Caryophyllene biotransformation
117
EXPERIMENTAL
data are listed in Tables 1 and 2. MS (re~z): [M] ÷ 252.1724
(0.5%) (252A725 calc. for ClsH2403), 43 (100).
The microorganisms were precultivated at 27° and 100 r.p.m.
in 100 ml Erlenmeyer flasks containing 20 ml of the following
medium: 1% glucose, 1% universal peptone (Merck), 2% malt
extract and 0.3% yeast extract. After 48 hr, 10 #1 of caryophyl-
lene (1) dissolved in 10 #1 of DMF were added to the cultures.
After 24, 48 and 72 hr samples were taken and analysed as
follows: to 1 ml of culture broth 0.2 ml of EtOAc were added and
the mixture was shaken for 2 rain prior to centrifugation. 10/~1 of
the extract were developed on HPTLC plates with
CH2CI2-Me2CO (9:1) [17]. The spots were made visible by
spraying with anisaldehyde-H2SO4 in HOAc and heating to
110° for 1 rain. For biotransformations on a preparative scale the
microorganisms were grown in 5 × 100 ml flasks, transferred,
after 48 hr, into five 2 1 flasks containing 400 ml of the medium
and incubated for another period of 24 hr. The substrate
(0.3 g/flask dissolved in 0.4 ml of DMF) was then added asepti-
cally.
589nm 578nm 546nm 436nm 365nm
-13.2 ° -13.8 ° -15.4 ° -23.9 ° --32.0
(c 1.00, MeOH).
(1R,4R,5R,7R,9S,11 R)-4,5-Epoxy-8(14)-caryophyllene-7,12-
diol (7). Colourless oil. R: 0.47 (B). ~H and ~3CNMR data are
listed in Tables 1 and 2. MS (re~z): [M] + 252.1724 (0.5%)
252.1725 calc. for C15H2aO3), 43 (100).
589nm 578nm 546nm 436nm 365nm
[~]
(c 1.00).
-63.9 ° -66.7 ° -75.2 ° _123.2 ° -180.6 °
(1R,4S,5R,9S, 11R)-4,5-Epoxy-8(14)-caryophyUene-12,15-diol
(8). Colourless viscous oil. R/0.42 (B). 1H and 13C NMR data are
listed in Tables 1 and 2. MS (re~z):[M-Me] + 237.1494 (1%)
(237.1491 calc. for C14H2103), 83 (82), 43 (100).
Biotransformation of 20 g caryophyllene was carried out in a
50 1 draft tube fermentor. The vessel was filled with 451 of the
above medium. After sterilization 1200 mt of a 48 hr preculture
was used as inoculum. Applying 120 r.p.m, the aeration was
satisfactory with 10 vols of air per 100 vols of culture, pH 5 was
obtained using a 15°/, soln of H3PO 4 and 15% soln of KOH.
Before adding substrate the culture was allowed to grow for a
total of 16 hr. Then 10 g of the substrate was added aseptically
and another 10g was administered 8 hr later. The curve of
conversion was followed by HPTLC and was terminated after
72 hr of contact.
Extraction and purification. Culture medium and mycelia were
separated by filtration and both extracted × 3 with EtOAc. The
solvent was evapd and the crude extract separated on Si-60
columns with a n-hexane-EtOAc gradient (changing from 19:1
to 1:1). When necessary the collected fractions were purified
further by prep. TLC.
The ~HNMR spectra were obtained at 400 MHz and the
3C NMR spectra at 75.5 MHz, CDC13 was the solvent if not
stated otherwise and TMS the int. standard [18]. IR spectra and
[~]D were measured in CHCI 3 unless otherwise stated. Mass
spectra were recorded with 70 eV. Mps: uncorr. TLC solvents: A
=CH2C12-Me2CO (4:1) or B=CH2C12-Me2CO (7:3). Bio-
transformation of 20 g caryophyllene (1) with Diplodia 9ossypina
ATCC 10936 in a 501 fermenter yielded, after 72 hr, 2 (65 mg), 3
(300 mg), 4 (900 mg), 5 (220 mg), 6 (180 mg), 7 (150 rag), 8 (60 mg),
9 (45 rag), l0 (35 rag), 11 (120rag), 12 (60mg), 13 (95 rag), 14
(25 mg), and 15 (15 rag).
589nm 578nm 546nm 436nm 365nm
[~] -21.5 ° -33.0 ° -36.7 ° -55.7 ° -72.4 °(el'00)"
(1R,4R,5R,7R,9S, I 1R)-4,5; 8,14-Diepoxycaryophyllane-7,12-
diol (9). Colourless crystals, mp 209°. R: 0.34 (B). 1H and
I3CNMR data are listed in Tables 1 and 2. MS (re~z):
[M-Me] + 237.1492 (2%) (237.1491 calc. for C14H2103), 161
(4), 55 (78), 43 (100).
