Synthesis of [3′-14C] coenzyme Q10

Article abstract of DOI:10.1002/jlcr.588

Radio-labelled coenzyme Q₁₀, labelled at the 3′-position with ¹⁴C, was synthesized starting from natural solanesol and ethyl [3-¹⁴C] acetoacetate. The radiochemical yield was 8.0% from ethyl [3-¹⁴C] acetoacetate

Full text of DOI:10.1002/jlcr.588

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 823–829.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.588
Research Article
Synthesis of [30-14C] coenzyme Q10
Kimio Hamamura1, Isao Yamatsu1, Norio Minami1, Yoji Yamagishi1,
Yuichi Inai1, Shizumasa Kijima1 and Tetsuya Nakamura2*
1 Eisai Research Laboratories, Eisai Co., Ltd., 4-6-10 Koishikawa, Bunkyo-ku,
Tokyo 112-8088, Japan
2 Department of Chemistry, Shibaura Institute of Technology, 307 Tameihara,
Hukasaku, Saitama-shi, Saitama 330-8570, Japan
Summary
Radio-labelled coenzyme Q10, labelled at the 30-position with 14C, was
synthesized starting from natural solanesol and ethyl [3-14C] acetoacetate.
The radiochemical yield was 8.0% from ethyl [3-14C] acetoacetate. The specific
radioactivity of the product was 44.8 mCi, 1.66 MBq/mg. The specific radio-
activity and radiochemical purity are sufficiently high to enable us to use this
labelled form of coenzyme Q10 in metabolic studies. Copyright # 2002 John
Wiley & Sons, Ltd.
Key Words: [30-14C]coenzyme Q10; ethyl [3-14C]acetoacetate; solanesol;
[3-14C]decaprenol; triethyl phosphonoacetate
Introduction
Radio-labelled coenzyme Q10 (Co Q10) is a particularly useful tool in the
investigation of the metabolic fate of Co Q10. This is because, while Co
Q10 exists endogenously, exogenous Co Q10 has been reported to have
beneficial effects under various conditions of oxidative stress. Therefore,
it is advantageous to be able to differentiate the source of Co Q10 in vivo
when discussing the role of exogenous Co Q10. The number of
*Correspondence to: T. Nakamura, Vitamin E Information Section, Eisai Co., Ltd, 4-6-10
Koishikawa, Bunkyo-ku, Tokyo 112-8088, Japan.
Copyright # 2002 John Wiley & Sons, Ltd.
Received 19 February 2002
Accepted 7 March 2002

