Biotransformation of caryophyllene oxide by Botrytis cinerea

Article abstract of DOI:10.1021/np980104b

Biotransformation of caryophyllene oxide (1) with B. cinerea afforded 15 products (2-16). Ten of these (3-5, 7, 9-11, and 14-16) are reported here for the first time. The main reaction paths involved stereoselective epoxidation at C-8/C-13 and hydroxylation at C-7. A rearranged compound was found, which was a cyclization product 16 possessing the caryolane skeleton.

Full text of DOI:10.1021/np980104b

J . Nat. Prod. 1999, 62, 41-44
41
Biotr a n sfor m a tion of Ca r yop h yllen e Oxid e by Botr ytis cin er ea
Rosa Duran, Elena Corrales, Rosario Herna´ndez-Gala´n, and Isidro G. Collado*
Departamento de Quı´mica Orga´nica, Facultad de Ciencias, Universidad de Ca´diz, Apdo. 40, 11510 Puerto Real, Ca´diz, Spain
Received March 18, 1998
Biotransformation of caryophyllene oxide (1) with B. cinerea afforded 15 products (2-16). Ten of these
(3-5, 7, 9-11, and 14-16) are reported here for the first time. The main reaction paths involved
stereoselective epoxidation at C-8/C-13 and hydroxylation at C-7. A rearranged compound was found,
which was a cyclization product 16 possessing the caryolane skeleton.
Botrytis species are serious pathogens toward a number
of commercial plants.1,2 In particular, Botrytis cinerea
attacks a wide range of plants, producing various leaf spot
diseases and gray powdery mildews on lettuce, tomatoes,
and grapes.
biotransformations of 1,7 while 12 and 13 were obtained
from diepoxycaryophyllene.7 Product 6 has also been
obtained from caryophyllene using two different microor-
ganisms, Chaetomium cochiolides7 and Diplodia gossypi-
na.8
Botrydial and structurally related compounds are char-
acteristic metabolites of Botrytis sp.3-5 Previous work5
yielded evidence that botrydial played a role in the in vivo
pathogenicity of B. cinerea. Biosynthetic studies suggest
that the sesquiterpenes botrydial and dihydrobotrydial are
formed from farnesyl pyrophosphate.6 The first stage in the
biosynthesis involves the formation and cyclization of a
caryophyllene cation. Accordingly, we have investigated
the biotransformation of caryophyllene oxide (1) in order
to elucidate the fate of compounds having the caryophyllene
skeleton.
Epoxidation at the double bond gave 3 and 5 as the main
biotransformation products. The ratio of 3 to 5 indicated
a selectivity toward diepoxide 3. NOE difference experi-
ments on 3 and 5 proved insufficient for the determination
of stereochemistry at C-8. The diepoxides 3 and 5 were
therefore reduced by treatment with LiAlH4 to give alcohols
4 and 17, respectively. Comparison of compounds 4 and
17 indicated that the C-8 sterochemistry of both 3 and 4
was R. Spectroscopic data (NMR, MS) of alcohol 4 were
identical to those obtained for compound 4 isolated from
the fermentation broth.
Diepoxides 3 and 5 could be the precursors of epimeric
alcohols 15 and 16, which can be obtained through reduc-
tive opening of the 8,13-epoxide. Their structures were
determined by comparison of their 13C NMR spectra, which
indicated the presence of a hydroxymethyl group in both
cases. The downfield shift of the signal corresponding to
H-9 in compound 16 indicated an R-orientation for the
hydroxymethyl group at C-13. This was confirmed by the
observation of an NOE effect between H-13 and H-9, which
is only possible for a hydroxymethyl in the R-orientation.
Aldehyde 137 probably arises from opening of the epoxide
and further oxidation of compound 3. Kobusone (12) was
Resu lts a n d Discu ssion
The fermentation of 1 using B. cinerea and isolation of
the biotransformation products was carried out as de-
scribed in the Experimental Section. The extracts of the
initial fermentation (2 days) contained products 2-9. A
second experiment was carried out under different condi-
tions in an attempt to increase the yield. A culture of B.
cinerea was grown for 3 days and then was filtered, and
the mycelia was transferred to a new medium without
glucose. The substrate was then added, and the mixture
was incubated for 1 day. Under the latter conditions, 13
products were isolated; six of these (3 and 5-9) were
previously obtained in the initial experiment along with
the additional compounds 10, 11, kobusone (12), and 13-
16. Products 2, 6, 8, and 12 were previously described in
* To whom correspondence should be addressed. Tel.: 34-56-830217.
