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S. A. S. Pope et al. / Bioorg. Med. Chem. 9 (2001) 1337±1343
A synthesis of (Æ)-a-CMBHC (5) was described by
Weichet et al. more than 30 years ago.15 However, this
report did not propose a-CMBHC (5) as a metabolite of
a-tocopherol and did not include NMR characterisa-
tion. The strategy relied upon the condensation of 6-
hydroxy-6-vinyl-2-methylheptanoicacid ( 7) with 2,3,5-
trimethylhydroquinone (8) (TMHQ) in the presence of a
mixture of zinchcloride and boron tri¯uoride±diethyl
etherate complex. The allylic alcohol (7) was obtained in
three steps by oxidative ring opening of dimethylcyclo-
hexanone (9), addition of acetylide to the resulting 6-
oxo-2-methylheptanoicaidc ( 10) and ®nal catalytic
16
hydrogenation of the propargylicalcohol intermediate.
In our synthesis of (Æ)-a-CMBHC (5), the ketoacid (10)
was prepared in a good yield by treatment of dimethyl-
cyclohexanone (9) with potassium permanganate
according to the procedure described by Weichet et al.15
However, we decided to synthesise the key intermediate
(7) by condensation of vinyl magnesium bromide with
6-oxo-2-methyl-heptanoicaicd ( 10) (Scheme 2). Pre-
liminary studies on a commercially available model
compound (6-oxo-heptanoic acid) suggested that the
Grignard condensation would be more eective on the
corresponding methyl ester (11). 6-Hydroxy-6-vinyl-2-
methyl-heptanoicacid methyl ester ( 12) was thus pre-
pared by condensation of vinyl magnesium bromide
with 6-oxo-2-methyl-heptanoicaicd methyl ester ( 11),
which was obtained from the corresponding acid (10). A
similar strategy was used by Wechter and Kantoci to
prepare the key intermediate, g-methyl-g-vinyl-butyro-
lactone, for their syntheses of a- and g-CEHC.10,13
Figure 1. The structures of the naturally occurring forms of vitamin E.
produced from naturally occurring vitamin E would be
present as single isomers. Indeed, the stereochemistry of
g-CEHC, derived from R,R,R-g-tocopherol, has been
shown to be S(+), meaning it is formed without epi-
merisation at C-2.13
In order to unambiguously con®rm the structure of this
new metabolite of a-tocopherol, we prepared a synthetic
standard of a-CMBHC (5) and showed that it had GC±
MS characteristics identical to those of the urinary
metabolite.
Condensation of 6-hydroxy-6-vinyl-2-methyl-heptanoic
acid methyl ester (12) with TMHQ (8) was performed
according to the conditions described by Kantoci et al.13
The allylicalcohol ( 12) was added at 110 ꢀC over 3 h to
a solution of TMHQ (8) and boron tri¯uoride±diethyl
etherate complex in dioxane to give a-CMBHC methyl
ester (13) in 80% yield. Saponi®cation of the methyl
ester (13) using aqueous sodium hydroxide aorded
(Æ)-a-CMBHC (5).
Results
In studies to elucidate the metabolism of a-tocopherol
we developed a new extraction procedure and GC±MS
method for human urinary vitamin E metabolites.11
This method, which requires trimethylsilyl-derivatisa-
tion of deconjugated metabolites, allowed us to analyse
a range of metabolites, including a-tocopheronolactone
(6) and a-CEHC (4). During these studies we observed a
minor, late eluting peak in human urine which had a
mass spectra displaying a major fragment ion (m/z 237)
in common with silylated a-CEHC (4), but with a
molecular ion of m/z 464, which is 42 daltons greater
than the molecular ion of silylated a-CEHC (Fig. 2).
After ingestion of d6-a-tocopherol,14 this unknown peak
increased in size and the mass spectrum showed an
increase in mass of 6 daltons for both the molecular ion
and the major fragment ion, con®rming the unknown
peak as a metabolite of a-tocopherol. This unknown
peak was tentatively identi®ed as a-CMBHC (5) based
on the GC±MS data and on the expected side-chain
metabolism of a-tocopherol. Although on biological
grounds a-CMBHC (5) was the most likely structure due
to the hypothesised b-oxidation of the phytyl side-chain,
other structures could display similar chromatography
and mass spectra. The structure of the unknown meta-
bolite has now been con®rmed by the unambiguous
synthesis of a-CMBHC (5).
Using the syntheticstandard ( Æ)-(5) in our GC±MS
analytical method, we con®rmed that the retention
times and mass spectra for the unknown metabolite and
synthetic a-CMBHC (5) were identical. This was
achieved by running the standard and urine extract
separately and then in combination to show co-elution
of the two peaks (Fig. 3). We assumed that the other
peaks observed in urine samples with similar mass spectra,
but with molecular and fragment ions 14 or 28 daltons
less, correspond to g- and d-CMBHC respectively. The
mass spectrum observed for the presumed g-CMBHC
agrees with that reported by Parker et al.12
Discussion
Vitamin E, and in particular a-tocopherol (1), is the
major fat soluble antioxidant in vivo, protecting cellular
membranes and other lipids against oxidative damage
caused by oxygen-derived free radicals.2,3 Owing to the