Transformation of β-damascone to (+)-(S)-4-hydroxy-β-damascone by fungal strains and its evaluation as a potential insecticide against aphids Myzus persicae and lesser mealworm Alphitobius diaperinus Panzer

Article abstract of DOI:10.1016/j.catcom.2016.03.018

Microbial conversion of β-damascone (1) into 4-hydroxy-β-damascone (2) was studied. The results showed the potential of the tested biocatalysts for the regio- and enantioselective hydroxylation of substrate 1. The highest enantioselectivity was exhibited

Full text of DOI:10.1016/j.catcom.2016.03.018

Catalysis Communications 80 (2016) 39–43
Contents lists available at ScienceDirect
Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
Short Communication
Transformation of β-damascone to (+)-(S)-4-hydroxy-β-damascone by
fungal strains and its evaluation as a potential insecticide against aphids
Myzus persicae and lesser mealworm Alphitobius diaperinus Panzer
a
b
b
c
Anna Gliszczyńska a, , Witold Gładkowski , Katarzyna Dancewicz , Beata Gabryś , Maryla Szczepanik
⁎
a
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
Department of Biology and Ecology, University of Zielona Góra, Szafrana 1, 65-516 Zielona Góra, Poland
Nicolaus Copernicus University, Department of Invertebrate Zoology, Lwowska 1, 87-100, Toruń, Poland
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Microbial conversion of β-damascone (1) into 4-hydroxy-β-damascone (2) was studied. The results showed the
potential of the tested biocatalysts for the regio- and enantioselective hydroxylation of substrate 1. The highest
enantioselectivity was exhibited by strain Mortierella isabellina AM212. β-Damascone (1) was a relatively good
deterrent towards Alphitobius diaperinus and it was behaviorally inactive to Myzus persicae. The racemic com-
pound 2 and its (+)-(S)-enantiomer were stronger antifeedants against A. diaperinus than β-damascone (1),
in the case of M. persicae only (+)-(S)-(2) exhibited the deterrent properties.
Received 1 February 2016
Received in revised form 24 March 2016
Accepted 25 March 2016
Available online 29 March 2016
Keywords:
Biooxidation
Fungi
© 2016 Elsevier B.V. All rights reserved.
Antifeedant activity
β-Damascone
Enantioselectivity
1. Introduction
4-hydroxy-β-damascone (2) was isolated in low yields in a mixture
with other hydroxyderivatives.
Damascones are a class of norisoprenoids defined as rose ketones,
which were discovered in the Bulgarian rose oil [1]. They are widely
used in fragrance and cosmetic industry [2,3]. In the center of commer-
cial interest are also the hydroxyderivatives of damascones, naturally
occurring in tobacco plants [4,5]. Chiral 4-hydroxy-β-damascone was
found to be suitable as a flavoring and plays a role of a building block
in the synthesis of biologically active compounds, for example decalins
[6,7]. Chemical synthesis of 4-hydroxy-β-damascone was the subject
of many studies, but turned out to be complicated due to the formation
of a variety of side products and lack of a selectivity. In this aspect, the
enzymatic transformation seems to be a powerful tool for the produc-
tion of optically pure or enantioenriched hydroxylated derivatives. Ad-
ditionally, the products obtained by the transformation of natural
compounds using microorganisms can be legally labeled also as ‘natural’
ones [8]. From that reasons the catalytic potential of fungi Botrytis, As-
pergillus, Botryosphaeria and Lasiodiplodia, and the isolated enzymes of
bacteria to the biooxidation of β-damascone (1) has been investigated
[6,9–12]. The microbial or enzymatic hydroxylation of β-damascone
(1) afforded mainly products of incorporation of a hydroxy group into
the cyclohexane ring at C-3 rather than products with a hydroxy
group in the allylic position. Moreover, during biooxidation processes
Presented studies make a substantial contribution to the research
area concerning the microbial hydroxylation of damascones. Our stud-
ies were focused on testing 15 microbial strains towards the hydroxyl-
ation reaction. 4-Hydroxy-β-damascone (2) found as a major product,
occurs in plants and contributes to the fragrance of many essential
oils. Evaluating of its antifeedant activity we tried to find an answer if
it plays other important role in a metabolism of plants. In 2010 Kaufman
et al. found that β-damascone (1) is a potential agent for controlling
multiple arthropod species [13]. Inspired also by the previous results
of biological tests for hydroxyderivatives of natural compounds e.g.