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K. HAMAMURA ET AL.
isoprenoid units in the side chain of Co Q homologues occurring in
animals differs among species.1 The homologue that occurs in man is Co
Q10 (ubidecarenone).2 The side chain has been confirmed to be of the
all-trans configuration.3 Using [30-14C]Co Q10 6, the behaviour of
exogenous Co Q10 has been reported in several papers.4–10 However, the
preparation procedure of the compound has not previously been
described. The purpose of this paper is to describe a detailed
preparation procedure for [30-14C]Co Q10 6.
Materials and methods
Solanesol (natural) was obtained from Nisshin Flour Milling Co.
(Tokyo, Japan). Ethyl [3-14C]acetoacetate (specific activity 50 mCi,
1.85 GBq/mmol) was purchased from Amersham International plc
(Buckinghamshire, UK). Co Q10 was supplied by Eisai Co. Ltd. (Tokyo,
Japan). Silica gel for chromatography, Wakogel C-200, was purchased
from Wako Chemical Industries, Osaka, Japan. Silica alumina was
purchased from Nikki Chemical Co. (Yokohama, Japan). Thin-layer
chromatography (TLC) was performed on plates of Silica Gel GF254
(Art 5715, Merck A. G., Darmstadt, Germany). The thickness of the
plates used for analysis was 0.25 mm. Lithium aluminium hydride
(LiAIH4) was purchased from Merck A. G. Radioactivity was measured
using a liquid scintillation counter, Aloka LSC-652 (Nihon Musen,
Tokyo, Japan). Counting efficiency was determined by external
standardization. All results were calculated as disintegrations per min,
and all determinations were performed twice. Radioactivity on TLC
plates was detected using a thin-layer chromatogram scanner, Aloka
TLC-2B (Nihon Musen, Tokyo, Japan).
Results and discussion
Position 30 of the side chain was selected for 14C labelling because of the
metabolic stability of this position. No metabolite of Co Q homologues
has been identified with less than five carbon atoms in the side chain.
The metabolic pathway of Co Q10 is considered to proceed via
o-oxidation and repeated b-oxidation and finally to the so-called Q
acid-I, which has seven carbon atoms in its side chain, and Q acid-II,
which has five carbon atoms in its side chain,10 just as is seen in vitamin
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF [30-14C] COENZYME Q10
825
K metabolism.11 Metabolic studies of Co Q10 have been carried out in
rats12, rabbits12 and guinea pigs.10 The synthesis of [30-14C]Co Q10 6 was
performed starting from natural solanesol (1, all-trans configuration)
and ethyl [3-14C]acetoacetate. The total synthetic process, which
includes five steps, is shown in Chart 1.
Chart 1. Synthesis of [30-14C] Coenzyme Q10
Treatment of natural solanesol 1 with phosphorus tribromide
followed by reaction of the bromide 2 with ethyl [3-14C]acetoacetate
gave [carbonyl-14C]solanesyl acetone 3. Wittig–Horner reaction of 3
with triethyl phosphonoacetate gave ethyl [3-14C]decaprenoate 4. The
reaction was employed as it is known to give highly stereoselective
formation of alkenes with the E-configuration.13,14 The elongated
alkene 4 was reduced with LiAIH4 to give [3-14C]decaprenol 5.
Condensation of 5 with Co Q0 hydroquinone borate followed by
hydrolysis and oxidation with lead dioxide gave a cis–trans mixture of
[30-14C]Co Q10 6. The crude product was purified by chromatography on
silica gel then recrystallized from cooled acetone. The overall radio-
chemical yield starting from ethyl [3-14C]acetoacetate was 8.0%. The
final product 6 was postulated to contain 1.5% of cis-isomer based on
analysis on high-performance liquid chromatography of the corre-
sponding deuterium-labelled compound ([2-CD3-10-CD2]Co Q10)
synthesized by the same procedure.15,16
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 823–829

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K. HAMAMURA ET AL.
Experiments
Solanesyl bromide 2
Solanesol 1 6.3 g (10 mmol) was dissolved in a mixture of 0.3 ml of
pyridine, 10 ml of n-hexane and 5 ml of ether and maintained at 58C. A
solution of phosphorus tribromide (0.6 ml) dissolved in 3 ml of n-hexane
was added dropwise to the mixture over a two-hour period. During this
time, the mixture was stirred and maintained at 58C. The disappearance
of the solanesol was checked by TLC. The reaction mixture was filtered.
To the filtrate 50 ml of iced water was added and extraction was carried
out with 100 ml of n-hexane. The extract was dried with sodium
sulphate and evaporated in vacuo. Crude solanesyl bromide 2 6.7 g was
obtained. The crude product thus obtained was used in the next reaction
without further purification.
[Carbonyl-14C]Solanesyl acetone 3
Solanesyl bromide 2 714 mg (1 mmol) was dissolved in a mixture of ethyl
[3-14C]acetoacetate, 132 mg (1 mmol, 50 mCi), and 2 ml of anhydrous
dioxane and maintained at 10–158C under stirring. Freshly prepared
sodium ethoxide (from 30 mg of sodium and 1 ml of ethanol) was added
dropwise to this solution. After completing the addition, the reaction
mixture was stirred for further 2 h. Then the reaction mixture was
warmed to 808C and 2 ml of 10% sodium hydroxide solution was
added. The reaction mixture was then stirred for further 4 h. The
reaction mixture was poured onto 50 ml of iced water and extracted
twice, each time with 50 ml of ether. The extract was washed with water
and dried over sodium sulphate. The solvent was evaporated in vacuo.
The crude product was purified by silica gel (25 g, 60–80 mesh)
chromatography. Product 3 was observed as a single peak on TLC using
a radio isotope (RI) scanner. Rf value: 0.47 (n-hexane/EtOAc, 9:1); 0.38
(benzene). Yield: 309 mg, 20 mCi (specific radioactivity: 64.7 mCi/mg).
Radiochemical yield: 40%.
Ethyl [3-14C]decaprenoate 4
Ethanol was completely removed from freshly prepared sodium
ethoxide, 136 mg in vacuo, and 5 ml of benzene was added to the
residue. To this suspension, Wittig reagent (triethyl phosphonoacetate)
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 823–829

