JP4773062B2 - NOVEL ELECTROCHEMICALLY RESPONSIBLE COMPOUND, PROCESS FOR PRODUCING THE SAME, AND METHOD FOR DETECTING BODY BETWEEN TARGET SUBSTANCE AND PROBE MOLECULE USING SAME - Google Patents

Description

  The present invention relates to a novel ferrocene compound having electrochemical responsiveness.

  In biological or medical research and clinical settings, in order to detect biomolecules such as nucleic acids and proteins such as DNA and RNA having a specific base sequence, substances that specifically bind to those biomolecules A method of using a probe is used. When such a method is used, a means for confirming whether or not the target biomolecule is bound to the probe molecule is required for detection of the target biomolecule.

  As a means for confirming the presence or absence of binding, a method of labeling a target molecule is well known, and a fluorescent dye or the like is used. For example, in order to analyze gene expression in a cell, the RNA contained in the cell is fluorescently labeled and hybridized with a probe cDNA immobilized on a solid phase carrier under appropriate conditions, and then on the solid phase carrier. Methods for detecting fluorescence are widely used. However, since the fluorescent material fades with time, it needs to be detected quickly.

  As a detection method instead of fluorescence, there is a method of observing the interaction between molecules by labeling a biomolecule with a conductive substance and detecting this substance electrochemically.

や、下記式（３）

で表されるフェロセン化ナフタレンジイミド誘導体等が利用されている。
特開平９−２８８０８０号公報 ＷＯ０２／０５３５７１ Anne, A. et al.; Bioconjugate Chem.,12, 396-405 (2001) As a method of labeling a biomolecule with a conductive substance, a method using a conductive intercalator has been proposed (see, for example, Patent Documents 1 and 2). An intercalator refers to a molecule having the property of intercalating between nucleic acid bases. Since intercalators are regularly sewn into double-stranded nucleic acids, a correlation can be obtained between the amount of intercalator that binds to the amount of double-stranded strands, and hybridization can be measured quantitatively. . As an intercalator having conductivity, the following formula (2)

Or the following formula (3)

The ferrocene naphthalene diimide derivative etc. which are represented by these are utilized.
JP-A-9-288080 WO02 / 053571 Anne, A. et al .; Bioconjugate Chem., 12, 396-405 (2001)

  The electrochemical detection method can easily perform more accurate quantitative measurement in real time without fading like a fluorescent dye. However, until now, it was mainly used for the detection of double-stranded nucleic acids using a conductive intercalator, and it has hardly been detected by electrochemical methods for interactions between biomolecules other than nucleic acid hybridization. It was.

  Also, a method for detecting nucleic acid hybridization using a conductive intercalator is required to be a more sensitive intercalator that is less affected by background current than conventional methods and can detect minute currents.

  Thus, the present invention is a novel electrochemically responsive compound that can be directly bound to a target molecule or a probe molecule and can be used to synthesize an intercalator, and can be detected with high sensitivity. The purpose is to provide.

で表される化合物、中でも、式（１）においてｍ＝ｎ＝０、Ｒ¹がエチレン基である化合物は、アミノ基を持つ物質と結合することができるので他の分子に標識として結合させやすいこと、および１４０ｍＶ前後と低い電位で最大電流を流す性質があるので、バックグラウンド電流の影響を受けにくく小さい電流を検出しやすいことを見出し、本発明を完成するに至った。 As a result of intensive studies in view of the above circumstances, the present inventor has obtained the following formula (1).

In particular, a compound represented by the formula (1), in which m = n = 0 and R ¹ is an ethylene group, can be bonded to a substance having an amino group, so that it can be easily bonded to other molecules as a label. And the fact that it has the property of flowing a maximum current at a low potential of around 140 mV, it has been found that it is difficult to be influenced by the background current and a small current can be easily detected, and the present invention has been completed.

That is, this invention is a manufacturing method of the compound represented by following formula (1 '),

(Wherein R ¹ Represents an alkylene group having 2 to 6 carbon atoms. )
  As shown in the following reaction formula (A), Br-R is added to diethyl malonate. ^Four -CO ₂ C ₂ H _Five (Where
R ^Four Is R ¹ Represents an alkylene group having 1 fewer carbon atoms. ) To obtain compound (a)
This is reacted with ferrocenylmethyltrimethylammonium iodide to give compound (b).
The first step to be obtained and the compound (c) by hydrolyzing the compound (b) as shown in the reaction formula (B)
And decarboxylation to obtain compound (d), and as shown in reaction formula (C),
A third step of reacting compound (d) with N-hydroxysuccinimide to obtain the desired product.
The present invention relates to a manufacturing method.

