Journal of Natural Products
Article
NMR (100 MHz, CDCl3) δ 199.5 (CO), 162.0 (C-1), 143.5 (C-
8), 126.9 (C-2), 113.7 (C-10), 54.0 (C-4), 30.5 (C-6), 27.8 (C-5),
24.4 (C-7), 20.8 (C-9); HRMS C10H14ONa [M + Na+] calcd
173.0942, found 173.0936.
22.8 (C-9 cis), 21.0 (C-5 cis), 19.5 (C-9 trans). Data matched those
reported.8
cis/trans-Isopiperitenol p-Nitrobenzoates (20/19). Isopiper-
itenol (18/2) (70 mg, 0.45 mmol) was dissolved in pyridine (3.2 mL)
and cooled to 0 °C. To the solution was added p-nitrobenzoyl
chloride (150 mg, 0.808 mmol), and the solution was stirred at room
temperature for 3 h. The mixture was poured into H2O and extracted
with DCM (10 mL). The organic layer was washed with 2 M HCl (3
× 5 mL), saturated bicarbonate solution (2 × 5 mL), and brine (5
mL). The organic phase was dried (MgSO4) and filtered, and the
solvents were removed in vacuo. Flash column chromatography
(hexanes/Et2O, 96:4, v/v) afforded 19/20 as a waxy solid (107 mg,
78%). The cis and trans isomers were separated by preparative HPLC
(ACE silica column (250 × 4.5 mm id); 10 mg/mL; injected 100 to
500 μL; 0.5% to 3% EtOAc/hexane; cis 28.3 min and trans 29.7 min).
trans-19: 1H NMR (400 MHz, CDCl3) δ 8.27 (d, 2H, J 9.2 Hz, ArH),
8.18 (d, 2H, J 9.2 Hz, ArH), 5.65 (d, 1H, J 8.8 Hz, H-3), 5.45 (br s,
1H, H-2), 4.76 (m, 2H, H-10a, H-10b), 2.54−2.48 (m, 1H, H-4),
2.2−2.11 (m, 1H, H-6a), 2.04−1.98 (m, 1H, H-6b), 1.86−1.76 (m,
2H, H-5a, H-5b), 1.85−1.78 (m, 3H, J 4.4 Hz, H-9), 1.57 (s, 3H, H-
Piperitenone (17). NaIO4 (2.09 g, 9.76 mmol) was added to a
mixture of 2-phenylselenyl-trans-isopulegone (1.50 g, 4.88 mmol) and
NaHCO3 (0.41 g, 4.88 mmol) in EtOH (100 mL) at 0 °C, and the
reaction mixture was stirred at room temperature for 3 days. The
mixture was diluted with DCM (200 mL) and washed with H2O (3 ×
50 mL) and brine (1 × 50 mL). The organic layer was dried over
MgSO4, filtered, and concentrated under reduced pressure. The
resultant residue was purified by flash column chromatography
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(hexanes/EtOAc, 95:5) to give a pale yellow oil (0.24 g, 33%): H
NMR (400 MHz, CDCl3) δ 5.87 (1H, app. sext., J 1.3 Hz, H-2), 2.64
(2H, br t, J 6.2 Hz, H-5), 2.28 (2H, tq, J 6.3, 0.7 Hz, H-6), 2.08−2.06
(3H, m, CH3-10), 1.91 (3H, dt, J 1.6, 0.8 Hz, CH3-7), 1.85−1.83
(3H, m CH3-9); 13C NMR (100 MHz, CDCl3) δ 191.8 (CO, C-1),
159.9 (C-6), 142.7 (C-3), 129.1 (C-8), 128.9 (C-2), 32.0 (C-4), 28.1
1
(C-5), 23.9 (C-7), 23.0 (C-10), 22.6 (C-9); H and 13C data match
those reported;29 HRMS C10H14ONa [M + Na+] calcd 173.0942,
found 173.0942.
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7). cis-20: H NMR (400 MHz, CDCl3) δ 8.25 (d, J 9.2 Hz, 2H,
ArH), 8.14 (d, J 9.2 Hz, 2H, ArH), 5.75- 5.73 (br d, J 6.0 Hz, 1H H-
3), 5.61−5.59 (m, 1H, H-2), 4.84−4.79 (m, 2H, H-10a, H-10b),
2.31−2.28 (m, 1H, H-4), 2.17−2.15 (m, 2H, H-6a, H-6b), 1.84−1.83
(m, 1H, H-5a), 1.79−1.79 (m, 1H, H-5b), 1.78 (s, 3H, H-9), 1.76 (s,
3H, H-7).
