Microbial Modifications of Androstane and Androstene Steroids by Penicillium vinaceum

Article abstract of DOI:10.3390/molecules25184226

The biotransformation of steroid compounds is a promising, environmentally friendly route to new pharmaceuticals and hormones. One of the reaction types common in the metabolic fate of steroids is Baeyer-Villiger oxidation, which in the case of cyclic ketones, such as steroids, leads to lactones. Fungal enzymes catalyzing this reaction, Baeyer-Villiger monooxygenases (BVMOs), have been shown to possess broad substrate scope, selectivity, and catalytic performance competitive to chemical oxidation, being far more environmentally green. This study covers the biotransformation of a series of androstane steroids (epiandrosterone and androsterone) and androstene steroids (progesterone, pregnenolone, dehydroepiandrosterone, androstenedione, 19-OH-androstenedione, testosterone, and 19-nortestosterone) by the cultures of filamentous fungus Penicillium vinaceum AM110. The transformation was monitored by GC and the resulting products were identified on the basis of chromatographic and spectral data. The investigated fungus carries out effective Baeyer-Villiger oxidation of the substrates. Interestingly, introduction of the 19-OH group into androstenedione skeleton has significant inhibitory effect on the BVMO activity, as the 10-day transformation leaves half of the 19-OH-androstenedione unreacted. The metabolic fate of epiandrosterone and androsterone, the only 5α-saturated substrates among the investigated compounds, is more complicated. The transformation of these two substrates combined with time course monitoring revealed that each substrate is converted into three products, corresponding to oxidation at C-3 and C-17, with different time profiles and yields.

Full text of DOI:10.3390/molecules25184226

molecules
Article
Microbial Modifications of Androstane and
Androstene Steroids by Penicillium vinaceum
´
Anna Panek *, Paulina Łyczko and Alina Swizdor
Department of Chemistry, Wrocław University of Environmental and Life Sciences, 50-375 Wrocław, Poland;
´
paulina.lyczko@upwr.edu.pl (P.Ł.); alina.swizdor@upwr.edu.pl (A.S.)
* Correspondence: anna.panek@upwr.edu.pl
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Rafal R. Sicinski and Akihito Yokosuka
Received: 30 July 2020; Accepted: 13 September 2020; Published: 15 September 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The biotransformation of steroid compounds is a promising, environmentally friendly
route to new pharmaceuticals and hormones. One of the reaction types common in the metabolic
fate of steroids is Baeyer-Villiger oxidation, which in the case of cyclic ketones, such as steroids,
leads to lactones. Fungal enzymes catalyzing this reaction, Baeyer-Villiger monooxygenases (BVMOs),
have been shown to possess broad substrate scope, selectivity, and catalytic performance competitive
to chemical oxidation, being far more environmentally green. This study covers the biotransformation
of a series of androstane steroids (epiandrosterone and androsterone) and androstene steroids
(progesterone, pregnenolone, dehydroepiandrosterone, androstenedione, 19-OH-androstenedione,
testosterone, and 19-nortestosterone) by the cultures of filamentous fungus Penicillium vinaceum AM110.
The transformation was monitored by GC and the resulting products were identified on the basis
of chromatographic and spectral data. The investigated fungus carries out effective Baeyer-Villiger
oxidation of the substrates. Interestingly, introduction of the 19-OH group into androstenedione
skeleton has significant inhibitory effect on the BVMO activity, as the 10-day transformation
leaves half of the 19-OH-androstenedione unreacted. The metabolic fate of epiandrosterone
and androsterone, the only 5α-saturated substrates among the investigated compounds, is more
complicated. The transformation of these two substrates combined with time course monitoring
revealed that each substrate is converted into three products, corresponding to oxidation at C-3 and
C-17, with different time profiles and yields.
Keywords: biotransformations; steroids; 19-hydroxyandrostenedione; 19-nortestololactone; lactonization;
Baeyer-Villiger oxidation; Penicillium vinaceum
1. Introduction
The diversity of chemicals used by contemporary industry, medicine, and agriculture requires
a constant search for new or more efficient synthetic routes and catalysts. Microbial enzymes and
whole-cell catalytic systems seem to be an almost infinite reservoir of catalysts, with the benefit of
relative ease of maintenance, mild reaction conditions, and low environmental track. Moreover,
microbes exist in virtually all environments on Earth able to sustain life, from the polar regions [
through dry deserts, to marine hydrothermal vents. Enzymes from such microorganisms must be able
to operate in very diverse conditions of temperature (ranging from Arctic cold [ ] to boiling water [ ]),
1]
2
3
salinity, pH, or pressure. Although the last two decades have brought some shift in our approach
to the marine, extreme, or isolated environments, from the use of natural products towards the
isolation and identification of enzymes or microorganisms with desired biotransformation properties,
a recent review [4] points out that the potential of catalytic activity found in these environments is
still underexplored.
