Microbial Baeyer-Villiger oxidation of 5α-steroids using Beauveria bassiana. A stereochemical requirement for the 11α-hydroxylation and the lactonization pathway

Article abstract of DOI:10.1016/j.steroids.2014.01.006

Beauveria bassiana KCH 1065, as was recently demonstrated, is unusual amongst fungal biocatalysts in that it converts C₁₉ 3-oxo-4-ene and 3β-hydroxy-5-ene as well as 3β-hydroxy-5α-saturated steroids to 11α-hydroxy ring-D lactones. The Baeyer-Villiger monooxygenase (BVMO) of this strain is distinguished from other enzymes catalyzing BVO of steroidal ketones by the fact that it oxidizes solely substrates with 11α-hydroxyl group. The current study using a series of 5α-saturated steroids (androsterone, 3α-androstanediol and androstanedione) has highlighted that a small change of the steroid structure can result in significant differences of the metabolic fate. It was found that the 3α-stereochemistry of hydroxyl group restricted "normal" binding orientation of the substrate within 11α-hydroxylase and, as a result, androsterone and 3α-androstanediol were converted into a mixture of 7β-, 11α- and 7α-hydroxy derivatives. Hydroxylation of androstanedione occurred only at the 11α-position, indicating that the 3-oxo group limits the alternative binding orientation of the substrate within the hydroxylase. Only androstanedione and 3α-androstanediol were metabolized to hydroxylactones. The study uniquely demonstrated preference for oxidation of equatorial (11α-, 7β-) hydroxyketones by BVMO from B. bassiana. The time course experiments suggested that the activity of 17β-HSD is a factor determining the amount of produced ring-D lactones. The obtained 11α-hydroxylactones underwent further transformations (oxy-red reactions) at C-3. During conversion of androstanedione, a minor dehydrogenation pathway was observed with generation of 11α,17β-dihydroxy-5α-androst-1-en-3-one. The introduction of C1C2 double bond has been recorded in B. bassiana for the first time.

Full text of DOI:10.1016/j.steroids.2014.01.006

Steroids 82 (2014) 44–52
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Microbial Baeyer–Villiger oxidation of 5a-steroids using Beauveria
bassiana. A stereochemical requirement for the 11a-hydroxylation
and the lactonization pathway
⇑
´
Alina Swizdor , Anna Panek, Natalia Milecka-Tronina
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 August 2013
Received in revised form 24 December 2013
Accepted 20 January 2014
Available online 30 January 2014
Beauveria bassiana KCH 1065, as was recently demonstrated, is unusual amongst fungal biocatalysts in
that it converts C19 3-oxo-4-ene and 3b-hydroxy-5-ene as well as 3b-hydroxy-5
11 -hydroxy ring-D lactones. The Baeyer–Villiger monooxygenase (BVMO) of this strain is distinguished
from other enzymes catalyzing BVO of steroidal ketones by the fact that it oxidizes solely substrates with
11 -hydroxyl group. The current study using a series of 5 -saturated steroids (androsterone, 3 -andro-
stanediol and androstanedione) has highlighted that a small change of the steroid structure can result in
significant differences of the metabolic fate. It was found that the 3 -stereochemistry of hydroxyl group
restricted ‘‘normal’’ binding orientation of the substrate within 11 -hydroxylase and, as a result, andros-
terone and 3 -androstanediol were converted into a mixture of 7b-, 11 - and 7 -hydroxy derivatives.
Hydroxylation of androstanedione occurred only at the 11 -position, indicating that the 3-oxo group
limits the alternative binding orientation of the substrate within the hydroxylase. Only androstanedione
and 3 -androstanediol were metabolized to hydroxylactones. The study uniquely demonstrated prefer-
ence for oxidation of equatorial (11 -, 7b-) hydroxyketones by BVMO from B. bassiana. The time course
experiments suggested that the activity of 17b-HSD is a factor determining the amount of produced ring-
D lactones. The obtained 11 -hydroxylactones underwent further transformations (oxy-red reactions) at
C-3. During conversion of androstanedione, a minor dehydrogenation pathway was observed with gen-
eration of 11 ,17b-dihydroxy-5 -androst-1-en-3-one. The introduction of C1AC2 double bond has been
recorded in B. bassiana for the first time.
a-saturated steroids to
a
a
a
a
Keywords:
Steroidal lactone
Androstanedione
a
a
a
a
a
3a-Androstanediol
a
17b-Hydroxysteroid dehydrogenase
Fungal 1-dehydrogenase
Beauveria bassiana
a
a
a
a
a
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
treatment of androgen dependent diseases. Steroidal testolactone
as an inhibitor of steroidal aromatase [3] is used in the clinical
treatment of breast cancer [3], and several forms of precocious
puberty [4].
A number of microorganisms, especially fungal species of the
Penicillium [5–9], Aspergillus [10–17] and Fusarium [18–20] genera,
Steroidal compounds are an important class of natural lipo-
philic products which have high possibilities to penetrate cells
and interact with membrane receptors. Research on modification
of steroids performed by oxidative enzymes is being continued
for development of processes exploitable by the pharmaceutical
industry, and also for the purpose of preparation of potentially use-
ful steroid analogues, which are otherwise inaccessible [1]. Due to
the important bioactivity of steroidal lactones, the research de-
voted to modification of steroids has, in recent years, provided sev-
eral reports on microbial Baeyer–Villiger oxidation. It was found
are known to transform steroids to
D-ring lactones. Until now,
however, only one of these species, i.e. Aspergillus tamarii, has been
the subject of systematic research into the area of understanding
structure–activity relationships in lactonization pathways with re-
spect to a broad spectrum of steroidal substrates [12–17].
