JPH0371437B2 - - Google Patents
Info
- Publication number
- JPH0371437B2 JPH0371437B2 JP19769085A JP19769085A JPH0371437B2 JP H0371437 B2 JPH0371437 B2 JP H0371437B2 JP 19769085 A JP19769085 A JP 19769085A JP 19769085 A JP19769085 A JP 19769085A JP H0371437 B2 JPH0371437 B2 JP H0371437B2
- Authority
- JP
- Japan
- Prior art keywords
- formula
- fluorescent
- integer
- general formula
- nucleotide
- 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
Links
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 239000002777 nucleoside Substances 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 11
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 7
- 238000000034 method Methods 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 4
- 125000003835 nucleoside group Chemical group 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- WEVJJMPVVFNAHZ-UHFFFAOYSA-N 4-amino-1-[4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-iodopyrimidin-2-one Chemical compound C1=C(I)C(N)=NC(=O)N1C1OC(CO)C(O)C1 WEVJJMPVVFNAHZ-UHFFFAOYSA-N 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 2
- 229940092714 benzenesulfonic acid Drugs 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000001226 triphosphate Substances 0.000 description 2
- LRPBXXZUPUBCAP-WOUKDFQISA-N (2r,3s,4r,5r)-2-(hydroxymethyl)-5-imidazo[2,1-f]purin-3-yloxolane-3,4-diol Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CN2C3=NC=C2)=C3N=C1 LRPBXXZUPUBCAP-WOUKDFQISA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- QSKPIOLLBIHNAC-UHFFFAOYSA-N 2-chloro-acetaldehyde Chemical compound ClCC=O QSKPIOLLBIHNAC-UHFFFAOYSA-N 0.000 description 1
- MVIVDSWUOGNODP-UHFFFAOYSA-N 2-iodobenzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1I MVIVDSWUOGNODP-UHFFFAOYSA-N 0.000 description 1
- 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 1
- CKTSBUTUHBMZGZ-ULQXZJNLSA-N 4-amino-1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-tritiopyrimidin-2-one Chemical compound O=C1N=C(N)C([3H])=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-ULQXZJNLSA-N 0.000 description 1
- LYYOFOQFTRXUPP-UHFFFAOYSA-N 4-nitro-2h-triazole;2,4,6-trimethylbenzenesulfonic acid Chemical compound [O-][N+](=O)C=1C=NNN=1.CC1=CC(C)=C(S(O)(=O)=O)C(C)=C1 LYYOFOQFTRXUPP-UHFFFAOYSA-N 0.000 description 1
- DUCZQUFMCSTEHH-PEBGCTIMSA-N 6-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazo[1,2-c]pyrimidin-5-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N2C=CN=C2C=C1 DUCZQUFMCSTEHH-PEBGCTIMSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 238000007259 addition reaction Methods 0.000 description 1
- 150000003838 adenosines Chemical class 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- FIMJSWFMQJGVAM-UHFFFAOYSA-N chloroform;hydrate Chemical compound O.ClC(Cl)Cl FIMJSWFMQJGVAM-UHFFFAOYSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- -1 dimethoxytrityl group Chemical group 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- WEVJJMPVVFNAHZ-RRKCRQDMSA-N ibacitabine Chemical compound C1=C(I)C(N)=NC(=O)N1[C@@H]1O[C@H](CO)[C@@H](O)C1 WEVJJMPVVFNAHZ-RRKCRQDMSA-N 0.000 description 1
