Biotransformations of bicyclic trimethylcyclohexane chloro-, bromo-and iodolactones using fungal strains

Article abstract of DOI:10.3109/10242422.2010.538688

Several fungal strains (Fusarium, Botrytis, Beauveria) were screened for their ability to transform three bicyclic halo-γ-lactones with a trimethylcyclohexane ring. Most of the micro-organisms carried out hydrolytic dehalogenation and transformed these la

Full text of DOI:10.3109/10242422.2010.538688

Biocatalysis and Biotransformation, September–December 2010; 28(5–6): 408–414
SHORT COMMUNICATION
Biotransformations of bicyclic trimethylcyclohexane chloro-,
bromo- and iodolactones using fungal strains
1
´
2
MAŁGORZATA GRABARCZYK & AGATA BIAŁONSKA
1
Department of Chemistry, University of Enviromental and Life Sciences, Norwida 25, 50-375Wrocław, Poland, and
Faculty of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383Wrocław, Poland
2
Abstract
Several fungal strains (Fusarium, Botrytis, Beauveria) were screened for their ability to transform three bicyclic halo-γ-lactones
with a trimethylcyclohexane ring. Most of the micro-organisms carried out hydrolytic dehalogenation and transformed these
lactones into two hydroxy-γ-lactones: cis (-)-2-hydroxy-4,4,6-trimethyl-9-oxabicyclo[4.3.0]nonan-8-one and trans (ꢀ)-2-
hydroxy-4,4,6-trimethyl-9-oxabicyclo[4.3.0]nonan-8-one. The structures of all substrates and products were established
on the basis of their spectral data and X-ray analysis. The method presented offers an alternative route to obtaining hydroxy-
lactones with high enantiomeric excess.
Keywords: Lactones, biotransformations, hydrolytic dehalogenation, fusarium species
Introduction
micro-organisms (fungal strains) for hydroxylation of
iodolactones (Grotowska & Wawrze n´ czyk 2002),
saturated (Gładkowski et al. 2004; 2006) and unsatu-
rated lactones (Gładkowski et al. 2006; 2007).
Here we present further examples of biohydroxy-
lation of chloro-, bromo- and iodolactones containing
a trimethylcyclohexane ring.These compounds were
chosen for two reasons. Some terpenoid halolactones
exhibit anti-feeding activity against insect pests, so
they could be useful for insect pest control (Paruch
et al. 2000;2001). Secondly, these lactones may be good
model substrates for finding micro-organisms which
are able to replace the halogen with a hydroxy group
in good yield.This would demonstrate the potential
for detoxification using model toxic compounds (halo-
lactone) by conversion to the environmentally safer
hydroxylactone.
Halogenated organic compounds are often met in
nature.They are used as herbicides, insecticides, pes-
ticides,fungicides,solvents and intermediates for chemi-
cal syntheses.These compounds can be toxic for the
environment, so biodegradation is an important topic
to study (Janssen et al. 1994; DeWit 2002; Erable et al.
2005; Segev et al. 2007; Kim et al 2009).
Natural terpenoids including a lactone ring and
hydroxy group in their structure are often isolated from
plants and animals. Natural hydroxylactones have many
specific biological activities like anti-malarial (Ortet
et al. 2008), anti-fungal (Vajs et al. 1999; Pujar et al.
2000), cytotoxic (Zhang & Feng 1997; El Hassany et al.
2
2
004; Liu et al. 2008), antibacterial (El Hassany et al.
004), anti-cancer (El-Gamal 2001) and phytotoxicity
(Fukushima et al. 1998).
There are many chemical routes to introduce a
hydroxy group into a lactone molecule, but these often
involve multiple steps (Kiegiel et al. 2000; Popsavin
et al.2003;Rodriguez et al.2006;Antoniotti & Du n´ ach
Materials and methods
Analysis
2009). A better method is direct biohydroxylation of
chemically produced lactones with different func-
tional groups. In our laboratory we have applied
The progress of chemical reactions and biotransfor-
mations as well as the purity of isolated products
Correspondence: Małgorzata Grabarczyk, Department of Chemistry, University of Enviromental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland.
Tel: ꢀ483205252. Fax: ꢀ48328576. Email: ma.grab@interia.pl
(
Received 21 June 2010; accepted 5 November 2010)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2010 Informa UK, Ltd.
