Synthesis of (6S)-5methyl-5,6,7,8-tetrahydrofolic acid

Abstract

A process for the large-scale chemoenzymatic production of (6S)-5-methyl-5,6,7,8-tetrahydrofolic acid, also known as (6S)-5-methylTHFA, the process comprising the steps of: (1) reducing folic acid (FA) so as to yield dihydrofolic acid (DHFA); (2) stereoselectively reducing DHFA with dihydrofolate reductase (DHFR) in the presence of NADP / NADPH, glucose and GluDH so as to yield (6S)-THFA; (3) converting (6S)-THFA to (6S)-5-methylTHFA; and (4) isolating (6S)-5-methylTHFA.

Description

REFERENCE TO PENDING PRIOR PATENT APPLICATION[0001]This patent application claims benefit of pending prior U.S. Provisional Patent Application Ser. No. 60 / 760,562, filed Jan. 20, 2006 by Gerald S. Jones, Jr. for SYNTHESIS OF (6S)-5-METHYL-5,6,7,8-TETRAHYDROFOLIC ACID (Attorney's Docket No. CHEMIC-6 PROV), which patent application is hereby incorporated herein by reference.FIELD OF THE INVENTION[0002]This invention relates to the chemical synthesis of tetrahydrofolic acid in general, and more particularly to a method for the large-scale chemoenzymatic production of (6S)-5-methyl-5,6,7,8-tetrahydrofolic acid, also sometimes referred to as (6S)-5-methylTHFA.BACKGROUND OF THE INVENTION[0003]Folic acid (FA), also known as pteroyl-L-glutamic acid, is a vital co-factor in enzymatic reactions necessary for the synthesis of nucleic acids, amino acids, and other biological molecules. The structure of folic acid is shown in FIG. 1, and again in FIG. 2 where n=1. Although many organisms are cap...

AI Technical Summary

Problems solved by technology

Although many organisms are capable of synthesizing folic acid, humans are unable to synthesize folic acid, and must depend on adequate dietary intake of this essential nutrient.

Without adequate intake of folic acid, humans may develop a folate deficiency.

It has also been found that insufficient folate levels during pregnancy correlate with the occurrence of neural tube defects in newborns.

Low folate levels may also lead to megaloblastic anemia, a disorder which results in inadequate production of red blood cells, particularly during pregnancy and in geriatrics.

Although folic acid is currently added to all commercial over-the-counter (OTC) vitamin preparations, and to some foods, folic acid is not the primary form of folate which is found naturally in fresh foods.

Although the importance of folic acid in the diet has been recognized, prior and widespread use of reduced folates as dietary supplements has been limited, in part by the stereochemistry of these compounds.

However, a low order of biological activity has been ascribed to the unnatural isomers and, more importantly, it appears that the unnatural isomers may have an inhibitory effect upon certain enzymatic processes.

Some of these methods produce large volumes of undesirable by-products which need to be removed, and thus negatively influence the economy and efficiency of the process.

Others of these methods require multiple fractionations / recrystallizations to achieve a product of high diastereomeric excess, and therefore can be time-consuming and costly.

In addition to the foregoing, there is an approach which uses the chromatographic separation of diastereomers, but it does not lend itself to large-scale production of pure folate isomers.

Furthermore, there is a method which synthesizes (6S)-THFA via the stereoselective catalytic hydrogenation of dihydrofolic acid (DHFA), but the cost of the exotic organometallic catalyst(s) is prohibitive for large-scale production.

A chemoenzymatic method has also been described for producing small quantities of (6S)-THFA and derivatives, but this latter method is typically regarded as unsuitable for commercial application due to its complexity (Tetrahedron 1986, 42, 117-136).

Thus, the foregoing approaches do not provide a cost-effective, large-scale method to produce the pure reduced folates (e.g., (6S)-THFA).

While the aforementioned enzymatic process of U.S. Pat. No. 4,929,551 is adequate to produce L-formylfolate, it is not adequate to produce L-methylfolate.

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