Efficient two-step synthesis of methylphytylbenzoquinones: Precursor intermediates in the biosynthesis of vitamin E

Article abstract of DOI:10.1016/S0040-4039(02)02564-9

Methylphytylbenzoquinone was synthesized from δ-tocopherol by a simple two-step sequence. Oxidative cleavage of the benzopyran ring with cerium sulfate followed by dehydration using Burgess reagent afforded the methylphytylbenzoquinone as a mixture of positional and geometric isomers which were separated by HPLC. The biological activity of the product corresponds to the natural biosynthetic precursor of vitamin E. The above method is a general procedure applicable to the preparation of any of the tocopherol derivatives.

Full text of DOI:10.1016/S0040-4039(02)02564-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 237–239
Efficient two-step synthesis of methylphytylbenzoquinones:
precursor intermediates in the biosynthesis of vitamin E
Satyamaheshwar Peddibhotla,a Zigang Cheng,b Dean DellaPennab and Jetze J. Tepea,*
aDepartment of Chemistry, Michigan State University, East Lansing, MI 48824, USA
bDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA
Received 16 October 2002; revised 9 November 2002; accepted 13 November 2002
Abstract—Methylphytylbenzoquinone was synthesized from d-tocopherol by a simple two-step sequence. Oxidative cleavage of the
benzopyran ring with cerium sulfate followed by dehydration using Burgess reagent afforded the methylphytylbenzoquinone as a
mixture of positional and geometric isomers which were separated by HPLC. The biological activity of the product corresponds
to the natural biosynthetic precursor of vitamin E. The above method is a general procedure applicable to the preparation of any
of the tocopherol derivatives. © 2002 Elsevier Science Ltd. All rights reserved.
Tocopherols are a class of lipid-soluble antioxidants
synthesized in higher plants and other oxygenic photo-
synthetic organisms such as cyanobacteria.1 The four
tocopherols synthesized (a, b, d, and g-tocopherol)
differ only in the number and position of methyl groups
on the chroman head group. Tocopherols are impor-
tant in limiting lipid peroxidation in a variety of organ-
isms and are an essential component of mammalian
diets as vitamin E (2R,4%R,8%R-[a]-tocopherol).2,3 The
production of tocopherols for animal nutrition and
various industrial purposes has sparked renewed inter-
est in the synthesis and enzymology of the tocopherol
biosynthetic enzymes.2,4–6 Although the tocopherol
pathway has been studied for many years,7 it is only
recently that pathway enzymes have been cloned,
providing new opportunities to study the substrate spe-
cificity and kinetics of the reactions.5,6,8,9
The pathway leading to the synthesis of a-tocopherol is
shown in Fig. 1. The committed intermediate in the
tocopherol biosynthetic pathway is 2-methyl-6-phytyl-
1,4-benzoquinone (MPBQ), the product of the conden-
sation of the aromatic compound homogentisate
(HGA) and the 20-carbon isoprenoid derived com-
pound, phytyl pyrophosphate.9,10 MPBQ methyltrans-
ferase catalyzes the first methylation reaction in
tocopherol synthesis and the methylation of the same
ring position of a related compound in plastoquinone
synthesis.11,12
Access to a variety of methylated phytylbenzoquinone
precursors and intermediates is required in order to
Figure 1. Biosynthesis of a-tocopherol.
* Corresponding author. Tel.: (517) 355-9715; fax: (517) 353-1793; e-mail: tepe@cem.msu.edu
0040-4039/03/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02564-9