  According to the present invention, a novel compound having electrochemical responsiveness that can be easily bonded to various substances including biomolecules such as nucleic acids and proteins can be provided. Since this substance has a peak at which a maximum current flows at a relatively low potential, it is not easily affected by the background current, can detect a minute current, and is a label for detecting a target substance in a test sample. Useful as a substance.

  Hereinafter, although the compound concerning this invention is demonstrated in detail based on the preferable embodiment, these do not limit the scope of the present invention at all.

  The compound according to the present invention is a novel ferrosense succinimide ester represented by the above formula (1) (hereinafter also referred to as “the compound of the present invention”). Since the compound of the present invention can be bound to a compound having an amino group and exhibits electrochemical responsiveness, it is useful as a labeling substance used for detecting a target substance in a test sample.

As shown in the above formula (1), the compound of the present invention is composed of a ferrocene part, a succinimide ester part, and an alkylene group R ¹ connecting both parts. Since a current flows between ferrocene molecules by applying a voltage, the ferrosense succinimide ester can be quantitatively detected by measuring this current. In addition, the succinimide ester portion reacts with an amine compound to produce an amide compound with a high yield, and thus can be used as a label by binding to a substance having an amino group.

The compound of the present invention preferably contains an unsubstituted ferrocenyl group in which m = n = 0 in the formula (1). However, the compound of the present invention is used in an aqueous solution as a labeling substance for electrochemical detection. When R ² and R ³ are used, compounds containing a group which becomes an electron donating group or an electron withdrawing group in an aqueous solution as R ² and R ³ are also preferable because the potential at the time of measuring the amount of current is changed by these groups.

In the above formula (1), when m or n is not 0 but 1 or 2, R ² or R ³ represents an electron donating group in an aqueous solution as a methyl group, a methoxy group, or an electron demanding group. Examples of the group serving as a pulling group include a methoxycarbonyl group, a nitro group, a carboxyl group, a dimethylaminomethyl group, and a dimethylaminoethylaminocarbonyl group. For example, in the case of a dimethylaminomethyl group, it is protonated in an aqueous solution and functions as a dimethylammoniummethyl group which is an electron withdrawing group.

In the formula (1), examples of the alkylene group represented by R ¹ include an ethylene group, a propylene group, a butylene group, a pentylene group, and a hexylene group.

  The compound of the present invention is not particularly limited by its production method, but is preferably produced as follows.

また、本発明の化合物において、特にＲ¹がエチレンである場合は、下記反応式（Ａ'）
に示すように、マロン酸ジエチルにヨウ化フェロセニルメチルトリメチルアンモニウムを
反応させてフェロセニルメチルマロン酸ジエチルエステルを得て、これを加水分解し２−
フェロセニルメチルマロン酸とし、次いで反応式（Ｂ'）に示すように、２−フェロセニ
ルメチルマロン酸を脱炭酸して３−フェロセニルプロピオン酸を得て、最後に反応式（Ｃ
'）に示すように、３−フェロセニルプロピオン酸とＮ−ヒドロキシスクシンイミドを反
応させることにより、目的物である本発明の化合物を得ることが好ましい。

ここで、前記反応式（Ａ'）に係る反応は、例えば、マロン酸ジエチルにＮａ^+-ＯＣ₂Ｈ
₅を反応させ、Ｎａ⁺ＣＨ（ＣＯＯＣ₂Ｈ₅）₂ ^-を得てからヨウ化フェロセニルメチルトリメ
チルアンモニウムを反応させ、次いでこれに水およびＫＯＨを加えて加熱することにより
加水分解し、Ｈ⁺を加えることによって２−フェロセニルメチルマロン酸を得ることがで
きる。 That is, as shown in the following reaction formula (A), diethyl malonate is added to Br—R ⁴ —CO ₂ C ₂ H ₅ (
In the formula, R ⁴ represents an alkylene group having 1 fewer carbon atoms than R ¹ . ) To give a compound (a
) Is reacted with ferrocenylmethyltrimethylammonium iodide to give a compound (
a first step for obtaining b) and a second step for obtaining compound (d) by hydrolyzing compound (b) to obtain compound (c) and decarboxylation as shown in reaction formula (B) As shown in the reaction formula (C), it is preferable to obtain the compound of the present invention which is the target product by reacting the compound (d) with N-hydroxysuccinimide to obtain the target product.

