer cells under oxidative stress conditions using the click reaction of acrolein and azide that was uniquely found (Non-patent Document 2). This could be visualized by direct action of a fluorescent azide probe on acrolein generated endogenously or exogenously from cells under oxidative stress conditions. At present, as a method for diagnosing an acrolein-related disease, a method for detecting 3-formyl-3,4-dehydropiperidine, which is an acrolein-amine adduct (Non-patent Document 3), is widely known. Detection of formyl dehydropiperidine (FDP) as a biomarker indirectly indicates the biological concentration of acrolein.
アクロレインの生理学的な役割を明らかにする研究に関して、千葉大学の五十嵐一衛教授をはじめとする、幾つかのグループが精力的に行ってきた。近年では、名古屋大学の内田浩二教授のグループが、タンパク質上のFDP構造を認識するモノクローナル抗体を作製し、タカラバイオ株式会社と共同してELISA-kitを市販化した(TaKaRa Acrolein-Lysine Adduct Competitive EIA Kit；非特許文献３〜７)。とりわけ、この手法は、動脈硬化、アルツハイマー病、癌、糖尿病、自己免疫疾患、高血圧といった様々な疾患・病状を査定するために使用されている。 Several groups, including Professor Kazue Igarashi from Chiba University, have been energetically engaged in research to clarify the physiological role of acrolein. Recently, a group of Prof. Koji Uchida from Nagoya University made a monoclonal antibody that recognizes the FDP structure on the protein, and in collaboration with Takara Bio Inc., marketed ELISA-kit (TaKaRa Acrolein-Lysine Adduct Competitive EIA Kit; Non-Patent Documents 3 to 7). In particular, this technique is used to assess various diseases and conditions such as arteriosclerosis, Alzheimer's disease, cancer, diabetes, autoimmune diseases, and hypertension.
そこで、本発明は、上述した実情に鑑み、酸化ストレスマーカーとして機能するアクロレイン-アミン付加体をより簡便に且つ効率的に検出する方法を提供することを目的とする。 Therefore, in view of the above situation, an object of the present invention is to provide a method for more easily and efficiently detecting an acrolein-amine adduct that functions as an oxidative stress marker.
また、非特許文献４及び６によれば、酸化ストレスマーカーとしてホルミルデヒドロピペリジン（FDP）構造を有する化合物（図１Ａ中、化合物１）だけでなく、その酸化構造であるピリジニウム（図１Ａ中、化合物２）も産生される。この酸化還元構造の発見は、FDP自身の酸化的芳香族化に伴う還元能力を示唆している。そのような例として、FDPとよく似たジヒドロピリジン環を有するニコチンアデニンヌクレオチド(NADH)は、天然の酸化還元剤として働くことが知られている(図１Ｂ；Aizpurua, J. M. et al. Mechanistic insights on the magnesium(II) ion-activated reduction of methyl benzoylformate with chelated NADH peptide beta-lactam models. J Org Chem 74, 6691-6702, doi:10.1021/jo901236d (2009)）。またvasicineのような含窒素環天然物も酸化的芳香族化に伴ってニトロ基に対する還元能を示すことが知られている(図１Ｃ；Sharma, S., Kumar, M., Kumar, V. & Kumar, N. Metal-free transfer hydrogenation of nitroarenes in water with vasicine: revelation of organocatalytic facet of an abundant alkaloid. J Org Chem 79, 9433-9439, doi:10.1021/jo5019415 (2014)）。もし、FDPも同じように還元能力を示すことができれば、非蛍光ニトロ化合物から蛍光のアミン化合物への変換に伴う蛍光スイッチングが実現できる(図１Ｄ）。これを活用することで、アクロレインのバイオマーカーを従来の抗体を使用せずに検出する手法へと展開できると考えた。 According to Non-Patent Documents 4 and 6, not only a compound having a formyl dehydropiperidine (FDP) structure (compound 1 in FIG. 1A) as an oxidative stress marker, but also pyridinium (a compound in FIG. 1A) that is an oxidized structure thereof. 2) is also produced. The discovery of this redox structure suggests the ability of FDP to reduce with oxidative aromatization. As such an example, nicotine adenine nucleotide (NADH) having a dihydropyridine ring similar to FDP is known to act as a natural redox agent (FIG. 1B; Aizpurua, JM et al. Mechanistic insights on the magnesium (II) ion-activated reduction of methyl benzoylformate with chelated NADH peptide beta-lactam models. J Org Chem 74, 6691-6702, doi: 10.1021 / jo901236d (2009)). Also, nitrogen-containing natural products such as vasicine are known to exhibit reducing ability for nitro groups with oxidative aromatization (FIG. 1C; Sharma, S., Kumar, M., Kumar, V. & Kumar, N. Metal-free transfer hydrogenation of nitroarenes in water with vasicine: revelation of organocatalytic facet of an abundant alkaloid. J Org Chem 79, 9433-9439, doi: 10.1021 / jo5019415 (2014)). If FDP can show the reducing ability in the same manner, fluorescence switching accompanying conversion from a non-fluorescent nitro compound to a fluorescent amine compound can be realized (FIG. 1D). By utilizing this, we thought that it was possible to develop a technique for detecting acrolein biomarkers without using conventional antibodies.
上記課題を解決するため鋭意研究を行った結果、FDP構造を含む化合物は、効率的に芳香族ニトロ化合物を対応するアニリン化合物へと還元することができ、この変換反応に伴う蛍光スイッチングに基づいてFDP構造を含む化合物を検出できることを見出し、本発明を完成するに至った。 As a result of diligent research to solve the above problems, a compound containing an FDP structure can efficiently reduce an aromatic nitro compound to a corresponding aniline compound. Based on the fluorescence switching accompanying this conversion reaction, The inventors have found that a compound containing an FDP structure can be detected, and have completed the present invention.
本発明は以下を包含する。
（１）サンプル中のFDP構造を含む化合物を検出する方法であって、
サンプルと下記一般式(I)：
R1は、独立に、置換若しくは無置換のアリール基、置換若しくは無置換のヘテロアリール基、及び電子求引性基から選ばれる基を表し、
mは、2〜5の整数を表し、
ただし、複数のR1のうち少なくとも1つは、電子求引性基、電子求引性基を有するアリール基、無置換のヘテロアリール基、及び電子求引性基を有するヘテロアリール基から選ばれる基を表す)
で示される化合物を反応させる工程と、
前記反応により生じる蛍光を検出する工程と、
を含む、前記方法。
The present invention includes the following.
(1) A method for detecting a compound containing an FDP structure in a sample,
Sample and the following general formula (I):
R 1 independently represents a group selected from a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, and an electron withdrawing group;
m represents an integer of 2 to 5,
However, at least one of the plurality of R 1 is selected from an electron withdrawing group, an aryl group having an electron withdrawing group, an unsubstituted heteroaryl group, and a heteroaryl group having an electron withdrawing group. Represents a group)
A step of reacting a compound represented by:
Detecting fluorescence generated by the reaction;
Said method.
（２）前記反応工程を金属イオンの存在下で行う、（１）に記載の方法。
（３）サンプルが生物学的サンプルである、（１）又は（２）に記載の方法。
(2) The method according to (1), wherein the reaction step is performed in the presence of a metal ion.
(3) The method according to (1) or (2), wherein the sample is a biological sample.
（４）一般式(I)：
R1は、独立に、置換若しくは無置換のアリール基、置換若しくは無置換のヘテロアリール基、及び電子求引性基から選ばれる基を表し、
mは、2〜5の整数を表し、
ただし、複数のR1のうち少なくとも1つは、電子求引性基、電子求引性基を有するアリール基、無置換のヘテロアリール基、及び電子求引性基を有するヘテロアリール基から選ばれる基を表す)
で示される化合物を含む、FDP構造を含む化合物の検出用組成物。
(4) General formula (I):
R 1 independently represents a group selected from a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, and an electron withdrawing group;
m represents an integer of 2 to 5,
However, at least one of the plurality of R 1 is selected from an electron withdrawing group, an aryl group having an electron withdrawing group, an unsubstituted heteroaryl group, and a heteroaryl group having an electron withdrawing group. Represents a group)
A composition for detection of a compound comprising an FDP structure, comprising the compound represented by:
（５）一般式(I)：
R1は、独立に、置換若しくは無置換のアリール基、置換若しくは無置換のヘテロアリール基、及び電子求引性基から選ばれる基を表し、
mは、2〜5の整数を表し、
ただし、複数のR1のうち少なくとも1つは、電子求引性基、電子求引性基を有するアリール基、無置換のヘテロアリール基、及び電子求引性基を有するヘテロアリール基から選ばれる基を表す)
で示される化合物を含む、FDP構造を含む化合物の検出キット。
（６）金属イオンをさらに含む、（５）に記載のキット。
(5) General formula (I):
R 1 independently represents a group selected from a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, and an electron withdrawing group;
m represents an integer of 2 to 5,
However, at least one of the plurality of R 1 is selected from an electron withdrawing group, an aryl group having an electron withdrawing group, an unsubstituted heteroaryl group, and a heteroaryl group having an electron withdrawing group. Represents a group)
The detection kit of the compound containing FDP structure containing the compound shown by these.
