Chemical and enzymatic synthesis of tritium labelled coenzymes

Article abstract of DOI:10.1002/jlcr.688

Details of the synthesis of tritium labelled co enzymes-nicotinamide adenine dinucleotide and coenzyme A by isotope exchange and enzymatic reactions are reported. It was established, that among the investigated chemical reactions, the most effective is solid state isotope exchange with gaseous tritium. This method was used to produce [³H] nicotinamide adenine dinucleotide (111 Ci/ mmol), [³H] coenzyme A (3.9 Ci/mmol) and D-[G-³H] pantothenic acid (43 Ci/ mmol). It was shown that most of the tritium in the labeled nicotinamide adenine dinucleotide and coenzyme A was localized in the nicotinamide (98%) and adenine (89%) sites, respectively. For synthesis of coenzymes labelled with tritium at other sites we developed enzymatic methods which used labelled precursors. Optimum conditions for enzymatic synthesis of [adenine-³H] nicotinamide adenine dinucleotide from [2,8-³H] ATP and [pantothenate-³H] coenzyme A from D-[G-³H] pantothenic acid were determined. The tritium labelled acetyl coenzyme A was synthesized by acetylation of labelled coenzyme A with acetic anhydride. The methods chosen allow one to produce tritium labelled coenzymes at high specific activity. Copyright

Full text of DOI:10.1002/jlcr.688

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 465–473.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.688
Research Article
Chemical and enzymatic synthesis of tritium
labelled coenzymes
G. V. Sidorov*, Yu. B. Zverkov, S. I. Shram, T. Yu. Lazurkina and
N. F. Myasoedov
Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Sq. 2,
123182, Moscow, Russia
Summary
Details of the synthesis of tritium labelled co enzymes-nicotinamide adenine
dinucleotide and coenzyme A by isotope exchange and enzymatic reactions are
reported. It was established, that among the investigated chemical reactions,
the most effective is solid state isotope exchange with gaseous tritium. This
method was used to produce [3H] nicotinamide adenine dinucleotide (111 Ci/
mmol), [3H] coenzyme A (3.9 Ci/mmol) and d-[G-3H] pantothenic acid (43 Ci/
mmol). It was shown that most of the tritium in the labeled nicotinamide
adenine dinucleotide and coenzyme A was localized in the nicotinamide (98%)
and adenine (89%) sites, respectively. For synthesis of coenzymes labelled with
tritium at other sites we developed enzymatic methods which used labelled
precursors. Optimum conditions for enzymatic synthesis of [adenine-3H]
nicotinamide adenine dinucleotide from [2,8-3H] ATP and [pantothenate-3H]
coenzyme A from d-[G-3H] pantothenic acid were determined. The tritium
labelled acetyl coenzyme A was synthesized by acetylation of labelled
coenzyme A with acetic anhydride. The methods chosen allow one to produce
tritium labelled coenzymes at high specific activity. Copyright # 2003 John
Wiley & Sons, Ltd.
Key Words: tritium; hydrogen isotope exchange; nicotinamide adenine di-
nucleotide; coenzyme A; pantothenic acid; enzymatic synthesis
*Correspondence to: G. Sidorov, Institute of Molecular Genetics, Russian Academy of Sciences,
Kurchatov Sq. 2, Moscow 123182, Russia. E-mail: sidgv@img.ras.ru
Copyright # 2003 John Wiley & Sons, Ltd.
Received 3 May 2002
Revised 11 November 2002
Accepted 18 November 2002

466
G. V. SIDOROV ET AL.
Introduction
Nicotinamide adenine dinucleotide (NAD, Figure 1a) and coenzyme A
(CoA, Figure 1b) are universal molecules in the living world. They
play an important role in the metabolism of low-molecular
compounds and in energy accumulation processes. NAD+ is the
major acceptor of electrons at the oxidation of ‘fuel molecules’,
such as glucose and fatty acids. Another important function of NAD
is its involvement in reactions of mono- and poly(ADP-ribosyl)ation,
which ensure the regulation of various cellular processes. CoA
plays a key role in the processes of oxidation and synthesis of
fatty acids.
For studying processes which involve the participation of coenzymes
the labelled analogues are sometimes necessary. The objective of this
work was to develop chemical and enzymatic methods of selective
tritium labelling of NAD and CoA. For this purpose we studied
hydrogen isotope exchange reactions of coenzymes with tritium gas
both in solution and the solid phase. We also develop enzymatic
methods of synthesis with the use of labelled precursors. [2,8-3H] ATP
and d-[G-3H] pantothenic acid were chosen for the synthesis of NAD
and CoA, respectively. The tritium labelled acetyl coenzyme A was
synthesized by acetylation of labelled coenzyme A with acetic
anhydride.
