【発明の詳細な説明】本発明は（２’−５’）オリチアゾニル酸の製造方法に
係る。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing (2'-5') orithiazonilic acid.
最近、インターフエａン処理した細胞中ＶＣ′Ｍ白合成
阻害物質が発見され氏、この物質の構造はｙ−トリホス
ホリルアデニリル−（２’−５’）−アヂニリルー（２
’−５’）−アデノシンであるとされてシシ、更にその
後の研究によれば、これより重合度の高い（２’−５’
）オリチアゾニル酸を骨格とする化合物にも生理活性が
認められている。こうしたこと力・らヌクレオチドのオ
リｆ−ｒ−に関心が高オリ、特Ｋ＜’２’−５’）オリ
デヌクレオチドの製造について種々研究が行なわれてき
ている。Recently, a substance that inhibits VC'M white synthesis in cells treated with interferon a was discovered, and the structure of this substance is y-triphosphoryladenyl-(2'-5')-adinilyru(2).
'-5')-adenosine, and later research has shown that it has a higher degree of polymerization (2'-5')-adenosine.
) Compounds with an orithiazonil acid skeleton have also been found to have physiological activity. In view of this, various studies have been conducted on the production of oligonucleotides with high oligonucleotides (especially K<'2'-5') due to the interest in the ori f-r- of nucleotides.
従来知られている（２’−５’）オリチアゾニル酸の製
造方法としては、例えば、先ずアデノシンの６位のア建
ノ基をアシル基やトリチル基で又ｙ位の水酸基なＯ−ニ
トロペンシル基で保護して得られるＮ６−アシル−又は
トリチル−３’−０−（ｏ−二トロベンジル）−アデノ
シン−５′−モノリン酸な脱水剤・と反応させてオリゴ
マー構造を形成させ、続いて保護基な離脱させるととに
より合成する方法がある（４１１Ｆ開昭５６−３４’６
９６、特開昭５６−３４６９７）。しかしヌクレオチド
の特定の基を保験して反応することは、その工程が複緒
であシ、合成に長時間な要し、又その収率も満足すべき
ものではない。As a conventionally known method for producing (2'-5') orithiazonylic acid, for example, first, the adenosine group at the 6-position of adenosine is converted into an acyl group or trityl group, and the hydroxyl group at the y-position is converted into an O-nitropenyl group. An oligomer structure is formed by reacting with a dehydrating agent such as N6-acyl- or trityl-3'-0-(o-nitrobenzyl)-adenosine-5'-monophosphoric acid obtained by protection with There is a method of synthesis by separating the
96, Japanese Patent Publication No. 56-34697). However, reacting while preserving a specific group of nucleotides requires complicated steps, takes a long time to synthesize, and the yield is also unsatisfactory.
本発明者もオリイヌクレオチ「に関心を抱き、′その合
成について研究を進め、ウリジンの特定の基を保護する
ことなく、即ち簡便な工程でオリビラリジル酸を合成す
る方法を見出し、先ＫＴ＊ｔｒａｈ＠ａｒｏｎ ＩＪ＠
ｔｔｅｒｓ、ｖｏ１２１．ｐ ２７１７〜２７２０．１
９８０年に発表した。。この方法は、ウリジンのｙ−位
の水酸基を保額することなく、ビリジンーテトラヒＦロ
フラン混合溶媒中でトリー積状ホスホイミダ・戸−ル免
マ生成し、続いてこれが直ちに他のウリシン−２’ 、
３’−環状ホスホイ書ダゾール又は原料ウリジンのう
位の水酸基と反応して環状エステル結合を有するオリゴ
マー１生成するので、このオリゴマーを含む溶液に＠素
および水を作用させることにより亜燐酸エステルの酸化
と２′位又は３′位エステル結合の開裂とを行なわせ、
（３’−５’）リン酸ジエステル結合と（２’ −５’
）リン酸ジエステル結合が混在するウリジンオリゴヌ
クレオチドを得る方法であった。The present inventor was also interested in oriinicleotide, conducted research on its synthesis, and discovered a method for synthesizing orivilaridylic acid without protecting a specific group of uridine, that is, in a simple process. aron IJ@
tters, vol121. p 2717-2720.1
Published in 1980. . In this method, the hydroxyl group at the y-position of uridine is not preserved, and the tri-shaped phosphoimida-trivalent polymer is produced in a biridine-tetrahydrofuran mixed solvent, which is then immediately used to form other uridine-2 hydroxyl groups. ',
Oligomer 1 having a cyclic ester bond is produced by reacting with the 3'-cyclic phosphorydazole or the hydroxyl group of the raw material uridine, so the phosphorous ester is oxidized by reacting @ and water with a solution containing this oligomer. and cleavage of the ester bond at the 2' or 3' position,
(3'-5') phosphodiester bond and (2'-5'
) This was a method to obtain a uridine oligonucleotide containing a mixture of phosphodiester bonds.
この方法では２′位又は５′位のいずれか一方のみを選
択的に開裂させることは困難であり、ウリジンオリゴヌ
クレオチドの場合は、７位のエステル結合の開裂が３２
１．３′位のエステル結合の開裂が６８−の割合であっ
た、この場合、生成オリゴマー中の（２’−５’）リン
酸ジエステル結合の含量を高めるためＫは必ず開裂規制
物質例えば特定のＩリヌクレオチＶや金属イオンを共存
させる必要があった。In this method, it is difficult to selectively cleave only either the 2'-position or the 5'-position, and in the case of uridine oligonucleotides, the ester bond at the 7-position is cleaved at the 32-position.
