Biotransformation of 6α-santonin and 1,2-dihydro-α-santonin by Acremonium chrysogenum PTCC 5271 and Rhizomucor pusillus PTCC 5134

Article abstract of DOI:10.1016/j.molcatb.2014.09.003

Biotransformation of 6α-santonin (1) and 1,2-dihyro-α-santonin (2) by two fungal strains Acremoniumchrysogenum (Cephalosporium chrysogenum) and Rhizomucor pusillus has been investigated for the firsttime. After 8 days of incubation of 1 by A. chrysogenum, four known metabolites including 1,2-dihydro-α-santonin (2) (30%), 8α-hydroxyl-α-santonin (3) (22%), 15-hydroxy-α-santonin (4) (15%) and 4,5-dihydro-α-santonin (5) (10%) were obtained. Incubation of 1 by R. pusillus afforded two metabolites 2 (45%) and 3(20%). Biotransformation of 1,2-dihyro-α-santonin by A. chrysogenum produced tetrahydro-α-santonin(6) with 52% yield and tetrahydroartemisin (7) with 33% yield. By R. pusillus, the yields of 6 and 7 were32% and 21%, respectively. The structures of the products were identified on the basis of spectroscopic data.

Full text of DOI:10.1016/j.molcatb.2014.09.003

Journal of Molecular Catalysis B: Enzymatic 110 (2014) 59–63
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biotransformation of 6˛-santonin and 1,2-dihydro-˛-santonin by
Acremonium chrysogenum PTCC 5271 and Rhizomucor pusillus
PTCC 5134
Somayyeh Gandomkar, Zohreh Habibi∗
Department of Pure Chemistry, Faculty of Chemistry, Shahid Beheshti University, G.C., Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2014
Received in revised form 7 September 2014
Accepted 7 September 2014
Available online 17 September 2014
Biotransformation of 6˛-santonin (1) and 1,2-dihyro-˛-santonin (2) by two fungal strains Acremonium
chrysogenum (Cephalosporium chrysogenum) and Rhizomucor pusillus has been investigated for the first
time. After 8 days of incubation of 1 by A. chrysogenum, four known metabolites including 1,2-dihydro-˛-
santonin (2) (30%), 8˛-hydroxyl-˛-santonin (3) (22%), 15-hydroxy-˛-santonin (4) (15%) and 4,5-dihydro-
˛-santonin (5) (10%) were obtained. Incubation of 1 by R. pusillus afforded two metabolites 2 (45%) and 3
(20%). Biotransformation of 1,2-dihyro-˛-santonin by A. chrysogenum produced tetrahydro-˛-santonin
(6) with 52% yield and tetrahydroartemisin (7) with 33% yield. By R. pusillus, the yields of 6 and 7 were
32% and 21%, respectively. The structures of the products were identified on the basis of spectroscopic
data.
Keywords:
Acremonium chrysogenum (Cephalosporium
chrysogenum)
Rhizomucor pusillus
Biotransformation
6˛-Santonin
© 2014 Elsevier B.V. All rights reserved.
1,2-Dihyro-˛-santonin
1. Introduction
[11]. Since metabolites in mammalian systems are not large in
quantities, they are difficult to identify. In contrast, by using micro-
Eudesmanolide compounds are biogenetic and chemical pre-
cursors of a range of sesquiterpene lactones [1]. They possess a
variety of biological and pharmaceutical activities such as cyto-
toxic, anti-cancer, anti-HIV and anti-tumor [2]. ˛-Methyl-ꢀ-lactone
and ˛,ˇ-unsaturated ester side chain adjacent to ˛-methylene-
ꢀ-lactone are structure-activity requirement of the sesquiterpene
lactones for high in vivo anti-tumor activity [3,4].
6˛-Santonin (1), the cheap and abundant sesquiterpene lactone,
is found in several species of genus Artemisia [3]. It has attracted
a great deal of interest from chemists owing to its wide range of
biological activities, such as cytotoxic, anti-tumor, immunosup-
pressive, insecticidal and anti-HIV activities [2,3,5,6]. 6˛-Santonin
is an important starting material on the account of many different
functional groups to obtain several naturally bioactive terpenoid
compounds bearing the guaiane or eudesmane skeleton [7–10].
Microorganisms catalyze various oxidative, reductive, conjuga-
tive and degradative reactions of many classes of natural products.
They have been used as in vitro models in mammalian systems
to mimic and predict the metabolic fate of pharmaceutical agents
bial transformations in large-scale, metabolites can be produced in
large quantities, which is easy to identify by spectroscopic methods.
