Fungal strains as catalysts for the biotransformation of halolactones by hydrolytic dehalogenation with the dimethylcyclohexane system

Article abstract of DOI:10.3390/molecules17089741

Bicyclic chloro-, bromo- and iodo-γ-lactones with dimethylcyclohexane rings were used as substrates for bioconversion by several fungal strains (Fusarium, Botrytis and Beauveria). Most of the selected microorganisms transformed these lactones by hydrolytic dehalogenation into the new compound cis-2-hydroxy-4,6-dimethyl-9-oxabicyclo[4.3.0]- nonan-8-one, mainly the (-)-isomer. When iodo-γ-lactone was used as the substrate, two products were observed: a hydroxy-γ-lactone and an unsaturated lactone. The structures of all substrates and products were established on the basis of their spectral data. The mechanism of dehalogenation of three halolactones was also studied.

Full text of DOI:10.3390/molecules17089741

Molecules 2012, 17, 9741-9753; doi:10.3390/molecules17089741
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Fungal Strains as Catalysts for the Biotransformation of
Halolactones by Hydrolytic Dehalogenation with the
Dimethylcyclohexane System
Małgorzata Grabarczyk
Department of Chemistry, University of Environmental and Life Sciences, Norwida 25,
50-375 Wrocław, Poland; E-Mail: magrab@onet.pl; Tel.: +48-71-3205-252; Fax: +48-71-3207-744
Received: 26 June 2012; in revised form: 30 July 2012 / Accepted: 1 August 2012 /
Published: 14 August 2012
Abstract: Bicyclic chloro-, bromo- and iodo-γ-lactones with dimethylcyclohexane rings
were used as substrates for bioconversion by several fungal strains (Fusarium, Botrytis and
Beauveria). Most of the selected microorganisms transformed these lactones by hydrolytic
dehalogenation into the new compound cis-2-hydroxy-4,6-dimethyl-9-oxabicyclo[4.3.0]-
nonan-8-one, mainly the (−)-isomer. When iodo-γ-lactone was used as the substrate, two
products were observed: a hydroxy-γ-lactone and an unsaturated lactone. The structures of
all substrates and products were established on the basis of their spectral data. The
mechanism of dehalogenation of three halolactones was also studied.
Keywords: lactones; biotransformation; hydrolytic dehalogenation; Fusarium species
1. Introduction
Halogenated organic compounds are often found in Nature. They are typically isolated from many
marine organisms such as red algae, bacteria, sponges and corals. Among them there are found
sesquiterpenoids [1–3], diterpenoids [4–6], lactones [7,8] and macrolides [9,10]. Halocompounds can
be used as therapeutic agents and they have different biological properties such as anticancer [11,12],
cytotoxic [13–17], antimicrobial [18] and antimalarial [19] activities. Halogenated organic compounds
are also used as solvents, as intermediates for chemical syntheses and also as pesticides or artificial
fertilisers. Such compounds are theoretically good because they protect our food from pests and they
have a positive effect on crop growth, but they can accumulate in soil or in water. As a consequence, in
large amounts, they can be poisonous to other animals or fish [20,21].

Molecules 2012, 17
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There are many different methods used for the biodegradation of halocompounds containing
aromatic rings using algae [22,23], bacteria [24–26], fungi [27–30]. There are fewer reports about
dehalogenation of non-aromatic compounds. Bacteria have been used to biodegrade different
aliphatic compounds [31–33]. Various fungal strains were used for biodegradation of terpenoid
halo-lactones [34–36]. These fungal strains are mainly able to introduce a hydroxy group in the place
of the halogen atom or to eliminate the halogen through the formation of a double bond.
Filamentous fungi of the genus Fusarium, Beauveria and Botrytis are known for their hydrolytic
properties in relation to miscellaneous compounds. Filamentous fungi of the genus Fusarium were often
used for biohydroxylation of different substrates such as sesquiterpenoids [37], diterpenoids [37–41],
steroids [42], sesquiterpene lactones [43–45], nitriles [46] and flavonoids [47]. An interesting example
of biohydroxylation is the use of Fusarium oxysporum for the production of α-terpineol (a
commonly used fragrance compound) [48,49]. The fungal strain Beauveria bassiana was used for
biohydroxylation of hydrocarbons [50], sesquiterpenes [50,51], diterpenes [52], lactone lovastatin [53]
amides [50]. The filamentous fungus Botrytis cinerea was also used to introduce hydroxy groups on
different substrates like monoterpenes [37], sesquiterpenoids [54,55], steroids [54] or halogenated
aromatic compound [56].
Bearing in mind that the fact that there are many reports about biological hydroxylation, but very
few of them concern the hydrolytic dehalogenation of halolactones, therefore, in this study, further
examples of the biohydroxylation of chloro-, bromo- and iodolactones with a dimethylcyclohexane
ring are shown. Halolactones have been chosen as model compounds because some of them exhibit
antifeeding activity against different insect pests and they can be used in insect pest control [57–62].
Additionally, halolactones are relatively simple to synthesise in good yield. Using these lactones as
substrates led me to select microorganisms which are able to eliminate a halogen atom from a
molecule. An additional advantage of this method is that fungal strains often introduce a hydroxy
group into the molecule, providing hydroxylactones which are different than those obtained using the
classical synthetic methods.
2. Results and Discussion
The substrates used for biotransformation were the racemic halolactones 3–5. They were all
synthesised from a known γ,δ-unsaturated ester 1 (Scheme 1). Iodolactone 5 was obtained by basic
hydrolysis of ester 1 and iodolactonisation of acid 2 according to the procedure described earlier [63].
Lactones 3 and 4 were also obtained in two steps: basic hydrolysis of ester 1 and bromo- or
chlorolactonization using N-bromosuccinimide (NBS) or N-chlorosuccinimide (NCS), respectively.
The structures of chloro-γ-lactone 3 and bromo-γ-lactone 4 were determined on the basis of their
13
spectral data (1H-NMR, C-NMR, COSY, HMQC and IR) and elemental analysis. The absorption
bands at 1788 cm−1 for 3 and 1789 cm−1 for 4 in their IR spectra confirmed the presence of a γ-lactone
ring in the structure of these compounds. The similarity between the spectral data (1H-NMR) of
chlorolactone 3, bromolactone 4 and the previously obtained iodolactone 5 [63] suggested that their
structures were identical. In all three cases, the cyclohexane ring was found in the chair conformation.
The signal of proton H-1 in the NMR spectra looked like a broad singlet. The multiplet of proton

