JPH0376594A - Production of dideoxynucleotide derivative - Google Patents
Production of dideoxynucleotide derivativeInfo
- Publication number
- JPH0376594A JPH0376594A JP21270689A JP21270689A JPH0376594A JP H0376594 A JPH0376594 A JP H0376594A JP 21270689 A JP21270689 A JP 21270689A JP 21270689 A JP21270689 A JP 21270689A JP H0376594 A JPH0376594 A JP H0376594A
- Authority
- JP
- Japan
- Prior art keywords
- fluoropurine
- dideoxynucleoside
- dideoxyriboside
- manufacturing
- dideoxyribose
- 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.)
- Pending
Links
- 238000004519 manufacturing process Methods 0.000 title claims description 22
- 239000005546 dideoxynucleotide Substances 0.000 title 1
- LGQVOKWMIRXXDM-UHFFFAOYSA-N 6-fluoro-7h-purine Chemical compound FC1=NC=NC2=C1NC=N2 LGQVOKWMIRXXDM-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 17
- 239000010452 phosphate Substances 0.000 claims abstract description 17
- 241000588722 Escherichia Species 0.000 claims abstract description 6
- 241000588923 Citrobacter Species 0.000 claims abstract description 5
- 241000186216 Corynebacterium Species 0.000 claims abstract description 5
- 241000588914 Enterobacter Species 0.000 claims abstract description 5
- 241000588698 Erwinia Species 0.000 claims abstract description 5
- 241000589565 Flavobacterium Species 0.000 claims abstract description 5
- 241000588731 Hafnia Species 0.000 claims abstract description 5
- 241000607142 Salmonella Species 0.000 claims abstract description 5
- 241000607720 Serratia Species 0.000 claims abstract description 5
- 241000589634 Xanthomonas Species 0.000 claims abstract description 5
- CJNBYAVZURUTKZ-UHFFFAOYSA-N hafnium(IV) oxide Inorganic materials O=[Hf]=O CJNBYAVZURUTKZ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000012736 aqueous medium Substances 0.000 claims abstract 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 30
- 244000005700 microbiome Species 0.000 claims description 20
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 17
- 150000003839 salts Chemical class 0.000 claims description 13
- 229960002656 didanosine Drugs 0.000 claims description 3
- LFRDGHVRPSURMV-YFKPBYRVSA-N (4s)-4,5-dihydroxypentanal Chemical compound OC[C@@H](O)CCC=O LFRDGHVRPSURMV-YFKPBYRVSA-N 0.000 claims description 2
- 239000003125 aqueous solvent Substances 0.000 claims 2
- 208000030507 AIDS Diseases 0.000 abstract description 5
- 239000003443 antiviral agent Substances 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 abstract 2
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 18
- 238000000034 method Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 11
- 239000000047 product Substances 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- WVXRAFOPTSTNLL-NKWVEPMBSA-N 2',3'-dideoxyadenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1CC[C@@H](CO)O1 WVXRAFOPTSTNLL-NKWVEPMBSA-N 0.000 description 3
- BTOTXLJHDSNXMW-POYBYMJQSA-N 2,3-dideoxyuridine Chemical compound O1[C@H](CO)CC[C@@H]1N1C(=O)NC(=O)C=C1 BTOTXLJHDSNXMW-POYBYMJQSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- OCLZPNCLRLDXJC-NTSWFWBYSA-N 2-amino-9-[(2r,5s)-5-(hydroxymethyl)oxolan-2-yl]-3h-purin-6-one Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1CC[C@@H](CO)O1 OCLZPNCLRLDXJC-NTSWFWBYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- BXZVVICBKDXVGW-NKWVEPMBSA-N Didanosine Chemical compound O1[C@H](CO)CC[C@@H]1N1C(NC=NC2=O)=C2N=C1 BXZVVICBKDXVGW-NKWVEPMBSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 229910001410 inorganic ion Inorganic materials 0.000 description 2
- 239000011785 micronutrient Substances 0.000 description 2
- 235000013369 micronutrients Nutrition 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 125000001477 organic nitrogen group Chemical group 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- XKKCQTLDIPIRQD-JGVFFNPUSA-N 1-[(2r,5s)-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)CC1 XKKCQTLDIPIRQD-JGVFFNPUSA-N 0.000 description 1
- -1 6-fluoropurine riboside Chemical class 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 239000012465 retentate Substances 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical class CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
６−フルオロプリン−２′，３’−ジデオキシリボシド
は強力な抗ウィルス作用を有している為、医薬、特に最
近世界的に問題となっているエイズ（ＡＩＤＳ）の治療
薬としての利用が期待されている。Detailed Description of the Invention (Field of Industrial Application) 6-Fluoropurine-2',3'-dideoxyriboside has a strong antiviral effect, so it has become a problem in medicine, especially in recent years worldwide. It is expected that it will be used as a treatment for AIDS (AIDS).