(+)-cis-Isopulegone (4) and (+)-Pulegone (5). (+)-cis-Iso-
pulegone (4) was synthesized by preparative scale bioreduction of
(−)-isopiperitenone (3) with IPR. The preparative scale biotransfor-
mation was performed in small multiples rather than as a single large
batch. These 58 × 25 mL vials, each with a total volume of 10 mL,
contained phosphate buffer (50 mM KH2PO4/K2HPO4 pH 6.4),
(−)-isopiperitenone (3) (8 mM, 250 mM stock in EtOH; total
substrate 700 mg), IPR (2.0 μM), dithiothreitol (1 mM), NADP+ (20
μM), glucose (30 mM), and glucose dehydrogenase (GDH; 10 U).
The vials were sealed and shaken at 25 °C and 130 rpm. After 4 h the
biotransformation was terminated by extraction with Et2O (2 × 100
mL), and the combined organic layer washed with brine (1 × 50 mL),
dried (MgSO4), and concentrated under reduced pressure.
Purification by flash column chromatography gave (+)-cis-isopulegone
(4) as a colorless oil (617 mg, 87%). (+)-Pulegone (5) (data matched
those reported)30 was obtained quantitatively from (+)-isopulegone
(4) by dissolving 4 (20 mg) in methanol (2 mL) and 10% aqueous
NaOH w/v (0.2 mL), the crude isolate being of sufficient purity for
direct biotransformation (vide infra). (+)-cis-Isopulegone (4): 1H
NMR (400 MHz, CDCl3) δ 4.97 (1H, app. sext., J 1.4 Hz, HAHB-10),
4.80−4.78 (1H, m, HAHB-10), 2.97 (1H, t, J 6.4 Hz, H-4), 2.42−2.36
(1H, m, Heq-2), 2.22−2.11 (3H, m, H-1 + Heq-5 + Hax-2), 1.90−1.77
(2H, m, Hax-5 + Heq-6), 1.74−1.72 (3H, m, CH3-9), 1.63−1.54 (1H,
m, Hax-6), 0.98 (3H, d, J 6.8 Hz, CH3-7); 13C NMR (100 MHz,
CDCl3) δ 212.1 (CO), 142.9 (C-8), 112.7 (C-10), 57.0 (C-4), 48.3
(C-2), 33.5 (C-1), 30.2 (C-6), 27.7 (C-5), 21.8 (C-9), 20.7 (C-7).
cis/trans-Isopiperitenol (18 and 2). A solution of (−)-iso-
piperitenone (3) (0.10 g, 0.66 mmol) in anhydrous Et2O (5 mL) was
added dropwise to a suspension of LiAlH4 (0.03 g, 0.85 mmol) in
anhydrous Et2O (5 mL) at 0 °C under N2 and stirred for 30 min. The
mixture was quenched using the Fieser method: H2O (0.04 mL) was
added slowly at 0 °C, followed by 1 M NaOH (0.15 mL) and then
H2O again (0.15 mL). The mixture was stirred for 30 min, MgSO4
was added, and the mixture was stirred for a further 15 min, filtered
through Celite, and concentrated under reduced pressure. The residue
was purified by flash column chromatography (hexanes/EtOAc, 95:5,
v/v) to give a mixture of cis/trans-isopiperitenol (18 and 2) (2:3,
(−)-trans-Isopiperitenol (2). trans-Isopiperitenol p-nitrobenzoate
(40 mg, 0.13 mmol) was dissolved in THF (1 mL) in a screw-top
sample vial and to this were added LiOH·H2O (13 mg, 0.33 mmol)
and H2O (160 μL). The vial was sealed, and the mixture was heated
to 60 °C for 4 h. The mixture was placed on ice, diluted with Et2O
(40 mL), and washed with H2O (3 × 1 mL). The organic phase was
dried (MgSO4) and filtered, and solvents were removed. Flash column
chromatography (hexanes/EtOAc, 9:1, v/v) provided (−)-trans-
isopiperitenol (2) (18 mg, 92% yield): 1H NMR (400 MHz,
CDCl3) δ 5.45−5.44 (m, 1H, H-2), 4.89−4.88 (m, 1H, H-10a),
4.85−4.84 (1H, H-10b), 4.13−4.10 (m, 1H, H-3), 2.10−2.04 (m, 2H,
H-4, H-6a), 1.95−1.93 (m, 1H, H-6b), 1.73−1.72 (m, 3H, H-9),
1.69−1.69 (m, 3H, H-7), 1.65−1.56 (m, 2H, H-5).
