JPS59167599A - Chemical phosphatization of inosine and guanosine - Google Patents
Chemical phosphatization of inosine and guanosineInfo
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- JPS59167599A JPS59167599A JP4099983A JP4099983A JPS59167599A JP S59167599 A JPS59167599 A JP S59167599A JP 4099983 A JP4099983 A JP 4099983A JP 4099983 A JP4099983 A JP 4099983A JP S59167599 A JPS59167599 A JP S59167599A
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- Prior art keywords
- guanosine
- inosine
- diphosphate
- phosphate
- added
- Prior art date
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Abstract
Description
【発明の詳細な説明】
本発明は、ヌクレオシドを化学的にリン酸化して化学調
味料として有用な5′−ヌクレオチドを工業的に、かつ
安価に製造する方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for industrially and inexpensively producing 5'-nucleotides useful as chemical seasonings by chemically phosphorylating nucleosides.
さらに詳しくは、本発明は、イノシンおよびグアノシン
を、その糖部分(リポース)の2′−および3′−位の
水酸基(2級水酸基)を保護することなく、5′−位の
水酸基(1級水酸基)を直接リン酸化するに際し、グア
ノシンとイノシンとの混晶を用いるもので、その目的と
するところは、グア/シアー5’−リン酸収率の向上と
、グアノシン−5′−シリン酸、グアノシン−2’(3
’) 、 5’−二リン酸等のグアノシン−ジホスフェ
ート類の副生を抑制することにある。More specifically, the present invention provides inosine and guanosine without protecting the 2'- and 3'-position hydroxyl groups (secondary hydroxyl groups) of the sugar moiety (lipose), and the 5'-position hydroxyl group (primary hydroxyl group) A mixed crystal of guanosine and inosine is used for direct phosphorylation of guanosine and inosine (hydroxyl groups), and its purpose is to improve the yield of guar/sheer 5'-phosphoric acid and to Guanosine-2'(3
'), to suppress the by-product of guanosine diphosphates such as 5'-diphosphate.
従来、5′−ヌクレオシドの製造法としては、リポヌク
レオシドを、そのリボフラノシル基の2′t3′−位を
アシル基あるいはアルキリデン基で保護した後、各種の
反応溶媒存在下で、種々のリン酸化剤を用いて、諸種の
条件でリン酸化し、次いで保護基を加水分解して脱離し
5′−リボヌクレオチドとする方法が知られているが、
このリン酸化方法は、■反応に多段階を要する、■リン
酸化剤の部用量が多い、■かならずしも収率が良好でな
いなどの欠点がある。Conventionally, in the production of 5'-nucleosides, liponucleosides are protected at the 2't3'-position of the ribofuranosyl group with an acyl group or alkylidene group, and then treated with various phosphorylating agents in the presence of various reaction solvents. A known method is to phosphorylate under various conditions using
This phosphorylation method has drawbacks such as (1) the reaction requires multiple steps, (2) the amount of phosphorylating agent is large, and (2) the yield is not always good.
また、これらの欠点を解決するために、従来、無保護の
ヌクレオシドを化学的にリン酸化する方法として、特定
の極性有機溶媒の存在下で、ヌクレオシドとリン酸化剤
を反応せしめる方法が知られている。このようなリン酸
化方法としては、(A)ニド1Jiv化合物を用いる方
法(特公昭42−20316 ) 、(B)有機酸エス
テルを用いる方法(特公昭42−21351 ) 、(
c)リン酸トリアルキルエステルを用いる方法(Tet
rahedronLetters Nch 50.
pp、 5065.1967iBulletin o
f the Chemical 5ociety
of Japan Vol、 4’2.3505
(1969) i特公昭42−1i071)、(D)リ
ン酸トリ(アμコキシアルキ/I/)エステルを用いる
方法(特開昭51−8.6.482 ) 、(E)エチ
レングリコールジアルキルエーテルを用いる方法(特公
昭46−31865)、および(F)極性有機溶媒と有
機アミンまたは有機アミンの無機酸塩を用いる方法(B
uiletin ofthe Chemical
5ociety of Japan、Vol。In addition, in order to solve these drawbacks, conventionally known methods for chemically phosphorylating unprotected nucleosides involve reacting nucleosides with a phosphorylating agent in the presence of a specific polar organic solvent. There is. Such phosphorylation methods include (A) a method using a Nido 1Jiv compound (Japanese Patent Publication No. 42-20316), (B) a method using an organic acid ester (Japanese Patent Publication No. 42-21351), (
c) Method using phosphoric acid trialkyl ester (Tet
rahedron Letters Nch 50.
pp, 5065.1967iBulletin o
f the Chemical 5ociety
of Japan Vol, 4'2.3505
(1969) I Japanese Patent Publication No. 42-1i071), (D) Method using phosphoric acid tri(ampkoxyalkyl/I/) ester (Japanese Unexamined Patent Publication No. 51-86.482), (E) Method using ethylene glycol dialkyl ether (F) a method using a polar organic solvent and an organic amine or an inorganic acid salt of an organic amine (B)
uiletin of the Chemical
5ociety of Japan, Vol.
48.2084 (1975)などが知られている。48.2084 (1975) and others are known.
しかし、これらの無保護リン酸化法をグアノシンのリン
酸化に適用した場合、目的とするグアノシン−5′−リ
ン酸への転換率は、いずれの方法によっても高々的90
%であシ、イノシンの場合、そのイノシン−5′−リン
酸への転換率が95%にも及ぶことと対比するならば、
工業的に満足すべきものではない。However, when these unprotected phosphorylation methods are applied to the phosphorylation of guanosine, the conversion rate to the target guanosine-5'-phosphate is at most 90%.
In contrast, in the case of inosine, the conversion rate to inosine-5'-phosphate is as high as 95%.