本発明は微生物を利用した６−フルオロプリン−２′，
３’−ジデオキシリボシドの製造方法に関するものであ
る。The present invention provides 6-fluoropurine-2' using microorganisms,
The present invention relates to a method for producing 3'-dideoxyriboside.
（従来の技術）
６−フルオロプリンリボシドの製造方法はＪ、Ｏｒｇ、
Ｃｈｅｍ、 、 ３４．１３９６ （１９６９）により
公知となっているが、収率が極めて低く工業的に満足す
る方法ではない。(Prior art) A method for producing 6-fluoropurine riboside is described by J. Org.
Chem, 34.1396 (1969), but the yield is extremely low and the method is not industrially satisfactory.
一方微生物を利用した２′，３’−ジデオキシヌクレオ
シドの製造方法は、Ａｐｐｌ、Ｅｎｖｉｒｏｎ、Ｍｉｃ
ｒｏｂｉｏｌ、。On the other hand, methods for producing 2',3'-dideoxynucleosides using microorganisms are published by Appl, Environ, and Mic.
robiol,.
ｎ、 ４１９（１９８９）で公知となっている。しかし
、６−フルオロプリン−２′，３’−ジデオキシリボシ
ドの製造方法は化学合成法及び微生物を利用する方法と
もに全（知られていない。n, 419 (1989). However, all methods for producing 6-fluoropurine-2',3'-dideoxyriboside, both chemical synthesis methods and methods using microorganisms, are unknown.
（発明が解決しようとする課題）
高収率で効率よく６−フルオロプリン−２′３′−ジデ
オキシリボシドを製造する方法が望まれてｌハゐ。(Problems to be Solved by the Invention) There is a need for a method for efficiently producing 6-fluoropurine-2'3'-dideoxyriboside with high yield.
（課題を解決するための手段）
本発明は上記課題を達成するべく鋭意検討の結果、化学
合成法により安定、安価に供給される２Ｔ、３Ｊ−ジデ
オキシヌクレオシド又は２．３−ジデオキシリボース−
１−リン酸を基質に用いて、微生物を作用させるこεに
点り、６−フルオロプリン−２′，３’−ジデオキシリ
ボシドを効率よく生産できることを見い出し、この知見
に基づいて本発明を完成するに至った。(Means for Solving the Problems) The present invention has been made as a result of intensive studies to achieve the above-mentioned problems. 2T, 3J-dideoxynucleoside or 2,3-dideoxyribose-
We have discovered that 6-fluoropurine-2',3'-dideoxyriboside can be efficiently produced by using 1-phosphoric acid as a substrate and allowing microorganisms to act on it, and based on this knowledge, we have developed the present invention. It was completed.
本発明を簡単に記すと以下の通りである。The present invention will be briefly described as follows.
核酸塩基
６−フルオロプリン
６−フルオロブリン
シド
２’１３”’−−ジデオキシリボ
即ち本発明は
■ ２．３−ジデオキシリボース−１−リン酸と６−フ
ルオロプリンに微生物を作用させて６−フルオロプリン
−２′，３’−ジデオキシリボシドを製造する方法、及
び
■２′，３’−ジデオキシヌクレオシドと無機リン酸又
はその塩と６−フルオロプリンに微生物を作用させて直
接６−フルオロプリン−２′，３’ジデオキシリボシド
を製造する方法に関する。Nucleic acid base 6-fluoropurine 6-fluoropurinside 2'13"'--dideoxyribo, that is, the present invention is: A method for producing purine-2',3'-dideoxyriboside, and (2) directly producing 6-fluoropurine by allowing microorganisms to act on 2',3'-dideoxynucleoside, inorganic phosphoric acid or its salt, and 6-fluoropurine. The present invention relates to a method for producing 2',3' dideoxyriboside.
もちろん、本発明には、２′，３’−ジデオキシヌクレ
オシドと無機リン酸又はその塩に微生物を作用させて、
目的とする６−フルオロプリン−２′，３’−ジデオキ
シリボシドの中間体である２、３−ジデオキシリボース
−１−リン酸を生成せしめる方法、も含まれる。Of course, the present invention includes the use of microorganisms acting on 2',3'-dideoxynucleoside and inorganic phosphoric acid or its salt.
Also included is a method of producing 2,3-dideoxyribose-1-phosphate, which is an intermediate of the target 6-fluoropurine-2',3'-dideoxyriboside.