(−)-cis-Isopiperitenol (18). cis-Isopiperitenol p-nitrobenzoate
(20) (40 mg, 0.13 mmol) was dissolved in THF (1 mL) in a
screw-top sample vial and to this were added LiOH·H2O (13 mg, 0.33
mmol) and H2O (160 μL). The vial was sealed, and the mixture was
heated to 60 °C for 4 h. The mixture was placed on ice, diluted with
Et2O (40 mL), and washed with H2O (3 × 10 mL). The organic
phase was dried over MgSO4 and filtered, and the solvent was
removed. Column chromatography (hexane s/EtOAc, 9:1, v/v)
provided 18 (18 mg, 90% yield). Data matched those reported.13 1H
NMR (400 MHz, CDCl3) δ 5.59−5.67 (m, 1H, H-2), 5.00−4.99 (m,
1H, H-10a), 4.81−4.81 (m, 1H, H-10b), 4.14−4.13 (m, 1H, H-3),
2.14−2.13 (m, 1H, H-4), 2.10−1.99 (m, 2H, H-6), 1.83 (s, 3H, H-9),
1.80−1.74 (m, 2H, H-5), 1.72 (s, 3H, H-7).
Menthone (7) and Isomenthone (8). A preparative scale
bioreduction of (+)-pulegone (5) with DBR from Nicotiana tabacum
(NtDBR-C-His6 in pET21b; Uniprot: Q9SLN8) was performed. The
preparative scale biotransformation was performed in three multiples
rather than as a single batch. Each of three 25 mL vials, with a total
volume of 10 mL, contained phosphate buffer (50 mM KH2PO4/
K2HPO4 pH 6.4), (+)-pulegone (5, 8 mM, 250 mM stock in EtOH,
total substrate 36 mg), DBR (2.0 μM), NADP+ (10 μM), glucose (15
mM), and GDH (10 U). The vials were sealed and shaken at 30 °C
and 130 rpm. After 4 h the biotransformation was terminated by
extraction with Et2O (2 × 100 mL), and the combined organic layer
washed with brine (1 × 50 mL), dried (MgSO4), and concentrated
under reduced pressure. Purification by flash column chromatography
(hexanes/EtOAc, 95:5, v/v) gave menthone (7) and isomenthone (8)
as clear oils (37% menthone, 31% isomenthone, total 68%). GC 10.4
and 10.6 min, respectively. Analytical data for both matched known
1
85%) as a colorless oil: H NMR (400 MHz, CDCl3) δ 5.69−5.66
(0.4H, m, H-2 cis), 5.45−5.43 (0.6H, m, H-2 trans), 5.01−4.99 (0.4,
m, HAHB-10 cis), 4.90−4.88 (0.6H, m, HAHB-10 trans), 4.85−4.84
(0.6H, m, HAHB-10 trans), 4.81−4.80 (0.4H, m, HAHB-10 cis), 4.15−
4.09 (1H, m, H-cis/trans), 2.14−1.88 (3H, H-+ CH2 cis/trans), 1.83−
1.82 (1.2H, m, CH3-9 cis), 1.78−1.54 (2H, m, CH2 cis/trans), 1.73−
1.71 (3H, m, CH3-9 trans + CH3-7 cis), 1.70−1.69 (1.8H, m, CH3-7
trans); 13C NMR (100 MHz, CDCl3) δ 146.7/146.6 (C-8 cis/trans),
139.9/136.9 (C-1 cis/trans), 124.4/122.5 (C-2 trans/cis), 112.5/111.9
(C-10 trans/cis), 68.8/64.0 (C-3 trans/cis), 51.1/46.3 (C-4 trans/cis),
31.3/30.4 (C-6 cis/trans), 26.3 (C-5 trans), 23.6/23.2 (C-7 cis/trans),
1
data, and GC matched standards. Isomenthone (8): H NMR (400
MHz, CDCl3) δ 2.30 (ddt, 1H, H-2a, J 13.1, 4.5, 1.2 Hz), 2.11 (dd,
F
J. Nat. Prod. XXXX, XXX, XXX−XXX