Synthesis of carbon-14 labeled [1-14C]-, and [2-14C]-L-tyrosine

Article abstract of DOI:10.1002/jlcr.483

Two labeled isotopomers of L-tyrosine, L-Tyr, have been synthesized using specific properties of the enzymes phenylanine ammonia lyase, PAL, and L-phenylalanine hydroxylase. In an intermediate step [1-¹⁴C]-, and [2-¹⁴C]-L-phenylalani

Full text of DOI:10.1002/jlcr.483

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 553–560.
DOI: 10.1002/jlcr.483
Synthesis of carbon-14 labeled [1-14C]-,
and [2-14C]-l-tyrosine
!
!
W. Augustyniak, R. Kanski and M. Kanska*
Department of Chemistry, Warsaw University, Pasteur 1 Street,
02-089 Warsaw, Poland
Summary
Two labeled isotopomers of l-tyrosine, l-Tyr, have been synthesized using
specific properties of the enzymes phenylanine ammonia lyase, PAL, and l-
phenylalanine hydroxylase. In an intermediate step [1-14C]-, and [2-14C]-l-
phenylalanine, l-Phe, have been obtained from [1-14C]-, and [2-14C]cinnamic
acid, prepared from potassium [14C]cyanate or [2-14C]malonicaicd, and by
addition of ammonia in the presence of enzyme PAL. The labeled isotopomers
of l-Phe have been converted into [1-14C]-, and [2-14C]-l-Tyr using the enzyme
l-phenylalanine hydroxylase. Copyright # 2001 John Wiley & Sons, Ltd.
Key Words: carbon-14; labeling; optical isotopomer; l-phenylalannine;
l-tyrosine; enzyme
Introduction
l-Tyrosine, l-Tyr, is important in a variety of biological functions. Its
metabolism plays a central role in relationships that occur in many
processes of living cells. In particular many studies have been concerned
with explaining the mechanism of the reversible reaction given in
Scheme 1. The enzyme b-tyrosinase (tyrosine phenol-lyase, EC. 4.1.99.1)
catalyzes the conversion of l-Tyr to phenol, pyruvate and ammonia.1–4
In some conditions this enzyme also participates in the reverse reaction
* 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: KBN; contract/grant number: KBN 3 T09A 046 17.
Copyright # 2001 John Wiley & Sons, Ltd.
Received 29 January 2001
Revised 12 March 2001
Accepted 11 April 2001
Published online \ \ \ \

