Synthesis, biotransformation and biological activity of halolactones obtained from β-ionone

Article abstract of DOI:10.1016/j.tet.2015.12.005

Commercially available β-ionone was used as a starting material for a four-step chemical synthesis of three new γ-halolactones. During these processes one known (β-ionol) and some new compounds (γ,δ-unsaturated ester, γ,δ-unsaturated acid, chloro-, bromo- and iodo-lactone) were obtained. In the last step, halolactones were converted into a hydroxylactone by several fungal strains (Fusarium species, Syncephalastrum racemosum, Botrytis cinerea). Most of the selected microorganisms transformed these lactones by the hydrolytic dehalogenation. The synthetic halolactones and the hydroxylactone obtained during biotransformation inhibited growth of some bacteria, yeasts and fungi and showed deterrent activity against aphids Myzus persicae.

Full text of DOI:10.1016/j.tet.2015.12.005

Tetrahedron 72 (2016) 637e644
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis, biotransformation and biological activity of halolactones
obtained from b-ionone
a
,
a
a
b
*
ꢀ
_
Ma1gorzata Grabarczyk , Katarzyna Winska , Wanda M a˛ czka , Barbara Zarowska ,
c
d
d
a
Gabriela Maciejewska , Katarzyna Dancewicz , Beata Gabry ꢀs , Miros1aw Anio1
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
a
b
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Chełmo nꢀ skiego 37/41, 51-630 Wrocław,
Poland
c
Faculty of Chemistry, Wrocław University of Technology, Wybrze z_ e Wyspia nꢀ skiego 27, 50-370 Wrocław, Poland
Department of Botany and Ecology, University of Zielona G oꢀ ra, Szafrana 1, 65-516 Zielona Gora, Poland
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 October 2015
Received in revised form 12 November 2015
Accepted 1 December 2015
Available online 18 December 2015
Commercially available
b
-ionone was used as a starting material for a four-step chemical synthesis of
three new -ionol) and some new compounds
-unsaturated ester, g,d-unsaturated acid, chloro-, bromo- and iodo-lactone) were obtained. In the last
g-halolactones. During these processes one known (b
(
g
,d
step, halolactones were converted into a hydroxylactone by several fungal strains (Fusarium species,
Syncephalastrum racemosum, Botrytis cinerea). Most of the selected microorganisms transformed these
lactones by the hydrolytic dehalogenation. The synthetic halolactones and the hydroxylactone obtained
during biotransformation inhibited growth of some bacteria, yeasts and fungi and showed deterrent
activity against aphids Myzus persicae.
Keywords:
b
-ionone
Lactones
Ó 2015 Elsevier Ltd. All rights reserved.
Hydrolytic dehalogenation
Fusarium species
Antimicrobial activity
9
,10
1
. Introduction
a few of them showed bioluminescent properties.
Other struc-
́
tural analogs of ionones, e.g.,
matic ring showed anti-ischemic activity. A very unusual product,
hydroxyionolactone, was obtained from -ionone following its
b-ionone substituted with an aro-
11
Ionones are an important group of natural compounds endowed
with interesting biological properties. Structural analogues of
ionone often occur in nature and are responsible for biological
properties of different plants and animals. Such compounds were
found, for example, in Greek tobacco or in brown algae Padina
tetrastromatica. Other compounds that are structural analogues of
b
-
b
12
oxidation by KMnO
Interesting products were also obtained by biotransformation of
-ionone. The most common one was 4-hydroxy- -ionone formed
when mutants of cytochrome P450 or recombinant Escherichia
4
.
1
b
b
2
13
14
b
-ionone possess a hydroxyl group or a hydroxyl group and oxirane
coli cells expressing cytochrome P450 were used as biocatalysts
or using PPL-catalyzed trans-esterification. Further derivatives
3
e5
15
ring. Some of these compounds showed an allelopathic activity.
The other
hexene ring and with a saturated side chain showed phytotoxic
activity.6 Two derivatives of
-ionone produced by a nudibranch
b
-ionone analogues with a hydroxyl group in the cyclo-
were obtained with plant and beetle biocatalysts. Caragana cham-
16
lagu cultures yielded (ꢀ)-5,6-epoxy-
b-ionone and larvae of Jap-
anese rhinoceros beetle (Allomyrina dichotoma) produced 4-keto-b-
b
1
7
Sclerodoris tanya showed ichthyotoxic activity against two species
ionol.
7
of predatory fish feeding on molluscs.
Lactones are another group of widely spread compounds pres-
ent in plants and sea organisms. Lactones with bromine or chlorine
atoms are usually found in marine organisms. These compounds
Derivatives of
b-ionone can be also obtained by chemical
methods. Some heterocyclic derivatives obtained synthetically
8
18e21
showed anti-inflammatory activity. A natural compound, luciferin,
exhibit diverse biological properties, e.g., they can be cytotoxic,
2
2
23
is responsible for the bioluminescence of the limpet-like snail Latia
neritoides. Some analogs of this compound were synthesized and
or antibacterial, they can reduce TNF-
a
-induced NF
k
B
or show
2
4
molluscicidal and ovicidal activity. Lactones containing a hy-
droxyl group are also characterized by important biological prop-
2
5
erties. They are often present in plants used in traditional folk
2
6
27e29
*
Corresponding author. Tel.: þ48 71 3205 252; fax: þ48 71 3207 744;
medicine. Hydroxylactones have anticancer,
cytotoxic,
e-mail address: magrab@onet.pl (M. Grabarczyk).
http://dx.doi.org/10.1016/j.tet.2015.12.005
0
040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

638
M. Grabarczyk et al. / Tetrahedron 72 (2016) 637e644
phytotoxic,30 antibacterial,31 and antiprotozoal32 properties and
they also protect mushrooms against predators, parasites, and
microorganisms.33
The acid (4) was used to obtain three halolactones: chloro- (5),
bromo- (6) and iodo-lactone (7). Chloro-, bromo-, and iodo-lacto-
nization were performed according to the procedures described
41,42
Combining our search for new derivatives of
a number of procedures for synthesis of various lactones,
decided to obtain halolactones from -ionone. These lactones were
b
-ionone and
earlier.
