Synthesis of tritium labeled isotopomers of L-tyrosine

Article abstract of DOI:10.1002/jlcr.582

The synthesis of four selectively labeled isotopomers of L-tyrosine, (L-Tyr), using chemical and enzymatic methods is reported. Four tritium labeled isotopomers of L-phenylalanine (L-Phe) - [2-³H]-, [2′,6′-³H₂]-, [3R-³H]- and [3S-³H]- have been synthesized using a combination of chemical and enzymatic methods. The labeled isotopomers of L-Phe have been converted into [2-³H]-, [2′,6′-³H₂]-, [3R-³H]-, and [3S-³H]-L-Tyr by using the enzyme L-phenyl-alanine 4′-monooxygenase. Copyright

Full text of DOI:10.1002/jlcr.582

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 559–567.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.582
Research Article
Synthesis of tritium labeled isotopomers
of L-tyrosine
!
!
W. Augustyniak, P. Suchecki, J. Jemielity, R. Kanski, and M. Kanska*
Department of Chemistry, Warsaw University, Pasteur 1 Street, 02-089 Warsaw,
Poland
Summary
The synthesis of four selectively labeled isotopomers of l-tyrosine, (l-Tyr),
using chemical and enzymatic methods is reported. Four tritium labeled
isotopomers of l-phenylalanine (l-Phe) – [2-3H]-, [20,60-3H2]-, [3R-3H]- and
[3S-3H]- have been synthesized using a combination of chemical and enzymatic
methods. The labeled isotopomers of l-Phe have been converted into [2 -3H]-,
[20,60-3H2]-, [3R-3H]-, and [3S-3H]-l-Tyr by using the enzyme l-phenyl-alanine
40-monooxygenase. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: tritium; labeling; optical isotopomer; phenylalanine; tyrosine;
enzyme
Introduction
The metabolism of l-tyrosine, l-Tyr, is a key step in many biological
processes of living organisms. A number of questions cannot be
answered without understanding the mechanism of the reversible
conversion of l-Tyr to phenol, pyruvate and ammonia, a reaction that
is catalyzed by the enzyme b-tyrosinase (tyrosine phenol lyase, EC
!
*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: contract/grant number: KBN3 T09A 046 17
Copyright # 2002 John Wiley & Sons, Ltd.
Received 13 December 2001
Revised 12 February 2002
Accepted 25 February 2002

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W. AUGUSTYNIAK ET AL.
Figure 1. Decomposition of L-Tyr catalyzed by enzyme b-tyrosinase
4.1.99.2).1–3 Under some conditions b-tyrosinase takes part in the
formation of l-Tyr4–6 (Figure 1). The two mentioned reactions are
multistep, involving several intermediates, and so complex that some
details, particularly with respect of the timing of the bonding changes,
are still unclear. The number of questions that arise can be minimized
by determining the kinetic isotope effects (KIE) for each atom involved
in the postulated rate determining step.7,8 The numerical values of the
KIE can provide a useful tool for distinguishing between the alternative
mechanisms. Such studies require different isotopomers of l-Tyr,
specifically labeled with tritium in the desired positions.
In the literature, there are several methods describing the synthesis of
tyrosine labeled with deuterium9–15 and tritium16–20 yielding the
different singly, multi and uniformly labeled isotopomers of l-Tyr.
Most of them are not useful for our proposed KIE studies.
The synthesis developed by us to obtain the desired isotopomers of l-
Tyr, specifically labeled with tritium consists of a combination of
chemical and enzymatic methods. First, the key intermediate i.e. the
corresponding tritium labeled isotopomer of l-phenylalanine, l-Phe,
was obtained which in turn was converted to isotopomers of l-Tyr by
using the enzyme l-phenylalanine hydroxylase (EC 1.14.16.1 – l-
penylalanine 40-mono-oxygenase) (Figure 2).
