JPS60120890A - Novel phosphorylation agent - Google Patents
Novel phosphorylation agentInfo
- Publication number
- JPS60120890A JPS60120890A JP22839583A JP22839583A JPS60120890A JP S60120890 A JPS60120890 A JP S60120890A JP 22839583 A JP22839583 A JP 22839583A JP 22839583 A JP22839583 A JP 22839583A JP S60120890 A JPS60120890 A JP S60120890A
- Authority
- JP
- Japan
- Prior art keywords
- nucleoside
- formula
- compound
- reacting
- product
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
【発明の詳細な説明】
本発明はヌクレオシド9の新規なホスホリル化剤に関す
るものであり、さらに詳しくは、一般式[1]で示され
る化合物及びその用途に関するものである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel phosphorylating agent for nucleoside 9, and more particularly to a compound represented by the general formula [1] and uses thereof.
〔CF3(CH2)no〕2POH〔l〕(式中nは1
〜3の整数である)
ヌクレオシドのホスホリル化剤は遺伝子工学上重装な出
発原料の一つであり現在までに種々のホスホリル化剤が
開発されて来たが、満足されるものは未だ得られていな
い。ヌクレオシド9のホスホリル化剤としては例えば4
−クロロフェニルホスホロジトリアゾール[R,W、A
damialc et al、 NucleicAci
dRess、3.3397(1976)]、ホスホロモ
ノテトラゾール[HoTakaku at aL Ch
em Lett192(1982)Eなどが公知である
が、これらのホスホリル化剤はグアノシンやウリジンの
塩基部へのホスホリル化が副反応として起こる。さらr
c縮合剤を用いたホスホリル化反応でtマ、縮合剤1と
してのアリールスルホニルがグアノシンやウリジンの塩
基部又は水酸基に導入される副反応が起こる(Pj(、
Br1dson et al、 J、O,S Obem
、 Ooram。[CF3(CH2)no]2POH[l] (in the formula, n is 1
Nucleoside phosphorylating agents are one of the most important starting materials for genetic engineering, and various phosphorylating agents have been developed up to now, but no satisfactory one has yet been obtained. Not yet. Examples of phosphorylating agents for nucleoside 9 include 4
-chlorophenylphosphoroditriazole [R, W, A
Damialc et al, NucleicAci
dRess, 3.3397 (1976)], phosphoromonotetrazole [HoTakaku at aL Ch
Lett 192 (1982) E and the like are known, but these phosphorylating agents cause phosphorylation of the base moiety of guanosine or uridine as a side reaction. Sarar
In the phosphorylation reaction using condensing agent c, a side reaction occurs in which arylsulfonyl as condensing agent 1 is introduced into the base moiety or hydroxyl group of guanosine or uridine (Pj (,
Br1dson et al., J.O.S.
, Ooram.
447.791(1977))。この様に現在用いられ
ている方法では何らかの副反応が起こっているので得ら
れる目的生成物の純度が悪(収率も低い欠点がある。そ
こで我々はヌクレオシド9のホスホリル化の際に副反応
の起こらないホスホリル化剤を開発するために1鋭意研
究を重ねた結果、ある踵の新規なジアルキルホスファイ
トがこの目的に適合することを見い出しに0このホスホ
リル化剤はトリアゾール、テトラゾールおよび縮合剤な
どの活性基を用(・ることかないので、副反応の生起を
心配する必猥がなく、しかも高収率でヌクレオシドのホ
スホリル化を達成することの出来る新規化合物である。447.791 (1977)). As described above, in the currently used method, some side reactions occur, resulting in poor purity of the target product (low yield). After extensive research to develop a phosphorylating agent that does not cause phosphorylation, it was discovered that a novel dialkyl phosphite is suitable for this purpose. Since it does not use active groups, there is no need to worry about side reactions, and it is a new compound that can achieve phosphorylation of nucleosides in high yields.
本発明のホスボリル化剤は一般式%式%(1
で表わされるジアルキルホスファイト誘導体である。こ
の一般式におけるnは1〜3の整数であり、好ましくは
n=1である。The phosphorylating agent of the present invention is a dialkyl phosphite derivative represented by the general formula % (1). In this general formula, n is an integer from 1 to 3, preferably n=1.