In all three cases, formation of a g-lactone ring was
3
4e37
we
observed and further confirmed by the presence of absorption
ꢁ
1
b
bands at 1786 (5), 1781 (6) and 1782 cm (7) in their IR spectra.
The coupling constants between H-7 and H-10 (10.6 Hz for 5 and
10.7 Hz for 6) as well as between H-10 and H-11 protons (9.9 Hz for
5 and 10.0 Hz for 6) indicated their mutual trans orientation, so
these protons were in an antiperiplanar conformation. It also
means that chlorine or bromine atoms were situated in the same
plane as the lactone ring. A narrow multiplet coming from the H-7
proton in both compounds indicated that the lactone ring was lo-
cated across the plane occupied by the cyclohexene ring. A signal
transformed into a hydroxylactone by fungal strains in the last step.
The microorganisms were used due to their stereoselectivity in the
introduction of a hydroxyl group in the place of a halogen atom by
the hydrolytic dehalogenation.
2
2
. Results and discussion
.1. Synthetic chemistry
A commercially available
coming from the CH
3
-12 group located at 1.55 ppm for (5) and
1.62 ppm for (6) also suggested its location in the same plane as the
b
-ionone was used as a substrate for
lactone ring. Iodolactone (7) presented us with a different situation.
The coupling constants between H-10 and H-11 protons of 2.3 Hz
indicated their anticlinal conformation. It means that the iodine
atom was located across the plane of the lactone ring. A wide
multiplet coming from the H-7 proton suggested that the lactone
ring was located in the same plane as the cyclohexene ring. A signal
a four-step chemical synthesis. This substrate was reduced to
ionol (2). The resulting allylic alcohol after Claisen rearrangement
gave -unsaturated ester (3). The ester was hydrolyzed to
unsaturated acid (4), which was subjected to lactonization and
yielded chlorolactone (5), bromolactone (6) and iodolactone (7)
b-
g,d
g,d-
(Scheme 1).
Scheme 1. Synthesis of halolactones 5e7.
Analysis of the 1H NMR spectra allowed us to determine the
structure of the compounds obtained during the synthesis. The
coming from the CH
group was heavily exposed and situated across the plane of the
lactone ring (Scheme 2). According to Snider and Jayaraman,
these differences in the structure of halolactones can be
explained by different mechanisms of chloro-, bromo- and iodo-
lactonization. During chloro- and bromo-lactonization, chlorine or
bromine atom attacks the double bond causing formation of
a chloronium (bromonium) ion. In the course of iodolactonization,
the carboxylate anion is attacked by the complex consisting of io-
dine and a double bond.
3
-12 group at 1.86 ppm suggested that this
4
4
45
coupling constant between the H-7 and H-8 protons (J¼16.4 Hz) in
b
-ionone (1) suggested their trans diaxial orientation. The same
situation was observed for
of -ionone (1) with lithium aluminum hydride in dry diethyl ether.
The coupling constant between the H-7 and H-8 protons in -ionol
2) was equal to 15.9 Hz. This observation also indicated trans ori-
b-ionol (2) obtained through a reduction
b
b
(
entation of the double bond in this molecule. Moreover, the cou-
pling constant between the H-8 and H-9 protons (J¼6.6 Hz)
suggested a trans position of the hydroxyl group located at C-9 in
relation to the H-8 proton. In the next step, allylic alcohol (2) was
subjected to JohnsoneClaisen rearrangement yielding an un-
saturated ester (3). A wide multiplet coming from H-7 suggested its
2.2. Biotransformation
Hydroxylactone was expected to be one of
b-ionone (1) de-
axial orientation, indicating that the CH
dicular to the plane of the ring. The unsaturated ester (3) was
reacted under basic conditions giving an unsaturated acid (4).
2
-11 group was perpen-
rivatives obtained during chemical synthesis. However, it was
found that obtaining hydroxylactone through chemical synthesis
gave no positive results. Therefore, an alternative method based on

M. Grabarczyk et al. / Tetrahedron 72 (2016) 637e644
639
Scheme 2. Spatial structures of lactones 5e7.
using whole cells of filamentous fungi as biocatalysts instead of
chemical reagents was employed. In the first step, nine fungal
strains (Fusarium culmorum AM10, Fusarium avenaceum AM11,
Fusarium oxysporum AM13, Fusarium tricinctum AM16, Fusarium
semitectum AM20, Fusarium equiseti AM22, Syncephalastrum race-
mosum AM105, Botrytis cinerea AM235, Cunninghamella japonica
AM472) were used for a screening procedure. This procedure was
performed to verify, which microorganisms were capable of con-
verting halolactones (5), (6) and (7) into the expected hydrox-
ylactone during hydrolytic dehalogenation. The results of GC
analyses of this step are presented in Table 1.
Fig. 1. The results of preparative biotransformation of lactone (6) (according to GC).
