JPH0557996B2 - - Google Patents
Info
- Publication number
- JPH0557996B2 JPH0557996B2 JP60044412A JP4441285A JPH0557996B2 JP H0557996 B2 JPH0557996 B2 JP H0557996B2 JP 60044412 A JP60044412 A JP 60044412A JP 4441285 A JP4441285 A JP 4441285A JP H0557996 B2 JPH0557996 B2 JP H0557996B2
- Authority
- JP
- Japan
- Prior art keywords
- monomer
- group
- pyridine
- oligodeoxyribonucleotides
- producing
- 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.)
- Expired - Lifetime
Links
- 238000000034 method Methods 0.000 claims description 14
- 229940124276 oligodeoxyribonucleotide Drugs 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 125000005843 halogen group Chemical group 0.000 claims description 2
- NSPMIYGKQJPBQR-UHFFFAOYSA-N 4H-1,2,4-triazole Chemical compound C=1N=CNN=1 NSPMIYGKQJPBQR-UHFFFAOYSA-N 0.000 claims 1
- 239000000178 monomer Substances 0.000 description 14
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 12
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- HMSCPNFZRAIJIP-BFHYXJOUSA-N 3-benzoyl-1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1N(C(=O)C=2C=CC=CC=2)C(=O)C(C)=CN1[C@H]1C[C@H](O)[C@@H](CO)O1 HMSCPNFZRAIJIP-BFHYXJOUSA-N 0.000 description 7
- 125000006239 protecting group Chemical group 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 239000000539 dimer Substances 0.000 description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 6
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000006482 condensation reaction Methods 0.000 description 5
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- -1 dimethoxytrityl group Chemical group 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 3
- 238000009833 condensation Methods 0.000 description 3
- 230000005494 condensation Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 125000003356 phenylsulfanyl group Chemical group [*]SC1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 3
- 150000008300 phosphoramidites Chemical class 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- FCQCLMZIGUFTQR-UHFFFAOYSA-N 3-benzoyl-5-methyl-1h-pyrimidine-2,4-dione Chemical group O=C1C(C)=CNC(=O)N1C(=O)C1=CC=CC=C1 FCQCLMZIGUFTQR-UHFFFAOYSA-N 0.000 description 2
- XVMSFILGAMDHEY-UHFFFAOYSA-N 6-(4-aminophenyl)sulfonylpyridin-3-amine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=N1 XVMSFILGAMDHEY-UHFFFAOYSA-N 0.000 description 2