Biotransformation introduced a regioselective and stereoselec-
tive alternatives for some of the enantiospecific synthesis of natural
sesquiterpene lactones [12]. Bioconversion of sesquiterpenes is
actually accepted as a preferable method, in combination with
chemical reactions, for the semi-synthesis of products of interest
with an enhanced bioactivity and low toxicity [9,13–15]. There have
been number of reports on the biotransformation of 6˛-santonin,
which many different results such as hydration, epoxidation, reduc-
tion, hydroxylation or combination of these reactions have been
concluded [1,12,16–21].
In this research, the ability of two fungi Acremonium chryso-
genum (Cephalosporium chrysogenum) and Rhizomucor pusillus in
the biotransformation of 6˛-santonin and 1,2-dihydro-˛-santonin
was investigated.
Reduction of the double bonds in 6˛-santonin by chemical syn-
thesis methods needs metal catalyst and hydrogen gas. In order
to reduce the production of harmful waste in chemical synthe-
sis methods, biotransformation was chosen as an environmentally
friendly alternative for reduction of the double bonds of 6˛-
santonin, but in addition to reduction of the double bonds in some
metabolites, hydroxylation was observed. Since biotransformation
∗
Corresponding author. Tel.: +98 21 2990 3110; fax: +98 21 2990 3110.
E-mail address: z habibi@sbu.ac.ir (Z. Habibi).
http://dx.doi.org/10.1016/j.molcatb.2014.09.003
1381-1177/© 2014 Elsevier B.V. All rights reserved.

60
S. Gandomkar, Z. Habibi / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 59–63
of 1,2-dihydro-˛-santonin has not been studied before by any
fungal strains, this compound was selected for investigating the
biotransformation ability of two mentioned strains.
In the previous reports on the biotransformation of 6˛-santonin
by fungal strains, the yield of the products was low. Despite var-
ious studies on bioconversion of 6˛-santonin, trying to find new
strains for its bioconversion and the production of pharmaceuti-
cal ingredients or precursors with good yield is still a great deal of
interest.
acetate. The extract was evaporated under reduced pressure on a
rotary evaporator. The concentrated residue was re-dissolved in
chloroform and loaded on preparative TLC with n-hexane/ethyl
acetate (4:6 v/v). All the purified metabolites were identified by
spectral data. Further support was obtained by comparison with
literature data.
2.6. Analytical methods
1H NMR spectra were recorded using a Bruker Avance-300 MHz
spectrometer with tetramethylsilane (TMS) as internal standard in
CDCl3. Chemical shifts (ı) are given in parts per million (ppm) rel-
ative to TMS. Mass (MS) spectra were obtained on a Finnigan MAT
TSQ-70 instrument by electron impact (EI) at 70 eV. Infrared (IR)
spectra were recorded using KBr disks on a Magna-IR 550 Nicolet
FTIR spectrometer. Thin layer chromatography (TLC) and prepar-
ative TLC was performed on 20 × 20 cm 0.25-mm thick layers of
silica gel G (Silica gel 60 GF254, Merck). Layers were prepared on
glass plates and activated at 110 ◦C 1 h before use.
2. Experimental
2.1. Materials
6˛-Santonin was purchased from Sigma–Aldrich (Taufkirchen,
Germany). Yeast extract and agar were purchased from Scharlau
(Barcelona, Spain). Inorganic salts, analytical grade reagents, sol-
vents and silica gel 60 GF254 fluorescent thin layer chromatographic
plates (TLC) were obtained from Merck (Darmstadt, Germany).
2.2. Microorganisms and cultural conditions
3. Result
The fungal strains A. chrysogenum PTCC 5271 and R. pusillus PTCC
5134 were obtained from the Persian type culture collection (Ira-
nian Research Organization for Science and Technology).
3.1. Biotransformation of 6˛-santonin by A. chrysogenum and R.
pusillus
R. pusillus was maintained on potato-dextrose agar plates (com-
position, g/L: glucose 20 g, diced potatoes 300 g, agar 15.0 g) and A.
chrysogenum was maintained on yeast extract agar plates (yeast
extract, 4.0 g; soluble starch, 15.0 g; K2HPO4, 1.0 g; MgSO4•7H2O,
0.5 g; agar, 15.0 g, distilled water, 1000.0 ml) at 4 ◦C and freshly
sub-cultured before using in the transformation experiment. All
media were sterilized by autoclaving at 121 ◦C for 20 min. Ten 250-
ml Erlenmeyer flasks, each containing 150 ml of liquid medium of
potato-dextrose broth for R. pusillus and ten 250 ml Erlenmeyer
flasks, each containing 150 ml of liquid medium of yeast extract
broth for A. chrysogenum, were inoculated with freshly obtained
spores from agar slope cultures and incubated 24 h at 26 ◦C in a
rotary shaker (120 rpm).
