Immobilized heterologous Rhizopus Oryzae lipase as an efficient catalyst in the acetylation of cortexolone

Article abstract of DOI:10.1002/ejoc.201200178

The enzymatic preparation of a monoacetyl derivative of the corticosteroid cortexolone, through a transesterification reaction, is described. The heterologous Rhizopus oryzae lipase, immobilized on three different supports, proved to be an efficient catalyst in the acylation reaction using a complex substrate such as cortexolone. Immobilization of the enzyme on Lewatit 1600 resin at pH = 7 and 25 °C was the best condition for catalysis of the acetylation reaction. The influence of various reaction parameters, such as the nature of the acetylating agent, the solvent, the temperature, and the ratios of acetylating agent to substrate, and enzyme to substrate, was evaluated. Using the response surface methodology and a central composite rotatable design, the specific yield of acetylated cortexolone was optimized by means of the study of the effect of the enzyme (E)/substrate (S) and the acylating agent (A)/substrate ratios. The ratios of 5 (E/S) and 31.6 (A/S) were predicted as the optimal values to reach the maximum specific yield of the product (P): 1.59 mmol P/mmol A·g E. The mild reaction conditions and low environmental impact make the biocatalytic procedure a convenient way to prepare the reported derivative of this biologically active steroid. The regioselective acetylation of cortexolone was achieved by using an immobilized heterologous Rhizopus oryzae lipase. The mild reaction conditions and low environmental impact make the biocatalytic procedure a convenient way to prepare the monoacetyl derivative of this biologically active steroid. Copyright

Full text of DOI:10.1002/ejoc.201200178

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DOI: 10.1002/ejoc.201200178
Immobilized Heterologous Rhizopus Oryzae Lipase as an Efficient Catalyst in
the Acetylation of Cortexolone
Paula G. Quintana,[a] Marina Guillén,[b] Marzia Marciello,[c] Francisco Valero,[b]
Jose M. Palomo,[c] and Alicia Baldessari*[a]
Keywords: Steroids / Acylation / Biocatalysis / Enzymes / Immobilization / Supported catalysts
The enzymatic preparation of a monoacetyl derivative of the
corticosteroid cortexolone, through a transesterification reac-
tion, is described. The heterologous Rhizopus oryzae lipase,
immobilized on three different supports, proved to be an ef-
ficient catalyst in the acylation reaction using a complex sub-
strate such as cortexolone. Immobilization of the enzyme on
Lewatit 1600 resin at pH = 7 and 25 °C was the best condition
for catalysis of the acetylation reaction. The influence of vari-
ous reaction parameters, such as the nature of the acetylating
agent, the solvent, the temperature, and the ratios of acet-
ylating agent to substrate, and enzyme to substrate, was
evaluated. Using the response surface methodology and a
central composite rotatable design, the specific yield of acet-
ylated cortexolone was optimized by means of the study of
the effect of the enzyme (E)/substrate (S) and the acylating
agent (A)/substrate ratios. The ratios of 5 (E/S) and 31.6 (A/S)
were predicted as the optimal values to reach the maximum
specific yield of the product (P): 1.59 mmol P/mmol A·g E.
The mild reaction conditions and low environmental impact
make the biocatalytic procedure a convenient way to prepare
the reported derivative of this biologically active steroid.
α-oxo aldehydes with arginine could lead to the formation
of modified proteins.[5] The hydroxy group at position 21
in corticosteroids is highly sensitive to oxidation by simple
exposure to air of a methanolic solution in the presence of
cupric acetate.[6] The preparation of an acyl derivative could
avoid this oxidation and, consequently, the undesired side
effects. It was observed that protection of the hydroxy
group at position 17 does not inhibit the oxidation, because
the same reaction carried out on corticosteroids with an
acylated 17α-hydroxy group, such as hydrocortisone-17α-
butyrate, also gave the expected α-oxo aldehydes.[7] There-
fore, the preparation of cortexolone-21-acetate seems to be
an interesting objective.
