Synthesis of [14C]-L-tryptophan and [14C]-5′-hydroxy-L-tryptophan labeled in the carboxyl group

Article abstract of DOI:10.1002/jlcr.685

The synthesis of selectively ¹⁴C-labeled L-tryptophan and its derivative 5-hydroxy-L-tryptophan using chemical and multienzymatic methods is reported. The mixture containing [1-¹⁴C[-DL-alanine, indole or 5-hydroxyindole has been converted to [1-¹⁴C]-L-tryptophan or 5′-hydroxy-[1-¹⁴C]-L-tryptophan, respectively, in a one-pot multienzymatic reaction using four enzymes: D-amino acid oxidase, catalase, glutamic-pyruvic transaminase and tryptophanase. Copyright

Full text of DOI:10.1002/jlcr.685

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 441–447.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.685
Research Article
Synthesis of [14C]-l-tryptophan and [14C]-
50-hydroxy-l-tryptophan labeled in the
carboxyl group
!
E. Boroda, R. Kanski and M. Kanska*
!
Department of Chemistry, Warsaw University, Pasteur 1 Street, 02-089 Warsaw,
Poland
Summary
The synthesis of selectively 14C-labeled l-tryptophan and its derivative 5-
hydroxy-l-tryptophan using chemical and multienzymatic methods is reported.
The mixture containing [1-14C[-dl-alanine, indole or 5-hydroxyindole has been
converted to [1-14C]-l-tryptophan or 50-hydroxy-[1-14C]-l-tryptophan, respec-
tively, in a one-pot multienzymatic reaction using four enzymes: d-amino acid
oxidase, catalase, glutamic-pyruvic transaminase and tryptophanase.
Copyright # 2003 John Wiley & Sons, Ltd.
14
Key Words: Cꢀlabeling; optical isotopomer; L-tryptophan; 50-hydroxy-
L-tryptophan; enzyme
Introduction
The enzyme tryptophanase, TPase, (l-tryptophan indole-lyase, EC
4.1.99.1) has been shown1–3 to catalyze the degradation of l-
tryptophan, l-Trp, and some of its analogues to indole, pyruvate and
ammonia (Scheme 1).
*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 number: KBN 3T09A 046 17
Copyright # 2003 John Wiley & Sons, Ltd.
Received 24 September 2002
Revised 29 October 2002
Accepted 30 October 2002