Molecules 2020, 25, 4226; doi:10.3390/molecules25184226
www.mdpi.com/journal/molecules

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One of the fungal species with such underexplored potential is Penicillium vinaceum, first described
in 1927 [ ], and present in soil and marine environments. This species was shown to produce
several known compounds of biochemical interest: cyclo (d-tryptophanyl-L-prolyl), citreoisocoumarin,
brevianamide F, indol-3-carbaldehyde, -cyclopiazonic acid, terretrione A [ ], and one product
specific to this fungus, a diketopiperazine alkaloid penicillivinacine with antimigratory activity
against the human breast cancer cells similar to terretrione A [ ]. On the other hand, little is
known of its biotransformation potential. P. vinaceum was shown to be ineffective in the reduction
of methyl cinnamate to 2-phenoxyethanol [ ], but its enzymatic system is able to carry out
effective transglycosylation leading to functionalized alkyl -d-galactopyranosides, potentially useful
biopolymers [ ]. Several patent applications related to the biotransformations with P. vinaceum were
filed: reduction of tetrahydrothiophen-3-one to (R)-tetrahydrothiophen-3-ol [10], conversion of eugenol
to coniferyl aldehyde [11], and production of 3 -hydroxy-17a-oxa-d-homo-androst-5-en-17-one [12].
5
α
6
7
8
α
9
β
The last reaction is an example of a process constituting the main research subject of the current study:
enzymatic Baeyer-Villiger oxidation of steroidal substrates.
Baeyer–Villiger oxidation (BVO), first reported in 1899 [13], involves the action of peroxides or
peroxyacids on ketones, with the insertion of an oxygen atom into the carbon chain at the carbonyl group.
Esters are formed from acyclic ketones, while cyclic substrates are converted to lactones. Industrial
applications of BVO are diverse, ranging from the production of monomers for polymerization to
obtaining antibiotic medicines, steroids, and pheromones [14]. The original oxidizers, peroxyacids,
are problematic in many aspects: they are unstable or even explosive, environmentally unfriendly
(each reacting peroxyacid molecule turns into a carboxylate anion which must then be disposed of),
the solvents frequently contain chlorinated hydrocarbons, and the reactions are far from being optimal
with regard to the substrate specificity, chemo-, regio-, or enantioselectivity. Large progress was
made with the use of hydrogen peroxide as a greener, environmentally friendly oxidizer for chemical
BVO [15], but even the most recent accomplishments which fine-tune chemical BVO to obtain high
selectivity and yields [16] use organic solvents as reaction media. On the other hand, the use of isolated
enzymes or whole-cell biocatalysts seems to be a tempting alternative able to solve the environmental
issues of chemical BVO. Several recent comprehensive reviews [17,18] summarize the research into the
microbial Baeyer-Villiger oxidation reactions since late 1940s, from pure research to the development
of bioprocesses. The latter review concludes with such statements as “the real break-through of the BV
biooxidation in industry has still not been realized. Hence, it is a prime time to seek out new targets for
BV biooxidations” [18]. Interestingly, many compounds considered nowadays as biotransformation
targets are steroids, the same class of substances found in the first reports on microbial Baeyer–Villiger
oxidation [19,20].
Importance of steroids for life is underlined by their diverse and ubiquitous roles in cellular
biochemistry: participation of cholesterol in cell membranes, digestion facilitated by bile acids,
and most importantly for medicinal applications, the hormonal activity of many steroid derivatives.
The last factor is crucial for the use of steroid derivatives in medicine (as anti-inflammatory and
anti-cancer medications) and sport (anabolic drugs). Recent reports have turned our attention especially
towards 19-hydroxy steroidal derivatives: 19-OH-androstenedione has been shown to participate
in neuroendocrine trans-differentiation of prostate cancer cells [21], while a new biocatalytic route
was devised [22] leading to 19-OH-cortexolone, 19-OH-androstenedione, and ultimately to numerous
C19-hydroxysteroids and 19-norsteroids with antitumor and insecticidal activities. Therefore we have
included representatives of 19-OH steroids in our biotransformation experiments. Biosynthetic routes
from 19-nortestosterone to 19-nortestololactone were established with the use of biocatalytic fungi
Aspergillus tamarii [23] and Penicillium notatum [24], but we were able to obtain in the current study
better final yields than those presented in the earlier reports.