Cortexolone-21-acetate proved to be a good starting ma-
terial in the biotransformation to hydrocortisone and hy-
drocortisone-21-acetate by parent and mutant strains of the
filamentous fungus Curvularia lunata[8] and Cunninghamella
blakesleeana.[9] It was observed that the position of the hy-
droxy group introduction, the selectivity of the bioconver-
sion process, and its efficiency, were strongly dependent on
the structure and substitution of the steroid molecule.
Moreover, the conjugation of cortexolone derivatives to cy-
tarabyme increased the stability of this nucleoside with anti-
cancer activity.[10]
Introduction
Corticosteroids constitute the most frequently used drugs
in the symptomatic treatment of inflammatory and prolifer-
ative skin diseases.[1] Among corticosteroids, cortexolone
(1) and its 17α-propionate showed a potent antiandrogenic
topical activity that is useful in the treatment of acne vul-
garis.[2] Despite their beneficial effects, corticosteroids ap-
plied topically have also been associated with allergic con-
tact dermatitis reactions.[3] This side effect results from the
formation of reactive intermediates during skin metabolism,
which are able to modify the side chain of nucleophilic
amino acids, and, therefore, lead to the formation of modi-
fied proteins that are recognized as foreign by the skin im-
mune system.[4] In this sense, Wilkinson and Jones sug-
gested that corticosteroid molecules could be oxidized into
their 21-dehydro derivatives and that the reaction of these
[a] Laboratorio de Biocatálisis, Departamento de Química
Orgánica y UMYMFOR, Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires,
Pabellón 2, Piso 3, Ciudad Universitaria,
C1428EGA, Buenos Aires, Argentina
Fax: +54-11-4576-3385
E-mail: alib@qo.fcen.uba.ar
alibhome@yahoo.com.ar
Regarding its synthesis, cortexolone-21-acetate was pre-
viously prepared from pregnenolone acetate in six steps.[11]
However, to obtain the 21-monoester in a purity that was
suitable for biological tests and pharmacological applica-
tions following this method, a complex and resource-inten-
sive purification procedure is necessary.
[b] Departament d’Enginyeria Química, EE, Universitat
Autònoma de Barcelona,
08193 Bellaterra, Barcelona, Spain
[c] Departamento de Biocatálisis, Instituto de Catálisis (CSIC),
Campus UAM Cantoblanco, 28049 Madrid, Spain
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200178.
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The application of enzymes in the synthesis of pharma- erated from the same lipase with very different catalytic
ceuticals and natural product derivatives is well known.[12] properties.[38,39]
Biocatalysis is a green alternative for the preparation of
In this work, we report the results obtained through li-
known and new compounds with physiological and thera- pase-catalyzed acetylation of cortexolone using an immobi-
peutic properties that are difficult to obtain by using con- lized recombinant Rhizopus oryzae lipase (ROL) as catalyst
ventional chemical methods.[13] Lipases have become at- (Scheme 1).
tractive as biocatalysts for chemo-, regio-, and stereoselec-
tive reactions under mild conditions. They have been inte-
grated into a variety of industrially interesting processes,[14]
in particular, the preparation of enantiomerically pure
pharmaceutical compounds.[15]
In the steroid field, enzyme catalysis can play an impor-
tant role in the mild and selective interconversion of func-
tional groups through regioselective transformations.[16–19]
Scheme 1. Enzyme-catalyzed acetylation of cortexolone.
Studies carried out in our laboratory on the esterification
After fixing some operational parameters, the optimal
and transesterification of polyfunctionalyzed steroids, have
conditions of enzyme/substrate ratio and acylating agent/
shown that lipases can act on substituents either on the A-
substrate ratio have been determined by a central composite
or D-ring.[20,21] In addition, in previous papers, we observed
rotatable experiment design using the response surface
methodology, to deliver the best immobilized derivative.