The data compiled in Table 1 indicated that the best results were
obtained for bromolactone (6), which was transformed by eight
microorganisms with a conversion degree between 50% and 95%.
Slightly less satisfactory results were observed for the trans-
formation of iodolactone (7)dfour strains were capable of con-
verting this compound with 50 %e79 % efficiency. The third
compound, chlorolactone (7), was converted by only one strain but
to a high degreedalmost 95%. During this step, a parallel experi-
ment consisting of using sterile medium (without microorganisms)
for checking the stability of halolactones (5e7) was performed. In
all cases, only the starting substrates were observed after 7 days in
the reaction mixture. Because during biotransformations the fungi
change the pH of the growth medium the stability of substrates
(
(
5e7) in acidic medium was also checked. The phosphate buffer
pH 4.5) was added to each halolactone dissolved in acetone and
Fig. 2. The results of preparative biotransformations of lactone (5) and lactone (7)
(according to GC).
the mixture was stirring for a week. In all cases in the reaction
mixture only the substrate was observed.
As the main objective of the screening procedure was to obtain
the new product with the greatest possible efficiency, bio-
transformation on a preparative scale used only these fungal strains
whose conversion degree of the substrate exceeded 50%. The re-
sults of these preparative transformations are presented in Figs. 1
and 2.
iodolactone (7) but the process efficiency was not very high
(51e70%). Chlorolactone (5) was converted by only one strain but
with very high yield of 98%, according to GC (Fig. 2). All these strains
transformed halolactones (5e7) into the same pro-
ductdhydroxylactone (8) (Scheme 3).
The best substrate for biotransformation was bromolactone (6),
which was converted into the product by eight strains (Fig. 1). The
degree of conversion was also very high. For almost all strains,
except for F. tricinctum AM16, the product yield ranged from 79% to
9
4% (according to GC). Four strains were capable of transforming
Scheme 3. Transformation of halolactones 5e7.
Table 1
Hydrolytic dehalogenation of lactones (5), (6) and (7) after 7 days of incubation
Our previous reports34e37,42 proved that a halogen atom present
in a lactone molecule was changed into a hydroxyl group through
a mechanism similar to S 2 substitution, usually with a simulta-
N
Microorganism
5
6
7
Fusarium culmorum AM10
Fusarium avenaceum AM11
Fusarium oxysporum AM13
Fusarium tricinctum AM16
Fusarium semitectum AM20
Fusarium equiseti AM22
Syncephalastrum racemosum AM105
Fusarium solani AM203
Botrytis cinerea AM235
(ꢁ)
(þþþ)
(þþþ)
(þþ)
(þþ)
(þþ)
(þ)
(ꢁ)
(ꢁ)
neous change in the molecule conformation. The same situation
(ꢁ)
(þþ)
(þ)
1
was observed in this study. An analysis of the H NMR spectrum of
(þþþ)
(ꢁ)
(þþþ)
(þ)
(þþ)
(þ)
hydroxylactone (8) revealed some changes. The coupling constants
between the H-7 and H-10 protons (7.0 Hz) and between the H-7
(þ)
(þ)
(þ)
(ꢁ)
(þþþ)
(þþþ)
(þþþ)
(þþ)
(þ)
proton and CH
2
-8 protons (11.5 Hz and 8.6 Hz) indicated that the H-
(ꢁ)
7
proton was situated across the plane of the lactone ring similarly
Cunninghamella japonica AM472
(þ)
(ꢁ)
to the halolactones. The signal of this proton moved from 2.9 Hz in
halolactones to 3.3 Hz in the hydroxylactone. This observation in-
dicated that the H-7 proton and OH group were located close to
each other. The signals coming from the H-10 and H-11 protons in
(
(
(
(
ꢁ)donly the substrates.
þ)dthe conversion between 20% and 49%.
þþ)dthe conversion between 50% and 79%.
þþþ)dthe conversion between 80% and 95%.

640
M. Grabarczyk et al. / Tetrahedron 72 (2016) 637e644
2.3. Biological tests
Continuing the research on the biological activity of the syn-
thetic halolactones (5e7) and the microbiologically obtained
hydroxylactone (8), we tested these compounds for their antibac-
terial and antifungal properties. Bioassays performed on selected
strains of bacteria (entries 1e5), yeasts (entries 6e10), and fungi
(entries 11e14) are presented in Table 5.
Fig. 3. Spatial structure of hydroxylactone 8.
The tests carried out on bacteria strains showed total growth
inhibition only in the Bacillus subtilis strain grown in the presence
of chlorolactone (5) and Staphylococcus aureus grown in the pres-
ence of hydroxylactone (8). The growth of the latter strain was also
significantly reduced by other compounds, and an increase in its
optical density amounted to 1.0e1.3, while in the control culture it
was 1.54. In the culture supplemented with iodolactone (7), a 20-h
adaptive phase was observed. There was also a significant increase
in the lag-phase (compared to the control culture), up to 31 h in the
cells of B. subtilis grown in the presence of bromolactone (6). In the
cells of E. coli grown in the presence of all the tested compounds,
diauxic growth was observed. This phenomenon was also reported
chloro- (5) and bromo-lactone (6) suggested they were perpen-
dicular to the plane of the lactone ring. A similar situation was
observed for the CH
trary to that, the CH
3
-12 group in iodolactone (7) molecule. Con-
-12 group in lactones (5) and (6) was located
3
parallel to the plane of the lactone ring. A similar situation was
observed for the H-10 and H-11 protons in lactone (7). In hydrox-
ylactone (8), H-10 and H-11 protons were located in the same plane
as the lactone ring, differently than in chloro- and bromo-lactone
but similarly to iodolactone. Finally, the CH -12 group in hydrox-
3
ylactone (8) was located as in chloro- and bromo-lactone but dif-
ferently than in iodolactone molecule (Fig. 3).