Previous studies carried out by our group had demonstrated
that Beauveria bassiana KCH 1065 is a unique species of the genus
Beauveria in its metabolism of steroids that results in Baeyer–
Villiger oxidations of the carbonyl group to the corresponding
that the steroidal lactones could inhibit the 5
a result of this, block the conversion of testosterone to 5
testosterone [2] and therefore have a therapeutic potential for the
a
-reductase, and as
a
-dihydro-
11
strain consists of two major steps that occur in a precise sequence:
11 -hydroxylation of the substrate and then its subsequent
a-hydroxylactones [21]. The metabolic pathway present in this
⇑
Corresponding author. Tel.: +48 71 3205252; fax: +48 71 3284124.
a
´
E-mail address: alina.swizdor@up.wroc.pl (A. Swizdor).
http://dx.doi.org/10.1016/j.steroids.2014.01.006
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

´
A. Swizdor et al. / Steroids 82 (2014) 44–52
45
BV oxidation. None of the tested substrates gave BVO products
without the 11 -hydroxyl group. These studies have highlighted
the powerful effects of a single functional moiety of C21 or C19
(C-11), 21.7 (C-15), 28.6 (C-6), 30.5 (C-7), 31.4 (C-12), 34.9 (C-8),
35.8 (C-16), 35.8 (C-10), 38.0 (C-2), 38.4 (C-1), 44.5 (C-4), 46.6
(C-5), 47.7 (C-13), 51.2 (C-14), 53.8 (C-9), 211.6 (C-3), 220.9 (C-17).
a
within the structure of substrates (3-oxo-4-ene or 3b-hydroxy-5-
ene) in directing their metabolite fate [21]. Only the 11
derivatives of 3-oxo-4-ene, 3b-hydroxy-5-ene as well as 3b-
hydroxy-5 -saturated 17-ketosteroids underwent oxidative
lactonization. The enzyme catalyzed oxidative esterification of
11 -hydroxyprogesterone – the first main metabolite of progester-
one, whilst pregnenolone was not susceptible to 11 -hydroxyl-
a
-hydroxy
2.2. Microorganism
a
B. bassiana KCH 1065 used in this study was taken from the col-
lection of Department of Chemistry, Wrocław University of Envi-
ronmental and Life Sciences (Wrocław, Poland). The fungus was
maintained on Sabouraud 4% dextrose-agar slopes at 4 °C and
freshly subcultured before use in the transformation experiments.
a
a
ation and was not metabolized at all. The activity of
17b-hydroxysteroid dehydrogenase (17b-HSD) had significant im-
pact on the amount of resulting 11
fore the formation of lactones, the oxidation of the C-17-ketone
must have occurred via 11 ,17b-diols (Fig. 1).
To determine whether this metabolic pathway can be general-
ized, we investigate within the current study the transformation
a
-hydroxy D-homo lactones. Be-
2.3. Conditions of cultivation and transformation
a
General experimental and fermentation details have been de-
scribed previously [6]. Each substrate was added to a 72-h-old cul-
ture of the microorganism as an acetone solution, in concentration
of 0.08 mmol/100 ml of medium. Each experiment was performed
with four replications.
of a series of 5
a-steroids containing 3a-alcohol (androsterone
and 3 -androstanediol) or 3-ketone (androstanedione). The differ-
a
ences between the tested compounds are related to the kind of
oxygen function at C-3 or C-17.
2.4. Isolation and identification of metabolites
The fungal mycelium was separated from the broth by filtration
under vacuum. The broth was then extracted three times with
chloroform. The organic extract was dried over anhydrous MgSO4
and the solvent was subsequently evaporated in vacuo to give
brown gum. That gum was analyzed by TLC and GC and then chro-
matographed on a column of silica with the same eluent as for thin
layer chromatography (see below). TLC was carried out with Merck
Kieselgel 60 F254 plates with the use of a mixture of acetone:chlo-
2. Materials and methods
2.1. Substrates
Androsterone (3
substrate for the transformation and as a starting material for the
preparation of 3 -androstanediol (5 -androstan-3 ,17b-diol) (5)
and androstanedione (5 -androstan-3,17-dione) (12), was pur-
a-hydroxy-5a-androstan-17-one) (1), used as a
a
a
a
a
roform (1:1.25 v/v) for transformation of androsterone (1) and 3a-
chased from Sigma–Aldrich Chemical Co. 11b-Hydroxyandrosten-
edione (16) was taken from the resources of the Department of
Chemistry, Wrocław University of Environmental and Life Sciences.
androstanediol (5), a mixture of hexane:acetone:ethyl acetate
(2:1.5:0.4 v/v/v) for transformation of androstanedione (12) or a
mixture of hexane:acetone (2:1 v/v) for transformation of 11b-
hydroxyandrostenedione (16) as eluents. In order to develop the
image, the plates were sprayed with solution of methanol in con-
centrated sulfuric acid (1:1 v/v) and heated to 120 °C for 3 min.
GC analysis was performed using Hewlett Packard 5890A Series
2.1.1. Preparation of 3
a-androstanediol (5)
3a-Androstanediol (5) was obtained by reduction of androster-
one (1) according to the procedure described earlier [22]. The prod-
uct was found to be in excess of 99.2% purity following GC and
elemental analysis, C19H32O2 calcd C, 78.03; H 11.03; found C,
78.10; H, 11.12%. 1H NMR: 0.74 (3H, s, 18-H), 0.79 (3H, s, 19-H),
II GC instrument (FID, carrier gas H2 at flow rate of 2 ml minÀ1
with a Thermo TR-5MS (5% phenyl polysilphenylene–siloxane) col-
umn, 30 m  0.25 mm  0.50 m film thickness (for transforma-
tion of 1 and 5) or HP-1 column cross-linked Methyl Siloxane,
30 m  0.53 mm  1.5 m film thickness (for transformation of
)
l
3.64 (1H, t, J = 8.4 Hz, 17a
-H), 4.03 (1H, t, J = 2.7 Hz, 3b-H). 13C
NMR: 11.1 (C-18), 11.2 (C-19), 20.3 (C-11), 23.3 (C-15), 28.5 (C-2),
29.1 (C-6), 30.6 (C-7), 31.5 (C-16), 32.2 (C-1), 35.5 (C-8), 35.8
(C-12), 36.1 (C-10), 36.7 (C-4), 39.2 (C-5), 43.2 (C-13), 50.9 (C-14),
54.4 (C-9), 66.5 (C-3), 82.0 (C-17).
l
12 and 16). The applied temperature program (for the HP-1 col-
umn): 220 °C/1 min, gradient 4 °C/min to 260° and then 30 °C/min
to 300 °C/2 min or 250 °C/1 min, gradient 3 °C/min to 300 °C/5 min
(for the Thermo column); injector and detector temperature was
300 °C. The NMR spectra were measured in CDCl3. The spectra were
recorded on a DRX 300 MHz Bruker Avance spectrometer with TMS
as internal standard. Characteristic shift values in the 1H NMR and
13C NMR spectra in comparison to the starting compounds were
used to determine structures of metabolites, in combination with
DEPT analysis to identify the nature of the carbon atoms (Tables 1
and 2). Partial signal assignment, where necessary, was achieved
by performing COSY, HSQC and HMBC experiments. The stereo-
chemistry of the hydroxyl group was deduced on the basis of NOESY
experiments. IR spectra (Table 3) were recorded in KBr disc on a
Mattson IR 300 Spectrometer. Elemental analysis was performed
on vario EL III analyzer.