DOI: 10.3109/10242422.2010.538688

Biotransformations of halolactones 409
2
were monitored by TLC on silica gel-coated alumin-
ium plates (DC–Alufolien Kieselgel 60 F254, Merck,
Germany) and by GC analysis which was carried out
on a Varian CP-3380 instrument using an HP-5
column (cross-linked methyl silicone gum, 30 m ꢁ 0.32
mm ꢁ 0.25 μm). The temperatures during GC
analysis were: injector 150°C,detector (FID) 300°C,
column temperature: 100°C held for 1 min, 100–
refined using all F data, as implemented by the XL
program (Bruker AXS 1999). Non-hydrogen atoms
were refined with anisotropic displacement param-
eters. All H atoms were placed at calculated posi-
tions. Before the last cycle of refinement all H atoms
were fixed and were allowed to ride on their parent
atoms. The Friedel pairs were merged before the
final refinement.
ꢂ1
ꢂ1
200°C at 10°C min , 200–300 at 50°C min , 300°C
held for 1 min. The enantiomeric excesses were
determined by GC analysis using the chiral column
CP-cyclodextrin-B-325, 30 m ꢁ 0.25 mm ꢁ 0.25 μm
under the following conditions: injector 200°C,
detector (FID) 250°C, column temperature: 140°C
Materials
Thesubstratesforbiotransformations,racemichalolac-
tones 3–5 were synthesized from known g,d-unsaturated
ester 1 (Grabarczyk et al. 2005) (Scheme 1). Lactone
ꢂ1
held for 45 min, 140–200°C at 20°C min , 200°C
3
was obtained from ester 1 in two steps: basic hydro-
held for 1 min. The products were purified by pre-
parative column chromatography on silica gel (Kie-
lysis and iodolactonization according to a procedure
described earlier (Grabarczyk et al. 2005). The two
known lactones 4 and 5 (Pisarski & Wawrze n´ czyk
2006) were also obtained in two steps: basic hydro-
lysis of ester 1 and bromo- or chlorolactonization.
The spectral data given below are useful for studying
biotransformation of the parent substrates.
1
selgel 60, 230–400 mesh). H NMR spectra were
recorded in CDCl solution on a Bruker Avance
3
DRX 300 spectrometer. IR spectra were determined
using a FTIRThermo-Mattson IR 300 Spectrometer.
Optical rotations were measured on an Autopol IV
automatic polarimeter (Rudolf Research Analytical,
NY, USA). All the melting points were measured on
a Boetius apparatus.
2
-Chloro-4,4,6-trimethyl-9-oxabicyclo[4.3.0]nonan-8-
one (3). A mixture of acid 2 (2.4 g, 0.013 mol) in 30 ml
ofTHF and NCS (3.36 g, 0.025 mol) was stirred for
2
4 h at room temperature.Then water was added to
X-ray crystallographic data
the mixture and the product diluted with diethyl ether.
TheorganiclayerwaswashedwithsaturatedNaHCO3
solution, brine and dried with anhydrous magnesium
sulphate. The crude product was chromatographed
on silica gel (hexane:acetone, 3:1) and 1.98 g (69%)
of chlorolactone 3 was obtained.The product is char-
acterized by the data given: mp 97–98°C, H NMR
(300 MHz, CDCl ):1.05 and 1.09 (two s, 6H, (CH )
C-4), 1.23 (s, 3H, CH C-6), 1.40 (d, J ꢃ 15.0 Hz,
X-ray data for 3 and 5 were collected on a Kuma KM4
CCD diffractometer (MoKα radiation; l ꢃ 0.71073
Å).The data were collected at 100 K using an Oxford
Cryosystem device. Data reduction and analysis were
carriedoutwiththeCrysAlice‘RED’program(Oxford
Diffraction 2001).The space groups were determined
using the XPREP program. The structures were
solved by direct methods using the XS program and
1
3
3 2
3
Scheme 1. Synthesis of halolactones 3-5.

4
10 M. Grabarczyk & A. Bialo n´ ska
1
H, one of CH -5), 1.56–1.70 (m, 2H, one of CH -5
Wrocław (Fusarium culmorum (AM 10), Fusarium ave-
naceum (AM 11), Fusarium oxysporum (AM 145),
Fusarium tricinctum (AM 395), Fusarium semitectum
(AM 20), Fusarium solani (AM 203), Botrytis cinerea
(AM 235), Beauveria bassiana (AM 278)).The micro-
organisms were cultivated on Sabouraud agar con-
taining aminobac (catalogue no.S-0002) 5 g,peptone
K (S-0011) 5 g, glucose (459560117) 40 g and agar
(S-0001) 15 g in distilled water (1 l) at 28°C and
stored in refrigerator at 4°C.