After 8 days of incubation of 6˛-santonin (1) at 26 ◦C by A.
chrysogenum, four metabolites 2–5 and by R. Pusillus, two metabo-
lites 2 and 3 were obtained. On the basis of the spectral data
including 1H NMR, FT-IR and MS, the structures of the products
were identified (Scheme 1). Their chemical and spectral data were
in a good agreement with those reported in the literature. The
observed bioconversion reactions were: C1–C2 double bond reduc-
tion in 2, C-8 hydroxylation in 3, 15-methyl hydroxylation in 4 and
C4–C5 double bond reduction in 5.
3.2. Biotransformation of 1,2-dihydro-˛-santonin by A.
chrysogenum and R. pusillus
Biotransformation of 1,2-dihyro-˛-santonin by A. chrysogenum
and R. pusillus produced tetrahydro-˛-santonin (6) and tetrahy-
droartemisin (7) by different yields (Scheme 2). The physical and
spectral data were in good agreement with literature. The observed
bioconversion reaction was reduction of C4–C5 double bond in
metabolite 6 and reduction of C4–C5 double bond and C-8 hydrox-
ylation in metabolite 7.
The obtained yields of all the metabolites from the biotransfor-
mation of 6˛-santonin and 1,2-dihyro-˛-santonin by two fungi and
a comparison of them with previously reported yields is shown in
Table 1. Chemical shifts of all metabolites are shown in Table 2.
2.3. Synthesis of 1,2-dihydro-˛-santonin
A mixture of 1 g of 6˛-santonin (4 mmol), 30 ml of dried CH2Cl2
and 150 mg of 2% Pd–C was stirred under 1 atm H2 at room tem-
perature for 10 h. Then the catalyst was removed and the filtrate
was concentrated under reduced pressure on a rotary evapora-
tor. The concentrated residue was re-dissolved in chloroform and
loaded on preparative TLC with n-hexane/ethyl acetate (7:3 v/v) for
further purification. Finally, 1,2-dihydro-˛-santonin was separated
and purified (60% yield) for using in biotransformation reaction.
2.4. General procedure for the biotransformation of 6˛-santonin
and 1,2-dihydro-˛-santonin using whole cells
4. Discussion
After preparation of fermentation media, 6˛-santonin (1 g) was
dissolved in acetone (10 ml); 1 ml of the solution was added to each
250-ml Erlenmeyer flask for each fungi. Higher concentrations of
substrate resulted in lower conversion and a decrease in metabo-
lites yields. The reaction was monitored by TLC. After 8 days of
incubation under the same conditions, the highest conversion was
achieved.
Metabolite 2: The MS spectrum of metabolite 2 showed a
molecular ion peak at m/z 248 and provided the molecular formula
C15H20O3, which was two units higher than 6˛-santonin suggest-
ing that double bond reduction was occurred. The optical rotation
of this metabolite was measured; [˛]D25 +90◦ (c 6.1, CHCl3) (lit. [14],
[˛]D +89◦). In 1H NMR spectrum of compound 2, elimination of
25
signals at ı 6–6.7 confirmed reduction of C1–C2 double bond. Dou-
blet at ı 4.67 was related to H-6. Addition of signals at ı 2.44 and
ı 2.52 related to H-2a and 2b confirmed the structure. Singlet at
ı 1.98 was related to Me-15. Chemical shift for this methyl group
showed olefinic structure at C-4.
2.5. Extraction of 6˛-santonin and 1,2-dihydro-6˛-santonin
transformation metabolites
At the end of incubation, all identical fermentation broths were
combined and extracted three times with equal volume of ethyl
Previously, 1,2-dihydro-˛-santonin (2) was obtained from
the incubation of 6˛-santonin with Cunninghamella bainieri,

S. Gandomkar, Z. Habibi / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 59–63
61
OH
14
1
9
O
O
2
3
8
7
10
O
O
A. chrysogenum, R. pusillus
5
13
O
O
O
4
6
11
O
(2)
15
(3)
12
O
(1)
O
O
H
O
O
HO
O
O
(4)
(5)
Scheme 1. The biotransformation of 6˛-santonin by A. chrysogenum (2–5) and R. pusillus (2 and 3).