本発明に使用される微生物は２′，３’−ジデオキシヌ
クレオシドと無機リン酸又はその塩を２．３−ジデオキ
シリボース−ニーリン酸に変換する酵素、並びに２，３
−ジデオキシリボース−１−リン酸と６−フルオロプリ
ンを６−フルオロプリン−２′，３’−ジデオキシリボ
シドに変換する酵素の両方の活性を有しているので、こ
れらの方法には同一の微生物を用いることができる。The microorganism used in the present invention contains an enzyme that converts 2',3'-dideoxynucleoside and inorganic phosphoric acid or its salt into 2,3-dideoxyribose-neelinic acid, and 2,3'-dideoxynucleoside.
-Dideoxyribose-1-phosphate and an enzyme that converts 6-fluoropurine to 6-fluoropurine-2',3'-dideoxyriboside, so these methods have the same activity. Microorganisms can be used.
これらの方法に使用される微生物は、エシェリヒア属、
フラボバクテリウム属、セラチア属、エンテロバクタ−
属、エルビニア属、シトロバクタ−属、コリネバクテリ
ウム属、ハフニア属、クルイヘラ属、サルモネラ属、及
びキサントモナス属のいずれかに属する。このような微
生物として、例えば、
を挙げることができる。The microorganisms used in these methods include Escherichia spp.
Flavobacterium, Serratia, Enterobacter
It belongs to any of the genera Erwinia, Citrobacter, Corynebacterium, Hafnia, Kluichella, Salmonella, and Xanthomonas. Examples of such microorganisms include:
これらの微生物を用いて目的とする６−フルオロプリン
−２′，３’−ジデオキシリボシド、又はその中間体で
ある２、３−ジデオキシリボース−１−リン酸を生成せ
しめる方法は、微生物の培養中に基質を添加する培養法
を用いても良いし、また培養した菌体あるいはこの処理
物を基質に作用させる酵素法を用いても良い。The method of producing the target 6-fluoropurine-2',3'-dideoxyriboside or its intermediate 2,3-dideoxyribose-1-phosphate using these microorganisms involves culturing the microorganism. A culture method may be used in which a substrate is added therein, or an enzymatic method may be used in which cultured microbial cells or a processed product thereof is allowed to act on a substrate.
培養法を用いる場合には、炭素源、窒素源、Ｐ、Ｓ ｓ
Ｆｅｓ Ｍｎ等の無機イオン、更に必要ならばビタミ
ン等の微量栄養素又は蛋白分解物、酵母エキスのような
有機窒素源を含有する通常の培地を基本とする。例えば
、中間体である２、３−ジデオキシリボース−■−リン
酸を生成する場合には、この基本培地に２′，３’−ジ
デオキシヌクレオシドと無機リン酸又はその塩を適宜添
加すればよい。When using the culture method, carbon source, nitrogen source, P, S s
It is based on an ordinary medium containing inorganic ions such as FesMn, and if necessary, micronutrients such as vitamins or protein decomposition products, and an organic nitrogen source such as yeast extract. For example, when producing the intermediate 2,3-dideoxyribose-■-phosphate, 2',3'-dideoxynucleoside and inorganic phosphoric acid or a salt thereof may be appropriately added to the basic medium.
そして、２，３−ジデオキシリボース−１−リン酸から
目的とする６−フルオロプリン−２′，３’−ジデオキ
シリボシドを生産するに際し、上記基本培地に２，３−
ジデオキシリボース−１−リン酸と６−フルオロプリン
を適宜添加すればよい。When producing the desired 6-fluoropurine-2',3'-dideoxyriboside from 2,3-dideoxyribose-1-phosphate, 2,3-
Dideoxyribose-1-phosphate and 6-fluoropurine may be added as appropriate.
更に、２′，３’−ジデオキシヌクレオシドから６−フ
ルオロプリン−２′，３’−ジデオキシリボシドを直接
生産する場合には、上記基本培地に２′，３’−ジデオ
キシヌクレオシドと無機リン酸又はその塩と６−フルオ
ロプリンを添加した培地を用いて培養すればよい。Furthermore, when producing 6-fluoropurine-2',3'-dideoxyriboside directly from 2',3'-dideoxynucleoside, 2',3'-dideoxynucleoside and inorganic phosphoric acid or Culture may be performed using a medium to which the salt and 6-fluoroprine are added.
上記基質の添加は培養初期でも培養途中でも構わない。The above-mentioned substrate may be added at the beginning of the culture or during the culture.
