Enzymatic synthesis of phenylpyruvic acid labeled with deuterium, tritium, and carbon-14

Article abstract of DOI:10.1002/jlcr.1530

The synthesis of isotopomers of phenylpyruvic acid, PPA, selectively labeled with hydrogen isotopes in the 3-position of the side-chain is reported. Three deuterium or tritium labeled isotopomers of L-phenylalanine, L-Phe, i.e. [(3S)-²H]-L-, [(

Full text of DOI:10.1002/jlcr.1530

Research Article
Received 1 April 2008,
Revised 6 June 2008,
Accepted 17 June 2008
Published online in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1530
Enzymatic synthesis of phenylpyruvic acid
labeled with deuterium, tritium, and
carbon-14
Ã
´
Katarzyna Skowera and Marianna Kanska
The synthesis of isotopomers of phenylpyruvic acid, PPA, selectively labeled with hydrogen isotopes in the 3-position of the
side-chain is reported. Three deuterium or tritium labeled isotopomers of L-phenylalanine, L-Phe, i.e. [(3S)-2H]-L-, [(3S)-3H]-
L-, and doubly labeled [(3S)-2H/3H]-L-Phe were synthesized using the enzyme phenylalanine ammonia lyase (EC 4.3.1.5). In
the second step these isotopomers of L-Phe were converted into [(3S)-2H], [(3S)-3H]-, and [(3S)-2H/3H]-isotopomers of PPA
using the enzyme L-phenylalanine dedydrogenase (EC 1.4.1.20). The isotopomer of PPA labeled with 14C in carboxylic
group, [1-14C]-PPA, was obtained in a two-step enzymatic reaction using [1-14C]-cinnamic acid as the starting substrate.
Keywords: carbon-14; deuterium; enzyme; phenylpyruvic acid; tritium
was used as an internal radiometric standard for the KIE assays.
For this purpose, PPA, labeled in the carboxylic acid group
Introduction
[1-14C]-PPA was chosen, as the 14C label is placed in a position
Phenylketonuria (PKU), a human genetic disease, named in
recognition of its unusual metabolic by-products, is inherited
with a high average birth incidence (10À4– one for 104 births) in
European and Asian populations.1 The disease is accompanied
by elevated levels of phenylalanine metabolites such as
phenylacetate, phenyllactate, and phenylpyruvate in body
fluids. Detailed knowledge of the mechanisms of enzymatic
conversion of L-Phe into its derivatives is essential for under-
standing PKU and the proper treatment and control of dietary
therapy of PKU patients. One of metabolic paths of conversion
of L-Phe into phenylpyruvic acid is NAD1 dependent, reversible,
oxidative deamination2,3 catalyzed by the enzyme L-phenylala-
nine dehydrogenase (EC 1.4.1.20), PheDH, Figure 1.
remote from the center of reaction.
In literature there are a few papers that describe the synthesis
of deuterium-,9 tritium-,10 and 14C-labeled11 isotopomers of PPA.
Unfortunately, most of them lead to isotopomers bearing the
label in positions not useful for our proposed studies.
In this paper, the synthesis of four isotopomers of PPA labeled
with deuterium and tritium in the 3-position, i. e. [3-2H]-PPA,
[3-3H]-PPA, doubly labeled [3-2H/3H]-PPA, and 14C-labeled in the
carboxylic group [1-14C]-PPA was reported.
Results and discussion
The addition of ammonia to cinnamic acid catalyzed by the
enzyme PAL (phenylalanine ammonia lyase, EC 4.3.5.1) carried
out in fully deuteriated ammonia buffer (1.8 M NH4Cl in D2O, pD
9.8) led to the formation of [(3S)-2H]-L-Phe (detailed protocol of
this step was earlier described12 by us). The similar way in which
this addition was carried out in tritiated ammonia buffer gave
[(3S)-3H]-L-Phe, as well as by using ammonia buffer composed of
deuteriated and tritiated water doubly labeled [(3S)-2H/3H]-L-
Phe was afforded. The extent of deuterium incorporation at
3-position of the obtained products were checked by H NMR
spectra (97% enrichment).