Baeyer-Villiger oxidation of steroids to steroidal lactones is one of the routes pursued in the
search for new bioactive compounds. The enzymes responsible for this process are Baeyer-Villiger
monooxygenases (BVMOs). While their sequences and phylogenetic tree have been intensively

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explored [18], much less is known about the structural aspects of BVMOs. Only recently were
some bacterial and fungal BVMOs with steroids in their substrate scope crystallized and their 3D
structures determined by crystallographic methods: a steroid monooxygenase from Rhodococcus
rhodochrous [25], and a versatile polycyclic ketone monooxygenase (PockeMO) from the thermophilic
fungus Thermothelomyces thermophile [26].
Microbial BVMOs have considerably broad substrate scope, for example the PockeMO can
oxidize both a and d rings of the steroidal substrates [26], and this versatility is related to the
structure of substrate binding pocket, able to accept bulky substrates in diverse configurations [26,27].
Such an idea of configurational flexibility of steroid substrates was proposed already in 1967 on
the basis of diverse metabolic fates of steroids biotransformed by Aspergillus tamarii cultures [28].
Some recent examples of the presence of steroidal BVMOs in fungal biotransformation pathways are:
lactonization of progesterone and 5-ene steroids [29
(DHEA), pregnenolone, and androstenedione by filamentous fungi of genus Penicillium [31
lactonization of DHEA by Aspergillus parasiticus [33]; ring-d lactonization of steroidal C-17 ketones
to 11 -hydroxy derivatives by Beauveria bassiana [34]; activity of Penicillium lanosocoeruleum in ring-d
,30]; lactonization of dehydroepiandrosterone
,
32];
α
lactonization of C19-steroids [35] and pregnene-based steroids [36]; biotransformation of DHEA into
hydroxylated steroid lactones Spicaria fumoso-rosea [37]; diverse biotransformation routes of steroids,
including ring-d lactonization, in the cultures of Penicillium notatum [24] and Aspergillus terreus [38];
formation of testololactone from diverse steroidal substrates with the use of a multifunctional strain of
Penicillium simplicissimum [39]; cascade of DHEA biotransformations by Beauveria species [40]; and the
formation of new derivatives of 3β-acetyloxy-5α-chloro-6,19-oxidoandrostan-17-one [41]. This rich
background of BVMO activity studies in fungal species, combined with the outlined above need for
further research in the field of biotransformations, prompted us to investigate in detail metabolic fates
of DHEA (
1
), epiandrosterone (
2
), androsterone (
3
), androstenedione (
4
), 19-OH-androstenedione
) in the cultures of
(5), testosterone (
6
), 19-nortestosterone (
7), progesterone (
8), and pregnenolone (9
Penicillium vinaceum AM110. The transformation progress was monitored by GC and TLC analysis and
the resulting products were identified on the basis of chromatographic and spectral data.
2. Results and Discussion
Biotransformations of the chosen substrates by Penicillium vinaceum AM110 proceed according
to one of two distinct schemes. Substrates
d, and no other detectable products are formed. On the other hand, transformation of compounds
and , the only 5 -saturated steroids in the investigated series of substrates, leads to a mixture of
products. For this reason we have carried out transformations of and with time course monitoring
1 and 4–9 undergo Baeyer-Villiger lactonization in ring
2
3
α
2
3
of the composition of the reaction mixture. These two schemes of biotransformation will be described
in separate subsections below. Scheme 1 presents the structures of the investigated compounds:
substrates 1–9 and the corresponding products.

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Scheme 1. Biotransformation of steroidal substrates
substrate and product structures.
1–9 by P. vinaceum AM110—overview of the
2.1. Baeyer-Villiger Oxidation of Steroidal Compounds by P. vinaceum AM110
Our earlier studies with biotransformations of steroids by the fungi of genus
Penicillium [31,32,35,36] have revealed large diversity of metabolic fates of the substrates. Baeyer-Villiger
oxidation, isomerizations, and hydroxylations with diverse product stereochemistry were found. On the
other hand, the biocatalyst used in the current study, Penicillium vinaceum AM110, prefers to carry
out only the BVO process for most of the substrates:
1, 4, and 6–9 (see Scheme 1 for a structural
overview, and Table 1 for the detailed results of the transformations; in some cases the products
were compounds for which we had no standard samples to compare with using the GC and TLC
experiments, and these compounds were identified on the basis of NMR spectroscopy—see Section 2.3).
In most cases the conversion ratio exceeds 90% in two days. An interesting exception is provided by

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19-hydroxyandrostenedione (
5
) where, after 10 days of transformation, only half of the substrate was
transformed. The parent compound of
in 48 h. The ring-d Baeyer-Villiger lactonization of
cultures of P. lilacinum [31] and P. camemberti [32]. Its 19-OH derivative
but it undergoes reduction at the same position C-17. Thus, introduction of the 19-OH function seems
5
, androstenedione (
was also the only process for this substrate in the
is not affected by the BVMO,
4), is converted in 95% into testololactone
4
5
to obstruct the action of the fungal BVMO but not the action of a steroid 17β-HSD (17β-hydroxysteroid
dehydrogenase).