The cleavage of the ester bond at the 1.3' position was at a rate of 68-.In this case, K must be used as a cleavage regulating substance, such as a specific It was necessary to coexist with the I-linucreotide V and metal ions.
本発明者は上記研究を更に発展させ（２’−５’）リン
酸ジエステル結合をもクオリデヌクレオチドの合ＭＫつ
いて研究した結果、環内にイミノ基を有す−る環状化合
、物かち得られるホスフィン誘導体（以下単にホスフィ
ン誘導体と略称丁：Ｊｂ）とアヂ（２’−５’）結合を
９０％以上含むアデノシンオリ？ヌクレオチｙが得られ
ることを見出し、本発明に至った・本発明の目的はアデノシンの特定の基を保護することな
く、又開裂規制物質を用いることなく（２’−５’）結
合ｖ９０−以上含むアデノシンオリ♂ヌクレオチ）’（
（２’−５’）オリゴアデニル酸〕を簡便に製造する方
法な提供することにある。The present inventor further developed the above research and studied the (2'-5') phosphodiester bond in the combined MK of qualidenucleotides. A phosphine derivative (hereinafter simply referred to as a phosphine derivative and abbreviated as Jb) and an adenosine oligomer containing 90% or more of adi(2'-5') bonds. We have discovered that nucleotide y can be obtained, leading to the present invention.The purpose of the present invention is to form a bond of v90- or more without protecting a specific group of adenosine or without using a cleavage-controlling substance (2'-5'). Containing adenosine ori♂ nucleoti)'(
(2'-5') oligoadenylic acid] is provided.
本発明で用いるホスフィン誘導体は一般式（１）〜（３
）；！’ｔ、Ｐ・・・・・・・・・・・・（１）ＲＰＸ、・
・・・・・・・・・・・（２）ＲＰＲ−細手１・・（３
）（式中、ｘ、Ｗは環内にイミノ基を有する環状化合物、
Ｘは塩素原子又は臭素原子）のいずれかでビロール、ビ
ロリン、ぎａリジン、ピラゾール、ピラゾリン、イミダ
ゾール、イミダゾリシン、トリアゾール、テトラゾール
勢の五員環化合物、ピペリジン、オ命すゾン、モルホリ
ン、チアジン郷の六員衰化合物、インｒ−ル、インｙリ
ン、イソインレール、イソインドリン、イン／ｆ−ル、
ペン・戸イ２ダゾール、ペン・戸トリアクール、ベンゾ
チアゾリン、プリン尋の二項化合物、カルバゾール、フ
ェノキサシン、フェノチアジン等の三穣化状化合物と三
塩化リン又は三臭化リンとを適轟な溶媒中で反応させる
ことにより得られる。即ち三塩化リン又は三臭化リンに
対し６倍モル（同時にハロゲン化水素捕集剤を用いると
きは５倍モルでよい）の環状化合物を用いて反応させる
と前記式（１１で表わされるホスフィン誘導体が得られ
、又２倍モル（同時に−ａ）ｆン化水素捕集剤を用いる
ときは１倍モルでよい）の積状化合物な用いて反応させ
るときは前記式（２）で表わされるホスフィン誘導体が
得られる。この式（２）で表わされるホスフィン誘導体
な先に合成して更に別の墳状化谷物と反応させると前記
式（３）で表わされるホスフィン誘導体を得ることがで
きる。この式（３）のホスフィン誘導体の場合にはＷは
Ｒよりも反応性に富む環状化合物を用いるのが好ましい
。このホスフィン誘導体の合成に際し、用いる溶媒とし
ては、アデノシンとホスフィン誘導体との反応忙用いる
ことのできら溶媒であることが、反応系より生成ホスフ
ィン誘導体を分離することなく、次の反応忙供すること
ができ好オしい。尚このホスフィン誘導体の媒に溶解し
、得られる溶液と前記ホスフィン誘導体とを反応させ、
この生成糸に酸化剤と水とを作用させることｋより直ち
Ｋ （２’−５’）オリデアヂニル酸な得る方法である
。The phosphine derivatives used in the present invention have general formulas (1) to (3).
); ! 't, P・・・・・・・・・(1) RPX,・
・・・・・・・・・・・・(2) RPR-Hoshite 1...(3
) (where x and W are a cyclic compound having an imino group in the ring,
X is either a chlorine atom or a bromine atom), and is a five-membered ring compound such as virol, viroline, alysine, pyrazole, pyrazoline, imidazole, imidazolysine, triazole, or tetrazole, piperidine, ozone, morpholine, or thiazine. six-membered decay compounds, inr-ru, inyrin, isoinler, isoindoline, in/f-ru,
When used in an appropriate manner, tricontaminated compounds such as pen-toi 2-dazole, pen-totriacur, benzothiazoline, purine dichotomous compounds, carbazole, phenoxacin, and phenothiazine are mixed with phosphorus trichloride or phosphorus tribromide. Obtained by reaction in a solvent. That is, when a cyclic compound is reacted with 6 times the mole of phosphorus trichloride or phosphorus tribromide (if a hydrogen halide scavenger is used at the same time, 5 times the mole is sufficient), the phosphine derivative represented by the formula (11) is reacted. is obtained, and when the reaction is carried out using 2 times the mole (when simultaneously using a hydrogen fluoride scavenger, 1 times the mole is sufficient) of the product compound, the phosphine represented by the above formula (2) is obtained. A derivative is obtained. If the phosphine derivative represented by the formula (2) is first synthesized and further reacted with another mounded material, the phosphine derivative represented by the formula (3) can be obtained. In the case of the phosphine derivative of formula (3), it is preferable to use a cyclic compound as W which is more reactive than R. When synthesizing this phosphine derivative, the solvent used should be one that can be used during the reaction between adenosine and the phosphine derivative, so that it can be used for the next reaction without separating the produced phosphine derivative from the reaction system. It's great. In addition, dissolving this phosphine derivative in a medium and reacting the obtained solution with the phosphine derivative,
A method for directly obtaining K (2'-5') orideadinilic acid is to allow an oxidizing agent and water to act on the produced yarn.