2
2
and H-3 ), 1.92 (ddd, J ꢃ 13.7, 4.0 and 2.2 Hz, 1H,
H-3 ), 2.17 and 2.74 (two d, J ꢃ 17.5 Hz, 2H, CH -7),
ax
eq
2
3.98 (ddd, J ꢃ 12.5, 8.8 and 4.0 Hz, 1H, H-2), 4.11
−
1
(d, J ꢃ 8.7 Hz, 1H, H-1); IR (KBr, cm ): 1786 (s),
1
455 (m), 1157 (s), 991 (m), 742 (m).
Crystal data for (3): C H ClO , Mr ꢃ 216.70,
1
1
17
2
colourless plate, crystal dimensions 0.31 ꢁ 0.14 ꢁ
0.03mm,orthorhombic,spacegroupP2 /c,aꢃ8.394(3),
1
b ꢃ 7.700(2), c ꢃ 17.215(4) Å (anstrem), Å ꢃ
3
95.21(3) °, V ꢃ 1108.1(6) Å , Z ꢃ 4, D ꢃ 1.299
c
ꢂ3
Mg m , T ꢃ 100(2) K, R ꢃ 0.045, wR2 ꢃ 0.085
(
2354 reflections with I ꢄ 2σ(I)) for 127 variables.
Biotransformations
CCDC No: 771511.
Screening procedure
2
-Bromo-4,4,6-trimethyl-9-oxabicyclo[4.3.0]nonan-8-
one (4). Acid 2 (2.1 g, 0.011 mol) was dissolved in
0 ml ofTHF and NBS (3.1 g, 0.092 mol) was added.
The strains were cultivated at 25°C in Erlenmeyer
flasks which contained 100 ml of medium consisting
of 3 g glucose and 1 g peptobac in water. After 4 days,
10 mg of biotransformation substrate in 1 ml of acetone
was added to the cultures. Incubation of the shaken
cultures with substrate was continued for 14 days.After
5, 9 and 14 days of incubation the products of biotrans-
formation were extracted with dichloromethane and
analyzed byTLC (silica gel, hexane:acetone 3:1) and
GC (HP-5 column). The results of GC analyses for
lactone 3–5 are presented inTable I. During the screen-
ing procedure the priority was to identify those organi-
sms which replaced the halogen with a hydroxy group
with the highest yield. At this stage we were interested
only in the degree of transformation of substrates into
products.
3
The mixture was stirred for 24 h at room tempera-
ture and then water was added. The product was
extracted with diethyl ether. The ethereal fractions
were combined, washed with saturated NaHCO solu-
3
tion, brine and dried with anhydrous magnesium
sulphate.The crude product was chromatographed on
silica gel (hexane:acetone, 3:1) giving 2.43 g (81%)
of bromolactone 4 with the following physical and
spectral data: mp 110–113°C; H NMR (300 MHz,
CDCl ): 1.03 and 1.09 (two s, 2H, (CH ) C-4),
1
3
3 2
1
.22 (s, 3H, CH C-6), 1.38 (d, J ꢃ 15.0 Hz, 1H, one
3
of CH -5), 1.70 (dd, J ꢃ 15.0 and 2.3 Hz, 1H, one
of CH -5), 1.72 (d, J ꢃ 13.4 Hz, 1H, H-3 ), 2.04
2
2
ax
(ddd, J ꢃ 13.4, 4.1 and 2.3 Hz, H-3 ), 2.15 and
eq
2
.77 (two d, J ꢃ 17.5 Hz, 2H, CH -7), 4.05 (ddd, J ꢃ
2
Preparative biotransformation
13.2, 9.2 and 4.1 Hz, 1H, H-2), 4.22 (d, J ꢃ 9.2 Hz,
ꢂ1
1H, H-1); IR (KBr, cm ): 1784 (s), 1454 (m), 1143
Halolactones 3–5 (100 mg dissolved in 10 ml acetone)
were added to 4-day old cultures prepared as described
in the screening procedure.The cultures were shaken
in 10 flasks with 100 ml of medium in each flask.After
(s), 1024 (s), 989 (s), 696 (m).