Table 1. The final outcome of the transformation of steroidal compounds by Penicillium vinaceum.
Rt
(min)
Time
(h/d)
Steroidal Compounds Identified in the
Mixture (%) a
Yield
Substrate
(%) a,c
4.20
7.79
24 h
DHEA
-
DHEA (1)
3β-Hydroxy-17a-oxa-d-homo-androst-5-
96
en-17-one b (10)
5.29
9.32
48 h
10 d
48 h
6 d
Androstenedione
5
95
Androstenedione (4)
19-Hydroxyandrostenedione (5)
Testosterone (6)
Testololactone b (11)
9.26
9.43
19-Hydroxyandrostenedione
17β,19-dihydroxyandrost-4-en-3-one (12)
54
46 (40)
5.61
9.32
Testosterone
-
Testololactone b (11)
100
5.19
9.09
19-nortestosterone
19-nortestololactone (13)
2
19-Nortestosterone (7)
Progesterone (8)
90 (85)
7.51
9.32
72 h
48 h
Progesterone
13
87
Testololactone b (11)
6.22
7.79
Pregnenolone
3β-Hydroxy-17a-oxa-d-homo-androst-5-
en-17-one b (10)
9
Pregnenolone (9)
91
a
b
Determined by GC analysis of the crude chloroform (chloride methylene for (
5
)) extracts. Identified in GC and
c
TLC on the basis of comparison with standard samples. Isolated yield in parentheses.
An interesting perspective on the inhibition of the BVMO by the presence of the 19-OH function
is provided by another pair of substrates, testosterone (
the 19-methyl group present in is replaced by a hydrogen atom in
the transformation kinetics: is totally converted into testololactone in two days, while
in minute amount (2%) after six days of transformation. However, both and are converted into the
6
) and 19-nortestosterone (
. This has only a moderate effect on
is still present
7). In this case,
6
7
6
7
6
7
corresponding lactones, and no traces of any reduced derivatives were observed. In the presented
studies, in the course of a six-day biotransformation 19-nortestololactone (13) was obtained with good
yield of 90%. An established biosynthetic route to 19-nortestololactone utilizes 72 h transformation of
19-nortestosterone by Aspergillus tamarii with 70% yield (16
example of transformation of 19-nortestosterone by Penicillium notatum, 19-nortestololactone was
obtained with 93% yield, but at lower substrate concentration (100 mg/300 mL medium) [24] than
×
50 mg/500 mL) [23]. In another known
in the current report. As a result of the 10-day transformation of 19-hydroxyandrostenedione (
17 ,19-dihydroxyandrost-4-en-3-one (12) (19-hydroxytestosterone) was obtained with 46% yield. In the
case of , the BVMO activity and formation of the ring-d lactone derivative were not observed.
To the best of our knowledge, we present the first described case of microbiological formation
of 19-hydroxytestosterone from 17 ,19-dihydroxyandrost-4-en-3-one in the culture of P. vinaceum
5),
β
5
β
(obtained chemically by Ehrenstein and Otto [42]).
These results confirm the preference of many steroid substrates to undergo lactonization, observed
in many studies [29
the substrates studied here are not lactones, but 17-hydroxy, 17-acetyl etc. derivatives. However,
the compounds differing only by the C-17 function are converted to the same product: as well as
yield 3 -hydroxy-17a-oxa-d-homo-androst-5-en-17-one, while 6, and yield testololactone. Thus,
–32,37]. This preference for lactonization includes also the fact that many of
1
9
β
4,
8

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successful BVMO activity requires also effective preceding steps leading to the cleavage or oxidation of
the substituent at C-17, which was noted already by Sebek et al. [43].
The performance of BVMOs of the Penicillium vinaceum AM110 strain is comparable to other species
of the genus Penicillium. For example, P. lanosocoeruleum converts progesterone to testololactone in 88%
yield in 72 h (the value very similar to the current study), but pregnenolone is converted into a mixture
of products, with only 44% yield of testololactone in 72 h [36] (in the current study on P. vinaceum,
testololactone is the sole detected product with 91% yield after 48 h). P. lanosocoeruleum converts DHEA
(1) solely to testololactone with 96% yield in 24 h [35], which is a different route, including isomerization,
than in the current study, where only BVO is observed. Interestingly, other Penicillium species also
differ in their ability of DHEA conversion: P. lilacinum AM111 transforms DHEA completely in 30 h
into the same product as in the current study, 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one [31],
while P. camemberti AM83 exhibits two distinct metabolic routes for DHEA, both however converging
into testololactone (85% yield after 24 h) [32].