We had to perform certain preliminary kinetic studies to find
the optimum reaction conditions for obtaining a high yield of
The metabolism of l-Phe, a key step in PKU disease, is not
clearly understood. In addition, the mechanism of the industrial
manufacture of L-Phe4 from PPA or phenyllactic acid has not
been understood to date. The production of phenylalanine as a
starting material for the sweetener aspartame has been a target
5–7
for industrial research;
new enzymatic methods for the synthesis of PPA.
this prompted research to seek for
The goal of our planned research was to investigate some
details of this reversible oxidative deamination reaction
presented in Figure 1 by applying the kinetic isotope effect
(KIE) and solvent isotope effect methods and determining
kinetic isotope values in the rate determining step.8 We assume
that in the course of this reaction some tautomerization of PPA
takes place, and in the process the stereospecific abstraction of
proton from the 3-position of PPA is involved.9 The numerical
values of the isotope effects allowed us to elucidate the intrinsic
details of the mechanism and were useful to draw a distinction
between alternative mechanisms. The aforementioned studies
require the use of isotopomers of PPA labeled with deuterium
and tritium in the 3-position. The 14C-labeled isotopomer PPA
1
Department of Chemistry, University of Warsaw, Pasteur Str. 1., 02-093 Warsaw,
Poland
*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
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´
K. Skowera and M. Kanska
the desired labeled isotopomers of PPA. We selected the UV-
spectrometric technique for monitoring the progress of
oxidative deamination of L-Phe, Figure 2. By observing the
absorbance of the NADH at 340 nm we studied the dependence
of the reaction yield on time, concentration of enzyme and
substrates. All the kinetic studies were carried out at room
temperature using glycine buffer.13 We have found that the
yield of PPA, depends on concentration of L-Phe, and this
increases to achieve a steady state after 30–40 min incubation
time. The maximal yield of PPA can be reached at 8 mM
concentration of L-phenylalanine in the reaction medium, Figure
3. A higher concentration of L-Phe does not improve the yield of
PPA. More detailed kinetics studies allowed us to elaborate the
optimal parameters for carrying out this key reaction step (pH
10.7, room temperature, reaction time ca 4 h, concentration of
buffer equal to 0.1 M, and quantity of enzyme ca 2 U) to afford
the highest possible chemical yield of PPA (ca 45% under these
experimental conditions).
Therefore, in a subsequent step these three isotopomers of
L-Phe were converted into corresponding isotopomers of PPA, i.
e. [(3S)-2H]-PPA, [(3S)-3H]-PPA, and [(3S)-2H/3H]-PPA using the
enzyme PheDH (L-phenylalanine dehydrogenase, EC 1.4.1.20).
[1-14C]-cinnamic acid, the substrate for the synthesis of
[1-14C]-L-Phe, was obtained by the method described earlier14,15
and subsequently 14C-labeled L-Phe (43% yield), and [1-14C]-PPA
(40% yield) were prepared as described above.
Experimental
Materials
Enzymes PAL (phenylalanine ammonia lyase, EC 4.3.1.5) from
Rhodotorula glutinis and PheDH (L-phenylalanine dehydrogen-
ase, EC 1.4.1.20) from Sporosacrina sp, cofactor NAD1 were
purchased by Sigma.
[1-14C]-cinnamic acid was produced from K14CN (supplied by
Polatom - Swierk, Poland) by a multistep synthesis according to
literature procedure.15 Deuteriated water (99.9% D) was
obtained from Aldrich. Tritiated water was purchased from INC
Pharmaceutical Inc., Irvine, CA, USA. Scintillation cocktail was
obtained from Rotiszint (Germany).
Silica gel plates were from Merck (silica gel 60 F254, catalog no
105554). Aluminum oxide for column chromatography was
obtained from POCh (Poland); Amberlit IR 120 was from Aldrich.
L-phenylalanine, phenylpyruvic acid, glycine, and other
chemicals needed for trial synthesis and kinetic measurements
were from Aldrich.
Methods
The radioactivity of all the samples was determined using a
liquid scintillation counter (LISA LSC PW470,Germany). The
extent of deuterium incorporation into 3S position of L-Phe and
PPA, and the presence of enol form of PPA were determined
from 1H NMR spectra recorded on a Varian Unity1200 MHz
spectrometer.