Molecules 2012, 17
9743
H-2 had very small coupling constants. This indicates that both H-1 and H-2 protons were in
trans-diequatorial positions.
Scheme 1. Synthesis of halolactones 3–5.
O
COOEt
COOH
O
I
I2, KI
KOH, EtOH
NaHCO3
1
2
5
NCS
Cl
NBS
O
O
O
O
Br
3
4
Biotransformations were carried out in two steps: first, the screening transformation and then the
preparative one. During the screening procedure, eight fungal strains of local origin: Fusarium
culmorum, Fusarium avenaceum, Fusarium oxysporum, Fusarium tricinctum, Fusarium semitectum,
Fusarium solani, Botrytis cinerea and Beauveria bassiana were tested for their ability to convert
halolactones into other products. The progress of all the biotransformations was monitored by means
of standard techniques (TLC and GC). All known products (1, 2, 5 and 7) obtained during syntheses or
biotransformation were analyzed on a HP-5 column on the basis of their retention times due to with
their known patterns obtained earlier in our team [63]. The retention times of new and expected
products (3 and 4) were compared with the analogous ones obtained previously [35]. During the
biotransformation of chloro- 3 and bromolactone 4 only two peaks coming from the substrate and the
product were observed in the chromatogram, whose relationship changed over time. The structures of
13
all obtained products were then confirmed by spectroscopic methods (1H-NMR, C-NMR, COSY,
HMQC and IR) and also elemental analysis.
The composition of the product mixture shows that, when chloro- 3 and bromolactone 4 were used
as substrates, hydroxylactone 6 was obtained as the only product. During the transformation of
iodolactone 5, the formation of the same hydroxylactone 6 was observed and in four cases (when
F. culmorum, F. solani, F. avenaceum and F. semitectum were used as biocatalysts) the unsaturated
lactone 7 [63] (Scheme 2, Table 1) was obtained too.

Molecules 2012, 17
9744
Scheme 2. Biotransformations of halolactones 3–5.
O
O
O
O
O
O
X
OH
microorganism
+
3: X=Cl
4: X=Br
5: X=I
6
3, 4, 5
7
Table 1. The composition (in % according to GC) of the product mixtures of screening
biotransformations of lactones 3, 4, 5.
Time of
Products of transformations (%)
Strain
incubation
6 (from 3)
6 (from 4)
6 (from 5)
7 (from 5)
(days)
5
Fusarium
culmorum
AM10
-
19.5
54.1
75.1
7.8
10.3
22.7
35.5
4.8
28.5
9
5.0
41.2
14
5
7.4
51.8
Fusarium
avenaceum
AM11
-
1.4
9
27.4
57.7
9.1
48.2
56.3
1.5
10.4
15.9
2.5
2.9
14
5
6.3
Fusarium
oxysporum
AM13
-
9
40.5
60.4
-
27.6
65.5
10.8
24.7
52.4
6.6
36.3
52.0
3.1
-
14
5
-
Fusarium
tricinctum
AM16
-
9
20.9
53.3
5.7
16.4
24.8
10.5
22.7
42.0
2.7
-
14
5
-
Fusarium
semitectum
AM20
11.0
9
16.7
51.4
1.8
30.3
55.9
27.7
48.7
61.1
3.6
20.0
14
5
9.3
Fusarium solani
AM203
-
9
33.0
76.6
3.3
35.8
54.7
7.3
6.5
14
5
7.7
Botrytis cinerea
-
-
-
-
-
-
AM235
9
84.1
85.3
6.1
56.6
95.0
3.3
24.3
27.7
3.5
14
5
Beauveria
bassiana
AM278
9
22.3
29.7
18.7
26.5
10.2
19.7
14
These results indicated that all three substrates, chloro- 3, bromo- 4 and iodolactone 5, were always
transformed into the same product—hydroxylactone 6. An additional product (unsaturated lactone 7)
was observed only for iodolactone 5, in three cases in small quantities and in one (when Fusarium
culmorum was used) in good yield. Two microorganisms, Fusarium oxysporum and Fusarium solani,
transformed all three substrates with good yields (respectively 52%–60% and 55%–77%). Using