2.1.2. Preparation of androstanedione (12)
Androstanedione (12) was obtained by oxidation of androster-
one (1) with CrO3 [23]. The product was found to be in excess of
99.5% purity following GC and elemental analysis, C19H28O2 calcd
C, 79.12; H 9.78; found C, 79.36; H, 9.84%. 1H NMR: 0.89 (3H, s,
18-H), 1.03 (3H, s, 19-H). 13C NMR: 11.4 (C-19), 13.8 (C-18), 20.7
2.6. Time course experiments
Time course experiments were conducted in order to determine
the metabolic pathways. Conditions were identical to those in the
main biotransformation experiments. One flask was harvested
after 3, 6, 9, 24 h, and every consecutive flask – after another
Fig. 1. Reaction sequence producing steroidal 11
through the transformation using Beauveria bassiana.
a-hydroxy D-homo lactones

´
A. Swizdor et al. / Steroids 82 (2014) 44–52
46
Table 1
13C NMR data for products transformations.
Carbon atoms
Compounds
2
3
4
6
7
8
10
31.9
11
40.0
13
39.9
14
40.1
15
17
35.0
1
2
3
4
5
6
7
8
33.8
29.5
66.0
39.6
39.0
29.0
30.9
34.5
60.4
37.4
68.9
43.1
48.1
50.3
21.8
35.8
219.1
14.7
12.7
32.1
28.9
66.0
35.4
36.3
38.7
74.7
42.9
52.6
35.6
20.1
31.5
48.3
51.1
24.9
36.2
31.9
28.9
66.3
35.4
31.6
36.4
66.8
39.1
46.1
36.3
19.9
31.2
47.6
45.9
21.3
35.8
34.0
29.1
66.2
38.4
39.1
29.4
30.6
34.8
60.5
37.6
68.6
47.6
43.4
50.0
23.5
31.4
81.5
11.8
11.5
32.0
28.5
66.1
36.0
36.2
38.6
73.8
43.7
52.5
35.5
20.3
35.6
44.1
50.0
26.4
31.7
80.8
11.1
11.2
33.9
29.2
66.0
36.0
38.4
28.5
30.5
36.9
59.3
37.9
68.9
49.9
81.6
45.5
21.7
29.1
162.3
126.1
200.1
41.3
44.1
27.9
30.7
34.8
56.2
40.4
69.4
48.6
43.5
50.1
23.2
30.6
81.1
12.3
13.2
29.0
65.9
35.1
35.6
39.4
73.7
47.3
52.2
35.5
19.9
39.6
83.9
45.5
22.0
29.2
170.9
20.5
11.2
38.2
211.5
44.8
46.7
28.8
30.0
36.8
58.7
37.2
68.7
50.0
81.4
45.4
20.0
28.4
171.0
21.1
11.5
38.3
211.7
45.1
47.3
29.1
30.3
34.1
60.0
37.4
68.7
43.0
47.9
50.2
21.8
35.8
219.0
14.6
11.8
38.3
212.1
45.1
47.4
29.3
31.1
34.9
60.0
37.3
68.9
48.6
43.4
50.0
23.4
30.5
81.2
12.1
11.8
33.8
199.5
122.3
172.1
32.2
32.0
31.4
56.5
39.2
68.0
46.3
41.8
51.9
23.4
30.3
82.1
13.5
20.9
9
10
11
12
13
14
15
16
17
18
19
221.5
14.0
11.3
221.1
13.5
10.1
171.7
21.0
11.3
Table 2
1H NMR data for products transformations.
Compounds
3-H
18-H3 19-H3 Other significant signals
3
3
3
3
3
3
a
a
a
a
a
a
,11
,7b-Dihydroxy-5
,7 -Dihydroxy-5
,11 ,17b-Trihydroxy-5
,7b,17b-Trihydroxy-5 -androstane (7)
,11 -Dihydroxy-17a-oxa-D-homo-5
-Dihydroxy-17a-oxa-D-homo-5
,7b-Dihydroxy-17a-oxa-D-homo-5
a
-Dihydroxy-5
-androstan-17-one (3)
-androstan-17-one (4)
-androstane (6)
a
-androstan-17-one (2)
4.02 (t, J = 2.7 Hz, 3b-H) 0.86
4.06 (t, J = 2.7 Hz, 3b-H) 0.88
4.08 (t, J = 2.7 Hz, 3b-H) 0.86
4.03 (t, J = 2.7 Hz, 3b-H) 0.77
4.02 (t, J = 2.7 Hz, 3b-H) 0.75
0.94
0.82
0.80
0.93
0.81
0.90
0.90
0.75
1.10
3.96 (dt, J = 5.1, 10.5 Hz, 11b-H)
3.47–3.56 (m, 7a-H)
3.92–3.98 (m, 7b-H)
a
a
a
a
a
3.65 (t, J = 8.5 Hz, 17
a-H); 3.94 (dt, J = 5.1, 10.5 Hz, 11b-H)
a
3.61 (t, J = 8.5 Hz, 17
a-H); 3.49–3.61 (m, 7 -H)
a
a
a
a
-androstan-17-one (8) 4.03 (t, J = 2.7 Hz, 3b-H) 1.33
-androstan-17-one (9) 3.59 (m, 3 -H) 1.28
-androstan-17-one (10) 4.08 (t, J = 2.7 Hz, 3b-H) 1.31
3.76 (dt, J = 5.0, 10.4 Hz, 11b-H)
3.75 (dt, J = 5.0, 10.4 Hz, 11b-H)
3.49–3.58 (m, 7a-H)
3.81 (dt, J = 4.8, 11.4 Hz, 11b-H); 2.86 (ddd, J = 2.4, 6.3,
13.6 Hz, 1b-H)
4.03 (dt, J = 5.1, 10.5 Hz, 11b-H); 2.77 (ddd, J = 2.4, 6.3,
13.6 Hz, 1b-H)
3.99 (dt, J = 5.1, 10.5 Hz, 11b-H); 3.68 (t, J = 8.5 Hz, 17
2.77 (ddd, J = 2.4, 6.3, 13.6 Hz, 1b-H)
3b,11
a
a
a
3a
11
11
11
11
a
a
a
a
-Hydroxy-17a-oxa-D-homo-5
a-androstan-3,17-dione (11)
1.33
0.90
0.78
0.81
1.04
-Hydroxy-5 -androstan-3,17-dione (13)
a
1.15
1.14
1.14
1.45
,17b-Dihydroxy-5a-androstan-3-one (14)
a
-H);
,17b-Dihydroxy-5a-androst-1-en-3-one (15)
4.11 (dt, J = 5.1, 10.5 Hz, 11b-H); 3.70 (t, J = 8.5 Hz, 17
5.80 (d, J = 10.5 Hz, 2-H); 8.29 (d, J = 10.5 Hz, 1-H)
4.39 (br s, 11a-H); 3.61 (t, J = 8.5 Hz, 17a-H); 5.68 (s, 4-H).