that, in androstanes and pregnanes, Candida rugosa lipase
showed a preference for C-3-hydroxy or -acyloxy groups,
whereas Candida antarctica catalyzed the reactions at the
D-ring.[22] Taking into account these properties, we have
Results and Discussion
prepared fatty acid derivatives of dehydroepiandro-
sterone,[23] 3,17-β-estradiol,[24] and a series of novel 20-suc-
Preliminary experiments showed that the acetylation of
cortexolone (1) using heterologous ROL as catalyst af-
cinates of pregnanes.[25]
Regarding glucocorticoids, we carried out the lipase-cat- forded cortexolone-21-acetate (21-acetoxy-17α-hydroxy-4-
alyzed synthesis of acyl derivatives of hydrocortisone,[26] pregnen-3,20-dione) as the only product (Scheme 1) ob-
and, in the field of corticosteroids, Ferraboschi et al. re- served by HPLC analysis (see Figure S1 in the Supporting
ported the synthesis of cortexolone-17α-propionate cata- Information).
lyzed by Candida rugosa lipase.[27]
The identity of the product was confirmed by its melting
The extracellular sn-1,3 regioselective, native and recom- point, which was in accordance with reported data,[11] and
binant, R. oryzae lipase (ROL) immobilized on different by spectroscopic methods. The acetylation on the hydroxy
supports has been successfully used as a biocatalyst for the group of C-21 was confirmed by observing the downfield
synthesis of human milk substitutes[28] and low caloric tri- shift in the H NMR signals of the 21-H resonance from δ
1
acylglycerol,[29] flavor,[30,31] enzymatic resolution[32] and bi- = 4.28 and 4.56 ppm in substrate 1 to δ = 4.86 and
odiesel production.[33]
5.09 ppm in product 2, respectively, and the appearance of
The use of extracellular recombinant lipases is a well- a new signal corresponding to CH3-23 of the acetyl group
referenced strategy that has been used to increase the pro- at δ = 2.17 ppm. There was no major variation in the
ductivity of the enzyme bioprocess production and to sim- methyl-18 chemical shift (moved from δ = 0.70 to
plify processes downstream of the enzyme. Pichia pastoris 0.72 ppm), the methyl-19 chemical shift (from δ = 1.18 to
is the preferred host yeast to produce recombinant lipases. 1.19 ppm) or the 4-H signal (from δ = 5.72 to 5.74 ppm).
The mature sequence of R. oryzae lipase has been produced In the 13C NMR spectrum, new signals of the carbonyl C-
in this cell factory under PAOX1 promoter using a strategy 22 (δ = 171 ppm) and the methyl C-23 (δ = 20.6 ppm) from
of mixed substrates.[34] The recombinant ROL has been the new acetate group could be observed.
characterized, and it showed a 40-fold higher specific ac-
To optimize the reaction conditions, several experiments,
using free and immobilized preparations of recombinant
tivity compared with the commercial native ROL.[35]
When a lipase is selected as a biocatalyst for a given ROL, were performed to study the variation of the reaction
reaction, its application is often hampered by some char- parameters such as solvent, temperature, enzyme/substrate
acteristics: difficult recovery and reuse, low selectivity (E/S) ratio, acylating nature and acylating/substrate (A/S)
towards non-natural compounds and low stability under ratio.
process conditions. Several strategies have been used to ad-
At first, we evaluated the behavior of free ROL (ROL0)
dress these drawbacks. In this context, conformational and three immobilized preparations named: ROL1 (immo-
engineering as been described as a very interesting ap- bilized on Octadecyl Sepabeads), ROL2 (immobilized on
proach that can be used to tune the lipase properties by Lewatit 1600) and ROL3 (immobilized on aldehyde-acti-
using different immobilization protocols.[36,37] Thus, im- vated Lewatit CNP 105).
mobilization of a particular lipase in different orientations,
Previous experiments showed that the performance of
with different rigidity, or in the presence of different mi- enzymes in the acetylation reaction was improved when
croenvironments permits a range of biocatalysts to be gen- they were dried before use. The water percentage in the
2
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An Efficient Catalyst in the Acetylation of Cortexolone
three immobilized ROL was variable: 56% in ROL1, 46% zyme is immobilized through the rich area of lysines, and
in ROL2, and 70% in ROL3. the open conformation is not fixed. Therefore, the “opening
The four lipase preparations were evaluated in the acety- of the lid” and the accessibility of the active site seems to
lation of 1 with ethyl, vinyl, and isopropenyl acetate. Only be blocked by this immobilization strategy.