This is not industrially satisfactory.
また、グアノシン−5′−リン酸との分離が困難なグア
ノシン−5′−二リン酸、グアノシン−2′(3’)
、 5’−二リン酸等のジホスフェート類が約10%以
上も副生ずるという欠点がある。In addition, guanosine-5'-diphosphate, which is difficult to separate from guanosine-5'-phosphate, and guanosine-2'(3')
The disadvantage is that about 10% or more of diphosphates such as 5'-diphosphoric acid are produced as by-products.
こうした状況のもとで、本発明者らは、従来の無保護リ
ン酸化法をグアノシンのリン酸化に適用する場合に起こ
る上述の障害を克服して、工業的に有利なグアノシン−
5′−リン酸の製造法を確立すべく広く検討した結果、
グアノシンとイノシンとの混晶を原料として用いること
により、イノシンだけでなくグアノ−シンもまた極めて
高収率で5′−モノホスフェートに導かれ、ジホスフェ
ート類の副生も著しく減少すると、と全発見し、さらに
検討して本発明を完成したものである。Under these circumstances, the present inventors have overcome the above-mentioned obstacles that occur when applying the conventional unprotected phosphorylation method to guanosine phosphorylation, and have developed an industrially advantageous guanosine phosphorylation method.
As a result of extensive research to establish a method for producing 5'-phosphoric acid,
By using a mixed crystal of guanosine and inosine as a raw material, not only inosine but also guanosine can be converted into 5'-monophosphate in an extremely high yield, and the by-product of diphosphates is also significantly reduced. This invention was discovered and further studied to complete the present invention.
すなわち、本発明は、極性有機溶媒中、グアノシンとイ
ノシンとの混晶にオキシハロゲン化リンもしくはその水
和物を作用させることを特徴とするグアノシン−5′−
リン酸とイノシン−5′−リン酸との混合物の製造法で
ある。That is, the present invention provides guanosine-5'-, which is characterized in that phosphorus oxyhalide or its hydrate is allowed to act on a mixed crystal of guanosine and inosine in a polar organic solvent.
This is a method for producing a mixture of phosphoric acid and inosine-5'-phosphoric acid.
本発明の製造法において、グアノシンとイノシンとの混
晶は、自体公知の方法で得られたものを用いることがで
きる。例えば、グアノシンおよびイノシンを含む水溶液
よシ通常溶質を析出させる手段によって晶出させて得た
混晶が用いられる(特公昭47−38199参照)。In the production method of the present invention, a mixed crystal of guanosine and inosine obtained by a method known per se can be used. For example, a mixed crystal obtained by crystallizing an aqueous solution containing guanosine and inosine by means of precipitating the solute is used (see Japanese Patent Publication No. 47-38199).
この場合、晶出の方法としては、例えば冷却、濃縮(水
の除去)、種晶の添加、グアノシンおよびイノシンを溶
解しない親水性溶媒(例えばアセトン)の添加、pH調
整(グアノシンおよびイノシンの溶解度が大きいpH3
以下の酸性域あるいはpH9以上のアルカリ性域からp
H3〜9の範囲に調整する)などおよびこれらの組合せ
の方法を適宜採用できる。このような方法でグアノシン
およりイノシンを含む溶液よりグアノシンとイノシンと
の混晶を析出させた後、吸引または加圧濾過、遠心分離
、遠心沈降等の通常用いられる方法で該混晶を分離し、
例えば減圧下に加熱乾燥する等によシ水などの溶媒を除
去し、次いでリン酸化° 反応に供するのが望ましい。In this case, methods of crystallization include, for example, cooling, concentration (removal of water), addition of seed crystals, addition of a hydrophilic solvent (e.g. acetone) that does not dissolve guanosine and inosine, pH adjustment (solubility of guanosine and inosine is big pH3
From the following acidic ranges or alkaline ranges of pH 9 or higher
Adjustment to a range of H3 to H9) and combinations thereof can be employed as appropriate. After precipitating a mixed crystal of guanosine and inosine from a solution containing guanosine and inosine in this manner, the mixed crystal is separated by a commonly used method such as suction or pressure filtration, centrifugation, or centrifugal sedimentation. ,
For example, it is desirable to remove the solvent such as water by heating and drying under reduced pressure, and then subjecting it to the phosphorylation reaction.
この際、分離された湿結晶をアセトン、メチルエチルケ
トンなどの親水性で、かつグアノシンおよびイノシンを
溶解しない溶媒で洗滌すると、低温短時間で乾燥するこ
とができるので有利である。At this time, it is advantageous to wash the separated wet crystals with a hydrophilic solvent such as acetone or methyl ethyl ketone, which does not dissolve guanosine and inosine, since it can be dried at low temperatures and in a short time.
また乾燥された混晶を適宜粉砕してから篩過し、例えば
100メツシユ以下に整粒して反応に供してもよい。Alternatively, the dried mixed crystal may be appropriately ground, passed through a sieve, and sized to, for example, 100 meshes or less, and subjected to the reaction.
本発明の製造法を適用するにあたってグアノシンとイノ
シンとの混晶中のイノシン含量が低過ぎたシ、また高過
ぎる場合は、グアノシンの5′−モノホスフェートへの
転換率の向上とジホスフェート類の副生抑制という本発
明の効果を奏し難いことがある。通常、イノシンのグア
ノシンに対する割合が約0.1〜10(モ/L//モ/
l/)、好ましくは約0,2〜4(モ)v/七ル)の混
晶を用いるのが有利である。When applying the production method of the present invention, if the inosine content in the mixed crystal of guanosine and inosine is too low or too high, it is necessary to improve the conversion rate of guanosine to 5'-monophosphate and to increase the conversion rate of diphosphates. It may be difficult to achieve the effect of the present invention of suppressing by-products. Usually, the ratio of inosine to guanosine is about 0.1 to 10 (Mo/L//Mo/
It is advantageous to use mixed crystals of about 0.2 to 4 (mol)v/7 l).