442
E. BORODA ET AL.
COOH
NH2
O
TPase (EC 4.1.99.1)
NH3
+
+
NH
COO-
pyruvate
NH
L-Trp
indole
Scheme 1. The reversible reaction catalyzed by enzyme TPase
At sufficiently high concentration of pyruvate and ammonia the
TPase also catalyzes the reverse reaction leading to formation3 of l-Trp.
This compound and its derivative 50-hydroxy-l-tryptophan, 5-OH-l-
Trp, are of special interest as the biological precursors for the synthesis
of dopamine and serotonin. It is therefore important to know the exact
mechanism behind the biological synthesis. Moreover, the above
multistep reaction involving hydrogen transfer is still not clear. A
number of mechanistic questions can be resolved4–6 by determining
kinetic isotope effects, KIE, using tritium attached to selected positions
of l-Trp and 5-OH-l-Trp. The numerical values of the KIE may be
useful in distinguishing between alternative mechanisms. Such studies
require l-Trp and 5-OH-Trp selectively 14C-labeled in the carboxyl
group. This label will serve as an internal radiometric standard
during the determination of the KIE for hydrogen isotopes.7–8 Such
studies require the use of only one biologically active enantiomer.
Therefore, enzymes are being widely used for introducing the
label as they exhibit high stereoselectivity and operate under mild
conditions. This paper reports on the multienzymatic one-pot synthesis
of l-Trp and 5-OH-l-Trp labeled with 14C in the carboxyl group. As a
source of 14C 14CO2 has used, which was converted to the starting
intermediate i.e., [1-14C]-dl-alanine on the chemical route.9,10 In the
literature there are several methods describing the synthesis of the
l-enantiomers of Trp and 5-OH-Trp selectively labeled with isotopic
carbon. The 13C-labeled indoles have been converted to the correspond-
ing isotopomers of l-Trp using transformed Esterechia coli cells
containing large amounts of enzyme tryptophan synthetase.11–13 The
11C-labeling of some isotopomers of l-Trp and its derivatives have been
reported; they are required for tumor diagnosis using positron emission
tomography.14–16 In addition several tedious chemical routes for
obtaining the different isotopomers of 14C-labeled DL-Trp have also
been described17.
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF [14C]-L–TRYPTOPHAN
443
Results and discussion
[1-14C]-l-Trp, 1, and 5-OH-[1-14C]-L-Trp, 2, specifically labeled with
14C in the carboxyl group, have been prepared using a combination of
chemical and multienzymatic methods. For this synthesis we applied, as
an intermediate, a racemic mixture of [1-14C]-dl-alanine, 3, which was
obtained in a multistep synthesis that has been previously reported.9,10
As a source of 14C-label 14CO2 (obtained from Ba14CO3) was used. It
has been converted in turn into [1-14C]-DL-alanine, 3, via [1-14C]pro-
pionic acid (carbonation of the Grignard reagent with 14CO2,
C2H5MgJ, and decomposition of the complex formed), 2-bromo[1-14C]-
propionic acid, followed by ammonolysis. In a one-pot multienzymatic
synthesis 3 was converted into [1-14C]-pyruvic acid, 4, using the enzymes
D-AAO/catalase and GPT.18,19 In turn 4 is coupled with indole or
5-hydroxyindole by the enzyme TPase giving 1 or 2, respectively
(Scheme 2). Enzyme D-AAO rapidly transforms D-alanine to pyruvic
acid only, therefore, to avoid the loss of half of radioactivity from the l-
enantiomer we also used the second enzyme, GTP, converting l-alanine
into pyruvic acid only. The medium also contained the enzyme catalase
(removing the H2O2 formed) and coenzymes for D-AAO and GPT, i.e.,
flavine adenine dinucleotide, FAD, and pyridoxal 5-phosphate, PLP,
respectively.
For this synthesis a reliable spectrophotometric method of determin-
ing the concentration of tryptophans was used. The enzyme TPase
14COOH
NH2
D-AAO/catalase
GPT
14COOH
O
[1-14C]-DL-alanine
3
[1-14C]-pyruvic acid
4
14COOH
NH2
R
R
NH
NH
[1-14C]-L-Trp
tryptophanase
or 5 -OH-[1-14C]-L-Trp
R = H or OH
1 or 2
Scheme 2. Enzymatic synthesis of [1-14C]-l-Trp or 5-OH-[1-14C]-l-Trp
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 441–447

444
E. BORODA ET AL.
decomposes l-Trp and its derivatives to pyruvic acid (Scheme 1). The
pyruvic acid is converted into lactic acid in the presence of the enzyme
lactic acid dehydrogenase, LDH, and nicotine amide dinucleotide,
NADH (Scheme 3). NADH gives a strong absorbance at 340 nm that is
easily registered spectrophotometrically. The concentration of NADH
enables the concentration of tryptophan in the measured sample to be
calculated.6,20
COOH
COOH
OH
NADH, H+
O
LDH
pyruvic acid
L-lactic acid
Scheme 3. Conversion of pyruvic acid into l-latic acid by enzyme LDH
Experimental
Materials
Ba14CO3 was obtained from Polatom (Poland). Scintillation cocktail
was purchased from Rotiszint (Germany). Silica gel TLC plates, 60 F256
were from Merck. The enzymes: D-amino acid oxidase (EC. 1.4.3.3), D-
AAO, from PORCINE KIDNEY; catalase (EC 1.11.1.6) from
BOVINE LIVER; glutamic-pyruvic tramsaminase (EC 2.6.1.2), GPT,
from PORCINE HEART; tryptophanase, Tpase (EC 4.1.99.1) from E.
coli, and lacic acid dehydrogenase (1.1.1.27), LDH, from RABBIT
MUSCLE were purchased from Sigma. Coenzymes, i.e., the oxidized
form of flavine adenine dinucleotide, FAD, pyridoxal 5-phosphate,
PLP, and the reduced form of nicotinamide adenine dinucleotide,
NADH, were also from Sigma. Other chemicals, needed for enzymatic
synthesis and control experiments, i.e., a-ketoglutarate, a-KG, TRIS/
HCl, l-tryptophan, dl-alanine, indole and 5-hydoxyindole were
products of Sigma.
Methods
The presence of l-Trp and 5-OH-l-Trp was checked qualitatively by
TLC using silica gel plates and developing solvent: acetic acid}
water}butanol (1 : 1 : 4, v/v). The concentration of the above-
mentioned compounds was determined indirectly by measuring the
concentration of NADH spectophotometrically. This procedure
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 441–447