the last substrate gave satisfactory results and was selected
as acylating agent. In the absence of biocatalyst no product ing was performed to establish the best solvent for this bio-
was obtained. catalytic acetylation. With the aim that the enzyme activity
Having established the optimal catalyst, a solvent screen-
Among the tested enzymes, ROL2 gave the most satisfac- and stability were affected as little as possible, three vari-
tory results. ROL1 and ROL3 showed a poor activity, and ous-polarity solvents [hexane, diisopropyl ether (DIPE),
with ROL0 cortexolone and isopropopenyl acetate did not and acetonitrile (MeCN)] were tested. The experiments
react at all (Table 1, Entries 1–12). In each case, only the C- (Table 1, Entries 2, 5, 8 and 11) clearly identified DIPE as
21 hydroxy group of cortexolone was acetylated.
the most efficient solvent for the reaction, and it was used
It was observed that the immobilized lipase ROL2 was in subsequent experiments for its ability to produce the
much more reactive than the free lipase. Immobilization of highest conversion.
the lipase fixes its open conformation, which can result in
Regarding the optimum temperature (Table 1, Entries 13
significant stabilization of the enzyme, especially for appli- and 14), we also performed the acetylation at 30 °C and,
cations in organic solvent. Furthermore, the large differ- even after longer periods of time, ROL2 performance de-
ences in reactivity between the immobilized and the native creased to 20.2% conversion showing that the ROL2 ac-
heterologous enzyme could be explained by considering tivity increased with an increase in temperature. Therefore,
that the native enzyme is probably a mixture of different 55 °C was chosen as the reaction temperature in subsequent
forms in organic solvent (aggregates, inactivated form for experiments.
the solvent, lipase molecule in closed conformation, lipase
To determine the best acylating agent/substrate (A/S) ra-
molecules in open conformation, etc.) whereas in the immo- tio, reactions with various concentrations of the acylating
bilized form, homogeneous distributed monomeric lipase agent were carried out (Figure 1).
molecules predominate.
An increase in conversion with an increase in A/S from
In the case of ROL2, immobilization of the lipase on 1 to 50 was observed. With A/S = 50 the conversion reached
Lewatit resin goes through a hydrophobic adsorption almost 100% under the present parameters.
mechanism that differs from, for example, the simple ad-
To evaluate the enzyme/substrate (E/S) ratio, we per-
sorption on hydrophobic Sepabeads supports (ROL1) that formed the acetylation reaction using various ROL2
was previously demonstrated.[40] Regarding ROL3 catalyst, amounts (Figure 2). From these experiments we observed
the immobilization is performed though a multipoint coval- that the use of E/S ratios between 15 and 50 gave almost
ent attachment on an aldehyde-activated support. The en- quantitative conversion.
Table 1. Optimization of reaction parameters for ROL-catalyzed acetylation of cortexolone (1).[a]
Entry
Enzyme
Solvent
Temperature [°C]
E/S ratio
A/S ratio
t [h]
Conversion [%]
Solvent
1
2
3
4
5
6
7
8
9
ROL0
ROL0
ROL0
ROL1
ROL1
ROL1
ROL2
ROL2
ROL2
ROL3
ROL3
ROL3
hexane
DIPE
MeCN
hexane
DIPE
MeCN
hexane
DIPE
MeCN
hexane
DIPE
55
55
55
55
55
55
55
55
55
55
55
55
30
30
30
30
30
30
30
30
30
30
30
30
25
25
25
25
25
25
25
25
25
25
25
25
48
48
48
48
48
48
48
48
48
48
48
48
n.d.
0.3
n.d.
n.d.
4.9
n.d.
23.6
52.1
n.d.
n.d.
5.0
10
11
12
MeCN
n.d.
Temperature
Time
13
14
ROL2
ROL2
hexane
DIPE
30
30
30
30
25
25
72
72
9.8
20.2
15
16
17
18
19
20
ROL 2
ROL 2
ROL 2
ROL 2
ROL 2
ROL 2
DIPE
DIPE
DIPE
DIPE
DIPE
DIPE
55
55
55
55
55
55
30
30
30
30
30
30
50
50
50
50
50
50
1.5
3
8
24
48
72
6.7
15.2
27.7
71.5
99.4
99.9
[a] Substrate concentration 1 mg/mL.
