Synthesis of tritium labelled [2′,6′]-L-tyrosine

Article abstract of DOI:10.1002/jlcr.886

The synthesis of a specifically ring labelled isotopomer of L-tyrosine, (L-Tyr), using a combination of chemical and enzymatic methods is reported. The tritium labelled [2′,6′]-L-Tyr has been synthesized via catalytic exchange of phenol with tritiated wat

Full text of DOI:10.1002/jlcr.886

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 977–981.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.886
Research Article
Synthesis of tritium labelled [20,60]-l-tyrosine
!
W. Augustyniak, R. Kanski and M. Kanska*
!
Department of Chemistry, Warsaw University, Pasteur 1 Street, 02-089 Warsaw,
Poland
Summary
The synthesis of a specifically ring labelled isotopomer of l-tyrosine, (l-Tyr), using a
combination of chemical and enzymatic methods is reported. The tritium labelled
[20,60]-l-Tyr has been synthesized via catalytic exchange of phenol with tritiated water
in the presence of K2PtCl4, reverse acid catalysed removal of tritium from the o-
and p-positions of phenol, and subsequent condensation of the resulting [30,5-3H2]-
phenol with S-methyl-l-cysteine using the enzyme b-tyrosinase from Citrobacter
freundii. Copyright # 2004 John Wiley & Sons, Ltd.
Key Words: tritium; deuterium; tyrosine; enzyme; optical isotopomer
Introduction
The enzyme b-tyrosinase (tyrosine phenol lyase, EC 4.1.99.2) has been
shown1–3 to catalyze the decomposition of l-Tyr to phenol, pyruvate and
ammonia. Under some conditions this enzyme also participates in the reverse
reaction leading to formation4–6 of l-Tyr (Scheme 1).
As the metabolism of l-Tyr is an important process of living cells, the
mechanism of the above reaction is of special interest, particularly biologists.
Scheme 1. Decomposition of l-tyrosine catalysed by enzyme b-tyrosinase
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*Correspondence to: M. Kanska, Department of Chemistry, Warsaw University, Pasteur 1 Street, 02-089
Warsaw, Poland. E-mail: mkanska@alfa.chem.uw.edu.pl
Contract/grant sponsor: Committee for Scientific Research (Poland); contract/grant number: KBN 4 T09A
039 23
Copyright # 2004 John Wiley & Sons, Ltd.
Received 14 June 2004
Revised 26 July 2004
Accepted 16 August 2004

