Microbial transformation of sesquiterpene lactones by the fungi Cunninghamella echinulata and Rhizopus oryzae

Article abstract of DOI:10.1021/np980520w

Incubations of the fungi Cunninghamella echinulata and Rhizopus oryzae with the sesquiterpene lactones (+)-costunolide (1), (+)-cnicin (2), (+)- salonitenolide (3), (-)-dehydrocostuslactone (4), (-)-lychnopholide (5), and (-)-eremantholide C (6) were performed. Incubation of 1 with C. echinulata afforded Δ(11(13))-dihydrogenation and Δ(1(10))-epoxidation products (7- 10). C. echinulata also hydrolyzed the side chain of 2, and transformed 4 into (+)-11α,13-dihydrodehydrocostuslactone (12), a new natural product. R. oryzae converted 4 into both Δ(11(13))-dihydrogenation and Δ(10(14))- epoxidation products (16 and 17). Both fungi transformed 5 into (-)-16-(1- methyl-1-propenyl)eremantholanolide (13), providing experimental evidence for the biosynthesis of the eremantholide hemiketal unit. Compounds 3 and 6 were not metabolized by either fungus under the test conditions.

Full text of DOI:10.1021/np980520w

726
J . Nat. Prod. 1999, 62, 726-729
Micr obia l Tr a n sfor m a tion of Sesqu iter p en e La cton es by th e F u n gi
Cu n n in gh a m ella ech in u la ta a n d Rh izopu s or yza e
Alejandro F. Barrero,*,† J . Enrique Oltra,† De´lio S. Raslan,‡ and Deˆnia A. Sau´de‡
Departamento de Quı´mica Orga´nica, Instituto de Biotecnologı´a, Facultad de Ciencias, Universidad de Granada,
Campus Fuentenueva s/ n, 18071-Granada, Spain, and Departamento de Qu´ımica, Instituto de Cieˆncias Exatas,
Universidade Federal de Minas Gerais, Av. Antoˆnio Carlos 6627, Belo Horizonte, MG, Brazil
Received November 18, 1998
Incubations of the fungi Cunninghamella echinulata and Rhizopus oryzae with the sesquiterpene lactones
(+)-costunolide (1), (+)-cnicin (2), (+)-salonitenolide (3), (-)-dehydrocostuslactone (4), (-)-lychnopholide
(5), and (-)-eremantholide C (6) were perfomed. Incubation of 1 with C. echinulata afforded ∆11(13)
-
dihydrogenation and ∆1(10)-epoxidation products (7-10). C. echinulata also hydrolyzed the side chain of
2, and transformed 4 into (+)-11R,13-dihydrodehydrocostuslactone (12), a new natural product. R. oryzae
converted 4 into both ∆11(13)-dihydrogenation and ∆10(14)-epoxidation products (16 and 17). Both fungi
transformed 5 into (-)-16-(1-methyl-1-propenyl)eremantholanolide (13), providing experimental evidence
for the biosynthesis of the eremantholide hemiketal unit. Compounds 3 and 6 were not metabolized by
either fungus under the test conditions.
As part of a program directed toward the enantiospecific
synthesis of natural sesquiterpene lactones with biological
activity,1,2 a microbiological alternative to improve the
regioselectivity and stereoselectivity of some chemical
reactions was considered. However, apart from biological
degradation of R-santonin, scant information about bio-
conversion of other sesquiterpene lactones was available.3
Therefore, microbiological transformation of some acces-
sible sesquiterpenoids (1-6), which can be useful as raw
materials for enantiospecific synthesis of bioactive lactones,
was investigated using the fungi Cunninghamella echinu-
lata and Rhizopus oryzae. The results from incubations
with (+)-costunolide4 (1), (+)-cnicin5 (2), (-)-dehydrocos-
tuslactone6 (4), and (-)-lychnopholide7 (5) are reported in
this paper. (+)-Salonitenolide5 (3) and (-)-eremantholide
C7 (6) were not transformed by either organism.
