Synthesis of deuterium-labelled coenzyme Q10

Article abstract of DOI:10.1002/jlcr.587

Deuterium-labelled coenzyme Q₁₀ ([2-CD₃-1′-CD₂]coenzyme Q₁₀, coenzyme Q₁₀-d₅) was synthesized by condensation of 2,3-dimethoxy-[5-CD₃]methyl-1, 4-hydroquinone with [1-CD₂]decaprenol. Five positions were selected for deuteration as replacement at these positions allowed examination of every step of the synthesis. This examination was carried out by a combination of ¹H- and ¹³C-nuclear magnetic spectrometry and mass spectrometry. Further, these positions have been proved to be metabolically stable. This reagent makes simultaneous quantification of the source of coenzyme Q₁₀ (exogenously supplied or endogenously supplied) possible in biological samples by measurements on gas chromatography-mass spectrometry. Copyright

Full text of DOI:10.1002/jlcr.587

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 831–839.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.587
Research Article
Synthesis of deuterium-labelled coenzyme
Q10
Kimio Hamamura1, Yoji Yamagishi1, Shizumasa Kijima1, Takeshi
Suzuki1, Yutaka Mori1, 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
Deuterium-labelled coenzyme Q10 ([2-CD3-10-CD2]coenzyme Q10, coenzyme
Q10-d5 ) was synthesized by condensation of 2,3-dimethoxy-[5-CD3]methyl-1,
4-hydroquinone with [1-CD2]decaprenol. Five positions were selected for
deuteration as replacement at these positions allowed examination of every
step of the synthesis. This examination was carried out by a combination of
1H- and 13C-nuclear magnetic spectrometry and mass spectrometry. Further,
these positions have been proved to be metabolically stable. This reagent
makes simultaneous quantification of the source of coenzyme Q10 (exogenously
supplied or endogenously supplied) possible in biological samples by
measurements on gas chromatography–mass spectrometry. Copyright #
2002 John Wiley & Sons, Ltd.
Key Words: [2-CD3-10-CD2]coenzyme Q10; [1-CD2]decaprenol; 2,3-dimethoxy-
[5-CD3]methyl-1,4-hydroquinone; LiAID4
Introduction
The effects of exogenous coenzyme Q10 (Co Q10) on isoprepoid
derivatives, including Co Q10, have long been of great interest.1 The
*Correspondence to: T. Nakamura,Vitamin E Technology 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

832
K. HAMAMURA ET AL.
differentiation of exogenous and endogenous Co Q10 in vivo is a crucial
problem in the discussion of the effectiveness of Co Q10 administration.
The contribution of exogenous Co Q10 to total Co Q10 levels in tissues
warrants investigation. Deuterium-labelled Co Q10 is an excellent tool
to resolve this issue. Simultaneous quantification of deuterium-labelled
and unlabelled compounds by gas chromatography–mass spectrometry
was established.2
Using deuterium-labelled Co Q10 (Co Q10-d5) 10 the behaviour of
exogenous Co Q10 has been reported in three papers.3–5 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 the synthesis of [2-CD3-10-CD2]Co Q10 10.
The synthesis of the corresponding radio-labelled compound, [30-14C]
Co Q10 was described in the previous article.6
Materials and methods
Solanesol (natural) was obtained from Nisshin Flour Milling Co.
(Tokyo, Japan). Ethyl decaprenoate (all-trans isomer: 95%) was
prepared followed the procedure described previously.6 3,4,5-Tri-
methoxybenzoic acid 3 was obtained from Tokyo Kasei Organic
Chemicals (Tokyo, Japan). Co Q10 was supplied by Eisai Co., Ltd.
Silica gel for chromatography (Wakogel C-200) and deuterium oxide
were purchased from Wako Chemical Industries, Osaka, Japan. Silica
alumina was purchased from Nikki Chemical Co. (Yokohama, Japan).
Lithium aluminium deuteride (LiAID4, Art 5664) was purchased from
Merck A. G. (Darmstadt, Germany). Thin-layer chromatography
(TLC) was performed using Kieselgel GF254 (Art 5715, Merck A. G.).