In the compound of the present invention, particularly when R ¹ is ethylene, the following reaction formula (A ′)
As shown in Fig. 2, ferrocenylmethylmalonic acid diethyl ester is obtained by reacting diethyl malonate with ferrocenylmethyltrimethylammonium iodide, which is hydrolyzed to give 2-
Ferrocenylmethylmalonic acid is obtained, and then 2-ferrocenylmethylmalonic acid is decarboxylated to give 3-ferrocenylpropionic acid as shown in reaction formula (B ′). Finally, reaction formula (C
As shown in '), it is preferable to obtain the target compound of the present invention by reacting 3-ferrocenylpropionic acid with N-hydroxysuccinimide.

Here, the reaction according to the reaction formula (A ′) is performed by, for example, adding diethyl malonate to Na ⁺ -OC ₂ H.
₅ is reacted to obtain Na ⁺ CH (COOC ₂ H ₅ ) ₂ ⁻ and then reacted with ferrocenylmethyltrimethylammonium iodide, which is then hydrolyzed by heating with addition of water and KOH. 2-ferrocenylmethylmalonic acid can be obtained by adding ⁺ .

  The reaction according to the reaction formula (B ′) can be performed by heating at 160 ° C., for example. After heating, separation / purification operations such as chromatography, solvent removal, and recrystallization are performed as necessary.

  The reaction according to the reaction formula (C ′) is performed, for example, by mixing metallocenylpropionic acid and N-hydroxysuccinimide and adding dropwise a solution obtained by dissolving DCC as a condensing agent in 1,4-dioxane. be able to. After the reaction, separation and purification operations such as chromatography, solvent removal under reduced pressure, and recrystallization are performed as necessary. By performing the separation / purification operation, the compound of the present invention can be obtained in higher yield with higher purity. Separation and purification operations can be easily performed.

  The use of the compound of the present invention is not particularly limited, and can be applied to various uses. In particular, it can be detected by measuring the amount of current flowing between ferrocene molecules by applying a potential, and the compound having an amino group. Since it can be easily combined, it can be used as various labels.

  For example, a probe molecule that specifically binds to a target substance contained in a test sample is first immobilized on the electrode surface, and then the target substance is labeled with the compound of the present invention, and then the test sample, the probe molecule, In contact and incubate. Then, by detecting an electrochemical signal from the compound of the present invention, it can be confirmed whether the compound of the present invention has bound to the probe molecule.

  The method according to the present invention is particularly suitable when at least one of a target substance and a probe molecule is a biomolecule, and is a compound that specifically binds to a biomolecule, nucleic acid hybridization, protein-protein interaction detection, or biomolecule. Can be used for screening and the like. In the present specification, the biomolecule is not particularly limited as long as it is a molecule derived from a living body. For example, the compound of the present invention can be bound to a nucleic acid to detect whether the nucleic acid has hybridized with a probe nucleic acid. Since the compound of the present invention can also bind to the amino group of a protein lysine residue, the amino group of an amino sugar, or the amino group contained in a lipid, these molecules can also be labeled with the compound of the present invention. It can be detected electrochemically.

  In addition, a double-stranded nucleic acid can be quantitatively detected by labeling an intercalator with the compound of the present invention. Cyclic voltammetry and differential pulse voltammetry (DPV) can be used for the detection of the electrochemical signal, and it is highly sensitive in real time whether the labeled substance has bound to the probe molecule immobilized on the electrode surface. High throughput can be measured.

で表される化合物は、電位１４０ｍＶ付近において最大電流を流す。１４０ｍＶは、従来のフェロセン化化合物に最大電流を与える電位よりも低く、バックグラウンド電流の影響を受けにくいので、小さい電流を検出しやすいという利点がある。 Among the compounds of the present invention, m = n = 0 and R ¹ is an ethylene group, the following formula (4)

The maximum current flows at a potential of about 140 mV. 140 mV is lower than the potential that gives the maximum current to the conventional ferrocene compound, and is less susceptible to the background current, so that it has the advantage that a small current can be easily detected.