Incubation of C. echinulata with (+)-cnicin (2) afforded
(+)-salonitenolide5 (3). No hydrogenation products or myce-
lium growth inhibition were observed. Selective hydrolysis
of the (+)-cnicin side chain to (+)-salonitenolide is of
synthetic interest because 2 is a widespread metabolite,
abundant in several species of the Centaurea genus5 as well
as in Cnicus benedictus,13 and 3 is a raw material useful
for the synthesis of (+)-stoebenolide,2 (+)-dehydro-
melitensin,2 and (+)-vernolepin derivatives.1
When (-)-dehydrocostuslactone (4) was added to a 24-h
culture of C. echinulata, complete inhibition of the myce-
lium growth was observed. Compound 4 was recovered
unchanged after the incubation period. However, incuba-
tion of a more developed biomass (48 h) with 4 provided a
guaianolide not previously described and identified as (+)-
1
11R,13-dihydrodehydrocostuslactone (12). In the H NMR
spectrum, a methyl doublet assignable to H-13 and signals
for only four olefinic protons were evident, indicating
saturation of the ∆11(13) double bond of 4. The coupling
constant between H-11 and H-7 (J ) 7.8 Hz) indicated the
â arrangement of the C-13 methyl group of 12. The NOE
observed between H-13 and H-6 confirmed this stereo-
chemistry.
Resu lts a n d Discu ssion
Incubations with C. echinulata were performed by add-
ing the exogenous substrate to a 24- or a 48-h culture.
When (+)-costunolide (1) was added to a 24-h culture,
strong inhibition of mycelium growth was observed. There-
fore, a second incubation was carried out using a more
developed biomass (48 h). Under 24-h. conditions, costuno-
lide was not completely transformed, but a hydrogenation
product, identified as (+)-11â,13-dihydrocostunolide4 (7),
was isolated. Incubation of 1 with 48-h biomass led to three
oxidation products, the well-known 1â-hydroxyeudesmano-
lides (+)-santamarine8 (8), (+)-reynosin9 (9), and (+)-1â-
hydroxyarbusculin A10 (10). Absolute stereochemistry of
(+)-costunolide (1) has been established.11 Therefore, trans-
formation of 1 into 8-10 indicates that the absolute
configuration of (+)-santamarine, (+)-reynosin, and (+)-
1â-hydroxyarbusculin A is as shown. Formation of 8-10
can be explained by enzymatic epoxidation of 1 to 1â,10R-
epoxicostunolide (11), and subsequent electrophilic opening
of the epoxide with concomitant rearrangement to the
eudesmanolide skeleton, as presumably occurs in plant
biogenesis of 1â-hydroxyeudesmanolides.12
Incubation of a 24-h mycelium of C. echinulata with (-)-
lychnopholide (5) afforded an eremantholide identified as
(-)-16-(1-methyl-1-propenyl)eremantholanolide14 (13). Bio-
genesis of 13 in plants, by reductive cyclization of the
corresponding 8R-angelate, was proposed previously,14,15
along the same line of an earlier hypothesis about the
biosynthesis of closely related structures called ereman-
tholides A and B.16 However, apart from the joint occur-
rence of 5 and 13 in plants of the Lychnophora, Piptolepis,
and Proteopsis genera,17 no other evidence confirming the
biogenesis of 13 has been reported so far. Transformation
of 5 into 13 by Cunninghamella lends experimental support
to the above-mentioned biogenetic hypothesis. To obtain
information on the mechanism involved in the intramo-
lecular cyclization to the hemiketal unit contained in 13,
chemical treatment of 5 with hydrides was tested. Reaction
with NaBH4 gave the alcohol 14, and treatment with Bu3-
SnH only causes isomerization of the lateral chain, leading
to 15.18 Formation of eremantholide 13 was not detected
in any case. Despite chemical hydride failure to mimic the
* To whom correspondence should be addressed. Tel. 34 958 24 33 18.
Fax: 34 958 24 84 37. E-mail: afbarre@goliat.ugr.es.
† Universidad de Granada, Spain.
‡ Universidade Federal de Minas Gerais, Brazil.