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Figure 1. Influence of enzyme/substrate ratio on the conversion of
cortexolone-21-acetate.
Figure 3. Diagram of the central composite rotatable design, using
the nomenclature (E/S);(A/S).
The reaction conditions were also fixed following the re-
sults obtained in the acetylation reaction. Therefore, the
biocatalyst was previously dried, temperature and shaking
were fixed at 55 °C and 200 rpm, respectively, isopropenyl
acetate was used as acetylating agent and DIPE as solvent.
The productivities of each acetylation reaction are shown
in Table 2.
Table 2. Specific yield of each acetylation reaction of the experi-
mental design.[a]
E/S
A/S
Specific yield
(mmol P/mmol A·g E)
5
50
35.8
30
35.8
50
64.18
70
64.18
50
1.56
1.34
0.92
0.72
0.51
0.33
0.46
0.84
0.81
0.81
0.81
0.81
7.18
12.5
17.8
20
17.8
12.5
7.18
12.5
12.5
12.5
12.5
Figure 2. Influence of enzyme/substrate ratio on the conversion of
cortexolone-21-acetate.
50
50
50
Taking into account the experiments mentioned above,
we selected the following preliminary standard conditions
for the biocatalytic acetylation of cortexolone with ROL2:
E/S ratio 30, solvent DIPE, temperature 55 °C, and isopro-
penyl acetate/cortexolone ratio 50.
Finally, the optimum reaction time was also studied, and
the results (Table 1, Entries 15–20) show that 48 h was opti-
mal.
Using the response surface methodology and a central
composite experimental design, the optimal values of E/S
and A/S were determined with the aim of maximizing the
specific yield defined as mmol of product (P) per mmol of
acylating agent and gram of enzyme (mmol P/mmol A·g E)
using ROL2. Taking into account the previous results, the
[a] Reaction conditions: Substrate (1 mg/mL), DIPE, isopropenyl
acetate, 55 °C, rotary shaking at 200 rpm, 48 h.
The obtained data was fitted to the mathematical model
expressed in Equation (1) and the values of each coefficient,
as well as the statistical parameter p value, are shown in
Table 3.
Therefore, the equation obtained for the specific yield is
expressed in Equation (1), and the corresponding response
surface is plotted in Figure 4.
Specific yield (mmol P/mmol A·g E) = 0.8091 – 0.3275·(E/S) –
0.1923·(A/S) + 0.099·(E/S)2 – 0.0738·(A/S)2
(1)
Firstly, the interaction effect of (E/S)·(A/S) had no sig-
selected enzyme/substrate ratio range was 5–50 and the acy- nificant effect on the specific yield, and the term E/S was
lating agent/substrate ratio was 30–70. The selected experi- the most significant one. Moreover, when the coefficients of
mental design is described in Figure 3.
E/S and A/S are compared, the former had higher values.
4
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An Efficient Catalyst in the Acetylation of Cortexolone
Table 3. Parameters of the empirical equations obtained for the sta-
bility response surfaces of specific yield (mmol P/mmol A·g E).
Finally, ROL2 is recyclable. Because the immobilized en-
zyme is insoluble in the organic reaction media, it is easily
removed by filtration at the end of the process. It can be
reused and, in this particular reaction, ROL2 kept 89% of
its activity after four reaction cycles (Figure 5).
Coefficient
Value
p value
a
b
c
d
e
f
0.8091
–0.3275
–0.1923
0.0990
Ͻ0.0001
Ͻ0.0001
0.0003
0.0134
0.0414
–0.0738
0.0272[a]
0.4781
[a] Not considered a significant factor (p Ͼ 0.05).
Figure 5. ROL2 reuse in the acetylation of cortexolone under stan-
dard conditions.
Conclusions
In this work we have studied the performance of three
immobilized heterologous Rhizopus oryzae lipases as cata-
lysts in the acetylation of cortexolone. The enzymatic reac-
tion was regioselective, and only cortexolone-21-acetate was
obtained. ROL immobilized on Lewatit 1600 (ROL2)
proved to be the best biocatalyst in the preparation of the
steroid derivative.