a
-H);
11b-Hydroxytestosterone (17)
24 h. Reaction mixtures were extracted and analyzed by GC and
TLC as in Section 2.4.
containing 100 ml of medium) and transformation was contin-
ued for further 2 days. After this time the mixtures were han-
dled as above.
2.6. Transformations using induced culture of B. bassiana
3. Results
(A) After 66 h of fungal growth, 0.006 mmol of epiandrosterone
3.1. Products isolated in the course of transformations
dissolved in 20 ll of acetone was added to the culture and
incubation was continued under the same conditions (tem-
perature, shaking) to those used for fungal growth. After
another 6 h, 0.08 mmol of androsterone (1) was added to
the culture and transformation was continued for further
4 days. The samples (5 ml) were taken after 6 h of reaction
and, further, every each 24 h, extracted and analyzed by
GC as in Section 2.4.
3.1.1. Transformation of androsterone (1)
After 48 h of transformation of 100 mg of 1 the isolates were:
3
a
,11
calcd C, 74.47; H, 9.87; found C, 74.51; H, 9.91%), 3
-androstan-17-one (3) (49 mg; C19H30O3: calcd C, 74.47; H, 9.87;
found C, 74.52; H, 9.89%) and ,7 -dihydroxy-5 -androstan-
a
-dihydroxy-5
a
-androstan-17-one (2) (21 mg; C19H30O3:
a
,7b-dihydroxy-
5a
3a
a
a
17-one (4) (13 mg; C19H30O3: calcd C, 74.47; H, 9.87; found C,
(B) After 48 h of fungal growth, 0.006 mmol of 3
a-androstane-
74.50; H, 9.89%).
diol (5) dissolved in 20 l of acetone was added to the cul-
l
ture and incubation was continued under the same
conditions to those used for fungal growth. After another 24 h,
3.1.2. Transformation of 3
After 24 h of transformation of 100 mg of 5 the isolates were: the
unreacted substrate (43 mg), ,11 -dihydroxy-5 -androstan-
a-androstanediol (5)
the total isolated yield of 3a,11a-dihydroxy-5a-androstan-
17-one (2) was evenly distributed between 2 flasks (each
3a
a
a

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A. Swizdor et al. / Steroids 82 (2014) 44–52
47
17-one (2) (12 mg),
(3 mg), ,7 -dihydroxy-5
,11 ,17b-trihydroxy-5 -androstane (6) (5 mg; C19H32O3: calcd C,
73.98; H, 10.46; found C, 73.94; H, 10.51%) and ,7b,17b-
trihydroxy-5 -androstane (7) (7 mg; C19H32O3: calcd C, 73.98; H,
10.46; found C, 74.11; H, 10.58%).
After 96 h transformation of 100 mg of 5 the additional isolates
were: 3 ,11 -dihydroxy-17a-oxa-D-homo-5 -androstan-17-one (8)
(7 mg; C19H30O4: calcd C, 70.77; H, 9.38; found C, 70.74; H, 9.27%),
3b,11 -dihydroxy-17a-oxa-D-homo-5 -androstan-17-one (9) (11 mg),
,7b-dihydroxy-17a-oxa-D-homo-5 -androstan-17-one (10) (8 mg;
19H30O4: calcd C, 70.77; H, 9.38; found C, 70.81; H, 9.44%), and
11 -hydroxy-17a-oxa-D-homo-5 -androstan-3,17-dione (11) (4 mg;
C19H28O4: calcd C, 71.22; H, 8.81; found C, 71.40; H, 8.85%).
3
a
,7b-dihydroxy-5
a
-androstan-17-one (3)
an equatorial proton. Lack of significant shift in either of the methyl
signals pointed towards substitution at C-7b. The stereochemistry
of C-7 OH group was supported on the basis of NOESY correlation be-
tween H-7 and H-9 (d 0.96 ppm). Additionally, the appearance of a
new methine carbon signal at dC 74.7 ppm, in combination with the
3
a
a
a-androstan-17-one (4), (12 mg),
3
a
a
a
3a
a
downfield shift for the C-6 (
carbon upfield signal shifts for C-5 (
and C-14 ( 0.4 ppm), was an important confirmation of 7b-hydrox-
ylation. These observations fully supported the structure of 3 as
,7b-dihydroxy-5
-androstan-17-one. The 1H NMR spectrum of 4
D
9.6 ppm) and C-8 (
D
7.8 ppm) and
c-
D
2.9 ppm), C-9 (
D
1.9 ppm)
a
a
a
D
a
a
a
3a
a
3
a
showed a new narrow hydroxyl-bearing methine proton signal at
dH 3.92–3.98 ppm indicating the introduction of an axial hydroxyl
C
a
a
group. Hydroxylation at C-7
field shift of the resonances assigned to C-6 (
4.0 ppm) and the -gauche shielding experienced by C-5 (
C-9 ( 8.4 ppm) and C-14 ( 5.6 ppm). The -stereochemistry of the
a
position was apparent from the down-
7.3 ppm) and C-8 (
7.6 ppm),
D
D
c
D
3.1.3. Transformation of androstanedione (5
a-androstan-3,17-dione)
D
D
a
(12)
OH group at C-7 was subsequently inferred from NOESY correlation
After 6 h transformation of 100 mg of 12 the following metabo-
lites were isolated: 11 -hydroxy-5 -androstan-3,17-dione (13)
(27 mg; C19H28O3: calcd C, 74.96; H, 9.27; found C, 74.93; H,
9.25%) and 11 ,17b-dihydroxy-5 -androstan-3-one (14) (30 mg;
19H30O3: calcd C, 74.47; H, 9.87; found C, 74.46; H, 9.85%).
