Chemistry of carbon compounds – Dipeptides – e.g. – aspartame – anserine – carnosine – etc.
Patent
1982-03-30
1984-10-23
Sutto, Anton H.
Chemistry of carbon compounds
Dipeptides, e.g., aspartame, anserine, carnosine, etc.
260941, C07F 940
Patent
active
044787626
DESCRIPTION:
BRIEF SUMMARY
This application is based upon, and claims the priority of, International Application No. PCT/US82/00402 which was filed Mar. 29, 1982.
TECHNICAL FIELD
This invention relates to organic chemical synthesis and, in particular, synthesis of phosphorlyating agents.
BACKGROUND ART
Phosphoenolpyruvic acid (PEP) represents a particularly efficient phosphorylation agent, especially for organic syntheses promoted by enzymatic catalysis in which it is desired to regenerate the ATP or adenosine triphosphate. It thus constitutes a useful alternative to acetyl phosphate (AcP), over which it has a number of advantages. In fact, PEP is recognized as a more powerful phosphorylation agent than AcP, it is more stable in solution, and it, therefore, is used more easily, particularly in semi-industrial applications.
This invention concerns a process of the preparation of phosphoenolpyruvic acid in the form of its potassium salt, or PEP-K. Since the potassium is inert towards most enzymatic systems, and its presence is even necessary, for example, to release the activity of pyruvic kinase, the salt thus obtained can be used directly in phosphorylation processes.
SUMMARY OF THE INVENTION
The process of the invention consists of two steps carried out consecutively, as follows: a dialkyl phosphate of enolpyruvic acid of the formula ##STR1## in which the symbols R, taken separately, represent a lower alkyl radical, or taken together, represent a lower alkylenyl radical, and presence of potassium hydroxide.
The process of the invention can be illustrated with the use of the following scheme: ##STR2##
The potassium salt thus obtained, insoluble in the reaction medium, can easily be separated from the reaction mixture by simple filtration and can thus be used directly as a phosphorylation reagent.
In this regard, in order to establish its activity, a sample of the product prepared by the process of the invention was used to synthesize glucose-6-phosphate. The method followed is specified in detail below.
A solution of 0.800 mole of glucose, 0.800 mole of PEP-K 35 mmoles of magnesium chloride, and 10 mmoles of 2-mercaptoethanol in 800 ml of distilled water was brought to pH 7.6 with solid KOH, and was then transferred into a 2-liter 3-necked flask equipped with a pH-measuring electrode and a magnetic stirrer. The solution was degassed by a stream of argon, and 1.20 mmoles of ATP and an aqueous suspension (0.78 liter) of pyruvic kinase (1260 U) and of hexokinase (863 U) (each enzyme being previously immobilized in a polyacrylamide gel--see J. Am. Chem. Soc. 102, 6324, [1980] --) were then added to it under an argon atmosphere. The reaction mixture, held at 20.degree. C. and at pH 7.5-7.6 by the occasional addition of several drops of 12 M HCl, was stirred for 8.5 days. The biochemical tests carried in accordance with the methods described by H. V. Bergmeyer in "Methods of Enzymatic Analysis", Verlag Chemie Weinheim, Academic Press, N.Y. (1974), showed the presence of 0.77 mole of glucose-6-phosphate G-6-P (yeild 96%).
As indicated above, the substituents R can represent either a lower alkyl radical, preferably methyl or ethyl, or a bivalent alkylenyl radical, such as --CH.sub.2 --CH.sub.2 --, for example. Thus, for example, trimethyl phosphite, triethyl phosphite, or methyl ethylene phosphite can be used as the trialkyl phosphite, with trimethyl phosphite being preferred.
As the halopyruvic acid, chloropyruvic or bromopyruvic acid can be used. These compounds can be obtained by known procedures.
The first step of the process of the invention is carried out in an organic medium, in the presence of a solvent such as an ether, diethyl ether, for example, an ester such as ethyl acetate, or an aromatic hydrocarbon, for example, toluene or benzene. The best yields have been observed when using diethyl ether.
The yields of the following step, which consists of the hydrolysis of the phosphate obtained, depend to a great extent on the quantity of potassium hydroxide used. Thus, the optimal value of the pH is approximately 1.7-2.8, whic
REFERENCES:
patent: 2685552 (1954-08-01), Stiles
Webster et al., "Biochem. J.", (1976), 155, pp. 433-441.
Kosolapoff et al., "Organic Phosphorus Compounds", vol. 6, (1974), pp. 260-261.
Hirschbein Bernard L.
Mazenod Francois P.
Whitesides George M.
Engellenner Thomas J.
Massachusetts Institute of Technology
Smith, Jr. Arthur A.
Sutto Anton H.
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