  When the intercalator is labeled with the compound of the present invention, as the intercalator, N, N-disubstituted naphthalenediimide, 1,5-, 2,6-, 9,10-disubstituted anthracene, 1,5-2, , 6-disubstituted anthraquinone, 1,5-, 2,6-, 4,9-disubstituted acridine derivatives, etc. can be used as stitched intercalators, and at the end (one end or both ends) of these intercalators The compounds of the present invention can be introduced.

反応式（Ｄ）に係る反応においては、例えばテトラヒドロフラン（ＴＨＦ）等の溶媒中
で反応物を加熱還流し、その後生成物を再結晶等により分離・精製する等通常の合成法で
行われる操作を必要に応じて行う。上記反応式（Ｅ）に係る反応においては、例えばまず
化合物（６）をトリフルオロ酢酸（ＴＦＡ）等の溶媒中で撹拌し、ブトキシカルボニル基
（Ｂｏｃ）で保護されたアミノ基の脱保護を行い、続いてクロロホルム、トリエチルアミ
ン等の溶媒中で反応物を撹拌し、減圧での溶媒除去や再結晶等により分離・精製する等の
通常の合成方法で行われる操作を必要に応じて行う。 Below, the outline of the process of manufacturing an intercalator using 1,4,5,8-naphthalene tetracarboxylic dianhydride is shown as an example. As shown in the reaction formula (D), 1, 4, 5
, 8-naphthalenetetracarboxylic dianhydride and a compound represented by the formula (5) to obtain a compound represented by the formula (6), and a formula as shown in the reaction formula (E) (6) It reacts with the compound (1) of this invention, and the process of obtaining the target object.

In the reaction according to the reaction formula (D), for example, an operation performed by a usual synthesis method such as heating and refluxing the reaction product in a solvent such as tetrahydrofuran (THF) and then separating and purifying the product by recrystallization or the like. Do as needed. In the reaction according to the above reaction formula (E), for example, the compound (6) is first stirred in a solvent such as trifluoroacetic acid (TFA) to deprotect the amino group protected with a butoxycarbonyl group (Boc). Subsequently, the reaction is stirred in a solvent such as chloroform or triethylamine, and an operation performed by a usual synthesis method such as solvent removal under reduced pressure or recrystallization is performed as necessary.

  In addition to the above-described stitched intercalators, the compounds of the present invention can be used in classical intercalators such as ethidium bromide derivatives and acridine derivatives, and in groove binder type intercalators such as destamycin derivatives and DNA-binding metal complexes. Can also be combined. FIG. 8 shows a structure in which the compound of the present invention is bonded to a classical intercalator, and FIG. 9 shows a structure in which the compound is bonded to a groove binder type intercalator.

In order to show the advantageous effects of the compounds according to the present invention, examples and test examples will be shown below, but these are illustrative and the present invention is in any case limited to the following specific examples. is not.
As an example of the compound of the present invention, ferrocenylpropionic acid succinimide in which m = n = 0 and R ¹ is an ethylene group in the above formula (1) was synthesized. The synthesis of ferrocenylpropionic acid was performed according to Non-Patent Document 1 and improved.

マロン酸ジエチル８．３５ｍｌ（５５ｍｍｏｌ）の無水エタノール５０ｍｌ溶液をスターラーで撹拌しながら、ナトリウムエトキシド３．７５ｇ（５５ｍｍｏｌ）の無水エタノール５０ｍｌ溶液を少しずつ加えた後、３分間撹拌した。この混合溶液にヨウ化フェロセニルメチルトリメチルアンモニウム１９．３ｇ（５０ｍｍｏｌ）を加え、オイルバスで９０℃、７２時間還流した。還流後、この溶液を超純水２００ｍｌで希釈し、１Ｎ ＨＣｌを用いてｐＨ試験しで確認しながら中和した。
この溶液を分液ロートによりジエチルエーテル１００ｍｌで２回抽出を行った。抽出液を飽和ＮａＨＣＯ₃水溶液で洗浄後（約１００ｍｌ）、ジエチルエーテル相を取り、ＭｇＳＯ４で乾燥後、減圧留去により残渣を得た。展開溶媒クロロホルム：ヘキサン＝１：１のシリカゲルカラムクロマトグラフィーにより、ＴＬＣ（クロロホルム：ヘキサン＝１：１）のＲｆ値０．１の橙色成分を分取し、減圧乾燥した。
得られた橙色固体の物質の¹Ｈ−ＮＭＲ測定およびＩＲ測定を行った。¹Ｈ−ＮＭＲスペクトルデータを表１に示す。