After isolation and purification of the hydroxylactones obtained
during all preparative biotransformations, it was possible to de-
termine their isolated yield. The degree of conversion was different
for all three halolactones (5e7) and depended on microorganism
used. Therefore, enantioselectivity of these processes might also be
different. For this reason, all the hydroxylactones were verified for
their enantiomeric excess and optical purity. These data are pre-
sented in Tables 2e4.
46e48
by other authors.
Yeast strain Candida albicans generally showed no sensitivity to
the test compounds. In the presence of hydroxylactone (8) only
a reduction in the growth rate was observed, and iodolactone (7)
induced diauxic growth. It is worth mentioning that in the cultures
supplemented with chloro- (5) and bromo-lactone (6), biomass
yield was higher than in the control.
Filamentous fungi used in the study showed no sensitivity to the
test compounds. Chlorolactone (5), iodolactone (7) and hydrox-
ylactone (8) actually stimulated the growth of Aspergillus niger and
Fusarium linii strains that was manifested by higher growth rate
and increased final biomass. The only exception was A. niger strain,
whose growth was completely inhibited by bromolactone (6).
The (ꢁ)-isomer of hydroxylactone (8) was usually preferentially
formed. Enantiomeric excesses were not too high, the best results
(
21.7 %e30.4 %) were noticed for a hydroxylactones obtained from
iodolactone (7), probably because its conversion degree was the
lowest.
With previous reports on insect feeding deterring activity of
49e51
lactones in mind,
we decided to investigate all the lactones
The enantiomeric excesses were measured for all hydrox-
ylactones independently from their degree of conversion. Accord-
described here for their activity against aphids. A settling bioassay
was applied. Aphids settle on a plant only when they accept it as
3
4e37
5
2
ing to our previous experiences
in the case of hydrolytic
a food source. Therefore, the number of aphids that settle and
feed on a given substrate is a good indicator of its suitability. The
aphid settling bioassay allows for studying aphid host preferences
under semi-natural conditions: the aphids are given free choice
between control leaves and the leaves treated with the investigated
compounds. Two of the experimental lactones chlorolactone (5)
and microbiologically obtained hydroxylactone (8) deterred set-
tling of Myzus persicae and the effect was durable, i.e., the aphids
dehalogenation reaction the degree of conversion of the substrate
does not directly impact on an enantiomeric excess of the product.
The value of the enantiomeric excess depends mainly on the
structure of the substrate. Moreover, as demonstrated by the data
presented in Table 4 even for degree of conversion of the substrate
amounting about 50% the product was obtained with ee of no more
than 30%.
Table 2
Data concerning hydroxylactone 8 obtained during preparative biotransformations of lactone 5
2
0
Entry
1
Strain
Yield (according to GC)
98.0
Isolated yield (g)/(%)
0.024/25.2
ee (%)
11.4
a
D
F. semitectum AM20
ꢁ7.175 (c¼1.89%, CHCl
3
)
Table 3
Data concerning hydroxylactone 8 obtained during preparative biotransformations of lactone 6
2
0
Entry
Strain
Yield (according to GC)
Isolated yield (g)/(%)
ee (%)
a
D
1
2
3
4
5
6
7
F. culmorum AM10
F. avenaceum AM11
F. oxysporum AM13
F. tricinctum AM16
F. semitectum AM20
B. cinerea AM235
C. japonica AM472
79.0
94.0
88.0
68.0
89.0
90.0
91.0
0.010/12.7
0.021/26.7
0.021/26.7
0.023/29.2
0.017/21.6
0.011/14.0
0.015/19.1
4.2
0
ꢁ3.21 (c¼1.82%, CHCl
d
3
)
5.2
1.8
5.3
18.3
14.3
ꢁ3.61 (c¼0.59%, CHCl
ꢁ1.57 (c¼1.20%, CHCl
ꢁ3.79 (c¼0.52%, CHCl
ꢁ9.64 (c¼0.84%, CHCl
3
)
3
)
3
)
3
)
ꢁ8.31 (c¼082%, CHCl
3
)

M. Grabarczyk et al. / Tetrahedron 72 (2016) 637e644
641
Table 4
Data concerning hydroxylactone 8 obtained during preparative biotransformations of lactone 7
2
0
Entry
Strain
Yield (according to GC)
Isolated yield (g)/(%)
ee (%)
a
D
1
2
3
4
F. culmorum AM10
F. avenaceum AM11
F. semitectum AM20
F. solani AM203
59.0
51.0
66.0
70.0
0.026/38.2
0.009/13.8
0.013/19.1
0.011/16.2
30.4
21.7
23.4
29.0
ꢁ12.96 (c¼0.43%, CHCl
ꢁ10.22 (c¼0.55%, CHCl
ꢁ10.88 (c¼0.61%, CHCl
ꢁ12.54 (c¼0.56%. CHCl
3
)
3
)
3
)
3
)
Table 5
Effects of lactones 5e8 on the bacterial, the yeast and the fungi growth
Entry
Strain
Control
Compound
5
6
7
8
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
1
2
3
4
5
6
7
E. coli
3.5
5.0
8.0
1.77
1.54
1.50
1.29
1.89
1.69
1.19
3.5
6.5
0.97
1.30
0
1.47
1.21
1.76
1.33
3.5
8.0
31.0
10.0
18.0
10.0
d
0.79
1.19
1.05
1.32
1.30
1.61
0
3.5
20.0
11.0
10.0
10.0
10.0
13.5
0.87
1.01
0.98
1.23
1.60
0.92
1.37
3.5
d
1.05
0
S. aureus
B. subtilis
C. albicans
Alternaria sp.
