Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-01-17
1998-01-27
Marschel, Ardin H.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
436501, 536 221, 536 231, 536 241, 536 243, 536 253, C12Q 168, G01N 33566, C07H 1900, C07H 2102
Patent
active
057120994
DESCRIPTION:
BRIEF SUMMARY
SUMMARY OF THE INVENTION
The invention relates as to a process for the production of arabinonucleosides of general formula I ##STR3## in which X represents a hydrogen atom or a fluorine atom, ##STR4## in which X has the above-mentioned meaning, and the groups these compounds.
As is generally known, the arabinonucleosides of general formula I, 9.beta.-D-arabinofuranosyl-9H-purine-6-amine (=vidarabine) and 9.beta.-D-arabinofuranosyl-2-fluoro-9H-purine-6-amine (=fludarabine) are pharmacologically active substances that are distinguished by an antiviral and cytostatic action (EP-A 317,728 and WO 9209604).
According to the known prior art, these compounds can be produced from the triacetates of general formula II by reacting an ethanolic ammonia solution with said triacetates (J. Org. Chem. 33, 1968, 432 ff; Can. J. Cem. 59, 1981, 2608 ff and Nucleic Acids Symp. Ser. 1981, 9, 61 ff). This process is not only quite expensive, but it also has the drawback that heavily contaminated process products are obtained, especially in the synthesis of fludarabine.
In contrast, the process according to the invention can be implemented at relatively low cost and produces the desired process products with high purity.
The enzymatic hydrolysis of the triacetates of general formula II takes place quantitatively even at high substrate concentrations. This is surprising to one skilled in the art since it is fairly well known that acetates of multivalent alcohols often are only partially saponified enzymatically (Tetrahedron 46, 1990, 6587-6611).
The process according to the invention can be implemented with commercially available carboxylic acid esterases or lipases. Such are, for example, about 130 U/mg of protein (1 U corresponds to the amount of enzyme that reacts 1 .mu.mol of butyric acid ethyl ester at 25.degree. C. and pH 8.0), U/mg of protein, about 230 U/mg of protein, 0.5 U/mg of solid, about 110-220 U/mg of prot. (olive oil substrate),
To implement the process according to the invention, the substrate and enzyme are dissolved in a suitable buffer solution (citrate buffer, phosphate buffer, tris buffer, etc.) and shaken or stirred at 30.degree. to 42.degree. C. until complete reaction is accomplished. In this reaction 1 to 10 g of substrate and 1 to 100 mg of enzyme per l are normally used. After the reaction has been completed (which can be determined in a simple way by chromatography such as HPLC or TLC), the isolation of the process product can be isolated in a simple way by concentrating the reaction mixture by evaporation.
To be able to use the esterase several times, it is advisable to immobilize the latter in an already known way (Tetrahedron 26 (no4), pp. 407-410 (1985)) or to use a commercially available immobilized esterase (such as, for example, that on Eupergit.RTM. (immobilized esterase from hog liver from the Fluka AG company)) to implement the reaction.
The following embodiments are used to explain the process according to the invention in more detail.
EXAMPLES
Example 1
100 ml of 0.25 molar aqueous tris-(hydroxymethyl)-aminomethane/HCl buffer of pH 8.7 is mixed with 0.500 g of (2',3',5'-tri-O-acetyl-9.beta.-D-arabinofuranosyl)-2-fluoro-9H-purine-6-am ine and 0.1 mg of hog liver-esterase (Boehringer Mannheim; 130 U/mg of protein) and stirred for 70 hours at 37.degree. C.
After HPLC, the reaction solution obtained is virtually free of 9.beta.-D-arabinofuranosyl-2-fluoro-9H-purine-6-aminomonoacetate or -diacetate. It is concentrated by evaporation at a maximum bath temperature of 50.degree. C. to about 5 ml, and the precipitated product is suctioned off, washed with water and dried in a vacuum at 100.degree. C. 9.beta.-D-Arabinofuranosyl-2-fluoro-9H-purine-6-aminine is obtained in a quantitative yield.
Example 2
Under the conditions of Example 1, 0.500 g of (2',3',5'-tris-O-acetyl-9.beta.-D-arabinofuranosyl)-2-fluoro-9H-purine-6-a mine and 0.1 mg of hog liver esterase (Sigma, Chem. St. Louis, USA 230 U/mg of protein) are reacted and worked up, and 9.beta.-D-arabinofuranosyl-2-fluoro-9H-purine-6-ami
Hummel-Marquardt Heidi
Kennecke Mario
Nickisch Klaus
Schmitz Thomas
Tisltam Ulf
Marschel Ardin H.
Riley Jezia
Schering Aktiengesellschaft
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