Fax: 34-56-834924. E-mail: isidro.gonzalez@uca.es.
10.1021/np980104b CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/19/1998

42 J ournal of Natural Products, 1999, Vol. 62, No. 1
Duran et al.
are uncorrected. Optical rotations were determined using a
Perkin-Elmer 241 polarimeter. IR spectra were recorded using
a Perkin-Elmer 881 spectrophotometer. 1H and 13C NMR
measurements (δ in ppm) were obtained using Varian Gemini
200 and Varian Unity 400 NMR spectrometers with SiMe4 as
the internal reference. Mass spectra were recorded using
VG12-250 and VG Autospec. spectrometers. HPLC was
performed using a Hitachi/Merck L6200 apparatus equipped
with differential refractometer detector (RI-71). TLC was
performed using Merck Kieselgel 60 F254, 0.2 mm layer
thickness. Silica gel (Merck) was used for column chroma-
tography. Purification by HPLC was achieved using a Si gel
column (LiChrospher Si 60 5 µm, 0.4 cm wide, 25 cm long).
Or ga n ism . The culture of B. cinerea 2100 employed was
obtained from the “Centro Espan˜ol de cultivos tipos” (CECT),
Facultad de Biologia, Universidad de Valencia, Spain, where
a culture of this strain is deposited.
Biot r a n sfor m a t ion E xp er im en t s. In procedure I, B.
cinerea was cultivated at 25 °C and 250 rpm in a 500 mL
Erlenmeyer flask containing Czapeck Dox medium. Biotrans-
fomation experiments were carried out in two different ways.
In the first experiment, a 48 h culture was used as the
inoculum. The organism was grown in 14 × 500 mL Erlen-
meyer flasks and transferred, after 48 h, into 50 × 500 mL
flasks containing 160 mL of Czapeck Dox medium. These were
then incubated for an additional 48 h. The culture medium
and mycelia was separated by filtration. The mycelia was
washed with 0.9% NaCl solution and distributed into 50 × 500
mL Erlenmeyer flasks, each containing 100 mL of Czapeck
Dox medium without glucose, 100 mL of phosphate buffer, and
the substrate (20 mg of 1 dissolved in 250 µL of EtOH).
After another 48 h, the culture medium and mycelia were
separated by filtration, and the medium was extracted with
pentane. The organic solvent was washed with water, dried
over Na2SO4, and evaporated. The crude extracts were
separated on Si-60 columns with a pentane/ethyl acetate
mixture. Fractions collected were purified further by semi-
preparative HPLC when necessary.
In the second experiment (procedure II) 31 × 500 mL
Erlenmeyer flasks, each containing 200 mL of Czapeck Dox
medium, were inoculated with B. cinerea and incubated for
72 h. The medium and mycelia were then separated by
filtration, and the mycelia was transferred to 31 × 500 mL
flasks, each containing 200 mL of Czapeck Dox medium
without glucose, and the substrate (20 mg of 1 dissolved in
250 µL of EtOH) was added and incubated for 24 h. The
culture medium and mycelia were then separated, and the
medium was extracted and purified as described previously
for experiment one.
Biotr a n sfor m a tion of Ca r yop h yllen e Oxid e (1). P r o-
ced u r e I. Biotransformation of 1 g of compound 1 with B.
cinerea 2100 gave, after 48 h, 27 (1 mg, 0.1%), 3 (10 mg, 1%),
4 (1 mg, 0.1%), 5 (1.5 mg, 0.15%), 67,8 (2 mg, 0.2%), 7 (1 mg,
0.1%), 87 (1.5 mg, 0.15%), and 9 (2 mg, 0.2%).
F igu r e 1. Selected NOE correlations observed for 14.
also found in the extract and is probably derived from the
8,13-diol by diol cleavage.
Compounds 6 and 9, products of hydroxylation at methyl
groups, were also isolated. Spectroscopic data for 6 were
the same as those described in the literature for the natural
product 15â-(hydroxymethyl)-4,5-epoxycaryophyllene (6).7
The structure of 9 was then assigned by comparison of their
NMR spectra.
Another transformation of 1 by B. cinerea was oxidation
at C-7. Compound 8, already known from biotransforma-
tion with C. Cochiolides,7 and compounds 7, 10, 11, and
14 were also isolated from the fermentation broth. Com-
pound 7 probably arises from 8 via an intramolecular
reaction. A detailed study using 2D-NMR methods solved
the structure of 7 unambiguously.