Spots were visualized under ultraviolet light in a dark room after
applying concentrated sulphuric acid spray followed by heating. All
yields given refer to isolated yields obtained after a final purification by
column chromatography or recrystallization. Nuclear magnetic reso-
nance (NMR) spectra were measured using CDCl3 solution with a
JEOL-GX200 spectrometer (Japan Electron Optics Laboratory, Tokyo,
Japan). Tetramethyl silane was used as an internal standard. The
conditions were as follows: 1H-NMR: 200 MHz. The data are expressed
in d (ppm). The NMR signals are abbreviated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet. The number of protons is shown in
parentheses. 13C-NMR: 200 MHz. The data are expressed in hertz (Hz).
Copyright # 2002 John Wiley & Sons, Ltd.
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DEUTERIUM-LABELLED COENZYME Q10
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Mass spectra were measured on a JEOL-JMS-D200. Assays for
individual determination of Co Q10 and [2-CD3-10-CD2]Co Q10 10 were
performed on a JEOL JMS-D300 gas chromatograph–mass spectro-
meter unit equipped with a computer system (JEOL, JMA-2000). The
ionizing voltage and ionizing current were 70 eV and 300 mA,
respectively. The chamber temperature was maintained at 3008C. The
sample temperature was raised linearly from 1008C to 4308C within
3 min. During this time, mass fragments from m=z 824 to m=z 900 were
scanned and integrated. The peak ratio was plotted against the internal
standard.
2,3,5-Trimethyl-6-decapenyl-p-benzoquinone7 was used as an internal
standard for quantitative analysis of Co Q10.2 For individual analysis, a
multiple ion detector was adjusted to m/z 869 ([M+2]+) for exogenous
Co Q10 ([2-CD3-10-CD2]Co Q10 10), m=z 864 ([M+2]+) for endogenous
Co Q10 and m=z 832 ([M+2]+) for the internal standard.3
Results and Discussion
The synthesis of [2-CD3-10-CD2]Co Q10 10 was performed by
condensation of deuterated decaprenol 2, and deuterated Co Qo,
hydroquinone (2,3-dimethoxy-[5-CD3]methyl-1,4-hydroquinone 9).
The total synthetic process, which includes eight steps, is shown in
Chart 1.
Basically, the route employed here for the synthesis of deuterium-
labelled Co Q10 was the same as that we established previously for
synthesizing radio-labelled Co Q10.6 [1-CD2]Decaprenol 2 was obtained
starting from natural solanesol (all-trans configuration) via ethyl
decaprenoate by the same synthetic route as shown previously.6 The
deuteration of decaprenol was performed by treating ethyl decaprenoate
1
1 with LiAID4, and then deuterium oxide. The H-NMR of 2 showed
no signal at d 4.24 (allylic methylene protons), even under conditions of
20-fold amplification
A
nuclear component 2,3-dimethoxy-[5-
CD3]methyl-1,4-hydroquinone 9 was synthesized starting from 3,4,5-
trimethoxybenzoic acid 3 in six steps. 3,4,5-Trimethoxybenzoic acid 3
was esterified with methanol to give methyl-3,4,5-trimethoxybenzoate 4.
Reduction of 4 with LiAlD4, gave 3,4,5-trimethoxy-[CD2]benzylalcohol
5. The alcohol 5 was brominated with PBr3 and then reduced with
LiAID4, giving 3,4,5-trimethoxy-[CD3]toluene 7. The deuteration ratio
of the compound was calculated to be 96.5% based on the residual
Copyright # 2002 John Wiley & Sons, Ltd.
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K. HAMAMURA ET AL.