以上の結果より、得られた化合物は下記式に示す、フェロセニルメチルマロン酸ジエチルエステルであることが確認された（収量１４．４ｇ、収率８１％）。

尚、上記構造式中のａ〜ｇは、表１中のケミカルシフト（δ値）に対応する水素（プロトン）それぞれの位置を示す。 (1) Synthesis of ferrocenylmethylmalonic acid diethyl ester Compound (b) was synthesized according to the following scheme.

While stirring a solution of 8.35 ml (55 mmol) of diethyl malonate in 50 ml of absolute ethanol with a stirrer, a solution of 3.75 g (55 mmol) of sodium ethoxide in 50 ml of absolute ethanol was added little by little, followed by stirring for 3 minutes. To this mixed solution was added 19.3 g (50 mmol) of ferrocenylmethyltrimethylammonium iodide, and the mixture was refluxed in an oil bath at 90 ° C. for 72 hours. After refluxing, this solution was diluted with 200 ml of ultrapure water and neutralized while confirming by pH test using 1N HCl.
This solution was extracted twice with 100 ml of diethyl ether using a separatory funnel. The extract was washed with a saturated aqueous NaHCO ₃ solution (about 100 ml), the diethyl ether phase was taken, dried over MgSO 4, and evaporated under reduced pressure to give a residue. The orange component of RLC value 0.1 of TLC (chloroform: hexane = 1: 1) was fractionated by silica gel column chromatography with developing solvent chloroform: hexane = 1: 1, and dried under reduced pressure.
The obtained orange solid substance was subjected to ¹ H-NMR measurement and IR measurement. ¹ H-NMR spectral data are shown in Table 1.

From the above results, it was confirmed that the obtained compound was ferrocenylmethylmalonic acid diethyl ester represented by the following formula (yield 14.4 g, yield 81%).

In addition, ag in the said structural formula shows the position of each hydrogen (proton) corresponding to the chemical shift (delta value) in Table 1.

（１）で合成したフェロセニルメチルマロン酸ジエチルエステル１３．５ｇ（３７ｍｍｏｌ）の無水エタノール２３ｍｌに、６Ｎ ＫＯＨ水溶液２．５ｍｌ（１１ｍｍｏｌ）を加え、６Ｎ ＫＯＨ溶液とし、２４時間還流した。還流後、この溶液を１５０ｍｌの超純水で希釈して、エバポレータでエタノールを減圧留去した。残った水層を分液ロートを用いてジエチルエーテル１５０ｍｌで２回洗浄した。水層を、６Ｎ ＨＣｌを用いてｐＨ試験紙で確認しながら中和し、ジエチルエーテル１５０ｍｌで２回洗浄した。水層にジエチルエーテル１５０ｍｌを加え、撹拌しながら１Ｎ ＨＣｌを水層が酸性になるまで加えた。ジエチルエーテル層を超純水で洗浄後、硫酸マグネシウムで乾燥し、減圧濃縮を行い、減圧乾固した。得られた黄色個体の¹Ｈ−ＮＭＲ測定およびＩＲ測定を行った。¹Ｈ−ＮＭＲスペクトルデータを表２に示す。

ＩＲ（ＫＢｒ）測定の結果は、３４００ｃｍ^-1（ＯＨ）、１７１０ｃｍ^-1（Ｃ＝Ｏ）であった。
以上の結果より、得られた化合物は下記式（７）に示す、２−フェロセニルメチルマロン酸であることが確認された。

尚、上記構造式中のａ、ｂ、ｃ、ｄおよびｅは、表２中のケミカルシフト（δ値）に対応する水素（プロトン）それぞれの位置を示す。 (2) Synthesis of 2-ferrocenylmethylmalonic acid According to the following scheme, 2-ferrocenylmethylmalonic acid was synthesized using the compound (b).