- AYGTXPWQTMVKIQ-XUVXKRRUSA-N [(2r,3s,5r)-5-(3-benzoyl-5-methyl-2,4-dioxopyrimidin-1-yl)-2-(hydroxymethyl)oxolan-3-yl] benzoate Chemical compound O([C@H]1C[C@@H](O[C@@H]1CO)N1C=C(C(N(C(=O)C=2C=CC=CC=2)C1=O)=O)C)C(=O)C1=CC=CC=C1 AYGTXPWQTMVKIQ-XUVXKRRUSA-N 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- PASDCCFISLVPSO-UHFFFAOYSA-N benzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1 PASDCCFISLVPSO-UHFFFAOYSA-N 0.000 description 2
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 2
- DCFKHNIGBAHNSS-UHFFFAOYSA-N chloro(triethyl)silane Chemical compound CC[Si](Cl)(CC)CC DCFKHNIGBAHNSS-UHFFFAOYSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- GYOZYWVXFNDGLU-XLPZGREQSA-N dTMP Chemical class O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 GYOZYWVXFNDGLU-XLPZGREQSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000921 elemental analysis Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000013638 trimer Substances 0.000 description 2
- OAUJBKXCQJYOTO-UHFFFAOYSA-N 1,3,5-tribromo-2-[chloro-(2,4,6-tribromophenoxy)phosphoryl]oxybenzene Chemical compound BrC=1C=C(Br)C=C(Br)C=1OP(=O)(Cl)OC1=C(Br)C=C(Br)C=C1Br OAUJBKXCQJYOTO-UHFFFAOYSA-N 0.000 description 1
- DXKZQNUSKZOLTC-UHFFFAOYSA-N 1,3,5-trichloro-2-[chloro-(2,4,6-trichlorophenoxy)phosphoryl]oxybenzene Chemical compound ClC1=CC(Cl)=CC(Cl)=C1OP(Cl)(=O)OC1=C(Cl)C=C(Cl)C=C1Cl DXKZQNUSKZOLTC-UHFFFAOYSA-N 0.000 description 1
- LOSXTWDYAWERDB-UHFFFAOYSA-N 1-[chloro(diphenyl)methyl]-2,3-dimethoxybenzene Chemical group COC1=CC=CC(C(Cl)(C=2C=CC=CC=2)C=2C=CC=CC=2)=C1OC LOSXTWDYAWERDB-UHFFFAOYSA-N 0.000 description 1
- QIUQZAQSDRSKSZ-UHFFFAOYSA-N 1-benzoyl-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C(=O)C1=CC=CC=C1 QIUQZAQSDRSKSZ-UHFFFAOYSA-N 0.000 description 1
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical compound CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- NIPKRLLKGZROTE-UHFFFAOYSA-N 4-methoxy-n-(2-oxo-1h-pyrimidin-6-yl)benzamide Chemical compound C1=CC(OC)=CC=C1C(=O)NC1=NC(=O)NC=C1 NIPKRLLKGZROTE-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- YTQGHFCRGPJUNX-UHFFFAOYSA-N n,n-diethylethanamine;pyridine;hydrate Chemical compound O.C1=CC=NC=C1.CCN(CC)CC YTQGHFCRGPJUNX-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- ACVYVLVWPXVTIT-UHFFFAOYSA-N phosphinic acid Chemical compound O[PH2]=O ACVYVLVWPXVTIT-UHFFFAOYSA-N 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- OENLEHTYJXMVBG-UHFFFAOYSA-N pyridine;hydrate Chemical compound [OH-].C1=CC=[NH+]C=C1 OENLEHTYJXMVBG-UHFFFAOYSA-N 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- UMSOKRFUOIKLNN-UHFFFAOYSA-M sodium;pyridine;hydroxide Chemical compound [OH-].[Na+].C1=CC=NC=C1 UMSOKRFUOIKLNN-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 238000005866 tritylation reaction Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Saccharide Compounds (AREA)