2
(
-Iodo-4,4,6-trimethyl-9-oxabicyclo[4.3.0]nonan-8-one
5). Melting point 99–100°C ; 1H NMR (300 MHz,
CDCl ), δ: 0.98 and 1.06 (two s, 6H, (CH ) C-4), 1.17
14 days of shaking the products were extracted with
3
3 2
dichloromethane.The organic solutions were dried over
(s, 3H, CH C-6), 1.37 (d, J ꢃ 15.0 Hz, 1H, one of
3
MgSO and the solvent evaporated in vacuo. The
CH -5), 1.73 (dd, J ꢃ 15.0, 2.4 Hz, 1H, one of CH -
4
2
2
products were separated by column chromatography
5), 1.95 (dd, J ꢃ 13.7, 13.5 Hz, 1H, H-3axial), 2.09
(
silica gel, hexane:acetone 3:1). The preparative
and 2.85 (two d, J ꢃ 17.6 Hz, 2H, CH -7), 2.13 (ddd,
2
biotransformations of lactones 3, 4 and 5 gave a mix-
ture of hydroxylactone 6 (in one case hydroxylactone
7) and untransformed substrate (Scheme 2). Results
of the preparative biotransformations are presented
in Tables II–IV. The physical and spectral data of
hydroxylactone6andhydroxylactone7(Wawrze n´ czyk
et al. 2003) are given below.
J ꢃ 13.7, 4.0, 2.4 Hz, H-3equatorial), 4.10 (ddd, J ꢃ
1
3.5, 9.9, 4.0 Hz, 1H, H-2), 4.32 (d, J ꢃ 9.9 Hz,
−1
1H, H-1), IR (film, cm ): 2976 (s), 1788 (s), 1468
(m), 1160 (s), 1028 (s).
Micro-organisms
The chemicals used for preparation of the growth media
were purchased from BTL (Poland), except from glu-
cose which was bought from POCh (Poland).
The fungal strains used came from the collection at
the Institute of Biology and Botany, Medical University,
Cis (-)-2-hydroxy-4,4,6-trimethyl-9-oxabicyclo[4.3.0]
nonan-8-one (6). mp 109–110°C, H NMR (300 MHz,
1
CDCl ): 0.96 and 1.02 (two ss, 2H, (CH ) C-4), 1.20
3
3 2
(d, J ꢃ 14.5 Hz, 1H, one of CH -5), 1.29 (s, 3H,
2

Biotransformations of halolactones 411
Table I. Composition (in percentage according to GC) of the product
mixtures of screening biotransformations of lactones 3, 4, 5.
a ꢃ 8.657(2), b ꢃ 10.632(2), c ꢃ 22.952(5) Å, V ꢃ
3
−3
2
112.5(8) Å , Z ꢃ 8, D ꢃ 1.247 Mg m , T ꢃ 100(2)
c
Products of transformations
K, R ꢃ 0.087, wR2 ꢃ 0.124 (2839 reflections with
I ꢄ 2σ(I)) for 253 variables. CCDC No: 771512.
(
%)
Time of
incubation
(days)
3
4
5
Trans (ꢀ)-2-hydroxy-4,4,6-trimethyl-9-oxabicyclo[4.3.0]
nonan-8-one (7). mp 109–110°C, H NMR (300 MHz,
Entry
1
Strain
3
6
6
4
6
5
6
1
F. culmorum
5
9
94
84 16
86 14
77 23
37 63
28 72
CDCl ): 1.05 and 1.09 (two s, 2H, (CH ) C-4), 1.23
3
3 2
81 19 53 47
63 37 35 65
85 15 57 43
49 51 28 72
43 57 25 75
95 5.0 62 38
36 64 33 67
35 65 24 76
78 22 59 41
52 48 32 68
46 54 27 73
86 14 45 55
85 15 32 68
78 22 28 72
63 37 50 50
47 53 27 73
40 60 24 76
(
s, 3H, CH C-6), 1.37 (d, J ꢃ 15.0 Hz, 1H, one of
3
14
5
9
14
5
9
14
5
9
14
5
9
14
5
9
14
5
9
14
5
9
14
CH -5), 1.61–1.70 (m 2H, one of CH -3 and one
2
2
2
3
4
5
6
7
8
F. avenaceum
F. oxysporum
F. tricinctum
F. semitectum
F. solani
of CH -5), 1.92 (ddd, J ꢃ 13.7, 4.1, 2.2 Hz, 1H, one
7
3
93
97
2
of CH -3), 2.18 and 2.74 (two d, J ꢃ 17.5 Hz, 2H,
2
60 40
32 68
31 69
36 64
27 73
13 87
26 74
17 83
12 88
80 20
28 72
25 75
CH -7), 4.00 (ddd, J ꢃ 12.6, 8.7, 4.1 Hz, 1H, H-2),
2
−1
4
.11 (d, J ꢃ 8.7 Hz, 1H, H-1), IR (film, cm ): 3460
(
s), 2968 (s), 1784 (s), 1164 (s), 1056 (s).
