Fungi-mediated biotransformation of the isomeric forms of the apocarotenoids ionone, damascone and theaspirane

Article abstract of DOI:10.3390/molecules24010019

In this work, we describe a study on the biotransformation of seven natural occurring apocarotenoids by means of eleven selected fungal species. The substrates, namely ionone (α-, β- and γ-isomers), 3,4-dehydroionone, damascone (α- and β-isomers) and theaspirane are relevant flavour and fragrances components. We found that most of the investigated biotransformation reactions afforded oxidized products such as hydroxy- keto- or epoxy-derivatives. On the contrary, the reduction of the keto groups or the reduction of the double bond functional groups were observed only for few substrates, where the reduced products are however formed in minor amount. When starting apocarotenoids are isomers of the same chemical compound (e.g., ionone isomers) their biotransformation can give products very different from each other, depending both on the starting substrate and on the fungal species used. Since the majority of the starting apocarotenoids are often available in natural form and the described products are natural compounds, identified in flavours or fragrances, our biotransformation procedures can be regarded as prospective processes for the preparation of high value olfactory active compounds.

Full text of DOI:10.3390/molecules24010019

molecules
Article
Fungi-Mediated Biotransformation of the Isomeric
Forms of the Apocarotenoids Ionone, Damascone
and Theaspirane
Stefano Serra * and Davide De Simeis
C.N.R. Istituto di Chimica del Riconoscimento Molecolare, Via Mancinelli 7, 20131 Milano, Italy;
dav.biotec01@gmail.com
* Correspondence: stefano.serra@cnr.it or stefano.serra@polimi.it; Tel.: +39-02-2399-3076
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 19 November 2018; Accepted: 20 December 2018; Published: 21 December 2018
Abstract: In this work, we describe a study on the biotransformation of seven natural occurring
apocarotenoids by means of eleven selected fungal species. The substrates, namely ionone ( -,
and -isomers), 3,4-dehydroionone, damascone ( - and -isomers) and theaspirane are relevant
α
β-
γ
α
β
flavour and fragrances components. We found that most of the investigated biotransformation
reactions afforded oxidized products such as hydroxy- keto- or epoxy-derivatives. On the contrary,
the reduction of the keto groups or the reduction of the double bond functional groups were observed
only for few substrates, where the reduced products are however formed in minor amount. When
starting apocarotenoids are isomers of the same chemical compound (e.g., ionone isomers) their
biotransformation can give products very different from each other, depending both on the starting
substrate and on the fungal species used. Since the majority of the starting apocarotenoids are often
available in natural form and the described products are natural compounds, identified in flavours
or fragrances, our biotransformation procedures can be regarded as prospective processes for the
preparation of high value olfactory active compounds.
Keywords: biotransformation; oxidation; apocarotenoids; flavours; fungi; ionone; damascone;
theaspirane
1. Introduction
In Nature, the oxidative degradation of the conjugated tetraterpene carotenoids (C40) produces a
plethora of smaller derivatives, called apocarotenoids [1], which possess a range of different chemical
structures and biological activities. Among these natural products, compounds having thirteen carbon
atoms in their frameworks are relevant flavours or fragrances and their manufacturing represents an
important economic resource for chemical companies [2]. The combination of the great diversity of the
carotenoids chemical structures with the different possible degradation pathways, gives rise to a huge
number of flavours and fragrances.
It is worth noting that the primary odorous C13 apocarotenoids, namely ionone, damascone
and theaspirane isomers (Figure 1), are cyclohexene derivatives and the possibility of three different
positions of the double bond, the presence of a stereogenic center in position 6 (carotenoids numbering)
and the eventual structural rearrangements, can give rise to a large number of isomers. In addition,
the latter volatile compounds are often accompanied by structural related apocarotenoids having
further oxygen atoms in their chemical framework.
Molecules 2019, 24, 19; doi:10.3390/molecules24010019
www.mdpi.com/journal/molecules

Molecules 2019, 24, 19
2 of 18
Figure 1. The formation of apocarotenoids through degradation of carotenoids and the seven C13
apocarotenoids (compounds 1–7) selected as substrates for the fungal biotransformation investigated
in the present study.
The first consequence of the introduction of a hydroxy- or keto- functional group on these
compounds is the decrease of the volatility and the increase of the so-called ‘substantivity’, namely
the long lasting odour of a substance having low vapor pressure. The aforementioned compounds
have been recognized as components of different natural flavours. For example 3-hydroxy and
3-keto-
α
-ionone, 4-hydroxy- and 4-keto-β-ionone and hydroxy-β-damascone isomers have been
identified in curry tree [
3
], eucalyptus honey [
4
], saffron [ ], black tea [
5
6
] and tobacco [ ] respectively,
7,8
whereas 3-keto-theaspirane (also known under the trade name theaspirone) is the character impact
compound of the black tea flavour [9].
All these derivatives are present in vegetables in very minute amounts and the extraction is not a
viable process for their production. Consequently, they are currently obtained by chemical synthesis
and are not commercially available in their natural form. Since flavours possessing ‘natural’ status are
usually hundreds times as expensive as their synthetic counterparts, any new procedure that provides
these compounds in their high value form can be very profitable.
In recent years, some new biocatalytic processes have provided a reliable access route to the most
common C13 apocarotenoids, such as α- and β-ionone. In addition, the genetic engineering of both
carotenoids biosynthesis and carotenoids cleavage pathways in the same microbial host [10] has laid
the foundation for the large-scale production of the C13 apocarotenoids in natural form.
According to the European and USA legislation, the biotransformation of a natural precursor
is a ‘natural method’ of synthesis [11]. Therefore, we singled out the compounds 1–7 as prospective
natural precursors to be used as starting materials for the biotechnological production of different
natural flavours.
From a biochemical standpoint, both prokaryotes and eukaryotes are able to degrade the
carotenoid frameworks. In spite of this fact, only a limited number of biotransformations of the
compounds of type
biotransformation goes back to 1950, when the oxidation of
1
–
7
have been reported to date. Although the first description of an ionone isomer
-ionone in rabbit was investigated [12],
β
only a limited number of studies on this topic took place in the following years. These researches
were based mainly on the fungi and bacteria-mediated chemical transformations whereas the
exploitation of some specific oxido-reductases were described only recently. In particular, Aspergillus
niger [13,14], Lasiodiplodia theobromae [15], Cunninghamella blakesleeana [16], Botrytis cinerea [17],
Aspergillus awamori [18], Pleurotus sapidus [19], Mortierella isabellina [20] and different Streptomyces
strains [21] have proved to be active biocatalysts for the transformation of these kind of compounds.
Concerning the use of isolated enzymes or the exploitation of a specific enzymatic activities, both

Molecules 2019, 24, 19
3 of 18
cytochrome P450 monooxygenases [22
,
23] and engineered whole cell biocatalysts expressing mutant
P450 monooxygenases [24], were used for the oxidation of different ionone isomers.
It is worth nothing that the biotransformation of some substrates such as -ionone, 3,4-dehydro-
-ionone and -damascone hasn’t been investigated yet. This paucity of scientific studies is often due
to the difficult availability of some apocarotenoids that can be either substrates or products of the
biotransformations. For example, -ionone is a rare natural isomer of ionone and can be obtained in
high isomeric purity only through demanding multistep syntheses [25 28]. Therefore is not surprising
γ
β
α
γ
–
that the fungus-mediated transformation of this compound hasn’t been studied yet. In addition,
whole cell biotransformations usually afford very complex mixtures of products whose chemical
identification, for example by GC or HPLC analysis, require the availability of the corresponding
reference standards. The aforementioned compounds are often not commercially available and have to
be prepared by specific and multistep chemical syntheses, thus hampering to perform a proper study
on the apocarotenoid’s biotransformation.
Taking advantage of our previous experience on the stereoselective synthesis of ionone and
damascone isomers [2,25–32], we decided to set up a comprehensive study on the biotransformation
of the seven natural substrates described above by means of eleven selected fungal species belonging
to the three more relevant phylums, namely ascomycota, zygomycota and basidiomycota. More
specifically, we selected Aspergillus niger and Mortierella isabellina because these microorganisms have
been already used for the biotransformation of some ionone isomers [13,14,20]. The remaining nine
strains were singled out among the plethora of the microorganisms described in the literature based on
their prospective biotransformation abilities. In effect, Nigrospora oryzae, different Penicillium species,
Rhizopus stolonifer, Curvularia lunata and Fusarium culmorum have been successful employed in the
biotransformation of terpenoids and steroids [33–35] whereas Geotrichum candidum was used for the
oxidation of the cyclohexanone derivatives [36]. Finally, we selected also the yeasts Yarrowia lipolytica
and Xanthophyllomyces dendrorhous since they are microorganisms of primary interest in the industrial
synthesis/degradation of lipids [37] and carotenoids [38], respectively.
The results obtained by our work, beside confirming and extending those described by some
previous researches, give new insights on the ability of fungi in the biotransformation of apocarotenoids,
establishing their prospective utility for flavour production.
2. Results and Discussion
As described in the introduction, each one of the selected apocarotenoid isomers was incubated
with a growing culture of each one of the eleven fungal strains. After a defined period of time
(see Experimental Section) the crude biotransformation mixtures were derivatized (by acetylation)
and analysed by GC-MS. To this end, several reference standard compounds were prepared by
chemical synthesis and then were used for the unambiguous identification of the compounds formed
in the biotransformation experiments. In spite of our efforts, a number of these derivatives were not
identified. Therefore, in order to spot the most relevant biochemical transformations that each fungal
strain is able to perform, we carried out the chromatographic isolation of the unknown metabolites
that were formed in relevant amount or that make up of the main part of the transformed derivatives.
The structures of these compounds were then elucidated through their comprehensive chemical
1
characterization. More specifically, the combined use of H-NMR, 13C-NMR, DEPT experiments,
GC-MS and ESI-MS spectroscopy allowed us to identify some compounds that haven’t been correlated
with any biotransformation experiment yet or that haven’t been described in the literature until now.
We first investigated the transformation of the ionone isomers
are very common in Nature, we deemed that it would be very interesting to also study the reactivity
of the rare -isomer. In effect, even if the latter three compounds differ only for the position of
1–3. Although only α- and β-ionone
γ
the cyclohexene double bond, they possess very different reactivity from each other. Therefore,
the identification of the derivatives obtained through biotransformation can help in understanding the
chemical processes involved in these fungi-mediated reactions.

