JP3598366B2 - Biologically active cyclic nucleotide derivative and method for producing the same - Google Patents

Description

【０００８】
（式中、Ｒ_１は−ＯＨ又は−ＮＨ_２を表し、Ｒ_２は水素又は−ＮＨ_２を表し、Ｒ_３は −ＮＨ_２又は−ＳＨを表す。また、ｎは２〜４の整数を表す。）
２．次の一般式（２）で表される環状ヌクレオチドを
【０００９】
【化７】

【００１０】
（式中、Ｒ_１は−ＯＨ又は−ＮＨ_２を表し、Ｒ_２は水素又は−ＮＨ_２を表す。）
式（３）で表される１，１’−カルボニルジイミダゾールと反応させて、
【００１１】
【化８】

【００１２】
得られる一般式（４）で表される環状ヌクレオチド中間体を、
【００１３】
【化９】

【００１４】
（式中、Ｒ_１及びＲ_２は上記と同じである。）
一般式（５）で表される化合物と反応させることを特徴とする、
Ｈ_２Ｎ（ＣＨ_２）_ｎＲ_３ （５）
（式中、Ｒ_３は −ＮＨ_２又は−ＳＨを表し、ｎは２〜４の整数を表す。）
一般式（１）で表される環状ヌクレオチド誘導体の製造方法。
【００１５】
【化１０】

【００１６】
（式中、Ｒ_１，Ｒ_２及びＲ_３は上記と同じである。）
【００１７】
【発明の実施の形態】
本発明の１例としてｃＡＭＰから目的とする２’−Ｏ−（２−アミノエチルカルバモイル）ｃＡＭＰを得る反応スキームを以下に示す。
【００１８】
【化１１】