Figure 4. Response surface, according to the experimental design,
corresponding to the specific yield of cortexolone-21-acetate under
different ratios of E/S and A/S. Reactions were carried out at 55 °C
and 200 rpm, using dry ROL2, DIPE as solvent and isopropenyl
acetate as acetylating agent. Reaction time was 48 h.
Thus, the effect on the specific yield of the enzyme/substrate
The reaction was performed at 55 °C, using isopropenyl
ratio was higher than the effect of the acylating agent/sub- acetate as acylating agent and diisopropyl ether as solvent.
strate ratio. The optimum values of E/S and A/S were 5 and By means of a response surface methodology and a central
31.6, respectively, entailing a predicted maximum specific composite rotatable design, the optimal values of enzyme/
yield of 1.59 mmol P/mmol A·g E.
substrate ratio and acylating/substrate ratio were deter-
Because the acylating agent is in excess, some acetic acid mined to maximize the specific yield. The optimal values of
could be produced by enzymatic hydrolysis of isopropenyl E/S and A/S were 5 and 31.6 respectively, lower than the
acetate. To test if acetic acid could act as a catalyst, we values obtained from individual experiments, saving both
performed the acetylation of cortexolone without enzyme, enzyme and acylating substrate.
using isopropenyl acetate and variable amounts of acetic
Finally, the enzymatic approach provides a simple and
acid (20, 40, 60, and 100 mm). In no case was any product mild alternative method for the synthesis of this cortico-
detected after 48 h of reaction, indicating that acetic acid steroid derivative. The application of ROL in an acetylation
does not catalyze the acetylation of cortexolone.
reaction on a steroid substrate such as cortexolone was not
Moreover, with the aim of studying the potential influ- previously reported, and the results show that this enzyme
ence of acetic acid on the enzymatic activity, acetic acid can be an efficient catalyst on complex and rigid substrates
(20 mm) was added to the enzymatic acetylation under the such as steroids, which are difficult to transform in a re-
standard conditions. In this case, we observed that the pres- gioselective way by using traditional synthetic procedures.
ence of acetic acid did not affect the enzyme activity; cor- Moreover, immobilized ROL kept 89% of its activity after
texolone-21-acetate was obtained as the only product in four reaction cycles.
100% conversion after 48 h of reaction. In conclusion, un-
The broad application of steroid derivatives and, particu-
der standard conditions the enzymatic acetylation of cor- larly, corticosteroids, as pharmaceuticals makes biocatalysis
texolone is only catalyzed by the lipase, and the possible an appropriate way to prepare them in high purity, which
presence of acetic acid does not influence the enzyme be- is an essential requisite for a product designed for human
havior.
consumption.
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A. Baldessari et al.
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by Bradford’s assay and the enzymatic activity assay described
above.
Experimental Section
General: All chemicals used in this work were of analytical grade
and purchased from Sigma–Aldrich. The free and immobilized en-
zymes were dried in a vacuum drying oven (0.1 kPa, 30 °C) over-
night before use. Enzyme/substrate ratio (E/S): enzyme amount
(ROL0, 1, 2 and 3) in mg/substrate amount in mg. Acylating agent/
substrate ratio (A/S): acylating agent amount in mg/substrate
amount in mg. Enzyme activity: ROL1: 9.4 UI/mg, ROL2: 160 UI/
mg, ROL3: 9.6 UI/mg. The enzymatic reaction was carried out with
an Innova 4000 digital incubator shaker, New Brunswick Scientific
Co., at 200 rpm. To monitor the reaction progress, aliquots were
withdrawn and analyzed by HPLC with a Waters 1515 chromato-
graph equipped with a UV detector set a 254 nm, and Symmetry
C18 (5 μm, 0.46 cm i.d. ϫ 15 cm) eluted at 30 °C with a methanol/
water mixture (80:20, v/v) at 0.5 mLmin–1. Retention times: cor-
texolone 4.4 min; cortexolone-21-acetate 5.7 min. Melting points
were determined with a Fisher Johns apparatus. NMR spectra were
recorded in CDCl3 as solvent with a Bruker AC-200 NMR instru-
ment operating at 200.1 and 50.2 MHz for 1H and 13C, respectively.