10.1021/np980520w CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/15/1999

Biotransformations of Sesquiterpene Lactones
J ournal of Natural Products, 1999, Vol. 62, No. 5 727
enzymatic cyclization to the hemiketal unit of 13, the
hydrogenating ability toward the R-methylene-γ-lactone
group, showed by the 24-h mycelium of Cunninghamella,
must be involved in the biogenesis of 13. In this way, when
5 was added to a 48-h mycelium (which lacks any hydro-
genating ability), no biotransformation products were
detected after incubation, and the exogenous substrate was
recovered unchanged.
Incubations of Rhizopus oryzae with lactones 4 and 5
were carried out by adding each lactone to a 48-h culture.
Microbial transformation of (-)-dehydrocostuslactone (4)
afforded two products identified as (+)-11â,13-dihydrode-
hydrocostuslactone (mokko lactone, 16)19 and (+)-11â,13-
dihydro-10,14-epoxydehydrocostuslactone (17). High-reso-
lution NMR data for 16 are included in the Experimental
Section, to facilitate further differentiation from its C-11
epimer (12). Previously, (-)-dehydrocostuslactone (4) and
(+)-mokko lactone (16) were chemically correlated with (-)-
R-santonin,20 whose absolute stereochemistry was deter-
mined by X-ray analysis.21 Thus, the absolute configuration
of 17 was established as shown. Compound 17 was previ-
ously found in the medicinal plant Vladimiria souliei, and
its structure was proposed on the basis of 1H NMR data.22
Biological synthesis of 17, from 4, confirms its structure
and the absolute stereochemistry of the (+)-enantiomer.
Unfortunately, optical rotation of the V. souliei metabolite
was not reported. For synthetic purposes, C. echinulata and
R. oryzae constitute a fungal pair useful for selecting a
given stereochemistry in ∆11(13)-hydrogenation processes on
4 and, presumably, other guaianolides. Microbial trans-
formation of lychnopholide (5) by R. oryzae, afforded the
same eremantholide (13) isolated from the Cunninghamella
culture.
Epoxidation reactions and hydrogenation of the C-11-
C-13 double bond seem to be the most common processes
in microbial transformation of sesquiterpene lactones by
the filamentous fungi C. echinulata and R. oryzae. Cyto-
toxic activity of sesquiterpene lactones lies chiefly in their
R-methylene-γ-lactone group.23 Hence, regioselective hy-
drogenation at ∆11(13) might be a defense mechanism of
these organisms against the fungistatic activity of some
lactones. From both the biochemical and the synthetic
points of view, cyclization to the hemiketal unit of ereman-
tholides is the most important feature of the biotransfor-
mations presented in this paper, due to its biogenetic
significance and the difficulties encountered achieving it
by chemical methods.
Aldrich, Merck, or Fluka and were used as received. All
solvents were purified and dried following standard proce-
dures.25
Exp er im en ta l Section
Sesqu iter p en e La cton es. Lactones 1 and 4 were isolated
from Costus Resinoid, a commercially available extract from
costus roots (Saussurea lappa), purchased from Pierre Chauvet
S. A., Seillans, France. The extract (5 g) was column chro-
matographed over 5% AgNO3-Si gel (150 g). Elution yielded
4 (0.92 g, hexane-t-BuOMe, 80:20) and 1 (0.47 g, hexane-t-
BuOMe, 60:40). Cnicin (2) was obtained from Centaurea
malacitana, as previously described.5 Salonitenolide (3) was
obtained by selective saponification of the side chain of 2.5
Compounds 5 and 6 were isolated from aerial parts of
Lychnophora trichocarpha.7 Physical data of these lactones,
including optical rotation, were consistent with published data.
Micr oor ga n ism s a n d Cu ltu r e Med ia . Cunninghamella
echinulata (NRRL3655) and Rhizopus oryzae (ATCC11145)
were purchased from Coleccio´n Espan˜ola de Cultivos Tipo
(CECT), Universidad de Valencia, Valencia (Spain). Stock
cultures of C. echinulata were stored on agar slants (potato
dextrose agar plus 2% yeast extract). R. oryzae was mantained
on agar slants prepared with a mixture of glucose (20 g),
L-asparagin‚H2O (2 g), KH2PO4 (5 g), MgSO4‚7H2O (0.5 g),
CaCl2 (0.028 g), tiamine‚HCl (0.001 g), citric acid‚H2O (0.002
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were determined on a Perkin-Elmer 141 polarimeter. IR
spectra were obtained as liquid films, between NaCl plates,
on a 983 G Perkin-Elmer apparatus. NMR spectra were
recorded on Bruker AM 300 and Bruker ARX 400 spectrom-
eters. Chemical shifts are reported in parts per million (δ)
relative to TMS, and coupling constants (J ) are in Hertz. 13C
NMR assignments are tentative unless otherwise stated.