238
S. Peddibhotla et al. / Tetrahedron Letters 44 (2003) 237–239
subjected to dehydration with Burgess reagent under
argon atmosphere to afford a mixture of methyl-
phytylquinones 2–6 (Scheme 1).17,18
elucidate the regulation and the activity of MPBQ/
MSBQ methyltransferase.11 Previously reported meth-
ods provide some of these substrates in relatively low
yields and as multiple isomers.12–14 The development of
a short and efficient synthetic method applicable to
generating a wide range of these substrates has there-
fore become an integral part of elucidation of the
biosynthetic pathway. We report herein an efficient
two-step synthesis of methylphytylbenzoquinones
(MPBQs) to study the kinetics and substrate specificity
of MPBQ methyltransferase.
The positional and geometric isomers 2–6 were isolated
by flash chromatography, followed by two rounds of
HPLC purification using a silica column with hexane/
isopropyl ether (992:8) as the eluant. Compounds 2–6
were isolated in a relative ratio of 1:2:1.1:1.5:2.2. The
positional and geometric isomers from the dehydration
will be further used as competitive inhibitors for evalu-
ating the reaction mechanism and kinetic parameters of
the MPBQ methyltransferase.11
The earlier reported chemical synthesis of MPBQ
involved oxidation of commercially available methyl-
ated phenols to methyl quinones followed by coupling
of the phytyl side chain in the presence of BF3·OEt2
resulting in a mixture of positional isomers.10 Oxidation
of the resulting quinols with Ag2O provided a mixture
of isomers, which were separated by repeated thin-layer
chromatography.
Purification of MPBQ and its isomers: The mixture of
isomeric methylphytylbenzoquinones was injected on a
ReliaSil Silica, 5 mm, 250×4.6 mm normal phase
column (Column Engineering, Ontario, CA) with a
guard column containing the same matrix. Chromato-
graphy was performed at 30°C with a mobile phase of
992:8 (v/v) hexane:isopropyl ether flowing at 1.0 mL/
min. For large-scale purification, a preparative column
of the same matrix (Column Engineering, Ontario, CA)
was used. The collected fractions were dried down
under vacuum before a small portion was injected onto
the analytical column to monitor the purity. The natu-
ral substrate 3 was eluted as the second HPLC peak.19
Unfortunately, due to the limited availability of the
required isophytol side chain, the generation of wide
mixtures of isomers, and corresponding low yielding
reactions, this procedure was quite limited as a routine
method for the large scale preparation of the many
different tocopherol precursors. We report here an
alternative preparation of MPBQ derivatives from the
naturally available tocopherols to provide more
efficient access to these biosynthetic precursors.
Enzyme Assay: Assays of MPBQ methyltransferase
activity were carried out in a 100 mL reaction contain-
ing 10 mL of solubilized E. coli extract (50 and 75 mg
protein) expressing the MPBQ methyltransferase of
Synechocystis PCC6803,11 50 mM Tris pH 8.0, 10 mM
DTT, 80 mM [14C-methyl]S-adenosylmethionine (56
mCi/mmol), 40 mM unlabeled S-adenosylmethioine,
and 100 mM each of the individual MPBQ compounds
2–6. After incubation at 30°C for 2 h, the reactions
were stopped by the addition of 200 mL of 0.9% (w:v)
NaCl, extracted with 450 mL of 2:1 (v:v)
methanol:chloroform, centrifuged, and the organic
phase collected and dried under a stream of nitrogen
gas. The resulting residue was resuspended in hexane
and fractionated on 250 m 60A KF6 silica gel TLC
plates (Whatman, Clifton, NJ, USA) developed with
Oxidative cleavage of a chroman ring with cerium
sulfate affords the quinone alcohol.15 The well estab-
lished FeCl3 oxidation as described by Cohen has also
successfully been used in the conversion of a-tocopherol
to a-tocopheroquinones.16 Cerium sulfate oxidation of
d-tocopherol resulted in a mixture of products from
which the quinone alcohol 1 was readily isolated in 30%
yield at multigram scale. The quinone alcohol 1 was
Figure 2. Efficacy of isomers 2–6 compared to natural MPBQ
as substrates for methyltransferase activity. The percent
efficacy of the isomers was based on the average of three
independent assays.
Scheme 1. Synthesis of methylphytylbenzoquinone.