この反応は次式により表わすと七ができる。This reaction can be expressed by the following formula:
尚このオリｆ−ｒ−の構成単位ＶＡｐ（ムはアデノシン
、ｐは燐ｌＩ）で表わすと、本発明方法ｆよれと（ムｐ
）３および（Ａｐ）ｒｌ二１Ａ （ｎ ＝ ２〜２０
） ｔ’製造することができる。In addition, when expressed as the structural unit VAp (mu is adenosine, p is phosphorus) of this ori f-r-, the method f of the present invention (mu p
)3 and (Ap)rl21A (n = 2-20
) t' can be produced.
を主として含む溶媒に溶解し、これをホスフィンｔｙ、
へ中サメチルホスホロアミｙ、又はジメチルスルホ中シ
ト等を意味し、更にこれらの少なくとも１ｓｖ主として
含むテトラヒドロフランとの混合溶媒も前記溶媒として
適している。反応に際し、アデノシンは予め前記溶媒に
溶解し、またホスフィン誘導体はテトラヒドロフラン又
は前記法＃＆に溶解した状態で用いるのが好ましい。重
合度の高い（２’−５’）オリｔアデニル酸を所望する
場合にはアデノシン溶液およびホスフィン誘導体溶液な
遂次反応系に追加供給することもある。反応時間は一般
に１０〜６０分で充分であるが、用いるホスフィン誘導
体のＳ炉中Ｐｉｒｗする重合度によってはこれに限定さ
れるものではなく、より短かい時間でよい場合も又より
長時間例えに１００〜５００分又はそれ以上の時間反応
させることもある。又反応雰囲気としては空気であって
もよい力（ホスフィン誘導体並びに生成ホスファイトの
酸化を避ける意味で窒素その他の不活性ガスとするのが
好ましい。is dissolved in a solvent mainly containing phosphine ty,
A mixed solvent containing at least 1 sv of these compounds with tetrahydrofuran is also suitable as the solvent. During the reaction, it is preferable to use adenosine dissolved in the above-mentioned solvent in advance, and the phosphine derivative dissolved in tetrahydrofuran or the above-mentioned method #&. If (2'-5') ori-t-adenylic acid with a high degree of polymerization is desired, it may be additionally supplied to the sequential reaction system such as an adenosine solution and a phosphine derivative solution. Generally, 10 to 60 minutes is sufficient for the reaction time, but it is not limited to this depending on the degree of polymerization of the phosphine derivative used in the S furnace. The reaction may be carried out for 100 to 500 minutes or more. The reaction atmosphere may be air (nitrogen or other inert gas is preferred in order to avoid oxidation of the phosphine derivative and the produced phosphite).
本発明ではアデノシンとホスフィン誘導体が反応して先
ず、７位および６′位で結合した環状亜リン酸エステル
（前記反応式中式Ｉで表わされる）が生成し、続いてこ
れが速やかに遂次的に縮合してオリイマー（前記反応式
中式■で表わされる）となる。これＫｒＩＲ化剤と水と
を作用させると亜リン酸エステルの酸化と３′位のエス
テル結合の選択的開裂がおこり、目的物である（２’−
５’）オリビアデニル酸を得ることができる。ここで酸
化剤としてはヨウ素、塩素、臭素などのハロゲンのほか
過酸化窒素、酸素、オナンな゛どが用いられ、好オしく
ｓｔ ｗｉつ素のテトラヒドロフランと水との混合溶
媒溶液を用いる。酸化剤と水とは同時に作用させるのが
好オしいが、酸化過程と開環過程を分離することもでき
る。この酸化と開環反応の反応温度は添加した水又は溶
液が液体として存在し得る温度であればよく１通常−５
〜７０℃で行い得、反応時間は１〜１０分程度である。In the present invention, adenosine and a phosphine derivative react to first form a cyclic phosphite (represented by Formula I in the above reaction formula) bonded at the 7- and 6'-positions, which is then rapidly and sequentially It condenses to form an oligomer (represented by formula (2) in the above reaction formula). When this KrIR agent interacts with water, oxidation of the phosphite ester and selective cleavage of the ester bond at the 3' position occur, resulting in the desired product (2'-
5') Oliviadenylic acid can be obtained. As the oxidizing agent, in addition to halogens such as iodine, chlorine, and bromine, nitrogen peroxide, oxygen, and onane are used. Preferably, a mixed solvent solution of two atoms in tetrahydrofuran and water is used. Although it is preferable that the oxidizing agent and water act simultaneously, it is also possible to separate the oxidation process and the ring-opening process. The reaction temperature for this oxidation and ring-opening reaction may be any temperature at which the added water or solution can exist as a liquid.1 Usually -5
It can be carried out at ~70°C, and the reaction time is about 1 to 10 minutes.