Results and discussion
The substrates for transformation were three racemic
halo-g-lactones (3, 4, 5) containing a trimethylcyclo-
hexane ring.Iodo-g-lactone 5 was obtained from known
g, d-unsaturated ester 1 as described earlier (Grabarczyk
et al. 2005). Two known halolactones (Pisarski &
Wawrze n´ czyk 2006), chloro-g-lactone 3 and bromo-
g-lactone 4, were also obtained from ester 1, which was
hydrolyzed in a 2.5% ethanolic solution of KOH to
give acid 2. g,d-Unsaturated acid 2 was a substrate for
chlorolactonization and bromolactonization using
N-chlorosuccinimide or N-bromosuccinimide inTHF
(Scheme 1).
B. cinerea
97
3
94
6
100
0
82 18
82 18
35 65 70 30
30 70 62 28
B. bassiana
98
2
99
1
6
96
91
4
9
90 10 94
84 16 88 12
88 12
CH C-6), 1.32 (dd, J ꢃ 14.5, 1.8 Hz, 1H, one of
3
CH -5), 1.43 (dd, J ꢃ 12.7, 12.4 Hz, 1H, one of CH -
The structures of chloro-g-lactone 3 and bromo-
2
2
3), 1.61 (ddd, J ꢃ 12.7, 4.5 and 1.8 Hz, one of CH2-
g-lactone 4 were determined on the basis of their
1
3
), 2.07 (s, 1H, OH), 2.37 (s, 2H, CH -7), 3.99 (ddd,
spectral ( H NMR and IR) data and X-ray analysis
2
−1
J ꢃ 12.4, 4.5 and 3.0 Hz, 1H, H-2), 4.31 (d, J ꢃ 3.0
(for 3).The absorption bands at 1786 cm for 3 and
Hz, 1H, H-1), 13C NMR (CDCl ): 23.84 (C-11),
−1
1784 cm for 4 in their IR spectra confirmed the
3
2
6.53 (C-10), 31.99, 40.24 (C-4 and C-6), 33.81
presence of the g-lactone ring in the molecule of these
compounds. X-ray analysis of 3 indicated that the
cyclohexane ring exists in a slightly deformed chair
conformation and the C-O bond of g-lactone ring and
C-Cl bond are situated in trans diequatorial posi-
tions. One of the methyl groups at C-4 is cis oriented
in relation to the C-O bond (Structure 1). The
(
(
C-9), 40.31 (C-3), 45.04 (C-5), 48.02 (C-7), 65.72
C-2), 85.96 (C-1), 175.64 (C-8), IR (film, cm ):
−1
3480 (s), 2966 (s), 1773 (s), 1177 (s), 1058 (s).
Crystal data for (6): C H O , Mr ꢃ 198.25,
11
18
3
colourless cut-needle, crystal dimensions 0.23 ꢁ 0.18 ꢁ
.14 mm, orthorhombic, space group P2 2 2 ,
0
1
1 1
Scheme 2. Biotransformations of halolactones 3-5.

4
12 M. Grabarczyk & A. Bialo n´ ska
Table II. Composition (in percentage according to GC) of the product mixtures of preparative biotransformations of lactone 3.
2
0
Entry
Strain
Time of incubation (days) 3 (%) 6 (%) 7 (%) Isolated yield (%) ee (%)
a
D
1
2
3
4
5
F. avenaceum
F. oxysporum
F. tricinctum
F. solani
14
10
14
14
14
51
22
40
48
38
—
78
60
52
62
49
—
—
—
—
44.2
40.4
48.0
39.0
47.0
92
24
51.7
55.9
27.5
ꢀ16.6° (c ꢃ 1.10%, CHCl3)
ꢂ11.4° (c ꢃ 1.11%, CHCl3)
ꢂ31.6° (c ꢃ 1.02%, CHCl3)
ꢂ38.2° (c ꢃ 1.20%, CHCl3)
ꢂ19.9° (c ꢃ 1.15%, CHCl3)
B. cinerea
1
−1
− 1
similarity between spectral data ( H NMR) of chloro-
lactone 3 and bromolactone 4 indicates that their
structures are identical.The same results were obtained
earlier for iodo-g-lactone 5 (Grabarczyk et al. 2005).
The first step of the biotransformation procedure
was screening using eight fungal strains of local origin:
F. culmorum, F. avenaceum, F. oxysporum, F. tricinctum,
F. semitectum, F. solani, B. cinerea and B. bassiana.The
progress of the biotransformation was monitored by
TLC and GC (Table I).These results indicated that
almost all strains transformed the three substrates
with a high or satisfactory conversion. Only B.bassiana
gave a low conversion (12–16%) after 14 days. Chlo-
rolactone 3 and bromolactone 4 were transformed by
five and six micro-organisms, respectively, with good
yields (49–78% and 57–75%).The best results were
obtained with F. oxysporum for 3 and F. culmorum,
F.avenaceum and F.tricinctum for 4. Iodolactone 5 was
transformed by six strains, giving the highest yields
bands at 1773 cm and 1784 cm ,respectively).Strong
ꢂ1
and broad bands found at 3480 cm for 6 and
3460 cm for 7 suggested the presence of a hydroxyl
group in both molecules.