F. linii
d
10.0
10.0
6.5
18.0
12.5
0.29
1.15
1.65
1.59
0.95
7.0
11.5
19.5
10.0
14.5
10.0
16.0
17.5
A. niger
avoided the treated leaves for at least 24 h after exposure. Attrac-
tiveness of iodolactone (7) increased over time (Table 6).
a preparative scale, the desired productdhydroxylactone was ob-
tained. The yield of these processes was 51%e98%. Hydroxylactone
(8) was obtained usually with low enantioselectivity, the best di-
3
. Conclusions
astereoisomeric excess was close to 30% when iodolactone (7) was
used as a substrate. In all cases (ꢁ)-diastereoisomer was preferred.
Biotransformation with filamentous fungi seems to be a good al-
ternative to chemical synthesis. Lactones (5)e(8) proved to be ef-
fective growth inhibitors of some tested bacteria, yeast and
filamentous fungi strains. Two of them, chlorolactone (5) and
hydroxylactone (8) were active aphid deterrents.
The
the four-step synthesis the known
-unsaturated ester (3), -unsaturated acid (4) and chloro- (5),
b
-ionone was used for obtaining new derivatives. During
a
-ionol (2) and new compounds,
g
,
d
g,d
bromo- (6) and iodo-lactone (7), were obtained. All the hal-
olactones were then converted into hydroxylactone (8) by fila-
mentous fungal strains. Knowing from past experiences
3
4e37,41
that
fungal strains are capable of replacing a halogen atom with a hy-
droxyl group, we also used them in this study. As a result of
screening biotransformations, eight strains were chosen to carry
out hydrolytic dehalogenation. Following biotransformation on
4
4
. Experimental
.1. General methods
Table 6
The progress of chemical reactions and biotransformation and
Effects of lactones (5e8) on aphids feeding
the purity of all products were checked on silica gel-coated alu-
minum plates (DC-Alufolien Kieselgel 60 F254, Merck) and by GC
analysis carried out on an Agilent Technologies 6890N instrument
Compound
Number of aphids
Avg (ꢀSD)
Avg (ꢀSD)
Avg (ꢀSD)
1
h
2 h
24 h
using
30 mꢂ0.32 mmꢂ0.25
ysis were as follows: injector 150 C, detector (FID) 280 C, column
a
DB-17 column (cross-linked methyl silicone gum,
(
5)
4.3 (ꢀ1.7)
6.9 (ꢀ2.1)
0.0020
0.2
4.9 (ꢀ3.2)
6.0 (ꢀ2.6)
0.2460
0.1
3.1 (ꢀ2.0)
5.9 (ꢀ3.2)
0.0120
0.3
m
m). The temperatures during the GC anal-
ꢃ
ꢃ
Control
P
DI
ꢃ
ꢃ
ꢃ
ꢃ
temperature: 120 C, 120e280 C (rate 25 C/min), 280 C (hold
min). All of the products were purified using preparative column
1
chromatography on silica gel (Kieselgel 60, 230e400 mesh). The
molar masses of obtained compounds were confirmed by high
resolution mass spectrometry analysis using a Waters LCT Premier
XE instrument (ESI ionization). The enantiomeric excess of the
products obtained during biotransformation was determined by GC
analysis using the chiral column CP-cyclodextrin-B-325
(
6)
5.8 (ꢀ4.0)
9.0 (ꢀ3.4)
0.1288
0.2
4.6 (ꢀ4.5)
7.4 (ꢀ)
0.1623
0.2
6.9 (ꢀ4.6)
8.8 (ꢀ2.6)
0.2309
0.1
Control
P
DI
(
7)
4.9 (ꢀ4.3)
6.9 (ꢀ2.9)
0.1712
0.2
4.6 (ꢀ3.1)
6.1 (ꢀ2.0)
0.1712
0.1
5.8 (ꢀ5.5)
3.4 (ꢀ2.2)
0.1358
Control
P
DI
(
30 mꢂ0.25 mmꢂ0.25
mm) under the following conditions: in-
ꢃ
ꢃ
ꢃ
L0.3
jector 200 C, detector (FID) 250 C, column temperature: 140
C
ꢃ
ꢃ
ꢃ
(hold 45 min), 140e200 C (rate 20 C/min), and 200 C (hold
(
8)
5.9 (ꢀ3.9)
10.1 (ꢀ2.9)
0.0504
5.8 (ꢀ1.9)
9.5 (ꢀ4.0)
0.0334
0.2
4.5 (ꢀ2.8)
8.3 (ꢀ2.7)
0.0141
0.3
1
min). Optical rotations were measured in chloroform solutions on
Control
P
DI
a Jasco P-2000 polarimeter. The melting points were determined on
a Boetius apparatus. Refractive index was measured on Carl Zeiss
Abbe and Pulfrich refractometer. NMR spectra were recorded in
a CDCl solution on a Bruker AvanceÔ 600 MHz spectrometer. IR
3
spectra were determined using FTIR on a Thermo-Mattson IR 300
0.3
AVGdthe average number of aphids on the leaf, SDdstandard deviation, Pdresults
of Student t-test, DIdindex of deterrence: CꢁT/CþT, Cdcontrol, Tdtest.