Results and discussion
[2-3H]-l-Tyr, 1, was synthesized from [2-3H]-l-Phe, 2, obtained
according to the reaction route shown in Figure 3. The tritium label
was introduced into the methylene group of malonic acid, 3, as a result
of isotope exchange between malonic acid and tritiated water at elevated
temperature.21 Knovenagel condensation of [2-3H]malonic acid, 3, with
benzaldehyde leads to [2-3H]cinnamic acid,22 4, which in turn was
converted23 by the enzyme PAL (Phenylalanine ammonia lyase, EC
4.3.5.1) to [2-3H]-l-Phe. Finally, as shown in Figure 2, tritiated l-Phe
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SYNTHESIS OF TRITIUM LABELED L-TYROSINE
561
Figure 2. Enzymatic oxidation of L-Phe to L-Tyr
Figure 3. Synthesis of [2-3H]-L-Phe
incubated in a medium containing the enzyme l-phenylalanine 40-
monooxygenase (EC 4.1.99.2) from rat liver, produces [2-3H]-l-Tyr.
The hydroxylation of l-Phe to l-Tyr was carried out in the presence of
a cofactor and the enzyme catalase that protects l-Tyr from hydrogen
peroxide formed during incubation. The general protocol of this step
was described earlier.24
The isotopomers [3R-3H]-, 7, and [3S-3H]-, 8, of l-Phe, intermediates
for synthesis of the corresponding isotopomers of l-Tyr, were obtained
as shown in Figures 4 and 5 according to the procedure described
earlier.23 These species, as well as [20,60-3H2]-l-Phe, 9, (commercial
product) were converted (Figure 2) to [2R-3H]-l-Tyr, 10, [2R-3H]-l-Tyr,
12, and [20,60-3H2]-l-Tyr, 13, respectively.
The presence of tritium in the 3R position of 7 and 10 was confirmed
by enzymatic transformation of 7 and 10 into [3-3H]cinnamic, 6, and p-
hydroxy[3-3H]cinnamic, 11 acids, respectively, employing the reverse
reaction catalyzed by the enzyme PAL (Figure 6). The obtained tritiated
cinnamic acids were separated, purified and radio assayed. The molar
activities of these labeled acids were the same as the molar activities of 7
and 10 used in the elimination reaction.
The same elimination reactions have been used to identify the
presence of tritium in the 3S position of 8 and 11. The obtained
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Figure 4. Synthesis of [3R-3H]-L-Phe
Figure 5. Synthesis of [3S-3H]-L-Phe
Figure 6. Reversible reaction catalyzed by enzyme PAL
cinnamic and p-hydroxycinnamic acids did not contain tritium. All the
tritium radioactivity was found in the water.
In KIE experiments only substrates of high radiochemical purity
should be used. To avoid contamination of starting tritiated l-Tyr
with products of its self-radiation formed during storage, a fresh sample
of the isotopomer of l-Tyr, sufficient for a single KIE run, was prepared.
Experimental
1. Materials: Tritiated water was obtained from INC Pharmaceutical
Inc., USA. Scintillation cocktails for aqueous and non-aqueous
samples were obtained from Rotiszint (Germany) and Sigma,
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respectively. Microcentrifuge filters NM-WL 10.000 and Amberlit
IR-120 (25–50 mesh) were purchased from Sigma and Aldrich,
respectively. Preparative TLC plates, PEI-Cellulose F, were
obtained from Merck. The enzymes, i.e. PAL (Phenylalanine
ammonia lyase, EC 4.3.1.5 from Rhodotorula glutinis) and catalase
(EC 1.11.1.6 from bovine liver) were obtained from Sigma.
2. Methods: l-Phenylalanine-40-monooxygenase (EC 1.14.16.1) was
isolated from rat liver according to the described procedure.25 The
obtained enzyme had an activity of 0.55 U/ml. The concentration of
l-Phe was determined indirectly by measuring the concentration of
its derivative, i.e. cinnamic acid, spectrophotometrically. This
general protocol was given earlier,23 and it consists of incubation
of l-Phe in the presence of enzyme PAL. The concentration of
cinnamic acid formed was determined spectrophotometrically by
measuring the absorbance at 290 nm (e290=10.000 for cinnamic
acid) on the UV–VIS spectrometer (Shimadzu-UV-102 CE-LV).