After 48 h transformation of 100 mg of 12 the additional
products were obtained: 11 -hydroxy-17a-oxa-D-homo-5 -andr
ostan-3,17-dione (11) (19 mg), 3b,11 -dihydroxy-17a-oxa-D-
homo-5 -androstan-17-one (9) (18 mg), and 11 ,17b-dihydroxy-
-androst-1-en-3-one (15) (9 mg; C19H28O3: calcd C, 74.96; H,
between the vicinal H-7 and H-8 (dH 1.42 ppm). Finally, 4 was iden-
a
a
tified as 3a,7a-dihydroxy-5a-androstan-17-one.
The 1H NMR spectra of 6 and 7 possessed resonances at dH
4.0 ppm (t, J = 2.7 Hz) and dH 3.6 ppm (t, J = 8.5 Hz), confirming that
a
a
C
the 3a,17b-dihydroxy skeleton of the substrate had been retained.
The characteristic hydroxyl-bearing methine proton signal (dt) at
dH 3.94 ppm visible in the spectrum of 6 suggested that that
a
a
a
metabolite was 3
supported by an oxygen-bearing methine carbon signal at dC
68.6 ppm, and downfield shifts for the b-carbons: C-9
6.1 ppm) and C-12 ( 11.8 ppm) that were consistent with refer-
ence shifts values [24]. 3 ,7b,17b-Trihydroxy-5 -androstane (7)
a,11a,17b-trihydroxy-5a-androstane. This was
a
a
5a
(D
9.27; found C, 75.02; H, 9.45%).
D
a
a
was identified by a new CH(OH) signal (m) at dH 3.49–3.61 ppm
indicating hydroxylation at an equatorial proton, and methine car-
bon resonance at dC 73.8 ppm. Further supporting evidence was
3.1.4. Transformation of 11b-hydroxyandrostenedione (16)
After 4 days transformation of 100 mg of 16, there were iso-
lated: the unreacted substrate (52 mg), and 11b-hydroxytestoster-
one (17) (41 mg; C19H28O3: calcd C, 74.96; H, 9.27; found C, 75.00;
H, 9.38%).
provided by downfield shifts for C-6
(
D
9.5 ppm) and C-8
8.3 ppm) together with upfield shifts for C-5 ( 3 ppm), C-9
2 ppm) and C-14 (
0.9 ppm) in 13C NMR [24]. The product 8
(D
D
(D
D
was identified by the characteristic shape and multiplicity of an
additional CH(OH) signal at dH 3.76 ppm (dt), the loss of signal at
dC 82.0 ppm assigned to C-17 in the 13C NMR spectrum of the start-
ing material 5 and its replacement with a signal at dC 171.7 ppm,
and a downfield shift of the C-13 resonance signal from dC
43.2 ppm to dC 81.6 ppm. This was coupled with the significant
3.2. Structural identification of metabolites
3a,11a-Dihydroxy-5a-androstan-17-one (2) was identified by the
significant downfield shift of the C-19 methyl group (
D
0.14 ppm),
the appearance of a new CH(OH) signal in the 1H NMR spectrum at
dH 3.96 ppm (dt, J = 5.1, 10.5 Hz) and of a new oxygen-bearing
methine carbon signal at dC 68.9 ppm in the 13C NMR spectrum.
The stereochemistry of the newly introduced hydroxyl group was
further supported by NOESY spectrum which showed correlation of
11b-H with the proton signals of C-18 (dC 14.7 ppm) and C-19
(dC 12.7 ppm) methyl groups. Downfield b-carbon shifts for C-12
downfield shift (
signal. All these results were consistent with 11
and oxygen insertion into ring-D, thereby structure of 8 was pro-
posed to be 3 ,11 -dihydroxy-17a-oxa-D-homo-5 -androstan-
17-one. Confirmation of structure of 3b,11 -dihydroxy-17a-oxa-
D-homo-5 -androstan-17-one (9) was achieved by comparison
D
0.59 ppm) of the 18-methyl protons resonance
a-hydroxylation
a
a
a
a
a
(
11
D
a
11.5 ppm) and C-9 (
D 5.9 ppm) confirmed hydroxylation at the
position. The 1H NMR spectrum of metabolite 3 revealed a
of spectroscopic data to that of an authentic sample isolated by
us during transformation of epiandrosterone [21]. The NMR
new broad signal at dH 3.47–3.56 ppm indicating hydroxylation at
Table 3
Significant IR absorption signals for products transformation.