Figure 1. Structures of nicotinamide adenine dinucleotide (a) and coenzyme A (b)
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CHEMICAL AND ENZYMATIC SYNTHESIS
467
Results and discussion
In the hydrogen isotope exchange between NAD and tritiated water
(Table 1, method a) or gaseous tritium (methods b and c) the highest
specific activity (Asp) was observed for the solid state isotope exchange
reaction with gaseous tritium (method c). High values of Asp and yield
of NAD were obtained both for hydrogen isotope exchange in solution
(method b) and solid state exchange (method c) with tritiated water
(method a). The distribution of tritium in the NAD molecule was
ascertained by acid hydrolysis. It was established by methods b and c
that only 2.2% and 1.3% of the radioactivity respectively was in the
adenine part of the NAD molecule, the majority being in the
nicotinamide part of the molecule.
We also studied the reaction of solid state catalytic isotope exchange
of coenzyme A and d-pantothenic acid (PA) with tritium (method c,
Tables 2 and 3). Table 2 shows the effect of different catalysts and
temperature on the yield and specified radioactivity of the CoA
produced by this reaction. Catalyst A is the first choice for the synthesis
of labelled CoA. Approximately 90% of the tritium in labelled CoA was
located in the adenine part of the molecule. The isotope exchange
reaction with PA was most efficient with catalyst C (Table 3). The
reaction of the barium salt of PA with carries-free tritium (catalyst C) at
temperature 1808C resulted in the formation of d-[G-3H] PA with Asp of
43 Ci/mmol and yield of 7.4%.
Table 1. Synthesis of tritium labelled NAD using different methods, Catalyst B
Methods of synthesis
Specific activity
(Ci/mmol)
Yield (%)
a.
b.
Isotope exchange with tritiated water
Isotope exchange with gaseous tritium in
solution
4.5
48.3
17.0
17.8
c.
Solid state isotope exchange with
gaseous tritium (1108C, 30 min)
111
12.6
Table 2. Solid state catalytic isotope exchange of coenzyme A using gaseous
tritium. Incubation time 30 min
Catalyst
T(8C)
Specific activity
(Ci/mmol)
Yield (%)
A
A
A
B
140
170
180
190
0.90
2.0
3.9
1.8
58
43
28
37
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G. V. SIDOROV ET AL.
Table 3. Solid state catalytic isotope exchange of pantothenic acid using gaseous
tritium-protium mixture (1:1000 v/v). Incubation time 30 min.
Catalyst
T(8C)
Characteristics of tritium labelled PA
Asp (Ci/mol)
Yield (%)
A
C
C
C
170
170
180
190
8.2
25
52
120
32
30
11
2.4
Selectively labelled adenine and pantothenate sites in NAD and CoA
are required for investigation of protein transferase reactions and for
other purposes. To produce these compounds, the one stage enzymatic
synthesis was studied. The enzymatic methods described earlier1–3 were
designed for the synthesis of higher quantities of coenzymes and gave
low yields of the final products.
The enzymatic synthesis of tritium labelled NAD was carried out with
[2,8-3H] ATP, nicotinamide monoculeotide (NMN), and enzyme
nicotinamide nucleotide adenylytransferase (NNAT) from baker’s yeast
(Saccharomyces cerevisiae):
Â
Ã
Â
Ã
2; 8-3H ATP þ NMN $ adenine-2; 8-3H NAD þ PPi
The reversibility of the reaction, enzyme inactivation and presence of
phosphodiesterase activity in the enzyme preparation are the main
limitation of this process. The high dependence of the NAD yield on the
initial concentrations of NMN, ATP, and enzyme were determined
(Table 4). We determined the optimum reaction conditions to be as
follows: NMN – 5 mM, ATP – 1 mM; enzyme – 3 mg/ml; incubation
time – 1 h. Under these conditions the yield, as based on a radioactivity,
was 85–90%.
For the synthesis of tritium labelled CoA we utilized permeable cells
from bacteria Brevibacterium ammoniagenes that catalysed complete
transformation of d-pantpthenic acid to CoA.
PA ! 40-phospho-PA ! 40-phosphopanthenoyl-L-cysteine !
40-phophopantetheine ! dephospho-CoA ! CoA
The total equation for the reaction with [G-3H] PA as initial compound
looks as follows:
½G-3HꢀPA þ L-cysteine þ 4 ꢁ ATP !