【００１９】
本発明で得られる一般式（１）で表される新規な環状ヌクレオチド誘導体は、例えばＣｙ３−ＯＳｕ（商品名：アマーシャム ファルマシア バイオテク社製）のような、環状ヌクレオチド（１）のＲ_３と反応性を有する公知の蛍光色素と縮合させて、Ｃｙ３−ＥＤＡ−ｃＡＭＰのような蛍光性環状ヌクレオチド誘導体を製造するのに使用される。この蛍光性環状ヌクレオチド誘導体は、細胞内における環状ヌクレオチドの機能動態を解析する研究の効率と質を向上させるのに有用である。
【００２０】
【実施例】
［２’−Ｏ−（２−アミノエチルカルバモイル）ｃＡＭＰの合成］
市販のｃＡＭＰ６．０ｇ（１８．２ｍｍｏｌ）をＭｏｌｅｃｕｌａｒ Ｓｉｅｖｅｓ ４Ａ（製品名：Ｋ，Ｎａアルミノシリケート）で乾燥したジメチルホルムアミド（ＤＭＦ）６００ｍｌに溶解し、トリブチルアミン４．３６ｍｌを添加後、１，１’−カルボニルジイミダゾール１６．８ｇ（１０４ｍｍｏｌ）を加えて室温で１時間攪拌した。さらに、ＤＭＦ６００ｍｌを加え、室温で３０分間攪拌後、加温し３０℃で１．５時間攪拌した。つぎに、エチレンジアミン１２ｍｌを添加し、３０℃で３時間攪拌後、濾過して不溶物を除去した。不溶物を高速液体クロマトグラフィー（ＨＰＬＣ）により分析したところ、殆どが未反応のｃ−ＡＭＰであった。
濾過した溶液を６０℃の水浴上で減圧濃縮し、微黄色の油状物３５．４ｇを得た。ジクロロエタン６００ｍｌを加えて室温で３０分攪拌し、結晶を濾取し、ジエチルエーテルで洗浄後、減圧乾燥して白色の粉末１３ｇを得た。
この粉末を３バッチに分けて水に溶解させ、カラムとしてＤａｉｓｏｇｅｌ ＳＰ−１２０−３０／５０−ＯＤＳ−Ｂ（５０ｍｍＩ．Ｄ．ｘ５００ｍｍ）を使用し、２５４ｎｍのＵＶで検出しながら、１０ｍＭ Ｔｒｉｂｕｔｙｌａｍｍｏｎｉｕｍ Ｂｉｃａｒｂｏｎａｔｅ（トリブチルアミン−重炭酸バッファー）−１０％メタノールで１５ｍｌ／ｍｉｎで溶出させた。目的とする画分を分取後、メタノールを減圧留去し、凍結乾燥した。得られた固体にメタノールを加えて減圧濃縮操作を３回繰り返し、白色固体２．２７ｇを得た。この固体のＨＰＬＣで測定した純度は９９．４％であり、^１Ｈ−ＮＭＲによって目的産物であることを確認した。
^１Ｈ−ＮＭＲ（Ｄ２Ｏ）：δ８．０６（ｓ，１Ｈ），８．０３（ｓ，１Ｈ），６．１５（ｓ，１Ｈ），５．３８（ｄ，１Ｈ，Ｊ＝７Ｈｚ），４．８２（ｍ，１Ｈ），４．３４（ｍ，１Ｈ），４．１〜４．２（ｍ，２Ｈ），３．２〜３．４（ｍ，２Ｈ），２．９５（ｔ，２Ｈ，Ｊ＝５．５Ｈｚ）
【００２１】
この環状ヌクレオチド誘導体が有効なアナログとして機能することは、粘菌の環状ＡＭＰに対する走化性応答によって明らかにした。細胞性粘膜（Ｄｉｃｔｙｏｓｔｅｌｉｕｍ ｄｉｓｃｏｉｄｉｕｍ）はｃＡＭＰを走化性物質（アトラクタント）とする。飢餓状態にして培養した粘菌細胞は細胞集塊となり子実体形成を開始する。この細胞塊はｃＡＭＰに対する感受性が高く細胞膜上にｃＡＭＰ受容体を数多く発現している。無栄養寒天培地に上記実施例で得られたヌクレオチド誘導体を１０^−８から１０^−５Ｍの濃度で加え、円形の培養器で固めた。この培地の中央に細胞塊（２μＬ）を載せて２０℃で３時間培養した。３時間後に細胞塊の広がりを測定して細胞の移動速度を求めた結果を図１に示す。この環状ヌクレオチド誘導体では、無修飾のｃＡＭＰに対する反応と同様の濃度と強度で細胞の移動速度の反応が得られた。
【００２２】
また、この環状ヌクレオチド誘導体に蛍光色素ｃｙ３−ＯＳｕを縮合させて蛍光性環状ヌクレオチド誘導体ｃｙ３−ＥＤＡ−ｃＡＭＰを合成し、同様にして細胞の移動速度を求めたところ、ｃＡＭＰと同様の反応を得た。（図１参照）
したがって、この蛍光性環状ヌクレオチド誘導体を使用すれば、細胞内の環状ヌクレオチドの機能動態を解析する研究の効率と質を向上させることができる。
【図面の簡単な説明】
【図１】本発明の環状ヌクレオチド誘導体及び該誘導体から得られる蛍光性環状ヌクレオチド誘導体を使用して、細胞の移動速度を求めた結果を示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel cyclic nucleotide derivative useful for studying the intracellular signal transduction mechanism and a method for producing the same.
[0002]
[Prior art]
Cyclic AMP (cAMP) and cyclic GMP (cGMP) are cyclic nucleotides with important roles in intracellular signaling. Studying how this cyclic nucleotide functions in cells or how it interacts with receptor proteins is not only for the intellectual value of cell biology, but also for the mechanism of disease and the development of drugs against it. Required for In such studies, a fluorescent cyclic nucleotide derivative in which a fluorescent probe is bound to a cyclic nucleotide is useful for improving the efficiency and quality of studies for analyzing the functional dynamics of cyclic nucleotides in cells.
[0003]
Conventional cyclic nucleotide derivatives have been developed mainly for pharmaceuticals, and have been synthesized as having a phosphodiesterase (PDE) inhibitory effect. However, in order to examine the dynamics of intracellular functions of cyclic nucleotides and their interaction with receptors on cell membranes, the modifications must not impair their biological activities. For this purpose, it is necessary to avoid modification to the adenine ring or the phosphate site and selectively carry out ribose modification which has the least effect on the physiological activity. In addition, when synthesizing a fluorescent cyclic nucleotide derivative, it is not easy for a general biological researcher to introduce a fluorescent probe into a cyclic nucleotide by an ordinary method.
[0004]
[Problems to be solved by the invention]
Therefore, the present invention is useful for investigating the intracellular functional dynamics of cyclic nucleotides and the interaction with receptors on the cell membrane, and a reaction precursor that can be easily condensed with a known dye having a fluorophore. It is an object of the present invention to provide a novel cyclic nucleotide derivative and a method for producing the same.
[0005]
[Means for Solving the Problems]
The present inventors have conducted intensive studies, and as a result, by introducing a highly reactive -NH ₂ group or -SH group to a hydroxyl group of ribose of a cAMP derivative by adding a linker having an appropriate length, a desired cyclic nucleotide is obtained. The inventors have found that a derivative can be obtained, and have completed the present invention.
That is, the present invention has the following configuration.
[0006]
1. A cyclic nucleotide derivative represented by the following general formula (1).
[0007]
Embedded image