Metabolite
–OH
>C@O
Lactone
3
3
3
3
a
a
a
a
,11
,7b-Dihydroxy-5
,7 -Dihydroxy-5
,11 ,17b-Trihydroxy-5
a
-Dihydroxy-5
-androstan-17-one (3)
-androstan-17-one (4)
-androstane (6)
a
-androstan-17-one (2)
3480
3465
3565
3488
3411
3390
3452
3450
3415
3390
1740
1742
1740
a
a
a
a
a
3a
3a
3a
,7b,17b-Trihydroxy-5
,11 -Dihydroxy-17a-oxa-D-homo-5
,7b-Dihydroxy-17a-oxa-D-homo-5
a-androstane (7)
a
a
-androstan-17-one (8)
1716
1713
1711
a
-androstan-17-one (10)
11
11
a
a
-Hydroxy-17a-oxa-D-homo-5
a-androstan-3,17-dione (11)
1658
1740
1655
1657
1687
1669
-Hydroxy-5 -androstan-3,17-dione (13)
a
11
11
a
a
,17b-Dihydroxy-5a-androstan-3-one (14)
3298
3330
3380
,17b-Dihydroxy-5a-androst-1-en-3-one (15)
11b-Hydroxytestosterone (17)

´
A. Swizdor et al. / Steroids 82 (2014) 44–52
48
spectra of compound 10 demonstrated a significant downfield shift
for the 18-methyl protons ( 0.57 ppm) and the presence of C-17
Table 4), whereas hydroxy derivatives of 3
were oxidized to 17-ketones and some of them (11
a
-androstanediol (5)
D
a- and 7b-
signal at dC 170.9 ppm which are fully indicative of lactonization
hydroxy) were further metabolized through BV oxidation (Fig. 3
and Table 4). Lactonization of the hydroxy derivatives followed be-
tween 24 and 48 h of incubation of the substrate. Formation of
in ring-D. This was supported in the 13C NMR spectrum with down-
field shifts for C-13 (
D 40.7 ppm) and C-18 (D 8.6 ppm). An addi-
tional multiplet at dH 3.49–3.58 ppm suggested equatorial
stereochemistry for the hydroxylation. Examination of the 13C
NMR spectrum of the product confirmed b-carbon downfield shifts
11a-hydroxy-lactones (11 and 9) from the starting androstanedi-
one (12) was observed earlier, between 6 and 24 h of reaction
(Fig. 4 and Table 4). Until the moment of appearance of the BVMO
for C-6 (
shifts for C-5
7b-hydroxylation. Thus, the product 10 was identified as
,7b-dihydroxy-17a-oxa-D-homo-5
-androstan-17-one. The 13C
D
10.3 ppm) and C-8 (
D
11.3 ppm) and
c
-carbon upfield
activity, a major portion of 11
the product formed during the first 6 h of incubation of 12 – was
reduced to the 11 ,17b-diol 14. On the other hand, comparison
a-hydroxy-androstanedione (13) –
(
D
3.6 ppm) and C-9
(D
2.2 ppm), indicating
a
3a
a
of composition of the product mixtures after 6 and 9 h incubation
NMR spectrum of 11 was similar to that of 8 with the exception
of signals of the A-ring carbons. The absence of the C-3 methine
signal at dC 66.0 ppm and the appearance of an additional
quaternary carbon signal at dC 211.5 ppm indicated the oxidation
of the C-3 hydroxyl group to a carbonyl group. Also, the signal of
3b-proton disappeared in the 1H NMR spectrum. This data led to
of androstanedione (12) has shown that part of the formed
11a,17b-dihydroxy-5a-androstan-3-one (14) was re-oxidized to
the C-17-ketone 13 and subsequently metabolized by BV oxida-
tion. The minor metabolic pathway of 14 was 1-dehydrogenation
leading to 11a,17b-dihydroxy-5a-androst-1-en-3-one (15). The
insertion of the ring-A double bond was observed 24 h after incu-
the identification of 11 as 11a-hydroxy-17a-oxa-D-homo-5a-
bation of the substrate.
androstan-3,17-dione.
The obtained results (Table 4) indicated that enzymes catalyz-
ing oxidation reaction are inducible. The reaction mixture, after
6 h incubation of androsterone (1), contained 33% of hydroxy
derivatives, and after further 3 h, their amount increased to 89%.
Similarly, increase in the amount of the hydroxylated metabolites
in the reaction mixtures harvested after subsequent periods was
observed for the remaining substrates. Inducing properties of the
substrates towards hydroxylase(s) decreased in the order:
Comparison of 1H NMR spectrum of the product 13 to that of the
substrate 12 revealed a new signal at dH 4.03 (dt, J = 5.1, 10.5 Hz),
indicating monohydroxylation. The characteristic shape, multiplic-
ity and downfield shifts (
suggested that the hydroxyl group was introduced at 11
The spectral data of this metabolite was in agreement with that
reported in the literature for 11 -hydroxy-5 -androstan-3,17-
D
0.12 ppm) for the 19-H methyl protons
a-position.
a
a
dione [25]. The 1H NMR spectrum of 14, in comparison to the
spectrum of 13, had an additional signal at dH 3.68 ppm (t) indicat-
ing that the C-17 ketone had been reduced to a C-17b-alcohol. The
evidence of the reduction was provided by the loss of the resonance
signal at dC 219.0 ppm, and the appearance of a methine carbon
signal at dC 81.2 ppm in the 13C NMR spectrum. The 1H NMR spec-
trum of 15, in comparison to that of 14, showed new one-proton
signals at dH 5.80 ppm (d, J = 10.5 Hz) and dH 8.29 ppm (d, J =
10.5 Hz) indicating the insertion of a new double C@C bond, no sig-
nificant shifts for the methyl groups and absence of the 1b-H signal
at dH 2.77 ppm. The insertion of double C@C bond was confirmed by
a DEPT experiment which demonstrated the disappearance of two
methylene signals at dC 40.1 and 38.3 ppm visible in the 13C NMR
spectrum of 14, and replacing them by two methine resonances
at dC 162.3 and 126.1 ppm. These and all other 13C NMR signals
androstanedione (12), androsterone (1) and 3
(5). In order to determine whether the alcohols obtained from
-hydroxy-substrates can be products of the same hydroxylase
(11 -), an experiment was devised in which androsterone (1)
was added to a culture of B. bassiana induced 6 h previously with
epiandrosterone (rationale: epiandrosterone was effective inducer
a-androstanediol
3a
a
of the B. bassiana enzyme catalyzing 11
the mixture obtained after 6 h transformation, mutual ratios of
the products 11 -OH/7b-OH/7 -OH were not significantly
a-hydroxylation [21]). In
a
a
changing, but the substrate consumption increased from 36% to
51% (Table 5). The main component of both mixtures was
7b-hydroxy-androsterone (3). After 4 d incubation of androsterone
in the induced cultures, the lactones were also not identified.