Cunninghamella echinulata, Mucor plumbeus and Rhizopus stolonifer
[14]. This metabolite was also obtained through biotransformations
of 6˛-santonin with Aspergillus niger, Cunninghamella blaksleeana,
Streptomyces aureofaciens, Bacillus subtilis, Bacillus cereus, C. ech-
inulata, Rhizopus oryzae and Pseudomonas sp. [12,14,18,22]. The
reported yield of this metabolite after incubation by Absidia
officinalis was 2.9% but in this study, the obtained yields were 45%
and 30% by R. pusillus and A. chrysogenum, respectively [6].
one new oxy-methylene at ı 4.80 was appeared. The optical rota-
tion of this metabolite was measured; [˛]25D −90.2◦ (c 0.11, CHCl3)
(lit [6], [˛]25 −92.0◦). Therefore, the structure of 4 was identified
D
to be 15-hydroxy-˛-santonin.
This metabolite was reported in 2005 by biotransformation of
6˛-santonin by cell suspension cultures of five plants (Catharan-
thus roseus, Ginkgo biloba, Platycodon grandiflorum, Taxus cuspidate
and Phytolacca asinosa). Biotransformation of 1 by cell cultures of
Abisidia coerulea afforded this metabolite with 1.0% yield but in our
research, the yield was 15% by A. chrysogenum [6,15].
Metabolite 5: The MS spectrum of metabolite 5 showed a
molecular ion peak at m/z 248 and provided the molecular formula
C15H20O3, which was two units higher than 6˛-santonin suggesting
that double bond reduction was occurred.
Metabolite 3: The MS spectrum of metabolite 3 showed a
molecular ion peak at m/z 262 consistent with the molecular
formula of C15H18O4, suggesting that hydroxyl group may be
introduced. The 1H NMR of 3 also showed the existence of a new
oxymethine proton signal at ı 4.15. The optical rotation of this
metabolite was measured, [˛]D −40.6◦ (c 0.51, CHCl3) (lit. [6],
20
[˛]D −41.1◦), it was confirmed ˛-stereochemistry for OH by com-
In comparison between 1H NMR of 5 and 6˛-santonin, a sig-
nificant difference for the resonances of H-4, H-5 and Me-15 was
observed. The C-15 methyl protons appeared as a doublet at ı 1.57
and it showed vicinal couplings with a C-4 methine proton (ı 2.60),
which was confirmed the reduction of C4–C5 double bond. H-5
appeared at ı 2.31. Stereochemistry was established by compar-
ison of optical rotation of this metabolite and published data in the
20
parison with the literature. Accordingly, the structure of 3 was
determined as 8˛-hydroxy-˛-santonin.
The fact of 8˛-hydroxylation of 6˛-santonin enables the for-
mation of 8,12-eudesmanolide instead of 6,12-eudesmanolide and
some other useful modification at C-8 position.
This metabolite was obtained by incubation of 6˛-santonin with
the cell cultures of Abisidia coerulea in a low yield (2%) but in this
study by R. pusillus and A. chrysogenum, the yields were 20% and
22%, respectively [6].
Metabolite 4: The MS spectrum of metabolite 4 showed a
molecular ion peak at m/z 262, consistent with the molecular
formula of C15H18O4, suggesting that a hydroxyl group may be
introduced. The 1H NMR of 4 was similar to that of 6˛-santonin
except that the signal of H-15 at ı 2.07 was disappeared, however,
literature, [˛]D +73.5◦ (c 9.7, CHCl3) (lit. [14], [˛]D +75◦).
Previously, metabolite 5 was obtained by R. stolonifer [14]. Bio-
transformation of 6˛-santonin by cell suspension cultures of five
plants (Catharanthus roseus, Ginkgo biloba, Platycodon grandiflorum,
Taxus cuspidate and Phytolacca asinosa) were also produced this
metabolite [15]. Incubation of 6˛-santonin by A. officinalis pro-
duced this metabolite by yield of 5.8% but in our research by A.
chrysogenum, the yield was increased to 10% [6].
25
25
OH
A. chrysogenum, R. pusillus
O
O
O
H
H
O
O
O
O
O
O
(2)
(7)
(6)
Scheme 2. The biotransformation of 1,2-dihydro-˛-santonin by A. chrysogenum and R. pusillus.

62
S. Gandomkar, Z. Habibi / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 59–63
Table 1
was confirmed ˇ-stereochemistry for H-4 by comparison with the
literature.
Obtained yields from biotransformation of 6˛-santonin and 1,2-dihydro-˛-santonin
by A. chrysogenum and R. pusillus and reported yields in literature.
This metabolite was obtained by incubation of 6˛-santonin by
Phanerochaete chrysosporium [1]. To the best of our knowledge, it is
the first report of this compound by microbial method, previously
6 was synthesized from 6˛-santonin by using hydrogen gas over
10% palladium on carbon [24,25].