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明はホスホトリエステル法によるオリゴデ
オキシリボヌクレオチドの製造法に関するもの
で、更に詳しくは縮合剤として一般式〔〕
（式中、Ｘはハロゲン原子を表わす）
で示されるビス（２，４，６−トリハロフエニ
ル）ホスホロクロリデート（以下、THPと称す
る）および３−ニトロ−１，２，４−トリアゾー
ル（以下、NTと称する）を用いてインターヌク
レオチド結合を形成させることからなるオリゴデ
オキシリボヌクレオチドの製造法に関する。本発
明により得られるオリゴデオキシリボヌクレオチ
ドは遺伝子工学における重要な素材として有用な
化合物である。
（従来の技術）
現在、オリゴデオキシリボヌクレオチドの製造
法としては、有機化学的方法と酵素的方法の二つ
の方法が開発されており、有機化学的方法として
はホスホトリエステル法のほか、ホスホロアミダ
イト法〔テトラヘドロンレターズ（Tetrahedron
Letters），22，1859（1981）および24，245
（1983）〕が知られている。ホスホロアミダイト法
は、インターヌクレオチド結合が迅速にかつ定量
的に得られる利点を有する反面、ホスホロアミダ
イト試薬が不安定であり、また各縮合反応の後に
酸化工程を必要とするなどの欠点を有している。
（発明が解決しようとする問題点）
ホスホトリエステル法によるオリゴデオキシリ
ボヌクレオチドの製造法は、直接ホスホトリエス
テル結合が得られる実際的な方法であり広く利用
されているが、ホスホロアミダイト法に比して縮
合速度が遅いという欠点を有する。この縮合速度
を促進させる手段としては、触媒としてテトラゾ
ールを用いる方法〔カナデイアン ジヤーナル
オブ ケミストリー（Canadian Journal of
Chemistry），54，670（1976）、ヌクレイツク ア
シツド リサーチ（Nucleic Acids Research），
８，5445（1980）〕やＮ−メチルイミダゾールを用
いる方法〔テトラヘドロン レターズ
（Tetrahedron Letters），23，961（1982）、ケミ
カル エンド フアーマシユーテイカル ブリテ
ン（Chemical and Pharmaceutical Bulletin），
30，3951（1982）〕などが知られ、また縮合温度を
高温にすることで縮合反応を促進する方法〔ヌク
レイツク アシツド リサーチ（Nucleic Acids
Research），10，5605（1982）〕も知られている
が、縮合剤についてはあまり検討されていないの
が現状である。
（問題点を解決するための手段）
本発明者はホスホトリエステル法によつてオリ
ゴデオキシリボヌクレオチドを製造する際のイン
ターヌクレオチド結合形成の迅速化の手段として
縮合剤に着目し、種々検討を加えた結果、THP
がすぐれた効果を有することを見い出し、本発明
を完成したものである。
本発明は一般式〔〕
（式中、Ｂは保護基を有する塩基残基を、
DMTrはジメトキシトリチル基を、SPhはフエニ
ルチオ基を表わす）
で示されるホスホジエステルのトリエチルアンモ
ニウム塩（以下、単量体（）と称する）と一般
式〔〕
【式】または【式】
（式中、Bzはベンゾイル基を、Ｂ，DMTrお
よびSPhは前記と同一の意味を表わす）
で示されるヒドロキシ成分（以下、単量体（）
と称する）とを、ピリジン溶媒中でTHP例えば
ビス（２，４，６−トリクロロフエニル）ホスホ
ロクロリデート（以下、TCPと称する）または
ビス（２，４，６−トリブロモフエニル）ホスホ
ロクロリデート（以下、TBPと称する）の存在
下に反応させる。なお、この際NTを併用するこ
とにより縮合反応はすみやかに進行し、一般式
〔〕
【式】または
【式】
（式中、Ｂ，DMTr，SPhおよびBzは前記と
同一の意味を表わす）
で示される２量体が85％以上の高収率で得られ
る。本発明の縮合反応は通常は室温で進行し、５
〜20分で完了するが、塩基残基の種類などにより
多少変動するのでこれらに限定されるものではな
い。反応終了後は通常の後処理操作により単離精
製することができる。更にまた、より長鎖のオリ
ゴデオキシリボヌクレオチドを得る場合は、一般
式〔〕で示される２量体を5Mホスフイン酸
（ピリジン溶液）−トリエチルアミン（２：１，
Ｖ／Ｖ）で処理することにより3′−位リン酸から
１個のフエニルチオ基を選択的に脱離させて２量
体のトリエチルアンモニウム塩とし、これを
THPおよびNTの存在下に単量体（）と反応
させれば３量体が得られ、あるいは一般式〔〕
で示される２量体を２％トリフルオロ酢酸／クロ
ロホルム中で処理して5′−位水酸基の保護基であ
るジメトキシトリチル基を脱離させたのち、これ
にTHPおよびNTの存在下に単量体（）を反
応させれば３量体が得られるので、これらの操作
を繰り返すことにより、目的とするオリゴデオキ
シリボヌクレオチドを得ることができる。
本発明における単量体（）や単量体（）の
塩基残基中のアミノ基やイミノ基は保護基により
保護することが必要であり、通常はアシル基が保
護基として用いられる。これらの保護基（アシル
基）は酸性（80％酢酸および２％トリフルオロ酢
酸／クロロホルム）および中性条件下では安定で
あるが、室温でトリエチルアミン−水−ピリジン
（２：１：２，Ｖ／Ｖ／Ｖ）で処理すると徐徐に
