Modified monoterpenes from biotransformation of (-)-isopiperitenone by suspension cell culture of Mentha piperita

Article abstract of DOI:10.1021/np970388k

The biotransformation of (-)-(4R)-isopiperitenone (1) by suspension cell culture of Mentha piperita yielded three new hydroxylated derivatives, 4-6, and two new epoxidized derivatives, 7 and 8. (-)-7-Hydroxyisopiperitonone (2) and its glucoside were previously isolated from the culture. The structures of 4-8 were elucidated using spectral methods, and their absolute stereochemistry was established by NMR experiments and correlation with compounds of known configuration.

Full text of DOI:10.1021/np970388k

354
J . Nat. Prod. 1998, 61, 354-357
Mod ified Mon oter p en es fr om Biotr a n sfor m a tion of (-)-Isop ip er iten on e by
Su sp en sion Cell Cu ltu r e of Men th a piper ita
Si-Hyung Park† and Soo-Un Kim*
Division of Applied Biology and Chemistry and The Research Center for New Bio-Materials in Agriculture,
Seoul National University, Suwon 441-744, Korea
Received August 14, 1997
The biotransformation of (-)-(4R)-isopiperitenone (1) by suspension cell culture of Mentha
piperita yielded three new hydroxylated derivatives, 4-6, and two new epoxidized derivatives,
7 and 8. (-)-7-Hydroxyisopiperitenone (2) and its glucoside were previously isolated from the
culture. The structures of 4-8 were elucidated using spectral methods, and their absolute
stereochemistry was established by NMR experiments and correlation with compounds of known
configuration.
Several studies have been reported to produce es-
sential oils by plant cell cultures.1,2 In the case of
Mentha piperita L. (Lamiaceae), there have been at-
tempts to produce major components of peppermint
oils through biotransformations of biosynthetic inter-
mediates.3-5 However, feeding of (-)-isopiperitenone
(1), an intermediate at the branching point leading
either to menthol or piperitone in M. piperita, to the
cell culture of the plant results in 7-hydroxylation6,7 and
subsequent glucosylation of the hydroxylated terpene8
to yield 2 and 3 with some stimulated conversion into
the desired products.6
There has been considerable interest in producing
novel structures from natural products through biotrans-
formation using plant cell culture.9 The above finding
with Mentha cells indicates that the cells possess the
capacity to transform exogenously fed monoterpenes.
This paper reports the isolation and identification of
three new hydroxylated (4-6) and two epoxidized
compounds (7 and 8) from cell culture of M. piperita fed
with 1. These compounds account for a significant
portion of the total biotransformation products.
Resu lts a n d Discu ssion
Compounds 1 and 4 showed the same base peak at
m/z 82, which was assigned to fragment [C5H6O]+
derived from retro-Diels-Alder reaction of the cyclo-
specific optical rotation value of +104°, contained (1R)-
and (1S)-4-hydroxymenthone with an unknown config-
uration at C-4. The optical rotation sign of (1S,4S)- and
(1R,4S)-4-hydroxymenthone is positive and that of
(1S,4R)- and (1R,4R)-4-hydroxymenthone is negative.10-12
Therefore, the reduced mixture with positive optical
rotation must be composed of (1R,4S)- and (1S,4S)-4-
hydroxymenthone (9 and 10), confirming the configu-
ration of compound 4 at C-4 as S.
1
hexenone ring. The H and 13C NMR spectra of 4 were
similar to those of 1 and showed peaks corresponding
to 14 protons and 10 carbons. The disappearance of the
signal at 3.04, corresponding to H-4 of 1, and the
appearance of a signal at 3.78 revealed that a hydroxyl
group had been introduced at C-4. This conclusion was
supported by the appearance of a new carbon signal at
77.14 in the 13C NMR spectra.
The stereochemistry of C-4 in 4 was deduced to be S
by comparison with optical rotation values of 4-hydroxy-
menthones as follows. Compound 4 was reduced to a
mixture of 4-hydroxymenthones. The mixture, with a
Compound 5 showed the H-2 proton signal at 5.88 and
the base peak, due to retro-Diels-Alder reaction, at m/z
98, suggesting that a hydroxyl group was present at C-6.
1
In the H-1H COSY spectrum, the doublet of doublets
at 3.05 (J ) 13.6, 4.5 Hz) assigned to H-4 was coupled
with the H-5 signals at 2.14 and 2.31. These two H-5
signals were assigned to H-5ax and H-5eq, respectively,
based on analysis of coupling with the axial H-4. The
C-6 stereochemistry in 5 could be easily determined
if the H-6 signal had been resolved into multiplets.
