title: Preparation of (6R)-tetrahydrofolic acid and (6R)-5-formyltetrahydrofolic acid of high stereochemical purity.
abstract: Commercially available 5-formyltetrahydrofolate (5-CHO-H4PteGlu) is chemically prepared in a reaction that introduces an asymmetric center at the 6 carbon, and hence is the mixture of diastereomers differing in chirality about this position. (6R)-5-CHO-H4PteGlu, the diastereomer that is not normally found in vivo, was prepared from folic acid. Folic acid was chemically reduced and (6R)-tetrahydrofolate (H4PteGlu) was obtained from the resultant (6R,S)-H4PteGlu by enzymatic consumption of the natural diastereomer of (6R,S)-5,10-CH2-H4PteGlu (reversibly formed from (6R,S)-H4PteGlu in the presence of formaldehyde) with Lactobacillus casei thymidylate synthase. The 5 position of purified (6R)-H4PteGlu was directly formylated in a carbodiimide-catalyzed reaction. The level of contamination of these preparations with the corresponding 6S diastereomers was estimated using the binding of fluorodeoxyuridylate to thymidylate synthase promoted by folate cofactor (for H4PteGlu) and by the growth of folate requiring bacteria (for 5-CHO-H4PteGlu). Purified preparations of (6R)-H4PteGlu promoted the binding of fluorodeoxyuridylate to L. casei thymidylate synthase (in the presence of formaldehyde) only at concentrations greater than 1000-fold higher than equiactive levels of (6S)-H4PteGlu. Likewise, the (6R)-5-CHO-H4PteGlu made by this method was 600 times less active as a growth factor for Pediococcus cerevisiae than was authentic (6S)-5-CHO-H4PteGlu. Hence, the minimum stereochemical purity of these preparations was 99.9% for (6R)-H4PteGlu and 99.8% for (6R)-5-CHO-H4PteGlu.
mesh_terms: D005468:Fluorodeoxyuridylate / Q000378:metabolism; D007780:Lacticaseibacillus casei / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development; D002955:Leucovorin / Q000138:chemical synthesis* / Q000494:pharmacology; D010084:Oxidation-Reduction; D010376:Pediococcus / Q000187:drug effects / Q000254:growth & development; D011485:Protein Binding / Q000187:drug effects; D013237:Stereoisomerism; D013763:Tetrahydrofolates / Q000138:chemical synthesis* / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000378:metabolism
chemical_list: D013763:Tetrahydrofolates; D005468:Fluorodeoxyuridylate; C030371:5,6,7,8-tetrahydrofolic acid; D013940:Thymidylate Synthase; D002955:Leucovorin