½pantothenate-3HꢀCoA þ CO2 þ 3ꢁADP þ PiþPPi
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CHEMICAL AND ENZYMATIC SYNTHESIS
469
Table 4. The effect of reagent concentration on the yield of tritium labelled NAD
in the enzymatic synthesis. Incubation time 1 h at 378C
Concentration of reagent
Yield (%)
ATP (mM)
0.1
NMN (mM)
0.5
NNAT (mg/ml)
0.3
1.0
1.0
3.0
2.0
6.0
3.0
10.0
28.9
49.5
55.3
55.9
71.3
72.2
85.2
79.5
0.3
0.6
1.0
1.5
3.0
5.0
Table 5. The effect of reagent concentration on the yield of tritium labelled CoA
in the enzymatic synthesis
Concentration of reagent
Total product
yield (%)
Yield of labelled
CoA (%)
Enzyme preparation
(mg/ml)a
5
20
100
1
2
4
68.7
90.7
91.0
94.8
84.0
76.9
73.7
34.4
ND
0.6
23.8
11.0
23.5
28.5
3.2
Pantothenic acid
(mM)b
7
10
ND
Incubation 15 h at 378C.
a PA – 1.4 mM; Cys – 5 mM; ATP – 10mM.
b B. Ammoniagenes – 100 mg/ml; the molar ratio of PA, Cys and ATP – 1:2:6.
The final yield depended in a complicated manner on the concentrations
of permeable cells and PA (Table 5). We identified the optimum
concentrations of reagents to be as follows: PA – 4 mM; ATP – 30 mM;
l-cysteine – 15 mM; B. ammoniagenes – 100 mg/ml. Under these
conditions the yield of CoA reached 35%.
The acetylation reaction of [pantothenate-3H] CoA (see Experimen-
tal) allowed us to produce acetyl-[pantothenate-3H] CoA with a high
yield (92.8%) and Asp of 39.6 Ci/mmol.
Experimental
The catalysts used were: 5% Pd/BaSO4 (A) and 5% Pd/CaCO3 (B)
(Fluka), 5% Pd/CaCO3 (C) (Aldrich) and PdO (Merck).
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G. V. SIDOROV ET AL.
Table 6. HPLC of tritium labelled compounds. Nucleosil 120-5, C18,
4.6 ꢂ 250 mm
Compound
NAD
Mobile phase
Retention time (min)
10.2
2%CH3CN in 0.1 M TEAB
(pH 7.0), 0.5 ml/min
CoA
5% CH3CN in 220 mM NaH2PO4
(pH 5.3) and 0.45 M dithiothreitol
(DTT), 1.0 ml/min
24.0
Acetyl-CoA
10% CH3CN in 0.1 M TEAB
(pH 6.5), 1.0 ml/min
2% CH3CN in 0.1 M TEAB
(pH 6.2), 1.0 ml/min
14.3
16.4
Pantothenic acid
All the labelling procedures were carried out in an installation
suitable for manipulations with gaseous tritium.4
TLC was performed on PEI-cellulose plates (Merck), solvents –
1 mol/l NaCl or distilled water and on Silufol silicagel plates in butanol–
acetic acid–amonia–acetone–water (35:15:3:25:15 v/v/v/v).
We carried out the purification of [3H] NAD in two stages: (1) by
chromatography on DEAE-cellulose column (16 ꢂ 250 mm), elution
(60 ml/h) by linear gradient 0.05–0.15 M (8 h) of triethylammonium
bicarbonate (TEAB) pH 7.5; and (2) by HPLC (see Table 6). The acetyl-
[3H] CoA was also purified by this procedure (Table 6).
UV absorption spectra were measured using a Specord M-40
spectrophotometer (Germany). Sample radioactivity was measured on
a b-radioactivity counter using LC-8 as scintillation liquid (NPO
‘Monocrystallreactiv’, Russia).
Isotope exchange reaction with high-specific activity tritiated water:
The tritiated water was synthesized by a method described previously.5
The tritiated water was placed in a reaction vial containing 10 mg of
catalyst and 1.5 mg of NAD. The mixture was stirred at room
temperature for 16 h. After the incubation, the catalyst was separated
by centrifugation and the solvent removed in vacuum. Labile tritium
was removed by dissolving residue in 10 ml of 50% ethanol and further
evaporation. This operation was repeated twice.
Isotope exchange with gaseous tritium in solution: 2.2 mg of NAD in
100 ml of water and 10 mg of catalyst A were placed in a glass reaction
vial. The vial was connected to a tritium gas line. The vial was then
frozen in liquid nitrogen, evacuated and filled with gaseous tritium up to
the pressure of 300 mm Hg. After defrosting, the reaction mixture was
stirred at room temperature. After removal of gas, the catalyst was
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CHEMICAL AND ENZYMATIC SYNTHESIS
471
separated by filtration. Labile tritium was removed by evaporation at
35–378C. The dry residue was dissolved in water, and the labelled
compound isolated.