* To whom correspondence should be addressed. Phone: (331) 290-
2401. Fax: (331) 293-8608. E-mail: soounkim@plaza.snu.ac.kr.
† Present address: Korea Research Institute of Bioscience and
Biotechnology, Natural Product Biosynthesis R. U., P.O. Box 115,
Yusong, Taejon 305-600, Korea.
S0163-3864(97)00388-1 CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/24/1998

Modified Monoterpenes from (-)-Isopiperitenone
J ournal of Natural Products, 1998, Vol. 61, No. 3 355
Nevertheless, the stereochemistry at C-6 was deter-
mined to be R on the basis of an 1H NMR coupling
pattern analysis at H-5 as follows. The H-5ax signal had
three large coupling constants, one (J ) 12.4 Hz) due
to the geminal coupling, the other (J ) 13.6 Hz) due to
coupling with H-4, so the last (J ) 10.2 Hz) must then
be due to coupling with H-6. The coupling constant
between H-5ax and H-6 was in the range expected for
axial-axial proton coupling. Assuming axial orienta-
tion of H-4 due to preference of propenyl side chain
toward equatorial, we concluded that H-6 was axial.
Therefore, the absolute configuration at C-6 was R. This
conclusion was supported by molecular modeling cal-
culations by PCMODEL for Windows (Serena Software,
configuration of compound 7 was finally determined to
be (4R,8R).
The stereochemistry of 8 was determined in the same
manner as 7. The physicochemical properties of 16 were
identical to those of (4R,8R)-8,9-epoxy-p-menth-1-ene,13
with the opposite sign of optical rotation. Therefore,
compound 16 was identified as (4S,8S)-8,9-epoxy-p-
menth-1-ene. Compound 16 was then oxidized to yield
(4R,8S)-epoxyisopiperitenone, whose physicochemical
properties were identical to those of compound 8.
Nicotiana cell culture is the most extensively studied
plant cell biotransformation system. Nicotiana cell
cultures possess catalytic capacity for reduction,15 hy-
droxylation,16 and epoxidation17 of monoterpenes. Bio-
transformation of piperitone into oxygenated products
such as 4-hydroxypiperitone via hydroxylation using cell
suspension cultures of Catharanthus roseus has also
been reported.18 The report, however, lacks information
on absolute stereochemistry of the products. The present
paper describes the complete structural elucidation of
products as well as demonstrating the versatile meta-
bolic capability of Mentha cells.
IN) which predicted J
to be 12.3 Hz and
H-5ax-H-4
J
11.1 Hz.
H-5ax-H-6
Spectral data of 6 were very similar to 5 and indicated
that 6 was a stereoisomer of 5. The H-6 proton signal
of 6 appeared at 4.35 (br s) and was shown to couple
with H-5 in 1H-1H COSY. Here again, the coupling
between H-6 and H-5 was determined indirectly as in
the case of compound 5. Assuming axial orientation of
H-4, the coupling constant of 9.1 Hz could be assigned
to axial-axial coupling between H-4 and H-5ax. Start-
ing from this, the coupling constant of 4.2 Hz then must
be due to axial-equatorial coupling between H-5ax and
H-6. Therefore, the configuration of the hydroxyl group
at C-6 was concluded to be S. Molecular mechanics
There have been few reports on the nature of the
enzymes responsible for biotransformation reactions
except for a report describing evidence for the involve-
ment of cytochrome P-450 monooxygenase.18 Thus, it
is also possible that the described bioconversion by M.
piperita cell culture was catalyzed by a cytochrome
P-450 monooxigenase.
calculations gave 2.81 Hz for J H-5 -H-6, but the S
ax
configuration was still indicated from this value.
Exp er im en ta l Section
HREIMS of both 7 and 8 indicated molecular formu-
las of C10H14O2. The IR spectra of compounds 7 and 8
showed an asymmetric C-O-C stretching peak instead
of the characteristic O-H stretching peak, which sug-
Gen er a l Exp er im en ta l P r oced u r es. Optical rota-
tions were measured on an Autopol polarimeter. UV-
vis spectra were obtained on a HP8452A diode-array
spectrophotometer. The IR spectra were measured on
a Magna 550 infrared spectrophotometer. 1H NMR and
13C NMR spectra were recorded with a J EOL LA400
FT NMR at 400 and 100 MHz, respectively, in CDCl3
using TMS as an internal standard. EIMS spectra were
obtained with a J EOL AX505WA mass spectrometer.