The concentration of l-Tyr was also determined spectrophotome-
trically24 using a sensitive and reproducible method developed for
assay of tyrosine in biological media.26 Under appropriate condi-
tions tyrosine reacts with 1-nitroso-2-naphthol to yield a stable
yellow product, the concentration of which can be determined by
measuring the absorbance at 450 nm.
The identities and purity of l-Tyr and l-Phe were also verified by TLC
on cellulose and silica gel, respectively. In the preliminary studies with
inactive compounds the optical rotation of phenylalanine and tyrosine
.
were checked on the polarimeter (P 3002, A. KRUS Optronic,
Germany).The radioactivity of all the samples (aqueous and non-
aqueous) was determined using liquid scintillation counting (LISA LSC
PW470 – Raytest, Germany).
3. Synthesis of [2-3H]-l-Tyr, 1:
(a) [2-2H]Malonic acid, 3. This compound was synthesized by isotope
exchange21 between malonic acid and tritiated water. To the glass
ampoule 480 mg (4.1 mmol) of malonic acid and 1 ml of tritiated
water with a total activity of 5.55 GBq were added. The ampoule
was frozen with liquid nitrogen, degassed and sealed, and
thermostated at 808C for 8 h. Water was removed by lyophilization
and crude the 3 obtained was used for the synthesis of cinnamic acid.
(b) [2-3H]Cinnamic acid, 4. Knovenagel condensation22,26,27 of the
crude 3 with benzaldehyde (5 mmol) yielded 318 mg (2.2 mmol) of 4
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W. AUGUSTYNIAK ET AL.
with a total radioactivity of 15.7 MBq, this compound was used
directly for the synthesis of [2-2H]-l-Phe.
(c) [2-3H]-L-Phe, 2, The general procedure for the enzymatic synthesis
of l-Phe from cinnamic acid in ammonia buffer containing an
enzyme PAL has been early described previously.23,24,27,28 In this
case 50 mg of 4 (0.34 mmol) of 2.42 MBq total radioactivity with
0.45 ml of enzyme PAL (4.4 U/cm3) were added to 9 ml of 1.6 M
ammonia buffer (pH 9.8) and the mixture incubated at 308C for 5
days. As a result a sample of 22.9 mg (0.138 mmol) of 2 was isolated
with a total radioactivity of 0.98 MBq and used in the next step to
synthesize tritiated tyrosine. The concentration of 2 was determined
spectrophotometrically as described in Methods.
(d) [2-3H]-L-Tyr, 1. The general method of enzymatic oxidation of l-
Phe to l-Tyr, and procedure for its isolation and purification has
been reported previously.24 The stoppered glass vial with the
reaction mixture containing the obtained sample of 2 (0.138 mmol),
4 mg of dl-6-methyl-5,6,7,8-tetrahydropterine (cofactor), 10.2 mg of
dl-dithiothreitol (HSCH2(OH)CH2(OH)CH2SH), 500 ml of enzyme
catalase (EC 1.11.1.6) from bovine liver (300 000 U/ml) and 3 ml of
the enzyme l-phenylalanine 40-monooxygenase (0.55 U/ml) in
0.1 M phosphate buffer (pH 6.8) of 20 ml total volume was
incubated at 258C for 3 days. After isolation and purification24
a
17 mg (0.1 mmol) sample of 1 was obtained with total radioactivity
of 0.715 MBq (specific activity 7.15 MBq/mmol). The concentration
of 1 was determined spectrophotometrically as described in
Methods.
4. Synthesis of [3R-3H]-L-Tyr, 10: The intermediates, tritiated
benzaldehyde, 5, [3-3H]cinnamic acid, 6 and [3R-3H]-L-Phe, 7, were
obtained (Figure 4) according to procedures described earlier.23
(a) [3R-3H]-L-Phe, 7: The enzymatic conversion23 of 6 to 7 proceeded
as described in (3c). As a result, from 50 mg (0.3 mmol) of sample of
6 (total radioactivity of 1.6 MBq) 20 mg (0.13 mmol) of 7 was
obtained (total radioactivity 0.61 MBq) and this was used for the
synthesis of 10.
