Enzymatic synthesis of tritium-labelled isotopomers of L-DOPA

Article abstract of DOI:10.1002/jlcr.919

The synthesis of two isotopomers of L-DOPA labelled selectively with tritium is reported. In the intermediate step [3S-³H]-, and [3′,5′-³H₂]-L-tyrosine, have been obtained using a combination of chemical and enzymatic meth

Full text of DOI:10.1002/jlcr.919

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 235–240.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.919
Note
Enzymatic synthesis of tritium-labelled isotopomers
of l-DOPA
!
M. Kozłowska, R. Kanski and M. Kanska*
!
Department of Chemistry, University of Warsaw, Pasteur Str. 1, 02-093 Warsaw,
Poland
Summary
The synthesis of two isotopomers of l-DOPA labelled selectively with tritium is
reported. In the intermediate step [3S-3H]-, and [30,50-3H2]-l-tyrosine, have been
obtained using a combination of chemical and enzymatic methods. The labelled
isotopomers of l-tyrosine, l-Tyr, have been converted into, [3S-3H]-, and [50-3H]-l-
DOPA using the enzyme mushroom tyrosinase (monophenol oxidase, EC 1.14.18.1)
from Neurospora crassa. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: DOPA; enzyme; labelling; tritium; tyrosine
Introduction
l-DOPA (30,50-l-dihydroxyphenylalanine) and its precursor l-Tyr are
important in a variety of biological functions.1 The former is a starting point
in the synthesis of melanins (natural humans and animals pigments), in
addition it is involved in the synthesis of catecholamines i.e., adrenaline,
noradrenaline and dopamine. l-Tyr is one of the most important neuro-
transmitters in the nervous system2–4 (Scheme 1).
l-DOPA is a drug used in pharmacotherapy as the precursor of dopamine.
Deficiency in dopamine causes the disruption of proper brain functions,
leading to many health problems such as Parkinson’s disease.5,6 l-DOPA,
specifically labelled with b+-emitters, is used in PET diagnostics to monitor
the synthesis and decomposition of dopamine in the brain.7–10 Selectively
labelled l-DOPA can also be used to elucidate the reaction mechanisms
associated with the formation of dopamine (Scheme 1). Despite numerous
!
*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 # 2005 John Wiley & Sons, Ltd.
Received 27 October 2004
Revised 26 November 2004
Accepted 30 November 2004

236
M. KOZŁOWSKA ET AL.
NH2
COOH
NH2
COOH
NH2
tyrosinase
DOPA
decarboxylase
EC1.14.18.1
HO
HO
OH
dopamine
OH
OH
L-DOPA
L-Tyr
CH3
HN
NH2
HO
HO
Phenylethanolamine
N-methyl transferase
dopamine
hydroxylase
(PNMT)
HO
HO
OH
adrenaline
OH
noradrenaline
Scheme 1. Synthesis of catecholamines
literature reports, the mechanism of this reaction is not completely under-
stood. Therefore, it would be interesting to investigate several reaction steps
leading to the formation of dopamine, especially to characterize the active
complex which is formed in the oxidation reaction of l-tyrosine to l-DOPA
and in the decarboxylation of l-DOPA to dopamine. Thus, the kinetic
isotopic effect method (KIE) can be used. This technique relies on obtaining
the ratios of rate constants using the lighter and heavier isotope. The results
obtained by this method will allow one to elucidate the rate determining step.11
In the literature there are several methods describing the enzymatic
conversion of [3H]-l-tyrosine to tritium-labelled l-DOPA. For this purpose
the enzymes extracted from hamster melanoma,3 adrenal glands12 or rat
brains13 were used.
This paper reports on the enzymatic synthesis of two selectively tritium-
labelled isotopomers of l-DOPA, i.e., [50-3H], and [3S-3H]-l-DOPA from the
appropriate isotopomers of l-Tyr.
Tyrosinase from mushrooms Neurospora crassa (EC 1.14.18.1) was the
enzyme14 used in this experiment. It catalyses the hydroxylation of l-Tyr to l-
DOPA, and immediately mediates the oxidation of l-DOPA to dopaqui-
none.15 However, in the presence of ascorbic acid, the oxidation of l-DOPA is
a reversible process.16 Ascorbic acid reduces dopaquinone to l-DOPA, and
itself undergoes oxidation to dehydroascorbic acid (Scheme 2).
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 235–240

SYNTHESIS OF TRITIUM-LABELLED L-DOPA
237
COOH
COOH
NH2
COOH
NH2
NH2
tyrosinase
ascorbic acid
HO
O
OH
O
OH
L-Tyr
DOPA quinone
L-DOPA
COOH
NH2
OH
O
OH
O
O
HO
O
+
HO
+
L-DOPA
O
O
O
HO
OH
O
dehydroascorbic
acid
DOPA quinone
ascorbic
acid
Scheme 2. Enzymatic conversion of l-tyrosine into l-DOPA
The intermediate substrate for the synthesis of labelled l-DOPA, i.e.,
isotopomer [30,50-3H2]-l-Tyr has been obtained by acid-catalysed H/T isotope
exchange at elevated temperature. This synthetic step17,18 has been tested by
using deuterium as the label. The position and degree of incorporation of
deuterium in the resulting product, i.e., [30, 50-2H2]-l-Try has been ascertained
by proton NMR spectroscopy. The second intermediate needed, i.e., [3S-3H]-
l-Tyr was prepared in our research group by the procedure described
elsewhere.19,20
Experimental
Materials
Tritiated water was purchased from ICN Pharmaceutical Inc., Irvine, CA,
USA. Deuteriated water (99.9% deuterium) was obtained from Polatom
(Poland). Scintillation cocktail was purchased from Rotiszint (Germany).
Silica gel TLC plates, 60 F256, were from Merck. The enzyme tyrosinase (EC
1.14.18.1) isolated from Neurospora crassa, and other chemicals, needed for
the enzymatic synthesis and control experiments, i.e., l-tyrosine, l-DOPA,
ascorbic acid were also obtained from Sigma.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 235–240