- 229960000374 ibacitabine Drugs 0.000 description 1
- JBFYUZGYRGXSFL-UHFFFAOYSA-N imidazolide Chemical compound C1=C[N-]C=N1 JBFYUZGYRGXSFL-UHFFFAOYSA-N 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- MPSJHJFNKMUKCN-OUCADQQQSA-N n-[1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-oxopyrimidin-4-yl]benzamide Chemical class C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(NC(=O)C=2C=CC=CC=2)C=C1 MPSJHJFNKMUKCN-OUCADQQQSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000002718 pyrimidine nucleoside Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940048102 triphosphoric acid Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
本発明は、蛍光を発するヌクレオシド又はヌク
レオチドに関する。
生体高分子、特に蛋白質や核酸における構造と
機能の相関を明らかにするために、蛍光プローブ
を利用した研究が広く行なわれている。そして核
酸の研究において、核酸中に存在する蛍光性の微
量塩基をプローブとして用いる方法や、核酸へ蛍
光分子を化学的に導入してこれをプローブとして
用いる方法がある。蛍光性塩基を有するヌクレオ
シドの例としては、アデノシン類やシチジン類を
クロルアセトアルデヒドで化学修飾して得られ
る、下記式で示されるような蛍光性のエテノ誘導
体がある。
The present invention relates to fluorescent nucleosides or nucleotides. Research using fluorescent probes has been widely conducted to clarify the relationship between structure and function in biological macromolecules, especially proteins and nucleic acids. In research on nucleic acids, there are methods that use a small amount of fluorescent bases present in nucleic acids as probes, and methods that chemically introduce fluorescent molecules into nucleic acids and use them as probes. Examples of nucleosides having fluorescent bases include fluorescent etheno derivatives shown by the following formula, which are obtained by chemically modifying adenosines and cytidines with chloroacetaldehyde.
【式】１，N6−エテノアデノシン[Formula] 1,N 6 -ethenoadenosine
【式】３，N4−エテノシチジン
特に、エテノアデノシン類は、中性で強く蛍光
を発することから、これをプローブとして用いて
種々の研究が行なわれてきた。しかし、これらは
塩基対形成能を有しない。
本発明者らは、蛍光性を有しグアニンあるいは
アデニンとの塩基対形成が可能な、ビリミジンヌ
クレオシド又はヌクレオチド誘導体を得るべく鋭
意研究を行ない、本発明に到達した。
即ち、本発明は、
一般式（）で表わされる蛍光性ヌクレオシド
又はヌクレオチドである。
〔式（）において、X1とY1は各々
[Formula] 3,N 4 -ethenocytidine In particular, ethenoadenosine is neutral and emits strong fluorescence, so various studies have been conducted using it as a probe. However, these do not have base pairing ability. The present inventors have conducted extensive research to obtain pyrimidine nucleoside or nucleotide derivatives that have fluorescence and are capable of forming base pairs with guanine or adenine, and have arrived at the present invention. That is, the present invention is a fluorescent nucleoside or nucleotide represented by the general formula (). [In formula (), X 1 and Y 1 are each
【式】を表わす（ｎは０，１，２又は
３の整数を示す）。Z1はＨ−又は
[Formula] (n represents an integer of 0, 1, 2, or 3). Z 1 is H- or