Carbon substitution degrees were established by DEPT mul-
tipulse sequence. LRMS were determined on a 5988A Hewlett-
Packard instrument, and HRMS were measured on an Au-
tospec-Q VG-Analytical (FISONS) mass spectrometer. Thin-
layer chromatography (TLC) was performed on precoated 0.25-
mm thick Merck plates of Si gel 60 F254, using a 7%
phosphomolybdic acid solution (EtOH) to visualize spots.
Column chomatography was carried out on Si gel, eluting with
hexane-t-BuOMe mixtures of increasing polarity, unless
otherwise stated; and flash chromatography was performed
as described previously.24 Reagents were purchased from

728 J ournal of Natural Products, 1999, Vol. 62, No. 5
Barrero et al.
g), Fe(NO3)3‚9H2O (0.0015 g), ZnSO4‚7H2O (0.001 g), MnSO4‚
H2O (300 µg), CuSO4‚5H2O (50 µg), Na2MoO4‚2H2O (50 µg),
yeast extract (1 g), and agar (15 g) in tap water, q. s. 1 L.
Liquid culture medium for C. echinulata contained glucose (20
g), yeast extract (5 g), peptone (5 g), NaCl (5 g), and K2HPO4
(5 g) in 1 L of distilled H2O. The culture medium used for R.
oryzae was the broth stock medium.
Biotr a n sfor m a tion of Lych n op h olid e (5). (-)-Lychno-
pholide (430 mg) was evenly distributed among 23 flasks
containing 24-h-old biomass (A) and 20 flasks containing 48-h
biomass (B). Biotransformations were stopped either after 4
days (A) or after 64 h (B). Extracts A (289 mg) and B (365 mg)
were obtained. Column chromatography of extract A afforded
5 (27 mg, hexane-EtOAc 85:15) and eremantholide 13 (14 mg,
hexane-EtOAc 85:15). Column chromatography of extract B
yielded 9 mg of 5, and no eremantholides were detected.
Com p ou n d 13: 1H NMR data matched those described by
Zdero et al.;14 13C NMR (CDCl3, 75 MHz) δ 205.6 (s, C-1), 187.2
(s, C-3), 176.0 (s, C-12), 175.8 (s, C-1′), 134.8 (d, C-5), 130.1 (s,
C-4), 127.9 (d, C-2′), 108.1 (s, C-16), 104.6 (d, C-2), 90.2 (s,
C-10), 81.5 (d, C-6), 78.9 (d, C-8), 62.0 (d, C-7), 61.3 (s, C-11),
43.7 (t, C-9), 22.0 (q, C-4′), 21.4 (q, C-13), 20.6 (q, C-14), 20.4
(q, C-15), 15.4 (q, C-3′).
In cu ba tion s w ith Cu n n in gh a m ella ech in u la ta . To
prepare a homogeneous suspension of biomass, C. echinulata
was first grown in two 500-mL culture flasks, each containing
100 mL of liquid medium, at 26 °C, on a rotatory shaker (200
rpm), for 48 h. The biomass in suspension obtained was
innoculated (2 mL per flask) in several 500-mL flasks, each
containing 100 mL of fresh liquid medium. The inoculated
flasks were submitted to a first stage of incubation on a
rotatory shaker (200 rpm), at 26 °C, either for 24 h (condition
A) or for 48 h (condition B). After the first stage, the exogenous
substrates were evenly distributed (10 mg in 0.2 mL of THF
per flask) among the 24- and/or 48-h-old cultures, and incu-
bated at 26 °C, on a rotatory shaker (200 rpm), for an
additional time (second stage of incubation). During the second
stage, aliquots from cultures were taken daily and analyzed
by TLC and 1H NMR in order to determine the degree of
transformation of substrates. In all experiments, two control
flasks without biomass (for substrate stability) and two flasks
without exogenous substrate (for endogenous metabolites)
were used.
