Enzyme-catalysed transformations of ent-kaurane diterpenoids

Article abstract of DOI:10.1002/ejoc.200400862

Several acetyl derivatives of linearol, atractyligenin and atractylitriol were obtained through enzyme-catalysed acetylation and deacetylation reactions. In most reactions lipases showed regio- and stereoselective behaviour, allowing a family of novel compounds to be prepared. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005.

Full text of DOI:10.1002/ejoc.200400862

FULL PAPER
Enzyme-Catalysed Transformations of ent-Kaurane Diterpenoids
Leandro N. Monsalve,[a] Sergio Rosselli,[b] and Maurizio Bruno,[b]Alicia Baldessari*[a]
Keywords: Enzyme catalysis / Acetylation / Deacetylation / Diterpenoids / ent-Kauranes
Several acetyl derivatives of linearol, atractyligenin and
atractylitriol were obtained through enzyme-catalysed acety-
lation and deacetylation reactions. In most reactions lipases
showed regio- and stereoselective behaviour, allowing a
family of novel compounds to be prepared.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
derivatives suggested that the position of the ester group
dramatically influences the activity. We tried to synthesise
C-7 monoesters and also to perform selective esterifications
on foliol (2). Unfortunately, we were not able to achieve
significant results by conventional chemical methods.
In previous works we reported the regioselective acyl-
ation of natural products as polyhydroxy compounds by
treatment with alkyl carboxylates or carboxylic acids with
catalysis by enzymes in anhydrous organic solvents.[21–23]
We have also performed enzyme-catalysed deacylation reac-
tions on various steroids such as cholestanes, androstanes
and pregnanes, which demonstrated the ability of enzymes
to discriminate between acetoxy substituents in these sub-
strates, furnishing deacetylation reactions at different posi-
tions in the steroid skeleton. While Candida rugosa lipase
removed acetyl groups situated in ring A, Candida antarc-
tica lipase was preferentially active on substituents located
in ring D.[24,25]
Introduction
A large number of ent-kaurane diterpenoids,[1] biogenet-
ically derived from (–)-kaur-16-ene (1), have been isolated
from plants belonging to several genus of different families
(Euphorbiaceae, Apiaceae, Rutaceae, Lamiaceae and Com-
positae). In the last few years, a considerable number of
studies on their interesting biological properties have been
published. In fact, antimicrobial activity has been reported
for kauren-19-oic acid,[2–4] kaur-15-en-19-oic acid[5] and fo-
liol (2),[6] the last isolated from some species of Sideritis
(Lamiaceae) used in Spanish folk medicine. Some products
have shown good cytotoxic,[7–11] anti-HIV,[12,13] hypoten-
sive[14] and antiinflammatory[15,16] activities. Recently we re-
ported on the anti-HIV[17] and the antifeedant[18] activities
of linearol (3), a common metabolite of genus Sideritis,[19,20]
and of several semisynthetic derivatives.
With the aims of extending the biocatalytic approach to
other natural products and of preparing a family of deriva-
tives difficult to obtain through traditional chemical meth-
ods, in order to study the structure/activity relationship, we
decided to apply an enzymatic approach to diterpenoids
such as ent-kauranes. We found that lipases and esterases
from several sources were able to catalyse regioselective
acetylation and alcoholysis reactions.
The presence of two free hydroxy groups in linearol (3)
allowed us to synthesise a large number of C-3 mono- and
C-3,C-7 diesters. A comparison of the activities of these
Results and Discussion
In this report we describe the results obtained by en-
zyme-catalysed acetylation and deacetylation reactions per-
formed on three related ent-kauranes: linearol (3), atractyli-
genin (8) and atractylitriol (11).
[a] Departamento de Química Orgánica y UMYMFOR, Facultad
de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Ciudad Universitaria, Pabellón 2 Piso 3,1428 Buenos Aires, Ar-
gentina
Reactions with Linearol (3)
Fax: +54-11-4576-3385
E-mail: alib@qo.fcen.uba.ar
[b] Dipartimento di Chimica Organica, Università di Palermo,
Viale delle Scienze, 90128 Palermo, Italy
Fax: +39-091-596825
Enzymatic Acetylation
The presence of the two hydroxy groups in linearol (3)
makes this compound an interesting model for enzymatic
E-mail: bruno@dicpm.unipa.it
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200400862
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Enzyme-Catalysed Transformations of ent-Kaurane Diterpenoids
FULL PAPER
transformation, so we began investigating the behaviour of
lipases from several sources in the acetylation of this ent-
kaurane. We used lipases from yeasts [Candida antarctica
lipase A (CAL A) and B (CAL B), Candida rugosa lipase
(CRL)], from fungi [Lipozyme (LIP)], from mammals [por-
cine pancreatic lipase (PPL)], and from bacteria [Pseudomo-
nas sp. lipase (PSL)].
As we had obtained good results with ethyl acetate as
acylating agent in previous work,[21,26] we also tried it in the
enzymatic acetylation of linearol (3). Accordingly, linearol
(3) was dissolved in ethyl acetate, serving both as acylating
agent and as solvent. The enzyme was then added and the
suspension was shaken at 30 °C for 24 h (Scheme 1).
The six lipases from various sources (Table 1, Entries 1
to 6) afforded the same product, which had an Rf value
lower than linearol’s when analysed by TLC. This product
was fully identified by spectroscopic methods as sidol (4), a
monoacetylated isomeric compound of linearol produced
by the migration of the acetyl group from carbon 18 to car-
bon 3, as clearly shown by the downfield shifts of H-3β (δ
= 4.91, dd) and C-3 (δ = 74.56) and the upfield shifts of the
H-18 protons (δ = 3.31, d and 2.98, d) and of C-18 (δ =
1
64.20) in the H NMR and 13C NMR spectra (Table 5 and
Table 7).
The reaction was not complete. Actually, linearol was
present even at longer reaction times, and by flash
chromatography separation of the products we observed a
sidol/linearol ratio of 2:1. The migration was also produced
in the absence of ethyl acetate and with use of other sol-
vents such as acetonitrile or tetrahydrofuran (Entries 8 and
9), so the presence of an acetylating agent such as ethyl
acetate did not seem to be necessary. On the other hand,
no migration product was detected without the enzymatic
participation (Entry 7).
Scheme 1.