Molecules 2019, 24, 19
4 of 18
As collectively described in Figure 2, the main part of the identified metabolites are the result
of four different biochemical reactions, namely the oxidation of the methylene functional groups,
the reduction of the conjugated double bond, the epoxidation of the 4,5-double bond and the reduction
of the keto group. The investigated fungal strains are able to perform both single step reactions and
sequential multi-steps transformations, in turn deriving from the combination of the aforementioned
four chemical reactions.
O
O
OAc
O
O
a, b
1
8
10
11
R
O
R'
R=H, R'=AcO 9a
9b
O
OAc
-ionone
R=AcO, R'=H
12
O
OAc
O
O
OAc
16
14
15
13
OAc
O
OAc
AcO
O
O
O
O
O
a, b
2
O
18
19
17
O
AcO
-ionone
20
OAc
OAc
O
21
22
O
O
O
O
O
R'
a, b
R
3
26
25
AcO
-ionone
R
R'
OAc
R=H, R'=AcO 23a
23b
24a
24b
R=H, R'=AcO
R=AcO, R'=H
R=AcO, R'=H
27
Figure 2. Compounds obtained through biotransformation of
α
-,
β- and γ-ionone by means of
the fungal strains employed in the present study. Reagents ◦and conditions: (
a
) fungal strain, malt
extract medium (MEM), aerobic conditions, 140 rpm, 20 or 25 C, 8–20 days; (
4-dimethylaminopyridine (DMAP) catalyst, room temperature (rt), 6 h.
b) Ac2O/pyridine (Py),
Overall, ionone isomers
in Table 1. A thorough perusal of these data shows considerable differences in the reactivity of the
three isomers. -Ionone was oxidized almost exclusively at the activated allylic methylene functional
1–3 were converted into derivatives 8–27 in relative amounts described
α
1
group, with the exception of Geotrichum candidum that is completely inactive and of Curvularia lunata
and Fusarium culmorum that are also able to oxidize the 4,5-double bond to give the corresponding
epoxy-ionone 10. The relative ratio of the obtained 3-keto-α-ionone 8 and of the diastereoisomeric
3-hydroxy- -ionones 9a and 9b changes significantly based on the fungal strain used. Overall,
α
the microorganisms that better performed the oxidation of 1 were Aspergillus niger, Nigrospora oryzae
and Fusarium culmorum.
The global amount of the 3-oxidized metabolites obtained using the latter fungal strains took
account of the 74%, 69% and 58% of the crude biotransformation mixture, respectively. On the contrary,
none of the tested strains showed notable reductive activity on α-ionone, as confirmed by the modest
formation of derivatives 11 and 12.

Molecules 2019, 24, 19
5 of 18
Table 1. Results of the fungi-mediated biotransformation of α-, β- and γ-ionone isomers.
Fungal Strains and Distribution of the Biotransformation Products 1
Biotransformation
Substrate
N.
oryzae
G.
Y.
P.
R.
P.
M.
C.
X.
F.
Products
A. niger
candidum
lipolytica roqueforti stolonifer corylophilum isabellina
lunata
dendrorhous culmorum
1
8
9a
9b
10
21
13
33
28
-
-
-
5
2
27
7
25
-
-
10
31
100
87
-
1
1
-
8
-
3
82
4
3
10
-
-
85
2
3
1
-
-
-
9
84
4
4
6
-
-
-
2
62
1
9
12
-
3
-
2
7
-
10
-
88
-
2
1
-
2
-
7
24
10
21
25
3
-
2
15
-
-
-
-
-
-
-
α-ionone (1)
11
12
-
1
-
13
7
74
N.D. 2
2
13
14
15
16
17
18
19
20
45
1
39
-
-
-
11
-
-
-
-
15
-
26
-
-
26
-
-
-
2
5
76
5
8
-
-
-
-
-
1
-
-
82
4
1
-
-
-
-
-
-
87
1
8
-
-
-
-
2
-
-
-
66
3
14
-
-
-
-
-
-
-
61
3
16
1
-
-
8
-
84
1
7
-
-
-
-
-
-
1
-
16
-
15
-
23
-
-
-
-
12
-
34
90
-
3
-
-
-
-
-
-
-
40
4
11
-
3
-
-
-
-
3
-
β-ionone (2)
-
3
-
21
11
1
1
22
-
17
-
7
N.D. 2
4
26
10
2
8
7
39
3
23a
23b
24
25
26
-
35
-
2
49
-
6
-
-
100
43
-
-
-
-
20
16
21
83
5
-
-
-
-
1
11
100
44
14
-
-
-
19
6
17
85
-
-
2
-
-
26
-
-
8
-
-
20
46
78
-
-
-
-
8
5
9
21
-
-
13
10
7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
γ-ionone (3)
4
2
2
86
27
-
14
2
11
-
49
N.D. 2
1
Percentage of the compound detected by GC-MS analysis of the biotransformation mixture, after extraction and chemical acetylation; 2 N.D. = not determined: the value indicates the
overall percentage of the compounds obtained by biotransformation whose chemical structure wasn’t assigned.

Molecules 2019, 24, 19
6 of 18
A more complex outcome were observed when
β-ionone
2
was used as substrate. In this case, even
if the latter ketone was oxidized mainly at the activated allylic methylene functional group (compounds
13 17), the biotransformations also afforded the 2-hydroxy- -ionone derivative 18 (Aspergillus niger
and Penicillium corylophilum) and a little amount of 3-hydroxy- -ionone derivative 19 (Penicillium
–
β
β
roqueforti). In addition, the partial oxidation of the ionone side chain is also possible, as shown by
the detection of trace (1%) of dihydroactinodiolide 20 in the biotransformation mixture of Geotrichum
candidum. Interestingly, all the tested fungal strains left unaffected the C(13) methyl group of both
α-
and -ionone isomers. In effect, we observed neither the formation of 13-hydroxy-derivatives nor the
β
presence of the epoxy-megastigmaen-9-one isomers that can arise from the intramolecular 1,4-addition
of the hydroxy-group to the conjugated double bond [31].
On the contrary, the reduction of the conjugated double bond and of the keto group are chemical
transformations of major significance in fungal
reactions can proceed also on intermediates deriving from the oxidation of the activated position
4 of the -ionone framework, affording a number of oxidized-reduced derivatives (15 17) besides
β-ionone biotransformation. The abovementioned
β
–
compounds obtained by simple reduction (i.e., 21 and 22).
Completely different results were observed for the biotransformation of γ-ionone where the
oxidation of the allyl methylene group, to afford derivative 24, appeared to be a path of minor relevance.
On the contrary, the oxidation of the positions 2 and 3 of the latter ionone isomer was efficiently
performed by Aspergillus niger that produced compounds 23a and 25 in high yield. Penicillium
corylophilum is also able to convert γ-ionone into the derivative 23a but for this microorganism,
as well as for Yarrowia lipolytica, the reductive steps are more relevant than the oxidative ones. In effect,
the reduction of the conjugated double bond and of the carbonyl functional group afforded the
compound 26 and 27, respectively. It is worth noting that Aspergillus niger, Penicillium roqueforti
and Penicillium corylophilum produced diastereoselectively cis-2-hydroxy-
of the microorganisms tested afforded its diastereoisomer, namely trans-2-hydroxy-
Differently, the fungi Nigrospora oryzae, Curvularia lunata and Fusarium culmorum transformed efficiently
-ionone, but the main part of the biotransformation reaction consisted in a mixture of unknown
compounds, most likely deriving by the extensive oxidative degradation of the ionone framework.
γ
-ionone (23a) and none
γ
-ionone (23b).
γ
The second group of the investigated apocarotenoids regards 3,4-dehydro-β-ionone (4) and
theaspirane (5, Figure 3).
Figure 3. Compounds obtained through biotransformation of 3,4-dehydro-β-ionone and theaspirane
by means of the fungal strains employed in the present study. Reagents and conditions: (
MEM, aerobic conditions, 140 rpm, 20 or 25 C, 14–20 days; (b) Ac2O/Py, DMAP catalyst, rt, 6 h.
a
) fungal strain,
◦
Although these compounds have different chemical structures, they both showed high reactivity
and the results of their biotransformation experiments were described together. More specifically,