Summarizing the ability of P. vinaceum to transform androstene substrates, we note that the
metabolic fate of these substrates, leading to the corresponding lactones, is rather less complicated
than in the other species of Penicillium genus, and the transformation kinetics and achieved yields are
very promising.
2.2. Transformations of 5α-Saturated Steroids in the Cultures of P. vinaceum AM110
The metabolic fate of androsterone (
-saturated steroids, turns out to be more complicated than the transformation of the unsaturated
steroids described above. Initial tests indicated that P. vinaceum is able to transform and into
a mixture of products oxidized at C-17 (BVMO activity, products 16 and 17), at C-3 (product 14
3) and epiandrosterone (2), representative biologically active
5
α
2
3
,
oxidation of the hydroxyl group to the corresponding ketone) or at both C-3 and C-17 (product 15).
See Scheme 1 for the structures of the substrates and products. This fact, already observed, e.g., for
P. lanosocoeruleum [35], prompted us to carry out transformations aimed at the time course analysis of
the transformation progress. The results are summarized in Table 2, Figures 1 and 2.
Table 2. The time course of the transformation of epiandrosterone and androsterone by Penicillium
vinaceum.
Time of Transformation (h)
Rt
(min)
Compounds Identified in the
Mixture (%) a
Substrate
24
48
72
96
8 d
4.51
4.98
Epiandrosterone
Androstanedione b (14)
17a-Oxa-d-homo-5α-androstan-3,
17-dione b (15)
94
2
91
2
87
3.5
82
4
77.5
5.5
Epiandrosterone (2)
8.95
8.43
2
2
3
4
4
4
5
3β-Hydroxy-17a-oxa-d-homo-5α-
5.5
10
12
androstan-17-one b (16)
4.68
4.98
Androsterone
Androstanedione b (14)
17a-Oxa-d-homo-5α-androstan-3,
17-dione b (15)
92
4
83
8
38
10
18
-
15
-
Androsterone (3)
8.95
8.48
2
2
3
6
12
40
22
60
32
53
3α-Hydroxy-17a-oxa-d-homo-5α-
androstan-17-one b (17)
a Determined by GC analysis of the crude chloroform extracts. b Identified in GC and TLC on the basis of comparison
with standard samples.

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100
80
60
40
20
0
2
14
15
16
0
2
4
6
8
Time of transformations (days)
Figure 1. The time course of the transformation of epiandrosterone (2) by Penicillium vinaceum.
100
80
3
60
14
15
40
17
20
0
0
2
4
6
8
Time of transformation (days)
Figure 2. The time course of the transformation of androsterone (3) by Penicillium vinaceum.
An interesting feature of the P. vinaceum enzymatic system is strong discrimination of the oxidation
kinetics on the basis of the C-3 stereochemistry. While , a 3 -hydroxy steroid, is not readily transformed
(even after eight days of transformation, still 77.5% of the unreacted is present in the reaction mixture),
its 3 -hydroxy stereoisomer is converted in 85%, and the transformation is especially fast on the
2
β
2
α
3
third day of the experiment. The oxidation kinetics is, however, generally noticeably slower than for
the 5-ene steroids described in Section 2.1.
While transformation of epiandrosterone (
grow monotonically, the case of androsterone (
derivative of , formed by oxidation of the hydroxyl group at C-3, undergoes between 72 and 96 h
further conversion, most probably BVO to the lactone 15. This assumption is supported by the
fate of 3 -hydroxy-17a-oxa-d-homo-5 -androstan-17-one (17), the product of the BVO retaining the
-hydroxy function. This compound is the predominant one within the experimental time scale,
2
) progresses slowly and concentrations of products
3) is more interesting. Androstanedione, the carbonyl
3
α
α
3
α

Molecules 2020, 25, 4226
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but its amount decreases in the final period of transformation, while new, corresponding amount of 15
is formed. This progression of product concentrations shows that the enzyme oxidizing the hydroxyl
group at C-3 to the ketone acts slower than the BVMO. Changing the configuration from 3
in epiandrosterone ( ) to 3 -hydroxy in androsterone ( ) slowed down the transformation process and
activity of Baeyer–Villiger monooxygenase.
It is also interesting to compare the transformation of DHEA (
analog—epiandrosterone ( ). The rapid conversion of to 3 -hydroxy lactone (10) with high yield
indicates that the substrate is the inducer of BVMO. In the case of transformation of , we observed a
β-hydroxy
2
α
3
1
) and its 5α-saturated
2
1
β
2
significant reduction in the transformation rate and the catalytic activity of BVMO, indicating that
epiandrosterone (2) may be the BVMO inhibitor.