Afterwards, when sidol (4) was treated under the same
In order to explain the role of lipases in this migration,
enzymatic conditions as linearol, we also obtained the sidol/ we decided to perform a molecular modelling analysis of
linearol mixture in the same 2:1 ratio, suggesting that the the conformations of linearol and sidol. This should let us
two compounds reach an equilibrium situation, though one make an estimation of the activation energy involved in the
only achieved through enzyme-catalysed interconversion.
intramolecular migration.
Table 1. Lipase-catalysed acetylation of linearol (3).
Entry
Enzyme
Acylating agent
Solvent
T (°C)
Time (h)
Product yields (%)
4
5
1
2
3
4
5
6
7
8
CAL B
CAL A
PSL
CRL
PPL
LIP
–
CAL B
CAL B
CAL B
CAL B
CAL B
CAL B
CAL B
CAL B
CAL A
ethyl acetate
ethyl acetate
ethyl acetate
ethyl acetate
ethyl acetate
ethyl acetate
ethyl acetate
–
AcOEt
AcOEt
AcOEt
AcOEt
AcOEt
AcOEt
AcOEt
CH3CN
THF
THF
THF
THF
THF
33
33
33
33
33
33
33
33
33
33
55
33
55
55
55
55
24
24
24
24
24
24
168
72
67
66
65
66
64
65
–
66
67
62
64
33
35
58
59
62
–
–
–
–
–
–
–
–
–
–
–
43
55
28
24
–
9
–
96
10
11
12
13
14
15
16
vinyl acetate
vinyl acetate
isopropenyl acetate
isopropenyl acetate
isopropenyl acetate
isopropenyl acetate
isopropenyl acetate
168
168
72
72
72
72
72
CH3CN
tBuOH
THF
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L. N. Monsalve, S. Rosselli, M. Bruno, A. Baldessari
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Our goal was to verify whether the acetyl migration from These calculations correlate well with our experimental re-
carbon 18 to carbon 3 could happen spontaneously without sults and suggest that the thermal energy of the system is
assistance of the enzyme.
not enough to favour the migration. This fact could explain
Conformational searches on compounds 3 and 4 were the absence of migration without enzyme.
performed by use of the semiempirical method AM1 ap-
proach as implemented in Hyperchem 7.5.
The enzyme-catalysed migration could occur if it were
possible to generate an acyl–enzyme complex between lin-
To begin with, molecular dynamic experiments were per- earol or sidol and the active site of the lipase. It would be
formed on the substrates to estimate local minima. Relative necessary that each of these substrates could be accommo-
distances between reactive groups were monitored during dated into the catalytic pocket. From the reported analysis
these experiments. The conformations obtained by the of the structures of lipase-binding sites carried out by
above method were then minimised and stored as probable Pleiss,[28] it is known that lipases have large hydrophobic
structures for the compound. The global minimum for lin- acyl binding pockets, their lengths varying considerably (be-
earol showed rings A and B in a chair conformation, as tween 7.8 Å and 22 Å). The sizes of the acyl binding sites
reported in the literature.[27] It can be also observed that in of all the lipases used in this paper would allow both sub-
this case, the acetyl group at the 18-position adopts an ex- strates 3 and 4 to fit to accomplish the migration of the
tended conformation relative to the structure of the mole- acetyl group from one to the other. As one example, the
cule and the distance between the acetyl group carbonyl CAL B binding site lies in a pocket some 9.5 Å deep and
carbon at 18 and the hydroxy group at carbon 3 is 4.99 Å. 4.5 Å wide, with Ser105 of the catalytic triad at its base.[29]
Similarly, the distance between the hydroxy group at car-
In conclusion, it could be assumed that the conforma-
bon 18 of sidol and the acetyl substituent at carbon 3 is tional change produced by the anchorage of substrate to
3.64 Å (Figure 1 and Figure 2). Moreover, the analysis of the active site of the enzyme could help in the acetyl group
the relative distances in molecular dynamics experiments migration between linearol and sidol.
showed no interaction between the groups.
Since ethyl acetate was not a good acylating agent of
linearol, we tried the enzymatic acetylation with vinyl ace-
tate and isopropenyl acetate (Table 1, Entries 10–16). Mi-
gration of the linearol acetyl group was slower in tetra-
hydrofuran, so it was used as solvent with these two acylat-
ing agents. Although treatment with vinyl acetate only pro-
duced sidol (4), we did obtain a monoacetylation product
when using isopropenyl acetate in THF. Its 1H and 13C
NMR spectra (Table 5 and Table 7) showed downfield shifts
of H-3β (δ = 4.83, dd) and C-3 (δ = 74.3) and upfield shifts
of C-2 (δ = 23.0) and C-4 (δ = 40.5), allowing us to assign
the structure of 3-acetyl-linearol (5) to this compound. The
best results were obtained with CAL B as biocatalyst and
at 55 °C (Entry 13). Sidol was the side product in every
case, but the acetylation reaction was highly regioselective
and 3-acetyl-linearol (5) was obtained as the only diacetyl
compound.
Considering the possibility that 5 could also arise from
acetylation of sidol we used CAL A, which appears to acet-
ylate primary alcohols, to increase the yield in 5, but
CAL A was inactive in this system (Entry 16).
Figure 1. Conformational energy minimum of linearol (3)
Enzymatic Alcoholysis
We next studied the lipase-catalysed alcoholysis reaction
of linearol by treating it with alcohols in the presence of
CAL A, CAL B and PSL.
In view of our previous work on enzymatic deacetylation
of steroids,[24,25] we began using octanol as nucleophile, but
there was no transformation in this case (Table 2, Entries 1–
3). Only ethanol gave satisfactory results in THF as solvent,
and we obtained foliol (2) in moderate yield with CAL B.
Figure 2. Conformational energy minimum of sidol (4)
The synchronous transit algorithm was used to estimate It is remarkable that we did not detect any migration of
the transition state based on the most probable structures the acetyl group of linearol to give sidol in the presence of
of linearol and sidol. The energy of activation for the intra- alcohols. In the case of octanol, linearol remained unal-
molecular migration was estimated to be 73 kcal mol–1. tered.
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Enzyme-Catalysed Transformations of ent-Kaurane Diterpenoids
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Table 2. Lipase-catalysed alcoholysis of linearol (3) and triacetyl-foliol (6).