脱離し、室温で１時間濃アンモニア水で処理する
と完全に脱離する。
以下、実施例および参考例により説明する。
（実施例および参考例）
参考例
塩基残基がN3−ベンゾイルチミジンである単
量体（）および単量体
ピリジン中でジイソプロピルエチルアミンの存
在下に、チミジン5mmolとトリエチルシリル
クロリド13mmolとを30分間処理したのち、塩化
ベンゾイル7.5mmolを加え１時間撹拌する。終了
後ピリジンを完全に除去し、残渣に５％トリフル
オロ酢酸／ジクロロメタン−メタノール（７：
３，Ｖ／Ｖ）50mlを加え20分間処理する。通常の
精製処理ののちシリカゲルカラムクロマトグラフ
イによりN3−ベンゾイルチミジンを87％の収
率で得た。
N3−ベンゾイルチミジン
1H−NMR（CDCl3−CD3OD）
δppm：7.88−7.31
（6H，ｍ，ArH＆６−Ｈ）
6.22（1H，ｔ，Ｊ＝6Hz，1′−Ｈ）
4.33（1H，ｍ，3′−Ｈ）
3.91（1H，ｍ，4′−Ｈ）
3.78（2H，ｍ，5′−Ｈ）
2.32（2H，ｍ，2′−Ｈ）
1.93（3H，ｓ，５−CH3）
13C−NMR（CDCl3−CD3OD）
δppm：169.15（Ｃ＝０）
163.24（４−Ｃ）
149.54（２−Ｃ）
136.81（６−Ｃ）
135.29（４−Ｃ（Bz））
131.44（１−Ｃ（Bz））
130.46（２，６−Ｃ（Bz））
129.28（３，５−Ｃ（Bz））
110.91（５−Ｃ）
87.34（4′−Ｃ）
85.82（1′−Ｃ）
70.81（3′−Ｃ）
61.77（5′−Ｃ）
40.42（2′−Ｃ）
12.46（５−CH3）
IRP（KBr）
γcm-1：1749（PhC＝０）
1700および1653
（イミドＣ＝０）
元素分析値（C17H18O6N2として）
Ｃ Ｈ Ｎ
計算値（％） 58.96 5.24 8.09
実測値（％） 58.47 5.40 7.93
次にN3−ベンゾイルチミジンの5′−位水酸
基をジメトキシトリチルクロリドを用いてトリチ
ル化したのち、ホスホリル化反応を行い、N3−
ベンゾイルチミジンユニツトが収率85％（対
）で得られた。なお、予め調製したチミジンユ
ニツトをピリジン中でジイソプロピルエチルア
ミンの存在下に塩化ベンゾイルと15時間反応する
ことにより収率91％でを得ることができる。
N3−ベンゾイルチミジンユニツト
1H−NMR（CDCl3）
δppm：7.22−7.96（25H，ｍ，ArH）
6.84（4H，ｄ，Ｊ＝8Hz，
４，５−Ｈ（DMTr））
6.36（1H，ｔ，Ｊ＝6Hz，1′−Ｈ）
5.36（1H，ｍ，3′−Ｈ）
4.10（1H，ｍ，4′−Ｈ）
3.78（3H，ｓ，CH3O（DMTr））
3.41（2H，ｍ，5′−Ｈ）
2.42（2H，ｍ，2′−Ｈ）
1.39（3H，ｓ，５−CH3）
元素分析値（C50H45O9N2S2Pとして）
Ｃ Ｈ Ｎ Ｓ
計算値（％） 65.78 4.97 3.07 7.02
実測値（％） 66.03 5.20 3.00 7.13
更に、N3−ベンゾイルチミジンユニツトを
5Mホスホフイン酸（ピリジン溶液）−トリエチル
アミン（２：１，Ｖ／Ｖ）により40℃で15分間処
理することにより、から１個のフエニルチオ基
を選択的に脱離させた塩基残基がN3−ベンゾイ
ルチミンである単量体（）が収率88％で得ら
れる。またを２％トリフルオロ酢酸／クロロホ
ルム中で０℃、５分間処理することにより5′−位
水酸基の保護基であるジメトキシトリチル基が脱
離し、塩基残基がN3−ベンゾイルチミンである
単量体（）が収率86％で得られる。
実施例 １
参考例により得られた単量体（）
0.24mmol単量体（）の3′−位水酸基をベン
ゾイル基で保護した3′−Ｏ，N3−ジベンゾイル
チミジン0.2mmolにNT0.26mmolを加え、無水
ピリジン中で脱水処理を行い無水としたのちピリ
ジン２mlに溶解する。これにTBP0.26mmolを加
え、室温で５分間反応後少量の水を加えて反応を
停止させる。反応終了後通常の方法により後処理
を行い、保護基により十分に保護されたチミジレ
ート２量体を収率95％で得た。チミジレート２
量体からの脱保護操作は、まず０℃で20分間
0.2M水酸化ナトリウム−ピリジン（１：１，
Ｖ／Ｖ）で処理したのち、ダウエツクス50W−
Xs（PyH+型）により中和し、室温で１時間濃ア
ンモニウム水で処理する。次いで室温で１時間ピ
リジン−水（２：１，Ｖ／Ｖ）中でヨウ素30当量
で処理したのち、ベンゼンで抽出して過剰のヨウ
素を除去し、更に室温で15分間80％酢酸で処理
後、DEAE−セフアデツクスＡ−25で精製するこ
とにより、純粋なチミジレート２量体である
TpTが103O.P.（260nm）が得られた（対２量体
14mg）。
実施例 ２〜６
単量体（）の塩基残基が実施例１と同じであ
るN3−ベンゾイルチミンかまたはN4−アニソ
イルシトシンをいずれも1.2当量用い、単量体
（）として実施例１と同じ3′−Ｏ，N3−ジベン
ゾイルチミジンかまたは参考例で得られた単量
体を用い、第１表記載の縮合剤の存在下に縮合
反応を行つて対応する２量体を第１表記載の収率
で得た。
【表】
（発明の効果）
ホスホトリエステル法によるオリゴデオキシリ
ボヌクレオチドを製造する際に、縮合剤として
THPおよびNTを用いることにより、短時間で
インターヌクレオチド結合が完結し、反応後の後
処理も容易であり、高収率でオリゴデオキシリボ
ヌクレオチドを製造することができるという効果