S. Peddibhotla et al. / Tetrahedron Letters 44 (2003) 237–239
2002, 511, 1–5.
239
3:7 (v:v) diethylether:petroleum ether. The TLC plates
were exposed on a Molecular Dynamics low energy
phosphor screen (Amersham) to determine the incorpo-
ration of 14C into the products.
12. Chenevert, R.; Courchesne, G. Tetrahedron Lett. 2002,
43, 7971–7973.
13. Soll, J. Methods Enzymol. 1987, 248, 383–387.
14. Henry, A.; Powls, R.; Pennock, J. F. Biochem. J. 1987,
242, 367–373.
15. Cohen, N.; Lopresti, R. J.; Saucy, G. J. Am. Chem. Soc.
1979, 101, 6710–6716.
16. Cohen, N.; Lopresti, R. J.; Neukom, C. J. Org. Chem.
1981, 46, 2445–2450.
Interestingly, MPBQ methyltransferase was found to
utilize all five MPBQ isomers (2–6), however the activ-
ity with the genuine substrate 3 (shown as fraction II in
Fig. 2) results in specific activity that is 10–100 times
higher than those of its isomers. The method described
in this report has also been successfully applied to
large-scale preparation of phytylquinone precursors of
a-tocopherol (data not shown) and is applicable to a
wide range of methylphytylbenzoquinone precursors.
17. Burgess, E. M.; Penton, H. R. J.; Taylor, E. A. J. Org.
Chem. 1973, 38, 26–31.
18. Procedure for the dehydration of methylphytyl benzoquinol
1: The quinone alcohol 1 (0.4 g, 0.9 mmol) was weighed
into a flame dried flask under argon atmosphere and dry
THF (50 mL) was added. To this solution was added
Burgess reagent (0.4 g, 1.8 mmol) and the mixture stirred
at room temperature overnight. The reaction mixture was
then diluted with ether (200 mL) and extracted twice with
water and the organic layer was dried over anhydrous
sodium sulfate and the ether layer was evaporated to
obtain a yellow residue. The residue was flash chro-
matographed on silica-gel (5% ether:hexane) to obtain the
methylphytylbenzoquinone as a mixture of five isomers
(0.16 g, 42%) They were further separated by HPLC.
References
1. Kamal-Eldin, A.; Appelqvist, L. A. Lipids 1996, 31,
671–701.
2. Fryer, M. J. Plant Cell Environ. 1992, 15, 381–392.
3. Topinka, J.; Binkova, B.; Sram, R. J.; Erin, A. N. Mutat.
Res. 1989, 225, 131–136.
4. Weber, P.; Bendich, A.; Machlin, L. J. Nutrition 1997, 13,
450–460.
5. Grusak, M. A.; DellaPenna, D. Annu. Rev. Physiol. Plant
1
19. Spectral data: 3; H NMR (500 MHz, acetone-d6) l 6.6
Mol. Biol. 1999, 50, 133–161.
(1H, td, J1=1.5 Hz, J2=4.5 Hz), 6.46 (1H, td, J1=1.5
Hz, J2=3.5 Hz), 5.22 (1H, qt, J1=1.5 Hz, J2=7.5 Hz),
3.14 (2H, d, J=7.5 Hz), 2.06–2.07 (3H, m), 2.02 (2H, d,
J=1.5 Hz), 1.66 (3H, s), 1.0–1.6 (18H, m), 0.85–0.9 (12H,
m); 13C NMR (125 MHz, acetone-d6) l 187.61, 148.56,
146.07, 139.55, 133.01, 132.04, 118.87, 39.85, 39.41, 37.43,
37.42, 37.31, 36.56, 32.81, 32.66, 28.82, 28.67, 27.99,
27.48, 25.25, 24.8, 24.43, 22.29, 22.2, 19.4, 15.36, 15.15;
IR; IR (neat): 1670 cm−1, 1600 cm−1. MS (EI): calculated
for C27H44O2 [M]+ 400.64, found [M]+ 400.33.
6. DellaPenna, D. Science 1999, 285, 375–379.
7. Trelfall, D. R.; Whistance, G. R. Aspects of Terpenoid
Chemistry and Biochemistry; Academic Press: London,
1971.
8. Garcia, I.; Rodgers, M.; Lenne, C.; Rolland, A.; Sailland,
A.; Matringe, M. Biochem. J. 1997, 325, 761–769.
9. Shintani, D.; DellaPenna, D. Science 1998, 282, 2098–
2100.
10. Soll, J.; Schultz, G. Phytochemistry 1980, 19, 215–218.
11. Shintani, D. K.; Cheng, Z.; DellaPenna, D. FEBS Lett.