酵素法を用いる場合の酵素源としては、炭素源、窒素源
、Ｐ％ Ｓｓ Ｆｅ、、Ｍｎ等の無機イオン、更に必要
ならばビタもン等の微量栄養素又は蛋白分解物、酵母エ
キスのような有機窒素源を含有する通常の培地で培養し
た培養液、洗浄菌体が使用できる他に、菌体処理物も使
用できる。菌体処理物としては、アセトン乾燥菌体、菌
体の磨砕物、菌体の超音波処理物、界面活性剤あるいは
トルエン等の処理菌体、リゾチーム等の酵素処理菌体、
菌体より抽出した後に塩析等により分離した菌体の蛋白
画分、本反応の酵素活性を有する蛋白画分の精製物、更
に本菌体および菌体処理物の固定化物等いずれもが使用
できる。Enzyme sources when using the enzyme method include carbon sources, nitrogen sources, inorganic ions such as P% Ss Fe, and Mn, and if necessary, micronutrients such as vitamins, protein decomposition products, and yeast extract. In addition to a culture solution cultured in a normal medium containing an organic nitrogen source and washed bacterial cells, a treated bacterial cell product can also be used. Examples of bacterial cell-treated products include acetone-dried bacterial cells, ground bacterial cells, sonicated bacterial cells, bacterial cells treated with surfactants or toluene, etc., bacterial cells treated with enzymes such as lysozyme, etc.
Protein fractions of bacterial cells separated by salting out after extraction from bacterial cells, purified protein fractions with enzyme activity from this reaction, and immobilized products of bacterial cells and processed bacterial cells are all used. can.
本発明において基質となる前述の２′，３’−ジデオキ
シヌクレオシドは、特に限定されるものではなく、２′
，３’−ジデオキシウリジン、２′３′−ジデオキシア
デノシン、２′，３’−ジデオキシイノシン、２′，３
’−ジデオキシグアノシン、２’１３′−ジデオキシチ
ミジン等、又はこれらの混合物が用いられる。また、同
様に基質さなる２゜３−ジデオキシリボース−１−リン
酸は、市販製品を用いてもよく、また、本発明で開示し
たように、２′，３’−ジデオキシヌクレオシＦから微
生物の作用により生産後、採取されたものを用いてもよ
い。また６−フルオロプリンについては一般的塩基であ
る已ポキザンチンから既知の化学合成法（Ｊ、Ａｍ、Ｃ
ｈｅｍ、Ｓｏｅ、、 出、６０７３（１９５４）、Ｊ、
Ｃｈｅ＋ｍ、５ｏｃ１（Ｃ） 、 ３９４２ （１９７
１）により容易に合成するここができる。The above-mentioned 2',3'-dideoxynucleoside that serves as a substrate in the present invention is not particularly limited, and the 2',3'-dideoxynucleoside is not particularly limited.
, 3'-dideoxyuridine, 2'3'-dideoxyadenosine, 2',3'-dideoxyinosine, 2',3
'-dideoxyguanosine, 2'13'-dideoxythymidine, etc., or a mixture thereof is used. Similarly, 2<3-dideoxyribose-1-phosphate, which is a substrate, may be a commercially available product, and as disclosed in the present invention, 2',3'-dideoxynucleoside F may be used as a substrate for microorganisms. You may use the one collected after production due to the action of. Furthermore, 6-fluoropurine can be synthesized using known chemical synthesis methods (J, Am, C
hem, Soe, 6073 (1954), J.
Che+m, 5oc1(C), 3942 (197
1) allows easy synthesis.
２′，３’−ジデオキシヌクレオシドあるいは２゜３−
・ジデオキシリボース−■・−リン酸の濃度は１１００
０ｍＭ程度が適当である。更に、無機リン酸又はその塩
の濃度は中間体の２，３−ジデオキシリボース−１−リ
ン酸生成の場合には２’＋３゛−ジデオキシヌクレオシ
ドと等モルあるいは等モル以上加えるのがよ（，１−１
０倍モル程度、更に６フルオロプリンー２′，３’−ジ
デオキシリボシド生成の場合には反め系でリン酸がリサ
イクルできるので２，３−ジデオ牛シリボースー１−リ
ン酸生戒の場合より少量でよく、０．０１−１０倍モル
程度が適当であゐ。無機リン酸の塩は反応の進行を大き
く阻書しないものであればいずれを用いても良く、例え
ばナトリウム塩、カリウム塩、アンモニウム塩、カルシ
ウム塩、マグネシウム塩等の無機塩、さらにはトリメチ
ルアンモニウム塩等の有機塩が用いられる。2',3'-dideoxynucleoside or 2゜3-
・Dideoxyribose-■・-The concentration of phosphoric acid is 1100
Approximately 0mM is appropriate. Furthermore, in the case of producing the intermediate 2,3-dideoxyribose-1-phosphate, the concentration of inorganic phosphoric acid or its salt should be equal to or more than the same mole as that of 2'+3'-dideoxynucleoside (, 1-1
0 times the mole, and in the case of 6-fluoropurine-2',3'-dideoxyriboside production, phosphoric acid can be recycled in the recycle system, so it is more effective than the case of 2,3-dideo-cow silibose-1-phosphate. A small amount is sufficient, and approximately 0.01 to 10 times the mole is appropriate. Any salt of inorganic phosphoric acid may be used as long as it does not significantly hinder the progress of the reaction, such as inorganic salts such as sodium salt, potassium salt, ammonium salt, calcium salt, magnesium salt, and even trimethylammonium salt. Organic salts such as
また、２ ’ 、３ ’−ジデオキシヌクレオシドから
６−フルオロプリン−２′，３’−ジデオキシリボシド
を直接生産する場合、２．３−ジデオキシリポ−４−１
−ＩＪン酸カら目的とする６−フルオロフ。In addition, when 6-fluoropurine-2',3'-dideoxyriboside is directly produced from 2',3'-dideoxynucleoside, 2,3-dideoxylipo-4-1
- 6-fluorophore from IJ acid.