Molecules 2019, 24, 19
7 of 18
we observed that all the investigated fungal strains were not able to oxidize the allylic positions of
compound . We identified as biotransformation products only compounds 28 30 (Table 2). These
4
–
three ionone derivatives originated from the oxidation of the conjugated diene functional group. Most
likely the first step is the epoxidation of the 3,4-double bond followed by the opening of the oxirane
ring by addition of a molecule of water. This two step mechanism could justify the formation of the
compound 28, possessing trans relative configuration of the two contiguous hydroxy groups, as the
major diastereoisomer. In addition, the following oxidation of the alcohol functional groups gave rise
to the keto derivatives. More specifically, the oxidation at position 4 or at both position 4 and 3 gave
compounds 29 and 30, respectively.
Otherwise, when a spirocyclic ether group replaces the conjugated carbonyl group, our selected
fungal strains become able to oxidize the theaspirane framework both at the allylic positions and at the
methine carbon linked to the ether oxygen atom. Overall, the main part of the compounds obtained by
biotransformation of 5 derive from allylic oxidation. In particular, the experiments performed using
Aspergillus niger, Rhizopus stolonifer and Fusarium culmorum showed a total content of the compounds 31
and 32 that accounts for at least 60% of the reaction mixtures. This result is noteworthy as theaspirone
31 is a relevant natural flavour and the corresponding alcohol (32 is the acetylated derivative) can
be regarded as its direct precursor. In effect, the preparation of 31 in natural form could be possible
by means of oxidation of the allyl alcohol, for example using a biocatalytic transformation involving
alcohol dehydrogenases.
Jointly with allylic oxidation, we observed also the oxidation of the ether functional group.
Hence, the latter moiety was transformed into the hemiketal and ketal groups as well in a completely
rearranged framework, as demonstrated by the isolation and characterization of compounds 34
,
36 and 35, respectively. Most likely, the latter derivatives are the result of a multistep oxidation
reaction. The transformation of the allyl methylene and methyl groups proceeds faster than the
oxidation of ether functional group, which is finally converted into a hemiketal functional group.
Accordingly, Nigrospora oryzae and Mortierella isabellina completely oxidized theaspirane (5) to give
two biotransformation mixtures containing 28% and 50% of compound 34, respectively. It is worth
mentioning that the latter compound is the direct precursor of 8,9-dehydrotheaspirone [32], a relevant
apocarotenoid flavour identified in white-fleshed nectarines [39].
The biotransformation experiments performed using Curvularia lunata gave results of more
complex interpretation affording a plethora (60% of the mixture) of undetermined compounds beside
the 3%, 8%, 12% and 9% of derivatives 32, 34, 35 and 36, respectively. Compound 35 could result
from a multistep transformation comprising both of oxidation and transposition reactions. The C(9)
oxidation is responsible for the formation of the hemiketal functional group that is in equilibrium
with the less stable open hydroxy-ketone form. The Baeyer-Villiger oxidation of the latter ketone
functional group could explain the formation of the primary alcohol acetate moiety whereas the 1,3-allyl
transposition of the tertiary hydroxy group could take account of the unexpected position of the C(4)
acetate group in compound 35. Similarly, the formation of compound 36 in Curvularia lunata–mediated
biotransformation could be explained by the hydroxylation of the carbons placed in position 3, 9 and
13 of the ionone framework.
The subsequent reaction of the obtained primary alcohol group with the hemiketal functional
group should afford the more thermodynamically stable tricyclic ketal 36. It is worth nothing that
a natural compound having an identical ketal structure, but devoid of the hydroxy group at the
C(4), is an aroma component of quince brandy [40]. The comparison of the NMR data measured for
36 with those reported for the natural flavour, allowed us to assign the above described chemical
structure suggesting a new synthetic approach to this chemical framework by fungal biotransformation
of theaspirane.

Molecules 2019, 24, 19
8 of 18
Table 2. Results of the fungi-mediated biotransformation of 3,4-dehydro-β-ionone and theaspirane.
Fungal Strains and Distribution of the Biotransformation Products 1
Biotransformation
Substrate
N.
oryzae
G.
Y.
P.
R.
P.
M.
C.
X.
F.
Products
A. niger
candidum
lipolytica roqueforti stolonifer corylophilum isabellina
lunata
dendrorhous culmorum
4
28
29
48
25
3
2
22
-
69
2
3
26
45
18
17
12
8
87
-
1
1
3
45
32
9
6
8
80
7
3
2
8
76
22
-
-
2
71
8
7
5
9
52
26
8
6
8
6
-
26
22
21
25
35
20
17
28
3,4-dehydro-
β-ionone (4)
30
N.D. 2
5
31
32
20
9
51
-
-
12
25
-
56
11
15
-
82
4
1
-
31
19
28
2
-
5
62
-
22
16
34
-
-
-
-
8
-
3
46
3
28
2
-
28
34
2
33
-
-
theaspirane
(5)
34
35
-
-
-
20
28
-
-
-
-
-
18
-
-
-
1
-
-
-
20
6
-
-
27
-
-
-
28
50
-
-
8
12
9
-
-
-
21
9
-
-
27
36
N.D. 2
35
50
60
1
Percentage of the compound detected by GC-MS analysis of the biotransformation mixture, after extraction and chemical acetylation; 2 N.D. = not determined: the value indicates the
overall percentage of the compounds obtained by biotransformation whose chemical structure wasn’t assigned.

Molecules 2019, 24, 19
9 of 18
A completely different reactivity was observed in the biotransformation of the
α
- and
β
-damascone isomers and (Figure 4). The latter apocarotenoids are isomers of - and β-ionone
6
7
α
respectively, as each one damascone isomer is interconvertible into the corresponding ionone isomer
by 1,3-shift of the enone moiety.
Figure 4. Compounds obtained through biotransformation of
α
- and
β-damascone by means of the
fungal strains employed in the present study. Reagents and conditions: (
conditions, 140 rpm, 20–25 C, 14–20 days; (b) Ac2O/Py, DMAP catalyst, rt, 6 h.
a) fungal strain, MEM, aerobic
◦
Despite the structural similarity of these compounds, none of the investigated fungal strains
was able to reduce the carbonyl functional group present in the damascone framework. In addition,
the fungi-mediated oxidative transformations of damascone isomers are restricted mainly to the allyl
methylene functional groups, as indicated by the formation of derivative 37 and 38 from
and derivatives 41 and 42 from -damascone (Table 3). The other position of the damascone framework
were unaffected with the exception of the 4,5-double bond that was oxidized by both Mortierella
isabellina and Xanthophyllomyces dendrorhous to produce a very minor amount of the epoxy- -damascone
39. Similarly, we did not record any reductive reactions with the exception of the conjugated double
bond of the -damascone isomer that was reduced by Penicillium corylophilum to give a small amount
of compound 40.
Overall, using the described fungal strains, we observed that the damascone isomers are less
reactive than the corresponding ionone isomers. This effect is more pronounced for the -isomer where
α-damascone
β
α
α
β
only Nigrospora oryzae and Fusarium culmorum produced a significant amount of the corresponding
4-hydroxydamascone, identified as acetyl derivative 42 (23 and 22%, respectively).
Our results seem in contrast to those reported in a recent study on the fungal biotransformation
of β-damascone [20], where a different Mortierella isabellina strain provides 4-hydroxydamascone in
a much higher yield. The recorded differences between these experimental data could be justified
considering both the substrate concentrations and the different biocatalytic activity among fungal
strains belonging to the same species.
In particular the substrate concentration seem to be the most relevant factor as apocarotenoid
derivatives show significant toxicity for many fungal strains and high concentration of these
compounds could inhibit their growth as well as their biocatalytic activity. We performed all the
investigated biotransformation experiments using a substrate concentration of about 2.5 g/L whereas,
in the above-mentioned work, the β-damascone concentration was set to 0.1 g/L. Our choice is justified
by the need of devising a preparative protocol for fungi-mediated ionone biotransformation. Since the
synthesis of this kind of flavours, in natural form, can show industrial significance only working with
substrate concentrations superior to 1 g/L, we set the above indicated concentration for all experiments.
As a consequence, it is reasonable that our selected fungal strains could have transformed better both
damascone as well as ionone and theaspirane isomers if they have been used in lower concentration.