がある。 DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing oligodeoxyribonucleotides by a phosphotriester method. (wherein, X represents a halogen atom) The present invention relates to a method for producing oligodeoxyribonucleotides, which comprises forming an internucleotide bond using NT (referred to as NT). The oligodeoxyribonucleotide obtained by the present invention is a compound useful as an important material in genetic engineering. (Prior art) Currently, two methods have been developed for producing oligodeoxyribonucleotides: an organic chemical method and an enzymatic method. Law (Tetrahedron Letters)
Letters), 22 , 1859 (1981) and 24 , 245
(1983)] is known. Although the phosphoramidite method has the advantage of rapidly and quantitatively obtaining internucleotide bonds, it has disadvantages such as the instability of the phosphoramidite reagent and the need for an oxidation step after each condensation reaction. are doing. (Problems to be Solved by the Invention) The method for producing oligodeoxyribonucleotides by the phosphotriester method is a practical method that can directly obtain a phosphotriester bond and is widely used, but it is not comparable to the phosphoramidite method. It has the disadvantage that the condensation rate is slow. One way to accelerate this condensation rate is to use tetrazole as a catalyst [Canadian Journal
of Chemistry (Canadian Journal of
Chemistry, 54 , 670 (1976), Nucleic Acids Research,
8, 5445 (1980)] and the method using N-methylimidazole [Tetrahedron Letters, 23 , 961 (1982), Chemical and Pharmaceutical Bulletin,
30, 3951 (1982)], and a method of accelerating the condensation reaction by increasing the condensation temperature [Nucleic Acids Research (Nucleic Acids Research)].
Research), 10 , 5605 (1982)], but the condensing agent has not been studied much at present. (Means for Solving the Problems) The present inventors focused on condensing agents as a means of speeding up the formation of internucleotide bonds when producing oligodeoxyribonucleotides by the phosphotriester method, and conducted various studies. As a result, THP
The present invention has been completed based on the discovery that this has excellent effects. The present invention is based on the general formula [] (In the formula, B represents a base residue having a protecting group,
DMTr represents a dimethoxytrityl group, SPh represents a phenylthio group) A triethylammonium salt of a phosphodiester (hereinafter referred to as monomer ()) represented by the general formula [] [Formula] or [Formula] (in the formula, Bz represents a benzoyl group, and B, DMTr and SPh represent the same meanings as above.