Chemical shifts are reported in δ units (ppm) relative to tetrameth-
ylsilane (TMS) set at δ = 0 ppm; coupling constants are given in
Hz.
Multipoint Covalent Immobilization of ROL on Aldehyde-Activated
Lewatit CNP 105 (ROL3): The commercial resin Lewatit CNP 105
was previously activated with aldehyde groups as reported in the
literature.[43] Aldehyde-activated Lewatit CNP 105 (2 g) was added
to sodium hydrogen carbonate buffer (20 mL, 100 mm, pH = 10.1)
containing lipase (25 mg; the amount of lipase was determined by
Bradford’s assay[41] of crude ROL). The mixture was then gently
stirred at 25 °C and 250 rpm on a Coulter stirrer for 24 h. When
the immobilization was finished (analyzed by enzymatic activity
assay described above), NaBH4 (20 mg) was added, and, after
30 min, the suspension was filtered through a sintered glass filter,
and the covalently immobilized lipase was washed several times
with abundant distilled water. The percentage of immobilization
was 55% (determined by the Bradford’s assay and the enzymatic
activity assay described above).
Enzymatic Acetylation of Cortexolone: To a solution of cortexolone
(0.1 g, 0.29 mmol) in diisopropyl ether (100 mL), isopropenyl acet-
ate (0.94 g, 9.16 mmol) and ROL immobilized on Lewatit 1600
(0.5 g) were added. The reaction mixture was incubated at 55 °C
and shaken at 200 rpm for 48 h. Once the reaction was finished,
the enzyme was filtered off, washed with DIPE (3ϫ10 mL), and
the solvent was removed under reduced pressure. The residue was
purified by column chromatography on silica gel (hexane/ethyl
acetate, 1:1, v/v). After solvent evaporation, the product was iso-
lated as a white solid (0.1 g, 0.26 mmol, 91% yield). M.p. 236–
238 °C (ref.[11] 235–238 °C). 1H NMR (200.1 MHz, CDCl3): δ =
0.72 (s, 3 H, CH3-18), 1.19 (s, 3 H, CH3-19), 2.17 (s, 3 H, CH3-23),
4.86 (d, J = 18 Hz, 1 H, CH2-21), 5.09 (d, J = 18 Hz, 1 H, CH2-
21), 5.74 (s, 1 H, =CH-4) ppm. 13C NMR (50.2 MHz, CDCl3): δ
= 14.5 (C-18), 17.4 (C-19), 20.6 (C-23), 68.0 (C-21), 123.9 (C-4),
171.0 (C-5), 171.2 (C-22), 199.7 (C-3), 205.3 (C-20) ppm. Reuse
experiments: the filtered and washed enzyme was used in the next
enzymatic acetylation under the same reaction conditions.
Enzyme Production: ROL was produced by the Bioprocess Engi-
neering and Applied Biocatalysis group of the Universitat Autò-
noma de Barcelona (UAB). This lipase is obtained by a mixed sub-
strates-fed batch cultivation of a recombinant P. pastoris strain
using methanol as inductor.[34] The culture broth was centrifuged
and microfiltered to remove the biomass. The supernatant was con-
centrated by ultrafiltration with a Centrasette Pall Filtron system
equipped with an Omega membrane of 10 kDa cut-off, and sub-
sequently dialyzed against 10 mm Tris-HCl buffer (pH = 7.5) and
thereafter lyophilized.[41]
Enzyme Immobilization
Standard Enzymatic Activity Assay Determination: To follow the
immobilization process, the activities of ROL0 and its immobilized
preparations (ROL1, ROL2 and ROL3) were analyzed spectropho-
tometrically measuring the increment in absorbance at 348 nm (ε
= 5.150 m–1 cm–1) produced by the release of p-nitrophenol (pNP)
in the hydrolysis of 0.4 mm pNPB in 25 mm sodium phosphate
buffer at pH = 7 and 25 °C. To initialize the reaction, 0.05–0.2 mL
of lipase solution (blank or supernatant) or suspension was added
to 2.5 mL of substrate solution.