To 23 ml of absolute ethanol of 13.5 g (37 mmol) of ferrocenylmethylmalonic acid diethyl ester synthesized in (1), 2.5 ml (11 mmol) of 6N KOH aqueous solution was added to form a 6N KOH solution, which was refluxed for 24 hours. After refluxing, this solution was diluted with 150 ml of ultrapure water, and ethanol was distilled off under reduced pressure with an evaporator. The remaining aqueous layer was washed twice with 150 ml of diethyl ether using a separatory funnel. The aqueous layer was neutralized with 6N HCl while checking with pH test paper, and washed twice with 150 ml of diethyl ether. 150 ml of diethyl ether was added to the aqueous layer, and 1N HCl was added with stirring until the aqueous layer became acidic. The diethyl ether layer was washed with ultrapure water, dried over magnesium sulfate, concentrated under reduced pressure, and dried under reduced pressure. The obtained yellow solid was subjected to ¹ H-NMR measurement and IR measurement. ^{The 1} H-NMR spectrum data is shown in Table 2.

The results of IR (KBr) measurement were 3400 cm ⁻¹ (OH) and 1710 cm ⁻¹ (C═O).
From the above results, it was confirmed that the obtained compound was 2-ferrocenylmethylmalonic acid represented by the following formula (7).

In the above structural formula, a, b, c, d and e represent the positions of hydrogen (protons) corresponding to chemical shifts (δ values) in Table 2.

２−フェロセニルメチルマロン酸２．８ｇ（９．３×１０^-3ｍｏｌ）を１００ｍＬナス型フラスコに入れ、１６０℃で３０分間脱炭酸反応を行い、ＴＬＣで反応終了を確認した。得られた粗生成物を展開溶媒（クロロホルム：エタノール＝９５：５）でシリカゲルカラムクロマトグラフィーを用いて精製した。Ｒｆ値が０．１の目的物を分取した。分取後、クロロホルム、メタノールを減圧留去し、減圧乾固した。黄色固体の物質が得られ、この物質を¹Ｈ−ＮＭＲで同定した。結果を表３に示す。 (3) Synthesis of 3-ferrocenylpropionic acid According to the following scheme, 2-ferrocenylmethylmalonic acid was decarboxylated to synthesize 3-ferrocenylpropionic acid.

2.8 g (9.3 × 10 ⁻³ mol) of 2-ferrocenylmethylmalonic acid was placed in a 100 mL eggplant type flask, decarboxylated at 160 ° C. for 30 minutes, and the completion of the reaction was confirmed by TLC. The resulting crude product was purified using silica gel column chromatography with a developing solvent (chloroform: ethanol = 95: 5). The target product having an Rf value of 0.1 was collected. After fractionation, chloroform and methanol were distilled off under reduced pressure and dried under reduced pressure. A yellow solid material was obtained, which was identified by ¹ H-NMR. The results are shown in Table 3.

以上の結果より、得られた化合物は下記式（８）に示す、３−フェロセニルプロピオン酸であることが確認された（収量２．１ｇ、収率８８％）。

尚、上記構造式中のａ、ｂ、ｃ、ｄおよびｅは、表３中のケミカルシフト（δ値）に対応する水素（プロトン）それぞれの位置を示す。

From the above results, it was confirmed that the obtained compound was 3-ferrocenylpropionic acid represented by the following formula (8) (yield 2.1 g, yield 88%).

Note that a, b, c, d, and e in the above structural formula indicate the positions of hydrogen (protons) corresponding to chemical shifts (δ values) in Table 3.

５０ｍｌのナス型フラスコに３−フェロセニルプロピオン酸０．４０ｇ（１．５６ｍｍｏｌ）、Ｎ−ヒドロキシスクシンイミド０．２１ｇ（１．８ｍｍｏｌ）を加え、これに１，４−ジオキサン８ｍｌを加えた。ＤＣＣ（縮合剤）０．３７ｇ（１．８ｍｍｏｌ）を溶かした１，４−ジオキサン１４ｍｌを用意し、これを滴下ロートで上記ナス型フラスコに滴下していった。滴下を約６０分かけて行い、滴定終了後、反応追跡をＴＬＣで行った。約１６時間撹拌後、溶液を吸引ろ過し固体物ＤＣＵｒｅａを除去した。得られた濾液から１，４−ジオキサンを減圧留去し、シリカゲルクロマトグラフィーで展開溶媒（クロロホルム：ｎ−ヘキサン＝１０：１）を用いて、Ｒｆ値０．１５の橙色成分を分取した。分取した溶液から展開溶媒を減圧留去後、約３時間減圧乾固し、黄色固体を得、この物質を¹Ｈ−ＮＭＲで同定した。結果を表４に示す。