THP such as bis(2,4,6-trichlorophenyl)phosphorochloridate (hereinafter referred to as TCP) or bis(2,4,6-tribromophenyl)phosphorochloridate (hereinafter referred to as TCP) in a pyridine solvent. The reaction is carried out in the presence of rochloridate (hereinafter referred to as TBP). At this time, by using NT in combination, the condensation reaction proceeds rapidly, and the general formula [] [Formula] or [Formula] (wherein, B, DMTr, SPh and Bz represent the same meanings as above) The dimer shown is obtained in a high yield of over 85%. The condensation reaction of the present invention usually proceeds at room temperature, and
The process is completed in ~20 minutes, but is not limited to this as it may vary depending on the type of base residue. After the reaction is completed, it can be isolated and purified by normal post-treatment operations. Furthermore, when obtaining a longer chain oligodeoxyribonucleotide, the dimer represented by the general formula [] is mixed with 5M phosphinic acid (pyridine solution)-triethylamine (2:1
V/V) to selectively remove one phenylthio group from the 3'-position phosphoric acid to form a dimeric triethylammonium salt, which is then
If reacted with monomer () in the presence of THP and NT, a trimer is obtained, or the general formula []
The dimer represented by was treated in 2% trifluoroacetic acid/chloroform to remove the dimethoxytrityl group, which is a protective group for the hydroxyl group at the 5'-position, and then the monomer was added to the dimer in the presence of THP and NT. Since a trimer can be obtained by reacting the compound (), the desired oligodeoxyribonucleotide can be obtained by repeating these operations. In the present invention, it is necessary to protect the amino group or imino group in the monomer () or the basic residue of the monomer () with a protecting group, and an acyl group is usually used as the protecting group. These protecting groups (acyl groups) are stable under acidic (80% acetic acid and 2% trifluoroacetic acid/chloroform) and neutral conditions, but at room temperature triethylamine-water-pyridine (2:1:2, V/ When treated with V/V), it is gradually desorbed, and when treated with concentrated aqueous ammonia for 1 hour at room temperature, it is completely desorbed. This will be explained below using Examples and Reference Examples. (Examples and Reference Examples) Reference Example Monomer () and monomer in which the base residue is N 3 -benzoylthymidine 5 mmol of thymidine and 13 mmol of triethylsilyl chloride were mixed in pyridine in the presence of diisopropylethylamine for 30 minutes. After the treatment, 7.5 mmol of benzoyl chloride was added and stirred for 1 hour. After completion, pyridine was completely removed and the residue was mixed with 5% trifluoroacetic acid/dichloromethane-methanol (7:
3. Add 50ml of V/V) and process for 20 minutes. After conventional purification treatment, N 3 -benzoylthymidine was obtained in 87% yield by silica gel column chromatography. N 3 -benzoylthymidine 1 H-NMR (CDCl 3 - CD 3 OD) δppm: 7.88-7.31 (6H, m, ArH & 6-H) 6.22 (1H, t, J = 6Hz, 1'-H) 4.33 (1H, m, 3'-H) 3.91 (1H, m, 4'-H) 3.78 (2H, m, 5'-H) 2.32 (2H, m, 2'-H) 1.93 (3H, s, 5-CH 3 ) 13 C-NMR (CDCl 3 - CD 3 OD) δppm: 169.15 (C = 0) 163.24 (4-C) 149.54 (2-C) 136.81 (6-C) 135.29 (4-C (Bz)) 131.44 ( 1-C (Bz)) 130.46 (2,6-C (Bz)) 129.28 (3,5-C (Bz)) 110.91 (5-C) 87.34 (4'-C) 85.82 (1'-C) 70.81 (3'-C) 61.77 (5'-C) 40.42 (2'-C) 12.46 (5-CH 3 ) IRP (KBr) γcm -1 : 1749 (PhC=0) 1700 and 1653 (imide C=0) Elemental analysis value (as C 17 H 18 O 6 N 2 ) C H N Calculated value (%) 58.96 5.24 8.09 Actual value (%) 58.47 5.40 7.93 Next, the 5′-position hydroxyl group of N 3 -benzoylthymidine was replaced with dimethoxytrityl chloride. After tritylation using N 3 −
Benzoylthymidine units were obtained in a yield of 85% (vs.). It can be obtained in a yield of 91% by reacting a previously prepared thymidine unit with benzoyl chloride in pyridine in the presence of diisopropylethylamine for 15 hours. N 3 -benzoylthymidine unit 1 H-NMR (CDCl 3 ) δppm: 7.22-7.96 (25H, m, ArH) 6.84 (4H, d, J = 8Hz, 4,5-H (DMTr)) 6.36 (1H, t , J=6Hz, 1'-H) 5.36 (1H, m, 3'-H) 4.10 (1H, m, 4'-H) 3.78 (3H, s, CH 3 O (DMTr)) 3.41 (2H, m , 5'-H) 2.42 (2H, m, 2'-H) 1.39 (3H, s, 5-CH 3 ) Elemental analysis value (as C 50 H 45 O 9 N 2 S 2 P) C H N S Calculation Value (%) 65.78 4.97 3.07 7.02 Actual value (%) 66.03 5.20 3.00 7.13 Furthermore, N 3 -benzoylthymidine unit