混晶中のイノシンとグアノシンとの量比は、前述の方法
において晶出原液中のこれらの濃度比あるいは(および
)晶出方法を適宜選ぶことによシ、任意に調節すること
が可能である。The quantitative ratio of inosine and guanosine in the mixed crystal can be arbitrarily adjusted by appropriately selecting the concentration ratio of these in the crystallization stock solution or (and) the crystallization method in the above-mentioned method. .
本発明の製造法で用いられる極性有機溶媒としては、ア
セトニトリル、10ビオニトリμ等のニトリル化合物、
酢酸エチル、酢酸メチル、プロピオン酸メチル等の有機
酸エステル類、リン酸トリメチル、リン酸トリエチル等
のリン酸トリ低級アルキμエステル類、リン酸トリ(メ
トキシエチ/L/)、リン酸トリ(エトキシエチ/L/
)等のリン酸トリ(アルコキシアルキ/L/)エステル
類、エチレングリコールジメチルエーテル、エチレング
リコールジエチルエーテル等のエチレングリコールジア
ルキμエーテμ類、テトラヒドロフラン、1.4−ジオ
キサン等の環状エーテ/l’類、ジクロロメタン、クロ
ロホ7レム、1.2−ジクロロエタン等ノハロゲン化炭
化水素類、ニトロメタン、ニトロエタン、ニトロベンゼ
ン等のニトロ化合物等が挙げられる。Examples of the polar organic solvent used in the production method of the present invention include acetonitrile, nitrile compounds such as 10 bionitrile μ,
Organic acid esters such as ethyl acetate, methyl acetate, and methyl propionate; tri-lower alkyl μ esters such as trimethyl phosphate and triethyl phosphate; tri(methoxyethyl/L/) phosphate; tri(ethoxyethyl/L/) phosphate; L/
), phosphoric acid tri(alkoxyalkyl/L/) esters such as ethylene glycol dimethyl ether, ethylene glycol diethyl ether, etc., cyclic ethers/L' such as tetrahydrofuran, 1,4-dioxane, etc. , dichloromethane, chlorophorem, 1,2-dichloroethane, and other halogenated hydrocarbons; and nitro compounds such as nitromethane, nitroethane, and nitrobenzene.
これらの極性有機溶媒は2種以上を混合して用いてもよ
く、また、これら以外の有機溶媒でも例えばベンゼン、
トルエン等の芳香族法化水素類、エチルエーテル等のエ
ーテル類のように反応に悪影響を及ぼさないものであれ
ば、これらの溶媒と混合して用いることができる。These polar organic solvents may be used in combination of two or more, and organic solvents other than these may also be used, such as benzene,
As long as it does not adversely affect the reaction, such as aromatic hydrogenated hydrogen compounds such as toluene and ethers such as ethyl ether, it can be used in combination with these solvents.
極性有機溶媒の使用量は、その種類によシ異なるが、通
常グアノシンおよびイノシンの量に対して約5〜約30
倍量の範囲で適宜選択され、好ましくは10〜20倍量
程度である。The amount of polar organic solvent used varies depending on the type, but is usually about 5 to about 30% of the amount of guanosine and inosine.
The amount is appropriately selected within the range of twice the amount, preferably about 10 to 20 times the amount.
また、上記溶媒に、ピリジン、γ−ピコリン、N、N−
−、Fメチμmアニリン等の3級アミン類もしくはその
無機酸塩をグアノシンおよびイノクン量に対し、約1〜
5倍モル程度、適宜添加することによシ、さらに反応促
進、収率向上等の好ましい効果をもたらすことが多い。In addition, pyridine, γ-picoline, N, N-
-, F Methy μm Tertiary amines such as aniline or their inorganic acid salts are added to the amount of guanosine and inocune from about 1 to
Appropriate addition of about 5 times the mole often brings about favorable effects such as further reaction acceleration and yield improvement.
本発明の製造法で用いられるリン酸化剤は、オキシ塩化
リン、オキシ臭化リン等のオキシハロゲン化リンである
。The phosphorylating agent used in the production method of the present invention is a phosphorus oxyhalide such as phosphorus oxychloride and phosphorus oxybromide.
オキシハロゲン化リンの使用量は通常、グアノシンおよ
びイノシンの約1〜5倍モ/L/量、好ましくは約16
5〜4倍モル量である。その使用量が少。The amount of phosphorus oxyhalide used is usually about 1 to 5 times the amount of guanosine and inosine, preferably about 16
It is 5 to 4 times the molar amount. Its usage is small.
な過ぎるとグアノシンおよびイノシンが未反応のまま残
シ、また、過度の使用量ではジホスフェート類の副生が
増えると同時に目的とする5′−モノホスフェートの収
率が下がるので好ましくない。If the amount is too low, guanosine and inosine will remain unreacted, and if it is used in an excessive amount, the by-product of diphosphates will increase and at the same time the yield of the desired 5'-monophosphate will decrease, which is not preferred.
通常、前記の使用範囲から、リン酸化剤あるいは溶媒の
種類等を考慮し適宜に選択される。Usually, it is appropriately selected from the above-mentioned range of use, taking into consideration the type of phosphorylating agent or solvent.
リン酸化に際し、オキシハロゲン化リンをそのまま反応
に供するよりも、一旦、部分水和物としてから用いる方
が、通常5′−モノホスフェート生成への選択性が高く
、2′または3′−モノホスフェート、ジホスフェート
類の副生量がすくなくなるので好ましい。During phosphorylation, rather than subjecting phosphorus oxyhalide to the reaction as it is, it is usually better to use it as a partially hydrated product, which has higher selectivity for the production of 5'-monophosphate, and to produce 5'-monophosphate. This is preferable because the amount of by-products of diphosphates is reduced.