Bold values signifies DI - index of deterrence is the most important indicator in-
cluded in this table.
spectrometer.

642
M. Grabarczyk et al. / Tetrahedron 72 (2016) 637e644
þ
4
.2. Organic synthesis
170.0 (C-9); HRMS (ESI): MH , found 271.1465. C15
71.1459.
2
H23ClO requires
2
The starting material, b-ionone (1), was purchased from Fluka.
All other products were obtained according to procedures de-
scribed below.
4.2.6. 10-(11-Bromoethyl)-7-(1,1,5-trimethylcyclohex-5-en-6-yl)di-
hydrofuran-2(3H)-one (6). Bromolactonization of acid 4 performed
4
2
according to the known procedure gave the title compound 5
(2.5 g, 74%) as a colorless oil; max(KBr) 2934, 1780, 1446, 1168,
) 0.98 (3H, s, CH -14), 1.09 (3H, s,
-15), 1.34e1.40 (1H, m, one of CH -2), 1.46e1.48 (1H, m, one of
-2), 1.53e1.58 (2H, m, CH -3), 1.62 (3H, d, J 6.2 Hz, CH -12), 1.68
-13), 1.95e2.01 (2H, m, CH -4), 2.75 (1H, dd, J 20.0,
2.6 Hz, H-8), 2.96 (2H, dd, J 20.0, 7.0 Hz, H-8), 2.94e3.01 (1H, m, H-
7), 4.30 (1H, dd, J 10.7, 10.0 Hz, H-10), 4.49 (1H, qd, J 10.0, 6.2 Hz, H-
11); (75.5 MHz, CDCl ): 19.1 (C-3), 20.9 (C-12), 21.1 (C-13), 28.2
4
(
CH
.2.1. (1,1,5-Trimethylcyclohex-5-en-6-yl)but-3-en-2-one
600 MHz, CDCl ) 1.05 (6H, s, CH -11, CH -12), 1.44e1.48 (2H, m,
-2), 1.57e1.65 (2H, m, CH -3), 1.74 (3H, d, J 0.8 Hz, CH -13), 2.06
-4), 2.28 (3H, s, CH -10), 6.08 (1H, d, J
(1).
d
H
n
ꢁ
1
3
3
3
998 cm
;
d
H
(600 MHz, CDCl
3
3
2
2
3
CH
CH
3
2
(
2H, dd, J 6.3, 6.1 Hz, CH
2
3
2
2
3
16.4 Hz, H-8), 7.25 (1H, dd, J 16.4, 0.7 Hz, H-7).
(3H, s, CH
3
2
1
4
.2.2. (1,1,5-Trimethylcyclohex-5-en-6-yl)but-3-en-2-ol (2). Lithium
aluminum hydride was used for the reduction of
according to the known procedure. The resulting the title com-
pound 2 (9.8 g, 97%) was characterized by the spectral data com-
b
-ionone
d
C
3
3
8
(C-15), 28.3 (C-14), 34.8 (C-4), 35.6 (C-6), 37.0 (C-8), 40.0 (C-2), 40.9
(C-7), 53.2 (C-10), 80.7 (C-11), 131.4 (C-1), 136.0 (C-5), 169.9 (C-9);
3
9
þ
parable with literature data:
n
max(KBr) 3360, 2926, 1457, 1361,
) 0.98 (6H, s, CH -12, CH -13), 1.31
-10),1.42e1.46 (2H, m, CH -2),1.55e1.61 (2H, m,
-3), 1.66 (3H, s, CH -11), 1.97 (2H, m, CH -4), 4.37 (1H, dd, J 6.6,
.3 Hz, H-9), 5.48 (1H, dd, J 15.9, 6.6 Hz, H-8), 6.05 (1H, d, J 15.9 Hz,
H-7).
HRMS (ESI): MH , found 315.0960. C15
H23BrO
2
requires 315.0954.