Molecules 2012, 17
9745
Botrytis cinerea led to products from chloro- and bromolactone with high yield (85% and 95%). The
other fungal strains converted one or two substrates with satisfactory yield (between 52% and 75%).
During these screening transformations, only the percentage of conversion was checked.
For all three lactones (compounds 3, 4 and 5), the abiotic control was carried out by adding each
one of them to a sterile medium containing 3 g glucose and 1 g peptobac in water (100 mL) and
shaking for two weeks. The samples were taken as in the case of the screening procedure. As a result
of the experiments, it was found that only the substrate was always in the reaction mixture. This means
that the hydrolytic dehalogenation of these halolactones does not occur without the presence of the
microorganisms. Another experiment was then carried out. The fungal strains were incubated for
14 days without the addition of substrate. This allowed me to find the secondary metabolites produced
by the tested strains.
The next step of preparative biotransformations was to find fungal strains which were capable of
converting the substrates in yields over 50%. Different Fusarium species and Botrytis cinerea were
chosen for this purpose. The results of these transformations are shown in Tables 2–4.
The structures of the two products 6 and 7 were established on basis of their spectral data. The IR
spectra showed that the γ-lactone ring had been retained during the biotransformation in both products
(absorption bands at 1788 cm−1 and 1779 cm−1, respectively). A strong, broad band found at
3292 cm−1 for 6 suggested the presence of a hydroxy group in the molecule.
Table 2. Results of preparative biotransformations of lactone 3 after 14 days.
Isolated
yield (%)
31.8
Isolated
yield (g)
0.029
D20
Strain
3 (%) 6 (%)
ee (%)
F. avenaceum AM11
F. oxysporum AM13
F. tricinctum AM16
F. solani AM203
43.2
39.0
25.8
12.2
16.2
56.8
61.0
74.2
87.8
83.8
6.7
+6.92 (c = 1.06, CHCl3)
−7.45 (c = 0.93, CHCl3)
−14.12 (c = 0.60, CHCl3)
−17.46 (c = 0.59, CHCl3)
−9.40 (c = 0.32, CHCl3)
26.3
0.024
15.8
25.8
33.8
16.7
36.2
0.033
37.3
0.034
B. cinerea AM235
37.3
0.034
Table 3. Results of preparative biotransformations of lactone 4 after 14 days.
Isolated
yield (%)
31.7
Isolated
yield (g)
0.024
D20
Strain
4 (%) 6 (%)
ee (%)
F. culmorum AM10
F. avenaceum AM11
F. oxysporum AM13
F. semitectum AM20
F. solani AM203
22.7
41.6
33.0
37.3
33.7
7.3
77.3
58.4
67.0
62.7
66.3
92.7
0
-
30.2
0.023
12.8
19.0
16.2
32.0
8.9
−4.56 (c = 0.59, CHCl3)
−9.44 (c = 0.77, CHCl3)
−7.11 (c = 0.94, CHCl3)
−33.42 (c = 1.00, CHCl3)
−2.20 (c = 0.99, CHCl3)
28.6
0.021
43.0
0.032
38.0
0.028
B. cinerea AM235
59.0
0.044

Molecules 2012, 17
Table 4. Results of preparative biotransformations of lactone 5 after 14 days.
9746
4
(%)
6
7
Isolated yield
Isolated yield
of 6 (g)/7 (g) 6 (%)/7 (%)
ee of
D20
Strain
(%) (%) of 6 (%)/7 (%)
F. culmorum
15.2 39.9 44.9
5.8/10.9
21.2/-
0.004/0.006
0.016/-
15.3/0
12.3/-
25.1/-
−15.83
(c = 0.28, CHCl3)/-
−11.22
(c = 0.21, CHCl3)
−33.42
AM10
F. oxysporum 40.2 59.8
-
-
AM13
F. solani
37.4 55.5
11.1/-
0.008/-
AM203
(c = 0.38, CHCl3)
During the earlier analysis of the structure of iodolactone 5, it was shown that the signals of the H-1
and H-2 protons were multiplets with very small coupling constants, indicating their trans diequatorial
positions and the trans diaxial positions of the iodine atom and the C-O bond.
1
The analysis of the H-NMR spectrum of hydroxy-γ-lactone 7 in comparison to halolactones 3, 4
and 5 led to the confirmation that the H-1 proton, which was a doublet with a small coupling constant
(J = 3.1 Hz), was located in an equatorial position. However, the H-2 proton gave a wide mutiplet,
which suggested its axial position.
For the determination of the changes in structure of the hydroxylactone 6 relative to the iodolactone
5, two additional 1H-NMR spectra were recorded: COSY and HMQC. The analysis of these spectra led
to the determination of differences between these two lactones. In the case of hydroxylactone 6, some
proton signals moved toward a higher field, especially the H-1, H-2 and H-3 protons. The signal of the
H-1 proton moved from 4.55 ppm (for iodolactone 5) to 4.25 ppm (for hydroxylactone 6), while the
signal of the axial H-3 proton moved from 1.59 ppm (for 5) to 1.22 ppm (for 7). The biggest difference
was visible for the signal from the H-2 proton, which changed its location from 4.70 ppm (for 5) to
3.80 ppm (for 6). The shape of the signal of the H-2 proton gave a wide mutiplet which suggested its
axial position. The doublet of the H-1 proton with a small coupling constant (J = 3.1 Hz) indicated its
equatorial position. In the case of the H-3 proton, the signal was a doublet of doublets of doublets for
lactone 5 and a doublet of doublets for lactone 6; the coupling constants changed from 15.3, 11.5 and
3.9 Hz for iodolactone 5 to 12.4 and 12.2 Hz for hydroxylactone 6.
Scheme 3. Equatorial location of hydroxy group in lactone 6 as the stereochemical
consequence of microbial hydrolytic dehalogenation of chloro- (3), bromo- (4) and
iodolactone (5) proceeding with the inversion at C-2.
O
O
O
H
O
7
7
5
5
1
1
6
dehalogenation
6
H
OH
H
3
3
4
2
4
3: X=Cl
2
4: X=Br
5: X=I
3, 4, 5
X
6
H
These observations confirmed that in hydroxylactone 6, the H-2 proton and the C-O bond were in
trans diaxial positions, while the H-1 proton and the OH group were situated in trans diequatorial

