Synthesis of Carbon-14-labeled Natural Folate and alpha-Tocopherol and Progress Towards a Synthesis of Carbon-14-labeled Lutein for in vivo Human Metabolic Studies

14C labeled compounds are valuable tools in elucidating their human metabolism. Towards this goal we completed a practical synthesis of 14C labeled 6-(S)-5-methyl tetrahydrofolic acid (5MTHF), the most abundant form of natural folate and [5-14CH3 ] - (2R, 4'R, 8'R)-α-tocopherol, the natural form of vitamin E.;[14C-U]-glutamate was conjugated to pteroyl azide to produce folic acid. The resulting folic acid was then 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 5MTHF with yields of 58% over-all in a one-pot reaction. 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.;[5-14CH3] -(2R, 4'R, 8'R)- α-tocopherol was synthesized in a one-pot two step reaction from α-(+)-tocopherol, C-14 formaldehyde and morpholine first by forming a methyl morpholino derivative followed by reduction with sodium cyanoborohydride. Reversed phase HPLC using 90% ethanol in water was carried out to purify the product. The identity of C-14 labeled 2R, 4'R, 8'R- α-tocopherol was confirmed by 1H and 13C NMR spectroscopy and the molecular mass determined by mass spectrometry. Specific activity was 46.8 mCi/mmol with a total yield of 90%.;In an effort to synthesize 14C lutein, we report an approach to hydroxylate and generate the â-ring of lutein from á-ionone through SeO2 oxidation. It is hypothesized to proceed through hydroxylation followed by isomerization. Likewise, a direct hydroxylation of α-ionone was carried out using a Streptomyces whole- cell culture to generate the other fragment containing the ϵ-ring. Both of the resulting racemic hydroxyionones were separated as R-Mosher’s esters in a R,R-Whelk-O2 column using methanol- isopropanol-water (64:4:32, v/v/v/) as mobile phase. Homologation by two carbons to generate the hydroxy ionylidene acetaldehyde was accomplished using Knoevenagel condensation with cyanoacetic acid in piperidine followed by DIBAL-H reduction.