554
W. AUGUSTYNIAK ET AL.
Scheme 1.
leading to the formation of l-Tyr. The two mentioned reactions are
multistep and some intrinsic details of the process are unclear.5–9
The goal of our planned research is directed at investigating the
mechanism of this reaction by applying the kinetic isotope effect (KIE)
method and determining KIE for each atom involved in the postulated
rate determining step.10 The studies require the synthesis of one para-
derivative of l-Phe, i.e., l-Tyr labeled with 14C in a particular position
of the side chain. In this paper, a synthesis of two isotopomers of
[1-14C], and [2-14C]-l-tyrosine is presented.
In the literature, there are several methods describing the synthesis of
selectively or uniformly [14C-]-labeled dl-Tyr11–18. [3-14C]-L-Tyr was
separated by diluting the racemic form with l-Tyr and recrystallizing
the product.18–19 Recently, the extensive use of positron emission
tomography has created a demand for [11C]-labeled l-Tyr. The
described synthesis of [11C]-l-Tyr used a combination of chemical and
enzymaticmethods to achieve the desired l-enantiomer in the shortest
possible time.20–23 In the literature, there are reports of the synthesis of
3
the different isotopomers of l-Tyr doubly labeled with H and 14C
obtained from labeled racemates by removing one enantiomer
enzymatically.24–25 The chemical and chemo-microbiological methods
have been also used to obtain the isotopomers l-Tyr, labeled with stable
2
isotopes H and 13C, which have then been used for spectroscopic and
26–28
metabolicstudies.
Results and discussion
The above-mentioned routes for the synthesis of specifically labeled l-
Tyr lead to the formation of racemates. The isolation of the l-
enantiomer has been achieved by resolution by chemical or enzymatical
methods. As a result half of the radioactive compound is wasted.
To avoid this loss we applied enzymes in the key steps of the synthetic
Copyright # 2001 John Wiley & Sons, Ltd.
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SYNTHESIS OF CARBON-14 LABELED
555
pathway, i.e. stereospecific reduction of a double bond and oxidation of
intermediate l-Phe to l-Tyr.
The two isotopomers of l-Tyr, specifically labeled with 14C in the
1- and 2-position of the side chain, have been prepared using a
combination of chemical and multienzymatic methods. For this
synthesis we used, as an intermediate, an isotopomer of [1-14C]-
or [2-14C]cinnamic acid, which has been converted into [1-14C]-,
or [2-14C]-l-phenylalanine, l-Phe, in the presence of the enzyme
PAL.29–32 In the next step labeled l-Phe was oxidized to l-Tyr
using an enzyme l-phenylalanine hydroxylase (EC 1.14.16.1 – l-
phenylalanine 40-monooxygenase) from rat liver.16 To stimulate
the hydroxylation of l-Phe to l-Tyr the reaction was carried out
in the presence of d,l-6-methyl-5,6,7,8-tetrahydropterine (cofactor)
and d,l-dithiothreitol (1,4-dithiobutane-2,3-diol
–
HSCH2(OH)-
CH2(OH)CH2SH). The medium also contained the enzyme
catalase that protects l-Tyr from H2O2 formed during the course of
incubation. The general route for the synthesis of labeled l-Tyr is
showed in Scheme 2.
Potassium cyanide, [14C]KCN, and commercial [2-14C]malonic
acid were used as the source of 14C-label. [14C]KCN was
converted into [1,10-14C1]malonic acid according to the literature33
procedure. The required intermediates, i.e. two isotopomers ([1-14C]-,
and [2-14C]-) of cinnamic acid have been previously obtained by
us by Knovenagel condensation of benzaldehyde with labeled
29–30
malonicacid.
Scheme 2.
Copyright # 2001 John Wiley & Sons, Ltd.
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556
W. AUGUSTYNIAK ET AL.
Experimental
1. Materials: [2-14C]Malonic acid was supplied by Moravek Biochem-
ical (USA). Potassium cyanide [14C]KCN was obtained from
Polatom (Poland). Scintillation cocktails for aqueous and nonaqu-
eous samples were obtained from Rotiszint (Germany) and Sigma,
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 livers according to a literature procedure.34 The
obtained enzyme had an activity equal to 0.55 U/ml. The
concentration of l-Phe was determined indirectly by measuring
the concentration of its derivative, i.e. cinnamic acid, spectro-
photometrically. This procedure consists of converting l-Phe into
cinnamic acid in the presence of enzyme PAL. The measured sample
of l-Phe was introduced into a medium containing 0.2 M borate
buffer (pH 8.8) and enzyme PAL and incubated at 308C for 30 min.
The concentration of cinnamic acid formed was determined by
measuring the absorbance at 290 nm (e290=104 for cinnamic acid)
using a SHIMADZU-UV-102 CE-LV spectrophotometer. The
concentration of l-Tyr was also determined spectrophotometrically
using a sensitive and reproducible method developed for assay of
tyrosine in biological media.35 Under appropriate conditions
tyrosine, like other substituted phenols, 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.
(Standards for calibration were prepared by treating known
amounts of tyrosine with nitrosonaphthol).
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 the obtained phenylalanine
.
and tyrosine were checked on the polarimeter (P 3002 – A. KRUS
Optronic– Germany). The radioactivity of all samples (aqueous and
nonaqueous) was determined using liquid scintillators (7 ml scintillation
vials) on the automaticocunter (LISA LSC PW470 – Raytest,