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W. AUGUSTYNIAK ET AL.
This multistep reaction involving hydrogen transport and formation or
rupture of the bond between the C1 ring and C3 side chain carbon atoms is still
not clear. Some mechanistic questions can be answered by determining kinetic
isotope effects (KIE) for each atom involved in the postulated rate determining
step.7,8 For this purpose the l-enantiomer form of specifically tritium labelled
tyrosine is needed. In the literature there are several papers which describe the
synthesis of deuterium9–15 and tritium16–20 labelled tyrosine. Unfortunately
most of them are not useful for our proposed KIE studies. In our earlier
paper21 we described the synthesis of [20,60-3H2]-l-Tyr by hydroxylation of
commercial [20,6-3H2]-l-phenylalanine using the enzyme l-phenylalanine
hydroxylase.21
Results and discussion
The new route to the synthesis of [20,60-3H2]-l-Tyr developed by us consists of
a combination of chemical and enzymatic methods. First, the key intermediate
i.e., [3,5-3H2]-phenol was obtained as a result of H/T exchange between phenol
and tritiated water. In turn it was condensed with S-methyl-l-cysteine using
the enzyme b-tyrosinase from Citrobacter freundii (Scheme 2).
The literature data22–26 show that phenol can be catalytically exchanged
with deuteriated or tritiated water selectively in the o- and p-positions
or per labelled. By the reverse acid catalysed exchange of uniformly
tritiated phenol, 2, with water it is possible to prepare the 3,5-tritiated
substrate, 3, needed for the enzymatic synthesis of the final product 4
(Scheme 2).
Scheme 2. Synthesis of [20,60-3H2]-l-tyrosine
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SYNTHESIS OF TRITIUM
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To check these expectations we have investigated the H/D exchange between
phenol and heavy water catalyzed with acid and a salt of divalent platinum.
The degree of incorporation and removal of deuterium from the aromatic ring
1
was determined using the H NMR spectra of samples of exchanged phenol.
Homogeneous, K2PtCl4 catalysed exchange gives phenol deuteriated in the o-
and p-positions22, 24–26 as well as the m-positions. Also the reverse acid
exchange of uniformly deuteriated phenol with water does not remove label
from the m-position, giving selectively labelled [3,5-2H2]-phenol.
Therefore, in the first step we prepared uniformly ring tritiated phenol, 2, by
H/T exchange with HTO in the presence of K2PtCl4 catalyst. In the next step
reverse acid catalysed exchange of 2 with water removes26 the tritium label
from the o- and p-positions of 2 leading to the [3,5-3H2] product 3.
Experimental
1. Materials: Tritiated water was obtained from INC Pharmaceutical Inc.,
USA. Deuteriated water was purchased from The Radioisotope Production
and Distribution Center, Poland. Scintillation cocktails were obtained from
Rotiszint (Germany). Amberlit IR-120 (2–50 mesh) and TLC plates were
the products of Aldrich and Merck, respectively. Cofactor i.e., pyridoxal 5-
phosphate, PLP, S-methyl-l-cysteine, mercaptoethanol (HSCH2CH2OH)
were purchased from Sigma. The enzyme b-tyrosinase (EC 4.1.99.2) from
Citrobacter Freundii was kindly given by Prof. R. Phillips from the
University of Georgia, Athens, USA.
2. Methods: The concentration of l-Tyr was determined spectrophotometri-
cally27 using a sensitive and reproducible method for assaying of tyrosine in
biological media.28 Under appropriate conditions tyrosine reacts with l-
nitroso-5-naphthol to yield a stable yellow product, the concentration of
which can be determined by measuring the absorbance at 490 nm using a
Shimadzu-UV-VIS-102 CE-LV spectrometer (Japan). The catalyst i.e.,
K2PtCl4, was prepared according to the literature.29 In the preliminary
deuterium labelling studies of phenol the degree and position of
1
incorporation of the label was determined from H NMR spectra using a
Varian 500 MHz NMR spectrometer. For the tritium work the radioactivity
was determined using a liquid scintillation counter (Lisa LSC470-Raytest,
Germany).
3. [G-3H]-phenol, 2. This intermediate was synthesized by isotope exchange
between phenol and tritiated water catalysed by the salt of divalent
platinum. To the glass ampoule 1 g (10.6 mmol) of phenol, 100 mg
(0.24 mmol) of K2PtCl4, 0.2 ml of concentrated HC1 and 0.5 ml of tritiated
water with a total activity 200 MBq were added. The ampoule was frozen
with liquid nitrogen, degassed, sealed and heated at 1008C for 24 h. After
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W. AUGUSTYNIAK ET AL.
cooling, the ampoule was opened and 5 ml of water was added to the post-
reaction mixture. Phenol was separated from the catalyst by extraction
(5 ꢀ 5 ml) with diethyl ether. Next, the combined organic layers were
washed (5 ꢀ 10 ml) with water. The solvent from the organic layer was
evaporated yielding 850 mg (about 9 mmol) of crude 2 of total radioactivity
36.8 MBq (about 85% chemical yield).
4. [3,5-3H2]-phenol, 3. The above crude sample of 2 dissolved in 12 ml of 2M
HCl was placed in an ampoule, degassed as above, sealed, and heated at
1008C for 24 h. The post reaction mixture was extracted with 5 ꢀ 5 ml of
diethyl ether. Combined organic layers were washed with 2 ꢀ 5 ml of water.
The solvent was evaporated off under reduced pressure. The phenol was
separated from the residue by sublimation (1108C, about 1 Pa). As a result
675 mgs (7.18 mmol) of 3 of total radioactivity 4.5 MBq (627 kBq/mmol
specific activity) were obtained (79.3% chemical yield).
5. [20,60-3H2]-l-tyrosine, 4. The enzymatic reaction was carried out in
an encapped glass vial. To this 4 ml of 0.1 M phosphate buffer (pH 8),
29.8 mg (221 mmol) of S-methyl-l-cysteine, 0.1 mmol of PLP, 1 mmol
of mercaptoethanol and 20 U of b-tyrosinase (EC 4.1.99.2 ) from
Citrobacter freundii were added. To this vial the solution of 15.3 mg
(163 mmol) of 3 dissolved in 0.5 ml of phosphate buffer was added in 5 equal
portions at the start and subsequently after 17, 25, 41 and 49 h of
the incubation. The incubation was carried out at 308C for 5 days. The
precipitated crystals of 4 were collected and washed with ethanol
and diethyl ether. As a result 5.3 mg (29 mmol) of 4 of total radioactivity
of 1.85 ꢀ 105 Bq (specific activity 627 MBq/mmol was obtained (18%
radiochemical and chemical yield).
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