The concentration of PPA was determined indirectly by
measuring the concentration of NADH spectrophotometri-
cally.16,17 The enzyme PheDH converts L-Phe into PPA in the
presence of coenzyme NAD1, Figure 1. The concentration
change of NADH was determined by measuring the absorbance
at 340 nm using a Shimadzu UV-102-CE-LV spectrometer.
The presence of phenylalanine and phenylpyruvic acid were
checked qualitatively by TLC using aluminum oxide plates and
developing solvents: butan-1-ol:acetic acid:water 4:1:5, v/v, and
acetonitrile:water, 4:1, v/v for phenylalanine and phenylpyruvic
acid, respectively (visualization by UV lamp or by exposing the
plates to iodine vapors).
The extent of deuterium incorporation at 3S-position of the
products was confirmed by disappearing the signal from 3S-
1
proton in H NMR spectrum (98% D enrichment).
The degree of incorporation of label, via stereospecifically
labeled L-Phe, into 3S- (or 3R-) positions of PPA can be affected
by the keto-enol tautomerism observed in keto acids, Figure 4.
We have confirmed by 1H NMR that under our experimental
conditions (synthesis, separation, and purification) the enol form
of PPA is present in a negligible amount (ca 1%).
COOH
O
COOH
NH2
Synthesis
Synthesis of [(3S)-2H]-phenylpyruvic acid, 3
Synthesis of [(3S)-2H]-l-phenylalanine, 3
EC. 1.4.1.20
+
NAD+
+
NADH
PPA
L-Phe
In a capped vial, to cinnamic acid (30 mg, 0.2 mmol) dissolved in
5.25mL of fully deuteriated 1.8 M ammonium buffer (NH4Cl
Figure 1. Enzymatic conversion of L-Phe into PPA catalyzed by enzyme
L-phenylalanine dehydrogenase.
COOH
COOH
O
COOH
NH2
H
R
H
R
D(T)
D(T)
S
S
PheDH
PAL
cinnamic
acid
[3(S)-D(T)]-L-Phe
PPA
=
14C-label
Figure 2. The enzymatic conversion of cinnamic acid into PPA.
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J. Label Compd. Radiopharm 2008, 51 321–324

´
K. Skowera and M. Kanska
collected as 5 mL fractions. The presence of 3 in eluted fractions
was checked by TLC as above. The fractions containing 3 were
concentrated to a volume of about 3 mL under reduced
pressure and lyophilized to dryness under vacuum. As a result
about 4.8 mg (0.029 mmol) of 3 was obtained (48% yield).
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Synthesis of [3-3H]-phenylpyruvic acid, 4.
Synthesis of [(3S)-3H]-l-phenylalanine, 5
3mM
4mM
7mM
9mM
In a capped vial, to cinnamic acid (8.6 mg, 0.058 mmol) dissolved
in ammonium buffer (1.5 mL, pH 9.4) were added a solution of
enzyme PAL (150 mL, 2 U) and 300 mL tritiated water with total
radioactivity of about 11 GBq. The incubation was carried out as
described in the section ‘synthesis of [(3S)-2H]-L-phenylalanine,
2’. Next, the tritiated water from post-reaction mixture was
removed by lyophilization. The tritiated 5 was extracted from
the residue five times by diethyl ether (10 mL) to separate it
from unreacted cinnamic acid and purified chromatographically
as described in the section ‘synthesis of [(3S)-2H]-l-phenylala-
nine, 2’. The remaining of HTO and tritium from labile position of
L-Phe was washed out with water, and in the next 5 was eluted
with 1 M NH3(aq), and collected as 5 mL fractions. From each
fraction 100 mL sample was taken for radioactivity assay. The
fractions containing 5 were combined and evaporated to
dryness under reduced pressure at 401C. A 3.8 mg (0.0232 mmol)
sample of 5 was obtained with total radioactivity 1.7 Â 106 Bq
(sp. activity 73.2 MBq/mmol, 40% yield).
0
10
20
30
40
50
60
time [min]
Figure 3. The dependence of conversion of L-Phe into PPA from its concentration
in the incubation medium.