jasmonates [14] carried out in our research group we expected that
the incorporation of a hydroxy group into the structure of β-
damascone (1) would confirm that the product (2) is useful in the con-
trol of pests. In biological tests we focused on two insect species of eco-
nomic importance that differ in the feeding habits and food preferences:
peach potato aphid (Myzus persicae) and lesser mealworm (Alphitobius
diaperinus). These insect pests represent two different groups with re-
spect to the practical application of feeding deterrents targeted at con-
tact chemoreceptory organs: the aphid possess sucking-piercing
mouthparts that lack external taste receptors, while the taste sensilla
of larvae and adults of A. diaperinus are located on their biting–chewing
mouthparts. Consequently, the aphids require the ingestion of plant sap
samples to evaluate food quality in contrast to the lesser mealworm
beetles and larvae that can examine food at the preingestional phase.
⁎
Corresponding author.
E-mail address: anna.gliszczynska@wp.pl (A. Gliszczyńska).
http://dx.doi.org/10.1016/j.catcom.2016.03.018
1566-7367/© 2016 Elsevier B.V. All rights reserved.

40
A. Gliszczyńska et al. / Catalysis Communications 80 (2016) 39–43
2. Materials and methods
2.1. Analytical methods
microorganisms were from the collection of Department of Chemistry,
Wroclaw University of Environmental and Life Sciences.
2.3. Microbial transformations
A course of microbial transformation was monitored by TLC tech-
nique on DC-Alufolien Kieselgel 60 F254 silica gel (0.2 mm; Merck).
Chromatograms were developed using hexane: acetone (2:1, v/v) sys-
tem. Visualization was effected with a solution of Ce(SO4)2 (10 g) and
H3[P(Mo3O10)4] (20 g) in 10% H2SO4 (1 L), followed by heating to
120–200 °C. The eluent used in the TLC technique was also used for
the preparative column chromatography performed on silica gel
(Kieselgel 60, 230–400 mesh ASTM, Merck). Gas chromatography
(GC) analysis was performed using an Agilent Technologies 6890N
(Network GC System) instrument fitted with a flame ionization detector
(FID) and HP-5 column (30 m × 0.32 mm × 0.25 μm) with hydrogen as a
carrier gas. Temperature program was as follows: injector 250 °C, detec-
tor (FID) 250 °C, column temperature: 100 °C, 100–300 °C (rate
30 °C min−1), 300 °C (hold 2 min). The chiral capillary column CP-
cyclodextrin-B (25 m × 0.25 mm × 0.25 μm) was used to determine
the enantiomeric compositions of the obtained products. Temperature
program was as follows: 70 °C, 100–200 °C (rate 2 °C min−1), and
200 °C (hold 2 min). Optical rotation was determined on JASCO P-
2000-Na polarimeter in a version with iRM controller using dichloro-
methane as a solvent, concentration denoted in g/100 mL. Nuclear mag-
netic resonance (NMR) spectra were recorded with a AMX 300-MHz
Bruker spectrometer and measured in CDCl3.
For the analytical tests the cultures were cultivated in Erlenmeyer
flasks (300 mL) containing 100 mL of medium (3 g glucose, 1 g
aminobac in distilled water (100 mL)). After full growth 10 mg of β-
damascone (1) dissolved in 1 mL of acetone was added to the shaken
cultures (150 rpm at 25 °C). After 1, 2, 4, 6 and 8 days 5 mL of the trans-
formation mixture were taken and the products were extracted with di-
chloromethane (3 × 4 mL). The extracts were dried over MgSO4 and
concentrated in vacuo. The residues were dissolved in 1 mL of acetone
and analyzed by TLC and GC. All experiments were repeated three
times. Additionally, two control flasks were used for each experiment:
one only with microorganism's culture in the medium for determining
the secondary metabolites and one with substrate in a growth medium
for determining the stability of substrate.