Molecules 2019, 24, 19
10 of 18
Table 3. Results of the fungi-mediated biotransformation of α- and β-damascone isomers.
Fungal Strains and Distribution of the Biotransformation Products 1
Biotransformation
Substrate
N.
oryzae
G.
Y.
P.
R.
P.
M.
C.
X.
F.
Products
A. niger
candidum
lipolytica roqueforti stolonifer corylophilum isabellina
lunata
dendrorhous culmorum
6
37
38
56
20
5
-
-
89
5
2
55
10
10
-
-
25
93
-
-
-
-
100
100
24
-
14
-
13
49
30
22
17
2
-
29
96
-
3
-
-
1
39
10
21
3
-
27
56
5
30
-
-
9
-
-
-
-
-
-
-
-
-
-
α-damascone
39
(6)
40
N.D. 2
19
4
7
7
41
98
-
1
70
2
23
95
-
1
95
-
1
100
88
1
2
95
-
-
95
-
2
92
-
7
95
-
1
57
4
22
-
-
-
β-damascone
42
(7)
N.D. 2
1
5
4
4
9
5
3
1
4
17
1
Percentage of the compound detected by GC-MS analysis of the biotransformation mixture, after extraction and chemical acetylation; 2 N.D. = not determined: the value indicates the
overall percentage of the compounds obtained by biotransformation whose chemical structure wasn’t assigned.

Molecules 2019, 24, 19
11 of 18
3. Materials and Methods
3.1. Materials and General Methods
All air and moisture sensitive reactions were carried out using dry solvents and under a static
atmosphere of nitrogen. All solvents and reagents were of commercial quality and were purchased
from Sigma-Aldrich (St. Louis, MO, USA). A large number of reference standard compounds were
synthesized in our laboratory and were used for the unambiguous identification of the compounds
formed in the biotransformation experiments.
α-Ionone, γ-ionone and α-damascone were used in
racemic form. Commercial theaspirane consists of an equimolar mixture of racemic diastereoisomers.
γ
-Ionone (
procedures previously described by us [26–28].
The keto derivatives: 3-keto- -ionone ( ), 4-keto-
-damascone (41), theaspirone (31), 3-keto-
prepared by oxidation of -ionone, -ionone,
11) and -ionol acetate (21), respectively. The oxidation reactions were performed using TBHP/MnO2
as oxidant according to our previously reported procedure [41].
The diastereoisomeric forms of 4,5-epoxy- -ionone (10), 4,5-epoxy-
theaspirane (33) as well as 5,6-epoxy- -ionone and 5,6-epoxy- -damascone were prepared by
epoxidation of -ionone, -damascone, theaspirane, -ionone and -damascone, respectively, using
m-chloroperbenzoic acid and CH2Cl2 as solvent.
-7,8-Dihydroionones, -7,8-dihydroionone (22) and
reduction of -, - and -ionone respectively, using hydrogen and Ni Raney as catalyst for
and Bu3SnH and (Ph3P)2PdCl2 as catalyst for - and -ionone [25 26]. -8,9-Dihydrodamascone (40
was prepared by reduction of -damascone using NaBH4 in methanol. -8,9-Dihydrodamascone
-cyclocitral followed by oxidation of the
3
) and 3,4-dehydro-
β
-ionone (
4
) were prepared starting from
α
-ionone, according to the
α
8
α
β
-ionone (13), 3-keto-
α
-damascone (37), 4-keto-
-ionol acetate (15) were
-damascone, theaspirane, -ionol acetate
β
-ionol acetate (12) and 4-keto-
-damascone,
β
α
β
α
β
α
(
β
α
α-damascone (39), 4,5-epoxy-
β
β
α
α
β
β
α
β
γ
-7,8-dihydroionone (26) were prepared by
-ionone [42
α
β
γ
α
]
β
γ
,
α
)
α
β
was prepared by addition of propylmagnesium bromide to
resulting carbinol using Dess-Martin periodinane [43].
β
β
-Ionol acetate (21, racemic),
-ionol acetates (27, as a mixture of two racemic diastereoisomers) and 3-acetoxy-theaspirane (32) (as a
mixture of four racemic diastereoisomers) were prepared by chemical acetylation (Ac2O/Py) of the
α-ionol acetate (11, as a mixture of two racemic diastereoisomers),
γ
corresponding alcohols, which were in turn obtained through the reduction of
theaspirone, respectively, using NaBH4 in methanol.
α-, β-, γ-ionone and
cis-2-Acetoxy-α-ionone, and cis-2-acetoxy-γ-ionone (23a) were prepared starting from 2,8,8-
trimethyl-6-oxabicyclo[3.2.1]oct-2-en-7-one and 8,8-dimethyl-2-methylene-6-oxabicyclo[3.2.1]octan-
7-one (kaharana lactone) respectively, according to the synthetic procedure developed by Audran [44].
The latter lactones were in turn prepared from racemic cis-2-hydroxy-
α-cyclogeraniol and cis-2-
hydroxy- -cyclogeraniol [45] by oxidation using BAIB and TEMPO as catalyst. In addition, the partial
γ
reduction of the two isomeric lactones afforded the corresponding lactols, whose condensation with
acetone [46] followed by acetylation (Ac2O/Py) of the crude reaction mixtures, gave the cis/trans
mixtures of acetoxy-
standards for the identification of the corresponding trans isomers.
Racemic 2-acetoxy- -ionone (18) was prepared starting from 2-hydroxy-β-cyclogeraniol [45] by
α-ionone and acetoxy-γ-ionone, respectively, that were used as GC-MS reference
β
selective oxidation of the primary alcohol functional group by MnO2 in CHCl3, condensation with
acetone [46] and acetylation (Ac2O/Py) of the obtained hydroxy-ionone derivative.
Samples of 3-acetoxy-
mixture) and of 3-acetoxy-
procedure described by Tu [47], by Serra [42] and Khachik [48], respectively. A sample of 3-acetoxy-
α
-ionone (
9
) (2:1 cis/trans mixture), of 4-acetoxy-
γ
-ionone (24) (4:1 cis/trans
β-ionone (19) were prepared starting from
α-ionone according to the
α
-
damascone (38) (1:1 cis/trans mixture) was prepared starting from ethyl 3-hydroxy-α-cyclogeraniate
according to the procedure described by Takei [49].
4-Acetoxy-β-ionone (14) and 4-acetoxy-
β-damascone (42) were prepared starting from 4,5-epoxy-
α
-ionone and 4,5-epoxy-
α
-damascone, respectively, by means of NaOMe mediated transposition

