Abstract
Publisher Summary This chapter discusses the method for preparation of N 5 -formyltetrahydrofolate labeled stereospecifically with deuterium or tritium at Carbon-6. N 5 -formyl-l,L-tetrahydrofolate has the naturally occurring configuration at carbon-6 of the pteridine ring and is known as citrovorum factor (CF), whereas synthetic preparations that consist of a mixture of diastereoisomers are known as leucovorin or folinic acid. In the method discussed in this chapter, dihydrofolate prepared, by dithionite reduction of folate is converted enzymically to 1,L-tetrahydrofolate, using dihydrofolate reductase and labeled nicotinamide adenine dinucleotide phosphate (NADP)H containing deuterium or tritium at carbon-4 on the A-side of the dihydronicotinamide ring. The labeled l,L-tetrahydrofolate is converted to N 5 -formyltetrahydrofolate, enzymically using N -formylglutamate and NS-formyltetrahydrofolate: glutamate transformylase. The CF prepared by this method was determined to be completely active biologically, by using it to regenerate tetrahydrofolate, with N -formyltetrahydrofolate: glutamate transformylase and assaying the tetrahydrofolate formed with thymidylate synthetase. The position of the label introduced by this procedure was determined, by preparing deuterium-labeled CF and examining the purified product, by proton magnet resonance (PMR) spectroscopy. The nuclear magnetic resonance results established unequivocally that the label is located at carbon-6 of the pteridine ring.
Published Version
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