(6) The kit according to (5), further comprising a metal ion.
（７）FDP構造を含む化合物と下記一般式(I)：
R1は、独立に、置換若しくは無置換のアリール基、置換若しくは無置換のヘテロアリール基、及び電子求引性基から選ばれる基を表し、
mは、2〜5の整数を表し、
ただし、複数のR1のうち少なくとも1つは、電子求引性基、電子求引性基を有するアリール基、無置換のヘテロアリール基、及び電子求引性基を有するヘテロアリール基から選ばれる基を表す)
で示される化合物とを反応させる工程を含む、下記一般式(II)で示される化合物の合成方法。
R 1 independently represents a group selected from a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, and an electron withdrawing group;
m represents an integer of 2 to 5,
However, at least one of the plurality of R 1 is selected from an electron withdrawing group, an aryl group having an electron withdrawing group, an unsubstituted heteroaryl group, and a heteroaryl group having an electron withdrawing group. Represents a group)
A method for synthesizing a compound represented by the following general formula (II), which comprises a step of reacting the compound represented by formula (II).
本発明によれば、酸化ストレスマーカーとして機能するアクロレイン-アミン付加体等のFDP構造を含む化合物を、安価に且つ簡便に検出することができる。さらに、検出したアクロレイン-アミン付加体に基づいて、アクロレインを発症因子とする様々な疾患を診断することができる。 According to the present invention, a compound containing an FDP structure such as an acrolein-amine adduct that functions as an oxidative stress marker can be detected inexpensively and easily. Furthermore, based on the detected acrolein-amine adduct, various diseases having acrolein as an onset factor can be diagnosed.
本発明に係るサンプル中のFDP構造を含む化合物を検出する方法(以下、「本方法」と称する)は、当該FDP構造を含む化合物を含有する、又は含有する可能性があるサンプルと下記一般式(I)：
R1は、独立に、置換若しくは無置換のアリール基、置換若しくは無置換のヘテロアリール基、及び電子求引性基から選ばれる基を表し、
mは、2〜5の整数を表し、好ましくは2であり、
ただし、複数のR1のうち少なくとも1つは、電子求引性基、電子求引性基を有するアリール基、無置換のヘテロアリール基、及び電子求引性基を有するヘテロアリール基から選ばれる基を表す)
で示される化合物を反応させる工程を含む方法である。当該反応によれば、サンプルがFDP構造を含む場合には、一般式(I)で示される化合物がFDP構造を含む化合物との反応により還元され、一般式(I)で示される化合物に対応する下記一般式(II)：
で示されるアニリン化合物が生じる。当該アニリン化合物の蛍光を検出し、当該蛍光を指標にFDP構造を含む化合物の有無を検出することができる。また、当該蛍光の強度に基づいて、FDP構造を含む化合物を定量的に検出することも可能である。
A method for detecting a compound containing an FDP structure in a sample according to the present invention (hereinafter referred to as “the present method”) contains a sample containing the FDP structure or may contain it, and the following general formula: (I):
R 1 independently represents a group selected from a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, and an electron withdrawing group;
m represents an integer of 2 to 5, preferably 2;
However, at least one of the plurality of R 1 is selected from an electron withdrawing group, an aryl group having an electron withdrawing group, an unsubstituted heteroaryl group, and a heteroaryl group having an electron withdrawing group. Represents a group)
It is a method including the process of making the compound shown by react. According to the reaction, when the sample includes the FDP structure, the compound represented by the general formula (I) is reduced by the reaction with the compound including the FDP structure, and corresponds to the compound represented by the general formula (I). The following general formula (II):
Is produced. The fluorescence of the aniline compound can be detected, and the presence or absence of the compound containing the FDP structure can be detected using the fluorescence as an index. It is also possible to quantitatively detect a compound containing an FDP structure based on the intensity of the fluorescence.
本方法において検出されるFDP構造を含む化合物としては、特に限定されず、合成された化合物や生体分子等に存在するアミノ基とアクロレインが反応して生成した化合物が挙げられる。 The compound containing the FDP structure detected in the present method is not particularly limited, and examples thereof include a compound formed by a reaction of an acrolein with an amino group present in a synthesized compound or a biomolecule.
具体的には、タンパク質又はペプチドに含まれるアミノ基にアクロレインが反応して生成した、下記一般式：
R3及びR4は、タンパク質もしくはペプチドのリジン残基に結合しているアミノ酸又はアミノ酸残基であるか、又は、R3は水素原子、アセチル基であり、且つR4は水酸基、ベンジルオキシ基、である)
で示されるアクロレイン-アミン付加体が挙げられる。
Specifically, the following general formula, which is generated by the reaction of acrolein with an amino group contained in a protein or peptide:
R 3 and R 4 are amino acids or amino acid residues bonded to a lysine residue of a protein or peptide, or R 3 is a hydrogen atom or an acetyl group, and R 4 is a hydroxyl group or a benzyloxy group )
An acrolein-amine adduct represented by
本方法によれば、サンプル(例えば、ヒト等の被験体由来の生物学的サンプル)において酸化ストレスマーカーとして機能するFDP構造を含む化合物を、安価に且つ簡便に検出することができる。また、FDP構造を含む化合物に基づいて、アクロレインを発症因子とする様々な疾患(アクロレイン関連疾患：例えば、動脈硬化、アルツハイマー病、癌、糖尿病、自己免疫疾患(全身性エリテマトーデス、慢性関節リウマチ、シェーグレン症候群、進行性全身性硬化症、多発性筋炎、皮膚筋炎、I型糖尿病等)、高血圧、脳梗塞等)を診断することができる。従って、本方法は、被験体由来の生物学的サンプルにおけるFDP構造を含む化合物を検出することによる、アクロレイン関連疾患若しくはアクロレイン関連疾患のリスクの診断、評価、検出若しくは判定方法又はアクロレイン関連疾患若しくはアクロレイン関連疾患のリスクを検出するためのin vitroにおけるデータ収集方法として使用することもできる。 According to this method, a compound containing an FDP structure that functions as an oxidative stress marker in a sample (for example, a biological sample derived from a subject such as a human) can be easily and inexpensively detected. In addition, based on compounds containing the FDP structure, various diseases with acrolein as an onset factor (acrolein-related diseases such as arteriosclerosis, Alzheimer's disease, cancer, diabetes, autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, Sjogren) Syndrome, progressive systemic sclerosis, polymyositis, dermatomyositis, type I diabetes, etc.), hypertension, cerebral infarction, etc.) can be diagnosed. Therefore, the present method comprises a method for diagnosing, evaluating, detecting or determining an acrolein-related disease or a risk of an acrolein-related disease or an acrolein-related disease or an acrolein by detecting a compound containing an FDP structure in a biological sample derived from a subject It can also be used as an in vitro data collection method for detecting the risk of related diseases.
本方法における生物学的サンプルとしては、例えば全血、血清、血漿、唾液、髄液、関節液、滑膜液、胸水、心膜液、腹膜液、尿等の体液サンプルが挙げられる。 Examples of the biological sample in this method include body fluid samples such as whole blood, serum, plasma, saliva, spinal fluid, joint fluid, synovial fluid, pleural effusion, pericardial fluid, peritoneal fluid, and urine.