Chart 1. Synthesis of Deuterium - labelled coenzyme Q10
signal intensity at d 2.3 (ring methyl protons) compared with ring
1
proton intensity at d 6.30 (2 H) on H-NMR spectra. The tri-deutero
compound 7 was oxidized by lead dioxide to give 2,3-dimethoxy-
[5-CD3]methyl-1,4-benzoquinone 8. The deuteration ratio of 8 was
calculated to be 96% based on the comparison of the signal intensity at
d 2.08 (ring methyl proton position) with those of ring protons (d 6.35)
on 1H-NMR spectra. The 13C-NMR spectrum of 8 showed no signal at
386 Hz corresponding to the ring methyl signal found in the spectrum of
the unlabelled compound. Apart from the differences mentioned above,
the labelled and unlabelled compounds were found to give similar
signals. Then the benzoquinone 8 was reduced with Na2S2O4 to give 2,3-
dimethoxy-[5-CD3]-methyl-1,4-hydroquinone 9. The 13C-NMR spec-
trum showed complete disappearance of the signal at 386 Hz, assigned
to ring methyl group. Condensation of 9 borate with [1-CD2]decaprenol
2, followed by hydrolysis and oxidation with lead dioxide gave a
cis–trans mixture of [2-CD3,-10-CD2]Co Q10 10. The crude product was
purified by chromatography on silica gel then recrystallized three times
Copyright # 2002 John Wiley & Sons, Ltd.
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DEUTERIUM-LABELLED COENZYME Q10
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from cooled acetone. The stable isotope chemical yield of the purified [2-
1
CD3-l0-CD2]Co Q10 10 from [1-CD2]decaprenol 2 was 22%. The H-
NMR spectrum of 10 showed no signal at d 3.28 (l0-methylene protons),
even under conditions of 20-fold amplification. The spectrum was
identical to that of the unlabelled Co Q10 except for the absence of peaks
corresponding to ring methyl and 10-methylene protons. On 13C-NMR
spectrum, no signal was detected at either 292 (ring methyl) or 637 Hz
(10-methylene). Stereospecificity of the final compound 10 was
confirmed by performing high-performance liquid chromatography
followed by the method described previously.8 After recrystallizing the
silica gel chromatography-purified product, the cis-isomer content was
found to be 0.5%. The deuteration ratio and stereospecificity were
sufficiently high to enable us to use this deuterium-labelled form of Co
Q10 in metabolic studies.
Experiments
[1-CD2]Decaprenol 2
Ethyl decaprenoate 1 25 g (34 mmol) and LiAlD4, 70 mg (17 mmol) were
suspended in 100 ml of absolute ether and 5 ml of deuterium oxide.
Reduction was carried out by adding 400 g of silica gel (60–80 mesh).
The crude product was purified by chromatography. [1-CD2]Decapre-
1
nol 2 21.7 g (yield: 92%) was obtained. H-NMR: 5.31 (s, 1 H,=CH),
5.01 (m, 9 H,=CH), 1.9–2.1 (m, 38 H, –CH2-), 1.62 (s, 3 H,=CCH3),
1.58 (s, 30 H, –CH3). 13C-NMR: Signal at 1490 Hz, observed for
corresponding unlabelled compound, disappeared completely. Mass
spectra: m=z 701 ([M+]), corresponding unlabelled compound: m=z 699,
m=z 682 ([M+]-DHO), corresponding unlabelled compound: m/z 681
([M+]-H2O).
Methyl-3,4,5-trimethoxybenzoate 4
3,4,5-Trimethoxybenzoic acid 3 was esterified quantitatively in metha-
nol in the presence of concentrated sulphuric acid under refluxing for
5 h. The reaction mixture was poured onto iced water to precipitate
needle crystals. After filtration, the crystals were recrystallized from
methanol. Methyl-3,4,5trimethoxybenzoate 4 was obtained as crystals.
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K. HAMAMURA ET AL.
M.p. 82–848C. Yield: 92%. Rf value: 0.56 (EtOAc/C6H6, 1:9); 0.18
(C6H6). IR spectra: 1700 cmꢀ1 (ester).
3,4,5-Trimethoxy-[CD2]benzylalcohol 5
Methyl-3,4,5-trimethoxybenzoate 4 60 g (265 mmol) was reduced with
8 g (190 mmol) of LiAID4, in 2 l of absolute ether under stirring and
refluxing for 4 h. Excess LiAID4 was decomposed with 20 ml of
deuterium oxide. The product was extracted with ether, and then dried.