Compound 14 had a caryolane skeleton. In this case, a
new bond had been formed between C-4 and C-13, which
resulted in a caryolane derivative. Compound 14 could be
formed by intramolecular attack of the exocyclic double
bond of 1 by the 4,5-epoxide. The structure was assigned
using 2D-NMR techniques, including long-range homo- and
heteronuclear correlation. The stereochemistry of the
compound was carefully established through NOE experi-
ments (Figure 1).
Alcohol 8 was oxidized further to give the ketone 11.
Comparison of the 13C NMR of 11 with that of 8 showed
that one hydroxylated methine carbon signal had disap-
peared and was replaced by a ketone signal (δ ) 214). An
identical signal was observed in the 13C NMR spectrum of
compound 10, which requires an additional epoxide at C-8/
C-13. The stereochemistry at C-8 was determined by
comparison of its NMR spectra with those of compounds 3
and 5.
The biotransformation of caryophyllene oxide with B.
cinerea produced a number of oxygenated compounds. The
major biotransformation pathways involved stereoselective
epoxidation at C-8/C-13 and hydroxylation at C-7. Inter-
estingly, position C-7 on the caryophyllene skeleton cor-
responds with C-15 on a botryane derivative. This position
usually bears an oxygen in the botryane skeleton. The
presence of kobusone (12) and aldehyde 13 in the fermen-
tation broth seems to indicate that another biotransfor-
mation path was hydrolysis of the 8,13-epoxide and a
further diol oxidation process. The low recovery of products
from the biotransformation may be related to their further
degradation, which could explain why a shorter incubation
(24 h) scheme yielded a much larger array of metabolites
(13) than the longer one (48 h, eight metabolites).
(4R,5R,8R)-4,5:8,13-Diep oxyca r yop h ylla n e (3): mp 72-
73 °C, [R]19 -67° (c ) 1, hexane); IR (film) νmax 2953, 2860,
D
1458, 1386 cm-1; 1H NMR (CDCl3, 400 MHz) δ 3.07 (1H, dd, J
) 10.2, 4.6 Hz, H-5), 2.66 (1H, dd, J 13′-13 ) 5.2 Hz, J ) 0.8
Hz, H-13), 2.55 (1H, d, J 13-13′ ) 5.2 Hz, H-13’), 2.16 (1H, m,
H-6â), 2.13-2.02 (3H, m, H-2, H-7′′ and H-9), 1.76 (1H, t, J )
9.6 Hz, H-1), 1.65 (1H, ddd, J 3R-3â ) 14.0 Hz, J ) 4.1, 3.9 Hz,
H-3R), 1.58-1.47 (3H, m, H-7â, H-10 and H-10’), 1.46-1.36
(1H, m, H-2’), 1.26 (s, 3H, H-12), 1.24 (1H, m, H-6R), 1.02 (1H,
ddd, J 3â-3R ) 14.0 Hz, J ) 13.2, 4.4 Hz, H-3â), 0.94 (s, 3H,
H-14), 0.92 (3H, s, H-15); 13C NMR data, Table 1; EIMS m/z
236 [M]+ (2), 221 [M - CH3]+ (5), 205 (4), 179 (6), 161 (6), 150
(8), 147 (14), 137 (26), 123 (32), 108 (100); HRMS 236.1769
(calcd for C15H24O2 236.1776).
(4R,5R,8R)-4,5-Ep oxyca r yop h ylla n -8-ol (4): mp 99 °C;
[R]20 -132° (c ) 1, hexane); IR (film) νmax 3476, 2952, 2858,