(b) [3R-3H]-L-Tyr, 10: This product was synthesized as in (3d).
Finally, 16.5 mg (0.094 mmol) of 10 was obtained with a total
radioactivity of 0.436 MBq (specific activity 4.64 MBq/mmol).
(c) Confirmation of 3R position of the tritium atom in l-Phe, 7: In a
preliminary experiment the sample of 7 was deaminated to 6 in the
presence of enzyme PAL (Figure 6). The conversion23 was carried
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SYNTHESIS OF TRITIUM LABELED L-TYROSINE
565
out in borate buffer solution at pH 8.8 at 308C over the course of
30 min. Under these conditions enzyme PAL stereospecifically
removes pro-S hydrogen from pro-S leaving the pro-R hydrogen
unaffected.29 The purified sample of cinnamic acid obtained after
deamination of 7 has the same specific activity as a starting 7 and 6
used for synthesis of [3R-3H]-l-Phe. This also means that during the
addition of ammonia to cinnamic acid the enzyme PAL introduces
hydrogen into position 3S.
(d) Confirmation of 3R position of the tritium atom in l-Tyr, 10: This
was performed similarly as in (4e). After one day of incubation of 10
in the presence of PAL in borate buffer only about 50% of tyrosine
was converted to p-[3-3H]hydroxycinnamic acid, 11. The isolated
and purified sample of 11 has the same specific activity as samples 7
and 10 and the sample of 6 used to synthesize of 7.
5. Synthesis of [3S-3H]-l-Tyr, 12:
(a) [3R-3H]-L-Phe, 8, the key intermediate was obtained according to
procedures described previously.23 The synthesis was carried out as
in (4c). The 50 mg (0.34 mmol) sample of cinnamic acid was
dissolved in 1.6 m ammonium buffer (pH 9.8) prepared from NH4Cl
and NaOH and 6 ml of HTO of total activity 5 GBq in an
incubation vial. To this solution 0.8 ml of enzyme PAL (4.4 U/ml)
was added and the mixture incubated for 5 days. As a result,
21.6 mg (0.14 mmol) of 8 with a total radioactivity of 4.3 ꢀ 107 Bq
(specific activity 3.07 ꢀ 107 Bq/mmol) was obtained.
(b) [3S-3H]-L-Tyr, 12 was prepared from the above sample of 8
according to the protocol given in (3d). After incubation and
purification a 19.2 mg (0.112mmol) sample of 12 of total radioactivity
3.5 ꢀ 107 Bq (specific activity 3.1 ꢀ 108 Bq/mmol) was obtained.
(c) Confirmation of 3S position of the tritium atom in l-Phe, 8 and l-
Tyr, 12: The confirmation was performed in the same manner as
described in (4c) and (4d). In this case the samples of cinnamic and
p-hydroxycinnamic acids obtained after deamination reaction of 8
or 12 in the presence of enzyme PAL were inactive. The
radioactivity was found in the water medium only.
6. [20,60-3H2]-l-Tyr, 13: This isotopomer of l-Phe was prepared in the
same manner as described in (3d). The starting substrate i.e. an
aqueous solution of [20,60-3H2]-l-Phe, 9, was a commercial product
from SIGMA. (specific activity – 1.0 mCi/ml, 50 Ci/mmol). To the
incubation vial containing 20 mg (0.13 mmol) of l-Phe (carrier)
dissolved in the same reaction mixture as given in (3d), a 30 ml of
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aqueous solution of 9 of total radioactivity of 1 ꢀ 106 Bq was added.
The reaction mixture was incubated at 258C for 3 days. After
separation and purification 16.7 mg (0.098 mmol) of 13 containing
7.2 ꢀ 105 Bq (7.3 MBq/mmol) was obtained.
Acknowledgements
The work was supported by grant KBN 3 T09A 046 17.
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