一般式(I)で示される化合物において、R1の置換位置としては、下記に示す位置が好ましい：
また、当該位置にある2つのR1は、両方とも電子求引性基、電子求引性基を有するアリール基、無置換のヘテロアリール基、及び電子求引性基を有するヘテロアリール基から選ばれる基であることが好ましい。
電子求引性基としては、例えばシアノ基、カルボキシル基、-SO2R2、-COR2、-COOR2(R2は炭素数1〜4のアルキル基若しくはフェニル基）等が挙げられ、好ましくはシアノ基である。
アリール基としては、例えばフェニル基、ナフチル基、ジフェニル基等が挙げられる。
ヘテロアリール基としては、例えばピロリル基、インドリル基、イミダゾリル基、ピリジル基、チエニル基等が挙げられる。
Further, the two R 1 at the positions are both selected from an electron withdrawing group, an aryl group having an electron withdrawing group, an unsubstituted heteroaryl group, and a heteroaryl group having an electron withdrawing group. It is preferable that it is a group.
Examples of the electron withdrawing group include a cyano group, a carboxyl group, —SO 2 R 2 , —COR 2 , —COOR 2 (R 2 is an alkyl group having 1 to 4 carbon atoms or a phenyl group), and the like. Is a cyano group.
Examples of the aryl group include a phenyl group, a naphthyl group, and a diphenyl group.
Examples of the heteroaryl group include a pyrrolyl group, an indolyl group, an imidazolyl group, a pyridyl group, and a thienyl group.
特に、一般式(I)で示される化合物としては、下記の一般式：
また、4-ニトロフタロニトリル等の一般式(I)で示される化合物は、例えばSigma-Aldrichや東京化成工業株式会社等から市販されているものであってよく、あるいは一般的に知られた化学合成法に準じて合成されたものであってよい。 Further, the compound represented by the general formula (I) such as 4-nitrophthalonitrile may be commercially available, for example, from Sigma-Aldrich, Tokyo Chemical Industry Co., Ltd. It may be synthesized according to a synthesis method.
本方法では、先ずFDP構造を含む化合物を含有するサンプルと一般式(I)で示される化合物を反応させ、還元反応を行う。当該還元反応は、金属イオンの存在下で行うことが好ましい。金属イオンとしては、例えばCu2+、In3+等の遷移金属のイオンや、Mg2+、Ca2+等のアルカリ土類金属のイオン等が挙げられ、好ましくはアルカリ土類金属のイオンであり、より好ましくはカルシウムイオンである。 In this method, first, a sample containing a compound containing an FDP structure is reacted with the compound represented by the general formula (I) to carry out a reduction reaction. The reduction reaction is preferably performed in the presence of a metal ion. Examples of the metal ions include transition metal ions such as Cu 2+ and In 3+ and alkaline earth metal ions such as Mg 2+ and Ca 2+ , preferably alkaline earth metal ions. Yes, more preferably calcium ions.
当該還元反応は、例えば1〜10mg(好ましくは1〜3mg)の一般式(I)で示される化合物(例えば4-ニトロフタロニトリル)及び1〜30mg(好ましくは2〜10mg)の金属イオン(例えば塩化カルシウム)を含む10〜300μL(好ましくは10〜100μL)の溶液と、10〜300μL(好ましくは10〜100μL)のFDP構造を含む化合物（例えばアクロレイン-アミン付加体)を含む生物学的サンプルとを混合し、例えば1〜10時間(好ましくは5〜7時間)、100℃の温度下で反応させることにより行われる。 The reduction reaction is performed, for example, by 1 to 10 mg (preferably 1 to 3 mg) of the compound represented by the general formula (I) (for example, 4-nitrophthalonitrile) and 1 to 30 mg (preferably 2 to 10 mg) of metal ions (for example, A biological sample containing a 10-300 μL (preferably 10-100 μL) solution containing calcium chloride) and a compound (eg, acrolein-amine adduct) containing 10-300 μL (preferably 10-100 μL) of FDP structure; For example, for 1 to 10 hours (preferably 5 to 7 hours), and reaction at a temperature of 100 ° C.
次いで、生じた一般式(II)で示されるアニリン化合物(例えば4-アミノフタロニトリル)の蛍光強度を、一般式(II)で示されるアニリン化合物の励起-蛍光波長(例えば4-アミノフタロニトリルの励起-蛍光波長[340-404 nm])に準じて読み取る。読み取った蛍光強度から生物学的サンプル中のFDP構造を含む化合物を検出する。あるいは、標準物を用いて作成した検量線から、読み取った蛍光強度に基づく生物学的サンプル中のFDP構造を含む化合物の量又は濃度を換算し、定量することもできる。また、アクロレイン関連疾患若しくはアクロレイン関連疾患のリスクの診断においては、アクロレイン関連疾患陽性であると判断されるFDP構造を含む化合物の量又は濃度の閾値レベル又はカットオフ値と比較することで、アクロレイン関連疾患若しくはアクロレイン関連疾患のリスクを判定することができる。 Next, the fluorescence intensity of the resulting aniline compound represented by the general formula (II) (for example, 4-aminophthalonitrile) is determined based on the excitation-fluorescence wavelength (for example, 4-aminophthalonitrile of the aniline compound represented by the general formula (II)). Read according to excitation-fluorescence wavelength [340-404 nm]). A compound containing an FDP structure in a biological sample is detected from the read fluorescence intensity. Alternatively, the amount or concentration of the compound containing the FDP structure in the biological sample based on the read fluorescence intensity can be converted and quantified from a calibration curve prepared using a standard. In the diagnosis of acrolein-related diseases or the risk of acrolein-related diseases, acrolein-related diseases are compared with the threshold level or cut-off value of the amount or concentration of a compound containing an FDP structure that is judged to be positive for acrolein-related diseases. The risk of a disease or an acrolein related disease can be determined.
また、本方法に準じて、本発明は、検出試薬として一般式(I)で示される化合物を含む、FDP構造を含む化合物の検出用組成物又は検出キットに関する。当該組成物又はキットは、アクロレイン関連疾患若しくはアクロレイン関連疾患のリスクの診断、評価、検出若しくは判定用組成物若しくはキット、又はアクロレイン関連疾患若しくはアクロレイン関連疾患のリスクを検出するためのin vitroにおけるデータ収集のための組成物若しくはキットとして使用することもできる。 According to the present method, the present invention also relates to a detection composition or detection kit for a compound having an FDP structure, which includes a compound represented by the general formula (I) as a detection reagent. The composition or kit is a composition or kit for diagnosis, evaluation, detection or determination of acrolein-related disease or risk of acrolein-related disease, or in vitro data collection for detecting the risk of acrolein-related disease or acrolein-related disease It can also be used as a composition or kit for.
当該キットは、一般式(I)で示される化合物以外に、金属イオン、検量線作成のための標準物、反応容器、FDP構造を含む化合物の検出のための取り扱い説明書等を適宜含むことができる。また、当該キットを、アクロレイン関連疾患若しくはアクロレイン関連疾患のリスクの診断に使用する場合には、当該キットは、アクロレイン関連疾患若しくはアクロレイン関連疾患のリスクの診断のための説明書を含んでもよい。 In addition to the compound represented by the general formula (I), the kit may appropriately include a metal ion, a standard for preparing a calibration curve, a reaction vessel, an instruction manual for detecting a compound containing an FDP structure, and the like. it can. Further, when the kit is used for diagnosis of an acrolein-related disease or a risk of an acrolein-related disease, the kit may include instructions for diagnosing the risk of an acrolein-related disease or an acrolein-related disease.
さらに、本発明は、本方法における還元反応工程を含む、上記一般式(II)で示される化合物の合成方法に関する。当該合成方法では、還元反応工程を上記に説明する本方法の反応工程に準じて行うことができる。 Furthermore, the present invention relates to a method for synthesizing the compound represented by the general formula (II), which includes a reduction reaction step in the present method. In the synthesis method, the reduction reaction step can be performed according to the reaction step of the present method described above.
以下、実施例を用いて本発明をより詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
１．材料及び方法
１−１．全般的な情報
全ての溶媒は、試薬グレードであった。全ての市販の化学製品を受け取ったまま使用した。
1. 1. Materials and methods 1-1. General Information All solvents were reagent grade. All commercial chemicals were used as received.