Metabolite Obtained yields by Obtained yields by Reported yields in
A. chrysogenum
R. pusillus
literature
2
30%
45%
aCell suspension of
Asparagus officinalis:
2.9%
Metabolite 7: The MS spectrum of metabolite 7 showed a
molecular ion peak at m/z 266 and provided the molecular formula
C15H22O4, suggesting that a hydroxyl group may be introduced and
also double bond reduction may be occurred. In 1H NMR of 7, elim-
ination of signals at ı 6– 6.7 confirmed reduction of C1–C2 double
bond. The upfield chemical shift value of C-15 indicated that this
methyl group was bonded to a sp3 hybridized carbon atom. These
data confirmed the reduction of double bond present between
C4–C5. The 1H NMR of 7 also showed the existence of a new
oxymethine proton signal at ı 3.15, which was related to H-8. The
stereochemistry of H-4 was determined to be ˇ-configuration by
bCell suspension of
Phytolacca asinosa: 62%
bCell suspension of
Taxus cuspidata: 7%
bCell suspension of
Catharanthus roesus:
<2%
3
4
22%
15%
20%
–
aAbsidia coerulea IFO
4011: 2.0%
aCell suspension of
Asparagus officinalis:
1.0%
bCell suspension of
Platycodon
comparison with optical rotation of this metabolite, [˛]D +50.1◦
14
(c 2.6, CHCl3) (lit. [24,26], [˛]D +50◦). In our knowledge, there
14
grandiflorum: 26%
bCell suspension of
Phytolacca asinosa: <2%
bCell suspension of
Catharanthus roesus:
<2%
is no report on 8˛-hydroxylation and double bonds reduction of
1,2-dihydro-˛-santonin by enzymatic approaches. Previously, this
compound was synthesized by chemical methods.
5
10%
–
aCell suspension of
Asparagus officinalis:
5.8%
5. Conclusion
aCell suspension of
Catharanthus roesus:
<2%
The ability of two fungal strains, R. pusillus and A. chrysogenum,
in bioconversion of 6˛-santonin (1) and 1,2-dihyro-˛-santonin (2)
has been studied for the first time. The observed bioconversion
reactions were C1–C2, C4–C5 double bonds reduction, C-8 hydrox-
ylation and 15-methyl hydroxylation in different metabolites of
these two substrates. In this research, in comparison with the pre-
vious reports on biotransformation of 6˛-santonin, yields of all
metabolites were improved. It is the first report of biotransforma-
tion of 1,2-dihydro-˛-santonin and the production of metabolite 7
and 8 by enzymatic approaches.
6
7
52%
33%
32%
21%
–
–
a
Ref. [16].
Ref. [15].
b
Table 2
1H NMR chemical shifts of metabolites 2–7.
Carbon
2
3
4
5
6
7
1H
1H
1H
1H
1H
1H
Acknowledgment
1
2
1.55
1.76
2.44
2.52
–
6. 67
–
6.27
–
–
–
4.82
1.91
4.15
–
1.45
2.06
2.65
1.26
1.35
2.18
6.70
–
6.27
–
–
–
4.81
1.86
1.69
2.05
1.56
1.93
2.41
1.26
1.34
4.80
6.67
–
5.36
–
1.26
1.46
1.82
1.88
2.69
1.52
3.85
1.56
1.61
2.01
1.17
1.35
2.24
1.20
1.00
1.22
1.28
1.46
1.82
1.88
2.69
1.50
3.85
1.87
3.15
–
1.53
1.91
2.24
1.20
1.00
1.22
This work was supported by Research Council of Shahid Beheshti
University, Tehran, Iran (Grant No. 600/4294) for which the authors
are thankful.
4
5
6
7
8
2.60
2.31
4.80
2.16
1.34
2.05
1.34
1.92
2.16
1.29
1.26
1.57
–
4.67
1.81
1.65
1.89
1.70
1.85
2.34
1.25
1.32
1.98
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Metabolite 6: The MS spectrum of metabolite 6 showed a
molecular ion peak at m/z 250 and provided the molecular
formula C15H22O3, which was two units higher than 1,2-dihydro-˛-
santonin suggesting that reduction of double bond was occurred. In
1H NMR of 6, elimination of signals at ı 6–6.7 confirmed reduction
of C1–C2 double bond. The upfield chemical shift value of C-15 indi-
cated that this methyl group was bonded to a sp3 hybridized carbon
atom. These data confirmed the reduction of double bond present
between C4–C5. The stereochemistry of H-4 was determined to be
ˇ-configuration. The optical rotation of this metabolite was mea-
sured, [˛]D +30.7◦ (c 0.24, CHCl3) (lit. [24], [˛]D +31◦), and it
25
25

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