ꢂ1
1H NMR spectra of hydroxy-g-lactone 6 gave very
interesting results. A small coupling constant (J ꢃ
3.0 Hz) of H-1 indicated that this proton was located
in an equatorial position. The signal from the H-2
proton at 3.99 ppm as a doublet of doublets (J ꢃ 12.2,
4.5 and 3.0 Hz) suggested that this proton was in an
axial position.The location of signals of the H-1 pro-
ton and CH C-6 group were shifted towards higher
fields, however signals of the CH -3 and CH -5 groups
moved in the opposite direction.The shape of the sig-
3
2
2
nal from the CH -7 group changed into singlet. All of
2
this suggested a change of conformation of the mol-
ecule during the biotransformation.
X-ray analysis of hydroxy-g-lactone 6 shows that the
cyclohexane ring adopted a chair conformation. In
this case, the OH group was in an equatorial position
and cis oriented in relation to the C-O bond of the
γ-lactone ring, which was in an axial position. One of
the methyl group at C-4 was trans oriented in rela-
tion to the C-O bond and cis oriented to the methyl
group at C-6 (Structure 2).
(
63–97%) of product after 14 days. The best results
were observed when F.avenaceum and F.semitectum were
used as biocatalysts. For all transformations only one
product was observed.
Preparative scale transformations of 3, 4, 5 were
performed using different Fusarium species, while
B. cinerea was used for 3 only (Tables II–IV). The
composition of product mixtures showed that,in almost
all cases, hydroxylactone 6 was obtained as the sole
product (Scheme 2). In a single case, when lactone
was transformed with F. avenaceum the alternative
hydroxylactone 7 was observed (Scheme 2).
The structures of products 6 and 7 were established
on the basis of their spectral and X-ray analysis data.
The IR spectra showed that the g-lactone ring had
been retained during the biotransformation (absorption
1H NMR spectra of hydroxy-g-lactone 7 and chlo-
rolactone 3 indicated a similarity between them. In
the case of lactone 7 a coupling constant (J ꢃ 8.7 Hz)
of H-1 indicated that this proton was located in an
axial position.The signal at 4.00 ppm as a doublet of
doublets (J ꢃ 12.6, 8.7 and 4.1 Hz) from the H-2 pro-
ton suggested that this proton was in an axial position.
The signal from the CH -7 group was present as two
doublets with coupling constant J ꢃ 17.5 Hz. In the
case of lactone 3 these signals and their multiplicity
3
2
Table III. Composition (in percentage according to GC) of the product mixtures of preparative biotransformations of lactone 4.
Time of
incubation
(days)
2
0
Entry
Strain
4 (%)
6 (%)
Isolated yield (%)
ee (%)
a
D
1
2
3
4
5
6
F. culmorum
F. avenaceum
F. oxysporum
F. tricinctum
F. semitectum
F. solani
14
10
14
10
10
10
19
25
33
26
37
20
75
75
67
74
63
57
25.1
59.5
52.7
69.0
29.0
41.4
0.4
81.0
74.4
70.0
0
ꢂ0.6° (c ꢃ 0.94%, CHCl3)
ꢂ48.9° (c ꢃ 0.85%, CHCl3)
ꢂ45.3° (c ꢃ 0.85%, CHCl3)
ꢂ42.3° (c ꢃ 1.22%, CHCl3)
0°
45.4
ꢂ29.4° (c ꢃ 1.18%, CHCl3)

Biotransformations of halolactones 413
Table IV. Composition (in percentage according to GC) of the product mixtures of preparative biotransformations of lactone 5.