Molecules 2012, 17
9747
positions. These results indicate that the OH group was cis oriented in relation to the lactone ring. It
suggested also that mechanism of the reaction of the hydrolytic dehalogenation is similar to the SN2
substitution (Scheme 3).
The second product of biotransformation of iodolactone 5 was an unsaturated lactone 7. It was
identified due to the presence of the characteristic signal from a double bond (between 5.80 and 5.95 Hz)
on the 1H-NMR spectra.
The optical activity of all hydroxylactones obtained from halolactones during the preparative
biotransformations was measured. The enantioselectivity of biotransformation is taken under
consideration to evaluate the ability of microorganisms to transform selected substrates into new
products. Enantiomeric excess was determined by GC using a chiral column (Beta Dex,
30 m × 0.25 mm × 0.25 μm). The results are shown in Tables 2–4. In almost every case, the (−) isomer
of lactone 6 was created. Only when chlorolactone 3 was transformed by F. avenaceum was the (+)
isomer of 6 observed; however, when bromolactone 6 was transformed by F. culmorum, the product
was a racemic mixture. The enantiomeric excess for all products was very low (from 6.7% to 33.8%).
The best result (ee = 25.1% to 33.8%) was observed when F. solani was used as the biocatalyst.
3. Experimental
3.1. General
The progress of chemical reactions and biotransformations and the purity of isolated products were
monitored by the TLC technique on silica gel-coated aluminium plates (DC-Alufolien Kieselgel
60 F254, Merck) 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 as follows: injector 150 °C, detector (FID) 300 °C, column temperature:
100 °C (hold 1 min), 100–200 °C (rate 10 °C/min), 200–300 (rate 50 °C/min), 300 °C (hold 1 min).
The enantiomeric compositions of the products obtained during biotransformation 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 (hold 45 min),
140–200 °C (rate 20 °C /min), 200 °C (hold 1 min). All products were purified by means of preparative
1
13
column chromatography on silica gel (Kieselgel 60, 230−400 mesh). H-NMR and C- NMR spectra
were recorded in a CDCl3 solution on a Bruker Avance DRX 300 spectrometer. The assignments of
13C-NMR chemical shifts were made by means of C/H correlation heteronuclear multiple quantum
coherence (HMQC). IR spectra were determined using FTIR on a Thermo-Mattson IR 300 spectrometer.
Optical rotations were measured on an Autopol IV automatic polarimeter (Rudolph). Elemental analysis
was done using Vario EL III CHNS automatic analyser from Elemental Analyzersysteme.
2-Chloro-4,6-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (3). A mixture of acid 2 (3.2 g, 0.019 mol) and
NCS (3.8 g, 0.029 mol) in THF (30 mL) was stirred at room temperature. After 24 h, water was added
to the mixture and the product was diluted with diethyl ether. The separated organic layer was washed
with a saturated NaHCO3 solution and brine, then dried with anhydrous magnesium sulfate. The crude
product was purified on silica gel (hexane-acetone, 3:1) and 2.1 g (yield 50%) of chlorolactone 3 was
obtained. 1H-NMR (CDCl3): 0.93 (d, J = 6.5 Hz, 3H, CH3C-4), 1.06 (dd, J = 13.1 and 13.0 Hz, one of

Molecules 2012, 17
9748
CH2-5), 1.39 (s, 3H, CH3C-6), 1.53 (m, 1H, one of CH2-5), 1.62 (ddd, J = 15.2, 12.0 and 3.5 Hz, 1H,
one of CH2-3), 1.92 (m, 1H, one of CH2-3), 2.05 (m, 1H, H-4), 2.26 and 2.48 (two d, J = 16.7 Hz, 2H,
13
CH2-7), 4.29 (m, 1H, H-1), 4.50 (m, 1H, H-2); C-NMR (CDCl3): 21.25 (C-9), 21.14 (C-4), 23.47
(C-10), 36.48 (C-3), 39.06 (C-6), 41.83 (C-5), 46.43 (C-7), 54.70 (C-2), 84.06 (C-1), 174.69 (C-8); IR
(KBr, cm−1): 2958 (s), 1788 (s), 1375 (m), 1207 (s), 1009 (m), 670 (m). Anal. Calcd. for C10H15ClO2
(202.68): C, 59.26; H, 7.46. Found: C, 59.18; H, 7.43.
2-Bromo-4,6-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (4). Acid 2 (3.2 g, 0.019 mol) was dissolved in
THF (30 mL), then NBS (4.5 g, 0.025 mol) was added. The mixture was stirred for 24 h at room
temperature and then water was added. The product was extracted with diethyl ether. The ether
fractions were combined, washed with a saturated NaHCO3 solution and brine, then dried with
anhydrous magnesium sulfate. The crude product was purified on silica gel (hexane-acetone, 3:1)
giving 2.3 g (yield 92%) of bromolactone 4. 1H-NMR (CDCl3): 0.95 (d, J = 6.5 Hz, 3H, CH3C-4), 1.07
(dd, J = 16.1 and 13.0 Hz, one of CH2-5), 1.45 (s, 3H, CH3C-6), 1.56 (dm, J = 16.1 Hz, 1H, one of
CH2-5), 1.70 (ddd, J = 15.2, 11.7 and 3.5 Hz, 1H, one of CH2-3), 1.97 (m, 1H, one of CH2-3), 2.09 (m, 1H,
H-4), 2.27 and 2.48 (two d, J = 16.7 Hz, 2H, CH2-7), 4.43 (m, 1H, H-1), 4.57 (m, 1H, H-2); 13C-NMR
(CDCl3): 21.30 (C-9), 21.88 (C-4), 24.15 (C-10), 37.52 (C-3), 39.15 (C-6), 40.68 (C-2), 41.95 (C-5),
46.62 (C-7), 85.33 (C-1), 174.38 (C-8); IR (KBr, cm−1): 2954 (s), 1789 (s), 1371 (m), 1204 (s), 1002
(s), 632 (m). Anal. Calcd. for C10H15BrO2 (247.13): C, 48.60; H, 6.12. Found: C, 48.57; H, 6.17.
1
2-Iodo-4,6-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (5). H-NMR (CDCl3): 0.97 (d, J = 6.6 Hz, 3H,
CH3C-4), 1.09 (dd, J = 13.2 and 12.9 Hz, one of CH2-5), 1.50 (m, 1H, one of CH2-5), 1.53 (s, 3H,
CH3C-6), 1.59 (ddd, J = 15.3, 11.5 and 3.9 Hz, 1H, one of CH2-3), 1.97 (m, 1H, one of CH2-3), 2.07
(m, 1H, H-4), 2.25 and 2.47 (two d, J = 16.8 Hz, 2H, CH2-7), 4.55 (m, 1H, H-1), 4.70 (m, 1H, H-2);
13C-NMR (CDCl3): 21.34 (C-9), 33.41 (C-4), 23.77 (C-2), 25.39 (C-10), 38.69 (C-3), 39.62 (C-6),
42.02 (C-5), 46.72 (C-7), 85.79 (C-1), 173.98 (C-8); IR (film, cm−1): 2924 (s), 1776 (s), 1453 (m),
1140 (s), 989 (s), 615 (s).
1
2-Hydroxy-4,6-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (6). H-NMR (CDCl3): 0.94 (d, J = 6.6 Hz,
3H, CH3C-4), 1.02 (d, J = 13.2 Hz, H-5ax), 1.19 (s, 3H, CH3C-6), 1.22 (dd, J = 12.4 and 12.2 Hz, 1H,
H-3ax), 1.41 (m, 1H, H-5eq), 1.60 (m, 1H, H-4), 1.83 (dm, J = 12.4 Hz, 1H, H-3eq), 2.00 (m, 1H, OH),
2.31 and 2.46 (two d, J = 16.5 Hz, 2H, CH2-7), 3.80 (m, 1H, H-2), 4.24 (d, J = 3.3 Hz, 1H, H-1);
13C-NMR (CDCl3): 21.65 (C-10), 21.39 (C-9), 26.88 (C-4), 37.41 (C-3), 40.54 (C-6), 41.21 (C-5),
46.25 (C-7), 68.07 (C-2), 85.81 (C-1), 1753.56 (C-8); IR (film, cm−1): 3340 (s), 2912 (s), 1788 (s),
1134 (s), 1052 (s). Anal. Calcd. for C10H15O3 (184.24): C, 65.20; H, 8.75. Found: C, 65.15; H, 8.71.
1
4,6-Dimethyl-9-oxabicyclo[4.3.0]non-2-en-8-one (7). H-NMR (CDCl3): 1.04 (d, J = 7.1 Hz, 3H,
CH3C-4), 1.15 (s, 3H, CH3C-6), 1.22 (dd, J = 13.0 and 6.4 Hz, 1H, one of CH2-5), 1.52 (dd, J = 13.0
and 4.9 Hz, 1H, one of CH2-5), 2.24 (m, 1H, H-4), 2.33 and 2.52 (dd, J = 17.2 Hz, 2H, CH2-7), 4.31
(d, J = 4.3 Hz, 1H, H-1), 5.83 (ddd, J = 10.0, 4.3 and 2.4 Hz 1H, H-3), 5.94 (d, J = 10.0 Hz, 1H,
H-2); IR (film, cm−1): 1780 (s), 1651 (m), 1212 (s), 999 (s).