1H及び13C NMRスペクトルを、JEOL RESONANCE AL400 NMR及びJEOL RESONANCE AL300 NMRスペクトロメーターにより取得した。溶媒のピークを内部標準としてケミカルシフトを帰属した。
高分解能質量分析を、micrOTOF-Q III-HCTM(BRUKER)を用いて行った。
1 H and 13 C NMR spectra were acquired on a JEOL RESONANCE AL400 NMR and JEOL RESONANCE AL300 NMR spectrometer. The chemical shift was assigned with the solvent peak as the internal standard.
High resolution mass spectrometry was performed using a micrOTOF-Q III-HC ™ (BRUKER).
全ての蛍光測定を、96ウェル平底プレート(Corning Inc)と共にJASCO FP6500スペクトロフルオロメーターを用いて行った。量に関する各値を、真正の標準の強度から算出した。 All fluorescence measurements were performed using a JASCO FP6500 spectrofluorometer with 96 well flat bottom plates (Corning Inc). Each value for the quantity was calculated from the intensity of the authentic standard.
実験動物に関する全ての処置は、RIKENの倫理委員会により承認された。また、実験は研究所及び国のガイドラインに準じて行われた。 All treatments for laboratory animals were approved by the RIKEN ethics committee. The experiment was conducted in accordance with the laboratory and national guidelines.
１−２．FDPによるニトロアレンの還元のための全般的な手順
ニトロアレン3d (25.0 mg, 0.10 mmol)及びCaCl2 (55.0 mg, 0.50 mmol)を含むDMF (0.2 mL)溶液に、N-ベンジル 3-ホルミル-3,4-デヒドロピペリジン (N-Bn FDP, 60.0 mg, 0.30 mmol)を100℃にて添加した。当該温度で数時間撹拌した後、混合物を真空で濃縮し、粘着性ガムとして粗混合物を得た。当該粗残留物を、NMRでモニターするか、又は溶出システムとしてヘキサン-EtOAc溶媒混合物を用いた分取TLC若しくはシリカゲルフラッシュカラムクロマトグラフィーにより精製し、所望のアニリン生成物4d (19.6 mg, 89％収率)を得た。
1-2. General procedure for the reduction of nitroallene with FDP To a solution of nitroallene 3d (25.0 mg, 0.10 mmol) and CaCl 2 (55.0 mg, 0.50 mmol) in DMF (0.2 mL), N-benzyl 3-formyl- 3,4-Dehydropiperidine (N-Bn FDP, 60.0 mg, 0.30 mmol) was added at 100 ° C. After stirring at that temperature for several hours, the mixture was concentrated in vacuo to give a crude mixture as a sticky gum. The crude residue was monitored by NMR or purified by preparative TLC or silica gel flash column chromatography using a hexane-EtOAc solvent mixture as the elution system to give the desired aniline product 4d (19.6 mg, 89% yield). Rate).
１−３．サンプルの調製及び検出手順
正常なラット血清(100 μL；Wako Pure Chemical Industry Ltd.)を100 μLのアクロレイン(血清タンパク質に対して>100倍等価な量)で、特定の日数(0、1、20又は60日)処理した。次いで、サンプルを蒸留水で1 mLに希釈した。
1-3. Sample Preparation and Detection Procedure Normal rat serum (100 μL; Wako Pure Chemical Industry Ltd.) with 100 μL acrolein (> 100-fold equivalent to serum protein) for a specific number of days (0, 1, 20 (Or 60 days). The sample was then diluted to 1 mL with distilled water.
新鮮な尿サンプル(100 μL)をRIKEN bio-resource centerのC57BL/6マウスから取得し、蒸留水で20倍に希釈した。 Fresh urine samples (100 μL) were obtained from C57BL / 6 mice at RIKEN bio-resource center and diluted 20-fold with distilled water.
ニトロアレンプローブ3k (1.7 mg, 10.0 μmol)及びCaCl2 (5.5 mg, 50.0 μmol)を含むDMF-H2O (50 μL)溶液に、所定の尿サンプルを添加し、100℃で5時間撹拌した後、粗反応混合物を濾過し、得られた濾液を、スペクトロフルオロメーターで340 nm/404 nmにて測定した。 A predetermined urine sample was added to a DMF-H 2 O (50 μL) solution containing a nitroallene probe 3k (1.7 mg, 10.0 μmol) and CaCl 2 (5.5 mg, 50.0 μmol), and the mixture was stirred at 100 ° C. for 5 hours. Thereafter, the crude reaction mixture was filtered, and the obtained filtrate was measured with a spectrofluorometer at 340 nm / 404 nm.
１−４．ELISAアッセイ
測定を、添付の取り扱い説明書に準じて、酵素結合免疫吸着アッセイ(ELISA)キットシステム(TAKARA, Acrolein-Lysine adduct competitive ELISA kit)を用いて行った(非特許文献４)。450 nmでの吸光度を、マイクロプレートリーダー(ImmunoMini NJ1000)を用いて測定した。データを、個々の実験から標準偏差と共に2回を超えるアッセイの平均値で表す。
1-4. ELISA assay The measurement was performed using an enzyme-linked immunosorbent assay (ELISA) kit system (TAKARA, Acrolein-Lysine adduct competitive ELISA kit) according to the attached instruction manual (Non-patent Document 4). Absorbance at 450 nm was measured using a microplate reader (ImmunoMini NJ1000). Data are expressed as the mean of more than two assays with standard deviation from each experiment.
１−５．化合物の特徴付け
1-((4-aminophenyl)sulfonyl)-1H-pyrrole (4d, 89%): 1H NMR (400 MHz, CDCl3) δ 7.63 (dd, 2H, J = 6.7, 2.0 Hz), 7.13 (apparent t, 2H, J = 2.3 Hz), 6.63 (dd, 2H, J = 6.8, 2.0 Hz), 6.26 (apparent t, 2H, J = 2.3 Hz), 4.21 (brs, NH2); 13C NMR (100 MHz, CDCl3) δ 151.6, 129.2(2C), 126.8, 120.6(2C), 114.1(2C), 113.1(2C); HRESI-MS m/z calcd for C10H10N2O2S [M+H]+ 223.0536, found 223.0534.
Methyl 4-aminobenzoate (4e, 69%): 1H NMR (400 MHz, CDCl3) δ 7.85 (dd, 2H, J = 8.7, 2.8 Hz), 6.64 (dd, 2H, J = 8.7, 2.8 Hz), 4.10 (brs, NH2), 3.87 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 167.2, 150.8, 131.2(2C), 119.7, 113.9(2C), 51.6; HRESI-MS m/z calcd for C8H10N1O2[M+H]+ 152.0706, found 152.0709.
4-aminoacetophenone (4f, 88%): 1H NMR (400 MHz, CDCl3) δ 7.79 (dd, 2H, J = 8.6, 1.4 Hz), 6.63 (dd, 2H, J = 8.7, 1.4 Hz), 4.23 (brs, NH2), 2.49 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 196,7, 151.3, 130.9(2C), 127.7, 119.7, 113.7(2C), 26.1; HRESI-MS m/z calcd for C8H9N1O1[M+H]+ 136.0762, found 136.0757.
4-aminobenzonitrile (4g, 82%): 1H NMR (400 MHz, CDCl3) δ 7.41 (dd, 2H, J = 8.6, 1.2 Hz), 6.64 (dd, 2H, J = 8.6, 1.2 Hz), 4.17 (brs, NH2); 13C NMR (100 MHz, CDCl3) δ 150.5, 133.9(2C), 120.2, 114.6(2C), 100.4; HRESI-MS m/z calcd for C7H6N2[M+H]+ 119.0604, found. 119.0605
aniline (4h, 18%): 1H NMR (400 MHz, CDCl3) δ 7.13 (ddd, 2H, J = 7.5, 7.5, 1.2 Hz), 6.74 (tt, 1H, J = 7.5, 1.2 Hz), 6.65 (dd, 2H, J = 7.5, 1.2 Hz), 3.51(brs, NH2); 13C NMR (100 MHz, CDCl3) δ 146.3, 129.2(2C), 118.4, 115.0(2C)
1-((4-aminophenyl)sulfonyl)-1H-indole (4j, 80%): 1H NMR (400 MHz, CDCl3) δ 7.97 (dd, 1H, J = 8.3, 1.2 Hz), 7.66 (dd, 2H, J = 9.1, 2.4 Hz), 7.55(d, 1H, J = 3.6 Hz), 7.52 (d, 1H, J = 8.3 Hz), 7.29 (ddd, 1H, J = 8.3, 8.3, 1.2 Hz), 7.20 (ddd, 1H, J = 8.3, 8.3, 1.2 Hz), 6.62 (d, 1H, J = 3.6 Hz) , 6.55 (dd, 2H, J = 9.1, 2.4 Hz), 4.11(brs, NH2); 13C NMR (100 MHz, CDCl3) δ 153.5, 151.3, 134.8, 130.7, 129.1 (2C), 126.3 (2C), 124.2, 122.9, 121.2, 113.9, 113.5, 108.4; HRESI-MS m/z calcd for C14H13N2O2S [M+H]+ 273.0692, found 273.0691.