オキシハロゲン化リンの水和物を得る方法は、オキシハ
ロゲン化リンを上記反応溶媒に加えて溶解してから、こ
れに少量の水またはメタノール、エタノ−μ、三級プク
ノール等のアルコール類、もしくはギ酸、酢酸等の有機
酸類を加えて反応させればよく、生成した部分水和物は
分離することなく、溶液として本発明のリン酸化反応に
供される。この際の水またはアルコ−/l/類、もしく
は有機酸の添加量はオキシハロゲン化リンのグアノシン
とイノシンとの混晶に対する仕込割合によって異なるが
、通常オキシハロゲン化リンに対して約0.1〜0.7
倍モ/I/量であや、好ましくは0.3〜0.6倍モ/
L’量である。The method for obtaining a hydrate of phosphorus oxyhalide is to add phosphorus oxyhalide to the above reaction solvent and dissolve it, and then add a small amount of water or an alcohol such as methanol, ethanol-μ, tertiary pucnol, or The reaction may be carried out by adding organic acids such as formic acid and acetic acid, and the generated partial hydrate is subjected to the phosphorylation reaction of the present invention as a solution without being separated. The amount of water, alcohol, or organic acid added at this time varies depending on the ratio of phosphorus oxyhalide to the mixed crystal of guanosine and inosine, but is usually about 0.1 to phosphorus oxyhalide. ~0.7
The amount is 0.3 to 0.6 times, preferably 0.3 to 0.6 times
It is the amount of L'.
ただ七、本発明の製造法で反応溶媒として用いられる上
記の極性有機溶媒類のうち、ニトリμ類、エチレングリ
コールシアpキルエーテμ類、環状エーテル類、ハロゲ
ン化戻化水素類、・もしくはニトロ化合物を用い、これ
にピリジン、γ−ピコリン、N、N−ジメチルアニリン
等の無機酸塩を添加して反応に供するときは、オキシハ
ロゲン化リンは部分水和する必要はなく、そのt−ま反
応に使用しても、部分水和物を用いる場合と同様に良好
な効果が得られる。However, among the above polar organic solvents used as reaction solvents in the production method of the present invention, nitri μ, ethylene glycol shea p-kylete μ, cyclic ethers, halogenated hydrogenated hydrogen, or nitro compounds When using phosphorus oxyhalide and adding an inorganic acid salt such as pyridine, γ-picoline, N,N-dimethylaniline, etc. to the reaction, the phosphorus oxyhalide does not need to be partially hydrated, and the t-reaction Even when used as a partial hydrate, good effects can be obtained as in the case of using a partial hydrate.
本発明の製造法は、約10℃以下に冷却しながら、前記
反応溶媒にリン酸化剤を加え、これに所定量の水(また
はアルコ−/l’類もしくは有機酸)を添加し、場合に
よってはさらに、3級アミン(またはその′無機酸塩)
を添加して溶解し、続いてグアノシンとイノシンとの混
晶を加え、攪拌、冷却下に反応させることにより行われ
る。In the production method of the present invention, a phosphorylating agent is added to the reaction solvent while cooling to about 10°C or less, and a predetermined amount of water (or alcohol/l's or organic acid) is added thereto. furthermore, a tertiary amine (or its inorganic acid salt)
is added and dissolved, then a mixed crystal of guanosine and inosine is added, and the reaction is carried out under stirring and cooling.
反応温度は一30’〜+50℃の範囲が実用的であり、
中でも一10°〜+3(lが特に好ましい。The reaction temperature is practically in the range of -30' to +50°C,
Among these, -10° to +3 (l) is particularly preferred.
この温度範囲において、反応時間は溶媒の種類、促進剤
添加の有無等によって異なるが、一般に30分〜10時
間である。当反応方法においては、グアノシンを単独に
リン酸化する従来の方法に比べ、反応は短時間で終了す
る。In this temperature range, the reaction time varies depending on the type of solvent, presence or absence of promoter addition, etc., but is generally 30 minutes to 10 hours. In this reaction method, the reaction is completed in a shorter time than in the conventional method of phosphorylating guanosine alone.
このようにして得られた反応生成物を常法によシ冷水と
混合することによって、未反応のリン酸化剤および生成
したヌクレオシドホスホハロゲネートを加水分解し、グ
アノシン−5′−リン酸およびイノシン−5′−リン酸
を含む溶液(加水分解液)を得る。By mixing the reaction product thus obtained with cold water in a conventional manner, the unreacted phosphorylating agent and the generated nucleoside phosphohalogenate are hydrolyzed, and guanosine-5'-phosphate and inosine A solution (hydrolysis solution) containing -5'-phosphoric acid is obtained.
このようにして得られたグアノシン−5′−ホスフェー
トおよびイノシン−5′−ホスフェートを精製する方法
としては、例えば、
1)、加水分解液のpHを水酸化ナトリウムで約1.5
に調整した後、活性炭で処理する方法、2)、反応溶媒
を他の有機溶媒を用いて抽出分離した後、水酸化ナトリ
ウムなどのアルカリで中和してから、吸着樹脂処理ある
いは晶析によシ精製する方法、
3)、反応溶媒を他の有機溶媒を用いて抽出分離した後
、活性炭で処理する方法、
などが挙げられる。次いで、これらの精製方法のいずれ
かを経た後、常法によジグアノシン−5′−リン酸二ナ
トリウムとイノシン−57−リン酸二ナトリウムとの混
晶として取得することができる。As a method for purifying the guanosine-5'-phosphate and inosine-5'-phosphate obtained in this way, for example, 1) the pH of the hydrolysis solution is adjusted to about 1.5 with sodium hydroxide;
2), the reaction solvent is extracted and separated using another organic solvent, neutralized with an alkali such as sodium hydroxide, and then treated with an adsorption resin or crystallized. 3), a method in which the reaction solvent is extracted and separated using another organic solvent, and then treated with activated carbon. Next, after passing through any of these purification methods, it can be obtained as a mixed crystal of diguanosine-5'-disodium phosphate and inosine-57-disodium phosphate by a conventional method.