2.3. Spectroscopic Identification of Products 12 and 13
The structures of the obtained and isolated products 12 and 13 were established by using
1
spectroscopic techniques (IR, H-NMR and 13C-NMR). The assumed structures were confirmed
by comparison of the characteristic shift values of selected, diagnostic signals of the products and
starting compounds.
The 1H NMR spectrum of 12 features a characteristic signal at δH 3.63 ppm (t, J = 8.5 Hz, 17
α-H)
from the proton at C-17. Further, in the 13C NMR spectrum of 12 a new signal appears at δC 81.5 ppm
and no signal at δC 220.3 ppm is observed (present in the substrate), which unanimously indicates
reduction of the ring-d carbonyl group to the hydroxyl group at 17
upfield shift of the C-18 methyl group signal ( 0.122 ppm) and downfield shift of the 4-H signal
0.013 ppm) in the 1H NMR spectrum. The signals at δH 3.90 ppm (d, J = 11 Hz) and at δH 4.06 ppm
β position, accompanied by an
∆
(
∆
(dd, J1 = 1 Hz, J2 = 11 Hz) are formed by two protons of the methylene group at C-19, and indicate the
close proximity of a hydroxyl group proton (19-CH2-OH). The signal appearing as a broad singlet at
δH 5.94 ppm points to the presence of a double bond at the C-4 carbon atom.
The analysis of the 1H NMR and 13C NMR spectral data indicates that the formed product 12 is
17β,19-dihydroxyandrost-4-en-3-one.
1
In the H NMR spectrum of 13 a significant downfield shift was observed for the 18-methyl
resonance signals in comparison with the substrate (from δH 0.81 to δH 1.36 ppm). It was consistent
with an oxygen atom insertion into the ring d. This was supported by the 13C NMR spectrum in
which the C-13 resonance signal had undergone a downfield shift of ca. 52 ppm (from δC 30.7 to δC
82.9 ppm). The lactonization via Baeyer–Villiger oxidation in the ring d of 19-nortestololactone was
confirmed by the appearance of the signal at δC ca. 171 ppm (C-17). The H-17α triplet characteristic for
19-nortestosterone (δH 3.66 ppm, J = 8.5 Hz) has disappeared, while the remaining signal of olefinic
proton δH ca. 5.84 ppm in the 1H NMR spectrum and a non-protonated signal at δC 199.5 ppm in the
13C NMR spectrum indicate the presence of 3-oxo-4-en moiety in the obtained product. The band
at 1725 cm−1 in the IR spectrum confirmed the formation of the
δ-lactone structure. These facts are
consistent with identification of 13 as 19-nortestololactone.
3. Materials and Methods
3.1. Chemicals and Microorganism
The substrates, namely DHEA (dehydroepiandrosterone, 3
epiandrosterone (3 -hydroxy-5 -androstan-17-one, ), androsterone (3
-one, ), androstenedione (androst-4-ene-3-one, ), 19-hydroxyandrostenedione (19-hydroxyandrost-
4-ene-3,17-dione, ), testosterone ( ), 19-nortestosterone ( ), progesterone ( ), and pregnenolone (
standards, were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA)
β-hydroxyandrost-5-en-17-one,
1),
β
α
2
α
-hydroxy-5 -androstan-17
α
3
4
5
6
7
8
9)
The fungal strain Penicillium vinaceum AM110 used for biotransformations was taken from the
collection of Department of Chemistry, Wrocław University of Environmental and Life Sciences,

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Wrocław, Poland. The strain was maintained on Sabouraud 4% dextrose-agar slopes and freshly
subcultured before use in the transformation experiments.
3.2. General Conditions of Cultivation and Biotransformation
General experimental and fermentation details were described previously [31]. After 4 days
of growth period of P. vinaceum AM110, a given substrate was added to each flask in an amount
necessary to obtain 0.30 g L−1 final substrate concentration (0.50 g L−1 final substrate concentration for
19-nortestosterone). The transformation was carried out at 25 ◦C in a rotary shaker (180 rpm) for eight
days (for 19-hydroxyandrostenedione the time was prolonged to 10 days, while for 19-nortestosterone,
the transformation lasted six days) with periodic checks of the presence of the substrate in the
reaction mixture and the stability of the obtained products (see below for the details of the checks).
Each experiment was performed with three replications (five replications for transformation of
19-hydroxyandrostenedione).