Biotr a n sfor m a tion of Costu n olid e (1). (+)-Costunolide
(200 mg) was evenly distributed among 20 flasks containing
24-h-old first-stage biomass. Incubation was stopped after 78
h of the second stage. Culture broth was filtered, and the
filtrate was extracted with EtOAc. The organic solvent was
removed, and the residue (138 mg) was chromatographed on
Si gel. Compounds 1 (34 mg, hexane-t-BuOMe 80:20) and 7
(6 mg, hexane-t-BuOMe 80:20) were obtained. Another 200
mg of 1 was dissolved in THF (4 mL) and evenly distributed
among 20 flasks containing 48-h-old first-stage biomass.
Incubation was stopped after 54 h, and the organic metabolites
were extracted from the medium. Column chromatography of
the organic extract (190 mg) afforded 8 (9 mg, hexane-t-
BuOMe 80:20), 9 (15 mg, hexane-t-BuOMe 70:30), and 10 (21
mg, hexane-t-BuOMe 30:70).
Biotr a n sfor m a tion of Cn icin (2). Incubation of (+)-cnicin
(200 mg) with 24-h-old biomass was carried out as described
above for costunolide. Biotransformation was stopped after 54
h. Column chromatography of the organic extract (181 mg)
yielded 3 (11 mg, CHCl3-Me2CO 80:20).
Biotr a n sfor m a tion of Deh yd r ocostu sla cton e (4). (-)-
Dehydrocostuslactone (220 mg) was evenly distributed be-
tween two flasks containing 24-h-old biomass (condition A) and
20 flasks containing 48-h-old biomass (condition B). Biotrans-
formations were stopped either after 4 days (condition A) or
after 2 days (condition B). Then, organic extracts A (35 mg)
and B (400 mg) were obtained. 1H NMR spectrum and TLC
analysis showed that extract A was mainly formed by com-
pound 4. Column chromatography of extract B afforded 4 (29
mg, hexane-t-BuOMe 90:10) and 12 (6 mg, hexane-t-BuOMe
85:15).
Red u ction of 5 w ith Na BH4. Lychnopholide (5) (200 mg,
0.56 mmol), in 5 mL of MeOH, was chilled down to 0 °C, and
NaBH4 was added. The solution was stirred at 25 °C for 1.5 h
after the hydride addition. Then, 20 mL of H2O were added,
and the mixture was neutralized with AcOH and extracted
with EtOAc. Flash chromatography (hexane-EtOAc 70:30) of
the organic extract afforded 30 mg of 14 as a gum: [R]25
D
-22.6° (c 1.83, CHCl3); IR (film) νmax 3426, 1763, 1708 cm-1
;
1H NMR (CDCl3, 400 MHz) δ 6.22 (1H, dd, J ) 2.4, 0.8 Hz,
H-13a), 6.16 (1H, qq, J ) 7.2, 1.5 Hz, H-3′), 5.65 (1H, dd, J )
2.1, 0.8 Hz, H-13b), 5.56 (1H, dq, J ) 4.4, 1.7 Hz, H-5), 5.37
(1H, m, H-6), 5.18 (1H, dt, J ) 10.6, 3.5 Hz, H-8), 4.38 (1H,
dd, J ) 10.7, 6.4 Hz, H-3), 4.16 (1H, m, H-7), 4.12 (1H, dd, J
) 9.4, 6.4, H-1), 2.54 (1H, dt, J ) 12.7, 6.4 Hz, H-2), 2.27 (1H,
dd, J ) 16.3, 3.5 Hz, H-9), 2.02 (3H, dq, J ) 7.2, 1.5 Hz, H-4′),
1.96 (3H, quin, J ) 1.5 Hz, H-5′), 1.75 (3H, t, J ) 1.7 Hz, H-15),
1.40 (3H, s, H-14); 13C NMR (CDCl3, 75 MHz) δ 170.2 (s, C-12),
166.9 (s, C1′), 140.0 (d, C-3′), 139.3 (s, C-4), 138.0 (s, C-11),
127.4 (d, C-5), 127.1 (s, C-2′), 124.5 (t, C-13), 82.5 (s, C-10),
79.7 (d, C-1), 77.2 (d, C-3), 76.6 (d, C-6), 74.3 (d, C-8), 49.3 (d,
C-7), 37.7 (t, C-2), 34.4 (t, C-9), 27.7 (q, C-14), 23.9 (q, C-15),
20.7 (q, C-5′), 15.9 (q, C-14); NMR data were assigned with
1
the aid of 2D NMR experiments H-1H homonuclear correla-
tion (COSY), 1H-13C direct (HETCOR), and long-range (HMBC)
heteronuclear correlations; CIMS m/z 363 [M + H]+ (1), 263
(3), 101 (81), 83 (50), 69 (28), 55 (51), 43 (100); HRFABMS
m/z 385.1628 (calcd for C20H26O6Na, 385.1627).
