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M. KOZŁOWSKA ET AL.
Methods
The presence of tyrosine and DOPA was checked qualitatively by TLC using
silica gel plates and developing solvents: (n-butanol–acetic acid–water – 4:1:1
v/v for tyrosine, and acetonitrile–water – 4:1 v/v for DOPA). The
concentration of the above-mentioned compounds was determined spectro-
photometrically as described elsewhere.21–24 The extent of deuterium
incorporation at 30 and 50 of phenyl ring in deuteriated l-Tyr was checked
by measuring the proton NMR spectra on a Varian 500 MHz Unity-Plus
spectrometer. The radioactivity of all samples was determined using an
automatic liquid scintillation counter, LSC, (LISA LSC PW470 – Raytest,
Germany).
Synthesis of [30,50-3H2]-l-Tyr, 1
To the glass reaction ampoule were added in turn: 200 mg (1.1 mmol) of l-
tyrosine, 0.2 ml of conc. HCl, and 1 ml of tritiated water with total activity
5.2 GBq. The contents of the ampoule were frozen with liquid nitrogen,
degassed under vacuum and the vial was sealed and thermostated at 1308C for
24 h. After cooling the ampoule has been opened and tritiated water was
removed by lyophylization. The residue was dissolved in a small amount of
water (about 1 ml) and loaded onto a column (Dowex 50WX-4-50 H+ form).
To remove the residual HTO and tritium incorporated into labile positions of
tyrosine (-NH2, -COOH groups) the column was washed with water up to the
moment when the radioactivity of eluted fractions was steady and close to
background. Next 1 was eluted with 0.5 M NH3(aq) and collected as 3 ml
fractions. For each fraction 10 ml samples were taken for the radioactivity
assay. The fractions containing 1 were combined and evaporated under
vacuum at 508C and the purity checked by TLC. As a result a sample of
137 mg (0.75 mmol) of 1 was obtained with a total activity of 4.16 MBq
(5.5 ꢀ 106 Bq/mmol specific activity) and chemical yield 75%.
Synthesis of [50-3H]-l-DOPA, 2
To a vial containing 10 ml of phosphate buffer (pH 6.1), 22 mg (122 mmol) of 1
of total radioactivity 6.7 ꢀ 105 Bq, 44 mg (0.25 mmol) of ascorbic acid, and
0.5 ml (1250 U) of the enzyme mushroom tyrosinase (EC 1.14.18.1) from
Neurospora crassa were added. The reaction mixture was incubated at room
temperature for 20 min with aeration and stirring. The volume of post-reaction
mixture was reduced to about 1 ml by lyophilization. The product obtained, 2,
was separated on the Dowex1X8 column (10 ꢀ 100) prepared as described
elsewhere.25 The column was eluted with water (about 100 ml) to wash out the
buffer salts and tritiated water up to the moment when the radioactivity of
eluted fractions was steady and close to background. First, unreacted
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 235–240