D
1456, 1367, 1263, 1119 cm-1; 1H NMR (C6D6, 400 MHz) δ 3.34
(1H, dd, J 5-6R ) 9.3 Hz, J 5-6â ) 5.6 Hz, H-5), 2.28 (1H, dddd,
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
measured using a Kofler block Reicher-J ung apparatus and
1H, J 6â-5 ) 5.6 Hz, J 6â-6R ) 12.9 Hz, J 6â-7R ) 5.4 Hz, J 6â-7â
)
5.0 Hz, H-6â), 1.95 (1H, ddd, J ) 12.3, 4.0, 2.5 Hz, H-3R), 1.83

Biotransformation of Caryophyllene Oxide
J ournal of Natural Products, 1999, Vol. 62, No. 1 43
Ta ble 1. 13C NMR Data (δ) of Compounds 3-5, 7, 9-11, and 14-17 (100 MHz, CDCl3, 4 in C6D6)
C
3
4
5
7
9
10
11
14
15
16
17
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
47.9
27.5
40.3
58.4a
61.8
25.5
30.4
57.9b
46.8
35.1
33.3
16.4
56.0
29.4a
21.6b
45.9
28.5
40.9
58.5
60.7
25.4
36.1
71.7
52.6
38.6
32.1
16.4
31.8
29.5a
22.6b
49.3
27.3
39.5
58.9a
62.6
24.8
31.3
59.8b
47.1
35.5
33.4
16.2
50.1
29.9a
21.9b
57.3
23.4
40.3
84.8
79.2
43.5
78.0
158.2
40.6
35.4
34.7
22.1
102.9
29.9a
21.8b
51.6
26.3
39.3
59.5
63.5
30.3
29.8
151.7
48.3
35.2
38.6
16.9
112.9
24.8
67.0
48.4
26.1
39.5
59.3
55.0
40.8
214.0
64.2
39.7
33.4
33.6
16.2
50.1
29.0a
21.7b
57.7a
26.3
39.1
50.8
27.7
39.8a
59.0
57.5
34.7
69.3
61.1
42.9
33.8b
32.9
16.3
45.0
30.0
22.7
46.1
27.8
40.1
59.3
61.6
28.0
29.9
53.1
45.5
39.5
34.1
16.3
66.4
29.8
21.8
42.4a
21.1
38.4
60.3
65.7
27.2
29.0
49.9
42.0b
34.2
35.1
17.5
66.4
29.9
21.3
47.2
25.0
40.5
58.4
60.1
29.0
35.5
74.1
52.5
40.7
31.8
16.5
20.7
23.3a
30.1b
59.3
57.8b
42.8
214.0
156.2
40.8
37.5
33.5
16.2
112.1
29.7
22.1
a,b
Assignments may be interchanged.
(1H, dd, J 1-9 ) 8.3 Hz, J ) 8.9 Hz, H-1), 1.57 (1H, dd, J 9-1
)
Biotr a n sfor m a tion of 620 m g of Ca r yop h yllen e Oxid e
(1). P r oced u r e II. Biotransformation of 620 mg of 1 with
B. cinerea 2100 gave, after 24 h, 3 (50 mg, 8%), 5 (4 mg, 0.6%),
7 (2 mg, 0.3%), 87 (8 mg, 1.2%), 9 (12 mg, 1.8%), 10 (1 mg,
0.15%), 11 (1 mg, 0.15%), kobusone (12,7 2 mg, 0.3%), 137 (1
mg, 0.15%), 14 (1.5 mg, 0.24%), 15 (8 mg, 1.2%), and 16 (8
mg, 1.2%).
8.3 Hz, J ) 9.5 Hz, H-9), 1.54-1.42 (3H, m, H-7R, H-10 and
H-10’), 1.45-1.39 (1H, m, H-2R), 1.29 (1H, ddd, J 7â-6â ) 5.0
Hz, J ) 14.0, 13.0 Hz, H-7â), 1.24-1.16 (2H, m, H-6R and
H-2â), 1.15 (3H, s, H-12), 1.04 (1H, m, H-3â), 0.88 (3H, s,
H-15â), 0.85 (3H, s, H-14R), 0.81 (3H, s, H-13R); 13C NMR data,
Table 1; EIMS m/z 223 [M - CH3]+, 220 [M - H2O]+ (0.1),
205 [M - H2O - CH3]+ (0.7), 177 (8), 149 (7), 141 (6), 121 (21),
109 (26), 95 (50) 43 (100); HRMS m/z 223.16675 (calcd for
(4R,5R,8R)-4,5:8,13-Diepoxycar yoph yllan -7-on e (10): col-
orless oil; [R]26D -16° (c ) 1, hexane); IR (film) νmax 2927, 2859,
1723, 1461, 1260 cm-1; 1H NMR (CDCl3, 400 MHz) δ 3.48 (1H,
C
14H23O2 223.1698).