Tr ea tm en t of 5 w ith Bu 3Sn H. A solution of Bu3SnH (0.09
mL, 0.031 mmol) and 2,2′-azobisisobutyronitrile (1 mg) in
toluene (1 mL) was added to a stirred solution of 5 (0.1 g, 0.28
mmol) in toluene (2 mL) at 80 °C under argon. The reaction
mixture was stirred for 4 h. The solvent was removed, and
the residue was flash chromatographed (hexane-EtOAc 80:
20) affording 43 mg of 15. IR, MS, and 1H NMR data of 15
matched those of a tiglate analogue of lychnopholide found in
Eremanthus bicolor.18
In cu ba tion s w ith Rh izopu s or yza e. R. oryzae was first
grown in two 500-mL flasks, each containing 100 mL of liquid
medium, on a rotatory shaker (200 rpm), at 28 °C, for 48 h.
The broth culture obtained, containing the developed biomass
in suspension, was inoculated (2 mL per flask) in 500-mL
flasks, each containing 100 mL of fresh liquid medium, and a
first stage of incubation was carried out in a rotatory shaker
(200 rpm), at 28 °C, for 48 h. Then, the exogenous substrates
were evenly distributed (30 mg in 0.2 mL of THF per flask)
among the first-stage cultures, and a second stage of incuba-
tion was carried out at 28 °C on a rotatory shaker. Monitoring
of the second-stage cultures was performed by TLC and 1H
NMR analysis of aliquot samples taken daily. In all the
experiments, two flasks without biomass (control for sub-
strate’s chemical stability) and two flasks without exogenous
substrate (control for endogenous metabolites) were used.
Biotr a n sfor m a tion of Deh yd r ocostu sla cton e (4). Lac-
tone 4 (450 mg) was evenly distributed among 15 flasks
containing 48-h-old biomass. Biotransformation was stopped
after 4 days. Broth culture was filtered, and the filtrate was
extracted with EtOAc. The organic solvent was removed, and
a crude residue (800 mg) was obtained. Column chromatog-
raphy of the residue afforded 4 (52 mg, hexane-t-BuOMe 90:
Com p ou n d 12: oil; [R]25 +13.7° (c 0.32, CHCl3); IR (film)
D
ν
max 1773 cm-1; 1H NMR (CDCl3, 400 MHz) δ 5.19 (1H, d, J )
2.1 Hz, H -15a), 5.05 (1H, d, J ) 2.0 Hz, H-15b), 4.86 (1H, br
s, H-14a), 4.77 (1H, br s, H-14b), 4.02 (1H, t, J ) 9.6 Hz, H-6),
2.86 (2H, m, H-1, H-5), 2.67 (1H, dq, J ) 7.8, 7.8 Hz, H-11),
2.49 (1H, br dt, J ) 12.0, 3.0 Hz, H-9â), 1.17 (3H, d, J ) 7.8
Hz, H-13); NOE-dif experiments, proton irradiated (NOEs
observed) H-15a (H-15b, H-6), H-15b (H-15a), H-14a (H-14b,
H-9â), H-14b (H-14a), H-6 (H-15a, H-13), H-13 (H-11, H-6);
13C NMR (CDCl3, 75 MHz) δ 152.0 (s, C-4), 150.0 (s, C-10),
111.8 (t, C-14), 109.5 (t, C-15), 85.3 (d, C-6), 52.3 (d, C-7), 47.2
(d, C-1), 45.0 (d, C-5), 39.5 (d, C-11), 37.6 (t, C-8), 32.6 (t, C-9),
29.8 (t, C-2), 28.9 (t, C-3), 11.5 (q, C-13); CIMS m/z 233 [M +
H]+ (41), 187 (35), 159 (100), 157 (17), 81 (21), 57 (24), 55 (22),
43 (36); HRFABMS m/z 255.1361 (calcd for C15H20O2Na,
255.1361).