Entry
Enzyme
Substrate
Nucleophile
Solvent
T (°C)
Time (h)
Product yield (%)
2
7
1
2
3
4
5
6
7
8
CAL A
CAL B
PSL
3
3
3
3
3
3
3
6
6
6
6
n-octanol
n-octanol
n-octanol
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
CH3CN
CH3CN
CH3CN
EtOH
THF
THF
CH3CN
THF
33
33
33
55
33
55
55
33
55
33
55
168
168
168
72
72
48
–
–
–
36
43
68
55
–
–
–
–
–
–
–
–
5
4
CAL B
CAL B
CAL B
CAL B
CAL A
CAL A
CAL B
CAL B
48
168
168
168
48
9
10
11
THF
THF
THF
–
–
–
12
32
EtOH
With the aim of extending the study of the enzymatic Enzymatic Acetylation on Atractyligenin (8)
alcoholysis reaction we prepared the triacetyl-foliol (6), ap-
In view of the previous results on linearol we tested four
lipases (PPL, CRL, CAL B and PSL) in the acetylation re-
action of the 18-nor-ent-kaurane atractyligenin (8)
(Scheme 2).
plying traditional chemical methodology, by treatment of 3
with an excess of acetic anhydride and pyridine. The triace-
tyl-foliol (6) was then subjected to the enzymatic alcoholysis
with CAL A and CAL B as biocatalysts, two temperatures
and various solvents (Entries 4–7). CAL A showed low ac-
tivity, even at 55 °C (Entries 8 and 9). The best result was
obtained with CAL B and THF as solvent at 55 °C (En-
try 11). The enzyme catalysed alcoholysis of the acetyl
group at C-3 in triacetyl-foliol, so it was possible to obtain
7-acetyl-linearol (7), the complementary diacetylated pro-
duct of (4) (Scheme 1). It seems that the deacetylation reac-
tion stopped at this point, since we did not detect any other
product even at longer reaction times.
This result is in agreement with our previous work with
pyridoxine as substrate,[21] in which CAL B was active at
the same position both in the alcoholysis and the acety-
lation reactions. The site of alcoholysis in every case was
unambiguously established by NMR spectroscopic analysis
(Table 5 and Table 7). In fact compound 7 showed down-
field shifts, with respect to linearol (3), of H-7α (δ = 4.77,
Scheme 2.
The reaction was performed with isopropenyl acetate as
t) and C-7 (δ = 79.53) and upfield shifts of C-6 (δ = 24.41) acylating agent and tetrahydrofuran as solvent (Table 3).
and C-8 (δ, 46.72), consistently with the β effect of acety-
CAL B and PSL gave the best results at 33 °C and 55 °C,
1
lation. Furthermore, the unexpected upfield shift of H-3β affording only one product. Its H and 13C NMR spectra
(δ = 3.32, dd) and the enlargement of the AB system of the (Table 6 and Table 8) showed downfield shifts of H-2β (δ =
H-18 protons (δ = 4.30, d and 3.56, d) was in full agreement 5.27, dddd) and C-2 (δ = 69.9) and upfield shifts of C-1 (δ
1
with the H NMR spectrum of 7,18-diacetyl-leucanthol,[30] = 46.6) and C-3 (δ = 34.9) with respect to atractyligenin
which has an identical substitution pattern at the decalin (8), allowing us to assign the structure of 2-acetyl-atractyli-
moiety.
genin (9) to this compound.
Table 3. Lipase-catalysed acetylation of atractyligenin (8) and 4-epi-atractyligenin (10).
Entry
Enzyme
Substrate
Solvent
T (°C)
Time (h)
Yield (%)
1
2
3
4
5
6
7
8
CRL
CAL B
PSL
PPL
PSL
CAL B
CAL B
CAL B
PSL
CAL B
CAL B
8
8
THF
THF
THF
THF
THF
33
33
33
55
55
55
33
55
55
55
55
168
72
72
168
48
48
48
48
168
168
168
–
38
12
–
23
57
34
46
–
8
8
8
8
THF
8
CH3CN
CH3CN
THF
THF
CH3CN
8
9
10
11
10
10
10
–
–
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L. N. Monsalve, S. Rosselli, M. Bruno, A. Baldessari
FULL PAPER
CAL B showed high regioselectivity in this reaction, with
the α-hydroxy group at carbon 15 remaining unaltered. No
acetylation product was obtained when the carbon 4 epimer
of atractyligenin – 4-epi-atractylgenin (10) – was treated
with CAL B in the same solvent and acylating agent, even
at 55 °C. This result shows that CAL B is also highly stereo-
selective in the acetylation of this type of substrates.[31]
Reactions with Atractylitriol (11)
Preparation and Enzymatic Acetylation
To find out more about the catalytic activity of enzymes
on this kind of substrate, we decided to synthesize atractyli-
triol (11), which contains three hydroxy groups. The syn-
thetic route involves the reduction of the corresponding al-
kyl esters. As lipases are very efficient in esterification reac-
tions,[26,32] we tested the enzymatic esterification of atrac-
tyligenin (8) and 4-epi-atractyligenin (10) with ethanol, bu-
tanol and octanol in the presence of CAL B. Unfortunately,
we did not obtain any satisfactory result.
We then obtained atractylitriol (11) by the previously re-
ported procedure.[33]
We attempted the enzymatic acetylation of 11 with iso-
propenyl acetate in THF. A mixture of acetyl derivatives
(Scheme 3) was obtained in different proportions depending
on the lipase used and the reaction conditions (Table 4).
Atractylitriol was soluble at concentrations appropriate
Scheme 3.
for the enzymatic acetylation only in THF and ethanol. As for 24 hours (Entries 5, 6 and 8) – but no reaction products
ethanol would compete with the substrate, we chose THF. were detected with CAL A under these conditions, while
At 55 °C and after 24 hours of reaction, both CAL B and CAL B and PSL afforded the mixture of the isomers 12, 13
PSL had afforded the diacetyl derivative at carbons 2 and and 14. Finally, at shorter reaction times (4 hours), we were
19 (12) and a mixture of monoacetyl derivatives of atractyli- able to avoid the formation of diacetylated compound (12)
triol in positions 2 (13) and 19 (14) (Entries 2 and 3). Only with CAL B and PSL. A 13/14 ratio of about 1:1 was main-
CAL A showed regioselectivity, and we were able to obtain tained under all reaction conditions. By TLC the 13/14 mix-
14 as the only product, but in low yield (Entry 1). At ture seemed to be a single product, but it was possible to
shorter periods of time – 4 hours reaction time with PSL, isolate them by column chromatography and they were
for example – the regioselectivity was no better. With the identified by spectroscopic methods.
aim of improving regioselectivity we performed the reac-
By comparison with the 1H and 13C NMR spectra
tions with the three enzymes at lower temperature – 33 °C (Table 6 and Table 8) of triacetyl derivative (15) (see below),
Table 4. Enzyme-catalysed acetylation of atractylitriol (11) and deacetylation of triacetyl-actractylitriol (15).