ꢁ1
1
060 cm
3H, d, J 6.3 Hz, CH
CH
;
d
H
(600 MHz, CDCl
3
3
3
(
3
2
4.2.7. 10-(11-Iodoethyl)-7-(1,1,5-trimethylcyclohex-5-en-6-yl)dihy-
2
3
2
drofuran-2(3H)-one (7). Iodolactonization of acid 4 according to the
4
2
6
known procedure gave the title compound 7 (1.98 g, 69%) as
ꢃ
a white solid, mp¼79e81 C;
n
max(KBr) 2928, 1782, 1457, 1159,
) 0.98 (3H, s, CH -14), 1.05 (3H, s,
-15), 1.37e1.42 (1H, m, one of CH -2), 1.46e1.49 (1H, m, one of
-2), 1.52e1.56 (2H, m, CH -3), 1.62 (3H, s, CH -13), 1.86 (3H, d, J
-12), 1.95e1.98 (2H, m, CH -4), 2.47 (1H, d, J 11.6 Hz, one
ꢁ1
1010 cm
;
d
H
(600 MHz, CDCl
3
3
4
.2.3. Ethyl (4E)-7-(1,5,5-trimethylcyclohex-5-en-6-yl)hex-4-enoate
CH
CH
3
2
(3). Alcohol 2 after Claisen rearrangement (orthoacetate modifi-
2
2
3
4
0
cation ) gave the title compound 3 (7.7 g, 61%) as a colorless liquid,
7.2 Hz, CH
of CH
of CH
H-10);
3
2
ꢁ1
n
D
1.4782;
CDCl ) 1.02 (6H, s, CH
.40e1.44 (2H, m, CH
m, CH -13), 1.64 (3H, d, J 6.3 Hz, CH
CH -4), 2.38 (1H, dd, J 15.2, 3.6 Hz, one of CH
5.2, 10.8 Hz, one of CH -11), 3.38e3.44 (1H, m, H-7), 4.14 (2H, q, J
.2 Hz, OCH CH ), 5.31e5.43 (1H, m, H-9), 5.53e5.56 (1H, m, H-8),
(75.5 MHz, CDCl ) 14.287 (OCH CH ),18.0 (C-13),19.5 (C-10), 21.5
C-3), 27.9 (C-15), 28.1 (C-14), 34.0 (C-4), 36.1 (C-1), 38.4 (C-7), 39.8
n
max(KBr): 2930, 1737, 1450, 1039 cm
-14, CH -15), 1.25 (3H, t, J 7.2 Hz, OCH
-2), 1.49e1.55 (2H, m, CH -3), 1.58e1.59 (3H,
-10), 1.89 (2H, dd, J 6.6, 5.7 Hz,
-11), 2.75 (1H, dd, J
;
d
H
(600 MHz,
2
-8), 2.93e2.95 (1H, m, H-7), 2.96 (1H, dd, J 21.6, 11.6 Hz, one
-8), 4.42 (1H, qd, J 7.2, 2.3 Hz, H-11), 4.51 (1H, dd, J 5.6, 2.3 Hz,
3
3
3
2
CH ),
3
2
1
2
2
d
C
(75.5 MHz, CDCl
3
) 19.2 (C-2), 20.8 (C-13), 21.7 (C-12), 28.7
3
3
(C-14), 29.4 (C-15), 29.3 (C-11), 34.4 (C-4), 36.0 (C-1), 38.2 (C-7),
2
2
38.7 (C-8), 88.5 (C-10), 131.2 (C-5), 139.0 (C-6), 175.8 (C-9); HRMS
þ
1
7
2
(ESI): MH , found 363.0821. C15
H23IO
2
requires 363.0815.
2
3
d
C
3
2
3
4.3. Biotransformation
(
(
C-2), 40.0 (C-11), 60.2 (OCH
2
CH
3
), 124.0 (C-9), 129.4 (C-6), 132.9 (C-
4.3.1. Microorganisms. The fungal strains which were used in this
study were obtained from a collection of the Institute of Biology
and Botany, Medical University, Wroc1aw: F. culmorum AM10, F.
avenaceum AM11, F. oxysporum AM13, F. tricinctum AM16, F. sem-
itectum AM20, Fusarium solani AM203, B. cinerea AM235, and
Beauveria bassiana AM278. All these strains are available in De-
partment of Chemistry, University of Environmental and Life Sci-
ences. The strains were cultivated on Sabouraud’s agar containing
0.5% of aminobac, 0.5% of peptone, 4% of glucose and 1.5% of agar
þ
8
C
), 138.8 (C-5), 173.2 (C-8); HRMS (ESI): MH , found 265.2168.
17 28 2
H O requires 265.2162.
4
.2.4. (4E)-7-(1,5,5-Trimethylcyclohex-5-en-6-yl)hex-4-enoic
acid
(4). Ester 3 subjected to basic hydrolysis according to the pro-
41
cedure described previously gave the title compound 4 (2.9 g, 75%)
ꢃ
as a white solid, mp 41e42 C;
n
max(KBr) 2930, 1701, 1430,
-14), 1.03 (3H, s,
-2), 1.50e1.54 (2H, m, CH -3), 1.58
-13), 1.66 (3H, d, J 6.3 Hz, CH -10), 1.88e1.92 (2H, m, CH
), 2.44 (1H, dd, J 15.7, 3.5 Hz, one of CH -11), 2.82 (1H, dd, J 15.7,
-11), 3.40e3.44 (1H, m, H-7), 5.37e5.47 (1H, m,
H-9), 5.54e5.61 (1H, m, H-8); (75.5 MHz, CDCl ) 18.0 (C-13), 19.4
C-10), 21.5 (C-3), 27.9 (C-15), 28.0 (C-14), 34.0 (C-4), 36.1 (C-1), 38.0
ꢁ1
1222 cm
;
d
H
(600 MHz, CDCl
3 3
) 1.02 (3H, s, CH
ꢃ
CH
3
-15), 1.40e1.44 (2H, m, CH
2
2
dissolved in distilled water at 28 C and stored in a refrigerator at
ꢃ
(
3H, s, CH
3
3
2
-
4 C.
4
2
10.8 Hz, one of CH
2
4.3.2. Screening procedure. The fungal strains which were used for
ꢃ
d
C
3
microbial transformations were cultivated at 25 C in 300 mL
(
(
Erlenmayer flasks filled with 100 mL of the medium containing 3 g
of glucose and 1 g of peptobac. After three days 10 mg of the sub-
strate dissolved in 1 mL of acetone was added to each flask of
culture. The shaken cultures were incubated with the substrate for
C-7), 39.3 (C-2), 40.0 (C-11), 124.2 (C-9), 129.7 (C-6), 132.7 (C-8),
ꢁ
138.7 (C-2), 178.5 (C-12); HRMS (ESI): MH found 235.1702.
C
H O
15 24 2
requires 235.1704.