COOH
O
COOH
OH
H
H
H
Synthesis of [(3S)-3H]-phenylpyruvic acid, 4
keto form
enol form
The whole obtained sample of 5 was dissolved in a vial
containing 0.1 M of glycine buffer (4.5 mL, pH 10.7), and 15 mg
NAD1, and enzyme PheDH (1,2 mg, 1.7 U) were added. The
reaction mixture was incubated, separated, and purified as
described in the section ‘synthesis of [3S-2H]-phenylpyruvic acid,
3’. As a result about 2.1 mg (0.013 mmol) sample of 4 was
obtained (56% yield) with total radioactivity 0.95 MBq (specific
activity 73.2 MBq/mmol).
Figure 4. The equilibrium between the keto and enol forms of phenylpyruvic acid.
dissolved in D2O and adjusted to pD 9.8 with 30% solution of
KOD in D2O) was added a solution of enzyme PAL (300mL, 2 U)
and the reaction mixture was incubated at 371C for 7 days. The
progress of reaction was monitored by TLC using butan-1-
ol:acetic acid:water (4:1:5, v/v) as developing solvent. Crude
deuteriated product, 2, was extracted five times by 10mL diethyl
ether and purified by chromatography. Therefore, the excess of
diethyl ether was evaporated to 2–3 mL volume, and the residue
was loaded onto Amberlite IR 120 H1 column (100Â 10mm) and
2 was eluted with 1 M NH3(aq) and collected as 5 mL fractions.
The fractions containing 2 were combined and evaporated to
dryness under reduced pressure at 401C. As a result a 12.5mg
(0.076mmol) sample of 2 was obtained with 38% yield. The
position and the extent of deuterium incorporation were verified
Synthesis of doubly labeled [(3S-2H/3H]-phenylpyruvic
acid, 6
Synthesis of [(3S)-2H/3H]-L-phenylalanine, 7
In a capped vial, to cinnamic acid (8.6 mg, 0.058 mmol) dissolved
in 1.8 M ammonium buffer (1.5 mL, pD 9.8) were added a
solution of enzyme PAL (150 ml, 2 U) and 300 mL of tritiated water
with total radioactivity of 5.2 GBq. The incubation and the
separation protocol were the same as described in the section
‘synthesis of [(3S)-2H]-l-phenylalanine, 2’. As a result 4.1 mg
(0.025 mmol) sample of 7 was obtained with total radioactivity
of 96 kBq (sp. activity 3.85 MBq/mmol, yield 43%)
1
by H NMR (97% D incorporation in 3-position).
Synthesis of [3S-2H]-phenylpyruvic acid, 3
In a capped vial, 10 mg (0.06 mmol) sample of 2 was dissolved in
0.1 M of glycine buffer (5 mL, pH 10.7), and 20 mg NAD1, and
1.2 mg of enzyme PheDH (1.6 U) was added. The reaction
mixture was incubated for 4 h at room temperature. Progress of
the reaction was monitored by TLC using acetonitrile:water (4:1,
v/v, visualization by UV light) as the mobile phase. Next, the
reaction mixture was loaded onto silica gel column
(100 Â 10 mm), and 3 was washed out with eluent composed
with diethyl ether, methanol, and formic acid (98:1:1, v/v) and
Synthesis of [3S-2H/3H]-phenylpyruvic acid, 6
The whole obtained sample of 8 was dissolved in a vial with
0.1 M of glycine buffer (3 mL, pH 10.7) and to this 16 mg NAD1,
and enzyme PheDH (1.6 mg, 2.6 U) were added. The incubation,
separation and purification of 6 were carried out as described
in the section ‘synthesis of [3S-2H]-phenylpyruvic acid, 3’. As a
result 1.9 mg (0.012 mmol) sample of 6 was obtained with total
J. Label Compd. Radiopharm 2008, 51 321–324
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´
K. Skowera and M. Kanska
radioactivity of 44 kBq (sp. activity of 3.7 MBq/mmol, yield
46%).
References
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`
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[3S-2H]-phenylpyruvic acid, 3’. As a result a 4.6 mg (0.028 mmol)
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This work was supported by grant 501/64-BST-123623.
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 321–324