Silica gel 60 (Merck, 230-400 mesh) was used for CC.
P la n t Cell Cu ltu r e a n d Biotr a n sfor m a tion P r o-
ced u r e. Suspension culture of M. piperita, established
in Lin and Staba medium20 with 2,4-D (2 mg/L), was a
gift from Professor H. J . Lee, Department of Food
Science and Technology, Seoul National University. The
culture was incubated in a 250 mL Erlenmeyer flask
containing 100 mL of the medium on a rotary shaker
(120 rpm) at 27 °C with a 16-8 h light-dark cycle. The
culture was subcultured monthly with the initial inocu-
lum of 1 g fresh cell per flask. A total of 800 mg of (-)-
isopiperitenone (1) was administered to 40 flasks con-
taining 3-week-old suspension cells, and these were
subsequently incubated for 48 h prior to isolation of
monoterpenes.
1
gested the presence of an oxirane ring. In the H NMR
spectrum, disappearance of the signal for H-9 and the
presence of the signal for H-2 at 5.87 revealed that an
oxirane ring had been introduced at C-8 and C-9. The
H-9 signals were split into doublets (J ) 4.5-4.6 Hz)
typical for methylene protons of a terminal oxirane ring.
Thus, compounds 7 and 8 were determined to be 8,9-
epoxyisopiperitenone isomers.
The stereochemistry of 7 and 8 was identical at C-4
but opposite at C-8. The chiral center at C-8, located
on a freely rotating side chain, rendered it impossible
to determine the stereochemistry at C-8 through an
NOE experiment. The stereochemistry of 7 and 8 was
thus determined by comparing the optical rotation
values and NMR data between the isolated products and
a synthetic sample of the known configuration. 1,2-
Dibromolimonene (11) was synthesized by the low-
temperature monobromination of (-)-limonene (12).
(1R,2R,4S)-Dibromolimonene (11) was epoxidized with
m-CPBA to yield a mixture of two epoxides 13 and 14.
One of the dibromoepoxides (13) was debrominated with
zinc powder to give an 8,9-epoxy-p-menth-1-ene (15).
The physicochemical data for 15 were identical to those
of (4R,8S)-8,9-epoxy-p-menth-1-ene,13 except for the sign
of optical rotation. From these results, the absolute
configuration of 15 was clearly identified as (4S,8R).
Compound 15 was oxidized with CrO3 and t-BuOOH14
to yield (4R,8R)-epoxyisopiperitenone, whose physico-
chemical properties were identical to those of the
biotransformed compound 7. Therefore, the absolute
Extr a ction a n d Isola tion . The cells were filtered
through filter paper and carefully washed with distilled
water. The filtrate was extracted with CH2Cl2 (500 mL
× 3). The CH2Cl2 layer was dried with anhydrous
MgSO4 and concentrated in vacuo. The oily residue was
chromatographed a on silica gel column (2 × 50 cm,
hexane-diethyl ether 4:1) and further purified by
HPLC. The HPLC conditions for isolation of monoter-
penes were as follows: column, LiChrosorb Si60 (Mer-

356 J ournal of Natural Products, 1998, Vol. 61, No. 3
Park and Kim
ck); 10 mm × 25 cm, solvent; hexane-diethyl ether 1:1,
flow rate; 3.0 mL/min, UV detector; at 240 nm. The
major products 2 and 3 have been described else-
where.7,8
m, H-5b), 2.35 (2H, m, H-6), 2.37 (1H, m, H-4), 2.53 (1H,
d, J ) 4.6 Hz, H-9a), 2.59 (1H, d, J ) 4.6 Hz, H-9b),
5.87 (1H, m, H-2); 13C NMR (CDCl3, 100 MHz) δ 21.31
(C-10), 24.12 (C-7), 24.75 (C-5), 30.67 (C-6), 50.89 (C-
4), 50.92 (C-9), 56.40 (C-8), 126.84 (C-2), 162.00 (C-1),
198.40 (C-3); EIMS m/z [M]+ 166 (19), [M - H2O]+ 148
(19), 136 (24), 121 (30), 108 (27), 93 (28), 82 ([C5H6O]+,
100), 67 (12); HREIMS m/z 166.0990 (calcd for C10H14O2
166.0994).