[0008]
(Wherein, R ₁ represents —OH or —NH ₂ , R ₂ represents hydrogen or —NH ₂ , R ₃ represents —NH ₂ or —SH, and n represents an integer of 2 to 4. .)
2. The cyclic nucleotide represented by the following general formula (2) is
Embedded image

[0010]
(Wherein, R ₁ represents —OH or —NH ₂ , and R ₂ represents hydrogen or —NH _2. )
By reacting with 1,1′-carbonyldiimidazole represented by the formula (3),
[0011]
Embedded image

[0012]
The obtained cyclic nucleotide intermediate represented by the general formula (4) is
[0013]
Embedded image

[0014]
(In the formula, R ₁ and R ₂ are the same as described above.)
Characterized by reacting with a compound represented by the general formula (5),
H ₂ N (CH ₂ ) _n R ₃ (5)
(In the formula, R ₃ represents —NH ₂ or —SH, and n represents an integer of 2 to 4.)
A method for producing a cyclic nucleotide derivative represented by the general formula (1).
[0015]
Embedded image

[0016]
(In the formula, R ₁ , R ₂ and R ₃ are the same as described above.)
[0017]
BEST MODE FOR CARRYING OUT THE INVENTION
As an example of the present invention, a reaction scheme for obtaining desired 2′-O- (2-aminoethylcarbamoyl) cAMP from cAMP is shown below.
[0018]
Embedded image