The monitoring of the time-dependent progress of the bioconversion and determination of the
metabolic pathways of substrates was carried out as follows. The 5-mL samples of the broth were
taken out at regular intervals (24, 48, 72, and 96 h from the start) from the reaction flask, extracted
with organic solvents (chloroform or methylene chloride), and analyzed by comparison of the GC and
TLC data with those of authentic samples. The final samples, taken at the end of transformation (6, 8,
or 10 days, depending on the substrate, as described above), served as the control of the extended
metabolic fate of the substrates and products.
3.3. Isolation and Identification of the Products
Briefly, 3
-androstan-17-one (16) and 17a-oxa-d-homo-5
in our laboratory by transformation by Penicillium lanosocoeruleum KCH 3012 as described in our
article [35]. 3 -Hydroxy-17a-oxa-d-homo-androst-5-en-17-one (10) and testololactone (11) were
obtained previously in our laboratory by transformation of DHEA by Penicillium lilacinum AM111 [31].
Androstanedione (5 -androstan-3,17-dione) (14) was obtained by the oxidation of androsterone (
α
-Hydroxy-17a-oxa-d-homo-5
α
-androstan-17-one (17), 3β-hydroxy-17a-oxa-d-homo-5α
α
-androstan-3,17-dione (15) were obtained previously
β
α
3)
with Jones’ reagent [35]. The aforementioned compounds were used as analytical standards for the
time course experiments.
Identification of the transformation products of substrates
GC and TLC (for : hexane/acetone (2:1 v:v); for : hexane/acetone (1:1 v:v); for
(2:1 v:v); for : hexane/acetone (2:1 v:v), for : methyl chloride/acetone (4:1 v:v), for ethyl acetate/methyl
chloride/acetone (3:1:1 v:v:v), for : ethyl acetate/methyl chloride/acetone (3:1:1 v:v:v)) analysis by
1
–9
was carried out on the basis of the
1
2
3: hexane/acetone
4
6
8
9
comparison of the retention times (Rt) with the Rt values of the standards available in our laboratory,
which have been already identified in our earlier studies [31,35].
1
Isolation and identification (by H NMR and 13C NMR) of the products were carried out
for 19-hydroxyandrostenedione (
Sigma-Aldrich Poland company, Poznan´, Poland.
The products of biotransformation were extracted three times with chloroform (for substrates
) or chloride methylene (for substrate ). The organic extracts were dried over anhydrous
magnesium sulfate, concentrated in vacuo, and analyzed by TLC and GC. Products were separated by
column chromatography on silica gel (Silica gel 60, 63–200 m, 70–230 mesh, Sigma-Aldrich, Buchs,
5) and 19-nortestosterone (7), the two substrates purchased from
1
–4
,
6–
9
5
µ
Switzerland) with hexane/acetone (2:3 v:v) for 19-hydroxyandrostenedione, and hexane/acetone (1:1 v:v)
for 19-nortestosterone, as eluents. TLC was carried out with Kieselgel 60 F254 plates (Merck, Darmstadt,
Germany) using the same eluents. In order to develop the image, the plates were sprayed with solution
◦
of methanol in concentrated sulfuric acid (1:1) and heated to 120 C for 3 min. GC analysis was
performed using Hewlett Packard 5890A Series II GC instrument (FID, carrier gas H2 at flow rate of
2 mL min−1, Hewlett-Packard Company, Wilmington, DE, USA) with a DB-5MS column cross-linked
phenyl-methylsiloxane, 30 m
×
0.32 mm
×
0.25 µm film thickness. The following program was used in

Molecules 2020, 25, 4226
10 of 13
the GC analysis: 220 ◦C/1 min, gradient 4 ◦C/min to 270 ◦C and 30 ◦C/min to 300 ◦C/3 min (for 1–6, 8,
substrates); 220 ◦C/1 min, gradient 4 ◦C/min to 260 ◦C and 30 ◦C/min to 300 ◦C/3 min (for
); injector
9
7
and detector temperatures were 300 ◦C. The NMR spectra (for obtained products of transformation
of 19-hydroxyandrostenedione and 19-nortestosterone) were measured in CDCl3 and recorded on a
DRX 600 MHz Bruker Avance spectrometer (Bruker Polska, Poznan´, Poland). Characteristic 1H- and
13C-NMR shift values in comparison to the starting compounds were used to determine structures
of metabolites, and in combination with DEPT analysis to identify the nature of the carbon atoms.
Elemental analysis was performed on vario EL III analyzer (Elementar Analysensysteme GmbH,
Hanau, Germany). Infrared spectra (IR) were recorded in KBr disc on a Mattson IR 300 Spectrometer
(Mattson-Garvin Company, Maitland, FL, USA). Melting points were determined on a Boetius melting
point apparatus and are uncorrected.