また必要ならば、例えば活性炭カラムを用いる分別溶出
法等によシ、グアノシン−5′−リン酸およびイノシン
−5′−リン酸を分離した後、所望のアルカリ金属の塩
としてそれぞれを純度の高い単一物の結晶として取得す
ることも可能である。これらの5′−ヌクレオチドまた
はその混合物は呈味性に冨み、化学調味料として有用な
物質である。If necessary, guanosine-5'-phosphoric acid and inosine-5'-phosphoric acid are separated by a fractional elution method using an activated carbon column, and then each is purified as a desired alkali metal salt. It is also possible to obtain it as a single crystal. These 5'-nucleotides or mixtures thereof are rich in taste and are useful substances as chemical seasonings.
本発明の製造法を適用することによりグアノシンの5′
−モノホスフェートへの転換率が向上し、またジホスフ
ェート類の副生量も大幅に下がるが、この場合のイノシ
ンの5′−モノホスフェートへの転換率は、イノシンを
単独に同条件でリン酸化した場合の転換率とほとんど変
わらない。By applying the production method of the present invention, the 5′ of guanosine
- The conversion rate to monophosphate is improved, and the amount of diphosphate by-products is also significantly reduced. The conversion rate is almost the same as the conversion rate.
以下、参考例および実施例を挙げて本発明をさらに具体
的に説明する。Hereinafter, the present invention will be explained in more detail with reference to Reference Examples and Examples.
参考例−1
アセトニトリ/’100m/、ピリジン37.9 gの
溶液に水冷下オキシ塩化リン67.5 (lを滴下し、
次に水4.5gをゆつくシ添加した。得られた混合液を
0〜2Cに保ち、攪拌しながらこれにグアノシン28.
3F/を加えた。この温度で6時間連続攪拌し反応した
後、反応液を700fの氷水と混合した。この水溶液を
高速液体クロマトで分析し、第1表の結果を得た。Reference Example-1 67.5 liters of phosphorus oxychloride was added dropwise to a solution of 37.9 g of acetonitrile/'100 m/pyridine under cooling with water,
Next, 4.5 g of water was slowly added. The resulting mixture was kept at 0-2C and 28% of guanosine was added to it while stirring.
3F/ was added. After reacting by stirring continuously at this temperature for 6 hours, the reaction solution was mixed with ice water at 700 f. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 1 were obtained.
グアノシン−5′−二リン酸 0.53
1.2グアノシンーグ、5′−二リン酸
1.24 2.8参考例−2
↓
つくシ添加した。得られた混合液10℃に保ち、攪拌し
ながらこれにグアノシン28.31加えた。Guanosine-5'-diphosphate 0.53
1.2 Guanosine, 5'-diphosphate
1.24 2.8 Reference Example-2 ↓ Tsukushi was added. The resulting mixture was maintained at 10° C. and 28.31 liters of guanosine was added thereto while stirring.
この温度で5時間連続して攪拌し反応した後、反応液を
500f/の氷水と混合した。この水溶液を高速液体ク
ロマトで分析し、第2表の結果を得た。After reacting by stirring continuously at this temperature for 5 hours, the reaction solution was mixed with 500 f/ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 2 were obtained.
第2表
グアノシン−5′−二リン酸 0.58
1.3グアノシン−2’、5’−二リン酸
1.37 3.1参考例−3
アセトニトリル200m1.ピリジン75.19の溶液
に水冷下オキシ塩化リン67.5 fを滴下し、次に水
8.9gをゆつくシ添加した。得られた混合液を0°〜
2℃に保ち、攪拌しながらこれにグアノシン28.3g
続いてイノシン26.31を加えた。Table 2 Guanosine-5'-diphosphate 0.58
1.3 Guanosine-2',5'-diphosphate
1.37 3.1 Reference Example-3 Acetonitrile 200ml. 67.5 f of phosphorus oxychloride was added dropwise to a solution of 75.19 g of pyridine while cooling with water, and then 8.9 g of water was slowly added. The obtained mixture was heated to 0°~
28.3 g of guanosine was added to this while stirring at 2°C.
Subsequently, 26.31 inosine was added.
この温度で6時間連続攪拌し反応した後、反応液を1,
4〜の氷水と混合した。この水溶液を高速液体クロマト
で分析し、第3表の結果を得た。After reacting by stirring continuously at this temperature for 6 hours, the reaction solution was
4. Mixed with ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 3 were obtained.
グアノシン−5′−リン酸 31.3 8
6.2グア/シン−ゴーニリン酸 0.51
1.1グアノシン−2’、5’−ニリン酸 1
.22 2.7グアノシンー3’、5’−ニリン
e 2.58 5.8参考例−4
リン酸トリエチ)’700m1、オキシ塩化リン++5
.0F!の混液に水冷下、水4.5yを滴下して加え、
次にこれにグアノシン28.3 g、続いてイノシン4
0.2fを加え5Cで6時間連続攪拌し反応させた。反
応液を1.2 kyの氷水と混合し、その水溶液を高速
液体クロマトで分析し、第4表の結果を得た。Guanosine-5'-phosphate 31.3 8
6.2 guar/singoniphosphoric acid 0.51
1.1 Guanosine-2',5'-nilinic acid 1
.. 22 2.7 Guanosine-3',5'-niline e 2.58 5.8 Reference example-4 Triethyl phosphate)'700ml, phosphorus oxychloride++5
.. 0F! Add 4.5y of water dropwise to the mixed solution under water cooling,
Next, this was followed by 28.3 g of guanosine, followed by 4 g of inosine.