Molecules 2012, 17
9749
3.2. Microorganisms
The fungal strains, which were used in this study, were obtained from the collection of the Institute
of Biology and Botany, Medical University, Wrocław (Fusarium culmorum AM10, Fusarium
avenaceum AM11, Fusarium oxysporum AM13, Fusarium tricinctum AM16, Fusarium semitectum
AM20, Fusarium solani AM203, Botrytis cinerea AM235 and Beauveria bassiana AM278). They all
are available in Department of Chemistry, University of Environmental and Life Sciences. These
strains were cultivated on Sabouraud’s agar containing 5 g of aminobac, 5 g of peptone, 40 g of
glucose and 15 g of agar dissolved in 1 L of distilled water at 28 °C and stored in refrigerator at 4 °C.
3.3. Screening Biotransformation
In all transformation experiments, the fungal strains were cultivated on a rotary shaker at 25 °C in
two Erlenmeyer flasks which contained 100 mL of medium containing 3 g of glucose and 1 g of
peptobac in water (100 mL). After 4 days, 10 mg of the substrate dissolved in 1 mL of acetone was
added to the culture in each flask. Incubation of the shaken cultures with the substrate was continued
for 14 days. After 5, 9 and 14 days of incubation, 30 mL of the reacting mixture including mycelium,
unreacted substrate and expected product was taken from the two flasks and extracted with 30 mL of
dichloromethane. After evaporated the solvent the residue was dissolved in 2 mL of acetone and
analyzed by TLC (silica gel, hexane-acetone, 3:1) and GC (HP-5 column).
3.4. Preparative Biotransformation
The halolactones 3–5 (100 mg) were dissolved in 10 mL of acetone and distributed between 10
Erlenmeyer flasks with the 4-day cultures of fungal strains prepared as described in the screening
procedure. The selected microorganisms were incubated with the substrates for 14 days, then the
products were extracted with dichloromethane (3 × 50 mL). The organic solutions were dried (MgSO4)
and the solvent was evaporated in vacuo. Mixtures containing the product, the unreacted substrate and
metabolites of the fungi were separated by column chromatography (silica gel, hexane:acetone 3:1) to
obtain pure products.
4. Conclusions
The filamentous fungal strains chosen for the conversion of halolactones 3, 4 and 5 showed very
high regio- and stereoselectivity. The OH group was introduced at C-2 in the equatorial position. Since
the halogen atom was in an axial position in all three substrates, this allowed us to conclude that the
hydrolytic dehalogenation reaction proceeded analogously to the mechanism of SN2 nucleophilic
substitution. Two substrates, chlorolactone 3 and bromolactone 4, were transformed by five and six
fungal strains, respectively, in good yield (conversions over 50%). The iodolactone 5 was transformed
by only three microorganisms in yields over 50%, and additionally this substrate gave an unsaturated
lactone 7 in some cases. The best microorganisms for the transformation of halolactones 3, 4 and 5 in
the best yields were F. solani, F. oxysporum and B. cinerea. Taking into account its ability to
transform substrates with high enantiomeric excess, the best biocatalyst was F. solani.