The mixtures of hydroxylactones obtained from the 3a-hydroxy
substrate 5 contained products with 3-oxo (11) and 3b-hydroxy
were consistent with the structure of 15 as 11
a,17b-
groups (9). Comparison of composition of the mixtures after 48
dihydroxy-5 -androst-1-en-3-one. The structure of 11b-hydroxy-
a
and 96 h incubation of 3
dihydroxy-lactone 9 is formed from the 3-oxo-11
11. All of the product mixtures harvested after various transformation
periods of the 3 -hydroxy substrate contained neither androstanedi-
one nor its 11 -hydroxy derivative. 3b,11 -Dihydroxy-lactone 9 was
identified also after transformation of the 3-oxo substrate 12. Its
share in the reaction mixtures increased together with elongation
of the substrate transformation time. The obtained results indicate
that transformations connected with oxidation and reduction at C-3
take place after introduction of the lactone moiety in the ring D of
the hydroxy derivatives of the substrates.
a
-androstanediol (5) suggests that 3b,11
a-
testosterone (17) was identified by a new triplet at dH 3.61 ppm,
which indicated that the C-17 ketone had been reduced to a
C-17b-alcohol. The 13C NMR spectrum confirmed this notion with
loss of the resonance signal at dC 219.0 ppm, and its replacement
with the resonance signal at dC 82.1 ppm. The 1H NMR data of this
compound are in agreement with those reported in the literature
[26].
a-hydroxy-lactone
a
a
a
3.3. Time course analysis of the transformation, determination of the
metabolic pathway and the order of hydroxylation and lactonization
reactions for substrates 1, 5 and 12
In order to investigate metabolic pathways of the substrates,
composition of mixtures sampled after various transformation
periods was studied. The results are compiled in Table 4. Their
analysis indicates that the first stage of the process was hydroxyl-
3.4. Further transformation of 3
a
,11
a
-dihydroxy-5
a-androstan-17-one
(2) and 11 -hydroxy-17a-oxa-
a
D
-homo-5a
-androstan-3,17-dione (11)
The 48 h transformation of 11
a-hydroxyandrosterone (2) in the
ation. 11
being transformed, but only the 3
yielded additionally 7 - and 7b-alcohols. Significant differences
a
-Hydroxylation was observed for all of the compounds
3a-androstanediol-induced cultures of the microorganism resulted
a-hydroxy substrates (1 and 5)
in formation of a mixture of three 11
a
-hydroxylactones: 8 (26%), 9
a
(3%) and 11 (8%).
in further course of transformations of the metabolites from the
first stage were observed. In the product mixtures resulting from
androsterone (1) no hydroxylactones were identified (Fig. 2 and
The transformation of 11
androstan-3,17-dione (11) resulted in a stereoselective reduction
of the 3-keto group of this lactone to a 3b-alcohol in the first
a
-hydroxy-17a-oxa-D-homo-5
a-

´
A. Swizdor et al. / Steroids 82 (2014) 44–52
49
Table 4
Composition of crude mixtures obtained in transformations by B. bassiana.
Substrate
Compounds present in the mixturea (%)
Time of transformation
3 (h)
6 (h)
9 (h)
24 (h)
48 (h)
96 (h)
Androsterone (1)
Androsterone (1)
100
–
–
64
8
18
7
7
–
–
3a
3a
3a
,11
,7b-Dihydroxy-5
,7 -Dihydroxy-5
a
-Dihydroxy-5
-androstan-17-one (3)
-androstan-17-one (4)
a
-androstan-17-one (2)
22
50
17
25
53
18
24
54
17
a
a
a
–
3a
-Androstanediol (5)
3
3
3
3
3
3
3
a
a
a
a
a
a
a
-Androstanediol (5)
,11 -Dihydroxy-5 -androstan-17-one (2)
,7b-Dihydroxy-5 -androstan-17-one (3)
,7 -Dihydroxy-5 -androstan-17-one (4)
,11 ,17b-Trihydroxy-5 -androstane (6)
,7b,17b-Trihydroxy-5 -androstane (7)
,11 -Dihydroxy-17a-oxa-D-homo-5 -androstan-17-one (8)
-Dihydroxy-17a-oxa-D-homo-5 -androstan-17-one (9)
,7b-Dihydroxy-17a-oxa-D-homo-5 -androstan-17-one (10)
-Hydroxy-17a-oxa-D-homo-5 -androstan-3,17-dione (11)
100
–
–
–
–
–
–
–
–
97
–
–
–
2
1
–
–
–
–
69
4
2
8
8
5
–
–
–
44
13
6
14
7
10
2
–
15
9
17
21
2
1
14
2
7
a
a
13
20
19
–
–
8
15
8
5
a
a
a
a
a
a
a
a
a
a
3b,11
3a
a
–
–
7
9
11
a
a
–
–
Androstanedione (12)
Androstanedione (12)
35
58
7
–
–
34
29
33
1
–
–
28
38
17
12
2
22
13
20
18
20
5
14
11
22
19
20
11
11a
11a
11a
-Hydroxy-5
,17b-Dihydroxy-5
-Hydroxy-17a-oxa-D-homo-5
-Dihydroxy-17a-oxa-D-homo-5
,17b-Dihydroxy-5 -androst-1-en-3-one (15)
a
-androstan-3,17-dione (13)
-androstan-3-one (14)
-androstan-3,17-dione (11)
-androstan-17-one (9)
a
a
3b,11a
a
11a
a
–
–
11b-Hydroxy-AD (16)
11b-Hydroxyandrostenedione (16)
11b-Hydroxytestosterone (17)
100
–
55
39
53
42
a
Determined by GC analysis.
Fig. 2. Transformation of androsterone by B. bassiana.
24 h time period affording 3b,11
-androstan-17-one (9).
All metabolites were easily identified by comparison of the GC
a
-dihydroxy-17a-oxa-D-homo-
‘‘normal’’ binding orientation of 1 within 11a-hydroxylase and
5a
the alternative substrate binding can occur, resulting in the inser-
tion of hydroxy group at C-7. The assumption that the steroidal
substrate is able to form the enzyme-substrate complex in more
than one orientation was proposed for the first time to explain
and TLC data with those of authentic samples.