Molecules 2019, 24, 19
12 of 18
followed by chemical acetylation (Ac2O/Py) of the obtained allyl alcohols. A sample of 4-acetoxy-
-ionol acetate (16) (1:1 mixture of diastereoisomers) was prepared from 4-keto- -ionol acetate by
reduction with NaBH4 in methanol followed by chemical acetylation (Ac2O/Py).
A sample of 4-acetoxy- -7,8-dihydroionone acetate (17) was prepared from 4-hydroxy-
β
β
β
β-ionone
by reduction with Ph3SiH followed by chemical acetylation (Ac2O/Py), according to the procedure
described by Pascual [50].
3,4-Diacetoxy-
according to the procedure described by Buschor [51]. The oxidation of 4-keto-
methanol [52] afforded 3-hydroxy-4-keto- -ionone that was further oxidated using oxygen in presence
of tBuOK [53] to give 3,4-diketo- -ionone. The acetylation (Ac2O/Py) of the latter two compounds
afforded 3-acetoxy-4-keto- -ionone (29) and 3-acetoxy-4-keto-2,3-dehydro- -ionone (30). Racemic
β
-ionone (28) (cis/trans mixture), was prepared from 3,4-dehydro-
β-ionone
β-ionone with IBDA in
β
β
β
β
dihydroactinodiolide (20) and 2-hydroxy-2,6,10,10-tetramethyl-1-oxaspiro[4.5]dec-6-en-8-yl acetate
(34) were prepared as described previously [32,54].
A comprehensive characterization of the above described reference standards is reported in the
Supplementary Materials section.
3.2. Analytical Methods and Characterization of the Products Deriving from the
Biotransformation Experiments
The crude biotransformation mixtures obtained according to the procedures described below
were then acetylated by treatment with pyridine/acetic anhydride (2 mL of a 2:1 mixture) and
catalytic DMAP (10 mg) for 6 hours at rt. The obtained acetylated mixture was analyzed by GC-MS.
The compounds whose chemical structure couldn’t be assigned only by GC-MS analysis were isolated
from the biotransformation mixtures by means of chromatographic separation and then characterized
by NMR analysis and GC-MS or ESI-MS analysis.
1H- and 13C-NMR spectra and DEPT experiments were recorded/performed at 400, 100 and 100
MHz, respectively, in CDCl3 solutions at rt using an AC-400 spectrometer (Bruker, Billerica, MA, USA);
13C spectra are proton decoupled; chemical shifts in ppm rel to internal SiMe4 (=0 ppm).
TLC: silica gel 60 F254 plates (Merck, Kenilworth, NJ, USA). Column chromatography: silica gel.
Melting points were measured on a Reichert apparatus, equipped with a Reichert microscope,
and are uncorrected.
Mass spectrum were recorded on a ESQUIRE 3000 PLUS spectrometer equipped with an ESI
detector (Bruker, Billerica, MA, USA) or by GC-MS analyses.
GC-MS analyses: HP-6890 gas chromatograph equipped with a 5973 mass detector, using a
HP-5MS column (30 m
with the following temp. program: 60◦ (1 min)—6◦/min—150◦ (1 min)—12◦/min—280◦ (5 min);
carrier gas, He; constant flow 1 mL/min; split ratio, 1/30; tR given in min: tR( ) 16.23, tR( ) 17.57,
tR( ) 16.53, tR( ) 17.53, tR( ) 13.40 and 13.77, tR( ) 15.44, tR( ) 15.91, tR( ) 20.42, tR(9a) 21.41, tR(9b
×
0.25 mm, 0.25 µm film thickness; Hewlett Packard, Palo Alto, CA, USA)
1
2
3
4
5
6
7
8
)
)
)
)
21.61, tR(10) 18.63 and 18.72, tR(11) 17.78, tR(12) 21.52, tR(13) 20.61, tR(14) 21.82, tR(15) 22.03, tR(16
22.56, tR(17) 21.59, tR(18) 22.18, tR(19) 22.23, tR(20) 18.61, tR(21) 18.55, tR(22) 16.51, tR(23a) 21.51, tR(24a
21.32, tR(24b) 21.60, tR(25) 21.79, tR(26) 15.67, tR(27) 17.47, 17.58, 18.01 and 18.08, tR(28) 24.10, tR(29
23.75, tR(30) 23.92, tR(31) 19.02 and 19.19, tR(32) 20.17, 20.33, 20.50 and 20.61, tR(33) 15.14 and 15.50,
tR(34) 19.37 and 19.67, tR(35) 21.78, tR(36) 21.64, tR(37) 20.15, tR(38) 21.17 and 21.30, tR(39) 17.84, tR(40
14.62, tR(41) 20.91, tR(42) 20.93, tR(7,8-dihydro- -ionone) 15.95, tR(7,8-dihydro- -damascone) 20.95,
)
α
β
tR(cis-2-acetoxy-α-ionone) 21.42, tR(trans-2-acetoxy-α-ionone) 21.36.
3.3. Microorganisms and Biotransformation Experiments
Geotrichum candidum (DSM 10452), Yarrowia lipolytica (DSM 8218), Rhizopus stolonifer (DSM
855), Xanthophyllomyces dendrorhous (DMS 5626), Curvularia lunata (CBS 215.54), Mortierella
isabellina (CBS 167.60), Aspergillus niger (CBS 626.26) were purchased from the DSMZ (Braunschweig,
Germany) or CBS-KNAW (Utrecht, The Netherlands) collections.

Molecules 2019, 24, 19
13 of 18
Penicillium corylophilum (MUT 5838), Nigrospora oryzae (MUT 5844), Penicillium roqueforti (MUT
5856) and Fusarium culmorum (MUT 5855) were isolated as axenic cultures in our laboratory, then
identified by the Mycotheca Universitatis Taurinensis (MUT) of the University of Turin and finally
deposited in the same institution under the collection number given in brackets.
All the biotransformations were carried out in triplicate and the presented results are the average
of three experimental runs.
3.3.1. Representative Procedures for Biotransformations
The experimental conditions used for the biotransformations are based on the type of
microorganism used. Here is described a general procedure depending on the different morphological
features regarding the various active grow mycelia. The main ones could be classified in yeast-shape
mycelia (Xanthophyllomyces dendrorhous, Geotrichum candidum and Yarrowia lipolytica) and spore-forming
mycelia (Aspergillus niger, Rhizopus stolonifer, Curvularia lunata, Penicillium corylophilum, Nigrospora
oryzae, Penicillium roqueforti, Mortierella isabellina and Fusarium culmorum). In the first case, a small
amount of the active mycelia grew previously in a petri dish, was suspend in 1 mL of sterile water and
then inoculated in a 100 mL conical Pyrex flask containing 40 mL of Malt Extract Medium (MEM)◦for
◦
2 days at 25 C and 140 rpm (with exception of Xanthophyllomyces dendrorhous that was grown at 20 C).
After this period, the cells were centrifuged 3 minutes, (rt, 3220
The cells (approx. 600 mg wet-weight) were suspended in 3 mL of sterile water than 350
·
g) and collected removing the media.
L of the same
µ
suspension were used for inoculating each biotransformation flask containing 40 mL of MEM. In order
to ensure aerobic conditions, the flasks were sealed with cellulose plugs. The microorganism was leave
to growth for 2 days and then was treated with a solution of 100 mg of substrate dissolved in 400 µL of
DMSO. Generally, after 14 days from the substrate injection using the growing condition described
above, the reaction media was filtered under vacuum through a celite pad then was extracted 3 times
with ethyl acetate. The organic phase was separated, dried on Na2SO4 and the solvent removed at
reduced pressure to give the crude biotransformation mixture.
In the case of the spore-forming mycelia, the spore were collected from a sporulated surface
cultures and suspended in 3 mL of sterile water. After that, 350 µL of the same suspension were used
for inoculating each biotransformation flask containing 40 mL of MEM. The subsequent steps are the
same of that described previously. The unique exception was carried out for Penicillium corylophilum,
in the case of γ-ionone. The toxicity of the compound forced us to keep its concentration lower than
the others (7.5 mM) and to block the biotransformation earlier (8 days). After this period, the most
important products are degraded. In the case of Fusarium culmorum the biotransformation was blocked
after 20 days instead of 14 days because the activity of the fungus did not stop in the prefixed time.
3.3.2. Preparative Biotransformations and Chemical Characterization of Compounds 23a, 25, 28,
35 and 36
For the main part of the strains tested, the GC-MS analysis of the crude biotransformation
mixtures indicated the presence of different compounds whose chemical structures could not be
assigned only on the basis of our reference standards. The unidentified peaks taking account of less
than 5% of the overall percentage of the compounds obtained by biotransformation were collectively
indicated as ‘not determined’. Otherwise, compounds 23a, 25, 28, 35 and 36 were isolated from the
fermentation broths by extraction and chromatographic separation and then submitted to chemical
characterization. Different reasons prompted us to undertake the isolation procedure. First of all, these
compounds were a relevant part of the biotransformation mixture and their MS fragmentations clearly
indicated a chemical structure deriving from the corresponding starting materials. The compounds
25 was not one of the references standard available from our laboratory. Hence, we identified this
compound only after its isolation and chemical characterization. Both the diastereoisomeric forms
of the compound 25 have been described in the literature [55] but only low resolution 1H-NMR data
was reported. Therefore, we were not able to assign the relative configuration to the compound