Entry
Enzyme[a]
Substrate
T (°C)
Time (h)
Product yield (%)
12
13
14
17
1
2
3
4
5
6
7
8
CAL A
CAL B
PSL
11
11
11
11
11
11
11
11
11
15
15
15
15
55
55
55
55
33
33
33
33
33
28
28
28
28
24
24
24
4
24
24
4
24
4
24
24
2
–
20
24
18
–
15
–
22
–
–
–
12
34
37
32
–
27
44
25
48
–
–
–
–
–
–
–
–
–
–
32
33
29
–
26
41
23
43
–
PSL
CAL A
CAL B
CAL B
PSL
9
PSL
10
11
12
13
PLE[b,c]
PLE[d]
PLE[e]
PLE[e]
–
16
28
30
24
[a] Solvent: THF with lipases. [b] No product was obtained by lipase-catalysed alcoholysis of 15 at 55 °C over 168 h with CAL A, CAL B
and PSL. [c] Solvent: acetone/water. [d] Solvent: phosphate buffer. [e] Solvent: acetone/phosphate buffer.
2110
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Enzyme-Catalysed Transformations of ent-Kaurane Diterpenoids
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run in the same solvent, we were able to assign structures CAL B it could be assumed that, in the case of linearol and
to compounds 12–14. The 1H NMR spectrum of com- activated acylating agents, the active site of the enzyme acts
pound 12 showed an upfield shift of H-15 (δ = 3.83, br. s) in the same position of the ent-kaurane skeleton both in
and although the chemical shift of C-15 was not changed acylation and in alcoholysis reaction. Conventional acylat-
in the 13C NMR spectrum, an usual downfield shift (β ef- ing agents based on chemical methodology are not highly
fect) was observed for C-16 (δ = 160.0). Apart from the regioselective, and polyacylated compounds are usually ob-
1
same shifts observed for compound 12, the H NMR spec- tained. These enzymatic reactions were always performed
tra of compounds 13 and 14 showed additional upfield under mild conditions and allowed us to obtain a family of
shifts for Hs-19 (δ = 3.64, d) and for H-2 (δ = 3.97, dddd). novel ent-kauranes with potential biological activity.
The shifts observed in the 13C NMR spectra of compounds
13 and 14 (with respect to compound 15) were in perfect
agreement with the proposed structure.
Experimental Section
General Remarks: All solvents and reagents were reagent grade and
were used without purification. Lipase from Candida rugosa CRL
(905 U mg–1 solid), and type II crude from porcine pancreas
(190 U mg–1 protein) were purchased from Sigma Chemical Co.;
Candida antarctica lipase A: Chirazyme l-5, c.-f. lyo (400 U g–1)
was purchased from Roche Diagnostics GmbH; Candida antarctica
lipase B: Novozym 435 CAL (7400 PLU g–1) and Lipozyme RM
(1 m, 7800 U g–1) were generous gifts from Novozymes Latinoamer-
ica Ltda and Novozymes A/S; Pseudomonas lipase: Lipase PS Am-
ano PSL (33,200 U g–1) was purchased from Amano Pharmaceuti-
cal Co. Hog liver esterase on Eupergit C® 450 U mg–1 was supplied
by Fluka. All enzymes were used “straight from the bottle”.
Chemical Acetylation of Atractylitriol (11)
By treatment of 11 with an excess of acetic anhydride
and pyridine at room temperature, we prepared the triace-
tylatractylitriol 15.
When 11 was treated with acetic anhydride in pyridine
at low temperature (–30 °C) a mixture of derivatives was
obtained. The main product was the 19-acetyl-atractylitriol
(14), with minor quantities of 12, 15 and of another diacetyl
derivative (16), the structure of which was determined by
analysis of spectroscopic data. The 1H NMR and 13C NMR
spectra of compound 16 showed (with respect to compound
15) upfield shifts of H-2 (δ = 3.96, ddd) and C-2 (δ = 64.2)
and downfield shifts of C-1 (δ = 49.1) and C-3 (δ = 37.6).
Enzymatic Reactions: Enzymatic reactions were carried out with an
Innova 4000 digital incubator shaker (New Brunswick Scien-
tific Co.) at 33 °C and 55 °C and 200 rpm. Enzymatic transesterifi-
cations were followed by TLC on Merck silica gel 60F-254 alumin-
ium sheets (0.2 mm thickness). For column chromatography, Merck
silica gel 60 (70–230 mesh) was used. IR spectra were obtained on a
Shimadzu FTIR-8300 spectrophotometer. 1H NMR and 13C NMR
spectra were recorded at 250 and 500 MHz with Bruker AC 250
and Bruker AM 500 spectrometers, respectively. DEPT experiments
were acquired on the same apparata. Chemical shifts are reported
in δ units relative to tetramethylsilane (TMS) set at 0 δ, and coup-
ling constants are given in Hz. Solvents are indicated. EI-MS were
obtained at 70 eV with TRIO-2 VG Masslab Shimadzu QP-5000
and Finnigan TSQ70 mass spectrometers, in m/z (%). Elemental
analysis was carried out with a Perkin–Elmer 240 apparatus. Op-
tical purities of products was determined by specific rotation with
Perkin–Elmer 343 and Jasco P-1010 polarimeters. Solvents are indi-
cated. GC-MS analyses were performed on a Shimadzu GCMS-
QP5050A gas chromatograph–mass spectrometer, on an ULTRA-
1 column (30 m×0.25 mm×0.25 μm). The following temperature
program was employed: 80 °C (1 min)/15 °C min–1/250 °C (1 min)/
5 °C min–1/270 °C (5 min).