反応生成物は重合度の異なる（Ａｐ）ｎ ’１１および
（ムｐ）ｎ−ｘム型オリｆマーの混合物として得られる
が、生成物中の（ムｐ）ｎ型および（Ａｐ）ｎ−ｘＡ型
の割合は原料アデノシンとホスフィン誘導体との量比シ
よび反応条件により規制できる。得られたオリ゛イマー
混合物の分離は例えばカラムクロｉトゲラフイーにより
行い得るが、その場合充填材としてＤＩムｌ１ｌ−Ｅｌ
ｅｐｈａａ＠ｘ Ａ ２５等が利用できる。The reaction product is obtained as a mixture of (Ap)n '11 and (p)n-x type oligomers with different degrees of polymerization, but the (p)n type and (Ap)n- The proportion of the xA type can be controlled by the ratio of the raw material adenosine to the phosphine derivative and the reaction conditions. The obtained oligomer mixture can be separated, for example, by column chromatography, in which case DIml-El is used as a packing material.
ephaa@x A 25 etc. can be used.
本発明の特長の第１は中間体である式Ｉの化合物の有す
る３つのエステル結合のうち、６′−位のエステル結合
が選択的に開裂することであり、第２はアデノシンの６
位のアミノ基を何ら保護する必要のないことである。The first feature of the present invention is that among the three ester bonds of the intermediate compound of formula I, the 6'-position ester bond is selectively cleaved, and the second feature is that the 6'-position ester bond is selectively cleaved.
There is no need to protect the amino group at this position.
本発明の目的とする生成物（２’−５’）オリ♂アデニ
ル酸は１つのアデノシンの２位と他のアデノシンの５′
位どの間にリン酸ジエステル結合をもつオリｆ−ｒ−で
ある。本発明方法の如く、ヌクレオシ「とホスフィン誘
導体を反応させるときは、中間に式■として示したよう
なヌクレオシｒの２位および３′位で結合した環状エス
テルが生成するため、この６′位のエステル結合を開裂
させることが必要となる。しかし、通常弐塁で示したよ
゛うなヌクレオシドの猥状エステル忙おいては、２′位
又はｙ位のエステル結合の１個のみを選択的に開裂させ
ることはできなかった。The target product of the present invention (2'-5') ori-adenylic acid is the 2-position of one adenosine and the 5'-position of the other adenosine.
It is an ori fr- having a phosphodiester bond between the positions. As in the method of the present invention, when a nucleosylated phosphine derivative is reacted with a phosphine derivative, a cyclic ester bonded at the 2- and 3'-positions of the nucleosylated r as shown in formula It is necessary to cleave the ester bond.However, in the case of obscene esters of nucleosides as shown in the second base, only one of the ester bonds at the 2' or y position is selectively cleaved. I couldn't do that.
例えば、齢述したように、ヌクレオシＶとしてウリシン
を用いて、ビリジ／−テトラヒドロフラン混合溶媒中で
トリー（イ叱ダシールー１−゛イ゛ル）ホスフィンと反
応させて得られる前記式■に相当するオリｆ −ｒ −
ｆヨウ素と水で酸化開裂させるとき、生成物中の（２’
−５’）結合と（！ｌ’−５’）結合の割合は前者が６
８１後者が３２−である。又ヌクレオシドとしてジチゾ
ンを用い、溶媒ＫＮ、Ｎ−ジメチルホルムアギドーテト
ラヒＰ ａフラン混合溶媒を用いたときのＣ２’−ｂ’
）結合の割合は７５−である。For example, as mentioned above, an oligomer corresponding to the formula f −r −
f When oxidatively cleaved with iodine and water, (2'
-5') bond and (!l'-5') bond ratio is 6 for the former.
81 the latter is 32-. In addition, C2'-b' when dithizone is used as the nucleoside and a mixed solvent of KN, N-dimethylformazidotetrahyPa and furan is used.
) The binding ratio is 75-.
然るに、アデノシンをアデノシン可溶の非プロトン性の
極性溶媒又はこれを主として含む溶媒に溶餌して用いる
本発明の場合には何らの開裂規制物質を用いることなく
、生成物中に於ける（２′−５′）結合の割合は９０チ
以上に遅し、後述する実施例にも示すように、前記シチ
ジンの場合と同じ混合溶媒を用いたときは９５チに達す
る。ここで（２’−５’）結合が９５ｇ６とは生成物中
に（２′又は５’−５′）結合か２０個存在するときｊ
Ｃ（２’−５’）結合が１９個あることを意味する。も
しく ２’ −５’ ）結合１００％のオリデアデニル
勢が望まれるときは、予め重合度が所望重合度よｐ幾分
高めのオリ♂マーを生成させておき、（３’−５’）結
合を選択的忙切断する肺臓リン酸ジエステラーぜ（ｓｐ
ｙ・ａｎｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ ）を作
用させることによシ効率よく得ることもできる。However, in the case of the present invention, in which adenosine is dissolved in an aprotic polar solvent that is soluble in adenosine or a solvent that mainly contains the same, no cleavage-controlling substance is used, and (2) '-5') The bonding rate slows down to 90 or more, and reaches 95 when the same mixed solvent as in the case of cytidine is used, as shown in the examples below. Here, 95g6 of (2'-5') bonds means that there are 20 (2' or 5'-5') bonds in the product.