589nm 578nm 546nm 436nm 365nm
-46.3 ° -48.6 ° -55.4 ° -96.9 ° -158.1 °
(c 1.00, MeOH).
(1R,4S,5R,8R,9S,11 R)-4,8-Epoxycaryophyllane-5,12,14-triol
(10). Colourless oil. Rf 0.11 (B). ~HNMR: 63.62 (1H, dd, J=8
and 3 Hz, 5-H), 3.39 (2H, s, t2-H), 3.28 (1H, d, J= 10 Hz, 14-H),
3.20 (1H, d, J=10Hz, 10'-H), 1.22 (3H, s, 15-H), 1.08 (3H, s,
13-H).
589nm 578nm 546nm 436nm
[ct]
(c 0.46).
-9.4 ° -9.6 ~ -10.7 ° -16.3 °
(1R,4R,5R,9S, 11R)-4,5-Epoxy-8-oxo-14-norcaryophyllan-12-
ol (11). Colourless oil. R: 0.44 (A). XH and 13CNMR data are
listed in Tables 1 and 2.
(1R,4R,5R,8R,9S,11 R)-4,5-Epoxycaryophyllane-12,14-diol
(12). Colourless oil. R: 0.29 (B). ~H and 13CNMR data are listed
in Tables 1 and 2. MS (m/z): [M-CHzOH] + 223.1698 (6%)
(223.1698 calc. for C14H2302), 43 (100).
(1R,4R,5R,9S,11R )-4,5-Epoxy-caryophyll-8(14)-en-12-oic acid
(4). Colourless viscous oil. R: 0.59 (A). 1H and ~3C NMR data are
listed in Tables 1 and 2. MS (m/z): [M-Me] + 223.1691 (5%)
(223.1698 calc. for C1,~H2302).
589nm 578nm 546nm 436nm 365nm
[~3
(c 1.00).
--36.3° --37.9° -42.7 ° -72.0 ° 112.7°
(1R,4R,5R,8S,9S, 11R)-4,5-Epoxycaryophyllane- 12,14-diol (13).
Colourless oil. R: 0.22 (B). 1H NMR: c53.31 (2H, m, 14-H), 3.27
(2H, m, 12-H), 3.06 (1H, dd, J = 10 and 5 Hz, 5-H), 1.29 (3H, s,
15-H), 0.99 (3H, s, 13-H). 13CNMR data are listed in Table 2.
MS (m/z): [M--CH2OH] + 223.1700 (3%) (223.1698 calc. for
C14H2302), 43 (100).
589nm 578nm 546nm 436nm 365nm
[~]
(c 1.00).
--55.5° --57.6° --64.6° _102.8 ° --145.1°
(1R,4R,5R,9S, 11R)-4,5-Epoxy- 12-nor-8(14)-caryophyllene-11-
ol (5). Colourless viscous oil. R: 0.53 (A). 1H NMR: 35.00 (IH,
brs, 14-H),4.89(lH, brs, 14'-H), 2.91 (1H, dd, J= lOand 4 Hz, 5-
H), 1.25 (3H, s, 15-H), 1.19 (3H, s, 13-H). 13C NMR data are listed
in Table 2. MS (re~z):207.1385 (1%) [M -Me] + (207.1385 calc.
for C13H1002), 43 (100).
589nm 578nm 546nm 436nm
[~] -44.2 ° -45.4 ° -51.4 ° -85.8 ° (c 1.00).
(1R,9S, 11R)-4,11-Bis(hydroxyrnethyl)-4,11-dimethyl-8-methyl-
ene-6-oxo-bicyclo[6.2.0]decane (14). Colourless oil. R: 0.68 (A).
IR Vmaxcm-X: 3640, 1710. ~H and 13CNMR data are listed in
Tables 1 and 2.
589nm 578nm 546nm 436nm 365nm
[~] -36.3 ° -37.8 ° -42.4 ° -67.0 ° -93.6 ° (c 1.00).
(1R,4R,8R,9S, 11R)-4,11 -Dimethyl-8-formyl-5-hydroxymethyl-
tricyclo[6.3.0.O]l"glundecan-5-ol (15). Colourless viscous oil. Rf
(1R,4R,5R,6R,9S, 11 R)-4,5-Epoxy-8( 14)-caryophyUene-6,12-
diol (6). Colourless viscous oil. R: 0.31 (B). 1H and 13CNMR
Abraham, Wolf-Rainer
