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

Introduction: Thymidylate synthase and folate receptors are well-developed targets of cancer therapy. Discovery of a simple and fast method for the conversion of 11CH3Ito[11C]-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.

Methods: In 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.

Results: The 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.

Conclusion: We 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.