For the preparative-scale biotransformation the substrate (150 mg
of β-damascone (1) dissolved in 10 mL of acetone) was added to the
cultures of selected strains cultivated in 2 L flasks (each containing
500 mL of the cultivation medium). After 2–8 days of incubation the
products were extracted with dichloromethane. Organic solutions
were dried over anhydrous MgSO4 and concentrated in vacuo. The
transformation products were separated by column chromatography.
All experiments were carried out in triplicates. Results in Fig. 1 are
reported as means of triplicate experiments standard deviations (SD).
Spectroscopic data of the obtained product, 4-hydroxy-β-damascone
(2) were in accordance with those presented by More and Bhat [19]:
1H NMR (300 MHz, CDCl3) δ: 1.02 (s, 6H, (CH3)2Cb), 1.25–1.54 (m, 4H,
CH2-3, CH2-2), 1.63 (s, 3H, CH3-13), 1.84 (s, 1H, OH) 1.92 (dd, J = 6.8
and 1.2 Hz, 3H, CH3-10), 3.99 (t, J = 4.9 Hz, 1H, H-4), 6.15 (dd, J = 15.7
and 1.8 Hz, 1H, H-8), 6.77 (dq, J = 15.7 and 6.8 Hz, 1H, H-9); 13C NMR
(151 MHz, CDCl3) δ: 201.3 (C-7), 146.6 (C-5), 143.6 (C-9), 135.1 (C-6),
133.9 (C-8), 69.0 (C-4), 34.7 (C-3), 34.6 (C-1), 29.8 and 27.7 (C-11 and
C-12), 28.6 (C-2), 18.5 (C-10), 17.9 (C-13); IR (film, cm−1): 3422 (s),
2928 (s), 2856 (s), 1716 (m), 1645 (s), 1442 (m).
2.2. Substrate and strains
The substrate used for biotransformation experiments was β-
damascone (1) purchased from Sigma Aldrich (90% purity). The micro-
organisms were maintained on Sabouraud 4% dextrose-agar slopes at
4 °C and freshly subcultured before use in the transformation experi-
ments. The following 15 fungal and yeast strains were used in the pre-
sented studies: Fusarium oxysporum AM13, Penicillium purpurogenum
AM80, Rhodotorula rubra AM82, Penicillium camemberti AM83,
Syncephalastrum racemosum AM105, Penicillium lilacinum AM111, Peni-
cillium chermesinum AM113, Mortierella vinaceae AM149, Mortierella
isabellina AM212, Absidia cylindrospora AM336, Aspergillus ochraceus
AM456, Cunninghamella japonica AM472, Aspergillus niger MB,
Chaetomium sp. KCh6651 and Didymosphaeria igniaria KCh6670. All
2.4. Biological studies
The lesser mealworm (A. diaperinus) culture and feeding deterrence
bioassays have been previously described [15,16]. Activity of the tested
Fig. 1. Time course of the transformation of β-damascone (1) to 4-hydroxy-β-damascone (2) by selected fungal strains.

A. Gliszczyńska et al. / Catalysis Communications 80 (2016) 39–43
41
this product was fully confirmed by its spectral data, which were in ac-
cordance with these reported previously by More and Bhat [19].
Wide absorption band at 3422 cm−1 in the IR spectrum confirmed
the presence of hydroxy group in the product 2 (S4 Fig. 3). The presence
of triplet at 3.99 ppm with coupling constant J = 4.9 Hz from proton H-4
shifted to higher field (compared to the spectrum of the substrate)
proved the location of hydroxy group.