7
days. After 3, 5 and 7 days of incubation, the mixture of unreacted
4
.2.5. 10-(11-Chloroethyl)-7-(1,1,5-trimethylcyclohex-5-en-6-yl)di-
substrate, products and mycelium was extracted with 15 mL of
dichloromethane. After evaporation of the solvent, the residue was
dissolved in 2 mL of acetone and analysed by TLC (silica gel, hex-
ane:acetone, 3:1) and GC (DB-17 column).
hydrofuran-2(3H)-one (5). Acid 4 subjected to the known pro-
cedure gave the title compound 5 (2.0 g, 69%) as a colorless oil;
4
2
ꢁ
1
n
max(KBr) 2931, 1786, 1455, 1142, 979 cm
.97 (3H, s, CH -14), 1.05 (3H, s, CH -15), 1.31e1.41 (2H, m, CH
.47e1.55 (2H, m, CH -3), 1.55 (3H, d, J 6.2 Hz, CH -12), 1.67 (3H, s,
CH -13), 1.97 (2H, dd, J 6.2, 6.0 Hz, CH -4), 2.74 (1H, dd, J 17.0,
.0 Hz, one of CH -8), 2.89e3.02 (1H, m, H-7), 2.98 (1H, dd, J 17.0,
.5 Hz, one of CH -8), 4.19 (1H, dd, J 10.6, 9.9 Hz, H-10), 4.38 (1H, qd,
J 9.9, 6.2 Hz, H-11); (75.5 MHz, CDCl ) 19. 2 (C-12), 19.9 (C-13),
1.1 (C-3), 28.0 (C-15), 29.2 (C-14), 34.6 (C-4), 35.6 (C-6), 36.7 (C-8),
;
d
H
(600 MHz, CDCl
3
)
0
1
3
3
2
-2),
2
3
4.3.3. Preparative transformations. 10 Erlenmeyer flasks with the 3-
day cultures of fungal strains were prepared as described in the
screening procedure. Then 100 mg of the halolactones 5e7 were
dissolved in 10 mL of acetone and distributed between these flasks.
After 7 days the products were extracted with dichloromethane
(3ꢂ40 mL). The combined organic solutions were dried over an-
hydrous magnesium sulfate and the solvent was evaporated in
3
2
9
7
2
2
d
C
3
2
4
0.0 (C-2), 40.6 (C-7), 60.6 (C-10), 80.3 (C-11), 87.5 (C-1),131.0 (C-5),

M. Grabarczyk et al. / Tetrahedron 72 (2016) 637e644
643
vacuo. Mixture containing the product, the unreacted substrate and
the fungal metabolites was separated by column chromatography
counted. Data were analyzed using a Student t-test. If the aphids
showed a clear preference for the leaf treated with the tested
compound (P<0.05), this compound was described as having at-
tractant properties. If the aphids settled mainly on the control leaf
(
silica gel, hexane:acetone 3:1) to obtain
a
pure pro-
ductdhydroxylactone 8.
(P>0.05), the compound was considered a deterrent. From the data
4
.3.3.1. 10-(11-Hydroxyethyl)-7-(1,1,5-trimethylcyclohex-5-en-6-
thus obtained, a relative index of deterrence (DI) was calculated
using the equation DI¼(CꢁT)/(CþT), where C was the number of
aphids that settled on the control leaf and T was the number of
aphids that settled on the leaf treated with the studied compound.
The value of DI may range between þ1 (ideal deterrent) and ꢁ1
(ideal attractant).
yl)dihydrofuran-2(3H)-one (8). The tile compound 8 was obtained as
ꢁ
1
a colorless oil;
n
max(KBr) 3450, 2921, 1772, 1162, 993 cm
0.98 (3H, s, CH -14), 1.01 (3H, s, CH -15), 1.36
-12),1.40e1.47 (2H, m, CH -2), 1.52e1.57 (2H, m,
-3), 1.62 (3H, s, CH -13), 1.94 (2H, dd, J 6.2, 6.0 Hz, CH -4), 2.56
1H, dd, J 18.8, 8.6 Hz, CH -8), 2.83 (1H, dd, J 18.8, 11.5 Hz, CH -8),
.35 (1H, dq, J 11.5, 8.6, 7.0 Hz, H-7), 3.77 (1H, qd, J 6.6, 1.3 Hz, H-11),
.41 (1H, dd, J 7.0,1.3 Hz, H-10). (75.5 MHz, CDCl ) 19.3 (C-3), 20.6
; d
H
(
(
CH
(
3
4
600 MHz, CDCl
3
)
d
3
3
3H, d, J 6.6 Hz, CH
3
2
2
3
2
2
2
Acknowledgements
d
C
3
(
3
1
C-12), 20.8 (C-15), 28.1 (C-14), 28.5 (C-1), 28.9 (C-13), 34.1 (C-4),
This project was financed by European Union from the European
Regional Development Fund. Grant No. POIG. 01.03.01-00-158/09.
5.1 (C-7), 37.5 (C-8), 39.6 (C-2), 67.3 (C-11), 88.6 (C-10), 130.6 (C-6),
þ
38.8 (C-5), 177.8 (C-9); HRMS (ESI): MH , found 253.1804.
C
15
H
24
O
3
requires 253.1798.
.4. Bioassay tests on microorganisms
.4.1. Bacteria strains. E. coli C1, B. subtilis B5, S. aureus, yeast strain
Supplementary data
4
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2015.12.005.
4
C. albicans KL-1 and also fungal strains: A. niger XP, F. linii 3A,
Alternaria sp. were tested.
References and notes
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€
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.5. Aphid settling bioassay
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ꢃ
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