Synthesis of isotopomers of dopamine labeled with deuterium or tritium

Article abstract of DOI:10.1002/jlcr.1123

The synthesis of four isotopomers of dopamine labeled with deuterium or tritium is reported. The ring labeled [2′,5′,6′- ²H₃]-, and [2′,5′,6′-³H ₃]-dopamine were obtained using acid catalyzed isotopic exchange b

Full text of DOI:10.1002/jlcr.1123

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 1061–1067.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1123
Research Article
Synthesis of isotopomers of dopamine labeled with
deuterium or tritium
M. Paja)k and M. Kanska*
´
Department of Chemistry, University of Warsaw, Pasteur Str. 1, 02-093 Warsaw,
Poland
Summary
The synthesis of four isotopomers of dopamine labeled with deuterium or tritium is
reported. The ring labeled [20,50,60-2H3]-, and [20,50,60-3H3]-dopamine were obtained
using acid catalyzed isotopic exchange between dopamine and heavy or tritiated
water respectively. Two selectively labeled isotopomers, i.e. [1R-2H]-, and [1R-3H]-
dopamine were synthesized by enzymatic decarboxylation of l-DOPA using the
enzyme tyrosine decarboxylase (EC 4.1.1.25) from Streptococcus faecalis. Copyright
# 2006 John Wiley & Sons, Ltd.
Received 27 June 2006; Revised 2 August 2006; Accepted 2 August 2006
Key Words: dopamine; deuterium; enzyme; labeling; tritium
Introduction
The biogenic dopamine, DA, plays an important role in many physiological
functions as a neurotransmitter in the nervous system of mammals.1–3 DA is
formed in the brain by decarboxylation of l-DOPA catalyzed by enzyme
Aromatic l-Amino Acid Decarboxylase4–7 (EC 4.1.1.28), Figure 1. DA is also
involved as a precursor in the synthetic enzymatic route of the other
catecholamines as adrenaline and noradrenaline,8–11 Figure 2.
In the recent years studies on the properties and pharmacological functions
of DA have drawn special interest, particularly among biologists and medical
personnel. DA is used as a drug in the treatment of many health problems,
*Correspondence to: M. Kanska, Department of Chemistry, University of Warsaw, Pasteur Str. 1, 02-093
´
Warsaw, Poland. E-mail: mkanska@alfa.chem.uw.edu.pl
Contract/grant sponsor: Committee for Scientific Research (Poland); contract/grant number: KBN 4 T09A
063 24
Copyright # 2006 John Wiley & Sons, Ltd.

´
M. PAJA)K AND M. KANSKA
1062
COOH
NH2
NH2
EC 4.1.1.28
PLP, pH 6.8
HO
HO
OH
OH
L-DOPA
DA
Figure 1. Enzymatic decarboxylation of l-DOPA
CH3
HN
NH2
NH2
HO
HO
Phenylethanolamine
N-methyl transferase
Dopamine
-hydroxylase
HO
(PNMT)
HO
HO
OH
OH
OH
DA
noradrenaline
adrenaline
Figure 2. Dopamine as a precursor of neurotransmitters
such as Parkinson’s disease.12–15 As the mechanism of decarboxylation of
l-DOPA (Figure 1), as well as the b-hydroxylation of DA (Figure 2) are not
well understood it is of interest to study these aspects using the kinetic isotope
effect method16,17 (KIE).
As the first step for starting such a study we concentrated on developing
methods for synthesizing deuterium and tritium ring labeled isotopomers of
DA i.e. [20, 50, 60-2H3]-, and [20, 50, 60-3H3]-DA, which can then be used for KIE
measurements. The acid-catalyzed exchange between aromatic compounds
and isotopic water is a very useful method for introducing deuterium or
tritium into non-labile positions of the desired molecules. This simple,
convenient and one step procedure has been widely used for the synthesis of
aromatic compounds labeled with isotopes of hydrogen.18–20 The kinetics and
1
degree of deuterium incorporation were easily monitored by H NMR signal
integration in the course of the exchange. The NMR approach is particularly
useful as the deuterium distribution of deuterium can be easily determined
over the course of the reaction. Investigation of H/D exchange also allows one
to optimize the reaction conditions for tritiation.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1061–1067
DOI: 10.1002/jlcr