This means that there are 19 C(2'-5') bonds. If 100% of the 2'-5') bond is desired, an oligomer with a polymerization degree slightly higher than the desired degree of polymerization is generated in advance, and the (3'-5') bond is selectively cleaves lung phosphodiesterase (sp
It can also be efficiently obtained by the action of y.amphosphodiesterase).
従来、アデノシンのｙ位の水酸基を保護するこなく（７
’−５′）結合を９０％以上有するオリイアデニル酸が
得られることは予期し得ぬものであった。Conventionally, the hydroxyl group at the y-position of adenosine was not protected (7
It was unexpected that an oligoadenylic acid having 90% or more of '-5') bonds could be obtained.
又、本発明においてはアデノシンの６位のアミノ基の保
酔を要しないことも大きな利点である。Another great advantage of the present invention is that it is not necessary to preserve the amino group at the 6-position of adenosine.
通常、ゾリン又はぎリミジン塙基部に活性な官能基を有
するヌクレオシ「をリン酸イビする場合忙は、予めその
官能基を保護することが行たわれるが、本発明方法にお
いてはアデノシンの６位のアミノ基の保護を要しない。Normally, when phosphorylating a nucleotide with an active functional group at the zoline or girimidine base, the functional group is protected in advance, but in the method of the present invention, the 6-position of adenosine is protected. No protection of amino groups is required.
塩基部の官能基を保護することなく（２’−５’）オリ
デアデニル酸が高収庫で得られることも予期し得ぬもの
であった、尤も工程が増加するがアミノ基を保護して反
応させても保護しない・場合と同様に（２’−５’）オ
リイアヂニル酸を得ることは可能ではある。It was also unexpected that (2'-5') orideadenylic acid could be obtained in high yield without protecting the functional group of the base moiety.Although it requires more steps, it is possible to react by protecting the amino group. It is possible to obtain (2'-5') oligoadinilic acid in the same manner as in the unprotected case.
以上述べた如く、本発明方法においては、従来公知の方
法と異かり、アデノシンの６位のアミノ基および３′位
の水酸基を保護することなく反応に供することができ、
従って短かい工程で、かつ、短時間の反応で、しかも途
中反応系より中間生成物を分離する工程も要せず（２’
−”／＞結合を９０−以上含むオリデアヂニル酸が得ら
れる特長を有する。As described above, in the method of the present invention, unlike conventionally known methods, the amino group at the 6-position and the hydroxyl group at the 3'-position of adenosine can be subjected to the reaction without protection,
Therefore, it is a short process and a short reaction time, and there is no need for a step to separate intermediate products from the reaction system (2'
It has the advantage that orideadinyl acid containing 90 or more -''/> bonds can be obtained.
以下実施例により具体的ｒｃＷ５２明する。The details of the rcW52 will be explained below with reference to Examples.
尚、以下の実施例に於て、生成オリテマーの結合様式即
ち（２’−５’）結合、（３′−５’）結合の決定は以
下のようにして行なつ几。In the following Examples, the bonding mode of the produced oligomers, ie (2'-5') bond and (3'-5') bond, was determined as follows.
生成オリイマーの有する２′位、６′位およびデ位の３
つのリン酸エステル結合のうち、（３’−５’）位のエ
ステル結合は肺臓リン酸エステル結合（５ｐｌｅｅｎ
ｐｈｏｓｐｈｏｄｉｅｓｔ＠ｒａａｅ ） ＫよＣＭ裂
する。3 of the 2', 6' and de positions of the oligomer produced
Of the three phosphate ester bonds, the ester bond at the (3'-5') position is the lung phosphate ester bond (5pleen).
phosphodiest@raae) K will break the commercial.
両末端のリン酸結合はアルカリホスファターゼで加水分
解する。又（２′−り結合並びＫ（５’ −５′）ＩＩ
Ｉｊ合は蛇毒リン酸ジェステラーゼ（５ｎａｋｅ ｖ・
ｎｏｎｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ ）とアル
カリで加水分解す６゜この３つの方法を適用することに
より、（２’−５’）結合と（３’−５’）結合の割合
、重合度並びに末端リン酸基の有無を決定した。The phosphate bonds at both ends are hydrolyzed by alkaline phosphatase. Also, (2'-rebond sequence K(5'-5') II
Ij is snake venom phosphate gesterase (5nake v.
Nonphosphodiesterase) and hydrolyzed with alkali6゜By applying these three methods, the ratio of (2'-5') bonds and (3'-5') bonds, the degree of polymerization, and the presence or absence of terminal phosphate groups can be determined. Decided.
実施例１１４．０オリモルのイミダゾールを１６−のテトラヒｒ
ロフラン（ＴＲＩＰ） Ｋ溶解し、これに２．３ミリモ
ルの三塩化燐を０℃で９素気流下に滴下した。Example 1 14.0 orimoles of imidazole was mixed with 16-tetrahydryl
Rofuran (TRIP) K was dissolved, and 2.3 mmol of phosphorus trichloride was added dropwise thereto at 0° C. under a stream of 9 atoms.