Con ver sion of 4 in to 4-Hyd r oxym en th on es 9 a n d
10. (+)-(4S)-4-Hydroxyisopiperitenone (4) (3 mg) was
dissolved in 2 mL of hexane, and 10 mg of Pd/C was
added. The suspension was stirred for 12 h under 1 atm
of H2. The suspension was filtered, and the filtrate
was evaporated. The residue contained a mixture of
(1R,4S)-4-hydroxymenthone (9) and (1S,4S)-4-hydroxy-
menthone (10). The structures of 9 and 10 were
identified by careful comparison of NMR data with
reported values.10-12
(+)-(4S)-4-Hyd r oxyisop ip er iten on e (4): colorless
oil (7 mg); [R]20 +136° (c 0.05, CH2Cl2); UV (MeOH)
D
λ
max (log ꢀ) 236 (4.3) nm; IR (neat) νmax 3475, 2930, 1670
cm-1; H NMR (CDCl3, 400 MHz) δ 1.84 (3H, s, H-10),
1.95 (1H, m, H-5a), 1.96 (3H, s, H-7), 2.30 (2H, m, H-6),
2.33 (1H, m, H-5b), 3.78 (1H, s, OH-4), 4.69 (1H, m,
H-9a), 4.94 (1H, m, H-9b), 5.98 (1H, m, H-2); 13C NMR
(CDCl3, 100 MHz) δ 18.09 (C-10), 24.22 (C-7), 29.97 (C-
6), 32.62 (C-5), 77.14 (C-4), 113.49 (C-9), 124.42 (C-2),
144.36 (C-8), 164.22 (C-1), 201.16 (C-3); EIMS m/z [M]+
166 (5), [M - H2O]+ 148 (59), 133 (32), 105 (46), 97 (22),
82 ([C5H6O]+, 100), 69 (16); HREIMS m/z 166.0974
(calcd for C10H14O2 166.0994).
1
(-)-(4R,6R)-6-Hyd r oxyisop ip er iten on e (5): color-
less oil (8 mg); [R]20D -7.2° (c 0.08, CH2Cl2); UV (MeOH)
Con ver sion of (-)-(4S)-Lim on en e (12) in t o
(4S,8R)- a n d (4S,8S)-Ep oxy-p-m en th -1-en e (15 a n d
16). Compounds 15 and 16 were prepared according
to the known procedure starting from 12.13 1H and 13C
NMR data of the intermediates (11, 13-16) were
identical to those of the enantiomers13 except for the
sign of optical rotation.
λ
max (log ꢀ) 231 (3.9) nm; IR (neat) νmax 3310, 2940, 1640
cm-1; H NMR (CDCl3, 400 MHz) δ 1.74 (3H, s, H-10),
2.06 (3H, s, H-7), 2.14 (1H, ddd, J ) 12.4, 13.6, 10.2
Hz, H-5ax), 2.31 (1H, ddd, J ) 12.4, 4.5, 4.7 Hz, H-5eq),
2.46 (1H, br s, -OH), 3.05 (1H, dd, J ) 13.6, 4.5 Hz,
H-4), 4.51 (1H, br. s, H-6), 4.81 (1H, m, H-9a), 4.98 (1H,
m, H-9b), 5.88 (1H, m, H-2); 13C NMR (CDCl3, 100 MHz)
δ 19.67 (C-7), 19.94 (C-10), 37.94 (C-5), 53.88 (C-4), 69.74
(C-6), 114.49 (C-9), 126.82 (C-2), 143.00 (C-8), 163.82
(C-1), 198.21 (C-3); EIMS m/z [M]+ 166 (8), [M - H2O]+
148 (27), 133 (16), 105 (22), 98 ([C5H6O2]+, 100), 69 (46);
HREIMS m/z 166.0990 (calcd for C10H14O2 166.0994).
1
(1R,2R,4S)-1,2-Dibr om o-p-m en th -8-en e (11): [R]20
-57.0° (c 0.2, CH2Cl2).
D
(1R ,2R ,4S ,8R )-1,2-Dib r om o-8,9-e p ox y-p -m e n -
th a n e (13): [R]20 -61.7° (c 0.6, CH2Cl2).
D
(1R ,2R ,4S ,8S )-1,2-D ib r o m o -8,9-e p o x y -p -m e n -
th a n e (14): [R]20 -50.8° (c 0.34, CH2Cl2).