以上の結果より、得られた化合物は式（９）に示す、フェロセニルプロピオン酸スクシンイミドであることが確認された（収量０．４６ｇ、収率８３％）。

尚、上記構造式中のａ、ｂ、ｃ、およびｄは、表４中のケミカルシフト（δ値）に対応する水素（プロトン）それぞれの位置を示す。
[試験例] (4) Synthesis of 3-ferrocenylpropionic acid succinimide ester According to the following scheme, 3-ferrocenylpropionic acid and N-hydroxysuccinimide were reacted to synthesize ferrocenylpropionic acid succinimide.

To a 50 ml eggplant-shaped flask, 0.40 g (1.56 mmol) of 3-ferrocenylpropionic acid and 0.21 g (1.8 mmol) of N-hydroxysuccinimide were added, and 8 ml of 1,4-dioxane was added thereto. 14 ml of 1,4-dioxane in which 0.37 g (1.8 mmol) of DCC (condensing agent) was dissolved was prepared, and this was dropped into the eggplant-shaped flask with a dropping funnel. The dropwise addition was performed over about 60 minutes, and the reaction was traced by TLC after completion of the titration. After stirring for about 16 hours, the solution was suction filtered to remove the solid substance DCUrea. 1,4-Dioxane was distilled off from the obtained filtrate under reduced pressure, and an orange component having an Rf value of 0.15 was fractionated by silica gel chromatography using a developing solvent (chloroform: n-hexane = 10: 1). The developing solvent was distilled off from the separated solution under reduced pressure, followed by drying under reduced pressure for about 3 hours to obtain a yellow solid, which was identified by ¹ H-NMR. The results are shown in Table 4.

From the above results, it was confirmed that the obtained compound was ferrocenylpropionic acid succinimide represented by the formula (9) (yield 0.46 g, yield 83%).

Note that a, b, c, and d in the above structural formula indicate the positions of hydrogen (protons) corresponding to chemical shifts (δ values) in Table 4.
[Test example]

  3-ferrocenylpropionic acid succinimide (hereinafter abbreviated as “Fcpro”) synthesized as described above was bound to an oligonucleotide, and hybridization with a probe DNA having a sequence complementary to this oligonucleotide was detected. went.

Ｈ₂Ｎ−ｄＴ２０のクロマトグラムを図１に、ＲＴ＝１１分のＵＶスペクトルを図２に示す。また、ｄＴ２０−Ｆｃｐｒｏのクロマトグラムを図３に、ＲＴ＝２１分のＵＶスペクトルを図４に示す。
ＨＰＬＣクロマトグラムより反応率１００％であることが確認された。また、ＵＶ−ｖｉｓ測定および濃度決定により収量は２６ｎｍｏｌ、収率は１００％であることが確認された。
ｄＴ２０−ＦｃｐｒｏのＭＡＬＤＩ−ＴＯＦ ＭＳによる測定結果を図５に示す。
測定条件を以下に示す。
Fc-dT20：m/z=6442
Laser intensity：1940 Polarity：Negative
Matrix：3-Hydroxypicolinic acid (1) Synthesis of oligonucleotide-Fcpro 3-ferrocenylpropionic acid succinimide ester 1 in 20 μl of Na ₂ CO ₃ / NaHCO ₃ buffer solution (pH 9.0) containing 25 nmol of H ₂ N-dT20 (thymine 20-mer) 6 μl of DMSO solution containing 3 μmol (0.43 mg) was added and shaken at room temperature for 24 hours. After shaking, 0.1M triethylamine-acetic acid (TEAA) buffer (pH 7.0) is added so that the total volume becomes 1 ml, and the oligonucleotide is added to the oligonucleotide using a NAP-10 column (Parmacia Sephadex G-25). The derived components were collected. At that time, the NAP-10 column was used after equilibrating in advance with 15 ml of 0.1 M TEAA buffer. The sample which was made up so that the total amount was 1 ml was charged to the column, and after 1 ml of the solution was eluted, 1.5 ml of 0.1 M TEAA buffer was charged. Immediately after this, 1.5 ml of the eluate was collected. The resulting solution was lyophilized overnight, 20 μl of sterile distilled water was added and analyzed by reverse phase HPLC. The HPLC-injected solution was diluted 1/40 and analyzed.
The HPLC conditions are shown below.
Column: Lichrospher RP-18 (Cica-MERCK) Flow rate: 1 ml / min
Detection wavelength: 260 nm Sample injection solvent: Ultrapure water
Eluent A: 0.1M TEAA buffer (pH 7.0), 10% CH ₃ CN solution
Eluent B: 0.1M TEAA buffer (pH 7.0), 40% CH ₃ CN solution Gradient conditions