4-aminobenzoic acid (4k, 61%): 1H NMR (400 MHz, d6-DMSO) δ 7.62 (d, 2H, J = 8.4 Hz), 6.54 (dd, 2H, J = 8.4 Hz), 5.89 (brs, NH2); 13C NMR (100 MHz, CDCl3) δ 167.6, 153.2, 131.3(2C), 116.9, 112.6(2C); HRESI-MS m/z calcd for C7H7N1O2[M+H]+ 138.0550, found 138.0539.
4-aminobenzaldehyde (4l, 40%): 1H NMR (400 MHz, d6-DMSO) δ 9.56 (s, 1H), 7.54 (d, 2H, J = 8.7 Hz), 6.62 (d, 2H, J = 8.7 Hz), 4.14; 13C NMR (100 MHz, d6-DMSO) δ 189.7, 155.3(2C), 132.2, 124.8, 113.1(2C); HRESI-MS m/z calcd for C7H7N1O1[M+H]+ 122.0600, found 122.0610
4-fluoroaniline (4m, 73%): 1H NMR (400 MHz, CDCl3) δ 6.84-6.78 (m, 2H), 6.56-6.51 (m, 2H), 3.51(brs, NH2); 13C NMR (100 MHz, CDCl3) δ 156.1(d, J = 235 Hz, C-F), 142.4, 115.8(2C), 115.3(2C); HRESI-MS m/z calcd for C6H6F1N1[M+H]+ 112.0557, found 112.0558.
4-chloroaniline (4n, 21%): 1H NMR (400 MHz, CDCl3) δ 7.09 (d, 2H, J = 8.6 Hz), 6.58 (d, 2H, J = 8.6 Hz), 3.45(brs, NH2); 13C NMR (100 MHz, CDCl3) δ 145.4, 129.2(2C), 123.2, 116.3(2C); HRESI-MS m/z calcd for C6H6N1Cl1[M+H]+ 128.0262, found 128.0263.
3-aminobenzonitrile (4o, 22%): 1H NMR (300 MHz, CDCl3) δ 7.16 (dd, 1H, J = 7.2, 7.2 Hz), 6.94 (d, 1H, J = 7.2 Hz), 6.83-6.77 (m, 2H), 3.82 (brs, NH2); 13C NMR (75 MHz, CDCl3) δ 147.0, 130.2(2C), 122.1, 119.3, 117.6, 113.1; HRESI-MS m/z calcd for C7H6N2[M+H]+ 119.0604, found. 119.0612
4-aminophthalonitrile (4p, 78%): 1H NMR (400 MHz, d6-DMSO) δ 7.64 (d, 1H, J = 8.7 Hz), 7.03 (d, 1H, J = 2.4 Hz), 6.68 (dd, 1H, J = 8.7, 2.4 Hz), 6.72 (brs, NH2); 13C NMR (100 MHz, d6-DMSO) δ 153.2, 135.1, 117.6, 117.3, 117.1, 116.5, 115.6, 97.9; HRESI-MS m/z calcd for C10H4N3[M+H]+ 166.0400, found 166.0380. Fluorescent excitation/emission: 304/404 nm, quantum yield Φ = 0.26.
1-5. Characterization of compounds
1-((4-aminophenyl) sulfonyl) -1H-pyrrole (4d, 89%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.63 (dd, 2H, J = 6.7, 2.0 Hz), 7.13 (apparent t , 2H, J = 2.3 Hz), 6.63 (dd, 2H, J = 6.8, 2.0 Hz), 6.26 (apparent t, 2H, J = 2.3 Hz), 4.21 (brs, NH 2 ); 13 C NMR (100 MHz , CDCl 3 ) δ 151.6, 129.2 (2C), 126.8, 120.6 (2C), 114.1 (2C), 113.1 (2C); HRESI-MS m / z calcd for C 10 H 10 N 2 O 2 S (M + H ] + 223.0536, found 223.0534.
Methyl 4-aminobenzoate (4e, 69%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.85 (dd, 2H, J = 8.7, 2.8 Hz), 6.64 (dd, 2H, J = 8.7, 2.8 Hz), 4.10 (brs, NH 2 ), 3.87 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 167.2, 150.8, 131.2 (2C), 119.7, 113.9 (2C), 51.6; HRESI-MS m / z calcd for C 8 H 10 N 1 O 2 [M + H] + 152.0706, found 152.0709.
4-aminoacetophenone (4f, 88%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.79 (dd, 2H, J = 8.6, 1.4 Hz), 6.63 (dd, 2H, J = 8.7, 1.4 Hz), 4.23 (brs, NH 2 ), 2.49 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 196,7, 151.3, 130.9 (2C), 127.7, 119.7, 113.7 (2C), 26.1; HRESI-MS m / z calcd for C 8 H 9 N 1 O 1 [M + H] + 136.0762, found 136.0757.
4-aminobenzonitrile (4g, 82%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.41 (dd, 2H, J = 8.6, 1.2 Hz), 6.64 (dd, 2H, J = 8.6, 1.2 Hz), 4.17 (brs, NH 2 ); 13 C NMR (100 MHz, CDCl 3 ) δ 150.5, 133.9 (2C), 120.2, 114.6 (2C), 100.4; HRESI-MS m / z calcd for C 7 H 6 N 2 [M + H] + 119.0604, found. 119.0605
aniline (4h, 18%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.13 (ddd, 2H, J = 7.5, 7.5, 1.2 Hz), 6.74 (tt, 1H, J = 7.5, 1.2 Hz), 6.65 (dd, 2H, J = 7.5, 1.2 Hz), 3.51 (brs, NH 2 ); 13 C NMR (100 MHz, CDCl 3 ) δ 146.3, 129.2 (2C), 118.4, 115.0 (2C)
1-((4-aminophenyl) sulfonyl) -1H-indole (4j, 80%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.97 (dd, 1H, J = 8.3, 1.2 Hz), 7.66 (dd, 2H, J = 9.1, 2.4 Hz), 7.55 (d, 1H, J = 3.6 Hz), 7.52 (d, 1H, J = 8.3 Hz), 7.29 (ddd, 1H, J = 8.3, 8.3, 1.2 Hz), 7.20 (ddd, 1H, J = 8.3, 8.3, 1.2 Hz), 6.62 (d, 1H, J = 3.6 Hz), 6.55 (dd, 2H, J = 9.1, 2.4 Hz), 4.11 (brs, NH 2 ); 13 C NMR (100 MHz, CDCl 3 ) δ 153.5, 151.3, 134.8, 130.7, 129.1 (2C), 126.3 (2C), 124.2, 122.9, 121.2, 113.9, 113.5, 108.4; HRESI-MS m / z calcd for C 14 H 13 N 2 O 2 S [M + H] + 273.0692, found 273.0691.
4-aminobenzoic acid (4k, 61%): 1 H NMR (400 MHz, d 6 -DMSO) δ 7.62 (d, 2H, J = 8.4 Hz), 6.54 (dd, 2H, J = 8.4 Hz), 5.89 ( brs, NH 2 ); 13 C NMR (100 MHz, CDCl 3 ) δ 167.6, 153.2, 131.3 (2C), 116.9, 112.6 (2C); HRESI-MS m / z calcd for C 7 H 7 N 1 O 2 [ M + H] + 138.0550, found 138.0539.