Time of
2
0
Entry
Strain
incubation (days)
5 (%)
6 (%)
Isolated yield (%)
ee (%)
a
D
1
2
3
4
5
F. avenaceum
F. oxysporum
F. tricinctum
F. semitectum
F. solani
10
10
14
14
14
10
26
25
11
15
90
74
75
89
85
85.6
47.1
60.7
79.0
52.9
4.0
26.6
22.2
14.7
13.4
ꢀ3.6° (c ꢃ 0.96%, CHCl3)
ꢂ25.7° (c ꢃ 0.85%, CHCl3)
ꢂ21.6° (c ꢃ 1.09%, CHCl3)
ꢂ11.0° (c ꢃ 1.03%, CHCl3)
ꢂ10.5° (c ꢃ 1.07%, CHCl3)
were identical. The signals from the other groups in
both lactones were also identical.This indicates that
the C-O bond of the g-lactone ring is cis oriented in
relation to axial methyl group at C-6.The OH group,
however, is in an equatorial position and trans oriented
in relation to both the axial methyl group at C-6 and
C-O bond of the g-lactone ring.
Products of biotransformation (6 and 7) were
optically active with the exception of lactone 6
derived from bromolactone 5 using F. semitectum.
Enantiomeric excess was determined by chiral GC
of the g-lactone ring. For that reason both products
(6, 7) show different stereo-selectivity for substrates
3 and 4 (Table II and III).
Conclusions
Most of the micro-organisms used for biotransfor-
mation transformed all three substrates into prod-
ucts with high yield.These micro-organisms showed
a very high region-selectivity, the OH group was
always introduced in an equatorial position at C-2.
In almost every case (for hydoxylactone 6) a change
in conformation of the cyclohexane ring was observed.
Only hydroxylactone 7, produced by transformation
of chlorolactone 3, retained its conformation.The best
substrate, giving product with the highest yield and
enantiomeric excess, was bromolactone 4. The best
micro-organism for transformation of halolactones
(
Beta Dex, 30 m ꢁ 0.25 mm ꢁ 0.25 μm). In almost
every case formation of the (-) isomer of lactone 6
was preferred (Tables II–IV). Only iodolactone 5
transformed by F. avenaceum gave the (ꢀ) isomer
of 6, but with a very small enantiomeric excess
(
4%). Lactone 7 obtained from chlorolactone 3 by
F. avenaceum was the (ꢀ) isomer with a good
enantiomeric excess (92%).
3-5 was F. avenaceum.
Only one micro-organism, F.avenaceum, gave two
different products during transformations of chloro-
lactone 3 and bromolactone 4, namely hydroxylactone
Declaration of interest: The authors report no
conflicts of interest.The authors alone are responsible
for the content and writing of the paper.
6
and 7, respectively.The lactone 6 had an equatorial
OH group cis oriented in relation to the axial C-O
bond of the g-lactone ring and trans oriented in rela-
tion to the axial methyl group at C-4 and the methyl
group at C-6. In the lactone 7 the OH group was also
in an equatorial position, but trans oriented in relation
to both the axial methyl group at C-6 and C-O bond
Structure 1. 2-Chloro-4,4,6-trimethyl-9-
oxabicyclo[4.3.0]nonan-8-one (3).
Structure 2. Cis (-)-2-hydroxy-4,4,6-trimethy-9-
oxabicyclo[4.3.0]nonan-8-one (6).

4
14 M. Grabarczyk & A. Bialo n´ ska
References
Kim KS, Lee SC, Kim KH, Shim WJ, Hong SH, Choi KH,
Yoon JH, Kim JG. 2009. Survey on organochlorine pesticides,
PCDD/Fs, dioxin-like PCBs and HCB in sediments from the
Han river, Korea. Chemosphere 75:580–587.
Liu JG, Zhang M, Zhang CF, Qi HY. 2008. Cytotoxic sesquit-
erpenes from Ligularia platyglossa. Phytochemistry 69:
Antoniotti S, Du n´ ach E. 2009. Tin(IV) triflimidate-catalyzed
cyclization of epoxy esters to functionalized d-hydroxy-g-lac-
tones. Tetrahedron Lett 50:2536–2539.
Bruker AXS. 1999. SHELXTL V5.1.
DeWit CA. 2002. An overview of brominated flame retardants in
the environment. Chemosphere 46:583–624.
El-Gamal AA. 2001. Sesquiterpene lactones from Smyrnium olu-
satrum. Phytochemistry 57:1197–1200.
El Hassany B, El Hanbali F, Akssira M, Mellouki F, Haidour A,
Barrero A. 2004. Germacranolides from Anvillea radiate.
Fitoterapia 75:573–576.
Erable B, Maugard T, Goubet I, Lamare S, Legoy MD. 2005.
Biotransformation of halogenated compounds by lyophilized
cells of Rhodococcus erythropolis in a continuous solid–gas bio-
filte. Proc Biochem 40:45–51.
Fukushima T, Tanaka M, Gohbara M, Fujimori T. 1998. Phyto-
toxicity of three lactones from Nigrospora sacchari. Phyto-
chemistry 48:625–630.