Biotransformations of Sesquiterpene Lactones
J ournal of Natural Products, 1999, Vol. 62, No. 5 729
(4) Rao, A. S.; Kelkar, G. R.; Bhattacharyya, S. C. Tetrahedron 1960, 9,
275-283.
(5) Barrero, A. F.; Oltra, J . E.; Rodr´ıguez, I.; Barraga´n, A. Fitoterapia
1995, 66, 227-230.
10), 16 (293 mg, hexane-t-BuOMe 90:10), and 17 (5 mg,
hexane-t-BuOMe 60:40).
1
Com p ou n d 16: H NMR (CDCl3, 300 MHz) δ 5.20 (1H, d,
J ) 2.0 Hz, H-15a), 5.03 (1H, d, J ) 2.0 Hz, H-15b), 4.89 (1H,
br s, H-14a), 4.78 (1H, br s, H-14b), 3.92 (1H, t, J ) 10.0 Hz,
H-6), 2.88 (1H, dt, J ) 8.0, 5.0 Hz, H-1), 2.79 (1H, br dd, J )
10.0, 8.0 Hz, H-5), 2.49 (1H, br dt, J ) 12.0, 3.0 Hz, H-9â),
2.21 (1H, dq, J ) 11.0, 8.0 Hz, H-11), 1.23 (3H, d, J ) 8.0 Hz,
H-13); NOE-dif experiments, proton irradiated (NOEs ob-
served) H-15a (H-15b, H-6), H-15b (H-15a), H-14a (H-14b,
H-9â), H-14b (H-14a, H-6), H-6 (H-15a, H-14b, H-11); 13C NMR
(CDCl3, 75 MHz) δ 179.5 (s, C-12), 151.8 (s, C-4), 150.0 (s,
C-10), 111.9 (t, C-14), 109.3 (t, C-15), 85.4 (d, C-6), 52.0 (d,
C-7), 50.0 (d, C-1), 47.2 (d, C-5), 42.2 (d, C-11), 37.7 (t, C-8),
32.6 (t, C-9), 30.3 (t, C-2), 29.1 (t, C-3), 13.3 (q, C-13).
(6) Mathur, S. B.; Hiremath, S. V.; Kulkarne, G. H.; Kelkar, G. R.;
Battacharyya, S. C. Tetrahedron 1965, 21, 3575-3590.
(7) Oliveira, A. B.; Sau´de, D. A.; Perry, K. S. P.; Duarte, D. S.; Raslan,
D. S.; Boaventura, M. A. D.; Chiari, E. Phytotherapy Res. 1996, 10,
292-295.
(8) Romo de Vivar, A.; J ime´nez, H.Tetrahedron 1965, 21, 1741-1745.
(9) Yoshioka, H.; Renold, W.; Fischer, N. H.; Higo, A.; Mabry, T. J .
Phytochemistry 1970, 9, 823-832.
(10) Clark, A. M.; Hufford, C. D. J . Chem. Soc., Perkin 1 1979, 3022-
3028.
(11) Bovill, M. J .; Cox, P. J .; Cradwick, P. D.; Guy, M. H. P.; Sim, G. A.;
White, D. N. J . Acta Crystallogr., Sect. B 1976, 32, 3203-3209.
(12) Barrero, A. F.; Oltra, J . E.; Morales, V.; AÄ lvarez, M. J . Nat. Prod.
1997, 60, 1034-1035.