Effect of Enzyme/Substrate Ratio and Acylating Agent/Substrate
Ratio on Specific Yield: The effect of enzyme/substrate (E/S) and
acylating/substrate (A/S) ratio on the specific yield of cortexolone-
21-acetate using ROL2 (ROL immobilized on Lewatit 1600) was
studied by means of a Box-Hunter experiment design[44] and the
response surface methodology. The applied central composite ro-
tatable design (CCRD) was formed by 12 experiments, based in
two variables having five levels each. The value of α was 1.41 and
four center points for replication were carried out. The enzyme/
substrate and acyl/substrate ratio ranges were selected by taking
into account the results obtained in previous enzymatic acetylation
reactions. The enzyme/substrate ratio range was 5–50 and the acyl-
ating/substrate ratio was 30–70. The values of both variables were
codified from –1.41 to 1.41. The empirical response surfaces were
built from the values of specific yield expressed as mmol P/
mmol A·g E, after 48 h of acetylation reaction. The data results fit
the empirical model expressed in Equation (2):
Adsorption of ROL on Octadecyl Sepabeads (ROL1): Sepabeads
(EC-OD) (2 g) were added to sodium dihydrogen phosphate buffer
(20 mL, 25 mm, pH = 7) containing lipase (25 mg; the amount of
lipase was determined by Bradford’s assay[42] of crude ROL). The
mixture was then gently stirred at 25 °C and 250 rpm on a Coulter
stirrer for 3 h. The solution was filtered through a sintered glass
filter, and the supported lipase was washed several times with abun-
dant distilled water. The percentage of immobilization was 80%
determined by the Bradford’s assay and the enzymatic activity as-
say described above.
Specific yield (mmol P/mmol A·g E) =
a + b·(E/S) + c·(A/S) + d·(E/S)2 + e·(A/S)2 + f·(E/S)·(A/S) (2)
Adsorption of ROL on Lewatit 1600 (ROL2): Lewatit VP OC1600
(2 g) was added to sodium dihydrogen phosphate buffer (20 mL,
25 mm, pH = 7) containing lipase (25 mg; the amount of lipase was
determined by Bradford’s assay[42] of crude ROL). The mixture was
then gently stirred at 25 °C and 250 rpm on a Coulter stirrer for
3 h. The solution was filtered through a sintered glass filter, and
the supported lipase was washed several times with abundant dis-
tilled water. The percentage of immobilization was 86% determined
The reaction conditions were fixed by taking into account the re-
sults obtained previously. Therefore, under standard conditions, the
immobilized biocatalyst was used dry, and temperature and shak-
ing were fixed at 55 °C and 200 rpm, respectively. DIPE was used
as a solvent, and cortexolone (2 mg in 2 mL) was acetylated by
using isopropenyl acetate.
6
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An Efficient Catalyst in the Acetylation of Cortexolone
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Acknowledgments
We thank the Universidad de Buenos Aires (grant UBA X010), the
Consejo Nacional de Investigaciones Científicas y Técnicas (grant
CONICET PIP) (112-200801-00801/09) and the Agencia Nacional
de Promoción Científica y Tecnológica (grant ANPCyT PICT
2005-32735) for partial financial support. A. B. is a Research Mem-
ber of the Science and Technology Research Council (CONICET).
This work was also supported by a project of the Spanish Ministry
of Science and Innovation (CTQ2010-15131) and the Reference
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Published Online: ᭿
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7

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/KAP1
Date: 13-06-12 16:45:37
Pages: 8
A. Baldessari et al.
FULL PAPER
Supported Catalysts
The regioselective acetylation of cor-
texolone was achieved by using an immobi-
lized heterologous Rhizopus oryzae lipase.
The mild reaction conditions and low en-
vironmental impact make the biocatalytic
procedure a convenient way to prepare the
monoacetyl derivative of this biologically
active steroid.
P. G. Quintana, M. Guillén, M. Marciello,
F. Valero, J. M. Palomo,
A. Baldessari* ................................... 1–8
Immobilized Heterologous Rhizopus Ory-
zae Lipase as an Efficient Catalyst in the
Acetylation of Cortexolone
Keywords: Steroids / Acylation / Biocatal-
ysis / Enzymes / Immobilization / Sup-
ported catalysts
8
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0