SYNTHESIS OF DOPAMINE LABELED WITH DEUTERIUM ON TRITIUM
1063
The next two isotopomers of DA specifically labeled with deuterium and
tritium, i.e. [1R-2H]-, and [1R-3H]-DA, were obtained by enzymatic
decarboxylation of l-DOPA catalyzed by the enzyme tyrosine decarboxylase
(EC 4.1.1.25) from Steptococcus faecalis carried out in a deuteriated or
tritiated medium respectively.
Results and discussion
The deuteriation of DA ꢀ HCl was carried out in heavy water acidified with
deuterochloric acid at 50oC. The incorporation of deuterium was monitored
1
by H NMR spectroscopy over the course of reaction. The control blank
experiment carried out at the same temperature for 5 days without acid added
shows that under these conditions H/D exchange is not occurring.
1
The interesting part of the H NMR spectrum of DA ꢀ HCl consists of
signals from the two -CH2- groups of the side chain and protons from the
aromatic ring.
1H-chemical shifts (d in ppm relative to TMS in D2O) and coupling
constants (J in Hz) for these exchangeable protons in DA are given below:
*
For the b-methylene group of the side chain; d = 2.859 (2 H, t, J = 8).
For the a-methylene group of the side chain: d = 3.209 (2 H, t, J = 7.5).
For the three protons in the aromatic ring: d = 6.739, 6.829 and 6.886.
*
*
No significant change of proton NMR signal integrations corresponding to
methylene group (-CH2-) have been noticed in the course of experiments. It
has been found that incorporation of deuterium take place only into the
aromatic ring of DA. Also the rates of H/D exchange are practically the same
for the protons in the 2, 5, and 6 ring positions.
1
In Figure 3 a plot of the natural logarithm of the normalized H NMR
signal integration, I, for ring hydrogen corresponding to deuterium exchange
in DA carried out in 6 M DCl/D2O at 508C is presented. It gives a good linear
dependence (correlation coefficient=0.98). The derived first-order rate
constant k has a value of 0.31 hꢁ1
.
The data from Figure 3 clearly indicate that the incorporation of deuterium
into the ring of DA is practically complete after 15 h. For tritium labeling of
DA we decided to carry out this reaction for 24 h.
Previous studies have shown that enzymatic decarboxylation of
l-amino acids occurs with retention of configuration at the a-carbon,21–23
Figure 4. This fact has been used to obtain two (R)-isotopomers of
dopamine labeled with deuterium or tritium. For enzymatic decarboxy-
lation of l-DOPA enzyme tyrosine decarboxylase23 was used. The reaction
carried out in a fully deuteriated medium produces [1R-2H]-DA, and
when carried out in tritiated water the tritium labeled isotopomer [1R-3H]-
DA is obtained.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1061–1067
DOI: 10.1002/jlcr

´
M. PAJA)K AND M. KANSKA
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6
5
4
3
2
1
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
t [h]
Figure 3. Semi-logarithmic plot of integrated signals Inl vs time for deuterium
exchange between dopamine and 6 M DCI/D2O
H
H
(S)
(S)
Decarboxylase
PLP, D2O
R
R
COOH
D
(R)
NH2
NH2
[1R-2H]- α amine
α amino acid
Figure 4. Retention of configuration in the course of enzymatic decarboxylation
of a amino acids
Experimental
Materials
Tritiated water (specific act. 185 GBq/g) was purchased from ICN Pharma-
ceutical Inc, Irvine Ca, USA. Deuteriated water (99.9% deuterium), solutions
of 37% DCl/D2O, 83% D3PO4/D2O, and 30% KOD/D2O needed for
preparation of fully deuteriated phosphate buffer were obtained from Polatom
(Poland). Scintillation cocktail was purchased from Rotiszint (Germany).
TLC plates (DC Plastikfolien Aluminiumoxid 60 F256, neutral, type E) were
from Merck. The enzyme tyrosine decarboxylase (EC 4.1.1.25) from S. faecalis
and coenzyme pyridoxal 5-phosphate, PLP, were purchased from Sigma.
DA ꢀ HCl, l-DOPA, and other chemicals, needed for the enzymatic synthesis
and control experiments, were obtained from Sigma.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1061–1067
DOI: 10.1002/jlcr