Enzymatic Deacetylation of Triacetyl-atractylitriol (15)
Lipase-catalysed alcoholysis of triacetyl-atractylitriol
(15) with PSL, CAL A and CAL B was unsuccessful, but
the hydrolysis of 15 in the 19-position with hog liver ester-
ase (PLE) supported on Eupergit C® in phosphate buffer/
acetone allowed us to obtain 2,15-diacetyl-atracytilitriol
(17), as clearly indicated by the upfield shifts of Hs-19 (δ =
3.67, d) and C-19 (δ = 61.9) in the 1H and 13C NMR spectra
with respect to the spectra of compound 15 (Table 6 and
Table 8).
Conclusions
This work describes application of enzymes to the prepa-
ration of specifically acylated derivatives of ent-kauranes.
Lipases from different sources and hog liver esterase exhib-
ited good performance as catalysts both in alcoholysis and
acylation reactions. Candida antartica B lipase and Pseu-
domnas sp. lipase gave the best results in both reactions. By
enzymatic acetylation and deacetylation reactions, various
mono- and diacetylated derivatives of linearol, atractytlig-
enin and atractylitriol have been regioselectively obtained;
some of these products had not been reported earlier in the
literature. Lipases have also catalysed acetyl migration in
linearol to produce sidol.
Isolation of Linearol (3) and Atractyligenin (8)
Linearol (3): was isolated from several species of Sideritis (La-
miaceae) and the purification procedures have been previously re-
ported (NMR: Tables 5 and 7.[19,20]
Atractyligenin (8): Isolated from Atractylis gummifera (Com-
positae) and the purification procedures have been previously re-
ported (NMR: Tables 6 and 8).[33]
4-epi-Atractyligenin (10) and Atractylitriol (11)
The syntheses of 4-epi-atractyligenin (10) and atractylitriol (11)
were performed by the previously reported procedure.[33]
Alcoholysis reactions afforded good results even in the
presence of short-chain alcohols such as ethanol and al-
lowed us to obtain complementary derivatives of some ent-
Atractylitriol (11): M.p. 217 °C (from benzene/EtOH). [α]2D5
=
1
–116.9 (c = 0.73, MeOH). H NMR ([D5]pyridine, 250 MHz): see
kauranes. With regard to the current results showed by Table 6. 13C NMR ([D5]pyridine, 62.7 MHz): see Table 8. IR (film)
Eur. J. Org. Chem. 2005, 2106–2115
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2111

L. N. Monsalve, S. Rosselli, M. Bruno, A. Baldessari
Enzymatic Acetylation of Linearol (3)
FULL PAPER
ν
max
= 3350, 1650, 1435, 1120, 1010, 903 cm–1. EIMS: m/z (%) =
˜
[M]+ (absent), 288 (33) [M – H2O]+, 273 (45), 257 (78), 239 (67),
189 (38), 132 (54), 105 (97), 91 (100). C19H30O3 (306.44): calcd. C
74.47, H 9.87; found C 74.51, H 9.86.
Linearol (3, 40 mg, 0.11 mmol) was dissolved in the indicated sol-
vent (3 mL). The acylating agent (0.25 mL) and an amount equiva-
1
Table 5. H NMR spectroscopic data of compounds 3–7 in CDCl3.
H
3
4
5
6
7
3β
7α
13
3.58 dd
3.56 t
2.68 s
4.91 dd
3.62 t
2.68 br. s
4.83 dd
3.60 t
2.68 br. s
4.74 dd
4.77 t
2.69 br. s
2.17 br. d (15a-H)
2.13 br. d (15b-H)
4.78 s
3.32 dd
4.79 t
2.70 br. s
15a, 15b
2.27 br. s (2 H)
2.27 br. s (2 H)
2.26 br. s (2 H)
2.27 br. s (2 H)
17a
17b
18a
18b
Me-19
Me-20
Ac
4.82 br. s
4.79 br. s
4.03 d
3.92 d
0.76
1.08
2.09 s
4.82 br. s
4.81 br. s
3.31 d
2.98 d
0.68 s
1.08 s
2.07 s
4.82 br. s
4.80 br. s
4.13 d
3.70 d
0.85 s
1.09 s
2.06 s
4.82 br. s
4.77 br. s
4.32 d
3.56 d
0.74 s
1.08 s
2.08 s
4.74 s
3.92 d
3.53 d
0.83 s
1.09 s
2.02 s
Ac
Ac
2.03 s
2.02 s
2.00 s
2.06 s
J (Hz)
2α,3β
2β,3β
6α,7β
6β,7β
15a,15b
18a,18b
11.3
5.1
2.7
2.7
–
11.3
5.1
2.7
2.7
–
11.2
5.2
3.8
3.8
–
11.2
5.2
3.8
3.8
17.0
11.8
11.2
5.2
3.5
3.5
–
11.3
12.3
11.6
12.0
1
Table 6. H NMR spectroscopic data of compounds 8, 9 and 11–17.