[0019]
The novel cyclic nucleotide derivative represented by the general formula (1) obtained in the present invention has a reactivity with R _{3 of the} cyclic nucleotide (1) such as Cy3-OSu (trade name: manufactured by Amersham Pharmacia Biotech). And used to produce a fluorescent cyclic nucleotide derivative such as Cy3-EDA-cAMP. This fluorescent cyclic nucleotide derivative is useful for improving the efficiency and quality of research for analyzing the functional dynamics of cyclic nucleotides in cells.
[0020]
【Example】
[Synthesis of 2'-O- (2-aminoethylcarbamoyl) cAMP]
6.0 g (18.2 mmol) of commercially available cAMP was dissolved in 600 ml of dimethylformamide (DMF) dried with Molecular Sieves 4A (product name: K, Na aluminosilicate), and 4.36 ml of tributylamine was added. 16.8 g (104 mmol) of -carbonyldiimidazole was added, and the mixture was stirred at room temperature for 1 hour. Further, 600 ml of DMF was added, and the mixture was stirred at room temperature for 30 minutes, and then heated and stirred at 30 ° C. for 1.5 hours. Next, 12 ml of ethylenediamine was added, and the mixture was stirred at 30 ° C. for 3 hours, and then filtered to remove insolubles. When the insolubles were analyzed by high performance liquid chromatography (HPLC), most were unreacted c-AMP.
The filtered solution was concentrated under reduced pressure on a water bath at 60 ° C. to obtain 35.4 g of a pale yellow oil. After adding 600 ml of dichloroethane and stirring at room temperature for 30 minutes, the crystals were collected by filtration, washed with diethyl ether, and dried under reduced pressure to obtain 13 g of a white powder.
This powder was divided into three batches, dissolved in water, and used as a column using Daisogel SP-120-30 / 50-ODS-B (50 mm ID × 500 mm), while detecting with UV at 254 nm, and using 10 mM Tributylammonium Bicarbonate (UV). It was eluted with tributylamine-bicarbonate buffer) -10% methanol at 15 ml / min. After fractionation of the desired fraction, methanol was distilled off under reduced pressure and freeze-dried. Methanol was added to the obtained solid, and the procedure of concentration under reduced pressure was repeated three times to obtain 2.27 g of a white solid. The purity of this solid measured by HPLC was 99.4%, and it was confirmed by ¹ H-NMR that it was the desired product.
¹ H-NMR (D2O): δ 8.06 (s, 1H), 8.03 (s, 1H), 6.15 (s, 1H), 5.38 (d, 1H, J = 7 Hz), 4. 82 (m, 1H), 4.34 (m, 1H), 4.1 to 4.2 (m, 2H), 3.2 to 3.4 (m, 2H), 2.95 (t, 2H, J = 5.5 Hz)
[0021]
The ability of this cyclic nucleotide derivative to function as an effective analog was demonstrated by the chemotactic response of slime mold to cyclic AMP. Cellular mucosa (Dictyostelium discoidium) uses cAMP as a chemotactic substance (attractant). The slime mold cells cultured under starvation become cell clumps and start to form fruiting bodies. This cell mass is highly sensitive to cAMP and expresses many cAMP receptors on the cell membrane. The nucleotide derivative obtained in the above example was added to the nutrient agar medium at a concentration of 10 ⁻⁸ to 10 ⁻⁵ M, and the mixture was solidified in a circular incubator. A cell mass (2 μL) was placed on the center of this medium and cultured at 20 ° C. for 3 hours. FIG. 1 shows the results obtained by measuring the spread of the cell mass after 3 hours to determine the moving speed of the cells. With this cyclic nucleotide derivative, a response at a cell migration rate was obtained at the same concentration and intensity as the response to unmodified cAMP.
[0022]
In addition, a fluorescent dye cy3-OSu was condensed with this cyclic nucleotide derivative to synthesize a fluorescent cyclic nucleotide derivative cy3-EDA-cAMP. When the cell migration rate was determined in the same manner, a reaction similar to that of cAMP was obtained. . (See Fig. 1)
Therefore, the use of this fluorescent cyclic nucleotide derivative can improve the efficiency and quality of research for analyzing the functional dynamics of cyclic nucleotides in cells.
[Brief description of the drawings]
FIG. 1 is a diagram showing the results of determining the cell migration rate using a cyclic nucleotide derivative of the present invention and a fluorescent cyclic nucleotide derivative obtained from the derivative.

Claims (2)

A cyclic nucleotide derivative represented by the following general formula (1).

(Wherein, R ₁ represents —OH or —NH ₂ , R ₂ represents hydrogen or —NH ₂ , R ₃ represents —NH ₂ or —SH, and n represents an integer of 2 to 4. .) A cyclic nucleotide represented by the following general formula (2)

(Wherein, R ₁ represents —OH or —NH ₂ , and R ₂ represents hydrogen or —NH _2. )
By reacting with 1,1′-carbonyldiimidazole represented by the formula (3),

The obtained cyclic nucleotide intermediate represented by the general formula (4) is

(In the formula, R ₁ and R ₂ are the same as described above.)
Characterized by reacting with a compound represented by the general formula (5),
H ₂ N (CH ₂ ) _n R ₃ (5)
(In the formula, R ₃ represents —NH ₂ or —SH, and n represents an integer of 2 to 4.)
A method for producing a cyclic nucleotide derivative represented by the general formula (1).

(In the formula, R ₁ , R ₂ and R ₃ are the same as described above.)

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