リン−２′，３’−ジデオキシリボシドを生産する場合
のいずれにおいても、６−フルオロプリンの添加量は２
′，３’−ジデオキシヌクレオシド又は２．３−ジデオ
キシリボース−１−リン酸と等モルあるいはそれ以上が
適当であり、通常１−１０倍モル程度が適当である。In all cases of producing phosphorus-2',3'-dideoxyriboside, the amount of 6-fluoropurine added is 2
Suitably, the amount is equivalent to or more than the molar amount of ',3'-dideoxynucleoside or 2,3-dideoxyribose-1-phosphate, and usually about 1 to 10 times the molar amount.
直接生産させる場合において、基質である２′３′−ジ
デオキシヌクレオシドが反応液に残ってもよい場合には
、６−フルオロプリンの添加量ば２′，３’−ジデオキ
シヌクレオシドの等モル以下でも良い。In the case of direct production, if the substrate 2'3'-dideoxynucleoside may remain in the reaction solution, the amount of 6-fluoropurine added may be equal to or less than the equivalent mole of 2',3'-dideoxynucleoside. .
さて、これらを含む水溶液に前記菌体、又はその処理物
を加え、ｐＨを４−１０の範囲に調製した後、２０−７
０℃、望ましくば４０−７０ ”Ｃで静置あるいは攬は
んしながら１０分−１ｏ日間保持すると反烏が進行し、
反め液中に目的とする６−フルオロプリン−２′，３’
−ジデオキシリボシド又は中間体の２．３−ジデオキシ
リボース−１−リン酸が著量蓄積される。Now, the above-mentioned bacterial cells or their treated products were added to an aqueous solution containing these, and after adjusting the pH to a range of 4-10, the pH was adjusted to 20-7.
If it is kept at 0℃, preferably 40-70''C for 10 minutes to 10 days while standing or being shaken, anti-corrosion progresses.
The target 6-fluoropurine-2',3' is in the retentate solution.
- Significant accumulation of dideoxyriboside or the intermediate 2,3-dideoxyribose-1-phosphate.
反烏液より６−フルオロプリン−２′，３’−ジデオキ
シリボシド又は中間体の２．３−ジデオキシリボース−
１−リン酸を採取する方法は、水等の溶媒に対する溶解
度差を利用したり、イオン変換樹脂や吸着樹脂を用いる
方法で行うことができる。6-fluoropurine-2',3'-dideoxyriboside or the intermediate 2,3-dideoxyribose-
1-Phosphoric acid can be collected by utilizing a difference in solubility in a solvent such as water, or by using an ion conversion resin or an adsorption resin.
またこれら生成物の定量は高速液体クロマトグラフィー
を用いる方法で行えばよい。In addition, these products may be quantified by a method using high performance liquid chromatography.
以下、実施例に基づいて、本発明を更に詳細に説明する
。Hereinafter, the present invention will be explained in more detail based on Examples.
（実施例）
実施例１
酵母エキス０．５ ｇ ／ ｄｉ、ペプトン１．０ｇ、
／７、肉エキス１．０ｇ／ｄｉおよびＮａＣ１４０゜５
ｇ／ｄ１を含む培地（ｐＨ７，０） ５０−を５００−
容肩付フラスコに分注し殺菌した。この培地に、ブイヨ
ン寒天培地にて３０℃、１６時間前培養した第１表に示
す微生物を１白金耳ずつ接種し、３０℃にて１６時時間
上う培養した。得られた培養液より菌体を遠心分離によ
り分離した後、０．０５ Ｍリン酸バッファー＜ｐ）Ｊ
７．０）で洗浄し、更に遠心分離することにより洗浄菌
体を調製した。(Example) Example 1 Yeast extract 0.5 g/di, peptone 1.0 g,
/7, meat extract 1.0g/di and NaC140°5
Medium containing g/d1 (pH 7,0) 50- to 500-
The mixture was dispensed into shoulder flasks and sterilized. This medium was inoculated with loopfuls of the microorganisms listed in Table 1 that had been precultured on a bouillon agar medium at 30°C for 16 hours, and cultured at 30°C for 16 hours. After separating the bacterial cells from the obtained culture solution by centrifugation, 0.05 M phosphate buffer <p)J
7.0) and further centrifugation to prepare washed bacterial cells.