本発明の化合物はいずれも文献未載の新規化合物であり
次式に従って製造される:
(OF 3 (CH2)nO〕2 POH〔1〕
(式中、nは1〜3の整数である)
すなわち溶媒中でt−ブタノールと三塩化リンを反応さ
せ、得られた化合物l0と任意のアルコールを反応させ
ろと高収率で本発明の化合物〔l〕が得られる。この様
にして得られたジアルキルホスファイトを用いてヌクレ
オシドへのホスホリル基の尋人を試みると、下記の反応
に従い好ましい結果が得られる:
VII
式中、Aft保両基、例えばモノメトキシトリチル基、
ジメトキシトリチル基を示し、Bは塩基であり、 f/
i+気はベンゾイルグアニン、アシルアデニン、イソジ
チルグアニン、インジイルアデニン、アシルアデニン、
アニソイルシトシン、(ンソイルシトシン、チミン、ウ
ラシルなどである。すなわち5′−水酸基を保護したヌ
クレオシド〔バ〕と 化合物[1〕を溶媒例えばピリジ
ン中で反応させると、目的とするヌクレオシド3′−ホ
スファイト[V]が得られる。これに酸化剤を加え処理
すると、対応するヌクレオノビ3′−ボスフエー)[、
V1]が商収率で得られる。この様にして得られるヌク
レオシド13′−ホスフェートを用いてオリゴマーの合
成を行うとジヌクレオシドが高収率で得られる。The compounds of the present invention are all novel compounds that have not been described in literature and are produced according to the following formula: (OF3(CH2)nO]2POH[1] (wherein, n is an integer from 1 to 3) The compound [1] of the present invention can be obtained in high yield by reacting t-butanol and phosphorus trichloride in a solvent and reacting the obtained compound 10 with any alcohol.The dialkyl compound thus obtained Attempting to attach a phosphoryl group to a nucleoside using a phosphite gives favorable results according to the following reaction: VII where the Aft conjugate group, such as the monomethoxytrityl group,
represents a dimethoxytrityl group, B is a base, f/
i + Qi is benzoylguanine, acyladenine, isoditylguanine, indiyladenine, acyladenine,
Anisoylcytosine, (nsoylcytosine, thymine, uracil, etc.) When the 5'-hydroxyl protected nucleoside [B] and compound [1] are reacted in a solvent such as pyridine, the desired nucleoside 3'- Phosphite [V] is obtained. When treated with an oxidizing agent, the corresponding nucleonobi-3'-bosphae) [,
V1] is obtained at a commercial yield. When oligomers are synthesized using the nucleoside 13'-phosphate thus obtained, dinucleosides can be obtained in high yield.
以上述べた打K、従来の方法と異なり活性基を用(・ず
にホスホリル化のできる本発明による新規なホスホリル
化試薬は従来技術において他にその例を見ないものであ
杓、副反応も起こらずヌクレオシドを選択的にホスホリ
ル化でき、かつ高収率で目的物の得られることは、核酸
の化学、煩薬の分野に重要な技術的進歩を与えるもので
ある。Unlike conventional methods, the novel phosphorylation reagent of the present invention, which can perform phosphorylation without using an active group, is unique in the prior art and does not cause side reactions. The fact that nucleosides can be selectively phosphorylated without phosphorylation and that the desired product can be obtained in high yield represents an important technological advance in the fields of nucleic acid chemistry and laxative medicine.
次に本発明による新規化合物の代表的な合成法および使
用法を実施例により詳述する。Next, representative methods of synthesizing and using the novel compounds of the present invention will be explained in detail with reference to Examples.
実施例1
ビス(2,2,2−トリフルオロエチル)ホスファイト
の合成
t−ツタ/−ル(10,6me、 0.125M )を
CH20e2に溶解し、これ[0℃K テCHzGe
2 (25ml ) K溶解したPGlh (10,9
rrLl、 0.125 M )を滴下する。滴下終了
後30分間反応させたのち、2.2.2−トリフルオロ
エタノール(2,5g、0.25M)をCH2Ce2
(25’ml) に溶解したものを徐々に加える。反応
系へ窒素ガスを吹込みながら約16時間室温で]が押し
たのち減圧蒸留すると、目的とするビス(2,2,2−
)リフルオロエチル)ホスファイト(b、p、 66〜
69℃/8mTlH,?)が2.8I得られる。Example 1 Synthesis of bis(2,2,2-trifluoroethyl)phosphite T-tutar (10,6me, 0.125M) was dissolved in CH20e2,
2 (25 ml) K-dissolved PGlh (10,9
rrLl, 0.125 M). After the reaction was completed for 30 minutes, 2.2.2-trifluoroethanol (2.5 g, 0.25 M) was added to CH2Ce2.