0.2f was added and the mixture was continuously stirred at 5C for 6 hours to react. The reaction solution was mixed with 1.2 ky of ice water, and the aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 4 were obtained.
(以下光・白)
第4表
グアノシン−5′−リン酸 30.8 8
4.8グアノシン−5′−二リン酸 0.55
1.2グアノシン−2’、5’−二リン酸 1
.32 3.0グアノシン−3’、5’−二リン
酸 2.90 6.5イノシン−5′−リン酸
49.7 95.2実施例−1
アセトニトリ/I/120耐、ピリジン46.7 gの
溶液に水冷下オキシ塩化リン83.Ofを滴下し、次に
水5.5yをゆつくシ添加した。得られた溶液を0〜2
℃に保ち、攪拌しながらこれにグアノシン28.3 f
およびイノシン6.2gを含む混晶を加えた。この温度
で5時間連続攪拌し反応させた後、反応液を900 w
tの氷水と混合した。この水溶液を高速液体クロマトで
分析し、第5表の結果を得た。(hereinafter referred to as light/white) Table 4 Guanosine-5'-phosphoric acid 30.8 8
4.8 Guanosine-5'-diphosphate 0.55
1.2 Guanosine-2',5'-diphosphate 1
.. 32 3.0 Guanosine-3',5'-diphosphoric acid 2.90 6.5 Inosine-5'-phosphoric acid 49.7 95.2 Example-1 Acetonitrile/I/120 resistant, pyridine 46.7 g Add 83% phosphorus oxychloride to a solution of 83% under water cooling. Of was added dropwise, and then 5.5 y of water was slowly added. The obtained solution is 0 to 2
℃ and add 28.3 f of guanosine to this while stirring.
and mixed crystals containing 6.2 g of inosine were added. After reacting by continuously stirring at this temperature for 5 hours, the reaction solution was heated to 900 w
t of ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 5 were obtained.
第5表
グアノシン−5′−二リン酸 0.18 0
.4グアノシン−2’、 5’−二リン酸 0.4
2 0.9グアノシン−3’、5’−二リン酸
1.02 2.3実施例−2
ニトロメタン140 mlにピリジン塩酸塩76.1g
を加え、これに水冷下オキシ塩化リン52.5fを滴下
した。得られた溶液を2°〜3℃に保ち、攪拌しながら
これにグアノシン28.3fおよびイノシン9.91を
含む混晶を加えた。この温度で5時間連続して反応させ
た後、反応液を600gの氷水と混合した。この水溶液
をエチルエーテルで抽出してニトロメタンを除き、抽残
液を高速液体クロマトで分析し、第6表の結果を得た。Table 5 Guanosine-5'-diphosphate 0.18 0
.. 4 guanosine-2', 5'-diphosphate 0.4
2 0.9 Guanosine-3',5'-diphosphoric acid 1.02 2.3 Example-2 76.1 g of pyridine hydrochloride in 140 ml of nitromethane
was added, and 52.5 f of phosphorus oxychloride was added dropwise thereto under water cooling. The resulting solution was maintained at 2° to 3° C., and a mixed crystal containing 28.3f of guanosine and 9.911 inosine was added thereto while stirring. After continuously reacting at this temperature for 5 hours, the reaction solution was mixed with 600 g of ice water. This aqueous solution was extracted with ethyl ether to remove nitromethane, and the raffinate was analyzed by high performance liquid chromatography to obtain the results shown in Table 6.
第6表
グ1アノシンー5′−リン酸 34,0 9
3.6グアノシンー5′−二リン酸 0.20
0.5グアノシン−2’、5’−二リン酸 0
.39 0.9グデン゛シン−3’、5’−二リ
ン酸 1.03 2.3イノシン−57−リ
ン酸 12.1 94.0実施例−3
ジグ1:I/l/メタン200gt、r−ピコリン44
.2Vの混液に氷冷下オキシ塩化リン133.61を滴
下し、次にメタノ−/l’+5.2fをゆつくシ添加し
た。得られた溶液を2°〜3℃に保ち、攪拌しながらこ
れにグアノシン28.3 fおよびイノシン26.3f
を含む混晶を加えた。この温度で5時間連続攪拌し反応
させた後、反応液を1400gの氷水と混合した。得ら
れた液のジクロμメタン層と水層を分液し、水層を高速
液体クロマトで分析し、第7表の結果を得た。Table 6 G1 Anosine-5'-phosphoric acid 34,0 9
3.6 Guanosine-5'-diphosphate 0.20
0.5 Guanosine-2',5'-diphosphate 0
.. 39 0.9 Gudensine-3',5'-diphosphoric acid 1.03 2.3 Inosine-57-phosphoric acid 12.1 94.0 Example-3 Jig 1: I/l/methane 200gt, r -Picoline 44
.. 133.61 liters of phosphorus oxychloride was added dropwise to the 2V mixed solution under ice cooling, and then 5.2 f of methanol/l'+ was slowly added. The resulting solution was kept at 2°-3°C and 28.3 f of guanosine and 26.3 f of inosine were added to it while stirring.
Added mixed crystals containing. After reacting by stirring continuously at this temperature for 5 hours, the reaction solution was mixed with 1400 g of ice water. The resulting liquid was separated into a dichloroμmethane layer and an aqueous layer, and the aqueous layer was analyzed by high performance liquid chromatography to obtain the results shown in Table 7.
第7表
生成物質名 生成量[) 生成率〔%〕グ
アノシンー5′−リン酸 33.7 92.
8グアノシン−5′−二リン酸 0.2.1
0.5グアノシン−2’、5’−二リン酸 0.