(13) Connolly, D.; Hill, R. A. Dictionary of Terpenoids; Chapman and
Hall: London, 1991; p 231.
Com p ou n d 17: [R]25 +21.0° (c 0.19, CHCl3), 13C NMR
D
(CDCl3, 75 MHz) δ 151.6 (s, C-4), 108.8 (t, C-15), 84.6 (d, C-6),
58.0 (s, C-10), 50.8 (d, C-7), 50.0 (t, C-14), 49.0 (d, C-1), 45.4
(d, C-5), 42.2 (d, C-11), 36.0 (t, C-8), 32.2 (t, C-9), 28.3 (t, C-2),
24.7 (t, C-3), 13.4 (q, C-13); HRFABMS m/z 271.1311 (calcd
for C15H20O3Na, 271.1310).
Biotr a n sfor m a tion of Lych n op h olid e (5). Compound 5
(450 mg) was evenly distributed among 15 flasks containing
48-h-old biomass. Biotransformation was stopped after 4 days.
Culture filtrate was extracted with EtOAc. Column chroma-
tography of the organic extract (300 mg) yielded 5 (15 mg,
hexane-EtOAc 80:20) and 1314 (10 mg, hexane-EtOAc 85:
15).
(14) Zdero, C.; Bohlmann, F.; Robinson, H.; King, R. M. Phytochemistry
1981, 20, 739-741. In this report, the structure of lychnopholide (5),
the putative biogenetic precursor of 13, was postulated to have a 6R-
ester side chain with lactone ring closure to the 8R-position. This
proposal was revised a year later.15
(15) Herz, W.; Goedken, V. L. J . Org. Chem. 1982, 47, 2798-2800. Lactone
ring closure to the 6R-position of lychnopholide, proposed in this
paper, is suported by the microbial transformation of 5 into 13.
(16) Le Quesne, P. W.; Levery, S. B.; Menachery, M. D.; Brennan, T. F.;
Raffauf, R. F. J . Chem. Soc., Perkin 1 1978, 1572-1580.
(17) Brown, D. S.; Paquette, L. A. Heterocycles 1992, 34, 807-834.
(18) Bohlmann, F.; Zdero, C.; King, R. M.; Robinson, H. Phytochemistry
1980, 19, 2663-2668. In this report, the structure of 15 was
postulated to have a 6R-tiglate side chain with lactone ring closure
to the 8R-position. The chemical correlation between
5 and 15
indicates a lactone ring closure to the 6R-position with an 8R-ester
side chain, confirming the proposal of Herz and Goedken.15
(19) Hikino, H.; Meguro, G.; Takemoto, T. Chem. Pharm. Bull. 1964, 12,
632-634.
(20) Ando, M.; Ono, A.; Takase, K. Chem. Lett. 1984, 493-496.
(21) Asher, J . D. M.; Sim, G. A. J . Chem. Soc. 1965, 6041-6055.
(22) Tan, R. X.; J akupovic, J .; Bohlmann, F.; J ia Z. J .; Schuster, A.
Phytochemistry 1990, 29, 1209-1212.
(23) Hoffmann, H. M. R.; Rabe, J . Angew. Chem., Int. Ed. Engl. 1985, 24,
94-110.
Ack n ow led gm en t. Our thanks go to J oaˆo E. Guimaˆraes
for his collaboration, to Martin J . Keane for his contribution
in the translation into English, to the Spanish CICYT for the
Research Program PB95/1192, and to the CNPq (Brazil) for
the grant provided to Deˆnia A. Sau´de.
Refer en ces a n d Notes
(1) Barrero, A. F.; Oltra, J . E.; Barraga´n, A. Tetrahedron Lett. 1995, 36,
311-314.
(24) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923-2925.
(25) Casey, M.; Leonard, J .; Lygo, B.; Procter, G. Advanced Practical
Organic Chemistry: New York, 1990.
(2) Barrero, A. F.; Oltra, J . E.; AÄ lvarez, M. Tetrahedron Lett. 1998, 39,
1401-1404.
(3) Lamare, V.; Furstoss, R. Tetrahedron 1990, 46, 4109-4132.
NP980520W