A chromatogram of H ₂ N-dT20 is shown in FIG. 1, and a UV spectrum at RT = 11 minutes is shown in FIG. Moreover, the chromatogram of dT20-Fcpro is shown in FIG. 3, and the UV spectrum at RT = 21 minutes is shown in FIG.
From the HPLC chromatogram, it was confirmed that the reaction rate was 100%. Further, it was confirmed by UV-vis measurement and concentration determination that the yield was 26 nmol and the yield was 100%.
FIG. 5 shows the measurement results of dT20-Fcpro by MALDI-TOF MS.
The measurement conditions are shown below.
Fc-dT20: m / z = 6442
Laser intensity: 1940 Polarity: Negative
Matrix: 3-Hydroxypicolinic acid

(2) Evaluation of electrochemical response using dT20-Fcpro As a probe DNA, dA20 (adenine 20-mer) was immobilized on an electrode, and 10 pmol / μl of dT20-Fcpro was hybridized. FIG. 6 shows a conceptual diagram of this process. This electrode was immersed in an electric field solution for 1 minute, and DPV measurement was performed.
The conditions for DPV measurement are shown below.
Scan Rate: 0.1V / sec, Pulse Amplitude: 50mV, Sample Width: 16.7msec,
Pulse Width: 50msec, Pulse Period: 200msec, Quiet Time: 2sec,
CE: Pt wire, RE: Ag / AgCl
The measurement results are shown in FIG. In FIG. 7, A is measured with a dA20 modified electrode only, B is dT20-Fcpro contacted with a SAM electrode (not modified with dA20 electrode) and incubated, and C is dA20 modified electrode contacted with dT20-Fcpro. D represents the change in the electrochemical signal at 0.14 V of the sample incubated and dA20 modified electrode in contact with dA20-Fcpro.
From this result, a peak current derived from ferrocene was obtained at Ep = 0.14V. Further, no peak current was obtained in any of the controls (A, B and D). From the above, the peak current obtained at Ep = 0.14V was shown to be a response derived from double-stranded formation, and it became clear that the compound of the present invention can be used as a label for DNA detection. There is some variation in peak current, which is caused by variation in the amount of probe immobilized by the electrodes.

It shows the chromatogram of H ₂ N-dT20. It shows the UV spectra of H ₂ N-dT20. The chromatogram of dT20-Fcpro is shown. 2 shows the UV spectrum of dT20-Fcpro. The measurement result by MALDI-TOF MS of dT20-Fcpro is shown. The conceptual diagram of hybridization experiment is shown. The electrochemical signal change at 0.14 V in the hybridization experiment is shown. Figure 2 shows the compound of the present invention bound to a classical intercalator. The structure which combined the compound of this invention with the groove binder type | mold intercalator is shown.

Claims (1)

A method for producing a compound represented by the following formula (1 ′),

(Wherein R ¹ Represents an alkylene group having 2 to 6 carbon atoms. )
  As shown in the following reaction formula (A), Br-R is added to diethyl malonate. ^Four -CO ₂ C ₂ H _Five (Where
R ^Four Is R ¹ Represents an alkylene group having 1 fewer carbon atoms. ) To obtain compound (a)
This is reacted with ferrocenylmethyltrimethylammonium iodide to give compound (b).
The first step to be obtained and the compound (c) by hydrolyzing the compound (b) as shown in the reaction formula (B)
And decarboxylation to obtain compound (d), and as shown in reaction formula (C),
A third step of reacting compound (d) with N-hydroxysuccinimide to obtain the desired product.
Manufacturing method.

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