41 0.9グアノシン−3’、5’−二リン酸
0.98 2.2イノシン−5′−リン酸
32.1 94゜1実施例−4
アセトニトリ/l/320ゴ、N、N−ジメチルアニリ
ン91.8fの混液に氷冷下オキシ塩化リン213.2
9を滴下し、次にギ酸29゜1gをゆつくシ添加した。Table 7 Name of produced substance Amount produced [) Production rate [%] Guanosine-5'-phosphoric acid 33.7 92.
8 Guanosine-5'-diphosphate 0.2.1
0.5 Guanosine-2',5'-diphosphate 0.
41 0.9 Guanosine-3',5'-diphosphoric acid 0.98 2.2 Inosine-5'-phosphoric acid 32.1 94゜1 Example-4 Acetonitrile/l/320 Go, N, N-dimethyl Add 213.2 phosphorus oxychloride to a mixture of aniline 91.8 f under ice cooling.
9 was added dropwise, and then 29.1 g of formic acid was slowly added.
得られた溶液を2°〜3℃に保ち、攪拌゛しながらこれ
にグアノシン28.31およびイノシン57.9 fを
含む混晶を加えた。この温度で4時間連続攪拌し反応さ
せた後、反応液を2.2 kqの氷水と混合した。この
水溶液を高速液体クロマトで分析し、第8表の結果を得
た。The resulting solution was maintained at 2 DEG -3 DEG C., and a mixed crystal containing 28.31 f of guanosine and 57.9 f of inosine was added thereto while stirring. After continuously stirring and reacting at this temperature for 4 hours, the reaction solution was mixed with 2.2 kq of ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 8 were obtained.
生成物質名 生成量CI〕 生成率〔%〕
グアノシンー5−二リン酸 0.17 0.4
グアノシン−2’、 5’−二リン酸 0.45
1.0グアノシン−3’、5’−二リン酸
?、00 2.3実施例−5
リン酸トリエチ)L/400txl、オキシ塩化リン5
9、7 fの混液に氷冷下、水2.31を滴下して加え
、次にこれにグアノシン2B、3fおよびイノシン8.
0yを含む混晶を加え10℃で4時間連続攪拌し反応さ
せた。反応液を600fの氷水と混合し、その水溶液を
高速液体クロマトで分析し、第9表の結果を得た。Name of produced substance Production amount CI] Production rate [%]
Guanosine-5-diphosphate 0.17 0.4
Guanosine-2', 5'-diphosphate 0.45
1.0 guanosine-3',5'-diphosphate
? , 00 2.3 Example-5 Triethyl phosphate) L/400txl, phosphorus oxychloride 5
2.31 of water was added dropwise to the mixture of 9 and 7f under ice cooling, and then guanosine 2B, 3f and inosine 8.31 were added to this mixture.
A mixed crystal containing Oy was added, and the mixture was stirred continuously at 10° C. for 4 hours to react. The reaction solution was mixed with ice water at 600 f, and the aqueous solution was analyzed by high performance liquid chromatography to obtain the results shown in Table 9.
第9表
グアノシン−5′−二リン酸 0.13 0
.3グアノシン−2’、5’−二リン酸 0.29
0.7グアノシンー3’、5’−二リン酸
0.71 1.6実施例−6
リン酸トリメチ/l’ 500 ml、オキシ塩化リン
82.9Fの混液に水冷下、水3.2gを滴下して加え
、次にこれにグアノシン2B、3gおよびイノシン21
.5Fを含む混晶を加え10℃で3時間連続攪拌し反応
させた。反応液を900gの氷水と混合し、その水溶液
を高速液体クロマトで分析し、第10表の結果を得た。Table 9 Guanosine-5'-diphosphate 0.13 0
.. 3 Guanosine-2',5'-diphosphate 0.29
0.7 guanosine-3',5'-diphosphate
0.71 1.6 Example-6 3.2 g of water was added dropwise to a mixed solution of 500 ml of trimethyl phosphate/l' and 82.9 F of phosphorus oxychloride under water cooling, and then 3 g of guanosine 2B and 3 g of guanosine 2B and Inosine 21
.. A mixed crystal containing 5F was added, and the mixture was stirred continuously at 10° C. for 3 hours to react. The reaction solution was mixed with 900 g of ice water, and the aqueous solution was analyzed by high performance liquid chromatography to obtain the results shown in Table 10.
この水溶液を1.2−ジクロルエタンで抽出してリン酸
トリメチμを除き、抽残液を炭未塔に通してグアノシン
−5′−リン酸等のヌクレオチドを吸着させ、よく水洗
し、次に2%水酸化ナトリウム水溶液で溶出した。溶出
液を塩酸でpH8,5に調整してから減圧下に水を留去
し、濃縮液を熱時脱色濾過した後メグノー7X/全添加
して晶析し、グアノシン−5−リン酸二ナトリウムおよ
びイノシン−5−リン酸二ナトリウムの混晶(含水塩、
純度98%)Σ得衣。This aqueous solution was extracted with 1,2-dichloroethane to remove trimethyl phosphate, and the raffinate was passed through a charcoal column to adsorb nucleotides such as guanosine-5'-phosphate, thoroughly washed with water, and then % aqueous sodium hydroxide solution. The eluate was adjusted to pH 8.5 with hydrochloric acid, water was distilled off under reduced pressure, and the concentrated solution was decolorized and filtered while hot, then Megno 7X/all was added to crystallize, resulting in disodium guanosine-5-phosphate. and mixed crystals of disodium inosine-5-phosphate (hydrated salt,
Purity: 98%)
グアノシン−5′−二リン酸 0.11 0.