(25'ml) and gradually add the solution. The desired bis(2,2,2-
) fluoroethyl) phosphite (b, p, 66~
69℃/8mTlH,? ) is obtained as 2.8I.
元素分析結果;C4H5F603P
実側値 計算値
C; 1946%、19.52%
H; 1.97%、 2.05%
工R; νC1n”−’、2485(P−H)、125
0(p=o )、1150(CF3)、
1080(CF3)、960(P−H)。Elemental analysis results; C4H5F603P Actual value Calculated value C; 1946%, 19.52% H; 1.97%, 2.05% Engineering R; νC1n''-', 2485 (P-H), 125
0 (p=o), 1150 (CF3), 1080 (CF3), 960 (P-H).
実施例2
5’−o−モノメトキシトリチルチミジン3’−()リ
フルオロエチル)ホスフェ−トノ合成
5’−o−モノメトキシトリ チルチミジン(MMTr
T) (0,515,9,1,0mM) をピリジン(
7ml)f溶解し、ビス(2,2,2−トリフルオロエ
テル)ホスファイト(0,46m1.3 mM) を加
え、室床ガス雰囲気下、50℃にて3時間反応させたの
ち、反応液を室温に冷却し、さらに0℃まで冷却し、m
−クロロ過安息香酸(1,04g、5mM)を加えて1
0分間処理すると生成物は酸化されホスフェートとなる
。反応液を氷水(5mJ)に加先塩化メチレン(10m
Aりで抽出したのち、有機層を5%NaHCO3(5m
lx 3 )、さらに水(smJx2)で洗浄する。塩
化メチレンを減圧除去し、さらに少量のトルエンを加え
ピリジン臭がなくなるまで減圧を(り返す。残渣を少量
の塩化メチレンに溶解し、檄しく攪拌しているヘキサン
−エーテル(10:1)中へ加えると白色の沈殿が生じ
、これを戸数すると、5’−o−モノメトキシトリチル
チミジン3’−()リフルオロエテル)ホスフェ−)
(m−1)、−r148−150℃)が0.69g得ら
れる。得られた生成物の物性値は次の通りである。Example 2 Synthesis of 5'-o-monomethoxytritylthymidine 3'-()rifluoroethyl)phosphate 5'-o-monomethoxytritylthymidine (MMTr
T) (0,515,9,1,0mM) in pyridine (
7 ml) f was dissolved, bis(2,2,2-trifluoroether) phosphite (0.46ml 1.3 mM) was added, and the mixture was reacted for 3 hours at 50°C under a room floor gas atmosphere. was cooled to room temperature, further cooled to 0°C, m
- Add chloroperbenzoic acid (1.04g, 5mM) to 1
When treated for 0 minutes, the product is oxidized to phosphate. The reaction solution was added to ice water (5 mJ) and diluted with methylene chloride (10 mJ).
After extraction with A, the organic layer was extracted with 5% NaHCO
lx 3 ) and further washed with water (smJx2). Remove methylene chloride under reduced pressure, add a small amount of toluene, and repeat under reduced pressure until the pyridine odor disappears. Dissolve the residue in a small amount of methylene chloride and pour into hexane-ether (10:1) with vigorous stirring. When added, a white precipitate is formed, which is converted into 5'-o-monomethoxytritylthymidine (3'-()rifluoroether)phosphate).
(m-1), -r148-150°C) is obtained. The physical properties of the obtained product are as follows.
Rf = 0.01 (c)(2ce2 :CH30H
= 9 : 1 ) pRf−0,97((CH3)2
00:H20=7:3);、 MeOH
UV、λ =265.230nm;
11aX
MeOH
λ −247,226nm 。Rf = 0.01 (c) (2ce2:CH30H
= 9:1) pRf-0,97((CH3)2
00:H20=7:3);, MeOH UV, λ = 265.230 nm; 11aX MeOH λ -247,226 nm.