(+)-(4R,6S)-6-Hyd r oxyisop ip er iten on e (6): white
D
(4S,8R)-8,9-Epoxy-p-m en th -1-en e (15): [R]20D -83.6°
(c 0.47, CH2Cl2).
powder (8 mg); [R]20 +17.4° (c 0.08, CH2Cl2); UV
D
(MeOH) λmax (log ꢀ) 231 (3.9) nm; IR (KBr) νmax 3410,
2920, 1660 cm-1; 1H NMR (CDCl3, 400 MHz) δ 1.77 (3H,
s, H-10), 2.05 (3H, s, H-7) 2.39 (1H, ddd, J ) 13.6, 9.1,
4.2 Hz, H-5ax), 2.10 (1H, ddd, J ) 13.6, 4.8, 5.6 Hz,
H-5eq), 3.30 (1H, dd, J ) 9.1, 4.8 Hz, H-4), 4.35 (1H, br
s, H-6), 4.76 (1H, m, H-9a), 4.96 (1H, m, H-9b), 5.88 (1H,
m, H-2); 13C NMR (CDCl3, 100 MHz) δ 20.96 (C-7), 21.08
(C-10), 36.11 (C-5), 50.01 (C-4), 67.28 (C-6), 113.73 (C-
9), 127.29 (C-2), 142.42 (C-8), 159.82 (C-1), 198.81 (C-
3); EIMS m/z [M]+ 166 (10), [M - H2O]+ 148 (58), 133
(35), 105 (47), 98 (C5H6O2+, 100), 69 (46): HREIMS m/z
166.0996 (calcd for C10H14O2 166.0994).
(4S,8S)-8,9-Epoxy-p-m en th -1-en e (16): [R]20D -77.7°
(c 0.14, CH2Cl2).
Con ver sion of 15 a n d 16 in to 8,9-Ep oxyisop ip -
er iten on es (7 a n d 8). t-BuOOH (90%, 1.3 mL) was
added to a solution of CrO3 (5 mg) in CH2Cl2 (5 mL).
The epoxide (152 mg, 15 or 16) in CH2Cl2 (1 mL) was
then added dropwise and stirred for 2 h at room
temperature. The solution was successively washed
with saturated aqueous NaHCO3 and brine and then
finally dried over MgSO4 and evaporated. The product
was separated using preparative TLC (hexane-diethyl
ether 5:1; multiple developments). Synthetic 8,9-ep-
oxyisopiperitenones (7 and 8) showed the same physi-
cochemical properties as the isolates from cell culture.
(-)-(4R,8R)-8,9-Ep oxyisop ip er iten on e (7): color-
less oil (10 mg); [R]20 -110.0° (c 0.70, CH2Cl2); UV
D
(MeOH) λmax (log ꢀ) 235 (4.1) nm; IR (neat) νmax 2930,
1670, 1200 cm-1; 1H NMR (CDCl3, 400 MHz) δ 1.26 (3H,
s, H-10), 1.97 (3H, s, H-7), 2.01 (1H, m, H-4), 2.04 (1H,
m, H-5a), 2.24 (1H, m, H-5b), 2.37 (2H, m, H-6), 2.73
(1H, d, J ) 4.5 Hz, H-9a), 2.82 (1H, d, J ) 4.5 Hz, H-9b),
5.87 (1H, m, H-2); 13C NMR (CDCl3, 100 MHz) δ 17.15
(C-10), 24.08 (C-7), 24.78 (C-5), 30.81 (C-6), 53.44 (C-
4), 55.77 (C-9), 56.57 (C-8), 126.45 (C-2), 162.40 (C-1),
197.82 (C-3); EIMS m/z [M]+ 166 (19), [M - H2O]+ 148
(19), 136 (24), 121 (30), 108 (28), 93 (30), 82 ([C5H6O]+,
100), 67 (13); HREIMS m/z 166.0984 (calcd for C10H14O2
166.0994).
Ack n ow led gm en t. This work was supported by a
grant from the Korea Science and Engineering Founda-
tion through The Research Center for New Bio-Materi-
als in Agriculture. S.U.K. thanks his wife Sang-Ok for
manuscript preparation.
Refer en ces a n d Notes
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(-)-(4R,8S)-8,9-Ep oxyisop ip er iten on e (8): color-
less oil (4 mg); [R]20 -41.2° (c 0.20, CH2Cl2); UV
D
(5) Galun, E.; Aviv, D.; Dantes, A.; Freeman, A. Planta Med. 1985,
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(MeOH) λmax (log ꢀ) 234 (4.1) nm; IR (neat) νmax 2930,
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s, H-10), 1.74 (1H, m, H-5a), 1.95 (3H, s, H-7), 2.08 (1H,
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Modified Monoterpenes from (-)-Isopiperitenone
J ournal of Natural Products, 1998, Vol. 61, No. 3 357
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NP970388K