4-aminobenzaldehyde (4l, 40%): 1 H NMR (400 MHz, d 6 -DMSO) δ 9.56 (s, 1H), 7.54 (d, 2H, J = 8.7 Hz), 6.62 (d, 2H, J = 8.7 Hz), 4.14; 13 C NMR (100 MHz, d 6 -DMSO) δ 189.7, 155.3 (2C), 132.2, 124.8, 113.1 (2C); HRESI-MS m / z calcd for C 7 H 7 N 1 O 1 [M + H] + 122.0600, found 122.0610
4-fluoroaniline (4m, 73%): 1 H NMR (400 MHz, CDCl 3 ) δ 6.84-6.78 (m, 2H), 6.56-6.51 (m, 2H), 3.51 (brs, NH 2 ); 13 C NMR (100 MHz, CDCl 3 ) δ 156.1 (d, J = 235 Hz, CF), 142.4, 115.8 (2C), 115.3 (2C); HRESI-MS m / z calcd for C 6 H 6 F 1 N 1 [M + H] + 112.0557, found 112.0558.
4-chloroaniline (4n, 21%): 1 H NMR (400 MHz, CDCl 3 ) δ 7.09 (d, 2H, J = 8.6 Hz), 6.58 (d, 2H, J = 8.6 Hz), 3.45 (brs, NH 2 ); 13 C NMR (100 MHz, CDCl 3 ) δ 145.4, 129.2 (2C), 123.2, 116.3 (2C); HRESI-MS m / z calcd for C 6 H 6 N 1 Cl 1 [M + H] + 128.0262, found 128.0263.
3-aminobenzonitrile (4o, 22%): 1 H NMR (300 MHz, CDCl 3 ) δ 7.16 (dd, 1H, J = 7.2, 7.2 Hz), 6.94 (d, 1H, J = 7.2 Hz), 6.83-6.77 (m, 2H), 3.82 (brs, NH 2 ); 13 C NMR (75 MHz, CDCl 3 ) δ 147.0, 130.2 (2C), 122.1, 119.3, 117.6, 113.1; HRESI-MS m / z calcd for C 7 H 6 N 2 [M + H] + 119.0604, found. 119.0612
4-aminophthalonitrile (4p, 78%): 1 H NMR (400 MHz, d 6 -DMSO) δ 7.64 (d, 1H, J = 8.7 Hz), 7.03 (d, 1H, J = 2.4 Hz), 6.68 (dd , 1H, J = 8.7, 2.4 Hz), 6.72 (brs, NH 2 ); 13 C NMR (100 MHz, d 6 -DMSO) δ 153.2, 135.1, 117.6, 117.3, 117.1, 116.5, 115.6, 97.9; HRESI- MS m / z calcd for C 10 H 4 N 3 [M + H] + 166.0400, found 166.0380. Fluorescent excitation / emission: 304/404 nm, quantum yield Φ = 0.26.
２．結果及び考察
実験結果を、下記の表１〜３及び図２〜６に示す。
2. Results and Discussion Experimental results are shown in Tables 1 to 3 and FIGS.
＜参考例＞
先ず、調製容易なN-Bn FDP 1a(Fuentes, L. et al. Direct chemical method for preparing 2,3-epoxyamides using sodium chlorite. J Org Chem 77, 5515-5524, doi:10.1021/jo300542d (2012))を還元剤として種々の基質を用いた検討を開始した。しかしながら、カルボニル基やカルボニル基で活性化された不飽和結合への還元反応は全く進行しなかった(表１Ａ）。一方で、スルホニルを含む芳香族ニトロ化合物3dに対して作用させたところ、アニリン誘導体が生成することを見出した(表１Ｂ）。この反応は60℃では全く進行せず、80℃でも痕跡量しか生成物が得られないが、100℃まで昇温した際に45％で目的のアニリン体4dが得られてくることを明らかにした(表１Ｂ、entry1-3）。さらに反応性を調べるため、幾つかの市販のニトロ化合物を用いて検討を行った。3dと同じように、電子求引基であるエステルを有する基質でも反応が35％で進行した(entry 4）。また、4-nitroacetophenon(3f)や4-nitrobenzonitrile(3g)でも還元反応がそれぞれ50％、73％で進行した(entry 5-6）。一方で、電子求引基をもたない3hや3iのような基質では反応が進行しなかった(entry 7-8)。
<Reference example>
First, N-Bn FDP 1a (Fuentes, L. et al. Direct chemical method for preparing 2,3-epoxyamides using sodium chlorite. J Org Chem 77, 5515-5524, doi: 10.1021 / jo300542d (2012)) The study using various substrates as a reducing agent was started. However, the reduction reaction to the carbonyl group or the unsaturated bond activated with the carbonyl group did not proceed at all (Table 1A). On the other hand, when it was made to act on the aromatic nitro compound 3d containing a sulfonyl, it discovered that an aniline derivative produced | generated (Table 1B). This reaction does not proceed at all at 60 ° C, and only a trace amount of product can be obtained at 80 ° C, but it is clear that the desired aniline 4d can be obtained at 45% when the temperature is raised to 100 ° C. (Table 1B, entry1-3). Furthermore, in order to investigate reactivity, it examined using some commercially available nitro compounds. Similar to 3d, the reaction proceeded at 35% with a substrate having an ester as an electron withdrawing group (entry 4). 4-nitroacetophenon (3f) and 4-nitrobenzonitrile (3g) also produced reduction reactions at 50% and 73%, respectively (entry 5-6). On the other hand, the reaction did not proceed with substrates such as 3h and 3i that did not have electron withdrawing groups (entry 7-8).
さらに、収率を向上させるために、ブレンステッド酸やルイス酸の触媒効果を想定した。生体内の還元試薬であるニコチンアミドアデニンジヌクレオシド(NADH)の還元機構をもとに(Aizpurua, J. M. et al. Mechanistic insights on the magnesium(II) ion-activated reduction of methyl benzoylformate with chelated NADH peptide beta-lactam models. J Org Chem 74, 6691-6702, doi:10.1021/jo901236d (2009))、3dをモデル基質として検討を開始した(表２)。塩酸、硫酸、トリフルオロ酢酸、ボロン酸等のブレンステッド酸では、元の条件より収率が低下した（表２、entry 2-8)。一方、ルイス酸として機能する銅やインジウム等の金属イオン種が収率を向上させることを見出すことができた(entry 9-19)。さらに、NADH還元と同じように(Aizpurua, J. M. et al. Mechanistic insights on the magnesium(II) ion-activated reduction of methyl benzoylformate with chelated NADH peptide beta-lactam models. J Org Chem 74, 6691-6702, doi:10.1021/jo901236d (2009))、マグネシウムやカルシウムといったアルカリ土類金属が有効ではないかと考えて検討を続けた(entry 20-25)。最終的に、5当量の塩化カルシウムを系内に添加することで、還元反応が効率的に進行することを見出した(entry 26)。 Furthermore, in order to improve the yield, the catalytic effect of Bronsted acid or Lewis acid was assumed. Based on the reduction mechanism of nicotinamide adenine dinucleoside (NADH), a reducing agent in vivo (Aizpurua, JM et al. Mechanistic insights on the magnesium (II) ion-activated reduction of methyl benzoylformate with chelated NADH peptide beta- lactam models. J Org Chem 74, 6691-6702, doi: 10.1021 / jo901236d (2009)), 3d was used as a model substrate (Table 2). For Bronsted acids such as hydrochloric acid, sulfuric acid, trifluoroacetic acid and boronic acid, the yield was lower than the original conditions (Table 2, entry 2-8). On the other hand, metal ion species such as copper and indium that function as Lewis acids were found to improve the yield (entry 9-19). Furthermore, as with NADH reduction (Aizpurua, JM et al. Mechanistic insights on the magnesium (II) ion-activated reduction of methyl benzoylformate with chelated NADH peptide beta-lactam models.J Org Chem 74, 6691-6702, doi: (10.1021 / jo901236d (2009)), and continued the study considering that alkaline earth metals such as magnesium and calcium are effective (entry 20-25). Finally, it was found that the reduction reaction proceeds efficiently by adding 5 equivalents of calcium chloride to the system (entry 26).
