Synthesis of 14C- labeled 6-(S)-5-methyltetrahydrofolic acid for in vivo human metabolic studies

Abstract

Determination of the in vivo human metabolism of 6-(S)- 5-methyltetrahydrofolic acid (5MTHF) would allow proper determination of the bioavailability of this most abundant natural form of folate found from vegetables and helps suggest proper recommended dietary requirements for folate. A quantitative metabolic study on 5MTHF is lacking. Using a ¹⁴C label on the stable glutamate position of 5MTHF as a metabolic tracer would allow one to quantify the total folate metabolism as a coenzyme and with ¹⁴C labelling facilitate true physiological dosing. Likewise, a comparison of the quantitative metabolism in healthy and diseased individuals like those with leukemia would allow one to quantify differences in folate utilization. It would also be interesting to see how one-carbon metabolism varies in healthy people and those with cancer considering that epigenetic changes contribute to carcinogenesis. The study aimed to develop a practical and efficient synthesis of ¹⁴C- labeled 6-(S)- 5- methyltetrahydrofolic acid (¹⁴C-5MTHF) suitable for metabolic studies. To develop a practical method for the purification and characterization of ¹⁴C 5MTHF. To synthesize ¹⁴C labeled 6-(S)-5-methyl tetrahydrofolic acid (¹⁴C5MTHF), [¹⁴C-U]-glutamate was conjugated to pteroyl azide to produce folic acid. The resulting folic acid was reduced to dihydrofolic acid using dithionite and further reduced to (6S) tetrahydrofolic acid using dihydrofolate reductase (DHFR) from chicken liver. Addition of formaldehyde solution followed by reduction with NaBH4 produced 14C- 5MTHF. High-Pressure Liquid Chromatography (HPLC) using a semi-preparative C-18 stationary phase and a 6-25 % acetonitrile gradient in 30 mM phosphate buffer pH 2.2 as mobile phase was done to purify the final product. Liquid scintillation counting (LSC) and mass spectrometry (MS) analysis were conducted to assess the radiochemical purity of synthesized 1 4C-5MTHF. A practical synthesis of 14C-5MTHF was accomplished through a onepot procedure of chemo enzymatic synthesis – from coupling of radiolabel up to the chemo-enzymatic reduction (five synthetic steps) to the final product. Overall yields of 58% through this one-pot reaction procedure was accomplished. Identity was confirmed by HPLC co-elution with a standard. The amount of C-14 incorporated was estimated to be 2.8 ± 0.4 % by MS analysis comparable to results of LSC. A practical one-pot chemo-enzymatic synthesis of 14C-5MTHF was reported. Purification was accomplished through semi-preparative HPLC on a C-18 column. The radiochemical purity was determined through LSC and MS analysis.