【式】を表わす（ｍは０，１，２又は
３の整数を示す）。W1はＨ−又はHO−を表わ
す。〕
一般式（）において特に好ましいのは、X1
とY1が共にHO−でZ1がＨ−又はHO−でW1がＨ
−の化合物、あるいはX1及び／又はY1が
[Formula] (m represents an integer of 0, 1, 2, or 3). W 1 represents H- or HO-. ] In the general formula (), particularly preferred is X 1
and Y 1 are both HO- and Z 1 is H- or HO- and W 1 is H
- or X 1 and/or Y 1 are
【式】（ｎは１，２又は３の整数を示
す）でZ1がＨ−又はHO−でW1がＨ−である。
水酸基が公知の適当な保護基で保護されているも
のも、本発明の範囲に含まれる。
一般式（）の化合物の製造法を、X1とY1が
共にHO−で、Z1とW1が共にＨ−の場合を例に
とつて説明する。
一つの方法は、糖部の水酸基を保護した５−ヨ
ードデオキシシチジンにＯ−ヨードベンゾイルク
ロライドを作用させ、得られた化合物をDMF中
で、２価のPdの存在下に加熱処理し、次いで保
護基を除去する方法である。
他の方法は、デオキシシチジンから容易に得ら
れるN4−ベンゾイルデオキシシチジン誘導体を、
メタノール中でヨウ素の存在下で光照射し、次い
で常法により脱保護を行なう方法である。
上記の如くして得られた蛍光性ヌクレオシド
は、その3′位又は5′位に後述の如き方法でリン酸
基を導入しヌクレオチドとすることができる。
かくして得らた一般式（）の化合物は、蛍光
性でありかつ核酸塩基のグアニンあるいはアデニ
ンと塩基対を形成する能力があるので、これを分
子中又は分子末端に有するオリゴ又はポリヌクレ
オチドは、蛍光プローブとして利用できる。ある
いは、これらの化合物は、DNA二重らせん中に
組み入れた際に、塩基部分は空間的に適合してお
り、塩基間の水素結合形成能力やスタツキング作
用を増強する可能性も考えられる。
従つて、本発明により、分子中又は分子末端
に、一般式（）で表わされる蛍光性ヌクレオチ
ドを、少なくとも１個有するオリゴ又はポリヌク
レオチドを得ることができる。
〔式（）において、X2はIn the formula (n is an integer of 1, 2 or 3), Z 1 is H- or HO- and W 1 is H-.
Those in which the hydroxyl group is protected with a known appropriate protecting group are also included within the scope of the present invention. The method for producing the compound of general formula () will be explained by taking as an example the case where both X 1 and Y 1 are HO-, and both Z 1 and W 1 are H-. One method is to allow O-iodobenzoyl chloride to act on 5-iododeoxycytidine with the hydroxyl group of the sugar moiety protected, heat-treat the resulting compound in DMF in the presence of divalent Pd, and then protect This is a method of removing groups. Another method uses N4 -benzoyldeoxycytidine derivatives, which are easily obtained from deoxycytidine, to
This method involves irradiation with light in methanol in the presence of iodine, followed by deprotection using a conventional method. The fluorescent nucleoside obtained as described above can be converted into a nucleotide by introducing a phosphate group into the 3' or 5' position by the method described below. The compound of the general formula () thus obtained is fluorescent and has the ability to form base pairs with the nucleic acid base guanine or adenine, so oligos or polynucleotides having this in the molecule or at the end of the molecule are fluorescent. Can be used as a probe. Alternatively, when these compounds are incorporated into a DNA double helix, the base moieties are spatially compatible, and it is possible that they enhance the ability to form hydrogen bonds between bases and the stacking effect. Therefore, according to the present invention, it is possible to obtain an oligo or polynucleotide having at least one fluorescent nucleotide represented by the general formula () in the molecule or at the end of the molecule. [In formula (), X 2 is