SYNTHESIS OF DOPAMINE LABELED WITH DEUTERIUM ON TRITIUM
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Methods
The proton NMR spectra were recorded in D2O using TMS as internal
standard on a Varian 500 MHz Unity-Plus spectrometer. The radioactivity of
all samples was determined using a liquid scintillation counter (LISA LSC
PW470, Germany):
1. Incorporation of deuterium into DA. For the deuteriation kinetics
experiments the 10 mg samples of DA ꢀ HCl were dissolved in 1 ml of
6 M solution of DCl in heavy water and placed in glass ampoules. The
ampoules were connected to vacuum, frozen with liquid nitrogen,
degassed, and after thawing, the cycle repeated. Next, the ampoules were
sealed under vacuum and after thawing they were immersed and shaken
for a short time in warm water (508C) until the solution became
homogeneous, and thermostated at 508C. After a fixed time the ampoule
was removed from the thermostat, cooled, opened, the contents
lyophilized under vacuum. From the dry residue a 5 mg sample was taken
for NMR assay.
2. Synthesis of [20,50,60-2H3]-DA ꢀ HCl, 1. A sample of 100 mg of dopamine
hydrochloride dissolved in 10 ml 6 M solution DCl/D2O was prepared and
H/D exchange allowed to occur for 24 h at 508C. The solvent was then
evaporated to about 5 ml under reduced pressure, and the rest lyophilized
to dryness leaving 93 mg of 1. The purity of 1 was checked by TLC
(n-butanol : acetic acid : water = 4:1:2 v/v).
3. Synthesis of [20,50,60-3H3]-DA ꢀ HCl, 2. To a glass ampoule 30 mg of
DA ꢀ HCl, 0.5 ml of HTO (with total radioactivity 1.35 ꢂ 109 Bq), and
0.5 ml of conc. HCl were added. The ampoule was sealed under vacuum
and thermostated at 508C for 24 h. Next the reaction mixture was
lyophilized to dryness under vacuum. The residue was dissolved in 1 ml of
water, loaded on Amberlite IRC 50 H+ column (100 ꢂ 10 mm), and
washed with water to remove the rest of the HTO. Then, 2 was eluted with
0.5 M HCl and collected as 6 ml fractions. For each fraction a 100 ml
sample was taken for radioactivity assay. The fractions containing 2 were
combined, evaporated under reduced pressure to about 5 ml volume and
extracted with n-butanol (4 ꢂ 4 ml). The organic layers were combined,
dried over anhydrous MgSO4, and evaporated under reduced pressure to
about 5 ml volume. The n-butanol was removed by lyophilization leaving
26.6 mg (0.14 mmol) of tritiated 2 (about 88% chemical yield) with a total
radioactivity 2.45 ꢂ 106 Bq (specific activity ꢁ1.75 ꢂ 107 Bq/mmol). The
purity of 2 was checked TLC.
4. Synthesis of [1R-2H]-DA ꢀ HCl, 3. To a vial containing 20 ml of
deuteriated phosphate buffer at pD 5.9 (0.1 M KD2PO4/D2O) 30 mg of
l-DOPA, 2.5 ml of 1 mM PLP/D2O and 5 U of enzyme tyrosine
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1061–1067
DOI: 10.1002/jlcr

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M. PAJA)K AND M. KANSKA
1066
decarboxylase were added. The mixture was incubated at room
temperature for 30 h. The enzyme was removed by centrifugation and
the reaction mixture loaded on to an Amberlite IRC-50 H+ column
(10 ꢂ 100 mm) previously equilibrated to pH 6.5 with 0.1 M KH2PO4.
Unreacted l-DOPA was washed out with 0.1 M KH2PO4 (pH 6.5) and
next 3 was eluted with 0.5 M HCl. The fractions containing 3 were
combined and lyophilized. 3 was extracted with 15 ml of n-butanol, which
in turn was lyophilized leaving 23.4 mg (0.123 mmol) of 3 (yield 81%). The
near 100% incorporation of deuterium into the 1R position was
1
ascertained from H NMR spectrum.
5. Synthesis of [1R-3H]-DA ꢀ HCl, 4. To a vial containing 3 ml of 0.2 M
phosphate buffer (pH 5.5),were added in turn: 5 mg of l-DOPA, 400 ml of
1mM PLP, 0.7 U of enzyme tyrosine decarboxylase, and 2 ml of HTO with
a total radioactivity of approximately 18.5 GBq. The mixture was
incubated at room temperature for 6 h. The enzyme was removed by
centrifugation, and the mixture lyophilized to dryness. The residue was
dissolved in 2 ml of water and loaded onto Amberlite IRC-50 column
previously equilibrated to pH 6.5 with 0.1 M KH2PO4. Unreacted
l-DOPA, the HTO, were washed out with 0.1 M KH2PO4 (pH 6.5). The
desired product, 4, was eluted with 0.5 M HCl and collected as 6 ml
fractions. The fractions containing 4 were pooled and lyophilized to
dryness. 4 was extracted with a 10 ml portion of n-butanol, which in turn
was lyophilized leaving 3.9 mg (0.02 mmol) of 4 with total radioactivity of
6.1 ꢀ 105 Bq (specific activity of 3.05 ꢂ 107 Bq/mmol. Chemical yield was
82%.
Acknowledgements
This was sponsored by Committee for Scientific Research (Poland); grant no
KBN 4 T09A 063 24.
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DOI: 10.1002/jlcr