By treating with 5M phosphoric acid (pyridine solution)-triethylamine (2:1, V/V) at 40°C for 15 minutes, the base residue from which one phenylthio group was selectively removed was converted into N 3 - Monomer (), which is benzoylthymine, is obtained with a yield of 88%. In addition, by treating it in 2% trifluoroacetic acid/chloroform at 0°C for 5 minutes, the dimethoxytrityl group, which is a protective group for the 5'-position hydroxyl group, is removed, and the monomer whose base residue is N 3 -benzoylthymine is removed. () is obtained in 86% yield. Example 1 Monomer obtained from Reference Example ()
0.26 mmol of NT was added to 0.2 mmol of 3'-O,N 3 -dibenzoylthymidine, in which the 3'-position hydroxyl group of the monomer () was protected with a benzoyl group, and the mixture was dehydrated in anhydrous pyridine to make it anhydrous. Then dissolve in 2 ml of pyridine. Add 0.26 mmol of TBP to this, react for 5 minutes at room temperature, and then add a small amount of water to stop the reaction. After completion of the reaction, post-treatment was carried out by a conventional method to obtain a thymidylate dimer sufficiently protected with a protecting group in a yield of 95%. Chimidylate 2
The deprotection operation from the polymer was first carried out at 0°C for 20 minutes.
0.2M sodium hydroxide-pyridine (1:1,
After processing with V/V), dowex 50W-
Neutralize with Xs (PyH + form) and treat with concentrated ammonium water for 1 hour at room temperature. It was then treated with 30 equivalents of iodine in pyridine-water (2:1, V/V) for 1 hour at room temperature, followed by extraction with benzene to remove excess iodine, and further treated with 80% acetic acid for 15 minutes at room temperature. , pure thymidylate dimer by purification with DEAE-Sephadex A-25
TpT of 103O.P. (260nm) was obtained (versus dimer
14mg). Examples 2 to 6 Examples using 1.2 equivalents of either N 3 -benzoylthymine or N 4 -anisoylcytosine, in which the base residue of the monomer () is the same as in Example 1, were used as the monomer (). Using the same 3′-O,N 3 -dibenzoylthymidine as in 1 or the monomer obtained in Reference Example, a condensation reaction was carried out in the presence of the condensing agent listed in Table 1 to obtain the corresponding dimer. It was obtained with the yield shown in Table 1. [Table] (Effects of the invention) When producing oligodeoxyribonucleotides by the phosphotriester method, it is used as a condensing agent.
By using THP and NT, the internucleotide bond is completed in a short time, post-reaction processing is easy, and oligodeoxyribonucleotides can be produced in high yield.
Claims (1)
シリボヌクレオチドを製造する際に、縮合剤とし
て一般式〔〕 （式中、Ｘはハロゲン原子を表わす） で示されるビス（２，４，６−トリハロフエニ
ル）ホスホロクロリデートおよび３−ニトロ−
１，２，４−トリアゾールを用いてインターヌク
レオチド結合を形成させることを特徴とするオリ
ゴデオキシリボヌクレオチドの製造法。[Claims] 1. When producing oligodeoxyribonucleotides by the phosphotriester method, the general formula [] is used as a condensing agent. (wherein, X represents a halogen atom) Bis(2,4,6-trihalophenyl)phosphorochloridate and 3-nitro-
A method for producing oligodeoxyribonucleotides, which comprises forming an internucleotide bond using 1,2,4-triazole.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60044412A JPS61204194A (en) | 1985-03-05 | 1985-03-05 | Production of oligodeoxyribonucleotide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60044412A JPS61204194A (en) | 1985-03-05 | 1985-03-05 | Production of oligodeoxyribonucleotide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61204194A JPS61204194A (en) | 1986-09-10 |
| JPH0557996B2 true JPH0557996B2 (en) | 1993-08-25 |
Family
ID=12690790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60044412A Granted JPS61204194A (en) | 1985-03-05 | 1985-03-05 | Production of oligodeoxyribonucleotide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS61204194A (en) |
-
1985
- 1985-03-05 JP JP60044412A patent/JPS61204194A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61204194A (en) | 1986-09-10 |
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