5,10-Methenyl-THF

To prepare 10-formyl TF1F (formyl donor for the formylation of fMet-tRNAfMet), 10 mg of 5-formyl-tetrahydrofolate (5-formyl THF) are dissolved in 1 ml of 0.1 MHC1 that has been flushed extensively with N2 and kept overnight at 4° in a closed vial. The resulting yellow suspension of 5,10-methenyl THF is agitated and an aliquot withdrawn for spectropho-tometric determination of the concentration (6 mM solution = 85 A355). The expected 5,10-methenyl THF concentration is approximately 20 mM. The cyclic methenyl THF can be stored for several weeks at —20°. The 10-formyl THF is stable for only a few weeks and is freshly prepared hydrolyzing the cyclic methenyl THF. For this purpose, the stock solution is diluted in 100 mM Tris-HCl (pH 8.0) extensively flushed with N2 and supplemented with 100 mM 2-mercaptoethanol. After 15 min incubation at 20°, the solution is divided in aliquots, which are stored at —20°. 

Figure 1 THF derivatives. 1, 10-formyl-THF 2, 5-formyl-THF 3, 5,10-methenyl-THF 4, 5-formimino-THF 5,...

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