Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1993-09-30
1995-12-05
Knode, Marian C.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 84, 435232, 536 187, 536 552, 536125, C12P 1902, C12P 1926, C07H 302, C12N 98
Patent
active
054728606
DESCRIPTION:
BRIEF SUMMARY
This a 371 of PCT/JP92/00757 filed Jun. 12, 1992.
FIELD OF THE INVENTION
The present invention relates to a method for preparing N-acetylneuraminic acid.
DISCLOSURE OF THE INVENTION
N-acetylneuraminic acid is an important substance in that it is a most ubiquitous substance in sialic acids, widely distributed in organs, body fluids and secreted fluid of the living body and that the relation of N-acetylneuraminic acid to hemagglutination, inter-cellular recognition, metabolism of serum proteins and the like are known.
N-acetylneuraminic acid, such an important substance, is hitherto prepared by hydrolysis of capsular polysaccharide of E. coli and also produced by hydrolysis of natural material, such as a nest of petrel, an egg, milk and the like.
The method of using natural material as a raw material has problems in that it is difficult to prepare a large quantity of N-acetylneuraminic acid whose demand is increasing yearly due to the limitation of absolute amount of raw material; separation and purification of N-acetylneuraminic acid from other contaminants after hydrolyzing natural material is not easy; and that preparation cost of the acid is high. An inexpensive mass production technique of N-acetylneuraminic acid is not fully developed.
To solve the problems, synthetic methods using an enzyme have been variously examined.
For example, Kim et al. [J. Am. Chem. Soc., 110, 6481-6486 (1988)] reported a method for preparing N-acetylneuraminic acid by reacting N-acetylmannosamine with pyruvic acid in the presence of N-acetylneuraminic acid lyase. However, this method is not practical in that the method uses N-acetylmannosamine as a starting material which is expensive and difficult to procure in a large quantity.
N-acetylmannosamine can be prepared by isomerizing N-acetylglucosamine in a strong alkaline condition, i.e., pH about 12 [Simon et al., J. Am. Chem. Soc., 110, 7159-7163 (1988)]. However, in this method the proportion of N-acetylglucosamine and N-acetylmannosamine is N-acetylglucosamine: N-acetylmannosamine=3: 1 in an equilibrium state so that the separating operation of N-acetylmannosamine is not easy because of a low proportion of N-acetylmannosamine.
A method for preparing N-acetylneuraminic acid by reacting N-acetylglucosamine with pyruvic acid in the presence of N-acetylneuraminic acid lyase and epimerase is also proposed [e.g., Kragl et al, Angew. Chem. Int. Ed. Engl., 30, 827-828 (1991)]. In this method, N-acetylglucosamine is converted into N-acetylmannosamine successively, and then N-acetylmannosamine is converted to N-acetylneuraminic acid by the action of N-acetylneuraminic acid lyase. However, acquisition of epimerase isomerizing N-acetylglucosamine is difficult, and also the conversion ratio from N-acetylglucosamine to N-acetylneuraminic acid is as low as 28%. This method is not practical either.
It is an object of the present invention to provide a simple and practical method for preparing N-acetylneuraminic acid.
The inventor has conducted extensive research to accomplish the object and found that reacting N-acetylglucosamine with pyruvic acid in the presence of N-acetylneuraminic acid lyase in an alkaline condition, that is, high pH region, which is not used hitherto so as to avoid denaturation of the enzyme and due to departing from optimum pH of N-acetylneuraminic acid lyase, converts N-acetylglucosamine into N-acetylneuraminic acid effectively under a repression of enzyme denaturation because of the protecting effect of starting materials, i.e., N-acetylglucosamine and pyruvic acid.
Thus, the present invention provides a method for preparing N-acetylneuraminic acid characterized in reacting N-acetylglucosamine with pyruvic acid in an alkaline condition by the action of N-acetylneuraminic acid lyase.
As used herein, "alkaline condition" means that the pH value of the reaction mixture is in the range of about 8 to about 12, preferably about 9 to about 12, more preferably about 10 to about 12, and most preferably about 10 to about 11. When the pH value of the reaction mixture is
REFERENCES:
patent: 5071750 (1991-12-01), Kragl et al.
patent: 5162513 (1992-11-01), Wong
Tanase et al., "Novel C-2 Epimerization of Aldoses and Stereosilective Uptake of One of the Epimeric Aldoses by Nickel (11) Complexes", J Chem Soc Commun pp. 1001-1003 1986.
Simon, Ethan S., et al., Jour. Amer. Chem. Soc. 1988, 110, 7159-7163; "Synthesis of CMP-NeuAc from N-Acetylglucosamine: Generation of CTP from CMP Using Adenylate Kinase.sup.1,2 ".
Kim, Mahn-Joo, et al., Jour. Amer. Chem. Soc., 1988, 110, 6481-6486; "Enzymes in Carbohydrate Synthesis: N-Acetylneuraminic Acid Aldolase Catalyzed Reactions and Preparation of N-Acetyl-2-deoxy-D-neuraminic Acid Derivatives".
Kragl, Udo, et al.; Ang. Chem. Int. Ed. Engl. 30 (1991) No. 7; "Enzymatic Two-Step Synthesis of N-Acetylneuraminic Acid in the Enzyme Membrane Reactor".
Ohta Yasuhiro
Tsukada Yoji
Knode Marian C.
Marukin Shoyu Co., Ltd.
Prats Francisco C.
LandOfFree
Method for preparing N-acetylneuraminic acid by N-acetylneuramin does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for preparing N-acetylneuraminic acid by N-acetylneuramin, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for preparing N-acetylneuraminic acid by N-acetylneuramin will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1373543