Molecules 2019, 24, 19
14 of 18
obtained by biotransformation. Differently, compound 36 is completely new and its analytic data
has not described yet. Concerning compound 28, we observed that its diastereoisomeric forms (trans
and cis isomers) have the same retention time by GC-MS analysis. As a consequence, the isolation
of compound 28 from the biotransformation mixture followed by its NMR analysis was necessary in
order to understand what was the main isomer formed. Finally, the case of compound 35 is singular.
It is the only compound obtained by biotransformation that was formed through a Baeyer-Villiger
oxidation. Consequently, acetate 35 was completely unexpected and the proper reference standard
was not synthesized.
Hereafter we reported the procedure for the preparative biotransformation experiments allowing
the isolation of compounds 23a, 25, 28, 35 and 36 as well as their main analytic data.
According to the procedure described before for the preparation of the inoculum of spore forming
mycelia, Aspergillus niger, Nigrospora oryzae and Curvularia lunata, were inoculated in three 1 L conical
pyrex flasks containing 400 mL of MEM. The microorganisms were left to grow at 25 ◦C and 140 rpm
for 2 days. Hence the cultures of Aspergillus niger, Nigrospora oryzae and Curvularia lunata, were treated
with a solution of 1 g of γ-ionone, 3,4-dehydro-β-ionone and theaspirane, respectively, each one
dissolved in 3 mL of DMSO. After 14 days from the substrate injection, using the growing condition
described above, the reaction media was filtered through a celite pad, the filter was washed with ethyl
acetate and the filtrate was extracted 3 times with the same solvent. The combined organic phases were
separated, were washed with brine, dried on Na2SO4 and the solvent was removed under reduced
pressure. The residue was then acetylated by treatment with pyridine/acetic anhydride (10 mL of
a 2:1 mixture) and catalytic DMAP (10 mg) for 6 h at rt. The acetylating mixture (Py/Ac2O) was
then removed under reduced pressure and the resulting oil was purified by chromatography using
n-hexane/AcOEt mixture as eluent.
The biotransformation of γ-ionone performed using Aspergillus niger allowed isolating 0.21 g (18%
yield) of compound 23a and 0.36 g (28% yield) of compound 25 as a single diastereoisomeric form
(configuration not determined):
cis-2-Hydroxy-
cyclohexyl acetate. H-NMR:
γ
-ionone acetate (23a) = (1SR,3RS)-2,2-Dimethyl-4-methylene-3-((E)-3-oxobut-1-en-1-yl)
1
δ = 6.97 (dd, J = 15.8, 9.9 Hz, 1H), 6.10 (d, J = 15.8 Hz, 1H), 4.88 (s, 1H), 4.74
(dd, J = 9.2, 4.0 Hz, 1H), 4.61 (s, 1H), 2.65 (d, J = 9.9 Hz, 1H), 2.41 (dt, J = 14.0, 5.4 Hz, 1H), 2.37–2.05 (m,
1H) 2.28 (s, 3H), 2.07 (s, 3H), 1.93–1.83 (m, 1H), 1.72–1.57 (m, 1H), 0.91 (s, 6H). 13C-NMR
= 197.9 (C),
170.4 (C), 146.0 (C), 145.5 (CH), 132.9 (CH) 111.1 (CH2), 77.8 (CH), 55.9 (CH), 39.0 (C), 31.2 (CH2), 27.6
(CH2), 27.3 (Me), 26.2 (Me), 21.1 (Me), 17.8 (Me). GC-MS (EI): m/z (%) = 250 [M+] (12), 235 [M+
Me]
(1), 208 (13), 190 (45), 175 (40), 165 (36), 147 (100), 131 (23), 122 (39), 109 (96), 91 (34), 79 (35), 71 (12).
δ
−
3-Hydroxy- -ionone acetate (25) = (E)-3,3-Dimethyl-5-methylene-4-(3-oxobut-1-en-1-yl)cyclohexyl acetate.
1H-NMR:
= 6.82 (dd, J = 15.8, 10.1 Hz, 1H), 6.13 (d, J = 15.8 Hz, 1H), 4.97 (s, 1H), 4.95–4.86 (m, 1H),
4.63 (s, 1H), 2,73 (dd, J = 12.6, 4.9 Hz, 1H), 2.58 (d, J = 10.1 Hz, 1H), 2.29 (s, 3H), 2.15–2.01 (m, 1H), 2.03
(s, 3H), 1.85 (dd, J = 12.6, 4.5 Hz, 1H), 1.45 (t, J = 12.1 Hz, 1H), 0.95 (s, 3H), 0.92 (s, 3H). 13C-NMR:
γ
δ
δ
=
197.8 (C), 170.3 (C), 145.3 (CH), 144.5 (C), 134.0 (CH), 112.5 (CH2), 69.8 (CH), 56.0 (CH), 45.5 (CH2),
41.1 (CH2), 35.8 (C), 30.4 (Me), 27.3 (Me), 21.4 (Me), 21.3 (Me). GC-MS (EI): m/z (%) = 250 [M+] (<1),
235 [M+
(14), 55 (8).
−
Me] (<1), 190 (28), 175 (29), 157 (14), 147 (100), 131 (32), 119 (20), 105 (39), 91 (27), 79 (15), 69
The biotransformation of 3,4-dehydro-
β-ionone performed using Nigrospora oryzae allowed
isolating 0.56 g (34% yield) of compound 28 as a 5:1 mixture of trans/cis isomers:
trans-3,4-Dihydroxy-
3-ene-1,2-diyl diacetate. H-NMR:
β
-ionone diacetate (28) = (1RS,2RS)-3,5,5-Trimethyl-4-((E)-3-oxobut-1-en-1-yl)cyclohex-
1
δ = 7.10 (dq, J = 16.4, 0.9 Hz, 1H), 6.11 (d, J = 16.4 Hz, 1H), 5.51 (d, J
= 7.8 Hz, 1H), 5.17–5.09 (m, 1H), 2.28 (s, 3H), 2.06 (s, 3H), 2.00 (s, 3H), 1.88–1.66 (m, 2H), 1.62 (br s, 3H),
1.17 (s, 3H), 1.07 (s, 3H). 13C-NMR:
= 197.8 (C), 170.7 (C), 170.4 (C), 141.0 (CH), 140.2 (C), 133.7 (CH),
δ