２０分後に生成した沈＃（イミダゾール塩散塩）を９素
雰囲気下で炉別し、トリー（イミダゾール−１−イル）
ホスフィンを含むＦ液を得た。このろ液１ｇ＋／（トリ
ー（イミダ・戸−ルー１−イル）ホスフィンｒ１．１４
ミリモルを含む）をアデノシン０．１４ミリモルを含む
Ｎ、Ｎ−ジメチルホルムアミド（ＤＭＦ）溶液１１Ｌｌ
中に＃下し、１１表に示した泥炭で１時間反応させ７′
２．にの反応溶液に０．１４ミリモルのヨウ素を含むＴ
ＨＰ−水（２：１）混合液１ｄを０℃で加え５分砕に過
剰の沃素を亜硫酸ナトリウム水溶液で処理し、乾燥した
。生成物はツ１表の如くであった、率で混在していた。The precipitate # (imidazole salt dispersion) produced after 20 minutes was separated in a furnace under an atmosphere of 9 elements, and tri(imidazol-1-yl)
A solution F containing phosphine was obtained. 1 g of this filtrate +/(tri(imida-to-ru-1-yl)phosphine r1.14)
11 L of a N,N-dimethylformamide (DMF) solution containing 0.14 mmol of adenosine
Pour # into the tank and react with the peat shown in Table 11 for 1 hour.
2. T containing 0.14 mmol of iodine in the reaction solution of
1 d of HP-water (2:1) mixed solution was added at 0° C., the mixture was crushed into 5 portions, excess iodine was removed with an aqueous sodium sulfite solution, and the mixture was dried. The products were as shown in Table 1, and were mixed at the following ratios.
実施例２７．０オリモルの一イミダゾールと７．０オリモルのト
リエチルア建ン１に１６ｍｌのＴＨＦに溶解し、２．３
ミリモルの三塩化リンを一２０°Ｃで窒素気流下に滴下
した。１０分後に沈澱を音素雰囲気下で戸別し、このト
リー（イミダゾール−１−イル）ホスフィンを含むＦ液
を下表に示す量を用いて、０．１４ミリモルのアデノシ
ンを１ｄのＤＭＦ−ＴＨＦ （３： １ ）混合溶媒に
溶解した溶液に一７８℃で滴下し、４０分反応させた。Example 2 7.0 orimoles of imidazole and 7.0 orimoles of triethylamine were dissolved in 16 ml of THF and 2.3
Millimole of phosphorus trichloride was added dropwise at -20°C under a nitrogen stream. After 10 minutes, the precipitate was separated from house to house in a phonetic atmosphere, and 0.14 mmol of adenosine was mixed with 1 d of DMF-THF (3 : 1) It was added dropwise to a solution dissolved in a mixed solvent at -78°C and reacted for 40 minutes.
この反応浴液（，０，１４（！Ｊモルのヨウ素を含むＴ
ＨＦ−水（１：１）混合液を室温で加え、５分砕に過剰
のヨウ素を亜硫酸ナトリウム水溶液で処理し乾燥した。This reaction bath solution (T containing ,0,14(!J moles of iodine)
A mixed solution of HF-water (1:1) was added at room temperature, and the mixture was crushed into 5 pieces, treated with an aqueous sodium sulfite solution to remove excess iodine, and dried.
生成物は第２表のごとくである。The products are shown in Table 2.
これらの生成物中オリ−？！−のリン酸ジエステル結合
様式は、２’ −５’型／３’−５’型−９５７５の比
率で混在していた。Ory in these products? ! The phosphodiester bonding modes of - were mixed at a ratio of 2'-5'type/3'-5' type -9575.
実施例３下表に示す各種ホスフィン誘導体をそれぞれ実施例２に
準じた方法で合成した。これを実施例１に準じた方法（
反応温度−７８℃）でアデノシンと反応させ第６表の結
果を得た。Example 3 Various phosphine derivatives shown in the table below were synthesized in a manner similar to Example 2. This was carried out using a method similar to Example 1 (
The results shown in Table 6 were obtained by reacting with adenosine at a reaction temperature of -78°C.
実施例４実施例１（反応塩度−７８℃）において、曹つ素のテＨ
ＩＦ−水混合物を用いる代りＫ、１．４ ｔ ＩＪモル
のＮ、０．のエーテル溶液１−を加えはげしく攪拌し、
５分後に水を加えた。生成物は実施例１の結果と同じで
あった。Example 4 In Example 1 (reaction salinity -78°C),
Instead of using an IF-water mixture, K, 1.4 t IJ mol N, 0. Add ether solution 1- of and stir vigorously.
Water was added after 5 minutes. The product was the same as in Example 1.
実施例５２ミリモルの三臭化リンを５−のＴＨ？ Ｋ溶解しこ九
に４ミリモルのイミダゾールを溶解したＴＨｙ５−を窒
素気流下ＫＯ℃で滴下反応させた。２０分後に沈澱（イ
ミダゾール臭素酸塩）を窒素雰囲気下で炉別し、インダ
ゾール−１−イルージプロモホスヴ４９／を含む液を得
た。Example 5 2 mmol of phosphorus tribromide was converted into 5-TH? THy5-, in which 4 mmol of imidazole was dissolved in the K solution, was added dropwise at KO° C. under a nitrogen stream to cause a reaction. After 20 minutes, the precipitate (imidazole bromate) was separated in a furnace under a nitrogen atmosphere to obtain a liquid containing indazole-1-iludipromofosv 49/.
この液１１Ｅ＃をアデノシン０，１５ミリモルを含むヘ
キサメチルホスホクアはド溶液５ｄＫ滴下し、−７８℃
で攪拌下１時間反応させた。この反応溶液に璽つ素１ミ
リモル含むＴＨＩＦ−水（２：１）混合液２ｗｔを０℃
で加え、５分後に過剰のヨウ素を亜硫酸す） ＋７ウム
水溶液で処理して後乾燥した。A 5 dK solution of hexamethylphosphoqua containing 0.15 mmol of adenosine was added dropwise to this solution 11E# at -78°C.