【式】又 は【Formula】Also teeth
【式】を表わし、X2が[Formula] is represented, and X 2 is
【式】
のときY2又はZ2は−Ｏ−を表わし（但し他方は
Ｈ−又はHO−を表わす），
X2が[Formula] When Y 2 or Z 2 represents -O- (however, the other represents H- or HO-), and X 2
【式】のときY2又はZ2は−Ｏ−又 はWhen [Formula], Y 2 or Z 2 is -O- or
【式】を表わす（但し他方はＨ−又
はHO−を表わす）（ｒは０，１，２又は３の整
数を示し、ｓは１，２又は３の整数を示す）。W2
はＨ−又はHO−を表わす。〕
一般式（）において特に好ましいのは、X2
が(wherein the other represents H- or HO-) (r represents an integer of 0, 1, 2 or 3, and s represents an integer of 1, 2 or 3). W 2
represents H- or HO-. ] In the general formula (), particularly preferred is X 2
but
【式】であり、Y2が−Ｏ−でW2がＨ−
である、オリゴ又はポリヌクレオチドである。
蛍光性ヌクレオシド又はヌクレオチドをDNA
オリゴマーあるはポリマーへ導入するには、有機
化学的に合成する方法と、酵素化学的に導入する
２通りの方法がとられる。
有機化学的に合成する方法は、蛍光性ヌクレオ
シド(F)を含むオリゴヌクレオチドを直接合成する
方法である。Itakuraらが開発した、固相リン酸
トリエステル法を用いて、第１図に示すサイクル
に従つて合成を行なつた。
すなわちステツプ１として、ベンゼンスルホン
酸（BSA）で、5′−水酸基のジメトキシトリチル
基（DNTr基）を除去し、ステツプ２で、ダイマ
ーブロツクをメシチレンスルホン酸ニトロトリア
ゾール（MSNT）で縮合し、固相に担持され
Ｆ含有オリゴマーの鎖を伸長した。また、目的と
異なる配列のオリゴマー生成を防ぐために、縮合
反応において未反応の5′−水酸基は、４−ジメチ
ルアミノピリジン（DMAP）存在下無水酢酸
（Ac2Ｏ）でキヤツピングを行なつた。
このサイクルをくり返すことによつて、目的と
するオリゴマーを合成した。オリゴマヌクレオチ
ドのポリマー支持体からの切り出しとリン酸の保
護基の除去は、室温でアンモニア水で処理するこ
とにより行なつた。
更にアンモニア水で加熱処理することにより、
塩基部のアシル基を除去した。次いで逆層のシリ
カゲルカラムクロマトグラフイーで精製し、必要
なフラクシヨンを分取し、5′末端のDMTr基を除
去し、更に高速液体クロマトグラフイーで精製し
た。
蛍光性ヌクレオシド又はヌクレオチドをDNA
オリゴマーあるいはポリマーに酵素化学的に導入
する方法としては、例えば、
（） DNAポリメラーゼを用いるニツクトラ
ンスレーシヨン
（Rigby，P.W.ら，J.Mol.Biol.，113，237
（1977）参照）
（） 末端デオキシヌクレオチドトランスフエ
ラーゼを用いる3′−末端付加反応
（F.J.Bollum，The Enzymes，（P.D.
Boyer，ed.），3rd Ed.Vol.10，pp.145−171，
Academic Press，New York，N.Y.（1974）
参照）
の２つが、通常用いられる。
いずれの酵素を用いる場合にも、基質として
は、好ましくは、ヌクレオシド5′−トリリン酸が
用いられる。蛍光性ヌクレオシドの5′−モノリン
酸の合成は、Yoshikawaら（Tetrahedron
Lett.，5065（1967））の方法に従つて合成できる。
また、5′−モノリン酸をイミダゾリドとした後ジ
リン酸と反応させる、5′−トリリン酸の合成は、
Ottoらの方法（J.An.Chem.Soc.，87，1785−
1788（1965））に従つて行なうことができる。
次に合成した5′−トリリン酸を基質として用
い、DNAポリメラーゼを用いて、ニツク・トラ
ンスレーシヨンを行なうと、シチジンの代りに、
蛍光性ヌクレオチドが導入され蛍光標識されたオ
リゴマーあるいはポリマーを調整することができ
る。また、末端デオキシヌクレオチドトランスフ
エラーゼを用いると、3′−末端に蛍光性ヌクレオ
チドポリマーを付加することができる。
以下、実施例、参考例により本発明を詳述す
る。
実施例 １
(1) ５−ヨードデオキシシチジン(a)500mgを無水
ピリジン10mlに、懸濁させ、これに１，１，
３，３−テトライソプロピル−１，３−ジクロ
ルジシロキサン0.57ml（1.2当量）を氷冷下で
加え、その後室温で２時間反応させた。
氷冷下で、水２mlを加えて反応を停止し、10
分間放置した後、溶媒を減圧下留去した。残渣
を水−クロロホルム系で抽出し、クロロホルム
層を無水硫酸ナトリウムで乾燥した。クロロホ
ルムを留去した後、シリカゲルクロマトグラフ
イーにより精製し、メタノールで再結晶して、
3′，5′−０−（１，１，３，３−テトライソプ
ロプルジシロキサン−１，３−ジイル）−５−
ヨードデオキシシチジン(b)750mgを得た。物性
値は次の通りであつた。
m.p. 210−211℃
UV λメタノ哀It is an oligo or polynucleotide of the formula: where Y 2 is -O- and W 2 is H-. fluorescent nucleosides or nucleotides to DNA