SYNTHESIS OF [14C]-L–TRYPTOPHAN
445
consists of eliminating pyruvic acid from tryptophans by the enzyme
Tpase. In the next step, pyruvic acid is converted into l-lactic acid in the
presence of the enzyme LDH and coenzyme NADH (Scheme 3). The
measured sample of l-Try or 5-OH-l-Trp was added to a medium
containing 0.2 M phosphate buffer (pH 8), enzymes TPase and LDH
and coenzyme NADH and incubated at room temperature for
45 min.The concentration change of NADH was determined by
measuring the absorbance at 340 nm using a Shimadzu-UV-102 CE-
LV spectrophotometer. In the control studies with inactive compounds
the optical rotation of the l-Trp and 5-OH-l-Trp was checked on the
.
polarimeter (P 3002- A. Krus Optronic}Germany). The radioactivity
of all samples (except of 14CO2, which was measured by an internally
.
filled Geiger–Muller counter) was determined using an automatic liquid
scintillation counter (LISA LSC PW470}Raytest, Germany).
Synthesis of [1-14C]-l-Trp, 1
Synthesis of dl-[1-14C]-alanine, 3. This compound was synthesized as
described elsewhere.9,10 From 100 mg of Ba14CO3 with a total radio-
activity of 3.7 ꢁ 107 Bq (specfic activity of 7.4 ꢁ 107 Bq/mmol) 1.66 g of
dl-[1-14C]alanine of 1.37 ꢁ 107 Bq radioactivity was obtained (specific
activity of 1.04 ꢁ 106 Bq/mmol).
Synthesis of 1. To the incubation vial containing 3 ml of 0.384 M
Tris/HCl buffer at pH 9, the following quantities of reactants were
added in turn:
(i) 50 ml of 0.2 M a-KG;
(ii) 10 ml of 0.1 M PLP;
(iii) 10 ml of 0.001 FAD;
(iv) 100 ml of 0.3 M aqueous solution of [1-14C]-[D,L]-alanine of
3.1 ꢁ 106 Bq radioactivity (sp. act. 1.04 ꢁ 106 Bq/mmol);
(v) 3 ml of catalase (1960 U/ml);
(vi) 50 ml of D-AAO (72 U/ml);
(vii) 50 ml of GPT (1400 U/ml);
(viii) 0.2 ml of 1,5 M (NH4)2SO4;
(ix) 100 ml of 0.4 M indole.
The reaction mixture was incubated at room temperature for 2 days.
The reaction was stopped by adjusting the pH to 5 with glacial acetic
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E. BORODA ET AL.
acid. Non-reacted indole was extracted with toluene (2 ꢁ 6 ml) and next
the aqueous layer was loaded on to a silica gel column (10 ꢁ 100 mm).
The 1 formed was eluted with a mixture of acetonitrile/water (4:1 by
volume). Fractions containing 1 were combined, concentrated under
vacuum at 40–458C and checked as described in Methods. As a result a
1.38 mg sample of 1 was obtained with total radioactivity of
6.8 ꢁ 103 Bq (sp. activity 1.03 ꢁ 106 Bq/mmol).
Enzymatic synthesis of 5-OH-[1-14C]-l-Trp, 2. This derivative of l-Trp
was prepared in the same manner as described in (3b) with the difference
that instead of indole the 5-hydroxyindole was used. A 1.92 mg sample
of 2 was obtained with total radioactivity of 8.9 ꢁ 103 Bq (sp. activity
1.02 ꢁ 106 Bq/mmole).
Acknowledgements
The work was sponsored by grant KBN 3 T09A 046 17
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