2231–2236.
Ortet R, Prado S, Mouray E, Thomas OP. 2008. Sesquiterpene
lactones from the endemic Cape Verdean Artemisia gorgonum.
Phytochemistry 69: 2961–2965.
Oxford Diffraction. 2001. Poland Sp z oo. CrysAlis Red V166.
Paruch E, Ciunik Z, Nawrot J, Wawrze n´ czyk C. 2000. Lactones
9. Synthesis of terpenoid lactones-active insect antifeedants.
J Agric Food Chem 48:4973–4977.
Paruch E, Nawrot J,Wawrze n´ czyk C. 2001. Lactones 11. Feeding-
deterrent activity of some bi- and tricyclic terpenoid lactones.
Pest Manag Sci 57:776–780.
Pisarski B, Wawrze n´ czyk C. 2006. Lactones 30. Reaction of
halolactones with trialkylphosphites. Tetrahedron Lett 47:
6875–6877.
Gładkowski W, Grabarczyk M, Konopka M, Wawrze n´ czyk C.
Popsavin V, Krstic I, Popsavin M. 2003. Enantiopure hydroxylac-
tones from d-xylose. A novel approach to the enantiodivergent
synthesis of (ꢀ)- and (ꢂ)-muricatacin suitable for the prepara-
tion of 7-oxa analogues. Tetrahedron Lett 44:8897–8900.
Pujar PP, Sawaikar DD, Rojatkar SR, Nagasampagi BA. 2000. A
new germacranolide from Artemisia pallens Fitoterapia 71:
2
004. Lactones 20. Biohydroxylation of saturated bicyclic
g-lactones with the substituted cyclohexane system. J MoL
Catal B: Enzym 29:13–18.
Gładkowski W, Grabarczyk M,Wi n´ ska K, Biało n´ ska A, Ciunik Z,
Wawrze n´ czyk C. 2006. Lactones 27. Transformations of
g-lactones fused to a dimethylcyclohexane ring in Absidia
cylindrospora cultures. J Mol Catal B: Enzym 39:31–39.
Gładkowski W, Grabarczyk M, Wi n´ ska K, Ratu s´ B, Biało n´ ska A,
Ciunik Z, Wawrze n´ czyk C. 2007. Lactones 26. Stereoselective
microbial epoxidation of unsaturated bicyclic g-lactones with
the alkylsubstituted cyclohexane system. J Mol Catal B: Enzym
590–592.
Rodriguez S, Kneeteman M, Izquierdo J, Lopez I, Gonzalez FV,
Peris G. 2006. Diastereoselective synthesis of g-hydroxy a,b-
epoxyesters and their conversion into b-hydroxy α-sulfenyl
g-butyrolactones. Tetrahedron 62:11112–11123.
4
9:79–87.
Segev O, Abeliovich A, Kushmaro A. 2007. Biodegradation of
dibromoneopentyl glycol by a bacterial consortium. Chemos-
phere 68:958–964.
Sheldrick GM. 2008. Acta Crystallogr Sect A:Found Crystallogr
A64:112-122 (instead Bruker AXS.1999. SHELXTL V5.1).
Vajs V, Todorovic N, Ristic M. 1999. Guaianolides from Centau-
rea nicolai: antifungal activity. Phytochemistry 52:383–386.
Wawrze n´ czyk C, Grabarczyk M, Biało n´ ska A, Ciunik J. 2003.
Lactones 16. Lactonization of g,d-epoxy esters with p-toluenesul-
fonic acid monohydrate. Tetrahedron 59:6595–6601.
Grabarczyk M, Szumny A, GładkowskiW, Biało n´ ska A, Ciunik Z,
Wawrze n´ czyk C. 2005. Lactones 18. Synthesis of bicyclic lac-
tones with methyl-, di- and trimethyl substituted cyclohexane
system. Polish J Chem 79:1763–1771.
Grotowska AK, Wawrze n´ czyk C. 2002. Lactones 13. Biotransfor-
mation of iodolactones. J Mol Catal B: Enzym 19–20:203–208.
Janssen DB,Van der Ploeg J, Pries F. 1994. Genetic and biochemistry
of dehalogenating enzymes. Annu Rev Microbiol 48:163–191.
Kiegiel J, Nowacki J,Tarnowska A, Stachurska M, Kurczak J. 2000.
A new asymmetric route to synthetically useful g-substituted
g-butyrolactones. Tetrahedron Lett 41:4003–4006.
Zhang J, Feng XZ. 1997. Sesquiterpene hydroxylactone from
Lactarius subvellereus. Phytochemistry 46:157–159.