(4R,5R,8S)-4,5:8,13-Diep oxyca r yop h ylla n e (5): mp 57-
dd, J 5-6 ) 8.8 Hz, J 5-6′ ) 6.6 Hz, H-5), 3.31 (1H, dd, J 6′-5
6.6 Hz, J 6′-6 ) 13.0 Hz, H-6), 2.85 (1H, d, J 13′-13 ) 4.6 Hz,
H-13), 2.77 (1H, q, J 9-1 ) 9.6 Hz, H-9), 2.70 (1H, d, J 13-13′
)
58 °C; [R]19 46° (c ) 1, hexane); IR (film) νmax 2922, 2862,
D
1461, 1386, 1370 cm-1; 1H NMR (CDCl3, 400 MHz) δ 2.87 (1H,
)
dd, J 5-6R ) 10.5 Hz, J 5-6â ) 4.4 Hz, H-5), 2.67 (1H, d, J 13′-13
)
4.6 Hz, H-13’), 2.13 (1H, ddd, J 3′-3 ) 12.9 Hz, J ) 4.2, 2.8 Hz,
H-3), 2.05 (1H, dd, J 6-5 ) 8.8 Hz, J 6-6′ ) 13.0 Hz, H-6), 1.80
(1H, t, J 1-9 ) 9.6 Hz, H-1), 1.59 (1H, m, H-2), 1.48 (1H, dd,
J 10′-9 ) 8.0 Hz, J 10′-10 ) 10.3 Hz, H-10), 1.41 (1H, m, H-2),
1.17 (3H, s, H-12), 1.17 (1H, dd, J 10-9 ) 10.5 Hz, J 10-10′ ) 10.3
Hz, H-10’), 1.00 (1H, m, H-3’), 0.97 (3H, s, H-14*), 0.92 (3H, s,
H-15*); 13C NMR data, Table 1; EIMS m/z 250 [M]+ (0.2), 235
[M - CH3]+ (5), 221 (52), 149 (24), 123 (24), 43(100).
4.6 Hz, H-13), 2.65 (1H, d, J 13-13′ ) 4.6 Hz, H-13’), 2.11 (1H,
m, H-9), 2.05 (1H, dddd, J 6â-5 ) 4.4 Hz, J 6â-6R ) 13.9 Hz, J 6â-7
) 9.7 Hz, J 6â-7′ ) 5.2 Hz, H-6â), 1.93 (1H, ddd, J 7′-6R ) 5.8
Hz, J 7′-6â ) 5.2 Hz, J 7′-7 ) 14.3 Hz, H-7), 1.76 (1H, ddd, J 7-6R
) 4.9 Hz, J 7-6â ) 9.7 Hz, J 7-7′ ) 14.3 Hz, H-7’), 1.65 (2H, m,
H-1 and H-2), 1.56 (1H, dd, J 10′-9 ) 9.5 Hz, J 10′-10 ) 11.9 Hz,
H-10) 1.43 (1H, dddd, 1H, J 6R-5 ) 10.5 Hz, J 6R-6â ) 13.9 Hz,
J 6R-7 ) 4.9 Hz, J 6R-7′ ) 5.8 Hz, H-6R), 1.25 (1H, dd, J 10-9
)
(4R,5R)-4,5-Ep oxyca r yop h yll-8(13)-en -7-on e (11): color-
11.0 Hz, J 10-10′ ) 11.9 Hz, H-10’), 0.99 (1H, m, H-3â), 0.95 (3H,
s, H-14*), 0.93 (3H, s, H-15*); 13C NMR data, Table 1; EIMS
m/z 236 [M]+ (0.1), 221 [M - CH3]+ (1), 175 (2), 163 (7), 133
(11), 121 (29), 108 (43), 107 (3), 95 (42), 93 (77); HRMS m/z
236.1774 (calcd for C15H24O2 236.1776).
less oil; [R]25 -40° (c ) 1, hexane); IR (film) νmax 2931, 2859,
D
1709, 1457, 1261 cm-1; 1H NMR (CDCl3, 400 MHz) δ 5.40 (1H,
bs, H-13), 5.24 (1H, bs, H-13’), 3.35 (1H, dd, J 5-6 ) 14.1 Hz,
J 5-6′ ) 6.6 Hz, H-5), 3.06 (1H, dd, J 6′-5 ) 6.6 Hz, J 6′-6 ) 9.3
Hz, H-6), 2.78 (1H, ddd, J 9-1 ) 4.6 Hz, J 9-10 ) 8.7 Hz, J 9-10′
)
(4R,5R,7S)-4,7-Ep oxyca r yop h yll-8(13)-en -5-ol (7): mp
9.6 Hz, H-9), 1.87 (1H, dd, J 10′-9 ) 9.6 Hz, J 10-10′ ) 11.1 Hz,
H-10), 1.79 (1H, dd, J 10-9 ) 8.7 Hz, J 10-10′ ) 11.1 Hz, H-10′),
1.61-1.31 (3H, m, H-1, H-2′ and H-3), 1.02 and 1.01 (3H each,
s, H-14* and H-15*), 0.92 (1H, m, H-3’); 13C NMR data, Table
1; EIMS m/z 219 [M - CH3]+ (2), 205 (10), 191 (11), 163 (10),
49 (11), 135 (13), 43(100); HRMS m/z 219.1388 (calcd for
129-130 °C; [R]25 -32° (c ) 1, ethyl acetate); IR (film) νmax
D
3410, 2926, 2864, 1462, 1105, 1068 cm-1; 1H NMR (CDCl3, 400
MHz) δ 4.90 (1H, dd, J 7-6R ) 8.2 Hz, J 7-6â ) 9.0 Hz, H-7), 4.58
(2H, bs, H-13), 3.81 (1H, t, J ) 5.1 Hz, H-5), 2.94 (1H, ddd, J
) 11.0, 10.7, 10.0 Hz, H-9), 2.31 (1H, ddd, J ) 14.0, 8.0, 7.8
Hz, H-6R), 2.06 (1H, ddd, J ) 9.0, 4.4 Hz, H-6â), 1.87 (1H,
ddd, J ) 14.2, 7.6, 6.4 Hz, H-3R), 1.65-1.57 (2H, m, H-10),
1.47 (1H, m, H-2), 1.36 (1H, dddd, J ) 13.7, 7.6, 1.9, 1.7 Hz,
H-2), 1.27 (3H, s, H-15R), 0.97 (3H, s, H-14â); 13C NMR data,
Table 1; EIMS m/z 236 [M]+ (18), 203 [M - H2O - CH3]+ (2),
108 (58), 93 (49), 79 (68), 43 (100); HRMS m/z 236.1748 (calcd
for C15H24O2 236.1776).