上記洗浄菌体を、２（ｌａＭの２．３−ジデオキシリボ
ース−１−リン酸と２０ｍＭ６−フルオロプリンを含む
０．０５Ｍ）リスバッファー（ｐＨ７，２） １０−
に５％になるように添加し、５０℃、２４時間反応させ
た。The washed bacterial cells were washed with 2 (0.05M containing laM 2.3-dideoxyribose-1-phosphate and 20mM 6-fluoropurine) lithium buffer (pH 7.2) 10-
was added to the solution at a concentration of 5% and reacted at 50°C for 24 hours.
反応液中に生成した６−フルオロプリン−２′３′−ジ
デオキシリボシドの濃度を高速クロマトグラフィーを用
いて測定し、その結果を第１表に示した。The concentration of 6-fluoropurine-2'3'-dideoxyriboside produced in the reaction solution was measured using high performance chromatography, and the results are shown in Table 1.
実施例２
実施例１と同様の方法により培養及び調製した第２表に
示す微生物の洗浄菌体を、２０−の２′３′−ジデオキ
シウリジン、２′，３’−ジデオキシアデノシン、２′
、３′−ジデオキシイノシン、２′，３’−ジデオキシ
グアノシン、又は２′，３’−ジデオキシチミジンのそ
れぞれと２０ｍＭの６−フルオロプリンを含む１００＋
ｗＭのリン酸バッファー （ｐＨ７，０） １０ ｔ
ａｌに５％になるように添加し、５０℃、２４時間反応
させた。各反応液中に生成した６−フルオロプリン−２
’ 、３ ’−ジデオキシリボシドを実施例１と同様の
方法で測定し、その結果を第２表に示した。Example 2 Washed bacterial cells of the microorganisms shown in Table 2, cultured and prepared in the same manner as in Example 1, were treated with 20-2'3'-dideoxyuridine, 2',3'-dideoxyadenosine, 2'
, 3'-dideoxyinosine, 2',3'-dideoxyguanosine, or 2',3'-dideoxythymidine and 20 mM 6-fluoropurine.
wM phosphate buffer (pH 7,0) 10 t
It was added to Al at a concentration of 5% and reacted at 50°C for 24 hours. 6-fluoropurine-2 produced in each reaction solution
',3'-dideoxyriboside was measured in the same manner as in Example 1, and the results are shown in Table 2.
実施例３
実施例１と同様の方法により培養及び調製した第３表に
示す微生物の洗浄菌体を、各々２０ｄの２′，３’−ジ
デオキシウリジン、２′，３’−ジデオキシアデノシン
、２′，３’−ジデオキシイノシン ２ｒ、３ｔ−ジデ
オキシグアノシン、又は２′３′−ジデオキシチミジン
を含む１００ｎ＋Ｈのリン酸バフファー（ｐＨ７゜０）
１０ｍ／に５％になるように添加し、５０℃、２４時間
反応させた。各反応液中に生成した２、３−ジデオキシ
リボース−１−リン酸の濃度を高速液体クロマトグラフ
ィーを用いて測定し、その結果を第３表に示した。Example 3 Washed bacterial cells of the microorganisms shown in Table 3, cultured and prepared in the same manner as in Example 1, were incubated with 20d of 2',3'-dideoxyuridine, 2',3'-dideoxyadenosine, and 2', respectively. , 3'-dideoxyinosine, 2r, 3t-dideoxyguanosine, or 100n+H phosphate buffer containing 2'3'-dideoxythymidine (pH 7°0)
It was added to 10 m/cm at a concentration of 5% and reacted at 50°C for 24 hours. The concentration of 2,3-dideoxyribose-1-phosphoric acid produced in each reaction solution was measured using high performance liquid chromatography, and the results are shown in Table 3.
実施例４
実施例１と同様の方法により培養及び調製したエシェリ
ヒアコリＡＴＣＣ１０７９８の洗浄菌体を、１００１の
２′，３’−ジデオキシウリジンと１００ｍＭの６−フ
ルオロプリンを含む１０１ｍＭのリン酸バッフｙ −（
ｐＨ７，０） １０ ｍｌに２％になるように添加し
、第４表に示す各一定温度条件下で２４時間反応させた
。各反応液中に生成した６−フルオロプリン−２′，３
’−ジデオキシリボシドを実施例１と同様の方法で測定
し、その結果を第４表に示した。Example 4 Washed cells of Escherichia coli ATCC 10798 cultured and prepared in the same manner as in Example 1 were added to a 101 mM phosphate buffer containing 1001 2',3'-dideoxyuridine and 100 mM 6-fluoropurine. (
The mixture was added to 10 ml (pH 7.0) at a concentration of 2%, and reacted for 24 hours under each constant temperature condition shown in Table 4. 6-fluoropurine-2',3 produced in each reaction solution
'-dideoxyriboside was measured in the same manner as in Example 1, and the results are shown in Table 4.