6b- and 11a-hydroxylation of steroids by Aspergillus tamarii [23],
4. Discussion
and was confirmed by studies using recombinant fission yeast with
gene CYP509C12 isolated from Rhizopus oryzae [29]. It is a com-
monly accepted model in the enzymatic hydroxylations of steroids,
and was demonstrated in a series of transformations by various
strains of microorganisms [1]. Recently, the hypothesis, that the
In the culture of the strain B. bassiana KCH 1065, the steroidal
5a
-dihydro substrates: androstanedione (12), 3
(5) and epiandrosterone (3b-hydroxy-5 -androstan-17-one) were
transformed to 11 -hydroxylactones. The metabolism of epian-
a-androstanediol
a
a
same enzyme can catalyze the hydroxylation at 11a- as well as
drosterone has been previously investigated [21]. Those studies
at C-7 explained the course of hydroxylation of the C19 steroids
in the cultures of Absidia coerulea [30] and Mortierella isabellina
[31]. The assumption that all hydroxy derivatives of androsterone
(1) may result from the activity of the same hydroxylase present
in B. bassiana is supported by results of the studies in which the
substrate was introduced after 6 h induction of the strain by epian-
have demonstrated that the substrate undergoes conversion in
the following sequence of reactions: 11
quent Baeyer–Villiger oxidation to a ring-D lactone (Fig. 1). In con-
trast, the incubation of 3 -hydroxy analogue of epiandrosterone 1
a-hydroxylation and subse-
a
did not result in the formation of the lactone and revealed only the
hydroxylating activity of this microorganism (Fig. 2 and Table 4). It
drosterone (epiandrosterone was transformed only to 11
derivatives [21]). After further 6 h reaction, the degree of substrate
conversion increased, but the percentage of 11 -hydroxyandroster-
a-hydroxy
is interesting that the 3
greater range of monohydroxylated products (11
alcohols) than the substrates with 3b-hydroxy group [21], which
underwent hydroxylation only at the 11 - position, the most com-
monly observed during the transformations by Beauveria genus
[27,28]. Almost 4-fold higher contents of the 11 - and 7b-alcohols
a-hydroxy compounds (1 and 5) gave a
a
-, 7 - and 7b-
a
a
one (2) in the extracts was similar to that observed in the standard
transformation (Table 5).
Interestingly, B. bassiana showed a significant difference in reac-
tivity towards the structurally close substrate 3a-androstanediol
a
a
(2+3) in the mixtures of products demonstrates the preference of
(5) (Fig. 3). Because the only difference between those two
substrates (1 and 5) is the oxo- and hydroxy-group at C-17, the
expected metabolism should be very similar. However, the
reaction mixture obtained after 48 h of transformation of 5
an equatorial stereochemistry of hydroxylation.
As the regiospecificity of hydroxylation depends on the correct
fit of the substrate in the active site of the enzyme, it is likely that
the axial stereochemistry of the 3a-hydroxy group restricts the

´
A. Swizdor et al. / Steroids 82 (2014) 44–52
50
Fig. 3. Transformation of 3a-androstanediol by B. bassiana.
contained, apart from hydroxy derivatives, 11
lactones 8–11 (Table 4). It could be speculated that for the 3
a
- and 7b-hydroxy-
-hy-
-steroids, the 17b-hydroxy functionality is necessary for
a
droxy-5
a
the activity of 17b-HSD and the stage of oxidation of the 17b-alco-
hol to the ketone is the decisive factor determining the amount of
produced D-lactones. It is remarkable that until the moment of
appearance of the BVMO activity, a major portion of 11
derivatives of epiandrosterone, DHEA (dehydroepiandrosterone)
and androstenedione [21] as well as 11 -hydroxyandrostanedione
a-hydroxy
a
(12) (Table 4) was transformed to the 17b-alcohols. None of the
identified metabolites of 1 underwent reduction of C-17 keto
group. It is quite likely that the reduction reactions of the formed
hydroxy derivatives of epimeric 3-hydroxy,17-ketones (androster-
one and epiandrosterone) are catalyzed by dehydrogenases with
different substrate specificity. 17b-HSD from B. bassiana did not ex-
hibit activity towards 3a-hydroxy C-17-keto steroids 2–4, the cor-
responding C-17b-alcohols were not created, the oxidation of the
C-17b-alcohols to C-17 ketones did not occur, and as a result
hydroxylactones were not identified. This observation may be sup-
ported by the results of experiments in which the transformation
of 11
with 17b-HSD activity (induced by the presence of 3
diol (5)). After 48 h incubation of the substrate, the reaction mix-
ture contained the 11 -hydroxy-lactones 8, 9 and 11, and their
a
-hydroxyandrosterone (2) was performed in the cultures
a-androstane-
Fig. 4. Transformation of androstanedione by B. bassiana.
a

´
A. Swizdor et al. / Steroids 82 (2014) 44–52
51
Table 5
synergism of steroidal lactonization and hydroxylation pathways,
B. bassana offers not only a model to study the relations between
the structure of the substrates and activity of the enzymes, but also
allows for the formation of new steroid derivatives with potential
biological activity.
Composition of crude mixtures obtained after 6 h of transformation of androsterone
by B. bassiana.
Compoundsa (%)
Transformation by
Standard
culture
Culture induced by
epiandrosteroneb
Acknowledgments
Androsterone (1)
64
8
49
12
3a
3a
3a
,11
a-Dihydroxy-5a-
androstan-17-one (2)
,7b-Dihydroxy-5
17-one (3)
This research was supported financially by the European Union
within the European Regional Development Found (Grant No.
POIG.01.03.01-00-158/09-02).
a
-androstan- 18
26
10
,7a
-Dihydroxy-5
a
-
7
androstan-17-one (4)
a
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Contents of compounds determined by GC analysis.
b
Culture with 11
a-hydroxylating activity confirmed on the basis of presence of
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The collected results uniquely demonstrated a preference for
´
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D
B. bassiana. Although the 3
a
,7 -dihydroxy-5 -androstan-17-one
a
a
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(4) was undoubtedly available to a culture with BVMO activity,
mixtures of products did not contain detectable amounts of the
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supported by the transformation of 11b-hydroxyandrostenedione
(16), which resulted only in a reduction pathway of substrate to
11b-hydroxytestosterone (17) (Table 4). The content of the
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[9] Swizdor A. Baeyer–Villiger oxidation of some C19 steroids by Penicillium
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11
96 h of incubation of 5 was close to 80%, and its main component
was 3b,11 -dihydroxy-17a-oxa-D-homo-5 -androstan-17-one (9).
The timed experiment suggested that the inversion of stereo-
chemistry at C-3 of 8 followed via 11 -hydroxy-17a-oxa-D-homo-
-androstan-3,17-dione (11). The same part of the metabolic
a-hydroxy lactones (8, 9 and 11) in the mixture of lactones after
a
a
a
5a
pathway of lactone 11 resulting in reduction of 3-ketone to 3b-alco-
hol was observed during the transformation of androstanedione
(12) (Fig. 4 and Table 4). Experiment, in which the isolated
3-keto-lactone 11 was subjected to transformation, confirmed that
this compound may be the precursor of 3b-hydroxy-lactone 9, and
the oxidation–reduction reactions at C-3 may occur in the presence
of a lactone function in ring-D of the steroid.
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Aside from oxidation and reduction, minor 1-dehydrogenation
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