C
14H19O2 219.1385).
(1S,9R,11R)-1,9-Ep oxyca r yola n -11-ol (14): oil; [R]25D -30°
(c ) 1, ethyl acetate); IR (film) νmax 3461, 2954, 2922, 2862,
1462, 1366, 1080, 1031, 903 cm-1; 1H NMR (CDCl3, 400 MHz)
δ 3.89 (1H, b dd, J 7-6 ) 8.2, 7.8 Hz, H-7), 2.96 (1H, d, J 13′-13
) 4.1 Hz, H-13), 2.90 (1H, d, J 13-13′ ) 4.1 Hz, H-13’), 2.77 (1H,
d, J ) 1.4 Hz, OH), 2.73 (1H, dd, J 5-6 ) 8.2, 7.1 Hz, H-5), 2.42
(1H, q, J 9-10â ) 9.5 Hz, H-9), 2.16 (1H, ddd, J 3R-3â ) 13.0 Hz,
J ) 3.7, 3.1 Hz H-3R), 1.93 (2H, dd, J 6-5 ) 8.2 Hz, J 6-7 ) 7.8
Hz, H-6), 1.67-1.47 (3H, m, H-2, H-10R, H-2’), 1.34 (3H, s,
H-12), 1.25 (1H, dd, J ) 11.9, 9.5 Hz, H-10â), 0.98 (3H, s,
H-14*), 0.94 (1H, m, H-3’), 0.93 (3H, s, H-15*); 13C NMR data,
Table 1; EIMS m/z 237 [M + 1]+ (0.1), 149 (1), 135 (3), 119 (3),
109 (11), 93 (10), 81 (14), 69 (18), 55 (40), 43 (100); HRMS m/z
237.1882 (calcd for C15H25O2 237.18545).
(4R,5R,11S)-4,5-Ep oxyca r yop h ylla n -8(13)-en -14-ol (9):
colorless oil; [R]26 -65° (c ) 1.7, ethyl acetate); IR νmax 3432,
D
2925, 2863, 1631, 1457, 1387, 1257, 1041, 909, 863, 758, 734
1
cm-1; H NMR (CDCl3, 400 MHz) δ 5.00 (1H, bs, H-13), 4.89
(1H, bs, H-13’), 3.70 (1H, d, J 14′-14 ) 10.8 Hz, H-14), 3.61 (1H,
d, J 14-14′ ) 10.8 Hz, H-14’), 2.85 (1H, dd, J ) 10.5, 4.4 Hz,
H-5), 2.66 (1H, dd, J ) 9.6, 9.3 Hz, H-9), 2.35 (1H, ddd, J 7′-7
) 12.6 Hz, J ) 7.4, 5.0 Hz, H-7), 2.23 (1H, m, H-6), 2.12-2.06
(2H, m, H-3 and H-7’), 1.94 (1H, m, H-1), 1.84 (1H, m, H-2â),
1.59 (1H, t, J ) 11.0 Hz, H-10), 1.53 (1H, m, H-2R), 1.32 (1H,
m, H-6’), 1.20 (3H, s, H-12), 1.07 (3H, s, H-15), 0.94 (1H, ddd,
J 3-3’ ) 13.4 Hz, J 3-2 ) 13.4, 5.0 Hz, H-3’); 13C NMR data, Table
1; EIMS m/z 236 [M]+ (0.5), 218 [M - H2O]+ (1), 205 [M -
CH2OH]+ (11), 193 (11), 187 (18), 177 (17), 161 (34), 121 (35),
107 (46), 93 (100); HRMS m/z 236.1768 (calcd for C15H24O2
236.1776).