H
8[a]
9[a]
11[b]
12[c]
13[c]
14[c]
15[b]
15[c]
16[c]
17[c]
1α
2.12 dd
0.64 dd
2.15 dd
0.69 dd
2.48 dd
1.05 dd
2.12 dd
0.80 dd
2.19 dd
0.83 dd
2.15 dd
0.73 dd
2.25 dd
0.76 dd
2.10 dd
0.79 dd
2.10 dd
0.75 dd
2.17 dd
0.83 dd
1β
2β
3α
4β
13
15
17a
17b
4.11 dddd 5.27 dddd 4.36 dddd 5.07 dddd 5.08 dddd 3.97 dddd 5.32 dddd
2.32 m
2.60 m
5.03 dddd 3.96 dddd 5.06 dddd
2.17 ddd
2.25 m
2.61 m
2.61 m
2.98 ddd
2.25 m
2.73 br. s
2.20 ddd
2.26 ddd 2.24 ddd
2.30 ddd
2.22 ddd 2.25 ddd
2.65 br. s
2.76 br. s
3.83 br. s
5.23 br. s
5.10 br. s
4.19 dd
(19a-H)
4.03 dd
(19b-H)
1.05 s
2.77 br. s 2.77 br. s
3.83 br. s 3.83 br. s
5.22 br. s 5.22 br. s
5.09 br. s 5.09 br. s
2.69 br. s
5.30 br. s
5.46 br. s
5.12 br. s
4.26 dd
(19a-H)
4.18 dd
(19b-H)
0.89 s
2.77 br. s
5.08 br. s
5.24 br. s
5.09 br. s
4.13 dd
(19a-H)
3.99 dd
(19b-H)
1.01 s
2.81 br. s 2.80 br. s
5.11 br. s 5.10 br. s
5.28 br. s 5.27 br. s
5.13 br. s 5.12 br. s
3.71 br. s 3.66 br. s 4.09 br. s
5.13 br. s 5.09 br. s 5.45 br. s
5.02 br. s 4.98 br. s 5.16 br. s
3.95 d
(2H)
3.64 d
(2H)
4.08 d
(2H)
4.07 d
(2H)
3.67 d
(2H)
19a, 19b
20
0.95 s
0.96 s
1.89 s
0.99 s
0.99 s
2.04 s
0.99 s
2.07 s
0.99 s
2.08 s
2.07 s
0.99 s
2.07 s
2.03 s
Ac
Ac
Ac
2.06 s
2.04 s
2.09 s
2.04 s
2.03 s
2.04 s
2.02 s
2.00 s
J (Hz)
1α,1β
1α,2α
1β,2α
2α,3α
2α,3β
3α,3β
3α,4β
4β,19
11.7
1.5
11.7
1.5
11.7
1.5
11.7
1.5
11.6
1.5
11.6
1.5
11.6
11.0
1.5
6.6
–
11.6
1.5
11.6
1.5
11.6
11.0
1.5
–
2.9
10.8
10.8
11.6
1.5
11.6
1.5
11.6
11.0
1.5
7.0
–
11.6
1.5
11.6
1.5
11.6
11.0
1.5
7.2
–
11.6
1.5
11.6
1.5
11.6
11.0
1.5
–
2.7
10.8
10.8
11.6
1.5
11.6
1.5
11.6
11.0
1.5
–
2.7
10.8
10.8
11.4
1.5
11.4
1.5
11.6
11.0
1.5
7.3
–
11.6
1.5
11.6
1.5
11.6
11.0
1.5
11.7
11.7
7.3
4β,19a
4β,19b
19a,19b
–
–
–
–
–
–
–
–
[a] In CD3OD solution. [b] In [D5]pyridine solution. [c] In CDCl3 solution.
2112 © 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org
Eur. J. Org. Chem. 2005, 2106–2115

Enzyme-Catalysed Transformations of ent-Kaurane Diterpenoids
Table 7. 13CNMR spectroscopic data of compounds 3–7 in CDCl3.
FULL PAPER
C
3
4
5
6
7
1
2
3
4
5
6
7
8
38.4 t
26.5 t
72.3 d
41.9 s
38.2 d
27.4 t
76.9 d
48.0 s
50.3 d
38.8 s
17.9 t
33.6 t
43.7 d
38.3 t
45.1 t
155.0 s
103.6 t
66.1 t
11.9 q
18.0 q
171.8 s
38.5 t
23.5 t
74.6 d
41.7 s
37.4 d
26.7 t
76.9 d
48.2 s
50.3 d
38.5 s
17.9 t
33.6 t
43.7 d
38.3 t
45.0 t
155.0 s
103.7 t
64.2 t
12.8 q
17.9 q
171.9 s
38.4 t
23.0 t
74.3 d
40.5 s
38.2 d
27.1 t
76.7 d
48.0 s
50.1 d
38.7 s
17.9 t
33.5 t
43.6 d
37.8 t
45.0 t
154.8 s
103.7 t
64.6 t
13.0 q
17.9 q
171.5 s
171.1 s
37.7 t
23.0 t
73.8 d
40.2 s
39.6 d
24.2 t
79.2 d
46.7 s
50.8 d
38.6 s
18.0 t
33.2 t
43.4 d
38.0 t
44.9 t
154.0 s
103. t
64.8 t
12.8 q
17.7 q
170.6 s
170.5 s
170.2 s
21.2 q
21.1 q
20.9 q
38.3 t
25.8 t
71.8 d
42.0 s
39.5 d
24.4 t
79.5 d
46.7 s
51.1 d
38.7 s
18.1 t
33.3 t
43.5 d
38.1 t
45.0 t
154.1 s
103.8 t
66.3 t
11.7 q
17.7 q
171.6 s
170.3 s
9
10
11
12
13
14
15
16
17
18
19
20
Ac
Ac
Ac
Ac
Ac
Ac
21.2 q
21.2 q
21.2 q
21.1 q
21.3 q
21.0 q
Table 8. 13C NMR spectroscopic data of compounds 8, 9 and 11–17.
C
8[a]
9[a]
11[b]
12[c]
13[c]
14[c]
15[b]
15[c]
16[c]
17[c]
1
2
3
4
5
6
7
8
50.4 t
65.1 d
38.3 t
44.9 d
50.4 d
26.7 t
36.2 t
48.9 s
54.5 d
41.8 s
19.2 t
33.6 t
43.7 d
37.2 t
83.6 d
160.3 s
109.1 t
178.8 s
17.3 q
46.6 t
69.9 d
34.9 t
44.8 d
50.2 d
26.6 t
36.1 t
48.9 s
54.3 d
41.9 s
19.2 t
33.5 t
43.6 d
37.2 t
83.5 d
160.2 s
109.1 t
178.6 s
17.0 q
172.4 s
50.5 t
63.7 d
38.5 t
45.8 d
49.6 d
24.9 t
35.9 t
48.1 s
53.8 d
40.5 s
18.4 t
32.9 t
42.7 d
36.7 t
82.7 d
160.9 s
107.8 t
61.6 t
19.0 q
45.1 t
67.9 d
33.6 t
40.0 d
48.7 d
24.0 t
36.2 t
47.5 s
52.9 d
40.1 s
18.0 t
32.3 t
42.4 d
34.6 t
82.7 d
160.0 s
108.6 t
64.0 t
18.1 q
171.1 s
170.5 s
45.2 t
68.4 d
33.1 t
44.2 d
48.8 d
24.0 t
36.3 t
47.5 s
52.9 d
40.1 s
18.0 t
32.3 t
42.2 d
34.7 t
82.7 d
160.0 s
108.6 t
61.6 t
18.5 q
170.8 s
49.2 t
64.2 d
37.6 t
40.3 d
48.6 d
24.0 t
36.3 t
47.5 s
52.9 d
40.1 s
18.0 t
32.4 t
42.2 t
34.7 t
82.7 d
160.0 s
108.6 t
64.4 t
18.8 q
171.3 s
44.7 t
67.6 d
33.5 t
40.1 d
47.9 d
23.6 t
36.9 t
47.0 s
52.2 d
39.9 s
17.9 t
32.2 t
42.3 d
33.9 t
82.6 d
155.4 s
109.9 t
63.7 t
17.9 q
170.4 s
170.3 s
169.9 s
20.9 q
20.7 q
20.5 q
44.9 t
67.6 d
33.4 t
39.8 d
48.2 d
23.7 t
37.1 t
47.1 s
52.3 d
40.0 s
18.0 t
32.2 t
42.2 d
34.0 t
82.8 d
154.6 s
110.2 t
63.9 t
18.4 q
171.0 s
170.9 s
170.4 s
21.3 q
21.2 q
20.9 q
49.1 t
64.2 d
37.6 t
40.3 d
48.4 d
23.9 t
37.3 t
47.2 s
52.5 d
40.2 s
18.1 t
32.5 t
42.4 t
34.2 t
83.0 d
155.0 s
110.3 t
64.3 t
18.7 q
171.1 s
170.5 s
45.1 t
68.1 d
33.2 t
44.1 d
48.6 d
24.0 t
37.2 t
47.2 s
52.5 d
40.2 s
18.2 t
32.4 t
42.4 t
34.2 t
83.0 d
155.0 s
110.3 t
61.9 t
18.5 q
171.0 s
170.7 s
9
10
11
12
13
14
15
16
17
19
20
Ac
Ac
Ac
Ac
Ac
Ac
21.3 q
21.4 q
21.1 q
21.5 q
21.1 q
21.2 q
21.0 q
21.5 q
21.3 q
[a] In CD3OD solution. [b] In [D5]pyridine solution. [c] In CDCl3 solution.
