Biotransformation of (ϩ)-menthol 81
appeared after 3 days’ incubation, while all transfor-
mation products 2–10 could be visualized by TLC
together with residual compound 1 after 7 days. Fur-
ther continuation of the experiment increased the
yields of transformed products until day 12.
(δ ϭ 1.81, 1.37 ppm) in the COSY 45° spectrum.
The β-stereochemistry of the hydroxyl group was
deduced by the multiplicity of the 6-αH signal
(δ ϭ3.10 ppm,dt,J6ax,5eq ϭ 4.3 Hz, J6ax,5ax/1ax ϭ10.4 Hz)
and the NOESY correlations of H-6α with H3-7
(δ ϭ 0.91 ppm) and H-4α (δ ϭ 1.42 ppm). Thus
compound 8 was characterized as 6R,8-dihydroxy-
menthol, which may be formed by the monohydrox-
ylation at C-8 or C-6β of compound 2 or 3,
respectively (Scheme 1).
Conclusions
M. phaseolina performed regio- and stereocon-
trolled hydroxylations of 1 at C-1β, C-2β, C-5β,
C-7, C-8 and C-9, which resulted in the produc-
tion of nine hydroxylated products 2–10.A hydroxyl
group at C-8 was common in all dihydroxylated
derivatives, indicating preferential oxidation at
this position in 1 by M. phaseolina. The dihydroxy-
lated derivatives, 6–10, were found to be new
compounds.
The HRFAB-MS of metabolite 9 displayed an
[M ϩ H]ϩ peak at m/z ϭ 189.1411 corresponding to
the formula C10H21O3 and indicating the incorpora-
tion of two oxygen moieties in 1.The 1H NMR spec-
trum of compound 9 showed three methyl singlets
at δ ϭ 1.25 (H3-7), 1.24 (H3-9) and 1.23 (H3-10)
ppm.The 13C NMR spectrum of 9 showed two qua-
ternary carbon signals at δ ϭ 71.5 and 75.6 ppm,
while the disappearance of C-1 and C-8 methine
carbons suggested that hydroxylation had taken
place at C-1 and C-8. The HMBC spectrum of 9
showed the interactions of H3-7 (δ ϭ 1.25 ppm),
H2-2 (δ ϭ 1.92, 1.40 ppm) and H2-6 (δ ϭ 1.61, 1.38
ppm) with C-1 (δ ϭ 71.5 ppm),while H3-9 (δ ϭ 1.24
ppm) and H3-10 (δ ϭ 1.23 ppm) and H-4 (δ ϭ 1.38
ppm) were correlated with C-8 (δ ϭ 75.6 ppm).The
NOESY spectrum showed the interaction of H3-7α
with H-4α, supporting a β-orientation of OH at C-1.
Thus the structure of 9 was deduced as 1R,8-dihy-
droxymenthol, formed by C-1β or C-8 hydroxylation
of compound 2 or 4, respectively (Scheme 1).
The [M ϩ H]ϩ ion of compound 10 was found
to be at m/z ϭ 189.1438, corresponding to the for-
Declaration of interest: The authors gratefully
acknowledge the financial support of the Higher
Education Commission (HEC) to various scholars
involved in this study. The authors report no con-
flicts of interest. The authors alone are responsible
for the content and writing of the paper.
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1
mula C10H21O3, in HRFAB MS (ϩve). The H
NMR spectrum of 10 showed an additional dou-
blet at δ ϭ 3.47 ppm (2H, J ϭ 6.2 Hz) and disap-
pearance of the H3-7 signal, which indicated
oxidation of the C-7 methyl to C-7 CH2OH. Sec-
ond, a 6H singlet at δ ϭ 1.21 ppm, due to the two
methyls of an isopropyl moiety, indicated that
another hydroxyl group was introduced at C-8.
The 13C NMR spectrum of 10 showed the disap-
pearance of the C-8 methine and C-7 methyl
carbon signals, in comparison to 1, while oxygen-
bearing quaternary and methylene carbons
appeared at δ ϭ 75.5 and 67.9 ppm, respectively.
The position of the OH at C-7 and C-8 was further
inferred by HMBC between H-4 (δ ϭ 1.41 ppm)
and H6-9/10 (δ ϭ 1.22 ppm) and with C-8
(δ ϭ 75.5 ppm), while H-1 (δ ϭ 1.51 ppm) showed
cross-peaks with C-7 (δ ϭ 67.9 ppm). The struc-
ture of compound 10 was deduced as 7,8-dihy-
droxymenthol, which may be formed by the C-7
hydroxylation of compound 2 (Scheme 1).
Time-course studies on compound 1 showed
that the monohydroxylated derivatives, 2 and 4,
Figueiredo AC, Almendra MJ, Barroso JG, Scheffer JC. 1996.
Biotransformation of monoterpenes and sesquiterpenes by