The mixture was reacted for 1 hour with stirring. To this reaction solution, 2 wt of a THIF-water (2:1) mixture containing 1 mmol of chlorine was added to
After 5 minutes, excess iodine was removed with a 7 um aqueous solution and then dried.
結果は第４表に示した。The results are shown in Table 4.
実施例６三項化リン２ミリモルを５ＷｌｌのＴＨＦ Ｋ溶解し、
これに４ミリモルのピラゾールを５−の’１’Ｈ７Ｆ
Ｋ溶解しこれを０℃で滴下反応させた。２０分後に沈澱
を炉別して得られるピラゾール−１−イルーシクａクホ
スフィンを含む溶液に、８ミリモルのイミダゾールを含
むＴＨＩＦ溶液５ｄを窒素気流下に滴下し、−２０℃で
反応させた。２０分後に沈澱を炉別して、ジ（イ２ダシ
ールー１−イル）−そノ（ピラゾール−１−イル）ホス
フィン溶液を得九このホスフィン誘導体１ミリモル含む
液をアデノシン０．８ミリモル含むＤＭＦ溶液５ｍ１Ｋ
滴下し、−７８℃で攪拌下６０分反応させた。この反応
液にヨウ票１．０ミリモル含むＴＨＦ−水（２：１）混
合液２ｄを０℃で加え、５分後に過剰のヨウ素をｉｗ＊
ナトリウム水溶液で処理し、乾燥した。Example 6 2 mmol of phosphorus trinomide was dissolved in 5 Wll of THF K,
To this, add 4 mmol of pyrazole to 5-'1'H7F.
K was dissolved and reacted dropwise at 0°C. After 20 minutes, the precipitate was separated in a furnace, and a THIF solution 5d containing 8 mmol of imidazole was added dropwise to a solution containing pyrazol-1-yl-cyclophosphine obtained under a nitrogen stream, and the mixture was reacted at -20°C. After 20 minutes, the precipitate was separated in a furnace to obtain a di(2dacyl-1-yl)-sono(pyrazol-1-yl)phosphine solution.9 A solution containing 1 mmol of this phosphine derivative was mixed with 5 ml of a DMF solution containing 0.8 mmol adenosine.
The mixture was added dropwise and reacted at -78°C for 60 minutes with stirring. To this reaction solution, 2 d of a THF-water (2:1) mixture containing 1.0 mmol of iodine was added at 0°C, and after 5 minutes, excess iodine was added to iw*.
Treated with aqueous sodium solution and dried.
結果は第４表に示した。The results are shown in Table 4.
実施例７実施例１におけるアゾリルホスフィン合成時の溶媒をＴ
ＨＦ ＫかえてＤＭＦを用いた（アデノシンとの反応温
度は０℃で行った）以外は実施例１と同様に行った。結
果は第４表に示した。Example 7 The solvent during azolylphosphine synthesis in Example 1 was T.
The same procedure as in Example 1 was performed except that DMF was used instead of HFK (the reaction temperature with adenosine was 0° C.). The results are shown in Table 4.
実施例８実施例５におけるアゾリルホスフィン合成時の溶媒（Ｔ
ＨＦ）をヘキサメチルホスホクアミドＫかえた以外は実
施例５と同様に行った、結果は第４表に示した。Example 8 Solvent (T
The same procedure as in Example 5 was carried out except that hexamethylphosphocamide K was used instead of HF). The results are shown in Table 4.
尚実施例５〜８において生成物中オリゴマーのリン酸ゾ
エステル結合様式ｚ−５／型／３’−５’型の比は夫々
９５１５．９７／３．９３／７及び９５１５であった。In Examples 5 to 8, the ratios of the phosphoric acid zoester bonding modes z-5/type/3'-5' type in the oligomers in the products were 9515.97/3.93/7 and 9515, respectively.
実施例９実施例１に記載した方法と同じ方法で合成したトリ（イ
ミダゾール−１−イル）ホスフィン２０きリモルを含む
ＴＨＦ溶液１０１１１およびアデノシン２０ミリモルを
含む１）ＭＩＦ溶液溶液１０智ｊ意し、反応器には予め
ＤＭＦ ５ ｄを仕込み一５０℃に冷却した、この反応
器に前記両溶液を０．５ Ｍｌ／ ＩＥｌｉ！１で滴下
し、滴下終了後頁ｒ−５０℃で６０分反応させた。この
反応溶液［２２ミリモルのヨウ素を含むＴＨＩＦ−水（
２：１）混合溶液な０℃で加え、５分後に過剰のヨウ素
を亜硫酸ナトＩＪウム水溶液で処理し乾燥した。Example 9 A THF solution containing 20 mmol of tri(imidazol-1-yl)phosphine and 1) MIF solution containing 20 mmol of adenosine synthesized in the same manner as described in Example 1. A reactor was charged with 5 d of DMF and cooled to -50°C. Both solutions were added to the reactor at a rate of 0.5 Ml/IEli! 1, and after the completion of the dropwise addition, the reaction was carried out at −50° C. for 60 minutes. This reaction solution [THIF-water containing 22 mmol of iodine (
A 2:1) mixed solution was added at 0° C., and after 5 minutes, excess iodine was removed with an aqueous solution of sodium sulfite and dried.
生成物であるオリゴアデニル酸の重合度ｎとその収率は
第５表のようであった。The degree of polymerization n of the oligoadenylic acid product and its yield are shown in Table 5.
