SITE-SPECIFIC DEUTERATION OF TYROSINE AND THEIR ASSAY BY SELECTIVE ION MONITORING METHOD

概要

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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°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 about 60%. The racemic mixture was resolved enzymatically. L-Tyr-d2 was prepared by refluxing the resultant Tyr-d4 with 5.5 N HCI. The deuterated amino acids were analyzed as the N, O-bis (trifluoroacetyl) methyl ester or 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/Tyr-do and Tyr-d2/Tyr-do 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.