The absolute configuration at C-4 was assigned as S by the compar-
ison of the specific rotation sign determined for this compound with
that reported by More and Bhat [19]. Depending on the fungus
employed the enantiomeric excesses and the yields of 4-hydroxy-β-
damascone (2) were different (Table 1).
Fig. 2. Hydroxylation of β-damascone (1) by selected fungal strains.
compounds was evaluated by the standard method of choice test. The
deterrence index (DI) was calculated using the formula: DI = (C −
T) / (C + T) × 100 where C and T are the weights of the control and
treated foods consumed by the insects, respectively [17]. Compounds
with deterrence index of 75–100 are classified as very strong deterrents,
those with values of 50–74 as good deterrents, whereas those with
values of 25–49 have medium activity. [18]. The mean values of the de-
terrence coefficients and percentage of consumption were compared by
means of one-way analysis of variance (ANOVA) followed by Tukey's
test at a level of P b 0.05.
Application of compounds, culture of aphids (M. persicae) and their
settling (choice-test) has been previously described by Gliszczyńska
et al. [14]. The data were analyzed using one way ANOVA (STATISTICA
6.1. package). If aphids showed clear preference for the leaf treated
with the tested compound (P b 0.05), the compound was described as
having attractant properties. If aphids settled mainly on the control
half of the leaf (P b 0.05), the compound tested in the respective
choice-test was stated as a deterrent. From the data thus obtained the
relative index of deterrence (DI) was calculated: DI = (C − T) /
(C + T) where C is the number of aphids settled on control half of the
leaf and T denotes the number of aphids settled on the treated half of
the leaf. The value of DI ranges between 1 (ideal deterrent) and −1
(ideal attractant).
3.3. The course of biotransformation of β-damascone by selected fungal
strains
Table 2 shows the reaction rates during hydroxylation of β-
damascone (1) by six active microorganisms. In the first 24 h of the in-
cubation of substrate with the selected strains the highest rate was ob-
served for the reaction catalyzed by M. isabellina AM212 (Table 2, entry
1). After that time the reaction mixture contained only 9% of unreacted
substrate and 91% of product 2 (Fig. 1). During the next day the sub-
strate 1 was fully converted to (+)-(S)-4-hydroxy-β-damascone (2) al-
though reaction rate decreased significantly. The product was isolated
in 35% yield and its enantiomeric excess (ee = 54%) was determined
by chiral GC (S3 Fig. 2B).
Lower reaction rate was observed for M. vinaceae AM149 (Table 2,
entry 2). After 24 h of incubation of substrate 1 the products mixture
contained 33% of unreacted substrate and 67% of product 2 (Fig. 1). In
the next days the reaction rate was reduced and conversion of substrate
reached 99% after four days. The (+)-(S)-hydroxyderivative 2 was iso-
lated in high yield (53%) but with significantly lower enantiomeric ex-
cess (ee = 12%).
The enzymatic system of A. cylindrospora AM336 transformed β-
damascone 1 into the product 2 within two days at nearly constant
rate (Table 2, entry 3). The process was carried out under the same ex-
perimental conditions as applied before but this time the racemic mix-
ture of product 2 (S3 Fig. 2A) was obtained in 17% yield.
3. Results and discussion
3.1. Screening experiments for biotransformations of β-damascone (1)
In our studies we applied fungi to obtain hydroxyderivatives of β-
damascone (1) with high enantiomeric excess. The screening of fifteen
fungal strains let us to select six of them: M. isabellina AM212,
M. vinaceae AM149, C. japonica AM472, S. racemosum AM105,
A. cylindrospora AM336 and D. igniaria KCh6670 that were able to catalyze
the conversion of β-damascone (1) (Fig. 1). It is worth to notice that in all
cases formation of only one hydroxylation product was observed which
evidence is a representative chromatograms obtained after biotransfor-
mation catalyzed by M. isabellina AM212 (S2 Fig. 1). No further oxidation
products were detected in the biotransformation mixtures even after
9 days of process. Nine strains did not produce any products and only sub-
strate was observed in the reaction mixture even after 12 days of reaction.