Solid state catalytic hydrogenation (SCH): A solid mixture of the
initial compound and the catalyst was place in a glass reaction vial that
was then connected to a system for handling gaseous tritium, evacuated
and tritium gas introduced into the vial at 300 mm Hg pressure. The vial
was thermostated at the chosen temperature, then cooled and the excess
tritium removed from the vial. The reaction products were washed with
water, and the catalyst was separated by filtration. Labile tritium was
removed by evaporation of the filtrate to dryness. The end product was
isolated by chromatography as described above.
Analysis of intramolecular distribution of tritium: The coenzymes were
hydrolysed under acid conditions. 0.25 Mg of coenzyme A (0.3 mmol)
was dissolved in 0.3 ml of NaH2PO4 (pH 4.4), placed in a sealed vial and
kept at 1008C for 45 h. 0.20 mg of NAD (0.3 mmol) was dissolved in
0.3 ml of 0.01 M HCl placed in the sealed vial and kept at 1008C for
30 min. After hydrolysis the AMP was isolated and its Asp was
determined.
Enzymatic synthesis of tritium labelled NAD: The enzymatic prepara-
tion of nicotiamide nucleotide-adenyliltransferase from S. cerevisiae
was prepared as described early.6 The synthesis of [adenine-3H]
NAD was carried out in the following way: 39 mCi of [2,8-3H] ATP
(34 Ci/mmol) was placed in a 50 ml flask, the solvent removed by
evaporation and the mixture (0.8 ml) of 0.1 M tris-HCl (pH 7.5) (500 ml),
1 M MgCl2 (150 ml), 50 mM NMN (100 ml) and deionized water (200 ml)
added. The mixture was pre-incubated for 5 min at 378C and 5 mg of
enzyme in 200 ml of 25 mM tris-HCl (pH 7.4) containing reduced
glutatione (2 mM) and ethylenediaminetetraacetic acid (1 mM) was
added. After 1 h incubation at 378C the vial was placed on a boiling
water bath for 3 min and then cooled to room temperature. Denaturated
proteins and other insoluble components were removed by centrifuga-
tion. NAD was purified by DEAE-cellulose and HPLC as described
above. Fractions with labelled NAD were combined and TEAB was
removed by evaporation. In this way 13.5 mCi of [adenine-3H] NAD
with Asp 13.6 Ci/mmol and radiochemical purity 98% was obtained.
The NAD was stored as a solution of 1 mCi/ml in 50% ethanol at
–208C.
Enzymatic synthesis of tritium labelled CoA: For the synthesis of
[pantothenate-3H] CoA from [G-3H] PA we utilized permeabilized cells
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G. V. SIDOROV ET AL.
of B. ammoniagenes as described in the literaure.3 The reaction mixture
contained the following reagents: 86 mCi (2 mmol) of d-[G-3H]
pantothenic acid; 750 ml of 0.5 M potassium-phosphate buffer (pH
6.5); 30 ml of 0.5 M MgSO4, 150 ml of 50 mM l-cysteine (30 ml), 150 ml of
100 mM ATP, and 420 ml of H2O. The mixture was carefully stirred and
pre-incubated for 10 min at 378C. Then 150 mg of permeable cells were
added and the mixture incubated for 16 h at 378C. After incubation the
mixture centrifuged for 20 min at 6000 rpm.The pellet was washed with
3 ml of water and centrifuged as described above. The supernatants were
combined and diluted with H2O to 5 ml. Then 2-mercaptoethanol was
added and the mixture kept at 308C for 1 h. The labelled CoA was
purified by HPCL. As a result we obtained 25.5 mCi of CoA with Asp of
40 Ci/mmol and purity 98%. The CoA prepared was stored as a
solution (1 mCi/ml) in 50% ethanol at –208C.
Acetylation of [pantothenate-3H]coenzyme A: For the preparation of
microquantity of labelled acetyl-CoA we modified the procedure
described in Ref.7 To 14 mCi of coenzyme A (0.35 mmol) in 44 ml of
deionized water was added 4 ml of 1 M KHCO3 and the mixture cooled
to 08C. Then 4.6 ml of freshly prepared 0.1 M acetic anhydried in water
(08C) was added and the mixture kept for 5 min at 08C. then the solution
was neutralized with 0.1 M TEAB and evaporated under vacuum.
TEAB was removed by twice dilution/evaporation procedure with 50%
ethanol as solution. The dry residue was dissolved in 0.5–1.0 ml of water
and fractionated on a DEAE-cellulose column. Fractions with acetyl-
[3H] CoA were combined and evaporated under vacuum. TEAB was
removed as previously described. The final purification was performed
by HPCL. After purification we obtained 13 mCi of acetyl-[3H] CoA
with Asp of 39 Ci/mmol.
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