(4R,5R,8R)-4,5-Ep oxyca r yop h ylla n -13-ol (15): mp 88 °C;
[R]25 -50° (c ) 3.5, ethyl acetate); IR (film) νmax 3236, 2950,
D
2882, 1456, 1068, 1034 cm-1
;
1H NMR (CDCl3, 400 MHz) δ
3.56 (1H, dd, J 13′-8 ) 3.2 Hz, J 13′-13 ) 10.5 Hz, H-13), 3.29
(1H, dd, J 13-8 ) 7.2 Hz, J 13-13′ ) 10.5 Hz, H-13’), 3.01 (1H, dd,
J ) 9.6, 5.2 Hz, H-5), 2.11 (1H, ddd, J 3′-3 ) 12.6 Hz, J ) 4.3,
2.7 Hz, H-3), 1.78 (1H, m, H-9), 1.64 (1H, dt, J 2′-2 ) 14.9 Hz,
J 2′-3 ) 3.6 Hz, H-2), 1.55 (1H, dd, J ) 8.7, 9.4 Hz, H-1), 1.48

44 J ournal of Natural Products, 1999, Vol. 62, No. 1
Duran et al.
(1H, m, H-8), 1.42 (1H, m, H-2’), 1.35 (1H, dd, J ) 9.8, 9.6 Hz,
H-10), 1.28 (3H, s, H-12), 1.28 (2H, m, H-10′ and H-12), 1.00
(1H, ddd, J 3-2 ) 13.5 Hz, J 3-3′ ) 12.6 Hz, J 3-2′ ) 3.6 Hz, H-3’),
0.93 and 0.92 (3H each, s, H-14 and H-15); 13C NMR data,
Table 1; EIMS m/z 223 [M - CH3]+ (0.4), 207 [M - CH2OH]+
(0.5), 177 [M - CH2OH - 2 × CH3]+ (0.5), 167 (4), 151 (9),
133 (15), 121 (18), 93 (100); HRMS m/z 233.1702 (calcd for
2 h. Water was then added, and the reaction mixture was
extracted in the usual way. Purification of the reaction
mixture by column chromatography yielded 32 mg of starting
material together with 43 mg of (4R,5R,8R)-4,5-epoxycaryo-
phyllan-8-ol (4) and 10 mg of (4R,5R,8S)-4,5-epoxycaryophyl-
lan-8-ol (17) (yield 98%).
(4R,5R,8S)-4,5-Ep oxyca r yop h ylla n -8-ol (17): mp 79-80
°C; [R]20D -91° (c ) 1, hexane); IR (film) νmax 3435, 2949, 1656,
1465, 1104, 1067 cm-1; 1H NMR (CDCl3, 400 MHz) δ 2.54 (1H,
dd, J 5-6R ) 9.5 Hz, J 5-6â ) 5.6 Hz, H-5), 1.96 (1H, dddd, J 6â-5
) J 6â-7â ) 5.6 Hz; J 6â-7R ) 5.9 Hz, H-6â), 1.90 (1H, ddd, J 3R-2
) 3.6 Hz, J 3R-2′ ) 3.3 Hz, J 3R-3â ) 12.8 Hz, H-3R), 1.78 (1H,
ddd, J 9-1 ) 8.3 Hz, J 9-10R ) 9.2 Hz, J 9-10â ) 10.2 Hz, H-9),
1.68 (1H, ddd, J 7â-6R ) 2.6 Hz, J 7â-6â ) 5.6 Hz, J 7â-7R ) 13.8
Hz, H-7â), 1.54 (1H, dd, J 10R-9 ) 9.2 Hz, J 10-10â ) 11.2 Hz,
H-10R), 1.49 (1H, ddd, J 7R-6R ) 5.3 Hz, J 7R-6â ) 5.9 Hz, J 7R-7â
C
14H23O2 223.1698).