The solvent was removed in vacuo. The residue was distilled under
reduced pressure. The purified compound 46.1 g was obtained by
vacuum distillation at 164–1678C/l mmHg. Yield: 87%. Rf value: 0.10
1
(EtOAc/C6H6, 1:9); 0.08 (C6H6). IR spectra: appearance of OH. H-
NMR: 6.32 (s, 2 H, Ar-H), 3.78 (s, 9 H, Ar-OMe). The signal at d 5.46
(benzyl protons) disappeared. The deuteration ratio could not be
determined due to overlap with the base line. Mass spectra: m/z 200
([M+]), m=z 185 ([M+]-CH3), m=z 183 ([M+]-OH).
3,4,5-Trimethoxy-[CD2]benzylbromide 6
3,4,5-Trimethoxy-[CD2]benzylalcohol 5 47 g (235 mmol) was dissolved
in 1 l of ether and 10 ml of pyridine was added and the temperature was
maintained at 0–58C. To the solution, 20 ml of PBr3 (350 mmol
equivalent) dissolved in ether (total volume 100 ml) was added
gradually. The reaction mixture became cloudy. After the completion
of addition, stirring continued for further 2 h at the same temperature.
After confirmation of the disappearance of the starting material by
TLC, the reaction mixture was poured into 1 l of diluted HCI and then
extracted twice with 1 l of ether each extraction. The extract was washed
with water, and then dried. The solvent was removed in vacuo. The
crude product 46 g was obtained as a pale yellow oil. Yield: 74%. As the
product was unstable, further purification was not attempted and this
crude product was directly applied to the next step. Rf value: 0.44
(EtOAc/C6H6, 1:9); 0.33 (C6H6). IR spectra: disappearance of OH.
3,4,5-Trimethoxy-[CD3]toluene 7
3,4,5-Trimethoxy-[CD2]benzylbromide 6 46 g (175 mmol) was reduced
with 3.7 g (88 mmol) of LiAID4 in 400 ml of absolute ether under
stirring and refluxing for 4 h. Excess LiAID4 was decomposed with
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DEUTERIUM-LABELLED COENZYME Q10
837
10 ml of deuterium oxide. The product was extracted with ether, and
then purified by silica gel chromatography. The purified compound 7
20.4 g was obtained as a pale yellow oil. Yield: 74%. The product could
not be differentiated from corresponding unlabelled compound by TLC
or IR spectra comparison. Rf value: 0.50 (EtOAc/C6H6, 1:9); 0.44
1
(C6H6). H-NMR: 6.30 (s, 2 H, Ar-H), 3.80 (s, 9 H, Ar-OCH3). The
deuteration ratio, calculated on the basis of the ratio of the residual
signal intensity at d 7.7 proton to the ring proton intensity at d 3.68
(2 H), was 96.5%. 13C-NMR: The signal at 546 Hz, observed for the
corresponding unlabelled compound, disappeared completely. Mass
specta: m=z 185 ([M+]), m=z 170 ([M+]-CH3).
2,3-Dimethoxy-[5-CD3]methyl-1,4-benzoquinone 8
3,4,5-Trimethoxy-[CD3]toluene 7 39.3 g (253 mmol) was dissolved in
200 ml of AcOH. To the solution, 10 ml of 10% sulphuric acid was
added. At room temperature, 80 ml of 40% peracetic acid was added
gradually to the solution. The colourless reaction mixture changed to
red and the temperature of the reaction mixture increased to 50–608C.
After completion of the addition of peracetic acid, stirring was
continued for further 16 h at 25–308C. The disappearance of peracetic
acid was checked with potassium iodide starch paper. The reaction
mixture was poured onto iced water. The product was extracted with
ether, washed with a diluted solution of sodium bicarbonate, and then
dried. The solvent was evaporated in vacuo. The residual solid was
purified by silica gel chromatography. The pure product 8 24.8 g was
obtained as reddish needle crystals. Yield: 54%. M.p.: 75–778C. The
product could not be differentiated from corresponding unlabelled
compound by TLC, or IR and UV spectra comparisons. Rf value: 0.36
1
(EtOAc/C6H6, 1:9); 0.36 (C6H6). H-NMR spectra: 6.35 (s, IH, Ar-H),
3.90 (s, 6 H, Ar-OCH3). Mass spectra: m/z 185 ([M+]), corresponding
unlabelled compound: m=z 182, m=z 170 ([M+]-CH3).