Germany).
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SYNTHESIS OF CARBON-14 LABELED
557
3. Synthesis of [1-14C]-L-Tyr, 1:
%
(a) [1,10-14C] Malonic acid, 2. This intermediate was synthesized from
%
0.85 g (13 mmol) of [14C]KCN, total activity 185 MBq, according to
a literature33 procedure. As a result 0.69 g (6.6 mmol, 94.3 MBq) of
crude 2 was obtained in 51% radiochemical.
%
(b) [1-14C]Cinnamic acid, 3. Knovenagel condensation18,29,30 of the
%
crude 2 with benzaldehyde yielded 0.65 g (4.4 mmol) of cinnamic
%
acid, 3, with a total activity of 30.5 MBq. Crude 3 was used directly
%
%
for the synthesis of [1-14C]-l-Phe.
(c) [1-14C]-L-Phe, 4. To a glass vial equipped with a cap about
%
30 mg (0.2 mmol) of 3 of 1.4 MBq total activity was added
%
and dissolved in 5.25 cm3 of 1.6 M ammonia buffer (pH 9.8). The
0.25 cm3 of enzyme PAL (4.17 U/cm3) was added and the
mixture incubated at 308C for 120 h. The enzyme was removed
by centrifugation using NM WL 10.000 filter (Sigma). The
filtrate was acidified to pH 1 with 6 M HCl and extracted
with diethyl ether to remove unreacted 3. Extraction was
%
repeated until the radioactivity of the etheral layer reached a
constant value close to background. The water layer was
loaded onto a column (100 ꢀ 10 mm) of Amberlite IR-120(H+).
The column was washed with water and then 4 was eluted with
%
0.3 M NH3(aq) and collected as 3 ml fractions. From each fraction
50 ml samples were taken for radioactivity measurements. The
fractions containing
4
were combined and evaporated
%
under vacuum at 408C and the purity checked by TLC (silica gel,
acetic acid–n-butanol–water – 1:4:5, v/v/v; Rf=0.2 and 0.7
for phenylanine and cinnamic acid, respectively) and enzymatically.
As a result a sample of 13.7 mg (0.083 mmole) of 4 was obtained
%
with a total activity of 5.75 ꢀ 105 Bq (6.9 MBq/mmol specific
activity), and used in the next step to synthesize labeled tyrosine.
The concentration of 4 was determined spectrophometrically as
%
described in Methods.
(d) [1-14C]-L-Tyrosine, 1 : The reaction mixture containing 13.7 mg
%
(0.083 mmol, 5.75 ꢀ 105 Bq) of 4, 2.4 mg (10 mmol) of dl-6-methyl-
%
5,6.7,8-tetrahydropterine, 6.16 mg (40 mmol) of dl-dithiothreitol,
330 ml of the enzyme catalase (EC 1.11.1.6) from bovine liver
(300 000 U) and 2 ml (0.55 U/ml) of a solution of the enzyme l-
phenylalanine 40-monooxygenase (EC 1.14.16.1) in 0.1 M phosphate
buffer (pH 6.8) of 20 ml total volume was incubated at 258C
for 3 days. The reaction was stopped by addition of 2 ml of 60%
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558
W. AUGUSTYNIAK ET AL.
HClO4 and 1 ml of concentrated HCl. The mixture was then
centrifuged to remove the precipitated proteins (7 krpm, 10 min,
48C), and supernatant was loaded onto a column (100 ꢀ 10 mm) of
Amberlit 120(H+). The column was washed with water to remove
the buffer salts, and then the amino acids (1 and unreacted 4 ) were
%
%
washed out with 0.3 M NH3(aq). The fractions with high radio-
activity were combined and evaporated under reduced pressure at
408C. The residue was dissolved and applied to a preparative
cellulose-coated glass plate (20 ꢀ 20 cm). The plate was developed by
ascending chromatography with the solvent system composed of
chloroform–methanol–25%NH3(aq)–water
58:32:8:2
(v/v/v/v;
Rf=0.25, and 0.8 for tyrosine and phenylalanine, respectively).
The plate was air dried and cellulose from the area corresponding to
1 was scraped off and washed with water. The water was then
%
evaporated off under reduced pressure at 408C and the residue again
dissolved in 1 ml of water. The purity of 1 was tested on the
%
analytical TLC cellulose plate using the same developing system as
for a preparative assay. The concentration of 1 was determined
%
spectrophotometrically as described in Methods. As a result 10.7 mg
(0.057 mmol) of 1 with a total activity of 3.9 ꢀ 105 Bq was obtained
(specific activity 6.8 MBq/mmol).
4. Synthesis of [2-14C]-L-Tyr, 5 :
%
(a) [2-14C]Cinnamic acid, 6. The synthesis of 6 was carried out in a
%
%
similar manner to that described in Section 3(b). As starting
material 0.5 g (4.8 mmol) of [2-14C]malonic acid, with a total activity
of 9.2 MBq, was used. As a result 0.6 g (4 mmol) of crude 6 with
%
total activity 7.6 MBq was obtained (83% radiochemical yield), and
this product was used to synthesize [2-14C]-l-Phe without further
purification.
(b) [2-14C]-L-Phenylalanine, 7 : The synthesis of 7 was carried out as
%
%
described in 3c. To the same incubation medium 30 mg (0.2 mmol)
of 6 of 3.8 ꢀ 105 Bq total activity was added. After chromatographic
isolation 12.5 mg (0.076 mmol) of 7 was obtained with a total
%
%
activity of 1.45 ꢀ 105 Bq (1.9 MBq/mmol specific activity) and used
directly in the next step.
(c) [2-14C]-L-Tyrosine, 5 : The enzymaticconversion of 7 to 5, isolation
%
%
%
and purfication of product, was carried out in the same manner as
described in Section 3(d) leading to 1. To the incubation medium
12.5 mg of 7 was introduced and 9.1 mg. (0.05 mmol) of 5 with a total
%
activity of 9.4 ꢀ 104 Bq (1.88 MBq/mmol specific activity) was obtained.
Copyright # 2001 John Wiley & Sons, Ltd.
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SYNTHESIS OF CARBON-14 LABELED
559
Acknowledgements
The work was supported by grant KBN 3 T09A 046 17.
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