Molecules 2019, 24, 19
15 of 18
128.5 (C), 74.7 (CH), 70.3 (CH), 41.4 (CH2), 36.4 (C), 29.7 (Me), 27.7 (Me), 27.5 (Me), 21.1 (Me), 20.8 (Me),
16.5 (Me). MS (ESI): 331.2 (M+ + Na).
The biotransformation of theaspirane performed using Curvularia lunata allowed isolating 95 mg
(7% yield) of compound 35 and 65 mg (5% yield) of compound 36:
1
2-(3-Acetoxy-2,6,6-trimethylcyclohex-1-en-1-yl)ethyl acetate (35). H-NMR:
δ = 5.13 (t, J = 4.6 Hz, 1H), 4.05
(dd, J = 9.4, 7.5 Hz, 2H), 2.46–2.37 (m, 2H), 2.06 (br s, 6H), 1.92–1.79 (m, 1H), 1.74–1.52 (m, 2H), 1.66 (s,
3H), 1.44–1.33 (m, 1H), 1.08 (s, 3H), 1.00 (s, 3H). 13C-NMR:
δ
= 171.0 (C), 171.0 (C), 139.5 (C), 128.2 (C),
72.3 (CH), 63.3 (CH2), 35.1 (C), 34.6 (CH2), 28.2 (Me), 28.0 (CH2), 26.8 (Me), 25.3 (CH2), 21.3 (Me), 21.0
(Me), 16.8 (Me). GC-MS (EI): m/z (%) = 268 [M+] (<1), 226 (13), 208 [M+
(17), 133 (100), 120 (28), 110 (36), 91 (16), 79 (8).
−
AcOH] (16), 166 (40), 148
1
3,6,6-Trimethyl-1,4,5,6,7,8-hexahydro-3H-3,5a-epoxybenzo[c]oxepin-8-yl acetate (36). H-NMR:
δ = 5.43–5.36
(m, 1H), 5.31 (s, 1H), 4.49 (dt, J = 14.2, 2.1 Hz, 1H), 4.20 (d, J = 14.2 Hz, 1H), 2.30–1.40 (m, 6H), 2.02 (s,
3H), 1.47 (s, 3H), 1.11 (s, 3H), 1.01 (s, 3H). 13C-NMR:
δ
= 170.5 (C), 138.7 (C), 116.3 (CH), 105.3 (C), 86.0
(C), 68.4 (CH), 63.7 (CH2), 40.7 (CH2), 35.3 (C), 34.3 (CH2), 30.5 (CH2), 24.6 (Me), 24.4 (Me), 23.3 (Me),
21.2 (Me). GC-MS (EI): m/z (%) = 266 [M+] (1), 236 (50), 224 (8), 207 [M+
164 (84), 153 (100), 136 (61), 121 (59), 107 (62), 91 (53), 79 (29).
−
AcO] (59), 195 (36), 178 (27),
4. Conclusions
Our work provides some relevant findings. First, we demonstrated that fungi are able to perform
different biotransformations on the isomeric forms of the C13 apocarotenoids ionone, theaspirane and
damascone. With respect to the eleven strains tested, we observed that the most common chemical
transformations are oxidation reactions that afford oxygenated products such as hydroxy- keto- or
epoxy-derivatives. On the contrary, the reduction of the keto groups or the reduction of the double
bond functional groups are less relevant transformations, occurring for few substrates and yielding a
minority amount of products.
A very significant feature of our study concern the prospective applicability of the fungi-mediated
biotransformation of apocarotenoids for the synthesis of high value natural flavours. Since some
ionone, damascone and theaspirane isomers are available in natural form and the biotransformation
of a natural precursor is considered a ‘natural method’ of synthesis, the flavours obtained by
means of the fungi-mediated reactions described above possess the natural status and could be
commercialized accordingly.
Finally, we would like to highlight that our microbial biotransformations allow the preparation
of many derivatives whose synthesis, using the classical chemical reactions, is very difficult. For
example, different fungal strains proved to be able to oxidize some inactivated positions of the
ionone or theaspirane framework, such as the position 2 and 3 of the
β- and γ-ionone and the
methine carbon linked to the theaspirane oxygen atom. By means of these microbial capabilities,
we isolated one new compound (compound 36) and we devised a new biocatalytic procedure for
the synthesis of 2-hydroxy-γ-ionone, 3-hydroxy-γ-ionone, 3,4-dihydroxy-β-ionone and 2,6,10,10-
tetramethyl-1-oxa-spiro[4.5]dec-6-ene-2,8-diol (identified as its monoacetate 34), which are natural
apocarotenoids or their direct precursors.
Supplementary Materials: The Supplementary Materials are available online.
Author Contributions: S.S. and D.D.S. equally contributed to the conceptualization of this study. S.S. and D.D.S.
equally contributed to conceive, design and perform the experiments as well as to analyze the data. S.S. wrote
the paper.
Funding: This research was funded by [Cariplo Fundation] grant number [2014-0568 INBOX (Innovative
Biocatalytic Oxidations)].
Acknowledgments: The authors thank Cariplo Foundation for supporting this study.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2019, 24, 19
16 of 18
References
1.
2.
3.
Walter, M.H.; Strack, D. Carotenoids and their cleavage products: Biosynthesis and functions. Nat. Prod. Rep.
2011, 28, 663–692. [CrossRef] [PubMed]
Serra, S. Recent advances in the synthesis of carotenoid-derived flavours and fragrances. Molecules 2015, 20,
12817–12840. [CrossRef] [PubMed]
Ma, Q.-G.; Wang, Y.-G.; Liu, W.-M.; Wei, R.-R.; Yang, J.-B.; Wang, A.-G.; Ji, T.-F.; Tian, J.; Su, Y.-L.
Hepatoprotective sesquiterpenes and rutinosides from Murraya koenigii (L.) Spreng. J. Agric. Food Chem.
2014, 62, 4145–4151. [CrossRef] [PubMed]
4.
5.
6.
7.
8.
9.
Schievano, E.; Morelato, E.; Facchin, C.; Mammi, S. Characterization of markers of botanical origin and other
compounds extracted from unifloral honeys. J. Agric. Food Chem. 2013, 61, 1747–1755. [CrossRef] [PubMed]
Li, C.-Y.; Wu, T.-S. Constituents of the stigmas of Crocus sativus and their tyrosinase inhibitory activity.
J. Nat. Prod. 2002, 65, 1452–1456. [CrossRef] [PubMed]
Ina, K.; Eto, H. 3-keto-β-ionone in the essential oil from black tea. Agric. Biol. Chem. 1971, 35, 962–963.
¯
[CrossRef]
Fujimori, T.; Kasuga, R.; Matsushita, H.; Kaneko, H.; Noguchi, M. Neutral aroma constituents in burley
tobacco. Agric. Biol. Chem. 1976, 40, 303–315. [CrossRef]
Bolt, A.J.N.; Purkis, S.W.; Sadd, J.S. A damascone derivative from Nicotiana tabacum. Phytochemistry 1983, 22,
613–614. [CrossRef]
Sato, S.; Sasakura, S.; Kobayashi, A.; Nakatani, Y.; Yamanishi, T. Flavor of black tea. Part VI. Intermediate
and high boiling components of the neutral fraction. Agric. Biol. Chem. 1970, 34, 1355–1367. [CrossRef]
10. Beekwilder, J.; van Rossum, H.M.; Koopman, F.; Sonntag, F.; Buchhaupt, M.; Schrader, J.; Hall, R.D.;
Bosch, D.; Pronk, J.T.; van Maris, A.J.A.; et al. Polycistronic expression of a -carotene biosynthetic pathway
-ionone production. J. Biotechnol. 2014, 192, 383–392. [CrossRef]
β
in Saccharomyces cerevisiae coupled to
β
[PubMed]
11. Serra, S.; Fuganti, C.; Brenna, E. Biocatalytic preparation of natural flavours and fragrances. Trends Biotechnol.
2005, 23, 193–198. [CrossRef] [PubMed]
12. Prelog, V.; Meier, H.L. Untersuchungen über organextrakte und harn. 18. Mitteilung. Über die biochemische
oxydation von β-jonon im tierkörper. Helv. Chim. Acta 1950, 33, 1276–1284. [CrossRef]
13. Mikami, Y.; Watanabe, E.; Fukunaga, Y.; Kisaki, T. Formation of 2S-hydroxy-β-ionone and 4-hydroxy-
β-ionone by microbial hydroxylation of β-ionone. Agric. Biol. Chem. 1978, 42, 1075–1077. [CrossRef]
14. Mikami, Y.; Fukunaga, Y.; Arita, M.; Kisaki, T. Microbial transformation of
β-ionone and
β-methylionone.
Appl. Environ. Microbiol. 1981, 41, 610–617. [PubMed]
15. Krasnobajew, V.; Helmlinger, D. Fermentation of fragrances: Biotransformation of
β-ionone by Lasiodiplodia
theobromae. Helv. Chim. Acta 1982, 65, 1590–1601. [CrossRef]
16. Hartman, D.A.; Pontones, M.E.; Kloss, V.F.; Curley, R.W.; Robertson, L.W. Models of retinoid metabolism:
Microbial biotransformation of -ionone and -ionone. J. Nat. Prod. 1988, 51, 947–953. [CrossRef] [PubMed]
α
β
17. Schoch, E.; Benda, I.; Schreier, P. Bioconversion of
α
-damascone by Botrytis cinerea. Appl. Environ. Microbiol.
1991, 57, 15–18.
18. Kakeya, H.; Sugai, T.; Ohta, H. Biochemical preparation of optically active 4-hydroxy-
β-ionone and its
transformation to (S)-6-hydroxy-α-ionone. Agric. Biol. Chem. 1991, 55, 1873–1876. [CrossRef]
19. Weidmann, V.; Kliewer, S.; Sick, M.; Bycinskij, S.; Kleczka, M.; Rehbein, J.; Griesbeck, A.G.; Zorn, H.;
Maison, W. Studies towards the synthetic applicability of biocatalytic allylic oxidations with the lyophilisate
of Pleurotus sapidus. J. Mol. Catal. B Enzym. 2015, 121, 15–21. [CrossRef]
20. Gliszczyn´ska, A.; Gładkowski, W.; Dancewicz, K.; Gabrys´, B.; Szczepanik, M. Transformation of
β-damascone
to (+)-(S)-4-hydroxy- -damascone by fungal strains and its evaluation as a potential insecticide against
β
aphids Myzus persicae and lesser mealworm Alphitobius diaperinus Panzer. Catal. Commun. 2016, 80, 39–43.
[CrossRef]
21. Lutz-Wahl, S.; Fischer, P.; Schmidt-Dannert, C.; Wohlleben, W.; Hauer, B.; Schmid, R.D. Stereo- and
regioselective hydroxylation of
[PubMed]
α-ionone by Streptomyces strains. Appl. Environ. Microbiol. 1998, 64, 3878–3881.