最適化した条件(塩化カルシウム5当量、100℃、5時間)により、還元反応の基質適用限界を調べることにした。表３Ａに示すように、電子求引基を有する基質群(3d-3g)を用いた際に、初期検討に比べて添加剤の効果によって収率が向上した(entry 1-4)。さらに、以前は還元反応が進行しなかった単純なニトロベンゼンにおいても還元反応が進行することを確かめた(entry 5)。一方で、電子供与基を有する3iを用いた際には反応が進行しなかった(entry 6)。また、天然物合成の中間体に用いられるスルホニルインドール骨格を持つ3jでは80％で還元が進行した(entry 7)。同様に4-nitrobenzoic acid(3k)や4-nitrobenzaldehyde(3l)も、それぞれ61％、40％で還元反応が進行した(entry 8-9)。同じく、4-fluoronitrobenzene(3m)では、置換反応等を起こさずに、73％で還元が進行した(entry 10)。4-chloronitrobenzene(3n)では収率が低いものの、副生成物を伴わずに還元が進行した(entry 11)。この置換基効果は、Hammett plot上からも支持されている(表３Ｂ)。 Based on the optimized conditions (calcium chloride 5 equivalents, 100 ° C., 5 hours), it was decided to investigate the substrate application limit of the reduction reaction. As shown in Table 3A, when the substrate group (3d-3g) having an electron withdrawing group was used, the yield was improved by the effect of the additive compared with the initial study (entry 1-4). Furthermore, it was confirmed that the reduction reaction proceeded even in simple nitrobenzene, where the reduction reaction did not proceed before (entry 5). On the other hand, when 3i having an electron donating group was used, the reaction did not proceed (entry 6). In addition, reduction progressed by 80% in 3j having a sulfonylindole skeleton used as an intermediate for natural product synthesis (entry 7). Similarly, reduction reactions of 4-nitrobenzoic acid (3k) and 4-nitrobenzaldehyde (3l) proceeded at 61% and 40%, respectively (entry 8-9). Similarly, with 4-fluoronitrobenzene (3m), reduction proceeded at 73% without causing a substitution reaction (entry 10). Although 4-chloronitrobenzene (3n) yield was low, the reduction proceeded without any by-products (entry 11). This substituent effect is also supported on the Hammett plot (Table 3B).
この還元反応を新たなアクロレイン・FDPの簡便な検出法として展開することとした。すなわち、アクロレインが生体組織(タンパク質アミノ基等)と反応し、はじめに還元剤となるFDPを与え、さらに還元反応によって非蛍光性ニトロプローブから蛍光性アニリン分子へと変換させることで蛍光検出を行うこととした。この検出形式では、蛍光強度が生成物のアニリンの量に比例するため、一定の割合で反応に関与するFDPの量を見積もることができる。ニトロプローブを適切に選択することで、様々な生体混合物中でもFDPレベルを測定することができる。そこで、必要となるニトロプローブを蛍光波長域や反応性に着目しながら探索することにした。 This reduction reaction was developed as a new simple detection method for acrolein and FDP. In other words, acrolein reacts with living tissues (protein amino groups, etc.), first gives FDP as a reducing agent, and further converts fluorescence from a non-fluorescent nitro probe to a fluorescent aniline molecule by a reduction reaction. It was. In this detection format, since the fluorescence intensity is proportional to the amount of product aniline, the amount of FDP involved in the reaction can be estimated at a certain rate. With proper selection of nitroprobes, FDP levels can be measured in various biological mixtures. Therefore, we decided to search for the necessary nitro probes while paying attention to the fluorescence wavelength region and reactivity.
＜実施例＞
１）．有機溶媒中での反応
上記の観点で、様々なニトロアレンをプローブ候補としてスクリーニングを行った。芳香環上に電子供与基と電子求引基の両方を持つようなpull-push型の蛍光基の分子構造に焦点を当て、ニトリル基とアミノ基の組み合わせに着目した(Oshima, J. et al. Extreme fluorescence sensitivity of some aniline derivatives to aqueous and nonaqueous environments: mechanistic study and its implication as a fluorescent probe. J Phys Chem A 110, 4629-4637, doi:10.1021/jp0570014 (2006))。また反応前後での励起-蛍光波長域の変化や量子効率の高さも重要な要素となる。4gや4iを含む数十種の候補化合物をスクリーンした結果、ジシアノニトロベンゼン3pを見出した(図２Ａ)。モデル反応では、ジシアノニトロベンゼン3pへの還元反応は78％で進行した(図２Ｂ)。さらに得られてくるアニリン体4pは励起-蛍光波長[340-404 nm]であることを確かめた(図２Ｃ)。さらに反応検討を行い、蛍光量の経時測定から5時間で反応が完結していることが明らかとなった(図２Ｄ)。
<Example>
1). Reaction in organic solvent From the above viewpoint, screening was performed using various nitroallenes as probe candidates. Focusing on the molecular structure of a pull-push type fluorescent group with both electron donating and electron withdrawing groups on the aromatic ring, we focused on the combination of nitrile and amino groups (Oshima, J. et al Extreme fluorescence sensitivity of some aniline derivatives to aqueous and nonaqueous environments: mechanistic study and its implication as a fluorescent probe. J Phys Chem A 110, 4629-4637, doi: 10.1021 / jp0570014 (2006)). In addition, changes in the excitation-fluorescence wavelength range before and after the reaction and high quantum efficiency are also important factors. As a result of screening several dozen kinds of candidate compounds including 4g and 4i, dicyanonitrobenzene 3p was found (FIG. 2A). In the model reaction, the reduction reaction to dicyanonitrobenzene 3p proceeded at 78% (FIG. 2B). Furthermore, it was confirmed that the obtained aniline body 4p has an excitation-fluorescence wavelength [340-404 nm] (FIG. 2C). Further, the reaction was examined, and it was revealed that the reaction was completed in 5 hours from the measurement of fluorescence over time (FIG. 2D).
２）．生体における酸化還元剤との反応性についての検討
還元反応を引き金とした「蛍光オン」のFDP検出システムを実現するため、3pをプローブとして実験系を確立していくこととした。生体サンプル中での直交性を保証するため、様々な生体の酸化還元剤で確かめていくこととした。先ず、FDPに対して、酸化剤としてH2O2やCu、Mg、Fe等を作用させたが、FDPは酸化されることなく回収された(図３Ａ)。またニトロプローブ3pに対して、還元剤としてシステインやグルタチオン(GSH)、あるいは硫化水素ナトリウム(NaSH)を反応させたところ(Wang, R. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J 16, 1792-1798, doi:10.1096/fj.02-0211hyp (2002))、生理学的濃度(1μM程度)では還元による蛍光増大は確認されなかった(図３Ｂ)。また、シスチンを作用しても影響がなかった（図３Ｂ)。ここから、生体サンプルの条件下でも本システムが機能することを確かめることができた。
2). Examination of reactivity with redox agents in living bodies In order to realize a “fluorescence-on” FDP detection system triggered by a reduction reaction, we decided to establish an experimental system using 3p as a probe. In order to guarantee the orthogonality in the biological sample, it was decided to check with various biological redox agents. First, H 2 O 2 , Cu, Mg, Fe, or the like was allowed to act on the FDP as an oxidizing agent, but the FDP was recovered without being oxidized (FIG. 3A). Nitroprobe 3p was reacted with cysteine, glutathione (GSH), or sodium hydrogen sulfide (NaSH) as a reducing agent (Wang, R. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter. FASEB J 16, 1792-1798, doi: 10.1096 / fj.02-0211hyp (2002)), and at physiological concentrations (about 1 μM), no increase in fluorescence due to reduction was confirmed (FIG. 3B). Moreover, there was no influence even if cystine acted (FIG. 3B). From this, we were able to confirm that this system works even under conditions of biological samples.
このシステムの定量性を確認するため、標品としてN-lys FDPを用いたモデル反応を行った(図４Ａ-４Ｂ)。様々な量のFDPを加えて、その蛍光量を測定した後に検量線を作成した。また従来の抗体ELISA法の検出限界(3.13 nmol/mL)に対して、本法では0.84 nmol/mLまで調べられることが明らかとなった。 In order to confirm the quantitative nature of this system, a model reaction using N-lys FDP as a standard was performed (FIGS. 4A-4B). After adding various amounts of FDP and measuring the amount of fluorescence, a calibration curve was prepared. In addition, the detection limit (3.13 nmol / mL) of the conventional antibody ELISA method was revealed to be 0.84 nmol / mL in this method.
３）．生体サンプルを用いた検討
上述のように確立した条件を用いて、プローブ3pや塩化カルシウム等の試薬類をキット化し、簡便な検出プロトコルを作成した(図５Ａ)。a) 先ず、サンプルとキット溶液を混合し、b) 100℃で5時間加熱後、c) 蛍光測定を行う手法を確立した。
3). Examination Using Biological Sample Using the conditions established as described above, reagents such as probe 3p and calcium chloride were made into a kit and a simple detection protocol was created (FIG. 5A). a) First, a method was established in which the sample and the kit solution were mixed, b) heated at 100 ° C. for 5 hours, and c) fluorescence measurement.