又このオリビマー中のリン酸ゾエステル結合様式は（２
’−５）型／（３’−５’）型−９５７５であった。In addition, the phosphoric acid zoester bonding mode in this oligomer is (2
'-5) type/(3'-5') type-9575.
実施例１０実施例９に記載の方法に於て、反応時間７６０分から３
００分に延長した結果、重合度１０〜２０に和尚するオ
リデマーの収率が６７−に達した。Example 10 In the method described in Example 9, the reaction time was 760 minutes to 3
As a result of extending the time to 00 minutes, the yield of oridemer with a degree of polymerization of 10 to 20 reached 67.
この分割部分を１０００Ｄ単位（波長２６０ ｎｍで測
定）／ｄの０．３モル酢酸アンモニウム（ｐ）！−６）
の１１１１溶液に調整し、と九に５００単位の肺臓リン
酸ゾエステラーゼ水溶液０．１ ｄを加え、３７℃で２
時間処理した結果、テ末端が水酸基である（ ２’−５
’）ホスホゾエステル結合のみを４つ（２’−５’）オ
リデアデニルａ！を得念。その収率は第６表の通りであ
った。This divided portion is 1000 D units (measured at a wavelength of 260 nm)/d of 0.3 mole ammonium acetate (p)! -6)
1111 solution, add 0.1 d of 500 units of lung phosphate zoesterase aqueous solution, and incubate at 37°C for 2 hours.
As a result of time treatment, the Te terminal is a hydroxyl group (2'-5
') Only 4 phosphozoester bonds (2'-5') orideadenyl a! I remember that. The yield was as shown in Table 6.
第６表代理人 川 口 義 雄Table 6Agent Yoshio Kawaguchi
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14576081AJPS5849399A (en) | 1981-09-16 | 1981-09-16 | Preparation of (2'-5') oligoadenylic acid |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14576081AJPS5849399A (en) | 1981-09-16 | 1981-09-16 | Preparation of (2'-5') oligoadenylic acid |
| Publication Number | Publication Date |
|---|---|
| JPS5849399Atrue JPS5849399A (en) | 1983-03-23 |
| JPS6365078B2 JPS6365078B2 (en) | 1988-12-14 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14576081AGrantedJPS5849399A (en) | 1981-09-16 | 1981-09-16 | Preparation of (2'-5') oligoadenylic acid |
| Country | Link |
|---|---|
| JP (1) | JPS5849399A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4981957A (en)* | 1984-07-19 | 1991-01-01 | Centre National De La Recherche Scientifique | Oligonucleotides with modified phosphate and modified carbohydrate moieties at the respective chain termini |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4981957A (en)* | 1984-07-19 | 1991-01-01 | Centre National De La Recherche Scientifique | Oligonucleotides with modified phosphate and modified carbohydrate moieties at the respective chain termini |
| Publication number | Publication date |
|---|---|
| JPS6365078B2 (en) | 1988-12-14 |
| Publication | Publication Date | Title |
|---|---|---|
| JPS6250479B2 (en) | ||
| JP2705913B2 (en) | In vitro oligonucleotide synthesis | |
| US5700919A (en) | Modified phosphoramidite process for the production of modified nucleic acids | |
| JPS63179889A (en) | Manufacture of oligonuleotde | |
| WO2006022323A1 (en) | Phosphoramidite compound and method for producing oligo-rna | |
| Gildea et al. | The synthesis of 2-pyrimidinone nucleosides and their incorporation into oligodeoxynucleotides | |
| EP0438203A2 (en) | Nucleoside derivatives and their use in oligonucleotide synthesis | |
| JPH03501128A (en) | Nucleoside and polynucleotide thiophosphoramidite and phosphorodithioate compounds and methods | |
| JP2001515086A5 (en) | ||
| WO2005005450A1 (en) | Method of synthesizing cyclic bisdinucleoside | |
| JP2794461B2 (en) | Phosphoramidite compounds and solid-phase synthesis of oligoribonucleotides using the same | |
| EP0124561B1 (en) | Process for producing oligonucleoside phosphonates | |
| WO2007097447A1 (en) | Method for removal of nucleic acid-protecting group | |
| JPS5849399A (en) | Preparation of (2'-5') oligoadenylic acid | |
| Grøtli et al. | Solid-phase synthesis of branched RNA and branched DNA/RNA chimeras | |
| JPH08245685A (en) | Production of 1-(2'-deoxy-2',2'-difluoro-d- ribopentofuranosyl)cytosine from 2-deoxy-2',2'-difluoro- beta-d-ribopentopyranose | |
| EP0064796A2 (en) | Phosphorylating agent and process for the phosphorylation of organic hydroxyl compounds | |
| Clivio et al. | Synthesis of deoxydinucleoside phosphates containing 4-thio substituted pyrimidine nucleobases | |
| JPS5841896A (en) | Decyanoethylation of protected nucleotide | |
| JPH07103149B2 (en) | Method for producing cytosine derivative | |
| EP0317207B1 (en) | Production of 2,3'-anhydro-2'deoxyuridine derivatives | |
| JPH069682A (en) | Polynucleotide phosphorodithioate as remedy against retroviral infection | |
| JPS60120890A (en) | Novel phosphorylation agent | |
| JPH10218893A (en) | Oligonucleotide containing 5-fluorouracil and its production | |
| Ioannidis et al. | Synthesis of 2′, 3′-Didehydro-2′, 3′-dideoxy-2′-C-methylsubstituted Nucleosides Using a Novel SN2′ Type Reaction |