There are two methods for introducing into oligomers or polymers: organic chemical synthesis and enzymatic chemical introduction. The organic chemical synthesis method is a method of directly synthesizing an oligonucleotide containing a fluorescent nucleoside (F). Synthesis was carried out using the solid-phase phosphotriester method developed by Itakura et al. according to the cycle shown in FIG. That is, in step 1, the dimethoxytrityl group (DNTr group) of the 5'-hydroxyl group is removed with benzenesulfonic acid (BSA), and in step 2, the dimer block is condensed with mesitylene sulfonate nitrotriazole (MSNT), and the solid phase is The chain of the F-containing oligomer was extended. Furthermore, in order to prevent the formation of oligomers having a different sequence than the intended one, unreacted 5'-hydroxyl groups in the condensation reaction were capped with acetic anhydride (Ac 2 O) in the presence of 4-dimethylaminopyridine (DMAP). By repeating this cycle, the desired oligomer was synthesized. Cleavage of the oligomer nucleotide from the polymer support and removal of the phosphoric acid protecting group were performed by treatment with aqueous ammonia at room temperature. Furthermore, by heat treatment with ammonia water,
The acyl group in the base part was removed. It was then purified by reverse phase silica gel column chromatography, the necessary fractions were collected, the DMTr group at the 5' end was removed, and further purified by high performance liquid chromatography. fluorescent nucleosides or nucleotides to DNA
Examples of methods for enzymatically introducing oligomers or polymers include () Nick translation using DNA polymerase (Rigby, PW et al., J. Mol. Biol., 113 , 237).
(1977)) () 3'-end addition reaction using terminal deoxynucleotide transferase (FJ Bollum, The Enzymes, (PD
Boyer, ed.), 3rd Ed.Vol.10, pp.145-171,
Academic Press, New York, NY (1974)
(see ) are commonly used. When using either enzyme, nucleoside 5'-triphosphate is preferably used as the substrate. The synthesis of the fluorescent nucleoside 5′-monophosphate was described by Yoshikawa et al.
Lett., 5065 (1967)).
In addition, 5'-triphosphoric acid can be synthesized by converting 5'-monophosphoric acid into imidazolide and then reacting it with diphosphoric acid.
Otto et al.'s method (J.An.Chem.Soc., 87 , 1785-
1788 (1965)). Next, using the synthesized 5'-triphosphate as a substrate and performing nick translation using DNA polymerase, in place of cytidine,
Fluorescently labeled oligomers or polymers into which fluorescent nucleotides have been introduced can be prepared. Furthermore, when terminal deoxynucleotide transferase is used, a fluorescent nucleotide polymer can be added to the 3'-end. Hereinafter, the present invention will be explained in detail with reference to Examples and Reference Examples. Example 1 (1) Suspend 500 mg of 5-iododeoxycytidine (a) in 10 ml of anhydrous pyridine, and add 1,1,
0.57 ml (1.2 equivalents) of 3,3-tetraisopropyl-1,3-dichlorodisiloxane was added under ice cooling, and the mixture was then reacted at room temperature for 2 hours. Under ice-cooling, add 2 ml of water to stop the reaction.