SYNTHESIS OF [30-14C] COENZYME Q10
827
452 mg (2 mmol) was added while the suspension was stirred and
maintained at below 108C. Stirring was continued until a clear solution
was obtained. This solution was added dropwise to the product of the
second step, [carbonyl-14C]solanesyl acetone 3 309 mg (0.46 mmol,
20 mCi) in benzene solution. After completion of the addition, the
mixture was stirred for further 4 h below 108C. The end of the reaction
was recognized by the disappearance of solanesyl acetone 3 as examined
by TLC. Water was added to terminate the reaction. The product was
extracted with n-hexane. The solvent was evaporated in vacuo. The oil
residue (290 mg) was purified by silica gel (25 g) chromatography with
n-hexane/isopropyl ether solvent system. The product 4 was observed as
a single peak on TLC using a RI scanner. Rf value: 0.51 (n-hexane/
EtOAc, 9:1); 0.48 (n-hexane/isopropyl ether, 9:1). Yield: 225 mg,
16.4 mCi (specific radioactivity: 59.6 mCi/mg). Radiochemical yield:
82%.
[3-14C]Decaprenol 5
Lithium aluminium hydride 20 mg (0.5 mmol) was suspended in
absolute ether. To this suspension, ethyl [3-14C]decaprenoate 4 225 mg
(0.30 mmol, 13.4 mCi) was slowly added. The reaction mixture began to
reflux and bubble. The mixture was allowed to react for 2 h under
stirring and refluxing. After the disappearance of ethyl decaprenoate 4
was confirmed by TLC, EtOAc and then water were added to
decompose the excess of LIAIH4. The product was extracted with
ether. The extract was evaporated in vacuo. The oil residue 217 mg was
purified by silica gel chromatography. Product 5 was observed as a
single peak on TLC using a RI scanner. Rf value: 0.26 (n-hexane/
EtOAc, 4:1). Yield:205 mg, 12.4 mCi. (specific radioactivity: 60.5 mCi/
mg). Radiochemical yield: 93%.
[30-14C]Coenzyme Q10
6
Co Q0 hydroquinone borate was prepared by allowing a mixture
of Co Q0 hydroquinone (2-methyl-5,6-dimethoxy-1,4-hydroquinone)
221 mg (1.2 mmol), boric acid 72 mg (1.2 mmol) and 3 ml of toluene to
react under stirring and refluxing. The toluene was then removed in
vacuo and 2 ml of benzene was added to the residue to substitute
the solvent. Silica alumina (400 mg) and 3 ml of n-hexane were
added dropwise to [3-14C]decaprenol 5 205 mg (0.29 mmol, 12.4 mCi)
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 823–829

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K. HAMAMURA ET AL.
at 40–458C under stirring. After completion of addition, the reaction
was allowed to continue for 2 h at the same temperature. The end of the
reaction was recognized by the formation of Co Q10 as examined by
TLC. The reaction mixture was then filtered. The filtrate was oxidized
by lead dioxide–acetic acid. The crude product was purified by silica gel
chromatography and crystallized by addition of a 10-fold excess of
cooled acetone (ꢀ28C) compared to the crude product, and recovering
the formed crystals. The final product was obtained as a yellow
crystalline powder. The product 6 was observed as a single peak on TLC
using a RI scanner. Rf value: 0.40 (benzene); 0.40 (n-hexane/EtOAc 4:1).
Yield: 73 mg, 3.27 mCi (specific radioactivity: 44.8 mCi, 1.66 MBq/mg).
Radiochemical yield: 26%.
Acknowledgement
Critical reading of the manuscript by Dr Ellen Beaumont is highly
appreciated.
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