第 ４ 表
実施例５
実施例１と同様の培地を用いて実施例１と同様の方法で
３７℃、１６時間培養したエシェリヒアコリＡＴＣＣ１
０７９８の培養液に、予め殺菌した２００Ｉｌ１Ｍの２
′，３’−ジデオキシウリジンと２００ｍＭの６−フル
オロプリンを含む５００ｍＭのリン酸バッファー（ｐＨ
７，０）５ ｍｌを添加し、更に１０時間培養を続けた
。この培養液中に生成した６−フルオロプリン−２′，
３’−ジデオキシリボシドを実施例１と同様の方法で定
量した結果、２）ｎ＋ｇ／ｄＪの６−フルオロプリン−
２′，３’−ジデオキシリボシドが生成した。Table 4 Example 5 Escherichia coli ATCC1 cultured at 37°C for 16 hours in the same manner as in Example 1 using the same medium as in Example 1.
0798 culture solution, pre-sterilized 200Il1M 2
',3'-dideoxyuridine and 200mM 6-fluoropurine in 500mM phosphate buffer (pH
7,0) was added, and the culture was continued for an additional 10 hours. 6-fluoropurine-2' produced in this culture solution,
As a result of quantifying 3'-dideoxyriboside in the same manner as in Example 1, 2) n+g/dJ of 6-fluoropurine-
2',3'-dideoxyriboside was produced.
（発明の効果）
本発明によれば、従来製造方法が公知でなかった６−フ
ルオロプリン−２′，３’−ジデオキシリボシドを微生
物を用いた簡便な方法でしかも高収率で製造できること
から、強力な抗ウィルス作用を有する６−フルオロプリ
ン−２′，３’−ジデオキシリボシドを、抗ウィルス剤
、特にエイズ（ＡＩＤＳ）の治療薬として用いる研究が
進展するものと期待される。(Effects of the Invention) According to the present invention, 6-fluoropurine-2',3'-dideoxyriboside, for which no production method was known in the past, can be produced by a simple method using microorganisms and at a high yield. It is expected that research on the use of 6-fluoropurine-2',3'-dideoxyriboside, which has a strong antiviral effect, as an antiviral agent, particularly as a therapeutic agent for AIDS (AIDS), will progress.
Claims (13)
ア属、エンテロバクター属、エルビニア属、シトロバク
ター属、コリネバクテリウム属、ハフニア属、クルイヘ
ラ属、サルモネラ属及びキサントモナス属のいずれかに
属し、２，３−ジデオキシリボース−１−リン酸及び６
−フルオロプリンから６−フルオロプリン−２′，３′
−ジデオキシリボシドを生成する能力を有する微生物を
、水性溶媒中で２，３−ジデオキシリボース−１−リン
酸及び６−フルオロプリンに作用せしめることを特徴と
する６−フルオロプリン−２′，３′−ジデオキシリボ
シドの製造方法。(1) Belongs to any of the genus Escherichia, Flavobacterium, Serratia, Enterobacter, Erwinia, Citrobacter, Corynebacterium, Hafnia, Kruichera, Salmonella, and Xanthomonas, 2,3 -dideoxyribose-1-phosphate and 6
-fluoropurine to 6-fluoropurine-2',3'
- 6-fluoropurine-2',3, characterized in that a microorganism capable of producing dideoxyriboside is allowed to act on 2,3-dideoxyribose-1-phosphate and 6-fluoropurine in an aqueous solvent. A method for producing '-dideoxyriboside.
シェリヒア属、フラボバクテリウム属、セラチア属、エ
ンテロバクター属、エルビニア属、シトロバクター属、
コリネバクテリウム属、ハフニア属、クルイヘラ属、サ
ルモネラ属、及びキサントモナス属のいずれかに属し、 イ）２′，３′−ジデオキシヌクレオシド及びロ）無機
リン酸又はその塩 とから２，３−ジデオキシリボース−１−リン酸を生成
する能力を有する微生物を、水性溶媒中でイ）２′，３
′−ジデオキシヌクレオシド及びロ）無機リン酸又はそ
の塩に作用せしめること により生産されたものである請求項（１）に記載の製造
方法。(2) 2,3-dideoxyribose-1-phosphate is present in Escherichia, Flavobacterium, Serratia, Enterobacter, Erwinia, Citrobacter,
2,3-dideoxyribose from a) 2',3'-dideoxynucleoside and b) inorganic phosphoric acid or a salt thereof, which belongs to the genus Corynebacterium, Hafnia, Kluihera, Salmonella, or Xanthomonas; - Microorganisms capable of producing 1-phosphoric acid are grown in an aqueous medium.