After standing for a minute, the solvent was distilled off under reduced pressure. The residue was extracted with a water-chloroform system, and the chloroform layer was dried over anhydrous sodium sulfate. After distilling off chloroform, it was purified by silica gel chromatography, recrystallized from methanol,
3',5'-0-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-5-
750 mg of iododeoxycytidine (b) was obtained. The physical property values were as follows. mp 210−211℃ UV λMethanosai
Claims (1)
ド又はヌクレオチド。 ［式（）において、X1とY1は各々
【式】を表わす（ｎは０，１，２又は ３の整数を示す）。Z1はＨ−又は
【式】を表わす（ｍは０，１，２又は ３の整数を示す）。W1はＨ−又はHO−を表わ
す。］ ２ 一般式（）において、X1とY1が共にH0−
でZ1がＨ−又はHO−でW1がＨ−である、特許
請求の範囲第１項記載の蛍光性ヌクレオシド。 ３ 一般式（）において、X1及び／又はY1が
【式】（ｎは１，２又は３の整数を示 す）でZ1がＨ−又はHO−でW1がＨ−である、
特許請求の範囲第１項記載の蛍光性ヌクレオチ
ド。[Claims] 1. A fluorescent nucleoside or nucleotide represented by the general formula (). [In formula (), X 1 and Y 1 each represent [formula] (n represents an integer of 0, 1, 2, or 3). Z 1 represents H- or [Formula] (m represents an integer of 0, 1, 2 or 3). W 1 represents H- or HO-. ] 2 In the general formula (), both X 1 and Y 1 are H0−
The fluorescent nucleoside according to claim 1, wherein Z 1 is H- or HO- and W 1 is H-. 3 In the general formula (), X 1 and/or Y 1 is [Formula] (n represents an integer of 1, 2 or 3), Z 1 is H- or HO-, and W 1 is H-,
Fluorescent nucleotide according to claim 1.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19769085A JPS6259293A (en) | 1985-09-09 | 1985-09-09 | Fluorescent nucleoside or nucleotide |
| JP3144090A JPH0798834B2 (en) | 1985-09-09 | 1991-05-21 | Fluorescent polynucleotide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19769085A JPS6259293A (en) | 1985-09-09 | 1985-09-09 | Fluorescent nucleoside or nucleotide |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3144090A Division JPH0798834B2 (en) | 1985-09-09 | 1991-05-21 | Fluorescent polynucleotide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6259293A JPS6259293A (en) | 1987-03-14 |
| JPH0371437B2 true JPH0371437B2 (en) | 1991-11-13 |
Family
ID=16378729
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP19769085A Granted JPS6259293A (en) | 1985-09-09 | 1985-09-09 | Fluorescent nucleoside or nucleotide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6259293A (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992005185A1 (en) * | 1990-09-20 | 1992-04-02 | Amoco Corporation | Probe compositions for chromosome identification and methods |
| WO1995005391A1 (en) * | 1993-08-18 | 1995-02-23 | Chromagen, Inc. | Applications of fluorescent n-nucleosides and fluorescent structural analogs of n-nucleosides |
| US5502177A (en) * | 1993-09-17 | 1996-03-26 | Gilead Sciences, Inc. | Pyrimidine derivatives for labeled binding partners |
| US6007992A (en) * | 1997-11-10 | 1999-12-28 | Gilead Sciences, Inc. | Pyrimidine derivatives for labeled binding partners |
| US6028183A (en) * | 1997-11-07 | 2000-02-22 | Gilead Sciences, Inc. | Pyrimidine derivatives and oligonucleotides containing same |
| EP1483280B1 (en) | 2002-03-08 | 2012-10-24 | Glen Research Corporation | Fluorescent nitrogenous base and nucleosides incorporating same |
| WO2004046147A1 (en) * | 2002-11-18 | 2004-06-03 | Otsuka Pharmaceutical Co., Ltd. | Polynucleotide derivative and use thereof |
-
1985
- 1985-09-09 JP JP19769085A patent/JPS6259293A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6259293A (en) | 1987-03-14 |
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