238
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

SYNTHESIS OF TRITIUM-LABELLED L-DOPA
239
[30,50-3H2]-l-Tyr, 1, was eluted with 0.2 M boric acid. Next, [50-3H]-l-DOPA,
2, was eluted with 0.75 M HCl. The presence of l-DOPA was also checked
using its colour reaction24 with KIO3. Radioactivity of the eluted fractions was
determined using a liquid scintillation counter. Radiochromatogram from this
synthesis is depicted in Scheme 3. As a result a 11.5 mg (63 mmol) sample of 2
was obtained with total radioactivity 1.7 ꢀ 105 Bq (sp. activity 2.7 MBq/mmol)
and chemical yield 48%. The purity of 2 obtained was also checked by TLC.
45000
40000
35000
DOPA
30000
25000
20000
15000
10000
5000
0
Tyr
HTO
0
10
20
30
40
50
fraction no (V = 7 ml)
Scheme 3. Radiochromatogram of the post-reaction mixture from synthesis of
[50-3H]-l-DOPA
Synthesis of [3S-3H]-l-DOPA, 3
To the incubation vial containing 7 ml of phosphate buffer (pH 6.1), 18 mg
(100 mmol) of [3S-3H]-l-tyrosine of total radioactivity 3.1 ꢀ 107 Bq, 36 mg
(0.2 mmol) of ascorbic acid and 0.1 ml (about 250 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 constant stirring,
and aerating. The separation procedure and purification of 3 was the same as
described in ‘Methods’ section. As a result a 10 mg (53 mmol) sample of 3 was
obtained with total radioactivity of 1.6 ꢀ 107 Bq (specific activity 3 ꢀ 108 Bq/
mmol) and radiochemical yield 53%.
References
1. Stryer L. Biochemistry (3rd edn). Freemann: New York, 1988.
2. Pomerantz SH. J Biol Chem 1963; 228: 2351–2357.
3. Pomerantz SH. Biochem Biophys Res Commun 1964; 16: 188–194.
4. Pomerantz SH, Warner MC. J Biol Chem 1967; 242: 5308–5314.
5. Gauthier G, Juge O, Birchler A. Eur Neurol 1974; 11: 133–157.
6. Nutt JG, Woodward WR, Hammerstad JP. N Engl J Med 1984; 310: 483–488.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 235–240

240
M. KOZŁOWSKA ET AL.
7. Bolster JM, Vaalburg W, Van Veen W, Van Dijk T, Van Der Molen HD,
Wynberg H, Woldring MG. Int J Appl Radiat Isot 1983; 34: 1650–1652.
8. Adam MJ, Grierson JR, Ruth TJ, Pedersen K, Pate BD. J Nucl Med 1987; 28:
1599–1603.
(
9. Bjurling P, Antoni G, Watanabe Y, Langstrom B. Acta Chem Scand 1990; 44:
.
178–182.
(
10. Bjurling P, Watanabe Y, Oka S, Nagasawa T, Yamada H, Langstrom B. Acta
.
Chem Scand 1990; 44: 183–188.
11. Huskey WP. In Enzyme Mechanism from Isotope Effects, Cook F (ed.). CRS
Press: Boca Raton, FL, 1991.
12. Nagatsu T, Levitt M, Udenfriend S. Anal Biochem 1964; 9: 122–126.
13. Coyle J. Biochem Pharmcol 1972; 21: 1935–1944.
14. Espin JC, Varon R, Fenoli LG, Gilabert MA, Garcia-Ruiz PA, Tudela J, Garcia-
"
Canovas F. Eur J Biochem 2000; 267: 1270–1279.
15. Marumo K, Waite JH. Biochim Biophys Acta 1986; 872: 98–103.
!
"
16. Ros JR, Rodriguez-Lopez JN, Garcia-Canovas F. Biochem J 1993; 295: 309–312.
17. Griffiths DB, Feeney J, Roberts GCK, Burgen ASV. Biochim Biophys Acta 1976;
446: 479–485.
18. Wishart DS, Sykes BD, Richards FM. Biochim Biophys Acta 1993; 1164: 36–46.
!
19. Augustyniak W, Kanski R, Kanska M. J Label Compd Radiopharm 2001; 44:
!
553–560.
!
20. Augustyniak W, Suchecki P, Jemielity J, Kanski R, Kanska M. J Label Compd
!
Radiopharm 2002; 46: 559–567.
21. Udenfriend S, Cooper JR. J Biol Chem 1952; 196: 227–233.
22. Nedergaard M. Pharm Acta Helv 1970; 45: 373–378.
23. Fatibello-Filho O, da Cruz Vieira I. Analyst 1997; 122: 345–350.
24. Abu-Nameh ESM, Helaleh MIH, Nabi SA. Acta Pol Pharm – Drug Res 1997; 54:
347–351.
25. Singh DV, Mukherjee PP. Ind J Biochem Biophys 1972; 9: 240–242.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 235–240