2. The production method according to claim 1, which is produced by reacting '-dideoxynucleoside and (b) inorganic phosphoric acid or a salt thereof.
′−ジデオキシウリジンである請求項（２）に記載の製
造方法。(3) 2',3'-dideoxynucleoside is 2',3
The manufacturing method according to claim (2), which is '-dideoxyuridine.
′−ジデオキシアデノシンである請求項（２）に記載の
製造方法。(4) 2',3'-dideoxynucleoside is 2',3
The manufacturing method according to claim (2), which is '-dideoxyadenosine.
′−ジデオキシイノシンである請求項（２）に記載の製
造方法。(5) 2',3'-dideoxynucleoside is 2',3
The manufacturing method according to claim (2), which is '-dideoxyinosine.
′−ジデオキシグアノシンである請求項（２）に記載の
製造方法。(6) 2',3'-dideoxynucleoside is 2',3
The manufacturing method according to claim (2), which is '-dideoxyguanosine.
′−ジデオキシチミジンである請求項（２）に記載の製
造方法。(7) 2',3'-dideoxynucleoside is 2',3
The manufacturing method according to claim (2), which is '-dideoxythymidine.
ア属、エンテロバクター属、エルビニア属、シトロバク
ター属、コリネバクテリウム属、ハフニア属、クルイヘ
ラ属、サルモネラ属、又はキサントモナス属に属し、 イ）２′，３′−ジデオキシヌクレオシド、ロ）無機リ
ン酸又はその塩、及び ハ）６−フルオロプリン、とから ６−フルオロプリン−２′，３′−ジデオキシリボシド
を生成する能力を有する微生物を、水性溶媒中で イ）２′，３′−ジデオキシヌクレオシド、ロ）無機リ
ン酸又はその塩、及び ハ）６−フルオロプリンに作用せしめることを特徴とす
る６−フルオロプリン−２′，３′−ジデオキシリボシ
ドの製造方法。(8) belongs to the genus Escherichia, Flavobacterium, Serratia, Enterobacter, Erwinia, Citrobacter, Corynebacterium, Hafnia, Kruichera, Salmonella, or Xanthomonas, a) 2′, A microorganism having the ability to produce 6-fluoropurine-2',3'-dideoxyriboside from 3'-dideoxynucleoside, b) inorganic phosphoric acid or its salt, and c) 6-fluoropurine, in an aqueous solvent. Among them, 6-fluoropurine-2',3'-dideoxyriboside, which is characterized by acting on a) 2',3'-dideoxynucleoside, b) inorganic phosphoric acid or its salt, and c) 6-fluoropurine. Method of manufacturing CID.
′−ジデオキシウリジンである請求項（８）に記載の製
造方法。(9) 2',3'-dideoxynucleoside is 2',3
The manufacturing method according to claim (8), which is '-dideoxyuridine.
３′−ジデオキシアデノシンである請求項（８）に記載
の製造方法。(10) 2′,3′-dideoxynucleoside is 2′,
The manufacturing method according to claim (8), which is 3'-dideoxyadenosine.
３′−ジデオキシイノシンである請求項（８）に記載の
製造方法。(11) 2′,3′-dideoxynucleoside is 2′,
The manufacturing method according to claim (8), which is 3'-dideoxyinosine.
３′−ジデオキシグアノシンである請求項（８）に記載
の製造方法。(12) 2′,3′-dideoxynucleoside is 2′,
The manufacturing method according to claim (8), which is 3'-dideoxyguanosine.
３′−ジデオキシチミジンである請求項（８）に記載の
製造方法。(13) 2′,3′-dideoxynucleoside is 2′,
The manufacturing method according to claim (8), which is 3'-dideoxythymidine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21270689A JPH0376594A (en) | 1989-08-18 | 1989-08-18 | Production of dideoxynucleotide derivative |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21270689A JPH0376594A (en) | 1989-08-18 | 1989-08-18 | Production of dideoxynucleotide derivative |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0376594A true JPH0376594A (en) | 1991-04-02 |
Family
ID=16627081
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP21270689A Pending JPH0376594A (en) | 1989-08-18 | 1989-08-18 | Production of dideoxynucleotide derivative |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0376594A (en) |
-
1989
- 1989-08-18 JP JP21270689A patent/JPH0376594A/en active Pending
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