The strains D. igniaria KCh6670 and C. japonica AM472 catalyzed the
process of biohydroxylation at significantly lower rates (Table 2, entries
4 and 5) than three cultures of microorganisms described above. After
24 h of the incubation of 1 with C. japonica AM472 the degree of conver-
sion was 32% and increased proportionally in the next two days. After this
time the reaction rate was reduced and the process proceeded slowly
affording finally 97% of the product 2 in the reaction mixture after eight
days (Fig. 1). Product 2 was isolated in 39% yield as (+)-(S)-enantiomer
with ee = 20%. Process of biotransformation carried out by D. igniaria
KCh6670 proceeded similarly (Fig. 1) and after eight days (+)-(S)-
enantiomer of product 2 was isolated in 22% yield with ee = 30%.
S. racemosum AM105 transformed substrate 1 with a lowest reaction
rate (Table 2, entry 6). After one day 23% of 4-hydroxy-β-damascone
(2) was identified in the reaction mixture (Fig. 1). In the next days the
rate of reaction was lower but the growing conversion of substrate 1
was observed to achieve 100% after 9 days. The product 2 was isolated
in 47% yield as enantiomerically enriched (+)-(S)-enantiomer with
ee = 48%.
3.2. Identification of transformations product
All selected microorganisms converted β-damascone (1) into
known 4-hydroxy-β-damascone (2), which was formed by oxidation
of the allylic C-4 position of cyclohexene ring (Fig. 2). The structure of
Table 1
Results of preparative biotransformation of β-damascone (1) by selected fungal strains.
Microorganism
Time of transformation (days)
Isolated yield of product (2) (%)
ee of product (2) (%)
[α]2D0 (lit. + 3.7 (c = 1.8, EtOH))
M. isabellina AM212
M. vinaceae AM149
A. cylindrospora AM336
D. igniaria KCh6670
C. japonica AM472
2
4
2
8
8
8
35
53
17
22
39
47
54
12
0
30
20
48
+14.74 (c = 1.3, CH2Cl2)
+3.98 (c = 1.4, CH2Cl2)
–
+8.19 (c = 1.5, CH2Cl2)
+5.46 (c = 1.6, CH2Cl2)
+13.10 (c = 1.3, CH2Cl2)
S. racemosum AM105

42
A. Gliszczyńska et al. / Catalysis Communications 80 (2016) 39–43
Table 2
The reaction rates of production of hydroxyderivative 2 by different strains.
Entry
Microorganism
Transformation
period [days]
Reaction rate
[mg/h]
of product
1
2
4
6
8
9
3.8 × 10−1
3.3 × 10−2
Full conversion
Full conversion
Full conversion
Full conversion
1
M. isabellina AM212
1
2
4
6
8
9
2.8 × 10−1
6.3 × 10−2
2
3
M. vinaceae AM149
3.5 × 10−2
2.1 × 10−3
Full conversion
Full conversion
Fig. 3. The effect of β-damascone and its derivatives on settling preferences of Myzus
persicae in the choice test. The data are expressed as values of indices of deterrence (DI).
The standard error is indicated on the bar *P b 0.05 (Student's t-test).
1
2
4
2.4 × 10−1
1.8 × 10−1
Full conversion
A. cylindrospora
β-Damascone (1) appeared to be a weak attractant for aphids during
the initial contact with the treated leaves in the choice test, as it was ob-
served that although the first probe was delayed in comparison to the
control, further probing was rarely interrupted. The exposure of
M. persicae to the racemic 4-hydroxy-β-damascone (2) resulted in the
initial avoidance of the treated leaves by freely moving aphids in the
choice-test. However, the antifeedant effect of racemic mixture of 4-
hydroxy-β-damascone (2) significantly decreased in the next hours.
The highest activity was observed for (+)-(S)-4-hydroxy-β-damascone
(2) with ee = 54%. Aphids clearly avoided leaves treated with this enan-
tiomer. The deterrent effect was observed 1 h after the exposure and it
was maintained until the end of the experiment (24 h) (Fig. 3).