Molecules 2019, 24, 19
17 of 18
22. Maurer, S.C.; Schulze, H.; Schmid, R.D.; Urlacher, V. Immobilisation of P450 BM-3 and an NADP+ cofactor
recycling system: Towards a technical application of heme-containing monooxygenases in fine chemical
synthesis. Adv. Synth. Catal. 2003, 345, 802–810. [CrossRef]
23. Litzenburger, M.; Bernhardt, R. Selective oxidation of carotenoid-derived aroma compounds by CYP260B1
and CYP267B1 from Sorangium cellulosum So ce56. Appl. Microbiol. Biotechnol. 2016, 100, 4447–4457.
[CrossRef] [PubMed]
24. Venkataraman, H.; Beer, S.B.A.d.; Geerke, D.P.; Vermeulen, N.P.E.; Commandeur, J.N.M. Regio- and
stereoselective hydroxylation of optically active α-ionone enantiomers by engineered cytochrome P450 BM3
mutants. Adv. Synth. Catal. 2012, 354, 2172–2184. [CrossRef]
25. Fuganti, C.; Serra, S.; Zenoni, A. Synthesis and olfactory evaluation of (+)- and (-)-gamma-ionone.
Helv. Chim. Acta 2000, 83, 2761–2768. [CrossRef]
26. Serra, S.; Fuganti, C.; Brenna, E. Synthesis, olfactory evaluation, and determination of the absolute
configuration of the 3,4-didehydroionone stereoisomers. Helv. Chim. Acta 2006, 89, 1110–1122. [CrossRef]
27. Serra, S.; Fuganti, C.; Brenna, E. Two easy photochemical methods for the conversion of commercial ionone
alpha into regioisomerically enriched gamma-ionone and gamma-dihydroionone. Flavour Fragr. J. 2007, 22,
505–511. [CrossRef]
28. Serra, S. An expedient preparation of enantio-enriched ambergris odorants starting from commercial ionone
alpha. Flavour Fragr. J. 2013, 28, 46–52. [CrossRef]
29. Brenna, E.; Fuganti, C.; Serra, S.; Kraft, P. Optically active ionones and derivatives: Preparation and olfactory
properties. Eur. J. Org. Chem. 2002, 967–978. [CrossRef]
30. Serra, S.; Fuganti, C. Synthesis of the enantiomeric forms of alpha- and gamma-damascone starting from
commercial racemic alpha-ionone. Tetrahedron Asymmetry 2006, 17, 1573–1580. [CrossRef]
31. Brenna, E.; Fuganti, C.; Serra, S. Synthesis and olfactory evaluation of the enantiomerically enriched forms
of 7,11-epoxymegastigma-5(6)-en-9-one and 7,11-epoxymegastigma-5(6)-en-9-ols isomers, identified in
passiflora edulis. Tetrahedron Asymmetry 2005, 16, 1699–1704. [CrossRef]
32. Serra, S.; Barakat, A.; Fuganti, C. Chemoenzymatic resolution of cis- and trans-3,6-dihydroxy-alpha-ionone.
Synthesis of the enantiomeric forms of dehydrovomifoliol and 8,9-dehydrotheaspirone. Tetrahedron Asymmetry
2007, 18, 2573–2580. [CrossRef]
33. Mazur, M.; Grudniewska, A.; Wawrzen´czyk, C. Microbial transformations of halolactones with p-menthane
system. J. Biosci. Bioeng. 2015, 119, 72–76. [CrossRef] [PubMed]
34. Simeo, Y.; Sinisterra, J.V. Biotransformation of terpenoids: A green alternative for producing molecules with
pharmacological activity. Mini-Rev. Org. Chem. 2009, 6, 128–134. [CrossRef]
35. Bhatti, H.N.; Khera, R.A. Biological transformations of steroidal compounds: A review. Steroids 2012, 77,
1267–1290. [CrossRef]
36. Carballeira, J.D.; Álvarez, E.; Sinisterra, J.V. Biotransformation of cyclohexanone using immobilized
Geotrichum candidum NCYC49: Factors affecting the selectivity of the process. J. Mol. Catal. B Enzym.
2004, 28, 25–32. [CrossRef]
37. Bankar, A.V.; Kumar, A.R.; Zinjarde, S.S. Environmental and industrial applications of Yarrowia lipolytica.
Appl. Microbiol. Biotechnol. 2009, 84, 847. [CrossRef]
38. Johnson, E.A. Phaffia rhodozyma: Colorful odyssey. Int. Microbiol. 2003, 6, 169–174. [CrossRef]
39. Knapp, H.; Weigand, C.; Gloser, J.; Winterhalter, P. 2-hydroxy-2,6,10,10-tetramethyl-1-oxaspiro[4.5]dec-6-
en-8-one: Precursor of 8,9-dehydrotheaspirone in white-fleshed nectarines. J. Agric. Food Chem. 1997, 45,
1309–1313. [CrossRef]
40. Näf, R.; Velluz, A.; Decorzant, R.; Näf, F. Structure and synthesis of two novel ionone-type compounds
identified in quince brandy (Cydonia oblonga Mil.). Tetrahedron Lett. 1991, 32, 753–756. [CrossRef]
41. Serra, S. MnO2/TBHP: A versatile and user-friendly combination of reagents for the oxidation of allylic and
benzylic methylene functional groups. Eur. J. Org. Chem. 2015, 2015, 6472–6478. [CrossRef]
42. Serra, S.; Lissoni, V. First enantioselective synthesis of marine diterpene ambliol-A. Eur. J. Org. Chem. 2015
2015, 2226–2234. [CrossRef]
,
43. Dess, D.B.; Martin, J.C. Readily accessible 12-I-5 oxidant for the conversion of primary and secondary
alcohols to aldehydes and ketones. J. Org. Chem. 1983, 48, 4155–4156. [CrossRef]
44. Audran, G.; Galano, J.M.; Monti, H. Enantioselective synthesis and determination of the absolute
configuration of natural (−)-elegansidiol. Eur. J. Org. Chem. 2001, 2293–2296. [CrossRef]

Molecules 2019, 24, 19
18 of 18
45. Serra, S.; Gatti, F.G.; Fuganti, C. Lipase-mediated resolution of the hydroxy-cyclogeraniol isomers:
Application to the synthesis of the enantiomers of karahana lactone, karahana ether, crocusatin C and
gamma-cyclogeraniol. Tetrahedron Asymmetry 2009, 20, 1319–1329. [CrossRef]
46. Kaiser, R.; Lamparsky, D. Inhaltsstoffe des Osmanthus-absolues. 1. Mitteilung: 2,5-epoxy-megastigma-6,8-dien.
Helv. Chim. Acta 1978, 61, 373–382. [CrossRef]
47. Tu, V.A.; Kaga, A.; Gericke, K.-H.; Watanabe, N.; Narumi, T.; Toda, M.; Brueckner, B.; Baldermann, S.;
Mase, N. Synthesis and characterization of quantum dot nanoparticles bound to the plant volatile precursor
0
of hydroxy-apo-10 -carotenal. J. Org. Chem. 2014, 79, 6808–6815. [CrossRef]
48. Khachik, F.; Chang, A.-N. Synthesis of (3S)- and (3R)-3-hydroxy-β-ionone and their transformation into (3S)-
and (3R)-β-cryptoxanthin. Synthesis 2011, 2011, 509–516. [CrossRef]
49. Takei, Y.; Mori, K.; Matsui, M. Synthesis of a stereoisomeric mixture of 3-hydroxy-
Chem. 1973, 37, 2927–2928. [CrossRef]
α
-damascone. Agric. Biol.
50. Pascual, A.; Bischofberger, N.; Frei, B.; Jeger, O. Photochemical reactions. 149th communication. Photochemistry
of 7,8-dihydro-4-hydroxy-β-ionone and derivatives. Helv. Chim. Acta 1988, 71, 374–388. [CrossRef]
0
0
0
0
51. Buschor, D.J.; Eugster, C.H. Synthese der (3S,4R,3 S,4 R)- und (3S,4R,3 S,4 S)crustaxanthine sowie weiterer
verbindungen mit 3,4-dihydroxy-β-endgruppen. Helv. Chim. Acta 1990, 73, 1002–1021. [CrossRef]
52. Irie, H.; Matsumoto, R.; Nishimura, M.; Zhang, Y. Synthesis of ( )-heritol, a sesquiterpene lactone belonging
±
to the aromatic cadinane group. Chem. Pharm. Bull. 1990, 38, 1852–1856. [CrossRef]
53. Cooper, R.D.G.; Davis, J.B.; Leftwick, A.P.; Price, C.; Weedon, B.C.L. Carotenoids and related compounds. Part
XXXII. Synthesis of astaxanthin, phoenicoxanthin, hydroxyechinenone, and the corresponding diosphenols.
J. Chem. Soc. Perkin Trans. 1 1975, 2195–2204. [CrossRef]
54. Serra, S.; Piccioni, O. A new chemo-enzymatic approach to the stereoselective synthesis of the flavors
tetrahydroactinidiolide and dihydroactinidiolide. Tetrahedron Asymmetry 2015, 26, 584–592. [CrossRef]
55. Oritani, T.; Yamamoto, H.; Yamashita, K. Synthesis of ( γ-ionylideneacetic acids, fungal
)-40-hydroxy-
±
biosynthetic intermediates of abscisic acid. Agric. Biol. Chem. 1990, 54, 125–130. [CrossRef]
Sample Availability: Samples of the compounds 23a, 25, 28, 35 and 36 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).