1n
実施例3
ジヌクレオシドの合成
5’−o−モノメトキシトリチルチミジン3′−(トリ
フルオロエチル)ホスフェ−)(0,6,9゜0.8m
M) と3′−〇−尽ノンシイルナミジン 0.17
&、 0.53mM) をピリジン(2,7ml)KM
Hし、8−キノリンスルホニル−3−二トロー 1.2
.4− )リアシーzu(0,83,9,2mM)を加
え室温で2時間反応させた後、通常の操作方法により処
理し、シリカゲルカラムクロマトグラフに充填し、溶離
液としてCH2Ce2−MeOH(98: 2 )を用
(・て#離すると目的とする化合物が溶出される。1n Example 3 Synthesis of dinucleoside 5'-o-monomethoxytritylthymidine 3'-(trifluoroethyl)phosphate) (0,6,9°0.8m
M) and 3'-〇-nonsilamidine 0.17
&, 0.53mM) in pyridine (2.7ml) KM
H, 8-quinolinesulfonyl-3-nitro 1.2
.. 4-) After adding Liashizu (0,83,9,2mM) and reacting at room temperature for 2 hours, it was processed according to the usual operation method, packed into a silica gel column chromatograph, and CH2Ce2-MeOH (98: 2) When released using (・#), the target compound is eluted.
溶出液を濃縮し、残渣をCH2Ce2に溶解し、激しく
攪拌しているヘキサン−エーテル(io:i)中へ滴下
すると白色の沈殿が生じる。この沈殿な戸数すると対応
するジヌクレオシド[鵬TrTp−(OCH2GF3)
TOBz ]が0.4’l得られる◎この生成物の物性
は次の通りである。The eluate is concentrated and the residue is dissolved in CH2Ce2 and added dropwise into vigorously stirring hexane-ether (io:i) resulting in a white precipitate. The number of precipitates corresponds to the dinucleoside [Peng TrTp-(OCH2GF3)
0.4'l of TOBz] is obtained. The physical properties of this product are as follows.
Rf=0.48 (CHzC6z:MeOH= 9 :
1 ) seOH−
λ −246nm。Rf=0.48 (CHzC6z:MeOH=9:
1) seOH-λ-246nm.
1lln
これらの生成物の化学構造は保護基を完全に脱保護した
のち、酵素分解し、その化学構造を確認した。なお上記
実施例2に準じる具体例を表1に侠約して示す:
表−1The chemical structures of these products were confirmed by complete deprotection of the protective groups and enzymatic decomposition. In addition, specific examples according to the above Example 2 are shown in Table 1: Table-1
Claims (1)
クレオシドのホスホリル化剤。[Claims] 1. A dialkyl phosphite brocade conductor represented by the general formula (1) % formula % (1 (in the formula, n is an integer from 1 to 3). 2. General formula (1) % formula % (wherein n is an integer from 1 to 3).
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP22839583A JPS60120890A (en) | 1983-12-05 | 1983-12-05 | Novel phosphorylation agent |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP22839583A JPS60120890A (en) | 1983-12-05 | 1983-12-05 | Novel phosphorylation agent |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS60120890A true JPS60120890A (en) | 1985-06-28 |
JPH0372235B2 JPH0372235B2 (en) | 1991-11-18 |
Family
ID=16875793
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP22839583A Granted JPS60120890A (en) | 1983-12-05 | 1983-12-05 | Novel phosphorylation agent |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS60120890A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0291088A (en) * | 1988-09-29 | 1990-03-30 | Central Glass Co Ltd | Phosphite and nucleoside-3'-phosphite derivative and synthesis of oligonucleotide using the same |
JPH0594874U (en) * | 1992-05-15 | 1993-12-24 | 桜井株式会社 | Label with copy |
-
1983
- 1983-12-05 JP JP22839583A patent/JPS60120890A/en active Granted
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0291088A (en) * | 1988-09-29 | 1990-03-30 | Central Glass Co Ltd | Phosphite and nucleoside-3'-phosphite derivative and synthesis of oligonucleotide using the same |
JPH0594874U (en) * | 1992-05-15 | 1993-12-24 | 桜井株式会社 | Label with copy |
Also Published As
Publication number | Publication date |
---|---|
JPH0372235B2 (en) | 1991-11-18 |
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Legal Events
Date | Code | Title | Description |
---|---|---|---|
LAPS | Cancellation because of no payment of annual fees |