AM336
6
8
9
Full conversion
Full conversion
Full conversion
1
2
4
6
8
9
1.9 × 10−1
1.2 × 10−1
4
5
6
D. igniaria KCh6670
C. japonica AM472
S. racemosum AM105
3.3 × 10−2
1.5 × 10−2
6.3 × 10−3
Full conversion
1
2
4
6
8
9
1.3 × 10−1
6.7 × 10−2
5.4 × 10−2
3.5 × 10−2
1.3 × 10−2
1.3 × 10−2
4. Conclusions
1
2
4
6
9.6 × 10−2
5.0 × 10−2
4.2 × 10−2
3.3 × 10−2
The selected fungal strains: M. isabellina AM212, M. vinaceae AM149,
C. japonica AM472, S. racemosum AM105 and D. igniaria KCh6670 con-
verted β-damascone (1) to (+)-(S)-4-hydroxy-β-damascone (2)
whereas only A. cylindrospora AM336 produced the racemic mixture.
The hydroxylation of β-damascone (1) was highly regioselective. The
most efficient transformation (53% isolated yield) was observed in the
case of M. vinaceae AM149 strain but the product obtained in the culture
of M. isabellina AM212 was characterized by the highest enantiomeric
excess (ee = 54%).
3.4. Antifeedant activity
Behavioral bioassays showed that the β-damascone (1) was a good
feeding deterrent towards the lesser mealworm. Both, larvae and adults
of A. diaperinus were eating in the choice test less than 30% of treated
food compared to the control (Table 3).
It is the first report on a regioselective microbial incorporation of a
hydroxy group into the structure of β-damascone (1) by fungi species:
The deterrent activity of oxyderivative of β-damascone (1)
depended on its enantiomeric purity. (+)-(S)-4-Hydroxy-β-
damascone (2) with ee = 54% exhibited significantly stronger activity
towards adults of lesser mealworm than its larvae. Racemic 4-
hydroxy-β-damascone (2) was characterized by very strong
antifeedant properties against both developmental stages of A.
diaperinus. Food treated with the racemic compound was eaten in
small quantities and made up only 7.29% (larvae) and 4.81% (adults)
of control consumption. In comparison with the starting β-damascone
(1), the significant improvement of activity was observed.
Mortierella,
Cunninghamella,
Syncephalastrum,
Absidia
and
Didymosphaeria that leads to the optically active product with increased
antifeedant activity. The biological studies have shown that the incorpo-
ration of a hydroxy group into the structure of β-damascone (1) in-
creases the antifeedant activity of the hydroxyderivative towards
A. diaperinus. It proves a high sensory sensitivity of this insect to a hy-
droxy group introduced into the molecule. Likewise, the transformation
of β-damascone (1) into 4-hydroxy-β-damascone (2), caused also a sig-
nificant change in the peach potato aphid behavior. While β-damascone
(1) appeared
a
behaviorally inactive compound, 4-hydroxy-β-
Table 3
Antifeedant activity of β-damascone (1) and its metabolites against A. diaperinusa.
Compound
Larvae
Adults
DIb SE
Consumptionc SE
DI SE
Consumptionc SE
β-Damascone (1)
(+)-(S)-4-hydroxy-β-damascone (ee = 54%)
55.59 3.41 a
45.83 2.27 a
86.46 1.63 b
28.73 2.89 a
37.25 2.18 a
7.29 0.95 b
56.94 4.49 a
82.81 4.52 b
90.89 2.06 b
27.82 4.04 a
9.61 2.76 b
4.81 1.15 b
(
)-4-hydroxy-β-damascone
Means within a column followed by the same letter are not significantly different (one-way ANOVA followed by Tukey's test: P b 0.05).
a
Deterrence index.
b
Values are the mean of the four replicates, each set up with ten larvae or adults (n = 40).
Data are expressed as percentage of control consumption.
c

A. Gliszczyńska et al. / Catalysis Communications 80 (2016) 39–43
43
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