2,3-Dimethoxy-[5-CD3]methyl-1,4-hydroquinone 9
2,3-Dimethoxy-[5-CD3]methyl-1,4-benzoquinone 8 24.8 g (134 mmol)
was reduced by the usual hydroreduction procedure with 50 g of
Na2S2O4, 300 ml of ether and 400 ml of water. The resulting mixture was
extracted twice, each time with 500 ml of ether and then dried. The
solvent was removed in vacuo. 2,3-Dimethoxy-[5-CD3]methyl-1,4-
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K. HAMAMURA ET AL.
hydroquinone 9, 16 g was obtained as colourless sand grain-sized
crystals. Yield: 64%. Rf value: 0.21 (EtOAc/C6H6, 1:9); 0.18 (C6H6);
0.13 (n-hexane/CHCl3, 1:1). A red spot was recognized on spraying
1
Emmerie–Engel reagent. H-NMR: d 6.45 (s, 1 H, Ar-H), 5.76 (s, 2 H,
Ar-OH), 3.84 (s, 3 H, Ar-OCH3), 3.01 (s, 3 H, Ar-OCH3). The 13C-
NMR spectrum showed complete disappearance of the signal at 386 Hz,
assigned to ring methyl group. Mass spectra: m=z 187 ([M+]), m=z 172
([M+]-CH3). The product was used immediately in the next reaction.
[2-CD3-10-CD2]Coenzyme Q10 10
2,3-Dimethoxy-[5-CD3]methyl-1,4-hydroquinone borate was prepared
by allowing a mixture of 2,3-dimethoxy-[5-CD3]methyl-1,4-hydroqui-
none 9 16 g (86 mmol), 5 g of boric acid and 85 ml of toluene to react
under stirring and refluxing until 2.3 ml of dehydrated water was
obtained. The toluene was then removed in vacuo and 65 ml of n-hexane
and 30 ml of benzene were added to the residue to substitute the solvent.
To the solution, [1-CD2]decaprenol 2 19.6 g (28 mmol) and 30 g of silica
alumina were added. The condensation reaction was carried out at
40–458C for 4 h under stirring. After completion of addition, the
reaction was allowed to continue for 2 hours at the same temperature.
The end of the reaction was judged by the appearance of the Co Q10
spot as examined by TLC. The reaction mixture was then filtered. The
filtrate was oxidized by 7 g of lead dioxide, 5 g of charcoal carbon and
5 ml of acetic acid. After the reaction, the reaction mixture was filtered
and the filtrate was washed with water, extracted with toluene and then
dried. The solvent was removed in vacuo. The red oily residue was
purified by chromatography using 800 g of silica gel. The chromato-
graphy purified product (14.5 g) was found to give a single spot on TLC.
Yield: 60%. After recrystallization three times, each time by addition of
a 5-fold excess of cooled acetone compared to the crude product
(ꢀ28C), [2-CD3-10-CD2]Co Q10 10 (6.4 g) was obtained. The final
product was obtained as a yellow solid. M.p.: 47.88C. Yield from [1-
CD2]decaprenol 2 was 22%. The cis-isomer content of the purified [2-
CD3-10-CD2]Co Q10 10 was 0.5%. The Co Q9 content of 10 was less
1
than 0.1%. Rf value: 0.44 (n-hexane-CHC13, 1:1); 0.46 (C6H6). H-
NMR: 6.78 (s, 3 H,=CCH3), 5.18 (m,10 H,=CH), 4.02 (s, 3 H,
Ar-OCH3), 4.00 (s, 3 H, Ar-OCH3), 1.9–2.1 (m, 38 H, -CH2-), 1.70
(s, 3 H,=CCH3), 1.60 (s, 27 H,=CCH3). Mass spectra: m=z 869
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DEUTERIUM-LABELLED COENZYME Q10
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([M+2]+), m=z 240 (pyrylium ion), m=z 202 (benzylium ion),
corresponding unlabelled compound: m=z 864, m=z 235, m=z 197.9
Acknowledgements
The authors are grateful to Mr T. Sato, Mr K. Hashimoto and Mr K.
Ema for their technical assistance in performing analyses. Critical
reading of the manuscript by Dr Ellen Beaumont is highly appreciated.
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