A fast chemoenzymatic synthesis of [11C]-N5,N10-methylenetetrahydrofolate as a potential PET tracer for proliferating cells

IntroductionThymidylate synthase and folate receptors are well-developed targets of cancer therapy. Discovery of a simple and fast method for the conversion of ¹¹CH₃Ito¹¹C]-formaldehyde (¹¹CH₂O) encouraged us to label the co-factor of this enzyme. Preliminary studies conducted on cell lines have demonstrated a preferential uptake of 11-¹⁴C]-(R)-N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (¹⁴CH₂H₄folate) by cancerous cell vs. normal cells from the same organ (Saeed M., Sheff D. and Kohen A. Novel positron emission tomography tracer distinguishes normal from cancerous cells. J Biol Chem 2011;286:33872–33878), pointing out ¹¹CH₂H₄folate as a positron emission tomography (PET) tracer for cancer imaging. Herein we report the synthesis of ¹¹CH₂H₄folate, which may serve as a potential PET tracer.MethodsIn a remotely controlled module, methyl iodide (¹¹CH₃I) was bubbled into a reaction vial containing trimethylamine N-oxide in N,N-Dimethylformamide (DMF) and heated to 70°C for 2 min. Formaldehyde (¹¹CH₂O) formed after the completion of reaction was then mixed with a solution of freshly prepared tetrahydrofolate (H₄folate) by using a fast chemoenzymatic approach to accomplish synthesis of ¹¹CH₂H₄folate. Purification of the product was carried out by loading the crude reaction mixture on a SAX cartridge, washing with water to remove unbound impurities and finally eluting with a saline solution.ResultsThe synthesis and purification of ¹¹CH₂H₄folate were completed within 5 min. High-performance liquid chromatography analysis of the product after SAX purification indicates that more than 90% of the radioactivity that was retained on the SAX cartridge was in ¹¹CH₂H₄folate, with minor (<10%) radioactivity due to unreacted ¹¹CH₂O.ConclusionWe present a fast (～5 min) synthesis and purification of ¹¹CH₂H₄folate as a potential PET tracer. The final product is received in physiologically compatible buffer (100 mM sodium phosphate, pH 7.0 containing 500 mM NaCl) and ready for use in vivo.