3.4. Spectral Data of the Isolated Transformation Products
17β,19-dihydroxyandrost-4-en-3-one (12) (60 mg, 40% mol): C19H28O3, Found: C, 74.89; H, 9.26.
Requires C, 74.96; H, 9.27. m.p. 200–202 ◦C (lit. 200-204 ◦C [42]). IR:
good agreement with [42].
υ
max (cm−1): 3410, 1660, 1615 in
1H NMR (CDCl3, 600 MHz,) δH: 0.77 (3H, s, 18-H), 3.63 (1H, t, J = 8.5 Hz, 17
α
-H), 3.90 (1H, d,
J = 11 Hz, 19-CH2-), 4.06 (1H, dd, J1 = 1 Hz, J2 = 11 Hz, 19-CH2-), 5.94 (1H, br s, 4-H); 13C NMR (CDCl3,
151 MHz) δC: 11.1 (C-18), 21.2 (C-11), 23.2 (C-15), 30.4 (C-16), 31.7 (C-7), 33.4 (C-6), 33.5 (C-2), 35.0 (C-8),
36.3 (C-12), 36.7 (C-1), 42.9 (C-13), 43.9 (C-10), 50.8 (C-14), 54.0 (C-9), 66.0 (C-19), 81.5 (C-17), 127.0 (C-4),
166.6 (C-5), 199.9 (C-3). The NMR spectra of 12 are attached as Supplementary Figures S1 and S2.
19-Nortestololactone (13) (127 mg, 85% mol): C18H24O3, Found: C, 74.90; H, 8.36. Requires C, 74.97;
H, 8.39; mp. 195–198 ◦C (lit. 196–199 ◦C [23]), IR: υmax (cm−1): 1725, 1670, 1610.
1H NMR (CDCl3, 600 MHz,) δH: 1.36 (3H, s, 18-H), 5.84 (1H, s, 4-H); 13C NMR (CDCl3,151 MHz):
19.8 (C-18), 20.0 (C-15), 26.3 (C-1), 27.0 (C-11), 28.5 (C-16), 29.7 (C-7), 35.0 (C-6), 36.4 (C-2), 38.9 (C-8),
42.3 (C-10), 42.6 (C-12), 45.03 (C-14), 48.2 (C-9), 82.9 (C-13), 124.8 (C-4), 164.7 (C-5), 171.2 (C-17), 199.5
(C-3). The spectroscopic data are in agreement with those reported in the literature [24]. The NMR
spectra of 13 are attached as Supplementary Figures S3 and S4.
4. Conclusions
Biotransformations of nine steroidal substrates in the cultures of the filamentous fungus Penicillium
vinaceum AM110 have been carried out. The series of androstene substrates underwent mostly
Baeyer–Villiger oxidation (lactonization), without side processes; the metabolic fate of these substrates
is therefore less complicated than in the other species of Penicillium genus, while the transformation
kinetics and achieved yields are very promising. Interesting inhibition of the BVMO activity was
observed when a hydroxyl function was inserted at the C-19 methyl group. Instead of any oxidation
process, the C-17 carbonyl group of 19-hydroxyandrostenedione was reduced to the hydroxyl group.
The fate of androstane steroids, epiandrosterone and androsterone, was more complicated.
Two possible sites of oxidation, the C-3 hydroxyl and the C-17 carbonyl groups, were oxidized
correspondingly to the ketone and to the δ-lactone with different rates. This allows for the potential
design of the biotransformation process to obtain preferentially only one of the oxidation products.
The transformation of epiandrosterone in comparison with its unsaturated analogue (DHEA) and
androsterone was much slower and occurred with much lower efficiency.
The presented results suggest usefulness of the Penicillium vinaceum AM110 strain in the
commercial preparation of biologically active
microbial Baeyer–Villiger oxidation.
δ-lactones of androstene and androstane series via

Molecules 2020, 25, 4226
11 of 13
Supplementary Materials: The following is available online, a PDF file containing Figures S1–S6: 1H and 13
NMR spectra of products 12 and 13, GC spectra of products 12 and 13 obtained during time course experiments.
C
´
´
Author Contributions: Conceptualization, A.P. and A.S.; methodology, A.P. and A.S.; investigation, A.P.,
´
´
´
P.Ł., and A.S.; writing—original draft preparation, A.P. and A.S.; writing—review and editing, A.P. and A.S.;
project administration, A.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: We acknowledge the help of MSc Student Kamila Gogola during part of the
biotransformation experiment.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 12 (17β,19-dihydroxyandrost-4-en-3-one) and 13 (19-nortestololactone)
are available from the authors.
©
2020 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/).