(4R,5R,8S)-4,5-Ep oxyca r yop h ylla n -13-ol (16): mp 72-
73 °C; [R]25 -31° (c ) 1.6, hexane); IR (film) νmax 3425, 2930,
D
2860, 1461, 1388, 1367, 1067, 1041 cm-1; 1H NMR (CDCl3, 400
MHz) δ 3.24 (2H, m, H-13), 2.92 (1H, dd, J ) 11.4, 3.5 Hz,
H-5), 2.29 (1H, m, H-9), 2.23 (1H, m, H-6â), 2.06 (1H, ddd,,
J 3′-3 ) 12.7 Hz, J 3′-2 ) 4.5, 2.1 Hz, H-3), 1.79 (1H, t, J ) 9.8
Hz, H-1), 1.70 (1H, ddd, J 2-2 ) 14.5 Hz, J 2-3 ) 6.0 Hz, J 2-3′
2.1 Hz, H-2), 1.66 (1H, b dd, J ) 13.9, 7.0 Hz, H-8) 1.45 (dd,
10â-10R ) 10.0 Hz, J 10â-9 ) 7.7 Hz, H-10â), 1.32 (m, 1H, H-2’),
)
J
) 13.8 Hz, H-7R), 1.47 (1H, dd, J 10â-9 ) 10.2 Hz, J 10â-10R
)
1.27 (3H, s, H-12), 1.26 (1H, dd, H-10R), 1.18 (1H, m, H-6R),
0.99 (1H, m, H-3’), 0.97 (3H, s, H-14R), 0.94 (3H, s, H-15â);
13C NMR data, Table 1; EIMS m/z 223 [M - CH3]+ (0.5), 207
[M - CH2OH]+ (0.5), 151 (7), 123 (7), 121 (17), 107 (23), 43
(100); HRMS m/z 223.1687 (calcd for C14H23O2 223.1698).
Ep oxid a tion of Ca r yop h yllen e Oxid e (1): Caryophyllene
oxide (1) (77 mg) dissolved in Et2O, was treated with 60 mg of
m-chloroperbenzoic acid. The reaction mixture was stirred at
room temperature for 7 h, a solution of Na2SO3 10% w/v was
then added, and the mixture was extracted three times with
Et2O. The organic layer was washed with brine and dried over
anhydrous Na2SO4, and the solvent was evaporated under
reduced pressure. The reaction mixture was purified by
column chromatography to give 43 mg of caryophyllene oxide
(1) and 28 mg of a mixture of (4R,5R,8R)-4,5:8,13-diepoxy-
caryophyllane (3) and (4R,5R,8S)-4,5:8,13-diepoxycaryophyl-
lane (5) (yield 71%). The epimeric compounds 3 and 5 were
separated by HPLC using hexane/ethyl acetate (95:5) to give
11 mg (28%) of 3 and 5 mg (13%) of 5.
11.2 Hz, H-10â), 1.33 (1H, m, H-2R), 1.30 (1H, m, H-1), 1.23
(1H, m, H-6R), 1.15 (1H, m, H-2â), 1.11 (3H, s, H-12), 1.00 (3H,
s, H-13), 0.86 (1H, m, H-3â), 0.83 (3H, s, H-14), 0.82 (3H, s,
H-15); 13C NMR data, Table 1; EIMS m/z 238 [M]+ (0.1), 177
(4), 149 (6), 121 (20), 95 (30), 91 (11), 81 (52); HRMS 238.1939
(calcd for C15H26O2 238.1933).
Refer en ces a n d Notes
(1) Whealer, B. E. J . An Introduction to Plant Diseases; J ohn Wiley:
London, 1969; pp 187-188.
(2) Coley-Smith, J . R.; Verhoell, K.; J arvis, W. R. The Biology of Botrytis;
Academic Press: London, 1980; pp 42-63.
(3) Collado, I. G.; Herna´ndez-Gala´n, R.; Dura´n-Patro´n, R.; Cantoral J .
M., Phytochemistry 1995, 38, 647-650
(4) Collado I. G.; Herna´ndez-Gala´n, R.; Prieto, V.; Hanson, J . R.;
Rebordinos, Phytochemistry 1996, 41, 513-517.
(5) Rebordinos, L. G.; Cantoral, M. J .; Prieto, M. V.; Hanson, J . R.;
Collado, I. G. Phytochemistry 1996, 42, 383-387.
(6) Hanson, J . R. Pure Appl. Chem. 1981, 53, 1155-62.
(7) Abraham, W. R.; Ernst, L.; Arfmann, H. A. Phytochemistry 1990, 29,
757-763.
(8) Abraham, W. R.; Ernst, L.; Stump, B. Phytochemistry 1990, 29,
115-120.
Red u ction of Diep oxid es 3 a n d 5. Both compounds were
treated with a 1 M solution of LiAlH4 in Et2O. In each case,
the reaction mixture was stirred under an N2 atmosphere for
NP980104B