Molecules 2012, 17
9750
Acknowledgments
This project was financed by European Union from the European Regional Development Fund.
Grant No. POIG. 01.03.01-00-158/09.
References
1. Vairappan, C.S.; Suzuki, M.; Ishii, T.; Okino, T.; Abe, T.; Masuda, M. Antibacterial activity of
halogenated sesquiterpenes from Malaysian Laurencia spp. Phytochemistry 2008, 69, 2490–2494.
2. Ji, N.Y.; Li, X.M.; Wang, B.G. Sesquiterpenes and Other Metabolites from the Marine Red Alga
Laurencia composita (Rhodomelaceae). Helv. Chim. Acta 2010, 93, 2281–2286.
3. Kladi, M.; Vagias, C.; Papazafiri, P.; Furnari G.; Serio, D.; Roussis, V. New sesquiterpenes from
the red alga Laurencia microcladia. Tetrahedron 2007, 63, 7606–7611.
4. Su, H.; Shi, D.Y.; Li, J.; Guo, S.J.; Li, L.L.; Yuan, Z.H.; Zhu, X.B. Sesquiterpenes from
Laurencia similes. Molecules 2009, 14, 1889–1897.
5. Rudi, A.; Benayahu, Y.; Kashman, Y. Negombins A-I, new chlorinated polyfunctional
diterpenoids from the marine sponge Negombata species. Org. Lett. 2007, 9, 2337–2340.
6. Sung, P.J.; Chiang, M.Y.; Tsai, W.T.; Su, J.H.; Su, Y.M.; Wu, Y.C. Chlorinated briarane-type
diterpenoids from the gorgonian coral Ellisella robusta (Ellisellidae). Tetrahedron 2007, 63,
12860–12865.
7. Diaz-Marrero, A.R.; Brito, I.; de la Rosa, J.M.; D’Croz, L.; Fabelo, O.; Ruiz-Perez, C.; Darias, J.;
Cueto, M. Novel lactone chamigrene-derived metabolites from Laurencia majuscule. Eur. J. Org.
Chem. 2009, 1407–1411.
8. Reed, K.A.; Manam, R.R.; Mitchell, S.S.; Xu, J.; Teisan, S.; Chao, T.H.; Deyanat-Yazdi, G.;
Neuteboom, S.T.C.; Lam, K.S.; Potts, B.C.M. Salinosporamides D-J from the marine
actinomycete Salinispora tropica, bromosalinosporamide, and thioester Derivatives are potent
inhibitors of the 20S proteasome. J. Nat. Prod. 2007, 70, 269–276.
9. Levenfors, J.J.; Hedman, R.; Thaning, C.; Gerhardson, B.; Welch, C.J. Broad-spectrum antifungal
metabolites produced by the soil bacterium Serratia plymuthica A 153. Soil Biol. Biochem. 2004,
36, 677–685.
10. Thaning, C.; Welch, C.J.; Borowicz, J.J.; Hedman, R.; Gerhardson, B. Suppression of Sclerotinia
sclerotiorum apothecial formation by the soil bacterium Serratia plymuthica: Identification of a
chlorinated macrolide as one of the causal agents. Soil Biol. Biochem. 2001, 33, 1817–1826.
11. Gutirrez-Cepeda, A.; Fernndez, J.J.; Gil, L.V.; Lopez-Rodriguez, M.; Norte. M.; Souto, M.L.
Nonterpenoid C15 Acetogenins from Laurencia marilzae. J. Nat. Prod. 2011, 74, 441–448.
12. Nam, S.J.; Gaudncio, S.P.; Kauffman, C.A.; Jensen, P.R.; Kondratyuk, T.P.; Marler, L.E.; Pezzuto,
J.M.; Fenical, W. Fijiolides A and B, inhibitors of TNF-α-induced NFκB activation, from a
marine-derived sediment bacterium of the genus Nocardiopsis. J. Nat. Prod. 2010, 73, 1080–1086.
13. Yin, S.; Boyle, G.M.; Carroll, A.R.; Kotiw, M.; Dearnaley, J.; Quinn, R.J.; Davis, R.A.
Caelestines A−D, Brominated quinolinecarboxylic acids from the Australian ascidian Aplidium
caelestis. J. Nat. Prod. 2010, 73, 1586–1589.
14. Lhullier, C.; Falkenberg; M.; Ioannou, E.; Quesada, A.; Papazafiri, P.; Horta, P.A.; Schenkel, E.P.;
Vagias, C.; Roussis, V. Cytotoxic halogenated metabolites from the Brazilian red alga Laurencia
catarinensis. J. Nat. Prod. 2010, 73, 27–32.

