Studies on Phenylalanine Metabolism by Use of Tracer Techniques

—Synthesis and Physicochemical Features of L-Phenylalanine [arom.-d₅] —

Abstract

Because of the reluctance to use radioactive isotopes in man no quantitative kinetic data are available for a wide variety of metabolic processes. However, recent developments in the use of gas chromatography/mass spectrometry (GC-MS) prompted this preliminary study to devise techniques using stable isotope (SI) labeled metabolites. Phenylalanine (Phe) was chosen as this prototype amino acid because its sensitive and specific assays are needed in the diagnosis of such inborn errors of metabolism as phenylketonuria. L-phenylalanine [arom.-d₅] was synthesized from benzaldehyde [arom.-d₅] ; yield being about 18% of theoretical.
Prior to the quantitative determination of deuterated metabolites we have to determine the isotopic purity of Phe-d₅. In IR of Phe-d₅, 740 and 690 cm^-1 band, which are assigned to the out-of-plane vibration mode of C_arom, -H of monosubstituted benzene derivatives, disappeared completely, and 2, 280 cm^-1 band, which is assigned to the stretching vibration mode of C_arom.-d, appeared. Moreover, in NMR of Phe-d₅, the signal of aromatic proton (7.25 ppm) was missing completely. From these data, we concluded Phe-d₅ obtained here did not contain a perceptible amount of hydrogen on its benzene ring. Phe and Phe-d₅ were measured as the enaminemethyl ester derivatives by GC-MS. Comparison of MS of the Phe-d₅ ester with that of the Phe ester suggested that neither isotope effect in the fragmentation between protio. and deuterio forms nor d-p exchange reaction in mass spectrometer was observed. On the mass fragmentogram retention time of deuterio form was shoter than that of protoo form. It suggests that the quantitative determination must be done by the use of MID.