3グアノシンーグ、5′−二リン酸 0.26
0.6グアノシンー3’、5’−二リン酸
0.68 1.5実施例−7
リン酸トリエチ)’700w1.オキシ塩化リン115
.0gの混液に水冷下、水4.5fを滴下して加え、次
にこれにグアノシン28.31!およびイノシン40.
2F!を含む混晶を加え5しで6時間連続攪拌し反応さ
せた。反応液を1.2 kyの氷水と混合し、その水溶
液を高速液体クロマトで分析し、第11表の結果を得た
。Guanosine-5'-diphosphate 0.11 0.
3 guanosine, 5'-diphosphate 0.26
0.6 guanosine-3',5'-diphosphate
0.68 1.5 Example-7 Triethyl phosphate)'700w1. Phosphorus oxychloride 115
.. Add 4.5 f of water dropwise to 0 g of the mixed solution under water cooling, and then add 28.31 g of guanosine! and inosine 40.
2F! A mixed crystal containing . The reaction solution was mixed with 1.2 ky of ice water, and the aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 11 were obtained.
この水溶液をトルエンで抽出してリン酸トリエチルを除
き、抽残液を炭末塔に通してグアノシン−5′−二リン
酸等のヌクレオチドを吸着させ、よく水洗し1.8、次
に1.5%塩化アンモニウム水溶液にアンモニア水を加
えてpH10,0にした液を通してイノシン−5′−リ
ン酸を溶出(第1溶出)させ、続いて2%水酸化すFl
lラム溶液を通してグアノシン−5′−リン酸等を溶出
(第2溶出)させた。This aqueous solution was extracted with toluene to remove triethyl phosphate, and the raffinate was passed through a charcoal tower to adsorb nucleotides such as guanosine-5'-diphosphate, thoroughly washed with water, and then 1.8. Inosine-5'-phosphoric acid was eluted (first elution) through a 5% aqueous ammonium chloride solution with aqueous ammonia added to pH 10.0, followed by 2% hydroxylated Fl.
Guanosine-5'-phosphoric acid and the like were eluted through the LAM solution (second elution).
第1溶出液に水酸化すIIウムを加えて濃縮し、放冷晶
析してイノシン−5′−リン酸ニナトリウムの結晶(8
水塩、純度99%)を得た。The first eluate was concentrated by adding II hydroxide, crystallized while standing to cool, and crystals of inosine-5'-disodium phosphate (8
water salt, purity 99%) was obtained.
グアノシン−5′−二リン酸 0.12 0
.3グアノシン−2’、5’−二リン酸 0.28
0.6グアノシンー3’、5’−二リン酸
0.70 1.6イノシンー5′−リン酸
49.6 95.0また、第2溶出液を塩酸でp
H8,5に調整してから濃縮し、放冷晶析して、グアノ
シン−5′・−リン酸ニナトリウムの結晶(7水塩、純
度98%)を得た。Guanosine-5'-diphosphate 0.12 0
.. 3 Guanosine-2',5'-diphosphate 0.28
0.6 guanosine-3',5'-diphosphate
0.70 1.6 inosine-5'-phosphate
49.6 95.0 Also, the second eluate was purified with hydrochloric acid.
After adjusting to H8.5, it was concentrated and crystallized while standing to cool to obtain crystals of guanosine-5'-disodium phosphate (heptahydrate, purity 98%).
実施例−8
リン酸トリメチ/1/ 1. l 12、オキシ塩化リ
ン184.91の混液に水冷下、水7.2gを滴下して
加え、次にこれにグアノシン2B、3fおよびイノシン
81.OFを含む混晶を加え5℃で4時間連続攪拌し反
応させた。反応液を2#の氷水と混合し、その水溶液を
高速液体クロマトで分析し、第12表の結果を得た。Example-8 Trimethyphosphate/1/1. 12, phosphorus oxychloride 184.91 was added dropwise to a mixture of 7.2 g of water under water cooling, and then guanosine 2B, 3f and inosine 81. A mixed crystal containing OF was added and the mixture was continuously stirred at 5° C. for 4 hours to react. The reaction solution was mixed with 2# ice water, and the aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 12 were obtained.
Claims (1)
シハロゲン化リンもしくはその水和物を作用させること
を特徴とするグアノシン−5′−リン酸とイノシン−5
′−リン酸との混合物の製造法。Guanosine-5'-phosphoric acid and inosine-5, which is characterized in that guanosine-5'-phosphoric acid and inosine-5 are treated with phosphorus oxyhalide or its hydrate on a mixed crystal of guanosine, inosine, and phosphorus in a polar organic solvent.
′-Process for producing a mixture with phosphoric acid.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4099983A JPS59167599A (en) | 1983-03-11 | 1983-03-11 | Chemical phosphatization of inosine and guanosine |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4099983A JPS59167599A (en) | 1983-03-11 | 1983-03-11 | Chemical phosphatization of inosine and guanosine |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS59167599A true JPS59167599A (en) | 1984-09-21 |
JPH0375560B2 JPH0375560B2 (en) | 1991-12-02 |
Family
ID=12596113
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP4099983A Granted JPS59167599A (en) | 1983-03-11 | 1983-03-11 | Chemical phosphatization of inosine and guanosine |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS59167599A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2055670A1 (en) * | 1992-07-08 | 1994-08-16 | Takeda Chemical Industries Ltd | Method for producing 5'-nucleotide |
-
1983
- 1983-03-11 JP JP4099983A patent/JPS59167599A/en active Granted
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2055670A1 (en) * | 1992-07-08 | 1994-08-16 | Takeda Chemical Industries Ltd | Method for producing 5'-nucleotide |
US5623069A (en) * | 1992-07-08 | 1997-04-22 | Takeda Chemical Industries, Ltd. | Method for producing 5'-nucleotide |
Also Published As
Publication number | Publication date |
---|---|
JPH0375560B2 (en) | 1991-12-02 |
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