lent to 1500 units of the indicated lipase were added. The suspen-
sion was shaken (200 rpm) at 33 °C or 55 °C and the progress of
the reaction was monitored by TLC (CH2Cl2/AcOEt, 3:2). After
the indicated time, the enzyme was filtered off, the solvent was
evaporated, and the crude residue was purified by flash chromatog-
raphy (eluent hexane/AcOEt), yielding sidol (4) and 3-acetyl-lin-
earol (5) (see Table 1).
Sidol (4): The spectroscopic and physical data were in perfect agree-
ment with those reported in the literature.[34] 1H NMR (CDCl3,
500 MHz): see Table 5. 13C NMR (CDCl3, 62.7 MHz): see Table 7.
3-Acetyl-linearol (5): Amorphous solid. [α]2D5 = –60.3 (c = 0.55,
EtOH). 1H NMR (CDCl3, 500 MHz): see Table 5. 13C NMR
(CDCl , 62.7 MHz): see Table 7. IR (film) νmax = 3442, 2936, 1721,
˜
3
Eur. J. Org. Chem. 2005, 2106–2115
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2113

L. N. Monsalve, S. Rosselli, M. Bruno, A. Baldessari
(CDCl , 62.7 MHz): see Table 8. IR (film) νmax = 3350, 2930, 1735,
FULL PAPER
1658, 1459, 1425, 1367, 1235, 1185, 1019 cm–1. EIMS: m/z (%) =
˜
3
404 (1) [M]+, 386 (1), 344 (1), 326 (19), 266 (28), 251 (12), 149 (28), 1358, 1243, 1026, 911 cm–1. EIMS: m/z (%) = [M]+ (absent), 330
121 (38), 93 (32), 43 (100).C24H36O5 (404.54): calcd. C 71.26, H (2) [M – H2O]+, 315 (1), 270 (10), 255 (6), 183 (5), 143 (11), 119
8.97; found C 71.30, H 8.93.
(16), 105 (8), 91 (31), 43 (100). C21H32O4 (348.48): calcd. C 72.38,
H 9.27; found C 72.40, H 9.30.
Synthesis of Triacetyl-foliol (6)
Low Temperature Acetylation of Atractylitriol (11)
Linearol (3, 300 mg,0.83 mmol) was dissolved in a mixture (5 mL)
of acetic anhydride and pyridine (1:1) and allowed to stand at room
temperature for 24 h to give, after the usual workup, triacetyl-foliol
(6, 340 mg, 92%).
Acetic anhydride (0.054 mL, 0.57 mmol) was added to a solution
of atractylitriol (11, 35 mg, 0.11 mmol) in dry pyridine (3 mL) at T
= –30 °C whilst stirring. After 1 h, the mixture was left standing at
0 °C for 3 h,ethyl acetate (10 mL) was then added,and the system
was washed in turn with cold HCl solution (5%) and H2O. The
organic layer was separated, dried (Na2SO4), concentrated and
chromatographed on a column (silica gel not deactivated, petro-
leum ether/ethyl acetate 4:1) to provide triacetyl-atractylitriol (15,
2 mg, 4%), 2,19-diacetyl-atractylitriol (12, 5 mg, 11%), 15,19-dia-
cetyl-atractylitriol (16, 3 mg, 7%), 19-acetyl-atractylitriol (14,
23 mg, 58%) and starting material (11, 7 mg, 20%).
Triacetyl-foliol (6): The spectroscopic and physical data were in
perfect agreement with those reported in the literature.[20] 1H NMR
(CDCl3, 500 MHz): see Table 5. 13C NMR (CDCl3, 62.7 MHz): see
Table 7.
Enzymatic Alcoholysis of Linearol (3) and Triacetyl-foliol (6): An
amount equivalent to 1500 units of the indicated lipase was added
to a solution of compound 3 or 6 (40 mg) in the indicated solvent
(10 mL) containing the indicated alcohol (5 mol equiv.). The sus-
pension was shaken (200 rpm) at 33 °C or 55 °C and the progress
of the reaction was monitored by TLC (CH2Cl2/AcOEt, 1:1). After
the indicated time, the enzyme was filtered off, the solvent was
evaporated, and the crude residue was purified by flash chromatog-
raphy (eluent hexane/AcOEt), yielding foliol (2) and 7-acetyl-lin-
earol (7) (see Table 2).
Triacetyl-atractylitriol (15): Amorphous solid. [α]2D5 = –19.7 (c =
1.44, CHCl3). 1H NMR (CDCl3, 250 MHz): see Table 6. 1H NMR
([D5]pyridine, 250 MHz): see Table 6. 13C NMR (CDCl3,
62.7 MHz): see Table 8. 13C NMR ([D5]pyridine, 62.7 MHz): see
Table 8. IR (film) ν
= 2931, 1732, 1369, 1238, 1028, 908 cm–1.