Molecules 2012, 17
9751
15. Han, B.; McPhail, K.L.; Gross, H.; Goeger, D.E.; Mooberry, S.L.; Gerwick, W.H. Isolation and
structure of five lyngbyabellin derivatives from a Papua New Guinea collection of the marine
cyanobacterium Lyngbya majuscula. Tetrahedron 2005, 61, 11723–11729.
16. Zhang, L.; An, R.; Wang, J.; Sun, N.; Zhang, S.; Hu, J.; Kuai, J. Exploring novel bioactive
compounds from marine microbes. Curr. Opin. Microbiol. 2005, 8, 276–281.
17. Zidorn, C.; Ellmerer, E.P.; Konwalinka, G.; Schwaiger, N.; Stuppner, H. 13-Chloro-3-O-β-D-
glucopyranosylsolstitialin from Leontodon palisae: The first genuine chlorinated sesquiterpene
lactone glucoside. Tetrahedron Lett. 2004, 45, 3433–3436.
18. Fehr, D.; Barlow, R.; McAtee, J.; Hemscheidt, T.K. Highly brominated antimicrobial metabolites
from a marine Pseudoalteromonas sp. J. Nat. Prod. 2010, 73, 1963–1966.
19. Yang, X.; Davis, R.A.; Buchanan, M.S.; Duffy, S.; Avery, V.M.; Camp, D.; Quinn, R.J.
Antimalarial bromotyrosine derivatives from the Australian marine sponge Hyattella sp.
J. Nat. Prod. 2010, 73, 985–987.
20. Särkkä, J.; Paasivirta, J.; Häsänen, E.; Koistinen, J.; Manninen, P.; Mäntykoski, K.; Rantio, T.;
Welling, L. Organic chlorine compounds in lake sediments. VI. Two bottom sites of Lake Ladoga
near pulp mills. Chemosphere 1993, 26, 2147–2160.
21. Koistinen, J.; Herve, S.; Ruokojärvi, P.; Koponen, J.; Vartiainen, T. Persistent organic pollutants
in two Finnish watercourses: Levels, congener profiles and source estimation by mussel
incubation. Chemosphere 2010, 80, 625–633.
22. Papazi, A.; Kotzabasis, K. Bioenergetic strategy of microalgae for the biodegradation of phenolic
compounds—Exogenously supplied energy and carbon sources adjust the level of biodegradation.
J. Biotechnol. 2007, 129, 706–716.
23. Papazi, A.; Kotzabasis, K. Inductive and resonance effects of substituents adjust the microalgal
biodegradation of toxical phenolic compounds. J. Biotechnol. 2008, 135, 366–373.
24. Zanaroli, G.; Pérez-Jiménez, J.R.; Young, L.Y.; Marchetti, L.; Fava, F. Microbial Reductive
Dechlorination of Weathered and Exogenous Co-planar Polychlorinated Biphenyls (PCBs) in an
Anaerobic Sediment of Venice Lagoon. Biodegradation 2006, 17, 19–27.
25. Hongsawat, P.; Vangnai, A.S. Biodegradation pathways of chloroanilines by Acinetobacter baylyi
strain. J. Hazard Mater. 2011, 186, 1300–1307.
26. Bao, H.; Gao, J.; Liu, Y.; Su, Y. A study of biodegradation/γ-irradiation on the degradation of p-
chloronitrobenzene. Radiat. Phys. Chem. 2009, 78, 1137–1139.
27. Katapodis, P.; Moukouli, M.; Christakopoulos, P. Biodegradation of indole at high concentration
by persolvent fermentation with the thermophilic fungus Sporotrichum. Int. Biodeterior.
Biodegradat. 2007, 60, 267–272.
28. Li, J.; Cai, W.; Zhu, L. The characteristics and enzyme activities of 4-chlorophenol
biodegradation by Fusarium sp. Bioresour. Technol. 2011, 102, 2985–2989.
29. Muzikar, M.; Kresinova, Z.; Svobodova, K.; Filipova, A.; Cvancarova, M.; Cajthamlova, K.;
Cajthaml, T. Biodegradation of chlorobenzoic acids by ligninolytic fungi. J. Hazard. Mater. 2011,
196, 386–394.
30. Wang, C.; Xi, J.Y.; Hu, H.Y.; We, X.H. Biodegradation of Gaseous Chlorobenzene by White-rot
Fungus Phanerochaete chrysosporium. Biomed. Enviro. Sci. 2008, 21, 474–478.
31. Hardman, D.J. Biotransformation of Halogenated Compounds. Crit. Rev. Biotechnol. 1991, 11, 1–40.
32. Janssen, D.B. Evolving haloalkane dehalogenases. Curr. Opin. Chem. Biol. 2004, 8, 150–159.

Molecules 2012, 17
9752
33. Janssen, D.B.; Oppentocht, J.E.; Poelarends, G.J. Microbial dehalogenation. Curr. Opin. Biotechnol.
2001, 12, 254–258.
34. Grotowska, A.K.; Wawrzeńczyk, C. Lactones 13. Biotransformation of iodolactones. J. Mol.
Catal. B: Enzym. 2002, 19–20, 203–208.
35. Grabarczyk, M.; Białońska, A. Biotransformations of chloro-, bromo- and iodolactone with
trimethylcyclohexane system using fungal strains. Biocatal. Biotransform. 2010, 28, 408–414.
36. Gładkowski, W.; Mazur, M.; Białońska, A.; Wawrzeńczyk, C. Lactones 35. Metabolism of
iodolactones with cyclohexane ring in Absidia cylindrospora culture. Enzyme Microb. Technol.
2011, 48, 326–333.
37. Borges, K.B.; Borges, W.S.; Durán-Patrón, R.; Pupo, M.T.; Bonato, P.S.; Collado, I.C. Stereoselective
biotransformations using fungi as biocatalysts. Tetrahedron: Asymmetry 2009, 20, 385–397.
38. Rodrigues-Filho, E.; Magnani, R.F.; Xie, W.; Mirocha, C.J.; Pathre, S.V; Hydroxylation of the Labdane
Diterpene Cupressic Acid by Fusarium graminearum. J. Braz. Chem. Soc. 2002, 13, 266–269.
39. Ghoumari, H.; Benajiba, M.H.; Garcia-Granados, A.; Fernandez, A.; Martinez, A.; Rivas, F.;
Arias, J.M. Biotransformations of ent-18-acetoxy-6-ketomanoyl oxides epimers at C-13 with
filamentous fungi. Phytochemistry 2006, 67, 2294–2302.
40. Garcia-Granados, A.; Fernandez, A.; Gutierrez, M.C.; Martinez, A.; Quiros, R.; Rivas, F.;
Arias, J.M. Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by
filamentous fungi. Phytochemistry 2004, 65, 107–115.
41. Tapia, A.A.; Vallejo, M.D.; Gouiric, S.C.; Feresin, G.E.; Rossomando, P.C.; Bustos, D.A.
Hydroxylation of dehydroabietic acid by Fusarium species. Phytochemistry 1997, 46, 131–133.
42. Świzdor, A.; Kołek, T. Transformations of 4- and 17α-substituted testosterone analogues by
Fusarium culmorum. Steroids 2005, 70, 817–824.
43. Kumari, G.N.K.; Masilamani, S.; Ganesh, M.R.; Aravind, S. Microbial transformation of
zaluzanin-D. Phytochemistry 2003, 62, 1101–1104.
44. Nobilec, E.; Aniol, M.; Wawrzeńczyk, C. Lactones 1. Hydroxylation of dihydro-β-
campholenolactone by Fusarium culmorum. Tetrahedron 1994, 50, 10339–10344.
45. Gładkowski, W.; Grabarczyk, M.; Konopka, M.; Wawrzeńczyk, C. Lactones 20:
Biohydroxylation of saturated bicyclic γ-lactones with the substituted cyclohexane system. J. Mol.
Catal. B Enzym. 2004, 29, 13–17.
46. Kaplan, O.; Nikolaou, K.; Pisvejcova, A.; Martinkova, L. Hydrolysis of nitriles and amides by
filamentous fungi. Enzyme Microb. Technol. 2006, 38, 260–264.
47. Bartmańska, A.; Huszcza, E.; Tronina, T. Transformation of isoxanthohumol by fungi. J. Mol.
Catal. B: Enzym. 2009, 61, 221–224.
48. Marostica, M.R., Jr.; Pastore, G.M. Production of R-(+)- α-terpineol by the biotransformation of
limonene from orange essential oil, using cassava waste water as medium. Food Chem. 2007, 101,
345–350.
49. Bicas, J.L.; de Quadros, C.P.; Neri-Numa, I.A.; Pastore, G.M. Integrated process for co-production of
alkaline lipase and R-(+)-α-terpineol by Fusarium oxysporum. Food Chem. 2010, 120, 452–456.
50. Grogan, G.J.; Holland, H.L. The biocatalytic reactions of Beauveria spp. J. Mol. Catal. B: Enzym.
2000, 9, 1–32.
51. Buchanan, G.O.; Williams, L.A.D.; Reese, P.B. Biotransformation of cadinane sesquiterpenes by
Beauveria bassiana ATCC 7159. Phytochemistry 2000, 54, 39–45.