本手法によって実際に生体検出できるかを調べるために、生体サンプルとして安価なラット血清を用いた検出試験を行った(図５Ｂ)。ラット血清存在下にアクロレインを加えてそれぞれ0、1、20、60日間保温したサンプルを調製後に、上述の操作で検出試験をそれぞれ行ったところ、また同じロットで標品FDPの蛍光量から求めた検量線から、1日間処理したサンプルで約7.7 nmol/mlのFDPを定量検出できた。また20日間処理したサンプルで約12.7 nmol/mlのFDPが観測され、アクロレイン処理時間に比例して蛍光強度が増大することが明らかとなった。また従来法であるELISAとも良い一致を示した。 In order to investigate whether or not a living body can actually be detected by this method, a detection test was performed using inexpensive rat serum as a biological sample (FIG. 5B). After preparing samples that had been incubated for 0, 1, 20, and 60 days with acrolein in the presence of rat serum, respectively, the detection test was carried out by the above-mentioned operation, and also obtained from the fluorescence amount of the standard FDP in the same lot. From the calibration curve, about 7.7 nmol / ml of FDP could be quantitatively detected in the sample treated for 1 day. In addition, about 12.7 nmol / ml FDP was observed in the sample treated for 20 days, and it became clear that the fluorescence intensity increased in proportion to the acrolein treatment time. Also, it is in good agreement with the conventional ELISA.
次に、生体サンプルとしてマウス尿サンプル(6週令)を用いた検出試験を行った(図６Ａ)。マウス尿を20倍希釈した生体サンプルを調製後に、検出試験を行ったところ、先ほどと同様に5.2 nmol/mlのFDPを定量検出できた(図６Ａ)。またこちらも従来法であるELISAとも良い一致を示した(非特許文献４)。 Next, a detection test using a mouse urine sample (6 weeks old) as a biological sample was performed (FIG. 6A). When a detection test was performed after preparing a biological sample in which mouse urine was diluted 20-fold, 5.2 nmol / ml of FDP could be quantitatively detected in the same manner as before (FIG. 6A). This also showed good agreement with the conventional ELISA (Non-Patent Document 4).
さらに本手法の簡便性を実証するため、様々な週令の10匹のマウスから採取した尿を用いた、一斉検出試験を行った(図６Ｂ)。再現性のためにサンプル毎に3回ずつ行うこととし、計30ロットの試験を行った。その結果、6時間で尿中FDPの含有量が判明した。以上のように、FDPの還元反応を基に、安価で簡便な酸化ストレスマーカー検出診断法を開発し、生体サンプルで実証した。 Furthermore, in order to demonstrate the simplicity of this technique, a simultaneous detection test was performed using urine collected from 10 mice of various ages (FIG. 6B). For reproducibility, the test was performed three times for each sample, and a total of 30 lots were tested. As a result, the content of FDP in urine was found in 6 hours. As described above, based on the reduction reaction of FDP, an inexpensive and simple oxidative stress marker detection diagnostic method was developed and demonstrated in biological samples.
３．結論
生体内の酸化ストレス産物であるFDPの還元反応性をはじめて明らかにし、塩化カルシウムをルイス酸として用いる、ニトロ基の効率的な還元法を開発した。さらにこの還元変換を、生体のFDPレベルを検出するための「蛍光スイッチングシステム」へと応用した。確立した検出系を、実際にモデル動物の尿や生体サンプルで評価し、検出精度を従来法と比較した。その結果、安価で簡便な酸化ストレスマーカー検出法を開発できた。本手法を活用することで、アクロレインを発症因子とする様々な病気の予前診断の普及に貢献することができる。
3. Conclusion We have clarified for the first time the reduction reactivity of FDP, an oxidative stress product in vivo, and developed an efficient method for reducing nitro groups using calcium chloride as a Lewis acid. Furthermore, this reduction conversion was applied to a “fluorescence switching system” for detecting FDP levels in living organisms. The established detection system was actually evaluated with urine and biological samples of model animals, and the detection accuracy was compared with the conventional method. As a result, we have developed an inexpensive and simple method for detecting oxidative stress markers. By utilizing this method, it is possible to contribute to the spread of prognosis of various diseases with acrolein as an onset factor.
Claims (7)
サンプルと下記一般式(I)：
R1は、独立に、置換若しくは無置換のアリール基、置換若しくは無置換のヘテロアリール基、及び電子求引性基から選ばれる基を表し、
mは、2〜5の整数を表し、
ただし、複数のR1のうち少なくとも1つは、電子求引性基、電子求引性基を有するアリール基、無置換のヘテロアリール基、及び電子求引性基を有するヘテロアリール基から選ばれる基を表す)
で示される化合物を反応させる工程と、
前記反応により生じる蛍光を検出する工程と、
を含む、前記方法。 A method for detecting a compound comprising a formyldehydropiperidine structure in a sample, comprising:
Sample and the following general formula (I):
R 1 independently represents a group selected from a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, and an electron withdrawing group;
m represents an integer of 2 to 5,
However, at least one of the plurality of R 1 is selected from an electron withdrawing group, an aryl group having an electron withdrawing group, an unsubstituted heteroaryl group, and a heteroaryl group having an electron withdrawing group. Represents a group)
A step of reacting a compound represented by:
Detecting fluorescence generated by the reaction;
Said method.
R1は、独立に、置換若しくは無置換のアリール基、置換若しくは無置換のヘテロアリール基、及び電子求引性基から選ばれる基を表し、
mは、2〜5の整数を表し、
ただし、複数のR1のうち少なくとも1つは、電子求引性基、電子求引性基を有するアリール基、無置換のヘテロアリール基、及び電子求引性基を有するヘテロアリール基から選ばれる基を表す)
で示される化合物を含む、ホルミルデヒドロピペリジン構造を含む化合物の検出用組成物。 Formula (I):
R 1 independently represents a group selected from a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, and an electron withdrawing group;
m represents an integer of 2 to 5,
However, at least one of the plurality of R 1 is selected from an electron withdrawing group, an aryl group having an electron withdrawing group, an unsubstituted heteroaryl group, and a heteroaryl group having an electron withdrawing group. Represents a group)
A composition for detecting a compound comprising a formyl dehydropiperidine structure, comprising the compound represented by the formula:
R1は、独立に、置換若しくは無置換のアリール基、置換若しくは無置換のヘテロアリール基、及び電子求引性基から選ばれる基を表し、
mは、2〜5の整数を表し、
ただし、複数のR1のうち少なくとも1つは、電子求引性基、電子求引性基を有するアリール基、無置換のヘテロアリール基、及び電子求引性基を有するヘテロアリール基から選ばれる基を表す)
で示される化合物を含む、ホルミルデヒドロピペリジン構造を含む化合物の検出キット。 Formula (I):
R 1 independently represents a group selected from a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, and an electron withdrawing group;
m represents an integer of 2 to 5,
However, at least one of the plurality of R 1 is selected from an electron withdrawing group, an aryl group having an electron withdrawing group, an unsubstituted heteroaryl group, and a heteroaryl group having an electron withdrawing group. Represents a group)
A detection kit for a compound containing a formyl dehydropiperidine structure, comprising the compound represented by:
R1は、独立に、置換若しくは無置換のアリール基、置換若しくは無置換のヘテロアリール基、及び電子求引性基から選ばれる基を表し、
mは、2〜5の整数を表し、
ただし、複数のR1のうち少なくとも1つは、電子求引性基、電子求引性基を有するアリール基、無置換のヘテロアリール基、及び電子求引性基を有するヘテロアリール基から選ばれる基を表す)
で示される化合物とを反応させる工程を含む、下記一般式(II)で示される化合物の合成方法。
R 1 independently represents a group selected from a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, and an electron withdrawing group;
m represents an integer of 2 to 5,
However, at least one of the plurality of R 1 is selected from an electron withdrawing group, an aryl group having an electron withdrawing group, an unsubstituted heteroaryl group, and a heteroaryl group having an electron withdrawing group. Represents a group)
A method for synthesizing a compound represented by the following general formula (II), which comprises a step of reacting the compound represented by formula (II).
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