˜
max
EIMS: m/z (%) = [M]+ (absent), 372 (7) [M –AcOH]+, 357 (2), 330
(7), 312 (18), 297 (12), 270 (14), 252 (34), 237 (27), 195 (10), 105
(18), 91 (22), 43 (100). C25H36O6 (432.55): calcd. C 69.42, H 8.39;
found C 69.45, H 8.41.
Foliol (2): The spectroscopic and physical data were in perfect
agreement with those reported in the literature.[34]
7-Acetyl-linearol (7): Amorphous solid. [α]2D5 = –46.2 (c = 0.68,
15,19-Diacetyl-atractylitriol (16): Amorphous solid. [α]2D5 = –27.6 (c
EtOH). 1H NMR (CDCl3, 500 MHz): see Table 5. 13C NMR
= 0.24, CHCl3). 1H NMR (CDCl3, 250 MHz): see Table 6. 13C
(CDCl , 62.7 MHz): see Table 7. IR (film) νmax = 3423, 2934, 1718,
˜
3
NMR (CDCl , 62.7 MHz): see Table 8. IR (film) νmax = 3353, 2930,
1657, 1462, 1425, 1368, 1230, 1190, 1020 cm–1. EIMS: m/z (%) =
404 (1) [M]+, 386 (2), 344 (2), 326 (28), 284 (9), 266 (38), 251 (18),
149 (30), 121 (35), 93 (37), 43 (100). C24H36O5 (404.54): calcd. C
71.26, H 8.97; found C 71.33, H 8.94.
˜
3
1732, 1360, 1243, 1020, 908 cm–1. EIMS: m/z (%) = [M]+ (absent),
375 (1), 330 (3), 312 (11), 270 (14), 255 (14), 252 (29), 237 (25), 195
(9), 143 (12), 119 (10), 105 (17), 91 (29), 43 (100). C23H34O5
(390.51): calcd. C 70.74, H 8.78; found C 70.78, H 8.75.
Enzymatic Acetylation of Atractyligenin (8)
Synthesis of Triacetyl-atractylitriol (15)
As described for 3.
Atractylitriol (11, 300 mg,0.98 mmol) was dissolved in a mixture
(5 mL) of acetic anhydride and pyridine (1:1) and allowed to stand
at room temperature for 24 h to give, after the usual workup, triace-
tyl-atractylitriol (15, 395 mg, 93%).
2-Acetyl-atractyligenin (9): Amorphous solid. [α]2D5 = –53.3 (c =
0.80, EtOH). 1H NMR (CD3OD, 500 MHz): see Table 6. 13C NMR
(CD OD, 62.7 MHz): see Table 8. IR (film) ν
= 3380, 3320,
˜
3
max
2910, 1730, 1695, 1655, 1450, 1350, 1195, 1045, 990 cm–1. EIMS:
m/z (%) = 362 (1) [M]+, 302 (23), 284 (10), 239 (3), 185 (5), 145
(10), 105 (22), 91 (35), 43 (100). C21H30O5 (362.46): calcd. C 69.59,
H 8.34; found C 69.53, H 8.37.
Preparation of 2,15-Diacetyl-atractylitriol (17)
Esterase from hog liver (160 mg, immobilized on Eupergit C®
450 U g–1) was added to a solution of triacetyl derivatives (15,
80 mg, 0.18 mmol) in acetone (0.2 mL)and phosphate buffer (pH
= 8.0, 2 mL, 20 mm). The mixture was stirred at 28 °C for 2 h and
H2O (10 mL) was then added. The mixture was extracted three
times with CH2Cl2 (10 mL). The combined organic layers were
dried (Na2SO4), concentrated and chromatographed by CC (silica
gel, petroleum ether/ethyl acetate 1:1) to give 17 (20 mg, 28%).
Enzymatic Acetylation of Atractylitriol (11)
As described for (3).
2,19-Diacetyl-atractylitriol (12): Amorphous solid. [α]2D5 = –29.7 (c
= 0.21, EtOH). 1H NMR (CDCl3, 500 MHz): see Table 6. 13C
NMR (CDCl , 62.7 MHz): see Table 8. IR (film) νmax = 3356, 2933,
˜
3
1736, 1365, 1241, 1026, 911 cm–1. EIMS: m/z (%) = [M]+ (absent),
330 (10), 315 (8), 270 (26), 255 (19), 212 (17), 183 (6), 143 (13), 105
(24), 91 (33), 43 (100). C23H34O5 (390.51): calcd. C 70.74, H 8.78;
found C 70.70, H 8.82.
2,15-Diacetyl-atractylitriol (17): Amorphous solid. [α]2D5 = +11.0 (c
= 0.25, EtOH). 1H NMR (CDCl3, 250 MHz): see Table 6. 13C
NMR (CDCl , 62.7 MHz): see Table 8. IR (film) νmax = 3352, 2935,
˜
3
1734, 1361, 1248, 1031, 907 cm–1. EIMS: m/z (%) = [M]+ (absent),
375 (1), 372 (4), 330 (6), 312 (15), 255 (9), 252 (25), 237 (12), 195
(14), 143 (11), 119 (15), 105 (11), 43 (100). C23H34O5 (390.51):
calcd. C 70.74, H 8.78; found C 70.71, H 8.81.
2-Acetyl-atractylitriol (13): Amorphous solid. [α]2D5 = +40.6 (c =
1
0.32, EtOH). H NMR (CDCl3, 500 MHz): see Table 6. 13C NMR
(CDCl , 62.7 MHz): see Table 8. IR (film) νmax = 3352, 2933, 1732,
˜
3
1360, 1243, 1026, 911 cm–1. EIMS: m/z (%) = [M]+ (absent), 330
(7) [M – H2O]+, 315 (5), 270 (8), 255 (5), 183 (3), 143 (7), 119 (11),
105 (9), 91 (26), 43 (100). C21H32O4 (348.48): calcd. C 72.38, H
9.27; found C 72.34, H 9.24.
Acknowledgments
19-Acetyl-atractylitriol (14): Amorphous solid. [α]2D5 = –72.7 (c =
We thank UBA (projects X022, X089), ANPCyT (project PICT
06–08293, Argentina) and Italian Government project PRIN 2003
1
2.01, EtOH). H NMR (CDCl3, 500 MHz): see Table 6. 13C NMR
2114
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Enzyme-Catalysed Transformations of ent-Kaurane Diterpenoids
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Received: December 03, 2004
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