Molecules 2012, 17
9753
52. Buchanan, G.O.; Reese, P.B. Biotransformation of diterpenes and diterpene derivatives by
Beauveria bassiana ATCC 7159. Phytochemistry 2001, 56, 141–151.
53. Qiao, L.; Xie, D.; Liu, Q.; Zou, J.; Shen, Z.; Dai, J. Microbial transformation of lovastatin by
Beauveria bassiana. Acta Pharm. Sinica B 2012, 2, 300–305.
54. Aleu, J.; Collado, I.C. Biotransformations by Botrytis species. J. Mol. Catal. B: Enzym. 2001, 13,
77–93.
55. Daoubi, M.; Duran-Patron, R.; Hernandez-Galan, R.; Benharref, A.; James, R.; Hanson J.R.;
Collado, I.C. The role of botrydienediol in the biodegradation of the sesquiterpenoid phytotoxin
botrydial by Botrytis cinerea. Tetrahedron 2006, 62, 8256–8261.
56. Bustillo, A.J.; Aleu, J.; Hernández-Galán, R.; Collado, I.C. Biotransformation of the fungistatic
compound (R)-(+)-1-(4′-chlorophenyl)propan-1-ol by Botrytis cinerea. J. Mol. Catal. B: Enzym.
2003, 21, 267–271.
57. Paruch, E.; Ciunik, Z.; Nawrot, J.; Wawrzeńczyk, C. Lactones 9. Synthesis of terpenoid
lactones-active insect antifeedants. J. Agric. Food Chem. 2000, 48, 4973–4977.
58. Paruch, E.; Nawrot, J.; Wawrzeńczyk, C. Lactones 11. Feeding-deterrent activity of some bi- and
tricyclic terpenoid lactones. Pest Manag. Sci. 2001, 57, 776–780.
59. Gabryś, B.; Szczepanik, M.; Dancewicz, K.; Szumny, A.; Wawrzeńczyk, C. Environmentally Safe
Insect Control: Feeding Deterrent Activity of Alkyl-Substituted γ- and δ-Lactones to Peach Potato
Aphid (Myzus persicae [Sulz.]) and Colorado Potato Beetle (Leptinotarsa decemlineata Say).
Polish J. Environ. Stud. 2006, 15, 549–556.
60. Wawrzeńczyk, C.; Grabarczyk, M.; Szumny, A.; Gabryś, B.; Dancewicz, K.; Nawrot, J.;
Prądzyńska, A.; Halarewicz-Pacan, A. Lactones 19. Synthesis and antifeedant activity of lactones
with methyl-, dimethyl- and trimethylcyclohexane system. In Chemistry for Agriculture;
Górecki, H., Dobrzański, Z., Kafarski, P., Eds.; Czech-Pol Trade: Wrocław, Poland, 2003;
pp. 117–129.
61. Szczepanik, M.; Grabarczyk, M.; Szumny, A.; Wawrzeńczyk, C. Feeding detergent activity of
lactones with di- and trimethylcyclohexane system against lesser mealworm, Alphitobius
diaperinus Panzer and Colorado potato beetle (Leptinotarsa decemlineata Say). J. Plant Prot. Res.
2003, 43, 87–96.
62. Dancewicz, K.; Gabryś, B.; Halarewicz-Pacan, A.; Grabarczyk, M.; Wawrzeńczyk, C. Effect of
terpenoid lactones with di- and trimethylcyclohexane systems on the behaviour of green peach
aphid Myzus persicae. Pesticydy (Pesticides) 2005, 4, 17–23.
63. Grabarczyk, M.; Szumny, A.; Gładkowski, W.; Białońska, A.; Ciunik, Z.; Wawrzeńczyk, C.
Lactones 18. Synthesis of bicyclic lactones with methyl-, di- and trimethyl substituted
cyclohexane system. Pol. J. Chem. 2005, 79, 1763–1771.
Sample Availability: Samples of the compounds 3–7 are available from the authors.
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