Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1992-11-05
1996-01-02
Rollins, John W.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 268, C07H 2102
Patent
active
054809827
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a crystalline potassium salt of thionicotinamide adenine dinucleotide phosphate (referred to as thio-NADP.K salt hereinafter) and the method for producing the same.
Conventionally known non-crystalline thio-NADP.Na salt (referred to as thio-NADP.Na salt hereinafter) shows distinctive hygroscopic property enough to deliquesce on contact with air, so that the storage thereof has been very difficult.
Because the thio-NADP.K salt in accordance with the present invention has no hygroscopic property to deliquesce on contact with air, the salt is extremely useful for the utilities as research reagents, diagnostic agents and the like.
PRIOR ART
It is generally known that thionicotinamide adenine dinucleotide phosphate (referred to as thio-NADP hereinafter), a product from non-natural origins, has a similar level of the coenzyme activity to that of .beta.-nicotinamide adenine dinucleotide phosphate (referred to as .beta.-NADP) as one of the redox coenzymes in living organisms. Based on the property, the utility thereof as a pharmaceutical agent such as a clinical diagnostic agent is under development, as well as the utility as a research agent for biochemistry and physiology. For the measurement of enzyme activity for clinical diagnostics and the analysis of substrate concentration for enzymatic analysis, thio-NADP is far more advantageous than .beta.-NADP in terms of sensitivity and in that reductive-type thio-NADP is colored. The increase in the purity and stability of thio-NADP is required.
A method for producing thio-NADP has conventionally been known, comprising generating thio-NADP through the catalytic activity of NAD nucleosidase (EC 3.2.2.5) in the presence of thionicotinamide and .beta.-NADP, purifying the thio-NADP on a Dowex column, and recovering the purified thio-NADP as an alcohol precipitate followed by drying in vacuum [Japanese Patent Publication No.47-14919; Biochemistry, 2, 1015-1017 (1963)].
However, the thio-NADP-Na salt thus obtained is amorphous, and shows an enriched hygroscopic property, resulting in the deliquescence thereof on contact with air, so that the salt requires special treatment for storage and transport, for example, air degassing and freezing at -20.degree. to -30.degree. C.
Even after crystallization process, the thio-NADP.Na salt described above is amorphous without crystallization and it is very difficult to store such salt because of the distinctive hygroscopic property thereof.
The present inventors have made investigations as to how to bring about the long-life shelf stability of thio-NADP. As a result, the inventors have found that thio-NADP can be prepared into the potassium salt thereof prior to the crystallization, whereby the resulting salt can be stored for a long period of time because the salt does not show nearly any hygroscopic property.
The present invention relates to a crystalline thio-NADP.K salt and the method for producing the same. The present invention is the first to crystalize thio-NADP as the potassium salt thereof.
Compared with conventional amorphous products, the crystalline thio-NADP.K salt in accordance with the present invention has many advantages in that it is stable because it is highly pure due to no inclusion of inhibitors and in that it has no hygroscopic property.
In accordance with the present invention, the thio-NADP in the form of free acids or salts is prepared into an aqueous solution of the potassium salt, which is then adjusted to pH 1.5 to 5.0, preferably pH 2.0, followed by addition of a hydrophlic organic solvent until the aqueous thio-NADP.K solution begins to get turbid to subsequently be left to stand under cooling. The crystalline thio-NADP.K salt is separated preferably after the completion of the crystallization thereof. As the hydrophilic organic solvent, preferably methanol is used at 0.15 to 2.0-fold, preferably at 0.3 to 1.0-fold the amount of the thio-NADP.K solution, and the above procedure is effected at a given temperature, preferably at room temperature,
REFERENCES:
patent: 4151349 (1979-04-01), Traeger et al.
Fluka Catalog, Chemika-BioChemika, Fluka Chemie AG, Buchs, Switzerland, 1993, p. 1270.
Abraham Stein et al, "The Thionicotinamide Analogs of DPN and TPN. I. Preparation and Analysis," Biochemistry, vol. 2, No. 5, pp. 1015-1017, Sep.-Oct. 1963.
Fujita Tsuyosi
Kitahara Tetsuo
Nonobe Masatsugu
Sugita Kouji
Crane L. Eric
Oriental Yeast Company, Ltd.
Rollins John W.
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