Site-specific deuteration of tyrosine and their assay by selective ion monitoring method

L-[2',3',5',6'-d]Tyrosine (Tyr-d4) and L-[2',6'-d[Tyr (Tyr-d2) were prepared. Tyr was heated in conc. DCl at 180 degrees C for 4 hr to obtain DL-[2,2',3',5',6'-d]Tyr (Tyr-d5), then the deuterium on the 2-position of the compound was successfully replaced with proton by refluxing with an acetic acid-acetic anhydride mixture. The isotopic purity of DL-Tyr-d4 was aobut 60%. The racemic mixture was resolved enzymatically. L-Tyr-d2 was prepared by refluxing the resultant Tyr-d4 with 5.5N HCl. The deuterated amino acids were analyzed as the N,)-bis (trifluoroacetyl) methyl ester of N,O-bis(heptafluorobutyryl) methyl ester derivatives by the gas chromatography-selective ion monitoring method (SIM). The calibration curves showed good linearity in the range where the molar ratios of Tyr-d4/Ttr-d0 and Tyr-d2/Tyr-d0 were above 5/1,000. The results indicate that the protein-bound Tyr-d4 may possibly be determined as Tyr-d2 after hydrolysis of protein.